Nitrogen-Containing Diterpenoids, Sesquiterpenoids, and Nor-Diterpenoids from Cespitularia taeniata

Abstract

Two new nitrogen-containing verticillene diterpenoids, cespilamides A and B (1 and 2), three new nitrogen-containing sesquiterpenoids, cespilamides C–E (3–5), and five new norverticillene and verticillene diterpenoids, cespitaenins A–E (6–10), were isolated from the Taiwanese soft coral Cespitularia taeniata. Compound 1 possesses an unusual oxazo ring system at C-10 while compound 2 displays an unprecedented C–C bond cleavage between C-10 and C-11 with an N-ethylphenyl group at C-10. Biogenetic pathways of 1 and 2 are proposed. The absolute configuration of 1 was confirmed by Mosher’s method and molecular mechanics calculations (MM2). The cytotoxicities of compounds 1–10 were evaluated against a small panel of human cancer cell lines.

1. Introduction

Marine invertebrates have been proven to secrete a number of secondary metabolites for self-defense, and those marine natural products usually show unexpected bioactivities. For example, sarcodictyins isolated from Bellonella albiflora and eleutherobins obtained from Eleutherobia aurea showed significant cytotoxicities [1,2]. Aberrarone discovered from gorgonian Pseudopterogorgia elisabethae possessed potent antibacterial effects [3]. Those compounds can benefit new drug development and also inspire drug design. Soft corals of the genus Cespitularia produce various types of terpenoids such as cembranes, neodolabellanes, cespitularanes, and verticillanes [4,5,6,7,8]. These compounds are reported to demonstrate cytotoxic and immune-modulatory activities [9,10,11,12,13,14]. In our continuous research of Taiwanese soft corals, a series of nor-verticillenes and nitrogen-containing verticillanes from C. taeniata were isolated and reported [10,11]. Those findings impel us to further investigate this benthos. In this paper, we describe the isolation and structural elucidation of ten new marine natural products including two nitrogen-containing verticillanes (1 and 2), three nitrogen-containing sesquiterpenes (3–5), two norverticillanes (6 and 7), and three verticillanes (8–10) from Taiwanese soft coral C. taeniata (Figure 1).

Figure 1. Structures of metabolites 1–10.

Figure 1. Structures of metabolites 1–10.

2. Results and Discussion

The EtOAc-MeOH (1:1) extract of C. taeniata was partitioned between H₂O and EtOAc to give an EtOAc-soluble fraction. Extensive column chromatography and HPLC purification allowed the separation of ten new compounds (1–10).

Cespilamide A (1), ${\left[\alpha \right]}_{\text{D}}^{25}$ −118.2 (CH₂Cl₂), had a molecular formula of C₂₂H₃₁O₃N as deduced from the NMR and HRESIMS (m/z 358.2380 [M + Na]⁺, calcd. 358.2382) data, indicating eight indices of hydrogen deficiency. The IR spectrum revealed the presence of hydroxy (3421 cm⁻¹) and conjugated amide (1695 cm⁻¹) moieties. The ¹H and ¹³C-NMR data (Table 1 and Table 2) showed the presence of an amidocarbonyl (δ_C 177.1), a trisubstituted olefinic unit [δ_C 134.8 (s), 132.3 (d); δ_H 5.50, d, J = 8.0 Hz], a tetrasubstituted olefinic moity (δ_C 166.2, 133.5), and an exomethylene group [δ_C 146.2 (s), 114.2 (t); δ_H 4.87, 4.83, each brs]. The DEPT NMR spectrum indicated an oxygenated quarternary carbon (δ_C 102.8), an oxygenated methine carbon (δ_C 68.5 d), eight methylene carbons (δ_C 17.9, 24.4, 32.3, 33.0, 42.5, 43.8, 46.6, 68.8), and three methyl groups (δ_H 1.47, 1.19, 1.59, each 3H and s; δ_C 17.2, 34.3, 35.2). The ¹H–¹H COSY experiment (Figure 2) showed three sets of correlations, H-1′′/H-2′′, H-7/H-6/H-5 and H-3/H-2/H-1/H-14/H-13, and the latter two sets of proton sequences were further connected by the HMBC correlations (Figure 2) of H-18/C-3 (δ_C 33.0), C-4 (δ_C 146.2), and C-5 (δ_C 43.8). Furthermore, the HMBC correlations of CH₃-16, CH₃-17/C-15 (δ_C 37.9), C-1 (δ_C 43.5), C-11 (δ_C 166.2) and H-13/C-12 (δ_C 133.5), C-20 (δ_C 177.1), C-11 indicated that compound 1 possesses a 2′,2′-dimethylcyclohexene moiety. The HMBC correlations of H-9/C-7 (δ_C 134.8), C-8 (δ_C 132.3), C-10 (δ_C 102.8), C-11 and CH₃-19/C-7, C-8, C-9 (δ_C 46.6) were used to establish the planar structure of compound 1, except for the C1′-C2′ moiety. Comparison of the ¹H- and ¹³C-NMR data of 1 with those of cespitulactam D revealed that they have similar verticillene skeletons [12]. ¹H–¹H COSY correlations of H-1′ (δ_H 3.84, m; 4.11, m)/H-2′ (δ_H 3.27, m; 3.90, m) and HMBC correlations of H-2′/C-10, C-20 and H-1′/C-10, C-11 suggested that there is an ethylene moiety between the C-10 oxygen function and the nitrogen of the amide moiety. The configuration of compound 1 was determined by NOESY correlations and the Mosher’s ester method. It was assumed that compound 1 has the same absolute configuration at C-1 as naturally-occurring verticillene diterpenoids, such as cespitulactams, cespitularines, and toxoids [10,12,13]. NOESY (Figure 2) correlations of H-1/Me-16, Me-17 and H-7/ Me-17 indicated the β-orientation of Me-16 and Me-17. Moreover, NOESY correlations of H-6/Me-19/H-9α (δ_H 2.83) and H-7/H-9β (δ_H 2.58) suggested that H-6 is α-oriented. The configuration of the hydroxy group at C-6 was further determined by Mosher’s reactions to yield products 1a and 1b. The results, illustrated in Figure 3, suggested that C-6 has the S configuration. A computer-generated MM2 structure for compound 1 calculated for the lowest energy is illustrated in Figure 3. The result also agreed with a S configuration at C-6. Due to lack of NOE interaction between H-7 and Me-19, the geometry of the 7,8-double bond in 1 was deduced to be E.

Table 1. ¹H-NMR data for compounds 1–10 ^a.

