Polyhydroxylated steroids from the bamboo coral Isis hippuris.

In previous studies on the secondary metabolites of the Taiwanese octocoral Isis hippuris, specimens have always been collected at Green Island. In the course of our studies on bioactive compounds from marine organisms, the acetone-solubles of the Taiwanese octocoral I. hippuris collected at Orchid Island have led to the isolation of five new polyoxygenated steroids: hipposterone M-O (1-3), hipposterol G (4) and hippuristeroketal A (5). The structures of these compounds were determined on the basis of their spectroscopic and physical data. The anti-HCMV (human cytomegalovirus) activity of 1-5 and their cytotoxicity against selected cell lines were evaluated. Compound 2 exhibited inhibitory activity against HCMV, with an EC(50) value of 6.0 μg/mL.

1. Introduction

The octocoral Isis hippuris, distributed widely in the western Pacific, has yielded a number of polyoxygenated steroids, including hippuristanol type [1-9], gorgosterol type [10-14], hippuristerone type [3,14,15], and hippuristerol type [3,14-16]. Those of the first type were originally reported as cytotoxins and later rediscovered as selective inhibitors against the translation factor eIF4A [17,18]. Some of the second types were reported to show cytotoxicity or a reversal of multidrug resistance activity [10]. The samples for previous studies on the secondary metabolites of Taiwanese octocoral I. hippuris were all collected at Green Island [5-7,12,14,15]. In our continued study of the bioactive metabolites from marine organism, the Taiwanese octocoral I. hippuris (Figure 1) collected at Orchid Island was selected for study since its acetone extract exhibited antiviral activity against HCMV. Bioactivity-guided fractionation resulted in the isolation of five polyoxygenated steroids: hipposterone M–O (1–3), hipposterol G (4), hippuristeroketal A (5) (Figure 2). We describe herein the isolation, structure elucidation, and biological activity of these compounds.

Figure 1

Bamboo coral Isis hippuris.

Figure 2

Structures of compounds 1–5.

2. Results and Discussion

The molecular formula C33H52O8 was assigned to hipposterone M (1) on the basis of positive HRESIMS (found m/z 599.3556 [M + Na]+), implying eight degrees of unsaturation. Its IR spectrum revealed the absorptions for hydroxyl (νmax 3454 cm−1), ketone carbonyl (νmax 1717 cm−1), and ester carbonyl (νmax 1733 cm−1) groups. NMR data (Tables 1 and 2) of 1 indicated the presence of a ketone (δC 211.7), two ester cabonyls, two oxygenated sp3 methines, an oxygenated sp3 methylene, three oxygenated sp3 quaternary carbons, two secondary methyls, four tertiary methyls, six non-oxygenated sp3 methines, eight non-oxygenated sp3 methylenes, and two non-oxygenated sp3 quaternary carbons. NMR signals (Table 1) at δC 80.0 (qC) and 67.1 (qC) suggested the existence of a tetrasubstituted expoxy. The quaternary carbon at δC 85.5, which has HMBC correlation (Figure 3) with tertiary methyl signals at δH 1.56 (s) and 1.43 (s) (Table 2) disclosed the presence of –OC(CH3)2. By extensive analysis of 2D NMR spectra, including COSY, HSQC, NOESY (Figure 4) and HMBC, 1 was shown to be a derivative of hippuristerone A [15]. HMBC correlations (Figure 3) from H2-18 (δH 3.94 and 3.75) to C-12, C-13, C-14, and C-17 established 1 as 18-hydroxyhippuristerone A. The stereochemistry of the side chain moiety was determined by comparison of the 1H and 13C NMR spectral data with those of hippuristerone A.

Table 1

13C NMR data for compounds 1–5.

