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A new taxol analogue from the leaves of Taxus cuspidata
Biochemical Systematics and Ecology 35 (2007) 704e708 www.elsevier.com/locate/biochemsyseco
A new taxol analogue from the leaves of Taxus cuspidata Changhong Huo a, Xiping Zhang a, Cunfang Li a, Yufang Wang a, Qingwen Shi a,*, Hiromasa Kiyota b a
Department of Medicinal Natural Product Chemistry, School of Pharmaceutical Sciences, Hebei Medical University, 361 Zhongshan East Road, 050017 Shijiazhuang, Hebei Province, PR China b Laboratory of Applied Bioorganic Chemistry, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan Received 10 December 2006; accepted 4 March 2007
Keywords: Taxus cuspidata; Taxaceae; Taxane; 7-O-b-xylosyl-10-deacetyltaxuspinanane A; Chemotaxonomy
1. Subject and source The leaves of Taxus cuspidata Siebold et Zucc. were collected in the autumn of 1997 in Toyama Prefecture, Japan, and identified by Prof. T. Oritani of Toyama Prefectural University, Toyama, Japan. A voucher specimen (No. NMC1997-1) has been deposited in the Laboratory of Applied Bioorganic Chemistry, Graduate School of Agricultural Sciences, Tohoku University, Japan. 2. Previous work Previous studies on T. cuspidata have led to the identification of more than 120 taxanes with a variety of skeletons (Shigemori and Kobayashi, 2004; Shi and Kiyota, 2005; Cao et al., 2006a,b,c). The presence of a sesquiterpenoid, steroids, lignans, flavonoids and other compounds such as (R)-taxiphylline, 3,5-dimethoxyphenol, 4-hydroxyphenylacetic acid, 2-hydroxyphenylacetic acid, hexadecanoic acid, linoleic acid and 5E,9E-octadecadienoic acid has also been reported in this plant (Cao et al., 2006c; Parmar et al., 1999). 3. Present study In our further investigation on the leaves of T. cuspidata, we report herein the isolation and characterization of a new compound, namely, 7-O-b-xylosyl-10-deacetyltaxuspinanane A 4, together with two 7-O-b-xylosyl-taxol analogues, such as 7-O-b-xylosyl-10-deacetyltaxol 2, 7-O-b-xylosyl-10-deacetyltaxol C 3 (Se´nilh et al., 1984), as well as taxol 1 (Appendino, 1995) from the leaves of T. cuspidata. * Corresponding author. Tel.: þ86 311 86265634. E-mail address: [email protected] (Q. Shi). 0305-1978/$ - see front matter Ó 2007 Published by Elsevier Ltd. doi:10.1016/j.bse.2007.03.005
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Air dried leaves of T. cuspidata (2.2 kg) were immersed in MeOH for one week, after filtering, fresh solvent was added and extracted for another week. After concentration in vacuo of the MeOH extract, part (21 g) of the residue was subjected to silica gel column chromatography eluting with acetoneehexane system (30e80%). Forty 500 mL fractions were obtained and pooled on the basis of TLC monitoring, to give 10 major fractions, designated FrAeJ. Column chromatography of combined fractions H and I, using hexaneeEtOAc (2:3e1:5) as mobile phase, afforded 15 subfractions, FrH-1 to FrH-15. Subfraction FrH-5 was subjected to semi-preparative HPLC (Whatman Partisil 10 ODS-2 Mag-9 semi-prep. Column, 9.4 250 mm, eluting solvent: a linear gradient of acetonitrile in water from 25% to
Table 1 1 H NMR spectral data of compounds 2e4 (d in ppm, 500 MHz, CDCl3) (o. ¼ overlapped) Position
2a
3 1
d ( H, mult, Hz) 1-OH 2 3 5 6a 6b 7 10 13 14a 14b 16 17 18 19 20a 20b OAc 2-OBz-o m p 20 30 30 -NH 30 -Ph-o m p 100 -Ph-o m p 200 a 200 b 300 a 300 b 400 500 a 500 b 600 700 1% 2% 3% 4% 5%a 5%b a
In acetone-d6.
