New Scalarane Sesterterpenoids from the Formosan Sponge Ircinia felix

Abstract

Five new scalarane sesterterpenoids, felixins A–E (1–5), were isolated from the Formosan sponge Ircinia felix. The structures of scalaranes 1–5 were elucidated on the basis of spectroscopic analysis. Cytotoxicity of scalaranes 1–5 against the proliferation of a limited panel of tumor cell lines was evaluated.

1. Introduction

Marine sponges belonging to the genus Ircinia (family Irciniidae, order Dictyoceratida, class Demospongiae, phylum Porifera) have been proven to be not only an important source of various interesting natural substances [1,2,3,4,5], but have also played an interesting role in marine ecology [6,7,8,9,10] and medicinal use [11,12]. In continuing research aimed at the discovery of new bioactive substances from marine organisms, an organic extract of the sponge identified as Ircinia felix (Duchassaing and Michelotti, 1864) (Figure 1) exhibited cytotoxicity toward MOLT-4 (human acute lymphoblastic leukemia) tumor cells (IC₅₀ < 6.25 μg/mL). We isolated five new scalarane sesterterpenoids, felixins A–E (1–5) from this organism (Figure 1). In this paper, we deal with the isolation, structure determination, and cytotoxicity of scalaranes 1–5.

Figure 1. The sponge Ircinia felix and the structures of felixins A–E (1–5) and 12-deacetyl-23-acetoxy-20-methyl-12-epi-deoxyscalarin (6) and scalarane 7.

Figure 1. The sponge Ircinia felix and the structures of felixins A–E (1–5) and 12-deacetyl-23-acetoxy-20-methyl-12-epi-deoxyscalarin (6) and scalarane 7.

2. Results and Discussion

Felixin A (1) was isolated as a white powder and the molecular formula for this compound was determined to be C₂₇H₄₂O₄ (seven unsaturations) using HRESIMS at m/z 453.29773 [M + Na]⁺ (calcd for C₂₇H₄₂O₄ + Na, 453.29753). Comparison of the ¹³C NMR and DEPT data with the molecular formula indicated there must be an exchangeable proton, which required the presence of a hydroxy group. The IR spectrum of 1 showed strong bands at 3480, 1731 and 1662 cm⁻¹, consistent with the presence of hydroxy, ester and α,β-unsaturated ketone groups. The ¹³C NMR and DEPT spectral data showed that this compound has 27 carbons (Table 1), including six methyls, nine sp³ methylenes (including an oxymethylene), four sp³ methines (including an oxymethine), four sp³ quaternary carbons, an sp² methine and three sp² quaternary carbons (including two carbonyls). Based on the ¹H and ¹³C NMR spectra (Table 1), 1 was found to possess an acetoxy group (δ_H 2.08, 3H × s; δ_C 170.2, C; 21.5, CH₃) and a ketonic carbonyl (δ_C 199.1, C-24). An additional unsaturated functionality was indicated by ¹³C resonances at δ_C 139.4 (CH-16) and 137.7 (C-17), suggesting the presence of a trisubstituted olefin. Thus, from the above data, three degrees of unsaturation were accounted for and 1 was identified as a tetracyclic sesterterpenoid analogue.

Table 1. ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data and ¹H–¹H COSY and HMBC correlations for scalarane 1.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	2.09 m; 0.52 ddd (12.8, 12.8, 3.2)	34.4, CH2	H2-2	C-3, -10, -22
2	1.51 m; 1.39 m	17.8, CH2	H2-1, H2-3	n. o. a
3	1.43 ddd (12.8, 4.0, 4.0)	41.7, CH2	H2-2	C-1, -4, -5, -19, -20
	1.17 ddd (12.8, 12.8, 4.8)
4		33.0, C
5	0.97 dd (12.0, 2.0)	56.9, CH	H2-6	C-3, -4, -6, -7, -9, -10, -20, -22
6	1.58 m; 1.45 m	18.3, CH2	H-5, H2-7	C-5, -8
7	1.81 ddd (12, 8, 3.2, 3.2); 1.05 m	41.9, CH2	H2-6	C-21
8		37.4, C
9	1.35 br d (13.2)	53.1, CH	H2-11	C-5, -7, -8, -10, -11, -12, -14, -21, -22
10		41.8, C
11	2.17 m; 1.96 m	25.2, CH2	H-9, H-12	C-9, -13
12	4.72 dd (3.6, 2.0)	77.1, CH	H2-11	C-9, -14
13		35.8, C
14	1.56 m	48.0, CH	H2-15	C-7, -8, -13, -15, -21, -23
15	2.34 m; 2.22 m	24.0, CH2	H-14, H-16	C-16, -17
16	6.86 m	139.4, CH	H2-15	C-14, -24
17		137.7, C
18	2.22 m; 1.92 m	35.1, CH2		C-13, -14, -16, -17, -23, -24
19	0.87 s	33.8, CH3		C-3, -4, -5, -20
20	0.77 s	21.9, CH3		C-3, -4, -5, -19
21	1.10 s	15.4, CH3		C-7, -8, -9, -14
22	4.03 d (11.6); 3.89 d (11.6)	63.0, CH2		C-1, -9, -10
23	0.87 s	19.6, CH3		C-12, -13, -14
24		199.1, C
25	2.28 s	25.2, CH3		C-17, -24
12-OAc		170.2, C
	2.08 s	21.5, CH3		Acetate carbonyl

^a n. o. = not observed.

