DOI: 10.3109/14756366.2015.1070843
Dehydroepiandrosterone benzimidazolyl derivatives as anti-androgens
3
7.9 (s, aromatic, 1H), 9.6 (s, formyl, 1H); 13C-NMR (300 MHz, Department of the General Hospital in Mexico City. The Ethical
CDCl3): d 73.3 (C3), 120.7 (C16), 120.6 (C-aromatic), 121.7 Committee of the General Hospital in Mexico City approved this
(C6), 123.4 (C-aromatic), 124.2 (C-aromatic), 140.1 (C-aro- protocol.
matic), 141.4 (C5), 174.9 (C¼O ester), 187.8 (C¼O formyl); and
The tissue was rinsed and immediately chilled in ice-cold
HRMS calculated for C33H38N2O3 (498.6557), found 499.6556.
150 mM NaCl and stored at ꢁ20 ꢂC. The frozen human prostate
3b-Cyclopentanoyloxy-17-(1H-bezimidazol-1-yl)-16-formylan- was thawed on ice, rinsed, and minced in buffer A (20 mM sodium
drosta-5,16-diene (5d in Figure 1). Yield: 97 mg (40.74%) white phosphate, pH 6.5, containing 0.32 M sucrose, 0.1 mM DTT
solid; m.p.: 165–166 ꢂC; IR (KBr) cmꢁ1: 1728, 1672, 1188; UV (Sigma-Aldrich, Mexico City, Mexico) with an IKAÕ A11 basic
(nm): 244, 281; 1H-NMR (400 MHz, CDCl3): d 1.1 (s, H-18, 3H), tissue mill (IKA Laboratory Equipment, Mexico City, Mexico).
1.1 (s, H-19, 3H), 4.6 (m, H-3, 1H), 5.4 (d, J ¼ 5.2 Hz, H-6, 1H), Unless otherwise specified, the following procedures were carried
7.4 (m, aromatic, 3H), 7.9 (s, aromatic, 1H), 7.9 (1H, s, aromatic), out at 4 ꢂC.
9.6 (s, formyl 1H); 13C-NMR (300 MHz, CDCl3): d 73.2 (C3),
120.6 (C16), 120.6 (C-aromatic), 121.6 (C6), 123.4 (C-aromatic),
124.0 (C-aromatic), 140.2 (C-aromatic), 141.4 (C5), 176.2 (C¼O
5ꢀ-R type 2 isozyme isolated from human prostate
Human prostate was used in this experiment because this tissue
is an abundant source of 5a-R2 for the study of the effect(s) of
5a–5f and 6, which were designed for inhibition of the activity
ester),187.8 (C¼O formyl); and HRMS calculated for
C35H40N2O3 (512.6823), found 513.6822.
3b-Cyclohexanoyloxy-17-(1H-bezimidazol-1-yl)-16-formylan-
of this enzyme in humans. In this tissue, 5a-R type 1 is not as
drosta-5,16-diene (5e in Figure 1). Yield: 61.5 mg (25.96%)
white solid; m.p.: 130–131 ꢂC; IR (KBr) cmꢁ1: 1725, 1674, 1195;
abundant as is type 2.
1
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 (1500 ꢃ g;
UV (nm): 244, 273, 280; H-NMR: (400 MHz, CDCl3): d 1.1 (s,
H-18, 3H), 1.1 (s, H-19, 3H), 4.6 (m, H-3, 1H), 5.4 (d, J ¼ 5.2 Hz,
H-6, 1H), 7.4 (m, aromatic, 3H), 7.8 (s, aromatic, 1H), 7.9 (s,
60 min)9 in a SW 60 Ti rotor (Beckman Instruments, Palo Alto,
aromatic, 1H), 9.6 (s, formyl, 1H); 13C-NMR: (300 MHz, CDCl3):
CA). The pellets were re-suspended in buffer A, and stored
at ꢁ70 ꢂC. This suspension had a final concentration of 5 mg
d 73.0 (C3), 120.6 (C16), 120.6 (C-aromatic), 121.7 (C6), 123.4
(C-aromatic), 124.0 (C-aromatic), 140.2 (C-aromatic), 141.4
protein/mL, as determined by the Bradford method10 and was
(C5), 175.6 (C¼O ester), 187.9 (C¼O formyl); and HRMS
used as source of 5a-R type 2 isozyme.
calculated for C37H42N2O3 (526.7089), found 527.7088.
