Gangan, V D

Pradhan, P

Sipahimalani, A T

NOPR

Indian Journal of ChemistryVo1.36B, September 1997, pp.787 - 792Phytoecdysones from Tinospora cordifolia: Structural elucidation ofEcdysterone and makisterone A by 2D NMR spectroscopyV 0 Gangan ', P Pradhan & A T SipahimalaniBio-Organic Division, Bhabha Atomic Research Centre. Bombay 400 085, IndiaReceived 10 July 1996; accepted 14 November 1996. The n-BuOH fraction of the methanolic extract ofT cordifolia stems. has been acetylated and subjected to exhaustivechromatography (column, radial and preparative TLC). Two phytoecdysones. viz. ecdysterone I and makisterone A 2have been isolated as their polyacetates. Their structures have been elucidated by extensive 10 and 20 NMR studies.Tinospora cordifolia Miers (Menispermaceae), animportant medicinal plant, cultivated throughout In-dian subcontinent has been used, through centuriesin Ayurvedic preparations, for treatment of variousailments'r'. More recently, Thatte and Dahanukar'have shown that the aqueous extracts of the plantwere associated with the stimulation of phagocyticand bactericidal capacity of neutrophils and macro-phages.Chemical investigations of the plant have indi-cated the presence of several diterpene furan lac-tones", phenolic lignarr', etc. Our previous studies,with the polar fraction of the plant, dealt with theisolation and characterisation of two phenyl propaneglycosides'', five norditerpene furan glycosides 7.8and two diterpene furan glycosides". This paper de-scribes the isolation and characterisation of-two phy-toecdysones, viz. ecdysterone I and makisterone A 2which were obtained as the minor components. SinceI and 2 were required for bio-evaluation, their struc-tures were determined essentially by non-destructiveprocedures, viz. high resolution 10 and 20 NMRspectroscopy. Although known from several plantsources'", this constitutes the first report about theiroccurrence in any species of Tinosporaceae familyand for their detailed characterisation based on 20NMR techniques. All the proton and carbon reso-nances were assigned unequivocally using 20 NMRspectroscopy.Following the procedure mentioned in our earlierpublication7, two phytoecdysones were isolated asPresent address: Hindustan CIBA-GEIGY Research Centre,Chemical Development, Aarey Road, Goregaon (E), Bombay400 (1)3. IndiaQAcOH ~'22u12Il H I." "-011AcO~',1AcO-LiuQAc011 ~I~C"" -".CI~ H26C1~II 011('II27 JIi011Ho 2Scheme Itheir polyacetates (La, lb and 2a) from the butanolfraction of the methanol ic extract of the stem of theplant.Compounds Ia {m.p. 145°, [a]26o + 63.8°(MeOH, c 0.170)}, Ib {m.p. 199°, [a]26o + 74.10(MeOH, c 0.130)} and 2a {m.p. 2080 [a]26o + 60.00(MeOH, c 0.160)} were colourless sol ids and showedsimilr colour reactions with SbC!] spray. Their UV788 INDIAN J.CHEM.SEC B, SEPTEMBER 1997spera showed an absorpion maxima at 241 nm (E 4.1)indicating the possibility of a, f3--- unsaturated car-bonyl system. The IR spectra exhibited a strongcharacteristic absorption for hydroxyl groups at3250-3600 em", several strong absorptions in thecarbonyl region (1660-1750 ern") and bands around1217.