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Cytotoxity of non-alkaloidal taxane diterpenes from Taxus chinensis against a paclitaxel-resistant cell line
Cancer Letters 158 (2000) 151±154
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Cytotoxity of non-alkaloidal taxane diterpenes from Taxus chinensis against a paclitaxel-resistant cell line Masao Fukushima a, Narihiko Fukamiya a,*, Masayoshi Okano a, Tatsuo Nehira a, Kiyoshi Tagahara b, Shun-Xiang Zhang c, De-Cheng Zhang d, Yoko Tachibana c, Kenneth F. Bastow c, Kuo-Hsiung Lee c a
Department of Interdisciplinary Studies of Natural Environment, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan b Faculty of Pharmaceutical Sciences, Kobe Pharmaceutical University, Kobe 658-8558, Japan c Natural Products Laboratory, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA d School of Pharmacy, Shanghai Medical University, Shanghai, China Received 15 May 2000; received in revised form 7 June 2000; accepted 7 June 2000
Abstract Seven taxane diterpenes were isolated from the EtOH extract of the aerial parts of Taxus chinensis, and evaluated for cytotoxicity against nine human cell lines, including a b-tublin mutant resistant to paclitaxel. Compound 2, a non-alkaloidtype taxane diterpene, showed signi®cant cytotoxicity in most cell lines, and notably, equipotent against both parental and b-tublin mutant tumor cell lines. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cytotoxicity; Non-alkaloid type taxane diterpene; b-Tublin mutant tumor cell line
1. Introduction Paclitaxel (Taxol w) is a most promising new antitumor agent [1,2], which was isolated from Taxus blevifolia. Consequently, paclitaxel's chemistry and pharmacology have been studied extensively, and many taxane diterpenes have been isolated from the Taxus species [3±5]. However, few isolated nonpaclitaxel-type compounds have been examined as potential antitumor agents. In addition, new class of taxane-type compounds must be developed that are effective in paclitaxel-resistant tumor cell lines. Accordingly, in the course of our studies of antitumor * Corresponding author. Tel.: 181-824-24-6537; fax: 181-82424-0757. E-mail address: [email protected] (N. Fukamiya).
agents from natural sources, we investigated seven taxane diterpene constituents of Taxus chinensis var. maire [6±9], for cytotoxicity against nine tumor cell lines, including a b-tublin mutant cell resistant to paclitaxel. 2. Result and discussion Table 1 shows the cytotoxicities of active tgacq 1±3 isolated from Taxus chinensis against a panel of human cancer cell lines. Prescreening tests with all compounds showed that 4±7 were inactive against KB (nasopharyngeal carcinoma) and MCF-7 (breast) cell lines. Among the active compounds, compound (2) demonstrated signi®cant (ED50 #4.0 mg/ml) cytotoxicity
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(00)00506-1
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M. Fukushima et al. / Cancer Letters 158 (2000) 151±154
3.2. Chemicals
Table 1 Cytotoxicities of taxoids 1±3 a Cell lines
KB A-549 HCT-8 CAKI-1 MCF-7 SK-MEL-2 1A9 1A9PTX10 U-87-MG
Compounds/ED50 (mg/ml) 1
2
3
Paclitaxel
5.98 15.30 15.60 .40 14.00 18.00 8.96 .40 26.20
2.39 4.42 3.02 4.93 .40 .40 3.28 3.86 .40
4.40 10.00 9.20 32.50 19.00 .40 7.37 7.06 .40
0.00137 0.0051 0.0111 ND ND ND 0.0017 0.0410 ND
a For signi®cant activity of the pure compounds, an ED50 ,4.0 mg/ml is required, ND, not determined
against human KB, A-549 lung carcinoma, HCT-8 colon tumor, CAKI-1 renal, 1A9 ovarian, and 1A9PTX10 b-tublin-mutant cell lines with ED50 values of 2.39, 4.42, 3.02, 4.93, 3.28, and 3.86 mg/ml, respectively. Compound (3) was less active but like (2), was equipotent against both the parental (1A9) and 1A9PTX10 b-tublin-mutant cell line. Compound (1) showed only weak cytotoxicity against most cell lines. From a structure-activity perspective, this result indicates that the ether linkage (C12±C17), the C-19 acetoxygroup, and the C-13 carbonyl group of (1) resulted in reduced cytotoxicity. Compounds 2 and 3 have non-paclitaxel-type, (i.e. they do not contain a C-13 N-benzoyl-3-phenylisoserine side chain and a C-4 and C-5 oxetane ring) and were less active than paclitaxel. However, (2) and (3) showed no loss of potency against the 1A9PTX-10 b-tublin-mutant paclitaxel-resistant cell line and, thus, may be useful lead compounds for the discovery of drugs active against paclitaxel-resistant tumor cells.
