438
SYNTHESIS OF SPECIFICALLY DEUTERIUM-LABELLED
PREGNANOLONE AND
PREGNANEDIOL SULPHATES FOR METABOLIC STUDIES IN HUMANS
T.A. Baillie (1) and J. Sjovall Department of Chemistry, Karolinska Institutet S-104 01 Stockholm 60, Sweden and J.E. Herz Department of Chemistry, Centro de Investigation y de Estudios Avanzados de1 IPN, Apartado Postal 14-740, Mexico 14, D.F.
Received:
B/4/75
ABSTRACT A synthesis is reported of 3B-hydroxy-5a-pregnan-ZO-one sulphate and the disulphate and 3-monosulphate of 5a-pregnane-3B,ZOa-diol, labelled specifically with deuterium in high isotopic purity for metabolic studies in humans. Base-catalyzed equilibration of 3B-hydroxy-5a-25R-spirostan-12-one (hecogenin, II) with deuterium oxide, followed by removal of the 12-keto group and degradation of the sapogenin side-chain afforded 38-hydroxy5a-[ll,ll-2H2]pregn-16-en-ZO-one (VII). Further deuterium atoms were introduced at the 3a and 2Op positions by reductions with sodium borodeuteride and lithium aluminum deuteride, respectively. These reactions led to 3B-hydroxy-5a-[3a ll,ll-2Hapregnan-ZO-one (X; isotopic purity 87.2%) and 5a-[3a,11,11,20f3-~H~pregnane-3B,20a-diol (XIV; isotopic purity 83.9%). The 3-sulphate of the pregnanolone and the 3,20-disulphate of the pregnanediol were prepared directly from the free alcohols, while the 3-monosulphate of the pregnanediol was obtained via 5a-[3a,ll,ll,ZOB-2H~pregnane-36,20a-diol 20-acetate (XVII). INTRODUCTION The occurrence and quantitative
importance in late pregnancy plasma
of isomeric pregnanolone sulphates and mono- and disulphated pregnanediols has been demonstrated by Sjijvall and co-workers (2,3). Although these compounds are assumed to be metabolites of progesterone, nothing is known about their pool sizes, turnover rates or metabolic interrela-
Vohme
26,
Number 4
S
TDROIDU
October, 2975
439
S
TDICOIDb
tionships. As an initial step in establishing the physiological significance of steroid sulphates in plasma of pregnant women, it was decided to carry out a detailed study of their metabolism in healthy subjects. For ethical reasons, it is not possible to administer radioactive tracers in such investigations. Since information was required both on the turnover of the steroid skeleton and on oxidoreduction of hydroxyl groups, labelling with deuterium atoms in specific positions and analysis by gas chromatography-mass
spectrometry
(GC-MS) was considered to be the method
of choice. This paper describes the synthesis of specifically-deuterated analogues of a series of structurally related C2,02 steroid sulphates encountered in pregnancy plasma, namely 3B-hydroxy-5o-pregnan-ZO-one phate, 5a-pregnane-3B,20a-diol
sul-
3-monosulphate and 5a-pregnane-3B,20a-diol
3,20-disulphate. EXPERIMENTAL SECTION GENERAL Melting points were determined on a Gallenkamp hot-stage apparatus and are corrected. Welting points quoted from the literature refer to the corresponding unlabelled compounds. Infrared spectra were recorded, either on solutions in carbon tetrachloride or on discs of potassium bromide, using a Perkin-Elmer 254 Infrared Spectrophotometer. Ultraviolet spectra were obtained with a Zeiss Model DMR 21 Recording Spectrophotometer. Elemental microanalyses were carried out at the Chemistry Department, University of Glasgow, Scotland. Thin-layer chromatography (TLC) was carried out using glass plates precoated with silica gel 60 F254 (Merck AG, Darmstadt, Germany). For analytical separations, 0.25 mm layers (5 x 20 cm plates) were used, while 2 mm layers (20 x 20 cm plates) were employed for preparative work. The following solvent systems were used (mixtures expressed as ratios by volume): system A, chloroform/methanol (95:5); system B, ethyl acetate/ chloroform/methanol (15:5:1); system C, ethanol/ethyl acetate/25% aqueous ammonium hydroxide (5:5:1). The compound zones were visualized by UV light ( A = 254 nm) or by exposure to iodine vapour. Analytical plates were subsequently sprayed with a vanillin-sulphuric acid solution and heated for 5 min at 1100.
S
440
~~EOID6
Liquid-gel chromatography was carried out using LipidexR-1000 and LipidexK-5000 stationary phases (Packard International, S.A., Zurich). Gas-liquid chromatography was performed with a Pye Series 104 instrument, equipped with 2 m x 3 mm i.d. glass columns packed with 1.5% SE-30 on Gas Chrom Q, loo-120 mesh (Applied Science Laboratories, Inc.). Flame ionization detectors were used, with nitrogen as carrier gas (flow rate 38 ml/min). Hydroxysteroids were analyzed as their trimethylsilyl (TMS) ether derivatives (4) and all retention times, tR, were measured relative to that of 5ol-cholestane (tR = 1.0). Mass spectrometry. A modified LKB 9000 instrument was used (5), equipped with a 3 m x 3 mm i.d. qlass column packed with 1.5% SE-30 on Gas Chrom Q, loo-120 mesh, with helium as the carrier gas. Analyses were carried out with an oven temperature of 2100, while the molecular separator and ion source were held at 2500 and 2900, respectively. The energy of the bombarding electrons was 22.5 eV and the ionizing current was 60 vamp. Multiple spectra were recorded on magnetic tape and average isotope content was determined by comparison with the unlabelled reference compounds using an IBM 1800 computer (6). Reagents. Deuterium-labelled solvents and reagents were as follows: deuterium oxide (99.8%; Norsk Hydro, Oslo, Norway), monodeuteromethanol (CH302H, 2 99%; Merck AG, Darmstadt, Germany), lithium aluminum deuteride ( 2 99%; Fluka AG, Buchs, Switzerland) and sodium borodeuteride ( 2 98%; Merck AG, Darmstadt, Germany). All other chemicals were used as purchased and were reagent grade where available. SYNTHESIS Hecogenin (3B-Hydroxy-5a-25R-spirostan-12-one;
II).
Hecogenin acetate (I; 13.2 g; 28.0 mmole) was hydrolyzed at room temperature overnight with 2% methanolic potassium hydroxide (400 ml) to give II (11.3 g; 94%). Recrystallization from methanol afforded large plates, m.p. 268-270° (lit. (7) 2680). Anal. Calcd. for C27H4204: C, 75.31; H, 9.83%. Found: C, 75.10; H, 9.7Or 3-Q,11,11-2H,
Hecogenin (III).
To a stirred mixture of monodeuteromethanol (500 ml) and deuterium oxide (12.3 ml) under a nitrogen atmosphere was added 1.31 g (57 mmole)of sodium metal in small pieces. Hecogenin (II; 11.2 g; 26.0 mmole) was added and the resulting mixture heated under reflux for 48 h. On cooling to 00, the product crystallized from solution. This material was not isolated but employed directly for tosylhydrazone formation. 11,11,23,23-ZH4
Hecogenin Tosylhydrazone
(IV).
The above slurry of labelled hecogenin (III) and monodeuteromethanol at Oo was made acidic by dropwise addition of acetyl chloride (7.4 ml). The resulting mixture was allowed to warm up to room temperature, treated with tosyl hydrazine (16.7 g; 82.6 mmole) and heated under reflux for 21 h when TLC analysis of the product indicated that reaction was complete (Rf of product 0.35, Rf of hecogenin 0.45 in system A). The mixture was then cooled to Oo and the tosylhydrazone IV filtered off and dried to
441
s
TPlICOIDb
afford 11.9 g (74%). A second crop (2.65 g) brought the yield of IV to 91%. An analytical sample, prepared by recrystallization from methanol, had m.p. 262-264O (lit. (8) 259-2600). UV(EtOH): Xmax = 226 nm ( E = 23,400). IR (KBr): 3615 (m), 3500 (m), 3212 (m), 3060 (w), 2215 (w), 2135 (w), 1648 (m), 1600 (m), 1450 (s), 1325 (s), 1170 (s), 1035 (s), 980 (s), 898 (m), 850 (m) and 820 cm-l (s). Anal. Calcd. for C34H462HqN205S: C, 67.77; H, 8.97; N, 4.65%. Found: C,r48; H, 8.99; N, 4.43%. ll,11,23,23-2H4 Tigogenin
(5a-nl,ll,23,23-2H4]-25R-Spirostan-3B-ol;
V).
The tosylhydrazone IV (4.54 g; 7.37 mmole) was dissolved in methanol (700 ml) and the solution treated with sodium borohydride (4.50 g; 119 mmole), added in portions with water-bath (room temperature) cooling. The reaction mixture was heated under reflux for 15 h, cooled and examined by TLC (system A). This indicated that only partial reduction of IV had taken place (two spots, with Rf 0.36 and 0.47), whereupon a further 4.5 g of sodium borohydride was added and reflux continued for a second period of 15 h. A third, and final, addition of sodium borohydride (4.5 g) and subsequent 15 h reflux was necessary to achieve complete reduction of IV, as judged by TLC (product gave single spot, Rf 0.47). The reaction product was concentrated in vacua until precipitation commenced, diluted with 2 N hydrochloric aciamml) and extracted with ether (3 x 350 ml). The combined ether extracts were washed with water, dried over magnesium sulphate and evaporated. Recrystallization of the crude product from acetone gave 1.73 g (56%) of V as needles, m.p. 203.5-2060 (lit. (7) 203-2060 . IR (CC14): 3625 (m), 2210 (w), 2130 (w), 1040 (s), 983 (m) and 888 cm- 1 (m). MS (TMS ether): m/e 492 (M+), 477, 421, 420, 376, 361, 347, 286, 271, 257, 141 (base peam 122 and 117. Anal. Calcd. for C27H402H403: C, 77.14; H, 11.40%. Found: C, 77.19; H, 117nr%. A further 9.86 g (16.0 mmole) of IV was similarly reduced, in two batches, and recrystallized as above to afford an additional 3.17 g (47%) of labelled tigogenin. 11,11,23,23-2H4 Tigogenin Acetate (VI). Acetylation of V (4.79 g; 11.4 mmole) was carried out at room temperature overnight with pyridinelacetic anhydride (l:l, 60 ml). The product (4.09 g; 78%) crystallized from ethanol as prisms, m.p. 212-2140 (lit. (7) 2032060). IR (CC14): 2210 (w), 2130 (w), 1735 (s), 1245 (s), 1040 (s), 982 (m), 902 (w) and 887 cm-l (m). MS: m/e 462 (M+), 447, 402, 401, 391, 390, 346, 331, 317, 286, 271, 257, 141 (base peak), 122 and 117. Anal. Calcd. for C2gH422H404: C, 75.32; H, 10.82%. Found: C, 75.08; H, llm. 38-Hydroxy-5a-bl,11-2HP]pregn-16-en-20-one
(VII).
