Introducing of potent cytotoxic novel 2-(aroylamino)cinnamamide derivatives against colon cancer mediated by dual apoptotic signal activation and oxidative stress

Article abstract of DOI:10.1016/j.bioorg.2020.103953

Curcumin and trans-cinnamaldehyde are acrolein-based Michael acceptor compounds that are commonly found in domestic condiments, and known to cause cancer cell death via redox mechanisms. Based on the structural features of these compounds we designed and synthesized several 2-cinnamamido-N-substituted-cinnamamide (bis-cinnamamide) compounds. One of the derivatives, (Z)-2-[(E)-cinnamamido]-3-phenyl-N-propylacrylamide 8 showed a moderate antiproliferative potency (HCT-116 cell line inhibition of 32.0 μM), no inhibition of normal cell lines C-166, and proven cellular activities leading to apoptosis. SAR studies led to more than 10-fold increase in activity. Our most promising compound, [(Z)-3-(1H-indol-3-yl)-N-propyl-2-[(E)-3-(thien-2-yl)propenamido)propenamide] 45 killed colon cancer cells at IC₅₀ = 0.89 μM (Caco-2), 2.85 μM (HCT-116) and 1.65 μM (HT-29), while exhibiting much weaker potency on C-166 and BHK normal cell lines (IC₅₀ = 71 μM and 77.6 μM, respectively). Cellular studies towards identifying the compounds mechanism of cytotoxic activities revealed that apoptotic induction occurs in part as a result of oxidative stress. Importantly, the compounds showed inhibition of cancer stem cells that are critical for maintaining the potential for self-renewal and stemness. The results presented here show discovery of covalently acting Michael addition compounds that potently kill cancer cells by a defined mechanism, with prominent selectivity profile over non-cancerous cell lines.

Full text of DOI:10.1016/j.bioorg.2020.103953

Journal Pre-proofs
Introducing of Potent Cytotoxic Novel 2-(Aroylamino)cinnamamide Deriva-
tives against Colon Cancer Mediated by Dual Apoptotic Signal Activation
and Oxidative Stress
Abdelsattar M. Omar, Moustafa E. El-Araby, Tamer M. Abdelghany, Martin
K. Safo, Mostafa H. Ahmed, Rio Boothello, Bhaumik B Patel, Mohamed S.
Abdel-Bakky, Azizah M. Malebari, Hany E.A. Ahmed, Radwan S. Elhaggar
PII:
S0045-2068(20)30718-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.103953
YBIOO 103953
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
29 March 2020
15 May 2020
16 May 2020
Please cite this article as: A.M. Omar, M.E. El-Araby, T.M. Abdelghany, M.K. Safo, M.H. Ahmed, R. Boothello,
B.B. Patel, M.S. Abdel-Bakky, A.M. Malebari, H.E.A. Ahmed, R.S. Elhaggar, Introducing of Potent Cytotoxic
Novel 2-(Aroylamino)cinnamamide Derivatives against Colon Cancer Mediated by Dual Apoptotic Signal
Activation and Oxidative Stress, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103953
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Graphical abstract
Introducing of Potent Cytotoxic Novel 2-(Aroylamino)cinnamamide Derivatives
against Colon Cancer Mediated by Dual Apoptotic Signal Activation and Oxidative
Stress

Introducing of Potent Cytotoxic Novel 2-(Aroylamino)cinnamamide Derivatives
against Colon Cancer Mediated by Dual Apoptotic Signal Activation and Oxidative
Stress
Abdelsattar M. Omar1,2*, Moustafa E. El-Araby1, Tamer M. Abdelghany3, Martin K.
Safo4, Mostafa H. Ahmed4, Rio Boothello5,6, Bhaumik B Patel5,6, Mohamed S. Abdel-
Bakky4, Azizah M. Malebari1, Hany E. A. Ahmed7,8*, Radwan S. Elhaggar9
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah
21589, Saudi Arabia
2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo
11884, Egypt
3 Department of Pharmacology, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
4 Department of Medicinal Chemistry, and the Institute for Structural Biology, Drug Discovery and
Development, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298, USA
5 Division of Hematology and Oncology, Department of Medicine, Virginia Commonwealth University,
and Massey Cancer Center, Richmond, VA 23298. USA
6 Hunter Holmes McGuire VA Medical Center, Richmond, VA 23249, USA
7 Pharmacognosy and Pharmaceutical Chemistry, College of Pharmacy, Taibah University, Al-Madinah Al-
Munawarah 47114, Saudi Arabia
8 Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Al-Azhar University,
Cairo 11884, Egypt
9 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Cairo 11790,
Egypt
* Correspondence: AMO; asmansour@kau.edu.sa, HEA; heahmad@taibahu.edu.sa,

ABSTRACT
Curcumin and trans-cinnamaldehyde are acrolein-based Michael acceptor
compounds that are commonly found in domestic condiments, and known to cause
cancer cell death via redox mechanisms. Based on the structural features of these
compounds we designed and synthesized several 2-cinnamamido-N-substituted-
cinnamamide (bis-cinnamamide) compounds. One of the derivatives, (Z)-2-[(E)-
cinnamamido]-3-phenyl-N-propylacrylamide 8 showed a moderate antiproliferative
potency (HCT-116 cell line inhibition of 32.0 µM), no inhibition of normal cell lines
C-166, and proven cellular activities leading to apoptosis. SAR studies led to more
than 10-fold increase in activity. Our most promising compound, [(Z)-3-(1H-indol-
3-yl)-N-propyl-2-[(E)-3-(thien-2-yl)propenamido)propenamide] 45 killed colon
cancer cells at IC50 = 0.89 µM (Caco-2), 2.85 µM (HCT-116) and 1.65 µM (HT-29),
while exhibiting much weaker potency on C-166 and BHK normal cell lines (IC50 =
71 µM and 77.6 µM, respectively). Cellular studies towards identifying the
compounds mechanism of cytotoxic activities revealed that apoptotic induction
occurs in part as a result of oxidative stress. Importantly, the compounds showed
inhibition of cancer stem cells that are critical for maintaining the potential for self-
renewal and stemness. The results presented here show discovery of covalently
acting Michael addition compounds that potently kill cancer cells by a defined
mechanism, with prominent selectivity profile over non-cancerous cell lines.
Keywords: Michael acceptor; safe covalent drugs; oxidative stress inducers; trans-
cinnamaldehyde; curcumin; colorectal cancer.