Table 1. ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data and ¹H–¹H COSY and HMBC correlations for scalarane 1.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	2.09 m; 0.52 ddd (12.8, 12.8, 3.2)	34.4, CH2	H2-2	C-3, -10, -22
2	1.51 m; 1.39 m	17.8, CH2	H2-1, H2-3	n. o. a
3	1.43 ddd (12.8, 4.0, 4.0)	41.7, CH2	H2-2	C-1, -4, -5, -19, -20
	1.17 ddd (12.8, 12.8, 4.8)
4		33.0, C
5	0.97 dd (12.0, 2.0)	56.9, CH	H2-6	C-3, -4, -6, -7, -9, -10, -20, -22
6	1.58 m; 1.45 m	18.3, CH2	H-5, H2-7	C-5, -8
7	1.81 ddd (12, 8, 3.2, 3.2); 1.05 m	41.9, CH2	H2-6	C-21
8		37.4, C
9	1.35 br d (13.2)	53.1, CH	H2-11	C-5, -7, -8, -10, -11, -12, -14, -21, -22
10		41.8, C
11	2.17 m; 1.96 m	25.2, CH2	H-9, H-12	C-9, -13
12	4.72 dd (3.6, 2.0)	77.1, CH	H2-11	C-9, -14
13		35.8, C
14	1.56 m	48.0, CH	H2-15	C-7, -8, -13, -15, -21, -23
15	2.34 m; 2.22 m	24.0, CH2	H-14, H-16	C-16, -17
16	6.86 m	139.4, CH	H2-15	C-14, -24
17		137.7, C
18	2.22 m; 1.92 m	35.1, CH2		C-13, -14, -16, -17, -23, -24
19	0.87 s	33.8, CH3		C-3, -4, -5, -20
20	0.77 s	21.9, CH3		C-3, -4, -5, -19
21	1.10 s	15.4, CH3		C-7, -8, -9, -14
22	4.03 d (11.6); 3.89 d (11.6)	63.0, CH2		C-1, -9, -10
23	0.87 s	19.6, CH3		C-12, -13, -14
24		199.1, C
25	2.28 s	25.2, CH3		C-17, -24
12-OAc		170.2, C
	2.08 s	21.5, CH3		Acetate carbonyl

^a n. o. = not observed.

From the ¹H–¹H COSY spectrum of 1 (Table 1), it was possible to establish the separate system that map out the proton sequences from H₂-1/H₂-2/H₂-3, H-5/H₂-6/H₂-7, H-9/H₂-11/H-12 and H-14/H₂-15/H-16. These data, together with the key HMBC correlations between protons and quaternary carbons (Table 1), such as H₂-3, H-5, H₃-19, H₃-20/C-4; H₂-6, H-9, H-14, H₃-21/C-8; H₂-1, H-5, H-9, H₂-22/C-10; H₂-11, H-14, H₂-18, H₃-23/C-13; H₂-15, H₂-18, H₃-25/C-17; and H-16, H₂-18, H₃-25/C-24, established the carbon skeleton of 1 as a 24-homo-25-norscalarane derivative [13]. The oxymethylene unit at δ_C 63.0 was correlated to the methylene protons at δ_H 4.03 and 3.89 in the HMQC spectrum. The methylene signals were ²J-correlated with C-10 (δ_C 41.8) and ³J-correlated with both C-1 (δ_C 34.4) and C-9 (δ_C 53.1), proving the attachment to a hydroxymethyl group at C-10 (Table 1). Thus, the remaining acetoxy group was positioned at C-12, an oxymethine (δ_H 4.72, δ_C 77.1) as indicated by analysis of the ¹H–¹H COSY correlations and characteristic NMR signals, although no HMBC correlation was observed between H-12 and the acetate carbonyl.

The relative stereochemistry of 1 was elucidated from the NOE interactions observed in an NOESY experiment (Figure 2). As per convention, when analyzing the stereochemistry of scalarane sesterterpenoids, H-5 and hydroxymethyl at C-10 were assigned to the α and β face, anchoring the stereochemical analysis because no correlation was found between H-5 and H₂-22. In the NOESY experiment of 1, H-9 showed correlations with H-5 and H-14 but not with H₃-21 and H₂-22. Thus, both H-9 and H-14 must also be on α face whilst Me-21 and the hydroxymethyl at C-10 must be located on the β face. Moreover, the correlations of H₃-23/H₃-21 and H₃-23/H-12, indicated the β-orientation of Me-23 and H-12 attaching at C-13 and C-12, respectively. The NOESY spectrum showed a correlation of H-16 with H₃-25, revealing the E geometry of the C-16/17 double bond. Based on the above findings, the structure, including the relative configuration of 1 was established unambiguously.

Figure 2. Selective NOESY correlations of 1.

Figure 2. Selective NOESY correlations of 1.

The HRESIMS of 2 (felixin B) exhibited a pseudomolecular ion peak at m/z 467.27707 [M + Na]⁺, with the molecular formula C₂₇H₄₀O₅ (calcd C₂₇H₄₀O₅ + Na, 467.27680), implying eight degrees of unsaturation. The IR absorptions of 2 showed the presence of hydroxy (3501 cm⁻¹), ester carbonyl (1733 cm⁻¹) and α,β-unsaturated ketone (1679 cm⁻¹) functionalities. The ¹³C NMR and DEPT spectrum of 2 exhibited for all 27 carbons: two ketones (δ_C 197.9, C-24; 197.7, C-16), an ester carbonyl (δ_C 170.2, acetate carbonyl), a trisubstituted olefin (δ_C 163.9, CH-18; 136.6, C-17), an oxymethylene (δ_C 62.7, CH₂-22), an oxymethine (δ_C 76.3, CH-12), six methyls, seven methylenes, three methines and four quaternary carbons. Both the ¹³C and ¹H NMR data for the rings A–C portions were essentially same as those of 1. It also contained an acetoxy (δ_H 2.05), an acetyl (methyl ketone, δ_H 2.42) and a hydroxymethyl (δ_H 4.04 and 3.87) groups as in 1. Analysis of ¹H–¹H COSY and HMBC data (Table 2) revealed the planar structure. The same stereochemistry was shown by coupling constant and NOE data (Figure 3). The NOESY spectrum showed correlations of H-18 with H-12 and H₃-23, revealing the Z geometry of the C-17/18 double bond.

