Cancer

Pereyra
et al. Cancer Cell Int (2019) 19:207

https://doi.org/10.1186/s12935-019-0925-8 Cancer Cell International
REVIEW Open Access
The diverse mechanisms and anticancer

potential of naphthoquinones
Carolina Escardó Pereyra1†, Rafael Ferreira Dantas1†, Sabrina Baptista Ferreira2, Luciano Pinho Gomes1
and Floriano Paes Silva‑Jr1*
Abstract
Cancer is one of the leading causes of death around the world and although the different clinical approaches have
helped to increase survival rates, incidence is still high and so its mortality. Chemotherapy is the only approach which
is systemic, reaching cancer cells in all body tissues and the search for new potent and selective drugs is still an attrac‑
tive field within cancer research. Naphthoquinones, natural and synthetic, have garnered much attention in the scien‑
tific community due to their pharmacological properties, among them anticancer action, and potential therapeutic
significance. Many mechanisms of action have been reported which also depend on structural differences among
them. Here, we describe some of the most relevant mechanisms of action reported so far for naphthoquinones
and highlight novel targets which are being described in the literature. Furthermore, we gather some of the most
impressive efforts done by researchers to harness the anticancer properties of these compounds through specifically
designed structural modifications.
Keywords: Naphthoquinones, Natural compounds, Multi-target agents, Chemotherapy, Cancer drug discovery
Background are among the disadvantages of the first two approaches,

Non-communicable diseases (NCDs) are non-trans- as well as the fatigue from radiotherapy which may last
mittable illnesses affecting all age groups and regions in for the duration of the treatment or months afterwards.
the world. Cancer is considered one of the four types of Chemotherapy is the only approach which is systemic,
NCDs—alongside cardiovascular diseases, chronic res- meaning that the drugs circulate through the blood
piratory diseases and diabetes- and on its own, is one stream reaching cancer cells in all body tissues. It can
of the leading causes of death globally, with approxi- annihilate both original tumor cells as well as metasta-
mately 9.6 million cases estimated to occur in 2018 alone. sized ones. The drawback of this approach is that it has
Approximately 70% of all cases occur in low- and middle- many side effects that affect the patient’s lifestyle and
income countries where cancer diagnostic and treatment also, resistance may be developed by cancer cells [2].
services are insufficient [1]. Natural compounds and their derivatives are a good
Cancer is associated to uncontrollable cell growth and source of molecules to be tested for their anticancer
the different types known to date are classified according properties. Among this group of compounds, quinones
to the cell type that was initially affected. Surgery, radia- are ubiquitous in nature, occurring in animals, plants and
tion and chemotherapy are the three main approaches microorganisms. They have a crucial role in the energy
to cancer treatment. Damage of healthy tissues and cells production of these organisms by providing essential
links in the respiratory chain of the cells [3]. For a long
*Correspondence: [email protected]
time, they have been a source of cytotoxic compounds,
†
Carolina Escardó Pereyra and Rafael Ferreira Dantas contributed equally being present in many drugs such as the anthracyclines
to this work
1
daunorubicin (1), doxorubicin (2) and mitoxantrone (3),
Laboratório de Bioquímica Experimental e Computacional de Fármacos,
Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Avenida Brasil 4365, Rio
used clinically in the therapy of cancer (Fig. 1). Many
de Janeiro, Rio de Janeiro 21040‑900, Brazil other pharmacological activities have been reported,
Full list of author information is available at the end of the article
© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Pereyra et al. Cancer Cell Int (2019) 19:207 Page 2 of 20
cell proliferation. This becomes of high importance in

