Kingianic Acids A–G, Endiandric Acid Analogues from Endiandra kingiana

A phytochemical investigation of the methanolic extract of the bark of Endiandra kingiana led to the isolation of seven new tetracyclic endiandric acid analogues, kingianic acids A–G (1–7), together with endiandric acid M (8), tsangibeilin B (9) and endiandric acid (10). Their structures were determined by 1D- and 2D-NMR analysis in combination with HRMS experiments. The structure of compounds 9 and 10 were confirmed by single-crystal X-ray diffraction analysis. These compounds were screened for Bcl-xL and Mcl-1 binding affinities and cytotoxic activity on various cancer cell lines. Compound 5 showed moderate cytotoxic activity against human colorectal adeno-carcinoma (HT-29) and lung adenocarcinoma epithelial (A549) cell lines, with IC50 values in the range 15–17 µM, and compounds 3, 6 and 9 exhibited weak binding affinity for the anti-apoptotic protein Mcl-1.


Introduction
In our pursuit to discover bioactive phytochemicals from the Malaysia flora [1][2][3] we recently reported a series of new natural pentacyclic polyketides, named kingianins A-L, isolated from the ethyl acetate extract of the bark of Endiandra kingiana Gamble (Lauraceae) [4]. Several kingianins showed strong binding affinity to the anti-apoptotic protein Bcl-xL, and can therefore be considered as potential anticancer agents [4]. In order to discover additional members of this chemical series or close analogues, we have embarked on the investigation on the methanolic extract of the bark of this species. E. kingiana is a medium-sized evergreen tree, distributed throughout Peninsular Malaysia and Borneo [5][6][7]. There are about 125 Endiandra species found throughout the tropical regions, including 10 species in Malaysia [5][6][7], but to our knowledge only three species: E. introrsa, E. anthropophagorum and E. kingiana, have been studied for their phytochemicals. The first one has been reported to produce interesting cyclic polyketides, named endiandric acids, possessing eight chiral centers, and usually isolated as racemic mixtures [8][9][10][11]. It was postulated by Black and co-workers that they could be formed by non-enzymatic cyclizations (8πe and 6πe electrocyclization followed by Diels-Alder reaction) of a phenylpolyene acid precursor [12][13][14]. In 1982, Nicolaou's group successfully synthesized the natural endiandric acids by implementing a biomimetic strategy based on Black's hypothesis [14][15][16][17]. Endiandric acids and their close derivatives, beilschmiedic acids, are the most characteristic type of natural products isolated from Beilschmiedia and Endiandra species. They were found to exhibit various biological activities, such as antibacterial [18][19][20], antiplasmodial [20], antitubercular [21], iNOS inhibitory activity [22], and anticancer properties [20,23]. Recently, Williams et al. reported the cytotoxic and antibacterial activities of a series of beilschmiedic acids isolated from a Gabonese Beilschmiedia species against NCI-H460 human lung cancer cells and a clinical isolate of methicillin-resistant Staphylococcus aureus, respectively [23]. Talonsti et al. have also reported recently the isolation of four beilschmiedic acid derivatives, cryptobeilic acids A-D and tsangibeilin B from the bark of Endiandra cryptocaryoides [20]. These compounds showed moderate antiplasmodial activity against the chloroquinone-resistant Plasmodium falciparum strain NF54, and antibacterial activities against Escherichia coli, Acinetobacter calcoaceticus and Pseudomonas stutzeri [20].
The chemical investigation of the methanolic extract of E. kingiana bark extract led to the isolation of seven new endiandric acids, kingianic acids A-G (1-7), together with endiandric acid M (8), tsangibeilin B (9) and endiandric acid (10) (compound 10 was only found in the "PubChem" database (CID 71521970) without an associated reference regarding its origin and its spectroscopic data, so this compound is fully described in the present manuscript). Herein, the isolation and structure elucidation of the new tetracyclic endiandric acids; kingianic acids A-G, and the cytotoxic activities, Bcl-xL and Mcl-1 affinities of compounds 1, 3, 5-9 are reported.

