Synthesis of 6-fluoro-1,4-dihydro-4-oxo-quinoline-3-carboxylic acid derivatives as potential antimicrobial agents

Article abstract of DOI:10.1016/S0223-5234(03)00151-X

In the present study, a series of 1-ethyl/benzyl-6-fluoro-7-(substituted piperazin-1-yl)1,4-dihydro-4-oxo-quinoline-3-carboxylic acid were synthesized and characterized by IR, ¹H-NMR, mass spectral and elemental analysis. The in vitro antibacterial and antifungal activities of the compounds were evaluated by paper disc diffusion method. The minimum inhibitory concentrations (MIC) of the compounds were also determined by agar streak dilution method. The in vivo antibacterial activity of the compounds against Escherichia coli was also evaluated by mouse protection test. All the compounds exhibited significant antibacterial and weak antifungal activities. The in vivo antibacterial activity (ED₅₀) against E. coli was 50-160 mg kg ^-1 in the order of 7<9<8<10. 1-Ethyl-6-fluoro-7-(2,5-dioxo- piperazin-1-yl)1,4-dihydro-4-oxo-quinoline-3-carboxylic acid (7) was found to exhibit the most potent in vitro antimicrobial activity with MIC of 4.1, 3.1, 3.1, 2.4, 1, 1, 25 and >100 μg mL^-1 against Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus, Bacillus cereus, E. coli, Klebsiella pneumoniae, Candida albicans and Aspergillus niger.

Full text of DOI:10.1016/S0223-5234(03)00151-X

European Journal of Medicinal Chemistry 38 (2003) 1001ꢀ1004
/
www.elsevier.com/locate/ejmech
Laboratory note
Synthesis of 6-fluoro-1,4-dihydro-4-oxo-quinoline-3-carboxylic acid
derivatives as potential antimicrobial agents
Natesh Rameshkumar *, Mohan Ashokkumar, Ekambaram Harihara Subramanian,
Raju Ilavarasan, Seshaiah Krishnan Sridhar
Department of Pharmaceutical Chemistry and Pharmacology, C. L. Baid Metha College of Pharmacy, Jyothi Nagar, Thorapakkam,
Chennai 600096, Tamilnadu, India
Received 17 March 2003; received in revised form 18 July 2003; accepted 29 July 2003
Abstract
In the present study, a series of 1-ethyl/benzyl-6-fluoro-7-(substituted piperazin-1-yl)1,4-dihydro-4-oxo-quinoline-3-carboxylic
acid were synthesized and characterized by IR, 1H-NMR, mass spectral and elemental analysis. The in vitro antibacterial and
antifungal activities of the compounds were evaluated by paper disc diffusion method. The minimum inhibitory concentrations
(MIC) of the compounds were also determined by agar streak dilution method. The in vivo antibacterial activity of the compounds
against Escherichia coli was also evaluated by mouse protection test. All the compounds exhibited significant antibacterial and weak
antifungal activities. The in vivo antibacterial activity (ED50) against E. coli was 50ꢀ
Ethyl-6-fluoro-7-(2,5-dioxo-piperazin-1-yl)1,4-dihydro-4-oxo-quinoline-3-carboxylic acid (7) was found to exhibit the most potent
in vitro antimicrobial activity with MIC of 4.1, 3.1, 3.1, 2.4, 1, 1, 25 and ꢀ
100 mg mLꢁ1 against Staphylococcus aureus,
/
160 mg kgꢁ1 in the order of 7B
/
9B
/
8B10. 1-
/
/
Staphylococcus epidermidis, Micrococcus luteus, Bacillus cereus, E. coli, Klebsiella pneumoniae, Candida albicans and Aspergillus
niger.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Fluoroquinolone; Antibacterial; Antifungal
1. Introduction
viral activities [2,7]. It was therefore envisaged that a
new series of 2,5-dioxo/2,5-dioxo-3,6-dimethyl-piperazi-
nyl fluoroquinolones with 1-ethyl/benzyl substitution
would result in compounds with potent antibacterial
and antifungal activities. In the present study, a series of
Most of the quinolone antibacterial research has been
focussed on the functionality at C-7 position since the
introduction of norfloxacin. Piperazine substitution at
C-7 position has resulted in a wide range of clinically
useful fluoroquinolone antibacterial agents namely ci-
profloxacin, perfloxacin, pefloxacin, ofloxacin, amiflox-
acin, fleroxacin, lomefloxacin, sparfloxacin, difloxacin,
enoxacin, enrofloxacin, levofloxacin, marbofloxacin and
orbifloxacin. Gatifloxacin and grepafloxacin exhibit
potent broad-spectrum activity including Streptococcus
pneumoniae.
