Biological Activity of Recently Discovered Halogenated Marine Natural Products

This review presents the biological activity—antibacterial, antifungal, anti-parasitic, antiviral, antitumor, antiinflammatory, antioxidant, and enzymatic activity—of halogenated marine natural products discovered in the past five years. Newly discovered examples that do not report biological activity are not included.

In the present review I have chosen to focus on halogenated marine natural products possessing demonstrated biological activity that were reported during the period 2011-2015. My organization is according to the type of observed activity, and many of these marine metabolites have multiple activities and therefore appear in more than one section.  [30,31].
Of these gemmacolides, N (1), O (2), and Q (4) show antibacterial activity against the Gram-negative bacterium E. coli in the agar diffusion assay, with the chlorinated gemmacolide O being the most active [30]. Antitumor and antifungal activities are discussed in the appropriate sections to follow.
The Antarctic soft coral Alcyonium roseum has yielded the two new illudalanes, alcyopterosins 44 and 45 (Figure 7) [41]. Although insufficient material was available for antibacterial testing, the authors believe that these metabolites may be feeding deterrents for the predatory sea star Odontaster validus and have antifouling activity, based on similar properties of related alcyopterosins. The soft-coral associated actinomycetes strain, Streptomyces sp. OUCMDZ-1703 has yielded the novel strepchloritides A (46) and B (47), which exhibit modest activity against E. coli, Pseudomonas aeruginosa, and S. aureus (Figure 7).  [41], and strepchloritides A (46) and B (47) from Streptomyces sp. OUCMDZ-1703 [42].
The South China Sea sponge Acanthella cavernosa contains eight new chlorinated diterpenoids, kalihinols M-T (100-107) ( Figure 18). In addition, seven previously isolated analogues were isolated [55].    While no new marinopyrroles were reported in the time frame for this review, it is important to cite an excellent survey of these antibacterial marine halogenated pyrroles [56] and an equally excellent report on their activity against methicillin-resistant S. aureus, including synthetic marinopyrrole analogues [57].
The ascidian Synoicum sp. collected from Korean waters was found to contain eudistomins Y2-Y7 (132-137) ( Figure 23) [65]. These known β-carbolines display a range of activity against both Gram-positive and Gram-negative bacteria (Table 3). This study also included the synthesis of several hydroxyl analogues via sodium borohydride reduction of the carbonyl group, but no improvement in antibacterial activity is observed. Although 132-137 were previously described, antibacterial activity was not reported [66].

Antifungal Activity
In addition to their often potent antibacterial activity (vide supra), many marine sponges contain halogenated metabolites with powerful antifungal properties. The new tetramic acid glycoside, aurantoside K (201), was isolated from a Fijian sponge belonging to the genus Melophlus ( Figure 34) [80]. Auranotoside K is a demethylated analogue of the previously known aurantoside I. Although devoid of antibacterial, antimalarial, and cytotoxicity in the assays examined, 201 displays broad antifungal activity towards Candida albicans (wild type ATCC 32354 and amphotericin-resistant ATCC 90873; MIC 31.25 and 1.95 µg/mL, respectively), Cryptococcus neoformans, Aspergillus niger, Penicillium sp., Rhizopus sporangia, and Sordaria sp. The Indonesian sponge Theonella swinhoei has yielded the new aurantoside J (202), which is an epimer of the previously known auranotoside G ( Figure 34) [81]. The new 202 differs from aurantoside G at the anomeric center C-1′ of the xylose sugar unit. Antifungal activity of 202 is negligible compared to that of aurantosides G and I. A Red Sea specimen of Theonella swinhoei contains the antifungal glycopeptide theonellamide G (203) (Figure 35), which is very similar to the known theonellamide A, lacking only a methyl group on the p-bromophenylalanine and a hydroxyl group in the α-aminoadipic acid group [82]. Theonellamide G shows potent antifungal activity against both wild and amphotericin B-resistant strains of Candida albicans; IC50 4.49 and 2.0 µM, respectively. The positive control amphotericin B had 1.48 µM against the wild type Candida albicans. Figure 35. Structure of theonellamide G (203) from the sponge Theonella swinhoei [82].
The New Zealand sponge Hamigera tarangaensis has yielded a suite of new hamigerans (204-211) (Figure 36), in addition to several known related hamigerans [83]. Hamigeran G (205) also exists as an enol tautomer, and hamigeran F (204) undergoes what appears to be an acid-catalyzed retro-aldol transformation (observed in a CDCl3 solution of 204). Hamigeran G selectively inhibits the growth of two strains of the yeast Saccharomyces cerevisiae.  [83].
A specimen of Verongula rigida from the coast of Columbia has afforded nine previously known bromotyrosines, and two of these, purealidin B and 11-hydroxyaerothionin, display selective antiparasitic activity at 10 and 5 µM against Leishmania panamensis and Plasmodium falciparum parasites, respectively [107]. The Australian sponge Iotrochota sp. contains the two antitrypanosomal compounds, iotrochamides A (281) and B (282) ( Figure 48). Both compounds exhibit moderate activity against Trypanosoma brucei brucei (IC50 3.4 and 4.7 µM, respectively) [108]. Another Australian marine sponge, Zyzzya sp., has furnished the new tsitsikammamine C (283), along with six previously known structurally related brominated alkaloids ( Figure 49) [109]. This novel bispyrroloiminoquinone displays extraordinarily potent in vitro antimalarial activity towards both chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum with values of IC50 13 and 18 nM, respectively. The selectivity index against HEK293 cells is >200. Known alkaloids makaluvamines J, G, and L are slightly less active than tsitsikammamine C in both screens.   [110].

