Synthesis, in-vitro and in-silico study of novel thiazoles as potent antibacterial agents and MurB inhibitors

Article abstract of DOI:10.1002/ardp.201900309

Efficient procedures are herein reported for the synthesis of novel hybrid thiazoles via a one-pot three-component protocol. The protocol involves the reaction of novel aldehyde, thiosemicarbazide and halogen-containing reagents in solvent- and catalyst-free conditions. The structures of the new thiazoles were elucidated by elemental analyses and spectroscopic data. The in-vitro antibacterial screening and MurB enzyme inhibition assays were performed for the novel thiazoles. The thiazol-4(5H)-one derivative 6d, with p-MeO, exhibits the best antibacterial activities with minimum inhibitory concentration values of 3.9, 3.9, 7.8, and 15.6 μg/ml against Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus mutans, and Escherichia coli, respectively, as compared to the reference antibiotic drugs. It also exhibits the highest inhibition of the MurB enzyme with an IC₅₀ of 8.1 μM. The structure–activity relationship was studied to determine the effect of the structures of the newly prepared molecules on the strength of the antibacterial activities. Molecular docking was also performed to predict the binding modes of the new thiazoles in the active sites of the E. coli MurB enzyme.

Full text of DOI:10.1002/ardp.201900309

Received: 17 October 2019
Revised: 20 December 2019
Accepted: 28 December 2019
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DOI: 10.1002/ardp.201900309
F U L L P A P E R
Synthesis, in‐vitro and in‐silico study of novel thiazoles as
potent antibacterial agents and MurB inhibitors
Sherif M. H. Sanad1
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Ahmed A. M. Ahmed1,2
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Ahmed E. M. Mekky1
1Department of Chemistry, Faculty of Science,
Cairo University, Giza, Egypt
Abstract
2Basic Science Department, Jouf University,
Sakaka, Kingdom of Saudi Arabia
Efficient procedures are herein reported for the synthesis of novel hybrid thiazoles
via a one‐pot three‐component protocol. The protocol involves the reaction of novel
aldehyde, thiosemicarbazide and halogen‐containing reagents in solvent‐ and
catalyst‐free conditions. The structures of the new thiazoles were elucidated by
elemental analyses and spectroscopic data. The in‐vitro antibacterial screening and
MurB enzyme inhibition assays were performed for the novel thiazoles. The thiazol‐
4(5H)‐one derivative 6d, with p‐MeO, exhibits the best antibacterial activities with
minimum inhibitory concentration values of 3.9, 3.9, 7.8, and 15.6 μg/ml against
Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus mutans, and Escherichia
coli, respectively, as compared to the reference antibiotic drugs. It also exhibits
the highest inhibition of the MurB enzyme with an IC50 of 8.1 μM. The
structure–activity relationship was studied to determine the effect of the structures
of the newly prepared molecules on the strength of the antibacterial activities.
Molecular docking was also performed to predict the binding modes of the new
thiazoles in the active sites of the E. coli MurB enzyme.
Correspondence
Sherif M. H. Sanad, Department of Chemistry,
Faculty of Science, Cairo University, Giza
12613, Egypt.
Email: sherif_hamed1980@yahoo.com
K E Y W O R D S
5‐(aryldiazenyl)thiazoles, in‐silico study, in‐vitro antibacterial activities, in‐vitro MurB inhibitory
activity, one‐pot three‐component protocol, structure–activity relationship
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1
INTRODUCTION
interface between α and γ subunits.[5] Attempts were made to
prepare derivatives containing the azo‐ moiety with enhanced
Resistance to antibiotic in‐vitro antibacterial activities arises
when bacterial strains change in response to the use of these
medicines. This harmful resistance results in more medical costs,
hospital stays, and so, more deaths.[1] Recently, attempts to
prepare new antibacterial agents to avoid antibiotic resistance
have gained more interest.[2]
antibacterial activities.[6]
On the contrary, several publications reported the synthesis of
2‐hydrazineylthiazole derivatives[7–9] because they exhibit an
important role in medicinal chemistry,[10] such as antibacterial and
antifungal agents,[11–13] as well as inhibitors for bacterial DNA
gyrase B.[14] Several thiazoles exhibit promising antibacterial
activities by their inhibition of the MurB enzyme, which is
responsible for the biosynthesis of bacterial cell walls.[15–17]
Moreover, thiazoles exhibit anti‐inflammatory and analgesic
activities.[18–20] Heterocyclic compounds incorporating the thiazole
moiety show fascinating antitumor and cytotoxic activities.[21,22]
Moreover, it has been reported that the presence of thioether
moiety in the tested derivatives increases their bioactivities such as
in‐vitro antimicrobial activity.[23–25]
It had been reported that azo‐ compounds exhibit interesting
biological activities such as in‐vitro antimicrobial activity.[3] In
general, azo‐ compounds exhibit good antibacterial activity against
Gram‐positive bacteria such as Staphylococcus aureus but they are
inactive against Gram‐negative bacteria such as Pseudomonas
aeruginosa.[4] This behavior is attributed to the structural similarity
of azo‐ compounds with stilbenes, and so, they could share the same
mechanism of action. They could inhibit ATP synthase binding at the
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Arch Pharm Chem Life Sci. 2020;e1900309.
wileyonlinelibrary.com/journal/ardp © 2020 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201900309

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In connection with the fascinating bioactivities of thiazoles and
our efforts regarding the preparation and characterization of
heterocyclic derivatives incorporating the sulfur atom,[26–32] the
work in this study was aimed to design a facile protocol for the
synthesis of hybrid arylazo‐ 4‐methylthiazoles and thiazol‐4(5H)‐
ones, bearing thioether moieties, as potent antibacterial agents
against both Gram‐positive and ‐negative bacterial strains. The
in‐vitro antibacterial screening, as well as in‐vitro MurB inhibitory
activity, were performed for the novel thiazoles. Molecular docking
was elucidated to predict the possible binding interaction modes
between the thiazoles and the active binding sites of the MurB
enzyme from Escherichia coli.
entries 6 and 12). After examining Table 1, it was clear that the
best yield of 4a was obtained by carrying out the one‐pot reaction
under both solvent‐free as well as catalyst‐free conditions (Table 1
and entry 14). Using the above protocol at 120°C, the conventional
heating method gave 4a in 82% yield, whereas the microwave
irradiation method gave 86% yield. It is worthy of note that
heating of the reaction mixture at 100 or 110°C gave an ununiform
melting of the reactants and so gave a mixture of unidentified
components. On the contrary, using 120°C as the temperature of
the reactants was suitable to obtain a uniform melting of all
reactants and so, 4a was isolated as a sole product. At 140°C or
more, a brown mass was obtained of unidentified products. This
may be attributed to the charring of some of the reactants.
Using the above optimum reaction conditions,
a variety of
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2
RESULTS AND DISCUSSION
Chemistry
4‐methyl‐5‐(aryldiazenyl)thiazole derivatives containing the thioether
moiety 4b–e were prepared by the one‐pot reaction of 2, thiosemi-
carbazide and hydrazonyl chlorides 4b–e under either conventional
heating or microwave irradiation (Scheme 3). The 1H‐NMR spectrum
of 4c, as a representative example of the prepared thiazoles, revealed
three singlet signals due to two p‐Me, thiazole‐CH3 and CH2 protons
at δ = 2.24, 2.58, and 4.13 ppm, respectively, and 11 aromatic protons
at δ = 6.87–7.67 ppm, in addition to three singlet signals due to
methine‐CH, OH, and NH protons at δ = 8.82, 10.70, and 10.78 ppm,
respectively (see Section 4). Similarly, the one‐pot three‐
component reaction of 2, thiosemicarbazide and hydrazonyl chlorides
5a–e gave the corresponding 5‐(2‐arylhydrazineylidene)-thiazol‐
4(5H)‐one derivatives 6a–e (Scheme 3). The 1H‐NMR spectrum of
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2.1
5‐(Chloromethyl)‐2‐hydroxybenzaldehyde 1 was prepared by the
reaction of a ternary mixture containing salicylaldehyde, formalde-
hyde and concentrated HCl.[33] Compound
1 reacted with
4‐methylbenzenethiol in ethanol containing an equivalent amount
of potassium hydroxide to give the novel 2‐hydroxy‐5‐((p‐tolylthio)-
methyl)benzaldehyde 2 (Scheme 1 and Section 4).
Benzaldehyde derivative 2 was used as a building block for the
synthesis of novel thiazoles, bearing both arylazo‐ and thioether
moieties, with potent biological activity via
a one‐pot three‐
component protocol. Initially, we investigated the reaction of 2,
thiosemicarbazide and hydrazonyl chloride 3a under different
reaction conditions to prepare the corresponding 4‐methyl‐5‐
(phenyldiazenyl)thiazole derivative 4a with maximum reaction yield
and minimum reaction time (Scheme 2 and Table 1).[34]
5‐(2‐phenylhydrazineylidene)thiazol‐4(5H)‐one derivative 6a, as a
representative example, revealed six singlet signals due to p‐Me,
CH2, methine‐CH, OH, and two NH protons at δ = 2.25, 4.13, 8.68,
10.54, 10.58, and 12.58 ppm, respectively, in addition to 12 aromatic
protons at δ = 6.87–7.64 ppm (see Section 4). The time of reactions
and their yields are shown in Table 2. Generally, using microwave
irradiation as an energy source gave better yields than conventional
heating.
Both piperidine and triethylamine were investigated as
reactive basic catalysts in the presence of polar or nonpolar
solvents and under either conventional heating or microwave
irradiation. Using water as a solvent gave only traces of 4a as
detected by thin‐layer chromatography (TLC) analysis (Table 1 and
entries 1 and 7), whereas using ethanol or 1,4‐dioxane gave
17–45% yields of 4a (Table 1 and entries 2, 3, 8, and 9). The action
of the above catalysts was also investigated in acetonitrile or
dichloromethane (as an example of nonpolar solvents) to give 4a in
25–38% yields (Table 1 and entries 4, 5, 10, and 11). The use of
glacial acetic acid as a dual solvent and catalyst was also studied. It
gave 4a in 20–28% yield (Table 1 and entry 13). Moreover, the
reaction was carried out using the above catalysts and under
solvent‐free conditions to give 4a in 45–54% yields (Table 1 and
The study also includes the reaction of a ternary mixture of
aldehyde derivative 2 with thiosemicarbazide and each of the
1‐chloropropan‐2‐one, 2‐bromo‐1‐phenylethan‐1‐one, 2‐bromo‐1‐
(4‐chlorophenyl)ethan‐1‐one, 3‐(2‐chloroacetyl)‐2H‐chromen‐2‐one,
or ethyl chloroacetate in both solvent and catalyst‐free conditions
to give the corresponding 4‐substituted thiazole derivatives 7–10
and thiazol‐4(5H)‐one derivative 11 (Scheme 4).[35,36] The time of
reactions and their yields are shown in Table 3. The 1H‐NMR
spectrum of 4‐(4‐chlorophenyl)thiazole derivative 9, as a represen-
tative example, revealed five singlet signals due to p‐Me, CH2,
methine‐CH, OH, and NH groups at δ = 2.24, 4.12, 8.27, 10.04, and
SCHEME 1 Synthesis of 2‐hydroxy‐
5‐((p‐tolylthio)methyl)benzaldehyde 2

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SCHEME 2 Synthesis of 4‐methyl‐5‐(phenyldiazenyl)thiazole 4a
12.09 ppm, respectively, in addition to 12 aromatic protons at
δ = 6.80–7.88 ppm (see Section 4).
S. mutans, K. pneumoniae, and E. coli, respectively, when compared
with inhibition zones of the reference antibiotic drugs. Its minimum
inhibitory concentration (MIC) values were 3.9 and 7.8 μg/ml against
S. aureus and S. mutans, respectively, when compared with standard
ampicillin (62.5 μg/ml). Moreover, its MIC values were 3.9 and
15.6 μg/ml against K. pneumoniae and E. coli, respectively, when
compared with standard gentamicin (62.5 and 31.25 μg/ml, respec-
tively). The analogue 6c, with the p‐Me group, was the second in
antibacterial strength against the above bacterial strains with MIC
values of 7.8, 15.6, 15.6, and 31.25 μg/ml against S. aureus, S. mutans,
K. pneumoniae, and E. coli, respectively, when compared with
reference drugs. The strong activities of both 6c and 6d could be
attributed to the presence of electron‐donating p‐Me or p‐MeO
groups. The p‐MeO group is more electron releasing than the p‐Me
group, therefore, compound 6d showed more enhanced antibacterial
activity than 6c. Compound 2b, with p‐Cl, exhibit moderate activities
against all the tested bacterial strains. Compound 2a or 2e, with
p‐H or p‐EtOCO, respectively, exhibited weak activities against
It is noteworthy that 4a and 6a could also be prepared by
the coupling of 4‐methylthiazole derivative 7 and thiazol‐4(5H)‐one
derivative 11 with benzene diazonium chloride in pyridine,
respectively (Scheme 4).
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2.2
Antibacterial screening
The in‐vitro antibacterial activities of the newly synthesized thiazoles
were evaluated against two Gram‐negative (Klebsiella pneumoniae
and E. coli) and two Gram‐positive bacterial strains (Staphylococcus
aureus and Streptococcus mutans; see Tables 4 and 5).
5‐(2‐(4‐Methoxyphenyl)hydrazineylidene)thiazol‐4(5H)‐one deri-
vative 6d was the most potent antibacterial derivative against all the
tested bacterial strains. It exhibited the maximum zones of inhibition
(63.0 1.0, 45.9 0.6, 62.5 1.0, and 51.2 1.0 mm) against S. aureus,
TABLE 1 Optimizing the yield of 4‐methyl‐5‐(phenyldiazenyl)thiazole 4a
Yield (%)a,b,c
Entry
1
Solvent
Temp. (°C)
120
100
120
100
60
Catalyst
Thermal heating
Microwave irradiation
Water
Piperidine
Trace
17
Trace
25
2
EtOH
Piperidine
3
1,4‐Dioxane
Acetonitrile
Dichloromethane
No solvent
Water
Piperidine
37
44
4
Piperidine
32
36
5
Piperidine
25
30
6
120
120
100
120
100
60
Piperidine
45
50
7
Triethylamine
Triethylamine
Triethylamine
Triethylamine
Triethylamine
Triethylamine
Glacial acetic acid
No catalyst
Trace
18
Trace
24
8
EtOH
9
1,4‐Dioxane
Acetonitrile
Dichloromethane
No solvent
Glacial acetic acid
No solvent
40
45
10
11
12
13
14
35
38
28
33
120
140
120
48
54
20
28
82
86
aThe reaction mixture was heated in an oil‐bath under constant stirring for 3 hr.
bThe reaction mixture was irradiated by microwaves under microwave irradiation of power 300 W for 30 min.
cThe reaction was followed by the thin‐layer chromatography analyses.

