Antibacterial modified diketopiperazines from two ascidians of the genus Didemnum

ARTICLE

Antibacterial modified diketopiperazines from two ascidians of the genus Didemnum^#

Miriam H. Kossuga^I; Simone P. Lira^I; Shayna McHugh^I; Yohandra R. Torres^{I, II}; Bruna A. Lima^III; Reginaldo Gonçalves^III; Katyuscya Veloso^IV; Antonio G. Ferreira^IV; Rosana M. Rocha^V; Roberto G. S. Berlinck^I,*

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

^IIDepartamento de Química, Universidade Estadual do Centro-Oeste, Rua Camargo Varela de Sá, 03, 85040-080 Guarapuava-PR, Brazil

^IIIDepartamento de Diagnóstico Oral, Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas, CP 52, 13414-018 Piracicaba-SP, Brazil

^IVDepartamento de Química, Universidade Federal de São Carlos, 13565-905 São Carlos-SP, Brazil

^VDepartamento de Zoologia, Setor de Ciências Biológicas, Universidade Federal do Paraná, Centro Politécnico, Jardim das Américas s/n, CP 19020, 81531-990 Curitiba-PR, Brazil

ABSTRACT

The chemical investigation of the crude extract of an ascidian of the genus Didemnumled to the isolation of the modified diketopiperazine rodriguesines A (1) and (2) as a mixture of homologues, which could be identified by analysis of spectroscopic data including MS/MS experiments. The investigation of a second Didemnumsp. led to the isolation of N-acetyl-rodriguesine A (3) and N-acetyl-rodriguesine B (4). The absolute configuration of compounds 1and 2could be established by hydrolysis and Marfey's analysis and comparison with literature data reported for compound 3, previously obtained as a synthetic product. The mixture of 1and 2displayed moderate antibiotic activity against a clinical isolate of Streptococcus mutansand against S. mutansUA159 and Staphylococcus aureusATCC6538.

Keywords: ascidian, marine, diketopiperazine, absolute configuration, antibiotic

RESUMO

A investigação química do extrato bruto de uma ascidia do gênero Didemnumlevou ao isolamento das dicetopiperazinas modificadas rodriguesinas A (1) e B (2) na forma de uma mistura de homólogos, os quais puderam ser identificados pela análise de seus dados espectroscópicos inclusive experimentos MS/MS. A investigação de uma segunda ascídia do gênero Didemnumforneceu a N-acetil-rodriguesina A (3) e a N-acetil-rodriguesina B (4). A configuração absoluta dos compostos 1e 2pode ser estabelecida por hidrólise e análise de Marfey e por comparação com dados da literatura do composto 3,previamente obtido como produto de síntese. A mistura de 1e 2apresentou atividade antibiótica moderada contra um isolado clínico de Streptococcus mutans, contra S. mutans UA159 e S. aureusATCC6538.

Introduction

Ascidians, or sea-squirts, are cosmopolitan, exclusively marine invertebrates, which constitute a rich source of biologically active secondary metabolites.¹ It has been estimated that ca. 85% of ascidian natural products are derived from amino acids, while the remaining 15% are derived from polyketide and/or terpenoid pathways.² Ascidians of the family Didemnidae are recognized as a particularly unique source of modified peptides and alkaloids.³ These include the well known tamandarins,⁴ didemnins, aplidine and related compounds,⁵ which display potent antitumor and immunosupressive activities, as well as many bioactive alkaloids such as the G2 cell cycle checkpoint inhibitors granulatimide and isogranulatimide.⁶

During several years we have collected distinct species of colonial didemnidae ascidians, in order to expand the diversity of our library of crude extracts.⁷ These animals are not easy to collect, since they frequently present encrusting, thin and soft bodied colonies. Two such species were collected during 1999, as red Didemnumspp. The two Didemnidae species gave crude extracts which displayed antituberculosis and cytotoxic activities. The investigation of these extracts led to the isolation of four modified diketopiperazines, which are the subject of the present report.

