Effect of dehydroepiandrosterone derivatives on the activity of 5α-reductase isoenzymes and on cancer cell line PC-3

Article abstract of DOI:10.1016/j.bmc.2014.08.019

It is well known that testosterone (T) under the influence of 5α-reductase enzyme is converted to dihydrotestosterone (DHT), which causes androgen-dependent diseases. The aim of this study was to synthesize new dehydroepiandrosterone derivatives (3a-e, 4a

Full text of DOI:10.1016/j.bmc.2014.08.019

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effect of dehydroepiandrosterone derivatives on the activity
of 5a-reductase isoenzymes and on cancer cell line PC-3
Eugene Bratoeff a, Mariana Garrido a, Teresa Ramírez-Apan b, Yvonne Heuze c, Araceli Sánchez c,
Juan Soriano d, Marisa Cabeza d,
⇑
a Department of Pharmacy, Faculty of Chemistry, National University of Mexico, Mexico, D.F., Mexico
b Institute of Chemistry, National University of Mexico, Mexico, D.F., Mexico
c Department of Biological Systems and Agricultural and Animal Production, Metropolitan University-Xochimilco, Mexico, D.F., Mexico
d Department of Pathology, General Hospital, Mexico, D.F., Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
It is well known that testosterone (T) under the influence of 5a-reductase enzyme is converted to dihy-
drotestosterone (DHT), which causes androgen-dependent diseases. The aim of this study was to synthe-
size new dehydroepiandrosterone derivatives (3a–e, 4a–i, 6 and 7) having potential inhibitory activity
Received 11 June 2014
Revised 10 August 2014
Accepted 18 August 2014
Available online xxxx
against the 5
roidal molecules. The results from this study showed that all compounds exhibited low inhibitory activity
for 5 -reductase type 1 and 2 enzymes and they failed to bind to the androgen receptor. Furthermore, in
a-reductase enzyme. This paper also reports the in vivo pharmacological effect of these ste-
a
Keywords:
5a-Reductase type 1 inhibitors
Prostate cancer
Benign prostatic hyperplasia
Dehydroepiandrosterone derivatives
the in vivo experiment, steroids 3b, 4f, and 4g showed comparable antiandrogenic activity to that of fin-
asteride; only derivatives 4d and 7 produced a considerable decrease in the weight of the prostate gland
of gonadectomized hamsters treated with (T). On the other hand, compounds 4a, f and h showed 100%
inhibition of the growth of prostate cancer cell line PC-3, with compound 4g having a 98.2% antiprolifer-
ative effect at 50
lM. The overall data indicated that these steroidal molecules, having an aromatic ester
moiety at C-3 (4f–h), could have anticancer properties.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
microsomal proteins that reduce the double bond at C-5 in the
steroidal skeleton.
In the prostate, androgens play a crucial role in the develop-
ment of benign prostatic hyperplasia (BPH) and prostate cancer
through androgen receptor regulation. Prostate cancer is still con-
sidered a leading cause of male death worldwide. It is estimated
that in 2009 around 27,360 men died from prostate cancer.1,2
BPH is the non-cancerous growth of the prostate; the incidence
of BPH afflictions is about 70% at 70 years of age and becomes
nearly universal with advancing age.3 Both diseases are associated
with increased levels of the androgen hormone dihydrotestoster-
one (DHT) and enhanced binding to the androgen receptor (AR).
It is well known that DHT binds with higher affinity to the AR than
testosterone (T).4 Testosterone is converted to DHT by the action of
There are three 5
-R type 1 (5 -R1), 5
which was recently detected. 5
epithelial cells and is related to the development of prostate can-
cer. 5 -R2 is mainly located in the stromal compartment and is
associated with BPH. 5
a
-reductase isoenzymes in the prostate tissue:
-R type 2 (5 -R2) and 5 -R type 3 (5 -R3),
-R1 is expressed in the prostate
5a
a
a
a
a
a
a
a
a-R3 can be found in the brain and pancreas
and is related to hormone-refractory prostate cancer (HRPC).5–7
None of the three isoenzymes have been purified due to their
OH
OH
5α-reductase isoenzymes
5a-reductase isoenzymes (Fig. 1), which are membrane-bound
H
H
H
H
H
H
O
O
⇑
Dehydrotestosterone
Testosterone
Corresponding author at present address: Departamento de Sistemas Biológ-
icos, Universidad Autónoma Metropolitana-Xochimilco, Calzada del Hueso No.
1100, México, D.F. C.P 04960, Mexico. Tel.: +52 (55) 5483 72 60x011; fax: +52 (55)
5483 72 60.
Figure 1. Conversion of testosterone to dehydrotestosterone by the action of 5a-
reductase isoenzymes.
E-mail address: marisa@correo.xoc.uam.mx (M. Cabeza).
http://dx.doi.org/10.1016/j.bmc.2014.08.019
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Bratoeff, E.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.08.019

2
E. Bratoeff et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
unstable nature, and as a result, 5
a
-reductase isoenzyme inhibitors
200 mL chloroform; after the solvent was removed under vacuum,
the crude product was purified by silica gel 60 (63–200 m)
have been designed by targeting their substrate. Since the cancer
cell lines show different mechanisms of action, it is important to
design drugs that have a specific activity for each target. The PC3
cell line shows 17b-hydroxydehydrogenase activity, whereas HeLa
l
column chromatography (Sigma–Aldrich) with ethyl acetate as
eluant. The melting point of the isolated Finasteride (252–254 °C)
was identical to that reported in the literature. Bio-gel
Hydroxyapatite (HAP) was provided by Bio-Rad Laboratories (CA,
USA).
cells display increased activity for type 1 5
Finasteride (F) is a 5 -R2 inhibitor with an IC50 value of 8.5 nM,
but it shows an IC50 value of 630 nM for 5 -R1; thus it is more spe-
cific for type 2 enzyme.8 Since 5
-R type 1 and 2 have been associ-
a-reductase.
a
a
a
2.1.2. Synthesis of the steroidal derivatives: overview
ated with prostate cancer development, this inhibition has been an
important target for the treatment of the disease. Previously, it has
been reported that some dehydroepiandrosterone derivatives with
For the synthesis of the new steroidal compounds 3a–i, 4a–i, 6
and 7 (Fig. 2), we used commercially available dehydroepiandros-
terone 1 and also its acetate 5. Compounds 2a–i and 6 were
prepared according to the procedure previously reported.8,13,14
The synthesis of the new compounds is briefly described below.
an ester moiety at C-3 exhibited 5a
-R inhibitory activity.8–10 There
are several publications which indicate that dehydroepiandoster-
one derivatives containing an azole group at C-17 showed low
5a-R inhibitory activity but high CYP17 inhibition; it is well known
2.1.2.1. Synthesis of 16-formyl-17-chloroandrosta-5,16-diene-
that the inhibition of one or both enzymes could be important for
improvement of HRPC-treatments.11,12 In recent studies, we have
demonstrated that the presence of electrophilic centers enhanced
3b-yl derivatives (3 in Fig. 2).
