Induction of Apoptosis in Hepatocellular Carcinoma Cell Lines by Novel Indolylacrylamide Derivatives: Synthesis and Biological Evaluation

Article abstract of DOI:10.1002/cbdv.202001037

Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer and one of the leading causes of cancer associated death worldwide. This is due to the highly resistant nature of this malignancy and the lack of effective treatment options for advanced stage HCC patients. The hyperactivity of PI3K/Akt and Ras/Raf/MEK/ERK signaling pathways contribute to the cancer progression, survival, motility, and resistance mechanisms, and the interaction of these two pathways are responsible for the regulation of cancer cell growth and development. Therefore, it is vital to design and develop novel therapeutic options for HCC treatment targeting these hyperactive pathways. For this purpose, novel series of trans-indole-3-ylacrylamide derivatives originated from the lead compound, 3-(1H-indole-3-yl)-N-(3,4,5-trimethoxyphenyl)acrylamide, have been synthesized and analyzed for their bioactivity on cancer cells along with the lead compound. Based on the initial screening, the most potent compounds were selected to elucidate their effects on cellular signaling activity of HCC cell lines. Cell cycle analysis, immunofluorescence, and Western blot analysis revealed that lead compound and (E)-N-(4-tert-butylphenyl)-3-(1H-indole-3-yl)acrylamide induced cell cycle arrest at the G2/M phase, enhanced chromatin condensation and PARP-cleavage, addressing induction of apoptotic cell death. Additionally, these compounds decreased the activity of ERK signaling pathway, where phosphorylated ERK1/2 and c-Jun protein levels diminished significantly. Relevant to these findings, the lead compound was able to inhibit tubulin polymerization as well. To conclude, the novel trans-indole-3-ylacrylamide derivatives inhibit one of the critical pathways associated with HCC which results in cell cycle arrest and apoptosis in HCC cell lines.

Full text of DOI:10.1002/cbdv.202001037

DOI: 10.1002/cbdv.202001037
FULL PAPER
Induction of Apoptosis in Hepatocellular Carcinoma Cell Lines by
Novel Indolylacrylamide Derivatives: Synthesis and Biological
Evaluation
Mohammed Hawash,a,1,2 Deniz Cansen Kahraman,b,1 Rengul Cetin-Atalay,b and
Sultan Nacak Baytas*a
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey,
e-mail: baytas@gazi.edu.tr
Cancer Systems Biology Laboratory, Graduate School of Informatics, METU, 06800 Ankara, Turkey
b
Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer and one of the leading causes of
cancer associated death worldwide. This is due to the highly resistant nature of this malignancy and the lack of
effective treatment options for advanced stage HCC patients. The hyperactivity of PI3K/Akt and Ras/Raf/MEK/ERK
signaling pathways contribute to the cancer progression, survival, motility, and resistance mechanisms, and the
interaction of these two pathways are responsible for the regulation of cancer cell growth and development.
Therefore, it is vital to design and develop novel therapeutic options for HCC treatment targeting these
hyperactive pathways. For this purpose, novel series of trans-indole-3-ylacrylamide derivatives originated from
the lead compound, 3-(1H-indole-3-yl)-N-(3,4,5-trimethoxyphenyl)acrylamide, have been synthesized and
analyzed for their bioactivity on cancer cells along with the lead compound. Based on the initial screening, the
most potent compounds were selected to elucidate their effects on cellular signaling activity of HCC cell lines.
Cell cycle analysis, immunofluorescence, and Western blot analysis revealed that lead compound and (E)-N-(4-
tert-butylphenyl)-3-(1H-indole-3-yl)acrylamide induced cell cycle arrest at the G2/M phase, enhanced chromatin
condensation and PARP-cleavage, addressing induction of apoptotic cell death. Additionally, these compounds
decreased the activity of ERK signaling pathway, where phosphorylated ERK1/2 and c-Jun protein levels
diminished significantly. Relevant to these findings, the lead compound was able to inhibit tubulin polymer-
ization as well. To conclude, the novel trans-indole-3-ylacrylamide derivatives inhibit one of the critical pathways
associated with HCC which results in cell cycle arrest and apoptosis in HCC cell lines.
Keywords: trans-indole-3-ylacrylamide derivatives, cancer, hepatocellular carcinoma, apoptosis, ERK signaling
pathway.
Introduction
patients is only 7%.[1] Chronic infections with hepatitis
Hepatocellular carcinoma (HCC), with about 900,000 B virus (HBV) or hepatitis C virus (HCV), aflatoxin
new cases each year is the sixth most common type of exposure, chronic alcohol intake and obesity are
cancer worldwide. The 5 year survival rate of HCC among the risk factors associated with HCC.[2,3] Liver
cancer cells are prone to acquire resistance to conven-
tional chemotherapy and radiotherapy. Early cases
1
usually undergo liver transplantation if available.
