NATURAL TOXINS 1:81-83 (1992)
PyrroIizidine Al kaIoids From Werneria nubigena Erhard Roeder, Thomas Bourauel, and lngeborg Theisen Pharmazeutisches lnstitut der Universitat Bonn (E.R., T.B.) and Botanisches lnstitut der Univenitat Bonn (I.T.) Bonn, Federal Republic of Germany
Four toxic pyrrolizidine alkaloids were isolated from Wemeria nub9ena and their structures ABSTRACT inc. determined by spectroscopic methods. 0 1992 WII~Y-LISS, Key Words: Asteraceae, Tribe Senecioneae, Retronine, Retrorsine-Noxide, Senecionine, lntegerrimine
INTRODUCTION
Plants containing hepatotoxic and mutagenic pyrrolizidine alkaloids (PAS) are of worldwide distribution. The most important genera are Senrcio (Asteraceae), Crotalaria (Fabaceae), Symphytum, and Heliotropium (Boraginaceae) [Bull et al., 1968; Mattocks, 19861. Toxic PAS within the family Asteraceae are restricted to the tribe Senecioneae and Eupcitorieae [Nordenstam, 1977; Dominguez, 19771. As Werneria nuhigena Humb., Bonpl. and Kunth, a plant species from the Andean highland areas of Ecuador belongs to the tribe Senecioneae, it may be presumed that PAS should be present in it, as well [Wagenitz, 19641. As Werneria species are used in Andean traditional medicine as a home remedy for inflammatory and gastrointestinal diseases, it is important to clarify the occurrence of PAS within this plant species [De Tommasi et al., 19921. However, PAS in W. nubigena have not been reported previously. We describe the separation and structural elucidation of alkaloids from this species. MATERIALS AND METHODS General
IH and I3C NMR spectra were determined in CDCI, with TMS as internal standard or D,O at 300 MHz for 'H and 75 MHz for I3C. 'H-'H-correlated and 'H-"Cheteronuclear shift correlated NMR spectra were collected using the pulse sequence HETCOR. High resolution EIMS were recorded at 70 eV, 300 PA, temperature 180°C. Mps are uncorr.
University of Bonn). A voucher specimen was deposited in the Herbarium of the Botanic Institute of University of Bonn (1. Meusel). Isolation of Alkaloids
Extraction of the plant material (whole plant, 26 g) was carried out as described by Roeder and Liu [1991]. Repeated prep. thin-layer chromotography (TLC) of the basic extract (silica gel, Merck plates F254,20 x 20 cm, CH2C12-MeOH-NH40H[25'%,],85: 14: 1) and the butanolic extract (A1203, Merck plates, 20 x 20 cm, nBuOH-AcOH-H20, 4: 1 : 5 , BuOH phase) yielded four PAS (alkaloid content: 3.3%): 129 mg (l), 705 mg (2), 8 mg (3), 2 mg (4). TLC detection was according to Dann [ 19601 and Mattocks [ 19671. Retrorsine (I )
Mp 216", [216-216.5": Bull et al., 19681. [U]D -23" (c 1; EtOH), [- 18" (c I ; EtOH): Bull et al., 19683. IR cm - I : 3565 (OH), 3050 (= C-H), 3000-2850 (CHalkyl), 2820 (NCH,), 1740 (satd CO, R), 17 10 (unsatd CO, R), 1655 (C=C), 1450 and 1375 (CH,), 1250 and 1230 (C-0). E I M S d z , (rel. int.): [MI" +,C1gH2sN06: found, 351.1680 (16.0), (calc. 351.1682); C17H25N04: 307 (2.1), Ci5H2oNOz: 246 (17.9), C13H18NOz: 220 (29.0), CgH,,NO: 138 (43.6),CgHllNO: 137 (26.9), Cg HioNO: 136 (98.5), CgHIlN: 121 (53.1), CgHioN: 120 (loo), CgH,N: 119 (88.1), CgHgN: 118 (14.9), CgH,N: 117 (4.5), CbHgN: 95 (48.7), CbHgN: 94 (62.7), C6H7N: 93 (75.9, C5H6N: 80 (28.3). 'H and 13C NMR (CDC13): Tables 1-111.
