Novel fluoroquinolones: Design, synthesis, and in vivo activity in mice against Mycobacterium tuberculosis H37Rv

Article abstract of DOI:10.1016/j.bmcl.2005.02.037

Novel 6,8-difluoro-1-alkyl-5-amino-1,4-dihydro-4-oxo-7-{4-substituted piperazin-1-yl}-quinoline-3-carboxylic acids, with the substituents at 4th position of piperazine being -[2(pyridine-4-carbonyl) hydrazono]propyl and -2 [(pyrazine-2-carbonyl) amino] ethyl, were synthesized and evaluated in vivo against Mycobacterium tuberculosis H₃₇Rv in Swiss albino mice. Test compounds exhibited activity comparable to that of sparfloxacin (survival rate, reduction of splenomegaly and reduced tubercular lesions) at a dose of 200 mg/kg.

Full text of DOI:10.1016/j.bmcl.2005.02.037

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1803–1806
Novel fluoroquinolones: design, synthesis, and in vivo activity
in mice against Mycobacterium tuberculosis H Rv
3
7
*
Anand V. Shindikar and C. L. Viswanathan
Department of Pharmaceutical Chemistry, The Bombay College of Pharmacy, Kalina, Santacruz (East), Mumbai 400098, India
Received 20 December 2004; revised 13 February 2005; accepted 14 February 2005
Abstract—Novel 6,8-difluoro-1-alkyl-5-amino-1,4-dihydro-4-oxo-7-{4-substituted piperazin-1-yl}-quinoline-3-carboxylic acids,
with the substituents at 4th position of piperazine being -[2(pyridine-4-carbonyl) hydrazono]propyl and -2 [(pyrazine-2-carbonyl)
amino] ethyl, were synthesized and evaluated in vivo against Mycobacterium tuberculosis H37Rv in Swiss albino mice. Test com-
pounds exhibited activity comparable to that of sparfloxacin (survival rate, reduction of splenomegaly and reduced tubercular
lesions) at a dose of 200 mg/kg.
Ó 2005 Elsevier Ltd. All rights reserved.
Tuberculosis (TB) is a serious health problem, due to
1
which three million people die every year worldwide.
chromosomal replication and interference with cell divi-
3
sion and gene expression.
The association of TB and HIV infection is so dramatic
that in some cases, nearly two-thirds of the patients
diagnosed with TB are also HIV seropositive. In addi-
tion to this, the increase in M. tuberculosis strains resis-
tant to front-line anti-mycobacterial drugs such as
rifampin (RIF) and isoniazid (INH) has further compli-
cated the problem, which clearly indicates the need for
more effective drugs for the efficient management of
As mycobacteria have lipid rich cell walls, lipophilicity is
an important consideration in the design and activity of
4
novel molecules. Similarly as C-7 substitution in the flu-
oroquinolones affect their pharmacokinetic profile and
5
the spectrum of activity, in the present work structural
modifications were mainly centered at the C-7 position.
2
tuberculosis.
An intermediate 1-(tert-butyl)-7-(piperazin-1-yl)-6-flu-
oro-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (vi)
is the intermediate for compounds I and III. It was syn-
thesized as shown in Scheme 1.
Among the quinolone class of anti-bacterial agents, flu-
oroquinolones have shown promise in curing TB. Oflox-
acin at a dose of 300–800 mg and Levofloxacin at a dose
of 250–500 mg daily when used in combination with
agents like p-aminosalicylic acid, ethionamide, and
cycloserine is effective in the treatment of multi-drug
resistant tuberculosis. These fluoroquinolones are found
to be highly concentrated in the host cells, which further
enhance their anti-mycobacterial action.
Equimolar amounts (0.171 M) of 3-chloro-4-fluoroani-
line (i) and diethylethoxymethylenemalonate (ii) were
condensed at 145 °C to get 3-chloro-4-fluoroanilino-
methylenemalonic acid diethyl ester (iii), which was then
cyclized by heating at 250 °C in diphenyl ether to get
ethyl 7-chloro-6-fluoro-4-oxo-1,4-dihydro-quinoline-3-
carboxylate (iv), which was then reacted with tert-butyl
bromide in N,N-dimethylformamide (DMF) in presence
of sodium hydride and the product formed was hydroly-
sed to get the free acid (v), which was then condensed
with piperazine in DMF to get vi (mp: 204 °C).
Fluoroquinolones act by interfering with the action of
the bacterial DNA gyrase, which results in the degrada-
tion of chromosomal DNA and leads to termination of
Reacting compound vi/x (0.02 M) with bromoacetone
(0.03 M) in presence of triethylamine in 120 ml of
Keywords: 7-{4-Substituted piperazin-1-yl}fluoroquinolones; Anti-
tubercular fluoroquinolones.
*
Corresponding author. Tel.: +91 022 26671027; fax: +91 022
6670816; e-mail: anandshindikar@yahoo.com
DMF at 80 °C for 2.5 h gave intermediates, which after
removal of excess bromoacetone were then reacted with
2
0
960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.037