However, when transplantation is not an option, or in
advanced HCC cases, sorafenib is used as a targeted
therapeutic agent. Sorafenib and Regorafenib are
multikinase inhibitors approved by the Food and Drug
Administration (FDA), and these drugs have both
Authors contributed equally to this work.
Present address: Department of Pharmacy, Faculty of
2
Medicine and Health Sciences, An-Najah National Univer-
sity, Nablus, Palestine.
Supporting information for this article is available on the
WWW under https://doi.org/10.1002/cbdv.202001037
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Chem. Biodiversity 2021, 18, e2001037
antiproliferative and anti-angiogenic effects on liver ported to show antiproliferative activities.[29–34] In our
cancer cells. Nevertheless, they only prolong patient previous work, starting from indole-3-carboxyaldehyde
survival for about 3–4 months.[4]
1, we first synthesized trans-indole-3-ylacrylic acid 2,
Mitogen-activated protein kinase (MAPK) pathways then, prepared a series of trans-indole-3-ylacrylamides
carry extracellular signals to the nucleus, and these (Scheme 1) and determined their cytotoxic activity
pathways are involved in important cellular processes, against five human cancer cell lines.[35] We found that
including growth, proliferation, differentiation, migra- the most potent derivative among these synthesized
tion, and apoptosis. The ERK pathway, which is a well- molecules, compound 3e was an inhibitor of tubulin
known member of MAPK pathways, is known to be polymerization and inhibited the binding of [3H]
deregulated in many human cancers. It is well-known colchicine to tubulin. Compound 3e caused cell cycle
that both PI3 K/Akt and Ras/Raf/MEK/ERK signaling arrest in HeLa and HL-60 cells and also induced
pathways play significant roles in the development apoptotic cell death by activation of caspase-3 activity.
and drug resistance of HCC.[5] Hyper activation of the Therefore, considering the limited availability of
components of these pathways is associated with poor targeted therapies in liver cancer, in continuation of
prognosis.[6] Therefore, development of novel drugs our previous work, we synthesized novel trans-indole-
targeting the components of these pathways is highly 3-ylacrylamide derivatives (4a–4u, 5a–5s) and eval-
critical for the treatment of patients with advanced uated their bioactivity properties on HCC cell lines and
HCC. ERK signaling pathway is also an important compared them to the lead compound 3e and its
player for the mitotic progression of cancer cells,[7,8] analogs. The compounds were also evaluated for their
and is associated with the kinetochore of mitotic effects on the cell cycle, apoptosis and ERK signaling.
spindles.[9–11] Previous studies have shown that micro-
tubule inhibitors cause inactivation of the ERK path-
way proteins and its downstream targets.[12–14] Recent Results and Discussion
studies that involve gene expression profiling empha-
size that microtubule-related cellular assembly and
Chemistry
organization is one of the most critical events in HCC A series of amide derivatives of trans-indole-3-ylacrylic
development,[15–18] which makes microtubules a po- acid were synthesized as illustrated in Scheme 1.
tential target for the therapy of HCC. Tubulin binding Briefly, indole-3-carbaldehyde was condensed with
agents, also called ‘spindle poisons’, induce dysregula- malonic acid in the presence of piperidine to generate
tion of the cell cycle, characteristically at the G2/M trans-indole-3-ylacrylic acid 2 by Knoevenagel condi-
phase,[19] and they can also be used as an alternative tions, as reported previously.[36] Amide derivatives of
method to synchronize mammalian cells.[20]
trans-indole-3-ylacrylic acid were synthesized with
A group of antimitotic compounds isolated from appropriate amines, mostly in the presence of EDC
the South African tree Combretum caffrum is known as and DMAP in CH2Cl2. Some of the final amide
Combretastatins.[21,22] Combretastatin A-4 is a well- derivatives (4f, 4g, 4h, 4i, 4k, 4m, and 4n) were
known natural tubulin binding molecule affecting prepared in the presence of triethylamine and ethyl
microtubule dynamics.[23] CA-4 has low water solubility chloroformate, which was used as the carboxylate
and leads to the synthesis of water-soluble derivatives. activator. Compounds were purified by automated
Combretastatin A-4 phosphate (CA-4P, Fosbretabulin) flash chromatography and checked for purity with TLC
has been investigated for the treatment of anaplastic and UPLC before being tested in biological assays
thyroid cancer.[24] Numerous studies have been carried (purity was >97%). The structures of these com-
out on CA-4 analogs, and their antitumoral effects pounds were confirmed by FT-IR, high resolution mass
1
have been demonstrated.[25,26] In a study conducted in spectrometry (HR-MS), H- and 13C-NMR spectral data.
