Isolation and Potential Biological Applications of Haloaryl Secondary Metabolites from Macroalgae

Macroalgae have been reported as an important source of halogenated aromatic secondary metabolites, being the majority of these derivatives isolated from red algae. Halophenols and haloindoles are the most common haloaryl secondary metabolites isolated from these marine organisms. Nevertheless, some halogenated aromatic sesquiterpenes and naphthalene derivatives have also been isolated. Most of these secondary metabolites showed interesting biological activities, such as antitumor, antimicrobial, antidiabetic, and antioxidant. This review describes in a systematic way the distribution and natural occurrence of halogenated aromatic secondary metabolites from extracts of red, brown, and green algae, as well as biological activities reported for these compounds.


Introduction
The search for bioactive compounds from marine organisms in recent decades has produced an abundance of secondary metabolites with pharmaceutical and industrial applications. Among these marine natural products, the isolation of halogenated derivatives from macroalgae has been exhaustively reported. This work describes in a systematic way the distribution, natural occurrence, and biological activities of aromatic secondary metabolites with halogens on the aromatic moiety.
Over the past four decades, reports about the isolation of haloaryl secondary metabolites from macroalgae have been increasing, describing about two hundred halogenated aromatic secondary metabolites. Among algae, macroalgae-including brown, green, and red algae-are an important source of these secondary metabolites, with red algae being responsible for the production of nearly 90% of these compounds identified thus far ( Figure 1).  The haloaryl secondary metabolites containing bromine are more common (176 compounds) than with chlorine (14 compounds) and iodine (9 compounds) (Table 1). Interestingly, the number of secondary metabolites with chlorine is very similar to that with iodine, which would not be expected because chloride and bromide are much more abundant than iodide in seawater [1]. According to  The haloaryl secondary metabolites containing bromine are more common (176 compounds) than with chlorine (14 compounds) and iodine (9 compounds) (Table 1). Interestingly, the number of secondary metabolites with chlorine is very similar to that with iodine, which would not be expected because chloride and bromide are much more abundant than iodide in seawater [1]. According to The haloaryl secondary metabolites containing bromine are more common (176 compounds) than with chlorine (14 compounds) and iodine (9 compounds) (Table 1). Interestingly, the number of secondary metabolites with chlorine is very similar to that with iodine, which would not be expected because chloride and bromide are much more abundant than iodide in seawater [1]. According to Lavoie et al. (2017), this disproportionately high number of iodinated compounds can be explained by the higher oxidation potential of iodide compared to bromide and chloride, allowing its faster oxidation by haloperoxidases, and their incorporation into the biosynthetic pathway of the secondary metabolites [2,3]. It is noteworthy that the halogenation degree is relatively higher for brominated metabolites than for chlorinated and iodinated metabolites (Table 1). Table 1. Degree of halogenation of macroalgae haloaryl secondary metabolites.
Among halophenols, the isolation of monoaryl bromophenols (70 compounds) and bromophenol dimers with a methylene bridge between the two phenyl rings is quite usual (25 compounds), as well as the description of bromophenol dimers with different linkers such as oxygen (5 compounds), oxybis(methylene) (4 compounds), ethylene (4 compounds), and a carbonyl group (1 compound). The isolation of halophenol trimers (3 compounds) and tetramers (1 compound) has also been reported (Table 1).
Although halophenols have been isolated from a wide range of genera, the isolation of halo-indoles, -sesquiterpenes and -naphthalene derivatives have been mainly reported from a restricted number of genera, namely, the Rhodophyllis and Laurencia genera.
Among haloindoles, the isolation of mono-indoles (39 compounds) is more common than dimers (only 7 compounds) and, in contrast to halophenols, the isolation of trimers or tetramers of haloindoles was not described (Table 1). Concerning the nature of the halogen, there are many more cases of indoles with chloro (13 compounds) or iodo (6 compounds) than in the halophenols class (Table 1). Nevertheless, the majority of the indoles presents two or three bromine atoms as happens with the phenol class.
Among halosesquiterpenes, the isolation of monoaryl sesquiterpenes (17 compounds) is more usual than dimers (only one example was found) and again the isolation of trimers or tetramers was not described (Table 1). Concerning the halogen, only bromosesquiterpenes have been found until now.
The naphthalene class is restricted to three bromonaphthalene derivatives. The structure, natural occurrence, and biological activities of haloaryl secondary metabolites isolated from macroalgae are presented in alphabetical order by clade and genus in the next sections. Further information is provided in Supplementary Table S1.
Liu et al. demonstrated that BDDE (17) has antifungal activity against several phytopatogenic fungi, namely, Valsa mali, Fusarium graminearum, Coniothyrium diplodiella, Colletotrichum gloeosporioides, and Botrytis cinerea [14]. Further studies revealed that 17 caused the disruption of the cell membrane integrity in Botrytis cinerea spores and newly formed germ tubes, as well as interacted with DNA via intercalation and minor groove binding [14]. These studies provided evidence that BDDE (17) has potential application in the control of gray mold after fruit harvest and could serve as a lead for the rational drug design of new antifungal agents [14].
This marine bromophenol (17) also has cytotoxic activity against several human tumor cell lines, through the interference with different cellular and molecular targets [10,16]. Liu et al. reported that compound 17 exhibited promising apoptotic activity in K562 cells via mitochondrial pathway and inhibited the activity of topoisomerase I, this effect being associated with the binding to the DNA minor groove [16]. Moreover, it was demonstrated that BDDE (17) acts as a potent antiangiogenesis agent both in vitro and in vivo [13]. In fact, this compound displayed in vitro antiangiogenesis ability through the inhibition of HUVEC cell proliferation, migration, and tube formation, and blocked in vivo subintestinal vessel formation in zebrafish embryos [13].

