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Piperidine alkaloid content of Picea (spruce) and Pinus (pine)
Phytahmurry,
Vol. 35, No 4. pp. 951.-9S3. I994 0 1994 Ekvicr !kha Led Printed m Chat BntJn. All ri&U ruwwd m31-9422p4 %.00+0.00
Pergamoa
PIPERIDINE
ALKALOID CONTENT OF PZCEA (SPRUCE) AND PZNUS (PINE)
FRANK R. STERMITZ,JEANNEN. TAWARA,MAXIMILIANEBOECKL,MARC POMEROY,TOMMASOA. FODERAROand FRED G. TODD Department
of Chemistry, Colorado
State University,
Fort Collins, CO 80523, U.S.A.
(Receioed 21 SepIember 1993) Key
Word Index-Picea;
Pinus; Pinaceae; piperidine alkaloids; pinidine; pinidinol; euphococcinine.
Abstract-The piperidine alkaloid content of the spruces Picea abies, P. glauca, P. pungens and P. sitchensis, and the pines Pinus edulis, P.Jexilis, P. jeffreyi, P. nigra, P. pinea, P. ponderosa and P. sylvestris was assessed by isolation and GCMS. The spruce species contained a variety of cis- and trans-2,6-disubstituted piperidine alkaloids, while the pines contained only cis-disubstituted piperidines. In some species the alkaloid patterns of individual plant parts were determined. Preliminary bioactivity of some of the piperidines is reviewed.
INTRODUCTION
4
n
For many years the only structurally confirmed alkaloids reported from the Pinaceae were a-pipecoline, 1 and (-)pinidine, 2 [l-3]. Recently, two additional piperidines were reported [4-61 and then an array of further 2,6disubstituted piperidines [A. In the course of our work we have examined alkaloid occurrence in several spruce and pine species and, in part, the alkaloid patterns in different plant parts. These results are presented here along with a review of preliminary data on biological activity of some of the alkaloids.
3ns
6
RESULTS AND DISCUSSION
The patterns of major and some minor alkaloid occurrences are shown in Table 1. GC-MS analysis of the total bases from several of the species indicated the presence of additional trace alkaloids, but the identification of these is awaiting synthesis or semisynthesis of standards for comparison. Complete plant part analyses were conducted for several, but not all of the species (Table 1). The widespread occurrence of such a variety of alkaloids in a family (Pinaceae) not known for alkaloids, and one which has been studied chemically for decades, is remarkable. Although many of the alkaloids occur in both spruce (Picea) and pines (Pinus), the pines contain only cis-2, 6-disubstituted piperdines, while both cisand trans-alkaloids were isolated from the spruces. Based upon structural relationships, we suggested [a that the biosynthesis in pines may proceed in the sequence 10,9,3, 2 with 6 being formed from 10 in a branch off the main path. The proposed biosynthesis does not account for the presence of the 2, 6-trans alkaloids, whose genesis in the spruces is at the moment unclear [6]. Reduction of a l,6imine, rather than a l,Zimine e.g. 9 or 10, is one possibility which could result in a /?. rather than an a substituent at C-6 (Scheme 1).
The data for Pinus nigra and P. sylvestris needles are interesting in that biosynthesis in the needles of these species did not proceed beyond formation of 9 and, presumably, cyclization of 10 to 6. This may indicate a lack of the enzyme which is involved with reduction of the imine bond. The needle content of Pinusjexilis is similar to that of P. nigra and P. sylvestris in that essentially all 10 goes to 6, and none is reduced to 9 and 3. The dominance of 3 in P. pinea, on the other hand, indicates the presence of an active imine reductase and marks off that species chemically from the other three pines. A recent detailed taxonomic analysis of seed proteins in Pinus [S] has confirmed affinities among certain ‘mountain pines’, including P. sylvestris and P. nigra, and established their separation from more typically Mediterranean species, including P. pinea. Our data would suggest affinities of the North American species P. Jexilis (limber pine) with 951
F. R. STERMITZet al.
952
Table
Piceu a&es (L.) Karsten: Bark (mature) Bark (young) Cones (mature) Needles Resin (sap) Roots Twigs Wood Piceo ylaucu
1. Alkaloids
3
5
+++ +++ +++ +++ +++ +++ +++ +++
++ +++ ++ ++ +++ ++ +++
+++ +++
+ ++
+ +++ +++ +++ ++ +++ +
+ +++ + + +++ +++ _
++ +++ +++
+ ++ ++ -
+++ +++ + ++ + ++ +++
-
of spruce (Picea) and pine (Pinus) species 2
_ _ 4-i
_ _
4
6
._ + _ ++ if ++ +
_
+ _. _.
