Novel ofloxacin derivatives: Synthesis, antimycobacterial and toxicological evaluation

Article abstract of DOI:10.1016/j.bmcl.2007.11.110

Thirty novel 9-fluoro-2,3-dihydro-8,10-(mono/di-sub)-3-methyl-8-nitro-7-oxo-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acids were synthesized from 2,3,4,5-tetrafluoro benzoic acid and evaluated for in vitro and in vivo antimycobacterial activities against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant Mycobacterium tuberculosis (MDR-TB), and Mycobacterium smegmatis (MC²) and also tested for the ability to inhibit the supercoiling activity of DNA gyrase from mycobacteria. Among the synthesized compounds, 10-[2-carboxy-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl]-9-fluoro-2,3-dihydro-3-methyl-8-nitro-7-oxo-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid was found to be the most active compound in vitro with MIC99 of 0.19 μM and 0.09 μM against MTB and MTR-TB, respectively. In the in vivo animal model also the same compound decreased the bacterial load in lung and spleen tissues with 1.91 and 2.91 - log 10 protections, respectively, at the dose of 50 mg/kg body weight. Compound 10-[(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)]-9-fluoro-2,3-dihydro-3-methyl-8-nitro-7-oxo-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid was found to be the most active in the inhibition of the supercoiling activity of DNA gyrase with an IC₅₀ of 10.0 μg/mL. The results demonstrate the potential and importance of developing new oxazino quinolone derivatives against mycobacterial infections.

Full text of DOI:10.1016/j.bmcl.2007.11.110

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1229–1236
Novel ofloxacin derivatives: Synthesis, antimycobacterial
and toxicological evaluation
a
a
a
Murugesan Dinakaran, Palaniappan Senthilkumar, Perumal Yogeeswari,
b
b
a,
*
Arnab China, Valakunja Nagaraja and Dharmarajan Sriram
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, Karnataka 560012, India
Received 8 October 2007; revised 23 November 2007; accepted 28 November 2007
Available online 4 December 2007
Abstract—Thirty novel 9-fluoro-2,3-dihydro-8,10-(mono/di-sub)-3-methyl-8-nitro-7-oxo-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-car-
boxylic acids were synthesized from 2,3,4,5-tetrafluoro benzoic acid and evaluated for in vitro and in vivo antimycobacterial activ-
ities against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant Mycobacterium tuberculosis (MDR-TB), and
2
Mycobacterium smegmatis (MC ) and also tested for the ability to inhibit the supercoiling activity of DNA gyrase from mycobac-
teria. Among the synthesized compounds, 10-[2-carboxy-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl]-9-fluoro-2,3-dihydro-3-
methyl-8-nitro-7-oxo-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid was found to be the most active compound in vitro with
MIC99 of 0.19 lM and 0.09 lM against MTB and MTR-TB, respectively. In the in vivo animal model also the same compound
decreased the bacterial load in lung and spleen tissues with 1.91 and 2.91 ꢀ log10 protections, respectively, at the dose of 50 mg/
kg body weight. Compound 10-[(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)]-9-fluoro-2,3-dihydro-3-methyl-8-nitro-7-oxo-
7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid was found to be the most active in the inhibition of the supercoiling activity
of DNA gyrase with an IC50 of 10.0 lg/mL. The results demonstrate the potential and importance of developing new oxazino quin-
olone derivatives against mycobacterial infections.
Ó 2007 Elsevier Ltd. All rights reserved.
