Targeting Several Biologically Reported Targets of Glioblastoma Multiforme by Assaying 2D and 3D Cultured Cells

Article abstract of DOI:10.1007/s10571-021-01072-9

Glioblastoma multiforme (GBM) is account for 70% of all primary malignancies of the central nervous system. The median survival of human patients after treatment is around 15?months. There are several biological targets which have been reported that can b

Full text of DOI:10.1007/s10571-021-01072-9

Cellular and Molecular Neurobiology
https://doi.org/10.1007/s10571-021-01072-9
ORIGINAL RESEARCH
Targeting Several Biologically Reported Targets of Glioblastoma
Multiforme by Assaying 2D and 3D Cultured Cells
Yudibeth Sixto‑López1 · Emilie Marhuenda2 · Juan Benjamin García‑Vazquez1
·
Manuel Jonathan Fragoso‑Vazquez3 · Martha Cecilia Rosales‑Hernández4 · Oscar Zacarías‑Lara1
·
David Méndez‑Luna1 · José Antonio Gómez‑Vidal5 · David Cornu6 · Bakalara Norbert2
José Correa‑Basurto1
·
Received: 5 August 2020 / Accepted: 1 March 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
Glioblastoma multiforme (GBM) is account for 70% of all primary malignancies of the central nervous system. The median
survival of human patients after treatment is around 15 months. There are several biological targets which have been
reported that can be pursued using ligands with varied structures to treat this disease. In our group, we have developed
several ligands that target a wide range of proteins involved in anticancer efects, such as histone deacetylase (HDACs),
G protein-coupled estrogen receptor 1 (GPER), estrogen receptor-beta (ERβ) and NADPH oxidase (NOX), that were
screened on bidimensional (2D) and tridimensional (3D) GBM stem cells like (GSC). Our results show that some HDAC
inhibitors show antiproliferative properties at 21–32 µM. These results suggest that in this 3D culture, HDACs could
be the most relevant targets that are modulated to induce the antiproliferative efects that require in the future further
experimental studies.
Keywords Glioblastoma multiforme · HDAC · GPER · ER · NOX · 3D cell culture
Introduction
years, in this study we selected some of them in order to
evaluate potential antimigratory or antiproliferative efect,
leading potentially to interesting therapy for GBM during
deeper investigations. It has been demonstrated that his-
tone deacetylases (HDACs), (able to remove acetyl groups
from histones protein on DNA) (Was et al. 2019; Lee et al.
2017), displays aberrant expression in cancer. HDACs
Glioblastoma multiforme (GBM) is one of the most com-
mon glial tumors, presenting poor prognosis, and less than
5% survival over the 5 years after initial diagnosis (Liu
et al. 2006; Kim et al. 2012; Singh et al. 2004). Multi-
ple therapeutic strategies and biomarkers have risen over
3
* Juan Benjamin García-Vazquez
Departamento de Química Orgánica, Escuela Nacional
benjagv_5202@hotmail.com
de Ciencias Biológicas, Instituto Politécnico Nacional
Prolongación de Carpio y Plan de Ayala S/N. Col. Casco de
Santo Tomas, 11340 Ciudad de México, Mexico
* José Correa-Basurto
corrjose@gmail.com
4
Laboratorio de Biofísica y Biocatálisis, Escuela Superior de
Bakalara Norbert
Medicina, Instituto Politécnico Nacional, Plan de San Luis y
Díaz Mirón s/n, 11340 Ciudad de México, Mexico
norbert.bakalara@enscm.fr
1
5
Laboratorio de Diseño y Desarrollo de Nuevos Fármacos y
Facultad de Farmacia, Departamento de Química
Productos Biotecnológicos, Sección de Estudios de Posgrado
e Investigación, Escuela Superior de Medicina, Instituto
Politécnico Nacional, Plan de San Luis y Díaz Mirón,
11340 México City, Mexico
Farmacéutica y Orgánica, Universidad de Granada, Campus
de Cartuja, 18071 Granada, Spain
6
Institut Europeen des Membranes, IEM, UMR-5635,
Univ. Montpellier, ENSCM, CNRS, Montpellier, France
2
Institut des Neurosciences de Montpellier, INM, U-1051,
Univ. Montpellier, CHU de Montpellier, ENSCM, INSERM,
Montpellier, France
Vol.:(0123456789)
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Cellular and Molecular Neurobiology
regulate the expression and activity of numerous proteins
involved in cancer initiation, proliferation and progression
and seem to be a potential target against GBM (Yelton
and Ray 2018; Lee et al. 2017). Some HDAC inhibitors
already developed exhibit hydroxamic acid groups that
chelate pivotal Zn2+ ions to the catalytic activity of the
enzyme (Rajak et al. 2013), but due to a lack of specifcity
of those compounds, other chemical strategies have been
employed to reach the nonpolar cavities of HDACs (Rajak
et al. 2013; Wang 2009).
