Synthesis and evaluation of novel 17-indazole androstene derivatives designed as CYP17 inhibitors

Article abstract of DOI:10.1016/j.steroids.2007.08.004

A series of novel 1H- and 2H-indazole derivatives of the commercially available dehydroepiandrosterone acetate have been synthesized and tested for inhibition of human cytochrome 17α-hydroxylase-C_17,20-lyase (CYP17), androgen receptor (AR) binding affinity, and cytotoxic potential against three prostate cancer (PC) cell lines.

Full text of DOI:10.1016/j.steroids.2007.08.004

steroids 7 2 ( 2 0 0 7 ) 939–948
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Synthesis and evaluation of novel 17-indazole androstene
derivatives designed as CYP17 inhibitors
Vaˆnia M.A. Moreiraa, Tadas S. Vasaitisb,
Vincent C.O. Njarb,∗∗, Jorge A.R. Salvadora,∗
a
´
´
ˆ
´
Laboratorio de Quımica Farmaceutica, Faculdade de Farmacia, Universidade de Coimbra,
Rua do Norte, 3000-295 Coimbra, Portugal
b
Department of Pharmacology & Experimental Therapeutics, University of Maryland School of Medicine,
Baltimore, 655W Baltimore Street, Baltimore, MD 21201-1559, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 1H- and 2H-indazole derivatives of the commercially available dehy-
droepiandrosterone acetate have been synthesized and tested for inhibition of human
cytochrome 17␣-hydroxylase-C17,20-lyase (CYP17), androgen receptor (AR) binding affinity,
and cytotoxic potential against three prostate cancer (PC) cell lines.
Received 4 July 2007
Received in revised form
20 July 2007
Accepted 9 August 2007
Published on line 15 August 2007
© 2007 Elsevier Inc. All rights reserved.
Keywords:
Indazole
CYP17
Prostate cancer
Androgen receptor
PC cell lines
1.
Introduction
can be further metabolized in steroidogenic tissues to more
potent androgens such as testosterone and dihydrotestos-
CYP17 is an endoplasmic reticulum membrane-bound mul-
terone (DHT).
tifunctional enzyme that exhibits 17␣-hydroxylase and
The testis and the adrenal cortex are the two sites thought
to produce most of the androgenic steroids in humans. The
testis are responsible for about 90–95% of circulating andro-
gens whereas the adrenals account for the remaining 5–10%
[6].
In the adult prostate, androgens act directly on epithe-
lial cells to maintain structural and functional viability. The
secretory epithelial cells express the AR and require chronic
androgenic stimulation for survival and functional integrity.
C17,20-lyase activities on a single active site, both of which
are crucial for human physiology [1–5]. The hydroxylase
activity is involved in the conversion of pregnenolone
to 17␣-hydroxypregnenolone and progesterone to 17␣-
hydroxyprogesterone whereas the lyase activity is responsible
for the side-chain cleavage of these hydroxy derivatives to
afford dehydroepiandrosterone (DHEA) and androstenedione
(AD), respectively. DHEA and AD are androgen precursors and
∗
∗∗
Corresponding author. Tel.: +351 239 859950; fax: +351 239 827126.
Corresponding author. Tel.: +1 410 706 5885; fax: +1 410 706 0032
E-mail addresses: vnjar001@umaryland.edu (V.C.O. Njar), salvador@ci.uc.pt (J.A.R. Salvador).
0039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2007.08.004

940
steroids 7 2 ( 2 0 0 7 ) 939–948
Glandular involution occurs as a result of cell apoptosis when
androgen levels drop below a threshold (as is the case in med-
ical or surgical castration) [7].
Androgen deprivation as therapy for advanced PC was
introduced by Huggins et al. in 1941 [8,9] and ever since
it has been the mainstay for advanced PC treatment. At
least 80% of the human prostate cancers show a favourable
response to androgen deprivation as evidenced by the dis-
appearance of symptoms or a decline of prostate specific
antigen (PSA) levels [10,11]. However, relapses are seen invari-
ably when tumors emerge as androgen-independent and
apoptosis-resistant [7]. Mechanisms that may mediate this
adaptation include AR amplification, AR mutation, alterations
in the balance between transcriptional coactivators and core-
pressors, and activation of signal transduction pathways that
by-pass the AR [12,13]. Gene amplification and amino acid
substitutions in the AR are detected at a high frequency in
recurrent tumors. These changes confer growth advantage to
the tumor cells due to either hypersensitivity of AR to low,
castrate-level androgens or a realignment of the receptor con-
formation, leading to altered ligand specificity that enables
antiandrogens, adrenal androgens and non-androgen steroids
to act agonistically to increase AR activity [7].
Enhanced intracellular conversion of adrenal androgens to
testosterone and DHT has also been reported as an important
mechanism for disease progression [14]. It may explain why
the available AR antagonists do not have substantial activity
against the androgen-independent tumor cells that emerge
subsequent to androgen deprivation therapy, seeing that the
AR antagonists will have a much lower affinity for the AR
than the natural substrates. Inhibition of CYP17 is therefore a
valuable approach for the treatment of androgen-dependent
diseases such as PC as a means of inhibiting androgen biosyn-
thesis both in the testis and adrenals. It should be noted
that PC is a leading cause of mortality being the second most
common cause of cancer-related death in both the USA and
Australia (behind lung cancer), and the third most common
cause of cancer-related death in the European Union (behind
lung cancer and colorectal cancer) [15–17].
Fig. 1 – CYP17 inhibitors.
gone Phase I clinical trials for PC treatment [33,34] and the
first set of results of an open-label Phase II clinical trial have
just been reported. Thus, 11 out of 18 patients have had PSA
declines ≥50% at 3 months with 5 patients having a PSA
decline ≥90% when 1000 mg of the drug were administered
orally once daily to chemotherapy-naive castration-resistant
PC (CRPC) patients, resistant to luteinizing hormone-releasing
hormone (LHRH) analogues, antiandrogens, and frequently
diethylstilbestrol (DES) and steroids [35].
Another class of interesting steroidal inhibitors has been
reported in which the azole group is attached to the C17
of the steroid nucleus through a nitrogen atom [36–38].
Both compound 3 (code named VN/85-1) (Fig. 1) and com-
pound 4 (VN/124-1) potently inhibit CYP17. VN/124-1 has also
been shown to have antiandrogenic properties against the
androgen-dependent LAPC4 human prostate tumor xenograft,
being actually more effective than castration in suppressing
its growth [38].
