A cardenolide tetraglycoside from Oxystelma esculentum

~~y~~~e~~sf~y, Vol. 30,No. 1,pp. 301303,1991 Printedin arcat Britain.

A CARDENOLIDE

~31.-9422~1s3.OO+o.m Pergamon Press

plc

FROM OXYSfl”ELMA ~~CU~~~TUM

TETRAGLYCOSIDE

SUMAN SRIVASTAVA, ANAKSHI KHARE and MAHESHWARE P. KHARE Department of Chemistry, Lucknow University, Lucknow, India (Received in revisedfinn Key Word ~~~-Ux~si~l~u

es~~~ent~

16 May 1990)

Asclepiada~e; roots; oxyline; steroid; cardenolide tetraglycoside.

Abstract-A new cardenolide tetraglycoside, oxyline, was isolated from the dried roots of Oxystelma esculentum. On the basis of chemical and spectroscopic evidence the structure of oxyline was established as 3-epi-uzarigenin-3-O-~-Dcymaropyranosyl-(1-+4)-O-B-D-thevetopyranosyl-( 1+4)-O-fl-D-cymaropyranosyl-( l-+4)-O-fi-n-digitoxopyranoside.

INTRODUCTION Cardenolide and pregnane derivatives are reported to be present in several species of the Asclepiadaceae [ 11. The presence of pregnane oligo~ycosides oxystine [2], oxysine [3] and esculentin [4] was reported in an earlier chemical investigation of the roots of Oxystelma esculentum. In continuation of these studies the structure elucidation of a new cardenolide glycoside, named oxyline, (1) is reported. RESULTS AND DISCUSSiON

Oxyline (1) mp 146-150” [e]n -t-4.2” C,,Hs,O,, was isolated from the combined chloroform-ethanol f4: 1 and 3 : 2) extract of 0. esculentum. The positive colour reaction with Kedde reagent [S] shown by 1, along with characteristic bands at 1750 cm-t and 1740 cm-* in its IR spectrum, suggested it contains a butenolide ring. In the ‘H NMR sptrum of 1 at 400 MHz the presence of an AB-system at 65.00 and 4.92 for H-21 (2H) of the butenolide ring, along with signals of four anomeric protons at 64.94 (2H), 4.49 (1H) and 4.35 (lH), indicated it to be a tetraglycoside of a cardenolide. The tetraglycosidic nature of 1 was further substantiated by the presence of four anomeric carbon signals at 6 103.3 (lC), 98.8 (1Ck 96.1 (1C) and 95.8 (1C) in the 13CNMR spectrum of I. To identify the sugar(s) and genin units in 1, it was subjected to mild acid hydrolysis with 0.25 M HaSO, [6] which afforded a crystalline genin 2 and a mixture of three sugars, 4 [x]n +49.8”, 5 [a]o +25.9” and 6 [aID +42.8” which were separated. The genin 2, mp 229232”, [x]n f l&9”, C23H34U4, appeared to be 3-epi~za~genin from comparison of its mp and rotation. This was confirmed by the comparison of its relative mobility on paper chromatography, taking an authentic sample of uzarigenin as the reference and the R, value of 2 was found to be comparable to the reported value for 3-epiuzarigenin [7]. Acetylation of 2 with acetic anhydride in pyridine yielded a known monoa~tate of 3epi~~genin, 3, mp 248-252” (lit. mp 249-254”) [aIn f 16.01, C&H,,O,, further lending support to the genin 2 being 3-epiuzarigenin, The three chromatographically pure sugars, 4-6 gave a pink colour with the xanthydrol reaction, confirming

