Design, synthesis, and biological evaluation of new challenging thalidomide analogs as potential anticancer immunomodulatory agents

Article abstract of DOI:10.1016/j.bioorg.2020.104218

Thalidomide and its analogs are immunomodulatory drugs that inhibit the production of certain inflammatory mediators associated with cancer. In the present work, a new series of thalidomide analogs was designed and synthesized to obtain new effective antitumor immunomodulatory agents. The synthesized compounds were evaluated for their cytotoxic activities against a panel of four cancer cell lines (HepG-2, HCT-116, PC3 and MCF-7). Compounds 33_h, 33_i, 42_f and 42_h showed strong potencies against all tested cell lines with IC₅₀ values ranging from 14.63 to 49.90 μM comparable to that of thalidomide (IC₅₀ values ranging from 32.12 to 76.91 μM). The most active compounds were further evaluated for their in vitro immunomodulatory activities via estimation of human tumor necrosis factor alpha (TNF-α), human caspase-8 (CASP8), human vascular endothelial growth factor (VEGF), and nuclear factor kappa-B P65 (NF-κB P65) in HCT-116 cells. Thalidomide was used as a positive control. Compounds 33_h and 42_f showed a significant reduction in TNF-α. Furthermore, compounds 33_i and 42_f exhibited significant elevation in CASP8 levels. Compounds 33_i and 42_f inhibited VEGF. In addition, compound 42_f showed significant decrease in levels of NF-κB p65. Moreover, apoptosis and cell cycle tests of the most active compound 42_f, were performed. The results indicated that compound 42_f significantly induce apoptosis in HCT-116 cells and arrest cell cycle at the G2/M phase.

Full text of DOI:10.1016/j.bioorg.2020.104218

BioorganicChemistry104(2020)104218
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis, and biological evaluation of new challenging thalidomide
analogs as potential anticancer immunomodulatory agents
⁎
Mohamed Ayman El-Zahabia, , Helmy Sakra, Khaled. El-Adla,b, Mohamed Zayeda,c,
⁎
Adel S. Abdelraheema, Sally I. Eissad, Hazem Elkadya,b, Ibrahim H. Eissaa,
a Pharmaceutical Medicinal Chemistry & Drug Design Department, Faculty of Pharmacy (Boys), Al-Azhar University, Cairo 11884, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Heliopolis University for Sustainable Development, Cairo, Egypt
c Pharmaceutical Sciences Department, Fakeeh College for Medical Sciences, Jeddah 21461, Saudi Arabia
d Pharmaceutical Medicinal Chemistry & Drug Design Department, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Nasser City, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Thalidomide and its analogs are immunomodulatory drugs that inhibit the production of certain inflammatory
mediators associated with cancer. In the present work, a new series of thalidomide analogs was designed and
synthesized to obtain new effective antitumor immunomodulatory agents. The synthesized compounds were
evaluated for their cytotoxic activities against a panel of four cancer cell lines (HepG-2, HCT-116, PC3 and MCF-
7). Compounds 33h, 33i, 42f and 42h showed strong potencies against all tested cell lines with IC50 values
ranging from 14.63 to 49.90 µM comparable to that of thalidomide (IC50 values ranging from 32.12 to
76.91 µM). The most active compounds were further evaluated for their in vitro immunomodulatory activities via
estimation of human tumor necrosis factor alpha (TNF-α), human caspase-8 (CASP8), human vascular en-
dothelial growth factor (VEGF), and nuclear factor kappa-B P65 (NF-κB P65) in HCT-116 cells. Thalidomide was
used as a positive control. Compounds 33h and 42f showed a significant reduction in TNF-α. Furthermore,
compounds 33i and 42f exhibited significant elevation in CASP8 levels. Compounds 33i and 42f inhibited VEGF.
In addition, compound 42f showed significant decrease in levels of NF-κB p65. Moreover, apoptosis and cell
cycle tests of the most active compound 42f, were performed. The results indicated that compound 42f sig-
nificantly induce apoptosis in HCT-116 cells and arrest cell cycle at the G2/M phase.
Anticancer
Apoptosis
Immunomodulators
Thalidomide
1. Introduction
(TNF-α) and can affect the production of the others, such as interleukin-
1b (IL-1b), IL-2, IL-4, IL-5, IL-6, IL-10, and interferon- γ (IFN- γ) [11].
Thalidomide was formerly recognized as a sedative and it was used
for the treatment of morning sickness especially in pregnant women
[1]. Nevertheless, its serious teratogenic side effects were reported in
1961 and 1962 [2]. Limb defects (amelia and phocomelia) were the
most well-known serious embryotoxic effects caused by the use of
thalidomide during early pregnancy [3]. Thalidomide was banned and
withdrawn from the market, but many studies have continued to reveal
the mechanism of its action [4–7]. Subsequently, thalidomide (1) has
been shown to be efficacious in the treatment of different diseases,
including cancer [8], acquired immunodeficiency syndrome (AIDS) [9],
leprosy [9], multiple myeloma [10], and other angiogenesis-dependent
disorders [11].
Moreover, thalidomide and thalidomide analogs inhibit the secretion of
both beta fibroblast growth factor (bFGF) and vascular endothelial
growth factor (VEGF) from cancer cells and bone marrow stromal cells,
leading to reduction of endothelial cell migration and proliferation and
induction of apoptosis [13].
The concept of using thalidomide in the treatment of cancer
emerged after the discovery of its antiangiogenic effects in the early
1990s [3]. Although the mechanism of thalidomide in cancer treatment
is not fully clear, its analogs have demonstrated immunomodulatory
and anti-inflammatory properties in addition to their anti-angiogenic
effects [14,15].
Second-generation thalidomide analogs, lenalidomide (2), is a po-
tent immunomodulator that is 50,000 times more potent than thali-
domide as an inhibitor of TNF-α [16]. Clinical studies have revealed
that lenalidomide demonstrates fewer side effects and almost neither
Thalidomide is considered as a prototype of the glutarimide-con-
taining immunomodulatory agents [12]. It inhibits the production of
many inflammatory mediators such as tumor necrosis factor-alpha
⁎ Corresponding authors.
E-mail addresses: malzahaby@yahoo.com (M.A. El-Zahabi), Ibrahimeissa@azhar.edu.eg (I.H. Eissa).
https://doi.org/10.1016/j.bioorg.2020.104218
Received 26 February 2020; Received in revised form 4 June 2020; Accepted 22 August 2020
Availableonline01September2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

M.A. El-Zahabi, et al.
BioorganicChemistry104(2020)104218
neurological toxicity nor teratogenicity, compared to thalidomide [17].
Structural modification of thalidomide (1) via the addition of an amino
group at the 4-position of the phthaloyl ring forming pomalidomide (3)
which was 10-fold more potent than lenalidomide (2) as a TNF-α in-
hibitor and interleukin-2 (IL-2) stimulator [18]. It also showed better
anti-angiogenic results than thalidomide (1) and lenalidomide (2) [19].
Thalidomide and its analogs have displayed promising activity
against a wide variety of diseases, including cancer and inflammatory
diseases [20]. The known immunomodulatory and anti-angiogenic
properties of thalidomide analogs provided the motivation to explore
these agents in the treatment of both hematologic malignancies and
solid tumors [8]. Numerous early-phase trials in solid tumors have
shown activity in prostate cancer [21], breast cancer [22], Kaposi’s
sarcoma [23], renal cell cancer [24], melanoma [24], neuroendocrine
tumors [25], hepatocellular carcinoma [26], lung cancer, and gliomas
[27]. Furthermore, thalidomide and thalidomide analogs are effective
agents for the treatment of both lymphoid and myeloid carcinoma with
significant activity in non-Hodgkin’s lymphoma [28]. Also, thalidomide
and thalidomide analogs showed promising activity against acute
myeloid leukemia [29] and myelofibrosis with myeloid metaplasia
[28].
β-aryl carboxamide derivative 4 and β-aryl acetate derivative 5 resulted
in significant inhibition of TNF-α by 500 times that exhibited by tha-
lidomide [48]. Other modifications were carried out by replacement of
the glutarimide moiety with phenyl ring to produce new compounds
with promising biological activity. For example, compounds 6 and 7
demonstrated improved anti-angiogenic and cancer activities [49,50].
Furthermore, variation of alkyl substituents strategy was performed to
get compound 8 as a modified thalidomide analog. Such compound
produced comparable TNF-α inhibitory effect to thalidomide [51].
Moreover, molecular hybridization of phthaloyl nucleus and 1,3,4-
thiadiazole moiety produced compound 9 which exhibited a significant
cytotoxic effect against HepG-2 cells comparable to thalidomide [52].
There is another pathway that has been taken to modify thalidomide
via the extension of extra functional groups strategy. Such extension
was carried out on the glutarimide moiety through the insertion of
dithiocarbamate derivative to produce compound 10. This compound
exhibited in vitro antitumor activity against an ascites carcinoma cell
line [53]. In 2009, Brana et al, synthesized compound 11 via ring
variation pathway, where the phthaloyl nucleus of thalidomide was
replaced by diphenylmaleimide moiety. Such compound showed a
strong inhibitory activity for TNF-α production [54].
The recent reported mechanism of thalidomide (1), lenalidomide
(2) and pomalidomide (3) as immunomodulatory agents is that these
compounds co-stimulate T cells via the degradation of two DNA tran-
scription factors known as Aiolos and Ikaros [30]. In the absence of
thalidomide, lenalidomide and pomalidomide, Aiolos and Ikaros are
expressed and act as repressors of the IL-2 promoter. The presence of
thalidomide, lenalidomide and pomalidomide increases the interaction
of cereblon (CRBN) with Aiolos and Ikaros, causing their ubiquitination
and degradation resulting in de-repression of the IL-2 promoter and
increased expression of IL-2 [30].
Dramatic modifications of thalidomide were carried out to give
compound 12, 13, and 14, where the phthaloyl nucleus was replaced by
4,9-dihydro-2H-[1,2,5]thiadiazolo[2,3-b]isoquinolin-3(3aH)-one 1,1-
dioxide in compound 12, 1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine
in compound 13, and 2-carboxybenzamide in compound 14. TNF-α
inhibitory effect and IL-10 stimulatory effect were reported in vitro on
account of these derivatives (Fig. 2) [55,56].
On the other side, bioisosteric modifications of thalidomide were
accomplished by replacement the glutarimide moiety by many bioi-
sosteres such as benzimidazole, benzothiazole, benzothiadiazole, urea,
thiourea, and thiazolidinone derivatives to afford compounds 15, 16,
17, 18, 19, and 20, respectively. Such derivatives showed promising
antiangiogenic potential, and compound 20 revealed im-
munosuppressive effect [57–59].
In this work, thalidomide (1) was selected a lead compound for the
synthesis of new anticancer agents with immunomodulatory activities.
The synthesized compounds were designed to have the main essential
pharmacophoric features of thalidomide and its analogs. The structures
of thalidomide analogs consist of glutarimide and phthalimide moieties.
It was found that, thalidomide can be a guide for our design, be-
cause it has the following criteria. i) Immunomodulation: It can co-
stimulate T-cells resulting in synthesis of specific T-killer cells which
target the cancer cells [31]. ii) The common side effects of cytotoxic
anticancer agents were not reported for thalidomide [32]. iii) Different
mechanisms were reported for thalidomide as anticancer drug [33].
Beside immunomodulation, it was reported to cause ubiquitination of
Ikaros and Aiolos, resulting in apoptosis and arresting cell life cycle
[34]. It also was reported to have anti-angiogenic activity by inhibition
of both VEGF and bFGF-2 [35]. Moreover, it inhibits the adhesion be-
tween tumor cells and stromal cells preventing signals which stimulate
tumor cells survival, growth and development of drug resistance [36].
iv) The teratogenicity of thalidomide was reduced in rabbit model via
structural modification to produce lenalidomide with higher anticancer
activity [12].
Additionally, the potential anticancer activity of some bioactive
moieties was explored. 1,3,4-Oxadiazole nucleus was proven to have
potential anticancer effect [60,61]. Compounds 21 and 22 as examples
of compounds containing 1,3,4-oxadiazole moiety showed in vitro an-
ticancer activity against breast cancer (MCF-7) cell line. Thiazolidine-
2,4-dione is another example of interesting bioactive moieties having
potential anticancer effect. Compounds 23, 24 and 25 incorporated
such moiety were reported to possess in vitro anti-breast cancer activity
(Fig. 2) [62].
Depending on the previously mentioned structural modification and
ligand-based drug design approach, we used thalidomide 1 as a lead
compound to get new effective immunomodulatory anticancer agents.
The rational of molecular design was based on molecular hybridization
strategy which involves the coupling of two or more groups with re-
levant biological properties to produce new compounds with promising
activities [63,64]. Accordingly, the aminopiperidinedione moiety was
hybridized with thiazolidinedione derivatives to produce (Scaffold-I),
and with oxadiazole derivatives to obtain (Scaffold-II) (Fig. 3).
The main core of our molecular design rationale comprised mole-
cular modification of thalidomide at three different positions (Fig. 4).
The first position was the phenyl ring; where different substituted
phenyl derivatives were used. The substituted phenyl derivatives may
be directly attached to heterocyclic ring as compounds 42a-i or sepa-
rated by one carbon-bridge as compounds 33a-j. The second position
was the 1H-pyrrole-2,5-dione ring. This five-membered ring was re-
1.1. Rationale of molecular design
For many years, our research group have many efforts to design and
synthesize new compounds with potential anticancer effect [37–47]. As
extension for such efforts, we carried out this work. Studying the
structure-activity relationship of thalidomide analogs (thalidomide (1),
lenalidomide (2) and pomalidomide (3)), it was found that they have
four common essential pharmacophoric features. These features in-
clude: i) hydrophobic domain, ii) five membered ring, iii) spacer, and
iv) glutarimide moiety (Fig. 1).
placed by different rings as thiazolidine-2,4-dione as compounds 33a-j
,
and 1,3,4-oxadiazole as compounds 42a-i. The third position was the
spacer region. The length of the spacer was modified to be three
(compounds 33a-j), or four (compounds 42a-i) atom bridges. Such
spacers were expected to increase flexibility of the designed com-
pounds.
By literature survey, it was found that there are many efforts to
modify thalidomide with the primary aim of obtaining more effective
new immunomodulatory agents free from harmful effects.
Briefly, replacement of the glutarimide moiety of thalidomide with
2

