New Oxidized Zoanthamines from a Canary Islands Zoanthus sp.

Abstract

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.

1. 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).

Figure 1. Structures of three new zoanthamine-type alkaloids isolated from Zoanthus sp.

Figure 1. Structures of three new zoanthamine-type alkaloids isolated from Zoanthus sp.

2. Results and Discussion

Colonies of Zoanthus sp. were collected by hand in the intertidal zone of Punta del Hidalgo, Tenerife, Canary Islands (28°34′35.06ʺ N; 16°19′43.64ʺ W) (Figure 2). Fresh specimens (120 g) were extracted with MeOH (3 × 2 L each time) at room temperature and the solvent removed in vacuo to give a brownish viscous oil (1.9 g). The extract was chromatographed on a Sephadex LH-20 column (3-cm internal diameter and 20-cm length) using MeOH and collecting 60 tubes of 10 mL. A second chromatographic step included a Lobar LiChroprep-RP18 column using MeOH:H₂O (7:3) as the eluent at 2 mL/min flux. Two fractions, obtained between 8–16 min and 17–25 min, were further purified by HPLC using a μ-Bondapack C-18 column and CH₃CN:MeOH:H₂O (2:1:1) or (2:1:2), respectively, as mobile phases at 2 mL/min. This procedure yielded 0.50 mg of 2-hydroxy-11-ketonorzoanthamide B (1), 0.45 mg of norzoanthamide B (2) and 0.30 mg of 15-hydroxynorzoanthamine (3), together with the previously known norzoanthamine, zoaramine and zoarenone [9].

Figure 2. Colonies of Zoanthus sp. (Photos courtesy of Alberto Brito).

Figure 2. Colonies of Zoanthus sp. (Photos courtesy of Alberto Brito).

2-Hydroxy-11-ketonorzoanthamide B (1) was isolated as an optically active powder [α]²⁵_D +9 (c 0.045, CHCl₃). Its molecular formula was established by ESI-HRMS as C₂₉H₃₅NO₈ (m/z 526.24340; calculated for 526.24354, C₂₉H₃₆NO₈, [M + H]⁺), requiring thirteen degrees of unsaturation. The ¹H NMR spectrum of 1 showed a number of signals characteristic norzoanthamine, including four methyl singlets at δ_H 1.01, 1.03, 1.34 and 2.01, one methyl doublet at δ_H 1.00, the AX system corresponding to CH₂-23 (δ_H 2.55 and 4.27, J = 20.6 Hz), as well as the distinctive olefinic methine proton at δ_H 5.90 (s). Analysis of the HSQC and HMBC experiments allowed us to classify the existent 29 carbon signals as five methyls, seven methylenes, five methines and twelve quaternary carbons, including two carboxylic and three carbonyl carbons at δ_C 168.1, 170.6, 195.8, 197.9 and 206.9 (Table 1).

Analysis of the COSY spectrum revealed the presence of three isolated ¹H-¹H spin-systems (I–III as showed in Figure 3). The first system (Fragment I) includes methyl CH₃-30 (δ_H 1.00, d, J = 6.0 Hz; δ_C 20.9), which is coupled with CH-4 (δ_H 2.05; δ_C 24.5) and this, in turn, with both CH₂-3 (δ_H 1.43/2.03; δ_C 38.0) and CH₂-5 (δ_H 1.15/2.05; δ_C40.3). Fragment II includes only methylenes CH₂-7 (δ_H 1.92/2.14; δ_C 28.7) and CH₂-8 (δ_H 1.70/1.86; δ_C 23.8). Finally, Fragment III was started from methylene CH₂-14 (δ_H 2.17/2.98; δ_C 32.7) that is coupled with CH-13 (δ_H 2.76; δ_C 46.7), in turn, connected with CH-18 (δ_H 2.70; δ_C 46.0) and CH₂-19 (δ_H 2.48/2.67; δ_C 42.7) (Figure 3). The significant number of quaternary carbons existent within this molecule made the HMBC experiment an essential tool to connect them with the previously described ¹H-¹H spin-systems. Thus, HMBC correlations of H₂-3 with carbon C-1 (δ_C 170.6) and C-2 (δ_C 100.6), together with the cross-peaks of H₂-5 and H₂-7 with C-6 (δ_C 90.2), allowed us to establish a seven-membered lactam ring. The chemical shifts of C-2 and C-6 indicated that both are ketal carbons and allowed us to build the azabicycle [3.2.1] system present in this new metabolite. In addition, the HMBC correlations observed from methines H-16 (δ_H 5.90, s) and H-21 (δ_H 2.89, s), as well as those from methyls CH₃-25, CH₃-27, CH₃-28 and CH₃-29 resulted in it being very valuable to build the polycyclic core of the molecule (Figure 3).

