Design and synthesis of new benzoxazole/benzothiazole-phthalimide hybrids as antitumor-apoptotic agents

Article abstract of DOI:10.1016/j.bioorg.2019.102978

Herein, we synthesized a series of twelve benzoxazole and benzothiazole derivatives incorporated with phthalimide core as anticancer agents. The most active compounds were 5a and 5g against HepG2 and MCF7 cell lines with IC₅₀ = 0.011 and 0.006 μM, respectively. They evaluated against EGFR and HER2 enzymes. From cell cycle analysis, it was observed that test compounds exerted pre G1 apoptosis and cell cycle arrest at G2/M phase. The achieved results suggested that apoptosis was due to activation of caspase-7 and caspase-9. EGFR was chosen as a biological target for carrying molecular modeling study for the newly synthesized compounds.

Full text of DOI:10.1016/j.bioorg.2019.102978

Accepted Manuscript
Design and synthesis of new benzoxazole/benzothiazole-phthalimide hybrids as
antitumor-apoptotic agents
John N. Philoppes, Phoebe F. Lamie
PII:
S0045-2068(18)31563-3
https://doi.org/10.1016/j.bioorg.2019.102978
102978
DOI:
Article Number:
Reference:
YBIOO 102978
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 December 2018
5 May 2019
7 May 2019
Please cite this article as: J.N. Philoppes, P.F. Lamie, Design and synthesis of new benzoxazole/benzothiazole-
phthalimide hybrids as antitumor-apoptotic agents, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/
j.bioorg.2019.102978
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Design and synthesis of new benzoxazole/benzothiazole-phthalimide
hybrids as antitumor-apoptotic agents
John N. Philoppesa,*, Phoebe F. Lamiea,*
aDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Beni-Suef
University, Beni-Suef 62514, Egypt
*Author to whom correspondence should be addressed; E-Mail: john_philoppes@yahoo.com
(J.N.P.); E-Mail: feby.farag@yahoo.com or feebi.lamey@pharm.bsu.edu.eg (P. F. L.) Tel.:
+20-82-266-76; Fax: +20-82-231-7958.
Abstract
Herein, we synthesized a series of twelve benzoxazole and benzothiazole derivatives
incorporated with phthalimide core as anticancer agents. The most active compounds were 5a
and 5g against HepG2 and MCF7 cell lines with IC50 = 0.011 and 0.006 μM, respectively.
They evaluated against EGFR and HER2 enzymes. From cell cycle analysis, it was observed
that test compounds exerted pre G1 apoptosis and cell cycle arrest at G2/M phase. The
achieved results suggested that apoptosis was due to activation of caspase-7 and caspase-9.
EGFR was chosen as a biological target for carrying molecular modeling study for the newly
synthesized compounds.
Keywords: Benzothiazole; Benzoxazole; Phthalimide; Antitumor; Synthesis
1

1. Introduction
Cancer is the second disease leading to death worldwide [1-3]. Hepatocellular carcinoma is
one form of cancer originates in liver cells and leads to high mortality rates in the world
today [4].
Due to cytotoxicity of most anticancer agents, researchers seek every day to develop more
effective, selective and safe chemotherapeutic agents [2].
By searching the literature, we found that azo compounds such as benzoxazole and
benzothiazole derivatives had different types of biological activities especially antitumor
activity [5-10].
Moreover, a number of benzothiazole derivatives displayed good anticancer activity and
apoptotic effect on HepG2 cells both in-vitro and in-vivo [11, 12].
3’-Substituted 2-(4-aminophenyl)benzothiazoles represent an important class of antitumor
agents by exhibiting inhibitory activity against certain cancer cell lines (e.g., breast MCF7,
MDA 468; renal TK 10; overian IGROV)[13, 14].
Structure-activity relationships for these compounds showed that substitution at 3’-position of
phenyl ring with either alkyl (CH3) or halogen (I, Br, Cl) groups increased the potency and
in-vitro activity against tumor cell lines, (IC50<0.0001μM) with bromo- and iodo- analogues
against MDA468 cell line [14, 15].
3’-Substituted 2-(4-aminophenyl)benzothiazoles are characterized by a unique biphasic dose-
response relationship in sensitive cell lines. Low nanomolar concentration of test compounds
for cell death, followed by low micromolar concentration for proliferative response in second
growth phase (SGP). This was attributed to inactivation of a metabolite to the bioactive
enzyme cytochrome P450 (CYP)1A1 [16-17].
On the other hand, imide derivatives have broad spectrum of pharmacological activities such
as anti-inflammatory, antiviral, antibacterial and antitumor [18].
As it is known, angiogenesis, the process by which new capillary blood vessels are formed,
plays an important role in growth and maintenance of solid tumors. Therefore, angiogenesis
has become an attractive area for approaching new therapeutic agents [19]. Thalidomide
2

(NKTA) – a phthalimide derivative- was used as a sedative and hypnotic drug in 1960, but it
was withdrawn from clinics due to its teratogenic disorders [20, 21]. In earlier 1990,
thalidomide was proved to be an anti-angeogenic agent and effective in treatment of different
diseases such as inflammation, immunodeficiency syndrome (AIDS) and cancer. It acts by
inhibiting the production of tumor necrosis factor alpha (TNF-α), interleukin-1b (IL-1b), IL-
2, IL-4, IL-5, IL-6, IL-10 and interferon-ɤ (IFN-ɤ) [20-26].
It has been showed that variation in thalidomide structure could eliminate its side effects like
teratogenicity, while still maintaining the promising anticancer activity. As a result, many
thalidomide analogs have been developed such as lenalidomide (Revlimid™) and
pomalidomide [27-29]. Merging both benzothiazole and phthalimide rings together in one
compound as in canthridin containing benzothiazole possessed antitumor properties in
hepatocellular carcinoma, breast cancer and non-small cell lung carcinoma [19, 25, 30].
Keeping in mind the molecular pharmacophores outlined above and in continuation to our
work in the field of designing and synthesis of anticancer agents [31-35],molecular
hypridization between phthalimide ring and benzothiazole or its isoster benzoxazole nucleus
has been designed and synthesized (figure 1). Their antitumor activity was evaluated against
HepG2 and MCF7 cells. The most active derivatives were subjected to kinase enzyme
activity determination on both EGFR and HER2 enzymes. Cell cycle analysis and apoptotic
inducers activity were also parts of this study. Molecular docking study was performed for all
the target compounds inside EGFR active site.
[Please, insert Figure 1 about here]
2. Results and discussion
2.1. Chemistry
The starting materials 1a&b, 2a&b, intermediates 3a-i, 4a&b, and final target compounds
5a-l were synthesized as shown in Scheme 1.
Cyclization of o-aminophenol 1a or o-aminothiophenol1b with 4-aminobenzoic acid
derivatives namely, 4-aminobenzoic acid,4-amino-3-methylbenzoic acid, 4-amino-3-
cyanobenzoic acid and 4-amino-3-chlorobenzoic acid in polyphosphoric acid at a high
temperature 180-220oC resulted in the formation of 2-phenylbenzoxazole and 2-phenyl
benzothiazole derivatives 3a-i according to precautions of reported method [14, 33].
3

Condensation of compounds 3a-i with phthalic anhydride 4a or phthalic anhydride-5-
carboxylic acid derivative 4b in glacial acetic acid afforded the target compounds 5a-l.
IR spectra of 5a-l showed the presence of two peaks for the two carbonyl groups of
phthalimide part at the range 1786-1774 and 1734-1703 cm-1. An additional C=O peak
(COOH) in compounds5d-f and 5j-l appearedat 1661-1687 cm-1. Moreover, a dragged peak
characterized for COOH group was observed in the later compounds at 3053-2543 cm-1.
Also, appearance of a peak due to CN group in 5b and 5h was observed at 2358 and
2234 cm-1, respectively.
1
Moreover, HNMR spectra of 5a and 5g revealed the presence of a singlet signal at δ 2.24-
2.28 ppm attributed to CH3 protons, in addition to eleven aromatic protons in the range of
aromatic region at δ 7.37-8.45 ppm.
A D2O exchangeable signal appeared in compounds 5d-f and 5j-l as a singlet signal due to
COOH proton at δ 13.75 to 13.84 ppm.
[Please, insert Scheme 1 about here]
2.2. Biological evaluation
2.2.1. In-vitro antiproliferative activity
Twelve newly synthesized benzoxazole/benzothiazole phthalimide hybrids were tested
through determination of their IC50 values (half maximal inhibitory concentration) against
two human cancer cell lines, liver HepG2, breast MCF7 and non-cancerous fibroblast cells
(WI-38). The reference drugs used in this study were 5-flurouracil (5-FU) and erlotinib
against HepG2and MCF7, respectively.
The obtained in-vitro results were summarized in Table 1. Deep insight to the tabulated
results showed that no cytotoxic activity was observed for targeted compounds on non-
cancerous fibroblast cells (WI-38), (IC50 = 28-82-122.31 µM). All the test compounds
exhibited potent anticancer activity against HepG2and MCF7 in nanomolar to micromolar
range with IC50 values between 0.011-0.036 μM and 0.006-0.468 μM, sequentially, except
oxazole derivatives5c, 5d, 5f and thiazole derivative 5h had IC50 values 0.132-0.724 μM
against HepG2 if compared to 5-FU (HepG2 reference) IC50 = 0.034 μM.
4

