Simplexidine, a 4-Alkylpyridinium Alkaloid from the Caribbean Sponge Plakortis simplex

Chemical analysis of the secondary metabolites of the Caribbean sponge Plakortis simplex, a source of many bioactive compounds, showed the presence of the new metabolite simplexidine (4), belonging to the extremely rare class of 4-alkyl-pyridinium alkaloids. The structural characterization of this molecule, based on spectroscopic methods, is reported.


Introduction
As part of an ongoing search for biologically active compounds from marine invertebrates, our research group has been devoting considerable efforts to the chemical investigation of the Caribbean sponge Plakortis simplex. This research project, started about ten years ago, has been strongly fostered by the discovery of a large array of structurally unique and biologically active metabolites. This richness of secondary metabolite production has been discussed in a review paper [1] and has been recently correlated to the wide presence of bacterial and fungal symbionts associated to the sponge host cells [2].
For example, P. simplex provided a series of promising lead compounds for drug development, including the immunosuppressive glycolipids plakosides [3] and simplexides [4] and a series of OPEN ACCESS endoperoxide-containing derivatives related to plakortin (1, Figure 1) [5,6], for which a potent antimalarial activity (in the nM range) has been discovered [7].
Structurally unique metabolites were also disclosed as components of the most polar fractions obtained from the organic extracts of P. simplex. Indeed, these fractions showed to contain the first natural betaine derivatives to be characterized by a iodinated indole ring [8,9], e.g. plakohypaphorine E (2, Figure 1), and the unique 4-alkylpyridinium alkaloid simplakidine (3, Figure 1) [10]. Careful examination of the polar fractions of P. simplex has now yielded to the isolation of a new 4-alkylpyridinium alkaloid that we have named simplexidine (4, Figure 1) and for which we report herein details about isolation and structural elucidation.

Results and Discussion
A specimen of the sponge Plakortis simplex (Demospongiae, order Homosclerophorida, family Plakinidae) was collected during the summer of 2002 along the coasts of The Bahamas and immediately frozen. After homogenization, the organism was exhaustively extracted, in sequence, with methanol and chloroform. The methanolic layer was partitioned between n-BuOH and water, and, subsequently, the organic phase, combined with the CHCl 3 extract, was subjected to chromatography over a reversed phase (RP 18 ) silica column eluted with a solvent gradient from H 2 O/MeOH 9:1 to MeOH and then to MeOH/CHCl 3 9:1. The most polar fractions were preliminarily separated over silica gel (gradient from EtOAc to MeOH) and then re-chromatographed by reverse-phase HPLC (eluent MeOH/H 2 O 4:6) to finally yield 2.0 mg of pure simplexidine (4).
The molecular formula C 15  The 1 H-NMR spectrum of simplexidine (Table 1) showed three signals (a singlet at δ H 8.81 and two doublets at δ H 8.63 and 7.91) in the aromatic region, three methine multiplets (δ H 5.48, 5.35, and 3.81) and a methyl singlet (δ H 4.34) in the midfield region, and a series of well resolved signals confined in the spectral region ranging from δ H 2.50 to 0.80, including two methyl triplets at δ H 0.93 and 0.89. The presence of an aromatic chromophore in the structure of simplexidine (4) was further suggested by the UV absorption at λ max 268 nm and by the presence of five signals between δ C 127 and 164 in the 13 C-NMR spectrum ( Table 1).
The 15 carbon signals present in the 13 C-NMR spectrum were assigned, with the aid of DEPT experiments, to three methyls, three methylenes, six methines and three unprotonated sp 2 type carbons. Among these latter resonances, the signal at δ C 168.6 could be ascribed to a carboxylate group, as suggested also by the IR absorption at ν max 1642 cm -1 . Association of the resonances of the 12 protonbearing carbons with those of the relevant protons was accomplished through the analysis of a 2D NMR gradient-HSQC spectrum. Inspection of the 1 H-1 H COSY NMR spectrum of simplexidine (4) allowed us to arrange all the proton multiplets within the two spin systems showed in red in Figure 2. The first fragment includes only the two mutually coupled aromatic doublets at δ H 8.63 and 7.91, while the second moiety is an eight-carbon fragment connecting the two methyl triplets (from H 3 -1 to H 3 -8) and comprising two coupled sp 2 methines (H-3 and H-4) and a single branching at the sp 3 methine C-6 (δ H 3.81).  With these data in our hands and taking into account the molecular formula, the assembly of the carbon framework of simplexidine (4) required the elucidation of an aromatic C 7 H 6 NO 2 subunit, probably linked at C-6 and comprising a carboxylate group. Interpretation of diagnostic gradient-HMBC cross-peaks (Figure 2) was of pivotal importance to resolve this issue and suggested the presence of a disubstituted N-methyl pyridinium ring. This assignment was corroborated by perfect agreement of 1 H-and 13 C-NMR resonances of this subunit with literature values [10,11].
Pyridinium alkaloids are frequently isolated from the polar extracts of marine invertebrates, mostly sponges; however, in spite of the wide diffusion, the chemical diversity within this class of compounds is somewhat limited and only two structural groups can be identified. The most common group of pyridinium derivatives includes oligomeric structures with alkyl linear chains linked at positions C-3 and N-1 of the pyridinium ring, e.g. the recently reported pachychaline C (5, Figure 3) [12]. These molecules are known to exhibit a range of bioactivities including cytotoxic [13] and anti-cholinesterase [14] properties. The second structural group includes the carboxyl-containing homarine (6, Figure 3) or trigonelline (7, Figure 3). substituted at C-3 or C-2, respectively, with short alkyl chains. The isolation of simplexidine (4) is particularly remarkable since it confirms the existence, within the Plakortis simplex biosynthetic machinery, of enzymes deputed to the attachment of polyketidederived carbon chains at the 4 position of pyridinium rings. In the case of simplakidine (3), this unique reaction led to the linkage between the trigonelline nucleus and a C 17 moiety that clearly shared the plakortin (1) carbon backbone. On the other hand, the biosynthetic origin of the C 8 group present in the structure of simplexidine (4) cannot be unambiguously predicted, although similarly to that postulated for plakortin derivatives [15], a polyketide origin can be hypothesized also in this case. Investigation of the role of microbial symbionts in the elaboration of the incredible pool of structurally unique secondary metabolites of Plakortis simplex is in progress in our lab. Simplexidine (4) exhibited very weak cytotoxicity toward murine macrophages (RAW 264-7) with 30% of growth inhibition at 80 μg/mL.

