Nitroimidazolyl hydrazones are better amoebicides than their cyclized 1,3,4-oxadiazoline analogues: In vitro studies and Lipophilic efficiency analysis

Article abstract of DOI:10.1016/j.ejmech.2013.03.034

Two series of compounds with hydrazone derivatives (HZ1-HZl2, series 1) and oxadiazoline derivatives (OZ1-OZ12, series 2) of the 2-methyl-5-nitro-1H- imidazole scaffold were designed and synthesized. Physicochemical properties and Lipophilic efficiency (LipE) analysis predicted higher intrinsic quality of the acylhydrazone derivatives (series 1) than their corresponding oxadiazoline analogues (series 2). In vitro antiamoebic results supported the above findings and validated that the acylhydrazone derivatives (HZ1-HZl2) show better activity than the oxadiazoline derivatives (OZ1-OZ12). MTT assay, using HepG2 cell line, revealed noncytotoxic nature of the compounds. The most promising results were observed for compounds HZ5 (IC₅₀ = 0.96 μM) and HZ9 (IC ₅₀ = 0.81 μM) both in silico and in vitro. Analysis of the Lipophilic efficiency (LipE) of the compounds provided new insight for the design of potent and selective amoebicides.

Full text of DOI:10.1016/j.ejmech.2013.03.034

Accepted Manuscript
Nitroimidazolyl hydrazones are better amoebicides than their cyclized 1,3,4-
oxadiazoline analogues: In vitro studies and Lipophilic efficiency analysis
Mohmmad Younus Wani, Abdul R. Bhat, Amir Azam, Fareeda Athar
PII:
S0223-5234(13)00194-3
10.1016/j.ejmech.2013.03.034
EJMECH 6072
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 December 2012
Revised Date: 20 March 2013
Accepted Date: 21 March 2013
Please cite this article as: M.Y. Wani, A.R. Bhat, A. Azam, F. Athar, Nitroimidazolyl hydrazones are
better amoebicides than their cyclized 1,3,4-oxadiazoline analogues: In vitro studies and Lipophilic
efficiency analysis, European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.03.034.
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Graphical Abstract
In vitro out comes and Lipophilic efficiency (LipE) analysis predicted higher intrinsic quality of
the acylhydrazone derivatives (series 1) than their corresponding oxadiazoline analogues (series
2), thus series 1 shows better lead-like properties.

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Nitroimidazolyl hydrazones are better amoebicides than their cyclized
1,3,4-oxadiazoline analogues: In vitro studies and Lipophilic efficiency
analysis
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Mohmmad Younus Wani1, Abdul R. Bhat2, Amir Azam3, Fareeda Athar1*
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1Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (Central
University), New Delhi-110025, India.
2School of Biotechnology,Yeungnum University, Gyeongshan, South Korea.
3Department of Chemistry, Jamia Millia Islamia (Central University), New Delhi-110025,
India
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ABSTRACT
Two series of compounds with hydrazone derivatives (HZ1-HZl2, series 1) and oxadiazoline
derivatives (OZ1-OZ12, series 2) of the 2-methyl-5-nitro-1H-imidazole scaffold were
designed and synthesized. Physicochemical properties and lipophilic efficiency (LipE)
analysis predicted higher intrinsic quality of the acylhydrazone derivatives (series 1) than
their corresponding oxadiazoline analogues (series 2). In vitro antiamoebic results supported
the above findings and validated that the acylhydrazone derivatives (HZ1-HZl2) show better
activity than the oxadiazoline derivatives (OZ1-OZ12). MTT assay, using HepG2 cell line,
revealed noncytotoxic nature of the compounds. The most promising results were observed
for compound HZ5 (IC50 = 0.96 µM) and HZ9 (IC50 = 0.81 µM) both in silico and in vitro.
Analysis of the Lipophilic Efficiency (LipE) of the compounds provided new insight for the
design of potent and selective amoebicides.
Keywords: acylhydrazone; oxadiazoline; Lipophilic efficiency; Entamoeba histolytica;
HepG2.
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*Corresponding author: Fareeda Athar, e-mail: fareeda.cirbs@jmi.ac.in, Phone+91-11-
26981717 Ext. 4492 Fax: +91-11-26980164

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1. Introduction
Design of a potential drug to treat infections caused by the intestinal protozoan parasite,
Entamoeba histolytica, has remained an active area of research since decades [1]. E.
histolytica primarily lives in the colon as a harmless commensal, but is capable of causing
devastating dysentery, colitis and liver abscess [2-4]. More than 50 million people are
estimated to suffer from the symptoms of amoebiasis such as hemorrhagic colitis and
amoebic liver abscess resulting in 100,000 deaths annually [5]. The 5-nitroimidazole drug
metronidazole has long been used for the treatment of this disease, but there are concerns
regarding its safety [6-9]. Although metronidazole and its derivatives are currently employed
in therapy, the paucity of effective drugs and potential clinical resistance necessitate the
development of a novel drug with a good therapeutic activity and without any side effects. In
this pursuit ample compounds have been synthesized and evaluated for their antiamoebic
potency, but until now no compound reached clinical trials or achieved approval which is due
to poor physicochemical properties associated with the compounds [1]. This emphasizes the
influence of physicochemical properties on the activity of the compounds and further
advocates that the attractiveness of increasing potency in medicinal chemistry optimizations
can be more detrimental than prosperous without the adequate control of physicochemical
parameters, especially log P or log D.
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In recent years the concepts of physicochemical properties [10] and Lipophilic efficiency
(LipE), [11, 12] which combines both “potency and lipophilicity,” have been shown to be
useful tools in the lead optimization process [13, 14]. Overall physicochemical properties of
drugs have been associated with bioavailability, however, to our knowledge, little has been
done to examine possible correlations between compound physicochemical properties and in
vitro outcomes. Within this study, we designed series of acylhydrazones and their cyclized
analogues (1,3,4-oxadiazolines), of the 2-methyl-5-nitro-1H-imidazole scaffold; which is the

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core ring of many antiprotozoal drugs, for the purpose of finding better antiamoebic leads
(Figure 1). Acylydrazones exhibit keto-enol tautomerism and –CO-NH-N< moiety of these
molecules is suggested to be responsible for their important biological activities [15].
Oxadiazoles on the other extreme are important bioisosters for esters and amides in drug
discovery and their biological properties are studied extensively [16]. Encouraged, we herein
report our efforts to identify better antiamoebic compounds, utilizing the concepts of LipE,
physicochemical properties and Lipinski’s rule of 5.
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2. Results and discussion
2.1. Chemistry
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Synthesis of acylhydrazone derivatives (series 1) was obtained in a three step reaction
procedure as outlined in Scheme 1. Target compounds were prepared from 2-methyl-5-nitro-
1H-imidazole (1). First of all, 2-methyl-5-nitro-1H-imidazo-ethylacetate (2) was prepared in
dry acetone from 2-methyl-5-nitro-1H-imidazole (1), addition of excess of hydrazine hydrate
in ethanol gave 2-methyl-5-nitro-1H-imidazo-acethydrazide (3). The acylhydrazone
derivatives (HZ1-HZl2) were obtained through a condensation reaction of 2-methyl-5-nitro-
1H-imidazo-acethydrazide (3) with different aromatic aldehydes in ethanol medium in 1:1
molar ratio. The structure of formed acylhydrazones was established by elemental analyses,
IR, NMR and ESI-MS spectra.
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For the synthesis of oxadiazoline derivatives (series 2), a reported procedure was adapted
[17]. The oxadiazoline derivatives were synthesized by refluxing the appropriate hydrazone
intermediates in acetic anhydride for 1-5 h under dry conditions (Scheme 1). The reaction
mixture was poured into ice-water, and the solid product was collected and washed with
water. Most products were found to be homogeneous by TLC and 300 MHz (300.13 MHz)
1H NMR analyses, but when needed, heterogeneous products were readily purified by silica