1. INTRODUCTION
Propenamide is a Michael acceptor (MA) moiety that is frequently employed in designing
drugs and drug candidates [1-3]. For instance, afatinib (Fig. 1) a target-specific
propenamide that selectively inhibits mutated HER2 kinase [4] has been recently approved
by the FDA for the treatment of non-small cell lung cancer [5]. Chidamide is a histone
acetylase (HDAC) inhibitor approved in China for the treatment of advanced peripheral T-
cell lymphoma. Chidamide incorporates 3-(3-pyridyl)acryloyl group which can be
considered bioisosteric to cinnamyl moiety [6]. Other examples, among many, include
ibrutinib (BTK inhibitor) and neratinib (HER-2 inhibitor) developed for B-cell cancers and
solid tumors, respectively [7, 8]. The efficacies of these drugs are, at least in part, depend
on the MA functionalities. There are anticancer Mechanistically, the MAs cause cancer cell
apoptosis by increasing the oxidative stress inside these cells. The cellular prooxidant
induction by MAs is attributed to covalently ligation of SH groups of certain targets
involved in regeneration of reduced glutathione (GSH). This result in accumulation of
reactive oxygen species (ROS) which cause cancer cell cycle exits and apoptosis [9].
F
O
HN
N
Cl
Acrolein
Carcinogen, Toxic
H
N
N
N
O
O
O
O
Afatinib
trans-Cinnamaldehyde
O
O
O
N
N
NH2
H
H3CO
HO
OCH3
OH
H
N
O
Curcumin
Chidamide
F
Fig. 1. Drugs and naturally occurring compounds containing Michael Addition moiety.

Acrolein (propenal, Fig. 1) is the simplest enone MA characterized by high reactivity and
non-selectivity to cellular nucleophiles and, therefore, is considered as a toxic
environmental pollutant with well-known carcinogenic activities [10]. Interestingly,
acrolein has also been reported to inhibit cancer cell growth. This is mediated by inhibition
of the β-subunit of NFκB, an anti-apoptotic protein, via covalent binding with the
nucleophilic Cys-61 and Arg-307 [11]. Replacement of the aldehyde group of acrolein with
amide group (acrylamide moiety) is known to attenuate the reactivity of the MA moiety
leading to less toxic derivatives [12]. In another direction, simple modification of acrolein
by adding a phenyl group to the β-carbon provides a moiety that is found in natural products
such as trans-Cinnamaldehyde (tCA) and curcumin (Fig. 1). These domestic condiments
are highly safe natural condiments with confirmed antiproliferative activities against
several cancer cell lines [9, 13, 14]. Driven by the lead pharmacophores presence in natural
food, attractive anticancer activities, and well-known safety profiles [9, 15-20], we
envisaged tCA and curcumin as building blocks to develop MA anticancer compounds with
superior pharmacologic properties (Fig. 2). We investigated structure activity relationship
of these novel compounds on the antiproliferative potency in colorectal cancer (CRC) cells
(CaCo-2, HCT-116 and HT-29). Our continued focus on CRC [21, 22] was influenced by
the prevalence of this malignant disease [23, 24], its high morbidity and mortality rate [25],
as well as the formidable problems of resistance to current available chemotherapeutic
agents [26].
The rationale for designing of these compounds depended on replacement of the
metabolically labile aldehyde group of tCA by a cinnamamide that extends to attach
another cinnamamide to form the bis-cinnamamide scaffold (Fig. 2). We investigated the

structure-activity relationship (SAR) in three phases (Fig. 2): the first generation was
testing the designed bis-cinnamamide scaffold as a close analogue to tCA, thus R1 and R2
were fixed as unsubstituted phenyl attached to two propenamide moieties and the variation
was limited to substituents on R3 that ranged from aliphatic, aromatic, heterocyclic groups.
In addition, a compound in this series was an ester (X = O). In the second generation, X-
R3 was fixed as NH-propyl and the distance between the two aromatic groups (R1 to R2)
was varied. In a third generation, the R1 and R2 were varied simultaneously by substituted
phenyl and heteraryl groups. This stepwise SAR studies helped to identify compounds with
high activity against three colon cancer cell lines.

X
O
R3
O
X
O
R3
O
N
H
N
H
R1
R2
Bis-(cinnamamide) scaffold
Designed based on tCA MA structure
First Generation (Entries 1-19, Table 1)
R = alkyl, aryl and heterocyclic
X = N, O
H
H
N
N
H
HN
HN
N
HN
O
O
O
O
O
O
8
20
21
Lead
HCT-116 IC50 = 32.0 M
Second Generation:
Scaffold Variants
Inactive against HCT-116
R2
HN
HN
H
H
N
N
HN
O
O
O
O
S
R1
Third Generation:
Fix R3 and vary R1 and R2
45
22 55
Compounds
-
HCT-116 IC50 = 2.85 M
Caco-2 IC50 = 0.89 M
HT-29 IC50 = 1.65 M
Fig. 2. General structure of bis-cinnamamide scaffold and its derivatives synthesized in
this research is illustrated (up and right). The colors in the structure indicate groups
equivalent to tCA and curcumin illustrated in Fig. 1. The SAR studies are illustrated. The
entries are detailed in Table 1 below.

