Eight New Diterpenoids and Two New Nor-Diterpenoids from the Stems of Croton cascarilloides

Abstract

From the stems of Croton cascarilloides, eight new diterpenoids, named crotocascarins A–H (1–8), having a crotofolane skeleton were isolated along with two new nor-diterpenoids (9 and 10), named crotocascarins α and β, derived through rearrangement of the crotofolane skeleton. The structures of these compounds were elucidated by means of extensive one- and two-dimensional NMR spectroscopic analyses. The absolute structures of the diterpene moiety were determined by application of the circular dichroism (CD) rule for the γ-lactone ring. The relative structures of the two crotofolanes (1 and 2) and one rearranged compound (9) were confirmed by X-ray crystallographic analyses. Compounds 1, 2 and 9 possessed 2-methylbutyric acid in their molecules, the absolute configuration of which was found to be 2S by comparison of its HPLC behavior with that of an authentic sample. Therefore, the absolute structures of these crotocascarins (1, 2 and 9) were unambiguously determined. The absolute structures of crotofolanes are reported for the first time in this paper.

Crotofolane-type diterpenoids have fused 5-, 6- and 7-membered rings, and are expected to be biosynthesized from cembranes via lathyrane through cross annular cyclization.¹⁾ In 1975, this type of diterpenoid was first found in Jamaican Croton species, C. corylifolius Lamarck as crotofolin A as shown in Fig. 1.^1,2) Only two other species, Kenyan C. dichogamus Pax³⁾ and Congolese C. haumanianus J. Léonard,⁴⁾ were also found to contain rare crotofolanes. Genus Croton (Euphorbiaceae) comprises about 600 species and is distributed in tropical areas of both hemispheres. Croton oil obtained from seeds of C. tiglium Linné has a strong purgative effect and the occurrence of cocarcinogenic agents; fatty acid esters of diterpene phorbol in this plant has attracted our interest to investigate this genus.⁵⁾ C. cascarilloides Räuschel is an evergreen shrubby tree that grows on elevated coral reefs of the Okinawa Islands, Taiwan, southern China, the Malay Peninsula and Malaysia. Leaves are oblong-lanceolate to oblong-oval, and their undersurface is covered by shiny white ramenta.⁶⁾ Our phytochemical investigation of the stems (14.5 kg) of C. cascarilloides collected in the Okinawa Islands led to the isolation of eight new crotofolane-type diterpenoids, given trivial names crotocascarins A–H (1–8), and two new nor-diterpenoids with a new skeleton, given trivial names crotocascarins α and β (9, 10). The absolute configuration of crotofolanes was first determined in this study.

Results and Discussion

Fig. 1. Structure of Crotofolin A, Isolated from C. corylifolius

Using several types of chromatography, diterpenoids (1–8) and nor-diterpenoids (9, 10) were isolated from a CH₂Cl₂-soluble fraction prepared from the MeOH extract of the stems of C. cascarilloides (Fig. 2).

Fig. 2. Structures of New Compounds Isolated

Crotocascarin A (1), [α]_D²⁶ +16.4, was isolated as colorless rods and its elemental composition was determined to be C₂₅H₃₂O₇ by high-resolution (HR)-electrospray ionization (ESI)-mass spectrometry. The IR spectrum showed absorption bands for ester carbonyl and lactone carbonyl groups (1763, 1739 cm⁻¹), and double bonds (1650 cm⁻¹). In the ¹H-NMR spectrum, signals for two singlet methyls, two doublet methyls and one triplet methyl together with ones for two olefinic protons (δ_H 5.06, 5.09) and two oxygenated methine protons (δ_H 3.17, 5.48) were observed (Table 1). The ¹H–¹H correlation spectroscopy (COSY) and heteronuclear multiple bond correlation (HMBC) spectra (Fig. 3) together with one-dimensional ones indicated the presence of a 2-methylbutanoic acid moiety, the remaining 20 ¹³C-NMR signals comprising those of three methyls, three methylenes, five methines, one tetra- and one disubstituted double bonds, three oxygenated tertiary carbons, one carbonyl carbon and one hemiketal carbon (δ_C 107.5) (Table 2). The ten degrees of unsaturation, based on the results of HR-ESI-MS, other than two carbonyl groups and two double bonds, required six ring systems in the skeleton. Precise inspection of two-dimensional NMR spectra led to the conclusion that compound 1 was a diterpenoid with an unusual carbon skeleton (Fig. 2). To confirm this, X-ray crystallographic analysis of 1 was performed and the relative stereostructure of 1 was established to be that of a derivative of crotofolane-type diterpenoid (Fig. 4). The positive Cotton effect observable in the circular dichroism (CD) spectrum [Δε +3.41 (251)] empirically indicated the absolute configuration at the 9-position of the α,β-unsaturated γ-lactone ring was S,^7,8) and chirality analysis of the 2-methylbutanoic acid moiety by HPLC with an optical rotation detector established the absolute configuration of 1 to be shown in Fig. 2. This is the first report of the absolute structure of a crotofolane diterpene being disclosed and the absolute configuration of the α,β-unsaturated γ-lactone portion, presumed based on the empirical rule for the CD spectra, was proved to be correct.