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SCHEME 3 Synthesis of 4‐methyl‐5‐(aryldiazenyl)thiazole 4b–e and 5‐(2‐arylhydrazineylidene)thiazol‐4(5H)‐one derivatives 6a–e
Other thiazole derivatives which lack arylazo‐ groups showed
Gram‐positive bacterial strains. Compounds 2a and 2e have no
decreased antibacterial activities when compared with
activities against Gram‐negative bacterial strains.
compounds 4 and 6. Therefore, compounds 7–9 and 11 exhibited
On the contrary, 5‐(aryldiazenyl)‐4‐methylthiazoles 4 showed a
weak or zero activities against all the tested bacterial strains.
similar pattern of antibacterial activities when compared with
Compound 10 exhibited moderate activities against all the tested
compounds 2. Therefore, the activities of compounds 4 follow the
bacteria. This may be attributed to the presence of coumarin
order: 4d (p‐MeO) > 4c (p‐Me) > 4b (p‐Cl) > 4a (p‐H) or 4e (p‐EtOCO)
moiety.
against all the tested bacteria.
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2.3
The in‐vitro enzyme inhibition of MurB
TABLE 2 Synthesis of 4‐methyl‐5‐(aryldiazenyl)thiazole 4b–e and
5‐(2‐arylhydrazineylidene)thiazol‐4(5H)‐one derivatives 6a–e under
both microwave irradiation and the conventional method
Peptidoglycan is an essential polymer of the cell wall of both Gram‐
positive and ‐negative bacterial strains. This polymer consists of
repeated units of disaccharide and pentapeptide, which are con-
nected by a lactyl ether bridge.[37] During its biosynthesis, the
UDP‐N‐acetylglucosamine‐enolpyruvate reductase (MurB) enzyme
carries out the reduction of enolpyruvyl uridine diphosphate
N‐acetylglucosamine to uridine diphosphate N‐acetylmuramic
acid.[38] Some antibiotics are able to kill bacteria by inhibiting the
role of the MurB enzyme in the biosynthesis of peptidoglycan.[39] It
has been reported that 4‐thiazolidinones act as inhibitors for MurB
enzyme and so they possess promising antibacterial activities.[17]
The enzyme inhibitory activity of the potential antibacterial
thiazoles 4c, 4d, 6c, and 6d were estimated against the MurB
enzyme. Each compound of the tested thiazoles was tested at six
different concentrations (5, 10, 25, 50, 75, and 100 µM).
4‐Thiazolidinone I, as an example of the potent antibacterial agents,
was taken as a reference drug[17] (see Figure 1).
Microwave
Thermal heatinga,c
irradiationb,c
Time
Time (hr) Yield (%) (min)
Yield
Entry Product
R
(%)
85
89
82
88
89
90
92
87
92
1
2
3
4
5
6
7
8
9
4b
4c
4d
4e
6a
6b
6c
6d
6e
Cl
3
78
74
70
79
80
76
77
74
81
30
15
30
45
15
15
30
30
30
Me
2.5
3
MeO
EtOCO
H
4
2.5
2.5
3
Cl
Me
MeO
EtOCO
3
3
aThe reaction mixture was heated in an oil‐bath at 120°C under constant
stirring.
bThe reaction mixture was irradiated by microwaves at 120°C under
IC50 values were estimated for all the tested thiazoles. All the
tested thiazoles exhibited good to excellent inhibitory activity
microwave irradiation of power 300 W.
cThe reaction was followed up by thin‐layer chromatography analyses.

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SCHEME 4 Synthesis of thiazoles 4a, 6a, 7–10, and 11
against the MurB enzyme (see Table 6). Thiazoles 4c, 4d, and 6c
exhibit good inhibitory activity against MurB enzyme with IC50
values in the range of 8.1–18.2 µM. The most potent thiazole in the
tested series was 6d. It exhibited a comparable inhibitory activity,
IC50 = 8.1 µM, when compared with the reference 4‐thiazolidinone I
(IC50 = 7.7 µM).
TABLE 3 Synthesis of 4‐substituted thiazole derivatives 7–10 and
thiazol‐4(5H)‐one derivative 11 under both microwave irradiation
and the conventional method
Thermal heatinga,c
Microwave irradiationb,c
Entry Product Time (hr) Yield (%) Time (min)
Yield (%)
1
2
3
4
5
7
8
3
78
74
70
79
82
30
15
15
45
30
89
89
82
88
92
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2.4
Molecular modeling of novel thiazole
2.5
2.5
4
derivatives: Docking and structure–activity
relationship study
9
10
11
3
Molecular docking, as one of the in‐silico study tools, is suitable to
obtain an optimized conformation, with minimum free energy, for
each of the newly prepared thiazoles as ligand molecules and the
target protein.[40,41] The thiazoles 4b, 4c, 4d, 6b, 6c, 6d, and 10
showed different antibacterial activities against E. coli. to investigate
aThe reaction mixture was heated in an oil‐bath at 120°C under constant
stirring.
bThe reaction mixture was irradiated by microwaves at 120°C under
microwave irradiation of power 300 W.
cThe reaction was followed by thin‐layer chromatography analyses.