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A. V. Shindikar, C. L. Viswanathan / Bioorg. Med. Chem. Lett. 15 (2005) 1803–1806
OC H5
O
O
2
Cl
OC H5
O
C
O
2
H
5
5
O
2
F
F
F
OC H5
2
+
145˚C
1 hr
Diphenyl ether
Reflux
O
C H
O
O
2
5
Cl
N
Cl
N
H
iv )
NH
O
2
C H
H
2
1
hr 15
(
(
i )
(
ii )
( iii )
1
) NaH, t-butyl bromide, DMF
Reflux, 5 hrs
O
O
2
) NaOH, Reflux, 30 min
O
O
F
OH
F
Piperazine, DMF
OH
N
N
1
40˚C, 8 hrs
Cl
N
HN
H C
CH3
3
H C
CH3
CH3
3
CH3
(
vi )
(
v )
1
1
-(tert- Butyl)-7-(piperazin-1-yl)-6-fluoro-4-oxo-
,4-dihydro-quinoline-3-carboxylic acid
N
O
O
Br
O
2) N
1
)
Br
N
N
H C
N
H
NH2
3
H
(vii)
(ix)
(viii)
DMF
DMF
DMF, 150 C, 1hr
0
0
0
8
0 C, 2.5 hrs
135 C, 1 hr
O
O
O
O
F
N
OH
F
OH
N
O
N
N
N
N
N
N
H C
CH3
HN
N
N
3
H C
CH3
3
CH3
O
N
H
CH3
H C
3
Compound I
Compound III
Scheme 1. Synthesis of compounds I and III.
NH2
O
N
O
O
NH2
O
O
F
Br
F
OH
CH3
OH
H C
3
(vii)
H C
3
N
N
N
O
HN
F
N
F
8
0˚C DMF
.5 hrs
2
H C
3
CH3
CH3
H N
2
(
xi)
(
x)
-cyclopropyl-5-amino-6,8-difluoro-7-[(3,5-dimethyl)
piperazin-1-yl]-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid
O
NH
1
1
35˚C
DMF
hr
K CO
O
2
3
N
N
Br
N
DMF
N
H
1
(viii)
150˚C
(
ix)
1
hr
NH2
O
O
NH2
O
N
O
F
F
OH
OH
O
H C
3
H C
N
N
3
O
N
N
N
H
N
F
N
N
N
F
N
N
H C
H
3
CH3
CH3
Compound II
Compound IV
Scheme 2. Synthesis of compounds II and IV.

