Novel 17 substituted pregnadiene derivatives as 5α-reductase inhibitors and their binding affinity for the androgen receptor

Article abstract of DOI:10.1248/cpb.52.535

The in vitro antiandrogenic activity of four new progesterone derivatives: 4, 5, 6 and 7 (8 is a known compound) was determined. These compounds were evaluated as 5α-reductase inhibitors as well as by their capacity to bind to the androgen receptor in gonadectomized hamster prostate. The IC₅₀ value was determined using increasing concentrations of 4, 5, 6, 7 and 8 in the presence of [³H]T and the microsomal fraction of the hamster prostate containing the 5α-reductase enzyme. In this paper we also demonstrated the effect of increasing concentrations of the novel steroids upon [³H]DHT binding to the androgen receptors from hamster prostate which produces competition for the androgen receptor sites. The in vitro studies showed that steroids 4, 5, 6, 7 and 8 had an inhibitory activity for the 5α-reductase with IC₅₀ of: 4 (0.17 μM), 5 (0.19 μM), 6 (1 μM), 7 (4.2 μM), and 8 (2.7 μM). On the other hand, the IC ₅₀ value for compounds 4, 5, 6, 7, 8 and DHT showed the following order of affinity for the androgen receptor: 6>7>5>DHT. Surprisingly compounds 4 and 8 did not bind to the androgen receptor. The overall data indicate that all synthesized compounds are inhibitors for the enzyme 5α-reductase present in the hamster prostate. In contrast, compounds 5, 6 and 7, which have a cyclohexyl group in the side chain showed a high affinity for the androgen receptor.

Full text of DOI:10.1248/cpb.52.535

May 2004
Chem. Pharm. Bull. 52(5) 535—539 (2004)
535
a
Novel 17 Substituted Pregnadiene Derivatives as 5 -Reductase Inhibitors
and Their Binding Affinity for the Androgen Receptor
Marisa CABEZA, ,a Eugenio FLORES,b Ivonne HEUZE,a Mauricio SÁNCHEZ,a Eugene BRATOEFF,b
*
c
Elena RAMÍREZ,b and Victor Alfonso FRANCOLUGO
a Department of Biological Systems and Animal Production, Metropolitan University-Xochimilco; Mexico D. F., 04960
Mexico: b Department of Pharmacy, Faculty of Chemistry, National University of Mexico City; Mexico D. F., 04510
Mexico: and c Department of Urology, Henri Dunant Hospital; Cuernavaca, 62270 Morelos, Mexico.
Received November 17, 2003; accepted January 13, 2004
The in vitro antiandrogenic activity of four new progesterone derivatives: 4, 5, 6 and 7 (8 is a known com-
pound) was determined. These compounds were evaluated as 5a-reductase inhibitors as well as by their capacity
to bind to the androgen receptor in gonadectomized hamster prostate. The IC50 value was determined using in-
creasing concentrations of 4, 5, 6, 7 and 8 in the presence of [3H]T and the microsomal fraction of the hamster
prostate containing the 5a-reductase enzyme. In this paper we also demonstrated the effect of increasing concen-
trations of the novel steroids upon [3H]DHT binding to the androgen receptors from hamster prostate which pro-
duces competition for the androgen receptor sites. The in vitro studies showed that steroids 4, 5, 6, 7 and 8 had an
inhibitory activity for the 5a-reductase with IC50 of: 4 (0.17 mM), 5 (0.19 mM), 6 (1 mM), 7 (4.2 mM), and 8 (2.7 mM).
On the other hand, the IC50 value for compounds 4, 5, 6, 7, 8 and DHT showed the following order of affinity for
the androgen receptor: 6ꢀ7ꢀ5ꢀDHT. Surprisingly compounds 4 and 8 did not bind to the androgen receptor.
The overall data indicate that all synthesized compounds are inhibitors for the enzyme 5a-reductase present in
the hamster prostate. In contrast, compounds 5, 6 and 7, which have a cyclohexyl group in the side chain showed
a high affinity for the androgen receptor.