1
2012, phase II clinical trials for the use of combretasta- In the H-NMR spectra of compounds 4a–4u and 5a–
tin A-4 phosphate (CA-4P) in ovarian, lung, and thyroid 5s, one of the olefinic protons (CH=CHÀ CO) was
cancers were performed.[27] Recent studies have observed as a doublet at about 7.56–8.02 ppm, while
reported 90–99% tumor necrosis with controlled the other (CH=CHÀ CO) was observed as a doublet at
doses of CA-4P, and phase III clinical trials are now about 6.41–6.99 ppm, with coupling constants of 15.6
being conducted.[28]
or 16.0, indicating the presence of the (E) isomer. The
Recently, a number of CA-4 analogs with potent more unshielded olefinic carbons (CH=CHÀ CO) were
activities have been developed. Among these analogs, observed 167.59–165.03 ppm, the other ones
indole group-containing compounds have been re- (CH=CHÀ CO) were observed 125.58–125.20 ppm. The
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Chem. Biodiversity 2021, 18, e2001037
carbonyl carbons were shifted 167.59–165.03 ppm. and 5-fluorouracil (5-FU) as well as multikinase inhib-
The synthetic procedure and spectral data of the itor Sorafenib (Table 1).
compounds (3a–3k) were reported previously by our
When the results of the initial screening experi-
ments were examined, the R1 moiety in the trans-
indole-3-ylacrylamide structure had a considerable
impact on cytotoxicity. In the amide section, the
phenyl ring directly attached to the amide nitrogen
was found to be required for activity. Activity was
research group.[35]
Cytotoxicity of trans-Indole-3-ylacrylamide Derivatives
on Cancer Cells and HCC Cell Lines
trans-Indole-3-ylacrylamide derivatives 3a–3k, 4a–4u, observed in the polymethoxyphenyl bearing deriva-
and 5a–5s were primarily screened against hepatocel- tives (3a–3f) at the amide site, while cell growth
lular (Huh7), breast (MCF7) and colon carcinoma inhibitory activity was lost in the polymethoxybenzyl
(HCT116) cells using the Sulforhodamine B (SRB) derivatives (3g–3j). Compound 4a from polymeth-
assay.[37] IC50 values of compounds were between 0.5– oxybenzyl amide derivatives inhibited cell growth in
40.6 μM and were compared to those of the well- Huh7 cells with an IC50 value of 9.3 μM. Compound 4a
known chemotherapeutic agents, doxorubicin (DOXO) carries a 3,4,5-trimethoxybenzyl group similar to the
precursor compound 3e bearing the 3,4,5-trimeth-
Scheme 1. Synthesis pathway to achieve the title compounds. Reagents and conditions: a) Malonic acid, piperidine, pyridine, 5 h,
°
40 C; b) i. amine derivative, EDC, DMAP, CH2Cl2, overnight, r.t. or ii. amine derivative, ethyl chloroformate, Et3N, CH2Cl2, overnight, r.t.
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Table 1. IC50 values of trans-indole-3-ylacrylamide derivatives on
Huh7, MCF7 and HCT116 cancer cells.
oxyphenyl group in the amide moiety. These results
show that the structure of 3,4,5-trimethoxyphenyl
directly bonded to amide nitrogen is important for its
activity. Compound 4l, bearing a tert-butyl group at
position 4 of the phenyl ring attached to the amide
nitrogen, showed significant activity against the three
cell lines (IC50 Huh7, 3.1 μM; MCF7, 2.0 μM; and
HCT116; 5.7 μM). Compound 4l is the compound with
activity closest to that of the lead compound 3e
among the synthesized compounds. Compound 4d,
carrying a 4-thiomethylphenyl group attached to the
amide nitrogen, also showed activity against the three
cell lines (IC50 Huh7, 7.9 μM; MCF7, 8.3 μM, and
HCT116, 16.6 μM). Weaker activity was observed in the
halogen substituted phenyl derivatives (4e–4j) bound
to the amide nitrogen, with IC50 values above 10 μM
(IC50 10.8–40.6 μM). A considerable decrease in activity
was observed with a methyl substitution in the phenyl
ring attached to the amide nitrogen (4k, IC50 31.4 μM).
When a benzyl group was attached to the amide
nitrogen, activity disappeared (4c). The methoxy group
placed in the 4 position of the benzyl group resulted
in low activity in the Huh7 cell lines (4b, IC50 33.1 μM).
When saturated, nitrogen containing, cyclic rings
replaced the amide nitrogen (4m, 4n, 4s), activity was
greatly reduced or disappeared. Compounds carrying
imidazole and pyridine rings linked by an aliphatic
intermediate chain in the amide moiety were also
synthesized, but activity was lost in these compounds
(4u–5f). Altogether, important data about structure-
activity relationships were obtained from these trans-
indole-3-ylacrylamide derivatives. For cytotoxic activ-
ity, in the amide portion of the trans-indole-3-
ylacrylamide derivatives, there is a requirement for a
phenyl ring directly linked to the amide nitrogen and
substituted with a bulky group.