Several steroidal and non-steroidal compounds have been
synthesized and evaluated as CYP17 inhibitors [18–23]. Out of
these compounds, ketoconazole 1 (Fig. 1), an imidazole fungi-
cide that has inhibitory activity towards CYP17 [24,25], has
been used clinically in high dose (400 mg, every 8 h) for the
treatment of advanced PC [26–28]. However, the fact that it
concomitantly inhibits other steroidal P450 enzymes causing
significant side effects [29] has limited its use. A recent investi-
gation of the efficacy of low dose ketoconazole (200 mg, three
times daily) found clinical benefit equal to high dose treat-
ment, with a reduction in side effects [30]. Ketoconazole is
still currently used alone or in combination with glucocorti-
coids as secondary hormonal therapy for hormone-refractory
PC (HRPC) [31].
A common approach to the synthesis of potent steroidal
inhibitors of CYP17 has been the design of substrate-like
molecules bearing a heterocycle at the C17 position with priv-
ileged heteroatoms (N, S, O) which can interact as the sixth
ligand with the heme iron of the enzyme. One of such com-
pounds, abiraterone acetate 2 (Fig. 1), reported to be a very
potent inhibitor of the enzyme [32], has successfully under-
Herein we report the synthesis and biological evaluation of
novel 17-indazole androstene derivatives designed as CYP17
inhibitors. Other than their CYP17 inhibitory potential, their
ability to bind to the AR, and cytotoxicity against three PC cell
lines has also been evaluated.
2.
Experimental
2.1.
Chemistry
2.1.1. General
Dehydroepiandrosterone acetate, indazole, bis(triphenyl-
phosphine)rhodium(I)carbonyl chloride, 1,3-bis(diphenyl-
phosphino)propane, aluminum isopropoxide, and N-
methylpiperidone were obtained from Sigma–Aldrich Co.
All solvents used were previously dried and purified accord-
ing to standard procedures. For TLC analysis, Kieselgel
60HF254/Kieselgel 60G was used. Melting points were deter-
mined using a BUCHI Melting Point B-540 apparatus and
are uncorrected. IR spectra were obtained using a JASCO

steroids 7 2 ( 2 0 0 7 ) 939–948
941
FT/IR-420 spectrophotometer. NMR spectra were obtained
using a Brucker Digital NMR-Avance 300 apparatus or a
Varian Inova 500 apparatus, in CDCl3 with MeSi4 as the
internal standard. Mass spectra were recorded on a Finnigan
Polaris Q GC/MS Benchtop Ion Trap mass spectrometer. HRMS
were determined on a Bruker 12T APEX-Qe FTICR-MS with an
Apollo II ion source and an Advion Triversa Nanomate system.
Elemental analysis was carried out on a Fisons Instruments
EA 1108 CHNS-O elemental analyzer.
washed with water (60 ml), dried with anhydrous MgSO4,
filtered, and concentrated under reduced pressure to give a
yellowish oil. This oil was subjected to flash chromatography
with chloroform/petroleum ether 40–60 ◦C (7:3) to give com-
pound 10 (700 mg; 62%): m.p. (acetonitrile/THF) 154–156 ◦C;
IR 1241, 1620, 1728 cm−1 1H NMR (CDCl3, 300 MHz): ı 1.09 (s,
;
3H, 18-H3), 1.18 (s, 3H, 19-H3), 2.04 (s, 3H, 3␤-OAc), 4.61 (m,
1H, 3␣-H), 5.43 (m, 1H, 6-H), 5.86 (m, 1H, 16-H), 7.18 (m, 1H,
aromatic-H), 7.39 (m, 1H, aromatic-H), 7.64 (m, 1H, aromatic-
H), 7.74 (m, 1H; aromatic-H), 8.07 (m, 1H, aromatic-H); 13C
NMR (CDCl3, 75 MHz): ı 124.3 (C3a), 139.9 (C7a), 140.1 (C5),
150.6 (C17), 170.5 (CH3CO); EI-MS m/z (%): 430 (20) M+, 370
(100), 355 (40), 195 (26), 157 (85), 144 (32), 119 (28), 91 (38); Anal.
calcd. for C28H34N2O2: C 78.1, H 7.96, N 6.51, found: C 78.40, H
7.60, N 6.36.
2.1.2. Synthesis of 17-(indazole) androstene derivatives
2.1.2.1. 17-Chloroandrosta-5,16-dien-3ˇ-yl acetate (6) and 17-
chloro-16-formylandrosta-5,16-dien-3ˇ-yl acetate (7). Details
of the synthesis of these compounds were reported previously
[37,39].
2.1.2.2. 16-Formyl-17-(1H-indazol-1-yl)androsta-5,16-dien-
3ˇ-yl acetate (8) and 16-formyl-17-(2H-indazol-2-yl)androsta-
5,16-dien-3ˇ-yl acetate (9). A mixture of 7 (2 g, 5.32 mmol),
indazole (943 mg, 7.98 mmol), and K2CO3 (2.2 g, 15.94 mmol) in
dry DMF (40 ml) was heated at 80 ◦C under N2 atmosphere for
6 h. The mixture was then concentrated under reduced pres-
sure. Water (60 ml) and dichloromethane (200 ml) were added
and the mixture was left under magnetic stirring for a couple
of hours. The aqueous phase was extracted another two
times with dichloromethane (2 × 50 ml). The organic phase
was then washed with water (60 ml), brine (60 ml), dried with
anhydrous MgSO4, filtered, and concentrated under reduced
pressure to give a yellowish oil. This oil was subjected to flash
chromatography with chloroform/petroleum ether 40–60 ◦C
(7:3) and afforded compound 8 (1.27 g; 52%): m.p. (acetone/n-
2.1.2.4. 17-(1H-Indazol-1-yl)androsta-5,16-dien-3ˇ-ol
(11).