Materials and Methods
Chemical Synthesis
All commercial grade reagents were used without further
purifcation. Analytical thin-layer chromatography (TLC)
was performed on silica gel 60 F254-coated aluminum
sheets (0.25 mm thickness) with a fuorescent indicator. Vis-
ualization was accomplished with UV light (254 nm). Flash
chromatography was performed using silica gel 60 (230–400
mesh). Melting points were determined in open capillary
tubes with an IA 91,000 electrothermal melting point appa-
ratus (Electrothermal, Bibby Scientifc, Stafordshire, ST15
OSA, UK) and were uncorrected. 1H and 13C NMR spectra
were recorded on either Bruker Avance III 750 or 400 MHz
spectrometers or on a 500 MHz Varian System using deuter-
ated dimethyl sulfoxide (DMSO-d6) or chloroform (CDCl3)
as the solvent and TMS as the internal standard. Chemical
shifts are given in ppm (δ) and are referenced to TMS as
the internal standard. Coupling constant values are quoted
in Hertz, with normal abbreviations (s, singlet; d, doublet;
t, triplet; br, broad; m, multiplet). For complete data assign-
ments, 2D HSQC and HMBC NMR spectra were used. IR
spectra were obtained on a Spectrum 2000 Perkin Elmer
FT-IR spectrometer. Only signifcant absorption bands are
shown with absorption values expressed as wavenumbers
(cm−1). Electrospray ionization (ESI) high-resolution mass
spectrometry was performed with a Bruker micrQTOF-Q II
instrument and an Agilent 6545 Q-TOF LC/MS. The chemi-
cal purity was determined by HPLC using an Agilent 1200
Infnity Series system.
G protein estrogen receptor (GPER) is implicated in
diverse biological efects, including regulation of immune,
endocrine, neuronal and cardiovascular functions. In can-
cer, GPER activation is already well described as regu-
lating cell growth and proliferation in several cell line,
migration and apoptotic cell death, but has not been stud-
ied in Glioma. Consequently, the use of GPER modulators
to explore his role in GBM proliferation and migration
appears as a great tool for in vivo studies, as well as pos-
sible target to treat GBM (Feldman and Limbird 2017).
Estrogens have shown implication in development of glio-
mas, and estrogens appear having neuroprotective efects
and improve glioblastoma outcome for women (Wan et al.
2018; Lan et al. 2017). Want et al. reported higher expres-
sion of ERs receptor in glioma cells in comparison with
glial cells (Wan et al. 2018) in which and ERβ appears as a
tumor suppressor in GBM (Liu et al. 2018). Consequently
ERβ also appears as a promising target to treat GBM (Lan
et al. 2017; Zhou et al. 2019).
Free radicals in GBM play an important role in cell
proliferation being the NADPH oxidase (NOX) as puta-
tive targets for cancer therapy. NOX family is the main
source of reactive oxygen species (ROS) (Meitzler et al.
2014) which could be an important mediator of neuroin-
fammation (Yang et al. 2020). The use of NOX inhibitors
has been reported to inhibit invadopodium formation, the
infammatory response and cancer cell migration in vitro.
Furthermore, NOXs as pharmacological targets appear as
an interesting strategy for GBM treatment (Munson et al.
2013; Nayernia et al. 2014).
Synthesis of NADPH Oxidase Inhibitors Ia‑Ib
3,3′‑Dimethoxy‑5,5′‑Dipropyl‑(1,1′‑Biphenyl)‑2,2′‑Diol (Ia)
The reaction was carried out as previously described by
our group (Macias Perez et al. 2017). Heptahydrate fer-
rous sulfate (0.075 g, 0.3 mmol) and potassium persulfate
(0.81 g, 3 mmol) were added to a solution of 2-methoxy-
4-propylphenol (1 g, 6 mmol) in 200 mL of hot water/
MeOH (3:1 v/v). The mixture was boiled and stirred for
1 h. After cooling to room temperature, the precipitate was
collected and dissolved in 100 mL of a 4 N aqueous solution
of NaOH. Subsequently, the crude material was precipitated
by the addition of 100 mL of a 6 N aqueous solution of
HCl. The resulting precipitate was fltered and washed with
hot (90 °C) deionized water (3×100 mL) and hot methanol
(1×100 mL). The resulting solid was purifed by fash col-
umn chromatography with hexane/EtOAc (1:1) to obtain Ia
(Scheme 1) in 66% yield. Full chemical characterization was
reported (Macias Perez et al. 2017).
In this study, we explored a diverse set of ligands contain-
ing a wide range of functional groups as possible therapeutic
agents to treat GBM. Then, 23 compounds were synthetized,
including NADPH oxidase inhibitors (Ia-Ib), ER modula-
tors (IIa-IId, IIIa-IIId), HDAC inhibitors (IVa-IVb, VIa-
VIg) and GPER inhibitors (Va-Vd). Then, we performed
a frst preliminary screening in 2D and 3D cell culture to
observe potential antiproliferative and/or antimigrating
efect on Glioblastoma Stem Cell (GSC). This study is a
frst step leading to a selection on compound for future and
deeper in vitro and in vivo investigations.
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Cellular and Molecular Neurobiology
(Z)‑3‑(4‑Bromophenyl)‑2‑(4‑Fluorophenyl)Acrylonitrile (IIc)
1,1′‑(6,6′‑Dihydroxy‑5,5′‑Dimethoxy‑(1,1′‑Biphenyl)‑3,3′‑Di
yl)‑Bis‑(Ethan‑1‑One) (Ib)
Application of the typical procedure using 2-(4-fuorophe-
nyl)acetonitrile (0.2 g, 1.48 mmol) and 4-bromobenzalde-
hyde (0.3 g, 1.63 mmol) gave acrylonitrile IIc (0.43 g, 96%).
The spectral data of IIc were identical to those reported
(Csuros et al. 1962).
