Antimycobacterial activities of novel 1-(cyclopropyl/tert-butyl/4- fluorophenyl)-1,4-dihydro-6-nitro-4-oxo-7-(substituted secondary amino)-1,8-naphthyridine-3-carboxylic acid

Article abstract of DOI:10.1021/jm700999n

Fifty-one 1-(cyclopropyl/tert-butyl/4-fluorophenyl)-1,4-dihydro-6-nitro-4- oxo-7-(substituted secondary amino)-1,8-naphthyridine-3-carboxylic acids were synthesized and evaluated for antimycobacterial in vitro and in vivo against Mycobacterium tuberculosi

Full text of DOI:10.1021/jm700999n

6232
J. Med. Chem. 2007, 50, 6232-6239
Antimycobacterial Activities of Novel 1-(Cyclopropyl/tert-butyl/4-fluorophenyl)-1,4-dihydro-
6-nitro-4-oxo-7-(substituted secondary amino)-1,8-naphthyridine-3-carboxylic Acid
Dharmarajan Sriram,*,† Palaniappan Senthilkumar,† Murugesan Dinakaran,† Perumal Yogeeswari,† Arnab China,‡ and
Valakunja Nagaraja‡
Medicinal Chemistry Research Laboratory, Pharmacy group, Birla Institute of Technology and Science, Pilani 333031, India, and Department
of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, Karnataka 560012, India
ReceiVed August 11, 2007
Fifty-one 1-(cyclopropyl/tert-butyl/4-fluorophenyl)-1,4-dihydro-6-nitro-4-oxo-7-(substituted secondary amino)-
1,8-naphthyridine-3-carboxylic acids were synthesized and 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 synthesized compounds, 1-tert-butyl-1,4-dihydro-
7-(4,4-dimethyloxazolidin-3-yl)-6-nitro-4-oxo-1,8-naphthyridine-3-carboxylic acid (10q) was found to be
the most active compound in Vitro with an MIC of 0.1 µM against MTB and MDR-TB and was 3 and 455
times more potent than isoniazid against MTB and MDR-TB, respectively. In the in ViVo animal model 10q
decreased the bacterial load in lung and spleen tissues with 2.39 and 3.89-log10protections respectively at
the dose of 50 mg/kg body weight.
Tuberculosis (TB), which is caused by Mycobacterium
tuberculosis (MTBa), is one of the most prevalent diseases and
is responsible for the deaths of about one billion people during
the last two centuries.1 MTB is a particularly successful
pathogen that latently infects about 2 billion people, about one-
third of world population. Each year, there are about 8 million
new TB cases and 2 million deaths worldwide. The increasing
emergence of drug-resistant TB, especially multi-drug-resistant
TB (MDR-TB, resistant to at least two frontline drugs such as
isoniazid and rifampin), is particularly alarming. MDR-TB has
already caused several fatal outbreaks2 and poses a significant
threat to the treatment and control of the disease in some parts
of the world, where the incidence of MDR-TB can be as high
as 14%. The standard TB therapy is ineffective in controlling
MDR-TB in high MDR-TB incidence areas. Fifty million people
have already been infected with drug-resistant TB.1 There is
much concern that the TB situation may become even worse
with the spread of HIV worldwide, a virus that weakens the
host immune system and allows latent TB to reactivate and
Figure 1. Standard fluoroquinolones.
makes the person more susceptible to reinfection with either
drug-susceptible or drug-resistant strains. In the last 50 years,
only a few drugs have been approved by the Food and Drug
Administration (FDA) to treat TB, reflecting the inherent
difficulties in discovery and clinical testing of new agents and
the lack of pharmaceutical industry research in the area.3 Hence,
faster acting and effective new drugs to better combat TB,
including multi-drug-resistant tuberculosis, are needed.
Several of the quinolone antibacterial have been examined
as inhibitors of MTB as well as other mycobacterial infections.4
As a result, gatifloxacin and moxifloxacin (Figure 1) are drugs
in pipeline and to be approved by FDA for the treatment of TB
by the year 2010. Quinolones inhibit bacterial type II topoi-
somerases, DNA gyrase, and topoisomerase IV,5 which are
essential enzymes that maintain the supercoils in DNA. The
incidence of mycobacterial resistance to fluoroquinolones is
relatively low at the present time, and there are no reports of
cross-resistance or antagonism with other classes of antimyco-
bacterial agents.6 A huge number of fluoroquinolones differing
in the nature of the R7 and R1 substituents were elaborated to
enable the establishment of structure/antituberculosis rela-
tionships7-11 and only a few R6- or/and R8-substituted quino-
lones are known, probably due to the difficult access to
precursors of these derivatives. Taking into account that
fluoroquinolones exhibit fairly good antimycobacterial activity
and having in mind the not fully explored antitubercular potential
of nitroquinolone pharmacophore, in the present work, effort
has been taken to study the effect of nitro substitution at sixth
position of naphthyridinone. The substitutions at the N1 position
include cyclopropyl/tert-butyl/4-fluorophenyl, in combination
with various unreported bulky secondary amino functions to
study the influence of lipophilic character at 7 position of
naphthyridone on activity against MTB. We report herein the
synthesis of newer 1-(cyclopropyl/tert-butyl/4-fluorophenyl)-
* Author to whom correspondence should be addressed. Tel: +91-1596-
244684; telefax: +91-1596-244183; e-mail: dsriram@bits-pilani.ac.in.
† Birla Institute of Technology and Science.
‡ Indian Institute of Science.
a Abbreviations: MTB: Mycobacterium tuberculosis; MDR-TB: MC2:
M. smegmatis; MIC: minimum inhibitory concentration.
10.1021/jm700999n CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/26/2007

Antimycobacterial Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6233
Scheme 1a
a (a) HNO3, (CH3CO)O; (b) CDI, C2H5OOCCH2COOK, MgCl2, (C2H5)3N; (c) (C2H5O)3CH, (CH3CO)2O; (d) R1NH2; (e) K2CO3; (f) HCl; (g) secondary
amines, K2CO3, MWI.
1,4-dihydro-6-nitro-4-oxo-7-(substituted secondary amino)-1,8-
naphthyridine-3-carboxylic acids and their antimycobacterial
activities together with toxicological results.
tography (TLC) and elemental analyses, and the structures were
identified by spectral data.
Biological Results and Discussion
Antimycobacterial Activity. The compounds were screened
for their in Vitro antimycobacterial activity against MTB, MDR-
TB, and M. smegmatis ATCC 14468 (MC2) by agar dilution
method13 for the determination of MIC in duplicate. The MDR-
TB clinical isolate was resistant to isoniazid, rifampicin,
ethambutol, and ofloxacin. The minimum inhibitory concentra-
tion (MIC) is defined as the minimum concentration of
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.
