Excavatoids O and P, new 12-hydroxybriaranes from the octocoral Briareum excavatum.

Two new 12-hydroxybriarane diterpenoids, designated as excavatoids O (1) and P (2), were isolated from the octocoral Briareum excavatum. The structures of briaranes 1 and 2 were established on the basis of extensive spectral data analysis. Excavatoid P (2) is the first metabolite which possesses a 6β -chlorine atom in briarane analogues.

1. Introduction

In our research on the chemical constituents of the marine invertebrates collected in Taiwan waters, a series of briarane-type diterpenoid derivatives had been isolated from various octocorals belonging to the genus Briareum (family Briareidae), Ellisella, and Junceella (family Ellisellidae), and the compounds of this type were proven to possess various interesting bioactivities [1-3]. Recently, our further chemical examination of Briareum excavatum has resulted in the isolation of two new highly oxidized briarane-type diterpenoids, excavatoids O (1) and P (2) (Scheme 1). The structures of compounds 1 and 2 were established by spectroscopic methods.

Scheme 1

The Structures of Excavatoids O (1) and P (2).

2. Results and Discussion

Excavatoid O (1) was obtained as a white powder and had a molecular formula C30H42O13, as determined by HRESIMS (C30H42O13 + Na, m/z found 633.2519; calculated 633.2523) indicating 10 degrees of unsaturation. The presence of hydroxy, lactone, and ester groups in 1 were evidenced by the IR absorptions at 3512, 1793, and 1741 cm−1. It was found that the 1H and 13C spectra of 1 in CDCl3 revealed mostly broad peaks when measured at 25 °C. In order to make more reliable assignments of NMR signals of the stabilized conformers, the 1H and 13C NMR spectra of 1 were measured at 0 °C in CDCl3. In the 13C spectrum of 1, five ester carbonyl resonances appeared at δC 173.6, 170.8, 170.1, 169.5, and 169.3 (5 × s) (Table 1). In the above carbonyl carbons, three were identified as acetate carbonyls by the presence of three methyl resonances in the 1H NMR spectrum at δH 2.18, 2.12, and 1.96 (each 3H × s) and one was identified as n-butyrate carbonyl by the presence of seven contiguous protons at δH 0.95 (3H, t, J = 7.2 Hz), 1.64 (2H, m), and 2.23 (2H, m) (Table 1). On the basis of the unsaturation data overall, 1 was concluded to be a briarane diterpenoid molecule possessing five rings. A tetra-substituted epoxide containing a methyl substituent was elucidated from the signals of two oxygenated quaternary carbons at δC 72.6 (s, C-8) and 63.3 (s, C-17); and further confirmed by the proton signal of a methyl singlet at δH 1.57 (3H, s, H3-18). In addition, a tri-substituted epoxide containing a methyl substituent was deduced from the signals of an oxymethine (δH 3.11, 1H, d, J = 8.8 Hz, H-6; δC 63.0, d, C-6), a quaternary oxygen-bearing carbon (δC 62.1, s, C-5), and a methyl singlet at δH 1.35 (3H, s, H3-16).

Table 1

1H and 13C NMR data for diterpenoids 1 and 2.

	1		2
Position	1Ha	13Cb	1Hc	13Cd
1		43.3 (s)f		44.0 (s)
2	5.81 d (2.0)e	69.3 (d)	4.62 s	88.2 (d)
3	5.13 br s	69.9 (d)	5.07 d (11.6)	68.8 (d)
4	2.25 m (2H)	33.7 (t)	5.82 s	70.9 (d)
5		62.1 (s)		77.8 (s)
6	3.11 d (8.8)	63.0 (d)	4.29 s	65.9 (d)
7	4.69 d (8.8)	78.3 (d)	5.26 s	75.7 (d)
8		72.6 (s)		67.5 (s)
9	5.76 s	68.5 (d)	5.36 d (8.4)	66.0 (d)
10	2.18 br s	45.3 (d)	3.52 dd (8.4, 4.4)	39.5 (d)
11	2.32 br s	34.5 (d)	2.54 m	37.3 (d)
12	3.96 br s	69.3 (d)	4.13 m	66.9 (d)
13	1.92 m (2H)	34.8 (t)	1.83 m (α)	30.3 (t)
			1.96 m (β )
14	5.16 br s	73.0 (d)	4.84 br s	80.8 (d)
15	1.52 s	18.2 (q)	0.86 s	18.2 (q)
16	1.35 s	21.1 (q)	1.55 s	22.3 (q)
17		63.3 (s)		60.6 (s)
18	1.57 s	11.1 (q)	1.63 s	10.0 (q)
19		170.8 (s)		170.0 (s)
20	1.19 d (7.2)	16.3 (q)	1.11 d (7.6)	9.0 (q)
OH-3			3.18 d (11.6)
OH-5			2.36 s
OH-12	n.o.g		2.17 br s
2-OAc		169.5 (s)		172.0 (s)
	2.12 s	21.1 (q)	2.03 s	21.1 (q)
9-OAc		169.3 (s)		170.4 (s)
	2.18 s	21.2 (q)	2.43 s	21.4 (q)
14-OAc		170.1 (s)		170.3 (s)
	1.96 s	21.1 (q)	2.17 s	21.3 (q)
3-OCOPr		173.6 (s)
	2.23 m (2H)	35.6 (t)
	1.64 m (2H)	17.8 (t)
	0.95 t (7.2)	13.6 (q)
4-OCOPr				173.9 (s)
			2.33 t (7.6) (2H)	36.3 (t)
			1.66 m (2H)	18.4 (t)
			0.98 t (7.6)	13.7 (q)