General
Optical rotations were measured in MeOH on a Perkin-Elmer 192 polarimeter equipped with a sodium lamp (λ = 589 nm) and a 10-cm microcell. IR (KBr) spectra were recorded on a Bruker model IFS-48 spectrophotometer. UV spectra were obtained in MeOH using a Beckman DU70 spectrophotometer. ESI-MS spectra were performed on a LCQ Finnigan MAT spectrometer. HR-FABMS were performed on a FISONS Prospec mass spectrometer using a glycerol matrix. 1 H (500 MHz) and 13 C (125 MHz) NMR spectra were measured on a Varian INOVA 500 spectrometer; chemical shifts are referenced to the residual solvent signal (CD 3 OD: δ H = 3.34, δ C = 49.0). The multiplicities of 13 C resonances were determined by DEPT experiments. Homonuclear 1 H connectivities were determined by using COSY experiments. One bond heteronuclear 1 H-13 C connectivities were determined with gradient-HSQC pulse sequence. Two-and three-bond 1 H-13 C connectivities were determined by gradient-selected HMBC experiments optimized for a 2,3 J of 7.0 Hz. Medium-pressure liquid chromatographies (MPLC) were performed using a Büchi 861 apparatus with RP 18 and SiO 2 (230-400 mesh) stationary phases. High performance liquid chromatography (HPLC) separations were achieved in isocratic mode on a Beckmann apparatus equipped with RI detector and LUNA (Phenomenex) columns (SI60, 250 × 4 mm).

Extraction and Isolation Procedure
A specimen of Plakortis simplex was collected in July 2002 along the coasts of The Bahamas. A voucher specimen is deposited at the Dipartimento di Chimica delle Sostanze Naturali, Italy with the ref. n° 02-10. The organism was immediately frozen after collection and kept frozen until extraction, when the sponge (43 g, dry weight after extraction) was homogenized and extracted with methanol (4 × 500 mL) and with chloroform (4 × 500 mL). The methanol extract was initially partitioned between H 2 O and n-BuOH and then the organic phase was combined with the CHCl 3 extract and concentrated in vacuo to afford a brown oil (22.1 g). This was subjected to chromatography on a column packed with RP 18 silica gel and eluted with 9:  Table 1.