Pilidiostigmin, a novel bioactive dimeric acylphloroglucinol derivative isolated from Pilidiostigma glabrum

Pilidiostigmin, a novel dimeric acylphloroglucinol derivative, was isolated from the leaves of the Australian plant species Pilidiostigma glabrum (Myrtaceae). Pilidiostigmin exists in the plant in the form of a sodium salt, which is rare in natural products. The elucidation of the structure and relative configuration was achieved by spectroscopic measurements with special emphasis on 1D and 2D NMR techniques. Pilidiostigmin was found to display biological activity; it significantly and dose-dependently inhibited the synthesis of nitric oxide in LPS-stimulated RAW 264.7 macrophages at low micromolar concentrations. 2013 Elsevier Ltd. All rights reserved. Pilidiostigma glabrum Burret (Myrtaceae), an endemic Australian plant commonly known as plum myrtle, is distributed in southeastern Queensland and north-eastern New South Wales. In the course of our screening work of native Australian plants with anti-inflammatory activity, the CHCl3 soluble part of the 95% EtOH extract of the leaves of P. glabrum was found to attenuate significantly nitric oxide (NO) production in vitro. Six dibenzofurans were subsequently isolated from the CHCl3 fraction in our previous investigation. In a systematic approach to identify the active compounds, we proceeded to further investigate the chemical constituents of the CHCl3 fraction. Herein, we report the isolation and structure elucidation of a novel acylphloroglucinol derivative named pilidiostigmin and the inhibitory effect of this compound on the synthesis of nitric oxide in LPS-stimulated RAW 264.7 macrophages. The chloroform fraction of the ethanol leaf extract was purified by repeated column chromatography over MCI gel (CHP20P) eluting with methanol/water (80–100%), C18 (Separa C18-E, 50 lm, Phenomenex) eluting with methanol/water (80–90%), and preparative HPLC using an acetonitrile–water gradient to afford pilidiostigmin (8 mg) (Fig. 1). Pilidiostigmin, obtained as a yellow oil, showed an [M+1] peak at 855.2622 (calcd 855.2840) and an [M 1] peak at 853.2707 (calcd 853.2684) according to high-resolution electrosprayll rights reserved. +61 2 66223459. ). ionization time of flight mass spectroscopy (HR-ESI-TOF-MS), corresponding to the molecular formula C44H48O16Na. Eight methyl groups with chemical shifts in the range of dH 1.24–1.35 were observed in the H NMR spectrum, showing correlations with four methine multiplets at dH 3.92, 4.09, 4.12, and 4.28 in the H–H COSY, which indicated the presence of four 2-methylpropionyl moieties. Four tertiary methyls were also observed at dH 2.31, 2.29, 1.88, and 1.73, and two of them were evidently aromatic methyls. Six downfield protons at dH 10.58, 11.78, 13.83, 13.88, 17.10, and 18.59 assignable to six hydrogen bonds of the OH groups chelated to carbonyl or OH groups were also observed. In addition, the C JMOD as well as the HSQC experiments showed eight methyl groups with chemical shifts in the range of dC 19.0–20.0 (dC 19.0, 19.2, 19.4, 19.5, 19.5, 19.5, 19.9, and 20.0), two aromatic methyls at dC 8.0 and 8.1, two tertiary methyls at dC 27.4 and 28.8, two oxygenated quaternary carbons at dC 70.3 and 73.4, and six ketone carbons at dC 212.6, 212.5, 208.9, 204.7, 202.0, and 196.6. The remaining carbon signals were quaternary carbons of the benzene rings and double bonds. These data, together with additional analysis of the 2D NMR spectra, revealed the presence of two units of similar magnetic resonance signals, which indicated the dimeric nature of the compound. In one fragment, the two hydroxyl protons at dH 10.58 and 13.88 were located on both sides of a 2-methylpropionyl moiety in a benzene ring, based on the formation of hydrogen bonds as well as the HMBC correlations (Fig. 2). In the HMBC spectrum, the correlations of the hydroxyl proton at dH 10.58 with dc 102.0