Results and Discussion

The hexane-defated MeOH extract of the ascidian Didemnumsp. (sample BA99ASCI-05) presented aromatic, nitrogen-bearing derivatives indicated by TLC and ¹H NMR analyses. The MeOH extract was separated by chromatography on Sephadex LH-20 and C₁₈ reversed-phase columns. Further HPLC separations using C₁₈-phenyl or cyanopropyl silica columns did not provide rodriguesines A (1) and B (2) as pure compounds, even using TFA, NH₄Cl or phosphoric acid buffers in the eluents. Subsequent analysis of 2D NMR and MS/MS analysis indicated that rodriguesines A (1) and B (2) were obtained as an inseparable mixture from the Didemnumsp. sample BA99ASCI-05.

NMR analysis of the fraction containing 1and 2indicated the presence of a phenylalanine residue (δ Hα 4.27, d CH₂-β 3.05 and 3.20, δ aromatic Hs 7.24-7.36), a glycine residue (δ 3.08 and two doublets at δ 3.55/3.56), a linear alkyl β-amino acid and a mono-acylated diamine moiety. These structural features were evident by analysis of the HSQC, COSY and HMBC spectra, of which key correlations are shown in Figure 1. The linear alkyl β-amino acid moiety was assigned from the carbonyl substituted methylene CH₂-18 (δ 2.27 and 2.38) coupled in the COSY spectrum to the broad methine signal at δ 4.65, itself correlated with a methylene group at δ 1.55 and 1.59 (CH₂-20) substituted with the alkyl chain (multiplet at δ 1.30). The diamino moiety was assigned from the amino-substituted methylene at δ 2.97 (CH₂-14) which showed couplings with methylene signals at δ 1.87 and δ 1.69. This methylene (CH₂-15 in 1)". showed a correlation with a methylene group at δ 3.31 and 3.28 (CH₂-16), while the methylene at δ 1.69 (CH₂-15 in 2) was coupled to a methylene group at δ 1.55 (CH₂-15a in 2), itself attached to a methylene at δ 3.23 (CH₂-16 in 2). Correlations between the methylene at δ 3.28 and 3.31 (CH₂-16), as well as the methylene CH₂-18 (δ 2.27 and 2.38), with the carbonyl groups at δ 172.9 and 173.6 established the connectivity between the diamino moiety and the β-amino acid residue. Couplings between H-3 and C-2 (δ 168.5/168.4) and C-5 (δ 168.6/168.4), as well as between CH₂-6 with the same carbonyl carbons, allowed us to assign the piperazine-2,5-dione moiety. The junction between the diketopiperazine residue with the β-amino acid moiety was established by a correlation observed between the glycyl doublet hydrogens of 1 and 2 at δ 3.55/3.56 with C-19.

Considering the planar structures assigned for 1and 2, a search in MarinLit and SciFinder led us to discover that these compounds were closely related to etzionin (5), previously isolated from the ascidian Didemnum rodriguesi. However, the spectroscopic data of etzionin was not very informative, since only broad, poorly-defined NMR signals have been reported for 5.⁸ A distinctive feature between 1and 2and etzionin (5) was that no evidence was obtained in the infrared spectrum of 1and 2for the presence of the unusual etzionin hydroxamate group. We observed only a broad ν_N-H band at 3283 cm^-1 but not a band associated with a ν_O-H at higher wavenumbers. Additionally, since the analysis of the acyl monosubstituted diamino moiety in the mixture of 1and 2indicated the presence of two amide carbonyls and two diamine alkyl chains, we suspected that two diamine homologues constituted the fraction isolated from the Didemnumsp. BA99ASCI-05. Indeed, analysis by HRMS/MS showed that the fraction containing 1and 2displayed two quasi-molecular ions [M+H]⁺ at m/z459.3335 (C₂₆H₄₃N₄O₃) and m/z473.3492 (C₂₇H₄₅N₄O₃), which, upon fragmentation, gave an identical fragment ion at m/z385.2500 (C₂₃H₃₃N₂O₃) corresponding to the lost of C₃H₁₀N₂ (1,3-diaminopropane) from 1and of C₄H₁₂N₂ (1,4-diaminobutane) from 2(Figure 2).The analysis of the MS/MS spectrum also indicated the formation of the corresponding oxazepine and oxazine ions at m/z238.2181 and 252.2352, respectively, as well as the formation of a diketopiperazinium at m/z343.2416 (Figure 2). Therefore, the complete planar structure of rodriguesine A (1) and B (2) was confirmed. Further support for the structures assigned for both 1and 2could be obtained after acetylation of this mixture and separation by HPLC. This procedure gave compounds 3and 4(see below) of which compound 3showed NMR data very similar to that of bis-acetyl etzionin (6).⁸

