May 2001
529
plied finasteride and NADPHϩ.
All P. crustosum culture media were prepared following Pitt’s proce-
dure.18) The materials were obtained from Baker/or Bioxon (Becton Dickin-
son), Mexico.
were carried out in order to test the radioactive purity of the isolated DHT.14)
All experiments were performed in duplicate. Radioactivity was determined
in a Packard tri-carb 3255 liquid scintillation spectrometer, using the stan-
dard solution (Packard, Downers Grove, IL, U.S.A.) as the counting solution.
The counting efficiency of 3H was 57%. The formation of 5a-DHT was cal-
culated and expressed as fmol/g of dry mycelium. The results were analyzed
with the analysis of variance (Anova) test, using the Epistat software.
Animals and Tissues Adult male Syrian Golden hamsters (150—200 g)
were obtained from the Metropolitan University-Xochimilco of Mexico. The
animals were kept in a room with controlled temperature (22 °C) and light–
dark periods of 12 h. Food and water were provided ad libitum.
Synthesis of the Steroid Compounds. 17a-Hydroxy-17b-methyl-
16b-phenyl-D-homoandrosta-1,4,6-triene-3,17a-dione (18) A solution
containing steroid 15 (1.0 g, 2.8 mmol), DDQ (2.2 g, 9.7 mmol) in dioxan
(36 ml) was allowed to reflux for 72 h. The reaction mixture was cooled to
0 °C and the precipitated dichlorodicyano hydroquinone was filtered off. The
organic phase was washed with an aqueous solution of sodium hydroxide
(3%) and dried over anhydrous sodium sulfate; the solvent was eliminated in
vacuum. The crude product was purified by silica gel column chromatogra-
phy. Hexane–ethyl acetate (8 : 2) eluted 0.5 g, 1.2 mmol (43%) of pure com-
pound 18. mp 280—282 °C. UV (nm): 256 (eϭ11600), 298 (eϭ26500). IR
Gonadectomies were performed under light ether anesthesia 15 d before
treatment. Animals were sacrificed by ether anesthesia. The effect of the
synthesized steroids was evaluated in the three different experimental mod-
els.
(KBr) cmϪ1: 3446, 3028, 1694, 1656, 1618, 1126. H-NMR (CDCl3) d: 1.0
1
(3H, s), 1.1 (3H, s), 1.4 (3H, s), 3.0 (1H, dd, J1ϭ3, J2ϭ2 Hz), 6.0 (1H, s), 6.3
(1H, s), 7.1 (2H, d. Jϭ3 Hz), 7.3 (5H, m). 13C-NMR (CDCl3) d: 12 (C-18),
16 (C-19), 24 (CH3 at C-17), 54 (C-16), 79 (C-17), 124 (C-4), 138 (C-1),
161 (C-2), 165 (C-5), 121, 124 (phenyl), 185 (C-3 carbonyl), 218 (C-17a-
carbonyl). MS (m/z) 402 (Mϩ). Anal. Calcd for C27H30O3: C, 80.60; H, 7.46;
O, 11.94. Found: C, 80.66;H, 7.41; O, 11.93.
Flank Organ Test The flank organ test was performed as previously re-
ported.16) The effect of steroids 7, 8, 13, 16—19 on the flank organs of male
hamsters, which were gonadectomized 15 d before the experiments, was de-
termined on 18 groups of 4 animals/experiment, selected at random.
Daily subcutaneous injections of 200 mg of the steroids 7, 8, 13, 16—19
(Figs. 2, 3) dissolved in 20 ml of sesame oil were administered for 4 d to-
gether with 200 mg of T. Three groups of animals were kept as control, one
was injected with 20 ml of sesame oil, the second with 200 mg of T for 4 d
and the third with 200 mg of finasteride.16)
After these treatments, the animals were sacrificed by ether anesthesia.
Both flank organs of the animals were shaven and the diameter of the pig-
mented spot was measured. The experiments were carried out in quadrupli-
cate on two different occasions. The results were analyzed using one-way
Anova with Epistat software.
17a-Acetoxy-17b-methyl-16b-phenyl-D-homoandrosta-1,4,6-triene-
3,17a-dione (19) A solution containing steroid 18 (1.0 g, 2.48 mmol), PTS
(50 mg), trifluoroacetic anhydride (0.6 ml) and glacial acetic acid (1.26 ml)
was stirred for 1.5 h at room temperature (nitrogen atmosphere). The reac-
tion mixture was diluted with chloroform (10 ml) and was neutralized with
an aqueous sodium bicarbonate solution to a pH of 7. The organic phase was
separated and dried over anhydrous sodium sulfate; the solvent was elimi-
nated in vacuum and the crude product was purified by silica gel column
chromatography. Hexane–ethyl acetate (8 : 2) eluted 0.6 g, 1.3 mmol (52%)
of the pure product 19. mp 240—242 °C. UV (nm): 254 (eϭ11800), 300
In Vitro Incorporation of [1,2-14C]Sodium Acetate into Lipids The
flank organs of the sacrificed animal were dissected around the pigmented
spot.15,16) To evaluate the effect of the hormone treatments on sodium acetate
incorporation into lipids under culture condition, the hamster flank organs
were placed in Erlenmeyer flasks (4 whole glands/flask) in a shaking water
bath with 3.7 mCi of [1,2-14C]sodium acetate specific activity 58.20 mCi/mmol.
To this solution were added 2 mM of nonradioactive glucose,16) albumin
16 mg/ml, biotin 0.36 mM, ATP 0.66 mM, nonradioactive sodium acetate
1.29 mM, NADPHϩ 0.0033 and 10 mg of each steroid. The cultures were in-
cubated in duplicate for 24 h at 37.5 °C in a final volume of 2 ml of Krebs–
Ringer buffer (pH 7.4) Incubations without tissue were used as a control.
