Omphalines A–E: ent-Rosane-Type Diterpenoids from the Madagascar Endemic Plant, Omphalea oppositifolia

Abstract

From the less polar fraction of the MeOH extract of the leaves and twigs of Omphalea oppositifolia, five new ent-rosane-type diterpenoids, named omphalines A–E (1–5), were isolated together with one known compound, 7-keto-ent-kaurane-16β,17-diol (6), by a combination of various kinds of chromatography. The structure of omphaline A (1) was elucidated to be 19-nor-ent-rosane-4,15-diene-2β,6α-diol-3-one. Omphalines B (2), C (3), D (4), and E (5) possessed two double bonds at 5- and 15-positions, and hydroxy functional groups at 3β-, 2α,3α-, 2α,3β-, and 2α,19-positions, respectively. The absolute configuration of 1 was determined by the comparison of the experimental electronic circular dichroism (ECD) spectrum and calculated ECD spectra.

Introduction

Madagascar is located close to the east coast of the African continent. However, the island was at the west coast of the Indian subcontinent before it moved to the current position, as explained by the continental drift theory of Alfred L. Wegener.¹⁾ Thus, many species of flora and fauna on the island developed independent of other regions of the world. About 15000 species of plants grow in Madagascar, of which 80% are endemic. Euphorbiaceae consists of about 7500 species in 300 genera and the Omphalea genus belonging to Euphorbiaceae is native to tropical parts of the Americas, the West Indies, Asia, Australia, and Africa, including Madagascar. Omphalea oppositifolia (Willd.) L.J. Gillespie is a plant endemic to the eastern part of Madagascar and leaves and twigs of O. oppositifolia were collected at Moramanga, Madagascar with the permission of the village chief and the Ministère de l’Environnement, de l’Ecologie et des Forêts. They were phytochemically investigated to afford five new ent-rosane-type diterpenoids, named omphalines A–E (1–5), together with a known compound, 7-keto-ent-kaurane-16β,17-diol (6), which has been isolated from Calibrachoa parviflora.²⁾ This paper deals with the structure elucidation of them.

Results and Discussion

The MeOH extract was separated by column chromatography on highly porous polymer, Diaion HP-20 and silica gel, and flash chromatography on octadecyl silanized silica gel (ODS). The final purification was performed by HPLC on an ODS column to give five new ent-rosane-type diterpenoids, named omphalines A–E (1–5), together with one known compound, 7-keto-ent-kaurane-16β,17-diol (6), isolated from Calibrachoa parviflora²⁾ (Fig. 1).

