Flavonoids from Inula viscosa M. Grande'- 2 . F. Piera 1 , A. Cuenca 1 , P. Torres and I. S. Bellido"' Received: March 18,1985; Accepted: June 25, 1985

Abstract: Phytochemical study of the aerial parts of Inula viscosa resulted in the isolation of sixteen flavonoids. One of them, 3-O-acetylpadmatin [(2i?,3R)-3-acetoxy-5,3' ,4'-trihydroxy-7-methoxyfIavanone], was isolated for the first time as a natural compound. Ferulic aldehyde, 2-methylphloroacetophenone, inuviscolide and 2-deacetoxyxanthinin were also isolated. 1 2 3

Dept. Química Orgánica, Facultad de Ciencias, Universidad de Alicante, Apartado 99, 03080 Alicante, Spain Address for correspondence Dept. Química Orgánica, Facultad de Farmacia, Universidad de Salamanca, 37007 Salamanca, Spain

Results and Discussion Inula viscosa (L.) Ait. (Tribus Inulea, Compositeae) is a herbaceous perennial plant, widespread in the Spanish Levante Region, which has been used for years in folk medicine in the Mediterranean area (1-3). Previous chemical works on the flavonoid content of flowers (4) or the whole plant (5-6) of I viscosa, reported the isolation of 3-(9-methylquercetin (7), 3-0-methylquercetin-7-glucoside, 3,3'-di-0-methylquercetin (8), 3-acetoxyhesperitin, quercetin (6) and 4'-0-methylkaempferol.

415

Flavonoids from Inula viscosa

Reexamination of the acetone extract of the arial parts of J. viscosa yielded, besides the already known flavonoids 6-8 twelve other flavonoids which were identified by spectral methods, comparison with authentic samples or literature data as apigenin (1), genkwanin (2), hispidulin (3), rhamnocitrin (4), 3-O-methylkaempferol (5), naringenin (9), sakuranetin (10), 7-O-methylaromadendrin (11), 3-O-acetylaromadendrin (12),3-0-acetyl-7-0-methylaromadendrin (13), 3-O-acetyltaxifolin (14), padmatin (15) and 3-O-acetylpadmatin (16).

OH OMe OH OMe OH OH OH OH

1 2 3 4 5 6 7 8

H H H OH OMe OH OMe OMe

H H OMe H H H H H

H H H H H OH OH OMe

^OH

f\ o -U r

"-R2

OH 0

9 IB 11 12 13 14 IS 16

R

R1

OH OMe OMe OH OMe OH OMe OMe

H H OH OAc OAc OAc OH OAc

H H H H H OH OH OH

The UV spectra of 1, 2 and 3, were typical of flavones; the shifts induced by addition of different reagents (7-9) (see Table I) and comparison of their physical and spectral data, with those of authentic samples, let us identify them as apigenin, genkwanin (10), and hispidulin (11,12) respectively. Compunds 4 and 5, were both kaempferol monomethyl derivatives, and the induced shifts in the UV spectra by A1C13, NaOAc and NaOEt (see Table I) let us conclude that the methoxy 1 groups were in 4 at C-7 and in 5 at C-3 (9,10,13,14). Compound 6 was identified as quercetin by comparison with an authentic sample; flavonols 7 and 8 are both quercetin deTable I.

rivatives , the induced shifts in the UV spectra (see Table I), let us conclude that 7 had three hydroxyl groups at C-3', C-4' and C-7, and 8 two hydroxyl groups at C-4' and C-7 (5,14,15). Compounds 9 and 10 are flavanones and they were identified as naringenin and its 7-OMe derivative, sakuranetin (M + = 272 and M + = 286, in agreement with the formulae Ci 5 H 12 O s and Ci 6 H H 0 5 , respectively). The 'H-NMR spectra of both flavanones showed the AA'BB' system, charateristic of a 1,4disubstituted aromatic ring (B ring) and that of 10 showed also one signal due to one methoxyl group; on acetylation, 9 yielded a triacetate and 10, a diacetate. Location of the OMe group at C-7 in 10, was easily deduced from the bathochromic shift of band II of the UV spectrum induced by NaOEt, characteristic of flavanones with one hydroxyl group at C-4' and one OMe group at C-7 (16) (see Table II); this was confirmed by the MS fragments (17) at miz (%) 193 (53), 167 (100), 166 (28) and 120 (47). The absolute configuration at C-2, was assigned as 25 from the CD curves, showing dichroic absorptions: 9: Ae2i5 = +14.37; Ae233 = +3.57; Ae247 = +1.94; Ae288 = -18.87; Ae325 = +4.25 10: Ae215 = + 10.70; A e ^ = +2.10; A e ^ = +2.00; Ae28g = - 12.40; Ae32g = +3.80 in agreement with bibliography data (18). Physical and other spectral data also agree with those communicated in the literature (19-21). Compound 11 was identified as a flavanonol because its *HNMR spectrum showed two doublets at 4.60 and 5.06 ppm (/ = 11 Hz), characteristic of trans H-2/H-3 protons in flavanonols (22), and by the bathochroic shift observed for band II of the UV spectrum when A1C13 was added, also characteristic of flavanonols (see Table II). The shifts induced by NaOEt, showed also the presence of one free hydroxyl group at C-4' and one OMe group at C-7. Other '-H-NMR signals (see experimental). showed the presence of one 1,4-disubstituted aromatic ring (B ring) and one methoxyl group. These spectral data and physical constants, allowed us to identify 11, as 7-0methylaromadendrin (23). Table II.

