RETRACTED ARTICLE: Three tetranortriterpenoids from Walsura yunnanensis

Abstract

Three tetranortriterpenoids, walsuranins A–C (1–3), as well as three known tetranortriterpenoids, anthothecol, 11β-hydroxydihydrocedrelone, and 11β-acetoxydihydrocedrelone, were isolated from the twigs and leaves of Walsura yunnanensis C. Y. Wu. Their structures were established on the basis of spectroscopic methods. Walsuranins B–C (2, 3) from this plant featured the typical rearranged A-ring structure of this compound class.

The genus Walsura (Meliaceae) comprising about 40 species and varieties is mainly distributed in the tropical areas of Asia [1]. In previous literature, many investigations on this genus have led to the isolation of quite a number of tetranortriterpenoids and triterpenoids that showed insect antifeedant activity, antimalarial activity, and cell protecting activity [2–7]. In the present paper, three new tetranortriterpenoids, walsuranins A–C (1-3), as well as three known tetranortriterpenoids, anthothecol [8], 11β-hydroxydihydrocedrelone [5], and 11β-acetoxydihydrocedrelone [5],were isolated from the leaves and twigs of W. yunnanensis. Their structures were established on the basis of spectroscopic methods. Walsuranins B–C (2, 3) featured the typical rearranged A-ring structure of this compound class. We present herein the isolation and structural elucidation of these compounds.

Walsuranin A (1), a white powder, was determined to have a molecular formula of C₂₇H₃₄O₇Na by the sodiated HR-ESI-MS ion at m/z 493.2197 [M + Na]⁺ (calcd for 493.2202), which is consistent with requiring 11 double-bond equivalents. The IR absorption bands at 3377, 1709, and 1678 cm^–1 indicated the presence of hydroxyl and carbonyl groups [5]. The UV spectrum indicated the presence of an α,β-unsaturated carbonyl group (212 nm, 279 nm). The ¹³C NMR (Table 2) showed 27 carbon resonances comprising two carbonyl groups (δ 197.5 and 213.4), six olefinic carbons, four of which were attributed to a β-substituted furan ring moiety, five tertiary methyl groups (δ_C 16.5, 20.7, 21.9, 22.9, 23.9), and four skeleton quaternary carbons [δ 47.7 (C-4), 45.1 (C-8), 44.0 (C-10), 41.2 (C-13)]. In addition, a trisubstituted epoxide (δ_H 4.03, s; δ_C 59.1 and 69.9) was further distinguished by analysis of its NMR data (Tables 1 and 2). One proton resonance at δ 6.35 (1H, s) showing no correlation with any carbons in the HSQC spectrum was assigned to the exchangeable protons of one hydroxyl group. The aforementioned data implied that compound 1 possesses the tetranortriterpenoid skeleton [5]. The ¹H and ¹³C NMR data of 1 were very similar to those of 11β-hydroxydihydrocedrelone [5], indicating that they are structurally related analogs.

Table 1 ¹H NMR Spectroscopic Data of 1–3 (400 MHz, CDCl₃, δ, ppm, J/Hz)

Table 2 ¹³C NMR Spectroscopic Data for 1–3 (100 MHz, CDCl₃, δ, ppm, TMS)

The structure of 1 was further demonstrated by analysis of 2D NMR spectra, especially HMBC. In combination with the analysis of its HMBC spectrum, the A, B, C, and D rings of 1 were readily constructed by comparison of the NMR data with those of the known 11β-hydroxydihydrocedrelone [5]. The HMBC correlations from H-1 to C-2, C-3, C-5, C-9, C-10, C-19, and OMe (δ_C 56.9) indicated the presence of an OMe-group substituted at C-1, and C-3 was a ketone moiety in the A-ring. One hydroxyl group resonated at δ 6.35 (1H, s), showing HMBC correlations with C-5 (δ_C 136.4), C-6 (δ_C 142.0), and C-7 (δ_C 197.5) was placed at C-6. In addition, correlations from H-30 and H-9 to C-7 also showed that the α,β-unsaturated carbonyl group was in the B-ring. The chemical shifts of C-14 (δ_C 69.9) and C-15 (δ_C 59.1) revealed the presence of a trisubstituted 14,15-epoxide, which was confirmed by the mutual HMBC correlations from H-15 to C-13, C-16, and C-17, and from H₃-18 and H₃-30 to C-14 in the D-ring. The remaining one oxygenated carbon was assigned to C-11 by the mutual HMBC correlations of H₃-18/C-12, H-11/C-8, and H-11/C-13 in the C-ring. The presence of a β-substituted furan ring at C-17 was also supported by the HMBC correlations of H-17/C-20, C-21, and C-22. Therefore, the planar structure of 1 was confirmed.

