Synthesis, anticancer activity and molecular modeling studies of 1,2,4-triazole derivatives as EGFR inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.07.024

A series of novel compounds carrying 1,2,4-triazole scaffold were prepared and evaluated for their antiproliferative activities against NCI 60 cell line. Compounds 10 (a, c), 11 (a-d), and 14 (a-e) were selected for evaluation at single concentration of 10 μM towards panel of sixty cancer cell lines. Some of nitric oxide (NO) donating triazole/oxime hybrids 11a-d showed antiproliferative activity better than their corresponding ketones. On the other hand, the thiazolo [3,2-b][1,2,4]-triazoles 14a-e showed remarkable antiproliferative activities against the same cell lines. Compound 14d was selected for five dose testing against the full panel of 60 human tumor cell lines. Compound 14d showed high selectivity against renal subpanel with selectivity ratio of 6.99 at GI₅₀ level. Compounds 11a-d, 10a-d and 14a-e were tested against four cell lines using MTT assay then compounds of the least IC₅₀ were evaluated against three known anticancer targets including EGFR, BRAF and Tubulin. The results revealed that compound 14d showed promising EGFR inhibitory activity of cancer cell proliferation and were also observed to be moderate BRAF and tubulin inhibitors. Moreover, cell cycle analysis and apoptosis assay were finished for compounds 14d and 14f. Finally molecular modeling studies were performed to explore the binding mode of the most active compounds to the target enzymes.

Full text of DOI:10.1016/j.ejmech.2018.07.024

Accepted Manuscript
Synthesis, anticancer activity and molecular modeling studies of 1,2,4-triazole
derivatives as EGFR inhibitors
Hany A.M. El-Sherief, Bahaa G.M. Youssif, Syed Nasir Abbas Bukhari, Ahmed H.
Abdelazeem, Mohamed Abdel-Aziz, Hamdy M. Abdel-Rahman
PII:
S0223-5234(18)30580-4
10.1016/j.ejmech.2018.07.024
EJMECH 10559
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 May 2018
Revised Date: 5 July 2018
Accepted Date: 9 July 2018
Please cite this article as: H.A.M. El-Sherief, B.G.M. Youssif, S.N. Abbas Bukhari, A.H. Abdelazeem, M.
Abdel-Aziz, H.M. Abdel-Rahman, Synthesis, anticancer activity and molecular modeling studies of 1,2,4-
triazole derivatives as EGFR inhibitors, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.07.024.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, anticancer activity and molecular modeling studies of 1,2,4-
triazole derivatives as EGFR inhibitors
1,3*
2,3
3
Hany A. M. El-Sherief , Bahaa G. M. Youssif , Syed Nasir Abbas Bukhari , Ahmed H.
5
1,6
4
Abdelazeem , Mohamed Abdel-Aziz *, Hamdy M. Abdel-Rahman
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Nahda University, Beni-suef,
2
Egypt; Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Assiut University,
3
Assiut 71526, Egypt; Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf
4
University, Aljouf, Sakaka 2014, Saudi Arabia; Department of Medicinal Chemistry, Faculty of
5
Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt; Department of Medicinal Chemistry,
6
Faculty of Pharmacy, Minia University, 61519-Minia, Egypt; Department of Medicinal Chemistry,
Faculty of Pharmacy, Assiut University, 71526-Assiut, Egypt.
Short running title: Synthesis, anticancer activity and mechanistic study of new 1,2,4-triazole
derivatives
*To whom correspondence should be addressed.
Mohamed Abdel-Aziz, Ph.D. Department of Medicinal Chemistry, Faculty of Pharmacy, Minia
University, 61519-Minia, Egypt.
Tel.:(002)-01003311327
E-mail address: Abulnil@hotmail.com
Hany A. M. El-Sherif, M.Sc. Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Nahda
University, Beni-Suef, Egypt
Tel.:(+966)-598031838
E-mail address: drhanyabdeltawab@yahoo.com

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Abstract
A series of novel compounds carrying 1,2,4-triazole scaffold were prepared and evaluated for
their antiproliferative activities against NCI 60 cell line . Compounds 10 (a, c), 11 (a-d), and 14
(a-e) were selected for evaluation at single concentration of 10 µM towards panel of sixty
cancer cell lines. Some of nitric oxide (NO) donating triazole/oxime hybrids 11a-d showed
antiproliferative activity better than their corresponding ketones. On the other hand, the
thiazolo[3,2-b][1,2,4]-triazoles 14a-e showed remarkable antiproliferative activities against the
same cell lines. Compound 14d was selected for five dose testing against the full panel of 60
human tumor cell lines. Compound 14d showed high selectivity against renal subpanel with
selectivity ratio of 6.99 at GI level. Compounds 11a-d, 10a-d and 14a-e were tested against
5
0
four cell lines using MTT assay then compounds of the least IC were evaluated against three
5
0
known anticancer targets including EGFR, BRAF and Tubulin. The results revealed that
compound 14d showed promising EGFR inhibitory activity of cancer cell proliferation and
were also observed to be moderate BRAF and tubulin inhibitors. Moreover, cell cycle analysis
and apoptosis assay were finished for compounds 14d and 14f. Finally molecular modeling
studies were performed to explore the binding mode of the most active compounds to the target
enzymes.
Keywords: 1,2,4-Triazoles, thiazolo[3,2-b][1,2,4]-triazoles
, antiproliferative, cell cycle,
apoptosis and EGFR inhibitors.