Fluoroquinolones exhibit potent in vitro and in vivo
1
There is also a considerable effort to discover and devel-
op newer fluoroquinolones, and some of them might
antimycobacterial activity. There is a significant effort
to include fluoroquinolones as new front-line agents
6
,7
have value in the treatment of TB. As a part of the
study attempting to further optimize the quinolones
(
(
ofloxacin and moxifloxacin) and second-line agents
2
ciprofloxacin) to combat tuberculosis (TB). Experts
8,9
against Mycobacterium tuberculosis (MTB)
multi-drug resistant M. tuberculosis (MDR-TB) as
and
from the World Health Organization favor a third-line
regimen containing four drugs (an aminoglycoside, ethi-
onamide, pyrazinamide, and ofloxacin) during the initial
phase and two drugs (ethionamide and ofloxacin) during
1
0–12
MTB is getting resistance to many quinolones,
here-
with we report the synthesis, antimycobacterial and
toxicological evaluation of novel 9-fluoro-2,3-dihydro-
8,10-(mono/di-sub)-3-methyl-8-nitro-7-oxo-7H-[1,4]oxaz-
ino[2,3,4-ij]quinoline-6-carboxylic acids.
3
the continuation phase. Quinolones inhibit bacterial
type II topoisomerase, DNA gyrase, and topoisomerase
4
IV, which are essential enzymes that maintain the
supercoiling of the DNA by carrying out two opposing
reactions. The incidence of mycobacterial resistance to
fluoroquinolones is relatively low at the present time,
and there are no reports of cross-resistance or antago-
Thirty new oxazinoquinolone carboxylic acids with var-
iation at C and C positions were synthesized from
2,3,4,5-tetrafluoro benzoic acid (1) according to the lit-
9
10
1
3
erature method
(Scheme 1). Briefly, compound 1 on reaction with 1,
with modification in some steps
5
nism with other classes of antimycobacterial agents.
0
1
-carbonyldiimidazole in tetrahydrofuran afforded the
corresponding imidazolide, which, in situ, was treated
with neutral magnesium salt of ethyl potassium malon-
ate in the presence of tri-ethyl amine to yield 82% of
Keywords: Oxazino[2,3,4-ij]quinoline-6-carboxylic acids; Antimyco-
bacterial activity; Tuberculosis.
*
Corresponding author. Tel.: +91 1596 244684; fax: +91 1596
44183; e-mail: dsriram@bits-pilani.ac.in
ethyl
3-(2,3,4,5-tetrafluorophenyl)-3-oxopropanoate
2
(2). Ethyl 2,3,4,5-tetrafluoro-a-[[(2-hydroxy-1-methyl-
0
960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.110

1230
M. Dinakaran et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1229–1236
O
O
O
F
O
F
O
F
O
F
O
OC H5
2
F
F
F
F
OH
OC H
F
F
a
2
5
b
OC H
c
d
2
5
F
F
F
HN
OC H5
2
F
F
1
2
F
3
4
CH3
H
OH
O
O
R
O
N
O
R
O
O
N
O
F
F
F
OC H
F
2
5
OH
e, f
g
OH
R1
N
N
F
F
5
O
6:R=H
7:R=NO2
8a-n
9a-n
10k,m
1
CH3
R
CH3
H
CH
3
H
OH
H
Scheme 1. Synthetic protocol of the compounds. Reagents and conditions: (a) CDI, EtOCOCH
-amino-1-propanol, EtOH/Et O (50:50); (d) DMSO, K CO ; (e) THF, 10% KOH; (f) H SO , KNO
h) H , Ran.Ni, DMF.
2
CO
2
K, MgCl
2
, Et
3
N; (b) (EtO)
3
2
CH, AC O; (c) (+)-
2
2
2
3
2
4
3
; (g) various secondary amines, DMSO, MWI;
(
2
ethyl)amino]methylene]-b-oxo-benzenepropanoic acid
4) was prepared by a two-step one-pot reaction. First
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.
(
treatment of the keto ester 2 with tri-ethyl orthoformate
in acetic anhydride gave the one-carbon homologue enol
ether intermediate ethyl a-(ethoxymethylene)-2,3,4,5-
tetrafluoro-b-oxo-benzenepropanoic acid (3) as an oil,
which on reaction with (S)-(+)-2-amino-1-propanol at
In the first phase of screening against MTB, all the com-
pounds showed excellent in vitro activity against MTB
with MIC99 less than 15 lM. Thirteen compounds
(9b, 9g, 9h, 9i, 8j, 9j, 8k, 9k, 9l, 8m, 9m, 8n, and 9n)
inhibited MTB with MIC99 of less than 2 lM and were
more potent than standard ofloxacin (MIC99: 2.16 lM).
