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Correspondence to: George J. Schroepfer, Jr..
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURES AND...DISCUSSIONREFERENCES |
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4,4-Dimethyl-5
-cholesta-8,14,24-trien-3ß-ol (I) from human follicular fluid and 4,4-dimethyl-5-cholesta-8,24-dien-3ß-ol (II) from bull testes have been reported to activate meiosis in mouse oocytes (Byskov et al., 1995. Nature. 374: 559–562). Described herein are new chemical syntheses of I, II, and the 
8(14),24 analog XXII. A critical step in these syntheses was a remarkably high yield side chain oxidation of 3ß-acetoxy-4,4-dimethyl-5-cholest-8(14)-en-15-one to the corresponding C24 24-hydroxy compound VI. Oxidation of VI to the aldehyde, followed by Wittig olefination gave 3ß-acetoxy-4,4-dimethyl-5-cholesta-8(14),24-dien-15-one. Reduction with sodium borohydride to the 15ß-hydroxysteryl ester, dehydration with sulfuric acid in CHCl3, and saponification furnished I in high purity. Reduction of VI with sodium borohydride to the 15-hydroxysteroid followed by dehydration gave 3ß-acetoxy-4,4-dimethyl-5-chola-8,14-dien-24-ol. Hydrogenation over Raney nickel gave the monounsaturated 8(14) and 8 compounds. Oxidation to the corresponding aldehydes followed by Wittig olefination and saponification gave II and XXII. Chromatographic, mass spectral, and 1H and 13C nuclear magnetic resonance spectral data have been presented for the synthetic sterols and their derivatives. I, II, XXII, and their 8,14 and 7,14 analogs, at 3 µg per ml, caused a resumption of meiosis in mouse oocytes in the presence of hypoxanthine (3.5 mM). Under the same conditions, 5 and 5,7 sterols were inactive.
—Ruan, B., S. Watanabe, J. J. Eppig, C. Kwoh, N. Dzidic, J. Pang, W. K. Wilson, and G. J. Schroepfer, Jr. Sterols affecting meiosis: novel chemical syntheses and the biological activity and spectral properties of the synthetic sterols. J. Lipid. Res. 1998. 39: 2005–2020.
Supplementary key words: mass spectrometry, nuclear magnetic resonance
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURES AND...DISCUSSIONREFERENCES |
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4,4 -Dimethyl-5-cholesta-8,14,24-trien-3ß-ol (I), 4,4-dimethyl-5-cholesta-8,24-dien-3ß-ol (II), and 4,4-dimethyl-5-cholesta-8,14-dien-3ß-ol (III) ( Figure 1) have recently been reported to activate meiosis in mammalian oocytes (1). I and II were samples isolated from human follicular fluid and bull testicular tissue, respectively, with chemical characterization based on gas chromatography–mass spectrometry (GC–MS) and, in the case of II, 13C nuclear magnetic resonance (NMR) spectroscopy in the absence of authentic standards. The 8,14,24-triene I and the 8,14-diene III are products of a cytochrome P-450 -dependent 14-demethylation of lanosterol and 24,25-dihydrolanosterol, respectively (2) (3) (4) (5) (6) (7). I, II, and III represent potential intermediates in the biosynthesis of cholesterol (8). The enzymatic formation of I from lanosterol in rat ovary has been recently reported to be under hormonal control (9), and I has also been reported to be a positive regulator of the nuclear orphan receptor LXR (10).
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Described herein are new approaches to the chemical syntheses of I ( Figure 2) and II ( Figure 3). Also presented are chromatographic, mass spectral (MS), and 1H and 13C NMR spectral properties of I, II, and III, their derivatives, and related compounds, as well as the results demonstrating the ability of the synthetic sterols to affect meiosis in mouse oocytes. Preliminary communications of portions of this work have been presented (11) (12).
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EXPERIMENTAL PROCEDURES AND RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURES AND...DISCUSSIONREFERENCES |
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General methods and materials
Melting points (MP) were measured in sealed, evacuated capillary tubes using a Thomas-Hoover apparatus. Ultraviolet (UV) spectra were recorded on a Shimadzu UV-1601 spectrophotometer using ethanol as solvent. Infrared (IR) spectra were recorded on a Mattson Galaxy 6020 Fourier-transform IR spectrometer with KBr pellets. Low resolution and high resolution MS were recorded on a VG ZAB-HF double sector instrument with an electron energy of 70 eV and direct inlet sample introduction. The composition and origins of ion B, ion B-CH3, and ion D in the mass spectra of C24 (13) (14) and C27 (15) (16) (17) (18) (19) 8(14)-15-ketosteroids have been presented previously. Ions designated with an asterisk (*) showed exact mass values on high resolution MS which were within 3 millimass units of the calculated values for the proposed ions. GC–MS was carried out on the same instrument coupled to an HP-5890-A GC unit using a 60 m DB-5MS column (0.25 mm ID; 0.1 µm film thickness; J&W Scientific Inc., Folsom, CA). The column temperature was 250°C and the temperatures of the injector and GC–MS interface were 290°C. Helium was used as the carrier gas with a head pressure of 20 psi. Trimethylsilyl ether derivatives were prepared by treatment of the steroids with a 1:1 mixture of bis(trimethylsilyl)trifluoroacetamide and pyridine for 2 h under nitrogen at 40°C, followed by evaporation to dryness at 40°C under nitrogen. NMR spectra were recorded on a Bruker AMX500 (500.1 MHz, 25°C for 1H, 22°C for 13C) spectrometer in CDCl3 solution (5–20 mM for 1H; 20–100 mM for 13C) and referenced to an internal standard of tetramethylsilane (1H) or CDCl3 at 77.0 ppm (13C).
Thin-layer chromatography (TLC) was carried out using aluminum-backed silica gel 60 plates (EM Science, Gibbstown, NJ). Components on the plates were visualized after spraying with 5% ammonium molybdate in 10% sulfuric acid and heating. Unless specified otherwise, medium pressure liquid chromatography (MPLC) was carried out on glass columns packed with silica gel (230–400 mesh), whereas silica gel (70–230 mesh) was used for routine column chromatography.
High performance liquid chromatography (HPLC) was carried out using a Rheodyne 7125 or Waters U6K injector and Waters 600 or 510 pumps with UV detection at 210 nm. Reversed phase HPLC was carried out using a Customsil ODS column (250 mm x 4.6 mm i.d.; Custom LC, Houston, TX) using methanol as solvent at a flow rate of 1 ml per min. Ag+-HPLC was carried out as described previously (20). In the current study 5 µm Nucleosil SA cation exchange (100 Å pore size) columns (250 mm x 4.6 mm, 300 mm x 3.2 mm, 300 mm x 4.6 mm, and 250 mm x 10 mm) were obtained from Alltech Associates (Deerfield, IL), and they were charged with silver ion and prepared for use as described previously (20). Flow rates for analytical and semipreparative (10 mm i.d.) columns were 1 ml per min and 3 ml per min, respectively.