A. V. Shindikar, C. L. Viswanathan / Bioorg. Med. Chem. Lett. 15 (2005) 1803–1806
1805
Table 1. Characterization data of test compounds
Compound Mp (°C) Yield (%) Spectral analysis
I
295–297 62.08
FT-IR (KBr): 3522 (O–H), 3067 (C–H, Ar), 1697 (C@O, acids), 1626 (C@O, amide), 1539 (C@N, imine),
253 ((CH –C)
H NMR (DMSO-d
s, 1H, –CONH–N@), 4.14, 2.50 (m, 8H, piperazine), 1.41 (s, 9H, t-butyl)
1
3 3
)
1
6
): 11.5 (s, 1H, O–H), 8.86 (s, 1H, C-2 of quinolone), 8.44, 8.12 (d, 2H, Ar), 5.32
(
II
304–308 62.31
FT-IR (KBr): 3481 (O–H), 3283 (N–H), 2980 (C–H aromatic), 2851 (C–H aliphatic), 1728 (C@O acids),
6135 (C@O amide), 1026 (C–H, cyclopropyl)
H NMR (DMSO-d ): 8.76 (s, 1H, C-2 of quinolone), 8.46, 7.80 (m, 4H, Ar) 7.20 (s, 2H, NH
m, 1H, cyclopropyl), 3.33 (m, 6H, 3,5-dimethyl of piperazine), 2.83–2.49 (m, 6H, piperazine), 1.05
m, 4H, cyclopropyl)
1
6
2
), 3.96
(
(
III
IV
210–212 74.33
FT-IR (KBr): 3499 (O–H), 3329 (N–H), 2930 (C–H, Ar), 2892 (C–H aliphatic), 1630 (C@O amide), 1572
N–H amide)
(
1
H NMR (DMSO-d
.6 (m, 1H, –CONH–), 4.08 (m, 2H, –CH2), 3.6, 2.5 (m, 8H, piperazine), 2.6 (m, 2H, –CH
t-butyl)
6
): 8.62 (s, 1H, C-2 of quinolone), 8.1, 8.0 (d, 2H, Ar), 7.7, 7.6, 7.4 (m, 2H, pyrazine),
5
2
), 1.23 (s, 9H,
226–228 72.61
FT-IR (KBr): 3545 (O–H), 3323 (N–H), 2930 (C–H, Ar), 2852 (C–H, –CH
2
–), 1631 (C@O, amide)
): 9.1 (s, 1H, O–H), 8.83 (s, 1H, C-2 of quinolone), 8.71, 8.58, 7.82 (m, 3H,
pyrazine), 7.3 (m, 2H, NH ), 5.6 (m, 3H, pyrazine), 4.06 (m, 1H, cyclopropyl), 4.03, 3.73 (m, 4H, –CH
.46 (m, 6H, 3,5-dimethyl of piperazine), 2.91–2.50 (m, 6H, piperazine), 1.63 (m, 4H, cyclopropyl)
1
H NMR (DMSO-d
6
2
2
–),
3
isonicotinic acid hydrazide (0.013 M) at 135 °C for 1 h.
Finally removal of DMF and recrystallization from eth-
anol–DMF (70:30) gave compounds I (Scheme 1) and II
(
Scheme 2), respectively.
Condensation of equimolar quantities (0.0086 M) of
compound vi/x with 2(pyrazinamido)-1-bromoethane
in presence of anhydrous potassium carbonate in
2
0 ml of DMF at 150 °C for 1 h followed by removal
of DMF, washing with water and recrystallization from
DMF–water (60:40) gave compounds III (Scheme 1)
and IV (Scheme 2), respectively.
Figure 1. Effect of test compounds I–IV and sparfloxacin on the
survival rate (determined on 30th day) and inhibition of CFUs
observed on Lowenstein–Jensen medium expressed as percentage.
Open columns represent survival rate and striped columns represent
Compounds ix and x were synthesized as per literature
,7
6
methods
points and spectral analysis. Characterization data for
and were characterized by their melting
%
inhibition of CFUs in spleen in mice.
*
Group 1-Infected mice without any drug treatment (Àve control); 2-
test compounds I–IV is given in Table 1.
Sparfloxacin treated (300 mg/kg) (+ve control); 3-Sparfloxacin treated
(
200 mg/kg) (+ve control); 4-Compound I treated (200 mg/kg); 5-
Compound II treated (200 mg/kg); 6-Compound III treated (200 mg/
kg); 7-Compound IV treated (200 mg/kg).
The test compounds were evaluated for anti-tubercular
8
activity in Swiss albino mice using sparfloxacin (SPFX)
as a standard (dose equivalents correlating anti-tubercu-
lar activity in mice: SPFX: 60 mg/kgꢀINH: 25 mg/
9
,10
kgꢀRIF: 20 mg/kg).
A group of five mice were used
for testing each compound. The mice were infected
6
intravenously with M. tuberculosis H Rv (ꢀ10 organ-
3
7
0
.3
.25
0.2
.15
.1
Splenomegaly
isms) and after 24 h, compounds I–IV were administered
orally six times a week for a period of four weeks. The
parameters studied were survival rate, spleen weights,
gross lung lesions, and colony forming units (CFUs) in
the spleen. Groups treated with III and sparfloxacin
exhibited 100% survival rate while those treated with I,
II, and IV gave a value of 80% (Fig. 1). All treated
groups exhibited reduction of splenomegaly (Fig. 2).
Groups treated with III, IV, and sparfloxacin showed
absence of lung lesions, while those with I and II showed
few lung lesions. Groups treated with II, III, and spar-
floxacin showed 55%, 75%, and 56% inhibition of
CFUs, respectively, at a dose of 200 mg/kg (Fig. 1).
0
0
0
0.05
0
1
2
3
4
5
6
7
8
group*
Figure 2. Effect of test compounds I–IV and sparfloxacin on spleen
*
enlargement observed during tuberculosis infection in mice. Group 1-
Infected mice without any drug treatment (Àve control); 2-Sparflox-
acin treated (300 mg/kg) (+ve control); 3-Sparfloxacin treated (200 mg/
kg) (+ve control); 4-Compound I treated (200 mg/kg); 5-Compound II
treated (200 mg/kg); 6-Compound III treated (200 mg/kg); 7-Com-
pound IV treated (200 mg/kg); 8-Non-infected mice.
Results of the study indicate potent anti-tubercular
activity for test compounds I–IV. Compound III with

