Metronidazole aryloxy, carboxy and azole derivatives: Synthesis, anti-tumor activity, QSAR, molecular docking and dynamics studies

Article abstract of DOI:10.1016/j.bmc.2018.12.003

A series of novel metronidazole aryloxy, carboxy and azole derivatives has been synthesized and their cytotoxic activities on three cancer cell lines were evaluated by MTT assay. Compounds 4m, 4l and 4d showed the most potent cytotoxic activity (IC₅₀s less than 100 μg/mL). Apoptosis was also detected for these compounds by flow cytometry. Docking studies were performed in order to propose the probable target protein. In the next step, molecular dynamics simulation was carried out on the proposed target protein, focal adhesion kinase (FAK, PDB code: 2ETM), bound to compound 4m. As, 4m showed a potent cytotoxic activity and an acceptable apoptotic effect, it can be a potential anticancer candidate that may work through inhibition of FAK.

Full text of DOI:10.1016/j.bmc.2018.12.003

Accepted Manuscript
Metronidazole Aryloxy, Carboxy and Azole Derivatives: Synthesis, Anti-tumor
Activity, QSAR, Molecular Docking and Dynamics Studies
Ehsan Faghih-Mirzaei, Salehe Sabouri, Leila Zeidabadinejad, Salman
AbdolahRamazani, Mehdi Abaszadeh, Arash Khodadadi, Mohadeseh
Shamsadinipour, Mandana Jafari, Somayeh Pirhadi
PII:
S0968-0896(18)31662-6
https://doi.org/10.1016/j.bmc.2018.12.003
BMC 14652
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 September 2018
27 November 2018
3 December 2018
Please cite this article as: Faghih-Mirzaei, E., Sabouri, S., Zeidabadinejad, L., AbdolahRamazani, S., Abaszadeh,
M., Khodadadi, A., Shamsadinipour, M., Jafari, M., Pirhadi, S., Metronidazole Aryloxy, Carboxy and Azole
Derivatives: Synthesis, Anti-tumor Activity, QSAR, Molecular Docking and Dynamics Studies, Bioorganic &
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.12.003
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Metronidazole Aryloxy, Carboxy and Azole Derivatives: Synthesis, Anti-tumor Activity,
QSAR, Molecular Docking and Dynamics Studies
Ehsan Faghih-Mirzaeia, Salehe Sabourib,c, Leila Zeidabadinejadd, Salman AbdolahRamazanie, Mehdi
Abaszadeh*f, Arash Khodadadif, Mohadeseh Shamsadinipourf, Mandana Jafaric and Somayeh Pirhadi*g
aDepartment of Medicinal Chemistry, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran
bDepartment of Pharmaceutical Biotechnology, Faculty of Pharmacy, Kerman University of Medical Sciences,
Kerman, Iran
cHerbal and Traditional Medicine Research Center, Kerman University of Medical Sciences, Kerman, Iran
dDepartment of Chemistry, Shahid Bahonar University of Kerman, Kerman, Iran
eMedicinal Chemistry Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
fPharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman
76175493, Iran
gMedicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
*corresponding authors
E-mail addresses: abaszadeh@kmu.ac.ir (M. Abaszadeh), s_pirhadi@sums.ac.ir (S. Pirhadi)
ABSTRACT: A series of novel metronidazole aryloxy, carboxy and azole derivatives has been
synthesized and their cytotoxic activities on three cancer cell lines were evaluated by MTT
assay. Compounds 4m, 4l and 4d showed most potent cytotoxic activity (IC50s < 100µg/mL).
Apoptosis was also detected for these compounds by flow cytometry. Docking studies were
performed in order to propose the probable target protein. In the next step, molecular dynamics
simulation was carried out on the proposed target protein, focal adhesion kinase (FAK, PDB
code: 2ETM), bound to compound 4m. As, 4m showed a potent cytotoxic activity and an
acceptable apoptotic effect, it can be a potential anticancer candidate that may work through
inhibition of FAK.
Keywords: Metronidazole, Cancer, Synthesis, QSAR, Docking, Molecular dynamics simulation,
MTT, Apoptosis
1. Introduction
The Cancer is a major health problem worldwide. In spite of being the choice method of
treatment, chemotherapy is still highly inadequate and there is a massive demand for discovering
new compounds with more potent anticancer activity with fewer side effects [1, 2]. To reach this
aim, many researchers are attempting to synthesis new compounds which specifically affect
molecular pathways in tumor cells. Metronidazole derivatives are a class of nitroimidazole
compounds with notable biological activities such as antiprotozoal,[3] antibacterial,[4]
antifungal,[5] anti-HIV,[6] antimycobaterial,[7] and anti-tumor activity [8-12]. The anti-tumor

activity, in particular, has attracted much attention due to their affinity for penetration and
accumulation in hypoxic tumors[13]. Nitroimidazoles can act as oxygen mimics especially in
hypoxic cells, so they can be used during irradiation to fix the radiation-induced damage in DNA
or other macromolecules [14]. Angiogenesis plays a very crucial role in tumor growth and
metastasis. Therefore, reducing the levels of angiogenic factors can be considered as a potential
therapeutic method for inhibition of tumor- specific angiogenesis. It is reported that some
derivatives of 2-Nitroimidazole inhibited tumor- specific angiogenesis by blocking the
production of angiogenic factors [15].
Additionally, there are other molecules proposed as targets of metronidazole derivatives. In
2010, Qian et al.[8] synthesized a series of cinnamic acid metronidazole ester derivatives. The
biological activity evaluation of the compounds indicated that some were potent inhibitors of
EGFR and HER-2. Also, in 2011, Luo et al.[10] synthesized a series of metronidazole acid acyl
sulfonamide. After biological activity assessment, it was revealed that these compounds were
potent anticancer agents and potential EGFR tyrosine kinase as well as HER-2 inhibitors. Later,
a series of 2-styryl-5-nitroimidazole derivatives were introduced by Duan et al. as focal adhesion
kinase (FAK) inhibitors with anticancer activity [11]. Another investigation by this research
group showed that 1-indolyl acetates 5-nitroimidazole targeted tubulin polymerization and
displayed a strong antiprolifrative activity [12]. Some of the pyridine ring containing
metronidazole derivatives synthesized by Yong et al. also showed quite good anticancer
activities [16].
In this study, we describe the synthesis and quantitative structure activity relationship of a
series of 26 novel aryloxy, carboxy, and azole derivatives of metronidazole. Biological
evaluation was also carried out for screening the potential cytotoxic activity by MTT assay. The
apoptosis study of three more potent of these compounds was performed by flow cytometry to
understand their groundwork mechanism of action. Molecular docking and dynamic studies were
also done based on the structure similarity of these compounds to previously reported
compounds that were potent EGFR tyrosine kinase inhibitors [10].
2. Results and discussion
2.1.Chemistry