The reaction was carried out as described elsewhere (Macias
Perez et al. 2017). Ferrous sulfate heptahydrate (0.075 g,
0.3 mmol) and potassium persulfate (0.81 g, 3 mmol) were
added to a solution of apocynin (1 g, 6 mmol) in 200 mL
of water (90 °C). The reaction mixture stirred in a boiling
water bath for 0.5 h. After cooling to room temperature, the
precipitate was collected and dissolved in 100 mL of a 4 N
aqueous solution of NaOH. Subsequently, the product was
precipitated by adding 100 mL of a 6 N aqueous solution
of HCl. The fnal precipitate was fltered and washed with
deionized water at 90 °C (3 × 100 mL) and hot methanol
(1 × 100 mL) to obtain Ib (Scheme 2) in 63% yield. Full
chemical characterization was reported (Macias Perez et al.
2017).
(Z)‑2‑(4‑Bromophenyl)‑3‑(4‑Fluorophenyl)Acrylonitrile (IId)
Application of the typical procedure using 2-(4-bromophe-
nyl)acetonitrile (0.2 g, 1.02 mmol) and 4-fuorobenzalde-
hyde (0.14 g, 1.12 mmol) gave acrylonitrile IId (0.26 g,
85%). The spectral data of IId were identical to those
reported (Buu-HoÏ et al. 1957).
Synthesis of Propanenitriles IIIa‑IIId
NaBH4 (1 equiv) was added to a 60–70 °C solution of acry-
lonitriles IIa-IId (1 equiv) in 10 mL of EtOH under a N2
atmosphere. After stirring for 2.5 h, the reaction was cooled
to room temperature and quenched with water. The reac-
tion mixture was acidifed with 6 M HCl, extracted with
EtOAc (3×25 mL), washed with brine, dried over Na2SO4
and concentrated in vacuo. The residue was purifed by fash
chromatography on silica (hexane/EtOAc 9:1) to give propi-
onitriles IIIa–IIId (Scheme 4).
Synthesis of Acrylonitriles IIa‑IId as ERβ Modulators
The synthesis of nitriles followed a previously reported
method (Vaccaro et al. 1996; Martinez-Archundia et al.
2018). A solution of 40% aqueous KOH (0.23 mL/mmol
nitrile) was diluted with EtOH (0.46 mL/mmol nitrile) and
added at room temperature to a solution of arylaldehyde (1.1
equiv) and arylacetonitrile (1.0 equiv) in EtOH (0.35 mL/
mmol nitrile). The resulting mixture stirred for 3 h at the
same temperature, quenched with water (5 mL) and 5%
aqueous HCl (1.5 mL), extracted with EtOAc (3×25 mL),
washed with brine, dried over Na2SO4 and concentrated in
vacuo. The residue was purifed by fash chromatography
on silica (hexane/EtOAc 9:1) to give acrylonitriles IIa-IId
(Scheme 3).
2,3‑Bis(4‑Fluorophenyl)Propanenitrile (IIIa)
Application of the typical procedure using acrylonitrile IIa
(0.2 g, 0.83 mmol) gave propanenitrile IIIa (0.18 g, 91%).
The spectral data of IIIa were identical to those reported
(Buschauer et al. 1992).
(Z)‑2,3‑Bis(4‑Fluorophenyl)Acrylonitrile (IIa)
2,3‑Bis(4‑Bromophenyl)Propanenitrile (IIIb)
Application of the typical procedure using 2-(4-fuorophe-
nyl)acetonitrile (0.2 g, 1.48 mmol) and 4-fuorobenzalde-
hyde (0.2 g, 1.61 mmol) gave acrylonitrile IIa (0.35 g, 97%).
The spectral data of IIa were identical to those reported
(Buu-HoÏ et al. 1957).
Application of the typical procedure using acrylonitrile IIb
(0.2 g, 0.55 mmol) gave propanenitrile IIIb (0.19 g, 95%).
The spectral data of IIIb were identical to those reported
(Dann et al. 1971).
3‑(4‑Bromophenyl)‑2‑(4‑Fluorophenyl)Propanenitrile (IIIc)
(Z)‑2,3‑Bis(4‑Bromophenyl)Acrylonitrile (IIb)
Application of the typical procedure using acrylonitrile IIc
(0.2 g, 0.66 mmol) gave propanenitrile IIIc (0.196 g, 97%)
as a white solid. Chemical purify 93.4% (HPLC). IR (KBr)
Ʋmax: 3326, 2928, 2850, 1741, 1610, 1573, 1473 cm−1. 1H
NMR (500 MHz, DMSO-d6)): δ 7.46 (2H, dm, J=8.4 Hz,
Hm’), 7.39 (2H, dd, J = 8.8, 5.3 Hz, Ho), 7.20 (2H, dd,
J=8.8, 8.8 Hz, Hm), 7.17 (2H, dm, J=8.4 Hz, Ho’), 4.52
(1H, dd, J=7.8, 7.7 Hz, CH), 3.12 (2H, d, J=7.8 Hz, CH2).
13C NMR δ (188 MHz, DMSO-d6): 162.1 (d, J=244.3 Hz,
Application of the typical procedure using 2-(4-bromophe-
nyl)acetonitrile (0.2 g, 1.02 mmol) and 4-bromobenzalde-
hyde (0.21 g, 1.12 mmol) gave acrylonitrile IIb (0.32 g,
87%). The spectral data of IIb were identical to those
reported (Dann et al. 1971).
