Synthesis and biological evaluations of putative metabolically stable analogs of VN/124-1 (TOK-001): Head to head anti-tumor efficacy evaluation of VN/124-1 (TOK-001) and abiraterone in LAPC-4 human prostate cancer xenograft model

Article abstract of DOI:10.1016/j.steroids.2011.06.002

In a continuing study of our clinical candidate 5 VN/124-1 (TOK-001) and analogs as potential agents for prostate cancer therapy, putative metabolites (10, 15 and 18) of compound 5 were rationally designed and synthesized. However, none of these agents were as efficacious as 5 in several in vitro studies. Using western blot analysis, we have generated a preliminary structure-activity relationship (SAR) of 5 and related analogs as androgen receptor ablative agents (ARAAs). In vivo using the androgen-dependent LAPC-4 prostate cancer xenograft model, we demonstrated for the first time that 5 is more efficacious than the 17-lyase inhibitor 3 (abiraterone)/4 (abiraterone acetate) that is currently in phase III clinical trials. In our desire to optimize the potency of 5, compounds 6 (3ξ-fluoro-) and 9 (3β-sulfamate-) designed to increase the stability and oral bioavailability of 5, respectively were evaluated in vivo. We showed, that on equimolar basis, compound 6 was ～2-fold more efficacious versus LAPC-4 xenografts than 5, but the toxicity observed with 6 is of concern. These studies further demonstrate the efficacy of 5 in a clinically relevant prostate cancer model and justify its current clinical development as a potential treatment of prostate cancer.

Full text of DOI:10.1016/j.steroids.2011.06.002

Steroids 76 (2011) 1268–1279
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and biological evaluations of putative metabolically stable analogs
of VN/124-1 (TOK-001): Head to head anti-tumor efficacy evaluation of VN/124-1
(TOK-001) and abiraterone in LAPC-4 human prostate cancer xenograft model
Robert D. Bruno c,1,3, Tadas S. Vasaitis c,2,3, Lalji K. Gediya a,c, Puranik Purushottamachar a,c
,
Abhijit M. Godbole a,c, Zeynep Ates-Alagoz d,e, Angela M.H. Brodie c, Vincent C.O. Njar a,b,c,
⇑
a Department of Pharmaceutical Sciences, Jefferson School of Pharmacy, Thomas Jefferson University, 130 South 9th Street, Philadelphia, PA 19107, USA
b Kimmel Cancer Center, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA
c Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 685 West Baltimore Street, Baltimore, MD 21201, USA
d Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Tandogan, Ankara 06100, Turkey
e Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of the Sciences, Philadelphia, PA 19104, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In a continuing study of our clinical candidate 5 VN/124-1 (TOK-001) and analogs as potential agents for
prostate cancer therapy, putative metabolites (10, 15 and 18) of compound 5 were rationally designed
and synthesized. However, none of these agents were as efficacious as 5 in several in vitro studies. Using
western blot analysis, we have generated a preliminary structure–activity relationship (SAR) of 5 and
related analogs as androgen receptor ablative agents (ARAAs). In vivo using the androgen-dependent
LAPC-4 prostate cancer xenograft model, we demonstrated for the first time that 5 is more efficacious
than the 17-lyase inhibitor 3 (abiraterone)/4 (abiraterone acetate) that is currently in phase III clinical
trials. In our desire to optimize the potency of 5, compounds 6 (3n-fluoro-) and 9 (3b-sulfamate-)
designed to increase the stability and oral bioavailability of 5, respectively were evaluated in vivo. We
showed, that on equimolar basis, compound 6 was ꢀ2-fold more efficacious versus LAPC-4 xenografts
than 5, but the toxicity observed with 6 is of concern. These studies further demonstrate the efficacy
of 5 in a clinically relevant prostate cancer model and justify its current clinical development as a poten-
tial treatment of prostate cancer.
Received 18 April 2011
Received in revised form 8 June 2011
Accepted 14 June 2011
Available online 24 June 2011
Keywords:
Androgen receptor
Androgen receptor ablative agents CYP17
CYP17 inhibitors
Prostate cancer therapy
VN/124-1 (TOK-001)
Abiraterone
Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction
that 192,280 new cases would be diagnosed and 27,360 patients
would die from the disease in the United States alone [1]. Despite
Prostate cancer (PCA)4 is the most common malignancy, and sec-
ond leading cause of cancer related deaths in men in the western
world. For the year 2009, the American Cancer Society estimated
advances in screening and treatment of localized disease, advanced
prostate cancer remains incurable [2]. Prostate cancer that is resis-
tant to all currently available endocrine therapies remains for the
most part driven by ligand-dependent or ligand-independent activa-
tion of androgen receptor signaling. Although strategies such as
surgical or chemical castrations, which can effectively reduce serum
testosterone levels have remained the most effective therapy for PCA
for over 65 years [3], recent studies clearly show that prostate can-
cers may generate intracrine androgenic steroids and also become
hypersensitive to low steroidal levels through AR gene mutations
and/or amplification supporting continued tumor growth [4–6].
Indeed, the limited prognosis of patients with castration-resistant
prostate cancer (CRPC) on all current types of therapies calls for
the urgent need for new and innovative drugs.
⇑
Corresponding author at: Department of Pharmaceutical Sciences, Jefferson
School of Pharmacy, Thomas Jefferson University, 130 South 9th Street, Philadel-
phia, PA 19107, USA. Tel.: +1 215 503 7468; fax: +1 215 503 9052.
E-mail address: Vincent.njar@jefferson.edu (V.C.O. Njar).
1
Present address: Mammary Stem Cell Biology Section, Mammary Biology and
Tumorigenesis Laboratory Center for Cancer Research National Cancer Institute, 37
Convent Drive, Bldg. 37, Room 1112, Bethesda, MD 20892, USA.
2
Present addresses: Department of Medicine, University of Maryland School of
Medicine, Baltimore, MD, USA and Department of Pharmaceutical Sciences, University
of Maryland Eastern Shore, Queen Anne, MD, USA.
3
These authors contributed equally to this work.
Abbreviations: AIPC, androgen-independent prostate cancer; AR, androgen recep-
4
The efficacy of androgen receptor blockade in the treatment of
prostate cancer [7] and the observation that the androgen receptor
activation is maintained and vital for disease progression has
prompted current interest in the development of new therapies to
tor; ARAAs, androgen receptor ablative agents; CRPC, castration-resistant prostate
cancer; DAST, diethylaminosulfur trifluoride; DHT, dihydrotestosterone, PCA, prostate
cancer; PK, pharmacokinetics; PSA, prostate specific antigen; SAR, structure activity
relationship; SCID, severe combined immunodeficient.
0039-128X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.06.002

R.D. Bruno et al. / Steroids 76 (2011) 1268–1279
1269
F
O
O
S
NC
O
O
O
N
CN
N
S
N
N
N
H
F C
3
CF
O
3
H
O
MDV3100 (1)
BMS-641988 (2)
N
N
N
Abiraterone (3), R = H
Abiraterone
acetate (4), R = COCH
VN/124-1 (TOK-001, 5)
RO
HO
3
Fig. 1. Chemical structures of MDV3100 (1), BMS-641988 (2), abiraterone (3), abiraterone acetate (4) and VN/124-1 (TOK-001, 5).
maximally suppress this vital androgenic pathway [8]. Current
investigations in both preclinical and clinical arena are focused on
discovery and development potent antiandrogens, CYP17 inhibitors
and androgen receptor ablative agents [9]. In these regard, there are
two antiandrogens (MDV3100 (1) and BMS641988 (2)) [10,11], one
CYP17 inhibitor (abiraterone/abiraterone acetate (3/4)) [12] and
the exciting novel multi-active agent VN/124-1 (5, 3b-hydroxy-17-
(1H-benzimidazole)androsta-5,16-diene, now called TOK-001)
[13,14] with antiandrogenic, CYP17 inhibitory and AR ablative activ-
ities in various phases of clinical trials (Fig. 1). Because of promising
results in clinical trials with abiraterone, Hartmann and colleagues
have designed, synthesized and evaluated several non-steroidal abi-
raterone analogs with promising androgen biosynthesis inhibitory
activities [15–18].