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1
. Introduction
Cancer is a life threatening disease and remains a major health problem around the globe. It is
the second most occurring disease after cardiovascular diseases. Thus, the development of
potent and effective novel antineoplastic drugs is one of the most intensely persuaded goals of
contemporary medicinal chemistry [1]. On the other hand, epidermal growth factor receptor
(EGFR) is a receptor tyrosine kinase (RTK) of the ErbB family [2]. EGFR is known to be a key
regulator of many complex biological processes, including cell motility, adhesion, and cell
cycle regulation, as well as angiogenesis, apoptosis, and metastasis [3, 4]. Aberrant activity of
EGFR has been observed in a wide variety of human cancers [5], such as non-small cell lung
cancer (NSCLC) [6], head and neck cancer [7], breast cancer [8], and colorectal cancer [9], and
the dysregulation of EGFR signaling is invariably associated with poor clinical outcomes.
Based on its critical role in the tumor progression, many antibodies and small molecule
inhibitors that target EGFR have been developed over the last three decades. Several of them
(e.g., Gefitinib and erlotinib)[10-13] have been approved by the US Food and Drug
Administration (FDA).
Furthermore, the chemistry of 1,2,4-triazoles and their fused heterocyclic derivatives have
received considerable attention owing to their synthetic and effective biological importance. For
example, a large number of 1,2,4-triazoles have been incorporated into a wide variety of
therapeutically interesting drug candidates possessing antimicrobial [14], anti-inflammatory
[
15], analgesic [16] and anticancer activities [17, 18]. We already reported the synthesis of a
series of acylated 1,2,4-triazole derivatives with potent anticancer and EGFR inhibitory
activities [19].

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Figure 1: Design of anticancer 1,2,4-triazoles-NO donors hybrids
Based on these finding, and in continuation of our search, we designed the synthesis of two
series of 1,2,4-triazoles –Nitric oxide (NO) hybrids to be evaluated for their anticancer activity
and EGFR inhibition. According to our published research [19], acylation on N-1 of 5-amino-
1
,2,4-triazoles enhanced their EGFR activity, Thus this work was designed to make alkylation
of 5-amino-1,2,4-triazoles at ring N-1 incorporation of NO donating moiety to further increase
the anticancer activity. The target compounds will incorporate both 1,2,4-triazole acetamide
structure as apoptosis inducers [20] connected to the NO donor moiety (Fig. 1). Already 1,2,4-
triazoles/nitric oxide (NO) hybrids were obtained by reaction of triazolylaminoacetophenones
with hydroxylamine hydrochloride (Fig. 1).

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During the course of our synthesis of the target compounds, the reaction of 1,2,4-triazole-3-
thiones with aromatic ketones in boiling acetic acid and catalytic amount of conc. H SO was
2
4
expected to afford the 1,2,4-triazolylthioacetophenones, however, the obtained products were
identified as substituted thiazolo[3,2-b][1,2,4]triazoles (Fig. 2). Furthermore, the cell cycle
activity will be determined for the most active compounds, to give an idea about the possible
phase in which those new compounds halt the growth of cancer cells. Finally, molecular
docking of the synthesized compounds inside the active site of EGFR will be performed in
order to confirm their possible mode with enzyme.
X
R
N
N
O
N
N
N
N
R2
N
R1
S
H
NH2
1
4a-f
Compounds 10a-d; X= O
1
1
4a: R = H, R = H
1
2
Compounds 11a-d; X= N-OH
4b: R = H, R = 4-Cl
1
2
1
1
1
1
0, 11a: R= CF3
14c: R = 4-OCH , R = H
1 3 2
0, 11b: R= C H
14d: R
14e: R
14f: R
1
= 4-OCH
= 3,4,5tri(OCH
= 3,4,5tri(OCH
3
, R
2
= 4-Cl
), R
), R
6
5
0, 11c: R= 4-Cl-C H
0, 11d: R= 3,4,5tri(OCH )-C H
6 2
1
3
2
= H
= 4-Cl
6
4
3
1
3
2
Figure 2: The final synthesized compounds
2
2
. Result and discussion
.1 Chemistry
The target compounds 11a-d were prepared according to Scheme 1 and 2. The key
intermediates 3 [21], 7a-c [22] [23] and compound 9 were prepared according to reported
literatures and their structures were confirmed by matching their physical characters with the
reported ones .
Compounds 10a-d were prepared in good yields by alkylation of compounds 3 and 7a-c with
compound 9 in acetonitrile using triethylamine as a base.

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1
13
IR, H NMR and C NMR studies as well as high resolution mass provide a confirm support
for the structural elucidation of compounds 10a-d. IR spectra of compounds 10a-d showed
-1
-1
-1
significant bands at 3450-3470 cm (NH), 1645-1650 cm (CO-NH), 1710-1720 cm (COCH )
3
-1
and forked peak at 3150-3200 cm corresponding to NH group indicating that alkylation of
2
1
amino triazole occurs on acidic NH not on NH . H NMR spectra showed singlet signals at 2.13
2
ppm (CH ), 4.89- 4.93 ppm (CH ), 6.43- 6.84 ppm (NH ) and 10.66-10.67 ppm (NH). In
3
2
2
addition to a doublet at 7.72-7.98 ppm (J = 8.4- 8.8 Hz) corresponding to aromatic protons of
1
3
N-acetylphenylacetamide which confirm cis configuration. C NMR spectra of compounds
1
0a-d showed different significant peaks at δ 56 and 60 ppm related to triOCH gp, 158, 166
3
and 199 ppm, representative for C=N, carbonyl of CONH and COCH , respectively. Moreover,
3
aliphatic groups CH and CH were detected in regions of 26 and 56 ppm, respectively.
3
2
Refluxing compounds 10a-d with hydroxylamine hydrochloride in ethanol to give the oximes
1a-d in good yields (Scheme 2). The structure assignment of oxime derivatives 11a-d is based
1
1
13
on IR, H NMR, C NMR spectral data and high resolution mass. IR spectra of compounds
-1
1
1a-d showed intense broad bands at 3000-3480 cm due to OH, NH and NH groups, strong
2
-1
absorption bands at 1645-1655 cm corresponding to (CO-NH) and disappearance of the ketone
-
1
1
peaks at 1710- 1720 cm (COCH ) which confirm oxime formation. The H NMR spectra of
3
compounds 11a-d showed a characteristic singlet signal at 10.40-10.42 ppm due to NH group.
The resonance of OH, CH and CH were observed as four singlets in the expected regions at
3
2
1
3
1
1.08-11.09 ppm, 2.13 ppm and 4.84-4.92 ppm, respectively. C NMR spectra of compounds
1
1a-d showed peaks at δ 56 and 60 ppm corresponding to triOCH gp, 158 and 166 ppm
3
signifying for C=N, and carbonyl of CONH, respectively. Moreover, detection of aliphatic
groups CH and CH at chemical shift 26 and 56 ppm with disappearance of the characteristic
3
2
peak of carbonyl (COCH ) indicate the formation of oxime.
3