3.78 5.65 3.93 4.93 2.68 1.88 4.20 5.35 6.18 2.34 2.15 1.16 1.13 1.89 1.45 4.15
(o.s) (d, 7.1) (d, 7.1) (dd, 9.7, 1.6) (ddd, 14.9, 9.7, 6.8) (m) (dd, 10.8, 6.8) (br.s) (br.t, 9.0) (br.dd, 15.4, 9.7) (dd, 15.4, 9.0) (s) (s) (br.s) (s) (o.m)
2.41 8.10 7.56 7.65 4.82 5.74 8.14 7.57 7.28 7.40 7.92 7.43 7.51
(s) (d, 8.5) (t, 7.5) (t, 7.5) (d, 4.9) (dd, 8.8, 4.9) (d, 8.5) (d, 8.0) (t, 7.5) (t, 7.3) (d, 7.5) (o.t) (t, 7.5)
4 1
d ( H, mult, Hz)
d (1H, mult, Hz)
5.65 3.89 4.89 2.73 2.02 4.06 5.17 6.18 2.27
(d, 7.2) (d, 7.2) (d, 9.9) (m) (m) (o.m) (s) (br.t, 9.0) (d, 9.3)
5.65 3.89 4.88 2.73 2.02 4.06 5.17 6.18 2.27
(d, 7.2) (d, 6.6) (d, 8.9) (dt, 15.9, 7.8) (m) (m) (br.s) (br.t, 9.4) (br.d, 9.7)
1.23 1.10 1.83 1.81 4.29 4.19 2.35 8.09 7.50 7.61 4.66 5.57 6.24 7.39 7.34 7.39
(s) (s) (br.s) (s) (d, 9.0) (d, 9.0) (s) (d, 8.1) (t, 7.7) (t, 7.2) (br.s) (dd, 9.5, 1.8) (d, 9.2) (m) (m) (m)
1.22 1.10 1.84 1.80 4.29 4.18 2.35 8.09 7.49 7.60 4.67 5.56 6.26 7.38 7.33 7.38
(s) (s) (s) (s) (d, 8.7) (d, 8.7) (s) (d, 7.4) (t, 7.7) (t, 7.3) (d, 2.3) (dd, 8.7, 2.3) (br.d, 8.5) (m) (m) (m)
2.23 2.18 1.62 1.37 1.29 1.27 1.09 0.80 0.81 4.06 3.23 3.48 3.67 3.95 3.25
(m) (o.m) (m) (m) (m) (m) (m) (o.t) (o.d) (o.d) (o) (br.t, 8.2) (br.m) (dd, 11.5, 4.7) (dd, 11.5, 9.7)
2.20 (t, 7.7) 1.55 (o.m) 1.25 (o.m) 1.25 (o.m) 0.83 (br.t, 8.0) 4.18 3.03 3.28 3.42 3.78 3.21
(d, 7.2) (br.t, 8.2) (br.t, 8.3) (m) (o.m) (dd, 11.5, 9.9)
4.06 3.24 3.48 3.68 3.96 3.25
(o.m) (o.m) (t, 8.0) (m) (dd, 11.7, 4.8) (dd, 11.7, 9.8)
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100% in 50 min at a flow rate of 3 mL/min) to yield taxane 1 (2.0 mg, tR ¼ 33.27 min). Subfractions FrH-11 to FrH-13 were pooled (195 mg) and were re-chromatographed over silica gel (20 g) eluting with hexaneeacetone (2:3 and 1:3) to yield five subfractions (FrH-11-1 to FrH-11-5). The fraction FrH-11-4 (19 mg) was further chromatographed using the same semi-preparative HPLC system, eluted with a linear gradient of acetonitrile in water from 25% to 100% in
Table 2 13 C NMR spectral data of compounds 2e4 (d 125 MHz, CDCl3) Carbons
2a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OAc
77.6 74.7 46.6 80.1 83.8 35.6 81.4 56.3 209.7 74.6 136.0 137.3 70.9 36.0 42.9 26.1 20.3 13.6 10.1 75.7 22.1 170.1
2-OBz C-1 o m p 10 20 30 30 -Ph o m p 100 CO 10 -Ph o m p 200 300 400 500 600 700 1% 2% 3% 4% 5% a
In acetone-d6.