Compound 10 (400 mg; 0.93 mmol) was dissolved in methanol
(7.5 ml) at room temperature under Ar. KOH (10%) in methanol
(2.5 ml) was added and the mixture was left under magnetic
stirring for 1.5 h. After this time the mixture was concen-
trated under reduced pressure. Dichloromethane (150 ml)
and water (20 ml) were added, and left to agitate for a cou-
ple more hours. The aqueous phase was extracted with
dichloromethane (2 × 100 ml). The organic phase was then
washed with water (20 ml), dried with anhydrous MgSO4,
filtered, and evaporated to dryness to give compound 11
(297.1 mg; 82%): m.p. (acetone) 189–191 ◦C; IR 1625, 3257 cm−1
;
1H NMR (CDCl3, 300 MHz): ı 1.08 (s, 3H, 18-H3), 1.18 (s, 3H,
19-H3), 3.55 (m, 1H, 3␣-H), 5.41 (m, 1H, 6-H), 5.86 (m, 1H,
16-H), 7.18 (m, 1H, aromatic-H), 7.39 (m, 1H, aromatic-H), 7.64
(m, 1H, aromatic-H), 7.74 (m, 1H; aromatic-H), 8.07 (m, 1H,
aromatic-H); 13C NMR (CDCl3, 75 MHz): ı 124.3 (C3a), 139.9
(C7a), 141.0 (C5), 150.6 (C17); EI-MS m/z (%): 388 (100) M+, 373
(35), 237 (29), 195 (24), 181 (14), 157 (26), 119 (22), 91 (17); Anal.
calcd. for C26H32N2O: C 80.37, H 8.3, N 7.21, found: C 80.46, H
8.07, N 7.0.
hexane) 185–189 ◦C; IR 1248, 163, 1661, 1721 cm−1 1H NMR
;
(CDCl3, 300 MHz): ı 1.08 (s, 3H, 18-H3), 1.27 (s, 3H, 19-H3), 2.04
(s, 3H, 3␤-OAc), 4.61 (m, 1H, 3␣-H), 5.43 (m, 1H, 6-H), 7.25
(m, 1H, aromatic-H), 7.45 (m, 2H, aromatic-H), 7.78 (m, 1H,
aromatic-H), 8.24 (m, 1H, aromatic-H), 9.72 (s, 1H, CHO); EI-MS
m/z (%): 458 (12) M+, 398 (26), 225 (25), 185 (39), 157 (100), 143
(16), 91 (22), 77 (14); Anal. calcd. for C29H34N2O3: C 75.95, H
7.47, N 6.11, found: C 75.89, H 7.29, N 5.90. A second fraction
afforded compound 9 (539 mg; 22%): m.p. (acetone/n-hexane)
2.1.2.5. 17-(1H-Indazol-1-yl)androsta-4,16-dien-3-one (12). A
mixture of 11 (470 mg; 1.21 mmol), N-methylpiperidone
(1.7 ml), and toluene (28 ml) was distilled off ca. 10 ml. Alu-
minum isopropoxide (453 mg; 2.22 mmol) was then added,
and the mixture was refluxed under N2 for 5 h. The mixture
was then concentrated under reduced pressure. Ethyl acetate
(20 ml) and water (50 ml) were added and the mixture was left
under magnetic stirring for a couple of hours. The aqueous
phase was further extracted with ethyl acetate (2 × 100 ml).
The organic phase was washed with 5% HCl (50 ml), 10%
NaHCO3 (50 ml), and water (50 ml), dried with anhydrous
MgSO4, filtered, and evaporated to dryness to afford com-
pound 12 (390 mg; 83%): m.p. (acetone/n-hexane) 196–197 ◦C;
190–192 ◦C; IR 1245, 1629, 1661, 1731 cm−1 1H NMR (CDCl3,
;
300 MHz): ı 1.09 (s, 3H, 18-H3), 1.28 (s, 3H, 19-H3), 2.04 (s, 3H,
3␤-OAc), 4.62 (m, 1H, 3␣-H), 5.43 (m, 1H, 6-H), 7.13 (m, 1H,
aromatic-H), 7.34 (m, 1H, aromatic-H), 7.68 (m, 1H, aromatic-
H), 7.72 (m, 1H, aromatic-H), 8.19 (m, 1H, aromatic-H), 10.15
(s, 1H, CHO); EI-MS m/z (%): 458 (32) M+, 398 (74), 383 (48),
293 (24), 225 (56), 185 (54), 157 (100), 130 (20); Anal. calcd. for
C29H34N2O3: C 75.95, H 7.47, N 6.11, found: C 76.02, H 7.53,
N 5.88.
2.1.2.3. 17-(1H-Indazol-1-yl)androsta-5,16-dien-3ˇ-yl acetate
(10). A mixture
carbonyl chloride
of
bis(triphenylphosphine)rhodium(I)
0.47 mmol) and 1,3-
IR 1621, 1672 cm−1 1H NMR (CDCl3, 300 MHz): ı 1.20 (s, 3H,
;
(325.4 mg,
18-H3), 1.24 (s, 3H, 19-H3), 5.76 (brs, 1H, 4-H), 5.85 (m, 1H, 16-
H), 7.17 (m, 1H, aromatic-H), 7.40 (m, 1H, aromatic-H), 7.64
(m, 1H, aromatic-H), 7.74 (m, 1H, aromatic-H), 8.07 (m, 1H,
aromatic-H); 13C NMR (CDCl3, 75 MHz): ı 124.4 (C3a), 139.9
(C7a), 150.4 (C17), 171.0 (C5), 199.5 (C3); EI-MS m/z (%): 386
(100) M+, 371 (75), 253 (22), 211 (16), 183 (21), 157 (30), 119
(18), 81 (14); HRMS calcd 387.2431 (C26H30N2OH+), found 387.
2435.
bis(diphenylphosphino)propane (420.7 mg, 1.02 mmol) in
dry xylenes (95 ml) was stirred at 80 ◦C under N2 for 15 min
when a fine yellow precipitate formed. Compound 8 (1.2 g,
2.62 mmol) was added, and the mixture was refluxed under
N2 for 10 days, then cooled, and concentrated under reduced
pressure. The resulting oil was dissolved in dichloromethane
(600 ml) and treated with charcoal. The organic phase was

942
steroids 7 2 ( 2 0 0 7 ) 939–948
from Sigma–Aldrich Co. Casodex was kindly provided by
Astra-Zeneca Inc. Scintiverse BD Cocktail (Scintanalyzed)
fluid was obtained from Fisher Scientific. VN/85-1 was
prepared as previously reported [37].
2.1.2.6. 17-(2H-Indazol-2-yl)androsta-5,16-dien-3ˇ-yl acetate
(13). The method followed that described for compound 10
but using compound 9 (700 mg; 1.53 mmol). The resulting
oil was subjected to flash chromatography with chloro-
form/petroleum ether 40–60 ◦C (7:3) which afforded compound
13 (308 mg; 47%): m.p. (acetone) 144–146 ◦C; IR 1243, 1620,
The synthetic androgen methyltrinolone [3H]R1881, with a
specific activity of 72 Ci/mmol, was purchased from Perkin-
Elmer. [21-3H3]-17␣-hydroxypregnenolone, with
a specific
1728 cm−1 1H NMR (CDCl3, 300 MHz): ı 1.09 (s, 3H, 18-H3),
;
activity of 13.61 ␮Ci/␮mol, was prepared as described by
Akhtar et al. [40].
The calcium phosphate transfection kit was purchased
from Promega (Promega Profection Mammalian Transfec-
tion System). Tox-2 kit (XTT based) was purchased from
Sigma–Aldrich Co.