Moreover, the IC50 values of selected compounds
(3e, 4d, 4l, 5p, and 5s) were comparable to those of
doxorubicin and sorafenib, which are agents, used
clinically for HCC conventional chemotherapy and
targeted therapy. These compounds were therefore,
chosen to be tested in a panel of HCC cells (Huh7,
HepG2, Mahlavu and SNU475). The inhibitory concen-
trations of the selected compounds were determined
using SRB assay and listed in Table 2.
The results have shown that compound 3e had IC50
values of 1 μM or less in the two more aggressive HCC
cell lines, Mahlavu and SNU475. Similarly, compound
4l, which exhibited the closest activity to the lead
compound 3e among the synthesized compounds,
showed strong inhibition of the growth of both
Mahlavu and SNU475 cells.
Compound
IC50 (μM)[a]
Huh7
MCF7[b]
HCT116
3a
3b
3c
3d
3e
3f
34.5�6.0
21.2�2.9
23.5�3.2
17.1�1.9
0.5�0.2
NI
–
–
–
–
–
NI
28.0�5.7
NI
24.0�0.7
1.1�0.7
–
NI
3g
3h
3i
NI
–
–
–
–
NI
NI
NI
NI
20.9�2.4
NI
3j
NI
3k
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4r
NI
–
NI
9.3�1.7
33.1�9.9
NI
NI
NI
NI
26.7�4.2
NI
NI
7.9�0.2
11.7�0.3
11.7�4.6
10.8�2.2
12.8�3.5
13.2�1.7
11.7�0.6
31.4�5.8
3.1�1.7
NI
8.3�1.0
16.6�1.4
12.1�1.0
19.6�1.2
25.4�4.5
26.4�1.9
13.1�1.0
12.7�0.2
40.6�3.4
29.0�1.5
15.1�1.2
18.3�3.5
14.9�1.9
20.4�3.7
NI
NI
2.0�0.4
5.7�1.4
NI
NI
NI
NI
NI
NI
NI
NI
NI
16.0�1.7
18.2�4.7
24.1�5.8
34.3�14.5
32.2�4.6
NI
NI
NI
NI
38.3�1.3
36.3�5.8
4s
4t
4u
5a
5b
5c
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5r
NI
NI
NI
NI
NI
NI
NI
NI
NI
10.7�2.8
18.9�2.3
24.2�3.1
NI
NI
NI
NI
26.9�4.6
NI
18.7�3.6
28.7�4.4
NI
NI
ND
ND
ND
ND
Nİ
NI
30.1�5.1
22.4�4.6
18.3�1.3
24.0�3.5
29.2�2.3
19.6�0.1
4.9�0.5
18.0�1.5
4.3�0.5
0.22�0.02
2.0�0.04
6.5�0.5
24.0�2.7
22.1�2.1
NI
NI
ND
ND
ND
ND
0.23�0.02
1.3�0.2
11�0.6
5s
DOX
5-FU
Sorafenib
0.14�0.05
1.5�0.1
14.6�0.2
[a] Data was represented as the mean �SD, n=3. [b] IC50 values for
compound 3a–3k in MCF7 cell lines could be found in our
previously published study,[35] NI: No inhibition; ND: Not deter-
mined.
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Table 2. IC50 values (μM) determined in a liver cancer cell panel for compounds 3e, 4d, 4l, 5p and 5s.
Compound
IC50 (μM)[a]
Huh7
HepG2
Mahlavu
SNU475
3e
4d
4l
5p
5s
0.5�0.2
7.9�0.2
3.1�1.7
4.9�0.5
4.3�0.5
3.6�0.6
1.0�0.1
12.2�0.8
5.3�0.5
32.0�0.3
6.6�1.8
0.3�0.1
25.3�1.1
8.4�1.0
7.6�0.5
10.7�0.7
11.5�0.8
4.8�0.8
>40
11.5�1.1
[a] Data was represented as the mean�SD, n=3.
Considering our previous work where our lead cleaved PARP, was observed in both well-differentiated
compound 3e was an inhibitor of tubulin polymer- (Huh7 and HepG2) and poorly differentiated (Mahlavu
ization and inhibited the binding of [3H]colchicine to and SNU475) cells,[39] after a 24 h treatment (Figure 2b)
tubulin,[35] we investigated the tubulin polymerization confirming that apoptotic cell death was induced by
inhibition and colchicine binding capacity of the these compounds.
compounds 4d, 4l, 5p, and 5s. We determined that
the compounds were inactive as anti-tubulin agents
G2/M Arrest Induced by the Selected trans-Indole-
(data not shown). Furthermore, when all the selected
3-ylacrylamide Derivatives 3e, 4d and 4l
compounds (3e, 4d and 4l) were screened against
normal human epithelial breast cell lines, MCF12A, all Because of the growth pattern of cells obtained in the
had relatively higher IC50 values (between 17.8– real-time cell growth assay, flow cytometry analysis
36.1 μM) (Table S2), indicating selective bioactivities of was performed by staining DNA with propidium iodide
these compounds towards cancer cells.
