The antimicrobial potential and pharmacokinetic profiles of novel quinoline-based scaffolds: synthesis andin silicomechanistic studies as dual DNA gyrase and DHFR inhibitors

Article abstract of DOI:10.1039/d1nj02838c

The resistance of pathogenic microbes to currently available antimicrobial agents has been considered a global alarming concern. Hence, close attention should be paid to the development of novel potent antimicrobials. Herein, we report the synthesis,in vitroantimicrobial evaluation, of two novel sets of quinoline derivatives as potential DNA gyrase and DHFR inhibitors. The design of new compounds depended on modifying the structural aspects of previously reported fluoroquinolones. In both sets, the methyl group replaced the fluorine atom at C-6. In the first set, the diverse heterocyclic fragments of reported antimicrobial potentials, including pyrazole, isoxazole, and pyrimidine, were attached to C-3 of the quinoline scaffold. In the second set, the quinolone ring was replaced with the pyrazolo[3,4-b]quinoline scaffold to examine the effect of this action on the antimicrobial activity and thein silicovirtual binding with DNA gyrase and DHFR. The preliminary antimicrobial activity of new compounds was assessed against a panel of pathogenic microbes including Gram-positive bacteria (Streptococcus pneumoniaandBacillus subtilis), Gram-negative bacteria (Pseudomonas aeruginosaandEscherichia coli), and fungal strains (Aspergillus fumigatus,Syncephalastrum racemosum, andGeotriucum candidum). Six derivatives displayed relatively potent antimicrobial activity with a percent activity range of 80-113% relative to ampicillin, gentamicin, and amphotericin B as reference antimicrobial agents. Molecular docking studies were conducted to predict the binding affinities of new compounds toward the active sites of DNA gyrase and DHFR as proposed therapeutic targets.

Full text of DOI:10.1039/d1nj02838c

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The antimicrobial potential and pharmacokinetic
profiles of novel quinoline-based scaffolds:
synthesis and in silico mechanistic studies as
dual DNA gyrase and DHFR inhibitors†
Cite this: New J. Chem., 2021,
45, 13986
a
a
a
Mohamed H. El-Shershaby,
Khaled El-Adl,
Ahmed A. Al-Karmalamy
Kamal M. El-Gamal, Ashraf H. Bayoumi,
bc
a
ad
Mohamed Alswah, Hany E. A. Ahmed,
e
ae
and Hamada S. Abulkhair
*
The resistance of pathogenic microbes to currently available antimicrobial agents has been considered a
global alarming concern. Hence, close attention should be paid to the development of novel potent
antimicrobials. Herein, we report the synthesis, in vitro antimicrobial evaluation, of two novel sets of
quinoline derivatives as potential DNA gyrase and DHFR inhibitors. The design of new compounds
depended on modifying the structural aspects of previously reported fluoroquinolones. In both sets, the
methyl group replaced the fluorine atom at C-6. In the first set, the diverse heterocyclic fragments of
reported antimicrobial potentials, including pyrazole, isoxazole, and pyrimidine, were attached to C-3 of
the quinoline scaffold. In the second set, the quinolone ring was replaced with the pyrazolo
[3,4-b]quinoline scaffold to examine the effect of this action on the antimicrobial activity and the in silico
virtual binding with DNA gyrase and DHFR. The preliminary antimicrobial activity of new compounds was
assessed against a panel of pathogenic microbes including Gram-positive bacteria (Streptococcus
pneumonia and Bacillus subtilis), Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli),
and fungal strains (Aspergillus fumigatus, Syncephalastrum racemosum, and Geotriucum candidum).
Six derivatives displayed relatively potent antimicrobial activity with a percent activity range of 80–113%
relative to ampicillin, gentamicin, and amphotericin B as reference antimicrobial agents. Molecular docking
studies were conducted to predict the binding affinities of new compounds toward the active sites of DNA
gyrase and DHFR as proposed therapeutic targets.
Received 9th June 2021,
Accepted 5th July 2021
DOI: 10.1039/d1nj02838c
rsc.li/njc
and the dwindling number of present effective antimicrobial
drugs as one of the alarming threats to human health. Also,
1
. Introduction
3
A wide-ranging panel of bacterial and fungal infections is problems of vancomycin-resistant and methicillin-resistant
becoming resistant to the effect of most frequently prescribed Staphylococcus aureus (VRSA MRSA), and fluconazole-
&
1
,2
antibiotics and antifungal medications. This resistance is the resistant Candida albicans have reached a disturbing level
main obstacle to the management of infectious diseases. worldwide. Consequently, the battle is still on, and it is
4
The World Health Organization has recognized this resistance essential to develop new medications with improved
antimicrobial potentials.
a
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy,
Al-Azhar University, Nasr City 11884, Cairo, Egypt.
DNA gyrase enzyme (EC number: 5.6.2.2) is a topoisomerase
II, that is crucial to DNA transcription and replication processes
E-mail: hamadaorganic@azhar.edu.eg
5
in eukaryotes. Consequently, inhibition of DNA gyrase has
b
c
Department of Medicinal Chemistry & Drug Design, Faculty of Pharmacy,
Al-Azhar University, Cairo, Egypt
long been considered as a striking goal for the development of
6
,7
antimicrobial agents against bacterial pathogens. Quinolone
antibiotics are the firstborn, and still the only, existing class of
agents that have been clinically used to inhibit bacterial DNA
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Heliopolis University for Sustainable Development, Cairo, Egypt
Pharmacognosy and Pharmaceutical Chemistry Department, Pharmacy College,
Taibah University, Al-Madinah Al-Munawarah 41477, Saudi Arabia
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Horus University -
Egypt, International Coastal Road, 34518, New Damietta, Egypt
Electronic supplementary information (ESI) available. See DOI: 10.1039/
d
e
8,9
synthesis. Nalidixic acid, (1) is a quinolone that formed the
basis for the development of improved analogues fluoroquinolone
antibiotics. Fluoroquinolones and their analogous naphthyridines
(Fig. 1) work as DNA gyrase poisons as they could inhibit bacterial
†
d1nj02838c
1
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Fig. 1 Fluoroquinolones, analogous naphthyridine antibiotics and DNA gyrase/DHFR inhibitors.
8
nucleic acid synthesis, thereby leading to cell death. Nowadays,
Quinoline is a vital pharmacophore ring system, presented
1
7,19
the use of fluoroquinolones has a place in the management of in a number of antifungal agents.
serious microbial infections such as bacterial pneumonia
and urinary tract infections triggered by susceptible pathogens, particularly as antimalarial,
including E. Coli, Enterobacter, and Klebsiella species. Over time, and antifungal.
the subsequent generations of novel fluoroquinolones with Other pharmacophoric heterocyclic ring systems are
Additionally, quinoline
10
derivatives possess
a
diverse pharmacological activity,
20,21
22,23
6,7,17
anticancer,
antibacterial,
11
17,18,24
improved efficacy were developed namely: norfloxacin (2), cipro- embedded in the core structures of reported antimicrobials
floxacin (3), levofloxacin (4), moxifloxacin (5), gemifloxacin (6), with DNA gyrase and/or DHFR degrading effect viz.
2
5–27
28–30
31,32
and delafloxacin (7). In addition, GS-K299423 (8) is mechanistically pyrazole,
isoxazole,
and pyrimidine.
All the latter
distinct from fluoroquinolones, which has recently been reported heterocyclic moieties were reported to have the optimum spatial
as a bacterial topoisomerase type II inhibitor and showed a potent configurations that enable them to interact with the DNA gyrase
3
3–35
inhibitory effect on DNA gyrase supercoiling in Streptococcus binding site.
Moreover, the a, the b-unsaturated ketonic
12
aureus. Furthermore, the design of quinolines over the last few fragment is also presented in a number of synthetic derivatives
years as antimicrobial and anticancer agents is continuing and with potent antimicrobial and DNA gyrase inhibitory
13,14
27,36,37
provides new derivatives with interesting activities.
activity.
Hybrid molecules constructed by joining more than
On the other hand, pathogenic fungi are one of the most one pharmacophore may exert better activity than the individual
3
8,39
harmful parasitic organisms that can cause serious health activity of each isolated one.
problems. Fungal infections also produce various toxins that
1
.1. Rationale and aim of the work
cause critical health problems, including disability and
1
5
death. Like bacteria, fungi can develop resistance when fungi Based on the aforementioned facts, inspired by the versatility of
are able to defeat drugs designed to exterminate them. Over the the quinoline moiety as an essential fragment in many
last few decades, the misuse of antifungal medications has FDA-approved antimicrobial agents, and in continuation of our
3
0,40,41
triggered a resistance to eradicate fungi, creating the efficacy of recent studies
of identifying new antimicrobial agents,
current traditional fungicides decline. Consequently, it is also syntheses of novel sets of 2-chloro-6-methylquinolin and
essential to develop novel effective fungicides to control those 6-methyl-1H-pyrazolo[3,4-b]quinoline derivatives were carried
fungal diseases.
out (Fig. 2) to get new molecules with good antimicrobial
The dihydrofolate reductase (DHFR; EC number: 1.5.1.3) is potency. The design of new compounds depended on modifying
an essential enzyme for the conversion of folic acid to its structural aspects of the previously reported fluoroquinolones to
reduced form, tetrahydro-folic acid (THF). The inhibition of evaluate their activities against pathogenic bacterial and fungal
DHFR interrupts the biosynthesis of THF, which is crucial for the strains. In the first set of compounds, methyl group replaced the
growth of both bacteria and fungi. Therefore, DHFR inhibition fluorine atom, and diverse heterocyclic fragments of reported
1
7,25
28,42
has long been a remarkable goal for the development of anti- antimicrobial potentials, including pyrazole,
microbial agents against both pathogen types. Over the last and pyrimidine,
isoxazole,
1
6
31,32,43
were attached to C-3 of the quinoline
decade, there were several reports on novel quinoline derivatives scaffold to investigate the effect of such substitution pattern on
as potential antimicrobial agents that target DNA gyrase and the antimicrobial activity of the designed compounds. In the
7,13,17,18
DHFR.
