Novel benzotriazole N-acylarylhydrazone hybrids: Design, synthesis, anticancer activity, effects on cell cycle profile, caspase-3 mediated apoptosis and FAK inhibition

Article abstract of DOI:10.1016/j.bioorg.2018.07.008

A series of novel benzotriazole N-acylarylhydrazone hybrids was synthesized according fragment-based design strategy. All the synthesized compounds were evaluated for their anticancer activity against 60 human tumor cell lines by NCI (USA). Five compounds: 3d, 3e, 3f, 3o and 3q exhibited significant to potent anticancer activity at low concentrations. Compound 3q showed the most prominent broad-spectrum anticancer activity against 34 tumor cell lines, with mean growth inhibition percent of 45.80%. It exerted the highest potency against colon HT-29 cell line, with cell growth inhibition 86.86%. All leukemia cell lines were highly sensitive to compound 3q. Additionally, compound 3q demonstrated lethal activity to MDA-MB-435 belonging melanoma. Compound 3e exhibited the highest anticancer activity against leukemic CCRF-CEM and HL-60(TB) cell lines, with cell growth inhibition 86.69% and 86.42%, respectively. Moreover, it exerted marked potency against ovarian OVCAR-3 cancer cell line, with cell growth inhibition 78.24%. Four compounds: 3d, 3e, 3f and 3q were further studied through determination of IC₅₀ values against the most sensitive cancer cell lines. The four compounds exhibited highly potent anticancer activity against ovarian cancer OVCAR-3 and leukemia HL-60 (TB) cell lines, with IC₅₀ values in nano-molar range between 25 and 130 nM. They showed 18–2.3 folds more potent anticancer activity than doxorubicin. The most prominent compound was 3e, (IC₅₀ values 29 and 25 nM against OVCAR-3 and HL-60 (TB) cell lines, respectively), representing 10 and 18 folds more potency than doxorubicin. The anti-proliferative activity of these four compounds appeared to correlate well with their ability to inhibit FAK at nano-molar range between 44.6 and 80.75 nM. Compound 3e was a potent, inhibitor of FAK and Pyk2 activity with IC₅₀ values of 44.6 and 70.19 nM, respectively. It was 1.6 fold less potent for Pyk2 than FAK. Additionally, it displayed inhibition in cell based assay measuring phosphorylated-FAK (IC₅₀ = 32.72 nM). Inhibition of FAK enzyme led to a significant increase in the level of active caspase-3, compared to control (11.35 folds), accumulation of cells in pre-G1 phase and annexin-V and propidium iodide staining in addition to cell cycle arrest at G2/M phase indicating that cell death proceeded through an apoptotic mechanism.

Full text of DOI:10.1016/j.bioorg.2018.07.008

Accepted Manuscript
Novel benzotriazole N-acylarylhydrazone hybrids: Design, synthesis, anticanc-
er activity, effects on cell cycle profile, caspase-3 mediated apoptosis and FAK
inhibition
Asmaa E. Kassab, Rasha A. Hassan
PII:
S0045-2068(18)30420-6
DOI:
https://doi.org/10.1016/j.bioorg.2018.07.008
YBIOO 2427
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Bioorganic Chemistry
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29 April 2018
7 July 2018
9 July 2018
Please cite this article as: A.E. Kassab, R.A. Hassan, Novel benzotriazole N-acylarylhydrazone hybrids: Design,
synthesis, anticancer activity, effects on cell cycle profile, caspase-3 mediated apoptosis and FAK inhibition,
Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.07.008
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Novel benzotriazole N-acylarylhydrazone hybrids: Design,
synthesis, anticancer activity, effects on cell cycle profile,
caspase-3 mediated apoptosis and FAK inhibition
a
a
Asmaa E. Kassab , Rasha A. Hassan
a
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy,
Cairo University, Cairo 11562, Egypt.
Address: 33 Kasr El-Aini Street, Cairo, Egypt.
1

Novel benzotriazole N-acylarylhydrazone hybrids: Design,
synthesis, anticancer activity, effects on cell cycle profile,
caspase-3 mediated apoptosis and FAK inhibition
Correspondence to: Asmaa E. Kassab, Pharmaceutical Organic
Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo
11562, Egypt.
Tel: 002023639307.
Fax: 002023635140.
E-mail: asmaa.kassab@pharma.cu.edu.eg
Abstract
A series of novel benzotriazole N-acylarylhydrazone hybrids was
synthesized according fragment-based design strategy. All the
synthesized compounds were evaluated for their anticancer activity
against 60 human tumor cell lines by NCI (USA). Five compounds: 3d,
3e, 3f, 3o and 3q exhibited significant to potent anticancer activity at low
concentrations. Compound 3q showed the most prominent broad-
spectrum anticancer activity against 34 tumor cell lines, with mean
growth inhibition percent of 45.80%. It exerted the highest potency
against colon HT-29 cell line, with cell growth inhibition 86.86%. All
leukemia cell lines were highly sensitive to compound 3q. Additionally,
compound 3q demonstrated lethal activity to MDA-MB-435 belonging
melanoma. Compound 3e exhibited the highest anticancer activity against
leukemic CCRF-CEM and HL-60(TB) cell lines, with cell growth
2

inhibition 86.69% and 86.42%, respectively. Moreover, it exerted marked
potency against ovarian OVCAR-3 cancer cell line, with cell growth
inhibition 78.24%. Four compounds: 3d, 3e, 3f and 3q were further
studied through determination of IC values against the most sensitive
50
cancer cell lines. The four compounds exhibited highly potent anticancer
activity against ovarian cancer OVCAR-3 and leukemia HL-60 (TB) cell
lines, with IC values in nano-molar range between 25 and 130 nM.
50
They showed 18 to 2.3 folds more potent anticancer activity than
doxorubicin. The most prominent compound was 3e, (IC values 29 and
50
25 nM against OVCAR-3 and HL-60 (TB) cell lines, respectively),
representing 10 and 18 folds more potency than doxorubicin. The anti-
proliferative activity of these four compounds appeared to correlate well
with their ability to inhibit FAK at nano-molar range between 44.6 and
80.75 nM. Compound 3e was a potent, inhibitor of FAK and Pyk2
activity with IC values of 44.6 and 70.19 nM, respectively. It was 1.6
50
fold less potent for Pyk2 than FAK. Additionally, it displayed inhibition
in cell based assay measuring phosphorylated-FAK (IC =32.72 nM).
50
Inhibition of FAK enzyme led to a significant increase in the level of
active caspase-3, compared to control (11.35 folds), accumulation of cells
in pre-G1 phase and annexin-V and propidium iodide staining in addition
to cell cycle arrest at G2/M phase indicating that cell death proceeded
through an apoptotic mechanism.
Key words: Benzotriazole; N- acylarylhydrazone; Fragment-based
design; Synthesis; Anticancer activity; FAK; Caspase-3; Cell cycle arrest
profile; Apoptosis.
1. Introduction
One of the vital hallmarks of cancer which distinguishes it from benign
tumors is its capability for tissue invasion and distant metastasis [1].
3

Focal adhesion kinase (FAK) is a multifunctional scaffolding protein
tyrosine kinase which is located at specialized subcellular structures
known as focal adhesions which link the extracellular matrix to the
cytoskeleton. FAK has been shown to be an important mediator of cell
adhesion, growth, proliferation, survival, angiogenesis and migration, all
of which are often disrupted in cancer cells [2-4]. Furthermore, FAK has
anti-apoptotic role via suppression of the activation of caspase-3 that
plays pivotal roles in apoptosis [5]. Normal tissues have low expression
of FAK, while FAK has been found to be overexpressed in most human
cancers, particularly in highly invasive cancer cells [6] such as ovarian
cancer [7]. FAK is encoded by the protein tyrosine kinase 2 (PTK2) gene,
which is located at human chromosome region 8q24.3. the region was
found to be commonly amplified in several types of cancer, such as
serous ovarian carcinoma [8]. Therefore, targeting FAK is a rational and
novel approach to retard cancer growth and block metastasis [9]. Several
FAK inhibitors have been developed and reported recently, such as NVP-
TAE-226, PF-562,271, PF-573,228 and FAK Inhibitor 14 (also known as
Y15) [10, 11]. These inhibitors have shown activity, either alone or in
combination with chemotherapy, both in vitro and in vivo, against
different types of cancer. Additionally, they demonstrated cell cycle
arrest in the G2/M phase and subsequently, cellular apoptosis [12].
Strategies targeting FAK inhibition using novel compounds have created
an exciting opportunity for anticancer therapy. Benzotriazole is a
privileged structure for the design and discovery of novel bioactive
molecules and drugs, since it is a structural isostere of purine nucleus,
which is found in naturally occurring nucleotides, such as ATP and other
naturally existent compounds. Moreover, it has three nitrogen atoms that
can act as H-bond acceptors. There-by, benzotriazole derivatives are
more ready to bind with catalytic binding sites of a variety of enzymes
4

