Intramolecular acyl migrations in taxanes from Taxus brevifolia

Phytochemistry, VoL 34, No. 2, pp. 473-476, 1993

0031-9422,/93 $6.00+0.00 Pergamon Press Ltd

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INTRAMOLECULAR ACYL MIGRATIONS IN TAXANES FROM TAXUS BREVIFOLIA ALEX CHU, LAURENCEB. DAVIN, JAROSLAVZAJICEK,* NORMAN G. LEWISt and RODNEY CROTEAUt Institute of Biological Chemistry, 467 Clark Hall, Washington State University, Pullman, WA 99164, U.S.A.; *University NMR Spectroscopy Center, 27 Fulmer Hall, Washington State University, Pullman, WA 99164, U.S.A.

(Received in revisedform 12 January 1993) Key Word Index--Taxus brevifolia; Taxaceae; yew; bark; diterpenoids; taxol; taxanes; 1-fl-hydroxybaccatin I; acyl group migration.

Abstract--Two new taxane epoxides were isolated from the bark of Taxus brevifolia and their structures were established as lfl,9~-dihydroxy-4fl,20-epoxy-2~,,5~,7fl,10fl,13~-pentaacetoxy-tax-ll-ene and lfl,7fl-dihydroxy-4/~,20epoxy-2~,5~,9~,10~,13u-pentaacetoxy-tax-ll-ene. These two compounds were found to readily isomerize via acyl migration upon standing in CDCI 3 solution and represent the first example ofintramolecular transesterification in this large family of taxane metabolites.

,,

INTRODUCTION

O

Taxol 1 is an highly promising antineoplastic drug obtained commercially from the bark of the Pacific yew (Taxus brevifolia) where it is found in trace amounts (<0.01% dry weight). Nothing is known about the regulation of taxol biosynthesis in plants or the sequence of steps beyond the presumed acyclic precursor, geranylgeranyl pyrophosphate [1]. As part of our continuing investigations on the biosynthesis of taxol 1 [2"1, ~ e taxanes 2 and 3 were isolated from T. brevifolia extracts. Each had spectroscopic properties very similar to that of lfl-hydroxybaceatin 1 4 I-3] and both readily underwent mild acid catalysed isomerization.

Ph

AcO

0

OH

~O~l,,"~O "

H~

6H E~ AcU

o~(~ 1

AcO

~R l

OR 2

RESULTS AND DISCUSSION

The epoxides 2-4 were isolated from concentrated methanol extracts of dry T. brevifolia bark (~ 1 kg), following partitioning between CHC! 3 and H20, and extensive chromatography of the CHCI 3 solubles. The 1H NMR spectra of 2-4 closely resembled each other, except that 2 and 3 contained one less acetoxy substituent than 4 (Table 1); determination of their structures was achieved as follows: FAB mass spectral analysis of 2 showed it to have a molecular formula of C 3 o H 4 2 0 1 3 . The connectivities of the protons in the taxane skeleton of 2 were determined by analysing the DQ-COSY spectrum. Interpretation of 1H and 13CNMR spectra (including HETCOR and HMBC analyses) permitted the positional assignment of functional groups. The spin system derived from H-5fl, H-6~, H-6fl and H-7~, was readily interpreted; the high-field H-5fl triplet at 64.10 was coupled with the tAuthors to whom correspondence should be addressed.

OH

~)Ac

2 : R 1 = H, R 2 = A C 3 : R 1 = AC, R 2 = H 4 : R 1 = R 2 = Ac

1H doublet of doublet of doublets at 61.74 (H-6~, Jsp. ~, = 2.8 Hz~ with the latter showing geminal coupling (J~,.ep = 14.3 Hz) to the 1H multiplet at 62.10 (H-6fl). Both H-6~ and H-6fl resonances were also coupled with the IH doublet of doublets at 65.47 assigned to H-7~, (Je,,7, = 4.4 Hz, J o . 7, = 12.1 Hz). Two doublet of doublets at 62.03 and 2.48 were assigned to the C-14 methylene 473

474

A. CHU et al.

Table 1. 1H chemical shifts (6) and scalar coupling constants (J) of epoxides 2~1 2 (Me2CO-d6)

3 (Me2CO-d6)

4 (CDCI3) [3]

