In vitro antimycobacterial activity and physicochemical characterization of diaryl ether triclosan analogues as potential inhA reductase inhibitors

Article abstract of DOI:10.3390/molecules25143125

Two sets of diphenyl ether derivatives incorporating five-membered 1,3,4-oxadiazoles, and their open-chain aryl hydrazone analogs were synthesized in good yields. Most of the synthesized compounds showed promising in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv. Three diphenyl ether derivatives, namely hydrazide 3, oxadiazole 4 and naphthylarylidene 8g exhibited pronounced activity with minimum inhibitory concentrations (MICs) of 0.61, 0.86 and 0.99 μg/mL, respectively compared to triclosan (10 μg/mL) and isoniazid (INH) (0.2 μg/mL). Compounds 3, 4, and 8g showed the InhA reductase enzyme inhibition with higher IC50 values (3.28-4.23 μM) in comparison to triclosan (1.10 μM). Correlation between calculated physicochemical parameters and biological activity has been discussed which justifies a strong correlation with respect to the inhibition of InhA reductase enzyme. Molecular modeling and drug-likeness studies showed good agreement with the obtained biological evaluation. The structural and experimental information concerning these three InhA inhibitors will likely contribute to the lead optimization of new antibiotics for M. tuberculosis.

Full text of DOI:10.3390/molecules25143125

molecules
Article
In Vitro Antimycobacterial Activity and
Physicochemical Characterization of Diaryl Ether
Triclosan Analogues as Potential InhA
Reductase Inhibitors
Tarek S. Ibrahim 1,2,
*
, Ehab S. Taher 3, Ebtihal Samir 4, Azizah M. Malebari 1,
Ahdab N. Khayyat 1 , Mamdouh F. A. Mohamed 5 , Riham M. Bokhtia 2,6
,
Mohammed A. AlAwadh 1, Israa A. Seliem 2,6, Hani Z. Asfour 7, Nabil A. Alhakamy 8
,
Siva S. Panda 6,
*
and Amany M. M. AL-Mahmoudy 2
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University,
Jeddah 21589, Saudi Arabia; amelibary@kau.edu.sa (A.M.M.); ankhayyat@kau.edu.sa (A.N.K.);
Malawadh@kau.edu.sa (M.A.A.)
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Zagazig University,
Zagazig 44519, Egypt; rbokhtia@augusta.edu (R.M.B.); isliem@augusta.edu (I.A.S.);
amanysinger77@gmail.com (A.M.M.A.-M.)
2
3
4
5
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Al-Azhar University,
Assiut 71524, Egypt; ehaborganic@yahoo.com
Physical Chemistry, Department of Analytical Chemistry, Faculty of Pharmacy, Deraya University,
New Minia 61519, Egypt; Ebtihal_samir@yahoo.com
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Sohag University, Sohag 82524, Egypt;
mamdouhfawzy3@yahoo.com
Department of Chemistry & Physics, Augusta University, Augusta, GA 30912, USA
Department of Medical Microbiology and Parasitology, Faculty of Medicine, King Abdulaziz University,
Jeddah 21589, Saudi Arabia; hasfour@kau.edu.sa
6
7
8
Department of Pharmaceutics, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia;
nalhakamy@kau.edu.sa
*
Correspondence: tmabrahem@kau.edu.sa (T.S.I.); sipanda@augusta.edu (S.S.P.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Rachid Skouta and Mai Antonello
Received: 28 January 2020; Accepted: 6 July 2020; Published: 8 July 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Two sets of diphenyl ether derivatives incorporating five-membered 1,3,4-oxadiazoles, and
their open-chain aryl hydrazone analogs were synthesized in good yields. Most of the synthesized
compounds showed promising in vitro antimycobacterial activity against Mycobacterium tuberculosis
H37Rv. Three diphenyl ether derivatives, namely hydrazide
8g exhibited pronounced activity with minimum inhibitory concentrations (MICs) of 0.61, 0.86
and 0.99 g/mL, respectively compared to triclosan (10 g/mL) and isoniazid (INH) (0.2 g/mL).
Compounds , and 8g showed the InhA reductase enzyme inhibition with higher IC50 values
(3.28–4.23 M) in comparison to triclosan (1.10 M). Correlation between calculated physicochemical
3, oxadiazole 4 and naphthylarylidene
µ
µ
µ
3, 4
µ
µ
parameters and biological activity has been discussed which justifies a strong correlation with respect
to the inhibition of InhA reductase enzyme. Molecular modeling and drug-likeness studies showed
good agreement with the obtained biological evaluation. The structural and experimental information
concerning these three InhA inhibitors will likely contribute to the lead optimization of new antibiotics
for M. tuberculosis.
Keywords: diphenyl ether; H37Rv; antitubercular agents; InhA inhibitors; molecular docking
Molecules 2020, 25, 3125; doi:10.3390/molecules25143125
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3125
2 of 18
1. Introduction
Tuberculosis (TB), caused mainly by Mycobacterium tuberculosis (MTB), remains the world’s
deadliest infection, having recently surpassed HIV as the leading cause of infectious disease mortality.
The risk of developing tuberculosis (TB) is estimated as 16–27 times greater in people living with
HIV than among those without HIV infection [1]. About 4500 people lose their lives, and close to
30,000 people fall ill each day with this preventable and curable disease [
2]. The treatment plan for
MTB infection is strenuous, requires long-term treatment of combination chemotherapy of two or more
drugs, like pyrazinamide, isoniazid (INH), rifampicin, streptomycin and/or ethambutol (Figure 1).
This combination usually accompanied by poor patient compliance, which leads to emerging of
multi-drug resistant (MDR-TB) strains [3].
Figure 1. Commercially available anti-tubercular drugs.
Although one possible long-term solution to this issue is a better vaccine, in the near future,
the major reliance will be on chemotherapy requiring an urgent development of potential, non-toxic,
and effective antitubercular agents against latent MTB. In the search for novel TB lead candidates,
the enoyl-acyl carrier protein, InhA reductase has become a target of interest by pharmaceutical
companies and academic institutions for the development of new anti-tubercular drugs. InhA is the
key enzyme that catalyzes NADH-dependent reduction of 2-trans-enoyl-ACP (acyl carrier protein)
to give up NAD+ and reduced enoyl thioester-ACP substrate, which in turn, aids in the synthesis
of mycolic acid. Mycolic acid is a unique signature fatty acid and considered as a core constituent
of the mycobacterial cell wall [4]. InhA enzyme has been identified as the key target of the most
currently existing anti-TB drugs isoniazid (INH), and ethionamide. Most cases of resistance to these
drugs result from mutations in the mycobacterial activating enzyme (catalase-peroxidase KatG for
isoniazid and EthA for ethionamide) [5–13]. Also, triclosan (Figure 2), the non-ionic lipophilic anti-TB
agent, has been identified as a promising reversible inhibitor of InhA. This diphenyl ether molecule
doesn’t require any bioactivation and capable of directly inhibit InhA. This approach probably allows
avoiding the dominant cause of the resistance to isoniazid consisting of KatG mutations. SAR studies
of triclosan revealed that chlorine atoms in ring B are not significant in the activity while ring B
itself is essential moiety for inhibition of InhA (Figure 2); it makes several hydrophobic contacts
with adenoyl acyl carrier protein reductase enzyme. The ether oxygen bond is also crucial for the
activity due to two reasons. Initially, it is necessary for maximizing the interaction of triclosan with
InhA and consequently better biological activity [14]. Secondly, being of critical in the formation
of the stable ternary InhA-triclosan-NAD+ complex. Replacement of this group by a sulfur atom

Molecules 2020, 25, 3125
3 of 18
abolishes the inhibitory activity [13
,
15]. Different literature studies have been carried out based on the
above hypothesis. Yang and coworkers carried out several of Des-chloro-diphenyl ether derivatives
such as A and B (Figure 2), in the absence of the phenolic group, to enhance pharmacokinetic and
pharmacodynamic properties thereby improving the druggability of triclosan. These compounds
showed good antitubercular potency against MTB strain H37Rv (Figure 2) [16].
Figure 2. Structures of triclosan, diphenyl ether, and hydrazone derivatives.
On the other facet, oxadiazoles represent an important class of 5-membered heterocyclic systems
that cover a broad range of biological activities such as antimycobacterial [17], pesticide [18],
anti-inflammatory and analgesic [19], antimicrobial [20] and antitumor [21,22], In the search of
more effective anti-tuberculosis, oxadiazole is one of the most versatile scaffolds against MTB strain.
Interestingly, extensive chemical and structural investigations have been performed by Kini and his
group through developing series of oxadiazole o/m/p-substituted diphenyl ether derivatives (C–E)
which displayed inhibition against the growth of the H37Rv strain of mycobacterium (Figure 2) [
The hydrazide hydrazone skeleton is also a class of privileged structures with a broad range of biological
activities such as antioxidant [23], anti-inflammatory [24], and antitubercular activities [25 26]. Recently,
9].
,
in a high throughput screening for inhibitors of M. tuberculosis H37Rv, several aryl hydrazine derivatives
(F–H, Figure 2) [27] were found to possess potent activity in combination with the relatively low
toxicity [27]. However, despite the promising in vitro activity of the currently existing diphenyl ethers
have poor bioavailability [28] and limited aqueous solubility [28], which is likely one reason why they
have limited in vivo efficacy [10].
In continuation of our previous work in parallel to the above findings and the observed relationship
between lipophilicity and in vivo efficacy, especially as it pertains to antimicrobial agents [5–12],
new diphenyl ether analogs have been designed as potential antitubercular inhibitors of InhA reductase
enzyme. This approach developed by keeping the known essential functional groups for the activity.
Modification has been made to the diphenyl ether ‘B’ ring of diphenyl ether through binding the B
ring with five-membered oxadiazole moieties either substituted or non-substituted ones; this might
enhance bioactivity, pharmacokinetics or pharmacodynamics properties and could be a good lead
for the generation of InhA reductase inhibitors. Furthermore, the substitution of the para position of
diphenyl ether ‘B’ ring with aryl hydrazine or arylidene moiety as an open-chain analog of oxadiazole
may improve solubility and enhance the antimycobacterial activity. Prediction of correlation between
antitubercular activity with both Clog P and IC50 of InhA inhibition has been represented. Also,
the correlation of the anti-TB activity with different thermodynamic, topological parameters was also
considered. Molecular modeling studies were carried out to better understand the observed activity
and determine the most important structural parameters participating in binding with InhA reductase
that may affect the biological activity.