The acetate VI (3.25 g; 7.02 mmole) was subjected to the three-step degradation sequence described by Wall et al. (9) in which pyridine hydrochloride is employed as catalyst for fie-@-eparation of the pseudosapogenin acetate. The crude product from this sequence of reactions was a pale yellow oil which solidified slowly on standing. Recrystallization from aqueous ethanol afforded VII (1.07 g; 48%) as small, yellowish crystals, m.p. 190-1950 (lit. (10) 200-2040). IR (CC14): 3620 (m) 3055 (w), 2210 (w), 2120 (w), 1672 (s), 1590 (m), 1233 (m) and 1041 cm-1 (s). An analytical sample of VII was prepared by preparative TLC (solvent system B) followed by two recrystallizations from aqueous ethanol to give colour-
S
442
TDROIDB
less needles, m.p. 201-2050. UV (EtOH):X max = 239 nm (& = 8,700). MS (TMS ether): m/e 390 (M+), 375, 347, 285, 257 (base peak) and 163. Anal. Calcd. for C2mO2H202: C, 79.24; H, 10.69%. Found: C, 79.25; H, 1Om. 3B-Hydroxy-5a-~l,ll,2H2]pregnan-20-one
(VIII).
The pregnenolone VII (196 mg; 0.616 mmole) in ethyl acetate (10 ml) was hydrogenated (7) for 1 h at 1 atm. pressure over 10% palladium-charcoal (196 mg). The catalyst was filtered off and the solvent evaporated to give VIII (188 mg; 95%). Recrystallization from ethanol afforded plates, m.p. 196-197.50 (lit. (9) 194-1960). IR (CC14): 3624 (m), 2215 (w), 2125 (w), 1708 (s) and 1042 cm-l (s). MS (TMS ether): m/e 392 (M+), 377 155. Anal. Calcd. (base peak , 335, 334, 302, 287, 263, 259, 245, 2rand for C2lH32 h H202: C, 78.75; H, 11.20%. Found: C, 79.13; H, 1l.m 5a-hl,ll-2H2]Pregnane-3,20-dione
(IX).
Oxidation of the pregnanolone VIII (168 mg; 0.525 mmole) with Jones reagent (11) afforded 158 mg (95%) of IX, which was recrystallized from acetone to give plates, m.p. 196.5-198.50 (lit. (12) 2000). IR (CC14): 2212 (w), 2122 (w) and 1709 cm-l (s). MS: m/e 318 (M+), 303, 300, 285, 274, 271, 260, 233 and 84 (base peak). AnarCalcd. for C21H302H202: C, 79.19; H, 11.30%. Found: C, 79.19; H, lTZY%. 3B-Hydroxy-5a-~a,ll,11-2H~pregnan-20-one
(X).
The pregnanedione IX (150 mg; 0.472 mmole) was reduced with sodium borodeuteride (19.7 mg; 0.471 mmole) in absolute ethanol (20 ml), according to the method of Mancera et al. (13). TLC analysis of the product indicated complete reduction ??Ffie starting material (Rf 0.62 in system A) to a mixture of the 3a and 36-pregnanolones (Rf 0.53 and 0.46, respectively), while trace amounts of pregnanediol isomers (Rf approx. 0.35) were also detected. The product was chromatographed on a column of LipidexR-5000 (40 g), prepared in and eluted with hexane/chloroform (8:2). Compound X (75 mg; 50%) was eluted between 175-220 ml of effluent and recrystallized from ethanol to give plates, m.p. 196-197.5O (lit. (9) 194-1960). IR (CC14): 3620 (m), 2210 (w), 2124 (w), 1708 (s) and 1064 cm-l (m). MS (TMS ether): 393 (M+), 378 (base peak), 336 335, 303, 288, 263, 260, 245, 218 and 156. Anal. Calcd. for C21H312H302: C, 78.50; H, 11.52%. Found: C, 78.77; H, TOGO%. Potassium 3f3-Hydroxy-5a-~a,ll,ll-2H~lpregnan-20-one
3-sulphate (XI).
The pregnanolone X (10.0 mg; 0.031 mmole) and N,N'-dicyclohexylcarbodiimide (37.8 mg; 0.183 mmole) were dissolved in-anhydrous dimethylformamide (0.27 ml) and the solution cooled to 00. 70 1.11 of a mixture of cont. sulphuric acid (35 ~1) and anhydrous dimethylformamide (0.60), which had been previously cooled to ice-bath temperature,was added and the mixture held at Oo for 1 h. The reaction mixture was then treated with 75% aqueous ethanol (2 ml), neutralized with 2 N potassium hydroxide (3 drops) and centrifuged. The supernatant was removed and the residue washed three times with aqueous ethanol (2 ml). The combined washings were added to the above supernatant and the mixture taken to dryness -in vacua. The residue was chromatographed on an 8 g column of
443
S
TDEOID1
Sephadex LH-20 (solvent system: methanol/chloroform, 1:1, saturated with potassium chloride (14))and 5 ml fractions were collected. TLC analysis indicated the presence of a monosulphate conjugate (Rf 0.40 in system C) in fractions 10-16, which were combined and taken to dryness. The residue was dissolved in ethanol (10.0 ml) and an aliquot (20 ~1) taken for solvolysis in acidified ethyl acetate (15). Following the addition of 5B-cholane-3a,24-dial (30 vg) as internal standard, the product from solvolysis was converted into its TMS ether derivative and analyzed by GLC. Quantitation of the solvolyzed steroid was based on GLC peak area measurements, which indicated a yield of the sulphate XI from X of 10.1 mg (74%). A sample of XI, free from potassium chloride, was obtained by chromatography on Amberlite XAD-2 (16), followed by recrystallization from methanol. This gave small needles, m.p. 229-231° (dec.) (lit. (17) 207-2100). IR (KBr): 2210 (w), 2120 (w), 1708 (s), 1250-1220 (s), 1072 (s), 1033 (s) and 955 cm-l (s). 5a-~11,11-2H~~Pregn-16-ene-3,20-dione
(XII).
The pregnenolone VII (301 mg; 0.947 mmole) was oxidized with Jones reagent (11) to give XII as a finely divided white solid (222 mg; 74%), m.p. 203-210° (lit. (18) 210-2120). IR (CC14): 3045 (w), 2215 (w), 2130 w), 1718 (s), 1672 (s) and 1591 cm-l (m). UV (EtOH): A max = 239 nm m/e 316 (M+), 301, 283, 273 (base peak), 255, 191 t E = 7,100). MS: -and 163. This material was not purified but used directly for reduction. 5a-~5,11,11,20~-2H4~Pregn-16-ene-3~,20~-diols
(XIII).
Reduction of XII (200 mg; 0.633 mmole) with lithium aluminum deuteride in anhydrous tetrahydrofuran (19) afforded the mixture of labelled pregnendiols XIII as an oil (230 mg; 113%). IR (CC14): 3620 (m), 3050 (w), 2210 (w), 2120 (w), 1264 (m) and 1060 cm-l (m). MS (TMS ether): m/e 466 (M+), 451, 376, 361, 271, 258, 157 (base peak) and 118. 5c(-~a,11,11,20!3-2H~Pregnane-3B,20a-diol
(XIV).
The mixture of Al6 -pregnenediols XIII (190 mg; 0.590 mmole) was reduced with platinum oxide (75 mg) in a Parr shaker under 3 atmospheres of hydrogen (20). Work-up afforded the crude product as a white solid (183 mg), which was chromatographed on a 50 g column of LipidexR-1000, packed in and eluted with methanol/water/chloroform (6:4:1). Pure XIV was eluted between 1,050-1,160 ml of effluent and recrystallized from aqueous ethanol to give 49.6 mg (0.153 mmole), m.p. 223-2240 (lit. (20) 2180). IR (KBr): 3380 (m),'2205 (w), 2140 (w), 2110 (w) and 1065 cm-l (m). MS (TMS ether): m/e 453 (M-15), 378, 363, 349, 273 and 118 (base peak). Anal. Calcd. for C2lH322H402: C, 77.77; H, 12.34%. Found: C, 77.38; H, 12.15%. Potassium 5a-~a,11,11,20B-2H4~Pregnane-3B,20a-diol
3,20-disulphate
(XV).
The labelled pregnanediol XIV (7.3 mg; 0.023 mmole) was converted into its disulphate XV by the method described above (cf. X -+X1) and the product chromatographed on a 4 g column of Sephadz LH-20. The column was first eluted with 100 ml of methanol/chloroform (l:l, saturated with potassium chloride), followed by 100 ml of methanol alone. Evaporation
S
444
9?IIROXDL)
of the methanol effluent gave the disulphate XV. Solvolysis of a small portion of this material, as previously described, and subsequent GLC analysis, with added internal standard, of the pregnanediol (XIV) so formed indicated the yield of XV to be 8.16 mg (65%). Further purification by chromatography on Amberlite XAD-2 gave the disulphate as a white solid which was homogeneous by TLC (Rf 0.17 in system C). IR (KBr): 2200 (w), 2118 (w), 1220-1245 (s), 1048 (s), 953 (m), 935 (m) and 915 cm-l (m). Attempts to prepare a crystalline sample of XV proved unsuccessful. 5a-~n,11,11,20B-2H4]Pregnane-3B,20~-diol
3,20-diacetate
(XVI).
The pregnanediol XIV (38.5 mg; 0.119 mmole) was acetylated at room temperature overnight with pyridine/acetic anhydride (1:l; 2 ml). Recrystallization of the product from methanol gave XVI as small plates (46.0 mg; 95%, m.p. 168-1690 (lit. (20) 1680). IR (CC14): 2205 (w), 2120 (w), 1728-1735 (s) and 1242-1265 cm-l (s). MS: m/e 348 (M-60; base peak), 333, 318, 288, 273, 233, 219, 218, 148 and 123. Anal. Calcd. for C25H362H404: C, 73.53; H, 10.78%. Found: C, 73.70; H, 10.82%. 5a-~3a,11,11,20B-2H4~Pregnane-3B,20~-diol
20-monoacetate
(XVIIl.
