Design of molecular hybrids of phthalimide-triazole agents with potent selective MCF-7/HepG2 cytotoxicity: Synthesis, EGFR inhibitory effect, and metabolic stability

Article abstract of DOI:10.1016/j.bioorg.2021.104835

This study reports an efficient and convenient click chemistry synthesis of a novel series of phthalimide scaffold linked to 1,2,3 triazole ring and terminal lipophilic fragments. Structures of newly synthesized compounds were well characterized by different spectroscopic tools. In vitro MTT cytotoxicity assay was performed comparing the cytotoxic effects of newly synthesized compounds to staurosporine using three different types: human liver cancer cell line (HepG2), Michigan cancer foundation-7 (MCF-7) and human colorectal carcinoma cell line (HCT116). The initial screening showed excellent to moderate anticancer activity for these newly synthesized compounds with high degree of cell line selectivity with micromolar (μM) half maximal inhibitory concentration (IC₅₀) values against tumor cells. The SAR analysis of these derivatives confirmed the role of molecular fragments including phthalimide, linker, triazole, and terminal tails in correlation to activity. In addition, enzymatic inhibitory assay against wild type EGFR was performed for the most active compounds to get more details about their mechanism of action. In order to further explore their binding affinities, molecular docking simulation was studied against EGFR site. The results obtained from molecular docking study and those obtained from cytotoxic screening were correlated. One of the most prominent analogs is (6f) with terminal disubstituted ring and amide linker showed selective MCF-7 cytotoxicity profile with IC₅₀ 0.22 μM and 79 nM to EGFR target. Extensive structure activity relationship (SAR) analyses were also carried out. The pharmacokinetic profile of (6f) was studied showing good metabolic stability and long duration behavior. This design offered a potent selective anticancer phthalimide-triazole leads for further optimization in cancer drug discovery.

Full text of DOI:10.1016/j.bioorg.2021.104835

Bioorganic Chemistry 111 (2021) 104835
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design of molecular hybrids of phthalimide-triazole agents with potent
selective MCF-7/HepG2 cytotoxicity: Synthesis, EGFR inhibitory effect, and
metabolic stability
a
,
*
b
b
,
*
b
Saleh K. Ihmaid , Shaya Yahya Alraqa , Mohamed R. Aouad , Ateyatallah Aljuhani ,
c
d
,
*
b
a,e
Hossein M. Elbadawy , Samir A. Salama , Nadjet Rezki , Hany E.A. Ahmed
a
Pharmacognosy and Pharmaceutical Chemistry Department, College of Pharmacy, Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia
Chemistry Department, College of Sciences, Taibah University, Al-Madinah Al-Munawarah 41477, Saudi Arabia
b
c
Pharmacology and Toxicology Department, College of Pharmacy, Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia
Division of Biochemistry, Department of Pharmacology, College of Pharmacy, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar University, 11884 Nasr City, Cairo, Egypt
d
e
A R T I C L E I N F O
A B S T R A C T
Keywords:
,2,3-triazoles
This study reports an efficient and convenient click chemistry synthesis of a novel series of phthalimide scaffold
linked to 1,2,3 triazole ring and terminal lipophilic fragments. Structures of newly synthesized compounds were
well characterized by different spectroscopic tools. In vitro MTT cytotoxicity assay was performed comparing the
cytotoxic effects of newly synthesized compounds to staurosporine using three different types: human liver
cancer cell line (HepG2), Michigan cancer foundation-7 (MCF-7) and human colorectal carcinoma cell line
1
Phthalimide analogues
Anticancer activity
EGFR kinases
Apoptosis study
(
HCT116). The initial screening showed excellent to moderate anticancer activity for these newly synthesized
compounds with high degree of cell line selectivity with micromolar (µM) half maximal inhibitory concentration
IC50) values against tumor cells. The SAR analysis of these derivatives confirmed the role of molecular fragments
(
including phthalimide, linker, triazole, and terminal tails in correlation to activity. In addition, enzymatic
inhibitory assay against wild type EGFR was performed for the most active compounds to get more details about
their mechanism of action. In order to further explore their binding affinities, molecular docking simulation was
studied against EGFR site. The results obtained from molecular docking study and those obtained from cytotoxic
screening were correlated. One of the most prominent analogs is (6f) with terminal disubstituted ring and amide
linker showed selective MCF-7 cytotoxicity profile with IC50 0.22 µM and 79 nM to EGFR target. Extensive
structure activity relationship (SAR) analyses were also carried out. The pharmacokinetic profile of (6f) was
studied showing good metabolic stability and long duration behavior. This design offered a potent selective
anticancer phthalimide-triazole leads for further optimization in cancer drug discovery.
1
. Introduction
The scientific researchers devote lots of efforts on the synthesis of
and the triazoles have been identified as one of the most promising
scaffold of drugs in pharmacology owing to their broad spectrum of
biological applications [4].
new drugs which have the beneficial effects on human health [1].
Nowadays, a variety of organic molecules are designed for this purpose
using different types of molecular hybridization protocols to attach
structural feature or modify functional groups into old or new chemical
moieties [2]. Among the different functional moieties employed for this
purpose, is various N-substituted heterocycles, which exhibited diverse
biological activities and have attracted a considerable attention of me-
dicinal chemists [3]. In particular, the benzoheterocyclic compounds
Phthalimide is a fused heterocyclic structure, and its derivatives are
an interesting class of compounds having a wide range of important
applications [5–7]. These compounds have been reported to have
interesting pharmacological proprieties such as antimicrobial [8,9],
anti-inflammatory [10], antitumor [11], and also antiviral [12].
Additionally, nitrogen heterocyclic compounds containing triazole
core play a key role in medicinal and pharmaceutical discoveries as
biologically active compounds. In particular, 1,2,3-Triazole derivatives
*
Corresponding authors.
E-mail addresses: sihmaid@taibahu.edu.sa (S.K. Ihmaid), aouadmohamedreda@yahoo.fr (M.R. Aouad), s.salama@tu.edu.sa (S.A. Salama).
https://doi.org/10.1016/j.bioorg.2021.104835
Received 1 December 2020; Received in revised form 10 March 2021; Accepted 17 March 2021
Available online 22 March 2021
0
045-2068/© 2021 Elsevier Inc. All rights reserved.

S.K. Ihmaid et al.
Bioorganic Chemistry 111 (2021) 104835
are considerable scaffolds exhibiting versatile biological properties such
as antimicrobial, antifungal, antitubercular, anti-inflammatory, anti-
cancer (I-IV) [13], analgesic, anticonvulsant and antityrosinase activ-
ities [6,14–19]. Recently, extensive research has been focused on the
molecular hybrids from bioactive chemical entities, which is a new
strategy, to enhance the drug design prototype [20,21]. In this regard,
many compounds have been designed bearing various biological activ-
ities by the covalent fusion of at least two or more pharmacophores
moieties in “one chemical structure”. Recently, there have been
numerous hybrid molecules already launched for clinical trials for the
treatment of various diseases [22]. Incorporating of 1,2,3-triazole
framework with other pharmacophores to generate the so-called
binding stability with liable thioether linker susceptible for metabolic
oxidation [36] that represent a pharmacokinetic problem.
Keeping in mind the molecular pharmacophores outlined in (Fig. 1)
including the phthalimide scaffold and 1,2,3, triazole as promising
motifs, and in continuation to our work in the field of designing and
synthesis of anticancer agents [19,25–35], molecular hybridization be-
tween phthalimide ring and triazole nucleus has been designed and
synthesized (Fig. 1). The main outlines for this study is the novel design
was introduced by replacement of benzothiazole ring with more prom-
ising phthalimide scaffold and thioether linker is replaced by one carbon
to adjust this problem and give metabolically stable agents. The novel
compounds were screened for their anticancer activity and detailed
mechanistic analyses, pharmacokinetic analysis, and molecular
modeling were reported.
“
hybrid molecule” has been proved to be effective in terms of
enhanced activity, when compared to that of individual pharmaco-
phores [4,23,24].
The view of these literature reports on the promising bio-activities of
phthalimides and substituted 1,2,3-triazole derivatives and in continu-
ation of our work in the molecular hybridization as promising approach
for producing novel range of bioactive compounds [19,25–35], the ef-
forts are made to designed and synthesized phalimide-1,2,3-triazole
hybrids incorporating different functionalities on the phthalimide ring
using a protocol based on click chemistry. In continuation to our pre-
vious work [33], we introduced novel design of triazole-based scaffold
with different biologically active benzothiazole and isatin linked to
polar acid sulphonamides which exhibited potent anticancer activity
through the inhibition of wild type-EGFR kinase. Based upon the
interaction analyses data observed, the benzothiazole offered least
2. Results and discussion
2.1. Chemistry
The targeted 1,2,3-triazoles-based phthalimide were successfully
synthesized through the Cu(I)-click chemistry approach as illustrated in
Schemes 1–3. The ‘‘click” 1,3-dipolar cycloaddition reaction requires
two coupling building blocks incorporating a terminal alkyne and an
azide side chain.
Initially, the commercially available phthalimide 1a and/or 5,6-
dichlorophtamide 1b reacted individually with propargyl bromide in
DMF using potassium carbonate as basic catalyst and afforded
Anticancer Triazole-based agents
OH
F
N
N
N
N
N
N
N
N
O
O
HO
O
N
S
O
O
N
N
S
O
N
N
O
NH2
O
O
N
IV
I
O
O
HO
O
O
O
O
II
III
Anticancer Phthalimide-based agents
O
O
O
NH
O
N
N
N
O
O
N
O
N
S
OCH3
O
O
O
Thalidomide
Canthridin
5a
Molecular hybridization of all pharmacophoric motifs
O
Cl
O
N
Cl
Cl
COCH
3
O
Cl
N
Non-linker
O
N
F
N
N
H
N
O
N
N
N
R
EGFR IC50, nM
79
O
Block of center metabolism
Cytotoxicity, µM
0.22, 15.02, 17.92
3 hours
F
N
H
Duration
Improved selective cytotoxicity
and EGFR inhibitory activity
Linker-based
Fig. 1. Design strategy of the target molecules with alignment to reported triazole/phthalimide-based anticancer agents.
2