1806
A. V. Shindikar, C. L. Viswanathan / Bioorg. Med. Chem. Lett. 15 (2005) 1803–1806
100% survival rate, absence of lung lesions, and 75%
inhibition of CFUs was most potent.
2. Bloch, A. B.; Cauthen, G. M.; Dansbury, K. D.; Kelly, G.
D. J. Am. Med. Assoc. 1994, 271, 665.
3. Cozarelli, N. R. Science 1980, 207, 643.
4. Brennan, P. J.; Nikaido, H. Annu. Rev. Biochem. 1995, 64,
Testing against clinical isolates of M. tuberculosis and
M. avium complex would establish their clinical efficacy
and potential for use in immunocompromised systems.
2
9.
. Renau, T. E.; Sanchez, J. P.; Gage, J. W. J. Med. Chem.
996, 39, 729.
. Miyamoto, T.; Matsumoto, J.; Chiba, K.; Egawa, H.;
Nishimura, T. J. Med. Chem. 1990, 33, 1645.
5
6
7
1
Acknowledgements
. Cortese, F. In Organic Syntheses, Coll. ; Blatt, A. H., Ed.;
John Wiley & Sons, New York, 1943; Vol. II, pp 91–
The authors are thankful to Amrut Mody Research
Foundation, The Bombay College of Pharmacy, Mum-
bai, for funding the project.
9
3.
8
9
. Lounis, N.; Ji, B.; Truffot-Pernot, C.; Grosset, J. Antimic-
rob. Agents Chemother. 1997, 41, 607.
. Lenaerts, A. M. J. A.; Chase, S. E.; Chmielewski, A. J.;
Cynamon, M. H. Antimicrob. Agents Chemother. 1999, 43,
References and notes
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10. Baohong, J.; Lounis, N.; Truffet-Pernot, C.; Grosset, J.
Antimicrob. Agents Chemother. 1995, 39, 1341.
1
. WHO/CDS/TB/2003.316 (www.who.int/gtb/publications/
tbrep_97/countries/brazil.htm).