Synthesis and Identification of Pregnenolone Derivatives as Inhibitors of Isozymes of 5α-Reductase

Article abstract of DOI:10.1002/ardp.201500220

Hyperplasia of the prostate gland and prostate cancer have been associated with high levels of serum 5α-dihydrotestosterone. This steroid is formed from testosterone by the activity of the enzyme 5α-reductase (5α-R) present in the prostate. Thus, inhibition of this enzyme could be a goal for therapies to treat these diseases. This study reports the synthesis and effects of five different 21-esters of pregnenolone derivatives as inhibitors of 5α-R types 1 and 2. The activity of these steroidal compounds was determined using in vivo and in vitro experiments. The results indicate that of the five steroids studied, the 21(p-fluoro)benzoyloxypregna-4,16-diene-3,6,20-trione derivative, whose structure has not yet been reported, has the best molecular conformation to inhibit the in vitro activity of both types of 5α-R. In addition, this steroid also displayed activity in vivo. Apparently, its pharmacological effect was increased by the presence of a keto group at C-6, because this group decreased the possibility that the steroid would be metabolized by hepatic enzymes. In addition, the double bond present at C-4 of this compound also enhanced its inhibitory activity on 5α-R, and the C-21 ester moiety increased its liphophilicity. Therefore, its solubility in the cell membrane and its pharmacological activity were both increased.

Full text of DOI:10.1002/ardp.201500220

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Arch. Pharm. Chem. Life Sci. 2015, 348, 808–816
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Full Paper
Synthesis and Identification of Pregnenolone Derivatives as
Inhibitors of Isozymes of 5a-Reductase
1
1
2
3
2
Alejandra Ch aꢀ vez-Riveros , Eugene Bratoeff , Yvonne Heuze , Juan Soriano , Isabel Moreno ,
2
2
Araceli S aꢀ nchez-M aꢀ rquez , and Marisa Cabeza
1
Departamento de Farmacia, Facultad de Qu ꢀı mica, Universidad Nacional Aut oꢀ noma de M eꢀ xico, Ciudad
de M eꢀ xico, Distrito Federal, M eꢀ xico, D. F., Mexico
Departamento de Sistemas Biol oꢀ gicos y de Producci oꢀ n Agr ꢀı cola y Animal, Universidad Aut oꢀ noma
Metropolitana-Xochimilco, Ciudad de M eꢀ xico, M eꢀ xico, D. F., Mexico
2
3
Departamento de Patolog ꢀı a, Hospital General de M eꢀ xico, M eꢀ xico, D. F., Mexico
Hyperplasia of the prostate gland and prostate cancer have been associated with high levels of serum
a-dihydrotestosterone. This steroid is formed from testosterone by the activity of the enzyme 5a-
5
reductase (5a-R) present in the prostate. Thus, inhibition of this enzyme could be a goal for therapies
to treat these diseases. This study reports the synthesis and effects of five different 21-esters of
pregnenolone derivatives as inhibitors of 5a-R types 1 and 2. The activity of these steroidal
compounds was determined using in vivo and in vitro experiments. The results indicate that of the
five steroids studied, the 21(p-fluoro)benzoyloxypregna-4,16-diene-3,6,20-trione derivative, whose
structure has not yet been reported, has the best molecular conformation to inhibit the in vitro
activity of both types of 5a-R. In addition, this steroid also displayed activity in vivo. Apparently, its
pharmacological effect was increased by the presence of a keto group at C-6, because this group
decreased the possibility that the steroid would be metabolized by hepatic enzymes. In addition, the
double bond present at C-4 of this compound also enhanced its inhibitory activity on 5a-R, and the
C-21 ester moiety increased its liphophilicity. Therefore, its solubility in the cell membrane and its
pharmacological activity were both increased.
Keywords: 5a-Reductase / Hamster prostate gland / Hamster seminal vesicles / (p-Fluoro)benzoyloxy
21-esters of pregnenolone derivatives / Steroidal esters
Received: July 1, 2015; Revised: August 19, 2015; Accepted: August 21, 2015
DOI 10.1002/ardp.201500220
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
long been known; the enzyme converts testosterone (T) to
5a-dihydrotestosterone (DHT) in the prostate gland.
4
The 5a-reductase enzyme (EC 1.3.99.5) converts D -3-ketoste-
roids to 5a-3-ketosteroids in androgen-dependent tissues.
The activity of this enzyme in androgen-dependent tissues has
Hyperplasia of the prostate gland and prostate cancer have
been associated with high levels of serum 5a-DHT [1–3].
Three types of 5a-reductase (5a-R) isozymes have been
described; type 2 5a-R plays a major role in prostate cancer
and benign prostatic hyperplasia as it is predominantly
expressed in this tissue. However, some evidence indicates
that type 1 is expressed in the prostate epithelial cells whereas
type 2 is mainly located in the stromal compartment [4, 5].
Type 1 5a-R is also located in the liver and skin and acts in a
neutral or basic medium, whereas type 2 is active in acidic
pH [4]. Recently 5a-R3 has been investigated; this isozyme can
Correspondence: Dr. Marisa Cabeza, Departamento de Sistemas
Biol oꢀ gicos, Universidad Aut oꢀ noma Metropolitana-Xochimilco,
Calzada Del Hueso No. 1100, M eꢀ xico, D. F., C.P. 04960, M eꢀ xico.