M.A. El-Zahabi, et al.
BioorganicChemistry104(2020)104218
O
O
O
O
O
O
NH
O
NH
NH
N
N
O
N
O
O
O
NH2
NH2
Lenalidomide (2)
Thalidomide (1)
(3)
Pomalidomide
Spacer
Hydrophobic
domain
Five membered
ring
Glutarimide moiety
Fig. 1. Pharmacophoric features of thalidomide, lenalidomide, pomalidomide.
As shown in Fig. 5, our target compounds have the main the
pharmacophoric features of the lead compound (thalidomide).
In general, to examine the cytotoxic effect of the synthesized com-
pounds, they were evaluated in vitro against HepG-2, HCT-116, PC3,
and MCF-7 cell lines. The reported activities of thalidomide analogs to
activate CASP8 and to inhibit TNF-α, VEGF, and NF-κB P65 drove us for
compounds 11b and 11d as representative examples exhibited mole-
cular ion peaks at 407 and 391, respectively.
Ester derivatives 35a-i were prepared according to the reported
procedures [73] by refluxing the appropriate acid derivatives (34a-i
)
namely, benzoic acid, 2-chlorobenzoic acid, 3-chlorobenzoic acid, 4-
chlorobenzoic acid, 4-nitrobenzoic acid, 4-aminobenzoic acid, picolinic
acid, nicotinic acid and/or isonicotinic acid in methanol in the presence
of sulfuric acid [73]. Reflux of 35a-i with hydrazine hydrate afforded
the corresponding acid hydrazides 36a-i. [73] Moreover, reflux of 36a-i
in alcoholic mixture of carbon disulphide and potassium hydroxide
produced 37a-i [73,74]. The treatment of compounds 37a-i with alco-
holic solution of potassium hydroxide afforded potassium salt of 5-(un)
substituted aryl-1,3,4-oxadiazole-2-thiol 38a-i [73,74]. On the other
hand, compound 41 was produced in two phase system (DCM and
water) in the presence of two molar equivalent of NaHCO3 in ice-salt
bath. Then, heating a mixture of compound 41 with 38a-i in dry DMF
further examinations to reach
a deep insight about the im-
munomodulatory effect of the synthesized compounds. Hence, the most
cytotoxic agents were examined for their immunomodulatory activities
via determination of their effect on TNF-α, CASP8, VEGF, and NF-κB
P65 in HCT-116. Moreover, the effect of the most active compound 42f
on apoptosis and cell cycle was investigated in HCT-116 cell line.
2. Results and discussion
2.1. Chemistry
yielded the corresponding target compounds 42a-i
, respectively
(scheme 2).
For synthesis of the target compounds, reactions sequence is clar-
ified in Schemes 1 and 2. At first, the starting material thiazolidine-2,4-
dione (TZD) 28 was synthesized according to the reported procedures
by the reaction of chloroacetic acid 26 with thiourea 27 under reflux in
conc. HCl [65]. Condensation of compound 28 with certain aromatic
aldehydes namely, benzaldehyde, 2-chlorobenzaldehyde, 4-chlor-
obenzaldehyde, 2-methoxybenzaldehyde, 4-methoxybenzaldehyde, 4-
fluorobenzaldehyde, 4-methylbenzaldehyde, 4-nitobenzaldehyde, 3-ni-
tobenzaldehyde and 2,6-dichlorobenzaldehyde in absolute ethanol with
catalytic amount of piperidine yielded the corresponding compounds
29a-j, respectively [65–67]. Subsequent treatment of compounds 29a-j
with potassium hydroxide produced the corresponding potassium salts
30a-j, respectively based on the reported procedure of preparation of
organic slats [68,69]. Reflux of 30a-j with methyl bromoacetate af-
forded the corresponding compounds 31a-j, respectively [65,66] which
in turn were hydrolyzed in acidic medium providing the corresponding
acids 32a-j in high yields [65,66]. The obtained compounds 32a-j were
allowed to react with ethyl chloroformate to give the corresponding
mixed anhydride. Then, 3-aminopiperidine-2,6-dione HCl reacted with
the obtained mixed anhydride to afford the final target compounds 33a-
j, respectively (scheme 1).
IR spectra of compounds 42a-i showed stretching bands at a range of
3360–3198 cm−1 corresponding to NH groups. 1H NMR spectra of
compounds 42a-i exhibited singlet signals for amidic protons at a range
of 8.72–8.74 ppm. Also, singlet signals appeared at range of δ
10.86–10.88 ppm corresponding to the imidic protons. Moreover, the
two protons of CH2CO appeared as two multiplet signals around 2.48
and 2.78 ppm due to enantiotropicity [70–72]. Mass spectroscopic
analyses for compounds 42b and 42h exhibited molecular ion peaks at
379 and 347, respectively.
2.2. Biological testing
2.2.1. In vitro anti-proliferative activity
The synthesized compounds have been tested for their in vitro an-
ticancer activities via MTT method [75], against four human tumor cell
lines; hepatocellular carcinoma (HepG-2), colorectal carcinoma (HCT-
116), prostate cancer (PC3) and mammary gland cancer (MCF-7). The
results of cytotoxicity were stated as IC50 values and summarized in
Table 1.
The cytotoxicity results indicated that the majority of the examined
compounds showed anti-proliferative activities against the tested cell
lines. In general, compounds 33h, 33i, 42f and 42h were the most active
members of this study, showing promising anti proliferative activities
against HepG-2, HCT-116, PC3 and MCF-7 with IC50 far less than the
The IR spectra of 33a-j showed no bands for carboxylic group and
demonstrated stretching bands at a range of 3419–3202 cm−1 corre-
sponding to NH groups. Moreover, 1H NMR of these compounds ex-
hibited neither signals for carboxylic proton nor signals for free NH2 of
3-aminopiperidine-2,6-dione. Instead, it showed a signal for single
proton at 8.72–8.74 ppm which indicates the presence of amidic proton
whereas the imidic proton of glutarimide ring appeared as a singlet
signal at a range of δ 10.86–10.88 ppm. Moreover, the two protons of
CH2CO appeared as two multiplet signals around 2.48 and 2.75 ppm
due to enantiotropicity [70–72]. Mass spectroscopic analyses for
reference compound, thalidomide, (IC50 = 61.10
76.91 4.2 and 45.76
cytotoxic activities with IC50 values of 21.34
42.96 2.9 and 19.62 1.6, respectively. Compound 33i had cy-
totoxic activities with IC50 values of 23.96 2.0, 36.07 3.2,
49.90 3.4 and 21.82 1.8 μM, respectively. Moreover, compound
3.8, 32.12
3.6, respectively). Compound 33h exhibited
1.8, 29.38 2.8,
3.2,
3

M.A. El-Zahabi, et al.
BioorganicChemistry104(2020)104218
O
F
F
O
F
F
O
O
O
N
N
O
N
O
O
O
O2N
NH2
O
O
4
( )
6
( )
5
( )
O
N
S
N
O
O
O
N
O
NH
S
N
O
N
O
O
O
HO
O
O
O
9
( )
8
( )
7
( )
N
O
O
S
N
NH
N
O
O
O
S
O
O
N
O
S
N
O
O
N
O
O
N
N
S
O
O
O
12
(
)
N
11
(
)
10
(
)
O
O
O
N
N
HN
O
N
N
N
N
H
H
N
H
S
O
CO2H
HO2C
O
(13)
15
(
)
14
(
)
S
O
O
O
S
N
N
O
O
NH2
NH2
NH
S
N
NH
N
N
N
N
O
O
O
O
19
)
(
16
(
)
18
( )
17
(
)
OH
O
N
N
O
NH
S
N
N
S
O
N
NH
N
O
O
N
N
O
O2N
n=6
O
22
(
)
21
( )
20
(
)
OMe
O
OMe
OMe
O
N
O
O
O
MeO
MeO
MeO
MeO
MeO
S
O
N
S
S
N
N
N
MeO
O
O
O
25
(
)
24
( )
23
(
)
Fig.2. Literature based structures of modified thalidomide analogues and anticancer agents.
42f revealed IC50 values of 16.48
and 14.63 1.3, respectively. Furthermore, compound 42h found to
have cytotoxic activities with IC50 values of 19.47 1.7,
1.6, 23.94
2.3, 28.43
2.0
32.60
2.9, 46.81
3.0 and 18.15
1.5 μM, respectively.
Moreover, several compounds such as 33a, 33f, 33j, 42e and 42i
showed moderate anti-proliferative activities with IC50 values ranging
4

M.A. El-Zahabi, et al.
BioorganicChemistry104(2020)104218
Fig. 3. Rationale concept to new thalidomide analogs.
from 28.13
2.2 to 72.65
4.6 µM against all cell lines. On the
2.2.2.1. Estimation of human tumor necrosis factor alpha (TNF-α) in HCT-
116 supernatant. In this test, the effect of the synthesized compounds
33h, 33i, 42f and 42h on TNF-α was examined. Thalidomide was used
as a positive control. Two negative controls were used, the first one was
untreated HCT-116 cells (control) and the second one was DMSO
treated HCT-116 cells (control-DMSO).
other hand, compounds 33c, 33d, 42a-d and 42g showed weak anti-
proliferative activities against the tested cell. Finally, compounds 33b,
33e and 33g were appeared to be inactive.
2.2.2. In vitro immunomodulatory assay
The data presented in Fig. 6 showed that, production of TNF-α was
significantly decreased in HCT-116 cells exposed to thalidomide and
the synthesized compounds 33h, 33i, 42f and 42h, compared with
control and control-DMSO cells. Comparing all the synthesized com-
pounds with thalidomide revealed that compounds 33h and 42f showed
Four compounds 33h, 33i, 42f and 42h which exhibited cytotoxic
activities higher than thalidomide against HCT-116 cancer cell line
were selected and evaluated for their inhibitory effect on TNF-α and
NF-κB P65 as well as their apoptotic and antiangiogenic activities.
Thalidomide was used as a positive control in these procedures.
O
O
H
N
N
O
Thalidomide
O
Different spacers
three or four atoms
Thiazolidine2,4-dione
1,3,4-Oxadiazole
Glutarimide moeity
remained intact
Selected ring
substituents
Fig. 4. Summary of structural modification modification of thalidomide.
5

M.A. El-Zahabi, et al.
BioorganicChemistry104(2020)104218
O
N
N
O
NH
H
O
N
O
Ar
N
O
S
NH
O
S
NH
O
X
O
O
(42a-i)
(33a-j)
Fig. 5. New target compounds having the same essential pharmacophoric features of thalidomide.
remarkable significant reduction in TNF-α levels while compounds 33i
and 42h represented insignificant increase in TNF-α levels in relation to
thalidomide.
significant elevation in CASP8 levels. Furthermore, insignificant
increase in CASP8 levels were presented after treatment with
compounds 33h and 42h as compared with thalidomide.
2.2.2.2. Estimation of human caspase-8 (CASP8) in HCT-116
supernatant. In this test, the effect of thalidomide and the synthesized
compounds 33h, 33i, 42f and 42h on CASP8 was examined. As
illustrated in Fig. 7, thalidomide and the synthesized compounds
showed a statistically significant increase in CASP8 levels compared
with control and control-DMSO cells. Comparing the tested compounds
with thalidomide revealed that compounds 33i and 42f exhibited
2.2.2.3. Estimation of human vascular endothelial growth factor (VEGF) in
HCT-116 supernatant. Assessment of the effect of thalidomide and the
synthesized compounds 33h, 33i, 42f and 42h on VEGF was
determined. The data in Fig. 8 indicated that, VEGF concentration
was significantly decreased in HCT-116 cells after exposure to
thalidomide and the synthesized compounds in comparison to the
control and control-DMSO cells. Compounds 33i and 42f induced
O
O
NH
S
O
i
ii
S
Cl
X
+
NH
H2N
NH2
OH
S
O
O
(29a-j
)
(26)
(27)
(28)
iii
O
N k
O
O
iv
S
S
O
X
X
N
O
O
(31a-j
)
(30a-j
)
v
O
NH
O
N
O
N
O
O
vi
S
NH
S
OH
O
X
X
O
O
(33a-j
)
a) X= H
b) X= 2-Cl
c) X= 4-Cl
d) X= 2-OCH3
e) X= 4-OCH3
f) X= 4-F
(32a-j
)
g) X= 4-CH3
h) X= 4-NO2
i) X= 3-NO2
j) X= 2,6-diCl
Scheme 1. General procedure for preparation of target compounds 33a-j; Reagents and conditions: (i) 4 N conc. HCl/reflux/10 h, (ii) absolute ethanol/piperidine/
reflux/10 h, (iii) absolute ethanol/KOH/reflux, (iv) methyl bromoacetate/KI/Dry DMF/heating in water bath/4h, (v) glacial acetic acid/HCl/reflux/5h, (vi) TEA/
ethyl chloroformate/30 min/TEA/3-aminopiperidine-2,6-dione HCl /r.t./12 h.
6

