The genus Aplysina, formerly known as Verongia and reclassified to Aplysina, is one of the richest in terms of secondary metabolites, described in 14 species of the family Aplysinidae, there are 2 species from the Mediterranean Sea, 8 from the Caribbean, 3 from the Pacific Coast of Mexico and 15 in the Brazilian coast. Of the above species, 8 have only been recently identified. From the Mediterranean Sea, the two described species of the genus Aplysina are: A. aerophoba (Schmidt, 1862) and A. cavernicola (Vacelet, 1959). From the Caribbean, among others we find A. fistularis insularis, A. fistularis form fulva, A. archeri, A. cauliformis and A. Lacunosa [33].

Like other genera of the order Verongida, Aplysina stands out for its unique biochemical characteristics. They show low terpene content, and possess a moderately high percentage of sterols, mostly within the aplystan skeleton. They also produce a significant series of brominated derivatives of tyrosine metabolites considered peculiar to species of this order. The sponges of this order are also known for their high phenotypic variability [34].

Marine organisms produce a cocktail of halogenated metabolites with potential commercial value. The structures found in these compounds go from linear chain acyclic, to complex polycyclic molecules [35,36]. The research of halogenated metabolites has been more focused on marine algae than on sea sponges [37–41]. Though many compounds have been discovered recently, many sponges species are poorly screened and the need for new drugs keeps this field open.

In a previous paper our research group evaluated crude algae, sponge extracts and chemically determined molecules from Northeastern Brazil [42–48] with database survey [49–62].

In this paper we review halogenated substances from the genus Aplysina. A compilation of the ¹³C NMR spectral data of the selected natural products is also provided. This type of genus and species investigation is helpful in the identification and capture of halogenated substances from the genus.

Although in the past, it was suspected that bromotyrosine compounds were not present in Brazilian Aplysina species [69], nowadays numerous studies have shown the presence of these chemical biomarkers, not only in Brazilian species, but in almost all the Verongida order.

In order to classify the large number of halogenated compounds reviewed in Table 2, for each sponge species, we listed the halogenated compounds under the correlated species. Considering the taxonomic species diagnosis of morphologic variation of spongin fibers is difficult [33], chemical composition can be used as a tool for a more accurate identification. The distribution of the halogenated compounds is widespread in Aplysina genre, and studies show that mainly bromoisoxazoline alkaloids have been found in almost all species. This family of metabolites was usefully employed as a chemical marker for the distinction of some taxa as Aplysina aerophoba and Aplysina cavernicola, two very physically similar species [63], but biochemically different. In another situation, majority of aerothionine was key to identify two subspecies of A. fistularis, which split into A. fulva and A. insularis [115].

The similarity between agelorins A and B, isolated from Agelas oroides and produced by Aplysina caissara, was essential to show the two genera, Aplysina and Agelas, have a phylogenetic relationship [76] and 11-epi-fistularin-3 was yielded by Aplysina fulva [98].

The presence of stereo metabolites isolated from Aplysina sponges as derivatives of fistularin-3 discussed by Rogers et al., 2005 [98], provides evidence that enzymatic pathways are non-stereoselective in these sponges.

As can be seen, each kind of genus adds a different profile of metabolites. However, even with a different chemical profile, Aplysina sp. has compounds that give a clue to their evolutionary origin.

The substances aplyzanzine A, aplysamine-1 and aplysamine-2 present a dibromotyramine structural portion, and probably originated in accordance with Evan et al. [82], by amidation with other bromotyrosinated radicals. Moloka’inamine [116] and purealidin C isolated from Psammaplysilla purea [90] are examples of metabolites isolated from sponges of the order Verongida, having dibromotyramine in their structures. According to Carney, free phenolic groups are important precursors nitrile phenolic [96], hence the similarity between methoxylated compound, aplysamine-2, and hydroxylated analogue, psammaplin-A [84], observed by Xynas and Capon, 1989, shows that psammaplin-A may be important precursor aplysimines as much of the fistularin and its derivatives

