In-vitro and in-silico evaluations of heterocyclic-containing diarylpentanoids as Bcl-2 inhibitors against lovo colorectal cancer cells

Article abstract of DOI:10.3390/molecules25173877

In the present study, we investigated the in-vitro anti-cancer potential of six diarylpentanoids against a panel of BRAF- and KRAS-mutated colorectal cancer cell lines including T84, SW620, LoVo, HT29, NCI-H508, RKO, and LS411N cells. Structure-activity relationship study suggested that the insertions of tetrahydro-4H-thiopyran-4-one and brominated phenyl moieties are essential for better cytotoxicity. Among the evaluated analogs, 2e has been identified as the lead compound due to its low IC₅₀ values of approximately 1 μM across all cancer cell lines and high chemotherapeutic index of 7.1. Anti-proliferative studies on LoVo cells showed that 2e could inhibit cell proliferation and colony formations by inducing G2/M cell cycle arrest. Subsequent cell apoptosis assay confirmed that 2e is a Bcl-2 inhibitor that could induce intrinsic cell apoptosis by creating a cellular redox imbalance through its direct inhibition on the Bcl-2 protein. Further molecular docking studies revealed that the bromophenyl moieties of 2e could interact with the Bcl-2 surface pocket through hydrophobic interaction, while the tetrahydro-4H-thiopyran-4-one fragment could form additional Pi-sulfur and Pi-alkyl interactions in the same binding site. In all, the present results suggest that 2e could be a potent lead that deserves further modification and investigation in the development of a new Bcl-2 inhibitor.

Full text of DOI:10.3390/molecules25173877

molecules
Article
In-Vitro and In-Silico Evaluations of
Heterocyclic-Containing Diarylpentanoids as Bcl-2
Inhibitors Against LoVo Colorectal Cancer Cells
Sze Wei Leong 1,*, Suet Lin Chia 1,2,
*
, Faridah Abas 3,4 and Khatijah Yusoff 1,2,5
1
Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia,
UPM Serdang 43400, Selangor Darul Ehsan, Malaysia; Kyusoff@upm.edu.my
UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia,
UPM Serdang 43400, Selangor Darul Ehsan, Malaysia
Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia,
UPM Serdang 43400, Selangor, Malaysia; Faridah_abas@upm.edu.my
Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia,
UPM Serdang 43400, Selangor Darul Ehsan, Malaysia
2
3
4
5
Malaysia Genome Institute (MGI), National Institute of Biotechnology Malaysia (NIBM), Jalan Bangi,
Kajang 43000, Malaysia
*
Correspondence: leongszewei@upm.edu.my (S.W.L.); suetlin@upm.edu.my (S.L.C.);
Tel.: +603-9769-7765 (S.W.L.); +603-9769-8295 (S.L.C.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Mai Antonello and Loredana Salerno
Received: 26 June 2020; Accepted: 2 August 2020; Published: 26 August 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: In the present study, we investigated the in-vitro anti-cancer potential of six diarylpentanoids
against a panel of BRAF- and KRAS-mutated colorectal cancer cell lines including T84, SW620, LoVo,
HT29, NCI-H508, RKO, and LS411N cells. Structure–activity relationship study suggested that the
insertions of tetrahydro-4H-thiopyran-4-one and brominated phenyl moieties are essential for better
cytotoxicity. Among the evaluated analogs, 2e has been identified as the lead compound due to its
low IC50 values of approximately 1 µM across all cancer cell lines and high chemotherapeutic index
of 7.1. Anti-proliferative studies on LoVo cells showed that 2e could inhibit cell proliferation and
colony formations by inducing G2/M cell cycle arrest. Subsequent cell apoptosis assay confirmed that
2e is a Bcl-2 inhibitor that could induce intrinsic cell apoptosis by creating a cellular redox imbalance
through its direct inhibition on the Bcl-2 protein. Further molecular docking studies revealed that
the bromophenyl moieties of 2e could interact with the Bcl-2 surface pocket through hydrophobic
interaction, while the tetrahydro-4H-thiopyran-4-one fragment could form additional Pi-sulfur and
Pi-alkyl interactions in the same binding site. In all, the present results suggest that 2e could be
a potent lead that deserves further modification and investigation in the development of a new
Bcl-2 inhibitor.
Keywords: diarylpentanoids; colorectal cancer; cytotoxicity; anti-proliferative; cell cycle; apoptosis;
Bcl-2 inhibitor; molecular docking
1. Introduction
Colorectal cancer (CRC) is the 3rd most common type of cancer and 2nd leading cause of cancer
death worldwide. According to statistics by Globocan, there were approximately 1.8 million new
CRC cases in 2018 and the number of CRC-related deaths has surpassed 850,000 in the same year.
Worse still, these numbers are estimated to increase to 3.1 and 1.3 million cases, respectively, by 2040 [1].
To date, chemotherapy remains as the mainstream in the management of CRC. Among the currently
available chemotherapy agents, oxaliplatin and 5-fluorouracil (5-FU) are the two most common drugs
Molecules 2020, 25, 3877; doi:10.3390/molecules25173877
www.mdpi.com/journal/molecules