In order to establish the absolute configuration of the phenylalanine residue, the mixture of 1and 2was subjected to hydrolysis followed by derivatization with Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-l-alanine amide) and LC-PDA-MS analysis. Only l-phenylalanine was detected. Moreover, since the absolute configuration of etzionin (5) was established after the total synthesis of its 4-desoxy-N-acetyl derivative (3),⁹ we decided to acetylate the mixture of 1and 2in order to separate both N-acetyl derivatives and compare the specific rotation of 4-desoxy-N-acetyl etzionine (3) derived from 1with the literature value reported for synthetic 3. The acetylated mixture of 3and 4could be separated by C₁₈ HPLC. Compound 3, obtained after acetylation of 1, showed [α]_D-37.0 (c0.026, CHCl₃), very close to the value reported for synthetic (-)-(3S,19R)-3, [α]_D -53.1 (c0.06, CHCl₃). The ¹H NMR spectrum of 3obtained from the acetylation of 1was identical to that of synthetic (-)-(3S,19R)-3.⁹ Since the absolute configuration of the phenylalanine residue in 3 derived from the acetylation of natural 1is S, the absolute configuration of C-19 must be R. After acetylation and purification, compound 4obtained from 2showed [α]_D -52 (c0.04, CHCl₃). Therefore, both rodriguesines A (1) and B (2) have the 3S, 19R absolute configuration.

Sample BA99ASCI-03 of the second Didemnumspecies gave, after a similar isolation procedure, small quantities of 3and 4as pure compounds. Analysis of the spectroscopic data recorded for N-acetyl rodriguesine A (3) clearly assigned the presence of an acetyl group (C=O at δ_C 173.4; CH₃ at δ_C 22.7 and δ_H 1.92), corroborated by the HRESIMS analysis which showed a [M+H]⁺ quasi-molecular ion at m/z501.3483 (C₂₈H₄₅N₄O₄), indicating an additional C₂H₂O fragment compared to the formula of 1. Analysis of the NMR data of the diamino moiety in 3 showed a coupling between the methylenes at δ 3.18 (t, 8.4 Hz, 6H, CH₂-14 and CH₂-16) and two carbonyl groups at δ_C 173.4 and 172.7. Since the methylene CH₂-18 (δ 2.20 and 2.32) confirmed a long-range coupling with the carbonyl group at δc 172.7 (C-17), and the methyl acetyl group showed a long range coupling with the carbonyl at δ_C 173.4, the acetyl group could be placed at the diamine terminus of compound 3. Analogous MS and NMR data recorded for N-acetyl rodriguesine B (4) showed a quasi-molecular ion [M+H]⁺ at m/z 515.3639 (C₂₉H₄₇N₄O₄) and quite similar NMR data for the diamide moiety (Tables 1 and 2), except for the presence of an additional methylene group (CH₂-15a at δ_H 1.50 and δ_C 27.8) in the diamino moiety. Comparison of ¹H, ¹³C NMR and MS data recorded for natural 3and 4with data recorded for 3and 4derived from the acetylation of 1and 2 proved them to be identical compounds. Therefore, the structure of the natural, minor N-acetylated compounds 3and 4, could be established. Compounds 3and 4should have the same absolute configuration as compounds 1and 2since their [α]_D, HPLC t_R, ¹H and MS data are practically identical to 3 and 4obtained from the acetylation of the mixture of 1 and 2.

