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

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

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

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inhibiting cells in 14, G2x/M phase. Cryptophycin 52, a chemical analog of cryptophycin 1, entered Mar. Drugs 2016, 3 of clinical 20 trials but produced only marginal activity [21]. cells in G2/M10 phase. Cryptophycin 52, a chemical that analog of cryptophycin entered trials butin the Dolastatin is cyanobacterial metabolite was synthesized1,by Pettitclinical 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 and toxicity of dolastatin 10 were investigated Phase II clinical in patients agent that affects microtubule assembly, which leads to cellindeath during the G2trials /M phase [24]. In with advanced prostate cancer but it was discontinued due to the development peripheral in 2005, the efficacy and toxicity of dolastatin 10 were investigated in Phase II of clinical trials inneuropathy patients 40% of theadvanced patients [25]. Many research have soughtdue aftertoSAR of the of synthetic analogs with prostate cancer but itgroups was discontinued the studies development peripheral of this compound, which are patients now classified as auristatins. Thishave group of compounds showed neuropathy in 40% of the [25]. Many research groups sought after SAR studies of the the most synthetic analogs of this compound, which are now classified as auristatins. This group amount of promise as antibody drug conjugates. For example, brentuximab vedotin (adcetris,of Seattle compounds the most amount of promise as antibody drug conjugates. For example, Genetics ) is nowshowed a FDA-approved drug against lymphoma. This compound is currently undergoing (adcetris, Seattle Genetics ) is now a FDA-approved drug againstand lymphoma. phasebrentuximab 3 studies tovedotin study its effect on cutaneous T-cell lymphoma, B-cell lymphomas, 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 thiazole, as wellasaswell a 3-hydroxy-7-mercaptohept-4-enoic contains a methylthiazoline to a thiazole, as a 3-hydroxy-7-mercaptohept-4-enoic acid acid unit, and aunit, thioester, which had previously not been observed in marine cyanobacterial natural products. and a thioester, which had previously not been observed in marine cyanobacterial natural This products. compound has been shown to selectively transformed over non-transformed cells [28,29], This compound has been shown to target selectively target transformed over non-transformed through inhibition of class I histone deacetylases (HDACs) [30,31].(HDACs) Largazole’s interesting structure cellsthe [28,29], through the inhibition of class I histone deacetylases [30,31]. Largazole’s interesting activity structurehave andattracted biologicalstrong activity have from attracted strong interest from community, the syntheticwhich and biological interest the synthetic chemistry community, which to seeks to establish routes to largazole to therapeutic investigate its seekschemistry to establish synthetic routes largazole and tosynthetic investigate its potential as a and cancer [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 called calothrixins, with an emphasis on emphasis their synthesis andsynthesis bioactivities. the natural products called calothrixins, with an on their 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 by BRickards [34]. Briefly, lyophilized of Calothrix strains Calothrixins A and (Figure 2)etareal.two cyanobacterial metabolites cells that were first isolated from were Calothrix in 1999 by Rickards et al. [34]. Briefly, lyophilized cellsethyl of Calothrix were using extracted extracted with dimethyl sulfoxide (DMSO) and then with acetatestrains (AcOEt) Soxhlet with dimethyl sulfoxide (DMSO) and then with ethyl acetate (AcOEt) using Soxhlet extraction extraction conditions. These extracts were fractionalized using a combination of bioassays, differential conditions. These extracts were fractionalized a combination of bioassays, differential solubility, and chromatography, which yielded the using relatively insoluble calothrixin 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 1 H– 1 H Correlation Spectra (EIMS), 1 H, 13 C and 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 with an assembly of quinoline, quinone, and indole possess an unique indolo[3,2-j]phenanthridine framework with an assembly of quinoline, quinone, pharmacophores. Calothrixin B Calothrixin is generallyB known to beknown the neutral whileanalog calothrixin and indole pharmacophores. is generally to be analog the neutral 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 and the promisethe they hold as lead compounds for cancer drug discovery. These scaffolds attracted interests of organic chemists 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.

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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. Research Group

Year

Ring Closure

Velu et al.

2014

B

Mn(OAc)3 mediated oxidative free radical reaction

Ishikura et al.

2011 & 2012

C

Palladium catalyzed tandem cyclization/cross-coupling

Dethe et al.

2014

C

LTA mediated rearrangement of o-hydroxy aryl hydrazone

[39]

Nagarajan et al.

2014

C

Friedel-Crafts hydroxyalkylation and directed o-lithiation

[40]

Mal et al.

2014

C

The anionic annulation of MOM-protected furoindolone

[41]

Kusurkar et al.

2012

D

Two one pot reaction sequences: a nucleophilic substitution and reduction

[42]

Nagarajan et al.

2013

D

Pd catalyzed intramolecular cross-coupling reaction via C–H activation

[43]

Mohanakrishnan et al.

2014

D

Electrocyclization of 2-nitroarylvinyl-3-phenylsulfonylvinylindole

[44]

Kumar et al.

2014

D

Pd mediated intramolecular C-X/C-H cross coupling reactions

[45]

Hibino et al.

2012

D

Allene mediated electrocyclic reaction of the 6π-electron system

[46]

Mohanakrishnan et al.

2013

C&D

FeCl3 mediated domino reaction of enamines

[47]

Key Step

Reference [36] [37,38]

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 B) 17 on a phenanthridine dione (ring C, D, and E) via a novel oxidative free radical Mar. Drugs 2016, 14, 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 of the et existing reports of calothrixin synthesis utilizes the late-stage indoleutilizes construction strategy. by Chuang al. [48–50]. None of the existing reports of calothrixin synthesis the late-stage Through this methodology, calothrixin B was synthesized in seven steps, with an overall yield 19% indole construction strategy. Through this methodology, calothrixin B was synthesized in of seven starting from steps, 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 A of calothrixin calothrixinsyntheses syntheseshave havebeen beenreported reported where ring C closure used as the A number number of where thethe ring C closure waswas used as the last last step in the construction of calothrixin scaffold. This includes the two syntheses reported step in the construction of calothrixin scaffold. This includes the two syntheses reported by Ishikura etby al. Ishikura et al. in 2011 and 2012 [37,38], and the three contributions made by Dethe et al. [39], in 2011 and 2012 [37,38], and the three contributions made by Dethe et al. [39], Nagarajan et al. [40], Nagarajan [40], and Mal et et al.al. [41] in and 2014.Mal et al. [41] in 2014. 2.2.1. 2.2.1. Ishikura’s Ishikura’s Synthesis Synthesis Ishikura al.[37,38] [37,38]was was one of the groups that designed a synthetic to calothrixins Ishikura etetal. one of the firstfirst groups that designed a synthetic route toroute calothrixins in which in C last wasascyclized as shown in ongoing Schemestudies 2. In oftheir ongoing studies of ringwhich C was ring cyclized shown inlast Scheme 2. In their trialkyl(indol-2-yl)borates, trialkyl(indol-2-yl)borates, they previously found that indolylborates show high cross-coupling reactivity in they previously found that indolylborates show high reactivity in palladium-catalyzed palladium-catalyzed cross-coupling reactions, such carbonylative cross-coupling andreactions. tandem reactions, such as carbonylative cross-coupling and as tandem cyclization/cross-coupling cyclization/cross-coupling reactions. This newAapproach the synthesis of calothrixins A and B This new approach for the synthesis of calothrixins and B wasfor demonstrated through a palladium-catalyzed was demonstrated through palladium-catalyzed cross-coupling of 1-methoxyindolylborate cross-coupling reaction of a1-methoxyindolylborate (generated reaction in situ from 1-methoxyindole by (generated in situn-BuLi from 1-methoxyindole by treatment withcompound n-BuLi and ) withthe thecompound intermediate treatment with and BEt3 ) with the intermediate 13BEt to 3form 15. compound to form the compound 15. Additional novelty synthetic route is the strategic Additional 13 novelty of this synthetic route is the strategic useofofthis Cu(OTf) .toluene complex for the 2 use of Cu(OTf)2.toluene complex for the 6π-elecrocyclization of the intermediate (16) to 6π-elecrocyclization of the hexatriene intermediate (16) to cyclize thehexatriene ring C to form the compound cyclize the ring synthesis C to form yielded the compound 17. Ishikura’s yielded calothrixin nine steps 17. Ishikura’s calothrixin B in ninesynthesis steps with an overall yield Bofin9% starting with overall yield of 9% starting from 2-iodoaniline. from an 2-iodoaniline.

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Scheme 2. Ishikura’s synthesis of calothrixins.

2.2.2. Dethe’s Synthesis

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

Scheme 3. Dethe’s synthesis of calothrixin B.

2.2.3. Nagarajan’s Synthesis

Scheme 3. Dethe’s synthesis of calothrixin B.

Schemea 3. Dethe’sfor synthesis of calothrixin B. Nagarajan et al. [40] outlined strategy the synthesis of calothrixin B featuring a directed o-metalation reaction as a key step, as outlined in Scheme 4. The synthesis began with the coupling 2.2.3. Nagarajan’s Synthesis 2.2.3.ofNagarajan’s Synthesis the 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 Nagarajanreaction et al. [40] outlined a as strategy forinthe synthesis ofsynthesis calothrixin B featuring a directed o-metalation a key step, outlined Scheme with theprocess coupling intermediate productaswith Dess-Martin periodinane (DMP)4.inThe CH2Cl2/AcOH.began This two-step

o-metalation as a key step, as outlined in Schemeethyl 4. The synthesis began with the coupling of the reaction commercially available reagents, indole-2-carboxylate (20) and of thequinoline-3-carboxaldehyde commercially available reagents, ethyl indole-2-carboxylate (20)followed and quinoline-3-carboxaldehyde (26) in the presence of TMG in MeOH by the oxidation of the (26)intermediate in the presence of TMG in MeOH followed by the oxidation of2/AcOH. the intermediate product with product with Dess-Martin periodinane (DMP) in CH2Cl This two-step process Dess-Martin periodinane (DMP) in CH2 Cl2 /AcOH. This two-step process yielded compound 27,

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yielded compound Mar. Drugs 2016, 14, x 27, which was then cyclized by an intramolecular directed o-lithiation reaction 7 of 20 using was lithium tetramethylpiperidide (LiTMP) too-lithiation form calothrixin B in lithium 48% yield. This synthetic which then cyclized by an intramolecular directed reaction using tetramethylpiperidide yieldedtocompound which was then by an directed o-lithiation reaction methodology offers a27, three-step route tocyclized calothrixin B intramolecular from commercially available reagents in an (LiTMP) form calothrixin B in 48% yield. This synthetic methodology offers a three-step route to using lithium tetramethylpiperidide (LiTMP) to form calothrixin B in 48% yield. This synthetic overall yield 38%. calothrixin B of from commercially available reagents in an overall yield of 38%. 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 This reagent was of prepared in three steps fromindololactone commercially et al. [41] reported5. a two-step synthesis calothrixin B fromsimple the N-protected as outlined in Scheme 5. This reagent was prepared in three simple steps from commercially available available Treatment compound 28 with 4-bromoquinoline (29) in the (28) as α-acetobutyrolactone outlined in Scheme 5. [51]. This reagent wasofprepared in three simple steps from commercially α-acetobutyrolactone [51]. Treatment of compound 28 with 4-bromoquinoline (29) in the presence of presence of α-acetobutyrolactone lithium diisopropylamide (LDA) in tetrahydrofuran (THF) at −78 °C afforded available [51]. Treatment of compound 28 with 4-bromoquinoline (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 of methoxymethylene presence of lithium diisopropylamide (LDA) in tetrahydrofuran (THF) at −78 °C afforded an of compounds 30a and 30b. However, after the removal of methoxymethylene (MOM) group using (MOM) groupmixture using HCl, it gave a mixture calothrixin B after (1b) and its regioisomer (31b) that could inseparable of compounds 30a andof30b. However, the removal of methoxymethylene HCl, it gave a mixture of calothrixin B (1b) and its regioisomer (31b) could be(31b) easily separated group using it gave mixture calothrixin B (1b)This and itsthat regioisomer that could be (MOM) easily separated in HCl, the ratio of a40% and of 6%, respectively. synthetic methodology afforded in the ratio of 40% and 6%, respectively. This synthetic methodology afforded calothrixin B with an be easily Bseparated in the ratio This synthetic methodology afforded calothrixin with an overall yieldofof40% 47%and for 6%, tworespectively. steps starting from compound 28. overall yield of 47% an foroverall two steps starting compound 28. from compound 28. calothrixin B with yield of 47% from for two steps starting