However, continuing bacterial and fungal resistance second set, the quinolone ring was replaced with a pyrazolo[3,4-b]
to present antimicrobials with no novel medications in the quinoline scaffold to which a number of other pharmacophoric
antimicrobial pipeline has driven intensive research in this area. tails were linked to C-3. All the synthesized compounds were
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Fig. 2 Rational design of the new pharmacophore-linked quinolines.
evaluated for their in vitro antimicrobial activity against a panel of to evaluate the potential of new compounds to build up as good
pathogenic microbes including Gram-positive bacteria (Strepto- drug candidates.
coccus pneumonia and Bacillus subtilis), Gram-negative bacteria
(Pseudomonas aeruginosa and Escherichia coli), and fungal
strains (Aspergillus fumigatus, Syncephalastrum racemosum, and
Geotriucum candidum). In addition, the structure–activity
relationship of the new compounds is discussed. As well, a
2
. Results and discussion
2.1. Chemistry
subsequent molecular docking study of the most active Synthetic approaches adopted for the synthesis of the starting
compounds was carried out to predict the binding affinity toward 2-chloro-6-methylquinoline-3-carbaldehyde (9) and 6-methyl-
the active site of DNA gyrase/DHFR enzymes as proposed ther- 1H-pyrazolo[3,4-b]quinolin-3-amine (11) are presented in
4
4
apeutic targets of their antimicrobial activity. Furthermore, Scheme 1. In the present work, Vilsmeier-Haack method
ADMET profiles of the highest effective derivatives were examined was selected to prepare the first intermediate 9. Quinolin-3-
Scheme 1 Synthetic protocol of starting quinoline and pyrazoloquinoline derivatives.
1
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carbaldehyde derivative 9 was employed as a starting material absorption value because of extended alkene conjugation
for the synthesis of the chalcone 12 and subsequent binucleo- with the carbonyl double bond. The absolute geometry of the
philic addition reaction products 13–17. Compound 10 was a,b-unsaturated carbonyl linker was assigned to be in the trans
obtained in good yield (80%), adopting Bell and Ackerman- form based on the coupling constant of alkene protons
4
5
modified protocol, where compound 9 was treated with ( J value = 15.0 Hz). In the present work, five different binucleo-
hydroxylamine hydrochloride at 70 1C for 2 h. Then, the philic addition reactions have been achieved. Namely, chalcone
in situ formed oxime was directly heated up to 110 1C to lose 12 was allowed to react with hydrazine hydrate, hydroxylamine
a water molecule and give the desired quinoline-3-carbonitrile hydrochloride, thiourea, guanidine hydrochloride and urea.
product. Treating the carbonitrile derivative 10 with hydrazine In general, all reactions proceeded smoothly, and final pro-
2
3,46,47
hydrate
amine (11), which was used as a starting material in the experimental part. First, to build a dihydropyrazole ring system,
synthesis of final pyrazoloquinoline derivatives (18–22). a mixture of chalcone 12 was heated up with hydrazine hydrate
gave 6-methyl-1H-pyrazolo[3,4-b]quinolin-3- ducts were obtained in relatively good yields as detailed in the
As depicted in Scheme 2, our convergent synthesis approach at reflux temperature to give the desired dihydropyrazole 13.
of final compounds 12–17 started with the preparation of 3-(2- The structure of 13 was established on the basis of its elemental
chloro-6-methylquinolin-3-yl)-1-(4-methoxyphenyl)prop-2-en-1-one and spectral data. The important band in the IR spectrum of
À1
by means of Claisen condensation of the aldehyde derivative 9 compound 13 was revealed at 3290 cm due to NH stretching
with p-methoxyacetophenone in the presence of sodium of the newly formed dihydropyrazole ring. The later NH also
48–50
1
hydroxide.
2
The produced chalcone was treated with a set of appeared on the H NMR spectrum as a broad D O-
five different binucleophiles in a series of binucleophilic addition exchangeable singlet within the aromatic region at 7.6 ppm.
reactions to obtain the final new compounds 13–17.
The structure of chalcone 12 was established based on its carbonyl characteristic absorption band at 1655 cm
elemental and spectral data. The IR spectrum is characterized confirmed the proposed structure of 13. Furthermore, the
As well, the disappearance of the conjugated a, b-unsaturated
À1
À1
1
by the presence of a strong absorption band at 1655 cm due appearance of three new signals in the H NMR spectrum at
to carbonyl ketone stretching, which appeared at a low d 15.3, 3.6, and 2.9 corresponding to pyrazole-H5, pyrazole-H4
Scheme 2 Synthetic route of new quinoline derivatives 12–17.
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Chart 1 The proposed mechanism for construction of compound 13.
axial and equatorial protons, respectively, further confirmed of three protons at 2.36 ppm due to CH
3
of quinoline. Similarly,
the structure. Collectively, the formation of the binucleophilic chalcones were reported to react with guanidine hydrochloride,
3
0
addition reaction products 13–17 was postulated to pass giving the corresponding 2-aminopyrimidines. According to
through two steps. The formation of 13, as a representative this procedure, 4-(2-chloro-6-methylquinolin-3-yl)-6-(4-methoxyphenyl)
example, includes in the first step a Michael-type addition on pyrimidin-2-amine (16) was synthesized. The structure of the
the carbonyl b-carbon, followed by protonation to afford isolated product was confirmed by spectral data, the IR spectrum
À1
b-hydrazinylpropanone intermediate. In this cyclization reaction, characterized by a band at 3300 cm due to NH stretching. The
2
2
the hydrazine nucleophile is firstly attacking the carbonyl b-carbon latter amino group at C-2 of the pyrimidine ring presented a D O-
1
of chalcone 12. A subsequent protonation of the anionic a-carbon exchangeable singlet signal at d 6.6 in the H NMR spectrum.
takes place to restore the trivalent state of nitrogen of the formed Unlike the previous four reactions in this series, a reaction with
hydrazinium transition state. The terminal amino group is then urea preceded first under basic conditions, but the yield was poor,
attacking the carbonyl carbon of the chalcone while carbonyl therefore we shifted towards the acidic medium. In brief, 6-(2-
oxygen gets hydroxylated. The unsaturation is finally located chloro-6-methylquinolin-3-yl)-4-(4-methoxyphenyl)pyrimidin-2(1H)-
between the carbonyl carbon and the adjacent a carbon via the one (17) was prepared, in a good yield, by allowing chalcone 12 to
51–53
elimination of a water molecule, as revealed in Chart 1.
react with urea in the presence of conc. hydrochloric acid at a
Next, to synthesize 2-chloro-3-[3-(4-methoxy-phenyl)-4,5-dihydro- reflux temperature. Under strong acidic media, an alternative
isoxazol-5-yl]-6-methyl-quinoline (14), a mixture of chalcone 12, product was also anticipated, where the 2-chloroquinoline was
hydroxylamine hydrochloride and NaOH in ethanol was heated expected to be alternatively hydrolyzed to afford the amide-
5
4,55
to reflux to give the desired compound according to the reported containing structure (Chart 2)).
The later structure was
17
procedure . The spectral data of the isolated product confirmed excluded based on the absence of its molecular ion peak from
structure 14, the H NMR spectrum showed a triplet of one proton the MS and the absence of a characteristic isotopic pattern of
1
at 5.7 ppm due to isoxazole-H5, which appeared downfield as chlorine-containing compounds. Other spectral and elemental
expected because C-5 of isoxazole is attached to the oxygen atom, data confirmed this assumption and confirmed the structure of
two doublets of the doublet, each equivalent for one proton at compound 17.
4.0 ppm and 3.6 ppm, which are attributed to isoxazole-H4 axial
Scheme 3 shows the adopted synthetic routes for the preparation
and equatorial protons, respectively. The most characteristic of new pyrazoloquinoline derivatives. The approaches depended
1
observation in the H NMR spectrum of compound 14 is the on the nucleophilic condensation reaction of 6-methyl-1H-
disappearance of the olefinic protons of chalcone 12.
pyrazolo[3,4-b]quinolin-3-amine with different acid anhydrides
7
7
Afterward, to prepare pyrimidine-2-thione derivative 15, a and acid halides. Chemical structures of isolated compounds
mixture of chalcone 12, thiourea and NaOH in ethanol was 18–22 were assigned based on their spectral and elemental
heated to reflux. The IR spectrum is characterized by an analyses. For instance, the lack of the biforked band character-
À1
absorption band at 3290 cm assignable to one NH stretching; istic to the primary amine of the starting material, compound 11,
1
the H NMR spectrum showed a singlet of one proton at from all IR spectra, confirms the consumption of the aminopyr-
11.98 ppm due to NH, which is D
2
O exchangeable, a singlet azoloquinoline and the reaction preceded to the completion.
of three protons at 3.8 ppm due to OCH
3
of phenyl and a singlet In addition, the appearance of a band corresponding to an
1
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Chart 2 Anticipated products upon the reaction of chalcone 12 with urea under acidic conditions.
Scheme 3 Synthetic route of new pyrazoloquinoline derivatives 18–22.
À1
amide carbonyl group between 1664 and 1683 cm also verifies derivatives were confirmed based on their IR, LC-MS,
1
13
tethering the new entity with the aminopyrazole ring of the
starting compound.
The progress of all chemical reactions was validated by
TLC methodology and final products were purified by column
H NMR, and C NMR spectral data.
