marine drugs Review

Mini-Review: Antifouling Natural Products from Marine Microorganisms and Their Synthetic Analogs Kai-Ling Wang 1,2,3 1

2 3 4 5 6

*

ID

, Ze-Hong Wu 4,5 , Yu Wang 1 , Chang-Yun Wang 2,3,6, * and Ying Xu 1, *

Shenzhen Key Laboratory of Marine Bioresource & Eco-environmental Science, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China; [email protected] (K.-L.W.); [email protected] (Y.W.) Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266200, China The Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen 518033, China; [email protected] Integrated Chinese and Western Medicine Postdoctoral research station, Jinan University, Guangzhou 510632, China Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China Correspondence: [email protected] (C.-Y.W.); [email protected] (Y.X.); Tel.: +86-532-82-031-536 (C.-Y.W.); +86-755-26-958-849 (Y.X.); Fax: +86-532-82-031-536 (C.-Y.W.); +86-755-26-534-274 (Y.X.)

Received: 19 May 2017; Accepted: 12 July 2017; Published: 28 August 2017

Abstract: Biofouling causes huge economic loss and generates serious ecological issues worldwide. Marine coatings incorporated with antifouling (AF) compounds are the most common practices to prevent biofouling. With a ban of organotins and an increase in the restrictions regarding the use of other AF alternatives, exploring effective and environmentally friendly AF compounds has become an urgent demand for marine coating industries. Marine microorganisms, which have the largest biodiversity, represent a rich and important source of bioactive compounds and have many medical and industrial applications. This review summarizes 89 natural products from marine microorganisms and 13 of their synthetic analogs with AF EC50 values ≤ 25 µg/mL from 1995 (the first report about marine microorganism-derived AF compounds) to April 2017. Some compounds with the EC50 values < 5 µg/mL and LC50 /EC50 ratios > 50 are highlighted as potential AF compounds, and the preliminary analysis of structure-relationship (SAR) of these compounds is also discussed briefly. In the last part, current challenges and future research perspectives are proposed based on opinions from many previous reviews. To provide clear guidance for the readers, the AF compounds from microorganisms and their synthetic analogs in this review are categorized into ten types, including fatty acids, lactones, terpenes, steroids, benzenoids, phenyl ethers, polyketides, alkaloids, nucleosides and peptides. In addition to the major AF compounds which targets macro-foulers, this review also includes compounds with antibiofilm activity since micro-foulers also contribute significantly to the biofouling communities. Keywords: antifouling; biofouling; marine microorganisms; marine natural products

1. Introduction Biofouling is defined as the undesirable colonization of submerged man-made surfaces by fouling organisms, including micro-organisms such as bacteria, algae and protozoa, and macro-organisms such as barnacles, bryozoans and tubeworms [1,2]. These fouling organisms not only cause huge material and economic loss in marine operations [3–5], but also create a series of environmental problems such

Mar. Drugs 2017, 15, 266; doi:10.3390/md15090266

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as the spread of invasive species [6,7]. Coating the substrata with antifouling (AF) paints containing antifoulants or AF compounds is the most commonly used strategy to prevent marine biofouling. However, due to their toxicities toward non-target organisms, some AF compounds have raised many environmental issues and consequently led to increasing regulation of their usage [8–10]. For instance, the organotin compounds have been prohibited worldwide by the International Maritime Organization (IMO) since September 2008. Moreover, some alternative AF biocides of tributyltin (TBT), such as Irgarol 1051 and diuron, have been banned by many European countries in recent years because of the increasing evidence of their environmental risks [10,11]. Thus, there is an urgent demand for search of novel AF compounds without causing environmental issues. Marine natural products are a potential source for the discovery of AF compounds. Over the last several decades, many compounds with AF EC50 values < 25.0 µg/mL (a standard established by the U.S. Navy program, actual EC50 values should be less than 5 µg/mL when selecting candidate AF compounds) have been isolated from seaweeds and marine invertebrates [12–17]. However, a major difficulty involved in commercialization of these active compounds for the marine coating industries is the supply issue. In contrast, marine microorganisms have recently attracted greater attention due to a number of benefits for industries, including the possibility of supplying large amounts of compounds through fermentation and genetic modification of the source organisms, as well as the ability of resource regeneration [18–20]. Indeed, many AF metabolites produced by marine microorganisms have been demonstrated as effective inhibitors of biofouling organisms, and some of these active substances have been considered as low- or even non-toxic antifoulants due to their high LC50 /EC50 ratios (a compound with LC50 /EC50 ratio > 15 is considered as a nontoxic antifoulant, but a much higher therapeutic ratio > 50 should be used when selecting candidate AF compounds) [15]. Although AF compounds have long been discovered from marine invertebrates, it was not until 1995 that the first publication describing AF compounds from marine microorganisms appeared [13,21]. In this paper, the AF metabolites were identified from the culture broth of a marine sponge Halichondria okada-associated bacterium Alteromonas sp. KK10304 [21]. Hence, this review describes 89 selected AF metabolites (EC50 values < 25.0 µg/mL) isolated from marine microorganisms during the period between 1995 and April 2017. Also included are 13 synthetic analogs of these AF natural products. All the compounds in this review, except the ones with antibiofilm activity, are sequentially introduced based on their chemical structures, including fatty acids, lactones, terpenes, steroids, benzenoids, phenyl ethers, polyketides, alkaloids, nucleosides and peptides. Potentials and challenges are also discussed with respect to the development of environmentally benign AF paints using natural products isolated from marine microorganisms. 2. Fatty Acids and Lactones Two fatty acids, 2-hydroxymyristic acid (HMA) (1) and cis-9-oleic acid (COA) (2) (Figure 1), were isolated from the chloroform extract of a marine bacterium Shewanella oneidensis SCH0402 and blocked the germination of green algae Ulva pertusa spores completely at concentrations of 10 and 100 µg/mL, respectively. More significantly, no attachment of micro-fouling or macro-fouling organisms occurred on the surface of panels treated with 10% (w/w) HMA or COA after being immersed for 1.5 years in seawater, suggesting that this class of metabolites might have great value in commercial application [22]. Subsequently, AF bioassay-guided isolation afforded an HMA-like homolog, 12-methyltetradecanoid acid (12-MTA) (3) (Figure 1), derived from a deep-sea actinomycete Streptomyces sp. This compound showed strong AF activity against the polychaete Hydroides elegans larvae with an EC50 value of 0.6 µg/mL, while its toxicity was very low (LC50 /EC50 ratio > 133.5). Studies on AF mechanism of 12-MTA against H. elegans revealed that the fatty acid mainly inhibited larval settlement through the down-regulation of the GTPase-activating gene and up-regulation of the ATP synthase gene [23]. (3R,5S)-3,5-dihydroxydecanoic acid (4) and a novel ester aureobasidin (5) (Figure 1) with an unusual 4,6-dihydroxydecanoic acid residue, obtained from the fermentation broth

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of a marine-derived fungus Aureobasidium sp., were demonstrated to display larval inhibition against the attachment of B. amphitrite, but the detailed data are not available [24]. Mar. Drugs 2017, 15, 266 3 of 20 O

O

OH

OH 1

OH

2 O

O

OH

OH 3

OH OH 4

OH OH O O O O OH

OH 5

Figure 1. Chemical Structures of Fatty Acids 1–5. Figure 1. Chemical Structures of Fatty Acids 1–5.

Five structurally similar butenolides (6–10) (Figure 2) were isolated from the crude extract of a