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Scheme 1: Synthesis of the key intermediates 3, 7a-c and 9.
Reagents and reaction conditions (i) 210°C, 2 h; (ii) Conc H SO / CH OH, reflux, 4-8 h; (iii)
2
4
3
NH NH .H O, EtOH, reflux, 2-4 h; (iv) S-methylthiourea, NaOH, RT, 72 h; (v) H O, reflux, 4
2
2
2
2
h; (vi) Bromoacetyl bromide, CH OH, K CO , RT, 4 h.
3
2
3

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Scheme 2: Synthesis of the target compounds 11a-d.
Reagents and reaction conditions (i) CH CN, triethylamine, reflux; (ii) NH OH.HCl, EtOH,
3
2
reflux.
Compounds 14a-f were prepared according to Scheme 3. Reaction of compounds 5a-c with
NH SCN under basic condition followed by cyclization with NaOH afforded 5-substituted-4H-
4
1
,2,4-triazole-3-thiones 12a-c [24]. Reaction of 12a-c with aromatic ketones 13a-b in boiling
acetic acid and catalytic amount of conc. H SO was expected to afford the 1,2,4-
2
4
triazolylthioacetophenones as reported [25]and afforded the cyclized products identified as the
,5-di(substitutedphenyl)thiazolo[3,2-b][1,2,4]triazoles 14a-f in one step with 63-84% yields
Scheme 3). The chemical structures of compounds 14a-f were elucidated on the basis of their
2
(
1
13
IR, H NMR, C NMR spectra and high resolution mass. IR spectra of compounds 14a-f
1
showed no peaks corresponding to NH , NH, CO and CONH groups. H NMR spectra of
2
compounds 14a-f showed singlet signal at δ 7.91- 8.01 ppm (1H) corresponding for CH of
thiazole. On the other hand, only compounds 14c-d derivatives exhibited doublet at 7.08 and
8
.07 ppm for p-methoxyphenyl moiety. Compounds 14e-f showed singlet signal at δ 7.42 ppm
(2H) of trimethoxyphenyl moiety. Appropriate signals for other aryl substituents were detected

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1
13
in the H NMR spectra of compounds 14a-f. C NMR spectra of compound 14a-f showed
different significant peaks at δ 56 and 60 ppm related to triOCH gp, 111, 134 and 166 ppm,
3
corresponding for, C-S, C-Cl and C=N, respectively.
Scheme 3: Synthesis of the final target compounds 14a-f.
Reagents and reaction conditions (i) NH SCN, dil. HCl, reflux; (ii) NaOH reflux, then 5M
4
HCl; (iii) CH COOH, Conc. H SO , Reflux.
3
2
4
2
2
.2 Biology
.2.1 Evaluation of in vitro antiproliferative activity for compounds selected by NCI
The newly synthesized compounds 10a-d, 11a-d and 14a-f were submitted to National Cancer
Institute (NCI, Bethesda, ML, USA; http://www.dtp.nci.nih.gov.), and twelve compounds 10 (a,
c), 11 (a-d), and 14 (a-e) were selected for evaluation at single concentration of 10 µM on the
basis of their structural variation towards panel of sixty cancer cell lines taken from nine
different tissues (lung, blood, CNS, colon, skin, kidney, ovary, prostate and breast). The results
for each compound were reported as the percent growth of treated cells compared to untreated
control cells. Compounds 11a-d possessing oxime group were more active than their parent
ketones 10a-c. This directly reflects the influences of such moieties on the antiproliferative
activity of the title scaffold. Compound 10c exhibited moderate cell growth inhibition activity
in opposition to all types of cancer cell lines, wherever it inhibited Non-Small Cell Lung Cancer
NCI-H522, Non-Small Cell Lung Cancer A549/ATCC and Ovarian Cancer SK-OV-3 with GI

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values of 28, 22 and 22 % respectively (Table 1, Appendix A). Compound 11d showed a
moderate cell growth inhibition activity against all types of cancer cell lines. It inhibited
Melanoma Cancer v and Non-Small Cell Lung Cancer NCI-H522 activity with GI value 36 and
2
9 %, respectively. Compounds 11a, 11c and 11d showed higher antiproliferative activity than
the corresponding phenyl analogues 11b. Compound 11d with electron donating trimethoxy
moiety has superior activity than the corresponding electron withdrawing chloro moiety against
most of all cell lines.
On the other hand, compounds 14a-e showed highly promising activities of all tested
compounds against all cancer cell lines. By comparing the effect of substituent attached to two
phenyl moieties on activity, it was found that compounds 14c and 14d possessing p-methoxy
and p-chloro substituent were more active than compounds 14a, 14b and 14e. Compound 14a
showed potent cell growth inhibition activity against all types of cancer cell lines with. It
inhibited Breast Cancer T-47D, Breast Cancer MCF7 and Renal Cancer UO-31 with (GI) values
of 63, 61 and 53 %, respectively (Table 2, Appendix A). Compound 14b showed powerful cell
growth inhibition activity against all types of cancer cell lines. It inhibited Breast Cancer T-
4
6
7D, Ovarian Cancer SK-OV-3 and Ovarian Cancer OVCAR-4with (GI) values of 74, 62 and
0 %, respectively (Table 2, Appendix A). P-methoxy derivatives 14c showed extremely
persuasive cell growth inhibition activity against all types of cancer cell lines. It has inhibitory
effect on Breast Cancer MDA-MB-468, Leukemia K-562, Breast Cancer MCF7, Melanoma
M14, Colon Cancer KM12, Colon Cancer HT29, Non-Small Cell Lung Cancer NCI-H522 and
Colon Cancer HCT-15 with (GI) values of 81, 76, 74, 71, 70, 70, 70 and 69 %, respectively. P-
methoxy derivatives 14c showed weak lethal effect on Melanoma MDA-MB-435 (Table 2,
Appendix A). Compound 14d showed the most potent cell growth inhibition activity of all
tested compounds. It has inhibitory effect on Renal Cancer SN12C, Ovarian cancer IGROV1,
Breast Cancer MDA-MB-468, CNS Cancer SF-539, Non-Small Cell Lung Cancer NCI-

