October 2003
1135
dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (2.2 g) in dioxane (50 ml) hydroxide solution (25 ml) was allowed to reflux for 30 min. Part of the
was allowed to reflux for 4 d. Upon cooling, the precipitated 2,3-dichloro- methanol was removed in vacuum and water (20 ml) was added to the result-
5
,6-dicyano-hydroquinone was filtered off. To the filtrate was added 3% ing solution . It was extracted with chloroform, the organic phase was sepa-
aqueous sodium hydroxide solution (100 ml) and chloroform (100 ml); the rated, washed with water and dried over anhydrous sodium sulfate. The sol-
mixture was stirred for 5 min. The organic phase was separated and washed vent was removed in vacuum; the crude product was recrystallized from
3
times with 3% aqueous sodium hydroxide solution and then with water. It
ethyl acetate–hexane. Yield 660 mg, 1.92 mmol (75%), mp 204—206 °C.
Ϫ1
1
was dried over anhydrous sodium sulfate and the solvent was removed in UV (nm): 254 (eϭ10800). IR (KBr) cm : 3436, 2947, 1702 and 1658. H-
vacuum. The crude product was purified by silica gel column chromatogra- NMR (CDCl ) d: 0.69 (3H, s), 1.22 (3H, s), 2.0 (3H, s), 2.00 (3H, s), 4.97
3
1
3
phy. Hexane–ethyl acetate (6 : 4) eluted 640 mg, 1.88 mmol (65%) of the (1H, d, Jϭ10 Hz) and 6.0 (1H, s). C-NMR (CDCl ) d: 15.4 (C-18), 17.1
3
ϩ
pure product 1, mp 198—2000 °C. UV (nm): 222, 255, 298 (eϭ14900, 1270 (C-19), 23.8 (C-21), 175.2 (C-3) and 211.5 (C-20). MS (m/z) 342 (M ).
Ϫ1
1
and 17900) respectively. IR (KBr) cm : 3387, 1705, 1656, 1602. H-NMR
6-Methylene-17a-toluoyloxypregn-4-ene-3,20-dione 4: A solution con-
(
(
CDCl ) d: 0.87 (3H, s), 1.1 (3H, s), 1.3 (3H, d, Jϭ4 Hz), 2.0 (3H, s), 6.0 taining steroid 3 (1 g, 2.92 mmol), PTSA (10 mg), trifluoroacetic anhydride
3
1H, s), 6.2 (1H, d, Jϭ2 Hz), 6.4 (1H, d, Jϭ3 Hz), 6.6 (1H, d, Jϭ3 Hz), 7.1 (8.29 g, 42.48 mmol) and p-toluic acid (1.36 g, 10 mmol) was stirred for two
13
(
2
3
1H, d, Jϭ2 Hz). C-NMR (CDCl ) d: 15.0 (C-18), 16.2 (CH at C-16), hours at room temperature (nitrogen atmosphere). The reaction mixture was
3
3
0.5 (C-19), 26.5 (C-21), 78.2 (C-17), 186.2 (C-3), 217.0 (C-20). MS m/z: neutralized with an aqueous solution of sodium bicarbonate to a pH of 7 and
ϩ
40 (M ).
diluted with chloroform (10 ml). The organic phase was separated and dried
1
6b-Methyl-17a-toluoyloxypregna-1,4,6-triene-3,20-dione 2: A solution over anhydrous sodium sulfate; the solvent was eliminated in vacuum. The
containing steroid 1 (1 g, 2.94 mmol), PTSA (10 mg) and trifluoroacetic an- crude product was purified by silica gel column chromatography.
hydride (8.29 g, 42.48 mmol) was stirred for 2 h at room temperature (nitro- Hexane–ethyl acetate (8 : 2) eluted 680 mg, 1.48 mmol (50%), mp 214—
Ϫ1
gen atmosphere). The reaction mixture was neutralized with an aqueous so- 216 °C. UV (nm): 242 (eϭ11000). IR (KBr) cm : 2948, 17816, 1672 and
1
lution of sodium bicarbonate to a pH of 7 and diluted with chloroform
(
1610. H-NMR (CDCl ) d: 0.74 (3H, s), 1.12 (3H, s), 2.1 (3H, s), 2.45 (3H,
3
10 ml) The organic phase was separated and dried over anhydrous sodium s), 4.96 (1H, t, Jϭ1.8 Hz) 5.1 (1H, d, Jϭ1.8 Hz) , 5.94 (1H, s), 7.27 (2H, m)
sulfate; the solvent was eliminated in vacuum. The crude product was puri- and 8.0 (2H,m). C-NMR (CDCl ) d: 15.2 (C-18), 16.8 (C-19), 24.1 (C-21),
fied by silica gel column chromatography. Hexane–ethyl acetate (8 : 2) eluted 165 (ester carbonyl), 195 (C-3) and 203 (C-20). MS (m/z) 460 (M ).