diseases such as cancer, in which a failure in the control
of this cycle or in the process of apoptosis (see below)
leads to the installment of the sickness [17].
Four clearly distinguishable phases can be recog-
nized in the cell cycle: G1, in which the cell prepares all
its machinery towards DNA replication, S, which is the
actual synthesis of new DNA identical to the one used
as template, G2, when the cell now prepares for division
and finally, M, known as mitosis, when the cell divides
itself into two identical daughter cells. These phases are
sequential and interdependent, i.e. they all need to take
place in an orderly fashion to allow for correct cell dupli-
cation [16]. As mentioned above, there is also a stage
Fig. 1 Chemical structures of anticancer drugs with the quinone
known as G0 or latent state that is not a direct part of
moiety
the sequence in the cycle. The entrance or the exit of the
cell from this state is fundamental to the depression of its
active functions or to direct it to duplication.
including their action as antiallergic [4, 5], antibacte- The proteins responsible for the progression of the
rial [6, 7], antifungal [6, 8], anti-inflammatory [4, 8], cell through the different stages are mainly kinases and
antithrombotic [9, 10], antiringworm [6], antiplatelet [4, phosphatases that act on each other producing their own
5] and antiviral agents [6, 10]. Cytotoxicity has been asso- activation or deactivation. Among kinases, the cyclin-
ciated to the inhibition of the human DNA topoisomer- dependent kinases (Cdks) play a fundamental role on
ase I and II [11–13] and production of reactive oxygen the activation of other key proteins in the cycle through
species (ROS) such as semiquinone and hydroxyl radicals phosphorylation. These are modulated by another set of
by spontaneous or enzymatic reduction [14, 15]. proteins known as cyclins [18]. This complexity allows
In this review, we will focus on the pharmacology and for the search of many therapeutic targets and markedly
the mechanisms of action reported up to date of one increases the possibility of finding chemical compounds
prominent type of quinones: naphthoquinones. Their that permit the manipulation of the cycle at different
anticancer activity has been widely reported in the sci- stages. They can bind directly to a protein, leading to
entific literature and many distinct mechanisms of action activation or blockage of its function as well downregu-
have been attributed to them. We will summarize some lating or upregulating its expression.
of the modifications which have been made to the core Checkpoints in the cycle are mechanisms to ensure the
structure of the naphthoquinones in order to enhance cell that preceding phases required for successful cellu-
their potential as anticancer agents and increase their lar replication have been completed correctly. There are
selectivity for cancer cells or for one particular molecular three key events that need to begin and finish flawlessly
target. for the cell to divide, which are: DNA replication, DNA
repair and chromosome segregation [19]. These are the
Main text stages at which many natural products can exert their
Cell cycle: checkpoints as therapeutic targets effect and the damage created by them can lead to cell
Many small compounds, and among them natural prod- cycle arrest [16].
ucts, can act at different stages of the cell cycle in order to Accordingly, there are three main checkpoints at which
stimulate cell replication, to arrest the cycle at one of its the cell cycle can be stopped. In the G1/S transition,
stages or to lead the cell into a state of depression, known the cell needs to make sure that its machinery is ready
as G0. In this state, the metabolic functions of the cell for the actual replication of DNA, particularly that the
become inactive, namely transcription and protein syn- template is not damaged. Eukaryotic cells cannot allow
thesis. Proteins that act as controllers, allowing the cell to division unless DNA replication and damage repair are
proceed or to halt its reproductive cycle have been prop- completed because these processes cannot occur after
erly identified and can therefore be used as therapeutic chromosome condensation has already happened and
targets [16]. the cell is entering mitosis [19]. A key protein here is the
A molecule that acts on cell cycle proteins, whether by mammalian p53 transcription factor and tumor suppres-
altering their levels or binding directly to them, can be a sor. This factor senses the extent of the damage in the
potential drug which could have a determining effect on DNA and decides the fate of the cell, whether the damage
is repairable and the cell can proceed to the next stage expressed in their proactive form in normal cells, cleav-
or the cell should undergo its death through apoptotic ing substrates at aspartic acid residues. They can acti-
mechanisms [17]. In almost every tumor, p53 is inacti- vate each other through their proteolytic activities
vated due to direct mutations, mostly of missense type, giving rise to a biochemical cascade of events [17].
in p53 gene (tumor protein p53, TP53) and/or to distur- The two major classes of caspases are the initiators,
bances in its regulatory pathways (e.g. overexpression of caspase-2, -8, -9 and -10, and the executioners or effec-
negative regulators). Thus, the components of p53 system tors, caspase-3, -6 and -7 [24, 25]. In the latter group,
represent potential targets for cancer therapy [20]. caspase-3 is the most important one since it can be
Another checkpoint is during the other gap transition, activated by any of the initiators, thus being an attrac-
G2, when the cell is preparing for division. Two enzymes tive target for anticancer therapy. There have been
play key roles in the correct replication of DNA: topoi- three other caspases identified, from which caspase-12
somerases (topo) I and II. They are responsible for the is of special interest for anticancer therapy. It mediates
regulation of the topology of DNA during replication, apoptotic pathways associated with endoplasmic retic-
hence, their names. Topo I is always active whereas the ulum stress that derives from oxidative stress [17], a
topo II is mostly expressed during the G2 and M phases phenomenon that could be caused by naphthoquinones
[16]. Pharmacological interference with any of these when considered as anticancer agents (see next topic).
enzymes could lead to cell cycle arrest and the subse- There are basically two signaling pathways in which
quent inhibition of cell division, representing another apoptosis could occur: the extrinsic and the intrin-
promising chemotherapeutic strategy. sic one. In the former, there are transmembrane death
Finally, there is a checkpoint at the mitosis stage, linked receptors involved, such as the tumor necrosis factor
to the accurate spindle formation. This causes a delay of (TNF) which brings the death signal from the exterior
the anaphase in the mitosis through a signal generated by to the interior of the cell [26]. The intrinsic pathway
kinetochores when spindle fibers fail to attach during cell depends on signaling inside of the cell and the events
division [21]. are mediated by the mitochondria. Signals could be of a
positive or negative nature. The positive ones could be
Apoptosis: signaling pathways and proteins involved external or internal factors that could induce cell death,
The term apoptosis for the process of programmed namely exposure to radiation, toxins, hypoxia, genera-
death carried out by the cell, was first coined by Kerr tion of free radicals, viral infections, etc. The negative
et al. [22]. It is a distinctive form of cell death, compared signals are the absence of certain molecules that are in
to necrosis, mainly because it is controlled and energy- charge of suppressing death programs, leading to the
dependent and no inflammation is associated to it [23]. initiation of the chain of events that ends in apopto-
Some authors even propose that the term necrosis should sis [17]. There is also the perforin/granzyme pathway,
not be used to refer to cell death since it is linked to the which is a form of cell death mediated by T-cells [27].
degenerative processes that take place after the cell has All three apoptotic pathways aforementioned meet at
already died. Additionally, apoptosis can occur on a sin- the execution phase, where executioners caspases acti-
gle cell within an organism without affecting other neigh- vate endonucleases and proteases leading to nuclear
boring cells. It is a process that normally takes place fragmentation and cytoskeletal degradation [17].
during the development of cells, to eliminate those that Focusing on the intrinsic pathway for anticancer
do not differentiate correctly, and also during aging [17]. therapy, the signals mentioned above cause changes
The morphology of an apoptotic cell involves shrink- in the inner mitochondrial membrane that lead to the
age, packing of organelles and pyknosis, a term used to loss of its transmembrane potential, loss of perme-
refer to chromatin condensation and segregation [22]. ability and the release of pro-apoptotic substances that
Also, there is the formation of apoptotic bodies which are normally retained into the cytosol [28]. These are
contain cytoplasm from the dying cell and its packed cytochrome c, Smac/DIABLO and HtrA2/Omi [29, 30].
organelles. These bodies may contain nuclear fragments Cytochrome c activates procaspase-9, one of the initia-
in them or not, and they are subsequently phagocytized tor caspases, and the other two exert their pro-apop-
by macrophages, parenchymal cells or neoplastic cells. totic action by inhibiting a group of proteins known as
All these features prevent inflammation from taking inhibitor of apoptosis proteins (IAP) [31, 32]. Another
place since the bodies are quickly removed and do not group of pro-apoptotic proteins that are released after-
release their content into their surroundings [17]. wards, once the cell is doomed to the path of irre-
The molecular processes that lead to apoptosis in versible death, are apoptosis-inducing factor (AIF),
a cell involve many proteins, being the family of the endonuclease C and caspase-activated DNase (CAD),
caspases the crucial ones. Caspases are proteases mediating DNA fragmentation and condensation of
chromatin. An excellent review of these events has UPR promotes cells survival, adaptation and resistance to
been reported by Elmore, 2007 [17]. death. In this scenario, ER homeostasis may be restored,
An important pro-apoptotic protein is p53. As men- and cell is kept alive. However, during chronic, severe
tioned above, its main functions include holding the cell and sustained ERS, UPR might be insufficient. In this sit-
cycle at the G1/S transition when it detects DNA dam- uation, the pro-death mechanisms of UPR prevails over
age, activating proteins that repair DNA and also leading pro-survival ones which ultimately provokes cell apopto-
the cell to its death if the damage is too severe [33]. This sis [41].
factor is mutated in 50% of human cancers, being the UPR participates in different aspects of cancer physi-
most mutated gene in humans with this disease [17]. opathology and treatment [41]. In the microenvironment
The Bcl-2 (B-cell lymphoma 2 protein) family of proof tumors, ERS may be induced by extrinsic (e.g. hypoxia
teins is also regulated by p53. These proteins are respon- and nutrient deprivation) and intrinsic (e.g. oncogene
sible for the regulation of the mitochondria-mediated activation and oxidative stress) factors [42]. In turn, cells
apoptotic pathway and they can be either pro or anti- activate UPR which ultimately leads to not only to cells
apoptotic. In a nutshell, they decide the fate of the cell, survival, but contributes for the development of many
whether it goes on and dies or aborts the process. It is cellular features that enable tumor growth and metas-
believed that Bcl-2 may exert its anti-apoptotic actions tasis dissemination (the so-called hallmarks of cancer),
through antioxidant mechanisms [34]. including genomic instability, invasion, proliferation and
angiogenesis [37, 43]. Such mechanisms have also been
Endoplasmic reticulum stress (ERS): pro‑survival associated with resistance to chemotherapy and radia-
and pro‑death signaling pathways tion. In this context, UPR modulation represent a useful
The endoplasmic reticulum (ER) is a specialized orga- approach to develop new anticancer drugs. In fact, sev-
nelle of eukaryotes that plays a major role in a myriad of eral natural and synthetic compounds induce apopto-
physiological functions, including lipid synthesis, Ca2+ sis of cancer cells by either blocking UPR pathway (e.g.
storage and detoxification. Moreover, ER is also respon- inhibiting PERK activity, decreasing the expression of
sible for the synthesis, folding and post-translational ERS-related proteins) or inducing chronic ERS [41, 42].
modifications of secreted and transmembrane proteins
[35]. ER homeostasis can be disturbed by several factors, Naphthoquinones: occurrence in nature and structural
such as nutrient deprivation, excessive protein synthesis, characteristics
imbalance in C a2+ and ROS homeostasis, hypoxia and Quinones could be chemically classified into three dif-
oxidative stress [35, 36]. If not resolved, these events may ferent classes regarding the type of aromatic system that
lead to ER stress (ERS), a condition characterized by the supports the quinone ring (Fig. 2): benzoquinones (a ben-
accumulation of misfolded proteins in ER lumen. ERS is zene ring) (4a and 4b), naphthoquinones (a naphthalene
associated to the physiopathology of many types of can- ring) (5a and 5b) and anthraquinones (an anthracene
cer [37] as well to metabolic [38], renal [39] and neurode- ring, linear or angular) (6). Depending on the position
generative disorders [40]. of the carbonyls within the ring system, one could have
In order to overcome ERS, cells rely on a multipath- different quinones. As an example, naphthoquinones
way system called unfolded protein response (UPR) could have two different arrangements for their carbonyl
whose main goal is to restore ER proteostasis (i.e. pro- groups: 1,2-naphthoquinones (5a) with neighboring
tein homeostasis). UPR is regulated by three ER-resident functional groups or 1,4-naphthoquinones (5b) with a
transmembrane proteins (“stress sensors”): inositol- space of two carbons between carbonyls. These isomers
requiring enzyme 1 (IRE1α), activating transcription fac- have notably different pharmacological actions due to the
tor 6 (ATF6) and pancreatic ER kinase (PERK). The difference in their physicochemical properties [13].
balance between pro-survival and pro-death mechanisms Quinones are highly reactive compounds with an appli-
of UPR defines cells fate during ERS. Under mild ERS, cation as natural or synthetic dyes. Widely distributed
Fig. 2 Basic structural cores within the quinone family