165.6 129.9 128.5 133.0 172.7 73.8 56.0 139.2 127.2 127.2 128.3 166.5 139.0 127.1 127.2 130.0
3 78.6 74.5 46.2 80.0 83.8 81.5 57.0 211.7 74.7 135.7 138.1 72.3 35.8 42.8 26.4 20.7 14.3 10.8 76.4 22.5 170.5
130.0 128.6 133.5 73.1 54.4 128.8 126.7 126.7 128.1
78.8 74.4 46.2 81.1 83.9 35.5 81.3 57.2 211.5 74.6 136.0 138.0 72.2 35.7 43.1 26.3 20.5 14.2 10.5 76.5 22.4 170.5 167.0 129.9 128.5 133.6 172.1 73.0 54.3
173.0
36.6 25.3 31.3 22.3 13.7 105.1 73.3 76.2 69.4 65.4
4
105.2 72.9 75.3 64.9
34.3 32.1 33.8 28.9 10.9 18.7 105.0 72.8 75.2 69.1 65.0
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50 min at a flow rate of 3 mL/min and finally provided taxanes 2 (3.5 mg, tR ¼ 25.65 min), 3 (4.5 mg, tR ¼ 27.29 min) and 4 (2.0 mg, tR ¼ 27.93 min), respectively. Compound 4, white amorphous powder, exhibited an HRFAB-MS spectral quasimolecular ion peak at m/z 990.3890 [M þ K]þ, corresponding to the molecular formula of C50H65NO17. Complete assignments of 1H and 13 C NMR signals were achieved (Tables 1 and 2) with the help of various NMR techniques such as 1He1H COSY, HMQC for direct HeC connectivities and HMBC for long-range HeC correlations. The 1H NMR spectrum of 4 showed the characteristic signals of four tertiary methyl groups at d 1.10, 1.22, 1.84 and 1.80 (each 3H, s), one acetyl group at d 2.35 (3H, s), which was further supported by carbon signals observed at d 22.4 and 170.5 in the 13C NMR spectrum, one benzoyl group at d 7.49 (2H, t, 7.7 Hz), 7.60 (1H, t, 7.3 Hz), 8.09 (2H, d, 7.4 Hz) and one phenyl group at d 7.33 (1H, m), 7.38 (4H, m) as well as one oxetane ring at d 4.18 and 4.29 mutually coupled with a coupling constant of 8.7 Hz. In addition, the 1H NMR spectrum of 4 displayed the featured signals of taxol such as H-2 at d 5.65 (1H, d, 7.2 Hz), H-3 at d 3.89 (1H, d, 6.6 Hz), H-5 at d 4.88 (1H, d, 8.9 Hz), H-7 at d 4.06 (1H, m), H-10 at d 5.17 (1H, br.s) and H-13 at d 6.18 (1H, br.t, 9.4 Hz) (Appendino, 1995). A xylosyl group was suggested by the signals of d 4.06 (1H, overlapped d, H-1%), 3.23 (1H, overlapped, H-2%), 3.48 (1H, br.t, 8.2 Hz, H-3%), 3.67 (1H, br.m, H-4%), 3.95 (1H, dd, 11.5, 4.7 Hz, H-5%a), 3.25 (1H, dd, 11.5, 9.7 Hz, H-5%b) (Se´nilh et al., 1984). The presence of a side chain similar to the C-13 side chain of taxol was suggested by the signals at d 4.67 (1H, d, 2.3 Hz, H-20 ), 5.56 (1H, dd, 8.7, 2.3 Hz, H-30 ), 6.26 (1H, br.d, 8.5 Hz, 30 -NH), 7.33e7.38 (5H, m, 30 -Ph). However, aliphatic signals were observed in
O NH 3'
AcO
18
O 19 OH
10
O
16
2'
7
O
1'
13 15
4
OH H
2
HO
OBz
O
OAc
20
1 Paclitaxel (Taxol ) ®
O 1'' NH 3'
HO
18
16
2' 1'
O 19 O
10
O
R1
O
13 15
4
OH H
2
HO
OAc
OBz
OH
R1 =
7
OH O 20
2
5''
3''
O 1'' NH
H3C R2
3'
18
HO 16
2' 1'
O 19 O
10
O O
7 13 15
4
OH HO
3 R2 = H
R1
H
2
4 R2 = CH3
Fig. 1. Structures of compounds 1e4.
