Hirtellanines A and B, a pair of isomeric isoflavonoid derivatives from Campylotropis hirtella and their immunosuppressive activities Qing Yao Shou a , Qing Tan a , Zheng Wu Shen a,b, * a School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, People’s Republic of China b Basilea Pharmaceutical China Ltd, Haimen, Jiangsu 226100, People’s Republic of China article info Article history: Received 27 February 2009 Revised 22 April 2009 Accepted 12 May 2009 Available online 18 May 2009 Keywords: Campylotropis hirtella (Franch.) Schindl. Hirtellanine A Hirtellanine B Immunosuppressive activity abstract A pair of isomeric isoflavonoid derivatives, Hirtellanines A (1) and B (2), has been isolated from the roots of Campylotropis hirtella (Franch.) Schindl. and their structures were elucidated on the basis of spectro- scopic methods, with special emphasis on 1D and 2D NMR techniques. The in vitro assay showed that Hirtellanine A had strong B lymphocyte suppression activity (IC 50 : 0.06 lM) and T lymphocyte suppres- sion activity (IC 50 : 0.92 lM). Hirtellanine B showed moderate B lymphocyte suppression activity (IC 50 : 3.00 lM) and T lymphocyte suppression activity (IC 50 : 9.55 lM). Due to the potent immunosuppressive activities and lower cytotoxicity, Hirtellanine A could be a promising lead towards novel immunosup- pressive agents. Ó 2009 Elsevier Ltd. All rights reserved. Campylotropis hirtella (Franch.) Schindl., which belongs to the plant family Leguminosaea, traditionally was used in Chinese folk- lore as a herb for activating blood circulation to treat diseases such as irregular menstruation, dysmenorrhea, metrorrhagia, metro- staxis as well as gastric ulcer. 1 It was reported that the Chinese herbs for activating blood circulation usually possessed immuno- suppressive activities. 2,3 Thus, C. hirtella was selected to initiate our studies for promising immunosuppressants. So far, tannins, steroids, triterpenes, flavonoids, coumarins, lign- ans, C-glycosylfavones and aromatic glycosides have been isolated from this plant. 4–8 Our efforts on identifying novel natural prod- ucts from the roots of C. hirtella led to the isolation of a pair of iso- meric isoflavonoid derivatives Hirtellanine A (1) and B (2). This letter describes the structure elucidation of 1 and 2 on the basis of spectroscopic methods, with special emphasis on 1D and 2D NMR techniques. Their immunosuppressive activities on mito- gen-induced splenocyte proliferations as well as their cytotoxicity on splenic lymphocytes were also described. The EtOAc extract of the dried roots (2 kg) of C. hirtella was puri- fied by repeated column chromatography over silica gel eluted with petroleum ether/acetone, petroleum ether/ethyl acetate and Pharmadex LH-20 eluted with methanol to afford Hirtellanines A (1) and B (2)(Fig. 1). 9 Hirtellanine A (1) 10 was obtained as a white amorphous powder and had an empirical formula, C 21 H 16 O 7 (calcd 380.0896), which was supported by HR-EIMS ([M] + m/z 380.0890). The UV spectro- scopic absorption at 270 nm and IR absorption at 1634 cm À1 sug- gested an isoflavone structure for 1. Moreover the absence of a characteristic proton due to H-2 (usually appearing at d ca 8) in the 1 H NMR spectrum was indicative of a coumaronochromone skeleton. 11 Three aromatic protons, each as singlet, were observed at d H 7.35, d H 7.05, and d H 6.91 in the 1 H NMR spectrum. One sin- glet (3H) was observed at d H 3.81, thus evidencing a methoxyl group. Two one-proton doublets (J = 10.0 Hz) at d H 6.66 and d H 5.91, and two methyl groups at d H 1.43 as a singlet indicated the presence of a dimethylchromene ring. 12 In addition, the 13 C NMR and DEPT experiments of 1 showed 21 carbon resonances containing three methyl groups (d C 28.4, 28.4, and 63.2), five methines (d C 99.5, 101.6, 106.5, 115.6, and 132.2), and 13 quaternary carbons (d C 78.5, 112.3, 113.7, 114.1, 114.1, 142.8, 144.5, 144.9, 155.5, 155.9, 157.3, 163.0, 172.3). The spectro- scopic data supported the presence of a coumaronochromone skel- eton with a dimethylchromene ring moiety. In the HMBC experiments (Fig. 2), the proton signal at d H 6.91 showed correla- 0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2009.05.043 * Corresponding author. Tel.: +86 513 82198001; fax: +86 513 82198003. E-mail address: jeff_shen_1999@yahoo.com (Z.W. Shen). O OH OH O O O O OH O O O OH 1 2 OMe OMe 1 2 3 4 5 6 7 8 2' 4' 3' 6' 5' 4" 5" 6" A B D E C Figure 1. Structures of Hirtellanines A (1) and B (2). Bioorganic & Medicinal Chemistry Letters 19 (2009) 3389–3391 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl