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Revised structure of the main alkaloid of Senecio adonidifolius
003 1 9422/92 $5.00 + 0.00 Q 1992 Pergamon Press plc
Phytochemistry, Vol. 31, No. 3, pp. 1027 -1028, 1992 Printed in Great Britain.
REVISED STRUCTURE
OF THE MAIN ALKALOID
OF SENECIO
ADONIDIFOLIUS LUDGER
WITTE, LUDGER ERNST,* VICTOR WRAY~ and THOMAS HARTMANN
Institut fiir Pharmazeutische Biologie, Technische Universitiit, W-3300 Braunschweig, Germany; *NMR-Laboratorium der Chemischen Institute, Technische Universitlt, W-3300 Braunschweig, Germany; tGBF-Gesellschaft fiir Biotechnologische Forschung mbH, W-3300 Braunschweig Germany
(Received23 July 1991) Key Ward Index-Senecio adonidi@ius; Compositae; pyrrolizidine alkaloids; adonifoline; revised structure.
Abstract-The structure of the main pyrrolizidine alkaloid (adonifoline) from Senecio adonidijblius is determined by GC-MS and ‘H and 13CNMR. ‘H-detected 2D long-range ‘H/13C-shift correlation proves a pentacyclic structure with a 1,5-dioxa-spiro[2.5]octane subunit. This requires the revision of two previous structures claimed for adonifoline.
INTRODUCTION
Table 1. “CNMR spectral data of compound 4
Senecio adonidifolius
Loisel. is a pyrrolizidine alkaloidproducing plant which grows on silicate soil meadows in the southern part of Europe (France, Spain, Italy). The alkaloid composition of S. adonidifolius was described by Urones et al. [ 11. They found florosenine (1) and another alkaloid which they claimed to be 12,13,19-trihydroxy15,20-epoxy- 15,20-dihydro-( 12S, 1SR,20R)-senecionan11,16-dione (2) (called adonifoline by Rader [2]) according to NMR data. Bohlmann et al. [3] described a pyrrolizidine alkaloid from the Peruvian S. dolichodoryius with practically identical NMR spectra; these authors suggested structure 3 for this alkaloid. In our group, GC-MS has been applied as a useful analytical tool for the rapid identification of plant pyrrolizidine alkaloids [4]. Preliminary GC-MS analyses of S. adonidfolius revealed data which are inconsistent with structures 2 and 3. This prompted us to reinvestigate the structure.
C
C 11 16 2 1 20 8
15 7 19
169.6 s 167.7 s 135.4 d 130.5 s 79.0 d 71.1 d 73.8 s 73.1 d 67.2 t
12 3 13 9 5
14 6 18 21
65.3 s’ 62.6 t 61.5 ss 60.3 t 53.6 t 40.4 t 35.0 t 15.4 q 14.4 q
*Interchangeable assignments.
Table 2. ‘H NMR spectral data of compound 4
RESULTSANDDISCUSSION
H
H
The GC-MS analysis of Zn/H+-reduced extracts of S. adonidifolius revealed the presence of two tertiary pyrrolizidine alkaloids. The minor component (ca 5%) was identified as the otonecine derivative 1 (RI = 2745, [M] + 423). The major alkaloid (ca 95%) showed an ion at m/z 365 as the highest mass in the spectrum. This ion could be explained by the loss of water from structure 2 or, as suggested by Bohlmann et al. [3], from structure 3. However, both NH&I and FAB mass spectra yielded [M +H] at m/z 366. This finding is inconsistent with either structures 2 or 3. Detailed NMR studies of the tertiary alkaloid were, therefore, performed to elucidate its structure. The 13C and ‘H NMR data of the main alkaloid from S. adonidifolius are given in Tables 1 and 2, respectively. The spectra were assigned by 2D experiments (H,HCOSY, C,H-COSY, C,H-COLOC, see e.g. [S]) and show the compound to be identical to the one described as 2 by Urones et nl. [1] and as 3 by Bohlmann et al. [3]. In
2 I 9a 8 9b 3a 19a 19b
6.13 m 5.58 t (3.0) 5.31 d (11.7) 4.43 m 4.28 br d (11.7) 4.07 br d (15.8) 3.76 dd (12.5, 2.1) 3.65 d (12.5)
3b 20 5a 5b 6a,b 14a,b 18 21
3.56 m 3.50 q (6.5) 3.48 rn 2.78 m 2.2-2.0 nl 2.2-2.0 m 1.58 s 1.40 d (6.5)
accordance with structures 2 and 3 we conclude from the NMR spectra that C-12, C-13, C-15, C-19 and C-20 are linked to oxygen. As the mass spectral data require, however, eight double bond equivalents and as the 13C NMR spectrum shows the presence of only one C=C and two C=O double bonds, the compound must be pentacyclic. According to the 13C chemical shifts of C-l 1,
1027
1028
L. WIITE
et d.