1-ethyl/benzyl-6-fluoro-7-(substituted
piperazin-1-
yl)1,4-dihydro-4-oxo-quinoline-3-carboxylic acid were
synthesized and screened for antibacterial and antifun-
gal activities.
2. Chemistry
Fluoroquinolones with 7-piperazinyl [1ꢀ
[9ꢀ13] and 1-benzyl [1,11,14] have been reported to
possess potent antibacterial [1ꢀ14], antifungal [7], anti-
/
8], 1-ethyl
In the present study, 3-chloro-4-fluoroaniline was
heated with diethyl-ethoxymethylenemalonate to form
diethyl-N-(3-chloro-4-fluoro-phenyl)aminomethylene-
malonate (1) which was cyclized to ethyl-7-chloro-6-
fluoro-4-hydroxy-quinoline-3-carboxylate (2). Ethyla-
tion/benzylation by treatment with ethyliodide or benzyl
chloride in the presence of anhydrous potassium car-
/
/
* Corresponding author.
E-mail address: clbmcpl@md4.vsnl.net.in (N. Rameshkumar).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(03)00151-X

1002
N. Rameshkumar et al. / European Journal of Medicinal Chemistry 38 (2003) 1001ꢀ1004
/
bonate formed ethyl-1-ethyl-6-fluoro-1,4-dihydro-4-
oxo-quinoline-3-carboxylate (3) and ethyl-1-benzyl-6-
fluoro-1,4-dihydro-4-oxo-quinoline-3-carboxylate (4),
respectively. The 1-ethyl/benzyl esters were hydrolysed
with aqueous sodium hydroxide to produce the corre-
sponding carboxylic acids (5 and 6) [1]. 1-Ethyl/benzyl-
6-fluoro-1,4-dihydro-4-oxo-quinoline-3-carboxylic acid
(5 and 6) were reacted with 2,5-dioxo-piperazine/2,5-
dioxo-3,6-dimethyl-piperazinyl to obtain the desired
5. Experimental protocols
5.1. Chemistry
The melting points were taken in open capillary tube
and are uncorrected. The IR spectra of the compounds
were recorded on ABB Bomem FTIR spectrometer
MB104 with KBr pellets. 1H-NMR spectra were re-
corded on 300 MHz-Bruker DPX 200. The chemical
shifts are reported as parts per million downfield from
tetra methyl silane. Mass spectra were recorded on
Shimadzu GC MS QP 5000. Microanalyses for C, H, N
were performed in Heraeus CHN Rapid Analyzer. All
products (7ꢀ10).
/
3. Biological investigation
the compounds gave satisfactory chemical analyses (9
/
0.4%). The purity of the compounds were checked by
TLC on precoated SiO2 gel (HF254, 200 mesh) alumi-
The in vitro antibacterial (Staphylococcus aureus
ATCC 9144, Staphylococcus epidermidis ATCC 155,
Micrococcus luteus ATCC 4698, Bacillus cereus ATCC
11778, Escherichia coli ATCC 25922 and Klebsiella
pneumoniae ATCC 11298) and antifungal (Candida
albicans ATCC 2091 and Aspergillus niger ATCC
9029) activities of the compounds were evaluated by
paper disc diffusion method. The minimum inhibitory
concentrations (MIC) of the compounds were also
determined by agar streak dilution method. The in
vivo antibacterial activity of the compounds against E.
coli was also evaluated by mouse protection test. Acute
oral toxicity test was performed for all the synthesized
compounds as per organization of economic co-opera-
tion and development (OECD) guidelines.
num plates (E Merck) using n-hexaneꢀ/ethyl acetate
(8:2) as mobile phase and visualized by iodine vapors.
