SYNTHESIS OF 3~4lYDROXY-5a-CHOLEST-8-EN-7-ONE AND 3/3-HYDROXY-5cx-CHOLEST-8-EN-li-ONE: EVALUATION AS POTENTIAL HYPOCHOLESTEROLEMIC AGENTS

Edward 2.

Parish, a* Venka a B. Seikel, Herbert H. Kohl, 5

B. Nanduri,a Jean g. and Kenneth E. Nusbaum

Departments of aChemistry and b Microbiology

(Veterinary Medicine) Auburn University, Alabama 36849-3501

Received April 1, 1986 Revised July 7, 1987

ABSTRACT

An efficient procedure for the chemical synthesis of 3B-hydroxy-5a-cholest-8-en-7.-one and 3B-hydroxy-5a- cholest-8-en-ll-one is described. These ketosterols have been shown to have possible significant hypocholesterolemic effects when fed to normal rats at a level of 0.15% in a laboratory chow diet. The diets containing the steroids caused significant decreases in food consumption whichwere associated with decreases in the rate of gain in body weight.

INTRODUCTION

Recent evidence has been presented that suggests

lowering plasma cholesterol levels can either reverse

or prevent certain cardiovascular diseases [l]. The

discovery of new agents in the treatment and prevention

of such diseases as atherosclerosis and hyperlipidemia

could be of considerable value in reducing the

incidence of these diseases. A search for inhibitors

of sterol biosynthesis has led to the discovery of new

STEROIDS 48 / 5-6 November-December 1986 (407-4 18) 407

408 Parish et al

steroid hypocholesterolemic agents [2]. Several of

these steroids had in common the CL, B-unsaturated

ketone functionality [2-41. These findings have led us

to consider the possibility that other steroidal

CX , B -unsaturated ketones may possess

hypocholesterolemic activity.

The purpose of this communication is to describe

the chemical synthesis of 3P-hydroxy-5a-cholest-a-en-

7-one(I) and 3B-hydroxy-5cl-cholest-8-en-ll-one(I1) and

to present evidence indicating the possible

hypocholesterolemic action of these compounds in rats.

RESULTS AND DISCUSSION

The synthesis of compounds I and II was performed

by developing a synthesis using 5a-cholest-7-en-3B-yl

benzoate(II1) as a starting material (prepared from

5,7-cholestadien-3B-ol[5]) as shown in Figure 1.

Introduction of the 7,9(11) double bonds, to form

IV from III, was accomplished using mercuric acetate

[61. Treatment of IV with m-chloroperoxybenzoic acid

resulted in the formation of the epoxide V. Similar

results have been obtained using the 7,14 [7,8] and

other 7,9(11) diene systems [g-111. A brief treatment

of V with hydrochloric acid resulted in the formation

of 7-oxo-5a-cholest-8-en-3B-yl benzoate(V1). Similar

rearrangements of *7,%11) -epoxides have been

SYNTHESIS OF CHOLESTEROL ANALOGS 409

410 Parish et a/

observed previously [ll-131. Further reaction with

Na2C03 in methanol [14] produced I in high yield. In

an alternate reaction, treatment of V with H2S04 in

ethanol gave I directly.

Rearrangement of epoxide V with BF3.Et20 and

subsequent treatment with acetic acid resulted in the

formation of ll-oxo-5n-cholest-8-en-3B-yl

benzoate(VI1). Similar rearrangements have been

observed with 14,15a-epoxy-5a-cholest-7-en-3S-y1

benzoate [15-171 and A 7'g(11) - epoxides

[11,13,18-201. Alkaline hydrolysis of VII gave

3 B-hydroxy-5a-cholest-8-en-11-one(I1) in high yield.

In a similar manner, II was produced directly from V by

reaction with BF 3.Et20 followed by hydrolysis with

H2S04 in ethanol.

A number of steroids with hypocholesterolemic

properties are CL, f3 -unsaturated ketones [3,4,21-261, and

we have recently developed new methodology to prepare

these compounds from the allylic alcohol by selective

oxidation [27-311.

However, not all unsaturated steroidal ketones

possess this property [26,32,33], and their failure may

be due to rapid metabolism by induced degradative

enzymes [341, by a lack of intestinal absorption [331,

or by other unknown factors.

SYNTHESIS OF CHOLESTEROL ANALOGS 4 1 I

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412 Parish et al

diet in the same amount consumed by the steroid group on the previous day. Blood was drawn from the rats by cardiac puncture (0.5-1.0 mL) between 8:15 AM and lo:30 AM, and serum was obtained by centrifugation. Serum cholesterol was assayed using an enzymatic diagnostic kit (#351, Sigma Chemical Company, St. Louis, MO).

