Novel derivatives of 5-amino-1-cyclopropyl-7-[(3R,5S)3,5- dimethylpiperazine-1-yl]-6,8-difluoro-4-oxo-quinoline-3-carboxylic acid: Their synthesis, antimicrobial, antifungal, and urease inhibitory studies

Article abstract of DOI:10.1007/s00044-013-0699-9

Sparfloxacin (SPFX) or 5-amino-1-cyclopropyl-7-[(3R,5S)3,5- dimethylpiperazine-1-yl]-6,8-difluoro-4-oxo-quinoline-3-carboxylic acid is an orally active synthetic, broad spectrum third generation quinolone, with excellent activity against Gram-positive bacteria with selectivity against anaerobes and atypical pathogens. Three derivatives of SPFX (2, 3, and 4) were synthesized by reacting different aromatic carboxylic acids with SPFX (1). Chemistry involved the formation of amide between reacting species through nucleophilic substitution reactions. The synthesized derivatives were then structurally characterized by IR, NMR, and mass spectroscopic techniques. The antimicrobial activities of these derivatives were evaluated against four Gram-positive, seven Gram-negative bacteria, and six fungi, using SPFX as a reference. Statistical analysis revealed these derivatives as active antimicrobial agents, and 2 was more potent antimicrobial agents than the parent drug as well other fluoroquinolones. Compounds 3 and 4 showed a significant activity against Fusarium solani. Moreover, these three derivatives were evaluated for inhibitory activities against enzyme urease, carbonic anhydrase II, and α-chymotrypsin. Results showed their selectivity against urease enzyme. Based on their nontoxic behavior, these derivatives may be potential agents for further studies.

Full text of DOI:10.1007/s00044-013-0699-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1248–1256
DOI 10.1007/s00044-013-0699-9
ORIGINAL RESEARCH
Novel derivatives of 5-amino-1-cyclopropyl-7-[(3R,5S)3,5-
dimethylpiperazine-1-yl]-6,8-difluoro-4-oxo-quinoline-3-
carboxylic acid: their synthesis, antimicrobial, antifungal,
and urease inhibitory studies
M. Saeed Arayne
•
Najma Sultana
•
Somia Gul Ajmal Khan
•
Received: 5 January 2013 / Accepted: 8 August 2013 / Published online: 30 August 2013
Ó Springer Science+Business Media New York 2013
Abstract Sparfloxacin (SPFX) or 5-amino-1-cyclopro-
pyl-7-[(3R,5S)3,5-dimethylpiperazine-1-yl]-6,8-difluoro-4-
oxo-quinoline-3-carboxylic acid is an orally active syn-
thetic, broad spectrum third generation quinolone, with
excellent activity against Gram-positive bacteria with
selectivity against anaerobes and atypical pathogens. Three
derivatives of SPFX (2, 3, and 4) were synthesized by
reacting different aromatic carboxylic acids with SPFX (1).
Chemistry involved the formation of amide between
reacting species through nucleophilic substitution reac-
tions. The synthesized derivatives were then structurally
characterized by IR, NMR, and mass spectroscopic tech-
niques. The antimicrobial activities of these derivatives
were evaluated against four Gram-positive, seven Gram-
negative bacteria, and six fungi, using SPFX as a reference.
Statistical analysis revealed these derivatives as active
antimicrobial agents, and 2 was more potent antimicrobial
agents than the parent drug as well other fluoroquinolones.
Compounds 3 and 4 showed a significant activity against
Fusarium solani. Moreover, these three derivatives were
evaluated for inhibitory activities against enzyme urease,
carbonic anhydrase II, and a-chymotrypsin. Results
showed their selectivity against urease enzyme. Based on
their nontoxic behavior, these derivatives may be potential
agents for further studies.
Keywords Sparfloxacin Á Derivatives Á
Antimicrobial activities Á Urease Á Enzyme inhibition
Introduction
The quinolones class of synthetic origin has a broad anti-
bacterial activity and used for the treatment of a wide variety
of infections (Haroon et al., 2012). Sparfloxacin (SPFX)
(Haroon et al., 2012) (Fig. 1) is an orally active broad
spectrum third generation quinolone, characterized by good
to excellent activity against Gram-positive cocci (notably S.
pneumoniae) and selectivity against anaerobes, and atypical
pathogens (Grady et al., 1997; Andersson and MacGowan,
2003). Functional groups at C-7 in the quinolones are
important for their antimicrobial activities (Foroumadi et al.,
2007; Sultana et al., 2009a, b). Therefore, the substituents at
C-7 position can alter the properties of quinolones, such as
antibacterial and pharmacokinetic properties as well as
adverse side effects (Domagala et al., 1988; Chu et al., 1985;
Shen et al., 1989). A five or six member cyclic amino moiety
M. S. Arayne (&)
Department of Chemistry, University of Karachi, Karachi,
Pakistan
e-mail: msarayne@gmail.com
(e.g., pyrrolidine or piperazine ring) is the most commonly
used substitution at C-7 position (De Sarro and De Sarro,
2
001; Anderson and Osheroff, 2001).
