Synthesis, β-Glucuronidase Inhibition, and Molecular Docking Studies of 1,2,4-Triazole Hydrazones

Article abstract of DOI:10.1007/s13738-018-1433-9

A series of 1,2,4-triazole hydrazones 1–25 has been synthesized and characterized using different spectroscopic techniques including FT-IR, ¹H-NMR, and ESI MS spectrometry. The synthetic derivatives were evaluated for their β-glucuronidase enzy

Full text of DOI:10.1007/s13738-018-1433-9

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-018-1433-9
ORIGINAL PAPER
Synthesis, β-Glucuronidase Inhibition, and Molecular Docking Studies
of 1,2,4-Triazole Hydrazones
1
1
2
3
4
Waqas Jamil · Darshana Kumari · Muhammad Taha · Muhammad Naseem Khan · Mohd Syukri Baharudin ·
7
5
6
5
Muhammad Ali · M. Kanwal · Muhammad Saleem Lashari · Khalid Muhammad Khan
Received: 27 March 2018 / Accepted: 7 June 2018
©
Iranian Chemical Society 2018
Abstract
A series of 1,2,4-triazole hydrazones 1–25 has been synthesized and characterized using different spectroscopic techniques
1
including FT-IR, H-NMR, and ESI MS spectrometry. The synthetic derivatives were evaluated for their β-glucuronidase
enzyme inhibition properties. Among them, 17 compounds demonstrated potential inhibitory activity towards β-glucuronidase
with IC values ranging between 2.50 and 53.70 µM. Compounds 1 having IC =2.50± 0.01 µM was found to be the most
5
0
50
active compound of the series and showed remarkable activity and found to be far more potent than the standard d-saccharic
acid 1,4-lactone (IC =48.4± 1.25 µM). Furthermore, the possible binding interaction of active compounds was explored
5
0
by in silico studies. These compounds can be used for anti-diabetic drug development process.
Keywords 1,2,4-Triazole hydrazone · β-Glucuronidase enzyme inhibition · Anti-diabetic · In silico studies
Introduction
as triadimefon, triadimenol, flusilazole, and flupoxam have
triazole scaffold [1] (Fig. 1).
Heterocyclic compounds have the top-most priority in drug
discovery and development due to their unique ability to
bind with various biomolecules, particularly the nitrogenous
heterocyclic compounds gained more attention in this field.
Among them, 1,2,4-triazole derivatives have been reported
as very important class of heterocyclic compounds with
diverse pharmacological, agricultural, as well as some
industrial applications. Many of the commercial drugs such
Previously, 1,2,4-triazole derivatives have been reported
for their antiparasitic [2], anti-inflammatory [3, 4], herbicidal
[5], antifungal [6, 7], antimicrobial [8], brassinosteroid bio-
synthesis inhibitory [9], and cytostatic activities [10]. Beside
this, the hydrazones (molecules containing –C=N– bonding)
are also molecules of interest due to their unique application
in many fields of chemistry [11]. Triazole hydrazones are
also reported for their phosphodiesterase inhibitory activity
[
12].
Diabetes mellitus (DM) is a syndrome of irregular car-
bohydrate and protein metabolism that produces some acute
chronic complications due to relative deficiency of insulin.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s13738-018-1433-9) contains
supplementary material, which is available to authorized users.
4
*
Waqas Jamil
Atta-ur-Rahman Institute for Natural Product Discovery,
waqas.jamil@usindh.edu.pk; waqasjam2@yahoo.com
Universiti Teknologi MARA (UiTM), Puncak Alam Campus,
Bandar Puncak Alam, 42300 Selangor, Malaysia
1
2
Institute of Advance Research Studies in Chemical Sciences,
University of Sindh, Jamshoro 76080, Pakistan
5
H. E. J. Research Institute of Chemistry, International Center
for Chemical and Biological Sciences, University of Karachi,
Karachi 75270, Pakistan
Department of Clinical Pharmacy, Institute for Research
and Medical Consultations (IRMC), Imam Abdul Rehman
Bin Faisal University, P.O. Box 1982, Dammam 31441,
Saudi Arabia
6
Department of Chemistry, University of Education, Campus
Dera Ghazi Khan, Lahore, Punjab, Pakistan
7
UoN Chair of Oman’s Medicinal Plants and Marine Natural
3
Department of Chemistry, COMSATS Institute
of Information Technology, University Road,
Abbottbad KPK 22060, Pakistan
Products, University of Nizwa, P.O. Box 33, Birkat Al-Mouz,
Nizwa 616, Oman, Oman
Vol.:(0
1
123456789)
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Journal of the Iranian Chemical Society
Fig. 1 Triazole ring containing
N
commercial drugs
O
N
F CF C
3 2
N
N
Cl
O
N
N
O
N
Cl
N
O
O
N
N
Si
N
Cl
NH2
N
OH
F
F
Flusilazole
Triadimefon
Triadimenol
Flupoxam
Hyperglycaemia is the most common marker of diabetics. It
has an adverse effect on vascular cells during the progression
of diabetes and creates vascular complications. Moreover,
individuals with diabetes may be more likely to develop peri-
odontal and cardiovascular diseases than healthy individuals.
These all functions may control by the lysosomal enzymes.
Defects in the lysosomal enzymes provoke an accumula-
tion of non-degraded molecules in the lysosomal system.
The lysosomal enzyme, β-glucuronidase is found in spleen,
lungs, biles, urine, etc. It is responsible for the catalytic deg-
radation of glucuronosyl-O-bonds and glycosaminoglycans,
including heparin, chondroitin, and dermatan sulphates. The
high expression of β-glucuronidase enzyme was reported in
many types of malignancies such as melanomas, bronchial
tumour, and breast gastrointestinal tract carcinoma. The ele-
vated levels of β-glucuronidase enzyme were found in bor-
derline tuberculoid and lepromatous patients. Besides these,
the low level of this enzyme may cause mucopolysacchari-
dosis type VII (MPS VII; Sly Syndrome). β-Glucuronidase
and some other enzymes have been found to be up-regulated
in many acute and chronic pathological conditions such as
trauma, sepsis, and diabetes mellitus [13–16].
we are reporting the β-glucuronidase inhibitory activities of
1,2,4-triazole hydrazone derivatives. The inhibitory activi-
ties were also supported by in silico studies (Fig. 2).