Fig. 3. ¹H–¹H COSY and Selected HMBC Correlation of 1

Fig. 4. ORTEP Drawing of 1

Table 1. ¹H-NMR Spectroscopic Data for Crotocascarins A–H (1–8), and α and β (9, 10) (400 MHz, CDCl₃)

H	1	2	3	4	5	6	7	8	9	10
1	5.48 d 5	5.38 d 5	5.34 d 5	5.49 d 5	5.43 d 5	5.48 d 5	5.39 d 5	5.35 d 4	5.78 d 5	5.82 d 5
2	2.18 m	2.18 dqdd 8, 7, 7, 5	2.18 m	2.21 m	2.16 m	2.20 m	2.18 m	2.18 m	2.22 m	2.22 m
3	2.47 dd 14, 7	2.49 dd 14, 8	2.50 dd 13, 7	2.48 dd 14, 7	2.48 dd 14, 7	2.47 dd 14, 7	2.50 dd 14, 7	2.50 dd 14, 7	1.56 dd 14, 10	1.52 dd 14, 10
	1.65 dd 14, 10	1.70 dd 14, 10	1.72 dd 13, 10	1.64 dd 14, 10	1.69 dd 14, 10	1.65 dd 14, 10	1.70 dd 14, 10	1.71 dd 14, 10	2.34 dd 14, 7	2.36 dd 14, 7
4	—	—	—	—	—	—	—	—	—	—
5	3.17 s	3.19 s	3.20 s	3.15 s	3.18 s	3.16 s	3.19 s	3.20 s	4.39 s	4.36 s
6	—	—	—	—	—	—	—	—	—	—
7	2.99 dd 13, 1	3.06 br d 12	3.00 dd 12, 1	3.10 d 13	2.95 dd 12, 1	2.99 d 13	3.06 d 13	2.99 dd 12, 1	2.92 d 13	3.01 d 13
8	—	—	—	—	—	—	—	—	—	—
9	—	5.14 ddd 13, 4, 2		4.85 dddd 11, 4, 2, 2	—	—	5.14 br d 11	—	—	—
10	2.46 m	2.73 ddd 13, 4, 4	2.76 dd 14, 3	2.51 m	3.04 dd 16, 2	2.44 m	2.73 ddd 14, 4, 4	2.75 dd 15, 3	2.07 dd 15, 10	2.17 m
	1.57 ddd 13, 13, 5	1.27 ddd 13, 13, 4	1.70 dd 14, 3	1.17 m	1.45 ddd 16, 5, 2	1.58 ddd 13, 13, 5	1.30 br d 14	1.71 ddd 15, 3, 2	2.46 dd 15, 7	2.36 m
11	2.48 m	4.53 ddd 4, 4, 2	4.61 dd 3, 3	2.52 m	4.43 ddd 5, 2, 2	2.44 m	4.52 dd 3, 3	4.60 ddd 3, 3, 2	4.17 br dd 10, 7	2.09 m
	2.14 ddd 13, 13, 5			2.44 m		2.11 ddd 13, 13, 5				2.17 m
12	—	—	—	—	—	—	—	—	—	—
13	3.05 d 13	3.15 d 13	3.45 d 12	2.56 d 13	3.52 d 12	3.04 d 13	3.15 d 13	3.45 d 12	3.11 br d 13	2.99 br d 13
14	—	—	—	—	—	—	—	—	—	—
15	—	—	—	—	—	—	—	—	—	—
16	—	—	—	—	—	—	—	—
17	1.90 3H d 1	1.90 3H br s	1.89 3H br s	1.91 3H br s	1.88 3H d 1	1.91 3H d 1	1.90 3H d 1	1.89 3H d 1	2.39 3H, s	2.39 3H s
18	5.09 s	5.20 s	5.28 s	5.09 s	5.23 s	5.09 s	5.21 s	5.28 s	5.23 br s	4.92 s
	5.06 br s	5.17 s	5.23 br s	5.07 br s	5.17 br s	5.06 br s	5.16 br s	5.23 br s	5.39 s	5.20 br s
19	0.92 3H d 7	0.98 3H d 7	1.00 3H d 7	0.92 3H d 7	0.96 d 7	0.91 3H d 7	0.97 3H d 7	0.99 3H d 7	0.92 3H, d 7	0.90 3H d 7
20	1.17 3H s	1.06 3H s	1.17 3H s	1.07 3H s	1.14 3H s	1.17 3H s	1.06 3H s	1.17 3H s	1.27 3H, s	1.29 3H s
1′	—	—	—					—	—	—
2′	2.43 m	2.49 qdd 7, 7, 7	2.49 qdd 7, 7, 7	2.49 m	2.47 qdd 7, 7, 7	2.62 septet 7	2.65 septet 7	2.66 septet 7	2.38 m	2.36 m
3′	1.72 dqd 14, 7, 7	1.74 dqd 14, 7, 7	1.72 dqd 14, 7, 7	1.73 dqd 14, 7, 7	1.75 dqd 14, 7, 7	1.21 3H s	1.21 3H s	1.20 3H s	1.43 dqd 14, 7, 7	1.42 dqd 14, 7, 7
	1.48 dqd 14, 7, 7	1.50 dqd 14, 7, 7	1.49 dqd 14, 7, 7	1.49 dqd 14, 7, 7	1.50 dqd 14, 7, 7	1.21 3H s	1.20 3H s	1.19 3H s	1.68 dqd 14, 7, 7	1.64 dqd 14, 7, 7
4′	0.94 3H t 7	0.93 3H t 7	1.19 3H t 7	0.94 3H t 7	0.93 3H t 7				0.90 3H t 7	0.89 3H t 7
5′	1.19 3H d 7	1.17 3H d 7	1.17 3H d 7	1.18 3H d 7	1.19 3H 6				1.12 3H d 7	1.10 3H d 7
–OCH3					3.57 3H s
9-OH						3.49 br s
11-OH		2.44 dd 2, 2			2.42 dd 2, 2			3.05 dd 2, 2		2.26 d 6

