ketones from “white snakeroot” eupatorium urticaefolium

Tetrahedron. 1962. Vol. 18. pp. 1295 to 1309. Pcrgamon Pres Ltd. Prinled in Northern Ireland

KETONES FROM “WHITE SNAKEROOT” EUPATORIUM URTICA EFOLIUM

W. A. BONNER and J. I. DEGRAW, Jr.’ Department of Chemistry, Stanford University, California

(Received 18 April 1962)

Abstract-“Tremetol”, the crude toxin of Euparorium urticaefolium, has been separated by partition chromatography into a Sterol Fraction and a Ketone Fraction. The Sterol Fraction was further separated by column chromatography into three pure components: Terpene I (&HZ,), Sterol (C,,H,,O) and Sterol II (C,,H,.O). The Ketone Fraction was likewise separated into four components three of which were characterized : Tremetone (48 %; C,,H,,O,), Dehydrotremetone (17 %; C,,H,,O,) and Hydroxytremetone (2.5%; C,,H,,O,). The three ketones proved toxic to goldfish, and the principal ketone, tremetone, was investigated structurally.

Ozone and hypoiodite reacted with tremetone to give formaldehyde and iodoform, respectively, indicating methylene and acetyl functions. Hydrogenation of tremetone yielded dihydrotremetone (C,,H,,O,) and a phenolic ketone hydrogenolysis product (C,,H,,O,). Oxidation of the methyl ether of the latter gave 4-methoxyisophthalic acid, while Beckmann rearrangement of its oxime, followed by hydrolysis, afforded an aminophenol. A decision was made among the twelve alternative dihydro- benzofuran and dihydrobenzopyran structures for tremetone which accord with these data by synthesis of one of the six possible hydrogenolysis products, 2-isoamyl4acetylphenol. The latter proved identical in all respects with the above hydrogenolysis product of tremetone, thus establishing the structure of tremetone as 2-isopropcnyl-2, 3-dihydro-5-acetylbenzofuran and that of dihydrotremetone as 2-isopropyl-2, 3dihydro-5-acetyl-benzofuran.

FROM the time of the revolutionary war until well into the nineteenth century a deadly pestilence called “milk sickness” claimed its share of lives among early settlers of the Old West (central United States). This illness, epidemic in the late summer months, frequently decimated entire populations in early frontier villages, and the mother of Abraham Lincoln perished in such an outbreak in the little community of Pigeon

Creek, Indiana in 1818 when young Abe was a boy of nine.2p3 The early pioneers

were also plagued by a cattle illness called “trembles” which depopulated their herds in those same areas and times of the year that milk sickness attacked their families.

The logical conclusion that milk products from diseased cattle contained a factor toxic to humans was early confirmed, but the infection of cattle was only gradually recognized as due to consumption of a widely distributed weed, “white snakeroot”

(Eupatorium urticuefofium). Cattle voluntarily consume this plant only when suitable forage is unavailable, which accounts for the occurrence of milk sickness only in the

late summer months or other periods of drought. This malady is no longer a problem

of public health due to recognition and eradication of the offending plant and to the

fact that occasionally contaminated milk is usually diluted at dairy pools before consumption. Trembles, however, still claims a small number of livestock each year and remains a problem of some agricultural concern. No cure for either disease has been reported.

’ The authors are indebted to the National Institutes of Health for a research grant (RG-6232)

which supported this research. * C. Sandburg, Ahruham Lincoln-The Pruirie Years Dell Publishing, New York, N.Y. (1959). a J. G. Nicolay and J. Hay. Abruham Lincoln-A History Vol. I. The Century Co., New York (1890).

1295

1296 W. A. BONNER and J. I. DEGRAW, Jr.

The chemical nature of the toxin of white snakeroot has received only cursory examination, the most significant investigations having been conducted by Couch at the Bureau of Animal Industry, U.S. Department of Agriculture in the late 1920’~.~-” By a complicated extraction and fractionation procedure, wherein all fractions were tested for trembles-producing activity against sheep, Couch isolated4 a straw-colored oil which he designated as “tremetol” and considered to be the responsible toxin. This product, looked upon as homogeneous,6 analyzed as C,,H,,O,, had [a],) -21’ (EtOH), appeared to contain two double bonds (Br, titration), distilled in L’UCUO only with decomposition and showed a typical color reaction (deep red interface) when dissolved in ligroin and layered on sulfuric acid, a color test shown by none of the non-toxic fractions. From these and other rather inconclusive data Couch somewhat

unjustifiably asserted tremetol to be an aromatic secondary alcohol with a doubly unsaturated side chain. These deductions have never been confirmed, and indeed this challenging problem has lain dormant since 1929. We have now undertaken’ to extend

the investigations of Couch into the active toxin of Eupatorium urticaefolium, responsible for trembles in cattle and milk sickness in humans.

White snakeroot plant was gathered in Illinoiss in August, 1959, crated in dry ice

and shipped by air-freight to Stanford, where it was processed within 30 hours after collection. Our original extraction and fractionation procedure followed precisely that of Couch4 and resulted in a 0.14% yield of crude tremetol, in agreement with the earlier literature.4 A year later another collection was made and we developed a simpler and more rapid extraction procedure employing methanol instead of ethanol, which resulted in a 0.32% yield of crude tremetol which appeared equivalent to our earlier sample. The crude product was separated by partition chromatography &groin-95% methanol) into a Sterol Fraction (31%) and a Ketone Fraction (49%; [a]g -48*8”(EtOH)), only the latter of which showed the sulfuric acid color test of

Couch.415*” Alumina chromatography of the Sterol Fraction resulted in 85 % recovery of three

principal and several minor components. The first main fraction @groin eluent),

designated Terpene I, showed an IR spectrum suggesting an unsaturated terpene hydrocarbon. The second fraction (ether-benzene) was partially crystalline. The crystalline portions of this fraction were designated Sterol I. The third main fraction (chloroform)

had an IR spectrum reminiscent of /?-sitosterol and was called Sterol II. The percent of each constituent in the original Sterol Fraction varied in our two preparations (I 959, 1960) as follows: Terpene I, 15, 44%; Sterol I, 32, 16%; Sterol II, 9, 8 “/ It is not clear whether these differences are attributable to variation of the plant sample or the

isolation procedure. Terpene I was purified further from two minor components by vapor-liquid par-

tition chromatography. The pure sample, [a]: f44.7” (CHCI,), proved to be C&H,, with one C-methyl group. Bromine titration, microhydrogenation and the UV

’ J. F. Couch, J. Agr. Rcs. 35, 547 (1927). See. this article for an extensive review of earlier literature. o J. F. Couch, J. Amer. Med. Assoc. 91, 234 (1928). B J. F. Couch, J. Amer. Chem. Sot. 51, 3617 (1929). ’ W. A. Bonner, J. I. DeGraw, Jr., D. M. Bowen and V. R. Shah, Tetrahedron Letters 12,417 (1961). 8 We are indebted to Prof. F. W. Slife, College of Agriculture, University of Illinois for collection

and shipment of our supply of white snakeroot. Prof. Slife informs us that 5 cows had died eating the weed in the patch from which our sample was obtained.

