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Cytisine-12-carboxy-ethylester, a quinolizidine alkaloid from Laburnum watereri and its occurrence in the leguminosae
Phytochemistry,
0031-9422po 53.00+0.00 PergamonPressplc
Vol. 29, No. 11, pp. 3553-3554, 1990.
Printedin Great Britain.
CYTISINE-12-CARBOXY-ETHYLESTER, A QUINOLIZIDINE ALKALOID FROM LABURNUM WATERER1 AND ITS OCCURRENCE IN THE LEGUMINOSAE ROLAND GREINWALD, PETER BACHMANN, LUDGER WITTE * and FRANZ-CHRISTIAN CZYGAN *t
Lehrstuhl fiir Pharmazeutische Biologie, University of Wiirzburg, Mittlerer Dallenbergweg 64, D-8700 Wiirzburg, F.R.G.; *Institut fiir Pharmazeutische Biologie, Technical University of Braunschweig, Mendelssohnstrasse 1, D-3300 Braunschweig, F.R.G. (Received
Key Word Index--Laburnum
watereri;
31 January 1990)
Leguminosae; quinolizidine alkaloid; cytisine-12-carboxy-ethylester.
Abstract-A new lupin alkaloid, cytisine-12-carboxy-ethylester, was isolated from suspension cultures of Laburnum watereri. Its structure was determined by spectroscopic data and by direct comparison with a synthetic sample. The distribution of the new alkaloid amongst different genera of the Leguminosae is reported.
As a result of screening plants belonging to the tribe Genisteae (Leguminosae) for lupin alkaloids, a new alkaloid, cytisine-12-carboxy-ethylester, was found as a minor compound in different plant parts of Laburnum watereri [l]. Further studies showed that tissue cultures of Laburnum watereri fed with cytisine produced this alkaloid in greater amounts (Greinwald, R. unpublished results). Therefore the new alkaloid was isolated from fed suspension cultures of Laburnum watered for its structural determination.
RESULTS AND DISCUSSION
The mass spectrum of the alkaloid displayed a CM]’ at m/z 262 together with fragment ions at m/z 160 and 146 (base peak), which are characteristic for lupin alkaloids
t Author to whom correspondence should be addressed.
containing an a-pyridone ring system such as cytisine [2]. According to these data and the fragment ion m/z 190, it was deduced, that we were dealing with a cytisine skeleton, having a side chain of mass 73. The fragments m/z 247 and 233 could be due to the elimination of a C,H,portion. The new compound could not be silylated by MSTFA (N-methyl-N-trimethylsilyl-trifluoracetamide) [3], which definitely excluded the presence of an OH- or NH-group. In addition, the IR spectrum lacked absorption bands at 3320 and 3250 cm-’ (NH). Hence the side chain must be situated at the N-12 position. The absorpto the absorption tion band at 1707 cm-‘, additionally (a-pyridone C=O), indicated the band at 1664cm-’ presence of an urethane group (NXO-0). Similar IR properties are reported for other alkaloids with a urethane structure as N’-carbomethoxynornicotine [4]. The IR and mass spectral data were compatible with the structure of a cytisine-12-carboxy-ethylester (1). This assumption was confirmed hydrolysing the new compound in methanolic KOH. A highly polar product
1OO.C
233
50.0
116
190 I 0
174 JL
217 200 1. 233 247 1)’ III’S I 200 250
Fig. 1. Mass spectrum of cytisine-12-carboxyethylester. PHYTO 29:11-L
3553
3554
R. GREINWALDet al. Table 1. Occurrence
Species Adenocarpus foliolosus A. mannii Argyrocytisus hattandieri Chamaecytisus austriacus Ch. purpureus Ch. ruthenicus Cytisophyllum sessilijolium Cytisus ardoini C. purgans C. scoparius C. villosus Echinospartum horridum Genista rorsica G. Lydia G. sagittalis Lahurnum anagyroides L. alpinum L. watereri Laburnocytisus adamii Petteria ramentacea Retama sphaerocarpa Spartium junceum Teline canariensis Teline gomerae T. monspessulana
of compound
(1)
Presence of cytisine-12-carboxy-ethylester + __ + + + + + + + + i+ + + +
Alkaloid extracts of plants were analysed by capillary GLC and GCMS. Cytisine-12-carboxy-ethylester was identified on account of its specific retention index and distinctive mass spectrum.
(cytisine-N-formiate) was formed, which could not be extracted from water with dichloromethane, giving support for the presence of a carboxyl-group. Further confirmation of the identity of the new alkaloid was obtained by comparison with a synthetic sample, prepared from cytisine and ethylchloroformate. The spectroscopic and chromatographic properties of the synthetic cytisine-l2carboxyethylester (1) were identical with the natural compound. The occurrence of cytisine-12-carboxy-ethylester (1) in different species of the tribe Genisteae is shown in Table 1. All species accumulating a-pyridone alkaloids as cytisine or N-methylcytisine as main alkaloids contain cytisine12-carboxy-ethylester (1) as the minor alkaloid. Compound 1 might be an intermediate in the biosynthesis of tetracyclic lupin alkaloids from cytisine or vice versa. EXPERIMENTAL
Zsolation of cytisine-12-carboxy-ethylester.
