Synthesis of new derivatives of 21-imidazolyl-16-dehydropregnenolone as inhibitors of 5α-reductase 2 and with cytotoxic activity in cancer cells

Article abstract of DOI:10.1016/j.bmc.2017.01.018

The aim of this study was to synthesize several 16-dehydropregnenolone derivatives containing an imidazole ring at C-21 and a different ester moiety at C-3 as inhibitors of 5α-reductase 1 and 2 isoenzymes. Their binding capacity to the androgen receptor (AR) was also studied. Additionally, we evaluated their pharmacological effect in a castrated hamster model and their cytotoxic activity on a panel of cancer cells (PC-3, MCF7, SK-LU-1). The results showed that only the derivatives with an alicyclic ester at C-3 showed 5α-R2 enzyme inhibition activity, the most potent of them being 21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-cyclohexanecarboxylate with an IC₅₀of 29?nM. This is important because prostatic benign hyperplasia is directly associated with the presence of 5α-R2. However, all the derivatives failed to inhibit 5α-R1 or bind to the AR. These alicyclic ester derivatives decreased the weight and size of androgen-dependent glands in the hamster, indicating they are very active in vivo and are not toxic. In addition, the 21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-acetate derivative showed the highest cytotoxic activity on the three cancer cell lines studied. It is therefore important in the synthesis of steroidal compounds to consider the size of the ester moiety at C-3 of the steroid skeleton, which is key in obtaining biological activity, as observed in this experiment.

Full text of DOI:10.1016/j.bmc.2017.01.018

Accepted Manuscript
Synthesis of new derivatives of 21-imidazolyl-16-dehydropregnenolone as in-
hibitors of 5α-reductase 2 and with cytotoxic activity in cancer cells
Aylin Viviana Silva-Ortiz, Eugene Bratoeff, Teresa Ramírez-Apan, Yvonne
Heuze, Juan Soriano, Isabel Moreno, Marisol Bravo, Lucero Bautista, Marisa
Cabeza
PII:
S0968-0896(16)31392-X
http://dx.doi.org/10.1016/j.bmc.2017.01.018
BMC 13497
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 December 2016
10 January 2017
13 January 2017
Please cite this article as: Silva-Ortiz, A.V., Bratoeff, E., Ramírez-Apan, T., Heuze, Y., Soriano, J., Moreno, I.,
Bravo, M., Bautista, L., Cabeza, M., Synthesis of new derivatives of 21-imidazolyl-16-dehydropregnenolone as
inhibitors of 5α-reductase 2 and with cytotoxic activity in cancer cells, Bioorganic & Medicinal Chemistry (2017),
doi: http://dx.doi.org/10.1016/j.bmc.2017.01.018
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1
Synthesis of new derivatives of 21-imidazolyl-16-dehydropregnenolone as inhibitors of
5α-reductase 2 and with cytotoxic activity in cancer cells
Aylin Viviana Silva-Ortiza, Eugene Bratoeff†, Teresa Ramírez-Apanb, Yvonne Heuzec,
Juan Sorianod, Isabel Morenoc, Marisol Bravoc , Lucero Bautistac, and Marisa Cabezac*
ꝉ RIP
a Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de
México, Av. Universidad 3000, Copilco Universidad, Coyoacán, 04510 Ciudad de México,
CDMX México
b Instituto de Quimica, Universidad Nacional Autónoma de México, Av. Universidad 3000,
Copilco Universidad, Coyoacán, 04510 Ciudad de México, CDMX México
c Departamento de Sistemas Biológicos y de Producción Agrícola y Animal, Universidad
Autónoma Metropolitana-Xochimilco, Calzada del Hueso 1100. Col. Villa Quietud, 04960
Ciudad de México. CDMX México
d
Departamento de Patología, Hospital General de México, Dr. Balmis 148, México,
CDMX
Pharmacy Department, Faculty of Chemistry, National Autonomous University of Mexico,
Avenida Universidad 3000, Ciudad de México, CDMX, 04510, México.
ASO: aylinsilva@hotmail.es
TRA: mtrapan@unam.mx
YH: ymheuze@gmail.com
JS: juansorianor@hotmail.com
IM: catrina_04@hotmail.com
MB: braru27@gmail.com
LB: guci_luz01@hotmail.com
MC: marisa@correo.xoc.uam.mx
*Address correspondence to:
Marisa Cabeza Ph.D.
Departamento de Sistemas Biológicos

2
Universidad Autónoma Metropolitana-Xochimilco
Calzada Del Hueso No. 1100
México, CDMX, C.P. 04960, México
Phone: [011-52-55] 5483-72-60
Fax: [011-52-55] 5483-72-60
E-mail: marisa@correo.xoc.uam.mx

3
Abstract
The aim of this study was to synthesize several 16-dehydropregnenolone derivatives
containing an imidazole ring at C-21 and a different ester moiety at C-3 as inhibitors of 5-
reductase 1 and 2 isoenzymes. Their binding capacity to the androgen receptor (AR) was
also studied.
Additionally, we evaluated their pharmacological effect in a castrated hamster
model and their cytotoxic activity on a panel of cancer cells (PC-3, MCF7, SK-LU-1).
The results showed that only the derivatives with an alicyclic ester at C-3 showed
5-R2 enzyme inhibition activity, the most potent of them being 21-(1H-imidazol-1-yl)-20-
oxopregna-5,16-dien-3β-yl-cyclohexanecarboxylate with an IC50 of 29 nM. This is
important because prostatic benign hyperplasia is directly associated with the presence of
5-R2. However, all the derivatives failed to inhibit 5α-R1 or bind to the AR. These
alicyclic ester derivatives decreased the weight and size of androgen-dependent glands in
the hamster, indicating they are very active in vivo and are not toxic. In addition, the 21-
(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-acetate derivative showed the highest
cytotoxic activity on the three cancer cell lines studied.
It is therefore important in the synthesis of steroidal compounds to consider the size
of the ester moiety at C-3 of the steroid skeleton, which is key in obtaining biological
activity, as observed in this experiment.

