Synthesis, molecular modelling and anticancer activities of new molecular hybrids containing 1,4-naphthoquinone, 7-chloroquinoline, 1,3,5-triazine and morpholine cores as PI3K and AMPK inhibitors in the metastatic melanoma cells

Article abstract of DOI:10.21577/0103-5053.20190096

Three molecular hybrids containing 1,4-naphthoquinones, 1,3,5-triazines, morpholine and 7-chloroquinoline, which have recognized contributions to the biological activity of many drugs, were synthesized in yields ranging from 43-84percent. All hybrids were obtained in three steps starting from readily available reactants: lawsone, cyanuric chloride, morpholine and 4,7-dichloroquinoline. A previous docking study was carried out to identify the binding energy and pharmacophore conformation of the promising anticancer compounds with PI3Kγ (phosphoinositide 3-kinase) and AMPK (5′ AMP-activated protein kinase). The cancer activity in human metastatic melanoma cells (SKMEL-103) were performed, and the synthetized compounds presented half maximal inhibitory concentration (IC₅₀) values around 25 μM. The expressions of PI3K and AMPK were also determined using western blotting technique, and all molecular hybrids negatively modulated both targets.

Full text of DOI:10.21577/0103-5053.20190096

http://dx.doi.org/10.21577/0103-5053.20190096
J. Braz. Chem. Soc., Vol. 00, No. 00, 1-14, 2019
Article
Printed in Brazil - ©2019 Sociedade Brasileira de Química
Synthesis, Molecular Modelling and Anticancer Activities of New Molecular
Hybrids Containing 1,4-Naphthoquinone, 7-Chloroquinoline, 1,3,5-Triazine and
Morpholine Cores as PI3K and AMPK Inhibitors in the Metastatic Melanoma Cells
,
a
b
b
b
Rodolfo G. Fiorot, * Regina Westphal, Bárbara C. Lemos, Rodrigo A. Romagna,
c
d
d
e
Paola R. Gonçalves, Maruska R. N. Fernandes, Carmen V. Ferreira, Alex G. Taranto
,
b
and Sandro J. Greco *
a
Instituto Federal de Ciências, Educação e Tecnologia do Rio de Janeiro,
2
1710-240 Rio de Janeiro-RJ, Brazil
b
Laboratório de Síntese Orgânica e Aplicada, Departamento de Química,
Universidade Federal do Espírito Santo, 29075-910 Vitória-ES, Brazil
c
Departamento de Ciências da Saúde, Centro Universitário Norte do Espírito Santo (CEUNES),
Universidade Federal do Espírito Santo, 29932-540 São Mateus-ES, Brazil
d
Departamento de Bioquímica e Biologia Tecidual, Instituto de Biologia,
Universidade Estadual de Campinas, 13083-970 Campinas-SP, Brazil
e
Laboratório de Química Farmacêutica, Universidade Federal de São João del Rei,
Campus Centro Oeste, 35501-296 Divinópolis-MG, Brazil
Three molecular hybrids containing 1,4-naphthoquinones, 1,3,5-triazines, morpholine and
7
-chloroquinoline, which have recognized contributions to the biological activity of many drugs,
were synthesized in yields ranging from 43-84%. All hybrids were obtained in three steps starting
from readily available reactants: lawsone, cyanuric chloride, morpholine and 4,7-dichloroquinoline.
A previous docking study was carried out to identify the binding energy and pharmacophore
conformation of the promising anticancer compounds with PI3Kγ (phosphoinositide 3-kinase)
andAMPK (5’AMP-activated protein kinase). The cancer activity in human metastatic melanoma
cells (SKMEL-103) were performed, and the synthetized compounds presented half maximal
inhibitory concentration (IC ) values around 25 μM. The expressions of PI3K and AMPK were
5
0
also determined using western blotting technique, and all molecular hybrids negatively modulated
both targets.
Keywords: molecular hybridization, anticancer activity, 1,4-naphthoquinone, 1,3,5-triazine,
7
-chloroquinoline
Introduction
simultaneously, co-formulation of two or more active agents
in a single tablet and development of hybrid molecular
entities capable to modulate multiple targets are the three
popular approaches for multidrug therapy. Hence, the
present form of polypharmacology is gaining popularity in
Continuous studies are essential to understand the
mechanisms of cancer evolution and to provide more efficient
and selective ways to eradicate it. Cancer is one of the
prominent causes of morbidity and mortality worldwide; it is
the second leading cause of death globally, thus, development
2
the form of hybrid molecules (multiple ligand approach).
Molecular hybridization (MH) is a strategy of rational
design of new prototypes, where the lead compound is
produced through an adequate fusion of pharmacophoric
sub-unities. MH is performed to achieve one of the
following objectives: (i) synergism of pharmacological
action, (ii) dual action therapy pharmacological or
1
of new drugs more selective and potent is necessary.
The polypharmacology is not a new concept and the
approaches adopted for administering the two or more
drugs together are varied. Taking two or more drugs
3
*e-mail: rodolfo.fiorot@ifrj.edu.br; grecosj@outlook.com
(iii) modulation of undesirable side effects.