M.A. El-Zahabi, et al.
BioorganicChemistry104(2020)104218
O
O
O
i
ii
NH2
Ar
N
Ar
OH
Ar
O
H
(34a-i
)
(36a-i
)
(35a-i
)
iii
H
N
O
O
Cl
N
O
+
Cl
N
Ar
O
NH2HCl
SH
(39)
(37a-i
)
(40)
v
iv
O
O
HN
Cl
N
O
N
N
H
Ar
O
S K
(41)
(38a-i
)
a) Ar
= phenyl
vi
b) Ar
c) Ar
d) Ar
e) Ar
f) Ar
2-chlorophenyl
3-chlorophenyl
4-chlorophenyl
=
=
=
N
N
O
H
Ar
N
O
S
NH
4-nitrophenyl
4-aminophenyl
pyridine-2-yl
pyridine-3-yl
=
=
O
O
(42a-i
)
g) Ar
h) Ar
i) Ar
=
=
=
pyridine-4-yl
Scheme 2. General procedure for preparation of target compounds 42a-i; Reagents and conditions: (i) conc.H2SO4/methanol/reflux/5h, (ii) NH2NH2·H2O/absolute
ethanol/reflux /6h, (iii) absolute ethanol/KOH/CS2/reflux/6h, (iv) absolute ethanol/KOH/reflux, (v) NaHCO3/DCM/H2O, (vi) KI/acetonitrile/stirring at r.t./3h.
significantly lower levels of VEGF while compound 33h induced
significantly higher level of VEGF in relation to thalidomide. In
addition, compound 42h showed insignificant increase in VEGF level
as compared to thalidomide.
Compound 33i exhibited decrease in NF-κB p65 level although it’s
statistically insignificant.
2.2.3. Effect on cell cycle progression
Flow cytometric analysis technique [76] was used to examine the
effect of compound 42f on cell cycle in HCT-116 cell line. The HCT-116
cells had been incubated for 24 h with 10 μM of compound 42f. Then,
cell cycle parameters of incubated cells were compared with untreated
control cells. The results revealed that HCT-116 cells accumulated at
G2/M phase with percent of 40.52%. Moreover, the tested compound
showed good ability to induce apoptosis at pre-G1 phase (Table 2,
Figs. 10 and 12).
2.2.2.4. Estimation of nuclear factor kappa-B P65 (NF-κB P65) in HCT-
116 cell lysate. The effect of thalidomide and the synthesized
compounds 33h, 33i, 42f and 42h on protein expression of NF-κB p65
was evaluated in the cell lysate.
According to the data in Fig. 9, there is a statistically significant
decrease in NF-κB p65 levels after exposure of HCT-116 cells to thali-
domide and the synthesized compounds. Comparing the synthesized
compounds with thalidomide itself revealed that compound 42f showed
significant decrease in level of NF-κB p65, in contrast, compounds 33h
and 42h demonstrated a significant increase in levels of NF-κB p65.
2.2.4. Induction of apoptosis
Annexin V/propidium iodide (PI) double staining assay method was
7

M.A. El-Zahabi, et al.
BioorganicChemistry104(2020)104218
Table 1
thalidomide as a lead compound. Some derivatives exhibited anti-pro-
Anti-proliferative activities of the target compounds against HepG-2, HCT-116,
PC3 and MCF-7 cell lines.
liferative activities higher than thalidomide against HepG-2, HCT-116,
PC3 and MCF-7 cell lines such as 33h (IC50 = 21.34
1.8,
Comp.
In vitro Cytotoxicity IC50 (µM)a
29.38
(IC50
2.8, 42.96
23.96
2.9 and 19.62
2.0, 36.07
1.6 μM, respectively), 33i
=
3.2, 49.90
1.8 μM, respectively), 42f (IC50 = 16.48
3.4 and
1.6,
HepG-2
HCT-116
PC3
MCF-7
21.82
23.94
2.3, 28.43
2.0 and 14.63
1.7, 32.60
1.3 μM, respectively) and
Thalidomide
33a
61.10
42.35
NAb
3.8
2.8
32.12
49.64
90.78
59.26
62.75
82.68
52.48
NAb
3.2
3.9
5.6
4.5
4.6
5.3
4.1
76.91
69.34
NAb
4.2
4.3
45.76
35.48
88.92
60.39
69.74
86.96
55.10
NAb
3.6
2.7
5.5
3.9
4.5
5.4
3.7
42h (IC50 = 19.47
2.9, 46.81
3.0 and
18.15
1.5 μM, respectively). Moreover, some compounds showed a
33b
33c
67.22
79.12
NAb
4.1
4.8
76.01
83.81
NAb
4.8
5.4
significant reduction in TNF-α (compounds 33h and 42f), VEGF (com-
pounds 33i and 42f) and NF-κB p65 (compounds 42f). Furthermore,
some compounds exhibited significant elevation in CASP8 levels as
compounds 33i and 42f). Additionally, Flow cytometry analysis de-
monstrated that compound 42f could significantly induce apoptosis
(21.43%) of HCT-116 cells at a concentration of 10 µM and can arrest
the cell cycle at G2/M phase. Structure-activity relationship study re-
vealed that oxadiazole derivatives were more active than thiazolidine-
2,4-dione ones. These results indicate that the designed compounds can
act as promising immunomodulators and effective anticancer agents.
Additionally, further optimization of these derivatives may result in
discovering more promising immunomodulators.
33d
33e
33f
63.04
NAb
4.0
72.65
NAb
4.6
33g
33h
33i
21.34
23.96
31.75
69.92
65.58
56.70
91.26
28.13
16.48
81.84
19.47
37.62
1.8
2.0
2.5
4.3
4.0
3.7
5.4
2.2
1.6
4.8
1.7
2.6
29.38
36.07
40.62
60.41
55.29
45.60
78.11
38.26
23.94
66.28
32.60
46.33
2.8
3.2
3.4
4.6
4.4
3.6
5.0
3.3
2.3
4.8
2.9
3.7
42.96
49.90
55.78
78.36
74.50
61.38
94.56
52.23
28.43
85.44
46.81
65.82
2.9
3.4
3.8
5.0
4.7
4.0
5.9
3.5
2.0
5.6
3.0
4.2
19.62
21.82
29.87
63.50
58.02
49.28
80.73
25.49
14.63
72.03
18.15
31.94
1.6
1.8
2.1
4.1
3.9
3.5
4.9
1.9
1.3
4.7
1.5
2.4
33j
42a
42b
42c
42d
42e
42f
42g
42h
42i
4. Experimental
a
b
Three independent experiments were performed for each concentration.
Compounds having IC50 value > 100 μM.
4.1. Chemistry
used to clarify the mechanism of induced cell death in HCT-116 cells at
pre-G1 phase. HCT-116 cells were treated with 10 μM of the tested
compound 42f. The results revealed that the percentage of total apop-
tosis induced by compound 42f was 21.34. In the early stage, there was
significant increase in the percentage of apoptotic cells from 0.42% for
control untreated cells up to 5.49%. Additionally, in the late phase,
there was an increase in the apoptotic cells to attain 13.1% in com-
parison to 0.51% in control HCT-116 cells. The obtained results re-
vealed that compound 42f induced pre-G1 apoptosis and arrested cell
cycle at G2/M phase (Table 3 & Figs. 11 and 12).
All melting points were performed by the open capillary method on
a Gallen kamp device. The infrared spectra were verified using the
potassium bromide disk technique on a pye Unicam SP 1000 IR spec-
trophotometer. Proton magnetic resonance 1H NMR spectra and 13C
NMR spectra were recorded on a BRUKER 400 MHZ- NMR spectro-
meter. Chemical shifts were measured in δ scale (ppm) and TMS was
used as internal standard. The mass spectra were recorded on Varian
MAT 311-A (70 e.v.). Elemental analyses (C, H, N) of the tested com-
pounds were carried out on a CHN analyzer. All compounds were
within
0.4 of the theoretical values. The reactions were monitored
by thin-layer chromatography (TLC) using TLC sheets precoated with
UV fluorescent silica gel Merck 60 F254 plates and were envisaged
using UV lamp and different solvents as mobile phases. Compounds 28,
29a-j, 30 a-j, 31 a-j, 32a-j, 35a-i, 36a-i, 37a-i, and 38a-i were synthesized
according to reported methods [65,66,73,74,77]
3. Conclusion
In summary, nineteen derivatives of 3-aminopiperidine-2,6-dione
derivatives have been designed and synthesized depending on
Fig. 6. Levels of TNF α in the cell supernatant after
exposure to 10 μM of thalidomide and the synthesized
compounds on HCT-116 cells. (a) Denotes group sta-
tistically significant from control cells, (b) Denotes
group statistically significant from control-DMSO
cells, (c) Denotes group statistically significant from
TNF
150
thalidomide. Results are expressed as mean
*(P < 0.05), **(P < 0.01),***(P < 0.001).
SEM.
100
a****
a****
50
b****
b****
c**
c*
0
Control
DMSO Thalidomide
33h
33i
42f
42h
Tested compounds against thalidomide, DMSO and control
8

M.A. El-Zahabi, et al.
BioorganicChemistry104(2020)104218
Fig. 7. Levels of CASP8 in the cell supernatant after
exposure to 10 μM of thalidomide and the synthesized
compounds on HCT-116 cells. (a) Denotes group sta-
tistically significant from control cells, (b) Denotes
group statistically significant from control-DMSO
cells, (c) Denotes group statistically significant from
CASP8
20
a****
b****
c**
a****
b****
c**
thalidomide. Results are expressed as mean
*(P < 0.05), **(P < 0.01),***(P < 0.001).
SEM.
15
10
5
0
Control
DMSO
Thalidomide
33h
33i
42f
42h
Tested compounds against thalidomide, DMSO and control
Fig. 8. Levels of VEGF in the cell supernatant after
exposure to 10 μM of thalidomide and the synthesized
compounds on HCT-116 cells. (a) Denotes group sta-
tistically significant from control cells, (b) Denotes
group statistically significant from control DMSO cells,
(c) Denotes group statistically significant from thali-
VEGF
500
400
300
200
100
0
domide. Results are expressed as mean
SEM.
*(P < 0.05), **(P < 0.01),***(P < 0.001).
a****
b****
c**
a****
b****
c***
Control
DMSO
Thalidomide
33h
33i
42f
42h
Tested compounds against thalidomide, DMSO and control
4.1.1. General procedure for synthesis of compounds (11a-j
)
m.p. = 266–268 °C. IR (KBr, cm−1): 3360, 3225 (NH), 3134 (CH
To a suspension of the appropriate acid 32a-j (3 mmol) in DCM
(10 mL), Et3N (0.33 g, 0.46 mL, 3.30 mmol) was added. The reaction
mixture was stirred in ice-salt bath for 10 min. To the previous clear
solution, ethyl chloroformate (0.35 g, 0.31 mL, 3.30 mmol) was added
in a drop wise manner over a period of 20 min. and the reaction mixture
was stirred for 1 h in the ice-salt bath. A solution of 3-aminopiperidine-
2,6-dione HCl (0.49 g, 3 mmol) and Et3N (0.33 g, 0.46 mL, 3.30 mmol)
in DCM (10 mL) was added to the reaction mixture. The whole mixture
was allowed to stir for 12 h at r.t. and the obtained precipitates were
filtered, washed with water followed by hot DCM and dried to furnish
the corresponding target compounds 33a-j, respectively.
aromatic), 2972 (CH aliphatic) and 1692 (C]O amide); 1H NMR
(DMSO‑d6)
δ ppm: 1.91–1.97 (m, 2H, CH2CH of piperidine),
2.48–2.52 (m, 1H, CH2CO of piperidine), 2.68–2.75 (m, 1H, CH2CO
of piperidine), 4.36 (s, 2H, CH2CONH), 4.40–4.62 (m, 1H, CH of
piperidine), 7.49–7.53 (dd, 1H, C-4 phenyl, J = 8.4 Hz & 8.2 Hz),
7.54–7.58 (dd, 2H, C-3 and C-5 phenyl, J = 8.4 Hz & 8 Hz), 7.64–7.66
(d, 2H, C-2 and C-6 phenyl, J = 8 Hz), 7.98 (s, 1H, eC]CH), 7.72–7.74
(d, 1H, J = 8.1 Hz, NHCO, D2O exchangeable), 10.87 (s, 1H, CONHCO,
D2O exchangeable); 13C NMR (DMSO‑d6, 100 MHz) δ (ppm): 24.71,
31.25, 43.85, 49.90, 121.58, 129.90(2C), 130.64(2C), 131.25, 133.35,
133.88, 165.61, 165.70, 167.51, 172.19, 173.32; Anal. Calcd. for
C
17H15N3O5S (373.38): C, 54.69; H, 4.05; N, 11.25. Found: C, 54.30;
H, 4.09; N, 11.51%.
4.1.1.1. 2-(5-Benzylidene-2,4-dioxothiazolidin-3-yl)-N-(2,6-
dioxopiperidin-3-yl) acetamide (33a). White crystal (yield, 75%);
9