4
Key words
16-dehydropregnenolone acetate derivatives, alicyclic ester moiety at C-3 of the steroidal
skeleton, 5--reductase, cytotoxic activity, cell lines: PC-3, MCF7, SK-LU-1.

5
1.
Introduction
Prostate cancer (PC) and benign prostatic hyperplasia (BPH) are two of the major
conditions that currently affect the male population over 45 years old. PC is the second
leading cause of cancer death, and BPH is the second cause of surgery in some countries;
for example the United States [1]. Both diseases are directly related to an excess of
androgens in the body.
The main androgens produced by the body are testosterone (T) and
dihydrotestosterone (DHT). Testosterone, the best known androgen, is converted to DHT, a
more potent androgen, by the action of 5α-reductase isoenzymes. The activity of these
isoenzymes in androgen-dependent tissues has long been known; DHT is responsible for
sending a signal to induce prostate growth, which in pathological conditions produces the
development of both diseases [2]. The three 5α-reductase isoenzymes are expressed in
different human tissues [3]. Type 1 isoenzyme (5-R1) is expressed in prostate epithelial
cells and is directly related to prostate cancer. Type 2 (5-R2) is associated with
development of benign prostatic hyperplasia [4] and is found mostly in the prostate,
seminal vesicles and genital skin. Type 3 (5-R3) is related to hormone-refractory prostate
cancer (HRPC) and is found in the brain and pancreas [5-6]. Unfortunately, these three
isoenzymes have not been purified and crystallized, due to their unstable nature, and as a
result, the 5-R inhibitors have been designed based on structure-activity studies. Two of
these compounds are finasteride and dutasteride. Both are 4-azasteroids currently used in
CP and BPH with undesirable secondary antiandrogen effects such as impotence, muscle
growth impairment, and gynecomastia [7]. Also, their use for long periods can have a
potential hepatoxic effect [8]. Many different compounds are currently being studied with

6
the aim of decreasing these unwanted effects and enhancing the therapeutic effect for a
successful prostate cancer treatment.
Nowadays a variety of many drugs used in the treatment of different steroid-
dependent and independent diseases, have a heterocyclic group in their structure, for
example the aminoglutethimide and its derivatives; esters of 4-pyridineacetic acid; bis-
chlorophenyl-pyrimidine analogues; as well as imidazole and triazole derivatives, such as,
liarozole, fadrozole, CGS 18320 B, vorozole and CGS 20267 [9-10]. In particular, various
biological studies have demonstrated that some pregnane 21 and 16-derivatives have
interesting activity, such as an antiproliferative effect, on different cancer cell lines and 5α-
reductase inhibition. [9-21].
On this basis, we synthesized and determined the biological activity of a novel
series of 21-(1H-imidazole)-5-16-pregnadiene-20-one-2-yl derivatives (8a−j; see below) as
inhibitors of type 1 and 2 5α-reductase isoenzymes. We determined their androgen receptor
(AR) binding capacity and their antiproliferative effect on the following cancer cell lines:
PC-3 (prostate), MCF7 (breast) and SK-LU-1 (lung). The pharmacological effect was
evaluated in a hamster model.
These experiments were designed to complement a previously reported study of a
molecule with a triazole group at C-21 on a pregnane skeleton [12, 15] and to compare the
importance of the azole group on the biological activity.

7
2.
Material and methods
2.1. Reagents
Reagents and solvents were purchased from commercial sources and were used
without further purification. Melting points (uncorrected) were determined on a Fisher
Johns melting point apparatus. Infrared spectra (IR) were recorded on a Perkin-Elmer 200
1
spectrometer (Perkin-Elmer Life and Analytical Science, Shelton CT, USA). H and 13C
NMR were taken on a Varian VRX-400 spectrometer (MR resources NC, USA) operating
at 400 (1H) and 100 (13C) MHz with tetramethylsilane (TMS) as internal standard (δ=0) in
CDCl3 (the abbreviations of signal patterns are as follows: s, singlet; d, doublet, t, triplet; m,
multiplet). High resolution mass spectra (HRMS) were obtained with a Thermo DFS
spectrometer by direct infusion and using the FAB+ ionization mode (Thermo Fisher
Scientific, San Jose, CA USA).
(1,2,6,7-3H) Testosterone [3H] T specific activity: 95 Ci/mmol and Mibolerone
(17a-methyl-3H) [3H] MIB 10 specific activity 70–87 Ci/mmol were provided by Perkin
Elmer Life and Analytical Sciences (Boston, MA).
Radioinert T, 5α-DHT, and MIB were supplied by Steraloids (Wilton, NH, USA).
Sigma Chemical Co. (St. Louis, MO) provided NADPH. Finasteride was obtained by
extraction from Proscar® (Merck, Sharp & Dohme). The tablets were crushed, extracted
with chloroform and the solvent was eliminated in vacuum; the crude product was purified
by silica gel 60 (63–200 µm) column chromatography (Sigma–Aldrich) with ethyl acetate
as eluent. The melting point of the isolated finasteride (252–254 °C) was identical to that
reported in the literature.

8
16-deydropregnenolone acetate and ketoconazole used as a reference compound
were purchased from Sigma Life Sciences.
2.2. Synthesis of 16-dehydropregnenolone derivatives (8a−j )
Steroids 8a−j (Fig. 1) were prepared from the commercially available 16-
dehydropregnenolone acetate (1) with the synthesis of their intermediate compounds being
previously reported by our group. [15] The synthetic pathways for the preparation of 8a–j
are briefly outlined in Fig. 1
Figure. 1. Figure. 1. Reagents and conditions: (i) H2O2, NaOH 4N; (ii) DHP, p-
toluensulphonic acid; CH2Cl2(iii) NaOH, C6H5I(OAc)2, MeOH; (iv) SOCl2, Py, CH2Cl2; (v)
1H-imidazole, Cs2CO3, 80 °C, 5 h; (vi) HCl, CrCl2, CH3COOH, 20 min.; (vii) a) acetic
anhiydride, Py; b-j) R-COOH, DCC, DMAP, CHCl3.