Table 2. ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data and ¹H–¹H COSY and HMBC correlations for scalarane 2.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	2.11, m; 0.53 ddd (13.2, 13.2, 4.0)	34.2, CH2	H2-2	C-2, -3, -10, -22
2	1.58–1.42 m	18.3, CH2	H2-1, H2-3	n. o. a
3	1.42 m; 1.19 m	41.6, CH2	H2-2	C-4, -20
4		33.0, C
5	0.96 m	57.0, CH	H2-6	C-3, -4, -6, -7, -9, -10, -20, -22
6	1.54 m; 1.42 m	17.7, CH2	H-5, H2-7	n. o.
7	1.78 ddd (12.8, 3.2, 3.2); 1.05 m	41.0, CH2	H2-6	C-21
8		37.2, C
9	1.31 br d (13.2)	53.1, CH	H2-11	C-5, -7, -8, -10, -11, -21, -22
10		41.7, C
11	2.29 ddd (13.6, 13.6, 2.4); 2.05 m	24.9, CH2	H-9, H-12	n. o.
12	4.97 dd (2.8, 2.8)	76.3, CH	H2-11	n. o.
13		41.4, C
14	2.11 m	48.9, CH	H2-15	C-8, -13, -21, -23
15	2.57–2.40 m	35.0, CH2	H-14	C-13, -14, -16
16		197.7, C
17		136.6, C
18	7.30 s	163.9, CH		C-12, -14, -17, -24
19	0.87 s	33.8, CH3		C-3, -4, -5, -20
20	0.76 s	21.8, CH3		C-3, -4, -5, -19
21	1.12 s	15.7, CH3		C-7, -8, -9, -14
22	4.04 d (12.0); 3.87 d (12.0)	62.7, CH2		C-1, -9, -10
23	1.17 s	18.4, CH3		C-12, -13, -14, -18
24		197.9, C
25	2.42 s	30.6, CH3		C-17, -24
12-OAc		170.2, C
	2.05 s	21.2, CH3		Acetate carbonyl

^a n. o. = not observed.

Table 2. ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data and ¹H–¹H COSY and HMBC correlations for scalarane 2.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	2.11, m; 0.53 ddd (13.2, 13.2, 4.0)	34.2, CH2	H2-2	C-2, -3, -10, -22
2	1.58–1.42 m	18.3, CH2	H2-1, H2-3	n. o. a
3	1.42 m; 1.19 m	41.6, CH2	H2-2	C-4, -20
4		33.0, C
5	0.96 m	57.0, CH	H2-6	C-3, -4, -6, -7, -9, -10, -20, -22
6	1.54 m; 1.42 m	17.7, CH2	H-5, H2-7	n. o.
7	1.78 ddd (12.8, 3.2, 3.2); 1.05 m	41.0, CH2	H2-6	C-21
8		37.2, C
9	1.31 br d (13.2)	53.1, CH	H2-11	C-5, -7, -8, -10, -11, -21, -22
10		41.7, C
11	2.29 ddd (13.6, 13.6, 2.4); 2.05 m	24.9, CH2	H-9, H-12	n. o.
12	4.97 dd (2.8, 2.8)	76.3, CH	H2-11	n. o.
13		41.4, C
14	2.11 m	48.9, CH	H2-15	C-8, -13, -21, -23
15	2.57–2.40 m	35.0, CH2	H-14	C-13, -14, -16
16		197.7, C
17		136.6, C
18	7.30 s	163.9, CH		C-12, -14, -17, -24
19	0.87 s	33.8, CH3		C-3, -4, -5, -20
20	0.76 s	21.8, CH3		C-3, -4, -5, -19
21	1.12 s	15.7, CH3		C-7, -8, -9, -14
22	4.04 d (12.0); 3.87 d (12.0)	62.7, CH2		C-1, -9, -10
23	1.17 s	18.4, CH3		C-12, -13, -14, -18
24		197.9, C
25	2.42 s	30.6, CH3		C-17, -24
12-OAc		170.2, C
	2.05 s	21.2, CH3		Acetate carbonyl

^a n. o. = not observed.

Figure 3. Selective NOESY correlations of 2.

Figure 3. Selective NOESY correlations of 2.

Felixin C (3) was isolated as a white solid. Its HRESIMS (m/z 469.29290 [M + Na]⁺) and NMR data (Table 3) established a molecular formula of C₂₇H₄₂O₅ (calcd C₂₇H₄₂O₅ + Na, 469.29245). The IR spectrum of 3 revealed the presence of hydroxy (ν_max 3480 cm⁻¹) ester (ν_max 1731 cm⁻¹) and α,β-unsaturated ketone (ν_max 1662 cm⁻¹) groups. By comparison of NMR data of 3 with those of 2 (Table 2 and Table 3), it was found that the ketone at C-16 in 2 (δ_C 197.7) was replaced by a hydroxy group (δ_C 63.3, δ_H 4.55, 1H, J = 3.6 Hz) in 3. Analyses of ¹H–¹H COSY and HMBC correlations established the planar structure of 3 (Table 3) as shown in Figure 1, which showed the C-16 positioning of the hydroxy group. Careful analysis of the NOESY spectrum of 3, in comparison with that of 2, allowed determination of the relative stereochemistry of A–C rings of felixin C (3) as shown in Figure 4. Moreover, the splitting pattern and J-value of proton at C-16 in 3, combined with the interactions observed between H-16 and both of the C-15 methylene protons revealed the α-orientation of the 16-OH. Furthermore, the correlations between the olefinic proton H-18/H₃-23 and H-18/H-12 assigned the E-configuration of the double bond between C-17 and C-18.