2
.2. Evaluation of biological activity
2.2.1. Antibacterial activity. All the newly synthesized com-
chromatography method. Structures and purity of new pounds were evaluated for their in vitro antibacterial activity
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against four bacterial pathogens: Streptococcus pneumonia and Table 2 Antifungal activity of new compounds
Bacillus subtilis as examples of Gram-positive bacteria;
Pseudomonas aeruginosa and Escherichia coli as examples of
Gram-negative bacteria. Results of the antibacterial activity of
new compounds are presented in Table 1. Agar-diffusion
a
Inhibition zone (mm)
Aspergillus
fumigatus
Syncephalastrum
racemosum
Geotriucum
candidum
Cpd no.
5
6
12
20.1 Æ 1.2
15.7 Æ 0.33
18.3 Æ 1.2
19.8 Æ 1.2
23.2 Æ 0.58
16.3 Æ 1.2
16.9 Æ 0.63
14.6 Æ 1.5
24.2 Æ 0.58
22.3 Æ 1.2
17.3 Æ 2.1
23.7 Æ 0.63
18.3 Æ 0.58
15.9 Æ 0.25
19.3 Æ 0.58
20.5 Æ 0.25
21.4 Æ 1.5
14.2 Æ 0.58
15.2 Æ 1.2
13.4 Æ 0.44
22.3 Æ 1.2
20.7 Æ 0.58
16.4 Æ 0.58
19.7 Æ 0.72
20.1 Æ 2.1
16.8 Æ 0.34
20.4 Æ 2.1
21.4 Æ 0.58
24.6 Æ 0.44
15.9 Æ 2.1
16.3 Æ 0.42
15.4 Æ 1.2
27.3 Æ 0.58
25.2 Æ 0.63
19.1 Æ 1.5
28.7 Æ 0.58
method
was used for the preliminary evaluation of
13
antibacterial activity following the directions of the Clinical 14
and Laboratory Standards Institute and results were listed as
the average diameter in mm of inhibition zones (IZs) of
bacterial growth. Ampicillin and gentamycin were used as 18
standard references for Gram-positive and Gram-negative 19
15
16
17
20
21
22
bacteria, respectively.
.2.2. Antifungal activity. The synthesized compounds were
2
tested in vitro for antifungal activity against three fungal pathogenic Amphotricin B
strains: Aspergillus fumigatus, Syncephalastrum racemosum, and
a
Mean zone of inhibition in mm Æ standard deviation for three
56
Geotriucum candidum. The Agar-diffusion method was also used experiments.
for the evaluation of the tentative screening of antifungal activity.
Amphotericin B and DMSO were used as positive and negative
controls, respectively. Results for each test compound were best activity against Streptococcus pneumonia and Bacillus
recorded as the average of inhibition zone diameter in mm of subtilis was observed in the case of the aminopyrimidine
fungal growth. Inhibition zone diameters of the test compounds derivative 16 with activity percentages of 102% and 80%,
and amphotericin B are shown in Table 2.
respectively, compared with ampicillin. This finding could be
2
.2.3. Structure–activity relationship study. From the above- supported by the documented potentials of analogous amino-
5
7
tabulated data, it is clear that the inhibition zone diameters pyrimidines as modulators of bacterial biofilm formation and
obtained for the new compounds revealed the significant as an antibacterial against Staphylococcus aureus, Bacillus
5
8
antimicrobial activity of new compounds against tested microbial subtilis, Escherichia coli, and Pseudomonas aeruginosa. The
pathogens. Most of the studied derivatives presented a better highest potency against Gram-negative strain, Escherichia coli,
activity toward the Gram-positive than that of Gram-negative was obtained by the action of the pyrimidin-2-thione 15, which
strains. As expected, there were no significant variations between presented an equipotent activity with that of gentamicin.
the antimicrobial activity of derivatives incorporating more than 2-Amino-4,6-disubstituted pyrimidines and pyrimidine-thiones
one pharmacophoric ring system (13–17). On the other hand, there were previously reported as effective antibacterials against Gram-
is a noted variation in the potency between the pyrazoloquinolines negative strains, such as Pseudomonas aeruginosa and Escherichia
5
9
derivatives. Inhibition zones in the antifungal activity evaluation coli. Regarding the antibacterial activity of the pyrazoloquino-
revealed the same trend as that of the antibacterial activity. line derivatives 18–22, the best activity against Gram-positive
Analyzing the antibacterial activity of derivatives with more bacterial strains, Streptococcus pneumoniae and Bacillus subtilis
than one pharmacophoric ring system 13–17 revealed that the were observed in the case of the ketonic derivatives 21 and 22
with activity percentages of 112% and 86% respectively (for
compound 21) and 95% and 83%, respectively, (for compound
22) compared with that of the positive control. The highest
Table 1 Antibacterial activity of new compounds against Gram-positive
potency against Gram-negative strain, Escherichia coli, was
and Gram-negative pathogens
gained under the effect of the halogenated ketonic derivative
a
Inhibition zone (mm)
22, which showed a much better activity than that of gentamicin
Streptococcus Bacillus
Pseudomonas Escherichia
(109%). Unfortunately, there was no detected activity against
Pseudomonas aeruginosa with any of the tested compounds. The
Cpd no.
pneumoniae
subtilis
aeruginosa
coli
b
12
13
14
15
16
17
18
19
20
21
22
16.2 Æ 0.15
16.3 Æ 1.2
19.3 Æ 0.58
21.3 Æ 0.44
24.3 Æ 2.1
19.3 Æ 0.58
16.8 Æ 0.58
17.1 Æ 1.5
16.3 Æ 1.5
26.8 Æ 1.63
22.8 Æ 2.1
23.8 Æ 0.2
19.8 Æ 0.42 NA
18.2 Æ 2.1 NA
15.3 Æ 0.53 assessment of halogenated quinolines as antibacterial and
15.4 Æ 0.72
biofilm-exterminating agents was reported in several studies.
Halogenated quinolines demonstrated more potent antibacterial
21.2 Æ 0.72 NA
22.1 Æ 0.63 NA
26.2 Æ 0.58 NA
21.2 Æ 0.72 NA
19.1 Æ 0.63 NA
19.6 Æ 0.63 NA
18.1 Æ 0.58 NA
28.1 Æ 0.58 NA
26.8 Æ 0.58 NAc
32.4 Æ 0.58 NT
NA
21.2 Æ 0.58
18.6 Æ 0.72 activity than nitroxoline against Staphylococcus. aureus and
NA
60
Staphylococcus epidermidis strains.
15.6 Æ 0.63
16.3 Æ 0.72
14.8 Æ 2.1
22.3 Æ 1.5
23.4 Æ 0.63
NT
Regarding the antifungal activity, the tabulated results
revealed moderate to good activity for most of the new derivatives.
Compounds 15, 16, 20, 21, and 22 were the most potent with
either an equipotent or even higher potency than that of the
Ampicillin
Gentamicin NT
NT
17.3 Æ 0.63
21.3 Æ 0.58 standard drug. The highest potent compound against Aspergillus
fumigatus was 20 followed by 16 with efficacy percentages of
a
Mean zone of inhibition in mm Æ standard deviation for three
b
c
experiments. NA = no activity. NT = not tested.
102% and 97%, respectively, compared with amphotericin-B.
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Pyrrolidinedione incorporating derivatives were previously the former and two codes for the latter. Based on the obtained
recognized as potent antifungal strains such as Aspergillus docking score values, 4DUH and 6DTC complexes were selected
61
fumigatus. The same activity pattern was observed against for molecular docking studies to understand the proposed
Syncephalastrum racemosum, where compounds 20 and 16 showed binding interactions of the highest active compounds inside
a more potent antifungal activity than the standard drug with the pockets of Escherichia coli DNA gyrase (EC number: 5.6.2.2)
6
5
efficacy percentages of 113% and 108%, respectively. Also, the as a target for antibacterial activity and Aspergillus flavus
succinimide derivative 20 revealed the best activity against the last dihydrofolate reductase (EC number: 1.5.1.3) as a target for
6
6
tested fungal species, Geotriucum candidum with an equipotent antifungal activity.
The in silico docking studies were
activity with that of the standard antifungal agent. A graphical performed using the MOE software, employing the flexible
summary of the structure–activity relationship of the most active docking protocol implemented in the MOE software. Docking
derivatives compared with standard antimicrobial agents is studies were validated in terms of the root mean square
presented in the ESI.† A graphical summary of the structure– deviation (RMSD). Poses possessing RMSD values within
6
7
activity relationship of the most active derivatives compared with 0–1.20 Å were only considered.
standard antimicrobial agents is presented in Charts 3 and 4.
2
.2.4. Molecular docking study. DNA gyrase and DHFR have
2
.2.4.1. Docking against Escherichia coli DNA gyrase. With two
6
,7,62
been defined as molecular targets for the antibacterial
antifungal activity
Accordingly, they were selected for computational studies to
rationalize the mechanism of action of the five most active
compounds. Based on PDB search for E coli DNA gyrase and
Aspergillus flavus DHFR, fifteen protein codes were obtained for
and
6
5
main interactions, the binding mode of the initial ligand, RLI
with the active pocket of DNA gyrase, exhibited binding energy
of À13.85 kcal mol (Fig. 3). These interactions include (a) two
1
6,63,64
of quinoline derivatives, respectively.
À1
hydrogen-bonding interactions between the carboxylate group
of RLI and both Arg76 and Arg136 residues; (b) another two
hydrogen bonds between NH and the S atom of the thiazole
ring of RLI and GLY101 residue. Free energy of binding,
hydrophobic interactions, and H-bonding interactions of the
highest potent derivatives (15, 16, 20, 21, and 22) and that of
RLI are presented in Table 3.
The behavior of new quinoline derivatives in the DNA gyrase
binding pocket is summarized in Fig. 4 and 5 and is almost like
that of RLI. The binding mode of compound 15 exhibited an
À1
affinity value of À10.98 kcal mol and almost obeyed the same
interaction pattern of RLI with the binding site of DNA gyrase.