Molecules 2020, 25, 3125
4 of 18
2. Results and Discussion
2.1. Chemistry
The target derivatives were prepared through the conversion of the starting phenoxybenzoic
into the corresponding ester using methanol and concentrated sulphuric acid under reflux
conditions [28 30]. Hydrazinolysis of the ester with hydrazine hydrate gives the hydrazide
(Scheme 1). Cyclocondensation of hydrazide through its reaction with triethyl orthoformate under
refluxing conditions led to the formation of the oxadiazole (Scheme 2). The reaction of the hydrazide
with the appropriate aromatic carboxylic acid derivatives 5a in the presence of phosphorus
oxychloride afforded the corresponding 5-aryl-substituted oxadiazoles 6a . The condensation reaction
of hydrazide with variant aromatic aldehydes 7a resulted in the formation of anticipated arylidenes
acid
1
2
–
2
3
3
4
3
–l
–l
3
–k
8a–k (Scheme 2). The prepared target compounds were characterized by spectral studies.
Scheme 1. Reagents and conditions: (i) Conc.H2SO4, MeOH, reflux, 8 h; (ii) NH2NH2.H2O, EtOH,
reflux, 3 h.
O
HN NH2
O
O
O
Ar
OH
5a-l
O
i
O
ii
3
O
iii
Ar
H
N
O
N
O
N
N
7a-k
O
O
Ar
4
6a-l
HN
O
N
O
Ar
8a-k
e
a
b
c
f
d
OMe
OMe
Ar
Ar
OMe
O
NO2
Cl
OMe
g
j
h
l
i
k
O
O
S
N
N
Scheme 2. Reagents and conditions: (i) Triethyl orthoformate reflux, 3 h; (ii) POCl3, reflux, 2–3 h;
(iii) EtOH, reflux, 3–6 h.

Molecules 2020, 25, 3125
5 of 18
2.2. Biology
2.2.1. Anti-Tubercular Activity
Compounds
3,
4
,
6a–
l
and 8a
–
k
were evaluated for antitubercular activity against
Mycobacterium tuberculosis H37Rv using the standard Microplate Alamar Blue Assay (MABA) protocol
(Table 1) [31 32]. The MIC reported as the lowest concentration ( g/mL) of targets inhibited the growth
of an organism. Triclosan and INH were used as reference standards. Among the tested compounds,
the results displayed that most of them i.e., 6h 6i 6l 8g, and 8k showed promising anti-tubercular
activity (MIC, 0.61–1.69 g/mL) in comparison to triclosan (MIC, 10 g/mL) and INH (MIC, 0.2 g/mL).
,
µ
3,
4,
,
,
,
µ
µ
µ
Table 1. The antitubercular activity of the diphenyl ether derivatives against H37Rv strain of
M. tuberculosis (MIC (µg/mL) and their inhibition for InhA reductase enzyme (IC50 µM).
Ar
No.
3
MIC (µg/mL)
IC50 (µM)
No.
4
MIC (µg/mL)
IC50 (µM)
- - - - - - - - - -
0.86
3.43
0.61
3.28
6a
2.74
>100
8a
34.54
73.23
6b
34.64
>100
8b
50.20
>100
6c
39.04
15.15
>100
8c
20.68
7.67
75.46
19.75
6d
68.44
8d
6e
6f
15.89
8.23
64.55
41.24
8e
8f
2.89
7.46
22.62
29.89
6g
6h
12.23
1.40
33.82
5.08
8g
8h
0.98
6.96
4.23
26.54
6i
6j
1.40
11.12
3.65
11.72
51.42
78.65
8i
8j
6.82
6.35
1.69
21.39
18.37
5.61
6k
8k
6l
1.17
5.63
INH
0.20
ND
1.10
- - - - - - - - - -
- - - - - - - -
- - - - - - - - - -
- - - - - - - - - -
Triclosan
10.00
The structure-activity relationship acquired suggested that installing the open-chain oxadiazole
moiety into the diphenyl ether caused a remarkable increase in activity as seen in compound
(MIC, 0.86 g/mL). Interestingly, the cyclic and unsubstituted oxadiazole (MIC, 0.61 g/mL) showed
3
µ
4
µ
experienced and superior activity than all the 5-substituted analogs. Noteworthy, the 5-phenyl 6a
and 5-benzyloxy-phenyl 6f derivatives experienced a slight decrease in activity with MIC of 2.74,
8.23
ring with a six-membered heterocyclic system showed a slight improvement in the activity as depicted
in compounds 6h and 6i (MIC, 1.40 g/mL) while alteration of this ring into 5-membered one exhibited
less activity such as 6k (MIC, 3.65 g/mL). Expectedly, bis(diphenylether) oxadiazole 6l was the most
active in this 5-substituted series (1.17 g/mL) that may be due to the incorporation of extra phenyl
µg/mL, respectively. It was noted, however, that replacement of the phenyl group at the oxadiazole
µ
µ
µ

Molecules 2020, 25, 3125
6 of 18
ether nucleus to the backbone. The substitutions on the 5-aryl groups have a deleterious effect on
biological activity especially the activating methoxy substituents in compounds 6b and 6c.
In a related vein, the arylidene group of compounds 8a–k was found comparably less active in vitro
where this situation may modify in vivo, as we know arylidenes are prodrugs. By comparing these
derivatives, it is clear that the introduction of the heterocyclic system into the diphenyl ether hydrazone
structure either five, compound 8j (MIC, 6.35
8h (MIC ~ 6.90 g/mL) caused a decrease in the anti-tubercular activity. Similarly, replacement of
the heterocyclic ring with substituted phenyl group i.e., compounds 8d (MIC, 7.67 g/mL) and 8e
(MIC, 2.89 g/mL) showed weak inhibition. Surprisingly, a remarkable increase in the activity was
observed for compounds 8g (0.98 g/mL; Ar = Napthyl) and 8k (MIC, 1.69 g/mL; Ar = thienyl).
µg/mL) or six-membered ring system, compounds
–
i
µ
µ
µ
µ
µ
Generally, the order of activity against MTB strain H37Rv of diphenyl ether bearing arylidene core was
Ar = zero > naphthyl > thienyl > p-Cl-phenyl > 3 pyridinyl ~ 4-pyridinyl > p-NO2-phenyl. All these
data suggested that the diaryl ether scaffold is very important for the antitubercular activity, removal
of the o-chloro group in triclosan, as well as the introduction of oxadiazole or its open-chain analog
hydrazide, increased activity than the parent triclosan. In conclusion, compounds
and 8k showed higher antitubercular activity than the reference standard triclosan. Three potential lead
candidates and and 8g were found to be the most active in which hydrazide oxadiazole or naphthyl
3, 4, 6h, 6i, 6l, 8g,
3
4
arylidine moieties are directly attached to an unsubstituted diphenyl ether core. Their structural
features could be further investigated for their potential use as new anti-TB drugs.
2.2.2. In Vitro InhA Reductase Enzyme Inhibition
The in vitro inhibitions data of the target compounds for the InhA reductase enzyme are presented
as IC50 µM values in Table 1. Among the tested compounds, the highly active hydrazide
and naphthyl arylidine 8g showed the highest inhibition of InhA reductase with IC50 of 3.43
3.28 M and 4.23 M, respectively, but still less than the reference triclosan (IC50, 1.10 M). On the
other hand, oxadiazole derivatives 6a and the corresponding arylidenes 8a showed deleterious
activity as inhibitors for InhA reductase. The oxadiazoles 6h 61 and arylidine 8k ether derivatives
3
and oxadiazole
4
µM and
µ
µ
µ
–
c
–c
,
experienced good inhibition activities with IC50 of 5.08, 5.63, and 5.61, respectively. In general, most of
the tested target compounds showed good inhibition for InhA reductase. Besides, other compounds
show promising activity against H37Rv strain of M. tuberculosis. We believe, additional modes of
mechanisms involved in the activity.
Moreover, calculated results showed a slight correlation between the molecular weight, Wiener
index, and topological diameter to biological activity with R = 0.308, 0.315, 0.259, respectively.
These results indicate the importance of the overall volume of a molecule on the biological activity,
but it is not the only factor affecting biological activity. On the other hand, it was found that all the
thermodynamic parameters (i.e., molar refractivity and Gibbs free energy and heat formation) have
a negative correlation with the biological activity where R =
(Table 2).
−
0.100,
−
0.602 and 0.683, respectively
−
The molecules that display a negative sign for the heat of formation parameter means that
binding of these molecules with their protein targets may be spontaneous and do not require energy.
The protein-ligand binding occurs only when the change in Gibbs free energy (∆G: is the free
energy that gives an indication about a binding affinity to the target site) of the system is negative.
Furthermore, Hydrogen bonding is one of the important factors which is responsible for determining
the three-dimensional structure of folded proteins including enzymes and the ligand molecule. Also,
it is involved in determining the polarity and permeability of compounds. Calculations showed that
antimycobacterial activity expressed in MIC (µg/mL) is negatively correlated with both hydrogen
bond donor and hydrogen bond acceptor count. Calculation of the number of hydrogen bond donors,
hydrogen bond acceptors, and other parameters of Lipinski’s (rule of five) showed that most of the
target compounds presented drug-like characters [33–35].

