Synthesis of novel benzohydrazone-oxadiazole hybrids as β-glucuronidase inhibitors and molecular modeling studies

Article abstract of DOI:10.1016/j.bmc.2015.10.037

A series of compounds consisting of 25 novel oxadiazole-benzohydrazone hybrids (6-30) were synthesized through a five-step reaction sequence and evaluated for their β-glucuronidase inhibitory potential. The IC₅₀ values of compounds 6-30 were found to be in the range of 7.14-44.16 μM. Compounds 6, 7, 8, 9, 11, 13, 18, and 25 were found to be more potent than d-saccharic acid 1,4-lactone (48.4 ± 1.25 μM). These compounds were further subjected for molecular docking studies to confirm the binding mode towards human β-d-glucuronidase active site. Docking study for compound 13 (IC₅₀ = 7.14 ± 0.30 μM) revealed that it adopts a binding mode that fits within the entire pocket of the binding site of β-d-glucuronidase. Compound 13 has the maximum number of hydrogens bonded to the residues of the active site as compared to the other compounds, that is, the ortho-hydroxyl group forms hydrogen bond with carboxyl side chain of Asp207 (2.1 ?) and with hydroxyl group of Tyr508 (2.6 ?). The other hydroxyl group forms hydrogen bond with His385 side chain (2.8 ?), side chain carboxyl oxygen of Glu540 (2.2 ?) and Asn450 side-chain's carboxamide NH (2.1 ?).

Full text of DOI:10.1016/j.bmc.2015.10.037

Accepted Manuscript
Synthesis of novel benzohydrazone-oxadiazole hybrids as β-glucuronidase in-
hibitors and molecular modeling studies
Muhammad Taha, Nor Hadiani Ismail, Syahrul Imran, Manikandan Selvaraj,
Abdul Rahim, Muhammad Ali, Salman Siddiqui, Fazal Rahim, Khalid
Mohammed Khan
PII:
S0968-0896(15)30115-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.10.037
BMC 12637
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 August 2015
22 October 2015
24 October 2015
Please cite this article as: Taha, M., Ismail, N.H., Imran, S., Selvaraj, M., Rahim, A., Ali, M., Siddiqui, S., Rahim,
F., Khan, K.M., Synthesis of novel benzohydrazone-oxadiazole hybrids as β-glucuronidase inhibitors and molecular
modeling studies, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.10.037
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis of novel benzohydrazone-oxadiazole hybrids as β-glucuronidase
inhibitors and molecular modeling studies
Muhammad Tahaa,b, Nor Hadiani Ismaila,b, Syahrul Imrana,b, Manikandan Selvarajc,d, Abdul Rahimb
Muhammad Alie, Salman Siddiquif, Fazal Rahimg, Khalid Mohammed Khanf
aAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM),
Puncak Alam Campus, 42300, Bandar Puncak Alam, Selangor, Malaysia
bFaculty of Applied Science Universiti Teknologi MARA (UiTM), 40450, Shah Alam, Selangor,
Malaysia
cIntegrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA (UiTM), Puncak
Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
dFaculty of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar
Puncak Alam, Selangor Darul Ehsan, Malaysia
eDepartment of Chemistry, COMSATS Institute of Information Technology, University Road,
Abbottabad-22060, KPK, Pakistan
fH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
gDepartment of Chemistry, Hazara University, 21300, Mansehra, Pakistan
Abstract:
A series of compounds consisting of 25 novel oxadiazole-benzohydrazone hybrids (6-30) were
synthesized through a five-step reaction sequence and evaluated for their β-glucuronidase inhibitory
potential. The IC50 values of compounds 6-30 were found to be in the range of 7.14 to 44.16 µM.
Compounds 6, 7, 8, 9, 11, 13, 18, and 25 were found to be more potent than D-saccharic acid 1,4-
lactone (48.4 ± 1.25 µM). These compounds were further subjected for molecular docking studies to
confirm the binding mode towards human β-D-glucuronidase active site. Docking study for compound
13 (IC50 = 7.14 ± 0.30 µM) revealed that it adopts a binding mode that fits within the entire pocket of
the binding site of β-D-glucuronidase. Compound 13 has the maximum number of hydrogens bonded
to the residues of the active site as compared to the other compounds, i.e., the ortho- hydroxyl group
forms hydrogen bond with carboxyl side chain of Asp207 (2.1 Å) and with hydroxyl group of Tyr508
(2.6 Å). The other hydroxyl group forms hydrogen bond with His385 side chain (2.8 Å), side chain
carboxyl oxygen of Glu540 (2.2 Å) and Asn450 side-chain's carboxamide NH (2.1 Å).
Keywords: Oxadiazole, Benzohydrazone, hybrids, β-Glucuronidase inhibition, Molecular docking

Correspondence and reprints
E-mail: taha_hej@yahoo.com and muhamm9000@puncakalam.uitm.edu.my, Tel: 0060182901765

1. Introduction
Heterocycles constitute among the largest division in medicinal chemistry and are of considerable
importance biologically and industrially. Oxadiazole, which is a heterocyclic moiety, is also of
immense importance in pharmaceutical industry due to its versatile biological actions. Compounds
having oxadiazole nucleus are reported to possess unique antioedema and anti-inflammatory activities
[1-4]. Besides that, compounds possessing oxadiazole ring have been reported for other activities such
as analgesic [3-4], antitubercular [5], anti-hepatitis B viral activities [6], antimicrobial [7], and
anticonvulsant [8]. The level of interest in oxadiazole for over the past few years is clearly indicated
by the number of patent applications that have been filed. Plenty of compounds containing oxadiazole
ring were being studied and are now in late stage of clinical trials such as zibotentan as an anticancer
agent. Raltegravir, which also has an oxadiazole ring, was being studied as antiretroviral drug to treat
HIV infection. Research has shown clearly that oxadiazoles impacted massively various drug
discovery programs including cancer [9], obesity [10] infections [11], diabetes [12], inflammation
[13], analgesic [14], antimitotic [15], anticonvulsive [16], antifungal [17] diuretic [18], and muscle
relaxants [19]. Due to these potentials, oxadiazole moiety has played pivotal role in drug discovery and
development as an essential or a part of the pharmacophore that could contribute to ligand binding
[20]. Oxadiazole moieties have also been employed as an aromatic linker to place substituents in the
right positions [21] as well as orientating molecular properties by locating them at the boundary of the
molecules [22]. A recent study showed that oxadiazole regioisomers significantly changes
thermodynamic properties by changing the water architecture within the active site of aldose reductase
enzyme [23]. Oxadiazoles have also been used as replacements for carbonyl containing compounds
such as carbamates, esters, hydroxamic esters and amides [24−26].
β-Glucuronidase is an enzyme that catalyzes cleavage of glucuronosyl-O-bonds [27]. This enzyme is
involved in diseases such as inflammation related to joints, like rheumatoid arthritis [35, 36]. While in
some cases, over-expression of this enzyme is reported to cause hepatic diseases and AIDS. Besides
that, β-glucuronidase also takes place in diseases such as infection of the urinary tract [28-30], epilepsy
[33], renal disease [31], rejection of transplantation [32], larynx and breast [34]. Various studies had
been carried out to show that bacterial β-glucuronidase inhibitor decreases carcinogen induced colonic
tumors [37].
Present work belongs to a simple and reliable synthetic protocol that has been developed for the
synthesis of a series of benzohydrazone-oxadiazole hybrid pharmacophores in an effort towards
developing more effective agents against β-glucuronidase enzyme. Evidence exists that hybridization
2

of two or more biologically active and new molecules with complementary pharmacophoric potentials
or with different mechanisms of action generally render synergistic effects. This combined
pharmacophore approach, in turn, may address the active site of different targets more effectively and
could offer the possibility to overcome the issue of drug resistance as well as unwanted side effects
[38]. The outcomes of this hybridization strategy have already been the subject of interest in finding
novel antibacterial and antimalarial agents to overcome the issue of drug resistance [39].
Encouraged by these efforts, we decided to develop benzohydrazone-oxadiazole hybrids in order to
reveal new perspectives in the biological potentials of newly synthesized hybrid molecules. β-
Glucuronidase potential of benzohydrazones and oxadiazoles that have already been reported are being
displayed in figure-1 [40-42]. However, the β-glucuronidase potential of benzohydrazone-oxadiazole
hybrids has never been reported before. Here in, we are unveiling the synthesis of benzohydrazone-
oxadiazole hybrids and their β-glucuronidase inhibitory potentials.
Figure-1 Comparison between hydrazide, oxadiazole, hydrazone, and hybrid compound 13.
2. Results and Discussion
2.1 Chemistry
The synthetic route towards hybrids 6-30 is shown in Scheme 1 and 2. Compound 2 was prepared by
refluxing methyl 2-hydroxybenzoate 1 with the excess of hydrazine hydrate. The compound 2 was then
reacted with methyl 4-formylbenzoate in methanol with catalytic amount of acetic acid to afford
compound 3. The resulting adduct 3 underwent oxidative cyclization using phenyliododiacetate
(PhI(OAc)2) in dry chloroform to form oxadiazole 4. The ester functional group of compound 4 was
further transformed into corresponding hydrazide 5 by treating with hydrazine hydrate.
3

Scheme-1. Synthesis of benzohydrazide having oxadiazole ring compound 5
In Scheme 2, the advanced intermediate hydrazide 5 was reacted with different aryl aldehydes in the
presence of acetic acid to form the targeted oxadiazole benzohydrazone derivatives 6-30 (Table-1).
The structures of oxadiazole benzohydrazone hybrids 6-30 were confirmed using spectroscopic
techniques such as NMR, MS and were further confirmed using CHN analysis.
Scheme-2. Synthesis of novel oxadiazole benzohydrazones 6-30
Table-1. Synthesis of oxadiazole benzohydrazones 6-30
Comp. no.
6
R
Comp. no.
19
R
4

7
20
21
8
9
22
23
24
10
11
25
26
27
12
13
14
15
28
29
30
16
17
18
2.2 β-glucuronidase inhibition
In continuation of our search on new enzyme inhibitor [43], we have evaluated (25) derivatives 6-
30 of oxadiazole bearing Schiff base linkage for their β–glucuronidase inhibition potential. The IC50
5

values for potent compounds were in the range of 7.14 to 44.16 µM (Table-2). Compound 13 that is a
trihydroxy substituted derivative displayed exceptionally potent single-digit activity with an IC50 value
of 7.14 ± 0.30 µM as compared to D-Saccharic acid 1,4-lactone. Compounds 6 (IC50 = 18.46 ± 0.65
µM), 7 (IC50 = 34.46 ± 0.85 µM), 8 (IC50 = 29.15 ± 0.75 µM), 9 (IC50 = 15.14 ± 0.55 µM), 11 (IC50 =
44.16 ± 1.20 µM, 18 (IC50 = 21.14 ± 1.05 µM) and 25 (IC50 = 42.26 ± 1.16 µM) also showed good
inhibitory potentials better than standard. It was observed that presence of hydroxyl group on aromatic
side chain enhanced the inhibitory potential of certain compounds against β-glucuronidase when
compared with the compounds without having the hydroxyl substituents. These findings corroborated
our previous results that presence of hydroxyl groups at particular sites are important and decisive
factors for compounds to be active against β-glucuronidase [41]. However, in current series, it is
observed that presence of an ortho- hydroxyl group on benzene ring attached to oxadiazole moiety is
not involved in the inhibitory potential of these compounds. For example, if we look at compounds 14-
17, 20, 22, 23, and 27-30, these all compounds lack hydroxyl group on benzene ring that is directly
attached with hydrazone part while all these compounds have an ortho- hydroxyl group on benzene
ring attached to oxadiazole part and were found all inactive. This observation suggests that presence of
ortho- hydroxyl group on aromatic ring attached with oxadiazole moiety is not imperative for these
compounds to be active. One could imagine the unavailability of this hydroxyl group since it may be
involved in hydrogen bonding with nearby oxygen atom of oxadiazole part.
To better understand the inhibition mechanism of active compounds and their binding modes with
enzyme active site, molecular modeling studies were performed. The compounds were docked into the
binding pocket of β-glucuronidase to further confirm the inhibition potential and also the docked
Goldscore is reported for newly synthesized 6-30 compounds (Table-2).
Table-2. In vitro β-glucuronidase inhibition activity of oxadiazole derivatives 6-30 and their docking
studies Goldscore results
External van
der waals
energy
External H-bond
energy
IC50 (M ±
Compound
Goldscore
SEMa)
46.02
48.25
49.80
45.29
50.28
49.11
49.98
45.93
18.46 ± 0.65
34.46 ± 0.85
29.15 ± 0.75
15.14 ± 0.55
64.12 ± 1.62
44.16 ± 1.20
82.16 ± 2.25
7.14 ± 0.30
6
7
64.78
69.07
73.77
68.78
68.97
66.02
71.99
72.89
11.10
11.04
11.07
11.13
7.89
8
9
10
11
12
13
9.84
6.90
13.74
6