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TABLE 4 Antibacterial activity of the novel thiazoles against different tested bacterial strains
Zone of inhibition (mm)
Gram‐positive bacteria
Staphylococcus aureus
16.5 0.5
36.3 0.6
51.2 1.0
55.7 1.0
14.8 0.5
18.5 0.5
37.1 0.6
59.4 1.0
63.0 1.0
16.7 0.5
15.2 0.5
15.0 0.5
−ve
Gram‐negative bacteria
Compound
Streptococcus mutans
14.7 0.5
22.7 0.5
34.7 0.6
38.4 0.6
−ve
Klebsiella pneumoniae
−ve
Escherichia coli
−ve
4a
4b
26.6 0.5
37.5 0.6
47.6 0.6
−ve
14.0 0.5
30.1 0.6
41.3 0.6
−ve
4c
4d
4e
6a
14.9 0.5
25.8 0.5
41.6 0.6
45.9 0.6
15.1 0.5
−ve
19.0 0.5
27.2 0.5
52.5 1.0
62.5 1.0
−ve
−ve
6b
25.0 0.5
46.6 0.6
51.2 1.0
−ve
6c
6d
6e
7
17.6 0.5
−ve
−ve
8
−ve
−ve
9
14.2 0.5
25.4 0.5
14.6 0.5
–
−ve
−ve
10
36.8 0.6
28.1 0.5
–
27.2 0.5
14.9 0.5
25 0.5
–
26.3 0.5
−ve
11
Gentamicin
Ampicillin
28 0.5
–
22 0.1
28 0.5
Note: Zone of inhibition is expressed as mm in the form of mean standard deviation. Well diameter (6 mm); 100 µl was tested. −ve means inactive
(inhibition zone < 8 mm).
TABLE 5 Minimum inhibitory concentration (MIC) of the tested thiazoles
Minimum inhibitory concentration (µg/ml)
Gram‐positive bacteria
Gram‐negative bacteria
Compound
Staphylococcus aureus
Streptococcus mutans
Klebsiella pneumoniae
Escherichia coli
−ve
4a
1,000
250
1,000
500
−ve
4b
500
125
31.25
−ve
1,000
250
4c
31.25
15.6
1,000
1,000
250
62.5
31.25
−ve
4d
62.5
−ve
4e
6a
1,000
250
1,000
500
15.6
3.9
−ve
6b
500
6c
7.8
15.6
7.8
31.25
15.6
−ve
6d
3.9
6e
1,000
1,000
1,000
−ve
1,000
−ve
−ve
7
1,000
−ve
−ve
8
−ve
−ve
9
1,000
250
−ve
−ve
10
250
500
1,000
62.5
–
500
11
500
1,000
–
−ve
Gentamicin
Ampicillin
–
31.25
–
62.5
62.5
Note: Stock concentration 1 mg/ml; concentrations unit of MIC are represented as µg/ml, MIC was recorded for tested compounds that showed inhibition
zone > 10 mm.

SANAD ET AL.
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compound 6d had tremendous bioactivity. Its docking pose showed
three favorable H‐bonding interactions between the S atom in the
4‐thiazolone ring and the residue of GLU 128 (3.01 Å, −0.9 kcal/mol),
the S atom in the thioether moiety and the residue of ASN 233
(2.57 Å, −2.5 kcal/mol) and the N atom in the azo group with GLU
128 (2.43 Å, −5.9 kcal/mol).
On the contrary, the docking of 4b, the least active antibacterial
agent in the tested thiazoles, with E. coli MurB, gave weak H‐bonding
interaction between the O atom in the hydroxy group and the
residue of ASN 233 (3.09 Å, −0.9 kcal/mol) and between the S atom
in the thiazole ring and the residue of GLU 325 (4.03 Å, −0.1 kcal/
mol; see Figure 3).
FIGURE 1 Structure of the potent 4‐thiazolidinone MurB
inhibitor I
TABLE 6 MurB IC50 (µM) of the tested thiazoles
The docking poses of the rest tested thiazoles showed
H‐bonding interactions with residues of different amino acids
as follows: between the S atom in the thiazole ring of 4d, 6b, and
6c with the residue of GLU 325, SER 229, and PRO 111; the S
atom in the thioether moiety of 4c, 6c, and 10 with the residue of
GLN 120, SER 229, and ARG 214; and the N atom in the arylazo‐
group of 4c, 4d, and 6c with GLU 334, ARG 214, PRO 111, and
ASN 51. In addition, other H‐bonding interactions were present
as follows: the O atom in the 4‐thiazolone ring of 6b with TYR
125; N atom in the hydrazinyl group of 6b with GLY 123; O atom
in the chromene‐CO group of 10 with LYS 262. Moreover, the
thiazoles 4d, 6b, and 10 form hydrophobic contacts (π–H or π–π)
with the residue of PRO 111, SER 229, TYR 125, and TYR 158
(see Supporting Information).
Compound
MurB IC50 (µM)
4c
18.2
13.8
11.0
8.1
4d
6c
6d
4‐Thiazolidinone I
7.7
the above thiazoles as potential MurB inhibitors, their calculated
structures were docked with the E. coli MurB enzyme (PDB ID:
1MBT). The binding energies of the above interactions were
calculated using computational docking studies.[42] The docking
results are listed in Table 7.
Among the 15 new thiazoles which were prepared in this study,
compound 6d, with the p‐MeO group, was found to be the most
potent antibacterial agent against all the tested bacterial strains.
Experimental results as well as docking studies revealed that
compound 10 exhibited moderate activities against all the tested
bacteria. This may be attributed to the presence of the coumarin
moiety which enhances its antibacterial activity.[43] Other
Figure
2 represents the two‐dimensional (2D) and 3D ligand
interactions of 6d with E. coli MurB. Our docking study showed that
TABLE 7 Docking results of thiazoles 4b, 4c, 4d, 6b, 6c, 6d, and 10 with the Escherichia coli MurB enzyme
Compound
4b
Ligand moiety
Site
Interaction
Distance (Å)
E (kcal/mol)
O 28
S 37
OD1 ASN 233 (A)
OE1 GLU 325 (A)
H‐donor
H‐acceptor
3.09
4.03
−0.9
−0.1
4c
4d
S 11
N 45
NE2 GLN 120 (A)
N GLU 334 (A)
H‐acceptor
H‐acceptor
2.87
3.35
−1.6
−3.0
S 37
OE1 GLU 325 (A)
NH2 ARG 214 (A)
CD PRO 111 (A)
H‐donor
H‐acceptor
π–H
2.98
3.03
3.78
−1.8
−3.6
−0.7
N 45
6‐ring
6b
6c
N 33
S 37
O 39
6‐ring
O GLY 123 (A)
OG SER 229 (A)
N TYR 125 (A)
N SER 229 (A)
H‐donor
H‐donor
H‐acceptor
π–H
3.52
3.51
3.61
4.39
−1.5
−0.2
−0.4
−0.5
S 11
S 37
N 42
N 41
OG SER 229 (A)
O PRO 111 (A)
O PRO 111 (A)
CB ASN 51 (A)
H‐donor
H‐donor
H‐donor
H‐acceptor
2.88
3.12
2.51
3.37
−2.1
−0.5
−5.0
−0.9
6d
10
S 37
N 42
S 11
OE1 GLU 128 (A)
OE1 GLU 128 (A)
ND2 ASN 233 (A)
H‐donor
3.01
2.43
2.57
−0.9
−5.9
−2.5
H‐donor
H‐acceptor
S 11
NH2 ARG 214 (A)
NZ LYS 262 (A)
H‐acceptor
H‐acceptor
π–H
3.32
3.46
4.74
3.94
−1.2
−0.8
−0.8
−0.0
O 56
5‐ring
6‐ring
CE1 TYR 158 (A)
6‐ring TYR 125 (A)
π–π