We first reported the CYP17 inhibitory, antiandrogenic and
antitumor properties of 5 in 2005 [13] and recently we discovered
that the agent is also a potent AR ablative agent in several human
prostate cancer in vitro and in vivo models [14,19]. In addition, the
compound caused marked reduction of circulating testosterone
levels in the male mouse, androgen-dependent organ weights,
anti-tumor efficacy and it is superior to castration or the clinically
used anti-androgen, bicalutamide. These promising anti-prostate
cancer activities justified its selection for clinical evaluation.
On the basis of previous pharmacokinetic studies of compound
5 in mice which showed extensive metabolism of 5 [13], we have
now synthesized and evaluated putative metabolically stable ana-
logs of the compound and have for the first time conducted a head
to head evaluation of 5 and the CYP17 inhibitor abiraterone (3/4)
that is currently undergoing phase III clinical trials in prostate
cancer patients [20,21]. These studies are the subject of this report.
A preliminary account of part of this work has been reported [22]
and patents to protect these novel and related compounds in the
United States and many countries are pending.
Inova 500 MHz spectrometer. High-resolution mass spectra (HRMS)
were determined on a Bruker12Tesla APEX-Qe FTICR-MSby positive
ionESImodebyMs. SusanA. Hatcher, FacilityDirector,CollegeofSci-
ences Major Instrumentation Cluster, Old Dominion University,
Norfolk, VA. 3b-Hydroxy-5a-androstan-17-one (trans-androster-
one) was purchased from Aldrich. Compound 5 was synthesized in
our laboratory as we previously described [13]. All other reagents
were purchased from Sigma–Aldrich. All compounds were stored
in an atmosphere of argon and in the cold (0–8 °C).
2.1.2. 3b-Hydroxy-17-(3-pyridyl)androsta-5,16-dien (3) and 3b-
acetoxy-17-(3-pyridyl)androsta-5,16-dien (4)
These compounds also known as abiraterone and abiraterone
acetate, prepared by a literature method [23], provided spectral
and analytical data identical with those reported [23,24]. However,
we developed a new purification procedure that resulted in
improved overall yield compared to the reported procedures.
2.1.3. 3b-Hydroxy-17-(1H-benzimidazol-1-yl)androsta-5,16-dien (5)
This compound, prepared as we have previously reported,
provided spectral and analytical as described [13].
2.1.4. 3n-Fluoro-17-(1H-benzimidazol-1-yl)androsta-5,16-dien (6)
Compound 5 (776 mg, 2 mmol) was dissolved in dichlorometh-
ane (30 ml) and to this added diethylaminosulfur trifluoride (DAST,
483 mg, 396 ll, 1.5 eq.) at room temperature. Reaction mixture
was stirred for 1 h and was filtered through basic alumina, washed
(H2O ꢁ 3), dried and evaporated. The crude product was purified
by FCC (dichloromethane:ethanol, 9:1) to afford 530 mg (68%) of
white solid: mp 140–142 °C. 1H NMR (400 MHz, CDCl3): d 1.05 (s,
3H, 18-CH3), 1.10 (s, 3H, 19-CH3), 4.11–4.23 (d, 1/2H, JHF = 48 Hz,
3a-H), 4.37–450 (d, 1/2H, JHF = 50 Hz, 3b-H), 5.46 (s, 1H, 6-H),
6.00 (s, 1H, 16-H), 7.32 (s, 1H, aromatic-H), 7.51 (s, 1H, aromatic-
H). 7.83 (s, 1H, aromatic-H), 7.98 (s, 1H, aromatic-H). HRMS calcd.
391.2544 (C26H30FN2 [M + H]+), found 391.2535.
2. Experimental
2.1. Chemistry
2.1.5. 3b-O-Mesyl-17-(1H-benzimidazol-1-yl)androsta-5,16-dien (7)
A stirring cold solution of 3b-hydroxy-17-(1H-benzimidazol-1-
yl)androsta-5,16-diene (5) (0.388 g, 1 mmol) in pyridne (5 ml) at
0 °C was treated with methane sulfonyl chloride solution (1.2 ml,
1.2 mmol) and was maintained at 0 °C for 30 min. The reaction
mixture was maintained at 0–8 °C for 16 h and then poured over
2.1.1. General
General procedures and techniques were identical with those
previously reported [13]. 1H NMR spectra were recorded in CDCl3
at 500 MHz with Me4Si as an internal standard using a Varian

1270
R.D. Bruno et al. / Steroids 76 (2011) 1268–1279
ice-water mixture (50 ml) with stirring. The crude product precip-
itate was filtered and purified by FCC, [petroleum ether/EtOAc
(9:1)] to give the titled compound 7 (0.37 g, 82.11%): mp 149–
150 °C. 1H NMR (500 MHz, CDCl3): d 1.03 (s, 3H, 19-CH3), 1.04 (s,
white product was filtered and dried under vacuum to give 5.5 g
of desired 3b-acetate: mp 113–115 °C. 1H NMR (500 MHz, CDCl3):
d 0.851 (3H, s, 18-CH3), 0.857 (3H, s, 19-CH3), 2.02 (3H, s, 3b-OAc),
4.68 (1H, m, 3a-H). A solution of 3b-acetoxy-5a-androst-17-one
3H, 18-CH3), 2.92 (s, 3H, 3b-SO2CH3), 4.30 (m, 1H, 3
1H, 6-H), 5.74 (s, 1H, 16-H), 7.15 (s, 1H, aromatic-H), 7.71 (s, 1H,
aromatic-H). 8.31(s, 1H, aromatic-H).
a
-H), 5.44 (s,
(2 g, 6.6 mmol) in dry chloroform (40 ml) was added drop wise
to a cold and stirred solution of phosphorus oxychloride (10 ml)
and dimethylformamide (10 ml). The mixture was allowed to
attain room temperature and then refluxed under argon for 5 h.
It was then concentrated under reduced pressure, poured onto
ice and then extraction with a mixture of ether and EtOAc (8:2,
v/v). The combined extracts were washed with brine and dried
(anhydrous Na2SO4), and solvent was removed under vacuum to
give a white solid (2.3 g). Purification by flash column chromatog-
raphy [FCC, petroleum ether/EtOAc (15:1)] gave the title com-
pound 12 (1.75 g, 77%): mp 155–157 °C; 1H NMR (500 MHz,
CDCl3) d 0.862 (3H, s, 18-CH3), 0.956 (3H, s, 19-CH3), 2.02 (3H, s,
2.1.6. 3a-Azido-17-(1H-benzimidazol-1-yl)androsta-5,16-dien (8)
Compound 7 (0.3 g, 0.77 mmol) was dissolved in DMF (5 ml)
and to this added sodium azide (0.1 g, 1.53 mmol) and heated at
80 °C for 48 h. The reaction mixture was diluted with ice-water
mixture (50 ml), extracted with ethyl acetate (3 ꢁ 25 ml) and sol-
vent was evaporated to obtain crude compound that was purified
by FCC [petroleum ether/EtOAc (7:3)] to give compound
8
(0.18 g, 65.7%): mp 120–122 °C. 1H NMR (400 MHz, CDCl3): d
1.03 (s, 3H, 18-CH3), 1.07 (s, 3H, 19-CH3), 3.92 (m, 1H, 3b-H),
5.47 (s, 1H, 6-H), 6.00 (s, 1H, 16-H), 7.31 (m, 2H, aromatic-H),
7.39 (s, 1H, aromatic-H). 7.84 (s, 1H, aromatic-H), 8.05 (s, 1H,
aromatic-H). HRMS calcd. 436.2471 (C26H31N5ꢂNa+), found
436.2469.
