Synthesis, antiamoebic activity and docking studies of metronidazole-triazole-styryl hybrids

Article abstract of DOI:10.1016/j.ejmech.2018.03.033

A series of 22 novel metronidazole-triazole-styryl hybrids were synthesized and evaluated for their in vitro antiamoebic activity against HM1: IMSS strain of Entamoeba histolytica. Some of the hybrids were found to be more active (IC₅₀ = 0.12–0

Full text of DOI:10.1016/j.ejmech.2018.03.033

Accepted Manuscript
Synthesis, antiamoebic activity and docking studies of metronidazole-triazole-styryl
hybrids
Beena Negi, Prija Poonan, Mohammad Fawad Ansari, Deepak Kumar, Sakshi
Aggarwal, Ramandeep Singh, Amir Azam, Diwan S. Rawat
PII:
S0223-5234(18)30276-9
10.1016/j.ejmech.2018.03.033
EJMECH 10298
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 July 2017
Revised Date: 14 October 2017
Accepted Date: 12 March 2018
Please cite this article as: B. Negi, P. Poonan, M.F. Ansari, D. Kumar, S. Aggarwal, R. Singh, A. Azam,
D.S. Rawat, Synthesis, antiamoebic activity and docking studies of metronidazole-triazole-styryl hybrids,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.033.
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ACCEPTED MANUSCRIPT
Synthesis, antiamoebicactivity and docking studies of metronidazole-
triazole-styryl hybrids
Beena Negi,a+ Prija Poonan,a Mohammad Fawad Ansari,b Deepak Kumar,a Sakshi
Aggarwal,c Ramandeep Singh,c Amir Azam,b Diwan S Rawata*
aDepartment of Chemistry, University of Delhi, Delhi 110007 (India)
bDepartment of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi (India)
cTranslational Health Science and Technology Institute, Vaccine and Infectious Disease
Research Centre, Faridabad-121001 (India)
+Current Address: Department of Chemistry, Gargi College, University of Delhi
*E-mail: dsrawat@chemistry.du.ac.in
Graphical Abstract
R1
N
N
N
N
N
N
CH3
N3
O2N
O2N
O2N
N
N
N
N
N
N
R
R
R
Metronidazole-triazole hybrids
Metronidazole-triazole-styryl
hybrids (Present study)

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Synthesis, antiamoebic activity and docking studies of metronidazole-
triazole-styryl hybrids
Beena Negi,a+ Prija Poonan,a Mohammad Fawad Ansari,b Deepak Kumar,a Sakshi
Aggarwal,c Ramandeep Singh,c Amir Azam,b Diwan S Rawata*
aDepartment of Chemistry, University of Delhi, Delhi 110007 (India)
bDepartment of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi (India)
cTranslational Health Science and Technology Institute, Vaccine and Infectious Disease
Research Centre, Faridabad-121001(India)
+Current Address: Department of Chemistry, Gargi College, University of Delhi
*E-mail: dsrawat@chemistry.du.ac.in
Abstract: A series of 22 novel metronidazole-triazole-styryl hybrids were synthesized and
evaluated for their in vitro antiamoebic activity against HM1: IMSS strain of Entamoeba
histolytica. Some of the hybrids were found to be more active (IC50 = 0.12 – 0.35 µM) than
the reference drug metronidazole (IC50 = 1.79 µM). The most active compounds were found
to be non-toxic (up to 50 µM) against the Vero cells showing a good safety profile of these
hybrids. The docking and ADMET studies were also conducted to investigate the probable
mode of action. Docking studies showed significant binding affinity in the active site of E.
histolytica thioredoxin reductase (EhTrR) protein.
Key Words: metronidazole, triazole, Entamoeba histolytica, docking studies, thioredoxin
reductase
*Corresponding author:
E. Mail: dsrawat@chemistry.du.ac.in
Phone No: 91-11-27662683
Fax No: 91-11-27667501

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1. Introduction
Protozoan parasites have been causing serious problems to human health and are
responsible for the high mortality and morbidity affecting more than 500 million people in
the world [1]. Among protozoal infections, amoebiasisis the third leading cause of death. It is
caused by the protozoa Entamoeba histolytica which is affects 50 million individuals per year
[2]. The parasite affects the gastrointestinal tract of humans and causes invasive infections
and induces tissue destruction, producing amoebic colitis, dysentery and liver abscesses [3].
The 5-nitroimidazole class of compounds has long been used for the treatment of
protozoal infections. Among the nitroimidazole class of compounds, metronidazole (MTZ) is
considered the drug of choice for the treatment of amoebiasis [4].The nitro group in the
metronidazole nucleus is crucial for its anti-amoebic activity. Reduction of the nitro group to
the nitro radical anion by electron carriers in an anaerobic environment leads to
decomposition to form toxic metabolites, which cause DNA damage and nonspecific
macromolecular damage leading to cell death [5]. However, this drug is associated with
certain serious side effects such as seizures, ataxia, gastric mucus irritation, genotoxicity,
carcinogenicity spermatozoid damage and hematuria [6-10]. Moreover, there are reports of
failure of treatment with metronidazole due to the development of resistance by the parasite
towards MTZ [11,12]. Thus there is a need to develop novel anti-amoebic agents with
increased efficacy and with no side effects. The two sites in the metronidazole moiety viz. the
ethylhydroxy side chain and the methyl group attached to the imidazole ring, can be
chemically modified to obtain a variety of compounds for the biological activity evaluation.
Recently the most promising and successful strategy to find a new drug is hybrid drug
concept which involves covalent linking of two molecules with their individual activity into a
single hybrid molecule [13]. In many cases the hybrid molecules have been found to have
more potency towards both drug sensitive and resistant strains with better pharmacokinetic
profile and less toxic effects [14-17]. Triazole moiety is one of the most widely studied
pharmacophore showing a wide range of biological activities such as antibacterial, antiviral,
antifungal, antitumor, anti-inflammatory and antiglycemic [18]. Considering the hybrid drug
concept in our mind, we had syntheiszed some hybrid molecules bearing triazole nucleus in
conjugation with the metronidazole (Fig. 1) which were found to exhibit remarkable
antiamoebic activities [19,20]. Encouraged by the antiamoebic activity of MTZ-triazoles we
designed and synthesised some novel metronidazole-triazole-styryl hybrids (Fig. 1).

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Compounds containing styryl scaffold are known to possess wide spectrum of biological
activities. The trans-cinnamic acid analogues have admirable pharmacological properties like
antioxidant, antibacterial and antitumor [21]. Rubner et al had reported the cyanine-styryl-
type dyes as bright non-covalent binders to RNA [22]. Hampannavar et al synthesised a
series of styryl hydrazine thiazole hybrids and evaluated them in vitro against M. tuberculosis
H37Rv strain [23]. Zaidi et al have reported antiamoebic activity of chalcones bearing N-
substituted ethanamine tail, these compounds have C=C-Ph linkage in their structure [24].
Pais-Morales et al investigated the effect of resveratrol (a compound with C=C-Ph linkage)
on E. histolytica trophozoites and the growth was arrested by 72 µM dose level of resveratrol
[25]. Abid et al have reported the synthesis of metronidazole thiosemicarbazone analogues
with styryl linkage and one of the compounds showed promising antiamoebic activity at IC50
0.56 µM [26]. Amalgamation of two biologically active pharmacophores (styryl and
nitroimidazole) in one molecular platform generates a new scaffold for antiamoebic
evaluation.
<Insert Fig. 1 here>
In order to understand the possible mechanism of action, molecular docking studies of
the most active compounds were performed in the binding site of E. histolytica thioredoxin
reductase (EhTrR) protein [27,28]. Leitsch et al identified five proteins, Trx, TrxR, SOD,
purine nucleoside phosphorylase, and a protein with a carbohydrate binding domain that are
affected by MTZ metabolites [29]. It has been shown that reduced MTZ binds covalently to
TrxR and Trx in E. histolytica, which leads to inhibition of the physiological disulfide-
reducing activity of the TrxR/Trx system. It has been suggested that the TrxR/Trx system is
an excellent target for anti-parasite chemotherapy. In order to better understand Trx-
dependent redox network of the parasite that could be indirectly affected by MTZ treatment,
Schlosser et al screened the target proteome of Trx using an active site mutant (cysteine to
serine) of Trx, which is capable of binding to interacting proteins but incapable to release its
reduced targets.
2. Chemistry
The synthetic pathway leading to target compounds (8-29) is depicted in Scheme 1.
Metronidazole (1) on reaction with p-toluene sulfonyl chloride yields tosyl derivative (2)
which further on reaction with sodium azide gets converted to azide derivative (3). The azide
derivative of MTZ on reaction with terminal alkynes yields MTZ-triazoles (4-7). Compounds

