A series of thiazol-4-one/thiophene-bearing pyrazole derivatives as pharmacologically attractive cores were initially synthesized using a hybridization approach. All structures were confirmed using spectra analysis techniques (IR, 1H NMR, and 13C NMR). In vitro antimicrobial activities, including the minimum inhibitory concentration (MIC), minimum bactericidal/fungicidal concentration (MBC/MFC), and time-kill assay, were evaluated for the most active derivatives 4a, 5a, 7b, 10, and 13. These derivatives were significantly active against the tested pathogens, with compound 7b as the most active derivative (MIC values range from 0.22 to 0.25 μg/mL). In the MBC and MFC, the active target pyrazole derivatives showed -cidal activities toward the pathogenic isolates. Further, the inhibition of biofilm formation of Staphylococcus aureus and Staphylococcus epidermidis was also carried out. Additionally, these derivatives displayed significant antibiofilm potential with a superior % reduction in the biofilm formation compared with Ciprofloxacin. The target derivatives behaved synergistically with Ciprofloxacin and Ketoconazole, reducing their MICs. Hemolytic results revealed that these derivatives were nontoxic with a significantly low hemolytic activity (%lysis range from 3.23 to 15.22%) compared with Triton X-100 and showed noncytotoxicity activity with IC50 values > 60 μM. In addition, these derivatives proved to be active DNA gyrase and DHFR inhibitors with IC50 ranging between 12.27-31.64 and 0.52-2.67 μM, respectively. Furthermore, compound 7b showed bactericidal activity at different concentrations in the time-kill assay. Moreover, a gamma radiation dose of 10.0 kGy was efficient for sterilizing compound 7b and enhancing its antimicrobial activity. Finally, molecular docking simulation of the most promising derivatives exhibited good binding energy with different interactions.
A series of thiazol-4-one/thiophene-bearing pyrazole derivatives as pharmacologically attractive cores were initially synthesized using a hybridization approach. All structures were confirmed using spectra analysis techniques (IR, 1H NMR, and 13C NMR). In vitro antimicrobial activities, including the minimum inhibitory concentration (MIC), minimum bactericidal/fungicidal concentration (MBC/MFC), and time-kill assay, were evaluated for the most active derivatives 4a, 5a, 7b, 10, and 13. These derivatives were significantly active against the tested pathogens, with compound 7b as the most active derivative (MIC values range from 0.22 to 0.25 μg/mL). In the MBC and MFC, the active target pyrazole derivatives showed -cidal activities toward the pathogenic isolates. Further, the inhibition of biofilm formation of Staphylococcus aureus and Staphylococcus epidermidis was also carried out. Additionally, these derivatives displayed significant antibiofilm potential with a superior % reduction in the biofilm formation compared with Ciprofloxacin. The target derivatives behaved synergistically with Ciprofloxacin and Ketoconazole, reducing their MICs. Hemolytic results revealed that these derivatives were nontoxic with a significantly low hemolytic activity (%lysis range from 3.23 to 15.22%) compared with Triton X-100 and showed noncytotoxicity activity with IC50 values > 60 μM. In addition, these derivatives proved to be active DNA gyrase and DHFR inhibitors with IC50 ranging between 12.27-31.64 and 0.52-2.67 μM, respectively. Furthermore, compound 7b showed bactericidal activity at different concentrations in the time-kill assay. Moreover, a gamma radiation dose of 10.0 kGy was efficient for sterilizing compound 7b and enhancing its antimicrobial activity. Finally, molecular docking simulation of the most promising derivatives exhibited good binding energy with different interactions.
Bacterial
infection is among the top 10 causes of death and the
first infectious disease-causing mortality worldwide.[1] The evolution of drug-resistant strains, viz., methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE), has unfortunately augmented both nosocomial and inherent
mortality rates, which poses an imperative threat to human health.[2] Nowadays, the development of new antibacterial
compounds to combat human bacterial infections has become a big challenge,
and that may be related to the explicit and repeated use of antibiotics.[3,4] Recently, only four new classes of antibiotics have been approved
by FDA over the past 17 years, while most current drugs have the same
well-understood target.[5,6] The thiazole moiety is found in
several substances, including thiamine (vitamin B1), thiamine pyrophosphate
(TPP), bacitracin, amoxicillin, cefotaxime, and sulfathiazole.[7−10] Additionally, thiazole derivatives have emerged as a new class of
potent antimicrobial agents, which are reported to inhibit bacteria
by many different mechanisms such as blocking the biosynthesis of
certain bacterial lipids[11] and inhibiting
DNA gyrase B[12,13] and dihydrofolate reductase.[14]Pyrazole is one of the most prevalent
nitrogen heterocyclic cores
in many biologically active natural and synthetic compounds, which
has received substantial attention in medicinal chemistry and drug
design research.[15,16] Some drugs with pyrazole pharmacophores
such as Celecoxib and Lonazolac (anti-inflammatory), Betazole (H2-receptor
agonist), and Fezolamine (antidepressant) have been used in clinical
applications.[17]Furthermore, persistent
biofilms are a key virulence factor, which
is recognized as the principal cause of chronic and frequent bacterial
infections ensuing an estimated 17 million new biofilm infections
and more than five hundred thousand deaths each year.[18] Biofilms make the bacteria resistant to conventional antibiotics
by around 1000-fold.[19] Subsequently, there
is an urgent need to counter biofilm formation and develop new antibacterial
agents to exert anti-biofilm activities.[20] This situation requires the development of new antibacterial drugs
with entirely distinct chemical structures, which may have a different
mode of action than currently available therapeutic medications.[21]DNA gyrase is composed of two subunits,
GyrA and GyrB, forming
a complex in the active form of the enzyme to which DNA binds. Both
subunits interact with DNA; the GyrA subunit contains the tyrosine
required for DNA cleavage, while the GyrB subunit contains the ATPase
active site.[22,23] The crucial role of DNA gyrase
in the bacterial and mycobacterial viability and its lack in the higher
eukaryotes make this enzyme a proper target for developing potential
drugs from the viewpoint of selective toxicity.[24−26]Sterility
of the different pharmaceutical products is a serious
aspect of ensuring their safe and effective use upon administration via the parenteral route. Sterilization by gamma irradiation
has shown significant applicability for a wide range of pharmaceutical
agents and has been approved by the European Pharmacopeia. Additionally,
gamma sterilization is a very attractive method for terminal sterilization,
considering its ability to attain a 10–6 probability
of microbial survival without causing excessive product heating or
exposure to toxic chemicals.[27]Hybridizing
two or more scaffolds in one molecule to create a new
hybrid molecule is useful for developing new therapeutic agents.[28−34] Based on these things mentioned above, herein, we report the design
and synthesis of novel series of pyrazole derivatives incorporating
thiazol-4-one/thiophene in one molecule via conventional
synthetic approaches to obtain new pyrazole derivatives (Figure ). The new hybrid
molecules were screened against three Gram-positive, three Gram-negative
bacteria, and two Candida albicans isolates.
The five most active derivatives were used to determine the minimum
inhibitory concentration (MIC), minimum bactericidal concentration
(MBC), and minimum fungicidal concentration (MFC) using the broth
microdilution technique, Staphylococcus biofilm mass
reduction, synergistic effect, time-kill kinetic assay, in
vitro cytotoxic activity against three normal cell lines,
and finally study of their mode of action using two different mechanisms
as DNA gyrase and DHFR inhibitors. A molecular docking study was performed
to confirm the binding mode and confirm the target’s action.
Figure 1
Structures
of the antibiotics containing pyrazole or thiazole/tetrahydrothiazole
moieties as bioactive cores and our designed derivatives.
Structures
of the antibiotics containing pyrazole or thiazole/tetrahydrothiazole
moieties as bioactive cores and our designed derivatives.
Results and Discussion
Chemistry
The synthetic strategy
pathway of the new pyrazole derivatives is depicted from Schemes –4. First, 2-(4-oxothiazolidin-2-ylidene)
derivatives (1a and 1b)[35] were used as precursor builders to synthesize many biologically
and pharmaceutically active heterocycles.[14,36] The thiazolidin-4-one derivatives 1a and 1b were coupled with 1,3-diaryl-1H-pyrazole-4-carboxaldehyde
derivatives 2a and 2b(37) in the presence of ethanol catalyzed with piperidine to
produce 4-oxo-5-((substituted-1H-pyrazol-4-yl)methylene)thiazolidin-2-ylidene
derivatives (3a[38,39] and 3b–d) and failed to obtain 2-(4-oxo-4,5-dihydrothiazol-2-yl)-3-(1-,3-(diaryl)-1H-pyrazol-4-yl)acryl derivatives 4a–d. The condensation reaction proceeds through the endocyclic
methylene rather than the exocyclic one.[40] The structures of 3a–d were confirmed
with the help of analytical and spectroscopic data. Thus, the IR spectrum
of pyrazole derivative 3b showed significant stretching
absorption bands at υ 3199, 2202, and 1718 cm–1 owing to NH, cyano (CN), and carbonyl (C=O) groups, respectively.
The 1H NMR spectrum of pyrazole derivative 3b showed characteristic singlet signals at δ 2.34 ppm, corresponding
to a methyl group, and δ 4.96, 7.92, 8.78, and 11.99 ppm due
to two methylinic protons (methine-H), pyrazole-H, and one exchangeable
NH proton. In addition, nine aromatic protons ranged between δ
7.41 and 8.09 ppm, which displayed two triplet and three doublet signals.
Also, the 13C NMR spectra of compound 3d assigned
significant signals at δ 20.81, 14.21, 59.55, and 166.68 ppm
corresponding to methyl, ethoxy, and carbonyl groups.
