Bromopyrrole Alkaloids from the Sponge Agelas kosrae

Two new sceptrin derivatives (1,2) and eight structurally-related known bromopyrrole-bearing alkaloids were isolated from the tropical sponge Agelas kosrae. By a combination of spectroscopic methods, the new compounds, designated dioxysceptrin (1) and ageleste C (2), were determined to be structural analogs of each other that differ at the imidazole moiety. Dioxysceptrin was also found to exist as a mixture of α-amido epimers. The sceptrin alkaloids exhibited weak cytotoxicity against cancer cells. Compounds 1 and 2 also moderately exhibited anti-angiogenic and isocitrate lyase-inhibitory activities, respectively.

During the course of our search for bioactive metabolites from tropical sponges, we encountered the purple elongated sponge Agelas kosrae from Kosrae Island, the Federated States of Micronesia, and the organic extract of this sponge exhibited moderate cytotoxicity (IC 50 279 µg/mL) against the diverse chromatographic methods led to the isolation of 10 bromopyrrole-bearing alkaloids similar to sceptrin and related structural classes including two new compounds. We report here the structural determination of dioxysceptrin (1) and ageleste C (2) by combinations of spectroscopic analyses ( Figure 1). These sceptrin alkaloids exhibited weak cytotoxicity against six cancer cell lines (K562, A549, HCT116, MDA-MB-231, SNU628, SK-Hep-1). In addition, compounds 1 and 2 moderately exhibited anti-angiogenic and isocitrate lyase (ICL)-inhibitory activities, respectively.

Results and Discussion
The molecular formula of dioxysceptrin (1) was deduced as C22H24Br2N10O4 by HRFABMS analysis (m/z [M + H] + 651.0432, calcd 651.0427) aided by isotopic clusters in both positive (m/z 651.0/653.0/655.0) and negative ion modes (m/z 648.9/650.9/652.9) with intensities in a 1:2:1 ratio, indicating a dibrominated compound ( Figure S13). However, an interesting phenomenon was found in the NMR spectra of this compound. That is, two sets of highly disproportionate signals existed in both the initial 1 H and 13 C NMR spectra. Then, during storage, the ratio between the intensities of these sets of peaks gradually reached equilibrium (from 6:1 to 1:1 according to the 1 H NMR spectrum). Since several attempts to separate these compounds under various HPLC conditions were not successful, 1 was thought to be a mixture of either epimers or conformational isomers (1a and 1b), and their structures were determined from the mixture.