them to be 2-deoxy sugars. The sugars 4-6 were identified as ~-cymarose f8], lilacinabiose [9] and D-digitoxosc [lO], respectively, by comparison with the authentic samples ([a&, PC). For further cha~cteri~ation 4 and 6 were oxidized with bromine water to their lactones, which on treatment with phenylhydr~ine yielded the known Dcymaronic acid phenylhydrazide [S] and D-digitoxonic acid phenylhydrazide [lo]. Compound 5 was oxidized with bromine water to the lactone which was subjected to acid hydrolysis under forcing condition using the Kiliani method [ 1l] to yield two components, a syrupy lactone and a free sugar. The sugar was identified as Dthevetose (3-0-methyl&deoxy-D-gluwse) [9J, [cr]n + 38.9” (PC and [a]& The syrupy lactone on treatment with phenylhydrazine yielded the known crystalline D-cymaronic acid phenylhydrazide [8] (mmp, TLC). Compound 5 was therefore characterized as the disaccharide of D-thevetose and D-cymarose [4-O-(3-U-methyl-6-deoxy-~-D-glucopyranosyl~D-c~arose], i.e. lil~nabjo~. A more direct chemical proof for 1 being a tetraglycoside comprised of cymarose, the disaccharide lilacinabiose and digitoxose, together with the determination of the sequence of the sugar units in 1, was provided by very mild acid hydrolysis (0.0025 M H,SO,) at room temperature. After six days, the hydrolysa~ contained cymarose (4) (PC, TLC) as the only sugar unit together with a new, vanillin-perchloric acid positive spot, presumably a triglycoside (7) and with some unreacted starting material 1. This indicated that cymarose was the terminal sugar unit in 1. After nine days two additional new spots (TLC) appeared, one of which was lilacinabiose (5) (PC, TLC) and the other was presumably the monoglycoside (S), indicating that lila~nabio~ was next in sequence after cymarose in 1. Finally after 12 days, two new spots (TLC) appeared, one identical in mobility with digitoxose (6) and the other comparable with 3-epiuzarigenin (2) (PC) indicating that digitoxose is directly linked to the genin moiety at the C-3 hydroxyl group, this being the only available secondary hydroxyl group in the genin moiety of 1. The EI mass spectrum of 1 did not exhibit a [M]’ but the mass ion peak recorded at m/z 374 corresponded to [M -tetrasaccharide unit]’ and was in agreement with the genin 2 (C,,H,@J. The subsequent losses of two

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S. SRWASTAVA et al.

302

RIO” k

1 R’= Rz t

R”=H

3

R’=Ac

MC

Me H,OH

H,OH

H,OH H

H

4

5

6

3_epiuurrigenin-3-O-P_D-Thev p fl-4)-O-~-D-Cymprl-4)-0-~~~.

D~tox~y~osi~

7 3-eplutarigenin-3-O-8- D - Dtgitoxopyranoside

8 Cym p =Cymaropymno~ ‘Ihev p = Thevetopyrsnose

water molecules from this ion giving ion peaks at m/z356 and 338 were in agreement with the presence of two hydroxy groups in its genin moiety. The ion peaks at m/z 322 and 292 originated from the tetrasaccharide moiety of the glycoside [12]. The lower mass region also contained the common fragment ion peaks of sugars and genin. The ‘H NMR spectrum of 1 at 400 MHz not only confirmed the derived structure but also helped in ascertaining the configuration of the glycosidic linkages. A two proton double doublet (J = 8 and 2.5 Hz) at 64.94, a doublet (J = 8 Hz) at 64.49 and a double doublet (J = 8 and 2.5 Hz) at 64.35 were assigned to the anomeric protons of the four sugars. The large coupling constants of these anomeric protons were typical of their axial configuration, suggesting that these sugar moieties were in the 4C, @)-conformation and joined to the aglycone through a &glycosidic linkage [13]. The spectrum also contained singlets of three protons each for the three methoxy groups at 63.67, 3.45 and 3.43 besides the downfield signal of one proton singlet at 65.88 of H-22 and an AB-system of two protons at 65.00 and 4.92 of H21 of the butenolide ring. In the light of the above evidence, the structure of oxyline (1) was established as 3-epi~a~g~in-3-O-~-~-cymaropyr~osyI(l+4)-0-~-Dthevetopyranosyi( 1~4)-~-~-~-~ymaropyranosyl (f-+4)0-B-D-digitoxopyranoside.