2
Synthesis, Molecular Modelling and Anticancer Activities of New Molecular Hybrids
J. Braz. Chem. Soc.
The synergistic action hybrid is obtained when
(for example, PI3K and mammalian target of rapamycin
3
2-35
pharmacophoric groups or whole drugs are covalently
linked. Both should have the same pharmacological
action, but should act on different biochemical targets. The
final objective of the hybrid will be to increase a single
(mTOR) inhibitor),
it is responsible to enhance the
solubility of 1,3,5-triazine intermediates, making it feasible
to proceed to the next steps in a synthetic route, that is,
36
characterization and biological evaluation.
4
pharmacological activity by different pathways.
Drug design and discovery has been a challenge task
that could demand a huge amount of time, reactants and
human resource. To overcome these issues, computational
chemistry provides helpful tools by gathering detailed
information regarding the mechanism of action in a
cheap and fast way. One of the most popular technique is
molecular docking due its ability to model the interaction
between drug candidates and their biomacromolecular
Recently, one of the most frequently used cancer
treatment approaches is to inhibit enzymes involved in the
5
,6
signaling pathway. This is a more selective and efficient
way to stop cancer development because cancerous cells
have signaling pathways that are overexpressed compared
7
,8
to healthy ones. PI3K (phosphoinositide 3-kinase) and
AMPK (5’AMP-activated protein kinase) kinases, which
are key players in autophagy, are highlighted as important
targets of cancer chemotherapy once they are strongly
37,38
targets. Several studies apply this technique to propose
a rational drug design and its probable mechanism of
9-12
39-41
associated to the cell reproduction. One example of a
cancer cell line with those overexpressed kinases is the
human metastatic melanoma (SKMEL), which is the most
aggressive skin cancer form. Pharmacological studies
action.
Based on the molecular hybridization concept and by
the participation of the cited groups in the suppression of
signaling pathways directly linked with carcinogenesis
(e.g., PI3K and AMPK cascades), herein we decided to
rationalize new molecules containing 1,4-naphthoquinone,
1,3,5-triazine and quinoline frameworks. Figure 1 depicts
the target molecular hybrids (1-3) designed for this
study. For 1,3,5-triazinic derivatives, the solubility of
1
3
14
reveal that PI3K andAMPK signaling cascades directly
impact different phenotypes of melanomas in its initiation,
15
progression, drug resistance and invasive capabilities.
The incidence of human metastatic melanoma is
increasing and unfortunately presents low survival rates.
Its treatment still poses a challenge, since currently
disubstituted intermediates is increased when it is attached
1
6
29,36
available therapies present poor efficacy. Therefore,
novel compounds that manage to decrease the viability of
melanoma cells are needed. In this context, one strategy
is to inhibit the autophagy process, which is linked to
to polar alicyclic groups.
Therefore, the morpholine
ring was chosen for this purpose and due to its contribution
to biological activity in substances with anticancer
3
4,35
activity and inhibition of PI3K.
1,3-Diaminopropane
17
overexpressed signaling pathways.
,4-Naphthoquinones, quinolines, 1,3,5-triazines and
was selected to connect the frameworks. 2-Hydroxy-
1,4-naphthoquinone, 2,4,6-trichloro-1,3,5-triazine and
4,7-dichloroquinoline were selected as starting materials
and are commercially available. Previously, molecular
docking was simulated with PI3K and AMPK (Protein
1
morpholine were selected by our research group to design
new hybrids with potential anticancer activity. These
frameworks are present in drugs with recognized anticancer
activity and different mechanisms, including cytotoxic
and inhibitors of enzymes of signaling pathways. The
42
43
Data Bank (PDB) codes 1E7U and 2UV4, respectively)
targets to verify its potential inhibitor character. Finally,
we tested their cancer activity in human metastatic
melanoma cells (SKMEL-103) and modulation of PI3K
and AMPK expression.
1
,4-naphoquinones derivatives are widely studied due to
their cytotoxic activity (e.g., inhibition of topoisomerase I
1
8,19
20
and II,
of p53 and Cdc25 phosphatase), besides the
2
1
inhibition of PI3K. Compounds containing the quinoline
nucleus also demonstrate activity inhibitory of protein
kinases, such as PI3K, AMPK and the growth factor
Experimental
22
23
2
4
receptors (GFR) in addition to cytomechanisms like
General methods
2
5
deoxyribonucleic acid (DNA) intercalant, antimitotic
2
6
27
28
agents, inhibition of topoisomerases and telomerases.
All solvents and starting materials were commercially
available and used without any previous treatment.
Melting points were determined using Fisatom 430D
equipment. Infrared (IR) spectra were recorded on Bomem
FTLA2000-102-ABB spectrometer. For the analysis of
On the other hand, 1,3,5-triazine derivatives, in turn, exhibit
different targets in cancer chemotherapy, for example,
2
9
30
31
the inhibition of PI3K, topoisomerase and Hsp90.
Finally, morpholine group was chosen to compose the
final set of molecular hybrids for two important reasons:
besides its well-known contribution to anticancer activity
1
13
H nuclear magnetic resonance (NMR) and C NMR, the
spectra were acquired on a Vary VNMRS spectrometer

Vol. 00, No. 00, 2019
Fiorot et al.
3
Figure 1. Molecular hybrids containing 1,4-naphthoquinone, 1,3,5-triazine, morpholine and quinoline cores.
1
model 400 (400 MHz) with tetramethylsilane (TMS) as
internal standard. NMR analysis of compounds 7 and 10
were performed in chloroform deuterated (99.8%) with
Preparation of N -(7-chloroquinolin-4-yl)propane-
1,3-diamine (9)
A suspension of 4,7-dichloroquinoline 5 (2.0 g,
10.1 mmol) in 4.2 mL of 1,3-diaminopropane was prepared
heating the round bottom flask until 80 °C for 1 h with no
stirring.After that, the solution was stirred for 4 h at 120 °C
until full consumption of 4,7-dichloroquinoline, 5. At the
end of the reaction, the brute material was transferred to
a beaker and 20 mL of cold water with crushed ice was
added. The precipitated was filtered and washed with 20 mL
of cold water. In the following step, the solid was stirred
with 15 mL of ethyl acetate and filtered under vacuum,
furnishing a white solid in 87%; mp 146-148 °C (lit.:
1% (v/v) of TMS and stabilized with silver foil (Cambridge
Isotope Laboratories, Inc., Tewksbury, USA). For
compounds 1, 2, 3 and 9, dimethyl sulfoxide (DMSO-d6,
Sigma-Aldrich, 99.9%, São Paulo, Brazil) was used and
for compound 8, a mixture 1:1 DMSO-d :MeOD was used
6
(
Aldrich 99.8%, São Paulo, Brazil).
Mass spectra were recorded on a Bruker Daltonics
model 9.4 T Solarix ultra-high resolution mass spectrometer
HRMS) operated in the negative and positive ionisation
(
modes over a mass range of m/z 200-2000 using Fourier
transform ion cyclotron resonance mass spectrometry
-1
145-147 °C); IR (ATR) ν / cm 3264, 2936, 2865, 1586,
1541, 1478, 1316, 1221, 1080, 800; H NMR (400 MHz,
1
(
ESI-FT-ICR MS). ESI-FT-ICR MS spectra were acquired
with a resolving power of 50% ca. 500000, in which 50%
is the full peak width at the half-maximum peak height of
m/z 400 and a mass accuracy of < 1 ppm, which provides
an unambiguous molecular formula.
DMSO-d 1:1 MeOD) d 8.40 (d, J 5.4 Hz, 1H), 8.18 (d,
6
J 9.0 Hz, 1H), 7.80 (d, J 2.2 Hz, 1H), 7.46 (dd, J 9.0, 2.2 Hz,
1H), 6.59 (d, J 5.4 Hz, 1H), 3.44 (t, J 7.0 Hz, 2H), 2.81
13
(t, J 7.0 Hz, 2H), 1.95-1.86 (m, 2H); C NMR (101 MHz,
All the reactions were monitored by TLC (thin
layer chromatography) using precoated silica gel aluminum
plates containing a fluorescent indicator UV254 (Merck,
São Paulo, Brazil).
DMSO-d ) d 152.3, 150.5, 149.4, 133.7, 127.9, 124.4,
124.4, 117.91, 99.0, 30.9.
6
Preparation of molecular hybrid 2-((3-((7-chloroquinolin-
4-yl)amino)propyl)amino)-naphthalene-1,4-dione (1)
Synthesis
Aminoquinoline 9 (0.2360 g, 1 mmol) was solubilized
in 20 mL of methanol at 70 °C with magnetic stirring. The
temperature was reduced to 40 °C and 2-methoxylawsone 8
(0.1840 g, 1 mmol) was added with 140 µL of triethylamine.
After completion of reaction (48 h), the solid was filtered
and washed under vacuum with cold methanol. The product
was obtained as an orange solid in 84 %; mp 210-212 °C; IR
Preparation of 2-methoxylawsone (8)
A suspension of lawsone 4 (1.0 g, 5.7 mmol) in 50 mL
of absolute methanol with concentrated chloridric acid
(
0.8 mL) was stirred at reflux for 4 h. Then, the solution
was allowed to obtain 8 as a palid yellow solid in 90%;
mp 179-180 °C (lit.: 182-183 °C); IR (ATR) ν / cm 3050,
-1
-1
(ATR) ν / cm 3371, 3066, 2916, 2865, 1676, 1614, 1584,
1
1
2
991, 1680, 1643, 1603, 1214, 1044; H NMR (400 MHz,
1279, 1118, 800, 761, 726; H NMR (400 MHz, DMSO-d )
6
CDCl ) d 8.16-8.07 (m, 2H), 7.76 (m, 2H), 6.19 (s, 1H),
d 8.33 (d, J 5.4 Hz, 1H), 8.20 (d, J 9.0 Hz, 1H), 7.93 (dd,
J 7.6, 0.9 Hz, 1H), 7.89 (dd, J 7.6, 0.9 Hz, 1H), 7.78 (td,
3
3
.92 (s, 3H, OCH3).

