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Sesquiterpene alcohols from foliage of Fitzroya cupressoides
Phytochemistry, Vol. 42, No. 4, pp. 1015-1019, 1996
Pergamon
S0031-9422(96)00109-4
Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0031-9422/96 $15.00 + 0.00
SESQUITERPENE ALCOHOLS FROM FOLIAGE OF FITZROYA CUPRESSOIDES LAURENCEG. COOL University of California Forest Products Laboratory, 1301 S. 46th St, Richmond, CA 94804, U.S.A.
(Received 13 December 1995) Key Word Index--Fitzroya cupressoides; Cupressaceae; foliage; sesquiterpenoids; biosynthesis; himachal-4-en-l-ol; bulgar-4-en-l-ol; trans-sesquipiperitol; trans-muurol-5-en-4-ol.
A b s t r a e t - - A new trans-fused himachal-4-en-1-ol was isolated from the foliage of the a-longipinene-producing chemotype of Fitzroya cupressoides. It was characterized by I D and 2D NMR and by partial synthesis. cis-Bulgar-4-en-l-ot is the probable identity of a co-occurring minor component. Identification of (-)-transsesquipiperitol and two epimeric trans-muurol-5-en-4-ols from this species is also reported.
INTRODUCTION In our earlier study of the foliar terpenoid variability of
Fitzroya cupressoides (Mol.) Johnst. [1] we reported that one of the three sesquiterpene chemotypes (denoted Type 1) of the species produced large amounts of a-longipinene and lesser quantities of several related hydrocarbons (/3-1ongipinene, longifolene, /3-himachalene and a-ylangene), as well as two unknown sesquiterpene alcohols (Unknowns 1-1 and 1-2 in [1]). The latter alcohol was a major component in this chemotype, and the amount of the two alcohols correlated positively with a-longipinene and the related hydrocarbons, indicating a close biosynthetic relationship. Three other unidentified sesquiterpene alcohols (Unknowns 0-4a/b and 0-5), unrelated to the longipinenes, were present in most trees of the species. The identity of these five sesquiterpene alcohols is the subject of this study.
RESULTS AND DISCUSSION
The major longipinene-related alcohol (Unknown 1-2 in ref. [1]) was isolated and identified as the transfused l laH-himachal-4-en-1/3-ol (1). GC-mass spectrometry gave an M r of 222. The IR spectrum showed a broad OH absorption at 3480 cm -~. ~3C and DEPT NMR data (see Experimental) gave four methyls, five methylenes, three methines and three quatemary carbons. One of the quaternary signals (8 76.0) was attributable to an OH-bearing carbon atom, while the methine at 8 123.5 and the quaternary at 8 134.3 indicated a trisubstituted double bond. The ~H NMR data showed the corresponding vinyl proton (1H, 5.43, br m, J ~ 1.5 Hz) and methyl (3H, 8 1.72, br s) absorptions. Two other singlets (at 8 0.87 and 1.09, 3H
each) were obviously gem-methyls attached to the third quaternary carbon ('3C: 8 37.2). In a ~H-JH COSY experiment, a broad multiplet ( I H , ~ 1.93, J ~ l . 8 H z ) was coupling to the vinyl proton, and there was also a weak cross-peak due to long-range coupling to the vinyl methyl. The lack of any other couplings for the proton implied that this methine carbon was attached to the other two quaternary carbons as in fragment A. Fragments B and C were also clear, despite severe overlaps of the protons on C-8 and C-9. Of the four possible carbon skeleta resulting from assembling fragments A - C , biosynthetic considerations made the himachal-4-en-l-ol structure the obvious choice. Although none of the stereoisomers of 1 have been reported in nature, there is mention of the partial synthesis of two of them from/~-himachalene epoxide 2 [2]. Unfortunately the stereochemistry at C-11 was not determined, nor were the reported spectral data adequate for proving or disproving the identity of 1 with either of the synthetic isomers. A repetition of this work seemed a promising way to ascertain the relative and absolute stereochemistry of the compound from F.
cupressoides. (+)-fl-Himachalene-fl-epoxide (2) was isolated from Cedrus deodara Loud. wood oil, this compound having been reported from the chemically similar C. atlantica Manet [3, 4]. Although both epimers were reported in ref. [3], the authors of ref. [4] found only one epimer which was the same as that prepared by peracetic acid epoxidation of /3-himachalene. These authors assumed that the epoxy group is a-oriented because of supposed steric hindrance on the /3 side of the A ~"~-double bond, but the work described in ref. [2] corrects this by showing that lithium-ethylenediamine reduction of the epoxy group produces the
1015
1016
L.G. COOL 15 2
H
5 H ]~k7
14
12 13
4 A 1'2
1
2
5 A 1'6
3
6 A I,II
:. H .