Key words 5a-reductase; prostate; pregnadiene derivative; androgen receptor
Virilization in mammals is mediated by two steroidal hor-
The most extensively studied class of 5a-reductase in-
mones, testosterone 1 (T) and dihydrotestosterone 2 (DHT). hibitors is the 4-azasteroids,4,5) which includes the drug finas-
Both hormones bind to the typical steroidal hormone recep- teride 3. This compound is the first 5a-reductase inhibitor
tor, the androgen receptor, and this activates the genes con- approved in the USA for the treatment of BPH. This drug has
taining the androgen-responsive DNA sequence.
approximately a 100-fold greater affinity for type II-5a-re-
The notion that 2 (DHT) is a potent androgen with physio- ductase than for the type I enzyme. In humans, finasteride
logical roles distinct from those of 1 (T) came from two decreases prostatic DHT levels by 70—90% and reduces
facts. First, androgen target tissues contain an enzyme prostate size, while T tissue levels remain constant.4) The use
(steroid 5a-reductase) capable of reducing T to DHT,1) and of finasteride demonstrated a sustained improvement in the
second, the product of this enzyme is accumulated in the nu- treatment of androgen dependent diseases and a reduction in
clei of responsive cells, such as those of rat ventral prostate specific antigen (PSA) levels.5)
prostate.1,2) The subsequent study of an inborn error of male
Previously it has been stated that b-sitosterol significantly
phenotypical sexual differentiation, now termed steroid 5a- improves the symptoms and urinary flow parameters present
reductase type 2 deficiency, provides genetic proof for the in BPH,6) and it appears that it can inhibit the growth and mi-
role of DHT in androgen action.3)
gration of one type of prostate cancer cell and to slow the
The deficiency of 5a-reductase in males results in an in- growth of prostate tumors in laboratory mice.7) On the other
complete differentiation of the external genitalia at birth.1) At hand, it was reported that b-sitosterol inhibits 5a-reductases
puberty, these patients have normal to elevated levels of T in type I and II with IC50 ꢀ100.8)
plasma and virilization occurs, but the prostate remains
Recently our group synthesized several new steroidal de-
small. These individuals never have acne or temporal reces- rivatives (17-alfa-acyloyloxypregnadiene and pregnatriene)
sion of the hairline. This low level of DHT is typical of male that decrease the prostate growth produced by T.9—12)
pseudohermaphroditism.4) On the other hand, abnormally
In view of the fact that the endocyclic dienes and trienes
high 5a-reductase activity in humans results in excessively described above9—12) showed a high biological activity, we
high DHT levels in peripheral tissues, which is implicated in describe here the synthesis of four new progesterone deriva-
the pathogenesis of prostate cancer, benign prostatic hyper- tives having a conjugated exocyclic double bond: 6-methyl-
plasia (BPH), acne, and male patte baldness,5) suggesting ene-17a-(3-cyclopentylpropionyloxy)-pregn-4-ene-3,20-
that both the enzyme and DHT play important physiological dione 4; 6-methylene-17a-(4-cyclohexylbutyryloxy)-pregn-
and pathological roles in humans. Therefore, the inhibition of 4-ene-3,20-dione 5; 6-methylene-17a-(3-cyclohexylpropi-
androgen action by 5a-reductase inhibitors is a logical treat- onyloxy)-pregn-4-ene-3,20-dione 6 and 6-methylene-17a-(2-
ment for 5a-reductase activity disorders. Furthermore, by the cyclohexylacetoxy)-pregn-4-ene-3,20-dione 7. These com-
beginning of the past decade two types of 5a-reductase en- pounds were evaluated as 5a-reductase inhibitors as well as
zyme had been identified as types I and II.1,5) The identifica- antagonists for the androgen receptor.
tion of these two isozymes opened the door for specific and
selective inhibition of this enzyme.