UV Spectra of Compounds?'-16 (X, nm).

Compound

96 % EtOH

A1C13

NaOAc

NaOEt

9 10 11 12 13 14 15 16

290.328 287,334 290.328 293.325 290.331 291.335 288.325 290.329

307,377 306,378 310.382 310.377 310.379 310.379 309.374

287,334 290,328 332 290.330 290,330 288,325 290,330

289,364 291,368 328 288,361 325,379 299,368 287,361

UV Spectra of compounds 1-8 (X, nm).

Compound

96 % EtOH

1 2 3 4 5 6 7 8

267,337 269,338 274,337 269,362 268,352 256.372 256,358 253,357

A1CU

NaOAc

NaOEt

NaOAc/H 3 BO ;

278,300.329,382 300,353 270,301,350,418 277,303,345,396

269,338 278,297.347 267,365 275,301,565

276,398 275,387 279,334,404 274,367,425 276,329,404

Not modified

270,299,360,401 266,276,295,356,400

267,369 254.269.357

280.405 273,333,416

261,382

Planta Medica 1985

416 The absolute configuration as IR was deduced from the CD curve showing dichroic absorptions, A e ^ = +3.0; A e ^ = + 2.4; A629! = - 1 1 . 0 ; Ae330 = +3.5 (18), thus, the absolute configuration at C-3 must also be R. Compounds 12 (M + 330, C 17 H 12 0 7 ) and 13 (M + 344, CisHi 4 Ó 7 ) were both identified as flavanonols because the signals due to C-2 and C-3 protons in their 'H-NMR spectra (see experimental) were characteristic of trans protons in y-pyrone systems (22). They were both acetyl derivatives [IR 12, 1725, 1245 cm"1; 13,1710,1250cm-'; 'H-NMR: 12, 5 1.92 (3H, s); 13, 61.97(3H,s)]. The shifts induced in the UV sepctra by different reagents (see Table II) allowed us to conclude that 12 contained three free hydroxyl groups at C-4', C-5 and C-7, and 13, two free hydroxyl groups at C-4' and C-5, and one OMe group at C-7. The O Ac group must be located at C-3 (16). All these facts identified 12 and 13 as the 3-O-acetyl derivatives of aromadendrin and 7-O-methylaromadendrin, respectively (24-26). The absolute configurations were assigned as (2R, 3R), by comparison of their optical rotations and CD curves with those of 7-O-methylaromadendrin. Compounds 14,15 and 16, were all taxifolin derivatives; 14 was a monoacetylated flavanonol [IR: 1735, 1235 cm' 1 ; 1 HNMR: 6 1.95 (3H, s)]. The trans relative configuration of H-2 and H-3, was deduced from the characteristic AB system in the ^ - N M R spectrum (see experimental). The shift of band II of the UV spectrum, induced by NaOEt (see Table II), is typical of flavanonols with two hydroxyl groups at C-5 and C-7. As the B ring was 1, 3, 4-trisubstituted and gave a pentacetylated derivative, 14 was identified as 3-O-acetyltaxifolin (25, 27). The UV and ^ - N M R spectra of 15 were characteristic of a flavanonol with a OMe group at C-7 (see Table II and experimental) and by acetylation gave a tetracetyl derivative, so, its was identified as 7-O-methyltaxifolin or padmatin (28). The dichroic absorptions of CD curves of 15, confirmed the (2R, 3K) configuration: Ae225 = +6.5; Ae25o = +2.2; AET^ = - 6 . 3 ; Ae 330 = +2.6. The 13 C-NMR of 14 and 15 confirmed the proposed structures (see Table III) (29-31). Table III.

,3

C-NMR data for 14,15 and 16 (50.3 MHz)*.

Carbon**

14

15

16

C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-l' C-2' C-3' C-4' C-5' C-6' MeCO MeCO MeO

81.7 72.9 192.6 164.9 97.1 163.5 96.0 159.4 110.3 128.1 115.7 146.6 146.7 115.2 120.2 167.8 19.8

84.3 72.9 196.3 164.4 95.4 169.0 94.4 163.2 102.2 129.4 115.7 145.5 146.5 115.5 120.3

—

56.0

81.4 72.7 191.9 164.4 95.9 169.9 94.9 162.5 102.2 128.1 115.6 144.2 145.3 114.5 120.8 168.9 20.4 55.9

-

Solvents: acetone-d 6 (14,15) and CDC13 (16) Assignments for C-2' and C-5' and those for C-: and C-4' can be interchanged

The ^ - N M R spectrum of 16 was very similar to that 0Í padmatin, showing as the only singificant difference, the signals due to one acetoxy group at C-3 (see experimental). The 13CNMR data of 16 as compared with those of 14 and 15, are fully consistent with the proposed structure. On acetylation 15 and 16 gave the same tetracetyl derivative, so 16 was identified as 3-O-acetylpadmatin, which was for the first time isolated as a natural compound. From the same extract two sesquiterpene lactones, identified as inuviscolide and 2-deacetoxycanthinin (5), ferulaldehyde (32) and 2-methylphloroacetophenone (33) were also isolated.