The chlorine atom of 15 formed a hydrogen bond with ILE94
residue within 3.51 Å. Three additional hydrophobic interactions
are formed between the quinoline ring scaffold and the PRO79,
LYS103, and ASN46 residues (Fig. 4 and 5). These four desirable
noncovalent interactions of compound 15 might explain the
superior activity of such a derivative as an antimicrobial agent.
The aminopyrimidine derivative 16 revealed an affinity value of
Chart 3 A graphical comparison between the inhibition zones of com-
pounds 15, 16, 21, and 22 and standard drugs against three of tested
bacterial strains.
À1
À10.96 kcal mol and exhibited a different virtual binding
mode with the DNA gyrase enzyme. The quinoline ring of
compound 16 played as a backbone HB acceptor to form two
hydrogen bonds with GLY101 and GLU50 residues. Also, the
latter derivative exerted two hydrophobic interactions with the
amino acid residues PRO79 and LYS103. The pyrrolidine-dione
Chart 4 A graphical comparison between the inhibition zones of com-
pounds 15, 16, 20, and 21 and the standard drug against tested fungal
strains.
Fig. 3 2D and 3D interactions of the internal ligand, with the active site of
DNA gyrase.
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Table 3 Results of in silico docking for the most active compounds
Distinguishably, compound 21 showed three additional arene-H
hydrophobic interactions with amino acid residues ILE78, ILE78,
and PRO 79.
H-Bonding
interactions
Hydrophobic
interactions
DG
RMSD
(Å)
Distance
Residue (Å)
Distance
Residue (Å)
À1
Comp. (kcal mol
)
2.2.4.2. Docking against Aspergillus flavus dihydrofolate reductase.
The molecular docking study of the five most active synthesized
quinolines was performed against the three-dimensional structure
of Dihydrofolate Reductase (DHFR) of Aspergillus flavus to realize
their binding affinity and interactions with the potential enzyme
target of their antifungal activity. New ligands and the internal co-
crystallized one (H9G) were docked in the active site of modeled
DHFR. The binding mode of the redocked ligand, H9G with the
pocket of Aspergillus flavus DHFR enzyme, demonstrated binding
ARG76
3.06
RLI
À12.23
À10.98
0.91
ARG136 3.15
GLY101 3.63
GLY101 2.81
—
1
5
ILE94
3.51
PRO79 3.97
LYS103 3.98
1.00
ASN46
3.71
1
6
0
À10.96
À11.21
1.01
GLY101 3.27
GLU50 3.56
THR165 3.10
LYS103 2.84
VAL120 3.40
PRO79 3.83
LYS103 3.97
LYS103 3.88
2
À1
0
1
.52
.17
energy of À11.16 kcal mol . There are two main interactions
between H9G and the receptor-binding site (Fig. 6): (a) two
hydrogen-bonding interactions between the amino group at
position 9 of the 2,3-dihydrofuro[2,3-f ]quinazoline ring and N-3
of tetrazole ring in H9G and ILE10 and ARG80 residues,
respectively; (b) an arene-H interaction between the tetrazole
ring of H9G and the amino acid residue LEU77. An outline of
2
1
2
À10.33
À10.71
ASP73
2.94
ILE78
ILE78
4.53
3.99
PRO79 3.61
LYS103 4.04
LYS103 3.93
2
1.07
ASP73
2.90
derivative 20 revealed
(
a
much better affinity value, free energy of binding, H-bonding interactions, and hydrophobic
À1
À11.21 kcal mol ), and showed four different interaction interactions of selected compounds and that of the internal
patterns with the binding site of the DNA gyrase receptor. These co-crystallized ligand is shown in Table 4.
interactions involve three hydrogen bonds between both oxygens
The behavior of new quinoline derivatives in the Aspergillus
of the pyrrolidine-dione moiety and the quinoline ring of the flavus DHFR binding pocket is summarized in Fig. 7 and 8 and
target compounds with THR165, LYS103, and VAL120 residues, is almost similar to that of H9G. The binding mode of
À1
respectively. The fourth interaction is in the form of arene-H compound 15 exhibited an affinity value of À10.29 kcal mol
.
hydrophobic interaction between the benzene ring of the The methoxyphenyl moiety of 15 presented a desirable p–p
quinoline scaffold and the LYS103 residue. With À10.33 and stacking with PHE44 residue and occupied the hydrophobic
À1
À10.71 kcal mol
free energies of binding, the obtained pocket formed by ILE10, ILE156, ALA12, VAL11, ASP40, MET41,
docking results for the carbonyl-containing derivatives 21 and and THR66. The NH of the pyrimidine ring played as a
2 involve one hydrogen bonding between the secondary amide hydrogen bond donor with the amino acid residue SER69.
2
functionality in the target compounds and the amino acid In addition, an arene-H interaction within 4.15 Å is formed
residue ASP73. Quinoline rings of both the latter compounds between the quinoline ring and the amino acid residue VAL70.
presented a hydrophobic interaction with LYS103 residue. Similarly, obeying the same interaction pattern and occupying
Fig. 4 2D interactions of compounds 15 (upper left panel), 16 (upper right panel), 20 (lower left panel), and 21 (lower right panel) with the active site of
DNA gyrase enzyme.
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Fig. 5 3D interactions of compounds 15 (upper left panel), 16 (upper right panel), 20 (lower left panel), and 21 (lower right panel) with the active site of
DNA gyrase enzyme.
Fig. 6 2D and 3D interactions of H9G with the active site of Aspergillus flavus DHFR.
the same pocket, the binding mode of compound 16 exhibited Table 4 Results of docking for the most active compounds with the
À1
binding site of DHFR
an affinity value of À11.11 kcal mol . The primary NH
2
group
played as a hydrogen bond donor with SER69 residue. Additional
two arene–H interactions within distances of 4.02 and 4.57 Å are
formed between the pyrimidine and quinoline rings of 16 and
H-Bonding
interactions
Hydrophobic
interactions
DG
RMSD
) (Å)
Distance
Residue (Å)
Distance
Residue (Å)
À1
the amino acid residues LEU32 and VAL70, respectively. With no Comp. (kcal mol
hydrogen bonding interaction, the pyrrolidine-dione derivative
H9G
À11.16
À10.29
À11.11
1.03
1.18
1.09
SER69
SER69
SER69
2.99
2.99
2.80
VAL70
VAL70
LEU32
VAL70
LEU32
VAL70
4.15
À1
2
0 revealed an affinity value of À8.83 kcal mol and exhibited a 15
4.15
4.02
4.57
4.43
3.90
1
2
6
different virtual binding mode with the DHFR active site.
The quinoline and pyrazole rings of compound 20 displayed
two arene–H interactions within distances of 4.43 and 3.90 Å
between the quinoline and pyrazole rings and the amino
0
À8.83
0.52
—
—
2
2
1
2
À8.75
À8.45
0.94
1.02
TYR162 3.08
TYR162 2.98
GLY157 4.45
GLY157 3.16
acid residues LEU32 and VAL70, respectively. With À8.75 and
À1
À8.45 kcal mol
free energies of binding, respectively, the
obtained docking results for the ketonic derivatives 21 and 22
involved one hydrogen bonding between the NH of pyrazole ring
2.2.5. Pharmacokinetic study. In the present work, a
in the target compounds and the amino acid residue TYR162. computational study was conducted on compounds showing
An additional arene-H interaction formed between the pyrazole the best antimicrobial activity to determine their main physico-
ring of each compound and the GLY157 residue.
chemical properties according to the directions of Lipinski’s
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Fig. 7 2D interactions of compounds 15 (upper left panel), 16 (upper right panel), 20 (lower left panel), and 21 (lower right panel) with the active site of
DHFR.
Fig. 8 3D interactions of compounds 15 (upper left panel), 16 (upper right panel), 20 (lower left panel), and 21 (lower right panel) with the active site of
DHFR.
6
8,69
rule.
Lipinski stated that the intestinal absorption of a compounds agree with Lipinski’s rules. Additionally, ADMET
molecule is more likely to be good enough if it fulfills at least profiles of the new quinoline derivatives were tentatively
three rules of the following: (i) molecular weight o 500; (ii) assessed to evaluate their potential to develop new oral drug
number of H bond donors r 5; (iii) number of H bond candidates.
acceptors r 10; (iv) log P o 5. The bioavailability of medications
ADMET profiling study was conducted using the pkCSM
7
0
violating more than one is expected to be not good enough. descriptors algorithm protocol. The absorption of a drug is
While the reference antimicrobial drugs gentamicin and ampho- depending on a number of factors, including intestinal
tericin B violated two or more of Lipinski’s rules, all the highest absorption, membrane permeability, skin permeability, and
active derivatives in this study (12, 15, 16, 20, 21, and 22) P-glycoprotein substrate or inhibitor. Drug distribution is
gratifyingly, satisfied all the Lipinski’s rules except compounds depending on the volume of distribution (VDss), the blood–
12 and 15, which are only violating the logP. All the new ligands brain barrier permeability (logBB), and CNS permeability.
own acceptable numbers of hydrogen bond acceptors (between 3 Metabolism is predicted depending on the CYP models for a
and 5) and only two or lesser hydrogen bond donor groups. substrate or inhibition. Excretion is predicted based on the
These numbers of HB acceptors and donors in the new total clearance and the renal OCT2 substrate. The toxicity of the
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Table 5 ADMET profile of the most active derivatives and reference antimicrobial agents
Parameter
12
15
16
20
21
22
Amp.
Gent.