3b-OAc), 4.69 (1H, m,3a-H), 9.98 (1H, s, 16-CHO).
2.1.10. 3b-Acetoxy-17-(1H-benzimidazol-1-yl)-16-formyl-5
a-
androstan-16-ene (13)
A mixture of 3b-acetoxy-17-chloro-16-formyl-5a-androstan-
16-ene (12, 2.5 g, 6.65 mmol), benzimidazole (2.35 g, 19.9 mmol)
and K2CO3 (2.76 g, 23.9 mmol) in dry DMF (20 ml) was stirred at
approx. 80 °C under argon for 1.5 h. After cooling to room temper-
ature, the reaction mixture was poured onto ice-cold water
(250 ml) and the resulting precipitate was filtered, washed with
water and dried to give a crude white solid (ca. 2.9 g). Purification
by FCC [petroleum ether/EtOAc/Et3N (6:4:0.3)] gave 2.7 g (88.7%)
of pure compound 13: mp 217–218 °C; IR (CHCl3) 3691, 3024,
2951, 2359, 1725, 1670, 1604, 1491, 1452, 1375, 1253, 1032,
2.1.7. 3b-O-Sulfamoyl-17-(1H-benzimidazol-1-yl)androsta-5,16-dien
(9)
A stirring cold solution of 3b-hydroxy-17-(1H-benzimidazol-1-
yl)androsta-5,16-diene (5, 0.388 g, 1 mmol) in DMF (5 ml) at 0 °C
was treated with potassium tert-butoxide solution (1.2 ml,
1.2 mmol, 1 M in THF) and was maintained at 0 °C for 30 min.
Sulfamoyl chloride (5 ml, 5 mmol, 1 M in toluene) was added drop
wise over 30 min. The reaction mixture is allowed to attain room
temperature over 2 h, and was cooled to 0 °C, and then quenched
by sequential addition of saturated solution of ammonium chloride
(5 ml) and then water (30 ml). Following extraction with ethyl ace-
tate (3 ꢁ 25 ml), the organic layers were washed with brine
(3 ꢁ 25 ml), dried over anhydrous sodium sulfate and then evapo-
rated to give pure compound 9 as white solid (0.34 g, 72.2%): mp
159–160 °C. 1H NMR (500 MHz, CDCl3): d 1.02 (s, 3H, 18-CH3),
897, 852, 818, 700, 657, 618, 576, 565, 550, 529, 511, 476 cmꢃ1
1H NMR (500 MHz, CDCl3) d 0.88 (s, 6H, 18- and 19-CH3), 2.02 (s,
3H, 3b-OCH3), 4.70 (m, 1H, 3 -H), 7.35 (br. s, 2H, aromatic-Hs),
;
a
7.85 (s, 1H, aromatic-H), 7.87 (s, 1H, aromatic-H), 7.97 (s, 1H, 21-
H) and 9.58 (s, 1H, 16-CHO).
2.1.11. 3b-Acetoxy-17-(1H-benzimidazol-1-yl)-5a-androsta-16-ene
1.03 (s, 3H, 19-CH3), 4.25 (m, 1H, 3a-H), 5.44 (s, 1H, 6-H), 5.74
(14)
(s, 1H, 16-H), 7.15 (s, 1H, aromatic-H), 7.71 (s, 1H, aromatic-H).
8.31 (s, 1H, aromatic-H). HRMS calcd. 490.2134 (C26H33N3O3SꢂNa+),
found 490.2124.
A
solution of 3b-Acetoxy-17-(1H-benzimidazol-1-yl)-16-for-
a-androsta-16-ene (13, 2.04 g, 4.45 mmol) in dry benzoni-
myl-5
trile (10 ml) was refluxed in the presence of 10% palladium on
activated charcoal (1.02 g, i.e., 50% weight of 3) for 5 h. After
cooling to room temperature, the catalyst was removed by filtra-
tion through a Celite pad. The filtrate was evaporated, and the
residue was purified by FCC petroleum ether/EtOAc/Et3N
(7.5:3:0.5)] to give 1.41 g (73.8%) of pure compound 14: mp
159–160 °C; IR (CHCl3) 3687, 2947, 2854, 2358, 2340, 1725,
1633, 1609, 1557, 1489, 1454, 1373, 1291,1253, 1195, 1136,
2.1.8. 3b-Hydroxy-17-(1H-benzimidazol-1-yl)androsta-5-ene (10)
To a solution of 5 (0.388 g, 1 mmol) in ethanol (25 ml) was
added hydrazine hydrate (1.6 ml, 5 mmol) and acetic acid (1 ml)
and reaction mixture was heated at 80 °C while a stream of air
was passed through the solution. The reaction mixture was cooled
to room temperature after 18 h, concentrated to approximately
5 ml under vacuum and poured onto ice cold water. Treatment of
this solution with drop-wise addition of saturated aqueous solu-
tion of sodium bicarbonate resulted in a crude product precipitate.
The crude product was purified by FCC [dichloromethane/MeOH
(9:1)] to give pure compound 10 (0.280 g, 71.9%): mp
143–145 °C; 1H NMR (500 MHz, CDCl3) d 0.67 (s, 3H,18-CH3),
1031, 985, 910, 839, 735, 665, 590, 544, 533,513, 502, 488 cmꢃ1
1H NMR (500 MHz, CDCl3) d 0.876 (s, 3H, 18-CH3), 0.974 (s, 3H,
19-CH3), 2.02 (s, 3H, 3b-OCH3), 4.70 (m,1H, 3 -H), 5.95 (s, 1H,
16-H), 7.30 (m, 2H, aromatic-Hs), 7.49 (s, 1H, aromatic-H), 7.81
(s, 1H, aromatic-H), and 7.94 (s, 1H, aromatic-H).
;
a
0.81 (s, 3H, 19-CH3), 4.69 (m, 1H, 3
a
-H), 7.29 (m, 2H, aromatic-
2.1.12. 3b-Hydroxy-17-(1H-benzimidazol-1-yl)-5a-androsta-16-ene
Hs), 7.43 (s, 1H, aromatic-H), 7.79 (s, 1H, aromatic-H), 8.09 (s,
1H, 21-H). HRMS calcd. 391.2743 (C26H33N2O [M + H+]), found
391.2741.
(15)
The acetate 14 (0.432 g, 1 mmol) was dissolved in methanol
(10 ml) under an inert argon atmosphere, and the resulting solu-
tion was treated with 10% methanolic KOH (4 ml). The mixture
was stirred at room temperature for 1.5 h and then concentrated
under reduced pressure at approximately 40 °C to a volume of
5 ml. This solution was poured into ice water (50 ml), and the
resulting white precipitate was filtered, washed with water and
dried to obtain 15 (0.310 g, 79.48%): mp 189–190 °C. 1H NMR
(500 MHz, CDCl3) d 0.675 (s, 3H, 18-CH3), 0.805 (s, 3H, 19-CH3),
2.1.9. 3b-Acetoxy-17-chloro-16-formyl-5a-androstan-16-ene (12)
First, 3b-acetoxy-5 -androstan-17-one was synthesized from
a
trans–androstane. Thus, trans-androstane (11, 5.8 g, 20 mmol)
was dissolved in 15 ml of pyridine and cooled to 0 °C and to this
added acetic anhydride (7.2 g, 6.6 ml, 3 eq.) over period of
30 min. Reaction mixture was kept in refrigerator for 16 h and then
poured over ice-water (50 ml) under stirring. The precipitated
3.61 (m, 1H, 3a-H), 7.30 (m, 2H, aromatic-Hs), 7.42 (s, 1H,

R.D. Bruno et al. / Steroids 76 (2011) 1268–1279
1271
aromatic-H), 7.78 (s, 1H, aromatic-H), and 8.08 (s, 1H, aromatic-H).
