Synthesis of new nitrofluoroquinolone derivatives with novel anti-microbial properties against metronidazole resistant H. Pylori

Article abstract of DOI:10.3390/molecules22010071

One of the major therapeutic approaches to preventing relapse and accelerating the healing of duodenal and gastric ulcers is the eradication of Helicobacter pylori. Due to the emergence of antibiotic resistance among clinical strains of H. pylori, alternative approaches using newly discovered antimicrobial agents in combination with the standard regimens for the treatment of H. pylori are increasingly needed. The purpose of the present study was to investigate the effect of newly synthesized 8-nitroflouroqunolone derivatives when used either alone or when combined with metronidazole against metronidazole-resistant H. pylori. Based on the standard antimicrobial susceptibility testing methods and checkerboard titration assay, all of the tested compounds showed interesting antimicrobial activity against 12 clinical strains of H. pylori, with the best in vitro effect for compound 3c. In addition, synergistic and additive activities of some of the tested compounds were observed when combined with metronidazole. Furthermore, among the tested nitroflouroquinolone derivatives, compound 3b showed significant urease inhibition activity with IC₅₀ of 62.5 μg/mL. These results suggest that 8-nitroflouroquinolone derivatives may have a useful role in combination with anti-H. pylori drugs in the management of H. pylori-associated diseases.

Full text of DOI:10.3390/molecules22010071

molecules
Article
Synthesis of New Nitrofluoroquinolone Derivatives
with Novel Anti-Microbial Properties against
Metronidazole Resistant H. pylori
Luay Abu-Qatouseh 1,*, Mohammad Abu-Sini 2,3, Amal Mayyas 4, Yusuf Al-Hiari 2,
Rula Darwish 2 and Talal Aburjai 2
1
Faculty of Pharmacy, University of Petra, 11914 Amman, Jordan
Faculty of Pharmacy, University of Jordan, 11914 Amman, Jordan;
2
mohammad.abusini@zuj.edu.jo (M.A.-S.); hiary@ju.edu.jo (Y.A.-H.);
rulad@ju.edu.jo (R.D.); aburjai@ju.edu.jo (T.A.)
Faculty of Pharmacy, Al-Zaytoonah University of Jordan, 11733 Amman, Jordan
3
4
Faculty of Health Sciences, American University of Madaba, 11821 Madaba, Jordan; a.mayyas@aum.edu.jo
*
Correspondence: labuqatouseh@uop.edu.jo; Tel.: +962-79-701-2082
Academic Editor: Diego Muñoz-Torrero
Received: 6 November 2016; Accepted: 28 December 2016; Published: 4 January 2017
Abstract: One of the major therapeutic approaches to preventing relapse and accelerating the healing
of duodenal and gastric ulcers is the eradication of Helicobacter pylori. Due to the emergence
of antibiotic resistance among clinical strains of H. pylori, alternative approaches using newly
discovered antimicrobial agents in combination with the standard regimens for the treatment of
H. pylori are increasingly needed. The purpose of the present study was to investigate the effect of
newly synthesized 8-nitroflouroqunolone derivatives when used either alone or when combined
with metronidazole against metronidazole-resistant H. pylori. Based on the standard antimicrobial
susceptibility testing methods and checkerboard titration assay, all of the tested compounds showed
interesting antimicrobial activity against 12 clinical strains of H. pylori, with the best in vitro effect for
compound 3c. In addition, synergistic and additive activities of some of the tested compounds were
observed when combined with metronidazole. Furthermore, among the tested nitroflouroquinolone
derivatives, compound 3b showed significant urease inhibition activity with IC50 of 62.5 µg/mL.
These results suggest that 8-nitroflouroquinolone derivatives may have a useful role in combination
with anti-H. pylori drugs in the management of H. pylori-associated diseases.
Keywords: nitrofluoroquinolones; H. pylori; urease inhibition
1. Introduction
Helicobacter pylori has been increasingly emerging as a major cause of chronic gastritis and
peptic ulcer worldwide [
major risk factor in the pathogenesis of gastric cancer and gastric mucosa-associated lymphoid tissue
(MALToma) [ ]. The eradication of H. pylori and/or attenuating its associated virulence factors
can therefore contribute to improving the clinical conditions of patients infected with this bacterium,
including the acceleration of peptic ulcer healing and minimizing the recurrence of gastric cancer [ ].
Current approaches for the effective eradication of H. pylori include the use of at least two antibiotics
and a proton pump inhibitor (triple and more recently quadruple therapy) [ ]. However, due to the
emergence of antibiotic resistance among H. pylori clinical strains—particularly against metronidazole
and clarithromycin—higher rates of treatment failure of triple therapy have been reported [ 10].
1,2]. Furthermore, H. pylori colonization of the gastric mucosa constitutes a
3–5
6
7,8
9,
Therefore, a search for new approaches with high efficacy and safety against H. pylori infection
is necessary.