In the first phase of screening against MTB, all the com-
pounds showed excellent in Vitro activity against MTB with an
MIC of less than 12 µM. Eleven compounds (9d, 10d, 9g, 10g,
9h, 10m, 9o, 10o, 8q, 9q, and 10q) inhibited MTB with an
MIC of less than 1 µM and were more potent than the standard
fluoroquinolone gatifloxacin (MIC: 1.04 µM). When compared
to isoniazid (MIC: 0.36 µM), two compounds (9q and 10q)
were found to be more active against MTB. 1-tert-Butyl-1,4-
dihydro-7-(4,4-dimethyloxazolidin-3-yl)-6-nitro-4-oxo-1,8-naph-
thyridine-3-carboxylic acid (10q) was found to be the most
active compound in Vitro with an MIC of 0.1 µM against MTB
and was 3.6 and 10.4 times more potent than isoniazid and
gatifloxacin, respectively. Subsequently, some of the compounds
were evaluated against MDR-TB, and among the thirty three
compounds screened, all of them inhibited MDR-TB with an
MIC ranging from 0.08 to 6.19 µM and were found to be more
active than isoniazid (MIC: 45.57 µM) and gatifloxacin (MIC:
8.34 µM). Fifteen compounds (9a, 9d, 10d, 9g, 10g, 9h, 9j,
8k, 9k, 10m, 9o, 10o, 8p, 9q, and 10q) inhibited MDR-TB with
an MIC of less than 1 µM. 7-(2-Carboxy-5,6-dihydroimidazo-
[1,2-a]pyrazin-7(8H)-yl)-1-(4-fluorophenyl)-1,4-dihydro-6-nitro-
4-oxo-1,8-naphthyridine-3-carboxylic acid (9o) was found to
be the most active compound in Vitro with an MIC of 0.08 µM
against MDR-TB and was 104 and 570 times more potent than
Chemistry
The synthesis of the titled compounds 8-10a-q was
accomplished as outlined in Scheme 1. The 2,6-dimethoxyni-
cotinic acid (1) was converted to 2,6-dimethoxy-5-nitronicotinic
acid (2) by treatment with nitric acid in presence of acetic
anhydride. Compound 2 on reaction with 1,1′-carbonyldiimi-
dazole in tetrahydrofuran afforded the corresponding imida-
zolide, which, in situ was treated with the neutral magnesium
salt of ethyl potassium malonate in the presence of triethylamine
to yield ethyl 3-(2,6-dimethoxy-5-nitropyridin-3-yl)-3-oxopro-
panoate (3). Ethyl 1-(cyclopropyl/tert-butyl/4-fluorophenyl)-7-
methoxy-6-nitro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carbox-
ylates (6a-c) were prepared by a three-step one-pot reaction.
First, treatment of the keto ester 3 with triethyl orthoformate in
acetic anhydride gave the one-carbon homologue enol ether
intermediate ethyl 2-[(2,6-dimethoxy-5-nitropyridin-3-yl)car-
bonyl]-3-ethoxyacrylate (4), which upon evaporation to dryness
was allowed to react with a slight excess of appropriate amines
under nitrogen atmosphere in a mixture of ether and ethanol at
0 °C. This gave the enamino ester ethyl 3-(substituted aminom-
ethylene)-2-[(2,6-dimethoxy-5-nitropyridin-3-yl)carbonyl]acry-
late (5), and base-catalyzed cyclization of 5a-c with potassium
carbonate in DMSO yielded naphthyridone 6a-c. Ethyl esters
were finally hydrolyzed in acidic condition to yield 1-(cyclo-
propyl/tert-butyl/4-fluorophenyl)-1,4-dihydro-7-methoxy-6-ni-
tro-4-oxo-1,8-naphthyridine-3-carboxylic acid (7a-c). The titled
compounds 8-10a-q were prepared by treating 7a-c with
appropriate secondary amines in presence of potassium carbon-
ate under microwave irradiation in DMSO. When compared to
conventional method12 of a 2-3 h process, microwave-assisted
synthesis was performed with short reaction times (2-3 min)
with ease and was environmentally friendly. The purity of the
synthesized compounds was monitored by thin layer chroma-

6234 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Sriram et al.
Table 1. Physical Constants, in Vitro Antimycobacterial Activities, and Cytotoxicity
a Cyclopropyl. b 4-Fluorophenyl. c tert-Butyl. d Same as above.
gatifloxacin and isoniazid respectively. The compounds were
also evaluated against MC2 in which all the compounds inhibited
MC2 with an MIC ranging from 1.47 to 173.59 µM, and 45
compounds were found to be more active than isoniazid (MIC:
45.57 µM).
With respect to the structure-MTB activity relationship, the
results demonstrated that the antimycobacterial activity imparted
by the in N1 substituent was in the order of tert-butyl >
4-flurophenyl > cyclopropyl. This result was in contrast to the
other antibacterial fluoroquinolones, where the cyclopropyl
group was the favorable substituent. This result correlates well
with the previous report on the importance of tert-butyl group
as the antimycobacterial pharmacophore.14 The enhanced activity
by the tert-butyl group could be supported by its better electron-
donating property and its higher hydrophobicity than by the
cyclopropyl group, which provides support for the cooperative-
binding model for the inhibition of DNA gyrase proposed by
Shen, in which two quinolone molecules self-associate by π-π
ring stacking and tail-to-tail hydrophobic interaction between
the N1 substituents.15 At the C7 position we have studied various
substituted piperazines (8-10a-f), (thio)morpholines (8-
10g,h), substituted piperidines (8-10i-n), fused piperazines
and piperidines (8-10o,p), and oxazolidine (8-10q). A com-
parison of the substitution pattern at C7 demonstrated that the
order of activity was oxazolidine > fused piperazines and
piperidines > (thio)morpholines > substituted piperazines g
substituted piperidines. The results demonstrated that the
contribution of the C7 position to antimycobacterial activity was
dependent on the substituent at N1 and was in the order of
oxazolidine > fused piperazines and piperidines > (thio)-
morpholines > substituted piperidines > substituted piperazines
> (thio)morpholines when N1 was cyclopropyl; oxazolidine >

Antimycobacterial Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6235
Table 2. In ViVo Activity Data of 10q, Gatifloxacin, and Isoniazid
against M. tuberculosis ATCC 35801 in Mice
DNA Gyrase Inhibition. The naphthyridone derivatives
synthesized and studied in this report were tested for their ability
to inhibit supercoiling activity of DNA gyrase. The bacterial
targets for quinolones and fluroquinolones are the type II DNA
topoisomerases, DNA gyrase and topoisomerase IV. These ATP-
dependent enzymes act by a transient double-stranded DNA
break, followed by strand passage and religation reactions to
facilitate DNA transactions processes.17 DNA gyrase is unique
in catalyzing the negative supercoiling of DNA and is essential
for DNA replication, transcription, and recombination. In all
species of mycobacteria including MTB, DNA gyrase is the
sole type II topoisomerase carrying out the reactions of both
type II topoisomerases. Further, our earlier studies have revealed
that DNA gyrase from M. tuberculosis and M. smegmatis are
highly similar in protein level, antigenic properties, and catalytic
activities.18 The supercoiling assay results with various com-
pounds using M. smegmatis DNA gyrase is presented in Figure
2. The IC50 values presented in Table 3 show that the compounds
8p, 9d, 9q, 10g, and 10m inhibit DNA gyrase at 50 µg/mL or
lower concentrations (56.54-116.72 µM). The other com-
pounds, viz. 9g, 9h, 9o, and 10q, have MIC values greater than
50 µg/mL (202.27-256.15 µM) and yet show comparable
antimicrobial activity to those which inhibit DNA gyrase at
lower concentrations (Table 1).