HRMS calcd. 391.2743 (C26H33N2O [M + H]+), found 391.2742.
and data acquisition and management were achieved with a
Waters millennium chromatography manager.
2.1.13. 3b-Acetoxy-17-(1H-benzimidazol-1-yl)androstane (16)
To a solution of 14 (0.432 g, 1 mmol) in ethanol (30 ml) was
added hydrazine hydrate (1.6 ml, 5 mmol) and acetic acid (1 ml)
and the reaction mixture was heated at 80 °C while a stream of
air was passed through the solution. The reaction mixture was
cooled to room temperature after 18 h, concentrated to 5 ml under
vacuum and poured onto ice cold water. Treatment of this solution
with drop-wise addition of saturated aqueous solution of sodium
bicarbonate resulted in a crude product precipitate. The crude
product was purified by FCC [dichloromethane/MeOH (9.5: 0.5)]
to give 0.350 g (73.8%) of pure compound 14: mp 159–160 °C; IR
(CHCl3) 3687, 2947, 2854, 2358, 2340, 1725, 1633, 1609, 1557,
1489, 1454, 1373, 1291,1253, 1195, 1136, 1031, 985, 910, 839,
2.2. Biology
2.2.1. General
The human prostate cancer cell line LNCaP was obtained from
the American Type Culture Collection (Rockville, MD). 293T cells
were the gift of Dr. Yun Qiu (University of Maryland, Baltimore),
and LAPC4 cells were kindly provided by Dr. Charles L. Sawyers
of UCLA School of Medicine. The RPMI 1640 medium, Dulbeccos
Modified Eagle Medium (DMEM), trypsin/EDTA (0.25%/0.02%),
penicillin/streptomycin (P/S), geneticin (G418), and Lipofectamine
were purchased from Gibco-BRL (Grand Island, NY). Phenol red
free IMEM and trypsin/versene were purchased from Biofluids
Inc. (Rockville, MD). Fetal Bovine Serum (FBS) and steroid free
FBS were obtained from Biofluids Inc. (Rockville, MD). Calcium
Phosphate transfection kit was purchased from Invitrogen (Carls-
bad, California).
The 293T and 293T-CYP17 cells were routinely maintained in
DMEM supplemented with 10% fetal bovine serum, 1% penicillin/
streptomycin solution. LNCaP cells were grown in RPMI 1640 med-
ium supplemented with 10% FBS and 1% penicillin/streptomycin
solution. LAPC4 cells were grown in IMEM supplemented with
15% FBS, 1% penicillin/streptomycin solution, and 10 nM DHT.
735, 665, 590, 544, 533,513, 502, 488 cmꢃ1
CDCl3) d 0.67 (s, 3H,18-CH3), 0.81 (s, 3H, 19-CH3), 2.02 (s, 3H, 3b-
OCH3), 4.69 (m,1H, 3 -H), 7.29 (m, 2H, aromatic-Hs), 7.43 (s, 1H,
aromatic-H), 7.79 (s, 1H,aromatic-H), and 8.09 (s, 1H, aromatic-H).
;
1H NMR (500 MHz,
a
2.1.14. 3b-Hydroxy-17-(1H-benzimidazol-1-yl)-5a-androstane (17)
The acetate 16 (1.3 g, 3.02 mmol) was dissolved in methanol
(20 ml) under an inert argon atmosphere, and the resulting solu-
tion was treated with 10% methanolic KOH (8 ml). The mixture
was stirred at room temperature for 1.5 h and then concentrated
under reduced pressure at approximately 40 °C to a volume of
10 ml. This solution was poured into ice water (300 ml), and the
resulting white precipitate was filtered, washed with water and
dried. Crystallization from EtOAc/MeOH gave 15 (1.10 g, 94%),
mp 189–190 °C; IR (CDCl3) 2934, 2339, 1609, 1490, 1453, 1291,
2.2.2. Transfection
Calcium Phosphate transfections were carried out as described
by the manufacturers protocol (Promega Profection Mammalian
Transfection System). Briefly, 100 mm plates were coated with
poly-L-lysine for 30 min, rinsed twice with distilled water and
1040, 837, 808, 705, 663, 608, 578, 550, 517 cmꢃ1
(300 MHz, CDCl3) d 0.675 (s, 3H, 18-CH3), 0.805 (s, 3H, 19-CH3),
3.61(m, 1H, 3 -H), 7.30 (m, 2H, aromatic-Hs), 7.42 (s, 1H, aro-
;
1H NMR
allowed to dry for 2 h. The 293T cells were plated in a 100 mm
plate the day before transfection at a density sufficient for approx-
imately 60% confluency on the day of transfection. Three hours
prior to transfection the media was replaced with fresh growth
a
matic-H), 7.78 (s, 1H, aromatic-H), and 8.08 (s, 1H, aromatic-H).
HRMS calcd. 415.2719 (C26H36ON2ꢂNa+), found 415.2714.
media. The pCDNA3Hmod17His(4) (10
deionized water, vortexed briefly, and then 62
added to bring the final volume to 500 l. This solution was added
drop-wise to 500 l HBS solution, and incubated at room temper-
ature for 30 min. The solution was vortexed again, and distributed
evenly across the 293T cell monolayer. Media was changed 18 h
later, and enzyme activity was assayed as described below 48 h
after transfection. LNCaP-ARR2-Luc transfections were carried out
utilizing LipofectAMINE 2000 transfection reagent (Invitrogen)
according to the manufacturer’s protocol.
l
g) was added to sterile,
l
l 2 M CaCl2 was
2.1.15. 17-(1H-Benzimidazol-1-yl)androsta-4,16-dien-3-one (18)
To a mixture of compound 17 (660 mg, 1.70 mmol), N-methyl-
morpholine-N-oxide (NMO) (2.5 ml) and dichloromethane
(40 ml), was added tetrapropylammonium perruthenate (TPAP)
(521 mg, 2.55 mmol) and molecular sieves (0.5 g), and the mixture
was stirred under argon for 3 h. The mixture was filtered and
diluted with EtOAc (50 ml), washed successively with 5% aqueous
NaHCO3 (x3) and brine (x2) and then dried (Na2SO4). The solvent
was evaporated, and the crude product was purified by FCC
[CH2Cl2/EtOH (25:1)] to give the title compound 16 (544 mg,
82%): mp 215–216 °C; IR (CHCl3) 2946, 2858, 1622, 1611, 1490,
1453, 1376, 1291,1270, 1228, 1189, 893, 850, 837, 722, 662, 615,
l
l
2.2.3. Acetic acid releasing assay for CYP17 activity
The 293T cells were transfected with the human CYP17 (293T-
CYP17) as described in transfections. The 293T-CYP17 cells were
grown in a 100 mm dish for 24 h and then evenly divided into 6
well tissue culture plates and incubated for 24 h. The cells were
washed with Dulbecco’s Phosphate Buffered Saline (DPBS), and
568, 553, 537,519 cmꢃ1 1H NMR (500 MHz, CDCl3) d 0.84 (s, 3H,
;
18-CH3),1.02 (s, 3H, 19-CH3), 7.27(m, 2H, aromatic-Hs), 7.41 (m,
1H, aromatic-H), 7.79 (s, 1H, aromatic-H), and 8.09 (s, 1H, aro-
matic-H). HRMS calcd. 391.2743 (C26H33ON2 [M + H]+), found
391.2735.
