Yezo’otogirins D–H, Acylphloroglucinols and Meroterpenes from Hypericum yezoense

Abstract

Investigation of the methanolic extract from the aerial parts of Hypericum yezoense resulted in the isolation of three new acylphloroglucinols, yezo’otogirins D–F (1–3), and two new meroterpenes, yezo’otogirins G (4) and H (5). The structures of 1–5 were assigned on the basis of spectroscopic data. Yezo’otogirin D (1) is an acylphloroglucinol with a monoterpene moiety linked through an ether bond, while yezo’otogirins E (2) and F (3) are polyprenylated acylphloroglucinols possessing a tricyclic core. Yezo’otogirins G (4) and H (5) are linear meroterpenes with an enolized β-diketone moiety. Yezo’otogirin E (2) exhibited antimicrobial activity against Escherichia coli and Staphylococcus aureus.

The plants belonging to the genus Hypericum (Hypericaceae) are distributed widely in temperate regions and have been used as traditional remedies in various parts of the world.^1,2) Structurally interesting acylphloroglucinols and meroterpenes possessing diverse biological activites such as antidepressant, antimicrobial, antiviral, and cytotoxic activities have been isolated from Hypericum plants.^3,4) In our continuing search for structurally unique natural products from Hypericum plants,^4–8) we have reported the isolation of tricyclic meroterpenes, yezo’otogirins A–C,⁹⁾ from the aerial parts of H. yezoense. Further investigation on the constituents of this species afforded three new acylphloroglucinols, yezo’otogirins D–F (1–3), and two new meroterpenes, yezo’otogirins G (4) and H (5) (Chart 1). In this paper, the isolation and structure elucidation of 1–5 are described.

Chart 1. Structures of Yezo’otogirins D–H (1–5)

2a/3a and 2b/3b are major and minor tautomers of yezo’otogirins E (2) and F (3), respectively.

The methanolic extract of the aerial parts of H. yezoense was partitioned with n-hexane and water. Repeated chromatographic separations of the n-hexane-soluble fraction with a silica gel column, an octadecylsilyl (ODS) column, and a Sephadex LH-20 column gave the fractions containing acylphloroglucinols and meroterpenes, which were purified by ODS HPLC to give yezo’otogirins D (1, 0.00012%), E (2, 0.00042%), F (3, 0.00082%), G (4, 0.00062%), and H (5, 0.00012%).

Yezo’otogirin D (1) was obtained as an optically active pale yellow oil {[α]_D+10.8 (c 0.06, MeOH)}, and its molecular formula was elucidated to be C₂₁H₃₀O₆ by the high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) (m/z 401.19327 [M+Na]⁺, Δ−0.19 mmu). The IR spectrum displayed absorptions at 3303 and 1731 cm⁻¹, indicating the existence of hydroxy and carbonyl functionalities, respectively. The ¹H-NMR spectrum showed the resonances due to one vinyl group, one isopropyl group, one oxygenated sp³ methine, two sp³ methylenes, and four tertiary methyls as well as the signals of three D₂O-exchangeable protons (Table 1). The ¹³C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra suggested the presence of one fully substituted benzene ring, one carbonyl carbon, and two quaternary carbons. These observations implied 1 to be an acylphloroglucinol derivative with a C₁₀ unit.

Table 1. ¹H- and ¹³C-NMR Data for Yezo’otogirin D (1) in CDCl₃

Position	δC	δH (J in Hz)
1	160.3	—
2	103.0	—
3	155.6	—
4	122.0	—
5	151.2	—
6	102.6	—
7	7.4	2.05 (3H, s)
8	210.2	—
9	39.0	3.91 (1H, sept, 6.8)
10	19.4	1.18 (3H, d, 6.8)
11	19.2	1.18 (3H, d, 6.8)
1′	112.4	5.30 (1H, d, 16.4), 5.11 (1H, d, 11.1)
2′	142.2	5.92 (1H, dd, 16.4, 11.1)
3′	84.8	—
4′	36.9	2.07, 1.85 (each 1H, m)
5′	26.7	2.04, 1.99 (each 1H, m)
6′	85.2	4.16 (1H, t, 7.8)
7′	84.1	—
8′	24.9	1.38 (3H, s)
9′	21.6	1.28 (3H, s)
10′	26.2	1.48 (3H, s)
1-OH	—	13.76 (1H, s)
3-OH	—	7.99 (1H, br s)
5-OH	—	7.93 (1H, br s)

