The marine sponge Agelas citrina as a source of the new pyrrole–imidazole alkaloids citrinamines A–D and N-methylagelongine

Summary The chemical investigation of the Caribbean sponge Agelas citrina revealed four new pyrrole–imidazole alkaloids (PIAs), the citrinamines A–D (1–4) and the bromopyrrole alkaloid N-methylagelongine (5). All citrinamines are dimers of hymenidin (6) which was also isolated from this sponge as the major metabolite. Citrinamines A (1) and B (2) are derivatives of the PIA dimer mauritiamine (7), whereas citrinamine C (3) is derived from the PIA dimer nagelamide B (8). Citrinamine D (4) shows an uncommon linkage between the imidazole rings of both monomeric units as it is only observed in the benzocyclobutane ring moiety of benzosceptrins A–C (9–11). Compound 5 is the N-methyl derivative of agelongine (12) which consist of a pyridinium ring and an ester linkage instead of the aminoimidazole moiety and the common amide bond in PIAs.


Results and Discussion
The crude extract of the sponge Agelas citrina (collected in the Bahamas in March 2001) was investigated by a standard separation scheme. The MeOH/CH 2 Cl 2 extract of the sample was partitioned between n-hexane, n-BuOH, and H 2 O. The n-BuOH soluble fraction was further purified by size exclusion chromatography (Sephadex LH-20) and preparative reversed-phase HPLC yielding five new compounds (1)(2)(3)(4)(5). The isolation and structure elucidation of compounds 1 to 5 are discussed in detail.
(6.10 ppm) to C-11´ (122.5 ppm), and H-10´ to C-11´] as well as by the absence of the H-15´ signal in the 1D 1 H NMR spectrum. The missing signal for H-15 in the 1D 1 H NMR spectrum and a sp 2 quaternary carbon (δ C 171.7 ppm) in the 1D 13 C spectrum verified the oxidation of one aminoimidazole ring at position C-15 like in mauritiamine (7) and nagelamide P (18).   to the E configuration. The same double bond geometries were also observed for mauritiamine (7) and nagelamide P (18). Citrinamine A (1) is the 2,2´-didebromo derivative of mauritiamine (7) and as in the original publications of 7 and 18 no chiroptical effect was observed for 1. Synthetic studies on mauritiamine (7) [14] demonstrated the formation of similar racemic products by a chemical oxidative dimerization which could be an alternative origin of these metabolites.
The molecular formula of citrinamine B (2) was established by HR-ESIMS (m/z 740.0337, [M + H] + , monoisotopic) and the pseudomolecular ion peaks at m/z = 740/742/744 (1:2:1) to be C 24 H 28 Br 2 N 11 O 5 S. The 1D 1 H and 13 C NMR spectra of 2 were similar to those observed for 1, except for the additional signals of one amine and two methylene groups ( Table 2). The analysis of the 1 H, 1 H-COSY and the 1 H, 13 C-HMBC spectra linked these new signals to an aminoethyl chain and the comparison with the molecular formula indicated the existence of a taurine moiety in compound 2. This structural proposal for citrinamine B (2) was proven by the 1 H, 13   The structure of 2 is very similar to nagelamide H (20) [15] which is a mauritiamine derivative with a taurine residue in position C-15. There is probably an assignment error of the carbons C-8 to C-11 and C-8´ to C-11´ in the original publication of nagelamide H (20). All mauritiamine derivatives have comparable chemical shifts of these moieties and therefore a mixing up is plausible.
Based on NOESY correlations H-8 (3.92 ppm) to H-9/10 and H-8´ (4.15/3.91 ppm) to H-10´ (6.26 ppm) as well as the corresponding 3 J HH coupling constant H-9´ (6.14 ppm)/H-10( 16.1 Hz), both of the double bonds of 2 were assigned to E configuration. Citrinamine B (2) is the 2,2´-didebromo derivative of nagelamide H (20). We could not observe a chiroptical effect for 2 as it was also described in the original work of 20.  Table S1).  The 1 H, 13 C-HMBC correlations of citrinamine C (3) from H-10 (4.09 ppm) to C-11´ (121.3 ppm) and C-15´ (117.9 ppm), suggested a connection of C-10 (34.5 ppm) with the imidazole ring of the second subunit as it was also described for nagelamide B (8) [15]. The structure of citrinamine C (3) was elucidated to be the 2,2´-didebromo derivative of nagelamide B (8) with an additional methylation of the hydroxy group at C-9 (80.8 ppm) and an oxidation of the imidazole ring at position C-13 (154.6 ppm) (an urea instead of a guanidine moiety). The relative configuration of the stereogenic centers C-9 and C-10 was identical as described for nagelamide B (8).

