Antiangiogenic and antitumoral activity of LQFM126 prototype against B16F10 melanoma cells

Article abstract of DOI:10.1016/j.cbi.2020.109127

Inhibition of mouse double minute 2 homolog (MDM2)-p53 interaction and reactivation of p53 signaling have been explored as effective anticancer therapeutic strategy. The potent and specific antitumor activity shown by Nutlins, first class of MDM2-p53 inhibitors discovered, has made these compounds potential antitumor candidates. To this end, we synthesized Nutlin-1 and Nutlin-2 analogs through molecular simplification and selected the compound with the most efficient antitumoral activity. Cytotoxicity of Nutlin-2 analog LQFM126 on B16F10 melanoma cells induced intense cytoplasmic vacuolization, reduction of cell size, chromatin condensation, cytoplasmic degeneration and nuclear fragmentation. LQFM126 antiproliferative effects mediated cell cycle retention in G₀/G₁ phase and increased the levels of cell cycle regulatory proteins p21 and p27. This Nutlin analog increased mitochondrial membrane potential, activated caspase-8, -9 and -3/7 and reduced VEGF levels in B16F10 cells. Therefore, LQFM126 promoted alterations suggestive of apoptosis, G0/G1 cell cycle arrest and suppression of angiogenesis through modulation of VEGF expression in B16F10 cells. Additionally, LQFM126 was classified as UN GHS category 4 (LD50 > 300–2000 mg/kg), suggesting it has low acute systemic toxicity. LQFM126 can be a promising prototype for anticancer therapy.

Full text of DOI:10.1016/j.cbi.2020.109127

Chemico-BiologicalInteractions325(2020)109127
Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Antiangiogenic and antitumoral activity of LQFM126 prototype against
B16F10 melanoma cells
Artur Christian Garcia da Silvaa, Bruna dos Santos Rodriguesa, Wanessa Machado Andradea,
Thaís Rosa Marques dos Santosa, Flávio Silva de Carvalhoc, Germán Sanzd, Boniek G. Vazd,
Luciano M. Liãoc, Ricardo Menegattib, Marize Campos Valadaresa,∗
a Laboratory of Education and Research in In Vitro Toxicology (Tox In), Faculty of Pharmacy, Universidade Federal de Goiás, Goiânia, Goiás, Brazil
b Laboratory of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Universidade Federal de Goiás, Goiânia, Goiás, Brazil
c Institute of Chemistry, Universidade Federal de Goiás, Goiânia, Goiás, Brazil
d Laboratory of Chromatography and Mass Spectrometry, Institute of Chemistry, Universidade Federal de Goiás, Goiânia, Goiás, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Antitumor drugs
Apoptosis
Nutlins
LQFM126
Inhibition of mouse double minute 2 homolog (MDM2)-p53 interaction and reactivation of p53 signaling have
been explored as effective anticancer therapeutic strategy. The potent and specific antitumor activity shown by
Nutlins, first class of MDM2-p53 inhibitors discovered, has made these compounds potential antitumor candi-
dates. To this end, we synthesized Nutlin-1 and Nutlin-2 analogs through molecular simplification and selected
the compound with the most efficient antitumoral activity. Cytotoxicity of Nutlin-2 analog LQFM126 on B16F10
melanoma cells induced intense cytoplasmic vacuolization, reduction of cell size, chromatin condensation, cy-
toplasmic degeneration and nuclear fragmentation. LQFM126 antiproliferative effects mediated cell cycle re-
tention in G0/G1 phase and increased the levels of cell cycle regulatory proteins p21 and p27. This Nutlin analog
increased mitochondrial membrane potential, activated caspase-8, -9 and -3/7 and reduced VEGF levels in
B16F10 cells. Therefore, LQFM126 promoted alterations suggestive of apoptosis, G0/G1 cell cycle arrest and
suppression of angiogenesis through modulation of VEGF expression in B16F10 cells. Additionally, LQFM126
was classified as UN GHS category 4 (LD50 > 300–2000 mg/kg), suggesting it has low acute systemic toxicity.
LQFM126 can be a promising prototype for anticancer therapy.
Melanoma
Molecular simplification
1. Introduction
damage and ribosomal stress leading to apoptosis, cell cycle arrest, DNA
repair, differentiation and senescence. It is believed that the abnormal
Melanoma is the most lethal skin cancer type, and although surgical
management is considered the gold standard treatment, im-
munotherapy and targeted molecular therapy has emerged as a pro-
mising strategy for the metastatic disease [1,2]. Reactivation of p53
gene through inhibition of mouse double minute 2 (MDM2)-p53
binding has been a potential strategy in drug development for cancer
treatment, including melanoma [3,4]. The transcription factor p53 is
the most frequently altered protein in human tumors and loss of its
function has demonstrated to be strictly related to tumorigenesis pro-
cess [5,6]. Under stress conditions, p53 regulates the signaling trans-
duction pathways to reverse protooncogenes activation, cellular DNA
function of p53 is due to incorrect p53 regulation or defective signaling
in the p53 pathway [7]. One mechanism related to p53 suppression
involves its negative regulator MDM2 protein, which is frequently
overexpressed in many human tumors [8,9].