H-2 H-3

5.38 (d, J2a, 3# = 3.4 Hz) 3.17 (d, J = 3.4 Hz)

5.49 (d, J2tt,3# = 3.0 Hz) 3.11 (d, d = 3.0 Hz)

H-5 H-6~t

4.10 (t, Js#,6, = Js#.6# = 2.8 Hz) 1.74 (ddd, Js#.6~ = 2.8 Hz, J6~.7a = 4.4 Hz, J 6 ~ , ~ # = 14.3 Hz) 2.10 (m) 5.47 (dd, J6a, 7a m 4.4 HZ, JaB.Ta = 12.1 Hz) 4.44 (d, J9#, 1o~ = 10.7 Hz) 6.07 (d, J = 10.7 Hz) 6.03 (m) 2.03 (m)

4.07 (t, Js#.6, = J5#,6# = 3.0 Hz)

5.46 (d, J2a,3# = 3.5 Hz) 3.15 (d, J = 3.5 Hz) 4.18 (bs)

H-6fl H-7

H-9 H-10 H-13 H-14a H-14fl

2.48 (dd, Jla#, 14# = 9.6 Hz, J14~,1,~# = 14.9 Hz) H-16 1.19 (s) H-17 1.49 (s) H-18 2.22 (d, J13#,ls = 1.5 Hz) H-19 1.40 (s) H-20 2.29 (d, J = 5.2 Hz) H-20' 3.60 (d, J = 5.2 Hz) COMe 1.92 (s) COMe 1.95 (s) COM__~¢ 2.02 (s) COMe 2.12 (s) COMe 2.23(s) COMe - -

1.84 (m) 2.00 (m) 4.24 (dd, J6a, 7a ~ 5.5 HZ, J6p, Ta = 11.0 Hz) 6.20 (s) 6.20 (s) 6.03 (m) 1.84 (m) 2.50 (dd, Jla#. 14# = 9.5 Hz, J14~,14# = 14.5 Hz) 1.16 (s) 1.63 (s) 2.19 (d, J13#,ls = 1.5 Hz) 1.21 (s) 2.27 (d, J = 5.0 Hz) 3.60 (d, J = 5.0 Hz) 1.96 (s) 2.02 (s) 2.12 (s) 2.14 (s) 2.24 (s) --

protons, H-14~t and H-14fl, respectively, based on their geminal coupling (J14~,~4#= 14.9Hz) and coupling (J~3#, 14~ = 6.8 Hz, J13p. 14# = 9.6 Hz) to the H-13fl multiplet at 6 6.03. The C-20 methylene protons of the oxirane bridge were observed as a characteristic AX system at 62.29 and 3.60 (J20,20, = 5.2 Hz). The H-3ct doublet at 63.17 was correlated with the signal at 65.38 assigned to H-2fl (J2p, 3, = 3.4 Hz). The isolated spin system comprised of doublets at 64.44 and 6.07 was attributed to H9 / / a n d H-10-, with the large vicinal coupling (Jg#,10, = 10.7 Hz) indicative of a t r a n s - o r i e n t e d configuration. Importantly, the H-9fl resonance at 64.44 in 2 was shifted upfield (,-, 1.6 ppm) from that observed for 4, strongly suggesting a free hydroxyl group at C-9 in 2. By contrast, all of the other resonances for protons adjacent to acetate groups at C-2, C-5, C-7, C-10 and C-13 in both 2 and 4 were essentially identical. That 2 contained five acetate groups was determined both by methyl proton resonances at 61.92, 1.95, 2.02, 2.12 and 2.23, and carbon signals at 621.0-21.9 and 6169.6-171.0 corresponding to five methyl and carbonyl groups, respectively. The three singlets at 61.19, 1.40 and 1.49 corresponded to methyl groups at C-19, C-16 and C-17, whereas the 3H doublet at 62.22 corresponded to the C-18 methyl group based upon its long-range coupling with H-13fl at 66.03 (H3#.l s = 1.5 Hz).