Experimental Apparatus Melting points were taken with a Kofler hot stage or in a Büchi 510 and are uncorrected. UV spectra were recorded in 96 % EtOH on a Bausch & Lomb spectronic 2000, IR spectra on a Philips PYE UNI CAM SP3200 spectrometer. 'H-NMR spectra of 1-13 were measured on a Varían EM 360 ('H, 60 MHz) spectrometer and 14-16 on a Bruker WP-200 ( ! H, 200 MHz; 13 C, 50.3MHz) spectrometer, using TMS as internal standard; 6 in ppm. MS were obtained on a Hewlett-Packard 5993 at 70 eV. CD curves were recorded on a Jobin-Yvon Dichrograph Mark III and optical rotations on a Optical Activity AA-100 polarimeter. Material, extraction and isolation Inula viscosa Ait., was collected at the end of October of 1982 in the spot known as Peña délas Águilas, Elche (Alicante, Spain). The plant material was identified by Dr. A. Escarré, from Biology Department of the University of Alicante. The aerial parts of the plant air-dried and finely grounded (7185 g), were extracted in a Soxhlet with acetone (6 + 6 L), yielding 345 g of crude extract (4.8% of the dry plant weight). The extract was suspended in H 2 0/MeOH and successively extracted with hexane (125 g, 36.2 % ) , CHC13 (112 g, 32.6 %) and n-butanol (82 g, 23.8 % ) . The CHCI3 fraction was constituted by a gummy residue, (80 g) which appeared while the extraction with hexane, and the genuine chloroform extract (32 g), constituted mainly by flavonoids. The gummy portion was chromatographed on silica gel with hexane-EtO Ac mixtures yielding the lactones inuviscolide and 2-deacetoxyxanthinin and the flavonoids 1-12,14 and 15. The fractions containing one or two predominant flavonoids were further chromatographed with CHCI3EtOH mixtures, to give pure 1(17 mg), 2 (20 mg), 3 (70 mg), 4 (35 mg), 5 (50 mg). 6 (20 mg), 7 (35 mg), 8 (90 mg), 9 (210 mg), 10 (93 mg), 11 (600 mg). V. (90 mg), 14 (21 mg), and 15 (18 mg). The second chloroform fraction, which showed in TLC nearly the same components as the gummy portion, was chromatographed on silica gel with CHCl^-EtOH mixtures and afterwards with hexane-EtOAc to give ferulaldehyde, (20 mg). 2-methylphoroacetophenone (20 mg), 13 (67 mg) and 16 (55 mg). Apigenin (5.7,4'-Trihydroxyflavone)

(1)

Was eluted with hexane-EtOAc ( 1 : 1) and then purified by CC with CHCl 3 -EtOH (98:2) and crystallization; m.p. 328-330° C (EtOHH ; 0 ) . IR (KBr): 3600-2000, Í640, 1550, 1480, 900. 820. 735 cm"1. >HNMR(DMSO.<4): 6 6.23 (1H, d, / = 2 Hz, H-6), 6.50 (1H, d , J = 2 Hz, H-8). 6.79 (1H. s. H-3), 6.98 (2H, d, / = 9 Hz, H-3' andH-5'), 7.96 (2H, d, J = 9 Hz. H-2' and H-6'), 13.15 (1H, s, 5-OH). MS, mlz (%): 270 (100. M"), 269 (17), 242 (18), 153 (16), 152 (10), 124 (9), 121 (11). 118(8). Acetylation of 1. (Ac 2 0/py) gave a triacetate of m.p. 185-186° C (EtOH). IR (KBr): 3090, 1760. 1635, 1200, 1180, 1130. 1080, 1030, 910, 890. S35 cm"1. 'H-NMR (CDC13): 62.31 (6H, s,2Ac), 2.43 (3H, s, Ac), 6.64 (1H, s. H-3). 6.87 (1H, d, J = 2 Hz, H-6). 7.27 (2H, d, J = 9 Hz, H-3' and H-5'). 7.36 (1H, d, / = 2 Hz, H-8), 7.89 (2H. d, / = 9 Hz, H2' and H-6').

