Journal of Natural P&s VOI.56, NO.I I , p p . 1999-2002,Nowmber 1993
I999
(+)-TERMISWE, A NOVEL LUPINE ALKALOID FROM THE SEEDS OF LUPINUS TERMIS MAHMOLJD HAMED MOHAMED
Department of Pharmacognosy, Faruhy ofpharmacy, AI-Azhar University, Arsiut 71524, Egypt and ABDEL-NASSER AHMED EL-SHORBAGI* Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Assiut Uniw'sty, Assiut 7 I5 15, Egypt ABSTFACT.-A novel lupine alkaloid, (?)-termisine 111, was isolated from the EtOH extract of the crushed seeds of Lapinus tennis. The structure of 1 was assigned on the basis of its spectroscopic data, in addition to chemical and semisynthetic methods.
In our continuing studies on lupine alkaloids in Egyptian plants (1-5), we have previously reported the presence of ( -)-A5-dehydromultiflorine, (-)-A'dehydroalbine, and nine lupine alkaloids isolated from the viable seeds of Lupinus termis Forsk. (Leguminosae)( 1,2). L. termis is widely cultivated in the Mediterranean area and Egypt for its edible seeds (6). The seeds are also used in traditional medicine for the treatment of diabetes and ectema(7-11). Recently, we demonstrated the hypoglycemic effect of (-)multiflorine, which was isolatedas amajor compound from L. termis seeds, on streptototocin-induced diabetic mice ( 5 ) . Now, we report the isolation and structure determination of the novel lupine alkaloid, (?)-termkine 111. From the 75% EtOH extract of the crushed seeds, (+)-termkine 117was isolated (0.08%fresh wt) by Si gel chromatography. The eims of 1 showed the same fragment ion peaks with almost the same intensities as those of (?)-lupanine 121 (1,12,13). An additional peak was found at mlz 264 (2%), i.e., 16 units more than thatof( +)-lupanine({b€j+ mlz248).Fabms measurements, using glycerol in one experiment and m-nitrobenzoic acid in another, showed peaks at mlz 297 (CM+ 11' , 2%), and at d z 279 (CM-OW', 100%) suggesting a mol wt of 296. The fragmentation pattern in the eims indicated a possible lupanine skeleton.
The ir spectrum of 1 (KBr) showed a broad intense band at 3300 cm-' (0-H stretching, intramolecular, hydrogen bonding). Multiple bands at 3100 cm-' extending to 2500 cm-' suggested quaternary nitrogen and combination overtones, embodying the C-Hstretching the Bohlmann's bands charactersticfor quinolizidine-type alkaloids (14). A strong, sharp absorption band at 1580 cm-' and a moderate one at 1405 cm-', due to a carboxylate anion, were also observed (15 ) . Thecarboxylateanionhastwostrongly coupled carbon-to-oxygen bonds, with bond strengths intermediate between C=O and C -0 (15 ) , which give rise to the two bands at 1580and 1405 cm-'. In addition, the ammonium band in the 3100-2600 cm-' region gave further evidence for the carboxylate anion, as it can make an endo-salt with the tertiary nitrogen of the compound.