293T and 293T-CYP17 cells were routinely maintained in
DMEM supplemented with 10% FBS and 1% P/S solution.
LNCaP and PC-3 cells were grown in RPMI 1640 medium sup-
plemented with 10% FBS and 1% P/S solution. LAPC4 cells were
grown in RPMI supplemented with 15% FBS, 1% P/S solution,
and 10 nM DHT.
The pCDNA3Hmod17(His)4 construct was designed as previ-
ously reported [41].
Radioactivity measurements were performed in a Tri-carb
2100 TR liquid scintillation analyzer. Absorbance and lumines-
cence measurements were made using a Victor 1420 Multilabel
counter.
1.19 (s, 3H, 19-H3), 2.04 (s, 3H, 3␤-OAc), 4.62 (m, 1H, 3␣-H),
5.43 (m, 1H, 6-H), 6.14 (m, 1H, 16-H), 7.06 (m, 1H, aromatic-
H), 7.27 (m, 1H, aromatic-H), 7.64 (m, 1H, aromatic-H), 7.71 (m,
1H; aromatic-H), 8.11 (m, 1H, aromatic-H); 13C NMR (CDCl3,
75 MHz): ı 121.7 (C3a), 140.0 (C5), 148.9 (C7a), 151.7 (C17),
170.5 (CH3CO); EI-MS m/z (%): 430 (14) M+, 370 (100), 355
(56), 209 (18), 195 (24), 157 (57), 105 (16), 91 (13); Anal. calcd.
for C28H34N2O2: C 78.1, H 7.96, N 6.51, found: C 78.5, H 7.6,
N 6.2.
2.1.2.7. 17-(2H-Indazol-2-yl)androsta-5,16-dien-3ˇ-ol
(14).
The method followed that described for compound 11 but
using compound 13 (255.8 mg; 0.59 mmol) to afford compound
14 (216.8 mg; 94%): m.p. (acetonitrile/THF) 224–226 ◦C; IR 1629,
3352 cm−1 1H NMR (CDCl3, 300 MHz): ı 1.08 (s, 3H, 18-H3), 1.19
;
(s, 3H, 19-H3), 3.55 (m, 1H, 3␣-H), 5.40 (m, 1H, 6-H), 6.17 (m,
1H, 16-H), 7.07 (m, 1H, aromatic-H), 7.27 (m, 1H, aromatic-H),
7.64 (m, 1H, aromatic-H), 7.71 (m, 1H; aromatic-H), 8.11 (m,
1H, aromatic-H); 13C NMR (CDCl3, 75 MHz): ı 121.7 (C3a), 141.2
(C5), 148.9 (C7a), 151.70 (C17); EI-MS m/z (%): 388 (100) M+, 373
(68), 369 (23), 223 (22), 209 (26), 195 (32), 181 (18), 157 (86); Anal.
calcd. for C26H32N2O: C 80.37, H 8.3, N 7.21, found: C 80.2, H
8.16, N 7.15.
2.2.2. In vitro CYP17 assay (C17,20-lyase activity)
The in vitro C17,20-lyase inhibitory activities of the compounds
were evaluated using the acetic acid releasing assay (AARA)
[37,38,41–49] with 293T cells that were transfected with the
pCDNA3Hmod17(His)4 construct using the calcium phosphate
method (Promega Profection Mammalian Transfection Sys-
tem), to express the human enzyme.
2.1.2.8. 17-(2H-Indazol-2-yl)androsta-4,16-dien-3-one
(15).
The method followed that described for compound 12 but
using compound 14 (450 mg; 1.16 mmol) to afford com-
pound 15 (370 mg; 83%): m.p. (acetone) 169–172 ◦C; IR 1629,
Briefly, 100 mm plates were coated with poly-l-lysine
(0.05 mg/ml) for 30 min, rinsed twice with sterilized distilled
water, and allowed to dry for 2 h. 293T cells were then plated in
DMEM at a density sufficient for achieving approximately 60%
confluency on the following day, for transfection. Three hours
prior to transfection the DMEM was renewed on the plate. On
a small eppendorf flask, 10 ␮g of pCDNA3Hmod17(His)4 were
added to sterile, deionized water and vortexed briefly. A 2 M
CaCl2 solution (62 ␮l) was added to bring the final volume up
to 500 ␮l. This mixture was added drop-wise with light vortex
to 500 ␮l of HEPES solution and incubated at room tempera-
ture for 30 min. The solution was vortexed again and dripped
into the 100 mm plate. The medium was changed 18 h later
and enzyme activity was assayed as described below 48 h after
transfection.
1674 cm−1 1H NMR (CDCl3, 300 MHz): ı 1.21 (s, 3H, 18-H3),
;
1.25 (s, 3H, 19-H3), 5.77 (brs, 1H, 4-H), 6.14 (m, 1H, 16-H),
7.07 (m, 1H, aromatic-H), 7.27 (m, 1H, aromatic-H), 7.64
(m, 1H, aromatic-H), 7.72 (m, 1H; aromatic-H), 8.11 (m, 1H,
aromatic-H); 13C NMR (CDCl3, 75 MHz): ı 121.7 (C3a), 148.9
(C7a), 151.6 (C17), 170.6 (C5), 199.4 (C3); EI-MS m/z (%): 386
(72) M+, 371 (62), 223 (39), 195 (36), 157 (100), 145 (25), 119
(35), 91 (42); HRMS calcd 387.2431 (C26H30N2OH+), found
387.2431.
2.2.
Biology
2.2.1. General
The human PC cell lines LNCaP and PC-3 were obtained from
the American Type Culture Collection (Rockville, MD). 293T
cells were the gift of Dr. Yun Qiu (UMB, Maryland), and LAPC4
cells were provided by Dr. Charles L. Sawyers (UCLA School of
Medicine).
RPMI 1640 medium, Dulbecco’s Modified Eagle Medium
(DMEM), Dulbecco’s Phosphate Buffered Saline (DPBS),
trypsin/EDTA (0.25%/0.02%), and penicillin/streptomycin (P/S)
were obtained from Gibco-BRL. Fetal Bovine Serum (FBS),
charcoal-stripped serum (CSS), and trypsin/versene were
obtained from Biofluids Inc. Poly-l-lysine, triamcinolone ace-
tonide, ketoconazole, DHT, and MTT powder were obtained
293T-CYP17 cells were grown to 80% confluency and
divided evenly in 6-well plates. On the following day, cells
were washed with DPBS and incubated with clear DMEM (with
5% CSS and 1% P/S) containing a saturating concentration of
[21-3H3]17␣-hydroxypregnenolone. The test compounds were
then added in the desired concentrations and the plates were
left to incubate for 18 h at 37 ◦C. The steroids were extracted
with 2 ml of chloroform at 4 ◦C. After 2 h, the aqueous phase
was collected and charcoal suspension was added to a 2.5%
final concentration. Following a 30-min incubation at 4 ◦C, an
aliquot of the aqueous supernatant was removed and radioac-
tivity measured by liquid scintillation counting.

