Synthesis and biological evaluation of esters of 16-formyl-17-methoxy-dehydroepiandrosterone derivatives as inhibitors of 5α-reductase type 2

Article abstract of DOI:10.3109/14756366.2015.1103235

In this study, we investigated the in vitro effect of 16-formyl-17-methoxy dehydroepiandrosterone derivatives on the activity of 5α-reductase type 2 (5α-R2) obtained from human prostate. The activity of different concentrations of these derivatives was de

Full text of DOI:10.3109/14756366.2015.1103235

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and biological evaluation of esters of
16-formyl-17-methoxy-dehydroepiandrosterone
derivatives as inhibitors of 5α-reductase type 2
Araceli Sánchez-Márquez, Yazmín Arellano, Eugene Bratoeff, Yvonne Heuze,
Karen Córdova, Gladys Nieves, Juan Soriano & Marisa Cabeza
To cite this article: Araceli Sánchez-Márquez, Yazmín Arellano, Eugene Bratoeff, Yvonne
Heuze, Karen Córdova, Gladys Nieves, Juan Soriano & Marisa Cabeza (2015): Synthesis and
biological evaluation of esters of 16-formyl-17-methoxy-dehydroepiandrosterone derivatives
as inhibitors of 5α-reductase type 2, Journal of Enzyme Inhibition and Medicinal Chemistry,
DOI: 10.3109/14756366.2015.1103235
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1103235
Published online: 02 Nov 2015.
Article views: 10
View supplementary material
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ienz20
Download by: [New York University]
Date: 24 November 2015, At: 07:26

http://informahealthcare.com/enz
ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–7
2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1103235
!
RESEARCH ARTICLE
Synthesis and biological evaluation of esters of
16-formyl-17-methoxy-dehydroepiandrosterone derivatives
as inhibitors of 5a-reductase type 2
1
1,2
2
1
2
2
´
´
´
´
Araceli Sanchez-Marquez , Yazmın Arellano , Eugene Bratoeff , Yvonne Heuze , Karen Cordova , Gladys Nieves ,
Juan Soriano3, and Marisa Cabeza1
1
´
Departamento de Sistemas Biologicos y de Produccion Agrıcola y Animal, Universidad Autonoma Metropolitana-Xochimilco, Mexico, D.F., Mexico,
´
´
´
´
´
2 3
Departamento de Farmacia, Facultad de Quımica, Universidad Nacional Autonoma de Mexico, Mexico, D.F., Mexico, and Departamento de
´
´
´
´
´
´
´
Patologıa, Hospital General de Mexico, Mexico, D.F., Mexico
´
´
Abstract
Keywords
In this study, we investigated the in vitro effect of 16-formyl-17-methoxy dehydroepiandroster-
one derivatives on the activity of 5a-reductase type 2 (5a-R2) obtained from human prostate.
The activity of different concentrations of these derivatives was determined for the conversion
of labelled testosterone to dihydrotestosterone. The results indicated that an aliphatic ester
moiety at the C-3 position of these derivatives increases their in vitro potency as inhibitors of
5a-R2 activity compared to finasterideÕ, which is considered to be a potent inhibitor of 5a-R2.
In this case, the augmentation of the lipophilicity of these dehydroepiandrosterone derivatives
increased their potency as inhibitors of 5a-R2. However, the presence of cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl rings as the cycloaliphatic ester moiety at C-3
of the formyl methoxy dehydroepiandrosterone scaffold did not inhibit the activity of this
enzyme. This may be due to the presence of steric factors between the enzyme and the spatial
structure of these derivatives.
5a-Reductase inhibitors, 5a-reductase
type 2, 16-formyl-17-methoxy
dehydroepiandrosterone ester
derivatives, androgen receptor,
prostate cancer
History
Received 9 June 2015
Revised 8 September 2015
Accepted 17 September 2015
Published online 30 October 2015
Introduction
response genes in recurrent prostate cancer8. This expression is
strongly related to prostate preservation because these genes have
the ability to induce apoptosis of the prostate cells and are less
involved in cell proliferation throughout prostate development9.
Furthermore, the increased levels of intracellular DHT cause
higher cellular propagation and faulty differentiation10. These
facts could be relevant because an overabundance of DHT has
been implicated in the pathogenesis of benign prostatic hyper-
plasia7–10 (http://www-dep.iarc.fr/WHOdb/WHOdb.htm).
The WHO reported a frequency of global prostate and testicular
cancer of 250 000 new cases annually. The WHO also indicated
that in the Unites States alone, the number of prostate cancer
cases per year is 233 159, with a mortality rate of 10.3%. This
mortality rate indicates that an improvement in therapies is
presently still necessary. These numbers have inspired several
research groups to develop new steroidal and non-steroidal
molecules with therapeutic potency to relieve this illness1–5
.
Dihydrotestosterone (DHT, Figure 1) has been associated with
the development of androgen-dependent afflictions such as
prostate cancer, benign prostatic hyperplasia, acne and andro-
gen-dependent baldness. Various studies have demonstrated that
males with a genetic deficiency of the 5a-reductase type 2 (5a-
R2) isozyme have a smaller prostate gland due to a low level of
DHT6,7. Lack of DHT induces imperfect development of external
genitalia and the prostate, although the epididymis, seminal
vesicles and ducts deferens remain normal. However, the external
genitalia of these individuals look similar to those of females;
furthermore, they do not develop baldness or acne7. Studies also
suggest that DHT stimulates the expression of several androgen-
5a-R2 (Figure 1) plays an important role in prostate gland
development because it is responsible for intraprostatic DHT
levels when the physiological serum testosterone (T) concentra-
tion is low11. The evidences reported in the literature indicate that
decreasing of T production during ageing can be a with an
increase in prostate tumour growth (benign and malign)10,11
.
