Synthesis and antibacterial activity of novel 7-haloanilino-8- nitrofluoroquinolone derivatives

Article abstract of DOI:10.1007/s00044-011-9692-3

The synthesis of new 7-(halophenylamino)-1-cyclopropyl-6-fluoro-8-nitro-4- oxo-1,4-dihydroquinoline-3-carboxylic acid derivatives is described. Substitution of quinolonic compound 1a with chloro and fluoro aniline derivatives at position 7 produced target

Full text of DOI:10.1007/s00044-011-9692-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1734–1740
DOI 10.1007/s00044-011-9692-3
ORIGINAL RESEARCH
Synthesis and antibacterial activity of novel
7-haloanilino-8-nitrofluoroquinolone derivatives
•
Yusuf M. Al-Hiari Amjad M. Qandil Rufaida M. Al-Zoubi
•
•
•
•
•
Muhammed H. Alzweiri Rula M. Darwish Ghassan F. Shattat
Tariq M. Al-Qirim
Received: 14 February 2011 / Accepted: 1 June 2011 / Published online: 11 June 2011
Ó Springer Science+Business Media, LLC 2011
Abstract The synthesis of new 7-(halophenylamino)-1-
cyclopropyl-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-
3-carboxylic acid derivatives is described. Substitution of
quinolonic compound 1a with chloro and fluoro aniline
derivatives at position 7 produced target compounds 2–9
with relatively low yields. Alternatively, the product of
correspondent carboxylate ester 1b subsequent with acid
hydrolysis produced satisfactory yield. The prepared targets
have shown interesting antibacterial properties against
standard and resistant gram-positive strains. In particular,
2-chloro and 3-chloro aniline derivatives (3 and 4) depict
MIC values of 6.7 and 0.9 lg/ml, respectively against stan-
dard S. aureous. Both compounds showed good activity
against resistant strains of S. aureous.
Introduction
Many fluoroquinolone antibacterial agents have been
introduced into clinical use with significant improvement in
antibacterial spectrum and activity. A vast array of fluoro-
quinolones presents antiinfective armory of the clinicians
(for example, ciprofloxacin and norfloxacin, Chart 1),
because of their therapeutic efficacy and tolerability (Baker
et al., 2004; Ball, 2000; Felmingham et al., 1985; Grohe,
1993; Okada et al., 1993; Wise et al., 1983). However,
continual prescribing of this class has led to the recent
emergence of fluoroquinolone-resistant bacteria that has
necessitated the search for innovative drugs efficacious
against resistant strains (Baker et al., 2004; Ball, 2000; Chen
and Lo, 2003; Cohen, 2000). The present work aims to
develop new 7-haloanilino-fluoroquinolones with potential
activity, lower toxicity, lower resistance or all of these.
Keywords 7-Anilinofluoroquinolone ꢀ
8-Nitrofluoroquinolone derivatives ꢀ Fluoroquinolone ꢀ
Antibacterial activity
Results and discussion
The structure–activity relationship of different positions of
the fluouroquinolone system has been thoroughly discussed
(Ball, 2000; Cianchetta et al., 2004; Emami et al., 2005;
Foroumadi et al., 2003). It is well established that C-7
substituent is regarded as drug–enzyme interaction domain,
where most chemical modifications led to major influence
on activity. The inhibition of DNA gyrase and cell per-
meability of quinolones is greatly influenced by the nature
of C-7 substitution, Chart 1. C-7 piperazinyl group in
addition to C-6 fluorine substituent has antibacterial
potency far superior to that of earlier classical quinolones
against both gram-positive and gram-negative bacteria
(Cianchetta et al., 2004; Emami et al., 2005; Foroumadi
et al., 2003).