Spectra were recorded at 400 MHz at 0 °C;

Spectra were recorded at 100 MHz at 0 °C;

Spectra were recorded at 400 MHz at 25 °C;

Spectra were recorded at 100 MHz at 25 °C;

J values (in Hz) in parentheses;

Multiplicity deduced by DEPT and HMQC spectra and indicated by usual symbols;

n.o. = not observed.

From the 1H-1H COSY experiment of 1 (Table 2), it was possible to establish the separate spin systems that map out the proton sequences from H-2/H-3/H2-4, H-6/H-7, and H-9/H-10. These data, together with the HMBC correlations between H-2/C-1, -4; H-3/C-4; H2-4/C-3, -5, -6; H-7/C-5, -6; H-9/C-1, -7, -8, -10; and H-10/C-1, -2, established the connectivity from C-1 to C-10 in the 10-membered ring (Table 2). The methyl group at C-5 was confirmed by the HMBC correlations between H3-16/C-4, -5, -6. The methylcyclohexane ring, which is fused to the 10-membered ring at C-1 and C-10, was elucidated by the 1H-1H COSY correlations between H-10/H-11/H-12/H2-13/H-14 and H-11/H3-20 and by the HMBC correlations between H-2/C-14; H-9/C-11; H-10/C-11, -12, -14; H-11/C-10, -20; H2-13/C-1; H-14/C-1, -2; and H3-20/C-10, -11, -12. The ring junction C-15 methyl group was positioned at C-1 from the HMBC correlations between H-2/C-15; and H3-15/C-1, -2, -10, -14. In addition, the HMBC correlations also revealed that three acetates should be attached at C-2, C-9, and C-14, respectively. The remaining n-butyrate ester and hydroxy groups were positioned at C-3 and C-12 as indicated by analysis of 1H-1H COSY correlations and characteristic NMR signals analysis (δH 5.13, 1H, br s, H-3; δC 69.9, d, C-3; δH 3.96, 1H, br s, H-12; δC 69.3, d, C-12). These data, together with the HMBC correlations between H-7/C-17, -19 and H3-18/C-8, -17, -19, were used to establish the molecular framework of 1.

Table 2

The 1H-1H COSY and HMBC (H→C) correlations for diterpenoids 1 and 2.

	1		2
Position	1H-1H COSY	HMBC	1H-1H COSY	HMBC
H-2	H-3	C-1, -4, -14, -15, acetate carbonyl	H-3	C-1, -3, -4, -10, -14, acetate carbonyl
H-3	H-2, H2-4	C-4	H-2, H-4, OH-3	C-1, -5
H-4	H-3	C-3, -5, -6	H-3	C-2, -5, -16, n-butyrate carbonyl
H-6	H-7	n.o.	H-7	C-4, -5, -7, -8, -16
H-7	H-6	C-5, -6, -17, -19	H-6	C-5, -6, -9, -19
H-9	H-10	C-1, -7, -8, -10, -11, acetate carbonyl	H-10	C-7, -8, -10, -11, -17, acetate carbonyl
H-10	H-9, H-11	C-1, -2, -11, -12, -14	H-9, H-11	C-1, -8, -9, -11, -12, -15, -20
H-11	H-10, H-12, H3-20	C-10, -20	H-10, H-12, H3-20	C-1, -10, -12, -20
H-12	H-11, H2-13	n.o.	H-11, H2-13	C-20
H-13	H-12, H-14	C-1, -14	H-12, H-14	C-12
H-14	H2-13	C-1, -2, -12, -13, acetate carbonyl	H2-13	C-10, -12
H-15		C-1, -2, -10, -14		C-1, -2, -10, -14
H-16		C-4, -5, -6		C-4, -5, -6
H-18		C-8, -17, -19		C-8, -17, -19
H-20	H-11	C-10, -11, -12	H-11	C-10, -11, -12
OH-3			H-3	C-3
OH-5				C-4, -5, -16
OH-12	n.o.a	n.o.	H-12	C-11

: n.o. = not observed.

