Pim2 Inhibitors from the Papua New Guinean Plant Cupaniopsis macropetala ⊥ Rohan A. Davis, † Moana M. Simpson, † Ryan B. Nugent, † Anthony R. Carroll, † Vicky M. Avery, † Topul Rali, ‡ Huawei Chen, § Barbara Qurallo, § and Ronald J. Quinn* ,† Eskitis Institute, Griffith UniVersity, Brisbane, QLD 4111, Australia, BiodiVersity Research Ltd., P.O. Box 24, UniVersity Post Office, Port Moresby, Papua New Guinea, and AstraZeneca R & D Boston, 35 Gatehouse DriVe, Waltham, Massachusetts 02451 ReceiVed August 16, 2007 Bioassay-guided fractionation of an organic extract from the leaves of Cupaniopsis macropetala resulted in the isolation of a new alkaloid, galloyl tyramine (1), together with the known flavonoid glycoside quercitrin (2). The structure of 1 was determined following 1D and 2D NMR, IR, UV, and MS data analysis. Compounds 1 and 2 displayed IC 50 values of 161 and 25 μM, respectively, in a Pim2 enzyme assay. The Pim family of cytoplasmic serine/threonine kinases consists of three highly homologous genes, Pim1, Pim2, and Pim3. 1,2 These enzymes are increasingly being recognized as important regulators of apoptosis and cellular metabolism. Pim overexpression has been reported in various cancers, including B cell lymphoma, chronic lymphocytic leukemia, prostate cancer, and acute myelogenous leukemia. 3 Pim kinases have also been shown to be activated in retroviral-induced lymphomas, e.g., Moloney murine leukemia virus. 4,5 Thus, pharmacological inhibition of these Pim kinases could have applications in the treatment of diseases such as cancer, inflammatory disorders, and ischemic diseases. 3 High-throughput screening of ∼35 000 biota extracts led to the discovery that an extract from the leaves of the rainforest tree Cupaniopsis macropetala Radlk. (Sapindaceae) showed potent activity in a Pim2 enzyme assay. Bioassay-guided fractionation of an organic extract resulted in the isolation of a new alkaloid, galloyl tyramine (1), along with the previously reported flavonoid glycoside quercitrin (2). Herein we report the isolation and structure elucida- tion of galloyl tyramine (1) along with the Pim2 inhibitory activity of both natural products. The air-dried and ground leaves of C. macropetala were sequentially extracted with CH 2 Cl 2 and MeOH. Both extracts were combined and chromatographed using reversed-phase C 18 -bonded silica HPLC to yield galloyl tyramine (1, 82.5 mg, 1.914% dry wt) and quercitrin (2, 43 mg, 0.998% dry wt). The TFA salt of galloyl tyramine (1) was isolated as a brown gum and was assigned the molecular formula C 17 H 16 F 3 NO 7 on the basis of (+)-HRESIMS and NMR data. The 1 H NMR spectrum of 1 displayed six downfield exchangeable resonances [δ H 9.38 (s, 2H), 9.12 (s, 1H), and 7.84 (brs, 3H)], two aromatic resonances [δ H 7.32 (d, J ) 8.4 Hz, 2H), 7.17 (d, J ) 8.4 Hz, 2H)] indicative of a 1,4-disubstituted benzene ring, one aromatic singlet [δ H 7.08 (s, 2H)], and two mutually coupled methylenes [δ H 2.88 (brt, J ) 7.8 Hz, 2H) and 3.07 (brs, 2H)]. The 13 C NMR spectrum of 1 contained only 11 resonances, nine of which appeared between δ C 109 and 165. Analysis of the gHSQC data enabled all protonated carbons to be assigned. A HMBC correlation from δ H 7.32 (H-4, H-8) to the ethylene carbon at δ C 32.4 (C-2) and a strong ROESY correlation between H-4/H-8 and H-2 indicated that the ethylene moiety was attached to C-3 of the 1,4-disubstituted benzene ring. HMBC correlations from δ H 2.88 (H-2) to the quaternary carbon at δ C 134.5 (C-3) and the methine carbon at δ C 129.6 (C-4, C-8) further confirmed this assignment. A strong COSY correlation between δ H 3.07 (H-1) and the exchangeable signal at δ H 7.84 suggested a protonated terminal amine group was attached to C-1 (δ C 39.9). HMBC correlations from both δ H 7.32 (H-4, H-8) and 7.17 (H-5, H-7) to a quaternary oxygenated carbon at δ C 149.6 (C-6) indicated that 1 contained a tyramine residue. Thus, the remaining portion of 1 consisted of C 7 H 5 O 4 . Due to the symmetry of this unassigned unit, as inferred by the NMR data, this moiety could only be a phloroglucinol carboxylic acid or gallic acid derivative. HMBC correlations from the remaining aromatic methine at δ H 7.08 (H-3′, H-7′) to two oxygenated carbons at δ C 145.7 (C- 4′, C-6′) and δ C 139.2 (C-5′) and a strong 3 J CH correlation to a carbonyl at δ C 164.6 (C-1′) confirmed the presence of a gallic acid moiety in 1. Two- and three-bond HMBC correlations from the phenolic hydroxy resonances at δ H 9.38 (s, 2H) to carbons at δ C 109.1 (C-3′, C-7′), 145.7 (C-4′, C-6′), and 139.2 (C-5′) provided further evidence of the gallic acid structure. Although no ROESY or HMBC correlations were observed between the gallic acid and tyramine substructures of 1, these systems by default were linked via an ester group. A strong absorption in the IR spectrum at ν max 1710 cm -1 confirmed this linkage and allowed the structure of 1 to be assigned to galloyl tyramine. The known flavonoid glycoside quercitrin (2) was identified by comparison of NMR and optical rotation data with literature values. 6 Galloyl tyramine and quercitrin were tested for inhibition activity against Pim2. Both compounds 1 and 2 proved to be weak inhibitors of the kinase with IC 50 values of 161 and 25 μM, respectively. Interestingly, several non-glycosylated flavonoids related to 2 such as quercetagetin, gossypetin, and myricetin have been shown recently to inhibit Pim1 activity. 1 Quercetagetin proved to be a highly selective inhibitor of Pim1 compared to Pim2 and seven other serine-threonine kinases. 1 Plant metabolites containing galloyl derivatives are relatively common and have been shown to display a number of different biological activities, although no Pim activity has been reported. 7 ⊥ Dedicated to Dr. G. Robert Pettit of Arizona State University for his pioneering work on bioactive natural products. * 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. ‡ Biodiversity Research Ltd. § AstraZeneca R & D Boston. J. Nat. Prod. 2008, 71, 451–452 451 10.1021/np070431w CCC: $40.75  2008 American Chemical Society and American Society of Pharmacognosy Published on Web 12/29/2007