-1267 em" indicative of acetoxy carbonyls andthe possibility of a, f3unsaturated ketone system. TheFAB MS [Nar of the compounds la, 1b and 2ashowed molecular ions at 629 [M+Nar, 671[M+Nar and 643 [M+Nar, thus indicating molecu-lar weights of 606 [Mr, 648[Mr and 620 [Mrrespectively. Thus compared to la, 1b possessed anadditional acetyl group and 2a an additional methylgroup. The UV, IR and NMR eH, I3C) spectralevidence and comparison of the spectral data I0, II,indicated the compounds 1a and 1b to be identical toecdysterone 2,3,22- triacetate, ecdysterone 2,3,22,25-tetraacetate and compound 2a to makisterone A2,3,22- triacetate. The assignments of the protonsattached to carbons are based on I3C_1H HETCORexperiment and differentiation into number of meth-yls, methylenes, methines and quaternary carbonswere inferred from DEPT experiment. The unambi-guous assignments of various C and H positions,were achieved on the basis of2D experiments involv-ing 13 C-IH COLOC, IH)H COSY and IH-IHNOESY experiments.Ecdysterone 2,3,22 - triacetate 1a. The resonancesignal at OH5.89 showed a long range COSY inter-action with methine protons at OH2.42, 3.12 ascer-taining the position of former at C-7 (Oe 121.5) andthe latter at C-5 rs, 50.9), C-9 (oe 33.5), respectively.The two methylenes at C-I , C-4 and two methines atC-2, C-3 were fixed on the basis of a chain of spin-spin couplings observed in COSY spectrum betweenH-5 (oH2.42)~ H-4 (OH1.82;s,29.1) ~ H-3 (oH5.38;s, 67.0) ~ H-2 (OH5.09; s,68.5) ~ Ha,b-I (oH.aI.52,oH 1.82; 33.9).The proton H-l did not show furtherCOSY cross peaks and was therefore adjacentto quaternary C-IO rs, 38.2). The deshielding ofC-2and C-3 signified that these could be attached tooxygen bonded functionalities and thus supportingthe presence of two secondary hydroxyl groups atthese positions. The position of an angular methylgroup at C-19 was fixed on the basis of COLOCexperiment as cross interactions were observed be-tween C-IO (Oe38.2) ~ H-19 methyl (OH 1.01; oe23.8)"C-1 rs, 33.9) ~ H-19 and C-5 (be 50.9) ~H-19.COSY experiment wts also usefu 1 in estab-lishing the positions of two methylenes at C-II, C-12as scalr couplings were observed between H-9 (bH3.12) ~ Ha,b-1l (Ha 1.48, Hb 1.80; be 20.4) and Hb-l1(bH 1.80)eq ~ Hb-12 (bH 2.07; be 31.8). The proton12 did not show further COSY cross peaks and wastherefore adjacent to quaternary C-13 (oe 47.4) ..COLOC in conjunction with COSY experimentswere also instrumental in establishing the positionsof two methylenes at C-15, C-16 and methine at C-17as cross interactions were observed between C-8 (be164.5) ~H-15 (bH 1.85; be 31.0), H-15 (bH 1.85) ~H-16 (bH 1.82; be 20.4) ~ H-17 (bH 2.32; be 49.4).Proton H-15 and H-17 did not show further COSYcross peaks and were therefore adjacent to quaternaryC-14 (be 84.4) and C-13 (be 47.4), C-20 (be 77.0)respectively. The unusual downfield shift of C-14and C-20 signified that these could be attached tooxygen bonded functionalities and thus supportingthe possibility of tertiary hydroxyl groups at thesepositions. The positions of two angular methylgroups at C-18 and C-21 were assigned on the basisof