Hirseins A and B, Daphnane Diterpenoids from Thymelaea hirsuta That Inhibit Melanogenesis in B16 Melanoma Cells Yusaku Miyamae, † Myra Orlina Villareal, †,‡ Manef Ben Abdrabbah, § Hiroko Isoda, †,‡ and Hideyuki Shigemori* ,† Graduate School of Life and EnVironmental Sciences, UniVersity of Tsukuba, Tsukuba 305-8572, Japan, Alliance for Research on North Africa, UniVersity of Tsukuba, Tsukuba 305-8572, Japan, and Unite ´ de Recherche: “Physico-chimie et Mole ´culaire”, Institut Pre ´paratoire aux Etudes Scientifiques et Techniques, La Marsa, Tunis, 2070 Tunisia ReceiVed January 7, 2009 Two new daphnane diterpenoids, hirseins A (1) and B (2), were isolated from the aerial parts of Thymelaea hirsuta, and their structures were elucidated on the basis of spectroscopic data interpretation. Hirsein B (2) is an unusual daphnane in possessing a coumaroyl moiety. NOESY correlations of 2 implied that isomerization of the coumaroyl group in 2 was caused by equilibrium between the E (2e) and Z (2z) forms. Compounds 1 and 2 were found to inhibit melanogenesis in B16 murine melanoma cells. During research on bioactive compounds extracted from Tunisian medicinal plants, we have focused on Thymelaea hirsuta, which belongs to the family Thymelaeaceae and is native to North Africa. In Tunisia, this plant has been used traditionally as an antiseptic and anti-inflammatory agent and for the treatment of hypertension by external application. It was previously reported that five daphnane diterpenoids were isolated from T. hirsuta, 1 and extracts of this species from Algeria showed antioxidant activity. 2 However, the chemical constituents of this plant and their bioactivity have not been investigated in recent years. It was found that an extract of T. hirsuta decreased the synthesized melanin content in B16 murine melanoma cells without cytotoxicity. 3 The bioassay-guided fractionation of this plant implied that the major bioactive com- ponents are daphnane diterpenoids. Daphnane diterpenoids have been found in plants of the families Thymelaeaceae and Euphor- biaceae. 4 These compounds are known to have various bioactivities, such as antileukemic and neurotrophic effects. 4-6 In this paper, we report on the structures of two new daphnane diterpenoids, hirseins A (1) and B (2), from T. hirsuta, and their antimelano- genesis activities. The aerial parts of T. hirsuta (500 g) were extracted with MeOH. The MeOH extracts were partitioned between EtOAc and H 2 O. The EtOAc-soluble portion (9.9 g) was subjected to silica gel column chromatography, separation through a C 18 Sep-Pak cartridge, and reversed-phase HPLC to yield two new daphnane diterpenoids, hirseins A (1, 0.00044%) and B (2, 0.00032%). Hirsein A (1), [R] 25 D +21 (c 1.0, MeOH), showed a pseudomo- lecular ion peak at m/z 671 (M + Na) + in the ESIMS. The molecular formula of 1 was deduced as C 37 H 44 O 10 from HRESIMS [m/z 671.2810 (M + Na) + , Δ -2.2 mmu]. The IR spectrum indicated the presence of hydroxyl (3421 cm -1 ), ester carbonyl (1700 cm -1 ), and unsaturated carbonyl (1654 and 1637 cm -1 ) groups, whereas the UV absorption at 236 nm implied that 1 possesses an R,- unsaturated ketone. The gross structure of 1 was deduced by detailed analysis of the 1 H and 13 C NMR data (Table 1) aided by 2D NMR experiments ( 1 H- 1 H COSY, HMQC, and HMBC). The 13 C NMR data indicated that the molecule possesses one unsaturated carbonyl carbon, one ester carbonyl carbon, eight disubstituted olefin carbons, six aromatic carbons, one orthoester carbon, four oxymethines, one oxymethylene, three methines, four methylenes, four methyl groups, and four oxygenated quaternary carbons (Table 1). The HMBC correlations of H-19 to C-1, C-2, and C-3 revealed that a methyl group is attached at C-2. The 1 H- 1 H COSY connectivities of H-1 and H-10 and HMBC correlations of H-1 to C-3 and C-4, and H-19 to C-1, C-2, and C-3, indicated the presence of a cyclopentenone moiety (ring A). The 1 H- 1 H COSY connectivities of H-7 and H-8 and the HMBC correlations of H-5 to C-4, C-6, and C-7, H-7 to * To whom correspondence should be addressed. Tel & Fax: 81-29- 853-4603. E-mail: hshige@agbi.tsukuba.ac.jp. † Graduate School of Life and Environmental Sciences, University of Tsukuba. ‡ Alliance for Research on North Africa, University of Tsukuba. § Unite ´ de Recherche: “Physico-chimie et Mole ´culaire”, Institut Pre ´pa- ratoire aux Etudes Scientifiques et Techniques. Table 1. 1 H and 13 C NMR Data (500 MHz, CDCl 3 ) of Hirsein A(1) position δ C δ H , mult. (J in Hz) HMBC a 1 160.4, CH 7.59, d (2.3) C-3, 4, 10 2 136.9, C 3 209.5, C 4 72.2, C 5 72.1, CH 4.26, s C-4, 6, 7 6 60.5, C 7 64.4, CH 3.63, s C-6, 8 8 35.5, CH 3.66, brs C-7, 9, 14 9 78.3, C 10 47.5, CH 3.92, d (2.7) 11 44.2, CH 2.50, q (7.3) C-12, 13, 18 12 78.6, CH 5.10, s C-9, 13, 14, 18, 1′′ 13 84.2, C 14 80.8, CH 4.91, d (2.6) C-9, 1′ 15 142.9, C 16 113.7, CH 2 5.02, s C-13, 17 17 18.8, CH 3 1.87, s C-13, 15, 16 18 18.2, CH 3 1.38, d (7.3) C-9, 12 19 9.84, CH 3 1.78, d (1.4) C-1, 2, 3 20a 65.1, CH 2 3.96, d (12.4) C-7 20b 65.1, CH 2 3.80, d (12.4) C-6 1′ 117.8, C 2′ 128.1, CH 6.16, m C-1′ 3′ 145.9, CH 6.16, m C-2′,4′ 4′ 33.0, CH 2.15, m C-2′,3′ 5′ 31.3, CH 1.43, m C-4′ 6′ 28.3, CH 2 1.29, m C-7′ 7′ 22.4, CH 2 1.29, m C-6′ 8′ 13.9, CH 2 0.89, m C-6′,7′ 1′′ 166.0, C 2′′ 118.1, CH 5.74, d (15.2) C-1′′,3′′,5′′,9′′ 3′′ 146.3, CH 7.20, m b 4′′ 135.3, C 5′′ 128.0, CH 7.71, m C-7′′ 6′′ 128.6, CH 7.38, m C-4′′ 7′′ 129.6, CH 7.38, m 8′′ 126.0, CH 7.38, m C-4′′ 9′′ 128.0, CH 7.71, m C-7′′ a HMBC correlations are from proton(s) stated to the indicated carbon. b The signal of H-3′′ was overlapped with that of CHCl 3 . J. Nat. Prod. 2009, 72, 938–941 938 10.1021/np800808h CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy Published on Web 03/13/2009