Synthesis of 1,2,3-triazolo[4,5-h]quinolone derivatives with novel anti-microbial properties against metronidazole resistant Helicobacter pylori

Article abstract of DOI:10.3390/molecules22050841

Helicobacter pylori infection can lead to gastritis, peptic ulcer, and the development of mucosa associated lymphoid tissue (MALT) lymphoma. Treatment and eradication of H. pylori infection can prevent relapse and accelerate the healing of gastric and duo

Full text of DOI:10.3390/molecules22050841

molecules
Article
Synthesis of 1,2,3-Triazolo[4,5-h]quinolone
Derivatives with Novel Anti-Microbial Properties
against Metronidazole Resistant Helicobacter pylori
Mohammad Abu-Sini 1, Amal Mayyas 2, Nehaya Al-Karablieh 3, Rula Darwish 4,
Yusuf Al-Hiari 4, Talal Aburjai 4, Shereen Arabiyat 5 and Luay Abu-Qatouseh 6,
*
1
Faculty of Pharmacy, Al-Zaytoonah University of Jordan, Amman 11733, Jordan;
mohammad.abusini@zuj.edu.jo
2
Faculty of Health Sciences, American University of Madaba, Madaba 11821, Jordan; a.mayyas@aum.edu.jo
Hamdi Mango Center for Scientific Research, University of Jordan, Amman 11914, Jordan;
n.alkarablieh@ju.edu.jo
Faculty of Pharmacy, University of Jordan, Amman 11914, Jordan; rulad@ju.edu.jo (R.D.);
hiary@ju.edu.jo (Y.A.-H.); aburjai@ju.edu.jo (T.A.)
3
4
5
6
Salt College, Al-Balqa Applied University, As-Salt 19117, Jordan; Shereen@bau.edu.jo
Faculty of Pharmacy, University of Petra, Amman 11914, Jordan
*
Correspondence: labuqatouseh@uop.edu.jo; Tel.: +962-79-701-2082
Academic Editor: Derek J. McPhee
Received: 13 April 2017; Accepted: 15 May 2017; Published: 20 May 2017
Abstract: Helicobacter pylori infection can lead to gastritis, peptic ulcer, and the development of
mucosa associated lymphoid tissue (MALT) lymphoma. Treatment and eradication of H. pylori
infection can prevent relapse and accelerate the healing of gastric and duodenal ulcers as well
as regression of malignancy. Due to the increasing emergence of antibiotic resistance among
clinical isolates of H. pylori, alternative approaches using newly discovered antimicrobial agents
in combination with the standard antibiotic regimens for the treatment of H. pylori are of major
importance. The purpose of the present study was to investigate the effect of newly synthesized
8-amino 7-substituted fluoroquinolone and their correspondent cyclized triazolo derivatives when
either alone or combined with metronidazole against metronidazole-resistant H. pylori. Based on
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 best
in vitro effect for compounds 4b and 4c. Fractional inhibitory concentration (FIC) mean values
showed synergistic pattern in all compounds of Group 5. In addition, additive activities of some of
the tested compounds of Group 4 were observed when combined with metronidazole. In contrast,
the tested compounds showed no significant urease inhibition activity. These results support the
potential of new fluoroquinolone derivatives to be useful in combination with anti-H. pylori drugs in
the management of H. pylori-associated diseases.
Keywords: H. pylori; triazoloquinolone derivatives; synergism
1. Introduction
H. pylori is a gram negative, spiral shaped, microaerophilic bacterium [1,2]. Due to its unique growth
and virulence characteristics, such as production of as urease and CagA and VacA toxins, H. pylori
has been recently recognized as a major causative agent of different diseases such as chronic gastritis,
peptic ulcer, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALToma) [
3–6].
Eradication of H. pylori has been shown to result in ulcer healing, prevent peptic ulcer recurrence,
and reduce the prevalence of gastric cancer in high risk populations [7,8]. Treatment of H. pylori
Molecules 2017, 22, 841; doi:10.3390/molecules22050841
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Molecules 2017, 22, 841
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infections is routinely achieved by use of at least two antibiotics, especially amoxacillin and clarithromycin
or metronidazole, and a proton pump inhibitor (triple and more recently quadruple therapy) [ 11].
9–
Although the conventional treatment for eradication therapy of H. pylori allows obtaining high cure rates,
the eradication failure rate remains of 5–20% [12]. This can be explained by lack of patient compliance
with the drug regimens and the development of antibiotic resistance [13–15]. Therefore, a search for new
approaches with high efficacy and safety against H. pylori infection is necessary.