3b-Cycloheptanoyloxy-17-(1H-bezimidazol-1-yl)-16-formylan-
5ꢀ-R type 1 isozyme isolated from rat liver
drosta-5,16-diene (5f in Figure 1). Yield: 75 mg (31.67%) white
solid; m.p.: 156–157 ꢂC; IR (KBr) cm–1: 1726, 1673, 1186.; UV
(nm): 244, 273, 280; 1H-NMR: (400 MHz, CDCl3): d 1.1 (s, H-18,
3H), 1.1 (s, H-19, 3H), 4.6 (m, H-3, 1H), 5.4 (d, J ¼ 4.8 Hz, H-6,
1H), 7.4 (m, aromatic, 3H), 7.8 (s, aromatic, 1H), 7.9 (s, aromatic,
1H), 9.6 (s, formyl, 1H); 13C-NMR: (300 MHz, CDCl3):d 73.1
(C3), 120.6 (C16), 120.6 (C-aromatic), 121.6 (C6), 123.4 (C-
aromatic), 124.0 (C-aromatic), 140.2 (C-aromatic), 141.4 (C5),
176.5 (C¼O ester), 187.8 (C¼O formyl); and HRMS calculated
for C39H44N2O3 (540.7355), found 541.7354.
All procedures involving animals were approved by the
Institutional Care and Use Committee of the Metropolitan
University of Mexico (UAM; Xochimilco, Mexico). All animals
used in this study were obtained from the Animal Care Facility
at UAM.
Two adult (8-month-old) had been fasted overnight to decrease
glycogen levels before their livers were extirpated for use as a
source of 5a-R1, as recommend Levy et al.11 To prepare
microsomes, the livers (30 g) were minced in one volume of
buffer A, by using the tissue mill. Unless otherwise specified, the
following procedures were carried out at 4 ꢂC. The tissue was
homogenized; the suspension was centrifuged (10 000 ꢃ g;
30 min; 0 ꢂC) (Beckman L70K ultracentrifuge, Mexico City,
Mexico); and the pellet was discarded. The supernatant was
filtered through a nylon mesh filter (pore size 11 mm, distributed
by ‘‘OEM-Membrane Solution’’, Dallas, TX) and centrifuged
(100 000 ꢃ g; 60 min). The microsomal pellet was re-suspended in
five volumes of buffer A with a homogenizer, and the protein
concentration was determined by the Bradford method. The
suspension was re-centrifuged (100 000 ꢃ g; 30 min) and the
pellet was re-suspended in buffer A to give a final concentration
of 20 mg protein/mL. The microsomal suspension was stored at
ꢁ80 ꢂC prior to the preparation of the solubilized steroid 5a-R
type 1.
Derivative 6. Compound 6 (Figure 1) was prepared from 4 g as
following:
A
mixture of 3b-hydroxy-17-chloro-16-formylandrosta-5,16-
diene, 4 g (0.1 mg, 0.3 mmol), benzimidazole (0.071 mg,
0.6 mmol) and Cs2CO3 (0.195 mg, 0.6 mmol) in dry DMF was
stirred at 60 ꢂC under 2 h. After, the mixture was poured onto ice-
cold water (50 mL) and the obtained precipitate was filtered,
washed with water and dried in vacuum. The dirty yellow solid
was purified by column chromatography on florisil
(Hexane:EtOAc 50:50) to give the compound 6.
3b-Hydrloxy-17-(1H-benzimidazole-1-yl)-16-formylandrosta-
5,16-diene (6 in Figure 1)
Yield 52%, white solid; m.p.: 203–204 ꢂC mp; IR (FTIR-ATR):
1
3334, 3087, 2958, 1671, 1618, 737 cmꢁ1; H-NMR (CDCl3) d:
9.59 (s, 1H, CHO); 7.98 (s, 1H, imidazole-H); 7.85 (dd, J ¼ 7.0,
2.8 Hz, 2H, aromatic-Hs); 7.35 (dd, J ¼ 6.0, 2.9 Hz, 2H, aromatic-
Hs); 5.40 (s, 1H, H-6); 3.55 (m, 1H, H-3); 1.05 (s, 6H, angular-
Hs); 13C-NMR (CDCl3) d: (16-CHO); 141.25 (imidazole-C);
124.21 (aromatic-C); 123.66 (aromatic-C); 122.68 (aromatic-C);
120.92 (C-6); 120.63 (aromatic-C); 71.62 (C-3); 20.57 (C-19);
19.45 (C-18). FAB-MS calcd for C27H32N2O2 (amu) 416, found
417 [M + 1]+.
The solubilization of 5a-R type 1 isozyme from the rat liver
microsomes was carried out according to Levy et al.11
Gonadectomized male hamsters
For the experiments in vivo, 88 adult male golden hamsters (2.5-
month-old; 150–200 g) were used. After gonadectomies had been
performed on 80 hamsters under isoflurane anesthesia, the
castrated hamsters were allowed to recover for 30 d prior to
experimentation5,12. The castrated hamsters and the remaining
eight intact hamsters were housed in a room with controlled
Human an animal tissues and procedures
Four hours after a 57-year-old man had died from myocardial temperature (22 ꢂC) and light–dark periods of 12 h; the hamsters
infarction; his normal prostate was extirpated in the Pathology were fed with food and water ad libitum. Thirty days post-