COLOC interactions between .C-14 (be 84.4) ~H-18 methyl (bH 0.83;be 17.3), C-17 (be 49.4) ~H-21 methyl (bH 1.22; be 21.0) and C-20 (be 77.0) ~H-21 COSY experiment established the positions oftwo methylenes at C-23, C-24 and methine at C-22on the basis of scalar coupling observed betweenH-22 (bH4.82; be 79.7) ~ Ha,b-23 (Ha 1.56, Hb 1.78;be 24.6) ~ H-24 (bH 1.47; be 40.3). The deshieldingofC-22 suggested that it could be attached to oxygenbonded functionality and thus supporting the pres-ence of the third secondary hydroxyl group at thisposition. The proton H-24 did not show furtherCOSY cross peaks and was therefore adjacent toquaternary C-25 (be 70.5). The two tertiary methylgroups were assigned the positions C-26 and C-27 asCOLOC interactions were observed between quater-nary C-25 (be 70.5) ~ H-26 methyl (bH 1.20; be28.4) and C-25 ~ H-27 methyl (bH 1.21; be 30.3).The downfield shift of C-2S signified that it couldalso be attached to oxygen bonded functionality andthus supporting the presence of tertiary hydroxylgroup at C-25. Other COSY and COLOC interactionsare as given in the Table I.The relative stereochemistry of the compound wasfixed on the basis of IH-IH NOESY spectrum. Theimportant nOe interactions observed were betweenH-2 (bH 5.09) ~ H-9 (bH 3.12), H-3 (bH 5.38) ~ H-4(bH 1.82), H-5 (bH 2.42) ~ H-19 methyl (bH 1.01),GANGAN et al. : PHYTOECDYSONES FROM T1NOSPOR4 CORDIFOLIA 789Table I - ID and 2D NMR data of ecdysterone 2.3.22-triacetate 1a in CDCIJPos. bC DEPT bH' COSY NOESY COLOC33.9 -CH2- 1.52(m,Ha) H-2,Hb-1 H-191.82(m,Hb) H-2,Ha-12 68.5 >CH- 5.09(m,IH) H-3.Ha.b-1 H-9 Hb-I3 67.0 >CH- 5.38(br s. IH) H-2,H-4 H-44 29.1 -CH2- 1.82(m,2H) H-3,H-5 H-35 50.9 >CH- 2.42(m.IH) H-4,H-7( I r) H-19 H-196 202.0 >C=O H-197 121.5 -CH= 5.89(br s, IH) H-9 (I r).H-) ( 1r)8 164.5 =C< H-9.H-159 33.5 >CH- 3.12(m.IH) Ha,b-II ,H-7( I r) H-2\0 38.2 >C< H-19.Hb-111 20.4 -CH2- 1.48(m,Ha) H-91.8(m,Hb) H-9,Hb-1212 31.8 -CH2- 1.59(m,Ha) Hb-122.07(m.Hb)t Ha-12,Hb-11 H-1813 47.4 >C<14 84.4 >C< H-7.H-1815 31.0 -CH2- r.85(m,2H) H-1616 20.4 -CH2- 1.82(m.2H) H-15.H-1717 49.4 >CH- 2.32(m,IH) H-16 H-22 H-2I.Hb-1218 17.3 -CH3 0.83(s,3H) Hb-1219 23.8 -CH3 1.01(s.3H) H-520 77.0 >C< H-2121 21.0 -CH3 1.22(s,3H)22 79.7 >CH- 4.82(dd,8.6, I, IH) Ha,b-23 H-17.H-24 H-2123 24.6 -CH2- 1.56(m,Ha) H-22.H-241.78(m,Hb) H-22,H-2424 40.3 -CH2- 1.47(m,2H) Hab-23 H-2225 70.5 >C< H-26.H-2726 28.4 -CH3 1.20(s,3H)27 30.3 -CH3 1.21(s,3H)OCOMe 170.2 >C=O170.4 >C=O172.4 >C=OOCOMe 21.0x3 3xCH3 2.I(s.9H)·Protmv.fssignments are based on I3C - 'H HETCOR and 'H - 'H COSY spectra.t Signal overlapping with acetyl methyl protons.Hb-12 (OH 2.07) ~ H-IS methyl (OH 0.S3), and H-17(OH 2.32) ~ H-22 (OH 4.S2) BH-24 (OH 1.47). NonOe interactions were observed between (H-2, H-9),(H-3, H-4) pairs with (H-5, H-19), (Hb-12, H-1S),(H-17, H-22), (H-22, H-24) pairs, indicating thatformers were on one side of the molecule whereaslatters on other side of the molecule. The importantnOe interactions are as given in Table I.Ecdystero.ne 2,3,22,25-tetraacetate I b The assign-ments of methyls, methylenes and methines weremade on the basis of2D experiments. Compound