- 0
,.i#
O'
OH
1
0
2
c' (s 7
i
H
5
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Journal of Natural Products
The I3C-nmr spectrum of 1 showed the presence of 16 carbons, which could be assigned as shown in the Experimental section. Determination of the multiplicity was carried out by DEPT experiments, which revealed that 1 has one carbonyl [C=Ol at 6 182.3 ppm, four methine carbons, and eleven methylene carbons. One of these methylene groups is highly deshielded at 6 79.4 ppm. Based on empirical calculations, this signal was assigned as a CH,OH on a quarternary nitrogenatom(16,17). Thechemical shift of a carbonyl group at 6 182.3 pprn (s, CD,OD) could be assigned to a saturated carboxylic acid(18,19), givingadditional evidence for the presence of a carboxylic moiety in the structure. 'H-'H and 'H-I3C Correlation (COSY) experiments showed that the
Wol. 56, No. 11
methylene proton signals at 8 4.60 and 6 4.34 pprn (J=11.7 Ht) were coupled to the carbon at 6 79.4 pprn which belongs to the N-CH,OH group. 'H and 13C assignments based on the above experiments are shown in the Experimental section and Table 1and indicate that 1 is a tricyclic structure substituted at N-12 (CH,OH) and having a side chain of four carbons at C-11, the terminal carbon being a carboxylic group. The conformation of the B and A rings of (?)-termkine should be assigned as a boat-chair on the basis of 'H-nmr analysis in CDC1,. Of the two C-10attached protons, the pseudo-equatorial proton (more downfield)showed geminal coupling(J=12.1 Hz)andalargevicinal coupling to C-9 (J=12.9 Hz). The more upfield C-10 proton (pseudo-axial) ex-
TABLE 1. Correlation of I3C-and 'H-nmr Shifts of (+>Termisine 117 Measured in MeOD and CDC1,. Chemical shift (6 value, J in Hz) CD,OD
4.60 ( l H , d,J=11.7; H,-14) 4.34 ( l H , dd,J=11.8,2.6; H,-14) 4.12 (lH, dd,J=12.8,2.6; H,-13) 3.72 ( l H , d,J=12.8; H q - l l )
-
3.50 ( l H , d,J=13.4; H,-10) 3.36 ( l H , d,J=13.4; H,-10) 3.33 ( l H , td,J=12.2, 1.8; H,-13) 3.13 ( l H , t,j=9.5; H,-2) 3.10 (IH, t,J=6.4; Hq-6) 2.96 ( l H , td,J=13.0, 1.3; K - 2 ) 2.30 ( l H , dd,J=10.4, 3.1; H,-3) 2.24 ( l H , m; H-4') 2.19 ( l H , dd,J=7.0, 1.3; H-2') 1.98 ( l H , d,J=13.7; H,-4) 1.94 (IH, d,J=9.2; H,-3) 1.79 (2H, br s; H,-9, H,-8) 1.74 ( l H , m; H,-5) 1.73(1H, S; H,-7) 1.71 ( l H , m; H,A) 1.68 ( l H , m; H,-8) 1.60 ( l H , dd; J=6.4, 1.7; Hq-3') 1.54 (3H, m; H,-3' H,-4', H,-5)
cDc1, 5.30 ( l H , s; exchangeable OH) 4.05 ( l H , d,J=11.8) 3.95 ClH, d,J=12.1) 3.72 ( l H , dd,J=14.0, 12.1) 3.64 ( l H , d,J=11.3) 3.46 ( l H , br s; N'H exchangeable) 3.30 ( l H , t,J=21.1) 3.22 ( l H , d,J=12.9) 2.98 ( l H , d,J=11.9 Em' Em Em Em Em Em Inb In In In In In In In In In In In
~~
'Em=Embodied in a multiplet integrating five protons centered at 6 2.14. bIn=Included in multiple peaks at 6 1.45 to 6 1.98.
Corresponding c in "C
79.4 79.4 51.5 71.7
-
60.0 60.0 51.5 59.6 63.0 59.6 28.7 25.4 38.6 19.6 28.7 30.5 23.3 30.4 31.5 19.6 23.3 24.0 24.0 25.4 30.4
November 19931 Mohamed and El-Shorbagi: hibited ageminal interaction, but amuch smaller vicinal coupling. These values were compatible only with a boat conformation of ring B, in which the dihedral angle between the pseudo-equatorial H10 and the equatorial H-9 is small. Since compound 1 had been proved by spectral data to contain both basic (N) and acidic (COOH) groups, it is amphoteric and exists as a dipolar ion (20,21). The structure of 1 was confirmed by chemical synthesis from (?)-lupanine 123 (22,23). In the plant, (?)