E-mail: marisa@correo.xoc.uam.mx
Fax: þ52-55-54837260
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1-Esters of Pregnenolone Derivatives as 5a-R Inhibitors
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be found in the brain and pancreas and is related to hormone-
refractory prostate cancer (HRPC) [6, 7]. None of these three
isozymes have been purified, due to their unstable nature,
and as a result, 5a-reductase isozyme inhibitors have been
designed by targeting their substrate.
(p-fluoro)benzoyloxy-5a-hydroxypregna-16-en-3,6,20-trione 8,
21(p-fluoro)benzoyloxypregna-4,16-diene-3,6,20-trione 9.
Various 5a-R2 inhibitors have been developed for relieving
the symptoms produced in benign prostatic hyperplasia.
These 5a-R inhibitors reduce bladder obstruction present in
this disease by inhibiting the conversion of T to its more active
form DHT. Moreover, these inhibitors eliminate the need for
surgery and prevent further progression of the disease. The
most extensively studied 5a-R inhibitors are finasteride and
dutasteride [8]. Finasteride and dutasteride are 4-azasteroids
that have demonstrated low affinity for androgen receptors
and thus were not expected to produce undesirable
antiandrogen effects such as impotence, muscle growth
impairment, or gynecomastia [9]. However, when these drugs
are used for long periods, they could induce hepatoxicity [10];
thus, synthesis of new 5a-R inhibitors with low side effects has
been a goal for researchers.
Results
Chemistry
The target compound 21(p-fluoro)benzoyloxypregna-4,16-
diene-3,6,20-trione (9) was synthesized as shown in
Scheme 1. The starting material 16-dehydropregnenolone
acetate (1) was treated with hydrogen peroxide under basic
conditions to form the 16a,17a epoxy compound 2.
Protection of the alcohol at C-3 of compound 2 with tert-
butyldimethylsilyl chloride followed by oxidation of C-21
using (diacetoxyiodo) benzene afforded compound 4. The
alcohol 4 was esterified using the Steglich method and the
C-3 hydroxy was deprotected using acidic conditions to
obtain compound 5. To regenerate the double bond at C-
2
16, the epoxide was reduced using CrCl to give compound
In view of the fact that we have recently synthesized several
progesterone derivatives with an ester moiety at C-17, which
showed high activity as inhibitors of 5a-R2 and prostate cancer
cell growth [11, 12], in this paper we describe the biological
activityoffivenovelsteroidsbasedontheprogesteroneskeleton
and with an ester moiety at C-21. They are the
following steroidal compounds: 21(p-fluoro)benzoyloxy-
6. The double bond at C-5 of compound 6 was reacted with
m-chloroperoxybenzoic acid to afford the 5a,6a-epoxy
compound 7. Oxidation of the alcohol 7 with chromic acid
yielded the 3,6-diketo-5a-alcohol 8. Dehydration of the
hydroxyl group at C-4 in 8 with thionyl chloride yielded the
a,b-unsaturated dicarbonyl analog 9.
The configuration of the novel pregnenolone derivatives
whose geometry resulted from the spatial arrangement of its
bonds is shown in Fig. 1. The models of these structures were
1
6a,17a-epoxy-3b-hydroxypregna-5-en-20-one 5, 21(p-fluoro)-
benzoyloxy-3b-hydroxypregna-5,16-dien-20-one 6, 21(p-fluoro)
benzoyloxy-5a,6a-epoxy-3b-hydroxypregna-16-en-20-one 7,
1
obtained using the ChemBio3D program.
Scheme 1. Synthesis of compounds 2–9.
Reagents: (i) 30% H
2
O
2
, 4 N NaOH, MeOH;
ii) TBDMSCL, imidazole, DMF; (iii) C I-
OAc) , NaOH, MeOH, CH Cl ; (iv) p-fluoro-
Cl ; (v) HCl,
, acetic acid, acetone; (vii)
; (viii) CrO /H O, acetone; (ix)
Cl
(
(
6 5
H
2
2
2
benzoic acid, DCC, DMAP, CH
acetone; (vi) CrCl
m-CPBA, CH Cl
SOCl , Py, CH
2
2
2
2
2
3
2
2
2
2
.
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Figure 1. Configuration of the novel 21-esters of pregnenolone derivatives; their geometry resulted from the spatial arrangement of
their bonds.
For 6 days, the castrated hamsters received subcutaneous
injections of the various test materials, with the castrated
Activity of compounds 5–9 as inhibitors of 5a-R
1
and 2
control animals (V) being treated with vehicle only. The
experiment was carried out in duplicate. The asterisks show
the instances of statistically significant differences (p < 0.05)
between the group of hamsters treated with testosterone (T)
and those treated with T and with either finasteride (F) or
Figure 2 shows the IC50 values of 5–9 required to inhibit 50%
of the activity of 5a-reductase isoenzymes 1 and 2. Compared
to 5–8, steroid 9 exhibited the highest inhibitory activity
(
5
IC50 ¼ 1.0 mM) for isoenzyme type 1. For the type
2
a-reductase isoenzyme, derivative 9 also displayed the
with the synthesized steroids (5–9; see Scheme
1 for
highest inhibitory activity (IC50 ¼ 0.179 nM). Steroids 5–8
structures).
showed lower inhibitory potency than finasteride for 5aR2
(
higher IC50 value) (Fig. 2).