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Cellular and Molecular Neurobiology
Cp), 136.6 (Ci’), 132.1 (d, J = 3.0 Hz, Ci), 131.9 (2C,
Cm’), 131.6 (2C, Co’), 130.1 (2C, d, J=8.4 Hz, Co), 121.2
(Cp’), 120.7 (CN), 116.2 (2C, d, J = 21.6 Hz, Cm), 39.4
(CH2), 37.2 (CH). ESI-HRMS calculated for C15H11BrFN:
[M+Na]: 325.995, found: 325.994.
precursor for the chemical synthesis of GPER ligands Va-Vd
(Scheme 6) (Martínez-Muñoz et al. 2018).
Methyl‑(3aS,4R,9bR)‑4‑(6‑Bromobenzo[d][1,3]
Dioxol‑5‑yl)‑3a,4,5,9b‑Tetrahydro‑3H‑Cyclopenta[c]
Quinoline‑8‑Carboxylate, (Va)
2‑(4‑Bromophenyl)‑3‑(4‑Fluorophenyl)Propanenitrile (IIId)
To a stirred solution of G1-PABA (0.25 g, 0.6 mmol) in dry
THF (5 mL) and methanol (10 mL) at 0 °C, thionyl chloride
(1.32 mL, 1.8 mmol) was added dropwise. The resulting
solution was heated to refux overnight. The solvent and the
excess thionyl chloride were then removed under reduced
pressure. The resulting crude material was purifed by fash
chromatography using hexane/EtOAc (8:2) as the eluent,
giving the expected product Va as a yellow solid (0.23 g,
90% yield). Full chemical characterization was reported
(Zacarias-Lara et al. 2019; Dann et al. 1971).
Application of the typical procedure using acrylonitrile IId
(0.2 g, 0.66 mmol) gave propanenitrile IIId (0.19 g, 94%).
The spectral data of IIId were identical to those reported
(Jana et al. 2018).
Synthesis of Valproic Acid Derivatives IVa‑IVb
as HDAC Inhibitors
Thionyl chloride SOCl2 (3 mmol) was added dropwise to VPA
(3.5 mmol) at 0 °C under a nitrogen atmosphere with constant
stirring. The reaction mixture stirred for 1 h under the same
conditions and then overnight at room temperature. After this
time, Et3N (0.55 g, 5.45 mmol), the corresponding aniline
(2.8 mmol) and 5 mL of hexane were added, and the reaction
continued stirring for 3 h. At the end of the reaction time,
15 mL of EtOAc was added and followed by washing with a
saturated NaHCO3 solution (1×15 mL) and brine (1×15 mL),
and the organic phase was dried over anhydrous Na2SO4
(Scheme 5). The solvent was evaporated under reduced pres-
sure, and the residue was purifed by column fash chromatog-
raphy using an eluent mixture of hexane/EtOAc (8:2).
(3aS,4R,9bR)‑4‑(6‑(3‑Methoxyphenyl)Benzo[d][1,3]
Dioxol‑5‑yl)‑3a,4,5,9b‑Tetrahydro‑3H‑Cyclopenta[c]
Quinoline‑8‑Carboxylic Acid (Vb)
Compound Vb was obtained through a Suzuki Miyaura cross-
coupling reaction (Miyaura et al. 1979). A solution of K2CO3
(0.27 g, 1.92 mmol) in dimethylformamide (DMF, 5 mL) and
water (5 mL) was stirred for 15 min under a N2 atmosphere.
Subsequently, G1-PABA (0.2 g, 0.48 mmol), (3-methoxyphe-
nyl) boronic acid (0.09 g, 0.58 mmol), triphenylphosphine
(0.016 g, 0.058 mmol) and Pd(AcO)2 (0.004 g, 0.019 mmol)
were added. The reaction mixture was heated to 100 °C for
24 h. Once the reaction was complete, it was allowed to cool
to room temperature, and the resulting mixture was extracted
with EtOAc (3×25 mL). The organic layer was washed with
distilled water and brine, dried over anhydrous Na2SO4 and
concentrated under reduced pressure. Full chemical charac-
terization was reported (Mendéz-Luna D et al. 2021 2019).
N‑(3‑Fluorophenyl)‑2‑Propylpentanamide (IVa)
Application of the typical procedure using VPA (0.5 g,
3.5 mmol) and 3-fuoroaniline (0.31 g, 2.8 mmol) gave VPA
derivative IVa (0.56 g, 85%). The spectral data of IVa were
identical to those reported (Muñoz et al. 2020).
N‑(3‑Methoxyphenyl)‑2‑Propylpentanamide (IVb)
(3aS,4R,9bR)‑4‑(6‑(3‑Nitrophenyl)Benzo[d][1,3]dDi‑
oxol‑5‑yl)‑3a,4,5,9b‑Tetrahydro‑3H‑Cyclopenta[c]Quino‑
line‑8‑Carboxylic acid (Vc)
Application of the typical procedure using VPA (0.5 g,
3.5 mmol) and 3-methoxyaniline (0.35 g, 2.8 mmol) gave
valproic acid derivative IVb (0.56 g, 82%). The spectral data
of IVb were identical to those reported (Muñoz et al. 2020).
