Holophyllane A: A Triterpenoid Possessing an Unprecedented B-nor-3,4-seco-17,14-friedo-lanostane Architecture from Abies holophylla

A novel triterpenoid, holophyllane A (1), featuring a B-nor-3,4-seco-17,14-friedo-lanostane, along with its putative precursor, compound 2 were isolated from the methanol extract of the trunks of Abies holophylla. The 2D structure and relative configuration of 1 were initially determined via analysis of 1D and 2D NMR spectroscopic data and the assignment was confirmed by quantum mechanics-based NMR chemical shift calculations. The absolute configuration was established by comparison of the experimental and simulated ECD data generated at different theory levels. Compounds 1 and 2 exhibited moderate to weak cytotoxicity and significant inhibitory activity against nitric oxide (NO) production.

. The cyclopentane-based B-ring was identified based on the HMBC correlations of H-19/C-9, C-10, and C-5, and H-6/C-5 and C-8, and COSY correlation of H-5/H-6 ( Fig. 2). Such a B-nor-3,4-seco-17,14-friedo-lanostane scaffold is unprecedented among diverse triterpenoids. The COSY correlation of H-6/H-7 and HMBC correlation of H-5 and H-6/C-7 and H-7/C-6 corroborated that the formyl group was positioned at C-6 ( Fig. 2). The location of the methoxy group was confirmed by the HMBC correlation of OCH 3 -8/C-8 (Fig. 2). The relative configurations of the B-ring stereogenic centers were confirmed by the NOESY correlations between H-6/H-9, H 3 -19, H-28b, and H-29, H-5/H-1a and H-7, and H-7/OCH 3 -8 as depicted in Fig. 2. The NOESY correlations of H-30/H-20 and OCH 3 -8, H-16a/H-21, and H-16b/H-18 and H-21 (Fig. 2), and the similar NMR chemical shift values of C-20 -C-27 of 1 to those of the aforementioned analogue, defined the relative configurations of 1 8 . The presence of the conformationally flexible moieties in 1 and its mass limitation negated the application of X-ray crystallography. Instead, the NMR-based initial structural assignment of 1 was reaffirmed by the comparison of the experimental and computed NMR chemical shift values. The NMR deshielding properties were calculated employing gauge-invariant atomic orbital (GIAO)-based NMR chemical shift calculations at the B3LYP hybrid density-functional theory (DFT) method using the 6-31 + G(d,p) and B3LYP/6-311 + G(2d,p) level basis sets with PCM (CHCl 3 ) (Fig. 3) 9,10 . The predicted chemical shift data at the two different basis sets for 1 were plotted with the experimental data. The statistical analyses of the experimental and the calculated chemical shift values at the B3LYP/6-31 + G(d,p) and B3LYP/6-311 + G(2d,p) levels, generated the correlation slopes with r 2 values 0.9979 and 0.9981, respectively, verifying the 2D structure and relative configurational assignments.
The absolute configuration of 1 was established via comparison of the experimental and calculated ECD spectra (Fig. 4) 11 . The full array of conformers used for the aforementioned chemical shift computations at the B3LYP/6-31 + G(d,p) and B3LYP/6-311 + G(2d,p) levels (S1-S4) was employed for excited state DFT calculations in CHCl 3 . The excitation energies and rotational strengths of the respective conformers were Boltzmann-averaged based on the calculated Gibbs free energies and fitted to the Gaussian functions to simulate  ECD curves 12 . The experimental and computed ECD spectra of 1 at the two different levels were not matched well, particularly a UV range over 300 nm, presumably due to the weak chromophores and conformational changes in the molecule. However, the comparison clearly exhibited negative Cotton effects at ca. 210 nm characteristic of the π → π * electronic transitions of olefinic moieties and positive effects at ca. 280 nm originating from the n → π * transitions of the carbonyl functionalities. This, along with the consistent biosynthetic pathway towards the formation of the lanostane architecture, permitted the assignment of the absolute configuration of 1 as (5S, 6R, 8S, 9S, 10S, 13S, 17S, 20R, 23R, and 25R). . The HMBC correlation of OCH 3 -3/C-3 confirmed that the methoxy motif was attached to C-3. The full structural assignment of 2 was carried out by analyzing the COSY, HSQC, and HMBC data (Fig. S2). The relative configuration was determined by the NOESY correlations (Fig. S2) and the absolute configuration was confirmed to be identical to that of the aforementioned lanostane 8 based upon its specific rotation Holophyllane A (1) is a novel triterpenoid possessing an unprecedented B-nor-3,4-seco-17,14-friedo-lanostane scaffold. A proposed biosynthesis pathway of 1 is outlined in Fig. 5. A likely biogenetic precursor 2 could undergo oxidation at C-6 and C-7, and hydroxylation at C-8 with subsequent formation of the Δ 14(15) olefinic bond (the 1 st step in Fig. 5). The unique cyclopentane-based B-ring of 1 might be formed via a pinacol-pinacolone-type rearrangement (the 2 nd step) followed by methylation via S-adenosyl methionine (SAM) (the 3 rd step) to generate 1. This hypothesis is supported by our previous report of the two rearranged diterpenoids, holophyllins A and B, presumably biosynthesized via such a rearrangement from abietic acid 5 .
The inhibitory potential of compounds 1 and 2 on NO production levels in lipopolysaccharide (LPS)-stimulated murine microglia were also evaluated 14,15 . Compounds 1 and 2 impeded NO production with IC 50 values of 12.74 and 18.96 μ M, respectively, without significant cell toxicity at 20 μ M (Fig. 6). Thus, compounds 1 and 2 are slightly more potent than the positive control N G -monomethyl-L-arginine (L-NMMA) (IC 50 20.53 μ M) 16 . To investigate an underlying mechanism by which these compound hampered NO production, compounds 1 and 2 were evaluated for their inhibitory potentials against inducible nitric oxide synthase (iNOS) expression in LPS-stimulated BV2 cells. The tested compounds indeed inhibited the iNOS expression in a concentration-dependent manner and similar to that of the NO inhibition result, compounds 1 and 2 were more potent than the positive control in inhibition of the target protein expression (Fig. 6). This implies that the inhibitory activity of those bioactives against NO production is cohesively correlated to their capability capable of inhibiting the iNOS expression at the cellular level.
In this current study, we have demonstrated the identification of a novel architecture 1 and its biosynthetic precursor 2 from A. holophylla capable of modestly hampering the growth of several cancer cell lines as well as NO production. The discovery of the new bioactive architecture may provide a basis for the development of novel chemopreventive and anti-inflammatory drug prototypes. Also, QM-NMR coupled analyses have again been shown to possess significant utility in the full structural elucidation of new bioactive entities in cases where these metabolites are mass-limited and/or X-ray crystallography is not applicable.