: H
14"
5
" j~
14 A4'5 A6'7, ix-Me 10 ~-OH, a - M e 7
8
9
11 o~-OH, o~-Me 12
~-OH, ~-Me
13 or-OH, ~-Me
known compound himachalol (3), with a fl-hydroxy group. In the current study, the C. deodara component (which was homogenous by capillary GC, JH and ~3C NMR) had an IR spectrum identical to that given in ref. [4], and as found in ref. [2], reductive opening of the oxirane ring gave several compounds, the major one being identical (GC-mass spectrometry, IR, optical rotation) with the known himachalol 3 [5]. This confirms the depicted relative and absolute stereochemistry at C-6 and C- 11 (and by necessity C- 1) of 2. One of the other products of reductive ring opening of 2 had spectral data (GC-mass spectroscopy, IR, optical rotation) identical to that of the F. cupressoides component, thus confirming its carbon skeleton and the stereochemistry at C-1 and C-6. The remaining question, the orientation of the methyl group at C- 11, was answered by dehydration of 1 with phosphorus oxychloride in pyridine, which should predominantly yield hydrocarbon products derivable by anti-elimination of water. In this case, compound 1 should yield 4-6, the last of which is the well characterized fl-himachalene; the 11-epimer of 1 would produce little or no 6. The dehydration experiment on natural 1 gave two major products: fl-himachalene (identical by GC on two OH
Me
A
Me
Me
B
C
15 A4'5 A6'7, [3-Me 16 A3'4 A5'6, t~-Me 17 A4'14 A5'6, a - M e 18 A3'4 A5'6, ~-Me 19 A4'14 A5'6, ~l-Me
columns, and by GC-mass spectrometry, with the authentic substance) and what is presumably 4, in a 3 : 1 ratio. No 5 was detected, but there was a small peak with M r 202 which is probably an ar-himachalene resulting from oxidation of 5. These results confirm the stereochemistry at C-11 of 1. The minor unknown 1-1 could not be isolated in sufficient quantity to determine NMR data. However, its GC-mass spectral data [1] resembled that of an authentic sample of 1,10-diepicubenol (though its GC retention time was slightly different), suggesting that this compound also has a cadin-4-en-1-ol structure. The proposed identity of this unknown as cis-bulgar-4-en-1ol (7) is based on the observed parallel biogenesis of both o~-longipinene (1-11 cyclization) and a-ylangene (1-10 cyclization)* in Type 1 F. cupressoides (Scheme 1). In addition to these two hydrocarbons, alternate pathways also lead to the formation of alcohols 1 and 7, no doubt by a 1,2-hydride shift and anti-hydroxyl attack at C-l, as shown in Scheme 1. Thus it appears likely that a single enzyme in F. cupressoides produces large and approximately equal amounts of the 1-11 cyclization products (+)-a-longipinene and 1, and much smaller, but again comparable, amounts of the analogous 1-10 cyclization products (+)-t~-ylangene and 7. An unrelated alcohol, Unknown 0-5 in [1], was *The analogous single-enzyme biogenesis of (-)-longifolene (1-11 cyclization) and (-)-sativene (1-10 cyclization) has been demonstrated in the fungus Helminthosporium sativum [6].
Sesquiterpene alcohols from foliage of Fitzroya cupressoides
~ 7
"<,
yclization
o.',:
1017
cyclization /
/ o.-
Y
~-ylangene
1
a-longipinene Scheme 1. Suggested common biosynthesis of ~-longipinene, o~-ylangene, 1 and 7.
isolated and identified as (-)-trans-sesquipiperitol (8). Its ~H NMR data were identical with that given for the enantiomer, which was found in Argyranthemum adauctum ssp. jacobaeifolium (Compositae) [7]. The mass spectral data given in ref. [7] for ent-8 appear to be in error; see ref. [1] for our mass spectral data. Also, the relative stereochemistry in ref. [7] shown for C-7 of the bisabolone used to synthesize ent-8 (and therefore for ent-8 itself) does not agree with that reported by the same group in ref. [8] and is apparently in error. The amount of 8 in the 57 F. cupressoides trees originally analysed correlated positively with a minor compound not reported in ref. [1] which was subsequently identified as/3-sesquiphellandrene 9. A common biogenesis of 8 and 9 is quite reasonable. The remaining unknown alcohols found in significant amounts in F. cupressoides were designated unknowns 0-4a and 0-4b in ref. [1]. Their percentages in the 57 trees analysed correlated positively with a-copaene, ce-muurolene and cubebol, implying a close biosynthetic relationship with these compounds. Because of the lack of sufficient F. cupressoides foliage to isolate these unknowns, foliage oil of Cupressus duclouxiana Hickel, which contains comparable amounts of these same two alcohols, was used. Unfortunately, these unknowns were thermally labile (precluding preparative GC purification) and were impossible to fully resolve by HPLC from the co-occurring major compounds cubebol and cis-muurol-5-en-4-~-ol (12). However, a small amount of an HPLC fraction rich in unknown 0-4b (ca. 80% pure) was analysed by GC-mass spectrometry under conditions which caused about 5% of the alcohol to dehydrate in the injector. Three hydrocarbons (two major and one minor) were observed, the last of which had a retention time and mass spectrum identical to that of zonarene (14). The other two had mass spectra superimposible upon those of the previously identified 18 and 19, but with longer retention times; these two hydrocarbons are almost certainly 16
and 17. [GC-mass spectrometry of 12 under the same conditions gives similar proportions of 18, 19 and epizonarene (15)]. GC-mass spectrometry of a later HPLC fraction containing ca 50% pure unknown 0-4a gave the same three hydrocarbons, thus proving it to be an OH-epimer of unknown 0-4b. Therefore, the F. cupressoides Unknowns 0-4a and 0-4b are tentatively identified as the epimeric trans-muurol-5-en-4-ols 10 and 11, although there is of course no data to suggest which epimer is which. Although 10 and 11 are new natural products, there was a recent report of the presumed enantiomer of one of them from the liverwort Scapania undulata (L.) Dum. [9]. In that study, the relative and absolute stereochemistry at C-l, C-7 and C-10 were unproven, being deduced only by analogy with co-occurring compounds, while the stereochemistry at C-4 was left unspecified. Before discussing a possible biosynthetic route to 10 and 11, it is instructive to consider the case of 12 and 13, the C-10 methyl epimers of 10 and 11, which we reported in an earlier study of Cupressus bakeri Jeps. foliar terpenoids [10, 11]. The percentages of 12 and 13 in 63 C. bakeri trees correlated positively (albeit weakly, R ~ 0.4, P < 0.02) not only with other IR, 7S, 10S sesquiterpenoids but also with a-cadinol, which has the opposite stereochemistry at C-I. The implied common biosynthesis of 1S-a-cadinol and 1R 12/13 in C. bakeri can be explained as follows (Scheme 2A). A common precursor, IS, 6R, 7S-cadin-4-en-10-carbocation, is either (a) quenched at C-10 by OH- to give a-cadinol or is (b) subject to two syn-l,2 hydride shifts (from C-I to C-10 and from C-6 to C - l ) with a A4"5 to AS'~-double-bond shift and O H - quenching at C-4, to give 12 and 13. It will be noted that this plausible sequence of events accounts for the change from cadinane (IS) to muurolane (1R) stereochemistry at C-1 and also for the fl-orientation of the C-10 methyl group. The biogenesis of 12/13 via a direct 1,3-hydride
1018
A
L.G. COOL OFF
~x-cadinol
b OH cadin-4-en10-carbocation 12, 13
H :"
B
Y 10,11
muurol-4-en10-carbocation
"%.