4,4-Dimethylcholest-5-en-3ß-ol, 4,4-dimethylcholesta-5,7-dien-3ß-ol, 4,4-dimethyl-5-cholesta-8,14-dien-3ß-ol (III), and 4,4-dimethyl-5-cholesta-7,14-dien-3ß-ol (IV) were prepared as described previously (21) and, after chromatographic purification, showed purities of >99%, ~97%, ~95%, and ~96%, respectively, as judged by capillary GC and 1H NMR. (20R,22R)-cholest-5-ene-3ß,20,22-triol and (20S,22S)-cholest-5-ene-3ß,20,22-triol were prepared as described previously (22), and each showed a purity >99% on the basis of TLC, Ag+-HPLC, and 1H NMR. Samples of (22R)-cholest-5-ene-3ß,22-diol and (22S)-cholest-5-ene-3ß,22-diol, showing minor impurities on TLC (overloaded), were purchased from Sigma Chemical Company (St. Louis, MO). Steryl acetates were prepared by treatment of the free sterols with a mixture of acetic anhydride and pyridine (1:1). Bovine serum albumin (crystallized) was obtained from ICN Biochemical (Aurora, OH) and equine chorionic gonadotropin was from Dyosynth (Oss, Holland).
Assays of activation of meiosis
Oocyte-cumulus cell complexes were isolated from the ovaries of 22–24 day old B6SJLF1 mice 44 h after intraperitoneal injection of equine chorionic gonadotropin (5 IU). The culture medium was minimum essential medium with Earle's salts supplemented with crystallized bovine serum albumin (5 mg/ml). The medium also contained hypoxanthine (3.5 mM) to suppress the spontaneous resumption of meiosis (23). Cumulus cells were removed from oocytes by drawing the complexes in and out of a small-bore glass pipet. The denuded oocytes were incubated in 2.5 ml of medium with or without sterols for 15–17 h at 37°C in an atmosphere of 5% O2, 5% CO2, and 90% N2. Sterols were dissolved in ethanol at a concentration of 1 mg/ml. All sterols were tested at a concentration of 3 µg/ml. Control cultures received the same amount of ethanol as the sterol-treated groups. After the incubation period, the percentage of oocytes that underwent germinal vesicle breakdown, indicative of the resumption of meiosis, was determined. Approximately 40 cumulus cell-free oocytes were distributed to each group. Data are presented as the mean percentage of four independent experimental replicates; variation among experiments is illustrated using the standard error of the mean. For evaluation of the differences among groups, data were subjected to arcsin transformation and ANOVA. When a significant F-ratio was defined by ANOVA, groups were compared using the Fisher's PLSD post-hoc test using software; when P 
0.05, the difference was considered significant.
Syntheses of 4,4-dimethylsterols
3ß-Acetoxy-4,4-dimethyl-5-cholest-8(14)-en-15-one (V)
3ß-Acetoxy-4,4-dimethyl-5-cholest-8(14)-en-15-one was prepared by treatment of 3ß-hydroxy-4,4-dimethyl-5-cholest-8(14)-en-15-one, obtained by minor modifications of a previously described synthesis (21), with acetic anhydride and pyridine. The crude product was subjected to MPLC on a silica gel column (50 cm x 2.5 cm) using 2% ethyl acetate in hexane as the eluting solvent. Fractions (20 ml) were collected every 4.5 min. The contents of fractions 159–235 were combined and evaporated to dryness to give V as a white solid; MP 123.5–126°C; single component (Rf 0.9) on TLC (solvent, 25% ethyl acetate in hexane) and a single component (tR 9.8 min) on reversed phase HPLC; MS, 470 (100%; M+), 452* (7%, M–H2O), 410* (20%; M–CH3COOH), 395* (31%; M–CH3COOH–CH3), 367 (11%; C26H39O), 357* (3%; M–SC), 339* (17%, M–SC–H2O), 297* (11%; M–CH3COOH–SC), 276* (21%; ion B), 261* (22%; ion B–CH3), and 135* (91%; C10H15); high resolution MS, calcd. for C31H50O3 470.3760, found 470.3760; IR, 
max 2963, 2872, 2853, 1732, 1703, 1626, 1466, 1365, 1240, 1084, and 1026 cm-1; UV, 
max 259 nm (
14,000); 1H NMR, Table 1; 13C NMR, Table 2.
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3ß-Acetoxy-24-hydroxy-4,4-dimethyl- 5 -chol-8(14)-en-15-one (VI)
To a mechanically stirred mixture of trifluoroacetic anhydride (25 ml) and sulfuric acid (10 ml) maintained at -10°C was added a solution of 30% hydrogen peroxide (2.5 ml) dropwise over a period of 30 min. During the addition the temperature of the mixture was maintained at between -4°C and -8°C. Acetate V (1.00 g; 2.13 mmol) was, with continued vigorous stirring, added in one portion. The color of the reaction turned to orange and the temperature of the reaction mixture was increased to –2°C. Within 1 h, the mixture, maintained at -2°C, turned to a thick slurry. With continued vigorous stirring for 3 h, the slurry changed to a clear, light yellow-colored, mobile solution. TLC (solvent, 25% ethyl acetate in hexane) of an aliquot of the reaction mixture indicated completion of the reaction as judged by consumption of almost all of the starting material. The reaction mixture was poured into ice water (100 ml) containing sodium sulfite (200 mg). To the resulting mixture, potassium carbonate (~20 g) was slowly added and, after stirring for 30 min, the mixture was extracted 4 times with ethyl acetate (200 ml portions), and the combined organic extracts were washed 5 times with 10% KOH (80 ml portions), once with a saturated NaHCO3 solution (50 ml), twice with brine (50 ml), and dried over anhydrous sodium sulfate. The crude product (1.4 g), obtained upon evaporation of the solvent under reduced pressure, showing a single component on TLC, was dissolved in CHCl3 (2 ml) and applied to a silica gel column (25 cm x 2.5 cm). Using 5% acetone in hexane as the eluting solvent, fractions (20 ml) were collected every 3.5 min. The contents of fractions 37 to 47 were combined and, after evaporation of the solvent under reduced pressure, gave VI (690 mg; 73% yield), MP 218–220°C; single component (Rf 0.12) on TLC (solvent, 25% ethyl acetate in hexane) and a single component (tR 3.82 min) on reversed phase HPLC, and (as its TMS ether derivative) it showed one major (~98%) component (tR 48.12 min) on capillary GC analysis on a DB-5 column (60 m); GC–MS (TMS ether), 516 (100%; M+), 501 (16%; M–CH3), 498 (3%; M–H2O), 456 (14%; M–CH3COOH), 441 (16%; M–CH3COOH–CH3), 413 (6%; ion D), 339 (11%), 333 (4%), 322 (8%), 307 (7%), 305 (5%), 297 (7%), 279 (9%), 159 (11%), and 135 (62%); MS (free sterol), 444 (100%; M+), 426 (3%; M–H2O), 411 (7%; M–H2O–CH3), 384* (12%; M–CH3COOH), 369 (22%; M–CH3COOH–CH3), 341* (12%; C23H33O2), 339 (16%), 297 (8%; M–CH3COOH-SC), 279 (13%; M–CH3COOH–SC–H2O), 250 (22%; ion B), 235 (22%, ion B–CH3), 217 (8%), and 135* (91%; C10H15); high resolution MS, calcd. for C28H44O4 444.3239, found 444.3247; IR, max 3512, 2941, 2870, 1730, 1693, 1620, 1246, 1084, and 1034 cm-1; UV, max 259 nm ( 14,400); 1H NMR, Table 1; 13C NMR, Table 2.