The relative stereochemistry of 1 was completed by the analysis of ROESY experiment. The ROESY cross-peaks of H-9/H-1, H-11, H-18 and H-12/H-11 and H-18, H-11/H-1, and H-15/H-16 indicated that H-1, H-9, H-11, H-15, and H₃-18 were cofacial and randomly assigned in a α-configuration. In consequence, the ROESY correlation between H₃-19 and H₃-30 suggested that they were β-oriented. Thus, the structure of walsuranin A (1) was confirmed.

Walsuranin B (2), a white amorphous powder, was determined to have a molecular formula of C₂₈H₃₆O₆ by the sodiated HR-ESI-MS ion at m/z 491.2404 [M + Na]⁺ (calcd for C₂₈H₃₆O₆Na, 491.2410). The strong IR absorption at 3462 and 1740 cm^–1 revealed the presences of hydroxyl and carbonyl groups. In accordance with its molecular formula, all the 28 carbons were well resolved as 28 carbon resonances in the ¹³C NMR spectrum (Table 2), and were further classified by DEPT experiments as five methyls, five methylenes, 10 methines (three oxygenated and four olefinic ones), and eight quaternary carbons (one ester carbonyl, one oxygenated, one ketone, and two olefinic carbons). In addition, the ¹H and ¹³C NMR data of 2 also showed a furan ring group in the structure. Furthermore, one proton resonance at δ_H 2.28 (1H, s), which did not correlate with any carbons in the HSQC spectrum, was only attributable to the presence of a hydroxyl. The above information suggested that 2 had a different tetranortriterpenoid skeleton. Comparison of the ¹H and ¹³C NMR (Tables 1 and 2) of 2 with those of zumsenin [9] suggested that they were structural analogs.

Comprehensive analysis of the 1D and 2D NMR spectra, especially HMBC, allowed the establishment of the typical A₁, A₂, B, C, and D rings of the unusual tetranortriterpenoid skeleton [9–12]. In the HMBC spectrum, the presence of the special A₁–A₂ ring structure was confirmed on the basis of the HMBC correlations of H-1/C-2, C-3, C-19, H-2/C-10, H₂-19/C-9, C-10, H₃-28/C-4, C-5, H₃-29/C-4, C-5, and H-5/C-19. One hydroxyl resonating at δ_H 2.28 was assigned to C-7 in the B-ring by the key correlations from 7-OH to C-6 (δ_C 26.2), C-7 (δ_C 73.2), and C-8 (δ_C 45.2), from H-7 to C-5 (δ_C 52.7) and C-9 (δ_C 41.8), and from H-30 to C-7. Furthermore, the mutual HMBC correlations of H-15/C-8, C-13, C-14, C-16, and C-17, suggest the presence of a ∆¹⁴ double bond in the D-ring. The HMBC correlations from H-11 to an acetyl carbonyl (δ_C 170.5), C-8, and C-12, and from H₃-18 to C-12 and C-17 further validated that OAc was located on C-11.

The relative stereochemistry of 2 was established by the ROESY experiment. The ROESY correlations of H-1/H-5, H-1/H-9, H-1/H-11, H-9/H-18, and H-1/H-28 indicated that H-1, H-5, H-11, and H-28 were cofacial, and they were randomly assigned an α-orientation. Subsequently, the ROESY correlations of H₃-19/H-2β, H₃-19/H-29, H₃-19/H-30, H-7/H-15, and H-7/H-30 indicated that they were in the upside of the molecule and were β-oriented. Therefore, the structure of walsuranin B was thus fully assigned as depicted.

Walsuranin C (3) gave a molecular formula of C₂₈H₃₄O₆ as determined by HR-ESI-MS. Comparison of the ¹H and ¹³C NMR data (Tables 1 and 2) of 3 with those of 2 revealed that the two compounds were very similar, and the major differences between them were the absence of the A₂-ring to form the five-membered spirocyclic system of the A₁-ring at C-10, and the presence of an α,β-unsaturated carbonyl group (δ_C 203.5) in the A₁-ring, which was validated by the HMBC correlations from H-1 to C-3 (δ_C 203.5), C-5, C-10, and C-19, and from H-19 to C-9 and C-3. In the HMBC spectrum, an isopropyl moiety attached to C-5 was confirmed by the key correlations of C-5/H₃-28, H₃-29, and H-19, which supported the above deduction. In addition, the hydrogen signal of 7-OH was missing while C-6 and C-8 were downfield-shifted ∆δ 11 and ∆δ 7, indicating that maybe a keto carbonyl group was located at C-7 (δ_C 209.1) of 3 due to the electronattraction effect of the group, which was confirmed by the HMBC correlations from H-6, H-9, and H₃-30 to C-7. The remaining structure of 3 was exactly the same as that of 2. As the result, the planar structure of 3 was confirmed.

The relative stereochemistry of 3 was fixed by a ROESY experiment. The similar NMR patterns of the above compounds indicated that they share the same relative configuration in the skeleton core, which was confirmed by the observed key ROESY correlations of H-1/H-5, H-9 and H-11, H-9/H₃-18 and H-11, showing these protons were a-oriented, and correlations of H-4/H-19, H₃-30, implying H-4 and H-19 were β-directed. Therefore, walsuranin C was established as depicted.