-termishe 113 may possibly be biosynthesized by hydrolysis of (?)-lupanine 121followed by one carbon addition and hydroxylation of the Me group thus formed. EXPERLMENTAL GENERALEXPERIMENTAL PROCEDURES.Mp's are uncorrected. Ir spectra were recorded in CHCI, and KBr. 'H-nmr (500 MHz), "C-nmr (67.8 Mz), 2D 'H- 'H, and 13C- 'H COSY nmr spectra were recorded in CDCI, and CD,OD on a JEOL GSX 500 spectrometer, using TMS as internal standard. Eims was measured on a Hitachi M60 at 70 eV. Fabms using glycerol and mnitrobenzoic acid was measured at room temperature. TICwas carried out in Si gel (Merck)plates (5 mm) in the following sytems: CH,CI,-MeOH28% ",OH (90:9:1, 80:18:1, and 70:30:1). EXTRACTION AND ISOLATION OF ( ? ) [l].-The seeds of L. tennis were collected at the Medicinal Plant Experimental Stationat Assiut University, Assiut, Egypt. Avoucher specimen was identified by Prof. Dr. Kamal ElBatanony (Department of Systematic Botany, Faculty of Science, Cairo University, Cairo, Egypt) and is deposited in the herbarium of the Department of Pharmacognosy,Faculty of Pharmacy, AlAzhar University, Assiut, Egypt. TERMISINE
EXTFMTION AND ISOIATION OF ALKALOIE5.A total basic fraction (22 g, fraction A) was obtained from the 75% EtOH extracts of the airdried seeds( 1 kg) by a previously described method (1,2). T h e aqueous layer remaining was made strongly basic by addition of K,CO, under icecooling andwasextractedwirhCHzC12.~e CH,CI, extracts were combined, dried (K,CO,), and concentrated to dryness to yield fraction B (5.1 g). Fraction B (5.1 g) was chromatographed on a Si gel column (Merck, type 60, 230-400 mesh, 350 g) using CH,CI,/MeOH/28% ",OH to yield the alkaloids as follows: (If:)-lupanine 1800 mg, mp 98", [a12'D 0 ' ( ~ 0 . MeOH), 1 eluted by 4% MeOH/CH,CI,); (-)-multiflorine 1200 mg,
Lupine Alkaloid
2001
oil, Ec~]"D -299" ( ~ 0 . 1MeOH), , eluted by 8% MeOWCH,CI,]; (+)-angustifoline (150 mg, oil, [IX]~'D +5.2' ( ~ 0 . 1 MeOH), , eluted by 8% MeOWCH,CI,]; (-)-albine 1200 mg, oil [Cyl2'D -103" (r=O.l, MeOH), eluted by 10% MeOW CH,Cl,]; (+)-13-hydroxylupanine 1350 mg, colorless needles, mp 174', [UI2'D +45.5" ( F 0 . 1 , MeOH), eluted by 12% MeOWCH,CI,]; and (2)-termisine f800mg, yellow needles, mp 9899', [a]"~0" ( ~ 0 . 1MeOH), , eluted by 25% MeOH/CH,CI,]. ( ?)-TERMSINE[l].-Fine yellow crystals: mp 98-99"; [ c ~ ] ~ ' D'0 ( ~ 0 . 1MeOH); , eims mlz (%) 264 (21,249 (121,248 (651,247 (401,219 (9), 151(11), 150(42), 149(55), 148(18),136(100), 134(20),124(10), 112 (16), 110(26),98 (30),84 (26), 55 (28), 41 (24); ir (KBr) A max cm-' 3650, 3300 (OH), 3100-2500 RJ+ and C-H), 1580, 1405 [C(-O),-]; 'H nmr and 'H-"C COSY (CD,ODandCDCI,)seeTable 1; "Cnmr(CD,OD,
67.8MHz)8182.3(s,C-l'),79.4(t,C-14),71.7 (d,C-l1),63.0(d,C-6),60.0(t,C-10), 59.6(t,C2), 51.5 (t, C-13),38.6 (t, C-2'), 31.5 (d, C-7), 30.5 (d, C-9), 30.4 (t, C-5),28.7 (t, C-3), 25.4 (t, C-4'), 24.0 (t, C-3'), 23.3 (t, C-8), 19.6 (t, C-4); p y l (COOH)=1.8, p& (quaternary N P 9 . 4 and p K , (further part of the molecule)= 10.9; PI (the isoelectric point)=6.3 ( 2)-Termishe was detected by tlc as a minor spot in fraction A and as amajor spot in fraction B.
[I]FROM (?)opening of ( 5 ) Lupanine.+?)-Lupanine 121 (150 mg) was dissolved in HCI-saturated EtOH (30 ml). T h e reaction mixture was allowed to stir at room temperature for 72 h and then refluxed for 2 h. Usual workup by cc yielded 55 mg (35 %) of the openstructured intermediate, mp 138-140°, R, 0.35 [CHCI,-MeOH-NH4OH(8O:20: l)]. Lupanine 121, 80 mg (60%), R,,0.75 was recovered.