I
8
9
IO
_
_
-.
+
_
+ _
..
_ ._
_ _
densata
(Moench) Voss: Cone5 (mature) Needles/twigs Picea pungens Engelm.: Bark (mature) Bark (young) Cones (mature) Needles Roots Twigs Wood Picea sitchensis (Bong) Carr: Needles Roots Twigs Bark (mature) Pinus edulis Engelm.: Bark (mature) Bark (young) Needles New growth Roots Twigs Wood Pinus Jexilis James: Needles/twigs Pinus jefieyi Grev. and Ball.: Bark (young) Needles Twigs Pinus n&a J. F. Arnold: Needles Pinus pinea L.: Needles/twigs
_ _
+
+++
_
++ +++ +++ + t
++ ++ _ +++ +++ + _
+ + + + _
++
+
+
+
+
+ +++ ++
_.
++ +++
-. -. _
+++ +++ ++
-
++ +++
-.
_
++ +++
_
+++ +++ +++ +++ + it+ +++
__ -. + _.
t + +
++
+ t
_ _.. -..
.-
_. _.
_ ++ + _
_.
_
++ +
-
+++
_
._
_..
+ ++ + if ++
-
-
-
+ -
+
++ +
+
+++
_
+++
++
t + ._ ++t +
+++ ++ +++ ++ ++ +++
++ +++ +++ +++ ++
-
_
+++
Pinus pomierosa
Dougl. ex Laws.: Bark (mature) Bark (young) Cones (immature) Needles New growth Roots Twigs Wood Pinus syhesfris (L.): Needles + + + : Major:
+ +: moderate;
_
+++ ++ +
+
-
+++
_.
+
_
-.. _ _
+ if + +
t
-..
+
_
.._
+ +++
+: minor; 1: trace.
_
_ _ _.
+++
+++
Piperidine
alkaloids
of spruce and pine
953
NADH
NADH
R
H
Scheme
1.
H
Proposed piperidine alkaloid biosynthesis.
the ‘mountain pine’ group of Europe, but additional plant part analyses are necessary for all these species. The absence of minor or trace alkaloids from several of the species is probably not meaningful since we have not done extensive work on some of the taxa of Table 1. Only preliminary data are available on alkaloid variation among individual trees of the same species and seasonal or growth stage variations, but it is clear that there will be considerable quantitative and even qualitative differences within a species. Genetic variation among individual trees and variable climatic or soil conditions could also alter the data of Table 1. Several studies [9-133 have suggested that soil acidification from atmospheric pollution causes ammonia and ammonium imbalances in soil, as well as changes in plant uptake or foliar deposition of nitrogen sources. Such processes might be expected to strongly influence alkaloid production in conifers, as was demonstrated for polyamines in Picea abies [lo]. We reported [7] that a mixture of alkaloids from Pinus ponderosa needles was strongly teratogenic in a frog embryo test. Further work has now implicated pinidine, 2, as the major teratogen of the mixture [J. A. Bantle, Oklahoma State University, unpublished results]. A mixture of spruce alkaloids, composed mainly of 3 and 5 in an artificial diet was shown to reduce variegated cutworm (Peridromo saucia) growth to 15% of the controls at seven days [M. B. Isman, University of British Columbia, unpublished results]. A preliminary study was reported [6] to have shown moderate-high antifeedant activity against Eastern spruce budworm for an alkaloid mixture from Picen engelmannii. Euphococcinine, 6, is a component of blood secreted by Mexican been beetles when disturbed and is a deterrent against spiders and ants [14]. Thus, data are beginning to show that the conifer piperidine alkaloids have a variety of biological effects and could be important in plant-herbivore interactions.
