Xuetonglactones A–F: Highly Oxidized Lanostane and Cycloartane Triterpenoids From Kadsura heteroclita Roxb. Craib.

Xuetonglactones A–F (1–6), six unreported highly oxidized lanostane- and cycloartane-type triterpenoids along with 22 known scaffolds (7–28) were isolated from the stems of Kadsura heteroclita (Roxb.) Craib. Compared with previous congeners, xuetonglactone A (1), possesses an unprecedented 20,21-α-epoxide, and xuetonglactone D (4) features an unusual 19-α-hydroperoxyl moiety. The structures and the absolute configurations of the compounds were established by extensive one- and two-dimensional NMR, and electronic circular dichroism (ECD) spectroscopic analysis, with those of 1 and 5 confirmed by single-crystal X-ray diffraction technique. Compounds 1 and 2 exhibited inhibition of iNOS activity in LPS-induced macrophages with IC50 values of 22.0, and 17.0 μg/mL, respectively. While compounds 6, 7, 8, and 24 showed potent cytotoxic activities against human cervical cancer cell lines (HeLa) with the IC50 values of 4.0, 5.8, 5.0, and 6.4 μM, and against human gastric cancer cells (BGC 823) with the IC50 values of 2.0, 5.0, 2.5, and 2.0 μM, respectively. Moreover, plausible biogenetic pathways of (1–6) were also proposed.


INTRODUCTION
Schisandraceae family has contributed to the novel chemical scaffolds with an array of biological activities in past three decades. The family comprises of around fifty plant species belongs to genus Kadsura and Schisandra that are widely distributed in East, and Southeast Asia. The family has derived significant interest due to its highly oxygenated lanostane-and cycloartanetype triterpenoids, and dibenzocyclooctadiene lignans, along with schinortriterpenoids which are the characteristics isolates Shi et al., 2015). These constituents demonstrated potential pharmacological effects e.g., anti-hepatitis, anti-HIV, anti-inflammatory, anti-cancer, and inhibitory effect in cholesterol biosynthesis Liu et al., 2014;Hu et al., 2015;Su et al., 2015).
Kadsura heteroclita (Roxb.) Craib. of the genus Kadsura is a climbing species primarily grows in Southwestern China, has a long history of its folk use in Traditional Chinese Medicine (TCM) Liu et al., 2014). The stems of K. heteroclita traditionally known as "Xuetong" has long been consumed for the treatment of rheumatoid arthritis, traumatic injuries, deudenal ulcers, and cancers, particularly by Tujia people living in Wulin mountains area. In "Tujia" dialect "Xue" (blood) herbs are commonly used for the treatment of these diseases by activating the blood circulation, relieving pain and eliminating dampness for centuries (Liu et al., 2016(Liu et al., , 2018Cao et al., 2019). This study aimed to trace back the biologically active chemical constituents responsible for its clinical application contained within the plant species. Recently, we reported the identification of several new sesquiterpenoids, and lignanoids from K. heteroclita and other species of the same genus (Liu et al., 2018;Cao et al., 2019). Our previous pharmacological studies displayed this plant has very good anti-rheumatoid arthritis, antiinflammatory, and analgesic effects .
In course of our continuous efforts to crack the immense diversity in structural frameworks with untapped biological potential, herein four new lanostane-type triterpenoids xuetonglactones A-D (1-4), and two cycloartane-type triterpenoids xuetonglactones E-F (5-6) possessing differently highly oxidized sites, were reported from K. heteroclita. Structurally, xuetonglactones A and D (1, 4) exhibited unique oxidized functionalities, featuring unprecedented 20,21-αepoxy group in xuetonglactone A, and rare 19-α hydroperoxyl moiety in xuetonglactone D skeletons. The compounds were also evaluated for their cytotoxicity and anti-inflammatory activities. Hence, in this report, the details of isolation, structure elucidation, biological evaluation, and possible biosynthetic pathways of (1-6) were described. The spectroscopic data of 1-6 is presented in the Supplementary Material (Figures S1-S48).