The diacetate XVI was selectively hydrolyzed essentially as described by Butenandt and Schmidt (21). Thus, XVI (41.5 mg; 0.102 mmole) was dissolved in methanol (10 ml), 2.0 ml of 0.07 M aqueous sodium bicarbonate solution was added and the mixture heated under reflux for 2 h. After evaporation under reduced pressure, the product was treated with water (10 ml) and extracted into ether (4 x 20 ml). The combined ether extracts were washed, dried (magnesium sulphate) and evaporated to afford a colourless oil (45.5 mg), which was chromatographed on a 6 g column of LipidexR-5000. Elution with hexane/chloroform (8:2) gave unchanged starting material XVI (12.2 mg) between 12-18 ml of effluent and the desired monoacetate XVII (23.7 mg; 64%) between 24-36 ml. Further elution afforded the pregnanediol XIV (1.0 mg). The labelled monoacetate XVII was recrystallized from aqueous acetone to give thin plates, m.p. 147-1490. IR (CC14): 3618 (w), 2206 (w), 2120 (w), 1728 (s), 1256 (s) and 1248 cm-l (s). MS (TMS ether): m/e 438 (M+), 423 (base peak), 378, 363, 348, 288, 287, 273, 248, 219 and 218. Anal. Calcd. for C23H342H403: C, 75.40; 11.47%. Found: C, 75.70; H,7Tr70%. Potassium 5a-fia,11,11,20B-2H~Pregnane-3B,20a-diol
3-sulphate, 20-
acetate (XVIII). The pregnanediol monoacetate XVII (17.7 mg; 0.048 mmole) was sulphated as described above and the product purified by chromatography on Sephadex LH-20. This afforded 15.9 mg (68%) of XVIII, which was homogeneous by TLC (Rf 0.48 in system C). Solvolysis of a small portion of XVIII, which yielded the monoacetate XVII, confirmed the site of sulphoconjugation as C-3. Potassium 5a-~a,11,11,20B-2H4]Pregnane-3B,20~-diol
3-sulphate (XIX).
The pregnanediol 3-sulphate, 20-acetate XVIII (15.9 mg; 0.033 mmole) was treated with 0.3 M potassium hydroxide (15 ml) and the mixture
445
ar
-.#?PI&OTDI
heated under reflux for 2 h. Once cool, the product was carefully neutralized with 2 N hydrochloric acid and the solvent removed in vacua.
S
TIlEOXDrn
Chromatography of the residue on Amberlite XAD-2 afforded XIX (11.7 mg; 80%) as a white solid which gave a single spot on TLC (Rf 0.45 in system C). Recrystallization from methanol gave fine needles, m.p. 234-2360. IR (KBr): 3430 (s), 2200 (w), 2120 (w), 1225-1250 (s), 1032 (m), 955 (m) and 940 cm-l (m). Solvolysis of a portion of this material gave the labelled pregnanediol XIV, while acetylation of XIX followed by solvolysis yielded the monoacetate XVII. These findings verified the structure of XIX. RESULTS AND DISCUSSION To provide a means by which the turnover rate of the steroid skeleton in metabolic experiments could be determined, it was necessary to introduce deuterium atoms at a position remote from biological attack. Ring C appeared to be a suitable area for such labelling and since hydroxylation at the llB-position does not participate in the conversion of progesterone
to sulphated metabolites, C-11 was chosen for this purpose.
Takes -et al. (22) have described a procedure for the introduction of two deuterium atoms at the 11-position of a 12-ketosteroid by exchange with deuterium oxide. Based on this reaction, the sequence illustrated in Scheme 1 was developed. Hecogenin (II) was chosen as a readily available starting material, which on base-catalyzed equilibration with deuterium oxide afforded the corresponding
3-c,ll,ll-zH3 species (III).
This compound was not isolated, but converted -in situ into its 12-tosylhydrazone derivative
(IV). The acidic conditions of the latter reaction
led to reversible opening of ring F, resulting in the introduction of two deuterium atoms into the side-chain (23). Reduction of IV with a large excess of sodium borohydride
(24) gave the tetradeutero tigogenin
(V), whose mass spectrum (TMS ether) is shown in Fig. l(a). The spectrum of the corresponding
unlabelled sapogenin is reproduced in Fig. l(b)
for comparison, where fragment ions resulting from cleavage of the spiroketal moiety are designated by lower-case letters according to
ag
447
WDlicOXD6
SCHEME 1
I R1=Ac; R2=R3= IH II
R1=R2=R3=
‘H
III
R,=R2=R3=
2H
ROT VII
V
RdH
VI R=Ac Budzikiewicz
et --
al.
(25).
The mass shifts in the spectrum of the labelled
compound of ions CJ and 2 , which arise through loss of the side-chain, result from the deuterium incorporation at C-11. Fragments t and u, which are formed by cleavage through ring F, are similarly shifted two units to higher mass in Fig. l(a) and confirm C-23 as the site of deuteration. labelling at this position is responsible for the displacement of the
S
448
TDEOID-
E/F ring ions 0 and p to m/e 141 and 117 in the spectrum of the deuterated derivative. Values for deuterium excess of the molecule as a whole, and at C-23, are given in Table 1.
Table 1 Relative retention times (tR) mediates and products.
Compound
V TMS
VI
-tR
3.12
3.58
and iSOtOpiC purity Of synthetic inter-
Deuterium excess (atoms%)
Ion m/e 488(M+)
2H0
2H1
2H2
2H3
'H4
2H5
61.8
7.4
0.0
0.0
10.1
20.7
73.2
0.2 27.2
139
7.0
19.6
458(M+)
1.1
2.1
14.5
139
9.9
23.2
66.9
53.8
1.3
VII TMS
0.74
388(Pl+)
5.9
2.5
91.6
VIII TMS
0.79
390(M+)
5.5
4.3
90.1
0.1
-
IX
0.71
316(M+)
4.5
5.2
89.7
0.6
-
X TMS
0.79
390(M+)
1.0
4.4
7.4
87.2
-
XII
0.63
314(M+)
3.4
7.6
88.5
0.5
XIV TMS
1.15
449(M-15)
0.0
1.0
4.6
9.1
83.9
1.4
117
4.0
96.0
0.2
1.3
3.8
12.2
82.3
0.2
5.7
-
XVa TMS
1.15
449(M-15) 117
4.4
95.6
XVI
1.40
344(M-60)b
0.1
0.5
3.1
11.1
79.5
XVII TMS
1.18
359(M-60-15)b
0.0
1 .O
4.3
15.6
79.1
XIXa TMS
1.15
449(M-15)
0.0
0.9
4.8
9.6
83.0
117
4.3
95.7
a Determinations solvolysis.
1.7
carried out on the unconjugated steroid liberated by
b Isotope measurements indicate the occurrence of fragmentations to yield ions at (M-59) (compound XVI) and (M-59-15) (compound XVII), accompanied by transfer of deuterium from the nuclear to the uncharged species.
s
449
TIIPtOXDI
Of the known by-products arising from sodium borohydride reduction of 12-ketosteroid tosylhydrazone derivatives, insignificant amounts of olefinic compounds (26) were encountered
in the conversion of IV to
V. However, GC-MS analysis of the crude reaction product indicated the presence of approximately
20% of the rearranged C-nor-D-homosteroid
(27-29). The mass spectrum of the TMS ether of this compound (Fig. l(c)) exhibits a fragmentation pattern characteristic
of steroidal sapogenins
(25). The presence of the A13(17) double bond results in an intense ion at m/e 128, probably formed from the spiroketal moiety and not normally present in spectra of ring D-saturated sapogenins. The pure tigogenin (V) could be obtained by recrystallization acetone. Compound V was transformed into its acetate (VI)
from
and subjected
to side-chain degradation according to the procedure of Wall -et al. (9) to yield the labelled 3p-hydroxy-5a-pregn-16-en-20-one
(VII),
where 91.6%
of the molecules were dideuterated. This Cpl steroid served as a common precursor for the synthesis of the deuterated pregnanolone and pregnanediol sulphates. Scheme 2 indicates the sequence of reactions leading from the A16pregnenolone
(VII) to the labelled pregnanolone sulphate (XI). Cataly-
tic hydrogenation of VII over palladium/charcoal
(7) proceeded smoothly
to yield 3B-hydroxy-5a-~l,ll-2H2]pregnan-20-one
(VIII).
The requirement
that the synthetic end-products be labelled such that the extent of oxidoreduction
of hydroxyl groups could be followed during metabolic
experiments necessitated the introduction of a deuterium atom at each site bearing a free or sulphated hydroxyl group. These labels would thus serve as markers for the processes of oxidoreduction, followed by oxidoreduction,
and hydrolysis
respectively. Thus, compound VIII was oxi-
S
450
%'B&OlcD1
SCHEME 2
0
X
XI
dized with Jones reagent (l'i) to give 5~-~~,~l"2H2~pregnane-3,20-dione (IX), which was selectively reduced at C-3 with sodium borodeuteride (13). Straight-phase
chromatography
of the reaction product on the lipo-
philic Sephadex derivative LipidexR -5000 resulted in complete separation of the resulting pregnanolones, epimeric at C-3, and afforded the desired 3B-hydroxy-5a-~a~ll,?l-*H~pregnan-20-one
(X). Fig. 2 illustra-
tes the mass spectra of the TMS ether derivatives of compound X and the corresponding
unlabelled pregnanolone. Calculations of deuterium con-
tent indicated the isotope excess at the 3a-position to be 97 atoms%. Preparation of the steroid sulphate XI was carried out essentially as described by Mumma (30) and the product was purified as the potassium salt (in view of its relatively high solubility in water) by chromatography an Sephadex LH-20 (14).