Both benzoxazole and benzothiazole derivatives5a, 5b and 5k exhibited 1.7- to 3-folds more
potent anticancer activity against HepG2 cell line than 5-FU, the reference drug. Moreover,
compounds 5c and 5g were the most potent derivatives between the test compounds. They
showed IC50 values 9 and 6 nM with 79- and 118-folds more potent anticancer activity than
erlotinib against MCF7 cell line.
The structure activity relationship revealed that the antiproliferative activity of the test
compounds was in correlation with aryl and two heteroaryl rings of the hybride structure.
The presence of phthalimide ring in the structure of the test compounds resulted in the
formation of a hydrogen bond between its C=O group and SH group of cysteine back bone
amino acid of caspase-7 enzyme leading to induction of apoptosis [36].
Further analysis of the test compounds revealed that the presence of CN group in a hybrid
structure of benzoxazole and phthalimide core 5b led to increase activity against HepG2 cell
line (IC50 = 0.018 μM). Compounds bearing CH3 group, benzoxazole and phthalimide5a or
CH3 group, benzothiazole and carboxy substituted phthalimide 5k showed higher anticancer
activity against HepG2 (IC50 = 0.011 and 0.019 μM, respectively) if compared to 5-FU (IC50
= 0.034 μM).
While, CH3 group, benzoxazole and carboxy substituted phthalimide 5e and CH3 group,
benzothiazole and unsubstituted phthalimide5g resulted in slight decrease in anticancer
activity against HepG2 (IC50 = 0.056 and 0.098 μM, sequentially) comparing to 5-FU.
Introduction of other groups such as Cl in benzoxazole analogues 5c, 5f and CN in 5h
showed marked decrease in activity (IC50 = 0.132-0.724 μM). Also, the presence of
benzoxazole and phenyl ring merged with carboxy substituted phthalimide5d-5fexhibited
lower anticancer activity against HepG2 than benzothiazole analogues 5j-5l.
On the other hand, unsubstituted phthalimide core gave better anticancer activity against
MCF7 than phthalimide substituted with COOH group at 4-position.
Introducing Cl group 5c (IC50 = 0.009 μM) or CH3 group 5g (IC50 = 0.006 μM) at phenyl ring
with unsubstituted phthalimide core showed the highest anticancer activity against MCF7 cell
line.
5

The presence of CN group at phenyl ring 5b (IC50 = 0.211 μM) and 5h (IC50 = 0.468 μM)
resulted in slight decrease in antiproliferative activity between all the test compounds but still
more active than the reference erlotinib (IC50 = 0.71 μM).
Finally, we could conclude that benzoxazole and benzothiazole derivatives exerted a positive
effect on the anticancer activity against MCF7 cell line. While, benzothiazole derivatives
were more active than benzoxazole analogues against HepG2. The higher activity of most of
the test compounds was observed in those bearing unsubstituted phthalimide core than
carboxy substituted phthalimide ones.
The order of anticancer activity for the substituted groups at phenyl ring was, CH3> CN ≈ H
> Cl against HepG2 cell line, while, CH3> Cl > H > CN against MCF7 cell line.
[Please, insert Table 1 about here]
2.2.2. In-vitro enzyme inhibition assay
EGFR IC50 for the most potent compounds 5a and 5g and the reference drug erlotinib was
determined to be 0.23 and 0.35 μM relative to the reference erlotinib IC50 = 0.15 μM, (Table
2). One further enzyme that is overexpressed in breast cancer, HER2 was used to help in
exploring the mechanism of action of 5a and 5g. It was found that IC50 values of 5a and 5g on
HER2 were determined to be 0.45 and 0.33 μM, sequentially, (Table 2). From the tabulated
results, it was observed that the most active antiproliferative compounds against HepG2 and
MCF7 cell lines, 5a and 5g, demonstrated moderate effects against both kinases (EGFR and
HER2) at 10 μM. The antiproliferative activity of the synthesized compounds might be due to
apoptosis rather than tyrosin kinase inhibitory activity. Finally, we concluded that the mode
of action for the synthesized compounds might be on those enzymes to some extent and
further future studies should be considered to discover other enzymes participate in
increasing the anticancer activity of the synthesized derivatives.
[Please, insert Table 2 about here]
2.2.3. Caspase activation assay
Caspase-7 and caspase-9 are among of caspases, a family of cysteine-aspartic protease
enzymes which play important role in programmed cell death and can be used as indicators
for induction of apoptosis by anticancer agents. Caspase-9 is an initiator caspase which initate
6

the apoptosis signal while caspase-7 is one of executioner caspases which carry out the mass
proteolysis that leads to apoptosis [37].
In this study, human liver HepG2 and breast MCF7 cell lines were treated with compounds
5a (IC50 = 0.011μM) and 5g (IC50 = 0.006μM), respectively, and protein expression levels of
both caspase-7 and caspase-9 were determined in the treated cell lines. The obtained results
showed significant over-expression in caspase-7 and caspase-9 protein level in treated
samples (Table 3).
[Please, insert Table 3 about here]
Thus, from above results, it was found that caspase-7 concentration for 5a/HepG2 and
5g/MCF7 was 1.449 ng/ml and 1.841 ng/ml, respectively. Moreover, concentration of
caspase-9 for 5a/HepG2and 5g/MCF7 was 22.72 ng/ml and 27.56 ng/ml, sequentially
(Figure 2).
These achieved results suggested that apoptosis was due to activation of caspase-7 and
caspase-9.
[Please, insert Figure 2 about here]
2.2.4. Cell cycle analysis and apoptosis detection
Both HepG2 and MCF7 cell lines after treatment with the most potent compounds 5a (IC50 =
0.011 μM) and 5g (IC50 = 0.006 μM), sequentially was subjected to cell cycle analysis.
In both cell lines, HepG2 and MCF7 the percentages of cells in G0/G1 phase of the cell cycle
were 55.37% and 48.92% and decreased to be 31.22% and 33.54% in G2/M phase for 5a and
5g, respectively, (Figures3 & 4, Table 4).
Additionally, the percentage of apoptotic cells was increased from 1.65% in control untreated
HepG2 to 21.25% in cells treated with 0.011μM of compound 5a (12-fold) and from 1.92%
for control untreated MCF7 cells to be 18.26% when cells were treated with 0.006 μM of
compound 5g (9.5-fold), (Figure 5).
From the above obtained results, the test compounds exerted pre G1 apoptosis and cell cycle
arrest at G2/M phase.
[Please insert Figures 3, 4, 5 and Table 4 about here]
7

2.2.5. Evaluation of Bax and Bcl-2 expressions
Pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 (B-cell lymphoma 2) expression
levels were measured in both HepG2 cells treated with compound 5a and MCF7 cells treated
with compound 5g. The obtained results showed up regulation in the apoptotic protein, Bax.
On the other hand, it was found that the anti-apoptotic Bcl-2 protein expression was
decreased (Figure 6).
[Please, insert Figure 6 about here]
2.3. Docking study
One of potential keys in cancer treatment is blocking of receptor tyrosine kinases (TKs)
which are over expressed in cancer [38]. In this study, EGFR, was chosen as a biological
target for carrying molecular modeling study for the newly synthesized compounds 5a-l.
Using Protein Data Bank (PDB: ID: 1M17), the crystal structure of EGFR kinase with its
Inhibitor erlotinib (4-anilinoquinazoline) was obtained [39]. Molecular Operating
Environment (MOE, Version 2005.06, Chemical Computing Group Inc., Montreal, Quebec,
Canada) used for docking calculations. Erlotinib was docked first into the active site of
EGFR and then the new compounds 5a-l to compare their binding mode to that of erlotinib.
By inspecting the mode of binding of erlotinib, it was found that it interacts with Cys 773
amino acid via a hydrogen bond with its side chain oxygen atom and the docking score was -
11.0797 Kcal/mol at 2.6 Å. The most active derivatives 5a and 5g against HepG2 and MCF7
cell lines showed one hydrogen bond between C=O of phthalimide part and the backbone
amino acids Cys 773-the same as the reference erlotinib- or with Thr 830 with docking scores
-15.6589 and -19.6074, respectively.
On the other side, rest of compounds containing carboxylic acid group in their structures bind
to Lys 721 amino acid through a hydrogen bond with oxygen in COOH group rather than
C=O of phthalimide core.
Some of active compounds (either on HepG2 and/or MCF7) didn't interact with any of amino
acids inside the active site of EGFR enzyme. Specially those of unsubstituted phthalimide
scaffold bearing CN or Cl groups at their phenyl spacer of benzoxazole and benzothiazole
derivatives 5b, 5c, 5h and 5i. Their docking scores were in the range -17.4219 to -12.3223
Kcal/mol higher than that of erlotinib -11.0797 Kcal/mol.
8