OBz
OAc
O 20
O
OH
708
C. Huo et al. / Biochemical Systematics and Ecology 35 (2007) 704e708
place of signals for the N-benzoyl signals of taxol. The coupling connectivities analyzed by the 1He1H COSY experiment indicated that the N-acyl group was a 4-methylhexanoyl group. These results were in good agreement with the relative molecular mass of 4. The relative configurations at H-20 and H-30 were concluded to be 20 R, 30 S by the 1H vicinal coupling constants compared with taxol (J20 ,30 ¼ 2.3 Hz, J30 ,40 ¼ 8.7 Hz) (Morita et al., 1997). The relative configurations at C-2, C-7, C-10 and C-13 were established on the basis of chemical shifts, multiplet patterns and coupling constant values of corresponding protons as well as by comparison with data for taxol and its analogues. Taking all these spectral data into account, the structure of 4 was elucidated unequivocally as 7-O-b-xylosyl-10-deacetyltaxuspinanane A (Fig. 1). 7-O-b-Xylosyl-10-deacetyltaxuspinanane A (4): Amorphous powder; [a]24 D 37 (c 0.1, CHCl3); HRFAB-MS m/z: þ 1 13 990.3890 [M þ K] , calcd for C50H65NO17K 990.3884. For H and C NMR spectral data see Tables 1 and 2. 4. Chemotaxonomic significance Compound 4 is a new taxol analogue with a rare 4-methylhexanoyl functional group instead of the usual benzoyl group, while compounds 2 and 3 were isolated for the first time from T. cuspidata. All of the compounds 2e4 have the same skeleton as baccatin III and bear an O-linked xylosyl group at C-7. Although this kind of compound has been isolated previously from Taxus baccata (Se´nilh et al., 1984) and Taxus yunnanensis (Li et al., 2001, 2003), this type of taxane was isolated for the first time from T. cuspidata. It is of great interest to note that compound 4 obtained in the present study was characterized by a rare 4-methylhexanoyl functional group; this group also occurs in taxuspinanane A which was isolated previously from the stems of T. cuspidata (Morita et al., 1997). Such a difference may serve as a characteristic feature to distinguish T. cuspidata from other species of yew tree. Acknowledgements The work described in this paper was financially supported from the Foundation for Researching New Drugs of People’s Republic of China (No. 2003AA2Z3527) and Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry of P.R. China to QWS. We thank Dr. Takayuki Oritani, Emeritus Professor of Tohoku University, for providing crude extract for this work. References Appendino, G., 1995. In: Farina, V. (Ed.), The Chemistry and Pharmacology of Taxol and Its Derivatives, vol. 22. Elsevier Science, Amsterdam, pp. 1e53, pp. 55e101. Cao, C.M., Zhang, M.L., Wang, Y.F., Shi, Q.W., Yamada, T., Kiyota, H., 2006a. Chem. Biodivers. 3, 1153. Cao, C.M., Huo, C.H., Zhao, Y.M., Si, X.T., Li, Z.P., Shi, Q.W., 2006b. China J. Chin. Mater. Med. 31, 1510. Cao, C.M., Li, Z.P., Shi, Q.W., 2006c. Nat. Prod. Res. Dev. 18, 330. Li, S.H., Zhang, H.J., Yao, P., Sun, H.D., Fong, H.H.S., 2001. Phytochemistry 58, 369. Li, S.H., Zhang, H.J., Niu, X.M., Yao, P., Sun, H.D., Fong, H.H.S., 2003. Tetrahedron 59, 37. Morita, H., Gonda, A., Wei, L., Yamamura, Y., Takeya, K., Itokawa, H., 1997. J. Nat. Prod. 60, 390. Parmar, V.S., Jha, A., Bisht, K.S., Taneja, P., Singh, S.K., Kumar, A., Poonam Jain, R., Olsen, C.E., 1999. Phytochemistry 50, 1267. Se´nilh, V., Blechert, S., Colin, M., Gue´nard, D., Picot, F., Potier, P., Varenne, P., 1984. J. Nat. Prod. 47, 131. Shi, Q.W., Kiyota, H., 2005. Chem. Biodivers. 2, 1597. Shigemori, H., Kobayashi, J., 2004. J. Nat. Prod. 67, 245.
