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(+)-12α-hydroxylupanine, a lupin alkaloid from Lygos raetam
Ph~f~~em~~ry,
Vol. 31, No. 9, pp. 3251-3253,
Printedin GreatBritain.
0031-9422,‘92 $5.00+0.00 8 1992Pergamon PressLtd
1992
(+)-12a-HYDROXYLUPANINE,
A LUPIN ALKALOID
OSAMA B. ABDEL-HALIN,* TOSHIKAZU SEKINE, KAZUK SAITO, AHMED
FROM LYGOS RAETAM
F. HALIM,t HOSNY ABDEL-FATTAHt and
I$AMU MURAKOSHI$ Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-lcu, Chiba 263, Japan; tFaculty of Pharmacy, Mansoura University, Mansoura, Egypt (Received15 Nove&er 1991) Key Word Index-Lygos raetam; Leguminosae; quinolizidine alkaloid; lupin alkaloid; (+)-i2a-hydroxylupsnine; (f)-retamint; (-)-lupanine; absolute ~n~~~tio~ alkaloid content.
Ab&rati-A new lupin alkaloid, ( + all-hydroxylu~nine, was isolated from the aerial part of Lvgos metant, together with six known alkaloids: (+)-retamine, (+ )-sparteine, f -)-lupanine, (-)-anagyrine, (-)-cytisine and (-)_Nmethylcytisine. The structure of the new alkaloid including absolute confitiration was determined by spectroscopic methods and by its chemical transformation to ( +)-retamine.
The various species of Retama have been a subject of interest to several groups of investigators Cl-53 due to their rich alkaloidal content. Lygos raetam (Retama metam) var. sarcocarpa (Zoh) is a small leafless wild desert shrub [6,7], growing in Egypt. In the present paper, we report the isolation and structural elucidation of a new lupin alkaloid, (+)-12a-hydroxylupanine (I), and of six known lupin alkaloids ( +)-retamine (2), (+ )-sparteine, (-)-lupanine (3), (-))-anagyrine, (-)-cytisine and (-)-Nmethylcytisine.
1 OH 3 H
RESULTSAND DISCUSSION
From the 75% ethanolic extract of the dry aerial parts of L. raetam var. sarcocarpa 1 was isolated along with six known alkaloids: (+)-sparteine, (+)-&amine (Z), (-)lupanine (3), (-)-anagyrine, (-)-cytisine and (-)-lvmethylcytisine. All of these compounds were identified by their physicochemical properties (mp, [a&,, IR, MS, ‘H NMR, 13C NMR) and chromato~aphic behaviour (HPLC and GC). The HR-EI mass spectrum of 1 afforded the molecular formula C,,H,,N,Oa ([Ml’ m/z 264.1851, talc. 264.1839). The presence of a hydroxyl group was indicated by the fragment ions at m/z 247 (20) and 246 (base peak) in the EI mass spectrum. These correspond to [M -OH] + and [M - H,O] +, respectively. The El mass spectrum of 1 showed some fragments which resemble those of ( +)-retamine (Z),especially a fragment ion at m/z 207 (24). In the IR spectrum, the band at 34OOcrn-’ indicated the presence of hydroxyl group, the band at 1630 cm-’ a lactam C=O group and the two bands at 2850 and 2770 cm- ’ a trans quinol~~ne group. From these res*Permanent address: Faculty of Pharmacy, Mansoura University, Mansoura, Egypt. $Author to whom correspondence should be addressed.
ults, 1 could be presumed to be the sparteine-type lupin alkaloid containing lactam and hydroxyl groups in the molecule. The 13CNMR spectrum 1 showed the presence of 15 carbons which could be assigned as shown in Table 1. Determination of the multiplicity was carried out by DEPT experiments, which revealed that 1 has five methine carbons and nine methylene carbons. The substitution pattern could be deduced as follows: the carbonyl group is located in position 2 because C-2 has the same chemical shift (S171.5) as (-)-lupaaine (3) and also the hydroxyl group is likely to be located at position 12 because the signal corresponding to the carbinol carbon at 6 70.6 (la,C-12) is coincident with that of (+)-retamine (2). The 13CNMR data of 1 resembles those of (+)retamine (2) and (-)-lupanine (3), where the chemical shifts of the carbons on ring A and B of 1 are coincident with those of (--)&pa&e (3). On the other hand, the chemical shifts of the carbons on rings C and D are similar to those of (+)-retamine (2), as shown in Table 1. Hence