lbshowed similar types of cross peak patterns as indi-cated for la.On comparing the l3C chemical shift values of lawith those of lb at position C-25 indicated that.comparative downfield shift was observed in 1b (Sc790 INDIAN lCHEM.SEC B, SEPTEMBER 1997Table I - 10 and 20 NMR data of ecdysterone 2,3,22-triacetate la in COCI3Pos. ()c OEPT ()H" COSY NOESY COLOC33.9 -CH2- 1.52(m,Ha) H-2 Hb-II.S2(m,Hb) H-2,Ha-12 6S.5 >CH- 5.09(m,IH) H-3,Ha,b-1 H-9 Hb-I3 67.0 >CH- 5.3S(br s, lH) H-2,H-4 H-44 29.1 -CH2- I.S2(m,2H) H-3,H-5 H-35 50.9 >CH- 2.42(m,lH) H-4,H-7(lr) H-19 H-196 202.0 >C=O H-197 121.5 -CH= 5.S9(br s, lH) H-9 (lr),H-5 (Ir)S 164.5 =C< H-9,H-159 33.5 >CH- ~.12(m,lH) Ha,b-II ,H-7( I r) H-210 3S.2 >C< H-19,Hb-1II 20.4 -CH2- I.4S(m,Ha) H-9I.S(m,Hb) H-9,Hb-1212 31.S -CH2- 1.59(m,Ha) Hb-122.07(m,Hb)t Ha-12,Hb-11 H-IS13 47.4 >C<14 S4.4 >C< H-7,H-IS15 31.0 -CH2- I.S5(m,2H) H~1616 20.4 -CH2- I.S2(m,~H) H-15,H-1717 49.4 >CH- 2.32(m,lH) H-16 H-22 H-21,Hb-12IS 17.3 -CH3 0.S3(s,3H) Hb-1219 23.S -CH3 1.01(s,3H) H-520 77.0 >C< H-2121 21.0 -CH3 1.22(s,3H)22 79.7 >CH- 4.82(dd,S.6, I, lH) Ha,b-23 H-17,H-24 H-2123 24.6 -CH2- 1.56(m,Ha) H-22,H-241.78(m,Hb) H-22,H-2424 40.3 -CH2- 1.47(m,2H) Ha,b!23 H-2225 70.5 >C< H-26,H-2726 2S.4 -CH3 1.20(s,3H)27 30.3 -CH3 1.21(s,3H)OCOMe 170.2 >C=O170.4 >C=O172.4 >C=OOCOMe 21.0x3 3xCH3 2. I (s,9H)"Proton assignments are based on \3CIH HETCOR and IH-IH COSY spectra.t Signal overlapping with acetyl methyl protons.GANGAN et al. : PHYTOECDYSONES FROM TINOSPORA CORDIFOLIA81.7) as compared to Ia (Sc 70.5). This argumentcould be explained by considering that under normalacetylating condition, the tertiary OH at C-25 alsogets acetylated and responsible for causing suchdeshielding which was in agreement with literaturevalues'o.Makisterone A 2,3,22-triacetate 2a. The assign-ment of the structure 2a was also based on 10 and20 NMR data. Compound 2a also displayed similartypes of cross peak patterns as indicated for Ia andI b and shown in Table II.The only difference between Ia and 2a was thatthe latter contained an additional carbon as comparedto the former and was assigned C-24 in the form ofsecondary methyl group (8H 0.91, d,J= 6.2 Hz, 3H).Experimental SectionGeneral. Mps are uncorrected. Optical rotationswere recorded on a JASCO polarimeter, (DIP-370).IR spectra were scanned as KBr pellets on a Shi-madzu FTIR spectrometer (model-8101). UV spectrawere recorded on a Shimadzu UV -visible spectro-photometer (model Graphicord UV -240) using spec-troscopic grade MeOH as the solvent. IH and l3CNMR spectra were recorded at 200 and 50 MHzrespectively on a Bruker AC 200 spectrometer. Allthe 20 experiments were carried out with 0.00 I Msolutions in COCI). For OEPT, signal editings weredone by varying the pulse width of the last polarisa-.tion pulse as 14.5, 29 and 43.5 IlS. In COLOC, 512transients were recorded for each tl experiment of80increments. It was then zero-filling to 256W. In theW2 dimension (l3C) 4K data points were recordedwithout zero-filled. The delays used were: 01 = Is,D2 = 0.030s and D3 = 0.0 15s. The NOESY and COSYspectra were recorded with following