Phytochemistry, Vol. 31, No. 2, pp. 593 596, 1992 Printedin Great Britain. A REARRANGED 0 003 1 -9422/92 $5.00 + 0.00 1992 Pergamon Pressplc zyxwvutsr GERMACRANOLIDE AND OTHER SESQUITERPENE STEP%4 JUJUYENSIS LACTONES FROM zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJ ROBERTO R. GIL, ADRIANA DEL V. PACCIARONI, JUAN C. OBERTI, JESUS G. D~Az* and WERNER HERZ* Instituto Multidisciplinario de Biologia Vegetal (CONICET) and Facultad de Citncias Quimicas, Universidad National de Cbrdoba, 5016 Cbrdoba, Argentina; *Department of Chemistry, The Florida State University, Tallahassee, FL 32306, U.S.A. (Received 5 June 1991) Key Word Index--Sreuia zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA jujuyensis; Eupatorieae; Compositae; jujuyensolide; germacranolides; heliangolides; sesquiterpene lactones. Abstract-Extraction of the aerial parts of Stevia jujuyensis afforded a sesquiterpene skeleton apparently formed by biological rearrangement of a 4-epitansanine derivative derivative itself, two new heliangolides, a new germacradienolide and jaceosidin. ments INTRODUCTION In continuation of our work on Argentine Steoia species [l-7], we have studied Stevia jujuyensis Cabr., a taxon thought to be limited to the province of Jujuy in northwestern Argentina [8]. The new germacradienolide la, two new heliangolides 2a,b, the new 4-epitansanin derivative 3, a sesquiterpene lactone 4 with a new carbon skeleton derived from 3 and the flavone jeceosidin. RESULTS AND DISCUSSION Lactone la was clearly a 1,5-germacradienolide with a trans-fused lactone ring closed to C-6 as shown by the ‘H and 13CNMR spectra listed in Tables 1 and 2. Assign- “f R=Ac b R=H 1 a were verified lactone with a new carbon as well as the 4-epitansanin by spin decoupling. The coupling constants involving H-3 and H-8 showed that the s;bstr tuents at these centres were p-orientated. The S-desacetyl analogue zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM lb is known from Helianthus maximiliani [9]. La&one 2a was a heliangolide with a trans-fused lactone ring closed to C-6 as indicated by the ‘HNMR spectrum (Table l), in particular the value of the coupling constants involving H-7 and H-13a,b and the chemical shift of H-5. /3-Orientation of the ester substituent on H-8 followed from the values of J,,,, Js,9a and J8,9b while aorientation of the hydroxyl group on C-3 followed from the values of Jza.? and J,,.,. The 4’,5’-dihydroxy analogue has been reported from a Eupatorium a~tissirhm colfection [lo]. A second new heliangolide was the corresponding acetate 2b whose ‘HNMR spectrum (Table l), if OR CHzOH La R-H b R=Ac b R=H o Table 1. ‘H NMR spectra of compounds 1, Za,b, 3a and 4 (500 MHz, CDCI,) H 1 1 2a 2b 3 4 5a 5b 6 7 8 9a 9b 13a 13b 148 15* 3 3’* Sa 5’b AC* OH 5.58 br ~(8.5) 5.08 br t (8.5) br dd (12, 3.5) 5.06 br t (7) 4.92 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 3.13 d (14.5) 2.48 dd (14, 6, 3.5) 2.60 ddd (12, 7, 5) 2.8 m 2.33 ddd (14, 12, 10) 2.09 ddd (12, 11.5, 7) 2.3 m 3.08 