Q ?8
8
m
I”
000000 yrlul~nr .lNI ml3
IN33
t13d
S
452
WDEOIDI SCHEME
3
xv
XIV I
XIX
XVII Ri='H;R2=Ac
XVI
XVIII R1=S03K;R2=Ac
The synthetic route to the deuterated pregnanediol sulphates is summarized in Scheme 3. The target compounds were the 3,20-disulphate (XV) and the 3-monosulphate
(XIX) of 5a-~~,11,11,ZOB-2H4]pregnane-3~,
ZOa-diol (XIV). Although the 3B-hydroxy-5o:-[3a-*qconfiguration
is
readily accessible through treatment of 3-keto-5a-steroids with complex
453
m
TIlEOXDl
8
i-u
S
454
TDEOXDI
metal deuterides, such reduction of ZO-ketopregnanes
gives appreciable
amounts of the more hindered 208, rather than the required 2Oa, alcohol (31). Reductions of 20-ketosteroids with alkali metals and alcohols yield mixtures containing up to 69% of the 201~01, in addition to variable amounts of the 17a-pregnanes (32). However, treatment of A '6_20_ ketones with lithium aluminum hydride is reported to give almost exclusively the pregnan-20a-ol
Al6 -20~~01, which can be hydrogenated to the corresponding (19). We have recently shown that deuterium is intro-
duced at the 206-position
in high isotopic purity by the use of lithium
aluminum deuteride in the above reaction and that the label is retained, without scrambling, in the subsequent hydrogenation step (33). Based on these considerations, 3,20-dione
the pregnenolone
VII was transformed, via the
XII, into a mixture of isomeric 'H4-pregnenediols (XIII)
prior to saturation of the hydrogenation of
Al6 double bond. The major product from the
XIII was the desired 5a-[3a,11,11,20B-2H4~pregnane-38,
'ZOa-dial (XIV), which was purifi,ed by reversed-phase chromatography LipidexR-1000.
The mass spectrum
on
of the TMS ether derivative of com-
pound XIV is compared with that of the corresponding unlabelled pregnanediol in Fig. 3. Mass shifts of ions in the upper mass region confirm the presence of four deuterium atoms in compound
XIV, while incorpora-
tion of the label at the 208-position is clearly shown by the displacement of the intense side-chain ion at m/e 117 (34) to m/e 118. The isotope excess of the labelled pregnanediol
XIV indicated that deu-
terium had been introduced in 98.8 and 96.0% isotopic purity at the 3a and 208 positions, respectively. Synthesis and purification of the disulphate XV were carried out in a manner similar to that described above for the 'H3-pregnanolone
(X -XI).
S
455 Finally, the from
3-monosulphate
free diol
palan and
WmEOZDm
according to
(35), in
its acetate.
(XIX)
procedure described by
the ZOa-hydroxy group is
as
compound XIV was converted to its diacetate XVI
which was selectively hydrolyzed with sodium bicarbonate in methanol (21) to afford 5n-[3a,11,11,20B-2H~~pregnane-3~,20a-diol
20-monoacetate
(XVII). Sulphation, followed by removal of the acetate protecting group under strongly alkaline conditions, yielded the labelled pregnanediol 3-monosulphate XIX. Since the aim of this work was to obtain labelled steroid sulphates for experiments in human subjects, a high degree of purity of the endproducts was necessary. For this purpose, compounds XI, XV and XIX were purified by chromatography on Sephadex LH-20 in a system where potassium salts of steroid sulphates have a very characteristic mobility (14). Furthermore, potassium steroid sulphates, in contrast to the corresponding sodium salts, are sufficiently soluble in water to permit intravenous injections of the doses (5-10 I_rmole/lOml) required. Criteria for purity of the labelled sulphates were their homogeneity and mobility on thin-layer chromatography and on gas chromatographic analysis following solvolysis. Much of the purification of intermediates in the synthetic sequences was carried out using liquid-gel chromatography on hydrophobic LipidexR gels having properties very similar to those deet al. (36). These gels provide a mild environment scribed by Ellingboe -where the risk of deuterium exchange is minimized, and permit the use of either straight- or reversed-phase solvent systems. Steroids with the Al6 -2O-oxopregnane
structure may be transformed
by established procedures into a wide range of pregnane and androstane
S derivatives
456
TDEOTDI
(37). Thus, the common intermediate in each of the above
synthetic schemes, 3~-hydroxy-5w~1,11-*H~pregn-16-en-20-one
(VII),
could serve as a precursor for the preparation of a large number of both naturally occurring and synthetic steroids, labelled with deuterium at C-II. Several of the intermediates described in this paper may themselves be useful in studies of metabolic transformations of unconjugated steroids -in vivo and -in vitro. Thus, 5a-pregnane-3,20-dione (38,39) and 3B-hydroxy-5a-pregnan-20-one
(39) occur normally in plasma
of pregnant women. Furthermore, since high isotopic purity is obtained, the compounds containing 3 or 4 deuterium atoms could be employed as internal standards in quantitative analyses by selective ion monitoring. Results of studies on the metabolism of the labelled steroid sulphates in pregnant women will be reported in separate communications. ACKNOWLEDGEMENTS The skillful technical assistance of Mrs Kerstin Robertsson, Miss Irene Ferdman and Miss Marit Karls is gratefully acknowledged. We also wish to thank Professor B. Lindberg (Department of Organic Chemistry, Stockholm University) for use of hydrogenation facilities and Dr. C.J.W. Brooks (Chemistry Department, University of Glasgow) through worn elemental analyses were obtained. The work was supported by grants from the Swedish Medical Research Council (grants No. 13X-219 and 60R-4166) and by the World Health Organization. T.A. Eaillie was the holder of a Postdoctoral Fellowship from the Royal Society (London), under the European Science Exchange Programme. J.E. Herz was a Swedish Medical Research Council Visiting Scientist at the Karolinska Institute, 1973-74. REFERENCES Present address: Department of Clinical Pharmacology, Royal Postgraduate Medical School, London WI2 OHS, England. 2. Sjijvall, J., Sjovall, K. and Vihko, R. STEROIDS II, 703 (1968). Sjovall; K.-ANN. CLIN. RES. 2, 393-(1970). 43: Makita, M. and Wells, W.W. ANAL. BIOCHEM. 5, 523 (1963 5. Reimendal, R. and SjUvall, 3. ANAL. CHEM. 44, 21 (1972 6. Axelson, M., Cronholm, T., Curstedt, T., Reimendal, R. and Sjovall, J. CHROMATOGRAPHIA 7, 502 (1974). 7. Marker, R.E., Wagner, R.B., Ulshafer, P.R., Wittbecker ', E.L., Goldsmith, D.P.J. and Ruof, C.H. J. AM. CHEM. SOC. 69, 2167 (1947). 1.
1:
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. ;:: 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
:::
32. 33. 34. ::: 37.
Hirschmann, R., Snoddy, C.S., Hiskey, C.F. and Wendler, N.L. J. AM. ;+$. ;O;. 76, 4013 (1954). Kenney, H.E. and Rothman, E.S. J. AM. CHEM. SOC. 77, 5665'(1955;. F;;j:;, F.E. and Turner, D.L. J. AM. CHEM. SOC. 62, 3003 (1940). Heilbron, I.M., Jones, E.R.H. and Weedon, B.C.L. J. CHEM. sot. 3; (i446). Marker, R.E., Kamm, 0. and McGrew, R.V. J. AM. CHEM. SOC. 59, 616 (1937). Mancera, O., Ringold, H.J., Djerassi, C., Rosenkranz, G. and Sondheimer, F. J. AM. CHEM. SOC. 75, 1286 (1953). Sjovall, J. and Vihko, R. ACTA CHEM. SCAND. 20, 1419 (1966). But-stein, S. and Lieberman, S. J. BIOL. CHEM. 233, 331 (1958). Bradlow, H.L. STEROIDS 11, 265 (1968). Lieberman, S., Dobriner, K., Hill, B.R., Fieser, L.F. and Rhoads, C.P. J. BIOL. CHEM. 172, 263 (1948). Marker, R.E. and Rohrmann, E. J. AM. CHEM. SOC. 62, 898 (1940). Shapiro, E.L., Gould, D. and Hershberg, E.B. J. AM. CHEM. SOC. 77, 2912 (1955). Marker, R.E., Kamm, O., Wittle, E.L., Oakwood, T.S., Lawson, E.J. and Laucius, J.F. J. AM. CHEM. SOC. 59, 2291 (1937). Butenandt, A. and Schmidt, J. CHEM. BER. 67, 1893 (1934). Tijkes, L., Jones, G. and Djerassi, C. J. AM. CHEM. SOC. 90, 5465 (1968). Faul, W.H., Failli, A. and Djerassi, C. J. ORG. CHEM. 35, 2571 (1970). Fisher, M., Pelah, Z., Williams, D.H. and Djerassi, C. CHEM. BER. 98, 3236 (1965). Budzikiewicz, H., Djerassi, C. and Williams, D.H. STRUCTURE ELUCIDATION OF NATURAL PRODUCTS BY MASS SPECTROMETRY, Vol. 2. HoldenDay Inc., San Fransisco, 1964, chapter 22. Caglioti, L. and Magi, M. TETRAHEDRON 19, 1127 (1963). Hirschmann, R., Snoddy, C.S. and Wendler, N.L. J. AM. CHEM. SOC. 74, 2693 (1952). J. Hirschmann, R., Snoddy, C.S., Hiskey, C.F. and Wend1er;N.L. AM. CHEM. SOC. 76, 4013 (1954). Elks, J., Phillipps, G.H., Taylor, D.A.H. and Wyman, L.J. J. CHEM. sot. 1739 (1954). Mumma, R.O. LIPIDS 1, 221 (1966). Wheeler, D.M.S. and Wheeler, M.M. in ORGANIC REACTIONS IN STEROID CHEMISTRY, Vol. 1, Ed. Fried, J. and Edwards, J.A. Van Nostrand Reinhold, New York, 1972, chapter 2. J. CHEM. SOC. (C), 968 (1969). Kirk, D.N. and Mudd, A.J. J. LABELLED COMPOUNDS Baillie, T.A., Herz, J .E. and Sjovall, J. 10, 549 (1974). Adlercreutz, H., Luukkainen, T. and Taylor, W. EUR. J. STEROIDS 1, 117 (1966). Rajagopalan, M.S. and Turner, A.B. J. CHEM. SOC. (C) 1858 (1969). Ellingboe, J., Nystrom, E. and Sjiivall, J. J. LIPID RES. 11, 266 (1970). Fieser, L.F. and Fieser, M. STEROIDS,Reinhold Publishing Co., New York, 1959, chapters 17,and 19. Stda, K.F. and Bessesen, A. J. STEROID BIOCHEM. 6, 21 (1975). Axelson, M. and Sjbvall, J. J. STEROID BIOCHEM. 5, 733 (1974).