9
Synthesis of 21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3-yl derivatives (8a−j in
Fig. 1)
2.2.1.1.
Preparation of 21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-
acetate (8a in Fig. 1).
The synthesis of this compound was carried out starting with a solution of steroid 7 [15]
(0.1 g, 2.62 mmol) in pyridine (0.4 mL) and acetic anhydride (0.4 mL). This mixture was
stirred at room temperature for 1 h. After, a solution of water and salt was added and the
precipitate formed was filtered. The compound was washed with water 3 times.
Yield 0.10 g (90%), m.p. 105–108 °C, IR (KBr) cm-1: 2937, 1729, 1236 and 1031. 1H NMR
(400 MHz, CDCl3) δ: 1.10 (s, H-18, 3H), 1.30 (s, H-19, 3H), 2.03 (t, ester-H-2′, 3H), 3.90
(m, J = 3.2 Hz, H-3, 1H), 4.65−4.87 (ABq, J= 16.4 Hz, H-21, 2H), 5.35 (d, J = 5.9 Hz, H-
6, 1H), 6.73 (s, J = 6.7, C-16, 1H), 7.04 (s, H-Het, 1H), 7.32 (s, H-Het, 1H), 7.69 (s, H-Het,
1H). 13C NMR (100 MHz, CDCl3) δ: 15.40 (C-18), 20.30 (C-19), 21.0 (C-2′ of ester), 65.60
(C-21), 74.07 (C-3), 121.96 (C-6), 140.03 (C-Het), 140.10 (C-5), 144.21 (C-Het), 147.20
(C-16), 152.04 (C-Het), 156.00 (C-17), 170.67 (ester carbonyl), 199.27 (C-20). HRMS: m/z
[M+H]+ calculated for 422.2569 C26H34N2O3, found 422.2563.
2.2.1.2.
Preparation of 21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-
(8b) to 21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-
propionate
cycloheptanecarboxylate (8j). To a solution of steroid 7 (0. 1 g, 0.262 mmol), DCC (0.110
g, 0.524 mmol) and DMAP (0.064 g, 0.524 mmol) in chloroform (3 mL), the corresponding
acid (0.6 mmol) was added. The resulting solution was stirred at room temperature for 2 h.
Ethyl acetate (8 mL) was added and the precipitated dicyclohexyl urea formed was filtered.
The organic phase was washed three times with 10% aqueous hydrochloric acid, 5%

10
aqueous sodium bicarbonate, and then water. This organic phase was then dried over
anhydrous sodium sulfate. The crude ester was recrystallized from a mixture of chloroform
and methanol.
Seven esters were obtained from this reaction: (3S)-21-(1H-imidazol-1-yl)-20-oxopregna-
5,16-dien-3β-yl-propionate (8b); (3S)-21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-
yl-butyrate (8c); (3S)-21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-pentanoate
(8d), (3S)-21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-hexanoate (8e), (3S)-21-
(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-cyclopropanecarboxylate (8f), (3S)-21-
(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-cyclobutanecarboxylate (8g), (3S)-21-
(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-cyclopentanecarboxylate (8h), (3S)-21-
(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-cyclohexanecarboxylate (8i) and (3S)-
21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-cycloheptanecarboxylate (8j).
2.2.1.2.1.
Fig. 1).
21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-propionate (8b in
Yield 0.078 g (68%), m.p. 180–182 °C, IR (KBr) cm-1: 2927, 1731, 1568, 1270 and 1087.
1H NMR (400 MHz, CDCl3) δ: 0.90 (s, H-18, 3H), 1.20 (s, H-19, 3H), 1.10 (t, H-3′ of ester,
3H), 3.90 (m, J = 3.6, H-3, 1H), 4.65−4.87 (ABq, J= 16.4 Hz, H-21, 2H), 5.35 (d, J = 5.9
Hz, H-6, 1H), 6.73 (s, J = 6.7 Hz, C-16, 1H), 7.04 (s, H-Het, 1H), 7.32 (s, H-Het, 1H), 7.69
13
(s, H-Het, 1H). C NMR (100 MHz, CDCl3) δ: 11.30 (C-3′ of ester), 15.30 (C-18), 21.30
(C-19), 65.35 (C-21), 71.54 (C-3), 121.01 (C-6), 141.21 (C-5), 144.21 (H-Het), 147.80 (C-
16), 151.55 (C-Het), 157.09 (C-17), 194.64 (ester carbonyl), 206.09 (C-20). HRMS: m/z
[M+H]+ calculated for 436.2726 C27H36N2O3, found 436.2732.