Results and Discussion
The molecular formula of dioxysceptrin (1) was deduced as C 22 H 24 Br 2 N 10 O 4 by HRFABMS analysis (m/z [M + H] + 651.0432, calcd. 651.0427) aided by isotopic clusters in both positive (m/z 651.0/653.0/655.0) and negative ion modes (m/z 648.9/650.9/652.9) with intensities in a 1:2:1 ratio, indicating a dibrominated compound ( Figure S13). However, an interesting phenomenon was found in the NMR spectra of this compound. That is, two sets of highly disproportionate signals existed in both the initial 1 H and 13 C NMR spectra. Then, during storage, the ratio between the intensities of these sets of peaks gradually reached equilibrium (from 6:1 to 1:1 according to the 1 H NMR spectrum). Since several attempts to separate these compounds under various HPLC conditions were not successful, 1 was thought to be a mixture of either epimers or conformational isomers (1a and 1b), and their structures were determined from the mixture.
In the 13 C NMR spectrum of 1a, three carbons at δ C 174.3, 160.2 and 158.7 were thought to be amide carbonyl and/or guanidine carbons (Table 1). This interpretation was supported by the IR absorption bands at 1680 and 1635 cm −1 . Four additional carbons at δ C 126.6 (C), 121.3 (CH), 111.8 (CH), and 95.1 (C) in conjunction with the protons at δ H 6.95 (1H, br s) and 6.84 (1H, br s) in the 1 H NMR data were indicative of a substituted pyrrole moiety. The remaining carbons were the protonated ones in the more shielded region: δ C 60.3 (CH), 41.9 (CH 2 ), 38.2 (CH) and 37.1 (CH). A very similar set of carbon and proton signals was also found for 1b. Given this information, the planar structure of 1a was determined by a combination of 2D NMR experiments. First, all of the carbons were matched to their attached protons by an HSQC experiment. Then, a direct connection was found between an aromatic proton (H-2) and an NH proton (NH-1) at δ H 6.95 and 11.73, respectively, by a COSY experiment. The HMBC correlations of these protons and an additional proton at δ H 6.84 (H-4) with the neighboring carbons readily identified a 2,4-disubstituted pyrrole moiety (1-NH-C-5) ( Figure 2). The significant shielding of C-3 at δ C 95.1 confirmed the attachment of bromine at this position. Similarly, although it was not directly found from the HMBC data, the shift of C-5 at δ C 126.6 revealed the presence of a carbon substituent, possibly a carbonyl carbon, at this position.
The COSY data revealed a long proton spin system of alkyl protons with NH groups at both termini (7-NH-12-NH), and this assignment was supported by several HMBC correlations among the carbons and protons in this moiety. An amide linkage was found between this group and the previously identified bromopyrrole by an HMBC correlation between 6-NH and C-6 (δ C 126.6). Similarly, a guanidine carbon and a carbonyl carbon were placed at C-13 and C-15, respectively, at the other terminus by a series of HMBC correlations: H-10/C-15, H-11/C-13 and C-15, and 12-NH/C-13 and C-15. Although it was not directly found from the 2D NMR data, the characteristic chemical shifts of the carbons and protons of C-11-C-13 and C-15, as well as an isolated proton signal at δ H 9.16 (2H, br s), were indicative of an aminoimidazolinone moiety ( Figure 2). Thus, 1a was found to possess a C 11 bromopyrrole-aminoimidazolinone moiety.
The formula identified for 1a based on its NMR spectra accounted for C 11 H 12 BrN 5 O 2 , exactly half of the molecular formula. Furthermore, the methine groups at C-9 and C-10 required the attachment of additional groups at these positions. Overall, the dimerization of the bromopyrrole-imidazoline moiety Mar. Drugs 2018, 16, 513 4 of 11 through a cyclobutane group at C-9 and C-10 could easily account for the substituents missing from these positions. Thus, the planar structure of 1a was identified as a dimeric sceptrin-type alkaloid. By utilizing the same NMR experiments, the planar structure of the other constituent, 1b, was confirmed to be the same as 1a (Table 1). A literature survey showed that oxysceptrin from the sponge Agelas conifera had the same kind of oxidation pattern as was seen in one of the imidazoles of sceptrin [14]. The COSY data revealed a long proton spin system of alkyl protons with NH groups at both termini (7-NH-12-NH), and this assignment was supported by several HMBC correlations among the carbons and protons in this moiety. An amide linkage was found between this group and the previously identified bromopyrrole by an HMBC correlation between 6-NH and C-6 (C 126.6). Similarly, a guanidine carbon and a carbonyl carbon were placed at C-13 and C-15, respectively, at the other terminus by a series of HMBC correlations: H-10/C-15, H-11/C-13 and C-15, and 12-NH/C-13 and C-15. Although it was not directly found from the 2D NMR data, the characteristic chemical shifts of the carbons and protons of C-11-C-13 and C-15, as well as an isolated proton signal at H 9.16 (2H, br s), were indicative of an aminoimidazolinone moiety ( Figure 2). Thus, 1a was found to possess a C11 bromopyrrole-aminoimidazolinone moiety.
The formula identified for 1a based on its NMR spectra accounted for C11H12BrN5O2, exactly half of the molecular formula. Furthermore, the methine groups at C-9 and C-10 required the attachment of additional groups at these positions. Overall, the dimerization of the bromopyrrole-imidazoline moiety through a cyclobutane group at C-9 and C-10 could easily account for the substituents missing from these positions. Thus, the planar structure of 1a was identified as a dimeric sceptrin-type alkaloid. By utilizing the same NMR experiments, the planar structure of the other constituent, 1b, was confirmed to be the same as 1a (Table 1). A literature survey showed that oxysceptrin from the sponge Agelas conifera had the same kind of oxidation pattern as was seen in one of the imidazoles of sceptrin [14].
The nature of 1a and 1b, as well as the configurations at the cyclobutane and aminoimidazolinone stereocenters, were determined by 1D selective gradient ROESY experiments. First, conformers and diastereomers could be distinguished by NOE irradiation of paired protons The nature of 1a and 1b, as well as the configurations at the cyclobutane and aminoimidazolinone stereocenters, were determined by 1D selective gradient ROESY experiments. First, conformers and diastereomers could be distinguished by NOE irradiation of paired protons [15]. For these compounds, the irradiations of 7-NH (δ H 8.04) and H-11 (δ H 4.45) of 1a increased the signal intensities of only the protons in this compound, while those in 1b were unaffected. The same phenomenon was also observed for 1b; the irradiations of 7-NH (δ H 8.24) and H-11 (δ H 4.28) only changed the intensities of the signals of the protons in this compound ( Figure S14). In addition, variable-temperature NMR experiments showed that the relative intensities of the key protons of 1a and 1b remained constant ( Figure S15). Alternatively, the possibility of 1 as a mixture of carbonyl-enol tautomers was eradicated by the 1 H NMR spectrum in MeOH-d 4 in which signals of both H-11 and H-11 were clearly observed ( Figure S15). Thus, 1a and 1b must be epimers at either the cyclobutane or α-amide positions.
The relative configuration of the cyclobutane was assigned by ROESY experiments. The NOE cross-peaks of H 2 -8/H-10 and H-9/H-11 (also H 2 -8 /H-10 and H-9 /H-11 ) assigned the 9S*, 10R*, 9 S*, and 10 R* configurations for 1a. The same ROESY cross-peaks for 1b clearly indicated the bis-epimerization at the α-amido C-11 and C-11 positions between these molecules. However, due to the absence of reliable ROESY correlations, the configurations at these positions remained unassigned. Both the epimerization and the unassigned configuration at the α-amide positions were consistent with what has been reported for oxysceptrin [14]. The absolute configurations of the cyclobutane core were assigned by ECD calculations. Since 1a and 1b, bis-epimers at the C-11 and C-11 stereocenters, existed as a mixture (1:1 v:v) in 1, both the experimental and calculated ECD data were determined in their dimeric form. The comparison of their ECD profiles clearly assigned the 9S, 10R, 9 S, and 10 R absolute configurations, which are consistent with known sceptrins (Figure 3) [9,13]. Thus, the structure of 1, designated dioxysceptrin, was determined to be a mixture of 11,11 -dioxo derivatives of sceptrin alkaloids.
The absolute configurations of the cyclobutane core were assigned by ECD calculations. Since 1a and 1b, bis-epimers at the C-11 and C-11' stereocenters, existed as a mixture (1:1 v:v) in 1, both the experimental and calculated ECD data were determined in their dimeric form. The comparison of their ECD profiles clearly assigned the 9S, 10R, 9'S, and 10'R absolute configurations, which are consistent with known sceptrins (Figure 3) [9,13]. Thus, the structure of 1, designated dioxysceptrin, was determined to be a mixture of 11,11'-dioxo derivatives of sceptrin alkaloids. The molecular formula of ageleste C (2) was established to be C18H18Br2N4O6 (m/z [M+H] + 544.9677, calcd 544.9671) by HRFABMS analysis. The NMR data of this compound showed signals of nine carbons with attached protons, indicating a dimeric nature ( Table 1). Comparison of the 1 H and 13 C NMR data with those of 1 revealed that the signals of the imidazoline moiety had been replaced with those of a carboxylic group at δC 174.0 (C-9), while those of the bromopyrrole and cyclobutane were intact (Table 1). This interpretation was confirmed by a combination of 2D NMR analyses in which the oxidative cleavage of the two imidazole moieties to carboxylic acids was clearly observed ( Figure 2). Further supporting evidence was provided by comparing the spectroscopic data with those of congeners 3 and 4 in which the protons and carbons showed virtually identical chemical shifts. After the assignment of the relative configurations by ROESY experiments (Figure 2), the absolute configurations of the cyclobutane moiety were defined to be the same as those of their congeners by a comparison of their CD data ( Figure S16).
In addition to 1 and 2, eight known structurally-related bromopyrrole-bearing compounds were isolated and identified by combinations of spectroscopic methods. These compounds were ageleste A (3) [16], ageleste B (4) [16], nakamuric acid (5) [16,17], nakamuric acid methyl ester (6) Table 1). Comparison of the 1 H and 13 C NMR data with those of 1 revealed that the signals of the imidazoline moiety had been replaced with those of a carboxylic group at δ C 174.0 (C-9), while those of the bromopyrrole and cyclobutane were intact (Table 1). This interpretation was confirmed by a combination of 2D NMR analyses in which the oxidative cleavage of the two imidazole moieties to carboxylic acids was clearly observed (Figure 2). Further supporting evidence was provided by comparing the spectroscopic data with those of congeners 3 and 4 in which the protons and carbons showed virtually identical chemical shifts. After the assignment of the relative configurations by ROESY experiments (Figure 2), the absolute configurations of the cyclobutane moiety were defined to be the same as those of their congeners by a comparison of their CD data ( Figure S16).
Sceptrins and structurally-related alkaloids are known to exhibit a broad range of bioactivities, such as anticancer, antibacterial, antifungal, anti-inflammatory, and anti-biofilm activities [1,3,4]. In our measurement of cytotoxicity, sceptrins were incubated with cancer cells for 72 h to assess the anti-proliferative activity. Compound 1 exhibited the most potent anti-proliferative effects against six cancer cell lines (Table 2). For anti-angiogenic activity, compounds 1-4 showed no cytotoxicity in HUVEC cells when treated up to 40 µM for 24 h. Subsequently, in the tube formation assay using the non-cytotoxic concentrations range (5-20 µM), only 1 exhibited moderate anti-angiogenic activity comparable to sunitinib, a positive control. The anti-angiogenic activity of compound 1 was  (Figure 4a-c). In antimicrobial bioassay, all the compounds were inactive (MIC > 128 µM) against a variety of human pathogenic bacterial and fungal strains. In a subsequent bioassay, contrarily, compound 2 displayed moderate inhibition of Candida albicans-derived isocitrate lyase (ICL), a key enzyme in microbial metabolism.  Data are presented as the mean fold changes ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 by t-test.