When compared to isoniazid (MIC99: 0.36 lM), three
compounds (9j, 9k, and 9m) were found to be more ac-
tive against MTB. Compound 10-[2-carboxy-5,6-dihy-
droimidazo[1,2-a]pyrazin-7(8H)-yl]-9-fluoro-2,3-dihydro-3-
methyl-8-nitro-7-oxo-7H-[1,4]oxazino[2,3,4-ij]quinoline-
6-carboxylic acid (9m) was found to be the most active
compound in vitro with MIC99 of 0.19 lM against
MTB, and was 2 and 11 times more potent than isonia-
zid and ofloxacin, respectively. Subsequently some of
the compounds were evaluated against MDR-TB, and
among the twenty compounds screened, all the com-
pounds inhibited MDR-TB with MIC99 ranging from
0.09 to 8.30 lM and were found to be more active than
isoniazid (MIC99: 45.57 lM) and ofloxacin (MIC99:
34.59 lM). Six compounds (8j, 9j, 8k, 9k, 8m and 9m)
inhibited MDR-TB with MIC99 of less than 1 lM.
Compounds 8k, 9k and 9m were found to be the most
active compound in vitro with MIC99 of 0.09 lM
against MDR-TB and were 384 and 506 times more po-
tent than ofloxacin and isoniazid, respectively. The com-
0
°C affords 67% of 4 as an oily residue. Compound 4
on cyclization with the base potassium carbonate in
dimethylsulfoxide yielded 62% of ethyl 6,7,8-trifluoro-
1
olinecarboxylic acid (5), when compared to earlier re-
,4-dihydro-1-(2-hydroxy-1-methylethyl)-4-oxo-3-quin-
9
port, this step proceeds smoothly without using
violently reactive sodium hydride. Further cyclization
and hydrolysis of compound 5 was done by heating with
1
0% aqueous potassium hydroxide in tetrahydrofuran,
affording 9,10-difluoro-2,3-dihydro-3-methyl-7-oxo-7H-
1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (6) with
0% yield, which on nitration at C position with sulfu-
[
7
8
ric acid and potassium nitrate yielded 88% of 9,10-di-
fluoro-2,3-dihydro-3-methyl-8-nitro-7-oxo-7H-[1,4]oxaz-
ino[2,3,4-ij]quinoline-6-carboxylic acid (7). Compounds
6 and 7 on further reaction with various secondary
amines under microwave irradiation in dimethylsulfox-
ide afforded titled compounds 8, 9a–n. When compared
1
3
to conventional method of 12 h process, microwave
assisted synthesis was performed with short reaction
times (3–6 min), with ease, and was environment
friendly. Compounds 9k and 9m were further reduced
by catalytic reduction of the nitro group to yield 10k
and 10m. The purity of the synthesized compounds
was monitored by thin layer chromatography (TLC)
and elemental analyses, and the structures were identi-
2
pounds were also evaluated against MC in which all the
compounds inhibited MC with MIC99 ranging from
2
1
4
fied by spectral data.
0.66 to 114.29 lM; twenty-three compounds were found
to be more active than isoniazid (MIC99: 45.57 lM) and
six compounds were more active than ofloxacin (MIC99:
2.16 lM).
The compounds were screened for their in vitro antimy-
cobacterial activity against MTB, MDR-TB, and Myco-
2
bacterium smegmatis ATCC 14468 (MC ) by agar
dilution method for the determination of MIC99 in
With respect to structure–MTB activity relationship, the
results demonstrated that the antimycobacterial activity
was enhanced 1.1- to 8.8-fold by the introduction of ni-
tro group at C position. Reduction of nitro group at C
1
5
duplicate. The MDR-TB clinical isolate was resistant
to isoniazid, rifampicin, ethambutol, and ofloxacin.