11
2.2.1.2.2.
1).
21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-butyrate (8c in Fig.
Yield 0.071 g (60%), m.p. 184–187 °C, IR (KBr) cm-1: 2929, 1729, 1574, 1270 and 1088.
1H NMR (400 MHz, CDCl3) δ: 1.00 (t, ester-H-4′, 3H), 1.10 (s, H-18, 3H), 1.30 (s, H-19,
3H), 3.90 (m, J = 3.9 Hz, H-3, 1H), 4.65−4.87 (ABq, J= 16.4 Hz, H-21, 2H), 5.35 (d, J =
5.9 Hz, H-6, 1H), 6.73 (s, J = 6.7 Hz, C-16, 1H), 7.04 (s, H-Het, 1H), 7.32 (s, H-Het, 1H),
13
7.69 (s, H-Het, 1H). C NMR (100 MHz, CDCl3) δ: 12.30 (C-3′ of ester), 15.01 (C-18),
23.40 (C-19), 65.30 (C-21), 71.54 (C-3), 121.01 (C-6), 141.21 (C-5), 141.26 (C-Het),
144.89 (C-Het), 148.44 (C-16), 151.55 (C-Het), 157.12 (C-17), 196.64 (ester carbonyl),
206.09 (C-20). HRMS: m/z [M+H]+ calculated 450.2882 C28H38N2O3, found 450.2878.
2.2.1.2.3.
Fig. 1).
21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-pentanoate (8d in
Yield 0.067 g (55%), m.p. 172–175 °C, IR (KBr) cm-1: 2929, 1729, 1574, 1270 and 1087.
1H NMR (400 MHz, CDCl3) δ: 0.90 (t, ester-H-5′, 3H), 1.01 (s, H-18, 3H), 1.29 (s, H-19,
3H), 3.90 (m, J = 3.2 Hz, H-3, 1H), 4.65−4.87 (ABq, J= 16.4 Hz, H-21, 2H), 5.35 (d, J =
5.9 Hz, H-6, 1H), 6.73 (s, J = 6.7 Hz, C-16, 1H), 7.04 (s, H-Het, 1H), 7.32 (s, H-Het, 1H),
13
7.69 (s, H-Het, 1H). C NMR (100 MHz, CDCl3) δ: 12.01 (C-5′ of ester), 15.30 (C-18),
24.50 (C-19), 63.40 (C-21), 71.54 (C-3), 121.01 (C-6), 141.22 (C-Het), 144.89 (C-Het),
148.45 (C-16), 151.55 (C-Het), 155.71 (C-17), 196.64 (ester carbonyl), 206.09 (C-20).
HRMS: m/z [M+H]+ calculated for 464.3039 C29H40N2O3, found 464.3033.

12
2.2.1.2.4.
Fig. 1).
21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-hexanoate (9e in
Yield 0.067 g, (53%), m.p. 169–171 °C, IR (KBr) cm-1: 2931, 1730, 1575, 1270 and 1087.
1H NMR (400 MHz, CDCl3) δ: 0.67 (t, ester-H-6′, 3H), 0.90 (s, H-18, 3H), 1.02 (s, H-19,
3H), 3.90 (m, J = 3.9 Hz, H-3, 1H), 4.65−4.87 (ABq, J= 16.4 Hz, H-21, 2H), 5.36 (d, J =
5.8 Hz, H-6, 1H), 6.73 (s, J = 6.7 Hz, C-16, 1H), 7.04 (s, H-Het, 1H), 7.32 (s, H-Het, 1H),
7.69 (s, H-Het, 1H). 13C NMR (100 MHz, CDCl3) δ: 11.36 (C-6′), 15.30 (C-18), 22.13 (C-
19), 62.31 (C-21), 71.54 (C-3), 121.01 (C-6), 141.21 (C-5), 141.22 (C-Het), 144.89 (C-
Het), 148.19 (C-16), 151.55 (C-Het), 155.71 (C-17), 196.64 (ester carbonyl), 206.08 (C-
20). HRMS: m/z [M+H]+ calculated for 478.3195 for C30H42N2O3, found 478.3189.
2.2.1.2.5.
21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-
cyclopropanecarboxylate (8f in Fig. 1).
Yield 0.065 g, (55%), m.p. 187–189 °C, IR (KBr) cm-1: 2926, 1726, 1624, 1570, 1270 and
1086. 1H NMR (400 MHz, CDCl3) δ: 1.25 (s, H-18, 3H), 1.30 (s, H-19, 3H), 1.65 (m, ester-
H-1′, 1H), 3.47 (m, J = 3.5 Hz, H-3, 1H), 4.22−4.59 (ABq, J= 16.4 Hz, H-21, 2H), 5.35 (d,
J = 5.4 Hz, H-6, 1H), 6.72 (s, J = 6.7 Hz, C-16, 1H), 7.11 (s, H-Het, 1H), 7.35 (s, H-Het,
1H), 7.51 (s, H-Het, 1H). 13C NMR (100 MHz, CDCl3) δ: 10.10 (C-1′ of ester), 13.253 (C-
18), 15.00 (C-19), 62.45 (C-21), 73.82 (C-3), 119.23 (C-6), 139.99 (C-Het), 147.21 (C-16),
147.22 (C-Het), 157.00 (C-Het), 157.03 (C-17), 174.44 (ester carbonyl), 204.08 (C-20).
HRMS: m/z [M+H]+ calculated for 448.2726 for C28H36N2O3, found 448.2730.
2.2.1.2.6.
21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-
cyclobutanecarboxylate (8g in Fig. 1).

