marine drugs

Review
Cyanobacterial Metabolite Calothrixins: Recent
Advances in Synthesis and Biological Evaluation
Su Xu, Bhavitavya Nijampatnam, Shilpa Dutta and Sadanandan E. Velu *
Received: 5 August 2015; Accepted: 4 January 2016; Published: 12 January 2016
Academic Editor: Paul Long

Department of Chemistry, University of Alabama at Birmingham, 901, 14th Street South, Birmingham,
AL 35294-1240, USA; suxu@uab.edu (S.X.); snij@uab.edu (B.N.); shilpad@uab.edu (S.D.)
* Correspondence: svelu@uab.edu; Tel.: +1-205-975-2478; Fax: +1-205-934-2543

Abstract: The marine environment is host to unparalleled biological and chemical diversity, making it
an attractive resource for the discovery of new therapeutics for a plethora of diseases. Compounds that
are extracted from cyanobacteria are of special interest due to their unique structural scaffolds
and capacity to produce potent pharmaceutical and biotechnological traits. Calothrixins A and B
are two cyanobacterial metabolites with a structural assembly of quinoline, quinone, and indole
pharmacophores. This review surveys recent advances in the synthesis and evaluation of the
biological activities of calothrixins. Due to the low isolation yields from the marine source and the
promise this scaffold holds for anticancer and antimicrobial drugs, organic and medicinal chemists
around the world have embarked on developing efficient synthetic routes to produce calothrixins.
Since the first review appeared in 2009, 11 novel syntheses of calothrixins have been published in the
efforts to develop methods that contain fewer steps and higher-yielding reactions. Calothrixins have
shown their potential as topoisomerase I poisons for their cytotoxicity in cancer. They have also been
observed to target various aspects of RNA synthesis in bacteria. Further investigation into the exact
mechanism for their bioactivity is still required for many of its analogs.

Keywords: marine natural product; calothrixin; cyanobacteria; calothrix; antimicrobial activity;

anticancer activity; total synthesis

1. Introduction

1.1. Marine Natural Products

Dating back to ancient civilizations, natural products have played a vital role in drug discovery.
Modern day drugs such as penicillin, morphine, and paclitaxel (Taxol™), which are used for the
treatment of bacterial infections, pain, and cancer, respectively, are examples of natural products
that have successfully progressed through the drug development pipeline. Natural products are
abundant in several sources and are routinely extracted from plants, microorganisms, and animals.
While terrestrial plants and terrestrial microbes continue to be the primary contributors to the natural
product derived drug market, marine sources are an emerging resource that has been relatively
unexplored until recent times [1]. Marine organisms are considered to yield superior natural products
compared to terrestrial organisms in terms of novelty of structures and in producing potent bioactivities,
due to the distinctive environmental conditions in which marine organisms live [2–4]. Factors such as
predation, competition for space on highly populated coral reefs, and biochemical warfare between
organisms have greatly contributed to the evolution of compounds with unique structures and potent
biological effects [5].

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Mar. Drugs 2016, 14, x 2 of 20
Mar. Drugs 2016, 14, 17 2 of 21
1.2. Cyanobacteria
Microbial organisms in the marine environment have increasingly become one of the major
1.2. Cyanobacteria
focal points for investigations seeking to identify new chemical entities with novel structural
Microbial
backbones andorganisms
diverse in the marine
biological environment
activities. In thehave increasingly
1970s, Professor become
Richardone E.
of the majoratfocal
Moore the
points for investigations seeking to identify new chemical entities with novel
University of Hawaii began exploring the chemistry of marine cyanobacteria [6]. Cyanobacteria are structural backbones and
diverse
one of thebiological activities. Inonthe
oldest organisms 1970s,
Earth, Professor
with Richardrecord
an existence E. Moore
of atatleast
the University
2.7 billion of Hawaii
years [7]. began
These
exploring
prokaryotethe chemistry
organisms ofalso
are marine
known cyanobacteria
as blue green [6]. algae,
Cyanobacteria are one ofand
cyanoprokaryotes, the oldest organisms
cyanophytes due
on Earth, with an existence record of at least 2.7 billion years [7]. These prokaryote
to their blue-green pigment, c-PC (c-phycocyanin) [8]. This pigment is used for photosynthesis and organisms are
also known as to
is considered blue green
have algae,
played cyanoprokaryotes,
a crucial and cyanophytes
role in releasing oxygen intodue the to their blue-green
primitive pigment,
atmosphere [7,9].
c-PC (c-phycocyanin) [8]. This pigment is used for photosynthesis
Cyanobacteria possess a broad geographical distribution, ranging from limnic and marine and is considered to have played
aenvironments
crucial role intoreleasing
terrestrialoxygen
habitatsinto the primitive atmosphere [7,9]. Cyanobacteria possess a broad
[10,11].
geographical
A subclass distribution,
of marine ranging
natural from limnic andismarine
compounds producedenvironments to terrestrial
by cyanobacteria. Thehabitats [10,11].
most studied
A subclass of marine natural compounds is produced by cyanobacteria. The
species of marine cyanobacteria include Nostoc, Calothrix, Lyngbya, and Symploca [12]. Cyanobacteria most studied species of
marine
are known cyanobacteria
to produce include
potent Nostoc,
toxins. Calothrix,
SeveralLyngbya, haveSymploca
studies and [12]. Cyanobacteria
investigated are known
the pharmaceutical and
to produce potentpotential
biotechnological toxins. Several
of the studies
secondary have investigated
metabolites the pharmaceutical
isolated and biotechnological
from cyanobacteria. These studies
potential
revealed aofwide
the secondary metabolites
range of potent isolated from
pharmacological cyanobacteria.
effects These studies revealed
that include anti-inflammatory, a wide
antimalarial,
range of potentantimicrobial,
antiprotozoal, pharmacological effects that include
immunosuppressant, anti-inflammatory,
anticancer, antimalarial, anticoagulant,
anti-HIV, antibacterial, antiprotozoal,
antimicrobial, immunosuppressant,
antifungal, anti-tuberculosis, antiviral,anticancer, anti-HIV,
and antitumor antibacterial,
activities [12–14]. anticoagulant, antifungal,
A number of compounds
anti-tuberculosis, antiviral, and antitumor activities [12–14]. A number
that were isolated from aquatic cyanobacteria have showed promise as anticancer leads. Examples of compounds that wereof
isolated from aquatic
such promising cyanobacteria
compounds have showed
include apratoxin promise as anticancer
A, cryptophycin, dolastatin leads.
10, andExamples
largazole of such
(Figure
promising
1). compounds include apratoxin A, cryptophycin, dolastatin 10, and largazole (Figure 1).

Figure 1.
Figure 1. Biologically
Biologically active
active natural
natural products
products isolated
isolated from
from marine
marine cyanobacteria.
cyanobacteria.

Apratoxin A is a cyclodepsipeptide derivative of the apratoxin family of cytotoxins isolated

Apratoxin A is a cyclodepsipeptide derivative of the apratoxin family of cytotoxins isolated from
from a Lyngbya sp. collected from Guam [15,16]. It induces G1 phase cell cycle arrest and apoptosis,
a Lyngbya sp. collected from Guam [15,16]. It induces G1 phase cell cycle arrest and apoptosis, and
and has exhibited IC50 values ranging from 0.36 to 0.52 nM in vitro against 60 human tumor cell lines.
has exhibited IC50 values ranging from 0.36 to 0.52 nM in vitro against 60 human tumor cell lines.
However, in vivo studies revealed only marginal activity against early stage adenocarcinoma [17–19].
However, in vivo studies revealed only marginal activity against early stage adenocarcinoma [17–19].
Another class of highly potent anticancer agents produced by cyanobacteria is cryptophycins. For
Another class of highly potent anticancer agents produced by cyanobacteria is cryptophycins.
example, cryptophycin 1, which was isolated from Nostoc sp. GSV224, has an IC50 of 5 pg/mL against
For example, cryptophycin 1, which was isolated from Nostoc sp. GSV224, has an IC50 of 5 pg/mL
KB human nasopharyngeal cancer cells, and 3 pg/mL against LoVo human colorectal cancer cells
against KB human nasopharyngeal cancer cells, and 3 pg/mL against LoVo human colorectal cancer
[20]. Its mechanism of action is through the suppression of microtubule dynamics, thereby inhibiting
cells [20]. Its mechanism of action is through the suppression of microtubule dynamics, thereby
Mar. Drugs 2016, 14, 17 3 of 21

inhibiting cells
Mar. Drugs in 14,
2016, G2x/M phase. Cryptophycin 52, a chemical analog of cryptophycin 1, entered 3 of clinical
20
trials but produced only marginal activity [21].
cells in G2/M10
Dolastatin phase. Cryptophycin 52,
is cyanobacterial a chemical that
metabolite analog of cryptophycin
was synthesized1,by entered
Pettitclinical trials butin the
et al. [22,23]
produced only marginal activity [21].
1980s and then its origin was later confirmed when its direct isolation occurred from Symploca sp.
Dolastatin 10 is cyanobacterial metabolite that was synthesized by Pettit et al. [22,23] in the
Dolastatin 10 binds to tubulin on the rhizoxin-binding site and is an established antiproliferative agent
1980s and then its origin was later confirmed when its direct isolation occurred from Symploca sp..
that affects microtubule assembly, which leads to cell death during the G2 /M phase [24]. In 2005,
Dolastatin 10 binds to tubulin on the rhizoxin-binding site and is an established antiproliferative
the efficacy
agent thatand toxicity
affects of dolastatin
microtubule 10 were
assembly, whichinvestigated
leads to cellindeath
Phase II clinical
during the G2trials
/M phase in patients
[24]. In with
advanced prostate
2005, the cancer
efficacy but it was
and toxicity discontinued
of dolastatin 10 weredue to the development
investigated in Phase II of peripheral
clinical trials inneuropathy
patients in
40% ofwiththeadvanced
patients [25]. Many
prostate research
cancer but itgroups have soughtdue
was discontinued aftertoSAR
the studies of the of
development synthetic
peripheralanalogs
of this compound,
neuropathy which
in 40% are patients
of the now classified
[25]. Manyas auristatins.
research groups Thishave
group of compounds
sought showed
after SAR studies of the
the most
amount of promise as antibody drug conjugates. For example, brentuximab vedotin (adcetris,of
synthetic analogs of this compound, which are now classified as auristatins. This group Seattle
compounds
Genetics ) is nowshowed the most amount
a FDA-approved of promise
drug against as antibody
lymphoma. drug conjugates.
This compound For example,
is currently undergoing
phasebrentuximab
3 studies tovedotin
study its (adcetris,
effect onSeattle Genetics
cutaneous ) is now
T-cell a FDA-approved
lymphoma, drug againstand
B-cell lymphomas, lymphoma.
mature T-cell
This compound is currently undergoing phase 3 studies to study its effect on cutaneous T-cell
lymphomas [26,27].
lymphoma, B-cell lymphomas, and mature T-cell lymphomas [26,27].
The cyclic depsipeptide largazole, of the genus Symploca, is a marine natural product that contains
The cyclic depsipeptide largazole, of the genus Symploca, is a marine natural product that
a methylthiazoline linked to alinked
contains a methylthiazoline thiazole, as wellasaswell
to a thiazole, a 3-hydroxy-7-mercaptohept-4-enoic
as a 3-hydroxy-7-mercaptohept-4-enoic acid acid unit,
and aunit,
thioester, which had previously not been observed in marine cyanobacterial
and a thioester, which had previously not been observed in marine cyanobacterial natural natural products.
This products.
compound has
This been shown
compound to selectively
has been shown to target transformed
selectively over non-transformed
target transformed cells [28,29],
over non-transformed
through
cellsthe inhibition
[28,29], through of class I histone deacetylases
the inhibition (HDACs)
of class I histone [30,31].(HDACs)
deacetylases Largazole’s interesting
[30,31]. structure
Largazole’s
interesting activity
and biological structurehave andattracted
biologicalstrong
activity have from
interest attracted strong interest
the synthetic from community,
chemistry the syntheticwhich
seekschemistry
to establishcommunity, which to
synthetic routes seeks to establish
largazole and tosynthetic
investigate routes to largazole
its potential as a and
cancerto therapeutic
investigate its[32,33].
potential as a cancer therapeutic [32,33].
Thus, cyanobacterial metabolites have the potential for expanded utilization in drug discovery.
Thus, cyanobacterial metabolites have the potential for expanded utilization in drug discovery.
Despite their potent biological activities, very few cyanobacterial compounds have entered clinical
Despite their potent biological activities, very few cyanobacterial compounds have entered clinical
trials; one of the reasons is the complexity of synthesis of the natural products. The focus of this review
trials; one of the reasons is the complexity of synthesis of the natural products. The focus of this
is thereview
naturalis products
the natural called calothrixins,
products with an emphasis
called calothrixins, with an on emphasis
their synthesis andsynthesis
on their bioactivities.
and
bioactivities.
1.3. Calothrixins
1.3. CalothrixinsA and B (Figure 2) are two cyanobacterial metabolites that were first isolated
Calothrixins
from Calothrix in 1999
Calothrixins A and (Figure 2)etareal.two
by BRickards [34]. Briefly, lyophilized
cyanobacterial metabolites cells of Calothrix
that were strains
first isolated from were
Calothrix
extracted in 1999
with by Rickards
dimethyl et al. [34].
sulfoxide Briefly,
(DMSO) andlyophilized
then with cellsethyl
of Calothrix
acetatestrains were using
(AcOEt) extracted
Soxhlet
with dimethyl sulfoxide (DMSO) and then with ethyl acetate (AcOEt) using Soxhlet
extraction conditions. These extracts were fractionalized using a combination of bioassays, differential extraction
conditions.
solubility, These extracts were
and chromatography, which fractionalized
yielded the using a combination
relatively of bioassays,
insoluble calothrixin differential
A (1a) and its more
solubility, and chromatography, which yielded the relatively insoluble calothrixin A (1a) and its
soluble co-metabolite, calothrixin B (1b). Both structures were elucidated by Electron Impact Mass
more soluble co-metabolite, calothrixin B (1b). Both structures were elucidated by Electron Impact
Spectra (EIMS), 1 H, 13 C and 1 H– 1 H Correlation Spectroscopy (COSY) NMRs. Calothrixins possess an
Mass Spectra (EIMS), 1H, 13C and 1H–1H Correlation Spectroscopy (COSY) NMRs. Calothrixins
unique indolo[3,2-j]phenanthridine framework
possess an unique indolo[3,2-j]phenanthridine framework with an assembly of quinoline,
with an assembly quinone,
of quinoline, and indole
quinone,
pharmacophores. Calothrixin B Calothrixin
and indole pharmacophores. is generallyB known to beknown
is generally the neutral
to be analog whileanalog
the neutral calothrixin
while A is
known to be itsAN-oxide
calothrixin is knownanalog.
to be its N-oxide analog.

Figure 2. Structures of calothrixins.

Figure 2. Structures of calothrixins.
These scaffolds have attracted the interests of organic chemists and biologists alike due to their
unique structures have
These scaffolds and the promisethe
attracted they hold as lead
interests compounds
of organic for cancer
chemists drug discovery.
and biologists alike Herein,
due to their
we summarize the recent advances in synthetic work on calothrixins and their analogs.
unique structures and the promise they hold as lead compounds for cancer drug discovery. Herein, we
summarize the recent advances in synthetic work on calothrixins and their analogs.
Mar. Drugs 2016, 14, 17 4 of 21

2. Synthesis of Calothrixins
Due to the structural complexity and drug discovery potential, organic and medicinal chemists
around the world have embarked on developing efficient synthetic methodologies to produce these
natural products. Earlier synthesis of calothrixins was reviewed in 2009 by Satoshi Hibino et al. [35].
There has been a lot of progress made in the synthesis of calothrixins and their analogs since then.
Herein, the discussion will focus on the progress achieved within the past six years in developing new
synthetic routes to achieve the large-scale production of calothrixins.
The calothrixin scaffold consists of five rings (A–E), as shown in Figure 2. Multiple synthetic
strategies have been used to synthesize calothrixins starting from suitably substituted indole, quinoline,
or carbazole derivatives. The synthetic routes for calothrixins are categorized into three main groups
based on the strategy of the last ring closure step in the construction of the calothrixin scaffold (1) ring
B closure; (2) ring C closure; and (3) ring D closure (Table 1). No syntheses have been reported in which
either ring A or ring E are constructed as the last ring closure step. Recent reports on the synthesis of
calothrixins and the key reactions involved in these syntheses are summarized in Table 1.

Table 1. Summary of reported syntheses of calothrixin B since 2009.