Fluoroquinolones have gained extensive use owing to their favorable pharmacokinetics and
broad antimicrobial spectra [16]. Various studies have reported that fluoroquinolones have many
interesting biological activities like analgesic [17], antidiabetic [18], antibacterial [19,20], and antitumor
activities [21,22]. The widespread use of fluoroquinolones has had its consequences, as selective pressure
has increased resistance for some species over the years [23]. So far, low level of resistance against
fluoroquinolones among H. pylori clinical strains is reported [24
activities of a group of 8-nitro 7-substituted fluoroquinolone derivatives have been evaluated and strong
antibacterial activity against Escherichia coli and Staphylococcus aureus was observed [26 27]. In general,
,25]. In a previous work, the biological
,
the synthesis of novel 8-nitro 7-substituted fluoroquinolone derivatives was evaluated for biological
activity. In the present study, the antibacterial activity of previously described 8-nitro 7-substituted
fluoroquinolone derivatives [28] and two newly synthesized 8-nitro 7-substituted fluoroquinolone
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 esters 1, compounds
procedure with good yields [27 29]. Compound 5c was prepared as previously described [30]. Due to
the novelty and remarkable activity of 4b 5b, and 4c, we report these compounds for the first time in
this work. All compounds ) were prepared in this work for biological activity, and their
spectral data was reported previously [29 31 32]. Upon coupling the substituted aniline to cipro ester 1,
2, 3 (a, d, e) were conceded following a previously reported
,
,
2–5 (a, d, e
,
,
compounds 2a–e were synthesized, (Scheme 1).
O
O
F
CO2Et
R
F
CO2Et
R
, pyridine
NH2
HCl/ EtOH
Reflux, 80-85oC
Cl
N
N
H
N
DMSO, 70oC
NO2
NO2
2
1
O
O
N
5
3"
5
3"
6"
4
1
4
1
R
F
COOH
R
F
COOH
4"
4a
6
4"
4a
8a
6
2"
1"
2"
1"
3
2
3
2
SnCl2/ HCl
25oC
7
7
5"
8a
5"
N
H
N
8
N
H
8
6"
NO2
NH2
3
4
O
N
5
6
F
4
COOH
5a
3"
7
2"
4"
R
Entry
R
3a
a
b
c
d
e
NaNO2
HCl/H2O, 0-5oC
3
8
9a
N
Cl
F
H
Me OMe
1"
9b
9
5"
N
N
6"
2
1
5
Scheme 1.
General procedure for the synthesis of novel target compounds 4, 5 (a–e).
DMSO: dimethylsulfoxide.

Molecules 2017, 22, 841
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This step was followed by a tedious hydrolysis process of nitro ester 2 to generated nitro acid
targets 3. Reduction of nitro acids 3a using stannous chloride in acidic media yielded the 8-amino
–
e
acid derivatives 4a–e with reasonable yield. These targets were identified and characterized by infrared
spectroscopy (IR), mass spectrometry (MS), and 1H- and 13C-NMR spectroscopic analyses. The spectral
data of 4, 5 (b, c) are presented in the experimental part since they are most active and novel.
1
The proton H-NMR spectra of the reduced compounds (4a
–
e
) contained a doublet for H-5
(3JH-F = 10–13 Hz) at ~8.0 ppm due to coupling with fluorine atom at C-6. A singlet peak for H-2 at
~8–9.0 ppm established that the fluoroquinolone system 4 is there. Both methyl and methoxy have
1
shown singlets in the range of 2.3–3.7 in the H-NMR spectra. The aromatic protons peaks were
present in the ranges of 6.8–7.2 which confirm the presence of the aniline side chain. Furthermore,
the appearance of a broad singlet stands for 2 protons at 4.2–5.5 ppm indicated that the reduction
step had proceeded successfully to give compounds 4b and 4c. The disappearance of such broad
singlet confirmed that compounds 4b and 4c had undergone the diazotization and further cyclization
reactions to give compounds 5b and 5c, respectively.