The relative configuration of 1 was deduced by analysis of the ROESY spectrum and ¹H-¹H coupling constants values together with a comparison of its chemical shift values with those of norzoanthamine. Particularly important were the dipolar correlations between H-21 (δ_H 2.89) and H-13 (δ_H 2.76), H₃-25 (δ_H 1.01) and H₃-29 (δ_H 1.03) together with those of H-18 (δ_H 2.70) and H₃-28 (δ_H 1.34), which allowed us to propose a 2R*, 4R*, 6S*, 9S*, 10S*, 12S*, 13R*, 18S*, 21R*, 22S*, 23S* relative configuration for 1 (Figure 3).

Figure 3. (Left) HMBC correlations for 2-hydroxy-11-ketonoroanthamide B (1) are indicated by arrows. ¹H-¹H spin systems I–III are showed in bold blue lines; (Right) significant dipolar correlations observed in the ROESY spectrum are indicated by a dotted blue line.

Figure 3. (Left) HMBC correlations for 2-hydroxy-11-ketonoroanthamide B (1) are indicated by arrows. ¹H-¹H spin systems I–III are showed in bold blue lines; (Right) significant dipolar correlations observed in the ROESY spectrum are indicated by a dotted blue line.

Table 1. NMR data (CDCl₃) for 2-hydroxy-11-ketonorzoanthamide B (1).

2-Hydroxy-11-ketonorzoanthamide B (1)
C	δ 13C	δ 1H	J (Hz)	C	δ 13C	δ1H	J (Hz)
1	170.6, C	-	-	16	124.5, CH	5.90	s
2	100.6, C	-	-	17	197.9, C	-	-
3	38.0, CH2	1.43 (β); 2.03 (α)	11.2; 12.0 m	18	46.0, CH	2.70	5.8; 11.3; 12.0
4	24.5, CH	2.05	m	19	42.7, CH2	2.48 (β); 2.67 (α)	11.3; 13.3; 5.8; 13.3
5	40.3, CH2	1.15 (β); 2.05 (α)	12.3; 14.8 m	20	206.9, C	-	-
6	90.2, C	-	-	21	60.8, CH	2.89	s
7	28.7, CH2	1.92 (α); 2.14 (β)	2.9; 4.0; 13.7; 3.1; 12.1; 13.7	22	38.1, C	-	-
8	23.8, CH2	1.70 (β); 1.86 (α)	3.1; 4.0; 13.4; 2.9; 12.1; 13.4	23	34.6, CH2	2.55 (β); 4.27 (α)	20.6; 20.6
9	43.9, C	-	-	24	168.1, C	-	-
10	95.7, C	-	-	25	20.9, CH3	1.01	s
11	195.8, C	-	-	27	24.4, CH3	2.01	s
12	54.9, C	-	-	28	14.1, CH3	1.34	s
13	46.7, CH	2.76	3.6; 10.3; 12.0	29	17.7, CH3	1.03	s
14	32.7, CH2	2.17 (β); 2.98 (α)	10.3; 18.2; 3.6; 18.2	30	20.9, CH3	1.00	6.0
15	162.2, C	-	-

Table 1. NMR data (CDCl₃) for 2-hydroxy-11-ketonorzoanthamide B (1).

2-Hydroxy-11-ketonorzoanthamide B (1)
C	δ 13C	δ 1H	J (Hz)	C	δ 13C	δ1H	J (Hz)
1	170.6, C	-	-	16	124.5, CH	5.90	s
2	100.6, C	-	-	17	197.9, C	-	-
3	38.0, CH2	1.43 (β); 2.03 (α)	11.2; 12.0 m	18	46.0, CH	2.70	5.8; 11.3; 12.0
4	24.5, CH	2.05	m	19	42.7, CH2	2.48 (β); 2.67 (α)	11.3; 13.3; 5.8; 13.3
5	40.3, CH2	1.15 (β); 2.05 (α)	12.3; 14.8 m	20	206.9, C	-	-
6	90.2, C	-	-	21	60.8, CH	2.89	s
7	28.7, CH2	1.92 (α); 2.14 (β)	2.9; 4.0; 13.7; 3.1; 12.1; 13.7	22	38.1, C	-	-
8	23.8, CH2	1.70 (β); 1.86 (α)	3.1; 4.0; 13.4; 2.9; 12.1; 13.4	23	34.6, CH2	2.55 (β); 4.27 (α)	20.6; 20.6
9	43.9, C	-	-	24	168.1, C	-	-
10	95.7, C	-	-	25	20.9, CH3	1.01	s
11	195.8, C	-	-	27	24.4, CH3	2.01	s
12	54.9, C	-	-	28	14.1, CH3	1.34	s
13	46.7, CH	2.76	3.6; 10.3; 12.0	29	17.7, CH3	1.03	s
14	32.7, CH2	2.17 (β); 2.98 (α)	10.3; 18.2; 3.6; 18.2	30	20.9, CH3	1.00	6.0
15	162.2, C	-	-