EXPERIMENTAL
Sources of plant material were as described [7] with the following additions. Picea sitchensis, Sitka spruce, was identified and collected in Tillamook Co., Oregon with the assistance of Wayne Patterson, District Silviculturist, U.S. Forest Service, Hebo, Oregon. Pinus pexilis, limber pine, was collected near Red Feather Lakes, Larimer Co.,
Colorado and identified by R. D. Moench, Colorado State Forest Service, Fort Collins. Picea glauca densata, Black Hills spruce, was commercially grown at T and M Tree Farms, Fort Collins, Colorado and identified by V. Tawara of T and M Tree Farms. Pinus pinea, Italian stone pine, was also a commercial sample. Alkaloid isolations and identifications, and GC-MS analysis methods were previously described [73. Acknowledgemenrs-This work was supported by National Science Foundation grant CHE-9023608. J.N.T. was supported by Department of Education Fellowship and a Colorado State University Graduate Diversity Education Assistantship. REFERENCES
1. Tallent, W. H., Stromberg, V. L. and Horning, E. C. (1955) J. Am. Chem. Sot. 77, 6361. 2. Tallent, W. H. and Horning, E. C. (1956) J. Am. Gem. Sot. 78, 4467. 3 Hill, R. K., Chan, T. H. and Joule, J. A. (1965) Tetrahedron 21, 147. 4. Schneider, M. and Stermitz, F. R. (1990) Phytochemistry 29, 1811. 5. Stermitz. F. R., Miller, M. M. and Schneider, M. J. (1990) .I. Nat. Prod. 53, 1019. 6. Schneider, M. J., Montali, J. A., Hazen, D. and Stanton, C. E. (1991) J. Nat. Prod. 54, 905. 7. Tawara, J. N., Blokhin, A., Foderaro, T. A. and Stermitz, F. R. (1993) J. Org. Chem. 58, 4813. 8. Schirone, B., Piovesan, G., Bellarosa, R. and Pelosi, c. (1991) PI. Syst. Evol. 178, 43. 9. Schulze, E.-D. (1989) Science 244, 776. 10. Santerre, A., Markiewicz, M. and Villanueva, V. R. (1990) Phytochemistry 29, 1767. 11. McLeod, A. R., Holland, M. R., Shaw, P. J. A., Sutherland, P. M., Darrall, N. M. and Skefington, R. A. (1990) Nature 347, 277. 1 Nussbaum, S., von Ballmoos, P., Gfeller, H., Schlunegger, U. P., Fuhrer, J., Rhodes, D. and Brunold, C. (1993) Oecologia 94, 408. Langford, A. 0. and Fehsenfeld, F. C. (1992) Science 255, 581. 1, Eisner, T., Goetz, M., Aneshansley, D., FerstandigArnold, G. and Meinwald, J. (1986) Experientio 204.
Vol. 35, No 4. pp. 951.-9S3. I994 0 1994 Ekvicr !kha Led Printed m Chat BntJn. All ri&U ruwwd m31-9422p4 %.00+0.00
Pergamoa
PIPERIDINE
ALKALOID CONTENT OF PZCEA (SPRUCE) AND PZNUS (PINE)
FRANK R. STERMITZ,JEANNEN. TAWARA,MAXIMILIANEBOECKL,MARC POMEROY,TOMMASOA. FODERAROand FRED G. TODD Department
of Chemistry, Colorado
State University,
Fort Collins, CO 80523, U.S.A.
(Receioed 21 SepIember 1993) Key
Word Index-Picea;
Pinus; Pinaceae; piperidine alkaloids; pinidine; pinidinol; euphococcinine.
Abstract-The piperidine alkaloid content of the spruces Picea abies, P. glauca, P. pungens and P. sitchensis, and the pines Pinus edulis, P.Jexilis, P. jeffreyi, P. nigra, P. pinea, P. ponderosa and P. sylvestris was assessed by isolation and GCMS. The spruce species contained a variety of cis- and trans-2,6-disubstituted piperidine alkaloids, while the pines contained only cis-disubstituted piperidines. In some species the alkaloid patterns of individual plant parts were determined. Preliminary bioactivity of some of the piperidines is reviewed.
INTRODUCTION
4
n
For many years the only structurally confirmed alkaloids reported from the Pinaceae were a-pipecoline, 1 and (-)pinidine, 2 [l-3]. Recently, two additional piperidines were reported [4-61 and then an array of further 2,6disubstituted piperidines [A. In the course of our work we have examined alkaloid occurrence in several spruce and pine species and, in part, the alkaloid patterns in different plant parts. These results are presented here along with a review of preliminary data on biological activity of some of the alkaloids.