Therefore, 5a-R2 is still an important therapeutic target for the
development of better drugs than those currently available, i.e.
finasterideÕ and dutasterideÕ.
5a-R2 (EC 1.3.99.5) has previously been observed in different
species6, and the product of the reaction catalysed by this enzyme
accumulates in the nuclei of responsive cells and binds to
androgen receptors (ARs)12. These AR complexes interact with
coregulator proteins (coactivators or corepressors) to adapt
transcription of androgen target genes using specific sequences
in the DNA12.
Address for correspondence: Marisa Cabeza, PhD, Departamento de
´
´
Sistemas Biologicos, Universidad Autonoma Metropolitana-Xochimilco,
´
´
Calzada del Hueso # 1100, Mexico, D.F., CP 04960, Mexico. Tel: [011-
52-55] 5483-72-60. Fax: [011-52-55] 5483-72-60.
E-mail: marisa@correo.xoc.uam.mx
In this study, we describe the synthesis and biological
activity of different steroidal derivatives, which are shown in

´ ´
A. Sanchez-Marquez et al.
2
J Enzyme Inhib Med Chem, Early Online: 1–7
Figure 1. Conversion of testosterone (T) to
5a-dihydrotestosterone (DHT). This reaction
is catalysed by the enzyme 5a-reductase,
which is present in the prostate gland6.
Figure 2. Route of synthesis of dehydroepiandrosterone derivatives.
Reagents and conditions: Vilsmeier–Haack reaction: phosphorus oxychloride (POCl3), dimethylformamide (DMF), chloroform (CHCl3), reflux ().
Addition–substitution reaction: sodium methoxyde (NaOMe), methanol (MeOH), nitrogen (N2). Esterification reaction: dicyclohexylcarbodiimide
(DCC), dimethylaminopyridine (DMAP).
Figures 2 and 3. Their biological activities were determined in
vitro using human prostate as a source of 5a-R2.
(1,2,6,7-3H)-Testosterone ([3H] T) with a specific activity
of 95 Ci/mmol and Mibolerone (17a-methyl-3H) ([3H] MIB)
with a specific activity of 70–87 Ci/mmol were provided by
Perkin Elmer Life and Analytical Sciences, respectively (Boston,
MA). Non-labelled T, 5a-dihydrotestosterone and MIB
were supplied by Steraloids (Wilton, NH). Sigma Chemical Co.
Material and methods
Chemical and radioactive materials
Solvents were laboratory grade or better. Melting points were (St. Louis, MO) provided NADPH and Lubrol PX. Finasteride
determined using a Fisher–Johns melting point apparatus and are was obtained by extraction from ProscarÕ (Merck, Sharp &
uncorrected. 1H-NMR and 13C-NMR data were collected on Dohme, Kenilworth, NJ). The tablets were crushed and extracted
Varian Gemini 400 and VRX-300 equipment, respectively. with chloroform, and the solvent was eliminated under vacuum;
Chemical shifts are given in ppm relative to that of Me4Si the crude product was purified by silica gel column chromatog-
(d ¼ 0) in CDCl3 (the abbreviations of signal patterns are as raphy. The melting point of the isolated finasteride (252–254 ꢀC)
follows: s, singlet; d, doublet, t, triplet, m, multiplet). Mass was identical to that reported in the literature. Bio-gel hydroxy-
spectra (MS) were obtained using a Thermo DFS spectrometer by apatite (HAP) was provided by Bio-Rad Laboratories (Hercules,
direct infusion and using FAB ionisation mode.
CA).

DOI: 10.3109/14756366.2015.1103235 Synthesis and biological evaluation of 16-formyl-17-methoxy-dehydroepiandrosterone derivatives
3
Figure 3. Structures, molecular weight, and biological activities of different dehydroepiandrosterone derivatives.
All steroids exhibited more potency (lower IC50 value) than that of the finasteride to inhibit 5a-R2 enzyme.
IC50 values: concentration of compound required to inhibit 50% of the activity of 5a-reductase type 2 (5a-R2); RBA: relative binding activity to
androgen receptor (AR).
Synthesis of the steroidal derivatives
(305 mg, 2.50 mmol) in chloroform (15 mL) were added to the
corresponding carboxylic acid derivative (3.50 mmol). The
resulting solution was stirred at room temperature for 1 h. Ethyl
acetate (20 mL) was added, and the precipitated dicyclohexylurea
was filtered. Then, 20 mL of chloroform was added to the organic
phase, and the solution was washed with 10% aqueous hydro-
chloric acid (H2O: HCl 7:3, 40 mL ꢁ 6), 5% aqueous solution of
sodium bicarbonate (twice) and then water (once). The mixture
was dried over anhydrous sodium sulphate, and the solvent was
removed under vacuum. The crude ester was purified by flash
column chromatography (FCC, FlorisilÕ 60–100 hexane: AcOEt
85:15).