SYNTHESIS OF (?)-TERMISINE LLQANINE [2].-Acid induced ring
Fmykation and hydrolysis oftbe interndate.To the open-structured intermediate obtained from the above-mentioned reaction (35 mg) and dissolved in EtOH (10 ml), was added formalin solution (374, 2 ml). The reaction mixture was allowed to stir at room temperature overnight. HCl ( l N , 10 ml) was added, and the reaction mixturewasallowedtostirforafurrher6 hatroom temperature. After neutralization by ",OH (10%) and workup with CHCI, and H,O, the organic layer was concentrated, and the residue was purified by cc to yield 1 (30 mg, 88%). ACKNOWLEDGMENTS The authors are grateful to Prof. Dr. Isamu Murakoshi (Faculty of Pharmaceutical Sciences, ChibaUniversity, Japan) for determining the spectral data.
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M.D. Antoun and O.M.A. Taha, J. Nut. Prod., 44, 179 (1981). M.H. Mohamed, K. Saito, I. Murakoshi, 12. N.N. Jehle, H. Nesvadba, and G. Spiteller, H.A.Kadry,T.I.I(halifa,andH.A.Hassan, Monaisb. Chn.,95,687 (1964). J. Nat. Prod., 53, 1578 (1990). 13. M. Wink, ..3 Witte, and T. Hartmann, M.H.Mohamed,K.Saito,H.A.Kadry,T.I. Planta Med., 43, 342 (1981). Khalifa, H.A. Hassan, and I. Murakoshi, (1958). 14. F,Bohlmann,CbemBer.,91,2157 Pbytochemist*y, 30,3111 (1991). 15. R.M. Silverstein, C.G. Bassler, and T.C. T.Sekine,N. Arai, K. Sait0,M.H. Mohamed, Morrill, “Spectrometric Identification of M.A. Makbod, and I. Murakoshi, Thai J. Organic Compounds,” 3rded.,John Wiley P h . Sci., 16,267 (1992). &Sons,NewYork, 1974,pp. 91,101,109. M.H. Mohamed, H.A. Wry,T.I. Khdifa, 16. J.D. Roberts,F.J. Weigert,J.I. Kroschwitz, H.A.Ammar,T. Sekine,K. Saito, K.Ogata, andH.J. Reich,J.Am. Chn.Soc.,92,1338 and I. Murakoshi, AI-Azhar J. Nat. Prod., (1970). 8, l(1992). 17 E. Breitmaier and W . Voelter,“Carbon-13 5. M.H. Mohamed, H.A. Kadry,T.I. Khd& NMR Spectroscopy,” 3rd ed., VCH, New H.A. Hassan,Y. Fujii, F. Ikegami, K. %to, York, VCH, 1987, p. 315. and I. Murakoshi, presented at the 15th 18. J.B. Stothers and P.C. Lauterber, Can. J. International Congress of Pharmaceutical Cbem., 42,1563 (1964). Sciences of F.I.P., Washington, D.C., Ab19. H. BrouwerandJ.B. Stothers, Can.J.Chn., StraCt 4P-008, (1991). 50,601 (1972). 6. V. Tackholm,“Student’s Flora of Egypt,’’ 20. J, Schmidt, in: “Organic Chemistry.” Ed. 2nd ed.,Cairo University Press, 1974, p. by N. Campbell, Oliver and Boyd LTD, 224. London, 7th Ed., 1955, p. 232. 7. R. Helmi, S.A. El-Mahdy, H. Mi, and M.A. 21. R.G. Bates, “DeterminationofpH,”Znded., KhayyalJ.Egypt.MMad hsa.,52,538(1%9). John Wiley&Sons,NewYork, 1973,p.254. 8. J. Shani, A. Goldschmid, B. Joseph, Z. 22. A.C. &be and S.W. Fenton, J. Am. Chn. Ahronson, and F.G. Sulmaan, Arch. lnt. SKY, 73: 1668 (1951). P b a m o d y n . Ther., 27,210 (1974). 23. H.E. Zauggand W.B. Martin, in: “Organic 9. M.A. Riyad, S.A. Abdel Salam, and S.S. Reactions.” Ed. by R. Adams, John Wiley Mohamed, Phnta M e d , 54,286 (1988). &Sons, New York, 1965, Vol. 14, p. 52. 10. M.D. Antoun and O.M.A. Taha,]. Nat. Raceid 1 9 M a c b 1993 Prod., 40,337 (1977). LITERATURE CITED
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