Molecules 2017, 22, 71; doi:10.3390/molecules22010071
www.mdpi.com/journal/molecules

Molecules 2017, 22, 71
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Since their discovery, fluoroquinolones have been extensively considered as major successful
therapeutic antibacterial and anticancer agents [11 14]. However, a low level of resistance against
fluoroquinolones among H. pylori clinical strains has been reported [15 16]. In a previous work, the
–
,
biological activities of a group of 8-nitrofluoroquinolone derivatives have been evaluated, and strong
antibacterial effect against Escherichia coli and Staphylococcus aureus was observed [17,18].
In general, the synthesis of novel C-7-substituted derivatives of 1-cyclopropyl-6-fluoro-8-nitro-
4-oxo-1,4-dihydroquinoline-3-carboxylic acid having an 8-nitro substituent as an electron withdrawing
group was evaluated for biological activity. In the present study, the antibacterial activity of previously
described 8-nitrofluoroquinoline derivatives and two newlysynthesized 8-nitrofluoroquinolone
derivatives was tested against twelve clinical strains and one control strain of H. pylori which are
resistant to metronidazole. In addition, the effect of the potentially effective derivatives in combination
with metronidazole against metronidazole-resistant H. pylori strains was evaluated.
2. Results and Discussion
2.1. Synthesis of New Compounds
Synthesis of ester
yields [18]. Compounds 2a
compound under reflux (Scheme 1). Methoxy 2c was obtained in higher yields compared to other
derivatives.Hydrolysis of nitro ester generated nitro acid target . Compounds and 3a were
1
was conducted following a previously reported procedure with good
–e
were synthesized by the reaction of substituted anilines with
1
2
3
2
–e
1
identified and characterized by infrared spectroscopy (IR), mass spectrometry (MS), and H- and
13C-NMR spectroscopic analyses. The data for 2b
,c
and 3b
,
c
are presented in the experimental section,
since they were the most active.
O
O
F
CO2Et
R
F
CO2Et
R
, pyridine
NH2
Cl
N
N
H
N
DMSO, 70oC
NO2
NO2
2
1
O
N
5
3"
4
1
R
F
COOH
4"
4a
8a
6
2"
1"
3
2
HCl/ EtOH
Reflux, 80-85oC
7
5"
N
H
8
6"
NO2
3
Entry
R
a
b
c
d
e
Cl
F
H
Me OMe
Scheme 1.
General procedure for the synthesis of novel target compounds 3(a–e).
DMSO: dimethylsulfoxide.
The 1H-NMR spectra of all synthesized compounds (3a
–e) contained a doublet for H-5
(
3JH-F = 10–13 Hz) at ~8.0 ppm. The splitting of this signal was caused by the vicinal fluorine, and
indicated the presence of the fluoroquinolone nucleus in all of these compounds. Similarly, the
singlet for H-2 at ~9.0 ppm effectively confirmed that compound had been successfully formed.
The 1H-NMR spectra of the side chain of 3a
contained new singlets in the range of 2.3–3.7 ppm and
broad singlets in the range of 6.5–8.7 ppm, which were assigned to the aromatic methyl or methoxy
3
–
e

Molecules 2017, 22, 71
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and NH, respectively. The appearance of new peaks in the ranges of 6.8–7.2 ppm corresponding to
the aromatic side chain protons was further proof. These signals effectively confirmed that the aryl
amine side chains had been effectively incorporated into compound
3. The appearance of a new peak
above 12 ppm in accompanied by the parallel disappearance of the triplet, quartet pattern for ester
3
derivative 2 indicated the success of the hydrolysis step.
All of the carbons belonging to arylamine side chains were recognizable by their number, position,
and orientation in depth charts in the aliphatic region. These signals confirmed that the arylamine
chains had been successfully incorporated in compounds 3a
the appearance of methyl and methoxy peaks within aliphatic regions. For the fluoroquinolone part,
the 13C-NMR spectra of compounds 3a
contained a doublet (1JC-F = 250 Hz) at ~150 ppm for C-6,
–e, further confirmation was noticed by
–
e
which indicated the presence of the fluoroquinolone (FQ) nucleus in all of these compounds. This was
also confirmed from depth analysis. The splitting of the neighboring carbon signals at C-5 and C-7
into doublet peaks in these compounds (2JC-F~20 Hz) effectively confirmed that they were all vicinal
to a fluorine atom.
High- and low-resolution mass analysis was carried out for most compounds, and confirmed the
expected compounds. (M + 1) molecular peak was obtained in most cases. Some cases had witnessed
the appearance of (M + 2) molecular peaks due to bad tuning of the detector. This phenomenon can be
also referred to the presence of multiple nitrogen atoms of the expected compounds. Positive mode of
ionization adopted in the analysis procedure renders nitrogen atoms in their protonated form. Infrared
spectroscopy was carried out for all compounds. Characteristic peaks of carbonyl, hydroxyl, amino,
N=N, and C-X confirmed the identity of the expected compounds.