M.A. El-Zahabi, et al.
BioorganicChemistry104(2020)104218
Fig. 9. Levels of NF-κB p65 in the cell lysate after
exposure to 10 μM of thalidomide analogs on HCT-
116 cells. (a) Denotes group statistically significant
from control cells, (b) Denotes group statistically
significant from control DMSO cells, (c) Denotes
group statistically significant from thalidomide.
NF- B P65
25
20
15
10
5
Results are expressed as mean
**(P < 0.01),***(P < 0.001).
SEM. *(P < 0.05),
a****
b****
c*
0
33i
42f
33h
42h
Control
DMSO
Thalidomide
Tested compounds against thalidomide, DMSO and control
Table 2
4.1.1.2. 2-(5-(2-Chlorobenzylidene)-2,4-dioxothiazolidin-3-yl)-N-(2,6
dioxopiperidin-3-yl)acetamide (33b). Grayish white crystal (yield, 67%);
m.p. = 245–247 °C. IR (KBr, cm−1): 3346, 3249 (NH), 3135 (CH
aromatic), 2990 (CH aliphatic) and 1695 (C]O amide); 1H NMR
Effect of compound 42f on cell cycle progression in HCT-116 cells.
Sample
Cell cycle distribution (%)
%G0/G1
%S
%G2/M
%Pre-G1
(DMSO‑d6)
δ ppm: 1.88–1.97 (m, 2H, CH2CH of piperidine),
2.50–2.54 (m, 1H, CH2CO of piperidine), 2.68–2.78 (m, 1H, CH2CO
of piperidine), 4.37 (s, 2H, CH2CONH), 4.56–4.62 (m, 1H, CH of
piperidine), 7.53–7.55 (dd, 1H, C-4 phenyl), 7.61–7.65 (m, 2H, C-5 and
C-6 phenyl), 7.65–7.67 (d, 1H, C-3 phenyl), 8.06 (s, 1H, eC]CH),
8.72–8.74 (d, 1H, J = 7.9 Hz, NHCO, D2O exchangeable), 10.88 (s, 1H,
CONHCO, D2O exchangeable); MS (m/z): 409 (M++2, 4.58%), 407
(M+, 17.92%), 372 (100%, base peak); Anal. Calcd. for C17H14ClN3O5S
(407.83): C, 50.07; H, 3.46; N, 10.30. Found: C, 50.10; H, 3.26; N,
10.22%.
42f/HCT-116 cell
41.29
57.41
18.19
26.84
40.52
15.75
21.34
2.19
Cont. HCT-116 cell
Cell Cycle Analysis
70
60
50
40 %
30
20
10
0
42f / HCT-116 cell
Cont. HCT-116 cell
57.41
41.29
40.52
4.1.1.3. 2-(5-(4-Chlorobenzylidene)-2,4-dioxothiazolidin-3-yl)-N-(2,6-
dioxopiperidin-3-yl)acetamide (33c). White crystal (yield, 75%);
m.p. = 256–258 °C. IR (KBr, cm−1): 3431, 3284 (NH), 3102 (CeH
aromatic), 2907 (CeH aliphatic) and 1690 (C]O amide); 1H NMR
(DMSO‑d6) δ ppm: 1.93–1.97 (m, 2H, CH2CH of piperidine), 2.50–2.51
(m, 1H, CH2CO of piperidine), 2.70–2.74 (m, 1H, CH2CO of piperidine),
4.36 (s, 2H, CH2CONH), 4.55–4.62 (m, 1H, CH piperidine), 7.61–7.63
(d, 2H, C-3 and C-5 phenyl, J = 8 Hz), 7.67–7.69 (d, 2H, C-2 and C-6
phenyl, J = 8 Hz), 7.98 (s, 1H, eC]CH), 8.70–8.72 (d, 1H, J = 8.1 Hz,
NHCO, D2O exchangeable), 10.86 (s, 1H, CONHCO, D2O
exchangeable); Anal. Calcd. for C17H14ClN3O5S (407.83): C, 50.07; H,
3.46; N, 10.30. Found: C, 50.25; H, 3.12; N, 10.24%.
26.84
18.19
21.34
15.75
2.19
%Pre-G1
%G2-M
%S
%G0-G1
Cell cycle distribuꢀon (%)
Fig. 10. Cell cycle analysis and apoptosis effect in HCT-116 cell line when
treated with compound 42f.
Table 3
4.1.1.4. N-(2,6-Dioxopiperidin-3-yl)-2-(5-(2-methoxybenzylidene)-2,4-
dioxothiazolidin-3-yl)acetamide (33d). Yellow crystal (yield, 69%);
m.p. = 272–274 °C. IR (KBr, cm−1): 3311, 3202 (2NH), 3090 (CeH
aromatic), 2935 (CeH aliphatic) and 1693 (C]O amide); 1H NMR
(DMSO‑d6) δ ppm: 1.94–1.97 (m, 2H, CH2CH of piperidine), 2.50–2.51
(m, 1H, CH2CO of piperidine), 2.68–2.78 (m, 1H, CH2CO of piperidine),
3.90 (s, 3H, OCH3), 4.35 (s, 2H, CH2CO), 4.55–4.62 (m, 1H, CHCO),
7.10–7.14 (dd, 1H, C-5 phenyl, J = 8.1 Hz & 7.6 Hz), 7.17–7.19 (d, 1H,
J = 8.4 Hz, C-3 phenyl), 7.46–7.48 (d, 1H, C-6 phenyl, J = 8.1 Hz),
7.50–7.54 (dd, 1H, C-4 phenyl, J = 8.3 &7.1 Hz), 8.10 (s, 1H, eC]CH),
8.70–8.72 (d, 1H, J = 8.1 Hz, NHCO, D2O exchangeable), 10.89 (s, 1H,
Apoptosis and necrosis percent induced by compound 42f on HCT-116 cells.
Sample
Apoptosis
Necrosis
Total
Early
Late
42f/HCT-116
21.34
2.19
5.49
0.42
13.1
0.51
2.75
1.26
Cont. HCT-116
10

M.A. El-Zahabi, et al.
BioorganicChemistry104(2020)104218
Apoptosis Analysis
NMR (DMSO‑d6) δ ppm: 1.94–1.96 (m, 2H, CH2CH of piperidine),
2.48–2.53 (m, 1H, CH2CO of piperidine), 2.68–2.77 (m, 1H, CH2CO of
piperidine), 3.83 (s, 3H, OCH3), 4.35 (s, 2H, CH2CO), 4.55–4.61 (m,
1H, CHCO), 7.10–7.12 (d, 2H, C-3 and C-5 phenyl, J = 8.1 Hz),
7.60–7.62 (d, 2H, C-2 and C-6 phenyl, J = 7.8 Hz), 7.91 (s, 1H,
eC]CH), 8.70–8.72 (d, 1H, J = 8.2 Hz, NHCO, D2O exchangeable),
10.87 (s, 1H, CONHCO, D2O exchangeable); 13C NMR (DMSO‑d6,
100 MHz) δ (ppm): 21.59, 24.71, 31.24, 43.81, 49.89, 120.33,
130.52(2C), 130.60(2C), 130.72, 133.94, 141.63, 165.64, 165.76,
167.54, 172.18, 173.32; Anal. Calcd. for C18H17N3O6S (403.41): C,
53.59; H, 4.25; N, 10.42. Found: C, 53.48; H, 4.32; N, 10.51%.
42f / HCT-116
Cont. HCT-116
25
20
15
10
5
21.34
13.1
%
5.49
2.75
1.26
2.19
0.42
0.51
Late
0
Necrosis
Early
Total
4.1.1.6. N-(2,6-Dioxopiperidin-3-yl)-2-(5-(4-fluorobenzylidene)-2,4-
dioxothiazolidin-3-yl)acetamide (33f). White crystal (yield, 74%);
m.p. = 235–237 °C. IR (KBr, cm−1): 3289 (2NH), 3091 (CeH
aromatic), 2890 (CeH aliphatic) and 1688 (C]O amide); 1H NMR
(DMSO‑d6) δ ppm: 1.91–1.97 (m, 2H, CH2CH of piperidine), 2.48–2.52
(m, 1H, CH2CO of piperidine), 2.69–2.78 (m, 1H, CH2CO of piperidine),
4.35 (s, 2H, CH2CONH), 4.55–4.62 (m, 1H, CH piperidine), 7.38–7.42
(d, 2H, J = 8 Hz, C-3 and C-5 phenyl), 7.71–7.75 (d, 2H, J = 8 Hz, C-2
and C-6 phenyl), 7.99 (s, 1H, eC]CH), 8.71–8.73 (d, 1H, J = 8.2 Hz,
NHCO, D2O exchangeable), 10.87 (s, 1H, CONHCO, D2O
exchangeable); MS (m/z): 391 (M+, 61.85%), 151 (100%, base peak);
Anal. Calcd. for C17H14FN3O5S (391.37): C, 52.17; H, 3.61; N, 10.74.
Found: C, 52.33; H, 3.35; N, 10.49%.
APOPTOSIS
Fig. 11. Percentage of induced cell death by compound 42f on HCT-116 cells.
CONHCO, D2O exchangeable); MS (m/z): 391 (M+, 61.85%), 151
(100%, base peak); Anal. Calcd. for C18H17N3O6S (403.41): C, 53.59;
H, 4.25; N, 10.42. Found: C, 53.81; H, 4.09; N, 10.19%.
4.1.1.5. N-(2,6-Dioxopiperidin-3-yl)-2-(5-(4-methoxybenzylidene)-2,4-
dioxothiazolidin-3-yl)acetamide (33e). Yellowish white crystal (yield,
76%); m.p. = 276–278 °C. IR (KBr, cm−1): 3413, 3287 (NH), 3101
(CeH aromatic), 2926 (CeH aliphatic) and 1687 (C]O amide); 1H
A-
Control
Compound 42f
B-
Control
Compound 42f
Fig. 12. A) HCT-116 cells distribution upon treatment with compound 42f. B) Induced apoptosis on HCT-116 cells by compound 42f.
11