3ß-Acetoxy-15-oxo-4,4-dimethyl-5-chol-8(14)-en-24-al (VII )
To oxalyl chloride (172 µl; 1.97 mmol) in CH2Cl2 (6 ml) at -50°C under argon was slowly added dimethyl sulfoxide (310 ml; 4.37 mmol) in CH2Cl2 (1 ml). After stirring for 2 min, VI (320 mg; 0.72 mmol) in CH2Cl2 (2 ml) was added dropwise. After stirring of the mixture for an additional 1 h at -50°C, triethylamine (1.2 ml) was added. The mixture was maintained at -50°C for 5 min and, after warming to room temperature, a saturated NH4Cl solution (15 ml) was added. The resulting mixture was extracted with methyl tert-butyl ether (MTBE; 200 ml) and the organic extract was washed twice with a saturated NH4Cl solution (15 ml portions), 3 times with a saturated cupric sulfate solution (15 ml portions), twice with a saturated NaCl solution (15 ml portions) and then dried over anhydrous sodium sulfate. The residue obtained upon evaporation of the solvent under reduced pressure was subjected to MPLC on a silica gel column (30 cm x 2.5 cm). Using 5% acetone in hexane as the eluting solvent, fractions (22 ml) were collected every 4 min. The contents of fractions 27 to 60 were combined to give, after evaporation of the solvent under reduced pressure, the desired aldehyde VII as a white solid (286 mg; 90% yield), MP 199–200°C; single component (Rf 0.63) on TLC (solvent, 40% ethyl acetate in hexane); MS, 442 (85%; M+), 424* (37%; M–H2O), 409 (2%; M–H2O–CH3), 382* (13%; M–CH3COOH), 367* (17%, M–CH3COOH–CH3), 339* (20%; M–SC–H2O), 297* (7%; M–SC–CH3COOH), 279* (13%; M–SC–CH3COOH-H2O), 248* (18%, ion B), 233* (16%; ion B–CH3), 230* (13%; C16H22O), and 135* (100%; C10H15); high resolution MS, calcd. for C28H42O4 442.3083, found 442.3086; IR, max 2944, 2710, 1728, 1697, 1626, 1364, 1246, and 1032 cm-1; UV max 259 nm ( 14,400); 1H NMR, Table 1; 13C NMR, Table 2.
3ß-Acetoxy-4,4-dimethyl-5-cholesta-8(14),24-dien-15-one (VIII )
To a cold slurry of isopropyltriphenylphosphonium iodide (503 mg; 1.16 mmol) in tetrahydrofuran (3 ml) was added n-butyllithium (300 µl; 0.70 mmol) at 0°C under argon. The resulting red solution was stirred for 15 min and then a portion (70%) was added dropwise to a solution of VII (190 mg; 0.43 mmol) in tetrahydrofuran (THF; 2 ml) at –78°C. After stirring for 2 h at 0°C, the mixture was poured into water and extracted 3 times with ether (100 ml portions). The combined ether extracts were washed twice with brine (20 ml portions), dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The resulting residue was subjected to MPLC on a silica gel column (50 cm x 1 cm) using 2% acetone in hexane as the eluting solvent (fraction volume, 22 ml). The contents of fractions 4 to 19 were combined to give, after evaporation of the solvent under reduced pressure, VIII as a white solid (150 mg; 75% yield); MP 128.0–129.5°C; single component (Rf 0.90) on TLC (solvent, 25% ethyl acetate in hexane) and single component (tR 8.20 min) on reversed phase HPLC; MS, 468 (100%; M+), 453* (4%; M–CH3), 408* (15%; M–CH3COOH), 393* (25%; M–CH3COOH–CH3), 365* (9%, C26H37O), 339 (6%), 323 (2%), 297* (15%; M–CH3COOH–SC–2H), 274* (14%; ion B), 259* (8%; ion B–CH3), 243 (10%), 227 (5%), 165* (64%; C11H17O), 135* (80%; C10H15), 109 (35%), and 69 (43%); high resolution MS calcd. for C31H48O3 468.3603, found 468.3607; IR, max2947, 1732, 1703, 1694, 1622, and 1244 cm-1; UV, max 259 nm ( 14,700); 1H NMR, Table 1; 13C NMR, Table 2.
3ß-Acetoxy-4,4-dimethyl-5-cholesta-8(14),24-dien-15ß-ol (IX) and 3ß-acetoxy-4,4-dimethyl-5-cholesta-8(14),24-dien-15-ol (X)
To VIII (75 mg; 0.16 mmol) in ethanol (20 ml) was added sodium borohydride (120 mg; 3.2 mmol). After stirring at room temperature for 2 h, the mixture was poured into water (20 ml) and extracted 3 times with MTBE (100 ml portions). The combined organic extracts were washed twice with brine (25 ml portions), dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The resulting residue was subjected to MPLC on a silica gel column (50 cm x 1 cm) using 2% acetone in hexane as the eluting solvent; 22 ml fractions were collected. The contents of fractions 96 to 105 were combined and, after evaporation of the solvent under reduced pressure, gave the 15ß-hydroxy-8(14),24-diene IX (61 mg; 81% yield); MP 138.5-140.0°C; single component (Rf 0.72) on TLC (solvent, 25% ethyl acetate in hexane) and a single component (tR 7.22 min) on reversed phase HPLC; MS, 470 (0.2%; M+), 452* (100%; M–H2O), 437* (28%; M–H2O–CH3), 409 (3%), 392* (9%; M–CH3COOH–H2O), 381* (6%; C28H41), 367 (26%), 340* (19%; C23H32O2), 325 (6%), 288 (14%), 257* (11%; C19H29), 227* (6%; C17H23), 145* (15%; C11H13); high resolution MS calcd. for C31H50O3 470.3760, found 470.3751; 1H NMR, Table 1; 13C NMR, Table 2.
The contents of fractions 108–115 were combined to give, after evaporation of the solvent under reduced pressure, the 15-hydroxy-8(14),24-diene X (4 mg; 5% yield) which showed a single component (tR 6.83 min) on reversed phase HPLC; 1H NMR, Table 1.
3ß-Acetoxy-4,4-dimethyl-5-cholesta-8,14,24-triene (XI)
The 15ß-hydroxy-8(14),24-diene IX (55 mg; 0.12 mmol) in CHCl3 (10 ml) was treated with sulfuric acid (20 µl) at room temperature with stirring for 5 min. Sodium bicarbonate (~30 mg) was added, and the mixture was diluted with CH2Cl2 (100 ml) and washed 3 times with brine (10 ml portions), dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The resulting residue was subjected to MPLC on a silica gel column (25 cm x 1 cm) using 1% acetone in hexane as the eluting solvent; fractions (7 ml) were collected every 2 min. The contents of fractions 5–7 were combined to give, after evaporation of the solvent under reduced pressure, a white solid (50 mg) which showed a single component (Rf 0.79) on TLC (solvent, 10% ethyl acetate in hexane) and a single component (tR 24.69 min) on reversed phase HPLC. However, analytical Ag+-HPLC showed one major component (95%) accompanied by several minor, less polar components (1–2% each). Accordingly, the crude product was subjected to semipreparative Ag+-HPLC (250 mm x 10 mm column) using 5% acetone in hexane as the eluting solvent (3 ml per min) with multiple injections of ~10 mg per injection and UV monitoring (210 nm) to give the 8,14,24 steryl acetate XI (45 mg; 85% yield); MP 137–138°C (lit. 126–128.5°C (3), 139–140°C (24)); MS, 452* (100%; M+), 437* (25%; M–CH3), 392* (4%; M–CH3COOH), 377* (33%; M–CH3COOH–CH3), 367* (23%; C25H35O2), 340 (14%), 325 (7%), 288 (17%), 279* (8%; M–CH3COOH–SC–2H), 265* (7%; C20H25), and 159* (11%; C12H25); high resolution MS, calcd. for C31H48O2 452.3654, found 452.3653; IR, max 2961, 2936, 2676, 1736, 1640, 1464, 1451, 1366, 1246, 1032, 1001, 990, 974, 941, 826, and 801 cm-1; 1H NMR, Table 3; 13C NMR, Table 4. As noted above, the purity of XI was ~95% by Ag+-HPLC. 1H NMR analysis indicated a purity of ~90%, with small amounts of its 8,14,25 (3.5%), 6,8(14),24 (1%), and 7,14,24 (1–2%) isomers and two or three unidentified components (1–3% each). Repetition of the Ag+-HPLC (250 mm x 10 mm column; elution with 5% acetone in hexane; ~10 mg portions) showed multiple components with the following tR values (min): 10.8 (1.3%), 11.8 (0.7%), 26.4 (0.2%), 37.6 (0.2%), 49.0 (0.5%), and 58.6 (96%). The major component, corresponding to XI, showed a melting point (137–138°C) identical to that obtained prior to Ag+-HPLC.