2.2. Antimicrobial Activity against Metronidazole-Resistant H. pylori
Previous work on the antimicrobial activity of 8-nitrofluoroquinolones reported variation in
the potency of the tested compounds against Gram positive and Gram negative bacteria. It has
been suggested in our group that the variable activity of these compounds could be related to the
hydrophobic/hydrophilic properties of their structures, with no specific pattern [17,18].
H. pylori possesses unique biological characteristics among all eubacteria. Being a microaerophilic
slow growing microorganism would dictate different activities of the antimicrobial agents against this
pathogen, particularly in vivo. In this study, 8-nitrofluoroquinolone derivatives were evaluated for
their antimicrobial properties against clinical strains of H. pylori. The initial screening showed that all of
the tested compounds had antimicrobial activity against H. pylori, with a potent effect of compound 3c
(Table 1). The minimum inhibitory concentration (MIC) values of the tested compounds against the
clinical strains were in the range of 2–64 µg/mL, which are higher than ciprofloxacin (Table 2). The best
activity was reported for compound 3c, followed by compounds 3a and 3d, respectively.
Table 1. Zones of inhibition (mm) of synthesized compounds (1 mg/mL), ciprofloxacin, and
metronidazole against clinical strains of H. pylori (1–12) and control strain.
Zones of Inhibition (mm)
Tested Compound
Clinical Isolates
Control Strain
1
2
3
4
5
6
7
8
9
10
11
12
3a
3b
3c
3d
3e
CIP
MTZ
DMSO
10
12
20
10
10
20
10
0
20
12
24
10
12
40
0
15
0
20
8
0
0
10
15
16
12
15
40
0
20
11
24
13
15
50
0
11
16
20
11
14
45
10
0
11
13
18
14
12
50
0
17
15
25
15
16
0
9
11
15
9
9
0
15
12
20
14
15
45
0
13
14
23
13
15
50
0
15
13
25
12
14
45
0
18
12
25
12
13
45
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
CIP: ciprofloxacin; MTZ: metronidazole.

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Table 2. Minimum inhibitory concentration (MIC, µg/mL) of tested compounds and metronidazole
against clinical strains of H. pylori (1–12) and control strain by two-fold agar dilution method.
Minimum Inhibitory Concentration (µg/mL)
Compounds
Strain Number
Control Strain
1
2
3
4
5
6
7
8
9
10
11
12
3a
3b
3c
32
32
4
16
32
4
16
16
8
16
32
4
8
32
2
32
64
4
16
32
4
8
32
8
16
32
4
32
32
4
16
32
8
16
32
4
16
32
8
3d
3e
CIP
MTZ
32
16
0.3
64
16
32
0.6
64
16
32
0.3
64
16
16
0.6
32
32
32
0.3
128
32
32
0.04
64
16
16
0.6
128
16
32
32
32
32
32
16
16
0.6
128
32
32
0.6
64
16
32
0.6
128
0.04 0.08 0.04
32 64 64
In terms of the antimicrobial effects of the combinations of the tested compounds with
metronidazole against the metronidazole-resistant strains, our study reported interesting synergistic
activity of the metronidazole 3a and metronidazole 3d combinations with fractional inhibitory
concentration (FIC) indices of 0.328, although these compounds when tested alone showed moderate
activity against the tested strains. It could be suggested that these compounds might work on the
cell membrane of H. pylori, making it more permeable to metronidazole, since these compounds have
some hydrophilic properties. Metronidazole is reduced to disrupt energy metabolism of anaerobes by
hindering the replication, transcription, and repair process of DNA results in cell death (i.e., it inhibits
nucleic acid synthesis by disrupting the DNA of microbial cells). Although metronidazole is a low
molecular weight compound that diffuses across cell membranes, facilitating its entry and achieving
an adequate concentration at its site of action would result in better effects on the bacteria, and this is
done by using compoundswhich have some hydrophilic properties. Moreover, the combination of
metronidazole + 3c and metronidazole + 3e showed an additive effect, with FIC indices of 0.656 and
0.874, respectively (Table 3). The combination of metronidazole + 3b showed indifferent activity with
FIC indices of 1.312, providing that compound 3b reported the least antimicrobial effect against the
studied strains when tested alone.
Table 3. Fractional inhibitory concentration (FIC) values of tested compounds in combination with
metronidazole against clinical strains of H. pylori (11, 12) and control strain by standard checkerboard
titration method with two-fold serial agar dilutions.
FIC Values (Index) (MIC Combination)
Strain Number
Compounds
FIC Mean
11
12
Control Strain
0.328 (*)
(2.5)
0.328 (*)
(2.5)
0.328 (*)
(2.5)
0.328 (*)
1.312 (=)
0.656 (+)
0.328 (*)
0.874 (+)
3a-MTZ
3b-MTZ
3c-MTZ
3d-MTZ
3e-MTZ
1.312 (=)
(10)
1.312 (=)
(10)
1.312 (=)
(10)
0.656 (+)
(5)
0.656 (+)
(5)
0.656 (+)
(5)
0.328 (*)
(2.5)
0.328 (*)
(2.5)
0.328 (*)
(2.5)
1.312 (=)
(10)
0.656 (+)
(5)
0.656 (+)
(5)
(*): Synergism; (+): Additive; (=): Indifferent.

