Synthesis and characterization by 1H and 13C nuclear magnetic resonance spectroscopy of 17α-hexanoic derivatives of 5α-dihydrotestosterone and testosterone

Synthesis and characterization by ‘H and DC nuclear magnetic resonance spectroscopy of lira-hexanoic derivatives of 5wdihydrotestosterone and testosterone Elisabetb Mappus, MClanie Renaud, and Claude Y. Cuilleron

Marc Rolland de Ravel, Catherine

INSERM

Mole’culaire, H&pita1 Debrousse,

U 329, Pathologie

Hormonale

Grenot,

Lyon,

France

The synthesis and characterization of 17~(6’-hexanoic acid) derivatives of So-dihydrotestosterone and testosterone, useful as ligands for af$nity chromatography purification or as precursors for afJinitylabeling of androgen-binding proteins, is described. Alkynylation of 3-ethylenedioxy-, 3P-hydroxy-, and 3/3,5-dihydroxy-So-androstan-17-one precursors with the potassium derivative of 5-hexyn-I-01 led to the corresponding 17o-(6’-hydroxyhex-I’-ynyl) derivatives, which were hydrogenated over 10% Pt-C catalyst to give 17c+(6’-hydroxyhexyl) derivatives. Chromic acid oxidation of the primary hydroxy group of the 3-ethylenedioxy-I 7-hexyl intermediate into carboxylic acid followed by acid cleavage of the 3-ketal group gave 17~(5’-carboxypentyl)-5a-dihydrotestosterone, which was also obtained directly by chromic acid oxidation of the 3P-hydroxy intermediate. Chromic acid oxidation of the primary hydroxy group of the 3p,5cz-dihydroxy precursor resulted in a So-hydroxy-3-oxo intermediate, which was dehydrated to give 17~(5’-carboxypentyl)testosterone. The 1701 configuration of these derivatives and of synthetic precursors was established by comparing their molecular rotations and their ‘H and ‘-‘C nuclear magnetic resonance (NMR) spectra including solvent effects, with data reported for 17~ or 17/3-substituted steroid analogs as well as with ‘H and 13C NMR reference data recorded in this work for 17cu-ethynyltestosterone, 17a-ethynyl-19nortestosterone, 17a-ethyl-19-nortestosterone, 17~ methyltestosterone, and 17~methyl-5~dihydrotestosterone. (Steroids 57~122-134, 1992)

Keywords:

steroids; 17a-(5’-carboxypentyl)testosterone; 17w(5’-carboxypentyl)-5wdihydrotestosterone; I’Jwalkynyl steroid derivatives; ‘H nuclear magnetic resonance; “C nuclear magnetic resonance; nuclear magnetic resonance solvent effects

Introduction Several 17asubstituted ligands of testosterone and 5adihydrotestosterone have been found useful for affinity chromatography purification of androgen-binding macromolecules from different species. A 17~(2’-carboxyethynyl)-testosterone ligand was used for purification of sex hormone-binding globulins (SHBGs) of bovine serum,’ canine serum,2 and human pregnancy plasma,3 as well as for purification of androgen receptors from

Address reprint requests to Dr. C. Y. Cuilleron, INSERM U 329, Pathologie Hormonale Moleculaire, Hbpital Debrousse, 29, rue Soeur Bouvier, 69322 Lyon Cedex 05, France. Received April 24, 1991; accepted October 14, 1991.

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human foreskins or cultured fibroblasts.4*5 On the other hand, a 17a-(6’-hexanoic) derivative of Sa-dihydrotestosterone was used for affinity purification of the androgen-binding protein of rat epididymi&’ and for purification of SHBGs of human serums-‘0 and rabbit plasma.” A related 17~(2’,3’-epoxypropyl)-Ldihydrotestosterone ligand was also linked to thiopropylsepharose for the purification of the androgen receptor from rat uterine cytosol,12 whereas a 17a-carboxymethyl-%dihydrotestosterone ligand failed to bind to the androgen receptor from rat ventral prostate cytoso1. l3 The affinities of these ligands for the different androgen-binding macromolecules mentioned here depend on the testosterone or Sa-dihydrotestosterone structure of the steroid part as well as on the choice of

0 1992 Butterworth-Heinemann

‘Hand 13CNMR of 77walkyl 17-substituents. Two attempts have been proposed recentiy for establishing correlations with the stereoelectronic environment of C-17 position as estimated by infrared, ‘H and 13C nuclear magnetic resonance (NMR) spectroscopy of 17-substituted steroid ligands, and binding affinities for steroid receptors. A comparison of estrogen receptor binding properties of 17~ substituted derivatives of estradiol 3-methyl ether showed that a 17a-ethyl group strongly disturbed biological activity, in contrast to 17a-methyl, 17a-vinyl, or 17cw-ethynyl substituents, owing probably to steric hindrance of hydrogen bonding of the 17P-hydroxy group by the 17cl-ethyl substituent, as suggested by differences in the characteristic shifts of the 17-OH signals in infrared and ‘H NMR spectra.‘4,15 A recent study has proposed correlations of 13CNMR signals of C-17 carbon atoms of structural analogs of 17-ethynyl1Pnortestosterone with the relative binding affinities (RBAs) for progesterone receptor and with the progestogen/androgen RBA ratios. l6 This work was undertaken to prepare 17c+(6’-hexanoic acid) derivatives in both testosterone and Sa-dihydrotestosterone series, as well as to characterize these compounds and their 17a-(hex- 1‘-ynyl) and 17a-hexyl precursors by ‘H and 13CNMR. These steroid ligands should allow us to evaluate the relative influence of unsaturations, either in ring A or in the 17-side-chain, on the binding properties for SHBG or androgen receptors, thus facilitating the design of more specific affinity ligands or affinity-labeling reagents.

Experimental

T and DHT: Mappus et al.

accurate to 20.05ppm. The ‘H chemical shifts are estimated to be accurate to kO.01 ppm and coupling constants to 20.5 Hz. NMR spectra

were recorded

in both CDCl, and C,D,N

for all

steroids that were soluble enough in these two solvents. This allowed us to estimate solvent-induced shifts, which were used for confirming assignments as well as for facilitating correlations with spectra of compounds soluble in C,D,N only. The systematic use of distortionless enhancement by polarization transfer (DEPT) techniques led to unequivocal identification of methyl

or methine, methylene, and quarternary carbon atoms. 3,3’-Ethylenedioxy-Sa-androstan-17p-ol

(1)

A solution of 5a-dihydrotestosterone (10 g, 34.42 mmol) dissolved in 750 ml of toluene and 165 ml of ethylene glycol containing 1.65 g of dry pyridinium hydrochloride was stirred under reflux for 16 hours in a Dean-Stark water separator. The cooled reaction mixture was neutralized first with solid NaHCO, then with aqueous NaHCO,, and the toluene layer was separated. The toluene layer was washed three times with water and evaporated. The residue, analyzed by thin-layer chromatography (TLC) on silica gel (Rf 0.5, chloroform/ethyl acetate 3 : 1 v/v), was dissolved in dichloromethane and purified by flash chromatography on a column of silica gel 70-230 mesh (same solvent mixture as for TLC). The pure product (9.7 g, 84%) was then crystallized four times from a dichloromethane-methanol mixture containing 0.5% of pyridine to give white crystals: mp 161-163 C (reported” 166.5- 168 C recrystallized from hexane-chloroform-carbon tetrachloride); [(~]o = + 14.9” (CHCI,); umax (Ccl,): 3,610-3,380 broad (OH), 1,100 cm-’ (ketal); ‘H NMR (CDCl,) 6 0.73 (3H, s, 18-CH,), 0.82 (3H, s, 19-CH,), 3.62 (IH, t: J = 8.5 Hz, 17a-H), 3.93 (4H, s, OCH,CH,O); ‘H NMR (C,D,N) 8 0.80 (3H, s, 19-CH,), 0.96 (3H, s, 18-CH,), 3.89 (lH, t: J = 8.5 Hz, 17a-H), 3.91 (4H, s, OCH,CH,O).

General methods

3,3’-Ethylenedioxy-.5a-androstan-I

Testosterone, Sa-dihydrotestosterone, and 3/3-hydroxyandrost5-en-17-one (DHEA) were purchased from Roussel-UCLAF, Paris, France. Thin-layer chromatography was performed on fluorescent silica gel (Merck GF 254, Darmstardt, Germany). The petroleum ether fraction used had bp 45-65 C. Melting points were taken on a Leitz hot-stage microscope and are uncorrected. Specific rotations were measured in 1% solution on a PerkinElmer 241 polarimeter (Norwalk, CT, USA) at 22 C and were estimated to be accurate to ?2”. Molecular rotations of estrone 3methyl ether and 19-norandrost-4-ene-3-17-dione were estimated from the specific rotations ([an] = + 157” and + 140”, respectively) of commercial products from Steraloids, Wilton, NH, USA. IR spectra were recorded on a Perkin-Elmer 257 spectrometer and UV spectra on a UVIKON 860 spectrometer (Kontron Instruments, Zurich, Switzerland). Mass spectra were obtained using a VG Micromass 7070 spectrometer at the Centre Commun de Spectrom&rie de Masse, CNRS, Solaise-Vernaison, France. Interpretation of mass spectra was made according to data found in the literature.” Elemental analyses were determined by Service Central de Microanalyse du CNRS, Solaise-Vernaison, France. NMR spectra were obtained at the Centre Commun de RMN, Universite Claude Bernard, Villeurbanne, France. ‘H and 13C NMR spectra were recorded, respectively, at 300 MHz and 75.47 MHz on a Bruker AM-300 spectrometer equipped with an Aspect 3000 computer. Chemical shifts were measured relative to tetramethylsilane. Samples were prepared by dissolving from 10 mg (‘H NMR) to 40-50 mg (13C NMR) steroid in 0.75 ml of solvent. CDCl, (99.8%) and CSD,N (99.5%) were purchased from CEA, Saclay, France. The “C chemical shifts are estimated to be

A solution of 3-ethylene ketal 1 (10.0 g, 29.89 mmol) in 100 ml anhydrous dichloromethane was treated with a solution of anhydrous CrO, (12.0 g, 120 mmol), dissolved carefully under a dry nitrogen atmosphere in a mixture of anhydrous pyridine (20 ml, 247.28 mmol) and anhydrous dichloromethane (300 ml) for 30 minutes at 0 C. The reaction mixture was filtered through a column of 150 g of Florisil and evaporated to give the pure ketone (9.6 g, 97%). The product was analyzed by TLC on silica gel (Rf 0.7, petroleum ether/ethyl acetate 3 : 2 v/v). Three successive recrystallizations from a dichloromethane-methanol mixture containing 0.5% pyridine gave white crystals: mp 147-149 C (reported” 149-151 C recrystallized from ethanol); [(Y], = +80.3” (CHCl,); vmax (Ccl,): 1,740 (C=O), 1,100 cm-’ (ketal); ‘H NMR (CDCl,) 6 0.84 (3H, s, 19-CH,), 0.86 (3H, s, 18-CH,), 3.93 (4H, s, OCH,CH,O).