64.72
67.49
66.57
66.33
70.05
65.23
66.98
71.17
66.16
63.46
70.83
63.43
64.26
67.59
63.34
63.54
67.39
6.74
5.37
0.00
2.32
11.09
6.03
5.07
6.01
2.54
1.49
5.98
10.97
5.54
3.87
2.83
3.51
2.87
43.87
47.37
49.25
48.36
46.09
47.14
45.22
47.56
50.42
47.17
46.66
45.08
47.23
44.90
45.51
46.16
49.08
N.A
N.A
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
N.A
N.A
21.14 ± 1.05
86.12 ± 2.15
N.A.
88.14 ± 2.38
N.A.
N.A.
91.4 ± 2.45
42.26 ± 1.16
108.14 ± 3.14
N. A.
N. A.
N. A.
N. A.
D-Saccharic acid 1,4-
lactone
59.80
5.83
50.06
48.4 ± 1.25
The most active compounds Goldscore and external h-bond energy are highlighted in bold.
2.3
Docking study
Molecular docking studies were carried out on crystallographic structure of human β-
glucuronidase (PDB code: 1BHG). Synthetic compounds 6-30 were docked into active site of human
β-glucuronidase to reveal the binding mode of each compound. The binding mode of known substrate
p-nitrophenyl β-glucuronide on β-glucuronidase active site reveals that glycoside bond of p-
nitrophenyl β-glucuronide was perfectly oriented towards the catalytic residues Glu540 and Glu451
and acts as the acid/base catalyst where Glu540 functions as the nucleophilic residue. Docked binding
mode showed p-nitrophenyl β-glucuronide perfectly fitting into the active site of β-D-glucuronidase
forming hydrogen bonding with residues Glu451, His385 and Lys606 that could significantly interact
with the enzyme, which is also shown in our previous study [44].
β-D-Glucuronidase structure was employed in this docking study to reveal the favorable
binding mode of newly synthesized derivatives 6-30. Predicted binding modes for biologically active
and inactive compounds and their docked Goldscore are reported in Table-2.
7

The overall Goldscore (Mainly consist of external h-bonding energy and external van der waals
energy) for active molecules were expected to be high when compared to the least active and inactive
compounds. But by considering the reported Goldscore in this study as in Table-2, significant
relationship cannot be established between Goldscore and inhibitory activity. Our previous studies
showed that hydrophilic (h-bond) characteristic feature of β-D-Glucuronidase inhibitors are crucial for
the inhibitory activity. Therefore, the overall activity of these compounds mainly depends on h-
bonding and hence the activities of these compounds mainly depend on external h-bonding energy,
while the external van der waals energy (Table-2) may not significantly influence the activity profile.
From Table-2 it could be noted that, as the external h-bond energy value increases the inhibitory
activity profile of compounds also increases. For the most active compound 13, compound 9,
compound 6 and compound 18, the external h-bonding energy values were 13.74, 11.13, 11.10 and
11.09, respectively. For the inactive molecules these values were considerably low. Consequently, a
significant relationship could be established for all compounds in Table-2 based on external h-bond
energy value and inhibitory activity.
Docking analysis of the binding mode for the most active compound 13 (IC50 = 7.14 ± 0.30
µM) indicated that compound 13 adopts a binding mode that fits well the entire furrow of the binding
site of β-D-glucuronidase. Analysis of the best ranked binding pose of compound 13 discovered that it
has the maximum number of hydrogen bonded to the active site residues as compared to the other
compounds and thus making compound 13 most potent. The benzenetriol group at ortho position
forms hydrogen bond with side chain carboxyl oxygen of Asp207 (2.1 Å) and Tyr508 side chain
hydroxyl group (2.6Å). While the other hydroxyl group at the ortho position forms hydrogen bonding
with His385 side chain imidazole nitrogen (2.8 Å), Glu540 side chain carboxyl oxygen (2.2 Å) and
Asn450 side-chain's carboxamide NH at a distance of 2.1 Å. Similarly, the benzohydrazide nitrogen
forms hydrogen bond with Tyr504 side chain hydroxyl group (2.4Å), while the other NH forms
hydrogen bond with carboxyl oxygen (2.2 Å) of Glu540. Benzohydrazide oxygen forms hydrogen
bond with Tyr508 side chain hydroxyl group (2.6 Å). Predicted binding mode of compound 13 is
shown in Fig.2a. Additionally, the phenyl group of the Tyr504, Tyr508 and the side chain of Asn484
forms hydrophobic interaction with the compound 13 that stabilize the binding mode in the β-D-
glucuronidase active site.
8

Figure-2. Shows the putative binding mode of the potent compounds among the synthesized
derivatives in the active site of β-glucuronidase. The key interacting residues are shown in magenta
color line and compound in stick representation, while the hydrogen bonds are shown in yellow dashed
line. Compound 13 in split pea color (1a), Compound 9 in dark green color (1b), Compound 6 in
orange color (1c), Compound 18 in fluorescent color (1d), Compound 8 in pale yellow (1e),
9

Compound 7 in dirty violet color (1f), Compound 25 in blue white (1g) and Compound 11 in light
pink color (1h).
Similarly, compound 9 which is the second most active compound (IC50 = 15.14 ± 0.30 µM) in
the series revealed that the hydroxyl group at ortho position of dihydroxybenzylidene is involved in the
hydrogen bond interaction with Asn450 side-chain's carboxamide NH at a distance of 2.2 Å and with
Glu540 side chain carboxyl oxygen (2.2 Å). The benzohydrazide nitrogen forms hydrogen bonding
with Tyr504 side chain hydroxyl group (2.4 Å), whereas oxygen of benzohydrazide forms hydrogen
bond with Tyr508 side chain hydroxyl group (2.1Å). Benzohydrazide NH forms hydrogen bond with
carboxyl oxygen of Glu540 (2.2 Å). Additionally, Tyr504 phenyl ring, Tyr508 side chain phenyl ring
and Asp207 side chain methyl group forms hydrophobic interaction with the compound 9 that stabilize
the binding mode in the β-D-glucuronidase active site. Lack of hydroxyl group at the other ortho
position of compound 9 hinders the formation of hydrogen bonding with Asp207, hence showing
reduction in activity profiling (Fig.2b).
The third most active compound in the series is compound 6 (IC50 = 18.46 ± 0.65 µM), in
which the hydroxyl group at the para position of the hydroxybenzylidene forms hydrogen bond with
backbone carbonyl oxygen group of Glu451 at a distance of 2.9 Å. The side chain carboxyl oxygen of
Glu451 forms hydrogen bonding with the benzohydrazide nitrogen at a distance of 1.9 Å.
Benzohydrazide oxygen also forms hydrogen bonding with Tyr504 side chain hydroxyl group (2.5 Å).
The phenyl ring of Tyr504 and Tyr508 forms hydrophobic interaction with benzene ring of compound
6 (Fig.2c). The next active molecule in the list is compound 18 (IC50 = 21.14 ± 1.05 µM). Even though
the benzenetriol moiety for compound 18 has hydroxyl group at ortho, meta and para position, the
hydroxyl group at the ortho and meta position does not interact with any key residue. Hydroxyl group
at para position forms hydrogen bond with backbone carbonyl oxygen of Glu451 at a distance of 1.9
Å, while the oxygen of side chain carboxyl for residue Glu451 forms hydrogen bond with the
benzohydrazide nitrogen at a distance of 2.2 Å. Additionally, the benzohydrazide oxygen forms
hydrogen bond with Tyr508 side chain hydroxyl group (2.3 Å). On the other hand phenyl ring of
Tyr504 and Tyr508 form hydrophobic interaction with benzene of compound 18 (Fig.2d). As for
compound 3 which is the fifth active compound among the list (IC50 = 29.15 ± 0.75 µM), oxygen of
para hydroxyl group forms hydrogen bond with the backbone NH of Asp207 at a distance of 2.2 Å,
while the meta hydroxyl group forms hydrogen bond with carboxyl oxygen of Glu451 side chain (1.6
Å). Similarly, the benzohydrazide nitrogen forms hydrogen bond with Glu540 side chain carboxyl
oxygen (2.2 Å) and benzohydrazide oxygen hydrogen bond with Tyr508 side chain hydroxyl group
(2.0 Å). For compound 8, phenyl group of the Tyr504, Tyr508 and Tyr504 forms hydrophobic
interaction with the oxygen of benzohydrazide and the other 2 hydroxyl groups to stabilize the binding
mode (Fig.2e).
10