General procedure for the preparation of 17-chloro-16-formy-
landrost-5,16-dien-3ꢀ-yl acetate2
17-chloro-16-formylandrost-5,16-dien-3b-yl acetate (Figure 2)
1
(2). Yield: 86%; m.p. 135–136 ꢀC, H NMR (CDCl3): d 0.99 (s,
H-19, 3H), 1.06 (s, H-18, 3H), 2.03 (s, OCO-CH3, 3H), 4.60 (m,
H-3, 1H), 5.39 (d, J ¼ 5.6 Hz, H-6, 1H), 10.09 (s, -CHO, 1H); 13
C
NMR (CDCl3): d 14.96 (C-19), 19.20 (CH3- acetoxy), 21.39 (C-
18), 73.65 (C-3), 121.82 (C-6), 136.42 (C-16), 139.99 (C-5),
162.24 (C-17-Cl), 170.47 (C¼O ester); MS (FAB, m/z): 373
(M + 1, 100%).
Procedure for the preparation of 16-formyl-17-methoxyan-
drost-5,16-dien-3b-ol (4). To a solution of 17-chloro-16-formylan-
drost-5,16-dien-3b-yl acetate (Figure 2) (2) (200 mg, 0.53 mmol) in
dimethylformamide under nitrogen atmosphere, 0.5 M sodium
methoxyde in methanol (1.3 mL, 1.2 meq) was added, and the
mixture was stirred for 30 min at room temperature. The reaction
mixture was then added into 20 mL of ice, and the solid obtained was
filtered by vacuum and washed three times with cold water13. The
yellow crude product was purified by flash column chromatography
(FCC, FlorisilÕ 60–100 hexane: AcOEt 65:25)13.
16-formyl-17-methoxyandrost-5,16-dien-3b-ol (Figure 2)
(4). Yield: 94%; m.p. 167–169 ꢀC, 1H NMR (CDCl3): d 0.99
(s, H-19, 3H), 1.06 (s, H-18, 3H), 2.03 (s, OCO-CH3, 3H), 4.60
(m, H-3, 1H), 5.39 (d, J ¼ 5.6 Hz, H-6, 1H); 13C NMR (CDCl3): d
14.96 (C-19), 19.20 (CH3- acetoxy), 21.39 (C-18), 73.65 (C-3),
121.82 (C-6), 136.42 (C-16), 139.99 (C-5), 162.24 (C-17-Cl),
170.47 (C ¼ O ester); and MS (FAB, m/z): 373 (M + 1, 100%).
16-formyl-17-methoxyandrost-5,16-dien-3b-yl
acetate
(Figure 2) (5a). Yield: 91%; m.p. 150–152 ꢀC, 1H NMR
(CDCl3): d 1.00 (s, H-18, 3H), 1.05 (s, H-19, 3H), 2.03 (s,
CH3-acetoxy, 3H), 4.12 (s, 17-OCH3, 3H), 4.60 (m, H-3, 1H),
5.40 (d, J ¼ 4.2 Hz, H-6, 1H), 10.09 (s, -CHO, 1H); 13C NMR
(CDCl3): d 15.54 (C-19), 19.19 (C-18), 20.32 (CH3-acetoxy),
62.30 (CH3O), 73.74 (C-3), 116.12 (C-6), 122.13 (C-16), 139.80
(C-5), 170.50 (C ¼ O ester), 182.21 (C-17), 186.40 (CHO); MS
(FAB, m/z): 373 (M + 1, 100%).
16-formyl-17-methoxyandrost-5,16-dien-3b-yl propionate
(Figure 2) (5b). Yield: 48%; m.p. 140–141 ꢀC, 1H NMR
(CDCl3): d 0.91 (t, CH3-propiloxy, 3H), 0.99 (s, H-18, 3H),
1.04 (s, H-19, 3H), 2.30 (t, CH2-propyloxy, 2H), 4.11 (s, 17-
OCH3, 3H), 4.60 (m, H-3, 1H), 5.39 (d, J ¼ 4 Hz, H-6, 1H), 10.08
(s, -CHO, 1H); 13C NMR (CDCl3): d 9.30 (CH3-propyloxy),
19.37 (C-19), 20.49 (C-18), 30.33 (CH2-propyloxy), 62.45
(CH3O), 73.68 (C-3), 116.30 (C-6), 122.24 (C-16), 140.04 (C-
5), 174.09 (C ¼ O ester), 182.21 (C-17), 186.55 (CHO); MS
(FAB, m/z): 373 (M + 1, 100%).
General procedure for the preparation of esters from carboxylic
acid derivatives
16-formyl-17-methoxyandrosta-5,16-dien-3b-yl
(Figure 2) (5c). Yield: 43% (86.0 mg) white solid; m.p. 166–
butyrate
To a solution of 16-formyl-17-methoxyandrost-5,16-dien-3b-ol
(165 mg, 0.50 mmol) (4), DCC (618 mg, 3.0 mmol) and DMAP
1
168 ꢀC, H NMR (CDCl3): d 0.94 (t, CH3-butyroxy, 3H), 0.99

´ ´
A. Sanchez-Marquez et al.
4
J Enzyme Inhib Med Chem, Early Online: 1–7
(s, H-18, 3H), 1.04 (s, H-19, 3H), 1.63 (m, 20-CH2-butyroxy, 2H), 174.24 (C ¼ O ester), 182.03 (C17), 186.40 (CHO); MS (FAB,
2.24 (t, 1-CH2- butyroxy, 2H), 4.11 (s, 17-OCH3, 3H), 4.60 (m, H- m/z): 373 (M + 1, 100%).