Molecules 2020, 25, 3125
7 of 18
Table 2. Correlation between calculated physicochemical parameters and biological activity.
Molecular
Topology:
Wiener Index
Topological
Diameter
Bond (s)
Molar
Refractivity,
cm3/mol
Heat of
Formation
[kJ/mol]
Gibbs Energy
[kJ/mol]
Comp.
No.
MIC
(µg/mL)
Molecular
Mass
Balaban
Index
H Bond
Donor
H Bond
Acceptor
Lipinski’s
Rule
Clog p
Energy
3
4
0.86
0.61
2.74
34.64
39.04
15.15
15.89
8.23
12.23
1.40
1.40
11.12
3.65
1.17
34.54
50.20
20.68
7.67
2.89
22.62
1.40
6.96
6.82
6.35
1.69
-
228.250
238.250
314.340
404.422
344.370
359.340
348.770
420.470
364.404
315.332
315.332
304.305
320.062
406.440
384.390
474.470
414.420
414.420
418.840
490.519
434.450
385.380
385.383
374.356
390.417
0.308
118,297
121,561
400,250
110,779
59,322
586
682
11
11
15
17
17
17
16
21
17
15
15
14
14
20
19
21
21
21
20
25
20
19
19
18
18
0.260
6.589
6.656
9.167
11.018
9.784
9.778
2.231
2.753
4.806
4.427
4.679
ND
5.364
6.412
5.803
3.468
3.468
3.421
4.788
6.343
4.734
4.354
4.607
ND
5.292
6.339
5.730
3.396
3.396
3.349
4.715
0.152
−60.70
115.57
216.79
−276.20
52.46
176.14
339.72
493.02
174.39
386.81
ND
471.46
549.74
623.72
561.65
561.65
372.20
498.18
541.32
ND
ND
ND
ND
ND
ND
ND
ND
ND
2
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
3
4
4
7
5
5
4
5
4
5
5
5
4
5
6
9
7
7
6
7
6
7
7
7
6
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
No
No
Yes
Yes
Yes
-
6a
6b
6c
6d
6e
6f
6g
6h
6i
1561
2828
1990
2219
1763
3769
2391
1561
1561
1380
1380
3377
2849
4753
3497
3497
3158
6023
4015
2849
2849
2566
2566
0.315
71,066
ND
488,162
142,971
706,482
400,250
400,250
326,596
32,659
120,686
104,520
250,732
145,706
145,706
12,360
301,096
161,770
1,045,201
1,045,201
880,346
88,034
9.659
189.58
165.15
313.83
270.22
270.22
74.450
251.71
185.97
145.52
−347.47
−18.810
−18.81
118.31
93.88
242.56
198.95
198.95
3.180
180.44
−0.683
12.295
10.855
8.956
8.956
8.381
6j
6k
6l
8.976
11.831
10.994
12.845
11.611
11.610
11.485
14.122
12.682
10783
10.783
10.208
10.803
−0.100
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
R
ND
ND
−0.602
−0.179
−0.370
−0.360

Molecules 2020, 25, 3125
8 of 18
2.2.3. Cytotoxic Study
Compounds
3
,
4,
6h
,
6i,
6l
,
8g, and 8k displaying the lowest MIC values were selected for
cytotoxicity test using model eukaryotic cell line (Vero) at the concentration range from 0.1 to 200
µg/mL
with IC50 values more than 60
(Table 3).
µg/mL. Compounds did not show a high cytotoxic effect on Vero cells
Table 3. MIC, IC50, and SI values determined for the selected most promising compounds.
M. tuberculosis
Vero Cell Line
Compound
SI (IC50/MIC)
MIC, µg/mL
IC50 µg/mL ± SEM
3
4
0.86
0.61
1.40
1.40
1.17
0.98
1.69
80.7 ± 1.31
99.4 ± 1.79
91.4 ± 1.55
95.5 ± 1.36
92.6 ± 1.80
72.2 ± 1.87
68.6 ± 1.19
93.83
162.95
65.29
68.21
79.15
73.67
40.59
6h
6i
6l
8g
6k
Notably, compound
cytotoxicity to Vero cells at all tested concentrations (0.1–200
(99.4) and accordingly its selectivity index (SI) value more than 10 (162.95), providing its promising
potential in biomedical application. In the case of compounds 6h 6i, and 6l, a slight reduction in
cell viability was observed with IC50 ranging from 80.7–95.5 g/mL, whereas 8g and 8k showed the
more pronounced cytotoxic effect was with IC50 72.3 and 68.6 g/mL, respectively. Therefore, it may
be assumed that 6h 6i 6l 8g, and 8k derivatives, which were non-toxic or showed very low toxicity
4
which showed the lowest MIC value of 0.61
µg/mL also displayed very low
µg/mL) with IC50 values close to 100 µg/mL
3,
,
µ
µ
4
,
,
,
,
at tested concentration range, are very promising and potentially safe antimycobacterial compounds.
2.2.4. Molecular Docking
The inhibitory profile of triclosan and compounds 3, 8g (models for the higher inhibitory
activity) and 8b (model for the deleterious inhibitory activity) were chosen for further investigation by
docking into InhA reductase active site (PDB code: 3FNH) to gain insight into the potential binding
modes [36–38]. As a first step, for the validation of docking parameters, the co-crystal ligand (JPJ400)
was re-docked at the catalytic site of the protein. The RMSD between co-crystal and re-docked poses was
found to be 0.331 (<2A), which confirmed the validity of the docking parameters. Triclosan exploited
two major interactions. Firstly, the hydrophilic interaction has been noticed by the hydroxyl OH via two
hydrogen bonds i.e., the oxygen engaged with the hydrophilic side chainTyr158 while the hydrogen
forms the second hydrogen bond with NAD. Secondly, clusters of hydrophobic interactions including
pi—or thirteen—alkyl interactions with NAD, Phe97, Met161, Pro193, Ala198, Met199, and Ile202 were
also found. Further, pi-cation interaction with NAD, pi-sulfur interaction with Met103 and also, pi-pi
stacked interaction with Phe149 have been noticed as well (Figure 3).
By careful analysis of the docked poses of compounds
can occupy the same binding mode as Triclosan with binding interaction energy (
(for compound 3) to 17.148 kcal/mol (for compound 8g) compared to Triclosan (
The hydrophilic interaction has been noticed in compound via the carbonyl oxygen that forms a
3
and 8g, it was clear that both compounds
24.264) kcal/mol
22.507) kcal/mol.
−
−
−
3
hydrogen bond with Tyr158 side chain, while the hydrogen of amino group forms the second one
with NAD, but compound 8g was involved in only one i.e., N4 of the oxadiazole ring forms one
hydrogen bond with the hydrophilic residue Tyr 158, likely due to its bulkiness and this may explain
its low potency than compound 3 (Figures 4 and 5). The hydrophobic interactions in compound 3 were
incorporating three pi-alkyl interactions with Pro193, Met199 and Leu218. Moreover, two pi-sulfur
interactions with Met155 and Met199, one attractive charge interaction with NAD and two pi-pi
T-shaped interactions with Phe149 and Tyr158 have been observed. In the same mode, compound 8g

Molecules 2020, 25, 3125
9 of 18
displays its hydrophobic interactions with different amino acid residues that embodying five pi-pi
stacked interactions with NAD, Phe97, and Phe149. Also, eight pi-alkyl interactions with Phe149,
Met161, Ala198, Ala201, Ile215, and Leu218 were found.
A
B
Figure 3. Docking and binding mode of Triclosan into the active site of InhA reductase enzyme
(PDB code: 3FNH). (A) 3D structure of triclosan (pink), (B) 2D structure of Triclosan (pink).
A
B
Figure 4. Docking and binding mode of compound
3
into the active site of InhA reductase enzyme
(PDB code: 3FNH). (A) 3D structure of compound 3 (green), (B) 2D structure of compound 3 (green).
A
B
Figure 5. Docking and binding mode of compound 8g into the active site of InhA reductase enzyme
(PDB code: 3FNH). (A) 3D structure of compound 8g (blue), (B) 2D structure of compound 8g (blue).
It is worth pointing out that in compound 8b, the hydrogen of the hydrazide group showed
unfavorable donor-donor interaction (red color) with binding interaction energy (+11.145) kcal/mol.

Molecules 2020, 25, 3125
10 of 18
Also, the compound has not involved in any hydrogen bonds and forms only hydrophobic interaction
with some amino acid residues and this may reflect the low activity of the compound (Figure 6).
A
B
Figure 6. Docking and binding mode of compound 8b into the active site of the InhA reductase enzyme
(PDB code: 3FNH). ( ) 3D structure of compound 8b (yellow), ( ) 2D structure of compound 8b (yellow).
A
B
3. Material and Methods
3.1. Chemistry
Melting points were recorded on a Stuart SMP30 (Stuart, Cole-Parmer, Staffordshire, UK) melting
point apparatus. IR spectra (KBr, cm−1) were recorded on a Vector 22FT-IR Fourier Transform infrared
(FTIR) spectrometer (Bruker, Billerica, MA, USA). 1H-NMR and 13C-NMR spectra were recorded at
500 and 125 MHz, respectively, on an Inova 500 MHz spectrometer (Bruker, Billerica, MA, USA), using
dimethyl sulphoxide (DMSO-d6)/deuterated chloroform (CDCl3) as a solvent and tetramethylsilane
(TMS) as an internal standard (chemical shift in δ, ppm). Mass spectra were determined using a
Shimadzu GC/MS QP 1000 EX system (Shimadzu Corporation, Tokyo, Japan) with ionization energy
70 eV. Elemental analysis were determined using an Automatic Elemental Analyzer CHN Model 2400
(Perkin Elmer, Richmond, CA, USA) at the Micro-analytical Center, Faculty of Science, Cairo University
(Cairo, Egypt). All the results of elemental analyses corresponded to the calculated values within
experimental error. Progress of the reaction was monitored by thin-layer chromatography (TLC) using
precoated TLC sheets with ultraviolet (UV) fluorescent silica gel (60F254, Merck, Kenilworth, NJ, USA)
and spots were visualized by iodine vapors or irradiation with UV light (254 nm). All the chemicals
were purchased from Sigma-Aldrich Chemicals, St. Louis, MO, USA. Compounds 2, 3, 4, 6g, 6i–k and
8i–k were prepared according to the reported procedures [30,39,40].
Methyl 4-phenoxybenzoate (2). Concentrated H2SO4 (1.0 mL) was added dropwise to a solution of
4-phenoxybenzoic acid (3.3 g, 15.6 mmol) in methanol (25 mL). The reaction mixture was allowed to
reflux for 8 h. The reaction mixture was concentrated under reduced pressure. The obtained residue
was treated with 10% aqueous NaHCO3 (10 mL). Stirring was continued for 15 m. During which time,
the precipitate was formed then collected by filtration then dried and purified by crystallization using
methanol to afford white solid in 80% yield; m.p. 159–160 ◦C; 1H-NMR (CDCl3)
6.99 (d, J = 8.8 Hz, 2H, ArH), 7.07 (d, J = 7.8 Hz, 2H, ArH), 7.19 (t, J = 7.9 Hz, 1H, ArH), 7.39 (t, 2H,
J = 7.9 Hz, ArH), 8.00 (d, J = 8.4 Hz, 2H, ArH); 13C-NMR (CDCl3)
: 52.1 (OCH3), 117.4(C3, C5), 120.2
(C30, C50), 124.6 (C1), 130.2 (C20, C60), 131.8 (C2, C6), 155.8 (C4), 162.0 (C10), 166.7 (C40), 207.1 (C=O).
δ: 3.90 (s, 3H, OCH3),
δ
4-Phenoxybenzohydrazide ( ). Methyl 4-phenoxybenzoate (228 mg, 1 mmol) was added to a solution of
3
hydrazine hydrate 98% (0.5 mL) in ethanol (5 mL). The mixture was refluxed for 2 h. The reaction
mixture was evaporated under vacuum, water (10 mL) was added to the obtained residue. The obtained
mixture was extracted with ethyl acetate (2
with a saturated solution of sodium bicarbonate and (1
×
10 mL). The combined organic layers were then washed
10 mL) then brine (1 10 mL) before being
×
×