Five structurally similar butenolides (6–10) (Figure 2) were isolated from the crude extract of deep-sea-derived Streptomyces albidoflavus UST040711-291, among which 6–8 showed moderate AF a deep-sea-derived Streptomyces albidoflavus which 6–8 9.65 showed moderate activities against the larval settlement of B.UST040711-291, amphitrite with the among EC50 values of 14.81, and 8.67 AF activities the [25]. larval settlement of B.compound, amphitrite with the EC50 values of 9.65 and μg/mL,against respectively Another butenolide 10-methylundec-3-en-4-olide (9),14.81, isolated from the North Sea Streptomyces sp. strain GWS-BW-H5, was also inhibitory to B. amphitrite larvae 8.67 µg/mL, respectively [25]. Another butenolide compound, 10-methylundec-3-en-4-olide (9), with an EC 50 value of 4.82 μg/mL [25,26]. Based on the preliminary analysis of the structure–activity isolated from the North Sea Streptomyces sp. strain GWS-BW-H5, was also inhibitory to B. amphitrite relationship (SAR), the 2-furanone ring was suspected to be the functional group responsible for AF larvae with an EC50 value of 4.82 µg/mL [25,26]. Based on the preliminary analysis of the activities of these compounds, and the alkyl side-chain might affect their bioactivities by varying the structure–activity relationship (SAR), the 2-furanone ring was suspected to be the functional group lipophilicity of these compounds. A series of butenolide derivatives were then synthesized, among responsible forcompounds AF activities of these compounds, and active the alkyl side-chain their bioactivities which 10–13 (Figure 2) were highly in inhibiting themight larvalaffect settlement of B. by varying the lipophilicity of these compounds. A and series ofμg/mL, butenolide derivatives were then amphitrite with the EC50 values of 0.518, 0.663, 0.722 0.827 respectively, while their toxicityamong towardswhich the B.compounds amphitrite cyprids was very low (LC50/EC50 > 97, 61, 73 and the 63, larval synthesized, 10–13 (Figure 2) were highlyratios active in inhibiting respectively) [27]. In addition, compound 10 also displayed strong and non-toxic AF activity against settlement of B. amphitrite with the EC50 values of 0.518, 0.663, 0.722 and 0.827 µg/mL, respectively, the larval settlement of B. neritina and H. elegans with the EC50 values of 0.199 and 0.0168 μg/mL and while their toxicity towards the B. amphitrite cyprids was very low (LC50 /EC50 ratios > 97, 61, 73 and 63, LC50/EC50 ratios higher than 250 and 119, respectively, indicating its broad-spectrum AF effects. respectively) In addition, compound 10AF also displayed strong andofnon-toxic activity Indeed,[27]. compound 10 exhibited excellent activity at a concentration 5% (w/w) inAF field testing against the larval settlement of Hong B. neritina and elegans with EC50 values 0.0168 µg/mL [25]. Interestingly, and Cho [28]H. also isolated two the compounds 14 and of 15 0.199 (Figureand 2) belonging the butenolide fromthan a seaweed epibiotic Streptomyces violaceoruber SCH-09. Both and LCto higher 250 and 119,bacterium respectively, indicating its broad-spectrum AF 50 /EC 50 ratios class compounds displayed significant inhibition against the seaweed U. pertusa, the diatom effects. Indeed, compound 10 exhibited excellent AF activity at a concentration of 5% (w/w) in field Naviculaannexa, and theHong musseland Mytilus EC50 values from 0.02 0.1 μg/mL, testing [25]. Interestingly, Choedulis [28]with alsothe isolated tworanging compounds 14toand 15 (Figure 2) whereas these two compounds showed high therapeutic ratios (LC50/EC50 ratios > 92) for the three belonging to the butenolide class from a seaweed epibiotic bacterium Streptomyces violaceoruber organisms tested. As mentioned above, butenolides may be considered as promising candidate SCH-09.antifoulants Both compounds displayed inhibition againstinthe seaweed U. pertusa, the diatom due to their simple significant structures, strong bioactivities field assays, and non-toxicity Naviculaannexa, and the mussel Mytilus fouling edulis with the EC values ranging from 0.02 to 0.1 towards the larvae of representative organisms. Recently, Zhang et al. [29] and Qian et al.µg/mL, 50 [30] reported the larvae of B. amphitrite and B. neritina responded to this class of butenolides by whereas these two compounds showed high therapeutic ratios (LC50 /EC50 ratios > 92) for the three modulating energy- andabove, stress-related proteins. Another lactone with organisms tested. their As mentioned butenolides may be considered as 2-furanone promisingring, candidate maculalactone A (16) (Figure 2), was the most abundant secondary metabolite from a marine antifoulants due to their simple structures, strong bioactivities in field assays, and non-toxicity towards cyanobacterium Kyrtuthrix maculans, which showed a toxic effect on the naupliar larvae of the the larvae of representative fouling organisms. Recently, Zhang et al. [29] and Qian et al. [30] reported barnacles B. amphitrite , Tetraclita japonica and Ibla cumingii with the LC50 values ranging from 1.1 to the larvae of B. amphitrite and B. neritina responded this classalso of showed butenolides by modulating 5.2 μg/mL. In the preliminary field investigation, thistocompound some inhibitory their energyand stress-related proteins. Another lactone with 2-furanone ring, maculalactone effects on marine foulers, especially against the bivalves, at both concentrations: 0.1% and 1% (w/w) in a marine environment [31]. A (16) (Figure 2), was the most abundant secondary metabolite from a marine cyanobacterium Kyrtuthrix maculans, which showed a toxic effect on the naupliar larvae of the barnacles B. amphitrite, Tetraclita japonica and Ibla cumingii with the LC50 values ranging from 1.1 to 5.2 µg/mL. In the preliminary field investigation, this compound also showed some inhibitory effects on marine foulers, especially against the bivalves, at both concentrations: 0.1% and 1% (w/w) in a marine environment [31].

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O

O O

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6

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9

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OO

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n

N HO H

1111 n =n3= 3 1212 n =n4= 4 1313 n =n5= 5

O

R

R O

O

O

O

O O OO

O

14 R =

14 R =

15 R =

16

15 R =

O

16

O O

Figure 2. Chemical Structures of Lactones 6–16.

Figure 2. Chemical Structures of Lactones 6–16. Figure 2. Chemical Structures of Lactones 6–16.

3. Terpenes and Steroids

3. Terpenes Terpenes and 3. and Steroids Steroids

Lobocompactol (17) (Figure 3), an active AF diterpene, was isolated from a marine-derived

Lobocompactol (17) (Figure (Figure 3), an an active was aa marine-derived actinomycete Streptomyces cinnabarinus PK209 AF andditerpene, exhibited significant AF from activity against the Lobocompactol (17) 3), active AF diterpene, was isolated isolated from marine-derived macro-alga U. pertusa and the diatom N. annexa (EC 50exhibited values of 0.18 and 0.43 μg/mL, respectively) actinomycete Streptomyces cinnabarinus PK209 and significant AF activity against the actinomycete Streptomyces cinnabarinus PK209 and exhibited significant AF activity against the with a large therapeutic ratio (LC50/EC 50 ratios(EC of 46.2 and 71.6, respectively), indicating that it might with macro-alga U. pertusa and the diatom N. annexa values of 0.18 and 0.43 µg/mL, respectively) 50 50 values of 0.18 and 0.43 μg/mL, respectively) macro-alga U. pertusa and the diatom N. annexa (EC betherapeutic a promising ratio candidate as/EC a nontoxic antifoulant targeting algae specifically. Lobocompactol also be a a large (LC ratios of 46.2 and 71.6,71.6, respectively), indicating that that it might 50(LC with a large therapeutic ratio 5050 /EC 50 ratios of 46.2 and respectively), indicating it might inhibited the growth of the fouling bacteria Pseudomonas aeruginosa KNP-5 and Pseudomonas sp. promising candidate as a nontoxic antifoulant targeting algae specifically. Lobocompactol also inhibited be a promising as a nontoxic antifoulant algae specifically. KNP-8 withcandidate the MIC values of 66 μg/mL and 112 targeting μg/mL, respectively [32]. FourLobocompactol bisabolane-type also the growththe of the fouling bacteria Pseudomonas aeruginosa KNP-5 and Pseudomonas sp. KNP-8 with the inhibited growth of the fouling bacteria Pseudomonas aeruginosa KNP-5 and sesquiterpenoids were isolated from the fungus Aspergillus sp. associated with Pseudomonas the sponge sp. MIC Xestospongia values 66testudinaria. µg/mL and 112 µg/mL, respectively [32]. Four bisabolane-type sesquiterpenoids KNP-8 with of the MIC values of 66 μg/mL and 112 μg/mL, respectively [32]. Four bisabolane-type Among them, compound 18 (Figure 3) could inhibit the larval settlement of were isolated from the fungus associated with compound thesp. sponge testudinaria. sesquiterpenoids were isolated from thesp.of fungus Aspergillus associated sponge B. amphitrite completely at theAspergillus concentration 25.0 μg/mL while 19Xestospongia (Figurewith 3) wasthe highly Among them, compound 18 (Figure 3) could inhibit the larval settlement of B. amphitrite completely toxic to the larvae of B.Among amphitrite at this concentration [33]. Xestospongia testudinaria. them, compound 18 (Figure 3) could inhibit the larval settlement of at amphitrite the concentration of 25.0 µg/mL while compound 19 (Figure 3) was highly toxic to3) the larvae of B. completely at the concentration of 25.0 μg/mL while compound 19 (Figure was highly B. amphitrite at this concentration [33]. HO HOconcentration [33]. toxic to the larvae of B. amphitrite at this O

O

OH

OH

17

HO O

OH

OH

O

HO

18

OH Figure 3. Chemical Structures of Terpenes 17–19. OH

OH 19

OH 19

17steroids 20 and 21 (Figure 4), obtained 18 from the crude extract of a red alga epiphyte Two filamentous bacterium Leucothrix mucor, were found to inhibit the17–19. spore settlement of U. pertusa Figure Figure 3. 3. Chemical Chemical Structures Structures of of Terpenes Terpenes 17–19. zoospores (EC50 values of 1.2 and 2.1 μg/mL, respectively) and the growth of diatom (EC50 values of 5.2 and 7.5 μg/mL, respectively). Both compounds also inhibited the attachment of the Two (Figure 4), 4), steroids 20 and 21 (Figure obtained from the crude extract of a red alga epiphyte biofilm-forming bacterial strains P. aeruginosa KNP-3 (MIC values of 32 and 56 μg/mL, respectively) filamentous bacterium Leucothrix mucor, were Leucothrix mucor, were found found to to inhibit inhibit the spore settlement of U. pertusa and Alteromonas sp. KNS-8 (MIC values of 66 and 90 μg/mL, respectively) [34].

