Notes
New Triterpenoids from the Stems of Cornus walteri
2017 Volume 65 Issue 7 Pages 683-686
Details
2017 Volume 65 Issue 7 Pages 683-686
A new 28-norlupane triterpenoid, 3-acetate-28-norlup-20(29)-en-3β-hydroxy-17β-hydroperoxide (1) and a new tirucallane triterpenoid, cornusalterin M (2), together with one known triterpenoid, 3-acetate-28-norlup-20(29)-en-3β,17β-diol (3), were isolated from a MeOH extract of the stems of Cornus walteri. The chemical structures of the new compounds (1 and 2) were elucidated based on comprehensive one- and two-dimensional (1D and 2D) NMR spectroscopic experiments and high resolution-electrospray ionization (HR-ESI)-MS. Among the isolates, compound 1 was a relatively rare triterpenoid identified as a 28-norlupane-type triterpene with a 17β-hydroperoxide group and compound 3 was previously reported but only as a synthetic product. The cytotoxic activities of the isolated compounds 1–3 were evaluated by determining their inhibitory effects on human tumor cell lines (A549 (non-small cell lung carcinoma), SK-OV-3 (ovary malignant ascites), SK-MEL-2 (skin melanoma), and HCT-15 (colon adenocarcinoma)).
Counus walteri WAGNER (Cornaceae) is a deciduous tree that mainly grows in the valley areas of Korea and China. In Chinese traditional medicine, the fruits and leaves of C. walteri have been widely utilized for the alleviation of skin inflammatory symptoms and the treatment of glycosuria.1–3) In addition, the leaves have been used for the treatment of diarrheal symptoms as a traditional medicine in Korea.4) A variety of therapeutic activities including anti-inflammatory, anti-obesity, and anti-hyperglycemic effects have been reported for the extracts of C. walteri.5,6) Additionally, the anti-photoaging activity of this source was investigated in a recent study.2) As part of our continuing efforts to explore structurally novel and bioactive constituents from natural sources,7–14) our recent phytochemical studies of the stems of C. walteri reported the isolation and identification of bioactive compounds, tirucallane triterpenoids and δ-valerolactones with cytotoxic and anti-inflammatory activities.15,16) In our ongoing search for bioactive constituents from this source, we further investigated the MeOH extract of the stems of C. walteri through extensive chromatographic purifications, which resulted in the isolation of a new 28-norlupane triterpenoid, 3-acetate-28-norlup-20(29)-en-3β-hydroxy-17β-hydroperoxide (1) and a new tirucallane triterpene, (20α,24S)-20,24-dihydroxy-tirucall-25(26)-ene-3-one (2), along with one known triterpenoid, 3-acetate-28-norlup-20(29)-en-3β,17β-diol (3) (Fig. 1). In the present study, we report the isolation and structural elucidation of the isolated compounds and their cytotoxic activities.
Compound 1 was isolated as a white amorphous powder, and possessed the molecular formula of C31H50O4 as deduced from the positive-ion mode high resolution electrospray ionization (HR-ESI)-MS data at m/z 487.3792 [M+H]+ (Calcd for C31H51O4, 487.3787). The characteristic absorption band (3405 cm−1) obtained from the IR spectrum of 1 suggested the presence of a hydroxyl group. The 1H-NMR spectrum of 1 displayed a typical pattern of a lupane-type triterpene, with six tertiary methyls [δH 0.86, 0.87, 0.88, 0.96, 1.05, and 1.69 (each 3H, s)] including a vinylic methyl at δH 1.69 (s); an oxygenated methine [δH 4.50 (1H, dd, J=11.0, 6.0 Hz)]; and an olefinic methylene [δH 4.62 (1H, br s), 4.72 (1H, br s)]. The 13C-NMR data of 1, with the aid of heteronuclear single quantum coherence spectroscopy (HSQC) analysis, showed 31 carbon signals, which were classified as seven quaternary carbons (δC 37.4, 38.7, 41.1, 42.3, 91.8, 150.2, and 171.2); six methines (δC 36.9, 48.2, 49.4, 50.8, 55.7, and 81.2) including an distinguished oxygenated carbon (δC 81.2); eleven methylenes (δC 18.5, 21.2, 23.9, 25.5, 27.2, 27.5, 29.7, 32.4, 34.6, 38.0, and 110.0) including an olefinic