lungs
spleen
compound
control
gatifloxacin (50 mg/kg)
isoniazid (25 mg/kg)
10q (50 mg/kg)
(log CFU ( SEM)
(log CFU ( SEM)
7.99 ( 0.16
6.02 ( 0.23
5.86 ( 0.23
5.60 ( 0.08
9.02 ( 0.21
6.92 ( 0.07
4.71 ( 0.10
5.13 ( 0.16
(thio)morpholines > substituted piperazines > substituted
piperidines > fused piperazines and piperidines when N1 was
4-fluorophenyl; oxazolidine > substituted piperazines substituted
piperidines > (thio)morpholines > > fused piperazines and
piperidines when N1 was tert-butyl. In MDR-TB, the results
demonstrated that the antimycobacterial activity imparted by
the in N1 substituents was in the order 4-flurophenyl > tert-
butyl > cyclopropyl. Introduction of bulky lipophilic secondary
amines at C7 enhanced the antimycobacterial activity, which
might be due to more penetration of these compounds into
mycobacterial cells.
All the compounds were further examined for toxicity (IC50)
in a mammalian Vero cell line up to 62.5 µg/mL concentrations
by a serial dilution method. After 72 h of exposure, viability
was assessed on the basis of cellular conversion of MTT into a
formazan product using the Promega Cell Titer 96 nonradioac-
tive cell proliferation assay,16 and the results are reported in
Table 1. Fifteen compounds when tested showed IC50 values
ranging from 55.5 to 160.1 µM. A comparison of the substitution
pattern at C7 demonstrated that piperidine-substituted analogues
were more cytotoxic than the substituted piperazines. These
results are important, as the piperdine-substituted compounds
with their increased cytoliability are much less attractive in the
development of a quinolone for the treatment of TB. This is
primarily because 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 10q was found
to be nontoxic up to 62.5 µg/mL (160.1 µM) and showed a
selectivity index (IC50/MIC) of more than 1600.
Prior to animal screening, the maximum tolerated dose (MTD)
was performed for compound 51 using C57BL/6 female mice
by administration of a one-time dose/animal of an escalating
dose of drug (100, 300, and 500 mg/kg). The nine mice (three
mice/dose) in each study were observed for a total of 1 week.
Surviving mice were sacrificed and the organs examined for
signs of overt toxicity. Compound 10q showed no effect or
adverse reactions/toxicity at the maximum dose tested. Subse-
quently, compound 10q was tested for efficacy against MTB
at a dose of 50 mg/kg (Table 2) in CD-1 mice. In this model,11
the mice were infected intravenously with M. tuberculosis
ATCC 35801. Drug treatment by the intraperitoneal route began
after 10 days of inoculation of the animal with the microorgan-
ism and continued for 10 days. After 35 days postinfection, the
spleens and right lungs were aseptically removed, and the
number of viable organisms were 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 10q decreased the bacterial load in
lung and spleen tissues with 2.39 and 3.89-log10protections,
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, 10q decreased the bacterial
load with 0.42 and 1.79-log10protections in lung and spleen
tissues, respectively. Compound 10q was found to be less active
than isoniazid in the in ViVo study. This lower in ViVo activity
might be due to the low bioavailability of the compound.
All the molecules tested do not show inhibition of supercoil-
ing activity at very low concentrations. The IC50 values were
higher compared to that of moxifloxacin and ciprofloxacin, two
well characterized fluoroquinolones, used as positive controls.
However, in cell-based assays, most of the compounds exhibited
higher potency. Thus, there is an inverse correlation between
IC50 values and enzyme inhibition data. Generally the inhibitory
molecules that show very good inhibition in enzyme assays have
a lower degree of efficacy in cell-based in Vitro assays because
of poor permeability, intracellular instability.19 The difference
in the extent of inhibition found in enzyme assays versus in
ViVo susceptibility tests may be due to the following reasons:
(1) The molecules may act as prodrugs and upon entering the
cell undergo enzymatic modifications to become a much more
potent drug.20 (2) Other proteins and/or ionic environment in
ViVo may influence binding characteristics to better accom-
modate the compounds to the binding site in the enzyme. Since
selected compounds showed only weak inhibition activity
against the target enzyme, it is supposed that the mode of action
of these compounds is different from inhibition of DNA gyrase.
Phototoxic Evaluation. Quinolones in general have favorable
safety profiles; phototoxicity has become a significant factor
in the clinical use of some21quinolones. Indeed, the first
quinolone, nalidixic acid, caused light-induced dermal effects.
This type of response has now been demonstrated for almost
all fluoroquinolones,22 although the relative phototoxic potential
varies greatly among compounds. Phototoxicity is considered
to be an acute, light-induced irritation response characterized
by dermal inflammation, with 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.23 This photosensitized
chromophore may then transfer its absorbed photo energy to
oxygen molecules, creating an environment for the production
of reactive oxygen species such as singlet oxygen. These reactive
species are then thought to attack cellular lipid membranes,
initiating the inflammatory process. Six (9g, 10g, 9h, 9o, 8p,
and 10q) compounds were evaluated for potential phototoxicity
in a standardized in ViVo test system that has been used

6236 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Sriram et al.