2. Results
2.1. Chemical Synthesis of 2-cinnamamido-N-substituted-cinnamamide derivatives
The synthetic pathway for Final Compounds (1-56; Table 1 and Fig. 2), described in
scheme 1 utilized Erlenmeyer chemistry for azlactone synthesis [21]. In this scheme, non-
commercially available cinnamic acid analogues A2 were prepared by condensation of the
corresponding aldehyde with malonic acid [27]. After conversion to the hippuric acid
analogues (A3), the cyclo-condensation was affected by their reaction with the
corresponding aldehydes to afford the 2-arylvinyl-4-benzylidene-5-oxazolinone
derivatives (azlactones, A4) under Erlenmeyer conditions [21, 28]. The key azlactone
intermediate A4 was subsequently reacted with a variety of aliphatic and aromatic amines
to furnish the final compounds. Generally, the aliphatic and benzylic amines were reacted
with the azlactone smoothly at room temperature in ethanol. The less reactive aromatic
amines needed microwave heating in the presence of N,N-dimethylformamide (DMF) as a
solvent.
O
b or c
a
OH
CO2H
A2
R1
N
R1
R1
O
H
O
A3
A1
R2
R3
H
N
O
R1
O
O
OH
e or f
d
O
R1
N
R1
N
O
R2
H
O
A3
1-55
A4
Scheme 1. Synthesis of title compounds (a) malonic acid, pyridine, heat. (b) [i] oxalyl
chloride, DCM, DMF (cat.); [ii] glycine, sodium carbonate, THF, water (c) [i] ethyl
glycinate hydrochloride, Et3N, EDCI, DCM; [ii] NaOH, EtOH, H2O (d) R2CHO, acetic
anhydride, heat; (e) aliphatic amines, ethanol, room temperature (f) aromatic amines, DMF,
microwave heating.

The homologues 20 and 21 were prepared in similar ways starting with the appropriate
azlactones (see experimental section). All compounds were confirmed using proton nuclear
magnetic resonance (1H NMR), carbon-13 nuclear magnetic resonance (13C NMR) and
Fourier-transform infrared (FT-IR) spectroscopic analysis. Compounds were introduced to
biological screening if their purity were 95% or more in high-performance liquid
chromatography at 254 nm UV detection. Since our compounds contain more than one
aliphatic double bond, establishing the geometric isomerism was discussed during structure
confirmations. Most of compounds except 20 contain a a cinnamoyl moiety originated from
cinnamic acid or its analogues (R1). This double bond must be (E) or trans because we
1
started with E-cinnamic acids. In addition, the H NMR’s signals for these two protons
showed as doublets of J value = ~16 Hz. The benzylidene proton on the other trisubstituted
double bond was established as Z based on previous studies that discussed the isomerism
of 5(4H)-oxazolone synthesized by Erlenmeyer method [29]. The exception was the
compound 21 that have cinnamylidene group and it was identified also as (Z) isomers but
contains about 10% of (E) isomer [21].
2.2. Biological Screening
2.2.1. Antiproliferative activities of bis-cinnamamide derivatives
Compounds in this study (Fig. 2, Table 1) were tested for their cytotoxicity activities
against colorectal cancer cell line HCT-116, using sulforhodamine-B (SRB) assay [30].
The first nineteen compounds (1-19), having fixed phenyl groups at R1 and R2 positions
(Fig. 2), showed moderate to weak antiproliferative activities (Table 1). The compound
with the highest activity in this subset was the N-(1-adamantyl) analogue (4) with IC50 =
14.7 µM, but with poor solubility (ClogP 6.08), while compound 8 with moderate activities

(IC50 = 32.0 µM) showed better solubility profile (CLogP 4.48). Interestingly, 8 exhibited
significantly low cancer cell resistance at 100 µM (the highest concentration tested) as the
viability of these cells declined to 13% [21]. In addition, compound 8 very weak growth
inhibition against C-166 mouse skin fibroblasts as a representative of highly proliferative
normal cells (Fig. 3).
Table 1. Cytotoxic activities of final compounds on different colon cancer cell lines
R2
H
R1
N
O
O
R3
Entry
1
R1
Ph
Ph
Ph
Ph
Ph
Ph
R2
Ph
Ph
Ph
Ph
Ph
Ph
R3
NHPh
NH-CH2Ph
HCT-116* CACO-2*
HT-29*
ND
75.3±3.1
53.6±0.6
48.5±3.1
14.7±0.3
>100
ND
ND
2
3
4
5
6
7
ND
NH-cyclopentyl
NH-(1-adamantyl)
NH-(3-pyridyl)
NH2
ND
ND
ND
ND
ND
ND
>100
ND**
ND
NH-(4-methyl-1-
piperazinyl)
Ph
Ph
>100
ND
ND
8
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
NH-(n-Pr)
NH-(3-CN-Ph)
NH-(4-F-Ph)
NH-furfuryl
32.0±2.6
>100
38.0±0.87
ND
35.5±1.7
ND
9
10
11
12
91.9±4.4
>100
ND
ND
ND
ND
NH-(2-
morpholinoethyl)
Ph
Ph
Ph
Ph
>100
>100
ND
ND
ND
ND
13
NH-(2-hydroxyethyl)