Discussion
Weight of the prostate gland and seminal
vesicles of hamsters
After sacrificing the animals they were observed statistically
significant (p < 0.05) reductions in the weights of the prostates
andseminalvesiclesascomparedtoglandsfromintacthamsters
We synthesized and identified the biological activity of five
different (p-fluoro)benzoyloxy 21-esters of pregnenolone
derivatives. On the basis of the results obtained in these
experiments, the most accepted spatial conformation of both
isozymes of 5a-R was that in steroid 9. The presence of an
ester group at C-21 masks the polar moiety and facilitates its
crossing through the cell membranes. Thus, all these steroidal
ester derivatives can cross phospholipid membranes and once
they are in the bloodstream, they are hydrolyzed at C-21 by
esterases in the blood to revert to the corresponding free acid.
However, 9 was the only one which displayed a suitable steric
(
Fig. 3). However, treatment with 1 mg/kg (BW) of T significant-
ly reversed the effect of castration on these glands.
Finasteride (F) and the steroids 9, when administered
together with T, significantly decreased the weight of the
prostate and seminal vesicles (p < 0.05); however, compared
to T, compounds 5–8 did not display any statistically
significant effect (Fig. 3).
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1-Esters of Pregnenolone Derivatives as 5a-R Inhibitors
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Figure 2. Structures and biological activities of different (p-fluoro)benzoyloxy 21-esters of pregnenolone.
conformation to induce pharmacological activity. This obser-
vation is consistent with the results previously reported by our
group [13]. These previous data indicated that the
synthesized steroid pregna-4,6-diene-3,6,20-trione was an
inhibitor of 5aR-2, with an IC50 value 0.85 nM. This steroid also
exhibited slight pharmacological activity, decreasing prostate
weight in castrated hamsters stimulated with T [13]. On this
basis, we designed the more lipophilic compound 9 to
improve the pharmacological activity of this pregna-4,6-
diene-3,6,20-trione previously studied, and obtained better
results.
The presence of an OH group in the 5a-position in steroid 8
enables the formation of polar conjugates which can be
promptly eliminated in the urine, as has been previously
reported [14]. The introduction of a keto group at C-6 as in 9
apparently blocked the hepatic metabolism [14] of this
molecule and, as a consequence, an increased effect of this
steroid was observed.
The epoxy moiety at C-16 of 5 enhanced the inhibitory
effect of 5a-R2 and increased its potency (lower IC50 value) as
compared with 7, which has the epoxy moiety in C-5. Thus this
spatial arrangement of the molecule was appropriate for
inducing the inhibitory effect. However, this increase in
activity and potency (5) was not observed for the 5a-R1
isoenzyme.
The unsaturated points of these pregnenolone derivatives
could also be correlated with the ability of these steroids to
5
inhibit 5a-R activity. The presence of the double bond in D as
in 6 increased the inhibitory effect on 5a-R2 activity as
compared with that produced by 8 (which does not have a
double bond in D ). However, the position of this double bond
Figure 3. Weights of prostate and seminal vesicles glands (ꢀ
standard deviation) from castrated (V) and intact animals (C)
ꢁ
5
hamsters. Statistical significance.
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did not influence 5a-R1 activity (8), whereas the unsaturated
point at C-4 in steroid 9 enhanced the inhibitory effect on the
activity of both isozymes.
The presence of a keto group at C-3 makes the synthetic
steroid a competitive substrate for these both isozymes,
because their natural substrates possess this same group.
However, the inhibitory activity of compound 8 was not
enhanced by the presence of this keto group in C-3, as we
would have expected.
In conclusion, 9 proved to have the best molecular
conformation for acceptance by both types of 5a-R as a
likely substrate. In addition, this steroid also displayed activity
in vivo; apparently the pharmacological activity was increased
by the presence of a keto group at C-6. This could be an
advantage for administration of this steroid in the body. As 9
was not metabolized by the liver, this compound could be
administered orally. Future studies will enable us to test this
hypothesis.
Syntheses of the steroid derivatives: Overview
As starting material to prepare the steroids, we used
commercially available 16-dehydropregnenolone acetate,
purity 95%, melting point 172–175°C, Rot: ꢂ40°
(Sigma–Aldrich) (1 in Scheme 1). This steroid was hydrolyzed
by treatment with 4 N solution of NaOH in methanol. To this
mixture, a 30% hydrogen peroxide solution was added drop
by drop. The reaction was stirred at room temperature for 4 h,
then the solvent was evaporated, and water was added. The
precipitate was filtered and washed with water. The crude
was purified by recrystallization from methanol to afford
0.87 g of the pure compound.
16a,17a-Epoxy-3b-hydroxypregna-5-en-20-one 2
ꢂ1
Yield: 95%, mp 180–182°C. IR (cm ): 3454, 2936, 1642, 1042,
1
1692. H NMR (CDCl
3
) d: 1.05 (s, 3H, H-18), 1.02 (s, 3H, H-19), 2.2
(m, 2H, H-21), 2.3 (m, 1H, H-16), 3.5 (m, 1H, H-3), 3.68 (s, 1H,
1
3
3
–OH), 5.33 (m, 1H, H-6). C NMR (CDCl ) d: 15.1 (C-18), 19.3 (C-
Because the structure of 5a-reductase has not been
elucidated, this study contributes to an understanding of
the molecular interactions of these two types of 5a-R
enzymes.
19), 27.51 (C-21), 60.46 (C-16), 71.02 (C-17), 71.06 (C-3), 120.9
(C-6), 141.12 (C-5), 204.9 (C-20). FAB-MS m/z: C21
30
H O
3
[MþH]þ
calculated 331.2228, found 331.2220.