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H322M, Colon Cancer HT29 and Non-Small Cell Lung Cancer HOP-92 with (GI) values of 95,
9
3, 87, 85, 83, 83 and 79 %, respectively. P-methoxy derivatives 14d showed weak lethal effect
on Colon Cancer HCT-116, CNS Cancer SNB-19, CNS Cancer SNB-75, Renal Cancer UO-31
and Renal Cancer CAKI-1. Moreover, compound 14d showed moderate lethal effect on Non-
Small Cell Lung Cancer A549/ATCC, Non-Small Cell Lung Cancer NCI-H460, Renal Cancer
A498 and Renal Cancer TK-10. Also it exhibited potent lethal effect on CNS Cancer U251,
Ovarian cancer OVCAR-4, Renal Cancer 786-0 and Renal Cancer ACHN (Table 2, Appendix
A). Compound 14d was selected for five dose screening as promising compound. Compound
1
4e showed forceful cell growth inhibition activity against all types of cancer cell lines. It
inhibited Leukemia SR, Non-Small Cell Lung Cancer EKVX and Breast Cancer T-47D with
GI) values of 65, 63 and 55 %, respectively (Table 2, Appendix A). According to the above,
(
the following can be deduced, compounds 14a-e showed potent cytotoxic activity especially in
presence of electron withdrawing group (like chloro), if compared to compounds containing
electron donating groups. Among all target compounds, compound 14c and 14d showed the
most promising results. They exerted broad-spectrum antiproliferative activity against different
cell lines of different cancer types. Therefore, these compounds could be potential lead
compounds for the future development of broad spectrum anticancer agents.
2
.2.2 In vitro five dose full NCI 60 cell panel assay
Compound 14d was selected for the advanced five dose testing against the full panel of 60
human tumor cell lines. All the 60 cell lines representing nine tumor subpanels were incubated
at five different concentrations (0.01, 0.1, 1, 10, and 100 µM). The outcomes were used to form
log concentration versus % growth inhibition curves and three response parameters (GI , TGI,
5
0
and LC ) were calculated for each cell line. The GI value (growth inhibitory activity)
5
0
50
corresponds to the concentration of the compound causing 50% decrease in net cell growth, the
TGI value (cytostatic activity) is the concentration of the compound resulting in total growth

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inhibition (TGI) and LC50 value (cytotoxic activity) is the concentration of the compound
causing net 50% loss of initial cells at the end of the incubation period of 48 h [26]. Compound
1
4d exhibited noteworthy antiproliferative activity against the majority of the tested cell lines
representing nine different subpanels. Compound 14d showed high activities alongside most of
the tested cell lines with GI50 ranging from 0.766 to >50 µM (Table 3, Appendix A). The
principle for selectivity of compound depends upon the ratio obtained by dividing the full panel
MID (the average sensitivity of all cell lines toward the test agent) (µM) by their individual
subpanel MID (µM). Ratios between 3 and 6 refer to moderate selectivity; ratios >6 indicate
high selectivity toward the corresponding cell line, whilst compounds not meeting either of
these criterion rated nonselective. In this case, compound 14d was found to have potent cell
growth inhibition activity against most of the tumor subpanels tested with selectivity ratios
ranging between 0.30 and 6.99 at the GI50 level. Compound 14d was found to be broad-
spectrum cell growth inhibition activity against most of the tested tumor subpanels cell lines
with high selectivity against renal subpanel with selectivity ratio of 6.88 µM at GI50 level
(Table 3, Appendix A)
Selectivity index
.

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Figure 3: Dose-antiproliferative response of compound 14d against nine different cancer cell
lines
2
.2.3 Effect of synthetic compounds on cancer cell growth
In this study, propidium iodide (PI) fluorescence assay was performed to evaluate the effect of
new synthetic compounds on Panc-1(pancreas cancer cell line), PaCa-2 (pancreatic carcinoma
cell line), HT-29 (colon cancer cell line) and H-460 (lung cancer cell line), while Erlotinib
hydrochloride was used as a positive control.
The anticancer activity results are summarized in Table 4 and are presented as IC values. It is
5
0
obvious from the results, that the synthesized compounds demonstrated promising anticancer
activity almost against all the cell lines tested to a reasonable extent. Among the first series
(10a-d and 11a-d) there were two types of compounds, first class (10a-d) were ketone
intermediates containing 1,2,4 triazole linked with phenyl acetyl through an acetamide bridge