The mixture of 1and 2was evaluated in cytotoxic, antituberculosis and antibiotic bioassays against a panel of pathogenic bacteria. Surprisingly, rodriguesines A 1and B 2did not present cytotoxic or antituberculosis activities. The mixture of 1and 2was weakly active against the majority of the pathogen microbial strains tested (Table 3), except against Pseudomonas aeruginosaP1 (MIC at 4.3 µg mL^-1), obtained from a hospital environment. Interestingly, the mixture of 1and 2proved to be more active against antibiotic-resistant strains than against standard ATCC or NTCC strains (Table 3). The strains Streptococcus mutansclinical isolate 2.M7/4, S. mutansUA159, Staphylococcus aureusATCC6538, Escherichia coliNTCC861, Enterococcus faecalisATCC14506, Streptococcus sanguinisATCC15300, S. sobrinus ATCC27607 and Candida albicansATCC36801(serum type A) are all associated with muco-bucal diseases.

Conclusion

We reported here the isolation of four new modified diketopiperazines from two ascidians of the genus Didemnum. The mixture of 1and 2displayed moderate antibiotic activity against several human pathogenic bacteria.

Experimental

General experimental procedures

Optical rotations were measured on a Perkin Elmer 341 MC polarimeter at 20 ºC. IR spectra (film on Si plate) were recorded on a FT-IR Bomem MB102 infrared spectrometer. The NMR spectra were recorded on a Bruker ARX 9.4 T instrument, operating at 400.35 MHz for ¹H and 100.10 MHz for ¹³C, respectively. All NMR spectra were obtained at 25 ºC using TMS as an internal reference. Solvents used for extraction and column chromatography were glass distilled prior to use. HPLC-grade solvents were utilized without further purification in HPLC separations. TLC analyses were performed with precoated TLC sheets of Si gel on polyester, eluting with different mixtures of MeOH in CH₂Cl₂. Plates were observed under a UV lamp (λ _max 254 and 365 nm). Semi-preparative HPLC separations were performed with a Waters 600 quaternary pump and a double beam model 2487 UV detector monitored by Waters Millenium 32. High resolution mass spectra obtained for 1and 2were recorded on a Micromass Q-ToF Micro, in ES⁺ mode, using the following experimental conditions: capillary voltage 3.0 kV; sample cone 42.0 V; sample infused at 10 µL min^-1 in 1: 1 MeOH/H₂O. Accurate mass measurements were made using leucine enkephalin as an internal reference at [M+H]⁺ 556.2771. The MS/MS experiments were recorded using with a range of collision energies from 20 to 30 eV. High resolution mass spectra obtained for compounds 3and 4were recorded on a Samples (0.5 µg mL^-1) dissolved in MeOH/H₂O 1:1 were introduced in the electrospray source at 5 µL min^-1 with a direct infusion pump. MS/MS and MSⁿ experiments were performed using standard isolation and excitation procedures. Nitrogen was used as nebulising and collision gas with the collision energy set at 4 eV. An accurate-mass calibration was obtained with a post acquisition application of a calibration created from the MS/MS of monensin A [M-18+Na]⁺ under the same CID and cell conditions. The collision energy was adjusted until the intensity of the parent ion was set between 5 and 20% relative to the base peak. Capillary voltage was set to 3500 V.

