3438
SOTES
VOL. 29
of a sample of this oil resulted in the isolation of a colorless gel cause of the debate about the position and relative which was shown by thin layer chromatography, chloroformstatus of Ambrosia and its allies3 within the family methanol (3: I ) , to be free of starting compound but contaminated Compositae. This has propipted us to initiate a more by traces of other impurities; Cmsx (ern.?) 3420 and 3280 ( S H ) , thorough study of these genera.4 In the present note 2990, 2940, and 2840 (CH), and 1575 ("2). Methyl 5-Deoxy-2,3-0-isopropylidene-~-~-ribo-hexofuranosidewe report our work on the constituents of Ambrosia (VII).-Sodium nitrite (1.7 g., 2.50 mmoles) was added to a hispida Pursh., a species whose distribution in the stirred solution of methyl 6-amino-5,6-dideoxy-2,3-0-isopropyliUnited States is limited to South Florida. dene-0-D-ribo-hexofuranoside(13.6 g., 63 mmoles) in 570 acetic Extraction of A . hispida in the usual manner and acid (750 ml.) cooled in an ice bath. Additional sodium nitrite chromatography of the crude extract over silicic acid was added to the cold, stirred solution a t intervals of 2 , 6, 24, 32, 48, and 56 hr. Eighteen hours after the final addition of nitrite, furnished ambrosin (1, 0.75%), previously obtained the reaction mixture was filtered to remove the small amount of from A . maritima L.5and Parthenium incanum H.B.K.,' sugar olefin that formed in the reaction and the filtrate was conand damsin (2, 0.34%), a second constituent of A. centrated in U ~ C U Oto one-sixth volume. The resulting concenmaritima. The gross structures of these compounds trate was extracted with two 100-ml. portions of chloroform. The combined chloroform extracts were washed with water, sodium have been established'f6 by correlation with parthenin bicarbonate solution, and again with water before being dried (3) ; recent work reporting the optical rotatory disperover magnesium sulfate. After removal of the magnesium sulfate sion curves of model compounds' indicates that our by filtration, the chloroform was removed invacuo and the residual conclusions' concerning the stereochemistry of the ring light amber oil was dried in vacuo giving 9.3 g. (667,) of crude junction rest on a firm basis. product ( V I I ) . Distillation of crude VI1 through a short-path system gave 5 g. of a colorless oil (b.p. 82-90" a t 0.150.20 mm.). A second distillation through a short Yigreux column gave pure material, 3.1 g. (22%), b.p. 84-86' a t 0.15-0.2 mm. (bath temperature 135140'). Chromatographic homogeneity was established by thin layer chromatography on silica gel H (Merck) using chloroform(cm.-l) 3440 (OH), 2990, 2940, methanol (99: 1) as eluent; ,,s and 2835 (CH); 1370 (C-CH,); and 1110-1060 (COC); p.m.r. 3 0 SC-3-H), 5.63 t spectrum T 5.03 (C-1-H), 5.38 (C-2-HS 2 (C-4-H), 6.20 t (C-6-H), 6.63 (OCH,), 7.71 (OH), 8.17 doublet of triplets ( C - 5 H ) , and 8.5, 8.67 (isopropylidene); [alz3D-54.6 f 0.2" ( c 2.3 g./100 ml. of methanol). A third, more polar substance, m.p. 291-292', was A n a l . Calcd. for CloHlsOs: C, 55.02; H , 8.31. Found: C, eluted in 0.037% yield. I t s empirical' formula, Cle55.33; H, 8.50. HI2O6,ultraviolet [Ama, 277 and 338 mp (log E 4.04 and 5-Deoxy-~-ribo-hexose Phenylosazone (XI) .-Methyl 5-deoxy2,3-isopropylidene-p-~-ribo-hexofuranoside ( 2 g., 9.2 mmoles) 4.24) ] and infrared spectrum (broad bonded hydroxyl, wm dissolved in ethanol (10 ml.) and the solution was diluted 1660, 1615, 1580, and 1500 cm.