For 13C spectra, all carbon peaks of
in the charts. These signals confirmed that the aryl amine chain is fused in compounds
fluoroquinolone part, the 13C-NMR spectra of compound contained a doublet (1JC-F = 250 Hz) at
~150 ppm for C-6 (C4 for compound ); which indicated the presence of the fluoroquinolone nucleus in
all of these compounds. This was also confirmed from depth analysis. The splitting of the neighboring
4
and
5
were detectable by their number, position and orientation
). For the
4, 5 (a–e
4
5
carbon signals of 4 at C-5 and C-7 (5 and 3a in compound 5) into doublet peaks in these compounds
(2JC-F ~20 Hz) effectively confirmed that they were all vicinal to a fluorine atom. Appearance of two
symmetrical long signals in depth confirmed the aromatic side chain presence.
2.2. Antimicrobial Activity against Metronidazole-Resistant H. pylori
Previous work on the antimicrobial activity of different quinolones derivatives reported significant
variation in the susceptibility of the tested compounds against Gram positive and Gram negative
bacteria [33
,34]. This was relatively ascribed to the hydrophobic/hydrophilic properties of their
structures, with no specific pattern [27
–
29]. H. pylori clinical isolates exhibit unique biological
characteristics among all eubacteria. Being a microaerophilic slow growing microorganism would
provide a plasticity of its response toward the antimicrobial agents particularly in vivo. This study is
an attempt toward developing putative novel anti-H. pylori agents derived from 8-amino 7-substituted
and their correspondent cyclized triazoloquinolone derivatives. The results of initial screening showed
that all of the tested compounds have antimicrobial activity against H. pylori, with remarkable potent
effects of compound 4b, followed by compounds 4a and 4c, respectively (Table 1). Compounds
of Group 5 (7–8 triazole) showed good but lower activity compared to compounds of group 4.
The MIC values of the tested compounds against the clinical strains were in the range of 0.6–5
µg/mL
which are comparable to ciprofloxacin. Compounds 4a and 5b exhibited significant in vitro
–
e
–e
anti-H. pylori activity (Table 2). The highest antibacterial activity (lowest MIC values) was reported
for compound 4b, followed by compounds 4a and 4c, respectively. It is worth mentioning that our
compounds are more active than metronidazole on the resistant strains. This activity might be linked
to their lipophilicity [26,27].
In terms of the antimicrobial effects of the combinations of the tested compounds with
metronidazole against the metronidazole resistant strains, this work reported that Group 5b–e
developed synergistic pattern in all its compounds with fractional inhibitory concentration (FIC)
indices range of 0.283–0.349. Surprisingly, our results showed that all tested compounds of Group 4
lost synergistic activity with metronidazole combination and showed mainly indifferent activity of
metronidazole 4a, metronidazole 4b, and metronidazole 4c combination with fractional inhibitory
concentration (FIC) indices of 1.025, 1.192, and 2.212, respectively (Table 3), although these compounds
when tested alone showed high activity against the tested strains. It could be suggested that C-8 amino
group of these compounds was responsible for such phenomenon through physical interaction with

Molecules 2017, 22, 841
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metronidazole. Such interaction might be attributed to intermolecular hydrogen bond interaction of
C-8 primary amine in these compounds with metronidazole.
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
Clinical Isolates
Control
Strain
Compounds
1
2
3
4
5
6
7
8
9
10 11 12
4a
4b
4c
4d
4e
5b
5c
5d
5e
25 45 35 40 46 43 40 16 11 38 41 42
25 46 38 44 48 45 42 21 15 42 40 44
24 38 34 37 35 38 35 20 14 29 37 35
15 35 30 35 38 36 44 18 20 25 35 40
15 35 34 40 45 42 43 17 12 30 38 36
40
49
45
44
43
21
35
32
25
65
0
10 15 12 10 20 12 13 15
16 30 25 28 29 30 22 15
22 22 20 19 24 20 16 14
20 25 15 15 24 17 14 13
7
7
8
7
0
0
0
17 18 16
25 29 28
20 22 20
19 24 22
64 50 55
CIP
MTZ
DMSO
20 60
0
0
0
50 53 65 59
0
0
0
10
0
0
0
0
0
0
0
10
0
0
0
0
0
0
0
0
0
0
CIP: ciprofloxacin; MTZ: metronidazole.
