Synthesis of a 19-(O-carboxymethyl)oxime hapten of 7β-hydroxy-epiandrosterone

Horm Mol Biol Clin Invest 2011;7(1):295–301 � 2011 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/HMBCI.2011.108

2011/0108

Article in press - uncorrected proof

Synthesis of a 19-(O-carboxymethyl)oxime hapten

of 7b-hydroxy-epiandrosterone

Clotilde Ferroud1, Gilbert Revial1,*, ChristopheRicco1, Olivier Hennebert2 and Robert Morfin2

1 Laboratoire de Transformation Chimique etPharmaceutique, ERL3193 CNRS/ESPCIParisTech/Cnam,2 rue Conte, 75003 Paris, France

2 Laboratoire de Biologie EA3199 Conservatoire nationaldes arts et metiers, 2 rue Conte, 75003 Paris, France

Abstract

In order to develop an immunoassay of 7b-hydroxy-epian-drosterone, a stereoselective synthesis of a specific hapten,7b-hydroxy-19-oxo-androstan 19-(O-carboxymethyl)oxime(17), was performed. This synthesis was achieved in 16%overall yield starting from the well-known 3b-acetoxy-19-hydroxy-5-androsten-17-one (1). After coupling of the alkyloxime moiety, an allylic oxidation of the C-7 carbon undermild conditions followed by two selective reductions estab-lished all the functionalities of the final compound 17.

Keywords: anti-inflammatory compound; stereoselectivesynthesis; 19-(O-carboxymethyl)oxime hapten of 7b-hydroxy-epiandrosterone.

Introduction

The biological occurrence and effects of 7b-hydroxy-epian-drosterone (7b-hydroxy-EpiA) were described in animalsand humans. Native 7b-hydroxy-EpiA derives from a two-step enzymatic transformation of epiandrosterone (EpiA).Firstly EpiA is 7a-hydroxylated by CYP-7B1 w1x and sec-ondly, the 7a-hydroxy-EpiA produced is inter-converted in7b-hydroxy-EpiA by the 11b-hydroxysteroid dehydrogenasetype 1 w2x. Studies of 7b-hydroxy-EpiA effects in rats werecarried out in vivo on a model of dextran sodium sulfateinduced colitis and showed a high potency for triggering theresolution of inflammation through a shift from inflamma-tory prostaglandin synthesis (PGE2) to prostaglandins asso-ciated with cell protection and resolution of inflammation(PGD2, 15d-PGJ2) w3x. The same shift was obtained withhuman peripheral blood monocytes cultured in the presenceof TNF-a w4x. In addition, 7b-hydroxy-EpiA triggeredtamoxifen-like effects in cultures of several human breastcancer cell lines w5x. Nanomolar concentrations of 7b-hydroxy-EpiA were sufficient to raise these effects and thissuggested that the steroid bound to a specific receptor, which

*Corresponding author: Dr. Gilbert RevialE-mail: [email protected] March 25, 2011; accepted July 19, 2011;previously published online August 30, 2011

may be found among estrogen receptors w4, 5x. Therefore,the knowledge of 7b-hydroxy-EpiA production and levels inhumans could be of interest in clinical and pharmacologicalinvestigations and chemical tools should also be prepared forreceptor characterization. Immunoassays were developed forother 7-hydroxysteroids, such as 7b- and 7a-hydroxy-dehy-droepiandrosterone w6, 7x and were used in several investi-gations w8, 9x. Therefore, our final objective was to producea hapten of 7b-hydroxy-EpiA in order to both characterizeits receptor and to develop an immunoassay for 7b-hydroxy-EpiA. To this end, the whole specific structure of 7b-hydroxy-EpiA should be made available as an epitope to theimmunoglobulin paratope, and as a putative ligand for estro-gen receptors. This led to a search for the construction of7b-hydroxy-EpiA derivatives, which could be linked throughthe C-19 of the hapten, either to a protein moiety or to amatrix. Such an approach was published for other steroidsw10, 11x but needed adaptation for 7b-hydroxy-EpiA. Thehapten once coupled to inert matrixes could be used in affin-ity chromatography columns for characterization of the puta-tive receptor in estrogen receptor containing human breastcancer cells.

In this paper we report an efficient eight-step synthesis ofthe compound 17 derived from 7b-hydroxy-EpiA and bear-ing a linker at the C-19, which can be coupled to a proteinmoiety or bonded to inert matrixes.

Experimental

General remarks

The reactions were monitored by thin layer chromatographies (TLC)on aluminum plates (0.20 mm) precoated with a fluorescent silicagel using EtOAc/cyclohexane as eluent. Reaction components werevisualized under UV light and then dipped in iodine vapor. Silicagel (40–63 mm) was used for flash chromatography (FC) separa-tions and EtOAc/cyclohexane was the eluent. Gas chromatography-mass spectroscopy (GC-MS) was performed with an Agilent 6890N(equipped with a 12 m=0.20 mm dimethylpolysiloxane capillarycolumn) linked to a Model 5973N. Melting points were determinedwith a Kofler block. Optical rotations were measured with a Perkin-Elmer model 241. Infrared spectra (wavenumbers in cm–1) wererecorded on Nicolet apparatus model Avatar 320. 1H and 13C NMRspectra were recorded respectively at 400 and 100 MHz (CDCl3unless otherwise stated). Chemical shifts for hydrogen and carbonresonances are reported in ppm (d) relative to TMS. Reagents andmaterials were obtained from commercial suppliers and were usedwithout further purification.

3b-Acetoxy-19-hydroxy-5-androsten-17-one (1)

Compound 1. Prepared according to the literature w12x. Rf: 0.40(40% EtOAc in cyclohexane); mp 1618C (EtOAc/cyclohexane)

AUTHOR’S COPY | AUTORENEXEMPLAR


296 Ferroud et al.: Synthesis of a 19-(O-carboxymethyl)oxime hapten of 7b-hydroxy-epiandrosterone


(Ref. w12x mp 1618C–1648C (ether/hexane); waxD20q2.0 (c 1.23,

CHCl3); EIMS m/z 286 (M–60, 7%), 268 (2), 256 (39), 255 (100),237 (24), 159 (14), 145 (20), 143 (27); IR (KBr) 3464, 1731 cm–1;1H NMR 0.92 (s, 3H), 2.02 (s, 3H), 3.62 (d, Js11.5 Hz, 1H), 3.87(d, Js11.5 Hz, 1H), 4.63 (tt, Js11.5, 4.8 Hz, 1H), 5.78 (m, 1H);13C NMR 13.88 (CH3), 20.90 (CH2), 21.31 (CH3), 21.69 (CH2),28.00 (CH2), 30.14 (CH2), 31.66 (CH2), 32.86 (CH), 33.22 (CH2),35.76 (CH2), 38.15 (CH2), 41.67 (C), 47.75 (C), 50.40 (CH), 52.45(CH), 62.74 (CH2), 73.27 (CH), 127.2 (CH), 135.0 (CH), 170.4 (C),220.9 (C).