muurolenes, copaenes, cubebenes
Scheme 2. Suggested biosynthetic routes (A) to o~-cadinoland 12/13 in Cupressus bakeri foliage; and (B) to muurolanerelated sesquiterpenoids and 10/11 in Fitzroya cupressoides foliage.
shift (i.e. from C-6 to C-10) would not meet the case, since this would give the wrong stereochemistry at both C-1 and C-10 of 12 and 13. Applying the same hypothetical sequence to the biosynthesis of 10 and 11 in F. cupressoides (Scheme 2B), starting instead with IR, 6R, 7S-muurol-4-en-10carbocation, accounts for the presumed common biogenesis of (a) the muurolene-related compounds (by deprotonation or further cyclization of the carbocation) and (b) 10/11, where the two syn-l,2-hydride shifts now preserve the muurolane C-1 stereochemistry and establish the a-orientation of the C-10 methyl. In this case, however, unlike with C. bakeri compounds 12 and 13, a 1,3-hydride shift from C-6 to C-10 cannot be ruled out since this would give the same stereochemical result as the sequence shown in Scheme 2B. EXPERIMENTAL
Collection of foliage from six F. cupressoides populations in Chile was described previously [1]. Additional longipinene-rich material was obtained by harvesting foliage from rooted cuttings of Type 1 trees from this collection. Foliage of Cupressus duclouxiana Hickel for partial purification of 10 and 11 was from a tree of known provenance (Yunnan, China; accession No. 84-
0769) from the University of California Botanical Garden. Hydrodistillation (from satd NaCI with NaHCO3) of the LN:-ground F. cupressoides or C. duclouxiana foliage (ca 500 g in each case); flash LC of the oil (silica gel; hexane-EtOAc eluents); prep. GC (Carbowax 20M column, 190 ° for final purification of 1, 3 and 8); and FFIR of purified compounds were described previously [11]. Prep. HPLC (5# SiO2, 25 cm × 10 mm, hexane-EtOAc mixtures) was used for final purification of 2 (hexane-EtOAc, 96:4) from Cedrus deodara wood oil and preliminary purification of 1 and 3 (hexane-EtOAc, 90: 10) from the product mixture of Li reduction of 2. This column (Phase Separations 5# SiO 2 Spherisorb packing) was found to completely dehydrate alcohols 10-13 but pretreatment of the column with 0.25% pyridine in hexane-EtOAc (90: 10), neutralized the silica gel sufficiently to prevent dehydration of the alcohols. Repeated prep. HPLC of the relevant fr from Cupressus duclouxiana foliage oil on the treated column with hexane-EtOAc (94:6) gave frs containing ca 80% and ca. 50% pure Unknowns 0-4b and 0-4a. 11 aH-Himachal-4-en- 1/3-ol, ( 1S, 6R, 11 S)-4,7,7,11 tetramethylbicyclo[4.0.5]undec-4-ene-l-ol (1). Oil; KBr [a]~ 2 +16.5 ° (c 2.0, n-hexane). IR Um.~x cm ~: 3480 (OH), 1461, 1449, 1377, 1364, 1300, 1276, 1117, 1018, 990, 854, 814; ~H NMR (400.13 MHz, CDC13, ~ from TMS): 60.87 (3H, s, gem-Me), 1.01 (3H, d, J = 7.1Hz, Me-15), 1.09 (3H, s, gem-Me), 1.72 (3H, brs, Me-14), 1.93 (brm, J~-1.8 Hz, H-6), 5.43 (brm, J ~ 1.5Hz, H-5). ~3C NMR (100.6MHz, CDC13, 6 from TMS): CH 3- ~ 17.9, 23.5, 25.7, 32.1; CH 2- 23.8, 27.5, 27.7, 29.7, 38.8; CH- 40.7, 59.3, 123.5; C- 37.2, 76.0, 134.3; multiplicities by DEPT. Partial synthesis of 1 and 3./3-Himachalene epoxide (2) (25mg) ([a] 2z +135°; c 1.3, n-hexane) was isolated by prep. silica gel HPLC of the epoxide-containing flash LC fr from the wood oil of Cedrus deodara Loud. Reductive oxirane ring opening was by Li in ethylenediamine, as in ref. [2]. The product mixt. was separated by prep. HPLC, followed by prep. GC of the relevant frs giving an analytical sample (ca 0.2 rag) of 1, [a]~ 2 ~ +12°; c =0.2, n-hexane, identical