Article pubs.acs.org/jnp
Probing the Structure−Activity Relationship of the Natural Antifouling Agent Polygodial against both Micro- and Macrofoulers by Semisynthetic Modification Lindon W. K. Moodie,†,¶ Rozenn Trepos,‡ Gunnar Cervin,§ Lesley Larsen,⊥ David S. Larsen,⊥ Henrik Pavia,§ Claire Hellio,‡ Patrick Cahill,∥ and Johan Svenson*,†,# †
Department of Chemistry, UiT The Arctic University of Norway, Breivika, N-9037, Tromsø, Norway Biodimar LEMAR UMR 6539, Université de Bretagne Occidentale, 6 Avenue le Gorgeu, 29200 Brest, France § Department of Marine Sciences - Tjärnö, University of Gothenburg, SE-452 96 Strömstad, Sweden ⊥ Department of Chemistry, University of Otago, P.O. Box 56, Dunedin, New Zealand ∥ Cawthron Institute, 98 Halifax Street East, Nelson 7010, New Zealand # Department of Chemistry, Material and Surfaces, SP Technical Research Institute of Sweden, Box 857, SE-501 15 Borås, Sweden ‡
S Supporting Information *
ABSTRACT: The current study represents the first comprehensive investigation into the general antifouling activities of the natural drimane sesquiterpene polygodial. Previous studies have highlighted a high antifouling effect toward macrofoulers, such as ascidians, tubeworms, and mussels, but no reports about the general antifouling effect of polygodial have been communicated before. To probe the structural and chemical basis for antifouling activity, a library of 11 polygodial analogues was prepared by semisynthesis. The library was designed to yield derivatives with ranging polarities and the ability to engage in both covalent and noncovalent interactions, while still remaining within the drimane sesquiterpene scaffold. The prepared compounds were screened against 14 relevant marine micro- and macrofouling species. Several of the polygodial analogues displayed inhibitory activities at sub-microgram/mL concentrations. These antifouling effects were most pronounced against the macrofouling ascidian Ciona savignyi and the barnacle Balanus improvisus, with inhibitory activities observed for selected compounds comparable or superior to several commercial antifouling products. The inhibitory activity against the microfouling bacteria and microalgae was reversible and significantly less pronounced than for the macrofoulers. This study illustrates that the macro- and microfoulers are targeted by the compounds via different mechanisms. (SAR) studies focusing on the cytotoxic,19−21 antifeedant,22 larvicidal,23 antifungal,24 and odiferous properties25 of 1.
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olygodial (1) is the bioactive constituent of several species of terrestrial plants1−3 and has also been isolated from marine sponges4 and nudibranchs.5 This drimane-type sesquiterpene dialdehyde has exhibited antiallergic,6 antifeedant,7 antifouling,8 antihyperalgesic,2 anti-inflammatory,6 antimicrobial,9,10 antinociception,11 and vasorelaxation12 activities. Such a diverse range of biological effects infers multiple modes of action for 1. It has been demonstrated that polygodial acts as a nonionic surfactant to disrupt the cell membrane of microorganisms,13 yet this compound also appears to interact with intracellular components such as glutathione,14 ATPase,15 vanilloid receptors,16 and Lcysteine-containing materials.13 This plethora of biological effects makes 1 potentially useful as a lead compound for a range of applications. The chemistry and biological properties of the drimane sesquiterpenes, which include 1, have been the subject of two extensive reviews, with the most recent published in 2004.17,18 Chemical modifications of the dialdehyde and alkene motifs of 1 have resulted in a number of structure−activity relationship © 2017 American Chemical Society and American Society of Pharmacognosy
Compound 1 has been shown recently to display powerful antifouling properties against the marine macrofoulers Ciona savignyi (Ascidiacea), Spirobranchus caraniferus (Polychaeta), and Mytilus galloprovincialis (Bivalvia).8 The low nanomolar 50% effective concentrations (EC50) against larval metamorphosis of the marine macrofoulers imply that 1 is a potentially useful lead in the search for new antifouling compounds, especially as this substance also has been shown to display a low toxicity toward higher organisms and is not known to bioaccumulate.26,27 Received: November 16, 2016 Published: February 7, 2017 515
DOI: 10.1021/acs.jnatprod.6b01056 J. Nat. Prod. 2017, 80, 515−525
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Scheme 1. Generation of Polygodial (1) Analogues and Structure of Reference Compounds
Scheme 2. Synthetic Routes to Structural Analogues Based on Polygodial (1)