Results and discussion
Chemistry
The triazole derivatives 1–25 were synthesized by reflux-
ing 2-amino-1,2,4-triazole with different substituted aryl
carbonyl derivatives (aldehydes and ketones) (Scheme 1).
The periodical TLC analysis is used to monitor the reaction.
After refluxing and solvent evaporation, the crude product
was crystallized from ethanol and pure triazole hydrazones
1–25 were obtained in excellent yield (Table 1 and Fig. 3).
β‑glucuronidase inhibition activity
All the synthetic triazole hydrazone derivatives 1–25 with
different substitution on aryl part obtained from aldehyde or
ketone were evaluated for their β-glucuronidase inhibitory
activities and results were compared with the standard d-sac-
charic acid 1,4-lactone (IC =48.4± 1.25 µM) (Table 2).
Various approaches were used to control diabetes through
different modes of action such as gluconeogenesis inhibition,
insulin release stimulation, increase of glucose transporters,
and delaying glucose absorption in the intestine [17–19].
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Compounds 1–25 showed remarkable β-glucuronidase
inhibitory activity with IC values in the range of
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2
.50–33.50 µM. Out of 25, 17 triazole hydrazone deriva-
Rationale of the current study
tives 1 (IC = 2.50 ± 0.01 µM), 2 (9.40 ± 0.25 µM), 3
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(
4.60± 0.01 µM), 4 (3.60± 0.01 µM), 5 (24.30± 0.55 µM),
For the exploration of new lead compounds as antidiabetic
agents, earlier, we have reported many heterocyclic deriva-
tives such as hydrazones A, oxadiazoles B, and flavones
C with β-glucuronidase, antiglycation, and α-glucosidase
activities, respectively [20–24]. In addition to that the stand-
ard d-saccharic acid 1,4-lactone, having one five-member
heterocyclic ring with adjacent electron-donating substitu-
ents, i.e. hydroxyl and carboxylic groups. Both of these parts
may interact with the enzyme efficiently, in the same manner
our synthetic compounds also possess these two structural
features, i.e. heterocyclic triazole ring and substituted aryl
part (electron-donating/withdrawing substituents). So, it can
be hypothesized that these compounds may also be bound to
the protein as the standard. Therefore, in continuation of our
previous work and resemblance with the standard, herein,
6 (22.10 ± 0.50 µM), 7 (23.510 ± 0.40 µM),
8 (29.60 ± 0.50 µM), 9 (9.40 ± 0.20 µM), 10
(13.50± 0.30 µM), 12 (43.10± 0.70), 13 (14.20± 0.35 µM),
14 (34.10 ± 0.65 µM), 15 (32.20 ± 0.21 µM), 16
(33.50± 0.63 µM), and 17 (19.40± 0.30 µM) showed supe-
rior activity than the standard d-saccharic acid 1,4-lactone
(IC =48.4± 1.25 µM) (Table 2).
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Structure–activity relationship was evaluated in terms
of substitution pattern on aryl part obtained from carbonyl
derivatives. The most active compound of this series is com-
pound 1 (IC = 2.50 ± 0.01 µM) and it was derived from
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isatin. The structural similarity with the standard having
five-member ring with carbonyl at 2nd position with adja-
cent –NH moiety may be the reason for superb activity. In
the same manner, compounds 15 (IC =32.20± 0.21 µM)
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Journal of the Iranian Chemical Society
Fig. 2 Rationale of the current study
inhibition potential. It is worth noting that mixed chloro
substitution with electron-donating group, i.e. –OH as in
compound 4 (IC =3.60± 0.01 µM), showed an enhanced
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0
activity, while the addition of electron withdrawing such
as –NO group with chloro substitution reduced the activity
2
up to fivefolds as in compound 5 (IC =24.30± 0.55 µM)
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Scheme 1 Reaction of 1,2,4-triazole Schiff base derivatives 1–25
(
(
Fig. 5).
The 2,5-di-hydroxyl-substituted compound 9
IC = 9.41 ± 0.20 µM) showed higher activity while
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and 16 (IC =33.50± 0.63 µM) derived from furfural and
mono –OH derivatives 6 (IC = 22.10 ± 0.50 µM) and 7
5
0
50
5
-methyl furfural also marked for their magnificent activity.
(IC =23.510± 0.40 µM) showed lesser activity than com-
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These derivatives also have five-member ring system but
these derivatives were not as active as compound 1. The
suppression in activity might be due to aromatic nature of
five-member ring or non-availability of carbonyl group at
pound 9. The decline in activity was observed in compound
8 (IC = 29.60 ± 0.50 µM) containing –OH group with
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additional –OCH group at 3rd position. However, all these
3
derivatives showed higher activity than the standard (Fig. 6).
Among electron-withdrawing substituents nitro deriva-
tives, compounds 10 (IC = 13.50 ± 0.30 µM) and 12
2
nd position, but still they showed higher activity than stand-
ard (Fig. 4).
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Among chloro-substituted derivatives, the 2,4-dichloro-
substituted compound 3 (IC =4.60± 0.01 µM) was found
(IC = 43.10 ± 0.70 µM) containing 2 and 4 –NO group,
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2
respectively, showed better potential than the standard,
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to be most active compound. The 3-chloro-substituted
whereas compound 11 with 3-NO (IC =53.70± 0.80 µM)
2
50
compound 2 (IC =9.40± 0.25 µM) also showed excellent
substitution was found to be a less active compound (Fig. 7).