The minimum inhibitory concentration (MIC99) is
defined as the minimum concentration of compound
8
8
position to amino group (10k and 10m) reduces the

M. Dinakaran et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1229–1236
Table 1. Physical constants, in vitro antimycobacterial activities, and cytotoxicity
1231
R
O
O
F
OH
R1
N
O
Compound
R
R
1
Yield (%)
mp (°C)
IC50 (lM)
MIC99 (lM)
2
MTB
5.71
MDR-TB
MC
Cl
8a
H
68
168
NT
NT
45.62
N
N
Cl
9a
NO
2
62
>250
105.39
2.63
2.63
0.66
N
N
O
C
8
9
8
9
8
b
b
c
c
d
H
N
N
N
N
73
66
73
80
69
118
216
188
215
154
NT
7.09
0.80
6.50
2.96
14.29
NT
1.60
NT
5.95
NT
3.53
O
O
O
C
NO
2
64.25
NT
3.21
H
2
C
C
N
N
N
O
O
H
25.96
1.48
O
H
2
N
NO
2
118.72
NT
O
N
N
H
3
C
C
N
H
114.29
C
6
H
5
H
3
N
9
8
d
e
NO
2
80
70
211
172
129.54
3.23
3.23
NT
6.49
C H
6 5
O
O
C
H
NT
12.27
49.07
N
N
O
(continued on next page)

1232
M. Dinakaran et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1229–1236
Table 1 (continued)
Compound
R
R
1
Yield (%)
mp (°C)
IC50 (lM)
MIC99 (lM)
2
MTB
2.81
MDR-TB
MC
O
O
C
9e
NO
2
83
224
112.72
1.41
11.27
43.98
N
N
O
F
O
C
8
f
H
N
N
70
84
193
208
NT
10.99
NT
N
CH
3
O
F
F
O
C
9f
NO
2
N
N
101.87
5.10
NT
1.27
N
CH
3
O
F
8
9
8
g
g
h
H
S
S
O
N
77
81
73
163
>171.52
152.67
2.14
1.91
4.14
8.59
1.91
8.3
4.28
1.91
4.14
NO
2
N
N
>250
124
H
>166.06
9h
NO
2
O
N
84
>250
148.32
1.85
3.70
1.85
HO
HO
8
9
i
i
H
70
76
136
220
NT
6.62
1.51
NT
26.43
6.04
Cl
Cl
N
N
NO
2
60.35
3.01
N
N
8
9
8
j
H
67
78
64
178
205
152
>121.85
112.03
154.55
1.52
0.34
0.47
0.76
0.34
0.09
48.74
1.39
3.86
O
N
H
Cl
N
N
j
NO
2
O
N
H
Cl
O
O
k
H
N

M. Dinakaran et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1229–1236
1233
Table 1 (continued)
Compound
R
R
1
Yield (%)
69
mp (°C)
IC50 (lM)
MIC99 (lM)
MDR-TB
0.09
2
MTB
0.21
MC
9
1
8
k
NO
2
O
225
139.08
3.47
N
N
O
O
0k
NH
2
61
75
189
172
>149.02
126.64
3.72
3.16
3.72
3.16
11.16
50.66
O
N
l
H
O
C
3 3
NHC(CH )
N
9l
NO
2
82
>250
116.06
1.45
1.45
2.89
O
C
3 3
NHC(CH )
N
N
8
9
m
m
H
HOOC
HOOC
78
84
208
>145.90
132.03
0.91
0.19
0.44
0.09
29.18
6.61
N
N
N
N
NO
2
>250
N
1
8
0m
n
NH
2
HOOC
59
72
219
168
>140.96
>137.53
0.88
1.72
1.76
3.43
5.28
N
N
H
3
CO
H
13.75
N
H
H
3
CO
CO
3
9n
NO
2
75
191
62.5
1.56
3.12
12.51
N
H
3
CO
Oflox
INH
—
—
—
—
—
—
—
—
>155.3
>455.8
2.16
0.36
34.59
45.57
2.16
45.57
NT indicates not tested.