∗ To whom correspondence should be addressed. e-mail: marisa@cueyatl.uam.mx
© 2004 Pharmaceutical Society of Japan

536
Vol. 52, No. 5
Fig. 1. Steroidal Structures
Fig. 2. Synthesis of Compounds 4, 5, 6 and 7
Results
pounds 4, 5, 6, 7, 8 was determined in gonadectomized ham-
Synthesis of the Steroidal Derivatives 4, 5, 6 and 7 ster prostate glands homogenized and centrifuged to obtain
These compounds were prepared from the commercially the prostatic enzyme and cytosolic fractions.
available 17a-acetoxyprogesterone 9 (Fig. 2). The reaction
The activity of 5a-reductase was assessed incubating the
of steroid 9 in dioxane with trimethyl orthoformate for 1 h at enzymatic fraction with 2 nM [3H]T. The dichloromethane ex-
room temperature afforded the enol ether 10. Treatment of 10 tract from castrated male hamster prostates was subjected to
with phosphorous oxychloride in N,N-dimethylformamide TLC analysis. The zone corresponding to DHT standard (Rf
and methylene chloride yielded the 6-formyl enol ether 11 value of 0.67) of the experimental chromatogram was eluted
(the Vilsmeyer reaction). Reduction of the formyl group in and the radioactivity determined. This result was considered
11 with sodium borohydride in methanol afforded the 6-hy- to be 100% of the activity of 5a-reductase for development
droxymethylene derivative (not isolated) which dehydrated to of inhibition plots.
the desired exocyclic methylene derivative with a simultane-
Determination of 50% Inhibitory Concentration of the
ous hydrolysis of the enol ether to form the desired 17a-ace- Novel Compounds in Hamster Prostate The concentra-
toxy-6-methylenepregn-4-ene-3,20-dione 12. Upon treatment tion of compounds 4, 5, 6, 7, 8 required for inhibiting 5a-re-
with methanol and sodium hydroxide, 12 afforded the free al- ductase activity by 50% (IC50) was determined from the inhi-
cohol 13. This compound was esterified with the correspond- bition curves using different concentrations of the steroids
ing acid in the presence of trifluoroacetic anhydride and p- and are shown in Table 1.
toluenesulfonic acid to form the desired esters 4, 5, 6 and 7.
The IC50 value obtained for compounds 4 and 5 showed a
Biological Activity The biological activity of com- higher 5a-reductase inhibitory activity than the derivatives 6,

May 2004
537
7 and 8 (Table 1).
tase proceeds via a Michael type addition reaction of the nu-
Competition Analysis of Compounds 4, 5, 6, 7 and 8 for cleophilic portion of the enzyme (amino group) to the conju-
the Androgen Receptors The effect of increasing concen- gated double bond of the steroid. In this study, we also deter-
trations of non-radioactive steroids upon [3H]DHT binding to mined that bulky groups at C-17 inhibit this addition and as a
androgen receptors from male hamster prostate in two differ- consequence of this steric inhibition, compounds 5, 6 and 7
ent experiments is shown in Fig. 3. The IC50 value for com- have a lower activity.
pounds 4, 5, 6, and 7 and DHT shows the following order of
Compounds 5, 6 and 7 exhibited a higher affinity for the
affinity for the androgen receptors: 6ꢀ7ꢀ5ꢀDHT. Com- androgen receptor than steroid 4 (Fig. 3). Apparently, the
pounds 5, 6 and 7 showed high affinity for the androgen re- higher symmetry of the cyclohexyl substituent in 5, 6 and 7
ceptor, whereas steroids 4 and 8 did not form a complex with enhanced the binding affinity as compared to compound 4
the androgen receptor.
which has a cyclopentyl group in the side chain.
In this study we also reported the 5a-reductase inhibitory
activity of b-sitosterol 8 with an IC50 value of 2.7 mM. This
Discussion
In this study, we assessed the inhibitory potency of four compound failed to show a binding tendency for the andro-
novel steroids 4, 5, 6 and 7. The 5a-reductase inhibitory ac- gen receptor from hamster prostate cytosol. Although Berges
tivity of these compounds was determined by comparing the et al.6) indicated that compound 8 improved the symptoms of
individual IC50 values. Compound 4 with a cyclopentyl side benign prostatic hyperplasia, the low 5a-reductase inhibitory
chain is slightly more active (IC50 of 0.17 mM) than steroid 5 activity as shown by its IC50 value makes this steroid a poor
(IC50 of 0.19 mM) which has a longer side chain and a bulkier candidate for the treatment of androgen dependent diseases.