RESULTS AND DISCUSSION
Compound 1 was purified as a white crystalline solid, and the molecular formula was deduced to be C 32 H 40 O 7 from HRESI-MS spectrum (positive ion mode) on the basis of [M + Na] + ion at m/z 559.2671 (559.2672 calculated for C 32 H 40 O 7 + Na) indicating 13 degrees of unsaturation. A 3,4-secocyclolanostane skeleton was deduced from the 1 H-and 13 C-NMR chemical shifts data with two α, β-unsaturated lactone rings, one of them being a seven membered ring in this triterpenoidal skeleton. This deduction was also supported by IR absorptions at 1,720, and 1,685 cm −1 for six-and seven-membered unsaturated lactone carbonyls and by UV absorptions (λ max ) at 202, and 329 nm, respectively. The 1 H-NMR data of 1 (Table 1) showed the presence of six tertiary methyl singlets (3H each, δ H 0.71, 1.90, 1.53, 1.41, 1.39, and 2.12), four olefinic methines at δ H 6.65 (d, J 1, 2 = 12.3 Hz, H-1), 5.82 (d, J 2, 1 = 12.0 Hz, H-2), 6.14 (s, H-19), and 6.51 (broad d, J 24, 23 = 4.8 Hz, H-24), and two oxygenated methines at δ H 5.14 (d, J 12β, 11 = 7.4, H-12), and 4.49 (dd, J 22, 23β = 12.7 Hz; J 22,23α = 3.7 Hz, H-22). The 13 C-NMR data ( Table 2) displayed 32 carbon signals attributed to six tertiary methyls (δ C 18.6, 16.9, 26.2, 29.3, 27.9, and 21.6), eight methines including four olefinic (δ C 143.2, 118.3, 141.7, and 137.6), and two oxygenated methines (δ C 71.9, and 78.3), seven methylenes, and eight quaternary carbons, including four olefinic (δ C 151. 1, 126.4, 140.4, and 128.2), and two oxygenated (δ C 80.3, and 58.1) quaternary carbons. Furthermore, it also showed the presence of three carbonyl signals (δ C 171.0, δ C 166.3, and δ C 165.0) corresponding to an acetoxy (C-12), and two lactone (C-3, and C-26) moieties, respectively. The 1 H-and 13 C-NMR chemical shifts data of 1 showed resemblances with that of known compound heteroclitalactone D (17) (Wang et al., 2006b) with obvious distinctions observed for resonances at C-17, C-20, and C-21. The detailed analysis of the NMR data established the structure of compound 1 bearing unprecedented oxirane in the structure at C-20. The NMR data revealed the presence of an extra methylene (δ C 46.88, C-21) with a characteristic proton doublet (δ H 2.75; and 2.96, dd, J 21a, 21b = 3.3 Hz each, H-21) which supported the presence of additional ring at C-20 as epoxide to fulfill the unsaturation demand in skeleton of 1. Furthermore, the up-field shift of quaternary carbon (δ C 58.12) at C-20 suggested the presences of epoxide at this junction, which could be attributed to the steric shielding effect of the strained ring at this position. These assignments were unambiguously confirmed by HMBC experiments, in which the epoxide methylene protons appeared at δ H 2.75, and 2.96 (dd, J 21a, 21b = 3.3 Hz each, H-21) were correlated with C-20 and C-22, while C-17 methine proton at δ H 3.55 (dd, J 17, 16a = 10.9, J 17, 16b = 7.5 Hz) was correlated with C-12, C-14, C-16, C-18, C-20, and C-21 (Figure 2). This oxidized strained ring along with the presence of three conjugated double bonds, and other oxygenated moieties in the skeleton further supported the structure of 1 is based on a highly oxidized cyclolanostane type triterpenoidal skeleton (Chen et al., 2001;Wang et al., 2006a). Chemical shifts assignments were made on the basis of HSQC, HMBC, and 1 H-1 H COSY experiments to get the planner structure of 1. The α-configuration of the C-12 (acetoxy group) was concluded based on the ROESY cross peaks between H-12 and CH 3 -18 (Figure 3). In the ECD spectrum, compound 1 showed a positive Cotton effect at 255 nm ( ε = + 5.64) which was similar to that of schiglausin A (Zou et al., 2012), indicating an R configuration of C-22. Combining the observed ROESY correlations of H-5 with CH 3 -30, CH 3 -30 with H-17, H-17 with H-22, the ECD spectrum, and the Xray diffraction using Cu Kα radiation (Figure 4), the absolute stereochemistry of the seven chiral centers, were determined as 5S, 12S, 13R, 14S, 17R, 20S, and 22R. Thus, the structure of 1 was fully established as shown (Figure 1) and named xuetonglactone A. Compound 2 was obtained as a white amorphous solid. Its molecular formula was determined to be C 32 H 42 O 7 based on [M + Na] + ion at m/z 561.2828 (561.2829 calculated for C 32 H 42 NaO 7 ) and [M + Cl] − peak at m/z 573.2612 (573.2619 calculated for C 32 H 42 O 7 + Cl) from its HRESI-MS spectra (positive ion and negative ion modes), corresponding 12 degrees of unsaturation. The 1 H-NMR chemical shifts data ( Table 1) of 2 showed the presence of seven tertiary methyl singlets (3H each, δ H 0.96, 1.32, 1.33, 1.41, 1.54, 1.91, and 2.13). By comparing the NMR chemical shifts data of compound 2 with that of 1 which has a methylene at position C-21, hence the presence of a tertiary methyl (δ H 1.33, s, C-21) instead of epoxide, in the skeleton of 2 was inferred. Furthermore, relative configuration on ring C was same as of 1, based on similar ROESY correlations. While the absence of ROESY cross peaks between CH 3 -21 and CH 3 -18 thus appearance of cross peaks between CH 3 -21 and H-17/H-22 (Figure 3) suggested the β-orientation of OH-20. The assignment of the absolute configuration at C-22 was concluded to be R by the similar ECD measurement as that of 1. Therefore, the structure of 2 was established as shown ( Figure 1) and named xuetonglactone B accordingly.
