From aerial parts of Ophryosporus heptanthus, one known and three new ent-labbane diterpenoids, four known tremetone and chromene derivatives and two known flavonoids were isolated. Their structures were estabilished by spectroscopic methods

FERRACINI, V. L.

GAO, F.

MABRY, T. J.

ROEWER, I.

English

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Phytochemí.çtry, Vol. 28, No. 5, pp. 14fí3 1465, 1989. Printed in Great Britain. 0031 ··9422/89 $3.00 + 0.00 © 1989 Pergamon Press pie. ENT-LABDANE DITERPENOIDS, TREMETONE AND CHROMENE DERIVATIVES ANO FLAVONOIDS FROM OPHRYOSPORUS HEPTANTHUS VERA L. FERitACINI,* INGEBORG ROEWER, FENO GAO and TOM J. MABRYt Department of Botany, University of Texas at Austin, Austin, Texas 78713-7640, U.S.A. (Received 10 August 1988) Key Word lndex-Ophryosporus heptanthus; Compositae; ent-labdane diterpenoids; tremetone derivatives; chrom-ene derivatives; flavonoids. Abstract-From aerial parts of Ophryosporus heptanthus, one known and Chree new ent-labdane diterpenoids, four known tremetone and chromene derivatives and two known ftavonoids were isolated. Their structures were established by spectroscopíc met hods. INl'RODUCI'ION Earlier studies of the terpenoid chemistry of thc genus of Ophryosporus, family Compoilitae, tribe Eupatorieae, subtribc Critoniniae, yielded several prenylated hydroxy-acetophenones, ent-labdane derivatives, tremetone de-rivatives anda cinnamyl esterfrom O. angustifolius (1], O. chilca (2] andO. perut'ianus [2]. Jn the present report, we describc the isolation from O. heptanthus of one known and three new ent-labdane dit•~rpenoids, four known tremetone and chromene derivatives and two known flavonoíds, the 7-monomethyl and 7,4' -dimethyl ether of apígenin. RESUL TS AND DISCUSSION A dichloromethane extract of ground-leaves of Op-lrryospoms lteptanthus afforded compounds 1-·lO. Com-pound I was previously reported from another species of the family Compositae, namely, Corymbium villosum [3]. Compound 5 was reported from "White snakeroot'-Eupatorium urticaefolium [ 4], and 6 was reported from a member of Ophryosporus, O. angustifolius [1]. Com-pounds 7 and 8 were previously reported from Ageratina aromatica [5], while 9 and lO are widespread ftavonoids. Compound 1 was identified as a known natural prod-uct by comparing its 1 H NMR and MS data with those previously reported [3]. Our observation that 1 was difficult to acetylate, as previously reported [3], was useful for the structure elucidation of the new diterpen-oids reported here. An optical rotation result was too small to be reliable, therefore, an ent-labdane skeleton was assigned for 1 based on the 1H NMR, MS and the acetylation studies, which were in accord with those reported [3]. Moreover, an ent-labdane derivative, the deacetyl of 2 was isolated by Zdcro, Bohlmann and co-workcrs (6] and the two compounds exhibited very • Pcrmanent address: EMRRAPA/Centro Nacional de Pes-quisa de Defesa da Agricultura, Cair. a Postal 69, 13820 Jaguar-iuna SP, Brazil. t Author to w~om correspondence should be addressed. similar NMR patterns. In the following discussion, most sttuctural features of 2-4 are based on comparison of spectral data with those for 1 and thc deacetyl of 2 and with those for each other. The 1H NMR data for 2 indicated that it also had an ent-labdane-type structure (Table 1), and signals at c54.00 (IH, ddd, J=3,4, 12Hz) and b3.45 (IH, brd, J=2 Hz) further suggested that 2 had two secondary hydroxyl groups. A 500 MHz 2D-COSY expcriment contirmed that the two protons on the two carbon atoms bcaring the hydroxyl groups exhibited coupling and therefore were on adjacent carbon atoms. The coupling patterns estab-lished that the two hydroxyl groups· were at C-2 and C-3. Moreover, a P-orientation of both hydroxyl groups in 2 could be assigned by inspection of Dreiding models of an ent-labdane-type skeleton as found for 1. The CIMS of 2 exhibited a molecular íon at m/z 365 [M +H]+ (26%) as expected for a molecular formula of C22H 360 4 • Frag-ments for 2 at m/z 305 [M- HOAc] + and 287 [305 -H 20] + were in accord with fragmentations observed for 1 [3]. The 1 H NMR data for 3 (Table 1) indicated