Ring
Research Group Year Key Step Reference
Closure
Mn(OAc)3 mediated oxidative
Velu et al. 2014 B [36]
free radical reaction
Palladium catalyzed tandem
Ishikura et al. 2011 & 2012 C [37,38]
cyclization/cross-coupling
LTA mediated rearrangement of o-hydroxy
Dethe et al. 2014 C [39]
aryl hydrazone
Friedel-Crafts hydroxyalkylation and
Nagarajan et al. 2014 C [40]
directed o-lithiation
The anionic annulation of
Mal et al. 2014 C [41]
MOM-protected furoindolone
Two one pot reaction sequences:
Kusurkar et al. 2012 D [42]
a nucleophilic substitution and reduction
Pd catalyzed intramolecular cross-coupling
Nagarajan et al. 2013 D [43]
reaction via C–H activation
Electrocyclization of
Mohanakrishnan et al. 2014 D [44]
2-nitroarylvinyl-3-phenylsulfonylvinylindole
Pd mediated intramolecular C-X/C-H cross
Kumar et al. 2014 D [45]
coupling reactions
Allene mediated electrocyclic reaction of
Hibino et al. 2012 D [46]
the 6π-electron system
FeCl3 mediated domino
Mohanakrishnan et al. 2013 C&D [47]
reaction of enamines

2.1. Formation of Indole (Rings A and B) as the Last Step in the Construction of the
Indolo[3,2-j]Phenanthridine Framework

Velu’s Synthesis
In 2014, Velu et al. [36] reported a synthesis of calothrixins in which the indole ring is formed last
to construct the five-ring scaffold. This approach relies on the construction of the indole ring (rings A
and B) on a phenanthridine dione (ring C, D, and E) via a novel oxidative free radical reaction mediated
by manganese triacetate Mn(OAc)3 , as outlined in Scheme 1. The method of Mn(OAc)3 mediated
oxidative reaction of 2-cyclohexenone with quinones was originally developed by Chuang et al. [48–50].
Velu’s Synthesis
In 2014, Velu et al. [36] reported a synthesis of calothrixins in which the indole ring is formed
last to construct the five-ring scaffold. This approach relies on the construction of the indole ring
(rings A and
Mar. Drugs B) 17
2016, 14, on a phenanthridine dione (ring C, D, and E) via a novel oxidative free radical 5 of 21
reaction mediated by manganese triacetate Mn(OAc)3, as outlined in Scheme 1. The method of
Mn(OAc)3 mediated oxidative reaction of 2-cyclohexenone with quinones was originally developed
None
by of the et
Chuang existing reports
al. [48–50]. of calothrixin
None synthesis
of the existing utilizes
reports the late-stage
of calothrixin indoleutilizes
synthesis construction strategy.
the late-stage
indole construction strategy. Through this methodology, calothrixin B was synthesized in of
Through this methodology, calothrixin B was synthesized in seven steps, with an overall yield 19%
seven
starting
steps, from
with an2,4,5-trimethoxyindole.
overall yield of 19% starting from 2,4,5-trimethoxyindole.

Scheme
Scheme 1.
1. Velu’s
Velu’s synthesis
synthesis of calothrixins.

2.2.
2.2. Ring
Ring C
C Closure
Closure as
as the
the Last
Last Step
Step in
in the
the Construction
Construction of
of the
the Indolo[3,2-j]Phenanthridine
Indolo[3,2-j]Phenanthridine Framework
Framework
AA number
number of of calothrixin
calothrixinsyntheses
syntheseshave havebeen
beenreported
reported where
where thethe ring
ring C closure
C closure waswas used
used as the
as the last
last
step in the construction of calothrixin scaffold. This includes the two syntheses reported by Ishikura etby
step in the construction of calothrixin scaffold. This includes the two syntheses reported al.
Ishikura et al. in 2011 and 2012 [37,38], and the three contributions made by Dethe et
in 2011 and 2012 [37,38], and the three contributions made by Dethe et al. [39], Nagarajan et al. [40], al. [39],
Nagarajan
and Mal et etal.al. [40],
[41] in and
2014.Mal et al. [41] in 2014.

2.2.1.
2.2.1. Ishikura’s
Ishikura’s Synthesis
Synthesis
Ishikura
Ishikura etetal.
al.[37,38]
[37,38]was
was one
one of the
of the firstfirst groups
groups that designed
that designed a synthetic
a synthetic route toroute to calothrixins
calothrixins in which
in
ringwhich
C was ring
cyclizedC last
wasascyclized
shown inlastSchemeas shown in ongoing
2. In their Schemestudies
2. In oftheir ongoing studies of
trialkyl(indol-2-yl)borates,
trialkyl(indol-2-yl)borates, they previously
they previously found that indolylborates show found that indolylborates
high reactivity show high cross-coupling
in palladium-catalyzed reactivity in
palladium-catalyzed cross-coupling
reactions, such as carbonylative reactions, such
cross-coupling and as carbonylative
tandem cross-coupling andreactions.
cyclization/cross-coupling tandem
cyclization/cross-coupling reactions.
This new approach for the synthesis This newAapproach
of calothrixins and B wasfor the synthesis
demonstrated of calothrixins
through A and B
a palladium-catalyzed
was demonstrated
cross-coupling through
reaction of a1-methoxyindolylborate
palladium-catalyzed cross-coupling
(generated reaction of 1-methoxyindolylborate
in situ from 1-methoxyindole by
(generated in situn-BuLi
treatment with from 1-methoxyindole
and BEt3 ) with the by treatment
intermediate withcompound
n-BuLi and 13BEt ) withthe
to 3form thecompound
intermediate15.
compound
Additional 13 to form
novelty the compound
of this synthetic route15. Additional novelty
is the strategic useofofthis synthetic
Cu(OTf) 2 route
.toluene is the
complex strategic
for the
use of Cu(OTf)2.toluene
6π-elecrocyclization complex
of the for the
hexatriene 6π-elecrocyclization
intermediate of the
(16) to cyclize thehexatriene intermediate
ring C to form (16) to
the compound
cyclize the ring synthesis
17. Ishikura’s C to form yielded
the compound 17. Ishikura’s
calothrixin B in ninesynthesis
steps with yielded calothrixin
an overall yield Bofin9%nine steps
starting
with
from an overall yield of 9% starting from 2-iodoaniline.
2-iodoaniline.
Mar. Drugs 2016, 14, 17 6 of 21
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Scheme 2. Ishikura’s synthesis of calothrixins.

Scheme 2. Ishikura’s synthesis of calothrixins.
Scheme 2. Ishikura’s synthesis of calothrixins.
2.2.2. Dethe’s Synthesis
2.2.2. Dethe’s
2.2.2. Synthesis
Dethe’s
Dethe etSynthesis
al. [39] reported a concise five-step total synthesis of calothrixin B using LTA-mediated
Dethe et et
al.al.
rearrangement
Dethe [39]ofreported
[39] suitableaao-hydroxy
areported concise
concisefive-step total
aryl hydrazone
five-step synthesis
into the of
total synthesis ofcalothrixin
calothrixinBquinone
corresponding Busing
using LTA-mediated
as the key
LTA-mediated
step, as outlined
rearrangement of a in Schemeo-hydroxy
suitable 3. The synthesis
aryl began with the
hydrazone coupling
into the of N-PMB (p-methoxy
corresponding quinone benzyl)
as
rearrangement of a suitable o-hydroxy aryl hydrazone into the corresponding quinone as thethe key
key
step, protected
asasoutlinedindolyl hyrazide (21) and the quinolinone derivative (22) to form the intermediate
N-PMB (p-methoxy
step, outlined in Scheme3.3.TheThe
in Scheme synthesis
synthesis began began with
with the the coupling
coupling of N-PMB of (p-methoxy benzyl)
hydrazone 23. The hydrazone (23) was treated with Pb(OAc)4 to undergo the LTA-mediated
benzyl) protected
protected indolyl indolyl hyrazide
hyrazide (21) and
(21) and the the quinolinone
quinolinone derivative
derivative (22)(22)
to to form
form thethe intermediate
intermediate
oxidative rearrangement followed by a BF3.Et2O-mediated cyclization to form the PMB-protected
hydrazone
hydrazone TheThe
23. 23. hydrazone
hydrazone(23) (23)
was treated with Pb(OAc)
was treated with Pb(OAc)to undergo the LTA-mediated
4 to undergo oxidative
the LTA-mediated
calothrixin B (25). This synthesis is short and high yielding.4 The overall yield of the calothrixin B
rearrangement
oxidative followed
rearrangement by a BF
followed
3 .OEtby
2 -mediated
a BF cyclization
3.Et2O-mediated
synthesis from ethyl indole-2-carboxylate (20) is 39% for five steps.
to form
cyclizationthe PMB-protected
to form the calothrixin
PMB-protected B
calothrixin
(25). B (25). isThis
This synthesis synthesis
short and high is short
yielding.and high yielding.
The overall Theof
yield overall yield of the
the calothrixin calothrixinfrom
B synthesis B
synthesis
ethyl from ethyl indole-2-carboxylate
indole-2-carboxylate (20) is 39% for five (20)steps.
is 39% for five steps.

Scheme 3. Dethe’s synthesis of calothrixin B.

2.2.3. Nagarajan’s Synthesis

Scheme 3. Dethe’s synthesis of calothrixin B.
Schemea 3.
Nagarajan et al. [40] outlined Dethe’sfor
strategy synthesis of calothrixin
the synthesis B.
of calothrixin B featuring a directed
o-metalation
2.2.3. reaction
Nagarajan’s as a key step, as outlined in Scheme 4. The synthesis began with the coupling
Synthesis
2.2.3.ofNagarajan’s
the Synthesis
commercially available reagents, ethyl indole-2-carboxylate (20) and
Nagarajan et al. [40] outlined a strategy for the synthesis of calothrixin B featuring a directed
quinoline-3-carboxaldehyde (26) in the presence of TMG in MeOH followed by the oxidation of the
o-metalation et al. [40] outlined
Nagarajanreaction a as
strategy forinthe synthesis ofsynthesis
calothrixin B featuring a directed
intermediate productaswitha key step,
Dess-Martin outlined
periodinaneScheme
(DMP)4.inThe
CH2Cl2/AcOH.began with theprocess
This two-step coupling
o-metalation
of the reaction as a key step,
commercially as outlined
available in Schemeethyl
reagents, 4. The synthesis began with the
indole-2-carboxylate coupling
(20) and of
thequinoline-3-carboxaldehyde
commercially available reagents, ethyl
(26) in the indole-2-carboxylate
presence of TMG in MeOH (20)followed
and quinoline-3-carboxaldehyde
by the oxidation of the
(26)intermediate
in the presence of TMG
product in MeOH followed
with Dess-Martin by the
periodinane oxidation
(DMP) in CH2Clof2/AcOH.
the intermediate product
This two-step with
process
Dess-Martin periodinane (DMP) in CH2 Cl2 /AcOH. This two-step process yielded compound 27,
Mar. Drugs
Mar. Drugs 2016,
2016, 14,
14, 17
x 77of
of21
20

yielded compound
Mar. Drugs 2016, 14, x 27, which was then cyclized by an intramolecular directed o-lithiation reaction 7 of 20
using was
which lithium tetramethylpiperidide
then cyclized (LiTMP)
by an intramolecular too-lithiation
directed form calothrixin B in lithium
reaction using 48% yield. This synthetic
tetramethylpiperidide
yieldedtocompound
methodology
(LiTMP) offers
form a27, which
three-step
calothrixin was
B in then
route
48% tocyclized
yield. by an
calothrixin B intramolecular
This syntheticfrom directed
commercially
methodology o-lithiation
available
offers reaction
reagents
a three-step in an
route to
using lithium
overall yield
calothrixin B of
from tetramethylpiperidide
38%. (LiTMP) to form calothrixin B
commercially available reagents in an overall yield of 38%. in 48% yield. This synthetic
methodology offers a three-step route to calothrixin B from commercially available reagents in an
overall yield of 38%.

Scheme 4. Nagarajan’s synthesis of calothrixin B.

Scheme 4. Nagarajan’s synthesis of calothrixin B.
Scheme 4. Nagarajan’s synthesis of calothrixin B.
2.2.4. Mal’s Synthesis
2.2.4. Mal’s Synthesis
2.2.4.
MalMal’s
et al.Synthesis
[41] reported a two-step synthesis of calothrixin B from the N-protected indololactone
Mal et al. [41] reported a two-step synthesis of calothrixin B from the N-protected indololactone (28)
(28) as Mal
outlined in Scheme
et al. [41] reported5. This reagent
a two-step was of
synthesis prepared in three
calothrixin B fromsimple steps fromindololactone
the N-protected commercially
as outlined in Scheme 5. This reagent was prepared in three simple steps from commercially available
available
(28) as α-acetobutyrolactone
outlined in Scheme 5. [51]. Treatment
This reagent wasofprepared
compound 28 with
in three simple4-bromoquinoline (29) in the
steps from commercially
α-acetobutyrolactone [51]. Treatment of compound 28 with 4-bromoquinoline (29) in the presence of
presence
available of α-acetobutyrolactone
lithium diisopropylamide (LDA) in
[51]. Treatment tetrahydrofuran
of compound 28 with (THF) at −78
4-bromoquinoline °C afforded
(29) in thean
lithium diisopropylamide (LDA) in tetrahydrofuran (THF) at ´78 ˝ C afforded an inseparable mixture
inseparable mixture of compounds 30a and 30b. However, after the removal
presence of lithium diisopropylamide (LDA) in tetrahydrofuran (THF) at −78 °C afforded an of methoxymethylene
of compounds 30a and 30b. However, after the removal of methoxymethylene (MOM) group using
(MOM) groupmixture
inseparable using HCl, it gave a mixture
of compounds 30a andof30b.
calothrixin
However, B after
(1b) and its regioisomer
the removal (31b) that could
of methoxymethylene
HCl, it gave a mixture of calothrixin B (1b) and its regioisomer (31b)itsthat could be(31b)
easily separated
be (MOM) group
easily separated using
in HCl, it gave
the ratio of a40%
mixture
and of
6%,calothrixin B (1b)This
respectively. and regioisomer
synthetic methodology that could
afforded
in the ratio of 40%
be easily Bseparated and 6%, respectively.
in the ratio This synthetic methodology afforded calothrixin B with an
calothrixin with an overall yieldofof40%
47%andfor 6%,
tworespectively.
steps startingThis
fromsynthetic
compoundmethodology
28. afforded
overall yield of
calothrixin 47% an
B with foroverall
two steps starting
yield of 47% from compound
for two 28. from compound 28.
steps starting

Scheme
Scheme5. Mal’s
Mal’ssynthesis
synthesis of
of calothrixin
calothrixin BB andits
its regioisomer.
Scheme 5.5.Mal’s synthesis of calothrixin Band
and itsregioisomer.
regioisomer.

2.3. Ring DDClosure

Closureasasthe
theLast
LastStep
Stepininthe
theConstruction of the
the Indolo[3,2-j]Phenanthridine
Indolo[3,2-j]Phenanthridine Framework
2.3.2.3. Ring
Ring D Closure as the Last Step in the Construction of
Construction of the Indolo[3,2-j]PhenanthridineFramework
Framework
Another
Another strategy
strategyforforthe
thecalothrixin
calothrixin synthesis involves cyclizing
synthesis involves cyclizingring
ringDDasasthe thelast
laststep
stepin in
thethe
Another strategy for the calothrixin synthesis involves cyclizing ring D as the last step in the
construction
construction ofofcalothrixin
calothrixinscaffold.
scaffold.AA number
number of contributions
contributions have havebeen
beenmade madetotothis
this category
category of of
construction of calothrixin scaffold. A number of contributions have been made to this category of
syntheses.
syntheses. These
These includefour
include fourindependent
independent publications
publications by by Kusurkar
Kusurkaretetal.al.inin2012
2012[42], Nagarajan
[42], Nagarajan et et
syntheses. These include four independent publications by Kusurkar et al. in 2012 [42], Nagarajan et al.
al. in 2013 [43], Mohanakrishnan et al. in 2014 [44], Kumar et al. in 2014 [45], and Hibino
al. in 2013 [43], Mohanakrishnan et al. in 2014 [44], Kumar et al. in 2014 [45], and Hibino et al. in 2012 et al. in 2012
in 2013
[46].
[43], Mohanakrishnan et al. in 2014 [44], Kumar et al. in 2014 [45], and Hibino et al. in 2012 [46].
[46].
2.3.1. Kusurkar’s Synthesis
2.3.1.
2.3.1. Kusurkar’s
Kusurkar’s Synthesis
Synthesis
Kusurkar
Kusurkar
et al.
et
[42]
al.
have
[42]have
published
havepublished
a synthesis
publishedaasynthesis
synthesis of
of calothrixinsstarting
of calothrixins
startingfromfrom4-hydroxy
4-hydroxy carbazole
carbazole
Kusurkar
as outlined et
in Schemeal. [42]
6. The6.novelty of theirofsynthetic calothrixins
route is the starting
unprecedentedfrom 4-hydroxy carbazole
use of DMF-NaOMe
as outlined
as aoutlined in
in SchemeScheme The
6. The the novelty
novelty their
of their synthetic
synthetic route
route is the unprecedented
isa high-yielding
the unprecedented useuseof of
as reagent for
DMF-NaOMe thea reduction
as reagent forofthe aldehyde
reduction of group and thegroup
the aldehyde use ofand Pd-catalyzed
the use of a high-yielding
DMF-NaOMe
coupling as atoreagent
reaction constructfor the
the ring
reduction
D of theof the aldehydesystem.
group and the use ofthe a high-yielding
Pd-catalyzed coupling reaction to construct the pentacyclic
ring D of the pentacyclic For system.
example, treatment
For example, the of
Pd-catalyzed
compound coupling reaction to construct the ring D of the pentacyclic system. ˝ For example, the
treatment35 of with
compoundNaOMe 35in dryNaOMe
with dimethyl in formamide
dry dimethyl (DMF) and CuI
formamide (DMF)at 120andCCuI resulted
at 120in°Cthe
treatment
substitution of compound 35 with NaOMe in dry dimethyl formamide (DMF) and CuI at 120 °C
resulted inofthe bothsubstitution
bromine and of benzyloxy
both bromine groupsandwith methoxy
benzyloxy groups
groups withmethoxy
with concomitant
groupsreduction
with
resulted
of the in the substitution
aldehydereduction
resultingofinthe of both
thealdehyde
formationbromine and benzyloxy
of the compound groups with
36. Pd-catalyzed methoxy
cyclization groups with
of ring D of
concomitant resulting in the formation of the compound 36. Pd-catalyzed
concomitant
thecyclization reduction
scaffold toofform
ring D of
compoundthe aldehyde resulting
39 istoanother
of the scaffold in the formation
novel key39step
form compound of the
in thisnovel
is another compound
synthesis.
key stepThe 36. Pd-catalyzed
inoverall yield for
this synthesis.
cyclization
The overall
Kusurkar’s of ring D
yield
synthesis forof the scaffold
ofKusurkar’s
calothrixin Btowas
form
synthesis ofcompound
25% calothrixin 39 is another
B was
for nine steps. 25% fornovel
nine key step in this synthesis.
steps.
The overall yield for Kusurkar’s synthesis of calothrixin B was 25% for nine steps.
Mar. Drugs 2016, 14, 17 8 of 21
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Mar. Drugs 2016, 14, x 8 of 20

Scheme 6. Kusurkar’s total synthesis of calothrixin B.