Ketones from “white snakeroot” 1297

spectrum indicated the presence of two non-conjugated doubte bDnds, one of which was shown by ozonization to be terminal.

Sterol i was purified by recrystallization from ligroin (m.p. 186-190”) and from ethanol (m.p. 184*5-185+5”), [rxJ;4, .+-.57+2” (CHCI,). It yielded an acetate, m.p. 217- 218’, [XI”,” 1- 64.4” (CHCl,). a-AmyrinQ and its acetate,‘O with properties approximat- ing those of sterol I and its acetate, however, showed differing IR spectra and mixed m.p. depressions on comparison with the latter substances. Elemental analysis of Sterol I and its acetate corresponded to the unexpected molecular formula C,H,O for the free sterol: Oxidation of Sterol I gave a corresponding ketone whose mass spectrometrically determined molecular weight, however, indicated C,,H,,O. Wolff- Kishner reduction of this ketone yielded the parent hydrocarbon whose molecular weight, similarly determined, indicated C,&,. These data require Sterol I to be C.H,O.

Sterol II was purified by rec~stallization, m.p. 147-148”. [z];; -. 32.X” (CHCI,). It appeared identical with the sterot, m.p. 148’, isolated by Couch,* and its IR spectrum and that of its acetate, m.p. 134-l 35*5”, [z$ -37.5’ (CHCI,), were nearly identical to the spectra of ,%sitosterol and its acetate, suggesting that Sterot II belonged to the sitosterol family. I1 These three constituents of the Sterol Fraction have not yet been investigated further.

The above Ketone Fraction was likewise effectively separated by afumina chromatography into four main constituents. The first and fourth were purified by recrystallization from ligroin, while the second could not be crystallized. The small third component (2”?), likewise an oil, could not be readily purified from residual contamination with the fourth component. The physical properties of the purified ketones are indicated in Table 1, while Table 2 indicates the approximate quantity of each component obtained in our 1959 extraction of the white snakeroot pIant.

TABLE 1. KETONES FROM WHITE SNAKEROOT

Ketone Molecular formula M.p.

~ehydrotremeto~e Tremetonc Hydroxytremetone

G,HuO, 87.5-88.5” 0.0 C,,H,,O, Oil ‘. 59% G,H,,O, 70-71” - 50.7

The ketones in Table 1 have been named on the basis of tremetone, the most abundant constituent (48 %) of the Ketone Fraction. The name “tremetone” is based on Couch’s name “tremetof” for the crude precursor, a name derived in turn from the cattle disease trembles. Examination of the molecular formulas in Table I indicates

the Iogic on the names “dehydrotremetone” and “hydroxytremetone” for the two minor constituents of the Ketone Fraction. These three ketones all proved toxic to goldfish, all showed Couch’s red color test with sulfuric acid and were suspected of being the active toxins of white snakeroot. The structure elucidation of the principal ketone, tremetone, was accordingly undertaken as described below.

*J. Simonson and W. Ross, The Terpenes Vol. 5; p. 116. Cambridge University Press (1957). lo J. A. Goodson, J. Chem. Sot. 999 (1938). I1 L, F. Fieser and M. Fieser, Steroids p. 352. Reinhold, New York, N.Y. (1959).


Tremetone was first converted to two crystalline derivatives, the semicarbazone, m.p. 222”, and the 2,~dinitrophenyIhydrazone, m.p. 183*8-184*2”, whose elemental

TABLE 2. RELATIVE QUANTITIES OF WHITE SNAKEROOT CONSTITUENTS

wt. g %

1 White Snakeroot 22,500 2 Crude Tremetol 32.0

a. Sferol fraction 10.0 (1) Terpene I 1.5 (2) Sterol I 3.1 (3) Sterol II 0.9

b: Ketone j-action IS.6 (1) Dehydrotremetooe 2.6 (2) Tremetone 7.5 (3) 3rd Fraction 0.3 (4) Hydroxytremetone 0.4

- 0.140

31b 156 32 9

49b 17d 48

2 2-s

a Of 1; b Of2; c Ofa; d Ofb.

analyses placed the parent molecular formula, C,,H,IO,, on an unambiguous basis. Reaction of tremetone with sodium hypoiodite yielded iodoform and with ozone yielded form~dehyde, indicating, along with UV and IR spectral data, the presence of acetyl and methyiene substituents. Oxidation of tremetone with potassium permanganate Ied to complete destruction of the molecule and the isoIation of no aromatic acid a fact which suggested that the second oxygen atom was attached directly to an aromatic ring. Tremetone had two C-methyl groups.

The most revealing structural information resulted on catalytic hydrogenation of tremetone over palladium-charcoal, whereby two hydrogenation products were obtained. The first of these (25 “/,), dihydrotremetone, C$,HX602, [r]z -47.0” (EtOH) (oxime, m.p. 91-S93.5’; 2,~dinitrophenyihydrazone, m.p. 181-184”), was a neutral oil whose IR and UV spectra, elemental analysis and optical activity indicated it to be a simple monohydrogenation product of tremetone, wherein the terminal double bond of the latter had been reduced. The second hydrogenation product (75x), C1,H,,Oe, optically inactive (oxime, m.p. 104*5-105”; 2,4_dinitrophenylhydrazone, m.p. 202+5-203.5”), was phenolic in nature and cleariy a hydrogenolysis product of tremetone which must have arisen by scission of a fused 5 or B-membered heterocyclic ring.

The position of substituents in the above phenolic ketone hydrogenofysis product was next investigated by methyfation of its phenolic oxygen and oxidation of all carbon substituents to carboxyl. The product arising from this reaction sequence was 4-methoxyisophthalic acid (I), characterized (diazomethane) as its methyl ester (II), m-p. 97.5-98.5’. The identities of I and II were established unambiguously by comparison

I, R=H L m

II, R=CH,


with authentic samples obtained by the reaction sequence: 2,4-dimethyIphenoI-(O- methylation) -+ 2,4-dimethylanisol- (oxidation) --t I - (CH,N,)-+ II. These results, coupled with the above observations (CHI, and CH, = 0 formation), suggested that tremetone had the partial formulation III. The presence of an acetyl function at the indicated position in III was confirmed by Beckmann rearrangement of the oxime (LV) of our phenolic hydrogenolysis product. Hydrolysis of the rearranged product (V) afforded acetic acid and an aminophenol (VI). Acetyfation of the latter yielded an O,N-diacetate (VII) identical with that obtained by direct acetylation of the Beckmann rearrangement product (V).

We may now examine possible structures for tremetone (alternatives of III) which accord with the observations that (I) a methylene and two C-methyl groups are present, (2) hydrogenolysis followed by drastic oxidation yields I and (3) hydrogenotysis occurs with no loss of carbon atoms. Reasonable structures which meet these requirements are shown below in Chart I.