3P-Hydroxy-Swandrostan-17-one

7-one (2)

(3)

A stirred solution of DHEA (5.0 g, 17.33 mmol) in 450 ml of ethanol was hydrogenated over 1.0 g of 5% Pd-C catalyst at 22 C at atmospheric pressure. Hydrogen uptake was complete after 1 hour. The solution was filtered through Celite 545. Evaporation of the solvent gave a crude product (4.8 g, 95%), which was analyzed by TLC on silica gel (Rf 0.5, petroleum ether/ethyl acetate 1 : 2 v/v). Recrystallization from a dichloromethane-ether mixture gave a white crystalline solid (4.2 g, 83%): mp 163-166 C (reported 173-174 C for reference product from Steraloids); [c&, = +85.6” (CHCl,) (reported +84.6 for reference product); vmax (Ccl,): 3,600-3,450 broad (OH), 1,735 cm-’ (C=O); ‘H NMR (CDCl,) 6 0.83 (3H, s, 19-CH,), 0.86 (3H, s, 18-CH,), 3.60

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1992, vol. 57, March

123

Papers (IH, r-n,3r~-H); ‘H NMR (C,D,N) 6 0.80 (3H, \. 19CH,), (3H, s, 1X-CH1), 3.85 (1H. m, 3~H). 3/3,.5-Dihydroxy-Sar-androstan-17-one

0.78

(4)

This compound was prepared from DHEA, according to a reported method. I9 The crude product was analyzed by TLC on silica gel (Rf 0.25, chloroform/methanol 10 : 1 v/v) and recrystallized from a chloroform-methanol mixture to give white crystals of pure product: mp 259-264 C (reported” 277.5-278 C recrystallized from methanol); [a]n = +70.5” (EtOH), (reported [aIn = +83.6”): Vmax (CHCI,): 3,610-3600-3.460 broad (OH), 1,735 cm-’ (C=O); ‘H NMR (CDCL,) 6 0.87 (3H, s. IS-CH,), 1.02(3H, s, 19-CH,), 4.09 (lH, m, 3cr-H); ‘H NMR (C,D,N) 6 0.82 (3H, s. l&CH,), 1.05 (3H, s, 19-CH,), 4.74 (IH, m, 3a-H).

17w(6’-Hydroxyhex-I’-ynyl)-3,3’-ethylenedioxySa-androstan-I 7@01 (5) Small pieces of potassium metal (1.40 g, 35.90 mmol) were added under a dry nitrogen atmosphere to 160 ml of freshly distilled liquid ammonia cooled in a dry ice-acetone mixture and stirred for 25 minutes. A blue color developed. 5-Hexyn-l-01 (3.0 g, 30.61 mmol) was added dropwise. The blue color was rapidly discharged and the reaction mixture was stirred for IO minutes. A solution of 3,3’-ethylenedioxy-5a-androstan-17-one 2 (2.2 g, 6.62 mmol) in 50 ml of anhydrous pyridine was added dropwise in 1.5minutes. The cooling bath was then removed and the reaction mixture was stirred for 12 hours at 22 C. Water (IO ml) was added and the solvents were evaporated under reduced pressure. Remaining traces of solvents were removed by addition and evaporation of toluene. The crude residue was suspended in water, neutralized with concentrated HCI, dissolved in chloroform, and purified by preparative TLC on silica gel (chloroform/ ethyl acetate 3 : 1 v/v). Two fractions were isolated: the less polar fraction (Rf 0.7, 0.750 g. 2.26 mmol) corresponded to unreacted starting material. whereas the more polar fraction (Rf 0.4, 1.21 g, 42%,) was characterized as the 17-alkylated product. Three successive recrystallizatjons from a dichloromethane-ethyl acetate mixture gave white crystals: mp 149-152 C; [cy]n = -27.6” (CHCI,); umax (Ccl,): 3.625-3,605-3,400 broad (OH), 2,225 cm-’ (C=C); ‘H NMR (CDCI,) 6 0.820 (3H. s, 18- or 19-CH,). 0.822 (3H. s. lg- or I9-CH?), 2.28 (2H, t: J = 6.7 Hz, CH,C=C). 3.69 (2H, t: J = 6.3 Hz, CH,O), 3.93 (4H. s, OCH&H,O); ‘H NMR (C,D,N) 6 0.82 (3H, s, 19-CH,), 1.13 (3H, s, IS-CH,), 2.42 (2H, t: J = 6.8 Hz, CH@=C), 3.86 (2H, m transformed in a triplet J = 6-7 Hz. after exchange with DzO, CH$)), 3.90 (4H. s, OCH,CH,O); mass spectrum (70 eV) m/e (relattve intensity) 430 (M-, 3), 415 (31), 397 (38), 373 (3), 372 (7). 332 (M-97, 9). 291 (II), 275 t5), 255 (3), 125 (38), 112 (5). 99 (100). High-resolution mass spectroscopy (MS) calculated for C2,H,z0, (M ‘): 430.3072. Found: 430.3083.

17w(6’-Hydroxyhex-1 3/3,17@dioi (6)

‘-ynyl)-5wandrostane-

This compound was prepared as above from 3P-hydroxy-Saandrostan- 17-one 3 (1 .Og, 3.44 mmol) and purified by preparative TLC on silica gel (chloroform/ethyl acetate 1 : 3 v/v). Two fractions were isolated: the less polar fraction (Rf 0.6, 240 mg, 0.826 mmol) corresponded to unreacted starting material, whereas the more polar fraction (Rf 0.3. 620 mg, 46%) was characterized as the 17-alkylated product. Three successive recrystallizations from acetone gave white crystals: mp 153-154 C; [(~]n = -24” (CHCI,): [cx]D = -29.6” (EtOH); vmax (CHZC12): 3,580-3,400 (OH), 2,200 cm-’ (C-C); ‘H NMR (CDCI,) 6 0.82 (6H, s, 18-CH, and 19-CH,), 2.28 (2H. t: J = 6.8 Hz, CH,C=C), 3.59

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(IH. m. 3cr-H). 3.69 (2H. t: J = 6.2 Hz, CH,O); ‘H NMR (C’,D,N) s0.86(3H. s, 19-CH,), 1.13 (3H, s. lg-CH,), 2.40(2H, t: J = 6.7 Hz. CH,C-C), 3.84 (2H, m transformed in a triplet J = 6-7 Hz. after exchange with D>O, CH20), 3.8 (IH, m superimposed to signal at 3.84 ppm, 3a-H): mass spectrum (70 eV) m/e (relative intensity) 388 (M + , 7). 373 (IOO), 355 (9X), 345 (1). 33 (31, 330 (241, 315 (5). 290 (M-98. 6), 246 (19). 233 (8). 231 (S), 217 (14), 215 (19). High-resolution MS calculated for C,,H,,,O, (M-): 388.2977. Found: 388.2972.

17~(6’-Hydroxyhex-1 3/3,5,17/3-trio1 (7)

‘-ynylj-k-androstane-

This compound was prepared as described above from 3p,5dihydroxy-SLY-androstan-I 7 one 4 (I .O g, 3.26 mmol) and purified by preparative TLC on silica gel (chloroform/methanol IO: 1 v/v). Two fractions were isolated: the less polar fraction (Rf 0.3, 312 mg, I .02 mmol) corresponded to unreacted starting material, whereas the more polar amorphous fraction (Rf 0.15, 630 mg, 48%) was characterized as the 17-alkylated product: mp 92-96 C; [ulD = - 26.1” (dioxane); Vmax (CHCI,): 3,600-3,460 broad (OH), 2,240 cm-’ (C=C); ‘H NMR (C,D,N) 6 I. 13 (3H, s. 19-CH,), 1.17 (3H. s, 18-CH,), 2.28 (2H, t: J = 7.0 Hz, CH,CrC), 3.80 (2H. t: J = 7.0 Hz. CH>O), 4.73 ( 1H, m, 3a-H); mass spectrum (70 eV) m/e (relative intensity) 404 (M’, 5), 389 (90), 371 (100). 353 (13), 346 (12), 335 (5), 328 (61, 313 (4), 310 (3). 295 (.5), 271 (3), 246 (19). 231 (24), 228 (24), 215 (23). Analysis calculated for C,,H,,,O,: C, 74.22; H, 9.97. Found: C. 74.46; H. 9.81.