A series of novel metronidazole aryloxy and carboxy derivatives (4a-4m, 4n-4u) were
synthesized according to the scheme 1 and the route outline in scheme 2.
Scheme 1. Reagents and conditions: (a) CHCl3, Pyridine, 0 °C, 24 h; (b) K2CO3, DMF,
80 °C, 24 h.
Scheme 2. Reagents and conditions: K2CO3, DMF, 80 °C, 72 h (c); K2CO3, DMF,
80 °C, 24 h (d) Compound 4z was synthesized as the literary method (e).17
MOTS (2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethyl 4-methylbenzenesulfonate) was
synthesized from metronidazole (MTZ) by a classical tosylation reaction which has been
precedently reported by Wang et al. [4]. In this method, a mixture of p-Toluenesulfonyl chloride,
metronidazole and pyridine was stirred in chloroform at 0 °C for 24 h to provide the desired

MOTS (yield: 74%). Then, MOTS, related phenol and potassium carbonate were heated (24 h)
in DMF at 80 °C to form compounds 4a-4n. Whereas, for the formation of 4o-4x, related
aromatic carboxylic acid was added and a longer duration (~72 h) of heating was applied. The
last series (Azole derivatives, 4v-4y) were synthesized according to the route outline in scheme
2. The general condition was the same as for compounds 4a-4m, while the workup procedure
was different. Compound 4z was synthesized as the literary method [17]. Chemical structures of
the synthesized compounds are summarized in Table 1 and 2.
Table 1
Structures of compounds 4a-4m
NO2
N
N
Ar
O
4a-4m
Compound
Ar
Compound
Ar
4a
4h
*4i
4j
O2N
O2N
4b
4c
4d
F
Cl
NO2
Cl
4k
O2N
NO2
Cl
H3C
4e
4l
NC
Cl
H3C
*4f
*4m
F

Cl
*4g
F
*QSAR prediction set
Table 2
Structures of compounds 4n-4y
NO2
NO2
O
N
N
N
N
Ar
O
NH
4v-4y
4n-4u
Compound
Ar
Compound
Ar
4n
4u
O
N
*4o
*4p
4v
F
N
N
4w
N
Br
CH3
N
4q
4x
4y
Cl
N
N
*4r
N
H3C
H3CO
N
4s
4t
OCH3

*QSAR prediction set
2.2.Cell proliferation assay
The cytotoxicity of the compounds was evaluated on three cancer cell lines by MTT method.
Table 3 shows IC50 values of 26 newly synthesized aryloxy, carboxy, and azole derivatives of
metronidazole on three cancer cell lines. Data are presented as mean ± S.E.M.
Table 3
Compounds IC50 (µg/mL) for three different cell lines. Data are presented as mean ±
S.E.M.
Cell
IC50 (µg/mL)
line
Compound
4a
HT29
A549
MCF7
134.31±3.96
102.32±4.50
552.13±14.01
108.82±6.64
158.75±8.26
140.389
107.44±3.25
129.06±2.14
574.48±16.39
77.90±6.98
149.63±9.07
225.705
106.43±5.06
85.81±5.06
378.06±11.26
69.65±8.51
187.64±9.40
115.828
4b
4c
4d
4e
4f
4g
4h
4i
198.99±7.45
115.61±11.71
111.81±2.43
580.73±18.14
104.04±5.77
76.84±2.44
94.82±12.60
121.85±4.51
110.38±13.48
108.36±1.73
573.39±15.49
144.30±4.18
79.81±3.05
96.06±2.36
110.13±3.18
126.63±16.06
71.67±2.90
582.73±19.55
109.05±6.45
83.18±2.78
64.90±12.57
4j
4k
4l
4m

4n
4o
4p
287.67
145.85
277.33
280.90
169.60
194.01
244.65
262.36
271.38
4q
4r
4s
4t
279.23
358.81
293.07
282.60
286.54
254.89
183.97
281.17
289.08
167.32
380.46
255.80
428.09
447.14
206.60
4u
4v
114.62±8.57
142.03±2.07
148.00±4.80
537.97±18.73
310.78
239.97±7.38
195.46±5.31
219.43±6.33
701.25±20.13
300.49
461.97±7.80
157.23±4.65
103.16±5.43
384.45±14.35
480.11
4w
4x
4y
4z
MOTS
MTZ
MTX
327.50
196.27
159.83
278.08
223.78
347.96
47.82±2.50
39.33±1.96
31.48±2.14
The results show that compound 4l had the most potent cytotoxic effect on HT-29 cell line
(IC50 value about 77 µg/mL). Compounds 4d and 4m also showed acceptable cytotoxicity on this
cell line (IC50 about 91 and 108 µg/mL). Compounds 4m (IC50 about 96 µg/mL), 4l (IC50 about
80 µg/mL), and 4d (IC50 about 78 µg/mL) had the most powerful anti-proliferative effect on
A549 cell line. Compounds, 4b, 4i, 4m, 4d, and 4l were more cytotoxic for MCF7 cell line than
other compounds.
With respect to the SAR, the following can be noted: 1- The presence of electron withdrawing
groups (-F -Cl -NO2 -CN) at the para position on phenyl ring enhances activity (4b, 4h, 4l, 4k).
2- Substitution at the meta position is also tolerated and does not drastically affect potency (4g,
4e), whereas ortho-substitution is not tolerated and lead to dramatic loss of activity (4c, 4j). 3- A
more dramatic improvement in activity could be achieved by the presence of a proton donor
group (e.g. OH) at the para position (comp. 4l). 4- It seems that there is a correlation between the
lipophylicity and reactivity since compound 4m has a top ranked antiprolifrative activity and

compound 4d has a greater potency than 4g. 5- In the phenolic series, the more potent the
electron withdrawing group is the more cytotoxic the compound will be (e.g. 4f is more potent
than 4e).
2.3.Apoptosis assay
After determining the IC50 values of the compounds, three of them were chosen to be
investigated by flow cytometry. The percent of necrotic, late apoptotic, viable and early
apoptotic cells are seen in Q1, Q2, Q3, and Q4 parts respectively (Figure 1). The results show
that the compounds cytotoxicity is mostly via induction of apoptosis.
Figure 1. Percentages of healthy, apototic (early and late), and necrotic A549 cells after 16 hours treatment with
none (A), 4d (B), 4l (C), and 4m (D). The cells were treated with 90 µg/mL of each compound and stained with PE
annexin V and 7AAD and analyzed with flow cytometer.
2.4.QSAR analysis
MLR model building was carried out on ten fingerprints output from the GA variable selection
method. The statistical parameters of the best constructed model are presented in Table 4.
Table 4. Statistical parameters of the best MLR model
a
b
R2
R2
R2
RMSEPc
0.063
F
R2
randomization
0.558
calibration
cv
pred
0.978
0.956
0.943
44.054
aR2 : R2 cross validation
cv
bR2pred: R2 prediction
cRMSEP: Root mean square error of prediction