Into a round-bottomed flask
containing cold phosphorus oxychloride (36 mmol), fitted with a
magnetic stirrer, dimethylformamide (43 mmol) was added drop-
wise followed by a solution of the corresponding ester in chloro-
form (1 mmol) (5 mL). The mixture was refluxed for 5 h under N2
atmosphere. The reaction was quenched with a saturated solution
of sodium bicarbonate (100 mL). The compound was extracted
with chloroform, and the organic phase was washed with water
and dried over anhydrous sodium sulfate. The compound was
purified on silica gel chromatography.
5a
-R inhibitory activity.9
On the basis of these findings, we decided to synthesize several
steroidal derivatives that could be more specific for the inhibition
of 5 -R enzymes and could be used for the treatment of androgen-
a
dependent diseases. The structural requirements that we
considered include a chlorine atom or an imidazole moiety at
C-17 (electrophilic center), a formyl substituent at C-16 (
a,b
conjugated carbonyl group), and several ester functions at C-3.
In this paper, we report the synthesis of 16-formyl-17-chloro-
androsta-5,16-diene-3b-yl derivatives (3a–3e and 6) and
16-formyl-17-(1H-imidazole-1-yl)androsta-5,16-diene-3b-yl derivatives
(4a–4i and 7). These compounds were evaluated (in vitro as well as
in vivo) as 5a-R1, 5a-R2, and androgen receptor inhibitors and also
in their ability to inhibit the proliferation of prostate cancer cell
2.1.2.2. Derivatives 3a–i. 2.1.2.2.1. 16-Formyl-17-chloroandrosta-
5,16-diene-3b-yl-cyclopropancarboxylate (3a). Yield: 72%, mp
124–125 °C. UV (nm): 257 (e
= 10200). IR (KBr) cmꢀ1: 2928,
1717, 1674, 1589. 1H NMR (CDCl3) d: 0.9 (3H, s, H-18), 1.0 (3H, s,
H-19), 4.6 (1H, m, H-3), 5.3 (1H, dd, J1 = 4.0 Hz and J2 = 2.0 Hz,
H-6), 9.9 (1H, s, aldehyde). 13C NMR (CDCl3) d: 8.4, 13.1, 15.0,
19.3, 20.4, 27.8, 29.9, 30.3, 30.5, 30.8, 31.6, 32.8, 35.9, 38.1, 50.3,
50.6, 53.8, 73.6, 121.8, 136.5, 140.1, 162.3, 174.3, 188.1. HRMS
calcd for C24H31ClO3 402.1932; found 402.1922.
lines (PC-3).
2. Material and methods
2.1.2.2.2. 16-Formyl-17-chloroandrosta-5,16-diene-3b-yl-cyclob-
utancarboxylate (3b). Yield: 69%, mp 152–153 °C. UV (nm): 256
(e
= 10100). IR (KBr) cmꢀ1: 2941, 1719, 1671, 1586. 1H NMR
2.1. Chemical and radioactive materials
(CDCl3) d: 0.9 (3H, s, H-18), 1.1 (3H, s, H-19), 4.6 (1H, m, H-3),
5.4 (1H, dd, J1 = 4.2 Hz and J2 = 2.1 Hz, H-6), 9.9 (1H, s, aldehyde).
13C NMR (CDCl3) d: 14.6, 17.9, 18.9, 19.9, 24.9, 27.3, 28.1, 30.1,
30.4, 32.5, 35.5, 37.7, 37.9, 49.8, 50.5, 53.3, 72.9, 121.4, 136.1,
139.7, 161.9, 174.6, 187.8. HRMS calcd for C25H33ClO3 416.2188;
found 416.2189.
2.1.1. Reagents
Solvents were laboratory grade. Melting points (uncorrected)
were determined on a melting point apparatus (Fisher Johns,
Mexico City, Mexico); 1H NMR and 13C NMR were taken on a
Varian Gemini 200 and VRX-300 spectrometer, respectively (MR
resources NC, USA). Chemical shifts are given in ppm relative to
that of Me4Si (d = 0) in CDCl3 (the abbreviations of signal patterns
are: s, singlet; d, doublet; t, triplet; m, multiplet). Mass spectra
were obtained with an HP5985-B spectrometer (Avantes,
Apeldoorn, The Netherlands). IR spectra were recorded 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).
2.1.2.2.3. 16-Formyl-17-chloroandrosta-5,16-diene-3b-yl-cyclop-
entancarboxylate (3c). Yield: 70%, mp 180–181 °C. UV (nm): 258
(e
= 10300). IR (KBr) cmꢀ1: 2927, 1728, 1708, 1667, 1590. 1H
NMR (CDCl3) d: 0.9 (3H, s, H-18), 1.1 (3H, s, H-19), 4.6 (1H, m, H-
3), 5.4 (1H, dd, J1 = 4.4 Hz and J2 = 2.0 Hz, H-6), 9.9 (1H, s, formyl).
13C NMR (CDCl3) d: 14.6, 18.9, 19.9, 25.6, 27.3, 28.3, 29.7, 29.9, 30.5,
32.6, 36.4, 36.5, 37.7, 43.7, 49.9, 50.2, 53.3, 72.9, 121.4, 136.1,
139.8, 161.9, 175.8, 187.7. HRMS calcd for C26H35ClO3 430.2215;
found 430.2210.
(1,2,6,7-3H) T specific activity: 95 Ci/mmol was purchased from
Perking Elmer Life and Analytical Science. (Boston, MA). Labeled T
was purified by thin layer chromatography (TLC) on HPTLC
Keiselgel 60 F254 plates (Merck, Darmstadt, Germany); the solvent
system recommended by the manufacturer was used.
2.1.2.2.4. 16-Formyl-17-chloroandrosta-5,16-diene-3b-yl-cycloh-
exancarboxylate (3d). Yield: 71%, mp 166–167 °C. UV (nm): 257
(e
= 10100). IR (KBr) cmꢀ1: 2929, 1727, 1668, 1592. 11H NMR
(CDCl3) d: 0.9 (3H, s, H-18), 1.1 (3H, s, H-19), 4.6 (1H, m, H-3),
5.4 (1H, dd, J1 = 4.5 Hz and J2 = 2.4 Hz, H-6), 9.9 (1H, s, formyl).
13C NMR (CDCl3) d: 14.6, 18.9, 19.9, 24.4, 25.1, 25.5, 25.9, 27.3,
28.1, 28.8, 30.1, 30.5, 32.6, 33.3, 36.6, 37.6, 43.0, 49.9, 50.4, 53.4,
72.7, 121.4, 136.1, 139.8, 161.9, 175.2, 187.7. HRMS calcd for
Radioinert T, 5a-DHT, and mibolerone (MIB) were purchased
from Steraloids (Wilton, NH, USA) and Perkin Elmer Life Sciences,
Inc. (Boston, MA, USA). NADPH, Lubrol PX, DL-dithiothreitol
(DTT), protease inhibitors, (activated charcoal acid washed with
hydrochloric acid), and dextran (Mr: 70,000) were purchased from
Sigma–Aldrich (St. Louis, MO, USA). Finasteride was obtained by
extraction from ProscarÒ (Merck, Sharp & Dohm, Mexico City) as
follows: 180 Proscar tablets were crushed and extracted with
C27H37ClO3 444.2481; found 444.2487.