The acyl group of 1 was assigned as a 2-methylpropanoyl group by an heteronuclear multiple bond coherence (HMBC) cross-peak of H₃-11 to C-8 (Fig. 1). The downfield-shifted chemical shift of 1-OH implied the presence of a hydrogen bond between 1-OH and the carbonyl group (C-8), which allowed the assignment of the 2-methylpropanoyl group at C-6. HMBC correlations for 3-OH with C-2, 5-OH with C-6, and H₃-7 with C-1, C-2, and C-3 suggested the existence of a methyl group at C-7 and hydroxy groups at C-3 and C-5. The chemical shift of C-4 (δ_C 122.0) implied the presence of an oxygen function at C-4. In the C₁₀ unit (C-1′–C-10′), ¹H–¹H correlation spectroscopy (COSY) analysis revealed the presence of the spin system for C-4′ to C-6′, while HMBC analysis disclosed the connectivities among C-4′, the vinyl group (C-2′), and the methyl group (C-10′) via C-3′, and among C-6′ and the methyl groups (C-8′ and C-9′) via C-7′. The chemical shifts of C-3′ (δ_C 84.8), C-6′ (δ_C 85.2), and C-7′ (δ_C 84.1) indicated that these carbons were oxygenated. A nuclear Overhauser effect spectroscopy (NOESY) correlation for H-6′ with H-1′ suggested the presence of an ether linkage between C-3′ and C-6′, forming a tetrahydrofuran ring, as well as the anti relationship of the vinyl group at C-3′ and the substituent at C-6′. Given the molecular formula of 1, the connectivity of C-4 and C-7′ via an oxygen atom was deduced. Thus, the structure of yezo’otogirin D (1) was elucidated as shown in Chart 1.

Fig. 1. Selected 2D-NMR Correlations for Yezo’otogirin D (1)

Yezo’otogirin E (2) was isolated as an optically active pale yellow oil {[α]_D +21.4 (c 1.7, MeOH)}. The molecular formula of 2 was assigned as C₂₆H₃₅O₄ by the HR-ESI-MS (m/z 411.25470 [M+Na]⁺, Δ+0.68 mmu). In the ¹H-NMR spectrum of 2, a pair of downfield-shifted hydroxy signals due to keto–enol tautomerism were observed at δ_H 18.76 and 18.44 in the ratio of ca. 2 : 1, characteristic of a diketo enolic moiety of polyprenylated acylphloroglucinols found in Hypericum plants. Comparison of the one-dimensional (1D)-NMR data (for major tautomer 2a in Table 2; minor tautomer 2b in Experimental) with the data in the literature suggested that 2 is a tricyclic acylphloroglucinol derivative similar to ialibinone A¹⁰⁾ but with a different substituent at C-3. The substituent was assigned as 6-methylhepta-1,5-diene (C-15–C-22) by interpretation of the ¹H–¹H COSY and HMBC spectra (Fig. 2). The connectivity of C-3 to C-15 was elucidated by an HMBC correlation for H₂-16 with C-3. The relative stereochemistry of the tricyclic moiety of 2, including the relative configuration of C-3, was concluded to be the same as ialibinone A by resemblance of the chemical shifts and NOESY analysis. Similarly, the structure of yezo’otogirin F (3), which also showed a pair of the signals due to major (3a) and minor (3b) tautomers in the ratio of ca. 5 : 4 in the ¹H-NMR spectrum (CDCl₃), was assigned as a 3-epimer of 2 by 2D-NMR analysis and comparison of the spectral data with the spectral data for ialibinone B¹⁰⁾ (3-epimer of ialibinone A).