d (14.5) 4.32 dd (10, 6) 4.65 dd (11.5, 5) 5.61 dd (11.5, 5) 2.82 ddq (12, 3.5, 6.5) 5.2 br d (11) 4.80 ddq (10, - 1) 5.26 dd (11, 1.5) 2.20 ddd (14, 13, 3) 1.39 ddd (14, 12, 4) 5.18 dd (10, 8) 5.15 br d (11) 5.21 br d (11) 4.21 dd (11.5, 3) 2.90 dddd (8, 3.5, 3, 1) 2.95 br s 2.97 br s 3.00 br s 5.79 br dd (5, 2) 5.20 br dd (4,2) 5.22 br s 5.24 br s 2.70 dd (14, 3) 2.73 dd (14, 3) 2.83 br dd (14.5, 5) 2.71 dd (14, 5, 3) 2.31 dd (14.5, 2) 2.54 dd (14.5, 3) 2.36 dd (14, 2) 2.41 dd (14, 2) 6.29 d (3.5) 6.32 d (1.5) 6.33 d (2.5) 6.37 d (2.5) 5.60 d (3) 5.79 d (2) 5.74 d (1.5) 5.76 d (1.5) 1.78 br s 1.71 br s 1.49 br s 1.79 d (1.5) 1.79 d (7) 1.89 br s 1.78 br s 1.09 d (6.5) 7.13 q (7) 6.90 q (7) 6.93 q (7) 6.94 q (7) 1.96 d (7) 1.99 d (7) 1.93 d (7) 1.91 d (7) 4.82 d (12) 4.31 d (12) 4.32 d (12.5) 4.33 br st 4.80 d (12) 4.28 d (12) 4.29 d (12.5) 1.99 s 2.10 s 2.50 br *Intensity three protons. TIntensity two protons. 2a 2b 3a 4 5.68 dd (18.5, 10.5) 5.23 d (10.5) 5.18 d (18.5) 3.16 2.37 1.75 4.48 ddq (12, 5, 7) ddd (12, 5, 12, 5) td (12.5, 6) ddd (12.5, 8, 5) 3.02 dddd (7.5, 3.5, 25, 1) 5.74 dt (7, 1.5) 2.55 dd (16, 7) 2.48 dd (16, 1.5) 6.34 d (3.5) 5.63 d (2.5) 1.20 s 1.19 d (7) 6.81 q (7.5) 1.91 d (7.5) 4.30 br st P a E z f Sesquiterpene Table 2. ‘-‘CNMR spectra (CDCI,, of compounds 67.89 MHz)* lactones from Steoia jujuyensis 595 constants involving H-4 and H-5a,b and the chemical 1, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDC 2a, 3a and 4 shift of H-15 are approximately the same in 4 as in 3a and remains the same as well. A possible biogenetic route to 4 from 3a, probably under 2a acid catalysis with the carbonyl group of 3a protonated, is 4 3a (20.15 MHz) H zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 1 indicated by the arrows in formula 5. In the conformation previously deduced for 3b [ 1 l] which also holds for 3a 123.4 d” 120.0 d 141.2 d 129.0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA dt this would lead to the C-10 stereochemistry shown in 4. 33.4 t 42.5 t 116.1 t 2 35.3 t According to Cabrera [S], among the Steuia species of 67.3 d 207.1 s 215.1 s 77.6 d 3 the region S. jujuyensis is most closely related to S. 140.5 s 40.0 d 35.9 d-f 144.4 s 4 achalensis, from which it differs in its much larger leaves, 40.2 t 40.3 tt 125.5 d” 123.4 dt in an inflorenscence formed from single or few glomerules 75.7 dt 79.3 db 76.7 d 75.0 d 6 on the apices of slender almost sharp branches and in a d 46.1 d 49.0 dt 48.6 d 52.2 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA somewhat different pappus. However, the chemistries of 74.3 db 80.2 d 70.1 dt 70.2 d 8 the two species seem to differ considerably [1,12]. 