1.72 ( m ) 2.10 (m) 5.47 (dd, J6a, Ta = 4.0 Hz, J6#,7a = 11.5 Hz) 6.02 (d, J9#, lOs = 11.5 Hz) 6.19 (d, J = 11.5 Hz) 6.06 (m) 1.85 (dd, J13#,14# = 10.0 Hz, J14~,14# = 15.0 Hz) 2.50 (dd, J~3#,~4# = 10.0 Hz, J14~,l,t# = 15.0 Hz) 1.22 (s) 1.62 (s) 2.21 (d, Jla#,ls = 1.5 Hz) 1.21 (s) 2.28 (d, J = 5.0 Hz) 3.52 (d, J = 5.0 Hz) 1.97 (s) 2.02 (s) 2.03 (s) 2.06 (s) 2.09 (s) 2.20 (s)

H E T C O R analysis was used to assign carbon signals for all proton-bearing carbons. The resonances for C-2, C-5, C-7 and C-13 at 672.5, 78.6, 70.0 and 71.8, respectively, correlated exactly with the corresponding proton signals at 65.38, 4.10, 5.47 and 6.03, and the C-9 and C-10 resonances at 674.3 and 76.5 correlated with the corresponding H-9fl and H-10ct signals at 64.44 and 6.07, respectively. The signal at 675.8 did not show any correlation with any proton signal and could, therefore, be assigned as C-1. The signals at 631.9, 39.9 and 50.3 were assigned to C-6, C-14 and C-20 methylenic carbons due to their correlations with the corresponding proton signals. The signal at 615.1 assigned to the C-19 methyl group was coupled with the 3H singlet at 61.40, whereas the C-18 methyl resonance at 615.7 correlated with the 3H doublet at 62.22. The C16/C~7 signals at 628.6 and 22.8 were coupled with the methyl proton singlets at 61.19 and 1.49, respectively. The H M B C spectrum established three-bond coupling between C-3 (641.9) and the C-19 methyl singlet at 61.40, and this latter signal also exhibited two-bond coupling with the quaternary carbon C-8 at 647.1. Correlation between the proton resonances at 65.38 (H-2fl) and 6.07 (H-10ct) and the carbon signal at 644.2 permitted not only the assignment of this signal to the quaternary carbon C-15, but also the unambiguous distinction between H-9fl and H-10cc Additionally, the

Taxane epoxides H-10ct signal showed two- and three-bond correlations with both olefinic carbons C-11 (6137.7) and C-12 (6139.7), respectively, while the H-9fl signal showed only three-bond coupling with C-11. Lastly, the presence of a free hydroxyl group at C-9 was unambiguously established by defining three-bond correlations between carbonyl resonances and proton signals. Thus, three-bond correlations of acetoxy carbonyl groups (6169.5, 169.9, 170.3, 170.2 and 171.0) were demonstrated to occur with the respective proton signals at 65.38 (H-2f), 4.10 (H-5f), 5.47 (H-7ct), 6.07 (H-10~t) and 6.03 (H-13fl) (i.e. there was no acetoxy group at C-9). Stereochemistry of the molecule was deduced by comparison with 4. Therefore, the structure of 2 was established as 1//,9~t-dihydroxy-4f,20epoxy-2~t,5ct,7fl,10f,13ct-pentaacetoxy-tax-11-ene. FAB mass spectral analysis of 3 also revealed a molecular formula of C3oH42013. The 1H N M R spectrum of the upfield region (61.16-2.24) suggested the presence of four methyl and five acetoxy methyl groups, with the 3H doublet at 62.19 assigned to the C-18 methyl group based upon its long-range coupling with H-13f (J13#.lS = 1.5 Hz) as before. The C-20 methylene protons of the oxirane moiety were again observed as the characteristic AX system (J2o, 20, = 5.0 Hz) at 62.27 and 3.60, respectively. The 1H doublet at 63.11 (J2#.3~ = 3.0Hz) was assigned as H-3ct, being coupled with the H-2fl doublet at 65.49 as previously noted in 2. The H-5f resonance appeared as a 1H triplet at 64.07 (J5#,6= = J5#,6# = 3.0 Hz), and was coupled to both H-6~t and H-6f proton resonances at 61.84 and 2.00. The doublet of doublets at 64.24 was attributed to H-7~t based upon its coupling (J6~, 7= -- 5.5 Hz, J6#, 7~ --- 11.0 Hz) with the methylenic protons at C-6. As noted previously, the H-13f resonance was observed as a 1H multiplet (66.03), whereas by contrast, the signals for H-9f and H-10~t were observed as a 2H singlet at 66.20 indicating that both protons were resonating at the same frequency. However, when the ~H N M R spectrum was recorded using CDCI3 as a solvent, the two protons appeared as an AB system at 66.02 (H9fl) and 6.19 (H-10ct, J9#.lo~ = 11.5 Hz). The principal difference between the ~H NMR spectrum of 3 and 4 was the 1.23 ppm upfield shift of the H-7ct proton from 65.47 to 4.24 suggesting the presence of a free hydroxyl group at C-7. As before, the stereochemistry of 3 was deduced based on its comparison with 2 and 4. The struc-