Fig. 2. Selected 2D-NMR Correlations for Major Tautomer (2a) of Yezo’otogirin E (2)

Table 2. ¹H- and ¹³C-NMR Data for Major Tautomers (2a and 3a) of Yezo’otogirins E (2) and F (3) in CDCl₃

Position	2aa)		3aa)
	δC	δH (J in Hz)	δC	δH (J in Hz)
1	72.0	—	72.3	—
2	25.9	2.51 (1H, t, 13.3)	24.6	2.52 (1H, dd, 13.7, 7.0)
		2.16 (1H, dd, 13.3, 5.9)		2.13 (1H, m)
3	53.6	2.08 (1H, m)	57.6	2.50 (1H, m)
4	42.7	—	44.8	—
5	55.4	2.35 (1H, t, 9.4)	55.8	2.29 (1H, dd, 9.8, 5.8)
6	34.4	2.23 (1H, dd, 13.0, 9.4)	32.7	2.22 (1H, m)
		1.74 (1H, dd, 13.0, 9.4)		1.87 (1H, dd, 13.3, 5.8)
7	61.3	—	62.1	—
8	201.6	—	201.7	—
9	109.6	—	108.0	—
10	191.0	—	191.0	—
11	206.1	—	206.7	—
12	24.4	0.97 (3H, s)	16.4	0.58 (3H, s)
13	25.8	0.82 (3H, s)	27.2	0.94 (3H, s)
14	12.4	1.40 (3H, s)	12.4	1.38 (3H, s)
15	147.0	—	146.8	—
16	111.9	4.95, 4.88 (each 1H, s)	112.2	5.00, 4.88 (each 1H, s)
17	36.6	2.07, 1.97 (each 1H, m)	36.8	2.09, 2.00 (each 1H, m)
18	27.0	2.12 (2H, m)	27.1	2.13 (2H, m)
19	124.0	5.11 (1H, t, 6.7)	124.0	5.12 (1H, m)
20	131.7	—	131.8	—
21	17.7	1.61 (3H, s)	17.7	1.62 (3H, s)
22	25.6	1.69 (3H, s)	25.7	1.69 (3H, s)
23	208.6	—	209.6	—
24	34.8	4.01 (1H, sept, 6.7)	34.9	4.04 (1H, sept, 6.7)
25	18.4	1.15 (3H, d, 6.7)	18.6	1.18 (3H, d, 6.7)
26	19.0	1.15 (3H, d, 6.7)	18.9	1.14 (3H, d, 6.7)
8-OH	—	18.76 (1H, s)	—	18.85 (1H, s)

a) The data for minor tautomers (2b and 3b) of yezo’otogirins E (2) and F (3) are listed in Experimental.

Yezo’otogirin G (4) was isolated as an optically active pale yellow oil {[α]_D−10.8 (c 1.27, MeOH)}. The HR-ESI-MS revealed the molecular formula of 4 to be C₁₅H₂₄O₄ (m/z 291.15668 [M+Na]⁺, Δ 0.00 mmu). The ¹H- and ¹³C-NMR spectra showed the signals of one carboxy, one methoxy, one prenyl, one isopropyl, and one methyl groups as well as the characteristic resonances due to one enolized β-diketone moiety (C-3–C-5)¹¹⁾ (Table 3). Interpretation of the HMBC spectrum revealed the connectivities between the isopropyl group (C-6) and the β-diketone moiety (C-5), and among the β-diketone moiety (C-3), the methyl group, the prenyl group, and the methoxy carbonyl group via a quaternary carbon (C-2) (Fig. 3). Therefore, the structure of yezo’otogirin G (4) was assigned as shown in Chart 1.

