1 Introduction

Pseudolarix amabilis is a plant indigenous to the south-east of China [1]. The root and trunk barks of it, known as ‘Tu Jin Pi’ in traditional Chinese medicine, have been used to treat skin diseases caused by fungal infection [2, 3]. Previous phytochemical studies on the root barks and seeds of P. amabilis revealed a variety of bioactive compounds with novel structures, with the main chemical constituents being pseudolaric acid analogous and triterpenoids [4,5,6,7,8,9]. Some of them, such as psedolaric acids A and B [10], have shown potent antimicrobial and cytotoxic activities. Peudolarolide B, a triterpene lactone, has shown potent cytotoxic activity [8]. Novel nortriterpenoid lactone, pseudolarenone [11], as well as triterpenoid–diterpenoid dimers [12], have also been reported from the cones of this plant. In this paper, we describe the isolation and structure elucidation of four new 3,4-secocycloartane triterpenoids from the cones of P. amabilis.

2 Results and Discussion

The dried cones of P. amabilis, collected in Jiujiang, Jiangxi province, P. R. China, were extracted with 80% EtOH for three times at room temperature. The extract was separated by chromatography techniques to yield four new triterpenes, pseudolactones A–D (1–4) (Fig. 1). The structures of four new triterpenoids were determined by analysis of HRESIMS and NMR spectroscopic data.

Fig. 1
figure 1

Chemical structures of 1–4

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Compound 1 corresponds to the molecular formula C32H50O6 as established by the hydrogen adduct ion peak at m/z 531.3678 [M + H]+ (calcd. 531.3680 for C32H51O6+) in HR-ESI–MS spectrum, indicative of 8 degrees of unsaturation.

The 1H NMR spectrum (Table 1) of 1 showed four tertiary (δH 1.00 (s), 1.17 (s), 1.18 (s), 1.08 (s)) and two secondary (δH 0.86 (d, J = 6.4 Hz), 1.19 (overlap)) methyls. Moreover, an oxygenated proton signal was observed at δH 4.05. In 13C NMR spectrum (Table 2), there existed 32 carbon resonances, which were sorted into seven methyls, eleven methylenes, six methines (including one oxygenated methane at δC 77.4), and eight quaternary carbons (including two ester carbonyls at δC 174.6 and 179.7, an oxygenated quaternary carbon at δC 76.1, and a ketal carbon at δC 107.3), by DEPT NMR spectrum. The above evidences, combined with the characteristic methylene protons at δH 0.51 (d, J = 4.4 Hz) and 0.70 (d, J = 4.4 Hz) as well as the carbon resonances at δC 32.4 (t), 21.8 (s), and 27.6 (s), revealed a cyclopropyl motif. The diagnostic chemical shifts for two ester carbonyls at δC 174.6 (C-3) and 179.7 (C-26), one oxygenated methine at δC 77.4, and the ketal carbon at δC 107.3, implied that compound 1 might be a 3,4-secocycloartane triterpenoid with a unique 16,23-epoxy-23,26-spirolactone side chain.

