Fimbriatols A–J, Highly Oxidized ent-Kaurane Diterpenoids from Traditional Chinese Plant Flickingeria fimbriata (B1.) Hawkes

Fimbriatols A–J (1–10), ten new ent-kaurane diterpenoids possessing differently highly oxidized sites, were isolated from Flickingeria fimbriata (B1.) Hawkes. The structures of these new compounds were determined by HRESI-MS, NMR, CD spectra and X-ray diffraction analysis. Compound 1 displayed moderately inhibitory ratio (48.5%) compared with the positive compound NSC-87877 (81.6%) at the concentration of 0.022 μg/mL. Compounds 7–10 possess 3, 4-seco-ent-kaurane skeleton containing a disaccharide moiety with an unusual linkage at C-2′ to C-1′′ instead of the common linkage at C-6′ to C-1′′, and this is the first report in 600 more ent-kauranes found in nature, which might be originated from ent-kaurane diterpenoids through post-modified reactions of Baeyer-Villiger oxygenation and glycosylation.


Results and Discussion
CNMR, and HMQC data for 1 (Tables 1 and 2) revealed the presence of two methyls, ten methylene units (two oxygenated), three methines, and four quaternary carbons (one oxygenated), and one ketone group. These data accounted for all 1 H and 13 C resonances except for three exchangeable protons, and required 1 to be four cyclic systems. Interpretation of the 1 H-1 H COSY NMR data led to the identification of three isolated proton spin-systems corresponding to the C-1-C-2, C-5-C-6-C-7, and C-9-C-11-C-12-C-13-C-14 fragments of structure 1. The remaining connectivity was determined by HMBC correlations (Fig. 3). The correlations from CH 3 -18 and OCH 2 -19 to C-3, C-4, and C-5 confirmed that C-4 was connected with C-3, C-5, C-18 and C-19, and correlations from CH 2 -1 to C-3, and CH 2 -2 to C-3 and C-4 implied the keto group C-3 was connected with C-2 and C-4. Those correlations from CH 3 -20 to C-1, C-5, C-9, and C-10 revealed that C-10 was connected with C-1, C-5, C-9 and C-20, whereas the correlations of CH 2 -15 with C-7, C-8, C-9, and C-14 confirmed that C-7, C-9, C-14 and C-15 all were connected with C-8. In the HMBC spectra, the distinct cross peaks from OCH 2 -17 to C-13, C-15, and C-16 confirmed the  connectivity of C-16 with C-13, C-15 and C-17. Accounting for the molecular formula and chemical shift values of C-16, C-17 and C-19, it implied that these three carbons possessed hydroxyl groups, respectively. Thus the planar structure of 1 was characterized. SciFinder searching found a phyllocladane diterpenoid named 16,17,18-trih ydroxyphyllocladan-3-one possessing the same planar structure as that of 1 16 . Liu et al. ever summarized the diagnostic 13 C NMR chemical shifts of selected phyllocladanes and ent-kauranes, which revealed that chemical shift values of of C-13, C-14, C-15, C-16, C-17 and C-20 in these two member of diterpenoids changed regularly 17 . From the 13 C NMR chemical shifts of those carbons in compound 1, it implied that this structure should possess the skeleton of ent-kauranes not that of phyllocladanes. This hypothesis was further confirmed by NOESY correlations and CD spectra. The correlations from H 2 -17 to H-15a, H-15a to H-9, H-9 to H-5, H-5 to CH 3 -18 implied the β configuration of these protons, whereas the correlations of CH 3 -20 with H 2 -14 and OCH 2 -19 established the α configuration of these protons (Fig. 3), which established the relative configuration of compound 1 as kauranes. The absolute configuration was determined by CD spectra (Supporting Information), which displayed the negative conton effects at 240, 290 nm, and positive one at 320 nm opposite to (16R)-16,17-dihydroxy-phyllocladan-3-one. This result confirmed that the stereo-center of C-5 was S-configuration. Thus the absolute configuration of compound 1 was determined to 4R, 5S, 8S, 13R, 16R 17 , which revealed that compound 1 indeed possessed the skeleton of ent-kauranes.
The HRESIMS of 2 gave a pseudomolecular ion [M + Na] + peak at m/z 375.2061 (Δ + 8.6), indicating the molecular formula of 2 as C 20 H 32 O 5 (five degrees of unsaturation) with one more oxygen atom than that of 1. Analysis of NMR data revealed one additional oxygenated methine (δ C 65.86; δ H 4.29) present in NMR spectra in 2 compared with that of 1. The 1 H-1 H COSY and HMBC correlations confirmed that the CH 2 -6 in 1 was oxygenated to the corresponding oxymethine unit in 2. The relative configuration was established by analysis of coupling constant and NOESY correlations. The small coupling constant between H-5 and H-6 (J ≈ 0 Hz) together with the NOESY correlations from H-5, H-6 to CH 3 -18 implied the cis relationship of this two protons. The other NOESY correlations were same as those of 1 (Fig. 5). Thus the relative configuration of 2 was determined.