3, 1H), 5.39 (d, J ¼ 4.8 Hz, H-6, 1H), 10.09 (s, -CHO, 1H); 13C
16-formyl-17-methoxyandrosta-5,16-dien-3b-yl cyclohex-
NMR (CDCl3): d 13.77 (CH3- butyroxy), 15.70 (2-CH2- anoate (Figure 2) (5i). Yield: 70%; m.p. 175–178 ꢀC, 1H NMR
butyroxy), 18.66 (C-19), 19.36 (C-18), 30.33 (1-CH2- butyroxy), (CDCl3): 1.00 (s, H-18, 3H), 1.05 (s, H-19, 3H), 4.12 (s, 17-
62.44 (CH3O), 73.57 (C-3), 116.30 (C-6), 122.22 (C-16), 140.04 OCH3, 3H), 4.60 (m, H-3, 1H), 5.38 (s, H-6, 1H), 10.09 (s, -CHO,
(C-5), 173.22 (C ¼ O ester), 182.07 (C17), 186.48 (CHO); MS 1H); 13C NMR (CDCl3): d 15.57 (C-19), 19.25 (C-18), 25.45 (C-
(FAB, m/z): 373 (M + 1, 100%).
3, C-50), 25.62 (C40), 25.80 (C-2, C-60), 36.74 (C1), 62.32
16-formyl-17-methoxyandrosta-5,16-dien-3b-yl pentanoate (CH3O), 73.17 (C-3), 116.18 (C-6), 122.05 (C-16), 139.95 (C-5),
(Figure 2) (5d). Yield: 34%; m.p. 195–197 ꢀC, 1H NMR (CDCl3): 175.58 (C ¼ O ester), 181.94 (C17), 186.38 (CHO); MS (FAB,
d 0.92 (t, CH3-pentanoyloxy, 3H), 0.99 (s, H-18, 3H), 1.05 (s, m/z): 373 (M + 1, 100%).
H-19, 3H), 1.39 (m, 3-CH2-pentanoyloxy, 2H), 1.68 (m, 2-CH2-
16-formyl-17-methoxyandrosta-5,16-dien-3b-yl cyclohep-
pentanoyloxy, 2H), 2.41 (t, 1-CH2-pentanoyloxy, 2H), 4.12 (s, tanoate (Figure 2) (5j). Yield: 68%; m.p. 190–193 ꢀC, 1H NMR
17-OCH3, 3H), 3.47 (m, H-3, 1H), 5.39 (d, J ¼ 4.8 Hz, H-6, 1H), (CDCl3): 1.00 (s, H-18, 3H), 1.05 (s, H-19, 3H), 4.12 (s, 17-
10.09 (s, -CHO, 1H); 13C NMR (CDCl3): d 15.71 (CH3- OCH3, 3H), 4.57 (m, H-3, 1H), 5.39 (s, H-6, 1H), 10.10 (s, -CHO,
pentanoyloxy), 19.32 (C-19), 20.42 (C-18), 25.75 (3-CH2- 1H); 13C NMR (CDCl3): d 15.54 (C-19), 19.22 (C-18), 26.36 (C-
pentanoyloxy), 28.35 (2-CH2-pentanoyloxy), 34.08 (1-CH2-pen- 3, C-60), 30.18 (C-4, C-50), 30.67 (C-2, C-70), 45.16 (C1), 62.28
tanoyloxy), 60.18 (CH3O), 62.49 (C-3), 116.26 (C-6), 122.18 (CH3O), 73.16 (C-3), 116.14 (C-6), 122.01 (C-16), 139.93 (C-5),
(C-16), 141.07 (C-5), 156.63 (C ¼ O ester), 182.12 (C17), 186.53 176.48 (C ¼ O ester), 181.96 (C17), 186.34 (CHO); MS (FAB,
(CHO); MS (FAB, m/z): 373 (M + 1, 100%).
m/z): 373 (M + 1, 100%).
16-formyl-17-methoxyandrosta-5,16-dien-3b-yl hexanoate
1
(Figure 2) (5e). Yield: 43%; m.p. 198–199ꢀ, H NMR (CDCl3): Human and animal tissues and procedures
d 0.89 (t, CH3-hexanoyloxy, 3H), 0.99 (s, H-18, 3H), 1.04 (s,
Four hours after a 58-year-old man died from myocardial
H-19, 3H), 1.30 (m, 3-CH2- hexanoyloxy, 2H), 1.35 (m, 4-CH2-
hexanoyloxy, 2H), 1.63 (m, 2-CH2- hexanoyloxy, 2H), 2.26 (t, 1-
CH2- hexanoyloxy, 2H), 4.12 (s, 17-OCH3, 3H), 4.60 (m, H-3,
1H), 5.40 (d, J ¼ 4.8 Hz, H-6, 1H), 10.08 (s, -CHO, 1H); 13C
NMR (CDCl3): d 14.07 (CH3- hexanoyloxy), 15.72 (C-19), 19.38
(C-18), 22.46 (4-CH2- hexanoyloxy), 24.88 (2-CH2- hexanoy-
loxy), 29.84 (3-CH2- hexanoyloxy), 31.44 (1-CH2- hexanoyloxy),
62.48 (CH3O), 73.61 (C-3), 116.30 (C-6), 122.24 (C-16), 140.05
(C-5), 173.47 (C ¼ O ester), 182.31 (C-17), 186.61 (CHO); MS
(FAB, m/z): 373 (M + 1, 100%).
infarction, 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 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) using an IKAÕ A11 basic
tissue mill (IKA Laboratory Equipment, Mexico City, Mexico).