3. Experimental procedures 3.1. Cells The tested cell lines were, human KB, A549 lung carcinoma, HTC-8 colon tumor, CAKI-1 human renal, MCF-7 human breast, SK-MEL-2 human melanoma, 1A9 human ovarian, 1A9PTX10 b-tublinmutant, and U-87-MG glioblasotma.
The cell culture chemicals were purchased from Sigma±Aldrich Chemical Co., Inc. 1H- and 13Cnuclear magnetic resonance (NMR) spectra were determined on JEOL ALPHA-400 instruments in CDCl3 and C5D5N using tetramethylsilane (TMS) as an internal standard. Silica gel (Merck, type 60, 70±320 mesh) was used for cell culture (CC). Precoated Silica gel plates (Merck 60F254) of 0.25 mm thickness were used for analytical thin layer chromatography (TLC), and plates of 1 and 2 mm thickness were used for preparation TLC. Analytical high performance liquid chromatography (HPLC) was performed on a Tosoh liquid chromatograph equipped with a UV detector at 254 nm and a reverse-phase column (TSK-gel ODS-80Ts) using a mixed solvent of MeOH/H2O. Preparative HPLC was carried out on Tosoh or Gilson liquid chromatographs equipped with a reverse-phase column (Lichrosorb RP-18) at 254 nm using the same solvents as employed for analytical HPLC. 3.3. Test compounds The plant bark, twigs, and leaves of T. chinensis (airdried material, 6.2 kg) were extracted with EtOH, and the EtOH extract (507 g) was obtained by evaporation of the solvent. The extract was diluted with EtOH and H2O, and extracted with hexane to give a hexane extract (46 g). The EtOH/H2O layer was then extracted with CH2Cl2 to give a CH2Cl2 extract (120 g), and followed by extraction with 1-BuOH to afford a 1-BuOH extract (153 g), and a H2O soluble layer (189 g). Silica gel column chromatography of this extract eluting with benzene/EtOAc/hexane (14:5:6, v/v) (29 l) gave 11 fractions. Each fraction was checked by analytical TLC and HPLC. Fraction 8 (1.839 g) was subjected Sephadex LH20 column chromatography eluted with MeOH to afford three fractions, Fr-8-1(198.3 mg), Fr8-2(975.4 mg), and Fr8-3(267.9 mg). Puri®cation of Fr-8-1(198.3 mg) with prep. HPLC (MeOH/H2O, 68:32) afforded three known taxoids, (5) (1.6 mg), (4) (11.2 mg), and (3) (2.8 mg). Fr8-2 (975.4 mg) also gave ®ve known taxoids, (2) (8.3 mg), (4) (6.4 mg), (6) (2.5 mg), (7) (54 mg) by the separation of prep. HPLC (MeOH/ H2O,75:25).