activity indicating that favorable substitution at C was
8
the substitution pattern at C demonstrated that nitro
8
NO > H > NH . At C position we have studied with
2
group was more cytotoxic than the unsubstituted deriv-
atives. These results are important as the C nitro substi-
2
9
various substituted piperazines (8 and 9a–f) (thio) mor-
pholines (8 and 9g–h), substituted piperidines (8 and 9i–
j), fused piperazines, and piperidines (8 and 9k–n). A
8
tuted compounds with their increased cytoliability are
much less attractive in the development of a quinolone
for the treatment of TB. This is primarily due to the fact
that the eradication of TB requires a lengthy course of
treatment, and the need for an agent with a high margin
of safety becomes a primary concern. The IC50 value of
compound 9m was found to be 132.03 lM and showed
selectivity index (IC /MIC) of more than 1467.
comparison of the substitution pattern at C demon-
9
strated that the order of activity was fused piperazines
and piperidines > substituted piperidines P (thio) mor-
pholines > substituted piperazines.
Some compounds were further examined for toxicity
5
0
(
IC ) in a mammalian Vero cell line at 62.5 lg/mL con-
50
16
centrations. After 72 h of exposure, viability was as-
sessed on the basis of cellular conversion of MTT into
a formazan product and the results are reported in Table
Subsequently, compound 9m was tested for in vivo effi-
cacy against MTB at a dose of 50 mg/kg (Table 2) in
8
CD-1 mice. The mice were infected intravenously with
M. tuberculosis ATCC 35801. Drug treatment by intra-
peritoneal route began after 10 days of inoculation of
1
. Twenty-one compounds when tested showed IC50 val-
ues ranging from 62.35 to 171.52 lM. A comparison of

1234
M. Dinakaran et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1229–1236
Table 2. In vivo activity data of 9m, ofloxacin, and isoniazid against
M. tuberculosis ATCC 35801 in mice
lungs were aseptically removed, and the number of via-
ble 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 9m decreased the
bacterial load in lung and spleen tissues with 1.91 and
2.91 ꢀ log10 protections, respectively, and was consid-
ered to be promising in reducing bacterial count in lung
and spleen tissues. When compared to ofloxacin at the
same dose level 9m decreased the bacterial load with
0.53 and 1.5 ꢀ log10 protections in lung and spleen tis-
sues, respectively.
Compound
Lungs
logCFU ± SEM)
Spleen
(logCFU ± SEM)
(
Control
Ofloxacin (50 mg/kg)
Isoniazid (25 mg/kg)
7.99 ± 0.16
6.61 ± 0.15
5.86 ± 0.23
6.08 ± 0.10
9.02 ± 0.21
7.61 ± 0.16
4.71 ± 0.10
6.11 ± 0.23
9m (50 mg/kg)
the animal with microorganism and continued for 10
days. After 35 days postinfection the spleens and right
Gyrase
-
+
+
+
+
+
+
Controls
Compounds
-
-
DMSO Mox
-
8b
-
8h
-
8k
-
8g
-
-
R
S
1
2
3
4
5
6
7
Gyrase
-
+
+
+
+
Controls
Compounds
-
-
DMSO Mox
-
8k
-
8g
-
-
R
S
1
2
3
4
5
Gyrase
-
-
-
+
+
+
-
+
-
+
-
+
-
Controls
DMSO Mox
Compounds
-
-
9a
9l
9h
9g
R
S
1
2
3
4
5
6
7
Gyrase
-
-
-
+
+
+
-
+
-
+
+
-
+
-
Controls
DMSO Mox
-
Compound 9a
-
-
40 30 20
10
5
μg/mL
R
S
1
2
3
4
5
6
7
8
Figure 1. DNA gyrase inhibition. (a) Lane 1: relaxed circular DNA, lane 2: supercoiling reaction in the presence of 5% DMSO used as a solvent
control, lane 3: moxifloxacin at 5 lg/mL concentration is used as the positive control. Lanes 4–7: reactions in the presence of 50 lg/mL of compounds