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A similar reaction was carried out with sterol IX (190 mg; 0.40 mmol) in chloroform (15 ml) and sulfuric acid (5 µl), with stirring for 15 min at room temperature. Workup and MPLC, as described above, gave XI (~180 mg; ~98% yield) as a white solid which was characterized by 1H NMR. The purity of XI was ~95%, accompanied by its 7,14,24 isomer (4%) and ~1–2% of unidentified material. In this case, none of the 6,8(14),24 or 8,14,25 isomers were detected.
4,4-Dimethyl-5-cholesta-8,14,24-trien-3ß-ol (I)
The 8,14,24-steryl acetate XI (40 mg; 0.088 mmol) was heated with 10% KOH in 95% ethanol (3 ml) for 2 h at 70°C. Water (3 ml) was added and the resulting mixture was extracted 4 times with MTBE (20 ml portions). The combined organic extracts were washed twice with brine (10 ml portions), dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was subjected to MPLC on a silica gel column (50 cm x 1 cm) using 2% acetone in hexane as the eluting solvent. Fractions (~20 ml) were collected. The contents of fractions 29–46 were combined to give, after evaporation of the solvent under reduced pressure, I (38 mg; 105% yield) as a white solid; ~96% purity by Ag+-HPLC and ~91–93% purity by 1H NMR (containing the 6,8(14),24 (1%), 8,14,25 (3%), and 7,14,24 (2%) isomers along with unidentified material (1–2%). Further purification by Ag+-HPLC (250 mm x 10 mm column; elution with 10% acetone in hexane) gave I (tR 47.0 min) accompanied by three minor components (tR values of 14.2, 36.6, and 140 min). Recrystallization of I gave an analytical sample; MP 135-137°C (lit. 119–121°C (24)); single component (Rf 0.80) on TLC (solvent, 40% ethyl acetate in hexane), a single component (tR 15.5 min) on reversed phase HPLC, and a purity of 99% on Ag+-HPLC (tR 18.6 min) using 9.1% acetone in hexane as the eluting solvent and a purity of 98–99% by 1H NMR; MS, 410* (100%; M+), 395* (33%; M–CH3), 377* (19%; M–CH3–H2O), 328* (11%; C23H36O), 325* (26%; C23H33O), 313* (11%; C22H33O), 298* (10%; C21H30O), 283 (12%), 265 (4%), and 246 (18%); high resolution MS, calcd. for C29H46O 410.3549, found 410.3555; UV, max 249 nm (
18,600); 1H NMR, Table 3; 13C NMR, Table 4.
Partial 1H NMR data for the minor components 4,4-dimethyl-5-cholesta-6,8(14),24-trien-3ß-ol and 4,4-dimethyl-5-cholesta-8,14,25-trien-3ß-ol, obtained by the Ag+-HPLC described above, were as follows.
4,4-Dimethyl-5-cholesta-6,8(14),24-trien-3ß-ol (~80% purity): 
0.678 (s, H–19), 0.826 (s, H–29), 0.888 (d, 0.6 Hz, H–18), 0.958 (d, 6.6 Hz, H–21), 1.063 (s, H–28), 1.602 (br s, H–27), 1.684 (br q, ~1.2 Hz, H–26), 1.896 (br t, ~2.8 Hz, H–5), 1.997 (dt, 12.5, 3.4 Hz, H–12ß), 3.301 (dd, 11.1, 2.5 Hz, H–3), 5.095 (br t, ~6.8 Hz, H–24), 5.616 (dd, 10.1, 2.3 Hz, H–6), 6.232 (dd, 10.1, 3.2 Hz, H–7).
4,4-Dimethyl-5-cholesta-8,14,25-trien-3ß-ol (~80% purity): 0.810 (d, 0.4 Hz, H–18), 0.832 (s, H–29), 0.945 (d, 6.6 Hz, H–21), 1.017 (s, H–28), 1.033 (d, 0.7 Hz, H–19), 1.217 (dd, 12.5, 2.1 Hz, H–5), 1.715 (br dd, 1.4, 0.9 Hz, H–27), 1.848 (dt, 13.0, 3.6 Hz, H–1ß), 2.348 (ddd, 16.0, 7.3, 3.2 Hz, H–16), 3.244 (dd, 11.8, 4.6 Hz, H–3), 4.667 (m, W1/2 ~5 Hz, H–26), 4.689 (m, W1/2 ~5 Hz, H–26), 5.357 (br dd, ~3, ~2 Hz, H–15).
3ß-Acetoxy-4,4-dimethyl-5-chola-8,14-dien-24-ol (XII), 3ß-acetoxy-4,4-dimethyl-5-chola-7,14-dien-24-ol (XIII), 3ß-acetoxy-4,4-dimethyl-5-chola-6,8(14)-dien-24-ol (XIV), and 3ß-acetoxy-4,4-dimethyl-5-chola-8(14),15-dien-24-ol (XV)
To the 3ß-acetate derivative of the 4,4-dimethyl-8(14)-15-keto-C24-3ß,24-diol VI (170 mg; 0.38 mmol) in ethanol (40 ml) was added sodium borohydride (500 mg; 13 mmol). After stirring for 2 h at room temperature, the mixture was poured into water (50 ml) and extracted 3 times with ethyl acetate (100 ml portions). The combined organic extracts were dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure to give 3ß-acetoxy-4,4-dimethyl-5-chol-8(14)-ene-15
,24-diol, which showed one major (~99%) component (Rf 0.67) and a minor (~1%) component (Rf 0.57) on TLC (solvent, 25% acetone in hexane). The 15,24-diol was dissolved in CHCl3 (50 ml) and treated with sulfuric acid (40 µl) for 15 min at room temperature. NaHCO3 (400 mg) was added and, after stirring for 2 min, the mixture was filtered through a short silica gel column (2 cm x 1 cm) using CHCl3 (50 ml) and acetone (20 ml) as the eluting solvents. The collected eluate was evaporated to dryness under reduced pressure, and the resulting residue was subjected to MPLC on a silica gel column (50 cm x 1 cm). Using 5% ethyl acetate in hexane as the eluting solvent, fractions (20 ml) were collected every 5 min. The contents of fractions 62 to 103 were combined to give, after evaporation of the solvent under reduced pressure, a white solid (163 mg). The crude product showed one component (Rf 0.4) on TLC (solvent, 25% acetone in hexane) and it showed a single component (tR 87.2 min) on capillary GC analysis of its TMS ether derivative on a 60 m DB-5 column. However, analysis by Ag+-HPLC (300 mm x 3.2 mm), with 15% acetone in hexane as the eluting solvent, showed 4 components with the following retention times: 22.0 min (4%), 25.6 min (3%), 41.1 min (82%), and 45.9 min (10%). 1H NMR analysis of the product also showed the presence of 4 components as indicated by methyl and olefinic proton chemical shifts corresponding to the following isomers: 8,14 XII (87%), 7,14 XIII (9%), 8(14),15 XV (3.5%), and 6,8(14) XIV (0.5%).