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gel column chromatography using a methanol/chloroform eluent. Oxadiazoline product yields
ranged from 45-78%. The structure of all the compounds was confirmed using NMR, IR,
mass spectrometry, and elemental analysis.
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2.2. Physicochemical properties and Lipophilic efficiency (LipE) analysis
Poor solubility and poor permeability are amongst the main causes for failure during
drug development. It is therefore, important to determine the physicochemical properties
associated with a drug. It is generally believed that high molecular weight and high
lipophilicity (cLog P) are correlated with poor oral drug-like properties. An increased
microsomal clearance and pharmacological promiscuity are often associated with higher
cLog P values, while limited cell permeation and absorption are linked with low lipophilicity
[11]. To achieve a compromise between absorption and first-pass clearance, a cLog P value
between 2 and 3 is often considered optimal in an oral drug program. Many of the processes
of drug disposition depend on the capability to cross membranes and hence there is a high
correlation with measures of lipophilicity. Hydrophobic drugs with high partition coefficients
are preferentially distributed to hydrophobic compartments such as lipid bilayers of cells
while hydrophilic drugs (low partition coefficients) preferentially are found in hydrophilic
compartments such as blood serum. Hydrophobicity/lipophilicity plays a major role in
determining where drugs are distributed within the body after adsorption and as a
consequence in how rapidly they are metabolized and excreted. Moreover, many of the
proteins involved in drug disposition have hydrophobic binding sites further adding to the
importance of lipophilicity. Our library design criteria included cLog P and molecular weight
filters (cLog P between 0 and 5 and molecular weight (MW) below 500). Figure 2 shows the
distribution of cLog P versus MW for the two series of compounds. High in vitro potency
(pIC50) is a desirable attribute in drug as it reduces the risk of non-specific, off-target
pharmacology. Lipophilic Efficiency (LipE) has recently been introduced as a parameter that
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combines both potency and lipophilicity (cLog P, cLog D or measured Log D, if appropriate)
[11]. LipE is increased when the pIC50 is increased by more than the increase in cLog P. High
LipE compounds should outperform low LipE compounds as these will be compromised by
reduced potency or increased lipophilicity. Furthermore, keeping the log P or log D of
clinical candidates low will reduce the chances of seeing toxicity or side effects [11].
Empirical evidences suggest that quality drug candidates have the highest, or near highest,
LipE for the series. Whilst achievable LipE values are target and series specific, compounds
with LipE above 5 are usually considered highly optimized. Plotting cLog P against pIC50 for
a range of compounds allows ranking series and individual compounds (Figure 2). In this
study we found that the series 1 has clog P values in the range of 0.708-2.576, while series 2
has clog P values in the range of 1.235-2.963. However the pIC50 value for series 1 ranges
from 5.368-6.091 and for series 2 it ranges from 5.007-5.661. Plotting cLog P against pIC50
(Figure 3) for series 1 and 2 shows a distribution along lines of different LipE values. Some
derivatives of series 1 (HZ1-HZl2) have high LipE values (high potency and relatively low
cLog P). Whereas series 2 (OZ1-OZ12) has most of its derivatives having lower LipE values
i.e. high cLogP values and low potency. Thus the series 1 has better lead-like properties than
series 2. Interestingly, comparing acylhydrazone derivatives, (HZ1-HZl2 pink circles) and
oxadiazoline derivatives, (OZ1-OZ12 blue circles) indicates that a reduction of cLog P and an
increase in pIC50 favors high potency. Compounds showing lower cLog P and PIC50 values
were less potent, similarly compounds showing higher cLog P and PIC50 values were also
less potent. So for a compound to show better lead-like properties and better efficacy a lower
cLog P value and a higher PIC50 value is desirable, which corresponds to a LipE value of >5.
2.3. Rule of 5 properties
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Molecular properties such as membrane permeability and bioavailability is always
associated with some basic molecular descriptors such as log P (partition coefficient),

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molecular weight (MW), or hydrogen bond acceptors and donors count in a molecule [18].
Lipinski used these molecular properties in formulating his “Rule of Five” [19]. The rule
states that most molecules with good membrane permeability have log P ≤5, molecular
weight ≤500, number of hydrogen bond acceptors ≤10, and number of hydrogen bond donors
≤5. This rule is widely used as a filter for drug-like properties. According to Lipinski [19],
four parameters namely molecular weight, log P, the number of hydrogen bond donors and
acceptors are thought to be associated with solubility, permeability and binding efficiency of
drugs which are the basic requirements for any drug to have good pharmacokinetic
properties. A compound that fulfils at least three out of the four criteria is said to adhere to
Lipinski's `rule of 5'. A poor permeation or absorption is more likely when there are more
than 5H bond donors, 10 H-bond acceptors. The compounds under investigation have most of
the compounds possessing less number of hydrogen bond donors (<5) but do possess
considerable number of acceptors (≤10), except compounds HZl2 and OZl2 which have 11
hydrogen bond acceptors as shown in tables 1 and 2. Lipinski's rule was used as a filter to
choose the reasonable scaffolds as potential lead scaffolds. Tables 1 and 2 list the values of
these properties for the new scaffolds and suggest that the active compounds are reasonable
starting points for a drug discovery effort.
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2.4. Pharmacology
2.4.1. In vitro antiamoebic activity
All the acylhydrazone derivatives (HZ1-HZl2) and their cyclized products (OZ1-OZ12)
were screened in vitro against HM1:IMSS strain of Entamoeba histolytica by microdilution
method [20]. All the experiments were carried out in triplicate at each concentration level
and repeated thrice. The results are summarized in Tables 3 and 4. The data is presented in
terms of percent growth inhibition relative to untreated controls, and plotted as probit values
as a function of drug concentration. The antiamoebic activity of the synthesized compounds