1
3
3
ϩ
7
00 mg, 1.74 mmol (59%), mp 186—189 °C. UV (nm): 221, 255 and 300
17a-(p-Bromobenzoyloxy)-6-methylenepregn-4-ene-3,20-dione 5: A so-
lution containing steroid 3 (1 g, 2.92 mmol), PTSA (10 mg), trifluoroacetic
Ϫ1
(
1
(
eϭ14600, 12400 and 17300, respectively). IR (KBr) cm : 1720, 1707,
1
664 and 1604. H-NMR (CDCl ) d: 0.8 (3H, s), 1.0 (3H, d, Jϭ2 Hz), 1.3 anhydride (8.29 g, 42.48 mmol) and p-bromobenzoic acid (2 g, 10 mmol)
3
3H, s), 2.0 (3H, s), 6.0 (1H, s), 6.1 (1H, s), 6.3 (2H, m) and 7.0 (1H, d, was stirred for 2 h at room temperature (nitrogen atmosphere). The reaction
13
Jϭ10 Hz). C-NMR (CDCl ) d: 15.7 (CH at C-16), 17.6 (C-18), 20.6 (C- mixture was neutralized with an aqueous solution of sodium bicarbonate to a
3
3
1
9), 83.9 (C-17), 165 (C-5), 172 (ester carbonyl), 182 (C-3) and 212 (C-20). pH of 7 and diluted with chloroform (10 ml). The organic phase was sepa-
ϩ
MS (m/z) 458 (M ).
7a-Acetoxy-3-methoxypregna-3,5-diene-20-one 19: A solution contain- vacuum. The crude product was purified by silica gel column chromatogra-
ing steroid 18 (1 g, 2.68 mmol), PTSA (200 mg) and trimethyl orthoformate phy. Hexane–ethyl acetate (8 : 2) eluted 710 mg, 1.35 mmol (46%), mp
rated and dried over anhydrous sodium sulfate; the solvent was eliminated in
1
Ϫ1
(
5 ml, 45.7 mmol) was stirred for 1 h at room temperature; at this time, pyri- 213—216 °C. UV (nm): 247 (eϭ10750). IR (KBr) cm : 2948, 1718, 1671,
1
dine (5 ml) was added and the reaction mixture was stirred for an additional 1589 and 757. H-NMR (CDCl ) d: 0.7 (3H, s), 1.1 (3H, s), 2.1 (3H, s), 5.9
1
cipitated; this was filtered and dried. Yield 620 mg, 1.6 mmol (60%); mp 16.3 (C-19), 23.2 (C-21), 170 (ester carbonyl), 197 (C-3) and 205 (C-20).
3
1
3
0 min. It was poured into ice-water (200 ml) and the crude product 19 pre- (1H, s), 7.65 (2H, m) and 8.0 (2H, m). C-NMR (CDCl ) d: 15.4 (C-18),
3
Ϫ1
ϩ
1
54—156 °C. UV (nm): 238 (eϭ12000). IR (KBr) cm : 2975, 1736, 1711 MS (m/z) 524 (M ).
1
and 1651. H-NMR (CDCl ) d: 0.68 (3H, s), 1.2 (3H, s), 2.0 (3H, s), 3.5
Biological Activity of the Synthesized Compounds The biological ac-
3
1
3
(
1
2
3H, s), 5.8 (1H, s), 6.0 (1H, d, Jϭ2 Hz). C-NMR (CDCl ) d: 14.3 (C-18), tivity of steroids 1, 2, 3, 4 and 5 was determined in in vitro experiments
3
7.4 (C19), 26.3 (C-21), 50.8 (methyl ether), 170.7 (ester carbonyl), 204 (C- using prostate glands from gonadectomized adult male golden hamsters. The
ϩ
0). MS (m/z) 386 (M ).
7a-Acetoxy-6-formyl-3-methoxypregna-3,5-dien-20-one 20: A solution ilco of Mexico. Gonadectomies were performed under light ether anesthesia
containing N,N-dimethylformamide (0.6 ml, 7.7 mmol), phosphorous oxy- 30 d before the experiments.
chloride (0.5 ml, 5.4 mmol) and methylene chloride (5 ml) was cooled to The prostate glands were immediately removed, blotted and weighed prior
°C. Steroid 19 (200 mg, 0.52 mmol) dissolved in methylene chloride (2 ml) to their use. Tissues used in the metabolic experiment were homogenized
animals (150—200 g) were obtained from Metropolitan University-Xochim-
1
5
and pyridine (1.5 ml) was added, keeping the temperature at 5 °C. The reac- with a tissue homogenizer (model 985—370; variable speed 5000—
tion mixture was stirred for 1 h at 0 °C and for 2 additional hours at room 30000 rpm, Biospec Products, Inc.)