Table 
1 Families of higher plants from which potency towards cells which are drug-resistant and those
naphthoquinones have been isolated. Adapted from which are drug-sensitive, as further discussed in the next
López et al. [44] section. Furthermore, β-lapachone (12) also occurs natu-
Families of higher plants rally and is isolated from the bark of lapacho tree (Tabe-
buia avellanedae) [47] as well your positional isomer,
Ancistrocladaceae Gentianaceae α-lapachone (13) (Fig. 3). A good review on naphthoqui-
Avicenniaceae Iridaceae nones obtained from natural sources and their action was
Balsaminceae Juglandaceae published by Nematollahi et al. [48].
Bignoniaceaea Lythraceae
Boraginaceae Nepenthaceae Mechanisms of action of naphthoquinones as anticancer
Dioncophyllaceae Plumbaginaceae agents
Droseraceae Proteaceaea Naphthoquinones are considered privileged structures in
Ebenaceae Scrophulariaceae medicinal chemistry, a term coined by Evans et al. [49] to
Euphorbiaceae Verbenaceaea highlight substructures associated to diverse pharmaco-
a
Most common families logical activities while allowing easy chemical modifica-
tions. This makes them suitable scaffolds for the synthesis
of new compounds that reproduce the same known
in nature, they can be found in several families of higher actions or lead to new ones, also improving their absorp-
plants (Table 1). Naphthoquinones are particularly ubiq- tion, distribution, metabolism, excretion and toxicity
uitous in the families Bignoniaceae, Verbenaceae and Pro- (ADMET) properties and even their pharmacodynamics.
teaceae, though their major occurrence is on the former, The focus of this review is on the anticancer proper-
specifically on the genus Tabebuia [13]. They are also ties of naphthoquinones. Many mechanisms have been
present as secondary metabolites of microorganisms. reported in the literature about how this class of com-
Lawsone (7), plumbagin (8), lapachol (9), juglone (10) pounds exert their action on cancer cells and others have
and shikonin (11) are naturally-occurring naphthoqui- been proposed but not yet completely elucidated. Hence,
nones, isolated from plants, which show many biological it is likely that they kill or induce cell death by more than
and pharmacological properties as well as being used as one mechanism, which will be described in the present
models for further structural modifications, as we will see review.
later on this review [44]. Isolated from the root tissues
of Lithospermum erythrorhizon and known in Chinese Generation of ROS and oxidative stress (DNA damage)
traditional medicine for many years for the treatment In eukaryotic cells, ROS are generated as byproducts of
of sore throat, burns, cuts and skin diseases, shikonin metabolism in mitochondria, as well in other cellular
(11) acts in a variety of molecular targets that are asso- organs that consume high levels of oxygen. Some of the
ciated to the genesis of cancer, such as the upregulation most reactive ROS have unpaired electrons (free radi-
of p53 and increased expression of apoptosis regulator cals), such as O2− and ·OH. Although H2O2 is not a free
BAX (Bax) [45, 46]. Besides, shikonin (11) shows similar
O O O O
OH OH
(7) Lawsone
(8) Plumbagin
O OH O O OH O
(8) (9) Lapachol
(7) (9) (10)
(10) Juglone
OH O H OH O O
(11) Shikonin
O O
(12) Beta-lapachone
(13) Alpha-lapachone
OH O O O
(11) (12) (13)
Fig. 3 Examples of naturally-occurring naphthoquinones
radical, it has the ability to diffuse through cell mem- the detoxifying enzyme cytochrome P450 reductase and
branes, thus affecting sites other than the one where other flavoprotein enzymes [44]. As an alternative path-
it was produced. Moreover, it can be converted to ·OH way for the activation, two-electron reductions can take
by the so-called Fenton reaction. Low concentrations of place followed by its inactivation through subsequent
ROS are necessary for certain functions in the cell to take glucuronidation and/or sulfation or by conversion into
place normally, for example the folding of new proteins in an alkylating intermediate. This pattern is believed to be
the ER and the control of caspase activity during apopto- the preferred one in anaerobic conditions [53] and the
sis [2, 50]. enzyme responsible for them is NQO1 (NADPH: qui-
Under specific conditions ROS may have deleterious none oxidoreductase 1), which is abundantly expressed in
effects on cells. The damage depends on their intracel- cancer tissues [54].
lular concentration and on the equilibrium with endog- Cancer cells are believed to have greater concentra-
enous antioxidant species. If this equilibrium is lost, tion of ROS compared to normal cells. They also have
oxidative stress takes place in the cell causing damage to higher metabolic demands and activity, thus requiring
molecules such as proteins, lipids, DNA and RNA [50]. more ATP. This leads to a major formation of superoxide
Regarding DNA damage, which is the one mostly associ- radical anions through the electron transport chain. It is
ated with carcinogenesis, three types have been reported: also believed that this higher, but non-lethal concentra-
base modification, strand breakage and DNA–protein tion of ROS, promotes tumor growth through cancer cell
cross-linkage. The base 2-deoxyguanosine is particularly survival even when damaging DNA, since some of these
affected by the hydroxyl radical and is one of the major damages could be advantageous for the cancer cell [55].
damages that occurs in vivo causing mutations to DNA This imbalance could be used as a target for chemother-
[2]. The derivative 8-hydroxy-2′-deoxyguanosine is con- apy since the production of even higher amounts of radi-
sidered a biomarker of oxidative stress and carcinogen- cal species in the cell can make the situation even more
esis [51]. critical leading the cell to its death [2]. Here is where
Naphthoquinones bear the ability to accept one or two naphthoquinones can have one of their actions as anti-
electrons to form semiquinones and hydroxyquinones, cancer agents and this would also serve as an explanation
both highly reactive species (Fig. 4). These species can to their selectivity towards cancer cells rather than nor-
then be oxidized again by molecular oxygen generat- mal ones, since their oxidative stress should not be natu-
ing ROS. Its direct implication in apoptosis has been rally imbalanced. For some naphthoquinones, such as
reported by Stangel et al. [52]. Also, naphthoquinones β-lapachone (12), selectivity may also be due to overex-
can participate in biochemical reactions being reduced pression of NQO1 in cancer cells since these compounds
to the semiquinone, a free radical, and subsequently to require this enzyme to become fully reactive [56].
the hydroxyquinone (Fig. 4). Under aerobic conditions, A class of enzymes which have been reported to be
one-electron reductions predominate, resulting in free targeted by ROS are cysteine proteases. The thiol of
radical intermediates [53]. They are carried out mainly by the catalytic Cys residue in them would undergo oxida-
tion by ROS to the sulfenic acid leading to the inhibition
of their activity [57, 58]. Within this type of enzymes,
ubiquitin-specific proteases 1 and 2 (USP1 and USP2)
a are linked with DNA damage repair. Particularly, USP2
has been associated with apoptosis resistance by can-
cer cells through stabilization of Fas and cyclin D1 [59].
It has been reported that both enzymes are susceptible
to ROS and are inhibited by the natural ortho-quinone
b β-lapachone (12), suggesting that they are interesting tar-
gets for the development of compounds for cancer ther-
apy. Also, a correlation was established between redox
potentials of several ortho-quinones and their inhibition
Fig. 4 Redox properties of naphthoquinones. a One-electron effect on the enzyme. Interestingly, when they tested the
reduction of the naphthoquinone core, first to the radical best candidates of the series on DU145 prostate cancer
semiquinone and then to the hydroquinone. b Reduction of the cells, which overexpresses USP2, the compounds did