(PI). Our goal was to determine if the indole-3-
ylacrylamide derivatives altered the progression of the
cell cycle at a certain stage. After 48 h treatment,
compound 3e caused G2/M arrest substantially in all
four cell lines. Compound 4l caused a minor change in
Real-Time Cell Growth Monitoring of HCC Cells Treated
with 3e, 4d, and 4l
The effects of the compounds on growth of HCC cells the percentage of cells in G2/M phase, especially in
were evaluated using real-time cell electronic sensing mesenchymal-like cells (Mahlavu and SNU475). Com-
system (RT-CES). The growth patterns of all cells pound 4d did not cause any considerable change in
suggested that all compounds were able to decrease cell cycle distribution, except for a small increase in
the growth rate in a dose dependent manner (Fig- G2/M cells in the Mahlavu cells at 24 and 48 h
ure 1). In addition, the growth pattern of the HCC cells (Figure 3).
led to the hypothesis that these compounds caused
an arrest in the cell cycle, where cells neither grow in
Effects of trans-Indole-3-ylacrylamide Derivatives 3e, 4d,
number nor die completely after a 72 h treatment.[38]
and 4l on Akt, ERK and c-Jun
In contrast, DMSO treated control cells were able to
proliferate at their normal pace.
An epithelial-like cell lines, Huh7 and a mesenchymal-
like cell lines Mahlavu (with hyperactive Akt pathway
due to PTEN loss[40]) were chosen for further analysis
of the Akt and ERK pathway, which are associated with
the development of HCC and poor prognosis in
Apoptotic Cell Death Induced by the Selected Indole-
3-ylacrylamide Derivatives 3e, 4d, and 4l
For the evaluation of induction of cell death by indole- patients as mentioned previously. Western blot analy-
3-ylacrylamide derivatives 3e, 4d, and 4l, HCC cells sis revealed that the compounds had no significant
were treated with the compounds for 48 h, and their effect on the levels of active form of (Ser473
nuclei were stained and analyzed under fluorescent phosphorylated) Akt protein whereas, all the selected
microscopy. Condensed chromatin structure as well as compounds were able to decrease levels of the
nuclear blebbings were visible in all cells treated with activated form of (Thy204 phosphorylated) ERK1/2
compounds (Figure 2a), the effect observed with 3e protein. Compound 3e and 4d were able to reduce
being the greatest. Furthermore, the apoptotic marker, the levels of c-Jun proteins as well in both Huh7 and
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Figure 1. Real-time cell monitoring of HCC cell lines treated with compounds 3e, 4d, and 4l at IC50 and IC100 concentrations for
72 h. Growth of treated cells was compared to that of a DMSO control (0.1%).
Conclusions
Mahlavu cells (Figure 4). Thus, indole-3-ylacrylamide
derivatives able to hinder the activity of ERK protein With regard to our previous knowledge about our lead
and its downstream targets such as c-Jun these effects compound, which exhibits anti-microtubule activity
were independent of the Akt pathway activity and induces apoptosis, we aimed to expand our work
(phospho-AktSer473), which also controls c-Jun. This with the newly synthesized trans-indole-3-ylacrylamide
analysis indicates that the compounds are specifically derivatives. For this purpose, a new series of trans-
effective on the ERK protein.
indole-3-ylacrylamide derivatives were synthesized,
and their bioactivities were evaluated in different
cancer cell lines (HCC, breast, and colon carcinoma).
The cytotoxic bioactivities of the compounds were
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compared with those of the clinically used chemo- PARP cleavage. Western blot analysis has showed that,
therapeutic agents, doxorubicin, and 5-FU as well as compounds 3e and 4d reduced the levels of activated
the multikinase inhibitor sorafenib. Our results have ERK and its downstream signaling element c-Jun
revealed that trans-indole-3-ylacrylamides induce G2/ protein. As mentioned previously, since ERK signaling
M arrest (Figure 3) and apoptotic cell death as and cell cycle arrest characteristically at the G2/M
demonstrated with nuclear Hoechst staining and the phase is associated with inhibition of tubulin polymer-
Figure 2. Induction of apoptosis in HCC cell lines treated with selected compounds. a) Images of Nuclear Hoechst 33258 staining
after incubation of cells with IC50 concentrations of compounds 4l, 4d or 3e for 48 h taken at a 20× magnification; b) Western blot
analysis of cleaved PARP levels in well-differentiated (Huh7 and HepG2) and poorly differentiated (Mahlavu and SNU475) HCC cells
treated with the corresponding IC100 concentrations of the same compounds for 24 h.