C#	1, aδC, type	2, aδC, type	3, aδC, type	4, bδC, type	5, cδC, type
1	38.3, CH2	38.3, CH2	38.2, CH2	36.7, CH2	35.6, CH2
2	38.1, CH2	38.1, CH2	38.0, CH2	31.4, CH2	29.2, CH2
3	211.7, qC	211.7, qC	211.5, qC	71.2, CH	100.7, qC
4	44.5, CH2	44.5, CH2	44.5, CH2	38.0, CH2	36.2, CH2
5	46.5, CH	46.5, CH	46.4, CH	44.7, CH	43.0, CH
6	28.6, CH2	28.5, CH2	28.5, CH2	28.3, CH2	28.8, CH2
7	31.7, CH2	31.7, CH2	31.5, CH2	31.9, CH2	32.7, CH2
8	34.5, CH	34.4, CH	34.4, CH	34.5, CH	35.1, CH
9	53.1, CH	53.6, CH	53.4, CH	54.0, CH	55.1, CH
10	35.7, qC	35.7, qC	35.6, CH	35.5, qC	36.4, qC
11	21.0, CH2	21.0, CH2	21.5, CH2	21.4, CH2	22.0, CH2
12	30.6, CH2	31.0, CH2	32.4, CH2	32.2, CH2	33.3, CH2
13	46.8, qC	46.7, qC	45.6, qC	45.6, qC	46.5, qC
14	47.7, CH	48.7, CH	49.2, CH	48.7, CH	50.3, CH
15	33.3, CH2	33.3, CH2	33.5, CH2	33.4, CH2	34.4, CH2
16	70.0, CH	70.1, CH	70.3, CH	70.1, CH	71.1, CH
17	80.0, qC	79.7, qC	77.2, qC	77.7,qC	78.6, qC
18	61.9, CH2	61.9, CH2	63.5, CH2	63.5, CH2	64.3, CH2
19	11.3, CH3	11.4, CH3	11.4, CH3	12.2, CH3	12.0, CH3
20	67.1, qC	67.5, qC	66.7, qC	66.4, qC	67.3, qC
21	16.1, CH3	15.9, CH3	16.1, CH3	16.2, CH3	17.1, CH3
22	77.2, CH	77.2, CH	77.2, CH	77.2, CH	78.1, CH
23	33.5, CH	32.9, CH	32.5, CH	33.6, CH	33.6, CH
24	39.9, CH	41.7, CH	38.8, CH	40.1, CH	42.2, CH
25	85.5, qC	73.7, qC	74.2, qC	85.6, qC	73.5, qC
26	23.2, CH3	30.9, CH3	71.0, CH2	22.8, CH3	31.2, CH3
27	25.1, CH3	25.8, CH3	20.3, CH3	25.1, CH3	25.9, CH3
28	10.4, CH3	11.4, CH3	10.9, CH3	10.5, CH3	11.7, CH3
29	11.9, CH3	12.1, CH3	12.3, CH3	11.9, CH3	12.6, CH3
OAc	20.9, CH3	20.9, CH3	21.2, CH3	21.2, CH3	21.1, CH3
	171.6, qC	171.6, qC	171.1, qC	171.0, qC	170.6, qC
	22.6, CH3		21.1, CH3	21.0, CH3	20.9, CH3
	169.8, qC		171.3, qC	171.2, qC	171.4, qC
			21.1, CH3	22.7, CH3
			170.8, qC	169.9, qC
OMe					47.6, CH3
					47.5, CH3

Spectra were measured in CDCl3 (100 MHz);

Spectra were measured in CDCl3 (125 MHz);

Spectra were measured in C6D6 (125 MHz).

Table 2

1H NMR data for compounds 1–5.