Y. M. Al-Hiari (&) ꢀ R. M. Al-Zoubi ꢀ M. H. Alzweiri
Department of Pharmaceutical Sciences, Faculty of Pharmacy,
University of Jordan, Amman 11942, Jordan
e-mail: hiary@ju.edu.jo
A. M. Qandil
Faculty of Pharmacy, Jordan University of Science
and Technology, Irbid 22110, Jordan
R. M. Darwish
Department of Pharmaceutics and Pharmaceutical Technology,
Faculty of Pharmacy, University of Jordan, Amman 11942,
Jordan
G. F. Shattat ꢀ T. M. Al-Qirim
Faculty of Pharmacy, Al-Zaytoonah Private University
of Jordan, Amman 11733, Jordan
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Med Chem Res (2012) 21:1734–1740
1735
In the same vein, previous studies by our team for
introducing more lipophilic groups at C-7 such as anilines
revealed noticeable increase in gram-positive activity,
especially against resistant strains with significant loss of
gram-negative activity (Al-Hiari et al. 2007, 2011; Forou-
madi et al., 2003). As a continuation of previous research
findings, this research aims at further investigating
7-haloanilino derivatives 2–9 of the main nucleus 6-fluoro-
8-nitro-4-quinolone-3-carboxylic acid (1), Schemes 1 and
2 (Al-Hiari et al. 2007, 2011). Synthon 1 possesses an
electron withdrawing C-8 nitro group to facilitate coupling
of the substituted anilines.
O
5
8
4
F
COOH
6
7
3
2
R2
N
1
R1
ciprofloxacin (R1 = c-C3H5; R2 = N-piperazinyl)
norfloxacin (R1 = C2H5; R2 = N-piperazinyl)
Chart 1 Fluoroquinolone nucleous
Scheme 1 Synthesis of target
compounds by acid pathway 2–
9. *The values are calculated
according to best method option
of Chemdraw Ultra software
O
N
R4
O
N
F
CO2H
R3
R2
F
CO2H
substituted
anilines
Cl
DMF, Pyridine
reflux
N
H
NO2
R1
NO2
(1a)
Target Compounds 2-9
Compound R1
R2
H
H
Cl
H
H
H
H
F
R3
R4
H
H
H
H
Cl
H
H
H
Calculated LogP*
2
3
4
5
6
7
8
9
H
Cl
H
H
Cl
F
H
H
H
Cl
H
H
F
3.16
3.72
3.72
3.72
4.27
3.32
3.47
3.63
F
F
F
O
O
NH2
O
5
F
CO2Et
4
F
CO2H
F
CO2Et
6
3
+
DMSO, Pyridine
reflux
HCl, EtOH
reflux
2
HN
N
7
N
HN
Cl
N
1
8
X
NO2
NO2
NO2
(2-9)
Target acid
X
1b
X
intermediates ester
Scheme 2 Synthesis of target compounds by ester pathway 2–9
123

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Med Chem Res (2012) 21:1734–1740
Chemistry
atom with H5. Typical H5–F coupling with fluorine
(12 Hz ca) has disappeared and substituted with a new
doublet signal possess coupling constant around 8 Hz
representing H5–H6 coupling. H6 is also appeared at
expected position as doublet peak with J-value = 8 Hz.
Moreover, these findings are supported by MS and EA.
An alternative strategy using the ester derivative 1b was
used instead of the acid 1a. Coupling 1b with haloaniline
followed by hydrolysis of the ester was a successful
alternative approach to obtain the target compounds (2–9).
The haloaniline was reacted with the correspondent ester
1b to produce the target acids in satisfactory yield on
hydrolysis of the ester intermediate, Scheme 2. The target
compounds were produced in higher yields and minimal
side products. This might be related to better solubility of
ester derivatives in reaction solvent (DMSO) in comparison
with acids.
Clinically used fluoroquinolone antibiotics have C-7 sub-
stitutions of secondary amino group derivatives. However,
our research on primary amine substituents at C-7 position
proved that primary amino substitution produces also
active antibacterial compounds (Al-Hiari et al. 2007,
2011). From chemical point of view, primary amino groups
are weak nucleophiles to undergo nucleophilic aromatic
substitution reaction into C-7 position of quinolone
nucleus. This makes such reactions hard, low yielding and
involves many side products as proved in our initial
attempts. Therefore, the addition of nitro group into C-8
position of quinolone nucleus in the above compound 1
was intended to increase the electropositivity of C-7 carbon
atom (Al-Hiari et al., 2007).