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Figure 3. Cell cycle analysis of HCC cell lines Huh7, HepG2, Mahlavu (MV), and SNU475 (S475) treated with the IC100 concentrations
of compounds 3e, 4d, and 4l for 24 and 48 h. Bar graphs indicate the overall cell cycle distribution; tables present corresponding
values at indicated times for each cell lines.
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Figure 4. Representative images of Western blot analysis of Akt, ERK, and c-Jun proteins following treatment of the indicated cell
lines with 3e, 4d and 4l for 24 h at the IC100 concentrations. Phosphorylation of Akt (Ser473) is indicative of the levels of active Akt,
and phosphorylation of ERK (Thr204) results in its activation. c-Jun is a downstream protein of both the ERK and Akt pathways.
Calnexin was used as a loading control. Bar graphs indicate relative band intensities of treatments with respect to DMSO control
groups for each cell lines. Bar graphs indicate statistical analysis of band intensities of treatments with respect to DMSO control
groups for each cell lines from n=3 experiments (values are represented as mean �SD). Statistical analysis was performed using a
Student’s t-test, *P<0.05.
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ization, our results with compound 3e further sup-
ported the hypothesis that compound 3e acts as a
microtubule targeting agent, whereas compounds 4d
Synthesis of trans-Indole-3-ylacrylamide Derivatives (4a–
4u, 5a–5s)
and 4l were found to act on the ERK signaling Procedure 1. Amine derivatives (1.8 mmol), DMAP
pathway, independent of mechanisms involved in (0.3 mmol), and EDCI (1.65 mmol) were added in
tubulin polymerization (Figure 4). In addition, the lack sequence to a solution of the indole acrylic acid
of any alteration in Akt protein phosphorylation (1.5 mmol) in CH2Cl2 (5 mL) and dimethylformamide
(activation) in the presence of the compounds, dem- (DMF) (0.5 mL) under N2. The solution was stirred at
onstrated that the molecular action primarily of 3e as room temperature for 24–48 h. Progress of the
well as 4d and 4l involve the ERK protein.
reaction was followed by TLC. After completion of the
To conclude, this study has provided valuable reaction, the solvent was evaporated under reduced
information on the potential of compound 3e and pressure, the residue was dissolved in CH2Cl2, and the
newly synthesized trans-indole-3-ylacrylamide deriva- organic phase was washed with a 1% NaHCO3
tives 4d and 4l to inhibit the ERK pathway, one of the solution and brine, dried over Na2SO4, and evapo-
critical pathways involved in HCC occurrence, and rated under vacuum. The final residue was purified by
induce cell cycle arrest and apoptotic cell death in flash column chromatography (Combiflash® Rf) using
HCC cell lines. Yet, additional information about the DCM/MeOH and hexane/ethyl acetate solvent gra-
molecular targets of these compounds remains to be dients. Some derivatives were recrystallized using a
investigated through biochemical and computational different solvent system.
target identification approaches.
Procedure 2. To a solution of the acid derivative
(1.5 mmol) in CH2Cl2 were added triethylamine
Experimental Section
(3 mmol) and ethyl chloroformate (1.5 mmol), fol-
°
lowed by stirring at 0 C for 30 min. After the addition
Chemistry
of the appropriate amine derivative (1.8 mmol), the
°
Melting points were determined with an SMP-II mixture was stirred for an additional 1 h at 0 C. Then,
Digital Melting Point Apparatus and are uncorrected the reaction mixture was warmed to room temper-
(Schorpp Geaetetechnik, Germany). IR spectra were ature and stirred overnight. The progress of the
obtained using a Perkin Elmer Spectrum 400 FT-IR/ reaction was followed by TLC. After completion of the
FTNIR spectrometer equipped with a Universal ATR reaction, the solvent was evaporated under reduced
1
Sampling Accessory. H-NMR spectra were recorded pressure, the residue was dissolved in CH2Cl2, and the
in CDCl3 or (D6)DMSO on a Varian Mercury 400 MHz organic phase was washed with a 1% NaHCO3
High Performance Digital FT-NMR spectrometer at solution and brine, dried over Na2SO4, and evapo-
the NMR facility of the Faculty of Pharmacy, Ankara rated under vacuum. The final residue was purified by
University, and 13C-NMR spectra were recorded in (D6) flash column chromatography (Combiflash® Rf) using
DMSO on a Bruker Avance Ultrashield 300 MHz NMR DCM/MeOH and hexane/ethyl acetate solvents gra-
spectrometer at the NMR facility of the Faculty of dients, some derivatives being recrystallized by differ-
Science, Gazi University using tetramethylsilane as ent solvent systems.