4
Synthesis, Molecular Modelling and Anticancer Activities of New Molecular Hybrids
J. Braz. Chem. Soc.
J 7.5, 1.3 Hz, 1H), 7.73 (d, J 2.2 Hz, 1H), 7.68 (td, J 7.5,
(0.6 mmol) was added in a round bottom flask and
magnetically stirred for 24 h at room temperature. An
aliquot of 15 mL of distilled water was then slowly added,
furnishing the pure product as an orange solid in 61%; mp
1
1
1
.3 Hz, 1H), 7.61 (t, J 6.1 Hz, 1H), 7.40 (dd, J 9.0, 2.2 Hz,
H), 7.29 (t, J 5.3 Hz, 1H), 6.47 (d, J 5.5 Hz, 1H), 5.70 (s,
13
H), 1.94 (q, J 6.7 Hz, 2H); C NMR (101 MHz, DMSO-d )
6
-1
d 181.9, 181.7, 152.2, 150.4, 149.4, 148.9, 135.2, 133.8,
124-126 °C (lit.: 124-126 °C); IR (ATR) ν / cm 3355,
1
1
1
33.5, 132.5, 130.8, 127.8, 126.3, 125.7, 125.4, 124.4,
24.4, 117.8, 99.8, 99.1, 26.3.
2933, 1682, 1670, 1642, 1566, 1243; H NMR (400 MHz,
DMSO-d ) d 8.08 (dd, J 7.6, 0.9 Hz, 1H), 8.02 (dd, J 7.6,
6
0
.9 Hz, 1H), 7.70 (td, J 7.6, 1.3 Hz, 1H), 7.59 (td, J 7.6,
Preparation of 4,4-(6-chloro-1,3,5-triazine-2,4-diyl)
dimorpholine (10)
1.3 Hz, 1H), 6.83 (br s, 1H), 3.27 (dd, J 12.3, 6.4 Hz, 2H),
2.89 (t, J 6.4 Hz, 2H), 1.81 (q, J 6.4 Hz, 2H).
Cyanuric chloride 6 (9.22 g, 50 mmol), 50 mL of
acetone, crushed ice (250 g), triethylamine (15 g, 20 mL)
and morpholine 7 (8.70 g, 100 mmol) was added in a round
bottom flask under magnetic stirring at room temperature.
After 2 h, 100 mL of distilled water at room temperature
was added and the solid was filtered under vacuum and
washed with cold distilled water. The product was obtained
as a white solid in 93%; mp 171-172 °C (lit.: 175-176 °C);
Preparation of molecular hybrid 2-((3-((4,6-dimorpholine-
1,3,5-triazin-2-yl)amino)propyl)amino)naph-thalene-
1,4-dione (3)
The aminonaphthoquinone 11 (0.1150 g, 0.5 mmol)
was solubilized in 20 mL of acetonitrile under magnetic
stirring and at reflux in a round bottom flask. The
intermediate 10 (0.1430 g, 0.5 mmol) and 70 μL of
triethylamine was then added. After 16 h of reflux, the
content was filtered under vacuum. The brute product was
recrystallized in hot ethanol to furnish the pure product as
-1
IR (ATR) ν / cm 2852, 1566, 1486, 1448, 1243, 1113,
1
9
3
1
78, 797; H NMR (400 MHz, DMSO-d ) d 3.62 (m, 4H),
6
1
3
.56 (m, 4H); C NMR (101 MHz, DMSO-d ) d 169.1,
64.2, 66.2, 43.9.
6
-
1
a yellow solid in 47%; mp 210-212 °C; IR (ATR) ν / cm
3
337, 2979, 2855, 1679, 1606, 1547, 1446, 1252, 1106,
1
1
Preparation of molecular hybrid N -(7-chloroquinolin-
1006, 803; H NMR (400 MHz, DMSO-d ) d 7.97 (dd,
6
3
4
1
-yl)-N -(4,6-dimorpholine-1,3,5-triazin-2-yl)propane-
,3-diamine (2)
Aminoquinoline 9 (0.1180 g, 0.5 mmol) was solubilized
J 7.4, 6.6 Hz, 1H), 7.94 (m, 1H), 7.82 (td, J 7.4, 1.2 Hz,
1H), 7.72 (td, J 7.4, 1.2 Hz, 1H), 7.39 (br s, 1H), 6.73
(s, 1H), 5.65 (s, 1H), 3.57 (m, 8H), 3.53 (br s, 8H), 3.23
1
3
in a round bottom flask with 20 mL of acetonitrile under
(m, 4H), 1.81 (m, 2H); C NMR (101 MHz, DMSO-d )
6
reflux and magnetic stirring. The intermediate 10 (0.1430 g,
d 181.5, 181.1, 165.7, 164.7, 148.4, 134.8, 133.2, 132.1,
0
1
.5 mmol) and triethylamine 70 μL was then added. After
6 h, the solid was filtered under vacuum and washed
130.3, 125.8, 125.3, 99.2, 65.9, 43.1, 37.6, 27.4; HRMS
+
(ESI (+) FT-ICR MS) m/z, calcd. for C H N O [M + H] :
24
30
7
4
with cold distilled water, furnishing a white solid which
was purified with chromatographic column (silica gel
as stationary phase and 10% MeOH/AcOEt as eluent).
The pure product was obtained as a white solid in 43%;
480.23538, found: 480.23504 (error = 0.71 ppm);
+
calcd. for [C H N O + Na] : 502.21732, found:
2
4
29
7
4
502.21705 (error = 0.54 ppm); dimer calcd. for
+
C H Cl N O [2M + H] : 959.46348, found: 959.46281
4
8
59
2
14
8
-1
+
mp 230-231 °C; IR (ATR) ν / cm 3360, 2965, 2862, 1546,
(error = 0.70 ppm); calcd. for [C H N O + Na] :
48 58 14 8
1
1
477, 1446, 1259, 1259, 1108, 808; H NMR (400 MHz,
981.44543, found: 981.44450 (error = 0.95 ppm).
DMSO-d ) d 8.33 (d, J 5.4 Hz, 1H), 8.21 (d, J 9.1 Hz, 1H),
6
7
.74 (d, J 2.3 Hz, 1H), 7.41 (dd, J 9.1, 2.3 Hz, 1H), 7.29 (t,
J 5.5 Hz, 1H), 6.85 (t, J 5.9 Hz, 1H), 6.41 (d, J 5.5 Hz, 1H),
.54 (m, 4H), 3.44 (m, 4H), 3.27 (m, 4H), 1.82 (m, 2H);
HRMS (ESI (+) FT-ICR MS) m/z, calcd. for C H ClN O
Anticancer activity
3
Reagents
Compounds tested were obtained as described below.
Reagents for western blotting were purchased from Bio-
Rad (Hercules, CA, USA) and Sigma-Aldrich (St. Louis,
MO, USA). Primary and HRP-conjugated secondary
antibodies were purchased from Cell Signaling Technology
(Beverly, MA, USA), Santa Cruz (St. Louis, MO, USA),
Sigma-Aldrich (St. Louis, MO, USA) and BIOSS (Boston,
MA, USA). MTT (methylthiazolyldiphenyl-tetrazolium
bromide) was purchased from Sigma-Aldrich (St. Louis,
MO, USA).
23
30
8
2
+
[M + H] : 485.21802, found: 485.21749 (error = 1.09 ppm);
+
dimer calcd. for C H Cl N O [2M + H] : 969.42822,
found: 969.42783 (error = 0.40 ppm).
4
6
59
2
16
4
Preparation of 2-((3-aminopropyl)amino)naphthaleno-
1
,4-dione (11)
A suspension of 2-methoxylawsone 8 (0.0941 g,
.5 mmol) in 10 mL of methanol, 1,3-diaminopropane
0
(84 μL, 0.07472 g, 1.0 mmol) and 0.084 mL of triethylamine

