Briarenols I—K, New Anti-inflammatory 8,17-Epoxybriaranes from the Octocoral Briareum excavatum (Briareidae)

Abstract

Five 8,17-epoxybriaranes, including three new compounds—briarenols I–K (1–3), along with two known analogues, briaexcavatolide P (4) and briaexcavatin P (5), were isolated from the octocoral Briareum excavatum. The structures of briaranes 1–3 were elucidated by spectroscopic methods, including 1D and 2D NMR studies and (+)-HRESIMS. Briarane 4 exerted inhibition effects on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) release from RAW 264.7.

1. Introduction

Octocorals of the genus Briareum (family Briareidae) [1,2,3,4] are proven to be the most important source to produce briarane-type diterpenoids [5]. The compounds of this type are only found in marine invertebrates, particularly in octocorals and demonstrated a wide spectrum of bioactivities, such as anti-inflammatory activity [6] and cytotoxicity [7]. In our continuing research into the chemical constituents of an octocoral B. excavatum (Nutting 1911), which was distributed extensively in the coral reefs of Taiwan, have resulted in isolation of three previously unreported 8,17-epoxybriaranes–briarenols I–K (1–3) along with two known analogues, briaexcavatolide P (4) [8] and briaexcavatin P (5) [9], (Figure 1). In the current study, the comprehensive workflow of isolation, structure determination, and anti-inflammatory activity evaluation, was implemented on briaranes 1–5.

2. Results and Discussion

Briarenol I (1) was isolated as an amorphous powder and displayed a sodiated adduct ion at m/z 649.24677 in the (+)-HRESIMS, which indicated its molecular formula was C₃₀H₄₂O₁₄ (calculated for C₃₀H₄₂O₁₄ + Na, 649.24668; unsaturation degrees = 10). The IR spectrum revealed absorptions for hydroxy (ν_max 3524 cm^–1), γ-lactone (ν_max 1783 cm^–1), and ester carbonyl (ν_max 1736 cm^–1) moieties. Resonances in the ¹³C NMR of 1 at δ_C 172.9, 172.3, 170.5, 170.0, and 170.0 (5 × C) supported the presence of a γ-lactone and four esters (

Table 1. ¹³C NMR (δ_C 100 MHz, CDCl₃) data for briaranes 1–3.

Position	1	2	3
1	43.3, C a	43.7, C	47.7, C
2	87.4, CH	85.6, CH	74.9, CH
3	73.1, CH	73.6, CH	31.6, CH2
4	66.0, CH	65.9, CH	28.4, CH2
5	142.0, C	139.3, C	144.8, C
6	125.5, CH	124.3, CH	118.4, CH
7	74.1, CH	74.0, CH	74.9, CH
8	70.8, C	69.9, C	70.8, C
9	66.2, CH	67.1, CH	67.4, CH
10	40.5, CH	41.4, CH	49.0, CH
11	37.2, CH	36.4, CH	78.2, C
12	66.6, CH	67.0, CH	73.4, CH
13	30.2, CH2	30.4, CH2	30.2, CH2
14	80.5, CH	80.1, CH	74.8, CH
15	18.6, CH3	19.1, CH3	14.3, CH3
16	62.5, CH2	16.8, CH3	27.2, CH3
17	62.5, C	61.8, C	66.5, C
18	10.3, CH3	10.3, CH3	10.4, CH3
19	170.5, C	170.9, C	170.4, C
20	8.9, CH3	8.7, CH3	16.9, CH3
OAc-2	172.9, C		170.2, C
	21.2, CH3		21.2, CH3
OAc-4		169.5, C
		21.0, CH3
OAc-9	170.0, C	169.2, C	168.1, C
	21.5, CH3	21.1, CH3	21.5, CH3
OAc-14	170.0, C	170.0, C	170.4, C
	21.2, CH3	21.0, CH3	21.3, CH3
n-OC(O)Pr-4	172.3, C
	35.9, CH2
	18.2, CH2
	13.7, CH3

^a Multiplicity deduced by DEPT and HSQC spectra.

). Three of the esters were identified as acetates by the presence of three methyl singlet resonances in the ¹H NMR spectrum at δ_H 2.34, 2.15, and 2.08 (

Table 2. ¹H NMR (δ_H, 400 MHz in CDCl₃) data (J in Hz) for briaranes 1–3.