Compound 7 with activity (IC50 = 34.46 ± 0.85 µM) shows that the benzohydrazide nitrogen
forms hydrogen bonding with Glu540 side chain carboxyl oxygen (2.4 Å) and benzohydrazide oxygen
forms hydrogen bonding with Tyr508 side chain hydroxyl group (2.0 Å). While the hydroxyl
benzylidene meta positioned hydroxyl oxygen hydrogen bond with the backbone NH of Asp207 at a
distance of 2.2 Å. Aromatic ring of Tyr504 and Tyr508 forms hydrophobic interaction with benzene
ring of compound 7 (Fig.2f).
The binding mode observed of compound 25 (IC50 = 42.26 ± 1.16 µM) suggests that the
benzohydrazide NH forms hydrogen bonding with Glu540 side chain carboxyl oxygen (1.9 Å), while
the other nitrogen forms hydrogen bond with the Tyr504 side chain hydroxyl group (2.4 Å). It was also
observed that the benzohydrazide oxygen forms hydrogen bond with Tyr508 side chain hydroxyl
group (2.1 Å). The presence of pyridine ring did not play any key role in the activity profile, since
there were no key residues to interact with in the binding mode of compound 25. Hydrophobic
interaction was established by the aromatic ring of Tyr504 and Tyr508 with the benzene ring of
compound 25 (Fig.2g). Compound 11 being the last compound among the considerable activity series
with activity (IC50 = 42.26 ± 1.16 µM), shows that the benzohydrazide nitrogen forms hydrogen bond
with Tyr508 side chain hydroxyl group (2.3 Å).
In contrast, the presence of nitrobenzene moiety could not influence the activity profile since
there were no key residues to interact with nitro group in the binding orientation of compound 11
(Fig.2h). Furthermore, there were some compounds with marginal activity profile, like compound 10
(IC50 = 64.12 ± 1.62 µM), compound 12 (IC50 = 82.16 ± 2.25 µM), compound 19 (IC50= 86.12 ± 2.15
µM), compound 21 (IC50 = 88.14 ± 2.38 µM), compound 24 (IC50 = 91.4 ±2.45 µM), compound 25
(IC50 = 108.14 ±3.14 µM). Altogether these compounds could only partially stabilize the binding
interaction within the key residues of human β-D-glucuronidase, which ultimately results in low
inhibitory profile.
Among the 25 compounds, the order of inhibitory potency tends to increase with tri-substituted
position rather than di and mono substitution at benzylidene ring. Presence of the hydrogen bond donor
group at ortho, meta and para position gives higher inhibitory activity. In contrast, hydrophobic group
substituted at these positions show less inhibitory activity and thus elucidates the significance of
chemical moiety in chemical space for activity profiling. Alternatively, the top ranked binding poses of
inactive compounds i.e. compounds 14, 15, 16, 17, 20, 22, 23, 27, 28, 29, and 30 are shown in Fig.3a.
The binding mode instability clearly elucidates the inactive profiling of these compounds. As shown in
Fig.3a, these compounds do not make significant hydrogen bonding interaction with the crucial active
site residues. Binding mode analysis suggested that inactivity of these compounds is dependent upon
hydrophobic substituted group, while the active site residues are of hydrophilic in nature. The top
ranked binding poses of active compounds are shown in Fig.3b.
11

Figure-3. Shows the binding mode of the synthesized derivatives in the active site of β-glucuronidase.
(2a) inactive compounds binding modes and (2b) active compounds binding modes.
The binding mode of the potent compounds illuminates the activity profile, where the
hydrophilic nature of these compounds form stable hydrogen bond network with the key hydrophilic
residues in the active site. Consistently, the activity profile of the compounds directly depends on
magnitude of hydrogen bonding.
Conclusions
In conclusion, the molecular docking studies have evidently demonstrated the interaction pattern of the
synthetic compounds in correspondence with biological inhibitory assay. Analysis of the binding mode
clearly demonstrated that the presence of hydrophilic group, such as hydroxyl group on benzene ring
next to hydrazone part plays a key role in the activity profile. However, ortho- hydroxyl group of
benzene ring next to oxadiazole moiety did not seem to involve in inhibitory potentials.
Acknowledgment:
The authors would like to acknowledge Universiti Teknologi MARA for the financial support under
the Research Intensive Faculty grant scheme with reference number UiTM 600-RMI/DANA 5/3/RIF
(347/2012).
3. Experimental
3.1 General
NMR experiments were performed on UltraShield Bruker FT NMR 500 MHz, CHN analysis was
performed on a Carlo Erba Strumentazion-Mod-1106, Italy. Electron impact mass spectra (EI-MS)
were recorded on a Finnegan MAT-311A, Germany. Thin layer chromatography (TLC) was performed
12

on pre-coated silica gel aluminum plates (Kieselgel 60, 254, E. Merck, Germany). Chromatograms
were visualized by UV at 254 and 365 nm.
3.2 β-glucuronidase inhibition activity
β-glucuronidase (E.C. 3.2.1.31 from bovine liver, G-0251) inhibition activity of the compounds had
been evaluated by using method as reported in [41]. In this study, D-saccharic acid 1,4-lactone has
been used as a standard inhibitor [45].
3.3 Molecular Docking studies
In order to computationally demonstrate our experimental findings, molecular docking studies were
performed for all synthesized compounds to reveal their binding mode in human β-D-glucuronidase
active site. The human β-D-glucuronidase crystal structure was first retrieved from the protein data
bank (PDB code: 1BHG) [46] and the structure was optimized by removing the hetero atoms, B-chain
and co-factors. Hydrogen atoms, missing atoms, bonds and charges were computed using chimera
[47]. Similarly, the 3D chemical structures for all 25 synthesized compounds were built and optimized
using Marvin Sketch [48].
Docking studies were done using GOLD (Genetic Optimization for Ligand Docking) version 5.1 for
all the 25 synthesized compounds targeting the active site of human β-D-glucuronidase [49]. GOLD
uses genetic algorithm method for protein–ligand docking and it is well-known for its performance and
accuracy specifically for the protein targets with buried active site. [50]. GOLD software has four
scoring functions namely ChemPLP, Goldscore, Chemscore, and ASP (the Astex Statistical Potential)
which takes into account the hydrogen bonding, van der Waal and intramolecular energies. In our
previous GOLD docking study on human β-D-glucuronidase, Goldscore scoring function
outperformed well over the other scoring function [47]. Therefore, in the present study we chose
Goldscore scoring function. Docking was carried out with settings of population size (100); selection-
pressure (1.1); number of operations (10,000); number of islands niche size 2 operator ei hts
for i rate tate and cross-over he active site as defined ith a cavity 2
radius with specified centroid (x, y, z coordinates: 80.43, 84.41, 90.48) and with 100 GA run. Results
divergent by less than 0.75 Å in ligand-all atom RMSDs were clustered together. Top ranked score and
best cluster poses were saved and analyzed by PyMOL [51].
Prior to the docking study of the 25 synthetic compounds, our previous study docked bound state
conformation of the substrate p-nitrophenyl β-glucuronide on β-glucuronidase was considered as
reference in the present study [46].
13

3.4 Synthesis of 2-hydroxybenzohydrazide (2)
Methyl 2-hydroxybenzoate (1) (7.60 g, 53 mmol) and 20 ml of hydrazine hydrate were mixed in
methanol (50 mL). The mixture was refluxed for 6 hours. Methanol was then evaporated and the
product formed was being rinsed with plenty of water to remove excess hydrazine hydrate. The
product formed was left to dry at room temperature and yielded 7.21 g (94.9 %). White solid, m.p.149-
150 °C. 1H NMR (500 MHz, DMSO-d6):  4.10 (s, 2H, NH2); 6.96 (dd, J = 7.0, 2.0 Hz, 1H), 7.25 (t,
J = 7.0 Hz, 1H), 7.53 (t, J = 7.0 Hz, 1H), 7.82 (dd, J = 7.0, 2.0 Hz, 1H,), 9.98 (s, 1H, NH); 13C-NMR
(150 MHz, DMSO-d6): δ 167.8, 159.1, 133.6, 129.3, 119.7, 118.2, 115.7; Anal. Calcd for C7H8N2O2, C
= 55.26, H = 5.30, N = 18.41, found C = 55.28, H = 5.28, N = 18.42; EI MS m/z (% rel. abund.): 152
(M+, 47), 121 (100), 93 (31), 76 (28).
3.5 Synthesis of (E)-methyl 4-((2-(2-hydroxybenzoyl)hydrazono)methyl)benzoate (3)
A mixture of compound 2 (7.00 g, 46 mmol), methyl 4-formylbenzoate (7.56 g, 46 mmol) and catalytic
amount of acetic acid in methanol (50 mL) was refluxed for 3 hours. The solvent was evaporated and
the residue (3) was washed with diethyl ether, filtered, dried, and then crystallized from ethanol and
1
gives white solid, (12.8 g, 93%). m.p. 275-277 °C. H NMR (500 MHz, DMSO-d6):  3.88 (s, 3H,
CH3), 6.97 (d, J = 8.0 Hz. 1H)7.01 (t,1H, J = 8.0 Hz), 7.47 (t, J = 7.5 Hz, 1H), 7.90 (d, J = 8.0 Hz,
3H) 8.06 (d, J = 8.0 Hz, 2H), 8.52 (s, 1H) 11.97 (s, 1H, NH), 11.73 (s, 1H, OH); 13C-NMR (150 MHz,
DMSO-d6): δ 167.5, 161.6, 160.6, 149.5, 137.1, 134.5, 134.5, 129.8, 128.7, 128.7, 127.8, 127.8, 119.0,
118.8, 114.2, 52.0; Anal. Calcd for C16H14N2O4, C = 64.42, H = 4.73, N = 9.39, found C = 64.43, H =
4.74, N = 9.37; EI MS m/z (% rel. abund.): 298 (M+, 50.), 267 (30), 239 (40), 121 (100), 93 (20).
3.6 Synthesis of methyl 4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)benzoate (4)
A mixture of compound 3 (11.0 g, 37 mmol) and equivalent amount of PhI(OAc)2 was stirred in
dichloromethane (100 ml) at room temperature overnight [52]. The solvent was evaporated and the
residue (4) was washed with diethyl ether, filtered, dried, and then crystallized from ethanol to gives
1
white solid, (9.8 g, 89 %). m.p. 274-276 °C. H NMR (500 MHz, DMSO-d6): δ 3.91 (s, 3H, CH3),
7.54 (t, J = 7.5 Hz, 1H), 7.60 (t, J = 8.0 Hz, 1H), 8.14 (d, J = 8.5Hz, 1H), 8.16 (d, J = 8.0 Hz, 2H),
8.28 (d, J = 8.5 Hz, 2H,) 10.13 (s, 1H); 13C-NMR (150 MHz, DMSO-d6): δ 167.8, 165.3, 158.7, 155.1,
131.0, 130.5, 129.7, 129.7, 129.1, 128.7, 125.3, 125.3, 119.8, 118.0, 111.4, 52.3; Anal. Calcd for
C16H12N2O4, C = 64.86, H = 4.08, N = 9.46, found C = 64.87, H = 4.10, N = 9.48; EI MS m/z (% rel.
abund.): 296 (M+, 100), 265 (38), 237 (50), 161 (30), 93 (60).
14