Finally, we could conclude that hydrogen bonds between C=O group of phthalimide core
might be important for activity. Moreover, hydrogen bonds with Cys 773 and Thr 830
backbone amino acids played an important role in fixation of compounds inside the enzyme
active site led to increase in cytotoxic activity rather than Lys 721 amino acid and COOH
group.
All the binding modes, interactions and docking scores are tabulated in S1 (supporting
information), and pictured as in Figures 7 and 8.
[Please, insert Figures 7 and 8 about here]
[S 1, supporting information]
3. Conclusion
The newly synthesized benzoxazole/benzothiazole-phthalimide hybrids were evaluated for
cytotoxic activity against two human cancerous cell lines, liver HepG2 and breast MCF7 cell
lines, and non-cancerous fibroblast cells (WI-38) in-vitro. They possess potency against two
cancer cell lines, HepG2 and MCF7. The most active compounds 5a (on HepG2) and 5g (on
MCF7) were also able to activate caspase-7 and 9 enzymes led to induce apoptosis. Cell cycle
analysis represented evidence through apoptosis of the HepG2 and MCF7 cancer cells at pre
G1 and cell arrest at arrest at G2/M phase.
The prepared compounds had no cytotoxic activity on non cancerous cell line (WI-38) if
compared with the used cancerous cell lines HepG2 and MCF-7.
From the obtained results, we concluded that apoptosis was the mechanism for
antiproliferative activity of the synthesized compounds rather than kinase inhibition against
EGFR and HER2 enzymes. Benzoxazole derivative 5a and its benzothiazole analogue 5g
possessed the highest potency against HepG2 and MCF7 cell lines with IC50 = 0.011 and
0.006 μM, respectively. Similarly, they exerted the highest ability to activate caspase-7 and
caspase-9 enzymes required for apoptosis. Subsequently, they arrested the cell cycle by
45.54% and 33.94%, sequentially at the G2/M stage. They showed high scores in molecular
modeling study -15.65 and -19.60 and had one hydrogen bond with Cys773 and Thr830
amino acids in EGFR active site, respectively.
9

4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were determined using a Griffin apparatus and are uncorrected. Infrared
spectra (IR) were recorded, using KBr discs, on a Shimadzu IR-435 spectrophotometer and
1
13
values were represented in cm-1. H NMR and C NMR (DEPT-Q) were carried out using
the Bruker instrument at 400 MHz for 1H NMR and 100 MHz for 13C NMR
spectrophotometer, (Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt), in
DMSO-d6, D2O using TMS as an internal standard and chemical shifts were recorded in ppm
on the δ scale using DMSO-d6(2.5) as a solvent. Coupling constant (J) values were estimated
in Hertz (Hz). Splitting patterns are designated as follows: s, singlet; d, doublet, t, triplet; q,
quartet; dd, doublet of doublet; m, multiplet. The electron impact (EI) mass spectra were
recorded on a Hewlett Packard 5988 spectrometer (Palo Alto, CA). Microanalysis was
performed for C, H, N on Perkin-Elmer 2400 at the Microanalytical center, Cairo University,
Egypt and was within + 0.4% of theoretical values. Analytical thin layer chromatography
(TLC): pre-coated plastic sheets, 0.2 mm silica gel with UV indicator (Macherey-Nagel) was
employed routinely to follow the course of reactions and to check the purity of products. All
other reagents, solvents were purchased from the Aldrich Chemical Company (Milwaukee,
WI) and, were used without further purification.Benzoxazole3a, 3c and benzothiazole
derivatives 3f-i were prepared according to literature procedures [14, 33].
4.1.2. General procedure for synthesis of compounds 3b & 3d
A mixture of 2-aminothiophenol 1a (1.09 g, 0.01 mol) and 3-methyl-4-aminobenzoic acid 2b
or 3-chloro-4-aminobenzoic acid 2d (0.01 mol) was heated in polyphosphoric acid (PPA; 85
g) at 220 °C for 4 h, cooled, then poured into ice-cold 10% aqueous sodium carbonate. The
obtained solid was filtered, washed with water, and crystallized from 95% ethanol.
4.1.2.1. 4-(Benzo[d]oxazol-2-yl)-2-methylaniline (3b).Buff solid; Yield 72%; m.p. 186-
1
188ᵒC; IR (KBr) 3230,3117 (NH2) cm-1; H NMR (DMSO-d6, δ ppm) δ 2.12 (s, 3H, CH3),
6.53 (s, 2H, NH2, D2O exchangeable), 6.57 (d, J = 7.5 Hz, 1H, aminophenyl H-5), 7.35 (d, J
= 7.5 Hz, 1H, aminophenyl H-6), 7.39-7.41 (m, 2H, benzoxazoleH-4, H-7), 7.54 (s, 1H,
aminophenyl H-2), 7.74-7.83 (m, 2H, benzoxazole H-5, H-6); 13C NMR (DMSO-d6, δ ppm):
18.60 (CH3), 110.46 (benzoxazoleC-7), 113.60 (aminophenyl C-5), 116.03 (aminophenyl C-
1), 119.12 (benzoxazole C-4), 121.88 (aminophenyl C-3), 123.81 (benzoxazoleC-6), 124.18
(benzoxazoleC-5), 125.35 (aminophenyl C-6), 131.34 (aminophenyl C-2), 141.59
10

(benzoxazole C-3a), 150.68 (benzoxazole C-7a), 150.88 (aminophenyl C-4), 162.70
(benzoxazole C-2); Anal. Calcd for C14H12N2O (224.26): C, 74.98; H, 5.39; N, 12.49. Found:
C, 74.96; H, 5.58; N, 12.42.
4.1.2.2. 4-(Benzo[d]oxazol-2-yl)-2-chloroaniline (3d).Creamy solid; Yield 76%; m.p. 155-
1
157ᵒC; IR (KBr) 3232, 3120 (NH2) cm-1; H NMR (DMSO-d6, δ ppm) δ 6.49 (s, 2H, NH2,
D2O exchangeable), 6.63 (d, J = 7.5 Hz, 1H, aminophenyl H-5), 7.39-7.41 (m, 2H,
benzoxazoleH-4, H-7), 7.45 (d, J = 7.5 Hz, 1H, aminophenyl H-6), 7.73-7.81 (m, 2H,
benzoxazole H-5, H-6), 7.83 (s, 1H, aminophenyl H-2); 13C NMR (DMSO-d6, δ ppm):
110.34 (benzoxazoleC-7), 115.30 (aminophenyl C-5), 117.05 (aminophenyl C-3), 117.50
(aminophenyl C-1), 119.33 (benzoxazole C-4), 123.80 (benzoxazoleC-6), 124.15
(benzoxazoleC-5), 125.88 (aminophenyl C-5), 128.26 (aminophenyl C-2), 141.47
(benzoxazole C-3a), 147.71 (aminophenyl C-4), 150.09 (benzoxazole C-7a), 162.53
(benzoxazole C-2); Anal. Calcd for C13H9ClN2O (244.68): C, 63.81; H, 3.71; N, 11.45.
Found: C, 63.75; H, 3.78; N, 11.43.
4.1.3 General procedure for synthesis of compounds (5a-l)
A mixture of benzoxazole or benzothiazole derivative 3a-i (0.01 mol), and the appropriate
phthalic anhydride derivative 4a&b (0.01 mol), in glacial acetic acid (10 mL) was refluxed
for 10-12 h. (monitored by TLC). The precipitated that formed after cooling or on hot (in case
of benzothiazole derivatives) was filtered, dried and crystalized from a mixture of
methanol/chloroform (2:1) to afford 5a-l.
4.1.3.1. 2-[4-(Benzo[d]oxazol-2-yl)-2-methylphenyl]isoindoline-1,3-dione (5a).Brown
1
solid; Yield 71%; m.p. 252-254ᵒC; IR (KBr) 1774, 1717 (2C=O) cm-1; H NMR (DMSO-d6,
δ ppm) δ2.26 (s, 3H, CH3), 7.43-7.45 (m, 2H, benzoxazoleH-4, H-7), 7.68 (d, J= 8 Hz, 1H,
phenyl H-6), 7.78-7.83 (m, 2H, benzoxazole H-5, H-6),7.94-7.96 (m, 2H, isoindoline H-4, H-
7), 8.01-8.03 (m, 2H, isoindoline H-5, H-6), 8.23 (d, J = 8 Hz, 1H, phenyl H-5), 8.29 (s, 1H,
13
phenyl H-3); C NMR (DMSO-d6, δ ppm): 18.20 (CH3), 111.41 (benzoxazoleC-7), 120.32
(benzoxazole C-4), 124.07 (phenyl C-6), 125.48 (phenyl C-4), 125.52 (isoindoline C-4, C-7),
126.17 (benzoxazoleC-6), 128.18 (benzoxazoleC-5), 128.72 (phenyl C-5), 132.25
(isoindoline C-3a, C-7a), 132.35 (phenyl C-3), 135.25 (isoindolineC-5, C-6), 141.39
(benzoxazole C-3a), 141.88 (phenyl C-1), 150.66 (benzoxazole C-7a), 161.99 (benzoxazole
C-2), 167.41 (isoindoline 2 C=O); Anal. Calcd for C22H14N2O3 (354.36): C, 74.57; H, 3.98;
N, 7.91. Found: C, 74.69; H, 3.85; N, 7.88.
11