H#	1, δH (J in Hz) a	2, δH (J in Hz) a	3, δH (J in Hz) a	4, δH (J in Hz) b	5, δH (J in Hz) c
1	α: 1.39 m	α: 1.35 m	α: 1.32 m	α: 1.02 m	α: 1.33 m
	β: 2.02 m	β: 2.00 m	β: 1.97 m	β: 1.69 m	β: 1.06 m
2	α: 2.32 m	α: 2.31 m	α: 2.31 m	α: 1.82 m	α: 1.86 m
	β: 2.38 m	β: 2.39 m	β: 2.37 m	β: 1.41 m	β: 1.41 m
3				3.60 m
4	α: 2.12 dd ovl	α: 2.09 dd ovl	α: 2.12 dd ovl	α: 1.58 m	α: 1.86 dd (13.6, 3.6)
	β: 2.28 t (13.6)	β: 2.27 t (13.6)	β: 2.26 t (13.6)	β: 1.29 m	β: 1.41 dd ovl
5	1.56 m	1.54 m	1.55 m	1.54 m	1.34 m
6	1.38 m	1.38 m	1.39 m	1.34 m	1.08 m
7	1.79 m	1.78 m	1.82 m	1.78 m	1.54 m
	0.93 m	0.92 m	0.95 m	0.91 m	0.67 m
8	1.58 m	1.58 m	1.72 m	1.67 m	1.45 m
9	0.85 m	0.74 m	0.81 m	0.75 m	0.70 m
11	α: 1.66 m	α: 1.65 m	α: 1.63 m	α: 1.60 m	α: 1.48 m
	β: 1.48 m	β: 1.44 m	β: 1.33 m	β: 1.23 m	β: 1.23 m
12	α: 1.28 m	α 1.28 m	α: 1.34 m	α: 1.38 m	α: 1.44 m
	β: 2.44 m	β: 2.44 m	β: 2.16 m	β: 2.17 m	β: 2.28 m
14	1.36 m	1.18 m	1.23 m	1.31 m	1.28 m
15	α: 2.23 m	α: 2.24 m	α: 2.21 m	α: 2.21 m	α: 2.22 m
	β: 1.44 m	β: 1.46 m	β: 1.48 m	β: 1.46 m	β: 1.59 m
16	4.10 t (7.2)	4.13 t (7.6)	4.06 t (7.6)	4.04 dd (8.0, 7.5)	4.38 t (7.5)
18	3.75 t (10.4)	3.74 t (11.2)	4.23 d (11.6)	4.27 d (11.5)	4.55 d (11.5)
	3.94 d (11.6)	3.94 dd (11.6, 2.4)	4.30 d (11.6)	4.20 d (11.5)	4.49 d (11.5)
19	1.02 s	1.02 s	1.02 s	0.82 s	0.64 s
21	1.64 s	1.66 s	1.60 s	1.59 s	1.84 s
22	4.62 d (10.8)	4.60 d (10.8)	4.66 d (10.8)	4.67 d (11.0)	5.04 d (10.5)
23	2.28 m	2.47 m	2.50 m	2.29 m	2.43 m
24	1.97 q (8.0)	1.47 q (6.8)	1.64 q (6.8)	1.92 q (7.0)	1.55 q (7.5)
26	1.56 s	1.24 s	3.89 d (11.6)	1.56 s	0.88 s
			4.04 d (11.6)
27	1.43 s	1.21 s	1.18 s	1.46 s	0.78 s
28	0.90 d (8.0)	0.90 d (6.8)	0.88 d (6.8)	0.91 d (7.0)	0.65 d (7.5)
29	0.88 d (6.4)	0.86 d (6.4)	0.88 d (6.8)	0.87 d (7.0)	0.80 d (7.0)
OAc	2.14 s, 1.99 s	2.14 s	2.07 s, 2.13 s, 2.13 s	2.06 s, 2.00 s, 2.13 s	1.76 s, 1.69 s
OMe					3.12 s, 3.02s
OH-16	3.36 s	3.43 s	3.27 br s	3.19 br s	3.83 br s
OH-18	2.44 d ovl	3.46 d ovl

Spectra were measured in CDCl3 (400 MHz);

Spectra were measured in CDCl3 (500 MHz);

Spectra were measured in C6D6 (500 MHz).

Figure 3

COSY and HMBC correlations of compounds 1–5.

Figure 4

NOESY correlations of compounds 1 and 4.