3.7 Synthesis of 4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)benzohydrazide (5)
Compound 4 (9.00 g, 30 mmol) and 15 ml of hydrazine hydrate were mixed in methanol (50 mL). The
mixture was refluxed for 6 hours. Methanol was then evaporated and the product formed was being
rinsed with plenty of water to remove excess hydrazine hydrate. The product formed (5) was left to dry
1
at room temperature and yielded 8.50 g (94 %). m.p. 291-292 °C. H NMR (500 MHz, DMSO-d6): 
4.59 (br. s, 2H, NH2); 7.64 (dt, J = 2.0 Hz, 6.0 Hz, 1H), 7.71 (dt, J = 2.0 Hz, 6.0 Hz, 1H), 7.77 (d, J =
7.5 Hz, 1H), 8.06 (d, J = 8.5 Hz, 2H), 8.18 (dd, J = 2.0 Hz, 7.0 Hz, 1H), 8.20 (d, J = 8.5 Hz, 2H),
10.02 (s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6): δ 167.7, 165.4, 158.2, 155.6, 132.5, 132.5, 131.2,
130.1, 127.1, 127.1, 126.3, 126.3, 119.6, 118.8, 111.1; Anal. Calcd for C15H12N4O3, C = 60.81, H =
4.08, N = 18.91, found C = 60.82, H = 4.09, N = 18.89; EI MS m/z (% rel. abund.): 296 (M+, 30), 265
(100), 237 (30), 161 (20), 93 (40).
3.7.1 General procedure for synthesis of oxadiazole benzohydrazones (6-30)
Equimolar quantities (1 mmol) of compound 5 and substituted benzaldehydes (1 mmol) in methanol
(25 mL) were refluxed for 3 h, in the presence of catalytic amount of glacial acetic acid. The resulting
solid was filtered and recrystallized from methanol in good yields.
3.7.1.1. N'-(4-hydroxybenzylidene)-4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)benzohydrazide
(6)
1
Brown solid. Yield: 84.1%. m.p.: 274-275 °C. IR (cm-1, KBr): 3233.82 (N-H), 1646.10 (C=N). H
NMR (500 MHz, DMSO-d6 : δ 6 85 (d, J = 8.5 Hz, 2H), 7.06 (t, J = 7.5 Hz, 1H) 7.12 (d, J = 8.5 Hz,
1H), 7.50 (t, J = 7.0 Hz, 1H), 7.59 (d, J = 8.5 Hz, 2H), 7.96 (dd, J = 1.5 Hz, 8.0 Hz, 1H), 8.13 (d, J =
8.5 Hz, 2H), 8.24 (d, J = 8.0 Hz, 2H), 8.39 (s, 1H), 11.90 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ
165.7, 164.3, 158.4, 158.2, 155.1, 149.3, 133.1, 132.0, 131.7, 130.1, 129.2, 129.2, 128.3, 128.3, 126.2,
126.0, 126.0, 119.8, 118.8, 115.6, 115.6, 111.0; Anal. Calcd for C22H16N4O4, C = 66.00, H = 4.03, N =
13.99, Found C = 66.02, H = 4.01, N = 13.97; EI MS m/z (% rel. abund.): 400 (M+, 41), 280 (10), 265
(100), 161 (15), 93 (20).
3.7.1.2. N'-(3-hydroxybenzylidene)-4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)benzohydrazide
(7)
Yellowish white solid. Yield: 72.9%. m.p.: 254-255 °C. IR (cm-1, KBr): 3233.92 (N-H), 1656.93
(C=N); 1H NMR (500 MHz, DMSO-d6 : δ 6 85 (d, J = 7.5 Hz, 1H); 7.07 (t, J = 7.5 Hz, 1H), 7.12-7.15
(m, 2H), 7.23 (s, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.96 (d, J = 7.5 Hz, 1H), 8.14
(d, J = 8.0 Hz, 2H), 8.25 (d, J = 8.0 Hz, 2H), 8.40 (s, 1H), 9.67 (s, 1H), 12.01 (s, 1H); 13C NMR (150
MHz, DMSO-d6 : δ 165.6, 164.5, 158.8, 156.8, 155.5, 148.6, 137.3, 133.7, 132.0, 131.5, 130.9, 130.4,
15

128.38, 128.38, 126.7, 126.7, 120.0, 119.5, 119.4, 118.3, 114.6, 111.8; Anal. Calcd for C22H16N4O4, C
= 66.00, H = 4.03, N = 13.99, Found C = 65.98, H = 4.02, N = 14.01; EI MS m/z (% rel. abund.): 400
(M+, 26), 280 (15), 265 (100), 161 (21), 93 (17).
3.7.1.3.
N'-(3,4-dihydroxybenzylidene)-4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)benzo-
hydrazide (8)
1
Yellow solid. Yield: 80.9%. m.p.: 258-259 °C. IR (cm-1, KBr): 3241.92 (N-H), 1593.98 (C=N); H
NMR (500 MHz, DMSO-d6 : δ 6.96 (d, J = 6.5 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.12 (d, J = 6.0 Hz,
1H), 7.27 (s, 1H), 7.51 (t, J = 7.0 Hz, 1H), 7.96 (d, J = 7.5 Hz, 1H), 8.14 (d, J = 8.0 Hz, 2H), 8.25 (d, J
13
= 8.0 Hz, 2H), 8.30 (s, 1H), 9.27 (s, 1H), 9.41 (s, 2H), 11.78 (s, 1H); C NMR (150 MHz, DMSO-
d6): δ 165.7, 164.3, 158.4, 155.5, 148.6, 148.3, 145.6, 133.7, 132.1, 131.6, 130.4, 128.3, 128.3, 127.9,
126.0, 126.0, 121.02, 119.8, 118.4, 116.3, 115.7, 111.2; Anal. Calcd for C22H16N4O5, C = 63.46, H =
3.87, N = 13.46, Found C = 63.48, H = 3.89, N = 13.44; EI MS m/z (% rel. abund.): 416 (M+, 15), 398
(17), 265 (100), 161 (22), 93 (19).
3.7.1.4.N'-(2,5-dihydroxybenzylidene)-4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-
yl)benzohydrazide (9)
Pale yellow solid. Yield: 74.7%. m.p.: 268-269 °C. IR (cm-1, KBr): 3236.03 (N-H), 1654.27 (C=N); 1H
NMR (500 MHz, DMSO-d6 : δ 6 73-6.77 (m, 2H), 7.00 (s, 1H), 7.05 (t, J = 8.0 Hz, 1H), 7.12 (d, J =
8.5 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H), 7.96 (d, J = 7.5 Hz, 1H), 8.16 (d, J = 8.0 Hz, 2H), 8.24 (d, J =
8.0 Hz, 2H), 8.61 (s, 1H) 8.91 (s, 2H), 10.31 (s, 1H); 13C NMR (150 MHz, DMSO-d6 : δ 165.4, 164.8,
158.7, 155.2, 152.6, 151.7, 149.8, 133.2, 132.8, 131.1, 130.5, 128.8, 128.8, 126.5, 126.5, 122.7, 121.4,
119.6, 118.4, 118.4, 116.7, 111.5; Anal. Calcd for C22H16N4O5, C = 63.46, H = 3.87, N = 13.46, Found
C = 63.49, H = 3.85, N = 13.47; EI MS m/z (% rel. abund.): 416 (M+, 16), 398 (20), 265 (100), 161
(20), 93 (15).
3.7.1.5. 4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(3-nitrobenzylidene)benzohydrazide (10)
Dark brown solid. Yield: 78.2%. m.p.: 272-273 °C. IR (cm-1, KBr): 3236.24 (N-H), 1654.53 (C=N);
1H NMR (500 MHz, DMSO-d6): δ 6.87 (dd, J = 6.5, 2.0 Hz, 1H ), 7.14 (d, J = 7.0 Hz, 1H), 7.17 (t, J =
7.0 Hz, 1H), 7.24 (s, 1H), 7.11 (d, J = 8.5 Hz, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.33 (d, J = 8.5 Hz, 1H),
7.33 (dt, J = 7.5,2.0 Hz, 1H), 8.02 (dd, J = 7.0, 2.0 Hz, 1H), 8.16 (d, J = 8.0 Hz, 2H), 8.25 (d, J = 8.0
Hz, 2H); 8.41 (s, 1H) 9.66 (s, 1H, OH); 12..01 (s, 1H, NH); 13C NMR (150 MHz, DMSO-d6): δ 165.6,
164.3, 158.2, 155.2, 148.1, 147.7, 137.6, 133.8, 132.2, 132.2, 131.2, 130.5, 129.4, 128.3, 128.3, 126.7,
126.7, 124.8, 122.2, 119.5, 118.7, 111.3; Anal. Calcd for C22H15N5O5, C = 61.54, H = 3.52, N = 16.31,
16

Found C = 61.55, H = 3.57, N = 16.32; EI MS m/z (% rel. abund.): 429 (M+, 20), 383 (18), 265 (100),
161 (14), 93 (19).
3.7.1.6. 4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(4-nitrobenzylidene)benzohydrazide (11)
1
White solid. Yield: 74.4%. m.p.: 306-307 °C. IR (cm-1, KBr): 3236.30 (N-H), 1660.79 (C=N); H
NMR (500 MHz, DMSO-d6): δ 7.07 (t, J = 7.5 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 7.49-7.53 (t, J = 7.0
Hz, 1H), 7.96 (dd, J = 1.5 Hz, 7.5 Hz, 1H), 8.03-8.06 (m, 2H), 8.17 (d, J = 8.0 Hz, 2H), 8.27 (d, J =
8.0 Hz, 2H), 8.32 (d, J = 8.0 Hz, 2H), 8.59 (s, 1H), 10.39 (s, 1H), 12.33 (s, 1H); 13C NMR (150 MHz,
DMSO-d6): δ 165.3, 164.1, 158.8, 155.1, 149.4, 148.8, 139.1, 133.4, 132.4, 131.7, 130.1, 128.4, 128.4,
127.5, 127.5, 126.3, 126.3, 124.8, 124.8, 119.1, 118.6, 111.0; Anal. Calcd for C22H15N5O5, C = 61.54,
H = 3.52, N = 16.31, Found C = 61.56, H = 3.53, N = 16.29; EI MS m/z (% rel. abund.): 429 (M+, 25),
383 (40), 265 (100), 161 (19), 93 (22).
3.7.1.7.4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(2,4,6-
trihydroxybenzylidene)benzohydrazide (12)
Yellowish solid. Yield: 72.1%. m.p.: 314-315 °C. IR (cm-1, KBr): 3229.75 (N-H), 1637.93 (C=N); 1H
NMR (500 MHz, DMSO-d6 : δ 5 86 s 2H 7 7 (t, J = 8.0 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 7.51 (t,
J = 8.0 Hz, 1H), 7.96 (d, J = 7.5 Hz, 1H), 8.16 (d, J = 8.5 Hz, 2H), 8.25 (d, J = 8.0 Hz, 2H), 8.85 (s,
13
1H), 9.86 (s, 1H), 10.35 (s, 1H), 11.10 (s, 1H), 12.08 (s, 1H); C NMR (150 MHz, DMSO-d6 : δ
165.3, 164.8, 163.1, 161.3, 161.7, 158.5, 155.3, 144.5, 133.9, 132.4, 131.2, 130.9, 128.6, 128.6, 126.1,
126.1, 119.8, 118.7, 111.2, 106.4, 95.4, 95.4; Anal. Calcd for C22H16N4O6, C = 61.11, H = 3.73, N =
12.96, Found C = 61.13, H = 3.71, N = 12.94; EI MS m/z (% rel. abund.): 432 (M+, 5), 414 (20), 265
(100), 161 (22), 93 (19).
3.7.1.8.
N'-(3-hydroxy-4-methoxybenzylidene)-4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-
yl)benzohydrazide (13)
Yield: 75.5%. m.p.: 244-245 °C. IR (cm-1, KBr): 3242.90 (N-H), 1655.22 (C=N); 1H NMR (500 MHz,
DMSO-d6): δ 3.83 (s, 3H), 6.99 (d, J = 8.0 Hz, 1H); 7.06 (d, J = 8.0 Hz, 1H), 7.10 (d, J = 6.0 Hz, 1H),
7.13 (d, J = 8.5 Hz, 1H), 7.31 (s, 1H), 7.51 (t, J = 7.0 Hz, 1H), 7.97 (dd, J = 1.5 Hz, 8.0 Hz, 1H), 8.14
(d, J = 8.0 Hz, 2H), 8.24 (d, J = 8.0 Hz, 2H), 8.35 (s, 1H), 9.32 (s, 1H), 10.34 (s, 1H), 11.87 (s, 1H);
13C NMR (150 MHz, DMSO-d6): δ 165.1, 164.7, 158.3, 155.1, 149.9, 148.3, 146.7, 133.4, 132.2,
131.7, 130.1, 129.4, 128.3, 128.3, 126.5, 126.5, 120.2, 119.7, 118.1, 115.6, 115.6, 111.4, 56.7; Anal.
Calcd for C23H18N4O5, C = 64.18, H = 4.22, N = 13.02, Found C = 64.19, H = 4.24, N = 13.01; EI MS
m/z (% rel. abund.): 430 (M+, 15), 399 (18), 265 (100), 161 (20), 93 (22).
17

