New Cytotoxic 24-Homoscalarane Sesterterpenoids from the Sponge Ircinia felix.

Two new 24-homoscalarane sesterterpenoids, felixins F (1) and G (2), were isolated from the sponge Ircinia felix. The structures of new homoscalaranes 1 and 2 were elucidated by extensive spectroscopic methods, particularly with one-dimensional (1D) and two-dimensional (2D) NMR, and, by comparison, the spectral data with those of known analogues. The cytotoxicity of 1 and 2 against the proliferation of a limited panel of tumor cell lines was evaluated and 1 was found to show cytotoxicity toward the leukemia K562, MOLT-4, and SUP-T1 cells (IC50 ≤ 5.0 μM).

1. Introduction

The sponge Ircinia felix (Duchassaing and Michelotti, 1864) (family Irciniidae, order Dictyoceratida, class Demospongiae, phylum Porifera) (Figure 1) has been proven to be an important source of interesting natural substances [1,2,3,4,5], and the extract from this organism has also played interesting roles in marine ecology [6,7,8,9,10] and medicinal use [11,12]. In our previous study, five new scalarane analogues, felixins A–E, were isolated from I. felix [13], and several compounds showed cytotoxicity. Scalarane-type sesterterpenoids were proven to be active in a number of bioassays, particularly in cytotoxic activity, and played important roles in chemical markers and chemical ecology [14]. In the further study of this interesting organism, two new 24-homoscalarane analogues, felixins F (1) and G (2), were isolated (Figure 1). In this paper, we deal with the isolation, structure determination, and cytotoxicity of homoscalaranes 1 and 2.

Figure 1

The sponge Ircinia felix and the structures of felixins F (1), G (2) and 12α-acetoxy-22-hydroxy-24-methyl-24-oxoscalar-16-en-25-al (3).

2. Results and Discussion

Felixin F (1) was isolated as a white powder and the molecular formula for this compound was determined to be C26H40O5 (seven unsaturations) using high-resolution electron spray mass spectroscopy (HRESIMS) (C26H40O5 + Na, m/z 455.27662, calculated 455.27680). Comparison of the 13C NMR and distortionless enhancement by polarization transfer (DEPT) data with the molecular formula indicated that there must be two exchangeable protons, which require the presence of two hydroxy groups. The 13C NMR and DEPT data showed that this compound has 26 carbons (Table 1), including five methyls, eight sp3 methylenes (including one oxymethylene), six sp3 methines (including one oxymethine), four sp3 quaternary carbons, and three carbonyls. Thus, from the above data, three degrees of unsaturation were accounted for and 1 was identified as a tetracyclic sesterterpenoid analogue. From the 1H–1H correlation spectroscopy (COSY) of 1 (Table 1), it was possible to establish the separate system that maps out the proton sequences from H2-1/H2-2/H2-3, H-5/H2-6/H2-7, H-9/H2-11, and H-14/H2-15/H-16/H-17/H-18/H-25. These data, together with the key heteronuclear multiple bond connectivity (HMBC) correlations between protons and quaternary carbons (Table 1), such as H2-3, H3-19, H3-20/C-4; H-9, H2-11, H-14, H3-21/C-8; H-5, H-9, H2-22/C-10; H2-11, H3-23/C-12; H2-11, H-14, H2-15, H-18, H3-23/C-13; and H-17, H3-26/C-24, established the main carbon skeleton of 1 as a 24-homoscalarane analogue [14]. The oxymethylene unit at δC 62.7 was correlated to the methylene protons at δH 4.07 and 3.93 in the heteronuclear multiple quantum coherence (HMQC) spectrum and these methylene signals were 2J-correlated with C-10 (δC 42.7) and 3J-correlated with C-1 (δC 33.9), C-5 (δC 56.8), and C-9 (δC 61.8), proving the attachment of a hydroxymethyl group at C-10 (Table 1).