Scheme 6. Kusurkar’s total synthesis of calothrixin B.
Scheme 6. Kusurkar’s total synthesis of calothrixin B.
2.3.2. Nagarajan’s Synthesis
2.3.2. Nagarajan’s Synthesis
2.3.2.In addition toSynthesis
Nagarajan’s developing a synthetic route for calothrixins that involved the closure of ring C in
theInlast
addition to developing
step, Nagarajan a synthetic
et al. [43] route for calothrixins
has also accomplished that
a synthesis in involved
which ring the
D closure of ring
was closed last C
In addition to developing a synthetic route for calothrixins that involved the closure of ring C in
in the lastpreparation
in the step, Nagarajan et al. [43] has also
of the five-membered ringaccomplished
system, followeda synthesis in which ring
by the installation D necessary
of the was closed
the last step, Nagarajan et al. [43] has also accomplished a synthesis in which ring D was closed last
lastsubstitutions
in the preparation of thecalothrixin
to produce five-membered ring system,
B as shown followed
in Scheme 7. Thisbysynthesis
the installation
featuredofathe necessary
prominent
in the preparation of the five-membered ring system, followed by the installation of the necessary
Pd-catalyzed
substitutions to intramolecular
produce calothrixin cross-coupling
B as shown reaction via C-H
in Scheme activation
7. This synthesisusing an appropriately
featured a prominent
substitutions to produce calothrixin B as shown in Scheme 7. This synthesis featured a prominent
substituted intramolecular
Pd-catalyzed carbazole derivative to construct
cross-coupling the indolophenanthridine
reaction via C-H activation using core ring system of
an appropriately
Pd-catalyzed intramolecular cross-coupling reaction via C-H activation using an appropriately
calothrixins.
substituted This
carbazole is the first
derivative reported synthesis
to construct of calothrixin pentacycles
the indolophenanthridine without
core ring any
system protection on
substituted carbazole derivative to construct the indolophenanthridine core ringofsystem
calothrixins.
of
the
This indole N atom.
is the first This
reported The overall yield for calothrixin B in this Nagarajan’s synthesis is 35% for five
calothrixins. is thesynthesis of calothrixin
first reported synthesis of pentacycles
calothrixinwithout anywithout
pentacycles protection
anyon the indole
protection on N
stepsThe
atom. starting
overallfrom 4-methoxycarbazole.
yield foroverall
calothrixin B incalothrixin
this Nagarajan’s
the indole N atom. The yield for B in thissynthesis
Nagarajan’s is 35% for five
synthesis stepsfor
is 35% starting
five
from 4-methoxycarbazole.
steps starting from 4-methoxycarbazole.

Scheme 7. Nagarajan’s synthesis of calothrixins A and B.

Scheme 7. Nagarajan’s synthesis of calothrixins A and B.

2.3.3. Mohanakrishnan’s Synthesis
Scheme 7. Nagarajan’s synthesis of calothrixins A and B.
2.3.3.Mohanakrishnan
Mohanakrishnan’s et al. have reported a linear synthesis of calothrixin B using the thermal electro
Synthesis
2.3.3. Mohanakrishnan’s
cyclization Synthesis
of 2-nitroarylvinyl-3-phenylsulfonylvinylindole as the key step as outlined in Scheme 8
Mohanakrishnan et al. have reported a linear synthesis of calothrixin B using the thermal electro
[44]. In this key
Mohanakrishnan step, the 2,3-divinylidole intermediate generated
et al. have reported a linear synthesis of from compound 52 thermal
by treatment
cyclization of 2-nitroarylvinyl-3-phenylsulfonylvinylindole as calothrixin
the key stepBas
using the
outlined electro
in Scheme 8
with Me2SO4 was subjected to thermal cyclization in refluxing xylenes to afford the carbazole
cyclization of 2-nitroarylvinyl-3-phenylsulfonylvinylindole
[44]. In this as the key
key step, the 2,3-divinylidole intermediate generated stepcompound
from as outlined52inbyScheme 8 [44].
treatment
derivative 53. Mohanakrishnan’s total synthesis thus afforded calothrixin B from 2-methylindole in
In with
this key
Me2step, the 2,3-divinylidole
SO4 was subjected to thermalintermediate generated
cyclization fromxylenes
in refluxing compound 52 by the
to afford treatment with
carbazole
14 steps with an overall yield of about 8%.
Mederivative
2 SO4 was 53. Mohanakrishnan’s
subjected total synthesis
to thermal cyclization thus afforded
in refluxing xylenescalothrixin
to afford B from
the 2-methylindole
carbazole derivativein53.
14 steps with an overall
Mohanakrishnan’s yield of about
total synthesis 8%.
thus afforded calothrixin B from 2-methylindole in 14 steps with an
overall yield of about 8%.
Mar. Drugs 2016, 14, 17 9 of 21
Mar. Drugs 2016, 14, x 9 of 20

Mar. Drugs 2016, 14, x 9 of 20

Scheme 8. Mohanakrishnan’s synthesis of calothrixins A and B.

Scheme 8. Mohanakrishnan’s synthesis of calothrixins A and B.

2.3.4. Kumar’s Synthesis Scheme 8. Mohanakrishnan’s synthesis of calothrixins A and B.

2.3.4. Kumar’s Synthesis
Kumar
2.3.4. et al.Synthesis
Kumar’s [45] reported the total synthesis of calothrixin B via multiple palladium-mediated
Kumar
C-X/C-H et al. intramolecular
cross [45] reported the total synthesis
coupling reactions asof outlined
calothrixin B via multiple
in Scheme palladium-mediated
9. The salient feature of this
C-X/C-H cross
Kumar intramolecular
et al. [45] reportedcoupling
the total reactions
synthesis as
of outlined
calothrixin in
B Scheme
via multiple
synthesis is the two intramolecular C-X/C-H cross coupling reactions of the intermediates 9. The salient
palladium-mediated60feature
to form of
C-X/C-H cross
thiscompound
synthesis is intramolecular
the two coupling
intramolecular reactions
C-X/C-H as outlined
cross in Scheme
coupling 9.
reactions
61 and 63 to form the compound 64. The intermediate 60 was cyclized under The salient
of the feature of
intermediatesthis 60
synthesis
to form is
compound the two intramolecular
and 63 toreactionC-X/C-H
form the cross
compound coupling reactions of the
The intermediate intermediates
60 was cyclized 60 to form
intramolecular cross61 coupling condition using64.Pd(OAc) 2, PCy3, and JohnPhos to affordunder
the
compound cross
intramolecular 61 and 63 to form
coupling the condition
reaction compoundusing 64. The intermediate
Pd(OAc) , PCy , 60 was
and cyclized
JohnPhos to under the
afford
carbazole derivative 61. The cyclization of 63 was carried out using 2 Pd(OAc)
3 2, PCy3, and K2CO3 to
intramolecular cross coupling reaction condition using Pd(OAc)2, PCy3, and JohnPhos to afford the
carbazole derivative
afford the compound The
61.64. cyclization
Kumar’s of 63 was
methodology carried
thus out using
accomplished Pd(OAc)
the synthesis 2 , of
PCy 3 , and K2B
calothrixin CO
in 37 to
carbazole derivative 61. The cyclization of 63 was carried out using Pd(OAc)2, PCy3, and K2CO3 to
afford
stepsthe compound
starting 64. Kumar’s methodology in
from 2,5-dimethoxybenzaldehyde thus
an accomplished
overall yield ofthe
50%. synthesis of calothrixin B in
afford the compound 64. Kumar’s methodology thus accomplished the synthesis of calothrixin B in 7
7 steps starting from 2,5-dimethoxybenzaldehyde in an overall yield of 50%.
steps starting from 2,5-dimethoxybenzaldehyde in an overall yield of 50%.

Scheme 9. Kumar’s synthesis of calothrixins B.

Scheme 9. Kumar’s synthesis of calothrixins B.
Scheme 9. Kumar’s synthesis of calothrixins B.
Mar. Drugs 2016, 14, 17 10 of 21
Mar. Drugs 2016, 14, x 10 of 20
Mar. Drugs 2016, 14, x 10 of 20
Compound 60 in Kumar’s synthesis has also been converted to the key intermediate 64 in three
Compound 60 in Kumar’s synthesis has also been converted to the key intermediate 64 in
Compound
steps as shown 60 in Kumar’s
in Scheme synthesis
10. In this has also
synthesis, been
rings converted
B and to the
D of the key intermediate
calothrixin scaffold64 in three
were cyclized
three steps as shown in Scheme 10. In this synthesis, rings B and D of the calothrixin scaffold were
in onesteps
stepasbyshown in Scheme 10.
intramolecular In this synthesis,
C-X/C-H rings B and
cross coupling D of the
reaction calothrixin
using Pd(OAc)scaffold
2, PCywere cyclized
3, and JohnPhos
cyclizedonein step
one bystep by intramolecular C-X/C-H cross coupling reaction using Pd(OAc)2 , PCy3 , and
in theinpresence of Kintramolecular C-X/C-H cross coupling reaction using Pd(OAc)2, PCy3, and JohnPhos
2CO3 in DMF to afford the key intermediate 64.
JohnPhos in the presence of K CO in DMF
in the presence of K2CO3 in 2DMF3 to afford theto afford the key intermediate
key intermediate 64. 64.

Scheme 10. Alternate synthesis of compound 64.

Scheme 10.
Scheme Alternate synthesis
10. Alternate synthesis of
of compound
compound 64.
64.
2.3.5. Hibino’s Synthesis
2.3.5. Hibino’s
Hibino’s Synthesis
Synthesis
Hibino et al. [46] synthesized calothrixin B and other N-alkyl-calothrixins using a biomimetic
Hibino
approach
Hibino et featuring
et al. [46] synthesized
al. synthesized calothrixin electrocyclic
a key allene-mediated
calothrixin N-alkyl-calothrixins
B and otherreaction usingsystem,
of the 6π-electron
N-alkyl-calothrixins using a biomimetic
as
outlined in
approach featuringScheme
featuringa akey11.
keyThe synthesis
allene-mediatedof
allene-mediatedthe key intermediate,
electrocyclic indolo[2,3-a]carbazole
reaction
electrocyclic of theof
reaction 6π-electron (72), was carried
system, system,
the 6π-electron as outlined
as
out by
in Scheme
outlined an
11.allene-mediated
The 11.
in Scheme synthesis electrocyclic
of the of
The synthesis key reaction
the of MOM-protected
intermediate, compound 71 (72),
indolo[2,3-a]carbazole
key intermediate, in the(72),
indolo[2,3-a]carbazole presence
was of out
carried
was carried
by antBuOK in tBuOH-THF. This electrocyclization involved the two [b]-bonds of indole units and the
out by allene-mediated
an allene-mediated electrocyclic reaction
electrocyclic reaction ofof
MOM-protected
MOM-protectedcompound
compound71 71ininthe
the presence
presence of
allene double bond generated in situ. The overall yield for Hibino’s synthesis of calothrixin B from
tBuOK in tBuOH-THF. This electrocyclization
tBuOK electrocyclization involved the two [b]-bonds of indole units and the
2-bromoindole-3-carboxaldehyde is 22% for nine steps.
allene double
allene double bond
bond generated situ. The
generated in situ. The overall
overall yield
yield for
for Hibino’s
Hibino’s synthesis
synthesis of
of calothrixin
calothrixin B B from
2-bromoindole-3-carboxaldehyde is 22% for nine steps.

Scheme 11. Hibino’s synthesis of calothrixin B.

2.4. Simultaneous Closure of Rings C and D

Only one methodology of calothrixin
Scheme synthesis
11. Hibino’s hasofbeen
synthesis developed
calothrixin B. in which multiple rings
Scheme 11. Hibino’s synthesis of calothrixin B.
were cyclized in a one-pot synthesis. This report was published by Mohanakrisnan et al. [47] in 2013,
prior to their Closure
2.4. Simultaneous report inof2014 [44]Cinand
Rings which
D ring D was the last ring closure step.
2.4. Simultaneous Closure of Rings C and D
Only one methodology
Mohanakrishnan’s Synthesisof calothrixin synthesis has been developed in which multiple rings
Only one methodology of calothrixin synthesis has been developed in which multiple rings were
were cyclized in a one-pot
Mohanakrishnan synthesis.
et al. [47] This report was
reported published byofMohanakrisnan et al. [47] in 2013,
cyclized in a one-pot synthesis. This report another novel
was published synthesis calothrixinetBal.
by Mohanakrisnan involving a key
[47] in 2013, prior
prior FeCl
to their reportdomino
3 mediated in 2014reaction
[44] in which ring D derivative,
was the last ring closure step. 12. In this key step,
to their report in 2014 [44] in whichofring
an enamine
D was the last ringasclosure
outlined in Scheme
step.
FeCl3 was used to cyclize the key intermediate 79 to afford calothrixin B. Both rings C and D were
Mohanakrishnan’s Synthesis
Mohanakrishnan et al. [47] reported another novel synthesis of calothrixin B involving a key
FeCl3 mediated domino reaction of an enamine derivative, as outlined in Scheme 12. In this key step,
FeCl3 was used to cyclize the key intermediate 79 to afford calothrixin B. Both rings C and D were
Mar. Drugs 2016, 14, 17 11 of 21

Mohanakrishnan’s Synthesis
Mohanakrishnan et al. [47] reported another novel synthesis of calothrixin B involving a key FeCl3
mediated domino reaction of an enamine derivative, as outlined in Scheme 12. In this key step, 11 of 20 
Mar. Drugs 2016, 14, x 
FeCl3
was used to cyclize the key intermediate 79 to afford calothrixin B. Both rings C and D were created
in a sigle step in this synthesis. The overall yield of calothrixin B from the compound 75 is 45% for
created in a sigle step in this synthesis. The overall yield of calothrixin B from the compound 75 is 
six steps.
45% for six steps. 

 
Scheme 12. Mohanakrishnan’s synthesis of calothrixins B. 
Scheme 12. Mohanakrishnan’s synthesis of calothrixins B.

