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Taxane Diterpenoids From the Seeds ofChinese Yew, Taxus MaireiQing-Wen Shi a , Takayuki Oritani a , Takeyoshi Sugiyama a & TeikoYamada aa Laboratory of Applied Bioorganic Chemistry, Division of LifeScience, Graduate School of Agricultural Science, TohokuUniversity , 1-1 Tsutsumidori-Amamiya, Aoba-ku, Sendai, 981-8555,JapanPublished online: 04 Oct 2006.

To cite this article: Qing-Wen Shi , Takayuki Oritani , Takeyoshi Sugiyama & Teiko Yamada (1999)Taxane Diterpenoids From the Seeds of Chinese Yew, Taxus Mairei , Natural Product Letters, 13:3,179-186, DOI: 10.1080/10575639908048784

To link to this article: http://dx.doi.org/10.1080/10575639908048784

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TAXANE DITERPENOIDS FROM THE SEEDS OF

CHINESE YEW, TAXUS MAIREI

Qing-wen Shi, Takayuki Oritani, Takeyoshi Sugiyama, and Teiko Yamada Laboratory of Applied Bioorganic Chemistry, Division of Life Science,

Graduate School of Agricultural Science, Tohoku University, 1- 1 Tsutsumidori-Amamiya, Aoba-ku, Sendai 98 1-8555, Japan

(Received 17th December 1998)

Abstract: Nine taxane diterpenoids were isolated from the seeds of Tuxus mairei. One of them was a novel compound with the structure (1) which was established as 9 a, 1 Op ,13 a-t ri ace tox y- 5 a( 3' -me thy lami no- 3' - pheny 1) -prop ion y loxy -taxa-

4(20), I I-diene on the basis of 'H NMR data and HR-EI-MS analysis. Key words: Tlixus mairei, seeds, alkaloid, taxane diterpenoid, Taxaceae.

INTRODUCTION As a continuation of our ongoing search for taxane diterpenoids to provide

precursors for the semisynthesis of analogs of the anticancer drug paclitaxel (Tax01 ), we have investigated the constituents of the leave and bark of Chinese yew, Taxus muirei (1 ) . Owing to no report on the constituents of seeds of this plant,

recently we investigated the component of the seed. In this communication we describe the isolation of a novel taxane diterpenoid along with several known compounds from the seeds of 7: mairei.

8

RESULT AND DISCUSSION A methanolic extract of the seeds of I: mairei was processed as described in

the Materials and Methods section to afford nine taxane diterpenoids.

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180 QING-WEN SHl et a1

1 R,=H R:=CH3 R,+~Ac 4 R,=H RZ=H R~=OAC 2 R,=H R,=H R,=OAc 5 R,=OH RFH R p O 3 RI=OAC R?=CH3 RGO 6 R,=H RFAC R 3 = 0

7 8 9

Compound 1 was isolated as a white powder and its structure was

determined to be 7,2'-bisdeacetoxyaustrospicatine from its physical and spectral data, which were identical with the data of 7,2'-bisdeacetoxyaustrospicatine described in the literatures (2 ).

The HR-EI-MS of compound 2 revealed a molecular formula of C,6H,,08N. Intensive absorptions at 1735, 1720, and a weak absorption at 3330 cm-' implied that 2 possesses ester and amino groups. The 'H NMR spectrum of 2

showed four methyl groups at relatively high field: 0.7 1, 1.08, 1.26, and 2.02 ppm,

which were the characteristic signals of the taxane skeleton. The signals at 4.84, 5.18 ppm (each 1 H, br.s) in the 'H NMR spectrum, which showed weak correlation in the ' H - l H COSY spectrum, suggested the presence of an exomethylene moiety. Additionally, four oxygen-bearing one-proton signals appeared at lower field, of which a pair of one-proton doublets at 6.03 and 5.84 ppm with a coupling constant of 10.7 I Hz were assigned to H- 10a and H-9P, respectively. Large vicinal coupling indicated a rrms-oriented configuration of the H-9 and H-10. The signal at 5.79 ppm ( IH, br.t, J = 7.14 Hz), which showed a correlation with 18-CH3 in the 'H-'H COSY spectrum, was assigned to H-13; the signal at 5.33 ppm (lH, br.s) was

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I 37 1.̂ "

TAXANE DITERPENOIDS 181

n r

Fig. 1. EI-MS ion mode spectrum of compound 2 at 13 1.1 "c (3).

i d : 623

, d z 178 d z 444

d z 120 m/: 504

Fig. 2. Major fragmental fission patterns of 2 in the EI-MS spectrum.

characteristic to H-5a. The presence of a nor-Winterstein acid [3-(N-methylamino)- 3-phenylpropanoyl] moiety in 2 was suggested from the signals at 2.29 (3H, s), 2.73 (2H, d, J = 7.41 Hz), 3.95 (IH, t, J = 7.41 Hz), 7.33 (SH, m) ppm in the 'H NMR spectrum, which was in good agreement with the literature values (2). Further

support was provided by the fragment ions in the EI-MS at m/z 178 and 120 (both

base peaks) that was analyzed for C, &I, 202N and C8H, &J by HR-EI-MS, respectively. The location of a nor-Winterstein acid moiety was suggested at C-5 as in the caSe of all the other taxoids ever isolated (4), although HMBC data could notbe obtained to provide direct evidence for the connecting position. three-proton The signal at 2.29 ppm was assigned as of N-CH,. The remaining three three-proton

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182 QING-WEN SHI er al.