CHART I. STRUCTURAL ALTERNATTVES FOR TREMETONE

0 “‘.:Xri, 3 0

0

%

‘I \ 0

l?m ix X

For hydrogenofysis to accompany the simpIe catalytic reduction of tremetone it appeared reasonable that the hetero-oxygen atom of III be in an allylic position. This stipulation is met in VIII, X, XU and one of XIV. In addition, the benzofuran structures VIII-XIII were given slight preference to the remaining benzopyran structures in view of the similarity (possibly fortuitous) of the UV spectrum of tremetone to that

I* Parentheses indicate the interchangeabiiity of the positions of the methyl and methylene functions.

1300 W. A. E~NNER and J. I. DEGRAW, Jr.

of 2,3-dihydrobenzofurant3 f&,,,, 220,282,289 mp). None of the alternatives in Chart 1, however, could be conscientiously excluded at this stage.

Let us now consider (Chart It) the consequences of (1) hydrogenolysis of the heterocyclic ring followed by (2) methylene reduction for each of the alternatives VIII-XVI. Compound VIII leads to 2-isoamyl-4-acetylphenol (XVII); IX and XVI yield 2-(1,2- dimethylpropyl)-4-acetylphenol (XVIII); X, XII and XV give 2-(I-methylbutyl)-~ acetylphenol (XIX); XI and XIII give 2-( I-ethylprop~l)-4-acetylphenol (XX); and XIV affords 2-(2-methylbutyl)-4-acetylphenol (XXI).

t&ART 11. POSSIBLE HYDROGENOLYSIS PRODUCTS

Of the various alternatives for the structure of tremetone (Chart I), structure VIII appeared to us most likely in view of the isolation by Robertson and Kamthongi” of the skeletally similar compound Euparin (XXII) from a plant of the same family,

Eupalorium purpureum. A test of this hypothesis was accordingly undertaken by synthesis of 2-isoamyl-4-acetylphenol (XVII), the hydrogenolysis product of VIII.

Phenyl isovalerate (XXIV) was subjected to the Fries rearrangement at 165” to yield Zisovalerylphenol (XXV). The latter was reduced by the Clemmensen method,15 affording 2-isoamylphenol (XXVI), which was in turn converted to 2-isoamylphenyl acetate (XXVII) by action of acetyl chloride. The latter ester was subjected to a second Fries rearrangement, producing 2-isoamyl-4-acetylphenol (XVII). The synthetictrans- formations yielding XVII as well as the characterizing derivatives of the intermediates are summarized in Chart III.

1* J. I. Jones and A. S. Lindsay, J. Chem. SOC. 1836 (1950). 1’ A. Robertson and B. Kamthong, J. C’hern. Sot. 925 (1939). 15 C. E. Co&hard, J. Marshall and F. L. Pyman, J. Chem. Sot. 280 (1930).


CHARTIII. SYNTHESIS OF 2-IsOAMYL4ACETYLPHENOL

OH

oxime, mp. 103.5O xxnz

2.4 - Dimtrophenythydmzone. m.p. 167 - 169’

3.5 - Dinitrobenzoote, m.p. 86. 5 - 87.5’

I I

Zn- HCl

"\

xsm xxm xxm

CHART IV. DEGRADATIVE REACTIONS OF TREMETONE

(Chart III)

cH,=O CHI, 7 1”2NoH II Ip

(3) CH,N,

"'"q Ac~~T%~ AQo

IiLI XII

AcNH%

+ l-i* or OH-

CH ,COOH


The synthetic XVII in Chart III had an IR spectrum identical in all respects with that of the hydrogenolysis product from tremetone, and the oximes of the two samples had identical IR spectra and showed no mixed m.p. depression. This identity estab- lishes beyond question the conclusion that tremetone has the constitution 2-isoprop- enyl-2,3-dihydro-5-acetylbenzofuran (VIII) and that the simple hydrogenation product (accompanying XVII) of tremetone, dihydrotremetone, is 2-isopropyl-2,3-dihydro-5- acetylbenzofuran (XXIII). The ready hydrogenolysis of tremetone seems unusual, since prior reduction of its allylic double bond might be expected to occur rapidly and thus preclude hydrogenolysis, dihydrotremetone not being subject to such cleavage. Such phenomena are not without precedent, however, since similar compounds obtained by degradation ofrotenoneexhibitanalogous behaviour on hydrogenation.16s17 The interpretation of our degradative reactions of tremetone in terms of structure VIII is summarized in Chart IV. These conclusions have been further confirmed by an independent synthesis’ of dihydrotremetone XXIII, which will be described in a subsequent paper. The structure elucidation of the two accompanying ketones of

white snakeroot, dehydrotremetone and hydroxytremetone, will also be described in a future publication.

EXPERIMENTAL

Isolution of “tremetol”. White Snakeroot was gathered in the vicinity of Champaigne, Illinois, immediately refrigerated in dry ice and shipped by air to the San Francisco Airport, from where it was transported, still chilled, to our laboratories for immediate processing within 30 hr after its collection. Stems were discarded and the remainder of the plant (28.9 Kg) was chopped into 0.5” pieces then steeped in methanol (100 1.) overnight. 12.3 Kg of the sample was refluxed in methanol (50 1.) for 3 hr in a steam-jacketed copper kettle equipped with a lid and efficient reflux condenser. The liquid was drained and the residue was covered with more methanol (27 1.) which was refluxed for an additional 1.8 hr, then drained. The remainder of the plant was extracted in the same fashion with refluxing methanol, and the extracts (175 I.) were combined, concentrated to 60 1. by distillation at atm. press. and finally to 7 1. by distillation at red. press. The residue was chilled overnight and the brown aqueous layer was decanted from the dark green lipid layer, then was diluted with an equal volume of water and chilled for several hr. The lipid layer was diluted to 6 1. with water, stirred and chilled 4 hr, then decanted. The combined aqueous extracts (18 1.) were extracted with ether, and the residue from evaporation of the ether was combined with the water insoluble lipid residue. The latter was extracted with 2.3 1. of hot (65”) 50 % ethanol, filtered rapidly by suction and the filtrate was concentrated to 1.4 1. at red. press. and chilled for several days. The aqueous supernatant was decanted from the lipid and extracted with ether (100 ml). The ether was evaporated and the residue was added to the lipid fraction, estimated to weigh between 30@-400 g. The latter was heated with refluxing 5 oA methanolic potassium hydroxide (1200 ml) for 3 hr to achieve saponification, whereupon the mixture was evaporated in uucuo to a volume of 600 ml and diluted with water (I.5 I.), then extracted with ether (1.2 I.), allowing the layers to separate overnight. The aqueous layer was re-extmcted with ether (750 ml) and the extracts were combined and evaporated in vucuo, yielding 119 g of red syrup. A third extraction with I 1. ether afforded only an additional 7 g, total 126 g. This material was dissolved in ether (I 25 ml) and the solution was diluted with ligroin (66-75”; 700 ml) but no precipitate formed. The solution was evaporated to dryness in vawo and the residue was dissolved in 85 % ethanol (700 ml) at 70” and allowed to stand at room temp overnight. The solution was then filtered from a small amount of waxy material, and the filtrate was evaporated to dryness L C(ICUO, yielding 91.8 g (0.32 %) of crude “tremetol”.