Unless otherwise specified, the following procedures were carried
General procedure for the preparation of esters from alicyclic
carboxylic acids derivatives
ꢀ
out at 4 C.
The same procedure cited below was used; however, the white 5ꢁ-R2 enzyme isolated from human prostate
crude ester was purified by flash column chromatography (FCC,
Human prostate was used in this experiment because this tissue is
FlorisilÕ 60–100 hexane: AcOEt 65:35).
an abundant source of 5a-R2 for the study of the effect(s) of 5a-j,
which were designed for the inhibition of the activity of this
enzyme in humans.
16-formyl-17-methoxyandrosta-5,16-dien-3b-yl cyclopro-
pionate (Figure 2) (5f). Yield: 72%; m.p. 130–132 ꢀC, 1H NMR
(CDCl3): d 0.91 (t, CH3-propiloxy, 3H), 0.99 (s, H-18, 3H), 1.04
(s, H-19, 3H), 2.30 (t, CH2-propyloxy, 2H), 4.11 (s, 17-OCH3,
3H), 4.60 (m, H-3, 1H), 5.39 (d, J ¼ 4 Hz, H-6, 1H), 10.08 (s,
-CHO, 1H); 13C NMR (CDCl3): d 8.17 (C-2, C-3), 12.93 (C1),
19.37 (C-19), 20.49 (C-18), 30.33 (CH2-propyloxy), 62.45
(CH3O), 73.68 (C-3), 116.30 (C-6), 122.24 (C-16), 140.04 (C-
5), 174.09 (C ¼ O ester), 182.21 (C17), 186.55 (CHO); MS (FAB,
m/z): 373 (M + 1, 100%).
Human prostate tissue was homogenised in two volumes of
buffer A using an Ultra-Turrax IKA tissue homogeniser, T18 basic
(Wilmington, NC). The homogenates were centrifuged (1500 ꢁ g;
60 min)10 in a SW 60 Ti rotor (Beckman Instruments, Palo Alto,
CA). The pellets were resuspended in buffer A and stored
at ꢂ70 ꢀC. This suspension had a final concentration of 5 mg
protein/mL, as determined by the Bradford method14, and was
used as the source of 5a-R2 isozyme.
16-formyl-17-methoxyandrosta-5,16-dien-3b-yl cyclobuty-
rate (Figure 2) (5g). Yield: 72%; m.p. 163–165 ꢀC, 1H NMR
(CDCl3): 0.98 (s, H-18, 3H), 1.03 (s, H-19, 3H), 4.11 (s, 17-
Cytosol of rat prostate as source of AR
OCH3, 3H), 4.59 (m, H-3, 1H), 5.38 (d, J ¼ 4.9 Hz, H-6, 1H), Prostates were extirpated from 50 adult rats (8 months old; 500 g).
10.08 (s, -CHO, 1H); 13C NMR (CDCl3): d 15.54 (C-19), 18.32 In this study, we used rats because their prostate gland is large;
(C-3), 19.21 (C-18), 25.19 (C-2, C-40), 33.00 (C1), 62.28 (CH3O), there is no difference in the binding activity of the AR present in
73.36 (C-3), 116.14 (C-6), 122.04 (C-16), 139.91 (C-5), 174.91 the cytosol of rats and humans15.
(C ¼ O ester), 181.94 (C17), 186.33 (CHO); MS (FAB, m/z): 373
The rat prostates were blotted, weighed, and soaked in ice-cold
TEMD (40 mM Tris-HCl, 3 mM EDTA, 20 mM sodium molyb-
(M + 1, 100%).
16-formyl-17-methoxyandrosta-5,16-dien-3b-yl cyclopen- date, 0.5 mM DTT, 10% glycerol; pH 8) prior to use. Unless
tanoate (Figure 2) (5h). Yield: 59%; m.p. 165–168 ꢀC, 1H otherwise specified, all procedures were carried out at 0 ꢀC on a
NMR (CDCl3): 1.00 (s, H-18, 3H), 1.05 (s, H-19, 3H), 4.12 (s, 17- bed of chopped ice. Tissues were homogenised in one volume of
OCH3, 3H), 4.58 (m, H-3, 1H), 5.39 (d, J ¼ 4.9 Hz, H-6, 1H), TEMD plus protease inhibitors (2 mM phenylmethylsulfonyl
10.10 (s, -CHO, 1H); 13C NMR (CDCl3): d 15.57 (C-19), 19.25 fluoride, 10 mg antipain/mL, 5 mM leupeptin)16 using a tissue
(C-18), 25.82 (C-3, C40), 30.01 (C-2, C-50), 44.03 (C10), 62.32 homogeniser immersed in a bed of chopped ice. The homogenates
(CH3O), 73.33 (C-3), 116.14 (C-6), 122.03 (C-16), 139.94 (C-5), were centrifuged (140 000 ꢁ g; 60 min)5 in a SW 60 Ti rotor. The

DOI: 10.3109/14756366.2015.1103235 Synthesis and biological evaluation of 16-formyl-17-methoxy-dehydroepiandrosterone derivatives
5
final protein concentration of the supernatant containing the presence of six different concentrations (1 ꢁ 10ꢂ11, 1 ꢁ 10ꢂ10 M,
cytosolic fraction, as determined by the Bradford method, was . . . 1 ꢁ 10ꢂ3 M) of each of the 5a–j derivatives. Extraction and
6 mg protein/200 mL. This supernatant was stored at ꢂ70 ꢀC until purification of the DHT formed from these incubations were
its use.