M. Fukushima et al. / Cancer Letters 158 (2000) 151±154
After insoluble material of fraction 11 (1.14 g) was ®ltered out, the remaining substance (1.49 g) was subjected prep. HPLC (MeOH/H2O, 7:3). Repeated puri®cation of the fraction by the prep. HPLC gave (1) (5.6 mg). The spectral analysis of IR, UV, magnetic spectroscopy (MS), 1H- and 13C-NMR, and 2D-NMR disclosed the structures of the compounds (1±7) were 19-acetoxytaxagi®n (1) [10], 2-deacetoxytaxinine J (2) [11], yunnanxane (3) [12], taxupine X (4) [13], 2 0 -enolacetoxytaxupine X (5) [14], 2(3-20) abeotaxinine NN-1 (6) [15], and 19-benzoyloxytaxagi®n (7) [16]Fig. 1. 3.4. Cytotoxicity assay experiment The assay was performed following a standard published procedure using microtiter plate format and sulfurhodamine B staining at the endpoint [17]. Human tumor cell lines were exposed to the test taxoid compounds for three days. Cells were cultured in RPMI-1640 growth medium, supplemented with 25
153
mM HEPES, 2% (w/v) sodium bicarbonate, 10% (v/v) fetal bovine serum and 100 mg/ml kanamycin. The cell cultures, were maintained in humidi®ed 5% CO2 atmosphere at 378C, included the following human cell lines: KB (nasopharyngeal carcinoma), A549 (lung carcinoma), HCT-8 (colon tumor), CAKI-1 (renal), MCF-7 (breast), SK-MEL-2 (melanoma), 1A9 (ovarian), 1A9PTX10 b-tubulin-mutant derived from 1A9, and U-87-MG (glioblastoma). Acknowledgements The authors thank Drs M. Sugiura, K. Saiki, T. Sai, Kobe Pharmaceutical University for MS (EI and HREI) spectra. This investigation was supported by grant 10878093 from the Ministry of Education of Japan awarded to N. Fukamiya and by grant CA.17625 from the National Cancer Institute awarded to K.H. Lee. 1A9PTX10 cells were a generous gift of Dr P. Siannakalo (NCI, Bethesda, MD). References
Fig. 1. Structure of active taxoids.
[1] D.J.I. Kingston, A.A. Molinero, J.M. Rimoldi, The taxane diterpenoids, in: W. Herts, G.W. Kirby, R.E. Moor, W. Steglich, Ch Tamm (Eds.), Springer-Verlag, Wien, Vol. 61, 1993, pp. 1±206. [2] G.I. Georg, T.T. Chen, I. Ojima, D.M. Vyas (Eds.), Taxanes anticancer agents, basic science and current status, ACS Symp. Ser. 1995, pp. 1±353. [3] G. Appendiano, The phytochemistry of the yew tree, Nat. Prd. Rep. 12 (1995) 349±360. [4] E. Baloglu, D.G.I. Kingston, The taxane diterpenoids, J. Nat. Prod. 62 (1999) 1448±1472. [5] L.X. Xu, A.R. Liu, Determination of taxol in the extract of Taxus chinensis by reverse phase HPLC, Acta Pharm. Sin 24 (1989) 552±555. [6] K. Fuji, K. Tanaka, B. Li, T. Shingu, H. Sun, T. Taga, Nobel diterpenoids from Taxus chinensis, J. Nat. Prod. 56 (1993) 1520±1531. [7] K. Tanaka, K. Fuji, K. Yokoi, T. Shingu, L.I. Bo, Sun the structure of six new diterpenenoids, taxchinines F, H, L, I, K and taxchin B, Chem. Pharm. Bull. 42 (1994) 1539±1541. [8] Z. Zhang, Z. Jia, Taxanes from Taxus chinensis, Phytochemistry 30 (1991) 2345±2348. [9] S. Zhang, C.T.L. Lee, K. Chen, Y. Kawada, D.C. Zhang, A.T. Macphile, K.H. Lee, Structure and stereochemistry of taxuchin A, a new 11(15-1)abeo-taxanetype diterpene from Taxus cinensis, J. Chem. Soc. Chem. Commun. (1994) 1561±1562. [10] M. Fukushima, J. Takeda, N. Fukamiya, M. Okano, K. Tagahara, S.X. Zhang, D. Zhang, K.H. Lee, A new taxoid 19-acet-
154
[11] [12] [13] [14]
M. Fukushima et al. / Cancer Letters 158 (2000) 151±154 oxytaxagi®ne, from Taxus chinensis, J. Nat. Prod. 62 (1999) 140±142. J. Ling, Z. Min, M. Niwa, Studies on the diterpenes of Taxus mairei, Acta Chim. Sin. 46 (1988) 1053±1054. W.M. Chen, P.L. Zhang, B. Wu, Q.T. Zheng, Studies on the chemical constituents of Taxus yunnanensis, Acta Chim. Sin. 26 (1991) 747±753. H. Shigemori, X. Wang, N. Yoshida, J. Kobayashi, Taxupines X-Z, new taxoids from Japanease yew Taxus cuspidata, Chem. Pharm. Bull. 47 (1997) 1205±1208. Q.W. Shi, T. Oritani, T. Sugiyama, T. Yamata, Novel bicyclic taxoids from the needles of Taxus maire, Proceedings of the 42nd Symposium on the Chemistry of Terpenes, Essential Oils and Aromatics, Gifu, 1998 pp. 281±283.