3.7.1.9. 4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(2-methylbenzylidene)benzohydrazide
(14)
1
White solid. Yield: 82.2%. m.p.: 228-229 °C. IR (cm-1, KBr): 3239.07 (N-H), 1656.16 (C=N); H
NMR (500 MHz, DMSO-d6): δ 2.38 (s, 3H), 7.07 (t, J = 7.5 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 7.28 (d,
J = 7.5 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 7.52 (t, J = 7.0 Hz, 1H), 7.54 (d, J = 7.5 Hz, 1H), 7.60 (s,
1H), 7.96 (dd, J = 1.5 Hz, 7.5 Hz, 1H), 8.16 (d, J = 8.5 Hz, 2H), 8.25 (d, J = 8.5 Hz, 2H), 8.46 (s, 1H),
13
10.34 (s, 1H), 12.03 (s, 1H); C NMR (150 MHz, DMSO-d6): δ 165.4, 164.3, 158.4, 155.1, 145.8,
136.3, 133.4, 132.8, 132.2, 131.7, 130.9, 130.1, 128.3, 128.2, 126.9, 126.9, 126.0, 119.6, 118.8, 111.7,
20.5; Anal. Calcd for C23H18N4O3, C = 69.34, H = 4.55, N = 14.06, Found C = 69.36, H = 4.56, N =
14.08; EI MS m/z (% rel. abund.): 398 (M+, 36), 384 (6), 265 (100), 161 (22), 93 (21), 91 (22).
3.7.1.10. 4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(3-methylbenzylidene)benzohydrazide
(15)
1
White solid. Yield: 68.9%. m.p.: 248-249 °C. IR (cm-1, KBr): 3236.93 (N-H), 1650.89 (C=N); H
NMR (500 MHz, DMSO-d6 : δ 2 48 s 3H 7 7 t J = 7.5 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 7.28-
7.32 (m, 2H), 7.35 (d, J = 7.5 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.87 (d, J = 7.5 Hz, 1H), 7.97 (d, J =
7.5 Hz, 1H), 8.17 (d, J = 8.0 Hz, 2H), 8.26 (d, J = 8.0 Hz, 2H), 8.79 (s, 1H), 10.35 (s, 1H), 12.02 (s,
1H); 13C NMR (150 MHz, DMSO-d6 : δ 165.4, 164.3, 158.4, 155.8, 145.8, 136.1, 133.8, 133.5, 132.1,
131.5, 130.5, 130.1, 128.2, 128.2, 128.0, 126.7, 126.7 126.3, 125.4, 119.8, 118.1, 111.4, 20.2; Anal.
Calcd for C23H18N4O3, C = 69.34, H = 4.55, N = 14.06, Found C = 69.33, H = 4.53, N = 14.04; EI MS
m/z (% rel. abund.): 398 (M+, 32), 384 (5), 265 (100), 161 (20), 93 (20), 91 (18).
3.7.1.11. N'-(2-chlorobenzylidene)-4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)benzohydrazide
(16)
1
White solid. Yield: 61.2%. m.p.: 251-252 °C. IR (cm-1, KBr): 3216.59 (N-H), 1650.86 (C=N); H
NMR (500 MHz, DMSO-d6): δ 7.07 (t, J = 7.5 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 7.47-7.54 (m, 3H),
7.56 (d, J = 7.0 Hz, 1H), 7.97 (dd, J = 1.5 Hz, 6.5 Hz, 1H), 8.06 (d, J = 7.5 Hz, 1H), 8.18 (d, J = 8.0
13
Hz, 2H), 8.27 (d, J = 8.0 Hz, 2H), 8.92 (s, 1H), 10.35 (s, 1H), 12.27 (s, 1H); C NMR (150 MHz,
DMSO-d6): δ 165.7, 164.6, 158.2, 155.8, 149.4, 133.4, 132.4, 132.3, 131.7, 131.1, 130.5, 130.1, 128.8,
128.8, 128.6, 127.8, 127.2, 126.6, 126.6, 119.3, 118.4, 111.6; Anal. Calcd for C22H15ClN4O3, C =
63.09, H = 3.61, N = 13.38, Found C = 63.08, H = 3.59, N = 13.37; EI MS m/z (% rel. abund.): 420
(M++2, 10), 418 (M+, 20), 383 (12) 265 (100), 161 (19), 93 (18).
18

3.7.1.12. 4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(4-methylbenzylidene)benzohydrazide
(17)
1
White solid. Yield: 77.3%. m.p.: 270-271 °C. IR (cm-1, KBr): 3233.75 (N-H), 1655.19 (C=N); H
NMR (500 MHz, DMSO-d6 : δ 2.37 (s, 3H), 7.07 (t, J = 7.5 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 7.29 (d,
J = 8.0 Hz, 2H), 7.51 (t, J = 7.5 Hz, 1H), 7.65 (d, J = 7.5 Hz, 2H), 7.97 (d, J = 7.0 Hz, 1H), 8.15 (d, J
13
= 8.0 Hz, 2H), 8.25 (d, J = 8.0 Hz, 2H), 8.46 (s, 1H), 10.35 (s, 1H), 11.98 (s, 1H); C NMR (150
MHz, DMSO-d6 : δ 165.8, 164.7, 158.2, 155.6, 149.1, 138.0, 133.4, 132.9, 131.9, 131.7, 130.1, 129.5,
129.5, 128.2 128.2, 127.5, 127.5, 126.3, 126.3, 119.8, 118.1, 111.4, 21.2; Anal. Calcd for C23H18N4O3,
C = 69.34, H = 4.55, N = 14.06, Found C = 69.35, H = 4.57, N = 14.07; EI MS m/z (% rel. abund.):
398 (M+, 30), 384 (8), 265 (100), 161 (18), 93 (24), 91 (25).
3.7.1.13.4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(2,4,5-
trihydroxybenzylidene)benzohydrazide (18)
1
Yellow solid. Yield: 89.7%. m.p.: 296-297 °C. IR (cm-1, KBr): 3233.51 (N-H), 1626.54 (C=N); H
NMR (500 MHz, DMSO-d6): δ 6.36 (s, 1H), 6.93 (s, 1H), 7.07 (t, J = 7.0 Hz, 1H), 7.13 (d, J = 8.0 Hz,
1H), 7.51 (t, J = 7.5 Hz, 1H), 7.97 (d, J = 6.5 Hz, 1H), 8.15 (d, J = 8.5 Hz, 2H), 8.25 (d, J = 8.0 Hz,
13
2H), 8.51 (s, 1H), 8.57 (s, 1H), 9.56, (s, 1H), 10.35 (s, 1H), 10.53 (s, 1H), 11.98 (s, 1H); C NMR
(150 MHz, DMSO-d6): δ 165.5, 164.2, 158.6, 155.8, 153.3, 150.5, 149.2, 140.7, 133.1, 132.5, 131.6,
130.8, 128.6, 128.6, 126.5, 126.5, 119.7, 118.4, 117.4, 112.2, 111.1, 102.5; Anal. Calcd for
C22H16N4O6, C = 61.11, H = 3.73, N = 12.96, Found C = C = 61.10, H = 3.75, N = 12.97; EI MS m/z
(% rel. abund.): 432 (M+, 10), 414 (30), 265 (100), 161 (20), 93 (15).
3.7.1.14. N'-(4-chlorobenzylidene)-4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)benzohydrazide
(19)
1
White solid. Yield: 81.6%. m.p.: 278-279 °C. IR (cm-1, KBr): 3233.34 (N-H), 1655.06 (C=N); H
NMR (500 MHz, DMSO-d6): δ 7.06 (t, J = 7.5 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 7.50 (t, J = 8.5 Hz,
1H), 7.54 (d, J = 8.0 Hz, 2H), 7.79 (d, J = 8.0 Hz, 2H), 7.96 (dd, J = 1.5 Hz, 7.5 Hz, 1H), 8.15 (d, J =
8.0 Hz, 2H), 8.26 (d, J = 8.0 Hz, 2H), 8.49 (s, 1H), 10.34 (s, 1H), 12.12 (s, 1H); 13C NMR (150 MHz,
DMSO-d6): δ 165.5, 164.7, 158.1, 155.3, 149.6, 135.2, 134.7, 133.1, 132.8, 131.6, 130.1, 129.8, 129.8,
129.5, 129.5, 128.4, 128.4, 126.7, 126.7, 119.6, 118.0, 111.0; Anal. Calcd for C22H15ClN4O3, C =
63.09, H = 3.61, N = 13.38, Found C = 63.11, H = 3.63, N = 13.39; EI MS m/z (% rel. abund.): 420
(M++2, 8), 418 (M+, 16), 383 (10) 265 (100), 161 (17), 93 (14).
19

3.7.1.15.
(20)
4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(2-nitrobenzylidene)benzohydrazide
White solid. Yield: 83.1%. m.p.: 256-257 °C. IR (cm-1, KBr): 3191.20 (N-H); 1649.84 (C=N) 1H
NMR (500 MHz, DMSO-d6): δ 7.07 (t, J = 7.5 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 7.49 (t, J = 8.5 Hz,
1H), 7.71 (t, J = 7.5 Hz, 1H), 7.85 (t, J = 7.5 Hz, 1H), 7.96 (d, J = 1.5 Hz, 1H), 8.10 (d, J = 8.5 Hz,
1H), 8.18-8.20 (m, 3H), 8.26 (d, J = 8.5 Hz, 2H), 8.93 (s, 1H), 10.35 (s, 1H), 12.36 (s, 1H); 13C NMR
(150 MHz, DMSO-d6): δ 165.7, 164.2, 158.5, 155.9, 147.1, 142.4, 133.8, 133.5, 132.1, 131.3, 130.7,
130.1, 129.0, 128.6, 128.6, 128.0, 126.5, 126.5, 125.9, 119.3, 118.6, 111.7; Anal. Calcd for
C22H15N5O5, C = 61.54, H = 3.52, N = 16.31, Found C = 61.56, H = 3.53, N = 16.29; EI MS m/z (%
rel. abund.): (M+, 10), 383 (26), 265 (100), 161 (20), 93 (25).
3.7.1.16.
N'-(2-hydroxy-5-methoxybenzylidene)-4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-
yl)benzohydrazide (21)
1
White solid. Yield: 77.2%. m.p.: 282-283 °C. IR (cm-1, KBr): 3226.13 (N-H), 1649.59 (C=N); H
NMR (500 MHz, DMSO-d6): δ 3.75 (s, 3H), 6.88 (d, J = 9.0 Hz, 1H), 6.93 (dd, J = 3.0 Hz, 9.0 Hz,
1H), 7.06 (t, J = 7.5 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 7.17 (d, J = 3.0 Hz, 1H), 7.52 (t, J = 8.5 Hz,
1H), 7.97 (dd, J = 1.5 Hz, 7.5 Hz, 1H), 8.17 (d, J = 8.5 Hz, 2H ), 8.27 (d, J = 8.0 Hz, 2H), 8.69 (s,
13
1H), 10.34 (s, 1H), 10.61 (s, 1H), 12.26 (s, 1H); C NMR (150 MHz, DMSO-d6): δ 165.6, 164.2,
158.7, 155.5, 154.4, 152.2, 149.6, 133.4, 132.9, 131.9, 130.1, 128.5, 128.5, 126.3, 126.3, 121.0, 119.8,
118.7, 116.4, 116.1, 112.4, 111.5, 56.0; Anal. Calcd for C23H18N4O5, C = 64.18, H = 4.22, N = 13.02,
Found C = 64.20, H = 4.20, N = 13.03; EI MS m/z (% rel. abund.): 430 (M+, 20), 399 (21), 265 (100),
161 (22), 93 (19).
3.7.1.17. 4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(pyridin-3-ylmethylene)benzohydrazide
(22)
Brown solid. Yield: 84.7%. m.p.: 241-242 °C. IR (cm-1, KBr): 3414.14 (N-H), 1655.04 (C=N); 1H
NMR (500 MHz, DMSO-d6): δ 7.07 (t, J = 6.5 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 7.52 (t, J = 8.5 Hz,
1H), 7.97 (dd, J = 1.5 Hz, 8.0 Hz, 1H), 8.17-8.19 (m, 3H), 8.27 (d, J = 8.0 Hz, 2H), 8.56 (s, 1H), 8.64
13
(d, J = 4.0 Hz, 1H), 8.90 (s, 1H), 10.34 (s, 1H), 12.22 (s, 1H); C NMR (150 MHz, DMSO-d6): δ
165.6, 164.3, 158.7, 155.6, 149.5, 146.1, 145.7, 135.5, 133.1, 133.0, 132.9, 131.9, 130.1, 128.6, 128.6,
126.9, 126.9, 124.1, 119.6, 118.0, 111.7; Anal. Calcd for C21H15N5O3, C = 65.45, H = 3.92, N = 18.17,
Found C = 65.47, H = 3.93, N = 18.15; EI MS m/z (% rel. abund.): 385 (M+, 29), 265 (100), 161 (20),
93 (18), 77 (17).
20