carried out as described in ‘‘Experiments in vitro’’ section.
Control tubes (without inhibitors) were prepared with the same
incubation medium and under the same conditions (‘‘Experiments
in vitro’’ section) and were labelled T plus 5a-R2. DHT
transformations in the presence of 5a–j were calculated from
the lanes, taking into account the entire radioactivity in the plate,
and were plotted using SigmaPlot 12 software for inhibition
curves.
Biological activity of steroidal derivatives
Experiments in vitro. The activity of the 5a-R2 was determined
by following the conversion of T to DHT, as previously
described1,5. The reagent mixture (final volume, 1 mL) contained
1 mM DTT in 40 mM sodium phosphate buffer (pH 6.5), 2 nM
[1,2,6,7-3H] T, and 2 mM NADPH. The reaction, which was
carried out in duplicate, was started when this mixture was added
to the enzymatic fraction (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 to be the end point. As a control, this
procedure was carried out without the addition of the enzyme
fraction.
Determination of the 50% inhibitory concentration of steroids
5a-j on the activity of 5ꢁ-R2. To determine the 50% inhibitory
concentration (IC50) of steroids 5a-j on the activity of 5a-R2, the
inhibition plots obtained in ‘‘Experiments in vitro’’ section were
analysed with SigmaPlot 12 software.
AR competitive binding assay. For the AR competitive binding
studies, a series of tubes containing 1 nM [3H] MIB plus a range
of increasing concentrations (1 ꢁ 10ꢂ10, 1 ꢁ 10ꢂ9 M, . . . 1 ꢁ 10ꢂ7
M) of cold MIB or of one of the steroids 5a–j (Figure 1) in
ethanol, acetone or in the absence of competitor were prepared5.
To prevent the interaction of MIB with either glucocorticoid or
PR receptors that may be present in the cytosol from rat prostates,
200 nM triamcinolone (Sigma-Aldrich) in ethanol was added
to the incubation mixtures5. For each preparation, the solvent
was removed under vacuum, and the cytosol containing AR
(5 mg protein/200 mL), together with 300 mL of TEM (pH 8)
containing protease inhibitors, was placed into individual tubes.
The tubes were incubated (18 h; 4 ꢀC), and the bound MIB-AR
was separated from free radiolabeled MIB using HAP (Bio-Rad,
Mexico, D.F., Mexico), as previously described17. The
ethanolic fraction (0.8 mL) obtained by this method was added
to Ultima Gold scintillation liquid (10 mL) (Packard Instruments,
Mexico, D.F., Mexico) and counted in a Tri Carb 2100
TR scintillation counter (Packard Instruments, Meriden, CT).
The IC50 value of each compound was calculated according to
the plots of concentration versus percentage of binding using
SigmaPlot 12. The relative binding affinity (RBA) values of 5a-j
for AR were calculated using the following equation:
To extract and purify the DHT formed by the activity of 5a-
R2, after the individual reaction mixtures had been agitated
(1 min) using a Type 16700 mixer (Barnstead Thermoline,
´
´
Proveedor Cientıfico, Mexico, D.F., Mexico), the dichloro-
methane 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 then suspended in methanol (50 mL); each
preparation was spotted onto HPTLC Keiselgel 60 F254 plates. T,
DHT, and a mixture of both standards were 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 using phosphomolybdic acid reagent (Sigma-
Aldrich), and T was detected by fluorescence (UV lamp; 254 nm).
The radioactivity on the plate was scanned using a Bioscanner
AR2000 (Bioscan, Washington, DC). The zones that showed
chromatographic behaviour identical to that of the standards
(retention factor, Rf) were quantified as T or DHT. The DHT
transformation yields were calculated from the lanes, taking into
account the entire 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.
IC50 ½3Hꢃ labelled reference compound ꢁ 100
RBA ¼
The 5a-R2 activity was calculated, taking into consideration
the recovery, blank values, and the specific activity of [3H] T.
The Rf of the T standard was 0.56. The radioactive zone that
exhibited chromatographic behaviour identical to that of the
standard T corresponded to 70% of the radioactivity in the plate.
The radioactive compound contained in the zone of the experi-
mental chromatogram, 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 the total radioactivity accounted for
in the TLC. This result was considered to be 100% of the activity
of 5a-R2 for 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
behaviour to the non-labelled standard T.
IC50 of inhibitor
IC50: concentration of compound required to inhibit 50% the
activity of 5a-R2; RBA: relative binding activity to AR.