4.1.3.2. 5-(Benzo[d]oxazol-2-yl)-2-(1,3-dioxoisoindolin-2-yl)benzonitrile (5b).Green solid;
1
Yield 68%; m.p. 318-320 ᵒC;IR (KBr) 2358 (C≡N), 1786, 1703 (2C=O) cm-1; H NMR
(DMSO-d6, δ ppm) δ 7.42-7.48 (m, 2H, benzoxazole H-4, H-7), 7.83-8.15 (m, 7H,
benzoxazole H-5, H-6, dioxoisoindolin H-4, H-5, H-6, H-7 and benzonitrile H-5), 8.57 (d,
13
1H, J = 7.6 Hz, benzonitrile H-3), 8.88 (s, 1H, benzonitrile H-6); C NMR (DMSO-d6, δ
ppm): 111.57 (benzoxzoleC-7), 113.45 (phenyl C-1), 1116.21 (C≡N), 120.48 (benzoxazoleC-
4), 121.20 (phenyl C-3), 123.10 (phenyl C-5), 124.63 (isoindoline C-4, C-7), 125.59
(benzoxazoleC-6), 126.36 (benzoxazole C-5), 126.88 (phenyl C-6), 131.81 (isoindoline C-3a,
C-7a), 132.55 (phenyl C-4), 136.10 (isoindoline C-5, C-6), 141.12 (benzoxazole C-3a),
142.03 (phenyl C-2), 151.00 (benzoxazole C-7a), 165.80 (benzoxazole C-2), 166.31
(isoindoline 2 C=O);Anal. Calcd for C22H11N3O3 (356.34): C, 72.33; H, 3.03; N, 11.50.
Found: C, 72.57; H, 3.15; N, 11.48.
4.1.3.3. 2-[(4-Benzo[d]oxazol-2-yl)-2-chlorophenyl]isoindoline-1,3-dione (5c). Brown
solid; Yield 73%; m.p. 260-262ᵒC;IR (KBr) 1776, 1720 (2C=O) cm-1; 1H NMR (DMSO-d6, δ
ppm) δ 7.43-7.50 (m, 2H, benzoxazole H-4, H-7), 7.80-7.86 (m, 2H, benzoxazole H-5, H-6),
7.97-8.06 (m, 5H, dioxoisoindolin H-4, H-5, H-6, H-7 and chlorophenyl H-6), 8.35 (d, 1H, J
13
= 8.4 Hz, chlorophenyl H-5), 8.55 (s, 1H, chlorophenylH-3); C NMR (DMSO-d6, δ ppm):
111.56 (benzoxazoleC-7), 120.56 (benzoxazoleC-4), 124.32 (chlorophenyl C-6), 125.68
(isoindolineC-4, C-7), 126.57 (benzoxazole C-6), 126.90 (chlorophenyl C-4), 129.94
(benzoxazoleC-5), 130.59 (chlorophenyl C-5), 131.19 (chlorophenyl C-2), 131.60
(chlorophenyl C-3), 131.98 (isoindoline C-3a, C-7a), 135.60 (isoindoline C-5, C-6), 136.34
(chlorophenyl C-1), 141.78 (benzoxazole C-3a), 150.79(benzoxazole C-7a), 161.03
(benzoxazole C-2), 166.73 (isoindoline 2 C=O); Anal. Calcd for C21H11ClN2O3 (374.78): C,
67.30; H, 2.96; N, 7.47. Found: C, 67.51; H, 3.05; N, 7.43.
4.1.3.4. 2-[4-(Benzo[d]oxazol-2-yl)phenyl]-1,3-dioxoindoline-5-carboxylic acid (5d).Buff
solid; Yield 72%; m.p. 329-331ᵒC;IR (KBr) 3060-2550 (OH), 1784, 1723, 1687 (3C=O) cm-
1
1; H NMR (DMSO-d6, δ ppm) δ7.44-7.49 (m, 2H, benzoxazole H-4, H-7), 7.75 (d, 2H, J =
8.8 Hz, phenyl H-2, H-6), 7.82-7.86 (m, 2H, benzoxazoleH-5, H-6), 8.12 (d, 1H, J = 8 Hz,
dioxoisoindolin H-7), 8.35 (d, 2H, J = 8.8 Hz, phenyl H-3, H-5), 8.38 (s, 1H, dioxoisoindolin
H-4), 8.44 (dd, 1H, J = 1.2 Hz, J = 8Hz, dioxoisoindolin H-6), 13.80 (s, 1H, OH, D2O
exchangeable); 13C NMR (DMSO-d6, δ ppm): 111.51 (benzoxzoleC-7), 120.41
(benzoxazoleC-4), 123.97 (isoindoline C-7), 124.44(benzoxazoleC-6), 125.51(benzoxazoleC-
5), 126.25(isoindoline C-4), 128.17 (phenyl C-3, C-5), 128.30 (phenyl C-2, C-6), 131.94
12

(phenyl C-4), 132.47 (isoindolineC-3a), 135.24 (phenyl C-1), 135.29 (isoindoline C-5),
136.09 (isoindoline C-6), 137.09(isoindoline C-7a), 141.92 (benzoxazole C-3a), 150.78
(benzoxazoleC-7a), 162.13(benzoxazoleC-2), 166.29 (isoindoline 2 C=O), 166.46
(isoindolineCOOH);MS (m/z, %): 384 (M, 11.30), 68 (100.00); Anal. Calcd for C22H12N2O5
(384.34): C, 68.75; H, 3.15; N, 7.29. Found: C, 68.55; H, 3.18; N, 7.13.
4.1.3.5.
2-[4-(Benzo[d]oxazol-2-yl)-2-methylphenyl]-1,3-dioxoisoindoline-5-carboxylic
acid (5e).Redish-brown solid; Yield 70%; m.p. 299-301ᵒC;IR (KBr) 3162 (broad band, OH),
1780, 1727, 1680 (3C=O) cm-1; 1H NMR (DMSO-d6, δ ppm) δ 2.28 (s, 3H, CH3), 7.37-7.47
(m, 2H, benzoxazole H-4, H-7), 7.68 (d, 1H, J = 8 Hz, phenyl H-6), 7.78-7.82 (m, 2H,
benzoxazole H-5, H-6), 8.13 (d, 1H, J = 8 Hz, phenyl H-5), 8.24 (d, 1H, J = 8 Hz,
dioxoisoindolin H-7), 8.32 (s, 1H, phenyl H-3), 8.36 (s, 1H, dioxoisoindolin H-4), 8.45 (d,
1H, J = 8 Hz, dioxoisoindolin H-6), 13.75 (s, 1H, OH, D2O exchangeable); 13C NMR
(DMSO-d6, δ ppm): 18.17 (CH3), 111.40 (benzoxazoleC-7), 120.31 (benzoxazoleC-4),
124.01 (phenyl C-6), 124.48 (isoindoline C-7), 125.52 (benzoxazoleC-6), 125.75
(benzoxazoleC-4), 126.19 (benzoxazole C-5), 128.29 (isoindoline C-4), 128.59 (phenyl C-5),
132.21 (isoindoline C-3a), 132.42 (phenyl C-3), 132.75 (isoindoline C-5), 135.56 (phenyl C-
2), 136.02 (isoindoline C-6), 137.02 (phenyl C-1), 141.40 (isoindoline C-7a), 141.83
(benzoxazoleC-3a), 150.64 (benzoxazole C-7a), 161.95 (benzoxazole C-2), 166.35
(isoindoline 2 C=O), 166.71 (isoindolineCOOH);Anal. Calcd for C23H14N2O5 (398.37): C,
69.34; H, 3.54; N, 7.03. Found: C, 69.51; H, 3.48; N, 7.11.
4.1.3.6.
2-[4-(Benzo[d]oxazol-2-yl)-2-chlorophenyl]-1,3-dioxoisoindoline-5-carboxylic
acid (5f). Grey solid; Yield 73%; m.p.286-288ᵒC;IR (KBr) 3273 (broad band OH), 1785,
1734, 1679 (3C=O) cm-1; 1H NMR (DMSO-d6, δ ppm) δ 7.43-7.50 (m, 2H, benzoxazole H-4,
H-7), 7.81-7.85 (m, 2H, benzoxazole H-5, H-6), 7.99 (d, 1H, J = 8.4 Hz, chlorophenyl H-6),
8.17 (d, 1H, J = 7.6 Hz, dioxoisoindolin H-7), 8.36 (d, 1H, J = 8.4 Hz, chlorophenyl H-5),
8.39 (s, 1H, chlorophenyl H-3), 8.48 (d, 1H, J = 7.6 Hz, dioxoisoindolin H-6), 8.57 (s, 1H,
13
dioxoisoindolin H-4), 13.84 (s, 1H, OH, D2O exchangeable); C NMR (DMSO-d6, δ ppm):
111.57 (benzoxazoleC-7), 120.57 (benzoxazoleC-4), 124.26 (isoindoline C-7), 125.68
(benzoxazole C-6), 126.59 (benzoxazole C-5), 126.93 (chlorophenyl C-4), 130.05
(isoindoline C-4), 130.45 (chlorophenyl C-5), 130.96 (chlorophenyl C-2), 131.66
(chlorophenyl C-3), 132.42 (isoindoline C-3a), 135.18 (isoindoline C-5), 136.23
(chlorophenyl C-1), 136.36 (isoindoline C-6), 137.42 (isoindoline C-7a), 141.78
(benzoxazoleC-3a), 150.79 (benzoxazole C-7a), 160.99 (benzoxazoleC-2), 165.97
13