Cavernicolines are γ and δ-lactames formed by a residual halogenated tyrosine precursor [81] and also having a bi-cyclic system. The junction of the rings occurs in carbons C-3 and C-4 in ortho position, while in the 7-bromocavernicolenone and in the 7-chlorocavernicolenone, this junction occurs in carbons C-2 and C-4. They can be defined as haloperoxidases with the role of converting either the 3-chloro (V) ortho 3-bromotyrosine (IV) in residues of 3,5-dichloro (VIII) or 3,5-dibromotyrosine (VI) or 3-chloro-5-bromotyrosine (VII) or 3-bromo-5-chlorotyrosine (IX), respectively [80]. These substances have a chiral center at C-2 and their R and S enantiomers are obtained in racemic mixtures, or relatively pure from the genus Aplysina. In the formation of cavernicolines, as to the substitution pattern (ortho or para) it is suggested that the biosynthesis pathway has either a halotyrosine (XV, XVI, XVIII and XVIX) or a halo-dopa (XIV and XVVI) intermediary which will form a spirolactone precursor (XXII and XXIII), allowing the formation of intermediaries in racemic or quasi-racemic mixtures. The absence of control in the absolute stereochemistry of this class is intrinsic to phenol oxidative coupling [81,117]. It is noteworthy that experimental observations [118] show that 3,5-dibromo-4-methoxyphenylalanine methyl ester (XX) in reaction in an anodic oxidative medium form a more appropriate intermediary (XXI) than the spirolactone, originating derivatives similar to the stereoisomers of the cavernicolines with considerable yields (See Figure 1).

The hydroverongiaquinols are 2,6-bromotyrosine phenolic derivatives. Both the hydroverongiaquinols and the verongiabenzenoids are important mediators in biosynthesis of other classes of bromotyrosine metabolites. The verongiabenzenoids are part of the biosynthesis of isoxazoline alkaloids, and hydroverongiaquinols are important precursors in the formation of metabolites which need free phenolic groups to convert themselves into α-oximine substances, such as the phenolic nitriles. However, phenolic nitriles have not been found in the genus Aplysina, they are found in the genus Ianthella where substances like the bastadins, are important chemotaxonomic markers for the genus [119].

The bromotyrosineketals have a 3,5-dibromocyclohexa-2,5-dienyl ketal skeleton system. Literature shows that the dimethoxy and methoxy-ethoxy ketals (XXXII) isolated from Aplysina fistularis and from Aplysina cauliformis [92,93,120], are artifacts formed by the oxidation of dienone, since the dimethoxy form is obtained as a mixture of diastereoisomers [94,95], both showing antibacterial activity. Further evidence that they are artifacts is the formation of dimethoxy and methoxy-ethoxy ketals which can be explained as being formed from an arene (dienone intermediary XII), which suffers 1,4 additions of methanol, water or ethanol (Figure 2), and displayed a reaction described by Kasperek et al. [94,95,121]. However, the methoxy-butoxy and methoxy-pentoxy ketals isolated from Aplysina thiona [109] are not considered reaction products. Aplysinketal A was isolated only in the form of diastereoisomers, and the absence of the dimethoxy ketal indicates the non-existence of reactions during the extraction process [109]. It has been suggested that the formation of the C₄ and C₅ chains are formed via lysine and ornithine respectively (Figure 2) [104,114].

The bromotyrosine lactone derivatives, with the exception of aplysinimine which is an imine, are five member lactones condensated with 3,5-dibromotyrosine residues (XXIV). Aeroplysinin-2 is different from the others because it has a cyclodiene group instead of an aromatic ring, while aplysinadiene presents a cis-trans diene side chain. It is proposed that the biosynthesis of this class of substances has an imine-ether (XXXIV) as initial intermediary, a hydroxylated derivative of aplysinimine. This derivative will either suffer a tertiary alcohol dehydration to form aplysinimine [109,120] or will follow other biosynthetic pathways similarly to bromotyrosine lactones as aeroplysinin-2, or the verongiabenzenoids and verongiaquinols, in this case the intermediary suffers dehydroxylation and demethylation and can form artifacts such as dimethoxys and methoxy-ethoxy ketals. [122]. Aplysinolide is considered an artifact, since it possesses an α,β-unsaturated side chain which is uncommon to find linked to a lactone ring. In accordance to Cruz et al. [109] this substance can be formed by combining aplysinimine with Me₂CO during the purification process (see Figure 1).

Oxazolidones are very common in Aplysina, but just two types are found: diastereoisomers of bisoxazolidone and methoxy derivative. This derivative presents two chiral centers with different stereochemistries for different species. Studies show that these bisoxazolidone’s isomers have a relative configuration of 7S*, 11R* [97] and 11R*, 7S* [106], as determined by comparison with bisoxazolidone isolated from Ascidia Clavelina oblonga [123] and an absolute configuration R, R [64] obtained by X-ray crystallography. There are evidences that fistularin-3 degradation promotes bisoxazolidone and aeroplysinin-1 production [13].