Position	1 b	2 c	3 b	4 b	5 b	6 b	7 b	8 b	9 c	10 b
1	1.59, m	1.44, m	1.39, m	1.59, m	1.32, m	1.66, m	2.18, m	1.60, m	1.46, m	1.43, m
			1.46, m		1.43, m
2	1.54, m	1.25, m	1.56, m	1.66, m	1.61, m	1.50, m	1.12, m	2.24, m	2.30, m	2.27, m
		1.62, m				1.98, m
3	2.11, m	1.97, m	2.01, m	2.36, m	2.00, m	2.68, m	1.93, m	2.15, m	2.08, m	2.13, m
	2.30, m	2.13, m	2.33, m		2.31, m		2.25, m		2.18, m
5	2.38, m	2.19, m	2.18, m	2.18, m	2.11, m	2.28, m	2.73, dd (3.9, 12.6)	2.40, m	2.23, m	2.20, m
						2.50, m			2.65, m	2.60, m
6	4.37, m	4.44, dt (5.5, 8.5)	2.42, m	2.63, m	2.36, m	5.38, dt (8.4, 2.4)	4.55, dt (3.9, 9.6)	4.36, dt (3.9, 7.8)	4.50, dt (3.0, 8.5)	4.40, dt (3.0, 8.7)
			2.60, m		2.57, m
7	5.50, d (8.0)	5.28, d (8.5)				5.15, d (8.4)	5.56, d (9.3)	5.51, d (7.8)	5.45, d (8.5)	5.43, d (8.7)
9	2.58, d (13.8)	2.89, s	5.05, s	5.14, s	5.06, s	3.07, d (15.9)	2.84, d (13.5)	2.85, d (14.1)	2.51, d (14.5)	2.53, d (14.4)
	2.83, d (13.8)					3.40, d (15.9)	3.89, d (13.5)	3.02, d (14.1)	3.02, d (14.5)	3.01, d (14.4)
12		2.31, m				6.20, t (3.6)
		2.50, m
13	1.63, m	1.99, m	1.87, s	1.87, s	1.88, s	2.31, m	4.39, t (3.3)	1.47, m	1.59, m	1.63, m
	2.15, m								1.69, m
14	2.15, m	1.88, m	0.80, s	0.84, s	0.73, s	2.25, m	2.14, m	1.66, m	1.08, m	1.16, m
	2.35, m							2.20, m	1.86, m	1.92, m
15			4.61, s	4.61, s	4.59, s
			4.86, s	4.87, s	4.85, s
16	1.47, s	1.11, s				1.27, s	0.77, s	1.24, s	0.94, s	0.97, s
17	1.19, s	1.03, s				1.20, s	1.47, s	1.44, s	1.32, s	1.31, s
18	4.83, br s	4.81, s				4.80, s	4.92, s	4.83, s	4.92, s	4.92, s
	4.82, br s	4.86, s				4.77, s	4.96, s	4.84, s	4.92, s	4.92, s
19	1.59, s	1.67, s				1.76, s	1.89, s	1.56, s	1.82, s	1.84, s
20									4.46, s	4.56, s
1′	3.84, m	3.51, m	3.74, t (7.5)	3.72, t (7.5)	3.84, dt (7.2, 14.4)			3.43, m	3.50, m	3.56, m
	4.11, m							3.63, m	3.86, m	3.77, m
2′	3.27, m	2.81, t (6.5)	2.86, t (7.5)	2.78, t (7.5)	3.03, t (7.2)			1.20, t (6.9)	1.24, t (7.0)	1.15, t (6.9)
	3.90, m
4′		7.18, d (7.0)	7.17, d (6.6)	7.01, d (8.4)	7.02, d (1.5)
5′		7.22, t (7.0)	7.19, t (6.6)	6.74, d (8.4)	8.05, (N H)
6′		7.31, t (7.0)	7.26, t (6.6)
7′		7.22, t (7.0)	7.19, t (6.6)	6.74, d (8.4)	7.35, d (7.8)
8′		7.18, d (7.0)	7.17, d (6.6)	7.01, d (8.4)	7.18, t (7.2)
9′					7.10, t (7.2)
10′					7.59, d (7.8)
OAc						2.01 s

^a Chemical shifts are in ppm; J values (Hz) are in parentheses. ^b Recorded in CDCl₃ at 300 MHz. ^c Recorded in CDCl₃ at 500 MHz.

Table 1. ¹H-NMR data for compounds 1–10 ^a.

Position	1 b	2 c	3 b	4 b	5 b	6 b	7 b	8 b	9 c	10 b
1	1.59, m	1.44, m	1.39, m	1.59, m	1.32, m	1.66, m	2.18, m	1.60, m	1.46, m	1.43, m
			1.46, m		1.43, m
2	1.54, m	1.25, m	1.56, m	1.66, m	1.61, m	1.50, m	1.12, m	2.24, m	2.30, m	2.27, m
		1.62, m				1.98, m
3	2.11, m	1.97, m	2.01, m	2.36, m	2.00, m	2.68, m	1.93, m	2.15, m	2.08, m	2.13, m
	2.30, m	2.13, m	2.33, m		2.31, m		2.25, m		2.18, m
5	2.38, m	2.19, m	2.18, m	2.18, m	2.11, m	2.28, m	2.73, dd (3.9, 12.6)	2.40, m	2.23, m	2.20, m
						2.50, m			2.65, m	2.60, m
6	4.37, m	4.44, dt (5.5, 8.5)	2.42, m	2.63, m	2.36, m	5.38, dt (8.4, 2.4)	4.55, dt (3.9, 9.6)	4.36, dt (3.9, 7.8)	4.50, dt (3.0, 8.5)	4.40, dt (3.0, 8.7)
			2.60, m		2.57, m
7	5.50, d (8.0)	5.28, d (8.5)				5.15, d (8.4)	5.56, d (9.3)	5.51, d (7.8)	5.45, d (8.5)	5.43, d (8.7)
9	2.58, d (13.8)	2.89, s	5.05, s	5.14, s	5.06, s	3.07, d (15.9)	2.84, d (13.5)	2.85, d (14.1)	2.51, d (14.5)	2.53, d (14.4)
	2.83, d (13.8)					3.40, d (15.9)	3.89, d (13.5)	3.02, d (14.1)	3.02, d (14.5)	3.01, d (14.4)
12		2.31, m				6.20, t (3.6)
		2.50, m
13	1.63, m	1.99, m	1.87, s	1.87, s	1.88, s	2.31, m	4.39, t (3.3)	1.47, m	1.59, m	1.63, m
	2.15, m								1.69, m
14	2.15, m	1.88, m	0.80, s	0.84, s	0.73, s	2.25, m	2.14, m	1.66, m	1.08, m	1.16, m
	2.35, m							2.20, m	1.86, m	1.92, m
15			4.61, s	4.61, s	4.59, s
			4.86, s	4.87, s	4.85, s
16	1.47, s	1.11, s				1.27, s	0.77, s	1.24, s	0.94, s	0.97, s
17	1.19, s	1.03, s				1.20, s	1.47, s	1.44, s	1.32, s	1.31, s
18	4.83, br s	4.81, s				4.80, s	4.92, s	4.83, s	4.92, s	4.92, s
	4.82, br s	4.86, s				4.77, s	4.96, s	4.84, s	4.92, s	4.92, s
19	1.59, s	1.67, s				1.76, s	1.89, s	1.56, s	1.82, s	1.84, s
20									4.46, s	4.56, s
1′	3.84, m	3.51, m	3.74, t (7.5)	3.72, t (7.5)	3.84, dt (7.2, 14.4)			3.43, m	3.50, m	3.56, m
	4.11, m							3.63, m	3.86, m	3.77, m
2′	3.27, m	2.81, t (6.5)	2.86, t (7.5)	2.78, t (7.5)	3.03, t (7.2)			1.20, t (6.9)	1.24, t (7.0)	1.15, t (6.9)
	3.90, m
4′		7.18, d (7.0)	7.17, d (6.6)	7.01, d (8.4)	7.02, d (1.5)
5′		7.22, t (7.0)	7.19, t (6.6)	6.74, d (8.4)	8.05, (N H)
6′		7.31, t (7.0)	7.26, t (6.6)
7′		7.22, t (7.0)	7.19, t (6.6)	6.74, d (8.4)	7.35, d (7.8)
8′		7.18, d (7.0)	7.17, d (6.6)	7.01, d (8.4)	7.18, t (7.2)
9′					7.10, t (7.2)
10′					7.59, d (7.8)
OAc						2.01 s

^a Chemical shifts are in ppm; J values (Hz) are in parentheses. ^b Recorded in CDCl₃ at 300 MHz. ^c Recorded in CDCl₃ at 500 MHz.