Plant Material
Werneria nubigena Humb., Bonpl. and Kunth was collected at the westside of Chimborazo (altitude: 4,800 m, Ecuador) in the third week of March 1991 and authenticated by Prof. Dr. W. Barthlott (Botanic Institute of 0
1992 Wiley-Ltss, Inc.
Received March 26, 1992; accepted for publication Juiy 30, 1992. Address reprint requests to Dr. Ehard Roeder, Pharmazeutisches Institut, Univenitat Bonn, An der lmmenburg 4, D-5300 Bonn 1, Federal Republic of Germany.
82
ROEDER ET AL. TABLE 111. Coupling Constants of Alkaloids ( I ) and (2)
TABLE 1. 13C NMR Spectral Data of Alkaloids ( I ) and (2) (1)
(2)
132.41 136.60.1 62.75 52.90 34.62 74.96" 77.35" 60.85 175.52 8 I .46 35.58" 37.85 131.19 167.33 66.89 I 1.53" 134.48" 14.95"
133.22 134.47" 80.26 70.83 35.33 76.85a 97.98" 62.39 178.02 84.96 38.56" 40.02 130.95 171.55 69.06 13.47" 141.10" 17.49"
~~~
c-1 c-2 c-3 c-5 C-6 c-7 C-8 c-9 c-11 c-12 C-13 C-14 C-15 C-16 C-18 C-19 c-20 c-21
I .8
J2.3b
I .8 I .8 15.8 I .8 1.8 5.8 9.2 0.0 1.5 5.8 12.2 13.8 I .2 3.7 3.7 11.8 0.0 10.5 6.4 12.5 I .5 I .5 11.2 7.1
J2.R J3a.3b
J3a.S J3a,9b
J3b.8 J5s.5b J5a.6a
J5a.6b JSb.6a
J5b.6b J6a.6b
J6a.7 J6b.7
J7.x J9a,Yb
J l 3.14" J13.14b
6 in ppm. "APT negative.
JI3,lY
J14d.14b '14a.20
J14~.21
TABLE I I . IH NMR Spectral Data of Alkaloids ( I ) and (2)
Ji
nd. I
nb
J,",Zl ~
(1)
(2)
6.16 ( I H ) q 3.90 ( I H) dq 3.34 ( I H) ddd 3.22 ( I H) dd 2.49 ( I H) ddd 2.34 ( I H) ddd 2.08 (1 H) dddd 4.97 ( 1 H) td 4.23 (1 H) ddt 5.45 ( I H) d 4.05 ( I H) dd 1.63 ( I H) dq 2.16 ( I H) dquin 1.71 (IH) dd 3.67 ( I H) d 3.57 (1 H) d 0.80 (3H) d 5.68 (1 H) qd I .79 (3H) dd 3.65 (2H) s br
6.37 ( I H) q 4.71 ( I H ) ddd 4.45 (1 H) ddd 3.94 (1 H) td 3.88 (1 H) ddd 2.85 ( I H) dddd 2.60 ( I H) dddd 5.58 (1 H ) td 4.95 (1 H) dtd 5.52 ( I H) d 4.36 ( I H) d 1.77 ( I H) dqd 2.21 ( I H) dquind 1.87 ( I H ) dd 3.77 ( I H) d 3.72 ( I H) d 0.83 (3H) d 6.03 ( I H) qd I .82 (3H) dd -
~~
2-H 3-HA 3-Hi 5-H, 5-Hb 6-H, 6-Hb 7-H 8-H 9-H, 9-Hb 13-H 14-Ha 14-Hb 18-H, 18-Hb 19-H, 20-H 21-H3 OH 6 in ppm.
