New Abietane and Kaurane Type Diterpenoids from the Stems of Tripterygium regelii

Eleven new abietane type (1‒11), and one new kaurane (12), diterpenes, together with eleven known compounds (13–23), were isolated and identified from the stems of Tripterygium regelii, which has been used as a traditional folk Chinese medicine for the treatment of rheumatoid arthritis in China. The structures of new compounds were characterized by means of the interpretation of high-resolution electrospray ionization mass spectrometry (HRESIMS), extensive nuclear magnetic resonance (NMR) spectroscopic data and comparisons of their experimental CD spectra with calculated electronic circular dichroism (ECD) spectra. Compound 1 is the first abietane type diterpene with an 18→1 lactone ring. Compound 19 was isolated from the plants of the Tripterygium genus for the first time, and compounds 14–17 were isolated from T. regelii for the first time. Triregelin I (9) showed significant cytotoxicity against A2780 and HepG2 with IC50 values of 5.88 and 11.74 µM, respectively. It was found that this compound was inactive against MCF-7 cells. The discovery of these twelve new diterpenes not only provided information on chemical substances of T. regelii, but also contributed to the chemical diversity of natural terpenoids.


Introduction
Diterpenes are naturally-occurring 20-carbon terpenoids that display a wide array of potentially useful biological effects. Abietanes are a large group of diterpenoids, which have been isolated from a variety of terrestrial plants, such as families of Araucariaceae, Cupressaceae, Phyllocladaceae, Pinaceae, Podocarpaceae, Asteraceae, Celastraceae, Hydrocharitaceae, and Lamiaceae, etc. [1,2]. Furthermore, this class of diterpenes have been found from fungal species [2]. So far, it has been reported that some abietane type diterpenes displayed a broad spectrum of promising biological activities including anticancer [3], cytotoxic [4,5], antiviral [6][7][8][9], anti-inflammatory [10], and anti-oxidant [9] effects, and so on. For example, tanshinone IIA was regarded as a potent cytotoxic compound for human leukemia cells [11]. Carnosol has been found to possess favorable anticancer and chemo-preventive effects [12]. Triptolide is a promising lead compound to treat inflammatory, immunological and cancerous diseases [13]. Recently, it was reported that miltirone is an inhibitor of P-glycoprotein [14].
As a part of ongoing research work on bioactive constituents from Tripterygium regelii [15][16][17], the methanolic extract of the stems of T. regelii was further investigated, leading to the isolation and characterization of twenty three diterpenoids, including eleven new abietane (1−11) and one new kaurane type (12) diterpenes, as well as eleven known abietane compounds (13−23) (Figure 1). Herein, this paper reports the isolation and structural elucidation of these new diterpenes, as well as cytotoxic evaluation of seventeen diterpenes on three cancer cell lines.
kaurane type (12) diterpenes, as well as eleven known abietane compounds (13−23) (Figure 1). Herein, this paper reports the isolation and structural elucidation of these new diterpenes, as well as cytotoxic evaluation of seventeen diterpenes on three cancer cell lines.
