Piplartine analogues and cytotoxic evaluation against glioblastoma

Article abstract of DOI:10.3390/molecules23061382

Piplartine (1) is an alkamide extracted from plants of the genus Piper which shows several pharmacological properties, including antitumor activity. To improve this activity, a series of analogues based on 1 have been synthesized by esterification and amidation using the 3,4,5-trimethoxycinnamic acid-like starting material. During the study, the moieties 3-(3,4,5-trimethoxyphenyl)acrylate and 3-(3,4,5-trimethoxyphenyl)acrylamide were maintained on esters and amides respectively. Meanwhile, functional changes were exploited, and it was revealed that the presence of two aromatic rings in the side-chain was important to improve the cytotoxic activity against the U87MG cell line, such as the compound (E)-benzhydryl 3-(3,4,5-trimethoxyphenyl)acrylate (10), an ester that exhibited strong cytotoxicity and a similar level of potency to that of paclitaxel, a positive control. Compound 10 had a marked concentration-dependent inhibitory effect on the viability of the U87MG cell line with apoptotic and oxidative processes, showing good potential for altering main molecular pathways to prevent tumor development. Moreover, it has strong bioavailability with non-genotoxic and non-cytotoxic properties on human blood cells. In conclusion, the findings of the present study demonstrated that compound 10 is a promising agent that may find applications combatting diseases associated with oxidative stress and as a prototype for the development of novel drugs used in the treatment of glioblastoma.

Full text of DOI:10.3390/molecules23061382

molecules
Article
Piplartine Analogues and Cytotoxic Evaluation
against Glioblastoma
Flávio Rogério da Nóbrega 1, Ozlem Ozdemir 2 ID , Sheila Cristina S. Nascimento Sousa 3,
Joice Nascimento Barboza 1, Hasan Turkez 2,4 and Damião Pergentino de Sousa 1,
*
ID
1
Laboratory of Pharmaceutical Chemistry, Universidade Federal da Paraíba, João Pessoa 58051-085, Brazil;
frnobrega@hotmail.com (F.R.d.N.); joice_nascimentob@hotmail.com (J.N.B.)
Department of Molecular Biology and Genetics, Erzurum Technical University, Erzurum 25240, Turkey;
ozlem.ozdemir@erzurum.edu.tr (O.O.); hasanturkez@gmail.com (H.T.)
Programa de Pós-Graduação em Engenharia Química, Universidade Federal de Sergipe,
São Cristóvão 49100-000, Brazil; shcrisnascimento@yahoo.com.br
2
3
4
Department of Pharmacy, “G. d’Annunzio” University of Chieti-Pescara, Via dei Vestini 31,
66013 Chieti Scalo, CH, Italy
*
Correspondence: damiao_desousa@yahoo.com.br; Tel.: +55-83-3216-7347
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 27 April 2018; Accepted: 14 May 2018; Published: 8 June 2018
Abstract: Piplartine (
several pharmacological properties, including antitumor activity. To improve this activity,
a series of analogues based on have been synthesized by esterification and amidation
1) is an alkamide extracted from plants of the genus Piper which shows
1
using the 3,4,5-trimethoxycinnamic acid-like starting material. During the study, the moieties
3-(3,4,5-trimethoxyphenyl)acrylate and 3-(3,4,5-trimethoxyphenyl)acrylamide were maintained
on esters and amides respectively. Meanwhile, functional changes were exploited, and it was
revealed that the presence of two aromatic rings in the side-chain was important to improve
the cytotoxic activity against the U87MG cell line, such as the compound (E)-benzhydryl
3-(3,4,5-trimethoxyphenyl)acrylate (10), an ester that exhibited strong cytotoxicity and a similar
level of potency to that of paclitaxel, a positive control. Compound 10 had a marked
concentration-dependent inhibitory effect on the viability of the U87MG cell line with apoptotic
and oxidative processes, showing good potential for altering main molecular pathways to prevent
tumor development. Moreover, it has strong bioavailability with non-genotoxic and non-cytotoxic
properties on human blood cells. In conclusion, the findings of the present study demonstrated
that compound 10 is a promising agent that may find applications combatting diseases associated
with oxidative stress and as a prototype for the development of novel drugs used in the treatment
of glioblastoma.
Keywords: cancer; cytotoxic activity; antitumor; phenylpropanoid; alkaloid; natural product; esters;
oxidative stress; Piper; synthetic derivatives
1. Introduction
Glioblastoma (GBM) is the most fatal primary brain cancer found in humans, with about 3 in
100,000 people developing the disease every year [1,2]. It originates from glial cells, but rarely
disseminates outside the nervous system and grows diffusely into surrounding brain tissue. This
cancer is still incurable and is characterized by nuclear atypia, mitotic activity, intense microvascular
proliferation and necrosis. It occurs more frequently in males and there is no association with
smoking [3], consumption of cured meat [4] or electromagnetic fields [5], however some viruses
such as SV40 and HHV have a positive association [
6
,7
]. Currently treatment for the cancer involves
Molecules 2018, 23, 1382; doi:10.3390/molecules23051382
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1382
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a multimodal approach which include surgery (total removal of tumor), post-operative radiation
therapy radiotherapy (RT) and concurrent and adjuvant temozolomide (TMZ). The median survival
rate is only 14.6 months with less than 3% to 5% of sufferers surviving longer than five years. Without
treatment, the patient does not survive for more than 3 months [2].
The gliomagenesis occurs with a multistep process that exhibits complex patterns of abnormal
gene expression [8]. The primary GBMs exhibit epidermal growth factor receptor (EGFR) amplification,
phosphatase and tensin homolog (PTEN) mutation as well as the loss of chromosome 10. The secondary
GBMs exhibit p53 mutation. And, glioblastoma tumor growth has been found as suppressed via
AKT-mediated inhibition [9]. The ligation of EGFR by epidermal growth factor (EGF) induces
endogenous generation of intracellular ROS and H2O2 in the cancer cell line. It is also clear that
PIP3K/AKT pathways play an important role in the regulation of metabolism, apoptosis and survival
in normal cells [10].
Piplartine (1) is an alkamide commonly found in long pepper (Piper longum L.). Previous reports
indicated piplartine exhibited a wide range of pharmacological activities, including anti-diabetic,
anti-ulcer, anti-platelet aggregation, anti-atherosclerotic, anti-depressant, anti-bacterial and anti-fungal
properties [11
property, which protects against various types of tumors [15
piplartine kills HGG (high-grade glioma) cells by PRDX4 inactivation inducing endoplasmic reticulum
stress [10]. Therefore, the antitumor activity of piplartine ( ) in a type of cancer of the Central Nervous
–
14]. Moreover, this compound possesses a highly selective and effective antitumor
,
16]. It has recently been demonstrated that
1
System motivated us to investigate a collection of synthetic analogues of piplartine on human U87MG
glioblastoma cell line, as well as exploring the molecular genetic basis underlying their effects for the
first time.
2. Results
Chemistry
For this study, 18 analogues of
(E)-3-(3,4,5-trimethoxyphenyl)acryloyl in the esters and amides (Scheme 1). Side-chain modifications
were also evaluated by changing the radical (R) to: methyl ( ), ethyl ( ), propyl ( ), butyl ( ), pentyl ( ),
decyl ( ), 2-methoxyethyl ( ), iPr ( ), CHPh2 (10), carvacryl (11), phenethyl (12), 4-methylphenethyl (13
on esters and butyl (14), benzyl (15), 4-methylbenzyl (16), 4-methoxylbenzyl (17), 4-chlorobenzyl (18
1 (Figure 1) were synthesized, maintaining the similar moiety
2
3
4
5
6
7
8
9
)
)
and N,N-diethyl (19) on amides. Side-chain modifications are expected to increase lipophilicity and
antitumor efficacy of the esters and amides, as well as their mode of interaction with the receptor
(Scheme 1) [17–19].
Figure 1. Chemical structure of piplartine (1).
To synthesize the compounds, it starting materials were utilized like 3,4,5-trimethoxycinnamic
acid, ROH to esters and primary or secondary amines to amides. The reactions used were (a), (b), (c)
to synthesize esters and (d) to synthesize amides (Scheme 1). Reactions were performed in one step
and most products were obtained in high yield.