and receptors in biological systems, since these ATP binding sites contain
specific hydrogen-bond forming groups. In addition, benzotriazole is a
common structural motif of marketed anticancer drugs, such as vorozole
(Rivizor®) [13] and 4,5,6,7-tetrabromobenzotriazole (TBB) [14] (Figure
1).Compounds I [15] and II [16] (Figure 1) are potent FAK inhibitors.
The main structural features of these compounds are benzotriazole ring,
substituted at N1 with oxadiazole moiety carrying substituted phenyl ring.
N-acylarylhydrazone scaffold,was used as a core to synthesize several
anticancer agents as PAC-1 (Figure 1), as it can readily bind with various
enzymes and receptors through hydrogen bonds, as well as due to its
flexible skeleton [17-19]. The anticancer activity of PAC-1 is dependent
on the presence of the o-hydroxy N-acylhydrazone motif that played the
key role in the chelation of zinc, which is a powerful inhibitor of
procaspase-3 enzymatic activity. Such mechanism allows procaspase-3 to
process itself to the active form [20-22]. There is an increasing interest in
the discovery of lead agents that concomitantly address more than one
biological target for cancer treatment [23, 24]. One of the most exciting
new methods for lead generation is fragment-based discovery. Fragments
are small, low molecular weight molecules that usually form part of drugs
(also known as scaffolds or templates). These fragments are then
combined or optimized to generate lead compounds [25]. The rational
beyond this approach is to identify novel leads with improved affinity and
selectivity, [26] and to minimize the drug resistance, as each fragment
will have different biological target [27]. Inspired by all these findings,
we have constructed a ‘hybrid-design’ model (Figure 1) to satisfy the
pharmacophoric features required to induce FAK inhibition and
apoptosis. We have used benzotriazole core to synthesize a series of
hybrids substituted at N1 position with biologically active N-
acylarylhydrazone scaffold (an apoptosis inducer), moreover, this
5

scaffold mimics the aryl substituted oxadiazole ring in compounds I and
II. In our design strategy, we aimed to incorporate various aryl or
heteroaryl groups in the N-acylarylhydrazone core, targeting exploring
their impact on anticancer activity and identifying potent anti-
proliferative agents. We have introduced mono, di or tri substituted
phenyl rings with a diverse array of substitutions, including fluoro,
hydroxyl or methoxy groups. We have also incorporated monocyclic
heteroaryl moieties [pyrrole, furan or pyridine] or benzo fused heteroaryl
rings such as benzofuran, benzothiophene or indole. The anticancer
activity of all the newly synthesized compounds was evaluated against a
panel of 60 human tumor cell lines provided by National Cancer Institute
(
USA). Four potent compounds were selected to be further studied
through determination of their half maximal inhibitory concentration
IC ) values against ovarian cancer OVCAR-3 and leukemia HL-60 (TB)
(
50
cell lines. We anticipated that the designed hybrids might be active
against different biological targets. In order to explore the mechanistic
pathways of the anticancer activity of the synthesized compounds, we
chose the most potent compound 3e, to perform further investigations,
such as FAK assay, apoptosis marker (caspase-3) and cell cycle analysis.
Molecular docking was performed for compound 3e to investigate the
inhibitor interaction with FAK and explore the binding mode of this
compound at the active site.
2
2
. Results and discussion
.1. Chemistry
Seventeen novel benzotriazole N-acylarylhydrazone derivatives 3a-3q were
synthesized according to Scheme 1. Commercial benzotriazole was
alkylated via heating under reflux with an equimolar amount of ethyl
chloroacetate in presence of anhydrous K CO in dry acetone according to a
2
3
6

reported procedure [28] to give ethyl 2-(1H-benzo[d][1,2,3]triazol-1-
yl)acetate (1). The C=O stretching of the ester 1 was observed as a strong
-1
band at 1747 cm in IR spectrum. Heating ester 1 under reflux with three
molar equivalents of hydrazine hydrate in ethanol was reported [29] to yield
benzotriazole acetohydrazide 2 in 57% yield. IR spectrum of compound 2
-1
showed a decrease in the amidic C=O stretching frequency into 1662 cm .
New benzotriazole N-acylarylhydrazone derivatives 3a-3q were prepared
through heating under reflux the acetohydrazide 2 with different aromatic
aldehydes in ethanol along with a small amount of glacial acetic acid for 6 h.
The IR spectra of 3a-3q showed the amidic C=O stretching as a
-1
characteristic strong band in a range (1700-1670 cm ) and the C=N
stretching of the azomethine group (N=CH) as a weak band in the range
-1
1
(
1620-1600 cm ). H NMR spectra of these compounds were recorded in
DMSO-d6 and displayed the characteristic signals corresponding to different
1
aryl groups. For all the derivatives 3a-3q, the H NMR spectra showed that
the N-acylarylhydrazone is a mixture of two tautomers: tautomer A (keto
amide) and tautomer B (enol amide) (Figure 2). In all the spectra N-CH2
appeared as two singlet signals at the range of (δ 5.97-6.15 ppm) and the
range of (δ 5.57-5.66 ppm) corresponding to tautomer A and tautomer B,
respectively. The integration of these signals was different according to the
1
aromatic substitution and examining the integration pattern of the H NMR
spectra throughout the seventeen compounds 3a-3q proved that tautomer A
was the major in the mixture in all the derivatives. For example, derivative
3b has two singlets at δ 6.09 ppm and δ 5.63 ppm corresponding to tautomer
A and tautomer B, sequentially with integrations as 2.0 H and 0.50 H.
Moreover, the azomethine proton of the hydrazones 3a-3q appeared in all
1
the H NMR spectra as two singlet signals the major at the range (δ 7.92-
8
8
.41 ppm) corresponding to tautomer A and the minor at the range (δ 8.09-
1
.62 ppm) corresponding to tautomer B. In the H NMR spectrum of 3b the
7

two singlets were recorded at δ 8.10 ppm and δ 8.30 ppm corresponding to
the two tautomeric forms A and B, respectively with integrations as 1.0 H
and 0.25 H. Finally an extra D O exchangeable peak appeared in the range (δ
2
10.0-13.0 ppm) in most of the spectra and was identified as the C-OH in the
enol amide (tautomer B). For compound 3b, the D O exchangeable peak
2
1
appeared at δ 12.15 ppm and has integration of 0.25 H. Some of the H
NMR spectra of derivatives throughout 3a-3q showed also peaks
corresponding to aromatic protons and the substituents of the phenyl ring of
both tautomers A and B. For example compound 3h with 4-methoxyphenyl
substituent showed the OCH protons as two singlets, at δ 3.82 ppm (3H)
3
and δ 3.81 (0.9H) corresponding to tautomers A and B, sequentially. Also
two singlets at δ 6.04 ppm (2H) and δ 5.59 (0.6 H) corresponding to N-CH2
protons of A and B, respectively. The azomethine proton appeared as two
singlets at δ 8.04 ppm (1H) and δ 8.23 ppm (0.3H) corresponding to
13
tautomers A and B, sequentially. C NMR also supported the presence of
tautomeric mixture A and B. The resonance of the amidic carbonyl in
tautomer A was at the range (δ 166.8-168.3 ppm) while the enol amide group
C(OH)=N corresponding to tautomer B appeared as a small peak at the range
(
δ 161.8-163.3 ppm). Moreover, the N-CH of the two tautomers appeared at
2
the range of (δ 49.1-49.3 ppm) as major peak corresponding to tautomer A
and at the range (δ 49.5-49.7 ppm) as small peak corresponding to tautomer
B.
2. 2. Growth inhibition against a panel of 60 human tumor cell lines
In this study, all the newly synthesized compounds were selected by
National Cancer Institute (USA) for anticancer evaluation under the
Developmental Therapeutic Program (DTP) with respective NCI codes
NCS 801617, NCS 801618, NCS 801619, NCS 801620, NCS 801621,
NCS 801622, NCS 801623, NCS 801639, NCS 801640, NCS 801641,
NCS 801642, NCS 801643, NCS 801644, NCS 801645, NCS 801646,
8