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2.3. Inhibitory Effects of the Synthesized Compounds against H. pylori Urease Enzyme
One of the new potentials for the control of H. pylori infections is to reduce its pathogenicity by
targeting its virulence factors [19]. The urease enzyme is a major virulence factor of H. pylori required
for the optimal colonization and survival of this pathogen in the acidic conditions of the human
stomach [20]. In this study, 8-nitrofluoroquinolones were evaluated for their potential inhibitory
effect against H. pylori urease. The compounds were tested for their inhibitory effects in the range
of 0.5–500 µg/mL by the standard colorimetric method mentioned above, and the compounds that
showed less than 60% inhibition were considered to have no significant effect [20]. Our work showed
that significant urease inhibition effect was reported only for compounds 3b and 3c, with higher
inhibitory effect for 3c with IC50 value of 62.5 µg/mL (Table 4). This unique inhibitory effect of the
methyl and methoxy derivatives might be due to allosteric action against the enzyme, and needs to be
further investigated.
Table 4. Urease inhibition activity of the synthetic compounds. All compounds tested in the
concentration range of 0.5–500
µg/mL. The effect was considered significant if the inhibition was
more than 60%. Acetohydroximic acid (AHA) was used as positive control. NS = not significant.
Compound
IC50 (µM)
3a
3b
3c
3d
3e
NS
629
151
NS
NS
27
AHA
2.4. Structural Activity Relationship Studies
The results of this research reveal that 3c (para methoxy aniline substituents) exhibited significant
inhibitory activity against H. pylori. An attempt was carried out to understand the underlying structural
effect of these compounds. Esters
against clinical strains of H. pylori, demonstrating no activity. At this early stage of research, we can
suggest that activity of these FQs (3a ) lies mainly within the 4-oxo-pyridine-3-carboxylic acid system.
Losing the free COOH group in condensed the activity significantly. It was noticed that ester 2c is
higher in its activity than , which lacks any substituent at C7—although both have low activity against
1 and 2c were both evaluated for their antimicrobial properties
–
c
2
1
H. pylori. This finding designates that increasing lipophilicity through aniline substituents at C-7 can
significantly improve the activity against H. pylori. It is well known and was previously highlighted
by our group that highly lipophilic fluoroquinolone ligands improve antibacterial activity against
Gram positive bacteria in particular, and to some extent against Gram negative bacteria Therefore,
the activity of FQs 3a
other hand, our group has emphasized that Gram negative activity was boosted through C-7 and C-8
hydrogen bond network of fluoroquinolone systems. The activity of compounds 3a against Gram
–e could be explained to some extent by their high lipophilicity [17,18]. On the
–e
negative H. pylori could also be attributed also to the extra hydrogen bond network that might be
formed on C-7 (NH) and C-8 (NO2) groups in our FQs. The extraordinary activity of compound 3c
in particular against H. pylori can be explained by the additional H-B displayed by the para-methoxy
group oxygen in the anisidine substituent of 3c. Again, same structural features of 3c and extra H-B of
the methoxy can illuminate its unique inhibitory activity against H. pylori urease enzyme.
Fluoroquinolones 3a–e are derivatives of clinically used antibacterial quinolones such as the drug
ciprofloxacin. They exhibit the same nucleus—1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-
3-carboxylic acid with aniline substituent instead of piperazine at C-7. Infact, the aniline system is
known to be used in many drug interties as an analgesic drug. Therefore, we do expect them to suffer
similar side effects and toxicity to known commercial FQs.

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3. Materials and Methods
3.1. Materials and Instruments
All chemicals, reagents, and solvents were of analytical grade and used directly without further
purification. p-anisidine and p-toluidine were purchased from Fluka (Buchs, Switzerland). Reducing
agent—anhydrous stannous chloride crystals—was purchased from Fluka (Buchs, Switzerland).
Sodium nitrate was purchased from Sigma Aldrich (St. Louis, MO, USA). Melting points (mp)
were determined in open capillaries on a Stuart scientific electro-thermal melting point apparatus
(Stuart, Staffordshire, UK) and are uncorrected. Thin layer chromatography (TLC) was performed on
10 × 10 cm2 aluminum plates pre-coated with fluorescent silica gel GF254 (ALBET, Germany), and was
visualized using UV lamp (at 254 nm wavelength/short wavelength/ long wavelength). Mobile phase
mixtures were: 94:5:1 chloroform–methanol–formic acid (CHCl3–MeOH–FA) (system 1) and 50:50
(n-hexane–ethyl acetate) (system 2). Nuclear magnetic resonance spectra were recorded on a Varian
Oxford-300 (300 MHz) spectrometer (ALT, CO, USA), and a Bruker, Avance DPX-300 spectrometer,
a Bruker Avance-400 (400 MHz) Ultrashield spectrometer, and on a Bruker 500 MHz-Avance III
(500 MHz) (Bruker, Billerica, MA, USA). Deuterated dimethylsulfoxide (DMSO-d6) and deuterated
chloroform (CDCl3) were used as solvents in sample preparation, unless otherwise indicated.