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while on the other side second group (11a-d) obtained from ketones were also 1,2,4 triazole but
linked to phenyl ethanone oxime through acetamide bridge. Substitution pattern was same on
both groups of compounds for position three of triazole moiety and position five was containing
amino group in both groups of compounds. Compound 11a of oxime class showed highly
potent anticancer activity (IC50 3.5 µM) against pancreas cancer cell line. Significantly,
compound 11a exhibited highest activity against Panc-1cell line, which is 2 fold higher activity
than that of other compounds in same group of oximes. The other potent molecule in this series
is 10a with IC 3.7 µM for lung cancer cell line.
5
0
Interestingly, in this series two compounds 10b and 11b, with an unsubstituted phenyl moiety at
position of triazole core showed weak cytotoxicity against almost all the cell lines tested. On a
careful observation an interesting structure activity relationship can be observed between all
these tested compounds of these two groups. First of all on the basis of results included in table
4
1
a general comparison between compounds clearly shows that all compound oxime compound
1a bearing trifluoromethyl at position 3 of triazole was more active than compound 10a
bearing same substitution on same position but without oxime core ketone intermediates for
only pancreas cancer cell line while for other tested cancer cell lines results are more promising
for non oxime group of compounds. Some oxime triazole hybrids were found more active than
ketone triazole hybrids. It’s also proven recently that NO-donating oximes hybrids showed
pronounced gastroprotective activity better than their corresponding ketone intermediates that
may be attributed to the release of NO [27].
The substitution variations at position 3 of triazole can be correlated with the activity of
compounds. Presence of trifluoromethyl (10a & 11a) showed best activities as compared to
other substitution groups while presence of unsubstituted phenyl ring at same position (10b &
1
1b) exhibited lowest effects against the growth of cancer cell lines. Chloro substituted phenyl
group exhibited better activities as compared to unsubstituted phenyl ring and 3,4,5-tri methoxy

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substituted phenyl expressed more improved results as shown by compound 11d compared to
other phenyl substituted triazole oxime hybrids.
Previously reported similar class of compounds were found considerable anticancer agents [20]
as a series of compounds bearing 2-(4H-1,2,4-triazole-3-ylthio) acetamide structure have been
synthesized and were evaluated for their anti-HIV activity and against the full panel of 60
human cancer cell lines derived from nine different cancer types. Antiproliferative effects of the
selected compounds were demonstrated in human tumor cell lines K-562, A549 and PC-3.
These compounds inhibited cell growth assessed by MTT assay. Selected compounds exhibited
anticancer activity and showed a noteworthy boost in caspase-3 activity in a dose-dependent
manner. Our synthesized compounds were found more effective as compared to these reported
compounds [20].
Table 4: Antiproliferative activities of synthetic compounds 10a-d and 11a-d
Antiproliferative activity IC ± SEM (µM)
5
0
Code
0a
0b
0c
1a
1b
1c
1d
Panc-1
4.5±1.4
9.5±3.1
7.4±2.4
3.5±1.1
8.7±2.7
6.6±1.5
5.2±1.4
PaCa-2
4.7±1.5
8.5±1.3
8.1±2.4
4.9±1.2
5.7±1.7
8.4±1.8
5.7±2.9
HT-29
3.8±2.0
8.4±1.7
6.9±2.1
5.4±0.8
4.7±2.5
5.8±1.4
7.9±0.5
H-460
3.7±2.1
9.8±1.0
6.8±7.4
5.2±2.3
4.4±1.9
7.1±2.4
7.9±1.2
1
1
1
1
1
1
1
Panc-1(pancreas cancer cell line): PaCa-2 (pancreatic carcinoma cell line): HT-29 (colon cancer
cell line): H-460 (lung cancer cell line).
Other six compounds (14a-f) synthesized in this study were also evaluated for same cancer cell
lines by same method mentioned above and results are shown in Table 5. These all compounds
were bearing thiazolo-triazole moiety and on the both sides of main core, substituted phenyls

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made the difference for anticancer activities. This class of compounds was found more effective
as compared to other series of compounds having ketone and oxime groups. Three compounds
1
4 (a, c & e) were bearing unsubstituted phenyl ring on thiazole moiety while other three were
bearing 4-chloro substituted phenyl ring. A compound 14a bearing unsubstituted phenyl ring on
both sides of thiazolo-triazole core was found least effective against cancer cell lines.
Attachment of 4-chloro substituted benzene increased the considerable effect of compound 14b.
Other four derivatives 14c-f were bearing substituted phenyl at triazole moiety and strongly
increased the antiproliferative effects of these new compounds. It was observed that when only
methoxy substituted phenyl ring was attached to compounds (14c), the activity was increased
almost double as compared to compounds without any substituted phenyls 14a. Methoxy
substitution at position 4 of phenyl attached to triazole was even more potent than 4-chloro
substituted phenyl at thiazole core. Trimethoxy availability at phenyl linked to triazole also
showed noteworthy effects but most potent and best antiproliferative effects were exhibited by
compound 14d bearing combination of substituted phenyls as 4-chloro phenyl on thiazole and
4
-methoxy phenyl on triazole ring. Compound 14d lowest IC (1.60 µM) for H-460 cell line.
50
The mechanistic effects of most active antiproliferative compounds from thiazolo-triazole series
V600E
were investigated on tubulin polymerization, EGFR TK kinases and BRAF
.
Therefore, compounds 14d and 14f were subjected to further investigations to explore the
possible mechanism of action on A549 cell line.