methylene (δC 110.0); and seven methyls (δC 14.2, 16.4, 16.5, 16.7, 19.4, 21.5, and 28.2). Detailed inspection of its NMR data revealed that compound 1 was almost identical to 3-acetate-28-norlup-20(29)-ene-3β,17β-diol (3), except for the oxygenated quaternary carbon of C-17 (δC 91.8) in 1, instead of the corresponding one (δC 80.4) in 3.17) The existence of the hydroperoxide group at C-17 was confirmed based on the characteristic carbon chemical shift of C-17 (δC 91.8),18) along with heteronuclear multiple bond correlation (HMBC) correlations from H-13 (δH 1.85), H2-15 (δH 1.37 and 1.98), and H-19 (δH 2.61) to C-17 (δC 91.8) (Fig. 2). The gross structure of 1 was confirmed by the cross peaks in the 1H–1H correlation spectroscopy (COSY) and HMBC spectra (Fig. 2). The β-configuration of the hydroperoxide group of C-17 in 1 was unambiguously determined by comparing the chemical shift (δC 91.8) of C-17 with that of 28-norlup-20(29)-en-3β-hydroxy-17β-hydroperoxide [α-form=δC 93.4 (C-17); β-form=δC 91.6 (C-17) in CDCl3].18) The nuclear Overhauser spectroscopy (NOESY) correlations of H3-25/H3-26, H-13/H3-26, H-13/H-19, H-9/H3-27, H-18/H3-27, H-18/H2-29, H-5/H-9, and H-3/H-5 as well as aspects of the biosynthetic pathway suggested that the relative configuration of 1 was the same as compound 3. Accordingly, the chemical structure of 1 was determined to be 3-acetate-28-norlup-20(29)-en-3β-hydroxy-17β-hydroperoxide.
) and HMBC (→) Correlations of 1 and 2Compound 2 was isolated as a white amorphous powder with the molecular formula of C30H50O3 determined by the HR-ESI-MS data at m/z 459.3836 [M+H]+ (Calcd for C30H51O3, 459.3838). The IR spectrum of 2 suggested the existence of hydroxyl groups and a ketone moiety from the distinguished absorption bands (3410 and 1730 cm−1, respectively). The 1H-NMR data of 2 showed the characteristic seven tertiary methyls [δH 0.93, 0.97, 1.04 (×2), 1.07, 1.12, and 1.71 (each 3H, s)] including a vinylic methyl at δH 1.71 (s); an oxygenated methine [δH 3.96 (1H, t, J=6.0 Hz)]; and an olefinic methylene [δH 4.82 (1H, br s), 4.90 (1H, br s)]. The 13C-NMR spectrum revealed 30 carbon resonances, which were classified by HSQC data as seven quaternary carbons (δC 37.0, 42.4, 47.8, 50.2, 74.7, 147.8, and 220.0); five methines (δC 42.5, 49.9, 50.3, 55.3, and 76.3) including an oxygenated carbon (δC 76.3); eleven methylenes (δC 22.0, 24.1, 24.6, 27.5, 29.0, 31.1, 33.9, 34.5, 36.8, 40.3, and 110.5) including an olefinic methylene (δC 110.5); and seven methyls (δC 14.5, 15.4, 15.7, 19.6, 20.3, 26.1, and 27.6). The NMR data of 2 was similar to the data from cornusalterins A–E identified from C. walteri by our group,15) which suggested that compound 2 was a tirucallane-type triterpene. Detailed interpretation of COSY and HMBC data led to elucidation of the gross structure of 2 (Fig. 2). The hydroxyl group at C-20 was confirmed based on the down-field shifted carbon chemical shift (δC 74.7) of C-20 and HMBC correlations from H-17 (δH 1.52) and H3-21 (δH 1.12) to C-20 (δC 74.7) (Fig. 2). The hydroxyl group of C-20 was confirmed to be α-oriented based on the NOESY correlations of H-17/H3-21 and H3-18/H3-2119) (Fig. 3). In addition, the presence of a hydroxyl group at C-24 and Δ25,26-double bond was unambiguously identified by the HMBC correlations of H-24/C-25, H-24/C-26, H-24/C-27, H2-26/C-24, H2-26/C-27, H3-27/C-24, and H3-27/C-26 (Fig. 2). The absolute configuration of C-24 was assigned as 24S based on the comparison of the coupling constant and chemical shifts [δH 3.96 (1H, t, J=6.0 Hz); δC 76.3] of C-24 with those previously reported [24R=δH 4.26 (1H, t, J=6.8 Hz); δC 90.5 and 24S=δH 3.97 (1H, t, J=6.1 Hz) and δC 76.9 in CDCl3] in the related compound.20) Finally, the other relative configuration of 2 was determined by analysis of the NOESY data. Thus, the chemical structure of 2 was elucidated as (20α,24S)-20,24-dihydroxy-tirucall-25(26)-ene-3-one and named as cornusalterin M.