475

ture of 3 was, therefore, lf,7fl-dihydroxy-4fl,20-epoxy2~t,5ct,9~t,1Off,13~t-pentaacetoxy-tax-11-ene. Interestingly, an unusual facile acid catalysed isomerization between 2 and 3 was observed due to an intramolecular acyl migration between C-7 and C-9. Thus, when pure 2 was dissolved in CDCI 3 and allowed to stand for 30 rain at room temperature, the : H N M R spectrum of this sample revealed a ca 5:4 mixture of 2 and 3. A similar result was also obtained with 3, i.e. both compounds were capable of undergoing acid catalysed isomerization. (No effort was made to determine the rates and optimal conditions for this intereonversion.) Previous X-ray crystallographic studies [4, 5] and conformational calculations [6] for the taxane skeleton have suggested that the preferred conformation for the six-membered A ring is a distorted boat when fused in cisfashion to the boat-chair eight-membered B ring, which, in turn, is linked to a trans-fused distorted chair sixmembered C ring. Examination of a Drieding model with this preferred conformation reveals that the hydroxyl and acetyl functionalities at C-7f or C-9~t are in close proximity with each other, thereby facilitating acyl migration via the ortho-acid intermediate 5 as shown (Scheme 1). Neighbouring group rearrangements of this class (i.e. between vicinal gauche hydroxyl groups in a cis relationship to each other) are frequently observed in carbohydrates 17]. The fact that facile interconversion between 2 and 3 takes place upon mild acid catalysis is in agreement with both molecular model studies and observations on the carbohydrates. However, this phenomenon (i.e. an acid catalysed intramolecular transesterification via acyl migration) has only now been demonstrated to occur in the taxane skeleta, both in this paper and in the following one where migration of acetate moieties between C9 and C~o were observed [8]. Taken together, these observations emphasize the need for care in the isolation and structural identification of taxanes from various Taxus species. EXPERIMENTAL

Silica gel 60 (Merck 230-4(0 mesh) was used for all CC, and solvents were redistilled prior to use. N M R spectra were recorded at 500 MHz (IH), 300 MHz (DQ-COSY and HMBC) and 125.7 MHz (13C and HETCOR) using

HO

AcO

AcO

OH OAc

~'t/OA c

o~~ _

AC OH

~)Ae

2

-0~0

~H ~ , . 0"~" bAc 5

AcO

OAc OH

"~OA~Ac~"/OAc OH ~)Ac

3

Scheme 1. Acid catalysed isomerization between 2 and 3 via the ortho-acid intermediate 5.