Results and Discussion
The EtOAc-soluble part of the E. kingiana methanol extract was subjected to silica gel chromatography to afford eight fractions Fr.1-Fr.8. Fractions Fr.4 and Fr.5 were further purified using silica gel as well as semi-preparative HPLC leading to the isolation of the kingianic acid series 1-7, endiandric acid M (8), tsangibeilin B (9) and endiandric acid 10 ( Figure 1). All assignments of 1 H-and 13 C-NMR data were then established through in depth analysis of 2D-NMR; NOESY, COSY, HSQC and HMBC experiments. All compounds 1-10 were isolated as optically inactive, thus suggesting that they are racemic mixtures and their spectroscopic data were very similar. They all possess a 13 carbon atom fused rings system and they can be divided into two main skeletal types. Six of compounds (1)(2)(3)(4)(5)8) belong to the first type as can be seen in endiandric acid K and endiandramide A [22]. While compounds 6, 7, 9 and 10 belong to the second type, similar to tsangibeilins A and B and endiandramide B [22]. Kingianic acid A (1) was isolated as a colorless oil. The HRESIMS spectrum of 1 showed a pseudomolecular ion peak [M−H] − at m/z 323.1279 (calcd. 323.1284), consistent with a molecular formula C 20 H 20 O 4 with 11 degrees of unsaturation. Its UV spectrum showed absorption bands at 233 and 286 nm, suggesting the presence of a benzenoid moiety, and its IR spectrum indicated the presence of OH (3,431 cm −1 ), carbonyl (1,701 cm −1 ) and methylenedioxy (1,040 and 936 cm −1 ) groups [22]. The 13 C-NMR and DEPT spectra exhibited 20 signals, including 13 methines, three methylenes, and four quaternary carbons. The resonances of the methines at C-1 (δ C 41.8), C-2 (δ C 39.7), C-3 (δ C 38.8), C-4 (δ C 40.6), C-5 (δ C 39.8), C-7 (δ C 38.3), C-8 (δ C 48.8) and C-9 (δ C 34.8), including two olefinic carbons at C-10, C-11 and a methylene at C-6 observed in the DEPT spectrum, were characteristic of a tetracyclic endiandric acid like endiandric acid K [22]. The 1 H and 13  H spectra of 1 (Table 1) were also similar to those of endiandric acid K [22], except for the methylene proton signals [δ H 2.72 (m, H-1'a); 2.78 (m, H-1'b)]. This group is placed between C-4 of the tetracylic acid moiety and C-2' of the benzenoid moiety in 1, instead of three methylenes in endiandric acid K. The 1 H NMR of 1 revealed two cis olefinic proton signals at δ   Their location at C-4 and C-8, respectively, was deduced from COSY and HMBC correlations. The COSY correlation between H-4 and H 2 -1' on one hand, and HMBC correlations from H-8 (δ H 3.25) to C-12 (δ C 140.2), and from H-13 (δ H 6.61)/H-17 (δ H 6.54) to C-8 (δ C 47.9) on the other hand, confirmed the location of the acetic acid group and the methylenedioxyphenyl moiety at C-4 and C-8, respectively. The relative configuration of compound 5, named kingianic acid E, was fixed by a NOESY experiment as the same as that of compound 1.
The molecular formula C 21 H 22 O 2 of kingianic acid F (6) was determined by HRESIMS analysis; 305.1557 (calcd 305.1542), for which 10 degrees of unsaturation could be deduced. The UV spectrum of 6 showed characteristic absorption bands at 232 and 288 nm suggesting the presence of a benzenoid moiety, and its IR spectrum showed absorption bands at 3,432 cm −1 for an OH group and 1,696 cm −1 for a carbonyl group. The 13 C-NMR and DEPT spectrum, which showed 21 signals for two methylenes, 16 methines (nine olefinic), and two quaternary and a carbonyl carbon that were characteristic of the tetracyclic endiandric acid skeleton as seen in tsangibeilin B (9) [22]. The 1 H and 13 C-NMR spectra of compound 6 were reminiscent to those of 9, except for the absence of signal for a methylenedioxy group in 6 and the appearance of five aromatic proton and carbon signals, thus suggesting that the methylenedioxyphenyl moiety was replaced by a monosubstituted phenyl moiety. All carbon-carbon connectivities of compound 6 were determined through a thorough analysis of 2D NMR spectra (Figures 2 and 3), and comparison with those of tsangibeilin B (9). The location of the COOH group at C-6, and the phenyl moiety at C-11, was confirmed by HMBC correlation from H-6 to the carbonyl carbon at δ C 179.4, and from COSY correlations between H-11 and H 2 -1', respectively. The relative configuration of 6 was ascertained by the careful inspection of NOESY spectrum ( Figure 3) and biogenesis considerations of tsangibeilin B as reference [22]. Thus, the relative configuration of 6, named kingianic acid F or 11{1'-[phenyl]}tetracyclo[5.4.2.0 3,13 .0 10,12 ]trideca-4,8-dien-6-carboxylic acid (kingianic acid F), was assigned as rel-(1RS, 2RS, 3RS, 4SR, 5SR, 7SR, 8RS, 9SR), same as that of tsangibeilin B (9) [22].
Kingianic acid G (7)  respectively, were equally intense, the relative configuration at C-4 could not be deduced from the NOESY spectrum. However, comparison of NMR data of kingianic acid G (7) with data reported in the literature for beilschmiedic acids H and I allowed the assignment of the 4-OH configuration [23]. Indeed, since compound 7 possesses similar 13 C chemical shifts to beilschmiedic acid H for carbons C-3, C-4 and C-5 (δ C 44.1, 73.6, 145.6, respectively for 7; δ C 45.1, 74.5 and 145.8, respectively for beilschmiedic acid H), it can be deduced that the hydroxy group is -oriented, as was the case for beilschmiedic acid H [23]. In the case of beilschmiedic acid I, having a β-oriented hydroxy group at C-4, chemical shifts of C-3, C-4 and C-5 are more shielded; 43.1, 65.9 and 141.5 ppm, respectively [23]. Thus, kingianic acid G (7) was proposed to have the same relative configuration as beilschmiedic acid H.