17w(6’-Acetoxyhex-I Sa-androstan-17/3-ol

‘-ynyl)-3,3’-ethyEenedioxy(8)

The dihydroxy derivative 5 (22 mg, 0.05 11 mmo1) was acetylated overnight at 25 C in 1 ml of a pyridine/acetic anhydride 3 : 1 mixture. The reaction mixture was evaporated and last traces of acetic anhydride were eliminated by addition and evaporation of toluene. The crude product was dissolved in dichloromethane and purified by preparative TLC on silica gel (Rf 0.6, petroleum ether/ethyl acetate 2 : I v/v) to give an amorphous pure product (22 mg. 91%): [a], = -21.6” (CHCI,); umax (Ccl,): 3,600-3,500 (OH), 2,225 (C=C) 1,735 (ester), I 1100 cm-’ (ketal); ‘H NMR (CDCI,) 6 0.82 (6H, s. IS-CH, and 19-CH,), 2.06 (3H, s, CH,OCOCH,), 2.28 (ZH, t: J = 7.0 Hz, CH$-C), 3.93 (4H, s, OCH,CH20), 4.10 (2H, t: J = 6.6 Hz, CH20); ‘H NMR (CSD,N) 6 0.83 (3H, s, 19-CH,), 1.13 (3H, s, l&CH,), 2.02 (3H, s, CH20COCH,), 2.34 (2H, t; J = 6-7 Hz, CH,C-C), 3.90 (4H, s, 0CH2CH20), 4.08 (2H, t: J = 6-7 Hz, CH,O); mass spectrum (70 eV) m/e (relative intensity) 472 (M +, 5), 457 (14), 454 (2), 439 (2), 430 (l), 427 (l), 415 (5), 412 (9), 397 (72), 383 (l), 372 (15), 291 (12), 125 (6). 99 (100). High-resolution MS calculated for C1,,H+,05 (M’): 472.3189. Found: 472.3182.

17~(6’-Acetoxyhex-1 ‘-ynyl)-17P-hydroxy-.5cuandrostan-3/3-y! acetate (9) The trihydroxy derivative 6 (34 mg, 0.0875 mmol) was acetylated as above and purified by preparative TLC on silica gel (Rf 0.5, chloroform/ethyl acetate 4: I v/v) to give a pure product (37 mg, 89%). Two successive recrystallizations from a petroleum ether-ether mixture gave white crystals: mp 88-90 C; [(~]n = -36.3” (CHCI,); vmax (Ccl,): 3,605-3,500 broad (OH), 2,220 weak (C=C), 1,735 cm-’ (ester); IH NMR (CDCl,) 6 0.82 (3H, s, 18-CH,), 0.84 (3H, s, 19-CH,), 2.02 (3H, s, 3-OCOCH,), 2.06 (3H, s, CHzOCOCH-,, 2.29 (2H, t: J = 7.0 Hz, CH,C-CL 4.11

‘H and 13CNMR of 17walkyl (2H, t: J = 6.6 Hz, CH,O), 4.69 (lH, m, 3a-H); ‘H NMR (C,D,N) S 0.80 (3H, s, 19-CH,), 1.11 (3H, s, 18-CH,), 1.99 (3H, s, CH,O COCH,), 2.05 (3H, s, 3-OCOCH,), 2.31 (2H, t: J = 7.0 Hz, CH,C=C), 4.09 (2H, t: J = 6.6 Hz, CH,O), 4.83 (lH, m, ~CX-H); mass spectrum (70 eV) m/e (relative intensity) 472 (M+, 3), 457 (22), 454 (l), 439 (l), 430 (2), 415 (13), 412 (13), 397 (lOO), 372 (9) 357 (3), 355 (2), 337 (2), 332 (M-139, 3), 329 (2) 314 (l), 297 (l), 294 (l), 288 (9), 272 (4), 244 (l), 239 (2), 231 (4), 230 (5) 229 (5), 228 (6), 218 (6), 217 (8), 216 (7), 215 (17). High-resolution MS calculated for C,H,O, (M’): 472.3189. Found: 472.3187.

17w(6’-Acetoxyhex-1 ‘-ynyl)-5,17P-dihydroxy-Saandrostan-3/Syl acetate (10) The tetrahydroxy derivative 7 (80 mg, 0.198 mmol) was acetylated as above and purified by preparative TLC on silica gel (Rf 0.6, chloroform/ethyl acetate 1 : 3 v/v) which gave a pure product (84 mg, 87%). Four successive recrystallizations from a petroleum ether-ether mixture gave white crystals: mp 76-79 C; [(Y], = - 24.6” (CHCl,); vmax (CCL): 3,600-3,510-3,450 broad (OH), 1,735 cm-’ (ester); ‘H NMR (CDCI,) 6 0.82 (3H, s, 18-CH,), 1.02 (3H, s, 19-CHS), 2.02 (3H, s, 3-OCOCH,), 2.06 (3H, s, CH,OCOCH,), 2.29 (2H, t: J = 6.9 Hz, CH,C=C), 4.11, (2H, t: J = 6.6 Hz, CH,O), 5.16 (lH, m, 3~-H); ‘H NMR (C,D,N) S 1.07 (3H, s, 19-CH,), 1.18 (3H, s, 18-CH,), 1.98 (3H, s, CH,O COCJI,), 2.05 (3H, s, 3-OCOCH,), 2.21 (2H, t: J = 7.0 Hz, CH,C=C), 4.04 (2H, t: J = 6.5 Hz, CH,O), 5.76 (lH, m, 3a-H); mass spectrum (70 eV) m/e (relative intensity) 488 (M+, 2) 473 (17), 455 (2), 431 (9), 428 (8), 413 (lOO), 410 (lo), 395 (9) 392 (7), 388 (12), 377 (4), 370 (2) 368 (l), 353 (3), 350 (4), 335 (5), 330 (l), 317 (2), 310 (6), 295 (4), 288 (5), 228 (33) 215 (20). High-resolution MS calculated for Cr9H,0, (M+): 488.3138. Found: 488.3126.

17cz-(6’-Hydroxyhexyl)-3,3’-ethylenedioxy-5aandrostan-17p-ol (11) A stirred solution of 17a-hexynyl derivative 5 (500 mg, 1.16 mmol) in 25 ml of ethanol containing 0.5% of pyridine was hydrogenated over 100 mg of 10% Rt-C catalyst at 22 C at atmospheric pressure. Hydrogen uptake was complete after 1.5 hours. The solution was filtered through a Teflon membrane (diameter 0.5 pm, Waters Associates, Milford, MA, USA) and evaporated. The crude product (439 mg, 87%) was analyzed by TLC on silica gel (Rf 0.5, chloroform/ethyl acetate 2 : 3 v/v). Three successive recrystallizations from a dichloromethane-ethyl acetate mixture gave a white crystalline solid: mp 150-153 C; [(~]n = -4.0” (CHCI,); vmax (CHCl,): 3,610-3,600-3,450 broad (OH), 1,100 cm-’ (ketal); ‘H NMR (CDCI,) 6 0.83 (3H, s, 19-CH,), 0.85 (3H, s, 18-CH,), 3.65 (2H, t: J = 6.6 Hz, CH,O), 3.93 (4H, s, OCH,CH,O); ‘H NMR (C,D,N) 6 0.82 (3H, s, 19-CH,), 1.12 (3H, s, 18-CH,), 3.88 (2H, t: J = 6-7 Hz, CH,O, fully separated from ketal signal at 3.93 ppm after exchange with D,O), 3.92 (4H, s, OCH,CH,O); mass spectrum (70 eV) m/e (relative intensity) 434 (M+, 13), 416 (3), 401 (l), 389 (2), 371 (l), 333 (M-101, 22), 315 (5), 291 (46), 278 (ll), 125 (36), 112 (4), 99 (100). High-resolution MS calculated for C27H4604 (M+): 434.3384. Found: 434.3396.

17cz-(6’-Hydroxyhexy1)-5cr-androstane-3P,17/3diol (12) The 17a-hexynyl derivative 6 (550 mg, 1.42 mmol) was hydrogenated as above. The crude product (486 mg, 87%) was analyzed by TLC on silica gel (Rf 0.3, chloroform/ethyl acetate 1 : 3 v/v). Three successive recrystallizations from an ethanol-ether

T and DHT: Mappus

et al.

mixture gave a white crystalline solid: mp 152-155 C; [a]D = - 1.3” (EtOH); ‘H NMR (C,D,N) 6 0.88 (3H, s, 19-CH,), 1.13 (3H, s, 18-CH,), 3.88 (2H, m transformed in a triplet J = 6-7 Hz, after exchange with D,O, CH,O), 3.8 (lH, m superimposed to signal at 3.88 ppm, ~CX-H);mass spectrum (70 eV) m/e (relative intensity) 392 (M+, l), 374 (6), 359 (4), 341 (2), 291 (M-101, loo), 273 (ll), 248 (lo), 246(11), 233 (8) 231 (13), 217 (6), 215 (7), 203 (3) 157 (29), 149 (5). High-resolution MS calculated for C,,H,O, (M+): 392.3290. Found: 392.3283.

170c-(6’-Hydroxyhexyl)-Sa-androstane-3/3,5,17@ trio1 (13) The 17cY-hexynyl derivative 7 (525 mg, 1.30 mmol) was hydrogenated as above to give a pure white crystalline product (505 mg, 95%) analyzed by TLC on silica gel (Rf 0.3, chloroform/methanol 10 : 1 v/v): mp 197-200 C; [cu]D = +0.8” (EtOH); vmax (CHCI,): 3,620-3,600-3,440 broad (OH), 1,735 cm-’ (ester); ‘H NMR (C,D,N) 6 1.13 (3H, s, 19CH,), 1.17 (3H, s, 18-CH,), 3.88 (2H, t: J = 6-7 Hz, CH,O), 4.7 (lH, m, 3a-H); mass spectrum (70 eV) m/e (relative intensity) 390 (M+ - 18, 21), 372 (37), 357 (7), 354 (13), 339 (lo), 289 (M-18-101, 23) 271 (23) 264 (3), 253 (ll), 246 (loo), 228 (59), 215 (60). Analysis calculated for CrsH,,O,: C, 73.48; H, 10.85. Found: C, 73.25; H, 10.67.

