A new norditerpenoid lactone from the seeds of Podocarpus nakaii

Abstract

A new norditerpenoid lactone, nakaiilactone A was isolated from the seeds of Podocarpus nakaii together with a known one, nagilactone C and a known bisnorditerpenoid lactone, inumakilactone B. The structure of the new compound was determined by detailed analyses of the spectroscopic data, especially 1D- and 2D-NMR and HR-ESI-MS.

Introduction

The genus Podocarpus (Podocarpaceae) is composed of more than 100 species of evergreen coniferous trees or shrubs, mainly distributed in tropical and subtropical areas and the south temperate zone. ¹ The typical chemical constituents of the genus are structurally diverse diterpenoids, especially nor- and bisnor-diterpenoids.^2–4 These nor- and bisnor-diterpenoids show a wide range of bioactivities, such as cytotoxic,^4–7 anti-inflammatory ⁸ and antibacterial activities. ⁹ Podocarpus nakaii, a dicotyledonous shrub plant, is mainly distributed in the middle area of Taiwan. ¹ Previously, diterpenoids, ¹⁰ phenylpropanoids, ¹¹ steroids^11,12 and flavonoids ¹² were obtained from the leaves, stems and bark of this plant.

In the present study, a chemical investigation of the CHCl₃-soluble part of an EtOH extract of P. nakaii resulted in the isolation of a new norditerpenoid, nakaiilactone A (1) and two known norditerpenoids, i.e. nagilactone C (2) and inumakilactone B (3) (see Figure 1). The structure of 1 was determined by spectroscopic methods, including 2D NMR (¹H-¹H COSY, HMQC, HMBC and NOESY).

Figure 1.

Compounds from P. nakaii.

Results and discussion

Compound 1 was obtained as colorless needles. The molecular formula of 1 was assigned to be C₂₀H₂₂O₈ by HR-ESI-MS (m/z [M + Na]⁺: found: 413.1201; calcd: 413.1207), implying 10 degrees of unsaturation. The IR spectrum revealed the presence of OH (3448 cm⁻¹) and carbonyl (1750 and 1710 cm⁻¹) functions in 1. The ¹H NMR spectrum (Table 1) of 1 displayed three methyls: δ_H 1.25 (3H, s), 1.42 (3H, d, J = 7.0 Hz), 1.49 (3H, s) and one methoxy at δ_H 3.73 (3H, s). Two tri-substituted double bond protons at δ_H 6.16 (1H, m) and 6.20 (1H, d, J = 1.5 Hz) were also present. Twenty carbon signals corresponding to the 20 carbon atoms in the molecular formula were all resolved in the ¹³C NMR spectrum. They comprised seven quaternary carbons (three of them carbonyls at δ_C 163.2, 171.9 and 178.0), nine tertiary carbons and four methyls (one oxygenated). Since three carbonyl groups and two double bonds accounted for five degrees of unsaturation, the remaining five degrees of unsaturation were accounted for by the presence of a tetracyclic ring system and an epoxy group in 1. The data mentioned above indicated that 1 was probably a norditerpenoid.^5,6,13

Table 1.

¹H NMR (400 MHz) and ¹³C NMR (100 MHz) data of 1 in CDCl₃.

Position	δH (J in Hz)	δC
1	3.54 (1H, d, 4.0)	55.6
2	3.50 (1H, dd, 6.0, 4.0)	50.6
3	4.49 (1H, dd, 6.0, 1.5)	68.7
4	–	48.4
5	2.14 (1H, d, 5.5)	48.6
6	5.00 (1H, dt, 5.0, 1.5)	71.1
7	6.16 (1H, m)	121.4
8	–	133.5
9	–	155.7
10	–	36.5
11	6.20 (1H, d, 1.5)	114.4
12	–	163.2
14	5.30 (1H, m)	79.1
15	3.21 (1H, q, 7.2)	42.9
16	1.42 (3H, d, 7.0)	12.8
17	–	171.9
18	1.49 (3H, s)	25.3
19	–	178.0
20	1.25 (3H, s)	19.9
OMe	3.73 (3H, s)	52.5