N. Sultana Á S. Gul
In an effort to develop new analogs of quinolones as
Research Institute of Pharmaceutical Sciences, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, University of
Karachi, Karachi 75270, Pakistan
agents with better therapeutic profiles (Sultana et al.,
2009a, b, 2011a, b, 2012; Arayne et al., 2009a, b, 2010)
and analogs of known therapeutic agents with objectives to
achieve enhanced therapeutic profiles and minimum
adverse effects, we synthesized a series of SPFX deriva-
tives with various aromatic carboxylic acids. In this
A. Khan
H.E.J. Research Institute of Chemistry, International Center for
Chemical and Biological Sciences, University of Karachi,
Karachi 75270, Pakistan
1
23

Med Chem Res (2014) 23:1248–1256
1249
were purchased from various chemical suppliers. All the
solutions were prepared fresh before work.
Instruments
Melting points were measured on a Gallenkamp apparatus,
and are uncorrected. TLC spots were detected under UV
light. Infrared spectra were recorded in KBr pellets on
Fig. 1 Sparfloxacin (SPFX) (1)
1
13
Shimadzu 470 instrument. H and C NMR spectra were
obtained by using Bruker XWIN NMR spectrometer with
TMS as an internal standard. Mass spectra were obtained
by using MAT 312 mass spectrometer and Jeol MS
instrument (range 50–800). Derivatives were dissolved in
CDCl or CD OD for NMR measurement.
process, our main focus was nucleophilic substitution of
carboxylic acid group forming the amide of substrate.
Carboxylic acids reacted with phosphorous pentachloride
(
PCl ) or sulfonylchloride (SOCl ) to form acid chloride.
5 2
3
3
The amide formation proceeded via attack by the amino
group of SPFX, which acted as nucleophile, on the car-
boxyl carbon of the acid chloride (Boteva and Krasnykh,
General procedure for the preparation of derivatives
2
009). The derivatives obtained in 57–78 % yields were
subjected to biological activity evaluation. Urease (urea
amidohydrolase EC 3.5.15), an enzyme responsible for an
organism to use urea as nitrogen source (Mobley and
Hausinger, 1989) catalyzed the hydrolysis of urea to
ammonia and carbon dioxide (Olivera-Severo et al., 2006).
In systemic nitrogen transport pathways of plants, it is
also used as defensive protein (Polacco and Holland, 1993;
Sirko and Brodzik, 2000). Urease produced by Helico-
bacter pylori is one of the major causes of pathologies, and
thus, play an important role in the pathogenesis of gastric
and peptic ulcers, apart from cancer (Smoot et al., 1990). It
is directly involved in the pathogenesis of urolithiasis,
pyelonephritis, and hepatic encephalopathy, hepatic coma,
and urinary catheter encrustation (Mobley and Hausinger,
Acid chlorides of benzoic, salicylic, and anthranilic acids
were reacted with SPFX. In first case, benzoyl chloride was
used as starting material, while salicylic acid was first
reacted with thionyl chloride in presence of DMF using
benzene as a solvent. In order to obtain oxychloride of
anthranilic acid, amino group was first protected by tert-
butyloxycarbonylation of amino group (Sarkar et al.,
2011), and later removed by trifluoroacetic acid in
dichloromethane (Isidro-Llobet et al., 2009).
For the synthesis of derivatives of SPFX, equimolar
solutions of both the drug and respective acid chloride were
mixed individually in methanol (double distilled) and
refluxed on water bath till the completion of reaction,
monitored by TLC on pre-coated silica gel plates using
methanol:ethyl acetate as eluting solvent. Compounds were
recrystallized in methanol till constant melting point.
Physicochemical and spectroscopic properties of these
derivatives are as follows:
1
989; Smoot et al., 1990; Mobley et al., 1995).
In agriculture, by contrast, a hydrolysis of fertilizer urea
by soil urease, if too rapid, results in unproductive vola-
tilization of nitrogen and may cause ammonia toxicity or
alkaline-induced plant damage (Krajewska, 2002). Due to
the diverse functions of this enzyme, its inhibition by
potent and specific compounds could provide an effective
tool for the treatment of infections caused by urease-pro-
ducing bacteria (Amtul et al., 2004).
5
1
-Amino-1-cyclopropyl-7-((3R,5S)-3,5-dimethylpiperazine-
-yl)-6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (SPFX)
In this paper, we report synthesis, characterization,
antimicrobial, and anti-enzymatic activities of three SPFX
derivatives (compounds 2, 3, and 4), which showed potent
antibacterial and antifungal activities.
Yellow powder, m.p. 260 °C. IR (KBr) 3460, 2519, 1718,
1
636, 1581, 1454, 1394, 1352, 1317, 1294, 1180, 1104,
1
047, 1024. H NMR (400 MHz-CDCl ): 0.53–0.28 (3H-
1
3
cyclopropyl), 3.56–3.31 (m, 4H, piperazinyl ring protons),
.0 (NH ), 7.96 (1H-phenyl), 8.51 (1H-quinolone), 11 (1H–
4
2
1
OH, Carbonyl). C NMR Shifts (ppm): CH 33.3, CH 7.7,
3
Experimental
2
C 136.3, C 137.5, C 177.5, C 126.1, C 136.0, CH 146.9,
CH 47.7, CH2 53, C 132.2, C 99.8. Mass m/z for
C H F N O = 393. EIMS: m/z (rel. abundance %) 393
Material and reagents
1
9 22 2 4 3
?