Vol. 00, No. 00, 2019
Fiorot et al.
5
Cell culture
cell lysate was cleared by centrifugation and the protein
concentration was determined using Bradford reagent
(Sigma-Aldrich). An equal volume of 2 × sodium dodecyl
sulfate (SDS) gel loading buffer (100 mM Tris-HCl, pH 6.8,
200 mM dithiothreitol (DTT), 4% SDS, 0.1% bromophenol
blue and 20% glycerol) was added and the samples were
boiled for 5 min. The corresponding to 100 μg of protein
was resolved by SDS-polyacrylamide gel (8-12%)
eletrophoresis (PAGE) and transferred to polyvinylidene
difluoride (PVDF) membrane. The membranes were
blocked in bovine serum albumin (5%) in tris-buffered
Melanoma metastatic cells (SKMEL-103) were kindly
donated by Prof Silvya Stuchi Maria Engler from São
Paulo University (Brazil). Cells were routinely grown in
Dulbecco’s modified Eagle’s medium (DMEM) containing
-
1
-1
1
1
00 U mL penicilium, 100 μg mL streptomycin and
0% fetal bovine serum (FBS), in a 5% CO humidified
2
atmosphere, at 37 ºC.
Treatment of melanoma cells
®
saline (TBS)-Tween 20 (0.05%) and incubated overnight at
-
1
SKMEL-103 cells (100 μL of 7 × 104 cells mL
4 °C with appropriate primary antibody at 1:1000 dilution.
®
suspension) were cultured in a 96-wells microplate for
4 h, subsequently the cells were treated with different
After washing in TBS-Tween 20 (0.05%), the membranes
2
were incubated with anti-rabbit, anti-mouse and anti-goat
horseradish peroxide-conjugated secondary antibodies at
1:2000 dilutions for 2 h. The detection was performed by
using enhanced chemiluminescence in ImageQuantTM 350
(GE healthcare Life Sciences).
concentrations of the compounds for 24 h.A stock solution
of compounds was prepared in DMSO and control group
was treated with the equivalent amount of DMSO. Cell
viability was assessed by MTT reduction assay as described
below. Non-treated cell viability was set as 100% and
half maximal inhibitory concentration (IC ) values were
Statistical analysis
50
determined from three independent experiments.
Results are expressed as mean ± standard error of the
mean (SEM). The statistical analyses were performed using
analysis of variance (ANOVA) combined with Tukey’s or
Bonferroni’s tests. p value > 0.05 was considered to be
statistically significant with ***p < 0.001, **p < 0.01 and
*p < 0.05; n.s. was not significant.
MTT reduction assay
Treatment medium was removed and the cells incubated
-1
-1
with 100 µL well of MTT solution (0.5 mg mL , in FBS
free culture medium) for 2 h at 37 ºC. Hereafter, the MTT
solution was removed and the formazan produced was
dissolved with 100 µL of ethanol. Microplate was shaken
for 10 min and the absorbance of formazan was determined
at 570 nm in a microplate reader (Synergy HT, BioTek).
The measured absorbance at 570 nm was normalized
to percentage of control. This value was calculated by
multiplying the absorbance of a treated well by 100 and
dividing it by the average absorbance of control wells.
Results and Discussion
Docking studies
Docking studies were performed initially to check the
interactions between the target molecules and the enzymes,
followed by the chemical studies to synthesize the target
substances.
Western blotting
Crystallographic studies of a series of enzymes
complexes with their ligands have facilitated the rational
design of new inhibitors, and they are available from the
PDB. In our case, the PI3Kγ crystal structure associated
with the wortmannin ligand was obtained under code
-
1
SKMEL-103 (10 mL of 15 × 104 cells mL suspension)
were cultured in a 100 mm plates for 24 h, afterwards the
cells were treated with compounds solutions in 0.05%
DMSO for 24 h. The control group was treated with the
equivalent amount of DMSO.After the treatment, the cells
was removed from plates by using a rubber policeman,
centrifuged and lysed for 2 h on ice in cell lysis buffer
42
1E7U (with resolution of 2.00 Å), and AMPK with ligand
adenosine monophosphate, AMP, was obtained under code
43
2UV4 (resolution of 1.33 Å), crystal structures displayed
in Supplementary Information (SI) section.
(
0
(
1
1
50 mM Tris-HCl, pH 7.4 containing 1% Tween 20,
.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA
ethyleneglycotetraacetic acid), 1 mM o-vanadate,
mM NaF, 1 µg mL aprotinin, 10 μg mL leupeptin and
mM PMSF (phenylmethylsulfonyl fluoride)). Then, the
44
The accuracy of Autodock Vina was evaluated with
respect to pose identification and database enrichment. In
the pose identifications, crystallographic ligands were re-
docked against their respective receptors to evaluate the
-1
-1