Amph. B
Molecular properties
Molecular weight
Log P
Rotatable bonds
HB acceptors
337.806
5.10132
4
3
393.899
5.99181
3
4
376.847
4.91142
3
5
280.287
2.07292
1
4
240.266
2.37792
1
3
274.711
2.59682
2
3
349.412
0.3181
4
5
477.603
7À3.3275
924.091
0.7117
3
12
17
HB donors
Surface area
0
1
1
1
2
2
3
8
12
380.536
145.103
166.392
161.373
119.313
103.218
113.521
143.121
194.977
Absorption
Water solubility
Intestinal abs. (human)
Distribution
À5.939
À4.073
À3.926
À3.216
À3.078
À3.169
À2.396
À2.56
0À2.937
96.098
91.663
95.785
93.931
94.527
94.192
43.034
13.46
VDss (human)
CNS permeability
Metabolism
CYP3A4 substrate
CYP3A4 inhibitor
Excretion
0.176
À1.326
0.297
À1.389
0.261
À1.748
0.369
À2.444
0.384
À2.365
0.284
À2.417
À1.23
À0.967
À5.49
À0.37
À3.166
À3.718
Yes
No
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
No
No
No
No
No
Total clearance
Toxicity
AMES toxicity
À0.03
0.157
0.127
0.333
0.354
0.27
0.337
0.722
À1.495
No
Yes
Yes
No
Yes
Yes
No
No
No
Max. tolerated dose
hERG I inhibitor
hERG II inhibitor
Oral rat acute toxicity (LD50
Hepatotoxicity
Minnow toxicity
0.502
No
Yes
2.473
Yes
À1.895
0.392
No
Yes
3.089
Yes
À0.971
0.292
No
Yes
2.81
Yes
À1.721
0.016
No
No
2.4
Yes
1.513
0.298
No
No
2.39
No
1.471
0.375
No
No
2.489
Yes
1.063
0.952
No
No
1.637
Yes
4.232
0.694
No
No
2.016
No
5.959
0.292
No
No
2.518
No
11.261
)
drugs is predicted depending on AMES toxicity, hERG ADMET profile of new quinolines. In this regard, one critical
inhibition, hepatotoxicity, and skin sensitization. All of these drawback of four new quinolines in the present study is the
parameters were calculated for the six highest potent quinoline positive probability of AMES toxicity, which means that the new
derivatives as well as to reference marketed antimicrobial ligands are expected to be mutagenic and hence may act as
agents. After evaluation of ADMET properties (Table 5), we carcinogens. Additionally, as revealed in Table 5, ampicillin and
can propose that the new derivatives have the advantage of all the new ligands except 21 are sharing the disadvantage of
better intestinal absorption in humans over all reference drugs hepatotoxicity. Gratifyingly, all designed compounds are free from
(91.6–96.1) compared with zero in the case of amphotericin B, the cardiotoxic probability (no hERG I inhibitory effect) and
and 13.4–43.0 in case of ampicillin and gentamicin. This showed comparable tolerability (0.29–0.50) with that of ampicillin
advantage may be attributed to the superior lipophilicity of and gentamicin. Finally, the oral acute toxic doses of the new
the new compounds, which would make them able to go along compounds (LD ), are almost higher than those of all marketed
5
0
7
1
biological membranes. Therefore, they may have significantly reference antimicrobial agents.
good bioavailability after oral administration.
Studying the CNS permeability, the chalcone derivative 12
displayed the highest ability to penetrate CNS (CNS permeability =
À1.32), while reference antimicrobials showed lower abilities to
3
. Conclusion
penetrate (CNS permeability r À3.16). As well, it was clear that in This study reports the synthesis and in vitro antimicrobial
contradiction of the three reference drugs, three out of six of the evaluation of novel 6-methylquinoline derivatives attached with
new compounds could inhibit the main cytochrome involved in different pharmacophoric fragments at C-3 as well as novel
drug metabolism, cytochrome P3A. This ability may also be pyrazolo[3,4-b]quinoline derivatives as dual inhibitors of DNA
attributed to the higher lipophilicity of our constructed quino- gyrase and DHFR. The preliminary antimicrobial activity of new
lines. Excretion was assessed in the term of total clearance, a compounds was assessed against a panel of seven pathogenic
parameter related to drug bioavailability, and is substantial in bacterial and fungal microbes. Six of the new derivatives (12,
deciding dosing intervals. The tabulated results demonstrated 15, 16, 20, 21, and 22) displayed relatively potent antimicrobial
that the pyrazoloquinoline derivative 21 and gentamicin revealed activity with a relative potency range of 80–113%. As well,
the highest total clearance values (0.35 and 0.72, respectively), subsequent docking studies of the most active compounds
compared with other ligands, especially chalcone 12, and were conducted to rationalize the binding affinity of new
standard antifungal, which showed the lowest total clearance compounds to the active sites of DNA gyrase and Aspergillus
value (À0.03 and À1.49, respectively). Thus, 12 is expected to be flavus DHFR enzymes. Overall, this study led us to identify six
excreted faster, and consequently needs dosing intervals of novel quinolines with interesting antimicrobial activity and
shorter duration. Toxicity is the last parameter studied in the DNA gyrase/DHFR inhibitory potentials.
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4
. Experimental section
as a yellowish white solid. Yield: 80%; m.p. 129–131 1C.
À1
IR (KBr) cm : 3050 (CH aromatic), 2950 (CH aliphatic), 1655
4.1. General
1
(
CQO). H NMR (DMSO-d ) d ppm: 9.0 (s, 1H, quinoline-H4),
6
Melting points were measured using electrothermal (Stuart
SMP30) apparatus and were uncorrected. Infrared spectra were
recorded on Pye Unicam SP 1000 IR spectrophotometer at the
Pharmaceutical Analytical Unit, Faculty of Pharmacy, Al-Azhar
University. H NMR and C NMR spectra were recorded in
DMSO-d at 300 and 100 MHz, respectively, on a Varian
Mercury VXR-300 NMR spectrometer at NMR Lab, Faculty of
Science, Cairo University. TMS was used as an internal stan-
dard, chemical shifts were related to that of the solvent.
Chemical shift and coupling constant values are listed in
ppm and Hz, respectively. Mass spectra and elemental analyses
were carried out at the Regional Center of Mycology and
Biotechnology, Al-Azhar University, Cairo, Egypt. Reaction
progress was monitored with Merck silica gel IB2-F plates
8
1
.2 (d, 2H, J = 9.0 Hz, phenyl-H2,H6 protons), 8.1 (d, 1H, J =
5.0 Hz, CH alkene b proton), 7.9 (d, 1H, J = 15.0 Hz, CH alkene
a proton), 7.8 (d, 1H, J = 9.0 Hz, quinoline-H8), 7.5 (d, 1H,
quinoline-H7), 7.4 (s, 1H, quinoline- H5), 7.1 (d, 2H, J = 9.0 Hz,
1
13
phenyl-H3, H5 protons), 3.8 (s, 3H, phenyl-OCH
quinoline-CH ). C NMR (DMSO-d
3 6
(
3
) 2.3 (s, 3H,
, 100 MHz) d (ppm): 165.5
CQO), 156.1 (phenyl-C4), 143.4 (quinoline-C2), 145.2 (enone, b
carbon), 143.4 (quinoline-C8a), 136.7 (quinoline-C6), 135.6
quinoline-C4), 135.2 (quinoline-C7), 135.0 (phenyl-C2, C6),
6
13
(
1
1
27.6 (quinoline-C3), 127.4 (phenyl-C1), 127.2 (quinoline-C8),
26.4 (enone, carbon), 123.8 (quinoline-C4a), 119.8
), 20.9
, 1.7%, M + 2), 337
5%, M ), 302 (C20 78%), 271
13NO, 3.8%), 256 (C18 10NO, 23.7%). Anal. calc. for:
a
(
(
(
(
(
quinoline-C5), 104.5 (phenyl-C3, C5), 53.8 (OCH
CH ). MS (m/z): 339 (C20 16ClNO
C
C
3
3
H
2
+
20
H
H
16ClNO
2
,
2
H16NO ,
(0.25 mm thickness) and was visualized under a UV lamp using
19
H
different solvent systems as mobile phases. Reagents and
starting p-toluidine, phosphorusoxy chloride, p-methoxy-
acetophenone, hydrazine,hydroxylamine, thiourea, guanidine,
C H ClNO ) (M.W. = 337): C, 71.11; H, 4.77; N, 4.15%; found:
2
0
16
2
C, 71.19; H, 4.74; N, 4.21%.
urea and acid anhydride, and acid halide derivatives were 4.3. Synthesis of 2-chloro-3-[3-(4-methoxy-phenyl)-4,5-
purchased from Aldrich chemical company and were used as dihydro-2H-pyrazol-5-yl]-6-methyl-quinoline (13)
received. Compounds 9 and 11 were synthesized according to
2
3
directions of the previously reported procedures. For prepara-
tion of the starting 2-chloro-6-methylquinoline-3-carbaldehyde
(
9), DMF and POCl were allowed to react at 0 1C for 2 h, and
3
then p-methyl acetanilide was added to the reaction mixture.
The overall reactant ratio was found to be a critical issue to
obtain the desired product in good yield. Different ratios
have been tried and the optimum one was 1 : 3 : 12 (p-methyla-
cetanilide:DMF:POCl ). The reaction of the carbonitrile 10 with
3
hydrazine hydrate gave 6-methyl-1H-pyrazolo[3,4-b]quinolin-3-
amine (11).