steroids 7 2 ( 2 0 0 7 ) 939–948
943
1% P/S added) containing a saturation concentration (5 nM)
of [3H]R1881, triamcinolone acetonide (1 ␮M), and the desired
concentrations of the test compounds. Following a 2 h incu-
bation period at 37 ◦C, cells were washed twice with ice-cold
DPBS, and solubilized in DPBS containing 0.5% SDS and 20%
glycerol. Extracts were removed and cell associated radioac-
tivity counted in a scintillation counter. All results represent
an average of a minimum of three wells. To determine the
EC50 values of the test compounds, a minimum of eight con-
centrations of each test compound was used. EC50 values were
2.2.3. Competitive AR binding assay
Competitive binding assays with the synthetic androgen
methyltrienolone [3H]R1881 were performed essentially as
previously described [50,51]. LNCaP or LAPC4 cells were
transferred to clear RPMI medium (with 5% CSS and 1% P/S) 3
days before the start of the experiment. 24-Well plates were
coated with poly-l-lysine (0.05 mg/ml) for 30 min, rinsed with
sterilized distilled water, and dried for 2 h. The cells were
then plated (2–3 × 105 cells/well) and allowed to attach. The
following day the medium was replaced by clear RPMI (with
Scheme 1 – Synthesis of 17-indazole compounds. (i) POCl3-DMF, CH3Cl, Ar, reflux; (ii) indazole, K2CO3, DMF, N2, 80 ◦C; (iii)
(PPh3)2RhCOCl-Ph2P(CH2)3PPh2, xylenes, N2, reflux; (iv) KOH (10%) in methanol, N2, rt; (v) Al(i-PrO)3, N-methylpiperidone,
toluene, reflux.

944
steroids 7 2 ( 2 0 0 7 ) 939–948
Cl−] in refluxing xylenes [37,52], after the use of both the
Wilkinson catalyst [RhCl(PPh3)3] and 10% palladium on acti-
vated charcoal, other known methods of decarbonylation,
failed. Hydrolysis of the 3␤-acetoxy group to obtain the cor-
responding 3␤-hydroxy derivatives 11 and 14 was achieved
with KOH (10%) in methanol, in 82 and 94% yield. Modified
Oppenauer oxidation of substrates 11 and 14 using aluminum
isopropoxide and N-methylpiperidone afforded the final 3-
keto derivatives 12 and 15, again in good yields (83% in both
cases).
The peak pattern of the carbons seen on the 13C NMR
spectra of compounds 13–15 (2H-series) is consistent with
data reported in the literature for 2H-substituted compounds
whereas for compounds 10–12 (1H-series) the spectra cor-
related well with that of 1H-substituted derivatives [53]. In
the 2H-series, the two quaternary carbons of the indazole
ring C3a and C7a are seen at ppm values of 121–123 ppm
and 148–153 ppm, respectively. For our compounds C3a was
identified at 121 ppm and C7a at 148 ppm. The 1H-series typ-
ically has C3a at higher ppm values (123–126 ppm) whereas
C7a is seen at lower ppm values of 137–148 ppm. Thus,
in our 1H-series we identified C3a at 124 ppm and C7a at
139 ppm.
determined by non-linear regression with Graphpad Prism
software.
2.2.4. Cell culture and viability assay
To determine the effect of steroids and novel compounds on
cell proliferation on LNCaP and LAPC4 cells, each cell type was
transferred into clear RPMI medium (with 5% CSS and 1% P/S) 3
days prior to the start of the experiments. The cells were then
plated on previously coated 96-well plates (2.5 × 103 cell/well)
and after a 24 h attachment period, the medium was aspi-
rated and replaced by new medium with the novel compounds
(0.1–20 ␮M). The medium was changed every 3 days and the
number of viable cells was compared by XTT assay on the 7th
day. Briefly, a 20% solution of XTT in clear medium was added
to each of the wells in the plates and after a 4 h incubation
period, the plates were read at 450 nm on a Victor 1420 Multi-
label counter. All results represent the average of a minimum
of three wells.
For PC-3 cells, 1.5 × 104 cells/well were plated in 24-well
plates in RPMI medium (with 10% FBS and 1% P/S). After
a 24 h attachment period, the cells were treated with the
novel compounds (0.1–20 ␮M). The medium was changed
every 3 days and the number of viable cells was compared
by MTT assay on the 7th day. Briefly, 0.5 mg/ml MTT in clear
RPMI medium (with 5% CSS and 1% P/S) was added to each
well and incubated at 37 ◦C for 4 h. Following incubation,
the medium was aspirated completely with care taken not
to disturb the formazan crystals. DMSO (400 ␮l) was used
to solubilize these crystals. After slight shaking, the plates
were immediately read at 540 nm on a Victor 1420 Multil-
abel counter. All results represent an average of a minimum
of three wells. To determine the EC50 values of the test
compounds, PC-3 cells were incubated with a minimum of
eight concentrations of each test compound. EC50 values were
determined by non-linear regression with Graphpad Prism
software.
3.2.
Biology
3.2.1. CYP17 inhibition (C17,20-lyase)
The AARA uses [21-3H3]17␣-hydroxypregnenolone as sub-
strate and C17,20-lyase activity is measured by the amount of
[3H]acetic acid released during the side-chain cleavage of the
substrate to DHEA. This assay proved comparable in terms
of accuracy and reliability to the previous HPLC procedures
[41,42]. Moreover, kinetic analysis of CYP17 in human testic-
ular microsomes showed that the C17,20-lyase activity is half
that of the 17␣-hydroxylase activity thus implying that inhi-
bition of the C17,20-lyase activity will actually characterize the
inhibition of the entire enzyme [41]. Human kidney 293T cells
were chosen for the assay because they showed rapid growth
3.
Results and discussion
3.1.