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FIGURE 2 Two‐dimensional (2D) ligand interaction and 3D ligand interaction 6d with Escherichia coli MurB
thiazole derivatives, which lack arylazo‐ groups showed de-
creased antibacterial activities when compared with compounds
4 and 6. Therefore, compounds 7–9 and 11 exhibited weak or
zero activities against all the tested bacterial strains. This
suggests that the presence of arylazo‐ groups enhances the
antibacterial activities.
Although we prepared both 5‐(aryldiazenyl)‐4‐methylthiazoles
4 and 5‐(2‐arylhydrazineylidene)thiazol‐4(5H)‐ones 6 with the same
set of substituents attached to para‐position of arylazo‐ group, in
general, compounds 6 showed stronger antibacterial activities when
compared with compounds 4. This may be attributed to the presence
of 4‐thiazolone moiety in 6.[17]
Moreover, both the experimental and docking results re-
vealed differences in antibacterial activity between derivatives
with different substituents in the para‐position of the arylazo‐
group at position‐5 of thiazole derivatives, indicating the effect of
substituents on the resulting activity. The results showed that the
To elucidate the relationship between electronic properties of the
target thiazoles and the antibacterial activity, we compare the values of
electronic substituent constant (σp) [47] with the MIC values against each
of S. aureus and E. coli as examples of Gram‐positive and ‐negative
bacterial strains (Table 8). After examining Table 8, a clear relation is
observed between the values of σp and MIC values. As the substituent
becomes more electron‐donating, the antibacterial activity increases.[48]
Furthermore, docking studies revealed that most of the tested
thiazoles showed strong H‐bonding interactions between the S
atom in the thioether moieties and residues of amino acids, which
are present in the MurB enzyme. Hence, the inhibition ability of
the prepared thiazoles toward MurB enzyme could be increased
by the incorporation of the thioether moiety in their structure.
These results are in agreement with other publications that
report the increased in‐vitro antimicrobial activity of the
derivatives containing thioether moieties.[24]
antibacterial activities of the new thiazoles
4 and 6 were
enhanced when strong electron‐donating substituent was
a
attached to the p‐position of the arylazo‐ group. This may be
attributed to the electron density of these compounds. The
presence of the electron‐donating group is responsible for
increasing the electron density of azo‐nitrogen atoms and so
enhancing their antibacterial activities by lowering their binding
energies with residues of amino acids that are present in the
target MurB enzyme.[44–46] These results are another confirma-
tion of the role of arylazo‐ group in increasing the antibacterial
activities of the new thiazoles.
FIGURE 3 2D ligand interaction and 3D ligand interaction of 4b with Escherichia coli MurB

SANAD ET AL.
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|
TABLE 8 The relation between electronic substituent constants and minimum inhibitory concentration (MIC) values
MIC (µg/ml)
Compound
Substituent
σp
0.00
Staphylococcus aureus
Escherichia coli
−ve
4a
4b
4c
4d
4e
6a
6b
6c
6d
6e
H
1,000
250
Cl
0.23
−0.17
−0.27
0.45
1,000
250
Me
31.25
15.6
1,000
1,000
250
MeO
EtOCO
H
62.5
−ve
0.00
−ve
Cl
0.23
500
Me
−0.17
−0.27
0.45
7.8
31.25
15.6
MeO
EtOCO
3.9
1,000
−ve
|
3
CONCLUSION
GC‐MS‐QP1000EX spectrometer using the inlet type at 70 eV.
Microwave experiments were performed using a CEM Discover &
Explorer SP microwave apparatus (300 W), utilizing 35‐ml capped
glass reaction vessels automated power control based on tempera-
ture feedback. Compounds 1,[33] 3a–e,[49] and 5a–e[49] are prepared
according to the literature procedure.
This study describes the synthesis of novel thiazoles in various
reaction conditions. The optimized protocol involves the three‐
component reaction of novel aldehyde, thiosemicarbazide and
the appropriate halogen‐containing reagent in both solvent and
catalyst‐free conditions. The in‐vitro antibacterial screening
and enzyme inhibition of MurB assays were performed for the
novel thiazoles. Molecular docking was also performed to predict
the binding modes of the new thiazoles with the active sites of
the MurB enzyme. Both in‐vitro as well as in‐silico studies show
that the thiazol‐4(5H)‐one derivative 6d with p‐MeO was the
most potent antibacterial derivative against all the tested
bacterial strains with the highest inhibition of the MurB enzyme.
The original spectra of the investigated compounds are provided
as Supporting Information, as are their InChI codes together with
some biological data.
|
4.1.2
Synthesis of 2‐hydroxy‐5‐((p‐tolylthio)-
methyl)benzaldehyde (2)
A
ternary mixture of 5‐(chloromethyl)‐2‐hydroxybenzaldehyde
(1, 5 mmol), 4‐methylbenzenethiol (5 mmol) and potassium hydroxide
(5 mmol) in ethanol (20 ml) was heated at reflux for 1 hr. The reaction
mixture was cooled, filtered off, washed with cold water and then the
product was recrystallized from ethanol as colorless crystals (82%);
m.p. 94–96°C; IR (υ cm−1): 3,433, 2,905, 1,656, 1,577, and 1,484;
1H‐NMR (DMSO‐d6): δ 2.24 (s, 3H, CH3), 4.13 (s, 2H, CH2), 6.90–7.59
(m, 7H, Ar–H), 10.21 (s, 1H, CHO), 10.65 (s, 1H, OH); 13C‐NMR
(DMSO‐d6): δ 20.2, 36.9, 116.2, 126.3, 128.7, 129.0, 129.5, 129.9,
132.7, 135.9, 137.9, 161.9, and 194.0; MS m/z (%): 258 (M+, 60.2);
Anal. for C15H14O2S: C, 69.74; H, 5.46; S, 12.41. Found: C, 69.99; H,
5.61; S, 12.20%.
|
4
EXPERIMENTAL
Chemistry
|
4.1
|
4.1.1
General
All organic solvents were acquired from commercial sources and
used as received unless otherwise stated. All other chemicals were
acquired from Merck or Aldrich and used without further purifica-
tion. The melting points (m.p.) were measured on a Stuart melting
point apparatus and are uncorrected. Infrared (IR) spectra were
recorded on
a Smart iTR, which is an ultra‐high‐performance,
|
versatile, attenuated total reflectance‐sampling accessory on the
Nicolet iS10 FT‐IR spectrometer. 1H‐NMR and 13C‐NMR spectra
were recorded on Varian Mercury at 300 MHz and 100 MHz
spectrophotometers, respectively, using tetramethylsilane as an
internal standard and dimethyl sulfoxide‐d6 (DMSO‐d6) as a solvent
and chemical shifts were expressed as δ ppm units. Elemental
analyses were carried out on a EuroVector instrument C, H, N, S
analyzer EA3000 Series. Mass spectra (MS) were recorded on a
4.1.3
Synthesis of thiazoles 4, 7–10 and thiazol‐
4(5H)‐ones 6 and 11
Method I: A ternary mixture of aldehyde 2 (5 mmol), thiosemicarba-
zide (5 mmol) and each of the appropriate hydrazonyl chlorides 3a–e,
5a–e or the appropriate halogen‐containing reagents (5 mmol) was
heated in an oil‐bath at 120°C and the reaction was followed by TLC.
The reaction mixture was cooled, poured into 100 g of ice‐water,