6
Synthesis, Molecular Modelling and Anticancer Activities of New Molecular Hybrids
J. Braz. Chem. Soc.
methodology (Figure 2). As a result, the root-mean-square
deviation (RMSD) between docked and crystallographic
structures were 1.53 Å (Figure 2a) and 0.51 Å (Figure 2b)
for wortmannin and AMP, respectively. As can be
observed,AutodockVina could reproduce pharmacophore
conformation into the binding site of both molecular targets,
and the threshold value is 2.0 Å.
site of the receptor, theATP (adenosine triphosphate) cleft,
via the same method, furnishing the calculated interaction
energy ligand/receptor (Table 1). Next, compounds 1, 2
and 3 were docked into the binding site using the same
methodology.
44
44
Table 1. Ligand / receptor interaction energies of compounds against
respective molecular targets
Next, the methodology was evaluated as a database
enrichment. Hence, 300 decoys, false-positive compounds,
were docked for each molecular target obtained from
the “Database of Useful Decoys: Enhanced (DUD-E)”
database. As a result, the total area under receiver
operation characteristic curves (AUC) showed good
enrichment for both receptors (0.7). AUC values of 0.5
and below characterizes a random process and poor
enrichment, respectively. In other words, Autodock Vina
can discriminate false-positive from positive compounds
-1
Interaction energy / (kcal mol )
Compound
PI3Kγ
−8.7
−8.5
−
AMPK
−
Wortmannin
LY294002
−
4
5
AICAR
−6.5
−6.7
−6.2
−7.5
−8.1
GSK621
−
1
2
3
−8.8
−8.3
−9.2
PI3Kγ: phosphoinositide 3-kinase; AMPK: 5’AMP-activated protein
kinase; LY294002: 2-morpholin-4-yl-8-phenylchromen-4-one; AICAR:
-aminoimidazole-4-carboxamide ribonucleotide; GSK621: 6-chloro-
5-(2’-hydroxy-3’-methoxy-[1,1’-biphenyl]-4-yl)-3-(3-methoxyphenyl)-
(
Figures 2c and 3d, suggesting the 1E7U and 2UV4 as
molecular targets).
Sequentially,wortmannin andLY294002(2-morpholin-
5
42
4
2
1,5-dihydro-2H-pyrrolo[3,2-d]pyrimidine-2,4(3H)-dione.
4
-yl-8-phenylchromen-4-one) (control drugs for
PI3Kγ) and AICAR (5-aminoimidazole-4-carboxamide
ribonucleotide) and 6-chloro-5-(2’-hydroxy-3"-methoxy-
46
Table 1 shows that all designed hybrids (1, 2 and 3) had
interaction energies close or better than those exhibited by
the control drugs. Supposedly, these hybrids present the
same potential inhibitory activity of PI3Kγ and AMPK
of the control drugs, or even better, which means that the
[
2
1,1’-biphenyl]-4-yl)-3-(3-methoxyphenyl)-1,5-dihydro-
H-pyrrolo[3,2-d]pyrimidine-2,4(3H)-dione (GSK621)
4
7
(
control drugs forAMPK), after previous optimization with
47
molecular mechanics, were superimposed on the binding
4
4
Figure 2. Evaluation of Autodock Vina methodology (hydrogen atoms were omitted for a better visualization). Crystallographic ligands (yellow) and the
redocked structures (green); (a) wortmannin (PI3Kγ: 1E7U, PDB); (b)AMP (AMPK: 2UV4, PDB); (c) and (d) roc curve for 1E7U and 2UV4, respectively,
both with 0.7 value.