Molecules 2018, 23, 1382
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Scheme 1. Reagents and conditions: (a) ROH, H2SO4, reflux; (b) Et3N, RBr or RCl, acetone, reflux;
(c) SOCl2, reflux, ROH (d) RNH2 or (CH3CH2)2NH, BOP, Et3N, CH2Cl2, DMF.
To evaluate the cytotoxic potential of compounds
2–19 against human U87MG, glioblastoma cells
were used with several concentrations (0 to 50 g/mL). After 48 h of incubation, cell viability rates were
µ
established by MTT and LDH analysis. IC50 values of each analogue were determined due to observed
cell viability results after MTT assay. All tested analogues led to inhibition of cellular proliferation
rates as compared to untreated cells in a clear concentration and compound type dependent manners,
as illustrated in Table 1 and Figure 2. In this sense, the most potent cytotoxic agent among those tested
was the compound 10 against U87MG cells.
Table 1. Cytotoxic activity of the compounds 2–19 against proliferation of U87MG cells.
Esters
IC50 (µg/mL)
Amides
Compound
Compound
IC50 (µg/mL)
2
3
4
5
6
7
8
9
10
11
12
13
85.129 ± 0.78
100.030 ± 1.12
78.393 ± 0.69
57.871 ± 0.71
31.666 ± 0.48
34.926 ± 0.54
50.077 ± 0.62
81.433 ± 0.55
2.579 ± 0.35
26.701 ± 0.47
24.786 ± 0.37
30.336 ± 0.35
14
15
16
17
18
19
13.196 ± 0.32
33.128 ± 0.41
22.741 ± 0.34
22.654 ± 0.39
43.396 ± 0.48
36.072 ± 0.44
Positive control (paclitaxel): IC50: 2.119 ± 0.35 µg/mL.
Cell apoptosis induced by compound 10 was examined by Hoechst 33,258 staining and observed
under a fluorescence microscope at 400
×
magnification. U87MG cells were treated with compound 10
for 48 h at their IC50 concentrations. The nuclear fragmentation of treated cells was compared with the
control group and characteristic alterations of apoptosis including enhanced chromatin condensation
and nuclear fragmentation were detected in the compound 10 treated cells (Figure 3).

Molecules 2018, 23, 1382
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120
LDH
MTT
100
80
60
40
20
0
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
NC
PC
0.78 1.56 3.125 6.25
12.5
25
50
Concentrations of compound 10 (μg/L)
Figure 2. The in vitro effects of compound 10 on the cell viability of human U87MG glioblastoma cell
line. Data are presented as the mean S.D. of four independent experiments. NC: Negative control,
PC: Positive control (as paclitaxel). * p < 0.05, comparison to the NC.
±
Figure 3. Hoechst 33,258 staining in U87MG cells. (
compound 10 for 48 h at IC50 concentration (arrow shows nuclear fragmentation, magnification
A
) Negative control; (
B
) Cells treated with
400).
×
The total antioxidant capacity (TAC) and the total oxidant status (TOS) levels were evaluated in
samples from treated and untreated cultures using colorimetric measurement methods. As shown
from the results presented in Figure 4, concentrations (0.78 and 50 µg/mL) of compound 10 resulted
in a significant increase of TAC levels on U87MG cells compared with the controls. On the other
hand, treatments with compound 10 did not change the TOS levels in cultured U87MG cells for all the
concentrations (data not shown).
To find out the differences in the level of gene expression in U87MG cells exposed using
compound 10, a custom plate with 15 different genes involved in cancer related pathways was
used via qRT-PCR studies. According to the gene expression results, two genes (AKT1 and AKT2)
were significantly (>0.5 fold change) down-regulated and four genes (NF-kB1, NF-kB2, PTEN and
TP53) were up-regulated in the U87MG cells as compared to untreated subjects upon exposure to 10
However, there were slight and insignificant alterations (<0.5 fold change) of nine genes, including
BRAF, DVL1, EGFR, FOS, KRAS, NF-kB1A, RAF1, PIK3CA and PIK3R1 (Figure 5).
.
To evaluate biosafety of 10, its cytotoxic and genotoxic potential were investigated on cultured
peripheral human whole blood (PHWB) cells. Firstly, the cytotoxicity testing was performed by MTT
and LDH assays. The human blood cells exposed to 0.78 to 50 µg/mL of 10. The results of both assays
showed that 10 did not lead to any significant (p > 0.05) changes in cell proliferation. Afterwards,
genotoxic potential was assessed by SCE testing and determination of 8-OH-dG. There were not
significant differences in the frequencies of SCEs between the control group and 10 treated groups for

Molecules 2018, 23, 1382
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72 h (p > 0.05). Likewise, 8-OH-dG levels were not increased in the blood cells that were treated with
10 concentrations (Data not shown). Collectively, the 10 exhibited non-cytotoxic and non-genotoxic
properties in PHWB cell cultures.
120
LDH
MTT
100
80
60
40
20
0
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
NC
PC
0.78 1.56 3.125 6.25
12.5
25
50
Concentrations of compound 10 (μg/L)
Figure 4. The effects of compound 10 on total antioxidant capacity (TAC) levels in cultured U87MG
cells for 48 h. Data are expressed as the mean S.D. of four independent experiments. NC: Negative
±
control, PC: Positive control (as ascorbic acid, 10 µM). * p < 0.05, comparison to NC.
Figure 5. Gene expression alterations after treatment with compound 10 on U87MG cells for 48 h.
3. Discussion
3.1. Structure Activity Relationship (SAR)
Table 1 shows that the introduction of methylene groups into the radicals attached to the
(E)-3-(3,4,5-trimethoxyphenyl)acrylate in compounds
to the carbon chain size, with the exception of (IC50: 100.030
(IC50: 57.871 0.71 g/mL) with amide 14 (IC50: 13.196 0.32 g/mL), it was observed that the butyl
2–
6
provided IC50 values inversely proportional
3
±
1.12 g/mL). Comparing ester
µ
5
±
µ
±
µ
radical attached to the group 3,4,5-trimethoxycinnamoyl on amide might more effective than the same
radical attached to the group (E)-3-(3,4,5-trimethoxyphenyl)-acrylate on esters. The introduction of
methyl groups contributed to increase lipophilicity, but did not show an increase of cytotoxic activity
when the compound 12 (IC50: 24.786 ± 0.37) was compared with 13 (IC50: 30.336 ± 0.35) [20].