The spiroisoxazolines also known as isoxazoline alkaloids form the biggest group of Aplysina and Verongid order metabolites. They are divided into two structure types: mono-spirocyclohexadienylisoxazolines and bis-spirocyclohexadienylisoxazolines [124].

The chemical structure of mono-spirocyclohexadienylisoxazolines (nuclei G7, G8) have essentially one spirocyclohexadienylisoxazoline ring bonded to a 1–6 carbon side chain with exception of the spiroisoxazoline acid ester (nucleus G9) [83] considered an artifact of ethanolic condensation from spiroisoxazoline acid [125]. The bis-spirocyclohexadienylisoxazolines (nuclei G1, G2) can present the same ring, which in the more common example is bonded to a 3–11 carbon side chain.

This side chain is bonded to another spirocyclohexadienylisoxazoline ring [124]. In other spiroisoxazolines, the rings suffer oxidation of the methoxy group forming a cyclohexenone (nuclei G3, G4) [74,76,101]. Cyclohexenone also suffers hydroxyl or bromine oxidations originating cyclohexenone epoxide between carbons 1 and 2 and forming oxaspirocyclohexenonylisoxazolines (nuclei G5, G6) [100]

The biosynthetic pathway for spiroisoxazolines needs tyrosine intermediates with O-methyl groups (XXVII), which are metabolized to form oxime grouped intermediates (XXVIII) and shortly thereafter form other intermediaries with arene oxide (XXIX). The nucleophilic attack of the oxime over either the epoxide or the phenol originates by breaking the epoxide which forms the isoxazole ring (XXX) [96]. The C₄ or C₅ side chains that extend out of the ring such as in aerothionin and inhomoaerothionin are produced via ornithine and lysine respectively (Figure 2) [70,94,95,126]. However, when the side chains present a 4-aminobutanoic substituent it is suspected that the amino acid involved is glutamic acid (Figure 2) [99]. In some spiroisoxazolines such as fistularin-3, and araplysillin N⁹-sulfamate, there is a 3-amino-1-propanol connector which binds itself to other structures and probably has as a precursor of its biosynthesis a decarboxylated product of the uncommon amino acid, homoserine. This amino acid is an intermediary for the enzyme S-adenosylmethionine (SAM). It is suggested that the SAM is involved in SN2 substitutions of hydroxyl of 3,5-dibromotyrosine (DBT) (XXIV), making it susceptible to the formation of methoxyl groups by methyltransferase and O-alkylated bonds via other enzymes. The enzyme responsible for O-alkylations is putative C³-alanil-methyltransferase which allows 3-amino-1-propanol connector bonding to the DBT residue, forming spiroisoxazoline residue complexes with large molecular masses (XXV) which are then incorporated in the isoxazoline rings [99]. The alkaloid archerine, a dimer of two imidazole rings, is probably formed by oxidative coupling [1 + 1] of two aerophobin-2 molecules [73]. In sponges of the order Verongida, the spherule cells have the capacity of stocking and secreting isoxazoline alkaloids, which are modified by enzymes located in distinct locations [127,128]. The extracts of the majority of the sponges belonging to the genus Aplysina, when tested, show that their enzymes convert brominated isoxazoline alkaloids into aeroplysinin-1 and dienones. Sponges of other orders are unable to perform this biotransformation [129]. Puyana et al. [130] demonstrated that there is no aeroplysinin-1 and dienones production when there is a decrease in the amount of spiroisoxazolines. The ecological function of this enzymatic activation is microbial pathogen growth inhibition and the repellent odor, which decreases the predatory search by fishes [129].

Although the agelocaissarines A1, A2, B1 and B2 were initially considered production artifacts as pairs of stereoisomers, this was later modified by the observation of in vitro experiments showing the absence of substances with relative stereochemistries different to those found in the work [76].

In therapeutics these substances demonstrate tumor cell cytotoxic [22], antimicrobial [15] and antihistamine [73] activity.

Spiroisoxazolines vary in different species, but also inside the single species. While A. fulva produces aerothionin as its major component (0.11%) [34], this same substance is not present in A. insularis [74] and in A. fulva it appears with a larger amount (0.52%) [102]. Nuñez et al. [97], affirms that this chemical variation may be due to either different extraction and isolation techniques, or to biological diversity of the areas in which the sponges are collected. This chemical distinction led to reinforce the hypothesis that Aplysina aerophoba and Aplysina carvernicola have metabolic differences and that A. aerophoba, erroneously identified in the work of Cimino et al. [67], was in fact Aplysina cavernicola.

