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A 3,11-cyclotaxane from Taxus baccata
Phytochemistry, Vol. 31, No. 12,pp.4259-4262,1992 Printed in GreatBritain.
0031 9422/92$5.00+0.00 Q 1992Pergamon PressLtd
A 3,l l-CYCLOTAXANE
FROM TAXUS BACCATA
GIOVANNI APPENDINO,* PATRIZIA Lusso, PIERLUIGIGARIBOLDI,~ Ezra BOMBARDELLISand BRUNO GABETTA$ Dipartimentodi
Scienza e Tecnologiade1Farmaco, Via PietroGiuria 9, I-10125 Torino, Italy; fDipartimento di Scienze Chimiche. via S. Agostino 1, I-62032 &merino (MC), Italy; $Indena S.p.A., via ~~monti 99, I-20141 Milano, Italy; $Laboratori Ricerca e Sviluppo, Inverni della Bet%, via Ripamonti 99, I-20141 Milano, Italy (Received 9 March 1992) Key Word Index-Tams
baccuta;Taxaceaq diterpenoids; S-cinnamoyl-l~~tylphototax~ci~
taxi&s.
Abstract-A collection of yew needles afforded a taxane diester characterized by an additional bond between C-3 and C-11. The structure was elucidated by spectral methods, and further confirmed by synthesis from a known taxane derivative. A general procedure for the photocyclization of taxicins is reported.
~~ODUffrON
Although taxol (1) shows exceptionally promising antitumour properties, its development as a drug has been hampered by its limited availability and relative lipophili~ty; as a result the supply of 1 has been scarce and formulation difficult [l]. To circumvent these difficulties, semisynthetic analogues prepared from lO-deacetylbaccatin III (2) are currently being evaluated. Among these products, taxotere (3) emerged as the leading compound [l]; 3 is actually an analogue of IOdeacetyltaxol (4), bearing a tert-butoxycarbonyl group instead of a benzoyl group on the aminoacidic side-chain. We became interested in the isolation of lo-deacetyltaxol from the needles of the European yew (Tuxars buccata L.), the current source of lO-deacetylbaccatin III [l]. Preliminary analysis of some collections from the Northern part of Italy, showed that some samples contained 4. However, the content was low, and the final purification of 4 was difficult due to the presence of another taxane (5) having similar chromatographic features. We present here the structure elucidation and the partial synthesis of this compound.
The location of the acetyi was confirmed by the synthesis of 5 from a known taxane derivative. Irradiation of 5-cinnamoyl-lO-acetyltaxicin I (12) [4] with a lowpressure mercury lamp gave 5 in quantitative yield The phot~~i~tion of 12 was accompanied by partial isome&ration of the E-cinnamoyl residue, and the semisynthetic material was an unseparable mixture (ca 5 : 1) of E- and Z-isomers, whereas the natural product was stereochemically pure (E-isomer). Irradiation of other taxi&-I and -11 derivatives (l&15) gave, in quantitative yield, their corresponding 3,l l-cycloderivatives (9-l 1, respectively). In all cases the phototaxicins were obtained
BFSULXSAND DISCUSSION Column chromatography of the plant extract gave a ca 1: 1.5 mixture of IO-deacetyltaxol and 5 as a powder (yield: 0.~0% of dried needles). These compounds could be separated by repeated column chromatography or, more conveniently, by HPLC. Compound 5 showed spectral features different from those of the more common taxanes; in particular, one of the methyls gave rise to an NMR doublet, a feature characteristic of 3,l i-cyclotaxanes, a rare class of taxanes of which only three examples are known [2,3]. Oxidation of 5 (PDC) gave the diketone 6 and the triketone 7: these products showed well-resolved NMR spectra that allowed us to assign the structure 5 to the natural product. *Author to whom correspondence should be addressed. 4259
G. APPENDINOet ai.
4260
OR4
5
R’ OH
6
OH
OR4
R3
R2
R4
uo%PH =o
7
OH
=o
8 9 10 11
OH OH OH A
uO%SH
aOH,PH aOAc$H aOAc$H
Mutt.