Molecules 2020, 25, 3125
11 of 18
dried (MgSO4). The obtained filtrate was concentrated under reduced pressure, filtered off and the
precipitate was subjected to crystallization using EtOAc/petroleum ether. White solid, Yield: 84%; m.p.:
180–190 ◦C; 1H-NMR (CDCl3)
δ
: 4.49 (s, 2H, NH2), 7.00 (d, J = 8.0 Hz, 2H, ArH), 7.05 (d, J = 8.0 Hz, 2H,
ArH), 7.18 (t, J = 7.5 Hz, 1H, ArH), 7.36–7.40 (m, 2H, ArH), 7.41 (s, 1H, NH), 7.72 (d, J = 8.2 Hz, 2H,
ArH). 13C-NMR (CDCl3) : 118.0 (C3, C5), 120.1 (C30, C50), 124.6 (C1), 127.1 (C10), 128.9 (C20, C60),
130.2 (C2, C6), 155.9 (C4), 161.1 (C40), 168.3 (C=O).
δ
2-(4-Phenoxyphenyl)-1,3,4-oxadiazole ( ). A mixture of hydrazide 3 (1 mmol) and triethyl orthoformate
4
(2 mL) were refluxed for 3 h. The reaction mixture was concentrated under vacuum; the residue was
cooled and then treated with hexane (5 mL). The obtained precipitate◦was filtered off and recrystallized
from ethanol/water (1:1). White solid, Yield: 73%; m.p. 140–144 C [26]. 1H-NMR (DMSO-d6)
δ:
7.14–7.17 (m, 4H, ArH), 7.25 (t, J = 7.5 Hz, 1H ArH), 7.45–7.49 (m, 2H, ArH), 8.01–8.04 (m, 2H, ArH),
9.31 (s, 1H, CH-oxadiazole). 13C-NMR (DMSO-d6) : 117.8 (C3, C5), 118.3 (C30, C50), 119.9 (C1), 124.7
δ
(C10), 128.9 (C2, C6), 130.3 (C20, C60), 154.2 (C40), 155.1 (C4), 160.2 (C=N), 163.3 (C=N). Anal. Calcd.
for C14H10N2O2: C, 70.58; H, 4.23; N, 11.76. Found: C, 70.50; H, 4.48; N 11.37.
2-(4-Phenoxyphenyl)-5-phenyl-1,3,4-oxadiazole (6a). White solid, Yield; 70%; m.p. 233–235 ◦C. 1H-NMR
(CDCl3)
δ
: 7.01–7.07 (m, 5H, ArH), 7.18–7.22 (m, 1H, ArH), 7.38–7.56 (m, 5H, ArH), 7.85–7.88 (m, 3H,
ArH). 13C-NMR (CDCl3)
δ
: 118.0 (C3, C5), 119.1 (C30, C50), 120.3 (C3”), 122.9 (C5”), 124.8 (C1), 125.7
(C10, C1”), 127.5 (C2, C6), 129.1 (C20, C60), 129.5 (C2”), 130.3 (C6”), 131.6 (C4), 132.7 (C40), 161.7, (C4”),
164.3 (C=N), 164.5 (C=N). Anal. Calcd for C20H14N2O2: C, 76.42; H, 4.49; N, 8.91. Found: C, 76.54; H,
4.23; N 9.27.
2-(4-Phenoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazole (6b). White solid, Yield; 76%, m.p.
250–253 ◦C. 1H-NMR (CDCl3)
δ
: 3.92 (s, 3H, OMe), 3.97 (s, 6H, 2OMe), 7.05 (d, J = 8.0 Hz, 2H, ArH),
7.18–7.24 (m, 3H, ArH), 7.34 (s, 2H, ArH), 7.64–7.70 (m, 2H, ArH), 7.98 (d, J = 8.2 Hz, 2H, ArH).
13C-NMR (CDCl3) : 56.7 (OMe), 56.8 (OMe), 61.3 (OMe), 104.4 (C3, C5), 104.7 (C30, C50), 118.5 (C3”,
δ
C5”), 119.2 (C1, C10), 120.2 (C1”), 124.8 (C2, C6), 129.1 (C20, C60), 130.3 (C2”, C6”), 141.4 (C4), 153.9
(C40), 155.9 (C4”), 161.0 (C=N), 162.4 (C=N). Anal. Calcd for C23H20N2O5: C, 68.31; H, 4.98; N 6.93.
Found: C, 67.92; H, 5.02; N 7.05.
2-(4-Methoxyphenyl)-5-(4-phenoxyphenyl)-1,3,4-oxadiazole (6c). White solid, Yield; 72%, m.p.: 226–228 ◦C.
1H-NMR (CDCl3)
δ
: 3.87 (s, 3H, OMe), 7.00–7.02 (m, 2H, ArH), 7.10–7.20 (m, 4H, ArH), 7.17–7.20 (m,
1H, ArH), 7.37–7.40 (m, 2H, ArH), 8.03–8.07 (m, 4H, ArH). 13C-NMR (CDCl3,)
δ: 55.7 (OMe), 114.8 (C3,
C5), 116.7 (C30, C50), 118.5 (C3”, C5”), 118.7 (C1), 120.2 (C10), 124.7 (C2, C6), 128.89 (C20, C60), 128.93
(C2”), 130.27 (C6”), 156.0 (C4), 160.9 (C40), 162.6 (C4”), 1624.0 (C=N), 166.6 (C=N). Anal. Calcd for
C21H16N2O3: C, 73.24; H, 4.68; N 8.13. Found: C, 73.62; H, 4.93; N 8.06.
2-(4-Nitrophenyl)-5-(4-phenoxyphenyl)-1,3,4-oxadiazole (6d). White solid, Yield; 77%, m.p.: 226–228◦C.
1H-NMR (CDCl3)
δ
: 7.09–7.13 (m, 4H, ArH), 7.19–7.24 (m, 1H, ArH), 7.42 (dd, J = 15.8, 8.0 Hz, 2H,
ArH), 7.42 (dd, J = 16.0, 8.0 Hz, 2H, ArH), 8.28–8.45 (m, 4H, ArH). 13C-NMR (CDCl3)
δ: 118.5 (C3),
120.2 (C5), 120.4 (C30), 124.7 (C50), 124.8 (C3”), 125.0 (C5”), 128.0 (C1), 129.0 (C10), 129.3 (C1”), 129.7
(C2), 130.3 (C6), 130.4 (C20, C60), 149.7 (C2”, C6”) 155.6 (C4), 155.9 (C40), 161.0 (C4”), 161.6 (C=N), 162.8
(C=N). Anal. Calcd for C20H13N3O4: C, 66.85; H, 3.65; N, 11.69. Found: C, 66.73; H, 3.39; N 11.92.
2-(4-Chlorophenyl)-5-(4-phenoxyphenyl)-1,3,4-oxadiazole (6e). White solid, Yield; 72%, m.p.: 212–214 ◦C.
1H-NMR (CDCl3)
δ
: 7.09–7.11 (m, 4H, ArH), 7.20–7.21 (m, 1H, ArH), 7.40–7.43 (m, 2H, ArH), 7.51–7.53
(m, 3H, ArH), 8.06–8.09 (m, 4H, ArH). 13C-NMR (CDCl3)
δ
: 118.5 (C3), 120.2 (C5), 120.3 (C30), 122.4
(C50), 122.7 (C3”), 124.7 (C5”) 124.8 (C1), 128.4 (C10), 128.5 (C1”), 129.0 (C2), 129.1 (C6), 129.7 (C20),
129.8 (C60), 130.28 (C2”), 130.30 (C6”), 138.2 (C4), 138.4 (C40), 155.8 (C4”), 161.2 (C=N), 164.2 (C=N).
Anal. Calcd for C20H13N3O4: C, 68.87; H, 3.76; N, 8.03. Found: C, 68.63; H, 3.40; N 8.23.
2-(4-(Benzyloxy)phenyl)-5-(4-phenoxyphenyl)-1,3,4-oxadiazole (6f). White solid, Yield; 80%, m.p.: 226–229
◦C. 1H-NMR (DMSO-d6)
δ: 5.14 (s, 2H, OCH2-), 7.03 (d, J = 8.3 Hz, 2H, ArH), 7.06 (d, J = 8.1 Hz, 2H,