The statistical parameters of the model show that it is of a high quality. The plot of predicted
versus measured activity is shown in Figure 2.
Figure 2. The plot of predicted versus measured pIC50 of the QSAR model
The model chosen fingerprints, their SMARTS pattern, regression coefficients per each and
molecules that had value one for each of them is shown in Table 5.
Table 5. Dictionary based fingerprints along with their SMARTS
pattern, regression coefficients, and compounds that had value one for
them
Rank Fingerprint
SMARTS
Regression Molecules with bit
pattern
coefficient
value one
1
2
KR2695
[!#1]Oc1[cH][ - 0.90
cH][cH][cH]c
1[!#1]
4c, 4j, 4t
AD2D 393
Presence of C- 0.122
4i, 4j, 4k, 4n, 4q, 4s,
4t, 4u, 4m, 4o, 4p, 4r
O
at
topological
distance 6
The intercept value of developed QSAR model is 2.930. Fingerprints chosen in the
constructed model are KR 2695, CDK extended fingerprint (ExtFP) 607, 359, 935, 704, 161, and
CDK 271, 161, 539, 600 and atom pairs 2D fingerprint (AD2D) 393. The most effective
fingerprints in the biological activity based on regression coefficients are KR 2695, ExtFP 935,
and CDK 271 with weight values -0.90, 0.692, -0.507 respectively. Fingerprints ExtFP and CDK
are among the hashed fingerprints. Therefore, herein just fingerprints KR 2695 and AD2D 393

are interpretable. Fingerprint KR 2695 (Oxygen attached to an aliphatic atom with a double
bond) with coefficient value -0.90 plays an important role in the structure activity relationship.
KR2695 has an adverse effect on the biological activity: having this pattern leads to less
biological activity. It appeared in compounds 4c, 4j, and 4t with pIC50 values 2.872, 2.700, and
2.834 that are among the least active compounds of the activity list. In compounds 4c, 4j, and 4t,
oxygen attached to the phenyl ring is in the ortho ring position with another substituent, Cl, NO2
and COC respectively. Another pattern for the biological activity is fingerprint AD2D 393 with
coefficient value 0.122 which shows that its occurrence improves the biological activity. It
occurred in compounds 4i, 4j, 4k, 4n, 4q, 4s, 4t, 4u, 4m, 4o, 4p, and 4r with pIC50 values 3.61,
2.7, 3.491, 3.051, 2.911, 2.853, 2.834, 3.108, 3.6601, 3.048, 3.116, and 3.053 respectively.
Several of these compounds like 4m are among the active compounds in the set. It is the
presence of C-O at topological distance 6 and is suitable for the biological activity. This
fingerprint along with ExtFP 359, and CDK 600 are the least important fingerprints effective in
the biological activity with weights of -0.117, and -0.135 respectively.
2.5.Molecular Docking studies
Based on previous reports on anticancer activity of some metronidazole derivatives [8-12],
molecular docking was performed to identify which anticancer target protein the synthesized
compounds probably affect. The compounds were fitted into the active site of epidermal growth
factor receptor (EGFR, PDB code: 1M17), tubulin (PDB code: 1SA0) and focal adhesion kinase
(FAK, PDB code: 2ETM). The results have been plotted and presented in Figure 3.

Figure 3. Interaction energies of compounds 4a-4z with EGFR (PDB code: 1M17), Tubulin (PDB code: 1SA0) and
FAK (PDB code: 2ETM)
It showed that the interaction energy between the synthesized compounds and the focal
adhesion kinase is the lowest. The compound 4m had the best energy of the ligand-receptor
complex (ΔG = -10.72 Kcal/mol) with FAK (Figure 3). Also, 4m was the most potent compound
in terms of IC50 (64 µM on MCF7) in cytotoxicity evaluation. Afterwards, the binding modes of
compounds 4m and FAK were depicted in Figure 4a, b.

Figure. 4. Binding mode of compound 4m with FAK. Only interacting residues were labeled. Hydrogen bonding
interactions are shown in green dotted lines. This figure was made using VMD (a) and MOE (b).
In the binding mode, compound 4m is strongly bound to the ATP binding site of FAK via
multiple hydrophobic interactions and three hydrogen bonds as well as one π-cation interaction.
The NH group of Gln432 formed one hydrogen bond (distance= 2.69 Å) with oxygen atom of
NO2 moiety of 4m. Also, the NH group of Phe433 formed one hydrogen bond (distance=2.88 Å)
with the other oxygen atom of NO2 moiety of 4m. Meanwhile, another hydrogen bond was
formed between the end group Lys454 and oxygen of naphtoxy group (distance=2.66 Å) of 4m.
Also, the imidazole ring of 4m formed a π-cation interaction with Lys454 (Figure 4b).
2.6.Molecular Dynamics studies
Potential energy was calculated and the MD simulation results demonstrated that the system
was equilibrated during the first 30 ns. The results show that the potential energy decreases and
finally takes constant value soon after starting the simulation (Figure 5a).
Figure 5. Potential energy is shown as a function of time for 2ETM protein (a) root mean square deviation (RMSD)
is shown as a function of time for 2ETM protein during 30 ns (b), root mean square fluctuation (RMSF) of the Cα
atoms in 2ETM plotted against time from MD simulation (c) and time evolution of the radius of gyration (Rg)
during 30 ns MD simulation of ETM and 4m (d)

The scope of structural changes of protein respect to the initial structure during the MD
simulations was monitored by plotting the root mean square deviation (RMSD) of protein in
Figure 5b. The Figure 5b shows the structural stability of 2ETM after about 12 ns from the start
of MD simulations. For 2ETM, it is simply seen that the RMSD increases steadily up to 0.37 nm.
Local 2ETM mobility was analyzed by calculating the time-averaged root mean square
fluctuation (RMSF). The RMSF showed that molecular system was stable. It measured the
deviation between the positions of the 2ETM and some reference position, which indicates the
local changes in the 2ETM (Figure 5c). The peaks show the areas of 2ETM fluctuating during
the simulation. The radius of gyration (Rg) values of 2ETM and 4m-bound-2ETM complex were
determined and plotted as a function of time, as shown in Figure 5d. In this system, initially, the
Rg value of the 4m-bound 2ETM was 0.4 nm then its value was stabilized at about 30 ns,
indicating that the MD simulation achieved equilibrium after 12 ns.
3. Conclusions
The present study describes the synthesis, structure elucidations, and investigation of cytotoxic
activities of a series of novel metronidazole aryloxy, carboxy and azole derivatives on three
cancer cell lines evaluated by MTT assay. Also, QSAR, docking, and molecular dynamics
simulation studies were carried out to examine important fragments and interactions responsible
for biological activity. Among three investigated cancer targets, interaction of synthesized
compounds with FAK was the lowest so that compound 4m showed the best interaction energy
of the ligand-receptor complex with FAK. Also, 4m was the most potent compound (IC50=64
µM on MCF7) in cytotoxicity assessment while having an acceptable apoptotic effect. So, it can
be a potential anticancer candidate that may act through inhibition of FAK. Moreover, compound
4l was the most potent cytotoxic one on HT-29 cell line (IC50 value about 77 µg/mL).
Compounds 4m, 4l, and 4d had the most powerful anti-proliferative effect on A549 cell line.
Also, compounds, 4b, 4i, 4m, 4d, and 4l were more cytotoxic for MCF7 cell line in comparison
to other compounds. The results of apoptosis assay show that the cytotoxicity of these
compounds is mostly via induction of apoptosis.
4. Experimental
4.1. Materials and measurements