marine biofouling organisms and appears to be contact active, whereby it can inhibit settlement and metamorphosis when bound to a surface.8 Traditionally, antifouling formulations slowly release a bioactive agent, with high potential for collateral environmental harm.36 Eliminating the need to release a biocide into the sea makes polygodial a particularly attractive alternative to conventional antifouling biocides. However, the SAR of 1 in relation to marine antifouling has, to the best of our knowledge, not been explored. Neither has the antifouling effect against other common foulers such as barnacles, marine bacteria, and microalgae been assessed. Establishing a SAR for 1 against marine biofouling organisms would provide useful insights into the mode of action of this compound. Establishing the width of the marine bioactivity spectrum of 1 will guide the future development of 1 or potentially commercially viable analogues thereof for marine antifouling applications. In this study, semisynthetic modifications of 1 yielded a library of analogues that was used to probe the drimane scaffold SAR
Biofouling is the unwanted colonization and growth of organisms on materials and surfaces submerged in water.28−30 It involves both microfouling, by marine microorganisms such as bacteria and microalgae, and macrofouling by larger organisms, as typified by mussels, barnacles, ascidians, and macroalgae.31 All activities at sea, including shipping, aquaculture, and the ocean energy sector, face reduced efficiencies and increased costs due to the settlement and growth of marine organisms.32 The management of fouling in the marine environment is an ageold problem,33 with the demand for effective environmentally friendly antifouling technologies having accelerated in recent years due to traditional ecotoxic biocidal approaches facing heavy regulatory scrutiny.28 Natural products have been heralded as possessing particular potential in yielding new nontoxic antifouling solutions.28,34 Both stationary marine organisms that have evolved mechanisms to limit their colonization by fouling species and natural products elicited from terrestrial sources have been studied for their antifouling properties.31,35 Compound 1 potently inhibits settlement and metamorphosis of 516
DOI: 10.1021/acs.jnatprod.6b01056 J. Nat. Prod. 2017, 80, 515−525
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Figure 1. Metamorphosis inhibition of C. savignyi larvae exposed to 1, 3, and 5. Inhibition of metamorphosis is relative to corresponding blank controls, and values are means (n = 3) ± standard error.
Figure 2. Effects of 1, 4, and 6 on the settlement of B. improvisus cyprid larvae presented as percentages of settled (dark gray columns) and dead cyprids (light gray columns) and given as means ± standard error (n = 4). Remaining larvae were free swimming. Filtered seawater (FSW) and DMSO (0.1%, v/ v) were used as the negative control.
conditions yielded cinnamolide38 (5). The alkene of both 3 and 5 was reduced by hydrogenation, to probe the role of the unsaturated motif in bioactivity. The alkene of 3 provided a useful functional handle for the oxidation of the B ring, which underwent dihydroxylation (9) or epoxidation (10). Treatment of diacetate 8 with SeO2 induced a selective allylic oxidation to afford 11. Deprotection under basic conditions yielded triol 12. These transformations provided a series of drimane compounds with differing polarities. Additionally, the epimerized version of 1, epipolygodial (2), was also synthesized. Macrofouling Activity. Compound 1 has an established bioactivity against the larvae of tunicates, mussels, and tubeworms in the nanomolar range and has been investigated as a protective agent to prevent unwanted growth in aquaculture.39 In addition, 1 has also been shown to work as a contact active antifouling biocide.8,39 As such, the antifouling effect of a coating incorporating 1 does not rely on this compound being released into the surrounding environment, a particularly attractive scenario for developing marine antifouling coatings that release a minimum amount of bioactive compounds to the surrounding water.40 While the SAR activities of 1 have been investigated against fungi, Drosophila melanogaster,23,24 and other biological targets, no attempts have been reported to
against the macrofouling organisms C. savignyi and Balanus improvisus (Maxillopoda). The prepared compounds were also screened against a selection of microfouling organisms, including both marine bacteria and marine microalgae, to establish the general antifouling properties of the compounds. The biological effects of the prepared compounds were compared to structural and commercial antifouling reference compounds. The natural compound drimendiol (3), a close analogue of 1, was also included and evaluated as an antifoulant for the first time.