Table 1 1H NMR (400 MHz) spectroscopic data for compounds 1–4 in CDCl3 (J in Hz)
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Table 2 13C (100 MHz) NMR spectroscopic data of compounds 1–4 in CDCl3
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Comparison of the NMR spectroscopic data of compound 1 with those of known pseudolarolide C [8] indicated that they were structurally quite similar except that an additional ethoxy in compound 1 (δH 1.19 (3H), 4.05 (2H); δC 14.1, 60.2) replaced the methoxyl in pseudolarolide C, which was confirmed by key 1H-1H COSY correlations (Fig. 2) of H2-1′/H3-2′ and HMBC correlation from H2-1′ (δH 4.05) to carboxylic carbon C-3 (δC 174.6 ppm). The structure of 1 was further evidenced by key 1H-1H COSY correlations of H2-2/H2-1, H-5/H2-6/H2-7/H-8, H2-11/H2-12, H2-15/H-16/H-17/H-20 (H3-21)/H2-22 together with key HMBC correlations (Fig. 2) from H2-2, H2-19, H2-6 to C-10 (δC 27.6), from H2-11, H-15 to C-13 (δC 43.5), from H2-12, H-16 to C-14 (δC 47.3), from H2-22, H-16, H2-24 to C-23 (δC 107.3), and H3-27 to C-26 (δC 179.7). The relative configurations of 1 were elucidated to be identical with those of pseudolarolide C on the basis of the REOSY correlations (Fig. 2) of H-19 with H-8, H-8 with CH3-18, CH3-18 with H-16, H-16 with H-20, H-17 with CH3-28 and CH3-21, H-22 with H-24, and H-24 with CH3-27. In cycloartane triterpenoids, the C-20 position of the 17-side chain is usually R-configuration. From the biogenetic point of view, the C-20 position of 1 should be R-configuration, too. Zhao et al. reported a serial of similar 16,23-epoxy-26(23)-olide-3,4-secocycloartan-3-oic acid esters, and determined their absolute configurations by single crystal X-ray diffraction [13]. By comparing the Cotton effect (− 0.7 at 223 nm) of 1 in ECD spectrum with those known compounds [13], in combination with analysis of chemical shifts, the absolute configurations of C-23 and C-25 positions of 1 were proposed to be S- and R-configuration, respectively. Thus, the structure of 1 was elucidated as (20R,23S,25R)-4-hydroxy-16,23-epoxy-26(23)-olide-3,4-secocycloartan-3-oic acid ethyl ester, and named pseudolactone A.

Fig. 2
figure 2

Key 1H-1H COSY, HMBC, and NOESY correlations of compound 1

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Compound 2 was obtained as colorless oil. Its molecular formula was determined as C34H54O6, by HR-ESI–MS spectrum. The 1H and 13C NMR spectroscopic data (Tables 1 and 2) of 2 showed typical signals similar to those of 1, including two ester carbonyls (δC 174.0, 179.8), a ketal carbon at δC 107.3 (C-23), an oxygenated methine at δC 77.3 (C-16), a cyclopropyl at δH 0.42 (d, J = 4.8 Hz), 0.78 (d, J = 4.8 Hz) and δC 31.6 (t), one oxygen-bearing proton at δH 4.09 (td, J = 5.6, 11.2 Hz) and δC 77.3 (d), one oxygenated methylene at δH 4.03 (t, J = 6.8 Hz) and δC 64.2 (t), three singlet methyls at δH 1.04 (s), 1.12 (s), and 1.67 (s), two doublet methyls at δH 0.89 (d, J = 6.8 Hz) and 1.25 (d, J = 7.2 Hz), one triplet methyl at δH 0.93 (t, J = 7.6 Hz). These information revealed that compound 2 possessed the similar 16,23-epoxy-26(23)-olide-3,4-secocycloartane skeleton to that of 1. The main differences between compounds 2 and 1 are that the resonance signals for one terminal double bond (δH 4.73 (dd, J = 1.6, 2.0 Hz), 4.80 (d, J = 2.0 Hz); δC 149.2 (s), 111.6 (t)), and for one butoxy (δH 4.03, 1.59, 1.37, 0.93; δC 64.2 (t), 30.6 (t), 19.1 (t), 13.7 (q)) in 2, took place of the signals for the oxygenated quaternary carbon at δC 76.1 (s, C-4) and the methyl at δH 1.17 (s) and δC 25.8 (q), as well as for the ethoxy at δH 4.05, 1.19 and δC 60.2 (t), 14.1 (q).