NCS 801647 and NCS 801648. The selected compounds were evaluated
-5
at a single dose (10 M) against 60 different human tumor cell lines,
representing leukemia, melanoma and cancers of lung, colon, central
nervous system (CNS), ovary, kidney, prostate as well as breast. The
growth inhibition percentages obtained from the single dose test are
shown in Table 1. In light of the NCI-60 results, the following
observations could be outlined. Compound 3d exhibited the most potent
anticancer activity against ovarian OVCAR-3 and colon HCT-15 cancer
cell lines with growth inhibition percentages 71.17 and 70.14,
respectively. Regarding compound 3e, it had mean growth inhibition
percent of 53.30%. An interesting phenomenon was that all leukemia cell
lines were sensitive to that compound. It exhibited the highest anticancer
activity against leukemic CCRF-CEM and HL-60(TB) cell lines with cell
growth inhibition 86.69% and 86.42%, sequentially. Moreover, it exerted
marked potency against colon HCT-15, melanoma LOX IMVI, ovarian
OVCAR-3 and renal UO-31 cancer cell lines with cell growth inhibition
percentages 74.21, 77.62, 78.24 and 83.14, respectively. Compound 3f
showed significant to potent cell growth inhibition against the leukemic
CCRF-CEM, MOLT-4 and SR cell lines with 66.08%, 62.47% and
6
4.37% inhibition respectively, the non-small cell lung cancer HOP-62
and NCI-H460 cell lines with 64.81 and 63.54%, the colon cancer COLO
05 and HCT-15 cell lines with 75.23% and 67.85% inhibition
2
respectively. Finally, it showed potent anti-proliferative activity against
the ovarian OVCAR-3 cancer cell line with growth inhibition 74%. K-
5
3
3
62 and SR cell lines belonging to leukemia were sensitive to compound
o with growth inhibition 64.72% and 62.07%, respectively. Compound
o revealed the most potent anticancer activity against MDA-MB-435
melanoma cell line with growth inhibition percentage 80.89. It exhibited
significant growth inhibition against non-small cell lung cancer NCI-
9

H522, colon cancer HCT-15, HT29 and breast cancer MCF7cell lines
with growth inhibition 68%, 62.26%, 68.76 and 69.27 %, respectively.
Compound 3q showed the most potent anticancer activity at low
concentrations against 34 tumor cell lines with mean growth inhibition
percent of 45.80%. It exhibited broad spectrum anti-proliferative activity
against almost all human cancer types. It is worth mentioning, that all
leukemia cell lines were sensitive to compound 3q. It exerted the highest
potency against K-562 cell line with cell growth inhibition 84.79%. It
showed selective cell growth inhibition against almost all colon cancer
cell lines, HT29 cell line was the most sensitive with 86.86% inhibition.
Compound 3q exhibited potent anti-proliferative activity against lung
cancer NCI-H522, melanoma M14, ovarian cancer OVCAR-3 and breast
cancer MCF7 cell lines with 81.88%, 80.26%, 77.60% and 77.99%
growth inhibition, respectively. Moreover, it showed lethal anticancer
activity against MDA-MB-435 melanoma cell line. Compounds (3a-c,
3
2
g-n and 3p) exhibited no activity against most investigated cell lines.
.3. Detection of IC against ovarian cancer OVCAR-3 and leukemia
50
HL-60 (TB) cell lines
Four compounds 3d, 3e, 3f and 3q were selected to be further studied
through determination of their half maximal inhibitory concentration
(
IC ) values against the most sensitive cancer cell lines compared to
50
doxorubicin as reference anticancer drug. The results of the mean values
of experiments performed in triplicate were summarized in Table 2 and
represented graphically in Figure 3. The in vitro results showed that the
four test compounds exhibited highly potent anticancer activity against
test cell lines with IC values in nano molar range between 25 and 130
50
nM. They showed 18 to 2.3 folds more potent anticancer activity than
doxorubicin. The most prominent compound was 3e with IC values 29
50
10

and 25 nM against OVCAR-3 and HL-60 (TB) cell lines, respectively
representing 10 and 18 folds more potency than doxorubicin. Structural
activity relationship analysis revealed that the anti-proliferative activity of
the newly synthesized hybrids correlates well with the aryl or heteroaryl
ring of N-acylhydrazone core. The highly potent compounds were 3d,
3e, 3f and 3q having the acylhydrazone scaffold with phenyl ring bearing
hydroxyl group at ortho position (3d, 3e and 3f) or having benzene ring
with a nearby NH group (3q). Such a structural motif is well known with
its ability to participate in zinc chelation and consequently is essential for
procaspase-3 activation and induction of apoptosis. Further analysis of
these compounds clearly revealed that introduction of additional hydroxyl
group on the phenyl ring in compounds 3e and 3f improved the anticancer
activity. Compound 3e with the additional hydroxyl group at position 3 of
phenyl ring showed more potent broad spectrum anticancer activity than
compound 3f with the additional hydroxyl group at position 4. Another
interesting phenomenon is that compounds 3a-c and 3g-j with phenyl ring
substituted with fluoro, hydroxyl or methoxy groups and devoid the
presence of ortho hydroxyl group were inactive against most investigated
cell lines, confirming that ortho hydroxyl substitution of the phenyl ring
is essential for the anticancer activity. Replacement of the phenyl ring of
N-acylarylhydrazone core with monoheterocycle in compounds 3k-m
resulted in a dramatic loss in the anti-proliferative activity. Incorporation
of a benzo fused heterocycle containing NH group in compound 3q
resulted in broad spectrum and highly potent anticancer activity.
According to these findings, we concluded that combination of
benzotriazole scaffold with N-acylarylhydrazone core bearing phenyl ring
substituted with hydroxyl group at ortho position or having benzene ring
with a nearby NH group exerted a positive effect on the anticancer
activity.
11

2.4. Measurement of the effect of benzotriazole hybrids on FAK and
Pyk2 enzymes
2.4.1. In vitro FAK assay
Benzotriazole scaffold is a part of several potent FAK inhibitors.
Therefore, to confirm the FAK binding site as a target, we investigated
the inhibitory effect of compounds 3d, 3e, 3f and 3q on FAK in vitro.
The anti-proliferative activity of these four compounds appeared to
correlate well with their ability to inhibit FAK with IC values at nano-
50
molar range between 44.6 and 80.75 nM. Compound 3e showed the most
prominent FAK inhibitory activity with IC = 44.6 ±2.28 nM (Table 3
50
and Figures 4, 5).
.4.2. Cell-based FAK auto-phosphorylation assay
2
To test whether Compound 3e directly inhibits FAK auto-
phosphorylation, we performed in vitro kinase assay using FAK
(
Phospho-Tyr397) Cell-Based ELISA Kit. Compound 3e displayed
inhibition in cell based assay measuring phosphorylated-FAK (IC50
32.72±2.11 nM), which was comparable to GSK-2256098 with IC50
value of 20.32 ±1.17 nM.
=
2.4.3. In vitro Pyk2 assay
Since Pyk2 is a tyrosine kinase which is closely related to FAK, both are
implicated in cell migration, mitosis and tumor metastasis. We have
measured the inhibitory effect of compound 3e on Pyk2 in vitro.
12