Animal material

The ascidians Didemnumsp. BA99ASCI-03 and Didemnumsp. BA99ASCI-05 were collected in August/ September 1999, from the Baía de Todos os Santos, Salvador, Bahia state, Brazil and immediately frozen. Both samples were deposited in the ascidian collection of the Departamento de Zoologia, Setor de Ciências Biológicas, Universidade Federal do Paraná. BA99ASCI-03: (DZUP DID 210) - colony is reddish orange when alive, but looses the color in formalin, zooids with the sperm duct forming ten coils around the only follicle of the testis, larval trunk oval, 0.5 mm long with four ectodermal ampullae in each side, long and close to each other. The three adhesive papillae are close to each other and present a short stalk.

BA99ASCI-05 (DZUP DID 209) - colony is red when alive, but looses the color in formalin, zooids with sperm duct forming seven coils around the only follicle of the testis, larval trunk 0.4 mm long with four to five ectodermal ampullae in each side, shorter than the ones observed in former voucher. The three adhesive papillae have long stalks.

Extraction and isolation

The frozen ascidian sample BA99ASCI-05 (308.9 g, wet) was freeze dried and exhaustively extracted with MeOH. The MeOH extract was filtered, concentrated to 400 mL and partitioned with hexane (3 × 400 mL). TLC analysis (UV and Dragendorff) of the MeOH fraction indicated the presence of intense UV-absorbing compounds with amine groups. This fraction was evaporated to dryness (5.6 g) before separation by C₁₈ reversed-phase chromatography (gradient of MeOH in H₂O) in 0.5-1.0 g portions. This separation yielded eleven fractions (DaMSP-1 to Da-MSP-11), of which the eighth (Da-MSP-8, 1.87 g) presented most of the Dragendorff and UV positive compounds. The fraction Da-MSP-8 was then subjected to chromatography on Sephadex LH-20 (MeOH). Seven fractions were obtained (Da-MSP-8A to DA-MSP-8G), of which Da-MSP-8D (0.402 g) and Da-MSP-8E (0.532 g) presented aromatic nitrogen-bearing compounds. Both fractions were similarly purified, firstly by C₁₈ reversed-phase column chromatography (gradient of MeOH in H₂O) then by HPLC (phenyl-bonded silica gel,Waters µBondapak 10 µm, 125 Å, 7.8 × 300 mm; eluent: 65:35 MeOH/0.1% TFA in H₂O; λ _max 220 nm; flow rate: 1 mL min^-1; t_R 25.6 min) to give 0.148 g of the mixture of 1and 2(0.05%, wet).

The frozen ascidian sample BA99ASCI-03 (450.0 g) was freeze dried and exhaustively extracted with MeOH. The MeOH extract was concentrated to 400 mL and partitioned with hexane (3 × 400 mL). The alcoholic fraction was evaporated until an aqueous suspension was obtained. The H₂O fraction was first partitioned with EtOAc then with 3:2 CH₂Cl₂/MeOH. The CH₂Cl₂/MeOH fraction (2.0 g) was separated by chromatography on Sephadex LH20 (MeOH), to give five fractions (BA99AC-3A to BA99AC-3E). The last fraction obtained (Ba99AC-3E, 214 mg) was subjected to chromatography on a cyanopropyl-bonded silica cartridge (5 g), with a gradient of EtOAc in CH₂Cl₂. The first fraction obtained from this separation (BA99AC3E1, 134 mg) was separated by chromatography on a silica-gel cartridge (2 g) with a gradient of MeOH in CH₂Cl₂, to give three fractions (Ba99AC-3E1A to BA99AC-3E1C). The second one (BA99AC-3E1B, 38.0 mg) was separated by HPLC using a phenyl-bonded silica column (Inertsil, 250 × 4.6 mm, 5 µm) using 45:55 MeCN/H₂O as eluent, to give five fractions. Fractions BA99AC-3E1C2 (7.1 mg) and BA99AC-3E1C2 (4.6 mg) were purified using a C₁₈ reversed phase HPLC column (Phenomenex 5 µm), using 1:1 MeOH/0.1% HCO₂H in H₂O as eluent, to give N-acetylrodriguesine A (3, t_R = 19.8 min, 3.6 mg, 8.0×10^-4%) and N-acetyl-rodriguesine B (4, t_R = 21.2 min, 4.6 mg, 1.0×10^-3%).