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
4a
4b
4c
4d
4e
5b
5c
5d
5e
2.5
0.6
5
2.5
5
2.5
5
2.5
5
0.6
1.3
2.5
5
2.5
5
1.3
1.3
2.5
0.6
64
2.5
2.5
5
1.3
5
1.3
1.3
1.3
2.5
0.3
64
2.5
2.5
5
2.5
5
1.3
2.5
1.3
5
0.6
32
1.3
0.6
2.5
2.5
5
2.5
2.5
2.5
2.5
0.6
1.3
2.5
1.3
2.5
1.3
1.3
1.3
1.3
1.3
0.6
1.3
5
5
1.3
2.5
5
2.5
5
5
5
10
5
0.6
5
2.5
2.5
5
10
10
10
2.5
2.5
5
1.3
5
1.3
2.5
2.5
2.5
0.6
0.6
0.6
1.3
2.5
1.3
1.3
1.3
1.3
1.3
0.6
0.6
1.3
2.5
1.3
1.3
1.3
1.3
0.6
64
1.3
1.3
1.3
1.3
2.5
5
2.5
2.5
2.5
0.6
128
5
2.5
1.3
2.5
5
CIP
MTZ
0.3
64
0.3 0.04 0.6 0.04 0.08 0.04 0.6
128 64 128 32 64 64 128
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)
Compounds
Strain Number
FIC Mean
11
12
Control Strain
4a-MTZ
4b-MTZ
4c-MTZ
4d-MTZ
4e-MTZ
5b-MTZ
5c-MTZ
5d-MTZ
5e-MTZ
2.039 (=)
2.039 (=)
4.078 (−)
1.039 (=)
1.078 (=)
0.259 (*)
0.519 (+)
0.259 (*)
0.519 (+)
0.519 (+)
1.019 (=)
2.039 (=)
0.519 (+)
0.539 (+)
0.519 (+)
0.259 (*)
0.519 (+)
0.259 (*)
0.519 (+)
0.519 (+)
0.519 (+)
0.519 (+)
0.539 (+)
0.072 (*)
0.269 (*)
0.269 (*)
0.134 (*)
1.025 (=)
1.192 (=)
2.212 (=)
0.692 (+)
0.718 (+)
0.283 (*)
0.349 (*)
0.349 (*)
0.304 (*)
(*): Synergism. (+): Additive. (=): Indifferent. (−): Antagonism.

Molecules 2017, 22, 841
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Moreover, combination of metronidazole + 4d and metronidazole + 4e showed additive effect,
with FIC indices of 0.692 and 0.718, respectively (Table 3). The combination of metronidazole + 4c
showed antagonistic effect, with FIC indices of 4.078 when tested against strain 11. The Most active
compounds 4a, 4b, and 4c, suffered significant loss in antibacterial activity due to significant hydrogen
bond interaction possibly with both N-H bond at 8-amino leading to loss polar characters. On the
other hand, less active derivatives 4d and 4e showed minor change in FIC values, due to weak original
antibacterial activity. Group 5 showed best FIC with synergistic mode in all derivatives since they
were unable to form such intermolecular hydrogen bond and thus no effect on final lipophilicity and
consequently on net activity.
2.3. Inhibitory Effects of the Synthesized Compounds against H. pylori Urease Enzyme
H. pylori infection may be controlled by reduction of its pathogenicity through targeting its
virulence factors [35]. The most important virulence factor of H. pylori is the urease enzyme, which
is essential for the optimal colonization and survival of this pathogen in the acidic conditions of
the human stomach [35]. In this work, 8-amino 7-substituted and their correspondent cyclized
triazoloquinolone derivatives were evaluated for their potential inhibitory effect against H. pylori
urease. The compounds were tested for their inhibitory effects in the range of 0.5–1000 µg/mL by the
standard colorimetric method described in the materials and methods section, and the compounds
that showed less than 60% inhibition were considered to have no significant effect [36]. Our study
showed no significant urease inhibitory effect for compounds 4a, 4b, and 4c at 1000 µg/mL with
inhibitory percent 13, 10, and 33%, respectively. The other compounds were found to be inactive at the
specified concentrations.
2.4. Structural Activity Relationship Studies
The work discloses that both series of
H. pylori. The inhibitory activity of the reduced 8-amino derivatives 4 (a–d) were much stronger than
the triazoloqhinolones ). Compounds 4b and 4c exhibited comparable MIC ( g/mL) compared
can open the door
4 and 5 (a–e) exhibited significant inhibitory activity against
5
(
b–
e
µ
to ciprofloxacin against some clinical strains (Table 2). These data reveal that series
4
for strong inhibitors against H. pylori.
In previous work [26
the 4-oxo-pyridine-3-carboxylic acid system present in all compounds prepared (
COOH group in compounds reduced the inhibitory activity against H. pylori significantly [29].