43.8 t 44.3 t 40.0 tt 43.0 t 9 b the C-4 sterochemistry 139.3 s 54.0 s 137.7 s 135.7 s 169.2 s 168.5 s EXPERIMENTAL t t 124.7 t 122.8 t Extraction of Stevia jujuyensis. Aerial parts of S. jujuyensis 18.2 qb 22.2 q q q Cabrera (980 g) collected in Ciudad Universitaria, Departaq 18.1 qb 19.8 qt q mento Capital, Salta Province, in March 1984 and identified by s 166.1 s 166.1 s s Dr. Luis Ariza Espinar (voucher on deposit in the Museo s 131.3 s 131.6 s s 2’ BotBnico, Cordoba) were exhaustively extracted with CHCI, at d 142.6 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA d 142.5 d d 3’ room temp. to give 109.3 g of crude extract. This was shaken with q 14.3 q 14.3 q q 4 a mixture of MeOH (1.23 l), hexane (5.45 1) and H,O (0.41 1).The r 56.4 t t 56.7 t 5’ aq. layer was sepd, washed with hexane, coned to small vol. at s red. pres. and extracted with CHCl,.Evapn of the CHCl, extract q 2” yielded 46.9 g of gum which was absorbed on silica gel (90 g) and chromatographed over silica gel (850 g) packed in C,H,, 200 ml frs being collected. The polarity of the eluent was increased by *Multiplicities by DEPT pulse sequence. adding Me,CO every 1.51 in the following percentages: 2, 4, 8, TIdentified by heteronuclear decoupling. 12, 16, 20, 25, 30 and 50%. “,bAssignments in the same column may be interchanged. Trituration of fr. 30 with hexane-Me,CO produced solid material which was recrystallized from hexane-Me,CO to give 250 mg of la. Treatment of fr. 40 in the same manner furnished an additional 192 mg of la. Rechromatography of the material compared with that of 2a, exhibited the expected paramagnetic shift (ca 1 ppm) of the H-3 signal as well as the from the mother liquors of fr. l(l.635 g) over silica gel (80 g, dry pack, CHCl,-MeOH, 99: 1) gave in frs 15-21 261 mg of a lactone additional acetate methyl singlet. Comparison of the ‘H and 13CNMR spectra of a mixt. containing 2b, 4 and other lactones, and 73 mg of a fourth lactone 3a (Tables 1 and 2) with those of 4- complex mixt. of lactones which was not further investigated, while frs 22-26 (101 mg) on similar rechromatography gave 8 mg epitansanin (3b) from St&a grisebachii [l l] showed that ofjaceosidin. Extensive purification of ca 150 mg of the lactone it contains an additional b-orientated sarracenyloxy group on C-8 as indicated by the appearance of the H-8 mixt. over sephadex LH-20 eventually furnished ca 10 mg of 2b signal at 65.74 as a doublet of triplets (J,,, N 1, Js,,9a= 7, and ca 40 mg of 4. Frs 4143 of the original chromatogram were combined (10.33g) J 8 9b = 1.5 Hz) and the usual signals of the sarracemc acid over 320 g silica gel packed in C,H,, moiety. The chemical shifts of H-4 (62.82), H-5a,b (62.20 and rechromatographed 100 ml frs being collected. After 500 ml of C,H, the polarity was and 1.39) and H-15 (6 1.09) and the coupling constants increased by adding Me&O in the ratios 1: 49,l: 24,2: 23,3 : 22, involving H-4 and H-5 (12 and ca 3.5 Hz) corresponded to those in 3b, whose structure was established by X-ray 4:21, 1:4, 1:3, 3:7 and 7:13 every 5OOm1, frs 28-31 gave 24mg were crystallography [ 111, hence the C-4 stereochemistry of the of jaceosidin. Frs 31-37 (6.2 g) of the rechromatogram rechromatographed over silica gel (150 C&H,-Me&O, 9: 1, new lactone in as shown in the formula. 