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RESULTS AND DISCUSSION Compound Design. Although 1 and structurally related drimane natural products have been accessed by total synthesis,17,18 the high abundance of 1 in various plant species and its ease of isolation37 encouraged a semisynthetic approach to developing analogues. Following extraction from Drimys winteri (Winteraceae) and a single chromatographic step, 1 was isolated in a practical yield (of 2.5% of dried plant material). Compound 3, obtained by the NaBH4-mediated reduction of 1,23 was identified as a suitable starting point to access a diverse range of analogues of 1. The hydroxy group functional handles of 3 could be masked as either the corresponding methyl ethers (7) or acetates (8). Furthermore, the reaction of 3 under oxidative 517
DOI: 10.1021/acs.jnatprod.6b01056 J. Nat. Prod. 2017, 80, 515−525
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compounds produced share the bicyclic drimane framework, with the differences lying mainly within the degree of substitution and oxidation. While the compounds displayed a range of ClogP values, no apparent link between polarity and inhibitory effect was noticeable. Going away from the drimane scaffold appears to be detrimental, as shown by the lack of activity for 1,5-decalindol and the relatively high inhibitory activity of sclareol. Compound 1 is reported typically to have multiple modes of action for a single biological activity. The unusually high potency of 1 against C. savignyi could reflect that this compound interferes with biochemical targets linked to tail resorption and confers generally toxic effects from disruption of cell membranes by way of its surfactant properties. Comparing the potency of 1 (25-fold higher inhibitory activity in comparison to the most active analogues) with epi-polygodial (2) and analogues with a similar CLogP suggests that the superior bioactivity is reliant on both the presence and orientation of the dialdehyde motif and is not solely dictated by polarity. No prominent inhibitory activity was seen for the natural product drimendiol (3) in comparison to the other synthetic derivatives. A similar trend between 1 and 3 has been reported for antifeedant activity against the cotton worm Spodoptera littoralis.48 Activities observed against the barnacle B. improvisus ranged between 0.1 and >5 μg/mL, and a closer link between structure and activity was observed. Compound 1 was highly active, with an IC50 of 0.25 μg/mL. This represents an inhibitory activity higher than those reported for the large majority of previously investigated antifouling natural products.34,49 The effect of compound hydrophilicity on settlement inhibition is more pronounced against the balanide cyprids. All of the active compounds (200 0.2c
0.25 >5 0.5 1.5 1.5 0.1 >5 >5 >5 5 >5 >5 n.t.b n.t. 0.25
a Calculated using ChemBio3D Ultra 14.0. bNot tested. cData from ref 41 against Ciona intestinalis.41
All of the synthetic compounds examined were potent inhibitors of C. savignyi larval settlement and metamorphosis, with submicrogram/mL EC50 values (Table 1). The natural compound 1 was the most effective inhibitor, with an EC50 of 4 ng/mL, corresponding well with values reported in previous studies.8 Both 2 and 11 displayed an EC50 value of 0.1 μg/mL and were the most potent synthetic inhibitors, while the remaining compounds were effective between 0.1 and 1.0 μg/mL. The diterpenoid reference compound sclareol was also active, while 1,5-decalindol did not display significant inhibition at the concentrations tested. It has been shown previously that 1 halts the metamorphosis of C. savignyi at the tail-resorption stage.39 The tail-resorption stage is completed within 20 min of metamorphosis onset and is characterized by massive apoptosis of cells in the tail and reordering of structures in the trunk.42,43 A number of studies have investigated the biochemical drivers of ascidian metamorphosis,44 providing potential links between the known modes of action of 1 in other scenarios and inhibition of tail resorption. The established nonionic surfactant properties of 1 can confer generally toxic effects on C. savignyi larvae,45 but specific biochemical targets are also apparent. In particular, 1 is known to form covalent adducts intracellularly with amine groups.22,46,47 From a structural perspective, it is interesting that all of the compounds prepared displayed pronounced inhibitory activity against larval metamorphosis of C. savignyi. All the synthetic 518
DOI: 10.1021/acs.jnatprod.6b01056 J. Nat. Prod. 2017, 80, 515−525
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that the general “drimane effect” is sufficient to generate an antifouling effect similar to that recently reported for the commercial antifouling booster biocides chlorothalonil (EC50 0.1 μg/mL) and tolylfluanid (EC50 0.3 μg/mL).50 These inhibitory values for the commercial biocides are almost identical to those reported by Bellas41 against C. intestinalis, implying a comparable sensitivity of these two related species despite diverging from a common ancestor more than 180 million years ago.51 Bellas also reported an EC50 for Sea-nine at 0.15 μg/mL against C. intestinalis.41 This general effect does not appear to extend to B. improvisus, and several of the investigated compounds were inactive at 5 μg/mL. This suggests that the drimane scaffold alone is insufficient for a general inhibitory effect against barnacle larvae. Compounds 1, 3, and 6 were nevertheless as active as the commercial control Sea-nine (EC50 0.25 μg/mL), illustrating a high inhibitory effect for select chemistries within the drimane scaffold. Microfouling Activity. The capacity of compounds to interfere with microfouling was assessed by screening the prepared library against a range of species of marine microalgae and bacteria that are involved in the initial formation of the fouling biofilm and biocorrosion.29 It is of interest that the effect of 1 against marine microorganisms has not been reported previously. In total, four microalgae and eight marine bacterial strains were included. The effect on both adhesion and growth (A and G, respectively, in the tables) was studied, and the results expressed as the minimal inhibitory concentration (MIC) are summarized in Tables 2 and 3.
Table 3. MIC (in μg/mL) of Polygodial (1) and Analogues against Marine Bacteriaa S. putrefaciens
C. closterium 1 2 3 4 5 6 7 8 9 10 11 12 Sea-nine a b
H. cof feaformis
G
A
G
A
G
10 − 10 − n.t.c 1 − 10 − 1 − 10