Table 3. ¹H- and ¹³C-NMR Data for Yezo’otogirins G (4) and H (5) in CDCl₃

Position	4		5
	δC	δH (J in Hz)	δC	δH (J in Hz)
1	173.6	—	173.6	—
2	56.2	—	56.2	—
3	196.9	—	196.8	—
4	94.4	5.52 (1H, s)	94.4	5.53 (1H, s)
5	195.4	—	195.6	—
6	35.9	2.46 (1H, sept, 6.7)	36.0	2.47 (1H, sept, 7.0)
7	19.5	1.14 (3H, d, 6.7)	19.5	1.15 (3H, d, 7.0)
8	19.6	1.14 (3H, d, 6.7)	19.5	1.15 (3H, d, 7.0)
9	19.5	1.34 (3H, s)	19.5	1.35 (3H, s)
10	34.1	2.61, 2.50 (1H, dd, 14.3, 7.6)	34.0	2.63, 2.52 (1H each, dd, 14.3, 7.6)
11	118.2	4.98 (1H, t, 7.6)	118.3	4.99 (1H, t, 7.6)
12	135.6	—	139.1	—
13	17.9	1.60 (3H, s)	16.2	1.60 (3H, s)
14	26.0	1.69 (3H, s)	39.9	1.99 (2H, m)
15			26.6	2.04 (2H, m)
16			124.1	5.05 (1H, t, 5.9)
17			131.5	—
18			17.7	1.59 (3H, s)
19			25.7	1.67 (3H, s)
5-OH	—	15.30 (1H, s)	—	15.33 (1H, s)
1-OMe	52.4	3.70 (3H, s)	52.3	3.71 (3H, s)

Fig. 3. Selected 2D-NMR Correlations for Yezo’otogirin G (4)

The molecular formula of yezo’otogirin H (5) was assigned as C₂₀H₃₂O₄ in light of the HR-ESI-MS. Though the 1D NMR spectral feature of 5 was similar to that of 4, the signals due to the prenyl group for 4 were replaced by those of the geranyl group for 5 (Table 3). The geranyl group at C-2 for 5 was confirmed by an HMBC correlation for H₃-9 with C-10, while the geometry of the double bond at C-11 was elucidated to be E by a NOESY cross-peak of H-11 with H₂-14. Thus, the structure of yezo’otogirin H (5) was assigned as shown in Chart 1. The absolute configurations of C-2 for 4 and 5 remained unsolved. The structures of yezo’otogirins G (4) and H (5) are partially correlated with that of yojironin A,¹¹⁾ a meroterpene isolated from Hypericum yojiroanum. As in the case of yojironin A, a plausible biogenetic precursor (X) composed of valine and two acetate units might be generated followed by methylation and prenylation or geranylation to generate 4 and 5 (Chart 2).

Chart 2. Possible Biogenetic Pathway of Yezo’otogirins G (4) and H (5)

Yezo’otogirin E (2) exhibited moderate antimicrobial activity against Escherichia coli (MIC 4.0 µg/mL) and Staphylococcus aureus (MIC 8.0 µg/mL), while yezo’otogirins G (4) and H (5) showed weak antimicrobial activity against Bacillus subtilis (MIC 16 µg/mL) and Trichophyton mentagrophytes (IFM62679, IC₅₀ 16 µg/mL).

Experimental

General

Optical rotations and IR spectra were recorded on a JASCO P-1030 digital polarimeter and a JASCO FT/IR-230 spectrophotometer, respectively. NMR spectra were measured by a Bruker AMX-600 spectrometer and a JEOL ECA 500 spectrometer. The resonances of residual CHCl₃ (7.26 and 77.0 ppm) were used as internal references for ¹H- and ¹³C-NMR chemical shifts, respectively. HR-ESI-MS spectra were recorded on a Thermo Scientific Exactive spectrometer.

Plant Material

Hypericum yezoense was cultivated at the Experimental Station for Medicinal Plant Studies, Hokkaido University, and the aerial parts were collected in August 2012. Herbarium specimen (specimen number: HYJ201208) was deposited in Graduate School of Pharmaceutical Sciences, Tokushima University.