To resolve the individual isomers, the mixture was, in ~4-5 mg portions, subjected to semipreparative Ag+-HPLC (250 mm x 10 mm) using 15% acetone in hexane as the eluting solvent and to apply the sterol mixture to the column) at a flow rate of 3 ml per min. Elution of the individual components was monitored by UV (210 nm) and by analysis of aliquots of fractions (3 ml) on an analytical Ag+-HPLC column (300 mm x 3.2 mm) using 15% acetone in hexane as the eluting solvent. After a total of 40 injections, the following pure products were obtained (in order of elution): 6,8(14)-steryl acetate XIV (1 mg; 0.6% yield), 8(14),15-steryl acetate XV (5 mg; 3% yield), 8,14-steryl acetate XII (141 mg; 86% yield), and 7,14-steryl acetate XIII (14 mg; 8.5% yield). Each of the compounds showed a single component (99%) on analytical Ag+-HPLC (300 mm x 3.2 mm; solvent, 15% acetone in hexane). Retention times for XIV, XV, XII, and XIII were 22.0, 25.2, 41.1, and 45.1 min, respectively. Compound XII melted at 172.5-173°C. The MS of each of the compounds showed a molecular ion at m/z 428. High resolution MS for XIV, XV, XII, and XIII showed exact masses of 428.3288, 428.3294, 428.3291, and 428.3294, respectively; calcd. for C28H44O3 428.3290. The MS of XIV, XV, XII, and XIII, shown in Table 5, were similar in the high mass range, but with notable different relative abundances for a number of the ions. 1H NMR spectral data for each of the four isomers are presented in Table 1, and 13C NMR spectral data for XII and XIII are presented in Table 2.
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3ß-Acetoxy-4,4-dimethyl-5-chol-8(14)-en-24-ol (XVI), 3ß-acetoxy-4,4-dimethyl-5-chol-8-en-24-ol (XVII), and 3ß-acetoxy-4,4-dimethyl-5-chol-7-en-24-ol (XVIII)
The 8,14 diene XII (95 mg; 0.22 mmol) in ethyl acetate (20 ml; freshly distilled from K2CO3) was hydrogenated for 24 h at one atmosphere at room temperature over a freshly prepared (25) Raney nickel catalyst (5 g). The mixture was applied to a silica gel column (10 cm x 1 cm) which was washed with additional ethyl acetate (300 ml). The total eluant was evaporated to dryness under reduced pressure to give a white solid (92 mg) which showed a single component (Rf 0.4) on TLC (solvent, 25% ethyl acetate in hexane). GC–MS analysis of the TMS derivative of the crude product showed three components, each with molecular ions at m/z 502, with retention times of 87.6 min (50%), 92.3 min (43%), and 98.7 min (7%). Ag+-HPLC (300 mm x 3.2 mm; solvent, 9.1% 2-propanol in hexane at 1 ml per min) showed 3 components with retention times of 4.31 min, 6.15 min, and 7.16 min. 1H NMR analysis of the crude product showed the following chemical shifts for the upfield methyl region: 0.841 (8(14); 53%), 0.595 (8; 39%), and 0.523 (7; 8%). The crude product was subjected to semipreparative Ag+-HPLC (250 mm x 10 mm) using 5% acetone in hexane as the eluting solvent at 3 ml per min. Fractions (3 ml) were collected each min. The elution of the components was monitored by UV detection (210 nm) and by analytical Ag+-HPLC (300 mm x 3.2 mm) of aliquots of the fractions. After 25 injections, the 3 components were obtained in a pure state: the 8(14)-24-ol XVI (45 mg; 47% yield), MP 182.0–182.5°C; the 8-24-ol XVII (35 mg; 37% yield), MP 220.0–220.5°C; and the 7-24-ol XVIII (9 mg; 9% yield), MP 202.0–203.0°C. Compounds XVI, XVII, and XVIII each showed single components on Ag+-HPLC (300 mm x 3.2 mm; solvent 9.1% acetone in hexane) with tR values of 36.5 min, 40.9 min, and 48.9 min, respectively, and purities of 99% by 1H NMR. MS data for XVI, XVII, and XVIII are presented in Table 6, and high resolution MS showed 430.3460, 430.3450, and 430.3443, respectively (calcd. for C28H46O3 430.3447). 1H NMR and 13C NMR data for XVI, XVII, and XVIII are presented in Table 1 and Table 2, respectively.
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3ß-Acetoxy-4,4-dimethyl-5-chol-8-en-24-al (XIX) and 3ß-acetoxy-4,4-dimethyl-5-chol-8(14)-en-24-al (XX)
To a cooled (–50°C) solution of oxalyl chloride (0.5 ml; 1 mmol; 2 M in CH2Cl2) in CH2Cl2 (2 ml) was added dimethyl sulfoxide (0.15 ml; 2.1 mmol) in CH2Cl2 (0.2 ml) over 3 min under argon. After stirring for 2 min, the 8 24-ol XVII (29 mg; 0.068 mmol) in CH2Cl2 (1 ml) was added over 5 min. After stirring for 1 h, triethylamine (0.6 ml; 4.3 mmol) was slowly added. After stirring for 5 min, the mixture was warmed to room temperature, and a saturated solution of NH4Cl (3 ml) and MTBE (150 ml) was successively added. The separated MTBE layer was washed twice with a saturated NH4Cl solution (3 ml portions), three times with a saturated cupric sulfate solution (5 ml portions), and once with brine (5 ml), and then dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The resulting residue was applied to a silica gel column (10 cm x 1 cm) using hexane (10 ml) and then a mixture (3:10:87) of acetone, CH2Cl2, and hexane (100 ml) as the eluting solvents, and fractions (5 ml) were collected. The contents of fractions 7 to 10 were combined and evaporated to dryness under nitrogen to give the 8-C24-24-aldehyde XIX (26 mg; 90% yield); MP 213.5–214.5°C; single component (Rf 0.7) on TLC (solvent, 25% acetone in hexane), single component (tR 31.2 min) on Ag+-HPLC (300 mm x 3.2 mm; solvent, 3% acetone in hexane), and a purity of 99% by 1H NMR; MS, 428* (100%; M+), 413* (32%; M–CH3), 400* (23%; M–CO), 368* (28%; M–CH3COOH), 353* (44%; M–CH3COOH–CH3), 343* (24%; M–SC), 325 (22%), 301* (12%; C20H29O2), 283 (29%; M–SC–CH3COOH), 257 (22%), 241 (43%), 135* (71%; C10H15); high resolution MS, calcd. for C28H44O3 428.3290, found 428.3286; 1H NMR, Table 1; 13C NMR, Table 2.