(isoindoline 2 C=O), 166.20 (isoindolineCOOH);Anal. Calcd for C22H11ClN2O5 (418.79): C,
63.10; H, 2.65; N, 6.69. Found: C, 62.97; H, 2.72; N, 6.52.
4.1.3.7. 2-[4-(Benzo[d]thiazol-2-yl)-2-methylphenyl]isoindoline-1,3-dione (5g).Green
solid; Yield 77%; m.p. 260-262ᵒC;IR (KBr) 1779, 1709 (2C=O) cm-1; 1H NMR (DMSO-d6, δ
ppm) δ2.24 (s, 3H, CH3), 7.48 (t, 1H, J = 7.6 Hz, benzothiazole H-6), 7.56 (t, 1H, J = 7.6 Hz,
1H, benzothiazoleH-5), 7.63 (d, 1H, J = 8 Hz, phenyl H-6), 7.95-8.06 (m, 5H, isoindoline H-
4, H-5, H-6, H-7 and phenyl H-5), 8.12-8.18 (m, 2H, benzothiazole H-4, H-7), 8.21 (s, 1H,
phenyl H-3); 13C NMR (DMSO-d6, δ ppm): 18.07 (CH3), 122.93 (benzothiazoleC-4), 123.31
(benzothiazoleC-6), 124.05 (phenyl C-6), 126.13 (isoindoline C-4, C-7), 127.25
(benzothiazoleC-6), 128.15 (benzothiazoleC-5), 128.46 (phenyl C-5), 132.05 (phenyl C-4),
132.26 (phenyl C-3), 132.28 (isoindoline C-3a, C-7a), 132.47
(phenyl C-2),
134.94(benzothiazole C-7a), 135.23 (isoindolineC-5, C-6), 140.64 (phenyl C-1), 153.92
(benzothiazole C-3a), 166.73 (benzothiazole C-2), 167.46 (isoindoline 2 C=O); Anal. Calcd
for C22H14N2O2S (370.42): C, 71.33; H, 3.81; N, 7.56. Found: C, 71.27; H, 3.85; N, 7.47.
4.1.3.8. 5-(Benzo[d]thiazol-2-yl)-2-(1,3-dioxoisoindolin-2-yl)benzonitrile (5h). Yellow
solid; Yield 73%; m.p. 263-265ᵒC;IR (KBr) 2234 (C≡N), 1784, 1720 (2C=O) cm-1; 1H NMR
(DMSO-d6, δ ppm) δ 7.54 (t, 1H, J = 7.6 Hz, benzothiazole H-6), 7.62 (t, 1H, J = 7.2 Hz,
benzothiazole H-5), 7.95-8.26 (m, 7H, dioxoisoindolin H-4, H-5, H-6, H-7, benzothiazole H-
7 and benzonitrile H-5, H-6), 8.62 (d, 1H, J = 1.2 Hz, 8.4 Hz, benzothiazoleH-4), 8.75 (s, 1H,
benzonitrileH-3); 13C NMR (DMSO-d6, δ ppm): 113.34 (phenyl C-1), 116.10 (C≡N), 123.13
(benzothiazoleC-4), 123.80 (benzothiazole C-7a), 124.60 (phenyl C-3), 126.75 (isoindoline
C-4, C-7), 127.56 (benzothiazole C-6), 131.56 (benzothiazole C-5), 131.72 (phenyl C-5),
132.30 (phenyl C-6), 133.06 (phenylC-4), 134.76 (isoindoline C-3a, C-7a), 135.89
(isoindoline C-5, C-6), 136.91 (benzothiazole C-7a), 141.89 (phenyl C-2), 154.00
(benzothiazole C-3a), 164.95 (benzothiazole C-2), 166.37 (isoindoline 2 C=O);Anal. Calcd
for C22H11N3O2S (381.41): C, 69.28; H, 2.91; N, 11.02. Found: C, 69.39; H, 2.84; N, 10.95.
4.1.3.9. 2-[4-(Benzo[d]thiazol-2-yl)-2-chlorophenyl]isoindoline-1,3-dione (5i).Green solid;
Yield 77%; m.p. 263-265ᵒC;IR (KBr) 1781, 1712 (2C=O) cm-1; 1H NMR (DMSO-d6, δ ppm)
δ7.51 (t, 1H, J = 7.6 Hz, benzothiazole H-6), 7.59 (t, 1H, J = 6.8 Hz, benzothiazole H-5),
7.92-8.10 (m, 6H, benzothiazoleH-4, H-7 and dioxoisoindolin H-4, H-5, H-6, H-7), 8.21 (d,
1H, J = 7.6 Hz, chlorophenyl H-6), 8.27 (d, 1H, J = 7.6 Hz, chlorophenyl H-5), 8.48 (s, 1H,
chlorophenylH-3); 13C NMR (DMSO-d6, δ ppm): 123.03 (benzothiazoleC-4), 123.56
(benzothiazoleC-7), 124.30 (chlorophenyl C-6), 126.49 (isoindolineC-4, C-7), 126.93
14

(chlorophenyl C-4), 127.45 (benzothiazoleC-6), 129.87 (benzothiazoleC-5), 130.33
(chlorophenyl C-5), 131.19 (chlorophenyl C-2), 131.47 (chlorophenyl C-3), 132.02
(isoindoline C-3a, C-7a), 133.28(benzothiazole C-7a), 135.22 (chlorophenyl C-1), 135.58
(isoindoline C-5, C-6), 153.85(benzothiazole C-3a), 165.53 (benzothiazole C-2), 166.78
(isoindoline 2 C=O); Anal. Calcd for C21H11ClN2O2S (390.84): C, 64.53; H, 2.84; N, 7.17.
Found: C, 64.48; H, 2.91; N, 7.06.
4.1.3.10. 2-[4-(Benzo[d]thiazol-2-yl)phenyl]-1,3-dioxo isoindoline-5-carboxylic acid
(5j).Green solid; Yield 85%; m.p. 339-341ᵒC;IR (KBr) 3053-2543 (OH), 1774, 1720, 1676
1
(3C=O) cm-1; H NMR (DMSO-d6, δ ppm) δ 7.50 (t, 1H, J = 6.8 Hz, benzothiazole H-6),
7.58 (t, 1H, J = 7.6 Hz, benzothiazole H-5), 7.71 (d, 2H, J = 8.4 Hz, phenyl H-2, H-6), 8.10-
8.14 (m, 2H, benzothiazole H-4, H-7), 8.19 (d, 1H, J = 7.6 Hz, dioxoisoindolin H-7), 8.27 (d,
2H, J = 8.4 Hz, phenyl H-3, H-5), 8.34 (s, 1H, dioxoisoindolin H-4), 8.44 (dd, 1H, J = 1.2,
7.6 Hz, dioxoisoindolin H-6), 13.77 (s, 1H, OH, D2O exchangeable); 13C NMR (DMSO-d6, δ
ppm): 122.92 (benzothiazoleC-4), 123.48 (benzothiazoleC-7), 123.94 (isoindoline C-7),
124.41 (benzothiazoleC-6), 126.21 (benzothiazoleC-5), 127.26 (isoindoline C-4), 128.17
(phenyl C-3, C-5), 128.27 (phenyl C-2, C-6), 132.52 (phenyl C-4), 132.73 (isoindoline C-3a),
134.75 (phenyl C-1), 135.15 (isoindoline C-5), 135.36(benzothiazole C-7a), 136.05
(isoindoline C-6), 137.04 (isoindoline C-7a), 154.03 (benzothiazole C-3a), 166.28
(benzothiazole C-2), 166.49 (isoindoline 2 C=O), 166.90 (isoindolineCOOH);Anal. Calcd for
C22H12N2O4S (400.41): C, 65.99; H, 3.02; N, 7.00. Found: C, 65.78; H, 3.11; N, 7.15.
4.1.3.11. 2-[4-(Benzo[d]thiazol-2-yl)-2-methylphenyl]-1,3-dioxoisoindoline-5-carboxylic
acid (5k).Greyish-green solid; Yield 78%; m.p. 292-294ᵒC;IR (KBr) 3365-2606 (OH), 1779,
1716, 1661 (3C=O) cm-1; 1H NMR (DMSO-d6, δ ppm) δ 2.25 (s, 3H, CH3), 7.48 (t, 1H, J =
7.6 Hz, benzothiazole H-6), 7.56 (t, 1H, J = 7.2 Hz, benzothiazole H-5), 7.63 (d, 1H, J = 8
Hz, phenyl H-6), 8.05 (d, 1H, J = 8Hz, phenyl H-5), 8.12-8.18 (m, 3H, benzothiazole H-4,
H-7 anddioxoisoindolin H-7), 8.23 (s, 1H, phenyl H-3), 8.36 (s, 1H, dioxoisoindolin H-4),
8.45 (d, 1H, J = 7.6 Hz, dioxoisoindolin H-6), 13.75 (s, 1H, OH, D2O exchangeable); 13C
NMR (DMSO-d6, δ ppm): 18.05 (CH3), 122.92 (benzothiazoleC-4), 123.30 (benzothiazoleC-
7), 123.99 (phenyl C-6), 124.45 (isoindolineC-7), 126.14 (benzothiazoleC-6), 127.26
(benzothiazoleC-5), 128.27 (isoindolineC-4), 128.31 (phenyl C-5), 132.04 (phenyl C-4),
132.27 (isoindolineC-3a), 132.32(phenyl C-3), 135.63 (phenyl C-2), 135.98 (isoindoline C-
6), 136.94 (phenyl C-1), 140.63 (isoindoline C-7a), 153.90 (benzothiazole C-3a), 166.33
15