Intracellular levels and activity of p53 are majorly regulated by
MDM2 through autoregulatory feedback loop which both proteins
control mutually their cellular levels. Regulation of p53 by MDM2 in-
volves direct blockage of p53 transcription domain and promotes ubi-
quitin-dependent p53 degradation [10–12]. Therefore, restoring or
improving p53 functions through blockage of MDM2-p53 complex
corresponds to a promising therapeutic target in the development of
∗ Corresponding author. Faculdade de Farmácia, Universidade Federal de Goiás, Rua 240 esquina com 5a, Avenida, s/n, Setor Universitário, Goiânia, Goiás, CEP
74605-170, Brazil.
E-mail addresses: christianartur@hotmail.com (A.C. Garcia da Silva), bruna.srodrigues04@gmail.com (B.d.S. Rodrigues),
wanessa.essa@gmail.com (W.M. Andrade), santostrm@gmail.com (T.R. Marques dos Santos), flavioas8@hotmail.com (F.S. de Carvalho),
manger84@gmail.com (G. Sanz), boniek@gmail.com (B.G. Vaz), lucianoliao@yahoo.com.br (L.M. Lião), rm_rj@ufg.br (R. Menegatti), marizeufg@gmail.com,
marizecv@ufg.br (M.C. Valadares).
https://doi.org/10.1016/j.cbi.2020.109127
Received 11 March 2020; Received in revised form 15 April 2020; Accepted 3 May 2020
Availableonline11May2020
0009-2797/©2020PublishedbyElsevierB.V.

A.C. Garcia da Silva, et al.
Chemico-BiologicalInteractions325(2020)109127
effective antitumoral drugs.
points were assessed using a Marte melting point apparatus, and the
results were uncorrected. The organic solutions were dried over anhy-
drous sodium sulphate and organic solvents were removed under re-
duced pressure in a rotary evaporator. Mass spectra (MS) were obtained
with a micro TOF III (Brucker Daltonics Bremen, Germany). The sample
preparation for MS analysis consisted of diluting 1 μg of sample in 1 mL
of methanol. Toper form the analysis in the positive mode, 1 μL of
formic acid was added to the sample. The solution obtained was di-
rectly infused at a flow rate of 3 μL/min into the ESI source. ESI (+)
source conditions were as follows: nebulizer with nitrogen gas; pressure
of 0.4 bar and temperature of 200 °C; capillary voltage of −4 kV,
A library screening of synthetic compounds identified cis-imidazole
analogs, Nutlins, with ability to inhibit p53-MDM2 binding and displace
the recombinant p53 from MDM2 complex [13]. Nutlins bind to MDM2
blocking MDM2-p53 interaction resulting in p53 activation and con-
sequent cell arrest, apoptosis and tumor inhibition in tested animals
[13–16]. Based on extensive set of preclinical efficacy and safety stu-
dies, Nutlin analogs have advanced into clinical trials for treatment of
human cancers [10,14,17].
Considering the antitumor potential of Nutlins, our research group
synthesized Nutlin analogs through molecular simplification strategy
from Nutlin-1 and Nutlin-2 lead compounds and performed a screening
for antitumor activity on B16F10 melanoma cells. This strategy led to
the synthesis of chemical entities with low molecular complexity and
fewer chiral centers through a simple synthetic route giving a good
yield. Synthesized Nutlin analog LQFM126, designed from Nutlin-2,
was selected for investigation of the mechanisms behind the anti-
proliferative activity on B16F10 cells.
transfer capillary temperature of 200 °C; drying gas of 4 L min−1
;
endplate offset of −500 V; skimmer of 35 V and collision voltage of
−1.5 V. Each spectrum was acquired using 2 microscans. The resolving
power is: m/Δm 50% 16,500.00, where Δm 50% is the peak full width
at half-maximum peak height. Mass spectra were acquired and pro-
cessed with Data Analysis software (Brucker Daltonics, Bremen,
Germany).
LQFM126 (2) design consisted of replacing A and B scaffolds present
in Nutlin-2 (1) by1-(4-bromophenyl)-1H-pyrazol-4-yl scaffold, and the
maintenance of (2-hydroxyethyl)piperazine (C) scaffold, as illustrated
in Fig. 1. The study evaluated the effects of LQFM126 (2) treatment on
B16F10 morphology, colony formation as well as the involved cell
death mechanisms including changes in cell cycle progression, expres-
sion of cell cycle regulatory proteins (p21 and p27) and antiangiogenic
activity through quantification of vascular endothelial growth factor
(VEGF). Furthermore, we studied the apoptotic pathways involved in
LQFM126-induced cell death in B16F10 cells through quantification of
reactive oxygen species (ROS) production, changes in mitochondria
membrane potential and caspases activity. The suitability of the pro-
totype for further in vivo studies was performed as estimative of acute
oral toxicity.