3b-Acetoxy-17-oxo-5-androsten-19-oic acid (2)

To a stirred solution of 3.55 g (10.2 mmol) of 3b-acetoxy-19-hydroxy-5-androsten-17-one (1) in 220 mL of acetone at 08C, 16.4mL of Jones reagent (2.7 M) was added dropwise. After 3 h, 3 mLof 2-propanol was added and the reaction mixture was diluted withice cold water. 65 g of NaOAc was added and the mixture wasextracted with EtOAc. The organic layer was washed with H2O,brine and then dried (Na2SO4) and evaporated. The resulting crudeproduct was purified by flash chromatography (70% EtOAc incyclohexane) to afford 2.10 g (57% yield) of 2 w13x.

Compound 2. Rf: 0.25 (60% EtOAc in cyclohexane); mp 2648C(EtOAc/cyclohexane) (Ref. w13x mp 2538C); waxD

26–61 (c 2.4,CHCl3); EIMS m/z 256 (M–104, 73%), 254 (100), 239 (10), 226(8), 211 (11), 197 (33), 183 (14), 156 (30), 143 (38), 129 (48), 115(32), 91 (53); IR (KBr) 3417, 1727, 1702 cm–1; 1H NMR 0.84(s, 3H), 2.02 (s, 3H), 2.24 (dtd, Js17.4, 5.3, 2.2 Hz, 1H), 2.47(dd, Js19.3, 8.7 Hz, 1H), 2.57 (m, 2H), 4.64 (tt, Js11.5, 4.6 Hz,1H), 5.75 (d, Js5.6 Hz, 1H); 13C NMR 13.62 (CH3), 21.29 (CH3),21.66 (CH2), 22.43 (CH2), 29.04 (CH2), 29.84 (CH2), 31.31 (CH2),31.51 (CH), 33.25 (CH2), 35.68 (CH2), 40.03 (CH2), 47.55 (C),48.72 (CH), 50.64 (C), 51.28 (CH), 72.67 (CH), 125.4 (CH), 133.6(C), 170.6 (C), 179.3 (C), 220.5 (C).

Methyl 3b-acetoxy-17-oxo-5-androsten-19-oate (3)

To 1.90 g (5.27 mmol) of 3b-acetoxy-17-oxo-5-androsten-19-oicacid (2) dissolved in 20 mL of ether an excess of ethereal diazo-methane solution was added. After 1 h, the excess diazomethanewas destroyed by a few drops of acetic acid and the solvent wasremoved under reduced pressure. The resulting crude product waspurified by flash chromatography (40% EtOAc in cyclohexane) togive 1.45 g of 3 in 73% yield.

Compound 3. Rf: 0.5 (40% EtOAc in cyclohexane); mp 1888C(EtOAc/cyclohexane); waxD

24–60 (c 2.4, CHCl3); EIMS m/z 314(M–60, 27%), 299 (1), 286 (1), 271 (1), 256 (19), 255 (100), 254(23), 237 (20), 145 (31), 91 (16); IR (KBr) 1730 cm–1; 1H NMR0.81 (s, 3H), 2.00 (s, 3H), 2.23 (dtd, Js17.5, 5.5, 2.4 Hz, 1H), 2.43(dd, Js19.2, 8.6 Hz, 1H), 2.51 (ddd, Js12.9, 4.7, 2.4 Hz, 1H),2.61 (dt, Js13.5, 3.5 Hz, 1H), 3.72 (s, 3H), 4.61 (tt, Js11.5, 4.7Hz, 1H), 5.71 (d, Js5.8 Hz, 1H); 13C NMR 13.51 (CH3), 21.26(CH3), 21.66 (CH2), 22.53 (CH2), 29.08 (CH2), 29.85 (CH2), 31.28(CH2), 31.63 (CH), 33.56 (CH2), 35.69 (CH2), 40.05 (CH2), 47.47(C), 48.88 (CH), 50.90 (C), 51.27 (CH), 51.79 (CH3), 72.74 (CH),124.9 (CH), 134.1 (C), 170.4 (C), 173.9 (C), 220.2 (C).

Methyl 3b-acetoxy-17,17-ethylenedioxy-5-androsten-

19-oate (4)

A solution of 1.00 g (2.67 mmol) of methyl 3b-acetoxy-17-oxo-5-androsten-19-oate (3), 0.50 mL (9.0 mmol) of ethanediol and 20mg of PTSA (p-toluenesulfonic acid) in 25 mL of benzene washeated at refluxing temperature for 3 h in a Dean-Stark apparatus.

The mixture was then cooled to room temperature and quenched bytwo drops of triethylamine. The solvent was removed under reducedpressure and the resulting crude product was purified by flash chro-matography (30% EtOAc in cyclohexane) to give 820 mg (73%yield) of 4 as white crystals.


26–98 (c 2.0, CHCl3); EIMS m/z 358(M–60, 59%), 314 (1), 299 (13), 237 (37), 99 (100); IR (KBr) 1730,1720, 1633 cm–1; 1H NMR 0.72 (s, 3H), 1.93 (s, 3H), 2.06 (m, 1H),2.43 (ddd, Js12.8, 4.5, 2.3 Hz, 1H), 2.54 (dt, Js13.4, 3.4 Hz, 1H),3.65 (s, 3H), 3.72-3.88 (m, 4H), 4.55 (tt, Js11.4, 4.7 Hz, 1H), 5.62(d, Js5.7 Hz, 1H); 13C NMR 14.13 (CH3), 21.28 (CH3), 22.48(CH2), 22.76 (CH2), 29.08 (CH2), 30.28 (CH2), 30.36 (CH2), 32.24(CH), 33.60 (CH2), 34.10 (CH2), 40.10 (CH2), 45.68 (C), 48.61(CH), 50.16 (CH), 50.82 (C), 51.73 (CH3), 64.49 (CH2), 65.17(CH2), 72.86 (CH), 119.2 (C), 125.4 (CH), 133.8 (C), 170.4 (C),174.1 (C).

Methyl 3b-acetoxy-17,17-ethylenedioxy-7-oxo-5-

androsten-19-oate (5)

To 1.54 g (15.4 mmol) of CrO3, suspended in 10 mL of dry CH2Cl2

under argon at –258C, 1.54 g (16.0 mmol) of 3,5-dimethylpyrazolewas quickly added. After 30 min 430 mg (1.03 mmol) of methyl3b-acetoxy-17,17-ethylenedioxy-5-androsten-19-oate (4) in 5 mL ofdry CH2Cl2 was added dropwise and the solution was stirred at–158C for 4 h and then cooled to –258C overnight. After dilutionwith a mixture of benzene-ethyl acetate (7:3) the reaction mixturewas filtered through a short column of silica gel layered with celite.The column was washed with the same solvent mixture and thecombined organic layers were evaporated under reduced pressure.The oily residue was purified by flash chromatography (40% EtOAcin cyclohexane) to give 226 mg (51% yied) of 5.