2.1.2.2.5. 16-Formyl-17-chloroandrosta-5,16-diene-3b-yl-cyclo-
heptancarboxylate (3e). Yield: 70%, mp 215–216 °C. UV (nm):
257 (
e
= 10100). IR (KBr) cmꢀ1: 2946, 1719, 1671, 1585. 1H NMR
Please cite this article in press as: Bratoeff, E.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.08.019

E. Bratoeff et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
Cl
O
O
i
ii
H
H
H
O
O
O
H
H
H
H
H
1
H
R
O
O
R
O
HO
3
2
N
N
iii
f)
h)
d)
e)
R= a)
b)
F
Br
I
H
g)
i)
O
H
H
c)
Cl
R
iii
O
4
N
O
Cl
N
ii
H
H
H
O
H
H
O
O
H
H
O
O
O
H
H
O
O
6
5
7
Figure 2. Synthesis of steroidal derivatives. (i) DCC, DMAP, CHCl3, RCOOH; (ii) POCl3, DMF, CHCl3; (iii) imidazole, K2CO3, DMF. 4-Dimethylaminopyridine (DMAP), N,N0-
dicyclohexyl carbodiimide (DCC), dimethyl formamide (DMF), phosphorus oxychloride (POCl3).
(CDCl3) d: 0.9 (3H, s, H-18), 1.0 (3H, s, H-19), 4.5 (1H, m, H-3), 5.4
(1H, dd, J1 = 4.6 Hz and J2 = 2.1 Hz, H-6), 9.9 (1H, s, formyl). 13C
NMR (CDCl3) d: 15.0, 19.3, 20.4, 26.5, 27.7, 28.3, 28.4, 30.5, 30.8,
30.9, 32.9, 36.9, 38.1, 45.3, 50.3, 50.7, 53.8, 73.1, 121.7, 136.5,
140.1, 162.3, 176.5, 188.1. HRMS calcd for C28H39ClO3 458.2518;
found 458.2514.
and J2 = 2.4 Hz, H-6), 7.7 (2H, d, J = 8.8 Hz, H-aromatic), 7.9 (2H,
d, J = 8.4 Hz, H-aromatic), 10.0 (formyl). 13C NMR (CDCl3) d: 14.9,
19.2, 20.3, 27.7, 28.4, 30.4, 30.8, 32.9, 36.8, 36.9, 38.1, 50.2, 50.5,
53.7, 74.6, 100.5, 122.1, 130.2, 130.9, 136.4, 137.6, 139.8, 162.2,
165.4, 188.0. HRMS calcd for
565.4310.
C27H30ClIO3 565.4311; found
´
2.1.2.2.6. 16-Formyl-17-chloroandrostan-5,16-diene-3b-yl-4-flur-
obenzoate (3f). Yield: 63%, mp 194–195 °C. UV (nm): 231, 256. IR
(KBr) cmꢀ1: 2932, 1714, 1663, 1518, 1278. 1H NMR (CDCl3) d: 1.0
(3H, s, H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.4 (1H, dd,
J1 = 4.5 Hz and J2 = 2.3 Hz, H-6), 7.1 (2H, ddt, J1 = 8.8 Hz,
J2 = 8.4 Hz, J3 = 2.8 Hz, H-aromatic), 8.0 (2H, dd, J1 = 8.4 Hz,
J2 = 9.0 Hz, H-aromatic), 9.9 (formyl). 13C NMR (CDCl3) d: 14.9,
19.2, 19.9, 27.6, 28.5, 30.3, 30.9, 32.9, 36.9, 38.1, 50.1, 50.6, 53.4,
74.2, 115.2, 115.5, 122.5, 127.1, 131.4, 136.6, 139.7, 162.2, 164.4,
165.1, 166.4, 187.9. HRMS calcd for C27H30ClFO3 457.4022; found
457.4029.
2.1.2.3. Synthesis of 16-formyl-17-(1H-imidazole-1-yl)androsta-
5,16-diene-3b-yl derivatives (4 in Fig. 3). A mixture of imid-
azole (3 mmol), K2CO3 (6 mmol) and the corresponding 16-formyl-
17-chloroandrosta-5,16-diene-3b-yl derivative (1 mmol) in dry
DMF (5 mL) was heated at 50 °C under N2 atmosphere for 3 h. Cold
water was added and the precipitated was filtered. The compound
was purified by column chromatography on florisil.
2.1.2.4. Derivatives 4a–i. 2.1.2.4.1. 16-Formyl-17-(1H-imidazole-
1-yl)androsta-5,16-dien-3b-yl-cyclopropancarboxylate (4a). Yield:
83.5%, mp 135–137 °C. UV (nm): 256. IR (KBr) cmꢀ1: 2939, 1720,
1667, 1610. 1H NMR (CDCl3) d: 1.0 (3H, s, H-18), 1.1 (3H, s, H-
19), 4.6 (1H, m, H-3), 5.4 (1H, dd, J1 = 4.6 Hz and J2 = 2.3 Hz, H-6),
7.1 (1H, s, H-aromatic), 7.2 (1H, s, H-aromatic), 7.6 (1H, s,
H-aromatic) 9.7 (formyl). 13C NMR (CDCl3) d: 8.3, 13.1, 15.9, 19.2,
20.4, 27.7, 28.2, 30.2, 30.8, 33.8, 36.7, 36.8, 49.3, 49.9, 53.8, 73.5,
119.9, 121.7, 133.9, 137.8, 139.9, 160.5, 160.6, 174.2, 187.6. HRMS
calcd for C27H34 N2O3 435.2600; found 435.2601.
2.1.2.4.2. 16-Formyl-17-(1H-imidazole-1-yl)androsta-5,16-dien-
3b-yl-cyclobutancarboxylate (4b). Yield: 79.8%, mp 181–182 °C.
UV (nm): 256. IR (KBr) cmꢀ1: 2947, 1727, 1665, 1607. 1H NMR
(CDCl3) d: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.6 (1H, m, H-3),
5.4 (1H, dd, J1 = 4,2 Hz and J2 = 2.7 Hz, H-6), 7.1 (1H, s, H-aromatic),
7.2 (1H, s, H-aromatic), 7.6 (1H, s, H-aromatic) 9.7 (formyl). 13C
NMR (CDCl3) d: 8.0, 15.7, 18.1, 18.9, 24.9, 24.9, 27.4, 29.9, 30.5,
35.5, 36.0, 49.1, 49.7, 53.5, 72.9, 119.6, 121.3, 130.4, 133.6, 137.5,
139.8, 160.2, 174.6, 187.4.HRMS calcd for C28H36 N2O3 449.2726;
found 449.2723.
