Alkaloids from the Australian Rainforest Tree Ochrosia moorei Anthony R. Carroll, † Rama Addepalli, † Gregory Fechner, † Jill Smith, † Gordon P. Guymer, ‡ Paul I. Forster, ‡ and Ronald J. Quinn* ,† Eskitis Institute, Griffith UniVersity, Brisbane, Queensland, Australia 4111, and Queensland Herbarium, Brisbane Botanic Gardens, Toowong, Queensland, Australia 4006 ReceiVed NoVember 18, 2007 High-throughput screening of a plant and marine invertebrate extract library to find natural products that down-regulate expression of pro-inflammatory genes associated with the glucocorticoid receptor ligand complex led to the identification of bioactive CH 2 Cl 2 extracts from stems and leaves of the Queensland tree Ochrosia moorei. Bioassay-guided purification of the stem extract enabled the isolation of four alkaloids including two new compounds, ochrosamines A (1) and B (2), and the known compounds ellipticine (3) and 9-methoxyellipticine (4). The leaf extract also afforded 3 and 4 as well as apparicine (5) and desoxycordifoline (6). The structures of the two new compounds were assigned from interpretation of 2D NMR and high-resolution ESIMS data. Ellipticine and 9-methoxyellipticine were the most active components, and both displayed IC 50 values of 90 µM. Apparicine and desoxycordifoline were only very weakly active, and ochrosamines A and B were inactive. The glucocorticoid receptor (GR) is a member of a superfamily of ligand-inducible transcription factors, the nuclear hormone receptors. Glucocorticoids play an essential role in maintaining basal and stress-related homeostasis and display potent anti-inflammatory and immunosuppressive properties. 1,2 As a consequence, synthetic glucocorticoids are used widely as drugs to treat inflammatory conditions such as rheumatoid arthritis or dermatitis and as adjunct therapy for conditions such as autoimmune diseases. The GR functions as a transcription factor upon binding of the glucocorti- coids (GCS). The GR-GCS complex up-regulates transcription of genes containing the glucorticoid response element (GRE). This results in activation of genes encoding anti-inflammatory proteins, such as secretory leukoprotease inhibitor (SLPI),  2 -adrenoceptors, and CD163, but is also thought to lead to some of the side effects of glucocorticoids. The major anti-inflammatory effects of the GR-GSC complex are, however, a result of the down-regulation of genes via interaction of the complex with other transcription factors such as activator protein-1 (AP-1). The GR-GCS binds to AP-1 via a protein–protein interaction and prevents AP-1 binding to the TPA responsive element (TRE), causing down-regulation of pro-inflammatory genes that are usually activated by AP-1. 3,4 The discovery of compounds that selectively down-regulate pro- inflammatory genes may provide new and improved drug therapies to treat inflammatory conditions. A high-throughput screening campaign employing a cell-based reporter gene assay was used to find extracts that down-regulate pro-inflammatory genes. For down-regulation, multiple TRE ele- ments were linked to a minimal promoter driving the -galactosi- dase gene. Extracts from 7399 plants and 517 marine invertebrates collected in Queensland were screened, and CH 2 Cl 2 and MeOH extracts of the leaves and stems of Ochrosia moorei (F. Muell.) F. Muell. ex Benth. (Apocynaceae) showed activity in the assay. Bioassay-guided isolation of these extracts led to the isolation of two new alkaloids, ochrosamines A (1) and B (2), and four known alkaloids, ellipticine (3), 9-methoxyellipticine (4), apparicine (5), and desoxycordifoline (6). This paper reports on the isolation, structure determination, and biological activity of these alkaloids isolated from O. moorei. The ground stems of O. moorei were extracted exhaustively with CH 2 Cl 2 , and the extract was chromatographed on Si gel using a stepped gradient from CH 2 Cl 2 to MeOH. An early-eluting fraction contained a mixture of ellipticine (3) and 9-methoxyellipticine (4). Two later-eluting fractions were each partitioned between CH 2 Cl 2 and 1 M HCl, and the aqueous layers were basified with 5 N NH 4 OH and then extracted with CH 2 Cl 2 , yielding ochrosamine A (1) and ochrosamine B (2). The ground leaves of O. moorei were extracted exhaustively with CH 2 Cl 2 and MeOH. The CH 2 Cl 2 and MeOH extracts were combined and chromatographed on Sephadex LH-20 eluting with MeOH. An early-eluting fraction was chro- matographed on diol-bonded Si gel using a stepped gradient from CH 2 Cl 2 to MeOH, yielding two alkaloid-containing fractions. These fractions were further purified by HPLC, the first on aminopropyl- bonded Si gel, yielding apparicine (5), and the second on C 18 , yielding desoxycordifoline (6). A later-eluting fraction from the Sephadex LH-20 column was purified by HPLC on aminopropyl- bonded Si gel, to yield ellipticine (3) and 9-methoxyellipticine (4). The known compounds were identified by 2D NMR analysis and by comparison of spectroscopic data with published results. 5–8 Accurate mass measurement of the pseudomolecular ion in the (+) HRESIMS at m/z 323.1768 (Δ 4.3 ppm) allowed a molecular formula of C 20 H 22 N 2 O 2 to be assigned to ochrosamine A (1). IR absorption bands at 3402, 2920, and 1630 cm -1 suggested the presence of hydroxyl, amine, and R,-unsaturated ketone function- alities. UV absorbances at 253 nm, indicative of an indoline chromophore, and at 311 nm, suggestive of a -amino-R,- unsaturated ketone, were observed in the UV spectrum. 9 The 1 H NMR spectrum of ochrosamine A (1) (Table 1) exhibited signals characteristic for a 1,2-disubstituted phenyl group (δ H 6.72, * To whom correspondence should be addressed. Tel: 61 7 3735 6000. Fax: 61 7 3735 6001. E-mail: R.Quinn@griffith.edu.au. † Griffith University. ‡ Queensland Herbarium. J. Nat. Prod. 2008, 71, 1063–1065 1063 10.1021/np070655e CCC: $40.75  2008 American Chemical Society and American Society of Pharmacognosy Published on Web 04/16/2008