All chemicals and reagents used in current study were purchased from Merck and Sigma
companies. TLC was performed on the Aluminium-backed silica gel sheets (silica GF254) and
visualized in UV light (254 nm). Melting points (uncorrected) were determined on an
Electrothermal-9100 apparatus. IR spectra were recorded on a Bruker FTIR (Alpha model). All
the 1H NMR spectra were recorded on a Bruker AVANCE III 300 MHz model Spectrometer.
13C NMR spectra were recorded on the same instruments at 75 MHz, using TMS as an internal
standard. Elemental analyses were performed using a Heraeus CHN-O-Rapid analyzer.
4.2.Chemistry
4.2.1.General procedure for synthesis of MOTS
A mixture of p-Toluenesulfonyl chloride (9.39 g, 50 mmol), metronidazole (8.55 g, 50 mmol)
and pyridine (6mL, 74.5 mmol) in chloroform (16 mL) was stirred at 0 °C in an ice bath. After
being stirred for 24 h, the precipitate formed was filtered and washed with water and dried in air.
4.2.1.1.2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-methylbenzenesulfonate (MOTS)
White crystals, yield: 80%, m.p. 151-153 ºC. IR (KBr, vmax/cm-1): 3034, 2975, 1595, 1526,
1366, 1175. 1H NMR (DMSO-d6, 300 MHz) δppm: 7.93 (s, 1H, ArH), 7.62-7.59 (q, 3J=3Hz,
2H, ArH), 7.41-7.39 (d, 3J=6Hz, 2H, ArH), 4.56 (t, 3J=6Hz, 2H, OCH2), 4.40 (t, 3J=6Hz, 2H,
NCH2), 2.41(s, 6H, 2CH3). 13C NMR (DMSO-d6, 75 MHz) δppm: 152.02, 145.74, 138.82,
133.48, 133.90, 130.66, 127.73, 69.10, 45.10, 21.38, 14.43.
4.2.2. General procedure for the synthesis of 4a-4m
To a mixture of respected phenol (3a-3n, 10 mmol) and MOTS (3.24 g, 10 mmol) in DMF (10
mL) was added potassium carbonate (1.38 g, 10 mmol) and heated at 80 °C for 24 h. The
progress of the reaction was monitored by TLC and ethanol/ethyl acetate was used as an eluent.
After completion of the reaction the mixture was poured into ice water (100 mL). The resulting
precipitate was filtered, washed with water and dried in air.
4.2.2.1. 2-methyl-5-nitro-1-(2-phenoxyethyl)-1H-imidazole (4a)
Brown crystals, yield: 74%, m.p. 102-104 ºC. IR (KBr, vmax/cm-1): 3035, 2925, 1596, 1534,
1366, 1189. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.04 (s, 1H, ArH), 7.30-6.37 (m, 5H, ArH),
4.71(t, 3J=6Hz, 2H, OCH2), 4.32 (t, 3J=3Hz, 2H, NCH2), 2.53 (s, 3H, CH3). 13C NMR

(DMSO-d6, 75 MHz) δppm: 158.21, 152.31, 133.36, 130.03, 128.51, 125.97, 121.49, 114.76
66.63, 45.75, 14.62. For C12H13N3O3, calcd.: C, 58.29; H, 5.30; N, 16.99; Found: C, 58.85; H,
5.27; N, 16.43.
4.2.2.2. 1-(2-(4-fluorophenoxy)ethyl)-2-methyl-5-nitro-1H-imidazole (4b)
Brown crystals, yield: 83%, m.p. 84-86 ºC. IR (KBr, vmax/cm-1): 3021, 2937, 1580, 1526, 1365,
1144. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.03 (s, 1H, ArH), 7.10 (t, 3J=3Hz, 2H, ArH),
6.92-6.87 (m, 2H, ArH), 4.70 (t, 3J=3Hz, 2H, OCH2), 4.30 (t, 3J=3Hz, 2H, NCH2), 2.52 (s, 3H,
CH3). 13C NMR (DMSO-d6, 75 MHz) δppm: 155.66, 154.69, 152.27, 138.94, 133.33, 116.49,
116.05, 67.32, 45.71, 14.58. For C12H12FN3O3, calcd.: C, 54.34; H, 4.56; N, 15.84; Found: C,
54.05; H, 4.27; N, 15.43.
4.2.2.3. 1-(2-(2-chlorophenoxy)ethyl)-2-methyl-5-nitro-1H-imidazole (4c)
Yellow crystals, yield: 77%, m.p. 129-131 ºC. IR (KBr, vmax/cm-1): 3010, 2943, 1589, 1530,
1361, 1186. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.07 (s, 1H, ArH), 7.42- 6.94 (m, 4H, ArH),
4.79 (t, 3J=6Hz, 2H, OCH2), 4.43 (t, 3J=6Hz, 2H, NCH2), 2.62 (s, 3H, CH3). 13C NMR
(DMSO-d6, 75 MHz) δppm: 154.30, 152.71, 139.13, 133.48, 130.61, 128.71, 122.74, 121.47,
114.52, 68.09, 45.68, 14.73. For C12H12ClN3O3, calcd.: C, 51.16; H, 4.29; N, 14.92; Found: C,
51.55; H, 4.17; N, 14.43.
4.2.2.4. 1-(2-(3,4-dichlorophenoxy)ethyl)-2-methyl-5-nitro-1H-imidazole (4d)
Brown crystals, yield: 68%, m.p. 147-150 ºC. IR (KBr, vmax/cm-1): 3020, 2989, 1599, 1527,
1361, 1155. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.04 (s, 1H, ArH), 7.49 (d, 3J=3Hz, 1H,
ArH), 7.24-7.21 (m, 1H, CH), 6.94-6.90 (m, 1H, ArH), 4.70 (t, 3J=3Hz, 2H, OCH2), 4.37 (t,
3J=3Hz , 2H, NCH2), 2.52 (s, 3H, CH3). 13C NMR (DMSO-d6, 75 MHz) δppm: 157.62,
152.23, 133.35, 132.25, 132.10, 131.44, 123.25, 116.77, 115.96, 67.60, 45.73, 14.59. For
C12H11Cl2N3O3, calcd.: C, 45.59; H, 3.51; N, 13.29; Found: C, 45.05; H, 3.29; N, 13.03.
4.2.2.5. 1-(2-(4-chloro-3-methylphenoxy)ethyl)-2-methyl-5-nitro-1H-imidazole (4e)
Yellow crystals, yield: 73%, m.p. 107-109 ºC. IR (KBr, vmax/cm-1): 3072, 2946, 1577, 1530,
1371, 1174. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.03 (s, 1H, ArH), 7.26 (d, 3J=9Hz, 1H,
ArH), 6.90 (s, 1H, ArH), 6.74 (d, 3J=3Hz, 1H, ArH), 4.70 (t, 3J=3Hz, 2H, OCH2), 4.30 (t,