Table 1

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

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	2.09 m; 0.55 ddd (12.8, 12.8, 3.2)	33.9, CH2	H2-2	n.o.
2	1.54–1.37 m	17.8, CH2	H2-1, H2-3	n.o.
3	1.42 m; 1,16 m	41.5, CH2	H2-2	C-4, -20
4		33.0, C
5	0.94 dd (12.8, 2.4)	56.8, CH	H2-6	C-6, -10, -20, -22
6	1.54–1.37 m	18.2, CH2	H-5, H2-7	C-5
7	1.93 m; 1.15 m	30.0, CH2	H2-6	n.o.
8		38.2, C
9	1.28 (14.4, 2.4)	61.8, CH	H2-11	C-8, -10, -21, -22
10		42.7, C
11	3.24 dd (14.4, 14.4); 2.53 dd (14.4, 2.4)	38.6, CH2	H-9	C-8, -9, -12, -13
12		214.6, C
13		52.4, C
14	1.29 m	57.3, CH	H2-15	C-7, -8, -13, -15, -16, -23
15	1.90 m; 1.02 m	41.9, CH2	H-14, H-16	C-13, -14, -16, -17
16	3.57 ddd (10.8, 10.8, 4.8)	73.3, CH	H2-15, H-17	n.o.
17	2.91 dd (11.6, 10.8)	53.0, CH	H-16, H-18	C-16, -18, -24
18	3.18 d (11.6)	57.2, CH	H-17, H-25	C-13, -16, -23, -25
19	0.86 s	33.5, CH3		C-3, -4, -5, -20
20	0.75 s	21.8, CH3		C-3, -4, -5, -19
21	1.26 s	16.4, CH3		C-8, -9, -14
22	4.07 d (11.6); 3.93 d (11.6)	62.7, CH2		C-1, -5, -9, -10
23	1.19 s	15.6, CH3		C-12, -13, -14, -18
24		212.7, C
25	9.89 s	204.4, CH	H-18	C-13, -17, -18
26	2.37 s	33.8, CH3		C-17, -24

n.o. = not observed.

The relative stereochemistry of 1 was elucidated from the nuclear Overhauser effect (NOE) interactions observed in nuclear Overhauser effect spectroscopy (NOESY) (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, respectively, anchoring the stereochemical analysis because no correlation was found between H-5 and H2-22. In the NOESY experiment of 1, H-9 showed a correlation with H-5 but not with H3-21 and H2-22. Thus, H-9 must be on the α face while Me-21 and the hydroxymethyl at C-10 must be located on the β face. Moreover, the correlations of H-14 with H-16, but not with H3-21 and H3-23, indicated the β-orientations of Me-23 and the hydroxy group attaching at C-13 and C-16, respectively. H3-23 showed correlations with H-17 and H-25, and large coupling constants were recorded between H-16/H-17 (J = 10.8 Hz) and H-17/H-18 (J = 11.6 Hz), indicating that the dihedral angles between H-16/H-17 and H-17/H-18 are approximately 180° and H-17 and the aldehyde group at C-18 have β-orientations. Based on the above findings, the structure of 1 was established unambiguously.

Figure 2

Selective NOESY correlations of 1.

The HRESIMS of 2 (felixin G) exhibited a pseudomolecular ion peak at m/z 523.30321 [M + Na]+, with the molecular formula C30H44O6 (calcd. C30H44O6 + Na, 523.30301), implying nine degrees of unsaturation. The 13C NMR and DEPT spectra of 2 exhibited 30 carbons: one aldehyde (δC 200.8, CH-25), one ketone (δC 198.7, C-24), two ester carbonyls (δC 171.0, 169.9, 2× acetate carbonyls), one trisubstituted olefin (δC 142.6, CH-16; 137.2, C-17), one oxymethylene (δC 64.8, CH2-22), one oxymethine (δC 74.8, CH-12), seven methyls, seven methylenes, four methines, and four quaternary carbons. The 1H NMR spectrum showed seven methyls (δH 2.34, 3H, s, H3-26; 2.17, 2.04, 2 × 3H, each s, acetate methyls; 1.03, 3H, s, H3-21; 0.95, 3H, s, H3-23; 0.89, 3H, s, H3-19; 0.83, 3H, s, H3-20); one acetoxymethylene (δH 4.58, 1H, d, J = 12.0 Hz; 4.13, 1H, d, J = 12.0 Hz, H2-22); one oxymethine (δH 4.76, 1H, s, H-12); one olefinic proton (δH 7.09, 1H, dd, J = 2.5, 2.5 Hz, H-16); and one aldehyde proton (δH 9.41, 1H, d, J = 3.5 Hz, H-25). A typical 24-methylscalarane carbon system bearing acetoxymethylene and four methyl groups along rings A–D could be established by the HMBC correlations from the acetoxymethylene (CH2-22) and four methyl groups (Me-19, -20, -21, and 23) to the associated carbons and a 24-homoscalarane skeleton could be obtained on the basis of further HMBC and 1H–1H COSY correlations (Table 2).