A new taxol analogue from the leaves of Taxus cuspidata Changhong Huo a, Xiping Zhang a, Cunfang Li a, Yufang Wang a, Qingwen Shi a,*, Hiromasa Kiyota b a
Department of Medicinal Natural Product Chemistry, School of Pharmaceutical Sciences, Hebei Medical University, 361 Zhongshan East Road, 050017 Shijiazhuang, Hebei Province, PR China b Laboratory of Applied Bioorganic Chemistry, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan Received 10 December 2006; accepted 4 March 2007
Keywords: Taxus cuspidata; Taxaceae; Taxane; 7-O-b-xylosyl-10-deacetyltaxuspinanane A; Chemotaxonomy
1. Subject and source The leaves of Taxus cuspidata Siebold et Zucc. were collected in the autumn of 1997 in Toyama Prefecture, Japan, and identified by Prof. T. Oritani of Toyama Prefectural University, Toyama, Japan. A voucher specimen (No. NMC1997-1) has been deposited in the Laboratory of Applied Bioorganic Chemistry, Graduate School of Agricultural Sciences, Tohoku University, Japan. 2. Previous work Previous studies on T. cuspidata have led to the identification of more than 120 taxanes with a variety of skeletons (Shigemori and Kobayashi, 2004; Shi and Kiyota, 2005; Cao et al., 2006a,b,c). The presence of a sesquiterpenoid, steroids, lignans, flavonoids and other compounds such as (R)-taxiphylline, 3,5-dimethoxyphenol, 4-hydroxyphenylacetic acid, 2-hydroxyphenylacetic acid, hexadecanoic acid, linoleic acid and 5E,9E-octadecadienoic acid has also been reported in this plant (Cao et al., 2006c; Parmar et al., 1999). 3. Present study In our further investigation on the leaves of T. cuspidata, we report herein the isolation and characterization of a new compound, namely, 7-O-b-xylosyl-10-deacetyltaxuspinanane A 4, together with two 7-O-b-xylosyl-taxol analogues, such as 7-O-b-xylosyl-10-deacetyltaxol 2, 7-O-b-xylosyl-10-deacetyltaxol C 3 (Se´nilh et al., 1984), as well as taxol 1 (Appendino, 1995) from the leaves of T. cuspidata. * Corresponding author. Tel.: þ86 311 86265634. E-mail address: [email protected] (Q. Shi). 0305-1978/$ - see front matter Ó 2007 Published by Elsevier Ltd. doi:10.1016/j.bse.2007.03.005
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Air dried leaves of T. cuspidata (2.2 kg) were immersed in MeOH for one week, after filtering, fresh solvent was added and extracted for another week. After concentration in vacuo of the MeOH extract, part (21 g) of the residue was subjected to silica gel column chromatography eluting with acetoneehexane system (30e80%). Forty 500 mL fractions were obtained and pooled on the basis of TLC monitoring, to give 10 major fractions, designated FrAeJ. Column chromatography of combined fractions H and I, using hexaneeEtOAc (2:3e1:5) as mobile phase, afforded 15 subfractions, FrH-1 to FrH-15. Subfraction FrH-5 was subjected to semi-preparative HPLC (Whatman Partisil 10 ODS-2 Mag-9 semi-prep. Column, 9.4 250 mm, eluting solvent: a linear gradient of acetonitrile in water from 25% to
Table 1 1 H NMR spectral data of compounds 2e4 (d in ppm, 500 MHz, CDCl3) (o. ¼ overlapped) Position
2a
3 1
d ( H, mult, Hz) 1-OH 2 3 5 6a 6b 7 10 13 14a 14b 16 17 18 19 20a 20b OAc 2-OBz-o m p 20 30 30 -NH 30 -Ph-o m p 100 -Ph-o m p 200 a 200 b 300 a 300 b 400 500 a 500 b 600 700 1% 2% 3% 4% 5%a 5%b a
In acetone-d6.