3J=3Hz, 2H, NCH2), 2.52 (s, 3H, CH3), 2.26 (s, 3H, CH3). 13C NMR (DMSO-d6, 75 MHz)
δppm: 156.99, 152.28, 138.95, 137.05, 133.36, 129.95, 125.49, 117.46, 113.99, 67.06, 45.68,
20.13, 14.60. For C13H14ClN3O3, calcd.: C, 52.80; H, 4.77; N, 14.21; Found: C, 53.05; H,
4.29; N, 14.03.
4.2.2.6. 1-(2-(4-fluoro-3-methylphenoxy)ethyl)-2-methyl-5-nitro-1H-imidazole (4f)
Yellow crystals, yield: 70%, m.p. 95-97 ºC. IR (KBr, vmax/cm-1): 3042, 2954, 1599, 1526,
1363, 1185. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.05 (s, 1H, ArH), 7.04 (t, 3J=12Hz, 1H,
ArH), 6.82 (m, 1H, ArH), 6.72 (m, 1H, ArH), 4.72 (t, 3J=3Hz, 2H, OCH2), 4.29 (t, 3J=3Hz, 2H,
NCH2), 3.34 (s, 3H, CH3), 2.53(s, 3H, CH3). 13C NMR (DMSO-d6, 75 MHz) δppm: 154.34,
152.32, 133.38, 125.84, 125.40, 117.43, 116.00, 115.68, 112.94, 67.51, 45.68, 14.73. For
C13H14FN3O3, calcd.: C, 55.91; H, 5.05; N, 15.05; Found: C, 55.53; H, 5.19; N, 15.41.
4.2.2.7. 1-(2-(3-chloro-4-fluorophenoxy)ethyl)-2-methyl-5-nitro-1H-imidazole (4g)
Yellow crystals, yield: 75%, m.p. 136-139 ºC. IR (KBr, vmax/cm-1): 3041, 2946, 1594, 1528,
1363, 1187. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.04(s, 1H, ArH), 7.31(t, 3J=3Hz, 1H,
ArH), 7.16 (m, 1H, ArH), 6.89 (m, 1H, ArH), 4.70 (t, 3J=6Hz, 2H, OCH2), 4.34 (t, 3J=6Hz, 2H,
NCH2), 2.53( s, 3H, CH3). 13C NMR (DMSO-d6, 75 MHz) δppm:154.84, 152.29, 138.95,
133.36, 120.10, 117.69, 117.52, 116.34, 115.56, 67.69, 45.59, 14.60. For C12H11ClFN3O3,
calcd.: C, 48.09; H, 3.70; N, 14.02; Found: C, 48.57; H, 3.27; N, 14.41
4.2.2.8. 2-methyl-5-nitro-1-(2-(4-nitrophenoxy)ethyl)-1H-imidazole (4h)
Brown crystals, yield: 75%, m.p. 170-173 ºC. IR (KBr, vmax/cm-1): 3075, 2929, 1594, 1530,
1333, 1151. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.20-8.17 (m, 2H, ArH), 8.03 (s, 1H, ArH),
7.13-7.10 (m, 2H, ArH), 4.76 (t, 3J=6Hz, 2H, OCH2), 4.50 (t, 3J=6Hz, 2H, NCH2), 2.54 (s, 3H,
CH3). 13C NMR (DMSO-d6, 75 MHz) δppm: 163.55, 152.23, 141.72, 139.01, 133.36, 126.30,
115.51, 67.83, 45.47, 14.59. For C12H12N4O5, calcd.: C, 49.32; H, 4.14; N, 19.17; Found: C,
48.95; H, 4.29; N, 19.03.
4.2.2.9. 2-methyl-5-nitro-1-(2-(3-nitrophenoxy)ethyl)-1H-imidazole (4i)
Brown crystals, yield: 80%, m.p. 144-147 ºC. IR (KBr, vmax/cm-1): 3025, 2933, 1619, 1533,
1353, 1186. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.03 (s, 1H, ArH), 7.83-7.36 (m, 4H, ArH),

4.75 (t, 3J=6Hz, 2H, OCH2), 4.48 (t, 3J=6Hz, 2H, NCH2), 2.55 (s, 3H, CH3). 13C NMR
(DMSO-d6, 75 MHz) δppm: 158.77, 152.20, 149.15, 138.80, 133.22, 131.01, 122.36, 116.74,
109.29, 67.88, 45.49, 14.60. For C12H12N4O5, calcd.: C, 49.32; H, 4.14; N, 19.17; Found: C,
48.97; H, 4.27; N, 19.41
4.2.2.10. 2-methyl-5-nitro-1-(2-(2-nitrophenoxy)ethyl)-1H-imidazole (4j)
Brown crystals, yield: 71%, m.p. 84-87 ºC. IR (KBr, vmax/cm-1): 3024, 2993, 1609, 1524, 1369,
1191. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.05 (s, 1H, ArH), 7.83(d, 3J=3Hz, 1H, ArH),
7.63 (m, 1H, ArH), 7.35 (d, 3J=9Hz, 1H, ArH), 7.12 (m ,1H, ArH), 4.74 (t, 3J=6Hz, 2H, OCH2),
4.54 (t, 3J=6Hz, 2H, NCH2), 2.50 (s, 3H, CH3). 13C NMR (DMSO-d6, 75 MHz) δppm: 152.36,
150.91, 139.92, 138.84, 134.84, 133.56, 125.29, 121.55, 115.59, 68.37, 45.54, 14.33. For
C12H12N4O5, calcd.: C, 49.32; H, 4.14; N, 19.17; Found: C, 49.56; H, 4.77; N, 18.96
4.2.2.11. 1-(2-(2,4-dinitrophenoxy)ethyl)-2-methyl-5-nitro-1H-imidazole (4k)
Brown crystals, yield: 72%, m.p. 184-187 ºC. IR (KBr, vmax/cm-1): 3051, 2985, 1607, 1530,
1347, 1187. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.51 (d, 3J= 3Hz, 1H, ArH), 8.48 (d,
3J=3Hz, 1H, ArH), 8.06 (s, 1H, ArH), 7.61 (d, 3J=9Hz, 1H, ArH), 4.79 (t, 3J=6Hz, 2H, OCH2),
4.31 (t, 3J=6Hz, 2H, NCH2), 2.51 (s, 3H, CH3). 13C NMR (DMSO-d6, 75 MHz) δppm: 156.17,
153.02, 140.41, 139.14, 138.52, 133.47, 129.99, 121.44, 116.41, 69.71, 45.68, 14.44. For
C12H11N5O7, calcd.: C, 42.74; H, 3.29; N, 20.77; Found: C, 42.58; H, 3.17; N, 20.41
4.2.2.12. 4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)benzonitrile (4l)
Yellow crystals, yield: 75%, m.p. 147-150 ºC. IR (KBr, vmax/cm-1): 3020, 2942, 2228, 1603,
1512, 1366, 1174. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.05 (s, 1H, ArH), 7.78 (d, 3J=9Hz,
2H, ArH),7.08 (d, 3J=9Hz, 2H, ArH), 4.75 (t, 3J=6Hz, 2H, OCH2), 4.46 (t, 3J=6Hz, 2H, NCH2),
2.55 (s, 3H, CH3). 13C NMR (DMSO-d6, 75 MHz) δppm: 161.67, 152.26, 139.01, 134.70,
133.37, 126.06, 119.46, 116.00, 103.82, 67.32, 45.50, 14.61. For C13H12N4O3, calcd.: C,
57.35; H, 4.44; N, 20.58; Found: C, 57.55; H, 4.17; N, 20.43.
4.2.2.13. 2-methyl-1-(2-(naphthalen-2-yloxy)ethyl)-5-nitro-1H-imidazole (4m)
Brown crystals, yield: 73%, m.p. 110-113 ºC. IR (KBr, vmax/cm-1): 3057, 2943, 1597, 1529,
1357, 1180. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.05 (s, 1H, ArH), 7.83-7.77 (m, 3H, ArH),