50 values μg/mL, respectively) (EC50 50 values of zoospores (EC50 values of 1.2 and 2.1 µg/mL, respectively) and the growth of diatom (EC 5.2 and and7.57.5 μg/mL, respectively). Both compounds also the inhibited theofattachment of the µg/mL, respectively). Both compounds also inhibited attachment the biofilm-forming biofilm-forming strains P. (MIC aeruginosa KNP-3 (MIC ofrespectively) 32 and 56 μg/mL, respectively) bacterial strains P.bacterial aeruginosa KNP-3 values of 32 and 56values µg/mL, and Alteromonas sp. and Alteromonas sp. of KNS-8 (MIC values ofrespectively) 66 and 90 μg/mL, KNS-8 (MIC values 66 and 90 µg/mL, [34]. respectively) [34].

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5 of 21 5 of 20 5 of 20

O O

OH OH

HO HO

O O

HO HO HO HO

OH OH

HO HO

O O 20 20

OH OH 21 21

Figure Figure 4. 4. Chemical Chemical Structures Structures of of Steroids Steroids 20–21. 20–21. Figure 4. Chemical Structures of Steroids 20–21.

4. 4. Benzenoids Benzenoids and and Phenyl Phenyl Ethers Ethers 4. Benzenoids and Phenyl Ethers Until to our our best knowledge, knowledge, only four four natural benzenoids benzenoids with Until now, now, to with chlorine chlorine atom atom have have been been Until now, to our best best knowledge, only only four natural natural benzenoids with chlorine atom have been isolated from three marine fungal strains and displayed inhibitive effects on microor isolated from three marine fungal strains and displayed inhibitive effects on microor macro-foulers. isolated from three marine fungal strains and displayed inhibitive effects on micro- or macro-foulers. Themetabolite first recorded metabolite 3-chloro-2,5-dihydroxybenzyl (Figure The first recorded 3-chloro-2,5-dihydroxybenzyl alcohol (22) (Figurealcohol 5) was (22) isolated from5) macro-foulers. The first recorded metabolite 3-chloro-2,5-dihydroxybenzyl alcohol (22) (Figure 5)a was isolated from a marine fungus Ampelomyces sp. UST040128. This compound showed an marine fungus Ampelomyces sp. UST040128. This compound showed an inhibitive effect on the larval was isolated from a marine fungus Ampelomyces sp. UST040128. This compound showed an inhibitive the larval settlement B. amphitrite H. elegans with the3.192 EC50 values settlementeffect of B. on amphitrite and H. elegansof the EC50and values ranging from 3.812 ranging µg/mL inhibitive effect on the larval settlement ofwith B. amphitrite and H. elegans with the EC50to values ranging from 3.192 to 3.812 μg/mL and from 0.672 to 0.78 μg/mL, respectively. The LC 50 value of 22 was and from to 0.78 µg/mL, LCμg/mL, of 22 was calculated be 267 50 valuerespectively. from 3.1920.672 to 3.812 μg/mL andrespectively. from 0.672 toThe 0.78 The LC50tovalue of µg/mL, 22 was calculated to non-toxicity be 267 μg/mL, indicating its non-toxicity towards B. amphitrite cyprids. However, 5 indicating its towards B. amphitrite cyprids. However, 5 µg/mL of 22 killed all H. elegans calculated to be 267 μg/mL, indicating its non-toxicity towards B. amphitrite cyprids. However, 5 μg/mL of 22the killed all H. LC elegans larvae, the calculated LC50 value was 2.642 μg/mL. In addition, larvae, and calculated wasand 2.642 µg/mL. In addition, the results of antibacterial bioassay 50 value μg/mL of 22 killed all H. elegans larvae, and the calculated LC50 value was 2.642 μg/mL. In addition, the results of antibacterial bioassay usingwere a standard disc-diffusion method wereof encouraging. At using a standard disc-diffusion method encouraging. At the concentration 50 µg per disc, the results of antibacterial bioassay using a standard disc-diffusion method were encouraging. At the concentration of 50 μg per disc, 22 showed different levels of inhibitory effects to the growth of 22 showed different levels of inhibitory effects different to the growth bacterial effects strains to that either the concentration of 50 μg per disc, 22 showed levelsofof15inhibitory thewere growth of 15 bacterial strains that were either marine inductiveofstrains for the larval settlement of marine pathogens or inductive strains for pathogens the larval or settlement H. elegans with inhibition zones 15 bacterial strains that were either marine pathogens or inductive strains for the larval settlement of H. elegansfrom with inhibition ranging from 6.50Another to 0.50 mm in diameter [35]. Another halogenated ranging to 0.50zones mm in diameter [35]. halogenated benzenoid amibromdole (23) H. elegans with6.50 inhibition zones ranging from 6.50 to 0.50 mm in diameter [35]. Another halogenated benzenoid amibromdole (23) (Figure 5) was extracted from a soft coral-derived fungus Sarcophyton (Figure 5) was extracted (23) from(Figure a soft coral-derived fungus sp., and exhibited weak AF benzenoid amibromdole 5) was extracted fromSarcophyton a soft coral-derived fungus Sarcophyton sp., andagainst exhibited weak AFlarvae activity against amphitrite larvae with [36]. an EC 50 value of 16.70 μg/mL activity B. amphitrite with an ECB. value of 16.70 µg/mL The final two compounds, 50 amphitrite sp., and exhibited weak AF activity against B. larvae with an EC 50 value of 16.70 μg/mL [36]. The final two compounds, (±)-pestalachlorides E (24) and Ffrom (25) (Figure 5), were isolated from a ([36]. ±)-pestalachlorides E (24) and F (25) (Figure 5), were isolated a marine-derived fungal strain The final two compounds, (±)-pestalachlorides E (24) and F (25) (Figure 5), were isolated from a marine-derived fungal strain Pestalotiopsis ZJ-2009-7-6. Both them showed AF activity against Pestalotiopsis ZJ-2009-7-6. Both of them showed potent AFof againstpotent the larval settlement of marine-derived fungal strain Pestalotiopsis ZJ-2009-7-6. Both ofactivity them showed potent AF activity against the larval settlement of B.values amphitrite with 50 values of 1.65 and 0.55 μg/mL, respectively, B. amphitrite with the EC of 1.65 andthe 0.55EC µg/mL, respectively, meanwhile demonstrating no the larval settlement of 50B. amphitrite with the EC 50 values of 1.65 and 0.55 μg/mL, respectively, meanwhile demonstrating no toxicity with LC 50/EC50 ratios > 30.3 and 18.2, respectively [36]. toxicity with LC /EC ratios > 30.3 and 18.2, respectively [36]. 50 50 meanwhile demonstrating no toxicity with LC50/EC50 ratios > 30.3 and 18.2, respectively [36]. O O

HO HO OH OH HO HO

22 22

Cl Cl

O O

Cl Cl

Cl Cl

O O O O 23 23

O O O O

Cl Cl HO HO HO HO

CHO O CHO OH OH O H H H 24 H 24

OH OH

Cl Cl

Cl Cl

OH OH

CHO O CHO OH OH O H H H 25 H 25

OH OH

Figure 5. Chemical Structures of Benzenoids 22–25. Figure 5. Chemical Structures of Benzenoids 22–25. Figure 5. Chemical Structures of Benzenoids 22–25.

Six phenyl ethers were isolated as characteristic secondary metabolites of a marine-derived Six phenyl ethers were isolated as characteristic secondary metabolites of a marine-derived fungus Aspergillus sp. XS-20090066. These compounds, together with their seven synthetic phenyl fungus fungus Aspergillus sp. XS-20090066. XS-20090066. These These compounds, compounds, together together with with their their seven seven synthetic phenyl ether derivatives were evaluated for their AF activity using B. amphitrite larvae. All of these ether derivatives derivativeswere were evaluated for their AF activity B. amphitrite of these evaluated for their AF activity using B.using amphitrite larvae. Alllarvae. of theseAll compounds compounds showed moderate to strong AF activity with the EC50 values ≤ 14.11 μg/mL. It should be compounds showed moderate to strong AF activity with the EC 50 values ≤ 14.11 μg/mL. It should be showed moderate to strong AF activity with the EC50 values ≤ 14.11 µg/mL. It should be noted that the noted that the synthetic compounds 26–30 (Figure 6) exhibited strong AF activity with the EC50 noted that the synthetic compounds 26–30 (Figure strong AF50activity with EC50 synthetic compounds 26–30 (Figure 6) exhibited strong6) AFexhibited activity with the EC values ≤ 0.96the µg/mL, (EC50 values ≤ 0.96 μg/mL, which was even lower than that of the positive control Sea-Nine 211TM TM (EC50 values ≤ 0.96 μg/mL, wasof even than that Sea-Nine of the positive Sea-Nine 211µg/mL), which was even lowerwhich than that the lower positive control 211TMcontrol (EC50 value of 1.23 value of 1.23 μg/mL), and their toxicity was also low with the LC50/EC50 ratios > 20. The investigation value of 1.23 μg/mL), toxicity was also 50 low with> the 50 ratios > of 20.SAR The indicated investigation and their toxicity was and also their low with the LC ratios 20. LC The50/EC investigation that 50 /EC of SAR indicated that the ester group substitution at C-4 could increase AF activity for this class of of that the ester group substitution at C-4 could AFofactivity this class of theSAR esterindicated group substitution at C-4 could increase AF activity forincrease this class naturalfor phenyl ethers, natural phenyl ethers, and the introduction of bromine atoms played an important role in improving natural ethers,ofand the introduction of bromine atoms played an important role in improving and the phenyl introduction bromine atoms played an important role in improving the AF activity of these the AF activity of these synthetic derivatives [37]. the AF activity of these synthetic derivatives [37]. synthetic derivatives [37].