M.A. El-Zahabi, et al.
BioorganicChemistry104(2020)104218
4.1.1.7. N-(2,6-Dioxopiperidin-3-yl)-2-(5-(4-methylbenzylidene)-2,4-
dioxothiazolidin-3-yl)acetamide (33g). Off white crystal (yield, 68%);
m.p. = 230–232 °C. IR (KBr, cm−1): 3291, 3251 (2NH), 3105 (CeH
aromatic), 2950 (CeH aliphatic) and 1688 (C]O amide); 1H NMR
(DMSO‑d6) δ ppm: 1.94–1.95 (m, 2H, CH2CH of piperidine), 2.37 (s,
3H, CH3), 2.48–2.51 (m, 1H, CH2CO of piperidine), 2.68–2.76 (m, 1H,
CH2CO of piperidine), 4.35 (m, 2H, CH2CONH), 4.55–4.61 (m, 1H, CH
of piperidine), 7.37–7.39 (d, 2H, C-3 and C-5 phenyl, J = 7.8 Hz),
7.54–7.56 (d, 2H, C-2 and C-6 phenyl, J = 8.2 Hz), 7.93 (s, 1H,
eC]CH), 8.72–8.74 (d, 1H, J = 8 Hz, NHCO, D2O exchangeable),
10.87 (s, 1H, CONHCO, D2O exchangeable); 13C NMR (DMSO‑d6,
100 MHz) δ (ppm): 24.72, 31.25, 43.77, 49.90, 56.00, 115.49(2C),
118.24, 125.81, 132.80(2C), 133.88, 161.75, 165.69, 165.82, 167.59,
172.20, 173.33; Anal. Calcd. for C18H17N3O5S (387.41): C, 55.81; H,
4.42; N, 10.85. Found: C, 55.61; H, 4.63; N, 10.56%.
(2.26 g, 1.59 mL, 20.05 mmol) in DCM (10 mL) was added drop wise to
the previous solution over a period of 0.5 h while stirring in the ice
bath. The reaction mixture was allowed to stir at r.t. for an extra 0.5 h.
The obtained precipitate was filtered, washed with DCM and dried to
afford a white product. The filtrate was extracted with ethyl acetate
(10 mL × 3), dried over MgSO4 anhydrous and filtered. Upon con-
centration of ethyl acetate a second crop of white product was obtained
to give compound 41.
Shine white crystal (yield, 80%); m.p. = 148–150 °C. IR (KBr,
cm−1): 3316, 3189 (2NH), 2871 (CeH aliphatic), 1708 (C]O imide),
1659 (C]O amide); 1H NMR (DMSO‑d6) δ ppm: 1.92–1.97 (m, 2H,
CH2CH of piperidine), 2.50–2.51 (m, 1H, CH2CO of piperidine),
2.72–2.80 (m, 1H, CH2CO of piperidine), 4.16 (s, 2H, ClCH2CO),
4.56–4.63 (m, 1H, CHCO), 8.57 (d, 1H, J = 8.0 Hz, NHCO), 10.86 (s,
1H, CONHCO).
4.1.1.8. N-(2,6-Dioxopiperidin-3-yl)-2-(5-(4-nitrobenzylidene)-2,4-
dioxothiazolidin-3-yl)acetamide (33h). Shine yellow crystal (yield,
72%); m.p. = 240–242 °C. IR (KBr, cm−1): 3432, 3291 (NH), 3098
(CeH aromatic), 2938 (CeH aliphatic) and 1695 (C]O amide); 1H
NMR (DMSO‑d6) δ ppm: 1.91–1.96 (m, 2H, CH2CH of piperidine),
2.50–2.52 (m, 1H, CH2CO of piperidine), 2.69–2.76 (m, 1H, CH2CO of
piperidine), 4.33–4.42 (m, 2H, CH2CO), 4.53–4.62 (m, 1H, CHCO),
7.91–7.93 (d, 2H, C-2 and C-6 phenyl, J = 8.2 Hz), 8.09 (s, 1H,
eC]CH), 8.36–8.37 (d, 2H, C-3 and C-5 phenyl, J = 7.5 Hz),
8.72–8.75 (d, 1H, J = 12 Hz, NHCO, D2O exchangeable), 10.87 (s,
1H, CONHCO, D2O exchangeable); MS (m/z): 418 (M+, 95.58%), 55
(100%, base peak), 366 (53.41%), 290 (48.51%), 236 (53.42%), 178
(71.86%), 160 (48.74%), 120 (49.45%), 82 (54.04%); Anal. Calcd. for
4.1.3. General procedure for synthesis of compounds (42a-i)
A mixture of the appropriate potassium salt of 5-un(substituted)
aryl-1,3,4-oxadiazole-2-thiol (17a-i) (2 mmol) and 2-chloro-N-(2,6-di-
oxopiperidin-3-yl)acetamide (42) (0.49 g, 2.40 mmol) in the presence
of catalytic amount of potassium iodide in acetonitrile (20 mL) was
stirred at r.t. for 12 h. The obtained precipitates were filtered off, wa-
shed with water, dried and crystallized from methanol to afford the
corresponding acetamide derivatives (42a-i) respectively.
4.1.3.1. N-(2,6-Dioxopiperidin-3-yl)-2-[(5-phenyl-1,3,4-oxadiazol-2-yl)
thio]acetamide
(42a). Off
white
crystal
(yield,
85%);
m.p. = 185–187 °C. IR (KBr, cm−1): 3288, 3198 (NH), 3097 (CeH
aromatic), 2878 (CeH aliphatic), 1708 (C]O imide) and 1676 (C]O
amide); 1H NMR (DMSO‑d6) δ ppm: 1.94–1.97 (m, 2H, CH2CH of
piperidine), 2.48–2.51 (m, 1H, CH2CO of piperidine), 2.68–2.78 (m,
1H, CH2CO of piperidine), 4.20 (s, 2H, CH2CO), 4.58–4.64 (m, 1H,
CHCO), 7.59–7.61 (m, 3H, Ar-H), 7.97–7.99 (d, 2H, C-2 and C-6 phenyl,
J = 8 Hz), 8.72–8.75 (d, 1H, J = 12 Hz, NHCO, D2O exchangeable),
10.86 (s, 1H, CONHCO, D2O exchangeable); 13C NMR (DMSO‑d6,
100 MHz) δ (ppm): 24.60, 31.26, 36.04, 50.15, 123.50, 126.88(2C),
129.88(2C), 132.47, 163.72, 165.57, 166.68, 172.20, 173.30; Anal.
Calcd. for C15H14N4O4S (346.36): C, 52.02; H, 4.07; N, 16.18. Found: C,
52.41; H, 4.35; N, 16.38%
C
17H14N4O7S (418.38): C, 48.80; H, 3.37; N, 13.39. Found: C, 48.44; H,
3.00; N, 13.57%.
4.1.1.9. N-(2,6-Dioxopiperidin-3-yl)-2-(5-(3-nitrobenzylidene)-2,4-
dioxothiazolidin-3-yl)acetamide (33i). Yellow crystal (yield, 75%);
m.p. = 228–230 °C. IR (KBr, cm−1): 3292, 3248 (2NH), 3130 (CeH
aromatic), 3095 (CeH aliphatic) and 1696 (C]O amide); 1H NMR
(DMSO‑d6) δ ppm: 1.88–1.95 (m, 2H, CH2CH of piperidine), 2.48–2.52
(m, 1H, CH2CO of piperidine), 2.69–2.77 (m, 1H, CH2CO of piperidine),
4.38 (s, 2H, CH2CO), 4.53–4.66 (m, 1H, CHCO), 7.83–7.87 (dd, 1H, C-5
phenyl, J = 8.2 Hz & 7.1 Hz), 8.06–8.07 (d, 1H, C-6 phenyl,
J = 8.2 Hz), 8.15 (s, 1H, eC]CH), 8.31–8.33 (d, 1H, C-4 phenyl,
J = 7.1 Hz), 8.51 (s, 1H, C-3 phenyl), 8.72–8.74 (d, 1H, J = 8.1 Hz,
NHCO, D2O exchangeable), 10.87 (s, 1H, CONHCO, D2O
exchangeable); Anal. Calcd. for C17H14N4O7S (418.38): C, 48.80; H,
3.37; N, 13.39. Found: C, 48.64; H, 3.48; N, 13.53%
4.1.3.2. 2-[(5-(2-Chlorophenyl)-1,3,4-oxadiazol-2-yl)thio]-N-(2,6-
dioxopiperidin-3-yl)acetamide (42b). White crystal (yield, 82%);
m.p. = 198–200 °C. IR (KBr, cm−1): 3291 (br, 2NH), 3101 (CeH
aromatic), 2925, 2876 (CeH aliphatic), 1711 (C]O imide) and 1668
(C]O amide); 1H NMR (DMSO‑d6) δ ppm: 1.95–1.97 (m, 2H, CH2CH of
piperidine), 2.47–2.50 (m, 1H, CH2CO of piperidine), 2.69–2.77 (m,
1H, CH2CO of piperidine), 4.21 (s, 2H, CH2CO), 4.57–4.63 (m, 1H,
CHCO), 7.60–7.64 (dd, 1H, J = 8.2 Hz & 7.8 Hz, C-4 phenyl),
7.70–7.74 (dd, 1H, J = 8.2 & 8.0 Hz, C-5 phenyl), 7.94–7.96 (d, 1H,
J = 7.8 Hz, C-3 phenyl), 7.96–7.98 (d, 1H, J = 8.0 Hz, C-6 phenyl),
8.74–8.75 (d, 1H, J = 4 Hz, NHCO, D2O exchangeable), 10.85 (s, 1H,
CONHCO, D2O exchangeable); MS (m/z): 379 (M+, 26.04%), 111
(100%, base peak), 141 (92.13%), 271 (27.06%), 378 (21.31%), 75
(22.93%); Anal. Calcd. for C15H13ClN4O4S (380.80): C, 47.31; H, 3.44;
N, 14.71. Found: C, 47.52; H, 3.62; N, 14.45%.
4.1.1.10. 2-(5-(2,6-Dichlorobenzylidene)-2,4-dioxothiazolidin-3-yl)-N-
(2,6-dioxopipe- ridin-3-yl)acetamide (33j). White crystal (yield, 71%);
m.p. = 263–265 °C. IR (KBr, cm−1): 3353, 3242 (2NH), 3095 (CeH
aromatic), 2976 (CeH aliphatic) and 1699 (C]O amide); 1H NMR
(DMSO‑d6) δ ppm: 1.93–1.97 (m, 2H, CH2CH of piperidine), 2.50–2.53
(m, 1H, CH2CO of piperidine), 2.63–2.77 (m, 1H, CH2CO of piperidine),
4.36 (s, 2H, CH2CO), 4.37–4.61 (m, 1H, CHCO), 7.50–7.54 (dd, 1H, C-4
phenyl, J = 8.0 Hz & 7.8 Hz), 7.62–7.64 (d, 2H, C-3 and C-5 phenyl,
J = 8.0 Hz), 7.91 (s, 1H, eC]CH), 8.76–8.78 (d, 1H, J = 8 Hz, NHCO,
D2O exchangeable), 10.87 (s, 1H, CONHCO, D2O exchangeable); MS
(m/z): 442 (M+, 6.69%), 55 (100%, base peak), 405 (54.23%), 203
(32.26%), 166 (21.93%), 131 (20.87%), 122 (19.78%), 83 (44.55%).
Anal. Calcd. for C17H13Cl2N3O5S (442.27): C, 46.17; H, 2.96; N, 9.50.
Found: C, 46.41; H, 3.19; N, 9.72%.
4.1.3.3. 2-[(5-(3-Chlorophenyl)-1,3,4-oxadiazol-2-yl)thio]-N-(2,6-
dioxopiperidin-3-yl)acetamide (42c). Grayish white crystal (yield, 85%);
m.p. = 224–226 °C. IR (KBr, cm−1): 3289, 3199 (2NH), 3099 (CeH
aromatic), 2922, 2865 (CeH aliphatic), 1708 (C]O imide) and 1669
(C]O amide); 1H NMR (DMSO‑d6) δ ppm: 1.92–1.97 (m, 2H, CH2CH of
piperidine), 2.49–2.51 (m, 1H, CH2CO of piperidine), 2.68–2.71 (m,
1H, CH2CO of piperidine), 4.19 (s, 2H, CH2CO), 4.59–4.63 (m, 1H,
CHCO), 7.66–7.68 (d, 1H, C-5 phenyl, J = 8.2 Hz), 7.70–7.72 (dd, 1H,
C-4 phenyl, J = 8.2 & 7.8 Hz), 7.76–7.78 (d, 1H, C-6 phenyl,
J = 8.2 Hz), 7.95 (s, 1H, C-2 phenyl), 8.71–8.73 (d, 1H, J = 8.0 Hz,
4.1.2. 2-Chloro-N-(2,6-dioxopiperidin-3-yl)acetamide (41)
A mixture of 3-aminopiperidine-2,6-dione HCl (3 g, 18.23 mmol)
and NaHCO3 (3.06 g, 36.45 mmol) was dissolved in water (20 mL)
while stirring until complete dissolution. DCM (10 mL) was added to
the aqueous solution in an ice bath. A solution of chloroacetyl chloride
12

