Further dibromotyrosine-derived metabolites from the marine sponge Aplysina caissara

Abstracts

The re-investigation of the crude extract obtained from the sponge Aplysina caissara led to the isolation of five new dibromotyrosine derivatives, named agelocaissarines A1, A2, B1, B2 and caissarine C, along with the already known fistularin-3 and 11-hydroxyaerothionin. All compounds were identified by analysis of mono- and bidimensional NMR spectra, high resolution mass spectra, infrared and ultraviolet spectra. The relative stereochemistry of agelocaissarines could be established by analysis of ¹H NMR spectra and molecular modeling, while the absolute configuration of the spiroxazolidine moieties of fistularin-3, caissarine C and 11-hydroxyaerothionin was established by analysis of ¹H NMR and circular dichroism spectra. Fistularin-3 and 11-hydroxyaerothionin displayed moderate antibiotic activity against several pathogenic bacteria.

sponge; Aplysina caissara; dibromotyrosine; absolute configuration

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 mono- e bidimensionais, espectro de massas de alta resolução, infravermelho e ultravioleta. A configuração relativa das agelocaissarinas pôde ser estabelecida por análise dos espectros de RMN-¹H 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 11-hidroxiaerotionina apresentaram atividade antibiótica moderada contra várias linhagens de bactérias patogênicas.

ARTICLE

Further dibromotyrosine-derived metabolites from the marine sponge Aplysina caissara

Tatiana O. de Lira^I; Roberto G. S. Berlinck^{*, I}; Gislene G. F. Nascimento^II; Eduardo Hajdu^III

^IInstituto de Química de São Carlos, Universidade de São Paulo, CP 780, 13560-970 São Carlos-SP, Brazil

^IIFaculdade de Ciências da Saúde, Universidade Metodista de Piracicaba, Rodovia do Açúcar, km 156, 13400-901 Piracicaba-SP, Brazil

^IIIMuseu Nacional, Universidade Federal do Rio de Janeiro, Quinta da Boa Vista, s/n, 20940-040 Rio de Janeiro-RJ, Brazil

ABSTRACT

The re-investigation of the crude extract obtained from the sponge Aplysina caissara led to the isolation of five new dibromotyrosine derivatives, named agelocaissarines A1, A2, B1, B2 and caissarine C, along with the already known fistularin-3 and 11-hydroxyaerothionin. All compounds were identified by analysis of mono- and bidimensional NMR spectra, high resolution mass spectra, infrared and ultraviolet spectra. The relative stereochemistry of agelocaissarines could be established by analysis of ¹H NMR spectra and molecular modeling, while the absolute configuration of the spiroxazolidine moieties of fistularin-3, caissarine C and 11-hydroxyaerothionin was established by analysis of ¹H NMR and circular dichroism spectra. Fistularin-3 and 11-hydroxyaerothionin displayed moderate antibiotic activity against several pathogenic bacteria.

Keywords: sponge, Aplysina caissara, dibromotyrosine, absolute configuration

RESUMO

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 mono- e bidimensionais, espectro de massas de alta resolução, infravermelho e ultravioleta. A configuração relativa das agelocaissarinas pôde ser estabelecida por análise dos espectros de RMN-¹H 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 11-hidroxiaerotionina 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-H₂O 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 ºC, 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 ¹H and 100 MHz for ¹³C channels, respectively, or on a Bruker DRX300 7.05 T, operating at 300 MHz for ¹H and 75 MHz for ¹³C, respectively. All NMR spectra were obtained at 25 ºC 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 Si gel TLC sheets, eluting with different mixtures of MeOH in CH₂Cl₂. Plates were visualized under UV (l_max 254 nm). HPLC separations were performed either with a Waters quaternary pump 600, double beam UV detector 2487, and data module 746, or with a Waters autosampler 717, Waters 600 pump, Waters 2996 photodiode array detector monitored by Waters Millenium 32.