1
2
6a,17a-Epoxy-3b-tert-butyldimethylsilyloxypregna-5-en-
0-one 3
Experimental
16a,17a-Epoxy-3b b-hydroxypregna-5-en-20-one
2
(1 g,
3
mmol) in Fig. 1 was dissolved in methanol; tert-butyldime-
Chemistry
thylsilyl chloride (0.76 g, 6.8 mmol) and imidazole (0.450 g,
5 mmol) in 15 mL of N,N-dimethylformamide were added to
this solution. The mixture was stirred at room temperature for
2 h. After completion of the reaction, the methanol was
evaporated and water was added, and the precipitate was
filtered and washed with water. The crude was purified by
column chromatography using a mixture of 20% ethyl acetate
in hexane to give 1.3 g of the pure compound 3. Yield: 98%,
Chemical reagents
Reagents and solvents (laboratory grade) were purchased
from commercial sources and were used without further
purification. Melting points (uncorrected) were determined
on a melting point apparatus (Fisher Johns, Mexico City);
1
13
H NMR and C NMR were taken on Varian Gemini 200 and
VRX-300 spectrometer, respectively (MR resources NC, USA).
Chemical shifts are given in ppm relative to that of Me Si
(the abbreviations of signal patterns are as
ꢂ1
1
4
mp 126–127°C. IR (cm ): 2929, 1659, 1698, 1083. H NMR
(CDCl ) d: 0.58 (m, 6H, (CH Si), 0.89 (s, 9H, (CH CSi), 0.99 (s,
3H, H-18), 1.09 (s, 3H, H-19), 2.13 (s, 3H, H-21), 2.2 (m, 1H,
(
d ¼ 0) in CDCl
3
3
3
)
2
3 3
)
follows: s, singlet; d, doublet; t, triplet; m, multiplet). Mass
spectra were obtained with an HP5985-B spectrometer
13
H-16), 3.42 (m, 1H, H-3), 5.24 (d, J ¼ 4.9 Hz, 1H, H-6). C NMR
(
Avantes, Apeldoorn, Netherlands). IR spectra were recorded
(CDCl ) d: 19.7 (C-18), 22.8 (C-19), 25.0 (C-21), 30.5 ((CH CSi),
3
3 3
)
on a Perkin-Elmer 200s spectrometer (Perkin-Elmer Life and
Analytical Science, Shelton CT, USA). The UV lamp (254 nm)
was from UVP (Upland, CA).
32.14 (C-Si), 65.1 (C-16), 75.5 (C-17), 77.0 (C-3), 125.0 (C-6),
146.5 (C-5), 209.4 (C-20). FAB-MS m/z: C27
44
H O
3
Si [MþH]þ
calculated 445.3093, found 445.3102.
3
3
(
1,2,6,7- H)-Testosterone ([ H]T; specific activity: 95 Ci/
mmol) was purchased from Perkin-Elmer Analytical Sciences
Boston, MA). This labeled steroid was purified by thin layer
chromatography (TLC) on HPTLC Kieselgel 60 F254 plates
Merck, Darmstadt, Germany); the solvent system recom-
16a,17a-Epoxy-21-hydroxy-20-dimethoxy-3b-tert-
butyldimethylsilyloxypregna-5-en-20-one 4
(
16a,17a-Epoxy-3b-tert-butyldimethylsilyloxypregna-5-en-20-
one (3, 1 g, 2.25 mmol) was dissolved in 10 mL of dichloro-
methane; 20 mL of a methanolic solution of NaOH (1 g,
25 mmol) and (diacetoxyiodo)benzene (1.2 g, 3.72 mmol)
were added. The mixture was stirred for 18 h and then
the solvent was evaporated in vacuo, water was added, and
the precipitate was filtered and washed with water. The crude
was purified on silica gel using 10% ethyl acetate in hexane to
give 0.74 g of the pure compound 4. Yield: 65%, mp
(
mended by the manufacturer was used.
Radioinert T and 5a-DHT were purchased from Steraloids
(
Wilton, NH, USA), Lubrol PX, DL-dithiothreitol (DTT). F was
1
obtained by extraction from Proscar (Merck, Sharp & Dohm,
Mexico City) as follows: 180 Proscar tablets were crushed and
extracted with 200 mL chloroform; after the solvent was
removed under vacuum, the crude product was purified by
silica gel 60 (63–200 mm) column chromatography
ꢂ1
1
205–207°C. IR (cm ): 3596, 1666, 1070, 1033. H NMR (CDCl
3
)
(
Sigma–Aldrich) with ethyl acetate as an eluant [15].
d: 0.1 (s, 6H, (CH Si), 0.83 (s, 9H, (CH CSi), 0.97 (s, 3H, H-18),
3
)
2
3 3
)
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1-Esters of Pregnenolone Derivatives as 5a-R Inhibitors
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1
6
.2 (s, 3H, H-19), 2.2 (m, 1H, H-16), 3.24 (m, 1H, H-3), 3.25 (m,
H, CH
O–), 3.5 (s, 1H, –OH), 3.25 (d, J ¼ 16 Hz, 1H, H-21), 5.24
21(p-Fluoro)benzoyloxy-5a,6a-epoxy-3b-hydroxypregna-
16-en-20-one 7
3
13
(
(
(
m, 1H, H-6). C NMR (CDCl
(CH CSi), 31.57 (C-Si), 50.75 (C-16), 50.75 (CH
C-21), 74.22 (C-17), 77.08 (C-3), 125.27 (C-20), 125.27 (C-6),
3
) d: 17.1 (C-18), 20.7 (C-19), 30.51
OH–), 63.05
21(p-Fluoro)benzoyloxy-3b-hydroxypregna-5,16-dien-20-one
(6) (0.45 g, 1 mmol) and m-chloroperbenzoic acid (0.52 g,
3 mmol) dissolved in 15 mL of dichloromethane were stirred at
room temperature for 45 min. After this, a saturated aqueous
solution of sodium bicarbonate (30 mL) containing sodium
bisulfite (0.3 g) was added. The reaction mixture was
extracted with chloroform; the organic phase was washed
with water, dried over sodium sulfate, and the solvent was
removed under vacuum. The product was recrystallized from
methanol to give 0.36 g of pure compound 7. Yield: 77%, mp
3
)
3
3
þ
1
5
46.39 (C-5). FAB-MS m/z: C29
07.3461, found 507.3472.