S.K. Ihmaid et al.
Bioorganic Chemistry 111 (2021) 104835
O
O
R1
R2
R1
R2
K CO , DMF
2
3
N
H
+
N
Br
O
O
1
2
1
2
1
a R = R = H
2a R = R = H
1
2
1
2
1
b R = R = Cl
2b R = R = Cl
Scheme 1. Synthesis of propargylated phthalimides 2a-b.
O
N
O
O
R1
R2
R1
CuSO , Naascorbate
4
Ar
N
N
+
3
t-BuOH/H O (1:1)
2
R2
O
N
N
N
Ar
2a-b
3a-d
4a-f
Scheme 2. Click synthesis of phthalimide-1,2,3-triazole molecular hybrids carrying un/functionalized aromatic side chains 4a-f.
O
O
R1
R2
R1
R2
CuSO , Naascorbate
R3
N
4
N
+
3
N
t-BuOH/H O (1:1)
2
O
O
N
N
R3
N
2a-b
5a-e
6a-i
Scheme 3. Click synthesis of phthalimide-1,2,3-triazole molecular hybrids carrying ester/and amide side chains 6a-i.
exclusively the corresponding N-propargylated key intermediates
namely 2-(prop-2-yn-1-yl)isoindoline-1,3-dione 2a and 5,6-dichloro-2-
Table 1
Chemical structures and analytical data of 1,2,3-triazoles 4a-
(
prop-2-yn-1-yl)isoindoline-1,3-dione 2b in 92% and 90%, respec-
tively (Scheme 1).
The formation of the propargylated phthalimides 2a,b was
f.
1
13
confirmed based on different spectroscopic techniques (IR, H NMR,
C
NMR and elemental analysis). In the case of alkyne 2b, the IR spectrum
ꢀ
1
revealed the presence of two absorption bands at 2145 and 3290 cm
1
2
◦
–
–
Comp. No
R , R
Ar
Mp ( C)
Yield (%)
85
due to the propargyl C
of compound 2bwas further supported by H NMR analysis in which the
signal attributed to NCH protons appeared as a singlet at 4.37 ppm,
CH signal resonated as a singlet at 3.28 ppm. The signals
–
C and
–
CH groups, respectively. The structure
1
4
4
a
b
H, H
235–236
2
Cl, Cl
H, H
Cl, Cl
H, H
H, H
221–222
264–265
251–252
198–199
244–245
84
83
83
86
82
–
whereas
–
–
4c
4d
belonging to the same characteristic propargyl carbons NCH
resonated at 27.57 and 74.65–78.29 ppm, respectively.
2
and C C
–
As mentioned in Scheme 2, un/functionalized aromatic azides 3a-
d were prepared from the appropriate commercially available anilines,
by well-established diazotization followed by azidolysis reactions
4
4
e
f
[
37–39].
The freshly prepared alkynes 2a,b and aromatic azides 3a-d were
then legated to afford regioselectivity the desired 1,4-disubstituted-
,2,3-triazoles 4a-f via copper(I) catalyzed the 1,3-dipolar cycloaddi-
properties and ester based aliphatic side chain as well as substituted
phenyl acetamide side chains linked to the N-1 triazolyl moiety.
Thus, propargylated phthalimides 2a,b, ester and/or amide based
1
tion reactions. The click reactions were achieved using copper sulfate
and sodium ascorbate as catalysts in t-BuOH/water at room temperature
azides 5a-e and catalytic amount of copper sulfate (CuSO ) and sodium
4
(
Scheme 2). Consequently, by employing four different aromatic azides
ascorbate were stirred at room temperature in aqueous t-BuOH under
and two phthalimide-based alkynes, six 1,2,3-triazole scaffolds carrying
different N-functionalization 4a-f were synthesized in good to excellent
yields (82–86%). The structures of the click products 4a-f are reported in
Table 1.
click conditions via 1,3-dipolar cycloaddition to afford the desired 1,4-
disubstituted 1,2,3-triazole functionalized phthalimide derivatives 6a-i
in good to excellent yields (86–90%). The structures of the click products
6a-i are tabulated in Table 2.
The newly designed phthalimide-1,2,3-triazole hybrids reported in
Scheme 3 comprise three parts: regioselective 1,4-disubstituted 1,2,3-
triazole as a pillar fragment, un/substituted phthalimide to maximize
the targeted pharmacophoric potentialities with promising drug like
The structures of the newly designed 1,2,3-triazole hybrids 4a-f and
1
13
6a-i were established based on their spectral data ( H NMR, C NMR, IR
and elemental analysis), which were in agreement with the proposed
structures. From their IR spectra, the disappearance of the characteristic
3