parameters;TD, = 256W, SII = 512W and SI = TD = IK withsquare sine bell multiplications in both dimensions.For NOESY a mixing time of I s was used.Isolation of ecdysterone 2,3,22-triacetate Ia, ec-dysterone 2,3,22,25-tetraacetate Ib and makis-terone A 2,3,22-triacetate 2a. The plant material(fresh wt., 5.8 kg was collected from Trombay cam-pus, Bhabha Atomic Research Centre, Bombay andidentified by Dr V Abraham of the Nuclear Agricul-ture Division, BARe. Fresh stems were subjected tocold extraction with MeOH through percolation. Thenon-polar compounds were removed by extractionwith petrol and EtOAc. It was then extracted with79ln-BuOH and subjected to column chromatographyover silica gel, using increasing polarities of MeOHin CHCI). The crude fractions 2 and 3 were acetylatedusing AC20 and pyridine at room temperature for 48hr. Further fractionation was achieved by repeatedradial chromatography of the acylated crude frac-tions as well as by preparative TLC. This resulted inthe isolation of two more compounds, in addition topreviously reported cordifolisides A,B,C7, cordifo-lisides D,E8 and palmatosides C,F9. These were ec-dysterone I and makisterone A 2 and werecharacterized as ecdysterone 2,3,22 triacetate Ia,ecdysterone 2,3,22,25 - tetraacetate Ib and makis-terone A 2,3,22-triacetate 2a, respectively.Chromatographic system. Thin layer chromatog-raphy: TLC plates were prepared using silica gel G(Acme, Bombay). CHCI)- MeOH (97:3) was used asthe solvent system. Detection was done by sprayingwith 20% SbCI) (CHCI) and heating (100°C, 10min., yellowish spot under long UV).Radial chromatography: The circular glass platesof thickness 1 mm, were prepared by using silica gel(PF254, E. Merck, 50 g) in cold distilled water (105mL). For elution, gradually increasing concentra-tions of MeOH in CHCI) were employed:Ecdysterone 2,3,22 - triacetate la. Colourlesssolid (10 mg); Rr 0.36 (CHCI) - MeOH, 97:3);C33H5001O;m.p. 145°; [0.]026+63.80(MeOH, cO.170);IR (KBr): 3525,3496,2967,2876, 1750, 17~0, 1659,1626, 1383, 1368, 1318, 1267, 1250, 1146, 1125,1090,1048,1030,974,947,907,874,841,804,668,552, 521cm-l; UV (MeOH): 229.6 sh (E 3790), 241.2sh (E 3228) nm; 'HNMR (200 MHz, CDCb) and l3CNMR (50 MHz, CDCb): see Table I, FAB MS: m/z629 [M + Nat, 607 [M + Ht, 606 [Mt, 590, 589,571,511,493,460,437,409,387,327,307,289,283,213, 155, 154, l36, 120, 107,89,77,69,56.Ecdysterone 2,3,22,25 - tetraacetate lb. Colour-less solid (10 mg); Rr 0.77 (CHCb - MeOH, 97:3);C35H5201'; m.p. 199°; [0.]D26+ 74.1£ (MeOH, c0.l30); IR (KBr): 3450, 2934, 2855, 1740, 1661,1507, 1449, l372, 1320, 1244, 1217, 1146, 1048,992,949, 882 ern"; UV(MeOH): 226 sh (E 5533),246 sh (E 3494)nm; IH NMR (200 MHz, CDCb):0.83 (s, 3H, CH3, H-18), 1.01(s, 3H, CH3, H-19), 1.21(s, 3H, CH3, H- 21), 1.38 (s, 3H, CH3, H-26), 1.41(s,3H, CH3, H-27), 1.45 (m, Ha-23), 1.54 (m, Ha-l), 1.59(m, Hb-15), 1.60 (m, Hb-23), 1.61(m, Hb-12), 1.65(m, Ha-l1), 1.71(m, Hb-4j, !.76 (m, 2H, H-24), 1.8792 INDIAN J.CHEM.SEC B, SEPTEMBER 1997(m, Hb-15), 1.60 (m, Hb-23), 1.61(m, Hb-12), 1.65(m, Ha-11), 1.71(m, Hb-4), 1.76 (m, 2H, H-24), 1.8(m, Hb-ll), 1.81 (m, Ha-4), 1.84 (m, Hb-1), 1.87 (m,Ha-15), 1.96 (s, 3H), 1.98 (s, 3H), 2.09 (s, 3H), 2.1 0(s, 3H) (4 x-OCOCH3), 1.98 (m, Ha-12), 2.04 (m, 2H,H-16),2.33 (m, 1H), 2.38 (m, 1H), 3.12 (m, 1H), 4.82(dd,J = 9.8, 1.5 Hz, 1H), 5.07 (m, 1H), 5.33 (br s, 1H),5.84 (br s, 1H). Proton assignments are based on\3C-IH HETCOR and IH)H COSY spectra. \3C. NMR (50 MHz, CDCh): 17.4 (C-18), 20.3 (C-11),20.5 (C-16), 21.0 (3 X-OCOrn3), 21.l,(C-21), 22.3(1 x -ococno, 23.7 (C-19), 24.5 (C-23), 25.9 (C-26), 26.2 (C-27), 29.1 (C-4), 31.0 (C-15), 31.6 (C-12),33.5 (C-9), 33.9 (C-1), 37.4 (G·24), 38.2 (C-10), 247.4(C-13), 49.5 (C-17), 50.8 (C-5), 66.9 (G.3)" 68.5(C-2), 76.8(C-20), 79.3(C-22), 81.7 (C-25), 84.4(C- 314), 121.5(C-7), 164.5(C-8), 170.2, 170.5 (2 x >C =0), 172.3 (four acetate carbonyls -OCOCH3), 202.0 4(C-6); FAB MS: m/z 671 [M + Nat, 649 [M + Ht, 5631,589,571,553,529,511,469,493,469,447,429,409,387,363,334, 154, 137, 107, 77, 52.Makisterone A 2,3,22-triacetate 2a. Colourless 7solid (12 mg); Rr 0.33 (CBCh - MeOH, 97:3);C34Hs201O; m.p. 208°;[a12~ + 60.0° (MeOH, c 80.160); IR (KBr): 3525, 2971, 2944, 2878, 1736,1655, 1559, 1541, 1507, 1447, 1372, 1320, 1242, 91219, 1144, 1121, 1048, 1030,992,953,922, 874, .606 em"; UV (MeOH): 226 sh (E 3067), 246 sh (E 101631)nm; IHNMR(200MHz,CDCh)and \3CN~R(50 MHz, CDCh): see Table II; FAB MS m/z: 643[M + Nat, 621 [M + nr. 620 [Mt, 585, 567, 543, 11525,507,483,465,441,437,408,388,327,285,237,191, 165, 154, 136, 107,89, 77, 69, 56.AcknowledgementWe thank Dr 0 Seligmann and Prof Dr H Wagnerof the Institute of Pharmaceutical Biology, Univer-sity of Munchen, Germany for recording the FABMS. One of the authors (V D G) thanks the Depart-ment of Atomic Energy for the award of senior re-search fellowship.ReferencesKirtikar K R & Basu B D, In: Indian medicinal plants,edited by E Blatter, J R Cauis and K S Mhaskar Vol. I (LalitMohan Basu, Allahabad, India) 1933, p.77.6NayampalIi S, Ainapore S S & Nadkarni P M, Indian JPharmacol, 14, 1982,64.Thatte U M & Dahanukar SA. Phytotherapy Res, 3,1989,43.Bhatt R K & Sabata B K. Phytochemistry. 28.1989.2419.Hanurrran J B. Mishra A K & Sabata B K. J Chem Soc,Perkin Trans-I, 1986. 1181.Sipahimalani A T. Norr H & Wagner H. Planta Medica,(>0,1984,596.Gangan V D. Pradhan P. Sipahimalani A T & Banerji A,Phytochemistry, 37. 1994. 781.Gangan V D, Pradhan P. Sipahimalani A T & Banerji A,Phytochemistry. 39. 1995, 1139.Gangan V D, Pradhan P, Sipahimalani A T & Banerji A,Indian J Chem, 35B. 1996,630.Bandra B M R, Jayasinghe L, Karunaratne V, WannigamaG P, Bokel M, Kraus W & Sotheeswaran, Phytochemistry,28. 1989, 1073 and the references cited therein.Miller R W, Clardy J. Kozlowski J. Mikolajczak' K L,Platter R D, Powell R G. Smith R. Weisleder D & ZhengQi-Tai, P1ania Medica. 1984.40.

Phytoecdysones from Tinospora cordifolia: Structural elucidation of Ecdysterone and makisterone A by 2D NMR spectroscopy

http://nopr.niscair.res.in/bitstream/123456789/57322/1/IJCB%2036B%289%29%20787-792.pdf

Phytoecdysones from Tinospora cordifolia: Structural elucidation of Ecdysterone and makisterone A by 2D NMR spectroscopy

Abstract

Similar works

Full text

Available Versions

NOPR