Fig. 1. The Structures of Compounds Isolated

Omphaline A (1), [α]²³_D −16.0, was isolated as an amorphous powder and its elemental composition was determined to be C₁₉H₂₈O₃ by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). The strong IR absorption at 1670 cm⁻¹ and UV absorption at 249 nm suggested the presence of a conjugated ketone system in the molecule. ¹H-NMR spectrum exhibited the two singlet methyls and one doublet methyl [δ_H 1.94 (d, J = 2.2 Hz)] probably on an olefinic carbon. The presence of two hydroxy groups (δ_H 4.11 and 4.87) and monosubstituted double bond [δ_H 4.88 (dd, J = 10.7 and 1.1 Hz), 4.94 (dd, J = 17.5 and 1.1 Hz), and 5.81 (dd, J = 17.5, 10.7 Hz)] was suggested by the ¹H-NMR spectrum. ¹³C-NMR spectroscopic data with distortionless enhancement by polarization transfer spectrum (DEPT) indicated the presence of three methyls, five methylenes, two hydroxylated and nonsubstituted methines, two quaternary carbon atoms, tetra- and monosubstituted double bonds, and a ketonic functional group. These spectroscopic data suggested that compound 1 must have a terpenic scaffold with 19 carbon atoms, namely a mono nor diterpenoid, and the six degrees of hydrogen deficiency suggested a tricyclic carbon skeleton. The ¹H–¹H correlation (COSY) and heteronuclear single quantum correlation (HSQC) spectra exhibited three distinct proton chains, such as I: C(2)(δ_C 71.6) HOH-C(1) (δ_C 29.0) H₂-C(10) (δ_C 45.3) H, II: C(6) (δ_C 65.8) HOH-C(7) (δ_C 35.3)H₂-C(8) (δ_C 33.1) H-C(14) (δ_C 38.9) H₂, and III: C(11) (δ_C 33.3) H₂-C(12) (δ_C 32.0)H₂. Besides those, a further correlation between δ_H 1.94 (3H, d, J = 2.2 Hz) on δ_C 10.7 (C-18) and δ_H 2.72 on δ_C 45.3 (C-10) was observed (Fig. 2). In the heteronuclear multiple bond correlation (HMBC) spectrum, the methyl protons at δ_H 1.94 showed correlation peaks with two tetrasubstituted double bonds (δ_C 130.1 and 156.9) and the ketonic carbon at δ_C 201.4 (Fig. 2). Significant HMBC correlations between H-2 (δ_H 4.11) and C-3 (δ_C 201.4) and C-4 thus made up the structure of the A-ring as shown in Fig. 1. Further, the HMBC correlations between methyl protons (C-20) at δ_H 0.61 and C-8, C-9, C-10, and C-11 and (C-17) at δ_H 1.09 and C-12, C-14, and C-15 together with COSY correlations shown in Fig. 2 established the planar structure of omphaline A (1) as 19-nor rosane-type diterpenoid. The relative structure was investigated using the phase-sensitive nuclear Overhauser effect spectrometry (PS-NOESY). The significant NOESY correlations, H-2 and H-10, H-10 and H-8, and H-8 and H₃-17 shown in Fig. 3, placed these protons in the same face, while H₃-17 and H₃-20 must be in the antiparallel relationship. From the large coupling constant (J = 14.0 Hz) between H-2 and H-1 (δ_H 1.51), the hydroxy group at the 2-position was expected to be in the equatorial orientation. Based on the NOESY correlation of H-6 with both of H₂-7 protons and the small coupling constants to H₂-7 protons, H-6 proton must bisect the H₂-7 protons, thus the hydroxy group at the 6-position was placed in the axial orientation (Fig. 3). The absolute structure was determined by comparison of calculated and experimental electronic circular dichroism (ECD) curves. Black solid and dotted curves in Fig. 4 show calculated ECD curves for ent-type and normal-type omphaline A (1), respectively. Experimental ECD curve depicted in red shows a close resemblance to that of ent-type. Therefore, the structure of 1 was elucidated to be 19-nor-ent-rosane-4,15-diene-2β,6α-diol-3-one as shown in Fig. 1.

Fig. 2. The ¹H–¹H COSY and HMBC Correlations of Omphaline A (1)

Fig. 3. The PS-NOESY Correlations of Omphaline A (1)

Fig. 4. Experimental and Calculated ECD Spectra of Omphaline A (1)

Omphaline B (2), [α]²⁴_D +9.8, was isolated as an amorphous powder and its elemental composition was determined to be C₂₀H₃₂O by HR-field ionization (FI) GC-MS from the peak at 19.6 min. HR-Atmospheric Pressure Chemical Ionization (APCI)-MS gave a dehydrated ion [M−H₂O + H]⁺ as a sole prominent peak, due to volatility of 2. The IR and UV spectra indicated the absence of a ketonic functional group and a conjugated ketone system. The ¹H-NMR spectrum displayed four singlet methyl groups and four olefinic protons and the ¹³C-NMR together with DEPT spectra indicated the presence of four methyls, six methylenes, one oxygenated and two non-oxygenated methines, three quaternary carbons, and mono- and trisubstituted double bonds. The COSY and HSQC spectra exhibited three similar proton chains to those of 1, except that chain I was extended by one more carbon atom such as C(3)HOH-C(2)H₂-C(1)H₂-C(10)H. Both of the geminal methyl proton signals at δ_H 0.97 (H₃-18) and 1.15 (H₃-19) showed diagnostic correlation peaks with C-3 (δ_C 76.9) and C-5 (δ_C 145.0) in the HMBC spectrum, and further correlations between H-1 (δ_H 1.12 and 1.78) and C-5 and H-6 (δ_H 5.60) and C-10 (δ_C 46.3) together with the significant correlations from H₃-17 (δ_H 1.02) to C-12, C-14, and C-15 and H₃-20 (δ_H 0.65) to C-8, C-9, C-10, and C-11 shown in Fig. 5 established the connectivity of the cyclic system, rings A, B, and C. The H-3 proton appeared as a doublet of doublet of doublet signal at δ_H 3.25 with coupling constants of J = 11.5, 4.1, 3.6 Hz. Since one (J = 3.6 Hz) of the coupling constants was expected to be coupling with OH group, the remaining coupling constants enabled the H-3 proton to be placed in the axial position. From the NOE correlations, H₃-18/H-10, H-10/H-8, and H-8/H₃-17, H-1ax/H₃-20, and H-3/H₃-19 and H₃-19/H-6 observed in the PS-NOESY spectrum (Fig. 6), the relative structure of 2 was proposed as shown in Fig. 1. Co-occurrence of ent-rosane type diterpenoid, omphaline A (1) and omphaline B (2) must also be in the enantio series. Therefore, the structure of 2 was expected to be ent-rosane-5,15-dien-3α-ol.