The above represents a simplification of the extraction procedure described by Couch. In an earlier extraction we employed theexact procedure of Couch,‘using95 xethanol. From 22.3 kgof plant there resulted 31.8 g (0.14%) of crude “tremetol”. While the modified procedure employing methanol and

16 F. B. LaForge and H. L. Halter, J. Amer. Chem. SOC. 53,446O (1931). I7 F. B. LaForge and L. E. Smith, J. Amer. Chem. Sot. 52, 1091 (1930).


eliminating several of the steps recommended by Couch is quicker and more convenient, we have no indication whether the 2-fold increase in yield in our second extraction was due to the superiority of the procedure or the greater tremetol content of the plant extracted. On both occasions the plant was harvested in early Autumn, at the time when it had begun to flower.

Partition chromatography of “tremetol”. Celite (207 g; washed with cont. hydrochloric acid, then methanol) was treated with the lower phase (185 ml) after ligroin (55-85’) was equilibrated with 95 % methanol. An excess of the upper phase was added and the mixture was stirred vigorously, then poured into a 4 x 80cm chromatographic column, allowed to settle and eluted with upper phase until the column height remained constant. The above “tremetol” (5.07 g) in 15 ml of upper phase was loaded onto the column and eluted with upper phase, collecting 13 50-ml fractions from which 4.05 g (80%) of material was recovered. The first 4 fractions, 1.58 g, partially crystallized and showed an IR spectrum similar to those of common plant sterols. The combination was designated as the “Sterol Fraction”. The remaining fractions were combined to give 2.46 g oil whose IR spectrum suggested the presence of aromatic ketones. This was designated as the “Ketone Fraction”. The Sterol Fraction gave a negative color test, while the Ketone Fraction, [a]: -48.8” (c, I .54; &OH) gave a red color in the sulphuric acid test of Couch.’ When 435 mg of the Ketone Fraction was rechromatographed in the same way (32 g celite; 29 ml lower phase) 407 mg of product was recovered having an IR spectrum identical to that of the original material.

Alumina chromatography of sterol fraction. A solution of 9.66 g of the above Sterol Fraction in 50 ml of ligroin-benzene (5 : 1) was loaded onto a column of alumina (450 g; Alcoa) and eluted as 100~ml fractions with solvents and solvent mixtures of increasing polarity, from ligroin to chloroform. A total of 8.16 g (85 %) was recovered in the 32 fractions collected. The first 5 fractions were combined to give 1.47 g clear oil whose IR spectrum suggested it to be a terpene hydrocarbon, designated Terpene 1. Fractions 16-23 were combined to give 3.10 g partially crystalline material, designated Sterol I. Fractions 27-39 were combined to give 0.89 g crude Serol II.

Alumina chromatography of ketonef+action. A solution of 4.99 g of the above Ketone Fraction in 15 ml of ligroin-benzene (4: 1) was loaded onto an alumina column (300 g; Alcoa) and elutcd as 24 fractions with solvent mixtures of increasing polarity from ligroin-benzene (1 : 2) through methanolchloroform (3 :2); affording 3.74 g (75 %) recovered material. Fractions 4-8 were combined to give 0.84 g partially crystalline material, designated as Ketone I on the basis of its IR spectrum. Fractions 9-19 were combined to give 2.41 g oil designated Ketone II. Fraction 23, 0.110 g, was a viscous oil whose IR spectrum showed strong hydroxyl absorption as well as aromatic ketone bands; it was designated Ketone III. Fraction 25, 0.13 g, was a viscous oil which later crystallized and was designated as Ketone IV. Similar, but somewhat less satisfactory separation, was achieved using Fluorisil chromatogrpahy on the crude Ketone Fraction.

Terpene I. Gas chromatographic investigation of the above Terpene I indicated it to contain two main components comprising approximately 80 % and 15 %, respectively, of the total material. The crude terpene was subjected to separation on a Beckman Megachrome unit, and the major component was collected. Its IR spectrum showed it to be an oletinic hydrocarbon, and a strong band at 11~ suggested that one double bond was methylenic. The UV spectrum showed only end absorption. The specific rotation was [a] F +44*7’ (c, 3.57; CHCI,). Microhydrogenation: 1.84, 1.93 moles HP absorbed for M. Wt. 204. (Found: C, 88.35; H, 1160; M. Wt. (Rast), 205. C,,H,, requires: C, 88.16; H, 11.84%; M. Wt., 204).

A solution of 120 mg of Terpene I in carbon tetrachloride (1 ml) decolorized 1.40 ml of a solution (147 mg/ml) of bromine in carbon tetrachloride. This corresponds to 2.19 moles Br, uptake for a substrate of M., Wt., 204. Solvent removal at red. press. yielded a gum which could not be crystallized.

A solution of 213 mg of Terpene I in methylene chloride (15 ml) was ozonized at 0” for 1.5 hr, then stirred with 20% ferrous sulphate (10 ml) for 1 hr. The methylene chloride layer was extracted twice with water (30 ml) and the combined aqueous portions (40 ml) were divided in half. One half was distilled to 5 ml, chilling the distillate in ice and finally treating it with a solution of dimedon (200 mg) in 10% ethanol (10 ml). The resulting fluffy precipitate, 29 mg, m.p. 19t-192.5”, showed no mixed m.p. depression with formaldehyde dimedon.

Sferol I. The above Sterol I (3-10 g) was recrystallized from ligroin (77-110”) to give 1.32 g, m.p. 186190”. Repeated recrystallization from absolute ethanol gave white crystals, m.p. 184.5- 185.5”, whose IR spectrum showed prominent hydroxyl and hydrocarbon bands and whose UV spectrum showed only end absorption. The specific rotation was [a];’ +57.2” (c, 1.15; CHC&).

7

1304 W. A. E~ONNER and J. I. D&RAW. Jr.

(Crystals from ligoin, Found: C, 83.8; H, 11.70; 0,4.43; M. Wt. (Rast), 427. &H,,O requires: C, 83.73; H, 11.81; 0, 446%; M. Wt., 359. Crystals from ethanol, Found: C, 81.45; H, 11.26; 0, 7.01; M. Wt (Rast), 421. C,,H,,O. H,O requires: C, 81.02; H, 11.79; 0, 7.20%; M. Wt., 445).

