Further Dibromotyrosine-Derived Metabolites from the Marine Sponge Aplysina caissara

A re-investigação química do extrato bruto da esponja Aplysina caissara levou ao isolamento de cinco novos derivados da dibromotirosina, denominados agelocaissarinas A1, A2, B1, B2 e caissarina C, além dos já conhecidos fistularina-3 e 11-hidroxiaerotionina. Os compostos isolados tiveram suas estruturas determinadas pela análise de seus espectros de RMN monoe bidimensionais, espectro de massas de alta resolução, infravermelho e ultravioleta. A configuração relativa das agelocaissarinas pôde ser estabelecida por análise dos espectros de RMN-H e modelagem molecular, enquanto que a configuração absoluta do sistema espiroxazolidínico da fistularina-3, da caissarina C e da 11-hidroxiaerotionina pôde ser estabelecida pela análise de seus espectros de dicroísmo circular. A fistularina-3 e a 11hidroxiaerotionina apresentaram atividade antibiótica moderada contra várias linhagens de bactérias patogênicas.


Introduction
Marine sponges constitute a remarkable source of novel, potently bioactive secondary metabolites. 1,2 In particular, sponges of the order Verongida have been the source of a variety of biologically active dibromotyrosine-derived modified peptides and alkaloids. Recent examples of compounds belonging to this structural class are the purpurealidins A -D, F -H isolated from the sponge Psammaplysilla purpurea, 3 trisulfide psammalin A, (E,E)bromopsammalin A and bispsammalin A from the association of the sponges Jaspis wondoensis and Poecillastra wondoensis, 4

purpuroceratic acids A and B from
Pseudoceratina purpurea, 5 several new and known psammalins active as histone deacetylase and DNA methyltransferase inhibitors, from the sponge Psammaplysilla purpurea, 6 the moderately cytotoxic purealidin S and purpuramine J isolated from the sponge Druinella sp., 7 and nakirodin A from an unidentified Verongida sponge. 8 We have recently reported the isolation of two new members of the dibromotyrosine derivatives, caissarines A (1) and B (2), from the sponge Aplysina caissara. Both caissarines were identified by analysis of spectroscopic data. 9 However, due to the lost of the samples of both 1 and 2, we have been unable to measure their specific rotation and to evaluate their biological activities. Therefore, we have been interested to re-isolate the compounds in order to complete their characterization and to evaluate their biological activities. Surprisingly, a first recollection of the sponge A. caissara did not provide any dibromotyrosine derivative. 9 Since the recollected sponge was stored in EtOH at room temperature for several weeks, we considered that the sponge metabolites could suffer degradation under these conditions. It has been indeed reported that dibromotyrosine metabolites of the sponge A. aerophoba can degrade in the presence of alcohol-H 2 O mixtures, since the enzymatic activity is not completely suppressed under such conditions. 10 Nevertheless, similar experiments carried out with extracts of the sponges A. insularis and A. archeri did not presented similar results, since the dibromotyrosine metabolites of these sponges did not suffer degradation when stored in alcohol. 11 A second recollection of A. caissara, followed by immediate animal freezing at -20 o C, rapid transportation to laboratory, freeze-drying and extraction, yielded, after several chromatographic separations, no trace of caissarines A (1) and B (2), 9 but gave fistularin-3 (3), 12 11-hydroxyaerothionin (4), 13 and five unprecedented dibromotyrosine derivatives, named agelocaissarines A1 (5), A2 (6), B1 (7), B2 (8) and caissarine C (9). Herein we report the isolation and structure determination of the new dibromotyrosine derivatives 5-9 as well as the absolute configuration of the spiroxazolidine moiety of compounds 3, 4 and 9 isolated from A. caissara. We have also evaluated the antibiotic activity of fistularin-3 (3) and 11-hydroxyaerothionin (4) against several pathogenic bacteria.

Experimental
General experimental procedures UV spectra were recorded on a Hitachi U-3210 spectrophotometer. IR spectra (film on Si plate) were recorded on a FT-IR Bomem MB102 infrared spectrometer. Specific rotations were measured on a Perkin Elmer 241 polarimeter in MeOH. NMR spectra were recorded either on a Bruker ARX 9.4 T instrument, operating at 400 MHz for 1 H and 100 MHz for 13 C channels, respectively, or on a Bruker DRX300 7.05 T, operating at 300 MHz for 1 H and 75 MHz for 13 C, respectively. All NMR spectra were obtained at 25 o C using TMS as internal reference. Low and high resolution mass spectra were recorded either on a VG-7070 mass spectrometer, using electron impact at 70 eV, FAB or CI, or on a Bruker-Hewlett Packard 1100 Esquire-LC system mass spectrometer. Solvents used for extraction and flash chromatography were distilled prior to use. HPLC-grade solvents were utilized without further purification in HPLC separations. TLC analyses were performed with plastic- backed