Table 3. ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data and ¹H–¹H COSY and HMBC correlations for scalarane 3.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	2.08 m; 0.57 ddd (12.8, 12.8, 3.2)	34.1, CH2	H2-2	n. o. a
2	1.54 m; 1.39 m	17.8, CH2	H2-1, H2-3	n. o.
3	1.42 m; 1.16 m	41.7, CH2	H2-2	C-20
4		33.0, C
5	1.02 dd (12.8, 2.4)	56.8, CH	H2-6	C-4, -20, -22
6	1.54 m; 1.47 m	18.4, CH2	H-5, H2-7	n. o.
7	1.88 m; 1.11 m	41.3, CH2	H2-6	C-8, -21
8		36.8, C
9	1.45 m	53.5, CH	H2-11	C-10, -11, -21, -22
10		41.8, C
11	1.96–1.81 m	25.3, CH2	H-9, H-12	C-8, -10, -13
12	4.97 dd (2.8, 2.8)	76.5, CH	H2-11	n. o.
13		41.4, C
14	1.88 m	44.0, CH	H2-15	C-8, -13, -15, -16, -21, -23
15	1.88 m; 1.64 dd (14.0, 4.8)	25.3, CH2	H-14, H-16	C-8, -13, -16, -17
16	4.55 d (3.6)	63.3, CH	H2-15	C-14, -17, -18
17		138.2, C
18	6.59 s	152.2, CH		C-12, -13, -14, -16, -24
19	0.85 s	33.8, CH3		C-3, -4, -5, -20
20	0.76 s	21.8, CH3		C-3, -4, -5, -19
21	1.06 s	16.4, CH3		C-7, -8, -9, -14
22	4.04 d (12.0); 3.90 d (12.0)	62.8, CH2		C-1, -9, -10
23	1.06 s	19.5, CH3		C-12, -13, -14, -18
24		201.4, C
25	2.24 s	25.4, CH3		C-17, -24
12-OAc		170.9, C
	2.04 s	21.4, CH3		Acetate carbonyl

^a n. o. = not observed.

Table 3. ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data and ¹H–¹H COSY and HMBC correlations for scalarane 3.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	2.08 m; 0.57 ddd (12.8, 12.8, 3.2)	34.1, CH2	H2-2	n. o. a
2	1.54 m; 1.39 m	17.8, CH2	H2-1, H2-3	n. o.
3	1.42 m; 1.16 m	41.7, CH2	H2-2	C-20
4		33.0, C
5	1.02 dd (12.8, 2.4)	56.8, CH	H2-6	C-4, -20, -22
6	1.54 m; 1.47 m	18.4, CH2	H-5, H2-7	n. o.
7	1.88 m; 1.11 m	41.3, CH2	H2-6	C-8, -21
8		36.8, C
9	1.45 m	53.5, CH	H2-11	C-10, -11, -21, -22
10		41.8, C
11	1.96–1.81 m	25.3, CH2	H-9, H-12	C-8, -10, -13
12	4.97 dd (2.8, 2.8)	76.5, CH	H2-11	n. o.
13		41.4, C
14	1.88 m	44.0, CH	H2-15	C-8, -13, -15, -16, -21, -23
15	1.88 m; 1.64 dd (14.0, 4.8)	25.3, CH2	H-14, H-16	C-8, -13, -16, -17
16	4.55 d (3.6)	63.3, CH	H2-15	C-14, -17, -18
17		138.2, C
18	6.59 s	152.2, CH		C-12, -13, -14, -16, -24
19	0.85 s	33.8, CH3		C-3, -4, -5, -20
20	0.76 s	21.8, CH3		C-3, -4, -5, -19
21	1.06 s	16.4, CH3		C-7, -8, -9, -14
22	4.04 d (12.0); 3.90 d (12.0)	62.8, CH2		C-1, -9, -10
23	1.06 s	19.5, CH3		C-12, -13, -14, -18
24		201.4, C
25	2.24 s	25.4, CH3		C-17, -24
12-OAc		170.9, C
	2.04 s	21.4, CH3		Acetate carbonyl

^a n. o. = not observed.

Figure 4. Selective NOESY correlations of 3.

Figure 4. Selective NOESY correlations of 3.