3ns
6
RESULTS AND DISCUSSION
The patterns of major and some minor alkaloid occurrences are shown in Table 1. GC-MS analysis of the total bases from several of the species indicated the presence of additional trace alkaloids, but the identification of these is awaiting synthesis or semisynthesis of standards for comparison. Complete plant part analyses were conducted for several, but not all of the species (Table 1). The widespread occurrence of such a variety of alkaloids in a family (Pinaceae) not known for alkaloids, and one which has been studied chemically for decades, is remarkable. Although many of the alkaloids occur in both spruce (Picea) and pines (Pinus), the pines contain only cis-2, 6-disubstituted piperdines, while both cisand trans-alkaloids were isolated from the spruces. Based upon structural relationships, we suggested [a that the biosynthesis in pines may proceed in the sequence 10,9,3, 2 with 6 being formed from 10 in a branch off the main path. The proposed biosynthesis does not account for the presence of the 2, 6-trans alkaloids, whose genesis in the spruces is at the moment unclear [6]. Reduction of a l,6imine, rather than a l,Zimine e.g. 9 or 10, is one possibility which could result in a /?. rather than an a substituent at C-6 (Scheme 1).
The data for Pinus nigra and P. sylvestris needles are interesting in that biosynthesis in the needles of these species did not proceed beyond formation of 9 and, presumably, cyclization of 10 to 6. This may indicate a lack of the enzyme which is involved with reduction of the imine bond. The needle content of Pinusjexilis is similar to that of P. nigra and P. sylvestris in that essentially all 10 goes to 6, and none is reduced to 9 and 3. The dominance of 3 in P. pinea, on the other hand, indicates the presence of an active imine reductase and marks off that species chemically from the other three pines. A recent detailed taxonomic analysis of seed proteins in Pinus [S] has confirmed affinities among certain ‘mountain pines’, including P. sylvestris and P. nigra, and established their separation from more typically Mediterranean species, including P. pinea. Our data would suggest affinities of the North American species P. Jexilis (limber pine) with 951
F. R. STERMITZet al.
952
Table
Piceu a&es (L.) Karsten: Bark (mature) Bark (young) Cones (mature) Needles Resin (sap) Roots Twigs Wood Piceo ylaucu
1. Alkaloids
3
5
+++ +++ +++ +++ +++ +++ +++ +++
++ +++ ++ ++ +++ ++ +++
+++ +++
+ ++
+ +++ +++ +++ ++ +++ +
+ +++ + + +++ +++ _
++ +++ +++
+ ++ ++ -
+++ +++ + ++ + ++ +++
-
of spruce (Picea) and pine (Pinus) species 2
_ _ 4-i
_ _
4
6
._ + _ ++ if ++ +
_
+ _. _.
I
8
9
IO
_
_
-.
+
_
+ _
..
_ ._
_ _
densata
(Moench) Voss: Cone5 (mature) Needles/twigs Picea pungens Engelm.: Bark (mature) Bark (young) Cones (mature) Needles Roots Twigs Wood Picea sitchensis (Bong) Carr: Needles Roots Twigs Bark (mature) Pinus edulis Engelm.: Bark (mature) Bark (young) Needles New growth Roots Twigs Wood Pinus Jexilis James: Needles/twigs Pinus jefieyi Grev. and Ball.: Bark (young) Needles Twigs Pinus n&a J. F. Arnold: Needles Pinus pinea L.: Needles/twigs
_ _
+
+++
_
++ +++ +++ + t
++ ++ _ +++ +++ + _
+ + + + _
++
+
+
+
+
+ +++ ++
_.
++ +++
-. -. _
+++ +++ ++
-
++ +++
-.
_
++ +++
_
+++ +++ +++ +++ + it+ +++
__ -. + _.
t + +
++
+ t
_ _.. -..
.-
_. _.
_ ++ + _
_.
_
++ +
-
+++
_
._
_..
+ ++ + if ++
-
-
-
+ -
+
++ +
+
+++
_
+++
++
t + ._ ++t +
+++ ++ +++ ++ ++ +++
++ +++ +++ +++ ++
-
_
+++
Pinus pomierosa
Dougl. ex Laws.: Bark (mature) Bark (young) Cones (immature) Needles New growth Roots Twigs Wood Pinus syhesfris (L.): Needles + + + : Major:
+ +: moderate;
_
+++ ++ +
+
-
+++
_.
+
_
-.. _ _
+ if + +
t
-..
+
_
.._
+ +++
+: minor; 1: trace.
_
_ _ _.
+++
+++
Piperidine
alkaloids
of spruce and pine
953
NADH
NADH
R
H
Scheme
1.