naphthoquinone core in a biochemical context in the presence of
not exhibit potent activity [58]. The same group which
NADPH under aerobic conditions, with the resulting production
of the superoxide radical anion. It is important to consider that reported these results, later on published a second arti-
semiquinones cannot be reoxidized to quinones without oxygen and, cle focusing on para-quinones suggesting that they also
therefore, can accumulate in the cell in anaerobic conditions [2] inhibit USP2 through redox cycling [59]. This finding
was unexpected since para-quinones are not regarded as ular oxygen to form semiquinones, producing ROS which
redox cyclers and are believed to exert their action pre- can generate oxidative stress, or forming the semiqui-
cisely through different mechanisms of actions that do nones through comproportionation reactions [68]. The
not depend so much on ROS formation [60]. hydroquinone formed through this mechanism can act as
A recent work [61] investigated the molecular mecha- an alkylating agent for nucleophilic sites, including DNA,
nism and intracellular targets of plumbagin (8) related to which can lead to irreparable damage. NQO1-directed
its cytotoxic effect in HepG2 hepatocellular carcinoma anticancer agents are designed to target this issue. It is
cells. Target identification was carried out by similarity important to highlight that the stability of the final hyd-
ensemble approach (SEA), interestingly obtaining the roquinone produced will determine the role of NQO1 as
result that most of candidate target proteins are involved a detoxifier or bioactivating enzyme [65]. Also, on this
in redox signaling. Among these proteins, some of the note, it has been reported that some quinones can redi-
most significant were mitogen-activated protein kinases rect NQO1 to a ‘futile redox cycle’ in which the unstable
(MAPK), thioredoxin reductase and glutathione reduc- final hydroquinone re-oxidizes in a spontaneous manner
tase. On the last two, plumbagin (8) acted by prevent- into parental compounds. This results in very high levels
ing reaction with their intracellular targets and by direct of superoxide anion which can then be metabolized by the
inhibition of the protein, respectively. Ong et al. [62], enzyme superoxide dismutase into hydrogen peroxide,
studied the cytotoxicity of 2-methoxy-1,4-naphthoqui- causing oxidative stress and damage [69].
none (MNQ) (14) (Fig. 5) a naturally-occurring naphtho- NQO1 is expressed in many tissues, particularly in
quinone extracted from Impatiens balsamina, in A549 those which require a high level of antioxidant protec-
lung adenocarcinoma cells, reporting for the first time its tion. Nevertheless, it has been widely reported that
action on the JNK (c-jun-NH2-kinase) and MAPK signal- NQO1 is overexpressed in cancer tissues, which would
ing pathways, triggered through the generation of ROS. serve as a justification for the selectivity of agents tar-
geting this enzyme [70, 71]. NQO1 could serve as a
i. NQO1: its role in cancer and as quinone detoxi- target for cancer therapy through two different mecha-
fier NQO1 is a two-electron reductase mainly located nisms: its inhibition can lead to cell growth suppression
in the cytosol, which can use either nicotinamide adenine since the normal function of this enzyme in the cell is to
dinucleotide reduced (NADH) or nicotinamide adenine protect them from mutagenic, carcinogenic and cyto-
dinucleotide phosphate reduced (NADPH) as electron toxic effects derived from quinones. On the other hand,
donors. This enzyme has an important role in the protec- NQO1 can also be used as a tool for activating quinone-
tion of the cell against natural and exogenous quinones. like compounds through reduction. A recent study car-
It is a homodimer made up of monomers of 247 residues ried out by Pidugu et al. [63], reported for the first time
with two flavine adenine dinucleotide (FAD) cofactors at the direct interaction of a dimeric naphthoquinone, E6a
each active site of each monomer [63], and it is reported (16), with NQO1 through the determination of the crys-
to be inhibited by dicoumarol (15) [64]. When reduc- tal structure of the complex with this compound bound
ing quinones to hydroquinones in a single step, it yields to the enzyme, also providing evidence that this interac-
substrates for Phase II conjugation and subsequent elimi- tion directly affects the redox state of the FAD cofactors
nation and it also bypasses the toxic radical intermedi- (Fig. 6) [63].
ates that occur in the one-electron reduction [65]. Both, Photodynamic therapy is one of the approaches for
ortho- and para-quinones are believed to be substrates of cancer treatment which has been clinically approved
this enzyme [66]. For instance, β-lapachone (12) is one of and is gaining relevance in the last years. It involves
its substrates, being eliminated metabolically by NQO1 the administration of a compound which acts as pho-
through quinone reduction and subsequent glucuronida- tosensitizer (e.g. 5-aminolevulinic acid, and its methyl-
tion [67]. The hydroquinones produced by NQO1 reduc- ated derivative) followed by illumination with light of
tion are not always stable, being able to react with molec- a specific wavelength, locally. Selectivity derives from
the ability of the sensitizer to accumulate in the tumor
and the precise delivery of light specifically where it
is needed and one of its main advantages is that it is
minimally invasive. It has been reported that expres-
sion of NQO1 in human breast cancer MCF-7c3 cells
is induced after photodynamic therapy and that, con-
sequently, the sensitivity of cancer cells to treatment
with β-lapachone (12) increases [72]. These findings
Fig. 5 Structure of 2-methoxy-1,4-naphthoquinone (MNQ) suggest that the combination of this type of therapy
with β-lapachone (12), or any other substrate of NQO1, Maybe ROS generation by naphthoquinones depends
would be synergistic and a good field of study for the on the presence of the enzymes which reduce them to
development of new therapies. generate the actual amount of ROS which is lethal for
the cell. Would this be the case, overexpression of these
ii. Food for thought It has been established that one of enzymes would be crucial for naphthoquinones to exert
the main mechanisms of actions for naphthoquinones is their action, which would also serve as an explanation
the generation of ROS. What could be, then, the expla- towards their selectivity, since enzymes such as NQO1
nation for not finding direct correlations between redox are reportedly overexpressed in cancer tumors. On the
potentials calculated in vitro and the biological effect? same note, it is also probable that not all naphthoqui-
In our particular experience, those redox potentials nones work mainly through this mechanism, but they
were positive, which implies a spontaneous reduction of might prefer others depending specifically on the char-
the compounds, meaning that they would be producing acteristics of their chemical structure. This backs up the
ROS even in the absence of reductases, but probably this idea that not all naphthoquinones are substrates for the
amount of ROS is not enough for killing the cell. However, same enzymes.
it is important to consider that redox potentials calculated
from cyclic voltammetry cannot be considered as exact ERS and induction of apoptosis
indications of standard potentials and cannot be directly There is an interplay between the level of Ca2+ in the cell,
linked to the potential which would be observed for the ERS mediated by ROS generation and mitochondrial
same molecules under physiological conditions [75]. function that can lead to apoptosis. Gara et al. [45], have
One must bear in mind that the environment in which recently proven the role of shikonin (11) in inducing cell
the redox reaction takes place will influence its outcome death through ERS in prostate cancer cell lines (DU-145,