2.2. Synthesis of 2-(4-((1-(4-bromophenyl)-1H-pyrazol-4-yl)methyl)
piperazin-1-yl)ethanol(LQFM126)(2)
To a stirred heterogeneous mixture of 1-(4-bromophenyl)-1H-pyr-
azole-4-carbaldehyde (5) (251 mg, 1.0 mmol), 1-(2-hydroxyethyl)pi-
perazine (6) (130 mg, 1.0 mmol), ZnCl2 (68 mg, 0.5 equiv), in 5 mL of
MeOH was added NaBH3CN (31 mg, 0.5 equiv) in one portion. The
mixture was stirred at 70 °C for 2 h. In turn, MeOH was then evaporated
and the residue was partitioned between water and CH2Cl2. At the end
of the reaction, the residue was split between water and CH2Cl2. The
phases were separated and the aqueous layer was extracted into
3 × 15 mL with CH2Cl2 and the combined organic layers were dried
(Na2SO4), concentrated in vacuum, and the crude product was purified
by column chromatography (SiO2, hexane/AcOEt = 6:4) to 2-(4-((1-(4-
bromophenyl)-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethanol
(2)
2. Material and methods
(244 mg, 67%) as a beige solid, m.p. = 110-112 °C, Rf = 0.71 (CH2Cl2/
MeOH = 95:5) [18]. IRmax (KBr) cm−1: 3500–3000 (υ O–H); 2918 (υ
C–H), 2796 (υ C–H), 1497 (υ C = c), 812 (υ Ar-1,4); 1H NMR
(500.13 MHz) CDCl3/TMS (δ): 7.83 (1H, s, H-5′); 7.64 (1H, s, H-3′);
7.55 (4H, m, H-6); 7.55 (4H, m, H-5); 7.55 (4H, m, H-3); 7.55 (4H, m, H-
2); 3.60 (2H, t, j = 5.43, H-8″); 3.49 (2H, s, H-6′); 2.54 (2H, t, j = 5.43,
H-7″); 2.54 (8H, m, H-6″); 2.54 (8H, m, H-5″); 2.54 (8H, m, H-3″); 2.54
(8H, m, H-2″).2D NMR (HSQC/HMBC – 125.76 MHz) CDCl3/TMS (δ):
142.2 (C-3′); 138.9 (C-1); 132.2 (C-5); 132.2 (C-3); 126.2 (C-5′); 120.2
(C-6); 120.2 (C-2); 119.9 (C-4′); 119.4 (C-4); 59.2 (C-7″); 57.6 (C-8″);
52.5 (C-6″); 52.5 (C-5″); 52.5 (C-3″); 52.5 (C-2”); 52,3 (C-6’).MS: [M+
H]+m/z of 365.0926.
2.1. General experimental procedure for synthesis of Nutlins 1 and 2
analogs
Nutlin 1 and 2 analogs (LQFM088 – LQFM144) were synthesized at
Laboratory of Pharmaceutical Medicinal Chemistry of Prof Ricardo
Menegatti (Faculty of Pharmacy, Universidade Federal de Goiás,
Goiânia, GO, Brazil). Reactions were monitored by TLC using com-
mercially available pre-coated plates (Whatman 60 F254 silica) and the
developed plates were examined under UV light (254 nm and 365 nm).
1H and13C NMR spectra were recorded in the indicated solvent on
Bruker Avance III 500 MHz spectrometer. Chemical shifts are quoted in
parts per million down field of TMS and the coupling constants are in
Hertz. All assignments of the signals of 1H and 13C NMR spectra are
consistent with the chemical structures of the products described.
Infrared spectra were recorded on a PerkinElmer Spectrum Bx-II FT-IR
System spectrophotometer instrument as films on KBr discs. Melting
2.3. Cell culture
B16F10 melanoma cells and Balb/3T3 clone A31 fibroblast cells
were purchased from ATCC (Manassas, VA, USA). They were cultured
in Dulbecco's Eagle's medium (DMEM) 10% (v/v) fetal bovine serum
(FBS) (Sigma-Aldrich, St Louis, MO, USA) and 1% penicillin/strepto-
mycin (Sigma-Aldrich, St Louis, MO, USA). Cells were cultured at 37 °C
under a humidified atmosphere containing 5% CO2.
2.4. Antitumor activity screening of Nutlin analogs
Nutlin analogs were solubilized in dimethyl sulfoxide (DMSO) and
stored at −20 °C. Before use, the compounds were solubilized in
complete medium resulting in DMSO concentration under 0.5%.
Antitumor activity of Nutlin analogs were evaluated in B16F10 mela-
noma cells using MTT assay. About 1 × 104 cells/well were plated in
96-well plates 24 h prior to the assay. Cells were exposed to Nutlin
analogs (LQFM088 – LQFM144) at 100 μM for 48 h. Cell viability was
evaluated by adding 10 μl MTT (5 mg/ml) to each well. After 3 h of
Fig. 1. Structural design of LQFM126 (2) from Nutlin-2 (1).
2

A.C. Garcia da Silva, et al.
Chemico-BiologicalInteractions325(2020)109127
incubation, MTT solution was removed and DMSO was added. Optical
density was measured at 560 nm. Untreated cells were used as control
and cell viability was expressed as percentage of control.