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Table 1 Compounds 1–25 with yields and melting points
S. No.
R
M.P .°C Yield(%)
1
172
49
2
3
4
170
190
194
90
60
75
5
140
57
6
198
70
Fig. 3 General structure of 1,2,4-triazole Schiff bases 1–25
7
140
98
Table 2 β-GlucuronidaseInhibition activity of 1,2,4-triazole Schiff
bases 1–25
8
9
160
81
S. no.
IC50 ± SEM (µM)
220
120
164
68
79
60
1
2
3
4
5
6
7
8
9
1
1
1
1
1
2.50± 0.01
9.40± 0.25
4.60± 0.01
3.60± 0.01
24.30± 0.55
22.10± 0.50
23.51± 0.40
29.60± 0.50
9.41± 0.20
13.50± 0.30
53.70± 0.80
43.10± 0.70
34.10± 0.65
34.10± 0.65
32.20± 0.21
33.50± 0.63
19.40± 0.30
NA
NA
NA
NA
NA
NA
NA
NA
48.4± 1.25
1
1
0
1
1
1
2
3
240
210
130
73
75
90
1
4
0
1
2
3
4
1
1
1
5
6
7
122
78
36
42
90
200
15
1
6
1
8
110
80
17
1
8
1
2
9
0
100
130
60
90
19
2
0
21
2
2
2
1
121
64
23
2
2
4
5
2
2
2
2
3
4
212
250
240
88
70
74
d-saccharic acid 1,4-lactone
SEM standard error mean, NA not active
2
5
70
-
48
-
The compounds 13 (IC = 14.20 ± 0.35 µM) and 14
5
0
-
-
(IC = 34.10 ± 0.65 µM) with substitutions N,N-di-CH
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3
and SCH at 4th position, respectively, showed signifi-
3
cant β-glucuronidase inhibition potential. Compound 17
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Journal of the Iranian Chemical Society
Fig. 4 Structure–activity rela-
tionship of compounds 1, 15,
and 16
Fig. 5 Structure–activity relationship of compounds 2, 3, 4, and 5
Fig. 6 Structure–activity relationship of compounds 6, 7, 8, and 9
Fig. 7 Structure–activity rela-
tionship of compounds 10, 11,
and 12
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Journal of the Iranian Chemical Society
(
IC =19.40± 0.30 µM) with two triazole ring also showed
50
better activity than the standard. All other derivatives were
found to be inactive against β-glucuronidase (Fig. 8).
The above activity pattern suggested that substitution at
2
nd position of aryl part may help to enhance the activity,
as in most cases 2-substituted derivatives showed superior
activity than the standard, but some other factors such as
polarity, ability to form hydrogen bond, size, hydrophilic
or hydrophobicity and electron-donating or -withdrawing
nature of group may also be responsible for activity.
To understand the binding mode, some of the active com-
pounds were subjected for in silico studies and results are
discussed in following paragraphs.
Molecular docking studies
In silico studies were carried out to support the in vitro
β-glucuronidase enzyme inhibitory potential studies. The
docking of most active molecules 1–4, 9, and 11 with
β-glucuronidase enzyme discloses that all the inhibitor
ligands are displaying the bonding with different amino
acids in the active site (Fig. 9).
Fig. 9 PDB human β-glucuronidase enzyme’s structure (ID: 1BHG)
Structure–activity relationship (SAR) was evaluated by
three-dimensional human β-glucuronidase structure. For our
study, X-ray crystal structure of human β-glucuronidase was
downloaded from Protein Data Bank (http://www.rcsb.org/
pdb) (PDB ID: 1BHG) (Fig. 9). Before docking the syn-
thetic derivatives, the protein active site interaction was
studied by docking the standard substrate molecule with it
by Autodock Vina and Autodock Tools [25–27].
First, most active derivative 1 (IC = 2.50 ± 0.01 µM)
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was predicted for its binding conformations to the straight
binding groove of β-glucuronidase. Graphical investiga-
tion of the top-most graded pose of compound 1 showed
that the amine group of indolin-2-one mediated interac-
tion with the side chain carboxyl oxygen (Oε1) of Glu540
(2.43 Å) and hydroxyl oxygen (Oη) of Tyr504 (2.4 Å)
via strong hydrogen bonding. These two hydrogen bonds
could be the main reason of the high inhibition poten-
tial of compound 1. The phenyl ring of indolin-2-one is
involved in π–anion and π–π interaction with the Glu451
(3.57 Å) carbonyl residue and phenyl ring of Tyr504
(5.0 Å), respectively. Additionally, carbon–hydrogen
bond interaction is found between =CH– of triazole group
and the carbonyl oxygen of Tyr205 (3.71 Å) along with
π-donor hydrogen bond between triazole ring and amine
group of Asp207 (2.66 Å) (Fig. 10e). Other residues such
as Phe208, His385, Val410, Asn450, Asn484, Tyr508,
The modelled structure of d-saccharic acid 1,4-lactone-
bound with human β-glucuronidase displayed the accu-
rate orientation of substrate towards the catalytic residues
Glu451 and Glu540 (Fig. 10a). During catalysis in human
β-glucuronidase, it is proposed that Glu451 acts like acid/
base catalyst, whereas Glu540 has nucleophilic characteris-
tics [28]. As shown in Fig. 10a, the inhibitor precisely fits
into the active site and interacts via hydrogen bonds with
the receptor active site residues including Asp207, His385,
Asn450, Glu540 and Lys606. The binding models most
active compounds obtained after docking shown in Fig. 10.
Fig. 8 Structure–activity rela-
tionship of compounds 13, 14,
and 17
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Journal of the Iranian Chemical Society
Fig. 10 Interaction of a. d-Saccharic acid 1,4-lactone, b 3, c 4, d 2, and e 1, and f 9 with β-glucuronidase
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Journal of the Iranian Chemical Society
Fig. 10 (continued)
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Journal of the Iranian Chemical Society
Trp587, and Lys606 stabilize the most active compound
1
.
Binding mode of compound 4 which is the second-most
active derivative of the series (IC = 3.60 ± 0.01 µM)
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showed that one nitrogen atom of the triazole ring is inter-
acting through strong hydrogen bonding with the amino
residue (Hζ3) of Lys606 (2.45 Å). Other van der Waals,
π–π, π–anion and π–cation interactions of the phenyl
and triazole rings of 4 with the side residues especially
Glu451 and Glu540, can be visualized in Fig. 10c. These
interactions stabilize the second-most potent compound
4
in the active site of enzyme.