13
Yield 0.069 g, (57%), m.p. 101–103 °C, IR (KBr) cm-1: 2928, 1723, 1624, 1572, 1270 and
1087. 1H NMR (400 MHz, CDCl3) δ: 0.80 (s, H-18, 3H), 1.10 (s, H-19, 3H), 2.29 (m, ester-
H-1′, 1H), 3.90 (m, J = 3.7 Hz, H-3, 1H), 4.65−4.87 (ABq, J= 16.4 Hz, H-21, 2H), 5.35 (d,
J = 5.4 Hz, H-6, 1H), 6.73 (m, J = 6.7 Hz, C-16, 1H), 7.04 (s, H-Het, 1H), 7.34 (s, H-Het,
1H), 7.69 (s, H-Het, 1H). 13C NMR (100 MHz, CDCl3) δ: 13.40 (C-18), 19.01 (C-19),
30.21 (C1′ of ester), 62.32 (C-21), 73.54 (C-3), 121.91 (C-6), 124.60 (C-Het), 128.83 (C-
Het), 140.23 (C-5), 144.00 (C-16), 156.99 (C-Het), 157.00 (C-Het), 175.09 (ester
carbonyl), 190.36 (C-20). HRMS: m/z [M+H]+ calculated for 462.2882 for C29H38N2O3,
found 462.2886.
2.2.1.2.7.
21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-
cyclopentanecarboxylate (8h in Fig. 1).
Yield 0.056 g, (45%), m.p. 91–93 °C, IR (KBr) cm-1: 2928, 1724, 1625, 1574, 1270 and
1087. 1H NMR (400 MHz, CDCl3) δ: 1.10 (s, H-18, 3H), 1.30 (s, H-19, 3H), 2.31 (m, ester-
H-1′, 1H), 3.47 (m, J = 3.4 Hz, H-3, 1H), 4.65−4.87 (ABq, J= 16.4 Hz, H-21, 2H), 5.33 (d,
J = 5.4 Hz, H-6, 1H), 6.73 (m, J = 6.7 Hz, C-16, 1H), 7.04 (s, H-Het, 1H), 7.32 (s, H-Het,
1H), 7.69 (s, H-Het, 1H). 13C NMR (100 MHz, CDCl3) δ: 13.01 (C-18), 19.00 (C-19),
30.10 (C1′ of ester), 62.30 (C-21), 73.54 (C-3), 121.91 (C-6), 124. 60 (C-Het), 128.83 (C-
Het), 140.15 (C-5), 140.15 (C-5), 144.00 (C-16), 156.99 (C-Het), 157.09 (C-aromatic),
175.09 (ester carbonyl), 199.36 (C-20). HRMS: m/z [M+H]+ calculated for 476.3039 for
C40H40N2O3, found 476.3035.

14
2.2.1.2.8.
21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-
cyclohexanecarboxylate (8i in Fig. 1).
Yield 0.071 g, (55%), m.p. 193–195 °C, IR (KBr) cm-1: 2929, 1726, 1624, 1591, 1270 and
1080. 1H NMR (400 MHz, CDCl3) δ: 1.24 (s, H-18, 3H), 1.40 (s, H-19, 3H), 2.30 (m, ester-
H-1′, 1H), 3.45 (m, J = 3.7 Hz, H-3, 1H), 4.62−4.82 (ABq, J= 16.4 Hz, H-21, 2H), 5.35 (d,
J = 5.4 Hz, H-6, 1H), 6.73 (m, J = 6.7 Hz, C-16, 1H), 7.10 (s, H-Het, 1H), 7.35 (s, H-Het,
1H), 7.51 (s, H-Het, 1H). 13C NMR (100 MHz, CDCl3) δ: 20.01 (C-18), 21.00 (C-19),
30.10 (C1′ of ester), 62.21 (C-21), 73.91 (C-3), 121.02 (C-6), 141.21 (C-Het), 141.27 (C-5),
141.45 (C-16), 144.89 (C-Het), 154.41 (C-Het), 157.09 (C-17), 170.66 (ester carbonyl),
200.87 (C-20). HRMS: m/z [M+H]+ calculated for 490.3195 for C31H42N2O3, found
490.3199.
2.2.1.2.9.
21-(1H-imidazol-1-yl)-20-oxopregna-5,16-dien-3β-yl-
cycloheptanecarboxylate (8j in Fig. 1).
Yield 0.071 g, (53%), m.p. 180–182 °C, IR (KBr) cm-1: 2917, 1725, 1626, 1564, 1260 and
1115. 1H NMR (400 MHz, CDCl3) δ: 1.01 (s, H-18, 3H), 1.20 (s, H-19, 3H), 2.30 (m, ester-
H-1′, 1H), 3.49 (m, J = 3.4 Hz, H-3, 1H), 4.62−4.82 (ABq, J= 16.4 Hz, H-21, 2H), 5.35 (d,
J = 5.4 Hz, H-6, 1H), 6.75 (m, J = 6.7 Hz, C-16, 1H), 7.10 (s, H-Het, 1H), 7.35 (s, H-Het,
1H), 7.51 (s, H-Het, 1H). 13C NMR (100 MHz, CDCl3) δ: 19.40 (C-18), 20.00 (C-19),
30.11 (C1′ of ester), 62.10 (C-21), 73.91 (C-3), 121.02 (C-6), 121.12 (C-Het), 141, 27 (C-
5), 144.50 (C-16), 144.52 (C-Het), 154.51 (C-17), 157.09 (C-Het), 170.66 (ester carbonyl),
200.87 (C-20). HRMS: m/z [M+H]+ calculated for 504.3352 for C32H44N2O3, found
504.3358.

15
2.2. Animal and human tissues
2.2.1. Type 1 5-R isoenzyme 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).
Two adult (8-month old) rats were obtained by the Metropolitan University in
Xochimilco from Animal Care Facility, Mexico.
The adult rats weighing 500 g had been fasted overnight to decrease glycogen levels
before their livers were extirpated for use as a source of 5-R1 [22]. To prepare
microsomes, the livers (30 g) were minced in one volume of 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 basic tissue mill (IKA Laboratory Equipment, Mexico City,
Mexico). Unless otherwise specified, the following procedures were carried out at 4 °C.
The tissue was homogenized and the suspension was centrifuged (13,200 rpm; 20
min; 0 °C) (Beckman L70 K ultracentrifuge), and the pellet was discarded. The supernatant
was filtered through a nylon mesh filter (pore size 11 lm, distributed by OEM-Membrane
Solution, Dallas TX) and centrifuged again (10,000 rpm; 60 min, 4 °C); the microsomal
pellet was suspended in five volumes of buffer A with a homogenizer. The protein
concentration was measured by the Bradford method [23].
The suspension was re-centrifuged (13,200 rpm; 20 min, 4 °C) and the pellet was
suspended in buffer A to give a final concentration of 20 mg protein/mL. The microsomal
suspension was stored at −70°C prior to the preparation of the sample steroid.
2.2.2. Type 2 5-R isoenzyme isolated from human prostate