417

Flavonoids from Inula viscosa Genhvanin (5,4'-Dihydroxy-7-methoxyflavone)

(2)

Was eluted with hexane-EtOAc (3:2) and then CHCl3-EtOH (97:3), m.p. 287-288° 0 ( 0 ^ ) . IR(KBr): 3270,1660,1605,1590,1500,1430, 1375, 1340, 1290, 1255, 1220, 1205, 1190, 1180, 830 cm"1. 'H-NMR (DMSO-d6): 6 3.80 (3H, s, OMe), 6.30 (IH, d, / = 2 Hz, H-6), 6.70 (IH, d,7 = 2Hz, H-8), 6.80 (IH, s, H-3), 6.90 (2H, d, / = 9 Hz, H-3' and H-5'), 7.90 (2H, d, / = 9 Hz, H-2' and H-6'), 12.90 (IH, br s, 5OH). MS, mlz (%): 284 (100, M + ), 283 (19), 256 (11), 255 (51), 241 (20), 167 (23), 138 (34), 121 (20), 118(21). Diacetate: m.p. 198-199° C (EtOH). IR (KBr): 1740, 1630, 1605. 1490, 1430, 1370, 1345, 1290, 1205, 1190, 1150, 1100, 1085. 910, 836 cm"1. 'H-NMR (CDC13): 8 2.33 and 2.43 (6H, 2s, 2Ac), 3.90 (3H, s, OMe), 6.64 (IH, d, / = 3 Hz, H-6), 6.87 (IH, d, J = 3 Hz, H-8), 6.58 (IH, s, H-3), 7.27 (2H, d, J = 9 Hz, H-3' and H-5'), 7.92 (2H, d, / = 9 Hz, H-2'and H-6'). Hispidulin (4,7,4'-Trihydroxy-6-methoxyflavone)

(3)

Was firstly eluted with hexane-EtOAc ( 3 : 2) and then C H C l r E t O H (98:2), m.p. 292-293° C (EtOH). IR (KBr): 3300, 3080, 1650, 1605, 1570,1480,1455,1425,1375,1160,1090,1035, 990, 910, 820cm- 1 . 'HNMR (DMSO-á6): 6 3.76 (3H, s, OMe), 6.60 (IH, s, H-8), 6.76(IH, s, H-3), 6.93 (2H, d,J = 9 Hz, H-3' and H-5'), 7.93 (2H, d, J = 9 Hz, H-2' and H-6'), 13.00 (IH, s, 5-OH). MS, mlz (%): 300 (51, M + ), 285 (33), 282 (31), 257 (44), 167 (15), 153 (10), 139 (32), 121 (11), 119 (28), 89 (10), 69 (100). Triacetate: m.p. 168-169° C (EtOH). IR (KBr): 3040, 1755, 1625, 1470, 1355, 1200, 1165, 1150, 1065, 910, 870, 840 cm"1. 'H-NMR (Acetone-¿6): 8 2.30 (3H, s, Ac), 2.43 (6H, s, 2Ac), 3.90 (3H, s, OMe), 6.78 (IH, s, H-3), 7.30 (2H, d, J = 9 Hz, H-3' and H-5'), 7.57 (IH, s, H-8), 8.13 (2H, d, / = 9 Hz, H-2' and H-6'). Rhamnocitrin (3,5,4'' -Trihydroxy-7-methoxyflavone)

(4)

Was eluted with hexane-EtOAc (7:3), m.p. 218-219° C (EtOH). IR (KBr): 3500, 1650, 1585, 1275, 1230, 1160, 830, 820 cm"1. 'H-NMR (DMSO-¿6): 6 3.86 (3H, s, OMe), 6.30 (IH, d, J = 2 Hz, H-6), 6.73 (IH, d,7 = 2 Hz, H-8), 6.97 (2H, d, / = 9 Hz, H-3' and H-5'), 8.10 (2H, d, / = 9 Hz, H-2' and H-6'), 9.60 (IH, br s, OH), 10.13 ( l H . b r s, OH). MS, mlz (%): 300 (100, M + ), 299 (20), 257 (23), 167 (13). 150 (19), 121 (65). Triacetate: m.p. 205-207° C (MeOH). IR (KBr): 3070, 1755, 1620, 1310,1195,1165,870,855 cm"1. 'H-NMR (CDC13): 8 2.30 (3H, s, Ac), 2.32 (3H, s, Ac), 2.46 (3H, s, Ac), 3.90 (3H, s. OMe), 6.63 (IH, d , / = 2Hz, H-6), 6.85 (IH, á, J = 2Hz, H-8), 7.26 (2H. d , / = 9Hz. H-3' and H-5'), 7.83 (2H, d, / = 9 Hz, H-2' and H-6'). 3-O-Methylkaempferol

(5,7,4' -Trihydwxy-3-methoxyflavone)

(5)

Was firstly eluted with hexane-EtO Ac (3 :2) and then CHCl 3 -EtOH (96:4), m.p. 290° C (EtOH). IR (KBr): 3500-2100, 1650, 1595. 1555. 1355, 1255, 1215, 1180, 1160, 875 cm"1. 'H-NMR (DMSO-
(6)

Was eluted with hexane-EtO Ac (1:1), m.p. 298-300° C ( E t O H - H 2 0 ) . IR (KBr): 3600-2500, 1650, 1560, 1445 cm"1. 'H-NMR (Acetone-á 6 ): 6.30 (IH, d, / = 2 Hz, H-6), 6.52 (IH. d, J = 2 Hz, H-8), 7.03 (IH, d, J = 9 Hz, H-5'), 7.75 (2H, m, H-2' and H-6'). 12.10 (IH, br s. 5-OH). MS, mlz (%): 302 (100, M + ), 301 (23), 273 (7), 153 (3), 137 (3).