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

2.3. Ring DD Closure of the the Indolo[3,2-j]Phenanthridine Indolo[3,2-j]Phenanthridine Framework Ring Closureasasthe theLast LastStep Stepininthe theConstruction Construction of 2.3.2.3. Ring D Closure as the Last Step in the Construction of the Indolo[3,2-j]PhenanthridineFramework Framework Another strategy involves cyclizing cyclizingring ringDDasasthe thelast laststep step Another strategyfor forthe thecalothrixin calothrixin synthesis synthesis involves in in thethe Another strategy for the calothrixin synthesis involves cyclizing ring D as the last step in the construction ofofcalothrixin havebeen beenmade madetotothis this category construction calothrixinscaffold. scaffold.AA number number of contributions contributions have category of of construction of calothrixin scaffold. A number of contributions have been made to this category of syntheses. These includefour fourindependent independent publications publications by Nagarajan et et syntheses. These include by Kusurkar Kusurkaretetal.al.inin2012 2012[42], [42], Nagarajan 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 et al. in 2012 al. in 2013 [43], Mohanakrishnan et al. in 2014 [44], Kumar et al. in 2014 [45], and Hibino et al. in 2012 in 2013 [43], Mohanakrishnan et al. in 2014 [44], Kumar et al. in 2014 [45], and Hibino et al. in 2012 [46]. [46]. [46]. 2.3.1. Kusurkar’s Synthesis 2.3.1. Kusurkar’s Synthesis 2.3.1. Kusurkar’s Synthesis Kusurkar et al. [42] have published a synthesis of calothrixinsstarting startingfrom from4-hydroxy 4-hydroxy carbazole Kusurkar et al. [42]have havepublished publishedaasynthesis synthesis of of calothrixins carbazole Kusurkar et al. [42] calothrixins starting from 4-hydroxy carbazole as outlined in Scheme 6. The6.novelty of theirof synthetic route is the unprecedented use of DMF-NaOMe as outlined in Scheme The novelty their synthetic route is the unprecedented use as aoutlined in Scheme 6. The the novelty of their synthetic route isa high-yielding the unprecedented useof of as reagent for thea reduction aldehyde and thegroup use of Pd-catalyzed DMF-NaOMe as reagent forofthe reduction of group the aldehyde and the use of a high-yielding DMF-NaOMe as atoreagent for the the ring reduction of the aldehydesystem. group and the use ofthe a high-yielding coupling reaction construct D of the For system. example, treatment Pd-catalyzed coupling reaction to construct the pentacyclic ring D of the pentacyclic For example, the of Pd-catalyzed coupling reaction to construct the ring D of the pentacyclic system. For example, the ˝ compound NaOMe dryNaOMe dimethyl (DMF) and CuI at 120 resulted treatment35 of with compound 35in with in formamide dry dimethyl formamide (DMF) andCCuI at 120in°Cthe treatment of compound 35 with NaOMe in dry dimethyl formamide (DMF) and CuI at 120 °C substitution bothsubstitution bromine and groups methoxy groups withmethoxy concomitant reduction resulted inofthe of benzyloxy both bromine andwith benzyloxy groups with groups with resulted in the substitution of both bromine and benzyloxy groups with methoxy groups with of the aldehydereduction resultingofinthe thealdehyde formation of the compound 36. Pd-catalyzed cyclization of ring D of concomitant resulting in the formation of the compound 36. Pd-catalyzed concomitant reduction of the aldehyde resulting in the formation of the compound 36. Pd-catalyzed ring D of the scaffold form compound is another key step inoverall this synthesis. thecyclization scaffold toofform compound 39 istoanother novel key39step in thisnovel synthesis. The yield for cyclization of ring D the scaffold form 39 is another step in this synthesis. The overall yield forof synthesis ofcompound calothrixin B was 25% fornovel nine key steps. Kusurkar’s synthesis ofKusurkar’s calothrixin Btowas 25% for nine steps. The overall yield for Kusurkar’s synthesis of calothrixin B was 25% for nine steps.

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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 addition toSynthesis developing a synthetic route for calothrixins that involved the closure of ring C in 2.3.2.In Nagarajan’s addition to developing a synthetic route for calothrixins that the of ring theInlast step, Nagarajan et al. [43] has also accomplished a synthesis in involved which ring D closure was closed last C In addition to developing a synthetic route for calothrixins that involved the closure of ring C in in the lastpreparation step, Nagarajan et al. [43] has also a synthesis in which ring D necessary was closed in the of the five-membered ringaccomplished system, followed by the installation of the 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 five-membered ring system, followed the installation necessary to produce B as shown in Scheme 7. Thisbysynthesis featuredofathe prominent in the preparation of the five-membered ring system, followed by the installation of the necessary Pd-catalyzed cross-coupling reaction via C-H activation using an appropriately substitutions to intramolecular produce calothrixin B as shown in Scheme 7. This synthesis featured a prominent substitutions to produce calothrixin B as shown in Scheme 7. This synthesis featured a prominent substituted intramolecular carbazole derivative to construct the indolophenanthridine core ring system of Pd-catalyzed cross-coupling reaction via C-H activation using an appropriately Pd-catalyzed intramolecular cross-coupling reaction via C-H activation using an appropriately calothrixins. This is the first reported synthesis of calothrixin pentacycles without any protection on substituted carbazole derivative to construct the indolophenanthridine core ring system calothrixins. substituted carbazole derivative to construct the indolophenanthridine core ringofsystem of the indole N atom. The overall yield for calothrixin B in this Nagarajan’s synthesis is 35% for five This is the first This reported of calothrixin pentacycles anywithout protection the indole calothrixins. is thesynthesis first reported synthesis of calothrixinwithout pentacycles anyon protection on N stepsThe starting from 4-methoxycarbazole. atom. overall yield foroverall calothrixin B incalothrixin this Nagarajan’s is 35% for five stepsfor starting the indole N atom. The yield for B in thissynthesis Nagarajan’s synthesis is 35% 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. et al. have reported a linear synthesis of calothrixin B using the thermal electro 2.3.3.Mohanakrishnan Mohanakrishnan’s Synthesis 2.3.3. Mohanakrishnan’s Synthesis cyclization 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 step, the 2,3-divinylidole intermediate generated from compound 52 thermal by treatment Mohanakrishnan et al. have reported a linear synthesis of using the electro cyclization of 2-nitroarylvinyl-3-phenylsulfonylvinylindole as calothrixin the key stepBas outlined in Scheme 8 with Me2SO4 was subjected to thermal cyclization in refluxing xylenes to afford the carbazole cyclization of 2-nitroarylvinyl-3-phenylsulfonylvinylindole as the key stepcompound as outlined52inby Scheme 8 [44]. [44]. In this key step, the 2,3-divinylidole intermediate generated from treatment derivative 53. Mohanakrishnan’s total synthesis thus afforded calothrixin B from 2-methylindole in In with this key the 2,3-divinylidole intermediate generated fromxylenes compound 52 by the treatment with Me2step, SO4 was subjected to thermal cyclization in refluxing to afford carbazole 14 steps with an overall yield of about 8%. Mohanakrishnan’s total synthesis thus afforded calothrixin from 2-methylindole in53. Mederivative subjected to thermal cyclization in refluxing xylenes to afford B the carbazole derivative 2 SO4 was 53. 14 steps with an overall yield of about 8%. Mohanakrishnan’s total synthesis thus afforded calothrixin B from 2-methylindole in 14 steps with an overall yield of about 8%.

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Scheme 8. Mohanakrishnan’s synthesis of calothrixins A and B. 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 2.3.4. Kumar’s Synthesis Kumar et al.Synthesis [45] reported the total synthesis of calothrixin B via multiple palladium-mediated 2.3.4. Kumar’s Kumar et al. intramolecular [45] reported the total synthesis of outlined calothrixin B via multiple palladium-mediated C-X/C-H cross coupling reactions as in Scheme 9. The salient feature of this C-X/C-H cross intramolecular coupling reactions as outlined in Scheme 9. The salient Kumar et al. [45] reported the total synthesis of calothrixin B via multiple palladium-mediated synthesis is the two intramolecular C-X/C-H cross coupling reactions of the intermediates 60feature to form of C-X/C-H cross intramolecular coupling reactions as outlined in Scheme 9. The salient feature of this 60 thiscompound synthesis is the two intramolecular C-X/C-H cross coupling reactions of the intermediates 61 and 63 to form the compound 64. The intermediate 60 was cyclized under synthesis is the two intramolecular C-X/C-H cross coupling reactions of the intermediates 60 to form to form compound 61 coupling and 63 toreaction form the compound The intermediate 60 was cyclized intramolecular cross condition using64. Pd(OAc) 2, PCy3, and JohnPhos to affordunder the compound cross 61 and 63 to form the condition compoundusing 64. The intermediate 60 was cyclized under the intramolecular coupling reaction Pd(OAc) , PCy , and JohnPhos to afford carbazole derivative 61. The cyclization of 63 was carried out using Pd(OAc) 2, PCy3, and K2CO3 to 2 3 intramolecular cross coupling reaction condition using Pd(OAc)2, PCy3, and JohnPhos to afford the carbazole derivative 61.64. The cyclization of 63 was carried out using Pd(OAc) PCy CO afford the compound Kumar’s methodology thus accomplished the synthesis calothrixin in 37 to 2 , of 3 , and K2B carbazole derivative 61. The cyclization of 63 was carried out using Pd(OAc)2, PCy3, and K2CO3 to afford compound 64. Kumar’s methodology in thus synthesis of calothrixin B in stepsthe starting from 2,5-dimethoxybenzaldehyde an accomplished overall yield ofthe 50%. 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.

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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 60 in Kumar’s synthesis has also been converted to the key intermediate in three steps as shown in Scheme 10. In this synthesis, rings B and D of the calothrixin scaffold64 were cyclized three steps as shown in Scheme 10. In this synthesis, rings B and D of the calothrixin scaffold were shown in Scheme 10. In this synthesis, rings B and D of the calothrixin scaffold were cyclized in onesteps stepasby intramolecular C-X/C-H cross coupling reaction using Pd(OAc) 2, PCy 3, and JohnPhos cyclizedone in step one by step by intramolecular C-X/C-H cross coupling reaction using Pd(OAc)2 , PCy3 , and intramolecular C-X/C-H cross coupling reaction using Pd(OAc)2, PCy3, and JohnPhos in theinpresence of K 2CO3 in DMF to afford the key intermediate 64. JohnPhos in the presence of K CO in DMF to afford the key intermediate 64. in the presence of K2CO3 in 2DMF3 to afford the key intermediate 64.