Animal collection and identification. The sponge Aplysina caissara (Pinheiro & Hajdu, 2001) was collected during the summer of 2002 at the São Sebastião channel and immediately frozen at -20 ºC. A voucher specimen was deposited at the Museu Nacional, Universidade Federal do Rio de Janeiro.⁹

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 (CH₂Cl₂-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 CH₂Cl₂), by C₁₈ reversed phase column chromatography (gradient of MeOH in H₂O), followed by purifications by HPLC with either a C₁₈ 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-H₂O 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).

11-Hydroxyaerothionin (4).¹³ Amorphous solid, [a]_D²⁸ + 178º (c 0.0014, MeOH). IR (film on Si plate) n_max/cm^-1: 3374, 2936, 1659, 1602, 1545, 1436, 1314, 1272, 1108, 992, 918, 775, 664. HRESIMS m/z 854.8400 (Calc. for C₂₄H₂₆⁷⁹Br₃⁸¹BrN ₄O₉Na, 854.8310) corresponding to [M+Na]⁺.

Agelocaissarine A1 (5) and agelocaissarine A2 (6). Amorphous solid. IR (film) n_max/cm^-1: 3369, 1701, 1660, 1603, 1544, 1431, 1267, 1108, 1002, 912, 780, 669. HRFABMS m/z 804.81762 (Calc. for C₂₂H₂₃⁷⁹Br₃⁸¹BrN ₄O₉, 804.81780) corresponding to [M+H]⁺. ¹H and ¹³C NMR data: see Tables 1 and 2.

Agelocaissarine B1 (7) and agelocaissarine B2 (8). Amorphous solid. IR (film) n_max/cm^-1: 3348, 1705, 1654, 1602, 1538, 1441, 1268, 1107, 901, 740, 604. HRESIMS m/z 840.8303 (Calc. for C₂₃H₂₄⁷⁹Br₃⁸¹BrN ₄O₉Na, 840.8154) corresponding to [M+Na]⁺. ¹H and ¹³C NMR data: see Tables 1 and 2.

Caissarine C (9). Amorphous solid, [a]_D²⁸ + 175º (c 0.0021, MeOH). IR (film) n_max/cm^-1: 3351, 2935, 1663, 1596, 1543, 1439, 1309, 1270, 1220, 1106, 988, 911, 766, 739, 606. HRESIMS m/z 868.8544 (Calc. for C₂₅H₂₈⁷⁹Br₃⁸¹BrN ₄O₉Na, 868.8467) corresponding to [M+Na]⁺. ¹H and ¹³C NMR data: see Table 3.