5
2
d
4.76
5 68 6a 7a 7b 9 10 12 i4a 148 16 17 18 19 20a 20b 2:6 3’,4’,5 7 8 OAc
t (W
5.57
m m
2.16 1.70 1.90 1.30 4.38 5.40 3.48 3.03 2.35 1.10 1.38 1.29 1.38 5.84 5.59 7.55 7.38 7.66 6.39 2.15
m d” d :: d s ZI s s (br) s (W m 7 d s s s
R2
R3
R4
OH
H
H
AC
AC
aOH$H =o
12
AC
130HH
AC
aOAc,pH aOAc$H
AC
14 15
aOAc,pH aOAc,PH
AC
OH H
AcH AC
AC
AC
AC&AC
H
AC
1. ‘H NMR data for compounds
H
R’
aOH,PH
as mixtures of E- and Z-isomers at the cinnamoyl residue, but reisomerization of the Z-isomer could be brought about by irradiating the photocyclization mixture in the presence of diphenyldisulphide [SJ. Interestingly, if this irradiation was carried out in the presence of iodine, a mixture containing mainly the Z-isomer was obtained instead. The transannular photocyclization of taxicins was discovered by Nakanishi [2], and presumably takes place via a C-3, C-11 diradical, formed by hydrogen transfer from C-3 to C-12, the enone r- carbon 163. The reaction is
Table
OR3
6
7
5.63 2.30 1.60 2.12 0.96 4.32 d (br) 5.64 3.58 2.69 2.55 1.26 1.18 1.42 1.24 ‘5.59 5.05 7.58 7.39 7.66 6.35 2.19
5.66 * * x
remarkable, since in photocyclizations of this type hydrogen transfer to the enone p-carbon has generally been observed [WX]. The stereochemistry of the secondary methyl was confirmed by NOE experiments (see below), and is in accordance with this mechanism, since in taxicins H-3 is a-orientated 141. The facility of the photocyclization reaction raises the question of whether 5 is a natural product or an artefact
5-11 (300MHz, CDCI,, TMS as int. standard)
” 6.23 s 3.25 2.68 2.54 0.83 1.30 1.32 1.17 5.58 4.95 7.5t 7.33 7.65 6.39 2.22
8
9
10
11
4.82 5.60 2.18 1.76 1.76 1.26 5.64 5.64 3.45 3.03 2.36 1.08 1.50 1.26 1.36 5.88 5.61 7.54 7.38 1.65 6.38 2.04 2.04
4.72 5.44 * * *
5.63 5.56 * * *
1s.41 4.33 3.40 2.94 2.29 1.04 1.39 1.34 1.31 5.91 5.51 7.47 7.30 7.58 6.29 2.05
g.63 m 5.67 m 3.41 2.81 2.34 1.05 1.56 1.21 1.22 6.06 5.80 7.48 7.30 7.60 6.3t 2.06 1.99 1.98
ca ca * * * * ca ca
*The corresponding signals could not be located. foverlapped signals. J(Hz): most coupling constants were virtually the same for 5-11;those for 5 are given as representative: ~9.2; J 9.10--98. 1 J ,z,,s=7.2; J14a,14P=20.0; J,s,,.=16.4 Hz. For 11: J1,,,=7.1.
J,,,=
5.70 rnt 5.70 mt
5.70 mi 5.70 mt 3.54 2.58 2.53 dd 1.23 1.51 1.30 1.33 5.86 5.7t 7.56 7.38 7.86 6.40 2.08 2.06 2.06
1.8; J,,,,=
J5,68
A 3,11-cyclotaxane from Taxus baccata Table 2. 13C NMR data of compounds 5,6 and 8 (75.43 MHZ, CD&, TMS as int. standard)
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1’ 2’6 3’5’ 7 8 9 AC
Mult.
d
d d d
4 4
5
6
8
78.1 78.5 61.9 142.1 76.1 25.9 29.3 45.7 82.6 84.0 56.4 51.0 214.2 46.5 45.0 23.3 22.9 15.8 25.2 126.7 134.1 128.7 128.1 145.4 117.5 165.9 172.2
80.8 211.1 s 67.9 143.0 74.0 26.2 29.3 46.7 82.4 85.4 55.0 50.3 216.4 48.1 44.1 21.8 21.9 15.7 25.5 128.4 134.2 128.9 128.2 145.6 117.4 165.4 172.3
21.2
21.3
78.3 78.8 61.8 141.4 76.5 25.8 31.0 44.9 82.5 79.7 56.2 51.3 214.4 46.5 44.9 23.5 22.7 15.8 25.9 128.8 134.1 128.8 128.2 145.5 117.6 166.0 171.0 170.0 21.1 21.0
Long-range lH-‘% correlations found in the FLOCK experiment on 8 (only quaternary aliphatic carbons are shown): C-l: C-3: C-8: C-11: C-15:
H-2, H-14a, H-14/3, H-16, H-17 H-2, H-19, H-2Oa, H-20b H-2, H-19 H-9, H-10, H-12, H-16, H-17, H-18 H-10, H-12, H-14a, H-16, H-17
formed from 12,the major taxane present in the collection of yew we investigated [4]. We believe 5 is a natural product on account of the following facts: (i) the natural product is stereochemically pure, whereas the semisynthetic product is a mixture of E- and Z-cinnamates; (ii) the photochemical reaction required a low-pressure mercury lamp (A< 300 nm) and quartz tubes. In pyrex glassware, the material used for processing the extracts, and with light of lower frequency, no reaction took place. It is worth noting that the secondary hydroxyl at C-2 shows a remarkable difference in reactivity within the taxi&s and phototaxicins series; thus, treatment of phototaxicin 5 with an excess of Ac,O did not a&t the hydroxyl at C-2, and gave the C-9 monoacetate 8, whereas under the same conditions the corresponding taxi& I derivative 12 afforded a 2,9-diacetate [4]. Furthermore, the oxidation of 5 with PDC gave the stable