Vol. 00, No. 00, 2019
Fiorot et al.
7
anticancer mechanism of action could be by the inhibition
of these enzymes beyond other mechanisms possible for
the isolated groups already described.
between the enzyme and the ligands as well as the type of
interaction (van derWaals, electrostatic, hydrogen bonding,
among others).
18-35
The compound 1-3 bind strongly to receptors as
inferred by its minimum binding energy value (Table 1).
The best result was obtained to molecular hybrid of 3 (−9.2
Figure 3 depicts the intermolecular interaction
between ligands and 1E7U (PI3Kγ in 2D diagram). As
can be observed, the wortmannin control drug can bind
to PI3Kγ by van der Waals interactions with the amino
Asp950; electrostatic interactions with Met804, Pro810,
Ile831, Ile881, Ile879 and Ile963 amino acid residues; and
hydrogen bonding withVal882 andAsp964 amino acids. In
addition, an unfavorable interaction was carried out with
Lys833, as shown in Figure 3a.
-1
and −8.1 kcal mol ) in PI3Kγ and AMPK, respectively.
However, the other compounds also obtained good binding
energies, demonstrating the promising potential of these
hybrids. The results obtained from the docking studies
permitted us to evaluate the pharmacophoric conformation
of control drugs inside the binding site of the enzymes.
Therefore, it was possible to analyze which amino acids
residues are important to the interactions energy established
The pharmacophoric conformation of LY294002 is
demonstrated in Figure 3b. Van der Waals interactions are
Figure 3. 2D intermolecular interaction diagram among ligands and PI3Kγ. (a) Wortmannin; (b) LY294002; (c) hybrid naphthoquinone-quinoline 1; (d) 2;
e) 3. The distance is in angstroms (Å) and the hydrogens were omitted for a better visualization.
(

8
Synthesis, Molecular Modelling and Anticancer Activities of New Molecular Hybrids
J. Braz. Chem. Soc.
found in the binding site of the enzyme with the amino acids
residues (i.e., Ser806, Glu880, Lys833, Ile881, Val882,
Lys890,Asp950 and Phe961), and electrostatic interactions
were found with Pro810, Ile831, Ile879, Met953 and Ile963
amino acids residues. In addition, a π-sulfur interaction
was observed with Met804. In contrast, an unfavorable
interaction was carried out with Thr887. No hydrogen
bonding was observed in this case.
The control drug GSK demonstrated van der Waals
interactions with Ala202, Ile204, Ala205, Val225 and
Ile312 amino acids, electrostatic interactions with Thr200,
Asn203, His223, Ser314,Asp317 and Gln320amino acids.
Moreover, Ser314 and Gln320 performed two hydrogen
bonding. Finally, it was also found a π-π* stacking with
His223 (Figure 4b).
For the AMPK complex, compound 1 showed
the following interactions (Figure 4c): van der Waals
interactions with Ala205, Thr200, Ser314, Asp317
(centered on aliphatic chain) and electrostatic interactions
with Ile204, Val225 and Ile312 amino acids, principally
with the naphthoquinone core. Hydrogen bonding was
carried out with carbonyl and Ser226.
The hybrid naphthoquinone-quinoline 1 exhibited
-1
values for interaction energy of −8.4 kcal mol ; this was
similar to the control drugs wortmannin and LY294002,
-
1
which exhibited −8.7 and −8.5 kcal mol , respectively,
for PI3Kγ. The interactions for the complex hybrid
1
/PI3Kγ are displayed in Figure 3c, which consist of van
der Waals interactions with Met804, Lys807, Pro810,
Glu880, Lys833, Thr887, Lys890 and Phe961 amino
acids and electrostatic interactions with Ile831, Tyr867,
Ile879, Val882, Met953 and Ile963 amino acids. Asp950
and Asp964 also established hydrogen bonding with
the quinoline nitrogen and naphthoquinone carbonyl,
respectively.
The hybrid-containing morpholine ring gathered with
triazine and the naphthoquinone cores of compound 2 could
form a complex withAMPK by a van der Waals interaction
with His298; electrostatic interactions were apparent with
amino acids Thr200, Ile204 and Ile312 (Figure 4d).
Finally, inside the binding site of AMPK and 3, there
were van der Waals interactions with Thr200, Asn203,
Ser314 and Ser316 residues and electrostatic interactions
with Ile204, Ala205, Val225 and Ile312 amino acids. A
hydrogen bond was formed between the oxygen atom of
the morpholine ring His298 residue (Figure 4e).
In addition, in both cases, the hybrid compounds share
similar electrostatic and van der Waals intermolecular
interactions with each other and with control drugs. Thus,
the binding site cavity of both targets were highlighted with
compound 3 (Figure 5). As can be observed, it depicts a
close and open cavity for PI3Kγ and AMPK, respectively.
It is noteworthy that both share two distinct regions, one
hydrophobic in which the naphthoquinone ring binds, and
other hydrophilic region that the triazine and morpholine
rings bind. In other words, triazine and quinoline moieties
have become protonated in physiologic pH, addressing
these groups for the polar region of the binding site. These
findings explain the high affinity of hybrids for both PI3Kγ
and the AMPK molecular target.
In addition, the morpholine, triazine and quinoline
hybrid 2 containing moieties can perform intermolecular
interactions with PI3Kγ, which has the best interaction
-1
energy among the compounds studied (−9.1 kcal mol ).
For the complex with PI3Kγ, the observed interactions
included the following: van der Waals interactions with
Ser806, Lys807, Lys808, Pro810, Trp812, Ile831, Tyr867,
Glu880, Ile881,Val882, Met804, Lys833, Lys890,Asp950
and Phe961 amino acids and electrostatic interactions with
Ala805, Ile879 and Ile963 (Figure 3d).
Furthermore, the hybrid 3, like 1 and 2, carried out
van de Waals interactions with PI3Kγ. Furthermore, this
molecular target recognized that the hybrid 3, like 1 and
2
, carried out van der Waals interactions with PI3Kγ. This
molecular target recognized 3 through Ser806, Met804,
Lys808, Pro810, Lys833, Glu880, Ile881, Val882, Ile831,
Thr887, Lys890,Asp950,Asn951, Phe961 and Leu1090. In
addition, it could have performed electrostatic interactions
with Lys807, Met953 and Ile963 and a hydrogen bond with
Asp964. On the other hand, it carried out an unfavorable
interaction with Tyr867 amino acids (Figure 3e).
Chemical results
In a similar way to PI3Kγ enzyme, Figure 4 shows the
intermolecular interactions between AMPK and ligands.
The AICAR complex with AMPK through van der Waals
interactions with Ile204,Val225, Leu228 and Ile312 amino
acid residues. Electrostatic interactions are formed with
Thr200, Asn203, Ala205, Ser226, Ala227, Ser314, Ser316
and Asp317 amino acids. In addition, six hydrogen bonds
were formed between amide portion ofAICAR andAla205,
Ala227, Ser226 and finally with Asp317 (Figure 4a).
After the satisfactory results of the previous analysis
of ligand / enzyme interaction energy, the synthetic part
of the work was started. The starting materials chosen to
synthesize the target molecular hybrids (1, 2 and 3) were
lawsone 4, 4,7-dichloroquinoline 5, cyanuric chloride 6
and morpholine 7, which are all commercially available
(Figure 6).
Initially, the preparation of 2-methoxylawsone 8, the
electrophile precursor to achieve hybrid 1, was made