5a-Cholesta-7.9(11)-dien-38-vl Benzoate(IV) : To a stirred solution of 5a-cholest-7-en-3B-yl

benzoate (III, 5 g, 10.2 mmol) in chloroform (28 mL), was added a solution of mercuric acetate (7.25 g) in acetic acid solution (42 mL). The suspension was stirred for 24 h at room temperature (25'C) and filtered to remove mercurous acetate. The filtrate was concentrated to dryness at reduced pressure and the residue recrystallized four times from acetone-water to yield IV as a white crystalline solid (3.8g, 76.3%):mp 132-134'C (lit [19] 132.5-133.5'C); Jpv max 1720, 1600, 1580, 1450, 1370, 1120, 710 cm ; H-NMR 0.51 (s, 3H, C-18-CH ), 0.90 C-3-H), 6.33 (m,

(s, 3H, C-19-CH3), 4.85 (m, lH, 2H, C-7-H and C-11-H), 7.30 (m, 3H,

benzoate), 7.90 (m, 2H, benzoate); mass spectrum, m/z 488(9%, M), 473 (26%, M-CH ),

‘3 375 (4%, M-side chain),

351 (6%, M-CH3-benzoic aci ), 336 (5%, M-CH3-CH3-benzoic acid), 238 (4%, M-CH -side chain-benzoic acid), 105 (lOO%, benzoyl). ? he product showed a single component on TLC in three solvent systems (toluene, benzene, 50% toluene-hexane).

9.lla-Eooxv-5a-cholest-7-en-313-yl Benzoate (V): Diene IV (50.0 g, 102.4 mmol) was dissolved in

anhydrous ether (2000 mL) with gentle warming. The solution was placed in an ice bath and cooled to 18'C, at which time a solution of m-chloroperoxybenzoic acid (42.5 g) in ether (268 mL) was added. The gixture was stirred for 5 m&n and allowed to stand at 0 C for 5 h, and then at -15 C for 24 h. The material was precipitated by blowing a stream of nitrogen into the flask and reducing the solvent volume to approx 10%. The precipitate was filtered, washed with cold ether, and recrystallized from ecetone-water to giveo38.1 g (73.6%) of V: mp 169-171 C (lit [19] 152-154-E); IR umax1720,1600, 1450, 1280, 1110, 960, 710 cm ; H-NMR 0.58 (s, (m,

3H, c-18-cH3), 0.89 (s, 3H, C-19-CH3), 3.20 lH, C-3-H), 4.86 (m, lH, C-11-H), 5.59 (m, lH,

C-7-H), 7.44 (m, 3H, benzoate), 7.93 (m, 2H, benzoate); mass spectrum, m/z 504 (5%, M), 489 (25%, M-CH 1, 474 (2%, M-CH3-CH 1,

'2 391 (6%, M-side chain, 361 (62,

M-CH -CH3-sid 2

chain), 105 (100% benzoyl). The product show d a single component on TLC in three solvent systems (20% ether-toluene, chloroform, 35% ethyl acetate-chloroform).

SYNTHESIS OF CHOLESTEROL ANALOGS 4 13

7-0xo-5a-cholest-8-en-38Fvl Benzoate (VI): 9,lh-Epoxy-5 crcholest-7-en-3@-yl benzoate(V, 5.0

g, 9.9 mmol) was refluxed for 25 min with a solution consisting of chloroform (100 mL), methanol (300 mL), and concentrated hydrochloric acid (20 mL). The solution was concentrated to one-fourth its volume under reduced pressure, diluted with water, and thoroughly extracted with ether containing 5% CH2C12. The combined extracts were evaporated to dryness at reduced pressure and the residue was subjected to column chromatography on a silica gel column using an increasing gradient of ether in toluene as the eluting solvent. The product was recrystallized frog acetone- water to yield 3.2 g (64%) of VI: mp 149-151 C;_$Rv max 1720, 1670, 1600, 1570, 1300, 1100, 980, 710 cm ; H-NMR 0.85 (s, 3H, C-18-CH3), 1.25 (s, 3H, C-19-CH 1, 5.00 (m, lH, C-3-H), 7.40 (m, 3H, benzoate) 7.90 ?m, 2H, benzoatef; mass spectrum, m/z 504 (21%, M), 489 (2%, M-CH ), 382 (2%, M-benzoic acid), 391 chain), 332 (20%, M-CH CH -benzoic acid)

(6%, M-side

benzolrl); high resolutian &ass spectrum, ;O~!~6~k"?~~1c for C H 0 504.3603); The product showed a

a4 48 2 TLC in three solvent systems (20% single

compo ent 0 ether-toluene, chloroform, 35% ethyl acetate chloroform).