0. B. ABDEL-HALIMet al.
3252 Table 1. Comparison of ‘“GNMR data of (+~12a-hydroxyiupanine (1) (+)-retamine (2) and (-)-lupanine (3) (GDCI,, TMS as int. standard) G
1
2
2 3 4 5 6 I 8 9 10 11 12 13 14 1s 17
171.5 s 33.0 t 19.5 t 27.3 t 61.0d 31.9 d 27.9 t 32.1 d 47.0 t 66.7 d 70.6 d 30.9 t 19.8 t 55.0 t 52.5 t
56.2 t 25.8 t 24.6 t 28.8 t 66.4d 32.1 d 29.3 t 33.0 d 62.2 t 66.9d 70.9 d 31.3 t 19.9 t 54.8 t 52.8 t
3 171.3 s 32.8 t 19.4 t 26.6 t 60.7 d 34.6 d 27.3 t 32.0 d 46.5 t 63.9 d 33.4 d 24.2 t 24.9 t 55.4 t 52.7 t
the structure of 1 was considered to be 12-hydroxylupanine. In the ‘HNMR spectrum of 1, the downfield shifted protons resonate at 64.5 (lH, dt, J= 13.1,2.3 Hz, H-LO@ and at 3.3 (lH, m, H-6@) and resemble those of (-)lupanine (3) in chemical shift, multipli~ty and coupling constant. On the other hand, the proton in 1 which resonated at 6 3.6 (lH, br s, H-12/I), resembles the carbinol proton (H-128) in ($)-retamine (2). These data provided further evidence that the sparteine skeleton of 1 is substituted by carbonyl and hydroxyl group at C-2 and C-12, respectively. The assi~ents including all protons and carbons were confirmed by ‘H-‘H correlation spectroscopy (COSY) and 13C-lH COSY. The a-configuration of the hydroxyl group is supported by NOESY spectral data (Fig. l), with cross peaks being observed between H-128 at 6 3.6 and H-13& H-11fl and H-9a at 6 1.46, 1.78 and 2.12, respectively. Also there is a cross peak between H-l& and H-8a indicting that both protons have a 1-3 diaxial relationship. Conclusive evidence for the configuration of the hydroxyl group in compounds 1 and 2 was obtained by single X-ray analysis of (+)-retamine (2). It was possible to establish unequivocally that the secondary OH group at C-12 in ring D has axial a-configuration, a feature which had not been clarified exactly before for (+)-re!amine (2) [&-lo]. The full details of the diffraction analysis and ~onfo~ation description of (+)-retamine (2) were presented [ll]. These results confirm the NMR proposal studies for the structure of 1. The final confirmation of the structure of 1
1 has the same absolute configuration as (+)-retamine (2) (6$7R,9R,llR). From these results, it can, therefore, be concluded that the new alkaloid (1) is (+)-12ff-hydroxylupanine.
We have already found that L. raetam var. sarcocarpa contained both the (-f-lupanine-type and the (-)anagyrine-type alkaloids which have the same absolute configuration (7R,9R,llR) [14]. Okuda et al. [14] have generalized from their extensive stereochemical studies on the lupin alkaloids that the alkaloids having the same absolute configuration of the methylene bridge (C-7 and C-9) occur together in a plant and that (7&9S) and (7R,9R) alkaloids generally do not accumulate in the same plant, except in a few cases [15]. Our present studies showed that L. ruetam var. surcocarpa accumulates only one type of (7R,9R) alkaloid which is consistent with this generalization. As ( - )-lupanine and ( +)-retamine are present in Lygos raetam var. surcocarpa, compound 1 is presumed to be biosynthesized from the latter two precursors either by hydroxylation or oxidation, respectively.