The general procedures were the same as those reported earlier [i4] except t3C NMR spectra were determined in CDC!, with a 400 MHz spectrometer. Plant extraction. Shade dried powdered roots (10 kg) of 0. esculentum (voucher No. 68528, deposited in the National Botanical Research Institute, Lucknow, India) were extracted and fractionated as reported earlier [a]. Repeated CC of the combined CHCI,-EtOH (4: 1 and 3: 2) extract (3.5 g) over silica gel using CHCI,-MeOH (47:35 as eluent afforded oxyline 1 (48 r@. Oxyfine (1). Mp 146-150’ [ali +4.2’ (MeOH, cO.lI), (Found: C, 63.09; H, 8.44, C,,HsoO,, requires C, 63.02, H, 8.40%). It gave a pink colour in the xanthydrol and violet in the Kedde test. ‘H NMR (400 MHz): 65.88 (lH, s, H-22). S.OO(lH, d, J== 18 Hz, H-21), 4.94 (ZH, dd, J=8 and 2.5 Hz. H-l”, H-l”“), 4.92 (lH, d, J- I8 Hz, H-21). 4.49 (IH, d, f=8 Hz, H-l”‘), 4.35 flH, dd, J=8 and 2.5 Hz, H-l‘), 3.67 (3H, s, OMe), 3.40 (lH, t, J =6 Hz, H-2”‘), 3.45 (3H, s, OMef, 3.43 (3H, s, OMe), 2.76(lH, m, H-17), 2.14-2.08(3H, m, H-2’,2”,2”“e), 1.94-184(3H, m, H-2’, 2”, 2”“a), 1.8-1.55 (-CH,-aglycone), 1.35 (3H, d, 5=6 Hz, V-Me) 1.31 (6H, d, J=6 Hz, 6’-Me), 1.30 (3H, d, J=6 Hz, 6’-Me), 0.87 (3H, s, 18-Me), 0.78 (3H, s, 19-Me). r jC NMR 636.9 (C-l), 26.8 (C-2), 76.5 (C-3), 35.5 (C-4), 44.7 (CS),29.1 (C-S), 27.2(C-7),41.9(C-8), 49.4(C-9), 35.7&J-lo), 20.0&Z-

Cardenolide glycoside from Oxystelma esculentum 1l), 39.9 (C-12), 49.6 (C-13), 85.5 (C-14), 35.5 (C-15), 27.2 (C-16), 51.2 (C-17), 15.6 (C-18), 12.0 (C-19), 175.1 (C-20), 74.5 (C-21), 117.4(C-22), 174.7(C-23), 103.3(C-l’), 98.8 (C-l’), 96.1 (C-l’), 95.8 (C-l’), 18.6 (C-6’), 18.5 (3 x C-fX), 60.6 (-OMe), 58.2 (-OMe), 57.9 ( - OMe). MS m/z: [M]’ not observed, 374 CM+--sugar], 356 [374 -H20]+, 338 [356-H20]+, 228 [338-CeHa02-j+, 203 [356 -C,H,O,]+, 111 [CCaH,02]+, sugar fragments; 322 [tetrasaccharide ion-C,,H,,O, (disaccharide)]+, 292 [tetrasaccharide ion-C,_+H,,O, (disaccharide)]+. Mild hydrolysis of compound 1 with acid. To a soln of l(15 mg) in 80% aq. dioxane (1 ml) was added 0.5 M H,SO, (1 ml) and the sola was warmed for 30 min at SO*.Usual work-up as reported earlier [14] afforded crystalline genin 2 (3 mg) mp 229-232, [aJi5 + 16.9” (CHCI,; cO.l), Rvurircnin 1.29 and a mixture of three sugars, which were separated through CC affording 4 (2 mg), [@I;” +49.8” (H,O; ~0.14). 5 (3.4 mg), [a]$ +25.9” (H,O; c 0.12) and 6 (1.8 mg), [a],” + 42.8” (MeOH; E0.11). All sugars gave positive colouration in the xanthydrol and Keller-Kiliani reactions. The specific rotation, TLC and PC comparisons of 4-6 showed them to be identical to D-CyIWirOSe, lilacinabiose and Ddigitoxose, respectively. Oxidation of4 with bromine wuter. A soln of4 (1.8 mg) in H,O (0.4 ml) was mixed with Br, (6 ~1)and shaken in a stoppered flask in the dark for 24 ht at room temp. The excess of Br, was then removed under red. pres., the acidic mixture was made neutral with freshly pptd A&CO,, and the suspension was filtered. H,S was passed through the filtrate to remove Ag+ ions, and the suspension was again filtered. The filtrate was evapd to dryness under red. pres. yielding a syrupy lactone (1.2 mg) showing a violet spot with NH,OH-FeCI, spray reagent. Ox~a~ion of5 with bromine water. A soln of 5 (3.2 mg) in H,O (0.1 ml) was mixed with Br, (12 111)as in the oxidation of 4 affording a syrupy lactone (2.6 mg) showing a violet spot with NH,OH-FeCI, spray reagent. Oxidafion of6 with bromine water. A soln of6 (1.6 mg) in H,O (0.3 ml) was mixed with Br, (5 11) as in the oxidation of 4 affording syrupy lactone (1 mg) showing a violet spot with NH,OH-FeCI, spray reagent. D-CparOniC acid phenylhydrazide. A soln of cymaronolactone (1.2 mg) in absolute EtOH (0.04 ml) on heating with freshly dist. phenylhydrazine (0.04 ml) and usual work-up yielded crystalfine Dcymaronic acid phenylhydrazide from MeOH-Et,0 (0.6 mg) mp 150-154, lit. mp 155” [S]. D-Digitoxonic acid p~nyl~ydrazide. A soln of digitoxonolactone (1 mg) in absolute EtOH (0.03 ml) was mixed with freshly diit. phenylhydrazine (0.03 ml) as in cymaronolactone affording Ddigitoxonic acid phenylhydrazide crystallized from MeOH-Et,0 as needles (0.6 mg) mp 119-123” lit. mp 123” [lo]. Kiiiani hydrolysis of &tone of copout 5. Com~und S (2.6 mg) was dissolved in Kiliani mixture (0.25 rnk HOAc-H,O-HCS 7: 11: 2), and heated at loo” for 1 hr. Usual work-up [9] yielded D-cymarono-l,4-lactone (1.0 mg) and 3-Omethyl-ddeoxy-D-glucose (1.1 mg) 116-120” (MeOH-petrol) [alp +38.9” (H& ~0.12).

