1-O-Sulfatobastadins-1 and -2 from Ianthella basta (Pallas). Antagonists of the RyR1-FKBP12 Ca2+ Channel

Two new sulfate monoesters of hemibastadins-1 and -2 were isolated from the marine sponge Ianthella basta (Pallas) from Guam. A third new compound was tentatively assigned the structure 34-O-sulfatobastadin-9. The 1-O-sulfatohemibastadins-1 and –2 were antagonists of the RyR1-FKBP12 Ca2+ channel under conditions where the known compound bastadin-5 exhibits potent agonism (EC50 2 μM).


Introduction
The release of Ca 2+ from stores of the sarcoplasmic reticulum (SR) stimulates contraction of striated muscle fibers through ATP hydrolysis coupled to induced conformational changes of the actin-myosin protein complex. In 1994, we discovered that the known bromotyrosine tetramer, bastadin-5 (1) [1] from the marine sponge Ianthella basta (Pallas), is a potent modulator of Ca 2+ release from the SR [2]. Bastadin-5 stimulates release of Ca 2+ from the SR by binding to the RyR1-FKBP12 Ca 2+ channel, a tetrameric heterodimeric channel protein (~2000 kDa) that is associated with the smaller 12 kDa immunophilin FKBP12 [3]. The mechanism of action of 1 is not fully understood, but it is known that binding occurs at a site on the SR junctional protein that is distinct from those of known effector molecules, including ATP and caffeine [2]. Bastadin-5 and -10 [4] also have been shown to relate ryanodine-sensitive and -insensitive Ca 2+ efflux pathways in skeletal SR and BC 3 H1 cells [5]. Recently, we took the opportunity to examine the most polar fractions from extracts of the sponge Ianthella basta, collected in Guam, and found a complex mixture of bastadin O-sulfate esters from which we have isolated the new compounds 1-O-sulfatohemibastadin-1 (2) and 1-O-sulfatohemibastadin-2 (3), along with the known compound 32-O-sulfatobastadin-13 (4) [11] and a third new compound, the bastadin sulfate ester 34-O-sulfatobastadin-9 (5), The structures of the new compounds were established by interpretation of their spectral data and comparison with the parent phenols [6]. To our surprise, 2 and 3 exhibited antagonistic activity toward the RyR-1/FKBP12 complex (IC 50 13 and 29 µM, respectively). This is the first report of antagonism of the SR channel by a bastadin analog, and suggests a bimodal mechanism of action upon a common, but as-yet unidentified, effector site of the RyR1-FKBP12 channel complex.

