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The structure of methostenol and its distribution in rat tissues
ARCHIVES
OF BIOCHEMISTRY
The Structure
AND
(1959)
of Methostenol and Its Distribution in Rat Tissues1’2
D. H. Neiderhiser From
81, 300-308
BIOPHYSICS
and W. W. Wells
the Biochemistry Department, University of Pittsburgh, Medicine, Pittsburgh, Pennsylvania Received
November
School
of
12, 1958
The isolation (1,2) and synthesis of a new sterol from rat feces have been reported (3, 4). Evidence was presented which tentatively indicated the assignment of 4ru-methyl-A’-cholesten-3/3-ol to this compound (3), and we have suggested methostenol as the common name of this methyl-substituted cholestane derivative (4). In the present study, we wish to report additional evidence for the structure of methostenol as 4a-methyl-A’cholesten-30-01, and its isolation from the small intestine and skin of the rat. EXPERIMENTAL
Isolation
of Methostenol from Rat Feces
In previous reports, methods have been described for the separation of the neutral sterols of rat feces into several chromatographic fractions (1,2). Methostenol, located in zone D of these columns, was contaminated with considerable amounts of coprostanol. Separation of the mixture was achieved by chromatography of the p-phenyl azobenzoates on silicic acid-Celite columns. In a typical isolation, 1.7 g. of mixed zone D sterols were azoylated with 1.7 g. of p-phenylazobenzoyl chloride in dry pyridine according to Idler and Baumann (5), resulting in 2.2 g. of the mixed azoates. The esters were dissolved in a minimum of warm petroleum ether3 and placed on a large Zechmeister column (71 mm. diameter) containing 1 kg. of silicic acid-Celite (2:l). A clean separation into two bands occurred after 5 days’ development using approximately 60 1. of recycled petroleum ether. The column was extruded, and the lower band was eluted with ethanol-ether (1: 1). Recrystallization of the material from petroleum ether-benzene yielded 1.8 g. of orange needles, m.p. 1934°.4 Hydrolysis of 1 This work was supported by a research grant (H-2458 C, CS) from the National Institutes of Health, U. S. Public Health Service. 2 Preliminary reports of the structure and synthesis of methostenol and its distribution in rat tissue have been published (3, 4). 3 Skellysolve C (b.p. 905”). 4 The melting points are uncorrected. 300
301
METHOSTENOL
METHOSTENOL
V 3000
FIG.
1. Infrared
I
/
2000
moo
absorption
I
I
I
I
1600 1400 WAVENUMBER CM-’
1200
1000
000
I
spectra
of fecal
and synthetic
methostenol.
the azoate and recrystallization of the ensuing alcohol from acetone gave needles, m.p. lOGlo. The infrared spectrum was identical to that of coprostanol. A mixed melting-point determination with authentic coprostanol gave no depression. The more polar band was washed from the adsorbent with ethanol-ether (l:l), and the resulting solid was recrystallized from a mixture of petroleum ether and benzene. Orange needles (150 mg.) melting at 195-7” were obtained. Saponification followed by recrystallization of the resulting alcohol from absolute ethanol gave long fine needles, m.p. 147-V, [cx]~ O”,5 acetate, m.p. llO-12”, [alo +27.6”,6 and benzoate, m.p. 153-5”, [a]. +40.6”. The alcohol was fast-acting when treated with LiebermannBurchard reagent, and its extinction coefficient at 620 rnp was similar to that of known A%tenols (6). Further evidence for the presence of a single nuclear double bond at position 7 was obtained from hydrogenation studies. Methostenyl acetate on hydrogenation in glacial acetic acid over Adam’s catalyst absorbed no hydrogen, but was converted into a new isomerization product, m.p. 76-7”. This product absorbed one mole of hydrogen upon forced hydrogenation in the presence of HCl at 60” to yield a saturated sterol acetate, m.p. 130-l” (purified from the originally reported product m.p. !39-101” (3) by chromatography on silicic acid-Celite), [cx]. t-37.2”. Hydrolysis of the saturated acetate and recrystallization of the resulting alcohol from absolute ethanol gave needles, m.p. 153-5”. This compound was purified by chromatography on silicic acid-Celite columns and gave a melting point of 159-60”, [oL]~ $21.1”. The optical rotation and infrared spectrum (Fig. 1)’ of the new fecal sterol were those characteristic of A?-stenols (7, 8). Elemental analysis (calcd. for C&l&O: C, 83.93; H, 12.08. Found: C, 83.35; H, 12.11) did not contribute to the elucidation of structure. However, the chromatographic behavior of methostenol was indicative of a sterol of higher molecular weight than the C27 series. Accordingly the C-methyl content (9) of methostenyl acetate was compared with the acetates of cholesterol, A’-cholestenol, and 4,4-dimethylcholesterol (10) (Table I). Methostenyl acetate lib5 The optical rotation measurements were taken at 25” in chloroform. 6 Purified by silicic acid chromatography. Original m.p. 93-4” (3). 7 The infrared spectra of the sterols and their derivatives were measured
in Nujol.
302
NEIDERHISER
AND
TABLE C-Methyl
Determination
WELLS
I
of
Various
Sterol
Acetates Total moles acetic acid’
The
Cholesteryl AT-Cholestenyl 4,4-Dimethyl
acetate acetate cholestenyl
Fecal
acetate
sterol
2.90 3.00 3.60 3.60 3.48
acetate
0 Each C-methyl group was equivalent figures are averages of 6-7 individual * Calculated as 4,4-dimethyl-AT-cholestenyl c Calculated as 4-methyl-AT-cholestenyl
to approximately determinations. acetate. acetate.
f f f f f
0.6 mole
0.03 0.22 0.31 0.26” 0.27” acetic
acid.