Extraction and Isolation

The dried aerial parts of H. yezoense (850 g) were extracted with MeOH to afford the extract (88.5 g), which was partitioned with n-hexane and water. The n-hexane-soluble fraction (36.7 g) was loaded on a silica gel column (n-hexane : EtOAc, 95 : 5 to 0 : 100) to give 11 fractions (frs. 1–11). Fraction 2 was subjected to an ODS column chromatography (MeOH : H₂O, 70 : 30 to 100 : 0) and purified by ODS HPLC (COSMOSIL 5C₁₈-MS-II, Nacalai Tesque, 20×250 mm, MeOH : H₂O, 90 : 10) to give yezo’otogirin H (5, 1.0 mg). Fraction 4 was separated by an ODS column chromatography (MeOH : H₂O, 50 : 50 to 100 : 0) and a silica gel column chromatography (n-hexane : EtOAc, 95 : 5 to 0 : 100), and then purified by ODS HPLC (YMC-Pack ODS-AQ, YMC Co., Ltd., 10×250 mm, MeOH : H₂O, 90 : 10) to isolate yezo’otogirin G (4, 5.3 mg). A column chromatography of fr. 5 on silica gel (n-hexane : EtOAc, 95 : 5 to 0 : 100) gave five fractions (frs. 5.1–5). The MeOH-soluble part of fr. 5.2 was separated by an ODS column chromatography (MeOH : H₂O, 50 : 50 to 0 : 100) and ODS HPLC (YMC-Pack ODS-AQ, 20×250 mm, MeOH : H₂O, 85 : 15) to give yezo’otogirins E (2, 3.5 mg) and F (3, 7.0 mg). An ODS column chromatography (MeOH : H₂O, 50 : 50 to 0 : 100) of the MeOH-soluble portion of fr. 6 afforded eight fractions (frs. 6.1–8). Fraction 6.2 was subjected to a silica gel column chromatography (n-hexane : EtOAc 100 : 0 to 0 : 100) and a Sephadex LH-20 column chromatography (MeOH : H₂O, 80 : 20 to 100 : 0) to afford seven fractions (frs. 6.2.1–7). Yezo’otogirin D (1, 1.0 mg) was isolated from fr. 6.2.5 using ODS HPLC (COSMOSIL 5C₁₈-AR-II, 10×250 mm, MeOH : H₂O, 75 : 25).

Yezo’otogirin D (1)

Colorless amorphous solid, [α]_D+10.8 (c 0.06, MeOH), IR (KBr) ν_max 3303, 1731, and 1627 cm⁻¹, HR-ESI-MS: m/z 401.19327 [M+Na]⁺ (calcd for C₂₁H₃₀O₆Na, 401.19346), ¹H- and ¹³C-NMR data (Table 1).

Yezo’otogirin E (2)

Pale yellow oil, [α]_D+21.4 (c 1.7, MeOH), IR (KBr) ν_max 3308 and 1763 cm⁻¹, HR-ESI-MS: m/z 411.25470 [M–H]^– (calcd for C₂₆H₃₅O₄, 411.25408), ¹H- and ¹³C-NMR for major tautomer (2a) (Table 2), ¹H-NMR for minor tautomer (2b) δ_H 18.44 (1H, s, 10-OH), 5.11 (1H, t, J=5.9 Hz, H-19), 4.97 (1H, s, H-16a), 4.91 (1H, s, H-16b), 3.94 (1H, sept, J=6.7 Hz, H-24), 2.54 (1H, t, J=12.9 Hz, H-2a), 2.44 (1H, t, J=9.0 Hz, H-5), 2.23 (1H, m, H-2b), 2.12 (4H, m, H-3, H-6a, H-17a, H₂-18), 2.07 (1H, m), 1.97 (1H, m, H-17b), 1.69 (3H, s, H₃-22), 1.65 (1H, dd, J=13.7, 9.0 Hz, H-7), 1.61 (3H, s, H₃-21), 1.34 (3H, s, H₃-14), 1.12 (3H, d, J=6.7 Hz, H₃-25), 1.12 (3H, d, J=6.7 Hz, H₃-26), 0.95 (3H, s, H₃-12), and 0.85 (3H, s, H₃-13), ¹³C-NMR for minor tautomer (2b) δ_C 207.5 (C-23), 207.0 (C-11), 200.1 (C-10), 194.6 (C-8), 147.3 (C-15), 131.8 (C-20), 123.9 (C-19), 111.9 (C-16), 109.4 (C-9), 67.8 (C-1), 65.1 (C-7), 57.6 (C-5), 53.9 (C-3), 43.3 (C-4), 36.8 (C-17), 34.2 (C-24), 33.3 (C-6), 26.9 (C-18), 25.7 (C-13), 25.6 (C-22), 25.2 (C-2), 24.4 (C-12), 19.1 (C-26), 18.5 (C-25), 17.7 (C-21), and 13.1 (C-14).