In 1H-1H COSY spectrum (Fig. 3), a butoxy was determined on the basis of the correlations of H2-1′/H2-2′/H2-3′/H3-4′. The butoxy was linked to C-3 through key HMBC cross peak (Fig. 3) from H2-1′ (δH 4.03) to the ester carbonyl at δC 174.0. The terminal double bond was evidenced to be placed between C-4 and C-29 as shown by key HMBC correlations (Fig. 3) from H-5 (δH 2.41), H2-29 (δH 4.73 and 4.80), CH3-30 (δH 1.67) to C-4 (δC 149.2). In REOSY spectrum of 2, the observation of key NOE correlations (Fig. 3) of H-19 with CH3-30, H-8 (δH 1.53), of CH3-18 (δH 1.04) with H-8, H-16 (δH 4.09) and H-20 (δH 2.10), of H-17 (δH 1.47) with CH3-28 (1.12) and CH3-21 (δH 0.89), suggested that the relative configuration of 2 was identical to that of compound 1. From structure, compound 2 could be considered as a dehydrated analogue of 1, implying that two compounds may share same configuration. Also, compound 2 had a negative Cotton effect (− 1.5) at 201 nm. Consequently, the structure of 2 was identified as (20R,23S,25R)- 16,23-epoxy-26(23)-olide-3,4-secocycloartan-4(29)-en-3-oic acid n-butyl ester, and named pseudolactone B.

Fig. 3
figure 3

Key 1H-1H COSY, HMBC, and NOESY correlations of compound 2

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The molecular formula of compound 3 was assigned as C31H46O5 with nine degrees of unsaturation, based on the hydrogen adduct ion [M + H]+ at m/z 499.3414 (calcd. 499.3418 for C31H47O5+). Its molecular weight is 42 Da less than that of 2. The 1H and 13C NMR spectra (Tables 1 and 2) were quite close to those of 2. The only difference was that a methoxyl at δH 3.61 and δC 51.5 was observed in 3, instead of those signals for the butoxy (δH 4.03, 1.59, 1.39, 0.93; δC 64.2 (t), 30.6 (t), 19.1 (t), 13.7 (q)) in 2. Key HMBC correlation from the methoxyl proton at δH 3.61 to the ester carbonyl at δC 174.3 further confirmed the above deduction, and established the structure of 3 as (20R,23S,5R)-16,23-epoxy-26(23)-olide-3,4-secocycloartan-4(29)-en-3-oic acid methyl ester, and given the name pseudolactone C.

Compound 4 had a molecular formula C32H52O6 with seven degrees of unsaturation, as evidenced by positive HR-ESI–MS at m/z 555.3674 [M + Na]+. The 1HNMR spectroscopic data (Table 1) of 4 exhibited four singlet methyls at δH 1.00 (s), 0.92 (s), 1.25 (s), and 1.21 (s), two doublet methyls at δH 0.86 (d, J = 6.0 Hz) and 1.22 (d, J = 9.6 Hz), one triplet methyl at δH 1.25 (t, J = 6.8 Hz), and an oxygenated methylene at δH 4.11 (2H, q, J = 6.8 Hz). In addition, a typical AB coupling system was observed at δH 0.57 (1H, d, J = 4.4 Hz) and 0.68 (1H, d, J = 4.4 Hz). In 13C NMR spectrum (Table 2), thirty-two carbon resonances were observed, including 7 methyls (δC 18.5, 19.2, 16.9, 19.5, 31.6, 26.2, 14.2), 12 methylenes (including one oxygenated methylene at δC 60.3), 5 methines, and 8 quaternary carbons (including one ester carbonyl at δC 174.8, one carboxyl at δC 180.8, one ketone carbonyl at δC 209.3, and one oxygenated quaternary carbon at δC 76.3). Deducting three degrees of unsaturation accounted for one ketone carbonyl and two carboxylic carbon, the remaining four degrees of unsaturation were indicative of the tetracyclic ring system of 4. The NMR spectroscopic data (Tables 1 and 2) of 4 quite resemble those of known compound pseudolarnoid G ((25S)-4-hydroxy-3,4-seco-cycloartan-23-one-3,26-dioic acid methyl), previously reported from the seeds of the tilted plant [13]. The main differences are that compound 4 had an ethoxy ester and one carboxyl functionalities, while pseudolarnoid G had two methyl ester functionalities.