Table 2. NMR data (CDCl₃) for norzoanthamide B (2) and 15-hydroxynorzoanthamine (3).

C	Norzoanthamide B (2)			15-Hydroxynorzoanthamine (3)
	δ 13C	δ1H	J (Hz)	δ 13C	δ1H	J (Hz)
1	175.0, C	-	-	47.1, CH2	3.23; 3.25	m
2	77.0, CH	4.25	2.1; 3.6	74.2, CH	4.55	m
3	32.7, CH2	1.41 (β); 1.86 (α)	3.6; 12.0; 13.5; 2.1; 4.5; 13.5	38.8, CH2	1.46; 1.55	m
4	23.8, CH	2.22	4.2; 4.5; 11.1; 12.0	22.9, CH	2.27	6.0; 11.1
5	40.8, CH2	1.18 (β); 2.19 (α)	11.1; 13.2; 4.2; 13.2	44.4, CH2	1.08 (β); 2.08 (α)	11.1; 14.0; 6.0; 14.0
6	93.4, C	-	-	89.9, C	-	-
7	29.8, CH2	1.90 (α); 2.08 (β)	3.1; 4.2; 13.5; 5.6; 13.3; 13.5	29.9, CH2	1.76; 1.89	m
8	22.6, CH2	1.63; 1.65	m	23.7, CH2	1.55; 1.67	m
9	39.8, C	-	-	39.9, C	-	-
10	96.6, C	-	-	102.0, C	-	-
11	40.2, CH2	2.30; 3.52	15.2; 15.2	42.1, CH2	1.90 (β); 2.13 (α)	14.0; 14.0
12	39.4, C	-	-	35.8, C	-	-
13	53.3, CH	2.22	4.0; 12.0; 12.1	51.6, CH	2.35	m
14	31.6, CH2	2.31 (β); 2.44 (α)	12.1; 17.9; 4.0; 17.9	37.8, CH2	1.61 (β); 1.87 (α)	m 3.0; 13.4
15	160.8, C	-	-	73.7, C	-	-
16	125.3, CH	5.90	s	54.4, CH2	2.46; 2.54	13.8; 13.8
17	198.3, C	-	-	207.0, C	-	-
18	46.2, CH	2.70	5.5;11.3;12.0	50.4, CH	2.73	m
19	42.3, CH2	2.51 (β); 2.66 (α)	11.3; 13.7; 5.5; 13.7	42.3, CH2	2.34 (β); 2.73 (α)	m
20	208.7, C	-	-	209.6, C	-	-
21	58.5, CH	2.83	s	59.4, CH	2.91	s
22	36.1, C	-	-	36.4, C	-	-
23	36.1, CH2	2.43 (β); 3.70 (α)	20.6; 20.6	36.0, CH2	2.36; 3.62	20.4; 20.4
24	169.9, C	-	-	172.3, C	-	-
25	20.8, CH3	1.04	s	18.4, CH3	0.99	s
27	24.3, CH3	2.10	s	31.4, CH3	1.45	s
28	18.0, CH3	0.97	s	18.0, CH3	1.00	s
29	17.0, CH3	1.22	s	18.4, CH3	1.17	s
30	21.3, CH3	0.99	6.4	21.9, CH3	0.91	6.6

Table 2. NMR data (CDCl₃) for norzoanthamide B (2) and 15-hydroxynorzoanthamine (3).