4261
ketones 6 and 7, whereas attempts to oxidize 12 led to its complete degradation [4]. No ’ CNMR data have been reported for 3,11-cycletaxanes, and a thorough investigation on compounds $6 and 8 was thus undertaken. The analysis of the ‘H NMR spectra did not allow a straightforward assignment of all signals due to overlapping and the presence of several singlets which prevented us from ascertaining the proton connectivities. However, the 2D-C, H correlation spectra solved most ambiguities, allowing the unambiguous assignment of all protons with the exception of the methyl singlets (H-16, H-17 and H-19). This also allowed us to evaluate the coupling constant between H-9 and H-10 (9.0 Hz), that are isochronous in 8. As to the 13C NMR spectra, most resonances of the protonated carbons could be assigned unambigously by means of the 2D-C, H correlation spectra. The assignments of C-6, C-7, C-16, C17, C-19 and the quaternary carbons were obtained through the 2D-C, H correlation spectra via long-range (two and three bonds) C-H coupling constants. The FLOCK sequence [9] displaying good sensitivity and perfect supression of the lJ,_, cross-peaks was used. The relative stereochemistry and the conformation were assessed by chemical shift and coupling constant considerations, and by analysis of the ROESY spectra (Table 3). The downfield position of H-12 (3.4S3.50 ppm) and the presence of a ROE-effect between H-16 and H-18 are in accordance with a /?-orientation for the secondary methyl at C-12. A relevant downfield effect induced by the C-13 carbonyl group on H-20a { A6 (H-20a-H20b) = +0.25 ppm in 5; -0.10 ppm in 12 [4]} as well as diagnostic ROE effects (H-20a-H-12; H-lO-H-6cr; H-7’-H-12) and the coupling pattern of H-S showed that ring D, the only flexible part of the molecule, adopts a boat conformation (4*7B-type [lo]) with the cinnamoyl residue pseudo-axial. In taxicins derivatives, this ring adopts instead a chair conformation [%,-type (lo)] [4], and the cinnamoyl residue is axial. This is responsible for the different splitting pattern of H-5 within compounds of the two
Table 3. Cross-peaks detected in the ROESY spectrum of 5. The intensities of the peaks have been qualitatively evaluated as follows: s = strong m = mdium, w = weak; similar results were also found for 6 and 8 H-2: H-5: H-68: H-6X H-7a: H-7b: H-9: H-10: H-12: H-14a: H-14P: H-16: H-17: H-18: H-19: H-20a: H-20b: H-7’: H-8’:
H-17 (s), H-19 (s) H-6B (w) H-5 (w), H-6a (s) H-6/I (s), H-10 (m) H-7b (s) H-7a (s) H-17 (s), H-19 (s), &&CO- (m) H-6a (m), H-12 (s), H-18 (m) H-10 (s), H-20a (s), H-18 (s), H-7’ (s) H-14/I (s), H-20a (m) H-14~ (s), H-16 (w) H-148 (w), H-18 (m), H-17 (w) H-16 (w), H-9 (s), H-2 (m) H-lo(m), H-12 (s), H-7’ (w), H-16(m) H-9 (s) H-2Ob (s), H-14~1(m), H-12 (s) H-20a (8) H-12 (s), H-18 (w) H-2’ (s), H-6’ (s)
G. APPENDINO et al.
4262
classes (f, J = ca 9 Hz in phototaxicins; taxicins) [4].
t, J =
ca
2.5 Hz in
EXPERIMENTAL
Prep. HPLC: Waters microporasil column (0.8 x 30 cm), detection by a Waters differential refractometer 3401; NMR: see ref. [4]. PIant material. Yew needles were obtained from plants growing in a public garden in Monza (Mi, Italy), and were collected in February 1991. A voucher specimen is kept at the laboratory of the University of Torino. Isolation of5. Dried needles and small branches (500 kg) were extracted with EtOH at room temp., and the extract purified as described in ref. [4], to give 3.5 kg of residue. Part of this (330 g) was fractioned by CC on silica gel, using CHCI, containing increasing amounts of MeOH as eluant. Fractions eluted with CHCI,-MeOH (19: 1) contained 4 and 5. After two additional separations by CC (hexane-EtOAc, 3:7), 1.88 g (0.0040%) of a powder was obtained; this material was a 1: 1.5 mixture (‘H NMR, 200 MHz) of 4 and 5, that could be sepd by further CC (hexane-EtOAc, 1:9) or by HPLC (hexane_EtOAc, 1:9). 