16
Four hours after a 53-year-old patient 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.
Human prostate was used in this experiment because this tissue is an abundant
source of 5-R2, but not of 5-R1; the AR study in this model is very complex.
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 phosphate, pH 6.5, containing 0.32 M sucrose, 0.1 mM DTT; Sigma-
Aldrich, Mexico City, Mexico) with an IKA® A11 basic tissue mill (IKA Laboratory
Equipment, Mexico City, Mexico). Unless otherwise specified, the following procedures
were carried out at 4 °C. The 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 (1500g; 60 min) [24] in a SW 60 Ti rotor (Beckman
Instruments, Palo Alto, CA). The pellets were suspended in buffer A, and stored at −70°C.
This suspension had a final concentration of 5 mg protein/mL, as determined by the
Bradford method [23] and was used as source of 5-R2 isozyme.
2.2.3. Rat prostate cytosol as source of AR
In this study we used rats because the prostate gland is bigger than that of the
hamster and there is no difference in the binding activity of the AR between rat and hamster
cytosol [25].
In order to evaluate the binding of steroids 8a−j to the androgen receptor, the
prostates of 50 adult rats (8 months old; 500 g) were removed, blotted, weighed and soaked

17
in cold TEMD (40 mM tris–HCl, 3 mM EDTA and 20 mM sodium molybdate,
dithiothreitol 0.5 mM, 10% glycerol at pH 8) before use. All procedures were carried out in
an ice bath at 0 °C. The tissues were homogenized with a tissue homogenizer (Teckmar,
Cincinnati, OH) in one volume of buffer TEMD plus protease inhibitors (2 mM PMSF, 10
lg/mL antipain, 5 mM leupeptin) [26] in an ice bath with a tissue homogenizer;
subsequently the homogenates were centrifuged (14,000 rpm, 60 min) [24] in a SW 60 Ti
rotor (Beckman Instruments, Palo Alto, CA). The cytosolic fraction obtained from the
supernatant liquid of the rat prostate homogenate described above was stored at −70 °C.
The protein concentration (6 mg of protein in 200 lL) was determined by the Bradford
method.
2.2.4. In vivo experiments
For the experiments in vivo, adult male golden hamsters (2.5 months old; 150–200
g) were used. After gonadectomies had been performed on the hamsters under isoflurane
anesthesia, the castrated hamsters were allowed to recover for 30 days prior to
experimentation [25]. The castrated hamsters and a group of 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 groups consisting of four animals per group. The hamsters
were treated for six days, and thereafter sacrificed with CO2 [24]. This experiment was
carried out twice under the same conditions.

18
2.2.5. Cell line characteristics
The panel of cells used in this study was: PC-3 (human prostate cancer cell line),
MCF7 (human breast cancer cell line), and SK-LU-1 (human lung cancer cell lines),
supplied by the National Cancer Institute (USA).
2.3. Biological activity of 8a−j compounds
2.3.1. In vitro experiments
The effect of steroids 8a−j (Fig. 1) on the activity of 5-R1 and 5-R2 was
evaluated by following the conversion of T to DHT, under the same conditions that we
described previously [27].
The 50% inhibitory concentration (IC50) of 8a−j was determined using a range of
concentrations (1×10−10 to 1×10−4 M) of each derivative. The transformation of T to DHT in
the presence of 8a−j was calculated from radioactive compounds present in each sample,
observed in the lanes of the chromatographic plate. The data were plotted using SigmaPlot
software 12 inhibition curves [24].
2.3.2. In vivo experiments
2.3.3.1. Weight of the prostate, seminal vesicles and diameter of the pigmented spot
For six consecutive days, each of the steroid derivatives 8a−j (2 mg/kg body weight
(BW)) dissolved in 200 µL of sesame oil, together with 1 mg/kg (BW) of testosterone, was
administered by subcutaneous (SC) injection to a group of gonadectomized hamsters (four
animals per derivative). Three groups of gonadectomized animals were kept as control; the
first group was injected SC with 200 µL sesame oil, the second group with 1 mg/kg (BW)
of testosterone, and the third group with 1 mg T plus 1 mg/kg (BW) of finasteride also

19
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 and the diameters
of the flank organs were measured using a Vernier Caliper; the prostate and 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 using 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).
2.3.3. Competitive studies on AR
The competitive studies of steroids 8a−j on AR were carried out using the same
procedure described by our group previously [28]. The IC50 of each compound was
calculated from plots of concentration versus percentage of binding using SigmaPlot 12.
Mibolerone (MIB) was used as a positive control.
2.3.4. Cytotoxicity assay
Cytotoxic activity of the steroid derivatives (8a−j) on three cancer cell lines (PC-3,
MCF7, and SK-LU-1) was evaluated. The procedure has been previously reported [29].
Cell growth inhibition was calculated according to the following expression:

20
3.
Results
3.1. Chemistry
The yield for the formation of 8a from the reaction involving acetic anhydride and
pyridine was 90%, while compounds 8b–j were obtained in moderate yields of 45–68%
using the Steglish esterification method [30].
8b–j were isolated and characterized by 1H NMR, 13C NMR and mass spectrometry.
Also, results obtained from the melting point determination of each compound showed a
maximum two degree difference, indicating that these steroids were pure.
3.2. Effect of derivatives as inhibitors of the activity of 5-R isoenzymes
Table 1 shows the IC50 values for derivatives 8a−j required to inhibit 50% of the
activity of 5-reductase isoenzymes.
These results showed that the chain derivative with the smallest ester at C-3 and
alicyclic derivatives also present at C-3 of the steroidal skeleton showed greater selectivity
as 5-R2 inhibitors than the other derivatives studied. However their potency to inhibit this
enzyme was lower than that of finasteride (IC50 = 8.5 nM) in all cases. None of the prepared
compounds was able to inhibit the activity of the 5α-R1 enzyme.
3.3. Weight of the prostate, seminal vesicles and diameter of the pigmented spot
After castration, the weight of the hamster prostate and seminal vesicles decreased
(p < 0.05) compared to the normal weight of the respective glands, as did the diameter of
the pigmented spot. Treatment with vehicle did not change these effects. However,
treatment with 1 mg/kg of T for 6 days significantly increased (p < 0.05) the weight of
prostate, seminal vesicles and diameter of the pigmented spot as well as the weight of the
prostate and seminal vesicles (Table 2). When T (1 mg/kg) and finasteride were injected