The molecular formula of 3 was same as that of 2 by analysis of its HRESIMS. The NMR data especially the 2D spectra revealed that 3 possessed the same planar structure as 2. The NOESY correlation spectrum of 3 was similar with that of 2, except that H-6 had correlations with H 2 -19 and CH 3 -20, not correlation with CH 3 -18 in 2, leading to the trans configuration between H-5 and H-6, and this was also supported by the big coupling constant between H-5 and H-6 (J = 10.8) (Fig. 5).
Compound 4 possessed the same as molecular formula as that of 2 and 3 on the basis of HRESIMS (m/z 375.2169 [M + Na] + , Δ − 2.7) implying that 4 was an isomer of 2 and 3. The NMR data mainly including the 2D NNR experiments suggested that the hydroxyl group was attached at C-9 not at C-6 found in 2 and 3. The NOESY correlation revealed the same relative configuration as those of 1-3 except for the 9-OH.
The HRESIMS of 5 suggested the same molecular formula as compounds 2-4. The NMR spectra revealed the similar structure feature as those present in 1 except the additional hydroxyl group was connected at C-11, which was confirmed by H-1 H COSY and HMBC correlations. The small coupling constant (J ≈ 0 Hz) between H-9 (singlet) and H-11 implied that the dihedral angel was 90°. The NOESY correlations from H-11 with CH 3 -20, and H-1a revealed that the 11-OH possessed the β-configuration (Fig. 5). The HRESI MS (m/z 375.2134 [M + Na] + Δ + 0.8) of compound 6 afforded the molecular formula as C 20 H 32 O 5 same as that of compounds 2-5. The 1 H NMR spectra displayed three methyl groups present in 6, implying that the 19-methyl was not oxidized. The 1 H-1 H COSY and HMBC correlations confirmed that both C-1 and C-6 contained one hydroxyl group, respectively. The hydroxyl group for C-1 was determined to be α-configuration on the basis of the NOESY correlations from H-1 to H-5 and H-9, and hydroxyl group at C-6 was established as      Table 3). The NMR spectra displayed a carboxylic group (δ H 11.94; δ C 174.7) and a double of exocyclic olefinic carbons (δ H 4.83, 4.62; δ C 147.1, 113.1) signals, whereas the keto carbonyl (C-3) and the oxymethylene (C-19) were disappeared. These differences implied that the carbon-bond of C-3/C-4 might be oxidized to shape the corresponding carboxylic group, and then dehydration reaction at C-19 formed the exocyclic double bond. The 2D NMR experiments especially the HMBC correlations from CH 2 -1 and CH 2 -2 to C-3 (δ C 174.7) and from CH 3 -18 to C-4, C-5 and C-19 (exocyclic olefinic carbon) confirmed the above-mentioned postulation (Fig. 6). Thus the planar structure of 7 was determined. The relative configuration was established by NOESY correlations. Compound 7 possessed the same relative configuration as that of 1 depicted in Fig. 6.  (Table 3). In addition, the carboxylic acid group, and the two hydroxyls at C-16 and C-17 were disappeared. This difference implied that the diol group at C-16/17 might react with acetone to form the acetonide product, and the carboxyl group might shape the oxyethyl derivative with ethanol solvent, and this postulation was further supported by HMBC correlations. Because ethanol and acetone solvents were used in the process of isolation, compound 8 was postulated to be an artifact product from 7. We added 7 to acetone and ethanol solvent mixed with silica gel for a week. The mixture was then analyzed by TLC, which did produce compound 8 by comparison. This revealed that compound 8 should be the artifact product from 7.
The molecular formula of 9 was established as C 31 H 50 O 13 by analysis of its HRESIMS [m/z 653.3179 (M + Na) + ; Δ − 3.0 mmu] and NMR data ( Table 3). The 1 H and 13 C NMR spectra for 9 suggested the presence of the same structure fragment found in 7 and 8 except for two additional sugar moieties. The HMQC, 1 H-1 H COSY and HMBC correlations confirmed the two sugar moieties were glucose and apiose, respectively. The coupling constant of the anomeric proton in the glucose moiety (δ 4.37, 1H, d, J = 7.8 Hz) suggested a β-configured glucose unit, whereas the coupling constant for the apiose anomeric proton (δ 5.20, 1H, d, J = 3.0 Hz) and the chemical shift value for the anomeric carbon signal at δ 109.9 suggested the β-configured apiose unit. The linkage of the sugar moieties was determined by HMBC correlations. The HMBCcorrelations from CH 2 -17 to C-1′ , in turn, from H-1′ to C-17 implied the connection of C-17 with C-1′ by an oxygen atom. The linkage between C-2′ and C-1′ ′ by an ether bond was confirmed by HMBC correlations from H-1′ ′ to C-2′ , and from H-2′ to C-1′ ′ . The chemical shifts of the glucose and apiose moiety were assigned through 1 H-1 H COSY, HMQC and HMBC spectra. Thus the planar structure of 9 was established. The relative configuration of 9 was same as that of 7 by analysis of the NOESY correlations.