incubated with steroid-free DMEM (1 ml) containing 0.5–20
of [21-3H]17
-hydroxypregnenolone. This substrate is converted
to DHEA and [3H]acetic acid is released during cleavage of the
C-21 side chain. Cold 17 -hydroxypregnenolone (1 mM) was
lM
a
2.1.16. HPLC analysis for relative retention time of putative
metabolites
a
added to control wells for detection of non-specific substrate con-
version. After an 18 h incubation at 37 °C (16 h for LNCaP-CYP17)
medium was collected and the steroids were extracted by shaking
with 2 ml of chloroform at 4 °C. After 30 min, the aqueous phase,
which contains the [3H]acetic acid, was collected, and a charcoal
suspension (2.5% final concentration) was added to it. Following
a 30 min incubation at 4 °C, 1 ml of the supernatant was removed
and radioactivity measured by liquid scintillation counting. After
Chromatographic separations and identification of the putative
metabolites were achieved by a reverse phase HPLC method on a
Waters SymmetryÒ C18 Column (4.6 ꢁ 75 mm). Briefly, the HPLC
system used in this study consisted of Waters Alliance System
coupled with 2695 Separation Module and Waters 2998 PDA
detector operated at 254 nm. The mobile phase composition was
Water/MeOH/CH3CN (35:35:30, v/v/v + 200 ll of Et3N and 0.77 g
of NH4OAc per 1000 ml of mobile phase) at a flow rate of 1.0 ml/
min. The HPLC analysis was performed at ambient temperature,
determining the saturating concentration of [21-3H]17
pregnenolone (6.8 M), compounds were evaluated for IC50 values
a-hydroxy-
l

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R.D. Bruno et al. / Steroids 76 (2011) 1268–1279
by incubation with medium (1 ml) containing the saturating con-
centration of [21-3H]17
-hydroxypregnenolone (6.8 M) and
different concentrations (1–1000 nM) of the inhibitors. After an
18 h incubation, the medium was analyzed as described above
and IC50 values were determined with Graphpad Prism software
(GraphPad Software, Inc., San Diego, CA).
streptomycin solution, and 10 nM DHT. To determine the effect
of steroids and novel compounds on cell proliferation, each cell
type was transferred into steroid-free medium three days prior
to the start of the experiments (steroid-free medium consisted of
phenol red free RPMI supplemented with 5% dextran-coated,
charcoal treated serum, and 1% penicillin/streptomycin solution).
Growth studies were then performed by plating cells (1.5 ꢁ
104 cells/well) in 24-well multiwell dishes (Corning, Inc., Corning,
NY). After a 24 h attachment period, the medium was aspirated
and replaced with steroid-free medium containing vehicle or the
indicated concentrations of androgens and novel compounds
a
l
2.2.4. Androgen receptor competitive binding assay
Competitive binding assays were performed with the synthetic
androgen methyltrienolone [3H]R1881 essentially as described by
Wong et al. and Yarbrough et al. [25,26]. Wells in 24-well multi-
well dishes were coated with poly-L-lysine (0.05 mg/ml) for
(1 nM–5 lM). Control wells were treated with vehicle (ethanol).
30 min, rinsed with sterilized, distilled water, and dried for 2 h.
To determine the kinetics of [3H]R1881 binding to the LNCaP AR
and the wild-type AR, LNCaP and LAPC4 cells were plated
(2–3 ꢁ 105 cells/well) in 24 well multiwell dishes in steroid-free
medium and allowed to attach. The following day the medium
was replaced with serum-free, steroid free RPMI supplemented
with 0.1% BSA and containing [3H]R1881 (0.01–10 nM) in the
presence or absence of a 200 fold excess of cold DHT, to determine
The medium was changed every three days and the cell viability
was compared by XTT assay on the seventh day per the manufac-
turers protocol (Invitrogen, Carlsbad, California). All results
represent the average of a minimum of three wells. Plates were
read at 450 nM with
spectrophotometer.
a Victor 1420 scanning multi-well
2.2.7. Protein expression – gel electrophoresis and western blotting
Equal amounts of total protein (50–100 g) were subjected to
nonspecific binding, and 1
l
M triamcinolone acetonide to saturate
l
progesterone and glucocorticoid receptors. Following a 2 h incuba-
tion period at 37 °C, cells were washed twice with ice-cold DPBS
and solubilized in DPBS containing 0.5% SDS and 20% glycerol. Ex-
tracts were removed and cell associated radioactivity counted in a
scintillation counter. The data was analyzed, including Kd and Bmax
determination, by nonlinear regression using Graphpad Prism
software (GraphPad Software, Inc., San Diego, CA). When the
concentration of [3H]R1881 required to almost saturate AR in both
cell lines was established (5.0 nM), the ability of the test com-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–
PAGE 60 V, 3 h) and transferred (90 V, 1 h) to nitrocellulose mem-
branes (Hybond ECL, Amersham). Immunodetections were per-
formed using mouse monoclonal antibodies against human
androgen receptor (SC-7305 Santa Cruz Biotechnologies, Inc., Santa
Cruz, CA). Immunoreactive bands were visualized using the en-
hanced chemiluminescence detection reagents (Amersham Corp.,
Arlington Heights, IL) according to the manufacturer’s instructions
and quantified by densitometry using Bio-Rad software (Quantity
One™).
pounds (1 nM–10 l
M) to displace [3H]R1881 (5.0 nM) from the
receptors was determined as described above. The IC50 of each
compound was determined by nonlinear regression with Graphpad
Prism software (GraphPad Software, Inc., San Diego, CA).
2.2.8. Mouse xenograft studies
All animal studies were performed according to the guidelines
and approval of the Animal Care Committee of the University Of
Maryland School of Medicine. Male SCID mice 4–6 weeks of age
were obtained from the National Cancer Institute-Frederick Cancer
Research Center (Frederick, MD). The mice were housed in a path-
ogen-free environment under controlled conditions of light and
humidity and received food and water ad libitum. Sub-confluent
cells were scraped into DPBS, collected by centrifugation and re-
suspended in Matrigel (10 mg/ml) at 2.0 ꢁ 107 cells/ml. Each
mouse received subcutaneous inoculations in 1 site per flank with
2.2.5. Transcriptional activation – luciferase assay
LNCaP cells were transferred to steroid-free medium 3 days
before the start of the experiment, and plated at 1 ꢁ 105 cells/well
in steroid-free medium. The cells were dual transfected with ARR2-
Luc and the Renilla luciferase reporting vector pRL-null with
LipofectAMINE 2000 transfection reagent (Invitrogen, Carlsbad,
California) according to the manufacturer’s protocol. After a 24 h
incubation period at 37 °C, the cells were incubated with fresh
phenol-red free serum-free RPMI 1640 medium and treated with
DHT, ethanol vehicle and/or the selected compounds in triplicate.
After an 18 h treatment period the cells were washed twice with
ice-cold DPBS and assayed using the Dual Luciferase kit (Promega)
according to the manufacturer’s protocol. Briefly, cells were lysed
100 ll of cell suspension. Once tumors formed in enough mice and
had an average tumor volume of at least 90 mm3, mice were
grouped (10–12 mice per group) with equal average tumor
volumes. Doses were administered as outlined with all compounds
formulated in 0.3% hydroxypropyl cellulose (HPC) in 0.9% saline.
Tumors were measured twice weekly with calipers and tumor vol-
with 100
ll of luciferase lysing buffer, collected in a microcentri-
fuge tube, and pelleted by centrifugation. Supernatants (100
ll
ume was calculated by the formula 4=3
p
r21 ꢁ r2, where r1 is the
aliquots) were transferred to corresponding wells of opaque
96-well multiwell plates. Luciferin was added to each well, and
the light produced during the luciferase reaction was measured
in a Victor 1420 scanning multi-well spectrophotometer (Wallac,
Inc., Gaithersburg, MD). After measurement, Stop and Glo reagent
(Promega) was added to quench the firefly luciferase signal and
initiate the Renilla luciferase luminescence. Renilla luciferase
luminescence was also measured in the Victor 1420. The results
are presented as the fold induction, that is, the relative luciferase
activity of the treated cells divided by that of the control, normal-
ized to that of the Renilla.
smaller radius and r2 is the larger radius. Animals were also
weighed weekly and monitored for general health status and signs
of possible toxicity due to treatment. At the end of the treatment
period, the animals were sacrificed under isoflurane anesthesia;
tumors were excised and weighed. For in vivo comparison of the ef-
fects of 5 and 3, testis, seminal vesicles, and prostates were
removed and weighed upon sacrifice as well.