3. Bioactivities of Calothrixins 
3. Bioactivities of Calothrixins
The  most  therapeutically  relevant  biological  activity  reported  for  marine  cyanobacterial 
The most therapeutically relevant biological activity reported for marine cyanobacterial metabolites
metabolites is their cytotoxicity. While some antimicrobial studies were also conducted, calothrixins 
is their cytotoxicity. While some antimicrobial studies were also conducted, calothrixins were majorly
were majorly pursued to serve as novel anticancer molecules. Herein, we summarize all bioactivities 
pursued to serve as novel anticancer molecules. Herein, we summarize all bioactivities conducted on
conducted on calothrixins and their analogs. Following their isolation, calothrixins quickly gained 
calothrixins and their analogs. Following their isolation, calothrixins quickly gained recognition due
recognition due to their anticancer activity as preliminary studies revealed a high potency against 
to their anticancer activity as preliminary studies revealed a high potency against the human cervical
the human cervical cancer cell line, HeLa cells. Since then, calothrixins have been evaluated against 
cancer cell line, HeLa cells. Since then, calothrixins have been evaluated against several different
several  different  cell  lines  and  have  been  studied  for  their  mode  of  action.  Calothrixins  were  also 
cell lines and have been studied for their mode of action. Calothrixins were also evaluated for their
evaluated for their antiparasitic activity. A summary of their bioactivities is found below. 
antiparasitic activity. A summary of their bioactivities is found below.
3.1. Antiparasitic Activity 
3.1. Antiparasitic Activity
Along with
Along with their
their discovery
discovery in in 1999,
1999, Smith
Smith et al.  [34] also
et al. [34] also reported
reported that
that the
the cell
cell extracts
extracts from
from 
cyanobacteria Calothrix were found to inhibit the growth of malarial strains. The two calothrixins, 
cyanobacteria Calothrix were found to inhibit the growth of malarial strains. The two calothrixins,
calothrixin AA 
calothrixin  andand  B,  were 
B, were evaluated 
evaluated against 
against the  chloroquine 
the chloroquine (QC)‐resistant 
(QC)-resistant malarial 
malarial strain, strain,  P. 
P. falciparum
falciparum FCR‐3 strain (QC‐sensitive), and were found to be effective in a dose‐dependent manner. 
FCR-3 strain (QC-sensitive), and were found to be effective in a dose-dependent manner. The IC50
The  ICwere
values 50  values  were  observed  to  be  58  nM  and  180  nM,  respectively,  compared  with  83  nM  IC50 
observed to be 58 nM and 180 nM, respectively, compared with 83 nM IC50 value for
value for chloroquine in the same assay [34]. 
chloroquine in the same assay [34].
In an attempt to find a better lead against malaria, Hibino et al. [46] synthesized and evaluated 
In an attempt to find a better lead against malaria, Hibino et al. [46] synthesized and evaluated
several N‐alkyl calothrixin analogs for their activity against the chloroquine‐resistant strain (Table 
several N-alkyl calothrixin analogs for their activity against the chloroquine-resistant strain (Table 2).
2).  The 
The resultsresults  for  antimalarial 
for antimalarial activityactivity  were  compared 
were compared against  chloroquine. 
against chloroquine. CalothrixinCalothrixin  B  was 
B was observed
observed to be the most active (IC
to be the most active (IC50 = 120 nM),50 = 120 nM), concurring with the findings of Rickards et al. [34]. 
concurring with the findings of Rickards et al. [34]. Calothrixin A
Calothrixin A was found to be almost equally active (IC
was found to be almost equally active (IC50 = 185 nM ) as 50 = 185 nM ) as calothrixin B. Although all 
calothrixin B. Although all compounds
compounds showed antimalarial activity, substitution of the indole nitrogen atom with various alkyl 
showed antimalarial activity, substitution of the indole nitrogen atom with various alkyl groups led to
agroups 
decrease led 
into  a  decrease 
activity. Among in  activity.  Among 
the analogs, the  analogs, group
2-hydroxyethyl 2‐hydroxyethyl  group better
showed slightly showed  slightly 
activity in
better activity in comparison to the other alkyl substitutions. 
comparison to the other alkyl substitutions.
In addition to its antimalarial activities, Smith et al. [52,53] reported the antibacterial activity of
Table 2. Antimalarial activity calothrixin analogs against FCR‐3 strain. 
calothrixin A against Bacillus subtilis 168, where it was found to be inhibiting the bacterial growth
in a dose-dependent mannerIC without any cell lysis. A complete growth inhibition
50 against FCR‐3  was achieved at
IC50 against FCR‐3 
Compound  Compound 
16 µM concentration. Strain (nM)  Strain (nM) 

185  120 
 several N-alkyl
several calothrixin
N-alkyl analogs
calothrixin for their
analogs activity
for their against
activity the chloroquine-resistant
against the chloroquine-resistant strain (Table
strain 2). 2).
(Table
The The
results for antimalarial
results for antimalarialactivity werewere
activity compared
comparedagainst chloroquine.
against Calothrixin
chloroquine. Calothrixin B was observed
B was observed
to betothe
be most active
the most (IC50(IC
active = 120 nM),nM),
50 = 120 concurring
concurringwithwith
the findings of Rickards
the findings et al.et[34].
of Rickards Calothrixin
al. [34]. Calothrixin
A was found
A was to betoalmost
found equally
be almost active
equally (IC50(IC
active = 185 nM nM
50 = 185 ) as )calothrixin B. Although
as calothrixin B. Although all compounds
all compounds
showed
showedantimalarial activity,
antimalarial substitution
activity, of the
substitution of indole nitrogen
the indole atom
nitrogen withwith
atom various alkylalkyl
various groups led led
groups
Mar. Drugs 2016, 14, 17 12 of 21
to a to
decrease in activity.
a decrease Among
in activity. Among the analogs, 2-hydroxyethyl
the analogs, 2-hydroxyethyl group showed
group slightly
showed better
slightly activity
better in in
activity
comparison
comparison to the
to other alkylalkyl
the other substitutions.
substitutions.
Table 2. Antimalarial activity calothrixin analogs against FCR-3 strain.
Table 2. Antimalarial
Table activity
2. Antimalarial calothrixin
activity analogs
calothrixin against
analogs FCR-3
against strain.
FCR-3 strain.

Compound IC50IC
IC 50 against
against
50 against FCR-3
FCR-3FCR-3 Compound
IC50
ICagainst
50IC FCR-3
against FCR-3
50 against FCR-3
Compound
Compound Strain (nM) Compound
Compound Strain (nM)
Strain (nM)
Strain (nM) Strain (nM)
Strain (nM)

185185
185 120120 120
Mar. Drugs
Mar. Drugs
2016,2016,
14, x 14, x 12 of 12
20 of 20
Mar.
Mar. Mar.
Drugs
Mar. Drugs
Drugs 2016,
2016,2016,
Drugs 14,
14, xx
2016, x 14,
14, xx 12
12 of
of 12
20
20 of
12 of 20
20
Mar. Drugs
Mar.
Mar. 2016,
Drugs
Drugs 14,
2016,
2016, 14,
14, x
x 12 of 20
12
12 of
of 20
20