Position	1	2	3
2	4.53 s	3.45 d (10.4)	5.13 d (8.4)
3α/β	4.59 d (12.0)	4.27 d (10.4)	1.67 m; 2.60 ddd (16.0, 14.8, 6.0)
4/4′	6.14 s	6.05 d (1.2)	2.48 br d (16.0); 1.90 m
6	5.53 d (6.0)	5.29 dq (6.4, 1.6)	5.19 s
7	5.62 d (6.0)	5.71 d (6.4)	5.19 s
9	5.32 d (8.8)	5.26 d (9.2)	5.78 d (1.2)
10	2.64 dd (8.8, 4.8)	2.55 dd (9.2, 5.2)	2.14 br s
11	2.41 m	2.47 m
12	4.10 m	4.05 m	3.72 dd (12.4, 4.8)
13α/β	1.75 m; 2.01 m	1.69 m; 2.00 m	1.67 m; 2.04 m
14	4.88 dd (2.8, 2.8)	4.92 dd (2.8, 2.8)	4.79 dd (2.4, 2.0)
15	0.99 s	0.99 br s	1.21 s
16a/b	4.35 dd (13.6, 4.4); 4.04 dd (13.6, 9.6)	1.89 br s	1.99 s
18	1.66 s	1.66 s	1.77 s
20	1.12 d (6.8)	1.07 d (7.2)	1.15 s
OH-2		2.79 d (10.4)
OH-3	4.30 d (12.0)	2.87 d (10.4)
OH-12	1.49 d (4.0)	1.43 d (4.0)	-
OH-16	3.49 dd (9.6, 4.4)
OAc-2	2.08 s		2.00 s
OAc-4		2.14 s
OAc-9	2.34 s	2.32 s	2.22 s
OAc-14	2.15 s	2.16 s	2.03 s
n-OC(O)Pr-4	0.95 t (7.2)
	1.63 tq (7.2)
	2.30 t (7.2)

) and the remaining ester was found to be an n-butyroxy group based on ¹H NMR studies, including a correlation spectroscopy (COSY) experiment, which revealed seven contiguous protons (δ_H 2.30, 2H, t, J = 7.2 Hz; 1.63, 2H, tq, J = 7.2 Hz; 0.95, 3H, t, J = 7.2 Hz). From the COSY spectrum (Figure 2), the proton sequences from H-6/H-7, H-9/H-10/H-11/H-12/H₂-13/H-14, and H-11/H₃-20 were established. The hydroxy proton signals at δ_H 4.30 (1H, d, J = 12.0 Hz), 1.49 (1H, d, J = 4.0 Hz), and 3.49 (1H, dd, J = 9.6, 4.4 Hz) were found to correlate with H-3 (δ_H 4.59, d, J = 12.0 Hz), H-12 (δ_H 4.10, m), and H₂-16 (δ_H 4.35, 1H, dd, J = 13.6, 4.4 Hz; 4.04, 1H, dd, J = 13.6, 9.6 Hz), respectively. Thus, the hydroxy groups should be positioned at C-3, C-12, and C-16, respectively. Olefinic resonances in the ¹³C NMR at δ_C 125.5 (CH-6) and 142.0 (C-5) indicated the presence of a trisubstituted carbon–carbon double bond. On the basis of these data and the heteronuclear multiple bond correlation (HMBC) experiment (Figure 2), the connectivity from C-1 to C-14 was established. A hydroxymethyl group at C-5 was revealed by the HMBC between C-16 oxymethylene protons to C-4, C-5, and C-6. The C-15 methyl group at C-1 was confirmed by the HMBC between H₃-15/C-1, C-2, C-10, C-14, and H-10/C-15. The n-butyrate positioned at C-4 was confirmed from the connectivity between H-4 (δ_H 6.14) and the carbonyl carbon of n-butyrate group (δ_C 172.3). HMBC from the oxymethine protons at δ_H 4.53 (H-2), 5.32 (H-9), and 4.88 (H-14) to the acetate carbonyls at δ_C 172.9, 170.0, and 170.0, placed the acetoxy groups on C-2, C-9, and C-14, respectively. Thirteen of the fourteen oxygen atoms in the molecular formula of 1 could be accounted for from the presence of a γ-lactone, four esters, and three hydroxy groups. The remaining oxygen atom had to be placed between C-8 and C-17 to form a tetrasubstituted epoxide based on the ¹³C NMR evidences at δ_C 70.8 (C-8) and 62.5 (C-17) and the ¹H NMR chemical shift of a tertiary methyl at δ_H 1.66 (3H, s, H₃-18).