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2828

27 27 BrBr

BrBr OO

Br Br

O

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O O

2626 BrBr

O

HO HO

OH OH

HO HO

O O

OO BrBr BrBr 2929

OO

30 30

Figure6.6.Chemical Chemical Structures Structures of Figure ofPhenyl PhenylEthers Ethers26–30. 26–30.

5. Polyketides 5. 5. Polyketides Polyketides Polyketides arethe thelargest largest family family of of secondary secondary metabolites and have Polyketides metabolitesfrom frommicroorganisms, microorganisms, and have Polyketides are are the largest family of secondary metabolites from microorganisms, and have provided a large number of compounds with structural and bioactive diversity, including provided aalarge large number of compounds with structural and bioactive diversity,macrolides, including provided number of compounds with structural and bioactive diversity, including macrolides, tetracyclics, anthraquinones, and polyethers. Two ubiquinones, ubiquinone-0 (31) and macrolides, tetracyclics, anthraquinones, and polyethers. Two ubiquinones, ubiquinone-0 (31) tetracyclics, anthraquinones, polyethers. Two ubiquinone-0 (31) and vitamin K3 and (32) vitamin K3 (32) (Figure 7),and were the earliest AFubiquinones, polyketides reported by Konya et al. [21]. Both vitamin7),K3 (32)the (Figure 7), were the earliest AF by polyketides reported bycompounds Konya et al. [21]. Both (Figure were earliest AF polyketides reported Konya et al. [21]. Both were isolated compounds were isolated from the culture broth of Alteromonas sp. strain KK10304 associated with compounds were isolated from the culture broth of Alteromonas sp. strain KK10304 associated with from culture broth of Alteromonas sp. strain KK10304 associated with the sponge thethe sponge Halichondria okadai and could effectively inhibit the larval settlement ofHalichondria B. amphitriteokadai at the sponge Halichondria okadai and could effectively inhibit the larval settlement of B. amphitrite at and could effectively inhibit the larval settlement of B. amphitrite at concentrations of 1.3 and 2.5 µg/mL, concentrations of 1.3 and 2.5 μg/mL, respectively, whereas they were very toxic to the barnacle B. concentrations of 1.3 and 2.5 respectively, they were very toxic barnacle respectively, whereas they were toxic to the B. amphitrite cyprids with to theAthe LC values B. of amphitrite cyprids with the LCμg/mL, 30very values of 5.2 andbarnacle 2.5whereas μg/mL, respectively. Aspergilone (33) 30(Figure amphitrite cyprids with the LC 30 values of 5.2 and 2.5 μg/mL, respectively. Aspergilone A (33) (Figure 5.27), and 2.5 µg/mL, respectively. Aspergilone A (33) (Figure 7), acarbon novel skeleton, benzylazaphilone derivative a novel benzylazaphilone derivative with an unprecedented was isolated from 7),the a novel benzylazaphilone derivative with an unprecedented carbon was isolated from with an unprecedented carbon skeleton, was isolated from the gorgonian-derived fungus Aspergillus sp. of 7.68 μg/mL gorgonian-derived fungus Aspergillus sp. This compound showed anskeleton, EC50 value 50 value of 7.68 μg/mL theagainst gorgonian-derived fungus Aspergillus sp. This compound showed an EC the settlement ofan B.EC amphitrite larvae. However, the symmetrical dimer of 70 with a unique This compound showed value of 7.68 µg/mL against the settlement of B. amphitrite larvae. 50 against thethe settlement of B.dimer amphitrite the symmetrical dimer of 70B,with a unique methylene bridge, aspergilone B, of displayed AF activity [38]. However, symmetrical 70larvae. with no a However, unique methylene bridge, aspergilone displayed no methylene AF activity bridge, [38]. aspergilone B, displayed no AF activity [38].

Figure 7. Chemical Structures of Polyketides 31–33.

Figure 7. Chemical Structures of Polyketides 31–33.

Figure 7. Chemical Structures of Polyketides 31–33. A marine-derived fungal strain Xylariaceae sp. SCSGAF0086 could produce dicitrinin A (34) and phenol A acid (35) (Figure 8). Dicitrinin A was more active with an EC50 value of 1.76 μg/mL than A marine-derived sp.sp. SCSGAF0086 could produce dicitrinin A (34) marine-derivedfungal fungalstrain strainXylariaceae Xylariaceae SCSGAF0086 could produce dicitrinin A and (34) phenol A acid (EC50 value of 14.35 μg/mL) against the attachment of B. neritina larvae, and less toxic phenol A acid (35) (35) (Figure 8). Dicitrinin A was moremore active withwith an EC 50EC value of 1.76 μg/mL than and phenol A acid (Figure 8). Dicitrinin A was active an value of 1.76 µg/mL 50 with LC50/EC50 ratio > 56 than phenol A acid (LC50/EC50 ratio > 15) [39]. Four pairs of rare value of 14.35 μg/mL) against the attachment of B. neritina and less phenol A acid than phenol A (EC acid50(EC of 14.35 µg/mL) against the attachment of B. neritina larvae, andtoxic less 50 value dihydroisocoumarin derivatives, (±)-eurotiumides A–D, were isolated from a larvae, gorgonian-derived with LC50Eurotium /EC 50 /EC ratio >ratio 56 >than phenol A acid 50/EC 50 ratio > B. 15) [39]. of toxic with LC50 56 than A acid (LC >amphitrite 15) [39].Four Fourpairs pairs of rare 50 /EC 50 ratio fungus sp.50XS-200900E6, andphenol evaluated for(LC AF activity against larvae. All these dihydroisocoumarin derivatives, (±)-eurotiumides A–D, were isolated from a dihydroisocoumarin derivatives, ( ± )-eurotiumides A–D, were isolated from gorgonian-derived compounds could inhibit larval settlement with the EC50 values ranging from 0.7–23.2 μg/mL, and fungus and evaluated forDAF activity All these Eurotium sp.BXS-200900E6, against B. amphitrite larvae. (±)-eurotiumides (±36) and (±)-eurotiumides (±37) (Figure 8) were the most promising compounds with the ECμg/mL, 50 values ranging from 0.7–23.2 and compounds could inhibit larvalsettlement settlement with the EC ranging from 0.7–23.2 compoundscould with inhibit the EC50larval values of 1.5, 0.7, 2.3, and 1.9 respectively, and they wereμg/mL, notµg/mL, toxic 50 values (±)-eurotiumides B50/EC (±36) and D (±37) (Figure 8) 8) were and ±)-eurotiumides B50(± 36) and ±)-eurotiumides D (±37) (Figure werethe the most most promising to (the cyprids (LC ratios >(±)-eurotiumides 20)([40]. compounds with the EC50 50 values values of of 1.5, 1.5, 0.7, 0.7, 2.3, and 1.9 μg/mL, µg/mL,respectively, respectively, and and they they were not toxic to the cyprids (LC50 50/EC 20) [40]. /EC5050ratios ratios> > 20) [40].

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Figure 8. Chemical Structures of Polyketides 34–37.

Figure 8. Chemical Structures of Polyketides 34–37. Figure 8. Chemical Structures of Polyketides 34–37.

Anthraquinones and xanthones are the most prominent members of the polyketides family.