3.78 5.65 3.93 4.93 2.68 1.88 4.20 5.35 6.18 2.34 2.15 1.16 1.13 1.89 1.45 4.15
(o.s) (d, 7.1) (d, 7.1) (dd, 9.7, 1.6) (ddd, 14.9, 9.7, 6.8) (m) (dd, 10.8, 6.8) (br.s) (br.t, 9.0) (br.dd, 15.4, 9.7) (dd, 15.4, 9.0) (s) (s) (br.s) (s) (o.m)
2.41 8.10 7.56 7.65 4.82 5.74 8.14 7.57 7.28 7.40 7.92 7.43 7.51
(s) (d, 8.5) (t, 7.5) (t, 7.5) (d, 4.9) (dd, 8.8, 4.9) (d, 8.5) (d, 8.0) (t, 7.5) (t, 7.3) (d, 7.5) (o.t) (t, 7.5)
4 1
d ( H, mult, Hz)
d (1H, mult, Hz)
5.65 3.89 4.89 2.73 2.02 4.06 5.17 6.18 2.27
(d, 7.2) (d, 7.2) (d, 9.9) (m) (m) (o.m) (s) (br.t, 9.0) (d, 9.3)
5.65 3.89 4.88 2.73 2.02 4.06 5.17 6.18 2.27
(d, 7.2) (d, 6.6) (d, 8.9) (dt, 15.9, 7.8) (m) (m) (br.s) (br.t, 9.4) (br.d, 9.7)
1.23 1.10 1.83 1.81 4.29 4.19 2.35 8.09 7.50 7.61 4.66 5.57 6.24 7.39 7.34 7.39
(s) (s) (br.s) (s) (d, 9.0) (d, 9.0) (s) (d, 8.1) (t, 7.7) (t, 7.2) (br.s) (dd, 9.5, 1.8) (d, 9.2) (m) (m) (m)
1.22 1.10 1.84 1.80 4.29 4.18 2.35 8.09 7.49 7.60 4.67 5.56 6.26 7.38 7.33 7.38
(s) (s) (s) (s) (d, 8.7) (d, 8.7) (s) (d, 7.4) (t, 7.7) (t, 7.3) (d, 2.3) (dd, 8.7, 2.3) (br.d, 8.5) (m) (m) (m)
2.23 2.18 1.62 1.37 1.29 1.27 1.09 0.80 0.81 4.06 3.23 3.48 3.67 3.95 3.25
(m) (o.m) (m) (m) (m) (m) (m) (o.t) (o.d) (o.d) (o) (br.t, 8.2) (br.m) (dd, 11.5, 4.7) (dd, 11.5, 9.7)
2.20 (t, 7.7) 1.55 (o.m) 1.25 (o.m) 1.25 (o.m) 0.83 (br.t, 8.0) 4.18 3.03 3.28 3.42 3.78 3.21
(d, 7.2) (br.t, 8.2) (br.t, 8.3) (m) (o.m) (dd, 11.5, 9.9)
4.06 3.24 3.48 3.68 3.96 3.25
(o.m) (o.m) (t, 8.0) (m) (dd, 11.7, 4.8) (dd, 11.7, 9.8)
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100% in 50 min at a flow rate of 3 mL/min) to yield taxane 1 (2.0 mg, tR ¼ 33.27 min). Subfractions FrH-11 to FrH-13 were pooled (195 mg) and were re-chromatographed over silica gel (20 g) eluting with hexaneeacetone (2:3 and 1:3) to yield five subfractions (FrH-11-1 to FrH-11-5). The fraction FrH-11-4 (19 mg) was further chromatographed using the same semi-preparative HPLC system, eluted with a linear gradient of acetonitrile in water from 25% to 100% in
Table 2 13 C NMR spectral data of compounds 2e4 (d 125 MHz, CDCl3) Carbons
2a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OAc
77.6 74.7 46.6 80.1 83.8 35.6 81.4 56.3 209.7 74.6 136.0 137.3 70.9 36.0 42.9 26.1 20.3 13.6 10.1 75.7 22.1 170.1
2-OBz C-1 o m p 10 20 30 30 -Ph o m p 100 CO 10 -Ph o m p 200 300 400 500 600 700 1% 2% 3% 4% 5% a
In acetone-d6.