dramatically and when it comes to the cell, characteristics PC-3), having a direct effect on the production of ROS
such as lipophilicity, oxygen partial pressure, etc. might and an increase in the amount of Ca2+ in the cell.
vary a lot compared to aqueous solutions [53]. Neverthe- It has also been reported that shikonin (11) directly
less, there are some groups of scientists which have been inhibits tumor proteasome in studies performed
able to prove correlations between reduction/oxidation in vitro (murine hepatoma H22 and leukemia P388;
(redox) potentials of some naphthoquinones and the bio- human prostate cancer PC-3 cells) and in vivo (murine
logical effect observed [76, 77], but in our experience this H22 allografts and PC-3 xenografts) [78]. Proteas-
is not always the case. omes are protein complexes present in all eukaryotic
cells whose main function is to degrade damaged or
Fig. 6 Visualization of naphthoquinone E6a (16) and its interaction with NAD(P)H dehydrogenase (pdb code: 5EAI). a The naphthoquinone is
represented in green, FAD and the protein in cartoon and side chains of residues with a distance less than 5Å are highlighted. b Protein chains
highlighted in blue and gray and the surface of the naphthoquinone in colors per heteroatom. c Representation of the protein’s surface and the
binding site of the naphthoquinone together with FAD. The cyan surface represents the residues which belong to the binding site with the distance
criterion to the naphthoquinone (surfaces were calculated according to Sanner et al. [73]). Molecular modeling graphs were produced with the
package UCSF Chimera [74]
unfolded proteins by proteolysis. The action of shikonin Regulation of the tumor suppressor factors p53 and p73
(11) on the tumor proteasome could lead then to ERS Activation of p53 factor seems to be a potential target in
generated by an excess of unfolded/misfolded proteins anticancer therapy and an important aspect in the phar-
in the cell because of lack of their clearance [45]. macology of naphthoquinones, since the modulation of
An important recent study [79] has shown that while the factor itself or the molecules that it targets, can help
shikonin (11) triggers the ERS response in human glio- in the modulation of the apoptotic intrinsic pathway.
blastoma stem cells as a way of exerting its cytotoxic There is another tumor suppressor factor, p73, that can
effect, this can also generate the activation of pro- act on the targets of p53, particularly on PUMA, p21 and
survival pathways that may compromise its anticancer p16INKA4 [82]. This factor is usually kept at low levels
activity. They reported that while ERS is involved in in basal conditions and its concentration is augmented
the antitumor activity of shikonin (11) it also compro- when the cell is stressed [83]. In addition to this, inverted
mises its cytotoxicity via upregulation of the JNK/c-Jun CCAAT box binding protein of 90 kDa (ICBP90) is a
pathway and that inhibition of ERS in cancer cells could nuclear protein which promotes the activity of topo IIα
potentiate cytotoxic effects. by binding to part of a known sequence of its gene pro-
Recent studies demonstrated the role of NQO1 in moter [84]. This protein is regulated by both tumor sup-
the cytotoxicity of a dual therapy with β-lapachone pressor factors already mentioned, p53 and p73, and it is
(12) and ionizing radiation in N QO1+-MDA-MB-231 believed to be overexpressed in various types of cancer
human breast cancer cells, and the induction of mito- [82].
chondria-mediated cell death through ERS induced A recent study proved that shikonin (11) produces the
JNK pathway activation [80]. On this note, uridine up-regulation of the p73 factor in human cervical (HeLa)
diphosphate-glucuronosyltransferases (UGTs), are and breast (MCF-7) cancer cells, thus down-regulating
important phase II metabolic enzymes which help in the anti-apoptotic ICBP90 and re-expressing p16INK4A,
the elimination of the products of the two-electron one of the targets of p53 and also a pro-apoptotic fac-
reduction of NQO1 substrates, acting as detoxifiers. In tor [82]. Also, it has been reported that shikonin (11)
their absence, the compound could return to its origi- can activate p53 in response to DNA damage, reducing
nal form by oxidation, producing ROS in the way. Stud- the expression of cdk4, leading to apoptosis in human
ies with human colon cancer cells (HT29 and HCT116) malignant melanoma A375-S2 cells and also through the
exposed to β-lapachone (12) have proven that UGTs upregulation of Bax and downregulation of Bcl-2 [85].
glucuronidate the toxic catechol that is the product Plumbagin (8), isolated from the roots of Plumbago
of its reduction by NQO1. Hence, the action of UGTs scandens L., has been reported to upregulate the expres-
could be the reason why many drugs targeting NQO1 sion of p53 in a panel of human brain cancer cell lines,
have shown chemoresistance [67]. inducing cell cycle arrest at the G2/M stage, and altering
Another very recent study has reported that 2-meth- the Bax/Bcl-2 ratio [86]. Also, it increases the expression
oxy-6-acetyl-7-methyljuglone (MAM) (17) (Fig. 7), a of apoptosis markers caspase-3 and -7 in human HepG2
natural derivative of juglone (10), isolated from P. cus- hepatocellular carcinoma cells [61].
pidatum, induced apoptosis in a caspase-dependent
manner on some human cancer cell lines like MCF7 Inhibition of the DNA topoisomerases
(breast) and B16-F10 (melanoma) and necrosis in A549 As mentioned before, naphthoquinones have been iden-
(lung) cells. These effects were mediated by the pro- tified to inhibit both types of topoisomerases present
duction of hydrogen peroxide inducing the JNK/iNOS/ in the eukaryotic cells: I and II, being the latter mostly
NO (c-jun-NH2-kinase/inducible nitric oxide synthase/ associated with their cytotoxic activity and the target for
nitric oxide) pathway [81]. many anticancer agents. Both enzymes break DNA at the
phosphodiester bond using a catalytic tyrosine residue
O O
OH OH O
Br (15) Dicoumarol
O
(16) E6a
O O O OH O
OO O (17) MAM
HO
(15) (16) (17)
O
Fig. 7 Chemical structures of dicoumarol, dimeric naphtoquinone E6a and MAM (2-methoxy-6-acetyl-7-methyljuglone)
and are critical for the correct functioning of the cell. Any mechanisms proposed are described in detail in both
alteration in their balance is enough to induce apoptosis excellent publications on the topic by the same group in
[13]. the years 2000 and 2001 [87, 90].
The catalytic function of topo II is essential for the Type I topoisomerase has received less attention
maintenance of the topology of the DNA molecule dur- regarding the action of naphthoquinones and their inhi-
ing replication, transcription and recombination. The bition of the enzyme. The enzyme received its name
enzyme has the ability of introducing a transient double- because it introduces single-strand breaks into the
stranded break on the DNA molecule by binding non- DNA molecule [88]. It has been reported that shikonin
covalently to it and is attached in a covalent manner to (11) is an inhibitor of the enzyme at low concentrations
the 5′-phosphate of the DNA molecule. After ATP is (IC50 = 2 µM) in studies carried out by Zhang and col-
bound, a second strand of DNA passes through the break laborators, 2013 [91]. β-lapachone (12) was first reported
and the enzyme reseals it. These events are known as as a topo I inhibitor but studies on yeasts lacking expres-
pre-strand and post-strand passage cleavage, respectively sion of the enzyme showed that the compound could
[87]. still suppress their growth [87]. Some naphthoquinone
There are two classes of inhibitors according to their derivatives have been synthesized and their inhibition
mechanism. ‘Poisons’ are those which stabilize the cova- of the enzyme has been proven [11, 12], but most of the
lent intermediates of the enzyme, usually a ternary com- work that has been dedicated to it is in relation to camp-
plex involving DNA, the enzyme and the compound [88]. tothecin derivatives, a quinolone alkaloid, which finally