26–197 (c 2.3, CHCl3); EIMS m/z 432(Mq, 1%), 389 (1), 373 (9), 313 (5), 273 (11), 100 (55), 99 (100);IR (KBr) 1730, 1668, 1640 cm–1; 1H NMR 0.71 (s, 3H), 1.96(s, 3H), 2.39 (m, 1H), 2.48 (dd, Js12.8, 11.2 Hz, 1H), 2.63(ddd, Js13.5, 4.7, 2.4 Hz, 1H), 2.69 (dt, Js14.1, 3.7 Hz, 1H), 3.73(s, 3H), 3.73–3.87 (m, 4H), 4.66 (tt, Js11.5, 4.7 Hz, 1H), 5.85(d, Js1.7 Hz, 1H); 13C NMR 14.33 (CH3), 21.17 (CH3), 22.50(CH2), 24.65 (CH2), 28.75 (CH2), 29.52 (CH2), 33.09 (CH2), 34.06(CH2), 39.69 (CH2), 44.24 (CH), 45.59 (CH), 46.02 (C), 47.93 (CH),51.97 (C), 52.44 (CH3), 64.45 (CH2), 65.21 (CH2), 71.48 (CH),118.4 (C), 128.5 (CH), 155.5 (C), 170.2 (C), 171.5 (C), 200.8 (C).

Methyl 3b-acetoxy-17,17-ethylenedioxy-7-oxo-5a-

androstan-19-oate (6)

190 mg (0.44 mmol) of methyl 3b-acetoxy-17,17-ethylenedioxy-7-oxo-5-androsten-19-oate (5) and 78 mg of 10% Pd/C were stirredin 15 mL of EtOAc under hydrogen at room temperature for 1 h.The solution was then filtered and the solvent was evaporated andthe crude compound was purified by flash chromatography (40%EtOAc in cyclohexane) to give 156 mg (82% yield) of ketone 6.


20–35.8 (c 0.81, CHCl3); EIMS m/z 434(Mq, 1%), 419 (1), 361 (1), 335 (2), 275 (1), 253 (1), 215 (2), 155(2), 139 (3), 100 (55), 99 (100); IR (KBr) 1730, 1701 cm–1; 1HNMR 0.68 (s, 3H), 1.94 (s, 3H), 2.03 (dd, Js13.2, 4.4 Hz, 1H),2.20 (m, 1H), 2.41 (m, 1H), 2.49 (dt, Js14.5, 4.1 Hz, 1H), 3.28(t, Js13.5 Hz, 1H), 3.75 (s, 3H), 3.76–3.89 (m, 4H), 4.66 (tt, Js11.3, 5.0 Hz, 1H); 13C NMR 14.38 (CH3), 21.24 (CH3), 22.65 (CH2),23.73 (CH2), 29.12 (CH2), 29.54 (CH2), 32.75 (CH2), 33.99 (CH2),



Ferroud et al.: Synthesis of a 19-(O-carboxymethyl)oxime hapten of 7b-hydroxy-epiandrosterone 297


35.40 (CH2), 43.35 (CH), 45.02 (CH), 45.48 (C), 46.04 (CH2), 49.95(CH), 50.66 (C), 51.50 (CH3), 51.61 (CH), 64.42 (CH2), 65.18(CH2), 71.82 (CH), 118.5 (C), 170.4 (C), 173.6 (C), 210.7 (C).

Methyl 3b-acetoxy-17,17-ethylenedioxy-7b-hydroxy-

5a-androstan-19-oate (7)

To 144 mg (0.33 mmol) of methyl 3b-acetoxy-17,17-ethylenedioxy-7-oxo-5a-androstan-19-oate (6) and 186 mg (0.50 mmol) ofCeCl3,7H2O in 5 mL of MeOH at 08C, 29 mg (0.77 mmol) ofNaBH4 was added portionwise. After 2 h, 1 mL of acetone wasadded and the mixture was stirred for 30 min. After evaporation ofthe solvent under reduced pressure, 20 mL of water was added andthe residue was extracted with EtOAc. The combined organic layerswere dried over anhydrous MgSO4, the solvent was removed, andthe resulting product was purified by flash chromatography (50%EtOAc in cyclohexane) to afford 117 mg (81% yield) of alcohol 7.


23–2.7 (c 2.1, CHCl3); EIMS m/z 418(Mq–18, 2%) 403 (1), 376 (1), 356 (1), 335 (8), 316 (1), 275 (2),100 (24), 99 (100); IR (KBr) 3543, 1731 cm-1; 1H NMR 0.76 (s,3H), 1.96 (s, 3H,), 2.14-2.26 (m, 1H), 2.42–2.50 (m, 1H), 3.38 (ddd,Js10.8, 8.9, 5.4 Hz, 1H), 3.69 (s, 3H), 3.83 (m, 4H), 4.70 (tt,Js10.8, 5.4 Hz, 1H); 13C NMR 14.56 (CH3), 21.32 (CH3), 22.18(CH2), 25.58 (CH2), 29.24 (CH2), 30.15 (CH2), 33.62 (CH2), 34.20(CH2), 35.31 (CH2), 38.44 (CH2), 41.74 (CH), 43.83 (CH), 46.40(C), 49.72 (CH), 49.83 (C), 49.91 (CH), 51.10 (CH3), 64.45 (CH2),65.19 (CH2), 72.39 (CH), 74.92 (CH), 118.5 (C), 170.6 (C), 173.9(C).

Methyl 3b-acetoxy-7b-hydroxy-17-oxo-5a-androstan-

19-oate (8)

95 mg (0.22 mmol) of methyl 3b-acetoxy-17,17-ethylenedioxy-7b-hydroxy-5a-androstan-19-oate (7) and 8 mg of PTSA in a mixtureof 10 mL of acetone and 1 mL of water was stirred overnight atroom temperature. After evaporation of the solvent under reducedpressure, the residue was dissolved in 20 mL of EtOAc and theorganic layer was washed with saturated NaHCO3, water, brine anddried over anhydrous MgSO4. The solvent was removed underreduced pressure, and the resulting crude product was purified byflash chromatography (50% EtOAc in cyclohexane) to afford 60 mg(69% yield) of 8.