by IR with the natural product; and 1.4 mg of 3, [a] 22 +67°; c 1.4, n-hexane; IR spectrum identical with that given in ref. [5] for natural himachalol. Dehydration of 1. Approximately 0.2 mg of natural 1 was dissolved in 0.15 ml dry pyridine and 0.1 ml POC13 added. After 45 rain at room temp. the reaction mixt. was pipetted onto a small amount of ice, extracted with n-pentane, and the pentane phase dried with NazSO 4 and analysed by GC-MS. The major product was identical (GC-MS; GC on SE-54 and Carbowax 20M capillary columns) with an authentic sample of /3himachalene. trans-Sesquipiperitol (8). Oil; [ce]~2 - 1 9 ° (e 6.3; hexane); IR /Jmax Km cm ~: 3350 (OH), 1733, 1674, 1451, 1377, 1157, 1025, 984, 901, 827; ~3C NMR (100.6 MHz, CDCI 3, ~ from TMS): CH 3- ~ 14.4, 17.7, 23.1, 25.7; CH 2- 20.9, 26.2, 30.5, 35.5; CH- 31.0, 46.5,
Sesquiterpene alcohols from foliage of Fitzroya cupressoides 69.2, 124.8, 125.7; C- 131.2, 137.4; multiplicities by DEPT. ~-Sesquiphellandrene (9) was identified by GC-MS comparison of a F. cupressoides foliage extract with a sample of ginger oil which is a rich source of 9. trans-Muurol-5-en-4fl-ol, (IR, 4S, 7S, lOR)-7-isopropyl-4,10-dimethyl bicyclo [4.0.4]dec- 5-en-4-ol (10, tentative, for unknown 0-4a or -4b) and trans-Muurol5-en-4ce-ol, (1R, 4R, 7S, lOR)-7-isopropyl-4,10-dimethylbicyclo[4.0.4]dec-5-en-4-ol (11 tentative, for Unknown 0-4b or -4a). Unknown 0-4a: GC-MS 70 eV, m/z (rel. int.): 222 [M] + (4), 207 [ M - M e ] + (100), 204 [ M - H 2 0 ] + (16), 161 [M-HzO-isoPr]+(45), 137 (13), 135 (13), 123 (13), 121 (10), 119 (14), 109 (12), 107 (11), 105 (26), 95 (15), 93 (14), 91 (23), 81 (20), 79 (15), 77 (13), 71 (7), 69 (11), 67 (11), 55 (20), 43 (100). Unknown 0-4b: GC-MS 70eV, m/z (rel. int.): 222 [M] + (3), 207 [M - Me] + (99), 204 [M - H20] + (5), 161 [ M - H 2 0 - i s o P r ] + (38), 137 (10), 135 (13), 123 (13), 121 (12), 119 (14), 109 (11), 107 (15), 105 (24), 95 (14), 93 (23), 91 (19), 81 (21), 79 (16), 77 (12), 71 (14), 69 (30), 67 (14), 55 (25), 43 (100).
Acknowledgements--Sincere thanks to Young-Kyoon Kim for help with some of the NMR experiments, to Hern~in Verscheure and members of the Committee for the Defense of the Flora and Fauna of Chile for assistance with the original foliage collections, to Ariel Power for provision of collected and propagated foliage, to Holly Forbes and the University of California Botanical Garden for provision of Cupressus duclouxiana foliage, and to the late Charles Berolzheimer
1019
Sr. and the California Cedar Products Co. for a grant supporting this work. Deo gratias.
REFERENCES
1. Cool, L. G., Power, A. B. and Zavarin, E. (1991) Biochem. Syst. Ecol. 19, 421. 2. Narula, A. E S. and Dev, S. (1976) Tetrahedron
Letters, 813. 3. Adams, D. R., Bhatnagar, S. E and Cookson, R. C. (1975) J. Chem. Soc., Perkin I 1502. 4. Plattier, M. and Teisseire, E (1974) Contribution to the Knowledge of Atlas Cedarwood Oil in Recherches, p. 131. Soci6td Anonyme Roure Bertrand Dupont, Paris. 5. Bisarya, S. C. and Dev, S. (1968) Tetrahedron 24, 3861. 6. Cane, D. E. (1981) in Biosynthesis of lsoprenoid Compounds (Porter, J. W. and Spurgeon, S. L., eds), pp. 354-357. John Wiley and Sons, New York. 7. Bohlmann, F., Tsankova, E. and Jakupovic, J. (1984) Phytochemistry 23, 1103. 8. Bohlmann, F., Zdero, C. and Sch6neweiss, S. (1976) Chem. Ber. 109, 3366. 9. Nagashima, F., Suda, K. and Asakawa, Y. (1994) Phytochemistry 37, 1323. 10. Rafii, Z., Cool, L. G. and Zavarin, E. (1992) Biochem. Syst. Ecol. 20, 123. 11. Kim, Y.-K., Cool, L. G. and Zavarin, E. (1994) Phytochemistry 36, 961.