A mixture of chalcone 12 (3.37 g, 10 mmol) and hydrazine
hydrate (1 ml, 20 mmol) was stirred in ethanol (20 ml) and
heated at reflux for 7 hours. After the reaction was completed,
the mixture was concentrated by evaporating the solvent under
reduced pressure, and then poured onto ice water. The
obtained precipitate was filtered off, washed with water, and
recrystallized from ethanol to afford compound 13 as white
4.2. Synthesis of (E)-3-(2-chloro-6-methylquinolin-3-yl)-1-(4-
methoxyphenyl)prop-2-en-1-one (12)
À1
needles. Yield: 70%; m.p. 116–118 1C. IR (KBr) cm : 3290
1
(
(
9
NH), 3050 (CH aromatic), 2950 (CH aliphatic). H NMR
DMSO-d
.0 Hz, phenyl-H2,H6), 7.8 (d, 1H, J = 9.0 Hz, quinoline-H8), 7.6
s, 1H, NH, D O-exchangeable), 7.6 (d, 1H, J = 9.0 Hz, quinoline-
6
) d ppm: 8.4 (s, 1H, quinoline-H4), 7.8 (d, 2H, J =
(
2
H7), 7.5 (s, 1H, quinoline- H5), 6.9 (d, 2H, J = 9.0 Hz, phenyl-H3,
H5), 5.1 (t, 1H, J = 15.3 Hz, pyrazole-H5), 3.7 (s, 3H, OCH of
3
phenyl), 3.6 (dd, 1H, J = 16, 9,2 Hz, pyrazole-H4 axial proton),
To a stirred and ice-cooled aqueous solution of sodium 2.9 (dd, 1H, J = 16.4, 9.2 Hz, pyrazole-H4 equatorial proton)
1
3
hydroxide (10 mmol, 50% w/w) and absolute ethanol (15 ml), 2.5 (s, 3H, CH
-methoxyacetophenone (1.5 g, 10 mmol) was added followed (ppm): 168.4 (phenyl-C4), 158.9 (pyrazole-C3), 155.5 (quinoline-C2),
by 2-chloro-6-methylquinoline-3-carbadehyde (9, 2.05 g, 147.7 (quinoline-C8a), 147.1 (quinoline-C6), 146.2 (quinoline-C4),
3 6
of quinoline). C NMR (DMSO-d , 100 MHz) d
4
7
2
1
3
0 mmol). The reaction mixture was vigorously stirred for 143.5 (quinoline-C-7), 136.7 (quinoline-C3), 135.7 (phenyl-C1),
hours while the temperature was maintained below 25 1C till 135.3 (phenyl-C2, C6), 133.5 (quinoline-C8), 131.2 (quinoline-
the reaction mixture became thick. The reaction mixture was C4a), 127.4 (quinoline-C5), 127.1 (phenyl-C3, C5), 61.0
left in the refrigerator overnight. The formed precipitate was (pyrazole-C5), 60.0 (pyrazole-C4), 42.3 (OCH ), 20.8 (CH ). MS
3
3
filtered off under vacuum and washed with copious amounts of (m/z): 353 (C H ClN O, 9.4%, M + 2), 351 (C H ClN O,
2
0
18
3
20 18
3
+
water until the filtrates became neutral to litmus paper, washed 37.11%, M ), 175 (C10
with three repetitive portions of ice-cold ethanol (20 ml), and (C20 18ClN O) (M.W. = 351): C, 68.28; H, 5.15; N, 11.94%;
then finally recrystallized from ethanol to afford compound 12 found: C, 68.31; H, 5.39; N, 11.43%.
7
H ClN, 100%). Anal. calc. for:
H
3
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.4. Synthesis of 2-chloro-3-[3-(4-methoxy-phenyl)-4,5-
NJC
1
4
2950 (CH aliphatic). HNMR (DMSO-d
6
) d ppm: 11.9 (s, 1H, NH,
dihydro-isoxazol-5-yl]-6-methyl-quinoline (14)
D
2
O-exchangeable proton), 8.5 (s, 1H, quinoline-H4), 8.3 (d, 1H,
J = 15 Hz, quinoline-H8), 8 (d, 2H, J = 9.0 Hz, phenyl-H2,H6), 7.7
(
(
(
(
d, 1H, J = 15 Hz, quinoline-H7), 7.3 (s, 1H, quinoline-H5), 7.2
s, 1H, pyrimidine-H5), 7.1 (d, 2H, J = 9.0 Hz, phenyl-H3,H5), 3.8
1
3
s, 3H, phenyl OCH
DMSO-d , 100 MHz) d (ppm): C NMR (DMSO-d
ppm): 187.7 (C = S), 187.7 (pyrimidine-C6), 163.1 (pyrimidine-C4),
3
) 2.36 (s, 3H, quinoline CH
3
.
C NMR
1
3
6
6
, 100 MHz) d
(
1
1
1
1
1
2
3
60.8 (phenyl-C4), 140.9 (quinoline-C2), 138.3 (quinoline-C8a),
36.9 (quinoline-C6), 133.0 (quinoline-C4), 131.3 (quinoline-C-7),
30.6 (quinoline-C3), 130.5 (phenyl-C2, C6), 127.9 (quinoline-C8),
25.9 (quinoline-C4a, C5), 123.8 (quinoline-C5), 119.0 (phenyl-C1),
15.0 (phenyl-C3, C5), 114.1 (pyrimidine-C5), 55.5 (OCH3),
A mixture of chalcone 12 (3.37 g, 10 mmol) and hydroxylamine
hydrochloride (0.69 g, 10 mmol) was stirred in ethanol (20 ml),
and then sodium hydroxide (0.8 g, 20 mmol) was added. The
reaction mixture was heated to reflux for 7 hours, and then the
solvent was evaporated under reduced pressure and poured
into ice water. The obtained precipitate was filtered off, washed
with a copious amount of water, and recrystallized from etha-
nol to afford the compound 14 as yellowish solid. Yield: 65%;
0.3 (CH
93 (C21
ClN). Anal. calc. for: (C21
C, 64.03; H, 4.09; N, 10.67%; found: C, 64.12; H, 4.16; N, 10.78%.
3
). MS (m/z): 395 (C21
H
16ClN
OS, 0.99%, M ), 200 (C12
16ClN O S) (M.W. = 393):
3
OS, 0.15%, M + 2),
+
H16ClN
3
7
H ClN, 100%), 175
(C
H
10 6
H
3
À1
m.p. 130–132 1C. IR (KBr) cm : 3050 (CH aromatic), 2950 (CH
1
aliphatic), 1590 (C = N). H NMR (DMSO-d ) d ppm: 8.3 (s, 1H,
6
4
.6. Synthesis of 4-(2-chloro-6-methyl-quinolin-3-yl)-6-
quinoline- H4), 7.8 (d, 1H, J = 9.0 Hz, quinoline-H8), 7.6 (d, 2H,
J = 9.0 Hz, phenyl-H2, H6), 7.3 (s, 1H, quinoline-H5), 7.4 (d, 1H,
J = 9.0 Hz, quinoline- H7), 7.2 (d, 2H, J = 9.0 Hz, phenyl-H3,H5),
(4-methoxy-phenyl)-pyrimidin-2-ylamine (16)
5
.7 (t, 1H, J = 14 Hz, isoxazole-H5), 4.0 (dd, 1H, J = 11, 5 Hz,
isoxazole-H4 axial proton), 3.7 (s, 3H, phenyl OCH ), 3.6 (dd,
H, J = 17, 4.8 Hz, isoxazole-H4 equatorial proton) 2.4 (s, 3H,
3
1
1
3
3 6
quinoline CH ). C NMR (DMSO-d , 100 MHz) d (ppm): 163.1
(
(
phenyl-C4), 160.8 (isoxazole-C3), 138.3 (quinoline-C2), 136.4
quinoline-C8a), 133.0 (quinoline-C6), 131.3 (quinoline-C4),
1
1
30.6 (quinoline-C-7), 130.5 (quinoline-C3), 127.9 (phenyl-C1),
25.3 (phenyl-C2, C6), 123.8 (quinoline-C8), 119.0 (quinoline-
A mixture of chalcone 12 (3.37 g, 10 mmol) and guanidine
hydrochloride (0.95 g, 10 mmol) was stirred in absolute ethanol
C4a), 115.0 (quinoline-C5), 114.1 (phenyl-C3, C5), 77.9
isoxazole-C5), 55.5 (OCH ), 42.2 (isoxazole-C4), 20.3 (CH ).
MS (m/z): 354 (C20 17ClN , 0.5%, M + 2), 352 (C20 17ClN
.73%, M ), 317 (C20 35.77%), 185(C12 11NO,
2.17%)). Anal. calc. for: (C H ClN O ) (M.W. = 352): C,
(
3
3
(20 ml), and then sodium hydroxide (0.8 g, 20 mmol) was
H
2
O
2
H
2 2
O ,
added. The reaction mixture was heated at reflux for 7 hours.
After completion of the reaction, as detected by TLC, the solvent
was concentrated under reduced pressure, and then poured
into ice water (50 ml). The obtained solid was filtered off,
washed and recrystallized from ethanol to afford the desired
+
1
1
6
H
17
N
2
O
2
,
H
2
0
17
2 2
8.09; H, 4.86; N, 7.94%; found: C, 68.13; H, 4.97; N, 7.87%.
4.5. Synthesis of 6-(2-Chloro-6-methyl-quinolin-3-yl)-4-
compound as yellow solid. Yield: 60%; m.p. 190–192 1C. IR
(
4-methoxy-phenyl)-1H-pyrimidine-2-thione (15)
À1
(
KBr) cm : 3300 (NH
2
), 3050 (CH aromatic), 2950 (CH aliphatic).