erated 3.48 f 0.27 equiv. of acetic acid (calcd. as 4-methyl-AT-cholestenyl acetate) or 3.60 f 0.26 (calcd. as 4,4-dimethyl-Ar-cholestenyl acetate). The acetates of 4,4dimethylcholesterol, cholesterol, and Ar-cholestenol gave 3.60 f 0.31, 2.90 f 0.03, and 3.00 f 0.22 equiv. of acetic acid, respectively. Since compounds possessing geminal methyl groups do not liberate more acetic acid by the Kuhn-Roth procedure than the corresponding monomethyl analogs, either 4- or 4,4-dimethyl-A’-cholesten-38-01 became an attractive possible structure for methostenol. A methyl group at position 14 was tentatively ruled out since the nuclear double bond migrated to an isomeric position [probably the 14(15)] during “super” hydrogenation. The gem-dimethyl structure was eliminated by two different kinds of evidence. 4,4-Dimethyl-ArJ4(1s)-cholestadienyl acetate, m.p. 123-5” was synthesized according to Woodward et al. (10). Forced hydrogenation of this ester in glacial acetic acid, HCl, over Adam’s catalyst at 60” led to formation of 4,4-dimethyl cholestanyl acetate, m.p. 135-6”. Hydrolysis and purification of the resulting stanol by chromatography and recrystallization from ethanol gave Liebermann-Burchard negative needles, m.p. 155-6”. The infrared spectrum did not correspond to that of methostanol. Mixed melting point determination of 4,4-dimethyl-cholestan-30-01 and methostanol resulted in a depression ranging from 12 to 15”. Studies of the Wagner-Meerwein rearrangement of ring A, which is known to yield the isopropylidene derivative of 3phydroxy-4,4-dimethyl compounds detectable by further oxidation of the diol intermediate to acetone (ll), were carried out. Under these conditions, methostenol gave rise to no detectable amounts of acetone, while authentic 4,4-dimethylcholesterol afforded significant amounts of this ketone (12). From the biogenetic standpoint, the 4.methyl derivative of A’-cholestenol then became the most likely structure for the new fecal sterol. Of the two possible isomers, the 4a-methyl was anticipated on the basis of its higher thermodynamic stability (13).
Synthesis of Methostenol (Fig. 2) Ten grams of recrystallized 7-dehydrocholesterol,s m.p. 147-V, was hydrogenated in dioxane over Raney nickel for 18 hr. (14). Recrystallization of the hydrogenation product from methanolethyl acetate gave 9.80 g. A7-cholesten-38-01; m.p. 1234”. Two grams of AT-cholesten-38-01 was heated at reflux with 28 ml. acetone, 57 ml. benzene, 8 Nutritional
Biochemicals
Company.
303
METHOSTENOL
Ho&&!p 3
3
Ip
Fro. 2. Synthesis via A’-cholesten-3-one
III
of 4a-methyl-A’-cholesten-3p-ol (II), and 4a-methyl-A’-cholesten-3-one
(IV)
from
A’-cholesten-38-01 (III).
(I)
and 3.6 g. aluminum t-butoxide for approximately 4 hr. (15). Chromatography of the resulting impure Ar-cholesten-3-one on a silicic acid-Celite (2: 1) column using petroleum ether as the sole developer furnished a separation of the ketone from unreacted alcohol; the latter remained on the column. Recrystallization from 95% alcohol afforded 1.00 g. A7-cholesten-3-one, m.p. 143-5” (literature (16), m.p. 146-7)‘. Methylation was accomplished by treatment of the ketone with 0.6 g. potassium in 46 ml. t-butanol and 2 ml. of freshly distilled methyl iodide. After refluxing with stirring for 1 hr., the reaction was stopped, and the material was recovered and recrystallized from chloroform-methanol giving 0.82 g. of mixed products. The ketone mixture was reduced with 3.6 g. LiAlH4 in ether, and the reaction product was purified through the digitonide and chromatographed on silicic acid-Celite (2: 1) using benzene-petroleum ether (5:l) as the developer. The dried adsorbent was extruded and streaked with Liebermann-Burchard reagent revealing four bands designated A, B, C, and D in descending order with band A being the most polar. Recovery of the material from band A with ether and subsequent recrystallization from methanol gave needles, m.p. 1.234”, identified as A?-cholestenol by mixed melting point and infrared spectrum. Band B furnished 100 mg. of fine needles from absolute ethanol, m.p. 147-S”, [LY]~ - 1.16” whose infrared spectrum was identical to isolated methostenol (Fig. 1), and the appropriate mixed melting point gave no depression. Isolation of the synthetic product was also accomplished by chromatography of the mixed methylated ketones. Methostenone, 4a-methyl-Ar-cholesten-3-one, m.p. 136-7”, was separated and subsequently reduced with LiAlHn to methostenol, m.p. 146-7”. Bands C and D have not been further identified although the polarity of these two sterols on silicic acid-Celite suggests that at least one of them may be a dimethyl derivative.
Methostanol
(da-Methylcholestun-Sp-ol)
The assignment of the 4cu-methyl-A’-cholesten-3&ol structure to methostenol is based upon comparison of the hydrogenation product of the natural material and its acetate with authentic samples of 4a-methylcholestan-3@-01, m.p. 16(r2”, [oI]~ +27”,
304
NEIDERHISER
I
I
3000
2000
FIG. 3. Infrared absorption genation of methostenol, and Sondheimer and Mazur.
I
1800
AND
I
WELLS
I
1600 1400 WAVENUMBER CM-’
I
1200
spectra of “natural methostanol” of authentic 4ol-methylcholestan-38-01
I
I
1000
800
prepared by hydrosynthesized by
and 4a-methylcholestan-3@-ol acetate, m.p. 130-2”, [CX]~ +40” [very generously supplied to us by Sondheimer and Mazur (16~) of the Weizmann Institute of Science]. Methostanol and its acetate were found to be identical to 4a-methylcholestan-30-01 and its acetate by mixed melting-point determination, optical rotation (slight differences in the alcohols), and infrared spectra (Fig. 3).