Sterol I (150 mg) was acetylated using pyridine (2 ml) and acetic anhydride (1 ml). After standing overnight the solution was evaporated to dryness and the residue was triturated with methanol, then recrystallized 4 times from a mixture of ethyl acetate and methanol to obtain 38 mg of solid, m.p. 217-218”, ]a)v +644’ (c, l-25; CHC&). (Found: C, 8060; H, 11.03; 0, 797; M. Wt. (Rast), 408. C,,H,,O, requires: C, 80.94; H, 11.07; 0.799%; M. Wt., 401).

A solution of 380 mg of Sterol I in 20 ml of 1: 1 ethyl acetate&her containing 40 mg of 10% palladized charcoal was placed in a volumetric apparatus and treated with hydrogen at room temp. and atm. press. No significant hydrogen absorption was noted on stirring overnight. The catalyst was removed by filtration and the filtrate was evaporated in uucuo, affording a white crystalline residue. This was recrystallized from absolute ethanol, m.p. 183.5-185”, mixed m.p. with Sterol I, 162-170”. Chromic acid oxidation of sterol I. Sterol I (200 mg) in boiling acetic acid (4 ml) was treated over 10 min with a solution of chromic acid (50 mg) in 90% acetic acid (1 ml). The green solution was diluted with water and chilled and the resulting precipitate (130 mg) was filtered, washed and dried. Two recrystallizations from methanolethyl acetate afforded a product, m.p. 160-162”, 23 mg, whose IR spectrum showed no hydroxyl absorption and a strong carbonyl band at 5.87~ and whose UV spectrum showed no significant absorption above 210 qc, indicating no conjugated carbonyl function. Its mass spectrometrically determined mol wt was 424, indicating CIOH1,,O. The product had [a]: +57*2” (c, 1.15; CHCI,) and its optical rotatory dispersion showed a positive Cotton effect curve with a peak of [a],oto 1-900” and a trough of [a]1,6,, +lOO”. These data suggest that the ketone product was not an ordinary 3-keto-steroid, that it probably contained agem-dimethyl group at the 4-position and that it belonged to the tetra- or pentacyclic triterpene family.l’*‘s

WoflKishner reduction of ketone from sterol I. A solution of the above ketone (100 mg), 95 % hydrazine (0.1 ml) and potassium hydroxide (0.05 g) in diethylene glycol (4 ml) was heated at 200” for 3 hr. then was cooled and diluted with water. The mixture was extracted with hexane and the extracts were washed with water, dried over anhydrous magnesium sulfate and stripped of solvents at red. press., affording 42 mg white product. This was recrystallized from absolute ethanol giving 18 mg white crystals, m.p. 166169” whose IR spectrum was devoid of functional group absorption and whose mass spectrometrically determined mol wt was 410, indicating the hydrocarbon to be C,,H,,. These data require that Sterol I have the composition CSOH1OO.

Sterol Il. The 0.89 g crystalline Sterol II obtained above was recrystallized twice from ligroin (77-110”) to give 0.41 g white solid, m.p. 139-142”. This was repeatedly recrystallized from absolute ethanol to give a product, m.p. 147-148”, [a]: - 32.8” (c. 1.58; CHCl& whose IR spectrum showed hydroxyl and hydrocarbon bands as well as strong bands at 10.3 and 10.5 p, and whose UV spectrum showed only end absorption. (Found: C, 82.95; H, 11.52 0,5.39; M. Wt. (Rast), 314. C&H,,0 requires: C, 83.87; H, 11.99; 0, 4.14%; M. Wt., 386. Discrepancy presumably accountable by partial solvation).

Sterol II (6Omg) was acetylated with acetic anhydride and pyridine as described above. The crude product was recrystallized thrice from methanol-ethyl acetate, m.p. 134135.5”, [a]g -37.5” (c, 0.75; CHCI,). (Found: C, 81.27; H, 11.13; 0,744; M. Wt., 394. C,,H,,O,requires: C, 81.25; H, 11.29; 0, 7.46%; M. Wt., 428). The IR spectrum of the acetate still possessed the 10.3 ,u band found in the parent sterol. The IR spectra of Sterol II and its acetate were nearly identical with those of /?-sitosterol and its acetate, except for the presence of the strong band at 10.3 p, whose intensity failed to changed on repeated recrystallization.

Ketone I; dehydrotremetone. The above partially crystalline Ketone I(O.96 g) was triturated with ligroin (77-l lo”) and chilled overnight, affording 0.43 g crystalline material. This was recrystallized twice from ligroin, 0.24 g, m.p. 87.5-88.5”, optically inactive. The IR spectrum showed bands at 6.02, 6.15, 6.25 (w), 6.32, 12.0 and 12.3~. The UV spectrum in ethanol showed maxima (mp) at 252 (6, 39000), 280 (19000) and 292 (15500). (Found: C, 7764; H, 6.30; 0, 16.11; CCH,, 1340; M. Wt. (Rast), 207. C,,H,,O, requires: C, 77.98; H, 6.04; 0,15.98; 2CCHS, 1496%; M.Wt. 200).

I8 Private communication, Prof. C. Djerassi, Stanford University. I9 C. Djerassi, J. Osiecki and W. Closson, J. Amer. Chem. Sot. 81,4587 (1959).


A mixture of dehydrotremetone (100 mg),absolute ethanol (1 ml),pyridine (0.7 ml) and hydroxylamine hydrochloride (125 mg) was heated under reflux for 2 hr and evaporated to dryness in wcuo.

The residue was stirred with water and the undissolved dehydrotremetone oxime was recrystallized 3 timesfromligroin-benzene, 14 mg, m.p. 131-132”. (Found: C, 72.16; H, 6.13; N, 643. CIIHIIOIN requires: C, 72.54; H, 6.09; N, 6.51%).

Ketone II; tremetone (VIII). The above liquid Ketone II defied attempts at crystallization. Rechromatographing on alumina failed to change its IR spectrum, which showed bands at 5.98, 6.10 (Sh), 6.22, 6.72, 11.05 and 12.20~. The UV spectrum in ethanol showed maxima (mp) at 227 (c, 11950), 280 (12600) and 285 (12300). The sample showed [a]: -59.6” (c, 5.52; abs. EtOH), ng 1.5658, 0:” 1.080. A vapor phase chromatogram (210”, “Ucon Polar”) indicated the product to be approximately 95 % pure. It gave a red color in the sulphuric acid test of Couch3 and showed a positive iodoform test in methanol. (Found: C, 76.00; H, 6.95; 0, 17.08; CCHS, 13.91, 13.92; M. Wt. (Rast), 186, 188. C,,H,,O, requires: C, 77.20; H, 6.98; 0, 15.82; ZCCH,, 14.86%; M. wt., 202).

A mixture of the above tremetone (300 mg), semicarbazide hydrochloride (300 mg), sodium acetate (400 mg), ethanol (3 ml) and water (60 drops) was heated on the steam bath for 10 min then chilled, affording 0.30 g (78 %) crude tremetone semicarbazone, m.p. 218*7-219.7”. Recrystallization from ethanol yielded the pure product, m.p. 222”, [a] E -56.2” (c, 0.84; CHC&). (Found: C, 65.01; H, 6.61; N, 16.49. C,,H,,O,N, requires: C, 64.84; H, 6.61; N, 16.21%).