that it had a structure similar to 1, but differed in two respects, namely, by not exhibiting a signal for an acetyl group, thus indicating a primary hydroxyl group at C-15; more-over, a signal at ô3.83 for a secondary hydroxyl group could only be assigned to C-2 based on the coupling pattem, which while appearing as tt is assigned as dddd, J = 4, 4, 11, 11 Hz. In support o f a C-2 hydroxy1 group was the observation that signals for the two C-4 germinai methyl groups overlapped, a phenomenon typical for this type of compound when there is not a C-3 hydroxyl group. Acetylation of 3 afforded a diacetate (Ja) which further supported the presence of two hydroxyl groups in 3, and also indicated that the secondary hydroxyl group was not at C-3; foUowing our's and other's [3] obser-vations (as noted above) that a C-3 hydroxyl group in this type of compound is diOicuh to acetylate~ Thc EIMS data (see Experimental) indicated ready loss o f water and the sidechain; this supported the structure assignment for 3. A 2/l-hydroxyt group was assigned on the basis of the coupling constants with respect to inspection ofDreiding models. 1463 1464 R' Me O 5 R 6 R OH O 9 Table J. H 2 3 7 14 15 16t 17t 18 19 20 OAc O Me A c V. L. FERRACJNI et ai. 1 2 3 Ja 4 R R' 7 8 MeO O Me R R' R2 H OH A c OH ou A c OH H H OAc H A c OH H A c R R' O Me O Me OH A c OH O 10 'H NMR data of compounds l-4 (360 MHz, CDCI 3 , TMS)• l 3 4 4.00 ddd 3.83 dddd 3.78 (3, 4, 12) (4, 4, 11, 11) 3.40 br s 3.45 br d (3) 5.37 br s 5.42 5.39 5.35 5.28 br t (7) 5.33 . 5.40 5.28 4.53 m 4.58 4.13 br d (1) 4.5j m 1.66 br s 1.69 1.70 1.64 1.68 br s 1.72 1.70 1.66 0.91 s 1.00 0.92 0.86 0.86 s 0.92 0.92 0.86 0.74 s 0.83 0.76 0.74 2.02 s 2.06 2.00 s •coupling pattems and coupling constants (J in Hz in parentheses) are not repeated if they are identical with the preceding column. lntegration is one-proton for signals for H-2, 3, 7 and 14, two protons for H-15 and three protons for the H-16, 17, 18, 19, 20 and OAc. t Assignments for H-16 and H-17 are interchangeable wit h in the column. -OH Compound 4 differed from 3 in that the primary hydroxyl group in 3 was acetylated in 4 (signal at ~ 2.00, 3H, s, for an acetyl group). Moreover, the chemical shift of the signals for the two C-15 protons (~4.53, 2H, m), when compared with the signals for the two C-15 protons in 3 (b4.l3, 2H, br d), indicated a C-15 acetyl function. The CIMS of 4 with a peak at m/z 349 [M +H]+ indicated a formula ofC22H360 3 , and the 13C NMR data supported two oxygenated carbon atoms (~61.3 and 65.3) and an acetyl group (b 171.2 and 21 .9). The remaining 13C NMR · signals as well as MS fragments supported the structure assignment. Again, the ent-labdane skeJeton was assigned on the basis of comparison ofthe data for I and its related compounds [3, 6] with the data for 4. A C-2P-hydroxyl Terpenoids from Ophryosporus heptanthus 1465 group was assigned by inspection of Dreiding models with respect to coupling constants. Finally, the structure of 4 was confirmed when acetylation of 4 gave the diacetate 3a. Compound 5 (hydroxytremetone) was isolated as a mixture of 5 and 7 (in a ratio ca 2: 1). We wcre able to subtract signals for 7 from both the 1 H NMR and MS spectra of the mixture because a pure sample of 7 was isolated in the study. Since earlier studies confirmed its structure by synthesis [ 4, 7], only some spectral data are provided here (Experimental) for comparison. EXPERIMENTAL Ophryo.oporus heptanthus (Wedd.) H. Rob. & King was collec-ted in June 1986 by M. Warnock ca 3-S km S ofTarata, Peru. A voucher specimen (Wamock 5077) is on deposit in the Plant Resources Center at the University of Texas at Austin. Dried leaves and flowerheads (965 g) were extracted with CH 2CI 2 (91, 10 min x 2). The combined extracts were evapd under red. pres. to yield a residue which was then dissolved in Me2CO and ttíe soln ~tored in a refrigerator overnight. The usual work-up procedure [8] yielded 64 g of gummy material. Part of this concentrate (27 g) was applied to a silica gel column which was then eluted with hexane with increasing amounts of EtOAc. Concentrates of fractions from the column were further