T h e b i n d i n g m o d e o f a n a l o g u e
3
(
IC = 4.60 ± 0.01 µM) showed that the amine group of
50
triazole ring interacts with the carboxyl oxygen (Oδ2)
counterpart of Asp207 (2.52 Å) via hydrogen bonding,
while one of the imine nitrogen of triazole ring is involved
in forming two hydrogen bonds with two side residues,
i.e. hydroxyl group of Tyr508 (2.18 Å) and amine hydro-
gen (Hζ2) of Lys606 (3.09 Å), respectively (Fig. 10b).
π–Anion interaction was also found between the triazole
ring and the carboxyl oxygen (Oε2) of Glu540 (3.63 Å).
Docking result of another active compound 2
Fig. 11 Hydrogen bonds in the binding pocket of β-glucuronidase
1BHG) and the distance (in Angstrom) of compound 11
(
Conclusion
Triazole hydrazones 1–25 with different functional groups
have been synthesized and their β-glucuronidase activity was
explored. Among them, 17 compounds showed remarkable
β-glucuronidase inhibition activity. The results were also
supported by molecular docking studies which identified
various structural features that interact with the active site of
enzyme. This work will enable us to identify new inhibitors
and structural features contributing towards β-glucuronidase
inhibition activity.
(
IC = 9.41 ± 0.25 µM) revealed that the imine nitrogen
50
is engaged in hydrogen bonding with the hydroxyl residue
of Tyr508 (2.45 Å), while one of the imine nitrogen of
triazole group was also interacting with the amine hydro-
gen (Hζ3) of Lys606 (2.66 Å). Carbon–hydrogen bond
interaction was spotted between hydrogen of imine group
and carboxyl oxygen (Oε1) of Glu451 (3.69 Å). Carboxyl
oxygen (Oε2) of Glu540 was also spotted in π–anion
interaction (3.49 Å) with the triazole ring of 2 (Fig. 10d).
Binding interaction of 9 (IC = 9.40 ± 0.20 µM)
Supplementary information
1
5
0
The H NMR spectra of most active compounds are available
showed that the imine nitrogen of triazole ring is engaged
with hydroxyl group of Tyr508 (2.24 Å) by hydrogen
bonding, while the triazole ring was found involved in
π–anion interaction with the carbonyl oxygen (Oε2) of
Glu540 (3.50 Å) (Fig. 10f).
in supplementary information.
Experimental
Compound 11 (IC = 53.70 ± 0.80 µM) showed lesser
Materials and methods
5
0
activity but still it showed good interaction with the bind-
ing site as its IC value is negligibly higher than the
The β-glucuronidase was purchased from Sigma-Aldrich,
USA. 3-Amino-4(H)-1,2,4-triazole, and various substituted
aryl aldehydes/ketone were purchased from Merck/TCI,
Germany/Japan. Dried ethanol was used in all experiments
and Avance Bruker AM 300, 500 MHz machines were used
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standard compound, i.e. d-saccharic acid 1,4-lactone
(
Fig. 10a)
Hydrogen bond surface of predicted docked poses
of all the selected compounds inside the active site of
β-d-glucuronidase was analyzed. The results show that
the ligand exactly fits into the active site of the enzyme
1
13
for H- and C-NMR experiments, respectively. Agilent
6330 Ion Trap instrument using positive or negative mode
was used for ESI MS determination. CHNS analyses were
performed on a Perkin Elmer, Milano, Italy. Kieselgel 60,
254, E. Silica gel-coated aluminium plates, Merck, Ger-
many, were used for thin-layer chromatography. UV light
source with wavelengths 254 and 365 nm were used for TLC
visualization.
(
Fig. 11).
The line with black dashes indicates different inter-
actions and the distances are in Angstrom. Amino acid
residues involved in interaction are shown in element col-
oured stick (Figs. 11, 12).
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Journal of the Iranian Chemical Society
Fig. 12 The Hydrogen bonding pattern of the d-saccharic acid 1,4-lactone (a) and 1,2,4-triazole derivatives with the of β-d-
glucuronidase’sactive site: compounds 3 (b), 4 (c), 2 (d), 1 (e), and 9 (f)
Typical method for synthesis of compounds 1–25
amount of 3-amino-1,2,4-triazole was added into it and
refluxed for 6 h. After refluxing, solvent was evaporated
under rotary evaporator; the obtained precipitates were
washed with water, dried, and characterized through vari-
ous spectroscopic techniques.
Variously substituted aryl aldehydes (0.03 moles) were
dissolved in 25 mL of ethanol with catalytic amount of
acetic acid, reaction mixture was stirred and equimolar
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Journal of the Iranian Chemical Society
3-(1H-[1, 2, 4]-Triazol-3-ylimino)-1,3-dihydro-indol-2-one
2-Chloro-5-nitro-benzylidene-(1H-[1, 2,
(1)
4]-triazol-3-yl)-amine (5)
1
1
Yield: 81%; H-NMR (DMSO-d ): δ 8.40 (s, 1H), 8.07
Yield: 57%; H-NMR (DMSO-d ): δ 9.13 (s, 1H), 8.51(s,
6
6
1
3
13
(
m, 2H), 7.48 (m, 2H); C-NMR (DMSO-d , 125 MHz):
1H), 7.60–7.12 (m, 3H); C-NMR (DMSO-d , 125 MHz): δ
6
6
δ 163.1, 161.3, 146.4, 146.3, 138.5, 131,3, 129.1,
163.3, 146.4, 146.3, 146.5, 140.2, 132.4, 129.6, 127.2, 125.1
+
+
+
−1
1
24.0, 123.2, 120.2: ESI MS (m/z): 214.1 [M + 1] ;
ESI MS (m/z): 254.0 [M+3] , 252 [M+1] ; FTIR (νcm ):
−
1
FTIR (νcm ): –NH– 3189.54, –C=O– 1724.12,
C=N– 1614.10, –C=C– 1482.73, –C–N– 1330.61; anal.
calcd. for C H N O, C = 56.32, H = 3.30, N = 32.84,
–NH– 3244.91, –N–C– 1342.28, –N=C– 1574.50, –NO
2
–
1523.34, –C=C– 1487.34, –C–Cl– 875.15; anal. calcd.
for C H ClN O , C = 42.96, H = 2.40, N = 27.83, found,
1
0
7
5
9
6
5
2
found, C = 55.31, H = 3.29, N = 32.87.