~~
J2.h
~~
~
0.8 0.8 0.8 17.0 2.0 0.0 2.0 12.0 12.0 6. I 8.5 3.0 15.0 4.8 2.5 4.8 12.2 I .o 10.8 6.4 13.0 0.8 I .o 11.7 7.0
J in Hz.
C l , H l x N 0 2 : 220 (29.2), C , H l z N O : 138 (43.7), CgHilNO: 137 (27.1). CXHIoNO: 136 (98.3), C8HllN: 121 (53.4), CRHloN: 120 (IOO), CgHgN: 119 (88.3), C x H x N : f 18 (15.1), C8H,N: 117 (4.9), C,H,N: 95 (48.9), C,H,N: 94 (62.3), C6H7N: 93 (75.1), C5i-i6N: 80 (28.1). ' H and 13C NMR (D20):Tables 1-111. Senecionine (3)
EIMS m / z , (rel. int.): [MI+", C 1 8 H 2 5 N 0 5found, : 335.1735 (8.8), (calc. 335.1733); CI,H2,NO3: 291 (1 1.9), CI,H22N02: 248 (11.3), C13HlgNO2: 220 (29.0), CgH,,NO: 138 (50.7), CgHIINO: 137(24.3), C8HloNO: 136 (90.6), CgHlIN: 121 (76.8), CgHioN: 120 (IOO), CgH9N: 119 (98.0), CgHgN: 118 (14.4), CXH7N: 117 (7.8), C6H9N: 95 (67.2), C6HsN: 94 (64.4), C,H,N: 93 (86.2), c5 H6N: 80 (28.0).
Retrorsine-N-Oxide (2)
lntegerrimine (4)
M p 145", [145": Rizk, 19911. [ a ] ~- 8 " (c I ; H,O), [-8" (c 1; H 2 0 ) : Rizk, 19911. IR vk,": cm-I: 3050 (= C-H), 3000-2850 (CH,,,,,), 2820 (NCH2), 1740 (satd C02R), 1715 (unsatd C 0 2 R ) , 1660 (C=C), 1450 and 1375 (CH,), 1250 and 1230 (C-0), 960 (N-0). EIMS m/z, (rel. int.): [MI" +,C 1 8 H 2 5 N 0 7 :found, 367.1629 (().I), (Cak. 367.1631); C18H25NO6: 351 (14.0), CLTH25N04: 307 (1.9), CIsH20NO2: 246 (17.5),
EIMS m / z , (rel. int.): [MI'", CI8H2,NO5: found, 335.1733 (3.3), (calc. 335.1733); CI7H25NO3: 291 (7.6), Cl5H22NO2: 248 (7.2), C13HigNO2: 220 (l8.1), CgHIzNO: 138 (43.4), Cg HIINO: 137 (16.1), C8HloNO: 136 (81.0), CgHIIN: 121 (82.0), CXHIoN: 120 (86.0), CgH9N: 119 (loo), CgHxN: 118 (ll.3), CgH7N: I17 (8.7), C6H9N: 95 (63.6), C6HsN: 94 (52.6), C6H7N: 93 (78.7), C S H ~ N80 : (12.3).