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was compared with the most widely used antiamoebic medication metronidazole with 50%
inhibitory concentration (IC50) of 1.80 ꢀM in our experiments.
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2.4.2. In vitro cytotoxicity studies
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MTT assay was used to assess the % cell viability of the newly synthesized compounds
against Human hepatocellular carcinoma cell line (HepG2) to ensure their toxic effect.
Metronidazole was used as a reference drug. A sub-confluent population of HepG2 cells was
treated with increasing concentration of these compounds and the number of viable cells was
measured after 48 h by MTT cell viability assay. The concentration range of all the
compounds was 3.13-100 µM. The cell viability (%) obtained with continuous exposure for
48 h are depicted in Figure 4. The cytotoxicity of all the compounds was found to be
concentration-dependent. Figure 4 depicts that all the compounds including the reference
compound metronidazole showed viability ranging from 98-100% at the concentration range
of 3.13 µM and up to a concentration of 25 µM all the compounds showed a viability of
≥88%. On increasing the concentration range up to 50 and 100 µM the compounds did not
show any remarkable cytotoxicity against the HepG2 cell line. All the tested compounds
showed a viability of ≥68% at a concentration of 100 µM as depicted in Figure 4. To further
investigate the selectivity of the compounds, the “safety index” (SI), defined as the toxicity
IC50/protozoal IC50, was calculated. This allows estimating the efficacy of compounds. The
results are summarized in Tables 3 and 4. Compound HZ5 and HZ9 showed higher safety
index values, better than metronidazole. From the results of antiamoebic activity and
cytotoxicity it can be inferred that all the tested compounds HZ5, HZ6, HZ9 and HZ10 are
least cytotoxic and excellent Entamoeba histolytica inhibitors as compared to the reference
drug metronidazole. These results also showed that the compound HZ5 and HZ9 despite of
being highly antiamoebic do not show any marked toxicity on human cell line and have
safety index values of >104.20 and >123.45 which is better than metronidazole (>55.55).
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2.4.3. Structure-activity relationship
The series 1 contains compounds HZ1-HZl2 with an acylhydrazone linkage which is cyclized
in the corresponding oxadiazoline derivatives (OZ1-OZ12) in series 2. The antiamoebic
activity of the test compounds seems to be position and substituent dependent. As depicted in
tables 3 and 4 compounds exhibit an interesting inhibition pattern on the tested HM1:IMSS
strain of Entamoeba histolytica. Among the synthesized compounds the series HZ1-HZl2
bearing an acylhydrazone moiety and having a free –NH and C=O group showed an
interesting inhibitory behavior which seems to be also guided by the presence of different
substituents on the phenyl ring of the acylhydrazone moiety. It is worthy to mention that the
cyclization of acylhydrazones (HZ1-HZl2) to their corresponding 1,3,4-oxadiazoline
analogues (OZ1-OZ12) resulted in precipitous decrease in the activity, which gives an
indication that the presence of free –NH and C=O group is important to activity (Figure 5).
The results of antiamoebic activity show that the compounds bearing electon withdrawing
groups at different positions of the phenyl ring of the acylhydrazone pendant show a
decreased activity than the compounds bearing electron donating groups. It is however
important to mention that the activity also depends on the strength of the group present at the
position-4 of the phenyl ring. Presence of weakly activating groups like methyl (HZ5)
showed less activity than the methoxy group (HZ9), which is strongly activating. A
simplified approach to understand this can be attributed to the electronic effects that
substituents can exert. Electron donating groups with lone pairs (OMe) on the atoms adjacent
to the p-system activate the aromatic ring by increasing the electron density on the ring
through a resonance donating effect. Methyl group without a lone pair however exerts an
inductive effect through the s-system due to electronegative effects. Similar exciting results
were obtained with a further chain extention from methyl (HZ5) to ethyl (HZ6) and methoxy
(HZ9) to ethoxy (HZ10). Electron donating groups increase the electron density which makes
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the compounds effective against microorganisms and enhances their activity. The stronger
electron donating groups however decrease the activity which corresponds to earlier reports
that high electron density causes more difficult diffusion through the cell membrane and
substantial activity loss may occur [21, 22]. Similar results were obtained for compounds
bearing electron donating groups (Me or OMe) at ortho and meta position of the phenyl ring.
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Compound HZ7 having methyl group at these positions showed moderate activity (IC50 =
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2.58 µM) comparable to the activity of the compound HZ10 (IC50 = 2.64 µM), having
methoxy group at the same positions. The presence of methoxy group at position 3, 4 and 5
of the phenyl ring in compound HZ12 further decreased the activity (IC50 = 4.12 µM). Thus
for a compound an optimum electron density is inevitable so as to gain a significant activity.
The compounds HZ2, HZ3 and HZ4 having electron withdrawing chloro group at ortho, meta
and para position respectively, showed a moderate activity however it was higher than the
compound HZ1 containing an unsubstituted phenyl ring (IC50 =4.28 µM). As discussed
earlier the antiamoebic activity decreased with the cyclization of acylhydrazone derivatives
(HZ1-HZl2) into their corresponding oxadiazoline analogues (OZ1-OZ12) however the
activity was position and substituent dependent following a similar pattern as that of their
parent acylhydrazone compounds, in the range of 2.18-9.82 µM. Based upon the results it
will be necessary to optimize the lead compounds by substitution at C-4 position of the
phenyl ring of the hydrazone pendant by electron donating groups, which seems to be very
important for antiamoebic activity, besides the position of substituents seems to be very
important factor behind the antiamoebic activity of the tested compounds. The results also
reveal that the presence of a free –NH and C=O group in the nitroimidazole based
acylhydrazones is very important for their activity. From the results it can be inferred that the
compounds HZ5, HZ6, HZ9, and HZ10 showed encouraging results with IC50 value in the
range of 0.81-1.65 µM, out of which compound HZ9 showed the most promising results with
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50% inhibitory concentration of 0.81µM. The results were also statistically evaluated by
analysis of variance. The null hypothesis was tested using t-test. The significativity of the
difference between the IC50 values of metronidazole and the compound HZ5 and HZ9 was
evaluated by t-test. The values of the calculated T were found higher than the Table value of
T at 5% level, thus concluding that the character under study is said to be significantly
influenced by the treatment.
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3. Conclusions
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In the present study, we have designed and synthesized two series of compounds with 12
hydrazone derivatives and 12 oxadiazoline derivatives of the 2-methyl-5-nitro-1H-imidazole
scaffold. LipE analysis of the synthesized compounds shows the higher intrinsic quality of
the acylhydrazone derivatives than their corresponding oxadiazoline analogues, and suggests
that these derivatives may provide us with a new class of potent and selective amoebicides. In
vitro antiamoebic results supported the above findings and validated that the acylhydrazone
derivatives (HZ1-HZl2) show better activity than the oxadiazoline derivatives (OZ1-OZ12).
Through a preliminary SAR campaign we found that the cyclization resulted in a precipitous
decrease in the activity of the compounds, which reveals that the presence of free -NH and
C=O group is important to activity, besides the presence and position of substituents played a
dominant role on the activity of the compounds, where activity seems to be guided by the
electron donating and withdrawing nature of the substituents. The most promising results
were observed for compound HZ5 (IC50 = 0.96 µM) and HZ9 (IC50 = 0.81 µM) both in silico
and in vitro. Thus the idea of appending the acylhydrazone moiety to the nitroimidazole
nucleus so as to combine the beneficial effects in a single structure by expecting some better
antiamoebic activity proved to be successful. In conclusion, the present study showed that the
synthesized compounds can be used as template for future development through modification
and derivatization to design more potent and selective antiamoebic agents.
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4. Experimental Protocol
4.1. Chemistry
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Solvents and organic reagents were purchased from Sigma Aldrich, Merck (Germany)
and Loba Chemie (India) and were used without further purification. Melting points (mp)
were performed using a Mel-temp instrument, and the results were uncorrected. Elemental
analyses were performed on HeraeusVario EL III analyzer at Central Drug Research Institute,
Lucknow, India. The results were within ± 0.4% of the theoretical values. IR spectra were
recorded on Perkin-Elmer model 1600 FT-IR RX1 spectrophotometer as KBr discs/ ATR
mode. 1H NMR and 13C NMR spectra were recorded on Bruker AVANCE 300 (300.13) MHz
spectrometer using DMSO-d6/ CDCl3 as solvent with TMS as internal standard. Splitting
patterns are designated as follows; s, singlet; d, doublet; dd, doublet of doublets; t, triplet; m,
multiplet. Chemical shift values are given in ppm. ESI-MS was recorded on a MICROMASS
QUATTRO II triple quadrupole mass spectrometer. Reactions were monitored using thin-
layer chromatography (TLC) using commercially available precoated plates (Merck Kieselgel
60 F254 silica). Visualization was achieved with UV light at 254 nm or I2 vapor staining.
Ethyl (2-methyl-5-nitro-1H-imidazol-1-yl)acetate (2)
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A mixture of 2-methyl-5-nitro-1H-imidazole 1 (1 mmol), ethyl chloroacetate (1 mmol) and
potassium carbonate (1.5 mmol) in dry acetone (5-10 mL) was refluxed for 50 h. The reaction
mixture was filtered hot and the solvent was distilled off from the filtrate. The crude ester
thus obtained was purified by recrystallization from ethanol.
Yield 85%; mp. 96 oC; Anal. Calc. For C8H11N3O4: C 45.07, H 5.20, N 19.71 %; found: C
45.40, H 5.33, N 20.01 %; ESI-MS m/z: [M++1] 213.15.
2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide (3)
A mixture of ethyl (2-methyl-5-nitro-1H-imidazol-1-yl)acetate 2 (2 mmol) and hydrazine
hydrate (2 mmol) in ethanol (10 mL) was refluxed for 10 h. The solution on cooling gave a