Furthermore, it was noted that FQs lack any aniline substituent at C7 as in compounds C3 in 5
–
29], we established that the activity of these FQs mendacities mainly within
3–5). Losing the free
2–5
3,
4
(
)
showed no or low activity against H. pylori. This conclusion entitles that increasing lipophilicity
through aniline substituents at C-7 can significantly improve the activity against H. pylori. Evidently,
most of our derivatives are lipophilic compound and have shown the reasonable inhibitory activity
against H. pylori [29]. These findings were emphasized previously by our group: highly lipophilic
fluoroquinolone ligands improve antibacterial activity against Gram positive bacteria in particular,
and to some extent against Gram negative bacteria [26
emphasized that Gram negative activity was boosted through C-7 and C-8 hydrogen bond network
of fluoroquinolone systems as represent by compounds of series . The activity of compounds 4a
–28]. Within the same vein, our group has
4
–e
against Gram 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 (NH2) groups in our FQs. The extraordinary activity
of compound 4b in particular against H. pylori can be explained by high lipophilicity of the methyl
substituent and H-B network enforced by NH2 group. Most possible that extra H-B imposed by the
8-amino group can leads to intermolecular hydrogen bond with the combined drug metronidazole
and hide the active group in both FQ and metronidazole from acting on their target in H. pylori.
This interaction between highly polar groups in both compounds might explain the low activity of the
combination against H. pylori. The cyclized triazoloquinolones of series 5 displayed lower inhibitory
activity possibly due to loss of free H-B amino group.

Molecules 2017, 22, 841
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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).
The 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
(m.p.) 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, Berlin, Germany)
and was visualized using UV lamp (at 254 nm wave length/short wave length/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 and a Bruker, Avance DPX-300 spectrometer, a Bruker
Avance-400 (400 MHz) Ultrashield spectrometer, and on 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 Shimadzu
8400F FT-IR spectrophotometer (Shimadzu, Kyoto, Japan). The samples were prepared as potassium
bromide (KBr) (Sigma, St. Louis, 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 2 µL/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 (USA), and an ion trap analyzer.
Molecular weight was recorded as (AMU) + 1, as the positive mode of ESI adds 1 AMU to molecular
ion peak. Some compounds were recorded as AMU + 2, as the positive mode of ESI adds 1 AMU to
molecular ion peak and so does the iontrap analyzer.
Synthesis of Novel Title Compounds 4b and 4c
Synthesis of Compound 1
Compound
procedure with slight modification. Compounds
reported, and their spectral data were reported therein [29]. Compounds
a similar procedure by our lab [31]. Compounds ) were prepared for the first time and reported
) are also new and sent for publication
1
was previously reported by our group [27]. It was synthesized following the same
and ) were also prepared and previously
were prepared following
2,
3
(
a,
d
e
4, 5
4, 5 (b, c
in this work for their activity, whereas compounds
for other activities [32].
4, 5 (a, d, e
Synthesis of New Compounds 4, 5 (b, c) (Scheme 1)
8-Amino-1-cyclopropyl-6-fluoro-7-[(4-methylphenyl)amino]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4b
)
(Scheme 1). A mixture of 1-cyclopropyl-6-fluoro-7-[(4-methylphenyl)amino]-8-nitro-4-oxo-1,
4-dihydroquinoli◦ne-3-carboxylic acid (3b, 0.38 mmol, 0.15 g) in 4 mL of 12 N HCl, left stirring
in ice bath (2–5 C) for 15 min. After that, the ice bath was removed, and (0.38 g, 1.7 mmol)
stannous chloride (SnCl2) was added portion wise and the reaction mixture left stirring overnight

Molecules 2017, 22, 841
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and monitored by TLC until completion. Then, the reaction mixture was poured on crushed ice to
precipitate product that is collected by ◦filtration and left to dry. Color of solid compound: yellow;
yield ≈ 70.7% (0.098 g); M.p.: 205–210 C (decomposition); Rf value in system 1 = 0.33. 1H-NMR
0
0
0
(300 MHz, DMSO-d6): 1.20 (m, 4H, H2-2 , H2-3 ), 2.20 (s, 3H, Ar-CH3), 4.52 (m, 1H, H-1 ), 5.69 (br s, 2H,
NH2), 6.59 (d, J = 7.9 Hz, 2H, H-200, H-600), 6.98 (d, J = 7.8 Hz, 2H, H-300, H-500), 7.35 (d, 3JH-F = 9.9 Hz,
1H, H-5), 7.70 (br s, 1H, NH, exchangeable), 8.75 (s, 1H, H-2), 14.67 (br s, 1H, COOH). 13C-NMR
(75 MHz, DMSO-d6): 10.61 (C-20, C-30), 20.61 (CH3), 39.91 (C-10), 98.39 (d, 2JC-F = 23 Hz, C-5), 106.60
(C-3), 114.86 (C-200, C-600), 115.09 (C-400), 121.25 (C-4a), 125.08 (C-8a), 127.86 (d, 2JC-F = 19.5 Hz, C-7),
129.74 (C-300, C-500), 139.04 (C-8), 150.05 (d, JC-F = 254 Hz, C-6), 151.08 (C-2), 158.53 (C-100), 166.30
1
(COOH), 172.21 (C-4). IR (KBr):
ν 3371, 3093, 2924, 2430, 1712, 1612, 1512, 1450, 1327, 2288, 1180, 1087,
1026 cm−1. HRMS (ESI, +ve): calc. for C20H18FN3NaO3 [M + Na]+ (390.12299). Found 390.12244. Anal.