a3
PYRROLlZl DINE FROM WERNERIA NUBIGENA
RESULTS AND DISCUSSION
Four alkaloids were isolated by preparatory TLC and their structures were determined by high resolution EIMS, IR, ‘H, and I3C NMR spectroscopic methods. 2D-NMR experiments were applied to gain reliable assignments of ‘H and I3C spectral data of all alkaloids. Alkaloid ( I ) was obtained as fine crystals, mp 216“, ( c = 1; EtOH). Its formula Cl8H2,NO6was [ a ] -23” ~ established by high resolution mass analysis, m / z 351.1680 ([MI” +), calcd for (I): 351.1682. The fragmentation pattern shows that (1) is a 12-membered macrocyclic PA. In addition, the IR spectrum gives the absorption bands of a saturated and an unsaturated ester. The ‘’C NMR spectrum exhibits signals at 6 132.41 and 6 136.60 which indicates a double bond between C-I and (2-2. The signals at 6 77.35 (C-8), 6 74.96 (C-7), and 6 34.62 (C-6) ensure the presence of a retronecine diester. The ‘ H NMR spectrum shows the characteristic signals of a isatinecic acid ester moiety at 6 3.67 (1 H, d, J = I I .2 Hz, 18-H,) and 6 3.57 (IH, d, J = 11.2 Hz, 18-Hb).An olefinic proton at 6 5.68 (IH, qd, J, = 7.1 Hz, J. = 1.5 Hz, 20-H) coupling with H-21 at 6 1.79 (3H, r / l , Jl = 7.1 Hz, J2 = I .5 Hz) and H- 14, at 6 2.16 ( 1 H, dqtiin. Jl = 12.5 Hz, J2 = 1.5 Hz) in addition with the carbon shift of C-I4 at 6 37.85 indicates that the double bond between C - I5 and C-20 has Z-configuration. Complete analysis of the 300 MHz ‘H NMR spectrum was accomplished using ‘ H and 13C 2D-spectra. Both were in complete accordance with the structural assignment. After interpretation of all data alkaloid ( I ) was identified as retrorsine [Segall and Dallas, 19831 (Fig. I). Alkaloid (2) was a glassy solid, mp 145”, [U]D-8” ( c = 1; H 2 0 ) . The formula CI8HZSNO, was established by high resolution mass analysis, m / z 367.1629 ([MI” +), calcd for (2): 367.1631. The ‘ H and I3C NMR spectra of alkaloid (2) are almost identical with alkaloid (I), but the shifted signals at 6 80.26 (C-3), 6 70.83 (C-5), and 6 97.98 (C-8) indicate that (2) is the N-oxide of retrorsine ( I ) [Segall and Dallas, 19831. The minor alkaloids (3) and (4) were obtained as fine needles and identified by comparison with authentic samples (high resolution EIMS and ‘H NMR) as senecionine (3) [Segall and Dallas, 1983; Roeder and Bourauel, 19921 and integerrimine (4) [Habermehl et al., 19881 (Fig. I). The ‘H NMR spectra of alkaloids (1) and (2) were scrutinized by NMR simulation. To our knowledge, this is the very first complete assignment of all coupling constants and multiplicities of the alkaloids (1) and (2). ACKNOWLEDGMENTS
We thank Prof. Dr. W. Barthlott (Botanic Institute of University of Bonn) for identification of plant material.
5
4
3
1
3
4
Fig. 1. Structures of the isolated alkaloids retrorsine (11, senecionine (3), and integerrimine (4)
REFERENCES Bull LB, Culvenor CCJ, Dick AT (1968): “The Pyrrolizidine Alkaloids.’’ Amstcrdam: North Holland Publishing Company. Dann AT (1960): Detection of N-oxides of the pyrrolizidine alkaloids. Nature 186:1051. De Tommasi N, Aquino R, De S h o n e F, Piacente S, Pizza C (1992): Diterpenes from Werneriu tlucrylophyllu. Phytochemistry 3 I :10421043. Dominguez XA ( I 977): Eupatorieae-chemical review. In “The Biology and Chemistry of the Compositae.” London, New York, San Francisco: Academic Press, vol I, pp 487-502. Habermehl GG, Martz W, Tokarnia CH, Doebereiner J, Mendez MC (1988): Livestock poisoning in South America by species of the Senecio plant. Toxicon 26275-286. Mattocks AR (1967): Detection of pyrrolizidine alkaloids on thin-layer chromatograms. J Chromatogr 27505-508. Mattocks AR (1986): “Chemistry and Toxicology of Pyrrolizidine Alkaloids.” London: Academic Press. Nordenstam B (1977): Senecioneae and Liabeae-systematic review. In “The Biology and Chemistry of the Compositae.” London, New York, San Francisco: Academic Press. vol 11, pp 800-830. Rizk AFM (1991): “Naturally Occurring Pyrrolizidine Alkaloids.” Boca Raton, Ann Arbor, Boston: CRC Press. Roeder E, Bourauel T (1992): Pyrrolizidine alkaloids from Melumpyrum prurense. Natural Toxins 1:35-37. Roeder E, Liu K (1991): Pyrrolizidine alkaloids from Senecio inregrijolius var. fauriri. Phytochemistry 30: 1734- 1137. Segall HJ, Dallas JL (1983): ’HNMR spectroscopy of pyrrolizidine alkaloids. Phytochemistry 22:1271-1273. Wagenitz G (1964):Compositae (Asteraceae). In “Engler’s Syllabus der Manzenfamilien,” 12th ed. Berlin-Nikolassee, Gebriider Borntraeger, vol 11, pp 484-497.