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solid mass of hydrazide 3, which was collected by filtration, and recrystallized from ethanol.
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Yield 60%; mp. 189 C; Anal. Calc. For C6H9N5O3: C 36.22, H 4.56, N 35.18 %; found: C
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36.03, H 4.35, N 35.42 %; ESI-MS m/z: [M++1] 199.12.
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Experimental procedure for the synthesis of acylhydrazones (HZ1-HZl2)
To a stirred solution of hydrazide 3 (1 mmol) and different aromatic aldehydes (1 mmol) in
ethanol (25 mL), water (5 mL) was added followed by dropwise addition of glacial acetic
acid (0.2 mL). The resulting mixture was refluxed for 5 h, after which the solution was
poured into ice water. The mixture was stirred until a precipitate formed, which was collected
using suction filtration and dried, followed by recrystallization in aqueous methanol, giving
acyl-hydrazones (HZ1-HZ12) in varying yields (70-85%).
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2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N'-[(1E)-phenylmethylene]acetohydrazide (HZ1)
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White solid; Yield 85%; mp 198 C; Anal. Calc. For C13H13N5O3: C 54.35, H 4.56, N 24.38
%; found: C 54.40, H 4.67, N 24.29 %; IR νmaxcm-1: 3375 (NH), 3078 (C-H stretch), 1665
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(C=O), 1621-1465 (C=N and C=C), 1548, 1365 (-NO2); 1H NMR (DMSO-d6) δ(ppm): 11.76
(1H, br, NH, hydrazone), 8.46 (1H, s, -N=CH-), 7.83 (1H, s, imidazole ring), 7.46-7.10 (5H,
m, Ar-H), 4.95 (2H, s, methylenic), 2.39 (3H, s, methyl); 13C NMR (DMSO-d6) δ(ppm):
167.9 (C=O), 164.0 (C=N), 145.5, 134.4, 130.5, 129.1, 128.5, 127.0, 125.0, 32.5, 25.5; ESI-
MS m/z: [M++1] 288.10.
N'-[(1E)-(2-chlorophenyl)methylene]-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide
(HZ2)
White solid; Yield 80%; mp 215-218 oC; Anal. Calc. For C13H12N5O3Cl: C 48.53, H 3.76, N
21.77 %; found: C 48.45, H 3.69, N 21.82 %; IR νmaxcm-1: 3358 (NH), 3075 (C-H stretch),
1661 (C=O), 1610-1460 (C=N and C=C), 1550, 1368 (-NO2), 788 (C-Cl); 1H NMR (DMSO-
d6) δ(ppm): 11.94 (1H, br, NH, hydrazone), 8.64 (1H, s, -N=CH-), 7.60 (1H, s, imidazole

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ring), 7.42-7.30 (4H, m, Ar-H), 4.81 (2H, s, methylenic), 2.45 (3H, s, methyl); 13C NMR
(DMSO-d6) δ(ppm): 168.0 (C=O), 165.0 (C=N), 159.5, 148.0, 135.0, 132.5, 129.5, 128.0,
127.5, 124.5, 31.5, 25.5; ESI-MS m/z: [M++1] 322.08.
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4
N'-[(1E)-(3-chlorophenyl)methylene]-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide
(HZ3)
5
6
White solid; Yield 82%; mp 222-225 oC; Anal. Calc. For C13H12N5O3Cl: C 48.53, H 3.76, N
21.77 %; found: C 48.42, H 3.62, N 21.89 %; IR νmaxcm-1: 3362 (NH), 3065 (C-H stretch),
1655 (C=O), 1614-1462 (C=N and C=C), 1548, 1365 (-NO2), 796 (C-Cl); 1H NMR (DMSO-
d6) δ(ppm): 11.77 (1H, br, NH, hydrazone), 7.98 (1H, s, -N=CH-), 7.69 (1H, s, imidazole
ring), 7.52-7.33 (4H, m, Ar-H), 4.78 (2H, s, methylenic) 2.43 (3H, s, methyl); 13C NMR
(DMSO-d6) δ(ppm): 169.0 (C=O), 165.5 (C=N), 159.0, 145.0, 134.5, 130.0, 129.5, 127.5,
127.0, 126.5, 37.0, 28.0; ESI-MS m/z: [M++1] 322.08.
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N'-[(1E)-(4-chlorophenyl)methylene]-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide
(HZ4)
White solid; Yield 72%; mp 236-239 oC; Anal. Calc. For C13H12N5O3Cl: C 48.55, H 3.75, N
21.77 %; found: C 48.58, H 3.70, N 21.82 %; IR νmaxcm-1: 3355 (NH), 3053 (C-H stretch),
1655 (C=O), 1611-1463 (C=N and C=C), 1545, 1370 (-NO2), 779 (C-Cl); 1H NMR (DMSO-
d6) δ(ppm): 11.76 (1H, br, NH, hydrazone), 8.23 (1H, s, -N=CH-), 7.85 (1H, s, imidazole
ring), 7.69-7.37 (4H, m, Ar-H), 4.74 (2H, s, methylenic), 2.63 (3H, s, methyl); 13C NMR
(DMSO-d6) δ(ppm): 168.0 (C=O), 166.5 (C=N), 162.5 (C=N), 150.5, 138.0, 135.0, 130.5,
129.0, 127.5, 125.0, 32.0, 28.5; ESI-MS m/z: [M++1] 322.07.
2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N'-[(1E)-(4-methylphenyl)methylene]acetohydrazide
(HZ5)