Table I . Spectral data of 2 and 5 (300 Hz for 'H, 125 MHz for 13C, CDCl3)

2 5

p o A o n IH J IH J 13c

I 2a

3 4 5 6a

7 a

9 I0 I I I2 13 I 4 a

IS 16 17 I X 19 20a 'Oh 2' 3' 3' 5 ' . 9 6'. K' 7' OAc

OAc

OAc

2P

6P

7P

I4P

N-CH3

1.81 rn 1.74 rn 1.67 rn 2.89 br s

5.33 br s 1.57 rn 1.67 rn 1.61 rn 1.61 rn 5.84 d 6.03 d

5.79 t 1.01 br ctl 2.63 cttl

1.08 s 1.26 s 2.02 d 0.71 s 5.18 br s 4.84 br s

2.73 d 3.9s I

7.30 rn 7.30 rn

2.05 5

2.01 \

1.73 s 2.29 s

4.67

10.7 I 10.71

7.14 14.90, 8.00 14.70. 9.30, 5.03

1.10

7.4 1 7.41

4.15 t 8.14

3.40 d 6.87

5.33 br s 2.03 rn 1.72 rn 2.02 m 1.74 m 5.90 d 10.30 6.09 d 10.30

2.69 s 2.69 s

1.66 s 1.27 s 2.26 s 0.98 s 5.48 br s 5.43 br s 6.38 d 16.10 7.65 d 16.10

7.75 rn 7.43 rn 7.43 rn 2.09 s

2.06 s

78.53 71.78

47. I9 144.12 78.44 29.30

28.22

76.05 73.65 153.33 140.09 200.36 44.87

42.86 18.19 20.6 1 14.39 34.82 118.91

I 18.08 153.31 134.98 129.07 129.55 131.06 21.51 170.7 1 21.35 170.29

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TAXANE DITERPENOIDS 183

signals at 2.05, 2.01, and 1.73 pprn should be the acetyl groups connected to C-9, C- 10, and C- I3 as deduced from the chemical shifts of the corresponding protons attached to them. Thus the structure was determined as 9 a , 1 Op, 1 3a-triacetoxy-Sa- (3'-methylamino-3'-phenyl)-propionyloxy-taxa-4(20), 1 1 -diene, i. e. a nor derivative of 7.2'-bisdeacetoxyaustrospicatine (2). It was reported that removal of one .methyl from the dimethylamino group of the Winterstein acid moiety caused a marked upfield shift (ca 8 ppm) of the remaining methyl (4b). Unfortunately, we couldn't isolate the sample sufficiently enough for running I3C NMR spectrum.

Compound 3 was isolated as a white gum in a 0.0002 % yield on the dried material. The structure was established as 1-dehydroxy-2-deacetyl taxine B (4c) on the basis of spectral analysis.

Compound 4 was isolated as a white powder. The structure was established as taxezopidine G ( 5 ) by means of 'H NMR spectral analysis.

Compound 5 was isolated as a colorless crystal. Detailed interpretation of 'H NMR. I3C NMR, 'H- 'H COSY, HMBC and HMQC spectra suggested its structure as taxicin I triester, which could be formed chemically from I-hydroxy taxicin with sodium borohydride to remove the acetyl group at C-2 chemoselectively (6). We report here the I3C NMR spectral data of taxicin I triester for the first time.

Compounds 6, 7, 8. and 9 were identified as taxinine (7). taxachitriene A

(8 1, 1 -hydroxy-7.9-dideacetyl-baccatin I (9), and 13-acetyl brevifoliol ( lo ) , respectively, on the basis of spectral analysis and by comparing with the reported physical and spectral data in the literatures.

MATERIALS AND METHODS Optical rotations were recorded on a Horiba SEPA-300 digital polarimeter.

UV spectra were recorded on a Shimadzu UV-I 600 spectrophotometer. IR spectra

were obtained on a Jasco IR-810 instrument. MS were measured on a Jeol JMS- 700 spectrometer using El modes. 'H NMR spectra were obtained on Varian

GEMINI 2000/300 spectrometer operating at 300 MHz for 'H nucleus, in CDCl, at 20 T, chemical shifts are reported in ppm scale relative to that of tetramethylsilane

(TMS. 6 = 0) as an internal standard, and coupling constants are given in Hertz.

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184 QING-WEN SH1 et al.