PyrroIizidine Al kaIoids From Werneria nubigena Erhard Roeder, Thomas Bourauel, and lngeborg Theisen Pharmazeutisches lnstitut der Universitat Bonn (E.R., T.B.) and Botanisches lnstitut der Univenitat Bonn (I.T.) Bonn, Federal Republic of Germany
Four toxic pyrrolizidine alkaloids were isolated from Wemeria nub9ena and their structures ABSTRACT inc. determined by spectroscopic methods. 0 1992 WII~Y-LISS, Key Words: Asteraceae, Tribe Senecioneae, Retronine, Retrorsine-Noxide, Senecionine, lntegerrimine
INTRODUCTION
Plants containing hepatotoxic and mutagenic pyrrolizidine alkaloids (PAS) are of worldwide distribution. The most important genera are Senrcio (Asteraceae), Crotalaria (Fabaceae), Symphytum, and Heliotropium (Boraginaceae) [Bull et al., 1968; Mattocks, 19861. Toxic PAS within the family Asteraceae are restricted to the tribe Senecioneae and Eupcitorieae [Nordenstam, 1977; Dominguez, 19771. As Werneria nuhigena Humb., Bonpl. and Kunth, a plant species from the Andean highland areas of Ecuador belongs to the tribe Senecioneae, it may be presumed that PAS should be present in it, as well [Wagenitz, 19641. As Werneria species are used in Andean traditional medicine as a home remedy for inflammatory and gastrointestinal diseases, it is important to clarify the occurrence of PAS within this plant species [De Tommasi et al., 19921. However, PAS in W. nubigena have not been reported previously. We describe the separation and structural elucidation of alkaloids from this species. MATERIALS AND METHODS General
IH and I3C NMR spectra were determined in CDCI, with TMS as internal standard or D,O at 300 MHz for 'H and 75 MHz for I3C. 'H-'H-correlated and 'H-"Cheteronuclear shift correlated NMR spectra were collected using the pulse sequence HETCOR. High resolution EIMS were recorded at 70 eV, 300 PA, temperature 180°C. Mps are uncorr.