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Table 5: Antiproliferative activities of synthetic compounds 14a-f
Antiproliferative activity IC ± SEM (µM)
5
0
Code
4a
4b
4c
4d
4e
4f
Panc-1
7.2±1.9
5.2±2.9
4.5±1.5
2.8±1.2
4.9±1.5
3.5±1.7
PaCa-2
9.2±2.2
6.5±0.2
4.9±1.7
2.6±0.5
4.7±1.2
3.9±1.2
HT-29
H-460
8.3±2.7
5.1±1.7
6.9±2.2
1.6±0.5
4.8±1.4
3.0±1.8
1
8.4±1.8
5.9±1.8
6.6±2.7
2.4±0.9
4.9±0.7
3.5±1.8
1
1
1
1
1
Panc-1(pancreas cancer cell line): PaCa-2 (pancreatic carcinoma cell line): HT-29 (colon cancer
cell line): H-460 (lung cancer cell line).
2
.2.4 Cell cycle analysis and apoptosis assay.
Compounds 14d and 14f were subjected to cell cycle analysis and apoptotic assay at
confirmatory diagnostic unit, VACSERA-EGYPT.
2
.2.4.1 Cell cycle analysis and apoptosis detection.
Once a cell goes through the cell cycle, it replicates two new indistinguishable cells. Each of the
two cells made from the first cell can go through this cell cycle again when new cells are
needed. The cell cycle includes four phases: G1 phase, S phase (synthesis), G2 phase and M
phase. During G1, cell enlargement and preparation of DNA duplication occurs. The S phase is
the stage of DNA replication and chromatid duplication. During G2, repairing of new DNA and
more growth occurs. In the M stage nuclear division takes place. Studies on the effect of
compounds 14d and 14f on cell cycle development and induction of apoptosis in the A549 was
done. A549 was incubated with IC concentration of compound 14d and 14f for 24 h. The cell
5
0
line was stained with PI/Annexin V and analyzed by flow cytometry using BD FASCC alibur.
Investigation of the results (Fig. 4) exposed that percentage of pre G1 apoptosis induced by

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compound 14d on A549 after 24 h incubation was 10.19%. A high percent of cell accumulation
was observed in G2/M phase in A549 treated with compound 14d after 24 h incubation
indicating arrest of cell cycle at G2/M phase. Compound 14f on A549 induced pre G1 apoptosis
by 14.87% after 24 h incubation. Also, A549 treated with compound 14f showed cell
accumulation at G1 phase and G2/M phase after 24 h incubation but more at G2/M. This
indicated that it arrested cell cycle also at G2/M phase.
Figure 4: Cell Cycle analysis results for compounds 14d and 14f
2
.2.4.2 Apoptosis assay.
Cell cycle analysis of A549 after treatment with compound 14d and 14f, respectively showed
presence of pre-G1 peak which is an indication of apoptosis. To confirm the ability of both
compounds to induce apoptosis, cells were stained with Annexin V/PI, incubated for 24 h and
analyzed. Analysis of early and late apoptosis showed that, compounds 14d and 14f were
certainly able to induce significant levels of apoptosis with necrosis percent 1.34 and 1.84,
respectively (Fig. 5). On the other hand, compounds 14d and 14f were able to induce late
apoptosis with a percent more than early apoptosis which making recovery of apoptic cell to be
healthy is more difficult.

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Figure 5: Apoptosis induction analysis using Annexin V/PI for compounds 14d and 14f.
Figure 6: Cell cycle analysis and Apoptosis induction analysis of compound 14d and 14f on
A549.

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2
.2.5 Tubulin Inhibitory Activity
In vitro tubulin polymerization inhibitory activity of most potent compound 14d selected was
evaluated, and it inhibited the assembly of tubulin. On comparison to model drugs docetaxel
and vincristine, compound 14d was found inhibitor as it decreased the arbitrary units as
compared to control but not more than vincristine. Previously new cis-restricted Combretastatin
A4 analogues containing 1,2,4-triazole in place of the olefinic bond were designed and
synthesized. The synthesized compounds were evaluated for their in vitro antiproliferative
activity and most potent compounds were selected for in vitro tubulin polymerization inhibitory
activity. The assay exposed that some 1,2,4-triazole derivatives showed a remarkable tubulin
inhibition [28].
2
.2.6 EGFR Inhibitory Activity
To evaluate the EGFR inhibitory potential of compounds, EGFR-TK assay was performed. The
findings of the cancer cell-based assays have been strongly complemented by the results from
this assay. All compounds showed potent EGFR inhibition with IC in the range of 1.5±0.6 to
5
0
9
.8±2.4 µM. The compound 14d displayed most potent activity (IC = 1.5±0.6 µM) among all
50
compounds. After 14d, compound 14f was also found to be more effective. Similar structure
activity relationship was observed for thiazolo-triazole compounds as observed for their
antiproliferative effects. This study demonstrates the compounds to be strong inhibitors of
EGFR and can probably be used as anticancer agents.
V600E
2
.2.7 BRAF
inhibitory activity
The RAS-RAF-MEK-ERK pathway is hyper-activated in roughly 30% of human cancers,
where it stimulates cell growth and survival. This hyper activation is motive in fraction by
mutations in receptor tyrosine kinases, the small G-proteins of the RAS family, and the serine
or threonine specific protein kinase BRAF [29]. A broad cancer genome mutation mapping has
exposed BRAF mutation in 7% of human cancers, and this verdict indicates BRAF as a prime

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oncogene, mainly in melanomas. These combined reasons render BRAF kinase as a potential
target for the development of anticancer drugs. In this study, most potent antiproliferative
compound 14d bearing combination of substituted phenyls as 4-chloro phenyl on thiazole and
V600E
4
-methoxy phenyl on triazole ring was subjected to in vitro BRAF
inhibitory assay and it
exhibited IC 4.7 µM.
5
0
V600E
Table 6: Effects of compounds 14d on Tubulin Polymerization, EGFR and BRAF
.
Tubulin inhibition BRAF inhibition
EGFR inhibition
Comp.
(arbitrary units)
IC ± SEM (µM)
IC ± SEM (µM)
5
0
50
1
1
1
1
1
1
4a
4b
4c
4d
4e
4f
-
-
9.8±2.4
7.2±2.9
5.6±1.9
1.5±0.6
2.8±1.0
1.7±0.5
0.07±0.03
-
-
-
-
-
1506±298
4.7±1.9
-
-
-
-
Erlotinib
DPBS
-
0.04±0.03
2880±237
722±249
4735±217
-
-
-
Vincristine
Docetaxel
-
-
2
.3 Molecular docking
The present docking simulation study was carried out in order to correlate the observed potency
and SAR of our newly synthesized thiazolotraizole fused system derivatives with their
behavior, binding modes and the possible interactions within the active site of EGFR kinase.
This study was performed using LIGDOCK, the hotspots-based docking protocol, embedded in
the Discovery Studio software 2.5 (San Diego, USA). The thiazolotraizole series 14a-f were