Chemistry
Table 1 – C17,20-lyase inhibition and AR binding for
17-indazole compounds
3.1.1. Synthesis of 17-indazole androstene derivatives
The synthesis of the new 17-indazole androstene derivatives is
outlined in Scheme 1. It started with the Vilsmeier-Haack reac-
tion of the commercially available dehydroepiandrosterone
acetate 5 with phosphorous oxychloride (POCl3) and dimethyl-
formamide (DMF), as reported previously [37,39]. The major
reaction product 17-chloro-16-formylandrosta-5,16-diene-3␤-
yl acetate 7 was then treated with indazole in the presence of
K2CO3 and DMF at 80 ◦C, under N2 to afford a mixture of the 1H-
8 and 2H-indazole 9 substituted compounds which were sep-
arated by flash chromatography on silica gel. The 1H-indazole
derivative 8 was found to be the major reaction product
being isolated in 52% yield (compound 9 was isolated in 22%
yield).
Compound
C17,20-lyase
IC50 293T
LNCaP
EC50 (nM)
LAPC4
EC50 (nM)
a
b
b
c
c
c
c
c
c
d
d
d
d
d
d
d
d
10
11
12
13
14
15
5000
d
d
5000
NT
NT
Ketoconazole
VN/85-1
Casodex
49 nM
1.29 nM
NTe
NT
NT
971
4500
a
IC50 is the concentration of inhibitor required to inhibit the
enzyme activity by 50%.
EC50 is the concentration of compound needed for a 50% displace-
ment of [3H]R1881 from the AR.
b
Each of the obtained 17-indazole compounds 8 and 9
was separately decarbonylated to afford compounds 10
and 13 in 62 and 47% yield, respectively. This decar-
bonylation was performed with in situ generated Rh(1,3-
c
d
e
Less than 30% effective at 10 ␮M.
Less than 30% binding at 5 ␮M.
NT = Not tested.
bis(diphenylphosphino)propane)2+Cl− catalyst [Rh(dppp)2
+

steroids 7 2 ( 2 0 0 7 ) 939–948
945
in culture medium and high transfection efficiency for the
human CYP17 plasmid.
Table 2 – Effect of the 2H-indazole series on PC-3 cell
proliferation
The synthesized compounds were found not to inhibit
C17,20-lyase significantly when compared to both ketocona-
zole and VN/85-1. Thus, all tested compounds (10–15) were
less than 30% effective at 10 ␮M whereas IC50 values of 49
and 1.29 nM were determined for ketoconazole and VN/85-1,
respectively, under the same assay conditions (Table 1). Rea-
sons that may account for this lack of inhibitory activity are
Compound
PC-3 EC50 (␮M)
13
14
15
5.4
8.3
1.9
Fig. 2 – Effect of selected compounds on (a) LNCaP, (b) LAPC4, and (c) PC-3 cell proliferation. The percentage (compared to
control) of growth inhibiton after 7 days of treatment was determined with the XTT (LNCaP and LAPC4) or MTT (PC-3) assay
using 0.1–20 ␮M of compound, as described in Section 2. One-way ANOVA with a Dunnet Post Test was used to analyze the
data: *p < 0.01.

946
steroids 7 2 ( 2 0 0 7 ) 939–948
the positioning of the N atoms in the heterocycle ring that may
not permit good interaction with the enzyme’s active site or
even the bulkiness of the indazole ring.
Acknowledgments
Vaˆnia M.A. Moreira thanks Fundac¸a˜o para a Cieˆncia e a Tec-
nologia for supporting this work (BD/12508/2003). Jorge A.R.
Salvador thanks Universidade de Coimbra for financial sup-
port.
Vaˆnia M.A. Moreira and Jorge A.R. Salvador also thank
Fundac¸a˜o Luso-Americana for sponsoring trips to the US that
have strongly contributed to this work.
3.2.2. AR binding
Following the observation that several compounds designed
as CYP17 inhibitors have been shown to bind to the AR and
interfere with its function [37,38,45,54], we decided to deter-
mine if the synthesized indazole androstene derivatives could
bind to the AR. LAPC4 and LNCaP cells were chosen which
express the wild-type (wt-) and mutated receptor, respectively.
Competitive binding of the compounds to the AR was eval-
uated using a saturating concentration of the radiolabeled
androgen [3H]R1881 in the presence of different concentra-
tions of test compound.
r e f e r e n c e s
[1] Nakajin S, Hall PF, Onoda M. Testicular microsomal
cytochrome P450 for C21 steroid side chain cleavage. Spectral
and binding studies. J Biol Chem 1981;256:6134–9.
[2] Nakajin S, Hall PF. Microsomal cytochrome P450 from
neonatal pig testis. Purification and properties of a C21
steroid side-chain cleavage system (17␣-hydroxylase-C17,20
lyase). J Biol Chem 1981;256:3871–6.
Compounds 10, 11, 13 and 14 showed less than 30% binding
to both types of AR at 5 ␮M (Table 1). However, compounds 12
and 15, having the ꢀ4-3-ketone system in common, bound to
the wt-AR with similar affinity when compared to Casodex,
an antiandrogen currently used in PC treatment, showing an
EC50 of 5 ␮M (4.5 ␮M for Casodex).
[3] Nakajin S, Shively JE, Yuan PM, Hall PF. Microsomal
cytochrome P450 from neonatal pig testis: two enzymatic
activities (17␣-hydroxylase and C17,20-lyase) associated with
one protein. Biochemistry 1981;20:4037–42.
[4] Zuber MX, Simpson ER, Waterman MR. Expression of bovine
17␣-hydroxylase cytochrome P450 cDNA in
nonsteroidogenic (COS 1) cells. Science 1986;234:1258–61.
[5] Hall PF. Cytochrome P450 C21scc: one enzyme with two
actions: hydroxylase and lyase. J Steroid Biochem Mol Biol
1991;40:527–32.
[6] Denis LJ, Griffiths K. Endocrine treatment in prostate cancer.
Semin Surg Oncol 2000;18:52–74.
[7] Chatterjee B. The role of the androgen receptor in the
development of prostatic hyperplasia and prostate cancer.
Mol Cell Biochem 2003;253:89–101.
[8] Huggins C, Hodges CV. Studies on prostatic cancer. I. The
effect of castration, of estrogen and of androgen injection on
serum phosphatases in metastatic carcinoma of the
prostate. Cancer Res 1941;1:293–7.
[9] Huggins C, Stevens RE, Hodges CV. Studies on prostatic
cancer. II. The effect of castration on clinical patients with
carcinoma of the prostate. Arch Surg 1941;43:209.
[10] Resnick MI, Grayhack JT. Treatment of stage IV carcinoma of
the prostate. Urol Clin North Am 1975;2:141–61, references
cited therein.
[11] Koivisto P, Kolmer M, Visakorpi T, Kallioniemi OP. Androgen
receptor gene and hormonal therapy failure of prostate
cancer. Am J Pathol 1998;152:1–9, references cited therein.