Table 2. ¹³C-NMR Spectroscopic Data for Crotocascarins A–H (1–8), and α and β (9, 10) (100 MHz, CDCl₃)

C	1	2	3	4	5	6	7	8	9	10
1	74.6	75.8	76.6	74.5	75.4	74.6	76.7	76.3	75.5	75.7
2	33.3	32.7	32.5	33.2	32.6	33.4	32.8	32.5	34.4	34.5
3	36.9	36.4	36.3	36.9	36.4	36.9	36.3	36.3	34.3	34.3
4	60.4	60.1	60.2	60.5	60.2	60.4	60.1	60.2	64.8	65.2
5	57.9	57.8	57.9	57.6	58.0	57.8	57.7	57.9	75.4	75.6
6	56.5	55.9	56.5	56.1	56.4	56.4	55.9	56.5	88.0	87.9
7	44.3	44.4	44.4	44.1	44.7	44.2	44.2	44.4	48.0	47.4
8	159.0	162.0	158.6	161.7	159.0	158.7	161.9	158.5	60.1	62.2
9	107.5	78.4	106.4	82.2	109.5	107.2	78.3	106.4	174.7	175.1
10	41.9	44.1	45.7	37.7	40.4	42.0	44.1	45.8	35.8	27.1
11	34.8	72.7	73.7	36.2	73.7	34.8	72.6	73.7	67.9	27.2
12	146.8	148.9	148.4	146.0	149.3	146.6	148.7	148.3	146.1	141.0
13	39.5	31.7	31.8	40.7	31.4	39.5	31.7	31.6	32.4	34.7
14	68.9	68.8	68.8	68.7	68.8	68.8	68.8	68.7	66.0	65.9
15	130.4	128.2	129.8	128.5	129.9	130.6	128.2	129.9	202.0	201.7
16	170.8	173.4	170.3	173.0	170.7	170.5	173.3	170.2
17	9.6	9.7	9.6	9.6	9.7	9.7	9.7	9.6	26.0	25.4
18	115.1	115.2	116.3	115.2	115.9	115.1	115.2	116.3	113.8	110.4
19	12.7	12.3	12.2	12.7	12.3	12.5	12.1	12.1	12.6	12.7
20	20.2	19.3	19.7	19.5	20.1	20.3	19.3	19.7	22.0	22.1
1′	175.4	178.0	178.8	175.2	178.1	175.6	178.1	179.0	175.5	175.0
2′	41.2	41.2	41.4	41.2	41.5	34.2	34.3	34.4	41.2	41.6
3′	26.7	26.6	26.6	26.8	26.5	19.3	19.3	19.2	26.6	26.6
4′	11.7	11.4	11.5	11.7	11.5	19.4	18.7	18.9	11.8	11.7
5′	17.2	16.2	16.6	17.1	16.5				16.9	17.3
–OCH3					52.8

Crotocascarin B (2), [α]_D²⁶ +81.8, was isolated as colorless plates and its elemental composition was determined to be C₂₅H₃₂O₇ by HR-ESI-MS. NMR spectroscopic data indicated that 2 was a similar compound to 1, except for the disappearance of the hemiketal signal, instead of which, two oxymethine signals (δ_H 5.14 on δ_C 78.4 and 4.53 on 72.7) were observed. Since these oxymethine protons were correlated in the ¹H–¹H COSY spectrum through methylene protons on C-10, the planar structure of 2 was established to be as shown in Fig. 2. To confirm this, X-ray crystallographic analysis of 2 was performed and the relative stereostructure of 2 was established to be that of a derivative of crotofolane-type diterpenoid⁹⁾ (Fig. 5). The positive Cotton effect observable in the CD spectrum [Δε +1.36 (249)] indicated the absolute configuration at the 9-position of the α,β-unsaturated γ-lactone ring was also S, and chirality analysis of the 2-methylbutanoic acid moiety by HPLC with an optical rotation detector established the absolute structure of 2 to be as shown in Fig. 2.