Open column chromatography was performed using Merck silica gel 60 (100-200

mesh). Thin layer chromatography was carried out with the precoated Merck silica

gel 60 F254 plates. Preparative TLC were performed using the same type of plates as

used for TLC but with 0.85 mm thickness and detected under W (254 nm) andor

by spraying with 10% sulfuric acid and then heating on a hot plate. The seeds of T m'rei were collected in Jinggangshan, in the southeast of

China, in October of 1995. The botanical identification was made by Prof. R. L. Liu, Zhangzhou Forestry School, China. A voucher specimen has been deposited in

our laboratory of the Graduate School of Agricultural Science, Tohoku University. Air dried seeds ( I . 1 kg) were extracted with hexane three times at r m m

temperature to remove most of undesired neutral component. The residue was

extracted twice with methanol (MeOH). The MeOH extracts were condensed to residue under reduced pressure. This residue was diluted with water and was extracted five times with EtOAc. The combined EtOAc layer was further extracted with 5% HCI. After neutralization, the aqueous layer was extracted three times with EtOAc. The combined W A C extract, upon evaporation, yielded 3 g of dark syrup,

which was subjected to preparative TLC repeatedly with the different developing solvents, and finally separated compounds 1, 2 (m 1 mg), 3, and 5. The organic layer after extracted with 5% HCI was condensed (9.5 g), and subjected to a silica gel column chromatography and eluted with hexane-acetone (4: 1 , 3: 1, 2: 1 , 1: 1 and 1:2). Five fractions were obtained. Frs. 1 and 2 were repeatedly separated and purified by means of preparative TLC on silica gel with hexane-acetone, hexane-

EtOAc. and CHC1,-MeOH as the solvent systems, finally afforded compounds 4 and 6-9.

Compound (2 ) . [a],24 +14.5' (c 0.003, CHCI,). IR (film, CHCI,) vmax:

3330, 3010. 2950, 2935, 1735, 1720, 1640, 1490, 1450, 1370, 1240, 1020, 960,

9 10, 890, 750 and 700 cm-' ; ELMS: m/z : 623 ([MI+), 608 ([M - CH31+), 564 ([M

+ H - AcOH]+), 548 ([M - CH, - AcOH]+), 504 ([M - MeNH+=CHPH]+), 444 ([M - HOCOCH,CH(MeNH)PH]+), 180, 179 ([HOCOCH2CH(MeNH)PH]+), and

I20 ([MeNH+=CHPH]+). HR-EI-MS: 623.342 1 (C,,H,,O,N, calcd for

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TAXANE DITERPENOIDS 185

623.3455), 609.3304 (C,SH,,08N, calcd for 609.3424), 444.25 13 (C,,H,,O,,

calcd for 444.2510), 178.0868 (C,,H,,O,N, calcd for 178.0867), 120.0814

(C,H , ,N, calcd for 120.081 3). The I H NMR spectral data see Table 1.

Compound (5) . +10.3' (c 0.005, CHCl,); IR (film, CHCl,) vmax : 3480, 3010,2950,2905, 2850, 1740, 1720, 1670, 1640, 1500, 1370, 1230, 1 160,

1100, 1020, and 760 crn-'; ELMS: d z : 580 ([MI'), 562 (M - H20)+, 520 ([M -

AcOH]+), 502 ([M - H,O - Acorn+), 460 ([M - AcOH]'), 432, 414, 390, 372,

(C33H4009, calcd for 580.2670). The 'H and 13C NMR spectral data see Table 1.

348, 312, 284, 265, 209, 177, 138, 131, 103, and 91. HR-EI-MS d.. 580.2672

ACKNOWLEDGEMENTS The authors are grateful to Mrs. Yuhko Sugiyama for measuring the NMR

spectra. One of the author would like to thank Professor R. L. Liu for collecting and identifying the plant material for this work. The financial support for this study from

the Ministry of Education, Science and Culture of Japan through a grant-in-aid for scientific research is highly appreciated.

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b) Q. W. Shi, T. Oritani, T. Sugiyama, and H. Kiyota, (1998) J. Naz. Prod., 6 1, 1437-1440. c) Q. W. Shi, T. Oritani, and T. Sugiyama, (1998) Nut. Prod.

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Plmtu Med., in press. e) Q. W. Shi, T. Oritani, and T. Sugiyama, (1998) Phytochemistty, in press. f) Q. W. Shi, T. Oritani, and T. Sugiyama, (1998) Biosci. Biochem. Biotech., 6 2, 2263-2266.

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3 . This technique provides a strong molecular ion ([MI') peak at m/z 623. AU the characteristic ions of the side chain and the skeleton are clearly observed.

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1 I . Full details of the isolation and characterization (including 'H NMR and 'H-lH

COSY spectra of the compounds 1-9, and I3C NMR, HMBC and HMQC spectra of the compound 5) of the above compounds are available on request

from the author of correspondence.

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