3.7.1.18. 4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(pyridin-4-ylmethylene)benzohydrazide
(23)
Yield: 80.1%. m.p.: 261-262 °C. IR (cm-1, KBr): 3229.73 (N-H), 1656.60 (C=N); 1H NMR (500 MHz,
DMSO-d6): δ 7.07 (t, J = 8.0 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 7.52 (t, J = 8.5 Hz, 1H), 7.70 (d, J =
4.0 Hz, 2H), 7.97 (dd, J = 1.5 Hz, 8.0 Hz, 1H), 8.17 (d, J = 7.5 Hz, 2H), 8.27 (d, J = 8.0 Hz, 2H), 8.50
13
(s, 1H), 8.68 (d, J = 4.0 Hz, 2H), 10.34 (s, 1H), 12.32 (s, 1H); C NMR (150 MHz, DMSO-d6): δ
165.4, 164.3, 158.4, 155.7, 150.4, 150.4, 149.1, 140.5, 133.8, 132.1, 131.4, 130.7, 128.5, 128.5, 126.7,
126.7, 122.5, 122.5, 119.8, 118.0, 111.4; Anal. Calcd for C21H15N5O3, C = 65.45, H = 3.92, N = 18.17,
Found C = 65.46, H = 3.94, N = 18.18; EI MS m/z (% rel. abund.): 385 (M+, 31), 265 (100), 161 (21),
93 (16), 77 (21).
3.7.1.19 N'-(2-hydroxybenzylidene)-4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)benzohydrazide
(24)
White solid. Yield: 46.8%. m.p.: 284-285 °C. IR (cm-1, KBr): 3228.83 (N-H); 1625.63 (C=N) 1H
NMR (500 MHz, DMSO-d6): δ 6.93-6.97 (m, 2H), 7.07 (t, J = 7.5 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H),
7.31 (t, J = 7.0 Hz, 1H), 7.50-7.54 (t, J = 8.5 Hz, 1H), 7.59 (d, J = 6.5 Hz, 1H), 7.97 (dd, J = 1.5 Hz,
8.0 Hz, 1H), 8.18 (d, J = 8.0 Hz, 2H), 8.27 (d, J = 8.5 Hz, 2H), 8.70 (s, 1H), 10.34 (s, 1H), 11.21 (s,
1H), 12.32 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ 165.3, 164.6, 158.9, 158.2, 155.4, 151.3, 133.8,
132.7, 131.6, 130.5, 129.7, 128.4, 128.4, 128.4, 126.7, 126.7, 121.9, 120.3, 119.8, 118.0, 117.3, 111.2;
Anal. Calcd for C22H16N4O4, C = 66.00, H = 4.03, N = 13.99, Found C = 66.01, H = 4.04, N = 14.02;
EI MS m/z (% rel. abund.): 400 (M+, 30), 280 (22), 265 (100), 161 (25), 93 (21).
3.7.1.20.4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(pyridin-2-ylmethylene)benzohydrazide
(25)
1
Brown solid. Yield: 84.7%. m.p.: 232-233 °C. IR (cm-1, KBr): 3213.98 (N-H), 1649.98 (C=N); H
NMR (500 MHz, DMSO-d6): δ 7.07 (t, J = 7.5 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 7.45 (t, J = 6.0 Hz,
1H), 7.49 (t, J = 7.0 Hz, 1H), 7.91 (t, J = 7.5 Hz, 1H), 7.96 (dd, J = 1.5 Hz, 8.0 Hz, 1H), 8.01 (d, J =
8.0 Hz, 1H), 8.17 (d, J = 8.0 Hz, 2H), 8.27 (d, J = 8.0 Hz, 2H), 8.53 (s, 1H), 8.64 (d, J = 4.0 Hz, 1H),
13
10.35 (s, 1H), 12.25 (s, 1H); C NMR (150 MHz, DMSO-d6): δ 165.7, 164.3, 158.3, 155.4, 154.9,
148.6, 146.1, 137.4, 133.2, 132.8, 131.9, 130.1, 128.3, 128.3, 126.4, 126.4, 123.0, 119.8, 119.8, 118.1,
111.0; Anal. Calcd for C21H15N5O3, C = 65.45, H = 3.92, N = 18.17, Found C = 65.43, H = 3.90, N =
18.16; EI MS m/z (% rel. abund.): 385 (M+, 22), 265 (100), 161 (18), 93 (21), 77 (16).
21

3.7.1.21.N'-(2-fluorobenzylidene)-4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)benzohydrazide
(26)
White solid. Yield: 69.2%. m.p.: 267-268 °C. IR (cm-1, KBr): 3235.10 (N-H); 1655.10 (C=N) 1H
NMR (500 MHz, DMSO-d6): δ 7.07 (t, J = 7.5 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 7.31 (t, J = 7.5 Hz,
1H), 7.49-7.62 (m, 4H), 7.96 (dd, J = 1.5 Hz, 7.5 Hz, 1H), 8.16 (d, J = 8.0 Hz, 2H), 8.26 (d, J = 8.0
13
Hz, 2H), 8.51 (s, 1H), 10.35 (s, 1H), 12.17 (s, 1H); C NMR (150 MHz, DMSO-d6): δ 165.4, 164.6,
160.5 (d, J = 262.0 Hz), 158.4, 155.5, 149.6, 149.1, 133.9, 132.0, 131.7, 130.7, 130.2, 130.1, 129.8,
129.0, 128.3, 128.3, 126.0, 126.0, 125.3, 125.3, 124.6, 124.5, 119.1, 118.7, 117.2, 117.0, 111.70;
Anal. Calcd for C22H15FN4O3, C = 65.67, H = 3.76, N = 13.92, Found C = 65.69, H = 3.75, N = 13.90;
EI MS m/z (% rel. abund.): 402 (M+, 27), 383 (18) 265 (100), 161 (23), 93 (20), 76 (11).
3.7.1.22. 4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(2-methoxybenzylidene)benzohydrazide
(27)
Yield: 78.6%. m.p.: 222-223 °C. IR (cm-1, KBr): 3233.17 (N-H), 1655.39 (C=N); 1H NMR (500 MHz,
DMSO-d6 : δ 3.84 (s, 3H), 7.05 (t, J = 8.5 Hz, 2H), 7.12 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 7.0 Hz, 2H),
7.40 (t, J = 8.0 Hz, 1H), 7.51 (t, J =8.0 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 8.15 (d, J = 8.0 Hz, 2H),
8.26 (d, J = 8.0 Hz, 2H), 8.48 (s, 1H), 10.39 (s, 1H), 12.07 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ
165.7, 164.4, 159.8, 158.6, 155.3, 150.1, 133.7, 132.4, 131.3, 130.6, 128.5, 128.5, 128.3, 127.9, 126.0,
126.0, 125.1, 121.5, 119.6, 118.8, 113.6, 111.0, 56.9; Anal. Calcd for C23H18N4O4, C = 66.66, H =
4.38, N = 13.52, Found C = 66.68, H = 4.36, N = 13.54; EI MS m/z (% rel. abund.): 414 (M+, 22), 383
(15) 265 (100), 161 (18), 93 (19), 76 (8).
3.7.1.23. N'-(4-fluorobenzylidene)-4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)benzohydrazide
(28)
1
White solid. Yield: 82.3%. m.p.: 225-226 °C. IR (cm-1, KBr): 3228.43 (N-H), 1651.10 (C=N); H
NMR (500 MHz, DMSO-d6 : δ 7 7 t J = 7.5 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 7.33 (t, J = 9.0 Hz,
2H), 7.51 (t, J = 8.5 Hz, 1H), 7.83 (t, J = 8.5 Hz, 2H), 7.96 (dd, J = 1.5 Hz, 8.0 Hz, 1H), 8.15 (d, J =
8.0 Hz, 2H), 8.26 (d, J = 8.5 Hz, 2H), 8.50 (s, 1H), 10.36 (s, 1H), 12.07 (s, 1H); 13C NMR (150 MHz,
DMSO-d6): 165.4, 164.3, 161.2 (d, J = 262.0 Hz), 158.4, 155.8, 149.5, 133.2, 132.9, 131.6, 130.8,
130.3, 130.3, 130.1, 128.3, 128.3, 126.0, 126.0, 119.6, 118.0, 115.7, 115.7, 111.4; Anal. Calcd for
C22H15FN4O3, C = 65.67, H = 3.76, N = 13.92, Found C = 65.68, H = 3.73, N = 13.94; EI MS m/z (%
rel. abund.): 402 (M+, 40), 383 (22) 265 (100), 161 (21), 93 (19), 76 (20).
22