Fig. 5. The ¹H–¹H COSY and HMBC Correlations of Omphaline B (2)

Fig. 6. The PS-NOESY Correlations of Omphaline B (2)

Omphaline C (3), [α]¹⁹_D −41.9, was isolated as an amorphous powder and its elemental composition was determined to be C₂₀H₃₂O₂ by HR-ESI-MS. The one- and two-dimensional NMR spectra indicated that 3 was analogous to 2, except for the presence of one extra hydroxylated methine carbon signal. The COSY spectrum indicated the hydroxylated methines were in the adjacent positions and the HMBC correlations from both the geminal methyl protons at C-18 and C-19 proposed that these hydroxy groups were placed at the 2- and 3-positions (Fig. 7). From 1,3-diaxial relationship correlations from H-2ax-H-10-H-11ax-H₃-17 observed in the PS-NOESY spectrum (Fig. 8), the hydroxy group at the 2-position was placed in the equatorial orientation and the one at the 3-position in the axial orientation from the small coupling constant between H-2 and H-3 (J = 2.7 Hz). Therefore, the structure of omphaline C (3) was elucidated to be ent-rosane-5,15-diene-2β,3β-diol as shown in Fig. 1.

Fig. 7. The ¹H–¹H COSY and HMBC Correlations of Omphaline C (3)

Fig. 8. The PS-NOESY Correlations of Omphaline C (3)

Omphaline D (4), [α]²⁰_D −70.3, was isolated as an amorphous powder and its elemental composition was determined to be C₂₀H₃₂O₂, which was the same as that of 3. The NMR spectra showed a close resemblance to those of 3 and the coupling constant of H-2 (δ_H 3.71) and H-3 (δ_H 2.99) protons was J = 9.2 Hz, indicating that these two protons were in the axial orientation. The same 1,3-diaxial relationship correlations from H-2ax-H-10-H-11ax-H₃-17 observed in the PS-NOESY spectrum proved the structure of 4 as shown in Fig. 1.

Omphaline E (5), [α]²⁶_D −25.8, was isolated as an amorphous powder and its elemental composition was determined to be C₂₀H₃₂O₂, which was the same as that of 3 and 4. Two hydroxy groups were, however, present as primary and secondary alcohols in the molecule and from the COSY, HMBC, and PS-NOESY spectral evidence, the secondary alcohol group was placed at the 2-position in the equatorial orientation. Since the methylene protons in the primary alcohol were correlated with C-3 and C-4 in the HMBC spectrum, one of the geminal methyl groups at the 4-position must be oxidized to the primary alcohol and the methyl protons at the C-4 position showed significant NOESY correlations with H-2 and H-10. Thus, the equatorial methyl group was expected to be replaced by a primary alcohol functional group. Therefore, the structure of omphaline E (5) was elucidated to be ent-rosane-5,15-diene-2β,19-diol, as shown in Fig. 1.

Closing Remarks

The relatively rare group of diterpenoid, rosane derivatives has been found in a range of plant species, such as in monocotyledons, Amaryllidaceae³⁾ and Alismataceae,⁴⁾ in dicotyledons, Euphorbaiceae,⁵⁾ Linaceae,⁶⁾ and Lamiaceae,⁷⁾ as well as in a fern family, Aspidaceae.⁸⁾ Some biological activities have also been reported in rosane-type diterpenoids, including as chemopreventive agents,⁹⁾ their inhibitory effects against lipase,¹⁰⁾ and the inhibition of Epstein–Barr virus lytic replication.¹¹⁾ Future expeditions to Madagascar will be conducted to collect a sufficient amount of O. oppositifolia for comprehensive chemical and biological investigations.