University of Bonn). A voucher specimen was deposited in the Herbarium of the Botanic Institute of University of Bonn (1. Meusel). Isolation of Alkaloids
Extraction of the plant material (whole plant, 26 g) was carried out as described by Roeder and Liu [1991]. Repeated prep. thin-layer chromotography (TLC) of the basic extract (silica gel, Merck plates F254,20 x 20 cm, CH2C12-MeOH-NH40H[25'%,],85: 14: 1) and the butanolic extract (A1203, Merck plates, 20 x 20 cm, nBuOH-AcOH-H20, 4: 1 : 5 , BuOH phase) yielded four PAS (alkaloid content: 3.3%): 129 mg (l), 705 mg (2), 8 mg (3), 2 mg (4). TLC detection was according to Dann [ 19601 and Mattocks [ 19671. Retrorsine (I )
Mp 216", [216-216.5": Bull et al., 19681. [U]D -23" (c 1; EtOH), [- 18" (c I ; EtOH): Bull et al., 19683. IR cm - I : 3565 (OH), 3050 (= C-H), 3000-2850 (CHalkyl), 2820 (NCH,), 1740 (satd CO, R), 17 10 (unsatd CO, R), 1655 (C=C), 1450 and 1375 (CH,), 1250 and 1230 (C-0). E I M S d z , (rel. int.): [MI" +,C1gH2sN06: found, 351.1680 (16.0), (calc. 351.1682); C17H25N04: 307 (2.1), Ci5H2oNOz: 246 (17.9), C13H18NOz: 220 (29.0), CgH,,NO: 138 (43.6),CgHllNO: 137 (26.9), Cg HioNO: 136 (98.5), CgHIlN: 121 (53.1), CgHioN: 120 (loo), CgH,N: 119 (88.1), CgHgN: 118 (14.9), CgH,N: 117 (4.5), CbHgN: 95 (48.7), CbHgN: 94 (62.7), C6H7N: 93 (75.9, C5H6N: 80 (28.3). 'H and 13C NMR (CDC13): Tables 1-111.
Plant Material
Werneria nubigena Humb., Bonpl. and Kunth was collected at the westside of Chimborazo (altitude: 4,800 m, Ecuador) in the third week of March 1991 and authenticated by Prof. Dr. W. Barthlott (Botanic Institute of 0
1992 Wiley-Ltss, Inc.
Received March 26, 1992; accepted for publication Juiy 30, 1992. Address reprint requests to Dr. Ehard Roeder, Pharmazeutisches Institut, Univenitat Bonn, An der lmmenburg 4, D-5300 Bonn 1, Federal Republic of Germany.
82
ROEDER ET AL. TABLE 111. Coupling Constants of Alkaloids ( I ) and (2)
TABLE 1. 13C NMR Spectral Data of Alkaloids ( I ) and (2) (1)
(2)
132.41 136.60.1 62.75 52.90 34.62 74.96" 77.35" 60.85 175.52 8 I .46 35.58" 37.85 131.19 167.33 66.89 I 1.53" 134.48" 14.95"
133.22 134.47" 80.26 70.83 35.33 76.85a 97.98" 62.39 178.02 84.96 38.56" 40.02 130.95 171.55 69.06 13.47" 141.10" 17.49"
~~~
c-1 c-2 c-3 c-5 C-6 c-7 C-8 c-9 c-11 c-12 C-13 C-14 C-15 C-16 C-18 C-19 c-20 c-21
I .8
J2.3b
I .8 I .8 15.8 I .8 1.8 5.8 9.2 0.0 1.5 5.8 12.2 13.8 I .2 3.7 3.7 11.8 0.0 10.5 6.4 12.5 I .5 I .5 11.2 7.1
J2.R J3a.3b
J3a.S J3a,9b
J3b.8 J5s.5b J5a.6a
J5a.6b JSb.6a
J5b.6b J6a.6b
J6a.7 J6b.7
J7.x J9a,Yb
J l 3.14" J13.14b
6 in ppm. "APT negative.