´
2.1.2.2.7. 16-Formyl-17-chloroandrostan-5,16-diene-3b-yl-4-chlo-
robenzoate (3g). Yield: 59%, mp 174–175 °C. UV (nm): 241.66. IR
(KBr) cmꢀ1: 2929, 1718, 1669, 1585. 1H NMR (CDCl3) d: 1.0 (3H, s,
H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.4 (1H, dd, J1 = 4.0 Hz
and J2 = 2.0 Hz, H-6), 7.4 (2H, d, J = 8.4 Hz, H-aromatic), 8.0 (2H,
d, J = 8.4 Hz, H-aromatic), 10.0 (formyl). 13C NMR (CDCl3) d: 14.9,
19.2, 20.4, 27.6, 28.6, 30.4, 30.8, 32.9, 36.8, 38.1, 50.2, 50.6, 53.6,
74.5, 121.9, 128.4, 129.1, 130.9, 135.7, 139.6, 139.9, 162.4, 165.1,
188.1. HRMS calcd for C27H30Cl2O3 473.2455; found 473.2451.
´
2.1.2.2.8. 16-Formyl-17-chloroandrostan-5,16-diene-3b-yl-4-bro-
mobenzoate (3h). Yield: 65%, mp 189–190 °C. UV (nm): 255. IR
(KBr) cmꢀ1: 2928, 1711, 1668, 1587, 1269. 1H NMR (CDCl3) d: 1.0
(3H, s, H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.4 (1H, d,
J = 4.2 Hz and J2 = 2.2 Hz, H-6), 7.6 (2H, d, J = 8.4 Hz, H-aromatic),
7.9 (2H, d, J = 8.8 Hz, H-aromatic), 10.0 (formyl). 13C NMR (CDCl3)
d: 14.9, 19.2, 20.3, 27.7, 28.4, 30.4, 30.1, 32.8, 36.7, 36.8, 38.1,
50.1, 50.6, 53.6, 74.6, 122.1, 22.4, 127.7, 127.8, 129.6, 129.8,
131.1, 131.6, 136.4, 139.8, 162.3, 165.2, 188.1. HRMS calcd for
C
27H30ClBrO3 517.2186; found 517.2186.
2.1.2.2.9. 16-Formyl-17-chloroandrostan-5,16-diene-3b-yl-4-iodo-
2.1.2.4.3. 16-Formyl-17-(1H-imidazole-1-yl)androsta-5,16-dien-
3b-yl-cyclopentancarboxylate (4c). Yield: 85.7%, mp 188–189 °C.
UV (nm): 261. IR (KBr) cmꢀ1: 2948, 1726, 1664, 1606. 1H NMR
(CDCl3) d: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.6 (1H, m, H-3),
5.4 (1H, dd, J1 = 4.1 Hz and J2 = 2.0 Hz, H-6), 7.1 (1H, s, H-aromatic),
´
benzoate (3i). Yield: 62%, mp 197–198 °C. UV (nm): 246. IR (KBr)
cmꢀ1: 2939, 1707, 1664, 1586, 1271. 1H NMR (CDCl3) d: 1.0 (3H, s,
H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.4 (1H, dd, J1 = 4.3 Hz
Please cite this article in press as: Bratoeff, E.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.08.019

4
E. Bratoeff et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 3. Weight of the prostate gland standard deviation from castrated hamsters receiving different treatments for 6 days. The control animals (C) were treated with
vehicle only.
7.2 (1H, s, H-aromatic), 7.6 (1H, s, H-aromatic) 9.7 (formyl). 13C
NMR (CDCl3) d: 15.1, 18.4, 19.6, 24.9, 26.8, 27.4, 29.2, 29.4, 29.9,
32.9, 35.9, 36.0, 27.2, 43.2, 48.5, 49.1, 52.9, 72.1, 119.1, 120.8,
129.9, 133.1, 136.9, 139.3, 159.9, 175.4, 186.8. HRMS calcd for
119.9, 122.0, 128.6, 129.1, 130.7, 130.9, 133.9, 137.8, 139.2,
139.8, 160.6, 139.8, 160.6, 165.6, 187.6. HRMS calcd for C30H33Cl
N2O3 505.3398; found 505.3394.
2.1.2.4.8. 16-Formyl-17-(1H-imidazole-1-yl)androsta-5,16-dien-
´
C29H38 N2O3 463.2622; found 463.2623.
3b-yl-4-bromobenzoate (4h). Yield: 91%, mp 249–250 °C. UV
2.1.2.4.4. 16-Formyl-17-(1H-imidazole-1-yl)androsta-5,16-dien-
(nm): 244 IR (KBr) cmꢀ1: 2937, 1710, 1662, 1604, 1267, 1238. 1H
NMR (CDCl3) d: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.9 (1H, m,
H-3), 5.5 (1H, dd, J1 = 4.2 Hz and J2 = 2.2 Hz, H-6), 7.1 (1H, s,
H-aromatic), 7.2 (1H, s, H-aromatic), 7.5 (2H, dd, J1 = 8.8 Hz,
J2 = 2.0 Hz, H-aromatic), 7.6 (1H, s, H-aromatic), 7.9 (2H, dd,
J1 = 8.8 Hz, J2 = 2 Hz, H-aromatic), 9.7 (formyl). 13C NMR (CDCl3)
d: 15.7, 18.9, 20.1, 27.4, 29.9, 30.5, 36.4, 36.5, 37.8, 49.1, 49.7,
53.5, 74.1, 119.7, 121.7, 127.6, 129.3, 130.4, 130.7, 131.6, 137.5,
3b-yl-cyclohexancarboxylate (4d). Yield: 80.5%, mp 226–227 °C.
UV (nm): 261. IR (KBr) cmꢀ1: 2948, 1726, 1664, 1606. 1H NMR
(CDCl3) d: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.6 (1H, m, H-3),
5.4 (1H, dd, J1 = 4.05 Hz and J2 = 2.2 Hz, H-6), 7.1 (1H, s,
H-aromatic), 7.2 (1H, s, H-aromatic), 7.6 (1H, s, H-aromatic) 9.7
(formyl). 13C NMR (CDCl3) d: 15.9, 19.2, 20.4, 25.4, 25.8, 27.6,
28.2, 29.0, 29.1, 30.2, 30.8, 33.8, 36.7, 36.8, 38.0, 43.3, 49.4, 49.9,
53.7, 73.0, 119.9, 121.6, 130.6, 133.9, 135.1, 137.8, 140.1, 160.6,
139.5, 160.3, 164.9, 187.3. HRMS calcd for
549.3219; found 549.3212.
C30H33BrN2O3
175.6, 187.6. HRMS calcd for
477.2999.
C30H40 N2O3 477.2990; found
2.1.2.4.9. 16-Formyl-17-(1H-imidazole-1-yl)androsta-5,16-dien-
´
2.1.2.4.5. 16-Formyl-17-(1H-imidazole-1-yl)androsta-5,16-dien-
3b-yl-cycloheptancarboxylate (4e). Yield: 81.3%, mp 216–218 °C.
UV (nm): 256. IR (KBr) cmꢀ1: 2926, 1729, 1661, 1610. 1H NMR
(CDCl3) d: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.6 (1H, m, H-3),
5.4 (1H, dd, J1 = 4.2 Hz and J2 = 2.3 Hz, H-6), 7.1 (1H, s, H-aromatic),
7.2 (1H, s, H-aromatic), 7.6 (1H, s, H-aromatic) 9.7 (formyl). 13C
NMR (CDCl3) d: 14.9, 18.2, 19.4, 24.4, 24.8, 26.6, 27.2,
28.0, 28.0, 29.2, 29.8, 32.8, 35.7, 35.8, 37.0, 42.3, 48.4, 48.9,
52.8, 72.0, 118.9, 120.6, 129.6, 132.9, 134.1, 136.8, 139.1, 159.6,
3b-yl-4-iodobenzoato (4i). Yield: 89%, mp 230–232 °C. UV (nm):
227. IR (KBr) cmꢀ1: 2939, 1709, 1606, 1273. 1H NMR (CDCl3) d:
1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.4 (1H, dd,
J1 = 4.1 Hz and J2 = 2.1 Hz, H-6), 7.1 (1H, s, H-aromatic), 7.2 (1H,
s, H-aromatic), 7.5 (2H, d, J = 8.8 Hz, H-aromatic), 7.6 (1H, s, H-aro-
matic), 7.8 (2H, d, J = 8.8 Hz, H-aromatic), 9.7 (formyl). 13C NMR
(CDCl3) d: 15.9, 19.2, 20.4, 27.7, 30.2, 30.8, 33.8, 36.7, 36.8, 45.5,
49.4, 49.9, 53.8, 68.3, 74.5, 100.5, 119.9, 122.0, 129.1, 130.1,
130.6, 130.9, 133.9, 137.6, 139.9, 159.9, 165.4, 187.6. HRMS calcd
for C30H33I N2O3 597.3816; found 597.3812.