Scheme 10. Alternate synthesis of compound 64.

Scheme 10. 10. Alternate Alternate synthesis synthesis of of compound compound 64. 64. Scheme

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

approach a key allene-mediated of the 6π-electron as Hibino et featuring al. [46] synthesized synthesized calothrixin electrocyclic N-alkyl-calothrixins usingsystem, Hibino et al. calothrixin B and otherreaction N-alkyl-calothrixins using a biomimetic outlined in Scheme 11. The synthesis of the key intermediate, indolo[2,3-a]carbazole (72), was carried allene-mediated electrocyclic reaction of theof 6π-electron system, system, as outlined approach featuring featuringa akey key allene-mediated electrocyclic reaction the 6π-electron as out by an electrocyclic reaction of MOM-protected compound 71 (72), in the(72), presence of out in Scheme 11.allene-mediated The 11. synthesis of the of key intermediate, indolo[2,3-a]carbazole was carried outlined in Scheme The synthesis the key intermediate, indolo[2,3-a]carbazole was carried tBuOK in tBuOH-THF. This electrocyclization involved the two [b]-bonds of indole units and the by an electrocyclic reaction ofof MOM-protected out by allene-mediated an allene-mediated electrocyclic reaction MOM-protectedcompound compound71 71in inthe 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 double bond bond generated generated in situ. situ. The The overall overall yield yield for for Hibino’s Hibino’s synthesis synthesis of of calothrixin calothrixin B B from allene 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 synthesis hasofbeen developed Scheme 11. Hibino’s synthesis 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 report inof2014 [44]Cinand which 2.4. Simultaneous Rings D ring D was the last ring closure step.

2.4. Simultaneous Closure of Rings C and D Mohanakrishnan’s Synthesis Only one methodology of 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 synthesis. This report was published byofMohanakrisnan et al. [47] in 2013, Mohanakrishnan et al. [47] reported novel synthesis calothrixinetBal. involving a key cyclized in a one-pot synthesis. This report another was published by Mohanakrisnan [47] in 2013, prior prior FeCl to their reportdomino in 2014reaction [44] in which ring D derivative, was the last ring closure step. 12. In this key step, 3 mediated an enamine outlined in Scheme to their report in 2014 [44] in whichofring D was the last ringasclosure 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

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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  FeCl3 Mar. Drugs 2016, 14, x  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 with their their discovery discovery in in 1999, 1999, Smith Smith et al.  [34] also also reported reported that that the the cell cell extracts extracts from from  Along et al. [34] 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  and  B,  were  evaluated  against  the  chloroquine  (QC)‐resistant  malarial  strain,  P.  calothrixin AA  and B, were evaluated against the chloroquine (QC)-resistant malarial strain, 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 50  values  were  observed  to  be  58  nM  and  180  nM,  respectively,  compared  with  83  nM  IC50  values 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  results  for  antimalarial  were  compared  against  chloroquine.  Calothrixin  B  was  The results for antimalarial activityactivity  were compared against chloroquine. Calothrixin 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 50 = 185 nM ) as calothrixin B. Although all  was found to be almost equally active (IC50 = 185 nM ) as 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 led  a  decrease  in  activity.  Among  the  analogs, group 2‐hydroxyethyl  group better showed  slightly  agroups  decrease into  activity. Among the analogs, 2-hydroxyethyl 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 was achieved at IC50 against FCR‐3  50 against FCR‐3  Compound  Compound  16 µM concentration. Strain (nM)  Strain (nM)  185 

120 

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

Table 2. Antimalarial activity calothrixin analogs against FCR-3 strain. Table 2. Antimalarial activity calothrixin analogs against FCR-3 strain.

Compound Compound Compound

FCR-3 IC50IC against FCR-3 50 IC 50 against against FCR-3 Strain (nM) Strain (nM) Strain (nM)

185185 185

Compound Compound Compound

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

120120 120

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12 of 12 20 of 20 12 20 12 of of 12 20 of 12 of 20 20 12 of 20 12 of 12 of 20 20