Those which work at any other stage of the catalytic cycle resulted in two well-known anticancer drugs: topotecan
are simply called “catalytic inhibitors”. Doxorubicin (2), (19) and irinotecan (20).
a well-known anthracycline used clinically for the treat- Apart from the inhibitors described so far, there is
ment of cancer, is classified as a “poison” [89] and so another class of compounds which act as dual inhibitors.
are the majority of the compounds which inhibit these They could be classified in three types: (i) drugs which
enzymes. Another example of a “poison” is mitoxantrone bind directly to DNA, usually by intercalation; (ii) hybrid
(3) which is depicted in Fig. 8 bound to topo II-β in the molecules rationally designed by linking inhibitors of
presence of DNA. both enzymes, type I and II, or by linking pure inhibitors
Eleutherin (18), a naturally-occurring compound iso- to DNA-interactive carriers, and (iii) compounds which
lated from the bulb of Eleutherine americana, has been recognize structural motifs present in both enzymes [92].
identified as a reversible catalytic inhibitor of topo II by Saintopin (21), a fungal secondary metabolite, was the
Krishnan and Barstow [87]. They also proved the in vitro first compound reported as a dual inhibitor of the type (i)
inhibition of topo II by α and β-lapachone (13, 12) and described above, binding weakly through intercalation to
classified them as irreversible catalytic inhibitors. The
Fig. 8 Representations of the complex human β topoisomerase II, mitoxantrone and DNA (pdb code: 4G0V). a Representation of the view on the
side of mitoxantrone which is colored by atomic type. Side chains of the residues highlighted and named with the criterion of distances less than
5Å; DNA in cartoon. b Representation of the top view of mitoxantrone (carbons in green) complexed with DNA and the side chains of the residues
(surfaces were calculated according to Sanner et al. [73]). Molecular modeling graphs were produced with the package UCSF Chimera [74]
Table 2 Table of quinone-like topoisomerase inhibitors microenvironment the tumor needs in order to survive
and progress [96]. It is suggested that the signal trans-
Topoisomerase II
ducer and activator of transcription (STAT) family pro-
Poisons Doxorubicin, MIX
teins, particularly STAT3, selectively induce and maintain
Reversible catalytic inhibitor Eleutherin
the inflammatory microenvironment, at the initiation as
Irreversible catalytic inhibitors α and β-lapachone
well as during progression of the tumor [97]. It is linked
Topoisomerase I
to inflammation-associated tumorigenesis initiated by
Shikonin, camptothecin derivatives
genetic alterations in cancerous cells as well as by many
Dual
other external factors such as chemicals, UV radiation,
Saintopin
stress, cigarette smoking and infection [96]. This type of
MIX mitoxantrone signaler undergoes phosphorylation at a specific tyrosine
residue (Tyr705 for STAT3) followed by homo/hetero-
dimerization, translocation to the nucleus and activation
substrates of both types of enzymes and inhibiting their
of the expression of specific genes. STAT3 works as a
catalytic activity [93].
homodimer or a STAT1-STAT3 heterodimer [98]. Three
Some of the problems related to the classic inhibitors
domains within the protein have been identified as suit-
are resistance developed by the cells, which causes clini-
able for targeting compounds: amino-terminal, DNA
cal failure in long-term therapies [94], and the fact that
binding and SH2, which is involved in receptor-bind-
some topo II inhibitors have caused secondary malignan-
ing and dimerization [99]. Kortylewski et al. reported
cies since they can trigger chromosomal translocations
that STAT3 could be used as a target for cancer therapy
which may lead to a specific type of leukemia [95].
and that its removal, even under chronic inflammatory
A summary of the types of inhibitors and the most
conditions, inhibits carcinogenesis and the growth of
notable molecules which have been developed/discov-
established tumors. Also, mice reconstituted with Stat3-
ered is on Table 2 and Fig. 9.
deficient immune cells can generate a potent anti-tumor
immune response. This group were also the first to report
Other targets and mechanisms of action reported:
that STAT3 is persistently activated in immune cells
recent advances in the anticancer pharmacology
associated to a tumor, which leads to the suppression of
of naphthoquinones
innate and adaptive immune responses [100].
Recently, novel targets for naphthoquinones as antican-
Bhasin and collaborators, 2013 [99], worked on the
cer agents have been reported in the pursue of a bet-
synthesis of anthraquinone and naphthoquinone deriv-
ter understanding of their action on cancer cells and
atives using as a model STA-21 (22), which was the first
the apoptotic pathways they may trigger. Inflammation
reported inhibitor of the SH2 domain of STAT3 [101],
has now been widely recognized as an important factor
and maintaining its quinone moiety. They tested their
in tumor initiation and progression, leading to onco-
antiproliferative activity in human cancer cells lines
genic transformation. Genetic and epigenetic changes
of prostate (DU-145) and colon (HT-29) obtaining
in cancerous cells also generate the inflammatory
N
O O
HO O
O
N
N
(18) Eleuthrin
(18) O O
(19) (19) Topotecan
OH O
N (20) Irinotecan
OH O OH OH (21) Saintopin
N O O
N
O
N
HO OH
(20) O
O
OH O (21)
Fig. 9 Chemical structures of quinone-like topoisomerase inhibitors
positive results, which suggests that STAT3 could be Lim et al. [107], embarked on the search of mucosa-
one of the many targets of this class of compounds and associated lymphoid tissue lymphoma translocation
a new line to exploit. protein (MALT1) inhibitors in order to find new drugs
Joo et al. [102], focused on the action of plum- for the treatment of diffuse large B-cell lymphoma, one
bagin (8) on the STAT3 signaling pathway in a gastric of the most aggressive types of cancer with unmet clini-
cancer cell line (MNK-28) and suggested that it nega- cal needs. They found that 1,2-amino-naphthoquinones
tively modulates its activity via dephosphorylation were good inhibitors but lacked the ability to inhibit
rather than protein degradation. They also suggested the proliferation of OCI-LY3 cells (human B-cell lym-
that plumbagin (8) inhibits cell proliferation in a time- phoma) in vitro. When testing β-lapachone (12), in the
and dose-dependent manner, as well as migration and search for new scaffolds, they interestingly found that it
invasion and induces apoptosis in MKN-28 cells. is a rather potent inhibitor of MALT1 (IC50 = 1.9 µM)
Mutation and activation of the epidermal growth fac- and, as mentioned before, is an important inhibitor of
tor receptor (EGFR) has been detected in many solid cell growth. They also suggested the formation of a cova-
tumors. Also, nuclear factor kappa B (NF-κB) has a cru- lent bond between the enzyme and the compound. When
cial role in the induction of the expression of inflamma- assessing derivatives of β-lapachone (12) synthesized by
tory mediators as well as being the main transcription the group, the addition of an electron-withdrawing sub-
factor in many immune responses [103]. Unsurpris- stituent at the C8 position led to an increase in activity.
ingly, signaling by STAT3 and NF-κB are highly inter- The initial hit for this discovery was obtained through
related [96]. Tian et al. [104], demonstrated the role high throughput screening and subsequently modified
of shikonin (11) on the inhibition of the EGFR-NF-κB through structure-based drug design strategies. Com-
signaling pathway on epidermoid carcinoma cells, pound 4d (24) (Fig. 10) exhibited good antiproliferative
A431. It decreased the phosphorylation of EGFR and activity, comparable to that of its precursor, and inhib-
STAT3 in a concentration-dependent manner (2.5 µM, ited the paracaspase activity of MALT1, showing that
5 µM and 10 µM were tested). Moreover, Zhao et al. the 1,2-naphthoquinone moiety could be a good starting