Moreover, two deoxoscalarin-like metabolites [13], felixins D (4) and E (5) were isolated from I. felix in this study. Felixin D (4) was isolated as white powder and its molecular formula was established as C₃₀H₄₆O₆ from the HRESIMS at m/z 525.31849 (calcd C₃₀H₄₆O₆ + Na, 525.31866). Eight degrees of unsaturation implied by the molecular formula were ascribed to five rings, a trisubstituted double bond (δ_C 141.2, C-17; 114.4, CH-16) and two ester carbonyl groups (δ_C 171.0, 170.9, 2 × C). The ¹H NMR spectrum showed seven methyls (δ_H 2.10, 2.05, 2 × 3H, s, acetate methyls; 1.26, 3H, d, J = 6.0 Hz, H₃-26; 0.98, 3H, s, H₃-23; 0.89, 3H, s, H₃-21; 0.83, 3H, s, H₃-22; 0.78, 3H, s, H₃-25); an acetoxymethylene (δ_H 4.59, 1H, d, J = 12.0 Hz; 4.16, 1H, d, J = 12.0 Hz, H₂-24); three oxymethines (δ_H 5.21, 1H, d, J = 3.2 Hz, H-19; 4.91, 1H, dd, J = 3.2, 2.4 Hz, H-12; 4.62, 1H, br s, H-20); and an olefinic proton (δ_H 5.35, 1H, br s, H-16). The ¹³C NMR and DEPT spectra exhibited 30 signals, including seven methyls, eight sp³ methylenes (including an oxymethylene), seven sp³ methines (including three oxymethines), an sp² methine, four sp³ quaternary carbons and three sp² quaternary carbons (including two ester carbonyls). A typical sesterterpenoid carbons system bearing an acetoxymethylene and four methyl groups along rings A–D could be established by the HMBC correlations from the acetoxymethylene (CH₂-24) and four methyl groups (Me-21, 22, 23 and 25) to the associated carbons and a deoxoscalarin skeleton could be obtained on the basis of further HMBC and ¹H–¹H COSY correlations (Table 4). The ¹H–¹H COSY correlations between H-18/H-19 and H-20/H₃-26 and the HMBC correlations from H-19/C-20 and H₃-26/C-17, -20, allowed the establishment of the hemiacetal ring E.

The relative stereochemistry of 4 was elucidated from the interactions observed in an NOESY experiment (Figure 5). In the NOESY experiment of 1, H-9 showed correlations with H-5 and H-14, but not with H₃-23 and H₂-24 at C-10. Thus, both H-5 and H-14 must be on α face whilst Me-23 and the acetoxymethylene at C-10 must be located on the β face. The correlations of H₃-25 with H₃-23 and H-12 indicated the β-orientation of Me-25 and H-12. H-18 correlated with H-14, but not with H-19, and H-19 correlated with H-12 and H₃-25, assuming that H-18 and H-19 were α- and β-oriented, respectively. H-16 showed correlations with H-20 and H₃-26, but not with H-18, revealing the E geometry of the C-16/17 double bond. It was found that the structure of 4 was similar with that of a known scalarane, 12-deacetyl-23-acetoxy-20-methyl-12-epi-deoxo- scalarin (6) [14], excepting the β-hydroxy group at C-12 in 6 was replaced by an α-acetoxy group in 4. The relative configuration of C-20 chiral carbon in 4 was elucidated by comparison the NMR data of CH-20 (δ_H 4.62, 1H, m; δ_C 74.0) of 4 with those of 6 (δ_H 4.67, 1H, m; δ_C 74.5), indicating H-20 in 4 was α-oriented.

Table 4. ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data and ¹H–¹H COSY and HMBC correlations for scalarane 4.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	1.98 dd (13.2, 2.4)	34.7, CH2	H2-2	C-5, -9, -24
	0.57 ddd (13.2, 13.2, 2.4)
2	1.56 m; 1.43 m	18.2, CH2	H2-1, H2-3	C-4
3	1.46 m; 1.15 m	41.6, CH2	H2-2	C-2, -4, -21, -22
4		33.0, C
5	1.04 dd (12.8, 2.0)	56.8, CH	H2-6	C-3, -4, -6, -7, -9, -10, -21, -22, -24
6	1.56 m; 1.38 dd (13.6, 3.2)	17.9, CH2	H-5, H2-7	n. o. a
7	1.79 ddd (12.8, 3.2, 3.2); 1.12 m	41.7, CH2	H2-6	C-5, -8, -9, -14, -23
8		37.7, C
9	1.46 m	52.9, CH	H2-11	C-1, -5, -8, -10, -11, -12, -14, -23, -24
10		40.1, C
11	2.05–1.89 m	25.1, CH2	H-9, H-12	C-8
12	4.91 dd (3.2, 2.4)	74.6, CH	H2-11	C-9, -14, acetate carbonyl
13		36.9, C
14	1.64 dd (11.2, 6.4)	50.4, CH	H2-15	C-7, -8, -9, -13, -15, -18, -25
15	2.16 m; 1.97 m	22.9, CH2	H-14, H-16	C-8
16	5.35 br s	114.4, CH	H2-15	n. o.
17		141.2, C
18	2.82 br s	54.7, CH	H-19	n. o.
19	5.21 d (3.2)	96.7, CH	H-18	C-20
20	4.62 m	74.0, CH	H3-26	n. o.
21	0.89 s	33.7, CH3		C-3, -4, -5, -22
22	0.83 s	21.9, CH3		C-3, -5, -21
23	0.98 s	15.4, CH3		C-7, -9, -14
24	4.59 d (12.0); 4.16 d (12.0)	64.9, CH2		C-1, -9, -10, acetate carbonyl
25	0.78 s	14.7, CH3		C-12, -14, -18
26	1.26 d (6.0)	17.6, CH3	H-20	C-17, -20
12-OAc		170.9, C
	2.10 s	21.5, CH3		Acetate carbonyl
23-OAc		171.0, C
	2.05 s	21.2, CH3		Acetate carbonyl

^a n. o. = not observed.