Dried and finely ground cell material from cell suspension cultures of Laburnum watereri fed with cytisine were extracted in a Soxhlet apparatus as previously described [S]. The crude alkaloid mixture was separated by prep. TLC on silica gel (Merck Nr. 5744,0.S mm): CHCl,-MeOH-28% NH&OH (100: 45 : 1). R, (cytisine): 0.35; R, (cytisine-12-carboxy-ethylester): 0.78. The separated alkaloids were further purified by microsublimation (190”; 0.06 Torr). Cytisine-12-carboxy-ethylester crystallized as needles.
Cytisine-12-carboxy-ethylester (1). UV 1::Y” nm: 232, 309, 1707 (N-CO-O), 1664 (a-pyridone C=O), 1547, IR v~~:crn-‘: 1587. EIMS (70 eV) m/z (rel. int.): 262 CM]’ (48), 247 (I), 233 (I), 217 (4), 190 (12), 160 (19). 147 (26), 146 (lOO), 122 (9), 116 (28), 44 (51). Hydrolysis. The unknown alkaloid was dissolved in 1 ml satd methanolic KOH and refluxed for about 6 hr. The MeOH was then evapd under red. pres., the residue dissolved in 1 ml H,O and extracted with CH,Cl,. Synthesis of cytisine-12-carboxy-ethylester (1). A mixture of cytisine (10 mg) and ethylchloroformate (150 ~1) in toluene (5 ml) was heated at 60” for 2 hr. After evaporating the solvent in uacuo, the residue was taken up in H,O, basified with NH, and extracted with CH,CI,. The yield was 95%, and the purity of the product was greater than 99% (by GLC).
REFERENCES
1. Greinwald, R., Schultze, W. and Czygan, F.-C. (1990) Biochemie und Physiologie der Pflanzen (in press). 2. Neuner-Jehle, N., Nesvadba, H. and Spiteller, G. (1964) Mh. Chem. 95, 687. 3. Witte, L., Miiller, K. and Arfmann, H.-A. (1987) Planta Med. 53, 192. 4. Hecht, S., Ornaf, R. and Hoffmann, D. (1974) J. Nat1 Cancer Inst. 54, 1237. 5. Greinwald, R., Witte, L., Wray, V. and Czygan, F.-C. (1990) Biochem. Syst. Ecol. (in press).
0031-9422po 53.00+0.00 PergamonPressplc
Vol. 29, No. 11, pp. 3553-3554, 1990.
Printedin Great Britain.
CYTISINE-12-CARBOXY-ETHYLESTER, A QUINOLIZIDINE ALKALOID FROM LABURNUM WATERER1 AND ITS OCCURRENCE IN THE LEGUMINOSAE ROLAND GREINWALD, PETER BACHMANN, LUDGER WITTE * and FRANZ-CHRISTIAN CZYGAN *t
Lehrstuhl fiir Pharmazeutische Biologie, University of Wiirzburg, Mittlerer Dallenbergweg 64, D-8700 Wiirzburg, F.R.G.; *Institut fiir Pharmazeutische Biologie, Technical University of Braunschweig, Mendelssohnstrasse 1, D-3300 Braunschweig, F.R.G. (Received
Key Word Index--Laburnum
watereri;
31 January 1990)
Leguminosae; quinolizidine alkaloid; cytisine-12-carboxy-ethylester.
Abstract-A new lupin alkaloid, cytisine-12-carboxy-ethylester, was isolated from suspension cultures of Laburnum watereri. Its structure was determined by spectroscopic data and by direct comparison with a synthetic sample. The distribution of the new alkaloid amongst different genera of the Leguminosae is reported.
As a result of screening plants belonging to the tribe Genisteae (Leguminosae) for lupin alkaloids, a new alkaloid, cytisine-12-carboxy-ethylester, was found as a minor compound in different plant parts of Laburnum watereri [l]. Further studies showed that tissue cultures of Laburnum watereri fed with cytisine produced this alkaloid in greater amounts (Greinwald, R. unpublished results). Therefore the new alkaloid was isolated from fed suspension cultures of Laburnum watered for its structural determination.