5-Cinnamoyl-lo-acetylphototaxicin I (5). Powder, mp 126”, [z]$” +7.5 (CH,Cl,: c 0.77); UV A%:” nm: 279, 217, 201; IR ~5:; cm -I: 3475,1700,1630,1450, tj70,1245,1~,9~,~, 770, 710; CIMS (IriH,) 140 eV, m/z (rel. int.): 556 [C,,H,,Os +NH‘,]+ [M+NHl]+ (100). Oxidation of5. A 100 mg (0.18 mM) sample of 5 was dissolved in 3 ml dry CH,Cl,, and excess PDC (210 mg, 3 mol. equiv.) was added. After stirring for 20 min at room temp., the reaction was diluted with Et,0 and filtered on a short column of celite. After removal of the sotvent, the reside was purified by CC (7.5 g silica gel, hexane-EtOAc, 3:2 as eluant) to give 9 mg 6 and 39 mg 7. 2-Dehydro-lo-acetyl-5-cinnamoylphototaxicin I (6). Powder, mp 73-76” [~}2’+44 (CH,Cl,; c 0.76); UV 22:” nm: 280,217, 201; IR YE:;cm-‘: 3460,1750,1710,1640,1450,1380,1240,1210, 1160, 1100, 1010, 715; CIMS (NH,) 140 eV, m/z (rel. int.): 554 [C31H3608+NH4]+ [M+NHd+ (100). 2,6-~isdekydro-lO-aeety~-S-c~n~amay~pkotota~~c~~ I (7). Oil, [~]g +4.h (CH,Cl,; c f.5); UV i.EzhlHnm: 282, 217, 203; IR “l$i” filmcm- 1: 1730, 1645, 1580,1450, 1370, 1020,990,940, 800,770,740, 710,690; CIMS (NH,) 140 eV, m/z (tel. int.): 552 [CJ1H3.+0s+NHI]+ [M+NHd+ (100). Acetylation of 5. A 200 mg sample of5 was dissolved in 2 ml dry pyridine, and 2 ml Ac,O were added. After stirring overnight at room temp., the reaction was worked up by the addition of MeOH and ice, and extracted with CH,C12. After washing with
dil. HCl, sat. NaHCO, and brine, the solvent was removed and the residue purified by CC (15 g silica gel, hexane-EtOAc, 3 : 7 as eluant) to give 170 mg 8 as a powder; mp 90”; UV 122” nm: 279, 217,203; IR vi:; cm-‘: 3450,1750,1710,1640,1580,1455,1375, 1250, 1160, 1050, 990,950, 770, 715, 590, CIMS (NH,) 140 eV, m/z (rel. int.): 598 [C31H3s08+NH4]+ [M+NH,]+ (100). Pkotocyclization qf taxicins. Reaction of 14 is reported as repr~ntative. In a quartz phot~hemical tube, a 105 mg sampie of 5-cinnamoyl-2,9,1O_tetraacetyltaxicin I (14) [4] was dissolved in 50 ml MeOH-EtOH (1: 5); the soln was flushed with N, for 5 min and then irradiated with a low-pressure Hg lamp in a Rayonet photochemical reactor. The reaction was followed by TLC (hexane_EtOAc, 3 : 7, R, 14: 0.55; R,10: 0.46). After 5 hr the reaction mixt. was worked-up by evapn of the solvent, and the residue @a 5: 1 mixture of E- and Z-isomers, ‘H NMR, 400 MHz) was redissolved in 7 ml EtOAc, and then 13 ml hexane and 37 mg (1 mol. equiv.) diphenyldisulphide was added. The soln was irradiated for 14 hr with a halogen lamp, and then evapd. The residue was mixed with silica gel (ca 300 mg) and charged at the top of a short column (25 g) of silica gel. Washing with hexane-EtOAc, 9: 1 removed diphenyldisulphide, and washing with hexane-EtOAc, (1: 1) gave 102 mg IO (yield 100%) as a stereochemically pure (‘H NMR control, 400 MHz) oil. Taxicins 12, 13 and 15 were photocyclized in the same way, giving the corresponding 3,iO~clotaxicins (5, 9 and 11) in quantitative yield.
REFERENCES 1. Borman, S. (1991) Chem Eng. News (21 Sept), 11. 2. Chiang, H. C., Woods, M. C., Nakadaira, Y. and Nakanishi, K. (1967) 3. Ckem. Sot., Ckem. Commun. 1201. 3. Ettouati, L., Ahond, A., Convert, O., Poupat, C. and Potier, P. (1989) Bull. Sot. Ckim. Fr. 687. 4. Appendino, G., Garibaldi, P, Pisetta, A., Bombardelfi, E. and Gabetta, B. (1992) Phytockemistry 31, 4253. 5. Miyata, O., Shinada, T., Ninomiya, I. and Naito, T. (1990)
Syntk~sis 1123. 6. Wolff, S., Schreiber, W. L., Smith III, A. B. and Agosta, C. (1972) J. Am. Ckem. Sot. 94, 7797. 7. Tobe, Y., Iseki, T., Kakiuchi, K. and Odaira, Y. (1984) Tetra~dron Letters 23, 3895. 8. Herz, W. and Nair, M. G. (1967) J. Am. Chem. 89, 5474. 9. Reynolds, W. F,, McLean, S., Perpick-Dumont, M. and Enriques, R. G. (1989) Magn. Res. Ckem. 27, f62. 10. Schwarz, J. C. P. (1973) J. Ckem. Sot., Ckem. Commun. 505.