EXPERIMENTAL General. Mps: uncorr; IR; thin films of GHCls and KBr; Optical rotations: 1Ocm path length in the solvents stated, ‘HNMR and 13CNMR: 500 and 125 MHz, respectively. TMS was used as int. standard in GDGl,. High and low resolution EIMS: 70 eV; TLC: silica gel Kieselgel60, F254) of 0.25 mm layer thickness in GH,Gl,-MeOH-28% NH,OH (43:6: 1) and Et,O-M&H-28% NH,OH (17:2: 1) and Al,O, (F254, type E) in G,H,--Me,GO-MeOH (34: 3: 3). The chromatograms were visualized by spraying with Dragendorf and iodoplatinate reagents. Analytical GG and HPLG were performed as described in the literature [16-181. Extraction and isolation of alkaloids. The aerial parts of Lygos raetam var. sarcocarpa wer,e collected in the Abo-Mady area (north of Egypt) in April, 1988. The plant was identified by Prof. N. El-Hadidy, Department of Botany, Faculty of Science, Cairo University and a voucher specimen has been deposited in the Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Egypt. The total alkaloid fraction from 75% EtOH extracts of the dry aerial parts was obtained in yields of 2.5% of the dry wt by a reported method [19]. The mixture of bases (lO.Og) was chromatographed on a silica gel column (Merck, type 60,230-400 mesh, 300 g, 2.5 x 54 cm) and gradient elution using MeOH in CH,Gl,-28% NH,OH (5OO:l) as reported in previous papers [ZO, 213. Fractions enriched in 1 (60 mg) were eluted with 3% MeOH in CH,Gl,-28% NH,OH (500: 1) together with (-)-lupanine (3). Pure 1 (35 mg) was obtained from these fractions by further purification on an Al,OJ column (Merck, type 94 mesh 70-230) with C,H,-MeOH-Me&O (100: 1.5: 1.5). (+~12a-~ydroxy~u~~i~ (1). Crystals; mp 173-175”; [a]k4 +2W (EtOH, c 0.15); HR-EIMS m/z (rel. int.): 264.1851 [MI’ (8) (calcd for G,,H,,N,O,, 264.1839), 247 (20), 246 (lOO),207 (24), 134 (24), 112 (20), 55 (14); IR ez” cm-‘: 3400 br (OH), 2850, 2770 (Bohlmann bands), 1630 (lactam C=O); ‘H NMR (CDCl,, 500 MHz): 64.5 (lH, dt, J= 13.1, 2.3, H-lOa), 3.6 (lH, br s, H12&3.3(1H,m,H-6a),2.85(1H, t,.I=11.2,H-17a),2.7(1H,d,.i =8.S, H-lSa), 2.53 (lH, d, J= 15.2, H-10&, 2.46 (lH, d, 1=11.3, H-30$, 2.33 (la, m, H-3fi), 2.12 (2H, br d, H-Sa, H-E)@), 2.09-2.05 (lH,m, H-7ol), 1.97(1H,d,J=3.48, H-178), 1.95(1H,d,J=3.48, H-15& 1.90-1.85 (2H, m, H-4a, H-14a), 1.82 (lH, m, H-13a), 1.79-1.75 (2H, m, H-Sa, H-lip), 1.62 (lH, m, H-4/Q 1.57-1.49 (lH, m, H-S@),1.47 (lH, d like, H-148), 1.44 (lH, d like, H-13B), 1.34 (lH, m, H-8gk 13GNMR (CDGI,, 125 MHz): Table 1. The other known alkaloids were eluted in the following order: (--)-anagyrine (95 mg), oil, [u]g - 162” (EtOH; c 0.8), eluted by
(+)-12a-Hydroxylupanine 1.5% MeOH in CH,Cl,-28% NH,OH (5OO:l); (--)-Nmethylcytisine (65 mg), needles, mp 137”, [a];’ -220” (EtOH; c O.lS), eluted by 2% MeOH in CH,Cl,-28% NH,OH (500: 1) (-)-lupanine (3) (9Omg), oil, [a]k4 -57.5” (EtOH; c 0.15); (-)cytisine (53 mg), needles, mp 154” [ali - 115” (EtOH; c 0.25), eluted by 3.5% MeOH in CH,Cl,-28% NH,OH (500: 1) (+)retamine (2) (2.5 g), prismatic crystals, mp 166”, [a]k4 +46” (EtOH, c 0.25), eluted by 5% MeOH in CH,Cl,-28% NH,OH (5oo:l); (+)-sparteine (1.2g), oil, [a]g+16.5” (EtOH, c l.O), eluted