Discovery of 1,4-Naphthoquinones as a New Class of Antiproliferative Agents Targeting GPR55

Article abstract of DOI:10.1021/acsmedchemlett.8b00333

A new series of 1,4-naphthoquinones, bearing various cyclic and aliphatic amines on C2, was designed and synthesized to identify antiproliferative agents for triple-negative breast cancer, which represents a clinical challenge without targeted therapies. Among naphthoquinones, 2a and 3a inhibited the proliferation of MDA-MB-231 cells (EC₅₀ = 1.6 and 2.7 μM, respectively), compared to primary human breast cells MCF10A. Furthermore, they did not affect the viability of peripheral blood mononuclear cells (PBMC), suggesting their potential safer use for cancer treatment. Recently, correlations have emerged between the expression of G protein-coupled receptor 55 (GPR55) and both triple-negative breast cancer development and invasion, making it a promising target for the development of targeted therapies. Based on this evidence, molecular docking studies supported the hypothesis of binding to GPR55, and pharmacological tests suggested that compound 3a could exert its antiproliferative activity acting as a GPR55 inverse agonist.

Full text of DOI:10.1021/acsmedchemlett.8b00333

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Letter
Discovery of 1,4-Naphthoquinones as a New
Class of Antiproliferative Agents Targeting GPR55
Mariateresa Badolato, Gabriele Carullo, Maria Cristina
Caroleo, Erika Cione, Francesca Aiello, and Fabrizio Manetti
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00333 • Publication Date (Web): 15 Feb 2019
Downloaded from http://pubs.acs.org on February 15, 2019
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Discovery of 1,4-Naphthoquinones as a New Class of Antiproliferative
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†
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†*
†*
Mariateresa Badolato, Gabriele Carullo, Maria Cristina Caroleo, Erika Cione, Francesca Aiello, and
Fabrizio Manetti
§*
† Department of Pharmacy, Health and Nutritional Sciences – Department of Excellence 2018-2022, University of Calabria,
Ed. Polifunzionale, 87036, Arcavacata di Rende (CS), Italy
§
Department of Biotechnology, Chemistry and Pharmacy – Department of Excellence 2018-2022, University of Siena, Via
Aldo Moro 2, 53100 Siena, Italy
KEYWORDS triple-negative breast cancer, naphthoquinone, antiproliferative, GPR55, molecular docking.
ABSTRACT: A new series of 1,4-naphthoquinones, bearing various cyclic and aliphatic amines on C2, was designed and synthesized
to identify antiproliferative agents for triple-negative breast cancer, that represents a clinical challenge without targeted therapies.
Among naphthoquinones, 2a and 3a inhibited the proliferation of MDA-MB-231 cells (EC50=1.6 μM and 2.7 μM, respectively),
compared to primary human breast cells MCF10A. Furthermore, they did not affect the viability of peripheral blood mononuclear
cells (PBMC), suggesting their potential safer use for cancer treatment. Recently, correlations have emerged between the expression
of G protein-coupled receptor 55 (GPR55) and both triple-negative breast cancer development and invasion, making it a promising
target for the development of targeted therapies. Based on this evidence, molecular docking studies supported the hypothesis of
binding to GPR55 and pharmacological tests suggested that compound 3a could exert its antiproliferative activity acting as a GPR55
inverse agonist.
Breast cancer is the most commonly diagnosed malignancy
among the female population and it is one of the leading causes
of cancer-related deaths in women all over the world. In
anticancer target. Most of them possess electronegative groups
and pendant aromatic or heterocyclic rings that make critical
hydrogen-bonding and aromatic interactions with specific
1
1
2-14
addition to surgery and radiotherapy, endocrine therapy usually
offers a hope to breast cancer patients. The triple-negative
breast cancer (TNBC) encompassed 15-20% of all diagnosed
cases and, compared to the other types of breast cancer, it is
characterized by a more aggressive nature and poorer
prognosis. Furthermore, the risk of relapses is high, and
residues in the receptor binding pocket.
Based on these data,
the design and the discovery of new potent ligands of GPR55
1
5
may be the key for TNBC targeted therapies.