2.2.9. Statistical analysis
Western blots were analyzed with a Kruskal–Wallis and Dunn’s
multiple comparison post-hoc. For the LAPC-4 xenograft study
comparing 5–3 and 4, statistical analysis was carried out as
follows. The tumor volume data were log transformed due to the
exponential growth of tumors in mice. The mixed-effects linear
models were used to model the data. The resulting multilevel
regression model included random effects associated with tumor
2.2.6. Cell proliferation – tetrazolium salt reduction
LNCaP cells were grown in RPMI 1640 medium supplemented
with 10% FBS and 1% penicillin/streptomycin solution. LAPC4 cells
were grown in IMEM supplemented with 15% FBS, 1% penicillin/

R.D. Bruno et al. / Steroids 76 (2011) 1268–1279
1273
side nested within mouse. The regression model had random ef-
fects for intercept and slope (day) at mouse level.
synthesized and test the 3b-sulfamate analog of compound 5. Thus,
5 was sulfamoylated to afford 3b-O-sulfamoyl-17-(1H-benzimid-
azole)androsta-5,16-diene (9) (Scheme 1) using tert-butoxide and
sulfamoyl chloride in DMF [32].
The tumor growth rate was estimated for each treatment group.
Average tumor growth rate was also compared between the
following groups: compound 5 and control, 5 and castration, 5
and 3, 5 and 3, 4 and control. Treatment means (average tumor vol-
umes) were also compared across groups on day 0 and day 31. As
intended, there were no differences in groups’ means on day 0,
p = 0.96.
It was also of interest to synthesize possible metabolites of com-
pound 5. The rationale for the three compounds (10, 15 and 18) syn-
thesized was based on the fact that the metabolite previously
isolated had a molecular mass of 390 amu [13]. This together with
the known metabolic transformations of steroidal molecules led us
to synthesize compounds with: (i) reduced D16 double bond (10),
(ii) reduced D5 double bond (15) and (iii) reduced D5 and
D
16 and
3. Results and discussion
oxidation of 3b-OH ? 3-oxo (18) as outlined in Schemes 1 and 2.
The saturated D-ring analog, 10 was readily prepared from 5 by
reduction with diimide as we have previously reported for closely
related 17-imidazole [33]. Compounds 15 and 18 (Scheme 2) were
also readily synthesized from the commercially available trans-
androsterone (11). The key intermediate in our synthesis of these
3.1. Chemistry
On the basis of the molecular mass (m/z 391 = M + H+) of the
metabolite of 5 in the mouse, we had tentatively identified it as
17-(1H-benzimidazol-1-yl)androst-3-one [13]. We reasoned that
the metabolite may have been formed from 5 via oxidation of
the 3b-OH ? 3-oxo, followed by reduction (reductases?) of both
D5 and D16 double bonds. Consequently, our studies focused on
generation analogs with modification of the 3b-OH of compound
5, via bioisostere replacement with known metabolically stable
functional groups, with specifically fluoro (F) or azido group (N3)
to afford compounds 6 and 8, respectively (Scheme 1). Both func-
tional groups are known to act as hydrogen bond acceptors just
as the hydroxyl group and are present in some approved drugs.
3n-Fluoro-17-(1H-benzimidazole)androsta-5,16-diene (6) was
readily synthesized in excellent yield from 5 following treatment
with diethylaminosulfur trifluoride (DAST) at room temperature
[27]. This compound was clearly shown by 1H NMR (integration
of 3-H signals) to be a stereoisomers mixture, consistent with
reported NMR data for related 3
pounds [28]. Several attempts to separate the two stereoisomers
were unsuccessful, and may constitute future studies. The 3 -azi-
do compound 8 was synthesized by reaction of 5 with methane
sulfonyl chloride to give the corresponding mesylate (7) that was
then treated with sodium azide in dimethylformamide (DMF) at
80 °C [29]. This reaction sequence is reported to give the final azide
with complete inversion of configuration [30], consistent with its
1H NMR data. Finally, because of improved oral bioavailability
reported for several steroidal compounds with sulfamate substitu-
two putative metabolites, 3b-acetoxy-17-chloro-16-formyl-5a-
androstan-16-ene (12) was prepared by our routine procedure as
previously described [13,33]. Treatment of 12 with benzimidazole
in the presence of K2CO3 in DMF at ꢀ80 °C gave the desired
3b-acetoxy-17-1H-benzimidazole 13 in near quantitative yield.
Compound 13 was smoothly deformylated with 10% palladium
on activated charcoal in refluxing benzonitrile to give 14 in excel-
lent yield, from which hydrolysis gave the required 3b-hydroxy-
D
5-dihydro 17-benzimidazole 15. Compound 14 was also
subjected to D-ring saturation as described above for 10, from
which hydrolysis gave 3b-hydroxy-
5,16-tetrahydro 17-benzimid-
D
azole 17. Finally, oxidation of 17 with tertapropylammonium
perruthenate (TPAP) in CH2Cl2 in the presence of N-methylmor-
pholine N-oxide (NMO) as co-oxidant [34] gave the desired
3-oxo-D
5,16-tetrahydro 17-benzimidazole 18 (Scheme 2).
a- and 3b-fluoro steroidal com-
Interestingly, we observed that these three putative mouse
metabolites (10, 15 and 18) of compound 5, with the same
molecular mass (390 amu) as the mouse metabolite of 5 exhibited
different reverse phase HPLC chromatographic profiles with differ-
ent retention times (tR) of 10.88, 22.67 and 15.02, minutes, for 10,
15 and 18, respectively (tR for 5 = 18.79 min) (Fig. 2). Of these three
compounds, 15 showed identical retention time as the metabolite
of compound 5, reported in our previous study. Clearly, further
studies are required to obtain adequate amounts of the metabolite
for rigorous identification by proton nuclear magnetic resonance
a
tions at C3 [31,32], we consider it
a
worthy endeavor to
N
N
N
N
N
N
i
ii
O
S
F
H N
2
O
HO
O
9
5
6
v
iii
N
N
N
N
N
N
H
iv
H
O
S
O
HO
N
3
O
8
7
10
Scheme 1. Synthesis of compound 5 analogs (6, 8, 9 and 10). aReagents and conditions: (i) DAST, DMC, rt, 1 h; (ii) t-BuOK, NH2SO2Cl, 0 °C – rt, 2 h; (iii) MeSO2Cl, py, 0–8 °C,
16 h; (iv) NaN3, DMF, 80 °C, 48 h; (v) H2NNH2ꢂH2O, AcOH, EtOH, air, reflux, 18 h.

1274
R.D. Bruno et al. / Steroids 76 (2011) 1268–1279
N
N
N
N
Cl
O
CHO
CHO
i, ii
iii
iv
AcO
AcO
HO
AcO
11
12
13
14
vi
v
N
N
N
N
N
N
N
N
H
vii
v
O
HO
AcO
HO
H
15
16
18
17
Scheme 2. Synthesis of compounds 15 and 18. aReagents and conditions: (i) Ac2O, py, 0–8 °C, 16 h; (ii) POCl3-DMF, CHCl3, Ar, reflux; (iii) benzimidazole, K2CO3, DMF, Ar,
80 °C; (iv) 10% Pd on activated charcoal, PhCN, reflux; (v) 10% methanolic KOH, Ar, rt; (vi) H2NNH2ꢂH2O, EtOH/AcOH, air, 80 °C, 18 h; (vii) TPAP, NMO, DMC, rt, 3 h.