-') suggested that it was a with 0.1 A' HC1 (10 ml.). The resulting solution was refluxed for previously unreported iiiethoxytrihydroxyff avone which 3 hr. before it was neutralized with sodium acetate (82 mg., 1 we have naiiied hispidulin. This was confirmed by its mmole) and evaporated t o dryness. A mixture of phenylhydraconversion to a triacetate, CzzHlg09, m.p. 168-70°, Amax zine hydrochloride (2.49.) and anhydrous sodium acetate (3.6 g.) in water (24 ml.) was added to the above sugar residue. The 265 and 364 mp (log E 4.29 and 4.33), whose n.m.r. resulting cloudy solution was heated with continuous stirring in a spectrum* exhibited one methoxyl and three acetate water bath for 1 hr. The dark red oil that precipitated was signals. collected by decantation and dissolved in 50% aqueous ethanol Methylation of the unknown substance furnished a (35 ml.). Dilution of this solution with an equal volume of water compound identical in all respects with the tetramethyl resulted in the precipitation of a crude phenylosazone as a gummy solid. The crude product was recrystallized from ethanolether (4b) of scutellarein (4a).9 The distribution of water-ether, then from ethyl acetate-benzene-petroleum ether, functional groups in 4b was also apparent from the and finally from benzene, m.p. 137-140", [aIz4Dno observable n.m.r. spectrum -(typical AzBz system of H-2', H-3', [mp ( e pH rotation (1.04 g./100 ml. of methanol); A, H-5', and H-6' at 6.95 and 7.76 p.p.m., singlets of H-3 1: 252 (13.2)and400(19.2); p H 7 : 250(15.5)and390(17.4). A n a l . Calcd. for ClsHzzN403: C, 63.14; H , 6.48; N ,16.36. and H-8 at (3.52 and 6.751°). Deniethylation of hispiFound: C, 63.09; H , 6.64; N , 16.10.
+
Acknowledgment.-The authors are indebted to Mr. Conrad Kussner for technical assistance, t'o Dr. W. J . Barret,t and members of the Analytical Section of Southern Research Institute who performed the spectra and analytical determinations, and to Dr. W. C. Coburn and Airs. 11. Thorpe for their int'erpret'ation of the proton magnetic resonance spectra. Constituents of Ambrosia hispida Pursh. lsa
WERNER HERZA N D YASUOS u m Department of Chemistry, The Florida State University, Tallahassee, Florada Receaved J u n e 19, 1964
The discovery' that certain Paifhenium and Ambrosia species elaborate very similar or identical "abnormally" constituted sesquiterpene lactones was of interest be-
':9 "'i
(1) Paper I11 of the series, Constituents of Ambrosia and Related Species. Previous papers: W. Herz and G . Hogenauer, J . OTE. Chem., 26, 5011 (1961) ; W. Herz, H. Watanabe, M. Miyazaki, and Y . Kishida, J . A m . Chem. Soc., 84, 2601 (1962). ( 2 ) Supported in part by a g r a n t from the U. S. Public Health Service (G RI-05814). (3) For surveys of the current s t a t u s of this problem, see W. Payne, A m . J . Botany, SO, 872 (1963); W. Payne, P. H. Raven, and D. W. Kyhos. ibid., 61, 419 (1964). (4) For a summary of our most recent results on Iva species, see W. Hem, G. Hogenauer, and A. Romo de Vivar, J . Or& Chem.. 29, 1700 (1964). (5) H. hbu-Shady and T. D. Soine, J . A m . Pharm. Assoc., 4'2, 387 (1953); 43, 365 (1954). (6) hf. Such$, V. Herout, and F. Sorm, Collection Czech. Chem. Commun.. 28, 2257 (1963). (7) C. Djerassi and J. F. Gurst, J . A m . Chem. Soc., 86, 1755 (1964). (8) S . m . r . spectra were run on a Varian A-60 spectrometer in deuteriochloroform with tetramethylsilane serving as internal standard. (9) J. Gripenberg in "The Chemistry of Flavonoid Compounds," T. A. Geissman, Ed., the Macmillan Company, New York, N. Y., 1962, p. 421. We are indebted to Professor T. R. Seshadri for an authentic sample of 4b. (10) Overlap in the chemical shifts exhibited by H-3 and H-8 of a large number of model compounds does not permit differentiation: J. Massico and J. P. Alarthe. Bull. mc. chim. France, 1962 (1962); see also T. Batterham and R . J. Highet. Auslralian J . Chem., 17, 428 (1964). However, in the n.m.r. spec-rum of the triaceta e, t h e signal a t lower field, Le., a'. 7.22 p.p.m.. is clearly ascribable to H-8, and that a t higher field, to H-3.