Molecules 2018, 23, 1382
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For research carried out by Example [21], piplartine (
1
) and other compounds were isolated
from the Piper cernuum plant and the pharmacological activity was made in several tumor cell lines.
The cell lines evaluated by the MTT test were: BF16F10-Nex2 (murine melanoma), U87MG (human
glioblastoma), HeLa (human cervical carcinoma), HCT (human colon carcinoma), HL-60 (human
leukemic cell) and A2258 (human melanoma). The same author showed that
results against these cancer cell lines (IC50: >200 g/mL in all tested strains), but that one of the
analogues, known as piplaroxide, did obtain moderate activity in the BF16F10-Nex2, HeLa and HL-60
strains. Piplaroxide differs from
by replacing the double bond between the 50 and 60 carbons by an
epoxide and absence of the methoxyl at the 3-position of the ring.
1 did not obtain significant
µ
1
The difference in chemical structure of 1 and piplaroxide is the oxygen atoms in the portion of
the epoxide at the 50 and 60 carbons on piplaroxide. This oxygen may have contributed to increased
piplaroxide antitumor activity as compared to 1 [21,22].
Comparing the side-chains R of compound
8
(IC50: 50.077
±
0.62 µg/mL) and 4 (IC50:
78.393 ± 0.69 µg/mL), it was found that oxygen is also determinant to increase antitumor activity in
the U87MG cell line. In accordance with the current research, the oxygen at the moieties provides a new
center for H-bond that could influence the binding of the analogue to its target site [20]. However, the
presence of the oxygen on methoxyl moiety on the para position on R1 of amide 17 (IC50: 22.654 ± 0.39
)
did not contribute to improve cytotoxic activity when compared to amide 16 (IC50: 22.741 ± 0.34),
with the presence of the methyl group in this position of the ring.
In general, the presence of aromatic rings on side-chain R of the esters and amides increased
antitumor activities against U87MG, but the trisubstituted aromatic ring present in the carvacryl
moiety of ester 12 did not provide more effectiveness in comparison to the other compounds with
aromatic rings on R.
The branch present on side-chains R of ester
activity compared with other compounds with side-chains R composed with saturated radicals.
However, the change of methyl groups to phenyl on branch radical of ester 10 (CI50: 2.579 0.35)
provide an IC50 more closely to a positive control paclitaxel (IC50: 2.119 0.35) and also appeared to
9
(IC50: 81.433
±
0.55) did not increase cytotoxic
±
±
be the most active synthesized compound against the U87MG cell proliferation. Therefore, further
experiments were performed with this compound.
3.2. Biological Activity
In cell biology, U87MG—an abbreviation for “Uppsala 87 Malignant Glioma”—is a primary
lineage of glioblastoma, which, immortalized, originated from a 44-year-old cancer patient. This
cell line is usually used to evaluate cytotoxic and genotoxic aspects of drugs and other molecules in
brain cancer cells, especially glioblastomas [23]. Present data revealed that tested analogues exhibited
anti-proliferative activity. Our findings also showed that compound 10 induced apoptosis in U87MG
cells via leading increases of chromatin condensation and nuclear fragmentation. There is scarce data
on the anti-glioblastoma actions of
the generation of ROS, and previous studies have demonstrated that
action via leading decreases of the expression of some key proteins (HER, JAK 1 and 2, STAT3, c-MET,
GSTP1, JNK) that are responsible for the development of various cancer types [24 26]. Again, in a
current investigation, it was determined that served as a microtubule-destabilising agent in MCF-7
breast cancer cells [27]. And, the apoptotic action by was associated to caspase-3-mediated poly
2–
19. However, the cytotoxic activities of
1 are associated with
1
exhibited antiproliferative
–
1
1
(ADP-ribose) polymerase (PARP) cleavage and cell cycle arrest at G2/M phase in cultured human
PC-3 prostate cancer cells [28].
Previous research revealed that
1 caused increases of intracellular level of reactive oxidative
species (ROS) and inhibited tumor growth [11]. Disparately, present results indicated that oxidative
alterations are not closely related to antitumor action by the novel and effective analogue, 10
In supporting our novel finding on in vitro oxidative effects of 10 analogue, it was determined that
most glioblastomas overexpressed ROS-degrading enzyme peroxiredoxin 4 (PRDX4) and suggested
.

Molecules 2018, 23, 1382
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that pharmaceutical PRDX4 inactivation effectively kills the cancer cells by increasing ROS levels [10].
Therefore, it can be concluded that analogue 10 induced oxidative stress that was restored by
overexpressed PRDX4 in cultured U87MG cells. Due to this,
1 was found to be less effective in
SF-295 human glioblastoma cells as compared to HCT-8 human colon carcinoma cells [29].
Our results firstly revealed that 10 led to down-regulation of AKT1 and AKT2 genes in U87MG
cells. Therefore, it was determined that the anticancer potential of 10 was largely associated with
the PI3K/AKT/mTOR pathway, since AKT was an important mediator of this intracellular signaling
pathway which is important in cell cycle regulation [30]. PI3K/AKT/mTOR was reported to play
key functions on tumor initiation and progression as well as angiogenesis. AKT2 was found to be
especially necessary for glioma cell migration [31]. In fact, the enhanced expression of AKT1 and
cyclin D1 were detected in cultured U87MG cells [32]. Likewise, AKT1 and AKT2 found to be related
to bcatenin/Tcf-4 signaling pathway that promoted malignant transformation in human LN229 glioma
cell line. Moreover, AKT1 was considered to be more effective than AKT2 in malignant transformation.
Thus, AKT1 and AKT2 were considered as valuable targets for glioblastoma therapy as well as a
rational strategy for restraining the metastasis of gliomas [33]. Our positive control agent paclitaxel was
known to down-regulate the MMP-9-mediated p38/JNK signaling pathway and block the proliferation
of U87MG cells [34]. However, it was determined that the combined use of paclitaxel with alkylating
agents supported a greater clinical efficacy of paclitaxel against malignant brain tumors [35]. But the
adverse effects of these combinations are still perturbative. At this point, present findings show that
10 as anti-tumoral agent or suppressor with a good biosafety level is interrelated to proliferation and
invasion of glioblastoma cells by inhibiting AKT1 signaling and 10 may be considered as an effective
therapeutic treatment against glioblastoma.
The present qRT-PCR analysis revealed up-regulation of the genes involving NF-kB1, NF-kB2,
PTEN and TP53 in vitro. Conversely, constitutive activation of NF-
of glioblastoma cells in human and mouse models [36]. However, it was determined that varied levels
of NF- B activation effected cancer propagation via several mechanisms. Thereof, it was considered
that the alterations of the NF- B signalling response in both upstream stimuli and the downstream
κB supported growth and survival
κ
κ
targets would be fundamental for gliomagenesis in the near future [37]. Similarly, the overexpression
of p53 and mutation in TP53 were associated with adverse outcomes in studies of malignant gliomas
from children [38]. On the other hand, the tumor suppressor gene PTEN was shown to regulate Akt
signalling, cellular growth and apoptosis [39]. Again, blocking the PTEN expression was linked to
metastasis in addition to both radio- and chemotherapy responses in patents with glioma and breast
cancer. Accordingly, PTEN is addressed as a main regulator of tumor sensitivity towards different
therapeutic options [40–47]. In a recent study [47], it was revealed that PTEN was not the primary
target of paclitaxel resistance and its regulator was evaluated as a substantive target for suppression of
miR-22, cyclin B1 or combining with inhibitor of Akt, providing a successful strategy for paclitaxel
therapy. In this regard 10 provides a rationale strategy against glioblastoma in single or combined use
with other antineoplastic agents. Supplementary materials are available on line.
The human lymphocytes were suggested as a serviceable experimental model for planning
chemotherapeutic strategies as well as dose selection from the point of genotoxic and cytotoxic damage
potentials of drug/drug candidates on healthy cells [48,49]. Consequently, cultured peripheral human
whole blood cells were used to evaluate the cytotoxic (MTT and LDH assays) and genotoxic (SCE and
8-OH-dG assays) potential of 10. Finally, the property of 10 as a non-cytotoxic and non-genotoxic agent
with high anti-cancer efficacy makes it a potential candidate for novel cancer therapy.
4. Materials and Methods
The 1H and 13C NMR, and IR signals assigned to analogues of
already published. The unpublished molecules such as 10
1
,
were comparing with signals
13 and 16 besides the same
6,
7,
8,
spectrophotometric methods used to other analogues, was used LS-MALDI TOF/TOF to confirm
the synthesis.