Compound 3e showed Pyk2 inhibitory activity, at the nanomolar level
(
IC value 70.19 ±0.39 nM).
50
2. 5. Molecular docking of compound 3e in the active site of FAK
Compound 3e showed highly potent activity in FAK enzyme inhibition
assay. At this stage, molecular docking study was carried out to
investigate its plausible binding pattern and its interaction with the key
amino acids in the active site of FAK. We used the 3D structure of FAK
that was downloaded from PDB (ID: 2ETM). The ability of compound 3e
to interact with the key amino acids in the ATP binding site of FAK
rationalized its excellent activity. The FAK protein catalytic region
formed three interaction bonds with 3e (Figures 6, 7). As can be seen,
compound 3e interacted by its two OH groups of phenyl ring as H-bond
acceptor with the key amino acid Lys 454. Also, N2 of benzotriazole
interacted as H-bond acceptor with Cys 502. Moreover, the superior
activity of compound 3e against FAK (IC50 value 44.6 nM) was
supported by its marked docking score (-13.76 kcal/mol).
2. 6. Effect of compound 3e on the level of active caspase-3 (key
executioner of apoptosis)
FAK enzyme has anti-apoptotic role via suppression of the activation of
caspase-3. As compound 3e showed highly potent FAK inhibitory
activity at the same time, it contains N-acylarylhydrazone moiety
carrying o-hydroxyl group, a structural motif known to participate in zinc
chelation, which is a powerful inhibitor of procaspase-3 enzymatic
activity. So the conversion of procaspase-3 to the active form was
suspected as mechanism of action of this compound. The level of active
caspase-3 was measured. Treatment of OVCAR-3 cells with compound
3e at concentration 29 nM produced a significant increase in the level of
13

active caspase-3 compared to control (11.35 folds) (Table 4 and Figure
8
).
2. 7. Cell cycle analysis and detection of apoptosis
The most active compounds 3e was selected to be further studied
regarding to its effect on cell cycle progression and induction of apoptosis
in the OVCAR-3 cell line. OVCAR-3 cells were exposed to compound 3e
at its IC values for 24h and its effect on the normal cell cycle profile and
50
induction of apoptosis was analyzed. Exposure of OVCAR-3 cells to
compound 3e resulted in an interference with the normal cell cycle
distribution of this cell line. Compound 3e induced significant increase in
the percentage of cells at pre-G1 by 14 folds, compared to control. Also,
it showed significant increase in the percentage of cells at G2/M phases
by 5 folds, compared to control (Table 5 and Figures 9, 10).
Accumulation of cells in pre-G1 phase which was confirmed by the
presence of sub-G1 peaks in the cell cycle profile analysis may result
from degradation or fragmentation of the genetic materials confirming a
role of apoptosis in compounds 3e induced cancer cell death and
cytotoxicity. While the accumulation of the cells in G2/M phase may
result from G2 arrest.
2. 8. Apoptosis determination by Annexin-V assay
To ensure the ability of compound 3e to induce apoptosis, a biparametric
cytofluorimetric analysis was performed using Propidium iodide (PI),
which stains DNA and enters only dead cells, and fluorescent
immunolabeling of the protein annexin-V, which binds to
phosphatidylserine (PS) expressed on the surface of the apoptotic cells
and fluoresces green after interacting with the labeled annexin-V [30] .
Dual staining for annexin-V and with PI permits discrimination between
14

live cells, early apoptotic cells, late apoptotic cells and necrotic cells. As
shown in Figures 11, 12 and Table 6, after 24 h of treatment of OVCAR-
3
cells with compound 3e at its IC concentration a decrease in the
50
percentage of the survived cells was observed. Moreover, a significant
increase in the percentage of annexin-V positive cells (almost 16 folds
more than control) occurred indicating an early apoptosis (lower right
quadrant). In addition, some treated cells were in a late apoptotic/
necrotic stage (upper right quadrant). This was indicated by the
significant increase in the percentage of annexin V positive, PI positive
cells (64 folds more than control).
3. Conclusion
In summary, a series of novel benzotriazole N-acylarylhydrazone hybrids
was synthesized according fragment-based design strategy. All the
synthesized compounds were evaluated for their anticancer activity by
NCI (USA) under DTP. Five compounds 3d, 3e, 3f, 3o and 3q exhibited
significant to potent anticancer activity at low concentrations.
Compounds 3e and 3q showed the most potent broad spectrum anti-
proliferative activity against several human cancer cell lines. Four
compounds 3d, 3e, 3f and 3q were further studied through determination
of IC values against the most sensitive cancer cell lines. The four
50
compounds exhibited highly potent anticancer activity against ovarian
cancer OVCAR-3 and leukemia HL-60 (TB) cell lines with IC values in
50
nano molar range between 25 and 130 nM. They showed 18 to 2.3 folds
more potent anticancer activity than doxorubicin. The most prominent
compound was 3e with IC values 29 and 25 nM against OVCAR-3 and
50
HL-60 (TB) cell lines, respectively representing 10 and 18 folds more
potency than doxorubicin. The anti-proliferative activity of these four
compounds appeared to correlate well with their ability to inhibit FAK at
15

nano-molar range between 44.6 and 80.75 nM. Compound 3e was a
potent, inhibitor of FAK and Pyk2 activity with IC values of 44.6 and
50
70.19 nM, respectively. It was 1.6 fold less potent for Pyk2 than FAK.
Additionally, it displayed inhibition in cell based assay measuring
phosphorylated-FAK (IC =32.72 nM). Inhibition of FAK enzyme leads
50
to a significant increase in the level of active caspase-3 compared to
control (11.35 folds), accumulation of cells in pre-G1 phase and annexin-
V and propidium iodide staining in addition to cell cycle arrest at G2/M
phase indicating that cell death proceeds through an apoptotic
mechanism. Benzotriazole N-acylarylhydrazone hybrids are promising
multi-targeted leads for the design and synthesis of potent anticancer
agents.
4
4
4
. Experimental
. 1. Chemistry
. 1. 1. General
Melting points were obtained on a Griffin apparatus and were
uncorrected. Microanalyses for C, H and N were carried out at the
Regional Center for Mycology and Biotechnology, Faculty of Pharmacy,
Al-Azhar University. IR spectra were recorded on Shimadzu IR 435
spectrophotometer (Shimadzu Corp., Kyoto, Japan) Faculty of Pharmacy,
-1 1
Cairo University, Cairo, Egypt and values were represented in cm . H
NMR spectra were carried out on Bruker 400 MHz (Bruker Corp.,
Billerica, MA, USA) spectrophotometer, Faculty of Pharmacy, Cairo
University, Cairo, Egypt. Tetramethylsilane (TMS) was used as an
internal standard and chemical shifts were recorded in ppm on δ scale and
1
3
coupling constants (J) were given in Hz. C NMR spectra were carried
out on Bruker 100 MHz spectrophotometer, Faculty of Pharmacy, Cairo
16

University, Cairo, Egypt. Progress of the reactions were monitored by
TLC using precoated aluminum sheet silica gel MERCK 60F 254 and
was visualized by UV lamp.
4. 1. 2. Ethyl 2-(1H-benzo[d][1,2,3]triazol-1-yl)acetate (1)[28]
A mixture of 1H-Benzotriazole (1.0 g, 0.0084 mol), ethyl chloroacetate
1.03 g, 0.0084 mol), anhydrous potassium carbonate (2.32 g, 0.0168
(
mol) and dry acetone (90 mL) was heated under reflux for 6 h. After
cooling the obtained precipitate was filtered, washed with ethanol, and
recrystallized from ethanol to afford the ethyl ester 1 as white crystals.
4. 1. 3. 2-(1H-Benzo[d][1,2,3]triazol-1-yl)acetohydrazide (2) [29]
A mixture of the ester 1 (2.05 g, 0.01 mol) and hydrazine hydrate (1.5 g,
.03 mol) in absolute ethanol (15 mL) was heated under reflux for 5 h.
0
After cooling the obtained precipitate was filtered, washed with water,
and recrystallized from ethanol to afford the corresponding hydrazide 2 as
white crystals.
4. 1. 4. General procedure for the preparation of compounds (3a-3q)
A mixture of 2-(1H-Benzo[d][1,2,3]triazol-1-yl)acetohydrazide (2) (0.2 g,
.001 mol) and the appropriate aldehyde (0.001 mol) was dissolved in
0
ethanol (5 mL) containing 0.5 mL of glacial acetic acid. The reaction
mixture was heated under reflux for 6 h. After cooling the separated solid
was filtered, washed with ethanol and crystallized from ethanol.
4. 1. 4. 1. 2-(1H-benzo[d][1,2,3]triazol-1-yl)-N'-(2-fluorobenzylidene)
o
acetohydrazide (3a) white solid: 61 % yield; mp 234-236 C; IR (KBr,
17