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4-7 were treated with substituted benzaldehydes in presence of strong base to generate MTZ-
triazole-styryl hybrids (8-29) (Scheme 1) [30]. The synthesized compounds were purified by
column chromatography using 20% ethyl acetate : hexane as eluent.
<Insert Scheme 1 here>
3. Results and discussion
A series of 22 MTZ-triazole-styryl hybrids were evaluated in vitro for their anti-amoebic
activity against HM1: IMSS strain of E histolytica. The MTZ-triazoles hybrids 4-7 have been
reported earlier by our group and have IC50 values 0.0223, 0.218, 0.0084 and 0.607 µM,
respectively [20]. Modifications at the methyl group of these triazoles 4-7 led to the desired
compounds 8-29 (Fig. 2). Modification of methyl group in compounds 4 (analogues 8-13), 5
(analogues 14-19) and 6 (analogues 20-24) by styryl group resulted into compounds with
decrease in activity, however the analogues 26 (4-F) and 27 (4-Cl) showed better activity
than the parent compound 7. This indicates that the incorporation of styryl group in place of
methyl group is well tolerated.
<Insert Fig. 2 here>
Table 1 show that the in vitro activity of these hybrids was retained. Four different series of
styryl derivatives (8-13, 14-19, 20-24 and 25-29) screened for in vitro antiamoebic activity
showed a similar trend in the activity due to the presence of various substituents on the
phenyl ring. In the styryl derivatives of compounds 4, 5, 6 and 7 incorporation of polar
groups like F, Cl and Br showed significant activity however a hydrophobic alky group such
as methyl or ethyl leads to diminished activity.
Fourteen compounds (8-11, 14-17, 21-23, and 26-28) were found to be more potent than the
reference drug MTZ. Compounds 15 (IC50 = 0.12 µM), 26 (IC50 = 0.28 µM) and 27 (IC50 =
0.35 µM) showed improved activity than the parent compounds 5 and 7. The compounds 9,
15, 21 and 26 containing fluoro substituents were found to be more active than non-
substituted analogues 8, 14, 20 and 25, respectively. Replacement of F with Cl or Br leads to
decrease in activity in compounds 10, 11, 16, 17, 22, 23, 27 and 28, indicating significance of
F towards biological activity of these compounds. However, compound 6 remains the most
active compound amongst both the MTZ-triazole and MTZ-triazole-styryl series of
compounds. Table 1 show that the change in the substituent present on the styryl moiety leads

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to change in IC50 values of the compounds synthesized, which clearly indicates that the styryl
linkage also plays a crucial role in the activity of these compounds.
Eleven compounds (8-10, 14-16, 21-23, 26 and 27) with IC50 > 1.35 µM, were determined for
cytotoxicity against Vero cell lines. The compounds tested were found to be non-toxic up to
50 µM, highest tested concentration.
<Insert Table 1 here>
Molecular docking studies of the six most active MTZ-triazole-styryl hybrids having
IC50 < 1 µM (9, 15, 21, 22, 26 and 27) were performed in the NADP binding site of E.
histolytica Thioredoxin reductase (EhTrR) (PDB ID:4CCQ) [32]. Thioredoxin reductase
(TrR) prevents, regulates and repairs the damage caused by oxidative stress in E. histolytica
[33]. Thioredoxin reductases enzymes are homodimeric proteins where each monomer
contains a NADPH binding domain, FAD domain and an active site containing a redox active
disulfide [34]. Thioredoxin reductase catalyzes the reversible transfer of reducing equivalents
between NADPH and thioredoxin [35].
The results of docking studies for the test compounds are illustrated in Table 2. These
docking results clearly indicate that the most active compounds in the study show substantial
binding affinities towards EhTrR as exhibited by good Glide energy (-52.48 kcalmol-1 to -
43.84 kcalmol-1) and GlideXP scores (-6.51 kcalmol-1 to -5.29 kcalmol-1). The docking
conformations of the protein-ligand complexes having high Glide energy and GlideXP scores
are presented in Figures 3 and 4 for compounds 15 and 21, respectively. Figure 3 and 4
shows the hydrogen bonding pattern along with cation-π interactions and hydrophobic
interactions of compounds 15 and 21 with the binding site residues. Interestingly, the
compounds are showing H-bond interactions with the conserved residues of the coenzyme
binding motif such as Arg183, Arg184, Arg188 and Gly160 similar to the native substrate
NADP [36,37]. However, NADP being a bulky molecule could occupy a larger volume of the
protein active site, hence forms several H-bond and hydrophobic interactions. This could be
attributed for higher GlideXP score (-9.43kcalmol-1) and Glide energy (-73.86kcalmol-1)
terms obtained for the docking results of NADP-EHTrR complex. Figure 3 shows the binding
pose for compound 15-EHTrR complex. Side chains of Arg183 and Arg188 is forming H-
bond interactions with the oxygen atom of nitro substituent of imidazole ring. Also side chain
amine of Arg183 is forming cation-π interaction with the triazole ring of the compound.

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Further main chain nitrogen of Gly 160 is involved in the H-bond interaction with imidazole
nitrogen atom. 2D and 3D docking pose of compound 21-EHTrR complex (Fig. 4) shows the
oxygen atoms of the nitro group of the compound forming H-bond interactions with side
chain amine group of Arg183 and Arg184. The imidazole nitrogen of the compound forms H-
bond with main chain nitrogen of Gly160. Further the π cloud of the triazole ring of
thecompound is involved in the cation-π interaction with the amine side chain of Arg183 (Fig. 4).
<Insert Table 2 here>
<Insert Fig. 3 here>
<Insert Fig. 4 here>
Lipinski’s rule of five and pharmacokinetic data of these compounds were calculated
through ADMET prediction using QikProp version 3.5. The most important of these
parameters together with its permissible ranges are listed in the Tables 3 and 4. The active
compounds were found to obey the Lipinski's rule of five, which is a preliminary test for
drug-likeness of compounds. An orally active compound should not have more than 4
violations of Lipinski's Rule. Table 3 clearly indicates that all the compounds showed no
violations of Lipinski’s rule of 5.
With the exception of compound 27, having QPlogPo/w > 5, remaining compounds have
good drug likeness properties (Table 4). Prediction of oral drug absorption (Percent Human
Oral Absorption) was highly satisfactory for all the test compounds. QPPCaco predictions for
all the test compounds showed high to moderately good values for Caco-2 cells permeability.
Further, QPlogKhsa, the prediction for human serum albumin binding and all inhibitors were
predicted lie within the expected range for 95% of known drugs (-1.5 to 1.5). Also, the
QikProp descriptor for brain/blood partition coefficient (QPlogBB) and the blood-brain
barrier mimic MDCK cell permeability (QPPMDCK) show satisfactory predictions for all the
test compounds and the reference compound. Further, aqueous solubility (QPlogS) parameter
for the test compounds were assessed and all the compounds were predicted to have QPlogS
values in permissible range. Furthermore, QPlogHERG descriptor for the prediction of IC50
value of HERG K+ channel blockage was predicted for the test compounds. With compound
23 (QPlogHERG = -4.863) and for MTZ (QPlogHERG =-3.156), having value in permissible
limits, all other test compounds were having QPlogHERG descriptor below the recommended
concerned value of -5.