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filtered off, washed with cold ethanol and then the product was
recrystallized from the proper solvent.
134.4, 134.8, 139.2, 144.4, 147.8, 150.0, 156.3, and 161.7; MS m/z
(%): 487 (M+, 74.1); Anal. for C26H25N5OS2: C, 64.04; H, 5.17; N,
14.36; S, 13.15. Found: C, 64.23; H, 5.01; N, 14.14; S, 13.11%.
Method II: A ternary mixture of aldehyde 2 (5 mmol), thiosemi-
carbazide (5 mmol) and each of the appropriate hydrazonyl chlorides
3a–e, 5a–e or the appropriate halogen‐containing reagents (5 mmol)
was prepared. The mixture was placed in process and was irradiated
2‐((2‐(5‐((4‐Methoxyphenyl)diazenyl)‐4‐methylthiazol‐2‐yl)-
hydrazineylidene)methyl)‐4‐((p‐tolylthio)methyl)phenol (4d)
by microwaves with
a
power of 300 W to reach
a
reaction
Red crystals (1,4‐dioxane); m.p. 178–180°C; IR (υ cm−1): 3,414, 3,224,
2,961, 1,601, and 1,486; 1H‐NMR (DMSO‐d6): δ 2.24 (s, 3H, CH3),
2.59 (s, 3H, CH3), 3.72 (s, 3H, OCH3), 4.14 (s, 2H, CH2), 6.86–7.68
(m, 11H, Ar–H), 8.82 (s, 1H, methine‐H), 10.42 (s, 1H, OH), and 10.61
(s, 1H, NH); Anal. for C26H25N5O2S2 (503.64): C, 62.01; H, 5.00; N,
13.91; S, 12.73. Found: C, 61.88; H, 4.82; N, 14.05; S, 12.81%.
temperature of 120°C under autogenerated pressure and the
reaction was followed by TLC. The reaction mixture was cooled,
poured into 100 g of ice‐water, filtered off, washed with cold ethanol,
and then, the product was recrystallized from the proper solvent.
Method III: A benzene diazonium chloride solution (5 mmol) was
prepared via the addition of sodium nitrite solution (0.5 g into 5 ml of
water) to aniline hydrochloride (5 mmol in 5 ml of concentrated HC1)
with stirring in an ice bath. The obtained solution was then poured to
a solution of each of compound 7 or 11 (5 mmol) in pyridine (15 ml)
with stirring for 1 hr at 0–5°C. The reaction mixture was stirred for
an additional 3 hr in an ice bath and then left for 12 hr at 4°C in a
refrigerator. The solid obtained was filtrated and recrystallized from
the appropriate solvent to give the corresponding 4a and 6a.
Ethyl 4‐((2‐(2‐(2‐hydroxy‐5‐((p‐tolylthio)methyl)benzylidene)-
hydrazineyl)‐4‐methylthiazol‐5‐yl)diazenyl)benzoate (4e)
Red crystals (ethanol/1,4‐dioxane mixture); m.p. 168–170°C; IR
(υ cm−1): 3,410, 3,220, 2,947, 1,739, 1,607, and 1,488; 1H‐NMR
(DMSO‐d6): δ 1.30 (t, J = 7.2 Hz, 3H, CH2CH3), 2.24 (s, 3H, CH3), 2.60
(s, 3H, CH3), 4.14 (s, 2H, CH2), 4.28 (q, J = 7.2 Hz, 2H, CH2CH3),
6.85–7.91 (m, 11H, Ar–H), 8.69 (s, 1H, methine‐H), 10.74 (s, 1H, OH),
and 10.99 (s, 1H, NH); Anal. for C28H27N5O3S2 (545.68): C, 61.63; H,
4.99; N, 12.83; S, 11.75. Found: C, 61.58; H, 5.14; N, 13.01; S, 11.59%.
2‐((2‐(4‐Methyl‐5‐(phenyldiazenyl)thiazol‐2‐yl)hydrazineylidene)-
methyl)‐4‐((p‐tolylthio)methyl)phenol (4a)
Red crystals (ethanol/1,4‐dioxane mixture); m.p. 164–166°C; IR
(υ cm−1): 3,411, 3,220, 2,963, 1,600, and 1,488; 1H‐NMR (DMSO‐
d6): δ 2.25 (s, 3H, CH3), 2.60 (s, 3H, CH3), 4.14 (s, 2H, CH2), 6.89–7.68
(m, 12H, Ar–H), 8.83 (s, 1H, methine‐H), 10.74 (s, 1H, OH), and 10.76
(s, 1H, NH); 13C‐NMR (DMSO‐d6): δ 14.6, 20.3, 36.8, 116.3, 120.6,
122.7, 128.0, 128.4, 129.3, 129.8, 130.5, 130.9, 132.9, 135.2, 137.7,
138.4, 144.3, 147.5, 151.6, 156.7, and 161.8; MS m/z (%): 473
(M+, 44.5); Anal. for C25H23N5OS2: C, 63.40; H, 4.90; N, 14.79;
S, 13.54. Found: C, 63.62; H, 5.07; N, 14.98; S, 13.36%.
2‐(2‐(2‐Hydroxy‐5‐((p‐tolylthio)methyl)benzylidene)hydrazineyl)‐5‐
(2‐phenylhydrazineylidene)thiazol‐4(5H)‐one (6a)
Yellow crystals (ethanol/1,4‐dioxane mixture); m.p. 284–286°C; IR
(υ cm−1): 3,434, 3,250, 2,917, 1,703, 1,639, 1,546, and 1,490; 1H‐NMR
(DMSO‐d6): δ 2.25 (s, 3H, CH3), 4.13 (s, 2H, CH2), 6.87–7.64 (m, 12H,
Ar–H), 8.68 (s, 1H, methine‐H), 10.54 (s, 1H, OH), 10.58 (s, 1H, NH),
and 12.58 (s, 1H, NH); 13C‐NMR (DMSO‐d6): δ 20.2, 36.8, 114.9, 116.4,
121.0, 124.8, 128.4, 129.1, 129.4, 129.8, 130.6, 132.3, 135.0, 136.2,
138.2, 143.0, 147.1, 156.0, 157.3, and 163.1; MS m/z (%): 475
(M+, 85.7); Anal. for C24H21N5O2S2: C, 60.61; H, 4.45; N, 14.73; S,
13.48. Found: C, 60.65; H, 4.28; N, 14.86; S, 13.30%.
2‐((2‐(5‐((4‐Chlorophenyl)diazenyl)‐4‐methylthiazol‐2‐yl)-
hydrazineylidene)methyl)‐4‐((p‐tolylthio)methyl)phenol (4b)
Red crystals (1,4‐dioxane); m.p. 190–192°C; IR (υ cm−1): 3,411, 3,221,
2,958, 1,601, and 1,484; 1H‐NMR (DMSO‐d6): δ 2.24 (s, 3H, CH3), 2.58
(s, 3H, CH3), 4.14 (s, 2H, CH2), 6.88–7.69 (m, 11H, Ar–H), 8.81 (s, 1H,
methine‐H), 10.70 (s, 1H, OH), and 10.78 (s, 1H, NH); 13C‐NMR
(DMSO‐d6): δ 14.5, 20.3, 36.7, 116.4, 120.4, 122.8, 128.6, 128.8, 129.3,
129.5, 130.9, 132.6, 134.2, 135.3, 137.2, 138.3, 144.5, 147.6, 150.9,
156.3, and 161.8; Anal. for C25H22ClN5OS2 (508.06): C, 59.10; H, 4.36;
N, 13.78; S, 12.62. Found: C, 58.87; H, 4.11; N, 13.89; S, 12.84%.
5‐(2‐(4‐Chlorophenyl)hydrazineylidene)‐2‐(2‐(2‐hydroxy‐5‐((p‐
tolylthio)methyl)benzylidene)hydrazineyl)thiazol‐4(5H)‐one (6b)
Yellow crystals (1,4‐dioxane); m.p. 280–282°C; IR (υ cm−1): 3,436,
3,243, 2,926, 1,702, 1,644, 1,548, and 1,491; 1H‐NMR (DMSO‐d6): δ
2.25 (s, 3H, CH3), 4.13 (s, 2H, CH2), 6.87–7.64 (m, 11H, Ar–H), 8.68
(s, 1H, methine‐H), 10.58 (s, 1H, OH), 10.62 (s, 1H, NH), and 12.65
(s, 1H, NH); Anal. for C24H20ClN5O2S2 (510.03): C, 56.52; H, 3.95; N,
13.73; S, 12.57. Found: C, 56.74; H, 4.10; N, 13.77; S, 12.49%.
2‐((2‐(4‐Methyl‐5‐(p‐tolyldiazenyl)thiazol‐2‐yl)hydrazineylidene)-
methyl)‐4‐((p‐tolylthio)methyl)phenol (4c)
2‐(2‐(2‐Hydroxy‐5‐((p‐tolylthio)methyl)benzylidene)hydrazineyl)‐5‐
(2‐(p‐tolyl)hydrazineylidene)thiazol‐4(5H)‐one (6c)
Red crystals (ethanol/1,4‐dioxane mixture); m.p. 186–188°C; IR
(υ cm−1): 3,414, 3,220, 2,957, 1,600, and 1,485; 1H‐NMR (DMSO‐
d6): δ 2.24 (s, 6H, 2CH3), 2.58 (s, 3H, CH3), 4.13 (s, 2H, CH2),
6.87–7.67 (m, 11H, Ar–H), 8.82 (s, 1H, methine‐H), 10.70 (s, 1H, OH),
and 10.78 (s, 1H, NH); 13C‐NMR (DMSO‐d6): δ 14.5, 20.2, 20.6, 37.0,
116.6, 120.4, 122.0, 128.1, 129.3, 129.6, 130.7, 131.1, 132.7, 133.9,
Yellow crystals (1,4‐dioxane); m.p. 290–292°C; IR (υ cm−1): 3,433, 2,918,
1,684, 1,636, 1,534, and 1,489; 1H‐NMR (DMSO‐d6): δ 2.24 (s, 6H,
2CH3), 4.14 (s, 2H, CH2), 6.84–7.70 (m, 11H, Ar–H), 8.69 (s, 1H, methine‐
H), 10.65 (s, 1H, OH), 10.78 (s, 1H, NH), and 12.64 (s, 1H, NH); MS m/z
(%): 489 (M+, 62.9); Anal. for C25H23N5O2S2: C, 61.33; H, 4.74; N, 14.30;
S, 13.10. Found: C, 61.54; H, 4.87; N, 14.24; S, 13.26%.