Compound Vc was obtained by following a previously
reported method (Liu et al. 2005). To a solution of K2CO3
Synthesis of GPER Ligands Va‑Vd
(3aS,4S,9bR)‑4‑(6‑Bromobenzo[d][1,3]
Dioxol‑5‑yl)‑3a,4,5,9b‑Tetrahydro‑3H‑Cyclopenta[c]
Quinoline‑8‑Carboxylic Acid (G1‑PABA)
A previous method reported by our research group (Mar-
tínez-Muñoz et al. 2018; Zacarias-Lara et al. 2018) was
used to obtain G1-PABA. This compound was used as a
Scheme 1 Synthesis of NADPH oxidase inhibitor Ia
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Cellular and Molecular Neurobiology
(0.27 g, 1.92 mmol) in a 1:1 mixture of MeOH/H2O
(15 mL) with constant stirring was added G1-PABA (0.2 g,
0.48 mmol), (3-nitrophenyl)boronic acid (0.1 g, 0.58 mmol),
PEG2000 (0.060 g, 0.10 mmol) and Pd(AcO)2 (0.004 g,
0.019 mmol) (see Scheme 6). The reaction mixture was
heated to refux at 70 °C for 5 h under a N2 atmosphere.
The solution was then cooled to room temperature, and the
resulting mixture was extracted with EtOAc (3 × 25 mL).
The organic phase was washed with distilled water and
brine, dried over anhydrous sodium sulfate (Na2SO4) and
concentrated under reduced pressure. The residue was puri-
fed by fash chromatography using a mixture of hexane/
EtOAc (7:3) as the eluent. Full chemical characterization
was reported (Mendéz-Luna et al. 2021, 2019).
26%). Full chemical characterization was reported (Sixto-
Lopez et al. 2020).
(S)‑N‑(1‑(Hydroxyamino)‑1‑Oxo‑3‑Phenylpro‑
pan‑2‑yl)‑2‑Propylpentanamide (VIb)
The procedure was followed starting with 0.12 mmol
of resin, HATU (0.19 g, 0.50 mmol), HOAt (0.068 g,
0.50 mmol), DIPEA (87 µL, 0.50 mmol), Fmoc-L-pheny-
lalanine (0.192 g, 0.50 mmol) and valproic acid (0.072 g,
0.50 mmol). The resulting compound was purifed by recrys-
tallization from DCM. A white solid was obtained (0.013 g,
36%). Full chemical characterization was reported (Sixto-
Lopez et al. 2020) (Scheme 7).
(3aS,4R,9bR)‑4‑(6‑Bromobenzo[d][1,3]Dioxol‑5‑yl)‑5‑(Tert
‑Butoxycarbonyl)‑3a,4,5,9b‑Tetrahydro‑3H‑Cyclopenta[c]
Quinoline‑8‑Carboxylic Acid (Vd)
(R)‑4‑Butyl‑N‑(1‑(Hydroxyamino)‑1‑Oxo‑3‑Phenylpro‑
pan‑2‑yl)Benzamide (VIc)
The procedure was followed starting with 0.14 mmol of
resin, DIC (57 µL, 0.57 mmol), HOAt (0.078 g, 0.57 mmol),
Fmoc-D-phenylalanine (1.22 g, 0.57 mmol) and 4-butylben-
zoic acid (0.102 g, 0.57 mmol). The compound was purifed
by recrystallization from DCM. A white solid was obtained
(0.025 g, 51%). Full chemical characterization was reported
(Sixto-Lopez 2013).
To a cold solution of G1-PABA in DMF (3.0 mL), triethyl-
amine (336 μL, 2.4 mmol) was added dropwise with continu-
ous stirring for 30 min at the same temperature. Subsequently,
di-tert-butyl dicarbonate (Boc2O, 0.53 g, 2.4 mmol) was
added, and the reaction mixture was allowed to reach room
temperature and continued overnight with vigorous stirring.
The progress of the reaction was monitored by TLC, and the
resulting mixture was extracted with EtOAc (3×25 mL). The
organic phase was washed with water and brine, dried over
anhydrous Na2SO4 and evaporated to dryness. The residue
obtained was purifed by fash chromatography using a mobile
phase of hexane/EtOAc (9:1). Full chemical characterization
was reported (Mendéz-Luna et al. 2021, 2019) (Scheme 6).
(S)‑4‑Butyl‑N‑(1‑(Hydroxyamino)‑3‑(Naphthalen‑2‑yl)‑
1‑Oxopropan‑2‑yl)Benzamide (VId)
The procedure was followed starting with 0.14 mmol of resin,
DIC (57 µL, 0.57 mmol), HOAt (0.078 g, 0.57 mmol), Fmoc-
3-(2-naphthyl)-L-alanine (0.251 g, 0.57 mmol) and 4-butyl-
benzoic acid (0.102 g, 0.57 mmol). The resulting compound
was purifed by recrystallization from DCM. A white solid
Synthesis of Hydroxamic Derivatives VIa‑VIg
4‑Butyl‑N‑(2‑(Hydroxyamino)‑2‑Oxoethyl)Benzamide (VIa)
The procedure started with 0.12 mmol of resin, HATU
(0.18 g, 0.48 mmol), HOAt (0.066 g, 0.48 mmol), DIPEA
(84 µL, 0.48 mmol), Fmoc-glycine (0.144 g, 0.48 mmol)
and 4-butylbenzoic acid (0.086 g, 0.48 mmol). The result-
ing compound was purified by flash chromatography
using EtOAc/MeOH/MeCN/H2O as the mobile phase
(70/2.5/1.25/1.25). A white solid was obtained (0.008 g,
Scheme 3 Synthesis of acrylonitriles IIa-IId
Scheme 4 Synthesis of propanenitriles IIIa–IIId
Scheme 2 Synthesis of NADPH oxidase inhibitor Ib
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Cellular and Molecular Neurobiology
according to a protocol used to derive NS from the
adult human spinal cord as described by Dromard et al.