1
(M
10.5), 392 (base peak 100), 375 (26.4), 361 (8.8), 321
SPFX (1) was obtained as a gift by Abbott Pharmaceuticals
(
(7.5), 305 (2.4), 291 (42.8), 276, 234, 18 (5.7).
Karachi), while solvents and chemicals of analytical grade
123

1
250
Med Chem Res (2014) 23:1248–1256
5
-Amino-7-((3S,5R)-4-(2-aminobenzoyl)-3,5-
dimethylpiperazine-1-yl)-1-cyclopropyl-6,8-difluro-4-oxo-
,2,3,4-tetrahydroquinoline-3-carboxylic acid (2)
Antibacterial activity
1
The synthesized derivatives of SPFX (1) were screened for
their antimicrobial activity against a series of Gram-posi-
tive (Bacillus subtilis, Micrococcus luteus, Staphylococcus
aureus, and Streptococcus features), and Gram-negative
(Salmonella typhi, Klebsiella pneumoniae, Proteus mira-
bilis, Pseudomonas aeruginosa, Escherichia coli, Citro-
bacter, and Shigella flexneri) organisms by the
conventional cylinder-plate method (Remington, 2006).
The solutions for soaking disks were made of different 5,
10, 20, and 40 ppm dilutions, using water:methanol (9:1
v/v) as solvent. To ensure that the solvent had no effect on
bacterial growth, a control test was performed with test
medium supplemented with methanol at the same dilutions
as used in the experiment. Nutrient agar was prepared,
autoclaved at 121 °C for 15 min, cooled and then poured in
Petri dishes. Streaking was done with the help of sterile
cotton swab, soaked disks of complex solutions were
placed in them and the dishes were incubated for 24 h at
37 °C. Finally, the zones of inhibition were carefully
measured with the help of Vernier’s caliper.
Yield: 0.39 g (78 %), shiny yellow powder, m.p. 228 °C.
IR (KBr) 1,636 cm (N–H bending), 1,715 cm (C=O),
-
1
-1
-
1
1
(Symmetrical N–H stretching). H NMR
3
,408 cm
(
400 MHz-CDCl ): d 1.050–1.23 (5H-cyclopropyl), 3.29-
3
3
.31 (m, 8H, piperazinyl), 1.18(3H, CH ), 8.82 (NH ),
3 2
6
.45–7.24 (m, 4H, phenyl), 8.62(NH)0.14.48 (s, 1H of
1
carboxylic acid); C NMR Shifts (ppm): CH 35.2, CH 5.6
3
2
(
cyclopropane), C 101.3, 118.9, 128.9, 131.2, 134.7, 136.5,
38.0, 149.5 (1-benzene), C189.9 (1-carbonyl), CH 45.2
1
2
(
aliphatic), CH, 46.4 (cyclohexane), CH 50.3 (cyclohex-
2
ane) CH 116.1, 118.5, 128.0, 130.5 (1-benzene), 59.4
aliphatic), C 178.1 (1-carboxyl), C 168.3 (1-amide), CH3
(
1
8.7 (aliphatic). Mass m/z for C H F N O = 511.
26 29 2 5 4
5
1
-Amino-7-((3S,5R)-4-benzoyl-3,5-dimethylpiperazine-
-yl)-1-cyclopropyl-6,8-difluro-4-oxo-1,2,3,4-
tetrahydroquinoline-3-carboxylic acid (3)
Yield: 0.365 g (73 %), yellow powder, mp. 222 °C, IR (KBr)
-
1
-1
1,718 cm (C=O),
1
,664 cm (N–H
bending),
-1
1
,291–3,417 cm (N–H stretching). H NMR (400 MHz-
3
Antifungal activity
CDCl ): d 0.97–1.09 (5H-cyclopropyl), 3.25–3.32 (m, 8H,
3
piperazinyl), 2.89–2.90 (t, CH ), 4.00–4.02 (NH ), 7.45–7.91
3
2
Same procedure as for antibacterial activity was carried out
against a number of fungi (Candida albicans, Fusarium
solani, Trichophyton rubrum, Aspergillus parasiticus, and
A. effusus). Sabraoud dextrose agar instead of agar was
used and the dishes were incubated for 48 h.
1
5H, phenyl), 8.49 (NH), 14.46 (s, 1H of carboxylic acid); C
3
(
NMR Shifts (ppm): CH 35.2, CH 56 (cyclopropane), 46.4
2
(
cyclohexane), C 101.3, 127.2, 128.9, 131.2, 134.7, 135.2,
36.5, 138.0 (1-benzene), C189.9 (1-carbonyl), CH 45.2
1
2
(
aliphatic), CH , 50.3 (cyclohexane), CH 59.4 (aliphatic), C
2
1
78.1 (1-carboxyl), C 171.7 (1-amide), CH3 18.7 (aliphatic).
Urease inhibition assay
Mass m/z for C H F N O = 496.
