New Phytoecdysteroid from the Needles of the Japanese Yew , Taxus cuspidata

Qing-Wen Shi,* Mei Dong, Chang-Hong Huo, Xiao-Hui Su, Xing Li, Teiko Yamada and Hiromasa Kiyota* Department of Natural Product Chemistry, School of Pharmaceutical Sciences, Hebei Medical University, 361 Zhongshan East Road, 050017, Shijiazhuang, Hebei Province, P. R. China Laboratory of Applied Bioorganic Chemistry, Division of Biocience & Biotechnology for Future Bioindustry, Graduate School of Agricultural Sciences, Tohoku University, 1-1 Tsutsumidori-Amamiya, Aoba-ku, Sendai 981-8555, Japan


Introduction
Phytoecdysteroids are steroidal molecules firstly recognized as hormones controlling mounting and metamorphosis in insects.Today it is realized that these steroids are present at all stages of insect development, regulating many biochemical and physiological processes. 1Ecdysteroids and their analogs are also of great interest for medicinal application. 2To date, more than 200 phytoecdysteroids were identified, 3,4 however, only four of them were isolated from the genus Taxus (yew trees). 5The Japanese yew, Taxus cuspidata (Taxaceae), is a low trailing bush or tree very popular in Hokkaido and Tohoku regions, widely used as ornamental plant in Japan.Previous phytochemical studies on this species let to the isolation of taxanes with various skeleton, and other classes of compounds. 5,6Here we report on the isolation and identification of a new phytoecdysteroids, 7,8β-dihydroponasterone A (2), as well as the known ponasterone A (1) from the needles of T. cuspidata.

Results and Discussion
A methanolic extract of the needles of Taxus cuspidata was processed as described in the experimental section to afford compounds 1 and 2 (Figure 1).Combined analysis of NMR 7 and FAB-MS data of compound 1, obtained as a white amorphous solid, established its structure as ponasterone A, which has been previously isolated from the bark of T. brevifolia, 8 T. cuspidata, 9 and T. yunnanensis. 10ompound 2 was obtained as a white amorphous solid with a molecular formula C 27 H 46 O 6 calculated from the peaks at m/z 489.3193 (M+Na) + and 505.2921 (M+K) + in the HR-FABMS.The spectroscopic data of 2 (Table 1) closely resembles those of 1, with the exceptions that in compound 2 the olefinic carbons and the olefinic proton were absent, the ketone resonance was shifted from δ C 206.4 to 215.1 ppm, and its UV spectrum lacked the characteristic absorption for the α,β-unsaturated ketone at 242 nm. 11,12ased on these features it was concluded that the structure of 2 corresponded to the 7,8-dihydro derivative of 1.
The relative stereochemistry of 2 was deduced from NOESY data and 1 H-1 H coupling constants of vicinal protons (Figure 2).cis-Fused chair conformation of the AB rings was elucidated from the coupling constants (J 1ax,2 12.3 Hz, and J 3,2 = J 3,4ax = J 3,4eq = 2.9 Hz), and the NOESY correlations of H-2 to H-4ax, H-2 to H-9, H-5 to H-19, and H-4ax to H-7ax.The hydroxy groups at C-2 and C-3 were to be β-oriented, judging from the small coupling constant between H-2 and H-3.The NOE correlations between H-7ax to H-9, H-8 to H 3 -18, H-8 to H 3 -19, and H-9 to H-12ax demonstrated the BC ring was trans-fused.trans-Fusion of the CD-rings was deduced from the similarity of the 1 H NMR chemical shifts and coupling constant values to 1 and the NOESY correlation of H-15β to H 3 -18.The β-orientation of the side chain C-20-C-27 was deduced from the NOE between H-12ax to H-17.C-20-OH and C-22-OH were assigned to be βand α-oriented, respectively, by comparing with the analogous compound. 13Consequently, the compound 2 was determined to be 7,8βdihydroponasterone.The corresponding 8α-diastereomer of 2 was previously synthesized by stereoselective catalytic hydrogenation of 1. 14 Compound 2 was formerly reported as a hypothetical CoMFA model compound to investigate QSAR of ecdysteroids. 15Phytoecdysteroids appeared to have agonistic activity at the insectsteroid receptor complex, 15,16 and participate in the defense of plant against nonadapted phytophagous invertebrates. 1,17The fact that yew trees exhibited a very strong resistance to insect pests was considered to be caused by taxanes such as 10deacetylbaccatin III and 10-deacetylbaccatin V. 18 The presence of 1 and 2 in the needles of the Japanese yew would also be responsible for insecticidal activity. 19

Plant material
The needles of Taxus cuspidata were collected in the autumn of 2000 at Tsutsuzigaoka park, Sendai city in the north-east of Japan.The botanical identification was made by Professor T. Oritani of Toyama Prefectural University, Toyama, Japan.Several voucher specimens have been deposited in the Laboratory of Applied Bioorganic

H 11e
Chemistry, Graduate School of Agricultural Sciences, Tohoku University, Japan.