The stereochemistry of 1 was deduced from an NOESY experiment (Figure 2) and biogenetic considerations. The NOE correlations of H-10/H-11, H-10/H-12, and H-11/H-12 indicated that these protons were situated on the same face of the structure and were assigned as the α protons since the C-15 methyl is the β-substituent at C-1. The NOE correlation between H₃-15 and H-14 implied that H-14 had a β-orientation. H-3 exhibited a correlation with H-10, and, as well as a lack of coupling constants were detected between H-2/H-3 and H-3/H-4, indicating the dihedral angles between H-2/H-3 and H-3/H-4 were approximately 90° and the 2-acetoxy, 3-hydroxy, and 4-n-butyroxy groups were β-, β-, and α-oriented, respectively. A correlation from H-4 to H-7, suggested that H-7 was β-oriented. The Z-configuration of C-5/6 double bond was confirmed based on the fact that the C-6 olefinic proton (δ_H 5.53) correlated to one of the C-16 hydroxymethyl protons (δ_H 4.04). H-9 was found to correlate with H-11, H₃-18, and H₃-20. From a consideration of molecular model, H-9 was found to be reasonably close to H-11, H₃-18, and H₃-20, thus, H-9 should be placed on the α face, and Me-18 was β-oriented in the γ-lactone moiety, and the 8,17-epoxy group should be α-oriented. It was found that the NMR signals of 1 were similar to those of a known briarane, briaexcavatolide P (4) (Figure 1) [8], except that the signals corresponding to the Me-16 vinyl methyl in 4 were replaced by signals for a hydroxymethyl group in 1. Additionally, as briaranes 1–5 were isolated along with a known briarane, excavatolide B (6) [6,10,11] from the same target organism, B. excavatum, and the absolute configuration of 6 was determined by a single-crystal X-ray diffraction analysis [6,11]. Therefore, on biogenetic grounds to assume that briaranes 1–5 had the same absolute stereochemistry as that of 6, tentatively, and the configurations of stereogenic carbons of 1 were determined as 1R,2R,3S,4R,7S,8S, 9S,10S,11R,12S,14S, and 17R (Supplementary Materials, Figures S1–S10).

Briarenol J (2) had a molecular formula C₂₆H₃₆O₁₂ by its (+)-HRESIMS at m/z 563.21007 (calculated for C₂₆H₃₆O₁₂ + Na, 563.20990). The IR spectrum showed bands at 3483, 1779, and 1727 cm⁻¹, consistent with the presence of hydroxy, γ-lactone, and ester groups, respectively, in 2. From the ¹³C and DEPT data (Table 2), one trisubstituted double bond was deduced from the signals of two carbons at δ_C 139.3 (C-5) and 124.3 (CH-6). A methyl-containing tetrasubstituted epoxy group was confirmed from the signals of two oxygenated quaternary carbons at δ_C 69.9 (C-8) and 61.8 (C-17), and from the chemical shift of a tertiary methyl (δ_H 1.66, 3H, s; δ_C 10.3, CH₃-18; Table 1 and Table 2). Four carbonyl resonances at δ_C 170.9, 170.0, 169.5, and 169.2 in the ¹³C spectrum confirmed the presence of a γ-lactone and three esters. All the esters were identified as acetates by the presence of three methyl singlet resonances in the ¹H NMR spectrum at δ_H 2.32, 2.16, and 2.14, respectively.

Coupling constants information in the COSY spectrum of 2 enabled identification of H-6/H-7, H-9/H-10/H-11/H-12/H₂-13/H-14, H-11/H₃-20, and H-6/H₃-16 (by allylic coupling; Figure 3), these data, together with the HMBC experiment (Figure 3), the molecular framework of 2 could be established. The HMBC also indicated that the acetoxy groups should be attached at C-4, C-9, and C-14, respectively. Thus, the remaining hydroxy groups have to be positioned at C-2, C-3, and C-12, as indicated by the COSY correlations between H-2/OH-2, H-3/OH-3, and H-12/OH-12.