the internal standard. All chemical shifts were
recorded as δ (ppm). High resolution mass spectra
Examples of spectra data of 4d and 4l are shown
data (HR-MS) were collected using a Waters LCT below, whereas data of rest of the final derivatives are
Premier XE Mass Spectrometer (high sensitivity listed in the Supporting Information.
orthogonal acceleration time-of-flight instrument)
using the ESI (+) or ESI (À ) methods. The instrument
Data
for
(2E)-3-(1H-Indol-3-yl)-N-[4-(meth-
was coupled to an AQUITY Ultra Performance Liquid ylsulfanyl)phenyl]prop-2-enamide (4d). Prepared
Chromatography system (Waters Corporation, Mil- using procedure 1. Purified by flash column chroma-
ford, MA, USA). Flash chromatography was performed tography (0%!6% methanol in CH2Cl2). Yield 22%.
À 1
with a Combiflash®Rf automated flash chromatogra- M.p. 224–225 C. IR (FT-IR/FTNIR-ATR): 1641 cm
°
1
phy system with RediSep columns (Teledyne-Isco, (C=O). H-NMR (400 MHz, (D6)DMSO): 11.62 (1H, s),
Lincoln, NE, USA) using DCM/MeOH, hexane/AcOEt, 9.97 (1H, s), 7.94–7.92 (1H, m), 7.80 (1H, d, J=2.8),
and AcOEt/DCM solvent gradients.
7.73 (1H, d, J=15.6), 7.66–7.63 (2H, m), 7.46–7.44
(1H, m), 7.23 (2H, d, J=8.4), 7.20–7.16 (2H, m), 6.76
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Chem. Biodiversity 2021, 18, e2001037
(1H, d, J=15.6), 2.43 (3H, s). 13C-NMR (300 MHz, (D6) with (MycoAlert, Lonza) mycoplasma test kit for
DMSO): 165.37, 137.94, 137.89, 135.21, 131.64, 131.58, mycoplasma contamination.
127.84, 125.30, 122.83, 120.96, 120.47, 119.97, 116.34,
112.87, 112.66, 16.16. HR-MS (m/z): [M+H]+ calc. for
C18H17N2OS 309.1062, found 309.1060.
Sulforhodamine B (SRB) Assay
SRB assay was performed as described previously.[41]
Data for (2E)-N-(4-tert-Butylphenyl)-3-(1H-indol- Briefly, cells were plated in 96-well plates (1000–3000
3-yl)prop-2-enamide (4l). Prepared using procedure cells/well) and grown for 24 h. Cells were treated with
1. Purified by flash column chromatography (0%! the compounds at different concentrations (40–
°
20% methanol in CH2Cl2). Yield 23%. M.p. 275–276 C. 2.5 μM) in triplicates. The final DMSO concentration
IR (FT-IR/FTNIR-ATR): 1643 cmÀ 1 (C=O). 1H-NMR was 0.1% in each well. After 72 h, cell fixation was
(400 MHz, (D6)DMSO): 11.63 (1H, s), 9.90 (1H, s), 7.96 done with 10% (v/v) trichloroacetic acid (MERCK) and
(1H, d, J=6.8), 7.82 (1H, s), 7.74 (1H, d, J=15.6), 7.61 cells were stained with SRB solution (50 μL of 0.4%
(2H, d, J=6.8), 7.47 (1H, d, J=6.4), 7.33 (2H, d, J=8.4), (w/v) SRB in 1% acetic acid). Unbound SRB was
7.24–7.18 (2H, m), 6.80 (1H, d, J=15.6), 1.27 (9H, s). removed by washing each well with 1% acetic acid
13C-NMR (300 MHz, (D6)DMSO): 165.28, 145.51, 137.93, followed by air-drying. Before absorbance measure-
137.71, 134.93, 131.47, 128.88, 125.83, 125.30, 122.79, ments at 515 nm, SRB was solubilized in 10 mM Tris-
120.91, 120.49, 119.08, 118.65, 116.60, 112.84, 112.68, base. Percent growth inhibition (% inhibition=100×
34.46, 31.60. HR-MS (m/z): [M+H]+ calc. for C21H23N2O (1À ODdrug/ODDMSO) vs. drug concentration values
319.1810, found 319.1797.
were used to generate dose-response curves to
calculate IC50 values for each compound.