(Dromard et al. 2008) and adapted by Guichet et al. for
Gli4 cells (Guichet et al. 2013). In proliferation condition
when GBM cells form NS, cells were cultivated in DMEM/
F12 medium supplemented with glucose, glutamine, insu-
lin, N2, epidermal growth factor and fbroblast growth
factor (proliferation medium). For migration, GSCs cell
culture was realized in DMEM/F12 medium supplemented
with glucose, glutamine, insulin, N2 and fetal bovine
serum (0.5%). Prior to cell seeding, 2D or NF were either
functionalized or not with poly-D-lysine added overnight
and then the addition of LN (sigma L2020) (0.05 mg/mL)
1 h at 37 °C (Scheme 6).
Scheme 5 Synthesis of valproic acid derivatives (IVa-IVb)
was obtained (0.039 g, 70%). Full chemical characterization
was reported (Sixto-Lopez et al. 2020) (Scheme 7).
(S)‑4‑Butyl‑N‑(3‑(4‑Fluorophenyl)‑1‑(Hydroxyamino)‑1‑Ox‑
opropan‑2‑yl)Benzamide (VIe)
For migration assay, same size GBM NS were performed
by seeding 5000 cells in round bottom wells and remained
in culture during 2 days until formation of single neuro-
sphere due to sedimentation. Compounds were prepared in
100% DMSO and diluted in media to obtain in each condi-
tion a fnal percentage of 0.3% DMSO. Then GSCs NS were
deposited on the top of NF+LN and let to migrate during
5 days in total, with 72 h in presence of either HDAC com-
pounds or 0.3% DMSO alone for control.
The procedure was followed starting with 0.14 mmol of
resin, DIC (57 µL, 0.57 mmol), HOAt (0.078 g, 0.57 mmol),
Fmoc-L-Phe(4-F)-OH (0.232 g, 0.57 mmol) and 4-butylben-
zoic acid (0.102 g, 0.57 mmol). The compound was purifed
by fash chromatography using EtOAc/MeOH/MeCN/H2O
as the mobile phase (70/2.5/1.25/1.25). A salmon colored
solid was obtained (0.102 g, 43%). Full chemical characteri-
zation was reported (Sixto-Lopez et al. 2020).
(S)‑4‑Butyl‑N‑(1‑(Hydroxyamino)‑3‑(4‑Iodophenyl)‑1‑Oxo‑
propan‑2‑yl)Benzamide (VIf)
Immunofuorescence
GSCs cultures were fxed by 4% PFA. Cell and brain sec-
tions were blocked and permeabilized using PBS—triton
0.5%—horse serum 5%. Fluorochrome-coupled phalloi-
din 546 was incubated 2 h at room temperature (dilution
1/500). Image acquisition was realized using Zeiss Axio-
imager Z1/Zen (with an apotome). Migration quantifca-
tions were done using ZEN 2012 software, and migration
capacity was quantifed by measuring an area of migration.
To measure migration areas, we subtracted the area of
the NS containing non-migrating cells from the total area
where cells were detected.
The procedure was followed starting with 0.14 mmol of
resin, DIC (57 µL, 0.57 mmol), HOAt (0.078 g, 0.57 mmol),
Fmoc-L-Phe(4-I)-OH (0.294 g, 0.57 mmol) and 4-butylben-
zoic acid (0.102 g, 0.57 mmol). The compound was purifed
by recrystallization from DCM. A white solid was obtained
(0.044 g, 66%). Full chemical characterization was reported
(Sixto-Lopez 2013) (Scheme 5).
(S)‑4‑Butyl‑N‑(1‑(Hydroxyamino)‑3‑(4‑Nitrophenyl)‑1‑Oxo‑
propan‑2‑yl)Benzamide (VIg)
The procedure was followed starting with 0.14 mmol of
resin, DIC (57 µL, 0.57 mmol), HOAt (0.078 g, 0.57 mmol),
Fmoc-L-Phe(4-NO2)-OH (0.248 g, 0.57 mmol) and 4-butyl-
benzoic acid (0.102 g, 0.57 mmol). The compound was puri-
fed by fash chromatography using EtOAc/MeOH/MeCN/
H2O as the mobile phase (70/2.5/1.25/1.25). A white solid
was obtained (0.03 g, 60%). Full chemical characterization
was reported (Sixto-Lopez et al. 2020).
Antiproliferative Activity: MTT Assay
The GBM stem cells are primary cell culture coming from
patient, gently donated by Luc Bauchet from the Hospital
Biological Evaluation
Cell Culture
Isolation and culture of GBM cells using the classical
nonadherent neurospheres (NS) method was performed
Scheme 6 Synthesis of GPER ligands (Va-Vd)
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Cellular and Molecular Neurobiology
Scheme 7 General solid-phase synthesis of hydroxamic acid derivatives VIa-VIg (Sixto-Lopez et al. 2020)
Guy de Chauliac and isolated by the Professor Jean-Philippe
Hugnot (Guichet et al. 2013; Saleh et al. 2019). Gli4 were
seeded into 96-well plates at 3000 cells per well in a 2D
system and 15,000 cells in a 3D system, 2D culture were
culture with laminin (2D+LN) or without laminin (2D-LN),
while 3D systems were also culture with laminin (3D+LN)
as single cells or without laminin (3D-LN) for collective
migrations. Once the cells were adherent, diferent con-
centrations of the compound were added at the µM range
(Table 1) to each well at a fnal DMSO concentration of
0.3%, and Phostine (PST3) was tested as positive control at
1 µM in 0.3% DMSO. The MTT assay was carried out as
previously described 72 h after the addition of compounds
(Mosmann 1983). Active compounds were tested in four
independent experiments, and each experiment was carried
out in triplicate for each condition.