380380
380 490490 490
380
380 380
380 380 490
490 490
490 490
380 490

380 380 220 220

380 380
380380
380 380 220220
220 220
220 220
380 220

320 320 640 640

320
320 320
320
320320 640640
640 640
640 640
320 640

250 250 330 330

250
250 250 330
330 330
250
250250
250 330330 330
330

180 180
180
180 180
180
180180
180
Chloroquine
Chloroquine
Chloroquine
Chloroquine
Chloroquine
Chloroquine
Chloroquine
Chloroquine
Chloroquine
In addition
In addition to itstoantimalarial
its antimalarial activities,
activities,
Smith Smith et al.et[52,53]
al. [52,53] reportedreported the antibacterial
the antibacterial activity
activity
of of
In
In addition
In In
addition
In A
addition addition
addition to its
itsto
toagainstto antimalarial
its antimalarial
antimalarial
itsBacillus
antimalarial activities,
activities,
activities, Smith
Smith
activities, Smith
Smith et
etit al.
al. et
et[52,53]
al.
[52,53]
al. [52,53]
[52,53]reported
reportedreported
reported the
the antibacterial
the
the antibacterial
antibacterial
antibacterial activity
activity
activity of
of
activity of
of
calothrixin In
calothrixin addition Ato
against itsto antimalarial
its
Bacillus antimalarial
subtilis activities,
subtilis
168, 168,
where Smith
activities,whereitSmith
was et al.
was et
found[52,53]
al.
foundto reported
[52,53] reported
betoinhibiting the
be inhibiting antibacterial
the antibacterial
bacterial
the bacterial activity
growth of
activity
growthin of in
calothrixin
calothrixin
3.2. Anticancer
calothrixin
calothrixin
calothrixin A
A against
Activity
A A
against
A
againstagainst
against Bacillus
Bacillus
Bacillus Bacillus
Bacillus subtilis
subtilis
subtilis 168,
168,
subtilis
subtilis 168, 168,
where
where
168,
where where
whereit
it
it was
was
was it
it was
found
found
was
found found
foundto
to
to be
be
be to
to inhibiting
be inhibiting
inhibiting
be inhibiting
inhibiting the
the
the bacterial
the bacterial
bacterial
the
bacterialbacterialgrowth
growth
growth growth
growthin
in
in in
in
calothrixin
a dose-dependent
a dose-dependent A against
manner manner Bacillus
without subtilis
withoutany any 168, where
cell lysis.
cell lysis. it
A completewas
A complete found growthto be
growth inhibiting
inhibition
inhibition the
was was bacterial
achieved
achieved growth
at 16at 16 in
aaa dose-dependent
aa dose-dependent
dose-dependent
dose-dependent
dose-dependent manner
manner
manner manner
manner without
without
without without
withoutany
any
any any
cell
cell
any
cell cell
lysis.
lysis.
cell
lysis. lysis.
A
A
lysis.
A complete
A
complete
A
complete complete
complete growth
growth
growth growth
growth inhibition
inhibition
inhibition
inhibition
inhibition was
was
was was
achievedachieved
achieved
was achieved
achieved at
at
at 16
16
16at
at 16
16
a
µAlong
Mµ dose-dependent
concentration.
Mwith concentration. manner without any cell lysis. A complete
their antimalarial activity, Rickards et al. [34] reported calothrixins to be potent against growth inhibition was achieved at 16
µ µMMµ concentration.
MM concentration.
concentration.
µ M µµ concentration.
concentration.
M concentration.
HeLa cells. The corresponding IC50 values for calothrixin A and B against the human cervical cancer
3.2. Anticancer
3.2. Anticancer Activity
Activity
3.2.
3.2. Anticancer
cell line,
3.2. 3.2.
HeLa Anticancer
Anticancer
3.2. cells,
Anticancer
Anticancer Activity
wereActivity
Activity
ActivityActivityobserved to be 40 nM and 350 nM, respectively. Calothrixins showing similar
3.2. Anticancer Activity
inhibitoryAlongAlong Along
effects with with
againsttheirtheir antimalarial
cancer antimalarial
cell linesactivity,
activity,
and Rickards
malarialRickards et al.et[34]
strains al. [34]
reported
indicated reported
thatcalothrixins
calothrixins
they mayto toshare
beto potent
be potent
a common
Along
Along Along
Along with
with
with with
their
their
with
their their
antimalarial
their antimalarial
antimalarial
antimalarial
antimalarial activity,
activity,
activity, Rickards
Rickards
activity,
activity, RickardsRickards
Rickards et
et al.
et al.
al.et
et[34]
al.
al. [34]
[34]
[34] reported
reported
[34]
reportedreported
reported calothrixins
calothrixins
calothrixins
calothrixins
calothrixins be
tohuman
to be
be to
to potent
be
potentpotent
becervical
potent potent
against
against Along
HeLa HeLa with
cells. cells.
The their
The antimalarial
corresponding
corresponding IC activity,
50 IC
values Rickards
50 values for calothrixin
for et al.
calothrixin [34]
A andAreported
and
B against
B calothrixins
against
the human
the to be
cervical potent
modeagainstofagainst
againstaction.
HeLa
HeLa
against Following
HeLa
HeLa cells.
cells. cells.
The
The
cells. The thiscorresponding
corresponding
corresponding
The report, inIC
corresponding IC 2003,
50 IC
50 values
values
IC 50 Waring
valuesfor
for
values et calothrixin
al. [54]A
calothrixin
for
calothrixin
for calothrixin Astudied
and
andAA and
B
B
and the
against
B
against
B effect
against
the
the
against of calothrixin
human
the
human
the human
human cervical
cervical
cervical
cervicalA on
against
against
cancercancerHeLa
cell cellHeLa cells.
line,line, HeLaThe
cells. HeLa corresponding
The
cells,corresponding
cells,
werewere IC
observed values
IC50
observed
50 tovalues
50
50 be for
to 40 calothrixin
befor nM40calothrixin
nM
and and A
350 Aand350
nM, B
and against
nM, B against
respectively. the human cervical
theCalothrixins
respectively. human cervical
Calothrixins
apoptosis
cancer
cancer
cancer
in
cancer
cancercell
cell
cell
humancell
line,
line,
cell
line,
Jurkat
line,
HeLa
HeLa
line,
HeLa HeLa
HeLa cells,cancer
cells,
cells,cells,
cells, were
were
cells,
were
cells.
were
observed
observed
were
observed
A well-known
observed
observed to be
tocancer
to be
be to
to 40
40
40be
becellnM
nM
nM40inducer
nM
40linesand
and and
nM
and and
350 of apoptosis,
350
nM,
350 malarial
and
350 nM,nM,
350
nM, nM,
respectively.
respectively.
nM, menadione,
respectively.
respectively.
respectively. Calothrixins
Calothrixins was
Calothrixins
Calothrixins
Calothrixins
used
cancer
showing
showing cell
similar line,
similar HeLa
inhibitory
inhibitory effects were
effects
against observed
againstcancer to
cell be lines40 nM
and malarial350 strains respectively.
strains
indicated
indicated Calothrixins
that that
theythey
showing
as a positiveshowing
showing
showing
showing similar
control
similar
similar similar
inhibitory
to inhibitory
compare
inhibitory
similar inhibitory
inhibitory effects
the
effects
effects effects
against
effects
against
effects
against against
of
againstcancer
cancer
cancer cancer
calothrixincell
cancercell
cell cell
lines
lines
cell
lines A lines
and
[55].
and
lines
and and
malarial
malarial
and
malarialmalarial
Calothrixin
malarial strains
strains
strains strains
A indicated
was indicated
indicated
strains observed
indicated
indicated that
that
that that
they they
to induce
they
that
they they
mayshowing
may
share share similar
a common a common inhibitory
mode mode effects
of action.
of action. against
Following cancer
Following this cell this
report, lines inand
report, in malarial
2003, 2003,
Waring Waring strains indicated
et al.et[54]al. [54]
studied that
studiedthethey the
may
cell death
may
may mayshare
via
share
may
share sharea
a
share common
apoptosis a
common
a common
common inmode
mode a mode
time-
mode of
of action.
of
and
action.
of action.
Following
Following
concentration-dependent
Following
action. Following this
this this
report,
report,
this report,
in
in
report, 2003,
in
in 2003,
Waring
manner,
2003, Waring
2003, Waring
Waringand et
et al.et
was
al.et [54]
al.
[54]
al. [54]
studied
found
studied
[54] studied
to
studiedthe
the more
be
the the
the
may
effecteffect ofa calothrixin
share
of calothrixin common
a common A on mode mode
Aapoptosis
on of action.
ofin
apoptosis Following
action.
human
in humanFollowing
Jurkat this
Jurkat report,
this
cancer cancercells.incells.
report, A2003,
in Waring
2003,
well-known
A Waring
well-known et al.et
inducer [54]
al.of
inducer studied
[54] ofstudied
apoptosis,
apoptosis, the
potenteffect effect
effect of
of
effect
than
effect calothrixin
of
of
menadione
of calothrixincalothrixin
calothrixin
calothrixin A
Ain
Ausedon
on
on A
A apoptosis
on
apoptosis
on apoptosis
apoptosis
anti-proliferative in
in human
in
human
in human
human Jurkat
Jurkat
activity, Jurkat
Jurkat
withcancer
cancer cancer
IC cells.
cells.
cancer cells.
valuesA
A
cells. well-known
A well-known
well-known
Aof well-known
1.6 µM inducer
inducer
and inducer
inducer
4.7 of
of
µM, apoptosis,
of apoptosis,
apoptosis,
of apoptosis,
respectively
effect
menadione,
menadione, of was
calothrixin
was
used asAaapoptosis
on
as aapoptosis
positive in human
positive
control in
controlto Jurkat
human to Jurkat
compare comparecancer
the cancercells.
effects
the
50 effectsAofwell-known
cells. Aofwell-known
calothrixin
calothrixin inducer
A inducer
[55].
A [55]. of apoptosis,
of apoptosis,
Calothrixin
Calothrixin A A
menadione,
(Table was3).menadione,
menadione,
menadione,
menadione,
wasIC50
menadione,
observed
was
was
was
values
observed
was
used
used
was
used
to was tofor
induce
used
as
as
used
as
used
induce
aa
a as
cell
positive
aa positive
positive
as
positive
calothrixin
as a positive
death
cell death
control
control
positive
control
viaA- control
and to
control
control
apoptosis
via to compare
to
compare
to compare
compare
tomenadione-induced
compare
apoptosis to
incompare
a in time-
the
athe
the time-
effects
the
effects
theand
effects
andthe
effects
of
of
effects
of
effects
calothrixin
of
of calothrixin
calothrixin
calothrixin
calothrixin
apoptosis
of calothrixin
concentration-dependent
concentration-dependent
A
A
A [55].
A
[55].
A 0.6
[55].
were A
[55].
Calothrixin
Calothrixin
Calothrixin
[55]. Calothrixin
Calothrixin
[55]. µM and
Calothrixin
manner, manner, and
AA
A12and A
A
µM
A
was
was
was was
observed
was observed
observed
observed
observed to
to
to induce
to
induce
to
induce induce
induce cell
cell
cell death
cell
death
cell
death death
via
via
death
via apoptosis
via apoptosis
apoptosis
via apoptosis
apoptosis in
in a
a in
time-
time-
in a
a time-
and
and
time- and
concentration-dependent
concentration-dependent
concentration-dependent
and concentration-dependent manner,
manner, manner,
manner, and
and and
and
was was
respectively. found observed
Both
found to be to more
calothrixin
tomore
be induce
potent A cell
potent and
than death
than via
menadione
menadione
menadione inin
apoptosis were a in time-a time-
observed
anti-proliferative
in and and
anti-proliferative concentration-dependent
concentration-dependent
toactivity,
induce
activity, cellwith
with cycle
IC 50 IC 50 manner,
arrest
values values
ofmanner,
in
1.6 and
the
of MGand
µ1.6 µM/M
was
was
was was
found
found
was
found found
found to
to
to be
be
be to
tomore
be
be more
more
more morepotent
potent
potent potent
potent than
than
than than
menadione
than menadione
menadione
menadione
menadione in
in
in anti-proliferative
in anti-proliferative
anti-proliferative
in anti-proliferative
anti-proliferative activity,
activity,
activity,
activity,
activity, with
with
with with
IC
IC
with
IC 50
50 IC
values
50 values
values
IC 50
values of
of
values
of 1.6
1.6
1.6of
ofµµ
µ1.6
M
M
1.6
M µµ2M
M
phase, and was
butand
4.7 µ
thefound
4.7M, µ to
concentrationbe
respectively
M, more
respectively potent
(Table (Table
required than
3). IC menadione
3).
for
50 IC
values values
calothrixin
50 for in anti-proliferative
calothrixin
for calothrixin
A was much A- and
A- activity,
and
menadione-induced
lower with
menadione-induced50 IC
than for menadione. 50
50 values of
apoptosis 1.6
apoptosis µ
The cellM
and
and and
4.7
4.7 µ
µ4.7
µ M, µrespectively
M, respectively (Table (Table
3).
3). IC3). IC
values
50 valuesfor calothrixin
for
for calothrixin A- and
A- and
menadione-induced
menadione-induced apoptosis
apoptosis
and µM,
4.7 Mµµrespectively
M, respectivelyM µ(Table 3).
(Table IC3). values
IC for
values calothrixincalothrixin A- and
A- menadione-induced
and menadione-induced apoptosis
apoptosis
50 50
and
were 4.7
and
were
0.6in4.7M,
0.6 respectively
µM,Mrespectively
and and
12 µphase12 (Table (Table
respectively.
M indicated IC
3).
respectively.50 values
IC
50Both 50Both
50 for
values
calothrixin calothrixin
for
calothrixin calothrixin
A and A-
Adamage,
and and
A-
menadione menadione-induced
and
menadione menadione-induced
were were
observedobserved apoptosis
apoptosis
tosupported
induce
to induceby
cyclewere arrest
were0.6 µ the
0.6
M G 2 /M intracellular DNA which was further
were
were
cell were0.6
were µ
0.6cycle
cycle
cell Mµ
M
0.6
µarrest
0.6
and
M
µµand
and
MM
arrest
and
12
12
and
12
inandtheinµ
µ
µG12
M
M
12
M2/M
12
the
µµrespectively.
M respectively.
µrespectively.
M2phase,
respectively.
GM /M respectively.
phase,
Both
but Both
respectively. Both
the
but
Both
calothrixin
calothrixin
calothrixin
Both calothrixin
calothrixin
Both calothrixin
concentration
the concentration
A
A and
andA
Arequired
andA
and
menadione
and
menadione
menadione
Arequired
and menadione
menadione
menadione
for calothrixin
were
werewere
were
for calothrixin
observed
observed
were
observed
were
A was
observed
observed
observed
A wasmuch
to
to induce
tomuch to
to induce
induce
induce
to
lower induce
induce
lower
directcell DNA
cell cycle
cell
cycle
cell damage
cycle
arrest
arrest
cycle arrest
in
in
arrest observed
the
thein
in G
the
Gthe22/M
/M G
G in
2phase,
/M
phase,
/M cell-free
phase,
but
but
phase, the
but
the
butexperiments.
concentration
the concentration
concentration
the concentration requiredrequired
required
required for
for calothrixin
for calothrixin
calothrixin
for calothrixin A
A was
wasAA was
much
much
was much
lower
lower
much lower
lower
cell
thancycle
cell
forcycle
than arrest
menadione.
for in the
arrest
menadione. inTheG
the 2/M G22phase,
The
cell2/M cell
cycle but
phase,
cycle the
arrestbut concentration
thethe
arrest
in concentration
in G the 2/M G2phase
/M required
required
phase for
indicated calothrixin
forintracellular
indicated calothrixin A was
intracellular ADNA much
wasDNA lower
much
damage, lower
damage,
Both
than
than
than than calothrixin
for
for
than
for menadione.
for menadione.
menadione.
for menadione.
menadione.
A and
The
The
The Themenadione
cell
cell
The
cell by cell
cycle
cycle
cell
cycle cycle
arrest
arrest
cycle
arrest
were
arrest
in
in
arrest
in the
the
the
found
in
in G
the
G
the
G 22/M
/M
2/M
G
G to be
22phase
/M
phase
/M redox
phaseindicated
indicated
phase active
indicated
indicated through
intracellular
intracellular
intracellular
intracellular the
DNA
DNA observation
DNA
DNA damage,
damage,damage,
damage, of
whichthan
which
was for was menadione.
further furthersupported The
supported cell cycle
direct
by directarrest
DNA DNA in damage
damage the G 22phase
/Mobserved
observed phaseindicated
indicated
in cell-free intracellular
in cell-free intracellular
experiments.
experiments.DNA DNA damage,damage,
which
additional
which
whichwhichwas
oxygen
was
which
was was
further
further
was
further further
intake
furthersupported
supported
supportedsupported
when
supported by
added
by direct
by
direct
by
by direct direct
to DNA
DNA
direct
DNA DNA
reductantdamage
DNAdamage
damage damage
damage observed
dithiothreitol
observed
observed observed
observed in
in cell-free
in
(DTT, cell-free
cell-free
in 2
cell-freeexperiments.
mM experiments.
at
experiments. pH
experiments. 8.0). Quinones can
which
Both Both was further
calothrixin
calothrixin Asupported
and A and menadioneby direct
menadione were DNA
were damage
found found to beto be in
observed
redox redoxcell-free
in active
active cell-free experiments.
through experiments.
through the observation
the observation of of
redox Both
cycle
Bothby
Both Both
calothrixin
one calothrixin
calothrixin orintake
twoA
A and
and A
A and menadione
electron menadione
menadione transfers, were werefound found
generating to be
beto
toredox
be
be redox
reactive active active
oxygen through through the
species observation
the observation
(ROS), byofofwhichof
additional Both
Both
additional oxygen calothrixin
calothrixin
calothrixin
oxygen Aintakeand A
when and
and when menadione
menadione
menadione
added added towere
were were
towere
reductant found
found
reductant found
found to be to
dithiothreitol redox
to dithiothreitol
redox
be redox
redoxactive
active
(DTT, 2through
active
active
(DTT, through
mM at the
through
2through
mM the
pH observation
the
the
at 8.0).
pH observation
observation
observation
8.0).
Quinones of
Quinones of
of
additional
additional
additional
additional
they additional
can redox
damage oxygen
oxygenDNAoxygen
oxygen intake
intake intake
intake
andintake when
when when
added
added
when added
addedto
to reductant
to reductant
reductant
to reductant dithiothreitol
dithiothreitol
dithiothreitol
dithiothreitol (DTT,
(DTT, (DTT,
(DTT,2
2 mM
mM 22 mM
at
at
mM pH
pHat
at 8.0).
pH
8.0).
pH 8.0).
Quinones
Quinones
8.0). Quinones
Quinones
can additional
can redox oxygen
cycle oxygen
cycle
by oneintake
by orinduce
one when
twoor whentwo apoptosis
added
electron added
electron [56].
to reductant
to
transfers,
transfers,It was
reductant
generating postulated
dithiothreitol
dithiothreitol
generating reactivereactivethat
(DTT, (DTT,
oxygen 2themM
oxygen 2ringmM
species structure
atspecies
pHat 8.0).
pH(ROS),
(ROS), 8.0).
byofwhich
Quinonescalothrixin
Quinones
by which
can
can redox
can
redox redox cycle
cycle cycle
by
by one
by
by one
one or
or two
twoor two
electron
electron
electron transfers,
transfers,
transfers, generatinggenerating reactivereactiveoxygen oxygen species species(ROS), (ROS),
by which
by which
might can
they can
redox
act
can
they
can redox
asredox cycle
adamage
can DNA cycle
by intercalator
cycle
damage one
by
DNA one
or
one
DNA two
andor and
or two
two
induceelectron
electron
that
electron
induce transfers,
might be generating
transfers,
transfers,
apoptosis
apoptosis generating
responsible
[56]. generating
generating
[56].
It was Itreactive
reactive
wasfor itsoxygen
reactive
reactive
postulated oxygen oxygen
anticancer
postulated oxygen
that species
species
that
thespecies(ROS),
(ROS),
activity.
species
the
ring (ROS),
(ROS),
ring by
by
structureInwhich
by
which
by which
addition,
structure which
of of
they
they
they they
can
can
they
can can
damage
damage
can
damage damage
damage DNA
DNA
DNA DNA
DNA and
and
and and
induce
induce
and
induceinduce
induceapoptosis
apoptosis
apoptosis
apoptosis
apoptosis [56].
[56].
[56]. [56].
It
It
[56].
It was
was
wasIt
It was
postulated
postulated
postulated
was postulated
postulated that
that
that that
the
the
that
the the
ring
ring
the
ring ring
structure
structure
structure
ring structure
structure of
of
of of
of
they can damage DNA a as
DNA a and
DNA induce
intercalator apoptosis
calothrixin A was observed to be cleaving DNA, though less effectively than menadione. Menadione caused
calothrixin
calothrixin might might
act as
act intercalator that that
might [56].
might be It was
responsible
be postulated
responsible for its
for that the
anticancer
its ring
anticancer structure
activity.
activity.
In of
In
calothrixin
calothrixin
calothrixin
calothrixin
calothrixin might
might
might might
act
act
might
act as
act
as
act
as a
a
a as
DNA
DNA
as
DNA aa DNAintercalator
DNA intercalator
intercalator
intercalator
intercalator that
that
that that
might
might
that
might might
might be
be
be responsible
be responsible
responsible
be responsible
responsible for
for
for its
for
its
for
its anticancer
its anticancer
anticancer
its anticancer
anticancer activity.
activity.
activity. In
In
activity.
activity. In In
In
calothrixin
addition,
addition, calothrixin might
calothrixin act
A was as
A was a DNA
observed intercalator
observed to betocleaving that
be cleaving might
DNA, DNA, be
thoughresponsible
though less less for
effectively its
effectively anticancer
thanthan activity.
menadione.
menadione. In
addition,
addition,
addition,
addition,
addition, calothrixin
calothrixin
calothrixin
calothrixin
calothrixin A was AA was
A significant
A was
was observed
observed
was
observed observed
observed to be
beto
tocleaving
to damage
to be be cleaving
cleaving
be10 cleaving
cleaving DNA,
DNA, DNA,
DNA, though
though though
though less less
effectively
lessincubation, effectively
effectively
less effectively than than
menadione.
thancalothrixin menadione.
menadione.
than menadione.
addition,
Menadione
Menadione calothrixin
caused caused significant A was DNAobserved
DNAdamage to be
at cleaving
atµ 10 MDNA, µ MDNA,
after though
after
60 minthough
60 minless effectively
less
incubation, effectively
whereas than
whereas menadione.
than menadione.
calothrixin A A
Menadione
Menadione
Menadione
Menadione
Menadione caused
caused
caused caused
caused significant
significant
significant
significant
significant DNA
DNA
DNA DNA
DNAdamage
damage
damage damage
damage at
at
at 10
10
10at
at µ
µ
µ 10
M
M
10
M µ
after
M
after
µ
afterM after
60
60
after
60 min
60
min
60
min min
incubation,
incubation,
incubation,
min incubation,
incubation, whereas
whereas
whereaswhereas
whereas calothrixin
calothrixin
calothrixin
calothrixin
calothrixin A
A A
A A
Menadione
exhibited
exhibited the same caused
the same effect significant
effect
at 500 at µ500 DNA
M.µ M. damage at 10 µ M after 60 min incubation, whereas calothrixin A
exhibited
exhibited
exhibited
exhibited the
the same the
the same
same sameeffect
effect effect
at
at 500
effect 500 at
at µ500
µ M.
M.µ
500 M.
exhibited
exhibited the same
the sameeffect at
effect 500 at µ M.
500 µµ M.
M.
Table
Table
3. Cell
3. cytotoxicities
Cell cytotoxicities
and apoptotic
and apoptotic
activities
activities
of calothrixin
of calothrixin
A andA menadione.
and menadione.
Table
Table
Table 3.
3. Cell
3.
3. Cell
cytotoxicities
Cell cytotoxicities
and apoptotic
and
and apoptotic
activities
activities
of calothrixin
of
of calothrixin
A and
A
A menadione.
and
and menadione.
Table 3. Cell
Table
Table
TableCell
3.
3.
cytotoxicities
cytotoxicities
Cell
Cell
and
and apoptotic
cytotoxicities
cytotoxicities
cytotoxicities and
and
activities
apoptotic
apoptotic
apoptotic
apoptotic
of
of calothrixin
activities
activities
activities
activities of
of
A
A and
calothrixin
calothrixin and
calothrixin
calothrixin A
A
menadione.
and
and menadione.
menadione.
menadione.
menadione.
Compound
Compound Structure
Structure IC50 IC
for50Cytotoxicity
for CytotoxicityIC50 IC
for50Apoptosis
for Apoptosis
Compound
Compound
Compound
Compound
Compound Structure
Structure
Structure
Structure
Structure IC 50 IC
IC50
IC for50Cytotoxicity
for50
IC
for for
for Cytotoxicity
CytotoxicityIC
Cytotoxicity 50 IC
IC50 for50Apoptosis
for50
IC for
for Apoptosis
Apoptosis
Apoptosis
Compound Structure 50 IC 50 Cytotoxicity
50 for CytotoxicityIC 50 for
IC 50 Apoptosis
50 for Apoptosis
Mar. Drugs 2016, 14, 17 13 of 21

significant DNA damage at 10 µM after 60 min incubation, whereas calothrixin A exhibited the same
effect at 500 µM.

Table 3. Cell cytotoxicities and apoptotic activities of calothrixin A and menadione.