0031 9422/92$5.00+0.00 Q 1992Pergamon PressLtd
A 3,l l-CYCLOTAXANE
FROM TAXUS BACCATA
GIOVANNI APPENDINO,* PATRIZIA Lusso, PIERLUIGIGARIBOLDI,~ Ezra BOMBARDELLISand BRUNO GABETTA$ Dipartimentodi
Scienza e Tecnologiade1Farmaco, Via PietroGiuria 9, I-10125 Torino, Italy; fDipartimento di Scienze Chimiche. via S. Agostino 1, I-62032 &merino (MC), Italy; $Indena S.p.A., via ~~monti 99, I-20141 Milano, Italy; $Laboratori Ricerca e Sviluppo, Inverni della Bet%, via Ripamonti 99, I-20141 Milano, Italy (Received 9 March 1992) Key Word Index-Tams
baccuta;Taxaceaq diterpenoids; S-cinnamoyl-l~~tylphototax~ci~
taxi&s.
Abstract-A collection of yew needles afforded a taxane diester characterized by an additional bond between C-3 and C-11. The structure was elucidated by spectral methods, and further confirmed by synthesis from a known taxane derivative. A general procedure for the photocyclization of taxicins is reported.
~~ODUffrON
Although taxol (1) shows exceptionally promising antitumour properties, its development as a drug has been hampered by its limited availability and relative lipophili~ty; as a result the supply of 1 has been scarce and formulation difficult [l]. To circumvent these difficulties, semisynthetic analogues prepared from lO-deacetylbaccatin III (2) are currently being evaluated. Among these products, taxotere (3) emerged as the leading compound [l]; 3 is actually an analogue of IOdeacetyltaxol (4), bearing a tert-butoxycarbonyl group instead of a benzoyl group on the aminoacidic side-chain. We became interested in the isolation of lo-deacetyltaxol from the needles of the European yew (Tuxars buccata L.), the current source of lO-deacetylbaccatin III [l]. Preliminary analysis of some collections from the Northern part of Italy, showed that some samples contained 4. However, the content was low, and the final purification of 4 was difficult due to the presence of another taxane (5) having similar chromatographic features. We present here the structure elucidation and the partial synthesis of this compound.
The location of the acetyi was confirmed by the synthesis of 5 from a known taxane derivative. Irradiation of 5-cinnamoyl-lO-acetyltaxicin I (12) [4] with a lowpressure mercury lamp gave 5 in quantitative yield The phot~~i~tion of 12 was accompanied by partial isome&ration of the E-cinnamoyl residue, and the semisynthetic material was an unseparable mixture (ca 5 : 1) of E- and Z-isomers, whereas the natural product was stereochemically pure (E-isomer). Irradiation of other taxi&-I and -11 derivatives (l&15) gave, in quantitative yield, their corresponding 3,l l-cycloderivatives (9-l 1, respectively). In all cases the phototaxicins were obtained
BFSULXSAND DISCUSSION Column chromatography of the plant extract gave a ca 1: 1.5 mixture of IO-deacetyltaxol and 5 as a powder (yield: 0.~0% of dried needles). These compounds could be separated by repeated column chromatography or, more conveniently, by HPLC. Compound 5 showed spectral features different from those of the more common taxanes; in particular, one of the methyls gave rise to an NMR doublet, a feature characteristic of 3,l i-cyclotaxanes, a rare class of taxanes of which only three examples are known [2,3]. Oxidation of 5 (PDC) gave the diketone 6 and the triketone 7: these products showed well-resolved NMR spectra that allowed us to assign the structure 5 to the natural product. *Author to whom correspondence should be addressed. 4259
G. APPENDINOet ai.
4260
OR4
5
R’ OH
6
OH
OR4
R3
R2
R4
uo%PH =o
7
OH
=o
8 9 10 11
OH OH OH A
uO%SH
aOH,PH aOAc$H aOAc$H
Mutt.