Figure 2. DNA gyrase supercoiling assay. The assays were performed as described in Experimental Section. DNA gyrase was incubated with
indicated concentrations of the compounds before the addition of rest of the components. Lane 1: relaxed DNA, lane 2: supercoiling reaction in
presence of 5% DMSO; ciprofloxacin (lane 3) and moxifloxacin at concentration of 5 µg/mL (lane 4) were used as the positive controls for
inhibition of enzyme. Lane 5: 30 µg/mL 8p, lane 6: 100 µg/mL 9o, lane 7: 50 µg/mL 9d, lane 8: 100 µg/mL 9h, lane 9: 100 µg/mL 9g, lane
10: 50 µg/mL 9q, lane 11: 40 µg/mL 10m, lane 12: 40 µg/mL 10g, lane 13: 100 µg/mL 10q. R and S indicate relaxed and supercoiled pUC18
DNA, respectively.
Table 3. IC50 values for DNA gyrase inhibition
2,6-Dimethoxy-5-nitropyridine-3-carboxylic Acid (2). This
compound was prepared according to the literature method12 with
slight modification. Briefly, to a mixture of concentrated nitric acid
(4 mL) (1) and acetic anhydride (12 mL) was added dropwise 2,6-
dimethoxynicotinicacid (16.3 mM) at 0-5 °C, and the mixture was
stirred for 3 h. The temperature was brought up to room temper-
ature, and the mixture was stirred for an additional 4 h. The resulting
precipitate was poured into ice-cold water and filtered. The
precipitate was dissolved in ether and extracted with saturated
sodium bicarbonate solution, and the aqueous layer upon acidifica-
tion with 2 N HCl yielded 2 with 80% yield; mp 236-238 °C (lit.
ref 230 °C).
compound
IC50 (µM)
8p
9d
9g
9h
9o
9q
10g
10m
56.54
99.31
>232.34
226.04
>202.27
116.72
101.93
92.50
10q
ciprofloxacin
256.15
15.09
Ethyl 3-(2,6-Dimethoxy-5-nitropyridin-3-yl)-3-oxopropanoate
(3). This compound was prepared from 2 according to the literature
method12 with 80% yield.
previously to assess quinolone antibiotics.24 The test compounds
(140 mg/kg) and the positive control lomefloxacin hydrochloride
(140 mg/kg) were evaluated for phototoxicity, and both ears of
each mouse were evaluated for changes indicative of a positive
response: erythema, edema, or a measurable increase in ear
thickness. Change from baseline was calculated separately for
each animal and time point and analyzed for statistical signifi-
cance and 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 to 96 h and from 24 to 96 h
when compared with time points and with the control, respec-
tively. The test compounds were found to show a significant
difference in ear thickness at various time-points when compared
with the predrug reading (0 h) but were less or not toxic when
compared with the negative (vehicle-treated) and positive
controls (lomefloxacin). No erythema occurred in mice dosed
with 140 mg/kg of 9g, 10g, 9h, and 8p throughout the 96 h
study, while compound 9o showed a significant erythema after
irradiation till 24 h only and compound 10q developed erythema
from 48 h.
Ethyl 1-(Cyclopropyl/tert-butyl/4-fluorophenyl)-7-methoxy-
6-nitro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylate (6a-
c). A mixture of 3 (1.00 equiv), triethyl orthoformate (1.5 equiv),
and acetic anhydride (2.5 equiv) were refluxed at 140 °C for 1 h.
The ethyl acetate produced as a byproduct was distilled simulta-
neously under atmospheric pressure. After completion of reaction,
the reaction mixture was concentrated under reduced pressure to
yield ethyl 2-[(2,6-dimethoxy-5-nitropyridin-3-yl)carbonyl]-3-
ethoxyacrylate (4). The above residue was dissolved in a mixture
of ether (20 mL) and ethanol (20 mL). The corresponding primary
amine (1.1 equiv) was added at 0 °C and stirred for 30 min under
nitrogen atmosphere, followed by distillation to yield ethyl 3-(sub-
stituted aminomethylene)-2-[(2,6-dimethoxy-5-nitropyridin-3-yl)-
carbonyl]acrylate (5a-c). The above crude solid (1.0 equiv) was
dissolved in DMSO (30 mL), and anhydrous potassium carbonate
(1.6 equiv) was added and refluxed at 60 °C for 3 h. After
completion of reaction, the mixture was diluted with ice cold water
and neutralized with 20% HCl to a pH of 5-6. The precipitate
thus formed was filtered and washed with water followed by
2-propanol to yield ethyl 1-(cyclopropyl/tert-butyl/4-fluorophenyl)-
7-methoxy-6-nitro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxy-
lates 6a-c.
Ethyl 1-cyclopropyl-7-methoxy-6-nitro-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxylate (6a). Yield: 92%; mp: 210-
212 °C; 1H NMR (DMSO-d6) δ ppm: 0.28-0.53 (m, 4H,
cyclopropyl), 1.32 (t, 3H, CH3 of OCH2CH3), 1.35 (m, 1H,
cyclopropyl), 3.74 (s, 3H, OCH3), 4.22 (m, 2H, OCH2), 9.26 (s,
1H, C5-H), 9.56 (s, 1H, C2-H); 13C NMR (DMSO-d6) δ ppm: 177.5,
165.3, 164.7, 159.9, 149.9, 135.2, 126.1, 112.2, 109.4, 61.4, 54.9,
36.0, 14.4, 5.6; Anal. (C15H15N3O6) C, H, N.
Experimental Section
Melting points were taken on an electrothermal melting point
apparatus (Buchi BM530) in open capillary tubes and are uncor-
rected. Infrared spectra (KBr disc) were run on Jasco IR Report
1
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 δ (ppm) relative to tetramethylsilane. 13C
NMR spectra were recorded on Bruker AC 200/DPX 400 MHz.
Elemental analyses (C, H, and N) were performed on Perkin-Elmer
model 240C analyzer, and the data were within (0.4% of the
theoretical values.