C	Norzoanthamide B (2)			15-Hydroxynorzoanthamine (3)
	δ 13C	δ1H	J (Hz)	δ 13C	δ1H	J (Hz)
1	175.0, C	-	-	47.1, CH2	3.23; 3.25	m
2	77.0, CH	4.25	2.1; 3.6	74.2, CH	4.55	m
3	32.7, CH2	1.41 (β); 1.86 (α)	3.6; 12.0; 13.5; 2.1; 4.5; 13.5	38.8, CH2	1.46; 1.55	m
4	23.8, CH	2.22	4.2; 4.5; 11.1; 12.0	22.9, CH	2.27	6.0; 11.1
5	40.8, CH2	1.18 (β); 2.19 (α)	11.1; 13.2; 4.2; 13.2	44.4, CH2	1.08 (β); 2.08 (α)	11.1; 14.0; 6.0; 14.0
6	93.4, C	-	-	89.9, C	-	-
7	29.8, CH2	1.90 (α); 2.08 (β)	3.1; 4.2; 13.5; 5.6; 13.3; 13.5	29.9, CH2	1.76; 1.89	m
8	22.6, CH2	1.63; 1.65	m	23.7, CH2	1.55; 1.67	m
9	39.8, C	-	-	39.9, C	-	-
10	96.6, C	-	-	102.0, C	-	-
11	40.2, CH2	2.30; 3.52	15.2; 15.2	42.1, CH2	1.90 (β); 2.13 (α)	14.0; 14.0
12	39.4, C	-	-	35.8, C	-	-
13	53.3, CH	2.22	4.0; 12.0; 12.1	51.6, CH	2.35	m
14	31.6, CH2	2.31 (β); 2.44 (α)	12.1; 17.9; 4.0; 17.9	37.8, CH2	1.61 (β); 1.87 (α)	m 3.0; 13.4
15	160.8, C	-	-	73.7, C	-	-
16	125.3, CH	5.90	s	54.4, CH2	2.46; 2.54	13.8; 13.8
17	198.3, C	-	-	207.0, C	-	-
18	46.2, CH	2.70	5.5;11.3;12.0	50.4, CH	2.73	m
19	42.3, CH2	2.51 (β); 2.66 (α)	11.3; 13.7; 5.5; 13.7	42.3, CH2	2.34 (β); 2.73 (α)	m
20	208.7, C	-	-	209.6, C	-	-
21	58.5, CH	2.83	s	59.4, CH	2.91	s
22	36.1, C	-	-	36.4, C	-	-
23	36.1, CH2	2.43 (β); 3.70 (α)	20.6; 20.6	36.0, CH2	2.36; 3.62	20.4; 20.4
24	169.9, C	-	-	172.3, C	-	-
25	20.8, CH3	1.04	s	18.4, CH3	0.99	s
27	24.3, CH3	2.10	s	31.4, CH3	1.45	s
28	18.0, CH3	0.97	s	18.0, CH3	1.00	s
29	17.0, CH3	1.22	s	18.4, CH3	1.17	s
30	21.3, CH3	0.99	6.4	21.9, CH3	0.91	6.6

Norzoanthamide B (2) was isolated as an optically active powder, [α]²⁵_D +10 (c 0.03, CHCl₃), and its molecular formula was established as C₂₉H₃₇NO₆ on the basis of the result obtained from ESI-HRMS (m/z 496.26918; calculated for 496.26936, C₂₉H₃₈NO₆, [M + H]⁺). MS data were further supported by analysis of NMR data. Thus, five CH₃, eight CH₂ and six CH, as well as ten quaternary carbons (including two carboxylic, two carbonyl and two olefinic carbons) were identified (Table 2). Comparative analysis of the NMR data of 2 with those of norzoanthamine allowed us to conclude that C-1 is now the carboxylic signal at δ_C 174.5. Further analyses of COSY, HSQC and HMBC spectra confirmed our previous conclusion. Consequently, an azabicycle [] system was built starting from methine CH-2 (δ_H 4.25; δ_C 77.0) and sequentially to CH₂-3 (δ_H 1.41/1.86; δ_C 32.7), CH-4 (δ_H 2.22; δ_C 23.8) and, finally, with both CH₂-5 (δ_H 1.18/2.19; δ_C 40.7) and methyl CH₃-30 (δ_H 0.99; δ_C 21.3). In addition, the HMBC connectivity of proton H-2 with the signal at δ_C 174.5 supported the existence of a lactam within this ring. The relative configuration of norzoanthamide B (2) was proposed to be 2R*, 4S*, 6S*, 9S*, 10S*, 12S*, 13R*, 18S*, 21R*, 22S*, 23S* based on the correlations observed in the ROESY experiment and supported by a comparison with the chemical shift data reported for norzoanthamine.