21
together, the weight of the prostate, seminal vesicles and diameter of the flank organs
decreased significantly (p < 0.05) (Table 2). This reduction in the weight of these glands
and diameter of the pigmented spot was comparable to that produced by finasteride (Table
2).
Table 2 shows that when T was administered together with finasteride or steroids
8g–j, the weight of the prostate and seminal vesicles decreased significantly (p < 0.05). In
addition, 8g–j showed a significant (p < 0.05) reduction in diameter of the pigmented spot.
The remainder of the compounds studied did not display any comparable effect (Table 2).
3.4. Competitive studies on AR
This study showed that compounds 8a−j did not displace the tritium-labeled MIB
from the AR. MIB is a very potent AR agonist and exhibited an IC50 value of 1 nM. Figure
2 below shows only the graphs of AR unbinding for compounds 8g–j to illustrate the
behavior of steroids that did not bind to this receptor.
These results show that the steroidal derivatives are not androgenic types and for
this reason cannot bind on the active site of the AR.

22
Figure 2
Mibolerone
8g
8h
8i
120
100
80
60
40
20
0
8j
0
2
20
100
200
2000
Steroid concentration [nM]
3.5. Cytotoxic activity on cancer cell lines (PC-3, MCF7 and SK-LU-1)
The effect of the synthesized compounds (8a−j) on the growth of three cancer cell
lines; PC-3 (prostate), MCF7 (breast) and SK-LU-1 (lung), was determined using the SBR
assay method. The values of the antiproliferative effect are shown in Table 1. Ketoconazole
and 16-dehydropregenolone acetate were used as controls in this study.
Ketoconazole was used as a positive control, because its interesting cytotoxic
activity in vitro studies has been previously reported [16; 31-34]. Compounds 7 [12,15] and
8a showed higher cytotoxic activity than 16-dehydropregenolone acetate in the three cancer
cell lines (Table 1). Furthermore, 8a showed higher toxicity than 8b−j in these three cancer
cell lines. This may be because these steroids do not dissolve well in the culture medium
containing a dimethyl sulfoxide (DMSO) concentration of 0.05%.This idea is supported by
the calculated log P value for each one of these derivatives. (Table 1)

23
Table 1. Biological activity of novel derivate compounds on growth inhibition in human cancer cell lines at 50 µM, IC50 value on 5α-R
isoenzymes and AR binding assay.
5α-R1
IC50
nM
RBA
AR
%
LogP
Calca
5α-R2
IC50 µM
Structure
Compound
R
PC-3
MCF-7
SKLU-1
−
−
1
88.48±3.9 95.16±4.8 73.39±1.6
−
−
−
−
−
4.00
Ketoconazole
Finasteride
16-DHP
NA
NA
NA
630
NA
0.0085
NA
NA
NA
4.51
21.4±11.5 25.5±1.2
12.8±3.5
3.11
96.2±1.3
89.4±2.3
74.8±1.5
NA
118.8
NA
−
7
3.82
4.18
4.74
5.24
5.75
4.48
-CH3
-C2H5
-C3H7
-C4H9
-C5H11
NA
NA
NA
NA
NA
8a
8b
8c
8d
8e
79.6 ± 8.7 77.9 ± 0.8 96.7 ± 1.3
NA
NA
NA
NA
NA
122
NA
NA
NA
NA
22.6 ± 5.2
0
0
0
16.8 ± 1.0
25.2 ± 0.1
23.7 ± 2.9 51.3 ± 8.3 39.2 ± 8.2
17.2 ± 3.6 28.1 ± 4.9 28.5 ± 7.4
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
8f
8g
8h
8i
NA
NA
NA
NA
NA
0.711
0.0764
0.035
0.0293
NA
4.65
5.39
5.90
6.40
8j
Results are expressed as a percentage of inhibition ±standard error. NA: This compound was not active.
a The LogP property was calculated by Molinspiration (http://www.molinspiration.com/).

24
Table 2. Weight of prostate, seminal vesicle glands, and diameter of the pigmented spot of
the flank organs ± standard deviation from castrated hamsters receiving different treatments
for 6 days.
Diameter of the
Pigmented Spot
Weight of Seminal
Vesicles
(mm)±standard
error
Weight of Prostate
(mm)±standard
error
Treatment
(mm)±standard
error
Control
T
50.4 ± 9.9
98 ± 12.6
96.11 ± 12.5
200.6 ± 55
2.03 ± 0.63
5.65 ± 1.12
3.57 ± 0.89
3.06 ± 0.08
1.80±0.38
1.98±0.8
T+F
69.6 ± 21.2
85.80 ± 19.41
88.40±16.10
83.43±8.46
80.93±6.06
71.85±16.06
77.1±17
161.4 ± 44.5
200.50 ± 32.79
231.70±25.08
217.45±59.28
198.33±22.20
198.33±22.20
160.9±15.8
T+8a
T+8b
T+8c
T+8d
T+8e
T+8f
T+8g
T+8h
T+8i
T+8j
2.94±0.99
3.36±0.40
3.4±0.47
71.3±9*
145.2±32.1*
143.7±50.66*
143.68±50.7*
143.6±29.89*
3.0±0.45*
2.0±0.4*
65.1±15.8*
54.3±4.9*
70.8±5.3
2.4±0.3*
2.8±0.5*
The control animals were treated with vehicle only.
* Statistically significant values.