Plant Material
The bark of Endiandra kingiana Gamble was collected at Reserved Forest Sg. Temau, Kuala Lipis, Pahang, Malaysia in May 2006. This plant was identified by T. Leong Eng Botanist University of Malaya. A voucher specimen (KL-5243) has been deposited at the Herbarium of the Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.

Extraction and Isolation
The air-dried bark of E. kingiana (1.5 kg) were sliced, ground and extracted with EtOAc (3 × 1.

Bcl-xL and Mcl-1 Binding Affinity Assays
The binding affinities of compounds for Bcl-xL and Mcl-1 were evaluated by competition against fluorescently labelled reference compounds, Bak and Bid, respectively, as described by Qian et al. [26]. Human 45 -84/ C37 Bcl-xL and mouse DN150/DC25 Mcl-1 proteins were recombinantly produced by N. Birlirakis at ICSN. Bak, 5-Carboxyfluorescein-Bak, Bid and 5-carboxyfluorescein-Bid peptides were synthetized by PolyPeptide Laboratories (Strasbourg, France). All sequences are available in the Supporting Information (S37). Unlabeled peptides were dissolved in DMSO (Carlo Erba, Val de Reuil, France) and labelled peptides were diluted in assay buffer, which contained 20 mM Na 2 HPO 4 (pH 7.4), 50 mM NaCl, 2 µM EDTA, 0.05% Pluronic F-68, without pluronic acid for storage at −20 °C. Liquid handling instrument, Biomek ® NX and Biomeck ® 3000 (Beckman Coulter, Villepinte, France), were used to add protein and fluorescein-labelled peptides. 15 nM labelled BH3 peptide, 100 nM protein, and 100 μM of unlabelled BH3 peptide or compound (first diluted in 10 mM DMSO and then buffer for final concentration from 10 −9 to 10 −4 M) into a final volume of 40 µL were distributed in a 96 well black polystyrene flat-bottomed microplate (VWR 734-1622). The microplate was then incubated at room temperature for 1 h and shaken before fluorescent polarization measure. Fluorescence polarization in millipolarization units was measured with a Beckman Coulter Paradigm ® using FP cartridge (λ ex 485 nm, λ em 535nm). The exposure time was 300 ms per channel. All experimental data were collected using the Biomek Software ® (Beckman Coulter, Inc, Brea, CA, USA) and analysed using Microsoft Excel 2010 (Microsoft, Redmond, WA, USA). Results are expressed as binding activity, i.e., percentage of inhibition of the binding of labelled reference compound, or as Ki, the concentration corresponding to 50% of such inhibition, and corrected for experimental conditions according to Kenakin rearranged equation [29], which is adapted from Cheng and Prusoff equation [30]. ABT-737, which was kindly provided by O. Nosjean (Institut de Recherche Servier, Croissy, France), and unlabeled peptides Bak and Bid were used as positives control. The performance of the assays was monitored by use of Z' factors as described by Zhang et al. [31]. The Z' factors for these assays are 0.8 (Bcl-xL/Bak) and 0.7 (Mcl-1/Bid) indicating that they should be robust assays.

Cell Viability Assay
Human cancer cell lines A549 (Lung adenocarcinoma epithelial), HT29 (Colorectal adenocarcinoma) and PC3 (Prostate adenocarcinoma) cells were obtained from the ATCC (Manassas, VA, USA). Cells were grown in RPMI-1640 or DMEM medium with 10% FBS supplemented with 4 mM L-glutamine and 1% penicillin-streptomycin. For experimental purposes, the cells growing exponentially and maintained at 70%-80% confluency were used. Cells were seeded into 96-well plates at 10 4 cells/well and allowed to adhere overnight; the medium was then removed. A stock solution of test compound in DMSO was diluted in medium to generate a series of working solutions. Aliquots (100 μL) of the working solutions were added to the appropriate test wells to expose cells to the final concentrations of compound in a total volume of 100 μL. Nine different concentrations (100 µL-0.4 µL) were tested, in triplicates. Cisplatin is used as a positive control and wells containing vehicle without compound were used as negative controls. Plates were kept for 48 h in a 37 °C, 5% CO 2 incubator. After incubation, viable cells were detected with the CellTiter 96 AQueous cell proliferation assay (Promega Corp., Madison, WI, USA). Plates were read in a microplate reader (Tecan Infinite ® 200 PRO series, Mannedorf, Switzerland) at 490nm. Then, dose-response curves were generated and the IC 50 values were determined using GraphPad Prism 5.04 software (La Jolla, CA, USA [32].

Conclusions
The phytochemical investigation of Endiandra kingiana methanolic bark extract has led to the isolation of seven new tetracyclic endiandric acid analogues, named kingianic acids A-G (1-7), together with endiandric acid M (8), tsangibeilin B (9) and compound 10. These compounds were screened for Bcl-xL and Mcl-1 binding affinities, and cytotoxic activity on various cancer cell lines. Compound 5 showed moderate cytotoxic activity, with IC 50 values in the range 15-17 µM, and compounds 3, 6 and 9 exhibited weak binding affinity for the anti-apoptotic protein Mcl-1. This is the first report of binding affinity toward Mcl-1 for endiandric acid analogues.