Isolation of compounds 3-9 from Aplysina caissara
The frozen sponge (1278 g) was freeze dried to give 317 g of dry material which was sequentially extracted with MeCN, acetone and MeOH. The MeCN and acetone extracts were separately filtered and evaporated to give brown gums. The MeOH extract was filtered, concentrated to 400 mL of an aqueous suspension, which was partitioned with EtOAc (3 × 400 mL). The organic layer was evaporated, solubilized in MeOH and filtered to eliminate inorganic salts, to yield a dark gum. The MeCN, acetone and EtOAC crude extracts have shown to be virtually identical by TLC analysis (CH 2 Cl 2 -MeOH 9:1), and were pooled to a single crude extract (9.25 g). This crude extract was subjected to a series of chromatographic separations by flash chromatography on silica gel (gradients of MeCN in CH 2 Cl 2 ), by C 18 reversed phase column chromatography (gradient of MeOH in H 2 O), followed by purifications by HPLC with either a C 18 reversed phase column (Waters μBondapak 7.8 x 300 mm, 10 m, 100 Å) or a phenyl reversed phase column (Waters μBondapak 7.8 × 300 mm, 10 m, 100 Å) eluting with MeCN-H 2 O 7:3 or 1:1. The compounds were obtained as amorph solids, caissarine C (9, 23.0 mg), fistularin-3 (3, 40.0 mg), 11-hydroxyaerothionin (4, 127.0 mg), agelocaissarines A1 and A2 (5 and 6, 4.0 mg) and agelocaissarines B1 and B2 (7 and 8, 5.0 mg). (4). 13 Table 3.
Considering the molecular formula established by HRFABMS, agelocaissarines A1 (5) and A2 (6) must present two spirobicyclic moieties joined through a 2hydroxy-1,4-diaminobutane bridge. The presence of the diamine chain was confirmed by analysis of the NMR data. The methylene group at δ 3.45 (m) and 3.36 (m) in the 1 H spectrum (δ C at 45.9) was assigned to CH 2 -10 for both compounds 5 and 6, the oxymethine at δ 3.75 (m) (δ C at 68.8) to CHOH-11, the methylene at δ 1.55 (m) and 1.70 (m) (δ C at 34.6) to CH 2 -12, and the methylene at δ 3.25 (m) and 3.31 (m) (δ C at 37.0) to CH 2 -13. The connection of the 2-hydroxy-1,4diaminobutane chain to the two spiroxazolidine moieties was established through the amide hydrogens, one of them (NH-9a) at δ 7.42 (bt) was vicinally coupled to the methylene CH 2 -10 (δ 3.45 and 3.36) in the COSY spectrum and showed a long range correlation to the amide carbonyl at δ 160.1. The other amide hydrogen (NH-9a') at δ 7.26 (bt) showed a vicinal coupling to the methylene CH 2 -13 at δ 3.25 and 3.31, and also showed a long range coupling to the amide carbonyl at δ 160.1. Since we have not observed a second distinct set of 1 H signals of the diamino moiety, except for the methylene CH 2 -10, we assumed that the structural difference between 5 and 6 was only the relative stereochemistry of their respective bicyclic spiroxazolidine moieties.
The relative stereochemistry of the major diastereomer agelocaissarine A1 (5) was established by analysis of the 1 H NMR spectrum and molecular modeling using both MM2 and MOPAC protocols of the Chem3D software. Considering the presence of three stereogenic centers, it is possible to consider four different relative stereochemistries for the spirobicyclic moieties of 5 and 6. Since the major diastereomer 5  CH 2 -14 at δ 3.35 (m) and 3.24 (m) (δ C 37.0) in the COSY spectrum. The remaining NMR data of compounds 7 and 8 was essentially identical to the corresponding assignments of 5 and 6, including the relative stereochemistries of the bicyclic spiroxazolidine systems.
The relative stereochemistry of agelocaissarines A1 (5) and A2 (6) as well as of agelocaissarines B1 (7) and B2 (8) are in agreement to the relative stereochemistry established for agelorines A and B, 14 11,17-dideoxyagelorins A and B, 15 and the monocyclic nitriles isolated from A. laevis. 