Scheme 1
Synthesis
of Pyrazole Derivatives Containing Thiazol-4-one Moieties
Scheme 4
Synthesis of 4-Oxothiazoles and Thiazolo[3,2-a]pyridine
Derivatives Having a Pyrazole Scaffold
Additionally, synthesizing thiazol-4-ones with bis-arylmethylidine
scaffolds 5a–c can happen via refluxing the 5-arylmethylidino-4-thiazolidinone derivative 3a with different aromatic aldehydes in the presence of ethanol
containing a catalytic amount of piperidine. The structures of 3-(aryl)-2-(5-((1H-pyrazol-4-yl)methylene)-4-oxo-4,5-dihydro-thiazol-2-yl)acrylonitrile 5a–c were established through correct
elemental analysis and spectroscopic data (Scheme ). The IR spectra of acrylonitrile derivatives 5a–c furnished characteristic absorption bands for
cyano and carbonyl groups at υ 2197–2199 and 1710–1721
cm–1 and absent bands at nearly 3135–3388
cm–1 for the NH group. The 1H NMR spectrum
of acrylonitrile derivative 5a (DMSO-d6) exhibited characteristic signals at δ 7.64, 7.94,
9.14, and 10.02 ppm corresponding to two methine-H, pyrazole-H, and
hydroxyl groups, respectively. Furthermore, the 13C NMR
spectrum of compound 5b displayed signals at δ
153.54, 154.17, 155.74, and 162.62 ppm attributed to C=C, C=N,
S–C=N, and C=O groups, respectively.The
formation of thiazolin-4-one derivatives 4a–d was
supposed to proceed by reaction of 2-((1,3-diaryl-1H-pyrazol-4-yl)methylene)malononitrile or ethoxy carbonyl malononitrile 6a–d(37) with sulfanylacetic
acid in dimethyl formamide (DMF) that contains catalytic amounts of
piperidine, but the reaction proceeds by two different pathways. Cyclization
of 2-((1,3-diaryl-1H-pyrazol-4-yl)methylene)malononitrile 6a and6b formed 3-(1,3-diaryl-1H-pyrazol-4-yl)-2-(4-oxo-4,5-dihydrothiazol-2-yl)acrylonitrile 4a and 4b, while ethyl 2-cyano-3-(1,3-diaryl-1H-pyrazol-4-yl)acrylate 6c and 6d formed ethyl 4-amino-2-(1,3-diaryl-1H-pyrazol-4-yl)thiophene-3-carboxylate 7a and 7b (the suggested mechanism is illustrated
in Scheme ).
Scheme 3
Suggested Mechanism with the Intermediates (A, B, and C) for the Synthesis of 4-Amino-thiophene-3-carboxylate
Derivatives Containing Pyrazole Moieties 7a and 7b
The structures of both 2-(4-oxo-4,5-dihydrothiazol-2-yl)acrylonitrile 4a and 4b and 4-amino-2-(substituted)thiophene-3-carboxylate 7a and 7b were confirmed by spectroscopic data
and elemental analysis. The IR spectra of compounds 4a and 4b displayed absorption bands at υ 2202–2210
and 1714–1720 cm–1 related to the cyano and
carbonyl of thiazolin-4-one. Conversely, the IR spectra of compounds 7a and 7b showed the absence of absorption bands
for the cyano and carbonyl of thiazolin-4-one and presented new bands
at υ 3345–3432, 1685–1686 cm–1 corresponding to the amino and carbonyl of ester groups. The 1H NMR spectrum of 2-(4-oxo-4,5-dihydrothiazol-2-yl)acrylonitrile
derivative 4b demonstrated four singlet signals at δ
2.49, 4.72, 8.80, and 8.85 ppm due to methyl, methylene groups, methine-H,
and pyrazole-H, respectively. On the other hand, the 1H
NMR spectra of the thiophene-3-carboxylate derivative 7b displayed triplet and quartet signals due to five protons at δ
1.30 and 4.31 ppm for the ethoxy group and three singlet signals at
δ 2.39, 8.09, and 9.13 ppm corresponding to methyl, two protons
of a methine group, and a pyrazole moiety. In addition, the singlet
signal at δ 7.49 ppm was related to an amino group that was
exchangeable with D2O and the absence of methylene singlet
signal at nearly δ 4.72 ppm. The 13C NMR spectra
of the thiophene-3-carboxylate 7a are characterized by
signals at δ 13.96, 62.13, 155.20, and 161.83 ppm, assignable
to an ethoxy group, C-NH2, and carbonyl group (C=O),
respectively.Moreover, it was interesting to synthesize a 4-oxothiazole
derivative
having 4-aminoantipyrine scaffold 8, and this happened via cyano acetylation of 4-aminoantipyrene[41] followed by cyclization with sulfanylacetic acid to afford N-(1H-pyrazol-4-yl)-2-(4-oxo-4,5-dihydrothiazol-2-yl)acetamide
derivative 10 (Scheme ). In a diazo coupling reaction, the diazonium salt
of 4-aminoantipyrine 11 acted as an electrophile and
reacted with malononitrile to form the pyrazolo-hydrazonoyl dicyanide
derivative 12(42) that underwent
cyclization with sulfanylacetic acid under reflux conditions to produce
N-substituted-4-oxo-4,5-dihydrothiazole-2-carbohydrazonoyl cyanide
derivative 13. The IR spectrum of compound 10 showed bands at υ 3436, 3061, 2917, 1688, and 1659 cm–1 due to NH, CH-aromatic, CH-aliphatic, and carbonyl
groups, respectively. The 1H NMR spectra of compound 10 revealed signals at δ 2.19 and 3.08 ppm for two methyl
groups of an antipyrine derivative and signals at δ 3.23 and
4.34 ppm owing to two methylene groups, in addition to the exchangeable
signal at δ 7.17 ppm corresponding to NH and five aromatic protons
that appeared between δ 7.36 and 7.50 ppm. Additionally, coupling
the 4-oxothiazole derivative 1a with the diazonium salt
of 4-aminoantipyrine 11(42) attacked
the endocyclic methylene group in position five to afford 2-(5-hydrazonyl-4-oxo-4,5-dihydrothiazol-2-yl)acetonitrile
derivative 14.Furthermore, the acetonitrile derivative 14 was condensed
with different aromatic aldehydes (1:1 molar ratio) in ethanol with
drops of piperidine to give the corresponding 3-aryl-2-(5-(2-substituted-hydrzono)-4-oxo-4,5-dihydrothiazol-2-yl)acrylonitrile
derivatives 15a and 15b. The IR spectra
of compounds 15a and 15b displayed bands
at υ 3181–3200, 2196–2205, and 1703–1708
cm–1 corresponding to NH, CN, and carbonyl groups,
respectively. The 1H NMR spectrum of compound 14 presented signals at δ 2.54, 3.31, 4.04, and 7.39 ppm corresponding
to the two methyl groups of an antipyrine moiety, methylene, and NH
groups. In a similar manner, the 1H NMR spectra of compound 15a presented four singlet signals at δ 2.49, 3.01,
7.35, and 8.37 ppm owing to two methyl groups of an antipyrine derivative,
a methylinic proton, and a NH group, in addition to 10 aromatic protons
ranging between δ 7.42 and 7.66 ppm. The 13C NMR
spectrum of compound 15a showed signals at δ 10.91
and 34.11 ppm related to two methyl groups and δ 143.37, 148.91,
157.84, and 174.93 ppm corresponding to C=N, S–C=N,
and two carbonyl groups, in addition to the aromatic carbons that
were observed between δ 105.89 and 134.52 ppm.Finally,
the cyclization of 3-aryl-2-(5-(2-substituted-hydrzono)-4-oxo-4,5-dihydrothiazol-2-yl)acrylonitrile
derivatives 15a and 15b with malononitrile
in catalyzed ethanol piperidine produced the 2,3-dihydro-7H-thiazolo[3,2-a]pyridine-6,8-dicarbonitrile
derivatives 16a and 16b. Similarly, the
thiazolo[3,2-a]pyridine-6,8-dicarbonitrile derivatives 16a and 16b were formed by either one-pot reaction
of 2-(5-hydrazonyl-4-oxo-4,5-dihydrothiazol-2-yl)acetonitrile derivative 14 with aromatic aldehyde and malononitrile or by refluxing
with arylidene-malononitrile in absolute ethanol having a little amount
of piperidine (three drops). The structures of the prepared compounds 16a and 16b were proven through accurate analysis.
The IR spectrum of 2,3-dihydro-7H-thiazolo[3,2-a]pyridine-6,8-dicarbonitrile derivative 16a revealed absorption bands in the zones of υ 3442, 3318, 3117,
2201, and 1700 cm–1 corresponding to an amino, imine,
cyano, and significant carbonyl for thiazolidine-4-one, respectively.
Its 1H NMR spectrum (DMSO-d6) showed a notable singlet signal of pyridine-H at δ 4.02 ppm,
in addition to two exchangeable signals at δ 5.24 and 7.35 ppm
for NH and NH2 protons. Moreover, the 13C NMR
spectrum (DMSO-d6) exhibited significant
singlet signals at δ 10.61, 34.37, 38.63, 144.63, 150.11, 154.24,
and 156.51 ppm for two methyl groups of an antipyrine derivative,
pyridine-H4, C=N, carbon attached to the amino group, and two
carbonyl groups, respectively (Scheme ).
Biological Activity
Antimicrobial Sensitivity Assay by an Agar
Well-Diffusion Method
Screening was carried out for the in vitro antimicrobial activity against three Gram +ve and
three Gram -ve bacteria as well as two C. albicans isolates. The broad-spectrum antibiotics, Ciprofloxacin, and Ketoconazole
were used as positive controls. The zones of inhibition are presented
in Table in mm. According
to the given data, five pyrazole derivatives 4a, 5a, 7b, 10, and 13 displayed
excellent activities with inhibition zones ranging from 25 to 33 mm
against the bacterial pathogens and ranging from 28 to 32 mm against
the fungal pathogens in comparison with the reference drugs. On the
other hand, the rest of the compounds showed moderate to no antimicrobial
activity.