25
4.
Discussion
Our research group previously synthesized several steroidal derivatives with an
ester moiety at C-3 and several azole derivatives. These steroids demonstrated high
antiproliferative activity on different cancer cell lines [28-29] and an inhibitory effect on
5-reductase type 1 and 2 activity [35].
Recently, we also demonstrated that the presence of a triazole group at C-21 with a
linear ester moiety at C-3 on the steroidal skeleton enhanced 5-R1 inhibitory activity. In
addition, compound 20-oxo-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-3-yl-acetate
presented a high cytotoxic effect on the SK-LU-1 cancer cell line. Banday et al. similarly
identified a series of novel D-ring substituted 1,2,3-triazolyl 20-keto pregnane derivatives
as antiproliferative agents on DU-145 and PC-3 cancer cell lines [19].
This report serves as an extension of our earlier study where it was demonstrated
that two pregnane derivatives with a triazole or imidazole moiety at C-21, inhibited the
three studied cancer cell lines proliferation; [15] particularly the 3β-hydroxy-21- (1H-
imidazol-1-yl) pregna-5,16-dien-20one; I-OH (7) showed a good PC3 cells antiproliferative
effect. However docking studies indicated this steroid is unable to binding to the pocket of
the AR, so that this activity cannot explained by this way. In order to understand the action
of imidazole derivatives in prostate tissue, here we report the in vivo and in vitro activity of
ten different novel 16-dehydropregnenolone derivatives with an imidazole group at C-21
and a linear or alicyclic ester moiety at C-3.
Although all these novel steroidal derivatives (8a−j) have an ester moiety at C-3,
they are noteworthy because they show a very different biological activity, allowing other

26
possible therapeutic applications. Interestingly, only 8a shows higher activity on PC-3,
which is satisfactory as the intent was to obtain specific compounds for each type of cancer.
Furthermore, only derivatives with an alicyclic ester at C-3 demonstrated 5α-R2
inhibitory activity, with compounds 8h and 8i showing the highest inhibitory activity on
this isoenzyme. The pharmacological activity of the compounds was also demonstrated by
a decrease in weight and size of the androgen-dependent glands studied. However, these
derivatives with an aliphatic group at C-3 were unable to bind to the androgen receptor as
docking studies had predicted, [15] hence the pharmacological effect observed was due to
their activity as inhibitors of 5α-R2 activity. This is important because prostatic benign
hyperplasia is directly associated with the presence of 5-R2. [4, 7] In addition, the fact
that these compounds were not able to bind the androgen receptor increases their
therapeutic value by generating the possibility of only causing minor side effects. The
overall data suggests that the size of the ester moiety could be related to the cytotoxic effect
of these compounds.
The antiproliferative activity of these compounds can also be attributed to the nature
of the azole group [36-37, 10]. This group has a sextet of µ-electrons, giving the azole a
higher nucleophilic character [38] that enables formation of a strong hydrogen bond
between the steroidal compound and active polar groups in the cells. Because of this, a
stronger cytotoxic effect is produced [39].
In conclusion, the results presented here indicate that 16-dehydropregnenolone
derivatives were successfully synthesized, and presented an inhibitory effect on 5-R2, as
well as an important pharmacological effect that was non-cytotoxic in the treatment-time.
Furthermore, the compounds showed interesting cytotoxic activities on different cancer cell

27
lines, especially on PC-3, an androgen-independent prostate cancer cell line [40-41],
showing therapeutic potential for cancer treatment.

28
Acknowledgements
We would like to thank DGAPA, project IN211312 (UNAM), CONACYT, and Project CB
2011/165049 for the financial support.
Conflict of Interest
The authors declare that that are no real or perceived conflicts of interest arising
from intellectual, personal, or financial circumstances of the research. Additionally, all
authors are aware of, and approve the contents and order of authorship of the manuscript.

29
References
1 Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer statistics CA. 2016, 66, 7.
2 Antognelli, C.; Del Buono, C. Baldracchini, F.; Talesa, V.; Cottini, E.; Brancadoro,
C.; Zucchi, A.; Mearini, E. Cancer Biol. Ther. 2007, 6, 1880.
3 Thomas, L.N.; Lazier, C.B.; Gupta, R.; Norman, R.W.; Troyer, D.A.; O’Brien, S.P.;
Rittmaster, R.S. Prostate 2005, 63, 231.
4 Russell, D.W.; Wilson, J.D. Annu. Rev. Biochem. 1994, 63, 25.
5 Ogishima, T.; Mitani, F.; Suematsu, M. J. Steroid Biochem. Mol. Biol. 2008, 111,
80.
6 Uemura, M.; Tamura, K.; Chung, S.; Honma, S.; Okuyama, A.; Nakamura, Y.;
Nakagawa, H. Cancer Sci. 2008, 99, 81.
7 Liang, T.; Cascieri, M. A.; Reynolds, G. F.; Rasmusson, G. H. Endocrinology 1985,
117, 571.
8 Brandt, M.; Levy, M.A. Biochemistry 1989, 28, 140.
9 Elmegeed, G.A.; Khalil, W.K.; Mohareb, R.M.; Ahmed, H.H.; Abd-Elhalim, M.M.;
Elsayed, G.H. Bioorg. Med. Chem. 2011, 19, 6860.
10 Elmegeed, G.A.; Yahya, S.M.M.; Abd-Elhalim, M.M.; Mohamed, M.S.; Mohareb,
R.M.; Elsayed, G.H. Steroids 2016, 115, 80.
11 Cabeza, M.; Sánchez-Márquez, A.; Garrido, M; Silva, A.; Bratoeff, . Curr. Med.
Chem. 2016, 23, 792.
12 Silva-Ortiz, A.V.; Bratoeff, E.; Ramírez-Apan, T.; Heuze, Y.; Sánchez, A.; Soriano,
J.; Cabeza, M. Bioorg. Med. Chem. 2015, 23, 7535.