Fig. 5. ORTEP Drawing of 2

Crotocascarin C (3), [α]_D²⁴ +88.9, and crotocascarin D (4), [α]_D²⁴ +2.6, were also similar compounds to the aforementioned ones with the respective elemental compositions of C₂₅H₃₂O₈ and C₂₅H₃₂O₆. In the NMR spectra of 3 and 4, a hemiketal carbon signal (C-9: δ_C 106.4) and an oxygenated carbon signal (C-11: δ_C 73.7) with δ_H 4.61 were observed in the former, and an oxygenated carbon signal (C-9: δ_C 82.2) and a methylene signal (C-11: δ_C 36.2) in the latter. A similar coupling pattern fot H-11 in 3 to that of 2 placed the hydroxy group at the 11-position in a β-orientation. Since the ¹H–¹H COSY spectrum of the former exhibited the proton chain from H₂-10 to H-11, along with the observation of a hemiketal carbon in the ¹³C-NMR spectrum, and that of the latter one from H-9 to H₂-11 through H₂-10, their structures were established to be as shown in Fig. 2. A similar coupling pattern for H-11 in 3 to that of 2 placed the hydroxy group at the 11-position in a β-orientation and positive Cotton effects at 252 nm (Δε +4.47, +1.17, respectively) substantiated that the absolute configurations of the diterpene moieties were the same as those of aforementioned compounds. The absolute configuration of 2-methylbutanoic acid in 3 and 4 must also be the same as that in 1 and 2, judging from the ¹³C-NMR chemical shifts of the acid moiety and the five-membered ring.

Crotocascarin E (5), [α]_D²⁵ +95.2, was isolated as an amorphous powder and its elemental composition was determined to be C₂₆H₃₄O₈ by HR-ESI-MS. The NMR spectroscopic data were essentially the same as those of crotocascarin C (Tables 1, 2), except for the presence of a methoxy signal [δ_H 3.57 (3H, s)], which crossed the ketal carbon one (δ_C 109.5) in the HMBC spectrum. Therefore, the structure of 5 was assigned, as shown in Fig. 1. The methoxy derivative is probably an artifact formed during the extraction and isolation processes.

Crotocascarins F (6), [α]_D²⁵ +16.8, and G (7), [α]_D²⁴ +81.7, were isolated as amorphous powders, and crotocascarin H (8), [α]_D²⁴ +94.2, as colorless needles, and their elemental compositions were determined to be C₂₄H₃₀O₇, C₂₄H₃₀O₇ and C₂₄H₃₀O₈, respectively, by HR-ESI-MS. The ¹³C-NMR spectral data for their diterpeneoid regions were essentially superimposable on those of crotocascarins A (1), B (2), and C (3), respectively. The common acyl moiety of these diterpenoids comprised four carbons, i.e., two doublet methyls, one methine, whose proton was coupled as a septet, and a carbonyl carbon. Therefore, the structure of the acyl moiety was expected to be isobutanoic acid and the gross structures of 6–8 were shown to be as in Fig. 1. The absolute configurations of 6, 7 and 8 were expected to be the same as those of crotocascarins A (1), B (2), and C (3), respectively, from similar respective optical rotation values and similar positive Cotton effect, [6: +4.66 (251), 7: +1.41 (248) and 8: +8.73 (252), respectively].

Crotocascarin α (9), [α]_D²⁶ +78.7, was isolated as colorless plates and its elemental composition was determined to be C₂₄H₃₂O₈ by HR-ESI-MS. In the IR spectrum, absorption bands for carbonyl groups (1761, 1721 cm⁻¹) and a double bond (1634 cm⁻¹) were observed. The NMR spectroscopic data indicated the presence of 2-methylbutanoic acid as an acyl substituent, thus the terpenoid region comprised 19 carbons, i.e., three methyls, two methylenes, three oxymethines, three methines, three oxygenated tertiary carbons, one quaternary carbon, an exomethylene moiety and two carbonyl carbons. The presence of the 2-methylbutanoic acid moiety and other functionalities, observed in one-dimensional NMR spectra, implied that crotocascarin α (9) was a compound related to crotofolanes. Since extensive examination of two-dimensional NMR data unfortunately did not lead to a structure which satisfied all the spectroscopic data, an attempt was made to solve the structure by X-ray crystallographic analysis and an ORTEP drawing of 9 is presented in Fig. 6.⁹⁾ Crotocascarin α (9) has a new skeleton and was probably derived from some crotofolane, like crotocascarin B (2), through several steps, such as decarboxylation, C–C bond migration, oxidation, etc. (Fig. 7). The absolute configuration of the 2′-position was determined to be S by the same method used for 1 and 2. Therefore, the structure of 9 is shown in Fig. 2, including the absolute one.