(benzothiazole C-2), 166.68 (isoindoline 2 C=O), 166.73 (isoindolineCOOH); Anal. Calcd
for C23H14N2O4S (414.43): C, 66.66; H, 3.40; N, 6.76. Found: C, 66.74; H, 3.45; N, 6.69.
4.1.3.12. 2-[4-(Benzo[d]thiazol-2-yl)-2-chlorophenyl]-1,3-dioxoisoindoline-5-carboxylic
acid (5l).Grey solid; Yield 75%; m.p. 319-321ᵒC;IR (KBr) 3068-2556 (OH), 1783, 1725,
1680 (3C=O) cm-1; 1H NMR (DMSO-d6, δ ppm) δ 7.51 (t, 1H,J = 7.6 Hz,benzothiazole H-6),
7.59 (t, 1H,J= 7.2 Hz,benzothiazole H-5), 7.93 (d, 1H,J = 8.4Hz,chlorophenyl H-6), 8.08 (d,
1H, J = 8 Hz, benzothiazole H-4), 8.16 (d, 1H, J = 8.4 Hz, chlorophenyl H-5), 8.20 (d, 1H, J
= 8 Hz, benzothiazole H-7), 8.27 (dd, 1H, J = 2.00, 8.4 Hz, dioxoisoindolin H-6), 8.39 (s,
1H, chlorophenyl H-3), 8.47 (d, 1H, J = 8.4Hz, dioxoisoindolin H-7), 8.49 (d, 1H, J = 2.00
Hz, dioxoisoindolin H-4), 13.86 (s, 1H, OH, D2O exchangeable); 13C NMR (DMSO-d6,
δppm): 123.07 (benzothiazoleC-4), 123.57 (benzothiazoleC-7), 124.21 (isoindoline C-7),
124.71 (chlorophenyl C-6), 126.46 (benzothiazoleC-6), 127.42 (benzothiazoleC-5),
129.95(isoindoline C-4), 130.15 (chlorophenyl C-5), 130.94 (chlorophenyl C-4), 130.96
(chlorophenyl C-2), 131.49 (chlorophenyl C-3), 132.45 (isoindoline C-3a), 133.29
(isoindoline C-5), 135.22 (chlorophenyl C-1), 135.48 (benzothiazole C-7a), 136.29
(isoindoline C-6), 137.32 (isoindoline C-7a), 153.38 (benzothiazole C-3a), 165.47
(benzothiazole C-2), 165.98 (isoindoline 2 C=O), 166.20 (isoindolineCOOH);Anal. Calcd for
C22H11ClN2O4S (434.85): C, 60.76; H, 2.55; N, 6.44. Found: C, 60.68; H, 2.47; N, 6.39.
4.2. Biological activity
4.2.1. Materials and methods
In this study, two human cell lines, hepatic cancer (HepG2), breast cancer (MCF7) and non-
cancerous fibroblast cells (WI-38) were supplied from American Type Cell Culture
Collection (ATCC, Manassas, USA) and was maintained at VACSERA, Giza, Egypt. The
reference anticancer drugs chosen in this study, against HepG2and MCF7 cell lines were5-
fluorouracil (5-FU)and erlotinib, respectively. Cells were cultured using DMEM
(Invitrogen/Life Technologies) supplemented with a mixture of 10% FBS (Hyclone,), 10
ug/ml of insulin (Sigma), and 1% penicillin-streptomycin. All of the other used chemicals
and reagents were obtained from Sigma, or Invitrogen. Cells were platedin a 96-well plate for
24 hours before the MTT assay as followed (cells density 1.2 – 1.8 × 10,000 cells/well) in a
volume of 100µL then complete growth medium + 100 uL of the tested compound per well
[40].
4.2.2. Measurement of cytotoxic activity by MTT assay
16

MTT (3-(4.5-dimrthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to
determine the cytotoxic activity of the test compounds according to the reported method
[40].Cells were plated in 96-multiwell plate in DMEM containing 10% FBS (fetal bovine
serum for 24 h. ThenDMSO was used as a vehicle for treatment of cells, test compounds and
reference drugs for 48 h. After that, the media was replaced with 200 μL DMEM containing
0.5mg/ml of MTT and were incubated for 2 h. Remove the supernatant and 200 μL of
DMSO was used to dissolve formazans precipitate. Finally, the absorbance was determined at
570 nm by usinga micro plate reader.
4.2.3. In vitro enzyme inhibitory assay
To investigate the mechanism of inhibition of EGFR and HER2 kinases,a cell-free assay was
used according to the reported method [41].Kit used for immune-assay was cloud clone
SEA757Hu 96 Tests.200 μM (EGFR) was used. A dose-response curve was obtained from
the values resulted from the following equation: E (%) = E max/ (1+ [I]/ID50), where E (%) is
the fraction of the enzyme activity measured in the presence of the inhibitor, E max is the
activity in the absence of the inhibitor, [I] is the inhibitor concentration and ID50 is the
inhibitor concentration at which E (%) = 0.5 E max. then, for each experiment,mean values
of two independent replicates were used for the interpolation.
4.2.4. Caspase-7 and 9 activation assays
HepG2 and MCF-7 Cell lines, pretreated with compounds 5a and 5g respectively, were
incubated in Lysis Buffer (conc.5 x106 cells/ml.) at room temperature for 60 minutes with
gentle shaking and then the extracts were transfered to microcentrifuge tubes and centrifuged.
Then, lysates were diluted over the range of the assay using Standard Diluent Buffer. After
that, standards or samples were added to the appropriate micro ELISA plate wells pre-coated
with antibodies specific to CASP7 or CASP9.Then a biotinylated detection antibody specific
for CASP7 or a polyclonal detection antibody specific for CAS9, captured by the first
antibody, was added to eachmicro plate well successively and incubated. Following
incubation, the unbound detection antibody was removed during a wash step and Avidin-
Horseradish Peroxidase (HRP) conjugate was added to each micro plate well to bind to the
Detection Antibody. The mixture was incubated again and washed to remove the unbound
HRB followed by addition of substrate solution reactive with HRP to the wells. A blue
colored conjugate was appeared and its optical density was directly proportional to the
amount of CAS7 or CAS9 present in the sample or standard. The enzyme-substrate reaction
was terminated by addition of a sulphuric acid and the optical density (OD) was measured
17

spectrophotometrically at a wavelength of 450 nm.CASP7 or CAS9 concentrations were
determined in the samples by comparing the O.D. of the samples to the standard curve.
4.2.5. Cell Cycle analysis
Both HepG2 and MCF-7 cells were incubated for 24 h after seeding into six-well plates
(density of 2 x 105 cells per well). Fetal bovine serum (FBS, 10%) was added to RPMI 1640
and the cells were cultured in it at 37oC and 5% CO2. Then the medium was replaced with
medium (final DMSO concentration, 1% v/v) containing compounds 5a and 5g (0.011 and
0.006 µM). Incubation continued for 24 h and 48 h. After that, the cell layer was trypsinized ,
washed with cold phosphate buffered saline (PBS) and fixed with 70% ethanol. PBS was
used to rinse the fixed cells and then the DNA fluorochrome PI in a solution containing
Triton X-100 and RNase were used as a staining solution for 15 minutes at 37oC according
the instruction manual. Then a FACS Caliber flow cytometer (Becton Dickinson & Co.,
Franklin Lakes, NJ) was used to analyze the samples. The number of cells analyzed for each
sample was chosen to be 10000 [42].
4.2.6. Measurement of apoptosis using Annexin-V-FITC apoptosis detection kit
Cells were stained with Annexin V-fluorescein isothiocyanate (FITC) and counterstaining
with propidium iodide (PI) using Annexin V-FITC/PI apoptosis detection kit to determine
apoptosis. 2 x 105 Cells were exposed to compounds 5a and 5g(0.011 and 0.006 µM) for 24 h
and 48 h. The trypsinized cells and washed twice with cold phosphate-buffered saline (PBS)
and stained with 5 μL Annexin V-FITC and 5 μL PI in 1 x binding buffer for 15 minutes at
37oC in the dark. Then, FACS Caliber flow cytometer (Becton Dickinson Biosciences & Co.,
Franklin Lakes, NJ) was used to analyze the samples. The number of analyzed cells for each
sample was 10000 [42].
4.2.7. Evaluation of Bax and Bcl-2 expressions
The cell culture samples were suspended in Lysis Buffer to concentration5 x106 cells/ml and
incubated at room temperature for 60 minutes with gentle shaking. Then the extracts were
centrifuged and the cleared lysates were transferred to clean microfuge tubules. Moreover,
lysates were diluted using Standard Diluent Buffer over the range of the assay. After that,
standards or samples were added to the appropriate micro ELISA plate wells pre-coated with
antibodies specific to Bax or Bcl-2 and were incubated at room temperature, on a microplate
shaker at 100 rpm if available, for 2 hours. The excess sample or standard were then washed
18