M.A. El-Zahabi, et al.
BioorganicChemistry104(2020)104218
NHCO, D2O exchangeable), 10.85 (s, 1H, CONHCO, D2O
exchangeable); Anal. Calcd. for C15H13ClN4O4S (380.80): C, 47.31; H,
3.44; N, 14.71. Found: C, 47.11; H, 3.22; N, 14.53%.
(C]O amide); 1H NMR (DMSO‑d6) δ ppm: 1.94–1.97 (m, 2H, CH2CH of
piperidine), 2.46–2.51 (m, 1H, CH2CO of piperidine), 2.67–2.77 (m,
1H, CH2CO of piperidine), 4.22 (m, 2H, CH2CO), 4.57–4.63 (m, 1H,
CHCO), 7.63–7.66 (dd, 1H, J = 4 Hz & 8 Hz, C-5 pyridine), 8.35–8.38
(d, 1H, J = 12 Hz, NHCO, D2O exchangeable), 8.74–8.76 (d, 1H,
J = 8 Hz, C-4 pyridine), 8.79–8.81(d, 1H, J = 8 Hz, C-6 pyridine), 9.15
(s, 1H, C-2 pyridine), 10.85 (s, 1H, CONHCO, D2O exchangeable); MS
(m/z): 347 (M+, 51.34%), 49 (100%, base peak), 276 (22.31%), 149
(31.08%), 143 (30.46%), 121 (59.73%), 104 (67.68%), 97 (50.39%),
79 (73.70%), 66 (69.14%); Anal. Calcd. for C14H13N5O4S (347.35): C,
48.41; H, 3.77; N, 20.16. Found: C, 48.52; H, 3.97; N, 20.29%.
4.1.3.4. 2-[(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)thio]-N-(2,6-
dioxopiperidin-3-yl)acetamide (42d). Off white crystal (yield, 89%);
m.p. = 234–236 °C. IR (KBr, cm−1): 3265 (br, 2NH), 3101 (CeH
aromatic), 2931, 2872 (CeH aliphatic), 1703 (C]O imide) and 1668
(C]O amide); 1H NMR (DMSO‑d6) δ ppm: 1.95–2.07 (m, 2H, CH2CH of
piperidine), 2.48–2.51 (m, 1H, CH2CO of piperidine), 2.68–2.77 (m,
1H, CH2CO of piperidine), 4.20 (s, 2H, CH2CO), 4.57–4.64 (m, 1H,
CHCO), 7.65–7.68 (d, 2H, C-3 and C-5 phenyl, J = 12 Hz), 7.98–8.00
(d, 2H, J = 8.2 Hz, C-2 and C-6 phenyl,), 8.73–8.75 (d, 1H, J = 8.0 Hz,
NHCO, D2O exchangeable), 10.86 (s, 1H, CONHCO, D2O
exchangeable); 13C NMR (DMSO‑d6, 100 MHz) δ (ppm): 24.58, 31.26,
36.01, 50.15, 122.37, 128.69(2C), 130.05(2C), 137.18, 164.01, 164.81,
166.66, 172.20, 173.30; Anal. Calcd. for C15H13ClN4O4S (380.80): C,
47.31; H, 3.44; N, 14.71. Found: C, 47.09; H, 3.49; N, 14.68%.
4.1.3.9. N-(2,6-Dioxopiperidin-3-yl)-2-[(5-(pyridin-4-yl)-1,3,4-oxadiazol-
2-yl)thio]
Acetamide
(42i). Bluish
crystal
(yield,
89%);
m.p. = 169–171 °C. IR (KBr, cm−1): 3295 (br, 2NH), 2998 (CeH
aromatic), 2917 (CeH aliphatic), 1708 (C]O imide) and 1653 (C]O
amide); 1H NMR (DMSO‑d6) δ ppm: 1.94–1.98 (m, 2H, CH2CH of
piperidine), 2.49–2.51 (m, 1H, CH2CO of piperidine), 2.68–2.75 (m,
1H, CH2CO of piperidine), 4.23 (s, 2H, CH2CO), 4.59–4.63 (m, 1H,
CHCO), 7.91–7.93 (d, 2H, C-3 and C-5 pyridine, J = 8.2 Hz), 8.74–8.76
(d, 1H, J = 8 Hz, NHCO, D2O exchangeable), 8.80–8.82 (d, 2H,
J = 8.1 Hz, C-2 and C-6 pyridine), 10.84 (s, 1H, CONHCO, D2O
exchangeable); Anal. Calcd. for C14H13N5O4S (347.35): C, 48.41; H,
3.77; N, 20.16. Found: C, 48.59; H, 4.02; N, 20.42%.
4.1.3.5. N-(2,6-Dioxopiperidin-3-yl)-2-[(5-(4-nitrophenyl)-1,3,4-
oxadiazol-2-yl)thio] Acetamide (42e). Faint green crystal (yield, 87%);
m.p. = 276–278 °C. IR (KBr, cm−1): 3277 (br, 2NH), 3095 (CeH
aromatic), 2928, 2864 (CeH aliphatic), 1707 (C]O imide) and 1661
(C]O amide); 1H NMR (DMSO‑d6) δ ppm: 1.92–1.97 (m, 2H, CH2CH of
piperidine), 2.47–2.50 (m, 1H, CH2CO of piperidine), 2.68–2.77 (m,
1H, CH2CO of piperidine), 4.22 (m, 2H, CH2CO), 4.57–4.64 (m, 1H,
CHCO), 8.23–8.25 (d, 2H, J = 8 Hz, C-2 and C-6 phenyl), 8.39–8.42 (d,
2H, C-3 and C-5 phenyl, J = 12 Hz), 8.75–8.77 (d, 1H, J = 8 Hz,
NHCO, D2O exchangeable), 10.85 (s, 1H, CONHCO, D2O
exchangeable); Anal. Calcd. for C15H13N5O6S (391.36): C, 46.04; H,
3.35; N, 17.90. Found: C, 46.32; H, 3.55; N, 18.21%.
4.2. Biological testing
4.2.1. In vitro anti-proliferative activities
Anti-proliferative activity screening of the synthesized compounds
was performed against four human cancer cell lines: hepatocellular
carcinoma (HepG-2), colorectal carcinoma (HCT-116), prostate cancer
(PC3), and mammary gland cancer (MCF-7), using MTT assay [78–81]
as follows:
4.1.3.6. 2-[(5-(4-Aminophenyl)-1,3,4-oxadiazol-2-yl)thio]-N-(2,6-
dioxopiperidin-3-yl)acetamide (42f). Orange crystal (yield, 87%);
m.p. = 255–257 °C. IR (KBr, cm−1): 3360, 3277 (2NH), 3092 (CeH
aromatic), 2884 (CeH aliphatic), 1706 (C]O imide) and 1635 (C]O
amide); 1H NMR (DMSO‑d6) δ ppm: 1.87–1.93 (m, 2H, CH2CH of
piperidine), 2.44–2.48 (m, 1H, CH2CO of piperidine), 2.65–2.87 (m,
1H, CH2CO of piperidine), 4.09 (s, 2H, CH2CO), 4.53–4.60 (m, 1H,
CHCO), 5.91 (s, 2H, NH2, D2O exchangeable), 6.62–6.64 (d, 2H, C-3
and C-5 phenyl, J = 8.2 Hz), 7.58–7.60 (d, 2H, C-2 and C-6 phenyl,
J = 8.3 Hz), 8.65–8.67 (d, 1H, J = 8.0 Hz, NHCO, D2O exchangeable),
10.82 (s, 1H, CONHCO, D2O exchangeable); 13C NMR (DMSO‑d6,
100 MHz) δ (ppm): 24.60, 31.25, 36.03, 50.13, 109.84, 114.00(2C),
128.42(2C), 152.81, 161.36, 166.42, 166.83, 172.20, 173.32; Anal.
Calcd. for C15H15N5O4S (361.38): C, 49.86; H, 4.18; N, 19.38. Found: C,
50.08; H, 4.39; N, 19.62%.
The cells were cultured in RPMI-1640 medium with 10% fetal bo-
vine serum. Penicillin (100 units/ml) and streptomycin (100 µg/ml)
were added at 37 C in a 5% CO2 incubator. The cells were seeded in a
96-well plate at a density of 1.0x104 cells/well. at 37 C for 48 h under
5% CO2. After incubation the cells were treated with different con-
centration of the synthesized compounds and incubated for 24 h. Then,
20 µL of MTT solution of a concentration of 5 mg/ml was added and
incubated for 4 h. DMSO (100 µL) was added into each well to dissolve
the formed purple formazan. The color intensity was measured and
recorded at absorbance of 570 nm using a plate reader (EXL 800, USA).
The percentage of cell viability in was calculated as (A570 of treated
samples/A570 of untreated sample) X 100. Results for IC50values of the
tested compounds were summarized in Table 1.
4.2.2. Supernatant preparation
4.1.3.7. N-(2,6-Dioxopiperidin-3-yl)-2-[(5-(pyridin-2-yl)-1,3,4-oxadiazol-
Cell culture supernatants were prepared from HCT-116 cell line.
Cells were cultured in RPMI 1640 medium supplemented with 10%
heat‐inactivated fetal bovine serum (FBS) having 100 U/ml of penicillin
and 50 μg/ml of streptomycin at 37 °C in a humidified 5% CO2/air
mixture. The candidate compounds and thalidomide were dissolved in
DMSO as a stock solution at 100 mmol/L and diluted with an FBS-free
medium to achieve the designated concentrations (10 μM). The same
concentration of DMSO without any compounds was used as a control.
The HCT-116 cells were cultured onto 6-well plates (1x106 cells/ well),
allowed to adhere for 24 h followed by treatment with the tested
compounds. After 72 h, the media was collected and centrifuged for
15 min at 5000 rpm and equal volume of cell culture supernatants were
collected and utilized for immunoassay using different kits [82].
2-yl)thio]
Acetamide
(42g). white
crystal
(yield,
82%);
m.p. = 173–175 °C. IR (KBr, cm−1): 3308, 3219 (2NH), 3091 (CeH
aromatic), 2910 (CeH aliphatic), and 1689 (C]O amide); 1H NMR
(DMSO‑d6) δ ppm: 1.95–1.98 (m, 2H, CH2CH of piperidine), 2.48–2.51
(m, 1H, CH2CO of piperidine), 2.68–2.79 (m, 1H, CH2CO of piperidine),
4.21 (s, 2H, CH2CO), 4.59–4.65 (m, 1H, CHCO), 7.58–7.62 (dd, 1H, C-5
pyridine, J = 8 Hz), 8.04–8.08 (dd, 1H, C-4 pyridine, J = 8 Hz),
8.11–8.13 (d, 1H, C-3 pyridine, J = 8 Hz), 8.70–8.72 (d, 1H, J = 8 Hz,
NHCO, D2O exchangeable), 8.72–8.74 (d, 1H, C-6 pyridine, J = 8 Hz),
10.84 (s, 1H, CONHCO, D2O exchangeable); Anal. Calcd. for
C
14H13N5O4S (347.35): C, 48.41; H, 3.77; N, 20.16. Found: C, 48.69;
H, 3.88; N, 20.31%.
4.1.3.8. N-(2,6-Dioxopiperidin-3-yl)-2-[(5-(pyridin-3-yl)-1,3,4-oxadiazol-
2-yl)thio] Acetamide (42h). Greenish white crystal (yield, 82%);
m.p. = 176–178 °C. IR (KBr, cm−1): 3286 (br, 2NH), 3048, 3004
(CeH aromatic), 2923 (CeH aliphatic), 1703 (C]O imide) and 1673
4.2.2.1. Estimation of TNF-α, CASP8, and VEGF in HCT-116
supernatant. The levels of TNF-α, CASP8, and VEGF in cell culture
supernatants were estimated by ELISA technique using commercially
available matched paired antibodies (R&D Systems Inc., Minneapolis,
13