Antiviral Compounds
In addition to harmful bacteria, fungi, and parasites, humans have to contend with lethal viruses, and the search for new antiviral compounds is intense. Although fewer in number than terrestrial sources, the marine environment has produced some antiviral active compounds. A review of antiviral lead compounds from sponges has appeared [115].

Antitumor Compounds
Of enormous concern to all mankind is cancer-the inexorable transformation of normal cells and the proliferation of cancerous cells into tumors. The marine environment provides an array of metabolites active against cancer cells.
Amongst all marine life, sponges have afforded the vast majority of anti-tumor compounds. The Vietnamese sponge Penares sp. contains the novel alkaloids, 322 and 323 (Figure 59), the former of which is moderately cytotoxic to the human tumor cell lines HL-60 (lung) and HeLa (cervix), IC50 16.1 and 33.2 µM, respectively, whereas 323 is inactive [126].    (Figure 62), along with nine previously known bromotyrosine analogues [129]. Whereas the ceratinamines are essentially devoid of cytotoxicity against a panel of human cancer cell lines, the psammaplysins are quite active (Table 10). Included in the table are some of the isolated known analogues and the positive control doxorubicin.
Callyspongiolide (337) is a novel macrolide characterized from the Indonesian sponge Callyspongia sp. (Figure 64) [131]. This metabolite exhibits potent cytotoxicity against L5178Y mouse lymphoma cells, human Jurkat J16 T and Ramos B lymphocytes with IC50 values of 320, 70, and 60 nM, respectively.       The sponge Stylissa sp. from the Derawan Islands in Indonesia has yielded four new brominated alkaloids, 342-345 (Figure 68), along with eight known analogues, including 346-353 [135]. All compounds were screened for their cytotoxicity towards mouse lymphoma cells L5187Y (Table 11)   An examination of the Thai sponge Smenospongia sp. gathered in the Andaman Sea has uncovered the novel 6′-iodoaureol (354) and the bromoindoles 355-359 (Figure 69), isolated from a natural source for the first time, along with several other known natural products [136]. The new compounds, 354-359, and the known 360-362 were screened against a battery of human cell lines for cytotoxicity (Table 12). Only 5,6-dibromotryptamine (362) shows good activity against MOLT-3 (human leukemia) and HeLa cells, with non-halogenated aureol (360) and 355 showing some modest cytotoxicity against HL-60 and HeLa, respectively.  Two studies of the chemical content of the Caribbean sponge Smenospongia aurea, collected in the Bahamas along the coast of Little Inagua, has led to the chlorinated smenamides A (363) and B (364), and smenothiazoles A (365) and B (366) ( Figure 70) [137,138]. Whereas the smenamides exhibit selectivity and nanomolar cytotoxic activity towards Calu-1 (lung) cancer cells, the smenothizoles are equally active and selective against A2780 (ovarian) cancer cells.  [137,138].
The South China Sea sponge Acanthella cavernosa has afforded the new cavernenes A-D (376-379), kalihinenes E (380) and F (381), and kalihipyran C (382) (Figure 74), in addition to several known analogues [142]. These metabolites were screened against several human cancer cell lines (Table 13).   A number of known marine organohalogens were examined for possible cytotoxicity against cancer cell lines during the period covered by this review. To conserve space, their structures are not shown. A review of the antitumor activity of the Jaspis sponges is available [143]. The Fascaplysinopsis sp. sponge metabolite fascaplysin displays excellent cytotoxicity against chemoresistant SCLC (small cell lung cancer) cell lines, by multiple mechanisms [144]. Other cell lines are also discussed. The Suberea sp. sponge alkaloids ma'edamines A and B display significant cytotoxicity against COLO 205 (human colon cancer), MCF-7 (human breast cancer), and A549 (human lung) with IC50 values of 7.9/10.3, 6.9/10.5, and 12.2/15.4 for ma'edamines A/B, respectively [145]. Synthetic analogues show activity against three breast cancer cell lines representing hormone receptor positive and HER2 positive breast cancer [146]. The bis-indole alkaloid 6″-debromohamacanthin A from a Spongosorites sp. sponge inhibits angiogenesis in human umbilical vascular endothelial cells and mouse embryonic cells [147]. The Pseudoceratina sp. alkaloids ceratamines A and B disrupt microtubule dynamics, which provides an explanation for their pronounced antimitotic activity (lower micromolar) [148]. The well known dibromo-dihydroxyoxocyclohexenyl acetonitrile has excellent activity against the K562 leukemia cell line (IC50 1.4 µg/mL) [149]. The known spirastrellolides A and B were isolated from the sponge Epipolasis sp. for the first time as free acids, and not as methyl esters. Both macrolides are cytotoxic to HeLa cells, with IC50 20 and 40 nM, respectively [150].
Larger marine animals like gastropod molluscs are known to produce biologically active metabolites, some of which contain halogen. The anticancer properties of the lamellarins, which were first isolated from a marine mollusc, have been reviewed [187].