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that have been widely used in treating CRC. Previous studies showed that the combination of these
compounds could simultaneously disrupt the synthesis and the replication of DNA, thereby effectively
suppressing the tumor growth [
used as a synergistic treatment against all stages of CRC, particularly metastasis [
2
,
3
]. Based on this, a mixture of oxaliplatin and 5-FU has been
]. Despite such
4
a cocktail successfully improving the CRC tumor response and prolonging the survival of CRC
patients, its associated side effects including nausea, cardiotoxicity and neurotoxicity remain as issues
of concern [5,6]. These undesirable side effects not only hinder the full therapeutic potential of the
respective chemotherapy but also reduce the quality of life of the cancer patients. On this account,
alternative effective anti-cancer agents with improved safety profile are urgently needed.
Turmeric is a yellow powder obtained from the dried rhizome of Curcuma longa Linn. that has
been widely used as a spice for food flavoring and coloring especially in Asian cuisines. Apart from
food preparation, turmeric has also been used in traditional remedies to treat several illness and injuries
including eye infections, skin rashes, indigestion, insect bites and burns [7]. Due to its medicinal values,
turmeric has received a great deal of attention by researchers in the past few decades. As the major
component of turmeric, curcumin (Figure 1) has been studied extensively for its biological activities
and therapeutic potential. Many studies revealed that curcumin possesses strong antioxidant and
inflammatory activities as it was found to scavenge several types of free radical species as well as
inhibit a wide range of pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-
interleukins, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) [ 11]. In addition,
curcumin was also found to exhibit prominent anti-cancer potential based on its growth-inhibiting
and apoptosis-inducing abilities against multiple cancer cell lines [12 15]. Interestingly, this naturally
occurring small molecule has displayed excellent safety profiles in both preclinical and clinical studies
which prompted it to be a promising candidate in the search for a safer chemotherapeutic agent [16 17].
Unfortunately, the practical use of curcumin is limited by its low bioavailability due to the instability of
the molecular structure. The -diketone moiety of curcumin has been reported to be highly reactive and
easily dissociated by several metabolic and detoxification enzymes such as aldehyde dehydrogenases
and aldo-keto reductases [18 20]. On this account, structural optimisation to remove the unstable
α),
8
–
–
,
β
–
β-diketone moiety of curcumin has been the focus of curcuminoid-based studies.
Figure 1. Chemical structure of curcumin.
Cyclic monocarbonyl diarylpentanoids are one of the bioactive families that are derived from
curcumin by eliminating the reactive
and metabolic stability, these diketone-free curcuminoids were also found to exhibit better medicinal
potential than that of curcumin, particularly as anti-cancer agents [21 22]. Many studies reported
β-diketone moiety. Apart from showing improved chemical
,
that cyclic monocarbonyl diarylpentanoids possess potent anti-cancer properties based on their
superior inhibitory activity against the growth of various cancer cell lines including breast, lung,
colon and prostate cancer cells [13,23–25]. Several downstream mechanistic studies revealed that
the remarkable anti-cancer properties of these diarylpentanoids are due to their anti-angiogenic
and apoptosis-inducing activities, which resulted from VEGF inhibition and caspases activation,
respectively [26
effective in-vivo based on their positive suppression against tumor growth in several mice xenograft
models [29 30]. Interestingly, recent studies demonstrated that this diarylpentanoid family possesses
a curcumin-like safety profile as they were shown to be non-toxic in acute toxicity evaluations of
several anti-inflammatory animal models [31 32]. Based on this, cyclic monocarbonyl diarylpentanoids
–28]. In addition, cyclic monocarbonyl diarylpentanoids were also found to be
,
,

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are therefore of interest and worthy for further investigation in the search of safer anti-cancer drugs.
Figure 2 depicts the general structures of previously reported cyclic monocarbonyl diarylpentanoids.
Figure 2. General structures of reported cyclic monocarbonyl diarylpentanoids.
To the best of our knowledge, previous studies were mainly focused on the cycloalkanone,
piperidone and tetrahydropyranone derivatives of diarylpentanoid as they were usually shown to
possess promising anti-cancer activity. Despite the respective tetrahydrothiopyranone derivatives
being rarely explored, their anti-cancer potential should not be underestimated as the cytotoxicity
enhancing effects of the tetrahydropyranone moiety has recently been reported [33,34]. On the
basis of this, the aim of the present study was to evaluate the anti-cancer potential of
tetrahydro-4H-thiopyran-4-one-containing diarylpentanoids and compare to that of their respective
cyclohexanone-based analogs.
2. Results and Discussions
2.1. Chemistry
Scheme 1 outlines the synthetic pathway of cyclohexanone- and tetrahydro-4H-thiopyran-
4-one-containing diarylpentanoids. As depicted, 2a
by reacting cyclohexanone or tetrahydro-4H-thiopyran-4-one with appropriate benzaldehydes in the
presence of 10 M sodium hydroxide. The formations of 2a 2e were confirmed by the detection of a
–2e were achieved through simple aldo-condensation
–
singlet at the chemical shift of 7.5–8.0 ppm in their respective proton NMR, where such signals represent
the presence of benzylidene vinylic groups. All synthesised compounds were purified by column
chromatography and characterized by 1H-NMR, 13C-NMR, and high-resolution electron impact-mass
spectrometry (HRMS). The melting point of known compounds (2a, 2b and 2f) were compared to
that of previously reported data [35 38]. Prior to assays, the purity of synthesized compounds was
–
confirmed to be greater than 95% based on their respective HPLC profiles (Supplementary Materials).
The synthesized compounds are listed in Table 1 and their respective chemical structures can be found
in both Table 1 and Figures 9–14.
Scheme 1. General synthetic steps for 2a– a) benzaldehyde, EtOH,
2e x. xReagents and conditions: (
10 M NaOH, RT (overnight).