Calculation of log p values. The calculation of the log P
parameter was carried out using the programme ACD/LABS
version 9. This value is calculated on the basis of the molecular
structure of the tested steroid and describes the relationship
between the lipophilicity of the drug, represented by log P, and the
minimum concentration of the drug (log1/c) necessary to produce
biological activity. A higher value of log P indicates a greater
solubility of the compound in the lipoprotein cell membrane with
a corresponding increase in the pharmacological activity. This
relationship is reliable up to a log P of 5.
The radioactivity contained in the zone corresponding to the
DHT standard (Rf: 0.67) of the control chromatogram was 1% of
the total radioactivity accounted for in the plate and was
considered to be an error; it was subtracted from the experimental
chromatograms.
Results
Chemistry
The synthetic strategy for compounds 5a–j is summarised in
Figure 2. The dehydroepiandrosterone derivatives 5a–5j were
synthesised using a previously published procedure with slight
modifications13. The new compounds 5a–j were obtained with
Effect of steroids derivatives on activity of 5ꢁ-R2. The effect of
steroids 5a–j on the activity of 5a-R2 was determined under the
same conditions in vitro as described in section 2.3.3.1 but in the

´ ´
A. Sanchez-Marquez et al.
6
J Enzyme Inhib Med Chem, Early Online: 1–7
Figure 4. Structures, molecular weights, and biological activities of different dehydroepiandrosterone derivatives.
None of these compounds was capable to inhibit the activity of 5a-R2 enzyme.
IC50 values: concentration of compound required to inhibit 50% of the activity of 5a-reductase type 2 (5a-R2); RBA: relative binding activity to
androgen receptor (AR).
moderate yields, and their structures and purities were confirmed Discussion
by spectroscopic and spectrometric methods and melting point
5a–5j steroids were designed to inhibit the activity of 5a-R2 and
(see experimental section).
this effect it can be demonstrated throughout this study.
The yields of the products after the various steps in the
Furthermore it was tried, in order to increase its therapeutic
syntheses were as follows: 86% for derivative 2 resulting from the
value as anti-prostate cancer drug, adding a formyl group at the C-
Vilsmeier–Haack reaction, 94% for the free alcohol resulting from
16 position of these derivatives. This formyl could convert 5a–5j in
2 being treated with sodium methoxyde-methanol in dimethyl-
a potential substrate of transformylase enzymes. These enzymes are
formamide, and 34%–91% for 5a–5j when the 16-formyl-17-
involved in the purine synthesis, which are essential for the cancer
methoxy-3b-ol was reacted with phosphorus oxychloride and
cells division19. Therefore, we hypothesise these steroidal deriva-
dimethylformamide and chloroform.
tives could be inhibitors of the activity of these transformylases20
All compounds were isolated in their crystalline form,
and thus could stop the prostate cancer cell growth21. Nevertheless,
and the melting points showed a maximum difference of two
the formyl group present at the C-16 5a–5j is toxic to the body so
degrees.
that an ester moiety was added in C-3 with the purpose to mask this
formyl group. The pharmacological results obtained in this
Activity of compounds 5a–5j as inhibitors of 5a-R2
experiment demonstrated that the compounds 5a–5j were not
Figure 3 shows the IC50 values of 4 and 5a–d required to inhibit toxic when applied to the hamsters for 6 consecutive days. Future
50% of the activity of 5a-R2. The steroids 4 and 5a–d showed investigation using these derivatives will be needed to determine
higher potency than that of the finasteride (the compound used as their effect on the nucleotide synthesis pathway.
a reference18) to inhibit 5a-R2 enzyme. Compared with 4, 5a, 5c
The lipophilicity that we added to steroids 5a–5d could be also
and 5d, 5b steroid (IC50 ¼ 0.044 0.012 nM) it was the most advantageous for improving the ability of these molecules to cross
potent of them and exhibited an effectiveness 200 times greater through cell membranes. However, it is well known the fact the
than finasteride (IC50 ¼ 8.5 2.5 nM) to inhibit 5a-R2 activity. presence of non-specific esterases in the bloodstream22, thus these
Nevertheless, derivatives 5f–j did not inhibit the activity of this esters derivatives could be hydrolysed by the esterases present in
enzyme (Figure 4).
the blood, and the resultant hydrolysed molecules may contain an
alcohol moiety at C-3, similar to that present in steroid 4. This
possibility was considered in this study, and we demonstrated that
steroid 4 is also an inhibitor of 5a-R2.
Competitive binding of compounds 5a–5j to the AR
In conclusion, since these esters can be regarded as prodrugs,
could be used to mask side effects and toxicity caused by the
formyl group23. Therefore, when these ester groups are
The results of the competitive binding assays (Figures 3–5)
indicated that these dehydroepiandrosterone derivatives (5a–5j)
did not replace labelled MIB of the AR.

DOI: 10.3109/14756366.2015.1103235 Synthesis and biological evaluation of 16-formyl-17-methoxy-dehydroepiandrosterone derivatives
7
Figure 5. Competitive binding of compounds
5a–5j to the androgen receptors (AR)
Unlabelled mibolerone (MIB) competed with
the labelled MIB by the binding to the AR;
however, the novel steroids did not remove
the labelled MIB from the AR. These results
support the idea that these steroids did not
bind to the AR.
10. Bosland MC. Chapter 2: the role of steroid hormones in prostate
carcinogenesis. JNCI Monographs 2000;2000:39–66.
hydrolysed by plasma esterases, the active drug is released
causing the desired response.