-1
1
cm ) 3410, 3387, 1685, 1606; H NMR (400 MHz, DMSO-d ) δ 5.63 (s,
6
0
7
.52 H, N-CH taut B), 6.08 (s, 2 H, N-CH ), 7.29-7.35 (m, 2H, ArH),
2
2
.40-7.44 (m, 1H, ArH), 7.49-7.60 (m, 2H, ArH), 7.84-7.88 (m, 1H,
ArH), 8.03-8.05 (m, 2H, ArH), 8.31 (s, 1H, N=CH), 8.51 (s, 0.26H,
13
N=CH, taut B), 12.03 (s, 1H, NH, D O exchangeable); C NMR (100
2
MHz, DMSO-d ) δ: 168.0, 162.9 (taut B), 161.2 (d, J= 248.3) 145.5,
6
1
1
4
37.8 (d, J= 4.6), 134.5, 132.5 (d, J= 8.6), 127.7, 127.0, 125.3 (d, J= 3.1),
24.3, 121.8 (d, J= 9.9), 119.4, 116.4 (d, J= 20.5), 111.5, 49.6 (taut B),
9.2. Anal. Calcd for C H FN O (297.29): C, 60.60; H, 4.07; N, 23.56,
15
12
5
found C, 60.87; H, 4.19; N, 23.80.
4. 1. 4. 2. 2-(1H-benzo[d][1,2,3]triazol-1-yl)-N'-(3-fluorobenzylidene)
o
acetohydrazide (3b) white solid: 92 % yield; mp 186-188 C; IR (KBr,
-1
1
cm ) 3448, 3421, 1678, 1606; H NMR (400 MHz, DMSO-d ) δ 5.63 (s,
6
0
7
.50 H, N-CH taut B), 6.09 (s, 2 H, N-CH ), 7.27-7.30 (m, 1H, ArH),
2
2
.40-7.46 (m, 1H, ArH), 7.50-7.61 (m, 3H, ArH), 7.66-7.69 (m, 1H,
ArH), 7.84-7.87 (m, 1H, ArH), 8.10 (s, 1H, N=CH), 8.30 (s, 0.25H,
N=CH, taut B), 11.98 (s, 1H, NH, D O exchangeable), 12.15 (s, 0.25H,
2
13
OH, D O exchangeable, taut B); C NMR (100 MHz, DMSO-d ) δ:
2
6
1
1
1
68.1, 162.9 (taut B), 162.8 (d, J= 242.2), 145.5, 143.6, 136.9 (d, J= 8.0),
34.4, 131.3 (d, J= 8.2), 127.3, 124.2, 124.2 (d, J= 2.4), 119.4, 118.3,
17.3 (d, J= 21.2), 113.3 (d, J= 22.6), 111.4, 49.6 (taut B), 49.3. Anal.
Calcd for C H FN O (297.29): C, 60.60; H, 4.07; N, 23.56, found C,
15
12
5
60.84; H, 4.21; N, 23.75.
4. 1. 4. 3. 2-(1H-benzo[d][1,2,3]triazol-1-yl)-N'-(4-fluorobenzylidene)
o
acetohydrazide (3c) white solid: 65 % yield; mp 254-255 C; IR (KBr,
-1
1
cm ) 3186, 3167, 1678, 1600; H NMR (400 MHz, DMSO-d ) δ 5.61 (s,
6
0
7
.55 H, N-CH taut B), 6.07 (s, 2 H, N-CH ), 7.30-7.33 (m, 2H, ArH),
2
2
.40-7.45 (m, 1H, ArH), 7.53-7.60 (m, 1H, ArH), 7.84-7.87 (m, 3H,
18

ArH), 8.06-8.08 (m, 1H, ArH), 8.09 (s, 1H, N=CH), 8.29 (s, 0.28H,
N=CH, taut B), 11.89 (s, 1H, NH, D O exchangeable), 12.03 (s, 0.26H,
2
OH, D O exchangeable, taut B). Anal. Calcd for C H FN O (297.29):
2
15 12
5
C, 60.60; H, 4.07; N, 23.56, found 60.79; H, 4.15; N, 23.78.
4. 1. 4. 4. 2-(1H-benzo[d][1,2,3]triazol-1-yl)-N'-(2-hydroxybenzylidene)
o
acetohydrazide (3d) white solid: 83 % yield; mp 218-220 C; IR (KBr,
-1
1
cm ) 3387-3100 br s, 3182, 1685, 1608; H NMR (400 MHz, DMSO-d )
6
δ 5.63 (s, 1.14H, N-CH taut B), 6.03 (s, 2 H, N-CH ), 6.84-6.89 (m,
2
2
1.11H, ArH, taut B), 6.90-6.94 (m, 2H, ArH), 7.24-7.24 (m, 0.51H, ArH,
taut B), 7.27-7.28 (m, 1H, ArH), 7.40-7.44 (m, 1H, ArH), 7.45-7.49 (m,
1
7
H, ArH), 7.53-7.55 (m, 0.55H, ArH, taut B), 7.57-7.60 (m, 1H, ArH),
.77-7.83 (m, 1H, ArH), 7.96 (d, 1H, J= 8.4, ArH), 8.06-8.09 (m, 1H,
ArH), 8.40 (s, 1H, N=CH), 8.51 (s, 0.54H, N=CH, taut B), 10.08 (s, 1H,
OH, D O exchangeable), 10.88 (s, 0.57H, OH, D O exchangeable, taut
2
2
B), 11.83 (s, 1H, NH, D O exchangeable). Anal. Calcd for C H N O
2
15 13
5
2
(295.30): C, 61.01; H, 4.44; N, 23.72, found C, 61.34; H, 4.60; N, 23.91.
4. 1. 4. 5. 2-(1H-benzo[d][1,2,3]triazol-1-yl)-N'-(2,3-dihydroxybenzyl-
o
idene)acetohydrazide (3e) whitish buff solid: 52 % yield; mp 256-258 C;
-1
1
IR (KBr, cm ) 3271, 3194, 3101, 1670, 1616; H NMR (400 MHz,
DMSO-d ) δ 5.64 (s, 1.25 H, N-CH taut B), 6.03 (s, 2 H, N-CH ), 6.66-
6
2
2
6
0
.70 (m, 0.58H, ArH, taut B), 6.71-6.75 (m, 1H, ArH), 6.82-6.84 (m,
.68H, ArH, taut B), 6.84-6.86 (m, 1H, ArH), 6.99-7.02 (m, 0.63H, ArH,
taut B), 7.22-7.28 (m, 1H, ArH), 7.39-7.43 (m, 1H, ArH), 7.45-7.49 (m,
0
7
.62H, ArH, taut B), 7.52-7.61 (m, 1H, ArH), 7.86 (d, 1H, J= 8.4, ArH),
.94-7.97 (m, 0.61H, ArH, taut B), 8.05-8.09 (m, 1H, ArH), 8.40 (s, 1H,
N=CH), 8.46 (s, 0.63H, N=CH, taut B), 9.36 (br s, 2H, OH, D O
2
exchangeable), 10.54 (s, 0.62H, OH, D O exchangeable, taut B), 11.81
2
13
(
s, 1H, NH, D O exchangeable);
C NMR (100 MHz, DMSO-d ) δ:
2
6
19

1
1
67.4, 162.6 (taut B), 1489.9, 146.1, 145.8, 145.5, 142.9, 134.5, 127.9,
27.6, 124.4, 124.2, 121.0,, 119.6,119.5, 119.4,112.1, 111.5, 49.5 (taut
B), 49.2. Anal. Calcd for C H N O (311.30): C, 57.87; H, 4.21; N,
15
13
5
3
22.50, found C, 58.13; H, 4.34; N, 22.74.
4. 1. 4. 6. 2-(1H-benzo[d][1,2,3]triazol-1-yl)-N'-(2,4-dihydroxybenzyl-
o
idene)acetohydrazide (3f) white solid: 53 % yield; mp 276-277 C; IR
-1
1
(
KBr, cm ) 3182, 3101, 3066, 1678, 1627; H NMR (400 MHz, DMSO-
d ) δ 5.60 (s, 1.45 H, N-CH taut B), 5.99 (s, 2 H, N-CH ), 6.30-6.32 (m,
6
2
2
1.40H, ArH, taut B), 6.34-6.36 (m, 2H, ArH), 7.34-7.37 (m, 0.72H, ArH,
taut B), 7.39-7.43 (m, 1H, ArH), 7.52-7.56 (m, 1H, ArH), 7.58-7.60 (m,
1
1
H, ArH), 7.85 (d, 1H, J=8.4, ArH), 8.05-8.09 (m, 1H, ArH), 8.27 (s,
H, N=CH), 8.37 (s, 0.7H, N=CH, taut B), 9.88 (s, 1H, OH, D O
2
exchangeable), 9.98 (s, 1H, OH, D O exchangeable), 11.02 (s, 0.70H,
2
OH, D O exchangeable, taut B), 11.60 (s, 1H, NH, D O exchangeable),
2
2
1
1.99 (s, 0.69H, OH, D O exchangeable, taut B). Anal. Calcd for
2
C H N O (311.30): C, 57.87; H, 4.21; N, 22.50, found C, 57.98; H,
15
13
5
3
4.39; N, 22.81.
4. 1. 4. 7. 2-(1H-benzo[d][1,2,3]triazol-1-yl)-N'-(3,4-dihydroxybenzyl-
o
idene)acetohydrazide (3g) white solid: 57 % yield; mp 276-278 C; IR
-1
1
(
KBr, cm ) 3479-3417,3298, 1678, 1612; H NMR (400 MHz, DMSO-
d ) δ 5.57 (s, 0.66 H, N-CH taut B), 6.01 (s, 2 H, N-CH ), 6.77 (s,
6
2
2
0
.36H, ArH, taut B), 6.81 (d, 1H, J=8.4, ArH), 6.93-6.96 (m, 0.33H,
ArH, taut B), 6.99-7.01 (m, 1H, ArH), 7.22 (s, 0.33H, ArH, taut B), 7.25
s, 1H, ArH), 7.39-7.45 (m, 1H, ArH), 7.52-7.59 (m, 1H, ArH), 7.86 (d,
(
1
0
1
1
H, J=8.4, ArH), 7.93 (s, 1H, N=CH), 8.06-8.08 (m, 1H, ArH), 8.09 (s,
.35H, N=CH, taut B), 9.43 (brs, 2H, OH, D O exchangeable), 11.62 (s,
2
13
H, NH, D O exchangeable); C NMR (100 MHz, DMSO-d ) δ: 167.3,
2
6
62.2 (taut B), 148.4, 146.1, 145.7, 145.5, 134.5, 127.7, 125.7, 124.2,
20