Fig. 2. HPLC chromatogram of 5 and its putative synthesized metabolites 10, 15 and 18. The retention times (tR) for 10, 18, 5 and 15 are 10.88, 15.02, 18.79 and 22.67 min,
respectively.
Table 1
CYP17, growth inhibitory activities and androgen receptor binding of 5 and analogs.
Compound
CYP17 IC50 (nM)a
AR binding EC50 (nM)b
Wild-type (LAPC-4)
Cell growth inhibition GI50 (l
M)c
Mutant (T877A, LNCaP)
LNCaP
LAPC-4
5
6
8
15
47
150
120
405
934
2460
–
1100
4300
1240
1600
4340
–
1280
971
6.0
8.0
7.0
17.6
–
3.2
16.0
19.0
–
–
–
–
*
18
For comparison: Casodex
–
–
a
b
c
IC50 is the concentration of inhibitor required to inhibit the CYP17 enzyme activity by 50%, each in duplicate.
EC50 is the concentration of compound required for a 50% displacement of [3H]R1881 from the androgen receptor.
GI50 is the concentration of compound required to cause 50% growth inhibition of LNCaP or LAPC-4 cells.
Less than 30% inhibition at 500 nM.
*
–
Not determined.
(1H NMR) spectroscopy and high resolution mass spectroscopy
(HRMS).
and 8, respectively (Table 1). However, they were less potent than
5 which has an IC50 of 47 nM [35], which suggest that the 3b-OH is
important for CYP7 inhibition. In sharp contrast, one of the puta-
tive metabolites of 5, that is, 18 exhibited weak inhibition of
CYP17 (less than 30% inhibition at 500 nM).
3.2. Inhibition of CYP17 and androgen receptor (AR) binding studies
In competitive binding studies, the ability to displace [3H]R1881
from both the wild-type AR (in LAPC-4 cells) and the mutant T877A
AR (in LNCaP cells) occurred in a dose-dependent manner (figure
These studies were conducted as we have previously reported
[13,14]. The new analogs of compound 5 still afforded strong
CYP17 lyase inhibition, with IC50 values of 150 and 120 nM for 6

R.D. Bruno et al. / Steroids 76 (2011) 1268–1279
1275
Fig. 3. Effect of compounds 6 and 8 on DHT stimulated transcription. LNCaP Cells were transfected with the ARR-2 reporter construct and treated with novel compounds in
the presence or absence of DHT. Blue bars represent treatment with DHT and the indicated concentration of compound. Yellow bars represent treatment with compound
only. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)
not shown). Compounds 6 and 18 with EC50 values of 934 and
1100 nM versus the wild-type AR were moderately reduced
(2.3- and 2.7-fold), respectively compared to 5. However, binding
of both compound to the mutant AR were not significantly differ-
ent from binding of 5. In contrast the binding affinities of
compound 8 to both receptors were greatly reduced (Table 1). As
shown in (Fig. 3) both compounds 6 and 8 were found to be pure
AR antagonists in transcriptional activation studies. However, nei-
ther was as potent as 5 at inhibiting androgen-induced AR
transcription.
3.3. Inhibition of prostate cancer cell proliferation
In cell viability assays, compounds 6 and 8 inhibited LNCaP
proliferation by 85% and 60% at 10
did not equal the effectiveness of 5. In DHT stimulated LNCaP cells,
6 had an IC50 of 8 M, while 8 was less effective with an IC50 of
16 M. Both compounds exhibited a similar pattern in DHT stimu-
lM, respectively, which also
l
l
lated LAPC4 proliferation, with IC50 values of 7 lM and 19 lM for 6
and 8, respectively (Table 1).
3.4. Effects of agents on AR down-regulation
Because we had previously demonstrated that some of our
novel CYP inhibitors/AR antagonists are also strong AR down-reg-
ulating agents [14], we assessed the abilities of the new analogs
to cause AR down-regulation in LNCaP cells. Compound 5 was still
the most effective compound in vitro with nearly complete (>95%)
Fig. 4. Densitometry analysis of AR protein expression in treated LNCaP Cells. Cells
were treated with test compounds for 24 h at the indicated concentrations. Cell
extracts were prepared and probed with anti-AR and anti-bactin antibodies.
Densitometry quantification was performed utilizing ImageQuant 5 software.
AR down-regulation at 15
in this study, 6 was able to down regulate AR expression by 50% at
15 M, the same effect was achieved with 10 M VN/124-1 in
LNCaP cells (Fig. 4). Compounds 8, 10, 15 and 18 did not cause
down-regulation of AR expression even at the highest concentra-
lM. Of the three new compounds tested
azido (N3) group completely abolished activity versus AR down-
regulation. Other modifications that also resulted in drastic reduc-
tion in AR down-regulation include, saturation of either or both D5
and D16 double bonds, introduction of 16-methyl alcohol or
replacement of 17-benzimidazole with substituted and unsubsti-
tuted purines (data not shown).
l
l
tion (15 lM) tested (data not presented). On the basis of our previ-
ous and present studies, we have derived a preliminary in vitro
structure–activity relationship (SAR) of our CYP17 inhibitors/AR
antagonists as androgen receptor ablative agents (ARAAs), summa-
rized in Fig. 5. There is no clear correlation of agents AR binding
affinities to AR down-regulating activities. Modifications of the
A- or B-ring resulted in significant decrease in AR down-regulating
activity, suggesting that 3b-OH-D5 moiety may be critical for
activity. While isosteric replacement of b-OH group at C-3 with
fluorine (F) significantly reduced activity, replacement with an
3.5. Evaluation of in vivo efficacy of compounds against LAPC4
xenografts
We have previously demonstrated the exceptional anti-tumor
efficacy of our lead compound 5 in several prostate cancer xeno-
graft models [13,14]. In the present study, we wanted to compare
the anti-tumor efficacy of 5 versus its putative metabolically stable

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R.D. Bruno et al. / Steroids 76 (2011) 1268–1279
N
N
Benzimidazole > imidazole;
purines not tolerated
Saturation not
tolerated
Methyl alcohol
not tolerated
4
3 -OH- 5 > 3-oxo-
Δ
HO
β
Δ
3 - OH > F >> N
β
Saturation not tolerated
3
Fig. 5. In vitro SAR of CYP17 inhibitors/antiandrogens as androgen receptor ablative
agents (ARAAs).
Fig. 6b. Effect of 5 and 3 on LAPC-4 tumor weights. Excised LAPC-4 tumors were
weighed following 31 days of treatment. ⁄p < 0.05 compared every other group.
excision, final tumor weights were also significantly reduced in
animals treated with compound 5 compared to animals treated
with control, 3, and castration (p < 0.05; Fig. 6b).
The effect of each treatment on the weights of the prostate,
seminal vesicles, and the testes was also measured (Fig. 6c). As
these tissues are sensitive to androgens, a reduction in their weight
is a biomarker for androgen deprivation. Compounds 5 and 5-ace-
tate significantly reduced the size of the seminal vesicles and
testes, and also reduced the size of the prostate, although the
reduction was not quite significant. Compound 3 failed to signifi-
cantly reduce the size of any of the organs, while 4 significantly
reduced the size of the seminal vesicles. None of the treatments
resulted in any noticeable toxicity, as exemplified by no significant
changes in body weight (data not shown).