SANAD ET AL.
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2‐(2‐(2‐Hydroxy‐5‐((p‐tolylthio)methyl)benzylidene)hydrazineyl)‐
5‐(2‐(4‐methoxyphenyl)hydrazineylidene)thiazol‐4(5H)‐one (6d)
Yellow crystals (ethanol/1,4‐dioxane mixture); m.p. 262–264°C; IR
(υ cm−1): 3,434, 3,241, 2,936, 1,688, 1,634, 1,549, and 1,495; 1H‐NMR
(DMSO‐d6): δ 2.25 (s, 3H, CH3), 3.72 (s, 3H, OCH3), 4.13 (s, 2H, CH2),
6.87–7.63 (m, 11H, Ar–H), 8.67 (s, 1H, methine‐H), 10.42 (s, 1H, OH),
10.60 (s, 1H, NH), and 12.50 (s, 1H, NH); 13C‐NMR (DMSO‐d6): δ 20.2,
36.9, 55.1, 114.2, 116.3, 118.1, 120.7, 128.8, 129.7, 129.8, 131.1,
132.6, 135.1, 136.6, 138.2, 140.0, 147.0, 155.2, 156.1, 157.6, and
163.3; Anal. for C25H23N5O3S2 (505.61): C, 59.39; H, 4.59; N, 13.85; S,
12.68. Found: C, 59.31; H, 4.45; N, 13.99; S, 12.74%.
128.4, 128.7, 129.6, 129.7, 130.0, 131.9, 132.1, 134.8, 135.1,
138.9, 147.3, 150.1, 156.6, and 167.8; Anal. for C24H20ClN3OS2
(466.01): C, 61.86; H, 4.33; N, 9.02; S, 13.76. Found: C, 62.04; H,
4.18; N, 9.17; S, 13.69%.
3‐(2‐(2‐(2‐Hydroxy‐5‐((p‐tolylthio)methyl)benzylidene)hydrazineyl)-
thiazol‐4‐yl)‐2H‐chromen‐2‐one (10)
Yellow crystals (1,4‐dioxane); m.p. 288–290°C; IR (υ cm−1): 3,433,
3,222, 2,916, 1,700, 1,578, and 1,491; 1H‐NMR (DMSO‐d6): δ 2.25
(s, 3H, CH3), 4.13 (s, 2H, CH2), 6.80–7.77 (m, 12H, Ar–H), 8.30 (s, 1H,
methine‐H), 8.54 (s, 1H, coumarin H‐4), 10.02 (s, 1H, OH), and 12.11
(s, 1H, NH); MS m/z (%): 499 (M+, 53.4); Anal. for C27H21N3O3S2: C,
64.91; H, 4.24; N, 8.41; S, 12.83. Found: C, 65.12; H, 4.14; N, 8.63;
S, 13.01%.
Ethyl 4‐(2‐(2‐(2‐(2‐hydroxy‐5‐((p‐tolylthio)methyl)benzylidene)-
hydrazineyl)‐4‐oxothiazol‐5(4H)‐ylidene)hydrazineyl)benzoate (6e)
Yellow crystals (1,4‐dioxane); m.p. 286–288°C; IR (υ cm−1): 3,438,
2,924, 1,738, 1,689, 1,638, 1,552, and 1,494; 1H‐NMR (DMSO‐d6): δ
1.30 (t, J = 7.2 Hz, 3H, CH2CH3), 2.24 (s, 3H, CH3), 4.13 (s, 2H, CH2),
4.27 (q, J = 7.2 Hz, 2H, CH2CH3), 6.87–7.92 (m, 11H, Ar–H), 8.69
(s, 1H, methine‐H), 10.55 (s, 1H, OH), 10.87 (s, 1H, NH), and 12.70
(s, 1H, NH); 13C‐NMR (DMSO‐d6): δ 14.1, 20.3, 36.8, 61.1, 114.7,
116.6, 121.4, 123.2, 128.5, 129.1, 129.5, 131.0, 131.4, 132.2, 135.4,
136.3, 138.2, 146.3, 146.7, 156.2, 157.6, 163.8, and 166.2; Anal. for
C27H25N5O4S2 (547.65): C, 59.22; H, 4.60; N, 12.79; S, 11.71. Found:
C, 59.07; H, 4.74; N, 12.85; S, 11.57%.
2‐(2‐(2‐Hydroxy‐5‐((p‐tolylthio)methyl)benzylidene)hydrazineyl)-
thiazol‐4(5H)‐one (11)
Beige crystals (ethanol); m.p. 260–262°C; IR (υ cm−1): 3,432,
2,919, 1,718, 1,640, and 1,487; 1H‐NMR (DMSO‐d6): δ 2.25
(s, 3H, CH3), 3.97 (s, 2H, thiazole‐CH2), 4.13 (s, 2H, CH2),
6.82–7.66 (m, 7H, Ar–H), 8.66 (s, 1H, methine‐H), 10.54 (s, 1H,
OH), and 10.54 (s, 1H, NH); MS m/z (%): 371 (M+, 39.9); Anal. for
C18H17N3O2S2: C, 58.20; H, 4.61; N, 11.31; S, 17.26. Found: C,
58.46; H, 4.73; N, 11.44; S, 17.35%.
2‐((2‐(4‐Methylthiazol‐2‐yl)hydrazineylidene)methyl)‐4‐((p‐tolylthio)-
methyl)phenol (7)
|
4.2
Biological assays
Pale beige crystals (ethanol); m.p. 146–148°C; IR (υ cm−1): 3,416,
3,224, 2,944, 1,602, and 1,488; 1H‐NMR (DMSO‐d6): δ 2.25 (s, 3H,
CH3), 2.59 (s, 3H, CH3), 4.13 (s, 2H, CH2), 6.57–7.70 (m, 8H, Ar–H),
8.81 (s, 1H, methine‐H), 10.70 (s, 1H, OH), and 10.75 (s, 1H, NH);
Anal. for C19H19N3OS2 (369.50): C, 61.76; H, 5.18; N, 11.37; S, 17.35.
Found: C, 61.80; H, 5.07; N, 11.52; S, 17.50%.
|
4.2.1
The in‐vitro antibacterial assay
The in‐vitro antibacterial activity of all tested molecules was
achieved using the agar well diffusion procedure.[50,51] The
antibacterial activity was evaluated against each of S. aureus
(ATCC: 6538) and S. mutans, (ATCC:25175) as Gram‐positive
bacterial strains (the reference drug was ampicillin, 10 µg
susceptibility disc; Oxford‐England) and Klebsiella pneumoniae
(ATCC:10031) and E. coli (ATCC: 9637) as gram‐negative bacterial
strains (the reference drug was gentamicin, 120 µg susceptibility
disc; Oxford‐England) using nutrient agar medium. The concentra-
tion of the tested molecules was 15 mg/ml against bacterial
strains. DMSO was used as the solvent. Negative control was the
wells containing only DMSO.
2‐((2‐(4‐Phenylthiazol‐2‐yl)hydrazineylidene)methyl)‐4‐((p‐tolylthio)-
methyl)phenol (8)
Beige crystals (ethanol/1,4‐dioxane mixture); m.p. 216–218°C; IR
(υ cm−1): 3,433, 3,321, 2,915, 1,618, 1,562, and 1,488; 1H‐NMR
(DMSO‐d6): δ 2.24 (s, 3H, CH3), 4.13 (s, 2H, CH2), 6.78–7.87 (m, 13H,
Ar–H), 8.26 (s, 1H, methine‐H), 10.02 (s, 1H, OH), and 12.05 (s, 1H,
NH); 13C‐NMR (DMSO‐d6): δ 20.3, 36.8, 106.4, 116.8, 120.4, 126.0,
128.5, 128.7, 129.0, 129.2, 129.5, 131.2, 132.1, 133.2, 134.6, 138.7,
147.6, 152.2, 156.5, and 167.7; MS m/z (%): 431 (M+, 70.8); Anal. for
C24H21N3OS2: C, 66.79; H, 4.90; N, 9.74; S, 14.86. Found: C, 66.94; H,
5.12; N, 9.85; S, 14.72%.
Sterilized media were poured onto Petri dishes, and
these then solidified at room temperature. The microbial
suspension was prepared in sterilized saline equivalent to
McFarland 0.5 standard solution (1.5 × 105 CFU/ml) with
a
2‐((2‐(4‐(4‐Chlorophenyl)thiazol‐2‐yl)hydrazineylidene)methyl)‐4‐((p‐
tolylthio)methyl)phenol (9)
turbidity of OD = 0.13 using a spectrophotometer at 625 nm.
After solidification of the media, 6‐mm diameter wells were
made. To each well, 100 μl of each the tested compounds were
added then the plates were incubated at 37°C for 24 hr to
observe the formation of inhibition zones. The above procedure
was repeated three times and the final reading of the inhibition
zones (in mm) was determined as the average.
Beige crystals (1,4‐dioxane); m.p. 232–234°C; IR (υ cm−1): 3,434,
3,312, 2,916, 1,617, 1,564, and 1,488; 1H‐NMR (DMSO‐d6): δ
2.24 (s, 3H, CH3), 4.12 (s, 2H, CH2), 6.80–7.88 (m, 12H, Ar–H),
8.27 (s, 1H, methine‐H), 10.04 (s, 1H, OH), and 12.09 (s, 1H, NH);
13C‐NMR (DMSO‐d6): δ 20.2, 36.6, 108.6, 116.7, 120.2, 127.6,