GIAO-based NMR chemical shift calculations. Conformational searches were performed using
MacroModel with the MMFF force field (gas phase), a 10 kcal/mol upper energy limit and 0.001 kJ (mol Å) − 1 convergence threshold on the rms gradient 9,10,17 . The geometries of all the conformers of 1 were optimized using the B3LYP hybrid DFT method not only with the 6-31 + G(d,p) basis set in the PCM with a dielectric constant representing CHCl 3 , but also with the 6-311 + G(2d,p) basis set in the gas phase. These two different basis sets were employed to probe into the effects of including a higher basis set. The GIAO magnetic shielding tensors were calculated at the B3LYP/6-31 + G(d,p) and B3LYP/6-311 + G(2d,p) levels in the PCM (CHCl 3 ) and averaged based on the Boltzmann populations of each conformer in the associated Gibbs free energy (Tables S2 and S4). The chemical shift values were calculated via an equation below where δ calc x is the calculated NMR chemical shift for nucleus x, σ o is the shielding tensor for the proton and carbon nuclei in tetra methylsilane calculated at the above-mentioned basis sets.
Scientific RepoRts | 7:43646 | DOI: 10.1038/srep43646 ECD simulation. ECD calculations were performed with the conformers described in the subsection immediately above. The generated excitation energies and rotational strengths were Boltzmann-averaged on the basis of the calculated Gibbs free energy of each conformer (Tables S2 and S4, Supplementary Information) and used for ECD visualization utilizing SpecDis 12 .

Measurement of NO production and cell viability in LPS-activated BV-2 cells. The inhibitory
effect of the test compounds on LPS-stimulated NO production was studied using BV2 cells. BV2 cells were seeded on a 96-well plate (4 × 10 4 cells/well) and treated with or without different concentrations of the compounds. These cells were stimulated with LPS (100 ng/mL) and incubated for 24 h. The concentration of nitrite (NO 2 ), a soluble oxidation product of NO, in the culture medium was measured using Griess reagent (0.1% N-1-napthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid). Fifty microliters of supernatant were mixed with an equal volume of the Griess reagent. Absorbance was measured after 10 min using a microplate reader (Emax, Molecular Devices, Sunnyvale, CA, USA) at 570 nm wavelength. L-NMMA, a nitric oxide synthase (NOS) inhibitor, was used as a positive control. Graded sodium nitrite solution was used as a standard to calculate nitrite concentrations. Cell viability was evaluated by the MTT assay.