The key HMBC correlation from H-1 (δ H 5.86) to C-18 (δ C 177.3) suggested a lactone formed between C-1 and C-18, accounting for the remaining one degree of unsaturation. Hydroxylation of C-3 was inferred from the HMBC correlations from H-1 (δ H 5.86), H 2 -2 (δ H 2.36 and 1.96) and H 3 -19 (δ H 1.85) to C-3 (δ C 74.3). It was deduced that the proton at C-1 and the hydroxyl group at C-3 should be a cis relationship due to the lactone between C-1 and C-3. Therefore, the proposed structure of 1 was established as a lactone derivative of triptoquinone A bearing 5S, 10S absolute configuration by X-ray crystallographic analysis [18] (Figure 2). C-ring [18,19], structurally similar to the known triptoquinone A (20) [18], an 18(4→3)-abeo-abietane quinone type diterpene, except for the A-ring. The Δ 4,5 double bond was inferred from the HMBC correlations from H2-2, H2-6 and H3-19 to C-4 (δC 130.2), from H-7 (δH 2.90), H3-19 and H3-20 to C-5 (δC 132.6). The oxygenated methine (δH 5.86; δC 78.4) was assigned to C-1 based on the HMBC correlations from H-1 proton (δH 5.86) to C-2 (δC 39.1), C-3 (δC 74.3), C-5 (δC 132.6), C-9 (δC 144.6), C-10 (δC 44.7), and C-20 (δC 23.7). The key HMBC correlation from H-1 (δH 5.86) to C-18 (δC 177.3) suggested a lactone formed between C-1 and C-18, accounting for the remaining one degree of unsaturation. Hydroxylation of C-3 was inferred from the HMBC correlations from H-1 (δH 5.86), H2-2 (δH 2.36 and 1.96) and H3-19 (δH 1.85) to C-3 (δC 74.3). It was deduced that the proton at C-1 and the hydroxyl group at C-3 should be a cis relationship due to the lactone between C-1 and C-3. Therefore, the proposed structure of 1 was established as a lactone derivative of triptoquinone A bearing 5S, 10S absolute configuration by X-ray crystallographic analysis [18] (Figure 2). However, the relative configuration of the substituents at the C-1 and C-3 could not be assigned by nuclear Overhauser effect spectroscopy (NOESY) experiment, owing to the fact that no any key NOE effects were observed ( Figure 3). Hence, electron circular dichroism (ECD) calculations were conducted to determine the absolute configuration of compound 1 by time-dependent density functional theory (TDDFT) with the B3LYP/DGDZVP method [20,21]. The calculated ECD of (1R, 3R)-1 matched well with the experimental CD spectrum ( Figure 4A) of 1. Therefore, compound 1 was determined as proposed, and given the trivial name of triregelin A. However, the relative configuration of the substituents at the C-1 and C-3 could not be assigned by nuclear Overhauser effect spectroscopy (NOESY) experiment, owing to the fact that no any key NOE effects were observed ( Figure 3). Hence, electron circular dichroism (ECD) calculations were conducted to determine the absolute configuration of compound 1 by time-dependent density functional theory (TDDFT) with the B3LYP/DGDZVP method [20,21]. The calculated ECD of (1R, 3R)-1 matched well with the experimental CD spectrum ( Figure 4A) of 1. Therefore, compound 1 was determined as proposed, and given the trivial name of triregelin A.       (Tables 1 and 2) indicated that 3 was structurally related to triptoquinone B (21) [18] except for the absence of the C-7 methylene in triptoquinone B and the presence of an additional hydroxyl proton (δ H 2.77) and an oxygenated methine (δ H 4.81, δ C 61.9) in 3. These data suggested hydroxylation of C-7 in 3, which was supported by HMBC correlation from the hydroxyl proton (δ H 2.77) to C-7 (δ C 61.9). The α-orientation of the hydroxyl group at C-7 was deduced from the NOESY correlation of H-7/H 3 -20. Thus, compound 3 was identified and named triregelin C.
Compound 4 gave a molecular formula of C 21 14.0142 atomic mass units (amu) more than that of 3 in the HRESIMS. The 1 H and 13 C NMR spectroscopic data (Tables 1 and 2) of 4 were closely similar to those of 3, except for the appearance of a methoxyl group. The methoxyl group was assigned at C-7, as evidenced from the observed HMBC correlation from the methoxyl protons (δ H 3.50) to C-7 (δ C 69.8). Thus, compound 4 was characterized and named triregelin D.