8b, 8h, 8k, and 8g, respectively. (b) Lane 1: relaxed circular DNA, lane 2: supercoiling reaction in the presence of 5% DMSO, lane 3: moxifloxacin
(5 lg/mL concentration). Lanes 4 and 5: reactions in the presence of 40 lg/mL of compounds 8k and 8g, respectively. (c) Lane 1: relaxed circular
DNA, lane 2: supercoiling reaction in the presence of 5% DMSO, lane 3: moxifloxacin (5 lg/mL). Lanes 4–7: reactions in the presence of 50 lg/mL of
compounds 9a, 9l, 9h, and 9g, respectively. (d) Lane 1: relaxed circular DNA, lane 2: supercoiling reaction in the presence of 5% DMSO, lane 3:
moxifloxacin at 5 lg/mL concentration. Lanes 4–8: reactions in the presence of 40, 30, 20, 10, and 5 lg/mL of compound 9a, respectively. R, relaxed
circular DNA substrate; S, supercoiled DNA product.

M. Dinakaran et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1229–1236
1235
Table 3. IC50 values for DNA gyrase inhibition
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 edema 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. Four
(9a, 8b, 9g, and 8k) compounds were evaluated for po-
tential phototoxicity in a standardized in vivo test sys-
tem that has been used previously to assess quinolone
antibiotics. The test compounds (140 mg/kg) and the
positive controls lomefloxacin and ofloxacin hydrochlo-
ride (140 mg/kg) were evaluated for phototoxicity and
both ears of each mouse were evaluated for changes
indicative of a positive response: erythema, edema or a
measurable increase in ear thickness. Change from base-
line was calculated separately for each animal and time
point and analyzed for statistical significance and is pre-
sented in Table 4. The drug and time factors were ana-
lyzed 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 sig-
nificantly different from zero. The results indicated that
lomefloxacin showed significant increase in ear thickness
from 4 to 96 h and 24 to 96 h when compared within
time points and with the control, respectively, but oflox-
acin did not show significant increase in ear thickness
when compared within time 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 or non-toxic when compared with the neg-
ative (vehicle-treated) and positive controls (lomefloxa-
cin and ofloxacin). No erythema occurred in mice
dosed with 140 mg/kg of 9a and 9g throughout the 96-
h study, while compound 8b showed a significant ery-
thema after irradiation untill 24 h only. Significant ery-
thema was observed in the ears of mice dosed with
compound 8k and this response was maximal after
Compound
IC50 (lg/ml)
8
8
8
8
9
9
9
9
b
h
k
g
a
l
>50
>50
50
40
10
50
h
g
>50
50
The quinolones and fluoroquinolones inhibit two en-
zymes viz., DNA gyrase and topoisomerase IV. The
reactions catalyzed by these ATP-dependent enzymes in-
volve a transient double-stranded break in DNA and
subsequent strand passage and religation reactions to
facilitate topological changes in DNA. Amongst the
two enzymes, DNA gyrase is unique in catalyzing the
negative supercoiling of DNA and has essential roles
during replication and transcription. Topoisomerase
IV is not found in many eubacteria including mycobac-
teria. Hence DNA gyrase is the sole target for quino-
1
8
lones
and
fluoroquinolones
in
mycobacteria.
Quinolones bind to the gyrase–DNA complex primarily
through their interaction with amino acid residues lo-
cated toward the amino terminus of GyrA subunit.