17w(6’-Acetoxyhexyl)-3,3’-ethylenedioxy-5aandrostan-17@01 (14) The dihydroxy derivative ll(80mg, 0.184 mmol) was acetylated overnight at 25 C in 1 ml of a pyridineiacetic anhydride 3 : 1 mixture. The reaction mixture was evaporated and last traces of acetic anhydride were eliminated by addition and evaporation of toluene. The residue was dissolved in dichloromethane and purified by preparative TLC on silica gel (Rf 0.6, petroleum ether/ ethyl acetate 2 : 1 v/v), which gave a pure product (86 mg, 98%). Four successive recrystallizations from a petroleum ether-ether mixture gave white crystals: mp 109-l 11 C; [a]D = + 12.1” -3.5” (CH,Cl,); [a]D = (CHCl,); vmax (Ccl,): 3,620-3,600-3,440 broad (OH), 1,735 (ester), 1,100 cm-’ (ketal); ‘H NMR (CDCl,) 6 0.83 (3H, s, 19-CH,), 0.85 (3H, s, 18-CH,), 2.05 (3H, s, CH,OCOCI-_I,), 3.93 (4H, s, OCH,CH,O), 4.06 (2H, t: J = 6.8 Hz, CH20); ‘H NMR (C,D,N) 6 0.83 (3H, s, 19-CH,), 1.13 (3H, s, 18-CH,), 2.02 (3H, s, CH,OCOCIi,), 3.93 (4H, s, OCH,CH,O), 4.12 (2H, t: J = 6.7 Hz, CH,O); mass spectrum (70 eV) m/e (relative intensity) 476 (M+, 17), 458 (4), 443 (2), 431 (2), 416 (l), 413 (2), 396 (l), 359 (13) 333 (M-142, 44) 315 (9), 291 (28), 290 (23), 278 (12), 199 (15), 125 (9), 112 (44), 99 (100). High-resolution MS calculated for C,,H,,O, (M+): 476.3502. Found: 476.3492.

17w(6’-Acetoxyhexyl)-3P,I 7@dihydroxy-Saandrostan-3p-yl acetate (15) The trihydroxy derivative 12 (34 mg, 0.0866 mmol) was acetylated as above and purified by preparative TLC on silica gel (Rf 0.5, chloroform/ethyl acetate 4 : 1 v/v), which gave a pure product (37 mg, 90%). Two successive recrystallizations from a petroleum ether-ether mixture gave white crystals: mp 133-135 C; [cw]D = - 11” (CHCI,); vmax (Ccl,): 3,620-3,600-3&O broad (OH), 1,735 cm-’ (ester); ‘H NMR (CDCl,) 6 0.84 (3H, s, 19-CH,), 0.85 (3H, s, 18-CH,), 2.02 (3H, s, 3-OCOCH,), 2.05 (3H, s, CH,OCOCH,), 4.06(2H, t: J = 6.8 Hz, CH,O), 4.61 (lH, m, 3a-H); ‘H NMR (C,D,N) 6 0.81 (3H, s, 19-CH,), 1.12 (3H, s, 18-CH,), 2.01 (3H, s, CH,OCOCH,), 2.06 (3H, s, 3-OCOCH,), 4.12 (2H, t: J = 6.6 Hz, CH,O), 4.88 (lH, m, 3a-H); mass

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1992, vol. 57, March

125

Papers spectrum (70 eV) m/e (relative intensity) 476 (M’. I), 458 (I), 443 (2), 416 (5). 398 (21). 383 (8). 333 (M-142, 100). 315 (ll), 288 (9), 273 (25), 255 (23). 245 (l), 230 (42), 217 (16), 215 (13), 199 (48), 186 (14). High-resolution MS calculated for t&H,,O, (M-): 476.3502. Found: 476.3496.

17w(6’-Acetoxyhexyl)-5,17@dihydroxy-Sczandrostan-3P-yl acetate (16) The tetrahydroxy derivative 13 (80 mg, 0.196 mmol) was acetylated as above and purified by preparative TLC on silica gel (Rf 0.6, chloroform/ethyl acetate I : 3 v/v) to give a pure product (83 mg, 86%). Four successive recrystallizations from a petroleum ether-ether mixture gave white crystals: mp 105-108 C; [(~]o = -9.4” (CHCI,); Ymax (WI,): 3,620-3,600-3,460 broad (OH), 1,735 cm-’ (ester): ‘H NMR (CDCI,) 6 0.85 (3H, s, 18CH,), 1.02 (3H, s, 19-CH,), 2.02 (3H, s, 3-OCOCH,), 2.05 (3H, s, CHIOCOC&), 4.06 (2H, t: J = 6.7 Hz, CH20), 5.15 (lH, m, 3a-H); ‘H NMR (C,D,N) 6 1.07 (3H. s, 19-CH,), 1.19 (3H, s, 18-CH,), 2.02 (3H, s, CH20COC&), 2.06 (3H, s, 3-OCOCH,), 4.13 (2H, t: J = 6.7 Hz, CH,O), 5.81 (lH, m, 3~H); mass spectrum (70 eV) m/e (relative intensity) 474 (M’ - 18, 3), 456 (3). 414 (40), 396 (48), 381 (25), 288 (22), 271 (34). 267 (12), 253 (43), 228 (IOO), 215 (28). 199 (46), 187 (11).

17cz-(5’-Carboxypentyl)-l7@hydroxy-5aandrostan-3-one (17) A stirred solution of 17~(6’-hydroxyhexyl)-3,3’-ethylenedioxySo-androstan-17P-ol 11 (200 mg, 0.46 mmol) in 30 ml of acetone at 20 C was treated dropwise with an 8 N chromic acid solution (26.72 g of CrO, in 23 ml of concentrated H$O, diluted to 100 ml with water) until an orange color persisted. The excess of oxidant was destroyed by addition of 3 ml of methanol and the reaction mixture was diluted with 50 ml of acetone and filtered. The organic phase was concentrated to 10 ml and poured into 200 ml of water. The precipitated solid was dissolved in 5 ml of dioxane and stirred for 3 hours at 22 C in the presence of 0.5 ml of water and 0.1 ml concentrated HCl. The reaction mixture was neutralized with aqueous NaHCO, and evaporated. The residue was taken up in water (5 ml) and the pH was brought to 2-3 with HCl. The precipitated acid was purified by preparative TLC on silica gel (Rf 0.5, chloroform/acetone/acetic acid 70: 25 : I v/v) Elution with a chloroform/methanol 10 : 1 mixture gave a pure product (134 mg, 72%). Three successive recrystallizations from an ethyl acetate-methanol mixture gave white crystals: mp 152-156 C (reported6 162-163 C recrystallized from ethyl acetate); [a]n = + 14.7” (EtOH); vmax (CHCl,): 3,600-3,500 broad (OH), 1,700 cm-’ (C=O); ‘H NMR (CDCI,) 6 0.88 (3H, s, 18CH,), 1.03 (3H, s, 19-CH,); 2.34 (4H, m, CH,C=O, signal superimposed to the low-field part of the signal for 2/4-CHz at 2.0-2.3 ppm); ‘H NMR (C,D,N) 6 0.95 (3H, s, 19-CH,), 1.11 (3H, s, 18-CH,); 2.55 (2H, t: J = 7.4 Hz, CH,C=O, signal distinct from the signal for 2/4-CH2 at 2.0-2.3 ppm); mass spectrum (70 eV) m/e (relative intensity) 404, (M+, I), 386 (8), 371 (IO), 289 (M115, loo), 271 (31), 247 (32), 231 (ll), 171 (21), 158(33), 153 (35). Analysis calculated for r&H,,,O,: C, 74.22; H, 9.97. Found: C, 74.15; H, 10.40. This hexanoic acid derivative 17 was also obtained from the trihydroxy derivative 12 by oxidation with chromic acid, as described above, but in lower and less reproducible yields.

17w[.5’-(Methoxycarbonyl)pentyl]-17/3-hydroxy5a-androstan-3-one (18) A solution of acid 17 (62 mg, 0.15 mmol) in 2 ml of chloroform was treated with an excess amount of an ethereal solution of

126

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1992, vol. 57, March

diazomethane for 5 minutes at 0 C. The residue upon evaporation of the solvent was purified by preparative TLC on silica gel (Rf 0.5, chloroform/ethyl acetate 3 : I v/v) to give the pure ester (53 mg, 83%). Three successive recrystallizations from a petroleum ether/ether mixture gave white crystals: mp 74-76 C; ]oc]n = +9.4” (CHCI,); [cY]D = + 13.6” (EtOH); umax (WI,): 3.620-3,600-3,500 broad (OH), 1,740 (ester), 1.715 cm-’ (C=O); ‘H NMR (CDCI,) 6 0.88 (3H, s, 18-CH,), 1.03 (3H, s, 19-CH,), 2.32 (4H, t: J = 7.5 Hz, side-chain CH,C=O, signal superimposed to the low-field part of the signal for 214 - CH, at 2.0-2.3 ppm), 3.67 (3H, s, COOCH,): ‘H NMR (C,D,N) 6 0.95 (3H, s, 19-CH,). 1.13 (3H, s, 18-CH,), 2.37 (4H, t: J = 7.4 Hz, side-chain CH&=O, signal superimposed to the low-field part of the signal for 2/4-CHz at 2.0-2.3 ppm), 3.62 (3H, s, COOCH,); mass spectrum (70 eV) m/e (relative intensity) 418 (M’, I), 400 (4), 385 (4), 369 (9), 289 (M-129, IOO), 271 (19), 247 (IO), 244 (IO), 231 (7). 229 (6), 217 (3). High-resolution MS calculated for GhH4?04 (M’): 418.3083. Found: 418.3086.