Isolation
of Methostenol from Rat S&n and Small Intestines
The presence of methostenol was next sought in adult rat small intestine since previous studies (I, 17) had shown that extreme dietary variations produced no significant change in the excretion of this sterol. It was necessary to collect the small intestines from over 500 adult male albino rats (Holtzman) for the direct isolation of methostenol. In a typical preparation, the small intestines from 211 adult male albino rats were removed immediately after sacrifice and washed free of intestinal contents by a strong stream of water. After draining, the combined intestines were extracted with acetone in a large Soxhlet extractor. The lipide (97.9 g.) was recovered and saponified with alcoholic KOH under nitrogen, and the nonsaponifiable matter (9.1 g.) was extracted with ethyl ether. The nonsaponifiable matter was dissolved in petroleum ether and chromatographed on silicic acid-Celite (2: 1) and developed with benzene:petroleum ether (5: 1). A Liebermann-Burchard positive “fast-acting” sterol fraction preceded the cholesterol-lathosterol band, and further purificatjon of this material on silicic acid-Celite columns furnished approximately 15 mg. needles with m.p. 13840”. The extinction coefficient and infrared spectrum identified the compound as methostenol. The low melting point suggested the presence of small amounts of impurities. Since “fast-acting” (Liebermann-Burchard) companions of cholesterol have been identified in rat skin (5, 18), attempts were made to isolate methostenol from this source. The dorsal skins (subcutaneous muscle removed) from 50 adult male albino rats (Holtpman,) were extracted with acetone in a large Soxhlet apparatus as described
METHOSTENOL
305
for intestines. The lipide was saponified, and the nonsaponifiable matter was chromatographed on silicic acid-Celite columnsin the samemannerasabove.After purification on several columns, methostenol was identified by extinction coefficient and infrared spectrum. The melting point of the preparation from skin, 140-l”, was lower than that of the best preparations from feces, 147-8”, but agreed in melting point with that of a skin sterol isolated by Franta et al. (18), 140-l”, [a], +28”. Apparently both our preparations and those of Frantz from rat skin are still impure as indicated by the low melting point and high positive rotation reported by Frantz. However, the infrared spectrum of Dr. Frantz’s sterol (private communication) was very similar to that of methostenol from feces having an additional absorption at 1030 cm.-1.9 By utilizing the extinction coefficients of methostenol, A’-cholestenol, cholesterol, and coprostanol (when present) (19, 2), the methostenol content of the following materials from adult male albino rat was estimated and is expressed as per cent of total sterol: feces from rats on a fat-free diet, 5.9%; dorsal skin, 10.2%; small intestine, .1-2%.
DISCUSSION
Although the alkylation of A4-3-one steroids occurs exclusively at the 4 position as a result of the cu,@unsaturation directional influence (20, 2l), methylation under alkaline conditions of cholestan-3-one leads only to the 2-mono- and 2,2-dimethyl derivatives (22). Thus it became essential to consi.der the possibility of a 2-methyl structure for methostenol. However, the probable structure of methostanol as a 4a-methylcholestane compound and hence also for methostenol demonstrates the very interesting directional influence of the A7 bond upon the 4 position of A7-cholesten-3-one.l” It is possiblethat at least one of the additional reaction products (band D) which traveled on the chromatogram considerably faster than methostenol is either 4,4-dimethyl-A’-cholestenol or the 2,2-dimethyl compound. Prior to the report of the isolation and synthesis of 4a-methyl-A’-cholesten-30-01 (3, 4), no other 4-monomethyl sterol had been found in nature, although a 4-monomethyl triterpenoid, cycloeucalenol, had been reported (23). A wider occurrence of 4-monomethyl sterols of the fast-acting A7 class in nature recently became evident when Djerassi et al. (24) reported the isolation of the samesterol from the cactus Lophocereusschottii. Samples of the sterol, which they have called lophenol, m.p. 149-51”, and its acetate, m.p. 119--21”, kindly supplied to us by Dr. Djerassi, gave infrared spectra which 9 It is interesting to note that in a paper presented to the University of Minnesota Hospitals Staff Meeting and abstracted in The University of Minnesota Medical Bulletin, Vol. 28, October 15, 1956, Dr. Frantz speculated that his sterol from skin was 4.methyl-AT-cholesten-36-01, but this suggestion has not been repeated in subsequent reports of the skin sterol from his laboratory. 10 Drs. Y. Mazur and F. Sondheimer have reinvestigated the methylation of A?cholcsten-3-one and have confirmed that the introduction of a AT-double bond into cholestan$one causes methylation to occur at C-4 instead of C-2 (presumably because the 3.keto group enoliaes toward C-4 instead of C-2) (private communication).
306
NEIDERHISER
AND
WELLS
Ho&o~~oaP CH,
3
LANOSTEROL
ZYMOSTEROL
DESMOSTEROL
lz!pGHO 3
METHOSTENOL
FIG.
4.
CHOLESTEROL
Speculative alternate routes of lanosterol to cholesterol.
in our laboratory were identical to those of methostenol and methostenyl acetate, respectively. The mixed melting point of methostenol and lophenol gave no significant depression, while the mixed melting point of the acetates (our purest acetate, m.p. 110-12”) gave only a slight depression.” Concurrently with the above communication, there appeared a report of the proof of structure of citrostadienol as 4ar-methyl-A 7J4(28)-stigmastadien-3P_01 by Sondheimer et al. (25). If methostenol is found to be an active link in the biosynthesis of cholesterol, then the picture of the sequential transformation from lanosterol to cholesterol becomes confused. For the presence of a methyl group at the 4 position and the double bond in the 7 position as well as the saturated side chain makes methostenol incompatible with a pathway to cholesterol which also includes zymosterol and desmosterol (26) (Fig. 4). With the removal of the 14-substituted methyl group of lanosterol (27), the possibility of a migration of the double bond from position 8(9) to 7 appears attractive. Since 4,4-dimethylcholesterol is not a precursor of cholesterol (27), it will be interesting to establish the activity of methostenol especially in light of the reported high rate of transformation of A’-cholestenol to cholesterol (28, 29). If methostenol is active, and preliminary results in this laboratory with radioactive acetate indicate that it is, then a possible alternate pathway from lanosterol to cholesterol might include 4,4-dimethyl-ATI1 The sample of methostenyl acetate which was sent to Dr. Djerassi for comparison was impure, m.p. 93-4” (3) and undoubtedly accounts for the depression of 10” of the mixed melting point of this sample and lophenyl acetate, m.p. 119-21” reported by these
workers
(24).