Table 2. ¹³C-NMR data for compounds 1–10 ^a.

Position	1 b	2 c	3 b	4 b	5 b	6 b	7 b	8 b	9 c	10 b
1	43.5 d	47.1 d	39.5 t	39.5 t	39.2 t	43.1 d	46.8 d	44.0 d	44.2 d	44.5 d
2	32.3 t	27.7 t	23.2 t	23.2 t	23.1 t	30.6 t	32.9 t	17.6 t	26.2 t	25.4 t
3	33.0 t	34.3 t	36.3 t	36.3 t	36.2 t	31.3 t	39.2 t	33.6 t	37.8 t	37.9 t
4	146.2 s	145.7 s	148.6 s	148.8 s	148.8 s	146.4 s	144.8 s	145.9 s	145.8 s	147.2 s
5	43.8 t	43.9 t	48.9 d	49.0 d	48.8 d	41.1 t	46.8 t	43.7 t	45.8 t	47.1 t
6	68.5 d	65.8 d	22.3 t	22.3 t	22.1 t	72.3 d	70.2 d	68.2 d	69.2 d	69.2 d
7	134.8 d	132.5 d	139.8 s	139.8 s	140.0 s	129.0 d	132.7 d	135.6 d	133.2 d	134.1 d
8	132.3 s	132.9 s	137.1 s	137.3 s	137.2 s	133.3 s	133.2 s	131.4 s	132.8 s	131.1 s
9	46.6 t	47.5 d	119.1 d	119.6 d	119.1 d	50.7 t	49.4 t	47.0 t	41.0 t	41.3 t
10	102.8 s	170.2 s	38.7 s	37.9 s	37.5 s	202.1 s	208.1 s	110.9 s	94.2 s	93.0 s
11	166.2 s	216.0 s	170.0 s	171.1 s	170.2 s	148.0 s	92.2 s	166.6 s	72.8 s	72.4 s
12	133.5 s	37.8 t	123.9 s	124.2 s	124.1 s	135.4 d	214.5 s	129.5 s	78.0 s	79.1 s
13	24.4 t	25.0 t	8.4 q	8.4 q	8.4 q	23.8 t	74.8 d	32.1 t	31.6 t	26.0 t
14	17.9 t	25.9 t	18.6 q	18.6 q	18.3 q	22.8 t	24.3 t	24.4 t	33.9 t	34.4 t
15	37.9 s	48.9 s	107.0 t	107.1 t	106.8 t	35.4 s	46.8 s	37.4 s	37.6 s	37.5 s
16	35.2 q	22.8 q				32.8 q	25.8 q	33.7 q	25.1 q	25.0 q
17	34.3 q	19.9 q				24.8 q	26.5 q	24.5 q	26.0 q	26.1 q
18	114.2 t	113.0 t				113.5 t	115.5 t	114.5 t	115.6 t	114.0 t
19	17.2 q	16.7 q				19.5 q	17.6 q	17.1 q	17.3 q	16.5 q
20	177.1 s							170.5 s	103.5 d	107.3 d
1′	68.8 t	40.5 t	41.0 t	41.1 t	39.9 t			58.8 t	65.2 t	65.4 t
2′	42.5 t	35.2 t	35.4 t	34.3 t	24.8 t			15.1 q	15.0 q	14.8 q
3′		138.7 s	139.2 s	130.8 s	113.3 s
4′		128.7 d	128.9 d	130.0 d	121.9 d
5′		126.5 d	126.4 d	115.4 d
6′		128.6 d	128.5 d	154.6 s	124.7 s
7′					111.1 d
8′					121.9 d
9′					119.3 d
10′					118.6 d
11′					127.6 s
OAc						170.1 s
						21.3 q

^a Multiplicities (s = C, d = CH, t = CH₂, q = CH₃) and assignments made by HMQC and HMBC techniques. ^b Recorded in CDCl₃ at 75 MHz. ^c Recorded in CDCl₃ at 125 MHz.

Table 2. ¹³C-NMR data for compounds 1–10 ^a.

Position	1 b	2 c	3 b	4 b	5 b	6 b	7 b	8 b	9 c	10 b
1	43.5 d	47.1 d	39.5 t	39.5 t	39.2 t	43.1 d	46.8 d	44.0 d	44.2 d	44.5 d
2	32.3 t	27.7 t	23.2 t	23.2 t	23.1 t	30.6 t	32.9 t	17.6 t	26.2 t	25.4 t
3	33.0 t	34.3 t	36.3 t	36.3 t	36.2 t	31.3 t	39.2 t	33.6 t	37.8 t	37.9 t
4	146.2 s	145.7 s	148.6 s	148.8 s	148.8 s	146.4 s	144.8 s	145.9 s	145.8 s	147.2 s
5	43.8 t	43.9 t	48.9 d	49.0 d	48.8 d	41.1 t	46.8 t	43.7 t	45.8 t	47.1 t
6	68.5 d	65.8 d	22.3 t	22.3 t	22.1 t	72.3 d	70.2 d	68.2 d	69.2 d	69.2 d
7	134.8 d	132.5 d	139.8 s	139.8 s	140.0 s	129.0 d	132.7 d	135.6 d	133.2 d	134.1 d
8	132.3 s	132.9 s	137.1 s	137.3 s	137.2 s	133.3 s	133.2 s	131.4 s	132.8 s	131.1 s
9	46.6 t	47.5 d	119.1 d	119.6 d	119.1 d	50.7 t	49.4 t	47.0 t	41.0 t	41.3 t
10	102.8 s	170.2 s	38.7 s	37.9 s	37.5 s	202.1 s	208.1 s	110.9 s	94.2 s	93.0 s
11	166.2 s	216.0 s	170.0 s	171.1 s	170.2 s	148.0 s	92.2 s	166.6 s	72.8 s	72.4 s
12	133.5 s	37.8 t	123.9 s	124.2 s	124.1 s	135.4 d	214.5 s	129.5 s	78.0 s	79.1 s
13	24.4 t	25.0 t	8.4 q	8.4 q	8.4 q	23.8 t	74.8 d	32.1 t	31.6 t	26.0 t
14	17.9 t	25.9 t	18.6 q	18.6 q	18.3 q	22.8 t	24.3 t	24.4 t	33.9 t	34.4 t
15	37.9 s	48.9 s	107.0 t	107.1 t	106.8 t	35.4 s	46.8 s	37.4 s	37.6 s	37.5 s
16	35.2 q	22.8 q				32.8 q	25.8 q	33.7 q	25.1 q	25.0 q
17	34.3 q	19.9 q				24.8 q	26.5 q	24.5 q	26.0 q	26.1 q
18	114.2 t	113.0 t				113.5 t	115.5 t	114.5 t	115.6 t	114.0 t
19	17.2 q	16.7 q				19.5 q	17.6 q	17.1 q	17.3 q	16.5 q
20	177.1 s							170.5 s	103.5 d	107.3 d
1′	68.8 t	40.5 t	41.0 t	41.1 t	39.9 t			58.8 t	65.2 t	65.4 t
2′	42.5 t	35.2 t	35.4 t	34.3 t	24.8 t			15.1 q	15.0 q	14.8 q
3′		138.7 s	139.2 s	130.8 s	113.3 s
4′		128.7 d	128.9 d	130.0 d	121.9 d
5′		126.5 d	126.4 d	115.4 d
6′		128.6 d	128.5 d	154.6 s	124.7 s
7′					111.1 d
8′					121.9 d
9′					119.3 d
10′					118.6 d
11′					127.6 s
OAc						170.1 s
						21.3 q