Oxidation of the 8(14)-C24-24-ol XVI was carried out in a similar fashion. Oxalyl chloride (1.05 ml; 2.1 mmol; 2 M in CH2Cl2) in CH2Cl2 (3 ml) was cooled to -50°C under argon. Dimethyl sulfoxide (0.31 ml; 4.4 mmol) was added over 3 min. XVI (38 mg; 0.088 mmol) in CH2Cl2 (1 ml) was added over 5 min. After stirring for 1 h, triethylamine (1.14 ml; 8.2 mmol) was slowly added. The mixture was worked up as described above and then subjected to silica gel column (10 cm x 1 cm) chromatography using hexane (10 ml) and 5% acetone in hexane (100 ml) as the eluting solvent (fraction size, 5 ml). The contents of fractions 7 to 9 were combined to give, after evaporation of the solvent, the 8(14)-C24-24-aldehyde XX (35 mg; 92% yield); MP 162.5-163.5°C; single component (Rf 0.7) on TLC (solvent, 25% acetone in hexane), single component (tR 22.74 min) on Ag+-HPLC (300 mm x 3.2 mm; solvent, 3% acetone in hexane), and a purity of 99% by 1H NMR; MS, 428* (100%; M+), 413* (28%; M–CH3), 400* (20%; M–CO), 368* (23%; M–CH3COOH), 353* (32%; M–CH3COOH–CH3), 343* (23%; M–SC), 325 (18%), 301 (9%), 283* (25%; M–SC-CH3COOH), 257 (19%), 241 (34%), 135* (65%; C10H15); high resolution MS, calcd. for C28H44O3 428.3290, found 428.3289; 1H NMR, Table 1; 13C NMR, Table 2.
3ß-Acetoxy-4,4-dimethyl-5-cholesta-8,24-diene (XXI)
To isopropyltriphenylphosphonium iodide (840 mg; 1.94 mmol) in THF (5 ml) was added n-butyllithium (0.6 mmol) in hexane (380 µl) at 0°C under argon. After stirring for 15 min, an aliquot (600 µl) of the resulting mixture was added to XIX (21 mg; 0.049 mmol) in THF (1 ml) at -78°C. The mixture was warmed to 0°C and, after stirring for 2 h, poured into water (30 ml) and extracted 4 times with MTBE (50 ml portions). The combined organic extracts were washed once with brine (20 ml), dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was applied to a silica gel column (8 cm x 0.8 cm) and the column was eluted with a 1:1:50 mixture of acetone, CH2Cl2, and hexane (fraction size, 4 ml). The contents of fractions 2 to 6 were combined and evaporated to dryness under nitrogen to give XXI (23 mg; 103% yield); MP 137–139°C (lit. 133–134°C (26), 128–132°C (27)); single component (Rf 0.9) on TLC (solvent 25% acetone in hexane), single component (tR 28.56 min) on Ag+-HPLC (300 mm x 3.2 mm; solvent, 3% acetone in hexane), and a purity of 99% by 1H NMR; MS, 454* (100%; M+), 439 (25%; M–CH3), 394* (7%; M–CH3COOH), 379* (15%; M–CH3COOH–CH3), 341 (5%; M–SC–2H), 259 (10%), 257 (9%), 245 (6%), 241 (15%), and 135 (30%); high resolution MS, calcd. for C31H50O2 454.3811, found 454.3809; 1H NMR, Table 3; 13C NMR, Table 4.
4,4-Dimethyl-5-cholesta-8,24-dien-3ß-ol (II)
The 8,24 steryl acetate XXI (11 mg; 0.024 mmol) was heated with 15% KOH in 95% ethanol (3 ml) for 2 h at 60°C. Water (3 ml) was added, and the resulting mixture was extracted 4 times with MTBE (12 ml portions). The combined organic extracts were washed with brine (3 ml), dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was subjected to MPLC on a silica gel column (8 cm x 0.8 cm) using 4% acetone in hexane as the eluting solvent (fraction size, 3 ml). The contents of fractions 3 to 5 were combined and evaporated to dryness under nitrogen to give II (10 mg; 100% yield) as a white solid; MP 158–159°C (lit. 139.5–140°C (26), 136–138.5°C (27), 128–129°C (24)); single component (Rf 0.4) on TLC (solvent, 25% acetone in hexane), and a purity of 99% by 1H NMR; MS, Figure 4; high resolution MS, calcd. for C29H48O 412.3705, found 412.3702; 1H NMR, Table 3; 13C NMR, Table 4.
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4,4-Dimethyl-5-cholesta-8(14),24-dien-3ß-ol (XXII)
The 8(14)-C24-24-aldehyde XX (29 mg; 0.068 mmol) was treated with the Wittig reagent and the crude product was isolated and subjected to silica gel column chromatography as described above for the preparation of the 8,24 diene XXI (except for the use of an older sample of the isopropyltriphenylphosphonium iodide employed in the preparation of the Wittig reagent). The crude product, a mixture of XXII and its acetate and acetoacetate derivatives (as indicated by 1H and 13C NMR), was heated with 15% KOH in 95% ethanol (3 ml) for 2 h at 60°C. Water (3 ml) was added and the resulting mixture was extracted 4 times with MTBE (12 ml portions). The combined organic extracts were washed with brine (3 ml), dried over anhydrous sodium sulfate, and evaporated to dryness under nitrogen to give XXII (10 mg; 36% yield) as a white solid; MP 140–141°C; single component (Rf 0.4) on TLC (solvent, 25% acetone in hexane), and a purity of 99% by 1H NMR; MS, Figure 4; high resolution MS, calcd. for C29H48O 412.3705, found 412.3701; 1H NMR, Table 3; 13C NMR, Table 4.
Chromatography of 3ß-acetate derivatives of 4,4-dimethylsterols
Presented in Table 7 are capillary GC and Ag+-HPLC data for the acetate derivatives of unsaturated 4,4-dimethylsterols with double bonds in the 5, 5,7, 8,24, 6,8(14), 7,14, 8,14, and 8,14,24 positions. Also presented are previously reported (28) TLC data for some of the same compounds on silica gel-AgNO3 plates. Whereas a number of the compounds showed clearly different retention times on GC, essentially no separation of the 8,14 and 5,7 isomers was observed and only partial resolution of the same compounds from the 7,14 isomer was observed. Ag+-HPLC was found to be considerably more powerful for the separation of the concerned compounds with the exception of the case of the 5,7 and 8,24 isomers. The value of the Ag+-HPLC over simple TLC on silica gel-AgNO3 plates is illustrated by the lack of any resolution of the 8,14 and 7,14 isomers in the latter system.
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As noted previously, Ag+-HPLC also proved extremely useful in the purification of the 3ß-acetate derivatives of unsaturated 3ß,24-dihydroxy C24 compounds. Retention times (min) for the 6,8(14), 8(14),15, 8,14, and 7,14 diunsaturated steroids on Ag+-HPLC (300 mm x 3.2 mm; solvent, 15% acetone in hexane) were 22.0, 25.2, 41.1, and 45.9, respectively. Using 9.1% acetone in hexane as the eluting solvent, retention times (min) for the 8(14), 8, and 7 monounsaturated isomers were 36.5, 40.9, and 48.9, respectively.