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<Insert Table 3 here>
<Insert Table 4 here>
4. Conclusion
The unification of nitroimidazole, styryl and triazole moieties is an effort to explore the
influence of structural hybridizations on the activity, hoping to find a new lead structure that
would have a promising antiamoebic activity. The nitro group, imidazole, triazole and styryl
contribute significantly towards the antiamoebic activity. The MTZ-triazole and their novel
styryl derivatives were effective against HM1: IMSS strain of E histolytica. Compound 6 (2-
{1-[2-(2-Methyl-5-nitro-imidazol-1-yl)-ethyl]-1H-[1,2,3]triazol-4-yl}-pyridine) was found to
be the most active against E. Histolytica amongst the MTZ-triazole series and MTZ-triazole-
styryl hybrid series. Thus we strongly believe that compound 6 could be considered as a lead
for the further development of anti-amoebic agents.
5. Experimental Section
5.1. In vitro antiamoebic activity
All the synthesized metronidazole-triazole-styryl hybrids (8-29) were screened for their in
vitro antiamoebic activity against HM1: IMSS strain of E. histolytica by microdilution
method [31]. E. histolytica trophozoites were cultured in culture tubes by using Diamond
TYIS-33 growth medium [38]. The test compounds (1mg) were dissolved in DMSO (40 µL,
level at which no inhibition of amoeba occurs) [39]. The stock solutions of the compounds
were prepared freshly before use at a concentration of 1 mg/mL. Two-fold serial dilutions
were made in the wells of 96-well microtiter plate. Each test includes MTZ as a standard
amoebicidal drug, control wells (culture medium plus amoeba) and a blank (culture medium
only). All the experiments were carried out in triplicate at each concentration level and
repeated thrice. The amoeba suspension was prepared from a confluent culture by pouring off
the medium at 37°C and adding 5 mL of fresh medium, chilling the culture tube on ice to
detach the organisms from the side of flask. The number of amoeba/mL was estimated with
the help of a haemocytometer, using trypan blue exclusion to confirm the viability. The
suspension was diluted upto 105 organism/mL by adding fresh medium and 170 µL of this
suspension was added to the test and control wells in the plate so that the wells were
completely filled (total volume, 340 µL). An inoculum of 1.7 x 104 organisms/well was
chosen so that confluent, but not excessive growth, took place in control wells. Plate was

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sealed and gassed for 10 min with nitrogen before incubation at 37 °C for 72 h. After
incubation, the growth of amoeba in the plate was checked with a low power microscope.
The culture medium was removed by inverting the plate and shaking gently. Plate was then
immediately washed with sodium chloride solution (0.9%) at 37 °C. This procedure was
completed quickly and the plate was not allowed to cool in order to prevent the detachment of
amoeba. The plate was allowed to dry at room temperature and the amoeba were fixed with
methanol and when dried, stained with (0.5%) aqueous eosin for 15 min. The stained plate
was washed once with tap water, then twice with distilled water and then allowed to dry. A
200 µL portion of 0.1 N sodium hydroxide solution was added to each well to dissolve the
protein and release the dye. The optical density of the resulting solution in each well was
determined at 490 nm with a microplate reader. The % inhibition of amoeba growth was
calculated from the optical densities of the control and test wells and plotted against the
logarithm of the dose of the drug tested. Linear regression analysis was used to determine the
best fitting line from which the IC50 value was found.
5.2 Molecular docking studies
The 2D structures of all the compounds were generated on ChemBioDraw Ultra 12.0
(www.cambridgesoft.com). Ligprep module implemented in Schrödinger was used to
generate energy minimized 3D structures. Partial atomic charges were computed using the
OPLS_2005 force field. The correct Lewis structure, tautomers and ionization states (pH 7.0
+/− 2.0) for each of these ligands were generated and optimized with default settings
(Ligprep 2.5, Schrödinger, LLC, New York, NY, 2012).The 3D crystal structure of
Thioredoxin reductase from Entamoeba Histolytica(PDB ID:4CCQ; resolution 1.50 Å), was
retrieved from protein data bank (www.rcsb.org). The proteins were prepared for docking
using Protein Preparation Wizard (Maestro 9.3 Schrödinger, LLC, New York, NY, 2012).
Water molecules within 5 Å of the protein structures were considered. Bond order and formal
charges were assigned and hydrogen atoms were added to the crystal structure. Further to
refine the structure OPLS-2005 force field parameter was used to alleviate steric clashes and
the minimization was terminated when RMSD reached maximum cut off value of 0.30 Å.
The binding site was defined by a 20 Å × 20 Å × 20 Å box centered on the centroid of the
EHTrR natural substrate, NADP as the centroid of the ligands used for docking. Default
settings were used for all the remaining parameters and no constrains were specified
separately. All ligand conformers were docked to each of the receptor grid files EHTrR

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structures using Glide extra precision (XP) mode. Default settings were used for the
refinement and scoring.
5.3 In silico ADMET Prediction
The pharmacokinetic profile of compounds in the study were predicted by using programs
Qikprop v3.5 (Schrödinger, Inc., New York, NY, 2012) [40]. All the compounds were
prepared in neutralized form for the calculation of pharmacokinetic properties by QikProp
using Schrodinger’s Maestro Build module and LigPrep, saved in SD format. The programs
QikProp utilizes the method of Jorgensen [41] to compute pharmacokinetic properties and
descriptors such as octanol/water partitioning coefficient, aqueous solubility, brain/blood
partition coefficient, intestinal wall permeability, plasma protein binding etc.
5.4 Synthetic Methodology
All the chemicals were purchased from Sigma-Aldrich and were used without further
purification. Progress of the reactions was monitored by thin layer chromatography (Merck
Kiesel 60 F254, 0.2 mm thickness) and the compounds were purified by silica gel column
chromatography. IR spectra were recorded on Perkin-elmer FT-IR spectrophotometer using
1
13
KBr pellets and the values were expressed in cm-1. H NMR (400 MHz) and C NMR (100
MHz) spectra were recorded on Jeol ECX spectrospin instrument using CDCl3 or DMSO-d6
as solvent with TMS as internal reference. The chemical shift values were expressed on δ
scale and the coupling constant (J) in Hz. Melting points were recorded on EZ-Melt
automated melting point apparatus, Stanford Research Systems and are uncorrected. Mass
data were recorded in Jeol-Accu TOF JMS-T100LC and micromass LCT mass
spectrometer/Data system.
5.5 Typical procedure for the synthesis of (E)-(1-(2-(5-Nitro-2-styryl-1H-imidazol-1-yl)ethyl)-
1H-1,2,3-triazol-4-yl)methanol (8) and related compounds (8-29): To a stirred solution of
compound 6 (200 mg, 0.609 mmol) in DMSO (5 mL) benzaldehyde (64.6 mg, 0.609 mmol)
o
or substituted benzaldehydes was added. The reaction mixture was cooled to 0 C and a
solution sodium methoxide (98.7 mg, 1.829 mmol) in dry MeOH (5 mL) was added. The
o
reaction mixture was further stirred at 40 C for 12 h. The solid obtained was filtered and
purified by column chromatography to obtain compound 8-29.
5.5.1.
(E)-(1-(2-(5-Nitro-2-styryl-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methanol
o
(8):Yield 56%; mp 161-162 C; IR (film, cm-1): 2923, 2857, 1625, 1429, 1377, 1263, 1190,