Tremetone 2,4-dinitrophenylhydrwone was prepared (94%) in the usual way, m.p. 179.7-180.7”. The product was recrystallized twice from dioxane, m.p. 183.8-184.2”. (Found: C, 59.50; H, 4.61; N, 14.57. C,,H,,O,N, requires: C, 59.68; H, 4.74; N, 14.65%).

Ketone III. This substance was obtained as an orange viscous oil from alumina chromatography of the above crude Ketone Fraction. It often appeared as a mixture with Ketone IV below, requiring further chromatography on Florisil to complete its separation. Even after further chromatography the purity of this material remained questionable, however, particularly with regard to contamination by Ketone IV. Its IR spectrum was generally similar to that of Ketone IV, but it possessed a strong hydroxyl band in addition. Structure work on this ketone was not vigorously pursued because of small quantity and questionable purity. It failed to give satisfactory crystalline derivatives of either its alcohol or carbonyl functions. An amorphous 2,4_dinitrophenylhydrazone could be obtained, whose IR spectrum, however, indicated it probably to be merely an impure sample of this derivative Ketone IV. The question remains as to whether Ketone III was really a unique component or merely a mixture of Ketone IV and some unknown hydroxy compound.

Kerone IV; hydroxytremetone. The partially crystalline crude Ketone IV obtained above was recrystallized several times from ligroin, affording white crystals, m.p. 70-71”. The IR spectum showed no discernable hydroxyl absorption and was generally similar to that of tremetone. The UV spectrum in ethanol showed maxima (mr) at 236 (e, 38700), 280 (29100) and 326 (19300); in 0.1 N NaOH the maxima were 248 (38800). 280 (20300) and 359 (20000). The specific rotation was [&’ -50.7’ (c, 0.74; abs. EtOH) and the compound gave a purple-black color with alcoholic ferric chloride. (Found: C, 71.29; H, 6.37; 0, 21.77; CCH*, 13.47; M. Wt. (Rast), 212. C,,H,,O, requires: C, 71.54; H, 6.47; 0, 21.99; ZCCH,, 13.74%; M. Wt., 218).

The above hydroxytremetone (50 mg) was converted to its acetate using pyridine (0.5 ml) and acetic anhydride (0.5 ml) as before. The crude product was recrystallized from ligroin affording 22 mg solid, m.p. 89-89.5”. Its IR spectrum showed no hydroxyl absorption, a phenolic acetate band at 5.70 and a carbonyl band at 5.98 ,L The material gave a positive iodoform test in methanol. (Found: C, 68.49; H, 6.20; 0,2476. C,,H,,O, requires: C, 69.21; H, 6.20; 0,24.59x).

Ozonizuhon of tremetone. A solution of tremetone (152 mg) in methylene chloride (10 ml) was ozonized for 4 hr at o”, then stirred for 15 min with water (10 ml) containing ferrous sulfate (1 g). The organic layer was extracted with 3 lO-ml portions of water and the combined aqueous extract was distilled into an ice-cooled receiver, collecting 30 ml. Dimedon (300 mg) in ethanol (5 ml) was added to the distillate and the solution was warmed on the steam bath. On standing overnight 67 mg of solid, m.p. 193%194, was collected. Recrystallization from dil. ethanol gave a sample of formaldehyde dimedon, m.p. 191.5-192”, which showed no mixed m.p. depression with an authentic sample.

Hydrogenalion of bernetone. Tremetone (1.48 g) in absolute ethanol (15 ml) was treated with 10% palladized charcoal (100 mg) and the mixture was hydrogenated at room temp. and atm. press. in a volumetric apparatus. After 7.5 hr approximately 1.5 mols hydrogen had been absorbed and


the catalyst was filtered. The filtrate was evaporated to dryness in D(ICUO, alfording 1.41 g syrup whose IR spectrum showed strong hydroxyl absorption at 3.1 and two carbonyl peaks at 6.0 and 6.1 ,u. The syrup was dissolved in ether (25 ml) and the solution was extracted with 3 lo-ml portions of 1 N NaOH. The aqueous layer was acidified with 6 N HCI and the resulting white percipitate was extracted into ether (25 ml). Solvent removal after drying afforded 090 g viscous oil which crystallized. Its IR spectrum showed bands at 3.20, 6.10 and Il.05 11. Its UV spectrum in ethanol showed maxima (mp) at 225 (e, 16600) and 280 (14450), shifted in 0.1 N NaOH to 242 (10600) and 336 (28000). The material had m.p. 55-60”, but could not be readily recrystallized from organic solvents. This phenolic ketone was subsequently shown, as described below, to be 2-isoamy14 acetylphenol (XVII).

The original ether extract was dried, filtered and evaporated to give 0.33 g of liquid residue whose IR spectrum still showed the presence of some of the phenolic ketone XVII. The liquid (322 mg) was dissolved in benzene (1 ml) and chromatographed on alumina (15 g), collecting 15 ml fractions. The first two fractions were combined and evaporated, producing 242 mg of liquid whose IR spectrum was free of hydroxyl absorption and whose UV spectrum in ethanol showed maxima (w) at 231 (e, 39500) and 279 (18800). The specific rotation was [a]; -47.0’ (c, 1.78; abs. EtOH). As indicated above this compound, dihydrotremetone, was subsequently shown to be 2-isopropyl-2,3- dihydro-5-acetylbenzofuran (XXIII).

Dihydrotremefone oxime. The above dihydrotremetone (600 mg) and hydroxylamine hydrochloride (1.0 g) were heated in a refluxing mixture of absolute ethanol (7 ml) and pyridinc (5 ml) for 3.5 hr. The oxime was isolated in the usual way (0.57 g) and recrystallized thrice from ligroin m.p. 91.5-93.5”. (Found: C, 71.20; H, 764; N, 6.55; 0, 14.39; CCHs, 9.89. C1,H,,OIN requires: C, 71.20; H, 7.82; N, 6.39; 0, 14.59; ZCCH,, 13.70%).

Dihydrotremetone 2, 4dinitrophenylhydrazone. The above dihydrotremetone was converted to its 2, 4-dinitrophenylhydraone in the usual manner, and the crude product was recrystallized from a mixture of ethanol and ethyl acetate, m.p. 181-184”. (Found: C, 59.25; H, 5.11; N, 1464. C,,H,,O,N, requires: C, 59.37; H, 5.24; N, 14.58%).

2-Isoamyl4acetylphenol oxime (IV). The above phenolic ketone XVII (376 mg) was converted to its oxime in the manner described above. The crude product (456 mg) was recrystallized from dil. ethanol to produce 298 mg of white crystals, m.p. 104.5-105”. (Found: C, 70.32; H, 8.70; N, 6.32; 0, 14.55; CCH,, 11.8. C,,H,,O,N requires: C, 70.55; H, 8.65; N, 6.33; 0, 14.46; ZCCH,, 13.5 %).