Compound 8. Rf: 0.4 (60% EtOAc in cyclohexane); EIMS m/z392 (Mq, 5%), 374 (4), 342 (12), 314 (70), 300 (100), 282 (12),272 (10), 255 (60), 254 (87), 237 (19), 231 (24), 199 (36), 145 (68);IR (KBr) 3541, 1733 cm-1; 1H NMR 0.76 (s, 3H), 1.95 (s, 3H), 2.01(dd, Js19.1, 9.6 Hz, 1H), 2.18–2.32 (m, 2H), 2.36 (dd, Js19.4,8.7 Hz, 1H), 2.44 (dd, Js10.0, 3.5 Hz, 1H), 3.41 (ddd, Js10.5,10.4, 5.4 Hz, 1H), 3.67 (s, 3H), 4.66 (tt, Js11.0, 5.3 Hz, 1H); 13CNMR 13.97 (CH3), 21.31 (CH3), 22.06 (CH2), 24.95 (CH2), 29.19(CH2), 31.34 (CH2), 33.45 (CH2), 35.26 (CH2), 35.96 (CH2), 38.61(CH2), 41.79 (CH), 43.12 (CH), 48.10 (C), 49.81 (C), 50.08 (CH),50.96 (CH), 51.20 (CH3), 72.25 (CH), 74.31 (CH), 170.6 (C), 173.8(C), 221.2 (C).

Methyl 3b,7b-dihydroxy-17-oxo-5a-androstan-

19-oate (9)

0.2 mL of K2CO3 aqueous solution (1.5 M) was added to 52 mg(0.13 mmol) of methyl 3b-acetoxy-7b-hydroxy-17-oxo-5a-andros-tan-19-oate (8) in 5 mL of MeOH. The mixture was stirred for 1.5h at room temperature. The solvent was removed under reducedpressure and the residue was taken up in EtOAc and was purified

by flash chromatography (100% EtOAc) to afford 30 mg (66%yield) of 9.

Compound 9. Rf: 0.20 (EtOAc); mp 2018C (EtOAc/cyclohexane);waxD

22q105 (c 0.69, CHCl3); EIMS m/z 350 (Mq, 2%), 332 (3),318 (14), 314 (12), 301 (24), 300 (90), 285 (50), 272 (48), 255 (34),254 (38), 244 (33), 243 (23), 237 (27), 213 (38), 199 (52), 197 (52),185 (38), 157 (52), 145 (89), 131 (55), 105 (91), 91 (100); 1H NMR(DMSO-d6) 0.70 (s, 3H), 1.95 (dd, Js19.2, 9.6 Hz, 1H), 3.25 (m,1H), 3.41 (m, 1H), 3.63 (s, 3H), 4.32 (d, Js6.2 Hz, 1H), 4.50 (d,Js4.6 Hz, 1H); 13C NMR (DMSO-d6) 13.48 (CH3), 21.68 (CH2),24.76 (CH2), 31.14 (CH2), 33.17 (CH2), 33.23 (CH2), 35.46 (CH2),38.87 (CH2), 39.47 (CH2), 41.28 (CH), 42.42 (CH), 47.36 (C), 49.20(CH), 49.55 (C), 50.36 (CH), 50.78 (CH3), 68.41 (CH), 72.98 (CH),173.7 (C), 220.3 (C); HRMS-ESI m/z wMHqx calcd for C20H31O5:351.2171, found: 351.2206.

3b-Acetoxy-17,17-ethylenedioxy-19-hydroxy-5-

androstene (10)

3.60 g (10.4 mmol) of 3b-acetoxy-19-hydroxy-5-androsten-17-one(1), 1.5 mL (27 mmol) of ethanediol, and 60 mg of PTSA in 100mL of benzene were heated at refluxing temperature in a Dean-Stark apparatus for 3 h. The mixture was then cooled to room tem-perature and quenched by five drops of triethylamine. The solventwas removed under reduced pressure, and the resulting crude prod-uct was purified by flash chromatography (20% EtOAc in cyclo-hexane) to give 3.78 g (93% yield) of 10 w14x.

Compound 10. Rf: 0.40 (30% EtOAc in cyclohexane); mp 1428C(EtOAc/cyclohexane) wRef. w14x mp 1438C–1448C), waxD–84(CHCl3)x; waxD

20–81.6 (c 1.8, CHCl3); EIMS m/z 360 (M–30, 2%),330 (12), 300 (23), 299 (18), 238 (65), 237 (71), 100 (26), 99 (100);IR (KBr) 3519, 1733, 1711 cm–1; 1H NMR 0.91 (s, 3H), 1.13 (td,Js14.0, 4.2 Hz, 1H), 2.02 (s, 3H), 2.27 (m, 1H), 2.42 (ddd,Js13.0, 4.9, 2.2 Hz, 1H), 3.61 (dd, Js11.4, 9.0 Hz, 1H), 3.80–3.96(m, 5H), 4.64 (tt, Js11.4, 4.8 Hz, 1H), 5.76 (m, 1H); 13C NMR14.60 (CH3), 21.15 (CH2), 21.38 (CH3), 22.55 (CH2), 28.07 (CH2),30.58 (CH2), 30.82 (CH2), 33.15 (CH2), 33.54 (CH), 34.18 (CH2),38.18 (CH2), 41.58 (C), 45.90 (C), 50.12 (CH), 51.37 (CH), 62.73(CH2), 64.53 (CH2), 65.17 (CH2), 73.36 (CH), 119.4 (C), 127.9(CH), 134.6 (C), 170.5 (C).

3b-Acetoxy-17,17-ethylenedioxy-19-oxo-5-androstene

(11)

14 mL (173 mmol) of pyridine was added dropwise to a suspensionof 5.7 g (57 mmol) of CrO3 and 8.0 g of anhydrous MgSO4 in 100mL of anhydrous CH2Cl2 and the mixture was stirred at 08C underargon. After 20 min, 3.70 g (9.47 mmol) of 3b-acetoxy-17,17-ethy-lenedioxy-19-hydroxy-5-androstene (10) in 30 mL of anhydrousCH2Cl2 was added and the mixture was stirred at 08C for 4 h. Theorganic medium was then diluted with 120 mL of ether and filteredthrough a column of alumina, which was then washed with a mix-ture of CH2Cl2/ether (1/1). The combined organic filtrates wereevaporated under reduced pressure. The crude product was purifiedby flash chromatography (30% EtOAc in cyclohexane) to give 2.19g (60% yield) of 11 as white crystals w14, 15x.