C=42.93, H=2.38, N=27.80.
2
-[(1H-[1,2,4]-Triazol-3-ylimino)-methyl]-phenol (6)
(
3-Chloro-benzylidene)-(1H-[1, 2, 4]-triazol-3-yl)-amine (2)
1
Yield: 70%; H-NMR (DMSO-d ): δ 9.92 (s, 1H), 7.99 (d,
6
1
Yield: 90%; H-NMR (DMSO-d ): δ 9.43 (s, 1H),
1H, J = 8.5 Hz), 7.59 (m, 2H), 7.08 (d, 1H, J = 8.4 Hz),
6
1
3
8
.57 (s, 1H), 7.92 (d, 1H, J = 8.3 Hz), 7.49 (dd, 1H,
6.92 (s, 1H); C-NMR (DMSO-d , 125 MHz): δ 163.4,
6
1
3
J = 7.9 Hz), 7.04 (d, 1H, J = 8.3 Hz); C-NMR (DMSO-
157.6, 146.5, 146.4, 132.1, 130.2, 121.1, 118.0, 115.3; ESI
+
− 1
d , 125 MHz): δ 163.5, 146.4, 146.3, 133.6, 132.4, 131.1,
MS (m/z): 189.0 [M + 1] ; FTIR (νcm ):–OH– 3251.93,
–NH– 3123.01, –C=N– 1612.18, –N–C– 1353.24; anal.
calcd. for C H N O, C=56.44, H=4.28, N=29.75, found,
6
+
1
30.1, 129.2, 127.3: ESI MS (m/z): 219.1 [M + 3] , 207
+
−1
[
M+1] ; FTIR (νcm ): –NH– 3233.02, –N=C– 1586.27,
C–N– 1329.00, –C=C– 1477.10, –C–Cl– 835.74; anal.
calcd. for C H ClN , C = 51.29, H = 3.40, N = 26.12,
9
8
4
–
C=56.41, H=4.26, N=28.75.
9
7
4
found, C = 51.28, H = 3.40, N = 26.09.
3-[(1H-[1,2,4]-Triazol-3-ylimino)-methyl]-phenol (7)
1
Yield: 98%; H-NMR (DMSO-d ): δ 9.17 (s, 1H), 8.52 (s,
6
(
(
2,4-Dichloro-benzylidene)-(1H-[1, 2, 4]-triazol-3-yl)-amine
3)
1H), 7.93 (d, 1H, J = 9.1 Hz), 7.72 (m, 1H), 7.40 (d, 1H,
1
3
J = 9.4 Hz); C-NMR (DMSO-d , 125 MHz): δ 163.2,
6
1
57.6, 146.5, 146.4, 132.2, 130.1, 121.6, 118.5, 116.1; ESI
1
+
−1
Yield: 60%; H-NMR (DMSO-d ): δ 9.02 (s, 1H), 8.27
MS (m/z): 189.0 [M+1] ; FTIR (νcm ): OH/NH– 3239.31,
6
(
s, 1H) 7.81 (d, 1H, J = 8.4 Hz), 7.34 (d, 1H, J = 8.2 Hz),
–C=N– 1582.11, C=C– 1493.92, –C–N– 1338.33; λ
max
1
3
6
1
.92 (s, 1H); C-NMR (DMSO-d , 125 MHz): δ 163.2,
(nm): 310. anal. calcd. for C H N O, C=57.43, H=4.27,
6
9
8
4
46.5, 146.4, 137.3, 135.5, 131.3, 129.2, 129.1, 126.9;
N=29.78, found, C=57.41, H=4.25, N=28.76.
+
+
ESI MS (m/z): 245.0 [M + 5] , 243 [M + 3] , 243
+
− 1
[
M + 1] ; FTIR (νcm ): NH– 3123.27, –C=N– 1605.13,
C=C– 1440.63, –N–C– 1319.34, –C–Cl– 799.70; anal.
calcd. for C H Cl N , C = 44.82, H = 2.50, N = 23.23,
3-Methoxy-4-[(1H-[1,2,4]-triazol-3-ylimino)-methyl]-phenol
(8)
–
9
6
2
4
1
found, C = 44.79, H = 2.45, N = 23.19.
Yield: 81%; H-NMR (DMSO-d ): δ 8.43 (s, 1H), 8.30 (s,
6
1
H), 8.29 (s, 1H), 8.27 (d, 1H, J = 7.6 Hz), 7.47 (d, 1H,
1
3
J=7.3 Hz), 1.32 (s, 3H); C-NMR (DMSO-d , 125 MHz):
6
5-Chloro-2-[(1H-[1,2,4]-triazol-3-ylimino)-methyl]-phenol
δ 163.7, 162.5, 160.1, 146.3, 146.2, 131.1, 109.4, 108.7,
+
− 1
(4)
101.2, 56.2; ESI MS (m/z): 219 [M + 1] ; FTIR (νcm ):
–
OH/NH– 3104.36; –C=C– 1538.80, –C=N– 1579.53,
1
Yield: 75%; H-NMR (DMSO-d ): δ 7.46 (d, 1H,
–C–N– 1330.07; anal. calcd. for C H N O C = 55.05,
10 10 4 2,
6
J = 8.2 Hz), 7.42 (d, 1H, J = 8.1 Hz), 7.40 (s, 1H), 7.06
H=4.63, N=25.69, found, C=55.03, H=3.58, N=24.64.
1
3
(
s, 1H), 7.04 (s, 1H); C-NMR (DMSO-d , 125 MHz):
6
δ 163.1, 159.5, 146.4, 146.6, 137.3, 131.7, 121.3, 116.4,
2-[(1H-[1,2,4]-Triazol-3-ylimino)-methyl]-benzene-1,4-diol
(9)
+
+
1
16.1; ESI MS (m/z): 225.0 [M + 3] , 223 [M + 1] ;
−
1
FTIR (νcm ): –OH/NH– 3111.30, –N=C– 1605.87,
C=C– 1569.32, –C–N– 1355.70, –C–Cl– 844.03; anal.
calcd. for C H ClN , C = 51.30, H = 3.40, N = 26.10,
1
–
Yield: 68%; H-NMR (DMSO-d ): δ 8.48 (s, 1H), 7.73 (s,
6
1H), 8.43 (s, 1H), 7.08 (d, 1H, J = 7.3 Hz), 7.01 (d, 1H,
9
7
4
1
3
found, C = 51.27, H = 3.37, N = 26.08.