Results and Discussion

Agelocaissarines A1 (5) and A2 (6) were obtained as an inseparable mixture, which displayed a quasi-molecular cluster [M+H]⁺ at m/z 803, 805, 807, 809 and 811 by FABMS, indicating the presence of four bromine atoms in the structures. A HRFABMS measurement at m/z 805 (measured: m/z 804.81762; calculated: 804.81780) indicated the molecular formula C₂₂H₂₃⁷⁹Br₃⁸¹BrN ₄O₉ for both diastereomers. The ¹³C NMR spectrum of the diastereomeric mixture displayed thirteen signals of the major stereoisomer agelocaissarine A1 (5) and eight signals of the minor stereoisomer agelocaissarine A2 (6) (Table 1). Five ¹³C signals were assigned to sp² carbons: one a,b-unsaturated ketone carbonyl (C-3 and C-3') at d 184.1 and one amide carbonyl (C-9 and C-9') at d 160.1 to both 5 and 6, one imine oxide carbon (C-8 and C-8') at d 154.8 to 5 and at d 155.5 to 6, a vinylic methine (C-5 and C-5') at d 149.3 to 5 and at d 146.2 to 6, as well as a quaternary bromine-substituted sp² carbon (C-4 and C-4') at d 122.7 to 5 (d 123.0 to 6). The ¹³C chemical shifts of the a,b-unsaturated ketone system were clearly reminiscent to those of agelorins A and B isolated from Agelas oroides,¹⁴ of 11,17-dideoxyagelorins A and B isolated from Suberea aff. praetensa,¹⁵ of (1'R, 5'S, 6'S)-2-(3',5'-dibromo-1',6'-dihydroxy-4'-oxocyclohex-2' -enyl)acetonitrile and its corresponding (1'R, 5'R, 6'S)-epimer isolated from Aplysina laevis,¹⁶ and of an unnamed compound isolated from Aplysina archeri.¹⁷ Indeed, the remaining carbon resonances assigned to the 4-(4',5'-dihydroisoxazolyl)- 2,6-dibromo-5-hydroxycyclohex-2-enone moiety of agelocaissarines A1 and A2 were very similar to the corresponding structural moieties of the preceding mentioned natural products. These included the spiro carbon C-6 at d 91.5 assigned to 5 (d 90.5 to 6), the oximethine carbon C-1 at d 74.5 for 5 (d 74.0 for 6) and the bromine substituted methine C-2 at d 57.2 for 5 (d 55.0 for 6). Analysis of the ¹H, ¹³C and HMQC NMR spectra of 5 and 6 (Table 2) enabled us to assign the ¹H resonance at d 4.28 (dd, J 2.2 and 11.4 Hz) of compound 5 to H-1 (at d 4.39 as a broad singlet in 6), at d 4.80 (dd, J 1.3 and 11.5 Hz) of 5 to H-2 (at d 5.19 also as a broad singlet in 6), and the ¹H resonance at d 7.51 (s) to H-5 in compound 5 (d 7.32, s, in compound 6). The H-7a/H-7b diastereotopic methylene observed at d 3.71 (1.3 and 18.0 Hz) and d 3.15 (dd, J 2.2 and 18.0 Hz) in 5 and at d 3.76 (dd, 1.3 and 18 Hz) and d 3.29 (dd, J 2.8 and 18.0 Hz) in 6 completed the ¹H assignments of the spiroxazolidine moiety of both compounds 5 and 6.

Analysis of the ¹H-¹H COSY spectrum confirmed these assignments, since H-1 and H-2 were vicinally coupled in 6, while H-1 showed couplings with both H-2 and H-7a/H-7b (long-range) in compound 5. Additional support for the spirobicyclic moiety assignments of both 5 and 6 was obtained by analysis of the HMBC spectra, which showed long range couplings between H-1 and C-2, C-6 and C-7, between H-2 and C-1, C-3 and C-6, between H-5 and C-1, C-3, C-4 and C-7 (weak), as well as between H-7a/H-7b and C-1, C-5, C-6 and C-8 for compound 5. The same HMBC spectra indicated similar long range couplings between H-5 and C-1, C-3, C-4 and C-7, as well as between H-7a/H-7b and C-1, C-5 and C-6 for compound 6, but no such couplings were observed for both H-1 and H-2.

Considering the molecular formula established by HRFABMS, agelocaissarines A1 (5) and A2 (6) must present two spirobicyclic moieties joined through a 2-hydroxy-1,4-diaminobutane bridge. The presence of the diamine chain was confirmed by analysis of the NMR data. The methylene group at d 3.45 (m) and 3.36 (m) in the ¹H spectrum (d _C at 45.9) was assigned to CH₂-10 for both compounds 5 and 6, the oxymethine at d 3.75 (m) (d_C at 68.8) to CHOH-11, the methylene at d 1.55 (m) and 1.70 (m) (d_C at 34.6) to CH₂-12, and the methylene at d 3.25 (m) and 3.31 (m) (d_C at 37.0) to CH₂-13. The connection of the 2-hydroxy-1,4-diaminobutane chain to the two spiroxazolidine moieties was established through the amide hydrogens, one of them (NH-9a) at d 7.42 (bt) was vicinally coupled to the methylene CH₂-10 (d 3.45 and 3.36) in the COSY spectrum and showed a long range correlation to the amide carbonyl at d 160.1. The other amide hydrogen (NH-9a') at d 7.26 (bt) showed a vicinal coupling to the methylene CH₂-13 at d 3.25 and 3.31, and also showed a long range coupling to the amide carbonyl at d 160.1. Since we have not observed a second distinct set of ¹H signals of the diamino moiety, except for the methylene CH₂-10, 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 ¹H 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 showed H-1 and H-2 vicinally coupled with an 11 Hz coupling constant, the corresponding dihedral angle (f) H-1/C-1/C-2/H-2 in 5 must be either close to 0º or 180º. Molecular modeling analysis indicated a dihedral angle f close to 161º, with a J_calc 10.9 Hz, for the 1(R*), 1'(R*), 2(S*), 2'(S*), 6(S*), 6'(S*) stereoisomer, which indicated a H-1/H-2 pseudo-trans relationship in compound 5. For compound 6, both H-1 and H-2 were observed as broad singlets in the ¹H NMR spectrum (see Figure 1), indicating a dihedral angle close to 90º between these hydrogens. Molecular modeling analysis indicated a dihedral angle close to 80º, with a J_calc 1.0 Hz, corresponding to the 1(R*), 1'(R*), 2(R*), 2'(R*), 6(S*), 6'(S*) stereoisomer, in which H-1 and H-2 in 6 must present a pseudo-axial/pseudo-equatorial relationship.