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Table 1. Chemical Structures of 2a–2e.
Cmpd
2a
Structure
Cmpd
Structure
Cmpd
Structure
2b
2c
2d
2e
2f
Cmpd = Compound.
2.2. Biological Studies
2.2.1. In-Vitro Cytotoxicity Analysis
The in-vitro cytotoxic activity of the synthesized compounds on seven human CRC cell lines
including KRAS (T84, SW620, LoVo) and BRAF (HT29, NCI-H508, RKO and LS411N) mutated cells
were determined by MTT assay as previously described [39]. The IC50 values obtained are summarized
in Table 2 in which 5-FU and normal human dermal fibroblasts (NHDF) were used as controls.
Meanwhile, a heatmap (Figure 3) was also generated using GraphPad Prism v7.0 (GraphPad Software,
La Jolla, CA, USA) to visualize the cytotoxic trends of synthesized compounds graphically.
Table 2. Cytotoxicity of 2a–2e on T84, SW620, LoVo, HT29, NCI-H508, RKO, LS411N and normal
human dermal fibroblast (NHDF) cell lines at 72 h post-treatment.
IC50 (µM)
Chemo-
Therapeutic
Index
KRAS
BRAF
Compound
X
R
NHDF
T84 SW620 LoVo HT29 NCI-H508 RKO LS411N
5-FU
2a
2b
2c
2d
2e
2f
-
-
>10
>10
>10
3.3
>10
1.6
>10
>10
8.9
3.2
>10
1.4
3.3
>10
>10
3.3
6.9
1.0
>10
>10
>10
5.4
>10
2.1
8.6
>10
7.8
3.3
5.3
1.1
6.2
2.9
>10
>10
2.6
7.1
1.2
4.6
>10
>10
3.7
5.8
1.3
>20
>20
>20
12.9
9.7
-
-
-
C
C
C
S
S
S
2-OMe
2-Br
3,4-F
2-OMe
2-Br
2.3–5.0
1.4–1.8
3.4–7.1
0.3–0.8
7.1
2.0
3,4-F
>10
2.9
5.1
>10
2.4
5.9
Figure 3. Overall IC50 distribution of 2a–2e against T84, SW620, LoVo, HT29, NCI-H508, RKO,
LS411N and NHDF cell lines.
As shown in Table 2 and Figure 3, 2c and 2e displayed excellent cytotoxicity effects against all
tested CRC cell lines (<5 µM) in which 2e was found to be the most promising candidate based

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on its high chemotherapeutic index of 7.1 and low IC50 values ranging from 1.0 to 2.1
µM. On top
of this, 2e displayed at least 2.5-fold better cytotoxicity than that of the positive control, 5-FU,
which further highlighted its potential to be developed as a chemotherapeutic agent. On the other
hand, 2f was found to be another strong cytotoxic agent as it successfully inhibited several CRC cell
lines with IC50 values ranging from 2.4 to 6.2 µM. However, although 2f exhibits a certain degree of
anti-cancer activity, its therapeutic potential is completely offset by its undesirable cytotoxic effects
on normal human cells (NHDF). Of note, all synthesized compounds displayed similar cytotoxic
effects against both KRAS- and BRAF-mutated CRC cell lines, which implies that both cyclohexanone-
and tetrahydro-4H-thiopyran-4-one-containing diarylpentanoids may not be BRAF- or KRAS-specific
inhibitors. A subsequent SAR study revealed that the tetrahydro-4H-thiopyran-4-one fragment is
crucial for improving cytotoxicity of diarylpentanoids, as both 2d and 2e displayed much better
cytotoxicity than their respective cyclohexanone analogs (2a and 2b). Interestingly, substitution of
halogen at the ortho position of phenyl rings appeared to be essential for cytotoxicity enhancement
as 2b and 2e, the two ortho-brominated diarylpentanoids, displayed stronger activity than their
ortho-methoxylated analogs (2a and 2d). In contrast, the 3,4-difluorophenyl group was found to be
undesirable as compounds with such a moiety displayed significantly higher cytotoxicity against
normal human cells. These observations are consistent with our previous studies in which we reported
that insertion of the ortho-halogenated phenyl moiety could improve the cytotoxicity of diarypentanoids
while the presence of the 3,4-difluorophenyl moiety may increase compounds’ cytotoxicity against
normal human cells [39,40]. Thus, in view of its potent cytotoxicity and high chemotherapeutic index,
2e was selected for further mechanistic investigation.
2.2.2. In-Vitro Anti-Proliferative Analysis
To identify the potential cytotoxic mechanisms of 2e, morphological inspection, colony formation
and cell cycle analyses were performed. The results obtained are summarized and compiled into
Figure 4.
Figure 4. Effects of 0.625, 1.25, 2.5 and 5
µM of 2e on cells morphology at 24 h post-treatment (
A),
colony formation at 7 days post-treatment (
B
) and cell cycle ( and ) of LoVo cells at 24 h post-treatment.
C
D
* is representing the level of significant difference. The more it has, the higher the significant difference
it is compared to the control.
As depicted in Figure 4A, the area of the LoVo cell islands was reduced upon the treatments with
higher concentrations of 2e, while the morphology of 2e-treated LoVo cells was found to shrink from an
epithelial-like shape to a round shape. These observations suggest that 2e may exhibit anti-proliferative

