A new kaurane diterpenoid from Isodon inflexus

Abstract A new 7,20-epoxy kaurane diterpenoid, 15-acetyldemethylkamebacetal A (1) and six known kaurane diterpenoids (2–7) were isolated from the aerial parts of Isodon inflexus in nuclear transcription factor-κB (NF-κB)-dependent reporter gene assay-guided fractionation. Their chemical structures were determined on the basis of extensive spectroscopic analysis (UV, IR, MS, 1D- and 2D-NMR) and comparison with literature data. The isolated compounds were evaluated for their inhibitory effects on TNF-α-induced NF-κB activation, and all compounds exhibited NF-κB inhibitory activities with IC50 values ranging from 1.91 to 20.15 μM.


Introduction
The genus Isodon (Lamiaceae) is a rich source of diterpenoids, and many of them have various important bioactivities such as antibacterial, antitumour, anti-inflammatory and anti-feeding effects . Up to date, more than 600 diterpenoids have been identified from this genus since 1910, and most of them have ent-kaurane skeletons possessing highly oxygenated structures and show various biological activities (Hwang et al. 2001;Lee et al. 2002;Han et al. 2003;Yang et al. 2008;Liu et al. 2013;Chen et al. 2015). Isodon inflexus (Thunb.) Kudo [synonym, Rabdosia inflexa (Thunb.) Hara] is a perennial shrub widely distributed in China, Korea and Japan. Previous phytochemical investigations on this plant resulted in the isolation of several ent-kaurane diterpenoids (Fujita et al. 1982;Takeda et al. 1989aTakeda et al. , 1989bTakeda et al. , 1990Takeda et al. , 1993 and some of them exhibited cytotoxic activities and inhibitory effects on nitric oxide (NO) production (Lee et al. 2008;Xie et al. 2012).
Nuclear transcription factor-κB (NF-κB) plays a crucial role in the regulation of genes controlling the immune system, apoptosis, tumor cell growth and tissue differentiation. The activation of NF-κB is known to relate to multiple pathophysiological conditions such as cancer, arthritis, asthma, inflammatory bowel disease and other inflammatory conditions. Therefore, NF-κB and the signalling pathways that regulate its activity have become a focal point for intense drug discovery and development efforts (DiDonato et al. 2012). In our ongoing research for NF-κB inhibitors from this plant, a new kaurane diterpenoid, 15-acetyldemethylkamebacetal A (1) and six known compounds, demethylkamebacetal A (2), excisanin A (3), rabdokunmin C (4), kamebakaurin (5), reniformin A (6) and isodomedin (7), were isolated from the methanol extract of I. inflexus (Figure 1). Herein, we present the isolation, structure elucidation and NF-κB inhibitory activities of these compounds.  The IR spectrum showed absorption bands due to hydroxyl groups (3392 cm −1 ) and an exocyclic methylene moiety (1739 and 1671 cm −1 ). All protons and related carbons were assigned according to the DePT, HSQC and HMBC experiments, which provided information on the characteristic signals of three methines (δ C 49.8, 47.3 and 46.4 due to C-5, 9 and 13), three quaternary carbons (δ C 34.8, 53.4 and 43.6 assignable to C-4, 8 and 10), two methyls (δ C 32.8 and 21.1 attributable to C-18 and 19), an oxygenated methine (δ C 69.8 assigned as C-7) and a hemiacetal methine carbon (δ C 95.6 assigned as C-20), indicating 1 would be a 7,20-epoxy-kaurane diterpenoid (Han et al. 2003). The epoxy hemiacetal linkage between C-7 and C-20 was suggested by the HMBC spectrum of 1, in which the H-20 (δ H 5.77) was coupled to C-9 (δ C 47.3), C-7 (δ C 69.8), C-5 (δ C 49.8) and C-10 (δ C 43.6), and the relative configuration at C-20 was determined as S by the downfield shift of C-11 due to the δ-syn-axial effect between 20-OH and C-11 (Huang et al. 1989), which was supported by the NOeSY correlation of H-20 with Me-19. Comparison of the 1 H-and 13 C-NMR spectral data of 1 with those of 2 showed only two differences: two newly observed carbon signals at δ C 172.6 and 21.5 arised from an acetyl group, and upfield shift of the signal at δ C 207.6 of 2 to δ C 76.9 (Table S1). This was confirmed by the maximum absorption wavelength at 204 nm in UV spectrum and HMBC correlations of H-15 (δ H 5.59) with C-7 (δ C 69.8), C-9 (δ C 47.3), C-14 (δ C 73.5) and carbonyl carbon (δ C 172.6). The β-orientation of OAc-15 was determined by the upfield shift of C-9 (Δ-5.6 ppm) because of the γ-steric compression effect between OAc-15β and H-9β (Zhao et al. 1997), which was supported by the NOeSY correlation of OAc-15 with H-9β. The α-orientation of 1-OH was indicated by the large coupling constants of H-1β with H-2α (J = 11.2) and H-2β (J = 5.5 Hz) (Takeda et al. 1994). The substituents at C-7 and C-14 possessed α-and β-orientations according to the observed NOeSY correlations of H-7β with H-5β and of H-14α with H-11α and H-20. Consequently, compound 1 was elucidated as 1α,14β,20-trihydroxy-15β-acetoxy-7α,20-epoxy-kaur-16-ene.
All the isolates were examined for their dose-response effect on the TNF-α-induced NF-κB activation using the NF-κB mediated reporter gene assay system. HeLa cells, which were transiently transfected with a NF-κB-dependent reporter gene construct, were stimulated with TNF-α in the presence of various concentrations of compounds, and then the expression of reporter gene luciferase activity was measured. As a result, compounds 1-7 all showed inhibitory activity in the reporter gene expression, with IC 50 values of 20. 15, 7.36, 5.38, 17.86, 4.39, 2.88 and 1.91 μM, respectively ( Table 1).
The active compounds (2-7) were kaurane diterpenes containing an active centre (cyclopentanone conjugated with an exomethylene group), which can react by Michael-type reaction with biological nucleophile, especially sulfhydryl group of cysteine residue in the target proteins involved in the NF-κB signalling pathway (Lee et al. 2002;Leung et al. 2006). However, compound 1, which contains only the exomethylene group without a conjugated carbonyl group, also exhibited NF-κB inhibitory activity. It would be interesting to examine whether the structural difference of Michael acceptor in the diterpenes would compromise the target cysteine for the covalent modification in the various target proteins involved in the NF-κB signalling pathway.