[12] Chen CD, Welsbie DS, Tran C, Baek SH, Chen R, Vessella R, et
al. Molecular determinants of resistance to antiandrogen
therapy. Nat Med 2004;10:33–9.
3.2.3. PC cell toxicity
The potential of the synthesized compounds to inhibit cell
proliferation was studied on LNCaP, LAPC4 and PC-3 cell lines.
All compounds were screened at 0.1, 1, 10, and 20 ␮M and the
more active were chosen for EC50 calculation. The results are
depicted in Table 2 and Fig. 2.
We identified compounds 13–15 in the 2H-indazole series
that significantly inhibited the proliferation of LAPC4 and
LNCaP cells mostly at 10 and 20 ␮M (Fig. 2a and b), and were
concomitanly effective against PC-3 cell proliferation. Their
EC50 values for PC-3 cell proliferation were 5.4, 8.3, and 1.9 ␮M,
respectively (Table 2). In the 1H-indazole series, compound 10
was only active at 20 ␮M for all PC cell lines. Compounds 11
and 12 were exclusively cytotoxic towards PC-3 cells, showing
50% inhibition of cell proliferation at 10 and 20 ␮M, respec-
tively (Fig. 2c).
Thus, seeing that there is not a very high affinity of the com-
pounds towards the AR, they probably act on PC cells through
mechanisms other than the ones mediated by the AR such as
apoptosis or cell cycle arrest. This is more evident for PC-3
cells that are human PC cells derived from bone metastases
and do not express the AR at all.
In summary, the synthesized compounds were found not
to inhibit C17,20-lyase activity significantly when compared
to both ketoconazole and VN/85-1. They also did not display
affinity towards the LNCaP mutated AR at the concentrations
tested. Compounds 12 and 15, having the ꢀ4-3-ketone sys-
tem in common, bound to the wt-AR in the same extension
as Casodex. However, only moderate inhibition of LAPC4 cell
proliferation was seen with compound 15 at higher concen-
trations, suggesting that other mechanisms that are non-AR
mediated account for this effect. The 2H-indazole series
(13–15) was particularly effective against PC-3 cells whereas
compounds 11 and 12 of the 1H-indazole series were exclu-
sively toxic towards them. Because PC-3 cells lack the AR, it is
likely that mechanisms such as apoptosis or cell cycle arrest
account for inhibition of proliferation on this particular cell
line.
[13] Debes JD, Tindall DJ. Mechanisms of androgen-refractory
prostate cancer. N Engl J Med 2004;351:1488–90.
[14] Stanbrough M, Bubley GJ, Ross K, Golub TR, Rubin MA,
Penning TM, et al. Increased expression of genes converting
adrenal androgens to testosterone in androgen-independent
prostate cancer. Cancer Res 2006;66:2815–25.
[15] Black RJ, Bray F, Ferlay J, Parkin DM. Cancer incidence and
mortality in the European Union: cancer registry data and
estimates of national incidence for 1990. Eur J Cancer
1997;33:1075–107.
[16] Greenlee RT, Murray T, Bolden S, Wingo PA. Cancer statistics,
2000. CA Cancer J Clin 2000;50:7–33.
[17] Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer
statistics, 2007. CA Cancer J Clin 2007;57:43–66.

steroids 7 2 ( 2 0 0 7 ) 939–948
947
[18] Barrie SE, Jarman M. Inhibitors of cytochrome P45017␣
[36] Njar VC, Klus GT, Brodie AMH. Nucleophilic vinylic
“addition-elimination” substitution reaction of
(17␣-hydroxylase/C17,20-lyase). Endocr Relat Cancer
1996;3:25–39.
[19] Jarman M, Smith HJ, Nicholls PJ, Simons C. Inhibitors of
enzymes of androgen biosynthesis: cytochrome P45017␣ and
5␣-steroid reductase. Nat Prod Rep 1998;15:495–512.
[20] Njar VC, Brodie AM. Inhibitors of
3␤-acetoxy-17-chloro-16-formylandrosta-5,16-diene: a novel
and general route to 17-substituted steroids. Part 1.
Synthesis of novel 17-azolyl-ꢀ16-steroids; inhibitors of
17␣-hydroxylase/17,20-lyase (17␣-lyase). Bioorg Med Chem
Lett 1996;6:2777–82.
17␣-hydroxylase/17,20-lyase (CYP17): potential agents for
the treatment of prostate cancer. Curr Pharm Des
1999;5:163–80.
[37] Njar VC, Kato K, Nnane IP, Grigoryev DN, Long BJ, Brodie AM.
Novel 17-azolyl steroids, potent inhibitors of human
cytochrome 17␣-hydroxylase-C17,20-lyase (P45017␣): potential
agents for the treatment of prostate cancer. J Med Chem
1998;41:902–12.
[38] Handratta VD, Vasaitis TS, Njar VC, Gediya LK, Kataria R,
Chopra P, et al. Novel C-17-heteroaryl steroidal CYP17
inhibitors/antiandrogens: synthesis, in vitro biological
activity, pharmacokinetics, and antitumor activity in the
LAPC4 human prostate cancer xenograft model. J Med Chem
2005;48:2972–84.
[21] Hartmann RW, Ehmer PB, Haidar S, Hector M, Jose J, Klein
CD, et al. Inhibition of CYP 17, a new strategy for the
treatment of prostate cancer. Arch Pharm Pharm Med Chem
2002;335:119–28.
[22] Leroux F. Inhibition of P450 17 as a new strategy for the
treatment of prostate cancer. Curr Med Chem
2005;12:1623–9.
[23] Hakki T, Bernhardt R. CYP17- and CYP11B-dependent steroid
hydroxylases as drug development targets. Pharmacol Ther
2006;111:27–52.
[39] Siddiqui AU, Rao VUM, Maimirani M, Siddiqui AH.
Heterocyclic steroids: synthesis of
[24] Pont A, Williams PL, Azhar S, Reitz RE, Bochra C, Smith ER, et
al. Ketoconazole blocks testosterone synthesis. Arch Intern
Med 1982;142:2137–40.
androsteno[17,16-d]pyrazoles and
androsteno[17,16-e]pyrimidines. J Heterocycl Chem
1995;32:353–4.
[25] Santen RJ, Van den Bossche H, Symoens J, Brugmans J, De
Coster R. Site of action of low dose ketoconazole on
androgen biosynthesis in men. J Clin Endocrinol Metab
1983;57:732–6.
[26] Trachtenberg J, Pont A. Ketoconazole therapy for advanced
prostate cancer. Lancet 1984;2:433–5.