The presence of hemifistularin, isolated together with 11-oxofistularin-3, begs us to question whether the first is a precursor of 11-oxofistularin-3 biogenesis, or a degradation product [72]. Fistularin-3 shows another type of variability. Besides having stereoisomers of (+) fistularin-3, such as (+)-isofistularin-3 and 11-epi-fistularin-3, the chemical composition of sponges contains them in irregular proportions, which makes it difficult to determine through optical rotation. In order to determine the absolute configuration of fistularin-3 and its stereoisomers, a microscale analysis with Marfey’s reagent, has been used that led to the formation of stable reaction products analyzed by LC-MS [98].

The verongiabenzenoids are aromatic methoxylated substances which present a skeleton with a 2,6-dibromomethoxybenzene nucleus. Biogenetically methoxylated, some verongiabenzenoids can form isoxazolines [13] or epoxide intermediaries from the arene oxide, which leads to forming other verongiabenzenoids [96].

The chemical structure of the verongiaquinols is either a cyclohexadien-2-one or cyclohexadien-2,6-one system, with either bromine or chlorine substituents in positions 2 and 6 and hydroxyls on carbons C-3 and C-4. They also may suffer ramifications on carbon C-4. The verongiaquinols seem to be related to degradation steps of tyrosine metabolism, as is the case of dienone (XII). The degradation of bromotyrosine substances such as iso-fistularin-1 and aerophobin-2 after mechanical injury led to the formation of aeroplysinin-1 (XXXII) and (XII). It is also noteworthy that the extraction of frozen sponges and consequent exposure to alkaline sea water will form dienones (XII) [97,130,131]. Besides being integrated at the metabolic level, dienone and aeroplysinin-1 have an important defense role for sponges: cytotoxic, algicidic, molluscicide and antibacterial activity have been reported [131,132].

Other verongiaquinols such as 2,6-dibromoquinone have been reported to inhibit the enzyme RNA polymerase II, blocking the initiation of the chain, but not its elongation [133].

It is not known for sure if (1′R,5′S,6′S) and (1′R,5′R,6′S)-2-(3′-5′-dibromo-1′-6′-dihydroxy-4′-oxocyclohex-2′-enyl)-acetonitrile are simple artifacts, as aeroplysinin-1 is able to form them in the presence of acetone by keto-enol tautomerism. However, these acetonitriles are not normally produced as metabolites of aeroplysinin-1 (XXXII) in other species [27].

This group is comprised of two substances which present a 1,2-dihydroarene-1,2-diol and may have their biogenesis via an arene oxide (XXXI) in agreement with their stereochemistry [94,95]. Aeroplysinin-1 (XXXII) is a nitrilated substance found in dextro and levorotatory forms. The dextrorotatory isomer (+) aeroplysinin-1, has been obtained from Aplysina aerophoba [70], Aiolocroia crassa, Verongula rigida, Aplysina archeri [71] and Psammoposilla purpurea [134]. (−) Aeroplysinin-1 has been found in Ianthella ardis [135] and Verongula gigantean [71]. The metabolic degradation of bis-oxazolidone, isofistularin-3, aplysinamisin-1 and aerophobin-2 is known to be an important source of aeroplysinin-1 [13]. In terms of pharmacological activity, aeroplysinin-1 is a versatile substance which has demonstrated cytotoxic [134], antiprotozoal [136] and antiangiogenic [137] activities. Aplysifulvin is one of the most recently isolated substances from the sponge A. fulva [97]. It possesses only two methoxies and no ethoxies, and since no ethoxy derivatives were detected, the possibility that aplysifulvin is an artifact has been discarded. Hypothetically, the chemical structure of aplysifulvin suggests that the 3,3-dialkoxy ketals (with OMe and OEt groups) previously described are artifacts [97].

This section describes the compilation of the ¹³C chemical shifts of halogenated compounds of the genus Aplysina. All compounds compiled in this review—bromotyramines (1–4), cavernicolins (5–17), hydroverongiaquinols (18–20), bromotyrosineketals (21–24), bromotyrosine lactone derivatives (25–28), oxazolidones (29–32), spiroisoxazolines (33–81), verongiabenzenoids (82–86), verongiaquinols (87–99) and dibromocyclohexadienes (100–101)—have in common 3,5-halotyrosine or halophenylalanine derivatives.