Molecules 2018, 23, 1382
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All reagents were purchased from Aldrich and were of commercial grade. IR spectra were
recorded on Irprestige-21 Shimadzu Fourier Transform spectrophotometer (Shimadzu, Kyoto, Japan).
1H and 13C NMR spectra were recorded on a Varian Mercury spectrometer 200 MHz (1H) and 50 MHz
(13C), Varian (Palo Alto, CA, USA). Chemical shifts are reported relative to the solvent peak CDCl3 or
TMS. To HMRS was used the equipment ultraflex II MALDI TOF/TOF equipped with a Laser with
High Performance (λ = 355 nm) and reflector operating by software FlexControl 2,4 (Bruker Daltonics
G, bsH, Bremen, Germany) Melting points were measured on an equipment Tecnal PFM-II, 220 V.
4.1. General Procedure for Synthesis of Compounds 2–5, 8 and 9
To a solution of 3,4,5-trimethoxycinnamic acid (0.25 g) in 250 mL of ROH, 0.5 mL 96% (v/v) H2SO4
was added under stirring. The reaction mixture was refluxed for 3 h. Half of ROH was removed under
reduced pressure and the following solution was then diluted with 10 mL water and the product
extracted with ethyl acetate. The organic phase of each reaction, when gathered were successively
washed with 5% (w/v) NaHCO3 and water, dried over anhydrous Na2SO4 and filtered. After removal
of ethyl acetate under vacuum, the pure products of each of those reactions were obtained [50].
4.1.1. (E)-Methyl-3-(3,4,5-trimethoxyphenyl)acrylate (2)
◦
Yield 93%; white solid, m.p.: 135–136 C; MM: 252.10 g/mol; 1H NMR (200 MHz, CDCl3) δH 7.59
(d, J = 16.0 Hz, 1H), 6.73 (s, 2H), 6.33 (d, J = 16.0 Hz, 1H), 3.87 (s, 9H), 3.79 (s, 3H). 13C NMR (50 MHz,
CDCl3) δC 167.5, 153.5, 144.9, 140.1, 129.9, 117.1, 105.2, 61.1, 56.2, 51.8, IR
ν
(KBr, cm−1) 3003, 1697,
max
2943, 2837, 1632, 1040, 1005, 1582, 1506, 851 [51,52].
4.1.2. (E)-Ethyl-3-(3,4,5-trimethoxyphenyl)acrylate (3)
Yield 98.8%; white solid, MM: 266.12 g/mol, m.p.: 65–66 ◦C; 1H NMR (200 MHz, CDCl3) δH 7.53
(d, J = 16.0 Hz, 1H), 6.68 (s, 2H), 6.28 (d, J = 16.0 Hz, 1H), 4.19 (q, J = 6.0 Hz, 2H), 3.81 (s, 9H), 1.27 (t,
J = 6.0 Hz, 3H), 13C NMR (50 MHz, CDCl3) δC 166.8, 153.3, 144.5, 139.9, 129.8, 117.4, 105.1, 60.8, 60.4,
56.0, 14.3, IR νmax (KBr, cm−1) 2999, 2974, 2945, 2837, 1701, 1634, 1038, 997, 1583, 1506, 872 [52].
4.1.3. (E)-Propyl-3-(3,4,5-trimethoxyphenyl)acrylate (4)
Yield 93%, beige solid, MM: 280.13 g/mol, m.p.: 97–100 ◦C; 1H NMR (200 MHz, CDCl3) δH 7.58
(d, J = 16.0 Hz, 1H), 6.74 (s, 2H), 6.34 (d, J = 16.0 Hz, 1H), 4.15 (t, J = 6.0 Hz, 2H), 3.87 (s, 9H), 1.72 (dt,
J = 6.0, 8.0 Hz, 2H), 0.98 (t, J = 8.0 Hz, 3H), 13C NMR (50 MHz, CDCl3) δC 167.1, 153.5, 144.6, 140.0,
130.0, 117.6, 105.2, 66.2, 61.0, 56.2, 22.2, 10.6, IR
1585, 1506, 835 [53].
ν
(KBr, cm−1) 3003, 2941, 2841, 1697, 1632, 1005,
max
4.1.4. (E)-Butyl-3-(3,4,5-trimethoxyphenyl)acrylate (5)
◦
Yield 99.6%, brown solid, MM: 294.15 g/mol, m.p.: 74–75 C; 1H NMR (200 MHz, CDCl3) δH 7.57
(d, J = 16.0 Hz, 1H), 6.73 (s, 2H), 6.33 (d, J = 16.0 Hz, 1H), 4.19 (t, J = 6.0 Hz, 2H), 3.86 (s, 6H), 3.85
(s, 3H), 1.75–1.59 (m, 2H), 1.52–1.32 (m, 2H), 0.94 (t, J = 8,0 Hz, 3H), 13C NMR (50 MHz, CDCl3) δC
167.1, 153.5, 144.6, 140.1, 130.0, 117.6, 105.2, 64.5, 61.0, 56.2, 30.9, 19.3, 13.8, IR
2965, 2943, 2841, 1697, 1638, 1005, 1582, 1506, 845 [54].
ν
max (KBr, cm−1) 2990,
4.1.5. (E)-2-Methoxyethyl-3-(3,4,5-trimethoxyphenyl)acrylate (8)
Yield 97%, brown oil, MM: 296,13 g/mol, m.p.: 60–62 ◦C; 1H NMR (200 MHz, CDCl3) δH 7.61
(d, J = 16.0 Hz, 1H), 6.74 (s, 2H), 6.39 (d, J = 16.0 Hz, 1H), 4.35 (d, J = 4.0 Hz, 2H), 3.86 (s, 9H), 3.65 (t,
J = 4.0 Hz, 2H), 3.41 (s, 3H), 13C NMR (50 MHz, CDCl3) δC 167.0, 153.5, 145.2, 140.1, 129.9, 117.1, 105.3,
70.7, 63.6, 61.0, 59.2, 56.2, IR
ν
(KBr, cm−1) 3019, 2963, 2939, 2841, 1709, 1638, 1037, 1001, 1584, 1504,
max
LS-MALDI TOF/TOF m/z [M]+ 296.1244 (calcd. for C15H20O6, 296.1260).

