Brevifoliol: A structure revision

003l-9422/93 $6.00+ 0.00 Vol. 34, No. 1, pp. 269-271, 1993 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0 1993Pergamon PressLtd zyxwvutsrq Printed in GreatBritain. Phytochemistry, BREVIFOLIOL: A STRUCTURE REVISION ALEX CHU, JAROSLAVZAJICEK,* G. H. NEIL TOWER&~ CHANTAL M. SOUCY-BREAU,~ NORMAN G. LEWIS RODNEY CROTEAU~ and Institute of Biological Chemistry, 467 Clark Hall and *University NMR Spectroscopy Center, 27 Fulmer Hall, Washington State University, Pullman, WA 99164, U.S.A., tDepartment of Botany and $Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 2Bl zyxwvutsrqponmlkjihgfedcbaZYXWVUT (Received in revised form 2 M arch 1993) IN HONOUR OF PROFESSOR Key Word Index--Taxus breuifolia; JEFFREY Taxaceae; HARBORNE’S taxanes; SIXTY-FIFTH BIRTHDAY brevifoliol. Abstract-Brevifoliol is an abundant metabolite found in the needles of the Pacific yew (Tuxus breoifolia). Its structure has been revised such that the acetate and benzoate functionalities originally considered to be attached to C-10 and C7, respectively, are transposed, i.e. brevifoliol is lOB-benzoxy-78,9a-diacetoxy-18,5a,13cr-trihydroxy-taxa-4(20),1 ldiene. INTRODUcTION During an investigation of the taxanes present in Tams breuifolia needles, an abundant metabolite (ca 1.7 mg g- 1 fresh weight), trivially named brevifoliol, was isolated and identified as structure 1 [l]. As part of our continuing studies on the biosynthesis of taxol (3) and related metabolites [2, 31, taxanes from yew needles are being systematically isolated and identified. In this paper, evidence is provided for the revision of the structure of brevifoliol to 2, i.e. where the acetate and benzoate functionalities at C-10 and C-7 are transposed. 1 :R’ = COPh, R2 = AC 2 : R’ = AC, R2 = COPh RESULTSAND DISCUSSION The 1H and “C chemical shifts reported for brevifoliol (i.e. proposed structure 1) [l] suggested a taxa-4(20), 1ldiene skeleton with six oxygenated functionalities containing one benzoxy, two acetoxy and three hydroxyl groups, respectively. On the basis of proton chemical shifts, the hydroxyl groups were assigned to C-l, C-5 and C-13, with ester groups at C-7, C-9 and C-10. Although an extensive NMR study had been performed on brevifoliol which resulted in the proposed structure 1, assignment of the benzoate to C-7, and the two acetate moieties to C-9 and C-10 was arbitrary since the positions of these substituents could not be unambiguously assigned simply on the basis of ‘H chemical shift differences. Moreover, neither was it possible to make an unambiguous assignment of quaternary carbons using only one-bond HETCOR experiments; as a result, the assignments for the two quaternary centres C-8 and C-15 were also uncertain. In this study, the connectivities of protons in brevifoliol were determined by a DQ-COSY experiment as follows: Ph 3 the spin system derived from H-2a, H-2/I and H-3a was interpreted by using H-3x as a starting point. Thus, the H-3a doublet at 62.77 was coupled with the H-2B doublet of doublets at 62.36 (JZB,3U=8.8 Hz) which was also geminally coupled (.J2=.Zs= 13.0 Hz) to the H-2a doublet at 6 1.49 (Table 1).The broad singlet at 64.45 was assigned to H-5p. The two doublet of doublets resonances (6 1.84 and 2.02) were assigned to C-6 methylene protons, H-60! and H-68, respectively, based upon their geminal coupling Us.. 61= 14.0 Hz) and their coupling with the H-7a doublet of doublets at 65.57 (Jsn,,== 5.0 Hz, JeB,,. $Authors to whom correspondence should be addressed. 269 270 A. CHU et al. Table 1. Revised ‘H and 13C NMR assignments for brevifohol zyxwvutsrqponmlkjihgfedcbaZYXWVU C ‘H 1 2 75.9 29.1 1.49,Ha (m) 2.36,H/?(dd.J28.3.