out and specific biotinylated monoclonal antibodies were added and bound to Bax or Bcl-2
captured by the first antibodies. Following that, 100 μL of diluted Streptavidin conjugated
to Horseradish peroxidase (HRP) was added to all wells, including the blank well sand the
all wells were incubated at room temperature, on a microplate shaker at 100 rpm if available,
for 1 hour. HRP was bound to the biotinylated monoclonal Baxor Bcl-2 antibodies. Excess
conjugate was washed out and substrate was added. After a short incubation, the enzyme
reaction was stopped by addition of acid and the color generated was read at 450 nm. The
measured optical density is directly proportional to the concentration of Bax or Bcl-2 in either
standards or samples.
4.3. Statistical analysis
To analyze statistically data obtained, computerized Prism 5 program was used using one-
way ANOVA test and Tukey’s as post ANOVA for multiple comparison at P ≤ 0.05.
Representation of data was as mean ± SEM.
4.4. Docking study
The crystal structure of erlotinib inside EGFRactive site (PDB: ID: 1M17) was obtained from
the protein data bank at Research Collaboration for Structural Bioinformatics (RSCB) Protein
Database (PDB). Docking of the co-crystallized ligand was carried out to study the scoring
energy(s), root mean standard deviation (RMSD) and amino acid interactions. RMSD was
used to measure superposing for the lead compound to be 2.61Å. Both London dG force and
force field energy were used for docking study and refinement of the obtained results. 3D
structure for the synthesized compounds were built by Molecular Operating Environment
(MOE, Version 2005.06, Chemical Computing Group Inc., Montreal, Quebec, Canada)
preparing them for starting the docking procedure. Moreover, 3D protonation of the built
structures, conformational analysis, and selecting the least energetic conformer were applied
using the same docking protocol used with ligand and test compounds. Amino acid
interactions, number and lengths of the hydrogen bonds were determined.
5. Acknowledgments
Authors would like to thank all members of Pharmaceutical Organic Chemistry, Faculty of
Pharmacy, Beni-Suef University for their great help and support during the whole work.
Also, we gratefully acknowledge Dr. Esam Rashwan, Head of the confirmatory diagnostic
unit VACSERA-EGYPT, for carrying out the cell cycle analysis, apoptosis biomarkers, in-
vitro enzyme inhibitory assays
19

Conflicts of Interest
Authors have no conflict of interest to declare.
References
[1] X.Pu, Z.Tian, W.Liang, S.Chang-Yan, H.Jing, Z.Ying-Lan,Y.Li, Novel benzothiazole,
benzimidazole and benzoxazole derivatives as potential antitumor agents: synthesis and
preliminary in vitro biological evaluation,Molecules, 17(2012), 873-883.
[2] M.Y. Amal, M.Ahmed, H.B.Mona, Y.E. Rasha, S.S. Ahmed, Synthesis and anticancer
activity of novel benzimidazole and benzothiazole derivatives against HepG2 liver cancer
cells,Med. Chem., 8(2012), 151-162.
[3] X.Xilei, Y.Yu, Z.Ning,L.Gang,Benzothiazoles exhibit broad-spectrum antitumor activity:
their potency, structureactivity and structure-metabolism relationships,Eur. J. Med.
Chem.,76(2014),67–78.
[4] K.Melén, P.Keskinen, A.Lehtonen, I.Julkunen, Interferoninduced gene expression and
signaling in human hepatoma cell lines,J. Hepatol. 33(2000), 764-72.
[5] A.Stefania, W.Geoffrey, L.S.Erica, K.Hachemi, B.Rana,R.B. David, F.G.S.Malcolm,
S.M.Charles, D.B. Tracey,D.W. Andrew, Synthesis and biological properties of
benzothiazole, benzoxazole, and chromen-4-one analogues of the potent antitumor agent 2-
(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (PMX610, NSC 721648),J. Med. Chem.,
51(2008),5135–5139.
[6] H.Ian, A.J. Sharon, B.V.Rao, D.W. Andrew, F.G.S. Malcolm, Antitumor benzothiazoles.
synthesis and pharmaceutical properties of antitumor 2-(4-aminophenyl)benzothiazole amino
acid prodrugs,J. Med. Chem.45(2002),744-747.
[7]P.K.Jauhari, A.Bhavani, V.Subhash, S.Kiran, R.Prem, Synthesis of some novel 2-
substituted benzoxazoles as anticancer, antifungal, and antimicrobial agents,Med. Chem.
Res., 17(2008), 412–424.
[8] A.Ruhi,S.Nadeem, Biological aspects of emerging benzothiazoles: A short review. J.
Chem., 1-12(2013).
[9] S.Xuan-Hong, W.Zhao, X.Yong, Y.Ting-Hong, D.Mei, X.You-Zhi, W.Yu-Quan,Y.Luo-
Ting, Synthesis and biological evaluation of novel benzothiazole-2-thiol derivatives as
potential anticancer agents,Molecules, 17(2012), 3933-3944.
[10] X.Yong, L.Qian, Z.Yongxia, Y.Tinghong, W.Ningyu, L.Guobo, S.Xuanhong,
L.Yantong,
S.Bin,
Y.Tao,
Z.Lifeng,W.Wenshuang,
S.Xuejiao,
X.Ying,
20

W.Yuquan,Y.Luoting, SKLB316, a novel small-molecule inhibitor of cell-cycle progression,
induces G2/M phase arrest and apoptosis in vitro and inhibits tumorgrowth in vivo.,Cancer
Letters, 355(2014), 297–309.
[11] A.A.Yaseen, H.A.Haitham, S.Bahjat, H.J.Ihsan, O.B. Mohammad, A.A.Najim,
A.Tahsin, L.Paolo, B.Bernardetta, S. Tiziana,L.Roberta, Synthesis and in vitro
antiproliferative activity of new benzothiazole derivatives,ARKIVOC,xv(2008), 225-238.
[12] M.H.Pourgholami, L.Woon, R.Almajd, J.Akhter, P. Bowery,D.L. Morris, In vitro and in
vivo suppression of growth of hepatocellularcarcinoma cells by albendazole,Cancer Lett.,
165(2001),43-9.
[13] H.Ian, C.Mei-Sze, L.B. Helen, T.Valentina, D.B. Tracey, D.W. Andrew, F.G.S.
Malcolm, Antitumor benzothiazoles. Synthesis and in vitro biological properties of
fluorinated 2-(4-aminophenyl)benzothiazoles,J. Med. Chem., 44(2001),1446-1455.
[14] D.F. Shi, T.D. Bradshaw, S.Wrigley, C.J. McCall, P.Lelieveld, I.Fichtner,M.F.G.
Stevens, Antitumor benzothiazoles. Synthesis of 2-(4-aminophenyl)benzothiazoles and
evaluation of their activities against breast cancer cell lines in vitro and in vivo,J. Med.
Chem.39(1996), 3375–3384.
[15] T.D. Bradshaw, D.F. Shi , R.J. Schultz, K.D.Paull, L.Kelland, A. Wilson, H.H.Fiebig,
S.Wrigley,M.F.G. Stevens, Influence of 2-(4-aminophenyl)benzothiazoles on the growth of
human ovarian carcinoma cell lines in vitro and in vivo. Br. J. Cancer, 78(1998), 421-429.
[16] M.S. Chua, E. Kashiyama, T.D. Bradshaw, S.F. Stinson, E. Brantley, E.A. Sausville,
M.F.G. Stevens, Role of CYP1A1 in modulation of antitumour properties of the novel agent
2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495) in human breast cancer
cells,Cancer Res., 60 (2000), 5196-5203.
[17] L.CheeOnn, S.Marie, J.S. David, C.B. Michael, F.G.S. Malcolm, D.B. Tracey In vitro, in
vivo, and in silico analyses of the antitumor activity of 2-(4-amino-3-methylphenyl)-5-
fluorobenzothiazoles,Mol. Cancer Ther., 3(12) (2004), 1565-1575.
[18] A.A.-M. Abdel-Aziz, Novel and versatile methodology for synthesis of cyclic imides
and evaluation of their cytotoxic, DNA binding, apoptotic inducing activities and molecular
modeling study, Eur. Med. Chem., 42 (2007), 614-626.
21

[19] N.Shunmugam, M.Syamantak, S.Upendra, R.Saranya, K.Neeraj, C.Suvro,S.Bikram,
Synthesis and anti-angiogenic activity of benzothiazole, benzimidazole containing
phthalimide derivatives,Bioorg. Med. Chem. Lett., 23 (2013), 287–290.
[20] U.Sharma, P.Kumar, N. Kumar,B.Singh, Recent advances in the chemistry of
phthalimide analogues and their therapeutic potential,Mini-Rev. Med. Chem., 10 (2010),678-
704.
[21] M.C.Patrícia, P.C.Marcilia, A.C. Adriana, M.T.C.Suellen, V.O.C. Marcos,
B.O.F.Gevânio, A.V.Daniel, V.F.J.Francisco, C.L.L. Ana, O.M.Manoel, P. Claudia,M.P.F.
Paulo, Improvement of in vivo anticancer and antiangiogenic potential of thalidomide
derivatives,Chem.-Biol. Inter., 239(2015), 174–183.
[22] X.Meng, D.Han, S.Zhang, W.Guo, R.Cui,Z.Li,Synthesis and anti-inflammatory activity
of N-phthalimidomethyl
2,3-dideoxy-
and
2,3-unsaturated
glycosides,Carbohydr.
Res.,342(2007), 1169–1174.
[23] T.Noguchi, H.Fujimoto, H.Sano, H.Miyajima,Y. Hashimoto,Angiogenesis inhibitors
derived from thalidomide,Bioorg. Med. Chem. Lett., 15(2005), 5509–5513.
[24] H.Sano, T.Noguchi,A.Miyajima,Y. Hashimoto,H.Miyachi,Anti-angiogenic activity of
basic-type, selective cyclooxygenase (COX)-1 inhibitors,Bioorg. Med. Chem. Lett.,16(2006),
3068–3072.
[25] S.H.L.Kok, R.Gambari, C.H. Chui, M.C.W. Yuen, E.Lin, R.S.M. Wong, F.Y. Lau,
G.Y.M. Cheng, W.S. Lam, S.H. Chan, K.H. Lam, C.H. Cheng, P.B.S. Lai, M.W.Y. Yu,
F.Cheung, J.C.O. Tang, A.S.C. Chan, Synthesis and anti-cancer activity of benzothiazole
containing phthalimide on human carcinoma cell lines,Bioorg. Med. Chem., 16 (2008),
3626–3631.
[26] S.H. Chan, K.H. Lam, C.H. Chui, R.Gambari, M.C.W. Yuen, R.S.M. Wong, G.Y.M.
Cheng, F.Y.L. Lau, Y.K. Au, C.H. Cheng,P.B.S. Lai, C.W.Kan, S.H.L.Lung-Kok, J.C.O.
Tang, A.S.C. Chan, The preparation and in vitroantiproliferative activity of phthalimide
based ketones on MDAMB-231 and SKHep-1 human carcinoma cell lines,Eur. J. Med.
Chem.,44(2009), 2736–2740.
[27] K.M. Cook, W.D. Figg, Angiogenesis inhibitors – current strategies and future
prospects, CA Cancer J. Clin.,60(2010), 222–243.
22

