Amberinone, a new guaianolide from Amberboa ramosa

The Amberboa is a medicinally important genus present in the family Asteraceae; members of this genus are mainly distributed in Pakistan and India. It has been used in different systems of traditional medicines for different diseases. Amberinone (1), a new sesquiterpene lactone, has been isolated from the ethyl acetate (EtOAc) soluble fraction of Amberboa ramosa together with chrysin (2), quercitine (3), eriodictyol (4) and keamferol (5). This is the first report of these compounds from this species. The structures of the isolated compounds have been elucidated by 1D and 2D 1H 13C NMR spectroscopy.


Introduction
Guaianolides are naturally occurring sesquiterpene lactones that exhibit important biological activities. Their structure consists of the tricyclic 5, 7, 5-ring system. Plants with guaianolides as active constituents have been used in traditional medicine for treating of rheumatic pains, increase of bile production and pulmonary disorders (Devreese et al. 1980;Connolly & Hill 1991).

Results and discussion
Amberinone (1)  The IR spectrum showed characteristic absorption bands at 3447, 1755, 1735 and 1656 cm 21 for hydroxyl, g-lactone, ester and olefinic functionalities, respectively. The UV absorption maxima at 197 and 205 nm were typical of a g-lactone (Khan et al. 2005). Further spectral data showed close agreement with a guaianolide-type sesquiterpene (Marco et al. 1993;Ibrahim, Farooq et al. 2013;. The 1 H NMR spectrum showed the signals for four methyl protons at d 0.97 (s), d 1.35 (d, J ¼ 6.0 Hz), d 1.84 (s) and d 2.09 (s). It also showed signals for the oxymethine protons at d 5.08 (ddd, J ¼ 8.3, 6.0, 5.5 Hz) and d 5.27 (d, J ¼ 10.0 Hz). The latter was assigned to the proton geminal to the lactone oxygen atom. The band axial stereochemistry of this oxymethine proton was assigned based on larger coupling constant values, which is characteristic of all guaianolides of the genus Amberboa and Ixeris. It also showed COSY correlations with the vicinal proton at d 2.73 (ddd, J ¼ 10.0, 9.5, 8.3 Hz) which could subsequently be assigned to H-7. The larger coupling constants suggested trans-diaxial disposition among H-6 (b) and H-7, providing conclusive evidence for an a orientation of H-7 (Seto et al. 1986).
The coupling pattern of the proton signals for H-1 and H-9 supported the guaianolide structure (Youssef 1998). The entire sequence of protons attached to the guaianolide skeleton was established by correlation spectroscopy (COSY) and spin decoupling experiments. Irradiation of H-7 at d 2.73 simplified the doublet of quartet at d 2.34 into a quartet. Irradiation of H-11 at d 2.34 simplified the doublet of the methyl group at d 1.23 into a singlet, confirming the presence of a methyl group at C-11. The HMBC correlation between H-8 and C-16 was missing, this helped to place the O-acetyl group at C-10. This was further supported by the chemical shift values recorded for C-8 (d 72.2) and C-10 (d 83.0) ( Figure S2 reported with chemical shifts 78 -80 ppm (Ibrahim, Farooq et al. 2013;, while in this case it was observed at 72.2 ppm. Similarly C-10 having 'OH' is observed at 76 -78 ppm (Ibrahim et al. 2010), while in this case it was observed at 83.0. The down-field frequency of C-10 and a little up-field frequency for C-8 confirmed the presence of O-acetyl group at C-10.
The 13 C NMR spectrum (broad-band decoupled and DEPT) showed 17 signals comprising four methyl, two methylene, five machines and six quaternary carbons. The down-field frequency region showed five signals at d 210.2, 179.2, 171.2, 164.6 and 142.7, which could be assigned to ketone, lactone ester, O-acetyl and substituted olefinic carbons, respectively. One oxygenated quaternary and two oxygenated methine carbons resonated at d 83.0, 72.2 and 78.5, respectively. The positions of substituents were confirmed by HMQC, HMBC and COSY experiments.
The relative stereochemistry at various chiral centres of Amberinone (1) was assigned through NOESY experiments (Zdero et al. 1987;Zdero & Bohlmann 1990;Khan et al. 2004) ( Figure S1), which revealed trans/anti/cis-fusion of the a-methyl-g-lactone moiety, the sevenmember ring at C-7 and C-6, the five-member ring at C-5 and C-1. The a-orientation of the acetate group at C-10 was confirmed from the b-orientation of the methyl group based on key NOESY interactions between the methyl protons attached to a C-10 resonating at d 0.97 and H-1 at d 3.40.

Experimental 3.1. General experimental procedures
Melting points were determined on a Gallenkemp apparatus. IR spectra were measured on a JASCO 302-A spectrophotometer with KBr cells (Jasco, Tokyo, Japan). UV spectra were obtained on a Hitachi UV-3200 spectrophotometer (U-3200, Hitachi Instruments Inc., Tokyo, Japan). Optical rotations were measured on a JASCO DIP-360 polarimeter and the 1D and 2D NMR spectra were recorded on a Bruker AMX-400 spectrometer operating at 500 MHz for 1 H and 125 MHz for 13 C NMR (Bruker BioSpin, Rheinstetten, Germany). Electron impact (EI) mass spectra were recorded on JEOL JMS-HX-110 and Varian MAT-311-A mass spectrometers (Jeol, Tokyo, Japan). The HR-ESI-MS was recorded on a Jeol JMS 600H instrument (Jeol, Tokyo, Japan). Silica gel (230-400 mesh, E. Merck, Darmstadt, Germany) was used for column chromatography (CC).

Supplementary material
Supplementary material relating to this paper is available online, alongside Table S1 and Figures S1 -S2.