17w(5’-Carboxypentyl)-I en-3-one (19)

7&hydroxyandrost-4-

Chromic acid oxidation of 17a-(6’-hydroxyhexyl)-5a_androstane-3P,5,17/3-trio1 13 (100 mg, 0.245 mmol) was performed as described above to give the 17a-(5’~carboxypentyl)-5a, 17/3-dihydroxyandrostan-3-one intermediate. In order to eliminate the 5~ OH group, the precipitated acid was dissolved in 30 ml of t-butanol and stirred under reflux for 4 hours in the presence of 2 ml of water and 0.1 g KOH. The reaction mixture was neutralized with IN HCI and evaporated. The residue was dissolved in methanol and purified by preparative TLC on silica gel (Rf 0.4, chloroform/acetone/acetic acid 70 : 25 : I v/v). Elution with a chloroform/methanol 10: I mixture gave an amorphous pure product (75 mg. 76%): mp 85-120 C: [(~]o = +50.8” (CHCl,); hmax = 241-242 nm (E = 13,900); vmax (CDCI,): 3,600-3,400 broad (OH), 1,710 broad (COOH), 1,660 (conjugated C=O), 1,610 cm-’ (conjugated C=C); ‘H NMR (CDCl,) 6 0.91 (3H, s, 18-CH,), 1.20 (3H, s. 19-CH,), 2.37 (2H, t: J = 7.4 Hz, sidechain CH+Z=O, signal superimposed to the lower field part of the signal for 214~CHz at 2.0-2.3 ppm), 5.73 (IH, s, 4-H); ‘H NMR (C,D,N) 6 1.07(3H, s, 19-CH,), 1.13 (3H, s, 18-CH,); 2.56 (2H, t: J = 7.4 Hz, side-chain CH,C=O, signal distinct from the signal for 2/4-CH, at 2.0-2.3 ppm), 5.89 (lH, s, 4-H); mass spectrum (70 eV) m/e (relative intensity) 402 (M’, loo), 384 (23). 371 (8), 369 (7), 351 (9), 289 (41), 287 (M-115, 27), 285 (IS), 271 (21), 269 (19), 245 (69), 229 (83), 217 (13). 187 (8), 171 (15), 153 (24), 124 (28). High-resolution MS calculated for C,,H,,O, (M+): 402.2760. Found: 402.2770.

17w[5’-(Methoxycarbonyijpentylj-I hydroxyandrost-4-en-3-one (20)

7@

The carboxylic acid derivative 19 (50 mg, 0.124 mmol) in 2 ml of chloroform was esterified with diazomethane as above and purified by preparative TLC on silica gel (Rf 0.4, chloroform/ ethyl acetate 3 : 1 v/v) to give the amorphous pure ester (41 mg, 79%): [cy]n = +64.7” (CHCI,); Vmax (Ccl,): 3,620-3,600-3,500 broad (OH), 1,740 (ester), 1,675 (conjugated C=O), 1,620 cm- ’ (conj. C=C); ‘H NMR (CDCI,) 6 0.91 (3H, s, 18-CH,), 1.21 (3H, s, 19-CH,), 3.68 (3H, s, COOCH,), 5.75 (lH, s, 4-H); ‘H NMR (C,D,N) 6 1.07 (3H, s, 19-CH,), 1.13 (3H, s, 18-CH,), 2.36 (4H, t: J = 7.5 Hz, side-chain CH,C=O, signal superimposed to the lower field part of the signal for 2/4-CH, at 2.0-2.3 ppm), 3.62 (3H, s, COOCH,), 5.90 (lH, s, 4-H); mass spectrum (70 eV) m/e (relative intensity) 416 (M’, IOO), 398 (23), 385 (8), 381 (6),

‘H and 13CNMR of 17walkyd T and DHT: Mappus RI

0 c

R2 .a\*

@O

Fi

1 R,= OH, R,= H 2 R,,R2= 0

0

H#-

et al.

i

3

8 R=COCH3

R@_:

Rg=c(F Fi

0

14 R= COCH3

6 R=H 9 R= COCH3

k

15

R= COCH3

o&zR

Rg=cER@c 13 16

7 R=H 10 R= COCH, Scheme

367 (6), 312 (22), 287 (M-129,33), 283 (13), 269 (20), 245 (52), 242 (25), 229 (48), 187 (6), 185 (17), 172 (9), 159 (13), 157 (18), 153 (19), 124 (19). High-resolution MS calculated for C$H,O, (M+): 416.2927. Found: 416.2927.

Results and discussion The main synthetic purpose of this work was the preparation of 17a-(5’-carboxypentyl)-testosterone 19. However, two modified syntheses of the reported6~*~” 17~ (5’-carboxypentyl)-5a-dihydrotestosterone derivative 17 were also undertaken by a similar procedure involving alkynylation of 17-0~0 precursors, but starting either from a protected 3-ethylenedioxy derivative, which should facilitate further chemical transformations of the 17_substituent, or from a 3P-hydroxy precursor, which replaced the previously used 3cu-hydroxy-5cr-androstan-17-one isomer in order to favor 17a attack (Scheme 1). The three 17-oxosteroid precursors were prepared using standard methods. Dioxolanation of the 3-0~0 group of Sa-dihydrotestosterone led to the 3-ethylene ketal 1, which was oxidized with the CrO,-(pyridine), complex to give the 3-ethylenedioxy-5cw-androstan-17one precursor 2. Catalytic hydrogenation of DHEA gave 3fi-hydroxy-Sa-androstan-17-one 3. The synthesis of the 3/3,5a-dihydroxyandrostan-17-one precursor 4 was performed according to a reported method,r9 starting from DHEA. Alkynylation of these 17-oxo-steroid precursors with the potassium derivative of 5-hexyn-l01 was carried out using potassium in liquid ammonia,

R=H R= COCH3

19 R=H 20 R= CH3

1

in conditions essentially similar to those reported previously,6 to give the three corresponding 17a-alkynyl derivatives 5,6, and 7, which were all characterized as their monoacetate or diacetate derivatives 8,9, and 10. Catalytic hydrogenation of the three 17a-(6’-hydroxyhex- 1‘-ynyl) derivatives gave the corresponding saturated 17a-(6’-hydroxyhexyl) analogs 11, 12, and 13, which were also characterized as their monoacetate or diacetate derivatives 14, 15, and 16. The oxidation of the terminal hydroxyl group of the 3-ethylenedioxy17a-(6’-hydroxyhexyl) intermediate 11 into a carboxylic acid was performed in acetone with an 8 N solution of chromic acid which also oxidized simultaneously the 3/3-OH group of the trihydroxy or tetrahydroxy intermediates 12 and 13 into 3-ketone and Sa-hydroxy3-ketone, respectively. However, more reproducible yields were obtained for the chromic oxidation of the 3-ethylene ketal precursor 11 than for the 3/3-hydroxy precursor 12, owing possibly to differences in solubilities. Mild acidolysis of the 3-ethylenedioxy group gave 17a-(5’-carboxypentyl)-5a-dihydrotestosterone 17, which was also characterized as the corresponding methyl ester 18. The P-hydroxyketone intermediate obtained after oxidation of the tetrol13 was dehydrated in situ in the presence of KOH to give 17a-(5’-carboxypentyl)-testosterone 19, which was also characterized as the corresponding methyl ester 29. Alkynylations with the potassium derivative of 5-hexyn-l-01 were expected to proceed predominantly by attack of the reagent at the sterically less hindered a-side of the 17-0~0 group as generally observed with

Steroids,

1992, vol. 57, March

127

Papers

similar but shorter potassium alkynes.‘” However, the formation of 17P-hexynyl derivatives could not be excluded because a significant p-attack had been reported with potassium derivatives of propargyl aldehyde diethyl aceta120; steric influences of the 3_ethylenedioxy, 3/3-hydroxy-, and Sa-hydroxy groups of 17-0~0 precursors 2, 3, and 4 could also differ from that of the 3a-hydroxy group of the previously used 3a-hydroxy5a-androstan-17-one precursor,6,” thus maintaining the necessity to establish the homogeneity and the 17configuration of 17-hexynyl and 17-hexyl derivatives described in this work. The negative specific rotations of 17a-hexynyl derivatives 5, 6, and 7 and of their corresponding acetates 8, 9, and 10 suggested the expected 17~ rather than a 17/3-configuration when compared with the specific rotations, which have been reported for 17~ and 17/3-epimers of 17-(3’-hydroxy-l’-propynyI)-estra1,3,5(10)-triene-3,17-diol 3-methyl etheP ([(Y]~ = -5.9” and +66.1”, respectively), 17_ethynylestra1,3,5( IO)-triene-3,17-diol 3-methyl ether” ([a]‘-, = + 6” and + 71.7”), and 17-ethynyl-19-nortestosterone?’ (1~11~ = - 31.7” and +83.5”). The molecular rotation differences (AM,) between these reference 17~alkynyl derivatives (AM, = - 428 to - 476) or their 17/3-alkynyl isomers (AM’, = - 132 to - 224) and the corresponding 17-0~0 steroid precursors were compared with those found in this work between the 17~hexynyl derivatives 5 (AM, = -386), 6 (AM, = -343), and 7 (AM, = - 322), and their 17-oxo-precursors 2, 3, and 4. These three latter values were intermediate between those established above for the two pairs of 17~ and 17palkynyl reference compounds, and therefore could not be used as a definitive proof of 17-configuration in the absence of the other epimer. The ‘H NMR chemical shifts of the 18-methyl protons could not help to determine the 17-configuration in the absence of the other epimer, because only a small 0.02- to 0.04-ppm downfield shift was reported for the la-methyl signal of 17/3-alkynyl derivatives as compared with their 17a-epimers.20~” This small difference of chemical shifts of IS-methyl protons was confirmed more recently for the two 17-epimers of 17-ethynyl- l7hydroxyandrosta- 1,4-dien-3-one” and of 17-ethynyl9a,17-dihydroxyandrost-4-en-3-one.’3,’” The assignments of the two very close signals in CDCI,, for 18-methyl protons (6 0.82-0.85 ppm) and 19-methyl protons (6 0.82-0.84 ppm), of 17-hexynyl or 17-hexyl Sa-H-steroids 6, 9, 11,14, and 15were made by comparison with the corresponding signals of the 5a-hydroxy analogs 10 and 16, which were more readily identified owing to the selective deshielding of the 19methyl protons (6 1.02 ppm) by the Sa-hydroxy group. These assignments indicate that the reduction of the acetylenic bond induced a significant downfield shift of the 18-methyl proton signal in CDCl, (As = +0.03 ppm). Conversely, the 18- and 19-methyl proton signals of 17-hexynyl or 17-hexyl-5a-hydroxysteroids 7, 10, 13,and 16 were very close in CSDSN. The upfield signal in C,D,N was tentatively assigned to the 19-methyl protons, assuming that acetylation of the 3-hydroxyl