Determination of Incorporated Radioactivity in Lipids For the lipid
extraction, the glands were placed in Folch’s solution (4 glands/10ml)19) for
24 h at room temperature in reviously weighed glass vials. Immediately
thereafter, the glands were recovered from the solvent medium. Water, one
ml, was added to the organic phase to remove the free [1,2-14C] sodium ac-
etate. The aqueous phase was removed in vacuum and the extract was dried.
The extracted lipids were weighed. The incorporated radioactivity was deter-
mined with a Packard liquid scintillation spectrometer with Ultima gold
(Packard Downers Grove, IL, U.S.A.) as the counting solution. The counting
solution efficiency for 14C was 87%. The specific activity of the incorporated
radiolabeled sodium acetate in lipids was calculated and expressed as
nmol/gland/h8,9) and as a percentage of incorporation of the total radioactiv-
ity used. The results were analyzed using one-way Anova with Epistat soft-
ware.
1
(eϭ26400). IR (KBr) cmϪ1: 3018, 1698, 1650, 1622. H-NMR (CDCl3) d:
1.2 (3H, s), 1.3 (3H, s), 1.5 (3H, s) 2.2 (3H, s), 2.8 (1H, dd, J1ϭ3, J2ϭ2 Hz),
5.9 (1H, s), 6.4 (1H, s), 7.0 (2H, d, Jϭ3 Hz), 7.3 (5H, m). 13C-NMR (CDCl3)
d: 14 (C-18), 17 (C-19), 25 (CH3 at C-17), 56 (C-16), 80 (C-17), 122 (C-4),
140 (C-1), 160 (C-2), 165 (C-5), 175 (acetoxy), 192 (C-3 carbonyl), 212 (C-
17a-carbonyl). MS (m/z) 444 (Mϩ). Anal. Calcd for C29H32O4: C, 78.38; H,
7.18; O, 14.39. Found: C, 78.43; H, 7.18; O, 14.39.
In this study we observed that when the ketal 14 was hydrolyzed, an ex-
pansion of the D-ring took place, thus forming the D-homosteroid 15. This
expansion was due to the presence of the perchloric acid used for the hydrol-
ysis. We actually could never obtain compound 15 with the normal D-ring.
In view of the fact that 15 was a new compound, we were very anxious to
determine its pharmacological activity as 5a-reductase inhibitor as well as
an antiandrogen.
Evaluation of the Synthesized Steroids in P. crustosum Cultures The
fungus P. crustosum was isolated in a solid culture medium consisting of
potato dextrose agar (PDA) as described in references 13, 14. Species identi-
fication was performed following Pitt’s criteria,18) which uses three different
culture media, Czapek yeast autolysate agar (CYA; Pitt, 1973); malt extract
agar (MEA; according to Blakeslee, 1915) and 25% glycerol nitrate agar
(25Y; Pitt, 1973) as well as three incubation temperatures, 5°, 25° and 37 °C
during seven days (Pitt, 1979). One plate of each of CYA, MEA, and G25Y
was incubated at 25°, another plate of CYA at 37° and a third at 5 °C.
Conversion of T to DHT by P. crustosum The conversion of T to its
metabolite DHT was determined by the reverse isotope dilution technique;
TLC was used for the isolation and identification of DHT.14)
Acknowledgement We gratefully acknowledge the financial support of
DGAPA (PAIP 99) for the project IN 204198.
Incubation: 16 Erlenmeyer flasks containing 16 ml of sterilized potato
dextrose broth14) were inoculated with P. crustosum and stoppered with foam
plugs. The inoculated flasks were placed in a water bath at 25 °C and main-
tained under constant shaking for 24 h. Then, 100 ml of radiolabeled T [3H]T,
(5.4 mCi) in 95% ethanol as well as 0.5 mM of each one of the new steroids
were added to the medium. The flasks were kept under incubation conditions
for on additional 96 h; two Erlenmeyer flasks were prepared as a non-inocu-
lated control.
Extractions: the cultures were extracted with 5 ml of dichloromethane sat-
urated with water (3X). The solvent was evaporated to dryness in a rotary
evaporator (Caframo vv 2000).
The extract was spotted on a TLC plate in order to isolate and identify the
formed products. The unknown samples were run together with steroid car-
riers (T, DHT) using; chloroform/acetone, 9 : 1 (v/v) as solvent system. After
development, the T standard was identified under 254 nm UV light. DHT
was assayed on the same plate after spraying with phosphomolybdic acid
reagent (8% in methanol). Recrystallizations to constant specific activity
References
1) Bruchowsky N., Wilson J. D., J. Biol. Chem., 243, 2012—2021 (1968).
2) Chen C., Li X., Singh S. M., Labrie F., J. Invest. Dermatol., 111,
273—278 (1998).
3) Raynaud J. P., Azadian-Boulanger B., Bonne C., Perronnet J., Sakis E.,
Present trends In: Antiandrogen Research in Androgens and Antian-
drogens, Martini L, Motta M. Raven Press: New York, 1977, pp.
309—311.
4) Brooks J. R., Harris G. S., Sandler M., Smitter H. J. (eds.) “Design of
Enzyme Inhibitors as Drugs,” Vol. 2, Oxford University Press, Oxford,
1994, pp. 495—498.
5) Bratoeff E., Ramírez E., Murillo E., Flores G., Cabeza M., Curr. Med.
Chem., 6, 1107—1123 (1999).
6) Rassmusson G., Reynolds G., Steinberg N., J. Med. Chem., 29, 2298—
2315 (1986).
7) Bratoeff E., Herrera H., Ramírez E., Solórzano K., Murillo E., Quiroz
Cabesa