Molecules 2018, 23, 1382
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4.1.6. (E)-Isopropyl 3-(3,4,5-Trimethoxyphenyl)acrylate (9)
Yield 96%, brown oil, MM: 280.13 g/mol; 1H NMR (200 MHz, CDCl3) δH 7.54 (d, J = 16.0 Hz, 1H),
6.71 (s, 1H), 6.29 (d, J = 16.0 Hz, 1H), 5.09 (hept, J = 6.00, 1H), 3.83 (s, 9H), 1.29–1.19 (m, 6H), 13C NMR
(50 MHz, CDCl3) δC 166.5, 153.4, 144.4, 139.9, 130.0, 118.0, 105.2, 67.9, 61.0, 56.1, 22.0, IR
cm−1) 3019, 2978, 2938, 2841, 1703, 1638, 1584, 1505 [55].
ν
(KBr,
max
4.2. General Procedure for Synthesis of Compounds 6, 7, 10 and 13
To a solution of 3,4,5-trimethoxycinnamic acid (0.1 g, 0.42 mmol) dissolved in 5.0 mL of acetone,
0.22 mL (1.68 mmol) of Et3N and 0.43 mmol of alkyl halide (RBr or RCl) were added and stirred and
the reaction mixture was refluxed for 24 h, except for the reaction of synthesis of compound 10, which
was stirred for 48 h. After cooling at room temperature, the acetone of each reaction was removed
under reduced pressure. The remaining content of those reactions were then diluted with 10 mL
ethyl acetate and transferred to the separator funnel containing 10 mL of water. The products were
extracted with 10 mL ethyl acetate three times and the during the organic phase when gathered, were
successively washed with 5% (w/v) NaHCO3 and water, dried over anhydrous Na2SO4 and filtered.
After the removal of ethyl acetate under vacuum, the products were isolated with silica gel 60-column
chromatography (eluent: hexane-ethyl acetate) [56].
4.2.1. (E)-Pentyl 3-(3,4,5-trimethoxyphenyl)acrylate (6)
Yield 66.5%, white solid, MM: 308.16 g/mol, m.p.: 105–106 ◦C; 1H NMR (200 MHz, CDCl3) δH
7.56 (d, J = 16.0 Hz, 1H), 6.72 (s, 2H), 6.32 (d, J = 16.0 Hz, 1H), 4.16 (t, J = 6.0 Hz, 2H), 3.84 (m, 9H),
1.76–1.58 (m, 2H), 1.41–1.27 (m, 4H), 0.90 (t, J = 6,0 Hz, 3H), 13C NMR (50 MHz, CDCl3) δC 167.1,
153.4, 144.5, 140.0, 130.0, 117.5, 105.1, 64.7, 61.0, 56.1, 28.5, 28.2, 22.4, 14.0, IR
ν
(KBr, cm−1) 2997,
max
2957, 2934, 2857, 1711, 1638, 1009, 1584, 1505, 825, LS-MALDI TOF/TOF m/z [M]+ 308.1621 (calcd. for
C17H24O5, 308.1624).
4.2.2. (E)-Decyl-3-(3,4,5-trimethoxyphenyl)acrylate (7)
Yield 64.5%, white solid, MM: 378.24 g/mol, m.p.: 37–38 ◦C; 1H NMR (200 MHz, CDCl3) δH 7.58
(d, J = 16.0 Hz, 1H), 6.74 (s, 2H), 6.34 (d, J = 16.0 Hz, 1H), 4.18 (t, J = 6,0 Hz, 2H), 3.87 (s, 9H), 1.76–1.60
(m, 2H), 1.35–1.15 (m, 14H), 0.86 (t, J = 6.0 Hz, 3H). 13C NMR (50 MHz, CDCl3) δC 167.1, 153.5, 144.6,
140.1, 130.1, 117.6, 105.2, 64.6. 61.1, 56.2, 32.0, 29.6, 29.4, 28.8, 26.1, 22.8, 14.2, IR
ν
(KBr, cm−1) 2995,
max
2953, 2926, 2853, 1713, 1636, 1007, 1582, 1504, 827, LS-MALDI TOF/TOF: m/z [M]+ 378.2406 (calcd. for
C22H34O5 378.2406).
4.2.3. (E)-Benzhydryl 3-(3,4,5-trimethoxyphenyl)acrylate (10)
Yield 41.6% colorless oil, MM: 404.16 g/mol 1H NMR (200 MHz, CDCl3) δH 7.67 (d, J = 16.0 Hz,
1H), 7.41–7.22 (m, 10H), 7.02 (s, 1H), 6.76 (s, 2H), 6.48 (d, J = 16.0 Hz, 1H), 3.88 (s, 9H), 13C NMR
(50 MHz, CDCl3) δC 166.0, 153.5, 145.5, 140.2, 129.9, 128.6, 128.0, 127.2, 126.6, 117.3, 105.3, 77.1, 61.0, 56.2.
IR
ν
(KBr, cm−1) 3028, 3001, 2938, 2837, 1709, 1636, 1001, 1582, 1504, 825, LC-MALDI TOF/TOF:
max
m/z [M]+ 404.1627 (calcd. for C25H24O5, 404.1624).
4.2.4. (E)-4-Methylphenethyl 3-(3,4,5-Trimethoxyphenyl)acrylate (13)
Yield 30.6%, white solid, MM: 356.26 g/mol, m.p.: 105–106 ◦C; 1H NMR (200 MHz, CDCl3) δH
7.60 (d, J = 16.0 Hz, 1H), 7.14 (s, 4H), 6.75 (s, 2H), 6.35 (d, J = 16.0 Hz, 1H), 4.41 (t, J = 8.0 Hz, 2H), 3.90 (s,
9H), 2.96 (t, J = 8.0 Hz, 2H), 2.33 (s, 3H), 13C NMR (50 MHz, CDCl3) δC 166.9, 153.4, 144.8, 140.1, 136.1,
134.8, 129.9, 129.2, 128.8, 117.3, 105.2, 65.2, 61.0, 56.1, 34.8, 21.1 IR ν
max (KBr, cm−1) 3001, 2941, 2845,
1703, 1632, 1045, 1003, 1585, 1506, 820 LC-MALDI TOF/TOF m/z [M]+ 356.1643 (calcd. for C21H24O5,
356.1624).

