N-substituted piperazinopyridylsteroid derivatives as abiraterone analogues inhibit growth and induce pro-apoptosis in human hormone-independent prostate cancer cell lines

Article abstract of DOI:10.1111/cbdd.12195

Nine new 17-(piperazin-1-yl)pyridin-5-yl)steroids as abiraterone analogues were synthesized. Compounds 5d and 5g showed selective activities against 17α-hydroxylase/C17,20-lyase (CYP17A1) and aromatase (CYP19), respectively. IC₅₀ values of 5d were 5.09 and >50 μm, whereas these values for 5g were >50 μm and 7.40 μm, respectively, for CYP17A1 and CYP19. Molecular modelling highlighted that the inhibitor designed to bind cytochrome P450 haem iron is a necessary condition but not the only rationale to explain inhibitory activity. These abiraterone analogues were then evaluated on hormone-independent prostate cancer cell lines DU-145 and PC-3 and on hormone-dependent breast and prostate cancer cell lines MCF-7 and LNCaP, respectively. Compounds 5e, 5g and 5i have showed potent activities only on hormone-independent prostate cancer cell lines DU-145 and PC-3 with 60-85% inhibition of both cell viability and growth at 10 nm with pro-apoptotic mechanism as illustrated in PC-3 cells by DNA ladder assay and Western blotting of Bax, Casp-3 and its substrate, the poly (ADP-ribose) polymerase. We conclude that hybrid heterocycle steroids could be good lead compounds in the drug design especially against hormone-independent prostate cancer. Three derivatives showed potent activities only on hormone-independent prostate cancer cell lines DU-145 and PC-3 with 60-85% inhibition of both cell viability and growth at 10 nm with pro-apoptotic mechanism.

Full text of DOI:10.1111/cbdd.12195

Chem Biol Drug Des 2013; 82: 620–629
Research Letter
N-substituted Piperazinopyridylsteroid Derivatives as
Abiraterone Analogues Inhibit Growth and Induce
Pro-apoptosis in Human Hormone-independent
Prostate Cancer Cell Lines
Dominique Brossard1,2, Ying Zhang1,3, Shozeb M.
of Bax, Casp-3 and its substrate, the poly
4
4
5
(ADP–ribose) polymerase. We conclude that hybrid
heterocycle steroids could be good lead compounds
in the drug design especially against hormone-inde-
pendent prostate cancer.
ꢀ
Haider , Miriam Sgobba , Mohamed Khalid , Remi
Legay1,2, Martine Duterque-Coquillaud6, Philippe
Galera1,3, Sylvain Rault1,2, Patrick Dallemagne1,2
,
Safa Moslemi1,3 and Laıla El Kihel
*
1,2,
€
Key words: abiraterone analogues, CYP17A1, CYP19, hor-
mone-dependent cancer, hormone-independent cancer,
hybrid heterocycle steroids, prostate cancer, steroidal alkaloid
analogues
1Universitꢀe de Caen/Basse-Normandie, Esplanade de la
Paix, 14032, Caen cedex, France
2UFR des Sciences Pharmaceutiques, Centre d’Etudes et
de Recherche sur le Mꢀedicament de Normandie (CERMN),
CNRS INC3M – SFR ICORE 146, Bd Becquerel, F-14032,
Caen cedex, France
Received 14 November 2012, revised 7 June 2013 and
accepted for publication 11 July 2013
3UFR de Médecine, Laboratoire Microenvironnement
Cellulaire et Pathologies (MILPAT, EA 4652), SFR ICORE
146, Avenue de la Cte de Nacre, 14032 Caen cedex,
France
Hybrid nitrogenous heterocycle steroids including steroidal
alkaloids and their analogues continue to attract a great
pharmaceutical interest. Many of these heterosteroids pos-
sess a wide spectrum of important biological properties,
such as selective inhibition of 11b-hydroxysteroid dehydro-
genase (1), anti-Alzheimer (2), cholinesterase inhibitory (3),
antitumour (4,5), antileishmanial (6), antiplasmodial (7), anti-
fungal (8), antibacterial (9) and anti-inflammatory activities
(10).