Molecules 2020, 25, 3125
12 of 18
ArH), 7.12 (t, J = 11.5 Hz, 2H, ArH), 7.34–7.45 (m, 8H, ArH), 7.99 (d, J = 8.6 Hz, 2H, ArH), 8.10 (d,
J = 9.1 Hz, 2H, ArH). 13C-NMR (DMSO-d6) : 70.9 (OCH2-), 114.6 (C3, C5) 114.8 (C30, C50), 115.7 (C3”),
δ
118.0 (C5”), 118.6 (C3000, C5000), 121.3 (C1, C10), 127.4 (C1”, C1000), 128.2 (C2, C6), 128.8 (C20), 129.1 (C60),
131.2 (C2”, C6”), 136.7 (C2000, C6000), 153.5 (C4, C40), 155.2 (C4”), 157.3 (C4000), 165.7 (C=N), 166.4 (C=N).
Anal. Calcd for C27H20N2O3: C, 77.13; H, 4.79; N, 6.66. Found: C, 77.53; H, 4.60; N 6.24.
2-(Naphthalen-1-yl)-5-(4-phenoxyphenyl)-1,3,4-oxadiazole (6g). White solid, Yield; 70%, m.p.: 173–175 ◦C.
1H-NMR (CDCl3)
δ: 7.11–7.15 (m, 2H, ArH), 7.20–7.23 (m, 1H, ArH), 7.40–7.44 (m, 1H, ArH), 7.59–7.64
(m, 3H, ArH), 7.69–7.75, (m, 1H, ArH), 7.49–8.09 (m, 3H, ArH), 8.16 (d, J = 8.0 Hz, 1H, ArH), 8.26 (d,
J = 7 Hz, 1H, ArH), 8.35 (d, J = 7 Hz, 1H, ArH), 9.28 (d, J = 8.5 Hz, 1H, ArH), 9.38 (d, J = 8.5 Hz, 1H,
ArH). 13C-NMR (CDCl3)
δ: 118.6, 120.2, 120.8, 124.8, 125.1, 126.5, 127.0, 128.45, 128.50, 128.8, 128.9,
129.2, 130.3, 132.7, 132.9, 134.1, 134.2, 155.9, 161.1 (Ar-C), 164.1 (C=N), 164.6 (C=N). Anal. Calcd for
C24H16N2O2: C, 79.11; H, 4.43; N, 7.69. Found: C, 78.93; H, 4.40; N 8.01.
2-(4-Phenoxyphenyl)-5-(pyridin-3-yl)-1,3,4-oxadiazole (6h). White solid, Yield; 70%, m.p.: 143–144 ◦C.
1H-NMR (DMSO-d6)
δ
: 7.03 (d, J = 7.5 Hz, 2H, ArH), 7.06 (d, J = 8.2 Hz, 2H, ArH), 7.19–7.22 (m, 3H,
ArH), 7.39 (t, J = 9.0 Hz, 2H, ArH), 7.98 (d, J = 7.5 Hz, 2H, ArH), 8.83 (d, J = 8.0 Hz, 2H, ArH); 13C-NMR
(DMSO-d6) δ
: 114.9 (C3, C5), 115.1 (C30, C50), 118.3 (C5”-pyridyl), 120.5 (C1, C10), 121.6 (C1”-pyridyl),
123.5 (C2, C6), 127.3 (C20, C60), 129.4 (C6”-pyridyl), 131.2 (C4), 132.45 (C40-pyridyl), 151.07 (C2=N
pyridyl, C4=N-pyridyl), 162.92 (C=N), 165.62 (C=N). Anal. Calcd for C19H13N3O2: C, 72.37; H, 4.16;
N, 13.33. Found: C, 72.66; H, 4.39; N 13.65.
◦
2-(4-Phenoxyphenyl)-5-(pyridin-4-yl)-1,3,4-oxadiazole (6i). White solid, Yield; 70%, m.p.: 125–127 C.
1H-NMR (CDCl3)
δ
: 7.05–7.16 (m, 6H, ArH), 7.22–7.26 (m, 1H, ArH), 7.41–7.45 (m, 3H, ArH), 8.08–8.15
(m, 3H, ArH). 13C-NMR (CDCl3)
δ
: 117.5 (C3), 117.6 (C5), 117.9 (C30), 118.2 (C50), 118.5 (C2, C6-pyridyl),
120.4 (C1), 120.5 (C10), 120.7 (C1”-pyridyl), 124.9 (C2), 129.5 (C6), 130.3 (C20), 130.4 (C60), 131.1 (C4),
132.6 (C40), 133.1 (C3=N pyridyl), 134.4 (C5=N pyridyl), 162.7 (2C=N). Anal. Calcd for C19H13N3O2:
C, 72.37; H, 4.16; N, 13.33. Found: C, 72.54; H, 4.40; N 13.43.
◦
2-(Furan-2-yl)-5-(4-phenoxyphenyl)-1,3,4-oxadiazole (6j). White solid, Yield; 77%, m.p.: 129–132 C.
1H-NMR (CDCl3)
δ
: 6.65 (s, 1H, ArH), 7.11–7.13 (m, 4H, ArH), 7.23–7.25 (m, 2H, ArH), 7.44 (t, J = 8.01,
: 112.4 (C3), 114.2 (C5),
118.1 (C30), 118.5 (C50), 120.2 (C1), 120.3 (C10), 124.8 (C2-furfuryl), 129.0 (C5-furfuryl), 129.2 (C3, C4
furfuryl), 130.3 (C2, C6), 139.8 (C20), 145.9 (C60), 155.8 (C4), 157.5 (C40), 161.2 (C=N), 163.9 (C=N). Anal.
Calcd for C18H12N2O3: C, 71.05; H, 4.67; N, 9.21. Found: C, 71.36; H, 4.39; N 9.55.
2H, ArH), 7.70 (s, 1H, ArH), 8.10 (d, J = 8.0 Hz, 2H, ArH). 13C-NMR (CDCl3)
δ
2-(4-Phenoxyphenyl)-5-(thiophen-2-yl)-1,3,4-oxadiazole (6k). White solid, Yield; 75%, m.p.: 139–141 ◦C.
1H-NMR (CDCl3)
δ
: 6.80–6.93 (m, 4H, ArH), 7.09–7.11 (m, 2H, ArH), 7.19–7.22 (m, 1H, ArH), 7.47–7.35
(m, 1H, ArH), 8.07–8.09 (m, 1H, ArH), 7.81–7.83 (m, 1H, ArH). 13C-NMR (CDCl3)
δ: 118.2 (C3), 118.4
(C5), 120.2 (C30), 124.7 (C50), 125.4 (C1), 128.3 (C10), 128.9 (C2, C5-thienyl), 129.0 (C3, C4-thienyl), 129.8
(C2), 130.0 (C6), 130.19 (C20), 130.21 (C60), 130.4 (C4), 155.8 (C40), 161.0 (C=N), 163.9 (C=N). Anal.
Calcd for C18H12N2O2S: C, 67.48; H, 3.78; N, 8.74. Found: C, 67.35; H, 3.36; N 9.02.
◦
1
2,5-bis(4-Phenoxyphenyl)-1,3,4-oxadiazole (6l). White solid, Yield; 85%, m.p.: 232–234 C. H-NMR
(CDCl3) : 6.95–7.12 (m, 9H, ArH), 7.17–7.24 (m, 2H, ArH), 7.32–7.40 (m, 4H, ArH), 8.04–8.406 (m, 3H,
ArH). 13C-NMR (CDCl3)
: 118.1, 118.5, 118.6, 118.9, 119.3, 119.5, 120.2, 123.9, 124.7, 129.0, 129.5, 129.8,
δ
δ
130.0, 130.3, 155.9 (Ar-C) 160.9 (C=N), 164.2 (C=N). Anal. Calcd for C26H18N2O3: C, 76.83; H, 4.46; N,
6.89. Found: C, 77.01; H, 4.60; N 6.74.
(E)-N0-Benzylidene-4-phenoxybenzohydrazide (8a). White solid, Yield; 89%, m.p.: 163–165 ◦C. 1H-NMR
(DMSO-d6)
2H, ArH), 7.96 (d, J = 8.2 Hz, 2H, ArH), 8.46 (s, 1H, CH=N), 11.82 (s, 1H, NH). 13C-NMR (DMSO-d6)
: 117.5 (C3, C5), 119.6 (C30, C50), 124.4 (C3”, C5”), 127.1 (C1, C10), 127.9 (C1”), 128.8 (C2, C6), 128.9
δ: 7.08–7.12 (m, 4H, ArH), 7.12–7.24 (m, 1H, ArH), 7.44–7.47 (m, 5H, ArH), 7.73 (d, J = 6.9
δ