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4.3. General Procedure for Synthesis of Compounds 11 and 12
To a 50 mL round-bottom flask with 0.1 g (0.42 mmol) of 3,4,5-trimethoxycinnamic acid connected
to a reflux condenser was added 0.03 mL (0.42 mmol) of thionyl chloride, the reaction mixture was
stirred and refluxed for 1 h and the reaction was finalized after adding 0.42 mmol of respective ROH
(carvacrol or phenethyl alcohol), followed by an additional hour of stir and reflux. After cooling at
room temperature, the remaining content of those reactions was then diluted with 10 mL ethyl acetate
and transferred to the separator funnel containing 10 mL of water. The products were extracted with
10 mL ethyl acetate three times and the organic phase when gathered were successively washed with
5% (w/v) NaHCO3 and water, dried over anhydrous Na2SO4 and filtered. After the removal of ethyl
acetate under vacuum, the products were isolated with silica gel 60-column chromatography (eluent:
hexane-ethyl acetate) [57].
4.3.1. (E)-Carvacryl-3-(3,4,5-trimethoxyphenyl)acrylate (11)
Yield 52%, White solid, MM: 370.18 g/mol, m.p.: 145–146 ◦C; 1H NMR (200 MHz, CDCl3) δH
7.81 (d, J = 16.0 Hz, 1H), 7.18 (d, J = 8.0 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.94 (s, 1H), 6.83 (s, 2H), 6.58
(d, J = 16.0 Hz, 1H), 3.91 (s, 9H), 2.91 (Hept, J = 6.0 Hz, 1H), 2.18 (s, 3H), 1.26 (s, 3H), 1.23 (s, 3H), 13
C
NMR (50 MHz, CDCl3) δC 165.3, 153.6, 149.4, 148.2, 146.5, 140.5, 131.0, 129.8, 127.4, 124.3, 119.9, 116.5,
105.5, 61.1, 56.3, 33.7, 24.0, 16.0, IR
822 [58].
ν
(KBr, cm−1) 3022, 2997, 2959, 2833, 1722, 1632, 1005, 1582, 1504,
max
4.3.2. (E)-Phenethyl 3-(3,4,5-trimethoxyphenyl)acrylate (12)
◦
Yield 83%, white solid, MM: 342.15 g/mol, m.p.: 110–112 C; 1H NMR (200 MHz, CDCl3) δH 7.60
(d, J = 16.0 Hz, 1H), 7.32–7.24 (m, 5H), 6.75 (s, 2H), 6.34 (d, J = 16.0 Hz, 1H), 4.44 (t, J = 8.0 Hz, 2H), 3.88
(s, 9H), 3.03 (t, J = 8.0 Hz, 2H), 13C NMR (50 MHz, CDCl3) δC 167.0, 153.5, 144.9, 140.1, 137.9, 130.0,
129.0, 126.7, 117.3, 105.3, 65.1, 61.0, 56.2, 35.3, IR
1582, 1506, 843 [59].
ν
max (KBr, cm−1) 3007, 2945, 2843, 1707, 1632, 1042,
4.4. General Synthesis of Amides
To a round-bottom flask, 0.1 g (0.42 mmol) of 3,4,5-trimethoxycinnamic was dissolved in 0.84
mL of DMF and 0.06 mL (0.42 mmol) of trimethylamine. The solution was cooled in an ice water
bath and 0.42 mmol of amine were added, followed by a solution of 0.42 mmol of BOP in 0.84 mL of
◦
CH2Cl2. The mixture was stirred at 0 C for 30 min and then at room temperature for 3 h. After of the
removal of CH2Cl2 under reduced pressure, the solution was diluted with 10mL of ethyl acetate and
transferred to separator funnel containing 10 mL of water. The products were extracted with 10 mL
ethyl acetate three times and the organic phase when gathered were washed successively with 1 N
HCl, water, 1 M NaHCO3 and water, dried over Na2SO4, Filtered and evaporated. The residues were
purified on a silica gel 60 column chromatography (eluent: hexane-ethyl acetate) [60].
4.4.1. (E)-N-Butyl-3-(3,4,5-trimethoxyphenyl)acrylamide (14)
◦
Yield 80%, white solid, MM: 293.16 g/mol, m.p.: 225–226 C; 1H NMR (200 MHz, CDCl3) δH 7.50
(d, J = 14.0 Hz, 1H), 6.58 (s, 2H), 6.35 (d, J = 16.0 Hz, 1H), 6.08 (s, 1H), 3.81 (s, 9H), 3.35 (q, J = 6.0 Hz,
2H), 1.60–1.23 (m, 4H), 0.90 (t, J = 7.1 Hz, 3H), 13C NMR (50 MHz, CDCl3) δC 166.0, 153.4, 140.6, 139.4,
130.6, 120.4, 104.9, 61.0, 56.1, 39.6, 31.8, 20.2, 13.8, IR
1614, 1579 [61].
ν
max (KBr, cm−1) 3290, 3005, 2957, 2932, 2841, 1653,
4.4.2. (E)-N-Benzyl-3-(3,4,5-trimethoxyphenyl)acrylamide (15)
◦
Yied 99.7%, white solid, MM: 327.15 g/mol, m.p.: 180–181 C;1H NMR (200 MHz, CDCl3) δH 7.55
(d, J = 16.0 Hz, 1H), 7.27 (s, 5H), 6.67 (s, 2H), 6.45 (d, J = 16.0 Hz, 1H), 4.51 (d, 2H), 3.83 (s, 3H), 3.78