The diagnostic chemical shifts at δC 174.8 (ester carbonyl) and δC 76.3 (s) were indicative of oxidative cleavage of ring A between C-3 and C-4, and formed an ethyl ester at C-3 and an oxygenated isopropyl moiety at C-4. The deduction was evidenced by key 1H-1H COSY correlation (Fig. 4) between H2-1′ (δH 4.11) and H2-2′ (δH 1.25), and between H2-1 and H2-2, together with HMBC correlations (Fig. 4) of H2-1 and H2-1′ with C-3 (δC 174.8), of CH3-29 (δH 1.25), CH3-30 (δH 1.21), H-5 (δH 1.85), H2-6 (δH 0.69, 1.73) with the oxygenated quaternary carbon at δC 76.3 (C-4). Also, the ketone carbonyl at δC 209.3 was assigned to be C-23 on the basis of HMBC correlations from H2-22 and H2-24 to the ketone carbonyl. Key HMBC cross-peaks of H2-24 and CH3-27 (δH 1.22 (d, J = 9.6 Hz) supported the attribution of C-26 carboxyl. In the ROESY spectrum, key NOE correlations (Fig. 4) of H-20 with CH3-18, of H-8 (δH 1.30) with CH3-18 and H-19, and of CH3-29 with H-19 indicated that H-8, CH3-18, CH2-19, H-20, and 4-hydroxyl isopropyl are β-oriented, whereas H-17 and CH3-28 were α-oriented based on the NOE cross-peak of H-17 with CH3-28. With regard to the absolute configurations of two chiral centers (C-20 and C-25) at C-17 side chain, Zhao et al. had ever determined the absolute configurations of C-20 and C-25 of similar compound pseudolarnoid G as R and S, respectively, by single crystal X-ray diffraction. The ECD spectrum of compound 4 showed a negative Cotton effect − 1.5 at 285 nm, in accordance with that of pseudolarnoid G (− 1.23 (283 nm)), revealed that compound 4 had same absolute configuration as that of pseudolarnoid G. Therefore, the structure of 4 was identified as (20R,25S)-4-hydroxy-23-oxo-3,4-secocycloartan-26-oic acid-3-ethyl ester, and named pseudolactone D.

Fig. 4
figure 4

Key 1H-1H COSY, HMBC, and NOESY correlations of compound 4

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3 Experimental Section

3.1 General Experimental Procedures

Optical rotations were measured with a Perkin–Elmer 341 polarimeter. IR spectra were recorded with a Bruker Vector-22 Spectrophotometer with KBr discs. NMR spectra were recorded with Bruker DRX-400 spectrometer (400 MHz). The chemical shifts (δ) are given in ppm with TMS as internal standard and coupling constants (J) are given in Hz. MS spectra were recorded with a Agilent MSD-Trap-XCT (for ESI) and Waters Micro-mass Q-TOF mass spectrometer (for HR-ESI–MS), in m/z. Column chromatographic separations were carried out by using silica gel (200–300 mesh; Marine Chemical Factory, Qingdao, P. R. China), Sephadex LH-20 (Pharmacia Fine Chemicals, Piscataway, NJ, USA) as packing material. TLC was carried out on precoated silica gel GF 254 plates (Yantai Chemical Industrials) and the TLC spots were viewed at 254 nm and visualized by using 10% sulfuric acid in alcohol containing 10 mg/mL vanillin.

3.2 Plant Material

The cones of P. amabilis were collected in Jiu Jiang, Jiangxi province, P. R. China, in October 2010, and authenticated by Prof. Han-Ming Zhang of Second Military Medical University. A voucher specimen (No. 20101015) is deposited in School of Pharmacy, Second Military Medical University.

