New Oxidized Zoanthamines from a Canary Islands Zoanthus sp.

Three new norzoanthamine-type alkaloids, named 2-hydroxy-11-ketonorzoanthamide B (1), norzoanthamide B (2) and 15-hydroxynorzoanthamine (3), were isolated from Zoanthus sp. specimens collected at the Canary Islands. Their structures were determined by interpretation of NMR and HR-ESIMS data. Relative configurations of their chiral centers were proposed on the basis of ROESY spectra and by comparison of their spectroscopic data with those of the well-known compound, norzoanthamine.


Introduction
Marine zoanthids are widely dispersed throughout the temperate and tropical littoral regions of the Indic, Pacific and Atlantic Oceans. Diverse species of these organisms produce a wide array of metabolites, some of which have unique structures and possess significant biological activities [1]. In particular, from the genus Zoanthus (phylum Cnidaria, class Anthozoa, order Zoanthidea), different research groups have isolated a series of alkaloids, known as zoanthamines, characterized by a unique polycyclic backbone [2]. Zoanthamine, isolated in 1984 from polyps collected at the Visakhapatnam coast of India by Faulkner et al. [3], was the first example of these compounds. Afterwards, norzoanthamine was identified from a colony of polyps collected at the Ayamaru coast of the Amami Island, South Japan, by Uemura and co-workers [4]. Later, other research groups reported a significant number of related compounds, some of which have substantial structural modifications [5][6][7][8][9]. From a pharmacological point of view, different biological effects, including anti-inflammatory, antiplatelet and antiosteoporotic activities, have been investigated [10][11][12]. In particular, the increasing worldwide prevalence of osteoporosis and the novel mechanism of action of zoanthamines have set them up as promising drug candidates [13]. Accordingly, a number of synthetic approaches to obtain these molecules independently from organism collection have been published [14][15][16].
In this paper, we describe the isolation and structural characterization of three new oxidized norzoanthamine congeners from the genus Zoanthus collected at the coast of Tenerife. The structures of these new metabolites, 2-hydroxy-11-ketonorzoanthamide B (1), norzoanthamide B (2) and 15-hydroxynorzoanthamine (3), were determined primarily on the basis of MS and NMR data ( Figure 1).
15-Hydroxynorzoanthamine (3) was obtained as a solid, [α] 25 D +8 (c 0.03, CHCl3). The molecular formula of 3 was deduced by ESI-HRMS as C29H41NO6 (m/z 500.30050; calculated for 500.30066, [M + H] + ), therefore indicating the presence of an additional oxygen atom with respect to norzoanthamine. Comparison of the NMR data of 3 with those reported for norzoanthamine clearly revealed that the new metabolite shares the same carbon skeleton, but contains an additional oxygen atom attached to C-15 ( Table 2). This is in accordance with the absence of the characteristic olefin carbons C-15 and C-16, as well as with the presence of two new signals at δC 73.6 and δC 54.4 corresponding to a tertiary hydroxyl at C-15 and a new methylene at C-16 (δH 2.46/2.54, d, J = 13.8 Hz), respectively ( Table 2). These findings were supported by the absence of the α,β-unsaturated ketone distinctive absorption band in the UV spectrum. Analysis of the ROESY experiment confirmed that the relative configurations of all chiral centers in 3 are equivalent to those observed in norzoanthamine. The relative configuration of the new stereogenic center at C-15 was established as R* on the basis of the cross-correlation peak between CH3-27 (δH 1.45) and both CH2-14 (δH 1.61/1.87) and CH2-16 (δH 2.46/2.54) observed in the ROESY spectrum ( Figure 4).

General Methods
Optical rotations were measured at room temperature in CHCl3 using a sodium lamp. Low and high resolution ESI-MS were recorded on a Micromass LTC Premier XE system mass spectrometer (Waters, Milford, CT, USA). NMR spectra were recorded on a Bruker AVANCE III 600 MHz (Bruker, Rheinstetten, Germany) equipped with a 5-mm TCI inverse detection cryo-probe (Bruker, Rheinstetten, Germany). 1 H and 13 C NMR chemical shifts were referenced either to the CDCl3 peaks at 300 K. COSY, TOCSY, multiplicity-edited HSQC, HMBC and ROESY experiments were performed using standard pulse sequences. HSQC, TOCSY and ROESY experiments were performed in the phase-sensitive mode (States-TPPI or Echo-AntiEcho for quadrature detection in F1) and used gradient coherence selection. 3 JH,H values were measured from 1D 1 H NMR. TOCSY experiment was recorded using DIPSI during the 50 ms of the isotropic mixing period. The ROESY experiment was recorded using a spin-lock generated by two 180° hard pulses during 400 ms in order to avoid TOCSY artefacts. The HMBC was optimized to detect long-range correlations using a value of 6 Hz. Prior to Fourier transformation, zero filling was performed to expand the data to at least double the number of acquired data points. HPLC separations were carried out with a preparative silica column (10 μ, 19 × 150 mm) (Waters, Milford, CT, USA) and a photodiode array detector (Waters, Milford, CT, USA). TLC plates were visualized by spraying with Dragendorff reagent and phosphomolybdic acid (10% in EtOH), followed by heating.

Biological Material
Colonies of Zoanthus sp. were collected by hand in the intertidal zone of Puntal del Hidalgo, Tenerife, Canary Islands (28°34′35.06ʺ N; 16°19′43.64ʺ W). A specimen was deposited at the Department of Animal Biology of University of La Laguna, Tenerife, and classified by Alberto Brito at the University of La Laguna (La Laguna, Tenerife).