Table 1
Inhibition Zone (IZ) in mm ±
Standard Deviation of Pyrazole Derivatives and Reference Drugs against
the Pathogenic Microbesa
Gram +ve bacteria
Gram -ve bacteria
fungi
Cpd. No.
S. aureus
S. epidermidis
B. subtilis
E. coli
A. baumannii
K. pneumoniae
C. albicans100
C. albicans200
3a
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
3b
15 ± 0.63
17 ± 0.21
12 ± 0.50
16 ± 0.34
14 ± 0.50
16 ± 0.77
13 ± 0.22
15 ± 0.80
3c
13 ± 0.46
15 ± 0.70
14 ± 0.80
18 ± 0.33
12 ± 0.32
14 ± 0.60
15 ± 0.45
14 ± 0.30
3d
15 ± 0.78
16 ± 0.10
13 ± 0.47
15 ± 0.67
11 ± 0.23
13 ± 0.11
13 ± 0.60
16 ± 0.22
4a
31 ± 0.18
28 ± 0.13
29 ± 0.42
26 ± 0.33
26 ± 0.14
27 ± 0.16
29 ± 0.23
31 ± 0.22
4b
20 ± 0.20
19 ± 0.31
21 ± 0.50
22 ± 0.60
20 ± 0.30
21 ± 0.12
22 ± 0.10
23 ± 0.22
5a
29 ± 0.22
27 ± 0.24
25 ± 0.37
28 ± 0.14
25 ± 0.11
26 ± 0.20
28 ± 0.40
30 ± 0.60
5b
24 ± 0.20
21 ± 0.10
22 ± 0.55
22 ± 0.56
20 ± 0.42
21 ± 0.32
23 ± 0.26
25 ± 0.77
5c
22 ± 0.66
24 ± 0.28
23 ± 0.22
21 ± 0.45
19 ± 0.23
22 ± 0.76
21 ± 0.52
24 ± 0.13
7a
24 ± 0.40
21 ± 0.12
23 ± 0.11
22 ± 0.41
20 ± 0.12
22 ± 0.60
21 ± 0.32
25 ± 0.33
7b
33 ± 0.32
31 ± 0.16
28 ± 0.22
30 ± 0.50
29 ± 0.12
27 ± 0.30
31 ± 0.11
32 ± 0.32
10
27 ± 0.21
26 ± 0.4
26 ± 0.32
25 ± 0.22
26 ± 0.31
25 ± 0.66
28 ± 0.88
30 ± 0.55
12
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
13
27 ± 0.82
25 ± 0.45
26 ± 0.35
28 ± 0.55
27 ± 0.44
25 ± 0.4
29 ± 0.25
28 ± 0.36
14
16 ± 0.21
19 ± 0.23
14 ± 0.44
17 ± 0.35
15 ± 0.34
15 ± 0.44
18 ± 0.66
20 ± 0.88
15a
16 ± 0.59
18 ± 0.56
17 ± 0.23
15 ± 0.77
16 ± 0.33
14 ± 0.98
16 ± 0.44
20 ± 0.58
15b
18 ± 0.44
16 ± 0.25
17 ± 0.56
16 ± 0.85
13 ± 0.42
16 ± 0.67
16 ± 0.34
18 ± 0.76
16a
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
16b
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
CIP
23 ± 0.3
24 ± 0.12
22 ± 0.33
20 ± 0.24
21 ± 0.2
19 ± 0.11
NA
NA
KCA
NA
NA
NA
NA
NA
NA
21 ± 0.23
24
± 0.54
DMSO
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
CIP: Ciprofloxacin (no activity
(≤18 mm), moderate activity (19–24 mm), and strong activity
(≥25 mm)); KCA: ketoconazole (no activity (≤20 mm),
moderate activity (21–27 mm), and strong activity (≥28
mm)); DMSO: dimethyl sulfoxide; NA: non-applicable. The reference
drugs and compounds were prepared in a concentration of 100 mg/mL.
CIP: Ciprofloxacin (no activity
(≤18 mm), moderate activity (19–24 mm), and strong activity
(≥25 mm)); KCA: ketoconazole (no activity (≤20 mm),
moderate activity (21–27 mm), and strong activity (≥28
mm)); DMSO: dimethyl sulfoxide; NA: non-applicable. The reference
drugs and compounds were prepared in a concentration of 100 mg/mL.
Minimum
Inhibitory Concentration (MIC) and
Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) Determination
The MIC values of the most active pyrazole derivatives 4a, 5a, 7b, 10, and 13 were measured in vitro using the broth microdilution
technique, as presented in Table and Table S1. It was noticed
that the pyrazole containing thiophene-3-carboxylate derivative 7b exhibited the highest MIC value among the other derivatives
over all the tested pathogens, with MICs ranging between 0.22 and
0.25 μg/mL. Moreover, the pyrazolo-thiazolin-4-one derivatives 4a and 5a were equally potent with the reference,
Ciprofloxacin, against E. coli (MIC
= 0.45 and 0.46 μg/mL, respectively), and A.
baumannii (MIC = 0.44 and 0.48 μg/mL, respectively)
and exhibited a two-fold increase in the potency compared to the standard
Ketoconazole against the C. albicans200 (MIC = 0.22 and 0.24 μg/mL, respectively). Additionally,
pyrazolo-thiazolin-4-one derivatives 4a and 5a displayed MIC values of 0.45 and 0.43 μg/mL, respectively,
close to that of Ketoconazole (MIC = 0.49 μg/mL) against the C. albicans100. Furthermore, 1,5-dimethylpyrazol-3-one
incorporating thiazolin-4-one derivatives 10 and 13 demonstrated a good antibacterial activity against the
Gram-negative bacteria used in this study with MIC values 0.43–0.98
μg/mL and against Gram-positive bacteria with MIC = 0.95–0.98
μg/mL compared with Ciprofloxacin (0.46–0.49 μg/mL).
All the remaining derivatives demonstrated promising to moderate antimicrobial
activity. It was interesting to show the antimicrobial activity of
the tested derivatives considered as bactericidal or fungicidal depending
on whether the MBC or MFC is not more than four-fold the MIC value.[43,44] Regarding the MBC and MFC, our pyrazole derivatives had prominent
MBC/MIC and MFC/MIC that were approximately ≤4, indicating
their bactericidal/fungicidal effect.
Table 2
MIC Values
in μg/mL and Ratios
(MBC/MIC or MFC/MIC) of the Most Potent Pyrazole Derivatives 4a, 5a, 7b, 10, and 13 and Positive Controls (CIP and KCA) against the Tested
Pathogensa
MIC (mean
± SEM) (μg/mL) (MBC/MIC or
MFC/MIC)
Gram + ve bacteria
Gram -ve bacteria
fungi
Cpd. No.
S. aureus
S. epidermidis
B. subtilis
E. coli
A. baumannii
K. pneumoniae
C. albicans100
C. albicans200
4a
0.43 ± 0.4 (0.5)
0.48 ± 0.2 (1)
0.45 ± 0.5 (0.5)
0.45 ± 0.2 (0.5)
0.44 ± 0.3 (1)
0.49 ± 0.1 (0.5)
0.45 ± 0.3 (1)
0.22 ± 0.2 (0.5)
5a
0.48 ± 0.1 (1)
0.49 ± 0.4 (0.5)
0.49 ± 0.2 (1)
0.46 ± 0.3 (1)
0.48 ± 0.5 (0.5)
0.46 ± 0.2 (1)
0.43 ± 0.5 (0.5)
0.24 ± 0.4 (1)
7b
0.22 ± 0.3 (0.5)
0.24 ± 0.2 (0.5)
0.23 ± 0.6 (1)
0.23 ± 0.1 (0.5)
0.24 ± 0.1 (1)
0.25 ± 0.5 (1)
0.25 ± 0.2 (1)
0.23 ± 0.3 (0.5)
10
0.95 ± 0.2 (1)
0.97 ± 0.4 (1)
0.98 ± 0.1 (2)
0.46 ± 0.4 (1)
0.45 ± 0.3 (0.5)
0.45 ± 0.2 (1)
0.95 ± 0.3 (2)
0.47 ± 0.1 (1)
13
0.98 ± 0.2 (1)
0.99 ± 0.3 (1)
0.98 ± 0.5 (2)
0.49 ± 0.2 (0.5)
0.43 ± 0.3 (0.5)
0.98 ± 0.4 (2)
0.99 ± 0.2 (2)
0.98 ± 0.3 (2)
CIP
0.48 ± 0.1 (1)
0.47 ± 0.2 (1)
0.48 ± 0.3 (1)
0.46 ± 0.4 (0.5)
0.47 ± 0.2 (0.5)
0.49 ± 0.5 (1)
NA
NA
KCA
NA
NA
NA
NA
NA
NA
0.49 ± 0.3 (1)
0.45 ± 0.3 (0.5)
SEM: standard error mean; each value
is the mean of three values; CIP: Ciprofloxacin; KCA: Ketoconazole;
NA: non-applicable.
SEM: standard error mean; each value
is the mean of three values; CIP: Ciprofloxacin; KCA: Ketoconazole;
NA: non-applicable.