S.K. Ihmaid et al.
Bioorganic Chemistry 111 (2021) 104835
from the 13C NMR analysis which confirmed the disappearance of the
carbon signals belonging to the alkyne building block (C–
Table 2
Chemical structures and analytical data of 1,2,3-triazoles 6a-
C) and the
–
appearance of new characteristic signals in the aliphatic and aromatic
regions attributable to the alkyl and/or aryl residues bonded to the N-1
atom of the triazole ring. The 13C NMR spectra of the ester-based 1,2,3-
triazoles 6a,b were characterized by the appearance of new signals at
i.
1
4.40–14.41, 50.80–50.81 and 167.63–167.82 ppm assigned, respec-
1
2
3
◦
–
and C
–
Comp. No
R , R
R
Mp ( C)
Yield (%)
tively, to the CH
3
, CH
2
O carbons of the ethyl acetate side chain.
) and carbonyl (C–
O)
For compounds 6c-i, the acetyl methylene (CH
2
–
6
6
6
a
b
c
H, H
172–173
183–184
222–223
90
89
88
carbons were recorded at 52.35–52.76 and 166.06–167.83 ppm,
respectively.
Cl, Cl
H, H
2
.2. Cytotoxicity screening
6
6
d
e
Cl, Cl
H, H
243–244
215–216
87
88
The newly synthesized phthalimide-1,2,3-triazole conjugates were
evaluated for their in-vitro cytotoxicity against different cancer cell lines
such as, Michigan cancer foundation-7 (MCF-7), human colorectal car-
cinoma cell line (HCT116) and human liver cancer cell line (HepG2)
using MTT assay method and compared with a reference treatment with
well documented toxic effect to breast cancer cells staurosporine and
erlotinib are listed in (Table 3). It is observed from the cytotoxicity re-
sults that derivatives containing phthalimide nucleus and acetamide
with direct attached triazole ring showed potent and selective cytotox-
icity against MCF-7. Compounds 6f, and 6 h possessing Cl substitution
on phthalimide and triazole cores with acetamide linker, displayed
selectivity and most potent cytotoxic activity on MCF-7 with an
IC50 value of (0.22 ± 0.26, and 0.56 ± 1.25 µM), respectively (Table 3).
While compounds 4c, 4e, and 4f with no linker on triazole showed
selectivity and moderate inhibitory effect against the HepG2 with an
IC50 value of (5.92 ± 1.46, 3.86 ± 0.14, and 6.52 ± 0.02 µM), respec-
tively. However, compound 4b, with electron-donating groups on the
phenyl ring attached with triazole showed more potent inhibitory effect
and selective against theHepG2with an IC50 value of (0.12 ± 1.22 µM).
While 6a, 6c, 6e, 6i, and 6g with acetamide linker displayed selective
and moderate cytotoxicity against theMCF-7 with an IC50 value of (3.25
± 0.10, 7.60 ± 1.25, 4.94 ± 0.20, 10.05 ± 0.12, and 9.16 ± 0.25 µM),
respectively. Compound 4d with Cl substitution on phthalimide,
exhibited selectivity and moderate cytotoxic activity on MCF-7 with an
IC50 value of (2.30 ± 1.10 µM). On the other hand, compounds 4a and
6
6
6
6
f
Cl, Cl
H, H
Cl, Cl
H, H
229–230
196–197
212–213
255–256
86
88
87
88
g
h
i
–
–
CH) provides the main evidence
alkyne absorption bands (C
–
C and
–
for the involvement of the propargyl side chain in the click synthesis. In
addition, the 1H NMR spectra of all 1,2,3-triazoles 4a-f and 6a-i
confirmed the disappearance of the active terminal alkyne proton
–
(
–
CH) and the appearance of the characteristic triazolyl proton (C
5
-H)
as a distinct singlet at 8.11–9.02 ppm. The methylene protons of the
CH
2
CO group for compounds 6a-i were recorded as singlet at 5.28–5.39
ppm. The formation of the amide-based 1,2,3-triazoles 6c-i has been also
6
b were less potent against the three cell lines. The contribution of
evidenced by the appearance of a diagnostic downfield singlet at
halogenated phthalimide increases the lipophilicity tolerance in the
binding pocket and acetamide linker share with hydrogen bonding sta-
bilization that stand for the SAR of anticancer activity distribution of
1
0.37–11.05 ppm attributed to the acetamide NH proton.
Further evidences for the success of the click reactions were obtained
Table 3
Cytotoxicity activities of compounds 2a-b, 4a-f, and 6a-6h against MCF-7, HCT116, and HepG2 cell lines.
a
Cps.
Linker
R
1
R
2
IC50 (µM)
Selectivity analysis
Inactive
MCF-7
HCT116
HepG2
2
2
4
4
4
4
6
6
6
6
6
4
4
6
6
6
6
a
b
a
c
e
f
–
2H
2Cl
2H
45.51 ± 0.38
25.81 ± 0.70
13.52 ± 0.92
25.46 ± 0.22
18.27 ± 0.91
16.52 ± 0.29
3.25 ± ±0.10
7.60 ± ±1.25
4.94 ± ±0.20
10.05 ± 0.12
9.16 ± 0.25
20.39 ± 1.47
2.30 ± ±1.10
11.49 ± 0.81
4.78 ± ±0.41
0.22 ± ±0.26
0.56 ± ±1.25
12.04 ± 0.88
66.62 ± 4.36
51.58 ± 1.76
21.75 ± 7.2
13.55 ± 0.65
19.09 ± 0.11
21.19 ± 0.45
42.80 ± 2.14
33.14 ± 1.22
17.51 ± 0.48
18.77 ± 0.39
13.36 ± 0.12
13.85 ± 6.91
16.31 ± 1.17
11.62 ± 4.36
11.34 ± 1.41
15.02 ± 1.71
23.04 ± 2.80
3.15 ± 0.07
35.42 ± 1.39
31.10 ± 0.02
10.92 ± 0.97
5.92 ± ±1.46
3.86 ± ±0.14
6.52 ± ±0.02
13.77 ± 0.01
20.78 ± 0.04
15.81 ± 0.60
20.98 ± 0.49
22.53 ± 0.01
0.12 ± ±1.22
10.36 ± 0.03
16.31 ± 0.61
14.73 ± 0.45
17.92 ± 0.25
25.74 ± 0.02
7.92 ± 0.25
HepG2 selective
MCF-7 selective
a
c
e
i
Acetamide
g
b
d
b
d
f
–
2Cl
HepG2 selective
MCF-7 selective
Non-selective
Acetamide
MCF-7 selective
h
Staurosporine
–
Non-selective
a
IC50: indicate the inhibitory concentration capable of reducing cell proliferation by 50%. Data are represented as the mean of three independent experiments ±
standard error (SE) as estimated from the dose-response curves.
4

S.K. Ihmaid et al.
Bioorganic Chemistry 111 (2021) 104835
target compounds.
detected for tested compound (6f).
.3. EGFR TK wild typeInhibitory assay
2.5. Cell cycle analysis
2
For the reduction of possible nonspecific effects of drugs in cancer
treatment, the design and synthesis of novel chemotherapeutic agents
that can regulate cell cycle progression and apoptosis is an attractive
approach [42–44]. Therefore, further investigation of the effects of
compound 6f on regulating cell cycle progression of cancer cells was
explored using the Propidium Iodide Flow Cytometry Kit assay (Fig. 3A-
B). The cell cycle histograms of the MCF-7 target cancer cell treated with
potent selected compound (at IC50 cytotoxicity) and compared to that of
the untreated cells are illustrated in (Fig. 4). The percentages of com-
pound 6f treated MCF-7 cells in the Sand Pre-G1 phase were signifi-
cantly higher than those in the control group. The distribution of cells in
G0-G1 phase was notably decreased compared to the control cells as
consistent with that previously reported [45,46]. Previous studies
showed that triazole-based EGFR drugs aligned along the microtubular
network within the cells during the mitotic phase [47,48]. In addition,
intracellular EGFR inhibitors have been associated with antimitotic ac-
tion [49,50]. This might provide a possible explanation of cell cycle
arrest observed at Sand Pre-G1 phases in cells treated with novel active
The epidermal growth factor receptor (EGFR) kinase is highly
expressed in MCF-7 cancer cell line growth and as shown from the
profile activity of anticancer to target compounds, inhibition of wild
type EGFR kinase assay was therefore performed in vitro as an indicator
for anticancer activity for the most actives. All potent derivatives 4b-f,
6
d-i were tested and reference erlotinib was used as positive control,
(
Table 4). All tested compounds gave potent EGFR inhibition in nano-
molar ranges; 79–399 nM. Among them, (4b) and (6f) displayed obvious
inhibition, but their activities were more than reference drug. These
data showed that (4b) and (6f) are promising agents for further modi-
fication and investigation which can be used to construct more active
derivatives. Compound 6f displayed a significant inhibition with an IC50
value of 79 nM, which was almost slightly better than erlotinib. This
series of derivatives can be tested, further, on mutant EGFR expressed by
specific cell lines. This indicates that these designated compounds are
standing out candidates for additional investigations to explore their
anticancer activities.
6
f derivative.
2
.4. Cell apoptosis analysis
2
.6. DNA fragmentation
Apoptosis induction was studied in MCF-7 cells for only 6f active
compounds taking into consideration the results from the EGFR assay,
Table 4) and IC50 of values of the tested derivatives (Table 3). Using
DNA fragmentation and strand cleavage into pieces are tow pro-
(
cesses commonly reported in apoptotic cells [51]. Apoptosis results in
DNA fragmentation which can be detected using diphenylamine assay.
The extent of DNA fragmentation, measured quantitatively, is an indi-
cation of the apoptosis process. IN our hands, MCF-7 cells treated with
the compound 6f exhibited higher percentage of DNA fragmentation
compared to staurosporine or untreated controls (Fig. 4). These findings
can be correlated to the ability of compound 6f to induce apoptosis, as
shown earlier. The suggested sequence of events can be as follows;
compound 6f may have caused disruption of MCF-7 cell membranes
leading to the fragmentation of chromosomal DNA [52]. Overall, this
novel compound (6f) was shown to be cytotoxic and the cytotoxic
mechanism can occur by apoptosis through DNA fragmentation.
untreated MCF-7 cells as a control, selected compound (6f) were
investigated for their possible apoptotic activity. Apoptosis was
measured using Annexin/PI double staining flow cytometric assay and
the results are shown (Fig. 2A-B). The overall apoptosis measurement
showed that 29% total apoptosis was noted after treatment with the test
agent, compared to 1.36% in negative controls. Cell death by necrosis
usually cause the release of cell debris and other proteins which can
induce inflammation in the media and affect the surrounding cells [38].
However, we observed that minimum number of cells (10%) underwent
necrosis after being exposed to (6f) compound. This may indicate that
apoptosis, rather than necrosis, is responsible for the effect of 6f com-
pound in vitro. A general target for anticancer drugs is to shift the bal-
ance of proliferation and apoptosis towards the later. When cancer cells
commit to apoptosis, this target is achieved [37]. The results in our
hands also comes in agreement with previous studies demonstrating that
erlotinib is an efficient inhibitor of MCF-7 proliferation, and it does so by
a mechanism involving apoptosis and necrosis, [39–41] as clearly
2
.7. Determination of total ROS generation in MCF-7cells
As reported some drugs interact with several ROS metabolic enzymes
in MCF-7 and up-regulates intracellular ROS levels in human tumor cells
13,52,53]. Production of ROS can be an evidence for oxidative stress
[
which is linked to the regulation of apoptosis [54]. Oxidative stress can
be accompanied by endoplasmic reticulum stress, a mechanism that is
also linked to the contribution of the mitochondrial in apoptosis as a
result of the overproduction of ROS. We investigated the significance of
the increase in ROS levels by active compound 6f for cell growth in MCF-
Table 4
wild type
In vitro inhibitory EGFR
assay (IC50, nM) of new candidate
compounds.
7
cell line using ELISA coated with anti-ROS. The absorbance at 450 nm
is inversely proportional to the production of ROS. Farnesiferol C was
used as a positive control to be compared with our tested compound 6f.
For untreated cells the production of ROS was minimal, showing more
absorption (Fig. 5). It was evident that treatment with Farnesiferol or
compound 6f resulted in an increase in the production of ROS, indi-
cating that Farnesiferol C as well as 6f may have induced apoptosis via
enhancing intracellular ROS generation. Therefore, compound 6f may
exert some of its anticancer effect by an oxidative stress-related mech-
anism. Further investigations are offered to study the regulatory
mechanisms at molecular level.
Cpds.
IC50
LogP
4
4
4
4
6
6
6
6
6
6
b
d
e
f
86 ± 1.68
307 ± 6.52
262 ± 6.87
417 ± 7.14
128 ± 2.72
156 ± 3.12
79 ± 2.31
399 ± 8.44
103 ± 2.36
190 ± 3.93
84 ± 1.68
3.572
3.068
3.547
3.817
3.763
2.160
3.467
2.130
3.437
1.621
3.405
d
e
f
g
h
i
2.8. Inhibition of 6f to EGFR autophosphorylation in MCF-7 cells
Erlotinib
–
Data are presented as average IC50 ± SD (nM) values from at least three in-
Compound (6f) was furtherly investigated to determine if it acts
dependent experiments.
through an EGFR mediated mechanism. The protein expressions of the
5

