Antibacterial Modified Diketopiperazines from two Ascidians of the Genus Didemnum

A investigação química do extrato bruto de uma ascidia do gênero Didemnum levou 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 Didemnum forneceu a N-acetil-rodriguesina A (3) e a N-acetil-rodriguesina B (4). A configuração absoluta dos compostos 1 e 2 pode 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 1 e 2 apresentou atividade antibiótica moderada contra um isolado clínico de Streptococcus mutans, contra S. mutans UA159 e S. aureus ATCC6538.


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 Vol. 20, No. 4, 2009 encrusting, thin and soft bodied colonies. Two such species were collected during 1999, as red Didemnum spp. 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 Didemnum sp. (sample BA99ASCI-05) presented aromatic, nitrogen-bearing derivatives indicated by TLC and 1 H NMR analyses. The MeOH extract was separated by chromatography on Sephadex LH-20 and C 18 reversed-phase columns. Further HPLC separations using C 18 -phenyl or cyanopropyl silica columns did not provide rodriguesines A (1) and B (2) as pure compounds, even using TFA, NH 4 Cl 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 Didemnum sp. sample BA99ASCI-05.
NMR analysis of the fraction containing 1 and 2 indicated the presence of a phenylalanine residue (d Hα 4.27, d CH 2 -β 3.05 and 3.20, d aromatic Hs 7.24-7.36), a glycine residue (d 3.08 and two doublets at d 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 CH 2 4), as well as between CH 2 -6 with the same carbonyl carbons, allowed us to assign the piperazine-2,5dione 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 d 3.55/3.56 with C-19.
Considering the planar structures assigned for 1 and 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 1 and 2 and etzionin (5) was that no evidence was obtained in the infrared spectrum of 1 and 2 for 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 1 and 2 indicated the presence of two amide carbonyls and two diamine alkyl chains, we suspected that two diamine homologues constituted the fraction isolated from the Didemnum sp. BA99ASCI-05. Indeed, analysis by HRMS/MS showed that the fraction containing 1 and 2 displayed two quasi-molecular ions [ Figure 2). The analysis of the MS/MS spectrum also indicated the formation of the corresponding oxazepine and oxazine ions at m/z 238.2181 and 252.2352, respectively, as well as the formation of a diketopiperazinium at m/z 343.2416 ( Figure 2). Therefore, the complete planar structure of rodriguesine A (1) and B (2) was confirmed. Further support for the structures assigned for both 1 and 2 could be obtained after acetylation of this mixture and separation by HPLC. This procedure gave compounds 3 and 4 (see below) of which compound 3 showed 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 1 and 2 was 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 1 and 2 in order to separate both N-acetyl derivatives and compare the specific rotation of 4-desoxy-N-acetyl etzionine (3)  derived from the acetylation of natural 1 is S, the absolute configuration of C-19 must be R. After acetylation and purification, compound 4 obtained from 2 showed [α] D −52 (c 0.04, CHCl 3 ). Therefore, both rodriguesines A (1) and B (2) have the 3S, 19R absolute configuration.
Sample BA99ASCI-03 of the second Didemnum species gave, after a similar isolation procedure, small quantities of 3 and 4 as pure compounds. Analysis of the spectroscopic data recorded for N-acetyl rodriguesine A (3) (Tables 1 and 2), except for the presence of an additional methylene group (CH 2 -15a at d H 1.50 and d C 27.8) in the diamino moiety. Comparison of 1 H, 13 C NMR and MS data recorded for natural 3 and 4 with data recorded for 3 and 4 derived from the acetylation of 1 and 2 proved them to be identical compounds. Therefore, the structure of the natural, minor N-acetylated compounds 3 and 4, could be established. Compounds 3 and 4 should have the same absolute configuration as compounds 1 and 2 since their [α] D , HPLC t R , 1 H and MS data are practically identical to 3 and 4 obtained from the acetylation of the mixture of 1 and 2.
The mixture of 1 and 2 was evaluated in cytotoxic, antituberculosis and antibiotic bioassays against a panel of pathogenic bacteria. Surprisingly, rodriguesines A 1 and B 2 did not present cytotoxic or antituberculosis activities. The mixture of 1 and 2 was weakly active against the majority of the pathogen microbial strains tested (Table  3), except against Pseudomonas aeruginosa P1 (MIC at 4.3 µg mL -1 ), obtained from a hospital environment. Interestingly, the mixture of 1 and 2 proved to be more active against antibiotic-resistant strains than against standard ATCC or NTCC strains (

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

General experimental procedures
Optical rotations were measured on a Perkin Elmer 341 MC polarimeter at 20 o C. 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 1 H and 100.10 MHz for 13 C, respectively. All NMR spectra were obtained at 25 o C 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 CH 2 Cl 2 . 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 1 and 2 were 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/H 2 O. 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 3 and 4 were recorded on a Bruker Daltonics equipment (UltrO-ToF, MA, USA). Samples (0.5 µg mL -1 ) dissolved in MeOH/H 2 O 1:1 were introduced in the electrospray source at 5 µL min -1 with a direct infusion pump. MS/MS and MS n 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 Didemnum sp. BA99ASCI-03 and Didemnum sp. 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.

Acetylation of rodriguesines A (1) and B (2)
The mixture of compounds 1 and 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 C 18

Hydrolysis and Marfey's analysis of rodriguesines A (1) and B (2)
Two milligrams of the mixture of 1 and 2 was dissolved in 0.5 mL of 6 mol L -1 HCl and refluxed at 105 o C for 24 h. Then the reaction medium was evaporated until dryness. The hydrolyzed product was dissolved in H 2 O (40 µL) and a 0.5 mol L -1 NaHCO 3 solution was added, followed by 80 µL of a 1.4 equiv. of Marfey's reagent (2,4-dinitro-5fluoro-phenyl-l-alaninamide) in acetone. The reaction was stirred at 40 o C 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-phenylalaninederivatized standard. The hydrolysed and the standardderivatized samples were analyzed by HPLC-PDA-MS using the following conditions: Phenomenex C 18 Synergi-4 Fusion RP-80 column, 250 × 4.6 mm; eluent: gradient of MeCN in Et 3 N/H 2 PO 4 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. Briefly, bacteria were grown in BHI (Brain Heart Infusion) liquid medium at 37 °C and suspended, as recommended, to yield final inocula of approximately 10 6 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 H 2 O) and incubated 24 h at 37 o C in 10% CO 2 . 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.