Staphylococcus Biofilm
Mass Reduction
Biofilms formed by bacterial pathogens are
one of the major health problems. They are life-threatening blood
infections associated with both high morbidity and mortality rates,
as well as high treatment costs.[45]S. aureus and S. epidermidis are responsible mostly for several invasive infections due to their
capability to form biofilms. What worsens the case is that the traditional
antibiotics are not effective enough to disrupt the biofilms due to
their high resistance to most agents. It becomes essential to develop
new antimicrobials capable of disrupting these biofilms and therefore
overcome the increasing resistance.[46]In our study, the ability of the potent pyrazole derivatives to reduce
the biofilm formation was analyzed, and the findings are illustrated
in Figure . The data
obtained confirmed that the most promising pyrazole derivatives 4a, 5a, 7b, 10, and 13 all significantly disrupted the biofilm-forming Staphylococcal isolates at their MIC, reducing the S. aureus biofilm formation by 74.3, 89.1, 67, 55.6,
and 51.2%, respectively. At the same time, the S. epidermidis biofilm formations were reduced by 64.8, 79.5, 58, 53.3, and 47.8%,
respectively. These pyrazole derivatives showed superior antibiofilm
activity to the reference antibiotic, Ciprofloxacin, by 43.2% for S. aureus and 38% for S. epidermidis.
Figure 2
Reduction % of biofilm-forming S. aureus and S. epidermidis by the potent
compounds 4a, 5a, 7b, 10, and 13 compared with Ciprofloxacin.
Reduction % of biofilm-forming S. aureus and S. epidermidis by the potent
compounds 4a, 5a, 7b, 10, and 13 compared with Ciprofloxacin.
Single-Step Resistance
Assessment
Upon confirming the rapid -cidal activities of
pyrazole derivatives 4a, 5a, 7b, 10, and 13 against the different pathogenic
isolates tested, it was
essential to assess the probability of these pathogens to develop
resistance to these compounds. Table presents the mutation frequencies produced against
each tested agent. Interestingly, thiophene-3-carboxylate derivative 7b showed a mutation frequency range from 1.34 × 10–6 to 1.67 × 10–7 compared with
Ciprofloxacin (2.82 × 10–7 to 3.31 × 10–9) and Ketoconazole (3.19 × 10–8 to 3.42 × 10–9). Additionally, the mutation
frequency of the most active derivatives ranged as follows: for 4a (1.58 × 10–7 to 1.54 × 10–9), for 5a (2.21 × 10–7 to 2.71 × 10–8), for 10 (2.41
× 10–8 to 3.50 × 10–8), and for 13 (2.71 × 10–7 to
2.92 × 10–9).
Table 3
Single-Step
Resistance Frequencies
for Thiazole-Pyrazole Compounds 4a, 5a, 7b, 10, and 13, Ciprofloxacin, and
Ketoconazole against Clinical Pathogenic Isolatesa
The extensive
use of antibiotics increases the rate of resistance to pathogens.
The combination therapy of commercially used antibiotics with other
antimicrobials has been used in the medical system to reduce the emergence
of resistant isolates to these antibiotics.[47] The most potent pyrazole derivatives 4a, 5a, 7b, 10, and 13 were further
evaluated to act as an adjunct to potentiate a reduction in the MIC
of Ciprofloxacin and Ketoconazole against the clinical isolates. A
checkerboard dilution assay has been used to determine the active
compounds’ fractional inhibitory concentration index (FICI).
A ΣFICI ≤ 0.5 indicates synergism between the antibiotic
and the compound and shows partial synergism if greater than 0.5 and
less than 1.As shown in Table , we observed the ΣFICI range from 0.25 to 0.75
in the drug combinations reported, indicating synergistic and partial
synergistic effects between the compounds and the reference antibiotics.
There was a substantial reduction in the MIC of both Ciprofloxacin
and Ketoconazole against the pathogenic bacteria and fungi. Three
compounds 4a, 5a, and 7b exhibited
synergistic relationships with Ciprofloxacin and Ketoconazole against
all the tested pathogens with ΣFICI ≤ 0.5 ranging from
0.25 and 0.5 able to re-sensitize the pathogenic isolates to antibiotics,
reducing their MICs. The N-(1H-pyrazol-4-yl)-2-(4-oxo-4,5-dihydrothiazol-2-yl)acetamide
derivative 10 has behaved synergistically with Ciprofloxacin
against S. aureus, E.
coli, and A. baumannii and with Ketoconazole against C. albicans200. Also, the synergistic effect was observed between compound 13 and Ciprofloxacin against A. baumannii and with Ketoconazole against the two Candida isolates.
Table 4
Synergistic Effects of Compounds 4a, 5a, 7b, 10, and 13 with
Antibiotics Ciprofloxacin and Ketoconazole against
the Different Pathogensa
fractional
inhibitory concentration index (FICI)/effect
Gram +ve
bacteria
Gram -ve bacteria
fungi
Cpd. No.
S. aureus
S. epidermidis
B. subtilis
E. coli
A. baumannii
K. pneumoniae
C. albicans100
C. albicans200
4a
0.25/S
0.5/S
0.5/S
0.25/S
0.25/S
0.5/S
0.5/S
0.5/S
5a
0.5/S
0.5/S
0.25/S
0.5/S
0.5/S
0.25/S
0.25/S
0.5/S
7b
0.25/S
0.25/S
0.5/S
0.5/S
0.25/S
0.5/S
0.5/S
0.25/S
10
0.5/S
0.75/P
0.75/P
0.5/S
0.5/S
0.75/P
0.75/P
0.5/S
13
0.75/P
0.75/P
0.75/P
0.75/P
0.5/S
0.75/P
0.5/S
0.5/S
S: synergism; P: partial synergism.
S: synergism; P: partial synergism.
Hemolytic Assay
The development
of new antimicrobial agents requires the assurance of their safety
to be used. Many compounds can be biologically active, but they also
can exert several toxic effects such as hemolysis (i.e., rupture the RBCs), causing damage to vital organs such as the
kidney, liver, and heart.[48,49]In our study,
the results of the hemolytic activity of the five active compounds 4a, 5a, 7b, 10, and 13 in Table rendered that they were nontoxic with low hemolytic activity. Compound 7b was the least cytotoxic due to its lowest hemolytic activity
(3.23%) relative to standard Triton X-100 with 95.12% as the positive
control. Compounds 4a and 5a showed hemolytic
activities of 7.55 and 9.67%, respectively. For compound 10, hemolysis was observed with %lysis of 11.66, while compound 13 showed a %lysis of 15.22. CIP and KCA showed RBCs hemolysis
of 16.2 and 15.38%, respectively. The lowest hemolytic activity of
the active derivatives can be attributed to the position of the different
functional groups that might impact the hemolytic effect. It has been
postulated that the electron-withdrawing and electron-donating nature
of the attached groups could affect the hemolytic activity of different
compounds.[50]
Table 5
Hemolytic
Activity of Synthesized
Compound
Cpd. No.
%lysis of
RBCs
4a
7.55 ± 0.63
5a
9.67 ± 0.31
7b
3.23 ± 0.58
10
11.66 ± 0.231
13
15.22 ± 0.43
CIP
16.2 ± 0.5
KCA
15.38 ± 0.32
Triton X-100
95.12 ± 0.721
Inhibitory Effects against
DNA Gyrase and
DHFR Enzymes
The most active pyrazole derivatives 4a, 5a, 7b, 10, and 13 were evaluated for their inhibitory activities, and their mechanisms
against DNA gyrase and DHFR enzymes were explored compared with Ciprofloxacin
and Trimethoprim. The obtained results are depicted as IC50 values of enzyme inhibition in μM in Table .
Table 6
In Vitro Inhibitory
Activities of the Screened Compounds 4a, 5a, 7b, 10, and 13 against DNA
Gyrase and DHFR Enzymesa
IC50 (mean ± SEM) (μM)
Cpd. No.
DNA gyrase
DHFR
4a
18.25 ± 0.67
0.98 ± 0.11
5a
22.13 ± 0.30
1.11 ± 0.15
7b
12.27 ± 0.34
0.52 ± 0.20
10
28.88 ± 0.12
1.42 ± 0.23
13
31.64 ± 0.17
2.67 ± 0.14
CIP
47.68 ± 0.17
TMP
5.16 ± 0.23
SEM: standard
error mean; each value
is the mean of three values; DHFR: dihydrofolate reductase; CIP: Ciprofloxacin;
TMP: Trimethoprim.
SEM: standard
error mean; each value
is the mean of three values; DHFR: dihydrofolate reductase; CIP: Ciprofloxacin;
TMP: Trimethoprim.The order
of the inhibitory activity of the most active derivatives
against DNA gyrase and DHFR can be represented as 7b > 4a > 5a > 10 > 13.
All of the most active derivatives exhibited DNA gyrase inhibitory
potential with IC50 values ranging between 12.27 ±
0.34 and 31.64 ± 0.17 μM, lower than Ciprofloxacin (IC50 = 47.68 ± 0.17 μM). Also, the promising pyrazole
derivatives displayed inhibitory activity against DHFR with IC50 values 0.52 ± 0.20 to 2.67 ± 0.14 μM, lower
than Trimethoprim (IC50 = 5.16 ± 0.23 μM). As
shown in Table , pyrazole
derivatives 4a and 7b proved to be the highest
active inhibitors with IC50 values 18.25 and 12.27 μM
for DNA gyrase and with IC50 values of 0.98 and 0.52 μM
for DHFR, respectively. However, compounds 5a and 10 revealed a moderately high inhibitory activity for DNA
gyrase (IC50 = 22.13 and 28.88 μM, respectively)
and moderate inhibitory activity for DHFR with IC50 of
1.11 and 1.42 μM, respectively. Moreover, compound 13 expressed a DNA gyrase inhibitory activity with an IC50 of 31.64 μM and a DHFR inhibitory activity with an IC50 of 2.67 μM.Finally, the above results confirmed
the validity of our design,
and the hybridization of pyrazole derivatives containing thiazol-4-one
or thiophene could result in compounds with excellent antibacterial
activities.