Pergamon
S0031-9422(96)00109-4
Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0031-9422/96 $15.00 + 0.00
SESQUITERPENE ALCOHOLS FROM FOLIAGE OF FITZROYA CUPRESSOIDES LAURENCEG. COOL University of California Forest Products Laboratory, 1301 S. 46th St, Richmond, CA 94804, U.S.A.
(Received 13 December 1995) Key Word Index--Fitzroya cupressoides; Cupressaceae; foliage; sesquiterpenoids; biosynthesis; himachal-4-en-l-ol; bulgar-4-en-l-ol; trans-sesquipiperitol; trans-muurol-5-en-4-ol.
A b s t r a e t - - A new trans-fused himachal-4-en-1-ol was isolated from the foliage of the a-longipinene-producing chemotype of Fitzroya cupressoides. It was characterized by I D and 2D NMR and by partial synthesis. cis-Bulgar-4-en-l-ot is the probable identity of a co-occurring minor component. Identification of (-)-transsesquipiperitol and two epimeric trans-muurol-5-en-4-ols from this species is also reported.
INTRODUCTION In our earlier study of the foliar terpenoid variability of
Fitzroya cupressoides (Mol.) Johnst. [1] we reported that one of the three sesquiterpene chemotypes (denoted Type 1) of the species produced large amounts of a-longipinene and lesser quantities of several related hydrocarbons (/3-1ongipinene, longifolene, /3-himachalene and a-ylangene), as well as two unknown sesquiterpene alcohols (Unknowns 1-1 and 1-2 in [1]). The latter alcohol was a major component in this chemotype, and the amount of the two alcohols correlated positively with a-longipinene and the related hydrocarbons, indicating a close biosynthetic relationship. Three other unidentified sesquiterpene alcohols (Unknowns 0-4a/b and 0-5), unrelated to the longipinenes, were present in most trees of the species. The identity of these five sesquiterpene alcohols is the subject of this study.
RESULTS AND DISCUSSION
The major longipinene-related alcohol (Unknown 1-2 in ref. [1]) was isolated and identified as the transfused l laH-himachal-4-en-1/3-ol (1). GC-mass spectrometry gave an M r of 222. The IR spectrum showed a broad OH absorption at 3480 cm -~. ~3C and DEPT NMR data (see Experimental) gave four methyls, five methylenes, three methines and three quatemary carbons. One of the quaternary signals (8 76.0) was attributable to an OH-bearing carbon atom, while the methine at 8 123.5 and the quaternary at 8 134.3 indicated a trisubstituted double bond. The ~H NMR data showed the corresponding vinyl proton (1H, 5.43, br m, J ~ 1.5 Hz) and methyl (3H, 8 1.72, br s) absorptions. Two other singlets (at 8 0.87 and 1.09, 3H
each) were obviously gem-methyls attached to the third quaternary carbon ('3C: 8 37.2). In a ~H-JH COSY experiment, a broad multiplet ( I H , ~ 1.93, J ~ l . 8 H z ) was coupling to the vinyl proton, and there was also a weak cross-peak due to long-range coupling to the vinyl methyl. The lack of any other couplings for the proton implied that this methine carbon was attached to the other two quaternary carbons as in fragment A. Fragments B and C were also clear, despite severe overlaps of the protons on C-8 and C-9. Of the four possible carbon skeleta resulting from assembling fragments A - C , biosynthetic considerations made the himachal-4-en-l-ol structure the obvious choice. Although none of the stereoisomers of 1 have been reported in nature, there is mention of the partial synthesis of two of them from/~-himachalene epoxide 2 [2]. Unfortunately the stereochemistry at C-11 was not determined, nor were the reported spectral data adequate for proving or disproving the identity of 1 with either of the synthetic isomers. A repetition of this work seemed a promising way to ascertain the relative and absolute stereochemistry of the compound from F.
cupressoides. (+)-fl-Himachalene-fl-epoxide (2) was isolated from Cedrus deodara Loud. wood oil, this compound having been reported from the chemically similar C. atlantica Manet [3, 4]. Although both epimers were reported in ref. [3], the authors of ref. [4] found only one epimer which was the same as that prepared by peracetic acid epoxidation of /3-himachalene. These authors assumed that the epoxy group is a-oriented because of supposed steric hindrance on the /3 side of the A ~"~-double bond, but the work described in ref. [2] corrects this by showing that lithium-ethylenediamine reduction of the epoxy group produces the
1015
1016
L.G. COOL 15 2
H
5 H ]~k7
14
12 13
4 A 1'2
1
2
5 A 1'6
3
6 A I,II
:. H .