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1032; 1H NMR (400 MHz, DMSO-d6) δ: 4.23 (d, J = 5.8 Hz, 2H, CH2OH), 4.81 (t, J = 5.1
Hz, 2H, NCH2), 4.98 (t, J = 5.8 Hz, 2H, NCH2), 5.06 (t, J = 5.8 Hz, 1H, CH2OH), 6.60 (d, J =
16.1 Hz, 1H, CH=CH), 7.36-7.39 (m, 2H, ArH), 7.41-7.43 (m, 1H), 7.56 (d, J = 15.4 Hz, 1H,
13
CH=CH), 7.60 (d, J = 6.6 Hz, 2H, ArH), 7.90 (s,1H), 8.22 (s, 1H); C NMR (100 MHz,
DMSO-d6) δ: 45.24, 49.02, 54.83, 112.01, 123.55, 127.65, 128.75, 129.43, 134.99, 135.27,
138.22, 138.51, 148.27, 150.16; ESI-MS (m/z): 341.34 [M + H]+. Anal Calcd for
C16H16N6O3: C 56.47, H 4.74, N 24.69. Found: C 56.65, H 4.90, N 24.82.
5.5.2. (E)-(1-(2-(2-(4-Fluorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)
o
methanol (9): Yield 57%; mp 160-162 C; IR (film, cm-1): 2922, 2852, 1443, 1380, 1278,
1
1184, 970, 821, 764; H NMR (400 MHz, CDCl3) δ: 3.85 (s, 1H, OH), 4.58 (d, J = 3.6 Hz,
2H, CH2OH), 4.86-4.87 (m, 2H, NCH2), 4.93-4.97 (m, 2H, NCH2), 5.95 (d, J = 15.4 Hz, 1H,
CH=CH), 6.93 (d, J = 8 Hz, 2H, ArH), 7.08-7.13 (m, 1H, ArH), 7.41-7.45 (m, 2H, ArH), 7.69
(d, J = 15.4 Hz, 1H, CH=CH), 8.15 (s, 1H); ESI-MS (m/z):359.28 [M + H]+. Anal Calcd for
C16H15FN6O3: C 53.63, H 4.22, N 23.45. Found: C 53.46, H 4.12, N 23.61.
5.5.3. (E)-(1-(2-(2-(4-Chlorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)
o
methanol (10): Yield 51%; mp 221-222 C; IR (film, cm-1): 2919, 2850, 1433, 1380, 1264,
1
1226, 1189, 1091, 1007, 968, 813; H NMR (400 MHz, CDCl3) δ: 1.72 (t, J = 5.9 Hz, 1H,
CH2OH), 4.59 (d, J = 6.6 Hz, 2H, CH2OH), 4.85 (t, J = 5.9 Hz, 2H, NCH2), 4.96 (t, J = 5.9
Hz, 2H, NCH2), 6.10 (d, J = 15.4 Hz, 1H, CH=CH), 7.25 (s, 1H), 7.39 (dd, J = 8.8 Hz, 2.9
Hz, 4H, ArH), 7.68 (d, J = 15.4 Hz, 1H, CH=CH), 8.15 (s, 1H); ESI-MS (m/z): 375.22 [M +
H]+, 377.24 [M + 2]+. Anal Calcd for C16H15ClN6O3: C 51.28, H 4.03, N 22.42. Found: C
51.39, H 4.14, N 22.33.
5.5.4. (E)-(1-(2-(2-(4-Bromostyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)
methanol (11): Yield 61%; mp 222-223 oC; IR (film, cm-1): 2921, 1623, 1420, 1027, 996;1H
NMR (400 MHz, CDCl3) δ: 1.71 (t, J = 5.9 Hz, 1H, CH2OH), 4.59 (d, J = 5.9 Hz, 2H,
CH2OH), 4.85 (t, J = 5.9 Hz, 2H, NCH2), 4.96 (t, J = 5.9 Hz, 2H, NCH2), 6.12 (d, J = 15.4
Hz, 1H, CH=CH), 7.25 (s, 1H), 7.33 (d, J = 8.8 Hz, 2H, ArH), 7.54 (d, J = 8.0 Hz, 2H, ArH),
7.66 (d, J = 15.4 Hz, 1H, CH=CH), 8.14 (s, 1H); ESI-MS (m/z): 419.12 [M + H]+, 421.14 [M
+ 2]+. Anal calcd for C16H15BrN6O3: C 45.84, H 3.61, N 20.05. Found: C 45.96, H 3.52, N
20.16.