11. Raynaud JP. Prostate cancer risk in testosterone-treated men.
Proceedings of the 17th International Symposium of the Journal of
Steroid Biochemistry and Molecular Biology Recent Advances In
Steroid Biochemistry And Molecular Biology (Seefeld, Tyrol); 2006
May 31–June 3;102:261–6; Austria.
Declaration of interest
The authors declare that there are no real or perceived conflicts of
interest arising from intellectual, personal, or financial circum-
stances of this research. Additionally, all authors are aware and
approve of the contents and order of authorship of the manuscript.
This study was supported by grants from Consejo Nacional de
12. Lee HJ, Chang C. Recent advances in androgen receptor action. Cell
Mol Life Sci 2003;60:1613–22.
13. Njar VCO, Klus GT, Brodie AMH. Nucleophilic vinylic ‘‘addition-
elimination’’ substitution reaction of 3-acetoxy-17-chloro-16-for-
mylandrosta-5,16-diene: a novel and general route to 17-substituted
steroids. Part 1. Synthesis of novel 17-azolyl-D16-steroids; inhibitors
of 17a-hydroxylase/17,20-lyase (17a -lyase). Bioorg Med Chem
Lett 1996;6:2777.
´
Ciencia y Tecnologıa (CONACYT project number 165049) and
from Direccion General de Asuntos del Personal Academico
´
´
(DGAPA project number IN 211312).
14. Bradford MM. A rapid and sensitive method for the quantisation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem 1976;72:248–54.
15. Altschul SF, Wootton JC, Gertz EM, et al. Protein database searches
using compositionally adjusted substitution matrices. FEBS J 2005;
272:5101–9.
References
1. Arellano Y, Bratoeff E, Garrido M, et al. New ester derivatives of
dehydroepiandrosterone as 5a-reductase inhibitors. Steroids 2011;
76:1241–6.
´
2. Bratoeff E, Sanchez A, Arellano Y, et al. In vivo and in vitro effect
16. Hendry IWJ, Danzo BJ. Structural conversion of cytosolic steroid
receptors by an age-dependent epididymal protease. J Steroid
Biochem 1985;23:883–93.
of androstene derivatives as 5a-reductase type 1 enzyme inhibitors.
J Enzyme Inhib Med Chem 2013;28:1247–54.
´
3. Garrido M, Cabeza M, Cortes F, et al. Cytotoxic effect of novel
17. Mukherjee A, Kirkovsky LI, Kimura Y, et al. Affinity labelling of
the androgen receptor with nonsteroidal chemoaffinity ligands.
Biochem Pharmacol 1999;58:1259–67.
18. Bull HG, Garcia-Calvo M, Andersson S, et al. Mechanism-based
inhibition of human steroid 5a-reductase by finasteride: enzyme-
catalyzed formation of NADP-dihydrofinasteride, a potent bisub-
strate analog inhibitor. J Am Chem Soc 1996;118:2359–65.
19. Warren MS, Marolewski AE, Benkovic SJ. A rapid screen of
active site mutants in glycinamide ribonucleotide transformylase.
Biochemistry 1996;35:8855–62.
dehydroepiandrosterone derivatives on different cancer cell lines.
Eur J Med Chem 2013;68:301–11.
´
4. Cabeza M, Trejo KV, Gonzalez C, et al. Steroidal 5a-reductase
inhibitors using 4-androstenedione as substrate. J Enzyme Inhib Med
Chem 2011;26:712–19.
´
5. Bratoeff E, Garcıa P, Heuze Y, et al. Molecular interactions of
progesterone derivatives with 5a-reductase types 1 and 2 and
androgen receptors. Steroids 2010;75:499–505.
6. Russell DW, Wilson JD. Steroid 5 alpha-reductase: two genes/two
enzymes. Annu Rev Biochem 1994;63:25–61.
20. Desharnais J, Hwang I, Zhang Y, et al. Design, synthesis and
biological evaluation of 10-CF3CO-DDACTHF analogues and
derivatives as inhibitors of GAR Tfase and the de novo purine
biosynthetic pathway. Bioorg Med Chem 2003;11:4511–21.
21. Christopherson RI, Lyons SD, Wilson PK. Inhibitors of de novo
nucleotide biosynthesis as drugs. Acc Chem Res 2002;35:961–71.
22. Graham LP. Medicinal chemistry. Oxford: Oxford University Press;
2001, 226–53.
7. Imperato-McGinley J, Zhu YS. Androgens and male physiology the
syndrome of 5alpha-reductase-2 deficiency. Mol Cell Endocrinol
2002;198:51–9.
8. Dadras SS, Cai X, Abasolo I, Wang Z. Inhibition of 5alpha-
reductase in rat prostate reveals differential regulation of androgen-
response gene expression by testosterone and dihydrotestosterone.
Gene Expr 2001;9:183–94.
9. Wright AS, Douglas RC, Thomas LN, et al. Androgen-induced
regrowth in the castrated rat ventral prostate: role of 5alpha- 23. Heck HD, Casanova M, Starr TB. Formaldehyde toxicity-new
reductase. Endocrinology 1999;140:4509–15. understanding. Crit Rev Toxicol 1990;20:397–426.
Supplementary material available online
Supplementary Figures S1–12