7.45 (t, 3J=6Hz, 1H, ArH), 7.37-7.32 (m, 2H, ArH), 7.11-7.07 (m, 1H, ArH), 4.78 (t, 3J=6Hz,
2H, OCH2), 4.46 (t, 3J=6Hz, 2H, NCH2), 2.57 (s, 3H, CH3). 13C NMR (DMSO-d6, 75 MHz)
δppm: 156.04, 152.37, 134.57, 133.39, 129.90, 129.09, 127.98, 127.17, 126.95, 124.26, 118.76,
107.34, 66.88, 45.65, 14.65.
4.2.3. General procedure for the synthesis of 4n-4u
To a mixture of respected aryl carboxylic acid (3o-3x, 10 mmol) and MOTS (3.24 g, 10
mmol) in DMF (10 mL) potassium carbonate was added (1.38 g, 10 mmol) and heated at 80 °C.
After 3 days, the reaction mixture was poured into the ice water and the formed insoluble matter
was recovered by filtration. The insoluble matter was dissolved in hot ethanol, whereupon the
mixture was allowed to cool and crystals formed were collected and dried in air.
4.2.3.1.2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl benzoate (4n)
Yellow crystals, yield: 63%, m.p. 101-103 ºC. IR (KBr, vmax/cm-1): 3017, 2982, 1717, 1598,
1523, 1365, 1187. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.06 (s, 1H, ArH), 7.87-7.51 (m, 5H,
ArH), 4.75 (t, 3J=6Hz, 2H, OCH2), 4.65(t, 3J=6Hz, 2H, NCH2), 2.48 (s, 3H, CH3). For
C13H13N3O4, calcd.: C, 56.72; H, 4.76; N, 15.27; Found: C, 57.06; H, 4.61; N, 15.56
4.2.3.2. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 2-fluorobenzoate (4o)
Brown crystals, yield: 75%, m.p. 90-92 ºC. IR (KBr, vmax/cm-1): 3024, 2987, 1721, 1612, 1525,
1355, 1185. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.05 (s, 1H, ArH), 8.01-7.31 (m, 4H, ArH),
4.73 (t, 3J=6Hz, 2H, OCH2), 4.65 (t, 3J=6Hz , 2H, NCH2), 2.46 (s, 3H, CH3). 13C NMR
(DMSO-d6, 75 MHz) δppm: 163.60, 163.06, 152.00, 136.11, 135.99, 133.61, 132.11, 125.16,
117.75, 117.58, 64.10, 45.21, 14.31. For C13H12FN3O4, calcd.: C, 53.24; H, 4.12; N, 14.33;
Found: C, 52.85; H, 4.57; N, 14.76
4.2.3.3. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-bromobenzoate (4p)
Yellow crystals, yield: 63%, m.p. 157-159 ºC. IR (KBr, vmax/cm-1): 3035, 2977, 1734, 1618,
1525, 1353, 1188. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.04 (s, 1H, ArH), 8.00-7.34 (m, 4H,
ArH), 4.73 (t, 3J=3Hz, 2H, OCH2), 4.65 (t, 3J=3Hz , 2H, NCH2), 2.46 (s, 3H, CH3). 13C NMR
(DMSO-d6, 75 MHz) δppm: 165.13, 151.90, 139.05, 133.65, 132.48, 131.41, 128.73, 128.17,