Anthraquinones and xanthones are the most prominent members of the polyketides family. However, the AF activity of this class has been seldomly Only eight Anthraquinones and xanthones are of thepolyketides most prominent members of thereported. polyketides However, the AF38–46 activity of this9), class of polyketides hasfungi beenPenicillium seldomly reported. Only eightsp.family. metabolites metabolites (Figure mainly from marine sp. and Aspergillus were However, the AF activity of this class of polyketides has been seldomly reported. Only eight 38–46demonstrated (Figure 9), mainly from marine fungi Penicillium sp. and Aspergillus sp. were demonstrated to have inhibitory effects on the larval settlement of B. amphitrite [37,41–44]. metabolites 38–46 (Figure 9), mainly from marine fungi Penicillium sp. and Aspergillus sp. were to have inhibitory effects the activity larvaleffects settlement ofsix B. amphitrite [37,41–44]. Compared withand the AF Compared with theoninhibitory AF of on other compounds, sterigmatocystin (38) demonstrated to have the larval settlement of B. amphitrite [37,41–44]. activity of other six compounds, sterigmatocystin (38) and methoxysterigmatocystin (39), isolated methoxysterigmatocystin (39), isolated from the fungal strains of the geneus Aspergillus, were much Compared with the AF activity of other six compounds, sterigmatocystin (38) and from more active the Aspergillus, larval settlement B. amphitrite with EC50 Aspergillus, values < 0.125 the fungal strainsinofinhibiting the geneus were much more in inhibiting the larval settlement methoxysterigmatocystin (39), isolated from theof fungal strainsactive of thethe geneus wereμg/mL, much and could paralyze the larvae at the effective concentration [42]. Another two anthraquinones of B. more amphitrite the EC50thevalues 0.125 µg/mL, and could paralyze larvae at the effective activewith in inhibiting larval < settlement of B. amphitrite with the EC50 the values < 0.125 μg/mL, averufin and 8-O-methylnidurufin (41) from the fungus Aspergillus sp.,two together with one the and could(40) paralyze the two larvae at the effective concentration [42]. Another anthraquinones concentration [42]. Another anthraquinones averufin (40) and 8-O-methylnidurufin (41) from xanthone 6,8-di-O-methyl versiconol (42) from an unidentified mangrove fungus ZSUH-36, averufin (40) sp., and together 8-O-methylnidurufin (41) from the fungus Aspergillus sp., together one fungus Aspergillus with one xanthone 6,8-di-O-methyl versiconol (42) from anwith unidentified displayed weak AF activity versiconol against B. amphitrite larvae with the EC50mangrove values of 2.03, 3.39,ZSUH-36, and 5.30 xanthone 6,8-di-O-methyl (42) from an unidentified fungus mangrove fungus ZSUH-36, displayed weak AF activity against B. amphitrite larvae with the EC50 μg/mL, respectively [37,42–44]. FourB. anthraquinones 43–46 isolated a gorgonian coral-derived displayed weak AF activity against amphitrite larvae with the ECfrom 50 values of 2.03, 3.39, and 5.30 valuesfungus of 2.03, 3.39, andsp. 5.30 µg/mL, respectively [37,42–44]. Four anthraquinones 43–46 isolated Penicillium SCSGAF 0023 showed moderate AF activity against B. amphitrite larvae with from μg/mL, respectively [37,42–44]. Four anthraquinones 43–46 isolated from a gorgonian coral-derived a gorgonian coral-derived fungus Penicillium sp.respectively SCSGAF 0023 moderate AF activity against the EC50Penicillium values of 6.7, 17.9, and 13.7 μg/mL, [41].showed fungus sp.6.1, SCSGAF 0023 showed moderate AF activity against B. amphitrite larvae with B. amphitrite larvae with the EC values of 6.7, 6.1, 17.9, and 13.7 µg/mL, respectively [41]. 50 the EC50values of 6.7, 6.1, 17.9, and 13.7 μg/mL, respectively [41].

Figure 9. Chemical Structures of Anthraquinones and Xanthones 38–46. Figure 9. Chemical Structures of Anthraquinones and Xanthones 38–46.

Structures of Anthraquinones and Xanthones 38–46. were isolated Recently, aFigure series 9. of Chemical 14-membered resorcylic acid lactones, named cochliomycins, from the marine-derived fungi of the genus Cochliobolus [45,46]. This class of metabolites their Recently, a series of 14-membered resorcylic acid lactones, named cochliomycins, wereand isolated Recently, a series of 14-membered resorcylic acid lactones, named cochliomycins, were isolated from the marine-derived fungi of the genus Cochliobolus [45,46]. This class of metabolites and their

from the marine-derived fungi of the genus Cochliobolus [45,46]. This class of metabolites and their synthetic derivatives were demonstrated to have AF activity against the larval settlement of

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Mar. Drugs 2017, 15, 266 8 of 20 B. amphitrite. Shao et al. [45] firstly isolated seven cochliomycins from a gorgonian-derived fungus C. lunatus. Among them, compounds 47–52 (Figure 10) could inhibit the larval settlement with synthetic derivatives were demonstrated to have AF activity against the larval settlement of B. the EC ranging from 1.2 isolated to 17.9 seven µg/mL, among which A (47)fungus showed 50 values amphitrite. Shao et al. [45] firstly cochliomycins from cochliomycin a gorgonian-derived C. the best AF activity with an EC value of 1.2 µg/mL and a LC /EC ratio > 15. Analysis of 50 50 the larval 50 lunatus. Among them, compounds 47–52 (Figure 10) could inhibit settlement with the EC50 SAR of 47–49 suggested the introduction of the acetonide moiety in 47 might its best AF AF activity, values ranging from 1.2 to 17.9 μg/mL, among which cochliomycin A (47) improve showed the activity with groups an EC50 probably value of 1.2 μg/mL and a LC 50/EC 50 ratio > 15. Analysis of SARresorcylic of 47–49 acid and the hydroxy have a positive effect on the AF activity of these suggested the introduction of the acetonide moiety in 47 might improve its AF activity, and thefrom lactones. Subsequently, cochliomycins D–F, along with eight known analogues, were isolated hydroxy groups probably have a positive effect on the AF activity of these resorcylic acid lactones. the fungus C. lunatus associated with a sea anemone by Liu et al. [46]. In AF bioassays, compounds cochliomycins D–F, along with eight known analogues, were isolated from the fungus 53–58Subsequently, (Figure 10) exhibited AF activity at nontoxic concentrations with the EC50 values ranging from C. lunatus associated with a sea anemone by Liu et al. [46]. In AF bioassays, compounds 53–58 1.82 to 22.5 µg/mL, and LL-Z1640-2 (55) displayed the highest potency against B. amphitrite larvae (Figure 10) exhibited AF activity at nontoxic concentrations with the EC50 values ranging from 1.82 to with 22.5 an EC of 1.82 µg/mL. It was shown that the cis-enone moiety in 57 could significantly 50 value μg/mL, and LL-Z1640-2 (55) displayed the highest potency against B. amphitrite larvae with an improve the EC value approximately compared to thatinof5754could (EC50 value of 18.1 µg/mL) EC50 value of501.82 μg/mL. It was shown9-fold that the cis-enone moiety significantly improve with the trans-enone, indicating that the cis-enone functionality might contribute to the increase EC50 value approximately 9-fold compared to that of 54 (EC50 value of 18.1 μg/mL) withof AF activity. More interestingly, contrary to the functionality enhancement of AF activity tocaused by theofacetonide trans-enone, indicating that the cis-enone might contribute the increase AF activity. More interestingly,resorcylic contrary to the the enhancement AF activity caused by the acetonide functionality of 14-membered acid, acetonideof functionality led to the obvious decrease 14-membered resorcylic the acetonide functionality led the obvious of AFfunctionality activity forof the enone resorcylic acidacid, lactones. The above findings oftoSAR indicatedecrease that the AF of AF activity for the enone resorcylic acid lactones. The above findings of SAR indicate that thechanges, AF activity of these 14-membered resorcylic acid lactones should be sensitive to subtle structural activity of these 14-membered resorcylic acid lactones should be sensitive to subtle structural such as the enone, the acetonide functionalities, and the hydroxy configurations. The preliminary changes, such as the enone, the acetonide functionalities, and the hydroxy configurations. The AF mechanism revealed that the 14-membered resorcylic acid lactones mainly inhibited the larval preliminary AF mechanism revealed that the 14-membered resorcylic acid lactones mainly inhibited settlement of B. amphitrite by stimulating the NO/cGMP pathway [47]. the larval settlement of B. amphitrite by stimulating the NO/cGMP pathway [47].

Figure 10. Chemical Structures of 14-membered Resorcylic Acid Lactones 47–58. Figure 10. Chemical Structures of 14-membered Resorcylic Acid Lactones 47–58.