Fig. 6. ORTEP Drawing of 9

Fig. 7. Possible Biosynthetic Pathway from 2 to 9

Crotocascarin β (10), [α]_D²³ +35.0, was isolated as an amorphous powder and its elemental composition was determined to be C₂₄H₃₂O₇, which was one oxygen atom less than that of 9. The NMR spectroscopic data indicated that crotocascarin β (10) was a congeneric compound to 9 with three methylene carbons. One of the oxymethine protons at δ_H 4.17, which was observed in the NMR spectrum of 9 was obviously replaced by methylene protons, with H-1 and H-5 remaining intact. Therefore, the structure of 10 was elucidated to be as shown in Fig. 2, namely 11-deoxycrotocascarin α. The absolute configuration of 2-methylbutanoic acid must also be the same as that in 1, 2 and 8, judging from the ¹³C-NMR chemical shifts of the acid moiety and the five-membered ring.

Only seven crotofolane-type diterpenoids have been isolated so far, four from Jamaican C. corylifolius,^1,2) two from Kenyan C. dichogamus,³⁾ and one from Congolese C. haumanianus.⁴⁾ In these studies, without exception, the authors used an X-ray crystallographic method to come to a the final conclusion as to the relative structure. In this investigation on C. cascarilloides, a series of crotofolanes was isolated and two nor-diterpenes having a new skeleton probably derived from a crotofolane through the postulated biosynthetic scheme in Fig. 7. The structures of 1 and 2 were solved by X-ray crystallography using a direct method, and assignment of the absolute configuration of the acyl moiety, 2-methylbutanoic acid, obtained on chemical degradation of crotocascarins A (1) and B (2) as S provided information on the absolute structure of a crotofolane for the first time.

Experimental

General

Melting points were measured on a Yanagimoto micro melting point apparatus and are uncorrected. Optical rotations were measured on a JASCO P-1030 digital polarimeter. IR and UV spectra were measured on Horiba FT-710 and JASCO V-520 UV/Vis spectrophotometers, respectively. ¹H- and ¹³C-NMR spectra were taken on a JEOL JNM α-400 at 400 MHz and 100 MHz with tetramethylsilane as an internal standard. CD spectra were obtained with a JASCO J-720 spectropolarimeter. Positive-ion HR-ESI-MS was performed with an Applied Biosystems QSTAR XL NanoSpray™ System. Silica gel column chromatography (CC) was performed on Kiesel Gel (silica gel 60) (70–230 mesh) (E. Merck, Darmstadt, Germany) and reversed-phase octadecylsilanized (ODS) open CC on Cosmosil 75C₁₈-OPN (Nacalai Tesque, Kyoto, Japan) (Φ=50 mm, L=25 cm). HPLC was performed on an ODS column (Inertsil ODS-3; GL Science, Tokyo, Japan; Φ=6 mm, L=25 cm, 1.6 mL/min), and the eluate was monitored with UV (210 nm) and refractive index monitors. (S)-(+)-2-Methylbutanoic acid was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Plant Material

Stems of C. cascarilloides were collected at Okinawa in June 2004, and a voucher specimen was deposited in the Herbarium of the Department of Pharmacognosy, Graduate School of Biomedical and Health Sciences, Hiroshima University (04-CC-Okinawa-0628).

Extraction and Isolation

Stems (14.5 kg) of C. cascarilloides were extracted with MeOH (15 L×3) for a week at 25°C. The combined extract was concentrated to 6 L and then partitioned with n-hexane (6 L, n-hexane extract: 92.1 g). The methanolic layer was concentrated and the resulting residue was suspended in 6 L of H₂O. The H₂O layer was partitioned with 6 L each of CH₂Cl₂, EtOAc and 1-BuOH to give 39.1 g, 10.5 g and 52.2 g of the respective residues.

The residue (39.1 g) of the CH₂Cl₂-soluble fraction was subjected to silica gel CC (400 g) (Φ=60 mm, L=30 cm) with CHCl₃ (5 L), CHCl₃–MeOH (15 : 1, 7 L and 12 : 1, 5 L), and MeOH (2 L). Fractions of 500 mL were collected. The residue (3.11 g) in fraction 14 was separated by two runs of ODS open CC [H₂O–MeOH (1 : 1, 1 L)→(1 : 9, 1 L) and then H₂O–MeOH (1 : 9, 250 mL)→MeOH (250 mL)], fractions of 10 g being collected. The residue (116 mg) in fractions 137–152 obtained on the first run of ODS open CC was again subjected to silica gel CC (Φ=10 mm, L=40 cm) with n-hexane–EtOAc [(9 : 1, 100 mL), (17 : 3, 100 mL), (4 : 1, 100 mL), (7 : 3, 100 mL), (3 : 2, 100 mL) and (1 : 1, 100 mL)], and EtOAc (100 mL). Fractions of 2 mL were collected, and 3.1 mg of 5 was obtained in fractions 135–144.