: H
14"
5
" j~
14 A4'5 A6'7, ix-Me 10 ~-OH, a - M e 7
8
9
11 o~-OH, o~-Me 12
~-OH, ~-Me
13 or-OH, ~-Me
known compound himachalol (3), with a fl-hydroxy group. In the current study, the C. deodara component (which was homogenous by capillary GC, JH and ~3C NMR) had an IR spectrum identical to that given in ref. [4], and as found in ref. [2], reductive opening of the oxirane ring gave several compounds, the major one being identical (GC-mass spectrometry, IR, optical rotation) with the known himachalol 3 [5]. This confirms the depicted relative and absolute stereochemistry at C-6 and C- 11 (and by necessity C- 1) of 2. One of the other products of reductive ring opening of 2 had spectral data (GC-mass spectroscopy, IR, optical rotation) identical to that of the F. cupressoides component, thus confirming its carbon skeleton and the stereochemistry at C-1 and C-6. The remaining question, the orientation of the methyl group at C- 11, was answered by dehydration of 1 with phosphorus oxychloride in pyridine, which should predominantly yield hydrocarbon products derivable by anti-elimination of water. In this case, compound 1 should yield 4-6, the last of which is the well characterized fl-himachalene; the 11-epimer of 1 would produce little or no 6. The dehydration experiment on natural 1 gave two major products: fl-himachalene (identical by GC on two OH
Me
A
Me
Me
B
C
15 A4'5 A6'7, [3-Me 16 A3'4 A5'6, t~-Me 17 A4'14 A5'6, a - M e 18 A3'4 A5'6, ~-Me 19 A4'14 A5'6, ~l-Me
columns, and by GC-mass spectrometry, with the authentic substance) and what is presumably 4, in a 3 : 1 ratio. No 5 was detected, but there was a small peak with M r 202 which is probably an ar-himachalene resulting from oxidation of 5. These results confirm the stereochemistry at C-11 of 1. The minor unknown 1-1 could not be isolated in sufficient quantity to determine NMR data. However, its GC-mass spectral data [1] resembled that of an authentic sample of 1,10-diepicubenol (though its GC retention time was slightly different), suggesting that this compound also has a cadin-4-en-1-ol structure. The proposed identity of this unknown as cis-bulgar-4-en-1ol (7) is based on the observed parallel biogenesis of both o~-longipinene (1-11 cyclization) and a-ylangene (1-10 cyclization)* in Type 1 F. cupressoides (Scheme 1). In addition to these two hydrocarbons, alternate pathways also lead to the formation of alcohols 1 and 7, no doubt by a 1,2-hydride shift and anti-hydroxyl attack at C-l, as shown in Scheme 1. Thus it appears likely that a single enzyme in F. cupressoides produces large and approximately equal amounts of the 1-11 cyclization products (+)-a-longipinene and 1, and much smaller, but again comparable, amounts of the analogous 1-10 cyclization products (+)-t~-ylangene and 7. An unrelated alcohol, Unknown 0-5 in [1], was *The analogous single-enzyme biogenesis of (-)-longifolene (1-11 cyclization) and (-)-sativene (1-10 cyclization) has been demonstrated in the fungus Helminthosporium sativum [6].
Sesquiterpene alcohols from foliage of Fitzroya cupressoides
~ 7
"<,
yclization
o.',:
1017
cyclization /
/ o.-
Y
~-ylangene
1
a-longipinene Scheme 1. Suggested common biosynthesis of ~-longipinene, o~-ylangene, 1 and 7.
isolated and identified as (-)-trans-sesquipiperitol (8). Its ~H NMR data were identical with that given for the enantiomer, which was found in Argyranthemum adauctum ssp. jacobaeifolium (Compositae) [7]. The mass spectral data given in ref. [7] for ent-8 appear to be in error; see ref. [1] for our mass spectral data. Also, the relative stereochemistry in ref. [7] shown for C-7 of the bisabolone used to synthesize ent-8 (and therefore for ent-8 itself) does not agree with that reported by the same group in ref. [8] and is apparently in error. The amount of 8 in the 57 F. cupressoides trees originally analysed correlated positively with a minor compound not reported in ref. [1] which was subsequently identified as/3-sesquiphellandrene 9. A common biogenesis of 8 and 9 is quite reasonable. The remaining unknown alcohols found in significant amounts in F. cupressoides were designated unknowns 0-4a and 0-4b in ref. [1]. Their percentages in the 57 trees analysed correlated positively with a-copaene, ce-muurolene and cubebol, implying a close biosynthetic relationship with these compounds. Because of the lack of sufficient F. cupressoides foliage to isolate these unknowns, foliage oil of Cupressus duclouxiana Hickel, which contains comparable amounts of these same two alcohols, was used. Unfortunately, these unknowns were thermally labile (precluding preparative GC purification) and were impossible to fully resolve by HPLC from the co-occurring major compounds cubebol and cis-muurol-5-en-4-~-ol (12). However, a small amount of an HPLC fraction rich in unknown 0-4b (ca. 80% pure) was analysed by GC-mass spectrometry under conditions which caused about 5% of the alcohol to dehydrate in the injector. Three hydrocarbons (two major and one minor) were observed, the last of which had a retention time and mass spectrum identical to that of zonarene (14). The other two had mass spectra superimposible upon those of the previously identified 18 and 19, but with longer retention times; these two hydrocarbons are almost certainly 16