As depicted in Scheme 1, the synthesis of compounds
2–9 was carried out through direct interacting of 7-chloro-
6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (1a) with 2 molar excess of halogenated anilines in
DMF/pyridine (7:3 V/V), followed by heating the mixture
at 70–80°C for 2–3 days. On work up, the precipitate was
purified over plate chromatography to produce the targets
in low yields. Although different procedures and condi-
tions were attempted to improve the yields, minimal
amounts were obtained and this was because of many
factors. The main reason was because of the weak
nucleophilicity of halogenated anilines that permitted
other stronger nucleophiles to attack at the same position
leading to two major side products; 10 (7-hydroxy
derivative when DMF was used as solvent) and 11 (in
case of using ethanol as solvent) (Al-Hiari et al., 2007).
Other major side products isolated with DMF/pyridine
procedure were the defluorinated side product 12 and the
dicoupled 13, Chart 2. De-fluorination of side product 12
is confirmed by disappearance of NMR coupling of flour
IR, MS, EA and NMR spectral data of 2–9 are in
accordance with the assigned structures and are given in
the experimental part. MS spectra display molecular weight
ions [M?] in good agreement with the calculated values of
1
their molecular formulae. H NMR signals and their cou-
pling constants were informative enough for assigning each
hydrogen in target structures. Moreover, EA results were
correct and within calculated values.
Antimicrobial screening
In vitro antibacterial activity of a series of 7-(chloro
and fluoro phenyl-amino)-1-cyclopropyl-6-fluoro-8-nitro-
4-oxo-1,4-dihydroquinoline-3-carboxylic acids (2–9) were
evaluated against standard and resistant isolates of S.
aureus and E. coli. Target compounds were all tested using
broth dilution method to determine the minimum inhibitory
concentrations (MICs). Results of active antibacterial
compounds were reported in Table 1.
O
O
N
O
O
N
F
COOH
COOH
F
COOH
F
COOH
HO
N
HN
EtO
N
HN
NO2
NO2
Cl
NO2
NO2
13
12
10
11
HN
F
Chart 2 Major side products isolated
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Med Chem Res (2012) 21:1734–1740
1737
Chemicals involved in the preparation of synthone 1 are
2,4-dichloro-5-fluoro-3-nitrobenzoic acid, ethyl 3-(N,N-
dimethyl-amino) acrylate and cyclopropyl amine. They were
all purchased from Acros (Belgium). Primary halogenated
anilines used are bought from the following: Aniline from
Merck (Dermstadt, Germany). 2-Bromoaniline, 4-bromo-
aniline, 2,4-dibromoaniline, 4-fluoroaniline and 2,3,4-trif-
luorooaniline from Acros (Belgium). 3-Chloroaniline from
Aldrich Chemicals (England). 4-Chloroaniline, 2,5-dichlo-
roaniline, 2-fluoroaniline and 2,4-difluoroaniline from Fluka
(Switzerland).
Table 1 Minimum inhibitory concentrations (MIC, lg/ml) for active
compounds against standard and resistant isolates of S. aureus com-
pared with ciprofloxacin
Compound no.
MIC (lg/ml)
S. aureus (standard)
ATCC6538
S. aureus
(resistant isolates)
Ciprofloxacin
2.9
11.7
6.7
1.4
2
3
4
5
7
9
ND
6.7
0.9
7.0
6.7
9.8
Mobile-phase mixtures for TLC were adjusted to be
94:5:1 chloroform–methanol–formic acid (CHCl3–MeOH–
FA) (system 1) and 90:10:1 CHCl3–MeOH–FA (system 2).