26 28 2 4 4
In each well of 96-well plates, 25 lL of enzyme urease
5
-Amino-1-cyclopropyl-6,8-difluoro-7-((3S,5R)-4-(2-
hydroxybenzoyl)-3,5 dimethylpiperazine-1-yl)-4-oxo-
,2,3,4-tetrahydroquinoline-3-carboxylicacid (4)
(jack bean) solution and 5 lL of test compounds (0.5 mM)
were incubated with 55 lL of buffer containing 100 mM
urea for 15 min at 37 °C. Ammonia production was
determined as a urease activity through indophenol
method, as described by Weatherburn (1967). Final volume
was maintained as 200 lL by addition of 45 lL phenol
reagent (1 % w/v phenol and 0.005 % w/v sodium nitro-
prusside) and 70 lL of alkali reagent (0.5 % w/v NaOH
and 0.1 % NaOCl) to each well. Using a microplate reader
1
Yield: 0.285 g (57 %), green crystalline powder, mp 232 °C.
IR (KBr) 1,647 cm (N–H bending), 1,717 cm (C=O),
-1
-1
-1
1
,339–3,447 cm (N–H stretching). H NMR (400 MHz-
3
CDCl ): d 1.08–1.22 (5H-cyclopropyl), 2.48–3.16 (m, 8H,
piperazinyl), 3.99–4.01 (NH ), 6.64–7.66 (4H, phenyl), 8.52
2
3
13
(NH), 14.64 (s, 1H of carboxylic acid); C NMR Shifts (ppm):
(Molecular Devices, CA, USA), the increase in absorbance
CH 35.2, CH 5.6 (cyclopropane), C 101.3, 120.8, 128.9, 131.2,
2
was measured at 630 nm after 50 min at pH 6.8. By using
softMax Pro software (Molecular Devices), the result
(change in absorbance per min) was collected. Thiourea
was used as the standard inhibitor of urease. Percentage
inhibitions were calculated from the formula 100 -
(ODtestwell/ODcontrol) 9 100 (Arfan et al., 2010).
134.7, 135.2, 136.5, 138.0, 159.1 (1-benzene), C189.9 (1-car-
bonyl), CH 45.2 (aliphatic), CH 46.4, (cyclohexane), CH 50.3
2
2
(cyclohexane), CH 117.5,128.6, 121.1, 131.1 (1-benzene), CH
5
9.4 (aliphatic), C 178.1 (1-carboxyl), C 168.3 (1-amide), CH3
1
8.7 (aliphatic). Mass m/z for C H F N O = 512.
2
6 28 2 4 5
1
23

Med Chem Res (2014) 23:1248–1256
1251
Results and discussion
hydrogen of 1-butyl-3-methylimidazolium bis(trifluoro-
methylsulfonyl)imide from d 8.39 to 8.66 in the presence
of (Boc) O in the 1H NMR, and a drastic reduction of the
Derivatives of SPFX (1) were prepared by refluxing aro-
matic carboxylic acids with SPFX. Chemistry involves the
formation of amide between reacting species and parent
compound by nucleophilic substitution reactions. Carbox-
2
catalytic efficiency with 1-butyl-2,3-dimethylimidazolium
ionic liquids that are devoid of the C-2 hydrogen. The
synthesis of derivatives is shown in Fig. 2. All derivatives
were synthesized in good yields, and characterized by
spectroscopic techniques.
ylic acids react with phosphorous pentachloride (PCl ) and
5
sulfonylchloride (SOCl ) to form acid chlorides. The amide
2
formation proceeds via attack by the amino group of SPFX,
which acts as a nucleophile, on the carboxyl carbon of the
acid chloride. In case of reaction of anthranilic acid,
Antibacterial activity
1
-alkyl-3-methylimidazolium cation-based ionic liquid
As shown in Tables 1, 2, and 3 (Figs. 3, 4), the in vitro
antibacterial activities of these derivatives were evaluated
against a series of Gram-positive and Gram-negative bac-
teria, and compared with the parent drug as well as with
other relatively recently developed fluoroquinolones.