The stereochemistry of 2 was elucidated from the NOE interactions observed in a NOESY experiment (Figure 3) and by the vicinal ¹H−¹H coupling constant analysis. In the NOESY spectrum, correlations were observed between H-10 with H-3 and H-12; and H-12 correlated with H-11, indicating that these protons should be α-oriented. H-14 gave a correlation with H₃-15, confirming the β-orientation for this proton. H-2 showed a correlation with H-14, and a lack of coupling constant was detected between H-2/H-3, indicating the dihedral angle between H-2/H-3 is approximately 90° and the 2-hydroxy group was β-oriented. H-4 exhibited correlations with H-7 and 2-hydroxy proton, confirming the β-orientations for H-4 and H-7. H-9 was found to show correlations with H-11, H₃-18, and H₃-20, and from molecular models, H-9 and H₃-18 should be placed on the α- and β-face, respectively. The Z-configuration of C-5/C-6 double bond was elucidated by a correlation between H-6 and H₃-16. The NMR data of 2 were found to be similar to those of a known briarane, briaexcavatin P (5) [9]. It was found that the 2-acetoxy substituent in 5 was replaced by a hydroxy group in 2. By comparison of the proton and carbon chemical shifts, coupling constants, NOESY correlations, and rotation value of 2 with those of 5, the stereochemistry of 2 was confirmed to be the same as that of 5, and the configurations of the stereogenic centers of 2 were assigned as 1S,2R,3R,4R, 7S,8S,9S,10S,11R,12S,14S, and 17R (Supplementary Materials, Figures S11–S20).

Briarane 3 (briarenol K) was found to have a molecular formula of C₂₆H₃₆O₁₁ based on its (+)-HRESIMS peak at m/z 547.21514 (calculated for C₂₆H₃₆O₁₁ + Na, 547.21498). Its absorption peaks in the IR spectrum showed ester carbonyl, γ-lactone, and broad OH stretching at 1739, 1780, and 3468 cm⁻¹, respectively. The ¹³C NMR spectrum indicated that three esters and a γ-lactone were present, as carbonyl resonances were observed at δ_C 168.1, 170.2, 170.4, and 170.4, respectively (Table 1). The ¹H NMR data also indicated that presence of three acetate methyls at δ_H 2.22, 2.03, and 2.00 (each 3H × s; Table 2). It was found that the spectroscopic data of 3 were similar to those of a known briarane, briareolide B (7) [12]; however, by comparison of the ¹H and ¹³C NMR chemical shifts of CH-12 oxymethine (δ_H 3.72, 1H, dd, J = 12.4, 4.8 Hz; δ_C 73.4), CH₂-13 sp³ methylene (δ_H 1.67, 1H, m; 2.04, 1H, m; δ_C 30.2), C-11 oxygenated quaternary carbon (δ_C 78.2), and Me-20 tertiary methyl (δ_H 1.15, 3H, s; δ_C 16.9) of 3 with those of 7 (δ_H 3.56, 1H, m; δ_C 73.9, CH-12; δ_H 2.03, 1H, m; 2.12, 1H, m; δ_C 27.6, CH₂-13; δ_C 74.7, C-11; δ_H 1.16, 3H, s; δ_C 22.5, Me-20) [12] showed that the hydroxy group at C-12 in 3 was β-oriented. The locations of the functional groups were further confirmed by other HMBC and COSY correlations (Figure 4), and hence briarenol K was assigned the structure of 3. The NOESY spectrum exhibited a correlation from H-10 to H-12, further supporting that H-12 was α-oriented and the stereogenic centers of 3 were assigned as 1S,2S,7S,8S,9S,10S,11S,12S,14S, and 17R, by the correlations observed in a NOESY spectrum (Figure 4) and this compound was found to be the 12-epimer of briareolide B (7) [12] (Supplementary Materials, Figures S21–S30).