380380 380 380 380 380 380 380 380

490490 490

490 490 490 490 490 490

380 380 380 380 380 380 380380 380

220220 220 220 220 220

220 220 220

320 320 320 320 320 320 320320 320

640 640 640640 640 640 640

250 250 250 250 250 250 250250 250

330 330 330 330 330 330330

640 640

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] reported reported the antibacterial the antibacterial activity activity of of In In addition to its antimalarial its antimalarial activities, activities, Smith Smith et et al. [52,53] reported reported the the activity activity of of In addition addition toagainst itsto antimalarial activities, Smith etit al. al. [52,53] reported the antibacterial antibacterial activity of In A addition to itsBacillus antimalarial activities, Smith et[52,53] al. [52,53] reported the antibacterial antibacterial activity of In addition its antimalarial activities, Smith et al. [52,53] the antibacterial activity of In addition to its antimalarial activities, et al. [52,53] reported the antibacterial activity calothrixin calothrixin against Ato Bacillus subtilis subtilis 168, 168, where where itSmith was was found found to reported betoinhibiting be inhibiting bacterial the bacterial growth growth in of in calothrixin calothrixin A against A against Bacillus Bacillus subtilis subtilis 168, 168, where where it was it was found found to be to inhibiting be inhibiting the bacterial the bacterial growth growth in in 3.2. Anticancer Activity calothrixin A against Bacillus subtilis 168, where it was found to be inhibiting the bacterial growth in calothrixin A against Bacillus subtilis 168, where it was found to be inhibiting the bacterial growth in calothrixin A against Bacillus subtilis 168, where it was found to be inhibiting the bacterial growth in calothrixin A against Bacillus subtilis 168, where it was found to be inhibiting the bacterial growth in a dose-dependent a dose-dependent manner manner without without any any cell lysis. cell lysis. A complete A complete growth growth inhibition inhibition was was achieved achieved at 16at 16 aaa dose-dependent aa dose-dependent manner manner without without any any cell cell lysis. lysis. A complete A complete growth growth inhibition inhibition was was achieved achieved at 16 at 16 dose-dependent manner without any cell lysis. A complete growth inhibition was achieved at 16 dose-dependent manner without any cell lysis. A complete growth inhibition was achieved at 16 dose-dependent manner without any cell lysis. A complete growth inhibition was achieved at 16 a dose-dependent manner without any cell lysis. A complete growth inhibition was achieved at 16 µAlong Mµ concentration. Mwith concentration. their antimalarial activity, Rickards et al. [34] reported calothrixins to be potent against µ concentration. M µM Mµ concentration. M concentration. concentration. µ M concentration. µµ 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 Activity Activity cell line, HeLa cells, were observed to be 40 nM and 350 nM, respectively. Calothrixins showing similar 3.2. Anticancer Activity 3.2. Anticancer Activity 3.2. Anticancer Anticancer Activity 3.2. Anticancer Activity Along Along with with theirtheir antimalarial antimalarial activity, activity, Rickards Rickards et al.et[34] al. [34] reported reported calothrixins calothrixins toshare beto potent be potent inhibitoryAlong effects against cancer cell lines and malarial strains indicated that they mayto a common Along with with their their antimalarial antimalarial activity, activity, Rickards Rickards et et al. reported reported calothrixins calothrixins be to potent be potent Along with their antimalarial activity, Rickards et al. al. [34] reported calothrixins tohuman be potent Along with their antimalarial activity, Rickards et[34] al. [34] [34] reported calothrixins to becervical potent Along with their antimalarial activity, Rickards et al. [34] reported calothrixins to be potent Along with their antimalarial activity, Rickards et al. [34] reported calothrixins to be potent against against HeLa HeLa cells. cells. The The corresponding corresponding IC 50 IC values 50 values for calothrixin for calothrixin A and A and B against B against the human the cervical modeagainst ofagainst action. Following thiscorresponding report, inIC 2003, Waring et calothrixin al. [54]A studied the effect of calothrixin A on HeLa HeLa cells. cells. The The corresponding 50 values 50 values for calothrixin for and A and B against B against the human the human cervical cervical against HeLa cells. The corresponding IC 50 IC values for calothrixin A and B against the human cervical against HeLa cells. The corresponding IC 50 values for calothrixin A and B against the human cervical against HeLa cells. The corresponding IC 50 values for calothrixin A and B against the human cervical against HeLa cells. The IC50 and B against theCalothrixins human cervical cancer cancer cell cell line,line, HeLa HeLa cells,corresponding cells, werewere observed observed tovalues be to 40 befor nM 40calothrixin nM and and 350 A 350 nM, nM, respectively. respectively. Calothrixins 50 apoptosis in human Jurkat cancer cells. A well-known inducer of apoptosis, menadione, was used cancer cancer cell cell line, line, HeLa HeLa cells, cells, were were observed observed to be to 40 be nM 40 nM and and 350 350 nM, nM, respectively. respectively. Calothrixins Calothrixins cancer cell line, HeLa cells,cells, were observed tocancer be 40 nM and and 350 malarial nM,nM, respectively. Calothrixins cancer cell line, HeLa cells, were observed to becell 40lines nM and 350 nM, respectively. Calothrixins cancer cell line, HeLa cells, were observed to be 40 nM and 350 nM, respectively. Calothrixins cancer cell line, HeLa were observed to be 40 nM 350 respectively. Calothrixins showing showing similar similar inhibitory inhibitory effects effects against against cancer cell lines and malarial strains strains indicated indicated that that theythey showing showing similar similar inhibitory inhibitory effects effects against against cancer cancer cell cell lines lines and and malarial malarial strains strains indicated indicated that that they they as a positive control to compare the effects of calothrixin A [55]. Calothrixin A was observed to induce showing similar inhibitory effects against cancer cell lines and malarial strains indicated that they showing similar inhibitory effects against cancer cell lines and malarial strains indicated that they showing similar inhibitory effects against cancer cell lines and malarial strains indicated that they similar inhibitory effects against cancer lines strains indicated that mayshowing may share share a common a common mode mode of action. of action. Following Following this cell this report, report, inand 2003, in malarial 2003, Waring Waring et al.et[54] al. [54] studied studied thethey the may may share share a common a common mode mode of action. of action. Following Following this this report, report, in 2003, in 2003, Waring Waring et al. et [54] al. [54] studied studied the the may share a common mode of action. Following this report, in 2003, Waring et al. [54] studied the cell death via apoptosis in a timeand concentration-dependent manner, and was found to be may share a common mode of action. Following this report, in 2003, Waring et al. [54] studied the may share common mode of action. Following this report, incells. 2003, Waring et al.et [54] studied the more may share a common mode ofin action. Following this report, in 2003, Waring al.of [54] the effect effect of calothrixin ofa calothrixin A on Aapoptosis on apoptosis human in human Jurkat Jurkat cancer cancer cells. A well-known A well-known inducer inducer apoptosis, ofstudied apoptosis, effect effect of calothrixin of calothrixin A on A apoptosis on apoptosis in human in human Jurkat Jurkat cancer cancer cells. cells. A well-known A well-known inducer inducer of apoptosis, of apoptosis, effect of calothrixin A on apoptosis in human Jurkat cancer cells. A well-known inducer of apoptosis, effect of calothrixin A on apoptosis in human Jurkat cancer cells. A well-known inducer of apoptosis, potent than menadione in anti-proliferative activity, with IC values of 1.6 µM and 4.7 µM, respectively effect of calothrixin Aused on in human cancer cells. Aofwell-known inducer of apoptosis, effect of was calothrixin on in human cancer cells. Aofwell-known inducer of apoptosis, 50 effects menadione, menadione, was used asAaapoptosis as positive aapoptosis positive control control to Jurkat compare to Jurkat compare the effects the calothrixin calothrixin A [55]. A [55]. Calothrixin Calothrixin A A menadione, was was used used as aa positive aa positive control control to compare to compare the effects the effects of calothrixin of A [55]. A [55]. Calothrixin Calothrixin A A menadione, was used as positive control to compare effects of calothrixin A [55]. Calothrixin A menadione, was used as positive control to compare theand effects of calothrixin calothrixin A 0.6 [55]. Calothrixin A menadione, was used as a as positive control tomenadione-induced compare the effects of calothrixin A [55]. Calothrixin A menadione, used as a positive control to the effects of calothrixin A [55]. Calothrixin A (Table 3).menadione, IC50 values calothrixin Aand apoptosis were µM and 12and µM was was observed observed to was induce tofor induce cell death cell death via apoptosis via apoptosis incompare a in timeathe timeand concentration-dependent concentration-dependent manner, manner, and was was observed observed to induce to induce cell death cell death via apoptosis via apoptosis in a in timea timeand and concentration-dependent concentration-dependent manner, manner, and and was observed to induce cell death via apoptosis in a timeand concentration-dependent manner, and was observed to induce cell death via apoptosis in a timeand concentration-dependent manner, and was observed to induce cell death via apoptosis a in timeand and concentration-dependent and observed to more induce cell death via apoptosis a timeconcentration-dependent manner, respectively. Both calothrixin A and menadione were observed toactivity, induce cellwith cycle arrest in the /M was was found found to be tomore be potent potent than than menadione menadione inin anti-proliferative in anti-proliferative activity, with IC 50 IC values 50 manner, values of 1.6 of µ1.6 MGand µM was was found found to be to be potent potent than than menadione menadione in anti-proliferative in anti-proliferative activity, activity, with with IC 50 IC values 50 of 1.6 of 1.6 M µµ2M was found to be more potent than menadione in anti-proliferative activity, with IC 50 values of 1.6 µ M was found tomore be more more potent than menadione in anti-proliferative activity, with IC 50 values values ofµ 1.6 M was found to be more potent than menadione in anti-proliferative activity, with IC 50 IC values of 1.6 µ M was found to be more potent than menadione in anti-proliferative activity, with 50 values of 1.6 µ M and and 4.7 µ 4.7 M, µ respectively M, respectively (Table (Table 3). IC 3). 50 IC values 50 values for calothrixin for calothrixin Aand Aand menadione-induced menadione-induced apoptosis apoptosis 50 phase, but the concentration required for calothrixin A was much lower than for menadione. The cell and and 4.7 µ M, µrespectively M, respectively (Table (Table 3). 3). 50 IC values 50 for calothrixin for Aand Aand menadione-induced menadione-induced apoptosis apoptosis and 4.7 µ 3). IC 50 values for calothrixin Aand menadione-induced apoptosis and 4.7 M, respectively (Table 3). IC 50 values values for calothrixin calothrixin Aand menadione-induced apoptosis and 4.7 µ4.7 M, respectively (Table 3). IC IC 50Both values for calothrixin Aand menadione-induced apoptosis and 4.7 (Table 3). IC 50 values for calothrixin Aand menadione-induced apoptosis were were 0.6in µM, 0.6 Mµµrespectively µM, and Mrespectively and 12 µphase 12 M µ(Table respectively. M indicated respectively. calothrixin calothrixin A and Adamage, and menadione menadione were were observed observed tosupported induce to induceby 50Both cyclewere arrest the G /M intracellular DNA which was further 2 were 0.6 µ 0.6 M and M and 12 µ 12 M µµrespectively. M respectively. Both Both calothrixin calothrixin A and A and menadione menadione were observed observed to induce to were 0.6 µ M 12 M calothrixin A and menadione were observed to induce were 0.6 M and 12 M2phase, respectively. Both calothrixin Arequired and menadione were observed to induce induce were 0.6cycle µarrest Mµ and 12 µG M2/M respectively. Both calothrixin Arequired and menadione werewere observed tomuch induce were 0.6 µµand M 12 µrespectively. M respectively. Both calothrixin A and menadione were observed to induce cell cycle cell arrest inand the inµ the G /M phase, but Both the but concentration the concentration for calothrixin for calothrixin A was A was much lower lower direct DNA damage observed in cell-free experiments. cell cycle cell cycle arrest arrest in the in G the 22/M G 2phase, /M phase, but the but concentration the concentration required required for calothrixin for calothrixin A was A was much much lower lower cell cycle arrest in the G /M phase, but the concentration required for calothrixin A was much lower cell cycle arrest in the G 2 /M phase, but the concentration required for calothrixin A was much lower cell arrest in the G 2/M but the concentration required for calothrixin A was much lower cell arrest inThe the G22phase, /M phase, but thethe concentration required forintracellular calothrixin A was much lower thancycle than forcycle menadione. for menadione. The cell cell cycle cycle arrest arrest in in G the 2/M G2phase /M phase indicated indicated intracellular DNA DNA damage, damage, Both calothrixin A and menadione were found to be redox active through the observation of than than for menadione. for menadione. The The cell cell cycle cycle arrest arrest in the in G the 22/M G 22phase /M phase indicated indicated intracellular intracellular DNA DNA damage, damage, than for menadione. The cell cycle arrest in the G /M phase indicated intracellular DNA damage, than for menadione. The cell cycle arrest in the G /M phase indicated intracellular DNA damage, than for menadione. The cell by cycle arrest in the G 2/M indicated intracellular DNA damage, than for menadione. The cell cycle arrest in damage the G 22phase /Mobserved phase indicated intracellular DNA damage, which which was was further further supported supported direct by direct DNA DNA damage observed in cell-free in cell-free experiments. experiments. which which was was further further supported supported by direct by direct DNA DNA damage damage observed observed in cell-free in cell-free experiments. experiments. additional oxygen intake when added to reductant dithiothreitol (DTT, 2 mM at pH 8.0). Quinones can which was further supported by direct DNA damage observed in cell-free experiments. which was further supported by direct DNA damage observed in cell-free experiments. which was further supported by direct DNA damage observed cell-free experiments. which was further by direct DNA damage observed in active cell-free experiments. Both Both calothrixin calothrixin Asupported and A and menadione menadione were were found found to beto redox be in redox active through through the observation the observation of of Both Both calothrixin calothrixin A and A menadione menadione were were found found to be be active active through through the observation the observation of Both calothrixin A and menadione found to be redox active observation of redox cycle one orintake two electron transfers, generating reactive oxygen species (ROS), byof Both calothrixin A and and menadione were found toredox be redox redox active through the observation of Bothby calothrixin Aintake and menadione were found to dithiothreitol beto redox active through the observation ofwhich Both calothrixin A and menadione found to be redox active the observation of additional additional oxygen oxygen when when added added towere reductant towere reductant dithiothreitol (DTT, (DTT, 2through mM 2through mM at the pH at 8.0). pH 8.0). Quinones Quinones additional additional oxygen oxygen intake intake when when added added to reductant to reductant dithiothreitol dithiothreitol (DTT, (DTT, 2 mM 2 mM at pH at 8.0). pH 8.0). Quinones Quinones additional oxygen intake when added to reductant dithiothreitol (DTT, 2 mM at pH 8.0). Quinones additional oxygen intake when added to reductant dithiothreitol (DTT, 2 mM at pH 8.0). Quinones they additional can redox damage DNA andintake apoptosis [56]. It was postulated that structure ofwhich calothrixin oxygen intake when added to reductant dithiothreitol (DTT, 2the mM atspecies pH 8.0). Quinones additional oxygen added to reductant dithiothreitol (DTT, 2ring mM at pH(ROS), 8.0). Quinones can can redox cycle cycle by one by one orinduce two or when two electron electron transfers, transfers, generating generating reactive reactive oxygen oxygen species (ROS), by by which can redox can redox cycle cycle by one by or two or two electron electron transfers, transfers, generating generating reactive reactive oxygen oxygen species species (ROS), (ROS), by which by which can redox cycle by one or two electron transfers, species (ROS), by which can redox cycle by one one or and two electron transfers, generating reactive oxygen species (ROS), by which can redox cycle by intercalator one or two electron transfers, generating reactive oxygen species (ROS), by which can cycle by one or two electron transfers, generating reactive oxygen species (ROS), by which might act asredox adamage DNA that might be generating responsible for itsoxygen anticancer activity. In addition, they they can can damage DNA DNA and induce induce apoptosis apoptosis [56]. [56]. It was Itreactive was postulated postulated that that the the ring ring structure structure of of they they can can damage damage DNA DNA and and induce induce apoptosis apoptosis [56]. [56]. It was It was postulated postulated that that the the ring ring structure structure of of they can damage DNA and induce apoptosis [56]. It was postulated that the ring structure of they can damage DNA and induce apoptosis [56]. It was postulated that the ring structure of they can damage DNA and induce apoptosis [56]. It was postulated that the ring structure of they can damage DNA and induce apoptosis [56]. It was postulated that the ring structure of calothrixin calothrixin might might act as act a as DNA a DNA intercalator intercalator that that might might be responsible be responsible for its for anticancer its anticancer activity. activity. In In calothrixin A was observed to be cleaving DNA, though less effectively than menadione. Menadione caused calothrixin calothrixin might might act as act a as DNA a DNA intercalator intercalator that that might might be responsible be responsible for its for anticancer its anticancer activity. activity. In In calothrixin might act as a DNA intercalator that might be responsible for its anticancer activity. In calothrixin might act as a DNA intercalator that might be responsible for its anticancer activity. In calothrixin might act as a DNA intercalator that might be responsible for its anticancer activity. In calothrixin might act as a DNA intercalator that might be responsible for its anticancer activity. In addition, addition, calothrixin calothrixin A was A was observed observed to betocleaving be cleaving DNA, DNA, though though less less effectively effectively thanthan menadione. menadione. addition, addition, calothrixin calothrixin A was A observed observed to be be cleaving DNA, DNA, though though less less effectively effectively than than menadione. menadione. addition, calothrixin A significant was observed to damage beto cleaving DNA, though lessincubation, effectively thancalothrixin menadione. addition, calothrixin A was was observed tocleaving be10 cleaving DNA, though less effectively than menadione. addition, calothrixin A was observed to be cleaving though less effectively than menadione. addition, calothrixin A was observed to be cleaving though less effectively than menadione. Menadione Menadione caused caused significant DNA DNA damage at atµ 10 MDNA, after µ MDNA, after 60 min 60 min incubation, whereas whereas calothrixin A A Menadione Menadione caused caused significant significant DNA DNA damage damage at 10 at µ 10 M µ after M after 60 min 60 min incubation, incubation, whereas whereas calothrixin calothrixin A Menadione caused significant DNA damage at 10 µ M after 60 min incubation, whereas calothrixin A A Menadione caused significant DNA damage at 10 µ M after 60 min incubation, whereas calothrixin A Menadione caused significant DNA damage at 10 µ M after 60 min incubation, whereas calothrixin A Menadione caused significant DNA damage at 10 µ M after 60 min incubation, whereas calothrixin A exhibited exhibited the same the same effect effect at 500 at µ500 M.µ M. exhibited exhibited the the effect effect at at 500 M. M. exhibited the same same effect at 500 500 µ M.µ exhibited the same same effect at µ 500 M. exhibited the same effect at 500 µ M. exhibited the same effect at 500 µµ M. Table Table 3. Cell 3. cytotoxicities Cell cytotoxicities and apoptotic and apoptotic activities activities of calothrixin of calothrixin A and A menadione. and menadione. Table Table 3. Cell 3. cytotoxicities and apoptotic and activities activities of calothrixin of A and A and Table 3. cytotoxicities and activities of A menadione. Table 3. Cell Cell cytotoxicities cytotoxicities and apoptotic apoptotic activities of calothrixin calothrixin A menadione. and menadione. menadione. Table 3. Cell Cell cytotoxicities and apoptotic apoptotic activities of calothrixin calothrixin A and and menadione. Table 3. Cell cytotoxicities and apoptotic activities of calothrixin A and menadione. Table 3. Cell cytotoxicities and apoptotic activities of calothrixin A and menadione.