The susceptibility of bacterial strains toward SPFX, and
its derivatives were evaluated by measuring the zones of
(
IL) was used to catalyze N-tert-butyloxycarbonylation of
amine nitrogen with excellent chemoselectivity. This IL is
envisaged as ‘‘electrophilic activation’’ of di-tert-butyl
dicarbonate (Boc) O through bifurcated hydrogen bond
2
formation with the C-2 hydrogen of IL, and has been
supported by a downfield shift of the imidazolium C-2
Fig. 2 Synthesis scheme for
SPFX derivatives (2–4)
O
OCl
COOH
COOH
C
NH2
NHBoc
NHBoc
IL (2.5 mol %)
rt, 0.5 - 45 min
PCl5
(
Boc) O
2
+
77%
Anthranilic acid
Reacted with 1
NH2O
O
NH2O
F
O
F
OH
H3C
N
TFA in DCM
OH
N
F
H3C
N
N
O
N
O
N
F
H2N
CH3
BocHN
CH3
2
2
IL = ionic liquid; rt = room temperature; (Boc) = di-tert-butyl dicarbonate
NH2 O
O
F
OH
H3C
reacted with 1
N
N
F
O
N
Cl
O
CH3
3
NH2 O
O
F
HO
OH
H3C
HO
SOCl in DMF
2
OH reacted with 1
N
N
F
O
N
Benzene
O
Cl
O
HO
CH3
4
123

1
252
Med Chem Res (2014) 23:1248–1256
Table 1 Antibacterial activity of SPFX derivatives and their comparisons with other fluoroquinolones against Gram-positive organisms
Derivative
Zone of inhibition (mm)
B. subtilis
M. luteus
S. aureus
S. features
Concentration
ppm)
5
10
20
40
5
10
20
40
5
10
20
40
5
10
20
40
(
2
7
13
12
12
–
14
14
13
–
21
15
15
–
10
9
15
10
11
–
17
11
12
–
21
15
14
–
11
8
14
9
16
11
10
–
18
12
11
–
6
9
7
–
7
7
7
12
10
10
–
13
12
11
–
18
14
12
–
3
10
10
–
4
9
7
9
Control
Sparfloxacin
Gatifloxacin
Gemifloxacin
–
–
–
12
7
13
8
15
11
8
16
13
9
11
8
15
9
17
11
11
19
12
12
9
10
16
12
14
19
13
15
21
15
9
11
11
9
13
12
11
7
9
–
–
10
11
11
8
Table 2 Antibacterial activity of SPFX derivatives and their comparisons with other fluoroquinolones against Gram-negative organisms
Species
Zone of inhibition (mm)
Citrobacter K. pneumoniae P. mirabilis
P. aeruginosa
10 20 40
E. coli
10 20 40
S. flexneri S. typhi
10 20 40 5 10 20 40
Conc (ppm)
5
10 20 40
5
10 20 40
5
10 20 40
5
5
5
2
8
8
9
–
7
–
8
12 16 17
6
11 14 17
5
9
9
9
14 17
7
12 17 19 11 14 16 19
8 11 17 18 12 13 16 19
3
10 11 13 10 11 12 14
10 11 13 11 12 14
10 13 15 10 11 14 15 10 12 13 15 10 12 13 18 10 11 13 15
4
8
10 11 14
9
11 13 15 10 12 14 16 10 12 13 15
7
9
8
11 12 13
Control
Sparfloxacin
Gatifloxacin
Gemifloxacin
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
9
11 13 10 13 16 17 10 12 15 16 11 13 17 18
20 25 26 11 12 25 26 10 12 13 12 12 14 15 14 23 23 24 19 20 23 24
10 12 13 10 11 12 14 11 12 12 13 10 12 13 15
9
11 14 15
9
11 13 15
10 15 16
9
7
8
9
8
9
–
–
7
9
7
8
12 25 26
Table 3 Antifungal activity of SPFX derivatives and their comparisons with other fluoroquinolones against Fungi
Derivatives species
Zone of inhibition (mm)
F. solani
T. rubrub
C. albican
2
0
0
14
21
25
–
22
25
28
–
10
15
17
–
17
22
18
–
20
23
21
–
28
26
27
–
9
8
11
9
14
16
12
11
–
3
15
15
–
18
16
–
11
10
–
4
8
9
Control
Sparfloxacin
Gatifloxacin
Gemifloxacin
–
–
–
–
10
12
\5
11
18
\5
–
20
8
23
12
\5
24
14
\5
7
15
–
16
8
17
9
–
7
–
–
\5
\5
\5
\5
\5
\5
\5
\5
inhibition. The drug and the derivatives showed inhibition
diameters larger than 20 mm being recorded as highly
sensitive (Livermore et al., 2001; Simor et al., 1989).
These derivatives exhibited moderate to good activity
against a wide variety of Gram-positive and Gram-negative
bacteria. Comparison of antibacterial activity data sug-
gested that 2 is more potent antimicrobial agent, than the
parent drug, as well as other fluoroquinolones, against
Gram-positive and Gram-negative species, while 3 and 4
were found to be less active than the parent compound.
Derivative 2 was found to be most active against E. coli,
M. luteus, P. aeruginosa, S. typhi, and B. subtilis, and
displayed mild to moderate activity against S. aureus, S.
flexneri, and S. features. Derivatives 3 and 4 did not show
any significant activity with respect to parent drug.
Antifungal activity
SPFX (1) itself is not used as fungicidal agent (Sultana et al.,
2010), but its synthetic derivatives (2–4) showed very good
1
23

Med Chem Res (2014) 23:1248–1256
1253
Fig. 3 Antibacterial activity of
SPFX-derivatives against Gram-
positive bacteria
22
20
B. Subtilis
M. luteus
S. aureus
S. features
1
1
1
1
1
8
6
4
2
0
8
6
4
2
0
Sparfloxacin
Gemifloxacin
Compound 3
Gatifloxacin
Compound 2
Compound 4
Fig. 4 Antibacterial activity of
SPFX-derivatives against Gram-
negative bacteria
28
Citrobacter
K. pneumoniae
P.mirabilis
P. aeruginosa
E. coli
2
2
2
2
1
1
1
1
1
6
4
2
0
8
6
4
2
0
8
6
4
2
0
S. flexneri
S. typhi
Sparfloxacin
Gemifloxacin
Compound 3
Gatifloxacin
Compound 2
Compound 4
Table 4 Urease inhibitory activity of SPFX derivatives
activity against F. solani and T. rubrum (Table 4; Fig. 5).
None of them, including SPFX, showed activity against A.
parasiticus and A. effusus. In general, these derivatives can
also be studied as potent antifungal agents.