In Vitro Cytotoxic Activity
on Normal Cell Lines
In order to evaluate the safety of the
five synthesized active compounds 4a, 5a, 7b, 10, and 13 toward the
normal cells, in vitro cytotoxic activity evaluation
was carried out on three normal noncancerous cell lines (BNL, Vero,
and H9C2) to investigate the toxicity and selectivity of these new
derivatives, as presented in Table . To our delight, the five compounds were found to
be noncytotoxic with IC50 values > 60 μM to the
three
normal cell lines. The most active pyrazole derivatives revealed nontoxic
activity with IC50 value ranges of 79.53–103.49
μM, 70.06–86.93 μM, and 61.58–103.3 μM
against the three normal cell lines, namely, mouse normal liver cells
(BNL), green monkey kidney (Vero), and rat heart/myocardium (H9C2),
respectively. Furthermore, compounds 7b and 4a showed the highest IC50 values for the three cell lines
with selectivity index (SI) against DNA gyrase (8.41and 5.44) against
BNL, (6.42 and 4.76) against Vero, and (8.28 and 5.66) against H9C2.
Additionally, the two active derivatives 4a and 7b displayed the highest SI against DHFR, 101.44 and 198.65,
88.70 and 151.61, and 105.40 and 195.44 against the three noncancer
cell lines (BNL, Vero, and H9C2), respectively. The order of safety
and selectivity of these derivatives can be ordered as 7b > 4a > 5a > 10 > 13. Finally, the effect of the five derivatives on the noncancerous
cells revealed a large safety margin on the normal cells, as shown
by their SI values (Table ).
Table 7
In Vitro Cytotoxicity
(IC50 and SI) of Active Compounds 4a, 5a, 7b, 10, and 13 against
Normal Noncancerous Cell Linesc
IC50 (μM ± S.D)
and SI against DNA gyrase and DHFR
BNL
Vero
H9C2
Cpd. No.
IC50
SIa
SIb
IC50
SIa
SIb
IC50
SIa
SIb
4a
99.42 ± 1.04
5.44
101.44
86.93 ±
0.97
4.76
88.70
103.3 ± 1.50
5.66
105.40
5a
103.49 ± 1.44
4.67
93.23
80.24 ± 1.79
3.62
72.28
99.21 ± 2.29
4.48
89.37
7b
103.30 ± 1.51
8.41
198.65
78.84 ±
1.10
6.42
151.61
101.63 ± 1.39
8.28
195.44
10
98.9 ± 1.54
3.42
69.64
70.06 ± 0.34
2.42
49.33
99.78 ± 1.84
3.45
70.26
13
79.53 ± 1.67
2.51
29.78
74.82 ± 2.12
2.36
28.02
61.58 ± 1.64
1.94
23.06
Selectivity index
against DNA gyrase.
Selectivity
index against DHFR.
S.D.:
standard deviation, each value
is the mean of three values; BNL: mouse normal liver cells; Vero:
green monkey kidney; H9C2: rat heart/myocardium; selectivity index
= (IC50 of normal cell)/(IC50 of DNA gyrase
or DHFR).
Selectivity index
against DNA gyrase.Selectivity
index against DHFR.S.D.:
standard deviation, each value
is the mean of three values; BNL: mouse normal liver cells; Vero:
green monkey kidney; H9C2: rat heart/myocardium; selectivity index
= (IC50 of normal cell)/(IC50 of DNA gyrase
or DHFR).
Time-Kill Kinetic Assay
Determination
of the time-kill kinetics of the synthesized compounds is essential
to evaluate the efficacy and killing rate of these antimicrobial agents.
The time-kill kinetic profiles of pyrazole derivative 7b displayed bactericidal and fungicidal activities toward all the
pathogenic isolates tested, showing ≥3log10 reduction
in the viable cell count relative to the initial inoculum with the
reference antibiotics (Figures and 4). The bacterial cells of S. aureus, E. coli, and A. baumannii were rapidly reduced
at 2 × MIC after 8, 4, and 8 h, respectively, and after 12 h
for S. epidermidis, B. subtilis, and K. pneumoniae. For C. albicans100 and C. albicans200, the maximum killing rate
was observed after 16 h, respectively. The maximum reduction in the
viable cell counts was observed with Ciprofloxacin at the MIC after
20, 24, 24, 12, 24, and 20 h for S. aureus, S. epidermidis, B.
subtilis, E. coli, A. baumannii, and K. pneumoniae, respectively. Alternatively, the reduction in the colony-forming
units was detected against C. albicans100 and C. albicans200 with Ketoconazole after 48 h. At 1/2 × MIC, only S. aureus and E. coli showed a maximum reduction after 24 h. Contrariwise, the remaining
bacterial isolates and C. albicans200 showed continuous growth up to 24 h. Also, it was noticed
that Candida albicans100 showed growth reduction after 24 h followed by an increase after
48 h.
Figure 3
In vitro time-kill evaluation of (a) 7b and (b) CIP against clinical bacterial isolates at 2 × MIC.
Figure 4
In vitro time-kill evaluation of (a) 7b and (b) KCA against clinical Candida isolates
at
2 × MIC.
In vitro time-kill evaluation of (a) 7b and (b) CIP against clinical bacterial isolates at 2 × MIC.In vitro time-kill evaluation of (a) 7b and (b) KCA against clinical Candida isolates
at
2 × MIC.
Effect
of Gamma Sterilization on the Antimicrobial
Activity of the Active Compound
Gamma radiation is considered
a conventional sterilization method for a wide range of pharmaceuticals
either in their dry form or as raw materials.[51] It has been reported that gamma radiation doses up to 25.0 kGy were
sufficient to sterilize the pharmaceutical products without causing
any physico/chemical changes.[52] The active
pyrazole derivative 7b was exposed to an array of gamma
irradiation doses ranging from 1.0 to 20.0 kGy. The data obtained
revealed that the gamma irradiation dose of 15.0 kGy was the minimum
adequate dose to sterilize the target compound, i.e., yield SAL < 10–6. Table shows that the antimicrobial activity of
the derivative tested remained unchanged at gamma irradiation doses
1.0 and 5.0 kGy. Contrariwise, the activity was increased at gamma
doses of 10.0, 15.0, and 20.0 kGy.
Table 8
In Vitro Antimicrobial
Activity of Compound 7b Expressed as MIC in μg/mL
before and after Radiationa
gamma radiation doses in kGy MIC (mean ± SEM) (μg/mL)
microorganisms
0
1
5
10
15
20
S. aureus
0.22 ± 0.3
0.22 ± 0.2
0.22 ± 0.3
0.123 ± 0.3
0.124 ± 0.2
0.124 ± 0.4
S. epidermidis
0.24 ± 0.2
0.24 ± 0.3
0.24 ± 0.4
0.124 ± 0.5
0.124 ± 0.3
0.123 ± 0.5
B. subtilis
0.23 ± 0.6
0.23 ± 0.4
0.23 ± 0.5
0.124 ± 0.4
0.124 ± 0.6
0.124 ± 0.3
E. coli
0.23 ± 0.1
0.23 ± 0.2
0.23 ± 0.1
0.122 ± 0.2
0.122 ± 0.5
0.123 ± 0.3
A. baumannii
0.24 ± 0.1
0.24 ± 0.1
0.24 ± 0.1
0.123 ± 0.1
0.124 ± 0.3
0.124 ± 0.5
K. pneumoniae
0.25 ± 0.5
0.25 ± 0.5
0.25 ± 0.2
0.123 ± 0.3
0.125 ± 0.2
0.125 ± 0.3
C. albicans100
0.25 ± 0.2
0.25 ± 0.2
0.25 ± 0.2
0.124 ± 0.5
0.125 ± 0.4
0.125 ± 0.4
C. albicans200
0.23 ± 0.3
0.23 ± 0.3
0.23 ± 0.2
0.123 ± 0.2
0.125 ± 0.1
0.125 ± 0.2
SEM: standard
error mean; each value
is the mean of three values.
SEM: standard
error mean; each value
is the mean of three values.
Molecular Docking Study
Owing to
the favorable results obtained by previous studies, molecular docking
simulation of the most promising derivatives 4a, 5a, 7b, 10, and 13 were
performed inside the active sites of S. aureus DNA gyrase (PDB: 2XCT) and dihydrofolate reductase (PDB: 1DLS).[53] The docking
process was performed using Molecular Operating Environmental (MOE)
10.2008.[54,55] Results obtained from the docking study
are presented in Table and Figures a,b, 6, and 7a,b. These results
exhibited that the most promising pyrazole-thiazol-4-one or pyrazole-thiophene
derivatives can recognize the active site of the protein and make
different types of interactions (H-bond, arene–cation, and
arene–arene) with the key amino acids in the active site of
the pocket.
Table 9
Molecular
Docking Study of the Most
Promising Derivatives 4a, 5a, 7b, 10, and 13 inside the Active Sites of S. aureus DNA Gyrase (PDB: 2XCT) and Dihydrofolate
Reductase (PDB: 1DLS)a
Docking inside the S. aureus DNA Gyrase (PDB: 2XCT)
(a) 2D and 3D binding mode of compound 7b inside the
active site of DNA gyrase (2XCT). (b) 2D and 3D binding mode of compound 5b inside the active site of DNA gyrase (2XCT).
Figure 6
2D and
3D binding mode of compound 4a inside the active
site of DHFR (1DLS)
Figure 7
(a) 2D and 3D binding mode of compound 10 inside the
active site of DHFR (1DLS). (b) 2D and 3D binding mode of compound 7b inside
the active site of DHFR (1DLS).