120.7, 119.4, 116.0, 113.4, 111.5, 49.6 (taut B), 49.1. Anal. Calcd for
C H N O (311.30): C, 57.87; H, 4.21; N, 22.50, found C, 57.93; H,
15
13
5
3
4.45; N, 22.72.
4. 1. 4. 8. 2-(1H-benzo[d][1,2,3]triazol-1-yl)-N'-(4-methoxybenzyl-
o
idene)acetohydrazide (3h) white solid: 88 % yield; mp 196-198 C; IR
-1
1
(
KBr, cm ) 3444, 3421, 3190, 1678, 1604; H NMR (400 MHz, DMSO-
d ) δ 3.81 (s, 0.9H, OCH , taut B), 3.82 (s, 3H, OCH ), 5.59 (s, 0.6H, N-
6
3
3
CH taut B), 6.04 (s, 2 H, N-CH ), 7.00-7.04 (m, 2H, ArH), 7.40-7.43
2
2
(
m, 1H, ArH), 7.53-7.60 (m, 1H, ArH), 7.69-7.74 (m, 2H, ArH), 7.83-
7
0
0
.87 (m, 1H, ArH), 8.04 (s, 1H, N=CH), 8.06-8.09 (m, 1H, ArH), 8.23 (s,
.3H, N=CH, taut B), 11.76 (s, 1H, NH, D O exchangeable), 11.87 (s,
2
13
.29 H, OH, D O exchangeable, taut B); C NMR (100 MHz, DMSO-
2
d6) δ: 167.5, 162.4 (taut B), 161.3, 145.5, 145.0, 144.9, 134.5, 129.3,
29.1, 127.7, 126.8, 124.2, 119.4, 118.3, 114.8, 114.7, 111.5, 55.7, 49.6
taut B), 49.2. Anal. Calcd for C H N O (309.33): C, 62.13; H, 4.89;
1
(
16
15
5
2
N, 22.64, C, found 62.40; H, 5.03; N, 22.78.
4. 1. 4. 9. 2-(1H-benzo[d][1,2,3]triazol-1-yl)-N'-(4-hydroxy-3-methoxy-
benzylidene) acetohydrazide (3i) white solid: 75 % yield; mp 242-244
o
-1
1
C; IR (KBr, cm ) 3224, 3159, 1693, 1606; H NMR (400 MHz, DMSO-
d ) δ 3.80 (s, 0.9H, OCH , taut B), 3.84 (s, 3H, OCH ), 5.58 (s, 0.61H,
6
3
3
N-CH taut B), 6.05 (s, 2 H, N-CH ), 6.83-6.85 (m, 1H, ArH), 7.12-7.15
2
2
(
m, 1H, ArH), 7.39-7.43 (m, 2H, ArH), , 7.53-7.60 (m, 1H, ArH), 7.83-
7
0
1
5
.87 (m, 1H, ArH), 7.97 (s, 1H, N=CH), 8.06-8.08 (m, 1H, ArH), 8.16 (s,
.3H, N=CH, taut B), 9.58 (br s, 1H, OH, D O exchangeable), 11.70 (s,
2
H, NH, D O exchangeable). Anal. Calcd for C H N O (325.33): C,
2
16 15
5
3
9.07; H, 4.65; N, 21.53, found C, 59.31; H, 4.82; N, 21.79.
4. 1. 4. 10. 2-(1H-benzo[d][1,2,3]triazol-1-yl)-N'-(3,4,5-trimethoxy-
o
benzylidene)acetohydrazide (3j) white solid: 94 % yield; mp 213-214 C;
21

-1
1
IR (KBr, cm ) 3425, 3186, 1685, 1616 ; H NMR (400 MHz, DMSO-d )
6
δ 3.70 (s, 0.88H, OCH , taut B), 3.71 (s, 3H, OCH ), 3.82 (s, 1.57H,
3
3
OCH ), taut B), 3.85 (s, 6H, 2(OCH )), 5.61 (s, 0.57 H, N-CH taut B),
3
3
2
6
7
.09 (s, 2 H, N-CH ), 7.11 (s, 2H, ArH), 7.40-7.44 (m, 1H, ArH), 7.45-
2
.48 (m, 0.53H, ArH, taut B), 7.53-7.60 (m, 1H, ArH), 7.83-7.88 (m, 1H,
ArH), 8.01 (s, 1H, N=CH), 8.06-8.09 (m, 1H, ArH), 8.21 (s, 0.27H,
13
N=CH, taut B), 11.94 (s, 1H, NH, D O exchangeable); C NMR (100
2
MHz, DMSO-d ) δ: 167.8, 162.7 (taut B), 153.6, 145.5, 144.9, 139.6,
6
1
4
34.4, 129.8, 127.7, 124.3, 119.4, 111.4, 104.8, 60.6, 56.4, 49.6 (taut B),
9.3. Anal. Calcd for C H N O (369.38): C, 58.53; H, 5.18; N, 18.96,
18
19
5
4
found C, 58.26; H, 5.40; N, 19.23.
4. 1. 4. 11. 2-(1H-benzo[d][1,2,3]triazol-1-yl)-N'-(furan-2-ylmethylene)
o
acetohydrazide (3k) white solid: 63% yield; mp 216-217 C; IR (KBr,
-1
1
cm ) 3228, 3170, 1793, 1670, 1616; H NMR (400 MHz, DMSO-d ) δ
6
5
.60 (s, 0.67 H, N-CH taut B), 5.97 (s, 2 H, N-CH ), 6.66-6.67 (m, 1H,
2
2
ArH), 6.98 (d, 1H, J=3.2, ArH), 7.39-7.45 (m, 1H, ArH), 7.52-7.99 (m,
1
1
H, ArH), 7.83-7.87 (m, 2H, ArH), 7.99 (s, 1H, N=CH), 8.05-8.09 (m,
H, ArH), 8.17 (s, 0.33H, N=CH, taut B), 11.93 (s, 1H, NH, D O
2
exchangeable). Anal. Calcd for C H N O (269.26): C, 57.99; H, 4.12;
13
11
5
2
N, 26.01, found C, 58.13; H, 4.39; N, 26.25.
4
1
. 1. 4. 12. N'-((1H-pyrrol-2-yl)methylene)-2-(1H-benzo[d][1,2,3]triazol-
o
-yl)acetohydrazide (3l) white solid: 70 % yield; mp 264-265 C; IR
-1
1
(
KBr, cm ) 3190, 3101, 1678, 1620; ; H NMR (400 MHz, DMSO-d ) δ
6
5
.57 (s, 0.69 H, N-CH taut B), 6.03 (s, 2 H, N-CH ), 6.13-6.17 (m, 1H,
2
2
ArH), 6.49-6.52 (m, 1H, ArH), 6.99-7.00 (m, 1H, ArH), 7.40-7.44 (m,
H, ArH), 7.53-7.58 (m, 1H, ArH), 7.81-7.86 (m, 1H, ArH), 7.92 (s, 1H,
N=CH), 8.07 (d, 1H, J=8.4, ArH), 8.12 (s, 0.34 H, N=CH, taut B), 11.47
1
22