^a Multiplicities (s = C, d = CH, t = CH₂, q = CH₃) and assignments made by HMQC and HMBC techniques. ^b Recorded in CDCl₃ at 75 MHz. ^c Recorded in CDCl₃ at 125 MHz.

Figure 2. COSY (bold bond), HMBC (arrow) and selected NOESY correlations of 1.

Figure 2. COSY (bold bond), HMBC (arrow) and selected NOESY correlations of 1.

Figure 3. Mosher reaction products (1a, 1b), Data are difference values of Δ_S-R (ppm); and computer-generated perspective model of 1.

Figure 3. Mosher reaction products (1a, 1b), Data are difference values of Δ_S-R (ppm); and computer-generated perspective model of 1.

Cespilamide B (2), ${\left[\alpha \right]}_{\text{D}}^{25}$ −8.0 (CH₂Cl₂), was assigned a molecular formula of C₂₇H₃₉O₃N, as deduced from the HRESIMS (m/z 448.2825 [M + Na]⁺, calcd. 448.2827), indicating nine indices of hydrogen deficiency. The presence of hydroxy, amide, and benzyl functionalities was indicated by IR absorptions at 3371, 1701, and 1647 cm⁻¹. The ¹H and ¹³C-NMR spectra revealed the presence of a ketocarbonyl (δ_C 216.0), an amide carbonyl (δ_C 170.2), a trisubstituted olefin [δ_C 132.9 (s), 132.5 (d); δ_H 5.28, d, J = 8.5 Hz], a 1,1-disubstituted olefin (δ_C 145.7) with an exomethylene group (δ_C 113.0; δ_H 4.86, 4.81, each s), an oxygenated methine carbon (δ_C 65.8), and a phenyl group [δ_C 138.7 (s), 128.7 (d, 2C), 126.5 (d, 2C), 128.6 (d); δ_H 7.18, d, J = 7.0 Hz (2H), δ_H 7.22 t, J = 7.0 Hz, δ_H 7.31 t, J = 7.0 Hz (2H)]. Thus, eight degrees of unsaturation were counted, leaving one further ring to be elucidated. The ¹H–¹H COSY (Figure 4) correlations of H-7/H-6/H-5, H-3/H-2/H-1/H-14/H-13/H-12, NH (δ_H 5.71, brs)/H-1′/H-2′ and H-4′/H-5′/H-6′/H-7′/H-8′ revealed the sequences of three fragments including H-5 to H-7, H-3 to H-12 and a benzylethyl amine side chain. The HMBC correlations (Figure 4) of H-9/C-10, C-8, H-12/C-11, Me-16/C-11, Me-17/C-11 and H-1′/C-10 permitted assignment of the two carbonyls at C-10 and C-11. Also, it established the connectivity between C-10 and C-1′. The absence of HMBC correlations between H-9/C-11, and H-12/C-10 indicated that compound 2 represents an unusual C-20 norditerpenoid [13] with bond cleavage between C-10 and C-11. The relative configuration of compound 2 was determined by NOESY experiments (Figure 5) and computer-generated perspective models using the MM2 force field calculation. A NOESY correlation between Me-19 and H-6, and the lack of a correlation between Me-19 and H-7 suggested that the 7,8-double bond has an E geometry, similar to compound 1.

Figure 4. COSY (bold bond) and HMBC (arrow) correlations of 2.

Figure 4. COSY (bold bond) and HMBC (arrow) correlations of 2.

Figure 5. Selected NOESY correlations and computer-generated perspective model of 2.

Figure 5. Selected NOESY correlations and computer-generated perspective model of 2.

The HRESIMS determined the molecular formula of compound 3 as C₂₃H₂₇ON (m/z 356.1992 [M + Na]⁺, calcd. 356.1990) and indicated eleven degrees of unsaturation. The IR absorption of 1676 cm⁻¹ suggested the presence of a conjugated amide group. The ¹H, ¹³C (Table 1 and Table 2) and DEPT NMR spectroscopic data revealed the presence of an amide carbonyl (δ_C 170.0), a trisubstituted olefin [δ_C 137.1 (s), 119.1 (d); δ_H 5.05, s], an exomethylene group [δ_C 148 (s), 107.0 (t); δ_H 4.86, 4.61, each s], a tetrasubstituted olefin (δ_C 123.9, 139.8), a phenyl group [δ_C 139.2 (s), 128.9 (d, 2C), 126.4 (d, 2C), 128.5 (d); δ_H 7.17, d, J = 6.6 Hz (2H), δ_H 7.19, t, J = 6.6 Hz (2H), δ_H 7.26, t, J = 6.6 Hz], an aliphatic CH group (δ_H 2.18, m; δ_C 48.9), and four aliphatic CH₂ group (δ_C 39.5, 23.2, 36.2, 22.3). The above findings accounted for five of the eight degrees of unsaturation, indicating that compound 3 is a tricyclic sesquiterpene with a phenyl group. ¹H–¹H COSY spectrum of 3 showed four sets of correlations, H-1/H-2/H-3, H-5/H-6, H-1′/H-2′, and H-4′/ H-5′/ H-6′/ H-7′/ H-8′. The HMBC correlations (Figure 6) of H₂-15/C-2, C-4, C-5 confirmed an exocyclic double bond between C-3 and C-5. The HMBC correlations of CH₃-13/C-12, C-11, C-7; Me-14/C-10, C-1, C-9, C-5, and H-9/C-10, C-8, C-7 not only suggested the occurrence of double bonds between C-7/C-12 and C-8/C-9 but also assign the methyl group at C-10 and C-12. The presence of an α,β-unsaturated δ-lactam was inferred from the IR and HMBC spectra. Moreover, the HMBC correlations of H-1′/C-11, C-8 and H-2′/C-3′, C-4′, C-8′ indicated an amide carbonyl at C-11 and a phenylethyl side chain attached to a nitrogen atom. The relative configuration of 3 was determined on the basis of NOESY experiment and comparison with the optical rotation and NMR data of recent published compounds, taenialactams A and B, which were isolated from C. taeniata [14]. Assuming that H-5 possesses an α-orientation similar to that of taenialactams, the lack of NOESY correlation between H-5 and Me-14, suggested that Me-14 is β-oriented (Figure 7).