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and apoptosis-inducing activities. In addition to this, 2e was also found to inhibit the colony formation
of LoVo cells in a dose-dependent manner (Figure 4B), which thereby confirmed its anti-proliferative
activity. To further investigate the possible anti-proliferative mechanism, 2e was subjected to flow
cytometry-based cell cycle analysis and the results obtained are summarized in Figure 4C,D. Based on
the results obtained, 2e was found to induce G2/M-phase cell arrest in LoVo cells, as treatments with
2.5 and 5 µM of 2e successfully increased the intensity of the G2/M peak. Thus, taking morphological
inspection, colony formation and cell cycle analysis together, the present results strongly suggest that
2e is a potent anti-proliferative agent that could induce G2/M-phase cell arrest.
2.2.3. In-Vitro Cell Apoptosis Analysis
To further investigate the underlying cytotoxic mechanisms of 2e against LoVo cells, both flow
cytometry and Western blot analyses were performed. The results obtained are summarised and
compiled into Figure 5.
Figure 5.
(
A
) Apoptosis profiles of LoVo cells at 24 h, 48 h and 72 h of post-treatments with 0.625, 1.25,
M of 2e. ( ) Comparison of total apoptotic cells induced by 2e at 24 h, 48 h and 72 h of
M of 2e. ( ) Western blot analysis on cPARP, caspases and
Bcl-2 family proteins expression in 2e-treated (5 M) LoVo cells at 24 h, 48 h, and 72 h of post-treatments.
) Representative flow cytometry-based ROS profiles of 2e-treated (5 M) LoVo cells at 24 h, 48 h and
2.5 and 5
µ
B
post-treatments with 0.625, 1.25, 2.5 and 5
µ
C
µ
(D
µ
72 h of post-treatments. * is representing the level of significant difference. The more it has, the higher
the significant difference it is compared to the control.
As shown in the annexin V-based flow cytometry analysis (Figure 5A), treatment with 2.5 and 5 µM
of 2e shifted the density plots from left to right and upper right. This indicates that 2e could induce
cellular apoptosis in LoVo cells as such a shifting pattern reflects the onset and progression of cellular
apoptosis. Similar to that of the cell cycle study, 0.625 and 1.25
µM of 2e exhibited minimal insignificant
effects on LoVo cells (Figure 5B), which implied that 2.5 M could be the lowest in vitro effective dose
µ
of 2e against LoVo cells. In order to validate the apoptosis inducing ability of 2e, Western blot studies
against cleaved PARP and cleaved caspase-7 were carried out.
As depicted in Figure 5C, both cleaved PARP and caspase-7, a hallmark of apoptotic cell death,
were increased after 24 h of treatments, thus confirming the apoptotic nature of 2e [41]. This is in
consistent with that of the annexin V-based assay in which the apoptotic cells were detected as early
as 24 h post-treatment. To further uncover the underlying apoptotic mechanism of 2e, its effects on
initiator caspases and proapoptotic proteins were studied. According to the results, 2e was suggested to
be an intrinsic apoptosis inducer based on its ability to activate the cleavage of caspase-9 while leaving
caspase-8 unaffected (with no detection of cleaved caspase-8). This finding is further supported by the
up-regulation of Bad and Bax proteins, as they are the common proteins that are actively involved in the

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progression of intrinsic apoptosis. However, unlike cleaved caspase-7 and cleaved PARP, the changes
in Bad and Bax expression is only detectable after 48 h of post-treatment. This suggests that Bax and
Bad activation might not be the primary apoptotic pathway induced by 2e. Thus, in continuing our
efforts to reveal the underlying mechanism, effects of 2e on anti-apoptotic proteins including Bcl-2,
Bcl-xL and Mcl-2 were examined. As presented in Figure 5C, Bcl-2 was significantly inhibited by 2e
as early as 24 h post-treatment, while the expressions of both Bcl-xL and Mcl-1 remained unaffected,
even if the treatments were extended to 72 h. This finding suggests that Bcl-2 could be the primary
target of 2e as its down-regulation occurred earlier than the up-regulation of Bax and Bad.
In general, Bcl-2 is an anti-apoptotic protein that is well-known for its ability to prevent the
permeabilisation of the mitochondrial outer membrane induced by Bak and Bax, thus protecting
cells from apoptosis [42]. However, recent evidence indicates that Bcl-2 is not only an anti-apoptotic
protein but also a cellular ROS regulator whereby knock-down of Bcl-2 increased subjects’ oxidative
susceptibility and responses in both in-vitro and in-vivo models [43–45]. Based upon this, it is
reasonable to suggest that Bcl-2 inhibition may lead to redox imbalance and thus increases the cellular
ROS level. Therefore, with the aim to validate the role of Bcl-2 as a primary target of 2e, we further
examined the effects of 2e on cellular ROS through DCFH-DA-based analysis.
As shown in Figure 5D, 24 h of treatment with 2e shifted the histogram to the right as a whole,
implying a rapid increment in cellular ROS level. Since Bax and Bad were not activated at this time
point, such an increment is therefore evidence of redox imbalance, which resulted from Bcl-2 inhibition.
Based upon this, the role Bcl-2 as primary target of 2e is thus confirmed. Of note, when the treatment
was prolonged to 48 h and 72 h, an additional peak appeared at the rightmost point of the histogram,
which implies a secondary increment of cellular ROS. This secondary increment could be rationalised
by the stimulation of ROS, which resulted from the vicious cycle of oxidative stress induced by Bcl-2
inhibition. Taken together, the present results confirmed that 2e is a Bcl-2 inhibitor that can induce
intrinsic apoptosis by disrupting the cellular redox state. The overall apoptosis mechanism induced by
2e is summarised in Figure 6.
Figure 6. Illustration of the intrinsic apoptosis mechanism induced by 2e in LoVo cells.
2.2.4. In-Silico Molecular Modelling Analysis
In order to gain further insights into the binding mode of 2e in Bcl-2, flexible docking was
performed using Discovery Studio 3.1. Prior to docking 2e into Bcl-2, the docking parameters