3R-Hvdroxv-5a-cholest-8-en-7-one(I) from VI; 7-Oxo-5a-cholest-8-en-3B-yl benzoate (2.0 g, 3.96

mmol) was dissolved in methanol (650 mL) and saturated aqueous Na CO (35 mL) was added. The mixture was stirred fog 32 h at 3S°C, the volume was reduced to one-half by a stream of nitrogen, water (400 mL) was added, and the mixture cooled at 4'C for 12 h. The resulting precipitate was collected, dried, and subjected to column chromatography on a silica gel column using an increasing gradient of ether in toluene as the eluting solvent. The product was recrystallized from acgtate-water to yiel$ 1.36 g (86%) of VI: mp 122-124 C(lit [37] 122-123 C);_$R vmax 3350, 1660, 1590, 1378, 1268, 1050, 933 cm ; H-NMR 0.56 (s, 3H, C-18-CH talc 0.571, 1.19 (s, 3H, C-19-CH3, talc 1.21), 31.70 (m, lH, C-3-H); mass spectrum, m/z 400 (lOO%, M), 385 (27%, M-CH f, 382 f5%, M-H 01, 287 (36%, M-side chain), 272 (69%‘ &CH -side chain ?, 269 (27%, M-H O-side chain); UV h max

=fO 000 (lit [371 253 nm). ? ethanol) 253 nm, The product was found to

iave g purity of 98% upon GLC analysis and was found to be a single component upon TLC analysis in three solvent systems (50% ether-toluene, 50% ethyl acetate-toluene, 35% ethyl acetate-chloroform).

414 Parish et a/

Z-Hvdroxv-5n-cholest-8-en-7-one(I) from V: Epoxide V (2.0 g, 3.69 mmol) was dissolved in

ethanol (700 mL); water (40 mL) and H SO4 (120 mL) were added and the mixture refluxed for 24 ?l. The volume was reduced to one-half at reduced pressure; water (700 mL) was added, and the mixture thoroughly extracted with ether containing 5% CH2C12. The combined extracts were evaporated at reduced pressure and the resulting residue was subjected to column chromatography on a silica gel column using an increasing gradient of ether in toluene as the eluting solvent. The product was recrystallized from acetgne-water to yield 0.90 g (56.7%) of I: mp 122-124 C; the spectral and chromatographic properties were identical with those described above.

ll-Oxo-w-cholest-8-en-3f3-vl Benzoate(VI1) : 9,llct-Epoxy-5a-cholest-7-en-3B-y1 benzoate(V), (4.0

g, 7.38 mmol) was dissolved in tetrahydrofuran (40 mL), anhygrous ether (400 mL) was added, the solution cooled to 2 c, and boron trifluoride etherate (40 mL) was slowly added with stirring. After standing for 30 min, the mixture was poured into ice water and thoroughly extracted with ether. The combined extracts were evaporated at reduced pressure and the residue was refluxed with glacial acetic acid (130 mL) for 8 h. Water (1000 mL) was added and the mixture extracted with ether, washed with water and 5% aqueous NaHCO and the solvent removed under reduced pressure. 2' T e residue was subjected to column chromatography on a silica gel column using an increasing gradient of ether in toluene as the eluting solvent. The reaction product was recrystallized frog acetone-water to yield 2.2 g (55%) of VII: mp 122-124 C; IR vmax11720, 1670, 1600, 1580, 1450, 1275, 1120, 980, 710 cm - H-NMR 0.75 (s, 3H, C-18-CH3), 1.02(s, 3H, C-19-CH3 ), i.82 (m, lH, C-3-H), 7.30(m, 3H, aromatic), 8.0 (m, 2H, aromatic); mass spectrum, m/z 504 (148, M), 489 (15%, M-CH ), 391 (2%, M-side chain); 382 (28, M-benzoic acid), 327 (98, M-CH -benzoic acid), 352 (18%, M-CH -CH -benzoic acid), 105 ?lOO%, benzoyl); high resolutioa ma2.s spectrum, 504.3504 (talc for C H showed a single compa~e~@"an5~~,3~~3~~r~~es~~~~~~t systems (20% ether-toluene, chloroform, 35% ethyl acetate-chloroform).