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White solid; Yield 70%; Anal. mp 190-194 C; Calc. For C14H15N5O3: C 55.81, H 5.02, N
23.24 %; found: C 55.78, H 4.94, N 23.16 %; IR νmaxcm-1: 3365 (NH), 3060 (C-H stretch),
1658 (C=O), 1609-1458 (C=N and C=C), 1543, 1368 (-NO2); 1H NMR (DMSO-d6) δ(ppm):
11.47 (1H, br, NH, hydrazone), 7.98 (1H, s, -N=CH-), 7.82 (1H, s, imidazole ring), 7.60-7.26
(4H, m, Ar-H), 4.71 (2H, s, methylenic), 2.49 (3H, s, CH3 imidazole ring), 1.28 (3H, s, CH3);
13C NMR (DMSO-d6) δ(ppm): 169.0 (C=O), 168.0 (C=N), 164.0, 155.0, 142.5, 138.0, 134.7,
132.9, 129.0, 127.0, 30.5, 25.5, 20.5; ESI-MS m/z: [M++1] 302.12.
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N'-[(1E)-(4-ethylphenyl)methylene]-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide
(HZ6)
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Cream solid; Yield 84%; mp 230-233 C; Anal. Calc. For C15H17N5O3: C 57.13, H 5.43, N
22.21 %; found: C 57.24, H 5.35, N 22.35 %; IR νmaxcm-1: 3349 (NH), 3050 (C-H stretch),
1662 (C=O), 1611-1463 (C=N and C=C), 1545, 1366 (-NO2); 1H NMR (DMSO-d6) δ(ppm):
11.57 (1H, br, NH, hydrazone), 8.21 (1H, s, -N=CH-), 7.86 (1H, s, imidazole ring), 7.65-7.23
(4H, m, Ar-H), 4.73 (2H, s, methylenic), 2.72-2.59 (2H, m, CH2CH3), 2.44 (3H, s, CH3
imidazole), 1.27 (3H, t, ethyl ); 13C NMR (DMSO-d6) δ(ppm): 170.5 (C=O), 165.5 ,160.5
(C=N), 155.0, 145.5, 139.0, 135.7, 132.9, 130.0, 129.0, 127.0, 32.5, 20.5, 14.5; ESI-MS m/z:
[M++1] 316.15.
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N'-[(1E)-(2,5-dimethylphenyl)methylene]-2-(2-methyl-5-nitro-1H-imidazol-1-yl)aceto
hydrazide (HZ7)
o
White solid; Yield 70%; mp 198-200 C; Anal. Calc. For C15H17N5O3: C 57.12, H 5.43, N
22.20 %; found: C 57.05, H 5.26, N 22.09 %; IR νmaxcm-1: 3368 (NH), 3057 (C-H stretch),
1668 (C=O), 1609-1455 (C=N and C=C), 1544, 1364 (-NO2); 1H NMR (DMSO-d6) δ(ppm):
11.36 (1H, br, NH, hydrazone), 8.17 (1H, s, -N=CH-), 7.94 (1H, s, imidazole ring), 7.82 (1H,
s, Ar-H), 7.67-6.90 (2H, m, Ar-H), 4.70 (2H, s, methylenic), 2.58 (6H, s, CH3), 2.00 (3H, s,
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CH3 imidazole ring); 13C NMR (DMSO-d6) δ(ppm): 169.5 (C=O), 165.5 ,160.5 (C=N),
155.0, 145.5, 139.0, 135.7, 132.9, 130.0, 129.0, 127.0, 32.5, 20.5, 14.5; ESI-MS m/z: [M++1]
316.14.
N'-[(1E)-(4-isopropylphenyl)methylene]-2-(2-methyl-5-nitro-1H-imidazol-1-yl)
acetohydrazide (HZ8)
o
White solid; Yield 80%; mp 210-213 C; Anal. Calc. For C16H19N5O3: C 58.35, H 5.81, N
21.26 %; found: C 58.37, H 5.77, N 21.25 %; IR νmaxcm-1: 3362 (NH), 3060 (C-H stretch),
1665 (C=O), 1618-1453 (C=N and C=C), 1548, 1367 (-NO2); 1H NMR (DMSO-d6) δ(ppm):
11.41 (1H, br, NH, hydrazone), 7.96 (1H, s, -N=CH-), 7.84 (1H, s, imidazole ring), 7.61-6.94
(4H, m, Ar-H), 5.18 (2H, s, methylenic), 3.94-3.85 (1H, m, -CH(CH3)2), 2.58 (3H, s, CH3
imidazole ring), 2.24 (6H, s, -CH(CH3)2); 13C NMR (DMSO-d6) δ(ppm): 167.5 (C=O), 165.5,
162.0 (C=N), 150.5, 146.5, 138.0, 135.7, 132.9, 130.0, 129.0, 127.0, 40.5, 35.5, 30.0, 23.5;
ESI-MS m/z: [M++1] 330.15.
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N'-[(1E)-(4-methoxyphenyl)methylene]-2-(2-methyl-5-nitro-1H-imidazol-1-yl)
acetohydrazide (HZ9)
o
White solid; Yield 75%; mp 222-225 C; Anal. Calc. For C14H15N5O4: C 52.99, H 4.76, N
22.07 %; found: C 53.02, H 4.67, N 22.15 %; IR νmaxcm-1: 3370 (NH), 3059 (C-H stretch),
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1653 (C=O), 1608-1459 (C=N and C=C), 1543, 1364 (-NO2); 1H NMR (DMSO-d6) δ(ppm):
11.63 (1H, br, NH, hydrazone), 7.92 (1H, s, -N=CH-), 7.54 (1H, s, imidazole ring), 7.36-6.82
(4H, m, Ar-H), 4.76 (2H, s, methylenic), 2.90 (3H, s, OCH3), 2.41 (3H, s, CH3 imidazole
ring); 13C NMR (DMSO-d6) δ(ppm): 168.5 (C=O), 166.8, 162.0 (C=N), 148.5, 144.0, 139.5,
134.7, 132.9, 130.0, 129.0, 127.0, 40.5, 55.0, 25.5, 10.5; ESI-MS m/z: [M++1] 318.12.
N'-[(1E)-(4-ethoxyphenyl)methylene]-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide
(HZ10)

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White solid; Yield 78%; mp 227-230 C; Anal. Calc. For C15H17N5O4: C 54.38, H 5.17, N
19.32 %; found: C 54.40, H 5.18, N 19.35 %; IR νmaxcm-1: 3365 (NH), 3055 (C-H stretch),
1650 (C=O), 1618-1463 (C=N and C=C), 1545, 1360 (-NO2); 1H NMR (DMSO-d6) δ(ppm):
11.67 (1H, br, NH, hydrazone), 8.42 (1H, s, -N=CH-), 7.68 (1H, s, imidazole ring), 7.15-6.95
(4H, m, Ar-H), 4.54 (2H, s, methylenic), 3.95-2.87 (2H, m, OCH2CH3), 2.42 (3H, s, CH3
imidazole ring), 1.35 (3H, t, OCH2CH3); 13C NMR (DMSO-d6) δ(ppm): 168.5 (C=O), 166.8,
162.0 (C=N), 148.5, 144.0, 139.5, 134.7, 132.9, 130.0, 129.0, 127.0, 62.5, 24.5, 15.5, 10.5;
ESI-MS m/z: [M++1] 332.14.
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N'-[(1E)-(2,5-dimethoxyphenyl)methylene]-2-(2-methyl-5-nitro-1H-imidazol-1-yl)
acetohydrazide (HZ11)
10
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o
White solid; Yield 80%; mp 200-203 C; Anal. Calc. For C15H17N5O5: C 51.87, H 4.93, N
20.16 %; found: C 51.76, H 5.02, N 20.08 %; IR νmaxcm-1: 3365 (NH), 3068 (C-H stretch),
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1658 (C=O), 1610-1460 (C=N and C=C), 1547, 1365 (-NO2); 1H NMR (DMSO-d6) δ(ppm):
11.73 (1H, br, NH, hydrazone), 7.93 (1H, s, -N=CH-), 7.49 (1H, s, imidazole ring), 6.96 (1H,
s, Ar-H), 6.89-6.78 (2H, m, Ar-H), 4.77 (2H, s, methylenic), 3.90 (6H, s, OCH3), 2.83 (3H, s,
CH3 imidazole ring); 13C NMR (DMSO-d6) δ(ppm): 170.5 (C=O), 165.5, 164.0 (C=N),
144.5, 140.0, 138.5, 136.5, 132.5, 130.0, 129.0, 127.0, 58.5, 22.5, 10.5; ESI-MS m/z: [M++1]
348.13.
2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N'-[(1E)-(3,4,5-trimethoxyphenyl)methylene]
acetohydrazide (HZ12)
o
White solid; Yield 72%; mp 205-207 C; Anal. Calc. For C16H19N5O6: C 50.93, H 5.08, N
18.56 %; found: C 50.95, H 5.05, N 18.60 %; IR νmaxcm-1: 3372 (NH) 3068 (C-H stretch),
1660 (C=O), 1612-1456 (C=N and C=C), 1550, 1368 (-NO2); 1H NMR (DMSO-d6) δ(ppm):
11.54 (1H, br, NH, hydrazone), 8.58 (1H, s, -N=CH-), 7.97 (1H, s, imidazole ring), 7.52 (2H,
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s, Ar-H), 4.73 (2H, s, methylenic), 3.83 (9H, s, OCH3), 2.70 (3H, s, CH3 imidazole ring); 13C
NMR (DMSO-d6) δ(ppm): 173.5 (C=O), 164.5, 160.5 (C=N), 153.5, 150.0, 143.5, 141.5,
136.5, 132.5, 130.0, 129.0, 127.0, 110.5, 54.0, 24.5, 12.5; ESI-MS m/z: [M++1] 378.15.
Experimental procedure for the synthesis of oxadiazolines (OZ1-OZ12)
All the 1,3,4-oxadiazoline compounds (OZ1-OZ12) were synthesized by addition of acetic
anhydride (5 mL) to hydrazones (HZ1-HZ12) (1 mmol), and the resulting solution was heated
to reflux for 1-5 h (Completion of the reaction was monitored by TLC). The reaction mixture
was poured into ice water, and the resulting solid product was filtered and washed with
copious amounts of water, followed by drying, to give the oxadiazoline compounds (OZ1-
OZ12) in varying yields (45-78%); [Hexane/EtOAc (9:1)].
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3-acetyl-5-[(2-methyl-5-nitro-1H-imidazol-1-yl)methyl]-2-phenyl-2,3-dihydro-1,3,4-
oxadiazole (OZ1)
o
White solid; Yield 45%; mp 195 C; Anal. Calc. For C15H15N5O4: C 54.71, H 4.59, N 21.27
%; found: C 54.64, H 5.03, N 21.37 %; IR νmaxcm-1: 3025 (C-H stretch), 1681 (C=O), 1652
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(C=N), 1548, 1359 (-NO2), 1255 (C-O-C); H NMR (DMSO-d6) δ(ppm): 7.95 (1H, s,
imidazole ring), 7.28-7.15 (5H, m, Ar-H), 6.95 (1H, s, oxadiazole ring), 4.15 (2H, s,
methylenic), 2.72 (3H, s, COCH3), 2.45 (3H, s, CH3 imidazole ring); 13C NMR (DMSO-d6)
δ(ppm): 168.0 (C=O), 143.0, 140.0, 138.5, 128.6, 125.2, 123.5, 130.0, 89.5, 35.5, 25.0 10.0;
ESI-MS m/z: [M++1] 330.11.
3-acetyl-2-(2-chlorophenyl)-5-[(2-methyl-5-nitro-1H-imidazol-1-yl)methyl]-2,3-dihydro-
1,3,4-oxadiazole (OZ2)
White solid; Yield 50%; mp 228-230 oC; Anal. Calc. For C15H14N5O4Cl: C 49.53, H 3.88, N
19.25 %; found: C 49.48, H 3.92, N 19.30 %; IR νmaxcm-1: 3029 (C-H stretch), 1685 (C=O),
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24
1658 (C=N), 1540, 1353 (-NO2), 1248 (C-O-C), 793 (C-Cl); H NMR (DMSO-d6) δ(ppm):