H
O
50 5
Si [MþH] calculated
2
1(p-Fluoro)benzoyloxy-16a,17a-epoxy-3b-
hydroxypregna-5-en-20-one 5
6a,17a-Epoxy-21-hydroxy-20-dimethoxy-3b-tert-butyldime-
1
thylsilyloxypregna-5-en-20-one 4 (1.5 g, 3.05 mmol) was dis-
solvedinmethanol;DCC(3.95 g,19.75 mmol)andDMAP(2.37 g,
ꢂ1
1
213–215°C. IR (cm ): 3517, 2930, 1718, 1678, 1087. H NMR
(CDCl
1
9.75 mmol) in dichloromethane (60 mL) was added p-fluoro-
3
) d: 0.89 (s, 3H, H-18), 1.1 (s, 3H, H-19), 2.93 (d, J ¼ 4 Hz,
benzoic acid (2.7 g, 19.75mmol). The resulting solution was
stirred at room temperature for 4 h. Ethyl acetate (250 mL) was
added and the precipitated dicyclohexyl urea was filtered. The
organic phase was washed three times with 10% aqueous
hydrochloric acid, 5% aqueous sodium bicarbonate and water,
the solvent was dried over anhydrous sodium sulfate and
evaporated under vacuum. The crude ester was dissolved in
acetone (30mL), and to this solution hydrochloric acid (1 mL)
was added drop by drop and the mixture was stirred for 15 min,
then water was added and the precipitate was filtered and
washed with water. The crude was purified by column
chromatography using 10% ethyl acetate in hexane to afford
1H, H-6), 3.84 (s, 1H, –OH), 3.91 (m, 1H, H-3), 4.09 (d, J ¼ 16 Hz,
1H, H-21), 5.27 (d, J ¼ 16 Hz, 1H, H-21), 6.8 (s, 1H, H-16), 7.12 (t,
1
3
J ¼ 8 Hz, 2H, H-Ar), 8.11 (m, 2H, H-Ar). C NMR (CDCl
3
) d: 15.14
(C-18), 15.91 (C-19), 58.8 (C-6), 65.87 (C-21), 66.08 (C-5), 68.60
(C-3), 115.47 (C-Ar), 125.71 (C-Ar), 132.56 (C-Ar), 143.97 (C-16),
151.81 (C-17), 165.06 (ester carbonyl), 167.22 (C-Ar), 198.8 (C-
þ
20). FAB-MS m/z: C28
found 469.2339.
H33FO
5
[MþH] calculated 469.2346,
(p-Fluoro)benzoyloxy-5a-hydroxypregna-16-en-3,6,20-
trione 8
21(p-Fluoro)benzoyloxy-5a,6a-epoxy-3b-hydroxypregna-16-
en-20-one 7 (0.47 g, 1 mmol) was dissolved in 10 mL of
acetone. A solution of chromium trioxide (0.85 g, 8.5 mmol)
dissolved in 4.5 mL water at 0°C for 10 min was added
dropwise. The resulting mixture was allowed to warm up to
room temperature and after 30 min the same amount of
chromium trioxide was added in the same manner. The
mixture was diluted with cold water (75 mL) and the
precipitate was filtered and dried. The product was recrystal-
lized from methanol to afford 0.41 g of the pure compound 8.
1
.17 g of pure compound 5. Yield: 82%, mp 210–212°C. IR
ꢂ1
1
(
1
cm ): 3320, 3044, 2936, 1721, 1381, 1055. H NMR (CDCl
.02 (s, 3H, H-18), 1.13 (s, 3H, H-19), 3.48 (m, 1H, H-3), 3.87 (s, 1H,
OH), 4.85 (d, J ¼ 16 Hz, 1H, H-21), 4.94 (d, J ¼ 16Hz, 1H, H-21),
.33(s,1H,H-6),7.12(t,J ¼ 12Hz,2H,H-Ar),8.1(t,J ¼ 8 Hz,2H,H-
3
) d:
–
5
13
Ar). C NMR (CDCl
3
) d: 15.17 (C-18), 19.33 (C-19), 42.18 (C-4),
6
1
1.66(C-21),66.44(C-16),70.66(C-17),71.60(C-3),115.74(C-Ar),
20.91 (C-6), 125.54 (C-Ar), 132.43 (C-Ar), 141.13 (C-5), 165.54
þ
(
ester carbonyl), 167.26 (C-Ar). FAB-MS m/z: C28
H33FO
5
[MþH]
ꢂ1
calculated 469.2346, found 469.2338.
Yield: 86%, mp 135–136°C. IR (cm ): 3332, 2950, 1710, 1685.
