Carijoside A, a Bioactive Sterol Glycoside from an Octocoral Carijoa sp. (Clavulariidae)

Abstract

A new bioactive sterol glycoside, 3β-O-(3′,4′-di-O-acetyl-β-d-arabinopyranosyl) -25ξ-cholestane-3β,5α,6β,26-tetrol-26-acetate) (carijoside A, 1), was isolated from an octocoral identified as Carijoa sp. The structure of glycoside 1 was established by spectroscopic methods and by comparison with spectral data for the other known glycosides. Carijoside A (1) displayed significant inhibitory effects on superoxide anion generation and elastase release by human neutrophils and this compound exhibited moderate cytotoxicity toward DLD-1, P388D1, HL-60, and CCRF-CEM tumor cells.

1. Introduction

Previous studies on the chemical constituents from the octocorals belonging to the genus Carijoa (=Telesto) have yielded a series of bioactive substances including amide [1], steroid [1–4], and prostanoid analogs [3,5]. In our continuing studies on the chemical constituents of octocorals distributed in Taiwan waters, a new sterol glycoside, carijoside A (1) (Figure 1) was isolated from an octocoral identified as Carijoa sp. The structure of 1 was determined by spectroscopic methods and by comparison of spectral data with those of known sterols. In this paper, we describe the isolation, structure determination, and bioactivity of glycoside 1.

2. Results and Discussion

Carijoside A (1) was isolated as a white powder. The molecular formula of 1 was established as C₃₈H₆₂O₁₁ (eight degrees of unsaturation) from a sodiated molecule at m/z 717 in ESIMS and further supported by HRESIMS (m/z 717.4186, calcd. 717.4190, [C₃₈H₆₂O₁₁Na]⁺). The IR spectrum of 1 showed bands at 3466 and 1743 cm⁻¹, consistent with the presence of hydroxy and ester carbonyl groups. Analysis of 2D NMR experiments revealed that carijoside A (1) was a pentose glycoside derivative of a known trihydroxy sterol, cholestane-3β,5α,6β-tetrol-26-acetate (=25ξ-cholestane-3β,5α,6β,26-tetrol-26-acetate) (2), a cytotoxic steroid previously isolated from Telesto riisei, collected from Northeast Pass, Chuuk, Federated States of Micronesia [1] and Mangaratiba, Rio de Janeiro State, Brazil [4]. In addition to the pentose moiety, the ¹³C NMR and DEPT spectra of 1 showed that this compound has 29 carbons for the cholestane carbon with an acetoxy group (

Table 1. ¹H and ¹³C NMR Data for Steroids 1 and 2.

C/H	1		2a
	1Hb/δ	13Cc/δ	1Hd/δ	13Ce/δ
1	1.42, m; 1.60 m	32.2 (CH2)	1.40 m; 1.52 m	32.3 (CH2)
2	1.86 m (2H)	28.6 (CH2)	1.86 m	30.7 (CH2)
3	4.07 m	74.9 (CH)	4.08 m	67.6 (CH)
4	1.69 m; 2.10 m	37.3 (CH2)	1.63 m; 2.08 m	40.6 (CH2)
5		75.9 (C)		76.0 (C)
6	3.54 br s	76.0 (CH)	3.52 br s	75.8 (CH)
7	1.59 m (2H)	34.6 (CH2)	1.60 m	34.3 (CH2)
8	1.74 m	30.1 (CH)	1.72 m	30.2 (CH)
9	1.16 m	45.9 (CH)	1.25j	45.7 (CH)
10		38.4 (C)		38.2 (C)
11	1.38 m (2H)	21.1 (CH2)	1.37 m	21.1 (CH2)
12	1.16 m; 2.02 m	39.9 (CH2)	1.16 m; 1.98 m	39.9 (CH2)
13		42.7 (C)		42.7 (C)
14	1.08 m	55.9 (CH)	1.08 m	55.9 (CH)
15	1.08 m; 1.59 m	24.1 (CH2)	1.08 m; 1.56 m	24.1 (CH2)
16	1.24 m; 1.77 m	28.2 (CH2)	1.22 m; 1.83 m	28.2 (CH2)
17	1.10 m	56.2 (CH)	1.11 m	56.2 (CH)
18	0.68 s	12.1 (CH3)	0.67 s	12.1 (CH3)
19	1.20 s	17.0 (CH3)	1.17 s	17.0 (CH3)
20	1.36 m	35.7 (CH)	1.37 m	36.0 (CH)
21	0.90 d (6.0)f	18.7 (CH3)	0.92 d (7.0)	18.6 (CH3)
22	1.00 m; 1.33 m	36.0 (CH2)	1.00 m; 1.37 m	35.7 (CH2)
23	1.33 m (2H)	23.3 (CH2)	1.37 m	23.3 (CH2)
24	1.13 m; 1.27 mg	33.9 (CH2)	1.72 m	33.9 (CH2)
		33.7		33.7
25	1.76 m	32.5 (CH)	1.77 m	32.5 (CH)
		32.5		32.4
26a	3.82 dd (10.4, 1.6)	69.6 (CH2)	3.82 dd (2.5, 10.5)	69.9 (CH2)
	3.85 dd (10.4, 1.6)	69.4	3.84 dd (2.5, 10.5)	69.5
b	3.92 dd (10.4, 6.0)h		3.93 dd (6.0, 10.5)
	3.93 dd (10.4, 6.0)h		3.95 dd (6.0, 10.5)
27	0.92 d (6.8)	16.9 (CH3)	0.90 d (6.0)	16.8 (CH3)
	0.91 d (6.8)	16.8	0.91 d (6.0)
26-OAc		171.3 (C)		171.3 (C)
	2.05 s	20.9 (CH3)i	2.05 s	21.0 (CH3)
1′	5.06 d (4.0)	97.4 (CH)
2′	3.93 dd (10.4, 4.0)	67.3 (CH)
3′	5.13 dd (10.4, 3.2)	70.5 (CH)
4′	5.25 br s	69.4 (CH)
5′	3.65 dd (12.8, 2.0); 3.97 mh	60.9 (CH2)
3′-OAc		170.9 (C)
	2.07 s	21.0 (CH3)i
4′-OAc		170.4 (C)
	2.13 s	21.0 (CH3)i