Pentacetate: m.p. 198-199° C (EtOH). IR(KBr): 1770,1620,1190, 1120,1070, 1010, 900, 710, 690 cm"'. 'H-NMR (CDC13): 6 2.30 (12H, s,4Ac),2.43(3H,s,Ac),6.87(lH,d,J = 2Hz,H-6),7.33(lH,d,/ = 2Hz, H-8),7.33 ( l H , d , / = 9 H z , H-5'),7.75 (2H,m,H-2'andH-6'). 3-O-Methylquercetin (5,7,3',4'-Tetrahydroxy-3-meíhoxyflavone)

(7)

Was firstly eluted with hexane-EtOAc (3:2), then CHCl 3 -EtOH (97 :3) and prep-TLC CHCl 3 -EtOH (8:1), m.p. 274-276° C (CHC13EtOH). IR (KBr): 3600-2200, 1640, 1610, 1550, 1440, 1160, 970, 830, 800 cm"'. 'H-NMR (Acetone-d 6 ): 8 3.87 (3H, s, OMe), 6.26 (IH, d , / = 2Hz. H-6), 6.50 (IH, d, J = 2 Hz, H-8), 7.03 (IH, A, J = 9Hz, H-5'), 7.65 (2H. m. H-2' and H-6'), 8.90 (2H, br s, 2 0 H ) , 12.50 (IH, s, 5OH). MS, mlz (%): 316 (100, M"), 315 (78), 301 (5), 298 (15), 287 (11), 285 (6), 273 (24), 153 (7), 137 (5). Tetracetate: m.p. 187-189° C (EtOH). IR (KBr): 3090,1760, 1620, 1470, 1430, 1200, 1080, 1020, 900, 880, 730, 700 cm-'. 'H-NMR (CDC13): 6 2.30 (9H, s, 3Ac), 2.43 (3H, s, Ac), 3.76 (3H, s, OMe), 6.76 (IH, d, J = 2 Hz, H-6), 7.20 (IH, d, J = 2 Hz, H-8), 7.30 (IH, d , / = 9 Hz, H-5'). 7.95 (2H, m, H-2' and H-6'). 3,3'-Di-0-methylquercelin (8)

(5,7,4'-Trikydroxy-3,3'-dimethoxyflavone

Was eluted with hexane-EtOAc (3:2), m.p. 256-257° C (EtOH). IR (KBr): 3510, 3140, 1645, 1610, 1600, 1580, 1500, 1475, 1360, 1285, 1210, 1175, 1015, 810, 790 cm"'. 'H-NMR (DMSO-¿6): 8 3.83 (3H, s, OMe), 3.93 (3H, s, OMe), 6.23 (IH, d, J = 2 Hz, H-6), 6.48 (IH, d, J = 2 Hz, H-8), 7.00 (IH, d, J = 9 Hz, H-5'), 7.65 (2H, m, H-2' and H6'), 12.70 (IH, s, 5-OH). MS, mlz (%): 330 (100, M + ), 329 (53), 315 (45), 301 (13), 299 (17), 287 (56), 153 (33), 151 (43), 135 (27), 121 (15), 108 (30), 69 (51). Triacetate: m.p. 193-194° C (EtOH). IR (KBr): 3100, 1750, 1620, 1600, 1430,1360, 1275, 1200, 1120, 1070,1025, 900,830, 810cm-'. 'HNMR (CDC13): 8 2.33 (6H, s, 2Ac), 2.47 (3H, s, Ac), 3.82 (3H, s, OMe), 3.90 (3H, s, OMe), 6.82 (IH, d, J = 2 Hz ; H-6), 7.17 (IH, d, / = 9 Hz, H-5'), 7.30 (IH. d. J = 2 Hz, H-8), 7.67 (2H, m. H-2' and H6'). Naringenin [(2S)-5,7,4'-Trihydroxyflavanone]

(9)

Was firstly eluted with hexane-EtO Ac (6:4) and then CHCl 3 -EtOH (97:3). m.p. 245-246° C (EtOH), [a] D : -19.6° (c 1, MeOH). IR (KBr): 3500-2000, 1630, 1605, 1495, 1460, 1315, 1255, 1160, 1085, 1070, 895, 835 cm"1. 'H-NMR (DMSO-4): 8 2.65 (IH, dd. J = 17 and 4 Hz, Hoc), 3.25 (IH, dd, J = 17 and 12 Hz, H-3f), 5.45 (IH, dd, J = 12 and 4 Hz, H-2), 5.93 (2H, s, H-6 and H-8), 6.80 (2H. d, J = 9 Hz, H-3' and H-5'), 7.33 (2H, d, / = 9 Hz, H-2' and H-6'). 9.90 (IH, br s, OH), 12.18 (IH, s, 5-OH). MS, mlz (%): 272 (22, M + ). 271 (13), 179 (22), 153 (100). 152 (23). 124 (37), 120 (85), 119 (24), 91 (33). Triacetate: m.p. 93-94° C (EtOH). IR (KBr): 3090, 1765, 1685, 1615, 1575, 1505, 1435, 1365, 1190, 1125, 1075. 1055, 1020, 910 cm"'. ! H-NMR (CDCI3): 6 2.33 (6H, s, 2Ac), 2.40 (3H, s, Ac), 2.60-3.30 (2H, AB m, 2H-3), 5.45 (IH. dd,7 A X + J BX = 16 Hz, H-2). 6.60 (IH, d. J = 2 Hz. H-6), 6.80 (IH. d, J = 2 Hz, H-8), 7.20 (2H. d. J = 9Hz, H-3' and H-5'). 7.60 (2H, d. J = 9 Hz, H-2' and H-6'). Sakuranetin ¡(2S)-5,4'-Dihydroxy-7-methoxyflavanone]