Based on previous studies, all naturally occurring briarane-type diterpenoids have the C-15 methyl group as trans to H-10, and these two groups are assigned as β - and α-oriented, respectively, as shown in most briarane derivatives [1-3]. The relative stereochemistry of 1 was established from a NOESY experiment (Figure 1). In the NOESY experiment of 1, the correlations of H-10 with H-2, H-3, H-6, H-9, and H-11; and H-11 correlated with H-12, indicated that these protons are situated on the same face and were assigned as α protons since the C-15 methyl is the β -substituent at C-1. H-14 was found to exhibit a correlation with H3-15, revealing the β -orientation of this proton. The correlations between H3-16 and H-3, H-6, reflected the α-orientation of H3-16. H-7 correlated with H3-15, indicating this proton should be β -oriented. Furthermore, H3-18 showed a correlation with H-9. By detailed analysis of molecular models, H3-18 was found to be reasonably close to H-9 when H3-18 was placed on the β face in the γ-lactone moiety. Based on the above findings, the structure of 1 was elucidated unambiguously.

Figure 1

Selective NOESY correlations of 1.

The molecular formula of excavatoid P (2) was determined as C30H43ClO14 by its HRESIMS (m/z 685.2235, calculated for C30H43ClO14 + Na, 685.2239). The IR spectrum showed bands at 3472, 1784, and 1734 cm−1, consistent with the presence of hydroxy, γ-lactone, and ester groups in 2. From the 13C NMR data of 2 (Table 1), five carbonyl resonances appeared at δC 173.9, 172.0, 170.4, 170.3, and 170.0 (5 × s), confirming the presence of a γ-lactone and four esters in 2; three acetyl methyls (δH 2.43, 2.17, 2.03, each 3H × s) and an n-butyryl group (δH 0.98, 3H, t, J = 7.6 Hz; 1.66, 2H, m; 2.33, 2H, t, J = 7.6 Hz) were also observed. According to the above data, briarane 2 was found to be a tetracyclic compound with a γ-lactone, as no other unsaturated functional group could be found.

1H NMR coupling information in the 1H-1H COSY spectrum of 2 enabled identification of the C-2/-3/-4, C-6/-7, C-9/-10/-11/-12/-13/-14, and C-11/-20 units (Table 2), which were assembled with the assistance of an HMBC experiment (Table 2). The HMBC correlations between protons and quaternary carbons, such as H-2, H-3, H-10, H-11, H3-15/C-1; H-3, H-4, H-6, H-7, H3-16, OH-5/C-5; H-6, H-9, H-10, H3-18/C-8; H-9, H3-18/C-17; and H-7, H3-18/C-19, permitted elucidation of the carbon skeleton. A methyl at C-5 was established by the HMBC correlations between H3-16/C-4, -5, -6 and H-4, H-6, OH-5/C-16. The ring junction C-15 methyl group was positioned at C-1 from the HMBC correlations between H3-15/C-1, -2, -10, -14; and H-10/C-15. The acetate esters at C-2 and C-9 were established by correlations between H-2 (δH 4.62), H-9 (δH 5.36) and the acetate carbonyls observed in the HMBC spectrum of 2. The n-butyrate ester positioned at C-4 was confirmed from the HMBC correlation between H-4 (δH 5.82) and the carbonyl carbon (δC 173.9) of n-butyrate ester. Thus, the remaining acetoxy group was positioned at C-14 as indicated by analysis of 1H-1H COSY correlations and characteristic NMR signals (δH 4.84, 1H, br s, H-14; δC 80.8, d, C-14). The presence of hydroxy groups at C-3 and C-12 was deduced from the 1H-1H COSY correlations between the hydroxy protons (δH 3.18, OH-3 and δH 2.17, OH-12) and H-3 (δH 5.07) and H-12 (δH 4.13), respectively. The C-5 hydroxy group was also confirmed by the HMBC correlations between the hydroxy proton (δH 2.36, OH-5) and C-4, -5, -16. Thus, the remaining chlorine atom in 2 should be attached C-6 by the 1H-1H COSY correlation between H-6 (δH 4.29) and H-7 (δH 5.26) and further supported by the HMBC correlations between H-6/C-4, -5, -7, -8, -16 and H-7, H3-16/C-6.