Results and Discussion
Cancer cells are presenting numerous deregulation favor-
ing the overexpression of several proteins which could be
a target for drug design as GPER1 (Martínez-Muñoz et al.
2018; Zacarias-Lara et al. 2018) leading to cell proliferation
and migration. GBM is a disease with a poor survival rate
and limited therapeutic options. Therefore, a multitargeted
approach for drug screening is necessary in order to explore
several pharmacological options for GBM treatment (Shea
et al. 2016). Traditional 2D cell cultures (monolayers) are
widely used to assay antiproliferative activity as one of the
frst approaches to decide if a compound should be further
investigated as an anticancer drug (Shan et al. 2018; Ver-
jans et al. 2018). However, it is well admitted that the use
of this strategy is the cause of the failure to discover new
anticancer drugs because 2D cell cultures not mimic the
in vivo 3D fbrillar microenvironment, cell–cell interactions
and cell–extracellular matrix interactions (Lv et al. 2017).
Therefore, 3D cultures may be a powerful tool for drug dis-
covery, ofering a more realistic approach for drug screening
(Lv et al. 2017). Our group has previously focused work
on drug design employing in silico tools (Zacarias-Lara
et al. 2019; Sixto-Lopez et al. 2020). In this drug screening
preliminary study, compounds are chemically synthesized
Statistical Analyses
The experiments were carried out at least in triplicate,
depending on the number of independent variables. Stu-
dent’s or ANOVA statistical tests were applied using Serf
software (bram.org/serf/CellsAndMaps.php) and GraphPad.
EC50 and Ki values were calculated using the Hill equation
from the dose−log response curves.
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Cellular and Molecular Neurobiology
Table 1 Percent inhibition of cell proliferation of the tested com-
pounds obtained in 2D and 3D GBM cell cultures with or without
laminin
Table 2 Final list of compounds that showed antiproliferative activity
in 2D and 3D GBM cell cultures with or without laminin
Compound
% Inhibition
2D-LN
Compound Concentra-
tion tested
% Inhibition
3D-LN
2D+LN
3D+LN
2D-LN 3D-LN 2D+LN 3D+LN
(µM)
IIa
VIb
VIc
VId
VIe
VIf
Phostine
56.301
63.066
48.657
58.91
48.143
55.017
56.517
36.828
28.205
51.541
45.936
41.135
41.902
10.591
68.923
68.872
62.955
56.7
47.099
55.994
70.898
53.692
61.086
47.845
56.946
54.078
44.821
65.767
Ia
Ib
IIa*
IIb
IIc
10
10
10
10
10
10
10
10
10
10
42
40
23
22
22
19
40
32
29
26
28
21
26
1
7.030
−4.419
56.301
−91.405
−38.858
−29.136
−11.432
−27.736
−12.795
19.213
3.205 −25.811 37.878
4.8559 −7.297 2.518
11.724
−11.233
−22.627 −106.95 −16.014
2.733 −80.941
−12.222 −11.288
63.066
48.657
58.91
48.143
55.017
18.388 −9.329 −25.771
14.857
8.04
3.699
53.692
8.607
36.828 68.923
32.654 −24.236
14.843 −31.901
3.179
IId
3.223
23.33
10.921
24.519
37.053
8.204 −17.542
10.269 −22.214
IIIa
IIIb
IIIc
IIId
IVa
IVb
Va
Vb
Vc
Vd
VIa
VIb*
VIc*
VId*
VIe*
VIf*
VIg
Phostine*
2D-LN=2D cell culture without laminin; 2D+LN=2D cell cul-
ture with laminin; 3D-LN=3D cell culture without laminin;
3D+LN=3D cell culture with laminin
5.391
13.261
9.829 −24.944
7.376 −35.252
37.193
11.854
6.772
Table 1) appeared unefcient on the GSC for the: NADPH
oxidase inhibitors Ia-Ib, estrogen receptor modulators IId
and IIIa-IIId, valproic acid derivatives IVa-IVb, GPER
ligands Va-Vd and two hydroxamic acid derivatives VIa
and VIg (Table 1). Those compounds didn’t present any
cytotoxic or antiproliferative potential. Nevertheless, fve
HDACis hydroxamic acid derivatives (VIb-VIf) exhibited
equal antiproliferative efciency on GSC either in 2D or in
3D. The assays were performed in presence and absence of
laminin, in order to recapitulate collective and individual
migration mode (Saleh et al. 2019). The antiproliferative
efect of the compounds (VIb-VIf) targeting HDACs didn’t
present any specifcity against single or collective migratory
cells (Table 2) (Fig. 1d). ERβ, here targeted by the com-
pounds IIa, has been described in other studies as inhibiting
the proliferation of glioma cell lines (Sareddy et al. 2016).
However, the estrogen modulator IIa seems to present anti-
proliferative properties in 2D, but not in 3D, validating the
3D model choice as stringent drug screening model for can-
didate selection.