1
3
H NMR (CDCl ) d: 0.95 (s, 3H, H-18), 1.04 (s, 3H, H-19), 2.8 (m,
2
1(p-Fluoro)benzoyloxy-3b-hydroxypregna-5,16-dien-20-
one 6
1(p-Fluoro)benzoyloxy-16a,17a-epoxy-3b-hydroxypregna-5-
en-20-one (5) (1 g, 2.1 mmol) dissolved in 100 mL of acetone
containing CrCl (1.29 g, 10.5 mmol) and 2 mL of acetic acid
2H, H-4), 3.89 (s, 1H, –OH), 5.12 (d, J¼ 16 Hz, 1H, H-21), 5.26 (d,
J ¼ 16 Hz, 1H, H-21), 6.81 (s, 1H, H-16), 7.13 (t, J ¼ 8 Hz, 2H, H-
1
3
2
3
Ar), 8.12 (m, 2H, H-Ar). C NMR (CDCl ) d: 13.82 (C-18), 15.98
(C-19), 66.1 (C-21), 82.77 (C-5), 115.52 (C-Ar), 125.68
(C-Ar), 132.59 (C-Ar), 143.62 (C-16), 151.68 (C-17), 165.09
(ester carbonyl), 167.28 (C-Ar), 190.26 (C-20), 210.18 (C-3),
210.87
2
were stirred at room temperature for 45 min. A mixture of ice
in water was then added and the precipitate was filtered and
dried under vacuum. The crude was purified on silica gel using
(C-6). FAB-MS m/z: C28
found 483.2144.
H31FO
6
[MþH]þ calculated 483.2138,
1
6
1
3
5
7
0% ethyl acetate in hexane to afford 0.65 g pure compound
. Yield: 69%, mp 280–282°C. IR (cm ): 3325, 2933, 1723,
677. H NMR (CDCl
ꢂ1
1
3
) d: 0.95 (s, 3H, H-18), 1.03 (s, 3H, H-19),
21(p-Fluoro)benzoyloxypregna-4,16-diene-3,6,20-trione 9
A volume of 4 mL of a cold solution of (p-fluoro)benzoyloxy-
5a-hydroxypregna-16-en-3,6,20-trione 8 (0.48 g, 1 mmol) was
dissolved in dichloromethane. A mixture with 0.4 mL pyridine
was added dropwise with thionyl chloride (0.1 mL) under a
nitrogen atmosphere. The resulting solution was stirred at
room temperature for 2 h. Iced water (100 mL) was added and
it was extracted three times with ethyl acetate. The organic
phase was washed with 10% aqueous hydrochloric acid, 5%
.51 (m, 1H, H-3), 4.20 (s, 1H, –OH), 5.11 (d, J ¼ 16 Hz, 1H, H-21),
.25 (d, J ¼ 16 Hz, 1H, H-21), 5.34 (s, 1H, H-6), 6.82 (s, 1H, H-16),
13
.11 (t, J ¼ 8 Hz, 2H, H-Ar), 8.11 (t, J ¼ 8 Hz, 2H, H-Ar). C NMR
(
CDCl
3
) d: 15.88 (C-18), 19.29 (C-19), 66.09 (C-21), 71.68 (C-3),
1
15.67 (C-Ar), 120.93 (C-6), 125.7 (C-Ar), 132.45 (C-Ar), 141.34
(
C-5), 144.26 (C-16), 152.0 (C-17), 165.07 (ester carbonyl),
þ
1
67.21 (C-Ar), 190.31 (C-20). FAB-MS m/z: C28
H
33FO
4
[MþH]
calculated 453.2396, found 453.2403.
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A. Ch aꢀ vez-Riveros et al.
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aqueous sodium bicarbonate, and water. It was dried over
sodium sulfate and the solvent was evaporated under
vacuum. The product was recrystallized from 20% ethyl
acetate in hexane to give 0.38 g of pure compound 9. Yield:
out at 4°C. The tissue was homogenized, the suspension was
centrifuged (10000g; 30 min; 0°C) (Beckman L70K ultracentri-
fuge), and the pellet was discarded. The supernatant was
filtered through a nylon mesh filter (pore size 11 mm,
distributed by OEM-Membrane Solution, Dallas, TX) and
centrifuged (100000g; 60 min). The microsomal pellet was
resuspended in five volumes of buffer A with a homogenizer,
and the protein concentration was determined by the
Bradford method. The suspension was recentrifuged
(100000g; 30 min) and the pellet was resuspended in buffer
A to give a final concentration of 20 mg protein/mL. The
microsomal suspension was stored at ꢂ80 °C prior to
preparation of the solubilized type 1 5a-R steroid.
ꢂ1
1
8
(
2%, mp 182–185°C. IR (cm ): 2942, 1722, 1681. H NMR
CDCl
) d: 1.0 (s, 3H, H-18), 1.21 (s, 3H, H-19), 5.14 (d, J ¼ 16 Hz,
H, H-21), 5.26 (d, J ¼ 16 Hz, 1H, H-21), 6.2 (s, 1H, H-4), 6.8 (s,
3
1
1
13
3
H, H-16), 7.13 (s, 2H, H-Ar), 8.11 (s, 2H, H-Ar). C NMR (CDCl )
d: 15.9 (C-18), 17.4 (C-19), 66.0 (C-21), 115.5 (C-Ar), 115.7 (C-
Ar), 125.8 (C-4), 127.6 (C-Ar), 132.4 (C-Ar), 143.3 (C-16), 151.4
(
(
C-17), 160.4 (C-5), 165.0 (ester carbonyl), 167.0 (C-Ar), 190.1
þ
C-20), 199.2 (C-3), 201.3 (C-6). FAB-MS m/z: C28
H29FO
5
[MþH]
calculated 465.2033, found 465.2026.