476

A. CHU et al.

Me2CO-d 6 as a solvent unless otherwise stated. Chemical shifts of the taxanes are reported in 6 (ppm) relative to the residual H in perdeuterated Me2CO (6 = 2.04) for I H N M R and 6Me2CO-d 6 =29.8 for 13CNMR. All I H N M R spectra were analysed as first order. FT-IR spectra were obtained as thin films. Isolation of 2, 3, and 4. Dried Pacific yew ( T. brevifolia) bark tissue (1 kg) was ground to a fine powder (13 mesh) using a Wiley Mill. The resulting powder was steeped in MeOH (10 1) overnight, filtered and the organic solubles were coned in vacuo to give a dark brown syrupy residue. The residue was partitioned between H 2 0 (2.5 1) and CHC13 (2 × 2.51) with the CHCI3 solubles then combined, dried (Na2SO4), and coned in vacuo to a syrup (32 g). The resulting extract was reconstituted in MeOH (20 mi) and applied to a silica gel column (320 g, 5 × 31 cm), eluted with CHC13(21 ) and a gradient of CHCI3-MeOH (99.5:0.5-1:1). Eight frs (1.25 1) were obtained and each was evapd to dryness in vacuo. Ft. 6 (2.92 g) was reconstituted in acetone (5 ml) and applied to a silica gel column (210 g, 5 x 22 era) eluted with hexanes-Me2CO (3:1) to give the mixt. of 2, 3 and 4 (0.72 g) as a pale brown foam. Reehromatography of this mixt. (200 g, 5 × 21 era) eluted with Mc2CO-CH2C12 (8: 92) first gave 4 (93.5 rag) as colourless crystals, followed by the mixt. of 2 and 3 (83.0 rag). Further chromatography of the mixt. containing 2 and 3 (180 g, 5 × 19 cm) eluted with EtOAc-hexane (6:4) afforded pure 2 (15.4 rag) and 3 (14.6 rag) as amorphous, colourless powders. l ~,9~-Dihydroxy-4~,20-epo x y-2~,5~,7 p,108,13~-pentaacetoxy-tax-ll-ene 2. IH NMR (500 MHz, Table 1); 13C NMR (125.7 MHz, Me2CO-d6): 615.1 (C-19), 15.7 (C-18), 21.0 (COMe, C-7), 21.1 (COMe, C-2), 21.5 (COMe, C-13), 21.6 (COMe, C-10), 21.9 (COMe, C-5), 22.8 (C-17), 28.6 (C-16), 31.9 (C-6), 39.9 (C-14), 41.9 (C-3), 44.2 (C-15), 47.1 (C-8), 50.3 (C-20), 59.2 (C-4), 70.0 (C-7), 71.8 (C-13), 72.5 (C-2), 74.3 (C-10), 75.8 (C-I), 76.5 (C-9), 78.6 (C-5), 137.7 (C-11), 139.7 (C-12), 169.6 (COMe, C-2),

169.9 (COMe, C-5), 170.2 (COMe, C-10), 170.3 (COMe, C-7), 171.0 (COMe, C-13); FT-IR veto-l: 3483, 2189, 1739, 1437, 1373, 1240, 1138. FABMS (m/z): 611 [MH] +, 593 [ M H - H 2 0 ] +, 551 [ M H - H O A c ] +, 431 [ M H - 3 x H O A c ] +.

I~,7~-Dihydroxy-4~,20-ePo xy-2~,5~,9n,108,13c~-pentaacetoxy-tax-I l-ene 3. IH N M R (500 MHz, Table I);FTIR v cm- i:3522, 2989, 1739, 1436, 1373, 1239, 1138, 1049, F A B M S (m/z): 611 [ M H ] +, 593 [ M H - H 2 0 ] +, 551 [ M H - HOAc] +, 533 [ M H - H 2 0 - H O A c ] +, 491 [ M H - 2 x H O A c ] +. Acknowledgements--Financial support from N I H CA55254 and RR063141 (for the purchase of 500 MHz spectrometer) is gratefully acknowledged. The authors also wish to thank Dr Nicholas Wheeler, Weyerhauser, Centralia, WA, for providing live plant material and to Dr Bill Siems (WSU) for measurement of the mass spectra. REFERENCES

1. Gueritte-Voegelein, F., Guenard, D. and Potier, P. (1987) J. Nat. Prod. 50, 9. 2. Chu, A., Zajicek, J., Davin, L. B., Lewis, N. G. and Croteau, R. B. (1992) Phytochemistry 31, 4249. 3. Miller, R. W., Powdl, R. G., Smith, C. R., Arnold, E. and Clardy, J. (1981) J. Org. Chem. 46, 1469. 4. Shiro, M. and Koyama, H. (1971) J. Chem. Soc. B 1342. 5. Castellano, E. E. and Hodder, O. J. R. (1973) Acta Crystallogr. B 29, 2566. 6. Senilh, V., Gueritt¢, F., Guenard, D., Colin, M. and Potier, P. (1984) C.R. Acad. Sci. Ser. 2 299, 1039. 7. Haines, A. H. (1976) Adv. Carb. Chem. Biochem. 33, 100. 8. Chmurny, G. N., Paukstelis, J. V., Alvarado, A. B., McGuire, M. T., Shader, K. M., Muschik, G. M. and Hilton, B. D. (1993) Phytochemistry 34, 477.