14
15
16
17
18
19
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
4-morpholinyl
1-pyrrolidinyl
NH-(n-Bu)
NH-(sec-Bu)
NH-Et
>100
>100
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
67.2±6.3
52.4±7.1
> 100
N(Me)Et
28.8±1.0
H
N
O
Pr
O
20
21
>100
>100
ND
ND
N
H
Ph
Ph
H
N
O
Pr
O
ND
ND
N
Ph
H
Ph
22
23
24
25
26
NH-(n-Pr)
4-ClPh
4-MeOPh
4-MePh
2-thienyl
Ph
Ph
10.3±0.34
34.1±1.8
35.2±0.54
43.0±1.5
15.2±1.2
10.7±0.5
18.5±1.5
12.7±0.67
27.8±1.3
13.2±1.0
7.90±0.43
55.3±3.2
54.1±0.42
43.2±2.3
7.20±0.31
NH-(n-Pr)
NH-(n-Pr)
NH-(n-Pr)
NH-(n-Pr)
Ph
Ph
Ph
4-FPh
27
28
29
30
31
NH-(n-Pr)
NH-(n-Pr)
NH-(n-Pr)
NH-(n-Pr)
NH-(n-Pr)
4-ClPh
4-MeOPh
4-MePh
2-thienyl
Ph
4-FPh
4-FPh
4-FPh
4-FPh
3.80±0.24
29.3±1.2
2.60±0.23
45.7±1.2
4.00±0.14
86.5±2.9
6.70±0.36
17.3±0.13
6.95±0.46
5.10±0.17
10.7±0.68
4.24±0.09
11.80±0.18
7.68±0.05
6.81±0.05
4-(Me2N)Ph
32
33
34
35
36
NH-(n-Pr)
NH-(n-Pr)
NH-(n-Pr)
NH-(n-Pr)
NH-(n-Pr)
4-ClPh
4-MeOPh
4-MePh
2-thienyl
Ph
4-(Me2N)Ph
4-(Me2N)Ph
4-(Me2N)Ph
4-(Me2N)Ph
20.7±0.32
12.4±0.25
22.1±0.10
11.6±0.39
>100
17.0±0.8
15.3±0.05
29.4±0.08
27.7±3.7
6.32±0.02
9.52±0.42
36.9±0.25
39.6±0.22
66.5±0.10
9.14±0.23
3-MeO-4-OHPh
3-MeO-4-OHPh
37
38
39
40
41
NH-(n-Pr)
NH-(n-Pr)
NH-(n-Pr)
NH-(n-Pr)
NH-(n-Pr)
4-ClPh
37.8±1.8
38.4±2.1
26.3±1.3
24.8±1.4
1.65±0.05
6.14±0.44
18.0±1.2
20.9±1.1
28.3±1.3
2.1±0.03
5.47±0.34
48.6±0.4
15.1±0.67
48.3±1.9
2.36±0.04
4-MeOPh 3-MeO-4-OHPh
4-MePh 3-MeO-4-OHPh
2-thienyl 3-MeO-4-OHPh
Ph
3-indolyl
42
43
44
NH-(n-Pr)
NH-(n-Pr)
NH-(n-Pr)
4-ClPh
4-MeOPh
4-MePh
3-indolyl
3-indolyl
3-indolyl
3.51±0.21
4.35±0.29
3.39±0.10
3.35±0.05
2.07±0.03
2.3±0.08
3.41±0.03
2.49±0.04
3.12±0.06

45
46
NH-(n-Pr)
NH-(n-Pr)
2-thienyl
Ph
3-indolyl
3-pyridyl
2.85±1.5
0.89±0.04
1.65±0.07
73.0±2.5
66.0±2.2
>100
47
48
49
50
51
NH-(n-Pr)
NH-(n-Pr)
NH-(n-Pr)
NH-(n-Pr)
NH-(n-Pr)
4-ClPh
4-MeOPh
4-MePh
2-thienyl
Ph
3-pyridyl
3-pyridyl
3-pyridyl
3-pyridyl
4-NO2Ph
49.4±1.9
52.1±1.6
>100
47.3±1.9
38.1±2.0
27.0±1.8
41.8±1.4
44.2±2.7
36.0±1.7
72.7±6.4
>100
>100
68.8±6.9
23.2±1.9
17.3±1.3
52
53
54
55
NH-(n-Pr)
NH-(n-Pr)
NH-(n-Pr)
NH-(n-Pr)
4-ClPh
4-MeOPh
4-MePh
2-thienyl
4-NO2Ph
4-NO2Ph
4-NO2Ph
4-NO2Ph
>100
>100
>100
>100
>100
>100
36.9±1.5
74.3±3.6
>100
>100
34.2±1.1
>100
tCA
Dox
O
12.4 ± 1.2
ND
ND
0.613±0.10 0.147±.01
0.325±0.01
*IC50 values in µM±SE of Means (SEM). **ND, Not Determined.
Fig. 3. Dose-response curve of compound 8 on HCT-116 cell lines (blue) and C-166 cell line (brown).

Based on compound 8 favorable biological properties (see Supplementary Material,
Section S1 for more details on cellular mechanisms of this lead), it was selected as a new
lead for the second subset of compounds (22 - 55) with fixed R3 as propyl (based on 8
structure) but diversified at R1 and R2 (Fig. 2). The new compounds were tested against
three CRC cell lines (HCT-116, Caco-2 and HT-29). Some of these compounds showed
high potencies comparable to the reference drug doxorubicin (Table 1). For instance,
entries 27, 31, 41, 42, 43, 44 and 45 demonstrated excellent potencies (5.0 µM or less) with
compound 45 demonstrating the most significant growth inhibition against all tested cell
lines. The IC50 values for 45 against HCT-116, Caco-2, and HT-29 cells were 2.85, 0.89
and 1.65 µM, respectively; compared to doxorubicin 0.60, 0.14, and 0.30 µM, respectively.
Additionally, compound 45 was safe on baby hamster kidney (BHK) cell lines (IC50 = 77.6
µM) as a representative of normal (non-cancerous) cell lines. The cancer cells also had
resistance as low as 3.8%, 4.5% and 2.1% for HCT-116, Caco-2 and HT-29, respectively
at the highest tested concentration (100 µM). Also, notable is another potent compound 41,
which exhibited IC50 of 1.65, 2.1 µM and 2.36 against HCT-116, Caco-2, and HT-29 cells,
respectively.
Table 2. Cytotoxic activities of selected compounds against healthy (non-cancerous) BHK
(Baby hamster kidney) cell line.
Non-cancerous cell
Entry
line IC50 (µM)
27
31
21.2±1.93
75.7±1.64
89.9±10.4
>100
37
41
45
77.6±6.20
63.9±13.0
tCA