The chemical shifts were reported in ppm relative to tetramethylsilane (TMS), which was used as an
internal reference standard.1H-NMR data are reported as follows: chemical shift (ppm), multiplicity,
coupling constant (Hz), number of protons, the corresponding proton(s). Infrared (IR) spectra were
recorded using a Shimadzu 8400F FT-IR spectrophotometer (Shimadzu, Kyoto, Japan). The samples
were prepared as potassium bromide (KBr) (Sigma, St. Luis, MO, USA) disks. High-resolution mass
spectra (HRMS) were measured in positive ion mode using electrospray ionization (ESI) technique by
collision-induced dissociation on a Bruker APEX-4 (7 Tesla) instrument. The samples were dissolved
in acetonitrile, diluted in spray solution (methanol/water 1:1 v/v + 0.1 M formic acid) and infused
using a syringe pump with a flow rate of 2µL/min. External calibration was conducted using arginine
cluster in a mass range m/z 175–871. Low-resolution mass spectra (LRMS) were measured by Applied
Biosystems-MDS SCIEX API 3200 LC/MS/MS system (Applied Biosystems, Foster City, CA, USA),
employing the positive mode using an electrospray ionizer (ESI) which was operated at 5.0–5.5 kV, with
the capillary heater at 350 ◦C, sheath gas pressure 45 psi and an ion trap analyzer. Molecular weight
was recorded as atomic mass unit (AMU) + 1, as the positive mode of ESI adds 1 AMU to the molecular
ion peak. Some compounds were recorded as AMU + 2, as the positive mode of ESI adds 1 AMU to
the molecular ion peak and so does the iontrap analyzer.
3.1.1. Synthesis of Novel Compounds 3b and 3c
Synthesis of Compound 1
Compound
1 was previously reported by our group [18]. It was synthesized following the same
procedure, with slight modification.
Synthesis of New Compounds (Scheme 1)
Ethyl1-cyclopropyl-6-fluoro-7-[(4-methyl phenyl)amino]-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylate (2b
)
(Scheme 1). Three equivalents of p-toluidine (0.9 g, 8.4 mmol) were added into a solution
containing ethyl7-chloro-1-cyclopropyl-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylate
(1.1 g, ◦2.8 mmol) and 10 mL of DMSO as a solvent and drops of pyridine, and was then refluxed at
65–70 C under anhydrous conditions for 4–5 days. The reaction mixture was monitored until no
starting material remained, then was left to crystallize at room temperature, and the product was
filtered, soaked with petroleum ether, and left to dry in a dark place. Color of solid compound:
1
orange; yield
≈
50% (0.6 g); mp: 227–230 ◦C (decomposition); Rf value in system 2 = 0.54. H-NMR
0
0
(300 MHz, DMSO-d6): 0.97 (m, 4H, H2-2 , H2-3 ), 1.26 (t, J = 7.2 Hz, 3H, CH3CH2), 2.21(s, 3H, Ar-CH3),

Molecules 2017, 22, 71
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3.58 (m,1H, H-10), 4.22 (q, J = 7.2 Hz, 2H, CH2CH3), 6.85 (dd, J = 2 Hz, 8.0 Hz, 2H, H-200, H-600),
7.01 (dd, J = 2 Hz, 8.1 Hz, 2H, H-300, H-500), 8.00(d, 3JH-F = 11.7 Hz, 1H, H-5), 8.54 (s, 1H, H-2), 8.60
(br s, 1H, NH, exchangeable).13C-NMR (75 MHz, DMSO-d6): 10.72 (C-20,C-30), 14.91 (CH3CH2), 21.0
(Ar-CH3), 39.15 (C-10), 60.89 (CH2CH3), 112.11 (C-3), 115.48 (d, 2JC-F = 21.5 Hz, C-5), 119.02 ( C-200,
C-600), 124.15 (d, 3JC-F = 6 Hz, C-4a), 129.85 (C-300, C-500), 131.72 (d, 2JC-F = 16 Hz, C-7), 132.04 (C-8a),
133.43 (C-8), 135.89 (C-100), 140.41 (C-400), 151.99 (C-2), 152.83 (d, 1JC-F = 258 Hz, C-6), 164.30 (CO2Et),
170.90 (d, 4JC-F = 2 Hz, C-4). IR (KBr):
ν .
3430, 3346, 1731, 1625, 1523, 1460, 1319, 1180, 1029 cm−1
HRMS (ESI, +ve): calc. for C22H21FN3O5 [M + H]+ (426.13870). Found 426.14598. Anal. Calcd for
C22H20FN3O5 (425.41): C, 62.11; H, 4.74; N, 9.88 Found C, 62.45; H, 5.13; N, 9.72.