METHOSTENOL
307
cholestenol, 401-methyl-A’-cholestenol, A’-cholestenol, and 7-dehydrocholesterol. The finding of methostenol (lophenol) in the plant kingdom raises the interesting question of whether or not plant and animal sterols have similar pathways of metabolism involving such common intermediates as squalene [rich in certain animal sources, e.g., shark liver oil, sebum (30), etc. and in plant material, e.g., olive oil (31)] as well as 4cz-methyl-A’-cholestenol (rat tissue and cactus). At this point, the common pathway may diverge with the a,ddition of a two-carbon fragment at the 24 position exemplified by Subsequent loss of citrostadienol (4a-methyl-A 7,24(28)-stigmastadien-3/3-01), the 4-methyl group would lead to such a compound as A’-stigmastenol [isolated from wheat oil by Idler et al. (32)], and thence via 7-dehydrositosterol (33) to /3-sitosterol. Such speculation does not seemunreasonable in view of the recent demonstration that ergosterol is synthesized by yeast from cholesterol with the methyl group at the 24 position furnished by S-adenosylmethionine (34, 35). ACKNOWLEDGMENTS We are indebted to Drs. Y. Mazur and F. Sondheimer, Weiamann Institute of Science, for samples of 4a-methylcholestan-38-01 and its acetate; to Dr. C. Djerassi, Wayne State University, for samples of lophenol and lophenyl acetate; to Dr. Hans No11 for infrared spectral analysis (Beckman IR-4) ; to Mrs. E. Schwartz for measurements of optical rotation, and to Mrs. S. C. Anderson for valuable technical assistance. SUMMARY
Additional evidence has been presented for the identity of methostenol as 4ar-methyl-A’-cholesten-3/3-ol by comparison of the totally saturated methostanol with authentic 4a-methylcholestan-30-01. The synthesis of methostenol from A’-cholesten-3/3-ol is described. The content of methosteno1 in the feces of rats fed a fat-free diet, in rat skin, and in rat small intestines was found to be 5.9 %, 10.2 %, and l-2 % of total sterols, respectively. The possible biogenetic role of methostenol is discussed. REFERENCES 1. WELLS, W. W., COLEMAN, D. L., AND BAUMANN, C. A., Arch. Biochem. Biophys. 67, 437 (1965). 2. COLEMAN, D. L., WELLS, W. W., AND BAUMANN, C. A., Arch. Biochem. Biophys. 60, 412 (1966). 3. WELLS, W. W., AND NEIDERHISER, D. H., J. Am. Chem. Sot. 79, 6569 (1957). 4. WELLS, W. W., AND NEIDERHISER, D. H., Federation PTOC. 17,333 (1958). 5. IDLER, D. R., AND BAUMANN, C. A., J. Biol. Chem. 196, 623 (1952). 6. IDLER, D. R., AND BAUMANN, C. A., J. Biol. Chem. 203,389 (1953).
308
NEIDERHISER
AND
WELLS
in the Chemistry of Fats and other Lipids,” Vol. I, 7. BERGMANN, W., “Progress pp. 18-69. Pergamon Press Ltd., London, 1952. 8. IDLER, D. R., NICKSIC, S. W., JOHNSON, D. R., MELOCHE, V. W., SCHUETTE, H. A., AND BAUMANN, C. A., J. Am. Chem. Sot. 76, 1712 (1953). 9. WEISENBERGER, E., Mikrochem. ver. Microchim. Acta 33, 51 (1947). 10. WOODWARD, R. B., PATCHETT, A. A., BARTON, D. H. R., IVES, D. A., AND KELLY, R. B., J. Chem. Sot. 1967 1131. 11. RUZICKA, L., MONTAVON, M., AND JEGER, O., Helv. Chim. Acta 31, 818 (1948). 12. NEIDERHISER, D. H., M. S. Thesis, Univ. of Pittsburgh, 1958. 13. BARTON, D. H. R., AND COOKSON, R. C., Quart. Revs. (London) 10, 44 (1956). 14. FIESER, L., AND HERZ, J. E., J. Am. Chem. Sot. 76, 121 (1953). 15. OPPENAUER, R. V., Rec. trav. chim. 66, 137 (1937). 16. ANTONUCCI, R., BERNSTEIN, S., LITTELL, R., SAX, K. J., AND WILLIAMS, J. H., J. Org. Chem. 17, 1341 (1952). 16~. MAZUR, Y., AND SONDHEIMER, F., J. Am. Chem. Sot. 60, 5220 (1958). 17. COLEMAN, D. L., AND BAUMANN, C. A., Arch. Biochem. Biophys. 66, 226 (1957). 18. FRANTZ, I. D., JR., DAVIDSON, A. G., AND DULIT, E., Federation Proc. 16, 265 (1956). 19. MOORE, P. R., AND BAUMANN, C. A., J. Biol. Chem. 196, 615 (1952). 20. SONDHEIMER, F., AND MAZUR, Y., J. Am. Chem. Sot. 79, 2906 (1957). 21. ATWATER, N. W., J. Am. Gem. Sot. 79, 5315 (1957). 22. RINGOLD, H. J., AND ROSENKRANZ, G., J. Org. Chem. 21, 1333 (1956). 23. Cox, J. S. G., KING, F. E., AND KING, T. J., J: Chem. Sot. 1967, 290. 24. DJERASSI, C., MILLS, J. S., AND VILLOTTI, R., J. Am. Chem. Sot. 80, 1005 (1958). 25. MAZUR, Y., WEIZMANN, A., AND SONDHEIMER,F., J. Am. Chem. Sot. 60, 1007 (1958). 26. BLOCH, K., Vitamins and Hormones 16, 119-50 (1957). 27. GAUTSCHI, F., AND BLOCH, K., J. Am. Chem. Sot. 79, 684 (1957). 28. BIGGS, M. W., LEMMON, R. M., AND PIERCE, F. T., JR., Arch. Biochem. Biophys. 61, 155 (1954). 29. BROOKS, S. C., AND BAUMANN, C. A., J. Biol. Chem. 229, 329 (1957). 30. MACKENNA, R. M. B., WHEATLEY, V. R., AND WORMALL, A., Biochem. J. 62,161-g (1952). 31. FITELSON, J., J. Assoc. O&. Agr. Chemists 28, 282 (1945). 32. IDLER, D. R., KANDUTSCH, A. A., AND BAUMANN, C. A., J. Am. Chem. Sot. 76, 4325 (1953) 33. WUNDERLICH, W., 2. physiol. Chem. 241, 116-24 (1936). 34. DANIELSON, H., AND BLOCH, K., J. Am. Chem. Sot. 79, 500 (1957). 35. PARKS, L. W., J. Am. Chem. Sot. 30, 2023 (1958).