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5.5.5. (E)-(1-(2-(2-(4-Methylstyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)
o
methanol (12):Yield 59%; mp 200-201 C; 1H NMR (400 MHz, DMSO-d6) δ: 2.32 (s, 3H,
CH3), 4.23 (d, J = 5.8 Hz, 2H, CH2OH), 4.80 (t, J = 5.8 Hz, 2H, NCH2), 4.96 (t, J = 5.8 Hz,
2H, NCH2), 5.06 (t, J = 5.8 Hz, 1H, CH2OH), 6.54 (d, J = 15.4 Hz, 1H, CH=CH), 7.21 (d, J =
8 Hz, 2H, ArH), 7.49 (d, J = 8 Hz, 2H, ArH), 7.52 (d, J = 16.1 Hz, 1H, CH=CH), 7.89 (s,
13
1H), 8.20 (s, 1H); C NMR (100 MHz, DMSO-d6) δ: 21.00, 45.18, 49.00, 54.83, 110.91,
123.51, 127.63, 129.34, 132.55, 135.05, 138.29, 138.42, 139.25, 148.24, 150.39; ESI-MS
(m/z): 355.26 [M + H]+. Anal calcd for C17H18N6O3: C 57.62, H 5.12, N, 23.72. Found: C
57.73, H 5.02, N, 23.59.
5.5.6.
(E)-(1-(2-(2-(4-Ethylstyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)
o
methanol (13): Yield 48%; mp 180-181 C; IR (film, cm-1): 2920, 2850, 1458, 1378, 1263,
1189, 1219, 1032; 1H NMR (400 MHz, CDCl3) δ: 1.25 (t, J = 7.0 Hz, 3H, CH2CH3), 1.66 (t, J
= 5.9 Hz, 1H, CH2OH), 2.68 (q, J = 7.3 Hz, 2H, CH2CH3), 4.58 (d, J = 5.9 Hz, 2H, CH2OH),
4.85 (t, J = 5.1 Hz, 2H, NCH2), 4.95 (t, J = 5.1 Hz, 2H, NCH2), 6.08 (d, J = 15.4 Hz, 1H,
CH=CH), 7.23 (s, 1H), 7.26 (s, 2H, ArH), 7.39 (d, J = 8.0 Hz, 2H, ArH), 7.72 (d, J = 15.4 Hz,
1H, CH=CH), 8.15 (s, 1H); ESI-MS (m/z): 369.28 [M + H]+. Anal calcd for C18H20N6O3: C
58.69, H 5.47, N 22.81
5.5.7.
(E)-1-(2-(5-Nitro-2-styryl-1H-imidazol-1-yl)ethyl)-4-phenyl-1H-1,2,3-triazole
o
1
(14):Yield 63%; mp 230-231 C; IR (film, cm-1): 2922, 1440, 1380, 1261, 1187; H NMR
(400 MHz, CDCl3) δ: 4.91 (t, J = 5.1 Hz, 2H, NCH2), 4.99 (t, J = 5.1 Hz, 2H, NCH2), 6.20 (d,
J = 15.4 Hz, 1H, CH=CH), 7.25 (s, 1H, ArH), 7.29 (s, 1H, ArH), 7.30 (s, 1H, ArH), 7.32-7.35
(m, 3H, ArH), 7.39-7.41 (m, 2H, ArH), 7.49 (s, 1H), 7.55-7.57 (m, 2H, ArH), 7.67 (d, J =
15.4 Hz, 1H, CH=CH), 8.16 (s, 1H); ESI-MS (m/z): 387.25 [M + H]+. Anal calcd for
C21H18N6O2: C 65.27, H 4.70, N 21.75. Found: C 65.39, H 4.59, N 21.87.
5.5.8. (E)-1-(2-(2-(4-Fluorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-4-phenyl-1H-1,2,3-triazole
(15): Yield 53%; mp 181-182 oC; IR (film, cm-1): 3119, 3089, 2920, 1598, 1518, 1487, 1449,
1
1381, 1273, 1261, 1227, 1187, 1039; H NMR (400 MHz, CDCl3) δ: 4.91-4.92 (m, 2H,
NCH2), 4.96-5.0 (m, 2H, NCH2), 6.05 (d, J = 15.4 Hz, 1H, CH=CH), 7.30 (d, J = 7.3 Hz, 1H,
ArH), 7.34-7.37 (m, 2H, ArH), 7.42-7.45 (m, 2H, ArH), 7.48 (d, J = 8 Hz, 1H, ArH), 7.54-
7.56 (m, 1H, ArH), 7.60 (d, J = 15.4 Hz, 1H, CH=CH), 7.40-7.76 (m, 2H, ArH), 8.03 (s, 1H),
8.15 (s, 1H); ESI-MS (m/z): 405.28 [M + H]+. Anal calcd for C21H17FN6O2: C 62.37, H 4.24,
N 20.78. Found: C 62.48, H 4.33, N 20.65.

ACCEPTED MANUSCRIPT
5.5.9.
(E)-1-(2-(2-(4-Chlorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-4-phenyl-1H-1,2,3-
o
triazole (16): Yield 38%; mp 272-273 C; IR (film, cm-1): 3085, 2922, 2852, 1519, 1485,
1449, 1433, 1378, 1271, 1259, 1187, 1178, 1086, 1011; 1H NMR (400 MHz, DMSO-d6) δ:
4.87 (t, J = 5.8 Hz, 2H, NCH2), 5.03 (t, J = 5.1 Hz, 2H, NCH2), 6.64 (d, J = 15.4 Hz, 1H,
CH=CH), 7.23-7.25 (m, 1H), 7.26 (s, 1H, ArH), 7.28-7.30 (m, 2H, ArH), 7.31 (s, 1H, ArH),
7.44 (d, J = 16.1 Hz, 1H, CH=CH), 7.49-7.50 (m, 2H), 7.52-7.53 (m, 2H), 8.22 (s, 1H), 8.44
13
(s, 1H); C NMR (100 MHz, DMSO-d6) δ: 45.26, 49.41, 112.22, 122.22, 125.03, 127.68,
128.52, 128.62, 129.18, 130.34, 133.70, 134.01, 135.05, 136.73, 138.54, 146.62, 149.97;
ESI-MS (m/z): 421.28 [M + H]+, 423.29 [M + 2]+. Anal calcd for C21H17ClN6O2: C 59.93, H
4.07, N 19.97. Found: C 59.83, H 4.18, N 19.88.
5.5.10.
(E)-1-(2-(2-(4-Bromostyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-4-phenyl-1H-1,2,3-
o
triazole (17):Yield 64%; mp 270-271 C; IR (film, cm-1): 3409, 3111, 3083, 2922, 1521,
1487, 1450, 1431, 1381, 1271, 1199, 1073; 1H NMR (400 MHz, DMSO-d6) δ: 4.88 (t, J = 5.1
Hz, 2H, NCH2), 5.04 (t, J = 5.8 Hz, 2H, NCH2), 6.66 (d, J = 16.1 Hz, 1H, CH=CH), 7.24-
7.27 (m, 1H), 7.29-7.31 (m, 1H, ArH), 7.43 (d, J = 15.4 Hz, 1H, CH=CH),7.44 (s, 5H, ArH),
13
7.50-7.51 (m, 1H), 7.52-7.53 (m, 1H), 8.24 (s, 1H), 8.44 (s, 1H); C NMR (100 MHz,
DMSO-d6) δ: 45.28, 49.42, 112.69, 122.22, 122.51, 125.04, 127.68, 128.63, 129.42, 130.34,
131.45, 134.34, 135.06, 136.81, 138.55, 146.63, 149.97; ESI-MS (m/z): 467.24 [M + H]+,
469.26 [M + 2]+. Anal calcd for C21H17BrN6O2: C 54.21, H 3.68, N 18.06. Found: C 54.33, H
3.59, N 18.19.
5.5.11. (E)-1-(2-(2-(4-Methylstyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-4-phenyl-1H-1,2,3-triazole
(18): Yield 51%; mp 237-238 oC; IR (film, cm-1): 3082, 1516, 1449, 1430, 1378, 1275, 1260,
1
1176, 1199; H NMR (400 MHz, DMSO-d6) δ: 2.29 (s, 3H, CH3), 4.88 (t, J = 5.8 Hz, 2H,
NCH2), 5.03 (t, J = 5.8 Hz, 2H, NCH2), 6.58 (d, J = 16.1 Hz, 1H, CH=CH), 7.10 (d, J = 8 Hz,
2H, ArH), 7.24-7.62 (m, 1H, ArH), 7.28-7.32 (m, 2H), 7.41 (d, J = 8 Hz, 2H, ArH), 7.47 (d, J
= 15.4 Hz, 1H, CH=CH), 7.54 (s, 1H, ArH), 7.56 (d, J = 1.5 Hz, 1H, ArH), 8.22 (s, 1H), 8.46
13
(s, 1H); C NMR (100 MHz, DMSO-d6) δ: 20.99, 45.20, 49.41, 110.79, 122.24, 125.13,
127.59, 127.70, 128.67, 129.20, 130.42, 132.40, 135.16, 138.48, 139.17, 146.64, 150.47;
ESI-MS (m/z): 401.28 [M + H]+. Anal calcd for C22H20N6O2: C 65.99, H 5.03, N, 20.99.
Found: C 65.89, H 5.14, N, 20.88.
5.5.12.
(E)-1-(2-(2-(4-Ethylstyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-4-phenyl-1H-1,2,3-
o
triazole (19): Yield 56%; mp 230-231 C; IR (film, cm-1): 3081, 2924, 1448, 1384, 1272,