) Correlations of 2The known compound 3 was identified as 3-acetate-28-norlup-20(29)-en-3β,17β-diol by detailed comparison of its physical and spectroscopic data with previously reported values.17) To the best of our knowledge, the two norlupane-type triterpenes (1 and 3) had been not reported from C. walteri and compound 3 was reported as a natural product in this study for the first time since it was previously only reported as a synthetic product.
The cytotoxic activities of the isolated compounds 1–3 were evaluated by determining their inhibitory effects on human tumor cell lines (A549 (non-small cell lung carcinoma), SK-OV-3 (ovary malignant ascites), SK-MEL-2 (skin melanoma), and HCT-15 (colon adenocarcinoma)) using the sulforhodamine B (SRB) bioassay. However, the three tested compounds were inactive (IC50: >10.0 µM).
Optical rotations were measured on a Jasco P-1020 polarimeter. IR spectra were recorded on a Bruker IFS-66/S FT-IR spectrometer. UV spectra were recorded with a Shimadzu UV-1601 UV-visible spectrophotometer. ESI and HR-ESI mass spectra were recorded on a SI-2/LCQ DecaXP LC-MS. NMR spectra, including 1H–1H COSY, HSQC, HMBC, and NOESY experiments, were recorded on a Varian UNITY INOVA 500 NMR spectrometer operating at 500 MHz (1H) and 125 MHz (13C), with chemical shifts given in ppm (δ). Preparative HPLC was performed using a Gilson 306 pump with a Shodex refractive index detector. A silica gel 60 (Merck, 230–400 mesh) and an RP-C18 silica gel (Merck, 230–400 mesh) were used for column chromatography. Merck precoated silica gel F254 plates and RP-18 F254 s plates were used for TLC. Spots were detected on TLC under UV light or by heating after spraying with anisaldehyde–sulfuric acid.
Plant MaterialsThe stems of C. walteri were acquired from Jeju Island, Korea, in October 2014. The plant was identified by one of the authors (K.H.K). A voucher specimen (SKKU MC-2014) was deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea.
Extraction and IsolationThe stems of C. walteri (2.5 kg) were dried, mashed, and extracted with 80% MeOH (4L×3) twice at room temperature and filtered. The filtrate was evaporated in vacuo to obtain a slurry of the MeOH extract (220 g). The MeOH extract was dissolved in distilled water (800 mL) and then successively solvent-partitioned with n-hexane, CHCl3 and n-BuOH, affording dried residues weighing 9, 25, and 43 g. The n-hexane-soluble fraction (9 g) was subjected to a silica gel (230–400 mesh) column eluted with a gradient solvent system of n-hexane–EtOAc (3 : 1 to 1 : 1, v/v) to afford five fractions (H1–H5). Fraction H1 (3.3 g) was fractionated by an RP-C18 silica gel (230–400 mesh) column with an isocratic solvent system of 100% MeOH to give five subfractions (H11–H15). Subfraction H13 (750 mg) was loaded onto a silica gel (230–400 mesh) column with n-hexane–EtOAc (7 : 1, v/v) and then successively followed by semi-preparative reverse-phase HPLC (Econosil C18 column, 250×10.0 mm, 5 µm, flow rate: 2 mL/min) utilizing 100% MeOH to provide compounds 1 (6 mg) and 3 (5 mg). Fraction H5 (0.5 g) was fractionated by an RP-C18 silica gel (230–400 mesh) column with an isocratic solvent system of 85% MeOH to give five subfractions (H51–55). Compound 2 (10 mg) was purified from subfraction H54 (120 mg) by semi-preparative normal-phase HPLC (Apollo Silica column, 250×10.0 mm, 5 µm, flow rate: 2 mL/min) with an isocratic solvent system of CHCl3–MeOH (22 : 1, v/v).