Plant material
The aerial parts of I. inflexus were collected at Jeju, Korea, in September 2006, and identified by Dr Young Ho Kim at Chungnam National University. A voucher specimen (No. 060908) was deposited at Korea Research Institute of Bioscience and Biotechnology.

NF-κB activity assay
A pNF-κB-Luc plasmid for NF-κB luciferase reporter assay was obtained from Strategene (LaJolla, CA, USA). Transfections were performed using Lipofectamine2000 according to the manufacturer's protocol. NF-κB-dependent luciferase activity was measured using the Dual Luciferase Reporter Assay system. Briefly, HeLa cells (1 × 10 5 cells/well) were seeded in a 96-well plate for 24 h. The cells were then transfected with plasmids for each well and then incubated for a transfection period of 48 h. After that, the cell culture medium was removed and replaced with fresh medium containing various concentrations of compounds and TNF-α for 8 h. Luciferase activity was determined in Microlumat plus luminometer (eG&G Berthold, BadWildbad, Germany) by injecting 100 μL of assay buffer containing luciferin and measuring light emission for 10 s. Co-transfection with pRL-CMV (Promega, Madison, WI, USA), which expresses Renilla luciferase, was performed to enable normalisation of data for transfection efficiency (Lee et al. 2006).

Conclusions
In this study, a new kaurane diterpenoid, 15-acetyldemethylkamebacetal A (1) and six known compounds (2-7) were isolated from the aerial parts of I. inflexus. Their structures were elucidated by spectroscopic analysis including UV, IR, MS, 1D-and 2D-NMR experiments and comparison with literature values. Bioassay results showed that compounds 1-7 all exhibited NF-κB inhibitory activity in a dose-dependent manner with IC 50 values ranging from 1.91 to 20.15 μM.

Supplementary material
Supplementary material relating to this article is available online, alongside Table S1 and Figures S1-S10.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the Natural Science Foundation of Jilin Province [grant number 201215241] and grants from "1000 Talent Plan for High-Level Foreign experts".