.3qp/g 0
N
Hog/$$ H
\
i\
N
t\
N
2
3
I
I
4
C-12 and C-13, an epoxide ring is present at C-11/C-12, cf. the spectrum of erucifoline [2]. Formally, the additional ring closure could have been brought about by formation of an ether bridge between the OH-19 group in 3 on the one hand and the OH-20 group (to yield 4) or the OH-15 group (to yield 5) on the other. Of these two Spiro structures, the correct one for adonifoline is 4 as we proved by a 2D ‘H-detected ‘H/“C-shift correlation experiment [6]. This experiment, optimized for 2Jc,- and 3J,-coupling constants, yields a spectrum with cross peaks between the chemical shifts of C-20 and both H-19s and between those of C-19 and H-20. None of the structures 2, 3, and 5 should show any of these correlations. Instead, 5 should show C-15/H-19 correlations which are, however, absent. Thus, adonifoline possesses structure 4. NOE experiments did not allow conclusions as to the configurations of C-12, C-13, C-15 and C-20.
EXPERIMENTAL
Plant material and extraction of alkaloids. Flowers of Senecio adonidijblius were collected on pasture land in the Cevennes
mountains near the village of Cabrillac, France. The material was air-dried. Alkaloids were extracted with 0.1 M H,SO,, treated with Zn dust to reduce possibly existing N-oxides and prepurified by liquid-solid extraction as described previously E73. Gas chromatography-mass spectrometry. The alkaloid extracts were analysed by capillary GC-MS on a fused silica capillary column (WCOT, 30 m x 0.32 mm, coated with an immobilized methyl silicone stationary phase) directly coupled to a quadrupole mass spectrometer. Conditions: inj. 250”, split ratio 1: 20;
5
temp. prog. 1%300”, 6” mini, carrier gas He. Mass spectra were recorded at 40 eV. Retention indices were determined by the use of co-chromatographed standard hydrocarbons [S]. Nuclear magnetic resonnnce. Spectra were obtained of a solution of ca 3 mg of 4 in 0.55 ml CDCl, (‘H: 400.1 MHz, ref.: TMS; isC: 100.6 MHz, ref.: CDCI,, 677.05). ‘H-detected long-range ‘H/i%-shift correlation (‘H: 600.1 MHz, 13C: 150.9 MHz). The delay for the evolution of long-range .I,-,-coupling was chosen as 0.07 sec. Adonifoline (4). RI = 2530, EI-MS (GC-MS) m/z (rel. int.): 365 ([Ml’, 2), 350 (1). 320 (l), 294 (4), 222 (3) 208 (4), 138 (35), 136 (23), 120 (90), 119 (100) 94 (37) 93 (59), 80 (17) 43 (22). CI-MS (NH,, probe), 100 eV, m/z (rel. int.): 366 ([M + H] +, 100). FABMS (Xe, glycerol), m/z (rel. int.): 366 ([M+H]‘, lOO), 460 ([M+Gly+H]+, 7). For NMR data, see Tables 1 and 2. REFERENCES
1. Urones, J. G., Barcala, P. B., Sanchez Marcos. I., Femandez Moro, R., Lopez Esteban, M. and Fernandez Rodriguez, A. (1988) Phytochemistry 27, 1507. 2. Roeder, E. (1990) Phytochemutry 29, 11. 3. Bohlmann, F., Zdero, C., Jakupovic, J., Grenz, M., Castro, V., King, R. M., Robinson, H. and Vincent, L. P. D. (1986) Phytochemistry 25. 1151. 4. Witte, L., Ernst, L.. Adam, H. and Hartmann, T. (1992) Phytockemistry 31 (in press). 5. Derome, A. E. (1987) Modern N MR Techniques for Chemistry Research. Pergamon Press, Oxford.
6. Bax, A. and Summers, M. F. (1986) J. Am. Chem. Sot. 10% 2093. 7. Hartmann, T. and Toppel, G. (1987) Phytochemistry X,1639. 8. Wehrli, A. and Kovats, E. (1959) Heir. Chim. Acta 42, 2709.