4Centre for Cancer Research and Cell Biology, Queen’s
University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK
5
ꢀ
ꢀ
Faculte des Sciences et Techniques, Universite Hassan
Premier, Km 3, Route de Casablanca, BP 577, 26000
Settat, Morocco
6Institut Pasteur de Lille/IFR142, CNRS UMR 8161, Institut
ꢀ
de Biologie de Lille, Universite de Lille Nord, 1 rue du
Professeur Calmette, B.P.447, 59021 Lille cedex, France
*Corresponding author: Laïla El Kihel,
laila.elkihel@unicaen.fr
Among these heterocycle steroids, natural steroids con-
tain one or more heterocyclic ring(s), fused or attached
to A or D ring, and or formed by modifications of the
side chain, which are sufficiently plentiful to become
major sources of steroidal intermediates for the synthesis
of steroid hormones and pharmaceutical analogues, and
furostans (11), as well as steroidal alkaloids (12), all have
received much attention in view of their relevant biological
activities. Considerable effort has been made during the
past decade to develop steroidal moiety with pyrazole,
isoxazole, pyridine, dehydropiperazine, pyran, pyrrole, or
pyrimidine rings using various synthetic strategies
(13–16). A large number of steroid derivatives bearing
modifications in ring D with nitrogen coupled to hetero-
cycles such as imidazolyl, pyrazolyl, oxazolyl, isoxazolyl,
pyridyl, pyrimidyl, oxazinyl and tetrahydro-oxazinyl hetero-
cycles are efficient inhibitors of cytochrome P450
enzymes and exhibit promising effects as potential agents
in the treatment of patients with androgen-dependent
diseases (17–19).
Nine new 17-(piperazin-1-yl)pyridin-5-yl)steroids as
abiraterone analogues were synthesized. Compounds
5d and 5g showed selective activities against 17a-
hydroxylase/C17,20-lyase (CYP17A1) and aromatase
(CYP19), respectively. IC50 values of 5d were 5.09 and
>50 lM, whereas these values for 5g were >50 lM and
7.40 lM, respectively, for CYP17A1 and CYP19. Molec-
ular modelling highlighted that the inhibitor designed
to bind cytochrome P450 haem iron is a necessary
condition but not the only rationale to explain inhibi-
tory activity. These abiraterone analogues were then
evaluated on hormone-independent prostate cancer
cell lines DU-145 and PC-3 and on hormone-depen-
dent breast and prostate cancer cell lines MCF-7 and
LNCaP, respectively. Compounds 5e, 5g and 5i have
showed potent activities only on hormone-independent
prostate cancer cell lines DU-145 and PC-3 with 60–
85% inhibition of both cell viability and growth at
10 nM with pro-apoptotic mechanism as illustrated in
PC-3 cells by DNA ladder assay and Western blotting
620
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12195

Abiraterone Analogues and Anticancer Activity
For instance, analogues of androsta-5,16-diene-3b-ol
bearing pyridyl groups at C17 have been widely studied as
cytochrome P450 CYP17 inhibitor. Abiraterone (17-(3-pyr-
idyl) androsta-5,16-dien-3b-ol) was developed as a highly
selective and irreversible inhibitor of CYP17A1, a key
enzyme in prostate cancer treatment. Abiraterone acetate
is currently approved for use in the treatment for meta-
static castration-resistant prostate cancer in patients (20).
In view of its action to prevent downstream oestrogen syn-
thesis from androgen, abiraterone is also being tested in
postmenopausal women with advanced oestrogen-depen-
dent breast cancer (21). Besides its role in prostate cancer
treatment, it is important to note that abiraterone belongs
to the family of hetero-arylsteroid. Similar natural molecules
of this hybrid heterocycle steroid are steroidal alkaloids,
which show a wide range of biological activities (22–28).
However, and despite considerable effort made in the
past, steroidal alkaloids and their analogues remain spar-
sely described in the literature.
interesting class of heterocycles (33–35). Additionally, we
have previously reported a series of piperazinyl bile acid
derivatives that showed promising antitumour activities
(31). We are interested here in the design and synthesis
to study the structure–activity relationship of novel hybrid
heterocycle steroids where 3b-hydroxy-17-(2-chloropyri-
din-5-yl)androsta-5,16-diene was used as
a building
block for the synthesis of abiraterone piperazinyl deriva-
tives (Scheme 1).
Methods and Materials
Chemistry
All solvents were distilled prior to use. Reagents and mate-
rials were obtained from commercial suppliers and were
used without further purification. The reactions were moni-
tored by TLC on Kieselgel-G (Merck Si 254 F) layers
(0.25 mm thick). The spots were detected using a UV
lamp (254 nm) and by spraying with sulphuric acid/ethanol
(2:8) on TLC and subsequent heating. Column chromatog-
raphy was carried out using silica gel 60 (0.063–0.2 mm)
(Merck). Melting points were determined on a Kofler block.