Molecules 2020, 25, 3125
13 of 18
(C20, C60), 130.0 (C2”), 130.3 (C6”), 134.4 (C4), 147.5 (C40), 155.5 (C4”), 159.9 (C=O), 162.4 (C=N). Anal.
Calcd for C20H16N2O2: C, 75.93; H, 5.10; N, 8.86. Found: C, 75.54; H, 5.32; N 9.11.
(E)-4-Phenoxy-N0-(3,4,5-trimethoxybenzylidene)benzohydrazide (8b). White solid, Yield; 92%, m.p.: 191–192
◦C. 1H-NMR (DMSO-d6)
δ: 3.71 (s, 3H, OMe), 3.84 (s, 6H, 2OMe), 7.03 (s, 2H, ArH), 7.10 (d, J = 7.9 Hz,
4H, ArH), 7.21–7.24 (m, 1H, ArH), 7.45 (t, J = 7.9 Hz, 2H, ArH), 7.95 (d, J = 8.0 Hz, 2H, ArH), 8.39 (s,
1H, CH=N), 11.80 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ: 55.9 (OMe), 602.1 (2OMe), 104.3 (C3, C5), 117.5
(C30, C50), 119.6 (C3”, C5”), 124.4 (C1, C10), 128.0 (C1”), 129.9 (C2, C6), 130.3 (C20, C60), 139.2 (C2”,
C6”), 147.6 (C4), 153.2 (C40), 155.5 (C4”), 159.9 (C=O), 162.4 (C=N). Anal. Calcd for C23H22N2O5: C,
67.97; H, 5.46; N, 6.89. Found: C, 67.92; H, 5.32; N 7.02.
(E)-N0-(4-Methoxybenzylidene)-4-phenoxybenzohydrazide (8c). White solid, Yield; 92%, m.p.: 233–235 ◦C.
1H-NMR (DMSO-d6)
δ: 3.81 (s, 3H, OMe), 7.02 (d, J = 8.4, 2H, ArH), 7.08–7.12 (m, 4H, ArH), 7.22 (t,
J = 7.5 Hz, 1H, ArH), 7.45 (t, J = 8.0 Hz, 2H, ArH), 7.67 (d, J = 8.0 Hz, 2H, ArH), 7.95 (d, J = 8.2 Hz, 2H,
ArH), 8.40 (s, 1H, CH=N), 11.68 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ: 55.3 (OMe), 114.3 (C3, C5), 117.5
(C30, C50), 119.6 (C3”, C5”), 124.4 (C1, C10), 126.9 (C1”), 128.1 (C2, C6), 128.6 (C20, C60), 129.8 (C2”),
130.3 (C6”), 147.4 (C4), 155.5 (C40), 159.8 (C4”), 160.8 (C=O), 162.2 (C=N). Anal. Calcd for C21H18N2O3:
C, 72.82; H, 5.24; N, 8.09. Found: C, 72.72; H, 5.53; N 8.06.
(E)-N0-(4-Chlorobenzylidene)-4-phenoxybenzohydrazide (8d). White solid, Yield; 92%, m.p.: 184–186 ◦C.
1H-NMR (DMSO-d6)
δ: 7.06 (d, J = 7 Hz, 2H, ArH), 7.09 (d, J = 7.5 Hz, 2H, ArH), 7.22 (t, J = 11.5 Hz,
2H, ArH), 7.44 (t, J = 12.0 Hz, 3H, ArH), 7.99 (d, J = 8.4 Hz, 2H, ArH), 8.10 (d, J = 9.2 Hz, 2H, ArH), 8.77
(s, 1H, CH=N),00101.860(00s, 1H, NH). 13C-NMR (DMSO-d6)
δ
: 117.5 (C3, C5), 119.6 (C30, C50), 124.4 (C3”,
C5”), 127.8 (C3 , C5 ), 128.7 (C1, C10), 128.9 (C1”, C1000), 129.9 (C2, C6), 130.3 (C20, C60), 133.3 (C2”,
C6”), 134.4 (C2000, C6000), 146.2 (C4, C40), 155.4 (C4”, C4000), 160.0 (C=O), 162.5 (C=N). Anal. Calcd for
C20H15ClN2O2: C, 68.48; H, 4.31; N, 7.99. Found: C, 68.37; H, 4.40; N 8.33.
(E)-N0-(4-Chlorobenzylidene)-4-phenoxybenzohydrazide (8e). White solid, Yield; 92%, m.p.: 184–186 ◦C.
1H-NMR (DMSO-d6)
δ: 7.09–7.12 (m, 4H, ArH), 7.23 (t, J = 7.5 Hz, 2H, ArH), 7.45 (t, J = 7.5 Hz, 2H,
ArH), 7.97 (d, J = 8.0 Hz, 4H, ArH), 8.29 (d, J = 8.4 Hz, 2H, ArH), 8.54 (s, 1H, CH=N), 12.10 (s, 1H,
NH).13C-NMR (DMSO-d6) : 117.4 (C3, C5), 119.7 (C30, C50), 124.1 (C3”, C5”), 124.5 (C3000, C5000), 127.5
δ
(C1, C10), 127.9 (C1”, C1000), 130.1 (C2, C6), 130.3 (C20, C60), 140.7 (C2”, C6”), 145.0 (C2000, C6000), 147.8
(C4, C40), 148.8 (C4”), 155.4 (C4000), 160.2 (C=O), 162.6 (C=N). Anal. Calcd for C20H15ClN2O2: C, 68.48;
H, 4.31; N, 7.99. Found: C, 68.37; H, 4.40; N 8.33.
(E)-N0-[4-(Benzyloxy)benzylidene]-4-phenoxybenzohydrazide (8f). White solid, Yield; 93%, m.p.: 228–231
◦C. 1H-NMR (DMSO-d6)
δ: 5.17 (s, 2H, ArH), 7.08–7.12 (m, 6H, ArH), 7.21–7.24 (m, 1H, ArH), 7.33–7.48
(m, 7H, ArH), 7.67 (d, J = 8.7 Hz, 2H, ArH), 7.94 (d, J = 8.6 Hz, 2H, ArH), 8.39 (s, 1H, CH=N), 11.68 (s,
1H, NH). 13C-NMR (DMSO-d6) : 69. 4 (OCH2), 115.2 (C3), 117.5 (C5), 119.6 (C30, C50), 124.4 (C3”),
δ
127.1 (C5”), 127.8 (C1), 127.9 (C10), 128.1 (C1”), 128.5 (C2), 128.6 (C6), 129.8 (C20, C60), 130.3 (C2”, C6”),
136.8 (C4), 147.4 (C40), 155.5 (C4”), 159.9 (C=O), 162.2 (C=N). Anal. Calcd for C27H22N2O3: C, 76.76; H,
5.25; N, 6.63. Found: C, 76.56; H, 5.60; N 6.34.
(E)-N0-(Naphthalen-1-ylmethylene)-4-phenoxybenzohydrazide (8g). White solid, Yield; 88%, m.p.: 193–195
◦C. 1H-NMR (DMSO-d6)
δ
: 7.12–7.14 (m, 4H, ArH), 7.24 (t, J = 7.9 Hz, 2H, ArH), 7.45–7.48 (m, 2H,
ArH), 7.60–7.62, (m, 2H, ArH), 7.63–7.69, (m, 1H, ArH), 7.93–7.95 (m, 1H, ArH), 8.01–8.04 (m, 4H, ArH),
8.87–8.89 (m, 1H, ArH), 9.11 (s, 1H, CH=N), 11.90 (s, 1H, NH). 13C-NMR (DMSO-d6)
: 117.5, 119.6,
δ
124.2, 124.4, 125.6, 126.3, 127.3, 127.7, 127.9, 128.8, 129.6, 129.9, 130.2, 130.3, 130.5, 133.5, 147.4, 155.5
(Ar-C), 160.0 (C=O), 162.3 (C=N). Anal. Calcd for C24H18N2O2: C, 78.67; H, 4.95; N, 7.65. Found: C,
78.92; H, 4.60; N 8.04.
(E)-4-Phenoxy-N0-(pyridin-3-ylmethylene)benzohydrazide (8h). White solid, Yield; 90%, m.p.: 149–151 ◦C.
1H-NMR (DMSO-d6)
δ: 7.09–7.14 (m, 4H, ArH), 7.22–7.25 (m, 1H, ArH), 7.44–7.50 (m, 3H, ArH), 7.97
(d, J = 8.4 Hz, 2H, ArH), 8.14 (d, J = 7.0 Hz, 1H, ArH), 8.51 (s, 1H, CH=N), 8.62 (d, J = 8.3 Hz, 1H, ArH),

Molecules 2020, 25, 3125
14 of 18
8.86 (d, J = 8.3 Hz, 1H, ArH), 11.98 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ
: 117.5 (C3, C5), 119.6 (C30, C50),
124.0 (C5-pyridyl), 124.5 (C1), 127.7 (C10), 130.0 (C1-pyridyl), 130.3 (C2, C6), 133.4 (C20, C60), 144.8
(C6-pyridyl), 148.7 (C4, C40), 150.7 (C2=N pyridyl), 155.4 (C4=N pyridyl), 160.0 (C=O), 162.5 (C=N).
Anal. Calcd for C19H15N3O2: C, 71.91; H, 4.76; N, 13.24. Found: C, 71.46; H, 4.54; N 13.55.
(E)-4-Phenoxy-N0-(pyridin-4-ylmethylene)benzohydrazide (8i). White solid, Yield; 92%, m.p.: 175–177 ◦C.
1H-NMR (DMSO-d6)
δ: 7.10–7.13 (m, 4H, ArH), 7.24 (t, J = 8.2 Hz, 1H, ArH), 7.46 (t, J = 8.2 Hz, 2H,
ArH), 7.67 (s, 2H, ArH), 7.97 (d, J = 7.9 Hz, 2H, ArH), 8.44 (s, 1H, CH=N), 8.66 (s, 2H, ArH), 12.08 (s,
1H, NH). 13C-NMR (DMSO-d6) : 117.5 (C3, C5), 119.7 (C30, C50), 121.0 (C5-pyridyl), 124.5 (C1, C10),
δ
127.5 (C1-pyridyl), 130.1 (C2, C6), 130.3 (C20, C60), 141.5 (C6-pyridyl), 145.0 (C4, C40), 150.3 (C3=N
pyridyl), 155.4 (C4=N pyridyl), 160.2 (C=O), 162.6 (C=N). Anal. Calcd for C19H15N3O2: C, 71.91; H,
4.76; N, 13.24. Found: C, 71.64; H, 4.60; N 13.42.
(E)-N0-(Furan-2-ylmethylene)-4-phenoxybenzohydrazide (8j). White solid, Yield; 89%, m.p.: 192–194 ◦C.
1H-NMR (DMSO-d6):
δ
6.64 (s, 1H, ArH), 6.92 (s, 1H, ArH), 7.08–7.12 (m, 4H, ArH), 7.21–7.24 (m,
1H, ArH), 7.45 (t, J = 7.9 Hz, 2H, ArH), 7.85 (s, 1H, ArH), 7.93 (d, J = 7.9 Hz, 2H, ArH), 8.34 (s, 1H,
CH=N), 11.75 (s, 1H, NH). 13C-NMR (DMSO-d6) : 112.2 (C3), 133.4 (C5), 117.5 (C30, C50), 119.6
δ
(C3, C4–furfuryl), 124.4 (C1), 127.9 (C10), 129.9 (C2, C6), 130.3 (C20, C60), 137.3 (C2—furfuryl), 145.1
(C5–furfuryl), 149.5 (C4), 155.5 (C40), 159.9 (C=O), 162.3 (C=N). Anal. Calcd for C18H14N2O3: C, 70.58;
H, 4.61; N, 9.15. Found: C, 70.37; H, 4.32; N 9.50.
(E)-4-Phenoxy-N0-(thiophen-2-ylmethylene)benzohydrazide (8k). White solid, Yield; 92%, m.p.: 182–183 ◦C.
1H-NMR (DMSO-d6)
δ
: 7.08–7.24 (m, 6H, ArH), 7.44–7.47 (m, 3H, ArH), 7.67 (d, J = 7.5 Hz, 1H, ArH),
7.93 (d, J = 7.0 Hz, 2H, ArH), 8.66 (s, 1H, CH=N), 11.76 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ: 117.5 (C3,
C5), 119.6 (C30, C50), 124.4 (C3, C4 thienyl), 127.8 (C1) 128.8 (C10), 128.8 (C2, C6), 129.8 (C20), 130.3 (C60)
130.8 (C4), 139.2 (C40), 142.7 (C2–thienyl), 155.5 (C4–thienyl), 159.9 (C=O), 162.2 (C=N). Anal. Calcd
for C18H14N2O2S: C, 67.06; H, 4.38; N, 8.69. Found: C, 67.36; H, 3.39; N 9.04.
3.2. Biology
3.2.1. In Vitro Anti-Tubercular Activity
The MICs of Compounds
microplate Alamar blue assay (MABA) (Table 1) [31
3
,
4
,
6a
–
l
, and 8a
–
k
for M. tuberculosis H37Rv were determined by the
32]. Triclosan and INH were used as reference
,
standards. M. tuberculosis H37Rv (ATCC 27294; American Type Culture Collection, Rockville, MD,
USA) was grown to late log phase in Middlebrook 7H9 broth. Twofold dilutions of tested drugs were
prepared in 7H9 broth in a volume of 100
concentration of 0.2, 0.4, 0.8, 1.6, 3.12, 6.25, 12.5, 25, 50 and 100
(containing 2
104 CFU) was added, yielding a final testing volume of 200
(bacteria-only) control was added.
The plates were incubated at 37 ◦C; on the seventh day of incubation, 12.5
L of Alamar blue were added to all wells. After incubation at 37 ◦C for 16 to 24 h, the fluorescence
of the wells was read at an excitation of 530 nm and emission of 590 nm. The MIC was defined as the
µ
L in 96-well, clear-bottom microplates at a final testing
g/mL. 100 L of M. tuberculosis H37Rv
L. In each row, a drug-free
µ
µ
×
µ
µL of 20% Tween 80 and
20
µ
lowest concentration affecting a reduction in fluorescence of 90% relative to the mean of replicate
≥
bacteria-only controls.
Among the tested compounds, the results displayed that most of them i.e.,
3
,
4,
6h, 6i, 6l, 8g,
and 8k showed promising anti-tubercular activity (MIC, 0.61–1.69
(MIC, 10 µg/mL) and INH (MIC, 0.2 µg/mL).
µg/mL) in comparison to triclosan
3.2.2. In Vitro InhA Enzyme Inhibition
InhAenzymeassaywascarriedoututilizingaCary100Biospectrophotometer(AgilentTechnologies,
Santa Clara, California, USA) at 25 ◦C, through monitoring the oxidation of NADH to NAD+ at 340 nm.
Reactions were initiated via the addition of dodecenoyl-CoA (50 mm) substrate to mixtures containing