38-Hvdroxv-5a-cholest-8-en-ll-one(I1) from VII: ll-Oxo-5-cholest-8-en-38-vl benzoate (2.0 q, 3.46

mmol) was dissolved in methanol (650 mL) and saturated aqueous Na2COp (35 mL) was added. The reaction and product isola ion were conducted as described previously to vi eld 1 41 n (88 8%) of II: m13 75-77OC:

SYNTHESIS OF CHOLESTEROL ANALOGS 415

Oral administration of compounds I and II produced

significant, but not sustained, hypocholesterolemic

activity (Figure 2). A reduction of food consumption

and suppression of growth were also observed, an

observation similar to those previously reported during

the feeding of other hypocholesterolemic steroids

[4,21,22,26,35].

In related studies, compound I was

a key enzyme inhibit HMG-CoA reductase,

cholesterolgenesis, while

inhibitor [36].

II was found to a poor

EXPERIMENTAL

found to

in mammalian

Procedures for the recording of melting points (mp), infrared (IR) spectra, low resolution mass spectra, proton nuclear magnetic resonance (11 -NMR) , and ultraviolet (UV) spectra have been reported previously [21]. Similarly, details concerning the use of gas-liquid (GLC), thin-layer (TLC), and column chromatography have also been described [24,25]. High resolution mass spectra were obtained using a VG-Model 70 E, double focusing mass spectrometer. The preparation of 5a-cholest-7-en-3@-yl benzoate(II1) has been described [51.

Sprague-Dawley male rats (loo-125 g, Sprague- Dawley Farms, Madison, WI) were maintained on a light (7:OO AM-5:30 PM)-dark (5:30 PM-7:OO AM) cycle and fed a cholesterol-free diet (#901377, Nutritional Biochemicals, Cleveland, OH) for a period of 7 days prior to each experiment. The rats were divided into 3 groups with approximately the same mean serum cholesterol concentrations. The steroids (I or II) were thoroughly mixed with diet at final concentrations of 0.15% w/w and stored at 4'C. The 3 groups were defined as follows: (1) ad libitum clroup (N=8) with free access to the basal diet; (2) steroid sroup (N=8) with free access to the basal diet containing I or II; and (3) pair-fed group (N=8) with access to the basal

416 Parish et a/

IR V max 3400, 1670, 1455, 1375, 1030 cm-'; H-NMR 0.65 (s, 3H, C-18-CH3, talc 0.68), 1.07 (s, 3H, C-19-CH talc 1.09), 3.5 (m, lH, C-3-H); mass spectrum, m/z d?)O (lOO%, 385) (4%, M-CH -CH ), 382 (158, M-H20), 367

2873(152, M-side chain), 272 (15%,

have a purity of 98% upon GLC analysis and was found to be a single component upon TLC analysis in three solvent systems (50% ether-toluene, 50% ethyl acetate-toluene, 35% ethyl acetate-chloroform).

3 B-Hvdroxv-5cx-cholest-8-en-ll-one(I1) from V: Epoxide V (2g, 3.69 mmol) was dissolved in

tetrahydrofuran (920 mL), anhydrous ether (400 mL) was added, the solution cooled to 2'C, and borontrifluoride etherate (20 mL) was slowly added with stirring. After standing for 30 min, the mixture was poured into ice water and thoroughly extracted with ether. The combined extracts were evaporated at reduced pressure and the residue was refluxed in a mixture of ethanol (350 mL), water (20 mL), and sulfuric acid (60 mL) for 24 h. The reaction mixture was reduced to one-half its volume at reduced pressure, water added (700 mL), the mixture was thoroughly extracted with ether, washed with water and 5% aqueous NaHC03, the solvent removed under reduced pressure, and the resulting residue subjected to column chromatography on a silica gel column using an increasing gradient of ether in toluene as the eluting solvent, The product was recrystallized from acetone-water to yield 0.77 g (48.5%) of II: mp 75-77'C; the spectral and chromatographic properties were identical with those described above.

ACKNOWLEDGMENTS

This research was supported in part by a Schering-Plough Corporation Grant for Research Corporation and by Auburn University (Grant-in-Aid 82-179).

NOTES AND REFERENCES

*Author to whom all correspondence should be addressed.

1. Kerr, R. A., SCIENCE (Research News) 223, 381(1984).

2. Schroepfer, G. J., Jr., ANN REV BIOCHEM 50, 585 (1981), and references cited therein.

SYNTHESIS OF CHOLESTEROL ANALOGS 4 17

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and Quiocho, F. A., CHEM PHYS zor (1977).