^a:Data was reported by Maia et al. [4];

^b:Spectrum recorded at 400 MHz in CDCl₃ at 25 °C;

^c:Spectrum recorded at 100 MHz in CDCl₃ at 25 °C;

^d:Spectrum recorded at 500 MHz in CDCl₃;

^e:Spectrum recorded at 50 MHz in CDCl₃;

^f:J values (in Hz) in parentheses;

^g:The ¹H NMR data for these methylene protons were assigned by the assistance of Dept and HMQC spectra;

^h:Signals overlapping;

^i:Data exchangeable;

^j:The coupling pattern for this methine proton was not assigned.

), including five methyls, 12 sp³ methylenes, eight sp³ methines, three sp³ quaternary carbons, and an sp² quaternary carbon. From the ¹³C and ¹H NMR spectra (Table 1), the presence of four oxygenated C atoms at δ_C 74.9 (d, CH-3), 75.9 (s, C-5), 76.0 (d, CH-6), and 69.6 (t, CH₂-26) in the ¹³C NMR spectrum and two oxymethine protons at δ_H 4.07 (1H, m, H-3) and 3.54 (1H, br s, H-6) and a pair of oxygen-bearing methylene protons at δ_H 3.84 (1H, m) and 3.97 (1H, m) in the ¹H NMR spectrum were determined. From the ¹H-¹H COSY spectrum, several different structural units, including C-1/-2/-3/-4, C-6/-7/-8/-9/-11/-12, C-8/-14/-15/-16/-17/-20/-22/-23/-24/-25/-26(-27), and C-20/-21, were identified (Figure 2), which were assembled with the assistance of an HMBC experiment permitted elucidation of the cholestane carbon skeleton of 1. The ring junctions C-18 and C-19 methyl groups were positioned at C-13 and C-10 from the HMBC correlations between H₃-18/C-12, -13, -14, -17 and H₃-19/C-1, -5, -9, -10, respectively (Figure 2). An oxymethine unit at δ_C 76.0 correlated to the methine proton at δ_H 3.54 in the HMQC spectrum, proving the attachment of a hydroxy group at C-6. The remaining hydroxy and acetoxy groups at C-5 and C-26 in the cholestane moiety of 1 were indicated by analysis of HMBC correlations and characteristic NMR signals. However, the doubling of the 26-acetoxymethylene and H₃-27 signals indicate that 1 consists of a stereoisomeric mixture (25R/25S).

Furthermore, the proton NMR signals for the pentose pyranoside between δ_H 3.6–5.3 and by the corresponding ¹³C NMR signals between δ_C 60–71 and for the characteristic sugar anomeric carbon (δ_C 97.4) and its corresponding methine proton (δ_H 5.06) (Tables 1 and

Table 2. NMR Data for the 3′- and 4′-O-Acetyl-arabinopyranoside Components in Glycosides Carijoside A (1), Riisein A (3) and Riisein B (4).

C/H	1		3a		4a
	1H/δ	13C/δ	1H/δ	13C/δ	1H/δ	13C/δ
1′	5.06 d (4.0)	97.4 (CH)	5.04 d (4.5)	97.8 (CH)	5.01 d (3.5)	97.6 (CH)
2′	3.93 dd (10.4, 4.0)	67.3 (CH)	3.94 m	67.3 (CH)	3.80 dd (3.5, 10.5)	68.8 (CH)
3′	5.13 dd (10.4, 3.2)	70.5 (CH)	5.07 dd (3.0, 9.8)	73.1 (CH)	3.94 m	67.8 (CH)
4′	5.25 br s	69.4 (CH)	4.03 br s	68.3 (CH)	5.15 br s	71.1 (CH)
5′	3.65 dd (12.8, 2.0)	60.9 (CH2)	3.66 dd (2.0, 12.5)	62.6 (CH2)	3.68 br d (11.0)	60.5 (CH2)
	3.97 m		3.94 m		3.93 m
3′-OAc		170.9 (C)		170.9 (C)
	2.07 s	21.0 (CH3)	2.17 s	21.2 (CH3)
4′-OAc		170.4 (C)				170.9 (C)
	2.13 s	21.0 (CH3)			2.17 s	21.2 (CH3)

^a:Data was reported by Maia et al. [4].