The 1,4-naphthoquinone scaffold is often found in many
natural and synthetic compounds, and the presence of the two
conjugated carbonyl groups on C1 and C4 is responsible for the
2,3
metastases frequently occur with shorter overall survival.
16
biological relevance of this system. Moreover, the number of
TNBC owes its name to the lacking expression of three markers,
estrogen and progesterone receptors (ER and PR), and human
epidermal growth factor receptor 2 (HER2), common targets for
specific substituents and their position on the naphthoquinone
core are responsible for a wide range of biological activities,
17-21
including anticancer.
In particular, the antiproliferative
4
breast cancer therapies. The peculiar heterogeneity of TNBC
21
activity is favored by the presence of a hydroxyl group on C5.
has always hampered the development of specific targeted
Although a number of strides are being made to understand the
molecular mechanism of action of 1,4-naphthoquinones, their
therapeutic target remains still unknown.
5
therapies. Although recently an intense understanding of the
molecular mechanisms involved in the physiology of TNBC has
opened the perspective for alternative therapeutic approaches,
they are still under clinical trials and preclinical studies and no
targeted therapies for TNBC have been approved yet. Currently,
systemic chemotherapy is the only available option for patients
Herein, we report the synthesis of novel 5,8-dihydroxy and
5,8-dimethoxy derivatives of 1,4-naphthoquinone, bearing
pendant non-aromatic heterocycles or aliphatic amines on C2,
with the main purpose to identify new antiproliferative agents
of TNBC. The molecular mechanism of action of the new
derivatives was investigated to ascertain their target. Molecular
docking studies were also carried out to support the in vitro
results. The synthetic steps to obtain compounds 1-6a and 1-3b
are outlined in Scheme 1. According to the method of Hout and
6,7
with TNBC. Therefore, the identification of therapeutically
targeted molecules remains an attracting clinical challenge.
In the last years, the membrane-bound G protein-coupled
receptor 55 (GPR55), initially deorphanized as a cannabinoid
receptor, has emerged as a potential target for treating cancer,
8
including TNBC. In particular, GPR55 is highly expressed in
22
Brassard, 1a was prepared as tautomeric pair through Friedel-
TNBC cell lines and its activation correlates with cancer
Craft condensation of 1,4-dimethoxybenzene and 2,3-
dichloromaleic anhydride, in a melted mixture of aluminum and
sodium chlorides. It was easily purified by crystallization and
9
-11
proliferation and invasion. Despite increasing evidence, the
paucity of specific ligands of GPR55 has limited its study as
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isolated in high yield. Due to its high reactivity in nucleophilic various cyclic and aliphatic amines.
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substitutions, 2-6a were quickly obtained by reaction of 1a with
a
Scheme 1. Synthetic steps for compounds 1-6a and 1-4b
OH
8
O
2
R
ii
Cl
5
OH
O
O
O
OH
OH
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-6a
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1b
2-3b
aReagents and conditions: (i) 1. a: AlCl
DCM, rt, overnight, 17-28%; (iii) CH I, Ag
, NaCl, 140-150 °C; b: 170-175 °C, 5 min; 2. H
O, CHCl , rt, 22 h, 56%.
3
2 2 3
O, HCl, rt, overnight, 99%; (ii) amine, Na CO ,
3
2
3
Heterocyclic and diethylamino derivatives were obtained in
high yield, while the lengthening of the alkyl amine chain
resulted in a lower yield and formation of by-products. The 5,8-
dihydroxyl groups were replaced with methoxy substituents to
evaluate their influence on the antiproliferative activity and to
rule SAR considerations. Thus, 1b was obtained in a higher
yield compared to its isomer through methylation of 1a with
methyl iodide and silver oxide. Following the same reaction
described before, 2-3b were obtained by nucleophilic
substitutions of 1b with cyclic amines (see Supporting
Information for reaction procedures).
primary human breast cell line. The EC50 value of both 2a and
3a against MCF10A cells was higher, suggesting a slightly low
antiproliferative activity than that found towards TNBC cell
line (Table 2). Since most of chemotherapeutics are
administered intravenously, we aimed to investigate the
cytotoxicity of the selected compounds also against peripheral
blood mononuclear cells (PBMC) to evaluate their safety.