S.K. Ihmaid et al.
Bioorganic Chemistry 111 (2021) 104835
Fig. 2. A) Dot plot of Annexin V/PI double staining of MCF-7 control and treated with analogue 6f. B) Statistical analysis of the apoptosis percentage of MCF-7 cells
after incubation with compounds (6f) in conc. (0.22
μg/mL respectively) for 24 h. The data are reported as the mean ± SD of three independent experiments
in triplicate.
total- EGFR, p-EGFR, were investigated in MCF-7 cells using Western
blotting (Fig. 6). MCF-7 cells were treated with (6f) or erlotinib at 220
nM and 84 nM respectively for 1 h and then epidermal growth factor
2.10. Metabolic stability of 6f
The anticancer activity of the synthesized compounds is not the sole
factor for them to be considered successful candidates. The compounds
must possess a set of drug-like properties as the metabolic stability and
rate of clearance. Therefore, a certain stability analysis of compound 6f
was initially investigated using in vitro assays (Table 5). The most
promising derivative in this series, compound 6f, showed a high degree
of metabolic stability with an intrinsic half-life (t1/2) of more than 3 h
compared to the previous active benzothiazole derivatives 7 [33]. This
value was increased in the absence of NADPH, the cofactor of CYP-450,
indicating that dichloro-phthalimide-triazole 6f is mainly metabolized
by this class of metabolic oxidases. Furthermore, the compound 6f is
comparable to high and low clearance rates reference propranolol and
terfenadine drugs. Despite the increase in its half-life in the absence of
NADPH suggesting that the compound is mainly metabolized by the co-
factor CYP-450, compound 6f exhibited pronounced stability to hepatic
metabolism, with a clearance rate of less than 62 µl/min/mg. This result
endows the new series of phathalamide-1,2,3-triazole hybrids are
metabolic stable and have longer duration than the old generation of
benzothiazole-1,2,3-triazole-sulfa drugs that were rapidly metabolized
with a t1/2 of less than half an hour.
(
EGF) was added. Compared with the reference erlotinib, compound (6f)
significantly inhibited EGFR autophosphorylation in MCF-7 cells, as
compared to the positive control erlotinib but at a concentration slightly
higher (220 nM).
2
.9. SAR analysis of target compounds
From the interpretation of results, the structure-activity relationship
has been drawn and graphically represented in (Fig. 7). It is evident that
the central core, phathalamide-1,2,3-trizole is an essential requirement
for exhibiting the activity. The dichloro substitutions at the 4th and 5th
position of the phthalimide ring have shown to exert a great impact on
the activity as well as block the metabolism on aromatic ring. The
methylene linker is responsible for the metabolic stability, which leads
to the low of clearance. Moreover, the (N) quinozoline atom in the
erlotinib is replaced by the bioisosteric N3 of the triazole ring, resulting
in conserved hydrogen bond. It is observed that acetamide as linker is
important in the structure to increase the cytotoxicity activity and give
the compound stability in EGFR pocket. It is interesting to notice that the
increase in the aromatic stacking interaction increase Log p values.
6

S.K. Ihmaid et al.
Bioorganic Chemistry 111 (2021) 104835
Fig. 3. A) Histogram showing the distribution of DNA content in control and treated cells. B) Bar chart representation of the percentage of MCF-7 cells in each stage
of the cell cycle after incubation with compound 6f (0.22
μM) or in untreated controls for 24 h. The data are reported as the mean ± SD of three independent
experiments.
Fig. 4. Bar chart showing the percentage of DNA fragmentation after 24 h from
treatment with compound 6f (0.22 M) as compared to staurosporine and un-
Fig. 5. The histogram shows control cells (two column on the left) which were
not treated with drug, and test agents (two column on the right) denoting the
production of ROS by MCF-7 cells as detected using specific ELISA kit using
DCFH-DA-derived fluorescence. The data are reported as the mean ± SD of
three independent experiments in triplicate.
μ
treated controls. The data are reported as the mean ± SD of three independent
experiments in triplicate.
2
.11. Computational ADMET analysis of compound 6f
These parameters were calculated for potent 6f analog compared to
previous reported 7 and marketed erlotinib drug. After calculating the
ADMET properties (Table 6), we can verify that the ligands 6f, and the
reference erlotinib have almost similar Log P value (3.46, and 3.40)
The ADMET analysis properties of the targets compounds were
analysis using the algorithm of pkCSM ADMET descriptors [55]. These
descriptors are chemically and functionally are described in detail [55].
7

S.K. Ihmaid et al.
Bioorganic Chemistry 111 (2021) 104835
Fig. 6. Western blot analysis of Erlotinib/6f-treated and control MCF-7 cell lines. The cancer cell line MCF-7 was treated with 6f as potent derivative compared to
reference erlotinib drug at concentrations of 220 and 84 nM on the phosphorylation of EGFR in MCF-7 cells compared to control untreated cells. The abundance of
the proteins EGFR and P-EGFR was determined; β-Actin staining was used as loading control.
Fig. 7. Structural Activity Relationship (SAR) studies.
bioavailability also it easy to pass through the biological membranes
Table 5
[
56]. In addition to, the ligand 6f has ability to inhibit the P-glycopro-
Metabolic stability of the active derivative 6f.
tein I and II similar to the reference. The central nervous system (CNS)
parameter factor for 6f, and reference have showed good CNS penetra-
tion in the CNS (ꢀ 3.05, and ꢀ 3.21) respectively [56]. These data may
help that 6f present lower side effects in the (CNS) compared to 7
Cpds.
IC50
µM)
Mean CLint (
μL/
Mean t1/2
(min)
Notes
(
min/mg)
6
7
f
0.22 ±
.26
2.45 ±
.04
62.3
194
Low clearance
control
0
(
ꢀ 3.45), which offered a better tolerability among patients. Thus, the
*
105.1
578.3
48.2
15.8
High clearance
control
kidneys, requiring longer administration intervals to maintain desired
therapeutic concentrations, could not excrete 6f due to their high hy-
drophobicity, and resistance on metabolism rapidly.
0
Terfenadine
Propranolol
0.22
8.95
High clearance
control
0.22
143.80
Low clearance
control
*This compound was previously published [33].
respectively, although the compound 7 has high LogP value 4.37. The 6f
derivative has low percentage of Caco2 permeability (0.28%), while
compound 7 and the reference erlotinib have a high percentage of Caco2
permeability (0.60%, and 1.43%) respectively, may ligands 6f poor oral
8