5
2
d
4.76
5 68 6a 7a 7b 9 10 12 i4a 148 16 17 18 19 20a 20b 2:6 3’,4’,5 7 8 OAc
t (W
5.57
m m
2.16 1.70 1.90 1.30 4.38 5.40 3.48 3.03 2.35 1.10 1.38 1.29 1.38 5.84 5.59 7.55 7.38 7.66 6.39 2.15
m d” d :: d s ZI s s (br) s (W m 7 d s s s
R2
R3
R4
OH
H
H
AC
AC
aOH$H =o
12
AC
130HH
AC
aOAc,pH aOAc$H
AC
14 15
aOAc,pH aOAc,PH
AC
OH H
AcH AC
AC
AC
AC&AC
H
AC
1. ‘H NMR data for compounds
H
R’
aOH,PH
as mixtures of E- and Z-isomers at the cinnamoyl residue, but reisomerization of the Z-isomer could be brought about by irradiating the photocyclization mixture in the presence of diphenyldisulphide [SJ. Interestingly, if this irradiation was carried out in the presence of iodine, a mixture containing mainly the Z-isomer was obtained instead. The transannular photocyclization of taxicins was discovered by Nakanishi [2], and presumably takes place via a C-3, C-11 diradical, formed by hydrogen transfer from C-3 to C-12, the enone r- carbon 163. The reaction is
Table
OR3
6
7
5.63 2.30 1.60 2.12 0.96 4.32 d (br) 5.64 3.58 2.69 2.55 1.26 1.18 1.42 1.24 ‘5.59 5.05 7.58 7.39 7.66 6.35 2.19
5.66 * * x
remarkable, since in photocyclizations of this type hydrogen transfer to the enone p-carbon has generally been observed [WX]. The stereochemistry of the secondary methyl was confirmed by NOE experiments (see below), and is in accordance with this mechanism, since in taxicins H-3 is a-orientated 141. The facility of the photocyclization reaction raises the question of whether 5 is a natural product or an artefact
5-11 (300MHz, CDCI,, TMS as int. standard)
” 6.23 s 3.25 2.68 2.54 0.83 1.30 1.32 1.17 5.58 4.95 7.5t 7.33 7.65 6.39 2.22
8
9
10
11
4.82 5.60 2.18 1.76 1.76 1.26 5.64 5.64 3.45 3.03 2.36 1.08 1.50 1.26 1.36 5.88 5.61 7.54 7.38 1.65 6.38 2.04 2.04
4.72 5.44 * * *
5.63 5.56 * * *
1s.41 4.33 3.40 2.94 2.29 1.04 1.39 1.34 1.31 5.91 5.51 7.47 7.30 7.58 6.29 2.05
g.63 m 5.67 m 3.41 2.81 2.34 1.05 1.56 1.21 1.22 6.06 5.80 7.48 7.30 7.60 6.3t 2.06 1.99 1.98
ca ca * * * * ca ca
*The corresponding signals could not be located. foverlapped signals. J(Hz): most coupling constants were virtually the same for 5-11;those for 5 are given as representative: ~9.2; J 9.10--98. 1 J ,z,,s=7.2; J14a,14P=20.0; J,s,,.=16.4 Hz. For 11: J1,,,=7.1.
J,,,=
5.70 rnt 5.70 mt
5.70 mi 5.70 mt 3.54 2.58 2.53 dd 1.23 1.51 1.30 1.33 5.86 5.7t 7.56 7.38 7.86 6.40 2.08 2.06 2.06
1.8; J,,,,=
J5,68
A 3,11-cyclotaxane from Taxus baccata Table 2. 13C NMR data of compounds 5,6 and 8 (75.43 MHZ, CD&, TMS as int. standard)
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1’ 2’6 3’5’ 7 8 9 AC
Mult.
d
d d d
4 4
5
6
8
78.1 78.5 61.9 142.1 76.1 25.9 29.3 45.7 82.6 84.0 56.4 51.0 214.2 46.5 45.0 23.3 22.9 15.8 25.2 126.7 134.1 128.7 128.1 145.4 117.5 165.9 172.2
80.8 211.1 s 67.9 143.0 74.0 26.2 29.3 46.7 82.4 85.4 55.0 50.3 216.4 48.1 44.1 21.8 21.9 15.7 25.5 128.4 134.2 128.9 128.2 145.6 117.4 165.4 172.3
21.2
21.3
78.3 78.8 61.8 141.4 76.5 25.8 31.0 44.9 82.5 79.7 56.2 51.3 214.4 46.5 44.9 23.5 22.7 15.8 25.9 128.8 134.1 128.8 128.2 145.5 117.6 166.0 171.0 170.0 21.1 21.0
Long-range lH-‘% correlations found in the FLOCK experiment on 8 (only quaternary aliphatic carbons are shown): C-l: C-3: C-8: C-11: C-15:
H-2, H-14a, H-14/3, H-16, H-17 H-2, H-19, H-2Oa, H-20b H-2, H-19 H-9, H-10, H-12, H-16, H-17, H-18 H-10, H-12, H-14a, H-16, H-17
formed from 12,the major taxane present in the collection of yew we investigated [4]. We believe 5 is a natural product on account of the following facts: (i) the natural product is stereochemically pure, whereas the semisynthetic product is a mixture of E- and Z-cinnamates; (ii) the photochemical reaction required a low-pressure mercury lamp (A< 300 nm) and quartz tubes. In pyrex glassware, the material used for processing the extracts, and with light of lower frequency, no reaction took place. It is worth noting that the secondary hydroxyl at C-2 shows a remarkable difference in reactivity within the taxi&s and phototaxicins series; thus, treatment of phototaxicin 5 with an excess of Ac,O did not a&t the hydroxyl at C-2, and gave the C-9 monoacetate 8, whereas under the same conditions the corresponding taxi& I derivative 12 afforded a 2,9-diacetate [4]. Furthermore, the oxidation of 5 with PDC gave the stable