temperature. Sodium acetate (1 g) dissolved in methanol (70 ml) was added
and the reaction mixture was stirred for 15 min. It was extracted 3 times with Tris–HCl, 1.5 mM EDTA and 10 mM sodium Molybdate) at pH 7.4 and at
chloroform, the organic phase was separated and washed with an aqueous 4 °C with tissue homogenizer. Homogenates were centrifuged at
Tissues were homogenized in 3 volumes of buffer TEMD (20 mM
a
1
3)
sodium bicarbonate solution and then with water. It was dried over anhy-
drous sodium sulfate and the solvent was removed in vacuum. The crude
product was recrystallized from ethyl acetate–hexane. Yield 120 mg,
140000ϫg for 60 min in a SW 60 Ti rotor (Beckman Instruments, Palo
Alto, CA, U.S.A.). The pellets were separated, washed with 3 tissue volumes
of medium A (20 mM sodium phosphate, pH 6.5 containing 0.32 M sucrose,
2
1
.9 mmol (60%), mp 205—208 °C. UV (nm): 238 and 315 (eϭ10800,
0.1 mM dithiothreitol Sigma-Aldrich, Inc.) and centrifuged two additional
5600 respectively). IR (KBr) cm : 2946, 1731 and 1652. H-NMR times at 440ϫg at 0 °C for 10 min. The washed pellets were suspended in
Ϫ1
1
14)
(
(
2
CDCl ) d: 0.68 (3H, s), 1.2 (3H, s), 2.0 (3H, s), 2.2 (3H, s), 3.5 (3H, s), 6.2 medium A and kept at Ϫ70 °C. The suspension (6.8 mg protein/ml deter-
3
13 15)
1H, s) and 10.2 (1H, s). C-NMR (CDCl ) d: 14.3 (C-18), 18.9 (C-19), mined by the Bradford method) was used as a source of 5a-reductase.
3
ϩ
6.5 (C-21), 54.8 (CH enol ether), 177 (ester carbonyl). MS m/z 414 (M ).
1
Determination of 5a -Reductase Activity 5a-Reductase was assayed
3
16)
7a-Acetoxy-6-methylenepregn-4-ene-3,20-dione 21: A suspension con- as previously described. The reaction mixture contained a final volume of
taining steroid 20 (100 mg, 0.24 mmol), methanol (2 ml) and sodium borohy- 1 ml: 1 mM dithiothreitol, sodium phosphate buffer (40 mM), 2 mM, NADPH,
3
dride (20 mg, 0.53 mmol) was stirred at 5 °C for 30 min. Water (20 ml) was 2 nM [1, 2, 6, 7- H]T (specific activity 95 Ci/mmol). The reaction mixture in
added and the mixture was transferred to a separatory funnel. It was ex- duplicate was started when it was added to the enzymatic fraction (250 mg
tracted three times with ethyl acetate, the organic phase was separated, protein), incubated at 37 °C for 60 min, and stopped by mixing with 1 ml of
washed with water and dried over anhydrous sodium sulfate. The solvent dichloromethane; this was considered as the 100% point. The fraction of
was removed in vacuum and the resulting crude product was recrystallized dichlorometane was separated and the extraction was repeated 4 more times.
from methanol. Yield 64 mg, 0.15 mmol (70%), mp 244—246 °C. UV (nm): The extract was evaporated under a nitrogen stream until dryness and sus-
Ϫ1
2
60 (eϭ11000). IR (KBr) cm : 2955, 1730, 1708, 1673, 1370 and 1262. pended on 50 ml of methanol that was spotted on HPTLC Kieselgel 60 F254
1
H-NMR (CDCl ) d: 0.68 (3H, s), 1.20 (3H, s), 2.00 (3H, s), 2.20 (3H, s),
.95 (1H, d, Jϭ10 Hz), 5.1 (1H, d, Jϭ10 Hz), 6.1 (1H, s). C-NMR (CDCl ) veloped in chloroform–acetone (9 : 1). The plates were air-dried and the
plates. T and dihydrotestosterone were used as carriers and the plate was de-
3
1
3
4
3
d: 14.2 (C-18), 17.1 (C-19), 26.3 (C-21), 170.6 (ester carbonyl), 203.9 (C- chromatography was repeated 2 more times. The T standard was visualized
ϩ
2
0). MS (m/z) 384 (M ).
7a-Hydroxy-6-methylenepregn-4-ene-3,20-dione 3: A solution contain- molybdatophosphoric acid hydrate reagent with a posterior heating of the
ing steroid 21 (1 mg, 2.6 mmol), methanol (100 ml) and 2% aqueous sodium plate. Dihydrotestosterone develops a classic dark blue color. The dihy-
under UV light (254 nm) and dihydrotestosterone was detected using
1