174.6, 186.7. HRMS calcd for
491.3290.
C31H42 N2O3 491.2195; found
2.1.2.4.6. 16-Formyl-17-(1H-imidazole-1-yl)androsta-5,16-dien-
3b-yl-4-fluorobenzoate (4f). Yield: 88%, mp 260–261 °C. UV
2.1.2.5.
Synthesis
of
16-formyl-17-(1H-1,3-imidazole-1-
´
yl)androsta-5,16-dien-3b-yl-acetate (7 in Fig. 3).
Yield:
(nm): 228 IR (KBr) cmꢀ1: 2940, 1713, 1665, 1600, 1273. 1H NMR
(CDCl3) d: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3),
5.4 (1H, dd, J1 = 4.1 Hz and J2 = 2.1 Hz, H-6), 7.0 (2H, dd,
J1 = 6.4 Hz, J2 = 5.2 Hz, H-aromatic), 7.1 (1H, s, H-aromatic), 7.2
(1H, s, H-aromatic), 7.6 (1H, s, H-aromatic), 8.0 (2H, dd,
J1 = 9.2 Hz, J2 = 5.6 Hz, H-aromatic), 9.7 (formyl). 13C NMR (CDCl3)
d: 15.3, 18.6, 19.8, 27.1, 27.7, 29.7, 30.2, 36.1, 37.5, 48.7, 49.5,
53.0, 73.7, 114.6, 114.9, 119.3, 121.3, 126.2, 128.1, 129.9, 130.2,
131.3, 131.4, 133.4, 137.1, 139.2, 159.9, 164.3, 186.9. HRMS calcd
for C30H33F N2O3 489.2072; found 489.2078.
93%, mp 225–226 °C. UV (nm): 257. IR cmꢀ1: 2947, 1731, 1659,
1616, 1234. 1H NMR (CDCl3) d: 1.0 (3H, s, H-18), 1.1 (3H, s,
´
H-19), 2.0 (H-1), 4.6 (1H, m, H-3), 5.4 (1H, dd, J1 = 4 .3 Hz and
J2 = 2.1 Hz, H-6), 7.1 (1H, s, aromatic), 7.2 (1H, s, aromatic), 7.6
(1H, s, aromatic) 9.7 (aldehyde). 13C NMR (CDCl3) d: 15.8, 19.0,
20.2, 21.2, 27.5, 28.1, 30.1, 30.6, 33.6, 35.5, 36.6, 37.9, 49.2, 49.8,
53.6, 73.4, 119.7, 121.5, 130.6, 133.7, 137.6, 139.8, 160.4, 170.2,
187.2. HRMS calcd for C25H32 N2O3 409.2473; found 409.2476.
2.2. Human and animal tissues and procedures
2.1.2.4.7. 16-Formyl-17-(1H-imidazole-1-yl)androsta-5,16-dien-
´
3b-yl-4-chlorobenzoate (4g). Yield: 92%, mp 210–212 °C. UV
Four hours after a 53-year-old man had died from diabetes and
renal insufficiency, his normal prostate was extirpated in the
Pathology Department of the General Hospital in Mexico City.
The Ethical Committee of the General Hospital in Mexico City
approved this protocol.
The tissue was rinsed and immediately chilled in ice-cold
150 mM NaCl and stored at ꢀ20 °C. The frozen human prostate
was thawed on ice, rinsed, and minced in buffer A (20 mM sodium
(nm): 241 IR (KBr) cmꢀ1: 2937, 1710, 1663, 1604, 1267, 1239. 1H
NMR (CDCl3) d: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-
3), 5.5 (1H, dd, J1 = 4 Hz and J2 = 2 Hz, H-6), 7.1 (1H, s, H-aromatic),
7.2 (1H, s, H-aromatic), 7.4 (2H, dd, J1 = 8.4 Hz, J2 = 2.4 Hz,
H-aromatic), 7.6 (1H, s, H-aromatic), 7.9 (2H, dd, J1 = 8.8 Hz,
J2 = 2.4 Hz, H-aromatic), 9.7 (formyl). 13C NMR (CDCl3) d: 15.9,
19.2, 20.4, 27.7, 30.3, 30.9, 36.7, 36.8, 49.3, 49.9, 53.8, 74.5,
Please cite this article in press as: Bratoeff, E.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.08.019

E. Bratoeff et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
phosphate, pH 6.5, containing 0.32 M sucrose, 0.1 mM DTT
(Sigma-Aldrich, Mexico City, Mexico) with an IKAÒ A11 basic tissue
mill (IKA Laboratory Equipment, Mexico City, Mexico). Unless
otherwise specified, the following procedures were carried out at
4 °C.
2.2.4. Gonadectomized male hamsters
For the experiments in vivo, 120 adult male golden hamsters
(2.5 months old; 150–200 g) were used. After gonadectomies had
been performed on 112 hamsters under isoflurane anesthesia,
the castrated hamsters were allowed to recover for 30 days prior
to experimentation.20 The castrated hamsters and the remaining
eight intact hamsters were housed in a room with controlled tem-
perature (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 nine groups consisting of four
animals per group. The hamsters were treated for six days as
described in Section 2.3.1.1 and thereafter sacrificed with CO2. This
experiment was carried out twice under the same conditions.
2.2.1. 5a-R type2 isozyme isolated from human prostate
Human prostate was used in this experiment because this
tissue is an abundant source of 5
of 3a–e, 6, 4a–i and 7, which were designed for inhibition of the
activity of this enzyme in humans. In this tissue, 5 -R type 1 is
a-R2 for the study of the effect(s)
a
not as abundant as is type 2; AR is very difficult to study in this
model.
The human prostate tissue was homogenized in two volumes of
buffer A with a tissue homogenizer Ultra-Turrax IKA, T18 basic
(Wilmington, NC). Homogenates were centrifuged (1500ꢁg;
60 min)10 in a SW 60 Ti rotor (Beckman Instruments, Palo Alto,
CA). The pellets were resuspended in buffer A, and stored at
2.3. Biological activity of steroidal derivatives
2.3.1. Experiments in vitro
The activity of the 5a-reductase type 1 and 2 were determined
ꢀ70 °C. This suspension had
protein/mL, as determined by the Bradford method,15 and was
used as source of 5 -R type 2 isozyme.
a
final concentration of 5 mg
by following the conversion of T to DHT, as previously
described.21,22 The reagent mixture (final volume, 1 mL) contained
1 mM of DTT in 40 mM sodium phosphate buffer (pH 8.0 for type
a
1; pH 6.5 for type 2), 2 nM [1,2,6,73H] T, 6.31
lM T (for type 1),
and 2 mM NADPH. The reaction, carried out in duplicate, was
2.2.2. 5aR type 1 isozyme isolated from rat liver
All procedures involving animals were approved by the Institu-
tional 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.
started when this mixture was added to the enzymatic fraction
(for type 1, 90
lg protein/6.7
lL from the solubilized microsomes;
for type 2, 50
l
g/80 L from the membrane fraction of human
l
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, this procedure was also car-
ried out without the addition of the enzyme fraction.