165.6 129.9 128.5 133.0 172.7 73.8 56.0 139.2 127.2 127.2 128.3 166.5 139.0 127.1 127.2 130.0
3 78.6 74.5 46.2 80.0 83.8 81.5 57.0 211.7 74.7 135.7 138.1 72.3 35.8 42.8 26.4 20.7 14.3 10.8 76.4 22.5 170.5
130.0 128.6 133.5 73.1 54.4 128.8 126.7 126.7 128.1
78.8 74.4 46.2 81.1 83.9 35.5 81.3 57.2 211.5 74.6 136.0 138.0 72.2 35.7 43.1 26.3 20.5 14.2 10.5 76.5 22.4 170.5 167.0 129.9 128.5 133.6 172.1 73.0 54.3
173.0
36.6 25.3 31.3 22.3 13.7 105.1 73.3 76.2 69.4 65.4
4
105.2 72.9 75.3 64.9
34.3 32.1 33.8 28.9 10.9 18.7 105.0 72.8 75.2 69.1 65.0
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50 min at a flow rate of 3 mL/min and finally provided taxanes 2 (3.5 mg, tR ¼ 25.65 min), 3 (4.5 mg, tR ¼ 27.29 min) and 4 (2.0 mg, tR ¼ 27.93 min), respectively. Compound 4, white amorphous powder, exhibited an HRFAB-MS spectral quasimolecular ion peak at m/z 990.3890 [M þ K]þ, corresponding to the molecular formula of C50H65NO17. Complete assignments of 1H and 13 C NMR signals were achieved (Tables 1 and 2) with the help of various NMR techniques such as 1He1H COSY, HMQC for direct HeC connectivities and HMBC for long-range HeC correlations. The 1H NMR spectrum of 4 showed the characteristic signals of four tertiary methyl groups at d 1.10, 1.22, 1.84 and 1.80 (each 3H, s), one acetyl group at d 2.35 (3H, s), which was further supported by carbon signals observed at d 22.4 and 170.5 in the 13C NMR spectrum, one benzoyl group at d 7.49 (2H, t, 7.7 Hz), 7.60 (1H, t, 7.3 Hz), 8.09 (2H, d, 7.4 Hz) and one phenyl group at d 7.33 (1H, m), 7.38 (4H, m) as well as one oxetane ring at d 4.18 and 4.29 mutually coupled with a coupling constant of 8.7 Hz. In addition, the 1H NMR spectrum of 4 displayed the featured signals of taxol such as H-2 at d 5.65 (1H, d, 7.2 Hz), H-3 at d 3.89 (1H, d, 6.6 Hz), H-5 at d 4.88 (1H, d, 8.9 Hz), H-7 at d 4.06 (1H, m), H-10 at d 5.17 (1H, br.s) and H-13 at d 6.18 (1H, br.t, 9.4 Hz) (Appendino, 1995). A xylosyl group was suggested by the signals of d 4.06 (1H, overlapped d, H-1%), 3.23 (1H, overlapped, H-2%), 3.48 (1H, br.t, 8.2 Hz, H-3%), 3.67 (1H, br.m, H-4%), 3.95 (1H, dd, 11.5, 4.7 Hz, H-5%a), 3.25 (1H, dd, 11.5, 9.7 Hz, H-5%b) (Se´nilh et al., 1984). The presence of a side chain similar to the C-13 side chain of taxol was suggested by the signals at d 4.67 (1H, d, 2.3 Hz, H-20 ), 5.56 (1H, dd, 8.7, 2.3 Hz, H-30 ), 6.26 (1H, br.d, 8.5 Hz, 30 -NH), 7.33e7.38 (5H, m, 30 -Ph). However, aliphatic signals were observed in
O NH 3'
AcO
18
O 19 OH
10
O
16
2'
7
O
1'
13 15
4
OH H
2
HO
OBz
O
OAc
20
1 Paclitaxel (Taxol ) ®
O 1'' NH 3'
HO
18
16
2' 1'
O 19 O
10
O
R1
O
13 15
4
OH H
2
HO
OAc
OBz
OH
R1 =
7
OH O 20
2
5''
3''
O 1'' NH
H3C R2
3'
18
HO 16
2' 1'
O 19 O
10
O O
7 13 15
4
OH HO
3 R2 = H
R1
H
2
4 R2 = CH3
Fig. 1. Structures of compounds 1e4.