[105], proposed the use of shikonin (11) in combina- point for the development of new MALT1 inhibitors in
tion with erlotinib (23), a well-known anticancer drug, the future.
which competes for the ATP-binding site of the tyros-
ine kinase (TK) domain in EGFR. Examples of recently synthesized naphthoquinones
On a different note, Khaw et al. [86], focused their with enhanced anticancer properties
studies on plumbagin (8) and its telomerase activity in As noted in this review, naphthoquinones as anticancer
human glioblastoma multiforme cells [A172, KNS60, agents exhibit many mechanisms of action (Fig. 11) and it
U251MG(KO)] and medulloblastoma cells (ONS76). Tel- appears that there are more to be elucidated and/or clari-
omerase is a reverse transcriptase specialized in synthe- fied. Many groups of scientists around the world have
sizing telomeric DNA, thus, contributing to maintaining embarked on the search for new analogous compounds
functional telomeres [106]. It has differential expression which could enhance the action of the naturally-occur-
in normal and cancer cells, which makes it an attractive ring naphthoquinones and provide new information
new target for chemotherapy. Validation of mechanisms into the actual mechanisms, or combinations of them, in
already reported for plumbagin (8), such as cell cycle which they exert their antitumoral action. Here, we chose
arrest at the G2/M phase and report of repression of tel- some of the most notable and creative efforts found in
omerase activity and shortening of telomeres after incu- the literature, which provide an advantage in potency or
bation with the compound for 15 days, was carried out by selectivity (sometimes in both), or work as ligands of spe-
this group. cific targets described in previous sections. These mol-
ecules, summarized in Fig. 12, could be used as starting
O N O
O NC O (22) STA-21
O O N
O
CH (23) Erlotinib
O OH HN C
O
O (24) 4d
(22) (23) (24)
Fig. 10 Chemical structures of STA-21, erlotinib and 4d, a compound which acts as MALT1 inhibitor. A summary of all the mechanisms of action
gathered in this review for naphthoquinones as anticancer agents can be seen in Fig. 11
Fig. 11 Summary of the mechanisms of action for naphthoquinones as anticancer agents gathered in this review
points for modifications which could turn them into bet- methyl group on the position 2 of the naphthoquinones
ter candidates and help in the further understanding of moiety is vital for cytotoxic activity [109].
this pharmacological class. A series of novel 2-N-aminonaphthoquinones were
Compounds 25a and 25b were synthesized by Car- synthesized by de Moraes and collaborators, from which
doso et al. in 2014 [108] as part of a series of 1,2-fura- compound 27 was identified as the most relevant due
nonaphthoquinones linked to 1,2,3-1H-triazoles with to its yield in the synthesis (60%) and its high potency
the objective of investigating their antileukemic activity against a panel of different cancer cell lines (exhibit-
in lymphoid (MOLT-4 and CEM) and myeloid (K562 and ing 100% growth inhibition in MDAMB-435 breast car-
KG1) leukemia cell lines The former has good potential cinoma cells). It also showed good selectivity towards
for further studies since it turned out to be a potent and cancer cells when tested in normal peripheral blood
selective hit towards cancer cell lines also showing pref- mononuclear cells (PBMC), greater than some of the
erence against leukemia lymphoid cell lines. Compound natural naphthoquinones used as precursors, such as
25b is also a promising hit with high potency as a cyto- juglone [110] (10).
toxic agent (IC50 for leukemia cell lines range between Wang and collaborators synthesized a series of lipo-
0.05 and 11.07 µM). philic lawsone (7) and juglone (10) derivatives, being
To develop new compounds based on the structures of compound 28 the most potent cytotoxic agent of the
juglone (10) and plumbagin (8), Fiorito and collabora- series showing the highest effect on the human colorectal
tors synthesized a series of derivatives from which com- adenocarcinoma cell line HT-29 with an IC50 of 2.0 µM
pounds 26a and 26b resulted as the best candidates. 26a in 48 h. They also reported that 28 arrested the cell cycle
is an ether derivative of juglone (10) which showed more of these cells at the S phase and that it induced apoptosis
cytotoxicity than its parent compound in 3 out of 6 cell [111].
lines tested (A549, SKMEL-28 melanoma and U373 glio- Another novel series of aminonaphthoquinones was
blastoma). On the other hand, 26b is also an ether deriv- synthesized by Benites and collaborators in 2010, from
ative but of plumbagin (8), showing better potency than which compound 29 was the best hit from cytotoxicity
its precursor in all cell lines tested. To be noted, all the studies with human cancer cell lines of breast (MCF-7),
plumbagin (8) derivatives were more active than the ones prostate (DU145) and urinary bladder (T24), making it a
related to juglone (10) suggesting that the presence of the promising candidate for further investigation [112]. Years
O
O R O
R2
O
N N
O N
R= a)
OR1 O
O N N O R= (CH2)3Cl b)
H
O
R1= CH2CH=C(CH3)2
R2= H a); Me b)
O (25)
O (33)
N N
N O
(26)
O Ph O
Br (32)
H
N CO2Et
(31)
(27)
O O
O
Cl
O O
(30)
N (29) (28) OH
N N
H
O
O O
N
O
O
OH
O
N NH
O O
Fig. 12 Novel structures found in the literature aimed to enhance the natural properties of naphthoquinones. Green—potency enhanced. Red—
selectivity enhanced. Blue—enzyme inhibitors
later, in 2016, the same group reported that juglone (10) naphthoquinones for their action as inhibitors of USP2
and 29 can be used in combination with ascorbate to through ROS production. Until this finding, ortho-qui-
potentiate their oxidative stress action in vivo in Ehrlich nones were regarded as good inhibitors of this cancer-
ascites tumor-bearing mice, showed inhibition of tumor related enzyme as opposed to para-quinones, precisely
progression [113]. for their inability to behave as redox cyclers. This opens a
In the search for new anti-HIV agents, Carter-Coop- door in this particular field of study for the development
eret al. synthesized a novel series of amino dimeric naph- of new para-quinones which can target USP2 [59].
thoquinones with reportedly good anti-integrase and While looking for new trypanocidal agents derived
cytotoxic activity against a panel of leukemia cancer cell from quinone moieties, Bahia and collaborators syn-
lines [114]. Trying to increase their antitumoral proper- thesized a series of 1,2,3-triazoles and evaluated their
ties, they made some modifications to the original struc- cytotoxic activities in a panel of cancer cell lines from dif-
tures that led to a series of bis-aziridinyl compounds. The ferent human tissues as well on normal cells (peripheral
derivative 30 showed the best results against acute mye- blood mononuclear cells). Compound 32 was one of the
loid leukemia (AML) cell lines (MOLM-14, MV4-11 and most potent in various cancer cell lines with IC50 values
THP-1) with IC50 values ranging from 0.18 to 1.05 μM. ranging from 0.41 to 1.59 μM. It also showed good selec-
This compound also had moderate selectivity towards tivity with an SI of 0.8–3.0 in comparison to that pre-
normal cells from hematopoietic bone marrow, with sented by a well-known and widely-used anticancer drug,
selectivity index (SI) ranging from 1.5 to 2. doxorubicin (2), which was 0.8 [115].
The high potency of para-quinone 31 caught the Finally, deoxynyboquinone (33) was originally syn-
attention of Gopinath and collaborators when testing thesized while studying the antibiotic nybomycin [116]
O O
N3
+ H2N CH2Cl2 N3
N
H
(34) O O
CuSO4.5H2O
Sodium ascorbate
THF/H2O (1:1), rt
N N
N R1
N
(35a-b) H
O
35a R1 = -CH3; A549 (IC50 9.23 µM), DU-145 (IC50 7.81 µM), and MCF-
7 (IC50 12.67 µM)
35b R1 = -CH2CH3; A549 (IC50 9.19 µM), DU-145 (IC50 8.02 µM), and
MCF-7 (IC50 8.73 µM)
Fig. 13 Synthesis of menadione-1,2,3-triazole hybrids
and it has been reported to have a tenfold superior