cyclohexyl group. Several years ago we determined the 5a-
Experimental
reductase inhibitory activity of similar compounds and the
Chemical and Radioactive Material Solvents were laboratory grade or
results from this study showed very clearly that when the
length and the size of the side chain at C-17 increase, the 5a-
reductase inhibitory activity decreases.13) In view of the fact
that steroids 5, 6 and 7 have a bulkier side chain (cyclohexyl
group) than 4 (with a cyclopentyl group), this fact explains
very well the lower 5a-reductase inhibitory activity of com-
pounds 5, 6 and 7. Previous studies9) carried out in our labo-
ratory indicated that the inhibition of the enzyme 5a-reduc-
better. (1, 2, 6, 7-3H) Testosterone [3H]T specific activity: 95 Ci/mmol and
(1, 2, 4, 5, 6, 7-3H) dihydrotestosterone [3H]DHT specific activity
110 Ci/mmol, were provided by New England Nuclear Corp. (Boston, MA,
U.S.A.). Radioinert T and 5a-DHT were supplied by Steraloids (Wilton,
NH, U.S.A.). Sigma-Aldrich (ST. Louis, MO, U.S.A.) supplied NADPH and
pure b-sitosterol (8).
Synthesis of the Steroidal Compounds 17a-Acetoxy-3-methoxy-
pregna-3,5-diene-20-one 10: A solution containing steroid 9 (1 g, 2.68
mmol), PTSA (200 mg), dioxane (10 ml) and trimethyl orthoformate (5 ml,
4.57 mmol) was stirred for 1 h at room temperature; at this time, pyridine
(5 ml) was added and the reaction mixture was stirred for an additional
10 min. It was poured into ice-water (200 ml) the crude product 10 precipi-
tated and it was filtered and dried. Yield 620 mg, 1.6 mmol (60%), mp 154—
156 °C. UV (nm): 238 (eꢁ12000). IR (KBr) cmꢂ1: 2975, 1736, 1711 and
Table 1. IC50 Value Determined for Synthesized Steroids
Steroid
IC50 (mM)
1
1651. H-NMR (CDCl3) d: 0.68 (3H, s, CH3-18), 1.2 (3H, s, CH3-19), 2.0
4
5
6
7
8
0.17
0.19
1.00
4.2
(3H, s, CH3-21), 2.2 (3H, s, CH3-acetoxy), 3.5 (3H, s, CH3-enol ether), 5.8
(1H, s, H-4) and 6.0 (1H, d, Jꢁ2 Hz, H6). 13C-NMR (CDCl3) d: 14.3 (C-18),
17.4 (C-19), 26.3 (C-21), 50.8 (enol ether), 170.7 (ester carbonyl), 204 (C-
20). MS (m/z) 386 (Mꢃ).
2.7
17a-Acetoxy-6-formyl-3-methoxypregna-3,5-diene-20-one 11: A solu-
tion containing N,N-dimethylformamide (0.6 ml, 7.7 mmol), phosphorous
oxychloride (0.5 ml, 5.4 mmol) and methylene chloride (5 ml) was cooled to
IC50 value represents the concentration of the steroid that inhibits 50% of 5a-reduc-
tase activity and was determined as described in Experimental.
Fig. 3. Binding Specificity
Gonadectomized hamster prostate cytosol incubated for 18—20 h at 4 °C in the presence of 3.15 nM of [3H]DHT and increasing concentrations of radio-inert steroids.

538
Vol. 52, No. 5
5.07 (2H, d, Jꢁ1.8 Hz, CH2 at C-6) and 5.93 (1H, s, H-4). 13C-NMR
(CDCl3) d: 14.4 (C-18), 17.1 (C-19), 26.4 (C-21), 118 (exocyclic methylene
at C-6), 155 (C-6), 173.8 (ester carbonyl) and 199.8 (C-20). MS (m/z) 480
(Mꢃ).