SYNTHESIS OF SPECIFICALLY DEUTERIUM-LABELLED
PREGNANOLONE AND
PREGNANEDIOL SULPHATES FOR METABOLIC STUDIES IN HUMANS
T.A. Baillie (1) and J. Sjovall Department of Chemistry, Karolinska Institutet S-104 01 Stockholm 60, Sweden and J.E. Herz Department of Chemistry, Centro de Investigation y de Estudios Avanzados de1 IPN, Apartado Postal 14-740, Mexico 14, D.F.
Received:
B/4/75
ABSTRACT A synthesis is reported of 3B-hydroxy-5a-pregnan-ZO-one sulphate and the disulphate and 3-monosulphate of 5a-pregnane-3B,ZOa-diol, labelled specifically with deuterium in high isotopic purity for metabolic studies in humans. Base-catalyzed equilibration of 3B-hydroxy-5a-25R-spirostan-12-one (hecogenin, II) with deuterium oxide, followed by removal of the 12-keto group and degradation of the sapogenin side-chain afforded 38-hydroxy5a-[ll,ll-2H2]pregn-16-en-ZO-one (VII). Further deuterium atoms were introduced at the 3a and 2Op positions by reductions with sodium borodeuteride and lithium aluminum deuteride, respectively. These reactions led to 3B-hydroxy-5a-[3a ll,ll-2Hapregnan-ZO-one (X; isotopic purity 87.2%) and 5a-[3a,11,11,20f3-~H~pregnane-3B,20a-diol (XIV; isotopic purity 83.9%). The 3-sulphate of the pregnanolone and the 3,20-disulphate of the pregnanediol were prepared directly from the free alcohols, while the 3-monosulphate of the pregnanediol was obtained via 5a-[3a,ll,ll,ZOB-2H~pregnane-36,20a-diol 20-acetate (XVII). INTRODUCTION The occurrence and quantitative
importance in late pregnancy plasma
of isomeric pregnanolone sulphates and mono- and disulphated pregnanediols has been demonstrated by Sjijvall and co-workers (2,3). Although these compounds are assumed to be metabolites of progesterone, nothing is known about their pool sizes, turnover rates or metabolic interrela-
Vohme
26,
Number 4
S
TDROIDU
October, 2975
439
S
TDICOIDb
tionships. As an initial step in establishing the physiological significance of steroid sulphates in plasma of pregnant women, it was decided to carry out a detailed study of their metabolism in healthy subjects. For ethical reasons, it is not possible to administer radioactive tracers in such investigations. Since information was required both on the turnover of the steroid skeleton and on oxidoreduction of hydroxyl groups, labelling with deuterium atoms in specific positions and analysis by gas chromatography-mass
spectrometry
(GC-MS) was considered to be the method
of choice. This paper describes the synthesis of specifically-deuterated analogues of a series of structurally related C2,02 steroid sulphates encountered in pregnancy plasma, namely 3B-hydroxy-5o-pregnan-ZO-one phate, 5a-pregnane-3B,20a-diol
sul-
3-monosulphate and 5a-pregnane-3B,20a-diol
3,20-disulphate. EXPERIMENTAL SECTION GENERAL Melting points were determined on a Gallenkamp hot-stage apparatus and are corrected. Welting points quoted from the literature refer to the corresponding unlabelled compounds. Infrared spectra were recorded, either on solutions in carbon tetrachloride or on discs of potassium bromide, using a Perkin-Elmer 254 Infrared Spectrophotometer. Ultraviolet spectra were obtained with a Zeiss Model DMR 21 Recording Spectrophotometer. Elemental microanalyses were carried out at the Chemistry Department, University of Glasgow, Scotland. Thin-layer chromatography (TLC) was carried out using glass plates precoated with silica gel 60 F254 (Merck AG, Darmstadt, Germany). For analytical separations, 0.25 mm layers (5 x 20 cm plates) were used, while 2 mm layers (20 x 20 cm plates) were employed for preparative work. The following solvent systems were used (mixtures expressed as ratios by volume): system A, chloroform/methanol (95:5); system B, ethyl acetate/ chloroform/methanol (15:5:1); system C, ethanol/ethyl acetate/25% aqueous ammonium hydroxide (5:5:1). The compound zones were visualized by UV light ( A = 254 nm) or by exposure to iodine vapour. Analytical plates were subsequently sprayed with a vanillin-sulphuric acid solution and heated for 5 min at 1100.
S
440
~~EOID6
Liquid-gel chromatography was carried out using LipidexR-1000 and LipidexK-5000 stationary phases (Packard International, S.A., Zurich). Gas-liquid chromatography was performed with a Pye Series 104 instrument, equipped with 2 m x 3 mm i.d. glass columns packed with 1.5% SE-30 on Gas Chrom Q, loo-120 mesh (Applied Science Laboratories, Inc.). Flame ionization detectors were used, with nitrogen as carrier gas (flow rate 38 ml/min). Hydroxysteroids were analyzed as their trimethylsilyl (TMS) ether derivatives (4) and all retention times, tR, were measured relative to that of 5ol-cholestane (tR = 1.0). Mass spectrometry. A modified LKB 9000 instrument was used (5), equipped with a 3 m x 3 mm i.d. qlass column packed with 1.5% SE-30 on Gas Chrom Q, loo-120 mesh, with helium as the carrier gas. Analyses were carried out with an oven temperature of 2100, while the molecular separator and ion source were held at 2500 and 2900, respectively. The energy of the bombarding electrons was 22.5 eV and the ionizing current was 60 vamp. Multiple spectra were recorded on magnetic tape and average isotope content was determined by comparison with the unlabelled reference compounds using an IBM 1800 computer (6). Reagents. Deuterium-labelled solvents and reagents were as follows: deuterium oxide (99.8%; Norsk Hydro, Oslo, Norway), monodeuteromethanol (CH302H, 2 99%; Merck AG, Darmstadt, Germany), lithium aluminum deuteride ( 2 99%; Fluka AG, Buchs, Switzerland) and sodium borodeuteride ( 2 98%; Merck AG, Darmstadt, Germany). All other chemicals were used as purchased and were reagent grade where available. SYNTHESIS Hecogenin (3B-Hydroxy-5a-25R-spirostan-12-one;
II).
Hecogenin acetate (I; 13.2 g; 28.0 mmole) was hydrolyzed at room temperature overnight with 2% methanolic potassium hydroxide (400 ml) to give II (11.3 g; 94%). Recrystallization from methanol afforded large plates, m.p. 268-270° (lit. (7) 2680). Anal. Calcd. for C27H4204: C, 75.31; H, 9.83%. Found: C, 75.10; H, 9.7Or 3-Q,11,11-2H,
Hecogenin (III).
To a stirred mixture of monodeuteromethanol (500 ml) and deuterium oxide (12.3 ml) under a nitrogen atmosphere was added 1.31 g (57 mmole)of sodium metal in small pieces. Hecogenin (II; 11.2 g; 26.0 mmole) was added and the resulting mixture heated under reflux for 48 h. On cooling to 00, the product crystallized from solution. This material was not isolated but employed directly for tosylhydrazone formation. 11,11,23,23-ZH4
Hecogenin Tosylhydrazone
(IV).
The above slurry of labelled hecogenin (III) and monodeuteromethanol at Oo was made acidic by dropwise addition of acetyl chloride (7.4 ml). The resulting mixture was allowed to warm up to room temperature, treated with tosyl hydrazine (16.7 g; 82.6 mmole) and heated under reflux for 21 h when TLC analysis of the product indicated that reaction was complete (Rf of product 0.35, Rf of hecogenin 0.45 in system A). The mixture was then cooled to Oo and the tosylhydrazone IV filtered off and dried to
441
s
TPlICOIDb
afford 11.9 g (74%). A second crop (2.65 g) brought the yield of IV to 91%. An analytical sample, prepared by recrystallization from methanol, had m.p. 262-264O (lit. (8) 259-2600). UV(EtOH): Xmax = 226 nm ( E = 23,400). IR (KBr): 3615 (m), 3500 (m), 3212 (m), 3060 (w), 2215 (w), 2135 (w), 1648 (m), 1600 (m), 1450 (s), 1325 (s), 1170 (s), 1035 (s), 980 (s), 898 (m), 850 (m) and 820 cm-l (s). Anal. Calcd. for C34H462HqN205S: C, 67.77; H, 8.97; N, 4.65%. Found: C,r48; H, 8.99; N, 4.43%. ll,11,23,23-2H4 Tigogenin
(5a-nl,ll,23,23-2H4]-25R-Spirostan-3B-ol;
V).
The tosylhydrazone IV (4.54 g; 7.37 mmole) was dissolved in methanol (700 ml) and the solution treated with sodium borohydride (4.50 g; 119 mmole), added in portions with water-bath (room temperature) cooling. The reaction mixture was heated under reflux for 15 h, cooled and examined by TLC (system A). This indicated that only partial reduction of IV had taken place (two spots, with Rf 0.36 and 0.47), whereupon a further 4.5 g of sodium borohydride was added and reflux continued for a second period of 15 h. A third, and final, addition of sodium borohydride (4.5 g) and subsequent 15 h reflux was necessary to achieve complete reduction of IV, as judged by TLC (product gave single spot, Rf 0.47). The reaction product was concentrated in vacua until precipitation commenced, diluted with 2 N hydrochloric aciamml) and extracted with ether (3 x 350 ml). The combined ether extracts were washed with water, dried over magnesium sulphate and evaporated. Recrystallization of the crude product from acetone gave 1.73 g (56%) of V as needles, m.p. 203.5-2060 (lit. (7) 203-2060 . IR (CC14): 3625 (m), 2210 (w), 2130 (w), 1040 (s), 983 (m) and 888 cm- 1 (m). MS (TMS ether): m/e 492 (M+), 477, 421, 420, 376, 361, 347, 286, 271, 257, 141 (base peam 122 and 117. Anal. Calcd. for C27H402H403: C, 77.14; H, 11.40%. Found: C, 77.19; H, 117nr%. A further 9.86 g (16.0 mmole) of IV was similarly reduced, in two batches, and recrystallized as above to afford an additional 3.17 g (47%) of labelled tigogenin. 11,11,23,23-2H4 Tigogenin Acetate (VI). Acetylation of V (4.79 g; 11.4 mmole) was carried out at room temperature overnight with pyridinelacetic anhydride (l:l, 60 ml). The product (4.09 g; 78%) crystallized from ethanol as prisms, m.p. 212-2140 (lit. (7) 2032060). IR (CC14): 2210 (w), 2130 (w), 1735 (s), 1245 (s), 1040 (s), 982 (m), 902 (w) and 887 cm-l (m). MS: m/e 462 (M+), 447, 402, 401, 391, 390, 346, 331, 317, 286, 271, 257, 141 (base peak), 122 and 117. Anal. Calcd. for C2gH422H404: C, 75.32; H, 10.82%. Found: C, 75.08; H, llm. 38-Hydroxy-5a-bl,11-2HP]pregn-16-en-20-one
(VII).