Figure 6. COSY (bold bond), HMBC (arrow) correlations of 3.

Figure 6. COSY (bold bond), HMBC (arrow) correlations of 3.

Figure 7. Selected NOESY correlation of 3.

Figure 7. Selected NOESY correlation of 3.

The molecular formula of 4 was determined to be C₂₃H₂₇O₂N (Δ = 11) by HRESIMS data (m/z 372.1937 [M + Na]⁺, calcd. 372.1939). The IR spectrum revealed the presence of hydroxy (3421 cm⁻¹) and α,β-unsaturated γ-lactam (1695 cm⁻¹) moieties. The ¹H and ¹³C NMR spectra (Table 1 and Table 2) of compound 4 were similar to those of 3, suggesting structural similarity with the exception that compound 4 contains a para-hydroxyphenylethyl side chain [δ_H 7.01, d, J = 8.4 Hz (2H), 6.74, d, J = 8.4 Hz (2H); δ_C 154.6 (s), 130.8 (s), 130.0 (d), 115.4 (d), 41.0 (t), 34.3 (t)] on the nitrogen atom, rather than a phenylethyl group as found in compound 3. Interpretation of ¹H–¹H COSY and HMBC spectra of compound 4 also indicated the presence of a hydroxy group at C-6′. The relative configuration of compound 4 was determined by comparison with the NMR and the optical rotation of compound 3.

The molecular formula of compound 5 was shown to be C₂₅H₂₈ON₂ (Δ = 13), as deduced from HRESIMS at m/z 395.2099 ([M + Na]⁺, calcd. 395.2099). Spectroscopic data of compound 5 were found to be similar to those of 3 and 4 except for the evidence of an ethylindole moiety. The LRMS of compound 5 exhibited a peak at m/z 229 [M + H − C₁₀H₁₀N]⁺, also consistent with the presence of an ethylindole group. In the ¹H and ¹³C NMR spectra (Table 1 and Table 2), signals for a 3-ethylindole group [δ_H 3.84, dt, J = 14.4, 7.2 Hz, 3.03, t, J = 7.2 Hz, 7.02, d, J = 1.5 Hz, 7.35, d, J = 7.8 Hz, 7.18, t, J = 7.2 Hz, 7.10, t, J = 7.2 Hz, 7.59, t, J = 7.8 Hz, 8.05, s (NH); δ_C 39.9 (t), 24.8 (t), 113.3 (s), 121.9 (d), 124.7 (s), 111.1 (d), 121.9 (d), 119.3 (d), 118.6 (d), and 127.6 (s)] were also observed. The 3-ethylindole group on the tertiary nitrogen in 5 was revealed by detailed analysis of 2D NMR spectra (Figure 8). The HMBC correlations of H-1′/C-11 (δ_C 170.2), C-8 (δ_C 137.2) as well as correlations of H-2′/C-3′, C-4′ and C-11′ indicated that the phenylethyl side chain at the nitrogen in compound 3 was replaced by the 3-ethylindole group in compound 5. Assignment of the ¹H and ¹³C-NMR spectroscopic data of 5 were accomplished by application of ¹H–¹H COSY, HMQC, and HMBC correlations. The relative configuration of compound 5 was assigned the same as those of compounds 3 and 4.

Figure 8. COSY (bold bond) and HMBC (arrow) correlations of 5.

Figure 8. COSY (bold bond) and HMBC (arrow) correlations of 5.

Cespitaenin A (6) was isolated as a colorless, amorphous solid. The molecular formula, C₂₁H₃₀O₃, was established by the HRESIMS at m/z 353.2096 [M + Na]⁺ (calcd. 353.2093). The IR bands at 1720 and 1706 cm⁻¹ were attributed to an ester and a carbonyl group, which were confirmed by the presence of the acetate (δ_C 170.1) and ketocarbonyl (δ_C 202.1). The ¹³C-NMR (Table 2) and DEPT spectra of compound 6 revealed 21 carbons including three methyl carbons (δ_C 19.5, 24.8, and 32.8), six aliphatic methylene carbons (δ_C 30.6, 31.3, 41.1, 50.7, 23.8 and 22.8), a methine carbon (δ_C 43.1), an oxygenated methine carbon (δ_C 72.3), an aliphatic quaternary carbon (δ_C 35.4), two olefinic methine carbons (δ_C 129.0 and 135.4), an olefinic methylene carbon (δ_C 113.5), three olefinic quaternary carbons (δ_C 146.4, 133.3, and 148.0), and two additional carbonyl signals. The ¹H–¹H COSY spectrum showed the connectivities of H-7/H-6/H-5 and H-3/H-2/H-1/H-14/H-13/H-12. Resonances at δ_C 133.3 (C-8) and 129.0 (C-7) were correlated in the HMBC spectrum with proton signals at δ_H 5.15 (d, J = 8.4 Hz, H-7), and with the vinylic methyl protons at δ_H 1.76 (Me-19), and suggested that compound 6 contains an E-trisubstituted double bond bearing a methyl group [14]. In addition, a trisubstituted double bond [δ_C 148.0 (s), 135.4 (d), δ_H 6.30, t, J = 8.4 Hz] and a 1,1-disubstituted olefin (δ_C 144.7) with an exomethylene group (δ_C 115.5; δ_H 4.87, 4.95, each s) were also implied by interpretation of the HMBC data of compound 6. Moreover, HMBC correlations of δ_H 5.38 (dt, J = 8.4, 2.4 Hz, H-6) with δ_C 170.1 indicated that C-6 (δ_C 72.3) is attached to an acetoxy group (δ_C 21.3). HMBC correlations of H-12/C-11, C-10, C-15, H-9/C-10, C-11, Me-16/C-11, C-15, C-1 and Me-17/C-11, C-15, C-1, H-18/C-3, C-5 established the final structure of 6. The relative configuration of 6 was determined by NOESY analysis and comparison of the coupling constants of 6 with the data reported [14,15,16,17]. Assuming that H-1 is at the β position, the correlations between H-1/Me-16/Me-17 indicated the β-disposition of Me-16 and Me-17. The spin pattern and coupling constants of H-6, and NOESY correlations of H-6/Me-19/H-9α and H-7/H-9β agreed with a β-orientation of the acetoxy group at C-6.