J = 7.8 Hz); C-NMR (DMSO-d , 125 MHz): δ 163.2,
6
1
50.1, 150.3, 146.5, 146.4, 119.7, 119.2, 117.5, 117.1; ESI
1
3

Journal of the Iranian Chemical Society
+
−1
MS (m/z): 205 [M+1] ; FTIR (νcm ): –OH/NH– 3131.04,
C=N– 1621.17, –C=C– 1517.52, –C–N– 1313.69; anal.
calcd. for C H N O C=52.95, H=3.94, N=27.45, found,
(4-Methylsulfanyl-benzylidene)-(1H-[1, 2,
4]-triazol-3-yl)-amine (14)
–
9
8
4
2,
1
C=51.91, H=2.92, N=26.42.
Yield: 90%; H-NMR (DMSO-d ): δ 9.17 (s, 1H), 8.52 (s,
6
1
H), 7.93 (d, 2H, J=7.3 Hz), 7.40 (d, 2H, J=7.1 Hz), 2,53 (s,
13
1
H); C-NMR (DMSO-d , 125 MHz): δ 163.4, 146.5, 146.4,
6
(
2-Nitro-benzylidene)-(1H-[1, 2, 4] triazol-3-yl)-amine (10)
137.7, 129.1, 129.1, 126.3, 126.3, 18.7; ESI MS (m/z): 219.0
+
−1
[
M+1] ; FTIR (νcm ): –NH– 3140.76, –N=C– 1614.20,
1
Yield: 79%; H-NMR (DMSO-d ): δ 8.45 (s, 1H), 8.25
–C=C– 1590.21, –N–C– 1329.10, –C–S– 705.86; anal. calcd.
for C H N S, C=55.03, H=4.61, N=25.68; S=14.67,
6
(
s, 1H), 8,21 (d, 2H, J=9.2 Hz), 7.64 (d, 1H, J=9.3 Hz),
1
0
10
4
1
3
7
1
.47 (m, 2H); C-NMR (DMSO-d , 125 MHz): δ 163.5,
found, C=54.00, H=3.59, N=24.65, S=14.64.
6
48.7, 146.5, 146.4, 134.5, 131.4, 129.7, 126.2, 123.4; ESI
+
−1
MS (m/z): 218.0 [M+1] ; FTIR (νcm ): –NH– 3152.34,
Furan-2-ylmethylene-(1H-[1, 2, 4]-triazol-3-yl)-amine (15)
–
–
C=N– 1520.96, –C=C– 1445.74, NO - 1343.31,
2
1
C–N– 1309.86; anal. calcd. for C H N O C = 49.78,
Yield: 36%; H-NMR (DMSO-d ): δ 8.39 (s, 1H), 8.23
9
7
5
2,
6
H=3.27, N=32.26, found, C=48.74, H=2.59, N=31.21.
(s, 1H), 7.97 (d, 1H, J = 6.4 Hz), 7.28 (dd, 1H), 7.84 (d,
1
3
1
1
H, J = 6.7 Hz); C-NMR (DMSO-d , 125 MHz): δ
6
63.3, 146.5, 146.4, 143.1, 143.1, 110.3, 110.3; ESI MS
+
− 1
(
3-Nitro-benzylidene)-(1H-[1, 2, 4]-triazol-3-yl)-amine (11)
(m/z): 163.0 [M + 1] ; FTIR (νcm ): –NH– 3404.45,
–
C=N– 1622.50, –C=C– 1582.85, –C–N– 1295.75,
1
Yield: 60%; H-NMR (DMSO-d ): δ 7.60 (s, 1H), 7.29 (s,
–C–O– 1250.00; anal. calcd. for C H N O, C = 51.86,
7 6 4
6
1
H), 8.63 (s, 1H), 8.02 (bd, 2H), 7.75 (d, 1H, J =6.8 Hz);
H=3.74, N=34.56, C, 54.54; H, 4.58; N, 31.80 °C=50.83,
H=2.71, N=33.53.
1
3
C-NMR (DMSO-d , 125 MHz): δ 163.4, 148.5,
6
1
46.5, 146.4, 135.2, 132.4, 129.3, 125.7, 124.1; ESI MS
+
− 1
(
m/z): 218.0 [M + 1] ; FTIR (νcm ): –NH– 3233.78
(5-Methyl-furan-2-ylmethylene)-(1H-[1, 2,
4]-triazol-3-yl)-amine (16)
–
–
N=C– 1583.43, –C=C– 1524.85, –NO – 1478.41,
2
N–C– 1344.16; anal. calcd. for C H N O C = 49.79,
9
7
5
2,
1
H=3.26, N=32.27, found, C=48.75, H=2.23, N=31.23.
Yield: 42%; H-NMR (DMSO-d ): δ 8.41 (s, 1H), 8.01 (s,
6
1
H), 7.78 (d, 1H, J=6.3 Hz), 6.78 (d, 1H, J=6.4 Hz), 1.49
1
3
(
s, 3H); C-NMR (DMSO-d , 125 MHz): δ 163.3, 153.4,
6
(
4-Nitro-benzylidene)-(1H-[1, 2, 4]-triazol-3-yl)-amine (12)
146.5, 146.4, 141.2, 111.2, 106.7,14.5; ESI MS (m/z): 177.0
+
−1
[
M+1] ; FTIR (νcm ): –NH– 3130.14, C=N– 1621.48,
1
Yield: 73%; H-NMR (DMSO-d ): δ 8.28 (s, 1H), 7.29 (s,
–C=C– 1581.44, –C–N– 1249.88, –C–O– 1249.88,
–CH– 2934.86; anal. calcd. for C H N O, C = 54.55,
6
1
3
1
H), 7.83 (d, 2H, J=8.3 Hz), 7.54 (d, 2H, J=8.2 Hz); C-
8
8
4
NMR (DMSO-d , 125 MHz): δ 163.4, 150.4, 146.5, 146.4,
H=4.58, N=31.81, C=53.50, H=3.53, N=30.74.