Compound 11. Rf: 0.5 (40% EtOAc in cyclohexane); mp 1758C(EtOAc/cyclohexane) (Ref. w14x mp 1498C–1508C); waxD

23–280 (c2.34, EtOH); EIMS m/z 359 (M–29, 1%), 328 (11), 299 (17), 238(27), 237 (61), 143 (11), 100 (26), 100 (25), 99 (100); IR (KBr)3437, 1731, 1711 cm–1; 1H NMR 0.80 (s, 3H), 1.99 (s, 3H), 2.20(ddd, Js12.7, 5.5, 2.7 Hz, 1H), 2.49 (ddd, Js13.0, 4.5, 2.4 Hz,1H), 2.57 (dt, Js13.7, 3.8 Hz, 1H), 3.79–3.95 (m, 4H), 4.59(tt, Js11.6, 4.5 Hz, 1H), 5.87 (d, Js5.5 Hz, 1H), 9.66 (d, Js1.4





Hz, 1H); 13C NMR 14.10 (CH3), 21.26 (CH3), 21.68 (CH2), 22.41(CH2), 28.69 (CH2), 30.25 (CH2), 30.46 (CH2), 30.60 (CH2), 33.20(CH), 34.03 (CH2), 39.49 (CH2), 45.55 (C), 48.61 (CH), 50.34 (CH),53.53 (C), 64.52 (CH2), 65.20 (CH2), 72.63 (CH), 119.1 (C), 128.2(CH), 131.6 (C), 170.4 (C), 204.7 (CH).

3b-Acetoxy-17,17-ethylenedioxy-19-oxo-5-androsten

19-(O-methoxycarbonylmethyl)oxime (12)

1.82 g (16.7 mmol) of O-(carboxymethyl) hydroxylamine hemihy-drochloride and 2.16 g (5.56 mmol) of 3b-acetoxy-17,17-ethyle-nedioxy-19-oxo-5-androstene (11) in 30 mL of pyridine were stirredovernight at room temperature. Toluene (30 mL) was added and thesolvents were removed under reduced pressure. The residue wasdissolved in 350 mL of EtOAc and a solution of a 5% citric acid(175 mL) was added. The aqueous phase was extracted with EtOAcand the combined organic phases were washed with 5% citric acidsolution and water. The solvent was removed under reduced pres-sure and the residue was dissolved in 130 mL of ether and 65 mLof methanol. An excess of an ethereal solution of diazomethane wasadded at 08C until a slight yellow color of the medium persisted.Excess of diazomethane was then destroyed by a few drops of aceticacid and the solvent was removed under reduced pressure. Theresulting crude product was purified by flash chromatography (30%EtOAc in cyclohexane) to afford 2.53 g (95% yield, 2 steps) of 12as an amorphous solid (syn-anti mixture) w15x.

Compound 12. Rf: 0.4 (30% EtOAc in cyclohexane); mp 1008C(EtOAc/cyclohexane) (Ref. w15x mp 998C–1028C); waxD

22–151 (c2.0, CHCl3); EIMS m/z 385 (Mq–90, 1%), 325 (4), 263 (2), 237(2), 100 (20), 99 (100); IR (KBr) 1763, 1739 cm–1; 1H NMR 0.82(s, 3H), 2.00 (s, 3H), 2.33–2.42 (m, 2H), 3.75 (s, 3H), 3.80–3.95(m, 4H), 4.61 (m, 3H), 5.62 (m, 1H), 7.38 (s, 1H); 13C NMR 14.14(CH3), 21.35 (CH3), 21.41 (CH2), 22.55 (CH2), 28.27 (CH2), 30.36(CH2), 30.83 (CH2), 32.46 (CH), 33.07 (C), 34.07 (CH2), 38.56(CH2), 43.60 (C), 45.54 (C), 49.59 (CH), 50.13 (CH), 51.81 (CH3),64.51 (CH2), 65.19 (CH2), 70.43 (CH2), 73.31 (CH), 119.2 (C),125.5 (CH), 134.6 (C), 155.4 (CH), 170.5 (C), 170.6 (C).

3b-Acetoxy-17,17-ethylenedioxy-7,19-dioxo-5-

androsten 19-(O-methoxycarbonylmethyl)oxime (13)

To 8.5 g (85 mmol) of CrO3 (VI) trioxide suspended in 80 mL ofdry CH2Cl2 at –258C, 8.5 g (89.0 mmol) of 3,5-dimethylpyrazolewas quickly added. After 30 min, a solution of 2.46 g (5.17 mmol)of 3b-acetoxy-17,17-ethylenedioxy-19-oxo-5-androsten 19-(O-methoxycarbonylmethyl) oxime (12) in 20 mL of dry CH2Cl2 wasadded dropwise and the resulting mixture was stirred at –258C for48 h. The reaction medium was then diluted with a mixture oftoluene-EtOAc (7:3) and filtered through a short column of silicagel layered with celite. The column was washed with the samesolvent mixture, and the combined organic phases were evaporatedunder reduced pressure. The residue was purified by flash chro-matography (40% EtOAc in cyclohexane) to give 1.97 g (78%yield) of 13 as an amorphous solid (10/90 syn-anti mixture).

Compound 13. Rf: 0.45 (50% EtOAc in cyclohexane);waxD

20.5–177 (c 1.87, CHCl3); EIMS m/z 400 (Mq–89, 2%), 340(1), 314 (1), 267 (1), 252 (1), 240 (1), 213 (2), 139 (3), 117 (2),100 (30), 99 (100); IR (KBr) 1760, 1735, 1675 cm–1; 1H NMR 0.79(s, 3H), 1.99 (s, 3H), 2.27 (dd, Js11.5, 11.3 Hz, 1H), 2.34–2.44(m, 3H), 2.53 (ddd, Js13.8, 4.9, 2.2 Hz, 1H), 3.70 (s, 3H), 3.84(m, 4H), 4.58 (s, 2H), 4.69 (tt, Js11.5, 4.6 Hz, 1H), 5.81 (d, Js1.8Hz, 1H), 7.49 (s, 1H); 13C NMR 14.33 (CH3), 21.14 (CH2), 21.20(CH3), 24.83 (CH2), 27.85 (CH2), 29.63 (CH2), 33.12 (CH2), 34.07(CH2), 38.26 (CH2), 43.96 (CH), 44.94 (C), 45.37 (CH), 45.92 (C),

49.26 (CH), 51.90 (CH), 64.43 (CH2), 65.21 (CH2), 70.50 (CH2),71.72 (CH), 118.5 (C), 128.6 (C), 152.5 (C), 157.6 (C), 170.1 (C),170.3 (C), 200.7 (C); HRMS-ESI m/z wMqNaqx calcd forC26H35O8NNa: 512.22549, found: 512.22541.