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docked into the ATP-active sites of EGFR using the 3D protein structure (PDB ID: 1M17)
retrieved from protein data bank for EGFR [30, 31]. The superimposition of the top docking
poses was depicted in Fig. 7 where these poses demonstrated good shape complementarities
with the ATP-binding sites of EGFR kinase with various binding patterns and interactions.
Figure 7: (A) Superimposition of the top docked poses 14a-f into the EGFR kinase binding site
(
PDB code: 1M17) where the protein is represented as a secondary structure; (B)
Superimposition of the top docked poses 14a-f into the EGFR kinase binding site (PDB code:
M17) where the protein is represented as a solid surface colored according to atom charges.
1
The binding sites are depicted as transparent green solid surface.
From the analysis of the docking results, it was clear that compounds 14d and 14f with the
highest EGFR inhibitory activities of 1.5 and 1.7 µM, respectively adopted the best and same
binding mode among the tested compounds confirming their great potency with the highest
docking scores (LibDockScore) of 90.41 and 98.69, respectively, (Table 7). The
thiazolotraizole core in both compounds was leaned in the middle of the binding cavity forming
one H-bond interaction with Met769 amino acid residue. Meanwhile, the monomethoxy or

ACCEPTED MANUSCRIPT
trimethoxyphenyl moieties was directed near the DFG motif (Asp831, Phe832 and Gly833)
forming one more H-bond with Thr830 residue in case of 14d while, the trimethoxyphenyl of
1
4f was engaged in a network of H-bonds with Thr830 and Lys721 amino acids, Fig.8 (A and
B). Phenyl ring in both compounds also formed some hydrophobic interactions near the DFG
motif with Ala719, Thr766 and Leu764 amino acids. Moreover, compound 14d formed π-cation
interaction with Lys721 residue explaining the higher activity of 14d than 14f. However, the p-
chlorophenyl attached to the thiazole ring was extended towards the hinge region making some
hydrophobic interactions with the surrounding amino acids including Leu694, Leu768, Phe771,
Cys773 and Leu820 residues. Additionally, the p-chlorophenyl moiety formed a favorable π-
stacking interaction with phenyl ring of Phe771 amino acids.
Figure 8: (A) Docking and binding pattern of compound 14d (orange) into ATP-active pocket
of EGFR kinase (PDB code: 1M17); (B) Docking and binding pattern of compound 14f
(
yellow) into ATP-active pocket of EGFR kinase; The poses were rendered as green line
models. H-bond interactions were represented as dashed blue lines. All hydrogens were
removed for the purposes of clarity.
Despite compound 14e showed similar disposition to that of compounds 14d and 14f, it has an
intermediate activity comparing with 14d and 14f with IC50 = 2.8 µM. This decrease in the
potency could be attributed to the absence of the chloride atom and the important H-bond with
Met769 residue, Fig. 9(A and B).

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Figure 9: (A) Docking and binding pattern of compound 14e (green) into ATP-active pocket of
EGFR kinase (PDB code: 1M17); (B) Superimposition of 14d-f docked poses within the ATP-
active site of EGFR protein; The poses were rendered as green line models. H-bond interactions
were represented as dashed blue lines. All hydrogens were removed for the purposes of clarity.
On the contrary, the rest of the tested compounds against EGFR kinase 14a-c with the weak
activity ranging 5.6-9.8 µM showed an inverse orientation to that of compounds 14d-f where
the thiazole ring was directed inside towards DFG motif. The opposite orientation and the loss
of the significant H-bonds and other interactions in case of compound 14a due to the absence of
the methoxy and chloride substituents might be the reason behind the least inhibitory activity
(
IC50 = 9.8 µM) exhibited by this compound against EGFR, Fig.10(A). Interestingly, the
potency was slightly enhanced in compound 14b (IC = 7.2 µM) by engaging the nitrogen of
5
0
the triazole ring in the important H-bond with Met769 residue, Fig.10(B). It was observed that
compound 14c incorporating a methoxy group adopted similar binding pattern to that of active
compounds 14d-f and forming H-bond between the thiazole ring and Met-769 amino acids
owing to much improvement in the activity (IC50 = 5.6 µM) comparing with compound 14a,
Fig.10(C).

ACCEPTED MANUSCRIPT
Figure 10: (A) Docking and binding pattern of compound 14a (magenta red) into ATP-active
site of EGFR kinase (PDB code: 1M17); (B) Docking and binding pattern of compound 14b
(
cyan) into ATP-active site of EGFR kinase; (C) Docking and binding pattern of compound 14c
blue) into ATP-active site of EGFR kinase; (D) Superimposition of 14d-f docked poses within
(
the ATP-active site of EGFR protein. The poses were rendered as green line models. H-bond
interactions were represented as dashed blue lines. All hydrogens were removed for the
purposes of clarity.
Interestingly, the docking score in table 7 of compounds 14a-f was in consistency with their
EGFR inhibitory activities. Overall, the docking study, along with EGFR in vitro results,
confirmed that the presence of methoxy and chloride substitutions greatly contribute to the
enhancement of the inhibitory activities of these thiazolotraizole-based compounds through
contributing in formation of the significant H-bonds and other interactions inside the ATP-
binding cavity.