Rodriguesines A (1) and B (2)

Colorless, glassy solid; UV (MeOH) λ _max 258 nm; ¹H NMR (CD₃OD, 400 MHz), see Table 1; ¹³C NMR (CD₃OD, 100 MHz), see Table 2; Positive HRESIMS for 1m/z459.3341 [M+H]⁺, calc. for C₂₆H₄₃N₄O₃ 459.3335. Positive HRESIMS for 2473.3499 [M+H]⁺ calc. for C₂₇H₄₅N₄O₃ 473.3492. MS/MS experiments: m/z385.2500, corresponding to C₂₃H₃₃N₂O₃ (calc. 385.24912); m/z238.2181, corresponding to C₁₅H₂₈NO (calc. 238.21654); m/z252.2352, corresponding to C₁₆H₃₀NO (calc. 252.23219); m/z343.2416, corresponding to C₂₁H₃₁N₂O₂ (calc. 343.23800).

N-acetyl-rodriguesine A (3)

Colorless, glassy solid; [α]_D -37 (c0.026, CHCl₃); UV (MeOH) λ_max 256 nm; ¹H NMR (CD₃OD, 400 MHz), see Table 1; ¹³C NMR (CD₃OD, 100 MHz), see Table 2; Positive CID-MS/MS for 1m/z501.3483 [M+H]⁺, calc. for C₂₈H₄₄N₄O₄ 501.34408.

N-Acetyl-rodriguesine B (4)

Colorless, glassy solid; [α]_D -52 (c0.04, CHCl₃); UV (MeOH) λ_max 257 nm; ¹H NMR (CD₃OD, 400 MHz), see Table 1; ¹³C NMR (CD₃OD, 100 MHz), see Table 2; Positive CID-MS/MS for 1m/z515.3639 [M+H]⁺, calc. for C₂₉H₄₇N₄O₄ 515.35918.

Acetylation of rodriguesines A (1) and B (2)

The mixture of compounds 1and 2(40 mg) was stirred overnight with 1 mL of pyridine and 1 mL of acetic anhydride, at room temperature. The reaction medium was transferred to a round bottom flask and evaporated until dryness. The acetylated mixture was separated by C₁₈ reversed-phase HPLC (Inertsil, ODS 3 250 × 4.6 mm, 5 µm) with a linear gradient of MeCN in H₂O, from 7:3 to 95:5 during 30 min, to give 1.7 mg of pure N-acetyl-rodriguesine A (3) and 3.0 mg of pure N-acetyl-rodriguesine B (4), as well as 30.7 mg of an intermediate fraction containing both 3and 4. N-acetyl-rodriguesine A (3) showed [α]_D -37.0 (c0.026, CHCl₃) and ¹H NMR, HPLC retention time and MS analysis identical to natural 3. Similarly, N-acetylrodriguesine B (4) showed [α]_D -58 (c0.03, CHCl₃) and ¹H NMR, HPLC retention time and MS analysis identical to natural 4.

Hydrolysis and Marfey's analysis of rodriguesines A (1) and B (2)