3.3 Extraction and Isolation

The air-dried cones (12.0 kg) of P. amabilis were ground into powder and extracted with 80% EtOH for four times at room temperature to give a crude extract, which was further partitioned with petroleum ether (60–90 °C) (PE), CHCl3, and EtOAc, successively. The CHCl3-soluable extract was subjected to a silica gel column chromatography (CC) eluting with a gradient PE/EtOAc (from 30:0 to 0:1) to obtain eight fractions 1–8. Fraction 2 (58 g) was chromatographed over RP-18 column eluting with MeOH/H2O (from 3:7 to 10:0) to afford five subfractions. Subfraction 2 was further chromatographed on a silica gel column (CH2Cl2/PE, from 0:20 to 1:0, and MeOH) and purified by preparative TLC (cyclohexane/CH2Cl2/EtOAc, 20:1:1) to afford 2 (20 mg) and 3 (20 mg). Subfraction 4 was separated by repeated column chromatography on Sephadex LH-20 (CHCl3/MeOH, 1:1, and MeOH), and then purified by preparative TLC (cyclohexane/CH2Cl2/EtOAc, 15:1:1) to yield 1 (10 mg). Fraction 7 (70 g) was separated by RP-18 CC (MeCN/H2O, from 2:8 to 10:0) to afford 6 subfractions. Subfraction 3 was further chromatographed on a silica gel column (CHCl3/MeOH from 50:1 to 0:1) and purified by preparative TLC (PE/EtOAc/MeOH, 20:1:0.1) to afford 4 (8 mg).

3.3.1 Pseudolactone A (1)

Colorless oil, [α] 45.1 (c = 0.37, CH2Cl2). CD (c = 2.83 mmol/L, CH3CN, 20 °C) nm (Δε) 223 (-0.7). IR (KBr) νmax 1731, 1778, 2964 cm−1. For 1H and 13C NMR data (400 MHz, CDCl3), see Tables 1 and 2. ESI–MS: m/z 553.5 [M + Na]+, 529.3 [M − H]−. HR-ESI–MS: m/z 531.3678 [M + H]+ (calcd C32H51O6+, 531.3680).

3.3.2 Pseudolactone B (2)

Colorless oil, [α] 51.6 (c = 0.24, CH2Cl2). CD (c = 2.11 mmol/L, CH3CN, 20 °C) nm (Δε) 201 (− 1.5). IR (KBr) νmax 1727, 1770, 2974 cm−1. For 1H (400 MHz, CDCl3) and 13C NMR data (100 MHz, CDCl3), see Tables 1 and 2. ESI–MS: m/z 563.2 [M + Na]+, 539.1 [M − H]−. HRESIMS: m/z 541.3902 [M + H]+ (calcd C34H53O5+, 541.3888).

3.3.3 Pseudolactone C (3)

Colorless oil, [α] 51.1 (c = 0.23, CH2Cl2). CD (c = 2.61 mmol/L, CH3CN, 20 °C) nm (Δε) 201 (− 1.6). IR (KBr) νmax 1737, 1778, 2964 cm−1. For 1H (400 MHz, CDCl3) and 13C NMR data (100 MHz, CDCl3), sees Tables 1 and 2. ESIMS: m/z 521.2 [M + Na]+. HR-ESI–MS: m/z 499.3414 [M + H]+ (calcd C31H47O5+, 499.3418).

3.3.4 Pseudolactone D (4)

White amorphous powder, [α] 51.6 (c = 0.23, CH2Cl2). CD (c = 1.88 mmol/L, CH3CN, 20 °C) nm (Δε) 210 (− 1.9), 285 (− 1.5). IR (KBr) νmax 1712, 1735, 2873, 2925, 2962, 3440 cm−1. For 1H (400 MHz, CDCl3) and 13C NMR data (100 MHz, CDCl3), see Tables 1 and 2. ESI–MS: m/z 531.4 [M − H]−. HR-ESI–MS m/z 555.3674 [M + Na]+ (calcd C32H52O6Na+, 555.3656).