Molecules 2018, 23, 1382
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(s, 6H), 13C NMR (50 MHz, CDCl3) δC 166.0, 153.3, 141.0, 139.3, 138.2, 130.4, 128.6, 127.7, 127.4, 120.2,
104.8, 60.9, 56.0, 43.7, IR νmax (KBr, cm−1) 3269, 2990, 2957, 2934, 2835,1653, 1620, 1582, 1505, 820 [62].
4.4.3. (E)-N-(4-Methylbenzyl)-3-(3,4,5-trimethoxyphenyl)acrylamide (16)
◦
Yield 97%, white solid, MM: 341.16 g/mol, m.p.: 203–205 C; 1H NMR (200 MHz, CDCl3) δH 7.55
(d, J = 16.0 Hz, 1H), 7.15 (q, J = 8.0 Hz, 4H), 6.68 (s, 2H), 6.37 (d, J = 16.0 Hz, 1H), 6.24 (m, 1H), 4.48 (d,
J = 6.0 Hz, 2H), 2.31 (s, 3H), 13C NMR (50 MHz, CDCl3) δC 165.9, 153.4, 141.2, 139.5, 137.3, 135.2, 130.5,
129.4, 127.9, 120.1, 104.9, 61.0, 56.2, 43.7, 21.2, IR ν
max (KBr, cm−1) 3292, 3001, 2934, 2837, 1653, 1607,
1580, 1504, 831 LS-MALDI TOF/TOF m/z [M + H]+ 342.1679 (calcd. for C20H23NO4, 342.1706).
4.4.4. (E)-N-(4-Methoxybenzyl)-3-(3,4,5-trimethoxyphenyl)acrylamide (17)
◦
Yield 71%, White solid, MM: 357.16 g/mol, m.p.: 225–226 C; 1H NMR (200 MHz, CDCl3) δH 7.54
(d, J = 16.0 Hz, 1H), 7.28–7.16 (m, 2H), 6.88–6.79 (m, 2H), 6.68 (s, 2H), 6.34 (d, J = 14.0 Hz, 1H), 4.46 (d,
J = 4.0 Hz, 2H), 3.85 (s, 3H), 3.83 (s, 6H), 3.78 (s, 3H), 13C NMR (50 MHz, CDCl3) δC 165.9, 159.1, 153.4,
141.2, 139.6, 130.5, 130.3, 129.3, 120.0, 114.2, 105.0, 61.0, 56.1, 55.3, 43.4, IR
2957, 2934, 2837, 1653, 1618, 1581, 1508, 829 [63].
ν
(KBr, cm−1) 3318, 2990,
max
4.4.5. (E)-N-(4-Chlorobenzyl)-3-(3,4,5-trimethoxyphenyl)acrylamide (18)
Yield 91.2%, White solid, MM: 361.11 g/mol, m.p.: 215–218 ◦C; 1H NMR (200 MHz, CDCl3) δH
7.56 (d, J = 16.0 Hz, 1H), 7.40–7.11 (m, 4H), 6.70 (s, 2H), 6.38 (d, J = 16.0 Hz, 2H), 4.50 (d, J = 6.0 Hz, 2H),
3.85 (s, 3H), 3.83 (s, 6H), 13C NMR (50 MHz, CDCl3) δC 166.0, 153.4, 141.5, 139.9, 136.9, 133.3, 130.4,
129.2, 128.9, 119.8, 105.0, 61.0, 56.1, 43.1, IR
1582, 1506, 825 [64].
ν
max (KBr, cm−1) 3294, 3044, 2959, 2932, 2837, 1651, 1616,
4.4.6. (E)-N,N-Diethyl-3-(3,4,5-trimethoxyphenyl)acrylamide (19)
◦
Yield 85%, white solid, MM: 293.16 g/mol, m.p.: 197–198 C; 1H NMR (200 MHz, CDCl3) δH 7.58
(d, J = 14.0 Hz, 1H), 6.67 (d, J = 16.0 Hz, 3H), 3.86 (s, 6H), 3.83 (s, 3H) 3.46 (q, J = 8.0 Hz, 4H), 1.31–1.11
(m, 6H), 13C NMR (50 MHz, CDCl3) δC 165.7, 153.0, 142.5, 139.5, 131.1, 117.0, 105.0, 61.0, 56.2, 42.4, 41.1,
15.1, 13.3, IR νmax (KBr, cm−1) 2995, 2968, 2938, 2837, 1649, 1597, 1506, 856 [65].
4.5. Cell Cultures and Conditions
Human glioblastoma (GBM) cell line U87MG (American Type Culture Collection; Rockville, MD,
USA) within passage number 15–20 were grown in an incubator at 37 ◦C in a humidified atmosphere
of 5% CO2. They were grown in Eagle’s Minimal Essential Medium from Sigma-Aldrich (Saint Louis,
MO, USA), supplemented with 10% fetal bovine serum (FBS) and 100
antibiotics: penicillin and streptomycin [66].
µg/mL of each of the following
4.6. In Vitro Evaluation of Anticancer Activity by MTT and LDH Assays
4.6.1. MTT Assay
To examine the effects of compounds on cell viability, an MTT assay was performed. U87MG cells
were seeded at 1
104 cells per well in 96-well plate and allowed to attach for 24 h. Then the cells
were exposed to different concentrations of 19 (0.78, 1.56, 3.125, 6.25, 12.5, 25 and 50 g/mL) for
×
2–
µ
48 h. All the compounds were dissolved in dimethyl sulfoxide (DMSO), and diluted in serum-free
culture medium. After the incubation, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) solution was applied to cell cultures according to the manufacturer’s instructions (Cayman
Chemical Company®, Ann Arbor, MI, USA). After 3 h of incubation, MTT-formazan crystals were
dissolved in DMSO and absorbance was measured at 570 nm on a plate reader and compared with
control, untreated cells. The half-maximal inhibitory concentration values (IC50) were determined by
MTT assay performed in four replicates.

Molecules 2018, 23, 1382
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4.6.2. LDH Assay
Cellular viability was assessed by the assay of LDH leakage from the cell [67]. A LDH cytotoxicity
assay kit purchased from Cayman Chemical Company® (Ann Arbor, MI, USA) was used following
instructions provided by the manufacturer. Briefly, the cells were seeded at 106 cells/well in 48-well
plates, treated with compound 10 as described above for 48 h. At the end of incubation 100
µL of
culture medium was collected and transferred to a fresh 48-well plate. 100 L of reaction mixture
µ
was added to each well and incubated for 30 min at room temperature. Finally, the absorbance of the
cultures was measured at 490 nm using a microplate reader [68].
4.7. Total Antioxidant Capacity and Total Oxidant Status Assays
The commercially available kits were purchased from (Rel Assay Diagnostics®, Gaziantep, Turkey)
to estimate the level of TAS and TOS. As a summary, the cells were seeded at 106 cells/well in 48-well
plates, treated with compound 10 as described above. After 48 h incubation, plasma samples of treated
and untreated cultures were transferred to a new plate, mixed with reaction solution and incubate for
10 min at room temperature. After that, the absorbance was read spectrophotometrically and the level
of TAS and TOS were calculated according to the given equation.
4.8. Apoptosis Detection by Hoechst 33,258 Staining
The control and formulation treated cells were incubated for 48 h and were cultured in six-well
cell culture plates for 48 h. after incubation, the medium was removed and the cells were fixed with
4% paraformaldehyde in phosphate buffered saline at 4 ◦C for 30 min. Following the washing cells
three times with phosphate-buffered saline (PBS). The cells were then stained with 1
µL Hoechst 33,258
(5 g/mL; Sigma-Aldrich, Saint Louis, MO, USA) and incubated at room temperature in the dark for
µ
5 min. Changes in nuclear morphology were visualized under a fluorescent microscope (excitation,
350 nm; emission, 460 nm; Leica® DM IL LED, Wetzlar, Germany).
4.9. RNA Extraction and Quantitative Reverse-Transcription Polymerase Chain Reaction Analyses
5
×
106 cells were incubated with IC50 concentrations of compound 10 in 6-well plate in
2 mL growth medium for 48 h. Total RNA was isolated from cultured cells using PureLink®
RNA Mini Kit (Invitrogen, Carlsbad, MA, USA) following the instructions described by the
manufacturer. For quantitative reverse-transcription polymerase chain reaction (qRT-PCR), RNA
was reverse-transcribed to cDNA by using a High-Capacity cDNA Reverse Transcription kit (Applied
Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. Real-time PCR analyses
were performed using the resulting total cDNA on a TaqMan® custom plate (Applied Biosystems)
designed with 15 different genes (EGFR, AKT1, AKT2, NFκB1, NFκB1A, NFκB2, PTEN, KRAS, PIK3CA,
PIK3R1, TP53, RAF1, BRAF, DVL1, FOS). Results were normalized to the expression of 18SRNA.
qRT-PCR and data collection were performed on 7500 Fast Real-Time PCR (Applied Biosystems). Data
analyses were performed using the The Applied Biosystems 7500 Fast System SDS software (Foster
City, CA, USA).
4.10. Biosafety Evaluation
Human peripheral blood cells were cultured in order to evaluate the biosafety of 10. To assess
the cytotoxicity and genotoxicity potential of 10, MTT, LDH, Sister chromatid exchange (SCE) and
8-hydroxy-20-deoxyguanosine assays were assessed on cultured blood cells [69,70].
4.11. SCE Testing
◦
Peripheral blood samples were incubated with 10 (50
µ
g/mL) at 37 C for 72 h in culture medium
(PB-MAX Karyotyping Medium, Gibco®, Baecelona, Spain) with 5.0 mg/mL of phytohemagglutinin
(Sigma Aldrich, Steinheim, Germany). 5-Bromodeoxyuridine (BrdU) (10 g/mL) was added to cell
µ