Compound 5 showed a molecular formula of C 22 Table 2) displayed resonances for 20 carbons, which were ascribed to a tetrasubstituted benzene ring, an exocyclic double bond, an aliphatic quaternary carbon, three methines, five methylenes (including an oxygenated one) and three methyl groups. The 1 H and 13 C NMR spectroscopic data of 6 were similar to those of triptobenzene P [22], an 18 (4→3)-abeo-abietane diterpene previously isolated from T. wilfordii, except for the following two differences. One difference is the replacement of the methoxyl group at C-12 in triptobenzene P by a hydrogen in 6, which was supported by 1 H-1 H COSY correlation of H-11/H-12, and HMBC correlations from H-12 (δ H 7.04) to C-9 (δ C 145.8) and C-15 (δ C 26.9). The other difference is the downfield shift of C-14 (δ C 150.3) in 6 relative to that (δ C 123.8) in triptobenzene P, indicating hydroxylation of C-14 in 6. Thus, the planar structure of 6 was established as 12-demethoxy-14-hydroxy-triptobenzene P, which was confirmed by the 1 H-1 H COSY and HMBC data ( ; δ C 124.0), and a methoxyl group (δ H 3.80 (3H,s, OCH 3 -12); δ C 55.7). The double bond was assigned at between C-6 and C-7, which was supported by HMBC correlations from H-6 (δ H 5.86) to C-4 (δ C 53.0) and C-10 (δ C 37.7), and from H-7 (δ H 6.82) to C-9 (δ C 145.2) and C-14 (δ C 150.3). The methoxyl group was located at C-12, as deduced from the HMBC correlation from the methoxyl protons (δ H 3.80) to C-12 (δ C 158.3). The key NOE correlations of H-5α/H 3 -18 and H 3 -20/H 2 -19 were observed in the NOESY spectrum. Accordingly, compound 7 was elucidated as illustrated in Figure 1, and named triregelin G.  (Tables 2 and 3) of 8 were closely related to those of triptobenzene A (13) [23]. However, one of the key differences was the replacement of the methylene at C-7 in triptobenzene A by a keto carbonyl carbon (δ C 204.3) in 8, as evidenced from HMBC correlation from H-5 (δ H 2.64) to C-7. The other difference was the absence of a doublet aromatic proton and the presence of an additional hydroxyl proton (δ H 4.62) together with the downfield shift of C-11 (δ C 144.5) in 8 compared to that in triptobenzene A, which suggested hydroxylation of C-11 in 8. Therefore, compound 8 was assigned and named triregelin H.  (Tables 2 and 3) of 11 with neotriptonoterpene (14) [24] showed that both compounds were structurally comparable, except for the absence of the C-3 keto carbonyl in neotriptonoterpene (14) and the presence of an extra oxygenated methine (δ H 3.68; δ C 74.8) in 11. These suggested that the C-3 keto carbonyl group in neotriptonoterpene (14) was reduced to be a hydroxyl group in 11. The C-3 hydroxyl group was α-oriented, as inferred from the coupling constant (J 2,3 = 3.6 Hz) and the NOESY correlation between H-3 and H 3 -19. Accordingly, the compound 11 was characterized and named triregelin K.  Table 2) showed 20 carbon signals including a carbonyl group, an exocyclic double bond, three quaternary carbons, there methines, nine methylenes (including an oxygenated one) and two methyl groups. All the above NMR data indicated that 12 was a kaurane type diterpenoid, and structurally similar to (−)-ent-kaur-16-en-19-ol [25][26][27]. The distinct difference was that the C-12 methylene in (−)-ent-kaur-16-en-19-ol was oxidized to be a keto carbonyl group in 12, as deduced from the downfield shift of C-12 (δ C 211.5), and the HMBC correlations from H-9 (δ C 1.58) and H 2 -14 (δ H 2.40, 1.51) to C-12. Finally, the planar structure of 12 was confirmed on the basis of the 1 H-1 H COSY and HMBC experiments (Figure 2). In the NOESY spectrum, the correlations of H 3 -20/H 2 -19 and H 3 -20/H 2 -14 indicated that these protons were in the same face. In the same way, the other key NOE cross peaks of H-5/H-9 and H-9/H 2 -15 were also observed (Figure 3), suggesting H-5, H-9, and H 2 -15 were in the other face. However, 12 displayed a positive specific rotation ([α] 21 D +50.86 (c 0.50, MeOH)) in contrast to the negative one reported for (−)-ent-kaur-16-en-19-ol [27]. ECD curves for the two possible stereo-structures (4R, 5S, 8S, 9R, 10S, 13R-12 and 4S, 5R, 8R, 9S, 10R, 13S-12) were, therefore, calculated to determine the absolute configuration of 12. As illustrated in Figure 4B, the calculated profile of 4R, 5S, 8S, 9R, 10S, and 13R-12 were in good agreement with the experimental CD spectrum of 12. Therefore, compound 12 was identified and named triregelin L.

