Niphatoxin C, a Cytotoxic Tripyridine Alkaloid from Callyspongia sp. Malcolm S. Buchanan, † Anthony R. Carroll, † Rama Addepalli, † Vicky M. Avery, † John N. A. Hooper, ‡ and Ronald J. Quinn* ,† Eskitis Institute, Griffith UniVersity, Brisbane, Queensland 4111, Australia, and Queensland Centre for BiodiVersity, Queensland Museum, South Brisbane, Queensland 4101, Australia ReceiVed July 25, 2007 As part of our studies to discover P2X 7 receptor antagonists, the sponge Callyspongia sp. was investigated. A new tripyridine alkaloid niphatoxin C (1) was isolated and had P2X 7 receptor antagonism; however, cytotoxicity of THP-1 cells was the predominant biological effect at higher concentrations. Its structure was determined by 1- and 2-D NMR spectroscopy. Previously we reported the isolation of the tetrameric pyrrole- imidazole alkaloids stylissadines A and B, from Stylissa flabellata, as specific P2X 7 antagonists. 1 During our P2X 7 campaign (62 749 extracts) another extract that showed P2X 7 inhibition was the Australian marine sponge Callyspongia sp. (Callyspongiidae). Bioassay-guided purification of this extract yielded the tripyridine alkaloid niphatoxin C (1) as the bioactive constituent. The aim was to discover selective P2X 7 receptor antagonists for the treatment of the inflammatory diseases osteoarthritis, rheumatoid arthritis, and chronic obstructive pulmonary disease (COPD). Niphatoxin C (1) belongs to the 3-alkylpyridinium class of alkaloids, which are a well-known family of marine natural products. 2 The known compounds niphatoxins A and B, which have ichthyo- and cytotoxic activity, are also tripyridine alkaloids and have similar structures to niphatoxin C (1). 3 Sponges of the genus Callyspongia are known to produce polymeric pyridinium alkaloids, 4,5 but no small molec- ular weight pyridinium compounds have been reported. The isolation, structure elucidation, and biological activity of niphatoxin C(1) will be discussed here. Niphatoxin C (1) had a molecular formula determined to be C 36 H 50 N 3 + by HRESIMS (m/z 262.702930 [C 36 H 50 N 3 +H] 2+ , calcd 262.703601). The 1 H NMR data analysis (Table 1) showed resonances for a 1,3-disubstituted pyridinium ring, including four aromatic hydrogens at δ 8.97 (s, H2), 8.94 (d, 6.0 Hz, H6), 8.41 (d, 8.4 Hz, H4), and 8.08 (dd, 8.4, 6.0 Hz, H5) as well as methylene resonances at δ 4.57 (t, 7.5 Hz, H1′) and 3.59 (d, 7.2 Hz, H7). There were also resonances for two 3-alkylpyridine units with isochronous chemical shifts, including eight aromatic hydrogens at δ 8.60 (brs, H17/14′), 8.57 (brd, 6.0 Hz, H19/16′), 8.00 (brd, 8.4 Hz, H21/18′), and 7.62 (brdd, 8.4, 6.0 Hz, H20/17′) as well as a methylene resonance at δ 2.67 (m, H15/12′). The 1 H NMR spectrum contained a further four olefinic resonances [δ 5.61 (brdt, 10.8, 7.2 Hz, H9); 5.56 (brdt, 10.8, 7.2 Hz, H8); 5.35 (brdt, 10.8, 7.2 Hz, H6′); 5.30 (brdt, 10.8, 7.2 Hz, H5′)], and the remaining resonances were for upfield aliphatic protons that resonated between δ 1.28 and 2.12, many of which were overlapping. A thorough study of the 2D NMR data (Table 1) revealed the partial structures shown in Figure 1. The Z-geometry of the double bonds was obtained on the basis of the coupling constant of the H8-H9 (10.8 Hz) and H5′-H6′ (10.8 Hz) double bonds as well as the 13 C chemical shift values of the allylic methylenes (δ C 29.5, 26.7, 26.0, 26.5). 6 With one unit of partial structure A and two units of partial structure B, there remained four methylenes (δ 1.28, 8H; δ C 28.3, 4C) to incorporate into the structure and complete the molecular formula. These were assigned C12–C13 and C9′-C10′ and joined partial structure A to the two units of partial structure B. ESIMS data strongly supported this structure. Thus, under high-energy * To whom correspondence should be addressed. Tel: +61 7 3735 6006.Fax: +61 7 3735 6001. E-mail: r.quinn@griffith.edu.au. † Griffith University. ‡ Queensland Museum. Table 1. 1 H (600 MHz), 13 C (125 MHz), gCOSY, and gHMBC NMR Data for Niphatoxin C (1) in DMSO-d 6 position δ C δ H (mult, J Hz) COSY (H no.) 2,3 J CH HMBC (C no.) 2 143.7 CH a 8.97 (s) 3, 4, 6, 7, 1′ 3 141.8 qC 4 144.9 CH 8.41 (d, 8.4) 5 6, 7 5 127.7 CH 8.08 (dd, 8.4, 6.0) 4, 6 3 6 142.4 CH 8.94 (d, 6.0) 5 4, 5, 1′ 7 29.5 CH 2 3.59 (d, 7.2, 2H) 8 3, 4, 8, 9 8 125.0 CH 5.56 (brdt, 10.8, 7.2) 7 7, 10 9 133.0 CH 5.61 (brdt, 10.8, 7.2) 10 7, 10 10 26.7 CH 2 2.12 (q, 7.0, 2H) 9, 11 8, 9, 11 11 28.7 CH 2 1.34 (m, 2H) 10 12 28.3 CH 2 b 1.28 (m, 2H) c 13 28.3 CH 2 b 1.28 (m, 2H) c 14 30.2 CH 2 1.58 (m, 2H) d 15 15, 16 15 31.8 CH 2 2.67 (q, 7.0, 2H) 14 14, 16, 17, 21 16 139.5 qC 17 145.8 CH 8.60 (brs) 15, 16, 21 19 143.7 CH a 8.57 (brd, 6.0) 20 17, 20, 21 20 124.8 CH 7.62 (brdd, 8.4, 6.0) 19, 21 16 21 140.1 CH 8.00 (brd, 8.4) 20 17 1′ 60.6 CH 2 4.57 (t, 7.5, 2H) 2′ 2, 6, 2′,3′ 2′ 30.3 CH 2 1.90 (quin., 7.5, 2H) 1′ 1′,3′ 3′ 25.5 CH 2 1.29 (m, 2H) c 4′ 26.0 CH 2 2.01 (q, 7.5, 2H) 5′ 2′,3′,5′,6′ 5′ 128.7 CH 5.30 (brdt, 10.8, 7.2) 4′ 6′ 130.2 CH 5.35 (brdt, 10.8, 7.2) 7′ 7′ 26.5 CH 2 1.96 (q, 7.5, 2H) 6′ 5′,6′,8′ 8′ 28.9 CH 2 1.28 (m, 2H) c 9′ 28.3 CH 2 b 1.28 (m, 2H) c 10′ 28.3 CH 2 b 1.28 (m, 2H) c 11′ 30.2 CH 2 1.58 (m, 2H) d 12′ 12′, 13′ 12′ 31.8 CH 2 2.67 (q, 7.0, 2H) 11′ 11′, 13′, 14′, 18′ 13′ 139.5 qC 14′ 145.8 CH 8.60 (brs) 12′, 13′, 18′ 16′ 143.7 CH a 8.57 (brd, 6.0) 17′ 14′, 17′, 18′ 17′ 124.8 CH 7.62 (dd, 8.4, 6.0) 16′, 18′ 13′ 18′ 140.1 CH 8.00 (brd, 8.4) 17′ 14′ a–d Assignments with the same superscript are interchangeable. J. Nat. Prod. 2007, 70, 2040–2041 2040 10.1021/np070366q CCC: $37.00  2007 American Chemical Society and American Society of Pharmacognosy Published on Web 11/21/2007