Molecules 2018, 23, 1382
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cultures at initiation. At 1.5 h before harvesting, colcemid (0.5
µg/mL) were added to cells. Then
cells were treated with a hypotonic solution (0.075 M KCl), followed by three times fixation step in
methanol/acetic acid (3:1, v/v). Cell suspensions were dropped on slides and air-dried. Three days
later, the slides were stained according to the fluorescence plus Giemsa (FPG) procedure. Slides were
observed under a fluorescence microscope and scores were calculated as SCEs per cell.
4.12. Nucleic Acid Oxidation
To detect the level of 8OHdG released into the extracellular space, in accordance with the
provider’s manual, a 8-Hydroxy-20-deoxyguanosine assay kit (Cayman chemical, Ann Arbor, MI,
USA) was used and performed in accordance with the provider’s manual.
4.13. Statistical Analysis
All experiments with U87MGs were performed in four independent experiments and the results
were expressed as the mean
±
standard error of the mean (SEM). Statistical analysis of data obtained
from cell culture experiments was performed using to the statistical program SPSS software (version
20.0, SPSS, Chicago, IL, USA). One-way analysis of variance (ANOVA) followed by Duncan’s test
was used for statistical comparisons of quantitative data. A p-value less than 0.05 was regarded
as significant.
5. Conclusions
The assessment of the anticancer potential of piplartine analogues (2–19) has shown that changes
in trimethoxycinnamic structure may reveal novel drug candidate compounds, such as compound 10
,
which executed strong cytotoxic actions against the proliferation of U87MG cells. Compound 10 had a
marked concentration-dependent inhibitory effect on the viability of brain cancer cells. Furthermore,
in the mechanisms of cytotoxic activity of 10, apoptotic and oxidative processes were involved and
showed good potential for altering other main molecular pathways (such as Akt and NF-κB signaling)
and preventing tumor development. Moreover, 3-BTA exhibited non-genotoxic and non-cytotoxic
properties on healthy human cells. The obtained results are the first showing the potential antitumor
activity of the compound 10 in brain human cancer cells and giving directions for future investigations
concerning a detailed assessment of pharmacological action and safe formulations.
Supplementary Materials: Supplementary materials are available on line.
Author Contributions: F.R.d.N. and O.O. performed the research, analyzed the data and wrote the paper.
S.C.S.N.S. and J.N.B. helped in writing the paper. H.T. and D.P.d.S. designed the research and appraisal of
the paper.
Funding: This research received no external funding.
Acknowledgments: This work was supported by the Brazilian agencies: Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
Conflicts of Interest: The authors declare that there are no competing interests regarding the publication of
this paper.
Abbreviations
4-OH-E2
8-OH-dG
18SRNA
AChE
4-Hydroxy-Estradiol
8-Hydroxy-2 -deoxyguanosine
18S ribosomal Ribonucleic Acid
8-OH-dG-Acetylcholinesterase
Adenosine triphosphate
0
ATP
AKT1
AKT2
ANOVA
BpV-pic
Other denomination of protein kinase B (PKB) type 1
Other denomination of protein kinase B (PKB) type 2
Analysis of variance
Bisperoxovanadium-pic

Molecules 2018, 23, 1382
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BRAF
3-BTA
CT
B-Raf proto-Oncogene, Serine/Threonine Kinase
(E)-benzhydryl 3-(3,4,5-trimethoxyphenyl)acrylate
Cycle Threshold
DMSO
cDNA
DVL1
EGF
EGFR
FBS
Dimethyl sulfoxide
Complementary Deoxyribonucleic Acid
Dishevelled type 1
Epidermal Growth Factor
Epidermal Growth Factor Receptor
Fetal Bovine Serum
FPG
ER
Fluorescence Plus Giemsa
Endoplasmic Reticulum
FOS
Finkel Osteosarcoma
GBM
HGG
HHV
IC50
IGF-1
KRAS
LDH
MMC
MTT
NC
Glioblastoma
High-Grade Glioma
Human Herpes virus
The half maximal inhibitory concentration
Insulin-like Growth Factor-1
Kirsten Rat Sarcoma viral oncogene homolog
Lactate Dehydrogenase
Mitomycin C
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Negative Control
NFkB
NFkB1
NFkB1A
NFkB2
PBS
Necrosis Factor kappa B
Necrosis Factor kappa B1
Necrosis Factor kappa B inhibitor-α
Necrosis Factor kappa B2
Phosphate-buffered saline
Positive control
PC
PCR
PHWB
PPL
Polymerase Chain Reaction
Peripheral Human Whole Blood
Piplartine
PTEN
PIP3K
PIK3CA
PIK3R1
PRDX4
RAF1
ROS
Phosphatase and Tensin Homolog
Phosphatidylinositol-4,5-bisphosphate 3-kinase
Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic
Phosphatidylinositol-3-kinase regulatory subunit 1
Peroxiredoxin 4
Rapidly Accelerated Fibrosarcoma type 1
Reactive Oxygen Species
RT
Radiotherapy
RT-PCR
SAR
SCE
SH-SY5Y
SV40
TAC
Real Time-Polymerase Chain Reaction
Structure Activity Relationship
Sister Chromatid Exchange
Human Neuroblastoma cell model
Simian Vacuolating virus 40
Total Antioxidant Capacity
Total Antioxidant Status
TAS
TMZ
TOS
Temozolomide
Total Oxidant Status
TP53
Tumor Protein p53
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Sample Availability: Samples of the compounds not are available from the authors.
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