Agelocaissarines B1 (7) and B2 (8) were also isolated as an unseparable mixture which displayed a quasi molecular ion cluster at m/z 838, 840, 842, 844 and 846. A HRESIMS measurement at m/z 840.8303 (calc. 840.8154) indicated the formula C₂₃H₂₄⁷⁹Br₃⁸¹BrN ₄O₉Na of the corresponding sodium adduct. Therefore, compounds 7 and 8 were assigned as the higher homologues of compounds 5 and 6 at the 2-hydroxy diamino chain. This hypothesis was confirmed by analysis of NMR data of 7 and 8 (Tables 1 and 2), indicating the presence of the additional methylene group CH₂-13 at d 1.46 (m) and 1.55 (m) (d_C 26.0), which was vicinally coupled to methylenes CH₂-12 at d 1.26 (m) and 1.35 (m) (d_C 32.4) and CH₂-14 at d 3.35 (m) and 3.24 (m) (d_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 CH₂-7 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 ¹H NMR signals of each single diastereomer had consistent intensities (measured by the integration of ¹H 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 ¹H NMR spectra of compounds 5 and 6 and of compounds 7 and 8 the ¹H 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^14-17 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 C₁₈ 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 ºC 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,^14-17 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 C₂₅H₂₈⁷⁹Br₃⁸¹BrN ₄O₉Na, corresponding to the 11-hydroxyaerothionin higher homologue (or to the caissarine B lower homologue). This hypothesis was fully supported by analysis of the ¹H and ¹³C NMR (Table 3), ¹H-¹H COSY and HMQC spectra, as well as by comparison with data reported for 11-hydroxyaerothionin¹³ and for caissarine B.⁹

11-Hydroxyaerothionin (4) also was identified by analysis of spectroscopic data, including HRESIMS, ¹H, ¹³C, ¹H-¹H 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 ¹H NMR and circular dichroism spectra. The ¹H spectrum of 4 displayed the signals of H-1 (H-1') and H-6 (H-6') as broad singlets in MeCN-d₃. Molecular modeling indicated that a 1R*, 6R* configuration would present a W long range coupling between H-1 (H-1') and H-6 (H-6'), which was not observed in both ¹H and COSY spectra. Therefore, the relative configuration should be 1R*, 6S*, which was confirmed by analysis of the CD spectrum of 4 {[q]₂₈₄^max + 68,000, [q]₂₄₈^max + 78,000}, virtually identical to those of 11-epi-fistularin-3 and aerothionin, both of which have the 1R, 1'R, 6S, 6'S absolute configuration.^14,19 Therefore, the absolute configuration of both spiroxazolidine bicyclic moieties of 4 was assigned as 1R, 1'R, 6S, 6'S. Since the specific rotation of 11-hydroxyaerothionin (4) previously isolated from the sponge Pseudoceratina durissima was [a]_D +189º (c = 0.15, solvent not specified)¹³ and the specific rotation of 4 isolated from A. caissara was [a]_D +178º (c = 0.0014, MeOH), we can suggest the same absolute configuration for the spiroxazolidine bicyclic moieties of both compounds.