Compounds 2a and 3a did not affect the viability of PBMC
either at 24 or 48 hrs of incubation (see Supporting
Information), which was over 60% even at the highest test
concentration (100 μM, Table 2). These data confirm the
selective antiproliferative activity of the new compounds
against TNBC and the potential application for the treatment of
this deadly type of breast cancer in which combination therapies
To determine whether the new derivatives provide the
desired TNBC antiproliferative activity, their EC50 was
assessed against the MDA-MB-231 cell line, over-expressing
GPR55, using the cell viability assay (Table 1). Although the
small number of compounds, the in vitro results indicated the
impact of the different substituents on the antiproliferative
activity. The dihydroxyl derivative 1a, with two chlorine atoms
on C2-3 positions, did not inhibit the cell proliferation. On the
other hand, the presence of amino substituents on C2 showed
some cytotoxic activity. Particularly, the presence of a 6-
membered ring, as in 2a and 3a, resulted in a potent cytotoxicity
30
is emerging as strategy .
Docking simulations were carried out to investigate the
binding mode of 3a on GPR55. No three-dimensional structure
of GPR55 is currently available from experimental sources. The
same docking protocol, already described by us for GPR40
2
4
agonists and very similar to that applied by others to GPR55
2
3,13
simulations,
has been used to evaluate the putative binding
mode of the new 1,4-naphthoquinones on GPR55. The
complementarity of the molecular surface of both 3a and
GPR55 binding site is illustrated in Figure 2A. Compound 3a,
in its best docking pose, showed the oxygen atoms in positions
4 and 5 in hydrogen bond contacts with the terminal ammonium
group of Lys80 and the imidazole ring of His170 (Figure 2B).
The importance of the 5-OH substituent for compound activity
is in agreement with SAR on the naphthoquinones that shows a
crucial role of the phenolic group for cytotoxic activity. In fact,
docking simulations on methoxylated compounds 1b-3b
showed that they lacked the charge-reinforced hydrogen bond
constituted by the imidazolium group of His170 and the phenate
substituent at position 5 of the ligand.
(
Figure 1A and B). The analysis of dose-response curve profiled
compound 3a as ligand for GPR55. In any case, redox activity
of compound 2a cannot be ruled out. The morpholino derivative
3
a exhibited the best antiproliferative activity, probably due to
hydrophobic contacts not found in 1a (see docking results).
Substitutions with the 5-membered pyrrolidine ring or
dialkylamino groups resulted in a significant loss of potency.
Also in this case, reduction of the ring size and its opening into
a dialkyl amine led to the lack of hydrophobic contacts with the
binding site amino acids. Furthermore, dimethoxy derivatives
1
-3b did not affect the proliferation of TNBC cells, even if 6-
membered rings are attached on C2, as in 2a and 3a. These
results suggest that the presence of both 6-membered rings on
C2 and hydroxyl groups on C5 and C8 are crucial for the
antiproliferative activity.
The two hydrogen bonds of 3a mimic those found between
the phosphate group of the lysophosphatidylinositols (LPIs) and
the same amino acids (Figure 2C). Moreover, this interaction
pattern was also found in the complex between GPR55 and
The most potent compounds 2a and 3a were then evaluated
1
3
for their safety, testing their cytotoxicity against MCF10A, a
tetrahydromagnolol (THM), whose 2,2’-biphenyldiol moiety
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that such hydrophobic interactions played a fundamental role in
is reminiscent of the two 1,3-dioxygenated molecular pathways
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found in 3a (namely, the 1-carbonyl/8-hydroxyl and 4-
carbonyl/5-hydroxyl pairs).
defining compound activity. In fact, compounds bearing a small
substituent (Cl or a dialkyl amine side chains, such as in 1a, 5a,
and 6a, respectively) or a five-membered heterocycle (such as
in 4a) instead of the 6-membered heterocyclic rings of 2a and
3
a, showed a significant reduction or a complete lack of
hydrophobic interactions with the two hydrophobic amino acids
and with the aromatic cage (not shown), which are probably
responsible for their loss of activity. Interestingly, the 8-OH
substituent of 3a was superposed to the alcoholic groups of the
LPI sugar moiety, although clear electrostatic or hydrogen bond
interactions with GPR55 were not evidenced (Figure 2C).