Mar. Drugs 2016, 14, x

Mar. Drugs 2016, 14, x 13 of
13 of 20
20
Mar. Drugs 2016, 14, xCompound Structure IC50 for Cytotoxicity IC50 for Apoptosis 13 of 20
Mar. Drugs 2016, 14, x 13 of 20
Mar. Drugs 2016, 14, x 13 of 20
Mar. Drugs 2016, 14, x 13 of 20
CalothrixinA A
Calothrixin
Calothrixin A 1.6 µM
1.6
1.6 µM
µ M 0.6
0.6 µM
0.6 µM
µ M
Calothrixin A 1.6 µ M 0.6 µ M
Calothrixin A 1.6 µ M 0.6 µ M
Calothrixin A 1.6 µ M 0.6 µ M
Calothrixin A 1.6 µ M 0.6 µ M
Menadione
Menadione
Menadione 4.7 µM
4.7
4.7 µM
µ M 12
1212
µMµM
µ M
Menadione 4.7 µ M 12 µ M
Menadione 4.7 µ M 12 µ M
Menadione 4.7 µ M 12 µ M
Menadione
One of
One of the
the known
known modesmodes of of action
action for for anticancer 4.7 µ
anticancer activitiesM
activities of of quinones
quinones is 12by
is µM
by the generation
the generation of of
OneROS of the
ROS through known
through redox modes
redox cycling of
cycling [56]. action
[56]. In for
In 2004, anticancer
2004, Wilkes
Wilkes et activities
et al.
al. [57]
[57] carriedof quinones
carried out is
out aa comparative by the generation
comparative study of
study of
of ROSOne of the known
bioactivities
through modes
cyclingof
of calothrixins
redox calothrixins action
and In for
some anticancer
Wilkes et
structurally activities
al. [57]
related of quinones
quinones. This ais
was bythethe generation
first study of
structure–
One of the known
bioactivities of modes of[56].
action
and some 2004,
for anticancer
structurally activities
related carried
of quinones
quinones. out was
This iscomparative
bythethe generation
first structure– of
of
ROS One of theredox
through
activity known
relationship modes
cycling study of done
[56]. action forcalothrixins.
Instructurally
2004,
for anticancer
Wilkes activities
et The
al. [57] of quinones
carried
cytotoxic effectoutofthe is by the
a various
comparative generation
quinones study
was of
One
bioactivitiesof
activitythe
of known
calothrixins
relationship modesand
study of
some action
done for for anticancer
related
calothrixins.
ROS through redox cycling [56]. In 2004, Wilkes et al. [57] carried out a comparative study activities
Thequinones.
cytotoxic of quinones
This
effectwas of is by
first
various the generation
structure–activity
quinones was of
ROS through
bioactivities
evaluatedofredox cyclingand
calothrixins
against [56]. some
human In 2004, Wilkes related
structurally
cervical et cancer
al. [57] carried
quinones. outHeLa
This
cells,quinones awascomparative
theevaluated study
first structure–
cells, using of
ROS through
evaluated
relationship
bioactivities ofredox
study done cycling
against
calothrixins [56].
human
for calothrixins.
and someIn 2004, Wilkes related
Thecervical
cytotoxic
structurally eteffect
al. [57]
cancer of carried
quinones.cells,
various outHeLa
This awascomparative
was thecells, study
using
against
first structure– of
bioactivities
activity of calothrixins
relationship study and
done some
for
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium structurally
calothrixins. related
Thebromide quinones.
cytotoxic
bromide (MTT)effect This
assay was
of wasvarious
(Table the first structure–
quinones
4). first
Calothrixin was
A
human3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bioactivities
activity cervicalof calothrixins
relationship cancer cells,and
study HeLa
done somecells,
for structurally related (MTT)
quinones. assay
This
using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
calothrixins. The cytotoxic effect of (Table
various 4).
the Calothrixin
structure–
quinones Awas
activity
evaluated relationship
and calothrixin
against study
B showed
showed done
human for calothrixins.
cytotoxicity against HeLa
cervical HeLa The cytotoxic
cells
cancer withcells,
EC50 effect ofof
valuesHeLaofvarious
0.12 µ µM quinones
Mcells,
and 0.24
0.24 µ was
µusing
M,
bromideand calothrixin B cytotoxicity Aagainst cells with EC values 0.12 and M,
50
activity
evaluated relationship
(MTT) against study
assay (Table 4).done
human for calothrixins.
Calothrixin and calothrixin
cervical The
cancer cytotoxic
B showed cells,effect
cytotoxicity of various
HeLa against quinones
HeLa cells
cells, was
with
using
evaluated respectively.
againstCalothrixin human B and
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
respectively. Calothrixin B and ellipticine
cervical
ellipticine quinone,
quinone, which
cancer
bromide
which differ
(MTT)
differ by
cells,
by the
assay
the ring
HeLa
ring E,
(Table
E, showed
showed4). comparable
cells,
Calothrixin
comparable usingA
evaluated
EC50 values ofagainst
0.12 µM and human 0.24
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium µM, cervical
respectively. cancer
Calothrixin
bromide cells,
(MTT) Bringand
assay HeLa
ellipticine
(Table cells,
4).quinone,
Calothrixin using
which A
and activities,Bindicating
calothrixin indicating
showed that there might
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
activities, that there
cytotoxicity might be only
be
against only aa limited
HeLa limited
bromide
cells role
role
with for
forEC the
(MTT)
the50 ring
values E in
assay
E inofdictating
(Table
dictating
0.12 µ4).
M the
the bioactivity
Calothrixin
bioactivity
and 0.24 µ A
M,
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
differ
and by the
calothrixin ring
B
of calothrixins.
calothrixins. E,
showed showed
Introducing comparable
cytotoxicitythe MOMMOM against activities,
group HeLa
on thethe bromide
indicating
cells
indolewith (MTT)
EC
nitrogen that
50 assay
there
values
seemedoftoof(Table
might
0.12
to0.12 µ4).
be
decrease M Calothrixin
only
and a limited
0.24
the potency
potency µ A
M,
and of
calothrixin
respectively. B showed
Calothrixin Introducing
Bcytotoxicity
and the
ellipticine againstgroup
quinone, on
HeLa indole
cells
which with nitrogen
differ ECby seemed
50 values
the ring E, decrease
µ M and
showed the 0.24 µ M,
comparable
and calothrixin
role forof
ofthe
respectively. both
bothring B showed
E in dictating
Calothrixin
calothrixin
calothrixin
cytotoxicity
BBand the
andellipticine
and ellipticine against
bioactivity HeLa
quinone,
quinone of
with cells
calothrixins.
which
EC50 with EC
differof
values of by
50
0.42values
Introducing
theµMMring of
andthe 0.12
E, MOMµ
showed
0.37 µ M, M and
group 0.24
comparable
M, respectively.
respectively. µ
on theM,
that BtoB andellipticine be quinone with EC 50 values 0.42 µ and 0.37 µ
respectively.
activities, Calothrixin
indicating there ellipticine
might onlyquinone,
a limited whichrole differ
for theby ringthe Ering
in E, showed
dictating the comparable
bioactivity
respectively.
indole nitrogen
activities,
Further,
Further, Calothrixin
indicating seemedthat
replacement
replacement B and
decrease
there
of the
of the ellipticine
might the
be
heteroaromatic
heteroaromatic only quinone,
potencya of
limited
ring-D which
bothrole
with a differ
calothrixin
for the
carbocyclicbyringthe
B Ering
and in
aromatic E, showed
ellipticine
dictating
ring in the comparable
quinone with
bioactivity
ellipticine and
activities,
of indicating
calothrixins. that there
Introducing themight
MOM be only on
group aring-D
limited with
the indole role a carbocyclic
for the ring
nitrogen
aromatic
seemed
ring in ellipticine
E in dictating
toofdecrease thethe and
bioactivity
potency
activities,
EC its indicating
valuesN-methoxymethyl
of calothrixins. of 0.42 that
µM there
Introducinganalog
50 its N-methoxymethyl analog
and might
0.37
the MOM also be
µM,
also ledled
grouponly
to a a limited
decrease
respectively.
to a on the indole
decrease inrole for
potency,
Further,
nitrogen
in potency, the ring
replacement E in
benzocarbazoledione dictating
seemed to decrease
benzocarbazoledione the the
(0.43
(0.43the µbioactivity
M),
heteroaromatic
µ M), and
potency
and
of calothrixins.
both calothrixin Introducing
N-MOM-benzocarbazoledione the MOM
B and ellipticine (1.6group
quinone
µ M).
M). on the
with
MenadioneECindole
50 values
showednitrogen
ofactivity
0.42 seemed
µat
M3.7 and µto
M. decrease
0.37 µ M,
Absence the potency
respectively.
of activity
activity
of calothrixins.
ring-D
both with a Introducing
calothrixin carbocyclic the MOM
aromatic
B and ellipticine
N-MOM-benzocarbazoledione group
ring
quinone
(1.6 µ on
with the
inMenadione ECindole
ellipticine and
50 values
showed nitrogen
its
ofactivity
0.42 seemed
N-methoxymethyl
µat
M3.7 and µto
M. decrease
0.37 µ M,
Absence analog the potency
also led
respectively.
of
of both for
Further, calothrixin
replacement
uncyclized B and
of theellipticine
precursorsheteroaromatic quinonering-D
of benzocarbazoledione
benzocarbazoledione with EC with50 values
and of 0.42 aromatic
a carbocyclic µ M and 0.37
N-MOM-benzocarbazoledione ringµ inM,ellipticine
respectively.
indicated and
the
of
to both calothrixin
for
a decrease
Further, replacement in B
uncyclized and
potency,
of theellipticine
precursors of quinonering-D
benzocarbazoledione
heteroaromatic with EC with and
(0.43
50 valuesµM),ofand 0.42 aromatic
µ M and 0.37
N-MOM-benzocarbazoledione
a carbocyclic ringµinM,indicated
N-MOM-benzocarbazoledione respectively.
ellipticine the
and
Further,
its replacement
N-methoxymethyl
importance
importance of
of a
a of the
analog heteroaromatic
tetracyclic
tetracyclicalso led
quinone
quinone to a
ring
ring ring-D
decrease
structure.
structure. with
inIn
In a
case
casecarbocyclic
potency, of
of all
all the
the aromatic
benzocarbazoledione
quinones
quinones ring
with
with EC
EC in50 ellipticine
(0.43
<
< 1.6
1.6 µµ M),
M,
M, and
the
the
its N-methoxymethyl analog also led to a decrease in potency, benzocarbazoledione (0.43 µ M), and
Further,
(1.6 µM). replacement
Menadione of the
showed heteroaromatic
activity at ring-D
3.7 µM. with
Absence a carbocyclic
of activity aromatic
for ring
uncyclized in50 ellipticine
precursors of
its N-methoxymethyl
quinones were
N-MOM-benzocarbazoledione
quinones analog also
were observed
observed to beled
to(1.6
be µ to aMenadione
reduced
M).
reduced decrease
to their
to inshowed
their respectivepotency,
respective benzocarbazoledione
semiquinones,
activity
semiquinones, at though
3.7
thoughµ M. no
no direct
Absence
direct(0.43 µ M),
correlation
of and
activity
correlation
its N-methoxymethyl analog
benzocarbazoledione
N-MOM-benzocarbazoledione also
(1.6 led to aMenadione
and N-MOM-benzocarbazoledione
µ M). decrease inshowed potency, benzocarbazoledione
indicated
activity the
at 3.7importance
µ M. Absence (0.43
of a of µ M), and
tetracyclic
activity
for was observed
uncyclized
was observed
N-MOM-benzocarbazoledione between
precursors
between the
of the reduction
(1.6
benzocarbazoledione
reduction potentials and
µ M). potentials
Menadione and
and bioactivity.
showed activity at 3.7 µ M. Absence
N-MOM-benzocarbazoledione
bioactivity. of activity
indicated the
N-MOM-benzocarbazoledione
quinone
for ring structure.
uncyclized precursors In case (1.6
of allµtheM).quinones
of benzocarbazoledione Menadione with
andshowed
EC < activity
1.6 µM,
N-MOM-benzocarbazoledione
50 at
the 3.7 µ M.
quinones Absence
were of activity
observed
indicated to
the
for uncyclized
importance of aprecursors
tetracyclic of benzocarbazoledione
quinone ring structure. and
In caseN-MOM-benzocarbazoledione
of all the quinones with EC 50 indicated
< 1.6 µ M, the
for uncyclized
be reduced of
importance precursors
to atheir respective
tetracyclic of benzocarbazoledione
semiquinones,
quinone Tablering
Table 4. though
structure.
4. Cytotoxicity
Cytotoxicity and
of In
of case
quinones
quinones
N-MOM-benzocarbazoledione
no direct
of allcorrelation
against
against the
HeLa
HeLa cells. was
quinones
cells. observed
with indicated
EC50 < between
1.6 µ M, the the
importance
quinones of aobserved
were tetracyclic to quinone
be reduced ring structure. In case of all the quinones with noEC 50 < 1.6 µ M, the
importance
reductionwere
quinones of aobserved
tetracyclic
potentials andto quinone
bioactivity.
be reduced ringto their respective
tostructure.
their respectiveIn casesemiquinones,
of all the quinones
semiquinones,
though
though with noEC
direct correlation
50 < 1.6
direct µ M, the
correlation
quinones
was observedwerebetween
observedthe to reduction
be reduced
Compound
Compound to their respective
potentials Structure
and Structure semiquinones,
bioactivity. EC50though
EC 50 (µM)
(µM) HeLa no direct
HeLa Cells correlation
Cells
quinones
was observedwerebetween
observedthe to reduction
be reduced to their respective
potentials and bioactivity. semiquinones, though no direct correlation
was observed between the Table reduction potentialsofand
4. Cytotoxicity bioactivity.
quinones against HeLa cells.
was observed between the reduction potentials and bioactivity.
Calothrixin A
Table 4. Cytotoxicity
Calothrixin A 0.12 ±
of quinones against HeLa cells. 0.12 ± 0.01
0.01
Table 4. Cytotoxicity of quinones against HeLa cells.
Table
Compound 4. Cytotoxicity of quinones against
Structure HeLa cells.
EC (µM) HeLa Cells
Table 4. Cytotoxicity of quinones
Compound against HeLaEC
Structure cells.
50
50 (µM) HeLa Cells
Compound Structure EC50 (µM) HeLa Cells
Compound Structure EC50 (µM) HeLa Cells
Compound
Calothrixin B
Calothrixin B Structure EC50 (µM) ±HeLa
0.24 ±
0.24 0.04 Cells
0.04
Calothrixin
CalothrixinAA 0.12˘±0.01
0.12 0.01
Calothrixin A 0.12 ± 0.01
Calothrixin A 0.12 ± 0.01
Calothrixin A 0.12 ± 0.01
N-MOM-calothrixin B
N-MOM-calothrixin B 0.42 ±
0.42 ± 0.02
0.02
Calothrixin B 0.24 ± 0.04
Calothrixin
CalothrixinBB 0.24˘±0.04
0.24 0.04
Calothrixin B 0.24 ± 0.04
Calothrixin B 0.24 ± 0.04
Ellipticine quinone
Ellipticine quinone 0.15 ±
0.15 ± 0.09
0.09

N-MOM-calothrixin B 0.42 ± 0.02

N-MOM-calothrixin B 0.42 ± 0.02
N-MOM-calothrixin
N-MOM-calothrixin BB 0.42˘±0.02
0.42 0.02
N-MOM-calothrixin
N-MOM-ellipticineBquinone
N-MOM-ellipticine quinone 0.42 ±±0.08
0.37 ±
0.37
0.02
0.08

Ellipticine quinone 0.15 ± 0.09

Ellipticine quinone 0.15 ± 0.09
Ellipticine quinone
Benzocarbazoledione
Benzocarbazoledione
0.15 ±±
0.43˘±
0.43
0.09
0.01
0.01
Ellipticine quinone
Ellipticine quinone 0.15
0.15 ±0.09
0.09

N-MOM-ellipticine quinone 0.37 ± 0.08

N-MOM-ellipticine quinone
N-MOM-Benzocarbazoledione 0.37
1.6 ±±1.0
±± 0.08
N-MOM-ellipticine quinone
N-MOM-Benzocarbazoledione 0.37
1.6 0.08
1.0
N-MOM-ellipticine quinone 0.37 ± 0.08

Benzocarbazoledione 0.43 ± 0.01

Benzocarbazoledione 0.43 ± 0.01
Benzocarbazoledione 0.43 ± 0.01
Benzocarbazoledione 0.43 ± 0.01
 Calothrixin B 0.24 ± 0.04
Calothrixin B 0.24 ± 0.04
Calothrixin B 0.24 ± 0.04

Mar. Drugs 2016,N-MOM-calothrixin

14, 17 B 0.42 ± 0.02 14 of 21
N-MOM-calothrixin B 0.42 ± 0.02
N-MOM-calothrixin B 0.42 ± 0.02
Table 4. Cont.
Ellipticine quinone 0.15 ± 0.09
Ellipticine quinone
Compound Structure
0.15 ± 0.09
EC50 (µM)
Ellipticine quinone 0.15 HeLa
± 0.09Cells

N-MOM-ellipticine
N-MOM-ellipticinequinone
quinone 0.37˘±0.08
0.37 0.08
N-MOM-ellipticine quinone 0.37 ± 0.08
N-MOM-ellipticine quinone 0.37 ± 0.08

Benzocarbazoledione 0.43 ± 0.01

Benzocarbazoledione
Benzocarbazoledione 0.43˘±0.01
0.43 0.01
Benzocarbazoledione 0.43 ± 0.01

N-MOM-Benzocarbazoledione 1.6 ± 1.0

N-MOM-Benzocarbazoledione 1.6 ± 1.0
N-MOM-Benzocarbazoledione
N-MOM-Benzocarbazoledione 1.6˘±1.0
1.6 1.0
Mar. Drugs 2016, 14, x 14 of 20
Mar. Drugs 2016, 14, x 14 of 20
Mar. Drugs 2016, 14, x 14 of 20

Menadione
Menadione 3.7˘±0.3
3.7 0.3
Menadione 3.7 ± 0.3
Menadione 3.7 ± 0.3

Uncyclized precursor to
Uncyclized
Uncyclizedprecursor
precursor to
to >50
Uncyclized precursor
benzocarbazoledione to >50
>50
benzocarbazoledione
benzocarbazoledione >50
benzocarbazoledione

Uncyclized precursor to
Uncyclized precursor to >50
Uncyclized
Uncyclizedprecursor
precursor to
N-MOM-benzocarbazoledioneto >50
N-MOM-benzocarbazoledione >50
>50
N-MOM-benzocarbazoledione
N-MOM-benzocarbazoledione