6
1
37.2, 129.5, 129.5, 123.3, 123.3; ESI MS (m/z): 218.0
+
−1
[
M+1] ; FTIR (νcm ): –NH– 3100.35, –N=C– 1596.27,
C=C– 1516.14, –NO – 1470.29, –N–C– 1344.89; anal.
N-{2-[(1H-1,2,4-triazol-3-ylimino)methyl]phenyl}
methylidene-1H-1,2,4-triazol-3 amine (17)
–
2
calcd. for C H N O C=49.79, H=3.27, N=32.27, found,
9
7
5
2,
1
C=48.74, H=2.22, N=31.21.
Yield: 90%; H-NMR (DMSO-d ): δ (s, 9.77, 2H), 7.71
6
1
3
(
dd, 2H), 7.48 (d, 2H, J = 8.1 Hz); C-NMR (DMSO-d ,
6
1
25 MHz): δ 162.5, 163.3, 146.4, 146.4, 146.3, 146.3, 131.6,
(
4
4-Dimethylamino-benzylidene)-(1H-[1, 2,
]-triazol-3-yl)-amine (13)
131.5, 130.8, 130.6, 129.4, 129.3; ESI MS (m/z): 267.0
+
−1
[M+1] ; FTIR (νcm ): –NH– 3119.65, –C=N– 1647.49,
–
C–N– 1340.28, –C=C– 1557.76; anal. calcd. for C H N
12 10 8
1
Yield: 75%; H-NMR (DMSO-d ): δ 9.02 (s, 1H), 8.51
C=53.11, H=2.71, N=41.01, Found, C=53.08, H=2.72,
N=42.06.
6
(
s, 1H), 7.81 (d, 2H, J=7.6 Hz), 6.79 (d, 2H, J=7.8 Hz),
1
3
3
1
4
–
–
.39 (s, 6H); C-NMR (DMSO-d , 125 MHz): δ 163.4,
6
46.7, 146.5, 146.4, 129.7, 129.7, 120.5, 113.1, 113.1,
(2,4-Dimethyl-benzylidene)-(1H-[1, 2, 4]-triazol-3-yl)-amine
(18)
+
−1
3.3, 43.3; ESI MS (m/z): 216.0 [M+1] ; FTIR (νcm ):
NH– 3093.36, –C=N– 1581.58, –C=C– 1526.20,
C–N– 1327.57, anal. calcd. for C H N C = 61.37,
1
Yield: 80%; H-NMR (DMSO-d ): δ 9.42 (s, 1H), 8.43
1
1
13 5,
6
H=6.08, N=32.55, found, C=60.34, H=5.06, N=31.52.
(s, 1H), 7.97 (d, 1H, J = 8.4 Hz), 7.87 (s, 1H), 7.17 (dd,
1
3

Journal of the Iranian Chemical Society
1
3
+
−1
1
1
1
H), 2.33 (s, 6H); C-NMR (DMSO-d , 125 MHz): δ
[M+1] ; FTIR (νcm ): –NH– 3235.54, –C=N– 1689.92,
6
63.4, 146.5, 146.4, 139.5, 138.3, 130.1, 128.7, 128.7,
–C=C– 1586.64, –C–N– 1326.26; anal. calcd. for C H N
10 4,
13
+
26.2, 21.1, 14.7; ESI MS (m/z): 201.0 [M + 1] ; FTIR
C=70.26, H=3.54, N=24.21, found, C=70.24, H=3.51,
−
1
(
νcm ): –NH– 3149.08, –N=C– 1600.47, –C=C– 1437.95,
N=24.17.
–
N–C– 1322.23, –CH– 2965.42; anal. calcd. for C H N ,
11 12 4
C=65.97, H=6.05, N=27.97, found, C=64.95, H=5.01,
N=26.97.
Anthracen-9-ylmethylene-(1H-[1, 2, 4]-triazol-3-yl)-amine
(23)
1
(
(
3,4-Dimethyl-benzylidene)-(1H-[1, 2, 4]-triazol-3-yl)-amine
19)
Yield: 71%; H-NMR (DMSO-d ): δ 10.47 (s, 1H), 9.03
6
1
3
(s, 1H), 8.98 (m, 4H), 7.74 (m, 4H), 8.20 (s, 1H), C-
NMR (DMSO-d , 125 MHz): δ 163.4, 146.5, 146.4, 132.2,
6
1
Yield: 60%; H-NMR (DMSO-d ): δ 9.13 (s, 1H), 8.51 (s,
132.2, 131.7, 131.7, 128.7, 128.4, 128.1, 128.1, 128.0,
6
1
H), 7.55 (d, 1H, J = 7.8 Hz), 7.48 (s, 1H), 7.13 (d, 1H,
128.0, 125.2, 125.2, 125.2, 125.2; ESI MS (m/z): 273.0
1
3
+
−1
J=7.6 Hz), 1.90 (s, 6H); C-NMR (DMSO-d , 125 MHz):
[M+1] ; FTIR (νcm ): –NH– 3048.61, –N=C– 1621.87,
6
δ 163.4, 146.5, 146.4, 140.5, 138.4, 129.4, 129.2, 128.3,
–C=C– 1475.13, –N–C– 1342.41; anal. calcd. for C H N
12 4,
17
+
1
25.7, 14.6, 14.6 ; EI ESI MS (m/z): 201.0 [M+1] ; FTIR
C=74.97, H=4.44, N=20.59, found, C=73.96, H=3.42,
−
1
(
νcm ): –NH– 3198.28, –N=C– 1619.06, –C=C– 1605.59,
N=21.55.
–
N–C– 1344.23; anal. calcd. for C H N , C = 64.96,
1
1
12
4
H=5.02, N=27.96, found, C=64.94, H=6.01, N=27.92.