Hipposterone N (2) had a molecular formula of C31H50O7, as suggested by the NMR and HRESIMS data. Its IR spectrum also showed the absorptions for hydroxyl (νmax 3454 cm−1), ketone carbonyl (νmax 1715 cm−1), and ester carbonyl (νmax 1731 cm−1) groups. NMR data (Tables 1 and 2) of 2 revealed the presence of a ketone (δC 211.7), an ester cabonyl, two oxygenated sp3 methines, an oxygenated sp3 methylene, three oxygenated sp3 quaternary carbons, two secondary methyls, four tertiary methyls, six non-oxygenated sp3 methines, eight non-oxygenated sp3 methylenes, and two non-oxygenated sp3 quaternary carbons. NMR data (Tables 1 and 2) (Figure 3) of 2 resembled those of 1 except for a hydroxyl group replacing the tertiary acetoxyl in 1 [14]. HMBC correlations (Figure 3) from H3-26 (δH 1.24) and H3-27 (δH 1.21) to C-25 established 2 as a 25-deacetyl-18-hydroxy derivative of hippuristerone A. The stereochemistry of the side chain moiety was determined by comparison of the 1H and 13C NMR data with those of hippuristerones F, H, and I isolated from I. hippuris [16].

The positive HRESIMS of hipposterone O (3) established a molecular formula of C35H54O10. NMR data (Tables 1 and 2) of 3 showed the presence of a ketone (δC 211.5), three ester cabonyls, two oxygenated sp3 methines, two oxygenated sp3 methylene, three oxygenated sp3 quaternary carbons, two secondary methyls, three tertiary methyls, six non-oxygenated sp3 methines, eight non-oxygenated sp3 methylenes, and two non-oxygenated sp3 quaternary carbons. By comparison of NMR spectroscopic data (Tables 1 and 2) of 3 with those of hippuristerone J [14], the primary acetoxy group at C-21 was shift to C-18 on the basis of HMBC correlations (Figure 3) from H2-18 [δH 4.23 (1H, d, J = 11.6 Hz) and 4.30 (1H, d, J = 11.6 Hz)] to C-12, C-13, C-14, C-17, and carbonyl carbon of 18-OAc. The stereochemistry of the side chain moiety was determined by comparison of the 1H and 13C NMR spectral data with those of hippuristerones J and K previously isolated from I. hippuris [14].

Hipposterol G (4) was isolated as a white powder, and its molecular formula, C35H56O9, was determined by HRESIMS. Its IR spectrum revealed the functionalities of hydroxyl (νmax 3471 cm−1) and ester carbonyl (νmax 1734 cm−1). NMR data (Tables 1 and 2) of 4 indicated the presence of three ester cabonyls, three oxygenated sp3 methines, an oxygenated sp3 methylene, three oxygenated sp3 quaternary carbons, two secondary methyls, four tertiary methyls, six non-oxygenated sp3 methines, eight non-oxygenated sp3 methylenes, and two non-oxygenated sp3 quaternary carbons. NMR data (Tables 1 and 2) of 4 were similar to those of hippuristerone G [16] with the absence of the ketone carbon signal at δC 211.6 ppm and the presence of signal at δH 3.60 ppm NOE correlation H-3/H-5 and chemical shift values for C-1–C-7 nuclei. This is in agreement with the results reported for 5α-cholestan-3β-ol, which allowed us to propose a β orientation of OH group at C-3 (Figure 4). The stereochemistry of the side chain moiety was determined by comparison of the 1H and 13C NMR spectral data with those of hippuristerone A.

The molecular formula of hippuristeroketal A (5) was found to be C35H58O9, as deduced from HRESIMS data. Its IR spectrum revealed the absorptions for hydroxyl (νmax 3471 cm−1) and ester carbonyl (νmax 1731 cm−1) groups. NMR data (Tables 1 and 2) of 5 indicated the presence of a ketal (δC 100.7), two ester cabonyls, two oxygenated sp3 methines, an oxygenated sp3 methylene, three oxygenated sp3 quaternary carbons, two secondary methyls, four tertiary methyls, six non-oxygenated sp3 methines, eight non-oxygenated sp3 methylenes, and two non-oxygenated sp3 quaternary carbons. By comparison of the NMR spectroscopic data (Tables 1 and 2) of 5 resembled those of hippuristerone F [14] with the absence of ketone carbon at δC 211.6 and the presence of two methoxyl signals [δH 3.12 (3H, s), 3.02 (3H, s) and δC 47.6 (CH3), 47.5 (CH3)] in the molecule. The HMBC correlations (Figure 3) of the methoxyl protons with C-3 [δC 100.7 (qC)], suggesting that C-3 was substituted by two methoxy groups. The stereochemistry of the side chain moiety was determined by comparison of the 1H and 13C NMR spectral data with those of hippuristerones F, H, and I previously isolated from I. hippuris [16]. Compound 5 was not an artifact because 1H NMR signals for the dimethylketal were observed before MeOH treatment.