Experimental

General Experimental Procedure

The optical rotations were measured on a JASCO P-2200 digital polarimeter. IR spectra were measured on a JASCO FT/IR-6100 spectrophotometer. ¹H- and ¹³C-NMR spectra were taken on a Bruker Avance III spectrometer at 600 and 150 MHz, respectively, with tetramethylsilane as an internal standard. The UV and ECD spectra were obtained with a JASCO J-1100 spectropolarimeter. Positive-ion HR-ESI-MS was performed with a Thermo Fisher Scientific LTQ Orbitrap XL instrument (Waltham, MA, U.S.A.). Positive-ion HR-FI-GC-MS was carried out with a JMS-T100GCV “AccuTOF GCv 4G” gas chromatograph-mass spectrometer (JEOL Ltd., Tokyo, Japan) on a HP-5 capillary column (0.25 mm × 30 m, liquid layer thickness: 0.25 µm) (Agilent Technologies, Inc., Santa Clara, CA, U.S.A). Oven temperature was set at 50 °C and raised to 300 °C at a rate of 10 °C/min. Helium was used as a carrier gas and flow rate was 1 mL/min. Injection split ratio and ionization voltage were set at 20 : 1 and 70 eV (300 µA), respectively.

A highly porous synthetic resin, Diaion HP-20, was product of Mitsubishi Chemical Corp. (Tokyo, Japan). Flash silica gel CC was performed with an EPLCL-AI-580S 10V system on a silica gel (40 µm) column (YAMAZEN Corp., Osaka, Japan) [Φ = 30 mm, L = 20 cm, flow rate: 10 mL/min, CHCl₃ (150 mL), linear gradient: CHCl₃ (750 mL)→CHCl₃–MeOH (7 : 3, 750 mL), CHCl₃–MeOH (7 : 3, 150 mL), CHCl₃–MeOH (1 : 1, 300 mL) with 30 mL fractions being collected] and flash ODS CC with the EPLCL-AI-580S 10V system on an ODS-SM (30 µm) column [Φ = 23 mm, L = 12.3 cm, flow rate: 10 mL/min, MeOH–H₂O (1 : 9, 100 mL), MeOH–H₂O (20 : 80, 100 mL), MeOH–H₂O (3 : 7, 100 mL), MeOH–H₂O (2 : 3, 100 mL), MeOH–H₂O (1 : 1, 100 mL), MeOH–H₂O (3 : 2, 100 mL), MeOH–H₂O (7 : 3, 100 mL), MeOH–H₂O (4 : 1, 100 mL), MeOH–H₂O (9 : 1, 100 mL), MeOH (200 mL), fractions of each eluent being collected]. HPLC was performed on an ODS column (Inertsil; GL Sciences, Tokyo, Japan; Φ = 6.0 mm, L = 250 mm, flow rate: 1.6 mL/min), and the eluate was monitored with a refractive index monitor.

Plant Material

The plant material was collected in Moramanga, Alaotra-Mangoro, Madagascar in July 2017. The voucher specimen (ST1529) has been deposited in the Herbarium of CNARP, Antananarivo, Madagascar. The plant was identified by one (SRR) of the authors.

