Ugandenial A, a New Drimane-type Sesquiterpenoid from Warburgia ugandensis

One new drimane-type sesquiterpenoid, named ugandenial A (1), was isolated from the ethyl acetate extract of the bark of Warburgia ugandensis (Canellaceae) together with eight known drimane-type sesquiterpenoids: 11α-hydroxycinnamosmolide (2), warburganal (3), polygodial (4), mukaadial (5), dendocarbin A (6), 9α-hydroxycinnamolide (7), dendocarbin L (8) and dendocarbin M (9). Their structures were established by detailed spectroscopic analysis. In addition a keto-enol equilibrium was demonstrated for compound 1 through a detailed NMR analysis run in CD2Cl2 at 190 K. Cytotoxicity of the isolated compounds against KB cells was evaluated.


Introduction
In order to better understand the potential self-medicative behavior of chimpanzees, one of us (SK) has investigated since 2000 the bioactivities of plant species eaten by wild chimpanzees in Kibale National Park in Uganda. Among 300 samples collected so far and screened for different bioactivities, the EtOAc extract of Warburgia ugandensis bark exhibited potent cytotoxic activity on KB cell line (99 % and 64% inhibition at 10 and 1 μg/mL, respectively). This evergreen straight tree may reach 30 m in height. The leaves are short-stalked, simple, alternate, dotted with glands and lacking stipules. Berries, purple when dried contain several heart-shaped seeds in a cream-green pulp. All parts of the tree are known by local people to have a hot aromatic taste and the spicy leaves are used as a chili substitute [1]. Roots and bark are used in traditional medicine especially to treat worms, pains, cough, colds and malaria [2]. However, traditional healers are cautious and use only small quantities as it may cause violent vomiting [1,3].
In previous phytochemical investigations of W. ugandensis, a large number of drimane-type sesquiterpenoids have been isolated from the heart wood, like ugandensolide, ugandesidial, warburgin and warburgiadione [4] and from the stem bark [5,6,7], like muzigadiolide, deacetylugandensolide, cinnamolide, mukaadial, ugandensidial, muzigadial, waburganal and others, while flavonol glycosides [8] and monoterpenes [5] were characterized from W. ugandensis leaves. The chemical investigation of the EtOAc bark extract was undertaken since a strong cytotoxicity was found against KB cells. Bioassay-guided purification of this extract by repeated column chromatography, semi-preparative HPLC and preparative TLC led to the isolation of one new compound 1, together with eight known drimane-type sesquiterpenes: 9α,11α-dihydroxy,6β-acetyl-cinnamolide (2), warburganal (3), polygodial (4), mukaadial (5), 9α-hydroxycinnamolide (7), and dendocarbins A, L and M (6, 8 and 9, respectively) reported for the first time in this species. Their structures were established by detailed spectroscopic analysis. In addition a keto-enol equilibrium was demonstrated for compound 1 through a detailed NMR analysis run in CD 2 Cl 2 at 190 K. Cytotoxicity of the isolated compounds against a KB cell line was also evaluated.