NOTES
NOVEMBER, 1964 2'
3'
OR2 0
C ; Ri, Rz; R3, R4 = AC d, R1 CH3; Rz, Rs, R4 CHzCHs e, R,, Rz, R4 = CHZCHI;R3 = CHI f , R1, RZ, Rd = H ; R, = CHI
dulin gave scutellarein, further identified as its tetraacetate (&). That hispidulin had a free hydroxyl group a t the 4'position was suggested by the large bathochromic shift of band I1 in base." This conclusion was confirmed by the observation that its triethyl ether differed from 4'methoxy-5,6,7-triethoxyflavone(4d) I 2 and by the isolation of p-hydroxybenzoic acid from alkali fusion of hispidulin. Location of the methoxyl a t C-5 was ruled out by the brown-green ferric chloride testi3 and by the spectral changes observed on addition of aluminum chloride. l 4 That the methoxyl group was at C-6 and not a t C-7 was indicated by the solubility of hispidulin in sodium carbonate s ~ l u t i o nand ~ ~by~ the ~ ~ similarity of the ultraviolet spectrum in neutral solution and in base to those Finally, the of apigenin (5,7,4'-trihydro~yflavone).~~ properties of hispidulin triethyl ether corresponded to those of 6-methoxy-5,7,4'-triethoxyflavoneiz (4e). Therefore hispidulin is 6-methoxy-5,7,4'-trihydroxyflavone (4f). Experimental'? Isolation of Ambrosin, Damsin, and Hispidulin .-Ambrosia hispida Pursh. was collected in the Florida Keys when in the flowering stage in late August, 1963. The ground, whole plant was extracted with chloroform in the usual way. The crude gum, 172 g. from 4.08 kg. of dry plant, was dissolved in 500 ml. of benzene and chromatographed over 2 lb. of silicic acid, 1-1. fractions being collected. Fractions 12-18 (benzene and benzenechloroform) were combined. Trituration with ether furnished 14 g. of crude damsin, m.p. 98-105". Recrystallization from benzenepetroleum ether (b.p. 35-65') raised the m.p. to 105-107". The substance did not depress the melting point of an authentic sample of damsin, kindly furnished by Dr. V. Herout, and was identical with damsin by infrared and thin layer chromatographic criteria. Fractions 21-23 (chloroform) were recrystallized from benzenepetroleum ether, yielding 30 g. of crude ambrosin, m.p. 142-146". Further recrystallization raised the m.p. to 144-146"; identity with an authentic sample of ambrosin was established by thin layer chromatography, infrared spectroscopy, and mixture melting point. Fractions 3 7 4 0 (chloroform-&her) were combined and recrystallized from methanol, yielding 1.5 g., m.p. 291-292"; infrared bands a t 3400 (broad, bonded hydroxyl), 1660 (conjugated, hydrogen-bonded carbonyl), 1615,1580, and 1500 cm.-1 (phenyl). A n d . Calcd. for CI6Hl2O6:C, 64.00; H , 4.30; 0, 31.97. Found: C, 64.17; H, 3.98; 0, 32.01. (11) L. Jurd, ref. 9 , p. 124. (12) S. Rangaswami and F. Venkata Rao, Proc. Indian Acad. Sci., 64A, 51 (1961). (13) L. H.Brims and R. H. Looker, J . Chem. Soc.. 3136 (1951). (14) Ref. 1 1 , p . 119. (15) G. H.Stout and V. F. Stout, Tetrahedron, 14, 296 (1961). (16) Ref. 11. p. 109. (17) Melting points are uncorrected. Ultraviolet spectra were run in 95y0 ethanol; infrared spectra were run in chloroform unless specified otherwise. Analyses are b y Dr. F. Pascher, Bonn, Germany.