Metabolites 1–5 were not cytotoxic against P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma) tumor cells, and human embryonic lung (HEL) cells with IC50 values greater than 50 μg/mL. The anti-HCMV activity and cytotoxicity against of selected cell lines of 1–5 were evaluated. Compound 2 exhibited inhibitory activity against HCMV, with an EC50 values of 6.0 μg/mL.

3. Experimental Section

3.1. General Experimental Procedures

Optical rotations were determined with a JASCO P1020 digital polarimeter. Ultraviolet (UV) and infrared (IR) spectra were obtained on JASCO V-650 and JASCO FT/IR-4100 spectrophotometers, respectively. NMR spectra were recorded on a Varian MR 400 NMR spectrometer at 400 MHz for 1H and 100 MHz for 13C or on a Varian Unity INOVA 500 FT-NMR spectrometer at 500 MHz for 1H and 125 MHz for 13C, respectively. 1H NMR chemical shifts are expressed in δ (ppm) referring to the solvent peaks δH 7.27 and 7.15 for CDCl3 and C6D6, respectively, and coupling constants are expressed in Hz. 13C NMR chemical shifts are expressed in δ (ppm) referring to the solvent peaks δC 77.0 and 128.0 for CDCl3 and C6D6, respectively. ESI-MS were recorded by ESI FT-MS on a Bruker APEX II mass spectrometer. Silica gel 60 (Merck, Germany, 230–400 mesh) and LiChroprep RP-18 (Merck, 40–63 μm) were used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) and precoated RP-18 F254s plates (Merck) were used for thin-layer chromatography (TLC) analysis. High-performance liquid chromatography (HPLC) was carried out using a Hitachi L-7100 pump equipped with a Hitachi L-7400 UV detector at 220 nm together with a semi-preparative reversed-phase column (Merck, Hibar LiChrospher RP-18e, 5 μm, 250 × 25 mm).

3.2. Biological Material

The octocoral I. hippuris was collected by hand using scuba at Orchid Island, 70 km off the southeastern coast of Taiwan, in August 2008 at a depth of 9 m and stored in a freezer until extraction. The voucher specimen (LY-19) was identified by Prof. Chang-Feng Dai, National Taiwan University and deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Taiwan.