M.A. El-Zahabi, et al.
BioorganicChemistry104(2020)104218
MN) according to reported procedure [83,84].
Acknowledgement
Authors are thankful to the team of Pharmacology & Toxicology
Department, Faculty of pharmacy (Boys), Al-Azhar University, Cairo,
Egypt and the team of Biochemistry Department, Faculty of medicine,
Cairo University, Cairo, Egypt for providing laboratory facilities for
biological activity.
4.2.3. Cell lysate preparation
The tested compounds and thalidomide were incubated for 72 h
with HCT-116 cells. Then, the cells were treated with trypsin/EDTA
solution (0.25 mM trypsin and 1 mM EDTA dissolved in a phosphate
buffer). Cell lysate were washed three times with phosphate buffer
saline (PBS, Sigma Chemical Company, St. Louis, MO, USA) and lysed
by three repetitive freezing/thawing cycles (thawing at 37 °C for 2 min
and freezing at –80 °C for 15 min), followed by homogenization of the
cells by passing through a 20G needle [85].
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104218.
4.2.3.1. Estimation of nuclear factor kappa-B P65 (NF-κB P65) in HCT-
116 cell lysate. The cell lysate samples were applied to the microtiter
plates in a concentration of 50 µL/well. The plates were incubated at
37 °C for 1 h, then kept at 4 °C for 12 h in a humidified chamber. Next,
the cell lysate was extracted from the wells and the plates were washed
three times with a buffer (PBS/0.05%Tween-20). Blocking buffer (PBS/
0.05%Tween-20/5% FBS) (200 µL) was added to each well and
incubated at 37 °C for 1.5 h then washed. Anti-rabbit NF-κB P65
polyclonal antibody was dispensed as 50 µL/well and incubated for 2 h
at 37 °C. The plates were washed and incubated for 1 h with 50 µL/well
of diluted polyclonal goat anti-rabbit-peroxidase conjugate (1:1000).
After that, the plates were washed four times followed by addition of
TMB and H2O2 in equal volume (50 µL/well). After the development of
color, 50 µL of stopping buffer (1 M HCl) were added per well. The
absorbance was measured at 450 nm using the ELISA plate reader
(FLUOstar OPTIMA) [85].
References
[1] J.B. Marriott, G. Muller, A.G. Dalgleish, Thalidomide as an emerging im-
munotherapeutic agent, Immunol. Today 20 (12) (1999) 538–540.
[2] N. Vargesson, Thalidomide-induced teratogenesis: History and mechanisms, Birth
Defects Res. Part C: Embryo Today: Rev. 105 (2) (2015) 140–156.
[3] T. Ito, H. Ando, H. Handa, Teratogenic effects of thalidomide: molecular mechan-
isms, Cell. Mol. Life Sci. 68 (9) (2011) 1569–1579.
[4] T. Stephens, R. Brynner, Dark Remedy: The Impact of Thalidomide And its Revival
as A Vital Medicine, Basic Books, 2009.
[5] S. Sleijfer, W.H. Kruit, G. Stoter, Thalidomide in solid tumours: the resurrection of
an old drug, Eur. J. Cancer 40 (16) (2004) 2377–2382.
[6] J.H. Kim, A.R. Scialli, Thalidomide: the tragedy of birth defects and the effective
treatment of disease, Toxicol. Sci. 122 (1) (2011) 1–6.
[7] I. Ali, W.A. Wani, K. Saleem, A. Haque, Thalidomide: a banned drug resurged into
future anticancer drug, Curr. Drug Therapy 7 (1) (2012) 13–23.
[8] J.B. Bartlett, K. Dredge, A.G. Dalgleish, The evolution of thalidomide and its IMiD
derivatives as anticancer agents, Nat. Rev. Cancer 4 (4) (2004) 314.
[9] P.A. Haslett, P. Roche, C.R. Butlin, M. Macdonald, N. Shrestha, R. Manandhar,
J. LeMaster, R. Hawksworth, M. Shah, A.S. Lubinsky, Effective treatment of er-
ythema nodosum leprosum with thalidomide is associated with immune stimula-
tion, J. Infect. Dis. 192 (12) (2005) 2045–2053.
[10] M. Dimopoulos, K. Zervas, G. Kouvatseas, E. Galani, V. Grigoraki, C. Kiamouris,
E. Vervessou, E. Samantas, C. Papadimitriou, O. Ecomomou, Thalidomide and
dexamethasone combination for refractory multiple myeloma, Ann. Oncol. 12 (7)
(2001) 991–995.
4.2.4. Cell cycle analysis
HCT-116 cells were seeded at density of 2 × 105 cells per well and
incubated in six-well plates for 24 h. Fetal bovine serum (FBS, 10%)
was applied after incubation of cells at 37 °C and 5% CO2. The medium
was substituted with (DMSO 1% v/v) containing the compound 20f
10 μM, then incubated for 48 h, washed with saline buffered with cold
phosphate (PBS), fixed with 70% ethanol, rinsed with PBS then stained
with the DNA fluorochrome PI, kept for 15 min at 37 °C. Then samples
were analyzed with a FACS Caliber flow cytometer [86,87].
[11] J. Folkman, Angiogenesis-dependent diseases, Seminars in oncology, Elsevier,
2001, pp. 536–542.
[12] R. Knight, IMiDs: a novel class of immunomodulators, Seminars in oncology,
Elsevier, 2005, pp. 24–30.
[13] M. Melchert, A. List, The thalidomide saga, Int. J. Biochem. Cell Biol. 39 (7–8)
(2007) 1489–1499.
[14] K.M. Cook, W.D. Figg, Angiogenesis inhibitors: current strategies and future pro-
spects, CA Cancer J. Clin. 60 (4) (2010) 222–243.
[15] A.C.L. Leite, F.F. Barbosa, M.V. de Oliveira Cardoso, D.R. Moreira, L.C.D. Coêlho, E.
B. da Silva, G.B. de Oliveira Filho, V.M.O. de Souza, V.R.A. Pereira, L.d.C. Reis,
Phthaloyl amino acids as anti-inflammatory and immunomodulatory prototypes,
Medicinal Chem. Res. 23(4) (2014) 1701–1708.
4.2.5. Annexin V-FITC apoptosis assay
Annexin V-FITC / PI apoptosis detection kit was used to analyze the
effect of the most cytotoxic compound 42f on apoptosis induction. In
this experiment, HCT-116 cells were stained with Annexin V fluorescein
isothiocyanate (FITC) and counterstained with propidium iodide (PI).
Then, HCT-116 cells in a density of 2 × 105 per well were incubated
with compound 42f (10 µM) for 48 h. Next, the cells were trypsinized,
washed with phosphate-buffered saline (PBS), and stained for 15 min at
37 °C in the dark. Finally, FACS Caliber flow cytometer was used in
analysis process [88,89].
[16] A.L. Ruchelman, H.-W. Man, W. Zhang, R. Chen, L. Capone, J. Kang, A. Parton,
L. Corral, P.H. Schafer, D. Babusis, Isosteric analogs of lenalidomide and pomali-
domide: Synthesis and biological activity, Bioorg. Med. Chem. Lett. 23 (1) (2013)
360–365.
[17] S.K. Teo, Properties of thalidomide and its analogues: implications for anticancer
therapy, AAPS J. 7 (1) (2005) E14–E19.
[18] R. Suppiah, J.G. Srkalovic, M.A. Hussein, Immunomodulatory analogues of thali-
domide in the treatment of multiple myeloma, Clin. Lymphoma Myeloma 6 (4)
(2006) 301–305.
[19] E. Terpos, N. Kanellias, D. Christoulas, E. Kastritis, M.A. Dimopoulos,
Pomalidomide: a novel drug to treat relapsed and refractory multiple myeloma,
OncoTargets Therapy 6 (2013) 531.
[20] S. Zhou, F. Wang, T.-C. Hsieh, J.M. Wu, E. Wu, Thalidomide–a notorious sedative to
a wonder anticancer drug, Curr. Med. Chem. 20 (33) (2013) 4102–4108.
[21] W.D. Figg, W. Dahut, P. Duray, M. Hamilton, A. Tompkins, S.M. Steinberg, E. Jones,
A. Premkumar, W.M. Linehan, M.K. Floeter, A randomized phase II trial of thali-
domide, an angiogenesis inhibitor, in patients with androgen-independent prostate
cancer, Clin. Cancer Res. 7 (7) (2001) 1888–1893.
4.2.6. Statistical analysis
The statistical analyses were performed using GraphPad Prism 6.01
MM. Data were presented as means with corresponding SE.
Comparisons between different groups were performed by one-way
analysis of variance (ANOVA). Tukey’s test was used as a post-hoc test.
Correlation among variables was deter-mined using Pearson’s correla-
tion test. The level of significance was set at P < 0.05.
[22] C.M. de Souza, L.F. de Carvalho, T. da Silva Vieira, A.C.A. e Silva, M.T.P. Lopes,
M.A.N.D. Ferreira, S.P. Andrade, G.D. Cassali, Thalidomide attenuates mammary
cancer associated-inflammation, angiogenesis and tumor growth in mice, Biomed.
Pharmacother. 66 (7) (2012) 491–498.
[23] S. Ng, M. Brown, W. Figg, Thalidomide, an antiangiogenic agent with clinical ac-
tivity in cancer, Biomed. Pharmacother. 56 (4) (2002) 194–199.
[24] T. Eisen, C. Boshoff, I. Mak, F. Sapunar, M. Vaughan, L. Pyle, S. Johnston, R. Ahern,
I. Smith, M. Gore, Continuous low dose thalidomide: a phase II study in advanced
melanoma, renal cell, ovarian and breast cancer, Br. J. Cancer 82 (4) (2000) 812.
[25] K.A. Varker, J. Campbell, M.H. Shah, Phase II study of thalidomide in patients with
metastatic carcinoid and islet cell tumors, Cancer Chemother. Pharmacol. 61 (4)
(2008) 661–668.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
14