5 °C. Steroid 10 (200 mg, 0.52 mmol) dissolved in methylene chloride (2 ml)
and pyridine (1.5 ml) was added, keeping the temperature at 0 °C. The reac-
tion mixture was stirred for 1 h at 0 °C and for 2 additional hours at room
temperature. Sodium acetate (1 g) dissolved in methanol (70 ml) was added
and the reaction mixture was stirred for 15 min. It was extracted three times
with chloroform, the organic phase was separated and washed with an aque-
ous 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,
2.9 mmol (60%), mp 205—208 °C. UV (nm): 238 (eꢁ10800) and 315
(eꢁ15600). IR (KBr) cmꢂ1: 2946, 1731 and 1652. 1H-NMR (CDCl3) d:
0.68 (3H, s, CH3-18), 1.2 (3H, s, CH3-19). 2.0 (3H, s, CH3-21), 2.2 (3H, s,
CH3-acetoxy), 3.5 (3H, s, CH3-enol ether), 6.2 (1H, s, H-4) and 10.2 (1H, s,
H-formyl). 13C-NMR (CDCl3) d: 14.3 (C-18), 18.9 (C-19), 26.5 (C-21), 54.8
(CH3-enol ether), 177 (ester carbonyl), 190 (formyl) and 204 (C-20). MS
(m/z) 414 (Mꢃ).
17a-Acetoxy-6-methylenepregn-4-ene-3,20-dione 12: A suspension con-
taining steroid 11 (100 mg, 0.24 mmol), methanol (2 ml) and sodium borohy-
dride (20 mg, 0.53 mmol) was stirred for 30 min at 5 °C. Water (20 ml) was
added and the mixture was transferred to a separatory funnel. It was ex-
tracted three times with ethyl acetate, the organic phase was separated,
washed with water and dried over anhydrous sodium sulfate. The solvent
was removed in vacuum and the resulting crude product was recrystallized
from methanol. Yield 64 mg, 0.15 mmol (70%), mp 244—246 °C. UV (nm):
260 (eꢁ11000). IR (KBr) cmꢂ1: 2955, 1730, 1708, 1370 and 1262. 1H-
NMR (CDCl3) d: 0.68 (3H, s, CH3-18), 1.2 (3H, s, CH3-19), 2.0 (3H, s,
CH3-21), 2.2 (3H, s, CH3-acetoxy), 5.2 (2H, d, Jꢁ2.1 Hz, CH2 at C-6) and
5.8 (1H, s, H-4). 13C-NMR (CDCl3) d: 14.2 (C-18), 17.1 (C-19), 26.3 (C-
21), 120 (exocyclic methylene at C-6), 150 (C-6), 170.6 (ester carbonyl) and
203.9 (C-20). MS (m/z) 384 (Mꢃ).
17a-(Cyclohexylacetoxy)-6-methylenepregn-4-ene-3,20-dione 7: Yield
48%, mp 134—136 °C. UV (nm): 254 (eꢁ10700). IR (KBr) cmꢂ1: 2924,
1729, 1710, 1673 and 1226. 1H-NMR (CDCl3) d: 0.72 (3H, s, CH3-18), 1.09
(3H, s, CH3-19), 2.0 (3H, s, CH3-21), 2.3 (2H, m, CH2 a to ester carbonyl),
5.07 (2H, d, Jꢁ2 Hz, CH2 at C-6), 5.93 (1H, s, H-4). 13C-NMR (CDCl3) d:
14.3 (C-18), 17.1 (C-19), 26.0 (C-21), 114 (exocyclic methylene at C-6),
145 (C-6), 172.7 (ester carbonyl) and 199.8 (C-20). MS (m/z) 466 (Mꢃ).
Biological Activity of the Synthesized Compounds The biological ac-
tivity of steroids 4, 5, 6, 7 and 8, was determined in in vitro experiments
using the prostate glands from gonadectomized adult male golden hamsters.
The animals (150—200 g) were obtained from the Metropolitan University-
Xochimilco of Mexico. Gonadectomies were performed under pentobarbital
anesthesia 3 d before the experiments and the animals were sacrificed with
CO2.
The prostate glands were immediately removed, blotted and weighed prior
to their use. Unless specified, all procedures were carried out a 4 °C.Tissues
used in the metabolic experiment were homogenized with a tissue homoge-
nizer (model 985-370; variable speed 5000—30000 rpm, Biospec Products,
Inc.).