3.3. Extraction and Isolation

A specimen of octocoral I. hippuris (4.0 kg, wet weight) was minced and exhaustively extracted with acetone (3 × 3 L) at room temperature. The combined acetone extracts was then partitioned between H2O and EtOAc. The resulting EtOAc extract (25.6 g) was subjected to gravity silica gel 60 column chromatography (Si 60 CC) using n-hexane–EtOAc and EtOAc–MeOH of increasing polarity, to give 44 fractions. Fraction 28 (0.86 g), eluted with n-hexane–EtOAc (1:6), was further subjected to Si 60 CC (n-hexane–EtOAc, 5:3) to give 4 subfractions. A subfraction 28-2 (105 mg) was separated by a RP-18 flash column (MeOH–H2O, 75:25 to 100:0) to give four fractions. In turn, a subfraction 28-2-2, eluted with MeOH–H2O (80:20), was further purified by RP-18 HPLC (MeOH–H2O–MeCN, 80:20:5) to affford 1 (3.0 mg) and 4 (0.5 mg). Similarly, the subfraction 28-3 (112 mg) was further subjected to a RP-18 flash column (MeOH–H2O, 75:25 to 100:0) to give five subfractions. A subfraction 28-3-2 (112 mg), eluted with MeOH–H2O (70:30), was further purified by RP-18 HPLC (MeOH–H2O–MeCN, 75:25:5) to obtain 1 (0.2 mg) and 4 (0.3 mg). Likewise, the subfraction 28-3-3, eluted with MeOH–H2O (80:20), was purified by RP-18 HPLC (MeOH–H2O–MeCN, 75:25:5) to give 5 (1.2 mg). Fraction 29 (0.41 g), eluted with n-hexane–EtOAc (1:7), was subjected to Si 60 CC (n-hexane–EtOAc, 8:2 to 2:8) to give four subfractions. A subfraction 29-3 (309 mg), eluted with n-hexane–EtOAc (2:7), was further fractionated by a RP-18 flash column (MeOH–H2O, 60:40 to 100:0) to give four subfractions. A subfraction 29-3-2, eluted with MeOH–H2O (75:25), was further purified by RP-18 HPLC (MeOH–H2O, 70:30) to afford 3 (1.0 mg), 2 (1.2 mg), and 1 (0.2 mg).

Hipposterone M (1): White amorphous powder; [α]D 25 −8 (c 0.1, CHCl3); IR (neat) νmax 3454, 2954, 2922, 1733, 1717, 1558, 1456, 1374, 1238, 1152, 1019 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data in Tables 1 and 2; HRESIMS m/z 599.3556 [M + Na]+ (calcd for C33H52O8Na, 599.3560).

Hipposterone N (2): White amorphous powder; [α]D 25 −11 (c 0.1, CHCl3); IR (neat) νmax 3463, 2970, 2933, 1731, 1715, 1374, 1244, 1021, 735 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data in Tables 1 and 2; HRESIMS m/z 557.3452 [M + Na]+ (calcd for C31H50O7Na, 557.3454).

Hipposterone O (3): White amorphous powder; [α]D 25 −5 (c 0.1, CHCl3); IR (neat) νmax 3471, 2974, 2939, 1731, 1449, 1373, 1247, 1023, 739 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data in Tables 1 and 2; HRESIMS m/z 657.3616 [M + Na]+ (calcd for C35H54O10Na, 657.3614).

Hipposterol G (4): White amorphous powder; [α]D 25 +5 (c 0.1, CHCl3); IR (neat) νmax 3471, 2928, 2860, 1734, 1454, 1371, 1244, 1023, 736 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data in Tables 1 and 2; HRESIMS m/z 643.3819 [M + Na]+ (calcd for C35H56O9Na, 643.3822).

Hppuristeroketal A (5): White amorphous powder; [α]D 25 +21 (c 0.1, CHCl3); IR (neat) νmax 3471, 2974, 1731, 1373, 1248, 1041, 739 cm−1; 1H NMR (C6D6, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data in Tables 1 and 2; HRESIMS m/z 645.3975 [M + Na]+ (calcd for C35H58O9Na, 645.3978).

3.4. Cytotoxicity Assay

Cytotoxicity was determined on P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma), and A-549 (human lung epithelial carcinoma) tumor cells using a modification of the MTT colorimetric method according to a previously described procedure [19,20]. The provision of the P-388 cell line was supported by J.M. Pezzuto, formerly of the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago. HT-29 and A-549 cell lines were purchased from the American Type Culture Collection.

3.5. Anti-HCMV Assay

To determine the effects of natural products upon HCMV cytopathic effect (CPE), confluent human embryonic lung (HEL) cells grown in 24-well plates were incubated for 1 h in the presence or absence of various concentrations of tested natural products. Then, cells were infected with HCMV at an input of 1000 pfu (plaque forming units) per well of 24-well dish. Antiviral activity was expressed as IC50 (50% inhibitory concentration), or compound concentration required to reduce virus induced CPE by 50% after 7 days as compared with the untreated control. To monitor the cell growth upon treating with natural products, an MTT-colorimetric assay was employed [21].