ACCEPTED MANUSCRIPT
1
1182; H NMR (400 MHz, DMSO-d6) δ: 1.15 (t, J = 7.3 Hz, 3H, CH2CH3), 2.57 (q, J = 7.3
Hz, 2H, CH2CH3), 4.88 (t, J = 5.9 Hz, 2H, NCH2), 5.02 (t, J = 5.9 Hz, 2H, NCH2), 6.56 (d, J
= 15.4 Hz, 1H, CH=CH), 7.11 (d, J = 8.0 Hz, 2H, ArH), 7.21-7.25 (m, 1H, ArH), 7.27-7.31
(m, 2H, ArH), 7.41 (d, J = 8.0 Hz, 2H, ArH), 7.47 (d, J =15.4 Hz, 1H, CH=CH), 7.54 (s, 1H),
7.56 (d, J = 1.5 Hz, 1H, ArH), 8.22 (s, 1H), 8.47 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ:
15.38, 28.02, 45.23, 49.42, 110.84, 122.23, 125.13, 127.67, 127.99, 128.65, 130.42, 132.64,
135.15, 144.41, 146.65, 150.46; ESI-MS (m/z): 415.32 [M + H]+. Anal calcd for C23H22N6O2:
C 66.65, H 5.35, N 20.28. Found C 66.61, H 5.32, N 20.22.
5.5.13. (E)-2-(1-(2-(5-Nitro-2-styryl-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)pyridine
(20): Yield 62%; mp 208-209 oC; IR (film, cm-1): 3076, 2920, 2851, 1603, 1518, 1490, 1461,
1
1446, 1428, 1378, 1304, 1260, 1190, 1157, 1081, 1038, 996, 961, 821, 784; H NMR (400
MHz, CDCl3) δ: 4.93 (t, J = 5.1 Hz, 2H, NCH2), 5.00 (t, J = 5.1 Hz, 2H, NCH2), 6.20 (d, J =
15.4 Hz, 1H, CH=CH), 7.15-7.18 (m, 1H, ArH), 7.30-7.32 (m, 3H, ArH), 7.36-7.38 (m, 2H,
ArH), 7.59-7.61 (m, 1H, ArH), 7.65(d, J = 15.4 Hz, 1H, CH=CH), 7.84 (d, J = 8.0 Hz, 1H,
ArH), 7.94 (s, 1H, ArH), 8.16 (s, 1H), 8.49 (d, J = 3.7 Hz, 1H, ArH); ESI-MS (m/z): 388.26
[M + H]+. Anal calcd for C20H17N7O2: C 62.01, H 4.42, N 25.31. Found: C 62.06, H 4.45, N
25.28.
5.5.14. (E)-2-(1-(2-(2-(4-Fluorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-
yl) pyridine (21): Yield 32%; mp 188-189 oC; IR (film, cm-1): 2927, 1596, 1508, 1420, 1363,
1
1259, 1227, 1188, 1080, 1036; H NMR (400 MHz, CDCl3) δ: 4.92-4.94 (m, 2H, NCH2),
4.98-5.0 (m, 2H, NCH2), 6.06 (d, J = 15.4 Hz, 1H, CH=CH), 6.96-7.0 (m, 1H, ArH), 7.16-
7.19 (m, 1H, ArH), 7.32-7.36 (m, 2H, ArH), 7.58 (d, J = 15.4 Hz, 1H, ArH), 7.62-7.65 (m,
1H, ArH), 7.83-7.87 (m, 1H, ArH), 7.91 (s, 1H), 7.94 (d, J = 4.4 Hz, 1H, ArH), 8.15 (s, 1H),
8.55-8.56 (m, 1H, ArH); ESI-MS (m/z): 406.30 [M + H]+. Anal calcd for C20H16FN7O2: C
59.26, H 3.98, N 24.19. Found: C 59.35, H 3.88, N 24.28.
5.5.15. (E)-2-(1-(2-(2-(4-Chlorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-
o
1
yl) pyridine (22): Yield 80%; mp 210-211 C; H NMR (400 MHz, CDCl3) δ: 4.92-4.94 (m,
2H, NCH2), 4.98-4.99 (m, 2H, NCH2), 6.10 (d, J = 15.4 Hz, 1H, CH=CH), 7.17-7.20 (m, 2H,
ArH), 7.29 (s, 1H), 7.56 (d, J = 15.4 Hz, 1H, CH=CH), 7.61-7.65 (m, 1H, ArH), 7.82-7.84
(m, 1H, ArH), 7.91-7.95 (m, 1H, ArH), 8.07 (s, 1H), 8.14-8.16 (m, 1H, ArH), 8.48-8.49 (m,
1H, ArH), 8.55-8.56 (m, 1H, ArH); ESI-MS (m/z): 422.25 [M + H]+, 444.28 [M + H]+. Anal
calcd for C20H16ClN7O2: C 56.94, H 3.82, N 23.24. Found: C 56.83, H 3.92, N 23.35.

ACCEPTED MANUSCRIPT
5.5.16. (E)-2-(1-(2-(2-(4-Bromostyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-
yl) pyridine (23): Yield 40%; mp 212-213 oC; IR (film, cm-1): 3369, 3131, 3086, 1597, 1535,
1524, 1458, 1448, 1379, 1269, 1232, 1079; 1H NMR (400 MHz, CDCl3) δ: 4.92-4.93 (m, 2H,
NCH2), 4.98-4.99 (m, 2H, NCH2), 6.11 (d, J = 15.4 Hz, 1H, CH=CH), 7.20 (d, J = 8 Hz, 3H,
ArH), 7.41 (d, J = 8 Hz, 2H, ArH), 7.54 (d, J = 15.4 Hz, 1H, CH=CH), 7.62-7.66 (m, 1H,
ArH), 7.82 (d, J = 8 Hz, 1H, ArH), 7.93 (s, 1H), 8.15 (s, 1H), 8.48 (d, J = 2.9 Hz, 1H, ArH);
ESI-MS (m/z): 466.23 [M + H]+, 468.28 [M + 2]+. Anal calcd for: C20H16BrN7O2: C 51.52, H
3.46, N 21.03. Found: C 51.56, H 3.41, N 21.11.
5.5.17. (E)-2-(1-(2-(2-(4-Methylstyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-
yl) pyridine (24): Yield 58%; mp 237-238 oC; IR (film, cm-1): 3087, 2916, 2848, 1596, 1516,
1485, 1443, 1379, 1361, 1265, 1227, 1194, 1082, 1038, 996; 1H NMR (400 MHz, CDCl3) δ:
2.35 (s, 3H, CH3), 4.92 (t, J = 5.9 Hz, 2H, NCH2), 4.99 (t, J = 5.9 Hz, 2H, NCH2), 6.17 (d, J
= 15.4 Hz, 1H, CH=CH), 7.11 (d, J = 8.0 Hz, 2H, ArH), 7.15-7.18 (m, 1H), 7.28 (d, J = 8 Hz,
2H, ArH), 7.60-7.62 (m, 1H), 7.65 (s, 1H), 7.85 (d, J = 6.6 Hz, 1H, ArH), 7.94 (s, 1H), 8.15
13
(s, 1H), 8.49 (d, J = 3 Hz, 1H, ArH); C NMR (100 MHz, CDCl3) δ: 21.47, 45.88, 50.11,
108.34, 120.18, 122.96, 123.09, 127.66, 129.50, 131.99, 135.51, 136.72, 140.29, 141.45,
149.12, 149.30, 149.39, 151.21; ESI-MS (m/z): 402.25 [M + H]+. Anal calcd for C21H19N7O2:
C 62.83, H 4.77, N 24.42. Found: C 62.80, H 4.71, N 24.46.
5.5.18.
(E)-1-(2-(5-nitro-2-styryl-1H-imidazol-1-yl)ethyl)-4-(phenoxymethyl)-1H-1,2,3-
o
triazole (25):Yield 47%; mp 179-180 C; IR (film, cm-1): 2923, 2852, 1491, 1459, 1429,
1
1379, 1264, 1243, 1192, 1050; H NMR (400 MHz, CDCl3) δ: 4.87 (t, J = 5.1 Hz, 2H,
NCH2), 4.97 (t, J = 5.1 Hz, 2H, NCH2), 4.99 (s, 2H, OCH2), 6.18 (d, J = 15.4 Hz, 1H,
CH=CH), 6.82 (d, J = 7.3 Hz, 2H, ArH), 6.92-6.96 (m, 1H, ArH), 7.21-7.25 (m, 2H, ArH),
7.36-7.42 (m, 4H, ArH), 7.46-7.48 (m, 2H, ArH), 7.75 (d, J = 15.4 Hz, 1H, CH=CH), 8.15 (s,
1H); ESI-MS (m/z): 417.32 [M + H]+. Anal calcd for C22H20N6O3: C 63.45, H 4.84, N 20.18.
Found: C 63.41, H 4.80, N 20.12.
5.5.19. (E)-1-(2-(2-(4-Fluorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-4-(phenoxymethyl)-1H-1,2,3-
o
triazole (26): Yield 44%; mp 182-183 C; IR (film, cm-1): 2921, 2851, 1449, 1379, 1254,
1192, 1173, 1032; 1H NMR (400 MHz, CDCl3) δ: 4.87-4.88 (m, 2H, NCH2), 4.93-4.97 (m,
2H, NCH2), 5.0 (s, 2H, OCH2), 6.07 (d, J = 15.4 Hz, 1H, CH=CH), 6.81-6.84 (m, 2H, ArH),
6.90-6.96 (m, 2H, ArH), 7.08 (s, 1H), 7.22 (d, J = 8.8 Hz, 2H, ArH), 7.36 (d, J = 7.3 Hz, 1H,
ArH), 7.42 (d, J = 8.8 Hz, 1H, ArH), 7.45-7.47 (m, 1H, ArH), 7.69 (d, J = 15.4 Hz, 1H,