3.7.1.24. N'-(3-fluorobenzylidene)-4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)benzohydrazide
(29)
1
White solid. Yield: 61.8%. m.p.: 254-255 °C. IR (cm-1, KBr): 3219.38 (N-H), 1651.54 (C=N); H
NMR (500 MHz, DMSO-d6): δ 7.07 (t, J = 8.0 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 7.31-7.35 (m, 2H),
7.49-7.53 (m, 2H), 7.96-8.00 (dd, J = 1.5 Hz, 8.0 Hz, 2H), 8.17 (d, J = 8.0 Hz, 2H), 8.26 (d, J = 8.0
Hz, 2H), 8.76 (s, 1H), 10.35 (s, 1H), 12.17 (s, 1H); 13C-NMR (150 MHz, DMSO-d6): δ 165.7, 164.3,
162.7 (d, J = 265.0 Hz), 158.4, 155.1, 148.8, 138.5, 133.4, 132.9, 131.7, 130.1, 130.0, 128.7, 128.7,
126.5, 126.5, 121.4, 119.8, 118.0, 116.3, 114.1, 111.0; Anal. Calcd for C22H15FN4O3, C = 65.67, H =
3.76, N = 13.92, Found C = 65.65, H = 3.77, N = 13.93; EI MS m/z (% rel. abund.): 402 (M+, 25), 383
(15) 265 (100), 161 (20), 93 (18), 76 (8).
3.7.1.25. N'-(3-chlorobenzylidene)-4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)benzohydrazide
(30)
1
White solid. Yield: 82.6%; m.p.: 243-244 °C, IR (cm-1, KBr): 3240.01 (N-H), 1657.34 (C=N); H
NMR (500 MHz, DMSO-d6): δ 7.07 (t, J = 7.5 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 7.50-7.53 (m, 3H),
7.73 (s, 1H), 7.82 (s, 1H), 7.96 (dd, J = 1.5 Hz, 8.0 Hz, 1H), 8.16 (d, J = 8.0 Hz, 2H), 8.26 (d, J = 8.0
Hz, 2H), 8.48 (s, 1H), 10.35 (s, 1H), 12.20 (s, 1H); 13C-NMR (150 MHz, DMSO-d6): δ 165.4, 164.3,
158.4, 155.8, 148.8, 135.3, 134.0, 133.4, 132.0, 131.7, 130.1, 130.1, 128.7, 128.7, 128.2, 127.6, 126.7,
126.7, 125.3, 119.6, 118.0, 111.5; Anal. Calcd for C22H15ClN4O3, C = 63.09, H = 3.61, N = 13.38,
Found C = 63.07, H = 3.62, N = 13.40 EI MS m/z (% rel. abund.): 420 (M++2, 6), 418 (M+, 17), 383
(10) 265 (100), 161 (16), 93 (18).
References
1. A. Husain, M. Ajmal, Synthesis of novel 1,3,4-oxadiazole derivatives and their biological
properties, Acta Pharm. 59 (2009) 223-233.
2. E. Palaska, G. Sahin, P. Kelicen, N. T. Durlu, G. Altinok, Synthesis and anti-inflammatory activity
of 1-acylthiosemicarbazides, 1,3,4- oxadiazoles, 1,3,4-thiadiazoles and 1,2,4-triazole-3-thiones, II
Farmaco 57 (2002) 101-107.
3. B. Narayana, K.K. Vijayaraj, B.V. Ashlatha, N.S. Kumari, 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. 338 (2005) 373-377.
4. M. Amir, S. Kumar, Synthesis and evaluation of anti-inflammatory, analgesic and lipid
peroxidation properties of ibuprofen derivatives, Acta Pharm. 57 (2007) 31-45.
23

5. M.A. Ali, M.S. Yar, Oxadiazole Mannich bases: Synthesis and antimycobacterial activity, Bioorg.
Med. Chem. Lett. 17 (2007) 3314-3316.
6. T.M. Tan, Y. Chen, K.H. Kong, J. Bai, Y. Li, S.G. Lim, T.H. Ang, Synthesis and the biological
evaluation of 2-benzenesulfonylalkyl-5-substituted-sulfanyl-(1,3,4)-oxadiazoles as potential anti-
hepatitis B virus agents, Antiviral Res. 71 (2006) 7-14.
7. S.L. Gaonkar, K.M. Rai, B. Prabhuswamy, Synthesis and antimicrobial studies of a new series of
2-4-[2-(5-ethylpyridin-2-yl)ethoxy] phenyl_-5-substituted-1,3,4-oxadiazoles, Eur J Med Chem. 41
(2006) 841-846.
8. A. Zarghi, S.A. Tabatabai, M. Faizi, A. Ahadian, P. Navabi, V. Zanganeh, A. Shafiee, Synthesis
and anticonvulsant activity of new 2-substituted-5-(2-benzyloxyphenyl)-1,3,4-oxadiazole, Bioorg.
Med. Chem. Lett. 15 (2005) 1863-1865.
9. H.Z. Zhang, S. Kasibhatla, J. Kuemmerle, W. Kemnitzer, M.K. Ollis, L. Qiu, C, Crogan-Grundy,
B. Tseng, J. Drewe, S.X. Cai Discovery and str ct re−activity relationship of 3-aryl-5-aryl-1,2,4-
oxadiazoles as a new series of apoptosis inducers and potential anticancer agents. J. Med. Chem.
48 (2005) 5215-5223.
10. S.H. Lee, H.J. Seo, S.H. Lee, M.E. Jung, J.H. Park, J. Yoo, H. Yun, J. Na, S.Y. Kang, K.S. Song,
M.A. Kim. Biarylpyrazolyl oxadiazole as potent, selective, orally bioavailable cannabinoid-1
receptor antagonists for the treatment of obesity. J. Med. Chem. 51 (2008) 7216-7233.
11. D.M. Cottrell, J. Capers, M.M. Salem, K. DeLuca-Fradley, S.L. Croft, K.A. Werbovetz,
Antikinetoplastid activity of 3-aryl-5-thiocyanatomethyl-1,2,4-oxadiazoles. Bioorg. Med. Chem.
12 (2004) 2815-2824.
12. R.M. Jones, J.N. Leonard, D.J. Buzard, J. Lehmann, GPR119 agonists for the treatment of type 2
diabetes. Expert Opin Ther. Pat. 19 (2009) 1339-1359.
13. P.B. Unangst, G.P. Shrum, D.T. Connor, R.D. Dyer, D.J. Schrier, Novel 1,2,4-oxadiazoles and
1,2,4-thiadiazoles as dual 5-lipoxygenase and cyclooxygenase inhibitors. J. Med. Chem. 35 (1992)
3691-3698.
14. S.V. Bhandari, K.G. Bothara, M.K. Raut, A.A. Patil, A.P. Sarkate, V. J. Mokale, Design, synthesis
and evaluation of antiinflammatory, analgesic and ulcerogenicity studies of novel S-substituted
phenacyl-1, 3, 4-oxadiazole-2-thiol and Schiff bases of diclofenac acid as nonulcerogenic
derivatives. Bioorg. Med. Chem. 16 (2008) 1822-1831.
15. X. Ouyang, E.L. Piatnitski, V. Pattaropong, X. Chen, H.Y. He, A.S. Kiselyov, A. Velankar, J.
Kawakami, M. Labelle, L. Smith, J. Lohman, S.P. Lee, A. Malikzay, J. Fleming, J. Gerlak, Y.
24

Wang, R.L. Rosler, K. Zhou, S. Mitelman, M. Camara, D. Surguladze, J.F. Doody, M.C. Tuma,
Oxadiazole derivatives as a novel class of antimitotic agents: Synthesis, inhibition of tubulin
polymerization, and activity in tumor cell lines. Bioorg. Med. Chem. Lett. 16 (2006) 1191-1196.
16. A. Almasirad, S.A. Tabatabai, M. Faizi, A. Kebriaeezadeh, N. Mehrabi, A. Dalvandi, A. Shafiee,
Synthesis and anticonvulsant activity of new 2-substituted-5-[2-(2-fluorophenoxy) phenyl]-1, 3, 4-
oxadiazoles and 1, 2, 4-triazoles. Bioorg. Med. Chem. Lett. 14 (2004) 6057-6059.
17. P. Puthiyapurayil, B. Poojary, C. Chikkanna, S.K. Buridipad, Design, synthesis and biological
evaluation of a novel series of 1,3,4-oxadiazole bearing N-methyl-4-(trifluoromethyl)phenyl
pyrazole moiety as cytotoxic agents. Eur. J. Med. Chem. 53 (2012) 203-210.
18. J.W. Watthey, M. Desai, R. Rutledge, R. Dotson, Synthesis and diuretic profile of 3-(3-amino-1,
2, 4-oxadiazol-5-yl)-5-chloro-2, 6-pyrazinediamine, an amiloride-type diuretic. J. Med. Chem. 23
(1980) 690-692.
19. H.L. Yale, K. Losee, 2-Amino-5-substituted 1,3,4-oxadiazoles and 5-imino-2-substituted-1,3,4-
oxadiazolines. A group of novel muscle relaxants J. Med. Chem. 9 (1966) 478-483.
20. K. Ohmoto, T. Yamamoto, T. Horiuchi, H. Imanishi, Y. Odagaki, K. Kawabata, T. Sekioka, Y.
Hirota, S. Matsuoka, H. Nakai, M. Toda, J.C. Cheronis, L.W. Spruce, A. Gyorkos, M. Wieczorek,
Design and synthesis of new orally active nonpeptidic inhibitors of human neutrophil elastase. J
Med. Chem. 43 (2000) 4927-4929.
21. M. Ono, M. Haratake, H. Saji, M. Nakayama, Develop ent of novel β-amyloid probes based on
3,5-diphenyl-1,2,4-oxadiazole. Bioorg. Med. Chem. 16 (2008) 6867-6872.
22. B.S. Orlek, F.E. Blaney, F. Brown, M.S.G. Clark, M.S. Hadley, J. Hatcher, G.J. Riley, H.E.
Rosenberg, H.J. Wadsworth, P. Wyman, Comparison of azabicyclic esters and oxadiazoles as
ligands for the muscarinic receptor. J. Med.Chem. 34 (1991) 2726-2735.
23. J.E. Ladbury, G. Klebe, E. Freire, Adding calorimetric data to decision making in lead discovery:
a hot tip. Nat. Rev. Drug Discovery 9 (2010) 23-27.
24. G. A. Patani, E.J. LaVoie, Bioisosterism: A rational approach in drug design. Chem. Rev. 96
(1996) 3147-3176.
25. J.S. Warmus, C. Flamme, L.Y. Zhang, S. Barrett, A. Bridges, M. Kaufman, H. Tecle, R. Gowan, J.
Sebolt-Leopold, W. Leopold, R. Merriman, S. Przybranowski, H. Valik, J. Chen, J. Ohren, A.
Pavlovsky, C. Whithead, E. Ahang, 2-Alkylamino- and alkoxysubstituted 2-amino-1,3,4-
oxadiazoles O-Alkyl benzohydroxamate esters replacements retain the desired inhibition and
selectivity against MEK (MAP ERK kinase). Bioorg. Med. Chem. Lett. 18 (2008) 6171-6174.
25