(10)

Was eluted with hexane-EtOAc (7 : 3). m.p. 88° C (C 6 H 6 ), [a] D : - 14.0° (c 1, MeOH). IR (KBr): 3600-2400,1640,1580,1520,1450,1200,1160, 835, 810 cm"'. 'H-NMR (CDCU): 6 2.86 (2H. m . i = 18, 11 and 4 Hz. 2H-3). 3.76 (3H, s, OMe). 5.30 (IH, dd, J = 11 and 4 Hz, H-2), 6.15 (2H, s. H-6 and H-8), 6.88 (2H, d, J = 9 Hz, H-3' and H-5'), 7.30 (2H, d, J = 9 Hz, H-2' and H-6'). 12.00 (IH, s, 5-OH). MS. mlz (%): 286 (70, M-). 285 (45), 193 (53), 167 (100), 126 (28). 120 (47). 119(23), 94 IS), 91 (34), 65 (23). Diacetate: m.p. 97-98° C (EtOH). IR (KBr): 3080. 3040, 1760. 1610,1550,1500,1430.1365,1250.1185,1140.1060,890,830cm-'. 'HNMR (CDClj): 8 2.30 (3H, s, Ac), 2.37 (3H. s, Ac), 2.50-3.50 (2H, ABm, 2H-3), 3.87 (3H. s. OMe), 5.48 (IH, dd. / A X + / B x = 16 Hz, H2), 6.30 (IH, d, J = 2 Hz, H-6), 6.43 (IH, d, J = 2 Hz, H-8), 7.25 (2H. d, J = 9 Hz, H-3' and H-5'). 7.45 (2H, d, J = 9 Hz, H-2' and H-6').

Planta Medica 1985

418 7-O-Methylaromadendrin flavanone] (11)

[(2R,

3R)-3,5,4'-Trihydroxy-7-methoxy-

Was eluted with hexane-EtOAc (2:1), m.p. 182-184° C ( Q J V CHC13), [a] D : +29.2° (c 1.06, MeOH), [a] 436 : - 11.1° (c0.35, CHC13). IR (KBr): 3600-3000, 3020, 1620, 1560, 1510, 1440, 1200, 1180, 1150, 810 cm"1. "H-NMR (Acetone-¿ 6 ): 6 3.87 (3H, s, OMe), 4.60 (IH, d, J = 11 Hz, H-2), 5.06 (1H, d,J = 11 Hz, H-3), 6.00 (2H, s, H-6 and H-8), 6.80 (2H, d. / = 9 Hz, H-3' and H-5'), 7.30 (2H, d, / = 9 Hz, H-2' and H-6'), 11.67 (IH, s, 5-OH). MS, mlz (%): 302 (19, M + ), 273 (22), 179 (19), 167 (100), 166 (6), 151 (7), 136 (20), 107 (74). Triacetate: m.p. 137-138° C (EtOH). IR (KBr): 1750, 1695, 1620, 1510, 1440, 1370, 1220, 1150 cm"1. 'H-NMR (CDC13): 6 2.00 (3H, s, Ac), 2.29 (3H, s, Ac), 2.35 (3H, s, Ac), 3.80 (3H, s, OMe), 5.36 (IH, d, J = 11 Hz, H-2), 5.70 (IH, d, J = 11 Hz, H-3), 6.30 (IH, d, J = 2 Hz, H-6), 6.40 (IH, d, / = 2 Hz, H-8), 7.15 (2H, d, J = 9 Hz, H-3' and H5'), 7.53 (2H, d, / = 9 Hz, H-2' and H-6'). 3-O-Acetyiaromadendrin none] (12)

[(2R, 3R)-Acetoxy-5,7,4'