Phytochemistry, Vol. 31, No. 3, pp. 1027 -1028, 1992 Printed in Great Britain.
REVISED STRUCTURE
OF THE MAIN ALKALOID
OF SENECIO
ADONIDIFOLIUS LUDGER
WITTE, LUDGER ERNST,* VICTOR WRAY~ and THOMAS HARTMANN
Institut fiir Pharmazeutische Biologie, Technische Universitiit, W-3300 Braunschweig, Germany; *NMR-Laboratorium der Chemischen Institute, Technische Universitlt, W-3300 Braunschweig, Germany; tGBF-Gesellschaft fiir Biotechnologische Forschung mbH, W-3300 Braunschweig Germany
(Received23 July 1991) Key Ward Index-Senecio adonidi@ius; Compositae; pyrrolizidine alkaloids; adonifoline; revised structure.
Abstract-The structure of the main pyrrolizidine alkaloid (adonifoline) from Senecio adonidijblius is determined by GC-MS and ‘H and 13CNMR. ‘H-detected 2D long-range ‘H/13C-shift correlation proves a pentacyclic structure with a 1,5-dioxa-spiro[2.5]octane subunit. This requires the revision of two previous structures claimed for adonifoline.
INTRODUCTION
Table 1. “CNMR spectral data of compound 4
Senecio adonidifolius
Loisel. is a pyrrolizidine alkaloidproducing plant which grows on silicate soil meadows in the southern part of Europe (France, Spain, Italy). The alkaloid composition of S. adonidifolius was described by Urones et al. [ 11. They found florosenine (1) and another alkaloid which they claimed to be 12,13,19-trihydroxy15,20-epoxy- 15,20-dihydro-( 12S, 1SR,20R)-senecionan11,16-dione (2) (called adonifoline by Rader [2]) according to NMR data. Bohlmann et al. [3] described a pyrrolizidine alkaloid from the Peruvian S. dolichodoryius with practically identical NMR spectra; these authors suggested structure 3 for this alkaloid. In our group, GC-MS has been applied as a useful analytical tool for the rapid identification of plant pyrrolizidine alkaloids [4]. Preliminary GC-MS analyses of S. adonidfolius revealed data which are inconsistent with structures 2 and 3. This prompted us to reinvestigate the structure.
C
C 11 16 2 1 20 8
15 7 19
169.6 s 167.7 s 135.4 d 130.5 s 79.0 d 71.1 d 73.8 s 73.1 d 67.2 t
12 3 13 9 5
14 6 18 21
65.3 s’ 62.6 t 61.5 ss 60.3 t 53.6 t 40.4 t 35.0 t 15.4 q 14.4 q
*Interchangeable assignments.
Table 2. ‘H NMR spectral data of compound 4
RESULTSANDDISCUSSION
H
H
The GC-MS analysis of Zn/H+-reduced extracts of S. adonidifolius revealed the presence of two tertiary pyrrolizidine alkaloids. The minor component (ca 5%) was identified as the otonecine derivative 1 (RI = 2745, [M] + 423). The major alkaloid (ca 95%) showed an ion at m/z 365 as the highest mass in the spectrum. This ion could be explained by the loss of water from structure 2 or, as suggested by Bohlmann et al. [3], from structure 3. However, both NH&I and FAB mass spectra yielded [M +H] at m/z 366. This finding is inconsistent with either structures 2 or 3. Detailed NMR studies of the tertiary alkaloid were, therefore, performed to elucidate its structure. The 13C and ‘H NMR data of the main alkaloid from S. adonidifolius are given in Tables 1 and 2, respectively. The spectra were assigned by 2D experiments (H,HCOSY, C,H-COSY, C,H-COLOC, see e.g. [S]) and show the compound to be identical to the one described as 2 by Urones et nl. [1] and as 3 by Bohlmann et al. [3]. In
2 I 9a 8 9b 3a 19a 19b
6.13 m 5.58 t (3.0) 5.31 d (11.7) 4.43 m 4.28 br d (11.7) 4.07 br d (15.8) 3.76 dd (12.5, 2.1) 3.65 d (12.5)
3b 20 5a 5b 6a,b 14a,b 18 21
3.56 m 3.50 q (6.5) 3.48 rn 2.78 m 2.2-2.0 nl 2.2-2.0 m 1.58 s 1.40 d (6.5)
accordance with structures 2 and 3 we conclude from the NMR spectra that C-12, C-13, C-15, C-19 and C-20 are linked to oxygen. As the mass spectral data require, however, eight double bond equivalents and as the 13C NMR spectrum shows the presence of only one C=C and two C=O double bonds, the compound must be pentacyclic. According to the 13C chemical shifts of C-l 1,
1027
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L. WIITE
et d.