Vol. 00, No. 00, 2019
Fiorot et al.
9
Figure 4. 2D intermolecular interaction diagram among ligands and AMPK. (a) AICAR; (b) GSK621; (c) naphthoquinone-quinoline hybrid 1; (d) 2; (e) 3.
The distance is in angstroms (Å) and the hydrogens were omitted for a better visualization.
Figure 5. Hydrophobic receptor surface complex with hybrid compound 3. (a) With PI3Kγ and (b) with AMPK.
44
following a procedure that has been previously published.
The lawsone was solubilized in methanol containing
hydrochloric acid and then maintained under reflux
conditions for four hours to form compound 8 as a yellow
solid with 90%.
In the following step, 4,7-dichloroquinoline 5 was
converted into 9, the nucleophile for the further step,
Figure 6. Starting materials for synthesis of molecular hybrids 1, 2 and 3.

1
0
Synthesis, Molecular Modelling and Anticancer Activities of New Molecular Hybrids
J. Braz. Chem. Soc.
4
8
according to the procedure described by Carmo et al. The
reaction was performed with excess of 1,3-diaminopropane at
a temperature of 80-110 °C for 4 h to form the corresponding
aminoquinoline 9 in 87% yield as a solid white.
that, since the compounds are hydrophobic, the frequency
of effective collisions is enhanced once they are repelled
by water molecules and forced to approach (hydrophobic
effect), resulting in the diminish of reaction time due to
statistical reasons.
Finally, the intermediates 8 and 9 were submitted to
synthesize the target molecular hybrid 1. The conditions
are displayed in Figure 7. The temperature was maintained
between 40 and 50 °C with the purpose of keeping the
reactants as soluble as possible. It is noteworthy that the
best result in terms of yield was obtained when MeOH
entry 3, 84%) was used as the solvent. It may have
occurred because of its intermediate polarity and dielectric
constant compared to EtOH (entry 1, 15%) and H O
The mechanism for the formation of 1 should resemble
the mechanism that was recently proposed by our research
5
0
group (Figure 8). The tetrahedral species, commonly
suggested as an intermediate in addition-elimination
mechanism of substitution reactions, is not stable or may
decompose with low activation energy. Therefore, reactions
occur in either a single asynchronous step involving a
simultaneous addition-elimination process that describes
(
2
(entry 2, 14%), allowing a better solubility of the reactants.
an uncoupled S 2, with one step and two stages, in which
N
Surprisingly, the reaction time when water is used as the
solvent (entry 2) is drastically reduced considering the
other solvents. In a recent work of our research group,
we discussed the preparation of 1,4-naphthoquinonic
derivatives in aqueous media and its effects: it is possible
the second stage (break of the carbon-leaving group bond)
occurs with low or no activation energy.
For the next hybrid, morpholine-triazine-quinoline 2,
the intermediate nucleophilic 9, which was already
prepared, reacts with the electrophilic intermediate 10
49
Figure 7. Synthesis of target molecular hybrid 1.
Figure 8. Proposed mechanism for synthesis molecular hybrid 1.

Vol. 00, No. 00, 2019
Fiorot et al.
11
formed by the reaction between cyanuric chloride 6 and two
equivalents of morpholine 7. Therefore, cyanuric chloride
reacted with morpholine in presence of triethylamine and
acetone at 0 °C and held at room temperature for 2 h to form
The characterization of the final molecular hybrids 1,
2 and 3 proved their formation. For 1, Fourier transform
1
13
infrared (FTIR), H and C NMR spectroscopies were used
to show the coupling of naphthoquinone and quinoline
35
1
10 as a pure white solid in 93% yield. The bismorpholino-
cores. The H NMR spectrum showed all the signals
1
,3,5-triazine scaffold have an advantage, therefore, one
observed in the precursors 8 and 9, except the singlet at
morpholine gets metabolically oxidized, still leaving
another morpholine to make, a possible, H bond interaction
with the receptor proteins. Another important function of
morpholine is to increase the solubility of the intermediate
in usual organic solvents, facilitating the homogeneity of
d 3.92 ppm due to OCH from 2-methoxylawsone (8),
3
which was substituted in the reaction displayed in Figure 7.
For hybrids 2 and 3, however, high-resolution mass
spectrometry was necessary to prove the number and type of
substituents in the triazine core, i.e., to detect two morpholine
groups and one quinoline (in case of hybrid 2) or one
naphthoquinone (for hybrid 3). In spectra of hybrid 2, two
important peaks were found: one in 485.21749 m/z with error
of 1.09 ppm, which was related to the protonated structure
of 2, and 969.42783 m/z with error of 0.40 ppm, which was
associated with its protonated dimer. In spectra of hybrid 3,
two peaks were also found: one for the protonated monomer
(480.23504 m/z, with error of 0.71 ppm) and one for the
protonated dimer (959.46281 m/z with error of 0.70 ppm).
After obtaining the target molecular hybrids 1, 2 and
3, we attempted to reach a molecular hybrid containing
morpholine, naphthoquinone, quinoline and triazine cores
51,52
subsequent reactions.
Posteriorly, intermediate 10 reacted with 9 in CH CN in
3
presence of Et N under reflux for 16 h, furnishing the desired
3
molecular hybrid 2 with a yield of 43% (Figure 9).
Once the electrophilic intermediate 10 was synthesized,
compound 11 (the nucleophile to obtain the molecular
hybrid 3) was prepared following a procedure described by
49
our research group earlier (Figure 10). In the following
step, the target molecular hybrid 3 was obtained with yield
of 47% in 16 h (Figure 10). The synthesis of the molecular
hybrids 2 and 3 follows the same mechanistic purpose: a
nucleophilic aromatic substitution.
Figure 9. Synthesis of target molecular hybrid 2.
Figure 10. Synthesis of target molecular hybrid 3.