Compound Compound Compound Compound Compound Compound Compound Compound

Structure Structure Structure Structure Structure Structure Structure Structure

IC50 IC for50Cytotoxicity for CytotoxicityIC50 IC for50Apoptosis for Apoptosis IC for for for for IC50 50 IC for50 Cytotoxicity IC50 50 IC for50 Apoptosis IC 50Cytotoxicity for Cytotoxicity CytotoxicityIC IC 50Apoptosis for Apoptosis Apoptosis IC 50 for IC 50 for IC 50 for Cytotoxicity IC 50 for Apoptosis 50 Cytotoxicity 50 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 Mar. Drugs 2016, 14, xCompound Mar. Drugs 2016, 14, x Mar. Drugs 2016, 14, x Mar. Drugs 2016, 14, x CalothrixinA A A Calothrixin Calothrixin

Structure

Calothrixin A Calothrixin A Calothrixin A Calothrixin A

Menadione Menadione Menadione

IC50 for Cytotoxicity

IC50 for Apoptosis

1.6 µM µM M 1.6 1.6 µ

0.6 µM M 0.6 µM 0.6 µ

4.7 µM µM M 4.7 µ 4.7

12 µM M µ 1212 µM

1.6 µ M 1.6 µ M 1.6 µ M 1.6 µ M

13 of of 20 20 13

13 of 20 13 of 20 13 of 20 13 of 20

0.6 µ M 0.6 µ M 0.6 µ M 0.6 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 4.7 µ M 12by µM One of of the the known known modes modes of of action action for for anticancer anticancer activities activities of of quinones quinones is is by the generation generation of of One the One of the known modes of action for anticancer activities of quinones is by the generation ROS through through redox redox cycling cycling [56]. [56]. In In 2004, 2004, Wilkes Wilkes et et al. al. [57] [57] carried carried out out aa comparative comparative study of of ROS study One of the known modes action anticancer activities of quinones by generation bioactivities of calothrixins calothrixins and some structurally related quinones. This was thethe first structure– of ROS through redox cyclingof In for 2004, Wilkes et al. [57] carried out was ais study of of One of the known modes of[56]. action for anticancer activities of quinones iscomparative by the generation of bioactivities of and some structurally related quinones. This the first structure– One of theredox known modes of done action forcalothrixins. anticancer activities of quinones is by the generation of ROS through cycling [56]. Instructurally 2004, Wilkes et The al. [57] carried outofthe a various comparative study activity relationship study for cytotoxic effect quinones was One of the known modes of action for anticancer activities of quinones is by the generation of bioactivities of calothrixins and some related quinones. This was first structure–activity activity relationship study done for calothrixins. The cytotoxic effect of various quinones was ROS through redox cycling [56]. In 2004, Wilkes et al. [57] carried out a comparative study ROS through cyclingand [56]. In 2004, Wilkes related et cancer al. [57] carried outHeLa awas comparative study of bioactivities ofredox calothrixins some structurally quinones. This theevaluated first structure– evaluated against human cervical cells,quinones cells, using evaluated against human cancer cells, using ROS through cycling [56]. In 2004, Wilkes related eteffect al. [57] carried outHeLa awas comparative study of relationship study done for calothrixins. Thecervical cytotoxic of various was against bioactivities ofredox calothrixins and some structurally quinones. This thecells, first structure– bioactivities of calothrixins and some structurally related quinones. This was the first structure– activity relationship study done for calothrixins. Thebromide cytotoxic effect of was various quinones was 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Table 4). first Calothrixin A (MTT) assay 4). Calothrixin A bioactivities of calothrixins some structurally related quinones. This the structure– human3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium cervical cancer cells,and HeLa cells, using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium activity relationship study done for calothrixins. The cytotoxic effect of (Table various quinones was activity relationship study done for calothrixins. The cytotoxic effect ofof quinones was and calothrixin B showed showed cytotoxicity against HeLa HeLa cells withcells, EC50 50 valuesHeLa ofvarious 0.12 µ µM Mcells, and 0.24 0.24 µ µusing M, evaluated against human cervical cancer calothrixin B cytotoxicity cells with EC values 0.12 and M, activity relationship study done for calothrixins. The cytotoxic effect of various quinones was bromideand (MTT) assay (Table 4). Calothrixin Aagainst and calothrixin B showed cytotoxicity against HeLa cells with evaluated against human cervical cancer cells, HeLa cells, using respectively. Calothrixin B and ellipticine quinone, which differ by the ring E, showed comparable evaluated against human cervical cancer cells, HeLa cells, using respectively. Calothrixin B and ellipticine quinone, which differ by the ring E, showed comparable 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Table 4). Calothrixin A evaluated human cervical cancer cells, HeLa cells, using EC50 values ofagainst 0.12 µM and 0.24 µM, respectively. Calothrixin Bring and ellipticine which 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Table 4).quinone, Calothrixin A activities,Bindicating indicating that there might might be only only limited role for the E in inof dictating the bioactivity 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Table 4). Calothrixin A activities, that there be aa limited role for the E dictating the bioactivity and calothrixin showed cytotoxicity against HeLa cells with EC 50 ring values 0.12 µ M and 0.24 µ M, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Table 4). Calothrixin A differ by the ring E, showed comparable activities, indicating that there might be only a limited and calothrixin B showed cytotoxicity against HeLa cells with EC 50 values of 0.12 µ M and 0.24 µ M, of calothrixins. calothrixins. Introducing the MOM MOM group on the the indole nitrogen seemedofto to0.12 decrease the potency potency of Introducing the group on indole nitrogen seemed decrease the and calothrixin B showed cytotoxicity against HeLa cells with ECby 50 values µ M and 0.24 µ M, respectively. Calothrixin B and ellipticine quinone, which differ the ring E, showed comparable and calothrixin B showed cytotoxicity against HeLa cells with EC 50 values of 0.12 µ M and 0.24 µ M, role forof ring E in dictating the bioactivity of calothrixins. Introducing MOM group on the respectively. Calothrixin andellipticine ellipticine quinone, which differof by the ring E, showed comparable ofthe both calothrixin BBand and quinone with EC50 50 values values of 0.42 µM M andthe 0.37 µ M, M, respectively. respectively. both calothrixin with EC 0.42 µ and 0.37 µ respectively. Calothrixin B andellipticine ellipticine quinone, which differ the E, showed comparable activities, indicating that Bto there might be quinone only a limited role for theby ring Ering in dictating the bioactivity respectively. Calothrixin B and ellipticine quinone, which differ by the ring E, showed comparable indole nitrogen seemed decrease the potency of both calothrixin B and ellipticine quinone with Further, replacement of the heteroaromatic ring-D with a carbocyclic aromatic ring in ellipticine and activities, indicating that there might be only a limited role for the ring E in dictating the bioactivity Further, replacement of the heteroaromatic with a carbocyclic aromatic ring in ellipticine and activities, indicating that there be only on aring-D limited role for the ring E in dictating thethe bioactivity of calothrixins. Introducing themight MOM group the indole nitrogen seemed toofdecrease potency its N-methoxymethyl analog also led to a decrease in potency, benzocarbazoledione (0.43 µ M), and activities, indicating that there might be only a limited role for the ring E in dictating the bioactivity EC values of 0.42 µM and 0.37 µM, respectively. Further, replacement the heteroaromatic of calothrixins. Introducinganalog the MOM group the indole nitrogen seemed to decrease potency also led to a on decrease in potency, benzocarbazoledione (0.43the µ M), and 50 its N-methoxymethyl of calothrixins. Introducing the MOM group on the nitrogen seemed decrease the potency both calothrixin B and ellipticine quinone with ECindole 50 values ofactivity 0.42 µat M3.7 and 0.37 µ M, respectively. N-MOM-benzocarbazoledione (1.6 µ M). M). Menadione showed µto M. Absence of activity activity of calothrixins. the MOM group on the nitrogen seemed to decrease the potency ring-D with a Introducing carbocyclic aromatic ring inMenadione ellipticine and its N-methoxymethyl analog also led N-MOM-benzocarbazoledione (1.6 µ showed µ M. Absence of both calothrixin B and ellipticine quinone with ECindole 50 values ofactivity 0.42 µat M3.7 and 0.37 µ M, respectively. of both for calothrixin B and ellipticine quinonering-D with EC 50 values of 0.42 aromatic µ M and 0.37 respectively. Further, replacement of the heteroaromatic with a carbocyclic ringµ inM,ellipticine and uncyclized precursors of benzocarbazoledione benzocarbazoledione and N-MOM-benzocarbazoledione indicated the for uncyclized precursors of and N-MOM-benzocarbazoledione the of calothrixin and ellipticine quinonering-D with EC 50 values 0.42 aromatic µ M and 0.37 respectively. to both a decrease in B potency, benzocarbazoledione (0.43 µM),ofand N-MOM-benzocarbazoledione Further, replacement of the heteroaromatic with a carbocyclic ringµinM,indicated ellipticine and Further, replacement of the heteroaromatic ring-D with a carbocyclic aromatic ring in ellipticine and its N-methoxymethyl analog also led to a decrease in potency, benzocarbazoledione (0.43 M), importance of a tetracyclic quinone ring structure. In case of all the quinones with EC 50 < 1.6 µ M, the importance of a tetracyclic quinone ring structure. In case of all the quinones with EC 50 ellipticine < 1.6 µ M, the Further, replacement of the heteroaromatic ring-D with a carbocyclic aromatic ring in (1.6 µM). Menadione showed activity at 3.7 µM. Absence of activity for uncyclized precursors of its N-methoxymethyl analog also led to a decrease in potency, benzocarbazoledione (0.43 µ M), and its N-methoxymethyl analog also to aMenadione decrease inshowed potency, benzocarbazoledione (0.43 µ M), and quinones were were observed observed to(1.6 beled reduced to their their respective respective semiquinones, though no direct correlation N-MOM-benzocarbazoledione µ M). activity at 3.7 µ M. Absence of activity quinones to be reduced to semiquinones, though no direct correlation its N-methoxymethyl analog also led to aMenadione decrease inshowed potency, benzocarbazoledione (0.43 µ M), and benzocarbazoledione and N-MOM-benzocarbazoledione indicated the importance of a of tetracyclic N-MOM-benzocarbazoledione (1.6 µ M). activity at 3.7 µ M. Absence activity was observed observed between the reduction potentials and and bioactivity. N-MOM-benzocarbazoledione (1.6 µ M). potentials Menadione showed activity at 3.7 µ M. Absence of activity was between reduction bioactivity. for uncyclized precursors of the benzocarbazoledione and N-MOM-benzocarbazoledione indicated the N-MOM-benzocarbazoledione (1.6 M).quinones Menadione showed activity at 3.7 µ M. Absence of activity quinone ring structure. In case of allµthe with EC < 1.6 µM, the quinones were observed to for uncyclized precursors of benzocarbazoledione and N-MOM-benzocarbazoledione indicated the 50 for uncyclized precursors of benzocarbazoledione and N-MOM-benzocarbazoledione importance of a tetracyclic quinone ring structure. In case of all the quinones with EC 50 indicated < 1.6 µ M, the for uncyclized precursors of benzocarbazoledione and N-MOM-benzocarbazoledione indicated the be reduced of to atheir respective semiquinones, though no direct observed importance tetracyclic quinone structure. case of allcorrelation the quinones with EC50 < between 1.6 µ M, the Tablering 4. Cytotoxicity Cytotoxicity of In quinones against HeLa cells. was Table 4. of quinones against HeLa cells. importance of aobserved tetracyclic ring structure. In case of all the quinones with 50 < 1.6 µ M, the quinones were to quinone be reduced their respective though noEC direct correlation importance of aobserved tetracyclic quinone ringto In casesemiquinones, of all the quinones with 50 < 1.6 µ M, the reductionwere potentials andto bioactivity. quinones be reduced tostructure. their respective semiquinones, though noEC direct correlation Compound Structure EC 50 (µM) HeLa Cells quinones werebetween observedthe to reduction be reduced to their respective semiquinones, no direct was observed potentials and Structure bioactivity. Compound EC50though (µM) HeLa Cells correlation quinones werebetween observedthe to reduction be reduced to their respective semiquinones, though no direct correlation was observed potentials and bioactivity. was observed between the Table reduction potentialsofand bioactivity. 4. Cytotoxicity quinones against HeLa cells. was observed between the reduction potentials and bioactivity. Calothrixin A 0.12 ± ± 0.01 0.01 Table 4. Cytotoxicity of quinones against HeLa cells. 0.12 Calothrixin A Table 4. Cytotoxicity of quinones against HeLa cells. Table 4. Cytotoxicity of quinones against HeLa cells. Compound Structure EC (µM) HeLa Cells 50 Table 4. Cytotoxicity of quinones against HeLaEC cells. Compound Structure 50 (µM) HeLa Cells