B16F10 with LQFM126 (2) at 40 μM (CV80) for 48 h, caspase reagent
was added and incubated for 1 h. Quantification of caspase activity was
performed through flow cytometry. Caspase expression was expressed
as percentage of control.
2.5. Cytotoxicity assay
2.8.5. Expression of p21 and p27
Cytotoxic potential of LQFM124, LQFM126 (2) and LQFM127 was
evaluated in B16F10 cells using MTT assay [19]. About 1 × 104 cells/
well were seeded in 96-well plates 24 h prior to the assay. B16F10 cells
were exposed to aforementioned analogs at 25–200 μM for 48 h. Un-
treated cells were used as control. Cell viability was expressed as per-
centage of control.
B16F10 cells were exposed to LQFM126 (2) at 40 μM (CV80) for
48 h. Following the treatment, cells were washed twice with 0.1% al-
bumin in PBS. Thereafter, cells were fixed with cytofix/cytoperm (BD
Biosciences, San Jose, CA, USA) solution, and then washed twice with
0.05% Tween 20 in PBS. Monoclonal antibodies were incubated for
30 min in the dark at room temperature. Cells were washed twice with
0.05% Tween 20 in PBS and analyzed using flow cytometry.
2.6. Cell morphology examination
2.9. Antiangiogenic activity
Morphological changes induced by LQFM126 (2) in B16F10 cells
were evaluated using May-Grünwald-Giemsa dye and Hoechst 33,342.
About 1 × 105 cells were seeded in sterile round coverslips for 24 h.
Thereafter, cells were exposed to cell viability 80% (CV80) of LQFM126
(2) (40 μM) for 24, 48 and 72 h. For May-Grünwald-Giemsa staining,
cells were fixed with 4% paraformaldehyde for 30 min. May-Grünwald-
Giemsa dye (Merck, Darmstadt, Germany) was added for 1 min and,
subsequently, the slides were prepared and observed using light mi-
croscope (DM 2000, Leica Microsystems, Wetzlar, Germany). For
Hoechst 33,342 staining, cells were fixed with methanol for 5 min, then
washed with 0.05% Tween-20 in PBS for 5 min and incubated with
Hoechst 33,342 (10 μg/ml) for 20 min. The slides were prepared and
observed using fluorescence microscope (DMI 4000 B, Leica
Microsystems, Wetzlar, Germany).
B16F10 (1 × 105 cells/well) were seeded in 6-well plate 24 h prior
to the assay. Then, cells were exposed to LFQM126 (2) at 40 μM (CV80
for 48 h. The levels of vascular endothelial growth factor (VEGF) were
determined in the supernatant using Mouse VEGF Quantikine ELISA kit
(R&D Systems, Minneapolis, MN, USA) according to manufacturer's
instructions. Absorbance was measured at 540 nm.
)
2.10. Balb/c 3T3 cytotoxicity assay for estimation of acute oral systemic
toxicity class
Cytotoxicity of LQFM126 (2) was also evaluated in 3T3 fibroblasts
using neutral red uptake assay, according to the OECD Guideline
No.129 [21]. As described in the guideline, the half inhibitory con-
centration (IC50) of a compound upon Balb/c 3T3 cells enable to esti-
mate their median lethal doses (LD50) and starting doses for acute oral
systemic toxicity tests. Cells (3 × 103 cells/well) were seeded in 96-
well plate 24 h prior the assay. Then, they were exposed to LQFM126
(2) at 25–200 μM for 48 h. Neutral red solution (25 μg/ml) was added
in each well and, after 3 h of incubation, neutral red desorb solution
(50% ethanol, 1% acetic acid and 49% ultrapure water) was added for
20 min under gentle agitation. Absorbance was measured at 540 nm
and cell viability was determined as percentage of control. The obtained
IC50 values were used to estimate in vivo LD50 using the following
equation: logLD50 (mg/kg) = 0.372 logIC50 (mg/kg) + 2.024 [22].
2.7. Clonogenic assay
B16F10 and 3T3 cells (1 × 103 cells) were seeded in 35 mm plate
for 24 h prior to perform the assay and exposed to LQFM126 (2) at 10,
20 and 40 μM for 48 h. Subsequently, cells were washed with PBS and
incubated for further 7 days. Cells were fixed with methanol for 2 min
and stained with May-Grünwald-Giemsa for 10 min. Number of co-
lonies was analyzed and recorded using BioSpectrum® Imaging System,
UVP (Upland, Canada) and calculated using ImageJ software [20].
2.8. Flow cytometry analysis
B16F10 cells (1 × 105 cells/well) were seeded in 6-well plates 24 h
prior the assays. For each analysis, 10,000 events were recorded.
2.11. Statistical analysis
The results were expressed as mean
standard deviation of three
2.8.1. Cell cycle assessment
independent experiments. IC50 and CV80 values were determined
through non-linear regression. One-way ANOVA and “t” test evaluated
significant differences between control and treated group with
p < 0.05 and analysis were performed using the software GraphPad
Prism 5.0.