Ethyl 1-(4-fluorophenyl)-7-methoxy-6-nitro-4-oxo-1,4-dihy-
dro-1,8-naphthyridine-3-carboxylate (6b). Yield: 92%; mp:
>250 °C; 1H NMR (DMSO-d6) δ ppm: 1.32 (t, 3H, CH3 of OCH2-
CH3), 1.76 (s, 9H, t-butyl), 3.74 (s, 3H, OCH3), 4.22 (m, 2H, OCH2),
9.26 (s, 1H, C5-H), 9.56 (s, 1H, C2-H); 13C NMR (DMSO-d6) δ
ppm: 177.5, 167.4, 165.3, 164.7, 152.9, 146.6, 139.8, 135.2, 126.1,

Antimycobacterial Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6237
Table 4. Phototoxic Evaluation of the Titled Compounds
time (approximately) after start of irradiaton (h)a
ear thickness (mm)b
erythemac
group
controld
9g
10g
9h
9o
8p
10q
0
4
24
48
72
96
0
4
24
48
72
96
0.37 ( 0.03
0.25 ( 0.01
0.27 ( 0.01
0.26 ( 0.01
0.27 ( 0.01
0.33 ( 0.01
0.24 ( 0.01
0.31 ( 0.01
0.36 ( 0.02
0.27 ( 0.01
0.28 ( 0.02
0.25 ( 0.01
0.32 ( 0.01
0.34 ( 0.02
0.28 ( 0.02
0.40 ( 0.02
0.38 ( 0.03
0.26 ( 0.01
0.28 ( 0.02
0.28 ( 0.01
0.33 ( 0.03
0.34 ( 0.01
0.26 ( 0.01
0.48 ( 0.02
0.37 ( 0.03
0.26 ( 0.01
0.29 ( 0.02
0.27 ( 0.01
0.32 ( 0.02
0.38 ( 0.02
0.29 ( 0.02
0.53 ( 0.02
0.37 ( 0.03
0.29 ( 0.01
0.30 ( 0.02
0.3 ( 0.01
0.33 ( 0.01
0.36 ( 0.01
0.30 ( 0.02
0.64 ( 0.04
0.38 ( 0.03
0.29 ( 0.02
0.32 ( 0.02
0.3 ( 0.01
0.33 ( 0.02
0.33 ( 0.02
0.30 ( 0.02
0.60 ( 0.06
0
0
0
0
0
0
0
6
0
0
0
0
4
0
0
6
0
0
0
0
4
0
0
6
0
0
0
0
0
0
0
0
0
4
6
0
0
0
0
0
0
4
6
0
0
1e
4
lomefloxacin
6
a Time zero ) predose (mice exposed to UVA light immediately after dosing); 4 h ) end of irradiation period. b Mean ear thickness ( SEM; left and
right ears were averaged. c Number of mice with erythema. d Control ) 0.5% aqueous solution of sodium carboxymethylcellulose (4000 cps) dosed at 10
mL/kg. e Very slight erythema, not considered to be drug-related.
116.3, 117.9, 110.2, 109.4, 61.4, 54.9, 14.4; Anal. (C16H19N3O6)
C, H, N.
142.8, 140.9, 135.4, 131.8, 129.4, 128.3, 126.3, 118.8, 111.5, 73.6,
49.3, 49.6, 36.0, 5.6; Anal. (C29H26 ClN5O5) C, H, N.
Ethyl 1-tert-butyl-7-methoxy-6-nitro-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxylate (6c). Yield: 92%; mp: 204-206 °C;
1H NMR (DMSO-d6) δ ppm: 1.32 (t, 3H, CH3 of OCH2CH3), 3.74
(s, 3H, OCH3), 4.22 (m, 2H, OCH2), 6.45-6.74 (m, 4H, Ar-H),
9.26 (s, 1H, C5-H), 9.56 (s, 1H, C2-H); 13C NMR (DMSO-d6) δ
ppm: 177.5, 165.3, 164.7, 159.9, 149.9, 135.2, 126.1, 112.2, 109.4,
65.0, 61.4, 54.9, 28.5, 14.4; Anal. (C18H14N3O6) C, H, N.
1-(Cyclopropyl/tert-butyl/4-fluorophenyl)-1,4-dihydro-7-meth-
oxy-6-nitro-4-oxo-1,8-naphthyridine-3-carboxylic Acid (7a-c).
Compound 6a-c (1.0 equiv) was suspended in 6 N HCl (5 mL),
refluxed for 6 h, and then cooled to 0 °C. The precipitate obtained
was filtered and washed with water followed by 20% ethyl acetate
yielded 7a-c.
1-Cyclopropyl-1,4-dihydro-7-methoxy-6-nitro-4-oxo-1,8-naph-
thyridine-3-carboxylic Acid (7a). Yield: 92%; mp: 232-234 °C;
1H NMR (DMSO-d6) δ ppm: 0.28-0.46 (m, 4H, cyclopropyl),
1.38 (m, 1H, cyclopropyl), 4.28 (s, 3H, methoxy), 9.26 (s, 1H, C5-
H), 9.56 (s, 1H, C2-H), 14.6 (s, 1H, COOH); 13C NMR (DMSO-
d6) δ ppm: 177.5, 166.3, 164.1, 159.9, 152.7, 135.2, 126.0, 111.3,
109.4, 54.9, 36.0, 5.6; Anal. (C13H11N3O6) C, H, N.
1-4-Fluorophenyl-1,4-dihydro-7-methoxy-6-nitro-4-oxo-1,8-
naphthyridine-3-carboxylic Acid (7b). Yield: 92%; mp: 239-
241 °C; 1H NMR (DMSO-d6) δ ppm: 1.6 (S, 9H, tert-butyl), 4.32
(s, 3H, methoxy), 9.26 (s, 1H, C5-H), 9.54 (s, 1H, C2-H), 14.68 (s,
1H, COOH); 13C NMR (DMSO-d6) δ ppm: 177.5, 167.4, 166.3,
164.1, 152.9, 149.4, 139.8, 135.2, 126.0, 116.3, 117.9, 109.4, 54.9;
Anal. (C14H15N3O6) C, H, N.
1-tert-Butyl-1,4-dihydro-7-methoxy-6-nitro-4-oxo-1,8-naph-
thyridine-3-carboxylic Acid (7c). Yield: 92%; mp: 196-198 °C;
1H NMR (DMSO-d6) δ ppm: 4.34 (s, 3H, methoxy), 6.48-6.68
(m, 4H, Ar-H), 9.24 (s, 1H, C5-H), 9.56 (s, 1H, C2-H), 14.6 (s, 1H,
COOH); 13C NMR (DMSO-d6) δ ppm: 177.5, 166.3, 164.1, 159.9,
152.7, 135.2, 126.0, 111.3, 109.4, 65.0, 54.9, 28.5; Anal. (C16H10
FN3O6) C, H, N.
1-(Cyclopropyl/tert-butyl/4-fluorophenyl)-1,4-dihydro-6-nitro-
4-oxo-7-(substituted secondary amines)-1,8-naphthyridine-3-
carboxylic Acid (8-10a-q). Compound 7a-c (1.0 equiv) in
dimethyl sulfoxide (2.5 mL) and appropriate secondary amines (1.1
equiv) were irradiated in a microwave oven at an intensity of 80%
with 30 s/cycle. 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 2-propanol to give titled products.