.3qp/g 0
N
Hog/$$ H
\
i\
N
t\
N
2
3
I
I
4
C-12 and C-13, an epoxide ring is present at C-11/C-12, cf. the spectrum of erucifoline [2]. Formally, the additional ring closure could have been brought about by formation of an ether bridge between the OH-19 group in 3 on the one hand and the OH-20 group (to yield 4) or the OH-15 group (to yield 5) on the other. Of these two Spiro structures, the correct one for adonifoline is 4 as we proved by a 2D ‘H-detected ‘H/“C-shift correlation experiment [6]. This experiment, optimized for 2Jc,- and 3J,-coupling constants, yields a spectrum with cross peaks between the chemical shifts of C-20 and both H-19s and between those of C-19 and H-20. None of the structures 2, 3, and 5 should show any of these correlations. Instead, 5 should show C-15/H-19 correlations which are, however, absent. Thus, adonifoline possesses structure 4. NOE experiments did not allow conclusions as to the configurations of C-12, C-13, C-15 and C-20.
EXPERIMENTAL
Plant material and extraction of alkaloids. Flowers of Senecio adonidijblius were collected on pasture land in the Cevennes
mountains near the village of Cabrillac, France. The material was air-dried. Alkaloids were extracted with 0.1 M H,SO,, treated with Zn dust to reduce possibly existing N-oxides and prepurified by liquid-solid extraction as described previously E73. Gas chromatography-mass spectrometry. The alkaloid extracts were analysed by capillary GC-MS on a fused silica capillary column (WCOT, 30 m x 0.32 mm, coated with an immobilized methyl silicone stationary phase) directly coupled to a quadrupole mass spectrometer. Conditions: inj. 250”, split ratio 1: 20;
5
temp. prog. 1%300”, 6” mini, carrier gas He. Mass spectra were recorded at 40 eV. Retention indices were determined by the use of co-chromatographed standard hydrocarbons [S]. Nuclear magnetic resonnnce. Spectra were obtained of a solution of ca 3 mg of 4 in 0.55 ml CDCl, (‘H: 400.1 MHz, ref.: TMS; isC: 100.6 MHz, ref.: CDCI,, 677.05). ‘H-detected long-range ‘H/i%-shift correlation (‘H: 600.1 MHz, 13C: 150.9 MHz). The delay for the evolution of long-range .I,-,-coupling was chosen as 0.07 sec. Adonifoline (4). RI = 2530, EI-MS (GC-MS) m/z (rel. int.): 365 ([Ml’, 2), 350 (1). 320 (l), 294 (4), 222 (3) 208 (4), 138 (35), 136 (23), 120 (90), 119 (100) 94 (37) 93 (59), 80 (17) 43 (22). CI-MS (NH,, probe), 100 eV, m/z (rel. int.): 366 ([M + H] +, 100). FABMS (Xe, glycerol), m/z (rel. int.): 366 ([M+H]‘, lOO), 460 ([M+Gly+H]+, 7). For NMR data, see Tables 1 and 2. REFERENCES
1. Urones, J. G., Barcala, P. B., Sanchez Marcos. I., Femandez Moro, R., Lopez Esteban, M. and Fernandez Rodriguez, A. (1988) Phytochemistry 27, 1507. 2. Roeder, E. (1990) Phytochemutry 29, 11. 3. Bohlmann, F., Zdero, C., Jakupovic, J., Grenz, M., Castro, V., King, R. M., Robinson, H. and Vincent, L. P. D. (1986) Phytochemistry 25. 1151. 4. Witte, L., Ernst, L.. Adam, H. and Hartmann, T. (1992) Phytockemistry 31 (in press). 5. Derome, A. E. (1987) Modern N MR Techniques for Chemistry Research. Pergamon Press, Oxford.
6. Bax, A. and Summers, M. F. (1986) J. Am. Chem. Sot. 10% 2093. 7. Hartmann, T. and Toppel, G. (1987) Phytochemistry X,1639. 8. Wehrli, A. and Kovats, E. (1959) Heir. Chim. Acta 42, 2709.