The acetate VI (3.25 g; 7.02 mmole) was subjected to the three-step degradation sequence described by Wall et al. (9) in which pyridine hydrochloride is employed as catalyst for fie-@-eparation of the pseudosapogenin acetate. The crude product from this sequence of reactions was a pale yellow oil which solidified slowly on standing. Recrystallization from aqueous ethanol afforded VII (1.07 g; 48%) as small, yellowish crystals, m.p. 190-1950 (lit. (10) 200-2040). IR (CC14): 3620 (m) 3055 (w), 2210 (w), 2120 (w), 1672 (s), 1590 (m), 1233 (m) and 1041 cm-1 (s). An analytical sample of VII was prepared by preparative TLC (solvent system B) followed by two recrystallizations from aqueous ethanol to give colour-
S
442
TDROIDB
less needles, m.p. 201-2050. UV (EtOH):X max = 239 nm (& = 8,700). MS (TMS ether): m/e 390 (M+), 375, 347, 285, 257 (base peak) and 163. Anal. Calcd. for C2mO2H202: C, 79.24; H, 10.69%. Found: C, 79.25; H, 1Om. 3B-Hydroxy-5a-~l,ll,2H2]pregnan-20-one
(VIII).
The pregnenolone VII (196 mg; 0.616 mmole) in ethyl acetate (10 ml) was hydrogenated (7) for 1 h at 1 atm. pressure over 10% palladium-charcoal (196 mg). The catalyst was filtered off and the solvent evaporated to give VIII (188 mg; 95%). Recrystallization from ethanol afforded plates, m.p. 196-197.50 (lit. (9) 194-1960). IR (CC14): 3624 (m), 2215 (w), 2125 (w), 1708 (s) and 1042 cm-l (s). MS (TMS ether): m/e 392 (M+), 377 155. Anal. Calcd. (base peak , 335, 334, 302, 287, 263, 259, 245, 2rand for C2lH32 h H202: C, 78.75; H, 11.20%. Found: C, 79.13; H, 1l.m 5a-hl,ll-2H2]Pregnane-3,20-dione
(IX).
Oxidation of the pregnanolone VIII (168 mg; 0.525 mmole) with Jones reagent (11) afforded 158 mg (95%) of IX, which was recrystallized from acetone to give plates, m.p. 196.5-198.50 (lit. (12) 2000). IR (CC14): 2212 (w), 2122 (w) and 1709 cm-l (s). MS: m/e 318 (M+), 303, 300, 285, 274, 271, 260, 233 and 84 (base peak). AnarCalcd. for C21H302H202: C, 79.19; H, 11.30%. Found: C, 79.19; H, lTZY%. 3B-Hydroxy-5a-~a,ll,11-2H~pregnan-20-one
(X).
The pregnanedione IX (150 mg; 0.472 mmole) was reduced with sodium borodeuteride (19.7 mg; 0.471 mmole) in absolute ethanol (20 ml), according to the method of Mancera et al. (13). TLC analysis of the product indicated complete reduction ??Ffie starting material (Rf 0.62 in system A) to a mixture of the 3a and 36-pregnanolones (Rf 0.53 and 0.46, respectively), while trace amounts of pregnanediol isomers (Rf approx. 0.35) were also detected. The product was chromatographed on a column of LipidexR-5000 (40 g), prepared in and eluted with hexane/chloroform (8:2). Compound X (75 mg; 50%) was eluted between 175-220 ml of effluent and recrystallized from ethanol to give plates, m.p. 196-197.5O (lit. (9) 194-1960). IR (CC14): 3620 (m), 2210 (w), 2124 (w), 1708 (s) and 1064 cm-l (m). MS (TMS ether): 393 (M+), 378 (base peak), 336 335, 303, 288, 263, 260, 245, 218 and 156. Anal. Calcd. for C21H312H302: C, 78.50; H, 11.52%. Found: C, 78.77; H, TOGO%. Potassium 3f3-Hydroxy-5a-~a,ll,ll-2H~lpregnan-20-one
3-sulphate (XI).
The pregnanolone X (10.0 mg; 0.031 mmole) and N,N'-dicyclohexylcarbodiimide (37.8 mg; 0.183 mmole) were dissolved in-anhydrous dimethylformamide (0.27 ml) and the solution cooled to 00. 70 1.11 of a mixture of cont. sulphuric acid (35 ~1) and anhydrous dimethylformamide (0.60), which had been previously cooled to ice-bath temperature,was added and the mixture held at Oo for 1 h. The reaction mixture was then treated with 75% aqueous ethanol (2 ml), neutralized with 2 N potassium hydroxide (3 drops) and centrifuged. The supernatant was removed and the residue washed three times with aqueous ethanol (2 ml). The combined washings were added to the above supernatant and the mixture taken to dryness -in vacua. The residue was chromatographed on an 8 g column of
443
S
TDEOID1
Sephadex LH-20 (solvent system: methanol/chloroform, 1:1, saturated with potassium chloride (14))and 5 ml fractions were collected. TLC analysis indicated the presence of a monosulphate conjugate (Rf 0.40 in system C) in fractions 10-16, which were combined and taken to dryness. The residue was dissolved in ethanol (10.0 ml) and an aliquot (20 ~1) taken for solvolysis in acidified ethyl acetate (15). Following the addition of 5B-cholane-3a,24-dial (30 vg) as internal standard, the product from solvolysis was converted into its TMS ether derivative and analyzed by GLC. Quantitation of the solvolyzed steroid was based on GLC peak area measurements, which indicated a yield of the sulphate XI from X of 10.1 mg (74%). A sample of XI, free from potassium chloride, was obtained by chromatography on Amberlite XAD-2 (16), followed by recrystallization from methanol. This gave small needles, m.p. 229-231° (dec.) (lit. (17) 207-2100). IR (KBr): 2210 (w), 2120 (w), 1708 (s), 1250-1220 (s), 1072 (s), 1033 (s) and 955 cm-l (s). 5a-~11,11-2H~~Pregn-16-ene-3,20-dione
(XII).
The pregnenolone VII (301 mg; 0.947 mmole) was oxidized with Jones reagent (11) to give XII as a finely divided white solid (222 mg; 74%), m.p. 203-210° (lit. (18) 210-2120). IR (CC14): 3045 (w), 2215 (w), 2130 w), 1718 (s), 1672 (s) and 1591 cm-l (m). UV (EtOH): A max = 239 nm m/e 316 (M+), 301, 283, 273 (base peak), 255, 191 t E = 7,100). MS: -and 163. This material was not purified but used directly for reduction. 5a-~5,11,11,20~-2H4~Pregn-16-ene-3~,20~-diols
(XIII).
Reduction of XII (200 mg; 0.633 mmole) with lithium aluminum deuteride in anhydrous tetrahydrofuran (19) afforded the mixture of labelled pregnendiols XIII as an oil (230 mg; 113%). IR (CC14): 3620 (m), 3050 (w), 2210 (w), 2120 (w), 1264 (m) and 1060 cm-l (m). MS (TMS ether): m/e 466 (M+), 451, 376, 361, 271, 258, 157 (base peak) and 118. 5c(-~a,11,11,20!3-2H~Pregnane-3B,20a-diol
(XIV).
The mixture of Al6 -pregnenediols XIII (190 mg; 0.590 mmole) was reduced with platinum oxide (75 mg) in a Parr shaker under 3 atmospheres of hydrogen (20). Work-up afforded the crude product as a white solid (183 mg), which was chromatographed on a 50 g column of LipidexR-1000, packed in and eluted with methanol/water/chloroform (6:4:1). Pure XIV was eluted between 1,050-1,160 ml of effluent and recrystallized from aqueous ethanol to give 49.6 mg (0.153 mmole), m.p. 223-2240 (lit. (20) 2180). IR (KBr): 3380 (m),'2205 (w), 2140 (w), 2110 (w) and 1065 cm-l (m). MS (TMS ether): m/e 453 (M-15), 378, 363, 349, 273 and 118 (base peak). Anal. Calcd. for C2lH322H402: C, 77.77; H, 12.34%. Found: C, 77.38; H, 12.15%. Potassium 5a-~a,11,11,20B-2H4~Pregnane-3B,20a-diol
3,20-disulphate
(XV).
The labelled pregnanediol XIV (7.3 mg; 0.023 mmole) was converted into its disulphate XV by the method described above (cf. X -+X1) and the product chromatographed on a 4 g column of Sephadz LH-20. The column was first eluted with 100 ml of methanol/chloroform (l:l, saturated with potassium chloride), followed by 100 ml of methanol alone. Evaporation
S
444
9?IIROXDL)
of the methanol effluent gave the disulphate XV. Solvolysis of a small portion of this material, as previously described, and subsequent GLC analysis, with added internal standard, of the pregnanediol (XIV) so formed indicated the yield of XV to be 8.16 mg (65%). Further purification by chromatography on Amberlite XAD-2 gave the disulphate as a white solid which was homogeneous by TLC (Rf 0.17 in system C). IR (KBr): 2200 (w), 2118 (w), 1220-1245 (s), 1048 (s), 953 (m), 935 (m) and 915 cm-l (m). Attempts to prepare a crystalline sample of XV proved unsuccessful. 5a-~n,11,11,20B-2H4]Pregnane-3B,20~-diol
3,20-diacetate
(XVI).
The pregnanediol XIV (38.5 mg; 0.119 mmole) was acetylated at room temperature overnight with pyridine/acetic anhydride (1:l; 2 ml). Recrystallization of the product from methanol gave XVI as small plates (46.0 mg; 95%, m.p. 168-1690 (lit. (20) 1680). IR (CC14): 2205 (w), 2120 (w), 1728-1735 (s) and 1242-1265 cm-l (s). MS: m/e 348 (M-60; base peak), 333, 318, 288, 273, 233, 219, 218, 148 and 123. Anal. Calcd. for C25H362H404: C, 73.53; H, 10.78%. Found: C, 73.70; H, 10.82%. 5a-~3a,11,11,20B-2H4~Pregnane-3B,20~-diol
20-monoacetate
(XVIIl.