General Experimental Procedures
Optical rotations were measured using a JASCO P-1020 polarimeter (Easton, MD, USA) with a 1 cm cell. CD spectra were obtained using an Applied Photophysics Chirascan Plus spectrometer (Applied Photophysics Ltd., Leatherhead, Surrey, UK). UV spectra were acquired using a Hitachi U-3010 spectrophotometer (Tokyo, Japan). IR spectra were recorded on a JASCO 4200 FT-IR spectrometer (Easton, MD, USA) using a ZnSe cell. NMR spectra were recorded in DMSO-d6, with the solvent peaks (δH 2.50/δc 39.50) as internal standards, on a Bruker Avance 600 MHz spectrometer (Billerica, MA, USA). High-resolution FABMS spectrometric data were obtained at the National Center for Inter-university Research Facilities (NCIRF), Seoul National University and acquired using a JEOL JMS 700 mass spectrometer with 6 keV-energy, emission current 5.0 mA, xenon as inert gas, and meta-nitrobenzyl alcohol (NBA) as the matrix. HPLC separations were performed on a SpectraSYSTEM p2000 equipped with a refractive index detector (SpectraSYSTEM RI-150 (Waltham, MA, USA)) and a UV-Vis detector (Gilson UV-Vis-151 (Middleton, WI, USA)). All solvents used were in the presence of VEGF for 24 h was measured by MTT and compared to the control. Data are presented as the mean fold changes ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.005 by t-test.