Lithothamniaceae Family
Only one halophenol is known from this family. Lithothamnin A (18), the first bastadin-like analogue isolated from a red alga, was described in 2011 from Lithothamnion fragilissimum collected east of Lighthouse Reef, Palau Island ( Figure 3, Supplementary Table S1) [17]. Compound 18, with

Rhodomelaceae Family
Among all red algae, the Rhodomelaceae family is the main producer of haloaryl derivatives with a total of 150 secondary metabolites isolated from Callophycus, Laurencia, Odonthalia, Osmundaria, Polysiphonia, Rhodomela, Symphyocladia, and Vidalia species.

Rhodomelaceae Family
Among all red algae, the Rhodomelaceae family is the main producer of haloaryl derivatives with a total of 150 secondary metabolites isolated from Callophycus, Laurencia, Odonthalia, Osmundaria, Polysiphonia, Rhodomela, Symphyocladia, and Vidalia species.

Callophycus Genus
In 2017, four new iodinated and brominated meroditerpenes (iodocallophycols A to D, 19-22) with a unique structure were discovered from the Fijian red alga Callophycus sp. by Lavoie et al. (Figure 3, Supplementary Table S1) [3]. At 10 µM, none of the compounds revealed antibiotic activity against several wild-type and resistant bacterial strains [3].    Table S1.
In addition to haloindoles and aromatic sesquiterpenes, Laurentia sp. have also been an interesting source of other bioactive halogenated aromatic secondary metabolites, namely, naphthalene, benzophenone, and diphenyl ether derivatives. Highly brominated secondary metabolites 72-76 were isolated from red alga L. similis and evaluated for their inhibitory activity against protein tyrosine phosphatase 1B (PTP1B). All compounds displayed inhibitory effect in this enzyme, compounds 75 and 76 being the most potent, with IC 50 values of 2.66 µg/mL and 2.97 µg/mL, respectively [30].