B16F10 cells were exposed to LQF126 (2) at 40 μM (CV80) for 24 h
and, subsequently, fixed with ice-cold 70% ethanol and stored over-
night at 4 °C. Cells were incubated with RNase solution (200 μg/ml) and
propidium iodide (50 μg/ml) for 1 h, in the dark at 4 °C. Thereafter,
cells were analyzed using flow cytometry.
2.8.2. Reactive oxygen species (ROS)
3. Results
B16F10 cells were exposed to LQF126 (2) at 40 μM (CV80) for 24 h.
Thereafter, cells were incubated with 2,7-dichlorodihydrofluorescein
diacetate (DCFH-DA) (10 μM) for 1 h at 37 °C. The levels of ROS were
quantified by flow cytometry.
3.1. Synthesis of LQFM126 (2)
As illustrated in Fig. 2, the synthetic route began with 1-(4-bro-
mophenyl)-1H-pyrazole (4) and proceeded through the classical
method described by Finar and Hurlock, in 73% of yield [23]. Che-
moselective and region specific formulation of 1-(4-bromophenyl)-1H-
pyrazole (4) to1-(4-bromophenyl)-1H-pyrazole-4-carbaldehyde (5) was
performed under Duff's conditions in 57% of yield [24]. The synthesis
2.8.3. Mitochondrial membrane potential (ΔΨm)
B16F10 cells were exposed to LQFM126 (2) at 40 μM (CV80) for 24 h
and ΔΨm was evaluated after incubation of cells with rhodamine-123
(1 μg/ml) using flow cytometer.
of
2-(4-((1-(4-bromophenyl)-1H-pyrazol-4-yl)methyl)piperazin-1-yl)
2.8.4. Caspase 3/7, 8 and 9
ethanol (2) was carried out through conditions of reductive amination
in 67% of yield [18]. LQFM126 (2) was obtained after three synthetic
steps in 28% overall yield.
Caspase 3/7,
8 and 9 expressions were evaluated using situ
CaspaTag™ (Millipore™, Burlington, MA, USA). After incubation of
3

A.C. Garcia da Silva, et al.
Chemico-BiologicalInteractions325(2020)109127
Fig. 2. Synthetic route for the preparation of 2-(4-((1-(4-bromophenyl)-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethanol (2) – LQFM126.
Fig. 3. Antitumoral activity of Nutlin-1 analogs in B16F10 melanoma cells at
100 μM after 48 h of incubation. Cell viability was determined by MTT assay.
Fig. 5. Cytotoxic profile and IC50 of LQFM124, LQFM126 (2) and LQFM127 at
25–200 μM at 1.56–200 μM in B16F10 cells. Cell viability was determined using
MTT assay.
Data are expressed as mean
SD (n = 3).
3.2. Antitumor activity of Nutlin analogs
and LQFM127, we also investigated the cytotoxic profile of these ana-
logs in B16F10 cells and we determined the IC50, Fig. 5. IC50 of
LQFM126 (2) and LQFM127, determined using MTT assay, were
100.0 μM and 190.0 μM, respectively. While IC50 of LQFM124 was
above 200 μM, therefore it was not established in this assay.
We performed an antitumor screening of Nutlin-1 analogs
(LQFM106-LQFM110, LQFM135-144) and Nutlin-2 analogs (LQFM080,
LQFM111-LQFM134) against to melanoma cells. B16F10 melanoma
cells were exposed to Nutlin analogs at 100 μM for 48 h and cell vi-
abilities were described in Fig. 3 and Fig. 4, for cells exposed to Nutlin-1
and Nutlin-2 analogs, respectively. Nutlin-2 analogs LQFM124,
LQFM126 (2) and LQFM127 showed antitumor potential against
B16F210 cells demonstrated by the lower cell viability. Additionally,
LQFM126 (2) showed significantly (p < 0.05) lower cell viability as
compared to other Nutlin analogs.
3.4. Cell morphology examination
Morphology examination of B16F10 cells treated with LQFM126 (2)
(40 μM) in different incubation times revealed cell morphology changes
suggestive of cell death via apoptosis. The morphological changes ob-
served included intense cytoplasmic vacuolization, reduction of cell
size, chromatin condensation, cytoplasmic degeneration and nuclear
fragmentation (Fig. 6). Alterations in the nucleus of these cells were
observed through Hoechst 33,342 staining. The treatment with
3.3. Cytotoxicity assay
Considering the antitumoral potential of LQFM124, LQFM126 (2)
Fig. 4. Antitumoral activity of Nutlin-2 analogs in B16F10 melanoma cells at 100 μM after 48 h of incubation. Cell viability was determined by MTT assay. Data are
expressed as mean
SD (n = 3). Statistically significant (p < 0.05) differences are shown as (*).
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A.C. Garcia da Silva, et al.