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Figure 1. Dose-response curves of compound 2a (A) and 3a (B).
Table 2. Comparison of the in vitro cytotoxic activities of the
best derivatives against TNBC and PBMC cell lines.
Table 1. Summary of the in vitro cytotoxic activities of the
new 1,4-naphthoquinone derivatives.
Compound
MDA-MB-231a
MCF10Aa
2.5
PBMCb
65%
2
3
a
a
1.6
2.7
Compound
R
MDA-MB-231
> 20
3.7
70%
1
2
a
a
aEC50 values were determined by MTT assay after 24 h treatment
piperidin-1-yl
morpholin-1-yl
pirrolidin-1-yl
1.6
and are expressed as μM concentrations. The values are averages
3a
2.7
b
of triplicate (n=3) data and shown as the mean. 100 μM test dose.
4
5
6
a
a
a
> 10
Cell viability of PBMC was evaluated with trypan blue dye
exclusion.
-N(Et)
2
> 20
-N(Bu)
2
> 20
Although 3a was able to maintain the hydrogen bond
interaction with Lys80 that is the primary interaction site of the
1b
> 20
1
3
complexes between GPR55 and antagonist compounds, the
overall structure of 3a was not characterized by the presence of
the three structural elements described for GPR55 antagonists
2
3
b
b
piperidin-1-yl
> 10
morpholin-1-yl
> 10
(
namely, a head edge, a central portion, and a pendant aromatic
aEC50 values were determined by MTT assay after 24 h treatment
and are expressed as μM concentrations. The values are averages
of triplicate (n=3) data and shown as the mean.
13
ring). This results in the inability to predict if 3a could act as
a GPR55 agonist or antagonist. In this view, we performed a
pharmacological test using CID16020046 (thereafter referred to
as CID160), a selective antagonist of GPR55 in order to
characterize the pharmacodynamics profile of 3a.
In particular, a rough superposition between 3a and THM
clearly shows structural similarity (in size and shape) between
compounds and match between oxygenated substituents (Figure
2
D).
The morpholine ring of 3a was accommodated within an
aromatic cage comprised of Tyr101, Phe102, Tyr106, Phe190,
and Phe246 (Figure 2B), thus mimicking the pendant
heteroaromatic ring found in already known GPR55
1
3
antagonists. This heterocyclic ring was also pointing toward
Met105 that is one of the amino acid responsible for GPR55
activation. However, the overall size of 3a, that is significantly
smaller than other known GPR55 antagonists since a head
region is not present (see below), allows such a compound to
have a certain mobility within the binding site, as already
1
3
hypothesized for other smaller GPR55 antagonists. As a
consequence, molecular docking simulations cannot predict if
3
a is or not able to prevent the amino acid conformational
switch responsible for GPR55 activation. Given the uncertainty
about the most appropriate form of GPR55 to be used for
molecular modeling, docking simulations have been set starting
from either the inactive or active GPR55 and resulted in very
similar interaction patterns of 3a. In fact, the key hydrogen
bonds involving both Lys80 and His170 were maintained,
further supported by hydrophobic interactions with the side
chain of the aromatic cage above described.
Figure 2. A) Complementarity between the molecular surface of
3
a (blue) and that of the GPR55 binding site (grey). B) and C)
Graphical representation of the interactions between 3a and LPI
and the binding site of GPR55. The GPR55-3a complex (A) is
stabilized by two hydrogen bonds (blue dashed lines) involving the
ligand oxygens at 4 and 5 positions and by strong hydrophobic
The side chains of Val242 and Leu270 provided profitable
hydrophobic contacts that contributed to further stabilize the
complex (Figure 2B). Docking simulations strongly suggested
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contacts (green dashed lines) mainly involving the morpholine ring
between 3a (pink and atom type) and THM (green and atom type)
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at C2. Interestingly, LPI conserved the same hydrogen bonds (blue
dashed lines, panel B), while its alkyl edge is superposed to the
hydrophobic portion of morpholine of 3a. D) Rough superposition
shows a clear correspondence between the two phenolic hydroxyls
of THM and the 4-carbonyl and 5-phenolic group of 3a. Likewise,
the overall size and shape of both molecules are comparable.
and the binding site residues of GPR55. Further in vivo studies
are needed to elucidate the pharmacokinetics profile of 3a as
antiproliferative agents in tumors harboring aberrant expression
of GPR55, including TNBC.