Results and Discussion
The EtOAc bark extract (3 g) of W. ugandensis Sprague was subjected to column chromatography on silica gel, semi-preparative HPLC, and preparative TLC to afford one new compound 1, in addition with eight known ones 2-9, which were identified by comparison of the spectral data with the reported literature values.
Compound 1 was obtained as a white amorphous powder. Its molecular formula was determined as C 15 H 22 O 4 from the [M+Na] + pseudomolecular ion peak at m/z 289.1427 (calc. 289.1416) in the HRESIMS. The IR spectrum of compound 1 revealed a broad absorption at 3,440 cm -1 , characteristic of an OH stretch, and a strong absorption at 1,745 cm -1 , characteristic of an ester or lactone carbonyl stretch. The 1 H-and 13 C-NMR data ( Table 1) of 1 were closely comparable to those of 11αhydroxycinnamosmolide isolated from Warburgia salutaris [9]. In the HMBC spectrum of 1 in CD 2 Cl 2 at 190 K (Figure 2), correlations from the methine at 41.1 (C-5), the quaternary carbon at 75.7 (C-9) and the carbonyl at 168.7 (C-12) to the olefinic proton at 6.98 (brs, H-7) confirmed the presence of a conjugated γ-lactone moiety. On the basis of the above spectral data, as well as HMQC, 1 H-1 H COSY analyses and other HMBC correlations, compound 1 is a typical drimane sesquiterpene possessing a C-ring. However, due to a keto-enol equilibrium the signals for the H-11 and C-11 were not visible, either in CDCl 3 , CD 3 OD, CD 2 Cl 2 or DMF at room temperature. A NMR study at 190 K in CD 2 Cl 2 showed that a doublet at δ H 5.66 (J = 10 Hz), which was assigned to H-11 is observable in the 1 H-NMR spectrum; the doublet give a sharp singlet after irradiation of the OH-11 at δ H 5.90. Moreover, at 190 K, a cross peak between H-11 and C-9 at δ C 75.7 in the HMBC spectrum, and the HMQC correlation indicating the chemical shift of C-11 was 98.5 confirmed the proposed γ-hydroxy-γ-lactone motif. Finally, in the NOESY spectrum an exchange crosspeak between H-11 and an aldehyde proton at δ H 9.75 ppm confirmed the keto-enol equilibrium suggested for compound 1. This equilibrium was also observed for compound 2. A correlation between H-11 and Me-13 (δ H 0.80 s) revealed the α-configuration of the hydroxy group at C-11. Therefore, the structure of 1, named ugandenial A, was established as depicted in Figure 1.   Compounds 1-9 were subjected to a cytotoxic assay against KB cancer cell line. Only compounds 3-5 showed any cytotoxic activities, with IC 50 values of 0.3, 1.0, and 5.3 µM, respectively. The other compounds were inactive at concentrations up to 10 µM. These results confirm that the presence of the dialdehyde function is essential for a strong cytotoxicity [13]. Taxotere was used for a control experiment.

General
Optical rotations were measured at 25 °C on a JASCO P1010 polarimeter. IR spectra were measured on a Nicolet FTIR 205 spectrophotometer. ESIMS spectra were obtained on a Navigator Mass Thermoquest. HRESIMS were obtained on a MALDI-TOF spectrometer (Voyager-De STR; Perseptive Biosystems). NMR spectra were recorded on a Bruker spectrometer (500 MHz for 1 H, 125 MHz for 13 C and 2D NMR) at 25 °C and/or (600 MHz for 1 H, 150 MHz for 13 C and 2D NMR) at 190 K (1 and 2), using TMS as an internal standard. Chemical shifts were in ppm, and coupling constants J in Hz. HPLC was performed using a Waters Autopurification system equipped with a UVvis diode array detector (190-600 nm) and a Pl-ELS 1000 ELSD detector (Polymer Laboratory). Precoated silica gel plates (Merck) were used for TLC. Detection was done by spraying plates with 5% anisaldehyde-sulfuric acid, followed by heating.

Plant material
Bark of Warburgia ugandensis were collected in March 2007 in the high altitude scrubland in Kibale National Park, western Uganda, by one of us (J.K.). A voucher specimen (UFC-0078) is stored in the herbarium of Museum National d'Histoire Naturelle in Paris.

Cytotoxicity activity assay
The human tumor cell line KB (mouth epidermoid carcinoma) was originally obtained from the ATCC. The cytotoxicity assays were performed according to a published procedure [14].

Conclusions
A chemical investigation of Warburgia ugandensis bark was carried out in the framework of a global investigation on the feeding behavior of chimpanzees in Kibale National Park in Uganda. This study showed that the bark contained γ-lactone sesquiterpenoids, of which ugandenial A (1) is characterized for the first time, and cytotoxic dialdehyde drimane-type compounds. The presence of dendocarbins A, L and M (6, 8 and 9, respectively) was not previously reported in W. ugandensis.