Chemico-BiologicalInteractions325(2020)109127
Fig. 6. Morphology alterations induced in
B16F10 cells after treatment with LQFM126 (2)
(40 μM) analyzed through May-Grünwald-Giemsa
staining. A) Untreated cells showing nucleus with
dispersed chromatin (DC), organized plasma mem-
brane (OPM) and preserved intercellular commu-
nication (PIC). B) Cells treated with LQFM126 (2)
for 24 h showing cytoplasmic vacuolization (CV),
reduction of cell size (RCS) and chromatin con-
densation (CC). C) Cells treated with LQFM126 (2)
for 48 h showing cytoplasmic vacuolization (CV),
chromatin condensation (CC) and cytoplasmic de-
generation (CD). D) Cells treated with LQFM126 (2)
for 72 h showing cytoplasmic vacuolization (CV),
reduction of cell size (RCS), chromatin condensation
(CC) and nuclear fragmentation (NF). Scale bar:
20 μm.
Fig. 7. Nuclear alterations induced in
B16F10 cells after treatment with LQFM126 (2)
(40 μM) analyzed through Hoechst 33,342
staining. A) Untreated cells showing nucleus
with dispersed chromatin and evident nucleolus.
B) Cells treated with LQFM126 (2) for 24 h
showing nuclear fragmentation (NF), chromatin
condensation (CC) and discrete nuclear pleo-
morphism. C) Cells treated with LQFM126 (2)
for 48 h showing nuclear fragmentation (NF)
and chromatin condensation (CC). D) Cells
treated with LQFM126 (2) for 72 h showing
intense nuclear fragmentation (NF) and chro-
matin condensation (CC). Scale bar: 20 μm.
LQFM126 (2) (40 μM) induced intense nuclear pleomorphism and
chromatin condensation (Fig. 7). The treatment also reduced the
number of cells as compared to control group.
significantly (p
<
0.05) inhibited colony formation at 20 μM and
40 μM in 3T3 cells (Fig. 8). Results suggested a relative selectivity of
LQF126 (2) to cancer cells, which had the growth significantly
(p < 0.05) inhibited after the interruption of treatment, as compared
to inhibitory effect observed in basal cells.
3.5. Clonogenic assay
Survival of B16F10 and 3T3 cells was assessed after treatment with
LQFM126 (2) by a colony formation assay. Results revealed that sup-
pression of colony formation occurred in a concentration-dependent
3.6. Cell cycle assessment
After the treatment of B16F10 with LQFM126 (2) (40 μM) for 24 h,
we observed significant (p < 0.05) increase (13.5
manner. LQFM126 (2) significantly (p
<
0.05) suppressed colony
1.2%) in the
formation at 10, 20 and 40 μM in B16F10 cells. While this Nutlin analog
number of cells in G0/G1 phase and significant (p < 0.01) reduction
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A.C. Garcia da Silva, et al.
Chemico-BiologicalInteractions325(2020)109127
Fig. 8. LQFM126 (2) inhibits melanoma cancer cells growth. A) Colony formation assay of B16F10 and 3T3 cells after exposed to LQFM126 (2) at 10, 20 and 40 μM
treatment for 48 h followed by additional 7 days incubation. B) Quantification analysis of survival fractions as percentage of control. Survival fractions for control
groups were normalized to 100%. Data are expressed as mean
SD of three independent experiments (*p < 0.05, **p < 0.001 and ***p < 0.0001 vs control).
(54.9
6.5%) in the number of cells in S phase (Fig. 9). The latter
3.10. Expression of p21 and p27
suggested that LQFM126 (2) promoted the retention of cell cycle of
B16F10 at first stage of cell cycle and, consequently, reducing the
number of cells in the following phases.
The expression of p21 and p27 regulatory proteins significantly
(p < 0.05) increased 30.5
9.69% and 53.8
37.3%, respectively,
in B16F10 cells after the treatment with LQFM126 (2) (40 μM) for 48 h
as compared to the levels expressed by control cells (Fig. 10).
3.7. Reactive oxygen species (ROS)
Treatment of B16F10 with LQFM126 (2) (40 μM) for 24 h sig-
3.11. Antiangiogenic activity
nificantly (p
< 0.01) increased the intracellular levels of ROS
(4778.4
1477.7%) indicating that this compound induced an in-
VEGF levels in cell supernatant significantly (p < 0.05) reduced
tracellular oxidative stress (Fig. 10).
(70.2
3.85%) in B16F10 cells after treatment with LQFM126 (2)
(40 μM) for 24 h as compared to the levels of control cells (Fig. 10).
3.8. Mitochondrial membrane potential
3.12. Estimation of acute oral systemic toxicity
Incubation of B16F10 cells with LQFM126 (2) (40 μM) for 24 h
induced a significant (p < 0.01) increase (66.4
4.24%) in the re-
IC50 of LQFM126 in 3T3 fibroblasts was 125.0 μM (45.5 μg/mL)
which was determined using neutral red uptake assay (Fig. 11). Ac-
cording to the equation described in OECD Guideline No. 129, the es-
timated LD50 of LQFM126 was 437.7 mg/kg, classifying this compound
as category 4 (LD50 > 300–2000 mg/kg) of toxicity regarding the
United Nations Globally Harmonized System Classification and Label-
ling of Chemicals (UN GHS).
lative fluorescence of rhodamine-123 as compared to control cells
(untreated cells) leading to hyperpolarization of mitochondrial mem-
brane potential (Fig. 10).