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.16 To
prepare microsomes, the livers (30 g) were minced in one volume
of buffer A using the tissue mill. Unless otherwise specified, the fol-
lowing procedures were carried out at 4 °C. The tissue was homog-
enized; the suspension was centrifuged (10,000ꢁg; 30 min; 0 °C)
(Beckman L70 K ultracentrifuge); and the pellet was discarded.
The supernatant was filtered through a nylon mesh filter (pore size
In order to extract and purify DHT formed by the activities of 5
a
reductase types 1 and 2, after the individual reaction mixtures had
been agitated (1 min) by using a Type 16700 mixer (Barnstead
Thermoline, Proveedor Científico, Mexico City), the dichlorometh-
ane phase of each was placed into individual tubes. This extraction
was repeated four additional times. Each pooled dichloromethane
extract was evaporated to dryness under a nitrogen stream and
11 lm, distributed by OEM-Membrane Solution, Dallas TX) and
centrifuged (100,000ꢁg; 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 re-centrifuged (100,000ꢁg; 30 min)
and the pellet was resuspended in buffer A to give a final concen-
tration of 20 mg protein/mL. The microsomal suspension was
stored at ꢀ80 °C prior to the preparation of the solubilized steroid
then suspended in methanol (50 lL); each preparation was spotted
onto HPTLC Keiselgel 60 F254 plates. T, DHT, and a mixture of both
standards was applied to the plates 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 two additional times. DHT was detected by using
phosphomolybdic acid reagent (Sigma–Aldrich); and T, by fluores-
cence (UV lamp; 254 nm). The radioactivity on the plate was
scanned using a Bioscanner AR2000 (Bioscan, Washington D.C.).
The zones that showed chromatographic behavior identical to that
of the standards (retention factor, Rf) were quantified as T or DHT.
The DHT transformation yields were calculated from the lanes, tak-
ing into account the entire radioactivity in the TLC, and were plot-
ted 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.
5a-R type 1.
The solubilization of 5a-R type 1 isozyme from the rat liver
microsomes was carried out according to Levy et al.
2.2.3. Cytosol of rat prostate as source of AR
The prostate was extirpated from each of 50 adult rats (8-
month old; 500 g). In this study, we used rats because the prostate
gland is larger than in the hamster; there is no difference in the
binding activity of the AR present in the cytosol of rats and
hamsters.17
The 5a-R type 1 activity was calculated from the percentage of
The prostates of the rats were blotted, weighed, and soaked in
ice-cold TEMD (40 mM tris-HCl, 3 mM EDTA and 20 mM sodium
molybdate, 0.5 mM DTT, 10% glycerol; pH 8) prior to use. Unless
otherwise specified, all procedures were carried out at 0 °C on a
bed of chopped ice. Tissues were homogenized in one volume of
the labeled DHT that had been formed, taking into consideration
the recovery, blank values, specific activity of [3H] T, and the ratio
of added [3H] T to unlabeled T. The 5
a-R type 2 activity was calcu-
lated, taking into consideration the recovery, blank values, and the
specific activity of [3H] T.
TEMD plus protease inhibitors (2 mM PMSF, 10 lg antipain/mL,
The Rf of the T standard was 0.56. The radioactive zone that had
chromatographic behavior identical to that of the standard T corre-
sponded to 70% of the radioactivity accounted for on the plate. The
radioactivity contained in the zone of the experimental chromato-
gram, which had an Rf identical to that of the DHT standard (Rf:
0.67), was identified as the transformed DHT; it corresponded to
20–27% of total radioactivity accounted for in the TLC. This result
5 mM leupeptin)18 with a tissue homogenizer immersed in a bed
of chopped ice. Homogenates were centrifuged (140,000ꢁg;
60 min)19 in the SW 60 Ti rotor. The final protein concentration
of the supernatant containing the cytosolic fraction, as determined
by the Bradford method, was 6 mg protein/200 lL. This superna-
tant was stored at ꢀ70 °C until its use.
Please cite this article in press as: Bratoeff, E.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.08.019

6
E. Bratoeff et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
was considered to be 100% of the activity of 5
a-R (types 1 or 2) for
200 lL sesame oil, together with 1 mg T/kg (BW), was adminis-
the development of inhibition plots. Unmodified [3H]T, identified
(Rf: 0.56) from control incubations (i.e., those that had not
contained tissue), had identical chromatographic behavior to those
of non-labeled standard T.
tered by SC 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 SC with
200 lL sesame oil, the second group with 1 mg T/kg (BW), and
The radioactivity contained in the zone corresponding to
DHT standard (Rf: 0.67) of the control chromatogram was 1%
of the total radioactivity accounted for on the plate and was
considered as an error; it was subtracted from the experimental
chromatograms.
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 the intact control. On the seventh day, the animals
were sacrificed with CO2. The diameters of the flank organs were
measured using a vernier caliper; the prostate and seminal vesicles
of each hamster were dissected and weighed.20
2.3.1.1. Effect of steroid derivatives on activity of 5
a
-reductase
Two separate experiments were performed for each group of
steroid-treated hamsters. The results were analyzed by using
one-way analysis of variance and Dunnett’s method to compare
means, using JMP IN 5.1 software (JMP, Statistical Discovery, Cary,
NC, USA).
types 1 and 2.
activity of 5
The effect of steroids 3a–e, 6, 4a–i and 7 on the
-reductase type 1 or 2 was determined in the same
a
conditions in vitro as described in Section 2.3.1, but in the presence
of six different concentrations (1 ꢁ 10ꢀ11
,
1 ꢁ 10ꢀ10 M, . . .,
1 ꢁ 10ꢀ3 M) of each of the 3a–e, 6, 4a–i and 7 derivatives. Extrac-
tion and purification of the DHT formed from these incubations
were carried out as described in 2.3.1.