The ability of the newly synthesized quinolone deriva-
tives to inhibit DNA supercoiling by MC gyrase was
studied (Fig. 1). The IC50 values summarized in Table
2
3
compounds against the fixed amount of the enzyme in
were arrived at by taking various quantities of the
1
7
standard assay conditions. Amongst all the com-
pounds tested for the gyrase inhibition, compound 9a
showed lowest IC50 value of 10 lg/mL. Compound 8g
showed an IC50 value of 40 lg/mL and compounds 8k,
9
l, and 9g showed IC50 values of 50 lg/mL, respectively.
Rest of the compounds tested for gyrase inhibition, that
is, 8b, 8h, and 9h do not inhibit the enzyme effectively as
their IC50 values are higher (>50 lg/mL).
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-
Table 4. Phototoxic evaluation
c
Time (approximately) after start of irradiaton (h)
Group
a
Ear thickness (mm)
b
Erythema
0
4
24
48
72
96
0
4
24
48
72
96
d
Control
0.29 ± 0.02
0.28 ± 0.01
0.29 ± 0.02
0.25 ± 0.01
0.34 ± 0.01
0.29 ± 0.01
0.31 ± 0.01
0.29 ± 0.01
0.28 ± 0.01
0.30 ± 0.02
0.28 ± 0.02
0.37 ± 0.02
0.32 ± 0.01
0.40 ± 0.02
0.32 ± 0.02
0.28 ± 0.02
0.29 ± 0.01
0.29 ± 0.01
0.32 ± 0.01
0.29 ± 0.01
0.48 ± 0.02
0.31 ± 0.01
0.29 ± 0.01
0.29 ± 0.02
0.29 ± 0.01
0.31 ± 0.01
0.30 ± 0.01
0.53 ± 0.02
0.34 ± 0.03
0.29 ± 0.01
0.29 ± 0.02
0.28 ± 0.02
0.32 ± 0.01
0.35 ± 0.03
0.64 ± 0.04
0.34 ± 0.03
0.31 ± 0.01
0.30 ± 0.02
0.28 ± 0.01
0.35 ± 0.01
0.35 ± 0.04
0.60 ± 0.06
0
0
0
0
0
0
0
0
0
4
0
6
6
6
0
0
0
0
6
0
6
0
0
0
0
2
0
6
0
0
0
0
1
0
6
0
0
0
0
0
0
6
9
8
9
8
a
b
g
k
Ofloxacin
Lomefloxacin
a
b
c
Mean ear thickness ± SEM; left and right ears were averaged.
Number of mice with erythema.
Time zero = pre-dose (mice exposed to UV light immediately after dosing); 4 h = end of irradiation period.
Control = 0.5% aqueous solution of sodium carboxymethylcellulose (4 Ns/m2) dosed at 10 mL/kg.
d

1236
M. Dinakaran et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1229–1236
2
4 h but gradually subsided over 72 and 96 h of irradia-
4. Maxwell, A. Trends Microbiol. 1997, 5, 102.
5
6
. Bozeman, L.; Burman, W.; Metchock, B.; Welch, L.;
Weiner, M. Clin. Infect. Dis. 2002, 40, 386.
. 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,
tion. Significant erythema was seen in animals treated
with lomefloxacin throughout the 96-h study but oflox-
acin showed significant erythema only at 4 h which sub-
sided in next 24 h.
1
478.
Screening of the antimycobacterial activity of these no-
vel series identified newer potent antitubercular oxazi-
no[2,3,4-ij]quinoline-6-carboxylic acids endowed with
high activity toward MDR-TB, with MIC99 ranging
from 0.09 to 8.30 lM. In conclusion, it has been shown
that the potency, selectivity, and low cytotoxicity of
these compounds make them valid leads for synthesizing
new compounds that possess better activity. Further
structure–activity and mechanistic studies should prove
fruitful.
7
8
. Janin, Y. L. Bioorg. Med. Chem. 2007, 15, 2479.
. Sriram, D.; Yogeeswari, P.; Basha, S. J.; Radha, D. R.;
Nagaraja, V. Bioorg. Med. Chem. 2005, 13, 5774.