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6. Alkaloids In recent years, dozens of indole alkaloids were isolated from marine-derived bacteria, and some of them showed moderate to strong AF activities. A bacterial strain Acinetobacter sp. associated with the ascidian Stomozoa murrayi could produce two AF metabolites 6-bromoindole-3-carbaldehyde (59) and its debromo analog (60) (Figure 11) with the EC50 values of 5 and 28 µg/mL against B. amphitrite larvae, respectively [48]. Nostocarboline (61) (Figure 11), a carbonium alkaloid isolated from the freshwater cyanobacterium Nostoc sp., along with its five synthetic analogs 62–66 (Figure 11) were all active in inhibiting the growth of the toxic cyanobacterium Microcystis aeruginosa PCC7806, the nontoxic cyanobacterium Synechococcus PCC6911, and the green algae Kirchneriella contorta SAG 11.81. Among these six compounds, nostocarboline (61) showed strongest inhibitory activity against the growth of the cyanobacteria and algae with the MIC values of 0.22 µg/mL. The quaternary group and the replacement of the substituent on the 2-N atom appeared to be essential for increasing the anti-algal activity of this alkaloid class [49]. Eight bisindole products 67–74 (Figure 11) that belong to the di(1H-indol-3-yl)methane (DIM) family were isolated from a Red Sea ascidian-derived bacterial strain Pseudovibrio denitrificans UST4-50. All diindol-3-ylmethanes (DIMs) inhibited the larval settlement of B. amphitrite (EC50 values ranging from 0.63 to 5.68 µg/mL) with low toxicity (LC50 ratios > 15), of which DIM-Ph-4-OH (74) was the best AF compound with an EC50 value of 0.63 µg/mL. DIM (67) and DIM-Ph-4-OH also displayed significant AF activity against B. neritina larvae with the EC50 values of 0.62 and 0.42 µg/mL, respectively. Interestingly, the AF activity of DIM and DIM-Ph-4-OH against B. amphitrite cyprids was reversible. Moreover, DIM also showed comparable AF performance to the commercial AF biocide Sea-Nine 211™ in the field test over a period of five months, thus indicating that DIMs should be considered as promising candidates as useful antifoulants. Finally, in order to investigate the SAR of these DIMs, the acetylation of DIM-Ph-4-OH yielded the product DIM-Ph-4-OAc (75), which was less active as DIM-Ph-4-OH against the larval settlement of B. amphitrite and B. neritina. Based on the data of the AF activity of these natural metabolites and the acetylated derivative, it is suspected that the common moiety, di(1H-indol-3-yl)methylene, may serve as an important functional group for maintaining reversibly AF mechanism of DIMs, and that the phenolic hydroxyl group that presents at the Ph-C1000 of DIM-C1 will significantly enhance the AF activity of these bisindoles [50].

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Figure Figure 11. 11. Chemical Chemical Structures Structures of of Alkaloids Alkaloids 59–75. 59–75.

In addition addition to tomarine marinebacteria, bacteria,marine-derived marine-derivedfungal fungal strains Aspergillus Penicillium strains of of Aspergillus sp.sp. andand Penicillium sp. sp. have a rich source of similar structural indole alkaloids with AF activity. Compounds have alsoalso beenbeen a rich source of similar structural indole alkaloids with AF activity. Compounds 76–79 76–79 and (Figure 80–83 (Figure 12), obtained twofungi marine fungi Penicillium SCSIO 00258 and and 80–83 12), obtained from twofrom marine Penicillium sp. SCSIO sp. 00258 and Aspergillus Aspergillus sydowii SCSIO 00305, were evaluated for their AF and antibacterial activities. of the sydowii SCSIO 00305, were evaluated for their AF and antibacterial activities. All of the naturalAll products naturalfrom products 76–79 from the exhibited strain SCSIO 00258settlement exhibitedagainst antilarval settlement against 76–79 the strain SCSIO 00258 antilarval B. amphitrite with the ECB. 50 amphitrite with the EC 50 values of 12.3, 1.1, 6.2, and 17.5 μg/mL, respectively. Compounds 78 and values of 12.3, 1.1, 6.2, and 17.5 µg/mL, respectively. Compounds 78 and 80–82 displayed AF activity 80–82 displayed activity neritina with values of 2.1, 15.3, 8.4 and 8.2 against B. neritinaAF larvae withagainst the EC50B.values of larvae 2.1, 15.3, 8.4 the andEC 8.250µg/mL, respectively. Moreover, μg/mL, respectively. Moreover, 77 and 81 also showed modest activity compounds 77 and 81 also showedcompounds modest antibacterial activity towards the larvalantibacterial settlement inducing towards the larval settlement inducing bacterium Micrococcus luteus with MIC values of 200 and 300 bacterium Micrococcus luteus with MIC values of 200 and 300 µg/mL, respectively. Another indole μg/mL, containing respectively. Another indoleneoechinulin alkaloid containing diketopiperazine, neoechinulin Afungal (83), alkaloid diketopiperazine, A (83), mainly isolated from marine-derived mainlyof isolated from marine-derived fungal strains of the genus Aspergillus, also with inhibited the larval strains the genus Aspergillus, also inhibited the larval settlement of B. amphitrite the EC value 50 settlement of B. amphitrite with the EC50 value of 14.99 μg/mL [51]. of 14.99 µg/mL [51].

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Figure Figure 12. 12. Chemical Chemical Structures Structures of of Alkaloids Alkaloids 76–83. 76–83.

thesame sametime, time,many many other alkaloids were obtained marine bacteria and At the other AFAF alkaloids were also also obtained from from marine bacteria and fungi. fungi. Screening of the chemical extracts from bacteria differentincorporated bacteria incorporated marine paints Screening of the chemical extracts from different into marineinto paints resulted in resulted in theof discovery of anbacterium effective bacterium Pseudomonas sp. NUDMB50-11, wasfrom isolated the discovery an effective Pseudomonas sp. NUDMB50-11, which waswhich isolated the from the of Nudibranch. All petri dishes withofthe of the of broth culture of surface ofsurface Nudibranch. All petri dishes treated with treated the extracts theextracts broth culture NUDMB50-11 showed a significant indecrease the settling of barnacles and algal spores at at a NUDMB50-11 showed adecrease significant in the frequency settling frequency of barnacles and algal spores concentration The a concentrationofof2020mg/mL. mg/mL. Thebioassay-guided bioassay-guidedfractionation fractionationand andfurther furtherpurification purification of of the the crude extract of of NUDMB50-11 led discovery to the of discovery of among five metabolites, among which NUDMB50-11 led to the five metabolites, which phenazine-1-carboxylic phenazine-1-carboxylic acid (Figure was the of the tested fouling acid (84) (Figure 13) was the(84) most active13) against all most of theactive testedagainst foulingallbacteria with the MIC bacteria MIC values the ≤ 10detailed μg. Unfortunately, the detailed data of AFlarvae activities against values ≤with 10 µg.the Unfortunately, data of AF activities against barnacle and fouling barnacle larvae and fouling were not reported [52].(Figure Penispirolloid A (85a/b) (Figure 13), a algae were not reported [52].algae Penispirolloid A (85a/b) 13), a novel alkaloid possessing novel alkaloid a unique spiro imidazolidinyl skeleton, was isolated from Penicillium a halotolerant a unique spiropossessing imidazolidinyl skeleton, was isolated from a halotolerant fungus sp. fungus Penicillium sp. OUCMDZ-776, the absolute configuration of C-11 wasThis undetermined. OUCMDZ-776, though the absolute though configuration of C-11 was undetermined. compound This compound displayed significant AF activity against the larval B. neritina with an displayed a significant AFa activity against the larval settlement of settlement B. neritina of with an EC50 value EC 50 value of 2.40 μg/mL [53]. Ten cytochalasin werefrom isolated the fermentation broth of 2.40 µg/mL [53]. Ten cytochalasin alkaloids alkaloids were isolated the from fermentation broth of a soft of a soft coral Sarcophyton sp.-derived fungus A.ZJ-2008010, elegans ZJ-2008010, they were evaluated for AF coral Sarcophyton sp.-derived fungus A. elegans and theyand were evaluated for AF activity activity against B. amphitrite ofdetermined them, determined as aspochalasins (86), D J (87), (88), against B. amphitrite larvae. larvae. Four ofFour them, as aspochalasins I (86), JI (87), (88), D and H and H (89) (Figure 13), respectively, displayed to moderate AF with activity EC50ranging values (89) (Figure 13), respectively, displayed strong tostrong moderate AF activity the with EC50 the values ranging from 2.59 to 15.44 μg/mL. Aspochalasin D, bearing an α,β-unsaturated was from 2.59 to 15.44 µg/mL. Aspochalasin D, bearing an α,β-unsaturated ketoneketone moiety,moiety, was found found to most be theactive mostcompound, active compound, indicating the electrophilic α,β-unsaturated carbonyl to be the indicating that thethat electrophilic α,β-unsaturated carbonyl moiety moiety and double-bond C-19 and C-20be might be considerably important for increasing the AF and double-bond at C-19 at and C-20 might considerably important for increasing the AF activity activity of these cytochalasins. Additionally, this compound alsobroad exhibited broad of of these cytochalasins. Additionally, this compound also exhibited spectrum of spectrum antibacterial antibacterial especially pathogenic bacteria [54].alkaloids Six activity, especiallyactivity, against pathogenic bacteriaagainst [54]. Six dihydroquinolin-2-one-containing dihydroquinolin-2-one-containing alkaloids from a gorgonian Scopulariopsis from a gorgonian coral-derived fungus Scopulariopsis sp. in thecoral-derived South China fungus Sea could inhibit the sp. in the South China Sea could Among inhibit the larval settlement of B. amphitrite. Among these alkaloids, larval settlement of B. amphitrite. these alkaloids, particularly important are compounds 90–94 particularly important are compounds 90–94 (Figure 13), all of which were demonstrated to be highly active in antilarval settlement bioassay with the EC50 values of 0.007 pg/mL, 0.012 ng/mL,