Table 4. ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data and ¹H–¹H COSY and HMBC correlations for scalarane 4.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	1.98 dd (13.2, 2.4)	34.7, CH2	H2-2	C-5, -9, -24
	0.57 ddd (13.2, 13.2, 2.4)
2	1.56 m; 1.43 m	18.2, CH2	H2-1, H2-3	C-4
3	1.46 m; 1.15 m	41.6, CH2	H2-2	C-2, -4, -21, -22
4		33.0, C
5	1.04 dd (12.8, 2.0)	56.8, CH	H2-6	C-3, -4, -6, -7, -9, -10, -21, -22, -24
6	1.56 m; 1.38 dd (13.6, 3.2)	17.9, CH2	H-5, H2-7	n. o. a
7	1.79 ddd (12.8, 3.2, 3.2); 1.12 m	41.7, CH2	H2-6	C-5, -8, -9, -14, -23
8		37.7, C
9	1.46 m	52.9, CH	H2-11	C-1, -5, -8, -10, -11, -12, -14, -23, -24
10		40.1, C
11	2.05–1.89 m	25.1, CH2	H-9, H-12	C-8
12	4.91 dd (3.2, 2.4)	74.6, CH	H2-11	C-9, -14, acetate carbonyl
13		36.9, C
14	1.64 dd (11.2, 6.4)	50.4, CH	H2-15	C-7, -8, -9, -13, -15, -18, -25
15	2.16 m; 1.97 m	22.9, CH2	H-14, H-16	C-8
16	5.35 br s	114.4, CH	H2-15	n. o.
17		141.2, C
18	2.82 br s	54.7, CH	H-19	n. o.
19	5.21 d (3.2)	96.7, CH	H-18	C-20
20	4.62 m	74.0, CH	H3-26	n. o.
21	0.89 s	33.7, CH3		C-3, -4, -5, -22
22	0.83 s	21.9, CH3		C-3, -5, -21
23	0.98 s	15.4, CH3		C-7, -9, -14
24	4.59 d (12.0); 4.16 d (12.0)	64.9, CH2		C-1, -9, -10, acetate carbonyl
25	0.78 s	14.7, CH3		C-12, -14, -18
26	1.26 d (6.0)	17.6, CH3	H-20	C-17, -20
12-OAc		170.9, C
	2.10 s	21.5, CH3		Acetate carbonyl
23-OAc		171.0, C
	2.05 s	21.2, CH3		Acetate carbonyl

^a n. o. = not observed.

Figure 5. Selective NOESY correlations of 4.

Figure 5. Selective NOESY correlations of 4.

The HRESIMS of 5 (felixin E) exhibited a pseudomolecular ion peak at m/z 441.29739 [M + Na]⁺, with the molecular formula C₂₆H₄₂O₄ (calcd C₂₆H₄₂O₄ + Na, 441.29753), implying six degrees of unsaturation. The IR absorptions of 5 showed the presence of hydroxy (3421 cm⁻¹) and ketone (1701 cm⁻¹) functionalities. The ¹³C NMR and DEPT spectrum of 5 exhibited for all 26 carbons: a ketone (δ_C 219.0, C-12), a hemiacetal (δ_C 97.1, CH-19), two oxymethines (δ_C 78.1, CH-20; 72.0, CH-16), six methyls, seven methylenes, five methines, and four quaternary carbons (Table 5). The NMR data of 5 were similar with those of 4, except for the acetoxymethylene group at C-10 and acetoxy group at C-12 in 4 were replaced by a methyl and a ketone group in 5, respectively. The C-16/17 trisubstituted olefin in 4 was replaced by a hydroxy group at C-16 in 5. The stereochemical configuration was identical to that of other scalarane sesterterpenes based on NOESY cross-peaks at H-5/H-9, H-9/H-14, H-14/H-16, H-14/H-18, H-16/H-18, H-16/H-20, H-19/H₃-25, H₃-22/H₃-24, H₃-23/H₃-24 and H₃-23/H₃-25 (Figure 6). Furthermore, it was found that the structure of 5 was similar with that of known scalarane 7 [15], excepting the 12α-acetoxy group in 7 was replaced by a ketone group in 5.

Table 5. ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data and ¹H–¹H COSY and HMBC correlations for scalarane 5.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	1.56 m; 0.76 m	39.3 CH2	H2-2	C-5
2	1.64–1.34 m	18.3, CH2	H2-1, H2-3	n. o. a
3	1.80 m; 1.38 m	41.6, CH2	H2-2	n. o.
4		33.3, C
5	0.94 m	56.5, CH	H2-6	C-4
6	1.64–1.34 m	18.1, CH2	H-5, H2-7	n. o.
7	1.81 m; 0.94 dd (13.2, 4.0)	41.7, CH2	H2-6	C-5
8		37.8, C
9	1.19 m	61.4, CH	H2-11	C-8, -12, -14, -23
10		38.2, C
11	2.70 dd (14.0, 13.2); 2.32 dd (13.2, 2.4)	35.3, CH2	H-9	C-8, -9, -12
12		219.0, C
13		51.2, C
14	1.21 m	59.2, CH	H2-15	C-12, -18
15	1.95 ddd (12.8, 4.4, 2.4); 1.41 m	30.8, CH2	H-14, H-16	C-13
16	3.55 ddd (10.4, 10.4, 4.8)	72.0, CH	H2-15, H-17	n. o.
17	1.62 m	53.0, CH	H-16, H-18, H-20	n. o.
18	1.86 m	59.2, CH	H-17, H-19	C-13, -16, -19, -25
19	5.31 d (6.0)	97.1, CH	H-18	C-18, -20
20	4.10 qd (6.0, 3.2)	78.1, CH	H-17, H3-26	n. o.
21	0.85 s	33.2, CH3		C-3, -4, -5, -22
22	0.82 s	21.3, CH3		C-4, -21
23	1.06 s	16.9, CH3		C-7, -8, -9, -14
24	0.87 s	15.6, CH3		C-10
25	1.24 s	15.3, CH3		C-12, -13, -14, -18
26	1.38 d (6.0)	20.5, CH3	H-20	C-17, -20

^a n. o. = not observed.