Compound
IC50 ± SEM
2
3
4
421.76 ± 0.58
418.43 ± 2.88
393.9 ± 0.99
21 ± 0.011
Enzyme inhibitory activity
a
Thiourea
These derivatives (2–4) were evaluated for enzyme inhib-
itory activity against urease, carbonic anhydrase II, and
SEM standard error of the mean
a
The standard inhibitors of the enzyme thiourea
123

1
254
Med Chem Res (2014) 23:1248–1256
Fig. 5 Antifungal activity of
SPFX-derivatives against fungi
30
2
2
2
2
2
1
1
1
1
1
8
6
4
2
0
8
6
4
2
0
8
6
4
2
0
F. solani
T. rubrub
C. albican
Sparfloxacin
Gemifloxacin
Compound 3
Gatifloxacin
Compound 2
Compound 4
a-chymotrypsin. In case of urease, all derivatives showed a
moderate activity, while against carbonic anhydrase II and
a-chymotrypsin they were inactive. From these studies, it
is concluded that these derivatives are selective against
urease. The results are presented in Table 4, where in
thiourea was used as reference.
pyridone ring were at 4.0 ppm, and carboxylic protons at
11 ppm. Comparing the main peaks of derivatives with that
of SPFX, proton NMR spectrum showed a set of signals
which were almost identical to those of SPFX, except for an
up field of signals where new peaks are found at the range of
6.45–7.91 ppm indicated presence of new phenyl rings, and
shifting of peaks particularly at carboxylic protons as well as
protons of aromatic C–NH indicating that these targeted
moieties took part in the synthesis of derivatives.
IR spectra
The characteristic absorption in the IR spectra of the
derivatives is listed above. In general, IR spectra of original
drug indicates the presence of functional groups at
Mass spectra
-1
-1
1
,300 cm (C=O, ketone), 1,752–1,742 cm (C=O,
–
Mass spectra of these SPFX derivatives showed molecular
ion peaks along with isotopic peaks at m/z values, consis-
tent with their respective molecular formulas. All deriva-
tives showed easily distinguishable peaks at m/z values of
70, 278, and 322 along with characteristics fragmentation
pattern before carboxylic acid moiety and aromatic C–NH
moiety in their respective structure, indicating that the
changes are made at these sites of reacting species.
Furthermore, peaks appearing at m/z values at 103, 304,
and 75 in mass spectra of synthesized derivatives indicated
the presence of new moieties with different fragmentation
patterns but these fragmentations were unstable and
showed a molecular ion peak at 392.
-1
COOH), 3,400 cm (stretching bends of –NH ), etc. (Dyer,
2
1
974; Nakamoto, 2009; Silverstein et al., 1981). Spectra of
-1
all derivatives showed new peaks at 1,664–1,647 cm in
addition of original peaks, confirming the formations of
amide which indicated that the reaction took place between
–NH group of parent drug and CO–OH of reacting species.
It was further confirmed by shifting of peaks at
-1
3,460–3,400 cm
which indicated N–H stretching of
amide. These facts were further aided by nuclear magnetic
resonance (NMR) spectroscopy and mass spectroscopy.
NMR spectra
These facts were further established by nuclear magnetic
resonance (NMR) spectroscopy. In the reported spectra of
SPFX, the peaks due to 1 b-N-cyclopropyl ring protons were
at 0.53 and 0.28 ppm, while peaks of piperazinyl protons
were found at 3.56 and 3.31 ppm. Protonsof rings adjacent to
Statistical analysis
Statistical package for the Social Sciences (SPSS Inc.
Chicago, IL, USA) was used for antimicrobial data
examination. Statistical analysis was appraised by one-way
1
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Med Chem Res (2014) 23:1248–1256
1255
analysis of variance (ANOVA) with the level of signifi-
cance chosen at p \ 0.05 or p \ 0.01 (any values lesser
than 0.05 or 0.01 were measured as significant). Multiple
comparisons of data were done by Dunnett’s test.
Boteva AA, Krasnykh OP (2009) The methods of synthesis,
modification, and biological activity of 4-quinolones. Chem
Heterocycl Compd 45(7):757–785
Chu DT, Fernandes PB, Claiborne AK, Pihuleac E, Nordeen CW,
Maleczka RE Jr, Pernet AG (1985) Synthesis and structure-
activity relationships of novel arylfluoroquinolone antibacterial
agents. J Med Chem 28(11):1558–1564
De Sarro A, De Sarro G (2001) Adverse reactions to fluoroquino-
lones: an overview on mechanistic aspects. Curr Med Chem
Conclusion
8
:371–374
Domagala JM, Heifetz CL, Hutt MP, Mich TF, Nichols JB, Solomon M,
Worth DF (1988) 1-Substituted 7-[3-[(ethylamino)methyl]-1-pyr-
rolidinyl]-6,8-difluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic
acids. New quantitative structure-activity relationships at N1 for
the quinolone antibacterials. J Med Chem 31(5):991–1001
Dyer JR (1974) Application of absorption spectroscopy of organic
compounds. Prentice-Hall of India Pvt. Ltd, New Delhi, p 35
Foroumadi A, Firoozpour L, Emami S, Mansouri S, Ebrahimabadi
AH, Asadipour A, Amini M, Saeid-Adeli N, Shafiee A (2007)
Synthesis and antibacterial activity of N-[5-(chlorobenzylthio)-
Present study shows that revisiting some of the well known
and making certain changes in their structures can lead to
better therapeutic profile and safer activity. We synthesized
three SPFX derivatives in good yields. Gram-positive bac-
teria and fungi (F. solani, and T. rubrum) proved to be more
sensitive toward newly synthesized derivatives (2–4). The
result of statistical analysis reveals that the synthetic deriv-
atives are active antimicrobial agents, whereas 2 is more
potent than the parent drug (1) as well as other fluoroquin-
olones against Gram-positive and Gram-negative species.