Molecules 2020, 25, 3125
15 of 18
InhA (5 nm), NADH (100 mm), and inhibitor (1–10,000 nm). The IC50 values were determined from a
dose-response plot of enzyme fractional activity versus the concentration of inhibitor.
3.2.3. Cytotoxic Activity
The cytotoxicity test was carried out using Vero cell line (African green monkey kidney epithelial
cells), obtained from American Type Culture Collection (ATCC). The cells were cultured according to
ATCC recommendations as it was described earlier [41
,
42]. To evaluate cytotoxicity, 3
×
104 Vero cells
were seeded in th◦e wells of 96-multiwell plates in 100
µL of the complete culture medium. After 24 h
incubation at 37 C when the cells were well attached and achieved 90% confluence, the culture
medium was replaced with 100
µL of derivative solutions, which were tested at the concentration range
of 0.1–200 g/mL. The stock solutions of the synthesized derivatives were prepared in DMSO and the
µ
tested concentrations were prepared by nine serial 2-fold dilutions. Vero cells were exposed to the
compounds and rifampicin for 24 h and then cell viability was assessed by MTT assay (Sigma-Aldrich
Chemicals, St. Louis, MO, USA). The IC50 value, which is defined as the concentration of the derivative
causing reduction in cell viability by 50%, was calculated using GraphPad Prism 5, version 5.03 software.
Selectivity/safety index (SI) for tested agents was determined using the formula: SI = IC50/MIC.
3.2.4. Docking Methodology
Discovery Studio 2.5 software (Accelrys Inc., San Diego, CA, USA) was used for docking analysis.
Fully automated docking tool using “Dock ligands (CDOCKER)” protocol running on Intel (R) Core
(TM) i32370 CPU @ 2.4 GHz, RAM Memory 2 GB under the Windows 7.0 system. The crystal structures
of InhA reductase enzyme (PDB code: 3FNH) were downloaded from the protein data bank [36
–38].
See the Supplementary Materials.
4. Conclusions
A set of diphenyl ether hydrazides and their cyclized oxadiazole analogs were designed and
synthesized as triclosan analogs. The targeted compounds have been identified using spectroscopic
data and elemental microanalysis. The antitubercular activity of the un-substituted oxadiazole
3 and the
hydrazide experienced the highest activity than triclosan as a reference drug. Arylated oxadiazoles 6i
4
and 6l and arylidenes 8g and 8k showed potent antitubercular activity against Mycobacterium tuberculosis
H37Rv strains. The InhA reductase enzymatic study supports the experimental data. We believe
there is more than one mode of mechanisms are involved in the antimycobacterial property of the
synthesized diphenyl ether derivatives. The obtained results from the in silico pharmacokinetic
properties calculation showed that most of the target compounds exhibited drug-like characters.
Taken together, a docking study has boosted that the higher activity of compounds
be further investigated as potential lead antimycobacterial candidates.
3, 4, and 8g could
Supplementary Materials: The following are available online: general experimental data and NMR spectra.
Author Contributions: Conceptualization, T.S.I. and S.S.P.; Methodology, A.M.M.A.-M., E.S.T., A.M.M., A.N.K.,
R.M.B. and I.A.S.; Software, M.F.A.M.; Validation, T.S.I. and S.S.P.; Formal analysis, E.S., A.M.M., A.N.K., R.M.B.
and I.A.S.; Investigation, T.S.I., E.S.T., M.A.A., and S.S.P.; Resources, H.Z.A., N.A.A.; Data curation, T.S.I. and S.S.P.;
Writing—original draft preparation, T.S.I., E.S.T., M.F.A.M. and S.S.P.; Writing—review and editing, T.S.I., E.S.T.
and S.S.P.; Visualization, T.S.I. and S.S.P.; Supervision, T.S.I. and S.S.P.; Project administration, H.Z.A., N.A.A.;
Funding acquisition, H.Z.A., N.A.A. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by King Abdulaziz University, Jeddah, under grant No. (RG-009-166-38).
Acknowledgments: The authors gratefully acknowledge DSR, King Abdulaziz University, Jeddah, for technical
and financial support No. (RG-009-166-38).
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2020, 25, 3125
16 of 18
References
1.
2.
3.
4.
World Health Organization. Global Tuberculosis Report. 2015. Available online: www.who.int/tb/
publications/global_report/gtbr15_main_text.pdf (accessed on 27 January 2020).
World Health Organization. Global Tuberculosis Report. 2019. Available online: https://www.who.int/news-
room/events/detail/2019/03/24/default-calendar/world-tb-day-2019 (accessed on 27 January 2020).
Matteelli, A.; Roggi, A.; Carvalho, A.C. Extensively drug-resistant tuberculosis: Epidemiology and
management. Clin. Epidemiol. 2014, 6, 111–118. [CrossRef] [PubMed]
Chollet, A.; Maveyraud, L.; Lherbet, C.; Bernardes-Genisson, V. An overview on crystal structures of InhA
protein: Apo-form, in complex with its natural ligands and inhibitors, Eur. J. Med. Chem. 2018, 146, 318–343.
[CrossRef]
5.
6.
Ende, C.W.A.; Knudson, S.E.; Liu, N.; Childs, J.; Sullivan, T.J.; Boyne, M.; Xu, H.; Gegina, Y.; Knudson, D.L.;
Johnson, F.; et al. Synthesis and in vitro antimycobacterial activity of B-ring modified diaryl ether InhA
inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 3029–3033. [CrossRef] [PubMed]
Perozzo, R.; Kuo, M.; Sidhu, A.B.S.; Valiyaveetti, J.T.; Bittman, R.; Jacobs, W.R., Jr.; Fidock, D.A.;
Sacchettini, J.C.J. Structural Elucidation of the Specificity of the Antibacterial Agent Triclosan for Malarial
Enoyl Acyl Carrier Protein Reductase. Biol. Chem. 2002, 277, 13106–13114. [CrossRef] [PubMed]
Sharma, S.; Ramya, T.N.C.; Surolia, A.; Surolia, N. Triclosan as a Systemic Antibacterial Agent in a Mouse
Model of Acute Bacterial Challenge. Antimicrob. Agents Chemother. 2003, 47, 3859–3866. [CrossRef]
Chhibber, M.; Kumar, G.; Parasuraman, P.; Ramya, T.N.; Surolia, N.; Surolia, A. Novel diphenyl ethers:
Design, docking studies, synthesis and inhibition of enoyl ACP reductase of Plasmodium falciparum and
Escherichia coli. Bioorg. Med. Chem. 2006, 14, 8086–8098. [CrossRef]
7.
8.
9.
Mishra, S.; Karmodiya, K.; Parasuraman, P.; Surolia, A.; Surolia, N. Design, synthesis, and application of
novel triclosan prodrugs as potential antimalarial and antibacterial agents. Bioorg. Med. Chem. Lett. 2008, 16,
5536–5546. [CrossRef]
10. Butler, M.S.; Cooper, M.A.J. Antibiotics in the clinical pipeline in 2011. J. Antibiot. 2011, 64, 413–425. [CrossRef]
[PubMed]
11. Tonge, P.J.; Kisker, C.; Slayden, R.A. Development of Modern InhA Inhibitors to Combat Drug Resistant
Strains of Mycobacterium tuberculosis. Curr. Top. Med. Chem. 2007, 7, 489–498. [CrossRef]
12. Kini, S.G.; Bhat, A.R.; Bryant, B.; Williamson, J.S.; Dayan, F.E. Synthesis, antitubercular activity and docking study
of novel cyclic azole substituted diphenyl ether derivatives. Eur. J. Med. Chem. 2009, 44, 492–500. [CrossRef]
13. Kini, S.G.; Bhat, A.R.; Pan, Z.; Dayan, F.E. Synthesis and antitubercular activity of heterocycle substituted
diphenyl ether derivatives. J. Enzyme Inhib. Med. Chem. 2010, 25, 730–736. [CrossRef] [PubMed]
14. Sivaraman, S.; Sullivan, T.J.; Johnson, F.; Novichenok, P.; Cui, G.; Simmerling, C.; Tonge, P.J. Inhibition of the
Bacterial Enoyl Reductase FabI by Triclosan: A Structure Reactivity Analysis of FabI Inhibition by Triclosan
−
Analogues. J. Med. Chem. 2004, 47, 509–518. [CrossRef] [PubMed]
15. Heath, R.J.; Yu, Y.T.; Shapiro, M.A.; Olson, E.; Rock, C.O. Broad spectrum antimicrobial biocides target the
FabI component of fatty acid synthesis. J. Biol. Chem. 1998, 273, 30316–30320. [CrossRef] [PubMed]
16. Yang, Y.H.; Wang, Z.L.; Yang, J.Z.; Yang, T.; Pi, W.Y.; Ang, W.; Lin, Y.N.; Liu, Y.Y.; Li, Z.C.; Luo, Y.F.; et al.
Design, synthesis and evaluation of novel molecules with a diphenyl ether nucleus as potential antitubercular
agents. Bioorg. Med. Chem. Lett. 2012, 22, 954–957. [CrossRef] [PubMed]
17. Navarrete-Vazquez, G.; Molina-Salinas, G.M.; Duarte-Fajardo, Z.V.; Vargas-Villarreal, J.; Estrada-Soto, S.;
Gonzalez-Salazar, F.; Hernandez-Nunez, E.; Said-Fernandez, S. Synthesis and antimycobacterial activity of
4-(5-substituted-1,3,4-oxadiazol-2-yl)pyridines. Bioorg. Med. Chem. 2007, 15, 5502–5508. [CrossRef] [PubMed]
18. Khan, R.H.; Rastogi, R.C. Synthesis of some 2-[(5-aryl-1,3,4-oxadiazol-2-yl)amino]-1,3-heterazoles as potential
pesticides. J. Agric. Food Chem. 1990, 38, 1068–1071. [CrossRef]
19. Narayana, B.; Raj, K.K.V.; Ashalatha, B.V.; Kumari, N.S. Synthesis of Some New 2-(6-Methoxy-2-Naphthyl)
-5-Aryl-1,3,4-Oxadiazoles as Possible Non-steroidal Anti-inflammatory and Analgesic Agents. Arch. Pharm.
Chem. Life Sci. 2005, 338, 373–377. [CrossRef]
20. Rasras, A.J.M.; Al-Tel, T.H.; Al-Aboudi, A.F.; Al-Qawasmeh, R.A. Synthesis and antimicrobial activity of
cholic acid hydrazone analogues. Eur. J. Med. Chem. 2010, 45, 2307–2313. [CrossRef]