(
s, 1H, NH, D O exchangeable), 11.52 (s, 0.38 H, NH, D O
2
2
exchangeable, taut B), 11.99 (s, 1H, NH, D O exchangeable), 11.69 (s,
2
0
.35 H, OH, D O exchangeable, taut B). Anal. Calcd for C H N O
2
13 12
6
(268.28): C, 58.20; H, 4.51; N, 31.33, found C, 58.02; H, 4.83; N, 31.61.
4.
1.
4.
13.
2-(1H-benzo[d][1,2,3]triazol-1-yl)-N'-(pyridin-4-
ylmethylene) acetohydrazide (3m) White solid: 92 % yield; mp 230-231
o
-1
1
C; IR (KBr, cm ) 3132, 1701, 1600; H NMR (400 MHz, DMSO-d ) δ
6
5
.66 (s, 0.46 H, N-CH taut B), 6.12 (s, 2 H, N-CH ), 7.40-7044 (m, 1H,
2
2
ArH), 7.45-7.48 (m, 1H, ArH), 7.52-7.60 (m, 1H, ArH), 7.65-7.67 (m,
0
8
.46H, ArH, taut B), 7.71-7.76 (m, 2H, ArH), 7.84-7.88 (m, 1H, ArH),
.08 (s, 1H, N=CH), 8.29 (s, 0.23H, N=CH, taut B), 7.65 (d, 0.47H, ArH,
taut B), 8.66-8.68 (m, 2H, ArH), 12.16 (s, 1H, NH, D O exchangeable),
2
13
1
2.30 (s, 0.23H, OH, D O exchangeable, taut B). C NMR (100 MHz,
2
DMSO-d ) δ: 168.3, 163.2 (taut B), 150.6, 145.5, 144.5, 142.5, 141.5,
6
1
4
34.5, 127.7, 126.9, 124.2, 121.4, 119.4, 118.3, 111.4, 49.7 (taut B),
9.3. Anal. Calcd for C H N O (280.29): C, 59.99; H, 4.32; N, 29.98,
14
12
6
found C, 59.72; H, 4.59; N, 29.73.
4. 1. 4. 14. 2-(1H-benzo[d][1,2,3]triazol-1-yl)-N'-(benzofuran-2-
ylmethy- lene)acetohydrazide (3n) white solid: 57 % yield; mp 199-200
o
-1
1
C; IR (KBr, cm ) 3221, 3178, 1685, 1620; H NMR (400 MHz, DMSO-
d ) δ 5.66 (s, 0.65 H, N-CH taut B), 6.06 (s, 2 H, N-CH ), 7.32 (t, 1H,
6
2
2
J=6.4, ArH), 7.40 (s, 0.99H, ArH, taut B), 7.42-7.46 (m, 3H, ArH), 7.55-
7
7
.57 (m, 1H, ArH), 7.64-7.68 (m, 1H, ArH), 7.71-7.74 (m, 1H, ArH),
.86 (d, 0.32H, J=8.4, ArH, taut B), 7.89 (d, 1H, J=8.4, ArH), 7.95-7.97
(
m, 0.32H, ArH, taut B), 8.06-8.10 (m, 1H, ArH), 8.14 (s, 1H, N=CH),
8
.32 (s, 0.32H, N=CH, taut B), 12.16 (s, 1H, NH, D O exchangeable);
2
13
C NMR (100 MHz, DMSO-d ) δ: 167.9, 163.0 (taut B), 155.1, 151.1,
6
145.5, 135.1, 134.6, 128.1, 127.7, 126.8, 124.2, 124.0, 122.4, 119.4,
23

1
11.8, 111.6, 111.0, 49.7 (taut B), 49.1. Anal. Calcd for C H N O
17
13
5
2
(319.32): C, 63.94; H, 4.10; N, 21.93, found C, 64.21; H, 4.28; N, 22.15.
4. 1. 4. 15. N'-(benzo[b]thiophen-2-ylmethylene)-2-(1H-benzo[d][1,2,3]-
triazol-1-yl)acetohydrazide (3o) yellowish white solid: 89 % yield; mp
o
-1
1
2
20-222 C; IR (KBr, cm ) 3421, 3367, 1685, 1608; H NMR (400 MHz,
DMSO-d ) δ 5.63 (s, 0.74 H, N-CH taut B), 6.01 (s, 2 H, N-CH ), 7.41-
6
2
2
7
7
8
1
.44 (m, 3H, ArH), 7.54-7.61 (m, 1H, ArH), 7.83-7.90 (m, 3H, ArH),
.95-7.97 (m, 1H, ArH), 8.06-8.10 (m, 1H, ArH), 8.41 (s, 1H, N=CH),
.62 (s, 0.38H, N=CH, taut B), 12.04 (s, 1H, NH, D O exchangeable),
2
2.15 (s, 0.23H, OH, D O exchangeable, taut B). Anal. Calcd for
2
C H N OS (335.39): C, 60.88; H, 3.91; N, 20.88, found C, 60.72; H,
17
13
5
4.12; N, 20.72.
4
1
. 1. 4. 16. N'-((1H-indol-2-yl)methylene)-2-(1H-benzo[d][1,2,3]triazol-
o
-yl)acetohydrazide (3p) whitish buff solid: 79 % yield; mp 281-282 C;
-1
1
IR (KBr, cm ) 3286, 3221, 3178, 1685, 1608 ; H NMR (400 MHz,
DMSO-d ) δ 5.63 (s, 0.52 H, N-CH taut B), 6.15 (s, 2 H, N-CH ), 6.87
6
2
2
(
s, 1H, ArH), 7.01-7.06 (m, 1H, ArH), 7.20 (t, 1H, J=7.6 ArH), 7.42-7.49
m, 2H, ArH), 7.55-7.60 (m, 2H, ArH), 8.89 (d, 1H, J=8.4, ArH), 8.08 (d,
(
1
1
H, J=8.4, ArH), 8.12 (s, 1H, N=CH), 8.33 (s, 0.28H, N=CH, taut B),
1.50 (s, 1H, NH, D O exchangeable), 11.56 (s, 0.27H, NH, D O
2
2
exchangeable, taut B), 11.94 (s, 1H, NH, D O exchangeable). Anal.
2
Calcd for C H N O (318.34): C, 64.14; H, 4.43; N, 26.40, found C,
17
14
6
64.35; H, 4.59; N, 26.81.
4
1
. 1. 4. 17. N'-((1H-indol-3-yl)methylene)-2-(1H-benzo[d][1,2,3]triazol-
o
-yl)acetohydrazide (3q) white solid: 86 % yield; mp 253-255 C; IR
-1
1
(
KBr, cm ) 3248, 3228, 3190, 1670, 1616; ; H NMR (400 MHz, DMSO-
24

d ) δ 5.58 (s, 0.45 H, N-CH taut B), 6.09 (s, 2 H, N-CH ), 7.14-7.24 (m,
6
2
2
2
H, ArH), 7.40-7.48 (m, 2H, ArH), 7.53-7.57 (m, 1H, ArH), 7.85-7.90
m, 2H, ArH), 8.08 (d, 1H,J=8.8, ArH), 8.25-8.27 (m, 1H, ArH), 8.29 (s,
H, N=CH), 8.45 (s, 0.23H, N=CH, taut B), 11.56 (s, 1H, NH, D O
(
1
2
exchangeable), 11.61 (s, 1H, NH, D O exchangeable), 11.68 (s, 0.23H,
2
13
OH, D O exchangeable, taut B); C NMR (100 MHz, DMSO-d ) δ:
2
6
1
1
4
66.8, 161.8 (taut B), 145.5, 142.6, 137.5, 134.5, 131.3, 127.7, 124.4,
24.3, 123.1, 122.3, 121.2, 119.4, 112.3, 111.6, 111.6, 49.7 (taut B),
9.2. Anal. Calcd for C H N O (318.34): C, 64.14; H, 4.43; N, 26.40,
17
14
6
found C, 64.42; H, 4.65; N, 26.67.
4
4
. 2. Anticancer activity
. 2. 1. Measurement of anticancer activity against a panel of 60 cell
lines
Anticancer activity screening of the newly synthesized compounds was
measured in vitro utilizing 60 different human tumor cell lines provided
by US National Cancer Institute according to previously reported
standard procedure [31-33] as follows: Cells are inoculated into 96-well
microtiter plates in 100 mL. After cell inoculation, the microtiter plates
are incubated at 37
ꢀ C, 5% CO , 95% air and 100% relative humidity for
2
24 h prior to addition of experimental compounds. After 24 h, two plates
of each cell line are fixed in situ with TCA, to represent a measurement
of the cell population for each cell line at the time of drug addition (Tz).
25