Calcd. for C20H16FN3O3 (367.37): C, 65.39; H, 4.49; N, 11.44 Found C, 65.73; H, 4.82; N, 11.05.
8-Amino-1-cyclopropyl-6-fluoro-7-(4-methoxy-phenylamino)-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (4c
)
◦
(Scheme 1). A mixture of (3c, 0.4 g, 1 mmol) in 4 mL of 12 N HCl was left stirring in ice bath (0–5 C) for
15 min. After that, the ice bath was removed, and (0.75 g, 4 mmol) stannous chloride (SnCl2) was added
portion wise. The reaction mixture left stirring overnight and was monitored by TLC until completion.
Then, the reaction mixture was poured on crushed ice to precipitate a red product that is collected
◦
by filtration and left to dry. Yield = 0.34 g (
≈
61%). M.p.: 190–192 C; Rf value in system 1 = 0.37
.
1H-NMR (300 MHz, DMSO-d6): 1.04, 1.23 (2m, 4H, H2-20, H2-30), 3.73 (s, 3H, OCH3), 3.8 (m, 1H,
NCH-10), 4.15 (m, 2H, NH2), 7.07 (dd, Jo = 7.2 Hz, Jm = 1.8 Hz, 2H, H-200, H-600), 7.41 (dd, Jo = 9 Hz
,
,
3
J
m = 2.7 Hz, 2H, H-300, H-500), 7.65 (m, 1H, NH), 8.54 (d, JH-F = 11.4 Hz, H-5), 8.91 (s, 1H, H-2)
14.46 (br s, 1H, COOH)
.
13C-NMR (75 MHz, DMSO-d6): 7.94 (2C, C-20, C-30), 39.74 (C-10), 55.82
2
3
(OCH3), 98.53 (C-3), 109.85 (d, JC-F = 22.5 Hz, C-5), 115.19 (d, JC-F = 16.78 Hz, C-5), 115.9 (C-4a),
124.83 (2C, C-300, C-500), 125.2 (2C, C-200, C-600), 131.9 (C-8a), 132.53 (C-8), 140.82 (C-7), 147.2 (C-100),
148.03 (C-2), 149.4 (d, 1JC-F = 240 Hz, C-6), 157.28 (C-400), 166.4 (COOH), 177.5 (C-4). IR (KBr):
ν 3356,
2924, 2854, 2484, 2291, 1635, 1512, 1458, 1381, 1249, 1180, 1033 cm−1. HRMS (ESI, +ve): calc. for
C20H18FN3NaO4 [M + H]+ (406.11790). Found 406.11736. LRMS (ES, +ve) m/z calc. for C20H18FN3O4
(383.13): Found 383.3 (100%), 378.4 (36%), 376.6 (5%), 370.4 (2%), 367.3 (39%), 364.6 (20%), 354.0 (95%),
346.4 (8%), 339.3 (11%), 336.1 (80%), 321.1 (5%), 310.6 (61%), 295.5 (12%), 258.1 (2%), 149.1 (1%), 130.3
(3%), 78.9 (1%), 73.6 (12%), 59.0 (37%), 57.2 9 (2%). Anal. Calcd. for C20H18FN3O4 (383.37): C, 62.66;
H, 4.73; N, 10.96 Found C, 62.94; H, 4.98; N, 11.14.
9-Cyclopropyl-4-fluoro-6-oxo-3-p-tolyl-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinolone-7-carboxylic acid (5b
)
(Scheme 1). Compound 5b was synthesized through cyclization of preceding reduced acid 4b
.