efficacy compared to that of β-lapachone (12) in killing
different cancer cell lines in a NQO1-dependent man-
ner [117]. Kolossov and collaborators further studied
this compound to conclude that it is a more specific
substrate of NQO1 and offered proof that it does not
elicit off-target responses at effective concentrations,
Fig. 14 Chemical structure of MitoK3
unlike β-lapachone (12) [77].
Another simple naphthoquinone that exhibits anti-
cancer activity and is also widely used as starting Moreover, it also enhanced the cytotoxic properties of
material for the preparation of derivatives with cyto- doxorubicin (2) in A549 cells.
toxic potential is menadione (34). In 2018, Prasad and
co-authors [118] synthesized a series of novel mena- Are naphthoquinones as PAINful as they seem?
dione-based triazole hybrids (Fig. 13) and evaluated Not all the attention which naphthoquinones have drawn
their anticancer activity against five selected cancer to themselves has been on the bright side. Since the
cell lines including lung (A549), prostate (DU-145), publishing of a highly cited article written by Baell and
cervical (Hela), breast (MCF-7), and mouse mela- Holloway 7 years ago [120], which created many follow-
noma (B-16). They found that the majority of hybrid ers worldwide, naphthoquinones have been disregarded
compounds displayed significant anticancer activity and looked upon by a great portion of the scientific
with special attention for compounds (35a) and (35b), community.
which exhibited potent activity against all cell lines. In the referred article, the authors coined the term pan-
Oliveira and collaborators [119], also used mena- assay interference compounds (PAINs) to refer to com-
dione to prepare a mitochondrial-directed agent, pounds which would form aggregates, react with proteins
MitoK3 (36), which was developed by conjugating a or interfere in screening assays, leading to false positives
TPP (triphenylphosphonium) cation to the C3 posi- [120]. In an attempt to prevent scientists from pursuing
tion of menadione’s naphthoquinone ring, increasing studies with them, they created a set of computational
its selective accumulation in mitochondria as well as filters (alerts) which have been widely accepted by aca-
leading to alterations of its redox properties and con- demia and the pharmaceutical industry, that allows for
sequent biological outcome (Fig. 14). MitoK3 showed the recognition of certain substructures within molecules
cytotoxic activity towards human cancer cell lines that are regarded as PAINs. A set of 480 PAINs alerts
of liver (HepG2), breast (MCF-7) and lung (A549). were created aiming at identifying and discarding these
substructural features frequently found in them and
suggesting that they could be used to flag false screening role whether its parent compound can act as an artifact
hits and annotate suspect compounds in screening librar- or not [125].
ies [120]. Particularly, in an article published in 2016, Finally, something that cannot be overlooked is the
the main author maintained the idea, extending their potential application of this kind of compounds which
application to natural products and referred to quinones elicit various pharmacological actions, in the advancing
as compounds which tend to be redox active as well as field of polypharmacology. Accordingly to this concept,
reactive to nucleophiles such as the amino acids cysteine compounds which have the ability to interact with mul-
and lysine, present in the side chains of proteins. They tiple targets are actually desired, and this kind of versatil-
regarded their screening hits as PAINs and nonprogress- ity should be clearly distinguished from promiscuity that
able [121]. derives from non-selective activity [126].
Although what was proposed by Baell and Holloway
is to some extent, true, in the sense that when working
with compounds, which have some of these character- Conclusions
istics, scientists should be careful enough not to report Naphthoquinones act as anticancer agents through a
false/biased results in order to avoid irreproducibility in variety of mechanisms involved in all types of cancer.
research, ruling them out of any kind of structural opti- Some of the most important explored so far and which
mization campaign or from the pharmaceutical pipeline have been extendedly reported in the literature are: dam-
could lead to the loss of valuable chemical material. Still, age of DNA through generation of ROS, inhibition of
the question remains of which molecules should be con- topoisomerase II, regulation of the tumor suppressor
sidered as PAINs [122]. factor p53 and induction of apoptosis via ERS. In this
In an effort to ameliorate the blind application of these review, we summarized the most well-known mecha-
filters, some authors have published data that question nisms as well as those which have been recently described
their usefulness. One finding which is crucial is the fact in the literature, providing new interesting targets to be
that many PAINs substructures can also be found in researched in this pharmacological family. There is still a
drugs available in the market [123, 124], meaning that if lot to be elucidated about the ways in which these com-
the filters are used without rational thinking, potentially pounds exert their action as anticancer agents and how
good drug candidates can be disregarded. Phenotypic to enhance their activity in order to exploit their poten-
assays, which are regaining the value they used to have in tial to the maximum. Being privileged structures in phar-
the past for screening libraries of compounds since they macology and chemistry, naphthoquinones will keep on
take the biological setting as a whole, would be incredibly fascinating scientists around the globe and will be further
affected by the use of PAINs filters, preventing optimiza- studied and researched in the many fields they have the
tion of the structure, and again, leading to the loss of pos- potential to thrive.
sible candidates [123].
Also, Capuzzi et al., have recently published a study in Abbreviations
which they hypothesized that PAINs filters have limited ADMET: absorption, distribution, metabolism, excretion and toxicity; AIF:
apoptosis-inducing factor; AML: acute myeloid leukemia; ATF6: activating
extrapolative power. They based on the limitations of the transcription factor 6; Bax: apoptosis regulator BAX; Bcl-2: B cell lymphoma 2
original study, in which a relatively small library of com- protein; CAD: caspase-activated DNase; Cdks: cyclin-dependent kinases; EGFR:
pounds of a proprietary nature was used, meaning that epidermal growth factor receptor; ER: endoplasmic reticulum; ERS: endoplas‑
mic reticulum stress; FAD: flavine adenine dinucleotide; IAP: inhibitor of apop‑
complete chemical structures were not shown, and they tosis proteins; IC50: 50% cytotoxic concentration; ICBP90: inverted CCAAT box
were tested for just one type of activity (protein–protein binding protein of 90 kDa; iNOS: inducible nitric oxide synthase; IRE1α: ino‑
interaction inhibition) in only six HTS campaigns, using sitol-requiring enzyme 1; JNK: c-jun-NH2-kinase; MALT1: mucosa-associated
lymphoid tissue lymphoma translocation protein; MAM: 2-methoxy-6-acetyl-
a single detection technology, AlphaScreen [122]. They 7-methyljuglone; MAPK: mitogen-activated protein kinase; MIX: mitoxantrone;
could prove that many compounds which contain PAINs MNQ: 2-methoxy-1,4-naphthoquinone; NADH: nicotinamide adenine
alerts do not exhibit assay promiscuity and they urge the dinucleotide reduced; NADPH: nicotinamide adenine dinucleotide phosphate
reduced; NCDs: non-communicable diseases; NF-κB: nuclear factor kappa B;
scientific community not to use these filters without con- NO: nitric oxide; NQO1: NADPH-quinone oxidoreductase 1; PAINs: pan-assay
ducting orthogonal experimental assays. They concluded, interference compounds; PBMC: peripheral blood mononuclear cells; PERK:
through the use of public data, that many compounds pancreatic endoplasmic reticulum kinase; PUMA: Bcl-2-binding component 3;
ROS: reactive oxygen species; SEA: similarity ensemble approach; SI: selectivity
without PAINs alerts are actually more promiscuous than index; STAT: signal transducer and activator of transcription; TK: tyrosine kinase;
the ones identified by the filters [122]. These findings are TNF: tumor necrosis factor; TP53: tumor protein p53; TPP: triphenylphospho‑
supported by another recent study indicating that the nium; UGT: uridine diphosphate-glucuronosyltransferase; UPR: unfolded
protein response; USP: ubiquitin-specific proteases.
molecular environment and structural context in which
the putative PAIN substructure is can play a fundamental
Acknowledgements newly synthesized 1,4-naphthoquinone derivative. Biochem Pharmacol.

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Cancer

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Cancer

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Pereyra

et al. Cancer Cell Int (2019) 19:207

REVIEW Open Access

The diverse mechanisms and anticancer

Background are among the disadvantages of the first two approaches,

cell proliferation. This becomes of high importance in

Fig. 2 Basic structural cores within the quinone family

and it has been reported to have a tenfold superior

Acknowledgements newly synthesized 1,4-naphthoquinone derivative. Biochem Pharmacol.

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Cancer

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Cancer

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Copyright

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Cancer

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Pereyra

et al. Cancer Cell Int (2019) 19:207

REVIEW Open Access

The diverse mechanisms and anticancer

Background are among the disadvantages of the first two approaches,

cell proliferation. This becomes of high importance in

Fig. 2 Basic structural cores within the quinone family

and it has been reported to have a tenfold superior

Acknowledgements newly synthesized 1,4-naphthoquinone derivative. Biochem Pharmacol.

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Fig. 2 Basic structural cores within the quinone family