ACCEPTED MANUSCRIPT
Table 7: The docking score and interactions of the newly synthesized compounds with the
amino acid residues in the ATP-active pocket of EGFR kinase.
Number
of H-
Amino
Acid
LibDockS
core
Comp.
Other interactions
Bonds
involved
Hydrophobic, π-
stacking with Phe771
Hydrophobic, π-
stacking with Phe771
Hydrophobic, π-
stacking with Phe771
Hydrophobic, π-
stacking with Phe771
and π-cation with
Lys721
1
4a
83.32
84.93
87.52
0
1
1
None
1
4b
Met769
Met769
1
4c
Met769,
Thr830
1
4d
90.41
2
Lys721,
Met769,
Thr830
Lys721,
Met769,
Thr830
Hydrophobic, π-
1
4e
90.08
98.69
3
3
stacking with Phe771
Hydrophobic, π-
1
4f
stacking with Phe771

ACCEPTED MANUSCRIPT
3
. Conclusion
In conclusion, a new class of novel compounds carrying 1,2,4-triazole scaffold were
synthesized and screened for anticancer activity. Compound 14d displayed highest activity
among all new compounds against cancer cell growth and also exhibited a significant inhibitory
activity against EGFR. Compound 14d was selected for five dose test by NCI showing
selectivity ratios ranging between 0.30 and 6.99 at the GI level. All the thiazolo[3,2-b][1,2,4]-
5
0
triazoles 14a-e backbone derivatives were found better inhibitors of cancer cell growth as
compared to triazole/oxime hybrids 11a-d derivatives. Compounds 14d and 14f showed high
cell accumulation at G2/M phase, confirming their ability to cause cell cycle arrest at G2/M
phase. Compounds 14d and 14f were certainly able to induce significant levels of apoptosis
with necrosis percent 1.34 and 1.84, respectively.
4
4
. Experimental
.1. Chemistry
Reactions were monitored by TLC analysis using Merck 9385 pre-coated aluminum plate silica
gel (Kieselgel 60) with F254 indicator thin layer plates. Melting points were determined on
Stuart electrothermal melting point apparatus and were uncorrected. IR spectra were recorded as
1
KBr disks on a Shimadzu S8400 IR spectrophotometer. H NMR spectra were carried out on
4
00 MHz Brucker spectrometer, using TMS as an internal reference. Chemical shift (δ) values
are given in parts per million (ppm) relative to CDCl (7.29) or DMSO-d (2.5) and coupling
3
6
constants (J) in Hertz. Splitting patterns are designated as follows: s, singlet; d, doublet; t,
triplet; q, quartet; dd, doublet of doublet; m, multiplet. High resolution mass spectrometric data
were obtained using MicroTOF-Q mass spectrometer (Bruker), Malaysia. Compounds 3, 7a-c,
9
and 12a-c were prepared according to reported procedures [14, 23].

ACCEPTED MANUSCRIPT
4
.1.1 N-(4-acetylphenyl)-2-(5-amino-3-alkyl or aroyl-1H-1,2,4-triazol-1-yl) acetamide.
(10a-d)
General procedure
An equimolar mixture of compound 3, 7a–c and N-(4-acetylphenyl)-2-bromoacetamide 9 (0.25
g, 1 mmol) in acetonitrile (50 mL) and TEA (0.10 g, 1.2 mmol) was heated at reflux for 4–8 h.
The reaction mixture was evaporated to dryness. The residue was crystallized from aqueous
ethanol affording products 10a-d.
4
.1.1.1 N-(4-Acetylphenyl)-2-(5-amino-3-(trifluoromethyl)-1H-1,2,4-triazol-1-yl)acetamide.
(10a)
°
-1
White powder (0.25 g, 75 % yield), m.p. 240-242 C, IR (KBr) ʋmax (cm ) 3180- 3470 (NH,
1
NH ), 1715 (COCH ), 1650 (CONH); HNMR (400 MHz, DMSO-d ) δ (ppm): 2.54 (s, 3H,
2
3
6
CH ), 4.89 (s, 2H, CH ), 6.84 (s, 2H, NH ), 7.67 (d, 2H, J = 8.8 Hz, Ar-H), 7.93 (d, 2H, J = 8.8
3
2
2
1
3
Hz, Ar-H), 10.67 (s, 1H, NH); C NMR (100 MHz, DMSO-d ) δ = 26.91, 50.21, 118.91,
6
1
21.35, 123.20, 125.46, 130.08, 132.50, 143.24, 149.40, 149.78, 158.34, 165.43, 197.06;
-
HRMS (ESI) m/z: [M-H] calc = 326.0943; found 326.0876 for C H F N O .
1
3
12
3
5
2
4
.1.1.2 N-(4-Acetylphenyl)-2-(5-amino-3-phenyl-1H-1,2,4-triazol-1-yl)acetamide. (10b)
°
-1
Whitish brown powder (0.22 g, 67 % yield), m.p. 199-201 C, IR (KBr) ʋmax (cm ) 3200- 3450
1
(
NH, NH ), 1720 (COCH ), 1645 (CONH); HNMR (400 MHz, DMSO-d ) δ (ppm): 2.53 (s,
2
3
6
3
H, CH ), 4.89 (s, 2H, CH ), 6.44 (s, 2H, NH ), 7.33-7.42 (m, 3H, Ar-H), 7.74 (d, 2H, J = 8.8
3 2 2
Hz, Ar-H), 7.88 (d, 2H, J = 6.8 Hz, Ar-H), 7.98 (d, 2H, J = 8.8 Hz, Ar-H), 10.66 (s, 1H, NH);
1
3
C NMR (100 MHz, DMSO-d ) δ = 26.91, 50..01, 118.88, 119.27, 125.78, 128.88, 129.81,
6
-
1
30.07, 132.43, 143.44, 157.74, 157.94, 166.27, 196.99; HRMS (ESI) m/z: [M-H] calc =
3
34.1304; found 334.1307 for C H N O .
1
8
17
5
2