OF BIOCHEMISTRY
The Structure
AND
(1959)
of Methostenol and Its Distribution in Rat Tissues1’2
D. H. Neiderhiser From
81, 300-308
BIOPHYSICS
and W. W. Wells
the Biochemistry Department, University of Pittsburgh, Medicine, Pittsburgh, Pennsylvania Received
November
School
of
12, 1958
The isolation (1,2) and synthesis of a new sterol from rat feces have been reported (3, 4). Evidence was presented which tentatively indicated the assignment of 4ru-methyl-A’-cholesten-3/3-ol to this compound (3), and we have suggested methostenol as the common name of this methyl-substituted cholestane derivative (4). In the present study, we wish to report additional evidence for the structure of methostenol as 4a-methyl-A’cholesten-30-01, and its isolation from the small intestine and skin of the rat. EXPERIMENTAL
Isolation
of Methostenol from Rat Feces
In previous reports, methods have been described for the separation of the neutral sterols of rat feces into several chromatographic fractions (1,2). Methostenol, located in zone D of these columns, was contaminated with considerable amounts of coprostanol. Separation of the mixture was achieved by chromatography of the p-phenyl azobenzoates on silicic acid-Celite columns. In a typical isolation, 1.7 g. of mixed zone D sterols were azoylated with 1.7 g. of p-phenylazobenzoyl chloride in dry pyridine according to Idler and Baumann (5), resulting in 2.2 g. of the mixed azoates. The esters were dissolved in a minimum of warm petroleum ether3 and placed on a large Zechmeister column (71 mm. diameter) containing 1 kg. of silicic acid-Celite (2:l). A clean separation into two bands occurred after 5 days’ development using approximately 60 1. of recycled petroleum ether. The column was extruded, and the lower band was eluted with ethanol-ether (1: 1). Recrystallization of the material from petroleum ether-benzene yielded 1.8 g. of orange needles, m.p. 1934°.4 Hydrolysis of 1 This work was supported by a research grant (H-2458 C, CS) from the National Institutes of Health, U. S. Public Health Service. 2 Preliminary reports of the structure and synthesis of methostenol and its distribution in rat tissue have been published (3, 4). 3 Skellysolve C (b.p. 905”). 4 The melting points are uncorrected. 300
301
METHOSTENOL
METHOSTENOL
V 3000
FIG.
1. Infrared
I
/
2000
moo
absorption
I
I
I
I
1600 1400 WAVENUMBER CM-’
1200
1000
000
I
spectra
of fecal
and synthetic
methostenol.
the azoate and recrystallization of the ensuing alcohol from acetone gave needles, m.p. lOGlo. The infrared spectrum was identical to that of coprostanol. A mixed melting-point determination with authentic coprostanol gave no depression. The more polar band was washed from the adsorbent with ethanol-ether (l:l), and the resulting solid was recrystallized from a mixture of petroleum ether and benzene. Orange needles (150 mg.) melting at 195-7” were obtained. Saponification followed by recrystallization of the resulting alcohol from absolute ethanol gave long fine needles, m.p. 147-V, [cx]~ O”,5 acetate, m.p. llO-12”, [alo +27.6”,6 and benzoate, m.p. 153-5”, [a]. +40.6”. The alcohol was fast-acting when treated with LiebermannBurchard reagent, and its extinction coefficient at 620 rnp was similar to that of known A%tenols (6). Further evidence for the presence of a single nuclear double bond at position 7 was obtained from hydrogenation studies. Methostenyl acetate on hydrogenation in glacial acetic acid over Adam’s catalyst absorbed no hydrogen, but was converted into a new isomerization product, m.p. 76-7”. This product absorbed one mole of hydrogen upon forced hydrogenation in the presence of HCl at 60” to yield a saturated sterol acetate, m.p. 130-l” (purified from the originally reported product m.p. !39-101” (3) by chromatography on silicic acid-Celite), [cx]. t-37.2”. Hydrolysis of the saturated acetate and recrystallization of the resulting alcohol from absolute ethanol gave needles, m.p. 153-5”. This compound was purified by chromatography on silicic acid-Celite columns and gave a melting point of 159-60”, [oL]~ $21.1”. The optical rotation and infrared spectrum (Fig. 1)’ of the new fecal sterol were those characteristic of A?-stenols (7, 8). Elemental analysis (calcd. for C&l&O: C, 83.93; H, 12.08. Found: C, 83.35; H, 12.11) did not contribute to the elucidation of structure. However, the chromatographic behavior of methostenol was indicative of a sterol of higher molecular weight than the C27 series. Accordingly the C-methyl content (9) of methostenyl acetate was compared with the acetates of cholesterol, A’-cholestenol, and 4,4-dimethylcholesterol (10) (Table I). Methostenyl acetate lib5 The optical rotation measurements were taken at 25” in chloroform. 6 Purified by silicic acid chromatography. Original m.p. 93-4” (3). 7 The infrared spectra of the sterols and their derivatives were measured
in Nujol.
302
NEIDERHISER
AND
TABLE C-Methyl
Determination
WELLS
I
of
Various
Sterol
Acetates Total moles acetic acid’
The
Cholesteryl AT-Cholestenyl 4,4-Dimethyl
acetate acetate cholestenyl
Fecal
acetate
sterol
2.90 3.00 3.60 3.60 3.48
acetate
0 Each C-methyl group was equivalent figures are averages of 6-7 individual * Calculated as 4,4-dimethyl-AT-cholestenyl c Calculated as 4-methyl-AT-cholestenyl
to approximately determinations. acetate. acetate.
f f f f f
0.6 mole
0.03 0.22 0.31 0.26” 0.27” acetic
acid.