S.K. Ihmaid et al.
Bioorganic Chemistry 111 (2021) 104835
Table 6
was longer than that of 6f (bond length: 3.3 Å), which made 6f more
potent than 4b (Fig. 8 B-C). Moreover, the pivotal hydrogen bond sta-
Predicted ADMET properties of selective target compounds.
–
bilization through the C O of phthalimide with conserved backbone
–
Parameter
6f
7*
Erlotinib
Met769 (bond length: 3.3 Å) makes this derivative more potent than
others. The amide linker NH contributes strongly to prominent accom-
modation of compound in phosphate cage. In addition, these derivatives
were deeply embedded into EGFR via hydrophobic contacts through
consistent double aromatic stabilization with Leu694 and Val702 resi-
dues in hinge region, (Fig. 8B-C) contribute to the role of substituted
phthalimide ring in activity. All behaviors of novel derivatives 4b and 6f
in binding pocket were mapped to reference erlotinib drug as aligned
structures in (Fig. 8A). Encouragingly, the results of binding mode
predictions for 4b and 6f were fully consistent with our 2D fitting as the
underlying structure-based inhibitor design strategy and showed the
role of phthalimide and amide linker in SAR of compounds. The novel
scaffold was introduced for the first time as EGFR inhibitor and mapped
with reference drugs could be used as lead compound for design of low
resistance anticancer agents. Our theoretical results were consistent
with the experimental data in vitro MTT assay.
Molecular Weight
LogP
462.26
3.46
5
509.64
4.37
7
393.44
3.40
10
Rotatable Bonds
Acceptors
6
10
7
Donors
1
1
1
Water solubility
ꢀ 4.42
0.28
82.55
ꢀ 2.93
Yes
ꢀ 3.28
0.60
98.58
ꢀ 2.73
Yes
ꢀ 4.73
1.43
94.58
ꢀ 2.74
Yes
Caco2 permeability
Intestinal absorption (human)
Skin Permeability
P-glycoprotein I inhibitor
P-glycoprotein II inhibitor
VDss (human)
Yes
Yes
Yes
ꢀ 0.04
0.08
¡3.05
No
0.13
0.08
¡3.45
No
0.19
0.05
¡3.21
No
Fraction unbound (human)
CNS permeability
CYP2D6 substrate
CYP1A2 inhibitor
CYP2D6 inhibitor
Total Clearance
No
No
Yes
No
No
No
0.066
No
0.145
No
0.702
No
Renal OCT2 substrate
AMES toxicity
No
No
No
Max. tolerated dose (human)
hERG I inhibitor
¡0.055
No
0.545
No
0.839
No
3
. Conclusion
Oral Rat Acute Toxicity (LD50
)
2.27
No
2.79
No
2.39
No
Many of pathological disorders like cancer invasion, metastasis, and
Skin Sensitization
angiogenesis, are linked to expressing a lot of EGFR enzymes [64].
Accordingly, EGFR groups are attractive targets for cancer therapy
development. In this study, two series of phthalimide analogues bearing
http://biosig.unimelb.edu.au/pkcsm/prediction.
*
This compound was previously published [33].
1
,2,3 triazole were designed, synthesized and evaluated for anticancer
2
.12. Molecular docking of active compounds 4b and 6f in EGFR TK
activity and screened as selective cytotoxic and EGFR inhibitors
compared to reference erlotinib drug with prominent metabolic stabil-
ity. The design was established by insertion of phthalimide active moiety
and modification of linker to the triazole scaffold to one carbon and
addition of terminal stable amide bond. The anti-proliferative activities
of the news compounds against MCF-7, HCT-116, and HepG2 cells were
evaluated. Most of the compounds exhibited higher antiproliferative
activities than a positive control drug. Moreover, most of the synthesized
derivatives had comparable of higher activity than reference inhibitor in
EGFR inhibitory testing. Compound 6f was the most potent EGFR in-
hibitor with IC50 values of 79 nM. Further mechanistic studies were done
that proved compound 6f promoted apoptosis of MCF-7 cells, induced
cell cycle arrest at the S/Pre-G1 phases in MCF-7 cell line with induction
of DNA fragmentation. The docking analysis of the active compound in
EGFR target revealed the prominent structural features contributions to
SAR of study. Metabolic stability study was done for compound 6f
showed long duration clearance of it and less susceptibility for meta-
bolism. Based on our results, it can be concluded that compound 6f may
serve as a lead compound for the discovery of new anticancer agents
targeting EGFR. Furthermore, we will continue to optimize this com-
pound and testing it against panel of resistant EGFR types to obtain
chemical entities with highly anticancer activity.
The docking work is beneficial experiment in computational chem-
istry for the interpretation of experimental biological data, which is
considered promising strategy in targeted novel drug discovery pro-
cesses [57–59]. Based upon the information obtained from the assay of
inhibitory activity of target compounds against EGFR TK, the placement
of the active compounds within ATP binding pocket was done for un-
derstanding their mode of interaction. MOE [60] software in combina-
tion to Autodock [61] tools were applied for docking calculations using
crystal structure of EGFR TK in complex with the erlotinib inhibitor
(
PDB entry 1 M17) extracted from the Protein Data Bank [62] as a
template. To understand better the potency order of 4b and 6f against
MCF-7 cells with over expressed EGFR, the two compounds was then
positioned into the binding pocket of the EGFR, and the binding mode of
the EGFR was depicted in (Fig. 8A-C). Compounds 4b and 6f were built
using MOE interface and all required protocols were done using Auto-
dock package before the running of target experiments for optimization
of process. For validation of the docking work, re-docking of crystallo-
graphic erlotinib and compounds 4b and 6f and the most populated
clusters of low energy conformations were selected for analysis. For
EGFR target, the applied docking protocol closely reproduced the
experimental binding modes of re-docked inhibitors. Previous studies
[
57,63] have indicated that 4-anilinoquinazolines such as erlotinib
4
. Experimental
cause inhibition through binding to the ATP site. The inhibitor was
located well in the ATP pocket and anchored by backbone NH of Met769
in the Hinge region through a hydrogen bond of 3.4 A0 length to N
quinazoline part, and affording more stability by hydrophobic in-
teractions with Leu694, Leu820, Lys721, and Thr766 (hinge region;
Fig. 7) with binding energy = ꢀ 20.4 Kcal/mol. The binding mode of 4b
and 6f and the EGFR was nearly the same as the reference erlotinib
4
.1. General information
All reagents and solvents used were of the highest quality of
analytical reagent grade and were used without further purification.
Melting points were measured on a Stuart Scientific SMP1 and are un-
corrected. TLC was performed on UV fluorescent Silica gel Merck 60
F254 plates, and the spots were visualized using a UV lamp (254 nm).
SHIMADZU FTIR-Affinity-1S spectrometer was used for identification of
(
Fig. 8 A). It was observed that the active compounds 4b (binding en-
ergy = ꢀ 21.5 Kcal/mol) and 6f (binding energy = ꢀ 23.65 Kcal/mol)
bind as the same manner to reference erlotinib drug and showed strong
H bonding with conserved Met769 residue (3.6, 3.3 A0 lengths for 4b
and 6f respectively through the conserved novel N3 triazole ring. This
proves that the importance of triazole fragment as isostere for quina-
zoline ring. The only difference was that the length of the key hydrogen
bond between 4b (bond length: 3.6 Å) and the residue Met769 of EGFR
ꢀ 1
functional groups in the range of 400–4000 cm . The NMR spectra
were run with a Bruker spectrometer (400 MHz) with TMS as internal
reference. Elemental analyses were performed using a GmbH-Vario EL
III Elementar Analyzer.
9

S.K. Ihmaid et al.
Bioorganic Chemistry 111 (2021) 104835
Fig. 8. 3D and 2D representation of molecular docking interactions of compound 4b and 6f in the ATP-binding pocket of EGFR tyrosine kinase (1 M17 PDB). Possible
hydrogen bonding interactions between compounds and EGFR are indicated by dashed lines.
4
.2. Synthesis and characterization of propargylated phthalimides 2a,b
A mixture of the appropriate phthalimide 1a,b (10 mmol) and po-
2.01; N, 5.53.
4
.3. Synthesis and characterization of phthalimide-based 1,2,3-triazoles
tassium carbonate (11 mmol) in DMF (20 mL) was stirred at room
temperature for 2 h. Thereafter, propargyl bromide (12 mmol) was
4
a-f and 6a-i
◦
added, and the mixture was stirred at 60 C for 4 h until the consumption
A solution of copper sulfate (0.10 g) and sodium ascorbate (0.15 g) in
of the starting material; as indicated by TLC. The reaction mixture was
poured into crushed ice and the resulting solid was collected by filtration
and recrystallized from ethanol to afford the targeted propargylated
phthalimides 2a,b.
water (10 mL) was added with stirring to a solution of the appropriate
propargylated phthalimide 2a,b (1 mmol) in t-BuOH (10 mL). There-
after, the appropriate azide 3a-d, 5a-e (1 mmol) was added, and the
reaction mixture was stirred at room temperature for 6–10 h. The re-
action was monitored via TLC (hexane–ethyl acetate), and after the
completion of the reaction, iced-water was added in the mixture. The
precipitate thus formed was collected by filtration, washed with satu-
rated solution of ammonium chloride and recrystallized from ethanol/
DMF to give the targeted 1,2,3-triazoles 4a-f and 6a-i
4
.2.1. Characterization of 2-(prop-2-yn-1-yl)isoindoline-1,3-dione (2a)
◦
◦
Yield 92%, mp: 150–151 C (Lit. mp: 151–153 C) [65].
4
.2.2. Characterization of 5,6-dichloro-2-(prop-2-yn-1-yl)isoindoline-1,3-
dione (2b)
Yield 90%, mp: 143–144 ◦C. IR: 1560 (C
–
–
–
–
C), 1700 (C
O), 2145
–
ꢀ 1
–
CH). 1H NMR (400
4.3.1. Characterization of 2-((1-(4-acetylphenyl)-1H-1,2,3-triazol-4-yl)
(
C
–
C), 2920 (al-CH), 3030 (ar-CH), 3290 cm
(
–
–
methyl)isoindoline-1,3-dione (4a)
MHz, DMSO‑d
6
): δ
H
3.28 (s, 1H,
–
CH), 4.37 (s, 2H, NCH ), 8.21 (s, 2H,
H). C NMR (100 MHz, DMSO‑d 27.57 (NCH ); 74.65, 78.29
): δ
C); 165.41 (N
: C, 52.00; H, 1.98; N, 5.51.Found: C, 52.03; H,
2
ꢀ
1
1
1
3
IR: 1545 (C
–
C), 1705 (C
–
H
O), 2940 (al-CH), 3070 cm (ar-CH). H
Ar
–
–
6
–
C
2
–
–
–
–
NMR (400 MHz, DMSO‑d ): δ 2.63 (s, 3H, CH ), 4.96 (s, 2H, NCH ),
.89–7.93 (m, 4H, Ar
(
C
C); 126.00, 131.79, 138.04 (Ar
–
C
–
O). Anal.
6
3
–
): δ
2
7
–
H), 8.06–8.13 (m, 4H, Ar
H), 8.95 (s, 1H, CH-
Calcd. for C11 Cl NO
H
5
2
2
1
,2,3-triazole). 13C NMR (100 MHz, DMSO‑d
6
C 3
27.29 (CH ); 33.43
1
0