) d ppm: 8.6 (s, 1H, quinoline-H4), 8.1 (d, 2H,
J = 9.0 Hz, phenyl-H2,H6), 7.9 (d, 1H, J = 9.0 Hz, quinoline-H8),
1
H NMR (DMSO-d
6
7
7
6
.7 (s, 1H, pyrimidine-H5), 7.5 (d, 1H, J = 9.0 Hz, quinoline-H7),
.4 (s, 1H, quinoline-H5), 7.0 (d, 2H, J = 9.0 Hz, phenyl-H3, H5),
.6 (s, 2H, NH , D O-exchangeable protons), 3.8 (s, 3H, phenyl
2
2
1
3
OCH ), 2.4 (s, 3H, quinoline CH ). C NMR (DMSO-d , 100 MHz)
3
3
6
d (ppm): 187.7 (CQO), 187.7 (pyrimidine-C6), 163.1 (pyrimidine-
C4), 160.8 (phenyl-C4), 140.9 (quinoline-C2), 138.3 (quinoline-
A mixture of chalcone 12 (3.37 g, 10 mmol) and thiourea (0.76 g, C8a), 136.9 (quinoline-C6), 133.0 (quinoline-C4), 131.3
0 mmol) was stirred in ethanol (20 ml) and then sodium (quinoline-C-7), 130.6 (quinoline-C3), 130.5 (phenyl-C2, C6),
1
hydroxide (0.8 g, 20 mmol) was added to it. The mixture was 127.9 (quinoline-C8), 125.9 (quinoline-C4a, C5), 123.8
heated at reflux for 7 hours. After the reaction was completed (quinoline-C5), 119.0 (phenyl-C1), 115.0 (phenyl-C3, C5), 114.1
the solvent was concentrated by evaporation under reduced (pyrimidine-C5), 55.5 (OCH ), 20.3 (CH ). MS (m/z): 378
3
3
+
pressure and poured into ice water. The obtained precipitate (C H ClN O, 27.34%, M + 2), 376 (C H ClN O, 70.33%, M ),
2
1
17
4
21 17
4
17 4 4
was filtered, washed with water, and recrystallized from ethanol 342 (C21H N O, 100%). Anal. calc. for: (C21H17ClN O) (M.W. =
to give the titled compound as a dark yellow solid. Yield: 40%; 376): C, 66.93; H, 4.55; N, 14.87%; found: C, 66.8; H, 4.47;
À1
m.p. 140–142 1C. IR (KBr) cm : 3290 (NH), 3050 (CH aromatic), N, 14.75%.
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À1
4.7. Synthesis of 6-(2-chloro-6-methyl-quinolin-3-yl)-4-(4-
Faint brown solid. Yield: 70%; m.p. 290–292 1C. IR (KBr) cm
:
methoxy-phenyl)-1H-pyrimidin-2-one (17)
3426 (N–H), 3015 (C–H aromatic), 2892 (C–H aliphatic), 1645
1
(
CQO). H NMR (DMSO-d ) d ppm: 13.8 (s, 1H, NH, D O-
6
2
exchangeable), 8.8 (s, 1H, quinoline-H4), 8–7.9 (m, 5H, Ar
H’s), 7.8 (s, 1H, quinoline-H5), 7.6 (d, 1H, J = 9.0 Hz,
1
3
quinoline-H7), 2.1 (s, 3H, CH
00 MHz) d (ppm): 166.9 (CQO), 151.3 (pyrazole-C3), 136.7
quinoline-C2), 135.6 (quinoline-C8a), 134.2 (quinoline-C4),
33.8 (quinoline-C6), 133.0 (isoindole-C5, C6), 132.0
isoindole-C4a, C7a), 130.0 (quinoline-C7), 129.2 (quinoline-C-8),
28.1 (quinoline-C5), 127.6 (isoindole-C4, C7), 125.2 (quinoline-
C4a), 112.1 (quinoline-C3), 21.4 (CH ). MS (m/z): 328 (C H N O ,
3
).
6
C NMR (DMSO-d ,
1
(
1
(
1
A mixture of chalcone 12 (3.37 g, 10 mmol) and urea (0.6 g,
3
19 12 4 2
10 mmol) was stirred in ethanol (20 ml), and then hydrochloric acid
+
6
5.41%, M ), 182 (C H N , 5%), 167 (C H N , 2.9%), 141
11 8 3 10 5 3
(2 ml) was added. The mixture was heated at reflux for 7 hours.
9 5 2
(C H N ,
9 4
4.78%), 126 (C H N, 1.7%). Anal. calc. for:
After completion of the reaction, the solvent was concentrated
under reduced pressure and poured into ice water (50 ml). The
obtained precipitate was filtered off, washed with distilled water,
and finally recrystallized from ethanol to yield the titled com-
pound as a yellowish white solid. Yield: 60%; m.p. 178–180 1C.
(C
19
H
12
N
4
O
2
) (M.W. = 328): C, 69.51; H, 3.68; N, 17.06; found:
C, 69.60; H, 3.73; N, 17.28%.
.8.2. 1-(6-Methyl-1H-pyrazolo[3,4-b]quinolin-3-yl)-1H-
4
pyrrole-2,5-dione (19)
À1
IR (KBr) cm : 3290 (NH), 3050 (CH aromatic), 2950 (CH aliphatic)
1
1
679 (CQO). H NMR (DMSO-d
6
) d ppm: 11.98 (s, 1H, NH, D
2
O-
exchangeable proton), 8.5 (s, 1H, quinoline-H4), 8.3 (d, 1H, J =
5 Hz, quinoline-H8), 8.0 (d, 2H, J = 9.0 Hz, phenyl-H2,H6), 7.7
d, 1H, J = 15 Hz, quinoline-H7), 7.3 (s, 1H, quinoline-H5), 7.2 (s,
H, pyrimidine-H5), 7.1 (d, 2H, J = 9.0 Hz, phenyl-H3,H5), 3.8 (s,
1
(
1
13
3H, phenyl OCH ) 2.4 (s, 3H, quinoline CH ). C NMR (DMSO-d6,
À1
3
3
Faint brown solid. Yield: 65%; m.p. 230–232 1C. IR (KBr) cm :
1
1
(
(
(
(
(
00 MHz) d (ppm): 164.6 (pyrimidine-C4), 163.4 (pyrimidine-C6),
57.6 (CQO), 163.1 (phenyl-C4), 160.8 (quinoline-C2), 149.6
quinoline-C3), 138.4 (quinoline-C8a), 136.9 (quinoline-C6), 133.0
quinoline-C4), 131.3 (quinoline-C-7), 130.5 (phenyl-C1), 127.9
phenyl-C2, C6), 125.9 (quinoline-C8), 123.8 (quinoline-C4a), 119.0
quinoline-C5), 115.0 (phenyl-C3, C5), 114.1 (pyrimidine-C5), 55.5
OCH ), 20.2 (CH ). MS (m/z): 379 (C H ClN O , 2.72%, M + 2),
3
229 (N–H), 3051 (C–H aromatic), 2994 (C–H aliphatic), 1683
1
6
(CQO). H NMR (DMSO-d ) d ppm: 13.9 (s, 1H, NH), 8.9 (s, 1H,
quinoline-H4), 7.8 (s, 2H, pyrrole-H‘s), 7.6 (d, 1H, J = 9.0 Hz,
quinoline-H8), 7.5 (d, 1H, J = 9.0 Hz, quinoline-H7), 6.9 (s, 1H,
13
quinoline-H5), 2.1 (s, 3H, CH3). C NMR (DMSO-d , 100 MHz) d
6
(ppm): 168.9 (CQO), 151.9 (pyrazole-C3), 147.1 (quinoline-C2),
3
3
21 16
3
2
140.5 (quinoline-C8a), 133.7 (quinoline-C4), 133.0 (quinoline-
C6), 132.7 (pyrrolidinedione-C3, C4), 128.4 (quinoline-C7),
+
377 (C H ClN O , 4%, M ), 346 (C H ClN O, 6.76%), 270
21 16 3 2 20 13 3
(C H ClN O, 12.14%), 216 (C H ClN , 5.68%), 92 (C H , 100%).
14 9 3 12 9 2 7 8
127.4 (quinoline-C8), 123.8 (quinoline-C5), 110.4 (quinoline-
Anal. calc. for: (C21
H
16ClN
3
O
2
) (M.W. = 377): C, 66.76; H, 4.27; N,
+
C4a), 22.9 (CH
, 100%), 182 (C11
41 (C H N , 8.9%). Anal. calc. for: (C H N O ) (M.W. = 278):
3
). MS (m/z): 278 (C15
H
10
N
4
O
2
, 1.27%, M ), 198
11.12%; found: C, 66.84; H, 4.33; N, 11.21.
(C
11
H
10
N
4
H
8
N
3
, 5.24%), 167 (C10
5 3
H N , 8.9%),
1
9
5
2
15 10 4 2
4
.8. General procedures for the synthesis of pyrazolo[3,4-b]
C, 64.74; H, 3.62; N, 20.13; found: C, 64.81; H, 3.62; N, 20.15%.
.8.3. 1-(6-Methyl-1H-pyrazolo[3,4-b]quinolin-3-yl)
quinoline derivatives 18–22
4
A mixture of 6-methyl-1H-pyrazolo[3,4-b]quinolin-3-amine (11,
pyrrolidine-2,5-dione (20)
1.98 g, 10 mmol) and the appropriate acid anhydride or acid halide
derivatives (10 mmol) in acetic acid (50 ml) was heat to reflux for
23
1
–4 hours (Scheme 1). After completing the reaction, as observed
by TLC, the reaction mixture was filtered off while hot, and the
solvent was concentrated, the separated solid was filtered and
recrystallized from ethanol to afford the target final product.