128 Steroids,

1992, vol. 57, March

group should modify more significantly the I9-methyl signal (6 I .07 ppm compared with 1.13 ppm) than the IS-methyl signal. This IS-methyl signal was only slightly influenced (6 I. 18 ppm compared with I. 17 ppm) whether by acetylation of the primary terminal hydroxy group or by catalytic reduction of the acetylenit bond. The low-field angular methyl proton signals in CDCI, of 17-hexanoic acid and methyl ester derivatives of 5adihydrotestosterone 17 and 18 (6 I .03 ppm), and of their testosterone analogs 19 and 20 (6 1.20 and I .21 ppm), were unambiguously assigned to 19-methyl protons owing to the strong deshielding effects of 3-0~0 and 4-en-3-oxo groups on the l9-methyl signal. The comparison of the 18-methyl signals in CDCI, of 17-hexanoic derivatives of Sa-dihydrotestosterone 17 and 18 with those of the 3-ethylenedioxy precursor 11 and of the acetate derivatives 14 and 15 showed that the oxidation of the primary hydroxy group to a carboxylic acid induced a small downfield shift (A6 = + 0.03 ppm) of the 18-methyl signal. This resulted in assignments for 18-methyl signal (6 0.88 ppm) and 19-methyl signal (6 1.03 ppm) of compound 17, which differed from those previously reported ” only by interchanging their order, thus suggesting that the same product was probably obtained even though the substituents used at C3 position of the starting synthetic precursors were different. The assignment of the low-field signals in C,D,N (6 1. I I-l.13 ppm) to 18-methyl protons of 17hexanoic acid and methyl ester derivatives of Sa-dihydrotestosterone and testosterone 17,18,19, and 20 was based on correlations with the assignments described above for the 18-methyl protons of I7cu-hexyl precursors. The ‘H NMR spectra in C,D,N of 17-hexynyl derivatives 5-10and 17-hexyl derivatives 11-20 showed the presence of a characteristic strong downfield shift of the 18-methyl proton signals as compared with ‘H NMR spectra m CDCl, (As = 8CSD5N-6 (-D(.‘?= +0.22 to +0.36 ppm). A similar solvent effect was observed by measuring the corresponding chemical shifts of IS-methyl proton signals of commercially available 17-ethynyl or 17-alkyd reference compounds such as 17a-ethynyl-testosterone, 17~ethynyl-19-nortestosterone, l7cw-ethyl- 19-nortestosterone, 17cr-methyltestosterone, 17a-methyl-5a-dihydrotestosterone, and of their 17~H precursors (Table I). The pyridine-induced downfield shifts of 18-methyl protons of 17cr-hexyl derivatives and of 17cr-ethyl and 17a-methyl reference steroids (A6 = +0.19 to +0.36 ppm) established in this study were similar to those reported’” for 17~ methyl-5a-dihydrotestosterone (Aa = + 0.24 ppm) and 17a-methylandrostane-3p, 17/3-diol (As = + 0.27 ppm) but were very different from those found2” for the 17pmethyl epimers of the two latter compounds (As = + 0.03 and + 0.04 ppm), thus suggesting the same 17~ configuration for methyl, ethyl, and hexyl derivatives and, therefore, for the ethynyl or hexynyl analogs. The comparison of the proton signals in [‘H,]-DMSO (data not shown) and of the infrared stretching band frequencies in Ccl, of the tertiary l7P-OH group

‘H and 13CNMR of 17walkyl Table 1

‘H NMR data for 176-hydroxy,

17a-ethynyl,

17wethyl,

and 17wmethyl

18-CH, 6-CD& Testosterone 19-Nortestosterone 5a-Dihydrotestosterone 17~Ethynyltestosterone 17a-EthynyL19nortestosterone 17wEthyl-19nortestesterone 17a-Methyltestosterone 17~Methyl-5a-dihydrotestosterone

0.80 0.81 0.76

0.94 0.91 0.87

T and DHT: Mappus

reference steroids 17-C=CH or 17-(CH,)W,

19-CH,

&C,D,N 1.04 1.02 0.97 1.11 1.14 1.16 1.10 1.10

showed that the saturated 17a-hexyl groups of 17monohydroxy derivatives 14, 15, 18, and 20 (S OH at 3.8 ppm; v OH: two bands of similar intensities at 3,620-3,600 cm-‘) exerted a much stronger steric shielding of the 17/3-OH group than the 17a-hexynyl group of compounds 8 and 9 (6 OH at 5.1 ppm; v OH: 3,600 cm-‘). These shielding effects are similar to those reported” for 17a-ethyl and 17a-ethynyl derivatives of estradiol 3-methyl ether. The assignments of 13C NMR signals for carbon atoms of the steroid skeletons described in this study were based on data reported for testosteronez6s2’ and androst-4-ene-3,17-dione,26 17P-hydroxy-5cz-androstan-3-one, 3P-hydroxy-Sa-androstan-17-one and 5a-androstane-3, 17-dione28, 5a-androstan-3-one29, Sa-androstan-17/3-01 and Sa-androstan-3/3-oP”, Sa-androstane-3/3,5a-dio13’v32, DHEA acetate33, monobutyryl esters of Sa-androstane-3P, 17/3-diol and androst-5-ene-3/3, 17P-dio134, 17a-methyltestosterone2’, 17a-ethynyl-19-nortestosterone (norethisterand one).35s36 The assignments of the two pairs of close signals at 31.41 and 31.53 ppm in CDCl,, and at 32.47 and 32.19 ppm in C,D,N, respectively, for C-2 and C7 or C-12 carbon atoms of 3/3-hydroxyandrostan-17one 3 (Table 2), were based on the large solvent effect on C-4 signal (Aac_4 = 8cSDSN- a,,,-,, = + 1.24 ppm), which should also occur for the symmetrical C-2 carbon atom (ASc2 = + 1.06 ppm). The resonance positions for carbon atoms of the 17m-alkynyl (Table 3) and 17a-alkyl side-chains (Table 4) were readily identified by comparing their 13CNMR spectra with those of the corresponding unsubstituted 17P-hydroxy analogs (Table 5) and 17-0~0 precursors (Table 2), as well as from the invariability of signals for carbon atoms of the same 17-side-chain between the different steroid series. Assignments of the resonances for the six carbon atoms of the 17-side-chains were made according to data established in CS237, for C-2 and C-3 of linear hexynes, C-l and C-2 of I-hexanol, and C-2 and C-3 of linear methyl hexanoates, but the order of the resonances for the two acetylenic carbon atoms of 17-hexynyl side-chains and for C-2’, C-3’, and C-4’ of 17-hexyl side-chains could not be established. The assignments for the carbon atoms of rings A and

et al.

6-CDC13

&C,D,N

1.20

0.99

1.02

0.92 1.05

1.20 1.03

1.06 0.94

6-CDC13

6-CSDSN

3.49 3.47 0.98 (t: J = 7.3 Hz) 1.21 1.21

0.98 (t: J = 7.2 Hz) 1.41 1.41

B of 3-ethylenedioxy steroids 1, 2, 5, and 11,were made by comparing their signals with those of Sa-dihydrotestosterone and its 17-hexanoic derivatives, 17 and 18. Dioxolanation of the 3-0~0 group modified predominantly the resonances for the C-l to C-5 carbon atoms, which were all shifted upfield, with a maximum effect on C-3 (A6 = - 101 to - 103 ppm). Assignments of the other four carbon atoms were based on those made in CDCl, for the 3-ethylenedioxy derivative of Sa-hydrotestosterone 1. The DEPT technique allowed us to determine unequivocally the resonances for C-5 in the 3-ethyleneketal 1 and in Sa-dihydrotestosterone, at 43.74 ppm and at 46.73 ppm, respectively, thus showing that dioxolanation of the 3-0~0 group resulted in an upfield substituent shift (A6 = -2.99 ppm) of C-5.

Table 2

13C NMR data for 17-oxosteroid 2

precursors 3

4

Carbon

SCDCI,

6-CSDSN

&CDCIB

6-CSDSN

B-C,D,N

C-l c-2 c-3 c-4 c-5 C-6 c-7 C-8 c-9 c-10 C-l 1 c-12 c-13 c-14 c-15 C-16 c-17 C-18 c-19 C-3-ketal

35.97 31.09 109.22 37.93 43.68 28.25 31 .57a 35.03 54.15 35.63 20.46 30.758 47.80 51.42 21.78 35.85 221.31 13.83 11.40 64.15 64.15

36.22 31.67 109.28 38.47 43.81 28.53 32.13a 35.07 54.33 35.82 20.74 31.02= 47.77 51.40 21.91 35.87 219.56 13.82 11.45 64.33 64.39

36.93 31.41 71.09 38.04 44.82 28.38 31 .53a 35.03 54.42 35.63 20.49 30.88a 47.80 51.41 21.77 35.85 221.46 13.81 12.30

37.44 32.47 70.49 39.28 45.18 28.88 32.19= 35.14 54.64 35.93 20.79 31.128 47.83 51.46 21.95 35.93 219.83 13.85 12.43

31.64= 32.30a 66.69 45.63 73.99 35.02 25.31 34.81 45.81 39.43 21.05 32.46 47.99 51.43 21.97 35.97 219.89 13.94 16.12

‘Assignments for C-7 and C-12 of compounds C-l and C-2 of compound 4 may be reversed.