7-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-1-cyclo-
propyl-1,4-dihydro-6-nitro-4-oxo-1,8-naphthyridine-3-carboxy-
lic Acid 8a. Yield: 70%; mp: 146-148 °C; 1H NMR (DMSO-d6)
δ ppm: 0.28-0.48 (m, 4H, cyclopropyl), 1.35 (m, 1H, cyclopropyl),
2.59 (t, 4H, 3,5-CH2 of piperazine), 3.12 (t, 4H, 2,6-CH2 of
piperazine), 4.2 (s, 1H, CH of diphenylmethyl), 7.0-7.18 (m, 9H,
Ar-H), 9.26 (s, 1H, C5-H), 9.56 (s, 1H, C2-H), 14.6 (s, 1H, COOH);
13C NMR (DMSO-d6) δ ppm: 177.5, 166.3, 161.2, 160.7, 152.7,
1,4-Dihydro-1-(4-fluorophenyl)-7-(4-(2-furoyl)piperazin-1-yl)-
6-nitro-4-oxo-1,8-naphthyridine-3-carboxylic Acid 9b. Yield:
76%; mp: 174-176 °C; H NMR (DMSO-d6) δ ppm: 3.16 (t,
4H, 3,5-CH2 of piperazine), 3.26 (t, 4H, 2,6-CH2 of piperazine),
6.5-7.68 (m, 7H, Ar-H), 9.26 (s, 1H, C5-H), 9.56 (s, 1H, C2-H),
14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) δ ppm: 176.5, 168.7,
167.3, 161.0, 155.2, 152.9, 149.4, 147.1, 145.0, 139.8, 135.0, 119.0,
117.9, 116.3, 113.5, 111.4, 109.3, 48.5, 47.5; Anal. (C24H18FN5O7)
C, H, N.
1
1-tert-Butyl-7-(4-((benzo[d]1,3dioxol-6-yl)methyl)piperazin-1-
yl)-1,4-dihydro-6-nitro-4-oxo-1,8-naphthyridine-3-carboxylic Acid
10c. Yield: 75%; mp: 180-182 °C; 1H NMR (DMSO-d6) δ ppm:
1.76 (s, 9H, t-butyl), 2.82 (t, 4H, 3,5-CH2 of piperazine), 3.1 (t,
4H, 2,6-CH2 of piperazine), 3.6 (s, 2H, CH2 of piperanoyl), 5.86
(s, 2H, OCH2O), 6.42-6.62 (m, 3H, Ar-H), 9.26 (s, 1H, C5-H),
9.56 (s, 1H, C2-H), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) δ
ppm: 177.5, 166.3, 161.2, 160.7, 152.7, 148.5, 147.3, 135.4, 128.9,
122.2, 118.8, 113.9, 111.3, 101.2, 65.0, 60.4, 52.2, 49.0, 28.5; Anal.
(C25H27N5O7) C, H, N.
1-Cyclopropyl-1,4-dihydro-7-(4-methyl-3-phenylpiperazin-1-
yl)-6-nitro-4-oxo-1,8-naphthyridine-3-carboxylic Acid 8d.
Yield: 75%; mp: 245-247 °C; 1H NMR (DMSO-d6) δ ppm:
0.28-0.52 (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), 7.0-7.2 (m, 5H, Ar-H), 9.28 (s, 1H, C5-H), 9.56
(s, 1H, C2-H), 14.4 (s, 1H, COOH); 13C NMR (DMSO-d6) δ ppm:
177.5, 166.3, 161.2, 160.7, 152.7, 137.4, 135.4, 129.6, 128.5, 127.3,
118.8, 111.7, 63.4, 59.0, 52.2, 49.0, 40.5, 36.0, 5.6; Anal.
(C23H23N5O5) C, H, N.
7-(4-(2,3-Dihydrobenzo[b]1,4dioxin-2-oyl)(piperazin-1-yl)-1,4-
dihydro-1-(4-fluorophenyl)-6-nitro-4-oxo-1,8-naphthyridine-3-
1
carboxylic Acid 9e. Yield: 85%; mp: 176-178 °C; H NMR
(DMSO-d6) δ ppm: 3.26 (t, 4H, 2,6-CH2 of piperazine), 3.4 (t,
4H, 3,5-CH2 of piperazine), 4.6 (d, 2H, 3-CH2 of dihydrobenzo-
dioxinyl), 5.14 (t, 1H, 2-CH of dihydrobenzodioxinyl), 6.5-6.98
(m, 8H, Ar-H), 9.26 (s, 1H, C5-H), 9.56 (s, 1H, C2-H), 14.6 (s, 1H,
COOH); 13C NMR (DMSO-d6) δ ppm: 177.5, 168.7, 166.3, 160.7,
152.9, 149.4, 146.7, 139.8, 135.4, 121.0, 118.8, 117.9, 116.3, 115.0,
111.9, 109.3, 85.9, 66.2, 48.5, 47.4; Anal. (C28H22FN5O8) C, H, N.
1-tert-Butyl-7-(3-(2,6-difluorophenyl)-5-methylisoxazol-4-oyl)-
(piperazin-1-yl)-1,4-dihydro-6-nitro-4-oxo-1,8-naphthyridine-3-
carboxylic Acid 10f. Yield: 70%; mp: >250 °C; 1H NMR
(DMSO-d6) δ ppm: 1.74 (s, 9H, t-butyl), 2.3 (s, 3H, 5-CH3 of
isoxazolyl), 3.12 (t, 4H, 3,5-CH2 of piperazine), 3.28 (t, 4H, 2,6-
CH2 of piperazine), 6.82-7.08 (m, 3H, Ar-H), 9.28 (s, 1H, C5-H),
9.54 (s, 1H, C2-H), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) δ
ppm: 177.5, 173.0, 168.9, 166.3, 161.2, 160.7, 159.8, 152.7, 135.4,
132.4, 118.8, 111.9, 111.3, 65.0, 48.5, 47.1, 28.5, 6.5; Anal.
(C28H26F2N6O7) C, H, N.
1-Cyclopropyl-1,4-dihydro-6-nitro-4-oxo-7-(4-thiomorpholino)-
1,8-naphthyridine-3-carboxylic Acid 8g. Yield: 76%; mp: 200-

6238 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Sriram et al.
202 °C; 1H NMR (DMSO-d6) δ ppm: 0.28-0.54 (m, 4H,
cyclopropyl), 1.36 (m, 1H, cyclopropyl), 2.64 (t, 4H, 3,5-CH2 of
thiomorpholine), 3.38 (t, 4H, 2,6-CH2 of thiomorpholine), 9.28 (s,
1H, C5-H), 9.56 (s, 1H, C2-H), 14.4 (s, 1H, COOH); 13C NMR
(DMSO-d6) δ ppm: 177.5, 166.3, 161.2, 160.7, 152.7, 135.4, 118.8,
111.9, 111.3, 50.2, 36.0, 28.2, 5.6; Anal. (C16H16N4O5S) C, H, N.