by 7% MeOH in CH,Cl,-28% NH,OH (500: 1). These alkaloids were identified with the authentic compounds by EIMS, IR, ‘H NMR, ‘“C NMR, HPLC and GC. Catalytic hydrogenation of 1. Compound 1 (20 mg) was dissolved in HCl(2 M, 1.0 ml) and hydrogenated using PtO, under H, for 24 hr. The catalyst was filtered off, and the filtrate was basified with aq. NaOH (10%) and extracted with CH,Cl,. On evapn, 2 (15 mg) was obtained, which was crystallized from Et,O. Mp 166-167”, [alA +46” (EtOH; c 0.15) IR v= cm-‘: 3350 (OH), 2800,275O (Bohlmann bands). REFERENCES
1. Williaman, J. J. and Shubert, G. (1961) in A&&id Bearing Plants and Their Contained Alkaloids, Technical Bulletin, No. 1234, Washington, D.C. 2. Plugger, P. C. and Rauwerda (1896) Arch. Pharm. 234,685. 3. White, E. P. (1957) N.Z.J. Sci. Tech. 38B, 712. 4. Ahmed,A. F. and Rixk, A. M.(1963) J. Chem U.A.R.6,227. 5. Gonzalez Gonxalex, A., Toste, A. H. and Hemandez, B. R. (1959) Ann. Real Sot. Espan. Fis. y Quim Ser. B 55,607. 6. Tackholm, V. (1974) Student Flora ofEgypt, 2nd edn, p. 227.
Cairo University Press, Academic Press, Cairo.
from Lygos raetam
3253
7. Zohary, M. (1972) Flora Palaestina, 47. 8. Bohlmann, F., Overwien, H. and Schumann, D. (1965) Chem. Ber. 98, 659. 9. Shin, K. H., Fonzes, L. and Marion, L. (1965) Can. J. Chem. 43, 2012. 10. Southon, I. W. and Buckinghan, J. (1989) Dictionary of Organic Alkaloids, p. 915. Chapman & Hall, London. 11. 112th Annual Meeting of the Pharmaceutical Society of Japan (1992) Abstract 2-226, Fukuoka. 12. Nakano, T., Spinelli, A. C. and Mendez, A. M. (1974) J. Org. Chem. 39, 3584. 13. Ribas, I., Castedo, J. L. and Garcia, A. (1965) Tetrahedron Letters 36, 3181. 14. Okuda, S., Kataoka, H. and Tsuda, K. (1965) Cheat. Pharm. Bull. 13, 491.
15. Ohmiya, S., Otomasu, H., Haginiwa, J. and Murakoshi, I. (1984) Phytochemistry 23, 2665. 16. Murakoshi, I., Watanabe, M., Okuda, T., Kidoguchi, E., Haginiwa, H. J., Ohmiya, S. and Otomasu, H. (1985) Phytochemistry 24, 2707. 17. Saito, K., Kobayashi, K., Ohmiya, Otomasu, H. and Murakoshi, I. (1989) J. Chromatogr. 462, 333. 18. Ohmiya, S., Kubo, H., Otomasu, H., Saito, K. and Murakoshi, I. (1990) Heterocycles 30, 537. 19. Ohmiya, S., Otomasu, H., Murakoshi, I. and Haginiwa, J. (1974) Phytochemistry 13, 643. 20. Saito, K., Takamatsu, S., Sekine, T., Ikegami, F., Ohmiya, S., Kubo, H., Otomasu, H. and Murakoshi, I. (1989) Phytochemistry 28, 958. 21. Saito, K., Takamatsu, S., Ohmiya, S., Otomasu, H., Yasuda, M., Kano, Y. and Murakoshi, I. (1988) Phytochemistry 27, 3715.
Vol. 31, No. 9, pp. 3251-3253,
Printedin GreatBritain.
0031-9422,‘92 $5.00+0.00 8 1992Pergamon PressLtd
1992
(+)-12a-HYDROXYLUPANINE,
A LUPIN ALKALOID
OSAMA B. ABDEL-HALIN,* TOSHIKAZU SEKINE, KAZUK SAITO, AHMED
FROM LYGOS RAETAM
F. HALIM,t HOSNY ABDEL-FATTAHt and
I$AMU MURAKOSHI$ Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-lcu, Chiba 263, Japan; tFaculty of Pharmacy, Mansoura University, Mansoura, Egypt (Received15 Nove&er 1991) Key Word Index-Lygos raetam; Leguminosae; quinolizidine alkaloid; lupin alkaloid; (+)-i2a-hydroxylupsnine; (f)-retamint; (-)-lupanine; absolute ~n~~~tio~ alkaloid content.