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were first validated by the re-docking of the co-crystallised ligand into the active site of the protein.
The parameters are considered acceptable if the root mean square deviation (RMSD) value obtained
from the re-docked and native ligands does not exceed 2.0 Å [46].
As depicted in Figure 7, both redocked (yellow) and native (blue) ABT-263 were found to
interact with Bcl-2 in very similar conformations, of which the respective RMSD value obtained was
1.8 Å. This implies that the selected parameters are acceptable and could be used further for the
docking studies of 2e. In order to compare and contrast the binding interactions contributed by
tetrahydro-4H-thiopyran-4-one and cyclohexanone fragments, 2b was also included in the present
docking analysis. Figure 8 illustrates the binding interactions of 2b and 2e in Bcl-2.
Figure 7. Overlay of re-docked (yellow) and native (blue) conformations of ABT-263 in Bcl-2
(PDB ID: 4LVT).
Figure 8. Molecular docking model of 2e with the Bcl-2 protein (PDB: 4LVT). (
positions of 2b and 2e in the surface pocket of Bcl-2. ( ) Key interactions of 2e in the surface pocket of
Bcl-2. ( ) 2D graph of 2e in the surface pocket of Bcl-2. ( ) Key interactions of 2b at the surface pocket
of Bcl-2. (E) 2D graph of 2b in the surface pocket of Bcl-2.
A) 3D conformation
B
C
D
As shown in Figure 8A, both 2b and 2e bound to the surface pocket of bcl-2 protein in very
similar conformations. This implies that 2b and 2e might share some similarity in interacting with
the Bcl-2 protein. A closer look at their respective binding interactions (Figure 8B–D) shows that

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the bromophenyl fragments of both compounds could interact with the Bcl-2 surface pocket by
forming hydrophobic interactions with several amino acid residues including ALA-146, MET-112
and ARG-143. This rationalises the increased cytotoxicity seen by brominated analogs, as the
bromo group is more hydrophobic than methoxyl moiety. More importantly, the present docking
analysis revealed that the thio-moiety of 2e could form additional Pi-sulfur and Pi-alkyl interactions
with PHE-101 and TYR-105 residues, respectively, thus explaining the improved cytotoxicity of
tetetrahydro-4H-thiopyran-4-one-containing analogs. The in silico molecular docking results are
summarized in Table 3.
Table 3. Data resulted from the molecular docking of 2b and 2e in Bcl-2.
Interacting
Amino Acid Residue
Compound
Structure
Type of Interaction
Bonding Distance (Å)
ALA-146
MET-112
ARG-143
LEU-134
Alkyl
Pi-Alkyl
Pi-Cation
Alkyl
3.84
4.12
2.39
4.8
2b
Carbon
2.49
ALA-146
MET-112
Pi-Alkyl
Pi-Alkyl
Alkyl
5.20
4.76
4.75
3.93
3.67
5.14
5.24
4.78
ARG-143
Alkyl
2e
Pi-Cation
Pi-Alkyl
Pi-Alkyl
Pi-Sulfur
TYR-105
PHE-101
3. Materials and Methods
3.1. Chemistry
Chemicals, reagents and solvents were purchased from Sigma-Aldrich and Merck, and were used
without purification. The chemical reactions were routinely checked on 0.20 mm Merck TLC plate
silica gel 60 F254 using a solvent system of ethyl acetate/hexane (15:85). Compound purifications were
performed using column chromatography on Merck silica gel 60 (mesh 70–230). Melting points were
determined using the Fisher–Johns melting point apparatus and were uncorrected. High-resolution
electron ionisation-mass spectrometry (HREI-MS) was determined using a DFS high resolution GC/MS
(Thermo Scientific, San Jose, CA, USA). Nuclear magnetic resonance spectra were recorded on a Varian
500 MHz NMR Spectrometer.
General Procedure for the Synthesis of 2a–2e
Cyclohexanone or tetrahydro-4H-thiopyran-4-one (1 mmol) and appropriate aromatic aldehydes
(2 mmol) were reacted overnight in 20 mL of absolute ethanol in the presence of a catalytic amount of
10 M NaOH aqueous solution. Then, the reaction mixture was poured into a 250 mL beaker containing
100 mL of 3 M HCl followed by extraction with ethyl acetate. The ethyl acetate layer was collected and
dried over anhydrous magnesium sulfate followed by evaporation with a rotatory evaporator to give
the crude of the desired compounds. Purifications with chromatography using an ethyl acetate/hexane
(10:90) solvent system were then performed to obtain pure diarylpentanoids (Figures 9–14).
2,6-Bis(2-methoxybenzylidene)cyclohexanone (2a). (Figure 9). Yellow gummy solid; yield: 87%; m.p. found:
1
m.p. reported: Mass calculated: 334.1569; Mass found: 334.1588. H NMR (500 MHz, chloroform-d)
δ
ppm 1.74 (dt, J = 12.21, 6.24 Hz, 2 H) 2.84 (t, J = 5.49 Hz, 4 H) 3.85 (s, 6 H) 6.91 (d, J = 8.23 Hz, 2 H) 6.96
(t, J = 7.41 Hz, 2 H) 7.28–7.35 (m, 4 H) 7.98 (s, 2 H). 13C NMR (126 MHz, chloroform-d)
δ ppm 23.5, 28.8,
55.5, 110.6, 119.9, 125.1, 123.0, 130.3, 132.5, 136.5, 158.3, 190.6.