purified over Sephadex LH-20 columns (eluting with a 7:4: I mixture of cyclohexane-CH 2CI2-Me0H and/or prep. TLC (sílica gel devel-oped with hexane-EtOAc, 2: 1). Compounds l (130 mg), 2 (230 mg), 3 (240 mg), 4 (8 mg), 5 and 7 (3 mg, ca 2: I mixture based on 1 H NMR), 6 (22 mg), 7 (100 mg), 8 (150 mg), 9 (6 mg) and lO (8 mg) were obtained. Compounds I and 6-8 were characterized by comparison of their spectroscopic properties with literature values [I, 3, 5], while 9 and 10 were identified following standard procedures [9]. Attempted acetylation of I. Compound l (62 mg) was treated with Ac20-pyridine in the usual manner for 5 hr. 8oth TLC and 1 H NMR showed that no acetylation had occurred. 2{J,3{J-Dihydroxy-1 S-acetoxy-ent-labda-1,13E-diene (2). Colourless gum. CIMS, methane, m/z (rei. int.): 365 [M + Hr (C22 H360 4) (26), 305 [365-HOAc]+ (62), 287 [305-H20]+ (100). 2{J, 15-Dihydroxy-ent-/abda-1, 13E-diene (3). Colourless gum. EIMS (probe) 70 eV, m/z(rel. int.): 288 [M- H 20] + (C20H 3402 , 306) (1 ), 220 [288 -sidechain] + (98), 188 [288 -C6H 120] + (22), 121 [188-C5H 7]+ (44), 106 [121-Me]+ (48), 91 [106-Me]+ (52), 81 [C6 H9 ] + (100). Acetylation of 3. Compound 3 (20 mg) was acetylated with Ac20-pyridine in the usual manner for 1 hr to afford 21 mg of the diacetate 3a. 1H NMR (200 MHz, CDCI 3 , TMS) tS4.99 (IH, dddd,J =4,4, 12,12 Hz, H-2), 5.40(1H, br s, H-7), 5.35 (1H, br t,J =7Hz, H-14),4.59 (2H,brd, J =1Hz, H-15), 1.71 (6H, br s, H-16 and 17), 0.97 (3H, s, H-18), 0.92 (3H, s, H-19), 0.84 (3H, s, H-20), 2.06 (3H, s, OAc), 2.04 (3H, s, OAc). 2{J-H ydroxy-1 S-acetoxy-ent-/abda-1, 13E-diene (4). Colourless gum. CIMS MeOH, m/z (rei. int.): 349 [M +H]+ (C22H 360 3 ) (14), 289 [M-OAc]+ (65), 271 [289-H20]+ (100), 220 [M -HOAc-C5H 8]+ (8). 13C NMR (125 MHz, CDCI 3 , N and P were Attachcd Proton Test results) tS: 34.1 (P, C-1), 65.3 (N, C-2~ 48.1 (P, C-3), 38.4 (P, C-4~ 49.4 (N, C-5), 25.3 (P, C-6), 122.2 (N, C-7), 134.8 (P, C-8), 54.0 (N, C-9}, 51.0 (P, C-10), 23.5 (P, C-11~ 41.6(P, C-12), 142.4(P, C-13), 118.4 (N, C-14~ 61.3 (P, C-15), 22.7 (N, C-16), 16.5 (N, C-17), 33.0 (N, C-18), 21.0 (N, C-19), 14.3 (N, . C-20), 21.9 (N) and 171.2 (P}, (Ac). Acetylation of 4. Compound 4 (17 mg) was acetylated with Ac20-pyridine in the usual manner to yield 20 mg of 3a as determined by 1 H NMR. Mixture ofS·methoxy-6-hydroxytremetone (5) and precocene li (7). Colourlesa gum. EIMS (probe) 70 e V, m/z (rei. int.) (for 5): 206 [Mr (C12H 1403 )(30), 191 [M-Me]+ (16),173 (19l-H 20]+ (18). 161 [191-2xMer (18). (for 7): 220 [M]+ (C 13H 160 3 ) (79), 205 [220-Me]+ (JOO~ 189 [M-OMe]+ (32), 174 (189 -Me]+ (16). 1H NMR(360 MHz,CDCI3 , TMS)tS5.18(1H, brt, J =9Hz, H-2), 3.29(1H, brdd, J =9, I S Hz, H-3), 2.98 (IH, brdd, J =9, 15Hz, H-3'),6.73 (I H, br s, H-4), 6.47(1H,s, H-7), 5.07 (IH, br s, H-11),4.90(1H, br s, H-11 '),1.77 (3H, br s, H-12), 3.82 (3H, s, O Me). Acknowledgements-We thank CNPq for a research fellowship to V.L.F. This work was supported at the University ofTexas at Austin by the Robert A. Welch Foundation (Grant F-130) and the National Institutes of Health (Grant GM-35710). REFERENCES I. Bohlmann, F. and Zdero, C. (1979) Phytochemistry 18, 145. 2. Bohlmann, F., Wallmeyer, M., King, R. M. and Robinson, H. (1984) Phytochemistry 23, 1513. 3. Zdero, C. and Bohlmann, F. (1988) Phytochemistry 27, 227. 4. Bonner, W. A., De Graw, J. I., Bowen, O. M. and Shah, V. R. (1961) Tetrahedron Letters, 417. S. Bohlmann, F., Zitzkowski, P., Suwita, A. and Fiedler, L. (1978) Phytochemistry 17, 2101. 6. Zdero, C., Bohlmann, F., King, R. M. and Robinson, H. (1986) Phytochemistry 25, 2841. 7. DeGraw,J.I. and Bonner, W. A. (1962)J. Org. Chem. 27,3917. 8. Gao, F. and Mabry, T. J. (1987) Phytochemistry 26, 209. 9. Mabry, T. J., Markham, K. R. and Thomas, M. 8. (1970) The Systematic ldentification of Flavonoids. Springer, New York. 

Ent-labdane diterpenoides, tremetone and chromene derivates and flavonoids from Ophryosporus heptanthus.

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Ent-labdane diterpenoides, tremetone and chromene derivates and flavonoids from Ophryosporus heptanthus.

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