The solubilization of type 1 5a-R isozyme from rat liver
microsomes was carried out according to Levy et al. [18].
Human and animal tissues and procedures
Four hours after a 57-year-old man had died from myocardial
infarction, his normal prostate was extirpated in the
Pathology Department of the Mexico City General Hospital.
The Ethical Committee of the General Hospital in Mexico City
approved this protocol.
Gonadectomized male hamsters
For the experiments in vivo, 80 adult male golden hamsters
(2.5 months old; 150–200 g) were used. After gonadectomies
had been performed on 80 hamsters under isoflurane
anesthesia, the castrated hamsters were allowed to recover
for 30 days prior to experimentation [19]. The castrated
hamsters and the remaining eight intact hamsters were
housed in a room with controlled temperature (22°C) and
light-dark periods of 12 h; the hamsters were fed with food
and water ad libitum. Thirty days post-gonadectomy, the
hamsters were separated into ten groups consisting of four
animals per group. The hamsters were treated for 6 days as
described above and thereafter sacrificed with CO2. This
experiment was carried out twice under the same conditions.
The tissue was rinsed and immediately chilled in ice-cold
1
50 mM NaCl and stored at ꢂ20°C. The frozen human prostate
was thawed on ice, rinsed, and minced in buffer A (20 mM
sodium phosphate, pH 6.5, containing 0.32 M sucrose, 0.1 mM
DTT) (Sigma–Aldrich, Mexico City, Mexico) with an IKA A11
1
basic tissue mill (IKA Laboratory Equipment, Mexico City,
Mexico). Unless otherwise specified, the following procedures
were carried out at 4°C.
Type 2 5a-R isozyme isolated from human prostate
Human prostate was used in this experiment because this
tissue is an abundant source of 5a-R2 for the study of the
effect(s) of 5–9, which were designed for inhibition of the
activity of this enzyme in humans. In this tissue, type 1 5a-R is
not as abundant as type 2.
Biological activity of steroidal derivatives:
Experiments in vitro
The activities of types 1 and 2 5a-reductase were measured by
Human prostate tissue was homogenized in two volumes of
buffer A with a tissue homogenizer Ultra-Turrax IKA, T18
basic (Wilmington, NC). The homogenates were centrifuged
following the conversion of T to DHT, as previously
described [16, 18, 20, 21]. The reagent mixture (final volume,
1 mL) contained 1 mM of DTT in 40 mM sodium phosphate
3
(
1500g; 60 min) [16] in a SW 60 Ti rotor (Beckman Instruments,
buffer (pH 8.0 for type 1; pH 6.5 for type 2), 2 nM [1,2,6,7- H]T,
Palo Alto, CA). The pellets were resuspended in buffer A, and
6.31 mM T (for type 1), and 2 mM NADPH. The reaction, carried
out in duplicate, was started when this mixture was added to
the enzymatic fraction (for type 1, 90 mg protein/6.7 mL from
the solubilized microsomes; for type 2, 50 mg/80 mL from the
membrane fraction of human prostate). After incubation
(37.5°C; 60 min), the reaction was stopped by adding
dichloromethane (1 mL) and mixing; this was considered
the end point. As a control, the procedure was also carried out
without the addition of the enzyme fraction.
In order to extract and purify DHT formed by the activity of
types 1 and 2 5a-reductase, after the individual reaction
mixtures had been shaken (1 min) using a Type 16700 mixer
(Barnstead Thermolyne, Proveedor Cient ꢀı fico, M eꢀ xico, D. F.),
stored at ꢂ70°C. This suspension had a final concentration of
5
mg protein/mL, as determined by the Bradford method [17]
and was used as a source of type 2 5a-R isozyme.
Type 1 5a-R isozyme isolated from rat liver
All procedures involving animals were approved by the
Institutional Care and Use Committee of the Metropolitan
University of Mexico (UAM; Xochimilco, Mexico). All animals
used in this study were obtained from the Animal Care Facility
at UAM.
Two adult (8-month-old) rats had been fasted overnight to
decrease glycogen levels before their livers were extirpated
for use as a source of 5a-R1, as recommended by Levy
et al. [18]. To prepare microsomes, the livers (30 g) were
minced in one volume of buffer A, using the tissue mill. Unless
otherwise specified, the following procedures were carried
the dichloromethane phase of each was placed into individual
tubes. This extraction was repeated for four additional times.
Each pooled dichloromethane extract was evaporated to
dryness under a nitrogen stream and then suspended in
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1-Esters of Pregnenolone Derivatives as 5a-R Inhibitors
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3
methanol (50 mL); each preparation was spotted onto HPTLC
Kieselgel 60 F254 plates. T, DHT, and a mixture of both
standards were applied to the plate in distinct lanes on either
side of the spotted preparation samples. The plates were
developed in chloroform/acetone (9:1) and were air-dried; the
chromatography was repeated for two additional times. DHT
was detected using phosphomolybdic acid reagent
f
Unmodified [ H]T, identified (R 0.56) from control incuba-
tions (i.e., those that had not contained tissue), had identical
chromatographic behavior to that of the non-labeled
standard T.