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 δ_C 73.6 and δ_C 54.4 corresponding to a tertiary hydroxyl at C-15 and a new methylene at C-16 (δ_H 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 (δ_H 1.45) and both CH₂-14 (δ_H 1.61/1.87) and CH₂-16 (δ_H 2.46/2.54) observed in the ROESY spectrum (Figure 4).

Figure 4. Relevant dipolar correlations observed for 15-hydroxynorzoanthamine (3) (dotted blue line) and a selected fragment of the ROESY spectrum.

Figure 4. Relevant dipolar correlations observed for 15-hydroxynorzoanthamine (3) (dotted blue line) and a selected fragment of the ROESY spectrum.

3. Experimental Section

3.1. 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). ¹H and ¹³C 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. ³J_H,H values were measured from 1D ¹H 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.

3.2. 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).

3.3. Extraction and Chromatographic Separation

Fresh specimens (120 g) were extracted with MeOH (three times using 2 L each time) at room temperature and the solvent removed in vacuo to give a brownish viscous oil (1.9 g). The extract was chromatographed on a Sephadex LH-20 column (3 cm of internal diameter and 20 cm of length) (GE Healthcare, Buckinghamshire, UK) using MeOH, collecting 60 tubes of 10 mL, and subsequently with Lobar LiChroprep-RP18 (Merck, Darmstadt, Germany) with MeOH:H₂O (7:3) as the eluent at 2 mL/min flux. The fractions collected between 8 and 16 min were polled together and further purified by HPLC using a μ-Bondapack C-18 column (Waters, Milford, CT, USA), employing CH₃CN:MeOH:H₂O (2:1:1) as the mobile phase at 2 mL/min to obtain norzoanthamide B (2) (0.45 mg; 15–17 min). In addition, fractions polled from the Lobar LiChroprep-RP18 chromatographic step (Merck, Darmstadt, Germany) between 17 and 25 min were further purified by HPLC using a μ-Bondapack C-18 column, employing CH₃CN:MeOH:H₂O (2:1:2) as the eluent at 2 mL/min to yield 2-hydroxi-11-ketonorzoanthamide B (1) (0.5 mg; 15–18 min) and 15-hydroxynorzoanthamine (3) (0.3 mg; 22–24 min).

2-Hydroxy-11-ketonorzoanthamide B (1): white powder; [α]²⁵_D +9 (c 0.045, CHCl₃); UV λ_max (CHCl₃) 239.8 (ε 1,630) (log ε = 3.2); IRνmax (CHCl₃) 3384, 2927, 2857, 1742, 1723, 1669, 1383, 1220 and 1008 cm⁻¹; ESI-HRMS m/z 526.24340 (calculated for C₂₉H₃₆NO₈, 526.24354, [M + H]⁺); ¹H (600 MHz, CDCl₃) and ¹³C (150 MHz, CDCl₃) NMR chemical shifts are reported in Table 1.

Norzoanthamide B (2): white powder; [α]²⁵_D +10 (c 0.03, CHCl₃); UV λ_max (CHCl₃) 239.8 (ε 1630) (log ε = 3.2); IRνmax (CHCl₃) 3455, 2926, 2856, 1726, 1465, 1383 and 1246 cm⁻¹; ESI-HRMS m/z 496.26918 (calculated For C₂₉H₃₈NO₆, 496.26936, [M + H]⁺); ¹H (600 MHz, CDCl₃) and ¹³C (150 MHz, CDCl₃) NMR chemical shifts are reported in Table 2.

15-Hydroxynorzoanthamine (3): white powder; [α]²⁵_D +8 (c 0.03, CHCl₃); IRνmax (CHCl₃) 3414, 2924, 2854, 1715, 1665, 1377, 1240 and 1217 cm⁻¹; ESI-HRMS m/z 500.30050 (calculated for C₂₉H₄₂O₆N, 500.30066, [M + H]⁺); ¹H (600 MHz, CDCl₃) and ¹³C (150 MHz, CDCl₃) NMR chemical shifts are reported in Table 2.

4. Conclusions

The structures of three new congeners of norzoanthamine have been elucidated by means of MS and NMR spectroscopy. Despite, from our point of view, the new metabolites described here not providing new insights into the intriguing biogenetic origin of this class of molecules, their isolation does provide additional evidence of the currently accepted hypothesis regarding the biosynthesis of this family of natural products.