2.3.3. Cytotoxicity assay
2.3.3.1. Prostate cancer cell line (PC-3).
PC-3 human pros-
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 (Section 2.3.1). DHT
transformations in presence of 3a–e, 6, 4a–i and 7 were calculated
from the lanes, taking into account the entire radioactivity in the
plate, and were plotted using SigmaPlot 12 software for inhibition
curves.
tate cancer cell line was supplied by the National Cancer Institute
(USA). The ketoconazol used as a reference in this experiment was
purchased from Sigma Life Science.24 The human tumor cytotoxic-
ity of the synthesized compounds (3a–e, 4a–i, 6 and 7) was deter-
mined using the protein-binding dye sulforhodamine B (SRB) in
microculture assay to measure cell growth. The cell lines were cul-
tured in RPMI-1640 medium supplemented with 10% fetal bovine
serum, 2 mM
L
-glutamine, 100 units/mL penicillin G sodium,
g/mL amphotericin B
2.3.1.2. Determination of the 50% inhibitory concentration of
100 g/mL streptomycin sulfate, and 0.25 l
l
steroids 3a–e, 6, 4a–i and 7 on the activities of 5
types 1 and 2. To determine the 50% inhibitory concentration
(IC50) of steroids 3a–e, 6, 4a–i and 7 on the activities of 5 -reduc-
tase types 1 and 2, the inhibition plots obtained in Section 2.3.1.1
were analyzed with SigmaPlot 12 software.
a
-reductase
(Gibco) and 1% nonessential amino acids (Gibco). They were main-
tained at 37 °C in humidified atmosphere with 5% CO2. The viabil-
ity of the cells used in the experiments exceeds 95% as determined
with trypan blue. The cells were removed from the tissue culture
flask and diluted with fresh media. Of this cell suspension,
a
100 lL containing 5000 or 10000 cells per well (depending on
2.3.1.3. AR competitive binding assay.
For the AR competi-
the duplication time), were pipetted into microtiter plates (Costar)
and the material was incubated at 37 °C for 24 h in a 5% CO2 atmo-
sphere. Additionally, a plate was prepared with culture medium
only and it was incubated for 1 h. Subsequently, 100 lL of a solu-
tion of the test compounds obtained by diluting the stocks were
tive binding studies, a series of tubes containing 1 nM [3H] MIB
plus a range of increasing concentrations (1 ꢁ 10ꢀ10, 1 ꢁ 10ꢀ9 M,
. . ., 1 ꢁ 10ꢀ7 M) of cold MIB or of one of the steroids 3a–e, 6, 4a–
i and 7 (Fig. 1) in ethanol or acetone, or in the absence of compet-
itor were prepared.20
added to each well. The cultures were exposed for 48 h to the syn-
To prevent the interaction of MIB with either glucocorticoid or
PR receptors that could be present in the cytosol from rat prostates
200 nM triamcinolone (Sigma–Aldrich) in ethanol was added to
the incubation mixtures.23 For each preparation, the solvent was
removed under vacuum and the cytosol containing AR (5 mg pro-
thesized compounds at concentration of 50
tion period, the cells were fixed to the plastic substrate by the
addition of 50 L of cold 50% aqueous trichloroacetic acid. The
plates were incubated at 4 °C for 1 h, washed with tap water and
air-dried. The trichloroacetic-acid-fixed cells were stained by the
addition of 0.4% SRB. Free SRB solution was removed by washing
with 1% aqueous acetic acid. The plates were air dried, and the
bound dye was solubilized by the addition of 10 mM of unbuffered
lM; after the incuba-
l
tein/200lL), together with 300 lL of TEM (pH 8) containing prote-
ase inhibitors, were placed into an individual tube. Tubes were
incubated (18 h; 4 °C); then the bound MIB-AR was separated from
free radiolabeled MIB using hydroxyapatite (Bio-Rad, Mexico, City,
Mexico), as previously described.23 The ethanolic fraction (0.8 mL)
obtained by this method was added to Ultima Gold scintillation
liquid (10 mL) (Packard Instruments, Mexico City) and counted in
a Tri Carb 2100 TR scintillation counter (Packard Instruments).
The IC50 value of each compound was calculated according to
the plots of concentration versus percentage of binding using Sig-
maPlot 12. Relative binding affinity (RBA) of 3a–e, 6, 4a–i and 7
values for AR was calculated from the following equation:
Tris base (100 lL). The plates were placed on a vortex for 5 min,
and absorption at 515 nm was determined using an ELISA plate
reader (Bio-Tek Instruments). Percentage cell growth inhibition
was calculated according to the following expression:
Cell growth inhibition ð%Þ ¼ 100 ꢀ ðsample absorptionÞ ꢁ 100
ðvehicle absorptionÞ
2.3.3.2. Isolation and culture of primary peritoneal macro-
phages.
Isolation and culture of primary peritoneal macro-
RBA ¼ IC50 of ½3Hꢂ labeled reference compound ꢁ 100
phages were conducted as described elsewhere.25 Swiss female
mice, 25–30 g, were treated in accordance with the Animal Care
and Use Committee (Mexican Standard NOM-062-Z00-1999). Mice
IC50 of inhibitor
were injected intraperitoneally with 1 mL of 3% (wt volꢀ1
)
2.3.2. Experiment in vivo
2.3.2.1. Effect of steroid derivatives on prostate of castrated
thioglycollate 3 days before harvesting. Peritoneal exudate cells
were harvested, washed and suspended in DMEM. These cells were
seeded into 48 well plates (Becton Dickinson, Oxnard, CA, USA) at a
hamsters.
For six consecutive days, each of the steroid deriv-
atives 3a–e, 6, 4a–i and 7 (2 mg/kg body weight (BW)) dissolved in
Please cite this article in press as: Bratoeff, E.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.08.019

E. Bratoeff et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
density of 1 ꢁ 106 cells mlꢀ1, and then incubated for 2 h at 37 °C in
a 5% carbon dioxide incubator. Non-adherent cells were washed off
and cultured in DMEM supplemented with 10% FCS. Cell viability
was determined by the MTT colorimetric assay. Briefly, 10 mL
MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,3-diphenyltetrazolium bro-
mide) was added to the medium after 48 h incubation with the test
samples. After 4 h, the medium was removed and DMSO was
added to dissolve the formazan solution produced in the cells.
The optical density of the formazan solution was measured with
a microplated reader at 414 nm.25
were isolated in crystalline form and the melting points showed
a maximum of one degree difference.
3.2. Activity of compounds 3a–e, 6, 4a–i and 7 as Inhibitors of
5a-R1 and 2
The biological activity of compounds 2a–i and 3f–i was previ-
ously reported.8,26
Figure 4 shows the IC50 values of 3a–e, 6, 4a–i and 7 required to
inhibit 50% of the activity of 5a-reductase isoenzymes 1 and 2.
These results indicated that the steroidal derivatives of the
17-chlorine series (3a–e and 6) substantially inhibited the enzyme
3. Results
5a-reductase type 2. On the other hand, the C-17 imidazole
series (4a–i) failed to inhibit this enzyme. Surprisingly these
3.1. Chemistry
compounds inhibited the 5
lower degree.
a-reductase type 1 enzyme, albeit to a
In this synthesis for the preparation of the final compounds 4a–
e (cycloalkyl aliphatic esters; 4f–i (aromatic esters) all having a
formyl group at C-16, C-16 double bond and imidazole ring at C-
17 (compound 7, 3-acetoxyderivative was used as a reference),
we used the commercially available dehydroepiandrosterone 1.
This steroid was treated with the corresponding acid, dicyclohexyl-
carbodiimide and 4-dimethylaminopyridine (Steglich esterifica-
tion) to form the desired esters 2a–i. These compounds were
treated with phosphorus oxychloride in dimethylformamide and
chloroform (Vilsmeier reaction) to form the 16-formyl-17-chloro-
3b-acyloxy compounds 3a–i with yields ranging from 59% to
82%. The next step in this synthesis involved nucleophilic substitu-
tion of the 17-chlorine atom with imidazole. This reaction was car-
ried out with the 17-chloro-l6-formyl-3b ester derivatives 4a–i,
imidazole and potassium carbonate. The desired compounds were
obtained with a yield ranging from 80% to 92%. All compounds
3.3. Competitive binding of compounds 3a–e, 6, 4a–i and 7 to AR
In the competitive binding assays (Fig. 3), non-labeled MIB
competed with the labeled MIB for the androgen receptor and
exhibited an IC50 value of 1. However, steroids 3a–i, 4a–i, 6 and
7 did not inhibit the labeled MIB binding to the androgen receptor.