and 17. [GC-mass spectrometry of 12 under the same conditions gives similar proportions of 18, 19 and epizonarene (15)]. GC-mass spectrometry of a later HPLC fraction containing ca 50% pure unknown 0-4a gave the same three hydrocarbons, thus proving it to be an OH-epimer of unknown 0-4b. Therefore, the F. cupressoides Unknowns 0-4a and 0-4b are tentatively identified as the epimeric trans-muurol-5-en-4-ols 10 and 11, although there is of course no data to suggest which epimer is which. Although 10 and 11 are new natural products, there was a recent report of the presumed enantiomer of one of them from the liverwort Scapania undulata (L.) Dum. [9]. In that study, the relative and absolute stereochemistry at C-l, C-7 and C-10 were unproven, being deduced only by analogy with co-occurring compounds, while the stereochemistry at C-4 was left unspecified. Before discussing a possible biosynthetic route to 10 and 11, it is instructive to consider the case of 12 and 13, the C-10 methyl epimers of 10 and 11, which we reported in an earlier study of Cupressus bakeri Jeps. foliar terpenoids [10, 11]. The percentages of 12 and 13 in 63 C. bakeri trees correlated positively (albeit weakly, R ~ 0.4, P < 0.02) not only with other IR, 7S, 10S sesquiterpenoids but also with a-cadinol, which has the opposite stereochemistry at C-I. The implied common biosynthesis of 1S-a-cadinol and 1R 12/13 in C. bakeri can be explained as follows (Scheme 2A). A common precursor, IS, 6R, 7S-cadin-4-en-10-carbocation, is either (a) quenched at C-10 by OH- to give a-cadinol or is (b) subject to two syn-l,2 hydride shifts (from C-I to C-10 and from C-6 to C - l ) with a A4"5 to AS'~-double-bond shift and O H - quenching at C-4, to give 12 and 13. It will be noted that this plausible sequence of events accounts for the change from cadinane (IS) to muurolane (1R) stereochemistry at C-1 and also for the fl-orientation of the C-10 methyl group. The biogenesis of 12/13 via a direct 1,3-hydride
1018
A
L.G. COOL OFF
~x-cadinol
b OH cadin-4-en10-carbocation 12, 13
H :"
B
Y 10,11
muurol-4-en10-carbocation
"%.
muurolenes, copaenes, cubebenes
Scheme 2. Suggested biosynthetic routes (A) to o~-cadinoland 12/13 in Cupressus bakeri foliage; and (B) to muurolanerelated sesquiterpenoids and 10/11 in Fitzroya cupressoides foliage.
shift (i.e. from C-6 to C-10) would not meet the case, since this would give the wrong stereochemistry at both C-1 and C-10 of 12 and 13. Applying the same hypothetical sequence to the biosynthesis of 10 and 11 in F. cupressoides (Scheme 2B), starting instead with IR, 6R, 7S-muurol-4-en-10carbocation, accounts for the presumed common biogenesis of (a) the muurolene-related compounds (by deprotonation or further cyclization of the carbocation) and (b) 10/11, where the two syn-l,2-hydride shifts now preserve the muurolane C-1 stereochemistry and establish the a-orientation of the C-10 methyl. In this case, however, unlike with C. bakeri compounds 12 and 13, a 1,3-hydride shift from C-6 to C-10 cannot be ruled out since this would give the same stereochemical result as the sequence shown in Scheme 2B. EXPERIMENTAL
Collection of foliage from six F. cupressoides populations in Chile was described previously [1]. Additional longipinene-rich material was obtained by harvesting foliage from rooted cuttings of Type 1 trees from this collection. Foliage of Cupressus duclouxiana Hickel for partial purification of 10 and 11 was from a tree of known provenance (Yunnan, China; accession No. 84-
0769) from the University of California Botanical Garden. Hydrodistillation (from satd NaCI with NaHCO3) of the LN:-ground F. cupressoides or C. duclouxiana foliage (ca 500 g in each case); flash LC of the oil (silica gel; hexane-EtOAc eluents); prep. GC (Carbowax 20M column, 190 ° for final purification of 1, 3 and 8); and FFIR of purified compounds were described previously [11]. Prep. HPLC (5# SiO2, 25 cm × 10 mm, hexane-EtOAc mixtures) was used for final purification of 2 (hexane-EtOAc, 96:4) from Cedrus deodara wood oil and preliminary purification of 1 and 3 (hexane-EtOAc, 90: 10) from the product mixture of Li reduction of 2. This column (Phase Separations 5# SiO 2 Spherisorb packing) was found to completely dehydrate alcohols 10-13 but pretreatment of the column with 0.25% pyridine in hexane-EtOAc (90: 10), neutralized the silica gel sufficiently to prevent dehydration of the alcohols. Repeated prep. HPLC of the relevant fr from Cupressus duclouxiana foliage oil on the treated column with hexane-EtOAc (94:6) gave frs containing ca 80% and ca. 50% pure Unknowns 0-4b and 0-4a. 11 aH-Himachal-4-en- 1/3-ol, ( 1S, 6R, 11 S)-4,7,7,11 tetramethylbicyclo[4.0.5]undec-4-ene-l-ol (1). Oil; KBr [a]~ 2 +16.5 ° (c 2.0, n-hexane). IR Um.