Extraction and Isolation

The air-dried plant material (251 g) was extracted with MeOH (1 L × 2) and the solvent was evaporated to dryness to give 14.0 g of a MeOH extract. The MeOH extract was subjected to a Diaion HP-20 column (Φ = 60 mm, L = 45 cm), and eluted with H₂O (4 L), H₂O–MeOH (1 : 4, 4 L), MeOH (4 L), CHCl₃ (4 L), n-hexane (4 L), fractions of each eluent being collected. The MeOH eluent fraction (1.25 g) was separated by flash silica gel CC to give fr. 1 (151 mg) in fractions 1–18, fr. 2 (207 mg) in fractions 19–23, and fr. 3 (112 mg) in fractions 24–25. fr. 1 was subjected to flash ODS CC to give fr. 1–90 (45.3 mg) in MeOH–H₂O (9 : 1) eluent and it was purified by HPLC (H₂O–MeOH, 1 : 9) to afford 0.41 mg of 2 from the peak at 17.9 min. fr. 2 was subjected to flash ODS CC to give fr. 2–70 (14.1 mg) in MeOH–H₂O (7 : 3) eluent, fr. 2–80 (20.2 mg) in MeOH–H₂O (4 : 1) eluent, and fr. 2-90 (62.9 mg) in MeOH–H₂O (9 : 1) eluent. fr. 2–70 was purified via HPLC (H₂O–MeOH, 3 : 7) to afford 5.5 mg of 1 from the peak at 17.4 min. fr. 2–80 was purified via HPLC (H₂O–MeOH, 1 : 4) to afford 6.2 mg of 5 from the peak at 16.6 min. fr. 2–90 was also separated via HPLC (H₂O–MeOH, 1 : 9) to give 2.1 mg of 3 and 3.9 mg of 4 from the peaks at 9.6 min and at 10.6 min, respectively. fr. 3 was subjected to flash ODS CC to give fr. 3–80 (26.2 mg) in MeOH–H₂O (4 : 1) eluent. fr. 3–80 was separated via HPLC (H₂O–MeOH, 1 : 4) to give 4.0 mg of 6 and 3.8 mg of 5 from the peaks at 5.2 min and at 16.6 min, respectively.

Omphaline A (1) Colorless amorphous powder, [α]²³_D −16.0 (c 0.28, CHCl₃); IR ν_max (KBr) cm⁻¹: 3316, 2923, 2854, 1670, 1446, 1287, 1136, 1077; UV λ_max (MeOH) nm (log ε): 217 (3.39), 249 (3.83), 324 (1.81); ¹H-NMR (CDCl₃, 600 MHz): Table 1; ¹³C-NMR (CDCl₃, 150 MHz): Table 2; CD Δε (nm): −3.97 (221), +5.98 (252), −1.93 (324) (c 9.04 × 10⁻⁴ M, CH₃CN); HR-ESI-MS (positive-ion mode) m/z: 327.1932 [M + Na]⁺ (Calcd for C₁₉H₂₈O₃Na: 327.1931).

Table 1. ¹H-NMR Spectroscopic Data for Omphalines A–E (1–5) (CDCl₃, 600 MHz)