S.K. Ihmaid et al.
Bioorganic Chemistry 111 (2021) 104835
(
NCH
39.95, 144.55 (Ar
Calcd. for C19
2
); 120.10, 121.95, 123.74, 130.50, 132.16, 135.04, 136.81,
DMSO‑d
(NCH CO); 125.33, 125.91, 131.97, 137.94, 142.37 (Ar
167.63 (N
4.49; N, 17.83.Found: C, 57.35; H, 4.51; N, 17.80.
6
): δ
C 3 2 2
14.41 (CH ); 33.31 (NCH ); 50.80 (OCH ); 61.93
–
3
C); 167.83 (N
–
C
–
O); 197.39 (COCH
). Anal.
–C); 166.03,
1
3
2
–
C
–
O, COOEt). Anal. Calcd. for C15
H
4
N O
4
: C, 57.32; H,
H
14
N
4
O
: C, 65.89; H, 4.07; N, 16.18. Found: C, 65.86; H,
14
4
.09; N, 16.20.
4
.3.2. Characterization of 2-((1-(4-acetylphenyl)-1H-1,2,3-triazol-4-yl)
4.3.8. Characterization of ethyl 2-(4-((5,6-dichloro-1,3-dioxoisoindolin-2-
methyl)-5,6-dichloroisoindoline-1,3-dione (4b)
yl)methyl)-1H-1,2,3-triazol-1-yl)acetate (6b)
–
–
H
ꢀ 1
1
–
–
ꢀ 1
1
IR: 1570 (C
–
C), 1710 (C
): δ
H), 8.16 (d, 2H, J = 8.0 Hz, Ar
–
O), 2970 (al-CH), 3080 cm (ar-CH). H
2.63 (s, 3H, CH ), 4.95 (s, 2H, NCH ),
H), 8.27 (s,
H), 8.94 (s, 1H, CH-1,2,3-triazole). C NMR (100 MHz,
): δ 27.32 (CH ); 29.49 (NCH ); 120.10, 121.87, 125.96,
30.56, 132.12, 137.91 (Ar C);166.06 (N
Anal. Calcd. for C19 12Cl : C, 54.96; H, 2.91; N, 13.49. Found: C,
4.97; H, 2.93; N, 13.46.
IR: 1570 (C
NMR (400 MHz, DMSO‑d
(q, 2H, OCH ), 4.86 (s, 2H, NCH
1,2,3-triazole), 8.22 (s, 2H, Ar
14.40 (CH ); 33.70 (NCH ); 50.81 (OCH
125.19, 132.03, 135.10, 142.67 (Ar–
–
C), 1720 (C
–
O), 2975 (al-CH), 3045 cm (ar-CH). H
1.20 (t, 3H, J = 8.0 Hz, CH ), 4.13–5.18
), 5.35 (s, 2H, NCH ), 8.11 (s, 1H, CH-
H). 13C NMR (100 MHz, DMSO‑d
): δ
); 61.95 (NCH CO); 123.75,
C); 167.66, 167.82 (N
NMR (400 MHz, DMSO‑d
6
–
3
2
6
): δ
H
3
8
2
.05 (d, 2H, J = 8.0 Hz, Ar
H, Ar
–
2
2
–
2
1
3
–
6
C
DMSO‑d
6
C
3
2
3
2
2
2
–
4
–
C
–
–
O); 183.45 (COCH
).
– –
–
C O,
1
3
H
2
N
O
3
2 4 4
COOEt). Anal. Calcd. for C15H12Cl N O : C, 47.02; H, 3.16; N, 14.62.
5
Found: C, 47.05; H, 3.14; N, 14.61.
4
1
.3.3. Characterization of 4-(4-((1,3-dioxoisoindolin-2-yl)methyl)-1H-
4.3.9. Characterization of 2-(4-((1,3-dioxoisoindolin-2-yl)methyl)-1H-
,2,3-triazol-1-yl)benzoic acid (4c)
1,2,3-triazol-1-yl)-N-(2-fluoro-4-iodophenyl)-acetamide (6c)
–
–
ꢀ 1
1
–
–
IR: 1580 (C
MHz, DMSO‑d
d, 2H, J = 8.0 Hz, Ar
CH-1,2,3-triazole), 12.56 (bs, 1H, COOH).
DMSO‑d 30.39 (NCH ); 120.99, 122.30, 123.75, 126.00, 132.16,
): δ
35.07, 141.21, 144.87, 147.17 (Ar
COOH). Anal. Calcd. for C18
Found: C, 62.10; H, 3.45; N, 16.10.
–
6
C), 1725 (C
): δ 4.97 (s, 2H, NCH
H), 8.44 (d, 2H, J = 8.0 Hz, Ar
–
C), 2660–2340 cm (OH). H NMR (400
), 7.89–7.94 (m, 4H, Ar H), 8.21
H), 9.02 (s, 1H,
C NMR (100 MHz,
IR: 1540 (C
–
C), 1710 (C O), 2950 (al-CH), 3060 (ar-CH), 3270
–
cm 1 (NH). H NMR (400 MHz, DMSO‑d
(s, 2H, NCH
Ar
H), 7.87–7.91 (m, 4H, Ar
(d, 2H, J = 8.0 Hz, Ar
H), 10.37 (s, 1H, NH). 13C NMR (100 MHz,
DMSO‑d 33.37 (NCH ); 52.47 (NCH CO); 123.70, 123.83, 124.83,
): δ
125.42, 125.50, 126.14, 132.04, 132.04, 133.94, 135.07, 135.22,
ꢀ
1
): δ
CO), 7.53 (d, 1H, J = 8.0 Hz, Ar
H), 8.12 (s, 1H, CH-1,2,3-triazole), 8.25
4.87 (s, 2H, NCH
–
2
), 5.38
H
2
6
H
(
–
–
2
–
H), 7.70–7.75 (m, 2H,
1
3
–
–
–
6
C
2
–
12 4 4
C); 165.15, 167.81 (N
–
C
–
O,
1
6
C
2
2
H N O : C, 62.07; H, 3.47; N, 16.09.
–
5 3
C); 165.34, 167.81 (N
–
–
C O, CONH). Anal. Calcd. for
–
154.72 (Ar
13FIN
13.84.
C
19
H
O : C, 45.17; H, 2.59; N, 13.86. Found: C, 45.20; H, 2.63; N,
4
.3.4. Characterization of 4-(4-((5,6-dichloro-1,3-dioxoisoindolin-2-yl)
methyl)-1H-1,2,3-triazol-1-yl)benzoic acid (4d)
–
–
ꢀ 1
1
IR: 1555 (C
–
C), 1720 (C
): δ 4.94 (s, 2H, NCH
H), 8.26 (s, 2H, Ar
–
O), 2690–3300 cm (OH). H NMR (400
), 8.00–8.02 (m, 2H, Ar H),
H), 8.11 (s, 1H, CH-1,2,3-
triazole), 11.37 (s, 1H, COOH). C NMR (100 MHz, DMSO‑d 33.80
): δ
C); 166.05
: C, 51.82;
4.3.10. Characterization of 2-(4-((5,6-dichloro-1,3-dioxoisoindolin-2-yl)
MHz, DMSO‑d
6
H
2
–
methyl)-1H-1,2,3-triazol-1-yl)-N-(2-fluoro-4-iodophenyl)acetamide (6d)
–
–
IR: 1570 (C
–
C), 1700 (C
cm (NH). H NMR (400 MHz, DMSO‑d
(s, 2H, NCH
CO), 7.51–7.54 (m, 1H, Ar
8.12 (s, 1H, CH-1,2,3-triazole), 8.24 (s, 2H, Ar H), 10.39 (s, 1H, NH).
–
O), 2940 (al-CH), 3035 (ar-CH), 3300