(a) 2D and 3D binding mode of compound 7b inside the
active site of DNA gyrase (2XCT). (b) 2D and 3D binding mode of compound 5b inside the active site of DNA gyrase (2XCT).2D and
3D binding mode of compound 4a inside the active
site of DHFR (1DLS)(a) 2D and 3D binding mode of compound 10 inside the
active site of DHFR (1DLS). (b) 2D and 3D binding mode of compound 7b inside
the active site of DHFR (1DLS).CIP = Ciprofloxacin,
MTX = Methotrexate,
TMP: Trimethoprim; (-) arene–cation interaction, (--) arene–arene
interaction.First, the
most active derivatives 4a, 5a, 7b, 10, and 13 displayed
binding energies from S = −16.40 Kcal/mol
to S = −20.68 Kcal/mol compared to Ciprofloxacin
having S = −10.58 Kcal/mol. The 4-amino-2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)thiophen derivative 7b showed the lowest binding energy S = −20.68
Kcal/mol through one hydrogen bond donor between Glu1088 and amino
of thiophene (bond length = 3.14 Å), a hydrogen bond acceptor
between Lys1043 and nitrogen of pyrazole derivatives (bond length
= 2.98 Å), and arene–cation interaction between Arg1092
and phenyl at N1 of the pyrazole moiety Figure a.Additionally,
3-(1H-pyrazol-4-yl)-2-(4-oxo-4,5-dihydrothiazol-2-yl)acrylonitrile
derivative 4a revealed a good binding energy S = −18.90 Kcal/mol through one hydrogen bond sidechain
acceptor with Arg1048 with a bond length 3.11 Å and strength
26%. Additionally, the residue His1081 formed an arene–cation
interaction with a phenyl ring at C3 of pyrazole in compound 4a, as described in Figure b.On the other hand, pyrazolo-thiazolin-4-one
derivative 5a demonstrated a binding energy S = −17.39
Kcal/mol through only one hydrogen bond sidechain acceptor between
the residue Lys1043 and carbonyl of thiazoline-4-one. (All figures
of the docking study are shown in the Supporting Information).In addition, N-(1H-pyrazol-4-yl)-2-(4-oxo-4,5-dihydrothiazol-2-yl)acetamide
derivative 10 exhibited a binding energy S = −16.74 Kcal/mol through three hydrogen bond sidechain acceptors
with His1079 and Lys1043 and one arene–cation interaction between
Arg1092 with phenyl at N1 of the pyrazole moiety.
Furthermore, N-(1H-pyrazol-4-yl)-4-oxo-4,5-dihydrothiazole-2-carbohydrazonoyl
derivative 13 demonstrated the lowest binding energy S = −16.74 Kcal/mol through two hydrogen bond sidechain
acceptors between Lys1043 and carbonyl of pyrazole derivatives and
Gln1267 with the nitrogen of the cyano group with bond lengths 2.72
Å (strength = 16%) and 3.43 Å (strength = 12%), respectively.
In addition, there is one hydrogen bond backbone acceptor between
Ile1175 with the carbonyl of the thiazolin-4-one core with a bond
length 3.05 Å (strength = 12%). (All figures for the docking
study are shown in the Supporting Information).On the other hand, to study the activity of the most promising
derivatives 4a, 5a, 7b, 10, and 13 against the DHFR enzyme, a molecular
docking study inside the active site of dihydrofolate reductase (PDB: 1DLS) was performed.
Trimethoprim as another positive control (used in the in vitro study) displayed a binding energy S = −19.92
Kcal/mol through a two hydrogen bond donors between the residue Val115
and amino group at position four in the pyrimidine ring (backbone
donor) with a bond length 2.85 Å and strength 24%, in addition
to the residue Glu30 and the other amino group at position two in
the pyrimidine ring (side chain donor) with a bond length 2.49 Å
and strength 30%. Also, there is one hydrogen bond sidechain acceptor
between Ser59 and oxygen of a methoxy group with a bond length 2.94
Å and strength 38%. (All figures are shown in the SI).The most promising derivatives 4a, 5a, 7b, 10, and 13 exhibited
a good binding energy from S = −20.17 Kcal/mol
to S = −15.46 Kcal/mol with different types
of interactions in comparison to Methotrexate having S = −23.47 Kcal/mol and Trimethoprim having S = −19.92 Kcal/mol. 3-(1H-Pyrazol-4-yl)-2-(4-oxo-4,5-dihydrothiazol-2-yl)acrylonitrile
derivative 4a displayed a binding energy score S = −19.74 Kcal/mol. Additionally, compound 4a fitted inside the active site through two hydrogen bond
sidechain acceptors between Trp24 with the carbonyl of thiazolin-4-one
and Ala9 with the nitrogen of a cyano group with bond lengths 2.60
and 2.72 Å as well as hydrophobic interaction that appears on
phenyl of the pyrazole scaffold and cyano group Figure . Moreover, the docking results of 2-(5-((1H-pyrazol-4-yl)methylene)-4-oxo-4,5-dihydro-thiazol-2-yl)acrylonitrile
derivative 5a and N-(1H-pyrazol-4-yl)-2-(4-oxo-4,5-dihydrothiazol-2-yl)acetamide derivative 10 exhibited binding energies S = −18.83
and −16.21 Kcal/mol with one hydrogen bond and arene–arene
interactions, respectively. The interaction of compound 10 inside the active site of DHFR (PDB: 1DLS) is presented in Figure a. Meanwhile, the thiophene derivative 7b revealed an interesting interaction with a binding energy S = – 20.17 Kcal/mol by its ability to form two hydrogen
bond sidechain acceptors between Ile7 and Val115 with the amino of
thiophene with bond lengths 3.07 and 2.75 Å, respectively (Figure b). Also, the N-(1H-pyrazol-4-yl)-4-oxo-4,5-dihydrothiazole-2-carbohydrazonoyl
derivative 13 showed a binding energy S = −15.46 Kcal/mol through one hydrogen bond sidechain acceptor
between Tyr 121 with the nitrogen of a cyano group with a bond length
3.11 Å and strength 23%, as well as the arene–arene interaction
between Phe31 and phenyl of a pyrazole ring.Finally, the docking
results confirmed the validity of the most
promising derivatives 4a, 5a, 7b, 10, and 13 against DNA gyrase and dihydrofolate
reductase. In addition, the hybridization of thiazol-4-one or thiophene
with pyrazole could improve the antibacterial activities with a lower
binding energy and through different types of interactions such as
hydrogen bonds and arene–arene and arene–cation interactions
with bond lengths not exceeding 3.43 Å.
Conclusions
We herein reported a novel route for synthesizing
some new pyrazole
derivatives containing thiazol-4-one/thiophene. The newly synthesized
derivatives are based on the reaction of 1,3-diaryl-4-formyl pyrazole
or 4-amino antipyrine derivatives with different chemical reagents.
The formyl pyrazole derivatives 2a and 2b were condensed with thiazolidine-4-one derivatives 1a and 1b to form 5-arylmethylidine derivatives 3a and 3b that reacted with the further aromatic
aldehyde to produce bis-aryl methylidene derivatives 5a and 5c. The pyrazolo-arylidene derivatives 6a–d reacted with sulfanylacetic acid to produce thiazoline-4-one 4a and 4b and thiophene derivatives 7a and 7b depending on the active methylene of arylidene.
Further, 4-aminoantipyrine was used as a precursor substance for the
synthesis of thiazolo-pyrazole 10, 13, 14, 15a, and 15b depending on the
conversion of the amino group by cyanoacylation or diazotization and
then reacted with sulfanylacetic acid. Also, this article includes
the synthesis of some thiazolo[3,2-a]pyridine derivatives 16a and 16b that were designed and synthesized
by reacting the mono- and bis-aryl methylidene derivatives 14 and 15a and 15b with arylidene-malononitrile
or malononitrile, respectively. Additionally, all the synthesized
analogs were examined for their in vitro antimicrobial
activities against eight isolate pathogens. Among them, five pyrazole
derivatives 4a, 5a, 7b, 10, and 13 revealed the ability to inhibit the
growth of the screened panel of eight isolates with excellent MICs
and MBCs/MFCs values compared with Ciprofloxacin and Ketoconazole
with a low resistance rate and potential for resistance. Moreover,
the in vitro inhibitory activity for the biofilm
formation illustrated these derivatives as the most potent biofilm
inhibitors compared with the reference drugs against two strains, S. aureus and S. epidermidis. Furthermore, the most active derivatives 4a, 5a, 7b, 10, and 13 showed
synergistic effects when combined with Ciprofloxacin and Ketoconazole,
reducing their MICs and reducing their resistance frequency. The five
active compounds have low hemolytic activity compared with CIP, KCA,
and Triton X-100, which makes them safe to be used as antimicrobial
agents. The enzyme inhibitory activity of the active agents exhibited
their significant activity against the DNA gyrase and DHFR enzymes
with lower IC50 values compared with Ciprofloxacin and
Trimethoprim. Moreover, all the five promising derivatives showed
noncytotoxic activity against three normal noncancerous cell lines,
indicating their safety margin to the normal cells. In addition, the
results also showed that gamma radiation could be safely used in the
sterilization of antimicrobial agents and can enhance their antimicrobial
activity. Finally, the molecular docking simulation was performed
inside the active site of both DNA gyrase (2XCT) and dihydrofolate reductase (1DLS) and exhibited agreement
with in vitro DNA gyrase and DHFR inhibition results
with low binding energy.
Experimental Section
Melting points are uncorrected.