The radioactivity contained in the zone corresponding to
f
the DHT standard (R 0.67) of the control chromatogram was
1% of the total radioactivity accounted for in the plate and
was considered as an error; it was subtracted from the
experimental chromatograms.
(
Sigma–Aldrich), and T, by fluorescence (UV lamp; 254 nm).
The radioactivity on the plate was scanned by using a
Bioscanner AR2000 (Bioscan, Washington, DC). The zones that
showed chromatographic behavior identical to that of the
Effect of steroid derivatives on activity of types 1 and 2 5a-
reductase types
f
standards (retention factor, R ) were quantified as T or DHT.
The DHT transformation yields were calculated from the
lanes, taking into account the total radioactivity in the TLC,
and were plotted using SigmaPlot 12 software (Systat
Software, INC., San Jose, CA). For the control incubations,
the chromatographic separations and identifications were
carried out in the same manner.
The effect of steroids 5–9 on the activity of types 1 and 2 5a-
reductase was determined under the same conditions in vitro
as described above, but in the presence of six different
ꢂ11
ꢂ10
ꢂ3
concentrations ({1 ꢃ 10
,
1 ꢃ 10
M,. . .,1 ꢃ 10 M}) of
each one of derivatives 5–9. Extraction and purification of
the DHT formed from these incubations were carried out as
described above.
Type 1 5a-R activity was calculated from the percentage of
labeled DHT that had been formed, taking into consideration
Control tubes (without inhibitors) were prepared with the
same incubating medium and labeled T plus either type 1 or
type 2 5a-reductase, under the same conditions. DHT trans-
formations in the presence of 5–9 were calculated from the
lanes, taking into account the total radioactivity in the plate,
and inhibition curves were plotted using SigmaPlot 12
software.
3
the recovery, blank values, specific activity of [ H]T, and the
3
ratio of added [ H]T to unlabeled T. Type 2 5a-R activity was
calculated taking into consideration the recovery, blank
3
values, and specific activity of [ H]T.
The R
that had chromatographic behavior identical to that of the
standard corresponded to 70% of the radioactivity
f
of the T standard was 0.56. The radioactive zone
T
accounted for in the plate. The radioactivity contained in
the zone of the experimental chromatogram, which had an
Determination of the 50% inhibitory concentration of
steroids 5–9 on the activities of types 1 and 2 5a-reductase
To determine the 50% inhibitory concentration (IC50) of
steroids 5–9 and 6 on the activities of types 1 and 2 5a-
reductase, the inhibition plots obtained as we indicated
above were analyzed with the SigmaPlot 12.5 software
(Fig. 4).
R
f
identical to that of the DHT standard (R
identified as the transformed DHT; it corresponded to
0–27% of the total radioactivity accounted for in the
TLC. This result was considered to be 100% of the activity of
a-R (types 1 or 2) for the development of inhibition plots.
f
: 0.67), was
2
5
Figure 4. IC50 values of steroids 4–9 determined from the curves obtained by plotting the activity of 5a-R types 1 and 2 versus the
concentration. The program SigmaPlot was used for curve analysis.
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Arch. Pharm. Chem. Life Sci. 2015, 348, 808–816
A. Ch aꢀ vez-Riveros et al.
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[
[
4] D. W. Russell, J. D. Ann. Rev. Biochem. 1994, 63(1), 25–61.
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Effect of steroid derivatives on prostate and seminal
vesicles of castrated hamsters
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Mol. Biol. 2008, 111, 80–86.
For 6 consecutive days, each of the steroid derivatives 5–9
(
2 mg/kg body weight (BW)) dissolved in 200 mL sesame oil,
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together with 1 mg T/kg (BW), was administered by s.c.
injection to a group of gonadectomized hamsters (four
animals per derivative). Three groups of gonadectomized
animals were used as controls: the first group was injected s.c.
with 200 mL sesame oil; the second group, with 1 mg T/kg
(
BW); and the third group, with 1 mg T plus 1 mg F/kg (BW)
also prepared in sesame oil. Additionally, one group of four
intact hamsters was used as an intact control. On the seventh
day, the animals were sacrificed with CO
2
. The prostate and
[11] M. Cabeza, E. Bratoeff, I. Heuze, A. Rojas, N. Ter aꢀ n,
M. Ochoa, M. T. Ram ꢀı rez-Apan, E. Ram ꢀı rez, V. P eꢀ rez,
I. Grac ꢀı a, J. Enzym. Inhib. Med. Chem. 2006, 21,
seminal vesicles of each hamster were dissected and weighed.
Two separate experiments were performed for each group
of steroid-treated hamsters. The results were analyzed by
one-way analysis of variance and Dunnett’s method to
compare means, using JMP IN 5.1 software (JMP, Statistical
Discovery, and Cary, NC, USA).
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13] V. P eꢀ rez-Ornelas, M. Cabeza, E. Bratoeff, I. Heuze,
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Oxford University Press, Oxford 2001.
This study was supported by grants from Consejo Nacional de
Ciencia y Tecnolog ꢀı a (CONACYT project number 165049) and
from Direcci oꢀ n General de Asuntos del Personal Acad eꢀ mico
(
DGAPA project number IN 211312).
[
15] G. Trapani, L. Dazzi, M. G. Pisu, A. Reho, E. Seu, G. Biggio,
The authors declare that there are no real or perceived
conflicts of interest arising from intellectual, personal, or
financial circumstances of the research. Additionally, all
authors are aware, and approve, of the contents and order
of authorship of the article.
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[
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