Compounds 3 and 4 showed significant activity against F.
solani. Anti-enzymatic assay showed that these derivatives
to be selective inhibitors of urease enzyme. These deriva-
tives thus are good candidates for further study.
1
,3,4-thiadiazol-2-yl] piperazinyl quinolone derivatives. Arch
Pharm Res 30(2):138–145. http://link.springer.com/content/pdf/
10.1007%2FBF02977685
Grady FO, Harold PL, Roger GF, David GW (1997) Antibiotic and
chemotherapy anti-infective agent and their use in therapy, 7th
edn. Churchill Livingstone Inc., New York, pp 419, 451
Haroon U, Zuberi MH, Arayne MS, Sultana N (2012) New improved
quinolone derivatives against infection. In: Bobbarala V (ed)
Biochemistry, genetics and molecular biology—‘‘a search for
antibacterial agents’’, pp 235–248. ISBN 978-953-51-0724-8.
doi:10.5772/46048
Acknowledgments Mrs. Somia Gul acknowledges the financial
support of Higher Education Commission Pakistan (Indigenous 5000
Ph.D Fellowship Program) to complete her Ph.D.
Isidro-Llobet A, lvarez MA, Albericio F (2009) Amino acid-
protecting groups. Chem Rev 109(6):2455–2504. http://pubs.
acs.org/doi/abs/10.1021/cr800323s
Krajewska B (2002) Ureases: roles, properties and catalysis. Wiad
Chem 56:223–253. http://www.chemia.uj.edu.pl/zespol_en.php
References
?id=10112
Amtul Z, Rasheed M, Choudhary MI, Rosanna S, Khan KM, Atta-Ur-
Rahman (2004) Kinetics of novel competitive inhibitors of
urease enzymes by a focused library of oxadiazoles/thiadiazoles
and triazoles. Biochem Biophys Res Commun 319:1053–1063.
http://www.sciencedirect.com/science/article/pii/S0006291X040
Livermore DM, Carter MW, Bagel S, Wiedemann B, Baquero F,
Loza E, Endtz HP, van Den Braak N, Fernandes CJ, Fernandes
L, Frimodt-Moller N, Rasmussen LS, Giamarellou H, Giama-
rellos-Bourboulis E, Jarlier V, Nguyen J, Nord CE, Struelens MJ,
Nonhoff C, Turnidge J, Bell J, Zbinden R, Pfister S, Mixson L,
Shungu DL (2001) In vitro activities of ertapenem (MK-0826)
against recent clinical bacteria collected in Europe and Australia.
Antimicrob Agents Chemother 45(6):1860–1867. http://www.
ncbi.nlm.nih.gov/pmc/articles/PMC90558/
1
0125
Anderson VE, Osheroff N (2001) Type II topoisomerases as targets
for quinolone antibacterials: turning Dr. Jekyll into Mr. Hyde.
Curr Pharm Des 7:337–353
Andersson MI, MacGowan AP (2003) Development of the quino-
lones. J Antimicrob Chemother 51(Suppl S2):1–11. doi:10.1093/
jac/dkg212
Mobley HLT, Hausinger RP (1989) Microbial ureases: significance,
regulation, and molecular characterization. Microbiol Rev
53:85–108
Arayne MS, Sultana N, Haroon U, Rizvi SBS (2009a) Synthesis,
characterization, antibacterial and anti-inflammatory activities of
enoxacinmetalcomplexes. BioinorgChemAppl. doi:10.1155/2009/
Mobley HLT, Island MD, Hausinger RP (1995) Molecular biology of
microbial ureases. Microbiol Rev 59:451–480. http://www.ncbi.
nlm.nih.gov/pmc/articles/PMC239369/
9
14105, http://www.hindawi.com/journals/bca/2009/914105/ref/
Nakamoto K (2009) Infrared and Raman spectra of inorganic and
coordination compounds, applications in coordination, organo-
metallic, and bioinorganic chemistry, 6th edn. Wiley, New York,
pp 279–280
Olivera-Severo D, Wassermann GE, Carlini CR (2006) Ureases display
biological effects independent of enzymatic activity. Is there a
connection to diseases caused by urease-producing bacteria? Braz
J Med Biol Res 39:851–861. http://www.scielo.br/scielo.php?
pid=S0100-879X2006000700002&script=sci_arttext
Arayne MS, Sultana N, Haroon U, Ahmed Mesaik M, Asif M (2009b)
Synthesis and biological evaluation of enoxain carboxamide
derivatives. Arch Pharm Res 32(7):967–974. http://www.ncbi.
nlm.nih.gov/pubmed/19641876
Arayne MS, Sultana N, Haroon U, Zuberi MH, Rizvi BS (2010)
Synthesis, characterization and biological activity of a series of
carboxamide derivatives of ofloxacin. Arch Pharm Res 33(12):
1901–1909. doi:10.1007/s12272-010-1203-4, http://www.springer
link.com/content/711t150286152321/
Arfan M, Ali M, Ahmad H, Anis I, Khan A, Choudhary MI, Shah MR
(2010) Urease inhibitors from Hypericum oblongifolium WALL.
