Sundaicumones A and B, Polyprenylated Acylphloroglucinol Derivatives from Calophyllum sundaicum with Weak Activity against the Glucocorticoid Receptor Shugeng Cao, ² Kia-Ngee Low, Robert P. Glover, Sharon C. Crasta, Siewbee Ng, Antony D. Buss, and Mark S. Butler* MerLion Pharmaceuticals, 1 Science Park Road, The Capricorn #05-01, Singapore Science Park II, Singapore 117528 ReceiVed December 14, 2005 Bioassay-directed fractionation using a glucocorticoid receptor assay led to the isolation of two new, weakly active polyprenylated acylphloroglucinol derivatives, sundaicumones A (1) and B (2), from the leaves of Calophyllum sundaicum collected in Singapore. The structures of 1 and 2, which were established by spectroscopic methods, contain a 3-substituted hexanoic acid unit not previously reported in other polyprenylated acylphloroglucinols. Polyprenylated acylphloroglucinols are a class of biologically active secondary metabolites isolated from the plant family Clu- siaceae (Guttiferae), predominantly from the genera Hypericum, 1-3 Garcinia, 4-10 and Clusia 10-13 but also from Symphonia, 10 Allan- blackia, 14 and Calophyllum. 15 Polyprenylated acylphloroglucinols also have been isolated from Cuban bee propolis and are thought to be present through transfer from the floral resin of Clusia rosea. 16,17 The best known example of a polyprenylated acylphlo- roglucinol is hyperforin (3), 1,2 a metabolite of Hypericum perfo- ratum (St. John’s Wort), which has been reported to have antidepressant 18 and in vitro and in vivo anticancer activity. 19 Other biological activities ascribed to polyprenylated acylphloroglucinols include HIV inhibition, 10,14 cytotoxic effects against cancer cell lines, 6,7,9,17,19 anti-inflammatory, 5 choline acetyltransferase inhibi- tion, 4 and as ligand binders of the liver-X receptor. 8 The glucocorticoid receptor (GR) belongs to the superfamily of ligand-activated transcription factors, the nuclear hormone receptors, which along with other steroid receptors affect the body through regulation of gene transcription. 20 The GR agonist complex binds and inhibits pro-inflammatory transcription factors such as nuclear factor-κB and activation protein-1, resulting in anti-inflammatory effects. 21,22 Synthetic glucocorticoids such as dexamethasone and prednisolone have been used since the 1960s to treat chronic inflammatory diseases but have side effects due to inhibition of other steroid receptors. 23 A reporter gene assay was developed to identify novel GR agonists as anti-inflammatory therapy for asthma and other airway diseases. Screening of our natural product extract library in the GR assay identified an active extract from the leaves of Calophyllum sundaicum P.F. Stev. (Clusiaceae). Bioassay-guided isolation using a modified Kupchan solvent partition scheme 24 and preparative C 18 reversed-phase HPLC gave two polyprenylated acylphloroglucinol derivatives, sundaicumones A (1) and B (2), which showed EC 50 values of 173 and 75 µM, respectively, in the GR assay. The GR activity of these compounds was too weak to warrant any further biological investigation. Dexamethasone was used as a control in the GR assay and had an EC 50 of 1.0 ( 0.6 nM. Sundaicumone A (1) was obtained as a colorless oil and had a molecular formula of C 32 H 50 O 9 based on its HRESIMS. The 1 H NMR spectrum of 1 (Table 1) displayed signals for six methyl singlet resonances at δ H 0.90, 0.96, 1.01, 1.08, 1.11, and 1.21, one methyl doublet at δ H 0.98, and two methyl triplets at δ H 0.70 and 0.81. The 13 C and multiplicity-edited HSQC NMR spectra of 1 showed nine CH 3 , seven CH 2 , five CH, and 11 quaternary carbons, six of which were located at low field (δ C 208.5, 205.1, 192.2, 175.3, 174.0, and 116.5). Analysis of the COSY, TOCSY, HSQC, and HMBC NMR data indicated the presence of 2-methyl-1- oxobutyl (C-16 to C-20), 3-substituted-hexanoic acid (C-1′ to C-6′), 2-oxygenated-3-hydroxy-3-methylbutyl (C-2, C-3, and C-13 to C-15), and a dihydroxylated monoterpene (C-9 to C-11 and C-21 to C-27) moiety, which were attached to C-8, C-6, C-1/C-5, and C-1/C-8 of the acylphloroglucinol core, respectively. These data suggested that the structure of 1 is similar to garsubellin B (4) except for the 3-substituted hexanoic acid moiety at C-6 in 1 instead of an isoprene moiety. 4 A total synthesis of (()-garsubellin A (5) was reported recently, which confirmed the structure and relative stereochemistry originally proposed. 25 Although H-17 did not exhibit a correlation to C-8 in the HMBC spectrum of 1, the chemical shift of C-8 (δ C 82.2) was consistent with that assigned in garsubellins B (4) and A (5). 4 Therefore, the planar structure of 1 was determined as shown and attention was focused on the compound’s relative stereochemistry. In their paper on the structures of the sampsoniones, Hu and Sim discussed an empirical rule that can be used to establish the axial or equatorial orientation of C-10 side-chains in acylphloroglucinols. 3 In 1, the C-10 side-chain was assigned as having an equatorial orientation due to the 13 C NMR resonances of the C-9 gem-dimethyl groups (C-21, δ 16.2 and C-22, δ 22.3) and the 1 H NMR resonances and coupling constants of H-11  (δ 1.53, dd, J ) 13.8, 12.0 Hz) and H-11 R (δ 2.19, dd, J ) 13.8, 4.4 Hz). 2-4,10 Furthermore, ROESY correlations between H-11  and H 3 -21 confirmed the assigned relative stereochemistry * To whom correspondence should be addressed. Tel: +65-6829 5611. Fax: +65 6829 5601. E-mail: mark@merlionpharma.com. ² Current address: Department of Chemistry, M/C 0212, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.