Analysis of the ¹H NMR, ¹³C NMR and HMQC spectra of 1 enabled us to assign all the protons to their bonding carbons. The NMR spectra of 1 were similar to those of podolactone E, implying a structural similarity between 1 and podolactone E. ¹⁴ Further interpretation of the NMR showed that 1 contained the same pentacyclic ring system (including epoxy) as that in podolactone E and the main difference between them lay in the side chain at C-14. The analysis of the ¹H-¹H COSY spectrum of 1 revealed the presence of the spin system of H₃-16–H-15-H-14, which was confirmed by an HMBC spectrum (Figure 2). The location of OMe at C-17 was deduced by the HMBC correlation between C-17 and the proton signal at δ_H 3.73. The key cross-peaks H-14/C-17 and H-16/C-17 allowed the identification of the linkages between C-15 and C-17. The key NOESY correlations of H-5/H-1, H-5/H-3, H-5/H-18 and H-18/H-2 indicated that the relative configuration of 3-OH and the 1,2-epoxy group were all β-face. Indeed, the structure of nakaiilactone A was very similar to that of nubilactone A except for the configuration of the epoxy group. ¹⁸ In nubilactone A, the configuration of epoxy was α, while the configuration of the epoxy in nakaiilactone A was β. Moreover, their optical rotations were different. The optical rotation of nubilactone A was +43.5° (in C₅H₅N), ¹⁸ while the optical rotation of nakaiilactone A was −12.0° (in MeOH). Thus, the structure of 1 was assigned to be a new norditerpenoid lactone which we named nakaiilactone A.

Figure 2.

Selected 2D NMR correlations of 1.

The known compounds were identified as nagilactone C (2),^14,15 a norditerpenoid and inumakilactone B (3),^16,17 a bisnorditerpenoid, by comparison of their NMR, MS and rotation data with those reported in the literature.

Experimental

All solvents were of analytical grade (Hangzhou Gaojing Fine Chemical Plant). Thin layer chromatography: precoated SiO₂ GF₂₅₄ plates (Qingdao Haiyang Chemical Co., Ltd), visualized by UV light (254 and/or 360 nm) or by spraying with 10% H₂SO₄ reagent followed by heating at 110°C for 5−10 min. Column chromatography: silica gel (SiO₂, 200−300 mesh, Qingdao Haiyang Chemical Co., Ltd) and reversed-phase SiO₂ (ODS-A 12 nm S-150, YMC Co.). Optical rotations: Rudolph-AutoPolIV polarimeter. IR spectra: Nicolet-Avatar-370 spectrometer (ν in cm⁻¹). UV spectra: Shimadz-UV-2450 spectrometer (λ in nm). NMR spectra: Bruker AM-400 spectrometer ( ¹ H NMR at 400 MHz, ¹³C NMR at 100 MHz, δ in ppm relative to Me₄Si, J in Hz). ESI-MS: Agilent-6210-Lc/TOF mass spectrometer (in m/z).

Plant material was collected in Wuxi County of Jiangshu Province, P.R. China, in October 2017 and identified as P. nakaii by Prof. Yong-Hong Zhang of the Fujian Medical University, P.R. China. A voucher specimen (No. P20171008) was deposited in the Zhejiang University of Technology.

The seeds of P. nakaii (1.5 kg) were pulverized and extracted with 95% EtOH exhaustively at room temperature to give a crude extract (55 g). The ethanol extract was then partitioned between H₂O and CHCl₃. Removal of CHCl₃ under reduced pressure yielded a brown residue (18 g), which was subjected to silica column chromatography eluting with petroleum ether (boiling range: 60°C–90°C) containing increasing amounts of Me₂CO to afford two fractions. Fraction 1 (1.3 g) eluting with petroleum ether/Me₂CO (8:2) was applied to a reversed-phase silica gel column, elution of which with MeOH-H₂O (5:5−6:4) yielded compounds 1 (12 mg) and 3 (45 mg). Fraction 2 (2.8 g), which was eluted with petroleum ether/Me₂CO (4:1), was applied to a silica gel column, gradient elution of which with CHCl₃-MeOH (20:1−10:1) yielded compound 2 (120 mg).

Nakaiilactone A (1): Colorless needles; m.p. 282−284°C; [α]²⁵_D −12.0° (c 0.1, MeOH); UV (EtOH): 265 (4.30); IR (KBr): ν_max 3448, 2360, 1750, 1710, 1637, 1384, 1141, 992, 849 cm−1; ESI-MS (pos.): 413 [M + Na]⁺; HR-ESI-MS for calcd for C₂₀H₂₂NaO₈⁺: [M + Na]⁺: 413.1207; found: 413.1201; ¹H NMR and ¹³C NMR data: see Table 1.

Nagilactone C (2): Colorless needles; dec above 282°C; [α]²⁵_D +40.0° (c = 0.1, MeOH); ¹H NMR and ¹³C NMR data were identical to literature data;^14,15 ESI-MS (pos.): 385 [M + Na]⁺.

Inumakilactone B (3): Colorless needles; m.p. 287−288°C; [α]²⁵_D +10.2° (c = 0.1, MeOH); ¹H NMR and ¹³C NMR data were identical to literature data;^16,17 ESI-MS (pos.): 369 [M + Na]⁺.