Cespitaenin B (7), ${\left[\alpha \right]}_{\text{D}}^{25}$ −109 (CH₂Cl₂), was isolated as a colorless, amorphous solid. Its molecular formula was determined to be C₁₉H₂₈O₅ (Δ= 6) from HRESIMS at m/z 359.1837 [M + Na]⁺. Its IR bands showed the presence of a hydroxy (3397 cm⁻¹) and conjugated carbonyl (1697 cm⁻¹) groups. The ¹H and ¹³C-NMR spectroscopic (Table 1 and Table 2) and DEPT data indicated the presence of two ketocarbonyls (δ_C 214.5 and 208.1), a trisubstituted olefin [δ_C 133.4 (s), 132.7 (d); δ_H 5.56, d, J = 9.3 Hz], and an exocyclic double bond [δ_C 144.8 (s), 115.5 (t); δ_H 4.92, 4.96, each s). In the aliphatic region, a quaternary carbon (δ_C 46.8), two oxygenated methine carbons (δ_C 70.2 and 74.8), an oxygenated tertiary carbon (δ_C 92.2), five methylene carbons (δ_C 32.9, 39.2, 46.8, 49.4, and 24.3), and three methyl groups (δ_C 25.8, 26.5, and 17.6; δ_H 0.77, 1.47, and 1.89, each s) were observed. HMQC correlations of δ_H 4.55 (dt, J = 9.6, 3.9 Hz, H-6) with δ_C 70.2 (d, C-6) and δ_H 4.39 (t, J = 3.3 Hz, H-13) with δ_C 74.8 (d, C-13) suggested that C-6 and C-13 are hydroxylated. The ¹H–¹H COSY spectrum indicated the connectivities of H-7/H-6/H-5 and H-3/H-2/H-1/H-14/H-13 to be similar with those of compound 6 (Figure 9). The two ketocarbonyls assigned at C-10 and C-12, and the hydroxyl group assigned at C-11 were deduced from the interpretation of HMBC correlations of H-9/C-10, C-11; H-13/C-12, C-11; Me-16, Me-17/C-1, C-11, C-15; OH-11 (δ_H 3.13, br s)/C-11, C-10, C-12. The remaining HMBC correlations of Me-16/C-15, C-1, Me-17/C-15, C-1 also indicated that compound 7 has the same 6/12 bicyclic system as compound 6. The NOESY spectrum showed correlations of H-1/Me-16, Me-17, OH-11/Me-16 indicating that the hydroxy on C-11 is β-oriented, while H-6 is α-oriented due to the correlations of H-6/Me-19/H-9α (δ_H 3.89) and H-7/Me-17/H-9β (δ_H 2.84). The lack of correlations of H-13/H-1, Me-16, Me-17 was consistent with an α-orientation of H-13.

Figure 9. COSY (bold bond) and HMBC (arrow) correlations of 7.

Figure 9. COSY (bold bond) and HMBC (arrow) correlations of 7.

The molecular formula of cespitaenin C (8) was determined to be C₂₂H₃₂O₄, as derived from a quasi-molecular ion at m/z 361.2378 ([M + Na]⁺, calcd. 361.2379), and seven indices of hydrogen deficiency. The IR spectrum displayed absorption bands suggestive of hydroxyl (3385 cm⁻¹) and ester carbonyl (1738 cm⁻¹) moieties. The ¹H and ¹³C-NMR spectra (Table 1 and Table 2) exhibited an exomethylene double bond [δ_C 145.9 (s), 114.5 (t); δ_H 4.83, 4.84, each s], a trisubstituted double bond [δ_C 131.4 (s), 135.6 (d); δ_H 5.51, d, J = 7.8 Hz, H-7), a tetrasubstituted double bond (δ_C 166.6, C-11; 129.5, C-12), and an ester carbonyl (δ_C 170.5), accounting for four degrees of unsaturation. These findings implied that 8 is a tricyclic compound. The ¹H–¹H COSY correlations of H-7/H-6/H-5, H-3/H-2/H-1/H-14/H-13, and H-1′/H-2′, along with the HMBC correlations of H₂-9/C-10, C-11, H-13/C-12, C-11, C-20; Me-16/C-11, C-12; Me-17/C-11, C-12 clearly indicated that compound 8 contains a common verticillene skeleton. HMBC correlations of H-1′/C-10 suggested the ethoxy group at C-10 and thus a carbonyl at C-20 (δ_C 170.5). The relative configuration of compound 8 was deduced from the NOESY analysis and comparison with chemical shifts and coupling constants of cespihypotin V [18]. The NOESY correlations of H-1′/Me-16, H-1/Me-17/Me-17 and H-6/Me-19 indicated that Me-16, Me-17, H-1, and the OEt were β-oriented, while H-6 is α-oriented.

The HRESIMS data of cespitaenin D (9) established the molecular formula of C₂₂H₃₄O₅ (m/z 401.2306, [M + Na]⁺), and indicated six indices of hydrogen deficiency. The IR spectrum displayed an absorption band indicative of hydroxy (3444 cm⁻¹) group. The ¹H and ¹³C-NMR spectroscopic data (Table 1 and Table 2) showed an exomethylene double bond (δ_C 145.8 (s), 115.6 (t); δ_H 4.92, s, 2H), a trisubstituted double bond [δ_C 133.2 (d), 132.8 (s); δ_H 5.45, d, J = 8.5 Hz, H-7), and a tetrasubstituted double bond, revealing two degrees of unsaturation. This implied that compound 9 possesses a tetracyclic ring system. The similar ¹H, ¹³C-NMR, COSY, and HMBC data suggested that 9 should have the same verticillene skeleton as 8. However, HMBC correlations of H-1′/C-20; H-13/C-12, C-11, C-20; H-20/C-12, C-11; Me-16, Me-17/C-11 indicated an ethoxy group at C-20 (δ_C 103.5) and an epoxy ring at C-11 (δ_C 72.8) and C-12 (δ_C 78.0). The epoxy ring at C-11 and C-12 was tentatively assigned the α-configuration due to the steric hindrance of the two β-faced methyl groups (Me-16 and Me-17). NOESY correlations (Figure 10) among H-1/Me-16, Me-17, H-6/Me-19/H-9α (δ_H 3.01) and H-7/H-9β (δ_H 2.53), and lack of NOESY correlation between H-20 and Me-17 indicated the β-orientation of the ethoxy group at C-20 and the α-disposition of H-6.

Figure 10. Selected NOESY correlation of 9.

Figure 10. Selected NOESY correlation of 9.

Cespitaenin E (10) was found to have the same molecular formula, C22H34O5, as 9. It displayed as a sodium adduct ion at m/z 401.2305 ([M + Na]+) in the HRESIMS. There were very few differences between the 1H-NMR spectroscopic data (Table 1) of 9 and 10. Comparison of their 13C-NMR spectra (Table 2) revealed that the differences occurred in the chemical shifts of C-13 (δC 26.0, 10; 31.6, 9) and C-20 (δC 107.3, 10; 103.5, 9). Furthermore, the COSY and HMBC correlations were closely comparable (Supporting Information). The NOESY correlations of H-20/Me-17 in 10 confirmed the β-orientation of H-20. The only difference between 9 and 10 is the configuration of the ethoxy group at C-20. The optical rotations of 10 [${\left[\alpha \right]}_{\text{D}}^{25}$ 0.1 (CH2Cl2)] and 9 [${\left[\alpha \right]}_{\text{D}}^{25}$ −20.6 (CH₂Cl₂)] supported the conclusion to be made that compound 10 is the 20-epimer of cespitaenin D.

A postulated biosynthetic pathway for compounds 1 and 2 is illustrated in Scheme 1. Compound 1 is probably produced from cespitularin C [19] via intermediates a–d, involving steps of oxidation, serine transformation, lactamization, decarboxylation, hydroxylation, and dehydration. Compound 2 may be generated from the nor-verticillene a through intermediates e and f. These reactions deal with decarboxylation, cleavage of the double bond between C-10 and C-11 [19], and phenylalanine transformation leading to an amide formation.