OBz
OAc
O 20
O
OH
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place of signals for the N-benzoyl signals of taxol. The coupling connectivities analyzed by the 1He1H COSY experiment indicated that the N-acyl group was a 4-methylhexanoyl group. These results were in good agreement with the relative molecular mass of 4. The relative configurations at H-20 and H-30 were concluded to be 20 R, 30 S by the 1H vicinal coupling constants compared with taxol (J20 ,30 ¼ 2.3 Hz, J30 ,40 ¼ 8.7 Hz) (Morita et al., 1997). The relative configurations at C-2, C-7, C-10 and C-13 were established on the basis of chemical shifts, multiplet patterns and coupling constant values of corresponding protons as well as by comparison with data for taxol and its analogues. Taking all these spectral data into account, the structure of 4 was elucidated unequivocally as 7-O-b-xylosyl-10-deacetyltaxuspinanane A (Fig. 1). 7-O-b-Xylosyl-10-deacetyltaxuspinanane A (4): Amorphous powder; [a]24 D 37 (c 0.1, CHCl3); HRFAB-MS m/z: þ 1 13 990.3890 [M þ K] , calcd for C50H65NO17K 990.3884. For H and C NMR spectral data see Tables 1 and 2. 4. Chemotaxonomic significance Compound 4 is a new taxol analogue with a rare 4-methylhexanoyl functional group instead of the usual benzoyl group, while compounds 2 and 3 were isolated for the first time from T. cuspidata. All of the compounds 2e4 have the same skeleton as baccatin III and bear an O-linked xylosyl group at C-7. Although this kind of compound has been isolated previously from Taxus baccata (Se´nilh et al., 1984) and Taxus yunnanensis (Li et al., 2001, 2003), this type of taxane was isolated for the first time from T. cuspidata. It is of great interest to note that compound 4 obtained in the present study was characterized by a rare 4-methylhexanoyl functional group; this group also occurs in taxuspinanane A which was isolated previously from the stems of T. cuspidata (Morita et al., 1997). Such a difference may serve as a characteristic feature to distinguish T. cuspidata from other species of yew tree. Acknowledgements The work described in this paper was financially supported from the Foundation for Researching New Drugs of People’s Republic of China (No. 2003AA2Z3527) and Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry of P.R. China to QWS. We thank Dr. Takayuki Oritani, Emeritus Professor of Tohoku University, for providing crude extract for this work. References Appendino, G., 1995. In: Farina, V. (Ed.), The Chemistry and Pharmacology of Taxol and Its Derivatives, vol. 22. Elsevier Science, Amsterdam, pp. 1e53, pp. 55e101. Cao, C.M., Zhang, M.L., Wang, Y.F., Shi, Q.W., Yamada, T., Kiyota, H., 2006a. Chem. Biodivers. 3, 1153. Cao, C.M., Huo, C.H., Zhao, Y.M., Si, X.T., Li, Z.P., Shi, Q.W., 2006b. China J. Chin. Mater. Med. 31, 1510. Cao, C.M., Li, Z.P., Shi, Q.W., 2006c. Nat. Prod. Res. Dev. 18, 330. Li, S.H., Zhang, H.J., Yao, P., Sun, H.D., Fong, H.H.S., 2001. Phytochemistry 58, 369. Li, S.H., Zhang, H.J., Niu, X.M., Yao, P., Sun, H.D., Fong, H.H.S., 2003. Tetrahedron 59, 37. Morita, H., Gonda, A., Wei, L., Yamamura, Y., Takeya, K., Itokawa, H., 1997. J. Nat. Prod. 60, 390. Parmar, V.S., Jha, A., Bisht, K.S., Taneja, P., Singh, S.K., Kumar, A., Poonam Jain, R., Olsen, C.E., 1999. Phytochemistry 50, 1267. Se´nilh, V., Blechert, S., Colin, M., Gue´nard, D., Picot, F., Potier, P., Varenne, P., 1984. J. Nat. Prod. 47, 131. Shi, Q.W., Kiyota, H., 2005. Chem. Biodivers. 2, 1597. Shigemori, H., Kobayashi, J., 2004. J. Nat. Prod. 67, 245.