RESULTS AND DISCUSSION
The mass spectrum of the alkaloid displayed a CM]’ at m/z 262 together with fragment ions at m/z 160 and 146 (base peak), which are characteristic for lupin alkaloids
t Author to whom correspondence should be addressed.
containing an a-pyridone ring system such as cytisine [2]. According to these data and the fragment ion m/z 190, it was deduced, that we were dealing with a cytisine skeleton, having a side chain of mass 73. The fragments m/z 247 and 233 could be due to the elimination of a C,H,portion. The new compound could not be silylated by MSTFA (N-methyl-N-trimethylsilyl-trifluoracetamide) [3], which definitely excluded the presence of an OH- or NH-group. In addition, the IR spectrum lacked absorption bands at 3320 and 3250 cm-’ (NH). Hence the side chain must be situated at the N-12 position. The absorpto the absorption tion band at 1707 cm-‘, additionally (a-pyridone C=O), indicated the band at 1664cm-’ presence of an urethane group (NXO-0). Similar IR properties are reported for other alkaloids with a urethane structure as N’-carbomethoxynornicotine [4]. The IR and mass spectral data were compatible with the structure of a cytisine-12-carboxy-ethylester (1). This assumption was confirmed hydrolysing the new compound in methanolic KOH. A highly polar product
1OO.C
233
50.0
116
190 I 0
174 JL
217 200 1. 233 247 1)’ III’S I 200 250
Fig. 1. Mass spectrum of cytisine-12-carboxyethylester. PHYTO 29:11-L
3553
3554
R. GREINWALDet al. Table 1. Occurrence
Species Adenocarpus foliolosus A. mannii Argyrocytisus hattandieri Chamaecytisus austriacus Ch. purpureus Ch. ruthenicus Cytisophyllum sessilijolium Cytisus ardoini C. purgans C. scoparius C. villosus Echinospartum horridum Genista rorsica G. Lydia G. sagittalis Lahurnum anagyroides L. alpinum L. watereri Laburnocytisus adamii Petteria ramentacea Retama sphaerocarpa Spartium junceum Teline canariensis Teline gomerae T. monspessulana
of compound
(1)
Presence of cytisine-12-carboxy-ethylester + __ + + + + + + + + i+ + + +
Alkaloid extracts of plants were analysed by capillary GLC and GCMS. Cytisine-12-carboxy-ethylester was identified on account of its specific retention index and distinctive mass spectrum.
(cytisine-N-formiate) was formed, which could not be extracted from water with dichloromethane, giving support for the presence of a carboxyl-group. Further confirmation of the identity of the new alkaloid was obtained by comparison with a synthetic sample, prepared from cytisine and ethylchloroformate. The spectroscopic and chromatographic properties of the synthetic cytisine-l2carboxyethylester (1) were identical with the natural compound. The occurrence of cytisine-12-carboxy-ethylester (1) in different species of the tribe Genisteae is shown in Table 1. All species accumulating a-pyridone alkaloids as cytisine or N-methylcytisine as main alkaloids contain cytisine12-carboxy-ethylester (1) as the minor alkaloid. Compound 1 might be an intermediate in the biosynthesis of tetracyclic lupin alkaloids from cytisine or vice versa. EXPERIMENTAL
Zsolation of cytisine-12-carboxy-ethylester.
Dried and finely ground cell material from cell suspension cultures of Laburnum watereri fed with cytisine were extracted in a Soxhlet apparatus as previously described [S]. The crude alkaloid mixture was separated by prep. TLC on silica gel (Merck Nr. 5744,0.S mm): CHCl,-MeOH-28% NH&OH (100: 45 : 1). R, (cytisine): 0.35; R, (cytisine-12-carboxy-ethylester): 0.78. The separated alkaloids were further purified by microsublimation (190”; 0.06 Torr). Cytisine-12-carboxy-ethylester crystallized as needles.
Cytisine-12-carboxy-ethylester (1). UV 1::Y” nm: 232, 309, 1707 (N-CO-O), 1664 (a-pyridone C=O), 1547, IR v~~:crn-‘: 1587. EIMS (70 eV) m/z (rel. int.): 262 CM]’ (48), 247 (I), 233 (I), 217 (4), 190 (12), 160 (19). 147 (26), 146 (lOO), 122 (9), 116 (28), 44 (51). Hydrolysis. The unknown alkaloid was dissolved in 1 ml satd methanolic KOH and refluxed for about 6 hr. The MeOH was then evapd under red. pres., the residue dissolved in 1 ml H,O and extracted with CH,Cl,. Synthesis of cytisine-12-carboxy-ethylester (1). A mixture of cytisine (10 mg) and ethylchloroformate (150 ~1) in toluene (5 ml) was heated at 60” for 2 hr. After evaporating the solvent in uacuo, the residue was taken up in H,O, basified with NH, and extracted with CH,CI,. The yield was 95%, and the purity of the product was greater than 99% (by GLC).
REFERENCES
1. Greinwald, R., Schultze, W. and Czygan, F.-C. (1990) Biochemie und Physiologie der Pflanzen (in press). 2. Neuner-Jehle, N., Nesvadba, H. and Spiteller, G. (1964) Mh. Chem. 95, 687. 3. Witte, L., Miiller, K. and Arfmann, H.-A. (1987) Planta Med. 53, 192. 4. Hecht, S., Ornaf, R. and Hoffmann, D. (1974) J. Nat1 Cancer Inst. 54, 1237. 5. Greinwald, R., Witte, L., Wray, V. and Czygan, F.-C. (1990) Biochem. Syst. Ecol. (in press).