Ab&rati-A new lupin alkaloid, ( + all-hydroxylu~nine, was isolated from the aerial part of Lvgos metant, together with six known alkaloids: (+)-retamine, (+ )-sparteine, f -)-lupanine, (-)-anagyrine, (-)-cytisine and (-)_Nmethylcytisine. The structure of the new alkaloid including absolute confitiration was determined by spectroscopic methods and by its chemical transformation to ( +)-retamine.
The various species of Retama have been a subject of interest to several groups of investigators Cl-53 due to their rich alkaloidal content. Lygos raetam (Retama metam) var. sarcocarpa (Zoh) is a small leafless wild desert shrub [6,7], growing in Egypt. In the present paper, we report the isolation and structural elucidation of a new lupin alkaloid, (+)-12a-hydroxylupanine (I), and of six known lupin alkaloids ( +)-retamine (2), (+ )-sparteine, (-)-lupanine (3), (-))-anagyrine, (-)-cytisine and (-)-Nmethylcytisine.
1 OH 3 H
RESULTSAND DISCUSSION
From the 75% ethanolic extract of the dry aerial parts of L. raetam var. sarcocarpa 1 was isolated along with six known alkaloids: (+)-sparteine, (+)-&amine (Z), (-)lupanine (3), (-)-anagyrine, (-)-cytisine and (-)-lvmethylcytisine. All of these compounds were identified by their physicochemical properties (mp, [a&,, IR, MS, ‘H NMR, 13C NMR) and chromato~aphic behaviour (HPLC and GC). The HR-EI mass spectrum of 1 afforded the molecular formula C,,H,,N,Oa ([Ml’ m/z 264.1851, talc. 264.1839). The presence of a hydroxyl group was indicated by the fragment ions at m/z 247 (20) and 246 (base peak) in the EI mass spectrum. These correspond to [M -OH] + and [M - H,O] +, respectively. The El mass spectrum of 1 showed some fragments which resemble those of ( +)-retamine (Z),especially a fragment ion at m/z 207 (24). In the IR spectrum, the band at 34OOcrn-’ indicated the presence of hydroxyl group, the band at 1630 cm-’ a lactam C=O group and the two bands at 2850 and 2770 cm- ’ a trans quinol~~ne group. From these res*Permanent address: Faculty of Pharmacy, Mansoura University, Mansoura, Egypt. $Author to whom correspondence should be addressed.
ults, 1 could be presumed to be the sparteine-type lupin alkaloid containing lactam and hydroxyl groups in the molecule. The 13CNMR spectrum 1 showed the presence of 15 carbons which could be assigned as shown in Table 1. Determination of the multiplicity was carried out by DEPT experiments, which revealed that 1 has five methine carbons and nine methylene carbons. The substitution pattern could be deduced as follows: the carbonyl group is located in position 2 because C-2 has the same chemical shift (S171.5) as (-)-lupaaine (3) and also the hydroxyl group is likely to be located at position 12 because the signal corresponding to the carbinol carbon at 6 70.6 (la,C-12) is coincident with that of (+)-retamine (2). The 13CNMR data of 1 resembles those of (+)retamine (2) and (-)-lupanine (3), where the chemical shifts of the carbons on ring A and B of 1 are coincident with those of (--)&pa&e (3). On the other hand, the chemical shifts of the carbons on rings C and D are similar to those of (+)-retamine (2), as shown in Table 1. Hence