Research data shows that works from the decades of 1970, 1980 and 1990 show little or no information of ¹³C NMR as compounds (95–97) whose structural elucidation was done by mass spectrometry (MS) and ¹H NMR spectroscopy analysis and reactions of structural identification.

The bromotyramine family skeleton can be recognized by the typical ¹H-NMR signals, as for example, in the case of compound 1: a singlet for aromatic protons H-2 and H-6 (δ 7.62) and four triples (δ 3.02, 2H, 8.0 Hz; δ 3.50, 2H, 8.0 Hz; δ 4.12, 2H, 5.5 Hz; δ 3.22, 2H, 5.5 Hz) attributed to H-7, H-8, H-1’, H-3’ respectively [84]. When NH-3’ has low electron density as compound 1 positively charged and compound 2 close to electrophilic oxime group, the ¹³C-NMR signals shift to downfield compared to compounds 3 and 4,whose substituents are methyl groups [84].

Typical ¹H NMR data from the cavernicolin class are, in the case of compound 5, for H-3 δ 4.05 (dd, J = 10.1 Hz and J = 1.5 Hz), a singlet for H-5 at δ 7.3 and the signals for NH and OH at 6.9 (s) and 4.4 (s) respectively [83]. ¹³C NMR data shows that chlorinated carbon have their signals at downfield shifts in relation to bromine carbons as it can be seen, for example, comparing compounds 5 and 6 with 9 and compound 11 with 12.

Biosynthetically, verongiaquinol metabolites are the oxidized form of hydroverongiaquinols, considered as a hydroquinone precursor [124,138]. Therefore, the basic difference in the ¹³C NMR between these two classes is the downfield signal of ketone carbon C-1 (δ 172.6) of compound 91 compared with hydroxylated C-1 (δ 150.0) of compound 20 [97].

Data analysis of the ¹³C NMR bromotyrosine lactone family brominated aromatic show carbon signals have more shielded signals compared to the other aromatic signals of the ring. The more is the unsaturated side branching at C-7, more deshielded are the signals of the lactone ring, with the increasing order of introducing the compounds 28, 27 and 26 [64,109].

The carbons of the spiroisoxazolinic system of most compounds with cores G₁, G₂, G₇, G₈ and G₉ acquire values which become a standard set of values, with the exception of carbons C-2 and C4 values, which seem to be mistakenly exchanged one for another at δ 120 or δ 114, being the correct value of δ 120 for carbon C-2 due to the proximity of the hydroxyl and the C-4 for δ 114.

The mono and bis-spiroisoxazolinic ring systems could be distinguished by double ¹H-NMR shield and deshield signals for the two rings, for example, compound 50 shows signals at δ 4.16 (1H, d, J = 8.3 Hz) for H-1 and δ 4.58 (1H, d, J = 7.9 Hz) for H 1’. A typical methylene signal at δ 4.43; 3.47 (2H, ABq, J = 18.2) is attributed to the isoxazol ring protons H-7, 7’ for this compound [29]. Today the most used techniques to elucidate absolute stereochemistry of the rings are ¹H-NMR spectrum analysis and molecular modeling using both MM2 and MOPAC protocols of the Chem3D software [76], and also NOE-difference spectroscopy studies [100].

¹³C data reveals spiroisoxazoline ring systems have distinguished shifts. The difference between cyclohexadienone (G₁, G₂, G₇, G₈ and G₉) and cyclohexenone (G₃ and G₄) systems are two chemical shifts at downfield for C-3’ and C-5’ (54–55) and one at upfield for C-2’ in G₃ (56–61), and the same shifts for C-3, -3′, C-5, -5′, C-2, -2′ in G₄ (56–61). The epoxide group in G₅ and G₆ can be characterized by the same shifts plus two differences: a strong shift at downfield for C-3 and C-3′ in G₅ and C-3′ in G₆. The other difference involves three chemical shifts at upfield for C-1, 1′, C-2, 2′ and C-6, 6′ of 62 and for C-1′, C-2′ and C-6′ of 63. Some ¹³C data as δ 67.2 and δ 65.7 attributed to chiral C-11 of compound 42 still remain with its stereochemistry unsolved [76].

According to Kossuga, 2004 [123], to determine the configuration of the relative stereochemistry of bis-oxazolidone uses [α] of (7R,11R) bis-oxazolidone with absolute stereochemistry [64]. It was possible to determine the relative configuration of bis-oxazolidones (7*R,11*S) and (11*R,7*S) isolated in previous works as can be seen in Table 2 [63,65,109].