106.0
39.0
106.0
9.8
General procedure for the synthesis
Interestingly, chlorinated targets have shown stronger
activity against both standard and resistant isolates of S.
aureus gram-positive bacteria compared with fluorinated
ones. On the other hand, chlorinated aniline derivatives 3–5
and some fluorinated targets 9 have shown good activity
towards both standard and resistant isolates of S. aureus
bacteria with MIC values less than 10 lg/ml. MIC values
of 3-chloroaniline derivative 4 were excellent against both
standard S. aureus and resistant isolates of S. aureus, with
values of 0.9 and 7.0 lg/ml, respectively. The fluorinated
derivative 9 has shown better activity against resistant
isolates of S. aureus.
The title synthon 1, 7-chloro-6-fluoro-8-nitro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid, required in this work,
is prepared from 2,4-dichloro-5-fluoro-3- nitrobenzoic
acid, ethyl 3-(N,N-dimethylamino) acrylate and cyclopro-
pyl amine by following the stepwise synthetic procedures
reported in previous work (Al-Hiari et al., 2007).
Acid pathway
The target compounds were synthesized using synthone 1a
to produce the targeted 2–9 using the following general
procedure.
The most interesting phenomenon of some tested com-
pounds is their significant activity against resistant gram-
positive strains. This might be because of the increase of
lipophilicity ofthesecompounds (Table 1) in comparison with
ciprofloxacin (logP = 1.32) and norfloxacin (logP = 1.37).
Two molar equivalents of the halogenated aniline deriva-
tive was gradually added into a solution of 7-chloro-6-fluoro-
8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (1a,
0.5 g) in DMF/pyridine (7:3 V/V) and heated at 70–80°C. At
the end of the reaction (2–3 days), cold water was added and
the resulting precipitate was filtered, washed with water and
left to dry at room temperature. The resulting solid was then
dissolved in acetone and separated on TLC plates using sys-
tem 1 as eluent followed by second elution step using system
2. The band of interest was collected from the plate and the
silica was grounded and washed with acetone. The organic
layerwasthenevaporatedandthetargetwaslefttodryatroom
temperature (yields 10–20%).
Experimental
General
Melting points were determined on a Stuart scientific
electro-thermal apparatus, whereas infra red (IR) spectra
were recorded using Shimadzu 8400F-FT-IR spectropho-
1
tometer as KBr or NaCl discs. H- and 13C NMR spectra
were recorded on Varian Oxford-300 (300 MHz) spec-
trometer and Bruker Avance-400 (400 MHz) Ultrashield
spectrometer. Chemical shifts are expressed in ppm related
to tetramethylsilane (TMS) as the internal standard. Mass
spectra were recorded on Agilent 1100 series LC–MS,
under electro spray ionization (ESI) condition with nega-
tive-ion mode. Elemental analysis (CHN) were performed
on Euro Vector Elemental Model (EA 3000 A) and the
results were in good agreement with the calculated values
( 0.4%).
Ester pathway
Two molar equivalent of the haloaniline was gradually added
into a solution of ethyl 7-chloro-1-cyclopropyl-6-fluoro-8-
nitro-4-oxo-1,4-dihydroquinoline-3-carboxylate (1b, 0.5 g &
1.41 mmol) and 10 ml of DMSO and drops of pyridine. The
mixture was heated at 70–80°C under anhydrous conditions.
The reaction mixture was left to crystallize, then filtered and
the product was left to dry in dark place. The resulting solid
was re-crystallized from methanol/chloroform (39) to give
123

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Med Chem Res (2012) 21:1734–1740
fairly pure ester intermediate (25–65%). The intermediate
was further hydrolyzed on dissolving the solid in ethanol and
adding concentrated HCl (10 ml). The mixture was refluxed
at 70–80°C for 5 h, then target compound was filtered and
collected on drying as yellow solid (80–90%).