[15] M. Ando, J. Sakai, S. Zhang, K. Kosugi, Y. Watanabe, H. Minato, H. Fujisawa, H. Sasaki, T. Suzuki, H. Hagiwara, N. Hirata, K. Hirosa, simple method for the isolation of taxol and its analogs from the needles of Taxus cuspidata and production of taxoids by its callus calture, Proceedings of the 40th Symposium on the Chemistry of Natural Products, Fukuoka, 1998, pp. 353±357. [16] F. Yoshizaki, M. Fukuda, S. Hisamichi, Y. Ishida, Structure of taxane diterpenoids from the seeds of Japanese yew, Taxus cupsidata, Chem. Pharm. Bull 36 (1988) 2098±2102. [17] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahorn, D. Vistica, J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyd, New colorimetric cytotoxicity assay for anticancer-drug screening, J. Natl. Cancer Inst. 82 (1990) 1107±1112.
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Cytotoxity of non-alkaloidal taxane diterpenes from Taxus chinensis against a paclitaxel-resistant cell line Masao Fukushima a, Narihiko Fukamiya a,*, Masayoshi Okano a, Tatsuo Nehira a, Kiyoshi Tagahara b, Shun-Xiang Zhang c, De-Cheng Zhang d, Yoko Tachibana c, Kenneth F. Bastow c, Kuo-Hsiung Lee c a
Department of Interdisciplinary Studies of Natural Environment, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan b Faculty of Pharmaceutical Sciences, Kobe Pharmaceutical University, Kobe 658-8558, Japan c Natural Products Laboratory, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA d School of Pharmacy, Shanghai Medical University, Shanghai, China Received 15 May 2000; received in revised form 7 June 2000; accepted 7 June 2000
Abstract Seven taxane diterpenes were isolated from the EtOH extract of the aerial parts of Taxus chinensis, and evaluated for cytotoxicity against nine human cell lines, including a b-tublin mutant resistant to paclitaxel. Compound 2, a non-alkaloidtype taxane diterpene, showed signi®cant cytotoxicity in most cell lines, and notably, equipotent against both parental and b-tublin mutant tumor cell lines. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cytotoxicity; Non-alkaloid type taxane diterpene; b-Tublin mutant tumor cell line
1. Introduction Paclitaxel (Taxol w) is a most promising new antitumor agent [1,2], which was isolated from Taxus blevifolia. Consequently, paclitaxel's chemistry and pharmacology have been studied extensively, and many taxane diterpenes have been isolated from the Taxus species [3±5]. However, few isolated nonpaclitaxel-type compounds have been examined as potential antitumor agents. In addition, new class of taxane-type compounds must be developed that are effective in paclitaxel-resistant tumor cell lines. Accordingly, in the course of our studies of antitumor * Corresponding author. Tel.: 181-824-24-6537; fax: 181-82424-0757. E-mail address: [email protected] (N. Fukamiya).