H	1	2	3	4	5
1	1.51 (ddd 14.0, 11.5, 10.6)	1.12 (dddd 13.0, 12.1, 11.8, 3.9)	1.45 (ddd 13.2, 12.3, 11.9)	1.14 (ddd 13.4, 11.9, 11.8)	1.06 (ddd 13.2, 11.6, 11.6)
	2.43 (ddd 11.5, 6.2, 4.8)	1.78 (dddd 11.8, 4.1, 4.0, 3.6)	1.81 (ddd 12.3, 4.9, 4.7)	2.04 (ddd 11.8, 4.5, 4.5)	2.06 (dddd 11.6, 4.2, 3.7, 2.8)
2	4.11 (dd 14.0, 6.2)	1.55 (dddd 12.1, 11.7, 11.5, 4.0)	4.02 (ddd 11.9, 4.7, 2.7)	3.71 (ddd 11.9, 9.2, 4.6)	3.87 (dddd 11.4, 11.4, 4.2, 4.1)
		1.81 (dddd 11.7, 4.1, 4.1, 3.9)
3	—	3.25 (ddd, 11.5, 4.1, 3.6)	3.46 (d 2.7)	2.99 (d 9.2)	1.24 (dd 12.8, 12.4)
					1.88 (ddd 12.8, 4.0, 2.6)
6	4.87 (br s)	5.60 (ddd 5.7, 2.0, 1.9)	5.62 (ddd 5.2, 2.7, 2.4)	5.62 (ddd 5.7, 2.0, 1.9)	5.64 (ddd 5.8, 2.8, 2.2)
7	1.54 (ddd 14.6, 12.8, 3.1)	1.67 (dddd 17.7, 11.7, 3.6, 2.5)	1.72 2H (m)	1.68 (dddd 17.7, 11.2, 3.4, 2.0)	1.72 (dddd 17.9, 11.5, 3.8, 2.1)
	1.62 (ddd 14.6, 3.6, 2.7)	1.72 (dddd 17.7, 5.6, 5.4, 2.1)		1.74 (dddd 17.7, 5.7, 5.4, 1.8)	1.81 (dddd 17.9, 5.8, 5.4, 1.9)
8	2.13 (dddd 12.8, 12.8, 3.6, 3.6)	1.47 (m)	1.51 (m)	1.49 (m)	1.49 (dddd 11.5, 11.5, 5.4, 4.4)
10	2.72 (ddd 10.6, 4.8, 2.2)	1.91 (br d 13.0)	2.05 (br d 13.2)	2.07 (br d 13.4)	1.90 (br d 13.2)
11	1.42 (m)	1.29 (m)	1.30 (m)	1.29 (m)	1.25 (m)
	1.54 (m)	1.66 (m)	1.68 (ddd 13.5, 4.8, 4.1)	1.66 (m)	1.67 (ddd 13.7, 4.4, 3.6)
12	1.33 (m)	1.25 (m)	1.27 (m)	1.26 (m)	1.26 (m)
	1.44 (m)	1.47 (m)	1.49 (m)	1.47 (m)	1.48 (m)
14	1.11 (m)	1.14 (m)	1.14 (ddd 13.7, 3.7, 2.4)	1.14(m)	1.15 (ddd 13.7, 3.6, 2.6)
	1.30 (m)	1.28 (m)	1.30 (m)	1.28 (m)	1.28 (m)
15	5.81 (dd 17.5, 10.7)	5.81 (dd 17.6, 10.8)	5.81 (dd 17.5, 10.7)	5.81 (dd 17.5, 10.7)	5.81 (dd 17.5, 10.8)
16	4.88 (dd 10.7, 1.1)	4.85 (dd 10.8, 1.3)	4.86 (dd 10.7, 1.3)	4.85 (dd 10.7, 1.3)	4.86 (dd 10.8, 1.2)
	4.94 (dd 17.5, 1.1)	4.92 (dd 17.6, 1.3)	4.93 (dd, 17.5, 1.3)	4.92 (dd 17.5, 1.3)	4.93 (dd 17.5, 1.2)
17	1.09 3H (s)	1.02 3H (s)	1.01 3H (s)	1.02 3H (s)	1.00 3H (s)
18	1.94 3H (d 2.2)	0.97 3H (s)	1.05 3H (s)	0.98 3H (s)	1.10 3H (s)
19	—	1.15 3H (s)	1.19 3H (s)	1.16 3H (s)	3.23 (d 10.4)
					3.43 (d 10.4)
20	0.61 3H (s)	0.65 3H (s)	0.72 3H (s)	0.68 3H (s)	0.71 3H (s)
3-OH		1.35 (br d 3.6)

Letters and figures in the parentheses are multiplicities and coupling constants in Hz.

Table 2. ¹³C-NMR Spectroscopic Data for Omphalines A–E (1–5) (CDCl₃, 150 MHz)

C	1	2	3	4	5
1	29.0	23.5	28.2	31.9	35.4
2	71.6	30.3	68.4	71.0	66.8
3	201.4	76.9	79.1	81.4	45.1
4	130.1	41.7	41.5	41.6	42.9
5	156.9	145.0	140.2	143.6	138.9
6	65.8	118.7	121.7	119.5	123.0
7	35.3	30.2	30.3	30.2	30.2
8	33.1	36.0	36.3	36.1	36.4
9	38.3	34.7	34.8	34.7	34.7
10	45.3	46.3	45.0	45.2	46.3
11	33.3	34.3	34.2	34.3	34.1
12	32.0	32.3	32.3	32.3	32.3
13	36.3	36.4	36.3	36.4	36.2
14	38.9	38.9	38.9	38.9	38.9
15	150.6	151.4	151.2	151.2	151.1
16	109.2	108.7	108.8	108.8	108.9
17	22.6	22.4	22.3	22.4	22.3
18	10.7	21.7	27.8	22.8	24.1
19		24.3	25.5	24.6	68.2
20	12.2	12.6	12.5	12.5	12.2