1,4-Dihydro-1-(4-fluorophenyl)-7-(2,6-dimethylmorpholino)-
6-nitro-4-oxo-1 ,8-naphthyridine-3-carboxylic Acid 9h. Yield:
81%; mp: 105-107 °C; 1H NMR (DMSO-d6) δ 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), 6.4-6.7 (m, 4H, Ar-H), 9.26 (s,
1H, C5-H), 9.56 (s, 1H, C2-H), 14.6 (s, 1H, COOH); 13C NMR
(DMSO-d6) δ ppm: 177.5, 168.7, 166.3, 160.7, 152.9, 149.4, 139.8,
135.4, 118.8, 117.9, 116.3, 111.9, 109.3, 65.4, 69.5, 21.2; Anal.
(C21H19FN4O6) C, H, N.
1-tert-Butyl-7-(2-carboxy-5,6-dihydroimidazo[1,2-a]pyrazin-
7(8H)-yl)-1,4-dihydro-6-nitro-4-oxo-1,8-naphthyridine-3-car-
boxylic Acid 10o. Yield: 72%; mp: 212-214 °C; 1H NMR
(DMSO-d6) δ ppm: 1.74 (s, 9H, t-butyl), 3.1-3.8 (m, 6H, 3-CH2),
7.6 (s, 1H, CH), 9.28 (s, 1H, C5-H), 9.54 (s, 1H, C2-H), 12.12 (s,
1H, 2-COOH), 14.6 (s, 1H, 3-COOH); 13C NMR (DMSO-d6) δ
ppm: 177.5, 167.9, 166.3, 161.2, 160.7, 158.4, 152.7, 139.4, 135.4,
130.6, 118.8, 111.9, 111.3, 65.0, 57.2, 50.5, 37.4, 28.5; Anal.
(C20H20N6O7) C, H, N.
1-Cyclopropyl-1,4-dihydro-7-(8-(4-methoxybenzyl)-3,4,5,6,7,8-
hexahydroisoquinolin-2(1H)-yl)-6-nitro-4-oxo-1,8-naphthyridine-
1
3-carboxylic Acid 8p. Yield: 77%; mp: 101-103 °C; H NMR
(DMSO-d6) δ 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), 3.73 (s, 3H,
OCH3), 6.8-7.1 (m, 4H, Ar-H), 9.28 (s, 1H, C5-H), 9.56 (s, 1H,
C2-H), 14.4 (s, 1H, COOH); 13C NMR (DMSO-d6) δ ppm: 177.5,
166.3, 161.2, 160.7, 158.4, 152.7, 135.4, 131.9, 129.1, 123.8, 118.8,
114.1, 111.9, 111.3, 61.5, 56.4, 42.9, 37.9, 36.0, 30.4, 28.6, 25.5,
24.3, 5.6; Anal. (C29H30N4O6) C, H, N.
1-tert-Butyl-1,4-dihydro-6-nitro-4-oxo-7-(4-(piperidin-1-yl)pi-
peridin-1-yl)-1,8-naphthyridine-3-carboxylic Acid 10i. Yield:
1
76%; mp: 154-156 °C; H NMR (DMSO-d6) δ ppm: 1.5-1.6
(m, 10H, 5 CH2), 1.78 (s, 9H, t-butyl), 2.2 (t, 4H, 2 CH2), 2.7 (m,
1H, CH), 2.8 (t, 4H, 2 CH2), 9.28 (s, 1H, C5-H), 9.54 (s, 1H, C2-
H), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) δ ppm: 177.5,
166.3, 161.2, 160.7, 152.7, 135.4, 118.8, 111.9, 111.3, 65.0, 58.5,
52.8, 47.1, 28.5, 27.9, 26.7, 25.7; Anal. (C23H31N5O5) C, H, N.
7-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-1-cyclopro-
pyl-1,4-dihydro-6-nitro-4-oxo-1,8-naphthyridine-3-carboxylic Acid
1,4-Dihydro-7-(4,4-dimethyloxazolidin-3-yl)-1-(4-fluorophe-
nyl)-6-nitro-4-oxo-1,8-naphthyridine-3-carboxylic Acid 9q.
Yield: 81%; mp: 192-194 °C; 1H NMR (DMSO-d6) δ ppm: 1.16
(s, 6H, 2-CH3), 3.41 (s, 2H, 5-CH2 of oxazolidinyl), 4.6 (s, 2H,
2-CHof oxazolidinyl), 6.4-6.7 (m, 4H, Ar-H), 9.26 (s, 1H, C5-H),
9.56 (s, 1H, C2-H), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) δ
ppm: 177.5, 168.7, 166.3, 160.7, 152.9, 149.4, 139.8, 135.4, 118.8,
117.9, 116.3, 111.9, 84.4, 83.6, 65.5, 22.4; Anal. (C20H17FN4O6)
C, H, N.
MIC Determination. All compounds were screened for their
in Vitro antimycobacterial activity against MTB, MDR-TB, and MC2
in Middlebrook 7H11agar medium supplemented with OADC by
agar dilution method similar to that recommended by the National
Committee for Clinical Laboratory Standards for the determination
of MIC in duplicate.13 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.
1
8j. Yield: 79%; mp: 162-164 °C; H NMR (DMSO-d6) δ ppm:
0.28-0.46 (m, 4H, cyclopropyl), 1.32 (m, 1H, cyclopropyl), 2.0
(t, 4H, 3,5-CH2 of piperidine), 2.7 (t, 4H, 2,6-CH2 of piperidine),
7.1-7.18 (m, 4H, Ar-H), 9.26 (s, 1H, C5-H), 9.56 (s, 1H, C2-H),
10.0 (bs, 1H, OH), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) δ
ppm: 177.5, 166.3, 161.2, 160.7, 152.7, 138.1, 135.4, 131.5, 129.6,
118.8, 111.9, 111.3, 74.5, 42.6, 38.2, 36.0, 5.6; Anal. (C23H21-
ClN4O6) C, H, N.
