8610009-3130/13/4905-0861 �2013 Springer Science+Business Media New York

Chemistry of Natural Compounds, Vol. 49, No. 5, November, 2013 [Russian original No. 5, September–October, 2013]

A NEW TAXANE WITH A 4�,20-EPOXY RING FROMTHE ROOTED CUTTINGS OF Taxus canadensis

Gen-Dong Yao,1 Hong-Feng Zhang,1 Yun Huang,2 UDC 547.913.6Francoise Sauriol,3 Qing-Wen Shi,2* Hiromasa Kiyota,4*

and Yu-Cheng Gu5

A new taxane, 2�,5�,10�-triacetoxy-4�,20-epoxytax-11-ene-1�,7�,9�,13�-tetraol (1), was isolated fromthe rooted cuttings of the Canadian yew, Taxus canadensis.

Keywords: Taxus canadensis, Taxaceae, taxanes, yew, rooted cuttings.

Taxol® (paclitaxel), first isolated from the bark of the Pacific yew, Taxus brevifolia Nutt., is one of the most promisinganticancer agents. Since its discovery, much effort has been made to investigate analogous compounds, and more than 500taxane diterpenoids have been reported to date [1–7]. Among natural yews, the Canadian yew, T. canadensis, is a rich sourceof natural taxanes with unique structural features [8]. In continuation of our phytochemical studies on the Canadian yew, anovel taxane peroxide has been isolated from the methanol extract of the rooted cuttings [9]. Further investigation on the sameextract resulted in the isolation of a new taxane 1 and taxumairol B (2) [10].

Compound 1 and the known taxumairol B (2) were isolated from the same extract of rooted cuttings of Taxus canadensis,previously reported by us [9]. The molecular formula of 1, C26H28O11, was deduced from HR-FAB-MS data. Combinedanalysis of 1H and 13C NMR data (Table 1) revealed that 1 was a typical 6/8/6-membered taxane including four hydroxy, threeacetoxy, one epoxy, and one tetrasubstituted double bond. The geminal methylene signals at �H 2.38, 3.58 (each 1H, d, J = 5.3 Hz),and �C 51.0 indicated the presence of an epoxy ring, whose location at the 4,20-position was elucidated from the HMBCcorrelations of these proton signals to C-4 (Table 1). The 13C NMR and the HMBC data also indicated the positions of fourhydroxy (C-1, C-7, C-9, and C-13) and three acetoxy (C-2, C-5, and C-10) groups. The relative stereochemistry was elucidatedfrom a NOESY experiment (Table 1). The correlation of H-13 with H-14� and H-17; H-2 with H-9, H-16, and H-19; and H-3 withH-7, H-10, and H-18 revealed the 1�-OH, 2�-H, 3�-H, 7�-H, 9�-H, 10�-H, and 13�-H configurations. The 5�-H configurationwas deduced from the NOE correlations of H-5 with H-6�, and H-6� with H-7. The configuration of the 4�,20-epoxy groupwas estimated by comparing the chemical shift values of H-5, H-20a, H-20b, C-4, and C-20 with taxumariol B (2). Thus, thestructure of 1 was determined to be 2�,5�,10�-triacetoxy-4�,20-epoxytax-11-ene-1�,7�,9�,13�-tetraol (1).

1) Handan Central Hospital, 056001, Handan, Hebei Province, P. R. China; 2) School of Pharmaceutical Sciences,Hebei Medical University, 050017, Shijiazhuang, Hebei Province, P. R. China, e-mail: shiqingwen@hebmu.edu.cn; 3) Departmentof Chemistry, Queen�s University, Kingston, Ontario, K7L 3N6, Canada; 4) Laboratory of Applied Bioorganic Chemistry,Graduate School of Agricultural Science, Tohoku University, 981-8555, Aoba-ku Sendai, Japan; 5) Jealott�s Hill InternationalResearch Centre, Syngenta, Berkshire RG42 6EY, UK. Published in Khimiya Prirodnykh Soedinenii, No. 5, September–October,2013, pp. 741–742. Original article submitted August 6, 2012.

1: R = H2: R = Ac

OH

OOAc

OH

RO

H

AcO

HO OAc

1 3 57

911

18

13

20

1716

19

15

1, 2

862

EXPERIMENTAL

General. Optical rotation: JASCO DIP-370. HR-FAB-MS: JMS-Dx 305 HF. Preparative TLC: (0.25 mm, Kieselgel60 GF254). Preparative HPLC: Waters Delta Prep 3000, UV 2487 (220 nm), Whatman Partisil 10 ODS-2 Mag-9 (9.4 � 250 mm),50 min linear gradient of CH3CN in water (25% to 100% at 18 mL/min).