JI3,lY
J14d.14b '14a.20
J14~.21
TABLE I I . IH NMR Spectral Data of Alkaloids ( I ) and (2)
Ji
nd. I
nb
J,",Zl ~
(1)
(2)
6.16 ( I H ) q 3.90 ( I H) dq 3.34 ( I H) ddd 3.22 ( I H) dd 2.49 ( I H) ddd 2.34 ( I H) ddd 2.08 (1 H) dddd 4.97 ( 1 H) td 4.23 (1 H) ddt 5.45 ( I H) d 4.05 ( I H) dd 1.63 ( I H) dq 2.16 ( I H) dquin 1.71 (IH) dd 3.67 ( I H) d 3.57 (1 H) d 0.80 (3H) d 5.68 (1 H) qd I .79 (3H) dd 3.65 (2H) s br
6.37 ( I H) q 4.71 ( I H ) ddd 4.45 (1 H) ddd 3.94 (1 H) td 3.88 (1 H) ddd 2.85 ( I H) dddd 2.60 ( I H) dddd 5.58 (1 H ) td 4.95 (1 H) dtd 5.52 ( I H) d 4.36 ( I H) d 1.77 ( I H) dqd 2.21 ( I H) dquind 1.87 ( I H ) dd 3.77 ( I H) d 3.72 ( I H) d 0.83 (3H) d 6.03 ( I H) qd I .82 (3H) dd -
~~
2-H 3-HA 3-Hi 5-H, 5-Hb 6-H, 6-Hb 7-H 8-H 9-H, 9-Hb 13-H 14-Ha 14-Hb 18-H, 18-Hb 19-H, 20-H 21-H3 OH 6 in ppm.
~~
J2.h
~~
~
0.8 0.8 0.8 17.0 2.0 0.0 2.0 12.0 12.0 6. I 8.5 3.0 15.0 4.8 2.5 4.8 12.2 I .o 10.8 6.4 13.0 0.8 I .o 11.7 7.0
J in Hz.
C l , H l x N 0 2 : 220 (29.2), C , H l z N O : 138 (43.7), CgHilNO: 137 (27.1). CXHIoNO: 136 (98.3), C8HllN: 121 (53.4), CRHloN: 120 (IOO), CgHgN: 119 (88.3), C x H x N : f 18 (15.1), C8H,N: 117 (4.9), C,H,N: 95 (48.9), C,H,N: 94 (62.3), C6H7N: 93 (75.1), C5i-i6N: 80 (28.1). ' H and 13C NMR (D20):Tables 1-111. Senecionine (3)
EIMS m / z , (rel. int.): [MI+", C 1 8 H 2 5 N 0 5found, : 335.1735 (8.8), (calc. 335.1733); CI,H2,NO3: 291 (1 1.9), CI,H22N02: 248 (11.3), C13HlgNO2: 220 (29.0), CgH,,NO: 138 (50.7), CgHIINO: 137(24.3), C8HloNO: 136 (90.6), CgHlIN: 121 (76.8), CgHioN: 120 (IOO), CgH9N: 119 (98.0), CgHgN: 118 (14.4), CXH7N: 117 (7.8), C6H9N: 95 (67.2), C6HsN: 94 (64.4), C,H,N: 93 (86.2), c5 H6N: 80 (28.0).
Retrorsine-N-Oxide (2)
lntegerrimine (4)
M p 145", [145": Rizk, 19911. [ a ] ~- 8 " (c I ; H,O), [-8" (c 1; H 2 0 ) : Rizk, 19911. IR vk,": cm-I: 3050 (= C-H), 3000-2850 (CH,,,,,), 2820 (NCH2), 1740 (satd C02R), 1715 (unsatd C 0 2 R ) , 1660 (C=C), 1450 and 1375 (CH,), 1250 and 1230 (C-0), 960 (N-0). EIMS m/z, (rel. int.): [MI" +,C 1 8 H 2 5 N 0 7 :found, 367.1629 (().I), (Cak. 367.1631); C18H25NO6: 351 (14.0), CLTH25N04: 307 (1.9), CIsH20NO2: 246 (17.5),
EIMS m / z , (rel. int.): [MI'", CI8H2,NO5: found, 335.1733 (3.3), (calc. 335.1733); CI7H25NO3: 291 (7.6), Cl5H22NO2: 248 (7.2), C13HigNO2: 220 (l8.1), CgHIzNO: 138 (43.4), Cg HIINO: 137 (16.1), C8HloNO: 136 (81.0), CgHIIN: 121 (82.0), CXHIoN: 120 (86.0), CgH9N: 119 (loo), CgHxN: 118 (ll.3), CgH7N: I17 (8.7), C6H9N: 95 (63.6), C6HsN: 94 (52.6), C6H7N: 93 (78.7), C S H ~ N80 : (12.3).