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7.89 (1H, s, imidazole ring), 7.20-7.12 (4H, m, Ar-H), 6.58 (1H, s, oxadiazole ring), 4.10
(2H, s, methylenic), 2.62 (3H, s, COCH3), 2.38 (3H, s, CH3 imidazole ring); 13C NMR
(DMSO-d6) δ(ppm): 169.7 (C=O), 162.3, 160.5, 143.0, 138.5, 130.7, 128.6, 125.2, 123.5,
89.5, 35.5, 21.3, 12.5; ESI-MS m/z: [M++1] 364.08.
3-acetyl-2-(3-chlorophenyl)-5-[(2-methyl-5-nitro-1H-imidazol-1-yl)methyl]-2,3-dihydro-
1,3,4-oxadiazole (OZ3)
White solid; Yield 58%; mp 215-218 oC; Anal. Calc. For C15H14N5O4Cl: C 49.54, H 3.88, N
19.25 %; found: C 49.40, H 3.90, N 19.12 %; IR νmaxcm-1: 3033 (C-H stretch), 1681 (C=O),
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1663 (C=N), 1547, 1352 (-NO2), 1253 (C-O-C), 789 (C-Cl); H NMR (DMSO-d6) δ(ppm):
7.83 (1H, s, imidazole ring), 7.18-7.02 (4H, m, Ar-H), 6.73 (1H, s, oxadiazole ring), 4.16
(2H, s, methylenic), 2.58 (3H, s, COCH3), 2.35 (3H, s, CH3 imidazole ring); 13C NMR
(DMSO-d6) δ(ppm): 168.2 (C=O), 164.8, 161.5, 143.2, 136.8, 132.9, 128.7, 125.0, 123.6,
91.5, 38.0, 25.3, 12.0; ESI-MS m/z: [M++1] 364.07.
3-acetyl-2-(4-chlorophenyl)-5-[(2-methyl-5-nitro-1H-imidazol-1-yl)methyl]-2,3-dihydro-
1,3,4-oxadiazole (OZ4)
o
Off White solid; Yield 65%; mp 184-187 C; Anal. Calc. For C15H14N5O4Cl: C 49.55, H
3.89, N 19.24 %; found: C 49.50, H 3.93, N 19.09 %; IR νmaxcm-1: 3035 (C-H stretch), 1679
1
(C=O), 1660 (C=N), 1555, 1348 (-NO2), 1250 (C-O-C), 790 (C-Cl); H NMR (DMSO-d6)
δ(ppm): 7.87 (1H, s, imidazole ring), 7.22-7.07 (4H, m, Ar-H), 6.98 (1H, s, oxadiazole ring),
4.26 (2H, s, methylenic), 2.53 (3H, s, COCH3), 2.37 (3H, s, CH3 imidazole ring); 13C NMR
(DMSO-d6) δ(ppm): 168.4 (C=O), 162.9, 161.8, 142.8, 137.5, 131.8, 127.4, 124.8, 123.0,
88.9, 36.7, 26.8, 10.0; ESI-MS m/z: [M++1] 364.07.
3-acetyl-5-[(2-methyl-5-nitro-1H-imidazol-1-yl)methyl]-2-(4-methylphenyl)-2,3-dihydro-
1,3,4-oxadiazole (OZ5)

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White solid; Yield 78%; mp 182-185 C; Anal. Calc. For C16H17N5O4: C 55.97, H 4.99, N
20.40 %; found: C 56.03, H 4.83, N 20.47 %; IR νmaxcm-1: 3042 (C-H stretch), 1685 (C=O),
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1658 (C=N), 1547, 1352 (-NO2), 1255 (C-O-C); H NMR (DMSO-d6) δ(ppm): 7.86 (1H, s,
imidazole ring), 7.15-7.00 (4H, m, Ar-H), 6.95 (1H, s, oxadiazole ring), 4.02 (2H, s,
methylenic), 2.49 (3H, s, imidazole ring), 2.37 (3H, s, CH3 phenyl ring), 2.10 (3H, s,
COCH3); 13C NMR (DMSO-d6) δ(ppm): 168.8 (C=O), 162.0, 161.3, 142.0, 137.8, 131.0,
127.9, 124.3, 123.8, 90.2, 35.0, 25.3, 10.6; ESI-MS m/z: [M++1] 344.14.
3-acetyl-2-(4-ethylphenyl)-5-[(2-methyl-5-nitro-1H-imidazol-1-yl)methyl]-2,3-dihydro-
1,3,4-oxadiazole (OZ6)
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White solid; Yield 62%; mp 195-197 C; Anal. Calc. For C17H19N5O4: C 57.14, H 5.36, N
19.60 %; found: C 57.22, H 5.43, N 19.56 %; IR νmaxcm-1: 3035 (C-H stretch), 1683 (C=O),
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1655 (C=N), 1552, 1349 (-NO2), 1250 (C-O-C); H NMR (DMSO-d6) δ(ppm): 7.79 (1H, s,
imidazole ring), 7.26-7.18 (4H, m, Ar-H), 7.02 (1H, s, oxadiazole ring), 4.17 (2H, s,
methylenic), 2.98 (2H, q, CH2CH3) 2.39 (3H, s, imidazole ring), 2.08 (3H, s, COCH3), 1.98
(3H, s, CH2CH3); 13C NMR (DMSO-d6) δ(ppm): 167.9 (C=O), 163.8, 162.5, 142.8, 137.4,
131.6, 127.0, 123.8, 123.0, 87.9, 72.5, 35.0, 25.3, 14.8, 12.0; ESI-MS m/z: [M++1] 358.15.
3-acetyl-2-(2,5-dimethylphenyl)-5-[(2-methyl-5-nitro-1H-imidazol-1-yl)methyl]-2,3-
dihydro-1,3,4-oxadiazole (OZ7)
o
Off White solid; Yield 58%; mp 200-203 C; Anal. Calc. For C17H19N5O4: C 57.14, H 5.36,
N 19.60 %; found: C 57.26, H 5.43, N 19.48 %; IR νmaxcm-1: 3032 (C-H stretch), 1680
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(C=O), 1660 (C=N), 1557, 1348 (-NO2), 1252 (C-O-C); H NMR (DMSO-d6) δ(ppm): 7.83
(1H, s, imidazole ring), 7.32-7.22 (3H, m, Ar-H), 7.06 (1H, s, oxadiazole ring), 4.13 (2H, s,
methylenic), 2.43 (3H, s, imidazole ring), 2.35 (6H, s, CH3 phenyl ring), 2.17 (3H, s,