NMR Methodology. Most NMR spectra were obtained at room temperature (298K) on a Bruker AV-500 spectrometeroperating at 499.40 MHz for 1H and 125.57 MHz for 13C on a BBFO probe. For 1H NMR, 16 transients were recorded witha 20 ppm spectral window, 45� pulse and 1 s relaxation delay, 32 k data points and 3.17 s acquisition time. For processing1D 1H NMR, zero filling and a Traficant function was used to improve resolution. The magnitude g-COSY experiment (gradientenhanced) was run using 1 transient with 2K � 256 data points and a recycling delay of 1.8 s. The data were processed with aphase-shifted sine function. The gradient-enhanced (TPPI phase mode) NOESY experiment was performed using a mixingtime of 0.4–1.0 s and a relaxation delay of 2 s. The acquisition was repeated for 2 transients, and 2k � 256 increments wereacquired. The data were processed using the Qsine (squared sine) function. The gradient-enhanced HSQC experiment usedecho-antiecho pulses (shaped pulses were used as refocusing pulses). The recycling delay was set to 1 s. The acquisition wasrepeated 2–64 times and 2k by 256–512 fids were acquired. During acquisition of the proton spectrum, 13C broadband GARPdecoupling was applied. The data were processed with a Qsine function and with zero filling in the evolution domain (13C). Inthe HMBC experiment (gradient enhanced, no decoupling) the delay used to emphasize long-range coupling was set to

TABLE 1. 1H (500 MHz) and 13C NMR (125 MHz) Data for 1 (methanol-d4, �, ppm, J/Hz)

C atom �H (mult) �C* HMBC NOESY**

1 2 3 4 5

6�6�7 8 9

10 11 12 13

14� 14�15 16 17 18 19 20a 20b OAc

– 5.31 (d, J = 3.5)

3.12 (br.d, J = 3.5) –

4.15 (t, J = 2.9) 1.96 (m) 1.74 (m)

4.29 (dd, J = 11.6, 4.4) –

4.42 (d, J = 10.5) 6.17 (d, J = 10.5)

– –

4.80 (m) 2.38 (dd, J = 14.9, 9.9) 2.04 (dd, J = 14.9, 6.3)

– 1.47 (s) 1.10 (s)

2.19 (br.s) 1.31 (s)

3.58 (d, J = 5.3) 2.38 (d, J = 5.3)

2.14 (s)

2.09(s)

2.00 (s)

75.3 73.5 41.4 58.7 78.4 33.9

71.0 46.5 79.3 75.3

134.8 144.6 69.0 42.4

43.2 21.1 27.7 16.2 13.8 51.0

21.0

170.1 21.0

170.9 20.8

169.9

8, 170.1

4

7, 8, 10 9, 11, 12, 15, 170.9

1, 11, 15, 17 1, 11, 15, 16 13, 11, 12 3, 7, 8, 9

4 4

170.1

170.9

169.9

9s, 16s, 19m 7s, 10w, 18s

6�m, 20bm

5m 7s

3s, 6�s, 10s, 18m

2s, 16s, 19m 3w, 7s, 18s

14� s , 17s, 18w 13s

2s, 9s, 17s 13s, 16s

3s, 7m, 10s, 13w 2m, 9m 20bs

5m, 20as

______*The 13C chemical shifts were extracted from the HMQC experiment (� 0.2 ppm). The numbers in bold character representquaternary carbons whose chemical shifts were obtained from the HMBC experiment (� 0.2 ppm). **NOESY intensities aremarked as strong (s), medium (m), or weak (w).

863

0.075 ms for aromatic compounds and 0.1 ms for aliphatic compounds. The number of scans was usually 4–64 with 2k � 256increments in the evolution period.

Isolation Process. Source of the plant material and part of the isolation process were described in the previous report[9]. The combined fraction FrD-18-13 to FrD-18-15 (120 mg) was subjected to a preparative HPLC, and the material eluted attR = 23.3 min was further purified by preparative TLC (hexane–acetone 40:50) to yield 1 (1.8 mg, Rf 0.37) as an amorphoussolid, [�]22

D +33� (c 0.050, CHCl3). HR-FAB-MS m/z 527.2496 [M + H]+, calcd 527.2492. Similarly, purification of thecombined fraction FrD-18-11 to FrD-18-12 (210 mg) by preparative HPLC (tR = 28.2 min) and preparative TLC (hexane–acetone50:55) yielded taxumairol B (2, 2.0 mg, Rf 0.34) as an amorphous solid, [�]20

D +59� (c 0.050, CHCl3) {[�]25D +15� (c 0.6,

MeOH) [10]}.

ACKNOWLEDGMENT

The authors gratefully acknowledge financial support from National Natural Science Foundation of China (81072551,81241101), Key Projects of Science & Technology of Hebei Province (11276103D-89), and Scientific Research Foundationof Hebei Province (C2010000489). We also wish to extend our sincere thanks for the financial support from Syngenta Ltd.(2011-Hebei Medical University-Syngenta-03) and Japan Society for the Promotion of Science (Nos. 19580120 and 22580112).

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