To study the involvement of the ring structure and the mechanism of action in the inhibitory
To study the involvement of the ring structure and the mechanism of action in the inhibitory
Tothe
effects, study
same thegroup
involvement of the ring
[58] synthesized andstructure
analyzedand the mechanism
the activity of variousof simple
action in the inhibitory
quinone analogs
effects,Tothe
study
same the involvement
group of the ring
[58] synthesized andstructure
analyzedand the mechanism
the activity of various ofsimple
action quinone
in the inhibitory
analogs
effects,
of the sameB group
calothrixin (Table[58]5) synthesized
in 2007. The andcompounds
analyzed thewere activity of various
evaluated forsimple
their quinone
selectivityanalogs
and
effects, the same group [58] synthesized and analyzed the activity of various
of calothrixin B (Table 5) in 2007. The compounds were evaluated for their selectivity and simple quinone analogs of
of calothrixin B activity,
antiproliferative (Table 5) in 2007.
using an MTT Theassay,
compounds
against were
three evaluated
different cellfor lines:
their human
selectivity and
cervical
calothrixin B (Table
antiproliferative 5) in 2007.
activity, usingTheancompounds
MTT assay, were evaluated
against threefordifferent
their selectivity
cell lines:andhuman
antiproliferative
cervical
antiproliferative
cancer (HeLa) cells, activity,
murineusing
P388 an MTT assay,
macrophage against
cancer cells,three different
and simian cell lines: human
non-cancerous cervical
CV-1 cells.
activity,
cancer usingcells,
(HeLa) an MTTmurineassay,
P388 against three different
macrophage cell lines:
cancer cells, human
and simian cervical cancer
non-cancerous (HeLa)
CV-1 cells.cells,
cancer (HeLa) cells, murine P388 macrophage cancer cells, and simian non-cancerous CV-1 cells.
murine P388 macrophage cancer cells, and simian non-cancerous CV-1 cells.
Table 5. Antiproliferative activity of calothrixin B and analogs in MTT assay against HeLa cells, P388
Table
In this5.study,
Antiproliferative
calothrixin activity of calothrixin
B was found to be theB and
mostanalogs
potentin MTT assay
against HeLaagainst HeLa
cells (0.25 cells,
µM) P388 by
followed
Tableand
cells, 5. Antiproliferative
CV-1 cells. activity of calothrixin B and analogs in MTT assay against HeLa cells, P388
cells, and CV-1 cells.
indolophenanthrene-7,13-dione analog (1.5 µM), indicating the importance of nitrogen in ring D.
cells, and CV-1 cells.
EC (µM) EC (µM)
The activity was further decreased with the deletion of ring E of this analog to form benzoncarbzoledione
50 50 EC 50 (µM)
Compound Structure EC50 (µM) EC50 (µM) EC50 (µM)
(1.8 µM). The Compound
rest of the analogs showed Structure
moderate activity EC50 ranging
HeLa (µM)
Cells from EC50 (µM)
P388 7Cells EC50while
CV-1
to 13 µM, (µM)
Cells the
Compound Structure HeLa Cells P388 Cells CV-1 Cells
carbazole-1,4-dione analog was found to be inactive. The results HeLawere Cellsnot asP388 Cells for
consistent CV-1 Cellscell
the P388
line, where murrayaquinone
Calothrixin B (2.3 µM) and 2-methylcarbazoledione (1 µM) were
0.25 ± 0.05 9 ±found
2 to be2.4
more± 0.7active
Calothrixin B 0.25 ± 0.05 9±2 2.4 ± 0.7
compared toCalothrixin
calothrixinBB (9 µM) or other analogs, whereas0.25 ± 0.05 B and92-methylcarbazoledione
calothrixin ±2 2.4 ± 0.7
showed similar activity with 2.4 µM and 1.7 µM, respectively, against non-cancerous cell line CV-1.
The rest of the analogs had either very low activity or no measurable
Indolophenanthrene-7,13-dione 1.5 ± 0.3 activity.>50 These studies>50 indicated
theIndolophenanthrene-7,13-dione
importance of rings
Indolophenanthrene-7,13-dione A–D in the activity of calothrixins. 1.5Also,
± 0.3a selective
1.5 ± 0.3
>50
>50usage of >50
calothrixin
>50 B
against human cervical cancer cells was indicated by the 10-fold higher effectiveness against HeLa
cells (0.25 µM) as compared to CV-1 (2.4 µM) and 38 times more
Benzocarbzoledione 1.8compared
± 0.1 to P388
>50(9 µM). The>50 quinones
containing Benzocarbzoledione
tetra- and penta cyclic systems (calothrixin-B,1.8 ±indolophenanthrene-7,13-dione
0.1 >50 >50 and
Benzocarbzoledione 1.8 ± 0.1 >50 >50
benzocarbazole-1,4-dione) were found to be more active against HeLa cells as compared to p388
and CV-1 cells. However, the trend was reversed in the quinones containing bi- and tricylic systems
Carbazole-1,4-dione >50 >50 >50
Carbazole-1,4-dione
(murrayaquinone, 2-methylcarbazoledione, and isoquinoline-5,8-dione). >50 >50 >50
Carbazole-1,4-dione >50 >50 >50
In 2009, another group, Hecht et al. [59], published a study to determine the mechanism of
action and the stage of the cell cycle that is targeted by the calothrixins. Calothrixin A and B and the
Murrayaquinone 13 ± 1 2.3 ± 0.3 10 ± 2
Murrayaquinone 13 ± 1 2.3 ± 0.3 10 ± 2
Murrayaquinone 13 ± 1 2.3 ± 0.3 10 ± 2

2-Methylcarbazoledione 7±1 1.0 ± 0.1 1.7 ± 0.4

2-Methylcarbazoledione 7±1 1.0 ± 0.1 1.7 ± 0.4
2-Methylcarbazoledione 7±1 1.0 ± 0.1 1.7 ± 0.4
 Uncyclized precursor
Uncyclized precursor to
to
Uncyclized precursor
Uncyclized precursor to
to >50
>50
Uncyclized precursor
benzocarbazoledione
Uncyclized to
precursor to
benzocarbazoledione >50
>50
benzocarbazoledione
benzocarbazoledione >50
>50
benzocarbazoledione
benzocarbazoledione
Mar. Drugs 2016, 14, 17
Uncyclized
Uncyclized precursor to
precursor to 15 of 21
Uncyclized precursor
Uncyclized precursor to to >50
>50
Uncyclized precursor to
N-MOM-benzocarbazoledione
Uncyclized precursor
N-MOM-benzocarbazoledione to >50
>50
N-MOM-benzocarbazoledione
N-MOM-benzocarbazoledione >50
>50
N-MOM-benzocarbazoledione
N-MOM-benzocarbazoledione
N-methylated derivative were synthesized and tested against CEM leukemia cells to measure their
cytotoxicity using an MTT assay. The activity of calothrixin analogs were compared to camptothecin,
To study
To study the
the involvement
involvement of of the
the ring
ring structure
structure and
and the
the mechanism
mechanism of of action
action inin the
the inhibitory
inhibitory
To study
To
which study the to
the
is known involvement
involvement of the
of the ring
cause irreversible ring structure
structure
DNAstructure and the
and
damage and
duringthethe
mechanism
mechanism of action
of
S phase. Delay action in
in
in S in the due
the
phase inhibitory
inhibitory
to DNA
To
effects,
To
effects, study
the same
study
the samethe
the involvement
group [58]
involvement
group [58] of
of the
synthesized
the
synthesized ring
and
ring
and analyzed
structure
analyzed the
and
the the
the mechanism
activity of
mechanism
activity of of
various
of
various action
simple
action
simple in the
quinone
the
quinone inhibitory
analogs
inhibitory
analogs
effects,
effects,
damage the
the same
same
is group
group
associated [58]
[58]
with synthesized
synthesized
a block in and
and analyzed
analyzed
replication. the
the
The ICactivity
activity
value of
of various
various
for simple
simple
calothrixin quinone
quinone
A was analogs
analogs
found to be
effects,
effects,
of the same
of calothrixin
the sameB
calothrixin group
Bgroup [58]
(Table[58] synthesized
5)synthesized
in 2007.
2007. Theandcompounds
The
and analyzed the
compounds
analyzed thewere
activity
were
activity of various
of various
50 evaluated simple
forsimple quinone
their quinone analogs
selectivity and
analogs
of five-fold
of calothrixin
calothrixin B (Table
B
higher (Table
(Table
than
5)
5) in
5) in 2007.
in
campothecin2007.and
The
The thecompounds
compounds
other two were
were
analogs
evaluated
evaluated
evaluated
were
for
for their
for
observed their
their
to be
selectivity
selectivity
selectivity
much less
and
and
and
potent,
of
of calothrixin B
antiproliferative
calothrixin
antiproliferative B activity,
(Table 5)
activity,
(Table 5) in 2007.
using
in
using 2007.
an MTT
an Theassay,
MTT
The compounds
assay, were
against were
compounds
against evaluated
three evaluated
three for lines:
different cell
cell
for their human
lines:
their selectivity
human and
cervical
selectivity and
antiproliferative
antiproliferative
with activity,
activity,
N-methylcalothrixin using
using
B an the
an
being MTT
MTT assay,
assay,
least at 5 against
against
µM three different
three
compared different
different
to cell
cell
calothrixin lines:
lines:
B at 1 human
human
µM
cervical
(Tablecervical
cervical
6).
antiproliferative
cancer (HeLa)
(HeLa) cells,
antiproliferative
cancer activity,
cells,
activity,
murine using
murineusing an
P388 an MTT assay,
macrophage
MTT assay, against
cancer three
cells,three
against different
and simian
simian cell lines:
lines: human
non-cancerous
different cell human cervical
CV-1 cells.
cells.
cervical
cancer (HeLa)
cancer (HeLa) cells,
cells, murine P388
murine P388 macrophage
P388 macrophage
macrophage
cancer
cancer
cancer
cells,
cells, and
cells, and simian
and simian non-cancerous
non-cancerous
non-cancerous
CV-1
CV-1 cells.
CV-1 cells.
cancer (HeLa) cells, murine P388 macrophage cancer cells, and simian non-cancerous
cancer (HeLa) cells, murine P388 macrophage cancer cells, and simian non-cancerous CV-1 cells. CV-1 cells.
Table 5. Antiproliferative activity of calothrixin B and analogs in MTT assay against HeLa cells, P388
Table 5.
Table 5. Antiproliferative
Antiproliferative activity
activity of
of calothrixin
calothrixin B
B and
and analogs
analogs in
in MTT
MTT assay
assay against
against HeLa
HeLa cells,
cells, P388
P388
Table 5.and
cells,5.
Table 5. Antiproliferative
CV-1 cells.
Antiproliferative activity of
activity of calothrixin
calothrixin B
B and
and analogs
analogs in
in MTT
MTT assay
assay against
against HeLa
HeLa cells,
cells, P388
P388
Table
cells, and
Table and Antiproliferative
CV-1 cells.
cells.
5. Antiproliferative activity
activity of calothrixin
of calothrixin B
B and
and analogs
analogs in
in MTT
MTT assay
assay against
against HeLa
HeLa cells,
cells, P388
P388
cells,
cells, and CV-1
CV-1 cells.
cells, and
cells, and CV-1
CV-1 cells.
cells.
cells, and CV-1 cells.
EC
EC 50 (µM)
EC5050 (µM)
(µM) EC
EC 50(µM)
EC5050 (µM)
(µM) EC50
EC (µM)
(µM)
50 (µM)
Compound
Compound
Compound Structure
Structure
Structure EC50
EC
HeLa (µM)
50 (µM) EC50
EC (µM)
50 (µM) EC50
EC (µM)
50 (µM)
50 Cells
Cells P388 50Cells CV-1 Cells
EC (µM) EC (µM) EC 50 (µM)
Compound
Compound Structure
Structure HeLa
EC 50
(µM)
HeLa P388
EC 50 Cells CV-1
(µM) EC 50 Cells
50 (µM)
Compound
Compound Structure
Structure
50
HeLa Cells
HeLa Cells
Cells
50 P388
P388 Cells
P388 Cells
Cells
50 CV-1
CV-1 Cells
CV-1 Cells
Cells
HeLa
HeLa Cells
Cells P388
P388 Cells
Cells CV-1
CV-1 Cells
Cells
Calothrixin BB
Calothrixin
Calothrixin B 0.25˘±
0.25
0.25 ±0.05
0.05 9˘±
99 ±222 2.4˘±
2.4 ± 0.7
0.7
Calothrixin
Calothrixin B
B 0.25 ± 0.05
0.25 ± 0.05
0.05 99 ±
± 22
±
2.4 ± 0.7
2.4 ±
2.4 0.7
0.7
Calothrixin B
Calothrixin B 0.25 ± 0.05
0.25 ± 0.05 9 ± 22
9 2.4 ± 0.7
2.4 ± 0.7

Indolophenanthrene-7,13-dione
Indolophenanthrene-7,13-dione
Indolophenanthrene-7,13-dione 1.5˘±
1.5
1.5 ± 0.3
0.3 >50
>50 >50
>50
Indolophenanthrene-7,13-dione
Indolophenanthrene-7,13-dione 1.5 ±0.3
1.5 ± 0.3
± 0.3
0.3 >50
>50
>50 >50
>50
>50
Indolophenanthrene-7,13-dione
Indolophenanthrene-7,13-dione 1.5
1.5 ± 0.3 >50
>50 >50
>50

Benzocarbzoledione
Benzocarbzoledione 1.8 ± 0.1
1.8 >50 >50
Benzocarbzoledione
Benzocarbzoledione
Benzocarbzoledione 1.8˘±±
1.8
1.8
0.1
±0.1
0.1
0.1
>50
>50
>50
>50
>50
>50
>50
>50
Benzocarbzoledione
Benzocarbzoledione 1.8 ±
1.8 ± 0.1
0.1 >50
>50 >50
>50

Carbazole-1,4-dione
Carbazole-1,4-dione >50
>50 >50
>50 >50
>50
Carbazole-1,4-dione
Carbazole-1,4-dione
Carbazole-1,4-dione >50
>50
>50 >50
>50
>50 >50
>50
>50
Carbazole-1,4-dione
Carbazole-1,4-dione >50
>50 >50
>50 >50
>50

Murrayaquinone
Murrayaquinone 13 ±
13 ±1 2.3 ±
± 0.3 10 ±
±2
Murrayaquinone
Murrayaquinone 13 ± 11
13˘±
±1
2.3
2.3 ± 0.3
2.3˘± 0.3
± 0.3
0.3
10
10 ± 22
10˘±
±2
Murrayaquinone
Murrayaquinone
Murrayaquinone 13
13
13 ±111 2.3
2.3
2.3 ±0.3
0.3 10
10
10 ± 222

2-Methylcarbazoledione
2-Methylcarbazoledione 777 ±
± 11 1.0 ±
1.0 ± 0.1
0.1 1.7 ±
1.7 ± 0.4
0.4
2-Methylcarbazoledione
2-Methylcarbazoledione 7±± 11 1.0 ±
1.0 ± 0.1
0.1 1.7 ±
1.7 ± 0.4
0.4
2-Methylcarbazoledione
2-Methylcarbazoledione
2-Methylcarbazoledione 7˘±
77 ±111 1.0˘±
1.0
1.0 ±0.1
0.1
0.1 1.7˘±
1.7
1.7 ± 0.4
0.4
0.4

Isoquinoline-5,8-dione
Isoquinoline-5,8-dione 12 ±
12 ± 11 999 ±
± 22 >50
>50
Isoquinoline-5,8-dione
Isoquinoline-5,8-dione 12 ±
12 ± 11 9±± 22
± >50
>50
Isoquinoline-5,8-dione
Isoquinoline-5,8-dione
Isoquinoline-5,8-dione 12˘±111
12
12 ± 9
99˘±22 2 >50
>50
>50