Experimental compounds are solubilized in dimethyl sulfoxide at 400-
fold the desired final maximum test concentration and stored frozen prior
to use. At the time of compound addition, an aliquot of frozen concentrate
is thawed and diluted to twice the desired final maximum test
concentration with complete medium containing 50 mg/mL gentamicin.
Aliquots of 100 mL of the compounds dilutions are added to the
appropriate microtiter wells already containing 100 mꢁ of medium,
resulting in the reꢂuired final compound concentration. Following
compound addition, the plates are incubated for an additional 48 h at 37
ꢀ
ꢀ
C, 5% CO , 95% air, and 100% relative humidity. For adherent cells, the
2
assay is terminated by the addition of cold trichloroacetic acid (TCA).
ꢃells are fixed in situ by the gentle addition of 50 mꢁ of cold 50ꢄ (wꢅv)
ꢆꢃꢇ (final concentration, 10ꢄ ꢆꢃꢇ) and incubated for 60 minutes at 4
C. The supernatant is discarded, and the plates are washed five times with
tap water and air dried. Sulforhodamine B (SRB) solution (100 mL) at
0.4% (w/v) in 1% acetic acid is added to each well, and plates are
incubated for 10 minutes at room temperature. After staining, unbound
dye is removed by washing five times with 1% acetic acid and the plates
are air dried. Bound stain is subsequently solubilized with 10 mM trizma
base, and the absorbance is read on an automated plate reader at a
wavelength of 515 nm. For suspension cells, the methodology is the same
except that the assay is terminated by fixing settled cells at the bottom of
the wells by gently adding 50 mL of 80% TCA (final concentration, 16%
TCA). Using the absorbance measurements [time zero, (Tz), control
growth, (C), and test growth in the presence of compound (Ti)], the
percentage growth is calculated for each compound. Percentage growth
inhibition is calculated as:
[
(Ti – Tz)/ (C – ꢆz)] x100 for concentrations for which ꢆi ˃ꢅ= ꢆz.
(Ti – ꢆz)ꢅ ꢆz] x100 for concentrations for which ꢆi ˂ ꢆz.
[
26

4
. 2. 2. Measurement of IC against ovarian cancer OVCAR-3 and
50
leukemia HL-60 (TB) cell lines
. 2. 2. 1. Cell Culture
4
Cell Line cells were obtained from American Type Culture Collection,
cells were cultured using Dulbecco's Modified Eagle's medium (DMEM)
(Invitrogen / Life Technologies) supplemented with 10% fetal bovine
serum (FBS) (Hyclone), 10 μg/mL of insulin (Sigma), and 1% penicillin-
streptomycin. All of the other chemicals and reagents were from Sigma,
or Invitrogen. Plate cells (cells density 1.2 – 1.8 × 10,000 cells/ well) in a
volume of 100µL complete growth medium and 100 μL of the tested
compound per well in a 96-well plate for 24 h before the MTT assay.
4. 2. 2. 2. Cell culture protocol
Remove culture medium to a centrifuge tube. Briefly rinse the cell layer
with 0.25% (w/v) Trypsin 0.53 μM ethylenediaminetetraacetic acid
(EDTA) solution to remove all traces of serum which contains Trypsin
inhibitor. Add 2.0 to 3.0 mL of Trypsin EDTA solution to flask and
observe cells under an inverted microscope until cell layer is dispersed.
Add 6 to 8 mL of complete growth medium and aspirate cells by gently
pipetting. Transfer the cell suspension to the centrifuge tube with the
medium and centrifuge for 5 to 10 minutes. Discard the supernatant.
Resuspend the cell pellet in fresh growth medium. Add appropriate
aliquots of the cell suspension to new culture vessels. Incubate cultures at
37°C for 24 h. After treatment of cells with the serial concentrations of
the compound to be tested incubation is carried out for 48 h at 37ºC, then
the plates are to be examined under the inverted microscope and proceed
for the MTT assay.
4. 2. 2. 3. MTT assay protocol
27

The 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MTT) method [34] of monitoring in vitro cytotoxicity is well suited for
(
use with multiwell plates. The assessment of cell population growth is
based on the capability of living cells to reduce the yellow product MTT
to a blue product, formazan, by a reduction reaction occurring in the
mitochondria. The five cell lines were incubated for 24 h in 96-microwell
plates. The number of living cells in the presence or absence (control) of
the various test compounds is directly proportional to the intensity of the
blue color, measured by spectrophotometry using (ROBONIK P2000
Spectrophotometer) at a wavelength of 570 nm. Measure the background
absorbance of multiwell plates at 690 nm and subtract from the 570 nm
measurement. Five concentrations ranging from 0.01μM to 100 μM (with
semi-log decrease in concentration) were tested for each of the
compounds under study. Each experiment was carried out in triplicate.
The IC values [the concentration required for 50% inhibition of cell
50
viability] were calculated using sigmoidal dose response curve-fitting
models.
4. 2. 3. Measurement of inhibitory activity against FAK
Compounds 3d, 3e, 3f and 3q were selected to be evaluated against FAK
enzyme using ab187395–FAK Human Simple Step ELISA® Kit
according to manufacturer’s instructions. In brief, prepare all reagents,
working standards, and samples. ꢇdd 50 μꢁ of all sample or standard to
appropriate wells. ꢇdd 50 μꢁ of the antibody cocktail to each well. Seal
the plate and incubate for 1 h at room temperature on a plate shaker set to
400 rpm. Wash each well with 3 x 350 μꢁ wash buffer. Wash by
aspirating or decanting from wells then dispensing 350 μꢁ wash buffer
into each well. ꢇdd 100 μꢁ of 3, 3', 5, 5'-Tetramethylbenzidine (TMB)
28

substrate to each well and incubate for 10 minutes in the dark on a plate
shaker set to 400 rpm. ꢇdd 100 μꢁ of stop solution to each well. Shake
plate on a plate shaker for 1 minute to mix. Record the OD at 450 nm.
Subtract average zero standard from all readings. Average the duplicate
readings of the positive control dilutions and plot against their
concentrations. Draw the best smooth curve through these points to
construct a standard curve. Interpolate protein concentrations for
unknown samples from the standard curve plotted.
4. 2. 4. Cell-based FAK auto-phosphorylation assay
FAK (Phospho-Tyr397) Cell-Based ELISA Kit was used to monitor FAK
protein phosphorylation and expression profile in cells treated with
compound 3e according to manufacturer’s instructions. Seed 200 μL of
20,000 adherent cells in culture medium in each well of a 96-well plate.
Incubate the cells for overnight at 37°C, 5% CO . Remove the cell culture
2
medium and rinse with 200 μLof 1x (Tris Buffered Saline) TBS, twice.
Fix the cells by incubating with 100 μL of fixing solution for 20 minutes
at room temperature. Remove the fixing solution and wash the plate 3
times with 200 μL 1x wash buffer for five minutes each time. Add 100
μL quenching buffer and incubate for 20 minutes at room temperature.
Wash the plate 3 times with 1x wash buffer for 5 minutes at a time. Add
2
00 μL of blocking buffer and incubate for 1 h at room temperature.
Wash 3 times with 200 μL of 1x wash buffer for 5 minutes at a time. Add
0 μL of 1x primary antibodies (Anti-FAK (Phospho-Tyr397) antibody,
5
anti-FAK antibody and/or anti-GAPDH antibody) to the corresponding
wells and incubate for 16 h at 4°C.Wash 3 times with 200 μL of 1x wash
buffer for 5 minutes at a time. ꢇdd 50 μL of 1x secondary antibodies
(
HRP-Conjugated Anti-Rabbit IgG and/or HRP-Conjugated Anti-Mouse
IgG) to corresponding wells and incubate for 1.5 h at room temperature.
29