Tissues were homogenized in 3 volumes of buffer TEMD (40 mM
Tris–HCl, 3 mM EDTA and 20 mM sodium molybdate, dithiotreitol 0.5 mM,
20% glicerol) at pH 7.5 and at 4 °C with a tissue homogenizer. Homogenates
were centrifuged at 140000ꢄg for 60 min11) 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 con-
taining 0.32 M sucrose, 0.1 mM dithiothreitol; Sigma-Aldrich, Inc.) and cen-
trifuged two additional times at 440ꢄg at 0 °C for 10 min.14,15) The washed
pellets were suspended in medium A and kept at ꢂ70 °C. The suspension
(6.8 mg protein/ml determined by the Bradford method16)) was used as a
source of 5a-reductase.
Determination of 5a-Reductase Activity The 5a-reductase activity
was assayed as previously described.17) The reaction mixture contained a
final volume of 1 ml: 1 mM dithiothreitol, sodium phosphate buffer (40 mM),
2 mM, NADPH, 2 nM [1,2,6,7-3H]T (specific activity 95 Ci/mmol). This mix-
ture in duplicate was started when it was added to the enzymatic fraction
(250 mg protein), incubated at 37 °C for 60 min, and stopped by mixing with
1 ml of dichloromethane; this was considered as the 100% point. The frac-
tion of dichloromethane was separated and the extraction was repeated 4
more times. The extract was evaporated under a nitrogen stream until dry-
ness and suspended on 50 ml of methanol that was spotted on HPTLC
Keiselgel 60 F254 plates. T and DHT were used as carriers and the plate was
developed in chloroform : acetoneꢁ9 : 1. It was air-dried and the chromatog-
raphy was repeated 2 more times. The T standard was visualized under UV
lights (254 nm) and DHT was detected using phosphomolibdic acid reagent
with a posterior heating of the plate. DHT developed a classical dark blue
color. The DHT containing areas were cut, the strips were soaked in 5 ml of
Ultima Gold (Packard) and the radioactivity was determined in a Tri Carb
2100TR liquid scintillation analyzer.
Determination of 50% Inhibitory Concentration Steroids 4, 5, 6, 7
and 8 in Gonadectomized Hamster Prostate In order to calculate the
IC50 values (the concentration of steroids 4, 5, 6, 7 and 8 required to inhibit
5a-reductase activity by 50%), three series of tubes containing increasing
concentrations of 4, 5, 6, 7 and 8 (10ꢂ11, 10ꢂ4 M) were incubated in duplicate
as detailed above.
Competitive Studies For competitive studies, tubes containing 3.15 nM
of [3H]DHT (specific activity 110 Ci/mmol) plus a range of increasing con-
centrations (10ꢂ6—10ꢂ3 M) of cold DHT and compounds 4, 5, 6, 7 and 8
were prepared.14,16)
17a-Hydroxy-6-methylene-4-ene-3,20-dione 13: A solution containing
steroid 12 (1 g, 2.6 mmol), methanol (100 ml) and 2% aqueous sodium hy-
droxide solution (25 ml) was allowed to reflux for 30 min. Part of the
methanol was removed in vacuum. The reaction mixture was extracted with
chloroform, the organic phase was separated, washed with water and dried
over anhydrous sodium sulfate. The solvent was eliminated in vacuum; the
crude product was recrystallized from ethyl acetate–hexane. Yield 660 mg,
1.92 mmol (75%) mp 204—206 °C. UV (nm): 254 (eꢁ10800). IR (KBr)
1
cmꢂ1: 3436, 2947, 1702 and 1658. H-NMR (CDCl3) d: 0.69 (3H, s, CH3-
18), 1.22 (3H, s, CH3-19), 2.0 (3H, s, CH3-21), 5.2 (2H, d, Jꢁ2.1 Hz, CH2 at
C-6) and 6.0 (1H, s, H-4). 13C-NMR (CDCl3) d: 15.4 (C-18), 17.1 (C-19),
23.8 (C-21), 122 (exocyclic methylene at C-6), 155 (C-6), 175.2 (C-3) and
211.5 (C-20). MS (m/z) 342 (Mꢃ).
Preparation of Compounds 4, 5, 6 and 7 These esters were prepared
according to the following general procedure.