d6): d 0.88–0.98 (m, 4H, H2-20/H2-30), 3.62 (m, 1H, H-10),
7.17 (d,d, J = 4, 8 Hz, 1H, Ar–H: H-400), 7.56 (d,
J = 8 Hz, 1H, Ar–H: H-300), 7.63 (d, 3JH–F = 12.0 Hz, 1H,
H-5), 8.39 (br s, 1H, NH, D2O exchangeable), 8.57 (s, 1H,
H-2), 8.68 (d, J = 4 Hz, 1H, Ar–H: H-600), 13.25 (s, 1H,
CO2H); MS (CI/ESI -ve): m/z (%): calc. for C19H12
Cl2FN3O5 (452.22): 454 (8), 453 (12), 452 (57), 451 (19),
450 (100), 437 (48), 436 (83), 401 (3), 400 (7), 265 (7);
EA: Calculated for C19H12Cl2FN3O5, C, 50.46; H, 2.67; N,
9.29. Found: C, 50.55; H, 2.74; N, 9.32.
7-(2-Chloro-phenylamino)-1-cyclopropyl-6-fluoro-8-nitro-
4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (3)
Colour: bright yellow. Yield: 6% acid pathway,; mp
279–281°C (decomp); Rf value in system (2) = 0.60; IR
(KBr): m 3,456, 3,435, 2,951, 2,630, 2,734, 2,510, 1,731,
1,676, 1,663, 1,605, 1,543, 1,506, 1,473, 1,440, 1,355,
1-Cyclopropyl-6-fluoro-7-(2-fluoro-phenylamino)-8-nitro-
4-oxo-1,4-dihydroquinoline-3-carboxylic acid (7)
1
1,232, 799, 773 cm-1; H-NMR (300 MHz, DMSO-d6): d
0.90–0.96 (m, 4H, H2-20/H2-30), 3.61 (m, 1H, H-10), 7.10
(d,d,d, J = 1.2, 6.6, 7.5, 1H, Ar–H: H-400), 7.34 (d,d,d,
J = 2.0, 7.5, 8.4, 1H, Ar–H: H-500), 7.52 (d,d, J = 1.0,
Colour: green. Yield 9%; mp 292–294°C (decomp); Rf value
in system (2) = 0.60; IR (KBr): m 3,420, 3,064, 2,920, 2,856,
1,675, 1,624, 1,601, 1,538, 1,506, 1,491, 1,462, 1,368, 1,319,
;
1,227, 1,095 cm-1 1H-NMR (300 MHz, DMSO-d6): d
3
7.2 Hz, 1H, Ar–H: H-600), 7.64 (d, JH–F = 12.3 Hz, 1H,
H-5), 8.48 (s, 1H, H-2), 8.55 (d,d, J = 1.0, 5.7 Hz, 1H, Ar–
H: H-300), 8.66 (br s, 1H, NH, D2O exchangeable), 13.01
(br s, 1H, CO2H); MS (CI/ESI –ve): m/z (%): calcd. for
C19H13ClFN3O5 (417.8): 420(2), 719(8), 418 (37), 417
(27), 416 (100), 397(1), 381(2), 360(3), 359 (16), 325 (3),
306(6), 265(35), 217(1), 197(2),161 (2); EA: Calculated for
C19H13ClFN3O5, C, 54.62; H, 3.14; N, 10.06. Found: C,
54.65; H, 3.15; N, 9.99.
0.84–0.96 (m, 4H, H2-20/H2-30), 3.61 (m, 1H, H-10), 7.10
(d,d,d, J = 4, 8, 16 Hz, 1H, Ar–H: H-600), 7.19 (d,d, J = 8,
16 Hz, 1H, Ar–H: H-400), 7.30 (d,d,d, J = 4, 8, 16 Hz, 1H,
Ar–H: H-300), 7.64 (d, 3JH–F = 12.0 Hz, 1H, H-5), 7.94 (br s,
1H, NH, D2O exchangeable), 8.49 (d,d, J = 8, 16 Hz, 1H,
Ar–H: H-500), 8.56 (s, 1H, H-2), 12.97 (br s, 1H, CO2H); MS
(CI/ESI -ve): m/z (%):calcd. for C19H13 F2N3O5 (401.32):
402 (4), 401 (22), 400.0 (100), 247 (4), 212 (4), 157 (6); EA:
Calculated for C19H13F2N3O5, C, 56.86; H, 3.27; N, 10.47.