Compound Compound Compound Calothrixin B B Calothrixin Calothrixin CalothrixinAA Calothrixin A Calothrixin A Calothrixin A

N-MOM-calothrixin B B N-MOM-calothrixin

Calothrixin B CalothrixinBB Calothrixin Calothrixin B Calothrixin B

Ellipticine quinone quinone Ellipticine

N-MOM-calothrixin B N-MOM-calothrixin B N-MOM-calothrixin N-MOM-calothrixin B B N-MOM-calothrixin N-MOM-ellipticineBquinone quinone N-MOM-ellipticine

Structure Structure Structure

EC50 (µM) HeLa Cells EC50 (µM) HeLa Cells EC50 (µM) 0.24 ± ±HeLa 0.04 Cells 0.24 0.04 0.12˘±0.01 0.01 0.12 0.12 ± 0.01 0.12 ± 0.01 0.12 ± 0.01 0.42 ± ± 0.02 0.02 0.42

0.24 ± 0.04 0.24 ± 0.04 0.24 ± 0.04

0.24 0.24˘±0.04 0.04

0.15 ± ± 0.09 0.09 0.15

0.42 ± 0.02 0.42 ± 0.02

0.42˘±0.02 0.02 0.42 0.42 0.02 0.37 ± ±±0.08 0.08 0.37

Ellipticine quinone Ellipticine quinone Ellipticine quinone Benzocarbazoledione Benzocarbazoledione Ellipticine quinone Ellipticine quinone

0.15 ± 0.09 0.15 ± 0.09 0.15 0.09 0.43˘± ±± 0.01 0.43 0.01 0.15 ±0.09 0.09 0.15

N-MOM-ellipticine quinone N-MOM-ellipticine quinone N-MOM-Benzocarbazoledione N-MOM-ellipticine quinone N-MOM-Benzocarbazoledione N-MOM-ellipticine quinone

0.37 ± 0.08 0.37 ±1.0 0.08 1.6 ± ±± 0.37 0.08 1.6 1.0 0.37 ± 0.08

Benzocarbazoledione Benzocarbazoledione Benzocarbazoledione Benzocarbazoledione

0.43 ± 0.01 0.43 ± 0.01 0.43 ± 0.01 0.43 ± 0.01

Calothrixin B Calothrixin B Calothrixin B

0.24 ± 0.04 0.24 ± 0.04 0.24 ± 0.04

Mar. Drugs 2016,N-MOM-calothrixin 14, 17

B N-MOM-calothrixin B N-MOM-calothrixin B

14 of 21

0.42 ± 0.02 0.42 ± 0.02 0.42 ± 0.02 Table 4. Cont.

Ellipticine quinone Ellipticine quinone Compound Ellipticine quinone

0.15 ± 0.09 0.15 ± 0.09

Structure

EC50 (µM) 0.15 HeLa ± 0.09Cells

N-MOM-ellipticine N-MOM-ellipticinequinone quinone N-MOM-ellipticine quinone N-MOM-ellipticine quinone

0.37˘±0.08 0.08 0.37 0.37 ± 0.08 0.37 ± 0.08

Benzocarbazoledione Benzocarbazoledione Benzocarbazoledione Benzocarbazoledione

0.43 ± 0.01 0.43 ± 0.01

0.43˘±0.01 0.01 0.43

N-MOM-Benzocarbazoledione N-MOM-Benzocarbazoledione N-MOM-Benzocarbazoledione N-MOM-Benzocarbazoledione

1.6 ± 1.0 1.6 ± 1.0

1.6 1.6˘±1.0 1.0

Mar. Drugs 2016, 14, x Mar. Drugs 2016, 14, x Mar. Drugs 2016, 14, x

Menadione Menadione Menadione Menadione

14 of 20 14 of 20 14 of 20

3.7˘±0.3 0.3 3.7

3.7 ± 0.3 3.7 ± 0.3

Uncyclized precursor to Uncyclized Uncyclizedprecursor precursor to to Uncyclized precursor to benzocarbazoledione benzocarbazoledione benzocarbazoledione benzocarbazoledione

>50 >50

>50 >50

Uncyclized precursor to Uncyclized precursor to Uncyclized N-MOM-benzocarbazoledione Uncyclizedprecursor precursor to to N-MOM-benzocarbazoledione N-MOM-benzocarbazoledione N-MOM-benzocarbazoledione

>50 >50

>50 >50

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 study thegroup involvement of the ring the mechanism of simple action in the inhibitory effects, same [58] synthesized andstructure analyzedand the activity of various quinone analogs Tothe study the involvement of the ring the mechanism ofsimple action quinone in the inhibitory effects, same group [58] synthesized andstructure analyzedand the activity of various analogs effects, the sameB group andcompounds analyzed thewere activity of various analogs of calothrixin (Table[58] 5) synthesized in 2007. The evaluated forsimple their quinone selectivity and effects, the same group [58] synthesized and analyzed the activity of various simple quinone analogs of of calothrixin B (Table 5) in 2007. The compounds were evaluated for their selectivity and of calothrixin B activity, (Table 5) in 2007. Theassay, compounds for lines: their human selectivity and antiproliferative using an MTT against were three evaluated different cell cervical calothrixin B (Table 5) in 2007. Thean compounds were evaluated their selectivity andhuman antiproliferative antiproliferative activity, using MTT assay, against threefordifferent cell lines: cervical antiproliferative activity, MTT assay, against different cell lines: human cervical cancer (HeLa) cells, murineusing P388 an macrophage cancer cells,three and simian non-cancerous CV-1 cells. activity, usingcells, an MTT assay, against three different cell lines: human cervical cancer (HeLa) cancer (HeLa) murine P388 macrophage cancer cells, and simian non-cancerous 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 Antiproliferative of calothrixin B and MTT assay HeLa cells, P388 by In this5.study, calothrixin activity B was found to be the mostanalogs potentin against HeLaagainst cells (0.25 µM) followed Tableand 5. Antiproliferative activity of calothrixin B and analogs in MTT assay against HeLa cells, P388 cells, CV-1 cells. 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 50 (µM) EC 50 (µM) EC 50 (µM) The activity was further decreased with the deletion of ring E of this analog to form benzoncarbzoledione EC50 (µM) EC50 (µM) EC50 (µM) Compound Structure EC50 ranging (µM) EC50 (µM) EC50while (µM) HeLa Cells from P388 CV-1 Cells the Compound Structure (1.8 µM). The rest of the analogs showed moderate activity 7Cells to 13 µM, Compound Structure HeLa Cells P388 Cells CV-1 Cells HeLawere Cellsnot as P388 Cells for CV-1 Cellscell carbazole-1,4-dione analog was found to be inactive. The results consistent the P388 line, where murrayaquinone (2.3 µM) and 2-methylcarbazoledione (1 µM) were to be2.4 more Calothrixin B 0.25 ± 0.05 9 ±found 2 ± 0.7active Calothrixin B 0.25 ± 0.05 9±2 2.4 ± 0.7 ± 0.05 B and92-methylcarbazoledione ±2 2.4 ± 0.7 compared toCalothrixin calothrixinBB (9 µM) or other analogs, whereas0.25 calothrixin 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 These studies>50 indicated Indolophenanthrene-7,13-dione 1.5 ± 0.3 activity.>50 1.5Also, ± 0.3a selective >50 >50 theIndolophenanthrene-7,13-dione importance of rings A–D in the activity of calothrixins. usage of calothrixin B Indolophenanthrene-7,13-dione 1.5 ± 0.3 >50 >50 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 to P388 quinones Benzocarbzoledione 1.8compared ± 0.1 >50(9 µM). The>50 Benzocarbzoledione 1.8 ±indolophenanthrene-7,13-dione 0.1 >50 >50 and containing tetraand penta cyclic systems (calothrixin-B, 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 >50 >50 >50 (murrayaquinone, 2-methylcarbazoledione, and isoquinoline-5,8-dione). 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 2-Methylcarbazoledione 2-Methylcarbazoledione

7±1 7±1 7±1

1.0 ± 0.1 1.0 ± 0.1 1.0 ± 0.1

1.7 ± 0.4 1.7 ± 0.4 1.7 ± 0.4

Uncyclized precursor precursor to to Uncyclized Uncyclized precursor precursor to to Uncyclized Uncyclized precursor to benzocarbazoledione Uncyclized precursor to benzocarbazoledione benzocarbazoledione benzocarbazoledione benzocarbazoledione benzocarbazoledione