The same 1(R), 1'(R), 6(S), 6'(S) absolute stereochemistry of the spiroxazolidine moieties was assigned for fistularin-3 (3) and caissarine C (9) isolated from A. caissara, based on the analysis of the ¹H NMR and CD spectra of 3 {[q]₂₉₀^max + 67,500, [q]₂₄₈^max + 76,000} and of 9 {[q]₂₉₀^max + 90,000, [q]₂₄₈^max + 87,000}. It is worth of mention that the absolute stereochemistry at C-1, C-1', C-6 and C-6' of fistularin-3 (3) isolated from A. caissara is identical to the same compound recently reported from A. cauliformis.¹⁸ Therefore, if we consider 11-hydroxyaerothionin (4) as the direct biogenetic precursor of agelocaissarines A1 (5) and A2 (6), and caissarine C (9) the precursor of agelocaissarines B1 (7) and B2 (8), the relative stereochemistry assigned for each component of the diastereomeric pairs may be considered as their respective absolute configurations. No attempts have been made to establish the absolute stereochemistry at C-11 of compounds 4-9.

The dibromotyrosine derivatives fistularin-3 (3) and 11-hydroxyaerothionin (4) displayed moderate antibiotic activity against Escherichia coli ATCC 25922 (MIC at 300 µg mL^-1 for 3, while 4 was inactive), Pseudomonas aeruginosa ATCC 27853 (MIC at 300 µg mL^-1 for 4, while 3 was inactive), oxacillin resistant Staphylococcus aureus strain 8 (MIC at 600 µg mL^-1 for both 3 and 4), oxacillin resistant S. aureus strain 108 (MIC at 50 µg mL^-1 and 80 µg mL^-1 for 3 and 4, respectively), and no activity at all when tested against Pseudomonas aeruginosa and Candida albicans. Compounds 5, 6, 7 and 8 were not tested since they were isolated as mixtures.

In conclusion, we described five new dibromotyrosine derivatives from the sponge A. caissara, agelocaissarines A1 (5), A2 (6), B1 (7) and B2 (8), isolated as pairs of diastereomers, and caissarine C (9). The relative stereochemistry of the bicyclic spiroxazolidine moiety of compounds 5-8 could be assigned by ¹H NMR and molecular modeling analysis. The concurrent isolation of fistularin-3 (3) and 11-hydroxyaerothionin (4) enabled us to establish the absolute configuration of the spiroxazolidine moiety in 4 and 9 by ¹H NMR and circular dichroism analysis. Compounds 3 and 4 displayed moderate antibiotic activity against different pathogenic bacteria.

Acknowledgments

The authors thank to Prof. José Carlos de Freitas and the technical staff of CEBIMar-USP for the many facilities during the sponge collection. The authors are also gratefully indebted to Dr. Norrie Pearce and Prof. Brent R. Copp (University of Auckland, Auckland, New Zealand) and to Dr. David E. Williams and Dr. Prof. Raymond J. Andersen (University of British Columbia, Vancouver, Canada) for their assistance in the MS analyses, as well as to Prof. Gil V. J. Silva and Virginia H. B. Glass (Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo) in obtaining the NMR spectra. Financial support to RGSB was funded by FAPESP grant 01/03095-5. EH received grants and/or fellowships from CNPq and FAPERJ. T.O.L. thanks CAPES for a MSc scholarship.

Supplementary Information

Supplementary Information is avaliable free of charge at http://jbcs.sbq.org.br, as PDF file.

References

1. Faulkner, D. J.; Nat. Prod. Rep. 2002, 19, 1.

2. Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.; Princep, M. R.; Nat. Prod. Rep. 2006, 23, 26.

3. Tilvi, S.; Rodrigues, C.; Naik, C. G.; Parameswaran, P. S.; Wahidhulla, S.; Tetrahedron 2004, 60, 10207.

4. Park, Y.; Liu, Y.; Hong, J.; Lee, C. O.; Cho, H.; Kim, D. K.; Im, K. S.; Jung, J. H.; J. Nat. Prod. 2003, 66, 1495.

5. Kijjoa, A.; Bessa, J.; Wattanadilok, R.; Sawangwong, P.; Nascimento, M. S. J.; Pedro, M.; Silva, A. M. S.; Eaton, G.; Van Soest, R.; Herz, W.; Z. Naturforsch., B: Chem. Sci. 2005, 60, 904.