The molecular formula of 10 was established as C 32 H 52 O 13 by analysis of its HRESIMS [m/z 667.33214 (M + Na) + ; Δ − 1.4 mmu] with 14 mass units more than that of compound 9. The 1 H and 13 C NMR spectra of 10 displayed resonances nearly identical to those of 9 except for an additional OMe group, suggesting that 10 was the oxymethyl derivative of 9. Analysis of HMBC data confirmed the above observations, and permitted the OMe unit to be connected with the carboxyl group (C-3). The relative configuration of 10 was same as those of 7 and 9 by analysis of its NOESY correlations. Due to the different groups found in compounds 7-10, the CD spectra of compunds 7-10 were different from those compounds 1-6 (supporting information), which could not determine the absolute configurations of 7-10 by CD spectra. From the biosynthetic view, these compounds were originated from same diterpene biogenetic pathway. Thus compunds 7-10 were postulated to possess the same absolute configurations as those 1-6. The configurations of two sugar moieties were determined to be D-glucose and D-apiose by hydrolysis and GC-MS methods 18 .
Protein phosphorylation as posttranslational modification plays a vital role to regulate cell activities. In cells, the protein kinases and phosphatases control the protein phoshorylation level 19 . Dual-specificity phosphatase 26 (DUSP26) is a heterogeneous group of protein phosphatases, and can dephosphorylate both phosphotyrosine and phosphoserine/phosphothreonine residues 20 . Due to the fact that DUSP26 is located at 8p, a chromosomal region that has been shown to be amplified in anaplastic thyroid cancer (ATC), this enzyme has emerged as a potential target for the treatment of human cancers by dephosphorylating p38 MAPK, thereby inhibiting p38-mediated In addition, compounds 1-10 were also tested against the activities of several plant pathogens, acetylcholinesterase, xanthinoxidase (XOD) and cytotoxic activities without any effects.
Up to date, more than 600 ent-kaurane diterpenoids with diverse structurally features have been isolated from different plant 1-3 . Compounds 1-6 are different from all known analogues by possessing highly oxidized sites, whereas 7-10 contain the 3, 4-seco-ent-kaurane skeleton, which is the first report in all ent-kaurane diterpenoids. Mono-glycosylated ent-kaurane diterpenoids are ever isolated from different resource, whereas 9 and 10 contained a disaccharide moiety at C-17 with the unusual linkage at C-2′ to C-1′ ′ not the common linkage at C-6′ to C-1′ ′ , which was the first report in all known ent-kaurane diterpenoids. Recently, Hu et al. confirmed that a flavin-dependent monooxygenase catalyzed a keto group to the corresponding ester moiety by Baeyer-Villiger reaction in Aspergillus clavatus 21 . Thus we postulated that compounds 7-10 might be biosynthesized from ent-kaurane diterpenoids firstly through Baeyer-Villiger oxidation reaction to cleave the carbon

General experimental procedures.
In vitro enzyme-based assay. DUSP26 expression and purification. The DNA fragment encoding DUSP26 was subcloned from human DUSP26 cDNA (Invitrogen, Carlsbad, CA, USA) into the expression vector pGEX-4T-2 as a fusion with a N-terminal GST-tag and a thrombin protease cleavage site. For proteins expression, Escherichia coli cells growing in 1 l LB media at OD 600 of near 0.8 were inducted with 0.5 mM IPTG at 16 °C for 16 h. Centrifuged culture pellets were extracted by sonication in PBS buffer with 100 mM PMSF. Extracts were clarified by centrifugation 13,000 rpm for 30 min at 4 °C to yield the soluble extract. GST-tagged proteins present in the soluble extracts were purified by GST-affinity chromatography and dialyzed overnight against buffer containing 50 mM Tris-HCl (pH 7.5), and 0.1 M NaCl.
Assays of DUSP26 inhibition. In vitro phosphatase assay used for determining whether compounds are DUSP26 inhibitors was established and optimized based on methods described previously 22,23 . The activity of DUSP26 was measured using the substrate p-nitrophenyl phosphate (pNPP; Sigma, St. Louis, MO) at the concentration using a 96-well microtiter plate. NSC-87877 (positive inhibitor; Calbiochem, San Diego, CA) and pNPP were solubilized in ddH 2 O. The compound 1 were solubilized in HPLC grade DMSO with concentration at 0.022 μ g/mL. All reactions were performed at a final concentration of 1% DMSO, while the enzyme activity was not affected by this concentration of DMSO.
The purified DUSP26 (10 nM), and a candidate inhibitor were incubated in the reaction mixture containing 50 mM Tris-HCl (pH 7.5), 1% DMSO, for 15 min at 37 °C. Reactions were initiated by addition of pNPP and the incubation time was 30 min at 37 °C. This enzymatic reaction was stopped with the addition of 0.2 M NaOH. The phosphatase activities were then monitored by measuring the absorbance changes due to the hydrolysis of the substrate at 405 nM. The activities of DUSP26 with and without candidate inhibitors were represented as Ai and A0. The value of Ai/A0 × 100% was the residual activity (R.A.) of DUSP26. All experiments were performed in triplicate.