3.9. Caspase 3/7, 8 and 9
After 48 h of incubation of B16F10 with LQFM126 (2) (40 μM), we
observed significant (p < 0.05) increase in expression of executioner
4. Discussion
caspase-3/7 (19.4
(15.1
1.98) as well as initiator caspase-8
5.16%) (Fig. 10).
0.92%) and caspase-9 (19.8
The important role of p53 in preserving and controlling the
6

A.C. Garcia da Silva, et al.
Chemico-BiologicalInteractions325(2020)109127
Fig. 9. Effects of LQFM126 (2) on cell cycle progression in B16F16 cells. A) Representative histograms of cell cycle analysis in B16F10 cells following treatment with
LQFM126 (2) (40 μM) for 24 h. C) Percentage of B16F10 cells in the sub G1, G0/G1, S and G2/M phases after 24 h treatment with LQFM126 (2) (40 μM). Data are
expressed as mean
SD of three independent experiments (*p < 0.05 and **p < 0.001 vs control).
proliferative cellular processes makes this nuclear transcription factor
an important target on cancer treatment [6,7]. Recent studies have
reported effective anticancer activity of compounds that block p53 in-
teraction to MDM2, which is a primary endogenous negative regulator
of p53 [9,14,17]. Nutlin family of MDM2 inhibitors was the first class of
p53-MDM2 inhibitors discovered and has proven the anticancer ther-
apeutic opportunities through this strategy [11–13,25]. Showing potent
and selective MDM2 inhibition, Nutlin analog compounds have pro-
gressed into clinical trials for treatment of human cancers [14,17,26].
Inspired by the p53 reactivation and antiproliferative properties of
Nutlin compounds, our group designed Nutlin-1 and Nutlin-2 analogs
through molecular simplification. Such drug design approach shortens
the synthetic routes yielding compounds structurally simpler, while
keeping or enhancing the biological activity of active prototype [27].
Initially, we performed a screening of antitumor activity in B16F10
melanoma cells of synthesized Nutlin analogs and selected the most
potent compound. The study was followed by investigation of
LQFM126 induced-cell death mechanisms in B16F10 cells and estima-
tion of acute oral systemic toxicity.
The present study demonstrated structural design and synthesis of
7

A.C. Garcia da Silva, et al.
Chemico-BiologicalInteractions325(2020)109127
Fig. 10. A) Intracellular ROS generation analysis in B16F10 cells treated with LQFM126 (2) (40 μM) for 24 h using a fluorescence probe, 2,7-dichlorodihydro-
fluorescein diacetate (DCFH-DA). B) Mitochondrial membrane potential changes in B16F10 cells after 24 h of LQFM126 (2) treatment (40 μM) determined using
rhodamine 123 staining. C) Caspase activity quantification in B16F10 treated with LQFM126 (2) for 48 h. D) Expression of cell cycle regulatory proteins p21 and p27
in B16F10 cells following treatment with LQFM126 (2) (40 μM) for 48 h. E) VEGF levels in supernatant of B16F10 cells following treatment with LQFM126 (2)
(40 μM) for 24 h. All data are expressed as mean
SD of two or three independent experiments (*p < 0.05 and **p < 0.01 vs control).
in G0/G1 phase checkpoint with increase in p21expression, which is a
cell cycle regulatory protein product of p53 gene transcription, and
slight increase in p27 expression, another cell cycle regulatory protein
that mediates cell cycle arrest [6]. These findings suggest that the an-
tiproliferative effects of designed Nutlin analog involved the activation
of p53 and cell cycle retention. Similarly to our findings, Nutlin analogs
have shown evidenced ability to mediate cell cycle arrest and increase
in p53, p21 and MDM2 expression both in vitro and in vivo
[12,16,29–31].
Analysis of mitochondrial function revealed a hyperpolarization of
mitochondrial membrane after treatment of cells with LQFM126 (2).
Despite mitochondrial membrane depolarization is commonly related
to apoptosis event; hyperpolarization has been described as an early
event in this process [32,33] suggesting the participation of mi-
tochondria in the cell death pathway activated by LQFM126 (2). Ad-
ditionally, we observed activation of initiator caspase-8 and -9 as well
as executioner caspase-3/7, which indicates the activation of intrinsic
and extrinsic caspase cascade. We hypothesize that hyperpolarization of
mitochondrial membrane would be followed by depolarization, release
of cytochrome c and activation of caspase-9. Cell surface pro-apoptotic
stimuli such as TNF (tumor necrosis factor) and Fas ligand might acti-
vate caspase-8. Both caspase-8 and -9 then activate caspase-3/7 leading
to cell death [34,35]. Our findings support earlier reports showing ac-
tivation of caspase-3 and -9 as main cell death pathway induced by
Nutlin analogs.
LQFM126 (2) antiproliferative activity included inhibition of VEGF
production in B16F10 cells, suggesting an antiangiogenic property.