-trihydroxyflava-

Was firstly eluted with hexane-EtOAc (3:2) and then CHCl 3 -EtOH (96:4), m.p. 236-237° C (hexane-EtOAc), [a] D : +50.1° (c 1.1, MeOH); CD: A E 2 I S 5 +17.3, Ae23S +3.0, A E ^ +1.9, A e ^ - 1 2 . 4 , AE 3 2 4 + 3 . 0 (c 1.26, MeOH). IR (KBr): 3330, 1725, 1630, 1610, 1490, 1445,1370,1290,1245,1170", 1090,1035,805 cm"1. 'H-NMR (DMSOd6): 6 1.92 (3H, s, Ac), 5.45 (IH, d, / = 11 Hz, H-2), 5.88 (IH, d, J = 11 Hz, H-3), 5.96 (2H, s, H-6 and H-8), 6.80 (2H, d, J = 9 Hz, H-3' and H-5'), 7.36 (2H, d, / = 9 Hz, H-2' and H-6'). MS, mlz (%): 330 (3, M + ), 270 (38), 153 (100), 136 (81), 134 (67), 124 (10), 123 (8), 121 (8), 43(42). Tetracetate: m.p. 81-82° C (EtOH), [a]D: +52° (c 0.51, MeOH). IR (KBr): 3070,1755, 1690, 1610, 1570, 1500, 1430,1360, 1190,1120, • 1060, 1020, 900, 840 cm"'. 'H-NMR (CDC13): 6 2.00 (3H, s, Ac), 2.26 (6H, s, 2Ac), 2.35 (3H, s, Ac), 5.43 (IH, d , / = 11 Hz, H-2), 5.73 (IH, d, / = 11 Hz, H-3), 6.60 (IH, d, 7 = 2 Hz, H-6), 6.80 (IH, d,7 = 2 Hz, H-8), 7.15 (2H, d, / = 9 Hz, H-3' and H-5'), 7.45 (2H, d, / = 9 Hz, H-2' and H-6'). 3-0-Acetyl-7-0-methylaromadendrin roxy-7-O-methoxyflavanone] (13)

[(2R, 3R)-3-Acetoxy-5,4' -dihyd-

Was firstly eluted with CHCl 3 -EtOH (98 : 2) and then hexane-EtOAc (75:25), m.p. 204-205° C (hexane-EtOAc), [a] D : +50.5° (c 0.7, MeOH). IR (KBr): 3400-2500, 1710, 1635, 1610, 1570, 1490, 1250 cm"'. 'H-NMR (DMSO-d 6 ): 6 1.97 (3H, s, Ac), 3.80 (3H, s, OMe), 5.50(IH, d, / = 12 Hz, H-2), 5.97 (IH, d, 7 = 12 Hz, H-3), 6.16 (2H, s, H-6 and H-8), 6.80 (2H, d, / = 9 Hz, H-3' and H-5'), 7.33 (2H, á,J = 9 Hz, H-2' and H-6'), 9.80 (IH, br s, OH), 11.50 (IH, br s, OH). MS, mlz (%): 344 (11, M + ), 302 (10), 285 (9). 284 (45), 273 (6), 168 (9), 167 (100), 166 (9), 138 (9), 134 (53), 107 (33), 95 (15), 77 (12), 43 (71). Gave the same triacetate than 11: m.p. 137-138° C (EtOH), [a] D : + 58.68° (c 1.03, MeOH). 3-O-Aceiyhaxifolin flavanone] (14)

[(2R,

3R)-3-0-Acetoxy-5,7,3',4'-tetrahydroxy-

Was firstly eluted with hexane-EtOAc (2:3), then CHCl 3 -EtOH (97:3) and then CHCI3-E1OH (99:1), m.p. 186-190° C (CHC1,EtOH), [a] D : +31.6° (c 0.75, MeOH). IR (KBr): 3600-2000, 1735, 1630, 1590, 1465, 1445, 1235, 865, 825, 800, 780, 740 cm"1. 'H-NMR (Acetone-d 6 ): 6 1.98 (3H, s, Ac). 5.36 (IH, d , / = 11.8 Hz, H-2). 5.83 (IH, d, J = 11.8 Hz, H-3), 5.99 (IH, d, J = 2.2 Hz, H-6), 6.02 (IH, d, J = 2.2 Hz, H-8), 6.98 (3H, m, H-2', H-5' and H-6'). MS, mlz (%): 346 (•14, M + ), 328 (10), 304 (13), 288 (7), 287 (20), 286 (100), 275 (11), 153 (55), 152 (24), 150 (20), 123 (18), 43 (14). Pentacetate: m.p. 85-87° C (EtOH-H 2 0), [a] D : +27.8° (c 1.6. MeOH). IR (KBr): 1760, 1690, 1610, 1420, 1360, 1200, 1120, 1070, 1030,1005, 895 cm"1. 'H-NMR (CDC13): ft 2.03 (3H, s, Ac). 2.30 (9H, s, 3Ac), 2.36 (3H, s, Ac), 5.40 (IH, d, 7 = 12 Hz, H-2), 5.68 (IH, d, 7 = 12 Hz, H-3), 6.61 (IH, d, 7 = 2 Hz, H-6), 6.80 (IH, d, 7 = 2 Hz, H-8), 7.33 (3H, m, H-2', H-5' and H-6').