): δ 4.86 (s, 2H, NCH ), 5.38
H), 7.70–7.74 (m, 2H, Ar H),
8
.08–8.10 (m, 2H, Ar
1
3
ꢀ 1
1
6
C
6
–
H
2
(
(
NCH
2
); 122.04, 125.95, 132.13, 137.90, 144.2 (Ar
–
–
N C O); 178.45 (COOH). Anal. Calcd. for C18H10Cl N O
–
2 4 4
–
2
–
–
1
3
H, 2.42; N, 13.43. Found: C, 51.85; H, 2.40; N, 13.46.
C NMR (100 MHz, DMSO‑d
6
): δ
C 2 2
33.76 (NCH ); 52.46 (NCH CO);
1
20.26, 124.54, 125.51, 125.93, 127.44, 132.01, 133.83, 133.97,
–
C); 165.34, 166.06 (N
–
C
–
O,
= ꢀ 122.50 to ꢀ 122.45 (m,
: C, 39.75; H, 1.93; N,
4
1
.3.5. Characterization of ethyl 4-(4-((1,3-dioxoisoindolin-2-yl)methyl)-
137.93, 142.22, 160.40, 161.32 (Ar
H-1,2,3-triazol-1-yl)benzoate (4e)
CONH). 19F NMR (377 MHz, DMSO‑d
6
): δ
2
F
–
–
H
ꢀ 1
1
1F, Ar–F). Anal. Calcd. for C19
IR: 1565 (C
–
C), 1720 (C
): δ
), 4.96 (s, 2H, NCH
–
O), 2940 (al-CH), 3070 cm (ar-CH). H
1.34 (t, 3H, J = 8.0 Hz, CH ), 4.34–4.37
), 7.88–7.94 (m, 4H, Ar H), 8.05–8.14
H), 8.94 (s, 1H, CH-1,2,3-triazole). C NMR (100 MHz,
14.80 (CH ); 33.42 (NCH ); 61.55 (OCH ); 120.23,
H
11Cl
FIN
5
O
3
NMR (400 MHz, DMSO‑d
6
3
12.20. Found: C, 39.72; H, 1.91; N, 12.21.
(
(
q, 2H, OCH
2
–
2
–
1
3
m, 4H, Ar
4.3.11. Characterization of 2-(4-((1,3-Dioxoisoindolin-2-yl)methyl)-1H-
DMSO‑d
6
): δ
C
3
2
2
1,2,3-triazol-1-yl)-N-(3-fluoro-4-methylphenyl)-acetamide (6e)
–
–
–
1
21.96, 123.73, 130.07, 131.36, 132.17, 135.03, 140.11, 144.59
IR: 1560 (C
–
C), 1710 (C
cm (NH). H NMR (400 MHz, DMSO‑d
2H, NCH ), 5.31 (2H, s, NCH CO), 7.24 (bs, 2H, Ar
Ar H), 8.15 (s, 1H, CH-1,2,3-triazole),
H), 7.89–7.94 (m, 4H, Ar
10.60 (s, 1H, NH). C NMR (100 MHz, DMSO‑d 14.05 (CH ); 33.18
): δ
(NCH ); 52.60 (NCH CO); 118.02, 123.74, 123.84, 125.35, 127.26,
127.78, 132.05, 132.09, 132.14, 134.88, 135.12, 142.64, 160.23,
O), 2950 (al-CH), 3015 (ar-CH), 3290
): δ 2.20 (s, 3H, CH ), 4.90 (s,
H), 7.51 (bs, 1H,
–
16
C); 165.28, 167.83 (N
–
C
–
O, COOEt). Anal. Calcd. for
ꢀ 1
1
(
Ar
6
H
3
C
20
H
4
N O
4
: C, 63.83; H, 4.29; N, 14. Found: C, 63.89; H, 4.32; N, 14.8.
2
2
–
–
–
1
3
4
.3.6. Characterization of 2-((1-(3-fluoro-4-methylphenyl)-1H-1,2,3-
6
C
3
triazol-4-yl)methyl)-isoindoline-1,3-dione (4f)
2
2
–
–
C), 2930 (al-CH), 3080 cm (ar-CH). 1H NMR (400
2.28 (s, 3H, CH ), 4.93 (s, 2H, NCH ), 7.48 (t, 1H, J
H), 7.75 (d, 1H, J = 12.0
ꢀ 1
IR: 1560 (C
MHz, DMSO‑d
): δ
8.0 Hz, Ar
H), 7.66 (d, 1H, J = 8.0 Hz, Ar
H), 7.88–7.92 (m, 4H, Ar H), 8.80 (s, 1H, CH-1,2,3-triazole).
C NMR (100 MHz, DMSO‑d 14.29 (CH ); 33.43 (NCH ); 107.66,
): δ
15.96, 121.80, 123.72, 125.30, 132.15, 133.16, 135.05, 136.05,
–
19
163.48 (Ar
DMSO‑d
): δ
16FN
17.78.
–
C); 165.18, 167.81 (N
= ꢀ 115.78 to ꢀ 115.72 (m, 1F, Ar
5 3
O : C, 61.07; H, 4.10; N, 17.80. Found: C, 61.10; H, 4.12; N,
–
C
O, CONH). F NMR (377 MHz,
6
H
3
2
=
–
–
6
F
–
F). Anal. Calcd. for
Hz, Ar
–
–
C
20
H
1
3
6
C
3
2
1
1
–
C); 167.81 (N
–
C
–
O). Anal. Calcd. for
4.3.12. Characterization of 2-(4-((5,6-dichloro-1,3-dioxoisoindolin-2-yl)
44.25, 162.27 (Ar
13FN : C, 64.28; H, 3.90; N, 16.66. Found: C, 64.26; H, 3.92; N,
6.64.
C
18
H
4
O
2
methyl)-1H-1,2,3-triazol-1-yl)-N-(3-fluoro-4-methylphenyl)acetamide (6f)
–
–
–
1
IR: 1560 (C
–
C), 1705 (C
cm 1 (NH). H NMR (400 MHz, DMSO‑d
2H, NCH ), 5.28 (2H, s, NCH
CO), 7.18–7.22 (2H, m, Ar
(m, 1H, Ar H), 8.12 (s, 1H, CH-1,2,3-triazole), 8.23 (s, 2H, Ar
10.58 (s, 1H, NH). C NMR (100 MHz, DMSO‑d
(NCH ); 52.61 (NCH CO); 106.24, 106.51, 115.24, 119.49, 119.66,
125.48, 125.92, 132.00, 137.93, 142.21, 159.48, 161.84, 164.74
O), 2920 (al-CH), 3060 (ar-CH), 3330
): δ 2.16 (s, 3H, CH ), 4.86 (s,
H), 7.45–7.48
H),
); 33.80
ꢀ
1
6
H
3
4
1
.3.7. Characterization of ethyl 2-(4-((1,3-dioxoisoindolin-2-yl)methyl)-
2
2
–
H-1,2,3-triazol-1-yl)acetate (6a)
–
–
–
–
–
ꢀ 1
1
13
IR: 1550 (C
NMR (400 MHz, DMSO‑d
q, 2H, OCH ), 4.87 (s, 2H, NCH
H, Ar H), 8.11 (s, 1H, CH-1,2,3-triazole). C NMR (100 MHz,
C), 1730 (C
–
O), 2920 (al-CH), 3060 cm (ar-CH). H
1.21 (t, 3H, J = 8.0 Hz, CH ), 4.14–4.19
), 5.35 (s, 2H, NCH
CO), 7.87–7.92 (m,
6
): δ
C 3
14.14 (CH
6
): δ
H
3
2
2
(
2
2
2
1
3
–
(Ar C); 165.05, 166.06 (N C O, CONH). F NMR (377 MHz,
– –
–
19
4
–
1
1