1.574 g of 3a. At first glance the presence in the ‘H NMR spectrum of 20 ml frs) to give in frs 2942 after recrystallization Several rechromatograms of frs 4748 of the original chromatothe remaining new lactone of an ABC system charactereventuistic of the terminal viny1 hydrogens of nerolidol sugges- gram (silica gel, various proportions of C,H,-Me&O) ted that it might be an elemanolide formed by Cope ally afforded 2a (280 mg) and unidentified material. (3S,6R,7R,8R)-3-Hydroxy-8-acetoxysarraceny~oxygermacrarearrangement of lb; however, the absence of a signal (la). Mp 118-119.5”; EIMS m/z corresponding to an aldehydic proton and the presence of 1(10),4,11(13)-trien-6,12-elide (3.9) 228 (2.4), 218 (1.9), 217 (2.2), the sequence H-4 (coupled to a methyl group) through H- (rel. int.): 246 [M-C,H,,O,]+ 213 (1.9X 141 (43.3), 99 (35.5), 81 (76), 53 (53.2), 43 (100); MS PC1 9a,b with C-5 carrying two hydrogen atoms quickly required abandoment of this notion. Because C-10 is 406 (21.1), 405 [M + 11’ (lOO), 247 (14,.5), 229 (15.4); ‘H and quaternary and the ’ 3C NMR spectrum (Table 2) exhib- 13CNMR Tables 1 and 2. (3R,6R,7R,8R)-3-Hydroxy-8-sarracenyloxyhe~ianga-1(10),4,11 ited the signal of a ketone carbonyl near 6215, the only (13)-trien-6,12-oIide (2a). Gum, EIMS m/z (rel. int.): 264 [M possible structure was 4 with the carbonyl group linking (3.2), 246 [M-C,H,O,]+ (21.3), 288 (8.8), 217 C-4 and C-10, thus producing a new sesquiterpene skel- -C5H602]+ (7.5), 99 (lOO), 81(86.7), 53 (35.3); PCIMS m/z (rel. int.): 363 [M eton which we have called jujuyane. As the coupling 10 11 12 13 14 15 134.3 136.4 169.3 121.0 18.8 12.0 164.9 127.4 145.7 14.4 57.1 170.3 20.4 s s s 133.8 137.2 169.6 124.6 18.2 17.0 166.1 131.5 142.5 14.2 55.8 s s s 596 R. R. GIL et al. Sosa, V. E., Gil, R. R., Oberti, J. C. Kulanthaivel, P. and + 11’ (28.1), 345 (85.4), 247 (48.0), 229 (100); ‘H and lJC NMR: Tables 1 and 2. Herz, W. (1985) J. Nat. Prod. 48, 340. Oberti, J. C. Gil, R. R., Sosa, V. E. and Herz. W. (1986) (3R,6R,7R,8R)-3-Acetoxy-8-sarracenyloxyhe~~unga-l (lo),&11 Phytochemistry 25, 1479. (Zb). Gum, PCIMS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA m/z (rel. int.): 405 [M (13)~hen-6,12-&de. Gil, R. R., Oberti, J. C., sosa, V. E. and Herz, W. (1987) + 11’ (28.4) 345 (100) 229 (45.7); ‘H NMR: Table 1. (4R,6R,7S,8R)-3-0xo-8-sarraceny[oxygermacru-l (lo),1 1(13)(3a). Mp 138-139”; EIMS m/z (rel. int.): 264 [M dien-6,12-olide -C,H,O,]+ (2.0), 246 [M+,H,O,]+ (S.l), 218 (3.3), 99 (lOO), 97 (18.3), 81 (60.9), 53 (33.0); PCIMS 364 (20.0) 363 [M+l]+ (lOO), 265 (25.3), 247 (21.8); ‘H and 13C NMR Tables 1 and 2. (4R,6R,7S,8R,lOS)-3-Oxo-8-sarroceny[oxyjujuye~so-l,11(13)dien-6,12-elide (4). Gum, PCIMS m/z (rel. int.) 363 [M+ 11’ (lOO), 345 (57.3), 265 (56.5), 247 (38.9 (245 (31.8), 229 (34.5) 197 (31.8); ‘H and i3CNMR: Tables 1 and 2. Acknowledgements-Work in Cordoba was supported by CONICOR and CONICET. J. G. 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Bohlmann, F., Zdero C, King, R. M. and Robinson, H. (1986) Liebigs Ann. 799