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(Figure 13), all of which were demonstrated to be highly active in antilarval settlement bioassay with the Mar. Drugs 2017, 15, 266 12 of 20 EC50 values of 0.007 pg/mL, 0.012 ng/mL, 0.001 ng/mL, 0.280 µg/mL and 0.219 µg/mL, respectively, while they non-toxic with the ranging from to 1200, suggesting 0.001were ng/mL, 0.280 μg/mL andLC 0.219 μg/mL, respectively, while57 they were thus non-toxic with thethat this 50 /EC 50 ratio 50/EC50 ratioare ranging from 57antifoulants to 1200, thus suggesting that this 95, class compounds are promising class of LC compounds promising [55]. Compound a of benzodiazepine alkaloid isolated antifoulants [55]. Compound 95, a benzodiazepine alkaloid isolated fromwas a deep sea-derived fungus from a deep sea-derived fungus Aspergillus westerdijkiae SCSIO 05233, reported as an inhibitor of Aspergillus westerdijkiae SCSIO 05233, was reported as an inhibitor of B. amphitrite larvae with an EC50 B. amphitrite larvae with an EC50 value of 8.81 µg/mL [56]. value of 8.81 μg/mL [56].

Figure 13. Chemical Structures of Alkaloids 84–95.

Figure 13. Chemical Structures of Alkaloids 84–95. 7. Nucleosides and Peptides

7. Nucleosides and Peptides

AF nucleoside derivatives have been rarely reported. Only one nucleoside compound

AFdiacetylkipukasin nucleoside derivatives been from rarely Onlyversicolor one nucleoside compound E (96) (Figurehave 14), isolated thereported. fungus Aspergillus associated with a diacetylkipukasin E (96) (Figure the fungus Aspergillus gorgonian Dichotella gemmacea,14), wasisolated found tofrom have weak AF activity against B.versicolor amphitrite associated larvae with with a an EC 50 value of 22.5 μg/mLwas [57].found to have weak AF activity against B. amphitrite larvae with an gorgonian Dichotella gemmacea, EC50 value of 22.5 µg/mL [57].

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O

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O NH O

HO

N NHO O

N

HO HO

O O

HO

O

O OO O O

96 O Figure14. 14.Chemical Chemical Structure of Nucleoside 96. Figure Structure 96 of Nucleoside 96. secondary metabolites 97–100 (FigureStructure 15) belonging totothe mixed polyketide–polypeptide Figure 14. Chemical of Nucleoside 96. FourFour secondary metabolites 97–100 (Figure 15) belonging the mixed polyketide–polypeptide structural family were produced by a benthic filamentous marine cyanobacterium Lyngbya majuscula structural family were produced by a benthic filamentous marine cyanobacterium Lyngbya majuscula Fourshowed secondary metabolites 97–100 (Figuresettlement 15) belonging to the mixed and they strong to moderate anti-larval activity against B. polyketide–polypeptide amphitrite with the EC50 and they showed strong to moderate anti-larval settlement activity against B.Lyngbya amphitrite with the structural family0.54, were produced a benthic filamentous marine cyanobacterium values of 0.003, 2.6, and 10.6by μg/mL, respectively. In addition, it was noteworthy that majuscula the most EC50active values of 0.003, 0.54, 2.6, and 10.6 µg/mL, respectively. In addition, it was noteworthy and they showed strong to moderate settlement activity B. amphitrite with the ECa50 that compound, dolastatin 16 (97), anti-larval appeared to be non-toxic to against the B. amphitrite cyprids with the most active compound, dolastatin 16 (97), appeared beofnon-toxic to16the B. amphitrite cyprids values of 0.54, 2.6, and 10.6 μg/mL, respectively. In to addition, it was noteworthy that the most LC 50/EC 50 0.003, ratio > 6000. More encouragingly, field testing dolastatin was conducted at with concentrations a LC50compound, /EC50 of ratio >1.0, 6000. encouragingly, testing dolastatin was conducted active dolastatin 16 appeared to befield non-toxic to of the B. amphitrite cyprids with a at 10.0, 0.1, More and(97), 0.01 μg/mL, respectively, and the barnacle counts16of all dolastatin LC50/EC50 plates ratio > 6000. More encouragingly, field testing and of dolastatin was suggesting conducted at 16-treated were than thatrespectively, of the negative controls after 416weeks, its concentrations of 10.0, 1.0,obviously 0.1, andlower 0.01 µg/mL, the barnacle counts of all dolastatin concentrations of 10.0, 1.0, 0.1, and 0.01 μg/mL, respectively, and the barnacle counts of all dolastatin potential application as an antifoulant [58]. Although its total synthesis was challenging due to its 16-treated plates were obviously lower than that of the negative controls after 4 weeks, suggesting 16-treated plates were obviously lower than thatAlthough of the negative controls after 4 was weeks, suggesting its complex structure, dolastatin 16 has been recently successfully by Casalme et al. [59]. The its potential application as an antifoulant [58]. itssynthesized total synthesis challenging due to potential dolastatin application16asshowed an antifoulant [58]. Although its total synthesis was challenging due to its synthetic potent antilarval settlement of B. amphitrite similar to that of natural its complex structure, dolastatin 16 has been recently successfully synthesized by Casalme et al. [59]. complex structure, 16 has been successfully synthesized byaCasalme etantifoulant. al. [59]. The dolastatin 16, whichdolastatin further supported thatrecently dolastatin 16 might be explored as candidate The synthetic dolastatin 16 showed potent antilarval settlement of B. amphitrite similar to that of natural synthetic dolastatin 16 showed potent antilarval settlement of B. amphitrite similar to that of natural dolastatin 16, which further supported that dolastatin 16 might be explored as a candidate antifoulant. dolastatin 16, which further O supported that dolastatin 16 might be explored as a candidate antifoulant. N O N O OO

O

O HN

O O

N O

O

HN

O O

O N O HN

O N O O HN

N

O N

NO

O

O

O

O

N97

O 99

O N

98

NH2

O O O

O

O

NH2

N

N

O

97

O

O

O

N

O O

N

N

O

O O NO

O

N Cl N Cl

O

N

O

O

O

O

98

O N

O

O O O N O HN

O O

O HN N

100 O

99Figure 15. Chemical Structure of Peptides 97–100. O

100

Two diketopiperazines (DKPs), namely (6S,3S)-6-benzyl-3-methyl-2,5-diketopiper-azine Figure 15. Chemical Structure of Peptides 97–100. Figure 15. Chemical Structure of Peptides 97–100. (bmDKP) (101) and (6S,3S)-6-isobutyl-3-methyl-2,5-diketopiperazine (imDKP) (102) (Figure 16), diketopiperazines (DKPs), (6S,3S)-6-benzyl-3-methyl-2,5-diketopiper-azine were Two isolated from a seaweed Undarianamely pinnatifida–derived Streptomyces praecox 291-11. Both Two diketopiperazines (DKPs), namely (6S,3S)-6-benzyl-3-methyl-2,5-diketopiper-azine (bmDKP) (bmDKP) (101) and zoospore (6S,3S)-6-isobutyl-3-methyl-2,5-diketopiperazine (imDKP) 16), compounds inhibited settlement of the seaweed U. pertusa with the EC50(102) values(Figure of 2.2 and (101) were and (6S,3S)-6-isobutyl-3-methyl-2,5-diketopiperazine (imDKP) (102) (Figure wereBoth isolated isolated from a seaweed Undaria pinnatifida–derived Streptomyces praecox 16), 291-11. from compounds a seaweed inhibited Undaria pinnatifida–derived Streptomyces 291-11. Both compounds zoospore settlement of the seaweed praecox U. pertusa with the EC50 values of 2.2inhibited and

zoospore settlement of the seaweed U. pertusa with the EC50 values of 2.2 and 3.1 µg/mL as well

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as the growth of the diotam N. of annexa with the EC50 values of EC 0.850and 1.1 of µg/mL, 3.1 μg/mL as well as the growth the diotam N. annexa with the values 0.8 andrespectively. 1.1 μg/mL, Compared to the low therapeutic ratios of positive controls (LC /EC ratios of 2 and 6 for respectively. Compared to the low therapeutic ratios of positive controls 2 and 6 50 (LC 5050/EC50 ratios of the the target organisms U. U. pertusa and N.N. annexa, higher for target organisms pertusa and annexa,respectively), respectively),bmDKP bmDKPand and imDKP imDKP showed higher therapeutic ratios ratios(LC (LC of and 17.7 21 and 21pertusa; to U. pertusa; to N. annexa), therapeutic 50/EC 50 ratios of 17.7 to U. 263 and 263 120.2and to N.120.2 annexa), indicating 50 /EC 50 ratios indicating that these two compounds might be environmentally friendly antialgal compounds [60]. that these two compounds might be environmentally friendly antialgal compounds [60]. Aspergillipeptide C C (103) (Figure 16), a cyclic tetrapeptide, tetrapeptide, was was isolated isolated from from aa culture culture broth broth of of a Aspergillipeptide gorgonian Melitodes Melitodes squamata-derived squamata-derived fungus fungus Aspergillus Aspergillus sp. sp. SCSGAF SCSGAF 0076 0076 and and showed showed inhibitory inhibitory gorgonian and aa LC LC5050/EC /EC ratio > 25 [61]. attachment against B. neritina larvae larvae with with an an EC EC50 50 value of 11 µg/mL μg/mL and 5050 ratio > 25 [61]. O OH

O

O

HN

HN

NH

NH

OH

HN

O

O

NH

HN

O

O 101

102

103

Figure Figure 16. 16. Chemical Chemical Structure Structure of of Diketopiperazines Diketopiperazines 101–103. 101–103.