100
75
50
25
0
BHK (IC50 = 77.66.2)
HT-29 (IC50= 2.36 0.04)
Caco-2 (IC50= 2.1 0.033)
HCT-116 (IC50 = 1.65 0.5)
-9
-8
-7
-6
-5
-4
-3
-2
Con (M)
Fig. 4. Cytotoxic effect of 45 against human colon adenocarcinoma cancer cells (HCT-
116, Caco-2 and HT-29) vs. normal baby hamster kidney cells (BHK). All cells were
exposed to different concentrations of 45 for 72 h. The cell viability concentration curves
were plotted, and the IC50 were determined. *denotes significance at P<0.001
2.2.2. Induction of apoptosis and apoptotic changes in HCT-116 cancer cell lines upon
treatment with 45.
The apoptotic effect of 45 on HCT-116 cells [31] was investigated by the DAPI staining
test after incubation of HCT-116 cells with the compound at 3.00 µM. The result clearly
shows that the ratio of cells with fragmented DNA and condensed nuclei are higher than
that of untreated cells (control), which significantly increased after 48 h compared to 24 h
(Fig. 5).
In a subsequent experiment, 45-treated HCT-116 cells (at 3.00 µM) were monitored for
apoptotic changes at 24 h and 48 h using flow cytometry analysis after staining with
Annexin V/Propidium iodide (Fig. 6). This dual staining allows discrimination between

live cells (annexin-V-/PI-), early apoptotic cells (annexin-V+/PI-), late apoptotic cells
(annexin-V+/PI+) and necrotic cells (annexin-V-/PI+) Compound 45 induced both early
and late apoptosis in HCT-116 cells in a time-dependent manner when compared to the
untreated control cells. At 24 h, there was very little change in cell viability, but decreased
to 72% (down from 99.9%) after 48 h. Notably, the cell populations at 48 h in both early
and late apoptosis increased from almost zero to 17.5% and 4.1%, respectively.
The compound 45 also caused increase levels of cleaved caspase 3 in a time-dependent
manner (24 and 48 h), indicating nuclear release of this apoptotic marker upon treatment
with 45 (Fig. 7).
Fig. 5. Morphological changes in HCT-116 cells following exposure to compound 45. The
cells were left untreated or treated with the IC50 for 24 and 48 h. Pictures at top show the
morphological changes examined under light microscope. Images at the bottom show the
nuclei stained with DAPI and visualized under fluorescence microscope. Arrows point to
apoptotic cells.

Fig. 6. Effect of 45 on HCT-116 cell cycle progression: Key, LL= Viable cells, LR= Early
apoptotic cells, UR= Late apoptosis.
Merge
DAPI
Cleaved - caspase-3
Control
24h
48h
Fig. 7. Effect of 45 on cleaved caspase-3 expression in HCT-116 cells. The cells were
stained with DAPI to visualize nuclei (blue) and with Alexa Fluor 488- and Cy3-coupled
secondary antibodies to visualize the distribution of caspase-3 (red). Scale bar, 100 µM

2.2.3. Elevation of oxidative stress indicators within HCT-116 cells upon treatment
with 45.
ROS release, as an indicator for increased oxidative stress, was monitored over time (24
and 48 h) by using 2`,7`-dichlorofluorescin diacetate (DCFDA) dye which detects various
ROS species and emit green fluorescence. It is clear that 45 causes accumulation of ROS
in HCT-116 cells, which is proportional to the time of exposure (Fig. 8).
Fig. 8. Effect of 45 on ROS production from HCT-116 cells using DCFDA staining. Green
fluorescence represents DCFDA staining and DAPI was used as counter nuclear staining.
Scale bar, 100 µM.

2.2.4. Effect of Pre-treatment with Antioxidants on Cellular Viability
To investigate the potential effects of compound 45 on ROS levels in HCT-116 colon
cancer cells, we employed a viability assay in which cells were pre-incubated with the
antioxidant N-acetyl cysteine (NAC) or the vitamin E derivative Trolox (6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid) prior to treatment with 45 (Fig. 1). It
worthy to mention that the antioxidant concentrations used in this test had no effect on
cell viability.
As shown in Fig. 9, pre-treatment with NAC for 1 h had a dramatic protective effect on
HCT-116 cells at the higher concentrations (50, 25 µM) of compound 45. Specifically,
NAC at 10 mM markedly increased the viable cells from 34% (45 alone, 50 µM) and
55% (45 alone, 25 µM) to 89% at 24 h, respectively. The increase in viability of cells
pre-treated with NAC became modest at 48 h (40% at 50 μM) and minimal at 72 h. No
significant increases in cell viability were noted when cells were pre-treated with Trolox.

Fig. 9. Effect of antioxidant pre-treatment on viability of HCT-116 cells treated with 45.
HCT-116 cells were seeded at a density of 5 x 104 cells/ ml in 96 well plates and left
overnight to adhere. Cells were then pre-treated with NAC (10 mM) or Trolox (100 μM)
for 1 h, followed by compound 45 (50, 25 µM). For 24 h (A), 48 h (B), 72 h (C). Cell

viability was expressed as a percentage of vehicle control [ethanol 1% (v/v)] and was
measured by MTT assay (average of three independent experiments).
2.2.5. Inhibition of cancer stem cell proliferation in HT-29 cancer cell lines upon
treatment with 41 and 45
We also evaluated 45, and another potent compound 41 for their ability to inhibit cancer
stem cell proliferation using a primary (1°) colonosphere formation assay. In this assay,
HT-29 colon cancer cells were grown in low adhesion plates to form spheres as previously
described.[32] Wells treated with our compounds were compared to vehicle treated cells
for primary sphere growth inhibition after 5 days of incubation. Results, as illustrated in
Fig. 4, showed a dose-dependent inhibition of colon cancer spheroids formation (> 50 µm).
The IC50 of the two compounds were 21.12 and 18.85 µM for 41 and 45 respectively,
reflecting similar closeness in potency as in HT-29 monolayer cell line assay (2.36 and
1.65 µM, respectively). Of note, monolayer growth conditions are ideally suited for
examining cellular proliferation, whereas spheroid growth conditions examine cancer stem
cell growth and self-renewal properties. Hence, differential potency of the molecules in the
two condition is reflective of their effects on two different phenotypes.