6. Pina, I. C.; Gautschi, J. T.; Wang, G. Y. S.; Sanders, M. L.; Schmitz, F. J.; France, D.; Cornell-Kennon, S.; Sambucetti, L. C.; Remiszewski, S. W.; Perez, L. B.; Bair, K. W.; Crews, P.; J. Nat. Prod. 2004, 68, 3866.

7. Tabudravu, J. N.; Jaspars, M.; J. Nat. Prod. 2002, 65, 1768.

8. Tsuda, M.; Endo, T.; Watanabe, K.; Fromont, J.; Kobayashi, J.; J. Nat. Prod. 2002, 65, 1670.

9. Saeki, B. M.; Granato, A. C.; Berlinck, R. G. S.; Magalhaes, A.; Schefer, A. B.; Ferreira, A. G.; Pinheiro, U. S.; Hajdu, E.; J. Nat. Prod. 2002, 65, 796; Saeki, B. M.; Granato, A. C.; Berlinck, R. G. S.; Magalhaes, A.; Schefer, A. B.; Ferreira, A. G.; Pinheiro, U. S.; Hajdu, E.; J. Nat. Prod. 2003, 66, 1039.

10. Ebel, R.; Brenzinger, M.; Kunze, A.; Gross, H. J.; Proksch, P.; J. Chem. Ecol. 1997, 23, 1451.

11. Puyana, M.; Fenical, W.; Pawlik, J. R.; Mar. Ecol. Prog. Ser. 2003, 246, 127.

12. Gopichand, Y.; Schmitz, F. J.; Tetrahedron Lett. 1979, 3921.

13. Kernan, M. R.; Cambie, R. C.; Bergquist, P. R.; J. Nat. Prod. 1990, 53, 615.

14. König, G. M.; Wright, A. D.; Heterocycles 1993, 36, 1351.

15. Kijjoa, A., Watanadilok, R., Sonchaeng, P., Silva, A. M. S., Eaton, G., Herz, W.; Z. Naturforsch., C: J. Biosci. 2001, 56, 1116.

16. Capon, R. J.; MacLeod, J. K.; Aust. J. Chem. 1987, 40, 341.

17. Ciminiello, P.; Dell'Aversano, C.; Fattorusso, E.; Magno, S.; Carrano, L.; Pansini, M.; Tetrahedron 1996, 52, 9863.

18. Rogers, E. W., Oliveira, M. F., Berlinck, R. G. S., König, G. M., Molinski T. F.; J. Nat. Prod. 2005, 68, 891; Oliveira, M. F., Oliveira, J. H. H. L., Galetti, F. C. S., Souza, A. O., Silva, C. L., Hajdu, E., Peixinho, S., Berlinck, R. G. S.; Planta Med. 2006, 72, 437, in which the absolute configuration of (+)-fistularin-3 and 11-deoxyfistularin-3 was correctly drawn but inversely indicated as 1(S), 1'(S), 6(R), 6'(R).

19. McMillan, J. A.; Paul, I. C.; Goo, Y. M.; Rinehart, Jr., K. L.; Krueger, W. C.; Pschigoda, L. M.; Tetrahedron Lett. 1981, 22, 39.

Received: February 28, 2006

Published on the web: August 11, 2006

FAPESP helped in meeting the publication costs of this article.

Supplementary Information

Figure S1 - Clique here to enlarge

Figure S2 - Clique here to enlarge

Figure S3 - Clique here to enlarge

Figure S4 - Clique here to enlarge

Figure S5 - Clique here to enlarge

Figure S6 - Clique here to enlarge

Figure S7 - Clique here to enlarge

Figure S8 - Clique here to enlarge

Figure S9 - Clique here to enlarge

Publication Dates

• Publication in this collection
  29 Jan 2007
• Date of issue
  Dec 2006

History

• Received
  28 Feb 2006
• Accepted
  01 Aug 2006