Results and Discussion
Samples of freshly collected Ianthella basta (Pallas) were directly extracted in solvent (1:1 CH 2 Cl 2 -MeOH) and the concentrated extracts purified by silica flash chromatography using a gradient of MeOH in CH 2 Cl 2 . Pooled extracts that eluted with 3:1 CH 2 Cl 2 -MeOH were further purified by HPLC (reversed phase, C 18 , MeOH-H 2 O followed by C 18 , 70:30:0.05 H 2 O-CH 3 CN-TFA) to provide two new sulfated hemibastadins, 2 and 3, the known 4 [11] and the novel 34-O-sulfatobastadin-9 (5). All compounds were readily soluble in MeOH and appreciably soluble in water, but insoluble in CHCl 3 .
Detailed MS analysis was used to secure compositions of the new compounds. For the purpose of clarity in the description that follows M is defined as the neutral acid, but the structures are depicted, here, as the Na + salts of the sulfate half-esters. MALDI HRMS of 2 (m/z 594.8815 [M-H+2Na] + ∆ mmu +6.0) showed the presence of two Br atoms and secured the formula of the neutral compound as C 17 H 16 Br 2 N 2 O 7 S, which suggested a structure of almost half the molecular mass of the macrocyclic bastadins (e.g. 1 [7]). Fragment ions in the ESI mass spectrum of 2 due to loss of SO 3 implicated the presence of O-sulfate esters. The 1 H-and 13 C-NMR spectra of 2, although displaying similar chemical shifts and couplings as those of bastadin-5 [8], contained about half of the expected signals of the macrodilactams ( Table 1). Lack of mirror symmetry in the NMR spectra, together with signals that accounted for a 3-bromo-4-tyramine and oxime-modified 3-bromotyrosine units suggested a 'hemibastadin' similar to the compounds from another sample of I. basta described by Capon and coworkers [6a]. The configuration of the ketoxime group in 2 was E, as is usually found in this series of compounds [9]. 1 H-13 C correlations observed in the HSQC and HMBC spectra of 2 were fully compatible with the proposed structure, including the position of the O-SO 3 H group which was assigned as follows. The 13 C-NMR signals of phenols undergo well-described local changes in δ upon O-sulfation. Ragan has documented that the ipso carbon of a phenol undergoes a diamagnetic shift  Critical 13 C-NMR assignments that distinguished the signals of the two aryl rings were made on the basis of HMBC and nOe experiments (Figure 1). Because the substitution pattern in each trisubstituted phenyl ring of 2 was the same, we could simply compare the assigned 13 C-NMR signals in each ring to determine which phenoxyl group was esterified as a sulfate ester.
For example, the isochronous proton pair H27/31 in 5 resonated at lower field (δ 7.55, 2H) than 6 (δ 7.42, s, 2H [11]) and the exceptionally high field aryl doublet in 5 (δ 6.20, d, J= 2.0 Hz) is not found in 6 (the closest signal is H-8, δ 6.50, d, J= 2.0 Hz). The exclusion of 6 leaves only three undescribed isomers -10-O-sulfatobastadin-13, 10-O-sulfatobastadin-9 and 34-O-sulfato-bastadin-9 -as remaining possibilities. The former two are eliminated because compound 5 lacks the downfield shifted 1 H-NMR signal expected for an H-11 or H-16 proton ortho to an O-sulfate ester (∆δ~ -0.7 ppm, c.f. 2). Thus, we tentatively assigned the structure 34-O-sulfatobastadin-9 to 5. Unfortunately, limited sample precluded acquisition of a complete 13 C-NMR spectrum, however, a partial set of HMBC correlations observed for 5 were consistent with the proposed structure. Compounds 1-3 were assayed for binding of [ 3 H]-ryanodine to the high-affinity site of the ryanodine receptor in the RyR1-FKBP12 complex [15,2]. Ryanodine binding is an indicator of the 'open state' of the SR Ca 2+ channel. Bastadin-5 (1) showed the expected stimulation of ryanodine binding to the channel in the open state (EC 50 2 µM) [2], however, both compounds 2 and 3 were antagonists and inhibited binding of [ 3 H]-ryanodine (IC 50 13 µM and 29 µM, respectively). This contrasts with 15,34-O-di-sulfatobastadin-7 and 4 which are weak agonists of the receptor (EC 50 13.6 µM and 100 µM, respectively) [8a]. Given the similarity of the structures of 2 and 3 and their resemblance to a 'truncated' northern hemisphere of 1, it is likely that all three compounds interact with the same site, however, the mode of action with respect to Ca 2+ channel opening are clearly distinct. Further investigations of these phenomena aimed at refinement of a model for bastadin-5-promoted Ca 2+ release are underway in our laboratories.

Conclusions
Two new hemibastadin sulfate esters isolated from Ianthella basta were characterized. The compounds suppressed ryanodine binding with IC 50 's 13 µM and 29 µM, respectively. This is the opposite effect of bastadin-5 (1), a potent agonist of Ca 2+ release from the SR. Although 1-3 have structural features in common, and most likely a common binding motif, the mode of action suggests 2 and 3 inhibit channel opening. Thus, a broader structure-relationship emerges for bastadins that reveals a bimodal mechanism of action.