5.1.1. General method of synthesis 7ꢀ10
/
The starting material (5, 6) for the synthesis of the title
compounds have been previously published [1]. A
mixture of 1-ethyl/benzyl-6-fluoro-1,4-dihydro-4-oxo-
quinoline-3-carboxylic acid (0.01 mol) (5, 6), 2,5-di-
oxo-piperazine/2,5-dioxo-3,6-dimethyl-piperazine (0.05
mol) and dimethyl formamide (20 mL) was heated to
130ꢀ140 8C for 10 h with stirring. The reaction mixture
/
was evaporated to dryness under reduced pressure and
water (20 mL) was added. The resulting precipitate was
4. Results and discussion
All the compounds exhibited highly significant anti-
bacterial and moderate antifungal activities. The com-
pounds were active against all the tested microorganism
compared to ciprofloxacin with a range of MIC values
of 4.1ꢀ
and 25ꢀ
/
25, 3.1ꢀ
/
25.4, 3.1ꢀ
/
20.2, 2.4ꢀ
/
25, 1ꢀ
/
10.2, 1ꢀ
/
10.9
50.2 mg mLꢁ1 against S. aureus, S. epidermidis,
/
M. luteus, B. cereus, E. coli, K. pneumoniae and C.
albicans, respectively. The compounds exhibited very
weak activity against A. niger (MIC: ꢀ
100 mg mLꢁ1).
/
1-Ethyl-6-fluoro-7-(2,5-dioxo-piperazin-1-yl)1,4-dihy-
dro-4-oxo-quinoline-3-carboxylic acid (7) was found to
exhibit the most potent in vitro antimicrobial activity
with MIC of 4.1, 3.1, 3.1, 2.4, 1, 1, 25 and ꢀ100 mg
/
mLꢁ1 against S. aureus, S. epidermidis, M. luteus, B.
cereus, E. coli, K. pneumoniae, C. albicans and A. niger.
The ED50 (in vivo antibacterial screening) of the
compounds against E. coli was 50ꢀ
/
160 mg kgꢁ1 in
10) did
the order of 7B
/
9B
/
8B
/
10. The compounds (7ꢀ
/
not cause mortality upto 2000 mg kgꢁ1 in acute oral
toxicity (OECD-423 guidelines) and were considered as
safe (x-unclassified).
Scheme 1.

N. Rameshkumar et al. / European Journal of Medicinal Chemistry 38 (2003) 1001ꢀ
/
1004
1003
filtered, washed with water, vacuum dried and recrys-
tallized using dimethyl formamide (Scheme 1).
nutrient agar medium (Hi-Media Laboratories, India).
The antifungal activity of the compounds was tested
against C. albicans and A. niger using sabouraud
dextrose agar medium (Hi-Media Laboratories, India).
5.1.1.1. 1-Ethyl-6-fluoro-7-(2,5-dioxo-piperazin-1-
yl)1,4-dihydro-4-oxo-quinoline-3-carboxylic acid (7).
Yieldꢂ
d: 8.95 (s, 1H, 2H), 7.88 (d, JH-F
7.13 (d, JHÃF 7.3 Hz, 1H, 8H), 4.59 (q, 2H, JHÃH
Hz, CH2), 3.26 (s, 2H, 6?-CH2), 2.86 (s, 2H, 3?-CH2),
1.42 (t, 3H, JHÃH
6.9 Hz, CH3). IR (KBr) cmꢁ1: 1713
(COOH), 1615 (CO), 1339 (CÃN), 1265 (CÃF), 808, 764
(ArÃ
H). EI MS m/z (Mꢃ): 347.20 (Calcd for
C16H14FN3O5: 347.29).
/
98%, m.p. 236ꢀ
/
238 8C. 1H-NMR (DMSO-d6)
5.2.1. Paper disc diffusion method
The sterilized [15] (autoclaved at 120 8C for 30 min)
ꢂ
/
13.5 Hz, 1H, 5H),
ꢂ
/
ꢂ/6.7
medium (40ꢀ50 8C) was innoculated (1 mL/100 mL of
/
medium) with the suspension (105 cfu mLꢁ1) of the
microorganism (matched to McFarland barium sul-
phate standard) and poured into a petridish to give a
ꢂ
/
/
/
/
depth of 3ꢀ4 mm. The paper impregnated with the test
/
compounds (200 mg mLꢁ1 in dimethyl formamide) was
placed on the solidified medium. The plates were
preincubated for 1 h at room temperature and incubated
at 37 8C for 24 and 48 h for antibacterial and antifungal
activities, respectively. Ciprofloxacin (100 mg/disc) and
ketoconazole (100 mg/disc) was used as standard for
antibacterial and antifungal activities, respectively. The
observed zone of inhibition is presented in Table I.