The residue (166 mg) in fractions 45–58 obtained on the second run of ODS open CC was again subjected to silica gel CC (Φ=10 mm, L=40 cm) with n-hexane (250 mL) → n-hexane–EtOAc (1 : 1, 250 mL), and then n-hexane–EtOAc (1 : 1, 250 mL). Fractions of 2 mL were collected. The residue (16.4 mg) in fractions 270–290 was finally purified by HPLC (H₂O–MeOH, 1 : 1) to give 3.5 mg of 9 from the peak at 35 min.

The residue (101 mg) in fractions 64–72 obtained on the second run of ODS open CC was again subjected to silica gel CC (Φ=10 mm, L=40 cm) with n-hexane (250 mL)→n-hexane–EtOAc (1 : 1, 250 mL), and then n-hexane–EtOAc (1 : 1, 250 mL). Fractions of 2 mL were collected. The residue (22.2 mg) in fractions 123–139 was purified by HPLC (H₂O–MeOH, 1 : 1) to give 12.3 mg of 7 from the peak at 76 min. From fractions 140–151, 10.5 mg of 8 was obtained in a crystalline state. The residue (12.2 mg) in fractions 152–151 was purified by HPLC [Inertsil (Ph-3), H₂O–MeOH, 1 : 1; 1.6 mL/min] to afford a further amount (5.1 mg) of 8 from the peak at 41 min.

The residue (155 mg) in fractions 73–85 obtained on the second run of ODS open CC was again subjected to silica gel CC (Φ=10 mm, L=40 cm) with n-hexane (250 mL)→n-hexane–EtOAc (1 : 1, 250 mL), and then n-hexane–EtOAc (1 : 1, 250 mL). Fractions of 2 mL were collected. The residue (2.3 mg) in fractions 106–113 was purified by HPLC (H₂O–MeOH, 2 : 3) to give 0.7 mg of 10 from the peak at 21 min. The residue (64.9 mg) in fractions 124–134 was purified by HPLC (H₂O–MeOH, 3 : 7) to yield 8.6 mg of 6 and 5.4 mg of 2 from the peaks at 10 min and 11 min. respectively. From fractions 135–147, 2.5 mg of 3 was obtained in a crystalline state.

The residue (126 mg) in fractions 86–95 obtained on the second run of ODS open CC was again subjected to silica gel CC (Φ=10 mm, L=40 cm) with n-hexane (250 mL)→n-hexane–EtOAc (1 : 1, 250 mL), and then n-hexane–EtOAc (1 : 1, 250 mL), fractions of 2 mL being collected. The residue (13.1 mg) in fractions 114–123 was purified by HPLC (H₂O–MeOH, 3 : 7) to give 6.5 mg of 1 and 2.9 mg of 4 from the peaks at 14 min and 15 min, respectively.

Crotocascarin A (1): Colorless rods (MeOH), mp 220–221°C, [α]_D²⁶ +16.4 (c=0.95, CHCl₃); IR ν_max (KBr) cm⁻¹: 3399, 2966, 2930, 1763, 1739, 1650, 1456, 1180, 1146, 1018, 802; UV λ_max (MeOH) nm (log ε): 223sh (3.87), 208 (4.21); ¹H-NMR (400 MHz, CDCl₃): Table 1; ¹³C-NMR (100 MHz, CDCl₃): Table 1; CD Δε (nm): +3.41 (251), −8.39 (224) (c=2.02×10^–5 m, MeOH); HR-ESI-MS (positive-ion mode) m/z: 467.2046 [M+Na]⁺ (Calcd for C₂₅H₃₂O₇Na: 467.2040).

Crotocascarin B (2): Colorless plates (2-PrOH), mp 152–153°C, [α]_D²⁶ +81.8 (c=1.52, CHCl₃); IR (KBr) ν_max cm⁻¹: 3478, 2972, 2929, 1769, 1739, 1659, 1457, 1185, 1143, 1014, 804; UV (MeOH) λ_max nm (log ε): 218 (4.00); ¹H-NMR (CDCl₃, 400 MHz): Table 1; ¹³C-NMR (CDCl₃, 100 MHz): Table 2; CD Δε (nm): +1.36 (249), −1.27 (210) (c 4.31×10^–5 m, MeOH); HR-ESI-MS (positive-ion mode) m/z: 467.2017 [M+Na]⁺ (Calcd for C₂₅H₃₂O₇Na: 467.2040).

Crotocascarin C (3): Colorless plates (MeOH), mp 203–205°C, [α]_D²⁴ +88.9 (c=0.82, CHCl₃); IR (KBr) ν_max cm^–1: 3448, 2974, 2938, 1759, 1734, 1649, 1140, 1081, 877; UV (MeOH) λ_max nm (log ε): 214 (3.85); ¹H-NMR (CDCl₃, 400 MHz): Table 1; ¹³C-NMR (CDCl₃, 100 MHz): Table 2; CD Δε (nm): +4.47 (252), −10.61 (226) (c=1.78×10^–5 m, MeOH); HR-ESI-MS (positive-ion mode) m/z: 483.1985 [M+Na]⁺ (Calcd for C₂₅H₃₂O₈Na: 483.1989).