0. B. ABDEL-HALIMet al.
3252 Table 1. Comparison of ‘“GNMR data of (+~12a-hydroxyiupanine (1) (+)-retamine (2) and (-)-lupanine (3) (GDCI,, TMS as int. standard) G
1
2
2 3 4 5 6 I 8 9 10 11 12 13 14 1s 17
171.5 s 33.0 t 19.5 t 27.3 t 61.0d 31.9 d 27.9 t 32.1 d 47.0 t 66.7 d 70.6 d 30.9 t 19.8 t 55.0 t 52.5 t
56.2 t 25.8 t 24.6 t 28.8 t 66.4d 32.1 d 29.3 t 33.0 d 62.2 t 66.9d 70.9 d 31.3 t 19.9 t 54.8 t 52.8 t
3 171.3 s 32.8 t 19.4 t 26.6 t 60.7 d 34.6 d 27.3 t 32.0 d 46.5 t 63.9 d 33.4 d 24.2 t 24.9 t 55.4 t 52.7 t
the structure of 1 was considered to be 12-hydroxylupanine. In the ‘HNMR spectrum of 1, the downfield shifted protons resonate at 64.5 (lH, dt, J= 13.1,2.3 Hz, H-LO@ and at 3.3 (lH, m, H-6@) and resemble those of (-)lupanine (3) in chemical shift, multipli~ty and coupling constant. On the other hand, the proton in 1 which resonated at 6 3.6 (lH, br s, H-12/I), resembles the carbinol proton (H-128) in ($)-retamine (2). These data provided further evidence that the sparteine skeleton of 1 is substituted by carbonyl and hydroxyl group at C-2 and C-12, respectively. The assi~ents including all protons and carbons were confirmed by ‘H-‘H correlation spectroscopy (COSY) and 13C-lH COSY. The a-configuration of the hydroxyl group is supported by NOESY spectral data (Fig. l), with cross peaks being observed between H-128 at 6 3.6 and H-13& H-11fl and H-9a at 6 1.46, 1.78 and 2.12, respectively. Also there is a cross peak between H-l& and H-8a indicting that both protons have a 1-3 diaxial relationship. Conclusive evidence for the configuration of the hydroxyl group in compounds 1 and 2 was obtained by single X-ray analysis of (+)-retamine (2). It was possible to establish unequivocally that the secondary OH group at C-12 in ring D has axial a-configuration, a feature which had not been clarified exactly before for (+)-re!amine (2) [&-lo]. The full details of the diffraction analysis and ~onfo~ation description of (+)-retamine (2) were presented [ll]. These results confirm the NMR proposal studies for the structure of 1. The final confirmation of the structure of 1
1 has the same absolute configuration as (+)-retamine (2) (6$7R,9R,llR). From these results, it can, therefore, be concluded that the new alkaloid (1) is (+)-12ff-hydroxylupanine.
We have already found that L. raetam var. sarcocarpa contained both the (-f-lupanine-type and the (-)anagyrine-type alkaloids which have the same absolute configuration (7R,9R,llR) [14]. Okuda et al. [14] have generalized from their extensive stereochemical studies on the lupin alkaloids that the alkaloids having the same absolute configuration of the methylene bridge (C-7 and C-9) occur together in a plant and that (7&9S) and (7R,9R) alkaloids generally do not accumulate in the same plant, except in a few cases [15]. Our present studies showed that L. ruetam var. surcocarpa accumulates only one type of (7R,9R) alkaloid which is consistent with this generalization. As ( - )-lupanine and ( +)-retamine are present in Lygos raetam var. surcocarpa, compound 1 is presumed to be biosynthesized from the latter two precursors either by hydroxylation or oxidation, respectively.