[27] De Coster R, Caers I, Coene MC, Amery W, Beerens D,
Haelterman C. Effects of high dose ketoconazole therapy on
the main plasma testicular and adrenal steroids in
previously untreated prostatic cancer patients. Clin
Endocrinol 1986;24:657–64.
[28] Muscato JJ, Ahmann TA, Johnson KM, Wilding W, Monaghan
G, Schlossman DM. Optimal dosing of ketoconazole (KETO)
and hydrocortisone (HC) leads to long responses in hormone
refractory prostate cancer. Proc Am Soc Clin Oncol
1994;13:229.
[29] Lake-Bakaar G, Scheuer PJ, Sherlock S. Hepatic reactions
associated with ketoconazole in the United Kingdom. BMJ
1987;294:419–22.
[30] Nakabayashi M, Xie W, Regan MM, Jackman DM, Kantoff PW,
Oh WK. Response to low-dose ketoconazole and subsequent
dose escalation to high-dose ketoconazole in patients with
androgen-independent prostate cancer. Cancer
2006;107:975–81.
[31] Lam JS, Leppert JT, Vemulapalli SN, Shvarts O, Belldegrun AS.
Secondary hormonal therapy for advanced prostate cancer. J
Urol 2006;175:27–34.
[32] Potter GA, Barrie SE, Jarman M, Rowlands MG. Novel
steroidal inhibitors of human cytochrome P45017␣
(17␣-hydroxylase-C17,20-lyase): potential agents for the
treatment of prostatic cancer. J Med Chem 1995;38:2463–71.
[33] O’Donnell A, Judson I, Dowsett M, Raynaud F, Dearnaley D,
Mason M, et al. Hormonal impact of the
[40] Akhtar M, Corina DL, Miller SL, Shyadehi AZ, Wright JN.
Incorporation of label from 18O2 into acetate during
side-chain cleavage catalyzed by cytochrome P45017␣
(17␣-hydroxylase-17,20-lyase). J Chem Soc Perkin Trans I
1994:263–7.
[41] Grigoryev DN, Kato K, Njar VC, Long BJ, Ling YZ, Wang X, et
al. Cytochrome P450c17-expressing Escherichia coli as a
first-step screening system for 17␣-hydroxylase-C17,20-lyase
inhibitors. Anal Biochem 1999;267:319–30.
[42] Ling YZ, Li JS, Liu Y, Kato K, Klus GT, Brodie A. 17-Imidazolyl,
pyrazolyl, and isoxazolyl androstene derivatives. Novel
steroidal inhibitors of human cytochrome C17,20-lyase
(P45017␣). J Med Chem 1997;40:3297–304.
[43] Njar VC, Klus GT, Johnson HH, Brodie AM. Synthesis of novel
21-trifluoropregnane steroids: inhibitors of
17␣-hydroxylase/17,20-lyase (17␣-lyase). Steroids
1997;62:468–73.
[44] Nnane IP, Kato K, Liu Y, Lu Q, Wang X, Ling YZ, et al. Effects
of some novel inhibitors of C17,20-lyase and 5␣-reductase in
vitro and in vivo and their potential role in the treatment of
prostate cancer. Cancer Res 1998;58:3826–32.
[45] Grigoryev DN, Long BJ, Nnane IP, Njar VC, Liu Y, Brodie AM.
Effects of new 17␣-hydroxylase/C17,20-lyase inhibitors on
LNCaP prostate cancer cell growth in vitro and in vivo. Br J
Cancer 1999;81:622–30.
[46] Nnane IP, Kato K, Liu Y, Long BJ, Lu Q, Wang X, et al.
Inhibition of androgen synthesis in human testicular and
prostatic microsomes and in male rats by novel steroidal
compounds. Endocrinology 1999;140:2891–7.
[47] Nnane IP, Njar VC, Liu Y, Lu Q, Brodie AM. Effects of novel
17-azolyl compounds on androgen synthesis in vitro and in
vivo. J Steroid Biochem Mol Biol 1999;71:145–52.
[48] Nnane IP, Long BJ, Ling YZ, Grigoryev DN, Brodie AM.
Anti-tumour effects and pharmacokinetic profile of
17-(5ꢀ-isoxazolyl)androsta-4,16-dien-3-one (L-39) in mice: an
inhibitor of androgen synthesis. Br J Cancer 2000;83:74–82.
[49] Zhu N, Ling Y, Lei X, Handratta V, Brodie AM. Novel P45017␣
inhibitors: 17-(2ꢀ-oxazolyl)- and 17-(2ꢀ-thiazolyl)-androstene
derivatives. Steroids 2003;68:603–11.
[50] Wong C, Kelce WR, Sar M, Wilson EM. Androgen receptor
antagonist versus agonist activities of the fungicide
vinclozolin relative to hydroxyflutamide. J Biol Chem
1995;270:19998–20003.
[51] Yarbrough WG, Quarmby VE, Simental JA, Joseph DR, Sar M,
Lubahn DB, et al. A single base mutation in the androgen
17␣-hydroxylase/C17,20-lyase inhibitor abiraterone acetate
(CB7630) in patients with prostate cancer. Br J Cancer
2004;90:2317–25.
[34] Attard G, Belldegrun AS, De Bono JS. Selective blockade of
androgenic steroid synthesis by novel lyase inhibitors as a
therapeutic strategy for treating metastatic prostate cancer.
BJU Int 2005;96:1241–6.
[35] Attard G, Reid AHM, Barrett M, Karavasilis V, Molife R,
Thompson E, et al. Inhibition of androgen synthesis results
in a high response rate in castration refractory prostate
cancer. In: American association for cancer research annual
meeting. 2007.

948
steroids 7 2 ( 2 0 0 7 ) 939–948
receptor gene causes androgen insensitivity in the testicular
feminized rat. J Biol Chem 1990;265:8893–900.
[52] Meyer MD, Kruse LI. Ergoline synthons: synthesis of
3,4-dihydro-6-methoxybenz[cd]indol-5(1H)-one
(6-methoxy-Uhle’s ketone) and
[53] Begtrup M, Elguero J, Faure R, Camps P, Estopa C, Ilavsky D,
et al. NMR studies in the heterocyclic series. Part 31. Effect of
N-substituents on the 13C NMR parameters of azoles. Magn
Reson Chem 1988;26:134–51.
[54] Long BJ, Grigoryev DN, Nnane IP, Liu Y, Ling YZ, Brodie AM.
Antiandrogenic effects of novel androgen synthesis
inhibitors on hormone-dependent prostate cancer. Cancer
Res 2000;60:6630–40.
3,4-dihydrobenz[cd]indol-5(1H)-one (Uhle’s ketone) via a
novel decarboxylation of indole-2-carboxylates. J Org Chem
1984;49:3195–9.