erated 3.48 f 0.27 equiv. of acetic acid (calcd. as 4-methyl-AT-cholestenyl acetate) or 3.60 f 0.26 (calcd. as 4,4-dimethyl-Ar-cholestenyl acetate). The acetates of 4,4dimethylcholesterol, cholesterol, and Ar-cholestenol gave 3.60 f 0.31, 2.90 f 0.03, and 3.00 f 0.22 equiv. of acetic acid, respectively. Since compounds possessing geminal methyl groups do not liberate more acetic acid by the Kuhn-Roth procedure than the corresponding monomethyl analogs, either 4- or 4,4-dimethyl-A’-cholesten-38-01 became an attractive possible structure for methostenol. A methyl group at position 14 was tentatively ruled out since the nuclear double bond migrated to an isomeric position [probably the 14(15)] during “super” hydrogenation. The gem-dimethyl structure was eliminated by two different kinds of evidence. 4,4-Dimethyl-ArJ4(1s)-cholestadienyl acetate, m.p. 123-5” was synthesized according to Woodward et al. (10). Forced hydrogenation of this ester in glacial acetic acid, HCl, over Adam’s catalyst at 60” led to formation of 4,4-dimethyl cholestanyl acetate, m.p. 135-6”. Hydrolysis and purification of the resulting stanol by chromatography and recrystallization from ethanol gave Liebermann-Burchard negative needles, m.p. 155-6”. The infrared spectrum did not correspond to that of methostanol. Mixed melting point determination of 4,4-dimethyl-cholestan-30-01 and methostanol resulted in a depression ranging from 12 to 15”. Studies of the Wagner-Meerwein rearrangement of ring A, which is known to yield the isopropylidene derivative of 3phydroxy-4,4-dimethyl compounds detectable by further oxidation of the diol intermediate to acetone (ll), were carried out. Under these conditions, methostenol gave rise to no detectable amounts of acetone, while authentic 4,4-dimethylcholesterol afforded significant amounts of this ketone (12). From the biogenetic standpoint, the 4.methyl derivative of A’-cholestenol then became the most likely structure for the new fecal sterol. Of the two possible isomers, the 4a-methyl was anticipated on the basis of its higher thermodynamic stability (13).
Synthesis of Methostenol (Fig. 2) Ten grams of recrystallized 7-dehydrocholesterol,s m.p. 147-V, was hydrogenated in dioxane over Raney nickel for 18 hr. (14). Recrystallization of the hydrogenation product from methanolethyl acetate gave 9.80 g. A7-cholesten-38-01; m.p. 1234”. Two grams of AT-cholesten-38-01 was heated at reflux with 28 ml. acetone, 57 ml. benzene, 8 Nutritional
Biochemicals
Company.
303
METHOSTENOL
Ho&&!p 3
3
Ip
Fro. 2. Synthesis via A’-cholesten-3-one
III
of 4a-methyl-A’-cholesten-3p-ol (II), and 4a-methyl-A’-cholesten-3-one
(IV)
from
A’-cholesten-38-01 (III).
(I)
and 3.6 g. aluminum t-butoxide for approximately 4 hr. (15). Chromatography of the resulting impure Ar-cholesten-3-one on a silicic acid-Celite (2: 1) column using petroleum ether as the sole developer furnished a separation of the ketone from unreacted alcohol; the latter remained on the column. Recrystallization from 95% alcohol afforded 1.00 g. A7-cholesten-3-one, m.p. 143-5” (literature (16), m.p. 146-7)‘. Methylation was accomplished by treatment of the ketone with 0.6 g. potassium in 46 ml. t-butanol and 2 ml. of freshly distilled methyl iodide. After refluxing with stirring for 1 hr., the reaction was stopped, and the material was recovered and recrystallized from chloroform-methanol giving 0.82 g. of mixed products. The ketone mixture was reduced with 3.6 g. LiAlH4 in ether, and the reaction product was purified through the digitonide and chromatographed on silicic acid-Celite (2: 1) using benzene-petroleum ether (5:l) as the developer. The dried adsorbent was extruded and streaked with Liebermann-Burchard reagent revealing four bands designated A, B, C, and D in descending order with band A being the most polar. Recovery of the material from band A with ether and subsequent recrystallization from methanol gave needles, m.p. 1.234”, identified as A?-cholestenol by mixed melting point and infrared spectrum. Band B furnished 100 mg. of fine needles from absolute ethanol, m.p. 147-S”, [LY]~ - 1.16” whose infrared spectrum was identical to isolated methostenol (Fig. 1), and the appropriate mixed melting point gave no depression. Isolation of the synthetic product was also accomplished by chromatography of the mixed methylated ketones. Methostenone, 4a-methyl-Ar-cholesten-3-one, m.p. 136-7”, was separated and subsequently reduced with LiAlHn to methostenol, m.p. 146-7”. Bands C and D have not been further identified although the polarity of these two sterols on silicic acid-Celite suggests that at least one of them may be a dimethyl derivative.
Methostanol
(da-Methylcholestun-Sp-ol)
The assignment of the 4cu-methyl-A’-cholesten-3&ol structure to methostenol is based upon comparison of the hydrogenation product of the natural material and its acetate with authentic samples of 4a-methylcholestan-3@-01, m.p. 16(r2”, [oI]~ +27”,
304
NEIDERHISER
I
I
3000
2000
FIG. 3. Infrared absorption genation of methostenol, and Sondheimer and Mazur.
I
1800
AND
I
WELLS
I
1600 1400 WAVENUMBER CM-’
I
1200
spectra of “natural methostanol” of authentic 4ol-methylcholestan-38-01
I
I
1000
800
prepared by hydrosynthesized by
and 4a-methylcholestan-3@-ol acetate, m.p. 130-2”, [CX]~ +40” [very generously supplied to us by Sondheimer and Mazur (16~) of the Weizmann Institute of Science]. Methostanol and its acetate were found to be identical to 4a-methylcholestan-30-01 and its acetate by mixed melting-point determination, optical rotation (slight differences in the alcohols), and infrared spectra (Fig. 3).