Table 2

1H (500 MHz, CDCl3) and 13C (125 MHz, CDCl3) NMR data and 1H–1H COSY and HMBC correlations for homoscalarane 2.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	1.98 m; 0.53 ddd (12.5, 12.5, 3.0)	34.7, CH2	H2-2	C-3
2	1.56 m; 1.41 m	18.1, CH2	H2-1, H2-3	C-1, -10
3	1.44 m; 1.18 m	41.5, CH2	H2-2	C-2, -19, -20
4		32.9, C
5	0.99 dd (17.0, 4.0)	56.8, CH	H2-6	C-4, -6, -10, -20, -22
6	1.54 m; 1.44 m	17.9, CH2	H-5, H2-7	C-5, -8
7	1.88 m; 1.18 m	41.9, CH2	H2-6	C-8, -9
8		37.8, C
9	1.39 m	51.9, CH	H2-11	C-1, -8, -10, -11, -12, -14, -21, -22
10		40.1, C
11	2.15–2.05 m	24.2, CH2	H-9, H-12	n.o.
12	4.76 s	74.8, CH	H2-11	n.o.
13		40.0, C
14	1.52 m	49.2, CH	H2-15	C-9, -15, -23
15	2.26–2.30 m	23.7, CH2	H-14, H-16	C-16, -17
16	7.09 dd (2.5, 2.5)	142.6, CH	H2-15	n.o.
17		137.2, C
18	3.53 broad s	53.0, CH	H-25	n.o.
19	0.89 s	33.7, CH3		C-3, -4, -5, -20
20	0.83 s	21.9, CH3		C-3, -4, -5, -19
21	1.03 s	16.1, CH3		C-7, -8, -9, -14
22	4.58 d (12.0); 4.13 d (12.0)	64.8, CH2		C-1, -9, -10, acetate carbonyl
23	0.95 s	15.2, CH3		C-12, -13, -14, -18
24		198.7, C
25	9.41 d (3.5)	200.8, CH	H-18	C-18
26	2.34 s	25.1, CH3		C-24
12-OAc		169.9, C
	2.17 s	21.2, CH3		Acetate carbonyl
22-OAc		171.0, C
	2.04 s	21.5, CH3		Acetate carbonyl

n.o. = not observed.

The relative stereochemistry of 2 was elucidated from the interactions observed in a NOESY experiment (Figure 3). In the NOESY experiment of 2, H-9 showed a correlation with H-5, but not with H3-21 and H2-22. Thus, H-5 and H-9 must be on α face while Me-21 and the acetoxymethylene at C-10 must be located on the β face. H-14 correlated with H-18, but not with H3-21 and H3-23, assuming that H-14 and H-18 were α-oriented. The correlation of H3-23 with H-12, but not with H-14, indicated the β-orientations of Me-23 and H-12. H-16 showed a correlation with H3-26, revealing the E geometry of the C-16/17 double bond. It was found that the structure of 2 was similar with that of a known 24-homoscalarane analogue, 12α-acetoxy-22-hydroxy-24-methyl-24-oxoscalar-16-en-25-al (3) [15], except that the 22-hydroxy group in 3 was replaced by an acetoxy group in 2.

Figure 3

Selective NOESY correlations of 2.