Crotocascarin D (4): Amorphous powder, [α]_D²⁴ +2.6 (c=0.19, CHCl₃); IR (KBr) ν_max cm⁻¹: 3463, 2972, 2932, 1794, 1748, 1651, 1457, 1161, 1112, 1062, 903; UV (MeOH) λ_max nm (log ε): 219 (4.00); ¹H-NMR (CDCl₃, 400 MHz): Table 1; ¹³C-NMR (CDCl₃, 100 MHz): Table 2; CD Δε (nm): +1.17 (252), −3.21 (212) (c=2.26×10^–5 m, MeOH); HR-ESI-MS (positive-ion mode) m/z: 451.2085 [M+Na]⁺ (Calcd for C₂₅H₃₂O₆Na: 451.2091).

Crotocascarin E (5): Amorphous powder, [α]_D²⁵ +95.2 (c=0.15, CHCl₃); IR (KBr) ν_max cm⁻¹: 3480, 2970, 2934, 1767, 1739, 1457, 1190, 1139, 1085, 802; UV (MeOH) λ_max nm (log ε): 216 (3.92); ¹H-NMR (CDCl₃, 400 MHz): Table 1; ¹³C-NMR (CDCl₃, 100 MHz): Table 2; CD Δε (nm): +4.23 (251), −12.68 (224) (c=3.06×10^–5 m, MeOH); HR-ESI-MS (positive-ion mode) m/z: 497.2129 [M+Na]⁺ (Calcd for C₂₆H₃₄O₈Na: 497.2145).

Crotocascarin F (6): Amorphous powder, [α]_D²⁵ +16.8 (c=0.22, CHCl₃); IR (KBr) ν_max cm⁻¹: 3466, 2973, 2932, 1765, 1739, 1651, 1459, 1337, 1191, 1154, 1066, 895; UV (MeOH) λ_max nm (log ε): 219 (3.88); ¹H-NMR (CDCl₃, 400 MHz): Table 1; ¹³C-NMR (CDCl₃, 100 MHz): Table 2; CD Δε (nm): +4.66 (251), −10.18 (225) (c=2.56×10^–5 m, MeOH); HR-ESI-MS (positive-ion mode) m/z: 453.1897 [M+Na]⁺ (Calcd for C₂₄H₃₀O₇Na: 453.1889).

Crotocascarin G (7): Amorphous powder, [α]_D²⁴ +81.7 (c=0.82, CHCl₃); IR (KBr) ν_max cm⁻¹: 3436, 1741, 1634, 1459, 1195, 1157, 1072, 887; UV (MeOH) λ_max nm (log ε): 220 (4.08); ¹H-NMR (CDCl₃, 400 MHz): Table 1; ¹³C-NMR (CDCl₃, 100 MHz): Table 2; CD Δε (nm): +1.41 (248), −4.22 (212) (c=1.91×10⁻⁵ m, MeOH); HR-ESI-MS (positive-ion mode) m/z: 453.1888 [M+Na]⁺ (Calcd for C₂₄H₃₀O₇Na: 453.1883).

Crotocascarin H (8): Colorless needles (CHCl₃), mp 242–244°C, [α]_D²⁴ +94.2 (c=0.33, CHCl₃); IR (KBr) ν_max cm⁻¹: 3440, 2978, 2938, 1760, 1739, 1648, 1444, 1183, 1141, 1082, 880; UV (MeOH) λ_max nm (log ε): 214 (4.16); ¹H-NMR (CDCl₃, 400 MHz): Table 1; ¹³C-NMR (CDCl₃, 100 MHz): Table 2; CD Δε (nm): +8.73 (252), −23.36 (226) (c=1.47×10^–5 m, MeOH); HR-ESI-MS (positive-ion mode) m/z: 469.1831 [M+Na]⁺ (Calcd for C₂₄H₃₀O₈Na: 469.1832).

Crotocascarin α (9): Colorless plates (CHCl₃), mp 202–203°C, [α]_D²⁶ +78.7 (c=0.13, CHCl₃); IR (KBr) ν_max cm⁻¹: 3479, 2968, 2926, 1761, 1721, 1634, 1461, 1193, 804; ¹H-NMR (CDCl₃, 400 MHz): Table 1; ¹³C-NMR (CDCl₃, 100 MHz): Table 2; HR-ESI-MS (positive-ion mode) m/z: 471.1973 [M+Na]⁺ (Calcd for C₂₄H₃₂O₈Na: 471.1989).

Crotocascarin β (10): Amorphous powder, [α]_D²³ +35.0 (c=0.04, CHCl₃); IR (KBr) ν_max cm⁻¹: 3467, 2926, 1741, 1714, 1654, 1460, 1162, 889; ¹H-NMR (CDCl₃, 400 MHz): Table 1; ¹³C-NMR (CDCl₃, 100 MHz): Table 2; HR-ESI-MS (positive-ion mode) m/z: 455.2044 [M+Na]⁺ (Calcd for C₂₄H₃₂O₇Na: 455.2040).