Found: C, 56.80; H, 3.30; N, 10.41.
7-(3-Chloro-phenylamino)-1-cyclopropyl-6-fluoro-8-nitro-
4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (4)
1-Cyclopropyl-7-(2,4-difluoro-phenylamino)-6-fluoro-8-
nitro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (8)
Colour: orange. Yield: 8% acid pathway; mp 240–243°C
(decomp); Rf value in system (2) = 0.45; IR (KBr): m
3,434, 3,390, 2,942, 1,735, 1,640, 1,535, 1,467, 1,365,
Colour: dark green. Yield: 80 mg,13%; mp 260–262°C
(decomp); Rf value in system (2) = 0.85; IR (KBr): m 3,501,
3,381, 2,942, 2,490, 1,679, 1,640, 1,604, 1,550, 1,532, 1,467,
1,210, 1,225 cm-1 1H-NMR (300 MHz, DMSO-d6): d
;
0.95, 1.02 (2 m, 4H, H2-20/H2-30), 3.71 (m, 1H, H-10), 7.16
(d,d, J = 1.0, 7.8 Hz, 1H, Ar–H: H-600), 7.39 (d,d, J = 7.8,
8.1 Hz, 1H, Ar–H: : H-500), 7.49 (d, J = 8.4 Hz, 1H, Ar–H:
H-400), 7.98 (m, J = 11.2 Hz, 2H, Ar–H: H-200 and H-5),
8.50 (br s, 1H, NH, D2O exchangeable), 8.76 (s, 1H, H-2),
12.95 (br s, 1H, CO2H); MS (CI/ESI -ve): m/z (%): calcd.
for C19H13ClFN3O5 (417.8): 419 (5), 418 (34), 417 (21),
416 (78), 383 (4), 360 (5), 359 (35), 339 (7), 325 (6), 311
(8), 293 (12), 265 (100), 191 (6), 161 (6); EA: Calculated
for C19H13ClFN3O5, C, 54.62; H, 3.14; N, 10.06. Found: C,
54.61; H, 3.20; N, 9.97.
1
1,356, 1,317, 1,225, 1,042, 957, 852, 803, 771 cm-1; H-
NMR (300 MHz, DMSO-d6): d 0.92, 0.97 (2 m, 4H, H2-20/
H2-30), 3.71 (m, 1H, H-10), 7.10 (d,d, J = 8.5, 8.9 Hz, 1H,
Ar–H: H-600), 7.39 (dd, J = 8.8, 11.0 Hz, 1H, Ar–H: H-300),
7.57 (br s, 1H, NH, exchangeable), 8.0 (d, 3JH–F = 11.5 Hz,
1H, H-5), 8.44 (dd, J = 9.1, 15.5 Hz, 1H, Ar–H: H-500), 8.76
(s, 1H, H-2), 12.40 (br s, 1H, CO2H); MS (CI/ESI -ve): m/z
(%): calcd. for C19H12 F3N3O5 (419.31): 419 (5), 418 (18),
417 (100), 339 (3), 293 (8), 256 (8), 265 (51), 113 (7); EA:
Calculated for C19H12F3N3O5, C, 54.42; H, 2.88; N, 10.02.
Found: C, 54.40; H, 2.81; N, 10.11.