7-(4-(6-Chloro-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)pip-
eridin-1-yl)-1,4-dihydro-1-(4-fluorophenyl)-6-nitro-4-oxo-1,8-
naphthyridone-3-carboxylic Acid 9k. Yield: 78%; mp: >250 °C;
1H NMR (DMSO-d6) δ ppm: 1.6-2.4 (m, 8H, 4 CH2 of piperidine),
4.1 (bm, 1H, CH of piperidine), 6.4-6.9 (m, 7H, Ar-H), 9.26 (s,
1H, C5-H), 9.56 (s, 1H, C2-H), 10.8 (s, 1H, NH), 14.6 (s, 1H,
COOH); 13C NMR (DMSO-d6) δ ppm: 177.5, 168.7, 166.3, 160.7,
152.9, 151.8, 149.4, 139.8, 135.4, 131.3, 130.1, 124.7, 123.4, 122.2,
118.8, 117.9, 116.3, 111.9, 109.3, 50.3, 46.3, 26.9; Anal. (C27H20-
ClFN6O6) C, H, N.
Cytotoxicity.
All the compounds were further examined for toxicity (IC50) in
a mammalian Vero cell line up to concentrations of 62.5 µg/mL16
by a serial dilution method. After 72 h of exposure, viability was
assessed on the basis of cellular conversion of MTT into a formazan
product using the Promega Cell Titer 96 non-radioactive cell
proliferation assay.
1-tert-Butyl-7-(3-(diethylcarbamoyl)piperidin-1-yl)-1,4-dihy-
dro-6-nitro-4-oxo-1,8-naphthyridine-3-carboxylic Acid 10l.
1
Yield: 70%; mp: >250 °C; H NMR (DMSO-d6) δ ppm: 1.2 (t,
6H, 2-CH3 of ethyl), 1.7 (s, 9H, t-butyl), 1.78-2.7 (m, 9H, H of
piperidine), 3.24 (q, 4H, 2-CH2 of ethyl), 9.28 (s, 1H, C5-H), 9.54
(s, 1H, C2-H), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) δ ppm:
177.5, 175.3, 166.3, 161.2, 160.7, 152.7, 135.4, 118.8, 111.9, 111.3,
65.0, 54.4, 51.0, 42.6, 41.3, 28.5, 28.2, 22.3, 12.9; Anal. (C23H31N5O6)
C, H, N.
In ViWo Studies.
One compound was tested for efficacy against MTB at a dose
of 25 mg/kg in six-week-old female CD-1 mice six per group. In
this model, the mice were infected intravenously through caudal
vein approximately 107 viable 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 postinfection, the spleens and right lungs were aseptically
removed and ground in a tissue homogenizer, and the number of
viable organisms was determined by serial 10-fold dilutions and
subsequent inoculation onto 7H10 agar plates. Cultures were
incubated at 37 °C in ambient air for 4 weeks prior to counting.
Bacterial counts were measured and compared with the counts from
negative controls (vehicle treated) in lung and in spleen.
DNA Gyrase Supercoiling Assay.
1-Cyclopropyl-1,4-dihydro-7-(1,4-dioxa-8-azaspiro[4.5]dec-8-
yl)-6-nitro-4-oxo-1,8-naphthyridine-3-carboxylic Acid 8m.
Yield: 78%; mp: 124-126 °C; 1H NMR (DMSO-d6) δ ppm:
0.28-0.52 (m, 4H, cyclopropyl), 1.38 (m, 1H, cyclopropyl), 1.78-
2.4 (m, 8H, 4-CH2 of azaspirodecane), 3.96 (m, 4H, 2-CH2 of
azaspirodecane), 9.28 (s, 1H, C5-H), 9.56 (s, 1H, C2-H), 14.4 (s,
1H, COOH);); 13C NMR (DMSO-d6) δ ppm: 177.5, 166.3, 161.2,
160.7, 152.7, 135.4, 118.8, 111.9, 111.3, 109.6, 64.3, 39.8, 36.0,
34.0, 5.6; Anal. (C19H20N4O7) C, H, N.
7-(1-(tert-Butylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-
1,4-dihydro-1-(4-fluorophenyl)-6-nitro-4-oxo-1,8-naphthyridine-
DNA gyrase was purified from M. smegmatis cells as described
previously.25 The compounds tested were dissolved in DMSO and
preincubated with gyrase. Supercoiling assays were carried out as
described previously,11 by incubating 400 ng of relaxed pUC18 at
37 °C in supercoiling buffer [35 mM Tris-HCl pH 7.5, 5 mM
MgCl2, 25 mM potassium glutamate, 2 mM spermidine, 2 mM
ATP, 50 µg/mL bovine serum albumin, and 90 µg/mL yeast t-RNA
in 5% (v/v) glycerol] for 1 h. Moxifloxacin at 5 µg/mL and
ciprofloxacin at 10 µg/mL final concentration were used as positive
1
3-carboxylic Acid 9n. Yield: 82%; mp: 128-130 °C; H NMR
(DMSO-d6) δ ppm: 1.3 (s, 9H, 3 CH3), 2.66-2.9 (m, 4H, 2 CH2
of isoquinoline), 4.85 (s, 1H, CH of isoquinoline), 6.7-7.1 (m,
8H, Ar-H), 9.26 (s, 1H, C5-H), 9.56 (s, 1H, C2-H), 10.2 (s, 1H,
NH), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) δ ppm: 177.5,
168.9, 168.7, 166.3, 160.7, 152.9, 149.4, 139.8, 138.1, 135.4, 129.5,
128.6, 127.9, 127.0, 118.8, 117.9, 116.3, 111.9, 109.3, 65.5, 48.3,
47.4, 30.6, 25.9; Anal. (C29H26FN5O6) C, H, N.

Antimycobacterial Agents
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controls, and a control reaction having 5% DMSO in absence of
compounds was also performed. The reaction samples were heat
inactivated and applied onto 1% agarose gel after electrophoresis
in Tris-acetate-EDTA buffer for 12 h. The gels were stained with
ethidium bromide and recorded by gel documentation.
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 photoirradiation period. Eighteen mice were
randomly distributed into three dosing groups. The first groups
received a single dose of screened compound at 140 mg /kg by
oral gavage. A second group received a single dose of 140 mg of
lomefloxacin HCl/kg. 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 methylcellulose
vehicle only. Test animals were exposed to UVA light in a manner
adapted from that described previously.24 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 evaluated for changes indicative of a positive re-
sponse: erythema, edema, or a measurable increase in ear thickness.
Acknowledgment. The authors are thankful to Council of
Scientific and Industrial Research [01 (1979)/05/EMR/-II] and
University Grant Commissions F.30-266/2004(SR) New Delhi,
India, for their financial assistance. The authors are also thankful
to Dr. Vanaja Kumar, Deputy Director, Tuberculosis Research
Center, Chennai, India, for his assistance in biological screening
and A. Nambi for technical assistance in gyrase assays. The
work in V.N.’s laboratory is supported by a COE grant from
Department of Biotechnology, Government of India.
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Supporting Information Available: Spectral and elemental
data and gel pictures of DNA gyrase inhibition studies of
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
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