The diacetate XVI was selectively hydrolyzed essentially as described by Butenandt and Schmidt (21). Thus, XVI (41.5 mg; 0.102 mmole) was dissolved in methanol (10 ml), 2.0 ml of 0.07 M aqueous sodium bicarbonate solution was added and the mixture heated under reflux for 2 h. After evaporation under reduced pressure, the product was treated with water (10 ml) and extracted into ether (4 x 20 ml). The combined ether extracts were washed, dried (magnesium sulphate) and evaporated to afford a colourless oil (45.5 mg), which was chromatographed on a 6 g column of LipidexR-5000. Elution with hexane/chloroform (8:2) gave unchanged starting material XVI (12.2 mg) between 12-18 ml of effluent and the desired monoacetate XVII (23.7 mg; 64%) between 24-36 ml. Further elution afforded the pregnanediol XIV (1.0 mg). The labelled monoacetate XVII was recrystallized from aqueous acetone to give thin plates, m.p. 147-1490. IR (CC14): 3618 (w), 2206 (w), 2120 (w), 1728 (s), 1256 (s) and 1248 cm-l (s). MS (TMS ether): m/e 438 (M+), 423 (base peak), 378, 363, 348, 288, 287, 273, 248, 219 and 218. Anal. Calcd. for C23H342H403: C, 75.40; 11.47%. Found: C, 75.70; H,7Tr70%. Potassium 5a-fia,11,11,20B-2H~Pregnane-3B,20a-diol
3-sulphate, 20-
acetate (XVIII). The pregnanediol monoacetate XVII (17.7 mg; 0.048 mmole) was sulphated as described above and the product purified by chromatography on Sephadex LH-20. This afforded 15.9 mg (68%) of XVIII, which was homogeneous by TLC (Rf 0.48 in system C). Solvolysis of a small portion of XVIII, which yielded the monoacetate XVII, confirmed the site of sulphoconjugation as C-3. Potassium 5a-~a,11,11,20B-2H4]Pregnane-3B,20~-diol
3-sulphate (XIX).
The pregnanediol 3-sulphate, 20-acetate XVIII (15.9 mg; 0.033 mmole) was treated with 0.3 M potassium hydroxide (15 ml) and the mixture
445
ar
-.#?PI&OTDI
heated under reflux for 2 h. Once cool, the product was carefully neutralized with 2 N hydrochloric acid and the solvent removed in vacua.
S
TIlEOXDrn
Chromatography of the residue on Amberlite XAD-2 afforded XIX (11.7 mg; 80%) as a white solid which gave a single spot on TLC (Rf 0.45 in system C). Recrystallization from methanol gave fine needles, m.p. 234-2360. IR (KBr): 3430 (s), 2200 (w), 2120 (w), 1225-1250 (s), 1032 (m), 955 (m) and 940 cm-l (m). Solvolysis of a portion of this material gave the labelled pregnanediol XIV, while acetylation of XIX followed by solvolysis yielded the monoacetate XVII. These findings verified the structure of XIX. RESULTS AND DISCUSSION To provide a means by which the turnover rate of the steroid skeleton in metabolic experiments could be determined, it was necessary to introduce deuterium atoms at a position remote from biological attack. Ring C appeared to be a suitable area for such labelling and since hydroxylation at the llB-position does not participate in the conversion of progesterone
to sulphated metabolites, C-11 was chosen for this purpose.
Takes -et al. (22) have described a procedure for the introduction of two deuterium atoms at the 11-position of a 12-ketosteroid by exchange with deuterium oxide. Based on this reaction, the sequence illustrated in Scheme 1 was developed. Hecogenin (II) was chosen as a readily available starting material, which on base-catalyzed equilibration with deuterium oxide afforded the corresponding
3-c,ll,ll-zH3 species (III).
This compound was not isolated, but converted -in situ into its 12-tosylhydrazone derivative
(IV). The acidic conditions of the latter reaction
led to reversible opening of ring F, resulting in the introduction of two deuterium atoms into the side-chain (23). Reduction of IV with a large excess of sodium borohydride
(24) gave the tetradeutero tigogenin
(V), whose mass spectrum (TMS ether) is shown in Fig. l(a). The spectrum of the corresponding
unlabelled sapogenin is reproduced in Fig. l(b)
for comparison, where fragment ions resulting from cleavage of the spiroketal moiety are designated by lower-case letters according to
ag
447
WDlicOXD6
SCHEME 1
I R1=Ac; R2=R3= IH II
R1=R2=R3=
‘H
III
R,=R2=R3=
2H
ROT VII
V
RdH
VI R=Ac Budzikiewicz
et --
al.
(25).
The mass shifts in the spectrum of the labelled
compound of ions CJ and 2 , which arise through loss of the side-chain, result from the deuterium incorporation at C-11. Fragments t and u, which are formed by cleavage through ring F, are similarly shifted two units to higher mass in Fig. l(a) and confirm C-23 as the site of deuteration. labelling at this position is responsible for the displacement of the
S
448
TDEOID-
E/F ring ions 0 and p to m/e 141 and 117 in the spectrum of the deuterated derivative. Values for deuterium excess of the molecule as a whole, and at C-23, are given in Table 1.
Table 1 Relative retention times (tR) mediates and products.
Compound
V TMS
VI
-tR
3.12
3.58
and iSOtOpiC purity Of synthetic inter-
Deuterium excess (atoms%)
Ion m/e 488(M+)
2H0
2H1
2H2
2H3
'H4
2H5
61.8
7.4
0.0
0.0
10.1
20.7
73.2
0.2 27.2
139
7.0
19.6
458(M+)
1.1
2.1
14.5
139
9.9
23.2
66.9
53.8
1.3
VII TMS
0.74
388(Pl+)
5.9
2.5
91.6
VIII TMS
0.79
390(M+)
5.5
4.3
90.1
0.1
-
IX
0.71
316(M+)
4.5
5.2
89.7
0.6
-
X TMS
0.79
390(M+)
1.0
4.4
7.4
87.2
-
XII
0.63
314(M+)
3.4
7.6
88.5
0.5
XIV TMS
1.15
449(M-15)
0.0
1.0
4.6
9.1
83.9
1.4
117
4.0
96.0
0.2
1.3
3.8
12.2
82.3
0.2
5.7
-
XVa TMS
1.15
449(M-15) 117
4.4
95.6
XVI
1.40
344(M-60)b
0.1
0.5
3.1
11.1
79.5
XVII TMS
1.18
359(M-60-15)b
0.0
1 .O
4.3
15.6
79.1
XIXa TMS
1.15
449(M-15)
0.0
0.9
4.8
9.6
83.0
117
4.3
95.7
a Determinations solvolysis.
1.7
carried out on the unconjugated steroid liberated by
b Isotope measurements indicate the occurrence of fragmentations to yield ions at (M-59) (compound XVI) and (M-59-15) (compound XVII), accompanied by transfer of deuterium from the nuclear to the uncharged species.
s
449
TIIPtOXDI
Of the known by-products arising from sodium borohydride reduction of 12-ketosteroid tosylhydrazone derivatives, insignificant amounts of olefinic compounds (26) were encountered
in the conversion of IV to
V. However, GC-MS analysis of the crude reaction product indicated the presence of approximately
20% of the rearranged C-nor-D-homosteroid
(27-29). The mass spectrum of the TMS ether of this compound (Fig. l(c)) exhibits a fragmentation pattern characteristic
of steroidal sapogenins
(25). The presence of the A13(17) double bond results in an intense ion at m/e 128, probably formed from the spiroketal moiety and not normally present in spectra of ring D-saturated sapogenins. The pure tigogenin (V) could be obtained by recrystallization acetone. Compound V was transformed into its acetate (VI)
from
and subjected
to side-chain degradation according to the procedure of Wall -et al. (9) to yield the labelled 3p-hydroxy-5a-pregn-16-en-20-one
(VII),
where 91.6%
of the molecules were dideuterated. This Cpl steroid served as a common precursor for the synthesis of the deuterated pregnanolone and pregnanediol sulphates. Scheme 2 indicates the sequence of reactions leading from the A16pregnenolone
(VII) to the labelled pregnanolone sulphate (XI). Cataly-
tic hydrogenation of VII over palladium/charcoal
(7) proceeded smoothly
to yield 3B-hydroxy-5a-~l,ll-2H2]pregnan-20-one
(VIII).
The requirement
that the synthetic end-products be labelled such that the extent of oxidoreduction
of hydroxyl groups could be followed during metabolic
experiments necessitated the introduction of a deuterium atom at each site bearing a free or sulphated hydroxyl group. These labels would thus serve as markers for the processes of oxidoreduction, followed by oxidoreduction,
and hydrolysis
respectively. Thus, compound VIII was oxi-
S
450
%'B&OlcD1
SCHEME 2
0
X
XI
dized with Jones reagent (l'i) to give 5~-~~,~l"2H2~pregnane-3,20-dione (IX), which was selectively reduced at C-3 with sodium borodeuteride (13). Straight-phase
chromatography
of the reaction product on the lipo-
philic Sephadex derivative LipidexR -5000 resulted in complete separation of the resulting pregnanolones, epimeric at C-3, and afforded the desired 3B-hydroxy-5a-~a~ll,?l-*H~pregnan-20-one
(X). Fig. 2 illustra-
tes the mass spectra of the TMS ether derivatives of compound X and the corresponding
unlabelled pregnanolone. Calculations of deuterium con-
tent indicated the isotope excess at the 3a-position to be 97 atoms%. Preparation of the steroid sulphate XI was carried out essentially as described by Mumma (30) and the product was purified as the potassium salt (in view of its relatively high solubility in water) by chromatography an Sephadex LH-20 (14).