Ethyl 1-cyclopropyl-6-fluoro-7-[(4-methoxyphenyl)amino]-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylate
(2c) (Scheme 1). Three molar equivalents of p-anisidine (3.1 g, 25.4 mmol) were added into a solution
containing (1.3 g, 8.46 mmol) and 5–10 mL of DMSO as a solvent and drops of pyridine, and was then
refluxed at 65–70 ◦C under anhydrous conditions for 2–3 days. The reaction mixture was monitored
until no starting material remained, then was left to crystallize at room temperature. The product
was filtered and left to dry in a dark place. Color of solid compound: red; yield
≈
86% (3.2 g); mp:
◦
245–247 C (decomposition); Rf value in system 2 = 0.48. 1H-NMR (300 MHz, DMSO): 0.92, 0.95
(2 m, 4H, H2-20, H2-30), 1.25 (t, J = 7.1 Hz, OCH2CH3), 3.56 (m, 1H, H-10), 3.68 (s, 3H, OCH3), 4.2
00
00
00
(q, J = 7.74 Hz, 2H, OCH2CH3), 6.8 (d, J = 8.88 Hz, 2H, H-2 , H-6"), 6.96 (d, J = 8.13 Hz, 2H, H-3 , H-5 ),
7.92 (d, 3JH-F = 12.3 Hz, 1H, H-5), 8.52 (br s, 1H, NH, exchangeable), 8.61 (s, 1H, H-2). 13C-NMR
0
0
0
(75 MHz, DMSO): 10.46 (2C, C-2 , C-3 ), 14.72 (OCH2CH3), 39.09 (C-1 ), 55.74 (OCH3), 60.7 (OCH2CH3),
111.91 (C-3), 114.44 (2C, C-200, C-600), 115.24 (d, 2JC-F = 21.15 Hz, C-5), 121.51 (2C, C-300, C-500), 122.85
(C-4a), 132.43 (d, 2JC-F = 14.8 Hz, C-7), 133.56 (C-8a), 135.35 (C-8), 135.38 (C-100), 151.77 (C-2), 151.9
(d, 1JC-F = 250.65 Hz, C-6), 155.95 (C-400), 164.11 (CO2Et), 170.66 (C-4). IR (KBr):
ν 3425, 3363, 1728,
1610, 1500, 1435, 1327, 1080, 1033 cm−1. HRMS (ESI, +ve): calc. for C22H20FN3NaO6 [M + Na]+
(464.12338). Found 464.12283. LRMS (ES, +ve) m/z calc. for C21H24FN3O5 (441.13): Found 443.4 (88%,
M+2), 442.0 (100%, M+1), 428.5 (2%), 412.6 (2%), 397.6 (7%), 396.6 (35%), 377.2 (1%), 335.2 (9%), 338.1
(1%), 322.1 (1%), 261.4 (1%), 102.1 (2%), 78.8 (1%), 74.3 (5%), 60.8 (1%), 59.2 (8%). Anal. Calcd for
C22H20FN3O5 (441.41): C, 59.86; H, 4.57; N, 9.52 Found C, 60.04; H, 4.32; N, 9.85.
1-Cyclopropyl-6-fluoro-7-[(4-methylphenyl)amino]-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3b
)
(Scheme 1). A vigorously stirred suspension of ethyl 1-cyclopropyl-6-fluoro-7-[(4-methyl
phenyl)amino]-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylate (2b, 0.28 mmol, 0.12 g) was dissolved
in a mixture of absolute ethanol (2 mL) and 12 N HCl (5 mL) under reflux at 80–90 ◦C for 24–48 h,
and the reaction was monitored by TLC. At the end of the reaction, the reaction mixture was poured
on crushed ice to precipitate a pure product that was collected by filtration and left to dry at room
◦
temperature. Color of solid compound: bright orange; yield
≈
55.4% (0.062 g); mp: 243–245 C.
1H-NMR (300 MHz, CDCl3): 0.87,1.14 (2 m, 4H, H2-20, H2-30), 2.29 (s,3H, Ar-CH3), 3.69 (m,1H,
H-10), 6.90 (d, J = 75 Hz, 2H, H-200,H-600), 7.09 (d, J = 7.8 Hz, 2H, H-300, H-500), 8.00 (br s, 1H, NH,
exchangeable), 8.12 (d,3JH-F = 12 Hz, 1H, H-5), 8.81 (s, 1H, H-2), 14.50 (br s, 1H, COOH). 13C-NMR
(75 MHz, DMSO-d6): 10.54 (C-20, C-30), 20.9 (CH3), 40.4 (C-10), 109.4 (C-3), 114.7 (d, 2JC-F = 21.83 Hz,
C-5), 119.2 (C-400), 120.0 (C-200, C-600), 120.6 (d, 3JC-0F0 = 7.8 Hz, C-4a), 129.7 (C-300, C-500), 133.0 (C-8a),
133.5 (d, 2JC-F = 15.4 Hz, C-7), 134.3 (C-8), 139.4 (C-1 ), 152.5 (C-2), 152.8 (d, 1JC-F = 253 Hz, C-6), 165.4
(COOH), 175.8 (C-4). IR (KBr):
ν .