Yezo’otogirin F (3)

Pale yellow oil, [α]_D+75.9 (c 1.7, MeOH), IR (KBr) ν_max 3315 and 1759 cm⁻¹, HR-ESI-MS: m/z 411.25453 [M−H]⁻ (calcd for C₂₆H₃₅O₄, 411.25408), ¹H- and ¹³C-NMR for major tautomer (3a) (Table 2), ¹H-NMR for minor tautomer (3b): δ_H 18.79 (1H, s, 10-OH), 5.12 (1H, m, H-19), 5.02 (1H, s, H-16a), 4.90 (1H, s, H-16b), 4.04 (1H, sept, H-24), 2.52 (1H, m, H-2a), 2.44 (1H, dd, J=10.1, 5.1 Hz, H-5), 2.44 (1H, m, H-3), 2.22 (1H, t, J=14.1 Hz, H-2b), 2.13 (2H, m, H₂-18), 2.11 (1H, m, H-6a), 2.09 (1H, m, H-17a), 2.00 (1H, m, H-17b), 1.78 (1H, dd, J=14.1, 5.1 Hz, H-6b), 1.69 (3H, s, H₃-22), 1.62 (3H, s, H₃-21), 1.32 (3H, s, H₃-18), 1.18 (3H, d, J=6.7 Hz, H₃-25), 1.13 (3H, d, J=6.7 Hz, H₃-26), 0.95 (3H, s, H₃-17), and 0.57 (3H, s, H₃-16), ¹³C-NMR for minor tautomer (3b) δ_C 209.0 (C-23), 207.3 (C-11), 200.3 (C-10), 193.6 (C-8), 146.6 (C-15), 131.7 (C-20), 123.9 (C-19), 112.4 (C-16), 107.9 (C-9), 68.2 (C-1), 66.2 (C-7), 58.2 (C-3), 57.7 (C-5), 45.1 (C-4), 36.8 (C-17), 34.6 (C-24), 31.1 (C-6), 27.2 (C-13), 27.1 (C-18), 25.7 (C-22), 25.2 (C-2), 18.8 (C-26), 18.7 (C-25), 17.7 (C-21), 16.9 (C-12), and 13.1 (C-14).

Yezo’otogirin G (4)

Pale yellow oil, [α]_D−10.8 (c 1.27, MeOH), IR (KBr) ν_max 1739 and 1602 cm⁻¹, HR-ESI-MS: m/z 291.15668 [M+Na]⁺ (calcd for C₁₅H₂₄O₄Na, 291.15668), ¹H- and ¹³C-NMR (Table 3).

Yezo’otogirin H (5)

Pale yellow oil, [α]_D−16.8 (c 0.19, MeOH), IR (KBr) ν_max 1733 and 1616 cm⁻¹, HR-ESI-MS: m/z 359.21921 [M+Na]⁺ (calcd for C₂₀H₃₂O₄Na, 359.21928), ¹H- and ¹³C-NMR (Table 3).

Antimicrobial Assay

Antimicrobial assay was carried out as previously described.¹²⁾

Acknowledgments

We thank Prof. T. Gonoi, Dr. A. Takahashi-Nakaguchi, and Dr. K. Sakai, Mycology Research Center, Chiba University, for evaluation of antimicrobial activity. This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of Interest

The authors declare no conflict of interest.

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