>50 >50 >50 >50 >50 >50

Mar. Drugs 2016, 14, 17 Uncyclized precursor to to Uncyclized precursor

15 of 21

Uncyclized precursor precursor to to >50 Uncyclized >50 Uncyclized precursor to to >50 N-MOM-benzocarbazoledione Uncyclized precursor >50 N-MOM-benzocarbazoledione >50 N-MOM-benzocarbazoledione >50 N-MOM-benzocarbazoledione 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 study the the involvement involvement of of the the ring ring structure structure and and the the mechanism mechanism of of action action in in the the inhibitory inhibitory To To study study the to involvement of the the ring ring structure and the thethe mechanism of action action in the due inhibitory To the involvement of structure and mechanism of in the inhibitory which is known cause irreversible DNAstructure damage and during S phase. Delay in S in phase to DNA To study the involvement of the ring the mechanism of action the inhibitory effects, the same group [58] synthesized and analyzed the activity of various simple quinone analogs To study the involvement of the ring structure and the mechanism of action in the inhibitory effects, the same group [58] synthesized and analyzed the activity of various simple quinone analogs effects, the same group [58] synthesized and analyzed the activity of various simple quinone analogs effects, the same group [58] synthesized and analyzed the activity of various simple quinone analogs damage is associated with a block in replication. The IC value for calothrixin A was found to be 50 evaluated effects, the same sameB group [58] synthesized andcompounds analyzed the thewere activity of various various simple quinone analogs of calothrixin calothrixin Bgroup (Table[58] 5)synthesized in 2007. 2007. The The compounds were forsimple their quinone selectivity and effects, the and analyzed activity of analogs of 5) evaluated for selectivity and of five-fold calothrixin B (Table (Table 5) in in 2007. 2007.and The compounds were evaluated for their their selectivity and of calothrixin B (Table 5) in The compounds were evaluated for their selectivity and higher than campothecin the other two analogs were observed to be much less potent, of calothrixin B B activity, (Table 5) 5) in 2007. 2007. Theassay, compounds were evaluated for lines: their human selectivity and antiproliferative activity, using an MTT MTT assay, against were three evaluated different cell cell lines: human cervical of calothrixin (Table in The compounds for their selectivity and antiproliferative using an against three cervical antiproliferative activity, using an the MTT assay, against three different different cell lines: human cervical antiproliferative activity, using an MTT assay, against three different cell lines: human cervical with N-methylcalothrixin B being least at 5 µM compared to calothrixin B at 1 µM (Table 6). antiproliferative activity, using an MTT assay, assay, against three different cell lines: lines: human human cervical cancer (HeLa) (HeLa) cells, cells, murineusing P388 an macrophage cancer cells,three and simian simian non-cancerous CV-1 cells. cells. antiproliferative activity, MTT against different cell cervical cancer murine cancer cells, non-cancerous CV-1 cancer (HeLa) (HeLa) cells, cells, murine P388 P388 macrophage macrophage cancer cells, and and simian simian non-cancerous CV-1 cells. cells. cancer murine P388 macrophage cancer cells, and non-cancerous CV-1 cancer (HeLa) cells, murine P388 macrophage cancer cells, and simian non-cancerous CV-1 cells. cancer (HeLa) 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 5. 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 Table 5.and Antiproliferative 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 cells,5. CV-1 cells. Table 5. Antiproliferative activity Table Antiproliferative activity of calothrixin calothrixin B B and and analogs analogs in in MTT MTT assay assay against against HeLa HeLa cells, cells, P388 P388 cells, and and CV-1 cells. cells. Table 5. Antiproliferative activity of cells, CV-1 cells, and CV-1 cells. cells, and and CV-1 CV-1 cells. cells. cells, cells, and CV-1 cells. EC EC (µM) EC5050 50 (µM) (µM) EC5050 50(µM) (µM) EC50 50 (µM) (µM) EC (µM) EC (µM) EC Compound Structure EC50 50 (µM) (µM) EC50 50 (µM) (µM) EC50 50 (µM) (µM) Compound Structure EC EC EC 50 Compound Structure HeLa CV-1 Cells EC 50 (µM) EC 50 (µM) EC 50 (µM) Compound Structure 50 Cells 50Cells 50 (µM) HeLa Cells P388 P388 Cells CV-1 Cells EC 50 (µM) EC 50 (µM) EC 50

Compound Compound Compound

Structure Structure Structure

HeLa HeLa Cells Cells HeLa Cells HeLa Cells HeLa Cells 0.25˘± ±0.05 0.05 0.25 0.25 0.25 ± ± 0.05 0.05 0.25 0.05 0.25 ± 0.05 0.25 ± 0.05

P388 P388 Cells Cells P388 Cells P388 Cells P388 Cells 9˘± ±222 99 ± 22 99 ± ± 9 9 ± 22

CV-1 CV-1 Cells Cells CV-1 Cells CV-1 Cells CV-1 Cells 2.4˘± ± 0.7 0.7 2.4 2.4 2.4 ± ± 0.7 0.7 2.4 0.7 2.4 ± 0.7 2.4 ± 0.7

Indolophenanthrene-7,13-dione Indolophenanthrene-7,13-dione Indolophenanthrene-7,13-dione Indolophenanthrene-7,13-dione Indolophenanthrene-7,13-dione Indolophenanthrene-7,13-dione Indolophenanthrene-7,13-dione

1.5˘± ± 0.3 0.3 1.5 1.5 1.5 ±0.3 0.3

>50 >50 >50 >50

>50 >50 >50 >50

Benzocarbzoledione Benzocarbzoledione Benzocarbzoledione Benzocarbzoledione Benzocarbzoledione Benzocarbzoledione Benzocarbzoledione

1.8 0.1 1.8˘±± ±0.1 0.1 1.8 1.8 0.1

1.8 ± 0.1 1.8 ± ± 0.1 0.1 1.8

>50 >50 >50 >50

>50 >50 >50

>50 >50 >50 >50

Carbazole-1,4-dione Carbazole-1,4-dione Carbazole-1,4-dione Carbazole-1,4-dione Carbazole-1,4-dione Carbazole-1,4-dione Carbazole-1,4-dione

>50 >50 >50 >50

>50 >50 >50

>50 >50 >50 >50

>50 >50 >50

>50 >50 >50 >50

Murrayaquinone Murrayaquinone Murrayaquinone Murrayaquinone Murrayaquinone Murrayaquinone Murrayaquinone

13˘± ±1 13 13 13 ±111

2-Methylcarbazoledione 2-Methylcarbazoledione 2-Methylcarbazoledione 2-Methylcarbazoledione 2-Methylcarbazoledione 2-Methylcarbazoledione 2-Methylcarbazoledione

7˘± ±111 77

1.0˘± ±0.1 0.1 1.0 1.0 0.1

1.7˘± ± 0.4 0.4 1.7 1.7 0.4

Isoquinoline-5,8-dione Isoquinoline-5,8-dione Isoquinoline-5,8-dione Isoquinoline-5,8-dione Isoquinoline-5,8-dione Isoquinoline-5,8-dione Isoquinoline-5,8-dione

12 ± ± 11 12 12 ± ± 11 12 12 ± 12˘±111 12

± 22 999 ± ± 22 9± ± 9 2 99˘±22

>50 >50 >50 >50 >50 >50 >50

Calothrixin BB Calothrixin Calothrixin B Calothrixin B Calothrixin B Calothrixin B Calothrixin B

1.5 ± ± 0.3 0.3 1.5 1.5 ± 0.3

13 ± ±1 13 13 ± 11

± 11 777 ± ± 11 7±

>50 >50 >50

2.3 ± ± 0.3 2.3 2.3 ± 0.3 0.3

2.3˘± ± 0.3 0.3 2.3 2.3 2.3 ±0.3 0.3

1.0 ± ± 0.1 0.1 1.0 1.0 ± ± 0.1 0.1 1.0

>50 >50 >50 >50 >50 >50 >50 >50 >50

10 ± ±2 10 10 ± 22

10˘± ±2 10 10 10 ± 222

1.7 ± ± 0.4 0.4 1.7 1.7 ± ± 0.4 0.4 1.7

In this 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 In this 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 In this study, calothrixin B was found to be the most potent against HeLa cells (0.25 µ M) followed by study, indolophenanthrene-7,13-dione analog (1.5 most µ M), M), indicating indicating the importance importance of(0.25 nitrogen In this calothrixin B was foundanalog to be the potent against HeLa cellsof µ M) followed by indolophenanthrene-7,13-dione (1.5 µ the nitrogen followed by indolophenanthrene-7,13-dione analog (1.5 µ M), indicating the importance of nitrogen followed byeffect indolophenanthrene-7,13-dione analog (1.5 µ M), M), indicating indicating the importance ofnocodazole, nitrogen The on the cell cycle was studiedanalog for these compounds using athe mitotic inhibitor followed by indolophenanthrene-7,13-dione (1.5 µ importance of nitrogen in ring ring D. D.by The activity was was further further decreased decreased with(1.5 theµ deletion of ring ringthe E importance of this this analog analog to form form followed indolophenanthrene-7,13-dione analog M), indicating of nitrogen in The activity with the deletion of E of to inwhich ring D. D. The activity was further decreased with the deletion of ring E of this analog to form in ring The activity was further decreased with the deletion of ring E of this analog to form blocks re-entry of cells into the G phase. Calothrixin B was observed to arrest the G1form phase 1the analogs in ring D. The activity was further decreased with the deletion of ring E of this analog to benzoncarbzoledione (1.8 µ M). The rest of showed moderate activity ranging from 7 to in ring D. The activity was further decreased with the deletion of ring E of this analog to form benzoncarbzoledione (1.8 µ M). The rest of the analogs showed moderate activity ranging from benzoncarbzoledione (1.8 µ µ M). M). The rest rest of the the analogs analogs showed moderate moderate activity activity ranging from 777atto tothe benzoncarbzoledione (1.8 The of showed ranging from to at 0.1 µM concentrations, whereas calothrixin A and N-methylcalothrixn B showed no effects benzoncarbzoledione (1.8 µ µ M). M). The The rest restanalog of the the analogs analogs showed moderate activity ranging from to 13 µ µ M, M, while while the the carbazole-1,4-dione carbazole-1,4-dione analog was found found to be bemoderate inactive.activity The results results were not77 as as benzoncarbzoledione (1.8 of showed ranging from to 13 was to inactive. The were not 13 µ M, while the carbazole-1,4-dione analog was found to be inactive. The results were not as 13same µ M, M, concentration. while the the carbazole-1,4-dione carbazole-1,4-dione analog was wascalothrixin found to to A beand inactive. The results results were were nottoas as At higher concentrations, N-methylcalothrixn B led cell 13 µ while analog found be inactive. The not consistent for the the P388 cell line, line, where where murrayaquinone murrayaquinone (2.3to µ M) M) and 2-methylcarbazoledione (1 µ µ M) M) 13 µ M, while theP388 carbazole-1,4-dione analog was found be inactive. The results were not as consistent for cell (2.3 µ and 2-methylcarbazoledione (1 consistent for the the P388 cell line, where murrayaquinone (2.3 µ M) and 2-methylcarbazoledione (1 µ M) consistent for P388 cell line, where murrayaquinone (2.3 µ M) and 2-methylcarbazoledione (1 µ M) accumulation in S and G /M phase. Compared to camptothecin, these effects were found to be readily 2line, consistent for the P388 cell cell where murrayaquinone murrayaquinone (2.3 µ M) M) and 2-methylcarbazoledione (1 µ µ M) M) were found foundfor tothe be more more active compared to calothrixin calothrixin B B (9 (9 µ M) M) orand other analogs, whereas whereas calothrixin calothrixin consistent P388 line, where (2.3 µ 2-methylcarbazoledione (1 were be active compared to or other analogs, were found to toCalothrixins be more more active compared to calothrixin calothrixin B activity (9 µµ µ M) M) or other analogs, whereas whereas calothrixin were found to be active compared to B (9 or other analogs, calothrixin reversible. were also evaluated for their against topoisomerase I. The effects of were found to be more active compared to calothrixin B (9 µ M) or other analogs, whereas calothrixin B and andfound 2-methylcarbazoledione showed similar similar activityBwith with 2.4 or µM Mother and analogs, 1.7 µ µ M, M, respectively, respectively, against were to be more active compared to calothrixin (9 µ M) whereas calothrixin B 2-methylcarbazoledione showed activity 2.4 µ 1.7 against B and and 2-methylcarbazoledione showed similar activity with 2.4control, µM M and andwhich 1.7 µ µ M, M, respectively, against B 2-methylcarbazoledione showed similar activity with 2.4 µ and 1.7 respectively, against calothrixins were studied keeping camptothecin as a positive is arespectively, known topoisomerase B and 2-methylcarbazoledione showed similar activity with 2.4 µ M and 1.7 µ M, against B and 2-methylcarbazoledione showed similar activity with 2.4 µ M and 1.7 µ M, respectively, 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 of of 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 derivative were synthesized and tested against CEM leukemia cells to measure their N-methylated 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 using an MTT MTT assay. The The activity of of calothrixin analogs analogs were compared compared to camptothecin, camptothecin, cytotoxicity cytotoxicity usingan an MTTassay. assay. Theactivity activity ofcalothrixin calothrixin analogswere were comparedto to camptothecin, which is known to cause irreversible DNA damage during the S phase. Delay in S phase due to to DNA which whichisisknown knownto tocause causeirreversible irreversibleDNA DNAdamage damageduring duringthe theSSphase. phase.Delay Delayin inSSphase phasedue due toDNA DNA damage is associated with a block in replication. The IC 50 value for calothrixin A was found to be 50 damage is associated with a block in replication. The IC 50 value for calothrixin A was found to damage is associated with a block in replication. The IC50 value for calothrixin A was found to be be five-fold higher than campothecin and and the other other two analogs analogs were observed observed to be be much less less potent, 16 of 21 Mar. Drugshigher 2016, 14,than 17 campothecin five-fold 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 N-methylcalothrixin B being the the least at at 5 µ M compared compared to calothrixin calothrixin B at 11 µ µ M (Table (Table 6). with with N-methylcalothrixinBB being being the least least at 55 µµM M compared to to calothrixinBBat at 1 µM M (Table6). 6). Table 6.6.Cytotoxicity effects against CEM leukemia cell line and topoisomerase I inhibition. Table6. Cytotoxicity effects against CEM leukemia cell line and topoisomerase I inhibition. Table effects against CEM leukemia cell IIinhibition. Table 6.Cytotoxicity Cytotoxicity effects against CEM leukemia cellline lineand andtopoisomerase topoisomerase inhibition. % Topo I DNA % NaCl Induced % Topo IIDNA Induced IC50 50 (µM) in CEM %% Topo DNA %NaCl NaCl Induced IC (µM) in CEM Topo I DNA % % NaCl Induced 50 IC Compound Structure Cleavage Reversibility IC50 50(µM) (µM)in inCEM CEM Compound Structure Compound Cleavage Reversibility Structure Leukemia Cells Compound Structure Cleavage Reversibility Leukemia Cells Cleavage at 5 µM Reversibility at 5 µM Leukemia at 5 µM at 5 µM LeukemiaCells Cells at at at55µM µM at55µM µM Camptothecin Camptothecin Camptothecin Camptothecin