Antioxidants and Antiinflammation
Because antioxidants can have anti-inflammatory activity, these two categories are combined. Like terrestrial phenolic compounds, marine phenols with antioxidant properties are well known, and several recent examples have appeared. The red alga Rhodomela confervoides from Liaoning Province, China, has afforded 19 bromophenols, six of which are new (538-543) ( Figure 104) [191].
Another study of Rhodomela confervoides has led to the discovery of five new nitrogen-containing bromophenols 546-550 ( Figure 105) in addition to nine known analogues such as 551 [192]. In the DPPH assay bromophenol 546 shows the strongest activity (IC50 5.22 µM) (BHT, IC50 82.1 µM), followed by 548 > 547 > 551. In the ABTS assay, 551 is the most active, more active than ascorbic acid. The antioxidant capacity of these bromophenols seems to be correlated with the number of hydroxyl groups (or phenolic rings).
A specimen of the red alga Symphyocladia latiuscula from the coast of Qingdao, Shandong Province, China, has furnished the new bromocatechols 552 and 553 ( Figure 106) [193]. Both are modest radical scavengers in the DPPH assay with IC50 14.5 and 20.5 µg/mL, respectively. Ascorbic acid shows IC50 7.82 µg/mL. The red alga Vertebrata lanosa, collected from Ullsfjorden, Norway, afforded the new bromocatechol 554 and the known 555-557 ( Figure 106) [194]. Their antioxidant capacity was screened using these assays: ORAC (oxygen radical absorbance capacity), CAA (cellular antioxidant activity), and CLPAA (cellular lipid peroxidation antioxidant activity). This study is the first to measure the cellular antioxidant activity of bromocatechols. The antioxidant activity is highest for 555 followed by 554, and then 556 and 557. At concentrations as low as 10 µg/mL, bromocatechol 555 inhibits 68% of oxidation in the CAA assay. By comparison, the known antioxidants quercetin and luteolin at this same concentration (10 µg/mL) inhibit the oxidation of the CAA substrate (2′,7′-dichlorofluorescin) to the extent of 92% and 58%, respectively.
Several marine sponges exhibit antioxidant behavior. The new 5,6-dibromo-L-hypaphorine (558) (Figure 107), along with four known bromoindoles, was isolated from the sponge Hyrtios sp. living in Fiji [195]. This new bromoindole displays significant antioxidant ability in the ORAC assay, only 4-fold less active than Trolox (a water-soluble analogue of Vitamin E). A study of the antioxidant activity of the known Zyzzya fuliginosa sponge metabolites, zyzzyanones and makaluvamines reveals that the presence of a phenolic ring is essential for maximum activity in both the ABTS and APPH assays, and that a p-hydroxystyryl unit as in the makaluvamines (e.g., 559) is more important than a simple phenolic ring as in the zyzzyanones (e.g., 560) ( Figure 107) [196].  [193,194].  [195,196].
The novel iodinated acetylenic acid sponge metabolites 561-564, isolated from the South Korean Suberites mammilaris (561 and 562) and Suberites japonicus (563 and 564), were examined for their antiinflammatory activity ( Figure 108) [197]. The methyl esters 561 and 562 strongly inhibit nitric oxide (NO) production from RAW 264.7 murine macrophase cells, with IC50 3.9 and 7.0 µM, respectively. However, in BV2 microglia cells, the methyl esters of 563 and 564 are the most active in NO inhibition: IC50 3.1 and 1.8 M, respectively. All four methyl esters attenuate the production of PGE2 (prostaglandin E2) from RAW 264.7 and BV2 cells as induced by LPS (lipopolysaccharide).