[28] M. Melchert,A.List, The thalidomide saga,Int. J. Biochem. Cell Biol.39(2007), 1489–
1499.
[29] C.I. Chen, Lenalidomide alone and in combination for chronic lymphocytic
leukemia,Curr.Hematol.Malig. Rep., 8(2013), 7–13.
[30] C.B.Yeh, C.J. Su, J.M. Hwang, M.C. Chou, Therapeutic effects of cantharidin
analogues without bridging ether oxygen on human hepatocellular carcinoma cells,Eur. J.
Med. Chem.,45(2010), 3981.
[31] F.L. Phoebe, Synthesis and antitumor activity of novel pyrazolo[3,4-d]pyrimidine and
their tricyclic derivatives,Int. J. Pharm. Sci. Rev. Res., 24(2)(2014), 208-214.
[32]M.K.Manal, M.R.Sameha, K.A.A.Eman,A.A. Mohamed, F.L.Phoebe,
Design,
synthesis and cytotoxic activity of some novel compounds containing pyrazolo[3,4-
d]pyrimidine nucleus,J. Chem. Sci., 125(5) (2013), 1029-1043.
[33] M.K.Manal, K.A.A.Eman,K.A. Mohammed, A.A. Mohamed, N.P. John, Synthesis and
in vitro antitumor activity of new benzothiazole and benzoxazole derivatives,J. Chem. Pharm.
Res., 5(8)(2013), 16-21.
[34] K.A.A.Eman,N.P. John,Synthesis and cytotoxic activity of new pyrazolo[1,5-
a]pyrimidinesand determination of pyrimidine regiospecific ring formation with 2D NMR,
ARKIVOC, (v)(2016), 210-224.
[35] B.L. Madlen, F.L. Phoebe, Design, synthesis and biological evaluation of novel
thiophene and theinopyrimidine derivatives as anticancer agents,Med. Chem. Res.
25(11)(2016), 2607-2618.
[36] H.R. Stennicke, G.S. Salvesen, Catalytic properties of the caspases, Cell death and
differentiation 6 (1999), 1054 – 1059.
[37]M.T. El Sayed, A.H. Nehal, A.O. Dalia,M.A.Khadiga, Indoles as anti-cancer
Agents,Adv. Mod. Oncol. Res.,1(1) (2015), 20–35.
[38] S.Ghavami, M.Hashemi, S.R.Ande, B. Yeganeh, W. Xiao, M. Eshraghi, C.J. Bus
K.Kadkhoda, E.Wiechec, A.J. Halayko, M.Los, Apoptosis and cancer: mutations
withincaspasegenes,J. Med. Genet., 46(2009), 497.
,
[39] http://www.rcsb.org/pdb/explore/explore.do?structureId=1M17
23

[40] J.M. Edmondson, L.S.Armstrong, A.O. Martinez, Manual of microbiologic
monitoring of laboratory animals,J. Tiss. Cult.Meth.11(1988), 5.
[41] F.Manetti, G.A.Locatelli, G.Maga,S.Schenone, M. Modugno, S. Forli, F. Corell,
M. Botta, Frontiers in drug design and discovery,J. Med. Chem., 49(2006), 3278.
[42] K.K. Lo, T.K. Lee, J.S. Lau, W.L. Poon, S.H. Cheng, Luminescent biological
probes derived from ruthenium (II) estradiol polypyridine complexes,Inorg. Chem., 47(1)
(2008), 200-208.
24

Figure 1.Reported biologically active compounds and a design for the target compounds 5a-
l.
Figure 2. Protein expression level of caspase-7 and caspase-9 in HepG2 and MCF-7 cell
lines treated with compounds 5a and 5g against control untreated cells.
Figure3. Cell cycle analysis and apoptosis effect of compounds 5a and 5g on MCF-7 and
HepG2 cell lines.
Figure 4. Cell cycle analysis using PI staining method by flow cytometryfor5a, 5g, control
MCF-7 and control HepG2.
Figure 5.Graphical representation of % apoptotic cell in case of treatment with compounds
6a and 5g on HepG2 and MCF7 cells, sequentially, against their control.
Figure 6.Effect of compounds 5a and 5g on Bax and Bcl-2 expression levels.
Figure 7.(A) 3D interaction, (B) 2D interaction of compound 5a inside EGFR active site.
Figure 8.(A) 3D interaction, (B) 2D interaction of compound 5g inside EGFR active site.
Scheme 1.Synthesis of the target compounds 5a-l.
Table 1. Cytotoxic activity (IC50) for test compounds 5a-l against HepG2, MCF7 and WI-38
cell lines.
Table 2.IC50 (μM) values of compounds 5a, 5ganderlotenibtowards EGFR and HER2
Table 3.Caspase-7 and caspase-9 concentrations in HepG2 and MCF-7 cell lines treated with
compounds 5a and 5g against control untreated cells.
Table 4.% apoptotic cell in case of treatment with compounds 5a and 5g on HepG2 and
MCF7 cells, sequentially, against their control.
25

Table 1. Cytotoxic activity (IC50) for test compounds 5a-l against HepG2, MCF7 and WI-38
cell lines.
O
R
Y
X
N
N
O
5a-l
Structure
R
*IC50 results (μM)
Compound
no.
X
Y
HepG2
MCF7
WI-38
97.06
84.11
54.71
122.31
35.43
38.33
28.82
42.14
31.04
42.91
45.94
40.09
O
O
O
O
O
O
S
CH3
CN
Cl
H
H
H
0.024
0.211
0.009
0.02
0.011
0.018
0.724
0.326
0.056
0.132
0.098
0.361
0.036
0.021
0.019
0.059
5a
5b
5c
5d
H
COOH
COOH
COOH
H
CH3
Cl
0.028
0.019
0.006
0.468
0.014
0.167
0.013
0.134
5e
5f
CH3
CN
Cl
5g
5h
5i
S
H
S
H
S
H
COOH
COOH
COOH
5j
S
CH3
Cl
5k
5l
S
0.034
--------
0.71
--------
--------
5-FU
---------
Erlotinib
26

Table 2.IC50 (μM)values of compounds 5a, 5ganderlotenibtowards EGFR and HER2
Compound No.
IC50 (μM)
(EGFR)
0.23
IC50 (μM)
(HER2)
0.45
5a
5g
0.35
0.33
0.15
0.27
Erlotenib
aValues are the mean of two independent replicates ± SD.
Table 3.Caspase-7 and caspase-9 concentrations in HepG2 and MCF7 cell lines treated with
compounds 5a and 5g against control untreated cells.
Compound
Results
Caspase-7
conc.ng/ml
Caspase-9
conc.ng/ml
Ser
Cpd. code.
1.449
1.841
22.72
27.56
1.082
1.364
1
2
3
4
5a/HepG2
5g/MCF7
0.3002
0.1994
cont.HepG2
cont.MCF7
27

Table 4. % apoptotic cell in case of treatment with compounds 5a and 5g on HepG2 and
MCF7 cells, sequentially, against their control.
Compound
Results
% G2-M
Comment
No.
5a
% G0-G1
28.39
% S
26.07
34.84
% Pre-G1
21.25
PreG1apoptosis,
Cell growth arrestatG2/M.
PreG1apoptosis,
45.54
33.94
5g
31.22
18.26
Cell growth arrestatG2/M.
Control
HepG2
Control
MCF-7
55.37
48.92
38.02
44.26
6.61
6.82
1.65
1.91
28

O
O
O
R
N
S
NH
N
S
N
O
N
O
H2N
OCF3
O
O
Canthridin containing benzothiazole
Thalidomide
R = H, CH3, Cl, Br, I
3'-Substituted 2-(4-aminophenyl)
benzothiazoles
Molecular hypridization
O
R
Y
N
X
N
O
R = H, CH3, Cl, CN
Y = H, COOH
Design for the target compounds
Figure 1.Reported biologically active compounds and a design for the target compounds 5a-
l.
29