0.04 ˘ ± 0.01 0.04 0.01 0.04 0.04±±0.01 0.01

100 100 100 100

100 100 100 100

Calothrixin A CalothrixinA A Calothrixin Calothrixin A

0.20 ˘ ± 0.02 0.20 0.02 0.20 0.20±±0.02 0.02

1818 18 18

17 17 17

17

Calothrixin B Calothrixin CalothrixinBB

1.05 ± 0.30 1.05 ±±0.30 1.05 0.30 1.05˘

13 13 1313

16 16 16

16

N-methycalothrixin B N-methycalothrixin N-methycalothrixinBB

5.13 ± 0.72 5.13 ±±0.72 5.13˘ 5.13 0.72

11 11 1111

13 13 13

13

The effect effect on the the cell cycle cycle was studied studied for these these compounds using using a mitotic inhibitor inhibitor The The effect on on the cell cell cycle was was studied for for these compounds compounds using aa mitotic mitotic inhibitor nocodazole, which blocks re-entry of cells into the G1 phase. Calothrixin B was observed to arrest the G111 phase 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 G G1 phase 0.1 medicinal μM concentrations, whereas potential calothrixinofAterrestrial and N-methylcalothrixn B showed no well Whileatthe and biosynthetic plants and microbes is fairly effects at the same concentration. At higher concentrations, calothrixin A and N-methylcalothrixn B effects the At concentrations, calothrixin BB effects at atcomparatively the same same concentration. concentration. At higher higher concentrations, calothrixin A and and N-methylcalothrixn N-methylcalothrixn studied, little is known regarding the chemistry and A biological activity of organisms in led to cell accumulation in S and G 2/M phase. Compared to camptothecin, these effects were found led to cell accumulation in S and G 2 /M phase. Compared to camptothecin, these effects were found 22/M phase. Compared to camptothecin, these effects were found led to cell accumulation in S and G the marine A unique group of oxygenic bacteria as cyanobacteria to be be readilyenvironment. reversible. Calothrixins Calothrixins were also also evaluatedphotosynthetic for their their activity againstknown topoisomerase I. to reversible. were evaluated for against topoisomerase I. to be readily readily reversible. Calothrixins were also evaluated for their activity activity against topoisomerase I. populate diverse habitats throughout the world. Their potential as a good source of new therapeutic The effects of calothrixins were studied keeping camptothecin as a positive control, which is The effects of calothrixins were studied keeping camptothecin as aa positive control, which isis aaa The effects of calothrixins were studied keeping camptothecin as positive control, which lead compounds has been realized the last few Calothrixins, which are cyanobacterial known topoisomerase I poison [60]. It Itduring was observed observed that decades. calothrixins A, B, B, and N-methylcalothrixin N-methylcalothrixin known known topoisomerase topoisomerase II poison poison [60]. [60]. It was was observed that that calothrixins calothrixins A, A, B, and and N-methylcalothrixin metabolites, have demonstrated a diversetopoisomerase range of bioactivities that include antimalarial, anticancer, B were were capable of stabilizing stabilizing the covalent covalent I/DNA complex complex at 18%, 18%, 13%, and and 11%, B B 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 properties. They have been observed to target various aspects of RNA synthesis in respectively, compared to 100% at 5 µ M concentration of camptothecin. respectively, respectively,compared compared to to100% 100%at at55 µµM Mconcentration concentration of of camptothecin. camptothecin. Calothrixins and their analogs were observed to be affecting different stages of cell cycle in a bacteria. Further investigation of the exact mechanism for their bioactivity is stillcell requiredinfor many Calothrixins Calothrixins and and their their analogs analogs were were observed observed to to be be affecting affecting different different stages stages of of cell cycle cycle in aa reversible manner, leading to low micromolar range cytotoxicities. Thus, calothrixins have shown aa analogs, which will be beneficial for the ongoing development and lead optimization. Several research reversible manner, leading to low micromolar range cytotoxicities. Thus, calothrixins have shown reversible manner, leading to low micromolar range cytotoxicities. Thus, calothrixins have shown a wide array array of developed activity including including antimalarial, anticancer, andnatural antibacterial. The This modes of action action of groups have synthetic routes toanticancer, obtain these products. review emphasized wide of antimalarial, and The of of wide array of activity activity including antimalarial, anticancer, and antibacterial. antibacterial. The modes modes of action of calothrixins’ bioactivities have not been fully deciphered yet. the syntheticbioactivities progress accomplished within the pastyet. six years in developing new synthetic routes. calothrixins’ have deciphered calothrixins’ bioactivities have not notbeen been fully fully deciphered yet.

4. Conclusions nocodazole, which blocks 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

Since 2009, 11 novel syntheses have been published to improve upon the efficiency in the production 4. Conclusions Conclusions 4. 4. calothrixins. Conclusions The number of steps in the various synthetic strategies ranges from 2 to 14, while the of 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 Ac2 O AcOEt AcOH AIBN

Acetyl chloride Acetic anhydride Ethyl acetate Acetic acid Azobisisobutyronitrile

Mar. Drugs 2016, 14, 17

AlCl3 BEt3 BF3 .OEt2 CAN CCl4 CH2 Cl2 CH3 CN COSY c-PC CSA CuI (CuOTf)2 .toluene DMAP DMF DMP DMSO DoM DTT Et2 NH Et3 SiH FeCl3 HCO2 H HDACs HIV H2 O2 K2 CO3 LDA LiTMP LTA MeOH Mn(OAc)3 MOM MTT NaCNBH3 NaH NaOH NaOMe NBS n-BuLi NH2 NH2 .H2 O Pb(OAc)4 PCC Pd/C PdCl2 (PPh3 )2 Pd2 (dba)3 Pd(dppf)Cl2 Pd(OAc)2

17 of 21

Aluminum chloride Triethylborane or triethylboron Boron trifluoride diethyl etherate Ceric ammonium nitrate Carbon tetrachloride Dichloromethane Acetonitrile Correlation spectroscopy c-Phycocyanin Camphorsulfonic acid Copper(I) iodide Trifluoromethanesulfonate toluene complex 4-Dimethylaminopyridine Dimethylformamide Dess-Martin periodinane Dimethyl sulfoxide Directed o-metalation Dithiothreitol Diethylamine Triethylsilane Iron(III) chloride Formic acid Histone deacetylases Human immunodeficiency virus Hydrogen peroxide Potassium carbonate Lithium diisopropylamide Lithium tetramethylpiperidide Lead tetracetate Methanol Manganese triacetate Methoxymethyl protecting group 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Sodium cyanoborohydride Sodium hydride Sodium hydroxide Sodium methoxide N-bromosuccinimide n-Butyllithium Hydrazine hydrate solution Lead(IV) acetate Pyridinium chlorochromate Palladium on carbon Bis(triphenylphosphine)palladium(II) dichloride Tris(dibenzylideneacetone)dipalladium(0) [1,11 -Bis(diphenylphosphino)ferrocene] dichloropalladium(II) Palladium acetate

Mar. Drugs 2016, 14, 17

Pd(TFA)2 PhCN (PhSe)2 PMB PMBCl P(o-tol)3 POCl3 PPh3 p-TSA PTT rac-BINAP ROS TBAD TBHP Tf2 O THF THP TMG TMP TMSI

18 of 21

palladium(II) trifluoroacetate Benzonitrile Diphenyl diselenide p-Methoxybenzyl p-Methoxybenzyl chloride Tri(o-tolyl)phosphine Phosphoryl chloride Triphenylphosphine p-Toluenesulfonic acid Polytrimethylene terephthalate Racemic 2,21 -bis(diphenylphosphino)-1,11 -binaphthalene Reactive oxygen species Bis(tetrabutylammonium) dichromate Tert-butyl hydroperoxide Trifluoromethanesulfonic anhydride Tetrahydrofuran Tetrahydropyran 1,1,3,3-Tetramethylguanidine 2,2,6,6-Tetramethylpiperidine Trimethylsilyl iodide

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