M.A. El-Zahabi, et al.
BioorganicChemistry104(2020)104218
[26] S.-F. Ang, S.-H. Tan, H.-C. Toh, D.Y. Poon, S.Y. Ong, K.-F. Foo, S.-P. Choo, Activity
of thalidomide and capecitabine in patients with advanced hepatocellular carci-
noma, Am. J. Clin. Oncol. 35 (3) (2012) 222–227.
derived via Heck cross coupling, Bioorg. Med. Chem. Lett. 17 (21) (2007)
5819–5824.
[52] T. Wang, Y.H. Zhang, S. Yu, H. Ji, Y.S. Lai, S.X. Peng, Synthesis and biological
evaluation of novel thalidomide analogues as potential anticancer drugs, Chin.
Chem. Lett. 19 (8) (2008) 928–930.
[27] R. Duncan, Polymer conjugates as anticancer nanomedicines, Nat. Rev. Cancer 6 (9)
(2006) 688.
[28] G. Barosi, M. Elliott, L. Canepa, F. Ballerini, P.P. Piccaluga, G. Visani, M. Marchetti,
G. Pozzato, F. Zorat, A. Tefferi, Thalidomide in myelofibrosis with myeloid meta-
plasia: a pooled-analysis of individual patient data from five studies, Leukemia
Lymphoma 43 (12) (2002) 2301–2307.
[53] M.A.-H. Zahran, T.A.-R. Salem, R.M. Samaka, H.S. Agwa, A.R. Awad, Design,
synthesis and antitumor evaluation of novel thalidomide dithiocarbamate and di-
thioate analogs against Ehrlich ascites carcinoma-induced solid tumor in Swiss al-
bino mice, Bioorg. Med. Chem. 16 (22) (2008) 9708–9718.
[29] M.B. Steins, T. Padró, R. Bieker, S. Ruiz, M. Kropff, J. Kienast, T. Kessler,
T. Buechner, W.E. Berdel, R.M. Mesters, Efficacy and safety of thalidomide in pa-
tients with acute myeloid leukemia, Blood 99 (3) (2002) 834–839.
[30] A.K. Gandhi, J. Kang, C.G. Havens, T. Conklin, Y. Ning, L. Wu, T. Ito, H. Ando,
M.F. Waldman, A. Thakurta, Immunomodulatory agents lenalidomide and poma-
lidomide co-stimulate T cells by inducing degradation of T cell repressors I karos
and A iolos via modulation of the E 3 ubiquitin ligase complex CRL 4 CRBN, Br. J.
Haematol. 164 (6) (2014) 811–821.
[54] M.F. Braña, N. Acero, L. Anorbe, D.M. Mingarro, F. Llinares, G. Domínguez,
Discovering a new analogue of thalidomide which may be used as a potent mod-
ulator of TNF-α production, Eur. J. Med. Chem. 44 (9) (2009) 3533–3542.
[55] C. Chaulet, C. Croix, D. Alagille, S. Normand, A. Delwail, L. Favot, J.-C. Lecron, M.-
C. Viaud-Massuard, Design, synthesis and biological evaluation of new thalidomide
analogues as TNF-α and IL-6 production inhibitors, Bioorg. Med. Chem. Lett. 21 (3)
(2011) 1019–1022.
[56] L. Mazzoccoli, S.H. Cadoso, G.W. Amarante, M.V. de Souza, R. Domingues,
M.A. Machado, M.V. de Almeida, H.C. Teixeira, Novel thalidomide analogues from
diamines inhibit pro-inflammatory cytokine production and CD80 expression while
enhancing IL-10, Biomed. Pharmacother. 66 (5) (2012) 323–329.
[57] S. Nagarajan, S. Majumder, U. Sharma, S. Rajendran, N. Kumar, S. Chatterjee,
B. Singh, Synthesis and anti-angiogenic activity of benzothiazole, benzimidazole
containing phthalimide derivatives, Bioorg. Med. Chem. Lett. 23 (1) (2013)
287–290.
[31] P. Bodera, W. Stankiewicz, Immunomodulatory properties of thalidomide analogs:
pomalidomide and lenalidomide, experimental and therapeutic applications,
Recent Pat. Endocr., Metab. Immune Drug Discov. 5 (3) (2011) 192–196.
[32] J.W. Adlard, Thalidomide in the treatment of cancer, Anticancer Drugs 11 (10)
(2000) 787–791.
[33] K.C. Anderson, Lenalidomide and thalidomide: mechanisms of action—similarities
and differences, Seminars Hematol. Elsevier (2005) S3–S8.
[34] Y. Dai, F. Jin, W. Li, J.G. Turner, Novel mechanisms of action for im-
munomodulatory drugs (IMiDs) against multiple myeloma: from a tragedy to a
therapy, Int. J. Hematol. Therapy 2 (1) (2015).
[58] P.M. Da Costa, M.P. da Costa, A.A. Carvalho, S.M.T. Cavalcanti, M.V. de Oliveira
Cardoso, G.B. de Oliveira Filho, D. de Araújo Viana, F.V. Fechine-Jamacaru, A.C.L.
Leite, M.O. de Moraes, Improvement of in vivo anticancer and antiangiogenic po-
tential of thalidomide derivatives, Chemico-biological Interact. 239 (2015)
174–183.
[35] T. Reske, M. Fulciniti, N.C. Munshi, Mechanism of action of immunomodulatory
agents in multiple myeloma, Med. Oncol. 27 (1) (2010) 7–13.
[36] A. Avigdor, P. Raanani, I. Levi, I. Hardan, I. Ben-Bassat, Extramedullary progression
despite a good response in the bone marrow in patients treated with thalidomide for
multiple myeloma, Leukemia Lymphoma 42 (4) (2001) 683–687.
[59] M.V. de Oliveira Cardoso, D.R.M. Moreira, G.B. Oliveira Filho, S.M.T. Cavalcanti,
L.C.D. Coelho, J.W.P. Espíndola, L.R. Gonzalez, M.M. Rabello, M.Z. Hernandes,
P.M.P. Ferreira, Design, synthesis and structure–activity relationship of phthali-
mides endowed with dual antiproliferative and immunomodulatory activities, Eur.
J. Med. Chem. 96 (2015) 491–503.
[37] W.M. Eldehna, M.F. Abo-Ashour, A. Nocentini, P. Gratteri, I.H. Eissa, M. Fares,
O.E. Ismael, H.A. Ghabbour, M.M. Elaasser, H.A. Abdel-Aziz, Novel 4/3-((4-oxo-5-
(2-oxoindolin-3-ylidene) thiazolidin-2-ylidene) amino) benzenesulfonamides:
Synthesis, carbonic anhydrase inhibitory activity, anticancer activity and molecular
modelling studies, Eur. J. Med. Chem. 139 (2017) 250–262.
[60] S. Bajaj, V. Asati, J. Singh, P.P. Roy, 1, 3, 4-Oxadiazoles: an emerging scaffold to
target growth factors, enzymes and kinases as anticancer agents, Eur. J. Med. Chem.
97 (2015) 124–141.
[38] A.A. Gaber, A.H. Bayoumi, A.M. El-morsy, F.F. Sherbiny, A.B. Mehany, I.H. Eissa,
Design, synthesis and anticancer evaluation of 1H-pyrazolo [3, 4-d] pyrimidine
derivatives as potent EGFR WT and EGFR T790M inhibitors and apoptosis inducers,
Bioorg. Chem. (2018).
[61] W. Caneschi, K.B. Enes, C.C. de Mendonça, F. de Souza Fernandes, F.B. Miguel, J. da
Silva Martins, M. Le Hyaric, R.R. Pinho, L.M. Duarte, M.A.L. de Oliveira, Synthesis
and anticancer evaluation of new lipophilic 1, 2, 4 and 1, 3, 4-oxadiazoles, Eur. J.
Med. Chem. 165 (2019) 18–30.
[39] A.M. El-Naggar, M.M. Abou-El-Regal, S.A. El-Metwally, F.F. Sherbiny, I.H. Eissa,
Synthesis, characterization and molecular docking studies of thiouracil derivatives
as potent thymidylate synthase inhibitors and potential anticancer agents, Mol.
Diversity 21 (4) (2017) 967–983.
[62] H. El-Kashef, G. Badr, N.A. El-Maali, D. Sayed, P. Melnyk, N. Lebegue, R.A. El-
Khalek, Synthesis of a novel series of (Z)-3, 5-disubstituted thiazolidine-2, 4-diones
as promising anti-breast cancer agents, Bioorg. Chem. (2020) 103569.
[63] C. Viegas-Junior, A. Danuello, V. da Silva Bolzani, E.J. Barreiro, C.A.M. Fraga,
Molecular hybridization: a useful tool in the design of new drug prototypes, Curr.
Med. Chem. 14 (17) (2007) 1829–1852.
[40] S.A. Elmetwally, K.F. Saied, I.H. Eissa, E.B. Elkaeed, Design, synthesis and antic-
ancer evaluation of thieno [2, 3-d] pyrimidine derivatives as dual EGFR/HER2
inhibitors and apoptosis inducers, Bioorg. Chem. 88 (2019) 102944.
[41] H.A. Mahdy, M.K. Ibrahim, A.M. Metwaly, A. Belal, A.B. Mehany, K.M. El-Gamal,
A. El-Sharkawy, M.A. Elhendawy, M.M. Radwan, M.A. Elsohly, Design, synthesis,
molecular modeling, in vivo studies and anticancer evaluation of quinazolin-4 (3H)-
one derivatives as potential VEGFR-2 inhibitors and apoptosis inducers, Bioorg.
Chem. (2019) 103422.
[64] M.A. El-Zahabi, E.R. Elbendary, F.H. Bamanie, M.F. Radwan, S.A. Ghareib,
I.H. Eissa, Design, synthesis, molecular modeling and anti-hyperglycemic evalua-
tion of phthalimide-sulfonylurea hybrids as PPARγ and SUR agonists, Bioorg. Chem.
91 (2019) 103115.
[65] G. Bruno, L. Costantino, C. Curinga, R. Maccari, F. Monforte, F. Nicolo, R. Ottana,
M. Vigorita, Synthesis and aldose reductase inhibitory activity of 5-arylidene-2, 4-
thiazolidinediones, Bioorg. Med. Chem. 10 (4) (2002) 1077–1084.
[66] R. Maccari, R. Ottanà, R. Ciurleo, M.G. Vigorita, D. Rakowitz, T. Steindl, T. Langer,
Evaluation of in vitro aldose redutase inhibitory activity of 5-arylidene-2, 4-thia-
zolidinediones, Bioorg. Med. Chem. Lett. 17 (14) (2007) 3886–3893.
[67] K.M. El-Gamal, A.M. El-Morsy, A.M. Saad, I.H. Eissa, M. Alswah, Synthesis, docking,
QSAR, ADMET and antimicrobial evaluation of new quinoline-3-carbonitrile deri-
vatives as potential DNA-gyrase inhibitors, J. Mol. Struct. 1166 (2018) 15–33.
[68] M.K. Ibrahim, I.H. Eissa, A.E. Abdallah, A.M. Metwaly, M. Radwan, M. ElSohly,
Design, synthesis, molecular modeling and anti-hyperglycemic evaluation of novel
quinoxaline derivatives as potential PPARγ and SUR agonists, Bioorg. Med. Chem.
25 (4) (2017) 1496–1513.
[42] I.H. Eissa, A.M. El-Naggar, M.A. El-Hashash, Design, synthesis, molecular modeling
and biological evaluation of novel 1H-pyrazolo [3, 4-b] pyridine derivatives as
potential anticancer agents, Bioorg. Chem. 67 (2016) 43–56.
[43] I.H. Eissa, A.M. El-Naggar, N.E. El-Sattar, A.S. Youssef, Design and discovery of
novel quinoxaline derivatives as dual DNA intercalators and topoisomerase II in-
hibitors, Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal
Chemistry-Anti-Cancer Agents) 18 (2) (2018) 195–209.
[44] M. Ibrahim, M. Taghour, A. Metwaly, A. Belal, A. Mehany, M. Elhendawy,
M. Radwan, A. Yassin, N. El-Deeb, E. Hafez, Design, synthesis, molecular modeling
and anti-proliferative evaluation of novel quinoxaline derivatives as potential DNA
intercalators and topoisomerase II inhibitors, Eur. J. Med. Chem. (2018).
[45] I.H. Eissa, A.M. Metwaly, A. Belal, A.B. Mehany, R.R. Ayyad, K. El-Adl, H.A. Mahdy,
M.S. Taghour, K.M. El-Gamal, M.E. El-Sawah, Discovery and antiproliferative eva-
luation of new quinoxalines as potential DNA intercalators and topoisomerase II
inhibitors, Arch. Pharm. 352 (11) (2019) 1900123.
[69] M.K. Ibrahim, I.H. Eissa, M.S. Alesawy, A.M. Metwaly, M.M. Radwan, M.A. ElSohly,
Design, synthesis, molecular modeling and anti-hyperglycemic evaluation of qui-
nazolin-4 (3H)-one derivatives as potential PPARγ and SUR agonists, Bioorg. Med.
Chem. 25 (17) (2017) 4723–4744.
[46] A.G.A. El-Helby, H. Sakr, I.H. Eissa, H. Abulkhair, A.A. Al-Karmalawy, K. El-Adl,
Design, synthesis, molecular docking, and anticancer activity of benzoxazole deri-
vatives as VEGFR-2 inhibitors, Arch. Pharm. 352 (10) (2019) 1900113.
[47] A.M. El-Naggar, I.H. Eissa, A. Belal, A.A. El-Sayed, Design, eco-friendly synthesis,
molecular modeling and anticancer evaluation of thiazol-5 (4 H)-ones as potential
tubulin polymerization inhibitors targeting the colchicine binding site, RSC Adv. 10
(5) (2020) 2791–2811.
[70] K. Gholivand, H. Mostaanzadeh, Z. Shariatinia, N. Oroujzadeh, Syntheses, crystal
structures and dynamic 1HNMR study of diastereotopic CH2 protons in several new
phosphoric triamides, Main Group Chem. Taylor & Francis (2006) 95–106.
[71] J.A. Aizina, I.B. Rozentsveig, G.G. Levkovskaya, A novel synthesis of chlor-
oacetamide derivatives via C-amidoalkylation of aromatics by 2-chloro-N-(2, 2, 2-
trichloro-1-hydroxyethyl) acetamide, Arkivoc 8 (2011) 192–199.
[72] J.A. DaSilva, C. Barríab, C. Jullianb, P. Navarreteb, L. NúñezVergaraa, J. Squellaa,
Unexpected diastereotopic behaviour in the 1H NMR spectrum of 1, 4-dihy-
dropyridine derivatives triggered by chiral and prochiralcentres, J. Braz. Chem. Soc.
16 (2005) 112.
[48] G.W. Muller, L.G. Corral, M.G. Shire, H. Wang, A. Moreira, G. Kaplan, D.I. Stirling,
Structural modifications of thalidomide produce analogs with enhanced tumor
necrosis factor inhibitory activity, J. Med. Chem. 39 (17) (1996) 3238–3240.
[49] S.M. Capitosti, T.P. Hansen, M.L. Brown, Thalidomide analogues demonstrate dual
inhibition of both angiogenesis and prostate cancer, Bioorg. Med. Chem. 12 (2)
(2004) 327–336.
[73] Z. Shi, Z. Zhao, M. Huang, X. Fu, Ultrasound-assisted, one-pot, three-component
synthesis and antibacterial activities of novel indole derivatives containing 1, 3, 4-
oxadiazole and 1, 2, 4-triazole moieties, C. R. Chim. 18 (12) (2015) 1320–1327.
[74] S.J. Gilani, S.A. Khan, N. Siddiqui, Synthesis and pharmacological evaluation of
condensed heterocyclic 6-substituted 1, 2, 4-triazolo-[3, 4-b]-1, 3, 4-thiadiazole and
1, 3, 4-oxadiazole derivatives of isoniazid, Bioorg. Med. Chem. Lett. 20 (16) (2010)
4762–4765.
[50] T. Noguchi, H. Fujimoto, H. Sano, A. Miyajima, H. Miyachi, Y. Hashimoto,
Angiogenesis inhibitors derived from thalidomide, Bioorg. Med. Chem. Lett. 15 (24)
(2005) 5509–5513.
[51] S.G. Stewart, D. Spagnolo, M.E. Polomska, M. Sin, M. Karimi, L.J. Abraham,
Synthesis and TNF expression inhibitory properties of new thalidomide analogues
[75] E. Borenfreund, J.A. Puerner, Toxicity determined in vitro by morphological
15

M.A. El-Zahabi, et al.
BioorganicChemistry104(2020)104218
alterations and neutral red absorption, Toxicol. Lett. 24 (2–3) (1985) 119–124.
C virus infection: correlation with disease severity, Viral Immunol. 23 (2) (2010)
[76] C. Riccardi, I. Nicoletti, Analysis of apoptosis by propidium iodide staining and flow
cytometry, Nat. Protoc. 1 (3) (2006) 1458.
151–157.
[84] mybiosource.com/, MyBioSource ELISA Test Kits, 2019. https://www.mybiosource.
com/human-elisa-kits/casp8/2513072#QLAPP_MBS2513072_TD (accessed May
2018)..
[77] A.G.A. El-Helby, H. Sakr, I.H. Eissa, A.A. Al-Karmalawy, K. El-Adl, Benzoxazole/
benzothiazole-derived VEGFR-2 inhibitors: Design, synthesis, molecular docking,
and anticancer evaluations, Arch. Pharm. 352 (12) (2019) 1900178.
[78] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1–2) (1983)
55–63.
[85] R. Talaat, W. El-Sayed, H. Agwa, A. Gamal-Eldeen, S. Moawia, M. Zahran, Novel
thalidomide analogs: Anti-angiogenic and apoptotic effects on Hep-G2 and MCF-7
cancer cell lines, Biomed. Aging Pathol. 4 (3) (2014) 179–189.
[86] J. Wang, M.J. Lenardo, Roles of caspases in apoptosis, development, and cytokine
maturation revealed by homozygous gene deficiencies, J. Cell Sci. 113 (5) (2000)
753–757.
[79] F. Denizot, R. Lang, Rapid colorimetric assay for cell growth and survival: mod-
ifications to the tetrazolium dye procedure giving improved sensitivity and relia-
bility, J. Immunol. Methods 89 (2) (1986) 271–277.
[87] W.M. Eldehna, G.S. Hassan, S.T. Al-Rashood, T. Al-Warhi, A.E. Altyar,
H.M. Alkahtani, A.A. Almehizia, H.A. Abdel-Aziz, Synthesis and in vitro anticancer
activity of certain novel 1-(2-methyl-6-arylpyridin-3-yl)-3-phenylureas as apop-
tosis-inducing agents, J. Enzyme Inhib. Med. Chem. 34 (1) (2019) 322–332.
[88] K.K.-W. Lo, T.K.-M. Lee, J.S.-Y. Lau, W.-L. Poon, S.-H. Cheng, Luminescent biolo-
gical probes derived from ruthenium (II) estradiol polypyridine complexes, Inorg.
Chem. 47 (1) (2008) 200–208.
[80] M.I. Thabrew, R.D. Hughes, I.G. Mcfarlane, Screening of hepatoprotective plant
components using a HepG2 cell cytotoxicity assay, J. Pharm. Pharmacol. 49 (11)
(1997) 1132–1135.
[81] H. Almahli, E. Hadchity, M.Y. Jaballah, R. Daher, H.A. Ghabbour, M.M. Kabil,
N.S. Al-shakliah, W.M. Eldehna, Development of novel synthesized phthalazinone-
based PARP-1 inhibitors with apoptosis inducing mechanism in lung cancer, Bioorg.
Chem. 77 (2018) 443–456.
[89] W.M. Eldehna, A. Nocentini, Z.M. Elsayed, T. Al-Warhi, N. Aljaeed, O.J. Alotaibi,
M.M. Al-Sanea, H.A. Abdel-Aziz, C.T. Supuran, Benzofuran-based carboxylic acids
as carbonic anhydrase inhibitors and antiproliferative agents against breast cancer,
ACS Med. Chem. Lett. (2020).
[82] T. Inoue, K. Kibata, M. Suzuki, S. Nakamura, R. Motoda, K. Orita, Identification of a
vascular endothelial growth factor (VEGF) antagonist, sFlt-1, from a human he-
matopoietic cell line NALM-16, FEBS Lett. 469 (1) (2000) 14–18.
[83] R.M. Talaat, Soluble angiogenesis factors in sera of Egyptian patients with hepatitis
16