General Experimental Procedures
Optical rotations were measured using a JASCO P-1020 polarimeter (Easton, MD, USA) with a 1 cm cell. CD spectra were obtained using an Applied Photophysics Chirascan Plus spectrometer (Applied Photophysics Ltd., Leatherhead, Surrey, UK). UV spectra were acquired using a Hitachi U-3010 spectrophotometer (Tokyo, Japan). IR spectra were recorded on a JASCO 4200 FT-IR spectrometer (Easton, MD, USA) using a ZnSe cell. NMR spectra were recorded in DMSO-d 6 , with the solvent peaks (δ H 2.50/δ c 39.50) as internal standards, on a Bruker Avance 600 MHz spectrometer (Billerica, MA, USA). High-resolution FABMS spectrometric data were obtained at the National Center for Inter-university Research Facilities (NCIRF), Seoul National University and acquired using a JEOL JMS 700 mass spectrometer with 6 keV-energy, emission current 5.0 mA, xenon as inert gas, and meta-nitrobenzyl alcohol (NBA) as the matrix. HPLC separations were performed on a SpectraSYSTEM p2000 equipped with a refractive index detector (SpectraSYSTEM RI-150 (Waltham, MA, USA)) and a UV-Vis detector (Gilson UV-Vis-151 (Middleton, WI, USA)). All solvents used were of spectroscopic grade or were distilled prior to use.