Biological Studies
Real-Time Cell Growth Surveillance by Electronic Sensing
(RT-CES)
Drugs and Chemicals
Sulforhodamine B (SRB) and Dimethyl sulfoxide Real-time cell growth monitoring of HCC cells was
(DMSO) was purchased from Sigma–Aldrich. 5-Fluo- performed using the xCELLigence System (Agilent) as
rouracil (5-FU), Doxorubicin (DOXO) and Sorafenib described previously.[41] Briefly, cells were seeded into
was obtained from Calbiochem. The stock solutions 96-well E-plates (1000–3000 cells/well) and monitored
(20 mM) of test compounds and drugs were prepared every 30 min until the cells reached their log (growth)
°
in DMSO and stored at À 20 C.
phase. Then, treatments with the compounds (IC50 and
IC100 conc.), and with DMSO (control) were done. The
cell index (CI) values were recorded every 30 min for a
total of 72 h. Time-zero normalized CI values were
Cell Culture
HCC cell lines (Huh7, HepG2, and Mahlavu, human used to demonstrate cell growth graphs for each cell
breast carcinoma cells (MCF7) and human colon lines.
carcinoma cells (HCT116) were grown in Dulbecco’s
Modified Eagles Medium (DMEM) supplemented with
10% fetal bovine serum (FBS) (Invitrogen, GIBCO) and
Nuclear Staining with Hoechst 33258
1% non-essential amino acids (Invitrogen, GIBCO). Cells were seeded onto coverslips placed in 6-well
SNU475 cells were grown in RPMI-1640 media plates. Following 24 h, cells were treated with the
(Invitrogen, GIBCO) supplemented with 10% FBS and compounds (IC50 conc. for 48 h). Then, cells were fixed
2 mM L-glutamine. Normal human epithelial breast with 100% cold methanol for 10 min. Cells were then
cells (MCF-12A) were grown in DMEM/HAM’s F12 washed with cold 1xPBS for several times before
medium supplemented with 10% fetal bovine serum nuclear staining. 1 μg/ml Hoechst 33258 dye was used
(FBS) and 1% non-essential amino acids, 10 ng/ml to stain nuclei and incubated for 5 min at room
EGF, 500 ng/ml hydrocortisone and 10 μg/ml insulin. temperature. De-staining was done with ddH2O for
All media were also supplemented with 100 units/mL 10 min to remove excess dye. Apoptotic nuclei were
penicillin and streptomycin, and cells were main- identified using a blue filter (340–380 nm) on a
tained in a humidified incubator with 5% CO2, at fluorescence microscope (Nikon Eclipse 50i).
°
37 C. Cell lines used in this study are short tandem
repeat (STR) authenticated and are regularly tested
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Chem. Biodiversity 2021, 18, e2001037
Development project (KanSiL_2016K121540), to R.C.A.
We would like to thank Tugce Oksakli for laboratory
Cell Cycle Analysis
Cells were seeded onto 10 cm culture dishes and technical assistance in KanSil.
grown for 24 h. Then, cells were treated with the
compounds (IC100 conc.) for 24 or 48 h. Cells were
°
fixed with ice-cold 70% ethanol at À 20 C for 3 h
Author Contributions Statements
before propidium iodide (PI) staining. MUSE Cell
Cycle Assay kit (Millipore) was used to stain cells, cell S.N.B designed the compounds and their synthesis.
cycle distributions was determined using MUSE Cell M.H. performed the chemical synthesis and character-
Analyzer (Millipore) according to the manufacturer’s ized the compounds. R.C.A. and D.C.K. designed the
recommendations.
biological experiments. D.C.K. performed the experi-
ments for biological evaluation of the compounds on
HCC cell lines. S.N.B., M.H., D.C.K. and R.C.A. contrib-
uted to the writing of the manuscript. All authors have
Western Blot Analysis
Cells were seeded onto 15 cm culture dishes and given approval to the final version of the manuscript.
treated with the compounds (IC100 conc.) or with
DMSO for 24 h. Mini-PROTEAN® Tetra Cell Systems and
TGX™ precast gels (loaded with 20–40 μg/well pro-
tein) were used for gel electrophoresis according to
the manufacturer’s instructions. Then proteins were
transferred to PVDF membrane using the Trans-Blot®
Turbo Transfer System (Bio-Rad). For immunoblotting,
α-calnexin (Cell Signaling, 2679), α-phospho-Akt (Cell
Signaling, 9271), α-Akt (Cell Signaling, 9272S), α-
phospho-Erk (Santa Cruz, 16982), α-Erk (Santa Cruz, sc-
135900), α-c-Jun (Santa Cruz, sc-45), α-PARP (Cell
Signaling, 9532S) and β-actin (Cell Signaling, 4967)
primary antibodies, followed by HRP-conjugated goat
anti-mouse IgG (Sigma-Aldrich, A0168), rabbit anti-
goat IgG (Sigma-Aldrich A8919), or goat anti-rabbit
IgG (Sigma-Aldrich, A6154) secondary antibodies were
used. Then, proteins of interest were identified using
the C-Digit® imaging system (Ll-COR). The intensity of
the bands was quantified using ImageJ program and
intensity values for each sample were normalized to
that of Calnexin.
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