3.4. Weight of the prostate gland
At sacrifice, there were statistically significant (p <0.05) reduc-
tions in the weights of the prostates and seminal vesicles in com-
parison to glands from intact hamsters (data not shown). However,
treatment with 1 mg/kg (BW) of T significantly reversed the effect
of castration on these glands (Fig. 4).
Figure 4. Biological activity of several compounds, the IC50 value is the concentration of the corresponding compound required to inhibit 50% of the activity of 5
isoenzymes as well as AR binding assay. Percentage of PC-3 and macrophages growth inhibition at 50 M. NA: non active compound. ND: non determined.
a-reductase
l
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8
E. Bratoeff et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
F and the steroids 3a–e, 4a–i and 7, when administered
skeleton confers 5a-R1 inhibitory activity. On the other hand, it
together with T, significantly decreased the weight of the prostate
(p <0.05); Surprisingly, compound 4d, having the same chemical
structure (the only difference is the cyclohexyl group at C-3), also
showed high activity compared to that of finasteride (Fig. 4).
is evident that the structure of the ester function at C-3 is very
important for the inhibition of this enzyme, since steroid 7 did
not inhibit the 5
a-R type 1. This is supported by the fact that com-
pounds 4a–i showed inhibitory activity only for the 5
a-R1 enzyme.
The advantage of having different specific inhibitors for each
enzyme is that it allows the possibility of better treatments with
fewer side effects than other approved molecules.
3.5. Cytotoxic activity: effect of the synthesized compounds on
PC-3 cell lines and peritoneal mouse macrophages
Apparently the imidazole substituent at C-17 on the androstane
The effect of the synthesized compounds (3a–e, 4a–i, 6 and 7)
on the growth of the PC-3 cancer cell line was determined using
the SRB assay method. These values for cell growth inhibition
and the results of the effect of the steroidal derivatives
(3a–e, 4a–i, 6 and 7) on the peritoneal mouse macrophages
(non-cancerous cell lines) are shown in Figure 4. Ketoconazole
and finasteride were used as reference compounds;26,27 finasteride
inhibited neither the growth of PC-3 line cells nor of the macro-
phages; however, ketoconazole hindered this cell proliferation
in PC-3 line cells.
Compounds 4a, 4f and 4h showed 100% inhibition for the
growth of prostate cancer cell line PC-3, with compound 4g
having an antiproliferative effect of 98.2% at 50 lM. Otherwise,
these compounds did not show the same effect for the PC-3
cancer cell line compared to that of non-cancerous cell lines
skeleton blocks complex formation with the active site of the 5
type 2.
a-R
The high cytotoxic activity of steroidal derivatives 4a, 4f–h
observed on PC-3 cell lines could be explained by considering the
steric effect of the ester moiety at C-3. Compound 4a, having a pla-
nar cyclopropyl group, shows 100% antiproliferative activity. Ste-
roids 4b–e, having bulky non planar ester moieties, lack any
effect. This is confirmed by the activity of compounds 4f–h having
a planar aromatic ester function, thus showing high cancer cell
growth inhibition rates of 100%, 98.2% and 100%, respectively.
Compound 4i, with a bulky iodine atom on the phenyl ring, lacks
any antiproliferative activity. It is important to note that these
compounds showed selectivity for the inhibition of cancer cell
growth compared to non-cancerous cell lines. The mechanism of
action of these steroids with this cell line is presently being
studied.
(peritoneal mouse macrophages) at 50
selectivity (Fig. 4).
lM, thus showing higher
The in vivo experiments demonstrated that steroids 3b (5
a-R2
inhibitor), 4d, 4f, 4g (5 -R1 inhibitors) and 7 were also capable of
a
inducing a decrease in the weight of the prostate gland. Compound
7 showed activity in vivo, which indicates that the methyl group in
the ester function probably plays a role in in vivo activity on other
4. Discussion
In this paper, we report the synthesis and biological activity of
several steroidal derivatives (3a–e, 4a–i, 6 and 7). Biological evalu-
ation of compounds 3f–i was not included in this paper; the results
are published in Ref. 8. Compounds 3a–e and 6, having a chlorine
atom at C-17 and a conjugated formyl group at C-16, exhibited an
inhibitory effect for 5a-R2 enzyme, with compounds 3a and d hav-
ing higher activity than that of finasteride. When the chlorine atom
at C-17 was replaced with an imidazole group, these steroidal
derivatives lost their inhibitory effect on the 5
targets than 5a-R and AR.
5. Conclusion
In conclusion, the results of this study demonstrated that the
steroidal derivatives having a chlorine atom at C-17 (3a–e and 6)
inhibited the enzyme 5a-R2. Surprisingly, when the chlorine atom
was replaced with an imidazole group (4a–i), these compounds
lost their inhibitory activity for this isoenzyme but showed an
inhibitory effect for the 5a-R1 enzyme. Of the compounds studied
in this paper, only 4a, 4f–h exhibited high antiproliferative activity
for PC-3 prostate cancer cell lines as compared to ketoconazole. In
spite of the fact that all compounds evaluated in vitro showed a
biological effect, the in vivo studies indicated that only compounds
4d and 7 exhibited a high antiandrogenic effect.
As a consequence of these results, this study could contribute to
better understanding of the design and synthesis of more active
molecules for the treatment of BPH and prostate cancer.
a
-R2 enzyme and
surprisingly acquired an inhibitory effect on the 5
a-R1 enzyme.
Apparently the rigidity of the imidazole ring does not allow the for-
mation of the complex with the reactive side of the type 2 enzyme
and as a consequence, no biological activity was observed.
It is a well known fact that the inhibitory effect of 5a-R2
enzyme consists of a Michael type 1,4 addition of the nucleophilic
part of the amino acids such as lysine, cysteine and serine on the
double bond conjugated with the carbonyl group,26–28 thus form-
ing an irreversible adduct, and as a result of this addition, an inhi-
bition of the enzyme 5a-R2 takes place. This explains the much
higher activity of the C-17 chlorine series compared to the C-17
imidazole derivatives. The chlorine atom, being very electronega-
tive, polarizes the C-17 carbon atom, thus creating a partial posi-
tive charge and makes this carbon atom more electrophilic. As a
consequence of this enhanced electrophilicity, the nucleophilic
amino acids add in a 1,4 type addition and inhibit the enzyme. In
the imidazole series 4a–i, the imidazole group cannot form a par-
tial positive charge on the C-17 atom, a Michael-type addition does
Acknowledgements
We would like to thank CONACYT project No 165049 and
DGAPA project IN 211312 (UNAM) for their generous financial
support.
References and notes
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important to add that this concept has never been studied with
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with the esterase enzyme and therefore the hydrolysis of the ester
moiety causes a loss their in vivo activity.
a-R2 enzyme inhibition is observed. It is
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