Molecules 2020, 25, 3877
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Figure 9. Chemical structure of 2a.
Figure 10. Chemical structure of 2b.
Figure 11. Chemical structure of 2c.
Figure 12. Chemical structure of 2d.
Figure 13. Chemical structure of 2e.
Figure 14. Chemical structure of 2f.
2,6-Bis(2-bromobenzylidene)cyclohexanone (2b) (Figure 10). Yellow solid; yield: 97 %; m.p. found: 106–107
◦C; m.p. reported: 120 ◦C; Mass calculated: 429.9568; Mass found: 429.9585. 1H NMR (500 MHz,
chloroform-d)
δ ppm 1.76 (quin, J = 6.04 Hz, 2 H) 2.75 (t, J = 5.21 Hz, 4 H) 7.17–7.22 (m, 2 H) 7.29–7.35
(m, 4 H) 7.64 (d, J = 8.23 Hz, 2 H) 7.85 (s, 2 H). 13C NMR (126 MHz, chloroform-d)
δ ppm 23.1, 28.3,
110.0, 125.1, 126.9, 129.7, 130.5, 132.9, 136.2, 136.4, 137.4, 189.6

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2,6-Bis(3,4-difluorobenzylidene)cyclohexanone (2c) (Figure 11). Brown solid; yield: 43%; m.p.: 90–91 ◦C;
1
Mass calculated: 346.0981; Mass found: 346.1094. H NMR (500 MHz, chloroform-d)
δ ppm 1.82 (quin,
J = 6.18 Hz, 2 H) 2.89 (t, J = 5.21 Hz, 4 H) 7.15–7.23 (m, 4 H) 7.25–7.32 (m, 2 H) 7.67 (s, 2 H). 13C NMR
(126 MHz, chloroform-d) d ppm 22.62, 28.19, 117.32, 117.46, 118.75, 118.88, 126.98, 127.00, 127.02, 127.05,
132.76, 132.79, 132.80, 132.83, 134.97, 136.45, 136.47, 149.04, 149.15, 149.24, 149.34, 151.02, 151.12, 151.25,
151.35, 189.6
3,5-Bis(2-methoxybenzylidene)tetrahydro-4H-thiopyran-4-one (2d) (Figure 12). Pale yellow solid; yield:
◦
83 %; m.p.: 120–121 C; Mass calculated: 352.1133; Mass found: 352.1145. 1H NMR (500 MHz,
chloroform-d) δ ppm 3.81 (s, 4 H) 3.86 (s, 6 H) 6.93 (d, J=8.23 Hz, 2 H) 6.97 (t, J = 7.41 Hz, 2 H) 7.20–7.28
(m, 2 H) 7.34 (t, J = 7.96 Hz, 2 H) 7.91 (s, 2 H). 13C NMR (126 MHz, chloroform-d) d ppm 30.3, 55.5,
110.8, 120.1, 124.3, 130.4, 130.4, 132.9, 134.3, 158.2, 189.3
3,5-Bis(2-bromobenzylidene)tetrahydro-4H-thiopyran-4-one (2e) (Figure 13). Pale yellow solid; yield: 78 %;
1
m.p.: 158–160 ◦C; Mass calculated: 447.9132; Mass found: 447.9140. H NMR (500 MHz, chloroform-d)
δ
ppm 3.73 (s, 4 H) 7.20–7.25 (m, 2 H) 7.27 (d, J = 5.49 Hz, 2 H) 7.32–7.37 (m, 2 H) 7.65 (d, J = 8.23 Hz, 2
H) 7.80 (s, 2 H). 13C NMR (126 MHz, chloroform-d)
δ ppm 29.7, 124.8, 127.2, 130.1, 130.4, 133.2, 135.1,
135.5, 136.3, 188.5
3,5-Bis(3,4-difluorobenzylidene)tetrahydro-4H-thiopyran-4-one (2f) (Figure 14). Pale yellow solid; yield:
55%; m.p. found: 124–125 ◦C; m.p. reported: 135–137 ◦C; Mass calculated: 364.0545; Mass found:
1
364.0558. H NMR (500 MHz, chloroform-d)
δ
ppm 3.87 (s, 4 H) 7.14 (dd, J = 8.23, 2.20 Hz, 2 H)
ppm 29.88, 76.76, 77.01, 77.27,
7.19–7.28 (m, 4 H) 7.66 (s, 2 H). 13C NMR (126 MHz, chloroform-d)
δ
117.65, 117.78, 118.67, 118.81, 126.57, 126.60, 126.62, 126.65, 131.88, 131.91, 131.92, 134.25, 134.26, 134.82,
149.18, 149.27, 149.49, 149.58, 151.16, 151.26, 151.50, 151.60, 188.21.
3.2. Biological Evaluations
3.2.1. Cell Culture
A panel of KRAS (T84, SW620 and LoVo) and BRAF (HT29, NCI-H508, RKO and LS411N) mutated
human CRC cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing
10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 ◦C under 5% CO2 while NHDF
cells were cultured in DMEM with 5% FBS under the same condition. The culture medium was
replenished every two days until the cells reached 80–90% confluency for further usage. All cell lines
(T84, ATCC®CCL-248
NCI-H508, ATCC®CCL-253
PCS-201-010 ) were purchased from American Type culture Collections (ATCC, Manassas, VA, USA).
™
; SW620, ATCC®CCL-227
™ ™
; LoVo, ATCC®CCL-229 ; HT-29, ATCC®HTB-38;
™
; RKO, ATCC®CRL-2577; LS411N, ATCC®CRL-2159; NHDF, ATCC®
™
3.2.2. MTT Assay
LoVo cells (1.5–5
5% CO2 atmosphere for 24 h. On the following day, the cell media were replaced with 100
compounds at various concentration. The final concentration of DMSO in the well is 0.1%. After 72 h
of incubation, culture medium was removed and 100 L of MTT (thiazoyl blue tertrazolium bromide)
×
103 cells/well) were seeded into a 96 well plate and incubated at 37 ◦C and
L of test
µ
µ
solution (0.5 mg/mL in culture media) was added. The resultant plates were then further incubated
at 37 ◦C for 4 h. Upon completion, the MTT solution was removed and the formazan salts formed
were dissolved in 100
µL of DMSO. The absorbance was recorded with a microplate reader at 570 nm.
The IC50 values of tested compounds were calculated from three independent experiments.