EXPERIMENTAL General. Mps: uncorr; IR; thin films of GHCls and KBr; Optical rotations: 1Ocm path length in the solvents stated, ‘HNMR and 13CNMR: 500 and 125 MHz, respectively. TMS was used as int. standard in GDGl,. High and low resolution EIMS: 70 eV; TLC: silica gel Kieselgel60, F254) of 0.25 mm layer thickness in GH,Gl,-MeOH-28% NH,OH (43:6: 1) and Et,O-M&H-28% NH,OH (17:2: 1) and Al,O, (F254, type E) in G,H,--Me,GO-MeOH (34: 3: 3). The chromatograms were visualized by spraying with Dragendorf and iodoplatinate reagents. Analytical GG and HPLG were performed as described in the literature [16-181. Extraction and isolation of alkaloids. The aerial parts of Lygos raetam var. sarcocarpa wer,e collected in the Abo-Mady area (north of Egypt) in April, 1988. The plant was identified by Prof. N. El-Hadidy, Department of Botany, Faculty of Science, Cairo University and a voucher specimen has been deposited in the Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Egypt. The total alkaloid fraction from 75% EtOH extracts of the dry aerial parts was obtained in yields of 2.5% of the dry wt by a reported method [19]. The mixture of bases (lO.Og) was chromatographed on a silica gel column (Merck, type 60,230-400 mesh, 300 g, 2.5 x 54 cm) and gradient elution using MeOH in CH,Gl,-28% NH,OH (5OO:l) as reported in previous papers [ZO, 213. Fractions enriched in 1 (60 mg) were eluted with 3% MeOH in CH,Gl,-28% NH,OH (500: 1) together with (-)-lupanine (3). Pure 1 (35 mg) was obtained from these fractions by further purification on an Al,OJ column (Merck, type 94 mesh 70-230) with C,H,-MeOH-Me&O (100: 1.5: 1.5). (+~12a-~ydroxy~u~~i~ (1). Crystals; mp 173-175”; [a]k4 +2W (EtOH, c 0.15); HR-EIMS m/z (rel. int.): 264.1851 [MI’ (8) (calcd for G,,H,,N,O,, 264.1839), 247 (20), 246 (lOO),207 (24), 134 (24), 112 (20), 55 (14); IR ez” cm-‘: 3400 br (OH), 2850, 2770 (Bohlmann bands), 1630 (lactam C=O); ‘H NMR (CDCl,, 500 MHz): 64.5 (lH, dt, J= 13.1, 2.3, H-lOa), 3.6 (lH, br s, H12&3.3(1H,m,H-6a),2.85(1H, t,.I=11.2,H-17a),2.7(1H,d,.i =8.S, H-lSa), 2.53 (lH, d, J= 15.2, H-10&, 2.46 (lH, d, 1=11.3, H-30$, 2.33 (la, m, H-3fi), 2.12 (2H, br d, H-Sa, H-E)@), 2.09-2.05 (lH,m, H-7ol), 1.97(1H,d,J=3.48, H-178), 1.95(1H,d,J=3.48, H-15& 1.90-1.85 (2H, m, H-4a, H-14a), 1.82 (lH, m, H-13a), 1.79-1.75 (2H, m, H-Sa, H-lip), 1.62 (lH, m, H-4/Q 1.57-1.49 (lH, m, H-S@),1.47 (lH, d like, H-148), 1.44 (lH, d like, H-13B), 1.34 (lH, m, H-8gk 13GNMR (CDGI,, 125 MHz): Table 1. The other known alkaloids were eluted in the following order: (--)-anagyrine (95 mg), oil, [u]g - 162” (EtOH; c 0.8), eluted by
(+)-12a-Hydroxylupanine 1.5% MeOH in CH,Cl,-28% NH,OH (5OO:l); (--)-Nmethylcytisine (65 mg), needles, mp 137”, [a];’ -220” (EtOH; c O.lS), eluted by 2% MeOH in CH,Cl,-28% NH,OH (500: 1) (-)-lupanine (3) (9Omg), oil, [a]k4 -57.5” (EtOH; c 0.15); (-)cytisine (53 mg), needles, mp 154” [ali - 115” (EtOH; c 0.25), eluted by 3.5% MeOH in CH,Cl,-28% NH,OH (500: 1) (+)retamine (2) (2.5 g), prismatic crystals, mp 166”, [a]k4 +46” (EtOH, c 0.25), eluted by 5% MeOH in CH,Cl,-28% NH,OH (5oo:l); (+)-sparteine (1.2g), oil, [a]g+16.5” (EtOH, c l.O), eluted by 7% MeOH in CH,Cl,-28% NH,OH (500: 1). These alkaloids were identified with the authentic compounds by EIMS, IR, ‘H NMR, ‘“C NMR, HPLC and GC. Catalytic hydrogenation of 1. Compound 1 (20 mg) was dissolved in HCl(2 M, 1.0 ml) and hydrogenated using PtO, under H, for 24 hr. The catalyst was filtered off, and the filtrate was basified with aq. NaOH (10%) and extracted with CH,Cl,. On evapn, 2 (15 mg) was obtained, which was crystallized from Et,O. Mp 166-167”, [alA +46” (EtOH; c 0.15) IR v= cm-‘: 3350 (OH), 2800,275O (Bohlmann bands). REFERENCES
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