3433, 3363, 3063, 2924, 1728, 1620, 1519, 1458, 1319, 1242, 1103 cm−1
HRMS (ESI, +ve): calc. for C20H16 FN3O5 [M + H]+ (397.10740). Found 397.10012. Anal. Calcd for
C20H16FN3O5 (397.36): C, 60.45; H, 4.06; N, 10.57 Found C, 60.86; H, 4.41; N, 10.79.
1-Cyclopropyl-6-fluoro-7-(4-methoxy-phenylamino)-8-nitro-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (3c
)
(Scheme 1). A vigorously stirred suspension of (2c, 0.6 g, 1.4 mmol) was dissolved in 12 mL mixture
◦
ofabsolute ethanol and 12 N HCl under reflux at 80–90 C for 24–48 h, and the reaction was monitored
by TLC. At the end of the reaction, the reaction mixture was poured on crushed ice to precipitate a pure
product that was collected by filtration and left to dry at room temperature. Color of product: deep

Molecules 2017, 22, 71
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◦
1
orange; yield
≈
69% (0.4 g); mp: 210–215 C; Rf value in system 1 = 0.5. H-NMR (300 MHz, DMSO-d6):
0
0
0
00
1.03, 1.21 (m, 4H, H2-2 , H2-3 ), 3.62 (m, 1H, NCH-1 ), 3.74 (s, 3H, OCH3 ), 7.05(d, J = 8.7 Hz, 2H, H-2 ,
00
00
00
H-6 ), 7.36 (d, J = 8.8 Hz, 2H, H-3 , H-5 ), 7.96 (d, 3JH-F = 11.7 Hz, 1H, H-5), 8.56 (s, 1H, H-2), 8.85 (br
s, 1H, NH, exchangeable), 14.66 (br s, 1H, COOH). 13C-NMR (75 MHz, DMSO-d6): 10.43 (2C, C-2 ,C-3 ),
39.85 (C-10), 55.73(OCH3), 106.84 (d, 2JC-F = 19 Hz, C-5), 109.35 (C-3), 114.13 (2C, C-200, C-600), 115.13
(2C, C-300, C-500), 122.65 (C-4a), 124.78 (C-8a), 132.45 (C-8), 134.46 (C-100), 140.33 (C-7), 148.01 (C-2),
0
0
152.3 (d, 1JC-F = 260 Hz, C-6), 156.65 (C-400), 166.59 (COOH), 176.45 (C-4). IR (KBr):
ν 3363, 3063, 2924,
2854, 1728, 1627, 1512, 1458, 1319, 1242, 1033 cm−1. HRMS (ESI +ve): calc. for C20H17FN3O6 [M + H]+
(414.11014). Found 414.10959. LRMS (ESI +ve): calc. for C20H17FN3O6 (413.1) Found 414.4 (5%, M + 1),
412.9 (76%), 384.2 (29%), 379.8 (1%), 367.4 (100%), 365.3 (13%), 361.3 (9%), 339.2 (58%), 331.7 (8%),
324.6 (15%), 321.2 (9%), 282.0 (3%), 240.9 (7%), 225.0 (4%), 217.4 (2%), 187.2 (1%), 145.0 (2%), 130.3
(13%), 123.4 (7%), 107.1 (1%), 101.0 (48%), 78.9 (93%), 74.5 (7%), 63.9 (13%), 59.0 (28%). Anal. Calcd for
C20H16FN3O6 (413.36): C, 58.11; H, 3.80; N, 10.17 Found C, 57.86; H, 4.11; N, 10.55.
3.2. Microbiological Methods and Anti-Microbial Assays
3.2.1. Bacterial Strains and Growth Conditions
Twelve H. pylori clinical isolates and one reference strain of H. pylori (NCTC 11916) were
used in this study. The clinical strains were isolated from gastric biopsy samples obtained by a
gastroenterologist of the Jordan University Hospital during a routine endoscopy. The gastric biopsy
material was processed according to the standard methodology. Briefly, each biopsy for culture
was homogenized using a tissue homogenizer (IKA, Staufen, Germany). Aliquots of 100 µL of the
homogenate were primarily cultured on Columbia blood agar (Oxoid, Hampshire, UK) supplemented
with 7% (v/v) horse blood and Dent selective supplement (Oxoid, Hampshire, UK). Subcultures of the
bacteria were performed using the same plates but without the Dent supplement. All of the plates
were incubated at 37 ◦C under microaerophilic conditions using CampyGen atmosphere generating
system (Oxoid, Hampshire, UK) in anaerobic jars for 5–7 days. Growth of H. pylori was confirmed
according to colony morphology, Gram staining, microaerophilic growth (at 37 ◦C), biochemical tests
(positive for oxidase, catalase, and urease), and subsequently by standard PCR of 16S ribosomal DNA
test [21]. H. pylori cultures were stored at
containing 10% (v/v) fetal calf serum (PAA, Pasching, Austria) and 15% glycerol.