3b-Acetoxy-17,17-ethylenedioxy-7,19-dioxo-5a-

androstan 19-(O-methoxycarbonylmethyl) oxime (14)

1.78 g (3.63 mmol) of 3b-acetoxy-17,17-ethylenedioxy-7,19-dioxo-5-androsten 19-(O-methoxy-carbonylmethyl)oxime (13) and 500 mgof 10% Pd/C in 50 mL of EtOAc were stirred under hydrogen atroom temperature for 3 h. After filtration and evaporation of thesolvent, the resulting crude product was purified by flash chroma-tography (50% EtOAc in cyclohexane) to give 1.45 g (81% yield)of 14 as an amorphous solid (10/90 syn/anti mixture).


20–48.9 (c 1.74, CHCl3); EIMS m/z 402 (Mq–89, 1%), 374 (1),341 (1), 139 (1), 113 (2), 100 (29), 99 (100); IR (KBr) 1750, 1734,1709 cm–1; 1H NMR 0.76 (s, 3H), 1.96 (s, 3H), 2.08 (dd, Js13.4,3.7 Hz, 1H), 2.40 (t, Js11.5 Hz, 1H), 2.75 (t, Js13.3 Hz, 1H),3.72 (s, 3H), 3.75–3.85 (m, 4H), 4.63 (s, 2H), 4.67 (tt, Js10.8, 5.4Hz, 1H), 7.69 (s, 1H); 13C NMR 14.37 (CH3), 21.27 (CH3), 21.35(CH2), 23.71 (CH2), 27.49 (CH2), 29.52 (CH2), 32.98 (CH2), 33.98(CH2), 34.19 (CH2), 43.06 (C), 43.33 (CH), 45.17 (CH), 45.40(CH2), 45.50 (C), 50.44 (CH), 51.93 (CH3), 53.08 (CH), 64.44(CH2), 65.20 (CH2), 70.54 (CH2), 72.30 (CH), 118.5 (C), 152.6(CH), 170.2 (C), 170.4 (C), 210.0 (C); HRMS-ESI m/z wMqNaxcalcd for C26H37O8NNa: 514.24114, found: 514.24113.

3b-Acetoxy-17,17-ethylenedioxy-7b-hydroxy-19-

oxo-5a-androstan 19-(O-methoxycarbonyl-methyl)

oxime (15)

To 1.30 g (2.64 mmol) of 3b-acetoxy-17,17-ethylenedioxy-7,19-dioxo-5a-androstan 19-(O-methoxycarbonylmethyl)oxime (14) in 20mL of MeOH 1.0 g (2.7 mmol) of CeCl3,7H2O was added. At 08C,150 mg (4.0 mmol) of NaBH4 was added portionwise. After 3 h,the reaction was quenched with 2 mL of acetone and the mixturewas stirred for 30 min. The solvent was removed under reducedpressure and the residue was taken up in 50 mL of water and extract-ed with EtOAc. The combined organic layers were dried over anhy-drous MgSO4 and evaporated under reduced pressure. The cruderesulting product was then purified by flash chromatography (60%EtOAc in cyclohexane) to give 920 mg (71% yield) of 15 as anamorphous solid (10/90 syn/anti mixture).

Compound 15. Rf: 0.5 (70% EtOAc in cyclohexane); waxD20–16.1

(c 1.6, CHCl3); EIMS m/z 404 (Mq–89, 2%), 386 (2), 341 (1), 237(1), 199 (1), 157 (1), 155 (1), 145 (1), 131 (2), 100 (25), 99 (100);IR (KBr) 3492, 1750, 1733 cm–1; 1H NMR 0.79 (s, 3H), 1.96(s, 3H), 3.42 (ddd, Js10.3, 10.2, 5.5 Hz, 1H), 3.72 (s, 3H), 3.75–3.85 (m, 4H), 4.60 (s, 2H), 4.67 (tt, Js10.9, 5.0 Hz, 1H), 7.58(s, 1H); 13C NMR 14.46 (CH3), 20.89 (CH2), 21.30 (CH3), 25.46(CH2), 27.67 (CH2), 29.96 (CH2), 32.63 (CH2), 34.16 (CH2), 34.47(C), 37.81 (CH2), 41.78 (CH), 42.40 (C), 43.96 (CH), 46.41 (C),49.72 (CH), 50.90 (CH), 51.83 (CH), 64.46 (CH2), 65.20 (CH2),70.48 (CH2), 72.90 (CH), 74.92 (CH), 118.5 (C), 152.8 (CH), 170.5(C), 170.6 (C); HRMS-ESI m/z wMqNax calcd for C26H39O8NNa:516.25679, found: 516.25668.

3b-Acetoxy-7b-hydroxy-17,19-dioxo-5a-androstan

19-(O-methoxycarbonylmethyl)oxime (16)

To 910 mg (1.84 mmol) of 3b-acetoxy-17,17-ethylenedioxy-7b-hydroxy-19-oxo-5a-androstan 19-(O-methoxycarbonylmethyl)oxime





Figure 1 7b-Hydroxy-epiandrosterone and one C-19 hapten.

(15) in 40 mL of acetone, 50 mL of water and 60 mg of PTSAwere added. The resulting mixture was then stirred at room tem-perature for 1.5 h. After removal of the solvent under reduced pres-sure, the residue was taken up in EtOAc and the organic solutionwas washed (NaHCO3, water and then brine) and dried (MgSO4).After evaporation of the solvent the resulting crude product waspurified by flash chromatography (50% EtOAc in cyclohexane) togive 755 mg (94%) of 16 as an amorphous solid (10/90 syn/antimixture).


20q59.5 (c 1.22, CHCl3); EIMS m/z 389 (Mq–60, 5%), 359(14), 342 (25), 300 (51), 282 (53), 272 (27), 254 (48), 240 (24),226 (40), 213 (21), 199 (28), 144 (50), 117 (52), 105 (80), 91 (100),79 (87); IR (KBr) 3432, 1736, 1631 cm–1; 1H NMR 0.84 (s, 3H),2.00 (s, 3H), 2.07 (dd, Js19.0, 9.7 Hz, 1H), 2.26 (m, 2H), 2.41(dd, Js19.0, 8.3 Hz, 1H), 3.52 (td, Js10.2, 5.4 Hz, 1H), 3.75(s, 3H), 4.63 (s, 2H), 4.69 (m, 1H), 7.63 (s, 1H); 13C NMR 13.92(CH3), 20.75 (CH2), 21.34 (CH3), 24.88 (CH2), 27.61 (CH2), 31.20(CH2), 32.59 (CH2), 34.41 (CH2), 35.96 (CH2), 38.09 (CH2), 41.76(CH), 42.40 (C), 43.19 (CH), 48.13 (C), 50.97 (CH), 51.00 (CH),51.89 (CH3), 70.41 (CH2), 72.76 (CH), 74.29 (CH), 152.5 (CH),170.5 (C), 170.7 (C), 221.4 (C); HRMS-ESI m/z wMqNax calcd forC24H35O7NNa: 472.23057, found: 472.23035.