Molecules 2020, 25, 3877
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3.2.3. Morphological Inspection
LoVo cells (5
×
104 cells/well) were seeded into a 12 well plate and incubated at 37 ◦C and 5%
CO2 atmosphere for 24 h. Next, the culture media were replaced with 1 mL of test compounds
at various concentration and further incubated for 24 h. The final concentration of DMSO in the
well is 0.1%. The morphological changes of compound-treated cells were photographed using a
fluorescent microscope.
3.2.4. Colony Formation Assay
LoVo cells (1
×
103 cells/well) were seeded into a 6-well plate and incubated at 37 ◦C and 5% CO2
atmosphere overnight. After incubation, the culture media were replaced with 3 mL of test compounds
at various concentration and further incubated for seven days. Upon completion, cells were washed
with PBS and fixed with 4% paraformaldehyde for 15 min followed by cell staining with 0.5% (w/v)
crystal violet for 20 min. The resultant plates were then washed with tap water and dried at room
temperature. Upon drying, the plate image was photographed.
3.2.5. Cell Cycle Analysis
LoVo cells (1
×
105 cells/well) were seeded into a 6-well plate and incubated at 37 ◦C and 5%
CO2 atmosphere overnight. The seeded cells were then treated with different concentrations of tested
compounds for 24 h. Upon completion, cells were trypsinized and washed with PBS followed by
◦
fixation with 70% ethanol overnight at 4 C. Then, the fixed cells were centrifuged at 160
×
g for
L of DNAse-free
RNAse A solution (1 mg/mL) was added to the cell-containing PBS and further incubated for 15 min.
Upon completion, 10 L of propidium iodide staining buffer (10 mg/mL) was added and the resulting
5 min and the resultant cell pellet was washed twice with PBS. Subsequently, 20
µ
µ
mixture was further incubated for 20 min at 4 ◦C in the dark. Lastly, samples were analyzed with a
flow-cytometer (ACEA Biosciences, Inc., San Diego, CA, USA) at the PE channel. The values presented
are the means ± SD of three independent experiments.
3.2.6. Cell Apoptosis Analysis
LoVo cells (1
×
105 cells/well) were seeded into a 6-well plate and incubated at 37 ◦C and 5%
CO2 atmosphere overnight. The seeded cells were then treated with different concentrations of
tested compounds for 24, 48 and 72 h. After treatments, cells were trypsinized and washed with
PBS followed by the addition of 1 binding buffer (500 µL), Annexin V-PE (5 µL) and 7-AAD (5 µL)
×
solutions. The resulting mixture was then incubated in the dark at 4 ◦C for 20 min. Upon completion,
the mixture was analysed with a flow-cytometer at the PE and APC channel. The values presented are
the means ± SD of three independent experiments.
3.2.7. Measurement of Intracellular Reactive Oxygen Species (ROS) Levels
LoVo cells (1
atmosphere overnight. After incubation, the seeded cells were treated with 1 mL of 10
in culturing media for 1 h. Upon completion, the cells were washed twice with PBS followed by
treatments with 5 M of selected compounds for 24, 48 and 72 h. Then, the treated cells were harvested,
×
105 cells/well) were seeded into a 6-well plate and incubated at 37 ◦C and 5% CO2
M DCFDA
µ
µ
washed and re-suspended in PBS followed by flow-cytometric analysis at the FITC channel.
3.2.8. Western Blot Analyses
Lovo cells (1.5
atmosphere overnight. The seeded cells were then treated with selected compounds at 5
24 h, 48 h, and 72 h. After treatment, the cells were harvested and lysed with phenylmethanesulfonyl
fluoride (PMSF)-containing cell lysis buffer. The lysates were then centrifuged at 13,300 g for
15 min and the supernatant was collected. Protein concentrations of all samples were determined
×
104 cells/cm2) were seeded in a T75 flask and incubated at 37 ◦C and 5% CO2
M of 2e for
µ
×