IR spectra were recorded on a Perkin-Elmer BX FT-IR
spectrometer. HRMS was recorded on a Jeol-GCmate
(GC-MS system) spectrometer with ionization energy from
The promising results obtained by us and other authors
fully deserve the necessity of developing these types of
molecules. In continuation of our previous work in devel-
oping new biologically active modified steroids (29–32),
we choose substituted nitrogenous heterocycles as
piperazine derivatives that are
a
pharmacologically
Scheme 1: Synthesis of 17-(piperazin-1-yl)pyridin-5-yl) steroid derivativesa. aReagents and conditions: (i) N2H4.H2O, AcOH, EtOH, reflux,
12 h; (ii) I2, 1,1,3,3-tetramethylguanidine, anhydrous THF, 50 °C, 24 h; (iii) 6-chloropyridin-3-yl boronic acid, Pd(OAc)2, PPh3, K2CO3,
DMF, 80 °C, 14 h; (iv) substituted piperazines, Pd(OAc)2, Binap, Cs2CO3, toluene, 100 °C, 15 h.
Chem Biol Drug Des 2013; 82: 620–629
621

Brossard et al.
30 to 40 eV. MS detection was performed by positive or
negative ESI. 1H NMR and 13C NMR spectra were
recorded, respectively, at 400 and 100 MHz (Jeol Lambda
400 spectrometer) using CDCl3 as solvent. Chemical shifts
are reported relative to TMS; J values are given in Hz. 13C
3b-hydroxy-17-iodo-5,16-androstadiene (3 g, 7.53 mmol)
and 6-chloropyridin-3-ylboronic acid (2.37 g, 15.06 mmol)
were added, and the solution was stirred at 80 °C for
14 h. The mixture was filtered on celite, which was
washed with diethylether, and the solvent was evaporated.
The crude product was purified by column chromatogra-
phy (silica gel, cyclohexane–ethyl acetate = 8:2) giving the
product 4 as a beige solid (1.66 g, 58%). mp: 210 °C; IR
(KBr): υ = 3384 (C-OH alcohol), 3036 (CH aromatic),
2966–2819 (CH alkane), 1669 (C=N), 1551 (C=C aromatic)
per cm; 1H NMR (400 MHz, CDCl3): d = 8.37 (s, 1H,
H6-pyr), 7.63–7.60 (dd, 1H, J = 7.79, 1.96 Hz, H4-pyr),
7.24 (s, 1H, H3-pyr), 6.00 (s, 1H, C16H), 5.39 (d, 1H,
J = 4.88 Hz, C6H), 3.54 (m, 1H, C3H), 1.07 (s, 3H, 19-
Me), 1.03 (s, 3H, 18-Me) ppm; 13C NMR (100 MHz,
CDCl3): d = 150.4, 149.4, 147.5, 141.1, 136.5, 131.9,
130.0, 123.6, 121.3, 71.7, 57.5, 50.3, 47.3, 42.2, 37.1,
36.7, 35.1, 31.8, 31.6, 31.5, 30.4, 20.8, 19.3, 16.5 ppm;
MS-ESI: 384.4 [M+H]+.
1
NMR spectra are H-decoupled.
3b-hydroxy-androst-5-en-17-hydrazone (2)
In
a
500-mL round-bottomed flask, DHEA (10 g,
34.67 mmol) was dissolved in EtOH (150 mL), monohy-
drate hydrazine (3.24 mL, 104.01 mmol) and acetic acid
(89 lL, 1.56 mmol) were added, and the mixture was re-
fluxing during 12 h. The solvent was evaporated, and the
crude product was dissolved in CH2Cl2 (150 mL), water
(150 mL) was added, and the precipitate was filtered on a
€
Buchner to give the product as a white amorphous solid
(10.49 g, quantitative). IR (KBr): υ = 3372 (C-alcohol),
2963–2857 (CH alkane) per cm; 1H NMR (400 MHz,
CDCl3): d = 5.36 (d, 1H, J = 5.2 Hz, C6H), 3.53 (m, 1H,
C3H), 1.03 (s, 3H, 19-Me), 0.87 (s, 3H, 18-Me) ppm; 13C
NMR (100 MHz, CDCl3): d = 166.1, 141.1, 121.1, 71.6,
53.9, 50.4, 43.8, 42.2, 37.2, 36.6, 34.1, 31.6, 31.3 (2C),
24.4, 23.4, 20.7, 19.4, 16.8 ppm; MS-ESI: 303.38 [M+H]+.