Animal Material
Specimens of the Agelas kosrae sponge (Demospongiae: Agelasida: Agelasidae) were collected by hand using SCUBA offshore of Kosrae Island in the Federated States of Micronesia at a depth of 15 m on 23 October 2013. The sponge had an elongated repent form with several branches and had dimensions of 6 cm wide and up to 20 cm long. The texture was firm and compressible, and the color was purple on the surface and beige in the choanosome. The skeleton was composed of spicules cored primary fibres, echinated secondary fibres, and very rare achinated tertiary fibres with diameters of 100-200, 30-60, and 10-20 µm, respectively. The spicules, acanthostyles, (110-140 × 6-8 µm) and acanthoxeas (150-170 × 6-8 µm) were identical to those in the literature [24]. A voucher specimen (registry No. spo. 80) was deposited at the Natural History Museum, Hannam University, Korea, under the curatorship of C.J.S.

ECD Calcualtions
All conformational searches were performed using Macromodel (Version 9.9, Schrodinger LLC. (New York, NY, USA)) software with "Mixed torsional/Low Mode sampling" in the MMFF force field. The searches were conducted in the gas phase with a 50 kJ/mol energy window limit and a maximum of 10,000 steps to thoroughly examine all low-energy conformers. The Polak-Ribiere conjugate gradient (PRCG) method was utilized for minimization processes with 10,000 maximum iterations and a 0.001 kJ (mol Å) −1 convergence threshold on the RMS gradient. Conformers within 10 kJ/mol of each global minimum for compounds 1a and 1b were used for gauge-independent atomic orbital (GIAO) shielding constant calculations without geometry optimization employing TmoleX Version 4.2.1 (COSMOlogic GmbH & Co. KG (Leverkusen, Germany)) at the B3LYP/6-31G(d,p) level in the gas phase. The CD spectra were simulated by overlapping each transition, where σ is the width of the band at 1/e height. ∆E i and R i are the excitation energies and rotatory strengths, respectively, for transition i. In the current work, the value of σ was 0.10 eV.

Anti-Proliferative Activity Assay
Anti-proliferative activity was evaluated using SRB staining assay in various cancer cell lines (K562, A549, HCT116, MDA-MB-231, SNU638, SK-Hep-1). Cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). They were cultured in media supplemented with 10% fetal bovine serum (FBS) and antibiotics-antimycotics (PSF; 100 units/mL penicillin G sodium, 100 ng/mL streptomycin, and 250 ng/mL amphotericin B). All cells were maintained at 37 • C under a humidified atmosphere containing 5% CO 2 . Briefly, cells were seeded in 96-well plates with various doses of compounds and incubated for 72 h. The cells were stained as previously described [25]. First, cells were fixed with 10% trichloroacetic acid and stained with 0.4% SRB in a 1% acetic acid solution. After washing and drying, dyes were dissolved in 10 mM Tris buffer (pH 10.0) and absorbance was measured at 515 nm. The percentage of cell proliferation was determined according to the following formula: cell proliferation (%) = 100 × ((A treated − A zero day)/(A control − A zero day)), where A is the average absorbance. The IC 50 values were calculated through non-linear regression analysis using TableCurve 2D v5.01 (Systat Software Inc., San Jose, CA, USA).

Anti-Angiogenic Activity Assay
Anti-angiogenic activity was evaluated using human umbilical vein endothelial cells (HUVEC) using previously described experimental methods [26]. HUVEC cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in EGM-2 (Lonza, Walkerswille, MD, USA) supplemented with 10% fetal bovine serum (FBS) and antibiotics-antimycotics (PSF; 100 units/mL penicillin G sodium, 100 ng/mL streptomycin, and 250 ng/mL amphotericin B). Briefly, HUVEC cells were mixed with tested compounds in 0.5% FBS EBM-2 media stimulated with or without VEGF (50 ng/mL) on matrigel-coated 96-well plates for 6 h at 37 • C under a humidified atmosphere containing 5% CO 2 . After the tube formation, cells were photographed using an inverted microscope (Olympus Optical Co. Ltd., Tokyo, Japan), then images were quantified with Angiogenesis Analyzer using Image J software. Tube formation activity was calculated using the following formula: (Total segment # (tested compound) − Total segment # (VEGF−))/(Total segment # (VEGF+) − Total segment # (VEGF−)) × 100. The IC 50 value was calculated through non-linear regression analysis using TableCurve 2D v5.01 (Systat Software Inc., San Jose, CA, USA). Cell viability with HUVEC cells were measured independently. First, HUVEC cells were seeded into a 96-well plate and the culture medium was replaced with a serum-free medium when it reached 60% confluency. After overnight starvation, cells were treated with samples and VEGF (50 ng/mL) in 2% FBS EBM-2 medium. Cells were further incubated for 24 h, and MTT assay was used to measure the cell viability. The formazan products were dissolved in dimethyl sulfoxide (DMSO). The absorbance was measured at 570 nm using VersaMax ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Isocitrate lyase (ICL) Activity Assay
A 1 mL aliquot of the reaction mixture contained 20 mM sodium phosphate buffer (pH 7.0), 1.27 mM threo-DL-(+)-isocitrate, 3.75 mM MgCl 2 , 4.1 mM phenylhydrazine, and 2.5 µg/mL of recombinant ICL. The reaction was immediately initiated following the addition of a substrate with or without a prescribed concentration of the inhibitor dissolved in DMSO (final concentration, 1%). Glyoxylate phenylhydrazone formation was spectrophotometrically assessed at 324 nm after incubation at 37 • C for 30 min. The percent inhibition of ICL enzyme activity for each compound was calculated relative to the inhibitor-free control and the IC 50 values were calculated using nonlinear regression analysis (percent inhibition versus concentration). 3-Nitropropionic acid was used as a positive control. Protein concentrations were measured using the Bradford method with the Bio-Rad protein assay kit (Bio-Rad) and bovine serum albumin as the standard.