The cytotoxicity of homoscalaranes 1 and 2 against CCRF-CEM (human acute lymphoblastic leukemia), HL-60 (human acute promyelocytic leukemia), K-562 (human chronic myelogenous leukemia), MOLT-4 (human acute lymphoblastic leukemia), SUP-T1 (human T-cell lymphoblastic lymphoma), U-937 (human histiocytic lymphoma), DLD-1 (human colorectal adenocarcinoma), LNCaP (human prostatic carcinoma), and MCF7 (human breast adenocarcinoma) tumor cells is shown in Table 3. Compound 1 was found to show cytotoxicity toward the leukemia K562, MOLT-4, and SUP-T1 cells (IC50 ≤ 5.0 μM). By comparing the cytotoxic data of 1 with those of 2 and the relative scalarane derivatives, flexins A−E, that we isolated previously [13], we find that 1 is more cytotoxic toward most tumor cells.

Table 3

Cytotoxic data of homoscalaranes 1 and 2.

Compounds	Cell Lines IC50 (μM)
	CCRF-CEM	HL-60	K-562	MOLT-4	SUP-T1	U-937	DLD-1	LNCaP	MCF7
1	NT a	NT	1.27	2.59	3.56	10.65	19.26	7.22	NT
2	7.90	6.50	19.9	NT	NT	13.08	27.08	17.14	NA b
Doxorubicin c	0.02	0.02	0.70	0.02	0.09	0.33	0.90	3.16	0.29

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

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); absorptions are reported in cm−1. NMR spectra were recorded on a Varian Mercury Plus 400 NMR spectrometer (Varian Inc.: Palo Alto, CA, USA) or a Varian Inova 500 spectrometer (Varian Inc.) using the residual CHCl3 signal (δH 7.26 ppm) as the internal standard for 1H NMR and CDCl3 (δC 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). A normal phase column (Supelco Ascentis® Si Cat #: 581515-U, 25 cm × 21.2 mm, 5 μm; Sigma-Aldrich: 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 self-containing underwater breathing apparatus (SCUBA) equipment off the coast of the Southern Taiwan (Johnson Outdoors Inc.: Racine, WI, USA), on 5 September 2012, and stored in a freezer until extraction. A voucher specimen (NMMBA-TWSP-12005) was deposited in the National Museum of Marine Biology & Aquarium, Pingtung, 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 H was chromatographed on silica gel and eluted using n-hexane/acetone (6:1–2:1) to afford 14 fractions H1–H14. Fraction H2 was separated by NP-HPLC using a mixture of dichloromethane (DCM) and EtOAc (5:1, flow rate: 2.0 mL/min) to afford 2 (1.4 mg, tR = 121 min). Fraction I was separated by NP-HPLC using a mixture of dichloromethane (DCM) and acetone (4:1, flow rate: 2.0 mL/min) as the mobile phase to yield 1 (1.8 mg, tR = 81 min).

Felixin F (1): white solid; mp 117−120 °C; +54 (c 0.4, CHCl3); IR (neat) νmax 3462, 1704 cm−1; 1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data, see Table 1; ESIMS: m/z 455 [M + Na]+; HRESIMS: m/z 455.27662 (calcd. for C26H40O5 + Na, 455.27680).

Felixin G (2): white solid; mp 121−124 °C; +43 (c 0.3, CHCl3); IR (neat) νmax 1738 cm−1; 1H (500 MHz, CDCl3) and 13C (125 MHz, CDCl3) NMR data, see Table 2; ESIMS: m/z 523 [M + Na]+; HRESIMS: m/z 523.30321 (calcd. for C30H44O6 + Na, 523.30301).

3.4. MTT Antiproliferative Assay

CCRF-CEM, HL-60, K-562, MOLT-4, SUP-T1, U-937, DLD-1, LNCaP, 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, St. Louis, MO, USA). 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

Our further studies on Ircinia felix for the extraction of natural substances have led to the isolation of five new 20-homoscalaranes, felixins F (1) and G (2) and compound 1 are potentially cytotoxic toward the leukemia K562, MOLT-4, and SUP-T1 cells. These results suggest that continuing investigation of novel secondary metabolites together with the potentially useful bioactivities from I. felix are worthwhile for future drug development.