The effect of both 5 and 3 on AR expression within tumors was
also compared (Fig. 7). Consistent with previous reports [14], 5
treatment resulted in an average 2-fold reduction in AR protein
levels with the majority of tumors analyzed not expressing any
detectable levels of AR. Compound 3, on the other hand, had no
significant effect on AR levels with an average expression found
to be slightly higher than that found in control tumors. The differ-
ence in the effect of each molecule on AR levels is striking and
represents a major distinction of compound 5’s therapy. It should
be noted that the in vivo efficacy of targeting AR expression to
block tumor growth and delaying tumor progression was first
demonstrated in 2006 by using short hairpin RNA (shRNA)-medi-
ated AR knockdown in an LNCaP hormone-dependent tumor
xenograft model [36]. Subsequently, the same group recently
(2009) demonstrated for the first time that this strategy is also a
viable therapeutic strategy for prostate C4–2 tumors that have
progressed to the castration-resistant state [37]. These important
findings provide strong proof-of-principle that inhibition of AR
Fig. 6a. Effect of 5 and 3/4 on the growth of LAPC-4 tumors in vivo. Male SCID mice
were inoculated with LAPC-4 cells and treated with either vehicle (0.3% HPC),
0.15 mmol/kg of 5 (s.c. b.i.d.), 3 (s.c. b.i.d.), 5-acetate (p.o. o.d. ) b.i.d), or 4 (p.o., o.d.
) b.i.d.). 5 b.i.d. treated tumors had a significantly reduced growth rate over
control, 3 and 4 (p < 0.0001, p = 0.025, p < 0.0001, respectively). " = marks day at
which p.o. dosing of 4 and 5-acetate were increased to b.i.d.
analog, 6, prodrug sulfamate 9 and abiraterone (3) (head-to-head
experiment between 5 and 3/4), a CYP17 inhibitor that is currently
undergoing phase III clinical trials n prostate cancer patients
[20,21]. However, for the sake of clarity, these experiments are
presented as three in vivo studies.
In the first in vivo study, the relative efficacies of 5 and 3 were
compared in vivo in an LAPC-4 xenograft model. Mice inoculated
with LAPC-4 tumors were treated subcutaneously with
0.15 mmol/kg of 3 or 5 twice daily. Because compound 3 is admin-
istered clinically orally as the acetate pro-drug, mice were also
treated with either 3-acetate (4) or 5-acetate 0.15 mmol/kg/o.d.
p.o. At day 24, oral dosing of 3 and 4 was changed to twice daily
because neither compound demonstrated any significant efficacy.
As shown in Fig. 6a, 5 s.c. b.i.d. was the most effective therapy in
inhibiting the growth of LAPC-4 tumors. Surprisingly, even tumors
in the castration group grew rapidly suggesting the LAPC-4 cells
used in the inoculation may have acquired some resistance to
androgen-deprivation therapy. Such acquired androgen indepen-
dence has been shown to occur after long-term culture of LNCaP
cells [19], and it is possible a similar effect has occurred here.
Nonetheless, mice treated with 5 had smaller average tumor vol-
ume on day 31 when compared to control (p = 0.0001), 3-acetate
(4) (p = 0.005), 3 (p = 0.053, of marginal statistical significance),
and 5-acetate (p = 0.006). Compound 5 treatment also significantly
reduced the growth rate of tumor growth compared to control, 3
expression represents
a therapeutically relevant strategy for
prostate cancer treatment.
In preclinical studies, we found that compound 5 is not orally
active. Because oral formulations of drugs are preferred clinically,
we synthesized a sulfamate prodrug analog of 5, that is, compound
9, with the hope that it would be orally active. Our motivation was
based on previous report of improved oral bioavailability for sev-
eral steroidal compounds with sulfamate substitutions at C3
[31,32]. However, studies with 9 administered orally as described
for 5 above did not show any significant effect on LAPC-4 tumor
growth (data not presented). It should be noted that formulated
(micronized) 5 is administered orally to patients in ongoing phase
I/II clinical trials.
In the third in vivo experiment, we tested the anti-tumor effi-
cacy of 5 versus its putative stable 3-fluoro analog, compound 6.
and
4 (p < 0.0001, p = 0.025, p < 0.0001, respectively). Upon

R.D. Bruno et al. / Steroids 76 (2011) 1268–1279
1277
Fig. 6c. Effect of 3, 4, 5 and 5-acetate on urogenital tissue weights. Following 31 days of treatment, urogenital tissues from mice in each treatment group were excised and
weighed. ⁄p < 0.05, ⁄⁄p < 0.01 compared to control.
Fig. 7. Effect of abiraterone (3) abiraterone acetate (4) and VN/124-1 (5) on AR protein levels in vivo. Tumors from each treatment group were excised and analyzed by
western blot for relative AR expression: (A) AR expression of representative tumors. (B) Average AR expression of at least 3 tumors from each treatment determined by
densitometry ⁄p < 0.05.
administration of 5. Once daily administration of 5 did not have
a significant effect on tumor growth. Unfortunately, treatment
with 6 resulted in the death of five mice in the once daily
administration group and two mice in the twice daily administra-
tion group. The mice began to die around day 25, with two mice
dying before the final measurement on day 31. The remaining mice
died following the last measurement but before necropsy (2 days
later). It is likely the drug accumulated overtime resulting in the
toxicities after extended treatment. Large deposits of compound
6 were discovered under the skin of the mice during necropsy. De-
spite the apparent toxicity, no changes in mouse weights were
observed (data not shown) and no overt signs toxicity were seen
during the internal examination of the mice. Clearly, additional
studies are warranted with this novel compound.
Fig. 8a. Effect of 5 and 6 on LAPC-4 tumor growth in vivo. Male SCID mice were
inoculated with LAPC-4 cells and treated with either vehicle (0.3% HPC), 0.15 mmol/
Tumor samples treated with 6 were analyzed to determine the
molecules effect on AR protein levels. As the AR was markedly re-
duced with treatment with 5, it was expected that twice daily
administration of 6 would also have an effect. However, as shown
in Fig. 8b and c, 6 had only a minor and statistically insignificant
effect on AR protein levels.
kg/b.i.d of
5 s.c., 0.15 mmol/kg/o.d. of 5 s.c., 0.15 mmol/kg/o.d. of 6 s.c., or
0.15 mmol/kg/b.i.d. of 6 s.c. Tumors were measured with calipers as described in
the materials and methods.
SCID mice were inoculated as before with the LAPC-4, and tumors
were allowed to form for 6 weeks until the average tumor volume
was ꢀ90 mm3. Mice were then grouped: 10 mice per group and
treated with once or twice daily doses of 0.15 mmol/kg 5 or 6.
As shown in Fig. 8a, once daily administration of 6 was as
effective in inhibiting LAPC-4 tumor growth as twice daily
4. Conclusions
The superior in vivo anti-tumor efficacy (prevention of tumor
formation and inhibition of tumor growth) of our investigational
new drug (IND) 5 in LAPC-4 tumor xenografts that express AR

1278
R.D. Bruno et al. / Steroids 76 (2011) 1268–1279
Fig. 8b and c. Effect of 6 on AR levels in vivo. (B) Tumors from each treatment group were excised and analyzed by western blot for relative expression of the AR. (C) Average
expression as determined by densitometry.
versus the PC-3 xenografts that lack AR, reinforces our previous
findings [14] that AR ablation is an important anti-cancer property
of compound 5. The present studies also provide compelling evi-
dence that 5 is a potent inhibitor of human prostate cancer tumor
growth and is remarkably more effective than castration or com-
pound 3 (4) that is currently undergoing phase III clinical trials
in prostate cancer patients. We also show that neither of the plau-
sible metabolites of 5 is as effective as the parent compound which
suggests that it may be possible to improve on its in vivo efficacy if
the site of metabolism is unequivocally identified and blocked. In
addition, because we strongly believe that the unique AR ablative
property of 5 is what distinguishes it from other closely related
compounds; our efforts are currently directed to develop new ana-
logs of compound 5 with nanomolar AR ablative activities. We also
showed, that on equimolar basis, compound 6 was ꢀ2-fold more
efficacious versus LAPC-4 xenografts than 5, but the toxicity ob-
served with 6 is of concern. Overall, these results strongly support
the ongoing clinical evaluation of compound 5 and we eagerly
await the phase I/II trial findings.
supported in part by US National Institutes of Health training grant
T32 AG000219.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.steroids.2011.06.002.
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