a3
PYRROLlZl DINE FROM WERNERIA NUBIGENA
RESULTS AND DISCUSSION
Four alkaloids were isolated by preparatory TLC and their structures were determined by high resolution EIMS, IR, ‘H, and I3C NMR spectroscopic methods. 2D-NMR experiments were applied to gain reliable assignments of ‘H and I3C spectral data of all alkaloids. Alkaloid ( I ) was obtained as fine crystals, mp 216“, ( c = 1; EtOH). Its formula Cl8H2,NO6was [ a ] -23” ~ established by high resolution mass analysis, m / z 351.1680 ([MI” +), calcd for (I): 351.1682. The fragmentation pattern shows that (1) is a 12-membered macrocyclic PA. In addition, the IR spectrum gives the absorption bands of a saturated and an unsaturated ester. The ‘’C NMR spectrum exhibits signals at 6 132.41 and 6 136.60 which indicates a double bond between C-I and (2-2. The signals at 6 77.35 (C-8), 6 74.96 (C-7), and 6 34.62 (C-6) ensure the presence of a retronecine diester. The ‘ H NMR spectrum shows the characteristic signals of a isatinecic acid ester moiety at 6 3.67 (1 H, d, J = I I .2 Hz, 18-H,) and 6 3.57 (IH, d, J = 11.2 Hz, 18-Hb).An olefinic proton at 6 5.68 (IH, qd, J, = 7.1 Hz, J. = 1.5 Hz, 20-H) coupling with H-21 at 6 1.79 (3H, r / l , Jl = 7.1 Hz, J2 = I .5 Hz) and H- 14, at 6 2.16 ( 1 H, dqtiin. Jl = 12.5 Hz, J2 = 1.5 Hz) in addition with the carbon shift of C-I4 at 6 37.85 indicates that the double bond between C - I5 and C-20 has Z-configuration. Complete analysis of the 300 MHz ‘H NMR spectrum was accomplished using ‘ H and 13C 2D-spectra. Both were in complete accordance with the structural assignment. After interpretation of all data alkaloid ( I ) was identified as retrorsine [Segall and Dallas, 19831 (Fig. I). Alkaloid (2) was a glassy solid, mp 145”, [U]D-8” ( c = 1; H 2 0 ) . The formula CI8HZSNO, was established by high resolution mass analysis, m / z 367.1629 ([MI” +), calcd for (2): 367.1631. The ‘ H and I3C NMR spectra of alkaloid (2) are almost identical with alkaloid (I), but the shifted signals at 6 80.26 (C-3), 6 70.83 (C-5), and 6 97.98 (C-8) indicate that (2) is the N-oxide of retrorsine ( I ) [Segall and Dallas, 19831. The minor alkaloids (3) and (4) were obtained as fine needles and identified by comparison with authentic samples (high resolution EIMS and ‘H NMR) as senecionine (3) [Segall and Dallas, 1983; Roeder and Bourauel, 19921 and integerrimine (4) [Habermehl et al., 19881 (Fig. I). The ‘H NMR spectra of alkaloids (1) and (2) were scrutinized by NMR simulation. To our knowledge, this is the very first complete assignment of all coupling constants and multiplicities of the alkaloids (1) and (2). ACKNOWLEDGMENTS
We thank Prof. Dr. W. Barthlott (Botanic Institute of University of Bonn) for identification of plant material.
5
4
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1
3
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Fig. 1. Structures of the isolated alkaloids retrorsine (11, senecionine (3), and integerrimine (4)
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