Table 5. ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data and ¹H–¹H COSY and HMBC correlations for scalarane 5.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	1.56 m; 0.76 m	39.3 CH2	H2-2	C-5
2	1.64–1.34 m	18.3, CH2	H2-1, H2-3	n. o. a
3	1.80 m; 1.38 m	41.6, CH2	H2-2	n. o.
4		33.3, C
5	0.94 m	56.5, CH	H2-6	C-4
6	1.64–1.34 m	18.1, CH2	H-5, H2-7	n. o.
7	1.81 m; 0.94 dd (13.2, 4.0)	41.7, CH2	H2-6	C-5
8		37.8, C
9	1.19 m	61.4, CH	H2-11	C-8, -12, -14, -23
10		38.2, C
11	2.70 dd (14.0, 13.2); 2.32 dd (13.2, 2.4)	35.3, CH2	H-9	C-8, -9, -12
12		219.0, C
13		51.2, C
14	1.21 m	59.2, CH	H2-15	C-12, -18
15	1.95 ddd (12.8, 4.4, 2.4); 1.41 m	30.8, CH2	H-14, H-16	C-13
16	3.55 ddd (10.4, 10.4, 4.8)	72.0, CH	H2-15, H-17	n. o.
17	1.62 m	53.0, CH	H-16, H-18, H-20	n. o.
18	1.86 m	59.2, CH	H-17, H-19	C-13, -16, -19, -25
19	5.31 d (6.0)	97.1, CH	H-18	C-18, -20
20	4.10 qd (6.0, 3.2)	78.1, CH	H-17, H3-26	n. o.
21	0.85 s	33.2, CH3		C-3, -4, -5, -22
22	0.82 s	21.3, CH3		C-4, -21
23	1.06 s	16.9, CH3		C-7, -8, -9, -14
24	0.87 s	15.6, CH3		C-10
25	1.24 s	15.3, CH3		C-12, -13, -14, -18
26	1.38 d (6.0)	20.5, CH3	H-20	C-17, -20

^a n. o. = not observed.

Figure 6. Selective NOESY correlations of 5.

Figure 6. Selective NOESY correlations of 5.

The cytotoxicity of compounds 1–5 against MOLT-4 (human acute lymphoblastic leukemia), SUP-T1 (human T-cell lymphoblastic lymphoma), DLD-1 (human colorectal adenocarcinoma), LNCaP (human prostatic carcinoma), T-47D (human ductal carcinoma) and MCF7 (human breast adenocarcinoma) tumor cells are shown in Table 6. The results showed that compounds 1–5 were found to exhibit cytotoxicity against DLD-1 tumor cells. By comparison with the structures and cytotoxicity of scalaranes 2 and 3, implying that the presence of 16-ketone would enhance the activity.

Table 6. Cytotoxic data of scalarane sesterterpenoids 1–5.

Compounds	Cell Lines IC50 (μM)
	MOLT-4	SUP-T1	DLD-1	LNCaP	T-47D	MCF7
1	NA b	NA	10.9	24.3	NA	NA
2	14.9	27.1	8.5	NA	32.2	23.0
3	18.5	NA	15.0	NA	NA	NA
4	12.8	31.6	7.9	21.5	20.2	NA
5	14.0	31.1	7.2	NA	22.7	24.3
Doxorubicin a	0.02	0.09	0.64	0.02	0.09	0.79

^a Doxorubicin was used as a positive control; ^b NA = not active at 20 μg/mL for 72 h.

Table 6. Cytotoxic data of scalarane sesterterpenoids 1–5.

Compounds	Cell Lines IC50 (μM)
	MOLT-4	SUP-T1	DLD-1	LNCaP	T-47D	MCF7
1	NA b	NA	10.9	24.3	NA	NA
2	14.9	27.1	8.5	NA	32.2	23.0
3	18.5	NA	15.0	NA	NA	NA
4	12.8	31.6	7.9	21.5	20.2	NA
5	14.0	31.1	7.2	NA	22.7	24.3
Doxorubicin a	0.02	0.09	0.64	0.02	0.09	0.79

^a Doxorubicin was used as a positive control; ^b NA = not active at 20 μg/mL for 72 h.

3. Experimental Section

3.1. General Experimental Procedures

Optical rotation values were measured with a Jasco P-1010 digital polarimeter (Japan Spectroscopic Corporation, Tokyo, Japan). IR spectra were obtained on a Jasco FT-IR 4100 spectrophotometer (Japan Spectroscopic Corporation, Tokyo, Japan); absorptions are reported in cm⁻¹. NMR spectra were recorded on a Varian Mercury Plus 400 NMR spectrometer (Varian Inc., Palo Alto, CA, USA) using the residual solvent (CDCl₃, δ_H 7.26 ppm for ¹H NMR and δ_C 77.1 ppm for ¹³C NMR) as the internal standard for ¹H NMR and CDCl₃ (δ_C 77.1 ppm) for ¹³C NMR. Coupling constants (J) are given in Hz. ESIMS and HRESIMS were recorded using a Bruker 7 Tesla solariX FTMS system (Bruker, Bremen, Germany). Column chromatography was performed on silica gel (230–400 mesh, Merck, Darmstadt, Germany). TLC was carried out on precoated Kieselgel 60 F₂₅₄ (0.25 mm, Merck, Darmstadt, Germany); spots were visualized by spraying with 10% H₂SO₄ solution followed by heating. Normal phase HPLC (NP-HPLC) was performed using a system comprised of a Hitachi L-7110 pump (Hitachi Ltd., Tokyo, Japan) and a Rheodyne 7725 injection port (Rheodyne LLC, Rohnert Park, CA, USA). Two normal phase columns (Supelco Ascentis^® Si Cat #: 581515-U, 25.0 cm × 21.2 mm, 5.0 μm and 581514-U, 25.0 cm × 10.0 mm, 5.0 μm, Sigma-Aldrich. Com. St. Louis, MO, USA) was used for HPLC.