18.861 −42.666
23.972 −21.05
1.157
10.793
1.617 −1.717
8.746
7.809
61.086
47.845
56.946
54.078
44.821
28.205 68.872
51.541 62.955
45.936 56.7
41.135 47.099
41.902 55.994
−2.397 −11.020 31.680 −16.718
56.517 10.591 70.898 65.767
*These compounds were selected for a second test. In the table, the
indicated value represents the average of the frst and second experi-
ments
2D-LN=2D cell culture without laminin; 2D+LN=2D cell cul-
ture with laminin; 3D-LN=3D cell culture without laminin;
3D+LN=3D cell culture with laminin
The HDAC antiproliferative efects are also already well
described (Was et al. 2019; Li et al. 2015). For instance,
HDAC8 inhibition by a specifc inhibitor produces DNA
damage, cell cycle arrest and decreases cell viability and
those effects were associated with the inhibition of the
O6-methyl-guanine DNA methyltransferase (Santos-Barri-
opedro et al. 2019). In its turn, HDAC6 inhibition decreases
GBM cell growth due to of SMAD2 phosphorylation inhibi-
tion and down expression regulation of p21 (Li et al. 2015).
HDACi (VIb-VIf) were previously designed and synthe-
sized depicting antiproliferative properties against a panel of
cancer cell line such as hepatocellular carcinoma (HepG2),
pancreatic cancer (MIA PaCa-2), breast cancer (MCF-7 and
HCC1954), renal cancer (RCC4-VHL and RCC4-VA) and
neuroblastoma (SH-SY5Y). According to molecular docking
and MD simulations studies, these compounds might block
targeting the 23 candidates potentially able to impair pro-
liferative and migrating behavior of GSCs (Zacarias-Lara
et al. 2019; Sixto-Lopez et al. 2020). (Rajak et al. 2013;
Wang 2009; Lan et al. 2017; Batistatou et al. 2004; Wan
et al. 2018; Liu et al. 2018; Zhou et al. 2019; Martinez-
Archundia et al. 2018; Feldman and Limbird 2017; Meitzler
et al. 2014; Munson et al. 2013; Nayernia et al. 2014). Those
targeted proteins are tackling several pathways, including
HDACs leading to epigenetic modulation, ROS production
and GPER and ER modulators associated in multiple can-
cer with cell proliferation and metastasis. First, to evaluate
the potential antiproliferative efect of the compounds, we
performed MTT test on GSC in 2D and 3D with or without
LN by adding the diferent compounds during 72 h. Dur-
ing this frst screening, the following compounds (from the
1 3

Cellular and Molecular Neurobiology
Fig. 1 a 5000 cells NS deposited on 3D+LN after 5 days of migra-
tion and percent survival of cell proliferation of the tested compounds
obtained from the 2D and 3D GBM cell cultures with (2D+LN or
3D+LN) or without (2D-LN or 3D-LN) laminin. VIf, VIc, VIe, VId,
VIb and IIa
the catalytic cavity of HDAC1, HDAC6 and HDAC8, which
could be due to their aromatic portion that is able to interact
with residues surrounding the catalytic tunnel (Sixto-Lopez
et al. 2020). Therefore, due to the previous promissory activ-
ity against several cancer cell lines, these HDACic were
tested against GBM cell lines in order to explore the anti-
proliferative and antimigratory activity against this disease.
GSCs during migration start differentiation process,
hence decreasing proliferation capacity. As HDACs com-
pounds are described as targeting proliferation, also follow-
ing the proliferation assay for the compounds (VIb-VIf), we
decided to evaluate HDACs inhibitor efects on migration
(Fig. 1a, b) and then potential impairment of cell viabil-
ity. We deposited same size neurosphere (NS) (5000 GSCs)
on those 3D matrices with LN, leave them to migrate dur-
ing 5 days and applied treatment during 72 h and quanti-
fed the NS size and the migration area around the NS after
subtracting the NS area from the total migration (Fig. 1c).
The HDACs inhibitor compounds (VIb-VIf) appear as not
decreasing the migrating capacity of the GSC comparing
to the control condition. We can hypothesize that HDACs
hydroxamic acid derivatives are not cytotoxic but impair
proliferation capacity without impairing migration in 3D
fbrillar matrix. Also, the compounds VIe and VIf show
1 3

Cellular and Molecular Neurobiology
only NS size decrease after 72 h treatment illustrating the
antiproliferative efect of the HDACs, independently of a
potential antimigration efect. Following those preliminary
experiment and for upcoming studies, we have, on frst hand,
to evaluate EC50 of the HDACs hydroxamic acid deriva-
tives and, on the other hand, evaluate in vivo efciency of
those compounds, potential cytotoxicity and side efects. In
fact, HDACs, are well described as having great therapeu-
tic potential but also pleiotropic efects at the cellular and
systemic levels and seems to present ability to modulate the
immune system afecting many cellular functions. Indeed,
their use is recently extended far beyond cancer treatment
in terms of their therapeutic capacity (Hull et al. 2016).
Consequently, it seems essential to perform in vivo trials as
next step for those HDACs inhibitor potential evaluation and
ultimate determine their action mechanism.
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Author Contributions YSL wrote the manuscript and synthetized
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MCRH synthetized compounds, wrote paper; OZL synthetized and
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Declarations
Conflict of interest No conficts of interest are present for any of the
authors.
Lee DH, Ryu HW, Won HR, Kwon SH (2017) Advances in epigenetic
glioblastoma therapy. Oncotarget 8(11):18577–18589. https://doi.
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Ethical Approval This article does not contain any studies with human
participants performed by any of the authors.
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