5.1.1.2. 1-Benzyl-6-fluoro-7-(2,5-dioxo-piperazin-1-
yl)1,4-dihydro-4-oxo-quinoline-3-carboxylic acid (8).
Yieldꢂ
d: 9.21 (s, 1H, 2H), 8.17 (d, JHÃF
7.87 (d, JHÃF 7.4 Hz, 1H, 8H), 6.99ꢀ
C6H5), 5.86 (s, 2H, NÃCH2), 3.15 (s, 2H, 6?-CH2), 2.89
(s, 2H, 3?-CH2). IR (KBr) cmꢁ1: 1717 (COOH), 1611
/
67%, m.p. 258ꢀ
260 8C. 1H-NMR (DMSO-d6)
/
ꢂ/10.3 Hz, 1H, 5H),
ꢂ
/
/7.61 (m, 5H,
/
(CO), 1342 (CÃ
/
N), 1268 (CÃ
/
F), 808, 754 (ArÃ
/
H). EI
MS m/z (Mꢃ): 409.25 (Calcd for C21H16FN3O5:
409.37).
5.2.2. Minimum inhibitory concentration (MIC)
MIC [16] of the test compounds were determined by
agar streak dilution method. A stock solution of the
synthesised compound (100 mg mLꢁ1) in dimethyl
formamide was prepared and graded quantities of the
test compounds were incorporated in specified quantity
of molten sterile agar (nutrient agar for antibacterial
activity and sabouraud dextrose agar medium for
antifungal activity). A specified quantity of the medium
5.1.1.3. 1-Ethyl-6-fluoro-7-(2,5-dioxo-3,6-dimethyl-
piperazin-1-yl)1,4-dihydro-4-oxo-quinoline-3-carboxylic
acid (9). Yieldꢂ
(DMSO-d6) d: 9.04 (s, 1H, 2H), 8.40 (d, JHÃF
Hz, 1H, 5H), 8.08 (d, JHÃF 5.9 Hz, 1H, 8H), 4.62 (q,
JHÃH 6.7 Hz, 2H, CH2), 4.23 (q, JHÃH 6.6 Hz, 1H,
6?-CH), 3.99 (q, JHÃH 6.6 Hz, 1H, 3?-CH), 1.53 (t,
JHÃH 6.8 Hz, 3H, CH2ÃCH3), 1.40 (d, JHÃH 6.7 Hz,
3H, 6?-CH3), 1.33 (d, JHÃH 6.8 Hz, 3H, 3?-CH3). IR
(KBr) cmꢁ1: 1717 (COOH), 1611 (CO), 1343 (CÃ
N),
H). EI MS m/z (Mꢃ): 375.25
/
57%, m.p. 242ꢀ
/
244 8C. 1H-NMR
9.2
ꢂ
/
ꢂ
/
ꢂ
/
ꢂ
/
(40ꢀ
/
50 8C) containing the compound was poured into a
ꢂ
/
petridish to give a depth of 3ꢀ/4 mm and allowed to
ꢂ
/
/
ꢂ
/
solidify. Suspension of the microorganism were pre-
pared to contain approximately 105 cfu mLꢁ1 and
applied to plates with serially diluted compounds in
dimethyl formamide to be tested and incubated at 37 8C
for 24 and 48 h for bacteria and fungi, respectively. The
MIC was considered to be the lowest concentration of
the test substance exhibiting no visible growth of
bacteria or fungi on the plate. The observed MIC is
presented in Table I.
ꢂ
/
/
1268 (CÃ
/
F), 808, 753 (ArÃ
/
(Calcd for C18H18FN3O5: 375.35).