=8.8Hz,J,,~,B=13.0Hz) 37.9 2.77 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA (d, J,,, 3a= 8.8 Hz) 149.0 4.45 (br s) 72.4 1.84,Ha (dd, J,,,,,=S.O Hz, Jsa,68= 14.0 Hz) 36.0 2.02,H/I (dd, J,,,,,=~L~Hz,J,,,,~=~~.OHZ) 70.1 5.57 (dd, J,,, ,== 5.0 Hz, J,,, 7o1 = 11.4 Hz) 45.0 6.06 (hd) 77.1 6.53 (d, J,,, ,,,.= 10.6 Hz) 70.2 133.9 151.5 76.7 4.38’0, J1ao,14a = J, 3p.1.q= 7.4 Hz) 47.3 2.46,Hp (dd. J13B,,4827.4 Hz, J141,148= 14.0 Hz) 1.29,Ha-(dd, J,,@ , 14.57.4 Hz, J,,,, 148= 14.0 Hz) 62.4 26.9 1.05 (s) 24.8 1.35 (s) 12.0 2.01 (s) 12.9 0.90 (s) 112.1 4.83,Ha (s) 5.19.Hb (s) 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OCOMe (C-7) OCOMe (C-7) OCOrV& (C-9) OCOMe (C-9) 1.75 (s) 20.7 169.9 2.08 (s) OCOPh (C-10) ipso-OCOPh o-OCOPh m-OCOPh p-OCOPh ‘3C 7.87 (d, J= 7.8 Hz) 7.43 (t. J = 7.8 Hz) 7.56 (t, J = 7.8 Hz) = 11.4 Hz). Next, an isolated spin system comprised of two sets of doublets at 66.06 and 6.53 was attributed to H-9P and H-lOa, where the magnitude of the vicinal coupling (Js,, 1oa= 10.6 Hz) indicated both protons were in a trans-oriented configuration relative to each other. The 1H doublet of doublets at 62.46 was assigned to H148 and also showed geminal coupling with the H-14c( doublet of doublets at 61.29 (J,4a,,4,r= 14.0 Hz). The stereochemistry of the H-14a and H-14/I was deduced from a 1D steady-state NOE experiment, in which irradiation of the signal at S 1.05 (H-16) caused an enhancement (9%) of the signal at 62.46 (H-148). Both protons were coupled to the H-13p triplet at 64.38 NJ1 sa, 148 N 7.4 Hz). The exocyclic methylenic (J138,14a protons of C-20 were observed as a characteristic pair of singlets at 64.83 and 5.19. The 2D NOE spectrum (mixing time 200 ms) showed a cross-peak between signals at 6 1.49 (H-21x) and 4.83, thereby allowing the signal at 64.83 to be assigned to the proton H-20a pointing towards the C-2 carbon atom, and the signal at 65.19 to the proton H-20b. HETCOR analysis was next used to assign carbon signals for all proton-bearing carbons. The upfield signal 21.4 170.5 164.3 129.3 129.4 128.7 133.2 at 6 12.0 was assigned to the C- 18methyl group as it could be correlated with the 3H singlet at 62.01, whereas the C19 methyl resonance at 612.9 correlated with the 3H singlet at 60.90. The carbon signals at 626.9 and 24.8 were coupled with the 3H singlets at 6 1.05 and 1.35 which were assigned to C-16 and C-17, respectively. The stereochemistry of the C-16 and C-17 methyl groups were assigned by interpretation of the 2D NOE spectrum, in which the signal at 6 1.05 (H-16) exhibited correlation with the H138 signal at 64.38. Heteronuclear multiple bond correlation (HMBC) experiments were next used to assign quaternary carbons and attachments of various ester functionalities. The HMBC spectrum established a threebond correlation between the C-19 methyl signal at 60.90 and the carbon signal at 637.9 (C-3). This same proton signal also exhibited a two-bond coupling with the carbon signal at 645.0 which was therefore assigned to the quaternary carbon at C-8. Interpretation of the HMBC spectrum revealed a three-bond correlation between the H-10~ (66.53) signal and the carbon signal at S62.4, thereby allowing the assignment of this carbon signal to the remaining quaternary centre C-15, i.e. both quaternary resonances for C-8 and C-15 have been unambigu- Brevifolioka structure revision 271 ously established and are now transposed with respect to Washington State University greenhouse facilities. the original proposed structure 1 for brevifoliol. The most Isolation of 2. Fresh T. breuifolia needles (150 g) were important correlation extracted from the information frozen (liquid N2) for 5 min, and subsequently homogencontained in the HMBC spectra was the cross-peak ized in MeOH (200 ml) using a Waring blender (10 min). between ‘H signals of the ortho-protons of the phenyl The resulting green paste was diluted with MeOH (1 1) ring (67.87) with the carbonyl absorption at 6164.3, stirred at