Fig. 10. Dose-response curve of HT-29 cell spheroids (expressed as % of vehicle treated
cells) by increasing concentration of 41 (blue) and 45 (red).
2.2.6. Lethal dose toxicity test for 45
Since compounds with electrophilic properties, e.g. MA are usually involved in variety of
toxic effects on mammalians, we decided to investigate the general acute toxicity of 45 by
determining the in vivo lethal dose (LD50) according to the Globally Harmonized
Classification System (GHS) and following the OECD guideline 423 (modified, adopted
March 23, 2006). As observed in our cellular assay, the compound safety was confirmed
to be in the highest category, as the LD50 for all compounds were between 2000-5000
mg/kg (Category 5) [33].
3. DISCUSSION
Toward the goal of developing novel chemotherapeutic agents, we designed and
synthesized several 2-cinnamamido-N-substituted-cinnamamide (bis-cinnamamide)
compounds. Some of these compounds showed high potency against three CRC cancer cell

lines, and in a few cases, were comparable to doxorubicin. The straight forward Erlenmeyer
chemistry enabled SAR studies in three stages. First set of compounds (Entries 1-20, Table
1) varied only at the R3 substituent. Despite the moderate to weak antiproliferative effects
of this series, it provided the intended outcome, a lead with confirmed activities against
cancer cell lines, clear SAR and weak activity against non-cancerous highly proliferative
cell lines. In a cell line inhibition assay against the CRC cell line HCT-116, it was clear
that hydrophobic R3 substituents (e.g. compounds 4 (NH-adamantyl), 8 (NH-n-propyl), 16
(NH-n-butyl), 17 (NH-sec-butyl) and 19 (N-(Me)(Et)) demonstrated superior activities
when compared to their hydrophilic counterparts (e.g. compounds 7 (NH-(4-methyl-1-
piprazinyl), 11 (NH-furfuryl), 12(NH-(2-morpholinoethyl), 13 (NH-(2-hydroxyethyl) and
14 (4-morpholinyl). This SAR aspect is highlighted by comparing the activities of
compounds 13 and 8, where the more hydrophilic compound 13 is relatively inactive while
8 shows moderate activity despite differing from 13 only by a terminal methyl instead of
the terminal hydroxyl group of 13. Compounds bearing aromatic hydrophobic amides (e.g.
compounds 1(NHPh), 2 (NH-CH2Ph) and 9 (NH-(3-CN-Ph) showed lower
antiproliferative activities compared to aliphatic hydrophobic compounds, likely due to
poor physico-chemical properties. The primary amide 6 also was among the weakly active
compounds.
The promising results of 8 suggested that this compound is a viable lead for further
optimization. In a limited set of only 2 compounds, the scaffold optimal size was
investigated that have the distance between the two aromatic rings were changed by
contracting (20) and expanding this distance (21) respectively. Both compounds showed
almost no activity at concentrations as high as 100 µM against HCT-116 cells, suggesting

that keeping the distance between the two aromatic rings in compound 8 at 6 atoms is
important for activity.
The third-generation compounds (Fixed R3 as n-propyl and varied at R1 and R2) also
showed a consistent SAR when tested against the CRC cell lines HCT-116, Caco-2 and
HT-29. For instance, the variation in R2 position between electron-deficient and electron-
withdrawing aromatic moieties demonstrated a strong correlation between electron density
over the R2 ring and activity. Rings with higher electron density (4-dimethylaminophenyl,
4-hydroxy-3-methoxyphenyl and 3-indolyl) as exemplified by compounds 27, 31, 37, 41-
45 exerted potent antiproliferative activities against cancer cells in the low micromolar
range (0.89 to 6.9µM). Some of these compounds exhibited low activity against the non-
cancerous cell lines BHK (Table 2). On the other hand, electron-deficient R2 aryl groups
showed almost no antiproliferative activity against the cancer cell lines, exemplified by
compounds 46 to 50 (Entries 46-50, Table 1) where R2 = 3-pyridyl; and 51 to 55 (Entries
51-55, Table 1) where R2 = 4-nitrophenyl. Notably, unlike R2, variations at the R1 position
affected anticancer activities less significantly. In this regard, all compounds bearing 3-
indolyl group at R2 were highly active regardless of the structure of the groups at R1.
Meanwhile, all compounds with electron withdrawing groups at R2 (such as 3-pyridyl and
4-nitrophenyl) were much less active, and this was also irrelevant to the structure of R1.
The most potent compounds in 2-cinnamamido-N-substituted-cinnamamide (bis-
cinnamamide) series were the 3-indolyl derivatives (41 to 45) which proved to be the most
active against all the three cell lines. Compounds 41 and 45 showed almost comparable
potency as doxorubicin (the positive control) on all of the tested cell lines. For instance,
Caco-2 IC50 for 45 was 0.89 µM compared to 0.14 µM for doxorubicin. The same

compound exhibited about 100 times weaker antiproliferative effect on C-166 (IC50 = 71
µM) compared to Caco2 cancer cell line (0.89 µM). The low toxicity of 45 was also
confirmed by a preliminary LD50 test.
Recently, models of anticancer activities are increasingly associated with activities of
compounds on cancer cell aggregates. Similar to normal stem cells, cancer stem cells
(CSCs) reside in niches characterized by hypoxia and low reactive oxygen species (ROS),
both of which are critical for maintaining the potential for self-renewal and stemness.[34]
Consistently, CSCs show low intracellular ROS levels, suggesting that maintaining a
reduced intracellular environment is associated with an undifferentiated state [35]. We,
therefore, tested the effect of 41 and 45 on CSCs in a cancer colonosphere formation assay,
which is designed to screen for molecules that selectively target colorectal CSCs [32].
Results of the CSC inhibition test showed clear concentration-dependent inhibition of
sphere size; corroborating results from other experiments.
Since the whole series of 2-cinnamamido-N-substituted-cinnamamide (bis-cinnamamide)
compounds was based on MA scaffold of tCA, cellular changes leading to cancer cell
growth inhibition confirmed that ROS elevation (Fig. 8) is a major contributor of 45 HCT-
116 cytotoxic activities. Moreover, the attenuation of 45 cytotoxicity by the antioxidant
NAC was another evidence of ROS involvement in the cell death process. Being
responsible for cancer recurrence after chemotherapy or radiotherapy, CSC targeting via
ROS holds a great promise in cancer therapy.
Annexin-V staining-flow cytometry method (Fig. 6) confirmed the apoptotic events upon
exposure to 45. The compound induced time-dependent increase in cell population
entrance to both early and late apoptosis. The cell apoptosis, induced by 45, was also