Compound 3 was obtained as a white amorphous solid and possess the same molecular formula with 2 as C 32 H 42 O 7 based on [M + Na] + ion at m/z 561.2836 (561.2828 calculated for C 32 H 42 O 7 + Na) from its HRESI-MS spectrum (positive ion mode). The spectroscopic data of 3 (Tables 1, 2) was quite similar with those of 2 except for a secondary methyl (δ H 0.98, J 21,20 = 7.0 Hz, H-21) instead of tertiary, and a broad doublet of oxymethine (δ H 4.56, J 23, 22 = 7.7 Hz, δ C 64.0, H-23) instead of methylene protons in the skeleton of 3. These observations were also confirmed by HMBC cross peaks of H-20 with C-23, and that of H-22 with C-17, C-23, and C-21, and C-24. Furthermore, αconfiguration of the acetoxy group was determined by the similar cross peaks between H-12 and CH 3 -18 in NOESY spectrum (Figure 3). A strong negative Cotton effect at 273 nm [ ε (273) = +0.41, MeOH] was observed in the experimental ECD spectrum of 3. The absolute configuration at C-22 was established as S by comparison of its ECD spectrum with analog colossolactone VIII (El Dine et al., 2008). Furthermore, the large coupling constant (9.4 Hz) between H-23 and H-22 indicated an anticonformation of these two protons (Lakornwong et al., 2014), hence the absolute configuration of these two chiral centers were found to be 22S, and 23R. Consequently, compound 3 was determined and given the trivial name xuetonglactone C.
Compound 4 was isolated as yellow, amorphous solid. Its molecular formula was deduced as C 32 H 42 O 8 on the basis of [M + Na] + ion peak at m/z 577.2772 in the HRESI-MS (577.2777 calculated for C 32 H 42 O 8 + Na), suggesting 12 degrees of unsaturation. Since the NMR resonances of 4 were similar to those of known compound 17 (Wang et al., 2006b) with some obvious discrepancies, therefore detailed comparison of the chemical shifts data revealed that both compounds have similar C/D/E rings system in the skeleton. However, different   Frontiers in Chemistry | www.frontiersin.org 1 H-and 13 C-NMR chemical shifts were observed for C-5, C-6, C-10, and C-19 suggesting the major skeletal difference of 4 corresponds to the rings A/B. The double bond between C-10 and C-19 in 17 shifted in between C-5 (δ C 153.1) and C-10 (δ C 133.7) in 4. This inference was further supported by the HMBC correlation of H-1 (δ H 6.58, d, J 1,2 = 12.2 Hz), H-19 (δ H 4.61, s), H 3 -29 (δ H 1.64, s), and H 3 -30 (δ H 1.67, s) with C-5, and of H-2 (δ H 6.21, d, J 2,1 = 12.0 Hz) and H-19 with C-10. Furthermore, C-19 was connected with an unusual hydroperoxyl group, which was supported by the carbon resonance observed at δ C 90.3 and the methine proton signal at δ H 4.61 (1H, s) (Song et al., 2013), which was also confirmed by HMBC cross peaks of H-1 and H-11 to C-19, and H-19 to C-1, C-5, C-8, C-9, C-10, and C-11. α oriented 12-acetoxyl group was ascertained on the basis of NOESY cross peak between H β -12 and CH 3 -18, the significant NOESY correlations between H β -12 /H β -11 (δ H 2.82, J Hβ−11/Hα−11 = 19.6, J Hβ−11/Hβ−12 8.0 Hz), and H β -11/H β -19 indicated that the hydroperoxyl group should be αorientated (Figure 3). The absolute configuration 22R could be delineated by similar ECD relationship (Wang et al., 2006b). Hence the structure of 4 was determined and given a trivial name xuetonglactone D accordingly.
Xuetongsu (schisanlactone E, 7) (Liu and Huang, 1991) was isolated as colorless crystal. The X-ray diffraction data of 7 was reported for the first time in this report (Figure 4). It was the major compound in "Xuetong" (Wang et al., 2006c). Biosynthetically, it might be the precursor of compounds 1-6, through series of oxidative cleavage via Baeyer-Villiger oxidation, ring expansion, hydroxylation, cyclization, acetoxylation, and epoxidation steps yielded compounds 1-6. A plausible biogenetic route for 1-6 was proposed as shown in Figure 5.
Anti-inflammatory activity of the compounds 1-6 were evaluated for their inhibitory effects against iNOS, and NF-κB activation. Compounds 1 and 2 showed inhibition of iNOS activity in LPS-induced macrophages with the IC 50 values of 22.0, and 17.0 µg/mL (Table 3), respectively, while parthenolide was used as control drug (Table 3), unfortunately no inhibitory effects found against NF-κB expression (Zhao et al., 2014). Additionally, cytotoxic activities of all the compounds against HeLa and BGC-823 cancer cell lines were also evaluated ( Table 4). Compounds 6, 7, 8, and 24 showed strong cytotoxicities against HeLa cancer cell lines with the IC 50 values of 4.0, 5.8, 5.0, and 6.4 µM, and against BGC 823 with the IC 50 values of 2.0, 5.0, 2.5, and 2.0 µM, respectively, while compared with paclitaxel as positive control ( Table 4; Hayon et al., 2003).