agents from natural sources, we investigated seven taxane diterpene constituents of Taxus chinensis var. maire [6±9], for cytotoxicity against nine tumor cell lines, including a b-tublin mutant cell resistant to paclitaxel. 2. Result and discussion Table 1 shows the cytotoxicities of active tgacq 1±3 isolated from Taxus chinensis against a panel of human cancer cell lines. Prescreening tests with all compounds showed that 4±7 were inactive against KB (nasopharyngeal carcinoma) and MCF-7 (breast) cell lines. Among the active compounds, compound (2) demonstrated signi®cant (ED50 #4.0 mg/ml) cytotoxicity
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(00)00506-1
152
M. Fukushima et al. / Cancer Letters 158 (2000) 151±154
3.2. Chemicals
Table 1 Cytotoxicities of taxoids 1±3 a Cell lines
KB A-549 HCT-8 CAKI-1 MCF-7 SK-MEL-2 1A9 1A9PTX10 U-87-MG
Compounds/ED50 (mg/ml) 1
2
3
Paclitaxel
5.98 15.30 15.60 .40 14.00 18.00 8.96 .40 26.20
2.39 4.42 3.02 4.93 .40 .40 3.28 3.86 .40
4.40 10.00 9.20 32.50 19.00 .40 7.37 7.06 .40
0.00137 0.0051 0.0111 ND ND ND 0.0017 0.0410 ND
a For signi®cant activity of the pure compounds, an ED50 ,4.0 mg/ml is required, ND, not determined
against human KB, A-549 lung carcinoma, HCT-8 colon tumor, CAKI-1 renal, 1A9 ovarian, and 1A9PTX10 b-tublin-mutant cell lines with ED50 values of 2.39, 4.42, 3.02, 4.93, 3.28, and 3.86 mg/ml, respectively. Compound (3) was less active but like (2), was equipotent against both the parental (1A9) and 1A9PTX10 b-tublin-mutant cell line. Compound (1) showed only weak cytotoxicity against most cell lines. From a structure-activity perspective, this result indicates that the ether linkage (C12±C17), the C-19 acetoxygroup, and the C-13 carbonyl group of (1) resulted in reduced cytotoxicity. Compounds 2 and 3 have non-paclitaxel-type, (i.e. they do not contain a C-13 N-benzoyl-3-phenylisoserine side chain and a C-4 and C-5 oxetane ring) and were less active than paclitaxel. However, (2) and (3) showed no loss of potency against the 1A9PTX-10 b-tublin-mutant paclitaxel-resistant cell line and, thus, may be useful lead compounds for the discovery of drugs active against paclitaxel-resistant tumor cells.
3. Experimental procedures 3.1. Cells The tested cell lines were, human KB, A549 lung carcinoma, HTC-8 colon tumor, CAKI-1 human renal, MCF-7 human breast, SK-MEL-2 human melanoma, 1A9 human ovarian, 1A9PTX10 b-tublinmutant, and U-87-MG glioblasotma.