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2 and 3 and for

1992, vol. 57, March

129

Papers Table 3

13C NMR data for 17a-hexynyl

and 17u-ethynyl

steroid derivatives

5

7

17~Ethynyltestosterone

17a-Ethynyl19nortestosterone

Carbon

6-CD&

&CsD,N

K-C,D,N

C-l c-2 c-3 c-4 c-5 C-6 c-7 C-8 c-9 C-IO c-11 c-12 c-13 c-14 c-15 C-16 c-17 C-18 c-19 C-3-ketal

36.08 31.14 109.42 37.92 43.71 28.42 31.51 36.12 53.88 35.51 20.92 31.86 46.96 50.48 23.10 39.09 79.98 12.97 11.45 64.10 64.13 85.75a 84.21a 18.59 25.13 32.88 62.29

36.37 31.77 109.42 38.53 43.85 28.80 32.02 36.53 54.34 35.85 21.47 32.89 47.65 50.95 23.73 40.19 79.34 13.75 11.62 64.31 64.37 86.55a 84.93a 19.10 26.24 33.75 61.49

31.74a 32.33a 66.76 45.60 74.25 35.23 26.12 36.20 45.94 39.45 21.78 32.96 47.89 50.97 23.75 40.29 79.40 13.87 16.35

35.90 34.39 198.35 124.12 170.51 32.75 31.83 36.31 53.71 38.73 21.06 33.31 47.28 50.34 23.52 39.92 79.03 13.41 17.22

26.79 36.82 198.70 124.68 166.28 35.47 31.03 41.16 49.56 42.42 26.47 33.29 47.46 49.34 23.36 39.84 79.03 13.37

86.62a 84.87a 19.03 26.28 34.01 61.57

90.10 74.37

90.07 74.36

CH(OH)-+C CH(OH)-C=C C=C-CH, C-&H,)& CH2-CH,OH CH~-CH~OH a Assignments

for the two acetylenic carbon atoms of compounds

This increment was then used to locate the resonance associated with C-l at 36.05 ppm ( - 2.51 ppm upfield shift) rather than at 37.96 ppm ( - 0.60 ppm upfield shift) and therefore to assign the resonances for C-2 at 3 1.14 ppm ( - 6.98 ppm upfield shift) and for C-4 at 37.96 ppm (-6.69 ppm upfield shift), assuming that the resonances for C-l and C-5, and C-2 and C-4 change symmetrically as the substituent at C-3 position is modified. The assignments for the C-7 and C-12 carbon atoms in 3/3-hydroxy-Sa-androstan-17-one 3 and 3-ethylenedioxy-5a-androstan-17-one 2 are shown as being interchangeable (Table 2), owing to the opposite orders that were found for these two carbon atoms either by comparison with assignments reported, in CDCl, only, for the derivative 3 and 5a-androstan-3, 17-dione2’ (C-7 signal upfield) or by correlation of the effects of 17@hydroxy and 17-0~0 substituents on C-7 and C-12 signals of steroid derivatives 1 and 2 with those reported for 17/Shydroxy-androst-4-en-3-one and androst-4ene-3, 17-dione26 (C-7 signal downfield). A correlation of C-2 and C-7 signals of 3-ethylenedioxy-5a-androstan-17/3-ol 1 in both CDCl, and C,D,N with those of 3-ethylenedioxy derivatives 5 and 11 suggests that the low-field resonance of these signals should correspond rather to C-7 than to C-2. This tentative assignment was confirmed by the similarity of the downfield shifts observed then, in CSDSN for C-2 (A6 = +0.05 130

Steroids,

1992, vol. 57, March

X,D,N

5 and 7 and for C-l and C-2 of compound

R-C,D,N

7 may be reversed.

ppm) and C-7 (A6 = +0.22 or +0.25 ppm, depending on the two alternative assignments mentioned in Table 4) after hydrogenation of the hexynyl derivative 5 to the hexyl derivative 11 with those readily established for their Sa-OH analogs 7 and 13 (A6 = +0.03 and t-O.31 ppm), owing to the strong deshielding of the C7 signal by the 5a-OH group. The close C-l, C-2, and C-12 signals of 3P,Scz-diol derivatives 4, 7, and 13 in C,D,N were assigned by correlation with the corresponding signals of their 3-ethylenedioxy analogs 2, 5, and 11 as well as by comparison of C-l or C-2 signals, which were only slightly influenced by changing the 17-function in each group of 3-substituted steroid derivatives. Catalytic hydrogenation of 17-hexynyl side chains led in both series to very small downfield shifts of the C-l or C-2 signals (A6 = + 0.02 to 0.05 ppm), whereas a significant upfield shift of the C-12 signal was observed. The signals for C-l and C-2 of derivatives 4, 7, and 13 in CSD,N were well separated in contrast to C-l and C-2 signals of the Sa-androstane-3&5a-diol reference compound3’ in CDCl,, but their relative order could not be established. The assignments for the C-l 1 to C-18 carbon atoms of rings C and D of 17cu-hexynyl steroids 5 and 7 were first made by comparison with those reported for 17~ propynylestradiol in C,D, and derived from heteronuclear ‘H-13C-shift correlated COSY experiments in

‘H and 13CNMR of 17walkyl Table 4

13CNMR

C-l c-2 c-3 c-4 c-5 C-6 c-7 C-8 c-9 C-IO c-11 c-12 c-13 c-14 c-15 C-16 c-17 C-16 c-19 C-3-ketal CH(OH)-CH, C-(C&l,-C -

CH2-CH*OH CH,-CH~~H CH,-COO coo OCH,

Table 5

11 6-CSDSN

13 6-CsDsN

36.41 31.82 109.47 38.58 43.97 28.90 32.248 36.71 54.38 35.88 21.40 32.27a 47.16 51.08 24.26 37.63 82.70 15.45 11.64 64.37 64.41 34.53 24.47 26.85 31.08 33.92 62.16

31.76' 32.36a 66.80 45.67 74.23 35.31 26.43 36.36 45.83 39.46 21.68 32.54 47.33 51.03 24.27 37.59 82.76 15.54 16.32

38.65 38.18 211.84 44.72 46.83 28.93 31.54 36.35 53.87 35.80 21.19 31.59 46.51 50.39 23.28 36.54 83.34 14.50 11.51

38.78 38.37 210.38 44.91 46.88 29.15 31.84 36.54 53.95 35.95 21.53 32.16 47.10 50.83 24.19 37.47 82.63 15.40 11.40

36.01 34.41 198.36 124.11 170.76 32.91 32.00 36.65 53.97 38.84 21.10 32.16 46.92 50.49 24.14 37.47 82.52 15.28 17.25

34.56 24.46 26.83 31.06 33.91 62.14

34.10 23.74 25.05 30.04

34.44 24.24 25.97 30.76

34.43 24.19 25.99 30.76

34.38 174.26 51.44

34.99 176.06

35.07 176.18

for C-7 and C-12 of compound

13C NMR

C-l c-2 c-3 c-4 c-5 C-6 c-7 c-a EO c-11 c-12 c-13 c-14 c-15 C-16 c-17 C-18 c-19 C-3-ketal

18 &CDCI,

11 and for C-land C-2 of compound

17 &CsD,N

19 &C,D,N

13 may be reversed.

data for 17P-hydroxysteroid derivatives

19-Nortestosterone Carbon

et al.

data for 17a-hexyl steroidderivatives

Carbon

aAssignments

T and DHT: Mappus

XDCI,, 26.56 36.38 200.06 124.52 166.86 35.47 30.66 40.44 49.70 42.55 26.11 36.49 42.99 49.56 23.17 30.33 al.59 11.07

Testosterone

6-CSDSN

S-CDC13

6-CsDsN

26.90 36.90 198.69 124.73 166.36 35.49 31.01 40.59 50.01 42.48 26.37 37.17 43.54 49.79 23.57 30.88 al.14 11.74

35.66 33.88 199.64 123.74 171.53 32.79 31.53 35.61 53.90 38.64 20.62 36.42 42.78 50.46 23.31 30.26 81.36 ii.08 17.38

35.91 34.29 198.17 124.03 170.57 32.76 31.85 35.68 54.16 38.71 20.90 37.09 43.25 50.75 23.65 30.81 80.99 11.69 17.16

5a-Dihydrotestosterone &CDCI, 38.56 38.12 212.12 44.65 46.73 28.79 31.25 35.42 53.91 35.72 21.03 36.65 42.98 50.82 23.38 30.41 81.71 11.16 ii.48

1

6-CSDSN

S-CDC13

6-CSDSN

38.76 38.35 210.33 44.90 46.84 29.04 31.56 35.63 54.17 35.90 21.40 37.42 43.54 51.15 23.80 30.97 al.28 ii.88 11.37

36.05 31.14 109.37 37.96 43.74 28.44 31.47 35.53 54.17 35.53 20.79 36.77 43.00 51.02 23.40 30.51 al.92 11.16 11.43 64.13 64.13

36.39 31.77 109.41 38.56 43.96 28.82 31.95 35.83 54.60 35.83 21.27 37.53 43.58 51.38 23.83 31.00 81.35 11.91 11.60 64.33 64.37

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1992, vol. 57, March

131

Papers

conjunction with DEPT experiments.-‘x These assignments were also compared with those reported for 17~ ethynylestradiol 3-methyl etherj9 and 17a-ethynyl-19nortestosterone3h in CDCI,, 17a-ethynyl-19-nortestosterone3s and 17a-ethynyl-5a-dihydro-IPnortestosterone40 in CD,OD, and 17~ and 17P-ethynyl derivatives of 9a, 17-dihydroxyandrost-4-en-3-onez3,14 in [‘HJDMSO. However, reverse orders of assignments have been mentioned for C-7 and C- 12 carbon atoms of 17~ ethynyl-19-nortestosterone between ‘%ZNMR spectra recorded in CD30D3’ (C-7 at 32.4 ppm and C-12 at 34.1 ppm) and in CDClj3” (C-7 at 32.3 ppm and C-12 at 30.6 ppm), although the chemical shift of the unequivocally assigned C-12 signals of 17a-ethynylestradiol 3-methyl ether had been found at 32.7 ppm in CDC1339and at 32.9 ppm for 17a-propynylestradiol in CgD6.38In this study, the comparison of i3C NMR spectra in C,D,N of 17cz-ethynyl-19-nortestosterone and 17~ethynyltestosterone (Table 3) with those of their respective 19-nortestosterone and testosterone precursors (Table 5) showed that in both series 17a-ethynylation changed exclusively the chemical shifts of carbon atoms of rings C and D without any significant modification of the C-7 signal. This C-7 signal could therefore correspond only to the unchanged upfield resonance of the two close C-7 and C-12 signals of 17a-hexynyl steroids 5, as compared with C-7 of its unsubstituted 17-OH precursor 1, thus confirming that the order of the assignments for C-7 and C-12 reported above in C,D,” or in CD30D35 is still valid in C,D,N. The substituent effects of unsaturated 17a-hexynylor 17a-ethynyl side-chains were estimated by comparing the 13C NMR spectra in C,D,N of the 17-hexynyl derivative 5 and of 17a-ethynyl-19-nortestosterone and 17a-ethynyltestosterone (Table 3) with those of their unsubstituted 17P-hydroxy analogs 1, 19-nortestosterone, and testosterone (Table 5). The largest effects concerned the resonances for C-12, C-13, C-16, C-17, and C-18 carbon atoms, which were modified similarly in the two 17a-hexynyl and 17a-ethynyl series, thus suggesting the same 17a-configuration. Upfield shifts were found for C-12 (As = - 3.78 to - 4.64 ppm) and C-17 (Aa = - 1.96 to - 2.11 ppm), and downfield shifts for C-16 (A6 = +8.96 to +9.19 ppm), C-13 (A6 = +3.92to +4.07ppm),andC-18(Aa = +1.63to +1.84 ppm) carbon atoms. This 17a-configuration was also suggested by the similarity of these effects with those estimated by comparing the corresponding resonances reported in 13C NMR spectra of 17a-propynylestradio13*, 17a-ethynylestradiol 3-methyl ether,” and estradiol. Much smaller modifications occurred for C-8 (A6 = +0.57 to +0.70 ppm), C-9 (As = -0.26 to -0.45 ppm), C-11 (As = +O.lO to +0.20 ppm), C-14 (A6 = -0.31 to -0.45 ppm), and C-15 (A6 = -0.10 to -0.21 ppm) signals, in both 17a-hexynyl and 17~ ethynyl series. Similar shifts were also observed in CDCl, for the 17a-hexynyl derivative 5. The 17cY-configuration of I7-hexynyl derivatives 5 and 7 and of 17a-ethynyltestosterone was confirmed by comparing C-17 and C-18 resonances with data reported in [*H,]-DMSO for 17~ and 17p-ethynyl deriva132