). ¹H NMR coupling constant analysis of the pyranose ring indicated the presence of a pyranoarabinoside sugar linked to the sterol by a β-glycoside linkage. The attachment of the sugar moiety at C-3 in 1 was based on the key HMBC correlations (Figure 2). The sugar anomeric carbon C-1′ (δ_C 97.4) and the aglycon carbon C-3 (δ_C 74.9) showed correlations with H-3 (δ_H 4.07) and H-1′ (δ_H 5.06), respectively. NMR data also indicated the presence of two additional acetate esters positioned at C-3′ (δ_H 5.13, 1H, dd, J = 10.4, 3.2 Hz; δ_C 70.5, CH) and C-4′ (δ_H 5.25, 1H, br s; δ_C 69.4, CH). Based on detailed analysis, the structure of 1 was found to be similar with those of two known cytotoxic sterol glycosides, riisein A (3β-O-(3′-O-acetyl-β-d-arabinopyranosyl)- 25ξ-cholestane-3β,5α,6β,26-tetrol-26-acetate) (3) and riisein B (3β-O-(4′-O-acetyl-β-d-arabinopyranosyl)-25ξ-cholestane-3β,5α,6β,26-tetrol-26-acetate) (4) [4], with the exception that the 4′-hydroxy group in 3 and 3′-hydoxy group in 4 was replaced by an acetoxy group in 1.

In anti-inflammatory activity testing, glycoside 1 displayed significant inhibitory effects on superoxide anion generation (IC₅₀ = 1.8 μg/mL) and elastase release (IC₅₀ = 6.8 μg/mL) by human neutrophils [6,7] and this compound exhibited moderate cytotoxicity towards DLD-1 (human colon adenocarcinoma), P388D1 (murine macrophage cells), HL-60 (human premyelocytic leukemia), and CCRF-CEM (human T-cell acute lymphoblastic leukemia) tumor cells (ED₅₀ = 9.7, 10.4, 12.0, and 13.1 μg/mL), respectively [8].

3. Experimental Section

3.1. General Experimental Procedures

Melting points were measured on a FARGO apparatus and were uncorrected. Optical rotation values were measured with a JASCO P-1010 digital polarimeter. The Infrared spectra were obtained on a VARIAN DIGLAB FTS 1000 FT-IR spectrophotometer. The NMR spectra were recorded on a VARIAN MERCURY PLUS 400 FT-NMR at 400 MHz for ¹H and 100 MHz for ¹³C, in CDCl₃, respectively, Proton chemical shifts were referenced to the residual CHCl₃ signal (δ_H 7.26 ppm). ¹³C NMR spectra were referenced to the center peak of CDCl₃ at δ_C 77.1 ppm. ESIMS and HRESIMS data were recorded on a BRUKER APEX II mass spectrometer. Gravity column chromatography was performed on silica gel (230–400 mesh, Merck, Darmstadt, Germany). TLC was carried out on precoated Kieselgel 60 F₂₅₄ (0.2 mm, Merck) and spots were visualized by spraying with 10% H₂SO₄ solution followed by heating.

3.2. Animal Material

Specimen of the octocoral Carijoa sp. were collected off the coast of Pingtung county, Southern Taiwan, in August 2008, and this organism was identified by comparison with previous descriptions [9]. The voucher specimen was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

3.3. Extraction and Isolation

The freeze-dried and minced material of Carijoa sp. (wet weight 1588 g, dry weight 422 g) were extracted with a mixture of MeOH and CH₂Cl₂ (1:1). The extract was partitioned between EtOAc and H₂O. The EtOAc layer was separated by silica gel and eluted using hexane/EtOAc (stepwise, 20:1–pure EtOAc) to yield 35 fractions. Fraction 17 was separated by silica gel and eluted using hexane/acetone (stepwise, 20:1–1:1) to afford 1 (1.6 mg, 2:1).

Carijoside A (1): white powder; mp 171–172 °C (decomp.); [α]_D²²-112 (c 0.06, CHCl₃); IR (neat) ν_max 3466, 1743 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) and ¹³C NMR (CDCl₃, 100 MHz) data, see Table 1; ESIMS m/z 717 (M + Na)⁺; HRESIMS m/z 717.4186 (calcd for C₃₈H₆₂O₁₁Na, 717.4190).

3.4. Human Neutrophil Superoxide Anion Generation and Elastase Release

Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Superoxide anion generation was carried out according to procedures described previously [10,11]. Briefly, superoxide anion production was assayed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. Elastase release experiments were performed using MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide as the elastase substrate.

3.5. Cytotoxicity Testing

The cytotoxicity of tested compound 1 was assayed using a modification of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric method. Cytotoxicity assays were carried out according to the procedures described previously [12,13].