26. M.D. McBriar, J.W. Clader, I. Chu, R.A. Del Vecchio, L. Favreau, W.J. Greenlee, L.A. Hyde,
A.A. Nomeir, E.M. Parker, D.A. Pissarnitski, L. Song, L. Zhang, Z. Zhao Discovery of amide and
heteroaryl isosteres as carbamate replace ents in a series of orally active γ-secretase inhibitors.
Bioorg. Med. Chem Lett. 18 (2008) 215-219.
27. B. Sperker, J.T. Backman, K. Kromer, The role of beta-glucuronidase in drug disposition and drug
targeting in humans. Clin. Pharm. 33 (1997) 18-31
28. E. Boyland, J.E. Gasson, D.C. Williams, Enzyme activity in relation to cancer, the urinary beta-
glucuronidase activity of patients suffering from malignant disease. Brit. J. Cancer. 11 (1957)
120-129.
29. J.C. Caygill, D.A. Pitkeathly, A study of beta-acetylglucosaminase and acid phosphatase in
pathological joint fluids. Ann. Rheum. Dis. 25 (1966) 137-144.
30. N. Bank, S.H. Bailine, Urinary Beta-Glucuronidase Activity in Patients with Urinary-Tract
Infection. New. Engl. J. Med. 272 (1965) 70-75.
31. A.P. Roberts, J. Frampton, S.M. Karim, R.W. Beard, Estimation of beta-glucoronidase activity in
urinary-tract infection. New. Engl. J. Med. 276 (1967) 1468-1470.
32. A.R. Ronald, F. Silverblatt, H. Clark, R.E. Cutler, M. Turck, Failure of urinary beta-glucuronidase
activity to localize the site of urinary tract infection. App Microbiol. 21 (1971) 990-992
33. A. Schapiro, W. Paul, H.J. Gonick. Urinary beta-glucuronidase in urologic diseases of the kidneys.
Urol. 100 (1968) 146-157.
34. H.A. Kallet, L.J. Lapco, Urine beta glucuronidase activity in urinary tract disease. Urol. 97 (1967)
3523-356.
35. H.C. Gonick, H.J. Kramer, A.E. Schapiro. Urinary -glucuronidase activity in renal disease. Arch.
Int Med 132 (1973) 63-69.
36. C.M. Plum, Beta-glucuronidase activity in serum, cerebrospinal fluid and urine in normal subjects
and in neurological and mental patients. Enzymol Biol Clin. 8 (1967) 97-112.
37. G. Weissmann, R.B. Zurier, P.J. Spieler, I.M. Goldstein, Mechanisms of lysosomal enzyme
release from leukocytes exposed to immune complexes and other particles. J Exp Med. 134
(1971) 149-165.
38. a) C. Hu, V. R. Solomon, G. Ulibarri, H. Lee, The efficacy and selectivity of tumor cell killing by
Akt inhibitors are substantially increased by chloroquine, Bioorg. Med. Chem. 2008, 16, 7888-
7893; b) C. Hu, V. R. Solomon, P. Cano, H. Lee, A 4-aminoquinoline derivative that markedly
26

sensitizes tumor cell killing by Akt inhibitors with a minimum cytotoxicity to non-cancer cells,
Eur. J. Med. Chem. 2010, 45, 705-709; c) C. T. Keith, A. A. Borisy, B. R. Stockwell,
Multicomponent therapeutics for networked systems, Nature Reviews Drug Discovery, 2005, 4,
71-78; d) Y. C. Mayur, G. J. Peters, V. V. S. R. Prasad, C. Lemos, N. K. Sathish, Design of New
Drug Molecules to be Used in Reversing Multidrug Resistance in Cancer Cells, Current Cancer
Drug Targets, 2009, 9, 298-306.
39. a) V. R. Solomon, C. Hu, H. Lee, Hybrid pharmacophore design and synthesis of isatin–
benzothiazole analogs for their anti-breast cancer activity, Bioorg. Med. Chem., 2009, 17, 7585-
7592; b) B. Meunier, Hybrid Molecules with a Dual Mode of Action: Dream or Reality?, Acc.
Chem. Res., 2008, 41, 69-77; c) C. Hubschwerlen, J. L. Specklin, D. K. Baeschlin, Y. Borer, S.
Haefeli, C. Sigwalt, S. Schroeder, H. H. Locher, Bioorg. Med. Chem. Lett., 2003, 13. 4229-4233;
d) S.K. Bhanot, M. Singh, N. R. Chatterjee, The Chemical and Biological Aspects of
Fluoroquinolones Reality and Dreams, Curr. Pharm. Des., 2001, 7, 311-335; e) J. Adamec, R.
Beckert, D. Weiss, V. Klimesova, K. Waisser, U. Mollmann, J. Kaustova, V. Buchta, Hybrid
molecules of estrone: New compounds with potential antibacterial, antifungal, and
antiproliferative activities, Bioorg. Med. Chem., 2007, 15, 2898-2906; f) V. V. Kouznetsov, A.
Gomez.-Barrio, Recent developments in the design and synthesis of hybrid molecules basedon
aminoquinoline ring and their antiplasmodial evaluation, Eur. J. Med. Chem., 2009, 44, 3091-
3113.
40. Khan, K. M., Shujaat, S., Rahat, S., Hayat, S., & Choudhary, M. I.. Beta-N-cyanoethyl acyl
hydrazide derivatives: a new class of beta-glucuronidase inhibitors. Chemical & pharmaceutical
bulletin 50 (2002) 1443-6.
41. W. Jamil, S. Perveen, S.A.A. Shah, M. Taha, N.H. Ismail, N. Ambreen, K.M. Khan, M.I.
Choudhary, Phenoxyacetohydrazide Schiff bases: β-Glucuronidase inhibitors. Molecules 19
(2014) 8788-8802.
42. Zawawi, N. K. N. A., Taha, M., Ahmat, N., Wadood A., Ismail N. H., Rahim F., Ali M., Abdullah
N, Khan, K. M. (2015). Novel 2, 5-disubtituted-1, 3, 4-oxadiazoles with benzimidazole backbone:
A new class of β-glucuronidase inhibitors and in silico studies. Bioorg. Med. Chem. 23 (2015)
3119-25.
43. a) Z.S. Saify, A. Kamil, S. Akhtar, M. Taha, A. Khan, K.M. Khan, S. Jahan, F. Rahim, S. Perveen,
M.I.Choudhary, 2-(2'-Pyridyl) benzimidazole Derivatives and their Urease Inhibitory Activity.
Med. Chem. Res. 23, (2014), 4447-4454; b) K.M. Khan, F. Rahim, A. Wadood, M. Taha, M.
Khan, S. Naureen, N. Ambreen, S. Hussain, S. Perveen, M.I. Choudhary. Evaluation of Bisindole
27

as Potent β-Glucuronidase Inhibitors: Synthesis and In Silico Based Studies. Bioorg. Med. Chem.
Lett. 24 (2014) 1825-1829; c) K.M. Khan, F. Naz, M. Taha, A. Khan, S. Perveen, M.I. Choudhary,
W. Voelter. Synthesis and In Vitro Urease Inhibitory Activity of N,N'-Disubsituted Thioureas.
Eur. J. Med. Chem. 74 (2014) 314-323; d) K.M. Khan, F. Rahim, A. Wadood, N. Kosar, M. Taha,
S. Lalani A. Khan, M.I. Fakhri, M. Junaid, W. Rehman, M. Khan, S. Perveen, M. Sajid, M.I.
Choudhary. Synthesis and Molecular Docking Studies of Potentn α-Glucosidase Inhibitors Based
on Biscoumarin Skeleton. Eur J Med Chem. 81 (2014) 245-252; e) F. Rahim, K. Ullah, H. Ullah,
A. Wadood, M. Taha, A. U. Rehman, M. Ashraf, A. Shaukat, W. Rehman, S. Hussain, K. M.
Khan Bioorg. Chem. 58 (2015) 81-87; f) M. Taha, N. H. Ismail, S. Lalani, M.Q. Fatmi, Atia-tul-
Wahab, S. Siddiqui, K.M. Khan, S. Imran, M.I. Choudhary, Synthesis of Novel Inhibitors of α-
Glucosidase Based on the Benzothiazole Skeleton Containing Benzohydrazide Moiety and their
Molecular Docking Studies. Eur J. Med. Chem. 92 (2015) 387-400; g) M. Taha N.H. Ismail, W.
Jamil , H. Rashwan, S.M. Kashif, A.A. Sain, M.I. Adenan, E.H. Anouar, M. Ali, F. Rahim, K.M.
Khan. Synthesis of novel derivatives of 4-methylbenzimidazole and evaluation of their biological
activities. Eur J Med Chem. 84 (2014) 731-738; h) K.M. Khan, Z. Shah, V.U. Ahmad, N.
Ambreen, M. Khan, M. Taha, F. Rahim, S. Noreen, S. Perveen, M.I. Choudhary, W. Voelter, 6-
Nitrobenzimidazole derivatives: potential phosphodiesterase inhibitors: synthesis and structure-
activity relationship. Bioorg. Med. Chem. 20 (2012) 1521-1526.
44. K.M. Khan, F. Rahim S.A. Halim, M. Taha, M. Khan, S. Perveen, Zaheer-Ul-Haq, M.A. Mesaik,
M.I. Choudhary, Synthesis of novel inhibitors of β-glucuronidase based on benzothiazole skeleton
and study of their binding affinity by molecular docking. Bioorg Med Chem. 19, (2011), 4286-
4294
45. A) O La ren Co rt M H Effect of the β‐glucuronidase inhibitor saccharolactone on
glucuronidation by human tissue microsomes and recombinant UDP‐glucuronosyltransferases. J.
Pharm. Pharmaco. 60 (2008) 1175-1182 b) K.M. Khan, S.M. Saad, N.N. Shaikh, S, Hussain, M.I.
Fakhri, S. Perveen, M.Taha, M.I. Choudhary. Synthesis and β-glucuronidase inhibitory activity of
2-arylquinazolin-4(3H)-ones. Bioorg. Med. Chem. 22 (2014) 3449-3454; c) K.M. Khan, N.
Ambreen, M. Taha, S.A. Halim, Zaheer-ul-Haq, S. Naureen, S. Rasheed, S. Perveen, S. Ali, M.I.
Choudhary. Structure-Based Design, Synthesis and Biological Evaluation of ß-Glucuronidase
Inhibitors. J. Comput. Aided Mol. Des. 28 (2014) 577-585; d) N.K.N.Z., Abdullah, M. Taha, N.
Ahmat, A. Wadood, N.H. Ismail, F. Rahim, M. Ali, N. Abdullah, K.M. Khan, Novel 2,5-
Disubtituted-1,3,4-Oxadiazoles ith Benzi idazole Backbone: Ne Class of β-Glucuronidase
Inhibitors and In Silico Studies Bioorg. Med. Chem. (2015) In press.
28

46. S. Jain, W.B. Drendel, Z.W. Chen, F.S. Mathews, W.S. Sly, J.H. Grubb. Nat. Struct. Biol. 3
(1996) 375-381.
47. E. F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng, T.E. Ferrin,
UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput Chem. 25
(2004) 1605-1612
48. Product version: MarvinSketch
http://www.chemaxon.com
V
5.2.6, copyright 1998-2009 ChemAxon Ltd,
49. G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, Development and validation of a genetic
algorithm for flexible docking. J. Mol. Biol. 267 (1997) 727-748.
50. G.T. Costakes, Overview of current docking programs. MMG 445 Basic Biotechnology e
Journal 1 (2006).
51. Schrödinger LLC (2010), The PyMOL Molecular Graphics System. 1.3 ed.
52. M. Taha, N.H. Ismail, S. Imran, K.M. Khan, 4-[5-(2-Methoxyphenyl)-1,3,4-oxadiazol-2-
yl]benzohydrazide. Molbank M826 (2014).
Captions of figures and tables
Figure-1 Comparison between hydrazide, oxadiazole, hydrazone, and hybrid compound 13.
Figure-2 Shows the putative binding mode of the potent compounds among the synthesized
derivatives in the active site of β-glucuronidase. The key interacting residues are shown in magenta
color line and compound in stick representation, while the hydrogen bonds are shown in yellow dashed
line. Compound 13 in split pea color (1a), Compound 9 in dark green color (1b), Compound 6 in
orange color (1c), Compound 18 in fluorescent color (1d), Compound 8 in pale yellow (1e),
Compound 7 in dirty violet color (1f), Compound 25 in blue white (1g) and Compound 11 in light
pink color (1h).
Figure-3 Shows the binding mode of the synthesized derivatives in the active site of β-glucuronidase.
(2a) inactive compounds binding modes and (2b) active compounds binding modes.
Scheme-1. Synthesis of benzohydrazide having oxadiazole ring compound 5
Scheme-2. Synthesis of novel oxadiazole benzohydrazones 6-30
Table-1. Structure of oxadiazole benzohydrazones 6-30
Table-2. In vitro β-glucuronidase inhibition activity of oxadiazole derivatives 6-30 and their docking
studies Goldscore results.
29