63.61, 45.13, 14.25. For C13H12BrN3O4, calcd.: C, 44.09; H, 3.42; N, 11.86; Found: C, 43.85;
H, 3.53; N, 11.61
4.2.3.4. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 2-chlorobenzoate (4q)
Pale yellow crystals, yield: 73%, m.p. 101-103 ºC. IR (KBr, vmax/cm-1): 3021, 2959, 1733,
1612, 1525, 1365, 1184. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.07(s, 1H, ArH), 7.70-7.44 (m,
4H, ArH), 4.74 (t, 3J=6Hz, 2H, OCH2), 4.65 (t, 3J=6Hz, 2H, NCH2), 2.45 (s, 3H, CH3). For
C13H12ClN3O4, calcd.: C, 50.42; H, 3.91; N, 13.57; Found: C, 50.06; H, 3.63; N, 13.76
4.2.3.5. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-methylbenzoate (4r)
Brown crystals, yield: 71%, m.p. 105-108 ºC. IR (KBr, vmax/cm-1): 3001, 2962, 1718, 1608,
1523, 1359, 1179. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.05 (s, 1H, ArH), 7.73(d, 3J=6Hz,
2H, ArH), 7.32 (m, 3J=6Hz, 2H, ArH) 4.73 (t, 3J=3Hz, 2H, OCH2), 4.62 (t, 3J=3Hz , 2H,
NCH2), 2.45 (s, 3H, CH3), 2.36 (s, 3H, CH3). 13C NMR (DMSO-d6, 75 MHz) δppm: 165.75,
151.87, 144.48, 139.04, 133.50, 129.86, 129.51, 126.76, 63.21, 45.13, 21.41, 14.33. For
C14H15N3O4, calcd.: C, 58.13; H, 5.23; N, 14.53; Found: C, 58.45; H, 5.57; N, 14.81
4.2.3.6. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-methoxybenzoate (4s)
Yellow crystals, yield: 67%, m.p. 112-114 ºC. IR (KBr, vmax/cm-1): 3028, 2993, 1718, 1531,
1363, 1182. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.04 (s, 1H, ArH), 7.74 (d, 3J=6Hz, 2H,
ArH), 7.42 (d, 3J=6Hz, 2H, ArH), 4.74 (t, 3J=6Hz, 2H, OCH2), 4.62 (t, 3J=6Hz, 2H, NCH2),
2.44 (s, 3H, OCH3), 2.35 (s, 3H, CH3). 13C NMR (DMSO-d6, 75 MHz) δppm: 165.83, 151.89,
139.08, 138.72, 134.65, 133.56, 129.91, 129.47, 129.18, 126.39, 63.21, 44.84, 21.39, 14.28. For
C14H15N3O5, calcd.: C, 55.08; H, 4.95; N, 13.76; Found: C, 55.38; H, 4.65; N, 13.47
4.2.3.7. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 2-methoxybenzoate (4t)
Yellow crystals, yield: 65%, m.p. 50-53 ºC. IR (KBr, vmax/cm-1): 3010, 2979, 1708, 1602,
1523, 1365, 1184. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.06 (s, 1H, ArH), 7.56-6.97 (m, 4H,
ArH), 4.70 (t, 3J=6Hz, 2H, OCH2), 4.57 (t, 3J=6Hz, 2H, NCH2), 3.75 (s, 3H, OCH3), 2.45 (s,
3H, CH3). 13C NMR (DMSO-d6, 75 MHz) δppm: 165.91, 158.70, 152.20, 138.50, 134.39,
133.56, 131.27, 120.48, 119.24, 112.96, 63.51, 56.05, 44.84, 14.31. For C14H15N3O5, calcd.:
C, 55.08; H, 4.95; N, 13.76; Found: C, 55.35; H, 4.55; N, 13.51

4.2.3.8. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 2-fluorobenzoate (4u)
Brown crystals, yield: 65%, m.p. 108-109 ºC. IR (KBr, vmax/cm-1): 3018, 2998, 1714, 1612,
1530, 1363, 1177. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.04 (s, 1H, ArH), 7.98 (s, 1H, ArH),
7.22 (s, 1H, ArH), 6.70 (s, 1H, ArH), 4.70 (t, 3J=6Hz, 2H, OCH2), 4.60 (t, 3J=6Hz , 2H, NCH2),
2.49 (s, 3H, CH3). 13C NMR (DMSO-d6, 75 MHz) δppm: 157.47, 151.91, 148.49, 143.45,
138.79, 133.53, 119.20, 112.89, 63.17, 45.19, 14.13. For C11H11N3O5, calcd.: C, 49.81; H,
4.18; N, 15.84; Found: C, 50.18; H, 4.55; N, 15.47
4.2.4. General procedure for synthesis of 4v-4y
To a mixture of respected azole (3ab-3ae, 0.68 g, 10 mmol) and MOTS (3.24 g, 10 mmol) in
DMF (10 mL) potassium carbonate was added (2.07 g, 15 mmol) and heated at 80 °C for 24 h.
The progress of the reaction was monitored by TLC and hexane/ethyl acetate was used as an
eluent. After completion, the reaction was cooled and diluted with 20 mL of ethyl acetate. The
resulting salt was filtered off and organic layer remains at room temperature to stand. After 24 h,
the precipitate formed was collected and recrystallized from acetone.
4.2.4.1. 1-(2-(1H-imidazol-1-yl)ethyl)-2-methyl-5-nitro-1H-imidazole (4v)
White crystals, yield: 72%, m.p. 176 ºC (dec). IR (KBr, vmax/cm-1): 1H NMR (DMSO-d6, 300
MHz) δppm: 8.05 (s, 1H, ArH), 7.44 (s, 1H, ArH), 7.03 (d, 3J=3Hz, 1H, ArH), 6.87 (d, 3J=3Hz,
1H, ArH), 4.58 (t, 3J=6Hz, 2H, CH2), 4.42 (t, 3J=6Hz, 2H, CH2), 1.79 (s, 3H, CH3). 13C NMR
(DMSO-d6, 75 MHz) δppm: 151.86, 138.75, 138.02, 133.69, 129.25, 120.23, 47.65, 46.00,
13.07. For C9H11N5O2, calcd.: C, 48.86; H, 5.01; N, 31.66; Found: C, 48.46; H, 5.27; N, 31.21
4.2.4.2. 2-methyl-1-(2-(2-methyl-1H-imidazol-1-yl)ethyl)-5-nitro-1H-imidazole (4w)
Brown crystals, yield: 65%, m.p. 215 ºC (dec). IR (KBr, vmax/cm-1): 3013, 2956, 1534, 1366.
1H NMR (DMSO-d6, 300 MHz) δppm: 8.06 (s, 1H, ArH), 6.82 (d, 3J=3Hz, 1H, ArH), 6.69 (d,
3J=3Hz, 1H, ArH), 4.56 (t, 3J=6Hz, 2H, CH2), 4.33(t, 3J=6Hz, 2H, CH2), 2.08 (s, 3H, CH3),
1.85 (s, 3H, CH3). 13C NMR (DMSO-d6, 75 MHz) δppm: 151.96, 144.29, 139.38, 133.78,
127.26, 120.75, 47.04, 45.42, 13.26, 12.36. For C10H13N5O2, calcd.: C, 51.06; H, 5.57; N,
29.77; Found: C, 51.46; H, 5.32; N, 30.13
4.2.4.3. 1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-benzo[d]imidazole (4x)