7b-Hydroxy-19-oxo-epiandrosterone

19-(O-carboxymethyl)oxime (17)

A mixture of 120 mg (0.27 mmol) of 3b-acetoxy-7b-hydroxy-17,19-dioxo-5a-androstan 19-(O-methoxycarbonylmethyl)oxime(16) in 6 mL of MeOH and 0.67 mL of 1 M aqueous NaOH (0.67mmol) was stirred at room temperature for 3 h. The reaction medi-um was then neutralized with Amberlite IR-120 (Hq) resin, stirredfor 1 h and filtered. The resin was washed with MeOH and thecombined filtrates were evaporated under reduced pressure to afford75 mg (71% yield) of compound 17.

Compound 17. Rf: 0.40 (RP-18, 50% H2O in acetonitrile);waxD

22q80 (c 0.83, MeOH); EIMS m/z 317 (Mq–76, 49%), 300(17), 290 (16), 271 (31), 254 (24), 242 (23), 226 (40), 212 (20),199 (31), 158 (39), 144 (49), 133 (46), 119 (57), 105 (84), 91 (89),79 (100), 67 (69), 55 (77); IR (neat) 3435, 1738 cm-1; 1H NMR0.85 (s, 3H), 2.01 (s, 3H), 2.07 (dd, Js18.8, 9.6 Hz, 1H), 2.27 (m,2H), 2.42 (dd, Js19.8, 8.7 Hz, 1H), 2.62 (m, 3H), 3.51 (td, Js10.1,5.3 Hz, 1H), 3.62 (dddd, Js10.8, 10.0, 5.9, 5.0 Hz, 1H), 4.63 (s,2H), 7.63 (s, 1H); 13C NMR 12.02 (CH3), 18.91 (CH2), 22.88 (CH2),22.98 (CH2), 29.33 (CH2), 29.70 (CH2), 31.04 (CH2), 36.41 (CH2),36.48 (CH2), 40.10 (CH), 40.51 (C), 41.34 (CH), 46.27 (C), 49.13(CH), 49.15 (CH), 68.37 (CH2), 68.56 (CH), 72.55 (CH), 150.9(CH), 168.9 (C), 219.7 (C); HRMS-ESI m/z wMq–Hx calcd forC21H30NO6: 392.20786, found: 392.20890.

Results and discussion

As mentioned in the Introduction, our synthetic objectivewas to produce a hapten of 7b-hydroxy-EpiA in order tocharacterize its receptor and to develop an immunoassay for7b-hydroxy-EpiA (Figure 1). We had to synthesize a steroidcompound bearing a linker at the C-19 position. Some pre-vious studies in the literature w16x as well as in our laboratorysuggested that the C-19 is much hindered by 1-3 interactionsof five axial neighboring hydrogen atoms and therefore dif-ficult to functionalize. We anticipated that it would be moreconvenient to begin the synthesis from a steroid already bear-

ing a C-19 hydroxyl function. Thus, our starting material wasthe steroidal alcohol 1, already described and readily acces-sible according to the literature w12x. In a first study, afteroxidation of the alcohol to ester, we established the alcoholfunction at the C-7 position and introduced the linker at theend of the synthesis.

As depicted (Figure 2), our first approach began by oxi-dation of the primary alcohol 1 with an excess of Jones’reagent in cool acetone solution to give the correspondingacid 2, which was then esterified with diazomethane to affordthe expected ester 3 in 41% overall yield. After protectionof the C-17 carbonyl group under standard conditions givingthe ester 4 (73% yield), an allylic oxidation with CrO3/3,5-DMP in CH2Cl2 at –258C afforded the enone 5 in 54% yield.The next steps involved two stereoselective reduction reac-tions, the CsC double bound and the carbonyl group. Ini-tially, reduction of the enone 5 was carried out with ahydrogen atmosphere over 10% palladium on charcoal inEtOAc to give the ketone 6. The a stereochemistry of the5-hydrogen (trans A/B stereochemistry) was attested by the1H NMR spectrum of 3a-hydrogen (4.66 ppm, tt, Js11.3,5.0 Hz; 2 axial-axial and 2 axial-equatorial couplingconstants).

To reduce this ketone 6 to the corresponding equatorialsecondary alcohol, we successfully used the Luche reactionconditions (NaBH4/CeCl3) to obtain 7 in a highly stereose-lective manner in 81% yield. The stereochemistry of the newchiral center was confirmed by the multiplicity in the 1HNMR of 7a-hydrogen (3.38 ppm, ddd, Js10.8, 8.9, 5.4 Hz,axial position of 7-hydrogen). The ketal function was easilyhydrolyzed in acidic acetone/water medium to give the cor-responding ketone 8 in 69% yield and the corresponding 3,7-diol 9 was obtained through a moderate basic treatment ofthe acetate 8 in 66% yield. Unfortunately, all attempts tomake the free corresponding acid derivative led to the for-mation of a lactone after cyclization with the alcohol func-tion at C-3 position. Therefore, we decided to change oursynthetic strategy to access the hapten.

The second approach (Figure 3) was based on the fact thatsome 19-(O-carboxymethyl)oxime hapten of steroids hasbeen previously described in the literature w10, 11x. The pro-tection of the C-17 carbonyl group as a dioxolan moiety wasthe first step of the synthesis and was carried out by a stan-dard process to give compound 10 in 93% yield w14x. Collinsoxidation type of the primary alcohol at C-19 was performedwith CrO3-pyridine at 08C to lead to the corresponding alde-hyde 11 isolated in 60% yield. The reaction of O-(carboxy-





Figure 2 Reaction conditions: (i) Jones reagent, acetone, 3 h, 08C; (ii) CH2N2, Et2O, 1 h, 08C; (iii) HOCH2CH2OH, TsOH, C6H6, reflux,3 h; (iv) CrO3-3,5-dimethylpyrazole, CH2Cl2, 15 h, –158C; (v) H2, 10% Pd/C, EtOAc, 1 h, room temperature; (vi) NaBH4, CeCl3, 7H2O,MeOH, 2 h, 08C; (vii) TsOH, H2O, acetone, 12 h, room temperature; (viii) K2CO3, MeOH, 1.5 h, room temperature.