8. Enzymes Enzymes During the the continuous continuous investigation investigation of of enzymatic enzymatic antifoulants, antifoulants, aa variety variety of of enzymes enzymes with with AF During activity have have been obtained. obtained. Many Many patents patentsof of those those promising promising enzymes enzymes have have been been applied applied [62]. [62]. activity According to Selvig et al. [63] and Polsenski & Leavitt [64], microorganism(s) alone or mixed with hydrolytic enzyme(s) enzyme(s) could could be be incorporated incorporated into into marine marine paints paints in in order order to to test test the the AF activity activity of hydrolytic microorganisms or or enzymes enzymes in field trials. A A novel novel protease protease from from a deep-sea derived bacterium microorganisms Pseudoalteromonasis sachenkonii sachenkonii UST041101-043 UST041101-043 showed showed strong strong inhibitive inhibitive activity activity against against the the larval Pseudoalteromonasis settlement of B. neritina with an EC value of 0.5 ng/mL. This enzyme also showed significant settlement of EC50 50 ng/mL. This enzyme also showed significant AF very low low effects on the settlement of the barnacle B. amphitrite and the bryozoan Schizoporella sp. at a very ng/mL[65]. [65]. concentration of 100 ng/mL 9. Biofilm Biofilm Inhibitors Inhibitors 9. In the the marine marine environment, environment, aa large large number number of of various various marine marine bacteria bacteria (especially (especially those those that that In induce settlement of fouling macro-foulers) communicate with each other by quorum sensing and form induce settlement of fouling macro-foulers) communicate with each other by quorum sensing and biofilms, which which has an has influence on marine adhesion [66]. Therefore, is promising form biofilms, an influence onmacroorganism marine macroorganism adhesion [66].itTherefore, it to is exploit those compounds with excellent anti-biofilm activity as potential antifoulants. Several marine promising to exploit those compounds with excellent anti-biofilm activity as potential antifoulants. invertebratesalgae-derived and their synthetic analogs, including halogenated Several marineor invertebratesor metabolites algae-derived metabolites and their synthetic analogs, including furanones, 2-amminoimidazole alkaloids, and flavonoids, have already been explored as candidate halogenated furanones, 2-amminoimidazole alkaloids, and flavonoids, have already been explored antifoulants However, only a handfulonly of natural products derived from marine with as candidate[67–71]. antifoulants [67–71]. However, a handful of natural products derivedmicrobes from marine antibiofilm activity have been reported at this reported. microbes with antibiofilm activity have been reported at this reported. A glycolipid glycolipid surfactant, surfactant, composed composed of of glucose glucose and and palmitic palmitic acid, acid, was was obtained obtained from from aa tropical tropical A marine bacterium Serratiamarcescens. It prevented, to different degrees, the adhesion of the pathogens marine bacterium Serratiamarcescens. It prevented, to different degrees, the adhesion of the Candida albicans BH,albicans and P. aeruginosa PAO1 as wellPAO1 as the as marine bacterium Bacillus pumilus pathogens Candida BH, and P. aeruginosa well biofouling as the marine biofouling bacterium TiO1 at concentrations ranging from 0.125 to 25 µg/mL. More interestingly, this glycolipid could Bacillus pumilus TiO1 at concentrations ranging from 0.125 to 25 μg/mL. More interestingly, also this efficiently disrupt the biofilm on glass surfacesper formed the tested bacteria at a concentration of glycolipid could also efficiently disrupt the biofilm on by glass surfacesper formed by the tested 50 µg/mL. detailed structure the glycolipid was yet to be determined [72]. 4-Phenylbutanoic bacteria at aThe concentration of 50 of μg/mL. The detailed structure of the glycolipid was yet to be acid (104) (Figure 17) was obtained from the crude extract of a marine bacterium pumilus S6-15ofby determined [72]. 4-Phenylbutanoic acid (104) (Figure 17) was obtained from theB.crude extract a Nithya et al. [73]. At concentrations ranging to 15Atµg/mL, this compound marine bacterium B. pumilus S6-15 by Nithyafrom et al.10[73]. concentrations rangingshowed from 10potent to 15 biofilm inhibitory activityshowed against potent all the 10 testedinhibitory bacterial strains of Gram-positive Gram-negative μg/mL, this compound biofilm activity against all the and 10 tested bacterial species. Scopel et al. [74] isolated a dipeptide, cis-cyclo(Leucyl-Tyrosyl) (105) (Figure from a strains of Gram-positive and Gram-negative species. Scopel et al. [74] isolated a 17) dipeptide, sponge-associated fungus(105) Penicillium sp.17) F37,from which was able to combat fungus ~80% biofilm formation of a cis-cyclo(Leucyl-Tyrosyl) (Figure a sponge-associated Penicillium sp. F37,

which was able to combat ~80% biofilm formation of a pathogenic bacterium Staphylococcus epidermidis at a concentration of 1.0 mg/mL without interfering with its growth. The search for new

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pathogenic bacterium Staphylococcus epidermidis at a concentration of 1.0 mg/mL without interfering inhibitors of theThe biofilm of Mycobacterium species led to of theMycobacterium discovery ofspecies three with its growth. searchformation for new inhibitors of the biofilm formation sesterterpenes namedofophiobolin K (106), 6-epi-ophiobolin K (107), and 6-epi-ophiobolin 6-epi-ophiobolin K G (107), (108) led to the discovery three sesterterpenes named ophiobolin K (106), (Figure 17) from the culture broth of a marine-derived fungal strain Emericella variecolor. All of these and 6-epi-ophiobolin G (108) (Figure 17) from the culture broth of a marine-derived fungal strain three compounds were of inhibiting biofilm formation of Mycobacterium with the Emericella variecolor. Allcapable of these three compounds were capable of inhibiting smegmatis biofilm formation MIC values of 1.58, 24.97, and 6.23 μg/mL, respectively. In addition, ophiobolin K could restore the of Mycobacterium smegmatis with the MIC values of 1.58, 24.97, and 6.23 µg/mL, respectively. antimicrobial activity of isoniazid against the tested bacterium by inhibiting its biofilm formation. In addition, ophiobolin K could restore the antimicrobial activity of isoniazid against the tested Ophiobolin K inhibiting also showed antibiofilm activityOphiobolin against Mycobacteriumbovis BCG with activity a MIC value of bacterium by its biofilm formation. K also showed antibiofilm against 3.15 μg/mL [75]. Mycobacteriumbovis BCG with a MIC value of 3.15 µg/mL [75].

OH

HO

O

O 104

105

H

OHC H O

H

H

H

O

HO

106

H

OHC H O

H

H

H HO

H N N H

H

OHC H O

107

108

Figure Figure 17. 17. Chemical Chemical Structure Structure of of Biofilm Biofilm Inhibitors Inhibitors 104–108. 104–108.

Conclusions and Future Perspectives 10. Conclusions

Generally, when exploring candidate compounds for further development as AF products, the and LC LC5050/EC /EC values areoften oftenused usedasasstandards standardsfor forthe theevaluation evaluationof ofthe the activity activity and and toxicity EC50 50 50 values are 50 and of a given candidate AF compound. Although a compound with an EC value < 25 µg/mL and a candidate AF compound. compound with an EC50 50 < μg/mL and /EC ratio> >1515isisoften oftenrecommended recommendedas asaanon-toxic non-toxic antifoulant, antifoulant, aa much lower EC50 value and LC50 5050 ratio 50 value 50/EC LC5050/EC /EC ratio is required when screening for candidate antifoulants. As pointed by 50 50 ratio is required when screening for candidate antifoulants. As pointed out byout Qian higher LC Qian et al. [15], only those small molecules which exhibit AF activities against a broad spectrum et al. [15], only those small molecules which exhibit AF activities against a broad spectrum of /EC5050ratio ratio>>5050should shouldbe beconsidered. considered.Meanwhile, Meanwhile, biofoulers with with an anEC EC50 50 value value