1-Cyclopropyl-7-(2,5-dichloro-phenylamino)-6-fluoro-8-
nitro-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (6)
1-Cyclopropyl-6-fluoro-8-nitro-4-oxo-7-(2,3,4-trifluoro-
phenylamino)-1,4-dihydroquinoline-3-carboxylic acid (9)
Colour: greenish-yellow. Yield 12%; mp 265–267°C
(decomp); Rf value in system (2) = 0.60; IR (KBr): m
3,720, 3,402, 2,905, 1,713, 1,605, 1,587, 1,539, 1,462,
Colour: green. Yield 11%; mp 330–332°C (decomp); Rf
value in system (2) = 0.60; IR (KBr): m 3,449, 3,278,
3,196, 3,063, 1,680, 1,623, 1,600, 1,541, 1,509, 1,462,
1
1,340, 1,295, 1,220 cm-1; H-NMR (400 MHz, DMSO-
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Med Chem Res (2012) 21:1734–1740
1739
1
1,317, 1,231, 1,051, 944, 779 cm-1; H-NMR (400 MHz,
DMSO-d6): d 0.92, 0.95 (2 m, 4H, H2-20/H2-30), 3.63 (m,
1H, H-10), 7.32 (d,d, J = 8, 16 Hz, 1H, H-600), 7.64 (d,
3JH–F = 11.0 Hz, 1H, H-5), 8.42 (s, 1H, H-2), 8.55 (br s, 1H,
NH, exchangeable), 11.55 (s, 1H, CO2H); MSEI m/z (%):
493 (M?, 100), 463, 401.
3
J = 12 Hz, 1H, H-500), 8.26 (d, JH–F = 8.0 Hz, 1H, H-5),
8.48 (br s, 1H, NH, D2O exchangeable), 8.55 (s, 1H, H-2),
13.14 (s, 1H, CO2H); MS (CI/ESI -ve): m/z (%): calc. for
C19H11 F4N3O5 (437.30); 436 (100), 435 (2), 419 (2), 418
(5), 417 (3), 416 (6), 402 (4), 401 (13), 400 (23), 359 (9),
255 (3); EA: Calculated for C19H11F4N3O5, C, 52.18; H,
2.54; N, 9.61. Found: C, 52.40; H, 2.55; N, 9.58.
In vitro antibacterial activity testing
Materials and instruments
Nutrient agar and nutrient broth were obtained from Himedia
(Mumbai, India); 0.5 McFarland suspension (prepared by
adding 0.5 ml of BaCl2 (1.175% w/v BaCl2ꢀ2H2O) to
99.5 ml of 0.36 N H2SO4 (1.0% v/v). Microbiology samples
were incubated at 37°C using WTC binder incubator, Tutt-
lingen (Germany). The 96-flat bottom microplates were used
in the conduction of broth dilution test. ELx 800UV uni-
versal microplate reader, Biotek instrument was used to
determine the turbidity in the wells. The in vitro antibacterial
activity for all targets (2–9) using broth dilution method was
carried out on both standard and resistant isolates of Staph-
ylococcus aureus and Escherichia coli. The used standard
strains were gram-positive bacteria (S. aureus ATCC 6538)
and gram-negative (E. coli ATCC-8739). The minimal
inhibitory concentration (MICs) of the chemical compounds
assays was carried out as described by Foroumadi et al.
(2003), with minor modification. Ciprofloxacin was used as
standard antibacterial agent.
*Compound 2 and 5 were prepared using acid procedure
in this research and their data were previously reported
(Al-Hiari et al., 2007) using different procedure.
*Compounds 10 and 11 were also cited in our previous
research (Al-Hiari et al., 2007).
7-(2-Chlorophenylamino)-1-cyclopropyl-8-nitro-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (12)
This side product was obtained during the workup of target
compound 3 from acid pathway. Data for 12 are reported as
model, although all defluorinated derivatives of targets 2–9
were also isolated and identified.
Colour: yellow; yield: 20%; mp = 278–280°C (decom-
position); Rf value in system (2) = 0.88; IR (KBr): m 3,466,
3,444, 2,950, 2,627, 2,710, 1,738, 1,655, 1,660, 1,611, 1,545,
1,512, 1,482, 1,454, 1,341, 1,233, 805, 782 cm-1; 1H NMR
(400 MHz, DMSO-d6): d 0.93, 1.01 (2 m, 4H, H2-20/H2-30),
3.62 (m, 1H, H-10), 7.18 (m, 1H, Ar–H: H-600), 7.39 (m, 1H,
Ar–H: H-400), 7.57 (m, 2H, Ar–H: H-300/500), 8.08 (d,
J = 8.0 Hz, 1H, H-6), 8.28 (br s, 1H, NH, D2O exchange-
able), 8.55 (d, J = 8.0 Hz, 1H, H-5), 8.82 (s, 1H, H-2), 12.55
(br s, 1H, CO2H). MS (CI/ESI -ve): m/z (%): 398 (12), 343
(10), 331 (29), 325 (8), 324 (30), 317 (15), 312 (88), 309 (11),
293 (20), 290 (37), 281 (38), 259 (25), 247 (16), 243 (21), 231
(30), 212 (100), 193 (17), calcd. for C19H14 ClN3O5 (399.78).