Omphaline B (2) Colorless amorphous powder, [α]²⁴_D +9.8 (c 0.02, CHCl₃); IR ν_max (KBr) cm⁻¹: 3384, 2924, 2854, 1653, 1457, 1101, 1071; ¹H-NMR (CDCl₃, 600 MHz): Table 1; ¹³C-NMR (CDCl₃, 150 MHz): Table 2; HR-APCI-MS (positive-ion mode) m/z: 271.2423 [M − H₂O + H]⁺ (Calcd for C₂₀H₃₁: 271.2420). HR-FI-GC-MS (positive-ion mode) t_R: 19.6 min; m/z: 288.2452 [M]⁺ (100%, Calcd for C₂₀H₃₂O: 288.2453), 270.2344 [M − H₂O]⁺ (36%, Calcd for C₂₀H₃₀: 270.2348).

Omphaline C (3) Colorless amorphous powder, [α]¹⁹_D −41.9 (c 0.10, CHCl₃); IR ν_max (KBr) cm⁻¹: 3363, 3276, 2922, 2877, 1652, 1454, 1178, 1074; ¹H-NMR (CDCl₃, 600 MHz): Table 1; ¹³C-NMR (CDCl₃, 150 MHz): Table 2; HR-ESI-MS (positive-ion mode) m/z: 327.2347 [M + Na]⁺ (Calcd for C₂₀H₃₂O₂Na: 327.2345).

Omphaline D (4) Colorless amorphous powder, [α]²⁰_D −70.3 (c 0.20, CHCl₃); IR ν_max (KBr) cm⁻¹: 3316, 2923, 2854, 1670, 1446, 1287, 1136, 1077; ¹H-NMR (CDCl₃, 600 MHz): Table 1; ¹³C-NMR (CDCl₃, 150 MHz): Table 2; HR-ESI-MS (positive-ion mode) m/z: 327.2295 [M + Na]⁺ (Calcd for C₂₀H₃₂O₂Na: 327.2295).

Omphaline E (5) Colorless amorphous powder, [α]²⁶_D −25.8 (c 0.31, CHCl₃); IR ν_max (KBr) cm⁻¹: 3244, 2913, 2874, 1654, 1457, 1119, 1066; ¹H-NMR (CDCl₃, 600 MHz): Table 1; ¹³C-NMR (CDCl₃, 150 MHz): Table 2; HR-ESI-MS (positive-ion mode) m/z: 327.2294 [M + Na]⁺ (Calcd for C₂₀H₃₂O₂Na: 327.2295).

Theoretical ECD Calculation for the Determination of Absolute Configuration of 1

Conformational analyses for 1 were performed with the Spartan’20 V1.0.0. program (Wavefunction, Inc., Irvine, CA, U.S.A.) on a commercially available personal computer [operating system: Microsoft 64-bit version of Windows 10 Home edition; 16-core central processing unit: Ryzen 9 5950X processor (Advanced Micro Devices, Inc., Santa Clara, CA, U.S.A.) run at 3.4 GHz; random access memory: 32 GB]. Stable conformers up to 40 kcal/mol for 1 were initially searched using the Merck molecular force field method. Then the aforementioned stable conformers were further optimized using the Hartree–Fock (HF)/3-21G and ωB97XD/6-31G* programs. The resulting conformers were subjected to ECD calculation, and the ECD calculations for these conformers were performed with Gaussian16 (Revision A.03 by Gaussian)¹²⁾ on the ChemPark cloud system.¹³⁾ The dominant conformers of 1 capable of covering >85% of the population according to Boltzmann’s distribution were selected. Time-dependent density functional theory calculations were conducted at the CAM-B3LYP/TZVP level for these conformers. The resulting rotational strength data were converted to Gaussian curves (bandwidth sigma = 0.6 eV) to obtain the ECD spectra of each conformer, and the spectra were combined after Boltzmann weighting according to their population contributions. The wavelength of the spectra was corrected (+10 nm) based on the absorptions of about 250 nm (referring to the experimental and calculated UV spectra) to give the corresponding theoretical ECD spectrum.

Acknowledgments

The measurements of HR-ESI-MS and HR-APCI-MS were performed with LTQ Orbitrap XL MS and HR-FI-GC-MS was performed with a JMS-T100GCV “AccuTOF GCv 4G” GC-MS at the Natural Science Center for Basic Research and Development (N-BARD), 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 (Nos. 15H04651, 17K08336, and 18K06740).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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