2-IsoamyUacetylphenol 2,4_dinitrophenylhydrazone. The phenolic ketone XVII (20 mg) was converted to its 2,4-dinitrophenylhydrazone in the usual way, and the resulting dark red crystals (26 mg) were purified by recrystallization from dil. ethanol, m.p. 202.5-203.5”. (Found: C, 58.67, 58.80; H, 4.66, 4.70; N, 14.66, 14.72. C,,H,,O,N, requires: C, 59.06; H, 5.74; N, 14.50%).

0-Methylarion of 2-isoamyI4acetylphenol. A mixture of the phenolic ketone XVlI (50 mg), methyl iodide (2 mJ) and silver oxide (250 mg) was stirred for 23 hr, then evaporated to dryness at red. press. The residue was thoroughly extracted with methylene chloride (10 ml) and filtered, and the filtrate was washed with 1 N NaOH and with water. The organic layer was dried, filtered and evaporated in cacao to yield 40 mg of crude 2-isoamyl-4-acetylanisol. The IR spectrum of this product showed no hydroxyl adsorption and a carbonyl band at 5.98 ,IL.

Permanganate oxidation of 2-isoamyl4acetylanisole. A mixture of the above methoxy ketone (98 mg), water (1 ml), 5 oA potassium permanganate solution (2.3 ml) and 10% sodium hydroxide solution (0.26 ml) was heated under reflux during 7 hr, adding additional 5 o/o potassium permanganate (14 ml) in l-ml portion periodically during the heating period. The mixture was filtered and the filtrate was acidified with cone hydrochloric acid, whereupon the turbid solution was extracted thoroughly with ether. Solvent evaporation yielded 51 mg of white residue. Recrystallization from hot water (1 ml) yielded 3.5 mg Cmethoxyisophthalic acid (I), m.p. 25&255”, whose IR spectrum was identical with that of the authentic sample described below.

Esterificarion of 4-methoxyisophthalic acid. The above acid I (3 mg) in methanol (0.5 ml) was treated with a slight excess of ethereal diazomethane. Solvent evaporation yielded a residue which was recrystallized twice from dil. methanol to yield pure methyl 4-methoxyisophthalic (II), m.p. 97.5-98.5’. mixed m.p. with the authentic sample below undepressed.

4-Methoxyisophthalic acid (I). A chilled (3”) solution of 2,4_dimethylphenol (3.05 g) in 2 N NaOH (12.5 ml) was treated dropwise with dimethyl sulphate (6.30 g) over a period of 8 min, the


temp remaining below 6’. The solution was allowed to warm to room temp, was heated under reflux for 1.3 hr, then was cooled and extracted twice with ether. The extract was washed with 10% sodium hydroxide solution and water, then dried. filtered and stripped of solvent, yielding 2.21 g crude 2,4_dimethylanisol whose. IR spectrum showed no hydroxyl adsorption. This product (1.36 g) was dissolved in boiling water (IO ml) and treated with a solution of potassium permanganate (6.5 g) in water (25 ml) gradually over a period of 2 hr. The mixture was heated under reflux an additional hr, filtered, and the filtrate extracted with ether. The aqueous layer was acidified with hydrochloric acid and the precipitate was recrystallized from hot water, yielding 0.14 g white crystals, m.p. 2735- 275”. Several different mps (from 255 to 277”) have been reported for this acid,‘LO-*‘) but that observed by Chattaway and Calvert (275-277”)= gave the best agreement with our product. The 1R spectrum of this acid was identical with that of the product obtained by oxidation of the above methoxy ketone.

Merhyl4-methoxyisophthulate (II). The above Cmethoxyisophthalic acid (50 mg) was converted to its methyl ester with diazomethane as described above. The product was recrystallized twice from dil. methanol, m.p. 96.5-97.5”, in agreement with the value reported (96”) by Wessely and SchinzeLLB The IR spectrum of this product was identical in a11 respects with that of the methyl ester II described above, and no mixed m.p. depression of the two products was noted.

2-lsocaproyl-4-methyIpheno1. This was synthesized early in our study for comparison with the phenolic ketone hydrogenolysis product XVII. A mixture ofp-cresol(2.70 g) and isocaproyl chloride (3.35 g) was heated on the steam bath for 2 hr then distilled at red. press. affording 4.0 g (78%) of pcresyl isocaproate, b.p. 91-93” (1 mm), whose IR spectrum showed a strong phenolic ester band at 5.69~. The latter product (3.5 g) was treated with anhydrous aluminum chloride (3.1 g), resulting in an exothermic reaction and nearly complete solution. The mixture was heated at 12&125” for 10 min, then cooled and treated with ice (10 g) and cone hydrochloric acid (3 ml) and finally was extracted twice with ether. The extracts were washed with water and extracted in turn twice with 1 N NaOH, a procedure which removed only 0.1 g material from the ether layer. The latter was dried, filtered and stripped of solvent to produce 2.7 g (80%) crude 2-isocaproyl-4-methylphenol whose IR spectrum showed weak chelated hydroxyl adsorption over the 3-4 p region and a carbonyl btind at 6.1 ,L. The product was characterized as its 2,4-dinitrophenylhydrazone which was prepared in the usual way. The crude derivative was recrystallized from ethanolxthyl acetate, m.p. 190-191”. (Found: C, 58.83; H, 6.03; N, 14.42. C,,H,,O,N, requires: C, 59.06; H, 5.74; N, 14.50”%).

Beckmunn reurrungement of 2-isoamyl-4-acetylphenol oxime (IV). The above oxime (IV) of the phenolic ketone (XVII, 269 mg) was dissolved in ether (I5 ml) and the solution was chilled to -5”, then treated with thionyl chloride (0.20 ml) over a period of 3 min. The mixture was stirred for 45 min at 0” then for 20 min at room temp, at which time nearly complete solution obtained. The ether layer was decanted from some tar into ice water (20 ml) and the mixture was stirred for 20 min, after which the layers were separated and the aqueous layer re-extracted with ether. The ether extract were dried, filtered and evaporated to dryness to yield 237 mg of dark syrup. This crude product (700 mg) was dissolved in benzenexther (4: 1; 3 ml) and chromatographed on Florisil (40 g). The first fraction (114 mg) was a dark oil and was discarded; the last fraction (485 mg) was nearly color- less and showed the IR spectrum anticipated for 2-isoamyl-4-acetylaminophenol (V). The amide could be crystallized from benzene solution but rapidly reverted to a syrup on standing in air.