The IR spectra were recorded on a Shimadzu 440 infrared spectrophotometer
(υ/cm–1) using the KBr technique (Shimadzu,
Japan). 1H NMR spectra were recorded on a Varian Gemini
spectrometer (δ/ppm) 300 and 400 MHz using TMS as an internal
standard. Mass spectra were recorded on a Jeol-JMS-600 mass spectrometer. 13C NMR spectra were run at 75 and 101 MHz. Micro analytical
data were obtained from the Micro Analytical Research Centre, Faculty
of Science, Cairo University. The reactions were monitored by thin
layer chromatography (TLC) using TLC sheets with UV fluorescent silica
gel Merck 60f254 plates using a UV lamp and different solvents as
mobile phases. 2-(4-Oxothiazolidin-2-ylidene)acetonitrile (1a) and ethyl-2-(4-oxothiazolidin-2-ylidene)acetate (1b) were prepared according to the reported method.[35] Furthermore, 1,3-diphenyl-1H-pyrazole-4-carbaldehyde
(2a), 1-phenyl-3-(p-tolyl)-1H-pyrazole-4-carbaldehyde (2b),[37] 2-cyano-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)acetamide (9),[41,56] and N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)carbonohydrazonoyldicyanide (12)[42] were also prepared.
Synthesis of 2-(4-Oxo-5-((1-phenyl-3-(aryl)-1H-pyrazol-4-Yl)methylene)thiazolidin-2-ylidene)acetonitrile
or Ethyl Acetate Derivatives (3a–d)
A
mixture of 1-phenyl-3-(aryl)-1H-pyrazole-4-carbaldehyde
derivatives (2a and 2b) (0.01 mol) in 20
mL of ethanol was refluxed for 2 h with thiazolidin-4-one derivatives
(1a and 2b) (0.01 mol) in the presence of
piperidine as a catalyst (three drops). The solid products formed
were collected by filtration and recrystallized from ethanol.
Synthesis of 2-(4-Oxo-4,5-dihydrothiazol-2-yl)-3-(1-,3-(diaryl)-1H-pyrazol-4-yl)acrylnitrile Derivatives (4a and 4b) and Ethyl 4-Amino-2-(1-phenyl-3-(aryl)-1H-pyrazol-4-yl)thiophene-3-carboxylate Derivatives (7a and 7b)
A mixture of 2-((1-phenyl-3-(aryl)-1H-pyrazol-4-yl)methylene)malononitrile (6a and 6b; 0.01 mol) and ethyl-2-cyano-3-(1-phenyl-3-(aryl)-1H-pyrazol-4-yl)acrylate (6c and 6d; 0.01 mol) was added to sulfanylacetic acid (0.01 mol) in 10 mL
of DMF that contains piperidine (0.5 mL), and finally, the reaction
mixture was heated under reflux for 2–4 h. The solid products
formed were collected by filtration and recrystallized from ethanol.
A mixture
of 5-arylmethylidine-4-thiazolidinone
derivative (3a) (0.01 mol) was added to different aromatic
aldehydes (0.01 mol) in 20 mL of absolute ethanol in the presence
of piperidine (three drops) as a catalyst, and the reaction mixture
was heated under reflux for 2–6 h. The solid products formed
were collected by filtration and recrystallized from ethanol.
A solution of thiazolidin-4-one derivative
(1a) (0.01 mol) in absolute ethanol (20 mL) was added
to a diazotized 4-aminoantipyrine solution (11) (0.01
mol). The mixture was stirred for 2 h at 0 °C and a further 2
h at room temperature. The solid product formed was collected by filtration
and recrystallized from ethanol.Brown powder (EtOH); Yield
70%; M.p.: 165–167 °C; IR (KBr, ν/cm–1): 3442, 3324 (2NH), 3065 (CH-arom.), 2990, 2940 (CH-aliph.), 2207(CN),
1720 (C=O), 1615 (C=N); 1H NMR (300 MHz,
DMSO-d) δ/ppm 2.54 (s, 3H, CH3), 3.31 (s, 3H, -N–CH3), 4.04 (s, 2H, CH2), 7.37 (d, J = 7.6 Hz, 2H, Ar-H), 7.39 (s,
1H, NH exchangeable by D2O), 7.57 (t, J = 7.6 Hz, 3H, Ar-H); 13C NMR (75 MHz, DMSO-d): δ/ppm 11.03 (CH3), 33.76 (CH2), 34.37 (N-CH3), 111.81, 122.28, 125.82, 125.97,
127.13, 127.74, 128.96, 129.24, 133.33, 149.16 (C=N), 155.36
(S–C=N), 157.36, 172.17 (2C=O); MS (EI, 70 eV):
m/z (%) = 354.04 (M+) (27.26%), 178.20 (100%); Anal. Calcd.
for C16H14N6O2S (354 .09):
C, 54.23; H, 3.98; N, 23.71; Found: C, 54.31; H, 4.11, N, 23.11.
Synthesis of 3-Aryl-2-(5-(2-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)hydr-zono)-4-oxo-4,5-dihydrothiazol-2-yl)acrylonitrile
Derivatives (15a and 15b)
A solution
of 2-(5-(2-hydrazono)-4-oxo-4,5-dihydrothiazol-2-yl)acetonitrile derivative
(14) (0.01 mol) in absolute ethanol (20 mL) having few
drops of piperidine with different aromatic aldehydes (0.01 mol) was
added to the reaction mixture, and then the solution was heated under
reflux for 6–8 h. The solid products formed were collected
by filtration and recrystallized from ethanol.
Synthesis
of 2,3-Dihydro-7H-thiazolo[3,2-a]pyridine-6,8-dicarbonitrile Derivatives
(16a and 16b)
Method
(1)
To a solution of 15a and 15b (0.01 mol) in absolute ethanol (20
mL) having few drops of piperidine, malononitrile (0.01 mol) was added.
The solution was refluxed for 2–4 h, and solid products formed
were collected by filtration and were recrystallized from ethanol.
Method (2)
To a solution of 2-(5-(2-hydrazono)-4-oxo-4,5-dihydrothiazol-2-yl)acetonitrile
derivative (14) (0.01 mol) in absolute ethanol (20 mL)
having few drops of piperidine, arylidine malononitrile derivatives
(0.01 mol) was added. The solution was heated under reflux conditions
for 4–8 h, and solid products formed were collected by filtration
and recrystallized from ethanol.
All the synthesized compounds were
individually tested against a panel of clinical Gram-positive [S. aureus (MDR00231), S. epidermidis (MDR00223), and B. subtilis (MDR00423)],
Gram-negative [E. coli (MDR00501), A. baumannii (MDR00521), and K. pneumoniae (MDR00244)] bacterial pathogens and two C. albicans (MDR00100) and (MDR00200) fungal isolates from different UTIs clinical
samples obtained from the Central Laboratories, El-Demerdash Hospital,
Ain-Shams University, Cairo, Egypt. The antimicrobial tests were carried
out by the agar well-diffusion method[45,57] using a suspension
containing 1 × 108 CFU/mL of each tested bacteria
and 1 × 106 CFU/mL of yeast on tryptic soya agar (TSA)
and Sabouraud dextrose agar (SDA) plates, respectively. Wells of 10
mm were made and loaded with 50 μL of each tested compound and
the reference drugs (100 mg/mL) dissolved in DMSO. The plates were
incubated at 37 °C for 24 h for bacteria and at 28 °C for
48–72 h for yeast. Ciprofloxacin and Ketoconazole were employed
as standard references for antibacterial and antifungal activities,
respectively. The antimicrobial activity was evaluated by measuring
the zones of inhibition (IZs), expressed in mm, against the pathogens
and compared with the standards. The test was carried out in triplicate,
and the average zone of inhibition (AZOI) was determined.
Minimum Inhibitory Concentration (MIC) and
Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) Determination
The MIC of the target compounds (having inhibition zones > 9
mm)
was then determined using the broth microdilution method.[58] Two-fold serial dilutions of the tested compounds
were prepared to give a concentration range 0.125–128 μg/mL.
Sterile test tubes containing the target compounds were placed in
sterile TSB medium for bacteria and sterile SDB for the fungi. The
TSB and SDB tubes with and without the compounds were used as controls.
The MIC was detected as the lowest concentration of the compound showing
no visible growth of the tested organisms. The experiment was performed
in triplicate, and the data was analyzed using the SPSS 20.0 software.
To determine the MBC and MFC for each test tube in the MIC determination,
a loopful of 5 μL was collected from those tubes that showed
no growth and inoculated on sterile TSA and SDA plates by streaking.
The plates were then incubated with bacteria and fungi at 37 °C
for 24 h and at 28 °C for 48 h, respectively. The lowest concentration
at which no visible growth was observed was noted as the MBC or MFC.[59] In addition, MBC/MIC and MFC/MIC ratios were
determined, and if the tested agent yields an MBC or MFC/MIC ratio
≤ 4, then it is considered to be -cidal, but if it yields a
ratio > 4, then it will be -static.[60]
Staphylococcus Biofilm
Mass Reduction Determination
The microtiter dish biofilm
formation assay[47,61] was employed to assess the ability
of the compounds to disrupt the biofilm formed by the Staphylococcal isolates (S. aureus and S. epidermidis). Each tested bacterial isolate was
transferred to TSB and incubated at 37 °C for 24 h[60] followed by further dilution using the TSB with
1% glucose to reach a concentration of 1:200. For each bacterial isolate,
the suspension was transferred onto the wells of a 96-well microtiter
plate and incubated to allow biofilm formation. The planktonic bacterial
cells were removed by washing the wells three times using PBS. The
synthesized compounds and Ciprofloxacin were added to the wells and
serially diluted. After incubation at 37 °C for 24 h, the plate
was washed three times using water, stained by 0.1% crystal violet,
and then left for 20 min before addition of 95% ethanol for decolorization.
Using a microtiter plate reader, the optical density of each well
at 595 nm was measured. The % of biofilm mass reduction was calculated
for each treatment compared with the control (wells with no treatment).