Molecules 2020, 25, 3125
17 of 18
21. Mohareb, R.M.; Fleita, D.H.; Sakka, O.K. Novel Synthesis of Hydrazide-Hydrazone Derivatives and Their
Utilization in the Synthesis of Coumarin, Pyridine, Thiazole and Thiophene Derivatives with Antitumor
Activity. Molecules 2011, 16, 16–27. [CrossRef]
22. Cascioferro, S.; Attanzio, A.; Di Sarno, V.; Musella, S.; Tesoriere, L.; Cirrincione, G.; Diana, P.; Parrino, B.
New 1,2,4-Oxadiazole Nortopsentin Derivatives with Cytotoxic Activity. Mar. Drugs 2019, 17, 35. [CrossRef]
23. Rollas, S.; Küçükgüzel, S.G. Biological Activities of Hydrazone Derivatives. Molecules 2007, 12, 1910–1939.
[CrossRef]
24. Asif, M. Pharmacologically potentials of hydrazonone containing compounds: A promising scaffold. Int. J.
Adv. Chem. 2014, 2, 85–103. [CrossRef]
25. Oliveira, P.F.M.; Guidetti, B.; Chamayou, A.; André-Barrès, C.; Madacki, J.; Korduláková, J.; Mori, G.;
Orena, B.S.; Chiarelli, L.R.; Pasca, M.R.; et al. Mechanochemical Synthesis and Biological Evaluation of Novel
Isoniazid Derivatives with Potent Antitubercular Activity. Molecules 2017, 22, 1457. [CrossRef]
26. Freundlich, J.S.; Wang, F.; Vilcheze, C.; Gulten, G.; Langley, R.; Schiehser, G.A.; Jacobus, D.R.; Jacobs, W.R.;
Sacchettini, J.C. Triclosan derivatives: Towards potent inhibitors of drug-sensitive and drug-resistant
Mycobacterium tuberculosis. Chem. Med. Chem. 2009, 4, 241–248. [CrossRef] [PubMed]
27. Ibrahim, H.R.; Tatsumoto, S.; Ono, H.; Van Immerseel, F.; Raspoet, R.; Miyata, T. A novel antibiotic-delivery
system by using ovotransferrin as targeting molecule. Eur. J. Pharm. Sci. 2015, 66, 59–69. [CrossRef] [PubMed]
28. Haga, N.; Takayanagi, H. Mechanisms of the Photochemical Rearrangement of Diphenyl Ethers. J. Org.
Chem. 1996, 61, 735–745. [CrossRef]
29. He, L.; Zhang, L.; Liu, X.; Li, X.; Zheng, M.; Li, H.; Yu, K.; Chen, K.; Shen, X.; Jiang, H.; et al. Discovering
Potent Inhibitors Against the β-Hydroxyacyl-Acyl Carrier Protein Dehydratase (FabZ) of Helicobacter pylori:
Structure-Based Design, Synthesis, Bioassay, and Crystal Structure Determination. J. Med. Chem. 2009, 52,
2465–2481. [CrossRef]
30. Ibrahim, T.S.; Al-Mahmoudy, A.M.; Elagawany, M.; Ibrahim, M.A.; Panda, S.S. Synthesis and Antiviral
Bioassay of New Diphenyl Ether-based Compounds. Chem. Biol. Drug Des. 2016, 88, 511–518. [CrossRef]
31. Collins, L.; Franzblau, S.G. Microplate alamar blue assay versus BACTEC 460 system for high-throughput
screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob. Agents
Chemother. 1997, 41, 1004–1009. [CrossRef]
32. Franzblau, S.G.; Witzig, R.S.; McLaughlin, J.C.; Torres, P.; Madico, G.; Hernandez, A.; Degnan, M.T.;
Cook, M.B.; Quenzer, V.K.; Ferguson, R.M.; et al. Rapid, low-technology MIC determination with clinical
Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay. J. Clin. Microbiol. 1998, 36,
362–366. [CrossRef]
33. Umaa, K.; Sudha Rani, S.; Sangeetha, G.; Maida Engels, S.E. Prediction of physiochemical parameters for
anti-tubercular potentials of Mannich and Schiff bases. J. Chem. Pharm. Res. 2013, 5, 52–59.
34. Lakshminarayana, S.B.; Huat, T.B.; Ho, P.C.; Manjunatha, U.H.; Dartois, V.; Dick, T.; Rao, S.P.S. Comprehensive
physicochemical, pharmacokinetic and activity profiling of anti-TB agents. J. Antimicrob. Chemother. 2015, 70,
857–867. [CrossRef] [PubMed]
35. Wiener, H. Structural determination of Paraffin boiling point. J. Am. Chem. Soc. 1947, 69, 17–20. [CrossRef]
36. Stec, J.; Vilchèze, C.; Lun, S.; Perryman, A.L.; Wang, X.; Freundlich, J.S.; Bishai, W.; Jacobs, W.R., Jr.;
Kozikowski, A.P. Biological evaluation of potent triclosan-derived inhibitors of the enoyl-acyl carrier protein
reductase InhA in drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis. Chem. Med.
Chem. 2014, 9, 2528–2537. [CrossRef] [PubMed]
37. Mohamed, M.F.A.; Youssif, B.G.M.; Shaykoon, M.S.A.; Abdelrahman, M.H.; Elsadek, B.E.M.; Aboraia, A.S.;
Abuo-Rahma, G.E.A. Utilization of tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinone as a cap moiety in design
of novel histone deacetylase inhibitors. Bioorg. Chem. 2019, 91, 103127. [CrossRef] [PubMed]
38. Youssif, B.G.M.; Mohamed, M.F.A.; Al-Sanea, M.M.; Moustafa, A.H.; Abdelhamid, A.A.; Gomaa, H.A.M.
Novel aryl carboximidamides and 3-aryl-1,2,4-oxadiazoles analogues of naproxen as dual selective COX-2 /
LOX inhibitors: Design, synthesis and Docking studies. Bioorg. Chem. 2019, 85, 577–584. [CrossRef] [PubMed]
39. Liu, Y.; Li, G.; Yang, L. Cu(I)-catalyzed Synthesis of Diaryl Ether Using a Simple Hydrazone as Promoter.
Chin. J. Chem. 2009, 27, 423–427. [CrossRef]
40. Bar-Yuan, H.; Kun-Ming, Y.; Yun, C. Synthesis of electroluminescent copoly(aryl ether)s containing alternate
1,4-distyrylbenzene derivatives and aromatic 1,3,4-oxadiazole or 3,3”-terphenyldicarbonitrile segments. J.
Polym. Sci. Pol. Chem. 2005, 43, 5009–5022.

Molecules 2020, 25, 3125
18 of 18
41. Sieniawska, E.; Sawicki, R.; Swatko-Ossor, M.; Napiorkowska, A.; Przekora, A.; Ginalska, G.;
Augustynowicz-Kopec, E. The effect of combining natural terpenes and antituberculous agents against
reference and clinical Mycobacterium tuberculosis strains. Molecules 2018, 23, 176. [CrossRef]
42. Trotsko, N.; Golus, J.; Kazimierczak, P.; Paneth, A.; Przekora, A.; Ginalska, G.; Wujec, M. Synthesis and
antimycobacterial activity of thiazolidine-2,4-dione based derivatives with halogenbenzohydrazones and
pyridinecarbohydrazones substituents. Eur. J. Med. Chem. 2020, 189, 112045. [CrossRef]
Sample Availability: Samples of the compounds 6a–l and 8a–k are available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).