The inhibition effects of briaranes 1–5 on the release of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein from lipopolysaccharides (LPS)-stimulated RAW 264.7 were assessed. The results showed that briarane 4 reduced the release of iNOS and COX-2 to 35.37% and 54.61% at a concentration of 10 µM, respectively (Figure 5 and

Table 3. Effects of briaranes 1–5 on LPS-induced pro-inflammatory iNOS and COX-2 protein expression in macrophages.

	iNOS	COX-2	β-Actin
	Expression (% of LPS)	Expression (% of LPS)	Expression (% of LPS)	n
Negative Control	1.71 ± 0.13	0.62 ± 0.09	120.48 ± 1.28	2
LPS	100.00 ± 4.53	100.00 ± 6.05	100.00 ± 3.09	4
1	60.27 ± 7.05	80.63 ± 2.32	100.29 ± 2.46	4
2	60.94 ± 4.89	79.65 ± 4.27	98.29 ± 3.35	4
3	66.64 ± 4.79	97.28 ± 5.66	100.49 ± 6.44	4
4	35.37 ± 4.94	54.61 ± 4.03	104.56 ± 2.83	4
5	62.36 ± 5.42	72.63 ± 2.6	104.79 ± 2.76	4
Dexamethasone	41.00 ± 2.63	3.73 ± 0.35	104.24 ± 5.82	2

Data were normalized to those of cells treated with LPS alone, and cells treated with dexamethasone were used as a positive control. Data are expressed as the mean ± SEM (n = 2–4).

). Briarane 1 was found to be weaker than those of 4 in term of reducing the expression of iNOS and COX-2, indicating that the hydroxy group at C-16 in 1 reduced the activity.

3. Materials and Methods

3.1. General Experimental Procedures

Optical rotation values were measured using a Jasco P-1010 digital polarimeter (Japan Spectroscopic, Tokyo, Japan). IR spectra were measured on a Thermo Scientific Nicolet iS5 FT-IR spectrophotometer (Waltham, MA, USA). NMR spectra were taken on a Jeol Resonance ECZ 400 S NMR spectrometer (Tokyo, Japan), using the residual CHCl₃ signal (δ_H 7.26 ppm) and CDCl₃ (δ_C 77.1 ppm) as the internal standard for ¹H and ¹³C NMR, respectively; coupling constants (J) are presented in Hz. ESIMS and HRESIMS were recorded using a Bruker 7 Tesla solariX FTMS system. Column chromatography was carried out with silica gel (230–400 mesh, Merck, Darmstadt, Germany). TLC was performed on plates precoated with Kieselgel 60 F₂₅₄ (0.25-mm-thick, Merck, Darmstadt, Germany), then sprayed with 10% H₂SO₄ solution followed by heating to visualize the spots. Normal-phase HPLC (NP-HPLC) was performed using a system comprised of a Hitachi L-7100 pump (Tokyo, Japan) and a Rheodyne 7725i injection port (Rohnert Park, CA, USA). Reverse-phase HPLC (RP-HPLC) was performed using a system comprised of a Hitachi L-2130 pump (Tokyo, Japan), a Hitachi L-2455 photodiode array detector (Tokyo, Japan), and a Rheodyne 7725i injection port (Rohnert Park, CA, USA). A semipreparative normal-phase column (YMC-Pack SIL, S-5 µm, 250 mm × 20 mm, Sigma-Aldrich, St. Louis, MO, USA) was used for NP-HPLC. A semipreparative reverse-phase column (Luna, 5 µm, C18(2) 100 Å, AXIA Packed, 250 mm × 21.2 mm; Phenomenex, Torrance, CA, USA) was used for RP-HPLC.

3.2. Animal Material

Specimens of B. excavatum were collected in June 2017 by hand with self-contained underwater breathing apparatus (SCUBA) divers off the coast of Lanyu Island (Orchid Island), Taiwan. The samples were then stored in a –20 °C freezer until extraction. A voucher specimen was deposited in the National Museum of Marine Biology and Aquarium, Taiwan (NMMBA-TW-SC-2017-418). Identification of the species of this organism was performed by comparison as described in previous publications [1,2,3,4].