Isolation
of Methostenol from Rat S&n and Small Intestines
The presence of methostenol was next sought in adult rat small intestine since previous studies (I, 17) had shown that extreme dietary variations produced no significant change in the excretion of this sterol. It was necessary to collect the small intestines from over 500 adult male albino rats (Holtzman) for the direct isolation of methostenol. In a typical preparation, the small intestines from 211 adult male albino rats were removed immediately after sacrifice and washed free of intestinal contents by a strong stream of water. After draining, the combined intestines were extracted with acetone in a large Soxhlet extractor. The lipide (97.9 g.) was recovered and saponified with alcoholic KOH under nitrogen, and the nonsaponifiable matter (9.1 g.) was extracted with ethyl ether. The nonsaponifiable matter was dissolved in petroleum ether and chromatographed on silicic acid-Celite (2: 1) and developed with benzene:petroleum ether (5: 1). A Liebermann-Burchard positive “fast-acting” sterol fraction preceded the cholesterol-lathosterol band, and further purificatjon of this material on silicic acid-Celite columns furnished approximately 15 mg. needles with m.p. 13840”. The extinction coefficient and infrared spectrum identified the compound as methostenol. The low melting point suggested the presence of small amounts of impurities. Since “fast-acting” (Liebermann-Burchard) companions of cholesterol have been identified in rat skin (5, 18), attempts were made to isolate methostenol from this source. The dorsal skins (subcutaneous muscle removed) from 50 adult male albino rats (Holtpman,) were extracted with acetone in a large Soxhlet apparatus as described
METHOSTENOL
305
for intestines. The lipide was saponified, and the nonsaponifiable matter was chromatographed on silicic acid-Celite columnsin the samemannerasabove.After purification on several columns, methostenol was identified by extinction coefficient and infrared spectrum. The melting point of the preparation from skin, 140-l”, was lower than that of the best preparations from feces, 147-8”, but agreed in melting point with that of a skin sterol isolated by Franta et al. (18), 140-l”, [a], +28”. Apparently both our preparations and those of Frantz from rat skin are still impure as indicated by the low melting point and high positive rotation reported by Frantz. However, the infrared spectrum of Dr. Frantz’s sterol (private communication) was very similar to that of methostenol from feces having an additional absorption at 1030 cm.-1.9 By utilizing the extinction coefficients of methostenol, A’-cholestenol, cholesterol, and coprostanol (when present) (19, 2), the methostenol content of the following materials from adult male albino rat was estimated and is expressed as per cent of total sterol: feces from rats on a fat-free diet, 5.9%; dorsal skin, 10.2%; small intestine, .1-2%.
DISCUSSION
Although the alkylation of A4-3-one steroids occurs exclusively at the 4 position as a result of the cu,@unsaturation directional influence (20, 2l), methylation under alkaline conditions of cholestan-3-one leads only to the 2-mono- and 2,2-dimethyl derivatives (22). Thus it became essential to consi.der the possibility of a 2-methyl structure for methostenol. However, the probable structure of methostanol as a 4a-methylcholestane compound and hence also for methostenol demonstrates the very interesting directional influence of the A7 bond upon the 4 position of A7-cholesten-3-one.l” It is possiblethat at least one of the additional reaction products (band D) which traveled on the chromatogram considerably faster than methostenol is either 4,4-dimethyl-A’-cholestenol or the 2,2-dimethyl compound. Prior to the report of the isolation and synthesis of 4a-methyl-A’-cholesten-30-01 (3, 4), no other 4-monomethyl sterol had been found in nature, although a 4-monomethyl triterpenoid, cycloeucalenol, had been reported (23). A wider occurrence of 4-monomethyl sterols of the fast-acting A7 class in nature recently became evident when Djerassi et al. (24) reported the isolation of the samesterol from the cactus Lophocereusschottii. Samples of the sterol, which they have called lophenol, m.p. 149-51”, and its acetate, m.p. 119--21”, kindly supplied to us by Dr. Djerassi, gave infrared spectra which 9 It is interesting to note that in a paper presented to the University of Minnesota Hospitals Staff Meeting and abstracted in The University of Minnesota Medical Bulletin, Vol. 28, October 15, 1956, Dr. Frantz speculated that his sterol from skin was 4.methyl-AT-cholesten-36-01, but this suggestion has not been repeated in subsequent reports of the skin sterol from his laboratory. 10 Drs. Y. Mazur and F. Sondheimer have reinvestigated the methylation of A?cholcsten-3-one and have confirmed that the introduction of a AT-double bond into cholestan$one causes methylation to occur at C-4 instead of C-2 (presumably because the 3.keto group enoliaes toward C-4 instead of C-2) (private communication).
306
NEIDERHISER
AND
WELLS
Ho&o~~oaP CH,
3
LANOSTEROL
ZYMOSTEROL
DESMOSTEROL
lz!pGHO 3
METHOSTENOL
FIG.
4.
CHOLESTEROL
Speculative alternate routes of lanosterol to cholesterol.
in our laboratory were identical to those of methostenol and methostenyl acetate, respectively. The mixed melting point of methostenol and lophenol gave no significant depression, while the mixed melting point of the acetates (our purest acetate, m.p. 110-12”) gave only a slight depression.” Concurrently with the above communication, there appeared a report of the proof of structure of citrostadienol as 4ar-methyl-A 7J4(28)-stigmastadien-3P_01 by Sondheimer et al. (25). If methostenol is found to be an active link in the biosynthesis of cholesterol, then the picture of the sequential transformation from lanosterol to cholesterol becomes confused. For the presence of a methyl group at the 4 position and the double bond in the 7 position as well as the saturated side chain makes methostenol incompatible with a pathway to cholesterol which also includes zymosterol and desmosterol (26) (Fig. 4). With the removal of the 14-substituted methyl group of lanosterol (27), the possibility of a migration of the double bond from position 8(9) to 7 appears attractive. Since 4,4-dimethylcholesterol is not a precursor of cholesterol (27), it will be interesting to establish the activity of methostenol especially in light of the reported high rate of transformation of A’-cholestenol to cholesterol (28, 29). If methostenol is active, and preliminary results in this laboratory with radioactive acetate indicate that it is, then a possible alternate pathway from lanosterol to cholesterol might include 4,4-dimethyl-ATI1 The sample of methostenyl acetate which was sent to Dr. Djerassi for comparison was impure, m.p. 93-4” (3) and undoubtedly accounts for the depression of 10” of the mixed melting point of this sample and lophenyl acetate, m.p. 119-21” reported by these
workers
(24).