Odonthalia Genus
From the Odonthalia genus, only bromophenols have been isolated. From these secondary metabolites, three are monoaryl bromophenols and six are bis-bromophenols. Kurihara (17) being the most potent [8]. As these compounds act as irreversible inhibitors, it was proposed that the α-glucosidase inhibition might result from the interaction of o-quinones which are oxidative products of o-diphenols, such as 17, to enzyme protein. Moreover, the same research group studied the effect of these bromophenols as well as structure-related compounds in yeast and rat intestinal α-glucosidase sucrase and maltase [31]. Bromophenols 17, 77, and 78 displayed activity for both sucrase and maltase enzymes, with IC 50 values between 1.1 and 3.5 mM [31]. Shoeib et al. identified by gas-liquid chromatography-mass spectrometry (GLC-MS) analysis, lanosol (77) and the methyl, ethyl, and n-propyl ethers of lanosol (81, 94, and 95, respectively), as well as the aldehyde of lanosol (89), in the chloroform fraction of red alga Polysiphonia lanosa, and all compounds showed in vitro cytotoxic activities against human colon cell lines DLD-1 and HCT-116 cells ( Figure 5, Supplementary Table S1) [35].   Figure 5. Naturally occurring haloaryl secondary metabolites 77-108. Further information is provided in Supplementary Table S1. More recently, from the same species of red alga collected along the coast of Sokcho, South Korea, six bromophenols (77 and 81-85) were isolated and evaluated for their effect as isocitrate lyase (ICL) inhibitors, a key enzyme in the glyoxylate cycle highly expressed during appressorium-mediated plant infection by the fungal pathogen of rice Magnaporthe grisea ( Figure 5, Supplementary Table S1) [32]. All compounds were ICL inhibitors, being bromophenols 83 (IC 50 = 2.1 ± 0.1 µM), 84 (IC 50 = 2.8 ± 0.2 µM), and 85 (IC 50 = 2.0 ± 0.1 µM) more potent than 3-nitropropionate, a well-known ICL inhibitor used as positive control. Interestingly, biarylbromophenols 83-85 displayed stronger ICL inhibitory activity than the simple brominated phenols such as 77, 81, and 82, and the debromination of all compounds resulted in the loss of the inhibitory effect upon ICL activity. Collectively, these data indicate that the diphenylmethane skeleton and bromine moiety of bromophenols are essential for potent inhibition of ICL activity [32].
As a result of the bioguided fractionation of red alga P. morrowii, compound 106 as well as the structure-related simple bromophenol 108 were identified as promising antiviral compounds against two fish pathogenic viruses, namely, infectious hematopoietic virus (IHNV) and infectious pancreatic necrosis virus (IPNV) ( Figure 5, Supplementary Table S1) [41]. For both compounds, the concentration causing a 50% inhibition of flounder spleen cell (FSP cell) proliferation (CC 50 ) and each viral replication (EC 50 ) were measured. Both compounds exhibited antiviral activity with selective index (CC 50 /EC 50 ) values of 20 and 42 against IHNV and IPNV, respectively. These results suggest the possible application of these compounds on the discovery of new beneficial agents against viral diseases of salmonid fish, which causes serious losses to the trout and salmon industries [41].

Symphyocladia Genus
Symphyocladia latiuscula (Harvey) Yamada is a member of the family Rhodomelaceae widely distributed along the coasts of northern China, Korea, and Japan [46]. This red alga is an important source of chemical diverse bromophenols, including monoaryl and diaryl secondary metabolites with antidiabetic, antioxidant, antifungal, and DNA polymerase inhibitory activities (Supplementary  Table S1).

Haloaryl Secondary Metabolites Isolated from Brown Algae
Brown seaweeds exhibit significant morphological diversity and are dominant in marine littoral zones from subpolar to equatorial regions. From algae of the Chordariaceae and Dictyotaceae families, 10 dimeric halophenols have been isolated.

Vidalia Genus
The only report about haloaryl secondary metabolites described in algae from Vidalia sp. concerns the isolation of two bromophenols, vidalols A (166) and B (167), from the Caribbean red alga Vidalia obtusaloba (Figure 7) [54]. Wiemer et al. described that these two compounds significantly reduced the edema when applied topically to phorbol ester (PMA)-induced swelling of the mouse ear [54]. Moreover, both compounds inhibited bee venom phospholipase A 2 (PLA 2 ), showing an inhibition percentage of 96% at 1.6 µg/mL, suggesting their potential as lead compounds to design new PLA 2 inhibitors [54]. According to Wiemer et al., the production of these bromophenols in V. obtusaloba could be important as a defense mechanism against some marine herbivores, an example being vidalol A (166) that has been shown to reduce the grazing of Thalassia testudinum by Caribbean herbivorous fishes [54,55].

Haloaryl Secondary Metabolites Isolated from Brown Algae
Brown seaweeds exhibit significant morphological diversity and are dominant in marine littoral zones from subpolar to equatorial regions. From algae of the Chordariaceae and Dictyotaceae families, 10 dimeric halophenols have been isolated. Compounds 110, 170, and 172 exhibited potent in vitro growth inhibitory activity against eight human cancer cell lines (A549, BGC-823, MCF-7, B16-BL6, HT-1080, A2780, Bel7402, and HCT-8) with an IC 50 value below 10 µg/mL, this effect being associated with a moderate inhibitory activity against protein tyrosine kinase (PTK) with over-expression of c-kit. Together, these results indicated that these bromophenol derivatives can be used as potent antitumor agents for PTK over-expression of c-kit [56].