3-Acetate-28-norlup-20(29)-en-3β-hydroxy-17β-hydroperoxide (1)White amorphous powder; [α]D25+17.9 (c=0.10, MeOH); IR (KBr) νmax 3405, 2947, 1723, 1632, 1354, 1032, 670 cm−1; 1H-NMR (CDCl3, 500 MHz) δ: 4.72 (1H, br s, H-29b), 4.62 (1H, br s, H-29a), 4.50 (1H, dd, J=11.0, 6.0 Hz, H-3), 2.61 (1H, m, H-19), 2.31 (1H, m, H-21α), 2.20 (1H, m, H-22α), 2.06 (3H, s, OAc), 1.98 (1H, m, H-15α), 1.86 (2H, m, H-1α, H-16α), 1.85 (1H, m, H-13), 1.69 (1H, m, H-2α), 1.69 (3H, s, H-30), 1.68 (1H, m, H-18), 1.66 (1H, m, H-12α), 1.65 (1H, m, H-6α), 1.61 (1H, m, H-2β), 1.48 (1H, m, H-11α), 1.43 (1H, m, H-21β), 1.41 (2H, m, H-7α, H-7β), 1.37 (1H, m, H-15β), 1.36 (1H, m, H-6β), 1.35 (1H, m, H-22β), 1.25 (1H, m, H-9), 1.19 (1H, m, H-12β), 1.17 (1H, m, H-11β), 1.08 (1H, m, H-1β), 1.07 (1H, m, H-16β), 1.05 (3H, s, H-26), 0.96 (3H, s, H-23), 0.88 (3H, s, H-27), 0.87 (3H, s, H-25), 0.86 (3H, s, H-24), 0.79 (1H, m, H-5); 13C-NMR (CDCl3, 125 MHz) δ: 171.2 (OAc), 150.2 (C-20), 110.0 (C-29), 91.8 (C-17), 81.2 (C-3), 55.7 (C-5), 50.8 (C-9), 49.4 (C-18), 48.2 (C-19), 42.3 (C-14), 41.1 (C-8), 38.7 (C-4), 38.0 (C-1), 37.4 (C-10), 36.9 (C-13), 34.6 (C-7), 32.4 (C-22), 29.7 (C-15), 28.2 (C-23), 27.5 (C-21), 27.2 (C-16), 25.5 (C-12), 23.9 (C-2), 21.5 (OAc), 21.2 (C-11), 19.4 (C-30), 18.5 (C-6), 16.7 (C-24), 16.5 (C-25), 16.4 (C-26), 14.2 (C-27); HR-ESI-MS (positive-ion mode) m/z: 487.3792 [M+H]+ (Calcd for C31H51O4, 487.3787).
Cornusalterin M (2)white amorphous powder; [α]D25−32.9 (c=0.10, MeOH); IR (KBr) νmax 3410, 2931, 1730, 1662, 1367, 1051, 679 cm−1; 1H-NMR (CDCl3, 500 MHz) δ: 4.90 (1H, br s, H-26b), 4.82 (1H, br s, H-26a), 3.96 (1H, t, J=6.0 Hz, H-24), 2.49 (1H, m, H-2β), 1.94 (1H, m, H-2α), 1.82 (1H, m, H-23b), 1.78 (1H, m, H-1β), 1.74 (2H, m, H-12β, H-16β), 1.73 (1H, m, H-6α), 1.71 (3H, s, H-27), 1.61 (1H, m, H-23a), 1.60 (1H, m, H-15β), 1.56 (1H, m, H-16α), 1.53 (1H, m, H-7α), 1.52 (1H, m, H-17), 1.51 (1H, m, H-11α), 1.50 (1H, m, H-6β), 1.44 (1H, m, H-8), 1.42 (2H, m, H-22), 1.35 (1H, m, H-9), 1.31 (2H, m, H-7β, H-15α), 1.30 (1H, m, H-1α), 1.25 (1H, m, H-12α), 1.12 (3H, s, H-21), 1.10 (1H, m, H-11β), 1.07 (3H, s, H-19), 1.04 (each 3H, s, H-28, H-29), 0.97 (3H, s, H-30), 0.93 (3H, s, H-18), 0.77 (1H, m, H-5); 13C-NMR (CDCl3, 125 MHz) δ: 220.0 (C-3), 147.8 (C-25), 110.5 (C-26), 76.3 (C-24), 74.7 (C-20), 55.3 (C-5), 50.3 (C-9), 50.2 (C-14), 49.9 (C-17), 47.8 (C-4), 42.5 (C-8), 42.4 (C-13), 40.3 (C-1), 37.0 (C-10), 36.8 (C-22), 34.5 (C-7), 33.9 (C-2), 31.1 (C-15), 29.0 (C-23), 27.6 (C-28), 27.5 (C-16), 26.1 (C-21), 24.6 (C-12), 24.1 (C-11), 22.0 (C-6), 20.3 (C-29), 19.6 (C-27), 15.7 (C-30), 15.4 (C-19), 14.5 (C-18); HR-ESI-MS (positive-ion mode) m/z: 459.3836 [M+H]+ (Calcd for C30H51O3, 459.3838).
Cytotoxicity AssayA sulforhodamine B (SRB) bioassay was used to determine the cytotoxicity of each isolated compound against four cultured human tumor cell lines.21–26) The cell lines used were A549, SK-OV-3, SK-MEL-2, and HCT-15. Doxorubicin was used as a positive control. The cytotoxicity of doxorubicin against the A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines was IC50 0.010, 0.001, 0.001, and 0.028 µM, respectively.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2015R1C1A1A02037383) and by the Ministry of Education (NRF-2012R1A5A2A28671860).
The authors declare no conflict of interest.