The relative configuration of 2 was elucidated from the interactions observed in a NOESY experiment and from vicinal proton coupling constant analysis. In the NOESY experiment of 2 (Figure 2), the correlations of H-10 with H-3, H-11, and H-12, indicated that these protons were situated on the same face and were assigned as α protons since the C-15 and C-20 methyls are β -substituents at C-1 and C-11, respectively. H-2 exhibited an interaction with H-3, and no coupling was found between H-2 and H-3, indicating that the dihedral angle between H-2/H-3 is approximately 90° and the acetoxy group at C-2 should be β -oriented. H-14 was found to exhibit a response with H3-15, showing that H-14 has a β -orientation. H-9 was found to show responses with H-11, H3-18, and H3-20. From modeling analysis, H-9 was found to be close to H-11, H3-18, and H3-20 when H-9 was α-oriented. Moreover, H3-16 exhibited correlations with H-3 and H-6, and no coupling constant was detected between H-6 and H-7, suggesting the α-orientation of H3-16 and H-6; and β-orientation of H-7. H-7 exhibited a correlation with H-4, and no coupling was found between H-3 and H-4, indicating that the dihedral angle between H-3 and H-4 is also approximately 90° and the n-butyrate ester group at C-4 was α-oriented. On the basis of the above results, the structure of 2 was elucidated. To the best of our knowledge, briarane 2 is the first briarane which possesses a 6β -chlorine atom.

Figure 2

Selective NOESY correlations of 2.

In the biological activity testing, briaranes 1 and 2 displayed 16.9 and 16.1% inhibitory effects on elastase release by human neutrophils at 10 μg/mL, resepectively [4].

3. Experimental

3.1. General Experimental Procedures

Melting points were determined on a FARGO apparatus and were uncorrected. Optical rotation values were measured with a JASCO P-1010 digital polarimeter at 25 °C. Infrared spectra were obtained on a VARIAN DIGLAB FTS 1000 FT-IR spectrometer. 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, at 25 or 0 °C, respectively. Proton chemical shifts were referenced to the residual CHCl3 signal (δH 7.26 ppm). 13C NMR spectra were referenced to the center peak of CDCl3 at δC 77.1 ppm. ESIMS and HRESIMS data were recorded on a BRUKER APEX II mass spectrometer. 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) and spots were visualized by spraying with 10% H2SO4 solution followed by heating. HPLC was performed using a system comprised of a HITACHI L-7100 pump, a HITACHI photodiode array detector L-7455, and a RHEODYNE 7725 injection port. A normal phase column (Hibar 250 × 10 mm, Merck, silica gel 60, 5 μm,) was used for HPLC.

3.2. Animal Material

Specimens of the octocoral Briareum excavatum were collected and transplanted in 0.6-ton cultivating tanks located in the NMMBA, Taiwan, in December 2003. This organism was identified by comparison with previous descriptions [5-7]. A voucher specimen was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

3.3. Extraction and Isolation

The organism (wet weight 1.0 kg) was collected and freeze-dried. The freeze-dried material was minced and extracted with EtOAc. The extract was separated by silica gel column chromatography, using hexane and hexane/EtOAc mixtures of increased polarity to yield 12 fractions. Fraction 3 was separated by normal phase HPLC, using a mixture of dichloromethane and acetone to afford briarane 2 (0.9 mg, 9/1). Fraction 2 was separated by normal phase HPLC, using a mixture of hexane and EtOAc to afford briarane 1 (13.2 mg, 1/1).

Excavatoid O (1): white powder; mp 137–138 °C; [α]D25 − 39 (c 0.4, CHCl3); IR (neat) νmax 3512, 1793, 1741 cm−1; 13C NMR (CDCl3, 100 MHz) and 1H NMR (CDCl3, 400 MHz) data, see Table 1; ESIMS m/z 633 (M + Na)+; HRESIMS m/z 633.2519 (Calcd for C30H42O13Na, 633.2523).

Excavatoid P (2): white powder; mp 154–155 °C; [α]D25 + 14 (c 0.05, CHCl3); IR (neat) νmax 3472, 1784, 1734 cm−1; 13C NMR (CDCl3, 100 MHz) and 1H NMR (CDCl3, 400 MHz) data, see Table 1; ESIMS m/z 685 (M + Na)+; HRESIMS m/z 685.2235 (Calcd for C30H43ClO14Na, 685.2239).

3.4. Human Neutrophil Elastase Release

Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Elastase release experiments were performed using MeO-Suc-Ala-Ala-Pro-Valp-nitroanilide as the elastase substrate [8,9].