Pyren-1-ylmethylene-(1H-[1, 2, 4] triazol-3-yl)-amine (24)
1
(
(
3-Methoxy-benzylidene)-(1H-[1, 2, 4]-triazol-3-yl)-amine
20)
Yield: 74%; H-NMR (DMSO-d ): δ 8.66 (s, 1H), 8.46 (s,
6
1H), 7.72 (m, 3H), 7.52 (d, 1H, J = 8.1 Hz), 7.49 (d, 1H,
J=8.3 Hz), 7.48 (d, 2H, J=8.5 Hz), 6.90 (d, 2H, J=8.1 Hz);
1
13
Yield: 90%; H-NMR (DMSO-d ): δ 8.85 (s, 2H), 8.34 (s,
C-NMR (DMSO-d , 125 MHz): δ 163.4, 146.5, 146.4
6
6
1
7
1
1
H), 8.18 (s, 1H), 7.83 (d, 1H, J=7.2 Hz), 7.53 (dd, 1H),
132.3, 132.1, 131.8, 130.2, 129.3, 128.3, 128.2, 126.7,
13
.15 (d, 1H, J=7.5 Hz), 3.88 (s, 3H); C-NMR (DMSO-d ,
126.5, 126.5, 126.5, 126.5, 126.1, 126.1, 122.7, 122.2; ESI
6
+
−1
25 MHz): δ 163.4, 162.3, 146.5, 146.4, 132.1, 129.7, 121.2,
MS (m/z): 297.0 [M+1] ; FTIR (νcm ): –NH– 3036.92,
+
16.2, 114.5, 56.1; ESI MS (m/z): 203.0 [M + 1] ; FTIR
–C=N– 1676.60, –C–N– 1647.48; anal. calcd. for C H N ,
19
12
4
−
1
(
νcm ): –NH– 3236.29, –N=C– 1584.34, –C=C– 1491.33,
C=77.02, H=4.09, N=18.90, found, C=77.01, H=3.05,
–
N–C– 1328.64; anal. calcd. for C H N O, C = 59.41,
10 10 4
N=17.88.
H=4.97, N=27.72, found, C=59.39, H=3.97, N=26.68.
2
-[1-(1H-[1,2,4]Triazol-3-ylimino)-ethyl]-phenol (25)
(
4
1H-[1, 2,
1
]-Triazol-3-yl)-(3,4,5-trimethoxy-benzylidene)-amine (21)
Yield: 48%; H-NMR (DMSO-d ): δ 8.25 (s, 1H), 8.45
6
(
d, 1H, J = 7.4 Hz), 7.64 (m, 2H), 7.40 (d, 1H, 7.6), 1.91
1
13
Yield: 64%; H-NMR (DMSO-d ): δ 9.87 (s, 1H), 8.50
(s, 3H); C-NMR (DMSO-d , 125 MHz): δ 163.4, 157.7,
6
6
1
3
(
(
s, 1H), 7.49 (s, 1H), 7.40 (s, 1H), 3.76 (s, 9H); C-NMR
146.5, 146.4, 132.1, 130.2, 121.1, 118.1, 115.7, 14.3; ESI
+
−1
DMSO-d , 125 MHz): δ 163.4, 148.7, 148.7, 146.5,
MS (m/z): 287.0 [M+1] ; FTIR (νcm ): –OH– 3403.16,
–NH– 3325.70, –C=N– 1636.31, –C=C– 1592.85,
–C–N– 1268.54; anal. calcd. for C H N O, C = 59.42,
6
1
46.4, 135.6, 125.2, 107.1, 56.2, 56.2, 56.2; ESI MS
+
− 1
(
m/z): 263.0 [M + 1] ; FTIR (νcm ): –NH– 3204.87,
1
0
10
4
–
N=C– 1682.54, –C=C– 1622.23, –N–C– 1354.88; anal.
H=4.97, N=27.72, found, C=58.37, H=3.96, N=26.69.
calcd. for C H N O C = 53.96, H = 5.38, N = 20.36,
1
2
14
4
3,
found, C=53.93, H=5.35, N=21.34.
β-Glucuronidase Inhibition Activity
Naphthalen-2-ylmethylene-(1H-[1, 2, 4]-triazol-3-yl)-amine
β-Glucuronidase (E.C. 3.2.1.31 from bovine liver, G-0251)
inhibition activity of reported compounds was evaluated by
previously reported method. In this study, d-saccharic acid
1,4-lactone was used as the standard drug [23, 24].
(22)
1
Yield: 88%; H-NMR (DMSO-d ): δ 9.77 (s, 1H), 8.81 (s,
6
1
H), 8.23 (s, 1H), 7.71 (d, 1H, J = 8.1 Hz), 7.70 (d, 1H,
J = 7.9 Hz), 7.49 (d, 2H, J = 7.6 Hz), 7.48 (m, 2H), 6.98
Molecular docking studies
1
3
(
d,1H, J = 7.8 Hz); C-NMR (DMSO-d , 125 MHz): δ
6
1
1
63.4, 146.5, 146.4, 135.8, 133.4. 128.5, 128.3, 128.2,
Autodock vina was used to study the positioning of inhibi-
28.2, 128.1, 126.3, 125.7, 125.7; ESI MS (m/z): 223.0
tors bound in the active site of β-glucuronidase enzyme.
1
3

Journal of the Iranian Chemical Society
PDBQT files of ligands and receptor to be used for dock-
ing were generated from their PDB files using Autodock
Tools. Protein structure of human β-glucuronidase (PDB
ID: 1BHG) as shown in Fig. 8 was taken from PDB (http://
www.rcsb.org/pdb). Missing atoms, contacts and bonds were
checked in the structure. B-chain and the hetero-atoms were
removed from the original file. Calculation of partial atomic
charges and addition of hydrogen atoms to the protein mol-
ecule were done using Autodock tools. Configuration file
containing parameters for a grid box of size 16×16×16 Å
cantered at the coordinates of X: 85.22, Y: 86.62, Z: 87.37
was used to cover the active site. Docking results were ana-
lyzed using Discovery Studio Visualizer [28].
8. M.A. Al-Omar, E.S. Al-Abdullah, I.A. Shehata, E.E. Habib, T.M.
Ibrahim, A.A. El-Emam, Molecules 15, 2526–2550 (2010)
9
. K. Yamada, Y. Yoshizawa, K. Oh, Molecules 17, 4460–4473
(
2012)
10. K. Benci, L. Mandic, T. Suhina, M. Sedic, M. Klobucar, S.K.
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1
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