Enzymatic and Molecular Activity
Overshadowed by the biological effects presented in the previous sections are molecular interactions between the marine natural products and the target molecules (enzymes, peptides, and other small biological molecules) that are the root cause of these effects. A review of the targeting of marine natural products to cytoskeletal proteins has appeared [205].
The sponge Xestospongia testudinaria has yielded five new brominated fatty acids, 612-616 ( Figure 118), which include testufuran A (612), similar to mutafuran H (594) (Figure 115) isolated from the same sponge. An additional 11 known brominated acetylenic acids were also characterized. Most of these 16 bromo carboxylic acids stimulated the secretion of the protein hormone adiponectin, which regulates glucose levels and fatty acid breakdown, from differentiated ST-13 preadipocytes. These compounds do not exhibit agonistic activity against PPAR-γ (the peroxisome proliferator-activated receptor) [216].
The ascidian Herdmania momus has yielded seven new herdmanines E-K (617-623) (Figure 119), some of which demonstrate significant PPAR-γ activation in Ac2F rat liver cells. The active examples are I (621) and K (623). For example, the latter herdmanine K exhibits strong PPAR-γ activation at 1 and 10 µg/mL concentrations, with greater potency than the antidiabetic drug rosiglitazone. The known (-)-leptoclinidamine B was also isolated from the ascidian and is only slightly less active than 623 [217].   The two rare iodinated polyacetylenes, placotylenes A (624) and B (625), were characterized in the Korean sponge Placospongia sp. (Figure 120). Placotylene A inhibits osteoclast differentiation of bone marrow-derived macrophages, perhaps by decreasing the expression of RANKL (receptor activator of nuclear factor-κB ligand). This marine polyacetylene could represent a lead compound for osteoporosis treatment [218]. The Caribbean sponge Chalinula molitba has afforded the novel chlorinated sterol disulfate, chalinulasterol (626) (Figure 120). Despite the resemblance of chalinulasterol to the known PXR (pregnane X receptor) agonist solomonsterol A (627), no activity is observed for the former sterol. This important receptor regulates expression of drug metabolizing and detoxifying enzymes [219].
A combined Curacao and Papua New Guinea collection of cyanobacteria has yielded five new vinylchloride metabolites, janthielamide A (628), kimbeamides A-C (629-631), and kimbelactone A (632) (Figure 121). Janthielamide A came from the collection at Jan Thiel Bay in Curacao, and the latter four metabolites came from the collection at Kimbe Bay, New Britain, Papua New Guinea. Janthielamide A (628) exhibits Na + channel blocking in murine Neuro-2a cells (IC50 11.5 µM), and also antagonizes induced Na + influx in neurons (IC50 5.2 µM). Kimbeamide A (629) displays similar Na + blocking activity at a concentration of 20 µg/mL, but it, along with the 630-632, undergoes oxidative decomposition [220].
The marine-derived fungus Aspergillus sp. SCSGAF0093 produces nine mycotoxins, four of which are new, aluminiumneoaspergillin (640), zirconiumneoaspergillin (641), aspergilliamide (642), and ochratoxin A n-butyl ester (643) (Figure 124). This is the first report of marine-based ochratoxins (ochratoxin and the methyl ester were also isolated), and the first discovery of a zirconium complex (641) in nature [223]. All nine compounds exhibit some toxicity to brine shrimp. The most toxic compounds in this assay are 643, ochratoxin A, and ochratoxin A methyl ester, with IC50 4.14, 13.74, and 2.59 µM, respectively. The innocent-looking, but ominous cone snails (genus Conus) comprise about 700 species and are widely distributed in the world's oceans [224]. It is estimated that these cone snails contain more than 50,000 distinct toxins, since the venom in each Conus species consists of 40-200 individual peptides with a unique biological action [225][226][227]. Many of these Conus sp. peptides contain 6-bromotryptophan [3], the function of which has been suggested to block proteolytic degradation since the large bromine makes the peptide a poor fit for docking in the active site of chymotrypsin [228]. Recent studies have established the binding site of α-conotoxin Vc1.1 from Conus victoria on the nicotinic α9α10 acetylcholine receptor, making this toxin a potential novel treatment for neuropathic pain [229]. A similar α-4/6-conotoxin TxID has been identified in Conus textile. It also blocks nicotinic acetylcholine receptors [230]. The conopeptide MVIIA (Ziconotide; Prialt) was approved by the U.S. FDA in 2004 for the treatment of severe pain.

Conclusions
Marine organisms possess an astonishing array of biological activities! The chemical compounds they produce proffer future medicinal developments in a multitude of human diseases. Of these compounds, organohalogen natural products frequently display the highest level of biological activity. The unceasing developments in aquatic exploration, organism collection, compound isolation and