4261
ketones 6 and 7, whereas attempts to oxidize 12 led to its complete degradation [4]. No ’ CNMR data have been reported for 3,11-cycletaxanes, and a thorough investigation on compounds $6 and 8 was thus undertaken. The analysis of the ‘H NMR spectra did not allow a straightforward assignment of all signals due to overlapping and the presence of several singlets which prevented us from ascertaining the proton connectivities. However, the 2D-C, H correlation spectra solved most ambiguities, allowing the unambiguous assignment of all protons with the exception of the methyl singlets (H-16, H-17 and H-19). This also allowed us to evaluate the coupling constant between H-9 and H-10 (9.0 Hz), that are isochronous in 8. As to the 13C NMR spectra, most resonances of the protonated carbons could be assigned unambigously by means of the 2D-C, H correlation spectra. The assignments of C-6, C-7, C-16, C17, C-19 and the quaternary carbons were obtained through the 2D-C, H correlation spectra via long-range (two and three bonds) C-H coupling constants. The FLOCK sequence [9] displaying good sensitivity and perfect supression of the lJ,_, cross-peaks was used. The relative stereochemistry and the conformation were assessed by chemical shift and coupling constant considerations, and by analysis of the ROESY spectra (Table 3). The downfield position of H-12 (3.4S3.50 ppm) and the presence of a ROE-effect between H-16 and H-18 are in accordance with a /?-orientation for the secondary methyl at C-12. A relevant downfield effect induced by the C-13 carbonyl group on H-20a { A6 (H-20a-H20b) = +0.25 ppm in 5; -0.10 ppm in 12 [4]} as well as diagnostic ROE effects (H-20a-H-12; H-lO-H-6cr; H-7’-H-12) and the coupling pattern of H-S showed that ring D, the only flexible part of the molecule, adopts a boat conformation (4*7B-type [lo]) with the cinnamoyl residue pseudo-axial. In taxicins derivatives, this ring adopts instead a chair conformation [%,-type (lo)] [4], and the cinnamoyl residue is axial. This is responsible for the different splitting pattern of H-5 within compounds of the two
Table 3. Cross-peaks detected in the ROESY spectrum of 5. The intensities of the peaks have been qualitatively evaluated as follows: s = strong m = mdium, w = weak; similar results were also found for 6 and 8 H-2: H-5: H-68: H-6X H-7a: H-7b: H-9: H-10: H-12: H-14a: H-14P: H-16: H-17: H-18: H-19: H-20a: H-20b: H-7’: H-8’:
H-17 (s), H-19 (s) H-6B (w) H-5 (w), H-6a (s) H-6/I (s), H-10 (m) H-7b (s) H-7a (s) H-17 (s), H-19 (s), &&CO- (m) H-6a (m), H-12 (s), H-18 (m) H-10 (s), H-20a (s), H-18 (s), H-7’ (s) H-14/I (s), H-20a (m) H-14~ (s), H-16 (w) H-148 (w), H-18 (m), H-17 (w) H-16 (w), H-9 (s), H-2 (m) H-lo(m), H-12 (s), H-7’ (w), H-16(m) H-9 (s) H-2Ob (s), H-14~1(m), H-12 (s) H-20a (8) H-12 (s), H-18 (w) H-2’ (s), H-6’ (s)
G. APPENDINO et al.
4262
classes (f, J = ca 9 Hz in phototaxicins; taxicins) [4].
t, J =
ca
2.5 Hz in
EXPERIMENTAL
Prep. HPLC: Waters microporasil column (0.8 x 30 cm), detection by a Waters differential refractometer 3401; NMR: see ref. [4]. PIant material. Yew needles were obtained from plants growing in a public garden in Monza (Mi, Italy), and were collected in February 1991. A voucher specimen is kept at the laboratory of the University of Torino. Isolation of5. Dried needles and small branches (500 kg) were extracted with EtOH at room temp., and the extract purified as described in ref. [4], to give 3.5 kg of residue. Part of this (330 g) was fractioned by CC on silica gel, using CHCI, containing increasing amounts of MeOH as eluant. Fractions eluted with CHCI,-MeOH (19: 1) contained 4 and 5. After two additional separations by CC (hexane-EtOAc, 3:7), 1.88 g (0.0040%) of a powder was obtained; this material was a 1: 1.5 mixture (‘H NMR, 200 MHz) of 4 and 5, that could be sepd by further CC (hexane-EtOAc, 1:9) or by HPLC (hexane_EtOAc, 1:9). 