A solution containing steroid 13 (1 g, 2.92 mmol), PTSA (10 mg), trifluo-
roacetic anhydride (8.29 g, 42.48 mmol) and the corresponding acid
(10 mmol) was stirred for 2 h at room temperature (nitrogen atmosphere).
The reaction mixture was neutralized with an aqueous solution of sodium
hydroxide to a pH of 7 and diluted with chloroform (10 ml). The organic
phase was separated, washed with water, dried over anhydrous sodium sul-
fate, filtered and the solvent was eliminated in vacuum. The crude product
was purified by silica gel column chromatography. Hexane–ethyl acetate
(8 : 2) eluted the pure ester.
17a-(3-Cyclopentylpropionyloxy)-6-methylenepregn-4-ene-3,20-dione 4:
Yield 40%, mp 140—143 °C. UV (nm): 264 (eꢁ10800). IR (KBr) cmꢂ1
:
2947, 1731, 1715, 1674 and 1264. 1H-NMR (CDCl3) d: 0.7 (3H, s, CH3-18),
1.1 (3H, s, CH3-19), 2.0 (3H, s, CH3-21), 2.2 (2H, m, CH2 a to carbonyl of
ester), 5.0 (2H, d, Jꢁ1.8 Hz, CH2 at C-6) and 5.9 (1H, s, H-4). 13C-NMR
(CDCl3) d: 14.4 (C-18), 17.1 (C-19), 26.4 (C-21), 118 (exocyclic methylene
at C-6), 150 (C-6), 173.4 (ester carbonyl) and 199.8 (C-20). MS (m/z) 466
(Mꢃ).
6-Methylene-17a-(4-cyclohexylbutyryloxy)-pregn-4-ene-3,20-dione 5:
The cytosolic fraction obtained from the supernatant liquid of the prostate
homogenate centrifuged at 140000ꢄg as described above was stored at
ꢂ70 °C. Aliquots of 200 ml of prostatic cytosol (2.4 mg protein, determined
by the Bradford method16)) were added and incubated (duplicate) for 18 h at
4 °C in the tubes as previously described. Eight hundred microliters of dex-
tran-coated charcoal in TEDAM buffer (containing dithiotreitol) was then
added and the mixture was incubated for 40 min at 4 °C. To prepare the dex-
tran-coated charcoal mixture, the dextran was agitated for 30 min before
adding the charcoal to the mixture. The tubes were vortexed and immedi-
ately centrifuged at 800ꢄg for 10 min; aliquots (200 ml) were taken and sub-
mitted for radioactive counting. The IC50 of each compound was calculated
according to the plots.
Yield 47%, mp 114—117 °C. UV (nm): 255 (eꢁ10600). IR (KBr) cmꢂ1
:
1
2922, 1730, 1710, 1674 and 1226. H-NMR (CDCl3) d: 0.71 (3H, s, CH3-
18), 1.1 (3H, s, CH3-19), 2.0 (3H, s, CH3-21), 2.3 (2H, m, CH2 a to carbonyl
of ester), 4.96 (2H, d, Jꢁ2 Hz, CH2 at C-6) and 5.9 (1H, s, H-4). 13C-NMR
(CDCl3) d: 14.4 (C-18), 17.1 (C-19), 26.3 (C-21), 120 (exocyclic methylene
at C-6), 152 (C-6), 173.6 (ester carbonyl) and 199.8 (C-20). MS (m/z) 494
(Mꢃ).
17a-(3-Cyclohexylpropionyloxy)-6-methylenepregn-4-ene-3,20-dione 6:
Yield 46%, mp 146—148 °C. UV (nm): 260 (eꢁ11000). IR (KBr) cmꢂ1
:
1
2923, 1705, 1673 and 1227. H-NMR (CDCl3) d: 0.7 (3H, s, CH3-18), 1.1
(3H, s, CH3-19), 2.0 (3H, s, CH3-21), 2.2 (2H, m, CH2 a to ester carbonyl),

May 2004
539
Acknowledgements We gratefully acknowledge the financial support of
82, 233—239 (2002).
CONACYT and DGAP for projects 33450-M and 200301.
9) Ramírez E., Cabeza M., Heuze I., Gutiérrez E., Bratoeff E., Membrillo
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