5.1.1.4. 1-Benzyl-6-fluoro-7-(2,5-dioxo-3,6-dimethyl-
piperazin-1-yl)1,4-dihydro-4-oxo-quinoline-3-carboxylic
acid (10). Yieldꢂ59%, m.p. 274ꢀ
275 8C. 1H-NMR
(DMSO-d6) d: 9.18 (s, 1H, 2H), 8.19 (d, JHÃF
Hz, 1H, 5H), 7.87 (d, JHÃF 6.6 Hz, 1H, 8H), 7.05ꢀ
(m, 5H, C6H5), 5.87 (s, 2H, NÃCH2), 4.01 (q, JHÃH
Hz, 1H, 6?-CH), 3.82 (q, JHÃH 7.1 Hz, 1H, 3?-CH),
1.35 (d, JHÃH 6.4 Hz, 3H, 6?-CH3), 1.26 (d, JHÃH 6.5
Hz, 3H, 3?-CH3). IR (KBr) cmꢁ1: 1714 (COOH), 1615
/
/
ꢂ
/
11.4
7.64
ꢂ
/
/
5.3. In vivo antibacterial activity
/
ꢂ7
/
ꢂ
/
5.3.1. Animals
ꢂ
/
ꢂ
/
Inbred male swiss albino mice (20ꢀ
the in vivo antibacterial activity. They were kept in
colony cages at 2592 8C, relative humidity 45ꢀ55%
/25 g) were used for
(CO), 1331 (CÃ
/
N), 1264 (CÃ
/
F), 808, 764 (ArÃ
/
H). EI
MS m/z (Mꢃ): 437.60 (Calcd for C23H20FN3O5:
437.43).
/
/
under 12 h light and dark cycle. The animals were fed
with standard animal feed and water ad libitum. All the
animals were acclimatized for a week before use. The
test compounds and the standard drugs were adminis-
tered orally by gavage in the form of a suspension (1%
carboxy methylcellulose as vehicle). Acute oral toxicity
test was performed for all the synthesized compounds
according to organization of economic co-operation and
5.2. In vitro antimicrobial activity
The antibacterial activity of the synthesised com-
pounds was tested against S. aureus, S. epidermidis, M.
luteus, B. cereus, E. coli and K. pneumoniae using

1004
N. Rameshkumar et al. / European Journal of Medicinal Chemistry 38 (2003) 1001ꢀ1004
/
Table I
Antimicrobial activity of the synthesized compounds
Compounds
In vitro activity*/zone of inhibition (MIC)
In vivo activity
(ED50
)
S. aureus S. epidermi- M. luteus B. cereus
dis
E. coli
K. pneumo-
niae
C. albi-
cans
A. niger
E. coli
ATCC
9144
ATCC 155
ATCC
4698
ATCC
11778
ATCC
25922
ATCC 11298 ATCC
2091
ATCC
9029
ATCC 25922
7
19(4.1)
18(5)
20(3.1)
13(4.1)
18(4.2)
10(25.4)
24(0.39)
20(3.1)
15(5.3)
18(4.2)
10(20.2)
23(0.1)
22(2.4)
15(3.2)
21(3)
22(1)
24(1)
17(3)
16(25)
13(ꢀ
11(ꢀ
12(ꢀ
7(ꢀ
/
100) 50
8
17(3.1)
19(2.1)
14(10.2)
25(0.2)
13(50.1)
16(30.4)
11(50.2)
/100) 100
9
10
17(4.5)
10(25)
20(2.6)
14(10.9)
26(0.1)
/100) 85
12(25)
25(0.3)
/
100) 160
Ciprofloxacin (100 mg/ 24(0.2)
disc)
ꢀ/
ꢀ/
ꢀ/
Ketoconazole (100 mg/
disc)
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
21(1)
19(6.1)
ꢀ/
Zone of inhibition in mm, MIC in mg mLꢁ1 and ED50 in mg kgꢁ1
.
development (OECD) guidelines. All the animal experi-
mentation was performed as per the recommendations
and the protocols of the institutional animals ethics
committee.
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/
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/1564.
/
/
/
and 2000 mg kgꢁ1
.
/
5.3.3. Mouse protection test
The in vivo antibacterial activity [7] of the compounds
against E. coli was determined in male swiss albino mice
/
[12] P.M. Carbateas, R.P. Brundage, K.O. Gelotte, M.D. Gruett,
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1863.
(nꢂ6). The mice were infected intraperitoneally with a
/
suspension (105 cfu mLꢁ1) containing an amount of E.
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(p.o.) with a specified amount of the synthesised
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calculated by extrapolation among survival rate in each
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dose of the compound (mg kgꢁ1) required to protect
50% of the mice from an experimentally induced lethal
systemic infection of E. coli.
/
/
/
Illustrated, Butterworth
247.
A Practical
/
/
/
/