3.3. Extraction and Isolation

The freeze-dried and sliced bodies (wet/dry weight = 1344/568 g) of the specimen were extracted with supercritical CO₂ to give 58.9 g of extract. Partial extract (36.4 g) was then applied on silica gel column and eluted with gradients of n-hexane/EtOAc to furnish fractions A−K. Fraction F was purified by NP-HPLC using a mixture of n-hexane/acetone (4:1) to yield fractions F1−F13. Fraction F6 was repurified by RP-HPLC, using a mixture of MeOH/H₂O (60:40; at a flow rate = 4 mL/min) to afford 4 (6.7 mg). Fraction G was separated by NP-HPLC, using a mixture of n-hexane/acetone (3:1) to yield fractions G1−G12. Fractions G6 and G7 were repurified by RP-HPLC using a mixture of MeOH/H₂O (60:40; at a flow rate = 4.0 mL/min) to afford 5 (1.3 mg) and 3 (1.0 mg), respectively. Fraction H was separated by NP-HPLC using a mixture of n-hexane and acetone (3:1) to yield fractions H1−H18. Fractions H12 and H15 were repurified by RP-HPLC, using a mixture of MeOH/ H₂O (60:40; at a flow rate = 4.0 mL/min) to afford 2 (2.1 mg) and 1 (0.6 mg), respectively.

Briarenol I (1): Amorphous powder; ${[\alpha ]}_{\mathrm{D}}^{22}$ + 207 (c 0.03, CHCl₃), IR (ATR) ν_max 3524, 1783, 1736, 1222, 891 cm⁻¹; ¹³C (100 MHz, CDCl₃) and ¹H (400 MHz, CDCl₃) NMR data (see Table 1 and Table 2); ESIMS: m/z 649 [M + Na]⁺; HRESIMS m/z 649.24677 (calculated for C₃₀H₄₂O₁₄ + Na, 649.24668).

Briarenol J (2): Amorphous powder; ${[\alpha ]}_{\mathrm{D}}^{26}$ + 140 (c 0.08, CHCl₃), IR (ATR) ν_max 3483, 1779, 1727, 1220, 890 cm⁻¹; ¹³C (100 MHz, CDCl₃) and ¹H (400 MHz, CDCl₃) NMR data (see Table 1 and Table 2); ESIMS: m/z 563 [M + Na]⁺; HRESIMS m/z 563.21007 (calculated for C₂₆H₃₆O₁₂ + Na, 563.20990).

Briarenol K (3): Amorphous powder; ${[\alpha ]}_{\mathrm{D}}^{23}$ + 37 (c 0.06, CHCl₃), IR (ATR) ν_max 3468, 1780, 1739, 1255, 892 cm⁻¹; ¹³C (100 MHz, CDCl₃) and ¹H (400 MHz, CDCl₃) NMR data (see Table 1 and Table 2); ESIMS: m/z 547 [M + Na]⁺; HRESIMS m/z 547.21514 (calculated for C₂₆H₃₆O₁₁ + Na, 547.21498).

Briaexcavatolide P (4): Amorphous powder; ${[\alpha ]}_{\mathrm{D}}^{24}$ + 182 (c 0.3, CHCl₃) (ref. [8], [α]27D + 167 (c 1.0, CHCl₃)), IR (ATR) ν_max 3513, 1783, 1731, 1218, 889 cm⁻¹; ¹H and ¹³C NMR data were found to be in agreement with previous study [8]; ESIMS: m/z 633 [M + Na]⁺.

Briaexcavatin P (5): Amorphous powder; ${[\alpha ]}_{\mathrm{D}}^{23}$ + 134 (c 0.05, CHCl₃) (ref. [9], ${[\alpha ]}_{\mathrm{D}}^{25}$ + 198 (c 0.08, CHCl₃)), IR (ATR) ν_max 3503, 1785, 1735, 1240, 889 cm⁻¹; ¹H and ¹³C NMR data were found to be in agreement with previous study [9]; ESIMS: m/z 605 [M + Na]⁺.

3.4. In Vitro Anti-inflammatory Assay

The proinflammatory suppression assay was employed to assess the activities of the isolated compounds 1–5 against the release of iNOS and COX-2 from macrophage cells as the literature reported [13,14,15].

4. Conclusions

B. excavatum was demonstrated to have a wide structural diversity of briarane-type diterpenoids that possessed various pharmacological properties, especially in anti-inflammatory activity. In our continued study on B. excavatum, three previously unreported briaranes, briarenols I–K (1–3), along with the known analogues, briaexcavatolide P (4) and briaexcavatin P (5), were isolated. In the present study, the anti-inflammatory activity of 1–5 was assessed using inhibition of pro-inflammatory iNOS and COX-2 release from macrophages. The results indicated that briaexcavatolide P (4) showed the most potent suppressive effect on iNOS release.