3439
Hispidulin exhibited the following color reactions: sulfuric acid, intense yellow with green fluorescence; sodium hydroxide, intense yellow; magnesium and hydrochloric acid, red; sodium amalgam and hydrochloric acid, magenta; ferric chloride, browngreen; Bargellini test and gossypetone reaction, negative; 5% sodium carbonate and saturated sodium bicarbonate, soluble (yellow color). Changes in the ultraviolet spectrum were as follows: in 0.002 M sodium ethoxide, band I1 at 280, band I a t 335 and 405 mp; in alcoholic sodium acetate, band I1 a t 278 (shoulder a t 305), band I1 a t 373 mp; in ethanol with aluminum chloride, band I1 shoulders a t 265 and 290 mp, band I a t 304 and 363 m p ; with boric acid-sodium acetate, band I1 a t 277, band I a t 340 mM. 6-Methoxy-5,7,4'-triacetoxyflavone.-A mixture of 0.15 g. of hispidulin, 0.1 g. of sodium acetate, and 5 ml. of acetic anhydride was refluxed for 4 hr., cooled, and poured into ice-water. The solid, 0.2 g., was recrystallized several times from met+nol, yielding 0.105 g.; m.p. 168-170"; Amax 265 and 304 r n w (log E 4.29 and4.33); n.m.r.signalsat6=7.18and7.81(J= 8.5c.p.s., H-2', H-3', H-5', and H-6'), 7.22 (HI, 6.53 (H-3), 3.85 (methoxy), 2.46, 2.35, and 2.29 p.p.m. (three acetate methyls). Anal. Calcd. for CZzH1809: C, 61.97; H , 4.22; 0, 33.80. Found: C, 61.21; H , 4.29; 0, 34.60. 5,6,7,4'-Tetramethoxyflavone (4b).-A solution of 0.2 g. of hispidulin in 30 ml. of acetone waa refluxed for 5 hr. with 3 g. of potassium carbonate and 1 ml. of dimethyl sulfate, concentrated to small volume, and poured into ice-water. The solid, 0.215 g., was recrystallized repeatedly from methanol and ethyl acetate: m.p. 163-164'; Am,, 267 and 322 mp (log e 4.13 and 4.33), n.m.r. signals a t 6 = 7.76 and 6.95 (AZBz, J = 9 c.P.s., H-2', H-3', H-5', and H-6'), 6.75and6.52 (H-3andH-8),3.98(6protons),3.85, and 3.83 p.p.m. (methoxyls). Admixture of an authentic sample of tetramethylscutellarein (m.p. 162-163") did not depress the melting point and both samples exhibited the same thin layer chromatographic behavior. 5,6,7,4'-Tetrahydroxyflavone (4a).-A suspension of 0.04 g. of hispidulin in 5 ml. of benzene was refluxed for 6 hr. with 0.4 g. of aluminum chloride, cooled, and poured into ice-hydrochloric acid. The yellow material which precipitated was warmed with 5 ml. of acetic acid-hydrochloric acid ( 1 : l ) , filtered, washed, and recrystallized from ethanol-water. The yellow needles, 0.021 g., did not melt below 310°, Amax 287 and 339 mp (log e 4.26 and 4.37), color reactions: sodium hydroxide, green turning to yellow; sodium carbonate, yellow turning to green; ethanol-sodium amalgam, green precipitate; ferric chloride, yellow-green; negative gossypetin reaction. Acetylation of the demethylated substancewithsodiumacetateacetic anhydride and recrystallization from ethanol furnished the tetraacetate 4c, m.p. 232-234", lit.18m.p. 235-237". Methylation of 0.06 g. of the demethylated substance furnished 0.08 g. of the tetramethyl ether 4b, m.p. 164-165', undepressed on admixture of authentic 4b. 6-Metboxy-S,7,4'-triethoxyflavone(4e).-A solution of 0.02 g. of hispidulin in 5 ml. of acetone was refluxed for 20 hr. with 1 g. of potassium carbonate and 0.4 ml. of dimethyl sulfate and poured into ice-water. The precipitate, 0.023 mg., was chromatographed over silicic acid. The benzene-chloroform (1: 1 ) fraction A as recrystallized from acetone-petroleum ether, yielding 0.01 g., m.p. 139-140°, lit.12m.p. 138-140". An authentic sample could not be obtained for comparison. Alkali Fusion of Hispidu1in.-A mixture of 0.1 g. of 4f, 1 g. of potassium hydroxide, and 2 drops of water was heated a t 210220" for 20 min., cooled, dissolved in water, acidified, and extracted with ether. The ether extract was dissolved in dilute base, saturated with carbon dioxide, and extracted with ether (this extract furnished a few milligrams of phenolic material which was not investigated further). The aqueous layer was acidified with hydrochloric acid and extracted with ether. Recrystallization of the ether extract furnished p-hydroxybenzoic acid, n1.p 214-215", m.p. of authentic sample 213", acetate m.p. 186-187", m.p. of authentic sample 186".
Acknowledgment.-We wish to thank Dr. Andre F. Clewell for plant collections and the Florida State University for a grant in aid. (18) G. Bargellini. GQZZ.chim. daZ., 46,69 (1915).