H
Proposed piperidine alkaloid biosynthesis.
the ‘mountain pine’ group of Europe, but additional plant part analyses are necessary for all these species. The absence of minor or trace alkaloids from several of the species is probably not meaningful since we have not done extensive work on some of the taxa of Table 1. Only preliminary data are available on alkaloid variation among individual trees of the same species and seasonal or growth stage variations, but it is clear that there will be considerable quantitative and even qualitative differences within a species. Genetic variation among individual trees and variable climatic or soil conditions could also alter the data of Table 1. Several studies [9-133 have suggested that soil acidification from atmospheric pollution causes ammonia and ammonium imbalances in soil, as well as changes in plant uptake or foliar deposition of nitrogen sources. Such processes might be expected to strongly influence alkaloid production in conifers, as was demonstrated for polyamines in Picea abies [lo]. We reported [7] that a mixture of alkaloids from Pinus ponderosa needles was strongly teratogenic in a frog embryo test. Further work has now implicated pinidine, 2, as the major teratogen of the mixture [J. A. Bantle, Oklahoma State University, unpublished results]. A mixture of spruce alkaloids, composed mainly of 3 and 5 in an artificial diet was shown to reduce variegated cutworm (Peridromo saucia) growth to 15% of the controls at seven days [M. B. Isman, University of British Columbia, unpublished results]. A preliminary study was reported [6] to have shown moderate-high antifeedant activity against Eastern spruce budworm for an alkaloid mixture from Picen engelmannii. Euphococcinine, 6, is a component of blood secreted by Mexican been beetles when disturbed and is a deterrent against spiders and ants [14]. Thus, data are beginning to show that the conifer piperidine alkaloids have a variety of biological effects and could be important in plant-herbivore interactions.
EXPERIMENTAL
Sources of plant material were as described [7] with the following additions. Picea sitchensis, Sitka spruce, was identified and collected in Tillamook Co., Oregon with the assistance of Wayne Patterson, District Silviculturist, U.S. Forest Service, Hebo, Oregon. Pinus pexilis, limber pine, was collected near Red Feather Lakes, Larimer Co.,
Colorado and identified by R. D. Moench, Colorado State Forest Service, Fort Collins. Picea glauca densata, Black Hills spruce, was commercially grown at T and M Tree Farms, Fort Collins, Colorado and identified by V. Tawara of T and M Tree Farms. Pinus pinea, Italian stone pine, was also a commercial sample. Alkaloid isolations and identifications, and GC-MS analysis methods were previously described [73. Acknowledgemenrs-This work was supported by National Science Foundation grant CHE-9023608. J.N.T. was supported by Department of Education Fellowship and a Colorado State University Graduate Diversity Education Assistantship. REFERENCES
1. Tallent, W. H., Stromberg, V. L. and Horning, E. C. (1955) J. Am. Chem. Sot. 77, 6361. 2. Tallent, W. H. and Horning, E. C. (1956) J. Am. Gem. Sot. 78, 4467. 3 Hill, R. K., Chan, T. H. and Joule, J. A. (1965) Tetrahedron 21, 147. 4. Schneider, M. and Stermitz, F. R. (1990) Phytochemistry 29, 1811. 5. Stermitz. F. R., Miller, M. M. and Schneider, M. J. (1990) .I. Nat. Prod. 53, 1019. 6. Schneider, M. J., Montali, J. A., Hazen, D. and Stanton, C. E. (1991) J. Nat. Prod. 54, 905. 7. Tawara, J. N., Blokhin, A., Foderaro, T. A. and Stermitz, F. R. (1993) J. Org. Chem. 58, 4813. 8. Schirone, B., Piovesan, G., Bellarosa, R. and Pelosi, c. (1991) PI. Syst. Evol. 178, 43. 9. Schulze, E.-D. (1989) Science 244, 776. 10. Santerre, A., Markiewicz, M. and Villanueva, V. R. (1990) Phytochemistry 29, 1767. 11. McLeod, A. R., Holland, M. R., Shaw, P. J. A., Sutherland, P. M., Darrall, N. M. and Skefington, R. A. (1990) Nature 347, 277. 1 Nussbaum, S., von Ballmoos, P., Gfeller, H., Schlunegger, U. P., Fuhrer, J., Rhodes, D. and Brunold, C. (1993) Oecologia 94, 408. Langford, A. 0. and Fehsenfeld, F. C. (1992) Science 255, 581. 1, Eisner, T., Goetz, M., Aneshansley, D., FerstandigArnold, G. and Meinwald, J. (1986) Experientio 204.