In this
In this study,
study, calothrixin
calothrixin B B was
was found
found to to be
be the
the most
most potent
potent against
against HeLa
HeLa cells
cells (0.25
(0.25 µ µ M)
M)
In this
In this study,
study, calothrixin
calothrixin B B was
was found
found to to be
be the
the most
most potent
potent against
against HeLa
HeLa cells
cells (0.25
(0.25 µ µ M)
M)
In
followed this
In this
followed by study,
by study, calothrixin B was
indolophenanthrene-7,13-dione found
calothrixin B was foundanalog
indolophenanthrene-7,13-dione to be
analog the most
(1.5 most
to be the
(1.5 µ M),
µ potent
M), indicating
indicatingagainst HeLa
the importance
potent against
the importance cells
HeLa cellsof (0.25
of(0.25 µ
nitrogenM)
µ M)
nitrogen
followed
followed Theby indolophenanthrene-7,13-dione
byeffect
indolophenanthrene-7,13-dione
on the cell cycle was studiedanalog analog
analog
for these(1.5
(1.5 µ M),
µ M), indicating
M), indicating
compounds indicating the
the
using athe importance
importance
mitotic inhibitor of nitrogen
ofnocodazole,
nitrogen
followed
in ring
ring D.
followed
in by
D.by indolophenanthrene-7,13-dione
The activity was
was further
further decreased
indolophenanthrene-7,13-dione
The activity decreased analog
with (1.5
with(1.5
theµ
the µdeletion of ring
M), indicating
deletion of ringthe importance
E importance
E of this
of this analog of
analog
of nitrogen
to form
form
nitrogen
to
inwhich
in ring D.
ring D. The
The
blocks activity
activity
re-entry was
was
of further
further
cells into decreased
decreased
the G with
with
phase. the
the deletion
deletion
Calothrixin B of
of
was ring
ring E
E
observed of
of this
this
to analog
analog
arrest the to
to form
form
G1form
phase
in
in ring
ring D.
D. The
benzoncarbzoledione
The
benzoncarbzoledione activity
activity was
(1.8
was
(1.8 µµ further
M). The
further
M). The decreased
rest of
decreased
rest of the with
1the analogs
with
analogs the
the deletion
showed
deletion
showed of
of ring
moderate
ring
moderate E
E of
of this
activity
this
activity analog
ranging
analog
ranging to
from
to
from 7 to
form
benzoncarbzoledione
benzoncarbzoledione
at 0.1 µM (1.8 µ
(1.8
concentrations, µ M).
M). The rest
The
whereas rest of the
of the analogs
calothrixin analogs
A and showed moderate
showed moderate activity
N-methylcalothrixn activity
B showedranging
ranging
no from 777atto
from
effects tothe
to
benzoncarbzoledione
13 µµ M,
M, while
while the
benzoncarbzoledione
13 (1.8 µ
(1.8 µ M).
M). The
the carbazole-1,4-dioneThe rest
carbazole-1,4-dione restanalog
of the
analog
of the analogs
analogs
was found
was showed
found
showed to be
to moderate
bemoderate activity
inactive.activity
inactive. ranging
The results
The results
rangingwere
werefrom
fromnot77 as
not to
as
to
13 µ
13same M,
µ M, while the
M, concentration.
while the carbazole-1,4-dione
the carbazole-1,4-dione
carbazole-1,4-dione analog
analog was
At higher concentrations, was found
wascalothrixin
found to to
to A be
beandinactive.
inactive. The results
The results were
results were
N-methylcalothrixn were
B lednot as
nottoas
as
cell
13
13 µ
consistent
µ while
for the
M, while
consistent for the P388
theP388 cell line,
line, where
carbazole-1,4-dione
cell analog
where murrayaquinone
murrayaquinone
analog found
was found (2.3to
(2.3 µ be
µ M)
M)
be inactive.
and
inactive.
and The
2-methylcarbazoledione
The results were
2-methylcarbazoledione not
(1 µ
not
(1 µ M)
M)
as
consistent
consistent for the
for
accumulation theinP388
P388
S andcell
cell
G line,
line,
/M where
where
phase. murrayaquinone
murrayaquinone
Compared to (2.3
(2.3
camptothecin, µ
µ M)
M) and
and 2-methylcarbazoledione
2-methylcarbazoledione
these effects were found to be(1
(1 µ
µ M)
M)
readily
consistent
were found
consistent
were for
foundfor the
tothe P388 cell
be more
more
P388 cell2line,
active
line, where murrayaquinone
compared
where murrayaquinone
to calothrixin
calothrixin B B (9(2.3
(9
(2.3 µ M)
µ M)
M)
µ M) and
orand 2-methylcarbazoledione
other analogs, whereas
whereas calothrixin
2-methylcarbazoledione (1 µ
µ M)
calothrixin
(1 M)
were
were found to
found
reversible. to
be
toCalothrixins
be more
be more active
active
active
were
compared
compared
compared
to
to calothrixin
to
also evaluatedcalothrixin
for their B (9 µµ
B activity
(9 µ M)
M) or
or
or
other
other
other
against
analogs,
analogs, whereas
analogs, whereas
topoisomerase calothrixin
calothrixin
I. The effects of
were
B and
were
B found
andfound to be more active
2-methylcarbazoledione compared
to be more active compared
2-methylcarbazoledione showed to
showed similar calothrixin
similar B (9
activityBwith
to calothrixin
activity with µ
(9 µ M)M) or
2.4 or
2.4 µµM other
Mother analogs,
and analogs,
1.7 µ
µ M, whereas
M, respectively,
respectively,calothrixin
against
whereas calothrixin
B and
B and 2-methylcarbazoledione
2-methylcarbazoledione
calothrixins were studied keeping showed
showed similar
similar
camptothecin activity
activity with
with
as a positive 2.4 µ M and
2.4control,
µM and
1.7
andwhich
1.7 µ
1.7 µ M,
M, respectively,
respectively,
is arespectively,
against
against
against
known topoisomerase
B and 2-methylcarbazoledione showed similar activity with 2.4 µ M and 1.7
B and 2-methylcarbazoledione showed similar activity with 2.4 µ M and 1.7 µ M, respectively, againstµ M, against
I poison [60]. It was observed that calothrixins A, B, and N-methylcalothrixin B were capable of
stabilizing the covalent topoisomerase I/DNA complex at 18%, 13%, and 11%, respectively, compared
to 100% at 5 µM concentration of camptothecin.
Calothrixins and their analogs were observed to be affecting different stages of cell cycle in
a reversible manner, leading to low micromolar range cytotoxicities. Thus, calothrixins have shown
a wide array of activity including antimalarial, anticancer, and antibacterial. The modes of action of
calothrixins’ bioactivities have not been fully deciphered yet.
action
action and
and the
the stage
stage ofof the
the cell
cell cycle
cycle that
that isis targeted
targeted by
by the
the calothrixins.
calothrixins. Calothrixin
Calothrixin A A and
and BB and
and the
the
N-methylated
N-methylated derivative were synthesized and tested against CEM leukemia cells to measure their
N-methylated derivative
derivative were were synthesized
synthesized and and tested
tested against
against CEM
CEM leukemia
leukemia cells
cells to
to measure
measure their
their
cytotoxicity using
cytotoxicity using an MTTMTT assay. The The activity of of calothrixin analogs
analogs were compared
compared to camptothecin,
camptothecin,
cytotoxicity usingan an MTTassay.
assay. Theactivity
activity ofcalothrixin
calothrixin analogswere were comparedto to camptothecin,
which
which is known to cause irreversible DNA damage during the S phase. Delay in S phase due to
to DNA
whichisisknown
knownto tocause
causeirreversible
irreversibleDNA DNAdamagedamageduring
duringthe theSSphase.
phase.Delay
DelayininSSphase
phasedue due toDNA
DNA
damage
damage is associated with a block in replication. The IC 50 value for calothrixin A was found to be
damage is associated with a block in replication. The IC50 value for calothrixin A was found to be
is associated with a block in replication. The IC 50
50 value for calothrixin A was found to be
five-fold
five-fold higher
Drugshigher than
2016, 14,than campothecin and
17 campothecin and the other
other two analogs
analogs were observed
observed to be be much lessless potent, 16 of 21
Mar.
five-fold higher than campothecin and the the other two two analogs were
were observed to to be much
much less potent,
potent,
with N-methylcalothrixin
with N-methylcalothrixin B being the the least at at 5 µ M compared
compared to calothrixin
calothrixin B at 11 µµ M (Table
(Table 6).
with N-methylcalothrixinBB being being the least
least at 55 µµM M compared to to calothrixinBBat at 1 µMM (Table6). 6).
Table 6.6.Cytotoxicity
Table6. Cytotoxicityeffects against
effects CEM
against CEMleukemia cell line
leukemia cell and
linetopoisomerase I inhibition.
and topoisomerase I inhibition.
Table
Table 6.Cytotoxicity
Cytotoxicity effects
effects against
against CEM
CEM leukemia
leukemia cell
cellline
lineand
andtopoisomerase
topoisomerase IIinhibition.
inhibition.
% Topo I DNA % NaCl Induced
IC50
IC 50 (µM) in CEM
(µM) in CEM %%%Topo
Topo
TopoIIDNA
I DNA %
DNA %NaCl
NaCl Induced
Induced
% NaCl Induced
Compound
Compound Structure
Structure IC
IC5050(µM)
50 (µM)in
inCEM
CEM Cleavage Reversibility
Compound
Compound Structure
Structure Leukemia
Leukemia Cells
Cells Cleavage
Cleavage
Cleavage at 5 µM Reversibility
Reversibility
Reversibility at 5 µM
Leukemia
LeukemiaCellsCells at 5 µM at 5 µM
at
at55µM
µM at
at55µM
µM

Camptothecin
Camptothecin 0.04 ˘
0.04 ± 0.01
0.01 100
100 100 100
Camptothecin
Camptothecin 0.04
0.04±±0.01
0.01 100
100 100
100

Calothrixin
CalothrixinAA
A 0.20 ˘
0.20 ± 0.02
0.02 1818 17 17
Calothrixin
Calothrixin A 0.20
0.20±±0.02
0.02 18
18 17
17

Calothrixin B 1.05 ± 0.30 13 16

Calothrixin
CalothrixinBB 1.05
1.05 ±±0.30
1.05˘ 0.30 13
1313 16
16 16

N-methycalothrixin B 5.13 ± 0.72 11 13

N-methycalothrixin
N-methycalothrixinBB 5.13 ±±0.72
5.13˘
5.13 0.72 11
1111 13
13 13

The effect
The effect on the the cell cycle
cycle was studiedstudied for these these compounds using using a mitotic inhibitorinhibitor
The effect on on the cellcell cycle was was studied for for these compounds
compounds using aa mitotic mitotic inhibitor
4. Conclusions
nocodazole,
nocodazole, which blocks
which blocks re-entry
re-entry of
of cells
cells into
into the
the G
G111 phase.
phase. Calothrixin
Calothrixin B
B was
was observed
observed to
to arrest
arrest the
the
nocodazole, which blocks re-entry of cells into the G1 phase. Calothrixin B was observed to arrest the
G111 phase
G phase at at 0.1
0.1 μM
μM concentrations,
concentrations, whereas whereas calothrixin
calothrixin A A and and N-methylcalothrixn
N-methylcalothrixn B B showed
showed no no
G1 phase
Whileatthe 0.1 medicinal
μM concentrations, whereas potential
and biosynthetic calothrixinofAterrestrial
and N-methylcalothrixn
plants and microbes B showed no well
is fairly
effects
effects at the same concentration. At higher concentrations, calothrixin A and N-methylcalothrixn B
effects at
studied, the
the same
same concentration.
atcomparatively concentration. At
At higher
little is known higher concentrations,
concentrations,
regarding the chemistrycalothrixin
and A
calothrixin A and
and N-methylcalothrixn
N-methylcalothrixn
biological activity of organisms BB in
led
led to
to cell
cell accumulation
accumulation in
in S
S and
and G
G 2/M phase. Compared to camptothecin, these effects were found
/M phase. Compared to camptothecin, these effects were found
led
the to cell
marine accumulation in S and G22/M phase. Compared to camptothecin, these effects were found
2
to be
to be readilyenvironment. A unique group
reversible. Calothrixins
Calothrixins of oxygenic
were also
also evaluatedphotosynthetic
for their
their activitybacteria
againstknown as cyanobacteria
topoisomerase I.
to be readily
populatereadily reversible.
reversible.
diverse habitatsCalothrixins
throughout were
were
the also evaluated
evaluated
world. Their for
for their activity
potential activity
as a against
goodagainst topoisomerase
sourcetopoisomerase
of new I.I.
therapeutic
The
The effects
effects of
of calothrixins
calothrixins were
were studied
studied keeping
keeping camptothecin
camptothecin as
as a positive
aa positive control,
control, which
which isis aaa
is
The
lead effects
compounds of calothrixins
has been were
realized studied keeping camptothecin as positive control, which
known
known topoisomerase I poison Itduring
[60]. It the last few
was observed
observed that decades.
calothrixins Calothrixins,
A, B, which are cyanobacterial
B, and N-methylcalothrixin
N-methylcalothrixin
known topoisomerase
topoisomerase II poison
poison [60].[60]. It was
was observed that that calothrixins
calothrixins A, A, B, and
and N-methylcalothrixin
metabolites,
B were
were capable have demonstrated
of stabilizing a diversetopoisomerase
stabilizing the covalent
covalent range of bioactivities that include
I/DNA complex
complex at 18%,
18%,antimalarial,
13%, and anticancer,
and 11%,
BB were capable
capable of of stabilizing the the covalent topoisomerase
topoisomerase I/DNA I/DNA complex at at 18%, 13%,
13%, and 11%, 11%,
and antibacterial
respectively,
respectively,
properties.
compared to 100% They
at 5 µ have
M been observed
concentration of to target
camptothecin. various aspects of RNA synthesis in
respectively,compared
compared to to100%
100%at at55 µµMMconcentration
concentration of of camptothecin.
camptothecin.
Calothrixins
bacteria. and their
Further investigation
Calothrixins analogs were
of the exactobserved to
mechanism be affecting different
for their bioactivity stages of cell
is stillcell cycle in
requiredinfor a many
Calothrixins and and their
their analogs
analogs werewere observed
observed to to be
be affecting
affecting different
different stages
stages ofof cell cycle
cycle in aa
reversible
analogs,
reversible manner,
which
manner,willleading
be
leading to
beneficial
to low
low micromolar
for the
micromolar range
ongoing
range cytotoxicities.
development
cytotoxicities. Thus,
and
Thus, calothrixins
lead optimization.
calothrixins have
have shown
Several
shown aa
research
reversible manner, leading to low micromolar range cytotoxicities. Thus, calothrixins have shown a
wide array
groups
wide array
have of developed
of activity including
including
syntheticantimalarial, anticancer,
routes toanticancer,
obtain these andnatural
antibacterial. The This
products. modes of action
review action of
emphasized
wide array of activity
activity including antimalarial,
antimalarial, anticancer, and
and antibacterial.
antibacterial. The
The modes
modes of
of action of
of
calothrixins’
the bioactivities
syntheticbioactivities
calothrixins’ have
progress accomplished
have not been fully
withindeciphered
the pastyet.yet.
six years in developing new synthetic routes.
calothrixins’ bioactivities have not
notbeen
been fully
fully deciphered
deciphered yet.
Since 2009, 11 novel syntheses have been published to improve upon the efficiency in the production
4. Conclusions
4. Conclusions
4. calothrixins.
of Conclusions The number of steps in the various synthetic strategies ranges from 2 to 14, while the
overall yield to construct calothrixin B ranges from 8% to 50%. Thus, significant progress has been
made in the last decade in attaining more efficient synthesis of calothrixins, which may aid to establish
calothrixins as a potential anti-cancer candidate in the future.

Acknowledgments: We thank the current and former members of Velu lab for their contributions to the research
projects that were cited in this review. Sadanandan E. Velu was supported by a Breast Spore pilot grant and
a Collaborative Programmatic Development grant from University of Alabama at Birmingham Comprehensive
Cancer Center. He was also supported by grant number 1UL1RR025777 from the NIH National Center for
Research Resources.
Author Contributions: Sadanandan E. Velu conceived the idea, Su Xu, Bhavitavya Nijampatnam and Shilpa Dutta
wrote the manuscript.
Conflicts of Interest: The authors declare no conflicts of interest.

Abbreviations
AcCl Acetyl chloride
Ac2 O Acetic anhydride
AcOEt Ethyl acetate
AcOH Acetic acid
AIBN Azobisisobutyronitrile
Mar. Drugs 2016, 14, 17 17 of 21

AlCl3 Aluminum chloride

BEt3 Triethylborane or triethylboron
BF3 .OEt2 Boron trifluoride diethyl etherate
CAN Ceric ammonium nitrate
CCl4 Carbon tetrachloride
CH2 Cl2 Dichloromethane
CH3 CN Acetonitrile
COSY Correlation spectroscopy
c-PC c-Phycocyanin
CSA Camphorsulfonic acid
CuI Copper(I) iodide
(CuOTf)2 .toluene Trifluoromethanesulfonate toluene complex
DMAP 4-Dimethylaminopyridine
DMF Dimethylformamide
DMP Dess-Martin periodinane
DMSO Dimethyl sulfoxide
DoM Directed o-metalation
DTT Dithiothreitol
Et2 NH Diethylamine
Et3 SiH Triethylsilane
FeCl3 Iron(III) chloride
HCO2 H Formic acid
HDACs Histone deacetylases
HIV Human immunodeficiency virus
H2 O2 Hydrogen peroxide
K2 CO3 Potassium carbonate
LDA Lithium diisopropylamide
LiTMP Lithium tetramethylpiperidide
LTA Lead tetracetate
MeOH Methanol
Mn(OAc)3 Manganese triacetate
MOM Methoxymethyl protecting group
MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
NaCNBH3 Sodium cyanoborohydride
NaH Sodium hydride
NaOH Sodium hydroxide
NaOMe Sodium methoxide
NBS N-bromosuccinimide
n-BuLi n-Butyllithium
NH2 NH2 .H2 O Hydrazine hydrate solution
Pb(OAc)4 Lead(IV) acetate
PCC Pyridinium chlorochromate
Pd/C Palladium on carbon
PdCl2 (PPh3 )2 Bis(triphenylphosphine)palladium(II) dichloride
Pd2 (dba)3 Tris(dibenzylideneacetone)dipalladium(0)
Pd(dppf)Cl2 [1,11 -Bis(diphenylphosphino)ferrocene] dichloropalladium(II)
Pd(OAc)2 Palladium acetate
Mar. Drugs 2016, 14, 17 18 of 21

Pd(TFA)2 palladium(II) trifluoroacetate

PhCN Benzonitrile
(PhSe)2 Diphenyl diselenide
PMB p-Methoxybenzyl
PMBCl p-Methoxybenzyl chloride
P(o-tol)3 Tri(o-tolyl)phosphine
POCl3 Phosphoryl chloride
PPh3 Triphenylphosphine
p-TSA p-Toluenesulfonic acid
PTT Polytrimethylene terephthalate
rac-BINAP Racemic 2,21 -bis(diphenylphosphino)-1,11 -binaphthalene
ROS Reactive oxygen species
TBAD Bis(tetrabutylammonium) dichromate
TBHP Tert-butyl hydroperoxide
Tf2 O Trifluoromethanesulfonic anhydride
THF Tetrahydrofuran
THP Tetrahydropyran
TMG 1,1,3,3-Tetramethylguanidine
TMP 2,2,6,6-Tetramethylpiperidine
TMSI Trimethylsilyl iodide

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