confirmed by increased levels of cleaved caspase-3 and its release through the nuclear
membrane (Fig. 7). In agreement with the above mentioned Annexin-V staining assay
result, 45 induces apoptosis via activation of caspases 3.
In conclusion, this work presents a model of molecular discovery of promising MA
compounds to be further optimized as anticancer agents especially for colorectal cancer.
These compounds showed selective toxicity against cancer cell lines and CSC over
noncancerous cells, low animal toxicity, and clear cell death mechanism. The two most
potent compounds, 41 and 45 will be subjected to more SAR investigations, as well as
antitumor activities in future studies.
4. MATERIALS AND METHODS
4.1. Chemical Synthesis
Solvents and reagents were purchased from Sigma-Aldrich (USA), VWR (USA) or Alfa
Aesar (UK). When needed, solvents were dried according procedures described in
literature. Unless stated otherwise, reactions were performed under inert atmosphere of
nitrogen. Microwave reactions (MW) were performed using Milestone StartSynth™
reactor (Milestone Inc., Italy). Melting points (Mp) were determined in open capillary tubes
using Electrothermal apparatus (Stuart, UK) and are uncorrected. NMR were recorded on
Bruker DPX-300 MHz (Bruker, Switzerland). HPLC-Mass Spectrometry were performed
on Agilent 1100 / ZQ MSD including C18 column and diod-array UV detector. The mobile
phase (containing 0.01 M ammonium acetate) was gradient starting from 20%

acetonitrile/80% water to 80% acetonitrile/20% water. Purities are reported according to
percentage of Peak Areas at wavelength 254 nm. According to LC/MS analyses, all
compounds in this study were confirmed to have 95% purity or higher. Infrared spectra
were recorded on a Thermo Scientific Nicolet iS10 Fourier transform (FT)-IR
Spectrometer. In this report, we only listed the important IR stretching bands, including
NH, OH, CH, C=O, C=N and/or C=C. In FT-IR, all samples were measured neatly
The previously reported azlactone A3 [36], the final 2-aminopropenamide
derivatives 1 [37], 2 [36], 6 [36] and 21 [21] were prepared according to procedures
mentioned below, and their physical and spectral properties were confirmed. Purity of
compounds were first assessed qualitatively using Thin Layer Chromatography (TLC), 1H
NMR and quantitatively using LC/MS (UV detection). The compounds were screened only
if purity was confirmed to be above 95%. The compounds subjected to all biological
screenings were used as a single Z isomer as detected by TLC, LC/MS and NMR except
compound 21 which contained 10% of the E isomer.
4.1.1. (Z)-2-cinnamamido-N,3-diphenylacrylamide (1) [37]
In the reaction tube of the microwave reactor (Milestone™, SynthLab), oxazolone A4 (2
mmol, 0.554 g) and aniline (2 mmol, 0.193 mL) were added to 5 mL of DMF. The reaction
was heated to 200°C with stirring for 10 min. After cooling, the mixture was added slowly
to ice-cold 1 M HCl. The resulting solid was collected by filtration, washed with water and

purified using silica gel chromatography (petroleum ether / DCM / MeOH, gradient;
petroleum ether 100% then petroleum ether : DCM (1 : 1) then DCM 100% and then 1-5%
MeOH in DCM). The final compound 1 was collected as a white solid (0.520 g, 70.6%),
Mp 129-132 C. 1H NMR (600 MHz, acetone-d6) δH ppm 9.50 (br. s, 1H, NH), 9.11 (br. s,
1H, NH), 7.80 (d, J = 7.5 Hz, 2H, Ar-H), 7.60-7.69 (m, 6H, Ar-H), 7.38-7.49 (m, 6H, Ar-
H + Ar-CH=CH-), 7.30-7.37 (m, 4H, Ar-H), 7.17 (s, 1H, -CH=C-), 7.06-7.12 (m, 1H), 7.02
(d, J = 15.8 Hz, 1H, Ar-CH=CH-).
4.1.2. (Z)-N-benzyl-2-cinnamamido-3-phenylacrylamide (2) [36]
Oxazolone A4 (10 mmol, 2.773 g) was dissolved in 50 mL ethanol, benzylamine (20 mmol,
2.18 mL) was then added, and the mixture was stirred for 2 h. The solvent was removed
under reduced pressure. The residue was treated with ice-cooled dilute HCl resulting in
precipitation of an off-white solid product. The crude product was purified by
crystallization from ethanol. The purified product 2 was an off-white solid, (0.45 g, 60%),
Mp. 198-199 C. 1H NMR (600 MHz, Acetone-d6) δH ppm 8.08 (br. s, 1H, NH), 7.57-7.68
(m, 5H, Ar-H), 7.36-7.48 (m, 8H, Ar-H+ Ar-CH=CH-), 7.29-7.35 (m, 4H, Ar-H + -CH=C-
), 7.24 (t, J = 7.3 Hz, 1H, Ar-H), 6.96 (d, J = 15.4 Hz, 1H, Ar-CH=CH-), 4.52-4.58 (m,
2H, Ar-CH2-), LC-MS (ESI), RT = 4.4 min, m/z Calculated, 382; found, 383.1 [M + H]+.
4.1.3. (Z)-2-cinnamamido-N-cyclopentyl-3-phenylacrylamide (3)
This compound was prepared according to the procedure described for the synthesis of 2
starting from oxazolone A4 (2 mmol, 0.554 g) and cyclopentylamine (2 mmol, 0.195 mL).
The product 3 was a white solid, (0.51 g, 73%), Mp 239 C. 1H NMR (600 MHz, acetone-
d6) δH ppm 8.85 (s, 1H, NH), 7.59-7.67 (m, 2H, Ar-H), 7.55 (d, J = 7.5 Hz, 2H, Ar-H),
7.40-7.47 (m, 3H, Ar-H+ Ar-CH=CH-), 7.37 (t, J = 7.5 Hz, 2H, Ar-H), 7.28-7.32 (m, 1H,