303

Very mild hydrolysis of compound 1 with acid. To a soln of 1 (15 mg) in 80% aq. l&dioxane (2.5 ml) was added 0.005 M H,SO, (2.5 ml) and the soln was kept at room temp. After 6 days TLC of the reaction mixture exhibited a spot due to cymarose (4) (R,,, 1.00, cymarose as reference) and two spots of mobilities (R,,, 2.03) and (RcYm1.55)presumed to be triglycoside (7) and the unhydrolysed starting material 1, respectively. After 9 days two additional new spots appeared, one comparable with the calculated mobility of lilacinabiose (5) (R_,, 0.64), and the other was presumed to be monoglycoside (8) (R,,, 2.68). The hydrolysis was complete in 12 days showing two additional new spots identical in mobilities with 3_epiuzarigenin, and digitoxose (6) (REy,,,0.2). The reaction mixture was then worked-up foilowed by CC affording a crystalline 3-epiuzarigenin (2) mp 229-232”, [a];’ + 17.1” (CHCI,; ~0.19) and three chromatographically pure reducing sugars as viscous syrups, viz 4 (2.1 me), S (3.2 mg) and 6 (1.9 mg) which were identified as cymarose, lilacinabiose, and digitoxose, respectively. Acetylation of compound 2. Compound 2 (2 mg) on acetyiation with Ac,O (0.3 ml) in pyridine (0.3 ml) at 100” for 4 hr and usual work-up afforded a crystalline monoacetate 3 (1.4 mg) mp 248-250”, [a];‘+ 15.9 ’ (MeOH, c 0.11) ‘H NMR (80 MHz) 2.08 (3H, s, OAc). Acknowledgement-One

of us (S. S.) is grateful to the C.S.I.R., New Delhi, for financial assistance.

1. Reichstein, T. (1967) Naburwissenschaffen 54, 53. 2. Trivedi, R., Khare, A. and Khare, M. P: (1988) Phytochemistry 27, 2297. 3. Trivedi, R., Khare, A. and Khare, M. P. (1989) P~ytoc~ernistry 2t3, 1211. 4. Trivedi, R., Khare, A. and Khare, M. P. (1990) Pkytochemistry 29, (in press). 5. Kedde, D. L. (1947), Pharm. Weekbl. 82,741. 6. Rang~wami, S. and Reichstein, T. (1949), Wetv. Chti. Acta 32,939. 7. Kuritzkes, A., Euw, I. V. and Reichstein, T. (1959) Helu. Chim. Acta 42, 1502. 8. Krasso, A. F., Weiss, E. and Reichstein, T. (1963) Hrlu. C&m. Acta 46, 1691. 9. Allgeier, H. (1968) Hero. Chim. Acta 51, 668. 10. Eppenberger, U., Kaufmann, H., St6cklin, W. and Reichstein, T. (1966) Helv. Chim. Acta 49, 1492. 11. Kiliani, H. (1930) Bar. E)eutscR.Chon. Ges. 63, 2866. 12. Khare, M. P. and Khare, A. (1987) J. Carbohydr. Ckm. 6, 523. 13. Allgeier, H. (1968) He/v. Chim. Acta 51, 311. 14. Kaur, K. J., Khan, M. P. and Khare, A. (1985) J. Nat. Pro&. 48,928.