Effects of synthetic sterols on meiosis
In the first experiment ( Figure 5, panel A), 49% of the oocytes underwent germinal vesicle breakdown (GVB) in medium containing 3.5 mM hypoxanthine. In contrast, 85% and 73% of the oocytes cultured under the same conditions, but treated at a concentration of 3 µg/ml, with the 4,4-dimethyl-8,14 sterol (III) or the 4,4-dimethyl-7,14 sterol (IV), respectively, underwent GVB. However, two other 4,4-dimethylsterols, i.e., 4,4-dimethylcholesta-5,7-dien-3ß-ol and 4,4-dimethylcholest-5-en-3ß-ol, did not promote the resumption of meiosis. Moreover, the 20R,22R- and 20S,22S- isomers of 20,22-dihydroxycholesterol and the 22R- and 22S-isomers of 22-hydroxycholesterol had no significant effect on meiosis under the same conditions. In the second experiment (Figure 5, panel B), both 4,4-dimethyl-5-cholesta-8,24-dien-3ß-ol (II) and 4,4-dimethyl-5-cholesta-8(14),24-dien-3ß-ol (XXII) were found to promote GVB. In the third experiment ( Figure 6), the 4,4-dimethyl-8,14-sterol (III) and the 4,4-dimethyl-7,14-sterol (IV), at 3 µg/ml, had a significant effect on meiosis, as did the 4,4-dimethyl-8,14,24-sterol (I). At this concentration, the extent of GVB was significantly higher with I than with III or IV.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURES AND...DISCUSSIONREFERENCES |
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In 1995, Byskov et al. (1) reported that four 4,4-dimethylsterols, i.e., 4,4-dimethyl-5-cholesta-8,14,24-trien-3ß-ol (I), 4,4-dimethyl-5-cholesta-8,24-dien-3ß-ol (II), 4,4-dimethyl-5-cholesta-8,14-dien-3ß-ol (III), and 4,4-dimethyl-5-cholest-8-en-3ß-ol, affected meiosis in cultured cumulus-enclosed and naked mouse oocytes. Compounds I and II were isolated from human follicular fluid and bull testicular tissue, respectively (see below). The other two sterols, containing 8,14 and 8 unsaturated moieties, were prepared by chemical synthesis and were reported to show purities of 98.5% and 97.8%, respectively, on the basis of reversed phase HPLC and MS data. These results alone would not, in our experience, preclude the presence of closely related double bond isomers. However, it was stated that 1H and 13C NMR spectral data were those expected. For evaluation of their actions on the cultured oocytes, the sterols were reported to have been sonicated (3 times for 1 min) in culture medium prior to addition to the cells. This procedure, which could easily result in the formation of oxidation products of the various unsaturated sterols, was used because "the material could not be dissolved in alcohol," the latter observation being, in our experience, unexpected for the concerned sterols (and confirmed herein). In determination of the meiosis activation by the various sterols, considerable experimental variation was encountered. Lanosterol, cholesterol, and ergosterol were reported to result in no activation of meiosis under the conditions studied. In 1998, Byskov et al. (29) reported that two other sterols, i.e., 4ß-methyl-5-cholesta-8,24-dien-3ß-ol and 5-cholesta-8,24-dien-3ß-ol, also affected meiosis in oocytes.
The 4,4-dimethylsterols I, II, and III, along with 4,4-dimethyl-5-cholest-8-en-3ß-ol are potential intermediates in sterol biosynthesis (8). I and III are products of the 14-demethylation of lanosterol and 24,25-dihydrolanosterol, respectively (2) (4) (5) (6) (7). Moreover, and of potential relevance to the matter of the reported effects of these sterols on meiosis, the conversion of lanosterol to I by rat ovary has recently been reported to be stimulated by gonadotropin of pregnant mare's serum (9). It should also be noted that I has been reported to activate gene expression for the nuclear orphan receptor LXR (10).
For exploration of these interesting and potentially important matters, we have pursued the chemical syntheses of 4,4-dimethylsterols I and II. We (21) and others (30) (31) have previously described the preparation of the 4,4-dimethyl-8,14-sterol III. The syntheses of I and II represent more formidable challenges, requiring the introduction of the 4,4-dimethyl functionality, the provision of the 8,14 and 8 double bonds of I and II, respectively, and the introduction of the 24 double bond. A number of synthetic routes to I and II can be envisioned with different orders of introduction of the various functionalities. However, considerations of economy and practicality dictate evaluation of the availability and costs of potential starting materials. Cholesterol, available at relatively low cost from commercial sources, can be readily obtained in a high state of purity. Ergosterol and various plant sterols are more costly and more difficult to obtain in a high state of purity.
Our synthesis of I (Figure 2) used the 4,4-dimethyl-8(14)-15-ketosteryl ester V, obtained from cholesterol, as the starting material (21). A critical step in the synthesis is based on our development of conditions permitting a remarkably efficient and specific oxidation of the saturated side chain of 3ß-acetoxy-5-cholest-8(14)-en-15-one with a mixture of trifluoroacetic anhydride, hydrogen peroxide, and sulfuric acid to give 3ß-acetoxy-24-hydroxy-5-chol-8(14)-en-15-one (14) (32), a C24 synthon already protected at C-3 from which the desired C8 side chain with various substituents in the side chain can be constructed (15) (19) (33). Oxidation of the 4,4-dimethyl-8(14)-15-ketosteryl ester V proceeded in gratifyingly high (73%) yield to give the C24 24-hydroxy-8(14)-15-ketosteryl acetate (VI) from which the corresponding aldehyde VII was prepared in high (90%) yield. Wittig olefination of VII with isopropyltriphenylphosphonium iodide and butyllithium gave the 8(14),24-15-ketosteryl ester VIII in 75% yield. Reduction of VIII with sodium borohydride gave a mixture which was resolved by silica gel MPLC to give the 15ß-hydroxy-8(14)-steryl ester IX (81% yield) and the corresponding 15-hydroxy isomer (5% yield). Acid treatment of IX gave, after semipreparative Ag+-HPLC, the 8,14,24-steryl acetate XI in 85% yield. Saponification of XI gave, after purification by silica gel MPLC, I in essentially quantitative yield.
The 4,4-dimethyl-8,24 sterol II was prepared as outlined in Figure 3. The C24 24-hydroxy-4,4-dimethyl-8(14)-15-ketosteryl acetate VI was reduced with sodium borohydride to give the corresponding 15,24-dihydroxy compound which, upon treatment with acid, gave a mixture of four dienes XII-XV as judged by Ag+-HPLC and 1H NMR. Purification by Ag+-HPLC resolved the desired 8,14 acetate XII (86% yield) and three other components, i.e., the 7,14 isomer XIII (8.5% yield), the 6,8(14) isomer XIV (0.6% yield), and the 8(14),15 isomer XV (3% yield). Catalytic hydrogenation of the 8,14 acetate XII gave a mixture of 3 isomeric components as judged by GC–MS, Ag+-HPLC, and 1H NMR. Ag+-HPLC permitted the isolation of the 3 components: 8 isomer XVII (37% yield), 8(14) isomer XVI (47% yield), and the 7 isomer XVIII (9% yield). The C24 8-24-hydroxy compound XVII was oxidized to the corresponding aldehyde XIX (90% yield) which, upon Wittig olefination, gave the 8,24 steryl acetate XXI in essentially quantitative yield. Saponification of XXI gave the desired 8,24 sterol II (100% yield). The overall yield for the conversion of the C27 4,4-dimethyl-8(14)-15-ketosteryl acetate V to the 4,4-dimethyl-8,24-sterol II was 21%.
Oxidation of the 8(14)-24-hydroxy compound XVI to the corresponding aldehyde XX followed by Wittig olefination gave, after saponification, the previously undescribed 4,4-dimethyl-8(14),24 sterol XXII.