General Experimental Procedures
Optical rotations were obtained using a Rudolph Research Analytical Autopol I automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). IR spectra were measured on an Agilent Cary 600 series FT-IR spectrometer (KBr) (Agilent, Santa Clara, CA, USA). Ultraviolet (UV) spectra were recorded on a Beckman Coulter DU ® 800 spectrophotometer (Beckman Coulter, Fullerton, CA, USA). HRMS spectra were carried out on an Agilent 6230 electrospray ionization (ESI) time-of-flight (TOF) mass spectrometer (Agilent, Santa Clara, CA, USA). Nuclear magnetic resonance (NMR) spectra were measured on a Bruker Ascend 600 NMR spectrometer at 600 MHz for 1 H NMR and 150 MHz for 13 C NMR (Bruker, Zurich, Switzerland). Chemical shifts were expressed in δ (ppm) with tetramethylsilane (TMS) as an internal reference, and coupling constants (J) were reported in hertz (Hz). Circular dichroism spectra were measured on a Jasco J1500 CD spectrometer (Jasco Corporation, Tokyo, Japan). Medium pressure liquid chromatography (MPLC) was conducted on a Sepacore Flash Chromatography System (Buchi, Flawil, Switzerland) by employing a flash column (460 mm × 36 mm, i.d., Buchi) packed with Bondapak Waters ODS (40-63 µm, Waters, Milford, MA, USA). Preparative high performance liquid chromatography (HPLC) was carried out on a Waters Xbridge Prep C 8 column (10 mm × 250 mm, 5 µm) by utilizing a Waters liquid chromatography system equipped with 1525 Binary HPLC Pump and 2489 UV/Visible detector (Waters, Milford, MA, USA). Semi-preparative HPLC was done on a Waters Xbridge Prep C 18 column (10 mm × 250 mm, 5 µm) by using an Agilent 1100 liquid chromatography system coupled with a quaternary pump and a diode array detector (DAD) (Agilent, Santa Clara, CA, USA). Column chromatography was conducted on silica gel (40−60 µm, Grace, Columbia, MD, USA) and Bondapak Waters ODS (40-63 µm, Waters). Thin layer chromatographies (TLCs) were performed on pre-coated silica gel 60 F 254 plates and TLC silica gel 60 RP-18 F 254S plates (200 µm thick, Merck KGaA, Darmstadt, Germany), which were used to monitor fractions. Spots on the TLC were visualized by UV light (254 nm) or heating after spraying with 5% H 2 SO 4 in ethanol.

Plant Material
The

Extraction and Isolation
The air-dried stems of T. regelii (8.0 kg) were powdered, and extracted three times with methanol (64 L) under ultrasonic-assisted extraction at room temperature for 1 h. The methanol extract was evaporated under reduced pressure to yield a dark brown residue, which was then suspended in H 2 O, and successively partitioned with n-hexane, ethyl acetate (EtOAc) and n-butanol. Then, the EtOAc-soluble extract (150.0 g) was fractionated over a silica gel column using   1 mg). Fraction 12 (9.0 g) was separated by MPLC using a gradient system of MeOH-H 2 O (5:95-100:0, 50 mL/min) to obtain six fractions (Fr.12-1-Fr.12-6). Fraction 12-5 was chromatographed over a silica gel column using CHCl 3 -MeOH (100:0-90:10, v/v) as solvent system, and then purified by semi-preparative HPLC using CH 3 CN-H 2 O (39:61, v/v) as mobile phase to give compound 9 (1.2 mg).   Tables 2 and 3 Tecan, Männedorf, Switzerland) was employed to determine the absorbance of each well at 570 nm. GraphPad Prism 6 software (Prism 6.0, GraphPad Software, Inc., La Jolla, CA, USA) was used to calculate the IC 50 values (concentration that suppresses 50% of cell growth) of all tested compounds. All assays were performed in triplicate in three independent experiments. Data was expressed as mean ± SD (n = 3).

Conclusions
To sum up, 23 diterpenoids were isolated from the Chinese herbal medicine T. regelii, including eleven new abietane, and one new kaurane, diterpenes. Importantly, triregelin A (1) represents the first abietane diterpene bearing an 18→1 lactone ring. Triregelin I (9) exhibited significant cytotoxic effects on A2780 and HepG2 cancer cells with IC 50 values of 5.88 µM and 11.74 µM, respectively, and was found inactive against MCF-7 cancer cells. Triregelin K (11) displayed a weak cytotoxic effect on MCF-7 cell with an IC 50 value of 26.70 µM.