Modulation of angiogenesis exerts an important role in cancer prog-
nostic and therapy, and VEGF represents the major key regulator of this
process. Likewise, Nutlin-3a, commercially available Nutlin-1 analog,
has also shown ability to suppress blood vessel formation and reduce
hemoglobin content in the tumoral mass in xenograft tumor models
Fig. 11. Cytotoxic profile of LQFM126 (2) at (25–200 μM) in 3T3 cells. Cell
viability was determined using neutral red uptake assay. All data are expressed
as mean
SD (n = 3).
new heterocyclic compound LQFM126 (2), a Nutlin-2 analog, which
showed enhanced antitumor activity in B16F10 cells as compared to the
other synthesized Nutlin analogs. The assumption of human drug effi-
cacy through in vitro nanomolar potency of anticancer agents has been
questioned especially due to the high rate failure and toxicity in clinical
trials [28]. Therefore, IC50 of new compounds should not be an ex-
cluding criterion used in evaluation of their efficacy likewise some
known antitumor agents used in anticancer therapy have in vitro IC50 in
the range of μM-mM.
LQFM126 (2) showed a concentration-dependent antiproliferative
effect on B16F10 cells leading to reduction of cell size, chromatin
condensation, cytoplasmic vacuolization and nuclear fragmentation.
These observations are suggestive of induction of cell death via apop-
tosis. To this end, the study focused on investigation of LQFM126 (2)
cell death pathways in B16F10. LQFM126 (2) promoted cell cycle arrest
8

A.C. Garcia da Silva, et al.
Chemico-BiologicalInteractions325(2020)109127
[36]. Additionally, Nutlin-3a has substantially reduced VEGF and hy-
poxia-inducible factor 1α (HIF-1α) production in neuroblastoma cells in
vitro and in vivo [37].
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LQFM126 (2) antiproliferative properties did not involve blockage of
MDM2-p53 complex formation (data not shown). Structural activity
studies as well as the effect on in vivo cancer animal models are needed
to detail the mechanisms of action of LQFM126 (2). The preliminary
toxicity study suggested the low-toxicity profile of LQFM126, once its
estimated LD50 yielded to UN GHS Category 4 (LD50 > 300–2000 mg/
kg) for acute oral systemic toxicity. Therefore, our findings showed the
molecular simplification as advantaged strategy for synthesis of de-
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CRediT authorship contribution statement
Artur Christian Garcia da Silva: Methodology, Investigation, Data
curation, Writing - original draft. Bruna dos Santos Rodrigues:
Writing - original draft. Wanessa Machado Andrade: Methodology,
Investigation, Data curation. Thaís Rosa Marques dos Santos:
Methodology, Investigation. Flávio Silva de Carvalho: Methodology,
Investigation. Germán Sanz: Methodology, Investigation. Boniek G.
Vaz: Formal analysis, Supervision. Luciano M. Lião: Formal analysis,
Supervision. Ricardo Menegatti: Conceptualization, Supervision.
Marize Campos Valadares: Conceptualization, Project administration,
Supervision, Funding acquisition.
[19] P.L. Benfica, R.I. de Ávila, B. dos S. Rodrigues, A.P. Cortez, A.C. Batista,
M.P.N. Gaeti, E.M. Lima, K.R. Rezende, M.C. Valadares, 4-Nerolidylcatechol:
apoptosis by mitochondrial mechanisms with reduction in cyclin D1 at G0/G1 stage
of the chronic myelogenous K562 cell line, Pharm. Biol. 55 (2017) 1899–1908,
https://doi.org/10.1080/13880209.2017.1311351.
[20] C.A. Schneider, W.S. Rasband, K.W. Eliceiri, NIH Image to ImageJ: 25 years of
image analysis, Nat. Methods 9 (2012) 671–675, https://doi.org/10.1038/nmeth.
2089.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
[21] OECD, Organization for Economic Cooperation and Development, Guidance
Document on using Cytotoxicity Tests to Estimate starting Doses for Acute Oral
Systemic Toxicity Tests vol. 129, (2010) Paris.
Acknowledgments
[22] ICCVAM, In Vitro Cytotoxicity Test Methods for Estimating Starting Doses for Acute
Oral Systemic Toxicity Test, (2006).
This work was supported by the Conselho Nacional de
Desenvolvimento Científico
e
Tecnológico (CNPq, Brazil), the
[23] I.L. Finar, R.J. Hurlock, The preparation of some trinitrophenylpyrazoles, 589, J.
Chem. Soc. (1957) 3024–3027, https://doi.org/10.1039/JR9570003024.
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V. De Oliveira, L.C. Cunha, F.G.F. Oliveira, J.L. Paz, M.N. Eberlin, R. Menegatti,
Chemoselective and regiospecific formylation of 1-phenyl-1H-pyrazoles through the
duff reaction, Synth. Commun. 43 (2013) 1633–1639, https://doi.org/10.1080/
00397911.2012.657764.
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES,
Brazil) and by Financiadora de Estudos e Projetos (FINEP, Brazil).
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