Four human cancer cell lines were chosen to test the in vitro cytotoxicity of compounds 1–10 (Table 3). Compound 5 exhibited cytotoxicity against human breast adenocarcinoma (MCF-7), medulloblastoma (Daoy), and cervical epitheloid carcinoma (Hela) cancer cells with IC₅₀ of 17.5, 22.3, and 24.7 μM, respectively. Compound 6 showed significant cytotoxicity against human breast adenocarcinoma (MCF-7) cancer cells with the IC₅₀ at 21.2 μM.

Scheme 1. A postulated biosynthetic pathway for compounds 1 and 2.

Scheme 1. A postulated biosynthetic pathway for compounds 1 and 2.

Table 3. Cytotoxicity of compounds 1–10 against human cancer cells (IC₅₀, μM) ^a.

Compound	Hela	Daoy	WiDr	MCF-7
3	30.9	34.8	49.5	30.6
5	24.7	22.3	34.1	17.5
6	28.5	31.5	36.4	21.2
mitomycin C	0.32	0.32	0.32	0.32

^a Hela: human cervical epitheloid carcinoma; Daoy: human medulloblastoma; WiDr: Human colon adenocarcinoma; MCF-7: human breast adenocarcinoma; ^b Compounds 1, 2, 4, 7–10 were inactive (>40 μM) in this assay system.

Table 3. Cytotoxicity of compounds 1–10 against human cancer cells (IC₅₀, μM) ^a.

Compound	Hela	Daoy	WiDr	MCF-7
3	30.9	34.8	49.5	30.6
5	24.7	22.3	34.1	17.5
6	28.5	31.5	36.4	21.2
mitomycin C	0.32	0.32	0.32	0.32

^a Hela: human cervical epitheloid carcinoma; Daoy: human medulloblastoma; WiDr: Human colon adenocarcinoma; MCF-7: human breast adenocarcinoma; ^b Compounds 1, 2, 4, 7–10 were inactive (>40 μM) in this assay system.

3. Experimental Section

3.1. General Experimental Procedures

Optical rotations were obtained on a JASCO DIP-1000 polarimeter. IR spectra were recorded using a Horiba FT-720 spectrophotometer. The ¹H and ¹³C-NMR spectra as well as 2D NMR spectra (¹H–¹H COSY, HSQC, HMBC, and NOESY) were recorded in CDCl₃ (or CD₃OD) using Bruker DRX NMR spectrometers operating at 300 or 500 MHz for ¹H and 75 or 125 MHz for ¹³C using the CDCl₃ solvent peak as internal standard (δ_H 7.26 for ¹H and δ_C 77.0 for ¹³C). Low-resolution ESIMS and HRESIMS were run on a JEOL JMS-HX 110 mass spectrometer. Silica gel 60 (Merck, Darmstadt, Germany) was used for column chromatography (CC). Precoated silica gel plate (Kieselgel 60 F-254, 1 mm, Merck, Darmstadt, Germany) was used for preparative TLC. Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden) was used for separation. LiChrospher Si 60 (5 μm, 250-10, Merck, Darmstadt, Germany) and LiChrospher 100 RP-18e (5 μm, 250-10, Merck, Darmstadt, Germany) were used for NP-HPLC and RP-HPLC (Hitachi, Tokyo, Japan), respectively.

3.2. Animal Material

Cespitularia taeniata was collected in Green Island, Taiwan, in March 2004. This soft coral was identified by one of the authors (Y.-C.S.). A voucher specimen (GSC-1) has been deposited in the School of Pharmacy, National Taiwan University, Taipei, Taiwan.

3.3. Extraction and Isolation

The whole animals of C. taeniata (dried, 1.1 kg) were extracted with EtOAc and CH₂Cl₂ (1:1, each 1 L × 3) at room temperature and concentrated under reduced pressure to yield a crude extract. The crude extract was partitioned between H₂O and EtOAc to yield an EtOAc-soluble fraction (100 g), which was chromatographed on a Si gel column (1 kg) and initially eluted with n-hexane (100%, 1 L), n-hexane/EtOAc (15:1 to 0:1, each 1 L), and finally MeOH (100%, 1 L) to give 12 fractions. Fractions six (3.1 g) and eight (1.7 g) were further separated on a Sephadex LH-20 column using CH₂Cl₂-MeOH (4:1) to furnish nine and five fractions (6-1~6-9, 8-1~8-5), respectively. Separation of fraction 6-5 (390 mg) was performed by a Si gel column (1.2 g) using a solvent mixture of n-hexane-CH₂Cl₂-MeOH (100:100:1~5:5:1) to yield six fractions (6-2-1~6-2-6). Fraction 6-5-3 (34 mg) was further purified with a NP-HPLC column (n-hexane-CH₂Cl₂-MeOH, 15:15:1) to give cespitaenin A (6, 2 mg). Fraction 6-5-4 (121 mg) and fraction 6-5-5 (68 mg) were separated with a NP-HPLC column (CH₂Cl₂-MeOH, 80:1) and then a RP-HPLC column was used (MeOH-H₂O-CH₃CN, 70:25:5) to yield cespitaenin C (8, 6 mg), cespitaenin D (9, 6 mg), cespilamide C (3, 5 mg) and cespitaenin E (10, 2.5 mg). Fraction 6-6 (310 mg) was purified with a NP-HPLC column (CH₂Cl₂-MeOH, 80:1) and with preparative TLC (n-hexane-BuOH, 12:1) to give cespilamide D (4, 9 mg). Fraction 6-8 (16 mg) was further purified with a RP-HPLC column (MeOH-H₂O-CH₃CN, 70:25:5) to yield cespilamide E (5, 5 mg). Fraction 8-4 (779 mg) and 8-5 (68 mg) were further separated with a NP-HPLC column (n-hexane-CH₂Cl₂-MeOH, 20:20:1) and with a RP-HPLC column (MeOH-H₂O-CH₃CN, 65:30:5) to yield cespilamide A (1, 1.5 mg), cespitaenin B (7, 3 mg) and cespilamide B (2, 3 mg).

3.4. Spectral Data

Cespilamide A (1): colorless, amorphous solid; ${\left[\alpha \right]}_{\text{D}}^{25}$ −118 (c 0.2, CH₂Cl₂); IR (neat) ν_max 3421, 2936, 1695 cm⁻¹; ¹H-NMR (CDCl₃, 300 MHz) and ¹³C-NMR (CDCl₃, 75 MHz) data, see Table 1 and Table 2, respectively; HRESIMS m/z 358.2380 ([M + Na]⁺, calcd for C₂₂H₃₁O₃NNa⁺, 358.2382).

Cespilamide B (2): colorless, amorphous solid; ${\left[\alpha \right]}_{\text{D}}^{25}$ −8.2 (c 0.2, CH₂Cl₂); IR (neat) ν_max 3371, 2929, 2360, 1701, 1647 cm⁻¹; ¹H-NMR (CDCl₃, 500 MHz) and ¹³C-NMR (CDCl₃, 125 MHz) data, see Table 1 and Table 2, respectively; HRESIMS m/z 448.2825 ([M + Na]⁺, calcd for C₂₇H₃₉O₃NNa⁺, 448.2827).