General Experimental Procedure
Optical rotations were measured on a PerkinElmer 341-MC digital polarimeter, UV spectra were recorded on a TU-1900 spectrophotometer; A Hitachi 260-30 spectrometer was used for scanning IR spectroscopy; Experimental ECD spectra were recorded on a JASCO J-815 Circular Dichroism (CD) Spectropolarimeter; NMR  spectra were performed on Bruker ARX-600 spectrometers, and on Agilent DD2-500 NMR spectrometer (500) MHz; HRESIMS were performed on a UPLC/xevo G2 Qtof spectrometer. Preparative RP-HPLC was conducted on Agilent 1260 Infinity Series equipped with quaternary pump with Eclipse XDB-C18 (5 µm 9.4 × 250 mm) column at flow rate of 2.5 mL/min, at 210 nm UV detection using single wavelength detector. While the separation conditions were optimized on semi-preparative Agilent 1260 HPLC equipped with DAD detector by using Eclipse XDB-C18 (5 µm 4.6 × 250 mm) at flow rate of 1 mL/min. Thin layer Chromatography was performed on TLC aluminum sheets pre-coated with silica gel GF254 (EMD Chemicals, Merck KGaA, Dermstadt, Germany), visualized under UV light of 254 and 365 nm followed by 5% vanilline-H 2 SO 4 reagent, and heat.