Q ?8
8
m
I”
000000 yrlul~nr .lNI ml3
IN33
t13d
S
452
WDEOIDI SCHEME
3
xv
XIV I
XIX
XVII Ri='H;R2=Ac
XVI
XVIII R1=S03K;R2=Ac
The synthetic route to the deuterated pregnanediol sulphates is summarized in Scheme 3. The target compounds were the 3,20-disulphate (XV) and the 3-monosulphate
(XIX) of 5a-~~,11,11,ZOB-2H4]pregnane-3~,
ZOa-diol (XIV). Although the 3B-hydroxy-5o:-[3a-*qconfiguration
is
readily accessible through treatment of 3-keto-5a-steroids with complex
453
m
TIlEOXDl
8
i-u
S
454
TDEOXDI
metal deuterides, such reduction of ZO-ketopregnanes
gives appreciable
amounts of the more hindered 208, rather than the required 2Oa, alcohol (31). Reductions of 20-ketosteroids with alkali metals and alcohols yield mixtures containing up to 69% of the 201~01, in addition to variable amounts of the 17a-pregnanes (32). However, treatment of A '6_20_ ketones with lithium aluminum hydride is reported to give almost exclusively the pregnan-20a-ol
Al6 -20~~01, which can be hydrogenated to the corresponding (19). We have recently shown that deuterium is intro-
duced at the 206-position
in high isotopic purity by the use of lithium
aluminum deuteride in the above reaction and that the label is retained, without scrambling, in the subsequent hydrogenation step (33). Based on these considerations, 3,20-dione
the pregnenolone
VII was transformed, via the
XII, into a mixture of isomeric 'H4-pregnenediols (XIII)
prior to saturation of the hydrogenation of
Al6 double bond. The major product from the
XIII was the desired 5a-[3a,11,11,20B-2H4~pregnane-38,
'ZOa-dial (XIV), which was purifi,ed by reversed-phase chromatography LipidexR-1000.
The mass spectrum
on
of the TMS ether derivative of com-
pound XIV is compared with that of the corresponding unlabelled pregnanediol in Fig. 3. Mass shifts of ions in the upper mass region confirm the presence of four deuterium atoms in compound
XIV, while incorpora-
tion of the label at the 208-position is clearly shown by the displacement of the intense side-chain ion at m/e 117 (34) to m/e 118. The isotope excess of the labelled pregnanediol
XIV indicated that deu-
terium had been introduced in 98.8 and 96.0% isotopic purity at the 3a and 208 positions, respectively. Synthesis and purification of the disulphate XV were carried out in a manner similar to that described above for the 'H3-pregnanolone
(X -XI).
S
455 Finally, the from
3-monosulphate
free diol
palan and
WmEOZDm
according to
(35), in
its acetate.
(XIX)
procedure described by
the ZOa-hydroxy group is
as
compound XIV was converted to its diacetate XVI
which was selectively hydrolyzed with sodium bicarbonate in methanol (21) to afford 5n-[3a,11,11,20B-2H~~pregnane-3~,20a-diol
20-monoacetate
(XVII). Sulphation, followed by removal of the acetate protecting group under strongly alkaline conditions, yielded the labelled pregnanediol 3-monosulphate XIX. Since the aim of this work was to obtain labelled steroid sulphates for experiments in human subjects, a high degree of purity of the endproducts was necessary. For this purpose, compounds XI, XV and XIX were purified by chromatography on Sephadex LH-20 in a system where potassium salts of steroid sulphates have a very characteristic mobility (14). Furthermore, potassium steroid sulphates, in contrast to the corresponding sodium salts, are sufficiently soluble in water to permit intravenous injections of the doses (5-10 I_rmole/lOml) required. Criteria for purity of the labelled sulphates were their homogeneity and mobility on thin-layer chromatography and on gas chromatographic analysis following solvolysis. Much of the purification of intermediates in the synthetic sequences was carried out using liquid-gel chromatography on hydrophobic LipidexR gels having properties very similar to those deet al. (36). These gels provide a mild environment scribed by Ellingboe -where the risk of deuterium exchange is minimized, and permit the use of either straight- or reversed-phase solvent systems. Steroids with the Al6 -2O-oxopregnane
structure may be transformed
by established procedures into a wide range of pregnane and androstane
S derivatives
456
TDEOTDI
(37). Thus, the common intermediate in each of the above
synthetic schemes, 3~-hydroxy-5w~1,11-*H~pregn-16-en-20-one
(VII),
could serve as a precursor for the preparation of a large number of both naturally occurring and synthetic steroids, labelled with deuterium at C-II. Several of the intermediates described in this paper may themselves be useful in studies of metabolic transformations of unconjugated steroids -in vivo and -in vitro. Thus, 5a-pregnane-3,20-dione (38,39) and 3B-hydroxy-5a-pregnan-20-one
(39) occur normally in plasma
of pregnant women. Furthermore, since high isotopic purity is obtained, the compounds containing 3 or 4 deuterium atoms could be employed as internal standards in quantitative analyses by selective ion monitoring. Results of studies on the metabolism of the labelled steroid sulphates in pregnant women will be reported in separate communications. ACKNOWLEDGEMENTS The skillful technical assistance of Mrs Kerstin Robertsson, Miss Irene Ferdman and Miss Marit Karls is gratefully acknowledged. We also wish to thank Professor B. Lindberg (Department of Organic Chemistry, Stockholm University) for use of hydrogenation facilities and Dr. C.J.W. Brooks (Chemistry Department, University of Glasgow) through worn elemental analyses were obtained. The work was supported by grants from the Swedish Medical Research Council (grants No. 13X-219 and 60R-4166) and by the World Health Organization. T.A. Eaillie was the holder of a Postdoctoral Fellowship from the Royal Society (London), under the European Science Exchange Programme. J.E. Herz was a Swedish Medical Research Council Visiting Scientist at the Karolinska Institute, 1973-74. REFERENCES Present address: Department of Clinical Pharmacology, Royal Postgraduate Medical School, London WI2 OHS, England. 2. Sjijvall, J., Sjovall, K. and Vihko, R. STEROIDS II, 703 (1968). Sjovall; K.-ANN. CLIN. RES. 2, 393-(1970). 43: Makita, M. and Wells, W.W. ANAL. BIOCHEM. 5, 523 (1963 5. Reimendal, R. and SjUvall, 3. ANAL. CHEM. 44, 21 (1972 6. Axelson, M., Cronholm, T., Curstedt, T., Reimendal, R. and Sjovall, J. CHROMATOGRAPHIA 7, 502 (1974). 7. Marker, R.E., Wagner, R.B., Ulshafer, P.R., Wittbecker ', E.L., Goldsmith, D.P.J. and Ruof, C.H. J. AM. CHEM. SOC. 69, 2167 (1947). 1.
1:
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. ;:: 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
:::
32. 33. 34. ::: 37.
Hirschmann, R., Snoddy, C.S., Hiskey, C.F. and Wendler, N.L. J. AM. ;+$. ;O;. 76, 4013 (1954). Kenney, H.E. and Rothman, E.S. J. AM. CHEM. SOC. 77, 5665'(1955;. F;;j:;, F.E. and Turner, D.L. J. AM. CHEM. SOC. 62, 3003 (1940). Heilbron, I.M., Jones, E.R.H. and Weedon, B.C.L. J. CHEM. sot. 3; (i446). Marker, R.E., Kamm, 0. and McGrew, R.V. J. AM. CHEM. SOC. 59, 616 (1937). Mancera, O., Ringold, H.J., Djerassi, C., Rosenkranz, G. and Sondheimer, F. J. AM. CHEM. SOC. 75, 1286 (1953). Sjovall, J. and Vihko, R. ACTA CHEM. SCAND. 20, 1419 (1966). But-stein, S. and Lieberman, S. J. BIOL. CHEM. 233, 331 (1958). Bradlow, H.L. STEROIDS 11, 265 (1968). Lieberman, S., Dobriner, K., Hill, B.R., Fieser, L.F. and Rhoads, C.P. J. BIOL. CHEM. 172, 263 (1948). Marker, R.E. and Rohrmann, E. J. AM. CHEM. SOC. 62, 898 (1940). Shapiro, E.L., Gould, D. and Hershberg, E.B. J. AM. CHEM. SOC. 77, 2912 (1955). Marker, R.E., Kamm, O., Wittle, E.L., Oakwood, T.S., Lawson, E.J. and Laucius, J.F. J. AM. CHEM. SOC. 59, 2291 (1937). Butenandt, A. and Schmidt, J. CHEM. BER. 67, 1893 (1934). Tijkes, L., Jones, G. and Djerassi, C. J. AM. CHEM. SOC. 90, 5465 (1968). Faul, W.H., Failli, A. and Djerassi, C. J. ORG. CHEM. 35, 2571 (1970). Fisher, M., Pelah, Z., Williams, D.H. and Djerassi, C. CHEM. BER. 98, 3236 (1965). Budzikiewicz, H., Djerassi, C. and Williams, D.H. STRUCTURE ELUCIDATION OF NATURAL PRODUCTS BY MASS SPECTROMETRY, Vol. 2. HoldenDay Inc., San Fransisco, 1964, chapter 22. Caglioti, L. and Magi, M. TETRAHEDRON 19, 1127 (1963). Hirschmann, R., Snoddy, C.S. and Wendler, N.L. J. AM. CHEM. SOC. 74, 2693 (1952). J. Hirschmann, R., Snoddy, C.S., Hiskey, C.F. and Wend1er;N.L. AM. CHEM. SOC. 76, 4013 (1954). Elks, J., Phillipps, G.H., Taylor, D.A.H. and Wyman, L.J. J. CHEM. sot. 1739 (1954). Mumma, R.O. LIPIDS 1, 221 (1966). Wheeler, D.M.S. and Wheeler, M.M. in ORGANIC REACTIONS IN STEROID CHEMISTRY, Vol. 1, Ed. Fried, J. and Edwards, J.A. Van Nostrand Reinhold, New York, 1972, chapter 2. J. CHEM. SOC. (C), 968 (1969). Kirk, D.N. and Mudd, A.J. J. LABELLED COMPOUNDS Baillie, T.A., Herz, J .E. and Sjovall, J. 10, 549 (1974). Adlercreutz, H., Luukkainen, T. and Taylor, W. EUR. J. STEROIDS 1, 117 (1966). Rajagopalan, M.S. and Turner, A.B. J. CHEM. SOC. (C) 1858 (1969). Ellingboe, J., Nystrom, E. and Sjiivall, J. J. LIPID RES. 11, 266 (1970). Fieser, L.F. and Fieser, M. STEROIDS,Reinhold Publishing Co., New York, 1959, chapters 17,and 19. Stda, K.F. and Bessesen, A. J. STEROID BIOCHEM. 6, 21 (1975). Axelson, M. and Sjbvall, J. J. STEROID BIOCHEM. 5, 733 (1974).