3.2. Animal Material

Specimens of the sponge Ircinia felix (Duchassaing and Michelotti, 1864) [16] were collected by hand using SCUBA equipment off the coast of the Southern Taiwan, in September 05, 2012 and stored in a freezer until extraction. A voucher specimen (NMMBA-TWSP-12005) was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

3.3. Extraction and Isolation

Sliced bodies of Ircinia felix (wet weight 1210 g) were extracted with ethyl acetate (EtOAc). The EtOAc layer (5.09 g) was separated on silica gel and eluted using a mixture of n-hexane and EtOAc (stepwise, 100:1–pure EtOAc) to yield 11 fractions A–K. Fraction F was separated by NP-HPLC using a mixture of n-hexane and EtOAc (3:1) as the mobile phase to yield 16 fractions F1–F16. Fraction F4 was purified by NP-HPLC using a mixture of n-hexane and acetone (3:1, flow rate: 1.0 mL/min) to afford 1 (1.3 mg, t_R = 50 min). Fraction G was chromatographed on silica gel and eluted using n-hexane/acetone (6:1–2:1) to afford four fractions G1–G4. Fraction G2 was separated by NP-HPLC using a mixture of dichloromethane (DCM) and EtOAc (5:1, flow rate: 2.0 mL/min) to afford 2 (5.8 mg, t_R = 210 min), 3 (5.3 mg, t_R = 324 min) and twelve subfractions G2A–G2L. Fraction G2L was further separated by NP-HPLC using a mixture of DCM and acetone (8:1) as the mobile phase to afford 4 (4.4 mg, t_R = 45 min). Fraction I was separated by NP-HPLC using a mixture of DCM and acetone (4:1) as the mobile phase to afford 5 (3.7 mg, t_R = 126 min).

Felixin A (1): white solid; mp 191–193 °C; ${[\mathrm{\alpha }]}_{\mathrm{D}}^{25}$ −84 (c 0.4, CHCl₃); IR (neat) ν_max 3480, 1731, 1662 cm⁻¹; ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data, see Table 1; ESIMS: m/z 453 [M + Na]⁺; HRESIMS: m/z 453.29773 (calcd for C₂₇H₄₂O₄ + Na, 453.29753).

Felixin B (2): white solid; mp 92–94 °C; ${[\mathrm{\alpha }]}_{\mathrm{D}}^{25}$ +34 (c 0.3, CHCl₃); IR (neat) ν_max 3501, 1733, 1679 cm⁻¹; ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data, see Table 2; ESIMS: m/z 467 [M + Na]⁺; HRESIMS: m/z 467.27707 (calcd for C₂₇H₄₀O₅ + Na, 467.27680).

Felixin C (3): white solid; mp 194–196 °C; ${[\mathrm{\alpha }]}_{\mathrm{D}}^{25}$ +35 (c 0.3, CHCl₃); IR (neat) ν_max 3480, 1731, 1662 cm⁻¹; ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data, see Table 3; ESIMS: m/z 469 [M + Na]⁺; HRESIMS: m/z 469.29270 (calcd for C₂₇H₄₂O₅ + Na, 469.29245).

Felixin D (4): white solid; mp 94–97 °C; ${[\mathrm{\alpha }]}_{\mathrm{D}}^{25}$ +22 (c 0.2, CHCl₃); IR (neat) ν_max 3441, 1738 cm⁻¹; ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data, see Table 4; ESIMS: m/z 525 [M + Na]⁺; HRESIMS: m/z 525.31849 (calcd for C₃₀H₄₆O₆ + Na, 525.31866).

Felixin E (5): white solid; mp 151–153 °C; ${[\mathrm{\alpha }]}_{\mathrm{D}}^{25}$ −5 (c 1.2, CHCl₃); IR (neat) ν_max 3421, 1701 cm⁻¹; ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data, see Table 5; ESIMS: m/z 441 [M + Na]⁺; HRESIMS: m/z 441.29739 (calcd for C₂₆H₄₂O₄ + Na, 441.29753).

3.4. MTT Antiproliferative Assay

MOLT-4, SUP-T1, DLD-1, LNCaP, T-47D and MCF7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine and antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified atmosphere of 5% CO₂. Cells were seeded at 4 × 10⁴ per well in 96-well culture plates before treatment with different concentrations of the tested compounds. The compounds were dissolved in dimethyl sulfoxide (less than 0.02%) and made concentrations of 1.25, 2.5, 5, 10 and 20 μg/μL prior to the experiments. After treatment for 72 h, the cytotoxicity of the tested compounds was determined using a MTT cell proliferation assay (thiazolyl blue tetrazolium bromide, Sigma-M2128). The MTT is reduced by the mitochondrial dehydrogenases of viable cells to a purple formazan product. The MTT-formazan product was dissolved in DMSO. Light absorbance values (OD = OD₅₇₀ − OD₆₂₀) were recorded at wavelengths of 570 and 620 nm using an ELISA reader (Anthos labtec Instrument, Salzburg, Austria) to calculate the concentration that caused 50% inhibition (IC₅₀), i.e., the cell concentration at which the light absorbance value of the experiment group was half that of the control group. These results were expressed as a percentage of the control ± SD established from n = 4 wells per one experiment from three separate experiments [17,18,19].

4. Conclusions

Sponges have been well-recognized as an important source of potential bioactive marine natural products. Our studies on Ircinia felix for the extraction of natural substances, have led to the isolation of five new scalaranes, felixins A–E (1–5) and compounds 1–5 are potentially cytotoxic toward DLD-1 tumor cells. These results suggest that continuing investigation of novel secondary metabolites together with the potentially useful bioactivities from this marine organism are worthwhile for future drug development.