5-Cinnamoyl-lo-acetylphototaxicin I (5). Powder, mp 126”, [z]$” +7.5 (CH,Cl,: c 0.77); UV A%:” nm: 279, 217, 201; IR ~5:; cm -I: 3475,1700,1630,1450, tj70,1245,1~,9~,~, 770, 710; CIMS (IriH,) 140 eV, m/z (rel. int.): 556 [C,,H,,Os +NH‘,]+ [M+NHl]+ (100). Oxidation of5. A 100 mg (0.18 mM) sample of 5 was dissolved in 3 ml dry CH,Cl,, and excess PDC (210 mg, 3 mol. equiv.) was added. After stirring for 20 min at room temp., the reaction was diluted with Et,0 and filtered on a short column of celite. After removal of the sotvent, the reside was purified by CC (7.5 g silica gel, hexane-EtOAc, 3:2 as eluant) to give 9 mg 6 and 39 mg 7. 2-Dehydro-lo-acetyl-5-cinnamoylphototaxicin I (6). Powder, mp 73-76” [~}2’+44 (CH,Cl,; c 0.76); UV 22:” nm: 280,217, 201; IR YE:;cm-‘: 3460,1750,1710,1640,1450,1380,1240,1210, 1160, 1100, 1010, 715; CIMS (NH,) 140 eV, m/z (rel. int.): 554 [C31H3608+NH4]+ [M+NHd+ (100). 2,6-~isdekydro-lO-aeety~-S-c~n~amay~pkotota~~c~~ I (7). Oil, [~]g +4.h (CH,Cl,; c f.5); UV i.EzhlHnm: 282, 217, 203; IR “l$i” filmcm- 1: 1730, 1645, 1580,1450, 1370, 1020,990,940, 800,770,740, 710,690; CIMS (NH,) 140 eV, m/z (tel. int.): 552 [CJ1H3.+0s+NHI]+ [M+NHd+ (100). Acetylation of 5. A 200 mg sample of5 was dissolved in 2 ml dry pyridine, and 2 ml Ac,O were added. After stirring overnight at room temp., the reaction was worked up by the addition of MeOH and ice, and extracted with CH,C12. After washing with
dil. HCl, sat. NaHCO, and brine, the solvent was removed and the residue purified by CC (15 g silica gel, hexane-EtOAc, 3 : 7 as eluant) to give 170 mg 8 as a powder; mp 90”; UV 122” nm: 279, 217,203; IR vi:; cm-‘: 3450,1750,1710,1640,1580,1455,1375, 1250, 1160, 1050, 990,950, 770, 715, 590, CIMS (NH,) 140 eV, m/z (rel. int.): 598 [C31H3s08+NH4]+ [M+NH,]+ (100). Pkotocyclization qf taxicins. Reaction of 14 is reported as repr~ntative. In a quartz phot~hemical tube, a 105 mg sampie of 5-cinnamoyl-2,9,1O_tetraacetyltaxicin I (14) [4] was dissolved in 50 ml MeOH-EtOH (1: 5); the soln was flushed with N, for 5 min and then irradiated with a low-pressure Hg lamp in a Rayonet photochemical reactor. The reaction was followed by TLC (hexane_EtOAc, 3 : 7, R, 14: 0.55; R,10: 0.46). After 5 hr the reaction mixt. was worked-up by evapn of the solvent, and the residue @a 5: 1 mixture of E- and Z-isomers, ‘H NMR, 400 MHz) was redissolved in 7 ml EtOAc, and then 13 ml hexane and 37 mg (1 mol. equiv.) diphenyldisulphide was added. The soln was irradiated for 14 hr with a halogen lamp, and then evapd. The residue was mixed with silica gel (ca 300 mg) and charged at the top of a short column (25 g) of silica gel. Washing with hexane-EtOAc, 9: 1 removed diphenyldisulphide, and washing with hexane-EtOAc, (1: 1) gave 102 mg IO (yield 100%) as a stereochemically pure (‘H NMR control, 400 MHz) oil. Taxicins 12, 13 and 15 were photocyclized in the same way, giving the corresponding 3,iO~clotaxicins (5, 9 and 11) in quantitative yield.
REFERENCES 1. Borman, S. (1991) Chem Eng. News (21 Sept), 11. 2. Chiang, H. C., Woods, M. C., Nakadaira, Y. and Nakanishi, K. (1967) 3. Ckem. Sot., Ckem. Commun. 1201. 3. Ettouati, L., Ahond, A., Convert, O., Poupat, C. and Potier, P. (1989) Bull. Sot. Ckim. Fr. 687. 4. Appendino, G., Garibaldi, P, Pisetta, A., Bombardelfi, E. and Gabetta, B. (1992) Phytockemistry 31, 4253. 5. Miyata, O., Shinada, T., Ninomiya, I. and Naito, T. (1990)
Syntk~sis 1123. 6. Wolff, S., Schreiber, W. L., Smith III, A. B. and Agosta, C. (1972) J. Am. Ckem. Sot. 94, 7797. 7. Tobe, Y., Iseki, T., Kakiuchi, K. and Odaira, Y. (1984) Tetra~dron Letters 23, 3895. 8. Herz, W. and Nair, M. G. (1967) J. Am. Chem. 89, 5474. 9. Reynolds, W. F,, McLean, S., Perpick-Dumont, M. and Enriques, R. G. (1989) Magn. Res. Ckem. 27, f62. 10. Schwarz, J. C. P. (1973) J. Ckem. Sot., Ckem. Commun. 505.