The starting material V and the intermediates and major byproducts in the syntheses of 8,14,24-, 8,24-, and 8(14),24-4,4-dimethylsterols (I, II, and XXII) were characterized by melting point, MS, high resolution MS, 1H and 13C NMR, and, when appropriate, UV spectral analysis. Minor byproducts were, in general, characterized only by NMR supplemented, in some cases, by MS.
The MS of compounds V, VI, VII, and VIII showed the expected ions in the high mass region along with ions characteristic of 8(14)-15-ketosteroids (13) (14) (15) (16) (17) (18) (19). The MS of the 3ß-acetates of the isomeric 4,4-dimethyl diunsaturated C24 24-alcohols containing double bonds in the 8,14 (XII), 7,14 (XIII), 6,8(14) (XIV), and 8(14),25 (XV) positions were very similar, differing only in the relative abundances of the individual ions with the 6,8(14) compound XIV being least similar in relative ion abundances to the other isomers (Table 5). The MS of the 3ß-acetates of the isomeric 4,4-dimethyl monounsaturated C24 24-alcohols containing double bonds in the 8(14) (XVI), 8 (XVII), and 7 (XVIII) positions were very similar, differing only in the relative abundances of the individual ions and with virtually identical MS for the 8(14) and 8 isomers (XVI and XVII, respectively). It is also noteworthy that the MS of the 3ß-acetates of 8(14) and 8 isomers of the 4,4-dimethyl monounsaturated C24 aldehydes (XIX and XX, respectively) were essentially identical as were the MS of the 8,24 and 8(14),24 isomers of the 4,4-dimethyl C27 sterols, II and XXII, respectively (Figure 4). These findings clearly demonstrate that MS alone provides no basis for distinguishing between the 4,4-dimethylsterols with double bonds in the 8,24 and 8(14),24 positions.
Table 1 Table 2 Table 3 Table 4 present high precision 13C and 1H NMR chemical shift data and assignments for each of the carbon atoms and protons of the starting material (V) and of each of the intermediates and major byproducts in the chemical syntheses of I, II, and XXII. These chemical shift data and individual assignments not only provide important structural information for the concerned compounds but also provide a basis for comparisons with previously reported data and with data to be obtained for isolated or synthetic sterols in this series.
In 1989, Dolle et al. (24) described the synthesis of I and II from the benzoate ester of 3ß-hydroxy-4,4-dimethylergosta-8(14),22-dien-15-one which itself can be prepared from ergosterol (34). The 8(14),22-15-ketosteryl ester was converted to I through an eleven-step synthesis, which involved production of the corresponding C-22 aldehyde by ozonolysis, elaboration of the desired C8 side chain with oxygen functions at C-22 and C-24, removal of the oxygen functionality at C-22, generation of the 8,14 diene system, and finally, introduction of the 24 double bond from the trifluoroacetate ester of the 4,4-dimethyl-8,14-24-hydroxysterol. The reported overall yield of I from the 4,4-dimethyl-8(14),22-15-ketosteryl ester was high (~53%). However, many intermediates were not isolated and characterized. Moreover, the melting point of I reported (119–121°C) was notably lower than that reported herein (135–137°C). Limited spectral data for I were presented. 1H NMR data, obtained at lower precision than those described herein (Table 3), were presented for the following protons: 3, 15, 18, 19, 21, 24, 26, 27, 28, and 29. The chemical shifts and assignments were in general agreement with those presented in Table 3 with the exception of an interchange of the assignments for H-18 and H-19. The synthesis of II described by Dolle et al. (24) was also a multistep synthesis in which many intermediates were not isolated and characterized. The overall yield of II from the 8(14),22-15-ketosteryl ester was ~18%. The reported melting point for II (128–129°C) was considerably less than that reported herein (158–159°C). Very limited spectral data were presented for II. 1H NMR data were reported only for the 3 ( 3.30) and 24 ( 5.21) protons which were not in good agreement with the data for II presented herein (Table 3). Maitra, Mohan, and Sprinson (35) previously described the preparation of labeled II by incubation of labeled mevalonate with 10,000 x supernatant fraction of rat liver homogenate preparations in the presence of added arsenite and lanosterol. The isolated biosynthetic II was characterized as its acetate derivative by its MS with major ions at m/z 454, 439, 394, 379, and 341 in the high region of the spectrum (as described herein) and by 1H NMR with reported peaks and assignments at 0.59 (18–CH3), 0.88 (19–CH3 and 4–CH3), 0.94 (21–CH3), 1.01 (4ß–CH3), 1.60, 1.68 (26– and 27–CH3), and 2.05 (CH3CO2). Our results (Table 3) indicate that the assignments of Maitra et al. (35) for H–19 and H–29 (4ß–CH3) should be reversed.
The 4,4-dimethyl-8,14,24 sterol I (designated by Byskov et al. (1) as FF-MAS) and the 4,4-dimethyl-8,24 sterol II (designated as T-MAS) were reported to have been obtained by n-heptane extraction of human follicular fluid and bull testicular tissue, respectively, followed by three HPLC purification steps (two reversed phase and one normal phase). Evidence for the structure of the 4,4-dimethyl-8,14,24 sterol I was limited to presentation of its mass spectrum, but with no comparison with that of an authentic sample. Insufficient material was available for NMR analysis. Evidence for the structure of the 4,4-dimethyl-8,24 sterol II was based upon the combination of its mass spectrum and the results of 1H and 13C NMR, again without comparisons with data for an authentic sample. The NMR data for II were reported to indicate the presence of two impurities (~10% lanosterol and 1% of material of unknown structure). Only 13C NMR data for II were presented. The assignment of structure to II was based upon comparison of its 13C NMR spectral data with our previously presented data for lanosterol (36) and zymosterol (5-cholesta-8,24-dien-3ß-ol) (37). The availability of synthetic II, as described herein, permits comparison of the 13C NMR data reported for the isolated sample of II (Table 4) with data obtained at higher precision for synthetic II, prepared in a high state of purity as described herein. The data for the two samples are in reasonable agreement with the exception of the signal at 29.7 which was assigned by Byskov et al. (1) to C-12. The signal at 29.7 corresponds to that observed in compounds with a long-chain alkane moiety.
As noted previously, Byskov et al. (29) reported that 4ß-methyl-5-cholesta-8,24-dien-3ß-ol activated meiosis in oocytes. This sterol was reportedly isolated after incubation of a yeast, Kluyveromyces bulgaricus A3410, in the presence of amphotericin B (29). The sterol in question was isolated by HPLC. Evidence for its identity was based upon MS, with its MS data reported as "identical with those of 4ß-methylzymosterol as recorded in the National Bureau of Standards library." To our knowledge, the only previous descriptions of the 4ß-methyl-8,24 sterol have been those concerning a 4-methyl-8,24 sterol isolated from the skin of rats treated with triparanol (38) (39) (40). It should be noted that a subsequent study (41) demonstrated that the physical data presented for the sterol isolated from skin corresponds to the 4-methyl isomer, i.e., 4-methyl-5-cholesta-8, 24-dien-3ß-ol.
The availability of the synthetic 4,4-dimethylsterols with double bonds in the 8,14,24, 8,24, 8(14),24, 8,14, 7,14, 5,7, and 5 positions permitted evaluation of their effects on meiosis in mouse oocytes. The synthetic 8,14,24 and 8,24 sterols (I and II, respectively) caused a resumption of meiosis as noted for the isolated sterols by Byskov et al. (1), as did the 4,4-dimethyl-8,14 sterol III. We also report, for the first time, that the corresponding 4,4-dimethyl-7,14 and 8(14),24 sterols (IV and XXII, res