S.K. Ihmaid et al.
Bioorganic Chemistry 111 (2021) 104835
DMSO‑d
14 Cl
N, 15.13.
6
): δ
2
F
= ꢀ 115.85 to ꢀ 115.80 (m, 1F, Ar
–
F). Anal. Calcd. for
cytometry analysis as described previously [33,70].
.8. DNA fragmentation
Quantitative analysis of DNA fragmentation was assessed using
C
20
H
FN
5 3
O : C, 51.97; H, 3.05; N, 15.15. Found: C, 52.01; H, 3.08;
4
4
1
.3.13. Characterization of 2-(4-((1,3-dioxoisoindolin-2-yl)methyl)-1H-
,2,3-triazol-1-yl)-N-(2,4,5-trifluorophenyl)-acetamide (6g)
diphenylamine (DPA) reagent according to the method of Boraschi and
Maurizi [71-73].
–
–
–
–
IR: 1555 (C
C), 1700 (C
cm (NH). H NMR (400 MHz, DMSO‑d
s, 2H, NCH
O), 2980 (al-CH), 3045 (ar-CH), 3340
): δ 4.87 (s, 2H, NCH ), 5.39
H), 7.87–7.91 (m, 4H, Ar H),
H), 8.12 (s, 1H, CH-1,2,3-triazole), 10.50 (s, 1H,
NMR (100 MHz, DMSO‑d ): 30.36 (NCH ); 52.35
2
CO); 106.74, 112.13, 122.78, 123.71, 125.41, 132.05, 137.93,
ꢀ
1
1
6
H
2
(
2
CO), 7.65–7.72 (m, 1H, Ar
–
–
4
.9. ROS assay
7
.98–8.05 (m, 1H, Ar
–
13
NH).
C
6
δ
C
2
MCF-7 cells were plated in 6-cm dishes. After 24 h of growth, the
(
NCH
46.97 (Ar
DMSO‑d
): δ
139.23 (m, 1F, Ar
12 3 5 3
Calcd. for C19H F N O
cells were treated with the compounds at the indicated concentrations
for 24 h. Cells were detashed using trypsin washed with PBS and
centrifuged. The pellet was resuspended in PBS then subjected to
ultrasonication three times. Samples and standards were added in 100 µl
per well of a 96 well ELISA (enzyme-linked immunosorbent assay) plate
–
C); 165.59, 167.83 (N
= ꢀ 141.70 to ꢀ 141.55 (m, 1F, Ar
F), ꢀ 125.96 to ꢀ 125.86 (m, 1F, Ar
requires C, 54.95; H, 2.91; N, 16.86. Found: C,
4.97; H, 2.94; N, 16.87.
–
C
–
O, CONH). F NMR (377 MHz,
F), ꢀ 139.34 to
F). Anal.
19
1
–
6
F
ꢀ
–
–
5
(
Amsbio, UK). PBS was added in the same volume to the control blank
wells. 50 µl of ROS-HRP conjugate is added to the deignated wells,
4
.3.14. Characterization of 2-(4-((5,6-dichloro-1,3-dioxoisoindolin-2-yl)
◦
incubated for and hour at 37 C before carryin out a washing step and
methyl)-1H-1,2,3-triazol-1-yl)-N-(2,4,5-trifluorophenyl)acetamide (6h)
then adding substrates (50 µl each) to all wells. After an incubation
–
–
IR: 1550 (C
–
C), 1690 (C
cm (NH). H NMR (400 MHz, DMSO‑d
s, 2H, NCH
CO), 7.65–7.73 (m, 1H, Ar
.12 (s, 1H, CH-1,2,3-triazole), 8.23 (s, 2H, Ar
–
O), 2950 (al-CH), 3055 (ar-CH), 3280
): δ 4.86 (s, 2H, NCH ), 5.39
H), 7.97–8.04 (m, 2H, Ar H),
H), 10.52 (s, 1H, NH).
33.75 (NCH ); 52.35 (NCH CO);
06.51, 112.06, 125.53, 125.93, 132.00, 137.93, 142.26
◦
period 15 min at 37 C the stop solution is added and the plate is read
ꢀ
1
1
6
–
H
2
using a microplate reader at 450 nm. Concentrations of ROS were
calculated from the standard curve plot of optical density against
concentration.
(
2
–
8
–
1
3
C NMR (100 MHz, DMSO‑d
6
): δ
C
2
2
1
–
C);165.58, 166.06 (N
): δ = ꢀ 141.66 to ꢀ 141.51 (m, 1F, Ar
F), ꢀ 125.96 to ꢀ 125.87 (m, 1F, Ar
: C, 47.13; H, 2.08; N, 14.46. Found: C,
–
C
–
–
O, CONH). 19F NMR (377 MHz,
F), ꢀ 139.32 to
F). Anal.
4
.10. Western blot assay
(
Ar
DMSO‑d
139.20 (m, 1F, Ar
Calcd. for C19 10Cl
7.12; H, 2.11; N, 14.42.
–
6
F
MCF-7 cells were plated in 6-cm dishes. After 24 h of growth, the
ꢀ
–
–
cells were treated with the compounds at the indicated concentrations
for 24 h. Cell lysates were collected using ice cold lysis buffer. Western
H
2 3 5 3
F N O
4
blot analyses were then conducted. Breifly, 50 g of total protein from
μ
each cornea homogenate was denatured by boiling for 5 min in 2% SDS
and 5% B-mercaptoethanol and loaded into separate lanes of the 12%
gel SDS–PAGE then electro-transferred to a nylon membrane using T-77
ECL semi-dry transfer unit, for 2 h. The membrane was blocked in TBS
buffer containing 0.05% Tween and 5% non-fat milk for one hour fol-
lowed by the incubation with the indicated antibodies, followed by a
peroxidase-conjugated rabbit secondary antibody. Rabbit monoclonal
anti-EGFR, rabbit monoclonal antibody specific EGFR and beta actin
4
1
.3.15. Characterization of 2-(4-((1,3-dioxoisoindolin-2-yl)methyl)-1H-
,2,3-triazol-1-yl)-N-(4-nitrophenyl)acetamide (6i)
–
–
–
IR: 1540 (C
–
C), 1705 (C
cm (NH). H NMR (400 MHz, DMSO‑d
s, 2H, NCH
CO), 7.65–7.72 (m, 1H, Ar
.87–7.92 (m, 4H, Ar H), 8.12 (s, 1H, CH-1,2,3-triazole), 8.25 (d, 2H, J
8.0 Hz, Ar H), 11.05 (s, 1H, NH). C NMR (100 MHz, DMSO‑d
0.37 (NCH ); 52.76 (NCH CO); 119.50, 123.71, 123.83, 125.42,
25.57, 132.05, 135.08, 135.22, 143.06, 144.94 (Ar C); 165.75,
: C, 56.16; H,
O), 2960 (al-CH), 3020 (ar-CH), 3310
): δ 4.89 (s, 2H, NCH ), 5.35
H), 7.80–7.83 (m, 2H, Ar H),
ꢀ
1
1
6
–
H
2
(
2
–
7
–
1
3
=
–
6
): δ
C
3
1
1
3
2
2
(
β-actin) rabbit polyclonal antibodies were obtained from Abcam (USA).
–
Semiquantitiative analysis was performed using densimetry, where
denstites of the bands were measured and plotted in a histogram.
–
14 6 5
67.82 (N C O, CONH). Anal. Calcd. for C19H N O
–
–
.47; N, 20.68. Found: C, 56.14; H, 3.43; N, 20.70.
4
.11. Meatbolic stability analysis
4
.4. In vitro cytotoxic screening
The metabolic stability of target compounds 6f and 7 were analyzed
The in vitro cytotoxic screening of target compounds was performed
as described in a previous study [74].
against different three cancer cell lines HepG2, MCF-7 and HCT116
using standard MTT assay as previously reported [33,66,67].
4
.12. Molecular docking
4
.5. EGFR inhibition assay
Molecular docking simulation was carried out using the program
AutoDock 4.0.1.34 (version
.0, Molecular graphics laboratories, La Jolla, CA, USA) with the
EGFR kinase activity of the target compounds was measured by HT
4
Scan EGFR kinase assay kits (Cell Signaling Technology, Danvers, MA,
USA) as reported [33].
graphical user interface AutoDock tools (ADT) [61] and Molecular
Operating Environment (MOE 2012) [60]. The active compounds were
docked into (3D) complex of the two biological targets: crystal EGFR
4
.6. Apoptosis quantification
(
PDB code: 1 M17) complexes with erlotinib at 2.6 Å resolution. The
methods of molecular and protein preparations with the placement
procedure are described prevously [33,57,59].
Annexin V-FITC apoptosis assay was done for the selected com-
pounds by using Annexin V-FITC/PI double staining detection kit (BD
Pharmingen, USA) as reported [68,69].
Declaration of Competing Interest
4
.7. Cell cycle analysis
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Cell cycle arrest and distribution was assayed, using the Propidium
Iodide Flow Cytometry Kit (ab139418, Abcam) followed by flow
1
2

S.K. Ihmaid et al.
Bioorganic Chemistry 111 (2021) 104835
Acknowledgment
[25] F.F. Al-Blewi, M.A. Almehmadi, M.R. Aouad, S.K. Bardaweel, P.K. Sahu, M.
Messali, N. Rezki, E.S.H. El Ashry, Design, synthesis, ADME prediction and
pharmacological evaluation of novel benzimidazole-1,2,3-triazole-sulfonamide
hybrids as antimicrobial and antiproliferative agents, Chem. Cent. J .2018, 12,
110–110.
We greatly thank Taif University for providing fund for this work
through Taif University Researchers Supporting Project number
[
26] M.R. Aouad, Click synthesis and antimicrobial screening of novel isatin-1,2,3-
triazoles with piperidine, morpholine, or piperazine moieties, Org. Prep. Proced.
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evaluation of new fluorinated 1,2,3-triazoles and their conversion into Schiff bases
Appendix A. Supplementary material
%
J, J. Braz. Chem. Soc. 26 (2015) 2105–2115.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104835.
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