Ar-H), 7.09 (br. s, 1H, -CH=C-), 6.96 (d, J = 15.8 Hz, 1H, Ar-CH=CH-), 4.24-4.32 (m,
1H, Pyrolidine-CH), 1.90-1.98 (m, 2H, Pyrolidine-CH2), 1.71 (br. s, 2H, Pyrolidine-CH2),
1.54-1.62 (m, 4H, 2-Pyrolidine-CH2).
4.1.4. (Z)-N-(1-adamantanyl)-2-cinnamamido-3-phenylacrylamide (4)
This compound was prepared according to the procedure described for the synthesis of 2
starting from oxazolone A4 (2 mmol, 0.554 g) and 1-aminoadamantane (2 mmol, 0.308 g).
The product 4 was a white solid (0.75 g, 88%), Mp 272 C. 1H NMR (600 MHz, acetone-
d6) δH ppm 8.86 (s, 1H, NH), 7.62-7.68 (m, 3H, Ar-H), 7.54 (d, J = 7.5 Hz, 2H, Ar-H),
7.40-7.47 (m, 3H, Ar-H+ Ar-CH=CH-), 7.37 (t, J = 7.5 Hz, 2H, Ar-H), 7.27-7.32 (m, 1H,
Ar-H), 7.05 (s, 1H, -CH=C-), 6.98 (d, J = 15.4 Hz, 1H, Ar-CH=CH-), 2.17 (m, 1H,
adamantly-CH), 2.13 (br. s, 6H, 3-adamantly-CH2), 2.00-2.03 (m, 1H, adamantly-CH),
1.95-1.97 (m, 1H, adamantly-CH), 1.73 (br. s, 6H, 3-adamantly-CH2), 1.57-1.59 (m, 1H,
adamantly-CH).
4.1.5. (Z)-2-[(E)-cinnamamido]-3-phenyl-N-(3-pyridyl)acrylamide (5)
In the reaction tube of a microwave reactor (SynthLab), oxazolone A4 (2 mmol, 0.554 g)
and 3-aminopyridine (2 mmol, 0.188 g) were mixed with 5 mL DMF. The reaction was
heated to 200°C while stirring in a Milestone microwave reactor for 10 min. After cooling,
the mixture was added slowly to ice-cold water. The resulting solid was collected by
filtration, washed with water and purified using silica gel chromatography (petroleum
ether/ dichloromethane (DCM)/MeOH, gradient; petroleum ether 100% then petroleum
ether : DCM (1 : 1) then DCM 100% and then 1-5% MeOH in DCM) to give 5 as a white
solid (0.206 g, 30%), Mp > 250C (dec). 1H NMR (600 MHz, Acetone-d6) δH ppm 9.79 (br
s, 1H, NH), 9.31 (br s, 1H, NH), 8.91 (s, 1H, Ar-H), 8.31 (dd, J = 6.5, 1.13 Hz, 1H, Ar-H),

8.27 (d, J = 8.7 Hz, 1H, Ar-H), 7.61-7.69 (m, 5H, Ar-H), 7.39-7.48 (m, 5H, Ar-H+ Ar-
CH=CH-), 7.38-7.32 (m, 2H, Ar-H), 7.21 (s, 1H, -CH=C-), 7.03 (d, J = 15.8 Hz, 1H, Ar-
CH=CH-), LC-MS (ESI), RT = 7.4 min, m/z Calculated, 369; found, 370.2 [M + H]+.
4.1.6. N-[(Z)-3-amino-3-oxo-1-phenylprop-1-en-2-yl]-(E)-cinnamamide (6)
Oxazolone A4 (2mmol, 0.554 g) was stirred in 5 mL solution of ammonia (2M) in ethanol
for 2h. The product was precipitated as white powder which was filtered, washed with
water several times followed by ethanol and dichloromethane. Crystallization from
aqueous methanol provided a white solid of 6 Mp >250 C (dec.) 1H NMR (DMSO-d6) dH
ppm 10.11 (br. s, 1H, NH), 7.75 (br. s, 1H, NH), 7.61 (d, J = 6.8 Hz, 2H, Ar-H), 7.55 (d, J
= 7.5 Hz, 2H, Ar-H), 7.35-7.53 (m, 6H, Ar-H+ Ar-CH=CH-), 7.31 (d, J = 7.2 Hz, 1H, Ar-
H), 7.16 (br. s, 1H, NH), 7.10 (s, 1H, -CH=C-), 6.91 (d, J = 15.8 Hz, 1H, Ar-CH=CH-).
4.1.7. (Z)-2-[(E)-cinnamamido]-N-(4-methyl-1-piperazinyl)-3-phenyl-acrylamide (7)
This compound was prepared according to the procedure described for the synthesis of 2
starting from oxazolone A4 (2 mmol, 0.554 g) and 1-amino-4-methylpiperazine (4 mmol,
0.49 mL). The product 7 was an off-white power, (305 mg, 39%), Mp. 190 °C; 1H NMR
(600 MHz, DMSO-d6) δH ppm 9.83 (s, 1H, NH), 9.58 (s, 1H, NH), 7.62 (d, J = 7.5 Hz, 2H,
Ar-H), 7.54-7.58 (m, J = 7.9 Hz, 2H, Ar-H), 7.52 (d, J = 15.8 Hz, 1H, Ar-CH=CH-), 7.44-
7.48 (m, 2H, Ar-H), 7.39-7.44 (m, 3H, Ar-H), 7.30-7.35 (m, 1H, Ar-H), 6.91 (d, J = 15.8
Hz, 1H, Ar-CH=CH-), 6.75 (br. s, 1H, -CH=C-), 3.43 (br. s, 2H, piprazinyl-CH2), 3.14-
3.22 (m, 4H, 2-piprazinyl-CH2), 3.10 (br. s, 2H, piprazinyl-CH2), 2.77 (br. s, 3H, N-CH3).
4.1.8. (Z)-2-[(E)-cinnamamido]-3-phenyl-N-propylacrylamide (8)
This compound was prepared according to the procedure described for the synthesis of 2
starting from oxazolone A4 (2 mmol, 0.554 g) and n-propylamine (2 mmol, 0.166 mL).