Conclusions and Perspectives
Marine macroalgae play an essential role in the marine environment for the production of oxygen and as a source of food for marine animals. Moreover, these organisms generate compounds and products utilized in many commercial fields, such as fertilizers, and help to obtain compounds with pharmaceutical, cosmetic, and industrial applications.
This review provides an overview of the most relevant haloaryl secondary metabolites isolated from macroalgae, including their distribution and biological activities. A total of 184 haloaryl secondary metabolites, including halophenols, indoles, aromatic sesquiterpenes, and naphthalene derivatives were isolated from macroalgae, with red algae currently being the most prominent source of these compounds, particularly several species of algae from the Rhodomelaceae family. Nevertheless, further biochemical analyses on green and brown macroalgae in the future may also result in the discovery of new compounds from other clades. Most of these halogenated compounds are brominated with a diverse degree of halogenation, as well as some examples of secondary metabolites with chlorine and iodine being described. The most abundant haloaryl derivatives are bromophenols, with most of them possessing at least one catechol group.

Haloaryl Secondary Metabolites Isolated from Green Algae
The isolation of haloaryl secondary metabolites from green algae is uncommon, with a description of only five compounds isolated from the Cladophoraceae and Dichotomosiphonaceae families.

Dichotomosiphonaceae Family
A total of four bromophenols were isolated from Avrainvillea sp., including the brominated diphenylmethanes 181-182, the monoaryl phenol 183, and the tetraarylphenol 184 ( Figure 8). According to Carte et al.,avrainvilleol (181) was identified from the ether extract of A. longicaulis, whereas its methyl ether was isolated from the methanol extract of the same species [59]. Studies of A. nigricans resulted in the isolation of not only avrainvilleol (181), but also the structure-related diaryl bromophenol 182, and the monoaryl phenol 183 (Figure 8). Compounds 181-183 showed inhibitory activity against Bacillus subtilis and Staphylococcus aureus, with 183 being also active against Pseudomonas aeruginosa, Escherichia coli, Serratia marcesens, and Candida albicans [60]. In addition, bromophenol 183 showed to be an in vitro growth inhibitor of the human KB cancer cell line with an ED 50 value of 8.9 µg/mL [60]. The secondary metabolite 184 exhibited HMG-CoA reductase inhibitory activity with an IC 50 value of 5 µM (Figure 8) [59].

Conclusions and Perspectives
Marine macroalgae play an essential role in the marine environment for the production of oxygen and as a source of food for marine animals. Moreover, these organisms generate compounds and products utilized in many commercial fields, such as fertilizers, and help to obtain compounds with pharmaceutical, cosmetic, and industrial applications.
This review provides an overview of the most relevant haloaryl secondary metabolites isolated from macroalgae, including their distribution and biological activities. A total of 184 haloaryl secondary metabolites, including halophenols, indoles, aromatic sesquiterpenes, and naphthalene derivatives were isolated from macroalgae, with red algae currently being the most prominent source of these compounds, particularly several species of algae from the Rhodomelaceae family. Nevertheless, further biochemical analyses on green and brown macroalgae in the future may also result in the discovery of new compounds from other clades. Most of these halogenated compounds are brominated with a diverse degree of halogenation, as well as some examples of secondary metabolites with chlorine and iodine being described. The most abundant haloaryl derivatives are bromophenols, with most of them possessing at least one catechol group.
The biological potential of the majority of haloaryl secondary metabolites has been exhaustively reported, as they are well known their antioxidant, antitumor, antimicrobial, and antidiabetic activities. Therefore, it is expected that some of these compounds may be used in the future in drug discovery. As the distribution of many of the macroalgae is rare in nature, strategies for securing the sustainable production of these secondary metabolites must be implemented. One strategy for overcoming this bottleneck is by using bioprocess technology to produce cell and tissue cultures of marine macroalgae. In fact, the bioprocess engineering of macroalgae for the production of secondary metabolites has been an emerging area of marine biotechnology. Several cell and tissue cultures derived from marine macroalgae have been developed, not only to facilitate the study of secondary metabolites biosynthesis, but also to allow the manipulation and controlled production of these compounds [61][62][63]. Other strategies may include the chemical synthesis of these or nature-inspired haloaryl compounds.
Among the bioactive compounds, bromophenols possessing 2,3-dibromo-3,4-dihydroxy phenyl rings, such as BDDE (17) and BDDPM (85), are the most promising. In fact, both compounds revealed to be quite active in a diverse array of biological activities, especially antitumor and antidiabetic. Taking these results into account, it will be interesting to develop new BDDE and BDDPM synthetic analogues in order to explore the potential of these compounds as leads for drug discovery.
Although several studies about the biological potential of these macroalgae natural products have been described, some unique indoles and aromatic sesquiterpenes have not been explored concerning their biological potential. Therefore, it is expected that the future exploitation of these haloaryl derivatives may contribute to medicinal chemistry in the discovery of innovative bioactive compounds.

Conflicts of Interest:
The authors declare no conflict of interest.