The cell culture chemicals were purchased from Sigma±Aldrich Chemical Co., Inc. 1H- and 13Cnuclear magnetic resonance (NMR) spectra were determined on JEOL ALPHA-400 instruments in CDCl3 and C5D5N using tetramethylsilane (TMS) as an internal standard. Silica gel (Merck, type 60, 70±320 mesh) was used for cell culture (CC). Precoated Silica gel plates (Merck 60F254) of 0.25 mm thickness were used for analytical thin layer chromatography (TLC), and plates of 1 and 2 mm thickness were used for preparation TLC. Analytical high performance liquid chromatography (HPLC) was performed on a Tosoh liquid chromatograph equipped with a UV detector at 254 nm and a reverse-phase column (TSK-gel ODS-80Ts) using a mixed solvent of MeOH/H2O. Preparative HPLC was carried out on Tosoh or Gilson liquid chromatographs equipped with a reverse-phase column (Lichrosorb RP-18) at 254 nm using the same solvents as employed for analytical HPLC. 3.3. Test compounds The plant bark, twigs, and leaves of T. chinensis (airdried material, 6.2 kg) were extracted with EtOH, and the EtOH extract (507 g) was obtained by evaporation of the solvent. The extract was diluted with EtOH and H2O, and extracted with hexane to give a hexane extract (46 g). The EtOH/H2O layer was then extracted with CH2Cl2 to give a CH2Cl2 extract (120 g), and followed by extraction with 1-BuOH to afford a 1-BuOH extract (153 g), and a H2O soluble layer (189 g). Silica gel column chromatography of this extract eluting with benzene/EtOAc/hexane (14:5:6, v/v) (29 l) gave 11 fractions. Each fraction was checked by analytical TLC and HPLC. Fraction 8 (1.839 g) was subjected Sephadex LH20 column chromatography eluted with MeOH to afford three fractions, Fr-8-1(198.3 mg), Fr8-2(975.4 mg), and Fr8-3(267.9 mg). Puri®cation of Fr-8-1(198.3 mg) with prep. HPLC (MeOH/H2O, 68:32) afforded three known taxoids, (5) (1.6 mg), (4) (11.2 mg), and (3) (2.8 mg). Fr8-2 (975.4 mg) also gave ®ve known taxoids, (2) (8.3 mg), (4) (6.4 mg), (6) (2.5 mg), (7) (54 mg) by the separation of prep. HPLC (MeOH/ H2O,75:25).
M. Fukushima et al. / Cancer Letters 158 (2000) 151±154
After insoluble material of fraction 11 (1.14 g) was ®ltered out, the remaining substance (1.49 g) was subjected prep. HPLC (MeOH/H2O, 7:3). Repeated puri®cation of the fraction by the prep. HPLC gave (1) (5.6 mg). The spectral analysis of IR, UV, magnetic spectroscopy (MS), 1H- and 13C-NMR, and 2D-NMR disclosed the structures of the compounds (1±7) were 19-acetoxytaxagi®n (1) [10], 2-deacetoxytaxinine J (2) [11], yunnanxane (3) [12], taxupine X (4) [13], 2 0 -enolacetoxytaxupine X (5) [14], 2(3-20) abeotaxinine NN-1 (6) [15], and 19-benzoyloxytaxagi®n (7) [16]Fig. 1. 3.4. Cytotoxicity assay experiment The assay was performed following a standard published procedure using microtiter plate format and sulfurhodamine B staining at the endpoint [17]. Human tumor cell lines were exposed to the test taxoid compounds for three days. Cells were cultured in RPMI-1640 growth medium, supplemented with 25
153
mM HEPES, 2% (w/v) sodium bicarbonate, 10% (v/v) fetal bovine serum and 100 mg/ml kanamycin. The cell cultures, were maintained in humidi®ed 5% CO2 atmosphere at 378C, included the following human cell lines: KB (nasopharyngeal carcinoma), A549 (lung carcinoma), HCT-8 (colon tumor), CAKI-1 (renal), MCF-7 (breast), SK-MEL-2 (melanoma), 1A9 (ovarian), 1A9PTX10 b-tubulin-mutant derived from 1A9, and U-87-MG (glioblastoma). Acknowledgements The authors thank Drs M. Sugiura, K. Saiki, T. Sai, Kobe Pharmaceutical University for MS (EI and HREI) spectra. This investigation was supported by grant 10878093 from the Ministry of Education of Japan awarded to N. Fukamiya and by grant CA.17625 from the National Cancer Institute awarded to K.H. Lee. 1A9PTX10 cells were a generous gift of Dr P. Siannakalo (NCI, Bethesda, MD). References
Fig. 1. Structure of active taxoids.
