3-Alkylpyridinium Alkaloids from the Pacific Sponge Haliclona sp. § Agostino Casapullo,* Oscar Cobar Pinto, † Stefania Marzocco, Giuseppina Autore, and Raffaele Riccio Dipartimento di Scienze Farmaceutiche, UniVersita ` di Salerno, Via Ponte don Melillo, 84084, Fisciano, Italy ReceiVed September 26, 2008 The analysis of the polar extracts of the Pacific sponge Haliclona sp. yielded new dimeric (1), trimeric (2), and polymeric 3-alkylpyridinium alkaloids. Their isolation and structural elucidation, based on NMR and MS data, are discussed in detail, along with their cytotoxic activity. Sponges belonging to the genus Haliclona and related genera are well known to contain alkaloid-like compounds. Several 3-alkylpyridinium alkaloids have been found during the last 30 years, often characterized by interesting biological activities, such as navenones, amphitoxins, halitoxins, niphatoxins, and cyclo- stellettamines. 1-16 A lyophilized sample of the Pacific sponge Haliclona sp., collected off the coast of Guatemala, whose crude extract was shown to be cytotoxic in preliminary tests, was extracted with MeOH, and the methanolic extract was subjected to a modified Kupchan partition procedure, thereby affording four extracts (see Experimental Section). On the basis of their chromatographic and biological profiles, the chloroform and butanol extracts were then submitted to successive fractionation steps. The chloroform resi- due was chromatographed by DCCC and by RP-HPLC to give pure compounds 1 and 2. The butanol extract, partly retaining the cytotoxic properties of the crude extract, was subjected to size exclusion chromatography on a Sephadex LH-20 resin (eluent methanol), and the bioactive fraction was then submitted to membrane ultrafiltration, yielding the following different molecular weight range fractions: A (0.9 mg, > 300 kDa), B (36.5 mg, 100-300 kDa), C (5.6 mg, 50-100 kDa), D (2.8 mg, 30-50 kDa), E (1.5 mg, 3-10 kDa). 1 H and 13 C NMR spectra in CD 3 OD of 1 and 2 were superim- posable. Chemical shifts and coupling constant patterns of the 1 H NMR resonances were suggestive of a 1,3-disubstituted alkylpy- ridinium moiety (Table 1): four aromatic protons at δ 8.89 (brs), 8.86 (d, J ) 6.1 Hz), 8.42 (d, J ) 8.0 Hz), and 8.03 (dd, J ) 6.1, 8.0 Hz) and the methylene protons at δ 4.61 (t, J ) 7.7 Hz). The 13 C and edited-HSQC NMR spectra indicated the presence of nine aliphatic methylene and two olefinic methine carbons within the side chains. An exhaustive analysis of 2D NMR spectra (COSY, edited-HSQC, and HMBC, see Table 1) allowed the connection of the 1,3-disubstituted pyridine ring with the alkyl chains. Key HMBC correlations H7/C2 and H7/C6, along with H4/C17, H17/C2, H17/ C3, and H17/C4, allowed the spin systems C7-C9 and C13-C17 to be connected to the nitrogen and C3 ring atoms, respectively. Owing to the absence of additional NMR connectivities caused by the overlap of H10, H11, and H12 resonances, C9 and C13 were tentatively connected through C10-C12 on the basis of their 1 H and 13 C chemical shifts. The Z geometry of the double bond was deduced from the 13 C chemical shift values of the allylic methylene (δ 28.3) and the H15/H16 J coupling (10.0 Hz). The structural hypotheses of the monomer subunit of 1 and 2 (Figure 1) were confirmed by ESIMS and MS/MS data, which also allowed the determination of their molecular formulas. The mo- lecular weight of 460.4 Da was attributed to 1 on the basis of the singly charged ion at m/z 459.4 [M - H] + and was consistent with the dimeric alkaloidic structure depicted. Pseudomolecular ion peaks corresponding to [M - H] + species have already been described for this kind of compound following a Hofmann-type rearrange- ment. 17 Moreover, the MS 2 spectrum contained a daughter ion at m/z 230.2 corresponding to the monomeric subunit, corroborating the structure assigned by NMR analysis. The presence in the ESI spectrum of 2 of a Hofmann-type doubly charged ion at m/z 344.7 accounted for a trimeric structure with a molecular weight of 690.5 Da. When submitted to collision-induced dissociation, this species produced the same singly charged ions at m/z 459.4 and 230.2 previously discussed. Taken together, this evidence was indicative of a trimeric structure for 2, formed by the same subunit as in Figure 1. The polymeric fractions A-E covered a wide molecular weight range (3 to 300 kDa) and showed many NMR signals reminiscent of those of 1 and 2, suggesting that also these polymers were composed of 3-alkylpyridinium monomers. In addition, 1 H NMR spectra of all polymeric species appeared superimposable, indicating that they differed only by the total number of monomeric subunits. The presence of two sets of signals in their 1 H and 13 C NMR spectra suggested the existence of two different types of monomeric subunits. One signal pattern was almost identical to that of 1 and 2, while the other was consistent with the absence of the side chain double bond, replaced by two additional methylenes resonating at δ Η 2.88 and 1.73. 2D NMR spectra confirmed this deduction. Moreover, the integration of peak areas in the proton spectrum established a 1:1 ratio between the two different subunits in all § Dedicated to the memory of Luigi Gomez-Paloma. * To whom correspondence should be addressed. Tel: +39 089 969243. Fax: +39 089 969603. E-mail: casapullo@unisa.it. † Faculdad de Ciencias Quimicas y Farmacia, Universidad de San Carlos de Guatemala, Ciudad Universitaria, Zona 12, 01012, Ciudad de Guatemala, Guatemala. Table 1. NMR Spectroscopic Data (600 MHz, CD 3 OD) for 1 and 2 a position δ C , mult. δ H (J in Hz) HMBC c 1 2 145.3, CH 8.89, br s 6, 7, 17 3 144.5, qC 4 146.5, CH 8.42, d (8.0) 2, 6, 17 5 129.1, CH 8.03, dd (8.0, 6.1) 2, 3 6 143.5, CH 8.86, d (6.1) 4, 5 7 63.0, CH 2 4.61, t (7.7) 2, 6, 8, 9 8 32.6, CH 2 2.03, m 9 27.2, CH 2 1.40, m 10 30.4 b CH 2 1.40, m 11 30.5 b CH 2 1.40, m 12 30.6 b CH 2 1.40, m 13 30.2, CH 2 1.40, m 14 28.3, CH 2 2.21, q (7.3) 15 135.4, CH 5.72, dt (10.0, 7.3) 17, 13 16 125.3, CH 5.60, dt (10.0, 7.3) 14 17 31.1, CH 2 3.69, d (7.3) 2, 3, 4, 15, 16 a All data for 1 and 2 are superimposable. b Assignments may be interchanged. c HMBC correlations, optimized for 7.5 Hz, are from proton(s) stated to the indicated carbon. J. Nat. Prod. 2009, 72, 301–303 301 10.1021/np800610p CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy Published on Web 01/09/2009