Polacco JC, Holland MA (1993) Roles of urease in plant cells. In:
Jeon KW, Jarvik J (eds) International review of cytology, vol
145. Academic Press, Inc., San Diego, pp 65–103
J Enzyme Inhib Med Chem 25:296–299. http://informahealth
care.com/doi/abs/10.3109/14756360903179385
Remington (2006) The science and practice of pharmacy, 21th edn,
vol 1. Lippincott Williams & Wilkins, Baltimore, p 561. ISBN
123

1
256
Med Chem Res (2014) 23:1248–1256
0
-7817-4673-6. http://books.google.com.pk/books?id=NFGSSS
Sultana N, Arayne MS, Rizvi B, Mesaik MA (2009b) Synthesis, char-
acterization and biological evaluation of a series of levofloxacin car-
boxamide analogues Bull Korean Chem Soc 30(10):2294–2298.
http://journal.kcsnet.or.kr/main/j_search/j_abstract_view.htm?code=
B091024&qpage=j_search&spage=b_bkcs&dpage=ar
Sultana N, Arayne MS, Gul S, Shamim S (2010) Sparfloxacin–metal
complexes as antifungal agents—their synthesis, characteriza-
tion and antimicrobial activities. J Mol Struct 975(1–3):285–291.
http://www.sciencedirect.com/science/article/pii/S00222860100
03911
Sultana N, Arayne MS, Rizvi SBS, Haroon U (2011a) Synthesis,
characterization and biological evaluations of ciprofloxacin car-
boxamide analogues. Bull Korean Chem Soc 32(2):483–488.
doi:10.5012/bkcs.2011.32.2.483, http://journal.kcsnet.or.kr/main/
j_search/j_abstract_view.htm?code=B110221&qpage=j_search
&spage=b_bkcs&dpage=ar
baWjwC&printsec=frontcover&source=gbs_atb#v=onepage&q&f=
false
Sarkar A, Roy SR, Parikh N, Chakraborti AK (2011) Nonsolvent appli-
cation of ionic liquids: organo-catalysis by 1-alkyl-3-methylimida-
zolium cation based room-temperature ionic liquids for chemose-
lective N-tert-butyloxycarbonylation of amines and the influence of
the C-2 hydrogen on catalytic efficiency. J Org Chem 76:7132–
7
140. http://www.organic-chemistry.org/abstracts/lit3/374.shtm
Shen LL, Mitscher LA, Sharma PN, O’Donnell TJ, Chu DW, Cooper
CS, Rosen T, Pernet AG (1989) Mechanism of inhibition of
DNA gyrase by quinolone antibacterials: a cooperative drug—
DNA binding model. Biochemistry 28(9):3886–3894
Silverstein RM, Bassler GC, Morrill TC (1981) Spectrometric
identification of organic compounds, 4th edn. Wiley, New York,
QD272.S6 S55
Simor AE, Ferro S, Low DE (1989) Comparative in vitro activities of
six new fluoroquinolones and other oral antimicrobial agents
against Campylobacter pylori. Antimicrob Agents Chemother
Sultana N, Hamza E, Arayne MS, Haroon U (2011b) Effect of metal
ions on the in vitro availability of enoxacin, its in vivo implica-
tions, kinetic and antibacterial studies. Quim Nova 34(2):186–189.
http://www.scielo.br/scielo.php?pid=S0100-40422011000200003
&script=sci_abstract
Sultana N, Arayne MS, Rizvi SBS, Haroon U, Mesaik MA (2012)
Synthesis, spectroscopic and biological evaluation of some
levofloxacin metal complexes. Med Chem Res. doi:10.1007/
s00044-012-0132-9
3
3(1):108–109
Sirko A, Brodzik R (2000) Plant ureases: roles and regulations. Acta
Biochim Pol 47(4):1189–1195. http://www.actabp.pl/pdf/4_2000/
1
189-1195s.pdf
Smoot DT, Mobley HLT, Chippendale GR, Lewison JF, Resau JH
1990) Helicobacter pylori urease activity is toxic to human
(
gastric epithelial cells. Infect Immun 58:1992–1994
Sultana N, Naz A, Khan B, Arayne MS and Mesaik MA (2009a)
Synthesis, characterization, antibacterial, antifungal and immu-
nomodulating activities of gatifloxacin derivatives. Med Chem
Res 19(9):1210–1221. doi:10.1007/500044-009-9264-y, http://
www.springerlink.com/content/727644w025665076/
Weatherburn MW (1967) Urea colorimetric endpoint determination
urease—berthelot reaction. Anal Chem 39:971. http://www.
multi-quimicos.com/plasmatecpdfs/UREACOL125.pdf
1
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