Two New Tetracyclic Triterpenoids from the Fresh Bark of Ailanthus altissima

Two new triterpenoids (1, 2) were isolated from the fresh bark of Ailanthus altissima. The structures were elucidated on the basis of spectroscopic analysis including 1D and 2D NMR techniques and high-resolution mass spectrometry.

Ailanthus altissima (Mill.) Swingle belongs to the Ailanthus genus of the Simaroubaceae family. It is commonly known as tree of heaven in China. The barks of the plant have been used as a traditional Chinese medicine for many years and the bioassay investigation has shown that the extract of this plant exhibited anti-inflammatory, fumigant toxicity, and phytotoxic activities [1]. In the previous study, we found a new triterpenoid in the fresh bark of Ailanthus altissima [2]. In this study, another two new tetracyclic triterpenoids were isolated from the same resource.

Compound 1 was obtained as white amorphous powder, exhibited a [M + Na]⁺ peak in the HR-ESI-MS at m/z 495.3448, which matched the molecular formula of C₃₀H₄₈O₄Na (calcd 495.3450). There are seven degrees of unsaturation. In the ¹³C NMR spectra (Table 1), together with HSQC and DEPT experiments, 30 carbon resonances were resolved and they were further sorted to eight Me, seven CH₂, six CH groups, and nine quaternary C atoms. One of the implied seven unsaturation degrees was attributed to the carbonyl group because the ¹³C NMR spectrum contained a low-field signal resonating at δ 216.5. In addition, two carbon-carbon double bonds at δ 118.4, 141.4, 145.7, and 116.1 were observed. No further sp²- or sp-hybridized carbon atoms implied that the remaining four degrees were a tetracyclic molecule.

TABLE 1. ¹H (700 MHz) and ¹³C (175 MHz) NMR Data of Compounds 1 and 2 (CDCl₃, δ, ppm, J/Hz)

The ¹H NMR spectrum of 1 displayed signals of seven tertiary Me groups, two of which are at δ 1.13 (s) and 1.06 (s) and mutually correlated in the HMBC experiment. Both exhibited long-range correlations with the C=O group at δ 216.5, a quaternary carbon at δ 47.8, and a tertiary carbon group at δ 50.1. According to this, a ketone group was located at C-3.

The HMBC technique showed the placement of the double bonds (Fig. 1): δ_H 1.16 (Me-19) correlated with δ_C 145.7 (C-9) and δ_H 0.88 (Me-30) correlated with δ_C 141.4 (C-8). Besides, there are two double bonds and HMBC at δ_H 5.23 (H-11) related to δ_C 36.1 (C-10), δ 38.3 (C-12), and δ_C 44.1 (C-13). Therefore, the two carbon-carbon double bonds are between C-7, C-8 and C-9, C-11.

Fig. 1.

Chemical structures of 1 and 2, key ¹H–¹H COSY and HMBC correlations of 1.

In addition to an oxygen atom in a ketone group, the remaining three oxygen atoms in the molecular formula must be hydroxyl groups; this conjecture was in accordance with observed chemical shifts in the ¹H NMR (δ 4.12, 3.17) and ¹³C NMR (δ 69.6, 74.9, and 73.3) spectra. ¹H and ¹³C NMR spectral data demonstrated that one quaternary and two tertiary carbons were substituted by hydroxyl groups. The 23-OH, 24-OH, and 25-OH substituents of the side chain were established by the ¹H–¹H COSY cross-peaks.

The NOESY experiment provided the relative configuration of the tetracyclic core. The correlations between H-2a (δ 2.83)/Me-19 and H-2a (δ 2.83)/Me-28 (δ 1.13) indicated that the Me-19 was upward equatorial. Me-19 and Me-30 had strong NOE, indicating that these were cis di-axial oriented. Me-30 and Me-18 had no NOE, suggesting that Me-18 might be downward equatorial. The NOESY correlation Me-18/Me-21 was observed, leading to (S)-configuration at C-20. Me-19/H-5 and Me-30/H-17 had no NOE, indicating that H-5 and H-17 were downward equatorial. The structure of 1 was very similar to ganodermanondiol [3], except for the hydroxyl group at C-23.