X-Ray Ccrystallographic Analysis of Crotocascarin A (1)

C₂₅H₃₂O₇, M=444.51, crystal size: 0.38×0.20×0.10 mm³, space group: orthorhombic, P2₁2₁2₁, T=90 K, a=6.1970(15) Å, b=15.124(4) Å, c=25.145(6) Å, V=2356.6(10) ų, Z=4, D_c=1.253 Mg/m³, F(000)=952. The data were measured using a Bruker SMART 1000 CCD diffractometer, using MoKα graphite-monochromated radiation (λ=0.71073 Å) in the range of 3.04<2θ<56.7. Of the 14241 reflections collected, 5548 were unique (R_int=0.0421, data/restraints/parameters 5548/0/298. The structure was solved by a direct method using the program SHELXTL-97.¹⁰⁾ The refinement and all further calculations were carried out using SHELXTL-97.¹¹⁾ The absorption correction was carried out utilizing the SADABS routine.¹⁰⁾ The H atoms were included at the calculated positions and treated as riding atoms using the SHELXTL default parameters. The non-H atoms were refined anisotropically using weighted full-matrix least-squares on F². Final goodness-of-fit on F²=1.078, R₁=0.0408, wR₂=0.0997 based on I>2σ(I), and R₁=0.0496, wR₂=0.1114 based on all data. The largest difference peak and hole were 0.645 and −0.402 eÅ⁻³, respectively.

X-Ray Crystallographic Analysis of Crotocascarin B (2)

C₂₈H₄₀O₈, M=504.60, crystal size: 0.50×0.30×0.15 mm³, space group: orthorhombic, P2₁2₁2₁, T=120 K, a=10.1775(10) Å, b=10.4348(10) Å, c=25.908(3) Å, V=2751.5(5) ų, Z=4, D_c=1.218 Mg/m³, F(000)=1088. Of the 13566 reflections collected in the range of 3.14<2θ<53.4, 3212 were unique (R_int=0.0224), data/restraints/parameters 3212/0/334. The structure was solved in a similar manner to as that for compound 1. Final goodness-of-fit on F²=1.048, R₁=0.0344, wR₂=0.0868 based on I>2σ(I), and R₁=0.0378, wR₂=0.0891 based on all data. The largest difference peak and hole were 0.335 and −0.228 eÅ⁻³, respectively.

Alkaline Hydrolysis of Crotocascarins A (1), B (2) and α (8)

Crotocascarins A (1) (2.3 mg), B (2) (2.5 mg) and α (2.0 mg), and authentic (S)-(+)-2-methylbutyric acid were (500 µL) each dissolved in 1 mL of a 1 : 1 mixture of 10% KOH in H₂O and 50% aqueous dioxane, and then heated for 3 h at 100°C. The cooled reaction mixtures were neutralized with Amberlite IR-120B (H⁺) and then the filtrates were evaporated. The four residues were analyzed by HPLC (column: Inertsil ODS-3, 6 mm×250 mm; solvent: 20% acetonitrile in H₂O containing 0.5% trifluoroacetic acid; flow rate: 1.6 mL/min) with a chiral detector (JASCO OR-2090plus) to give a peak of (S)-(+)-2-methylbutyric acid at 17.5 min with a positive optical rotation sign.

X-Ray Crystallographic Analysis of Crotocascarin α (10)

C₂₄H₃₂O₈, M=448.50, crystal size: 0.30×0.15×0.15 mm³, space group: monoclinic, P2₁, T=120 K, a=9.9294(12) Å, b=9.1267(11) Å, c=12.5443(15) Å, β=98.650(1)°, V=1123.9(2)ų, Z=2, D_c=1.325 Mg/m³, F(000)=1088. Of the 5560 reflections collected in the range of 3.28<2θ<54.1, 2416 were unique (R_int=0.0154), data/restraints/parameters 2416/1/296. The structure was solved in a similar manner to as that for compound 1. Final goodness-of-fit on F²=1.056, R₁=0.0315, wR₂=0.0794 based on I>2σ(I), and R₁=0.0335, wR₂=0.0809 based on all data. The largest difference peak and hole were 0.285 and −0.208 eÅ⁻³, respectively.

Supplementary Data

Supplementary X-ray crystallographic data for 1 (CCDC 894968), 2 (CCDC 761004), and 10 (CCDC 761005) can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, U.K.; fax: [phone]; or [e-mail]).

Acknowledgment

The authors are grateful for access to the superconducting NMR instrument (JEOL JNM α-400) at the Analytical Center of Molecular Medicine of the Hiroshima University Faculty of Medicine, and an Applied Biosystem QSTAR XL system ESI (Nano Spray)-MS at the Analysis Center of Life Science of the Graduate School of Biomedical Sciences, Hiroshima University. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Japan Society for the Promotion of Science. Thanks are also due to the Research Foundation for Pharmaceutical Sciences and the Takeda Science Foundation for the financial support.

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