Cespilamide C (3): colorless, amorphous solid; ${\left[\alpha \right]}_{\text{D}}^{25}$ 15.5 (c 0.2, CH₂Cl₂); IR (neat) ν_max 2926, 1676 cm⁻¹; ¹H-NMR (CDCl₃, 300 MHz) and ¹³C-NMR (CDCl₃, 75 MHz) data, see Table 1 and Table 2, respectively; HRESIMS m/z 356.1992 ([M + Na]⁺, calcd for C₂₃H₂₇ONNa⁺, 356.1990).

Cespilamide D (4): colorless, amorphous solid; ${\left[\alpha \right]}_{\text{D}}^{25}$ 18.2 (c 0.2, CH₂Cl₂); IR (neat) ν_max 3312, 2927, 1649 cm⁻¹; ¹H-NMR (CDCl₃, 300 MHz) and ¹³C-NMR (CDCl₃, 75 MHz) data, see Table 1 and Table 2, respectively; HRESIMS m/z 372.1937 ([M + Na]⁺, calcd for C₂₁H₃₀O₃Na⁺, 372.1939).

Cespilamide E (5): colorless, amorphous solid; ${\left[\alpha \right]}_{\text{D}}^{25}$ 23.6 (c 0.2, CH₂Cl₂); IR (neat) ν_max 2929, 1659, 1340 cm⁻¹; ¹H-NMR (CDCl₃, 300 MHz) and ¹³C-NMR (CDCl₃, 75 MHz) data, see Table 1 and Table 2, respectively; HRESIMS m/z 395.2099 ([M + Na]⁺, calcd for C₂₅H₂₈ON₂Na⁺, 395.2099).

Cespitaenin A (6): colorless, amorphous solid; ${\left[\alpha \right]}_{\text{D}}^{25}$ 9.7 (c 0.2, CH₂Cl₂); IR (neat) ν_max 1720, 1706 cm⁻¹; ¹H-NMR (CDCl₃, 300 MHz) and ¹³C-NMR (CDCl₃, 75 MHz) data, see Table 1 and Table 2, respectively; HRESIMS m/z 353.2096 ([M + Na]⁺, calcd for C₂₁H₃₀O₃Na⁺, 353.2093).

Cespitaenin B (7): colorless, amorphous solid; ${\left[\alpha \right]}_{\text{D}}^{25}$ −109 (c 0.2, CH₂Cl₂); IR (neat) ν_max 3397, 2359, 2339, 1697, 1276 cm⁻¹; ¹H-NMR (CDCl₃, 300 MHz) and ¹³C-NMR (CDCl₃, 75 MHz) data, see Table 1 and Table 2, respectively; HRESIMS m/z 359.1837 ([M + Na]⁺, calcd for C₁₉H₂₈O₅ Na⁺, 359.1834).

Cespitaenin C (8): colorless, amorphous solid; ${\left[\alpha \right]}_{\text{D}}^{25}$ −35.5 (c 0.2, CH₂Cl₂); IR (neat) ν_max 3385, 2924, 1738 cm⁻¹; ¹H-NMR (CDCl₃, 300 MHz) and ¹³C-NMR (CDCl₃, 75 MHz) data, see Table 1 and Table 2, respectively; HRESIMS m/z 361.2378 ([M + Na]⁺, calcd for C₂₂H₃₂O₄Na⁺, 361.2379).

Cespitaenin D (9): colorless, amorphous solid; ${\left[\alpha \right]}_{\text{D}}^{25}$ 0.1 (c 0.2, CH₂Cl₂); IR (neat) ν_max 3444, 2986, 2950, 1731 cm⁻¹; ¹H-NMR (CDCl₃, 500 MHz) and ¹³C-NMR (CDCl₃, 125 MHz) data, see Table 1 and Table 2, respectively; HRESIMS m/z 401.2306 ([M + Na]⁺, calcd for C₂₂H₃₄O₅Na⁺, 401.2304).

Cespitaenin E (10): colorless, amorphous solid; ${\left[\alpha \right]}_{\text{D}}^{25}$ −20.6 (c 0.2, CH₂Cl₂); IR (neat) ν_max 3390, 2930, 1757 cm⁻¹; ¹H-NMR (CDCl₃, 300 MHz) and ¹³C-NMR (CDCl₃, 75 MHz) data, see Table 1 and Table 2, respectively; HRESIMS m/z 401.2305 ([M + Na]⁺, calcd for C₂₂H₃₄O₅Na⁺, 401.2304).

3.5. Preparation of (S)- and (R)-MPTA Esters (1a and 1b) from 1

R-(−)- or S-(+)-MPTA chloride (one drop) was added to a solution of 1 (3 mg in 2 mL pyridine) and the solution was allowed to stand at room temperature for 12 h. After purification using preparative LC, the resultant ester (3 mg, 90% yield) was analyzed by ¹H NMR spectroscopic measurement, and ∆ = δ_S − δ_R was calculated. Compound 1a: ¹H-NMR (CDCl₃, 300 MHz) δ_H 5.578 (1H, dd, J = 8.9, 7.2 Hz, H-6), 5.542 (1H, overlap, H-7), 1.199, 1.466 (6H, s, H-16, 17), 4.788 (1H, s, H-18), 4.770 (1H, s, H-18), 1.597 (3H, s, H-19), 4.12 (1H, t, J = 6.6 Hz, H-1′), 3.92 (1H, t, J = 6.6 Hz, H-1′), 3.89 (1H, m, H-2″), 3.25 (1H, m, H-2″); Compound 1b: ¹H-NMR (CDCl₃, 300 MHz) δ_H 5.525 (1H, dd, J = 8.9, 7.2 Hz, H-6), 5.402 (1H, d, J = 8.9 Hz, H-7), 1.189, 1.444 (6H, s, H-16, 17), 4.871 (1H, s, H-18), 4.835 (1H, s, H-18), 1.586 (3H, s, H-19), 4.12 (1H, t, J = 6.6 Hz, H-1′), 3.92 (1H, t, J = 6.6 Hz, H-1′), 3.87 (1H, m, H-2″), 3.25 (1H, m, H-2″).

3.6. Cytotoxicity Assay

Cytotoxicity was tested against the MCF-7 (breast carcinoma), Daoy (medulloblastoma), DLD-1 (colon adenocarcinoma), and Hela (cervical epitheloid adenocarcinoma) human tumor cell lines. The assay procedure using MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide was carried out as previously described.[20] The cells were cultured in RPMI-1640 medium. After seeding of the cells in a 96-well microplate for 4 h, 20 µL of sample was placed in each well and incubated at 37 °C for three days, and then 20 µL MTT was added and allowed to stand for 5 h. Then the medium was removed and DMSO (200 µL/well) was added and the mixture was shaken for 10 min. The formazan crystals were redissolved and their absorbance was measured on a microtiter plate reader (MR 7000, Dynatech, Scottsdale, USA) at a wavelength of 550 nm. The ED₅₀ value was defined by a comparison with the untreated cells as the concentration of test sample resulting in 50% reduction of absorbance. Mitomycin C was used as the positive control.

4. Conclusions

This paper describes the first isolation of five novel nitrogen-containing diterpenoids and sesquiterpenoids, and five bicyclic verticillenes and nor-verticillenes from Taiwanese soft coral Cespitularia taeniata.