The amino◦ 4b
(4b, 0.6 g, 1.6 mmol) was treated with 20 mL aqueous HCl, then left stirring in ice
bath (0–5 C) for 15 min. NaNO2 (0.14 g, 1.6 mmol) dissolved in 10 mL H2O is added drop wise.
The reaction mixture was left stirring overnight. Progress of cyclization reaction was monitored by
TLC and was completed within 24 h. Thereafter, the reaction mixture was cooled, poured onto crushed
ice (250 g), and the resulting off-white precipitate was collected, washed with cold water (2 × 20 mL),
◦
and left to dry. Yield = 0.45 g (
≈
67%). M.p.: 280–282 C; Rf value in system 1 = 0.39. 1H-NMR
(300 MHz, CDCl3): 1.25 (m, 2H, CH2-20), 1.35 (m, 2H, CH2-30), 2.44 (s, 3H, CH3), 4.62 (m, 1H, H-10),
7.35 (d, J = 8 Hz,
2H, H-300, H-500), 7.68 (d, J = 8 Hz, 2, H-200, H-600), 8.24 (d, 3JH-F = 18.25 Hz, 1H, H-5),
8.8 (s, 1H, H-8), 14.9 (br s, 1H, COOH). 13C-NMR (75 MHz, CDCl3): 9.96 (2C, C-20, C-30), 22.15 (CH3),
41.84 (C-10), 108.56 (C-5), 114.85 (d, 2JC-F = 22.13 Hz, C-5), 115.1 (2C, C-300, C-500), 122.4 (C-5a), 125.6
(C-100), 127.75 (C-200, C-600), 129.5 (C-3a), 130.6 (C-9a), 135.2 (C-9b), 140.3 (C-400), 151.4 (d, 1JC-F = 248
Hz, C-4), 147.6 (C-8), 166.74 (COOH), 174.2 (C-6). IR (KBr):
ν 3368, 3044, 2820, 2310, 1609, 1516, 1432,
1176, 1044, 960 cm−1. LRMS (ES, +ve) m/z calc. for C20H15FN4O4 (378.11): Found 379.4 (100%, M + 1),
362.4(15%), 334.4 (8%), 323.3 (10%), 310.1 (4%), 146.2 (4%), 118.4 (15%), 80.1 (2%), 54.2 (4%). Anal.
Calcd. for C20H15FN4O3 (378.36): C, 63.49; H, 4.00; N, 14.81 Found C, 63.81; H, 3.74; N, 15.28.

Molecules 2017, 22, 841
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9-Cyclopropyl-4-fluoro-3-(4-methoxy-phenyl)-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-carboxylic
acid (5c) (Scheme 1). Compound 5c was synthesized through cyclization of preceding reduced acid 4c
.
The amino acid (4c, 0.8 g, 2.1 mmol) was treated with 20 mL aqueous HCl, and then left stirring
◦
in ice bath (0–5 C) for 15 min. NaNO2 (0.14 g, 2.1 mmol) dissolved in 10 mL H2O was added
drop wise to the mixture. The reaction mixture was left stirring overnight. Progress of cyclization
reaction was monitored by TLC and was completed within 20 h. Thereafter, the reaction mixture
was cooled, poured onto crushed ice (250 g), and the resulting off-white precipitate was collected,
washed with cold water (2 × 20 mL) and left to dry. Yield = 0.6 g (≈75%). M.p.: 280–282 ◦C; Rf value
1
0
0
00
in system 1 = 0.36. H-NMR (300 MHz, CDCl3): 1.45 (m, 2H, CH2-2 ), 1.56 (m, 2H, CH2-3 ), 3.78 (s, 3H,
0
00
00
00
OCH3), 4.59 (m, 1H, H-1 ), 7.24 (d, J = 8.2 Hz, 2H, H-3 , H-5 ), 7.8 (d, J = 8.2 Hz, 2H, H-2 , H-6 ), 8.17
(d, 3JH-F = 16.36 Hz, 1H, H-5), 8.92 (s, 1H, H-8), 15.2 (br s, 1H, COOH). 13C-NMR (75 MHz, CDCl3):
10.24 (2C, C-20, C-30), 41.84 (C-10), 56.17 (OCH3), 109.56 (C-7), 114.85 (d, 2JC-F = 22.13 Hz, C-5), 115.1
(2C, C-300, C-500), 122.4 (C-5a), 127.75 (C-200, C-600), 129.50(0C-3a), 130.6 (C-9a), 135.2 (C-9b), 140.6 (C-100),
ν 3359,
146.7 (d, 1JC-F = 258.52 Hz, C-4), 148.45 (C-8), 161.00 (C-4 ), 165.4 (COOH), 176.5 (C-6). IR (KBr):
3087, 2914, 2332, 1617, 1535, 1421, 1164, 1087, 1007 cm−1. LRMS (ES, +ve) m/z calc. for C20H15FN4O4
(394.11): Found 395.4 (100%, M + 1), 381.6 (6%), 377.3 (31%), 372.4 (1%), 361.4 (4%), 345.2 (1%), 333.4
(3%), 325.6 (9%), 317.4 (1%), 141.1 (1.2%), 122.4 (17%), 104.3 (2%), 83.3 (1%), 67.2 (1%), 59.2 (2%). Anal.