16 These compounds have a similar bicyclic spiroxazolidine system, or a monocyclic substituted ring, in which the CH 2 -7 methylene and the C-1 hydroxyl group have a cis relationship. The hypothesis that the two bicyclic spiroxazolidine moieties within 5 and 6 or in 7 and 8 may not have an identical relative stereochemistry was considered, but rulled out since the 1 H NMR signals of each single diastereomer had consistent intensities (measured by the integration of 1 H signals). In the spectrum of agelocaissarines B1 (7) and B2 (8), the minor diastereomer 8 corresponds to ca. 26% the amount of 7. In the case of agelocaissarines A1 (5) and A2 (6), the amount of the minor diastereomer 6 corresponds to ca. 50% of compound 5. Since in the 1 H NMR spectra of compounds 5 and 6 and of compounds 7 and 8 the 1 H signals of the bicyclo spiroxazolidine moiety with the 1(R*), 1'(R*), 2(R*), 2'(R*), 6(S*), 6'(S*) stereochemistry are consistently of lower intensity, it is clear that each compound of the pairs 5 and 6 as well as 7 and 8 did not present two bicyclic spiroxazolidine systems with different relative stereochemistry each. This fact is relevant, since it would be possible to consider these compounds as artifacts of isolation generated by acid-catalyzed hydrolysis of the methoxyl group. However, such a hypothesis is questionable due to the following reasons. Firstly, a related metabolite with a 11-keto functionality was isolated from the sponge Aplysina archeri as a single diastereomer. 17 Secondly, if the bicyclo spiroxazolidine system present in agelocaissarines A1, A2, B1 and B2 and related metabolites isolated from other sponges [14][15][16][17] would be chemically generated in vitro, we would expect to isolate compounds with each of the two bicyclo spiroxazolidine moieties presenting distinct relative stereochemistries. We have been unable to detect such compounds, which are likely to present a very close retention time to the agelocaissarines in the HPLC analysis. The mixture of 5 and 6 as well as the mixture of 7 and 8 have proven to be inseparable under several different HPLC separation conditions using reversed phase with C 18 or phenyl bonded columns, or using normal phase separation conditions using silica gel or phenyl bonded HPLC columns. Therefore, it seems that these compounds are true secondary metabolites, although it has been recently mentioned that prolonged standing of 11-epi-fistularin-3 at -20 o C led to the formation of both agelorins A and B. 18 A biogenetic pathway to the formation of the bicyclo spiroxazolidine system present in agelocaissarines A1, A2, B1 and B2, as well as in the related metabolites mentioned previously, [14][15][16][17] is proposed in Scheme 1. A possible enzyme-catalyzed methyl extrusion, followed by proton capture concomitantly to the enol ketolyzation gives the bicyclic spiroxazolidine system unique to this class of secondary metabolites, of which only seven related compounds are known up to the present. 14-17 The enzymatic formation of two different stereoisomers in different proportions suggest that this mechanism has a low stereospecificity, indicating that proton capture may not be enzymatically controlled.
The isolation of 11-hydroxyaerothionin (4) and caissarine C (9) from A. caissara during the present investigation is a support that 4 and 9 are the biogenetic precursors of each of the diastereomeric pairs, 5 and 6 and 7 and 8, respectively. Caissarine C (9) was obtained as an amorphous solid which gave a quasi-molecular sodium adduct [M+Na] + ion cluster at m/z 866/868/870/872/874. A HRESIMS measurement at m/z 868.8544 (calculated: 868.8467) indicated the formula C 25 H 28 79 Br 3 81 BrN 4 O 9 Na, corresponding to the 11-hydroxyaerothionin higher homologue (or to the caissarine B lower homologue). This hypothesis was fully supported by analysis of the 1 H and 13 C NMR (Table 3), 1 H-1 H COSY and HMQC spectra, as well as by comparison with data reported for 11hydroxyaerothionin 13 and for caissarine B. 9 11-Hydroxyaerothionin (4) also was identified by analysis of spectroscopic data, including HRESIMS, 1 H, 13 C, 1 H-1 H COSY, HMQC and HMBC NMR spectra, as well as by comparison with literature data. 19 The absolute stereochemistry of 4 isolated from A. caissara was established by analysis of 1 H NMR and circular dichroism spectra. The 1 H spectrum of 4 displayed the signals of H-1 (H-1') and H-6 (H-6') as broad singlets in MeCN-d 3 . Molecular modeling indicated that a 1R*, 6R* confi-Scheme 1. Postulated biogenetic pathway of the agelocaissarines.