~x cm ~: 3480 (OH), 1461, 1449, 1377, 1364, 1300, 1276, 1117, 1018, 990, 854, 814; ~H NMR (400.13 MHz, CDC13, ~ from TMS): 60.87 (3H, s, gem-Me), 1.01 (3H, d, J = 7.1Hz, Me-15), 1.09 (3H, s, gem-Me), 1.72 (3H, brs, Me-14), 1.93 (brm, J~-1.8 Hz, H-6), 5.43 (brm, J ~ 1.5Hz, H-5). ~3C NMR (100.6MHz, CDC13, 6 from TMS): CH 3- ~ 17.9, 23.5, 25.7, 32.1; CH 2- 23.8, 27.5, 27.7, 29.7, 38.8; CH- 40.7, 59.3, 123.5; C- 37.2, 76.0, 134.3; multiplicities by DEPT. Partial synthesis of 1 and 3./3-Himachalene epoxide (2) (25mg) ([a] 2z +135°; c 1.3, n-hexane) was isolated by prep. silica gel HPLC of the epoxide-containing flash LC fr from the wood oil of Cedrus deodara Loud. Reductive oxirane ring opening was by Li in ethylenediamine, as in ref. [2]. The product mixt. was separated by prep. HPLC, followed by prep. GC of the relevant frs giving an analytical sample (ca 0.2 rag) of 1, [a]~ 2 ~ +12°; c =0.2, n-hexane, identical by IR with the natural product; and 1.4 mg of 3, [a] 22 +67°; c 1.4, n-hexane; IR spectrum identical with that given in ref. [5] for natural himachalol. Dehydration of 1. Approximately 0.2 mg of natural 1 was dissolved in 0.15 ml dry pyridine and 0.1 ml POC13 added. After 45 rain at room temp. the reaction mixt. was pipetted onto a small amount of ice, extracted with n-pentane, and the pentane phase dried with NazSO 4 and analysed by GC-MS. The major product was identical (GC-MS; GC on SE-54 and Carbowax 20M capillary columns) with an authentic sample of /3himachalene. trans-Sesquipiperitol (8). Oil; [ce]~2 - 1 9 ° (e 6.3; hexane); IR /Jmax Km cm ~: 3350 (OH), 1733, 1674, 1451, 1377, 1157, 1025, 984, 901, 827; ~3C NMR (100.6 MHz, CDCI 3, ~ from TMS): CH 3- ~ 14.4, 17.7, 23.1, 25.7; CH 2- 20.9, 26.2, 30.5, 35.5; CH- 31.0, 46.5,
Sesquiterpene alcohols from foliage of Fitzroya cupressoides 69.2, 124.8, 125.7; C- 131.2, 137.4; multiplicities by DEPT. ~-Sesquiphellandrene (9) was identified by GC-MS comparison of a F. cupressoides foliage extract with a sample of ginger oil which is a rich source of 9. trans-Muurol-5-en-4fl-ol, (IR, 4S, 7S, lOR)-7-isopropyl-4,10-dimethyl bicyclo [4.0.4]dec- 5-en-4-ol (10, tentative, for unknown 0-4a or -4b) and trans-Muurol5-en-4ce-ol, (1R, 4R, 7S, lOR)-7-isopropyl-4,10-dimethylbicyclo[4.0.4]dec-5-en-4-ol (11 tentative, for Unknown 0-4b or -4a). Unknown 0-4a: GC-MS 70 eV, m/z (rel. int.): 222 [M] + (4), 207 [ M - M e ] + (100), 204 [ M - H 2 0 ] + (16), 161 [M-HzO-isoPr]+(45), 137 (13), 135 (13), 123 (13), 121 (10), 119 (14), 109 (12), 107 (11), 105 (26), 95 (15), 93 (14), 91 (23), 81 (20), 79 (15), 77 (13), 71 (7), 69 (11), 67 (11), 55 (20), 43 (100). Unknown 0-4b: GC-MS 70eV, m/z (rel. int.): 222 [M] + (3), 207 [M - Me] + (99), 204 [M - H20] + (5), 161 [ M - H 2 0 - i s o P r ] + (38), 137 (10), 135 (13), 123 (13), 121 (12), 119 (14), 109 (11), 107 (15), 105 (24), 95 (14), 93 (23), 91 (19), 81 (21), 79 (16), 77 (12), 71 (14), 69 (30), 67 (14), 55 (25), 43 (100).
Acknowledgements--Sincere thanks to Young-Kyoon Kim for help with some of the NMR experiments, to Hern~in Verscheure and members of the Committee for the Defense of the Flora and Fauna of Chile for assistance with the original foliage collections, to Ariel Power for provision of collected and propagated foliage, to Holly Forbes and the University of California Botanical Garden for provision of Cupressus duclouxiana foliage, and to the late Charles Berolzheimer
1019
Sr. and the California Cedar Products Co. for a grant supporting this work. Deo gratias.
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Letters, 813. 3. Adams, D. R., Bhatnagar, S. E and Cookson, R. C. (1975) J. Chem. Soc., Perkin I 1502. 4. Plattier, M. and Teisseire, E (1974) Contribution to the Knowledge of Atlas Cedarwood Oil in Recherches, p. 131. Soci6td Anonyme Roure Bertrand Dupont, Paris. 5. Bisarya, S. C. and Dev, S. (1968) Tetrahedron 24, 3861. 6. Cane, D. E. (1981) in Biosynthesis of lsoprenoid Compounds (Porter, J. W. and Spurgeon, S. L., eds), pp. 354-357. John Wiley and Sons, New York. 7. Bohlmann, F., Tsankova, E. and Jakupovic, J. (1984) Phytochemistry 23, 1103. 8. Bohlmann, F., Zdero, C. and Sch6neweiss, S. (1976) Chem. Ber. 109, 3366. 9. Nagashima, F., Suda, K. and Asakawa, Y. (1994) Phytochemistry 37, 1323. 10. Rafii, Z., Cool, L. G. and Zavarin, E. (1992) Biochem. Syst. Ecol. 20, 123. 11. Kim, Y.-K., Cool, L. G. and Zavarin, E. (1994) Phytochemistry 36, 961.