ACCEPTED MANUSCRIPT
CH=CH), 8.14 (s, 1H); ESI-MS (m/z): 435.27 [M + H]+. Anal calcd for C22H19FN6O3: C
60.82, H 4.41, N 19.35. Found: C 60.93, H 4.55, N 19.44.
5.5.20. (E)-1-(2-(2-(4-Chlorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-4-(phenoxymethyl)-1H-1,2,3-
o
triazole (27): Yield 51%; mp 211-212 C; IR (film, cm-1): 3079, 2918, 2850, 1450, 1383,
1
1271, 1243, 1194, 1172, 1049; H NMR (400 MHz, CDCl3) δ: 4.86 (t, J = 5.8 Hz, 2H,
NCH2), 4.97 (t, J = 5.1 Hz, 2H, NCH2), 4.99 (s, 2H, OCH2), 6.12 (d, J = 15.4 Hz, 1H,
CH=CH), 6.82 (d, J = 8 Hz, 2H, ArH), 6.94 (t, J = 7.3 Hz, 1H), 7.22 (d, J = 7.3 Hz, 2H,
ArH), 7.35 (d, J = 5.8 Hz, 3H, ArH), 7.39 (d, J = 8 Hz, 2H, ArH), 7.67 (d, J = 15.4 Hz, 1H,
CH=CH), 8.14 (s, 1H); ESI-MS (m/z): 451.23 [M + H]+, 453.24 [M + 2]+. Anal calcd for
C22H19ClN6O3: C 58.60, H 4.25, N 18.64. Found: C 58.72, H 4.37, N 18.57.
5.5.21. (E)-1-(2-(2-(4-Bromostyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-4-(phenoxymethyl)-1H-1,2,3-
o
triazole (28): Yield 52%; mp 223-224 C; IR (film, cm-1): 2921, 2851, 1516, 1458, 1377,
1
1263, 1181, 1059, 970; H NMR (400 MHz, CDCl3) δ: 4.86 (t, J = 5.8 Hz, 2H, NCH2), 4.96
(t, J = 5.8 Hz, 2H, NCH2), 4.99 (s, 2H, OCH2), 6.15 (d, J = 16.1 Hz, 1H, CH=CH), 6.82 (d, J
= 7.3 Hz, 2H, ArH), 6.92-6.96 (m, 1H, ArH), 7.22 (d, J = 7.3 Hz, 2H, ArH), 7.31 (s, 1H),
7.33 (d, J = 3.7 Hz, 2H, ArH), 7.50 (s, 1H), 7.52 (d, J = 1.5 Hz, 1H, ArH), 7.65 (d, J = 15.4
Hz, 1H, CH=CH), 8.13 (s, 1H); ESI-MS (m/z): 495.18 [M + H]+, 497.17 [M + 2]+. Anal calcd
for C22H19BrN6O3: C 53.35, H 3.87, N 16.97. Found: C 53.32, H 3.81, N 16.90.
5.5.22. (E)-1-(2-(2-(4-Methylstyryl)-5-nitro-1H-imidazol-1-yl)ethyl)-4-(phenoxymethyl)-1H-1,2,3-
o
triazole (29): Yield 53%; mp 214-215 C; IR (film, cm-1): 3111, 3077, 2917, 2850, 1725,
1626, 1601, 1528, 1515, 1496, 1450, 1435, 1406, 1382, 1365, 1301, 1265, 1245, 1195, 1213,
1173, 1078, 1042; 1H NMR (400 MHz, CDCl3) δ: 2.36 (s, 3H, CH3), 4.85 (t, J = 5.2 Hz, 2H,
NCH2), 4.95 (t, J = 5.2 Hz, 2H, NCH2), 4.98 (s, 2H, OCH2), 6.12 (d, J = 16.1 Hz, 1H,
CH=CH), 6.82 (d, J = 8.0 Hz, 2H, ArH), 6.92-6.96 (m, 1H, ArH), 7.18-7.22 (m, 2H, ArH),
7.23 (d, J = 1.5 Hz, 1H, ArH), 7.25 (s, 1H, ArH), 7.35-7.37 (m, 3H), 7.72 (d, J = 16.1 Hz,
1H, CH=CH), 8.14 (s, 1H); 13C NMR (100 MHz, CDCl3) δ: 21.48, 45.83, 50.00, 61.54,
108.27, 114.57, 121.26, 123.65, 127.71, 129.47, 129.73, 132.01, 135.51, 140.61, 141.50,
145.26, 151.19, 157.92; ESI-MS (m/z): 431.25 [M + H]+. Anal calcd for C23H22N6O3: C
64.17, H 5.15, N 19.52. Found: C 64.12, H 5.17, N 19.49.