Calcd. for C20H15FN4O4 (394.36): C, 66.91; H, 3.83; N, 14.21 Found C, 66.61; H, 4.07; N, 14.38.
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). 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 [37]. 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 s◦ubsequently by standard PCR of 16S ribosomal DNA test [38]. H. pylori cultures were
stored at
−
70 C in Trypticase soy broth (Oxoid, Hampshire, UK) containing 10% (v/v) fetal calf serum
(PAA, Pasching, Austria) and 15% glycerol.
3.2.2. Antimicrobial Susceptibility Testing and Minimal Inhibitory Concentration Determination
Bacterial suspensions were prepared to the 2 McFar
spread on a solid growth medium in a Petri dish. Sterile paper discs (6 mm in diameter; Oxoid,
Hampshire, UK) were impregnated with 25 L of the each compound, and were placed on the surface
←
land’s standard and subsequently uniformly
µ
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 compound were determined by the
two-fold agar dilution method as previously described [39]. 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
of visible colonies was determined after seven days of incubation at 37 C under microaerophilic
×
105 CFU) was applied on the surface of each plate, and the growth
◦

Molecules 2017, 22, 841
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conditions. MIC was recorded as the lowest concentration that inhibited visible growth of organisms;
and 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.
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 [40]. 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; 0.1–4.0, indifference;
3.3. Urease Inhibition Assay
Urease inhibition was performed as described elsewhere [41]. Briefly, 10
µ
L of 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 acetohdyroxamic acid
×
(Sigma Aldrich, St. Louis, MO, USA).
4. Conclusions
This study was successfully implemented and introduced new 8-amino fluoroquinolone
derivatives using a new procedure developed within the course of this study. The fluoroquinolone
derivatives were fully identified and characterized using NMR, IR, electrochemical analysis
(EA), and MS. Moreover, all of these new compounds have interesting antimicrobial activity
against metronidazole-resistant H. pylori. Losing synergistic activity between new derivatives
and metronidazole might be associated with increased lipophilicity of the new complex due to
inter-molecular hydrogen bond of the 8-amino group in these compounds and metronidazole. With the
emergence of H. pylori resistance to a number of antimicrobial agents (especially metronidazole), the
bacteria is becoming a major health problem and there is a need to develop novel agents to treat
and eradicate H. pylori infections. The results of this study suggest that all of the newer 8-amino
7-substituted fluoroquinolone and their correspondent cyclized triazolo derivatives (with their good
to excellent activities) may have potential to be used as anti-H. pylori, and this should be explored.
Acknowledgments: The authors wish to thank Deanship of Academic Research, Deanship of Post Graduate
Studies, and the Faculty of Pharmacy, University of Jordan, Jordan for providing necessary facilities and funds for
conducting this research. The authors also wish to thank the Faculty of Pharmacy, University of Petra, Jordan, and
the Jordan Company for Antibody Production (MONOJO) for their technical support and providing the H. pylori
control strain
Author Contributions: Mohammad Abu-Sini, Rula Darwish, and Luay Abu-Qatouseh conceived and
designed the microbiological and biological activities. Yusuf Al-Hiari, Shereen Arabiyat, and Talal Aburjai
designed the chemical synthesis and their corresponding experiments. Mohammad Abu-Sini, Amal Mayyas,

Molecules 2017, 22, 841
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and Nehaya Al-Karablieh 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. Mohammad
Abu-Sini and 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, or in the decision
to publish the results.
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Sample Availability: Samples of the compounds 4c, 5 (a–d) are 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/).