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4.2.2
Minimum inhibitory concentration
co‐crystallized ligands and other unsupported elements (e.g., Na, Mg,
SO4, etc.) were removed but the amino‐acid chain was reserved.[54,55]
The ligand’s structure in the PDB file format was created by Gaussian
03 software. The structure of the MurB enzyme from E. coli was
downloaded from PDB (https://www.rcsb.org/).
MIC is used to evaluate the minimum concentration of the tested
compound which prevents the growth of bacteria after overnight
incubation. The twofold serial dilution was used to estimate the
MIC values.[51,52]
For each bacterial strain, three to five isolated colonies from a fresh
agar plate were transferred into a 3‐ to 4‐ml sterile broth medium. The
bacterial suspension was incubated at 35–37°C for 2–6 hr until the
turbidity of the bacterial suspension became equal to or greater than
the turbidity of a McFarland Standard 0.5. The tested molecules were
diluted to 1,000 μg/ml, as a stock solution. Further dilutions for the
tested molecules were performed with the broth medium. Then, a fixed
volume of the prepared bacterial inoculum was added to each tube of
the different concentrations of the tested molecules and incubated for
16–20 hr at 37°C. By turbidity, the growth or a lack of growth in the
tested tubes was observed by comparison with the growth control
(which represents the original inoculum without any tested compounds).
In the same way, both ampicillin and gentamicin were screened under
the same conditions for comparison.
CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.
ORCID
Sherif M. H. Sanad
Ahmed A. M. Ahmed
Ahmed E. M. Mekky
http://orcid.org/0000-0002-0186-6418
http://orcid.org/0000-0002-4855-8850
http://orcid.org/0000-0002-8226-010X
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How to cite this article: Sanad SMH, Ahmed AAM, Mekky
AEM. Synthesis, in‐vitro and in‐silico study of novel thiazoles as
potent antibacterial agents and MurB inhibitors. Arch Pharm
Chem Life Sci. 2020;e1900309.
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https://doi.org/10.1002/ardp.201900309