By comparing the ¹H and ¹³C NMR chemical shifts of the side chain of 1 [δ_H 4.12 (1H, m, H-23), 3.17 (1H, s, H-24); δ_C 69.6 (C-23), 74.9 (C-24)] with those of (20S,23R,24S)-7α,23,24,25-tetrahydroxyapotirucallan-3-one [4] [δ_H 4.17 (1H, dd, J = 8.1, 5.1 Hz, H-23), 3.96 (1H, s, H-24); δ_C 69.6 (C-23), 75.2 (C-24)], we cannot get the configurational assignment of C-23 and C-24.

Compound 2 had a similar structure to 1, except for the 12-OH. The chemical shift at δ_H 4.06 (1H, d, H-12) and the correlation with δ_C 145.3 (C-9), 117.0 (C-11), 73.7 (C-12), 48.5 (C-13) showed that there is a hydroxyl group at C-12. The NOE between H-12/Me-21 and H-12/Me-18 indicated that the 12-OH had a different direction than the Me-18 and Me-21; we infer that the 12-OH is upward equatorial.

Experimental

General. Column chromatography (CC): silica gel (200–300 mesh); TLC: silica gel GF₂₅₄ (Qingdao Marine Chemical Factory, China); visualization under UV light (254 and 365 nm) and by spraying with a solution of 10% (v/v) H₂SO₄ in EtOH, followed by heating at 110°C; Sephadex LH-20 (Pharmacia), and ODS gel (50 mm; Merck). HPLC: Shimadzu LC-6AD equipped with an SPD-10A detector, with reversed-phase (RP) C₁₈ column (YMC-Pack, ODS-A; 20 × 250 mm, 5 mm). ¹H and ¹³C NMR spectra on a NEO-700 machine were taken at 700 MHz and 175 MHz, resp. in CDCl₃ with chemical shift (δ) in ppm as referenced to a residual solvent peak (CHCl₃) taken as an internal standard at 7.26 ppm in ¹H NMR and 77.0 ppm in ¹³C NMR. Full assignments of the proton and carbon signals were secured with the help of ¹H–¹H COSY, HSQC, and HMBC spectra. ESI-MS: Agilent-1100 LC/MSD trap mass spectrometer. HR-ESI-MS: Agilent Technologies 6250 Accurate-Mass Q-TOF LC/MS spectrometer.

Plant Material. The plant, collected in April 2017 in Anguo, Hebei Province, China, was kindly authenticated by Prof. J. H. Wang. A voucher specimen (2017-4-29-TH) is avaible for inspection at the Herbarium of the Department of Medicinal Natural Product Chemistry, School of Pharmaceutical Sciences, Hebei Medical University.

Extraction and Isolation. The fresh bark of Ailanthus altissima (20 kg) was cut into smaller pieces and extracted exhaustively by 95% EtOH at room temperature three times. The solvent was removed under vacuum to give a crude extract (705.9 g), which was then subjected to extraction successively by petroleum ether (PE), CH₂Cl₂, and EtOAc for three times, respectively. The crude CH₂Cl₂ extract (143.2 mg) was loaded onto a silica gel column eluting with a PE–EtOAc–MeOH gradient (5:1:0–1:1:0–0:1:0–0:0:1). The elution of No.1 to No. 76 (each 500 mL) was combined into 14 fractions (Frs. 1–14) on the basis of their TLC profiles. Fraction 4 (17.5 g) was applied to a silica gel column and eluted with a CH₂Cl₂–acetone (40:1–10:1–1:1) gradiently to give 9 fractions denoted Subfrs. 1–9. Subfraction 9 was purified by preparative reversed phase HPLC (MeOH–H₂O, 6:4–9:1; flow rate, 4.0 mL/min; 220 nm; t_R = 12.59 min) to afford 1 (1.23 mg). Fraction 10 (3.9 g) was applied to a silica gel column and eluted with a CH₂Cl₂–acetone (10:1–5:1–0:1) gradiently to yield 5 fractions denoted Subfrs. 1–5. Subfraction 5 was purified by preparative reversed phase HPLC (MeOH–H₂O, 6:4–9:1; flow rate, 4.0 mL/min; 220 nm; t_R = 11.46 min) to afford 2 (4.52 mg).

23-Hydroxyganodermanondiol (1), white powder. ¹H and ¹³C NMR data, see Table 1. HR-ESI-MS m/z 495.3448 [M + Na]⁺ (calcd for C₃₀H₄₈O₄Na, 495.3450).

12β,23-Dihydroxyganodermanondiol (2), white powder. ¹H and ¹³C NMR data, see Table 1. HR-ESI-MS m/z 511.3402 [M + Na]⁺ (calcd for C₃₀H₄₈O₅Na, 511.3399).