Experimental

The leaves and twigs of W. yunnanensis were collected from Xishuangbanna of Yunnan Province, People's Republic of China. All solvents used were of the highest commercially available quality.

FT Infrared (IR) spectra were recorded in KBr disks using an FD-5DX spectrometer. UV spectra were recorded on a Shimadzu UV-2550 spectrophotometer. Specific rotations were determined on a Perkin–Elmer 341 polarimeter. ESI-MS and HR-ESI-MS were obtained on an Esquire 3000 plus (Bruker Daltonics) and a Waters-Micromass Q-TOF Ultima Global electrospray mass spectrometer, respectively. ¹H NMR and ¹³C NMR spectra were obtained on a Varian INOVA-400 spectrometer with CDCl₃ as the solvent; TMS (δ 0.00 ppm) was used as an internal standard. All NMR chemical shifts are reported as δ values in parts per million (ppm), and coupling constants (J) are given in hertz (Hz). The splitting pattern abbreviations are as follows: s, singlet; d, doublet; br, broad peak; and m, multiplet. Semipreparative HPLC was carried out on a Waters 515 pump with a Waters 2487 detector (254 nm) and a YMC-Pack ODS-A column (250 × 10 mm, S-5 μm, 12 nm).

Extraction and Isolation. The air-dried powders of trunks of W. yunnanensis (5 kg) were extracted three times with 95% EtOH at room temperature to give an ethanolic extract (160 g), which was partitioned between EtOAc and water to obtain the EtOAc-soluble fraction (60 g). The fraction was separated on a column of MCl gel (MeOH–H₂O, 40:60 to 90:10, v/v) to afford eight fractions, A–H. Fraction C (6.15 g) was chromatographed on a silica gel column eluted with petroleum ether–ethyl acetate (100:1 to 5:1, v/v) to afford six major fractions. Fraction C6 (215 mg) was purified by semipreparative HPLC with 55% acetonitrile in water to yield compounds 1 (7 mg), 2 (10 mg), and 3 (5 mg). Fraction D (5.10 g) was chromatographed on a silica gel column eluted with petroleum ether–ethyl acetate (17:1 to 1:1, v/v) to afford seven major subfractions, D1–D7. Fractions D4 (58 mg) and D5 (132 mg) were purified by semipreparative HPLC with 55% acetonitrile in water to yield anthothecol (15 mg) and 11α-hydroxydihydrocedrelone (10 mg) from D4, and 11α-acetoxydihydrocedrelone (8 mg) and 11α-hydroxycedrelone (7 mg) from D5.

Walsuranin A (1). White amorphous powder, [α]²⁴ _D –16.0° (c 0.05, CH₃OH). UV (MeOH, λ_max nm) (log ε): 212 (4.08), 279 (4.58). IR (KBr, ν_max, cm^–1): 3377, 2933, 1709, 1678, 1458, 1352, 1257, 1086, 1034. For ¹H NMR (400 MHz, CDCl₃, δ, ppm) and ¹³C NMR (100 MHz, CDCl₃, δ) data, see Tables 1 and 2, respectively. Positive mode ESI-MS m/z 471 [M + H]⁺; HR-ESI-MS m/z 493.2197 [M + Na]⁺ ([calcd for C₂₇H₃₄O₇Na, 493.2202).

Walsuranin B (2). White powder, [α]_D ²⁴-64.0° (c 0.05, CH₃OH). UV (MeOH, λ_max nm) (log ε): 207 (4.30). IR (KBr, ν_max, cm^–1): 3462, 2920, 1740, 1458, 1383, 1236, 1036, 1001. For ¹H NMR (400 MHz, CDCl₃, δ, ppm) and ¹³C NMR (100 MHz, CDCl₃, δ) data, see Tables 1 and 2, respectively. Positive mode ESI-MS m/z 491 [M + Na]⁺; HR-ESI-MS m/z 491.2404 [M + Na]⁺ (calcd for C₂₈H₃₆O₆Na, 491.2410).

Walsuranin C (3). White powder; [α]²⁴ _D –114.0° (c 0.05, MeOH). UV (MeOH, λ_max, nm) (log ε): 205 (4.29), 257 (4.57). IR (KBr, ν_max, cm^–1): 3412, 2956, 1738, 1699, 1657, 1389, 1238, 1111, 1032. For ¹H NMR (400 MHz, CDCl₃, δ, ppm) and ¹³C NMR (100 MHz, CDCl₃, δ) data, see Tables 1 and 2, respectively. Positive mode ESI-MS m/z 489 [M + Na]⁺; HR-ESI-MS m/z 489.2248 [M + Na]⁺ (calcd for C₂₈H₃₄O₆Na, 489.2253).