Steroids,

1992, vol. 57, March

tives of 9c~,17-dihydroxyandrost-4-en-3-one. The C-IX resonance (6 13.41 ppm) of 17a-ethynyltestosterone in C,D,N was in better agreement with that mentioned for C-18 (6 12.03 ppm) of 90!,17-dihydroxy-17~ethynylandrost-4-en-3-onez3 than with that reported for C-18 (6 15.54 ppm) of 9a, 17-dihydroxy- I7P-ethynyiandrost-4en-3-one,24 thus supporting the 17a-configuration of the 17-ethynyltestosterone reference compound and therefore of the 17-substituted derivatives 5 and 7, which have similar chemical shifts for C-18 (6 13.75 and 13.87 ppm, respectively). A similar conclusion could be reached by comparing the C- 17 resonances of 17cw-ethynyltestosterone (6 79.03 ppm) and of compounds 5 and 7 (6 79.34 and 79.40 ppm, respectively) with resonances reported for C- 17 carbon atoms of 17~ and 17/3-ethynyl derivatives of 9a, 17-dihydroxyandrost-4-en-3-one (6 78.06 ppm and 87.35 ppm, respectively). However, the correlation of these two latter resonances with those reported4’ for C- 17 carbon atoms of 17~ and 17/%cyano analogs of 9a, 17-dihydroxyandrost-4-en-3-one (6 79.66 ppm and 76.70 ppm, respectively) in [*H,]-DMSO suggests that the above mentioned resonance at 87.35 ppm for 9~~,17-dihydroxy-l7p-ethynylandrost-4-en-3-one should be assigned rather to the acetylenic Ca than to C-17 carbon atom, which should therefore correspond to one of the resonances at 75.35 ppm or 76.55 ppm. This latter assignment does not modify the conclusions mentioned above concerning C- 17 configuration. The 17a-configuration of 17-hexyl derivatives was readily derived from that established for 17-hexynyl precursors, because catalytic hydrogenation is not expected to modify the orientation of the 17-side-chain. This 17a-configuration was also confirmed by the analogies between 17-substituent effects, established in CDCI, or in C,D,N, by comparing the 13CNMR spectra of the 17-hexyl derivatives 11and 13, and 17-hexanoic derivatives 17, 18, and 19 with those of commercially available 17a-ethyl-19-nortestosterone, 17a-methyltestosterone, and 17a-methyl-5a-dihydrotestosterone reference steroids. The largest 1‘I-substituent effects, in 17a-hexyl derivatives 11, 17, and 19 (Table 4) as compared with their unsubstituted 17@-hydroxy analogs 1, Sa-dihydrotestosterone, and testosterone (TableS), concerned the resonancesinC,D,N,forC-12,C-13,C-16,C-17,andC-18 carbon atoms. An upfield shift was found for C- 12 (As = - 4.93 to - 5.26 ppm), whereas downfield shifts were foundforc-17(A6 = + 1.35to + 1.53ppm),C-16(A6 = +6.50 to +6.66 ppm), C-13 (A6 = +3.56 to +3.67 ppm), and C-18 (A6 = +3.52 to +3.59 ppm). Much occurred for the C-8 smaller modifications (A& = +0.88to +0.97ppm),C-9(Aa = -0.19to -0.22 ppm), C-11 (A6 = +0.13 to +0.20 ppm), C-14 (A6 = - 0.26 to - 0.32 ppm), and C- 15 (A6 = + 0.36 to + 0.49 ppm) carbon atoms, whereas a weak characteristic effect in ring B was also found for the C-7 signal (A6 = +0.15 to +0.32 ppm), which was not observed in the case of 17-hexynyl substituents. Similar substituent effects were also observed in CDCI, for the 17a-hexanoic methyl ester 18 compared with Sa-dihydrotestosterone. These 17-substituent effects of hexyl side-chains

‘H and 13CNMR of 77cu-alkyl T and DHT: Mappus Table 6

13C NMR data for 17a-ethyl

and 17wmethyl

steroid derivatives 17a-Methyl-Scu-dihydrotestosterone

17a-Ethyl-19nortestosterone Carbon C-l c-2 c-3 c-4 c-5 C-6 c-7 C-8 c-9 c-10 C-l 1 c-12 c-13 c-14 c-15 C-16 c-17 C-18 c-19 CH,-17 CH3-CH2 CH,-CH,

et al.

6-CDC13

6-CSDSN

8-CDC13

26.56 36.49 199.99 124.47 166.88 35.52 30.91 41.27 49.45 42.55 26.16 31.25 46.39 49.21 23.43 33.36 83.27 14.48

26.90 36.92 198.70 124.72 166.42 35.57 31.31 41.48 49.66 42.52 26.44 31.90 47.03 49.65 23.90 33.54 82.60 15.31

35.70 33.94 199.59 123.81 171.38 32.84 31.68 36.45 53.79 38.66 20.66 31.40 45.34 50.13 23.20 38.81 81.41 13.91 17.39 25.81

7.78 28.78

8.52 29.47

were similar to those established for ethyl side-chains when comparing the corresponding resonances in CSD,N, of 17a-ethyl-19-nor-testosterone (Table 6) with those of 19nortestosterone (Table 5), except for the C-16, which was much less deshielded (A6 = +2.66 ppm compared with + 6.50 to 6.66 ppm). These effects were also similar to those established for methyl sidechains when comparing 13CNMR spectra in C,D,N, of 17a-methyltestosterone and 17a-methyl-Sa-dihydrotestosterone (Table 6) with those of testosterone and Sa-dihydrotestosterone (Table 5). However, the comparison of these two 17-methyl derivatives with the 17ethyl analog showed an increased shielding effect on C-16 (A6 = +8.51 to 8.57 ppm compared with +6.50 to 6.66 ppm) and an opposite deshielding effect on C17 (A6 = -0.56 to -0.72 ppm compared with + 1.35 to + 1.53 ppm). A comparison of 17-substituent effects in C,DSN between the 17cr-hexyl derivatives 11 or 13 and their 17o+hexynyl precursors 5 or 7 indicates that hydrogenation of the acetylenic bond resulted in significantly different effects on resonances for C-17 (A6 = + 3.36 ppm), C-16 (A6 = -2.56 to -2.70 ppm), and C-18 (A6 = + 1.67 to + 1.70 ppm). Smaller differences were observed for C-12 (A6 = -0.42 to -0.65 ppm), C-13 (A6 = -0.49 to -0.56 ppm), and C-15 (A6 = +0.52 to +0.53 ppm), whereas a small but characteristic downfield substituent effect was found for C-7 (A6 = + 0.22 to + 0.3 1 ppm). Similar observations were made by comparing the corresponding resonances of 17aethyl-19-nor-testosterone and 17a-ethynyl-19-nortestosterone (Table 6), except for C-16 resonance, which was shifted more upfield as compared with the hexyl

X,D,N 35.96 34.40 198.33 124.09 170.70 32.86 32.04 36.53 53.99 38.80 20.99 32.04 45.94 50.53 23.71 39.38 80.43 14.72 17.20 26.71

6-CDC13

&C,D,N

38.56 38.13 212.07 44.65 46.75 28.83 31.41 36.23 53.79 35.72 21.06 31.58 45.51 50.47 23.25 38.89 81.55 14.01 11.47 25.82

38.77 38.36 210.31 44.91 46.89 29.12 31.77 36.47 54.00 35.94 21.45 32.24 46.15 50.90 23.82 39.48 80.56 14.86 11.38 26.77

group (As = - 6.30 ppm compared to - 2.56 to - 2.70 ppm) owing probably to the stronger steric effect of the ethyl group. The 13C NMR data described in this work indicate that structural modifications of the 17-substituent of 17ar-alkynyl and I7a-alkyl derivatives of 19-nortestosterone, testosterone, and Sa-dihydrotestosterone result in characteristic stereoelectronic differences in the environment of C-17 position. The evaluation of the influence of these modifications on the relative binding affinities of these derivatives, as well as of other 17substituted analogs and their use for the analysis and purification of SHBG and androgen receptors are under investigation and will be presented in a future article.

Acknowledgments We thank 0. Miani, Dr. B. Fenet, and Dr. J. C. Duplan (Centre RMN, Universite Claude Bernard, Lyon) for NMR measurements, M. J. Bobenrieth, Dr. D. Fraisse, and Dr. J. Favre-Bonvin (CNRS Lyon-Solaise) for mass spectra, and Dr. J. Carew for help in editing the manuscript.

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37. 38.

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