Padmatin [(2R, 3R)-3,5,3'',4'-Tetrahydroxy-7-methoxyflavanone]

(15)

Was firstly eluted with hexane-EtOAc (2:3) and then CHCl3-EtOH (98:2), m.p. 184-186° C (CHCl 3 -EtOH), [a)D: +17.3° (c 1.1, MeOH). IR (KBr): 3600-2900,1630,1570,1500,1445,1000,820 cm"1. 'H-NMR (Acetone-d 6 ): 6 3.86 (3H, s, OMe), 4.64 ( I H , d, 7 = 11.5 Hz, H-2), 5.05 (IH, d,7 = 11.5 Hz, H-3), 6.05 (IH, d,7 = 2.2 Hz, H-6), 6.08 (IH, d, / = 2.2 Hz, H-8), 6.98 (3H, m, H-2', H-5' and H-6'). MS, mlz (%): 318 (69, M + ), 290 (14), 289 (84), 179 (17), 168 (9), 167 (100), 152 (16), 150 (28), 123 (26), 95 (8). Tetracetate: m.p. 87-89° C (EtOH), [a] D : +44.3° (c0.62, MeOH). IR (KBr): 1755, 1680, 1610. 1570, 1420,1360, 1200, 1145, 1100, 1070, 890, 830, 790, 745 cm' 1 . 'H-NMR (CDC13): ó 2.03 (3H, s, Ac), 2.27 (6H, s, 2Ac), 2.33 (3H, s, Ac), 3.80 (3H, s, OMe), 5.30 (IH, d, 7 = 12 Hz, H-2), 5.63 (IH, d, 7 = 12 Hz, H-3), 6.30 (IH, d, 7 = 2 Hz, H-6), 6.36 (IH. d, J = 2 Hz, H-8), 7.28 (3H, m, H-2', H-5' and H-6'). 3-O-Acetylpadmadn [(2R, 3R)-3-Acetoxy-5,3' ,4' -trihydroxy-7methoxyflavanone] (16) Was firstly eluted with CHCl 3 -EtOH (97:3) and then CHCl 3 -EtOH (98:2), m.p. 162-163° C (CHCI3-E1OH), [a] D : +41.0° (c 0.84, MeOH). IR (KBr): 3500-2200, 1715, 1630, 1565, 1520, 1490, 1440, 1230 cm"'. 'H-NMR (CDC13): 6 2.04 (3H, s, Ac), 3.81 (3H, s, OMe), 5.22 (IH, d, 7 = 11.8 Hz, H-2), 5.82 (IH, d, 7 = 11.8 Hz, H-3), 6.04 (IH, d, 7 = 2.1 Hz, H-6), 6.10 (IH, d, 7 = 2.1 Hz, H-8), 6.92 (3H, m, H-2', H-5' and H-6'), 11.46 (IH, s, 5-OH). MS, mlz (%): 360 (27, M + ), 318 (12), 301 (18), 300 (95), 289 (10), 271 (8), 168 (9), 167 (100), 152 (29), 150 (42), 123 (18), 43 (28). On acetylation with Ac 2 0/pyridine, gave the same tetracetate as IS. Ferulaldehyde Was firstly eluted with CHCl 3 -EtOH (99:1) and then hexane-EtOAc (85:15) as an oil. IR: 3350, 2720, 1650, 1580,1500, 1200, 1120, 1025, 805 cm"1. 'H-NMR (CDC13): 6 3.94 (3H, s, OMe), 6.10(IH, brs, OH), 6.60 (IH, dd,7 = 1 6 and 7.5 Hz, H-2), 7.06 (3H, rp, H-2', H-5' and H6'), 7.43 (IH, d, 7 = 16 Hz, H-3), 9.65 (IH, d, 7 = 7.5 Hz, H-l). MS, mlz (%): 178 (100, M + ), 177 (24), 163 (13), 161 (18), 147 (33), 145 (10), 135 (42), 124 (14), 118 (13), 109 (14), 107 (30), 89 (13), 78 (11), 77 (24), 63(12),53(10), 51(15). 2-Methylphloroacetophenone methoxyacetophenone)

(2,4-Dihydroxy-6-

Was firstly eluted with CHCl 3 -EtOH (98 : 2) and then hexane-EtOAc (8:2), m.p. 207-208° C (EtOH-H 2 0). IR (KBr): 3360, 3120, 1625, 1550, 1275, 1160, 1100, 825 cm"'. 'H-NMR (Acetone-d 6 ): 6 2.55 (3H, s, Me-1). 3.90 (3H. s. OMe), 6.02 (2H, m, H-3' and H-5'), 9.40 (IH, br s, OH), 13.40 (IH, s, OH). UV XE,i&H mM at 288.5 nm (E 11600). MS, mlz (%): 182 (32, M"), 167 (100), 152 (13), 124 (9), 69 (15), 43 (13).

Acknowledgements We wish to thank Prof. J. de Pascual Teresa and Dr. M. S. González, University of Salamanca, for high field 'H and 13C-NMR spectra; Prof. P. Molina and Dr. A. Guirado, University of Murcia, for MS measurements; Dr. J. D. Connolly. University of Glasgow, for samples of 12 and 13 and Dr. A. Escarré. University of Alicante, for plant material identification.

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