Clinical data identified LPIs as biomarkers of poor
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prognosis in cancer patients. LPIs were found to be
1
0
endogenous ligands of GPR55. Recently, the LPI/GPR55 axis
was demonstrated to enhance human breast cancer cell
2
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ASSOCIATED CONTENT
Supporting Information
migration independently of the TNBC phenotype. Therefore,
we tested 3a on both TNBC and hormone-responsive breast
cancer cell lines. The exposure of MDA-MB-231 and MDA-
MB-468 (Figure 3A and 3C), as well as MCF-7 and T47D
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The Supporting Information is available free of charge on ACS
Publications website.
(Figure 3B and 3D) to 3a led to a significant decrease of the
proliferation rate compared to DMSO-treated cell. The
evidence that CID160 caused a decrease of cell viability in 3 of
the 4 tested cell lines, could indicate the presence of
General experimental information; full synthetic procedures and
characterization data for all new compounds; NMR, GC-MS and
IR spectra; experimental procedure for PBMC isolation and cell
viability assays; statistical analysis; computational details (PDF)
26
endogenously produced ligand. In this view, the exposure of
MDA-MB-231, MDA-MB-468 and MCF-7 to CID160 for 30
min before treatment with 3a was able to reverse its effect. This
suggested that 3a acts as inverse agonist of GPR55. It is of note
that the GPCRs exist in an active and an inactive form. In the
physiological system, both forms are in equilibrium.27,28
Generally, agonists of GPCRs have higher affinity for the active
form. In addition, based on this, 3a is an inverse agonist since
it is able to evocate an antiproliferative effect.
AUTHOR INFORMATION
Corresponding Authors
* Francesca Aiello, PhD
Assistant Professor of Pharmaceutical and Toxicological
Chemistry, E-mail address: francesca.aiello@unical.it
Phone number: +390984493154
* Erika Cione, PhD
Assistant Professor of Applied Biochemistry and Molecular
Biology, E-mail address: erika.cione@unical.it
Phone number: +390984493193 office-+390984493147 lab
*
Fabrizio Manetti, PhD
Associate Professor of Pharmaceutical and Toxicological
Chemistry, E-mail address: fabrizio.manetti@unical.it
Phone number: +390577234330
ORCID
Francesca Aiello: 0000-0001-6846-5582
Erika Cione: 0000-0002-0562-0597
Fabrizio Manetti: 0000-0002-9598-2339
Mariateresa Badolato: 0000-0002-5892-9635
Gabriele Carullo: 0000-0002-1619-3295
Maria Cristina Caroleo: 0000-0001-6824-4616
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Figure 3. Activity of the GPCR55 selective antagonist
CID16020046 and 3a, alone and in combination on two TBNC cell
line MDA-MB-231 and MDA-MB-468 (A and C) and on two
hormone-responsive cell lines MCF-7 and T47D (B and D). Values
with the same letter are not significantly different, while values
with different letter are significantly different (*p<0.05 and **
p<0.001 one-way ANOVA followed by Bonferroni's Comparison
Test, as post-hoc Each sample was run in triplicate (n=3).
ACKNOWLEDGMENT
We are indebted with P. H. Reggio and D. Hurst (Department of
Chemistry and Biochemistry, University of North Carolina at
Greensboro, NC) who kindly provided us with the three-
dimensional coordinates of both the active and inactive structures
of GPR55 as obtained by homology modeling simulations.
In conclusion, a new class of 1,4-naphthoquinones was
designed and synthesized to identify potent antiproliferative
agents. On the basis of the data obtained in vitro, we
demonstrated that 3a is a GPR55 inverse agonist. Moreover,
molecular docking studies showed the interactions between 3a
ABBREVIATIONS
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DCM, dichloromethane; ER, estrogen receptor; GPR55, G
protein-coupled receptor 55; HER2, human epidermal growth
factor receptor 2; LPI, lysophosphatidylinositol; PBMC,
peripheral blood mononuclear cells; PR, progesterone receptor;
THM, tetrahydromagnolol; TNBC, triple-negative breast
cancer. Santa Chiara Lab (University of Siena) is also
acknowledged for the use of the Schrodinger suite.