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COCH3); 13C NMR (DMSO-d6) δ(ppm): 169.4 (C=O), 162.8, 140.7, 139.4, 132.6, 129.7,
124.2, 120.4, 89.3, 76.2, 38.1, 22.6, 10.8; ESI-MS m/z: [M++1] 358.15.
3-acetyl-2-(4-isopropylphenyl)-5-[(2-methyl-5-nitro-1H-imidazol-1-yl)methyl]-2,3-dihydro-
1,3,4-oxadiazole (OZ8)
Pale yellow solid; Yield 60%; mp 218-220 oC; Anal. Calc. For C18H21N5O4: C 58.21, H 5.70,
N 18.86 %; found: C 58.07, H 5.63, N 18.98 %; IR νmaxcm-1: 3042 (C-H stretch), 1676
1
7
8
(C=O), 1660 (C=N), 1563, 1352 (-NO2), 1250 (C-O-C); H NMR (DMSO-d6) δ(ppm): 7.92
(1H, s, imidazole ring), 7.26-7.06 (4H, m, Ar-H), 6.96 (1H, s, oxadiazole ring), 4.17 (2H, s,
methylenic), 3.30 (1H, s, CH(CH3)2), 2.48 (3H, s, imidazole ring), 2.27 (3H, s, COCH3), 1.63
(6H, s, CH(CH3)2); 13C NMR (DMSO-d6) δ(ppm): 168.3 (C=O), 162.9, 140.1, 138.2, 130.9,
128.2, 124.8, 120.6, 90.5, 74.3, 36.9, 35.0, 25.6, 10.2; ESI-MS m/z: [M++1] 372.17.
3-acetyl-2-(4-methoxyphenyl)-5-[(2-methyl-5-nitro-1H-imidazol-1-yl)methyl]-2,3-dihydro-
1,3,4-oxadiazole (OZ9)
9
10
11
12
13
14
15
o
Cream solid; Yield 45%; mp 223-225 C; Anal. Calc. For C16H17N5O5: C 53.48, H 4.77, N
19.49 %; found: C 53.29, H 4.80, N 19.32 %; IR νmaxcm-1: 3049 (C-H stretch), 1682 (C=O),
1
16
17
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19
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24
1663 (C=N), 1558, 1355 (-NO2), 1252 (C-O-C); H NMR (DMSO-d6) δ(ppm): 7.90 (1H, s,
imidazole ring), 7.08-6.89 (4H, m, Ar-H), 6.82 (1H, s, oxadiazole ring), 4.09 (2H, s,
methylenic), 3.73 (3H, s, OCH3), 2.48 (3H, s, imidazole ring), 2.18 (3H, s, COCH3); 13C
NMR (DMSO-d6) δ(ppm): 168.6 (C=O), 158.7, 151.9, 141.3, 132.7, 128.6, 128.0, 114.1,
89.2, 74.6, 35.8, 35.0,10.5; ESI-MS m/z: [M++1] 360.13.
3-acetyl-2-(4-ethoxyphenyl)-5-[(2-methyl-5-nitro-1H-imidazol-1-yl)methyl]-2,3-dihydro-
1,3,4-oxadiazole (OZ10)
o
Cream solid; Yield 68%; mp 203-205 C; Anal. Calc. For C17H19N5O5: C 54.69, H 5.13, N
19.49 %; found: C 54.65, H 5.18, N 19.50 %; IR νmaxcm-1: 3050 (C-H stretch), 1680 (C=O),

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1660 (C=N), 1562, 1350 (-NO2), 1252 (C-O-C); H NMR (DMSO-d6) δ(ppm): 7.81 (1H, s,
imidazole ring), 7.13-6.92 (4H, m, Ar-H), 6.86 (1H, s, oxadiazole ring), 4.10 (2H, s,
methylenic), 3.96-3.85 (2H, m, OCH2CH3), 2.46 (3H, s, imidazole ring), 2.20 (3H, s,
COCH3), 1.55 (3H, t, OCH2CH3); 13C NMR (DMSO-d6) δ(ppm): 169.2 (C=O), 155.1, 154.7,
142.3, 130.7, 128.2, 127.3, 112.7, 88.9, 72.7, 65.3, 32.8, 14.8, 10.0; ESI-MS m/z: [M++1]
374.15.
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4
5
6
7
3-acetyl-2-(2,5-dimethoxyphenyl)-5-[(2-methyl-5-nitro-1H-imidazol-1-yl)methyl]-2,3
dihydro-1,3,4-oxadiazole (OZ11)
8
o
9
White solid; Yield 70%; mp 210-214 C; Anal. Calc. For C17H19N5O6: C 52.44, H 4.92, N
10
17.99 %; found: C 52.38, H 4.87, N 18.03 %; IR νmaxcm-1: 3037 (C-H stretch), 1683 (C=O),
1
11
12
13
14
15
16
17
18
19
1665 (C=N), 1560, 1356 (-NO2), 1253 (C-O-C); H NMR (DMSO-d6) δ(ppm): 7.86 (1H, s,
imidazole ring), 7.03-6.89 (3H, m, Ar-H), 6.82 (1H, s, oxadiazole ring), 4.08 (2H, s,
methylenic), 3.79 (6H, s, OCH3 phenyl ring), 2.40 (3H, s, imidazole ring), 2.12 (3H, s,
COCH3); 13C NMR (DMSO-d6) δ(ppm): 168.9 (C=O), 162.4, 157.3, 155.7, 143.6, 128.5,
114.2, 112.6, 89.0, 67.8, 54.6, 37.8, 23.4, 12.6; ESI-MS m/z: [M++1] 390.14.
3-acetyl-2-(3,4,5-trimethoxyphenyl)-5-[(2-methyl-5-nitro-1H-imidazol-1-yl)methyl]-2,3-
dihydro-1,3,4-oxadiazole (OZ12)
o
Yellow solid; Yield 65%; mp 205-208 C; Anal. Calc. For C18H21N5O7: C 51.48, H 5.05, N
16.70 %; found: C 51.58, H 4.93, N 16.79 %; IR νmaxcm-1: 3029 (C-H stretch), 1676 (C=O),
1
20
21
22
23
24
1661 (C=N), 1557, 1352 (-NO2), 1248 (C-O-C); H NMR (DMSO-d6) δ(ppm): 7.89 (1H, s,
imidazole ring), 6.89 (1H, s, oxadiazole ring), 6.25 (2H, s, Ar-H), 4.13 (2H, s, methylenic),
3.80 (9H, s, OCH3 phenyl ring), 2.48 (3H, s, CH3 imidazole ring), 2.16 (3H, s, COCH3); 13C
NMR (DMSO-d6) δ(ppm): 169.7 (C=O), 160.8, 156.4, 141.5, 138.9, 137.2, 108.2, 89.7, 73.4,
56.2, 35.8, 23.6, 10.8; ESI-MS m/z: [M++1] 420.15.