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Bladon, P,, Henbest, H. B., Jones, E. R. H., Woods, G. W., Eaton, D. C., and Wagland, A. A., J CHEM SOC ,2916 (1953), and references cited therein. Chamberlin, E. M., Ruyle, W. V., Erickson, A. E., Chemerda, J. M., Aliminosa, L. M., Erickson, R. L ., Sita, G. E., and Tishler, M., J AM CHEM sot 35, 3477 (1953). Budziarek, R., Johnson, F., and Spring, F. S., J CHEM SOC , 3410 (19.521, and references cited therein. Heusser, H., Eichenberger, P. F., Dallenbach, H. R and Jeger, O., $51) *

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Heusser, H., Anliker, R., Eichenberger, K., and Jeger, O., HELV CHIM ACTA z, 936 (1952). Parish, E. J. and Schroepfer, G. J., Jr., J ORG CHEM E, 4034 (1980). Parish, E. J., Newcomer, M. E., Gilliland, G. L., Quiocho, F. A., and Schroepfer, G. J., Jr., TETRAHEDRON LETT 4401 (19761. Parish, E. J. and Schroepfer, G. J.,Jr., CHEM PHYS LIPIDS 19, 107(1977). Gilliland, G. L., Newcomer, M. E., Parish, E. J., Schroepfer, G. J., Jr., and Quiocho, F. A., ACTA CRYSTALLOGR m, 3117 (1977) t Blandon, P., Henbest, H. B., Jones, E. R. H., Lovell, B. J., Wood, G. W., WOOLS, G. F., Elks, J Evans, R. M., Hathway, D. E., Oughton, J. F., an; Thomas, G. H., J CHEM SOC , 2921 (1953). Heusser, H., Heusler, K., Eichenberger, K., Honegger, C. G., and Jager, O., HELV CHIM ACTA 35, 295 (1952). Akhtar, M., Freeman, C. W., Rahimtula, A. D., and Wilton, D. C., BIOCHEM J 129, 225 (1972).

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Raulston, D. L., Mishaw, C. O., Parish, E. J., and Schroepfer, G. J., Jr., BIOCHEM BIOPHYS RES COMMUN 7l, 948 (1976). Schroepfer, G. J., Jr., Monger, D., Taylor, A. S., Chamberlain, J. S., Parish, E. J., Kisic, A., and Kandutsch, A. A., BIOCHEM BIOPHYS RES COMMUN 78, 1227 (1977). Schroepfer, G. J., Jr., Parish, E. J., Kisic, A., Jackson, E. M., Farley, C. M., and Mott, G. E., PROC NATL ACAD SC1 USA 79, 3042 (1982) . Kisic, A., Monger, D., Parish, E. J., Sattlerfield, S., Raulston, D. L., and Schroepfer, G. J., Jr., ARTERY 3, 421 (1977). Kisic, A., Taylor, A. S. , Chamberlain, J. S., Parish, E. J., and Schroepfer, G. J., Jr., FED PROC 37, 1663 (1978). Schroepfer, G. J., Jr., Walker, V., Parish, E. J., and Kisic, A., BIOCHEM BIOPHYS RES COMMUN 93, 813 (1980). Parish, E. J. and Schroepfer, G. J., Jr., CHEM PHYS LIPIDS 27, 281 (1980). Parish, E. J. and Scott, A. D., J ORG CHEM 48, 4766, (1983). Parish E. J., Scott, A. D., Dickerson, J. R., and Dykes, W., CHEM PHYS LIPIDS 35, 315 (1984). Parish, E. J., Chitrakorn, S., and Lowery, S., LIPIDS 2, 550 (1984). Parish, E. J. and Chitrakorn, S., SYNTH COMMUN 15, 393 (1985). Langdon, R., El-Masry, S., and Counsell, R. E., J LIPID RES 18, 24 (1977). Gas, J. H. and Desai, B. N., J ORG CHEM 44, 1590, (1979). Erickson, S. K., Matsui, S. M., Shrewsbury, M. A., Cooper, A. D., and Gould, R. G., J BIOL CHEM 253, 4159 (1978). Kandutsch, A. A., Heiniger, A. J., and Chen, H. W., BIOCHIM BIOPHYS ACTA &Q&, 260 (1977). Parish, E. J., Nanduri, V. B. B., Kohl, H. H., and Taylor, F. R., LIPIDS 21, 27 (1985). Tsuda, M., Parish, E. J., and Schroepfer, G. J., Jr., J ORG CHEM 44, 1282 (1979).