room temp. for 3 hr, filtered and coned in uacuo which permitted the assignment of the carbonyl carbon of to give a green residue (14.5 g). This residue was partithe benzoate. This same carbonyl resonance also showed tioned between H,O (11) and CHCl, (1 1). The CHCl, a correlation with the ‘H signal at 66.53 (H-lox) proving solubles were dried (Na,SO,), filtered and coned in uacuo that the benzoxy moiety was attached to C-10 and not C- to afford a green gum (3.7 g). The residue was taken up in 7 as originally proposed [l]. The remaining two carbon a minimum amount of CHCI,, applied to a silica gel resonances at 6169.9 and 170.5 were assigned to the column (300 g, 5 x 31 cm) and eluted sequentially with signals derived from the acetoxy carbonyl carbons based CHCl, (2 1), MeOH-CHCI, (1:99, 2 I), MeOH-CHCI, upon their two-bond coupling with the 3H singlets at (2: 98,2 1),MeOH-CHCl, (5 : 95,2 1)and MeOHCHCl, 6 1.75 and 2.08, respectively. The attachment of both (50:50, 2 1) to give 80 frs (125 ml). acetoxy groups at C-7 and C-9 was again unambiguously Frs (3442) containing 2 were combined and coned to established by defining the three-bond correlations be- give a yellow foam (578.7 mg). The resulting foam was tween the acetoxy carbonyl resonances and proton sig- dissolved in a minimum amount of EtOAc and applied to nals. Thus, three-bond correlations of carbonyl resona silica gel column (310 g, 5 x 32 cm) eluted with ances (6 169.9 and 170.5) were demonstrated to occur with hexane-EtOAc (2:3). Frs containing 2 were combined the respective proton signals at 65.57 (H-7x) and 6.06 (H- and evapd to dryness, following which recrystallization 9/I), i.e. in the original structure zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 1 of brevifoliol, the acetate from hexane-Me&O afforded pure crystalline 2 and benzoxy groups at C-10 and C-7 are in fact reversed. (215 mg). [a&-28” (CHCl,; c 1.02); mp 200-205” The structure for brevifoliol was, therefore, established as (lit. 20&203” Cl]); FABMS m/z: 557 [MH]+, 539 [MH-PhCOOH]+, 417 lO~-benzoxy-7#?,9a-diacetoxy-l/I,5cc,l3cr-trihydroxy[MH-HzO]+, 435 375 [MH-PhCOOH taxa-4(20),1 I-diene (2). (A more comprehensive study on [MH-PhCOOH-H,O]+, the X-ray crystal structure of brevifoliol 2 by Piers, E., - HOAc] +, ‘H and ’ 3C NMR: Table 1; FTIR v,,, cm- ‘: Saucy-Breau, C. and Towers, G. H. N. will be discussed in 3367, 1733, 1652, 1602, 1585, 1451, 1373, 1264 and 1178. a later report.) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Acknowledgements-Financial support from NIH and RR063141 for the purchase of (CA55254) EXPERIMENTAL zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 500 MHz NMR spectrometer is gratefully acknowledged. Instrumentation and chromatography materials. Silica The authors also wish to thank Dr Nicholas Wheeler gel 60 (Merck 23&400 mesh) was used for all CC and (Weyerhauser Co., Centralia, WA) for providing live solvents were redistilled prior to use. NMR spectra were plant material and Dr William Siems (WSU) for measurerecorded at 500 MHz (‘H), 300 MHz (DQ-COSY and ment of the mass spectra. HMBC) and 125.7 MHz (13C and HETCOR) using CDCl, as a solvent. Chemical shifts of the taxanes are REFERENCES reported in 6 (ppm) using TMS as an int. standard. All Balza, F., Tachibana, S., Barrios, H. and Towers, G. H. ‘HNMR spectra were analysed as first order. FTIR N. (1991) Phytochemistry 30, 1613. spectra were obtained as thin films. Optical rotations Chu, A., Zajicek, J., Davin, L. B., Lewis, N. G. and were measured at 23” using a mercury lamp source at Croteau, R. B. (1992) Phytochemistry 31,4249. 577 nm. Chu, A., Davin, L. B., Zajicek, J., Lewis, N. G. and Plant materials. Taxus brevifolia plants, obtained from Croteau, R. B. (1993) Phytochemistry 34 (in press). Weyerhauser Co. (Centralia, WA), were maintained in