Pale yellow crystals, yield: 68%, m.p. 108-109 ºC. IR (KBr, vmax/cm-1): 3020, 2951, 1614,
1523, 1365. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.03 (s, 1H, ArH), 8.01(s, 1H, ArH), 7.67-
7.18 (m, 4H, ArH), 4.73 (t, 3J=3Hz, 2H, OCH2), 4.68 (t, 3J=3Hz, 2H, NCH2), 1.64 (s, 3H,
CH3). 13C NMR (DMSO-d6, 75 MHz) δppm: 151.49, 144.54, 143.76, 138.86, 134.05, 133.67,
123.22, 122.21, 120.03, 110.21, 46.32, 43.96, 13.17. For C13H13N5O2, calcd.: C, 57.56; H,
4.83; N, 25.82; Found: C, 57.18; H, 4.52; N, 25.56
4.2.4.4. 1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-benzo[d][1,2,3]triazole (4y)
Yellow crystals, yield: 66%, m.p. 158-159 ºC. IR (KBr, vmax/cm-1): 3020, 2951, 1616, 1590,
1365. 1H NMR (DMSO-d6, 300 MHz) δppm: 8.05-7.38 (m , 5H, ArH), 5.20 (t, 3J= 6Hz, 2H,
CH2), 4.82 (t, 3J=6Hz, 2H, CH2), 1.79 (s, 3H, CH3). 13C NMR (DMSO-d6, 75 MHz) δppm:
151.47, 145.39, 138.81, 133.75, 133.43, 128.11, 124.84, 119.65, 110.34, 47.22, 46.04, 13.04. For
C12H12N6O2, calcd.: C, 52.94; H, 4.44; N, 30.87; Found: C, 52.66; H, 4.63; N, 30.66
4.3. Cell proliferation assay
The cancer cell lines A549 (lung), MCF-7 (breast), and HT-29 (colorectal) were purchased
from the Iranian Biological Resource Center (IBRC, Tehran, Iran). The cells were grown in
DMEM culture media supplemented with 10% FBS (Gibco) and antibiotics (penicillin 100 U/mL
and streptomycin 100 µg/mL) at 37 °C in a CO2 incubator (5% CO2 and 95% relative humidity).
After the cells reached the exponential growth, 1 ×104 (for A549 and MCF-7) and 1.2×104 (for
HT-29) cells were seeded in each well of a flat-bottom microplate and incubated for 24 h at the
above mentioned conditions. The newly synthesized compounds were dissolved in dimethyl
sulfoxide (DMSO) and the desired concentrations of each compound ranging from 0.1 to 1000
µg/mL were prepared by dilution in DMEM (without FBS). The amount of DMSO was less than
0.5% for the highest concentration. Each concentration was added to 3 wells and the microtiter
plate was further incubated for 24h. Methotrexate (MTX) was used as the reference drug.
Another well was considered as the solvent control. To evaluate the effect of the compounds on
cell growth and proliferation, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide
(MTT) powder (Sigma, St. Louis, Mo.) dissolved in PBS (pH 7.2) to a concentration of 5 mg/mL
was added to each well and the plates were incubated for 3-4 h at 37°C in dark. Then, 100 μL
DMSO was added to each well and cell viability was determined by measuring the absorbance at
570 nm. The test was done 3 times for each compound and IC50 values were calculated.

4.4. Apoptosis assay
Detection of apoptosis in cells was carried out by flow cytometry using PI annexin V
apoptosis detection kit I (BD Pharmingen). A549 cells were treated with compounds 4d, 4l, and
4m (90 µg/mL) for 16h, then trypsinized and washed twice in ice-cold PBS. The washed cells
were suspended in binding buffer to a concentration of 1 × 106 cells/mL. Then, the cells were
stained with PE annexin V and 7-amino-actinomycin-D (7-AAD) based on manufacturer's
protocol and incubated in dark. The measurement was done by FACS using flow cytometer (BD
Biosciences, San Jose, CA, USA).
4.5. Quantitative structure activity relationship (QSAR)
28 compounds were used for QSAR model building. IC50 values in µg/mL were taken in
molar range and were expressed in negative logarithmic units, pIC50 (-logIC50). The test set of 7
molecules was chosen based on Kennard-Stone algorithm [18], and was used to determine the
external predictive ability of the resulting QSAR model. The remaining 21 compounds were
treated as the training set and used to derive the regression model. Molecular fingerprints were
utilized as molecular descriptors. Most fingerprints encode structural information in a binary bit
string and are quickly calculable from the 2D-structure without any need to get the 3D structure
of compounds or carry out the energy minimization. A set of 10066 descriptors from 8 types of
fingerprints including CDK (1024 bit standard hashed), CDK extended fingerprint (1024 keys),
CDK graph only fingerprint (1024 keys), MACCS (166 keys), Pubchem (881 keys), FP4 (307
keys), atom pairs 2D fingerprints (780 keys) and Klekota-Roth fingerprints (4860 keys) were
calculated using PaDEL-Descriptor software [19]. Except for CDK, CDK extended fingerprint,
and CDK graph only fingerprint, all the others were among dictionary based fingerprints in
which SMARTS patterns define functional groups.
Genetic algorithm (GA) as a highly efficient optimization algorithm was used to search the
large dimensional space of molecular features that are responsible for the biological activities of
these compounds [20, 21]. GA is based on the principle of evolution. The selected fingerprints
were introduced to MLR analysis. As, fingerprint descriptors consist of bit strings, just zero and
one, ten bits were finally selected for model building while all compounds have had at least one
bit on. The Genetic algorithm from QSAR-Tools (http://teqip.jdvu.ac.in/QSAR_Tools/) was used
to apply GA for data generated from PaDEL-Descriptor [22, 23]. A data treatment of removing

variables with variance cut-off value of 0.001 and inter correlation cut-off value of 0.97 was
carried out before running the variable selection. Default parameters of the GA program
containing initial population=100, total number of generations=100, cross over probability=1,
mutation probability=0.5, and number of best selected equations=20 were used to set up the GA
process while the run was repeated for at least 15 times and the best set of descriptors based on
R2 and R2cv was finally chosen for the model building. Chemoface software [24] then was used
to build the MLR model, make predictions and getting statistical parameters.
4.6. Docking simulations
Molecular docking of the synthesized compounds into the three dimensional X-ray structure of
protein, was performed using the program AutoDock 4.2 (ADT) [25]. The three-dimensional
structures of the above-mentioned compounds were built and energy minimized using the
Openbabel 2.3.2 software [26]. The crystal structures of the protein complex were obtained from
the RCSB Protein Data Bank (http://www.rcsb.org). All bound water molecules and ligands were
removed from the protein and polar hydrogens were added to the proteins. Gasteigere Huckel
charges were used for the ligand while Kollman charges were calculated for the protein.
Molecular docking of all compounds was then carried out using the AutoDock 4.2 software. The
grid box was focused, based on the cognate ligand with a spacing of 0.375 Å and 60×60×60
dimensions. Parameters of genetic algorithm were set to one hundred runs, 2,500,000 energy
evaluations and 150 population size. Cluster analysis was done on the docked results, using an
RMSD tolerance of 2 Å [27].
4.7. Molecular dynamics simulation
The molecular dynamics (MD) simulation has emerged as a modern tool to specify the
interactions between proteins and drugs. The 3D structure of the protein was obtained from
protein data bank (PDB) with PDB code of 2ETM for Adhesion Kinase with 4m inside of it. MD
simulation was conducted for modeled system in explicit solvent using the GROMACS 4.5.3
package (http://www.gromacs.org). The model was solvated by 23854 water molecules in a
water box having edges at a distance of 0.7 nm. The reference temperature for coupling was 300
K and 1 atm pressure was retained. The final MD was carried out for 30,000 ps (30 ns).
Acknowledgements

The authors express their great appreciation to the Pharmaceutics Research Center, Institute of
Neuropharmacology, Kerman University of Medical Sciences, for supporting this investigation.
We also sincerely thank Prof. Farzin Hadizadeh from Mashhad University of Medical Sciences
for valuable cooperation.
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