Bacterial suspensions were prepared in sterilized dis-
tilled water, in a concentration around 1 9 107 cfu/ml,
which was standardized according to 0.5 McFarland sus-
pension as described by the Clinical and Laboratories
Standards Institute (CLSI) 2007.
Determination of minimum inhibitory concentration
The MICs (lg/ml) of targets were determined by the broth
dilution method using the two serial dilution techniques with
concentrations in the range 250–0.244 lg/ml. The MIC is
defined as the average of two successive concentrations of
the tested compound showing growth and no growth.
Tested compounds (1,000 lg) were first dissolved in
200 ll DMSO, and then 800 ll of sterilized distilled water
was added to end up with a final concentration of the stock
solution of 1,000 lg/ml.
The MIC test was performed in 96-flat bottom microtiter
plates, 100 ll of previously prepared and sterilized broth
was added in each well followed by 100 ll of tested
compounds stock solution (1,000 lg/ml) that was added to
the first well, mixed with the broth, followed by twofold
serial dilution on to successive wells across the plate to end
up with 11 successive twofold dilutions for each tested
compound. Then, 10 ll of bacterial suspension was used to
inoculate each well.
1-Cyclopropyl-6-fluoro-7-[4-(4-fluorophenylamino)-
phenylamino]-8-nitro-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (13)
This side product was obtained during the preparation of
1-cyclopropyl-6-fluoro-7-(4-fluoro-phenylamino)-8-nitro-
4-oxo-1,4-dihydroquinoline-3-carboxylic acid that was not
successful using this acid pathway.
Colour: orange; mp: 282–283°C (decomposition); Rf
value in system (2) = 0.0.92. IR (KBr): m 3,356, 3,056,
1,678, 1,605, 1,588, 1,560, 1,506, 1,461, 1,407, 1,368, 1,334,
1,289, 1,207, 1,156 cm-1; 1H NMR (400 MHz, DMSO-d6):
d 0.60 (m, 4H, H2-20/H2-30), 3.32 (m, 1H, H-10), 6.60–6.71
(m, 4H, Ar–H: H-200/H-300/H-500/H-600), 6.80 (m, 2H, Ar–H:
H-2000/H-6000), 7.30 (m, 2H, Ar–H: H-3000/H-5000), 7.75 (d,
123

1740
Med Chem Res (2012) 21:1734–1740
toward the development of water soluble fluoroquinolones and
structure–activity relationships of quinoline-3-carboxylic acids.
J Med Chem 47:4693–4709
Control tests for each experiment were performed.
Positive growth control was performed by adding one drop
of each micro-organism suspensions to four wells in each
plate of the culture medium without the test compound.
Negative growth control was also performed using four
uninoculated wells of medium without the test compound.
Plates were incubated at 37°C for 24 h and were checked
for turbidity.
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Twofold serial dilutions were carried out in a similar
manner for DMSO (20% v/v in water) to test its antibac-
terial activity. Ciprofloxacin standard was also tested as
reference compound. The turbidity was determined visu-
ally and using microplate reader. The results are presented
in the Table 1.
Acknowledgments We wish to thank the University of Jordan
represented by the Deanship of Academic Research, Jordan Univer-
sity of Science and Technology and Al-Zaytoonah Private University
for providing research facilities.
Grohe K (1993) The importance of the cycloaracylation process for
the synthesis of modern fluoroquinolones. J Prakt Chem/Chem-
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