METHOSTENOL
307
cholestenol, 401-methyl-A’-cholestenol, A’-cholestenol, and 7-dehydrocholesterol. The finding of methostenol (lophenol) in the plant kingdom raises the interesting question of whether or not plant and animal sterols have similar pathways of metabolism involving such common intermediates as squalene [rich in certain animal sources, e.g., shark liver oil, sebum (30), etc. and in plant material, e.g., olive oil (31)] as well as 4cz-methyl-A’-cholestenol (rat tissue and cactus). At this point, the common pathway may diverge with the a,ddition of a two-carbon fragment at the 24 position exemplified by Subsequent loss of citrostadienol (4a-methyl-A 7,24(28)-stigmastadien-3/3-01), the 4-methyl group would lead to such a compound as A’-stigmastenol [isolated from wheat oil by Idler et al. (32)], and thence via 7-dehydrositosterol (33) to /3-sitosterol. Such speculation does not seemunreasonable in view of the recent demonstration that ergosterol is synthesized by yeast from cholesterol with the methyl group at the 24 position furnished by S-adenosylmethionine (34, 35). ACKNOWLEDGMENTS We are indebted to Drs. Y. Mazur and F. Sondheimer, Weiamann Institute of Science, for samples of 4a-methylcholestan-38-01 and its acetate; to Dr. C. Djerassi, Wayne State University, for samples of lophenol and lophenyl acetate; to Dr. Hans No11 for infrared spectral analysis (Beckman IR-4) ; to Mrs. E. Schwartz for measurements of optical rotation, and to Mrs. S. C. Anderson for valuable technical assistance. SUMMARY
Additional evidence has been presented for the identity of methostenol as 4ar-methyl-A’-cholesten-3/3-ol by comparison of the totally saturated methostanol with authentic 4a-methylcholestan-30-01. The synthesis of methostenol from A’-cholesten-3/3-ol is described. The content of methosteno1 in the feces of rats fed a fat-free diet, in rat skin, and in rat small intestines was found to be 5.9 %, 10.2 %, and l-2 % of total sterols, respectively. The possible biogenetic role of methostenol is discussed. REFERENCES 1. WELLS, W. W., COLEMAN, D. L., AND BAUMANN, C. A., Arch. Biochem. Biophys. 67, 437 (1965). 2. COLEMAN, D. L., WELLS, W. W., AND BAUMANN, C. A., Arch. Biochem. Biophys. 60, 412 (1966). 3. WELLS, W. W., AND NEIDERHISER, D. H., J. Am. Chem. Sot. 79, 6569 (1957). 4. WELLS, W. W., AND NEIDERHISER, D. H., Federation PTOC. 17,333 (1958). 5. IDLER, D. R., AND BAUMANN, C. A., J. Biol. Chem. 196, 623 (1952). 6. IDLER, D. R., AND BAUMANN, C. A., J. Biol. Chem. 203,389 (1953).
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AND
WELLS
in the Chemistry of Fats and other Lipids,” Vol. I, 7. BERGMANN, W., “Progress pp. 18-69. Pergamon Press Ltd., London, 1952. 8. IDLER, D. R., NICKSIC, S. W., JOHNSON, D. R., MELOCHE, V. W., SCHUETTE, H. A., AND BAUMANN, C. A., J. Am. Chem. Sot. 76, 1712 (1953). 9. WEISENBERGER, E., Mikrochem. ver. Microchim. Acta 33, 51 (1947). 10. WOODWARD, R. B., PATCHETT, A. A., BARTON, D. H. R., IVES, D. A., AND KELLY, R. B., J. Chem. Sot. 1967 1131. 11. RUZICKA, L., MONTAVON, M., AND JEGER, O., Helv. Chim. Acta 31, 818 (1948). 12. NEIDERHISER, D. H., M. S. Thesis, Univ. of Pittsburgh, 1958. 13. BARTON, D. H. R., AND COOKSON, R. C., Quart. Revs. (London) 10, 44 (1956). 14. FIESER, L., AND HERZ, J. E., J. Am. Chem. Sot. 76, 121 (1953). 15. OPPENAUER, R. V., Rec. trav. chim. 66, 137 (1937). 16. ANTONUCCI, R., BERNSTEIN, S., LITTELL, R., SAX, K. J., AND WILLIAMS, J. H., J. Org. Chem. 17, 1341 (1952). 16~. MAZUR, Y., AND SONDHEIMER, F., J. Am. Chem. Sot. 60, 5220 (1958). 17. COLEMAN, D. L., AND BAUMANN, C. A., Arch. Biochem. Biophys. 66, 226 (1957). 18. FRANTZ, I. D., JR., DAVIDSON, A. G., AND DULIT, E., Federation Proc. 16, 265 (1956). 19. MOORE, P. R., AND BAUMANN, C. A., J. Biol. Chem. 196, 615 (1952). 20. SONDHEIMER, F., AND MAZUR, Y., J. Am. Chem. Sot. 79, 2906 (1957). 21. ATWATER, N. W., J. Am. Gem. Sot. 79, 5315 (1957). 22. RINGOLD, H. J., AND ROSENKRANZ, G., J. Org. Chem. 21, 1333 (1956). 23. Cox, J. S. G., KING, F. E., AND KING, T. J., J: Chem. Sot. 1967, 290. 24. DJERASSI, C., MILLS, J. S., AND VILLOTTI, R., J. Am. Chem. Sot. 80, 1005 (1958). 25. MAZUR, Y., WEIZMANN, A., AND SONDHEIMER,F., J. Am. Chem. Sot. 60, 1007 (1958). 26. BLOCH, K., Vitamins and Hormones 16, 119-50 (1957). 27. GAUTSCHI, F., AND BLOCH, K., J. Am. Chem. Sot. 79, 684 (1957). 28. BIGGS, M. W., LEMMON, R. M., AND PIERCE, F. T., JR., Arch. Biochem. Biophys. 61, 155 (1954). 29. BROOKS, S. C., AND BAUMANN, C. A., J. Biol. Chem. 229, 329 (1957). 30. MACKENNA, R. M. B., WHEATLEY, V. R., AND WORMALL, A., Biochem. J. 62,161-g (1952). 31. FITELSON, J., J. Assoc. O&. Agr. Chemists 28, 282 (1945). 32. IDLER, D. R., KANDUTSCH, A. A., AND BAUMANN, C. A., J. Am. Chem. Sot. 76, 4325 (1953) 33. WUNDERLICH, W., 2. physiol. Chem. 241, 116-24 (1936). 34. DANIELSON, H., AND BLOCH, K., J. Am. Chem. Sot. 79, 500 (1957). 35. PARKS, L. W., J. Am. Chem. Sot. 30, 2023 (1958).