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4.2. In vitro antiamoebic assay
All the test compounds (HZ1-HZl2 and OZ1-OZl2) were screened in vitro for
antiamoebic activity against HM1:IMSS strain of Entamoeba histolytica by microdilution
method [20]. Entamoeba histolytica trophozoites were cultured in wells of 96-well microtiter
plate by using Diamond TYIS-33 growth medium [23]. The test compounds (1 mg) were
dissolved in DMSO (40 µL, level at which no inhibition of amoeba occurs) [24, 25]. The
stock solutions of the compounds were prepared freshly before use at a concentration of 1 mg
mL-1. Two-fold serial dilutions were made in the wells of 96-well microtiter plate (costar).
Each test includes metronidazole as a standard amoebicidal drug, control wells (culture
medium plus amoebae) 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 and adding 5 mL of fresh
medium, chilling the culture tube on ice to detach the organisms from the side of the flask.
The number of amoeba mL-1 was estimated with a haemocytometer, using trypan blue
exclusion to confirm the viability. The suspension was diluted to 105 cells mL-1 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 ×
104 organisms/well was chosen so that confluent, but not excessive growth, took place in
control wells. Plates were sealed and gassed for 10 min with nitrogen before incubation at
35.5 oC for 72 hr. 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 35.5 oC.
This procedure was completed quickly and the plate was not allowed to cool in order to
prevent the detachment of amoebae. The plate was allowed to dry at room temperature and
the amoebae were fixed with methanol and when dried, stained with (0.5%) aqueous eosin for
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15 min. The stained plate was washed once with tap water, then twice with distilled water and
allowed to dry. A 200 µL portion of 0.1N 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 amoebal
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. The IC50 values in µM
are reported in Table 3 and 4.
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4.3. Cytotoxicity studies (MTT assay)
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4.3.1. Cell culture
Human hepatocellular carcinoma cell line (HepG2) was cultured in Dulbecco’s
modified Eagle’s medium with 10% fetal bovine serum (heat inactivated), 100 units mL-1
penicillin, 100 ꢀg mL-1 streptomycin, and 2.5 ꢀg mL-1 amphotericin B, at 37 C in a
o
saturated humidity atmosphere containing 95% air/5% CO2 [26]. The cell lines were
harvested when they reached 80% confluence to maintain exponential growth.
4.3.2. MTT assay
The MTT assay is a standard colorimetric assay, in which mitochondrial activity is
measured by splitting tetrazolium salts with mitochondrial dehydrogenases in viable cells
only [27]. For viability testing, MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium
bromide, M2128 from Sigma) cell proliferation assay was carried out. The cell monolayers
in exponential growth were harvested using 0.25% trypsin and single-cell suspensions were
obtained by repeated pipetting. Only viable cells were used in the assay. Exponentially
growing cells were plated at 1.2 × 104 cells per well into 96-well plates (Costar, Corning, NY,
USA) and incubated for 48 hr before the addition of drugs to achieve the maximum
confluency of the cells. Stock solutions were prepared by dissolving the compounds in 10%

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(v/v) DMSO and further diluted with fresh complete medium to achieve 1M concentration.
Cells were incubated with different concentrations of metronidazole and test compounds for
48 hours at 37 oC in 5% CO2 humidified incubator together with untreated control sample. At
appropriate time points, cells were washed in PBS, treated with 50 ꢀL MTT solution (5
mgmL-1, tetrazolium salt) and incubated for 4 hr at 37 oC. At the end of the incubation period,
the medium was removed and pure DMSO 150 µL was added to each well. The metabolized
MTT product dissolved in DMSO was quantified by measuring the absorbance at 570 nm on
an Microplate reader (iMark, BIORAD, S/N 10321) with a reference wavelength of 655 nm.
All assays were performed in triplicate and repeated thrice. Percent viability was defined as
the relative absorbance of treated versus untreated control cells.
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Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by Department of Science and Technology (Grant no. SR/FT/LS-
069/2007). M.Y.Wani would like to thank UGC india for financial support. Authors are
thankful to Dr. S.N. Kazim, Centre for Interdisciplinary Research in Basic Sciences, JMI,
New Delhi, for providing Laboratory facilities for biological work.
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Caption Headings
Tables
3
4
Table 1: Physicochemical properties, Lipophilic efficiency analysis and Lipinski’s rule of 5
for acylhydrazone derivatives (HZ1-HZ12) series 1.
5
6
Table 2: Physicochemical properties, Lipophilic efficiency analysis and Lipinski’s rule of 5
7
8
for oxadiazoline derivatives (OZ1-OZ12) series 2.
Table 3: In vitro antiamoebic activity of compounds (HZ1-HZ12) against HM1: IMSS strain
of Entamoeba histolytica and toxicity profile.
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Table 4: In vitro antiamoebic activity of compounds (OZ1-OZ12) against HM1: IMSS strain
11
of Entamoeba histolytica.
12 Figures
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Figure 1: Antiprotozoal drugs and the designed target compounds with a common 2-methyl-
5-nitro-1H-imidazole scaffold.
Figure 2: cLog P versus molecular weight (MW) distribution of compounds, colored by
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series (series 1: pink dots, series 2: blue dots).
Figure 3: A plot of cLogP vs pIC50 for 2 series of compounds. Diagonal lines represent areas
of equal LipE. Analysis of this LipE plot shows that series 1 includes many
compounds with a high LipE, and thus may represent a better lead series for further
optimization.
Figure 4: Percentage of viable cells after 48 h pre-treatment of HepG2 cell line with
Metronidazole, compounds HZ5, HZ6, HZ9 and HZ10 as evaluated by MTT
assay.
Figure 5: Structural requirements for the antiamoebic activity of substituted/unsubstituted
nitroimidazole ring bearing acylhydrazones.
27 Schemes
28
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31
Scheme 1: Schematic representation of synthesis of acylhydrazones and their cyclized 1,3,4-
oxadiazoline products. Where “R” corresponds to different substituents as
mentioned in tables 3 and 4.

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Table 1: Physicochemical properties, Lipophilic efficiency analysis and Lipinski’s rule of 5
for acylhydrazone derivatives (HZ1-HZ12) series 1.
Compound
Lipophilic Hydrogen Hydrogen Lipinski’s
pIC50 C Log P M.W
efficiency
LipE
bond
donors
bond
acceptors
rule of 5
5.368
5.413
5.417
5.438
6.017
5.879
5.588
5.434
6.091
5.782
5.578
5.385
1.149
1.262
1.862
1.862
1.648
2.177
1.847
2.576
1.368
1.897
1.007
0.708
287.2
321.7
321.7
321.7
301.3
315.3
315.3
329.3
317.3
331.3
347.3
377.3
4.237
4.151
3.555
3.576
4.369
3.702
3.741
2.858
4.732
3.885
4.571
4.677
1
1
1
1
1
1
1
1
1
1
1
1
8
8
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
HZ1
HZ2
HZ3
HZ4
HZ5
HZ6
HZ7
HZ8
HZ9
HZ10
HZ11
HZ12
8
8
8
8
8
8
9
9
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5
M.W (Molecular Weight); Y: Yes (Lipinski’s rule of 5 obeyed or not)