Acid hydrolysis of 2-isoumyl-4-acetylaminophenol (V). The above amide V (230 mg) was heated under nitrogen in refluxing 3 N H,SOI (15 ml) for 6 hr. The solution was partially distilled, and the residue was extracted thoroughly with ether. The aqueous layer was adjusted to pH 8-9 and the resulting precipitate extracted into ether. The ether extracts were dried, filtered and evaporated to give 61 mg dark crystals. These were recrystallized twice from benzene and once from dil. ethanol, yielding an off-white sample of 2-isoamyl+aminophenol (VI), m.p. 118-119.5”, which rapidly darkened on standing in air. (Found: C, 72.52; H, 9.34; N, 7.71; 0, 10.32. CIIH,,ON requires: C, 73.70; H, 9.56; N, 7.81; 0, 8.93%).

ao F. We-ssely and E. Schinzel, Monutsh. 84,969 (1953). e1 L. S. Fosdick and 0. E. Fancher, J. Amer. Chem. Sot. 63, 1277 (1941). 22 W. Wenner, J. Org. Chem. 16,457 (1951). *a C. Schall, Ber. Dfsch. Chem. Ges. 12, 828 (1879). 2p F. D. Chattaway and F. Calvert, J. Chem. Sot. 2913 (1928).


The above aqueous distillate was neutralized to the phenolphthalein end point, silver nitrate solution was added and the resulting silver oxide was filtered. On standing the filtrate precipitated 2.7 mg white crystals which were identified as silver acetate by their IR spectrum.

Acetylation of 2-isoamyl4amitzophenol (VI). A mixture of the above chromatographed amide V (485 mg), methanol (1 ml) and 20% aqueous sodium hydroxide (4 ml) was heated to reflux under nitrogen for 3.5 hr, then cooled and diluted to 15 ml with water. The solution was acidified with sulfuric acid and filtered from some tarry material The filtrate was adjusted to pH 9 and the precipitate (285 mg) was collected, washed with water and directly acetylated. 153 mg of this product was treated with acetic anhydride (1 ml) and pyridine (1 ml), processing the reaction mixture in the usual way. The crude 2-isoamyl4acetylaminophenyl acetate (VII, 160 mg) was recrystallized twice from hexane-benzene (9: 1), affording 100 mg of pure product, m.p. w5-91.5”. (Found: C, 68.21; H, 786; 0.18.39; N, 5.28. C,,H,,O,N requires: C, 68.41; H, 8.04; 0, 18.23; N, 5.32%)

Acetylation of V. The above 2-isoamyl-4~acetylaminophenol (V’) from the Reckmann rearrangement (114 mg) was acetylated with 0.5 ml each of acetic anhydride and pyridine in the usual fashion. The crude diacetate VII. (95 mg) was recrystallized twice from hexane-benzene, 65 mg, m.p. 90-91”. A mixed m.p. of this product with the diacetate VII obtained above was undepressed.

2-Isooalerylphenol (XXV). Phenol (31.3 g) was added with cooling over 10 min to isovaleryl chloride (40.0 g). After the vigorous hydrogen chloride evolution ceased the mixture was warmed on the steam bath for l-5 hr, then was cooled and dissolved in ether (50 ml). The ether solution was washed with saturated sodium bicarbonate solution and with water, dried over anhydrous magnesium sulfate, filtered and evaporated at red. press. to yield crude phenyl isovalerate (XXIV). The latter was distilled, b.p. 54-55” (0.5 mm), 50.4 g (85 ‘A>. Autenrietha8 has prepared this ester using isovaleric anhydride and reported b.p. 224-226” at atm. press. Our product (40.0 g), showing a strong phenolic eater band at 5.71 p, was warmed to 70’ and treated with anhydrous aluminium chloride (36-O g). A vigorous exothermic reaction ensued, the temp. rising to 120”. The mixture was immediately placed in an oil bath and heated to 165” for 1.75 hr, then cooled and stirred with ice water (500 ml) and cone hydrochloric acid (30ml) and finally was extracted twice with ether (1OOml portions). The extracts were washed with water, dried and evaporated at red. press. The residue was distilled, b.p. 55-65” (O-5 mm), giving 23.9 g (60%) 2-isovalerylphenol (XXV). This product was characterized as ita 2,4-dinitrophenylhydrazone, prepared in the usual way. The crude derivative was recrystallized from ethanokthyl acetate, m.p. 187-189”. (Found: C, 57.14; H, 4.91; N, 15.31. CI,H,BOINI requires: C, 56.98; H, 5.06; N, 1564%).

24soamylphenof (XXVI). To a suspension of zinc amalgam (prepared from powdered zinc (60 g), water (80 ml), cone hydrochloric acid (1 ml) and mercuric chloride (1.5 g))*’ in 6 N HCl (120 ml) was added a solution of the above 2-isovalerylphenol (20.7 g) in ethanol (100 ml). The mixture was heated under reflex for 20 hr, after which a ferric chloride test was negative. The mixture was extracted 3 times with 75-ml portions of methylene chloride, and the extracts were washed with water, dried and stripped of solvent. The residue was distilled, b.p. 62-66” (0.5 mm), to produce 11.9 g (61%) of 2-isoamylphenol (XXVI), whose IR spectrum showed strong hydroxyl adsorption and was free of carbonyl adsorption. The product was characterized as its 3,5dinitrobenzoate, prepared by the action of 3,5dinitrobenzoyl chloride in pyridine. After 2 recrystallizations from ethanol, the product had m.p. 86.5-87.5”. (Found: C, 60.03; H, 5.01; N, 7.80. C,,H,,O,N, requires: C, 60.33; H, 5.06; N, 7.82%).

2-lsoamyl4acet~lphenol (XVII). The above 2-isoamylphenol (13.5 g) was treated slowly with acetyl chloride (9.5 g), and the solution was warmed on the steam bath for 2 hr, allowed to stand overnight and then evaporated to dryness in wcuo. The residue was dissolved in ether (60 ml) and the solution was washed with saturated sodium bicarbonate solution and water. The ether layer was dried, filtered and stripped of solvent to yield 13.8 g (82 %) crude 2-isoamylphenyl acetate (XXVII) This product (l-03 g) was chilled to 0” and treated with anhydrous aluminum chloride (0.80 g). The mixture was stirred at room temp overnight, then was heated for 1 hr on the steam bath, cooled, treated with water (5 ml) containing cone hydrochloric acid (0.5 ml) and finally was extracted with ether. The extract was washed with water and twice with IO-ml portions 5% sodium hydroxide solution. The combined aqueous layers were acidified with hydrochloric acid and the resulting

*a W. Autenrieth, Eer. Dtsch. Chem. Ges. 34, 181 (1901). *I E. L. Martin, J. Amer. Chem. Sot. 58, 1438 (1936).


precipitate was extracted into ether. The extracts were dried, filtered and evaporated to yield 0.37 g clear syrup which later crystallized. The IR spectrum of this product was identical in all respects with that of the 2-isoamyl4acetylphenol (XVII) obtained by hydrogenolysis of tremetone. The oxime of this product was prepared as before, m.p. 102-103.5”. Admixture of this sample with the oxime of XVII described above led to no mixed m.p. depression, 102-103.5”, and the IR spectra of the two samples were identical in all respects.

ketones from “white snakeroot” eupatorium urticaefolium

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