[1] D.J.I. Kingston, A.A. Molinero, J.M. Rimoldi, The taxane diterpenoids, in: W. Herts, G.W. Kirby, R.E. Moor, W. Steglich, Ch Tamm (Eds.), Springer-Verlag, Wien, Vol. 61, 1993, pp. 1±206. [2] G.I. Georg, T.T. Chen, I. Ojima, D.M. Vyas (Eds.), Taxanes anticancer agents, basic science and current status, ACS Symp. Ser. 1995, pp. 1±353. [3] G. Appendiano, The phytochemistry of the yew tree, Nat. Prd. Rep. 12 (1995) 349±360. [4] E. Baloglu, D.G.I. Kingston, The taxane diterpenoids, J. Nat. Prod. 62 (1999) 1448±1472. [5] L.X. Xu, A.R. Liu, Determination of taxol in the extract of Taxus chinensis by reverse phase HPLC, Acta Pharm. Sin 24 (1989) 552±555. [6] K. Fuji, K. Tanaka, B. Li, T. Shingu, H. Sun, T. Taga, Nobel diterpenoids from Taxus chinensis, J. Nat. Prod. 56 (1993) 1520±1531. [7] K. Tanaka, K. Fuji, K. Yokoi, T. Shingu, L.I. Bo, Sun the structure of six new diterpenenoids, taxchinines F, H, L, I, K and taxchin B, Chem. Pharm. Bull. 42 (1994) 1539±1541. [8] Z. Zhang, Z. Jia, Taxanes from Taxus chinensis, Phytochemistry 30 (1991) 2345±2348. [9] S. Zhang, C.T.L. Lee, K. Chen, Y. Kawada, D.C. Zhang, A.T. Macphile, K.H. Lee, Structure and stereochemistry of taxuchin A, a new 11(15-1)abeo-taxanetype diterpene from Taxus cinensis, J. Chem. Soc. Chem. Commun. (1994) 1561±1562. [10] M. Fukushima, J. Takeda, N. Fukamiya, M. Okano, K. Tagahara, S.X. Zhang, D. Zhang, K.H. Lee, A new taxoid 19-acet-
154
[11] [12] [13] [14]
M. Fukushima et al. / Cancer Letters 158 (2000) 151±154 oxytaxagi®ne, from Taxus chinensis, J. Nat. Prod. 62 (1999) 140±142. J. Ling, Z. Min, M. Niwa, Studies on the diterpenes of Taxus mairei, Acta Chim. Sin. 46 (1988) 1053±1054. W.M. Chen, P.L. Zhang, B. Wu, Q.T. Zheng, Studies on the chemical constituents of Taxus yunnanensis, Acta Chim. Sin. 26 (1991) 747±753. H. Shigemori, X. Wang, N. Yoshida, J. Kobayashi, Taxupines X-Z, new taxoids from Japanease yew Taxus cuspidata, Chem. Pharm. Bull. 47 (1997) 1205±1208. Q.W. Shi, T. Oritani, T. Sugiyama, T. Yamata, Novel bicyclic taxoids from the needles of Taxus maire, Proceedings of the 42nd Symposium on the Chemistry of Terpenes, Essential Oils and Aromatics, Gifu, 1998 pp. 281±283.
[15] M. Ando, J. Sakai, S. Zhang, K. Kosugi, Y. Watanabe, H. Minato, H. Fujisawa, H. Sasaki, T. Suzuki, H. Hagiwara, N. Hirata, K. Hirosa, simple method for the isolation of taxol and its analogs from the needles of Taxus cuspidata and production of taxoids by its callus calture, Proceedings of the 40th Symposium on the Chemistry of Natural Products, Fukuoka, 1998, pp. 353±357. [16] F. Yoshizaki, M. Fukuda, S. Hisamichi, Y. Ishida, Structure of taxane diterpenoids from the seeds of Japanese yew, Taxus cupsidata, Chem. Pharm. Bull 36 (1988) 2098±2102. [17] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahorn, D. Vistica, J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyd, New colorimetric cytotoxicity assay for anticancer-drug screening, J. Natl. Cancer Inst. 82 (1990) 1107±1112.