ACCEPTED MANUSCRIPT
Acknowledgements
D.S.R. thanks R&D Grant, University of Delhi for financial support. Authors are thankful to
CIF-USIC, University of Delhi, India for spectral data.
References
1. E. C. Keen, Future Microbiol. 8 (2013) 821-823.
2. J.D. Ospina-Villa, N.Guillén, C. Lopez-Camarillo, J. Soto-Sanchez, E. Ramirez-Moreno,
R. Garcia-Vazquez, C.A. Castañon-Sanchez, A. Betanzos, L.A. Marchat, J. Microbiol.
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Table 1: In vitro antiamoebic activity of MTZ-triazole-styryl conjugates
N
Compound
IC50
R'
N
O2N
(µM)
N
N
N
R
R
R’
H
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
C6H5
8
1.12
0.18
1.11
1.65
2.13
4.18
1.35
0.12
1.22
1.77
2.51
4.68
5.13
0.13
0.29
1.10
6.01
3.56
0.28
0.35
1.68
5.38
1.79
±
0.002
0.002
0.002
0.007
0.003
0.001
0.005
0.004
0.013
0.002
0.009
0.001
0.005
0.006
0.008
0.005
0.004
0.005
0.004
0.006
0.009
0.013
0.001
4-F
9
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
4-Cl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Ref
4-Br
4-CH3
4-CH2CH3
H
C6H5
4-F
C6H5
4-Cl
C6H5
4-Br
4-CH3
4-CH2CH3
H
C6H5
C6H5
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
CH2OC6H5
CH2OC6H5
CH2OC6H5
CH2OC6H5
CH2OC6H5
MTZ
4-F
4-Cl
4-Br
4-CH3
H
4-F
4-Cl
4-Br
4-CH3

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Table 2: Glide docking energies and XP docking scores of best active molecules along with
the reference compound NADP, bound to the binding site of EhTrR.
Compound
Glide Energy
(kcal mol-1)
-47.60
XP GScore
(kcal mol-1)
-6.17
9
-52.48
-6.51
15
-48.12
-6.38
21
-46.85
-5.89
22
-45.98
-6.16
26
-43.84
-5.29
27
-73.86
-9.43
NADP

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Table 3: Prediction of Lipinski's ‘rule of 5’ for the active test compounds
Comp.
#rotor
mol_MW
donorHB accptHB QPlogPo/w
Rule of Five
(0 – 15)
(< 500 amu) (< 5)
(< 10)
6.7
6.7
6.7
6.7
6.7
6.7
5
(- 2.0 to 6.5)
1.986
2.212
2.465
2.703
2.285
2.627
3.751
4.397
4.124
4.184
4.484
4.36
(< 4)
0
9
340.34
358.33
374.79
419.24
354.37
368.39
386.41
404.40
420.86
465.31
400.44
414.47
387.4
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
8
9
0
9
9
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
MTZ
9
0
9
0
10
7
0
0
7
5
0
7
5
0
7
5
0
7
5
0
8
5
0
7
6
3.729
3.783
4.04
0
7
405.39
421.85
466.30
401.43
416.44
434.43
450.88
495.33
430.47
171.16
6
0
7
6
0
7
6
3.437
3.873
4.677
4.26
0
7
6
0
10
10
10
10
10
4
5.75
5.75
5.75
5.75
5.75
4.2
0
0
5.159
4.378
4.178
-0.019
1
0
0
0

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Table 4: Calculated ADMET properties
Comp. #rotor
(0-15)
QPlogS
(-6.5-
0.5)
QPlogHERG QPPCaco
QPlogBB QPPMDCK
(-3.0-1.2) (<25 poor
>500 great)
QPlogKhsa Percent
PSA
(concern
below -5)
(<25 poor,
>500
(-1.5to1.5)
Human Oral
(7.0 –
200.0)
Absorption
(>80% high,
<25% poor)
76.934
great)
8
9
9
9
9
9
-3.045
-3.386
-3.748
-4.226
-3.595
-5.224
-5.097
-5.153
-5.612
-5.18
139.108
140.244
140.451
152.481
139.206
-1.778
-1.674
-1.641
-1.686
-1.838
58.67
-0.225
-0.189
-0.12
113.402
113.318
113.319
113.311
113.403
9
107.139
146.41
172.042
58.715
78.325
10
11
12
79.814
81.849
78.689
80.703
92.531
100
-0.059
-0.077
13
14
15
16
17
18
10
7
-3.949
-4.143
-5.422
-4.595
-4.664
-5.653
-5.299
-5.523
-6.421
-5.294
-5.284
-6.47
139.338
273.689
345.115
275.654
276.423
347.611
-1.947
-1.317
-1.271
-1.173
-1.16
58.775
0.027
0.355
0.517
0.448
0.467
0.643
113.405
91.721
89.478
91.635
91.6
121.925
283.558
250.827
268.334
157.88
7
7
94.767
95.145
100
7
7
-1.415
89.495
19
20
21
22
23
24
8
7
7
7
7
7
-4.839
-4.954
-4.833
-5.21
-5.396
-6.906
-6.362
-6.395
-4.863
-6.417
276.429
268.858
288.645
287.732
319.495
289.078
-1.413
-1.563
-1.352
-1.313
-1.042
-1.501
123.245
119.6
0.616
0.258
0.224
0.302
0.075
0.353
96.171
92.266
93.129
94.61
91.646
101.624
100.844
100.838
99.782
233.764
317.852
320.827
129.351
-3.629
-5.075
91.895
93.669
100.854
25
10
10
10
10
10
4
-5.535
-4.542
-6.345
-4.78
-7.413
-5.864
-7.348
-5.919
-5.838
-3.156
515.31
-1.503
-1.471
-1.405
-1.472
-1.604
-1.001
241.618
214.666
549.24
0.45
100
97.815
98.848
98.796
98.97
26
278.917
477.266
269.318
270.275
259.68
0.347
0.579
0.384
0.398
-0.689
95.658
92.14
96.075
94.935
70.045
27
28
247.833
120.282
115.193
29
-4.527
-1.25
98.95
MTZ
81.489

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Fig. 1: Design strategy of the novel metronidazole-triazole-styryl hybrids

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N
N
N
N
N
N
N
CH3
CH3
CH3
CH3
N
O2N
O2N
O2N
O2N
N
N
N
N
N
N
N
N
N
N
N
N
OH
O
N
4
5
6
7
IC50 = 0.0223
µ
M
IC50 = 0.218
µ
M
IC50 = 0.0084 µM
IC50 = 0.607
µM
Lead MTZ-triazole hybrids from our previous report20
R1
R1
R1
R1
N
N
N
N
N
N
N
N
O2N
O2N
O2N
O2N
N
N
N
N
N
N
N
N
N
N
N
N
O
OH
N
(8-13)
(14-19)
14, R1 = H; 15, R1 = 4-F
(20-24)
(25-29)
8,
9,
1 = H; R1 = 4-F
R
20, R1 = H; 21, R1 = 4-F
25, R1 = H; 26, R1 = 4-F
10, R1 = 4-Cl; 11, R1 = 4-Br
12, R1 = 4-Me; 13, R1 = 4-Et
16,
18,
17,
22,
24,
; 23,
R1 = 4-Cl R1 = 4-Br
R1 = 4-Me
R1 = 4-Cl;
R1 = 4-Me;
R1 = 4-Br
19,
27,
; 28,
R1 = 4-Cl
29, R1 = 4-Me
R1 = 4-Br
R1 = 4-Et
Fig. 2: Structures of MTZ-triazole-styryl hybrids

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Fig. 3: 2D and 3D docking pose showing interaction for compound 15 in the binding site of
EHTrR.

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Fig. 4: 2D and 3D docking pose showing interaction for compound 21 in the binding site of
EHTrR.

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Scheme 1: Reagents and conditions: (i) p-TsCl, Pyridine, RT, 12 h; (ii) NaN3, DMF at 0oC then 70–
o
80 C for 1 h; (iii) t-BuOH : H2O (1:1) , Sodium ascorbate, CuSO4.5H2O; (iv) Substituted
benzaldehydes, DMSO, Dry MeOH, NaOMe, 40 oC,12 h

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Highlights
•
•
•
Synthesis of novel metronidazole-triazole-styryl hybrids.
Antiamoebic activity against HM1: IMSS strain of Entamoeba histolytica.
Investigated molecular docking studies with EhTrR protein and ADMET properties.