Our investigation on
In contrast to citrinamine C (3), a different connection of the monomeric units was found for citrinamine D (4). Furthermore, 3 and 4 have an additional methoxy group at C-9 (80.8 and 78.1 ppm, respectively) and an oxidized imidazole ring compared to 1. The 1 H, 13 (12). Debromoagelongine (daminin (21) [16]) is the third compound in the agelongine family. All agelongine analogues were isolated from marine sponges.
The citrinamines A-D (1-4) were further evaluated for antimicrobial and cytotoxic activity. Classical agar diffusion assays were performed using the fungus Aspergillus niger, the yeast Saccharomyces cerevisiae as well as the Gram-negative bacterium Escherichia coli, and the Gram-positive bacterium Micrococcus luteus and Mycobacterium phlei as test organisms.
In agar diffusion assays with Mycobacterium phlei considerable inhibition zones were observed for citrinamines B, C, and D (2-4), while there was also an activity of citrinamine C (3) against Micrococcus luteus. All compounds (1)(2)(3)(4) showed no inhibition of cell proliferation of mouse fibroblasts.

Conclusion
The analysis of the marine sponge Agelas citrina revealed four new compounds of the pyrrole-imidazole alkaloid (PIA) family. Citrinamines A (1) and B (2) are closely related to mauritiamine (7) which can be seen as the most less complex dimeric PIA (the first published one) in which the monomeric units are only connected by one bond (C-11/C-15'). As it was already described for mauritiamine (7) and its congeners nagelamide P (18) and nagelamide H (20), compounds 1 and 2 were obtained as racemic mixtures. Citrinamines C (3) and D (4) show a different connection of the two monomeric hymenidin units (C-10/C-15' and C-15/C-15') compared to mauritiamine (7). Citrinamine C (3) is very closely related to nagelamide B (8) which are both hydroxylated at C-9 (methoxy in 3, hydroxy in 8). The C-15/C-15' linkage between the imidazole rings of both monomers in citrinamine D (4) is uncommon. This connection was only known from the benzosceptrins A-C (9-11) in which the two imidazole rings are connected by a benzene ring (benzocyclobutane ring system) and not by a single bond as in citrinamine D (4). Although, 3 and 4 have different connectivities, both compounds were also obtained as racemic mixtures. Finally, the pyrrole-pyridinium alkaloid N-methylagelongine (5) was also isolated from Agelas citrina. Compound 5 is the N-methylated pyrrole derivative of agelongine (12). The debromo compound is known as daminin (21). The citrinamines A-D (1-4) were tested against several pathogenic bacteria, fungi, and cultures of mice fibroblasts. Only minor antimicrobial activities were obtained for citrinamines B-D (2)(3)(4) whereas no activities were found in the cytotoxicity assay for citrinamines A-D (1-4).

Experimental
General experimental procedures 1 H, 13

Extraction and isolation
As already described in [17] the freeze-dried sponge tissue of Agelas citrina (120 g) was crushed with a mill and extracted exhaustively at room temperature with a 1:

Antimicrobial assay
As already described in [18] the antimicrobial activities were determined by agar diffusion tests using paper disks of 6 mm diameter soaked with 20 µL of the test compound in MeOH (1 mg/mL). The microorganisms were obtained from the HZI collection, grown on standard media, and cultured in liquid agar medium to a final OD of 0.01 (bacteria) or 0.1 (yeasts). Spores of fungi were collected from well-grown Petri dishes, which were rinsed with 10 mL of sterile H 2 O. One mL of the spore suspension was added to 100 mL of molten agar medium. Plates were incubated at 30 °C, and the diameters of resulting inhibition zones were measured after 1 and 2 days.

Cell proliferation assay
As already described in [18] L929 mouse fibroblasts were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and cultivated at 37 °C and 10% CO 2 in DME medium (high glucose) supplemented with 10% fetal calf serum. Cell culture reagents were purchased from Life Technologies Inc. (GIBCO BRL). Growth inhibition was measured in microtiter plates. Aliquots of 120 µL of the suspended cells (50000/mL) were added to 60 µL of serial dilutions of the test compounds. After 5 days the growth was determined using an MTT assay [19].

Supporting Information
Supporting Information File 1 NMR data.