(17) Janeczko, M.; Demchuk, O. M.; Strzelecka, D.; Kubiński, K.;
Masłyk, M. New family of antimicrobial agents derived from 1,4-
naphthoquinone. Eur. J. Med. Chem. 2016, 124, 1019-1025.
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(18) Kehelpannala, C.; Kumar, N. S.; Jayasinghe, L.; Araya, H.;
Fujimoto, Y. Naphthoquinone Metabolites Produced by
Monacrosporium ambrosium, the Ectosymbiotic Fungus of tea Shot-
Hole Borer, Euwallacea fornicates, in Stems of Tea, Camellia sinensis.
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19) Deniz, N. G.; Ibis, C.; Gokmen, Z.; Stasevych, M.; Novikov,
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D. L.; Madrigal, K. E.; Heynen-Genel, S.; Milan, L. B.; Chung, T. D.
Y.; Seltzman, H. H.; Bai, Y.; Caron, M. G.; Barak, L. S.; Croatt, M. P.;
Abood, M. E.; Reggio, P. H. Identification of the GPR55 Antagonist
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Ligands. Biochemistry. 2013, 52, 9456-9469.
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Gamage, T. F.; Sharir, H.; Gettys, K. E.; Hurst, D. P.; Kotsikorou, E.;
Shore, D. M.; Caron, M. G.; Rao, N.; Barak, L. S.; Abood, M. E.;
Reggio, P. H.; Croatt, M. P. Design, synthesis, and analysis of
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Scheme 1. Synthetic steps for compounds 1-6a and 1-4b a
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aReagents and conditions: (i) 1. a: AlCl
Na CO , DCM, rt, overnight, 17-28%; (iii) CH
, NaCl, 140-150 °C; b: 170-175 °C, 5 min; 2. H
I, Ag O, CHCl , rt, 22 h, 56%.
3
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O, HCl, rt, overnight, 99%; (ii) amine,
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Figure 1. Dose-response curves of compound 2a (A) and 3a (B).
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Figure 2. A) Complementarity between the molecular surface of 3a (blue) and that of the GPR55 binding site (grey). B) and C)
Graphical representation of the interactions between 3a and LPI and the binding site of GPR55. The GPR55-3a complex (A) is
stabilized by two hydrogen bonds (blue dashed lines) involving the ligand oxygens at 4 and 5 positions and by strong hydrophobic
contacts (green dashed lines) mainly involving the morpholine ring at C2. Interestingly, LPI conserved the same hydrogen bonds (blue
dashed lines, panel B), while its alkyl edge is superposed to the hydrophobic portion of morpholine of 3a. D) Rough superposition
between 3a (pink and atom type) and tetrahydromagnolol (THM, green and atom type). A clear correspondence between the two
phenolic hydroxyls of THM and the 4-carbonyl and 5-phenolic group of 3a appears. Moreover, the overall size and shape of both
molecules are comparable.
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97x209mm (300 x 300 DPI)
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Table 1. Summary of the in vitro cytotoxic activities of the new 1,4-naphthoquinone derivatives.
Compound
1a
R
MDA-MB-231
> 20
2
3
4
a
a
a
piperidin-1-yl
morpholin-1-yl
pirrolidin-1-yl
1.6
2.7
> 10
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5a
-N(Et)
2
> 20
6
1
2
a
b
b
-N(Bu)
2
> 20
> 20
piperidin-1-yl
morpholin-1-yl
> 10
3b
> 10
aEC50 values were determined by MTT assay after 24 h treatment and are expressed as μM concentrations. The values are averages
of triplicate (n=3) data and shown as the mean.
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Table 2. Comparison of the in vitro cytotoxic activities of the best derivatives against TNBC and PBMC cell lines.
Compound
2a
MDA-MB-231a
MCF10Aa
2.5
PBMCb
65%
1.6
2.7
3
a
3.7
70%
aEC50 values were determined by MTT assay after 24 h treatment and are expressed as μM concentrations. The values are averages
b
of triplicate data and shown as the mean ± standard deviation (SD), n=3. 100 μM test dose. Cell viability of PBMC was evaluated
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with trypan blue dye exclusion.
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