4
.8.1. 2-(6-Methyl-1H-pyrazolo[3,4-b]quinolin-3-yl)
Reddish white solid. Yield: 60%; m.p. 260–262 1C. IR (KBr)
isoindoline-1,3-dione (18)
À1
cm : 3265 (N–H), 3077 (C–H aromatic), 2918 (C–H aliphatic),
1
1
664 (CQO). H NMR (DMSO-d
6
) d ppm: 10.8 (s, 1H, NH), 8.9
(
(
(
(
s, 1H, quinoline-H4), 7.8 (d, 1H, J = 9.0 Hz, quinoline-H8), 7.6
d, 1H, J = 9.0 Hz, quinoline-H7), 7.5 (s, 1H, quinoline-H5), 2.9
1
3
s, 4H, 2CH ), 2.2 (s, 3H, CH ). C NMR (DMSO-d , 100 MHz) d
2
3
6
ppm): 177.1 (CQO), 169.0 (pyrazole-C3), 152.0 (quinoline-C2),
1
1
47.1 (quinoline-C8a), 140.5 (quinoline-C4), 133.7 (quinoline-C6),
32.9 (quinoline- C7), 132.7 (quinoline-C8), 128.4 (quinoline-C-5),
1
4000
|
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5
6
1
27.6 (quinoline-C4a), 123.8 (quinoline-C3), 29.32 (pyrroli- of Clinical and Laboratory Standards Institute.
dinedione-C3, C4), 21.4 (CH ). MS (m/z): 280 (C15 , 5.7%, suspensions of the selected microorganisms were uniformly
M ), 198 (C H N , 100%), 182 (C H N , 2.58%), 167 (C H N , spread using sterile cotton swabs on sterile Petri dishes of
Briefly,
3
12 4 2
H N O
+
11
10
4
11
8
3
10 5 3
4.15%), 141 (C H N , 3.97%). Anal. calc. for: (C H N O ) (M.W. = nutrient agar for bacteria and Sabouraud agar for fungi. For
9 5 2 15 12 4 2
280): C, 64.28; H, 4.32; N, 19.99; found: C, 64.32; H, 4.38; bacterial strains, suspensions of the microorganisms were
N, 20.02%.
prepared by inoculating fresh stock cultures into separate broth
tubes, each containing 7 mL of nutrient agar. The inoculated
tubes were incubated at 37 1C for 24 hours. Compounds to be
tested were prepared in the required concentrations (1 mg in
4.8.4. N-(6-Methyl-1H-pyrazolo[3,4-b]quinolin-3-yl)-
acetamide (21)
1
ml DMSO), ampicillin and gentamicin were prepared in the
same manner. Nutrient agar was dissolved and distributed in
5 ml quantities in 100 ml conical flasks and was sterilized in
2
an autoclave at 121 1C for 20 minutes. The medium was poured
in Petri dishes and allowed to set for 30 minutes at room
temperature. Cultures of bacterial strain were spread with dry
sterile swabs on the surface of the previously prepared plates.
Cups of 6 mm diameters at equal distances were aseptically
punched in each plate, one cup was used for the control
Yellowish-white solid. Yield: 78%; m.p. 230–232 1C. IR (KBr)
À1
cm : 3214 (N–H), 3077 (C–H aromatic), 2990 (C–H aliphatic),
1
1
675 (CQO). H NMR (DMSO-d ) d ppm: 10.8 (s, 1H, NH), 9.04
6
(s, 1H, NHCO), 8.9 (s, 1H, quinoline-H4), 7.8 (d, 1H, J = 9.0 Hz,
(DMSO) and two other cups for the standards (Ampicillin,
quinoline-H8), 7.6 (d, 1H, J = 9.0 Hz, quinoline-H7), 7.4 (s, 1H,
Gentamicin) where the remaining cups were used for the tested
compound. The plates were incubated at 37 1C for 24 h then the
plates were examined for inhibition zones.
For fungal strains, suspensions of the fungi were prepared
by inoculating fresh stock cultures into tubes containing
Sabouraud agar. The inoculated tubes were incubated at
1
3
quinoline-H5), 2.2 (s, 3H, quinoline-CH
NMR (DMSO-d , 100 MHz) d (ppm): 161.8 (CQO), 149.4
pyrazole-C3), 148.9 (quinoline-C2), 141.2 (quinoline-C8a),
3
), 1.8 (s, 3H, CH
3
).
C
6
(
1
1
1
36.4 (quinoline-C4), 133.0 (quinoline-C6), 129.7 (quinoline-C7),
28.4 (quinoline-C8), 127.3 (quinoline-C5), 125.3 (quinoline-C4a),
21.1 (quinoline-C3), 23.4 (CH ), 21.0 (CH ). MS (m/z): 240
3
3
25 1C for 48 h. Sabouraud agar was dissolved and distributed
+
(C H N O, 42.17%, M ), 225 (C H N O, 100%). Anal. calc. for:
13
12
4
12 9 4
in 25 ml quantities in 100 ml conical flasks and was sterilized
in an autoclave at 121 1C for 20 minutes. Afterward, the
medium was poured into Petri dishes and allowed to set for
13 12 4
(C H N O) (M.W. = 240): C, 64.99; H, 5.03; N, 23.32; found:
C, 65.12; H, 5.01; N, 23.41%.
.8.5. 2-Chloro-N-(6-methyl-1H-pyrazolo[3,4-b]quinolin-3-
4
30 minutes at room temperature. Cultures of each organism
yl)-acetamide (22)
were aseptically spread on the surface of the previously
prepared Petri dishes using a dry sterile swab. Cups of 6 mm
diameters at equal distances (20 mm) were made on each plate.
In each plate, one cup was used to introduce 75 mL of the
control and another one for Amphotericin B, while the other
cups were used to introduce equal volumes of compounds to be
tested. The plates were incubated at 25 1C for 48 h then were
examined to measure the inhibition zones.
À1
Yellowish-white solid. Yield: 93%; m.p. 270–272 1C. IR (KBr) cm
:
3
224 (N–H), 3035 (C–H aromatic), 2995 (C–H aliphatic), 1669
4.9.1. Determination of the inhibition zones. 75 mL
1
À1
(CQO). H NMR (DMSO-d ) d ppm: 13.1 (s, 1H, NH), 11.2 (s, 1H, solution of each test compound (1 mg ml in DMSO), was
6
NHCO), 8.9 (s, 1H, quinoline-H4), 7.8 (d, 1H, J = 9.0 Hz, quinoline- placed in a 6 mm-diameter cup in agar plate seeded with the
H8), 7.6 (d, 1H, J = 9.0 Hz, quinoline-H7), 7.61 (s, 1H, quinoline-H5), appropriate test pathogen in triplicate. Ampicillin, gentamicin,
1
3
À1
4
(
1
1
.4 (s, 2H, CH
ppm): 165.5 (CQO), 139.9 (pyrazole-C3), 139.5 (quinoline-C2), standards for Gram-positive, Gram-negative antibacterial, and
37.4 (quinoline-C8a), 134.2 (quinoline-C4), 133.4 (quinoline-C6), antifungal agents, respectively. DMSO as a negative control
33.2 (quinoline-C7), 130.1 (quinoline-C8), 128.5 (quinoline-C5), showed no inhibition zone (IZ). Plates were incubated at
27.0 (quinoline-C4a), 123.8 (quinoline-C3), 43.6 (CH ), 20.8 37 1C for 24 h (for bacteria) or at 25 1C for 48 h (for fungi).
2 3 6
), 2.48 (s, 3H, CH ). C NMR (DMSO-d , 100 MHz) d and amphotericin B (1 mg ml in DMSO each) were used as
1
2
(
CH ). MS (m/z): 276 (C H Cl N O, 2.72%, M + 2), 274
4.9.2. Docking studies. Molecular docking experiments
were conducted by MOE builder within the Molecular Operat-
3
13 11
4
+
(
C
13
H
11ClN
4
O, 7.76%, M ), 225 (C12
, 3.52%). Anal. calc. for: (C13
M.W. = 274): C, 56.84; H, 4.04; N, 20.40; found: C, 56.92; H, chemcomp.com/Products.htm) to evaluate the binding free
H
9
N
4
O, 9.86%), 198 (C11
H
9
N
4
,
1
(
00%), 182 (C10
H
6
N
4
4
H11ClN O) ing Environment (MOE) software suite (MOE2014, https://www.
4
.03; N, 20.53%.
energy and to discover the binding modes toward DNA
gyrase and DHFR enzymes (PDB IDs: 4DUH, Resolution:
4.9. In vitro antimicrobial evaluation
1.50 Å, https://www.rcsb.org/structure/4DUH; 6DTC: Resolution:
5
6
Agar-diffusion method was used for the determination of 2.00 Å, https://www.rcsb.org/structure/6DTC) and considered
antibacterial and antifungal activity following the directions as a target for docking simulation.
6
5,66
Molecular docking
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studies were conducted following our previously reported 12 B. D. Bax, P. F. Chan, D. S. Eggleston, A. Fosberry, D. R.
7
3–76
procedures.
Gentry, F. Gorrec, I. Giordano, M. M. Hann, A. Hennessy,
M. Hibbs, J. Huang, E. Jones, J. Jones, K. K. Brown,
C. J. Lewis, E. W. May, M. R. Saunders, O. Singh,
C. E. Spitzfaden, C. Shen, A. Shillings, A. J. Theobald,
A. Wohlkonig, N. D. Pearson and M. N. Gwynn, Nature,
Author contributions
K. El-Gamal, and H. S. Abulkhair were responsible for the
conception and rational design of the work. M. El-Shershaby,
M. Alswah and A. Bayoumi were responsible for the data
collection and synthesis of new compounds. K. El-Adl,
A. Al-Karmalawi, and H. Ahmed, performed the molecular
docking study. K. El-Gamal, and H. S. Abulkhair were
responsible for analyzing the spectral data. K. El-Adl and
H. Abulkhair performed the pharmacokinetic study. All authors
contributed to the writing and revision of the manuscript.
2010, 466, 935–940, DOI: 10.1038/nature09197.
1
1
1
1
1
1
3 K. Douadi, S. Chafaa, T. Douadi, M. Al-Noaimi and I. Kaabi,
J. Mol. Struct., 2020, 1217, 128305, DOI: 10.1016/
j.molstruc.2020.128305.
4 S. Ghosh, S. Pal, K. S. S. Praveena and J. Mareddy, J. Mol.
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128137.
5 Centers for Disease Control and Prevention, Antifungal
Resistance, https://www.cdc.gov/fungal/antifungal-resistance.
html, (accessed 11 May 2021).
6 C. DeJarnette, A. Luna-Tapia, L. R. Estredge and G. E.
Palmer, mSphere, 2020, 5(3), e00374–20, DOI: 10.1128/
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Conflicts of interest
The authors declare that there is no conflict of interest.
7 M. F. El Shehry, M. M. Ghorab, S. Y. Abbas, E. A. Fayed,
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The authors would like to thank the website of https://www.
dreamstime.com/ for providing free clipart tools that have been
used to design the graphical abstract.
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