Two milligrams of the mixture of 1and 2was dissolved in 0.5 mL of 6 mol L^-1 HCl and refluxed at 105 ºC for 24 h. Then the reaction medium was evaporated until dryness. The hydrolyzed product was dissolved in H₂O (40 µL) and a 0.5 mol L^-1 NaHCO₃ solution was added, followed by 80 µL of a 1.4 equiv. of Marfey's reagent (2,4-dinitro-5-fluoro-phenyl-l-alaninamide) in acetone. The reaction was stirred at 40 ºC for 1 h. Afterwards, 16 µL of 0.5 mol L^-1 HCl was added to the reaction medium followed by evaporation to dryness. The reaction product was dissolved in 1 mL of MeOH. An identical derivatization procedure was employed for the preparation of a l-phenylalanine-derivatized standard. The hydrolysed and the standard-derivatized samples were analyzed by HPLC-PDA-MS using the following conditions: Phenomenex C₁₈ Synergi-4 Fusion RP-80 column, 250 × 4.6 mm; eluent: gradient of MeCN in Et₃N/H₂PO₄ aqueous buffer; flow rate: 1 mL min^-1. The hydrolyzed and derivatized sample as well as the derivatized phenylalanine standard were injected separately and co-injected. Retention time of 2,4-dinitro-5-(l-phenylalanine)-phenyl-l-alaninamide: 14.85 min.

Antibiotic assay

Procedures for the antimicrobial assays against Staphylococcus aureusATCC 6538, S. aureusATCC 259223, Escherichia coliATCC861, E. coliATCC 259222, Pseudomonas aeruginosaATCC 27853, P. aeruginosa13, P. aeruginosaP1, Candida albicansATCC 10231, C. albicansATCC 36801 (serum type A), Enterococcus faecalisATCC 14506, Streptococcus sanguinisATCC 15300, Streptococcus sobrinusATCC 27607, Streptococcus mutansUA159, S. mutans(clinical isolate 2.M7/4) were performed using the broth microdilution assay, adapted from the National Committee for Clinical Laboratory Standards (USA) and from Oliveira et al.,¹⁰ as it follows.

Briefly, bacteria were grown in BHI (Brain Heart Infusion) liquid medium at 37 ºC and suspended, as recommended, to yield final inocula of approximately 10⁶ CFU/mL. Tests were performed in sterile 96-well microplates by dispensing into each well a total volume of 100 µL (40 µL of medium + 20 µL de inocula + 40 µL substance solution diluted in H₂O) and incubated 24 h at 37 ºC in 10% CO₂. Microorganism growths were determined by absorbance measurement at 550 nm in an automated microplate reader (Molecular Devices, Versa Max). The MIC was defined as the lowest concentration of drug that inhibited growth. As control microbial culture growths without the addition of the tested substance were used and all samples were assayed in replicate.

Acknowledgments

The authors thank Prof. Ian Castro-Gamboa (Instituto de Química, Universidade Estadual Paulista, Araraquara) for fruitful discussions, Michael LeBlanc (Department of Earth and Ocean Sciences, University of British Columbia) and Prof. Louis Barrows (Department of Medicinal Chemistry, University of Utah) for help in the ascidian collection, Dr. Tim S. Bugni and Prof. Chris M. Ireland (Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah, USA), as well as Prof. Norberto P. Lopes (Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Ribeirão Preto, Brazil) for assistance in obtaining MS analyses. Prof. Carlos Roque D. Corrêa (Instituto de Química, Universidade Estadual de Campinas) is acknowledged for assistance in the specific rotation measurements. Financial support was provided by the American Society of Pharmacognosy 1998 Research Starter Grant, NIH grant CA 67786 and FAPESP/BIOprospecTA grant 05/60175-2 to R.G.S.B., a CAPES scholarship to M.H.K., a Fulbright scholarship to S.M. and by CNPq scientific research awards to A.G.F., R.M.R. and R.G.S.B.

Supplementary Information

¹H NMR, ¹³C NMR and MS spectra of 1and 2, 3and 4are available free of charge at http://jbcs.sbq.org.br, as a PDF file.

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Received: November 11, 2008

Web Release Date: April 24, 2009

FAPESP helped in meeting the publication costs of this article.

Supplementary Information

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Figure S9 - Click to enlarge

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Dedicated to Prof. Vanderlan da Silva Bolzani, for her outstanding contributions to the development of natural product chemistry and related sciences in Brazil.

Publication Dates

• Publication in this collection
  10 June 2009
• Date of issue
  2009

History

• Accepted
  24 Apr 2009
• Received
  11 Nov 2008