−
70 ◦C in Trypticase soy broth (Oxoid, Hampshire, UK)
3.2.2. Antimicrobial Susceptibility Testing and Minimal Inhibitory Concentration Determination
Bacterial suspensions were prepared to the 2 McFarland’s standard and subsequently uniformly
spread on a solid growth medium in a Petri dish. Sterile paper disks (6 mm in diameter; Oxoid,
Hampshire, UK) were impregnated with 25 µL of each compound, and were placed on the surface
of each agar plate. Plates were incubated for 5–7 days under appropriate cultivation conditions.
Antibacterial activity was determined by the production of an inhibition zone around the impregnated
disc with the compounds. Disks impregnated with DMSO served as negative controls, and disks
with standard antibiotics (ciprofloxacin and metronidazole, Oxoid, Hampshire, UK) served as positive
controls. The minimal inhibitory concentrations (MICs) of each extract were determined by the
two-fold agar dilution method as previously described [22]. In brief, each compound was serially
diluted in DMSO and incorporated to molten Columbia blood agar plates supplemented with 7% (v/v)
horse blood. Spot of H. pylori (1
×
105 CFU) was applied on the surface of each plate, and the growth of
visible colonies was determined after 7 days of incubation at 37 ◦C under microaerophilic conditions.
MIC was recorded as the lowest concentration that inhibited the visible growth of organisms;
the plates with the standard antibiotics served as positive controls, and plates with DMSO served as
negative controls. Triplicates of each tested compound were performed, and the average of the results
was taken.

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3.2.3. Determination of in Vitro Interaction
Antimicrobial interactions between each compound and metronidazole against three strains
(two clinical isolates and one reference strain) of H. pylori were evaluated by the standard checkerboard
titration method [23]. Each compound and metronidazole was dissolved in DMSO and distilled water,
respectively. The bacterial inoculum, media, and culture conditions were the same as those described
for the MIC determination mentioned above. Experiments were performed in triplicate.
The fractional inhibitory concentrations (FICs) were calculated as follows:
•
•
FIC = (MIC of drug A in combination/MIC of drug A alone) + (MIC of drug B in combination/MIC
of drug B alone).
The FIC indices were interpreted as follows:
>4.0, antagonism.
≤
0.5, synergy; 0.5–1, additive; 1–4.0, indifference;
3.3. Urease Inhibition Assay
Urease inhibition was performed as described elsewhere [24]. Briefly, 10
µL of 1
×
108 CFU/mL
H. pylori suspension was incubated with equal amount of serially diluted compound in 96-well
◦
microplates for 30 min at 37 C. Subsequently, 200
µL of detection reagent composed of 50 mM
phosphate buffer, pH 6.8 containing 500 mM urea and 0.02% phenol red was added to each well.
The color development was measured at 555 nm in 5 min intervals. Controls included bacteria with
the reagent, and reagent with each compound. The percentage of inhibition was calculated by the
equation % inhibition = [(activity without compound
−
activity with compound)/(activity without
compound)] 100. The activity was compared to a reference urease inhibitor—acetohdyroxamicacid
×
(Sigma-Aldrich, St. Louis, MI, USA).
4. Conclusions
In conclusion, this work successfully introduced new 8-nitro-fluoroquinolone derivatives utilizing
a new procedure developed within the course of this work. The fluoroquinolone derivatives were fully
identified and characterized using NMR, IR, electrochemical analysis (EA), and MS. The antimicrobial
properties of all pure compounds were evaluated against H. pylori. Although the selected structures
did not follow a specific pattern (structure), it could be suggested that different hydrophilic/lipophilic
properties and different intra-cellular targets are considered for further investigation of the mechanism
of antibacterial action
The results clearly indicate that all new compounds have interesting antimicrobial activity against
metronidazole-resistant H. pylori. In addition, these compounds could be considered good candidates
for studying the combination effect with metronidazole in vivo for future potential as alternatives in
the triple therapy regimen used in clinical practice.
Acknowledgments: Authors wish to thank Deanship of Academic Research, Deanship of Post Graduate Studies
and the Faculty of Pharmacy, University of Jordan, Amman, Jordan for providing necessary facilities and funds
for conducting this research. Authors wish also to thank the Jordan Company for antibody production for their
technical support and providing us with the H. pylori control strain.
Author Contributions: Luay Abu-Qatouseh and Rula Darwish conceived and designed the microbiological
and biological activities, Yusuf Al-Hiari and Talal Aburjai designed the chemical synthesis and their
corresponding experiments; Mohammad Abu-Sini and Amal Mayyas performed the experiments. Rula Darwish
and Yusuf Al-Hiari analyzed the data; Talal Aburjai and Luay Abu-Qatouseh contributed by providing
reagents/materials/analysis tools; Luay Abu-Qatouseh wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.

Molecules 2017, 22, 71
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Sample Availability: SampleAvailability: Samples of the compounds are not available from the authors.
©
2017 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).