9. Sriram, D.; Aubry, A.; Yogeeswari, P.; Fisher, L. M.
Bioorg. Med. Chem. Lett. 2006, 16, 2982.
0. Ginsburg, A. S.; Grosset, J. H.; Bishai, W. R. Lancet
Infect. Dis. 2003, 3, 432.
1. Aubry, A.; Veziris, N.; Cambau, E.; Truffot-Pernot, C.;
Jarlier, V.; Fisher, L. M. Antimicrob. Agents Chemother.
1
1
2
2. Cheng, A. F.; Yew, W. W.; Chan, E. W.; Chin, M. L.;
006, 50, 104.
1
Hui, M. M.; Chan, R. C. Antimicrob. Agents Chemother.
2
004, 48, 596.
13. Mitscher, L. A.; Sharma, P. N.; Chu, D. T. W.; Shen, L.
L.; Pernets, A. G. J. Med. Chem. 1987, 30, 2283.
Acknowledgments
The authors are thankful to Dr. Vanaja Kumar, Deputy
Director, Tuberculosis Research Center, Chennai, India,
for his assistance in biological screening. A.C is a recipi-
ent of junior research fellowship from Council of Scien-
tific and Industrial Research, Government of India. This
work was supported by the grant from Department of
Biotechnology, Government of India, to V.N, D.S, and
P.Y (Centre of excellence in mycobacterial research).
14. Representative analytical data for compound 10-[2-car-
boxy-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl]-9-fluoro-
2
,3-dihydro-3-methyl-8-nitro-7-oxo-7H-[1,4]oxazino[2,3,4-
ij]quinoline-6-carboxylic acid (9m): Yield: %; mp °C; IR
ꢀ1
1
(
KBr) cm : 3400, 1750, 1660, 1590; H NMR (DMSO-d
d ppm: 1.60 (d, 3H, CHCH ), 3.1–3.8 (m, 6H, 3-CH ), 4.35
br s, 2 H, OCH ), 4.5 (m, 1H, CHCH ), 7.6 (s, 1H, CH),
6
)
3
2
(
2
3
8
.00 (s, 1H, C
5
–H), 12.12 (s, 1H, 2-COOH), 14.4 (s, 1H, 6-
): calcd C, 50.75; H, 3.41;
5 8
COOH); Anal. (C20H16FN O
N, 14.79; found C, 50.62; H, 3.47; N, 14.78.
5. National Committee for Clinical Laboratory Standards.
Antimycobacterial susceptibility testing for Mycobacte-
rium tuberculosis. Proposed standard M24-T. National
Committee for Clinical Laboratory Standards, Villanova,
PA, 1995.
6. Gundersen, L. L.; Meyer, N. J.; Spilsberg, B. J. Med.
Chem. 2002, 45, 1383.
7. Manjunatha, U. H.; Dalal, M.; Chatterji, M.; Radha, D.
R.; Visweswariah, S. S.; Nagaraja, V. Nucleic Acids Res.
1
References and notes
1
2
3
. Shandil, R. K.; Jayaram, R.; Kaur, P.; Gaonkar, S.;
Suresh, B. L.; Mahesh, B. N.; Jayashree, R.; Nandi, V.;
Bharath, S.; Balasubramanian, V. Antimicrob. Agents
Chemother. 2007, 51, 576.
. Nuermberger, E. L.; Yoshimatsu, T.; Tyagi, S.; O’Brien,
R. J.; Vernon, A. R.; Chaisson, R. E.; Bishai, W. R.;
Grosset, J. H. Am. J. Respir. Crit. Care Med. 2004, 169,
1
1
2
8. Mayne, T. N.; Johnson, N. J.; Kluwe, W. M.; Lencoski,
002, 30, 2144.
1
421.
D. L.; Polzer, R. J. J. Antimicrob. Chemother. 1997, 39,
6
. Veziris, N.; Truffot-Pernot, C.; Aubry, A.; Jarlier, V.;
Lounis, N. Antimicrob. Agents Chemother. 2003, 47, 3117.
7–73.