Molecules 2020, 25, 3877
13 of 16
using a BCA assay kit (Thermo Scientific Inc., Waltham, MA, USA). Equal amounts of protein were
electrophoresed in 12% and 20% SDS-polyacrylamide gel (SDS-PAGE), and electro-transferred onto a
0.22-µm polyvinyldene difluoride membrane. Each membrane was blocked with 5% (w/v) skim milk
at room temperature for 1 h followed by overnight incubation with specific primary antibodies (1:1000)
at 4 ◦C. Subsequently, the respective membrane was washed thrice with TBST (0.05% (v/v) of tween-20)
followed by 1 h incubation with secondary antibody (1:10,000). Upon completion, the membrane
was washed thrice with 1X Tris-Buffered Saline, 0.1% Tween® 20 Detergent (TBST) and the immune
reactive bands were developed using SuperSignal West Dura Extended Duration Substrate (Thermo
Scientific Inc., Waltham, MA, USA). Subsequently the blots were visualized with an Amersham
Imager 600 system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
β-actin served as an internal
control [47]. All primary antibodies (cPARP, #5625; cCas-7, #12827; B-actin, #4967; cCas-9, #9508; Cas-8,
#9746; Bad, #9239; Bax, #5023; Mcl-1, #94296) except anti-Bcl-2 and anti-Bcl-xL were purchased from
Cell signaling Technology (Beverly, MA, USA). Both anti-Bcl-2 (K200018M) and anti-Bcl-xL (K001595P)
primary antibodies were purchased from Solarbio Life Sciences (Beijing, China).
3.2.9. Molecular Docking
Molecular docking studies were carried out using Discovery Studio 3.1 (Accelrys, San Diego,
USA) on an Intel® (TM)2 Quad CPU Q8200 @2.33 GHz running under a Windows XP
Professional environment.
(1) Receptor preparation
The crystal structure of Bcl-2 (PDB ID: 4LVT) was first obtained from the Protein Data Bank.
Then, all water molecules and co-crystallized ligands were removed from the crystal structure to obtain
the Bcl-2 receptor. Prior to docking the selected compounds into Bcl-2, the Bcl-2 receptor underwent a
protein preparation protocol with CHARMm force field.
(2) Ligand preparation
Co-crystallized ligand (ABT-263) and 2e were drawn with ChemDraw Ultra 12.0. Then, the structures
were imported to the Discovery Studio 3.1 followed by the ligand preparation protocol with the default
setting recommended by Accelrys. The prepared ligands were then subjected to ligand minimization
with CHARMm force field before being used for docking analyses.
(3) Flexible docking
Minimized co-crystallized ligands were re-docked into their respective enzymes with several sets
of amino acids as flexible residues. The top ranked conformations resulted from the docking experiment
were compared to their original crystallographic confirmation in terms of RMSD. The parameters with
lowest RMSD values were selected for the flexible docking of 2e. The flexible docking results were
analyzed using Discovery Studio Visualizer v4.1.0.14169 (Accelrys, San Diego, CA, USA).
4. Conclusions
In summary, six diarylpentanoids were synthesized and investigated for their anti-cancer potential
against a panel of KRAS- and BRAF-mutated CRC cell lines. Compound 2e was found to be the most
potent candidate based on its low IC50 values and high chemotherapeutic index. The anti-proliferative
study on LoVo cells shows that 2e could inhibit cell proliferation by triggering cell cycle arrest at
the G2/M phase. Further cell apoptosis analysis revealed that 2e could induce intrinsic apoptosis by
disrupting the cellular redox balance through its direct inhibitory effects on Bcl-2 protein. A subsequent
molecular docking study suggested that the insertion of the thio group into the cycloalkane ring and
the substitution of a hydrophobic moiety on the phenyl ring could improve the binding interactions
between Bcl-2 and diarylpentanoids, thus leading to better anti-cancer activity. In conclusion, we believe
that tetrahydro-4H-thiopyran-4-one-containing diarylpentanoids could be a new class of Bcl-2 inhibitor
that deserves further investigation in the search for new anti-cancer agents.
Supplementary Materials: The following are available online. Figure S1: NMR Spectral and HPLC Profile of
Synthesized Compound 2a; Figure S2: NMR Spectral and HPLC Profile of Synthesized Compound 2b; Figure S3:

Molecules 2020, 25, 3877
14 of 16
NMR Spectral and HPLC Profile of Synthesized Compound 2c; Figure S4: NMR Spectral and HPLC Profile of
Synthesized Compound 2e; FigureS5: NMR Spectral and HPLC Profile of Synthesized Compound 2f.
Author Contributions: Conceptualization, S.W.L., S.L.C., F.A. and K.Y.; methodology, S.W.L., S.L.C.; software,
S.W.L.; validation, S.W.L., S.L.C., F.A. and K.Y.; data curation, S.W.L. and S.L.C. All authors wrote the manuscript.
All authors have read and agreed to the published version of the manuscript
Funding: This research received no external funding.
Acknowledgments: The authors thank the Newton-Ungku Omar Fund (MR/P012795/1) and Universiti Putra
Malaysia for supporting S.W.L. under the post-doctoral programme.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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