1
2
Synthesis, Molecular Modelling and Anticancer Activities of New Molecular Hybrids
J. Braz. Chem. Soc.
simultaneously in one hybrid (structures shown in SI
section). However, we faced problems with the solubility
of key intermediates (i.e., the disubstituted triazine with
morpholine and naphthoquinone or quinoline group),
which could drive the desired hybrid. Even using the one
pot methodology in which the nucleophiles were added at
different temperatures (0 °C, room temperature and reflux),
the hybrid could not be obtained.
degradation. This is a well-organized process that ensures
important quality control of the cytoplasmic components
along with the generation of building blocks to support cell
53,54
proliferation and survival.
This multistep catabolic process has been shown to
play an especially important role in melanoma acquisition
5
3
of resistance to anti-cancer therapies. Therefore, the
aberrant control of autophagy has been considered among
55,56
key hallmarks of cancer, including melanoma.
Anticancer activity
Intermediate 10 and molecular hybrids 2 and 3, which
display the 1,3,5-triazinic and morpholine core, decreased
the viability of SKMEL-103 (N-RAS mutated) with IC50
values around 25 μM. Temozolomide was used as the
control drug. The other compounds tested 1, 9 and 11 did
not obtain relevant cytotoxicity. These results point clearly to
the synergism between triazine, morpholine and quinoline to
compound 2 or naphthoquinone to 3. However, morpholine
and triazine appear to be essential for cytotoxic activity, since
intermediate 11, which does not have the naphthoquinone
and quinolinic portion, also showed increased cytotoxicity
in the cancer studied. The interaction energy obtained in
the docking studies corroborate with the experimental data
where the compound 3 was the most effective.
Interestingly, the molecular hybrids 2 and 3 and
intermediate 10 negatively modulate both signaling ways,
PI3K and AMPK, in the melanoma SKMEL-103 cells.
Expressions of PI3K and AMPK were determined using
western blotting and equal loading was confirmed by
reprobing blots for tubulin. Figure 11 summarizes the
relevant results of melanoma cells viability expressions of
PI3K and AMPK.
Malignant melanoma is the most aggressive form
of skin cancer. Most patients are diagnosed in early
stages when the disease is curable. However, the more
advanced or metastatic cases have always been a
challenge for physicians. The poor prognosis for patients
with melanoma has been changing since numerous of
4
8
promising therapeutic protocols have appeared recently.
Nevertheless, the development of new possible methods of
treatment is still needed with improved durable response
rates and wider target populations to treat metastatic
melanoma.
In this study, we show that it is possible to decrease
the viability of the metastatic melanoma cell line as well
as the activity and expression ofAMPK and PI3K kinases,
both of which positively modulate the autophagy process.
Autophagy is stimulated in response to both physiological
and pathological conditions such as starvation, hypoxia and
low energy as well as protein aggregates. This process is
a crucial adaptive response that initiates the sequestration
of damaged or ageing intracellular content and leads to its
Figure 11. (A) Results of (a) temozolomide; (b) intermediate 10; (c) and (d) molecular hybrids 2 and 3, respectively, on melanoma cells viability
SKMEL-103). SKMEL-103 cells were treated with the compounds for 24 h. Cell viability was measured by MTT assay and cell viability of non-treated
(
cells was considered as 100%. Data are shown as mean ± SEM (n = 6). **p < 0.01, ***p < 0.001 vs. control and n.s.: not significant, assessed withANOVA
followed by a Tukey’s test. (B) Results of intermediate 10 and molecular hybrids 2 and 3 downregulating (a) PI3K and (b) AMPK. Expressions of PI3K
and AMPK in SKMEL-103 cells were determined using western blotting. Equal loading was confirmed by reprobing blots for tubulin (c).

Vol. 00, No. 00, 2019
Fiorot et al.
13
Conclusions
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Molecular hybridization was accomplished by
gathering together four structures (morpholine,
naphthoquinone, quinolone and 1,3,5-triazine) with
renowned anticancer activity in three novel substances. We
started our work with a rational design of potential drugs,
investigating interaction energies between the hybrids
with PI3Kγ and AMPK enzymes by docking studies.
From this, the synergism of the triazine, morpholine
and naphthoquinone moieties was clearly observed by
the greater bind affinity of the complex formed between
hybrid 3 and biological receptor tested. For PI3Kγ, the
interactions are commonly electrostatic, mainly with
the amino acid residues Lys833 and Lys807 in its active
site. For AMPK, the main observed interactions were
hydrogen bonds with Ser314 and Ser226 amino acid
residues that also appeared in complex ligand / enzyme
of the control drugs. The preliminary positive results from
docking encouraged us to synthesize the novel molecular
hybrids 1, 2 and 3, which were obtained in yields ranging
from 43 to 84% in three steps. Biological results showed
that compounds 2, 3 and intermediate 10 decreased the
viability of melanoma cells and negatively modulated
the expression of PI3K, inhibiting AMPK as well. As
predicted by docking, the in vitro tests also showed that
compound 3 was the most cytotoxic in SKMEL-103 cells
evidencing the synergism of naphthoquinone associated
with morpholine and triazine cores.
4. Nepali, K.; Sharma, S.; Sharma, M.; Bedi, P. M. S.; Dhar, K. L.;
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Palavan-Ünsal, N.; Exp. Cell Res. 2015, 338, 10.
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1
8. Kennedy, S.; DiCesare, J. C.; Sheaff, R. J.; Biochem. Biophys.
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Supplementary Information
1
9. Boonyalai, N.; Sittikul, P.; Pradidphol, N.; Kongkathip, N.;
Biomed. Pharmacother. 2013, 67, 122.
Supplementary information is available free of charge
at http://jbcs.sbq.org.br as PDF file.
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2
Acknowledgments
2
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Ahmed, T.; Sabatini, D. M.; Gray, N. S.; J. Med. Chem. 2011,
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Authors acknowledge the Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq
4
49984/2014-1 and 305117/2017-3), the Coordenadoria de
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Aperfeiçoamento de Pessoal do Nível Superior (CAPES),
Fundação deAmparo à Pesquisa do Espírito Santo (FAPES)
and Fundação de Amparo à Pesquisa de Minas Gerais
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Photobiol., B 2009, 94, 13.
(
FAPEMIG APQ-00557-14 and APQ-02742-17) for their
financial support.
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