Single-Step Resistance Selection
The
frequency of single-step resistance of the most active pyrazole
compounds 4a, 5a, 7b, 10, and 13 and antibiotics (Ciprofloxacin and
Ketoconazole) for the tested bacterial and fungal isolates was determined
as reported.[47,62] In brief, cultures of 1 ×
109 CFU/mL were spread onto TSA and SDA plates containing
each compound and each antibiotic at 4 × MIC concentration. The
plates were incubated at 37 °C (for bacteria) and at 28 °C
(for fungi) for 48 h. the frequency of resistance was calculated by
counting the number of resistant colonies per inoculum.
Combination Therapy Analysis of Active Pyrazole
Derivatives with Antibiotics
The combination between active
pyrazolo-thiazole derivatives 4a, 5a, 7b, 10, and 13 and antibiotics (Ciprofloxacin
and Ketoconazole) was assessed via a standard checkerboard
assay[63] and represented as the fractional
inhibitory concentration index (ΣFICI) i.e., the sum of the FIC for each compound. Bacterial suspensions were
prepared in PBS equivalent to the 0.5McFarland standard. The bacteria
were then diluted in TSB to achieve a starting bacterial density of
1 × 105 CFU/mL. The MIC of each test compound in combination
with each antibiotic studied was determined as the lowest concentration
of each combination in which no visible growth was observed. The fractional
inhibitory concentration index (ΣFICI) was calculated for each
combination using the following equation:FICI ≤ 0.5 (synergism);
0.5 < FICI
<1 (partial synergism); 1 ≤ FICI <2 (indifference); and
FICI ≥2 (antagonism).
Hemolytic
Assay
Hemolytic activity
was studied according to the reported studies.[64,65] Three milliliters of fresh heparin-added human blood was obtained
from healthy donors, who contributed voluntarily and had their oral
informed consents before collecting the samples. Initially, the plasma
and WBCs were removed after centrifugation of the blood at 1000g for 5 min, and the blood cells were washed using fresh
sterile cold isosmotic phosphate-buffer saline (PBS) three times.
The RBCs were prepared to be 1 × 108 cells/mL for
each assay, and the active compounds 4a, 5a, 7b, 10, and 13 were prepared
at a concentration of 1.0 mg/mL. The assays of cells in the presence
of the active compounds were incubated at 37 °C for 35 min followed
by agitation after 10 min. All samples were held on ice for 5 min
followed by centrifugation at 1000g for 5 min. Aliquots
of 100 μL was taken from each tube and 10-fold diluted with
cold PBS. PBS and Triton X-100 (0.1% v/v, Sigma-Aldrich, MO, UK) were
used as negative and positive controls, respectively. Also, CIP and
KCA were also used as antibiotic controls. The %lysis of RBCs was
measured for each sample by noting the absorbance at 570 nm using
a UV/Vis spectrophotometer.
Inhibitory
Effects against DNA Gyrase and
DHFR Enzymes
The newly synthesized active targets 4a, 5a, 7b, 10, and 13 were assessed for their in vitro inhibition against
both DNA gyrase and dihydrofolate reductase (DHFR) enzymes using Ciprofloxacin
and Trimethoprim as reference drugs. The assay was carried out using
an E. coli DNA gyrase kit (TopoGEN
Inc., Port Orange, FL) and DHFR assay kit (Sigma-Aldrich, UK) following
the manufacturers’ procedures. The results are depicted as
IC50 values of enzyme inhibition.
In Vitro Cytotoxicity Activity
on Normal Cell Lines
The cytotoxic activity of the new active
derivatives was evaluated in vitro against three
normal noncancerous cell lines (BNL: mouse normal liver cells, Vero:
green monkey kidney, and H9C2: rat heart/myocardium). The cell lines
were obtained from Nawah Scientific Inc., Cairo, Egypt. The cells
were maintained in DMEM media supplemented with 100 mg/mL streptomycin,
100 units/mL penicillin, and 10% heat-inactivated fetal bovine serum
in a humidified 5% (v/v) CO2 atmosphere at 37 °C.
The cell viability was assessed by a colorimetric SulphoRhodamine-B
(SRB) assay. Aliquots of 100 μL cell suspension (5 × 103 cells) were seeded in 96-well plates and incubated in complete
media for 24 h. Cells were treated with another aliquot of 100 μL
media containing the compounds tested at various concentrations. After
72 h of compound exposure, cells were fixed by replacing media with
150 μL of 10% trichloroacetic acid (TCA) and incubated at 4
°C for 1 h. The TCA solution was removed, and the cells were
washed five times with distilled water. Aliquots of 70 μL SRB
solution (0.4% w/v) were added and incubated in a dark place at room
temperature for 10 min. Plates were then washed three times with 1%
acetic acid and allowed to air-dry overnight. Then, 150 μL of
Tris base (10 mM) was added to dissolve protein-bound SRB stain; the
absorbance was measured at 540 nm using a BMG LABTECH-FLUOstar Omega
microplate reader (Ortenberg, Germany). The viability was calculated
as (A540 of treated samples/A540 of untreated sample) × 100, and the inhibitory
concentration at 50% (IC50) was determined from the exponential
curve of viability vs concentration. Also, the selectivity index (SI)
was determined to estimate the safety and selectivity of the tested
compounds. The IC50 values, viability %, and SI for the
tested compounds were calculated as IC50 of the normal
cell lines/IC50 of DNA gyrase or DHFR. Three independent
experiments for each concentration were performed.
Time-Kill Assay
Time-kill assay
was performed using the broth microdilution method following the CLSI
guidelines. Each bacterial isolate at the exponential growth phase
(∼1 × 105 CFU/mL) was inoculated in TSB containing
compounds 7b, CIP, and KCA at 1/2 × MIC, MIC, and
2 × MIC. A growth control containing each bacterial and fungal
isolate without the tested compound was included in each trial. The
bacterial cells were incubated at 37 °C, and aliquots of 100
μL were removed at 0, 2, 4, 8, 12, 20, and 24 h. The aliquots
were 10-fold serially diluted and plated on TSA plates. Similarly,
each fungal isolate at the exponential growth phase (∼1 ×
104 CFU/mL) were inoculated in SDB containing the compound 7b at 1/2 × MIC, MIC, and 2 × MIC concentrations.
The tubes were incubated at 28 °C. Aliquots of 100 μL were
removed at 0, 4, 8, 12, 16, 24, and 48 h. The aliquots were 10-fold
serially diluted and plated on SDA plates. The number of both bacterial
and fungal colonies were counted after incubating the plates at 37
and 28 °C for 24 and 48 h, respectively. Data were analyzed as
killing curves by plotting the log10CFU/mL vs time (h)
as described previously.[63,66]
Effect of Gamma Sterilization on the Antimicrobial
Activity of Pyrazole-Thiophene Derivative 7b
The most active compound 7b was subjected to different
irradiation doses (1.0, 5.0, 10.0, 15.0, and 20.0 kGy) using the radioactive
source (60Co) located in the National Center of Research
and Radiation Technology (NCRRT), Egyptian Atomic Energy Authority
(EAEA), Cairo, Egypt. The radiation dose was 1.0 kGy/h at the time
of experiment. Changes in the chemical structure of the irradiated
derivative was detected using the UV–Vis spectrophotometer
at 570 nm. In addition, the effect of gamma radiation on the antimicrobial
activity of the active target was studied against the different bacterial
and fungal isolates by determination of their MICs values after exposure
to radiation, according to the CLSI protocol.[58] In the tests, microbial culture with a standardized O.D. was used,
and the assay was run in triplicate.
Molecular
Docking Study
First, the
docking process was performed using Molecular Operating Environmental
(MOE) 10.2008.[67−71] The most active pyrazole-thiazolinone derivatives 4a, 5a, 10, and 13, and pyrazole-thiophene
derivative 7b were built using Chembiodraw 2014 and exported
to MOE. The protein structures of DNA gyrase (PDB: 2XCT) and DHFR (PDB: 1DLS) were obtained from
the protein data bank (https://www.rcsb.org/). The docking process were performed inside the active site of S. aureus DNA gyrase (PDB: 2XCT) and dihydrofolate
reductase (PDB: 1DLS) according to the previously reported methods.[53,72]
Authors: Dustyn A Barnette; Mary A Schleiff; Arghya Datta; Noah Flynn; S Joshua Swamidass; Grover P Miller Journal: Toxicol Lett Date: 2020-11-27 Impact factor: 4.372
Authors: Marcelo Gomes Davanço; Anna Caroline Campos Aguiar; Leandro Alves Dos Santos; Elias Carvalho Padilha; Michel Leandro Campos; Cleverton Roberto de Andrade; Luiz Marcos da Fonseca; Jean Leandro Dos Santos; Chung Man Chin; Antoniana Ursine Krettli; Rosangela Gonçalves Peccinini Journal: PLoS One Date: 2014-08-18 Impact factor: 3.240
Authors: Irshad S Sharafutdinov; Elena Y Trizna; Diana R Baidamshina; Maria N Ryzhikova; Regina R Sibgatullina; Alsu M Khabibrakhmanova; Liliya Z Latypova; Almira R Kurbangalieva; Elvira V Rozhina; Mareike Klinger-Strobel; Rawil F Fakhrullin; Mathias W Pletz; Mikhail I Bogachev; Airat R Kayumov; Oliwia Makarewicz Journal: Front Microbiol Date: 2017-11-20 Impact factor: 5.640
Authors: Mahmoud Eldeeb; Eman F Sanad; Ahmed Ragab; Yousry A Ammar; Khaled Mahmoud; Mamdouh M Ali; Nadia M Hamdy Journal: Biomedicines Date: 2022-03-20
Authors: Samar A El-Kalyoubi; Ahmed Ragab; Ola A Abu Ali; Yousry A Ammar; Mohamed G Seadawy; Aya Ahmed; Eman A Fayed Journal: Pharmaceuticals (Basel) Date: 2022-03-20
Figure 1
Scheme 1
Scheme 4
Scheme 3
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7