Plant Material
The stems of K. heteroclita (Roxb.) Craib. were collected from Hupingshan mountainous region at elevation of 5,971 ft in Shimen County, Hunan, P. R. China, and identified by Prof. Wei Wang from School of Pharmacy, Hunan University of Chinese Medicine. The voucher specimen number (CEL 1280-KH) was deposited to TCM and Ethnomedicine Innovation & Development International Laboratory, School of Pharmacy, Hunan University of Chinese Medicine, Changsha, Hunan, P. R. China.

Extraction and Isolation
Air dried plant material (100 kg) was extracted three times by using 80% ethanol in water under refluxed condition for 3 h each to produce viscous extract. This whole extract was then sequentially partitioned by liquid-liquid extraction (LLE) using non-polar, moderate to high polar organic solvents (pet-ether, chloroform, and n-butanol) against water to obtain wide range of metabolites.
Chloroform extract (353 g) was then subjected to silica gel column chromatography (CC) by gradient elution of solvent system PE-EtOAc (100% PE, 25% EtOAc in PE, 50% EtOAc/PE, 75% EtOAc in PE, 100% EtOAc) followed by EtOAc-MeOH elution. Subsequently the collected fractions were compiled, under the continuous guidance of TLC monitoring system to afford 12 final fractions (Kh-A to Kh-L).
Fraction Kh-C (54.7 g) was subjected to a series of silica gel column chromatography by gradient elution of PE-EtAcO v/v% to afford Kh-C-I to Kh-C-VIII. Fraction Kh-C-I yielded needle like crystals of 12 (35 mg) eluted with 10% EtAcO/PE on silica gel column. Compound 18 (9.0 mg) was isolated as white scaly crystals by silica gel CC using 4% EtAcO/PE mobile phase from Kh-C-III. Fraction Kh-C-V after successive separation afforded white solid of 13 (18.2 mg) eluted with 10% acetone, and 16 (6.0 mg) eluted with 15% acetone in PE, respectively on silica gel column. While Kh-C-VII yielded 25 (150 mg), and 24 (13 mg) by 6% and 10% EtAcO/PE, respectively, thorough silica gel CC.
Fraction Kh-F (48.5 g) was subjected to series of silica gel CC using DCM/EtOAc followed by EtOAc/MeOH solvent system of increasing polarity. Compounds 11 (5.0 mg), and 17 (28.00 mg) were isolated on silica gel columns by 30% DCM/PE, and 20% acetone/PE, respectively, from sub-fraction. The resulting fraction Kh-F-X-e was separated on sephadex LH-20 CC using 50% MeOH in CHCl 3 to yield sub-fraction Kh-F-X-e-2 (88 mg), which was then further purified by semi-preparative RP-HPLC. The separation conditions were optimized on analytical HPLC equipped with DAD detector. Compound 2 (20.0 mg, retention time = t R 14.03 min), 10 (12.0 mg, retention time = t R 17.52 min), and compound 5 (6.2 mg, retention time = t R 20.53 min) were purified by 75% MeOH in H 2 O at flow of 2.5 mL/min using 210 nm of UV detection by using Eclipse XDB-C18 (5 µm 9.4 × 250 mm) column. While compound 28 (13.0 mg) was isolated from Kh-F-X-h eluted with 30% EtAcO/DCM on silica gel column.

X-Ray Crystallographic Analysis
X-ray crystallographic data of 1, 5, and 7 were obtained using a Bruker APEX-II CCD diffractometer with Cu K radiation, = 1.54178 Å The CCDC numbers for 1, 5, and 7 contain the supplementary crystallographic data, which can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html.

Inhibition of iNOS Activity
The assay was performed in mouse macrophages (RAW264.7) cultured in phenol red-free RPMI medium with 10% bovine calf serum, 100 U/mL penicillin G sodium, and 100 µg/mL streptomycin. The cells were seeded in 96-well plates at the density of 1 × 10 5 cells/well, and incubated for 24 h for a confluency of 75% or more. The cells were treated with the test compounds, and after 30 min of incubation, lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO, USA) (5 µg/mL) was added and further incubated for 24 h. The activity of iNOS was determined in terms of the concentration of NO by measuring the level of nitrite in the cell culture supernatant using Griess reagent (Sigma-Aldrich, St. Louis, MO, USA). Percent inhibition of nitrite production by the test compound was calculated in comparison to the vehicle control. IC 50 values were obtained from dose response curves. Parthenolide was used as the positive control (Zhao et al., 2014).

Cytotoxicity Assay
Cell viability was determined by a MTT assay (Roche Diagnosis, Indianapolis, IN). Briefly, BGC-823 and HeLa cell lines were seeded at 6 × 10 3 cells/well in 96-well plates. Cells were allowed to adhere for overnight, and then the cells were changed to fresh medium containing various concentrations natural compound dissolved in DMSO. After 48 h incubation, the growth of cells was measured. The effect on cell viability was assessed as the percent cell viability compared with untreated control group, which were arbitrarily assigned 100% viability. The compound concentration required to cause 50% cell growth inhibition (IC 50 ) was determined by interpolation from dose-response curves. All experiments were performed in triplicate, and paclitaxel was used as the positive control (Hayon et al., 2003).

CONCLUSIONS
To sum up, four new highly oxygenated lanostane-type triterpenoids xuetonglactones A-D (1-4) and two highly oxygenated cycloartane-type triterpenoids xuetonglactones E-F (5-6), along with 22 known compounds (7-28) were isolated from stems of K. heteroclita. To the best of our knowledge xuetonglactones A (1) endowed with unprecedented 20,21-αepoxide functionality, and xuetonglactones D (4) possessed rare 19-α hydroperoxyl moiety, their absolute configurations were determined by X-ray diffraction and ECD data analysis. Moreover, bioassays indicated that 1 and 2 showed inhibition of iNOS activity in LPS-induced macrophages, 6, 7, 8, and 24 showed potent cytotoxicities against HeLa and BGC 823 cancer cell lines. Notably, this study has further enriched the chemical diversity of highly oxygenated triterpenoidal skeletons, which might trigger research rigor among synthetic and medicinal chemistry community.