Figure 3 Reaction conditions: (i) HOCH2CH2OH, TsOH, C6H6, reflux, 3 h; (ii) CrO3MgSO4, CH2Cl2, 4 h, 08C; (iii) 1/ HCl-H2NOCH2COOH, pyridine, toluene, overnight, room temperature; 2/ CH2N2, Et2O, 1 h, 08C (iv) CrO3-3,5-dimethylpyrazole, CH2Cl2, 48 h,–258C; (v) H2, 10% Pd/C, EtOAc, 3 h, room temperature; (vi) NaBH4, CeCl3, 7H2O, MeOH, 3 h, 08C; (vii) TsOH, H2O, acetone, 1.5 h,room temperature; (viii) NaOH, MeOH, overnight, room temperature.





methyl)-hydroxylamine in pyridine medium, followed by theesterification of the resulting acid by the diazomethane,afforded the ester 12 in almost quantitative yield. The spec-troscopic data showed the presence of small quantities of Zisomer at the double bound CsN. The next steps of thesynthesis were the same as in the case of 7b-hydroxy-EpiA,previously described w17x: firstly allylic oxidation of theC-7 and then reductions of the double bond and the carbonylgroup.

The oxidation reaction was conducted under similar con-ditions (CrO3/3,5-DMP in CH2Cl2) at –258C to give theenone 13 in 78% yield. Again, the NMR spectroscopic dataexhibit the presence of a few quantities of Z isomer at theCsN double bond. The catalytic reduction of the doublebond was carried out with hydrogen over 10% palladium oncharcoal in EtOAc to give the ketone 14 in 81% yield. Thetrans A/B stereochemistry was confirmed by the multiplicityin the 1H NMR spectrum of 3a-hydrogen (4.67 ppm, tt,Js10.8, 5.4 Hz) characteristic of an axial position. Thereduction of the carbonyl group was achieved by the use ofNaBH4/CeCl3 mixture in MeOH to afford the equatorial sec-ondary alcohol 15 in 71% yield. The multiplicity of the 7a-hydrogen resonance (3.42 ppm, ddd, Js10.3, 10.2, 5.5 Hz)again was consistent with its axial position.

The final compound 17 was obtained in 67% overall yieldafter hydrolysis of the C-19 ketal group giving the ketone16 and double saponification of the C-3 acetate ester and themethyl ester under mild conditions.

Conclusion

We have described a stereoselective synthesis of a hapten of7b-hydroxy-epiandrosterone in 16% overall yield startingfrom 3b-acetoxy-19-hydroxy-5-androsten-17-one (1). Afterconversion of the C-19 aldehyde function into (carboxyme-thyl)oxime, an allylic oxidation of the C-7 was followed bytwo stereoselective reductions. However the required acid 17with two additional chiral carbon centers was obtained afterhydrolysis of the ketal function and saponification of the twoesters.

Finally, 17 was coupled to the available amine residues inovalbumin with use of the carbodiimide method, and thederivative is presently used in rabbits for the production ofpolyclonal antibodies.

References

1. Kim SB, Chalbot S, Pompon D, Jo DH, Morfin R. The humancytochrome P4507B1: catalytic activity studies. J Steroid Bio-chem Mol Biol 2004;92:383–9.

2. Hennebert O, Pernelle C, Ferroud C, Morfin R. 7a- and 7b-hydroxy-epiandrosterone as substrates and inhibitors for thehuman 11b-hydroxysteroid dehydrogenase type 1. J SteroidBiochem Mol Biol 2007;105:159–65.

3. Hennebert O, Pelissier MA, Le Mee S, Wulfert E, Morfin R.Anti-inflamatory effects and changes in prostaglandin patternsinduced by 7b-hydroxy-epiandrosterone in rats with colitis. JSteroid Biochem Mol Biol 2008;110:255–62.

4. Le Mee S, Hennebert O, Ferrec C, Wulfert E, Morfin R. 7b-hydroxy-epiandrosterone-mediated regulation of the prostaglan-din synthesis pathway in human peripheral blood monocytes.Steroids 2008;73:1148–59.

5. Niro S, Hennebert O, Morfin R. New insights into the protec-tive effects of DHEA. Hormone Molecular Biology and Clin-ical Investigation 2010;4:489–98.

6. Lapcik O, Hampl R, Hill M, Bicikova M, Starka L. Immuno-assay of 7-hydroxysteroids: 1. Radioimmunoassay of 7b-hydroxydehydroepiandrosterone. J Steroid Biochem Mol Biol1998;67:439–45.

7. Lapcik O, Hampl R, Hill M, Starka L. Immunoassay of 7-hydroxysteroids: 2. Radioimmunoassay of 7a-hydroxy-dehy-droepiandrosterone. J Steroid Biochem Mol Biol 1999;71:231–7.

8. Hampl R, Hill M, Starka L. 7-Hydroxydehydroepiandrosteroneepimers in the life span. J Steroid Biochem Mol Biol 2001;78:367–72.

9. Bicikova M, Ripova D, Hill M, Jirak R, Havlikova H, TallovaJ, Hampl R. Plasma levels of 7-hydroxylated dehydroepian-drosterone (DHEA) metabolites and selected amino-thiols asdiscriminatory tools of Alzheimer’s disease and vasculardementia. Clin Chem Lab Med 2004;42:518–24.

10. Pouzar V, Slavikova T, Cerny I. Synthesis of (19E)-3b,7a-dihy-droxy-17-oxoandrost-5-en-19-al 19-(O-carboxymethyl)oxime, anew hapten for 7a-hydroxydehydroepiandrosterone (3b,7a-dihydroxyandrost-5-en-17-one). Steroids 1998;63:454–8.

11. Cerny I, Pouzar V, Hill M, Havlikova H, Hampl R. Synthesesof 19-wO-(carboxymethyl)oximex haptens of epipregnanoloneand pregnanolone. Steroids 2006;71:120–8.

12. Terasawa T, Okada T. Convenient preparative routes to 19-hydroxy-, 19-oxo-, 19-oic-, and 19-nor-deoxycorticosterone.Tetrahedron 1986;42:537–45.

13. Tanabe K, Takasaki R, Hayashi R, Morisawa Y, Hashimoto T.Steroid Series. XVII. New synthetic routes to 19-norsteroids.Chem and Pharm Bull 1967;15:27–37.

14. Hauser FM, Philip A, Carrol FI. Synthesis of some 17-substi-tuted 3,10-ethano-5a-estranes. J Org Chem 1973;38:3696–700.

15. Pouzar V, Cerny I, Lapcik O, Hill M, Hampl R. Synthesis oftwo new haptens of 16a-hydroxy-dehydroepiandrosterone(3b,16a-dihydroxyandrost-5-en-17-one). Steroids 2003;68:149–58.

16. Rajagopalan MS, Smith DSH, Turner AB. Steroid conjugates.Part III. Syntheses of 19-oxygenated dehydro-epi-androsteronesulphates. J Chem Soc (C), 1971:646–50.

17. Ricco C, Revial G, Ferroud C, Hennebert O, Morfin R. Syn-thesis of 7b-hydroxy-epiandrosterone. Steroids 2011;76:28–30.



Synthesis of a 19-(O-carboxymethyl)oxime hapten of 7β-hydroxy-epiandrosterone

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