Antibacterial Activity Assay
Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 19433 and Enterococcus faecium ATCC 19434) and Gram-negative bacteria (Klebsiella pneumoniae ATCC 10031, Salmonella enterica ATCC 14028 and Escherichia coli ATCC 25922) were used for antibacterial activity tests. Bacteria were grown overnight in Mueller Hinton (MH) broth at 37 • C, harvested by centrifugation and washed twice with sterile distilled water. Stock solutions of the compound were prepared in DMSO. Each stock solution was diluted with MH broth to give serial two-fold dilutions in the range of 128 to 0.06 µg/mL. The final DMSO concentration was maintained at 1% by adding DMSO to the MB broth. Aliquots (10 µL) of the broth containing approximately 5 × 10 5 colony-forming units (cfu)/mL of the bacteria were added to each well of a 96-well plate. The plates were incubated for 24 h at 37 • C. The minimum inhibitory concentration (MIC) values were determined as the lowest concentration of the test compound that inhibited bacterial growth. Ampicillin and tetracycline were used as reference compounds.

Antifungal Activity Assay
Potato dextrose agar (PDA) was used to cultivate Candida albicans ATCC 10231. After incubation for 48 h at 28 • C, yeast cells were harvested by centrifugation and washed twice with sterile distilled water. Aspergillus fumigatus HIC 6094, Trichophyton rubrum NBRC 9185 and Trichophyton mentagrophytes IFM 40996 were plated on PDA and incubated for 2 weeks at 28 • C. Spores were harvested and washed twice with sterile distilled water. Stock solutions of the compound were prepared in DMSO. Each stock solution was diluted with RPMI 1640 broth (Difco) to give serial two-fold dilutions in the range of 128 to 0.06 µg/mL. The final DMSO concentration was maintained at 1% by adding DMSO to the broth. Aliquots (10 µL) of the RPMI 1640 broth containing approximately 10 4 cells/mL were mixed with the test compound solutions in each well of a 96-well plate. The plates were incubated for 24 h (for C. albicans), 48 h (for A. fumigatus) and 96 h (for T. rubrum and T. mentagrophytes) at 37 • C. A culture with DMSO (1%) was used as a solvent control, and a culture supplemented with amphotericin B was used as a positive control.

Disk Diffusion Assay
Gram-positive bacteria (S. aureus ATCC 25923 and Bacillus subtilis ATCC 6633) were used for disk diffusion assay. Bacteria were grown overnight in Mueller Hinton (MH) broth at 37 • C, harvested by centrifugation and washed twice with sterile distilled water. Stock solutions of the compound were prepared in DMSO. Bacterial cells (5 × 10 5 colony-forming units (cfu)/mL) were inoculated and 128 µg/disk of the compound was added to each agar plate. The plates were incubated for 24 h at 37 • C. Ampicillin was used as a reference compound.

Conclusions
Two new sceptrin derivatives (1,2) and eight structurally-related known bromopyrrole-bearing alkaloids were isolated from the tropical sponge Agelas kosrae. The structure elucidation of compounds 1 and 2 were established by combined spectroscopic methods. Dioxysceptrin (1) was also found to exist as a mixture of α-amido epimers. Absolute configurations were determined by comparison of the experimental and calculated ECD data. The sceptrin alkaloids exhibited weak cytotoxicity against cancer cell-lines. Compounds 1 and 2 also moderately exhibited anti-angiogenic and isocitrate lyase-inhibitory activities, respectively.