General procedure for the coupling of 3b-hydroxy-
17-(2-chloropyridin-5-yl)androsta-5,16-diene with
substituted piperazines (5e, 5g and 5i)
17-iodo-androsta-5,16-diene-3b-ol (3)
BINAP (0.16 g, 0.3 mmol) and Cs2CO3 (1.06 g, 3.3 mmol)
In
a
500-mL round-bottomed flask, iodine (22 g,
were added to
a solution of compound 1 (0.50 g,
86.68 mmol) was dissolved in anhydrous THF (50 mL)
under argon. At 0 °C, tetramethylguanidine (22.18 mL,
173.35 mmol) was added dropwise. Crude 3b-hydroxy-
androst-5-en-17-hydrazone 2 (10.49 g, 34.67 mmol) dis-
solved in anhydrous THF (50 mL) was added, and the mix-
ture was stirred at 0 °C during 2 h, then filtered and THF
was concentrated. The solution was then heated at 50 °C
during 24 h. THF was evaporated, and the crude mixture
was dissolved in CH2Cl2 (100 mL) and washed with 1M HCl
(3 9 100 mL), 10% NaHCO3 (3 9 100 mL), 1M Na2S2O3
(3 9 100 mL) and water (3 9 100 mL). The organic phase
was dried over MgSO4, and the solvent was evaporated
under reduced pressure. The crude product was purified by
recrystallization from ethanol to give 10.77 g of compound
3. IR (KBr): υ = 3254 (C-alcohol), 2967–2851 (CH alkane),
1.3 mmol), substituted piperazine (2.6 mmol) and Pd
(OAc)2 (0.03 g, 0.1 mmol) in toluene (10 mL). The reaction
mixture was stirred under N2 atmosphere at 100 °C for
15 h. The solution was filtered on celite, washed with tolu-
ene and then with water. The combined organic layers
were dried over MgSO4, and the solvent evaporated under
reduced pressure. The crude product was purified by
column chromatography (silica gel, eluting by cyclohex-
ane–ethyl acetate).
3b-hydroxy-17-(2-(4N-piperonyl-piperazin-1-yl)
pyridin-5-yl)androsta-5,16-diene (5e)
Brown solid (214 mg, yield : 29% and average yield:
13%). mp : 170 °C; IR (KBr): υ = 3381 (C-alcohol), 3035
(CH aromatic), 2924–2849 (CH alkane), 1664 (C=N), 1596
(C=C aromatic) per cm; 1H NMR (400 MHz, CDCl3):
d = 8.24 (s, 1H, H6pyr), 7.51–7.48 (dd, 1H, J = 8.7,
2.9 Hz, H4pyr), 6.89 (s, 1H, H5ar), 6.76 (s, 2H, H2ar and
H6ar), 6.58 (d, 1H, J = 8.7 Hz, H3pyr), 5.95 (s, 2H, -CH2-
piperazinyl), 5.80 (s, 1H, C16H), 5.39–5.38 (m, 1H, C6H),
4.90 (m, 1H, C3H), 3.53 (t, 4H, J = 4.8 Hz, 2 9 -CH2-pip-
erazinyl), 3.46 (s, 2H,-CH2-piperazinyl), 2.53 (t, 4H,
J = 4.8 Hz, 2 9 -CH2-piperazinyl), 2.31–2.18 (m, 2H,
C4H), 1.87–1.81 (m, 2H, C7H), 1.06 (s, 3H, 19-Me), 1.00
(s, 3H, 18-Me) ppm; 13C NMR (100 MHz, CDCl3):
d = 158.2, 151.7, 147.6, 146.7, 145.5, 141.1, 135.8,
128.5, 125.3, 122.6, 122.3, 121.4, 109.5, 107.9, 106.4,
100.9, 71.7, 62.8, 57.5, 52.7 (2C), 50.3, 47.1, 45.2 (2C),
43.4, 42.3, 37.1, 36.9, 35.4, 31.6, 31.5, 30.4, 30.1, 26.9,
20.9, 19.3, 16.5 ppm; HRMS-ESI: m/z [M+H]+ calcd for
1665 (C=C) cmÀ1 1H NMR (400 MHz, CDCl3): d = 6.16–
;
6.12 (m, 1H, C16H), 5.36 (d, 1H, J = 4.8 Hz, C6H), 3.53 (m,
1H, C3H), 1.04 (s, 3H, 19-Me), 0.76 (s, 3H, 18-Me) ppm;
13C NMR (100 MHz, CDCl3): d = 141.2, 137.5, 121.2,
112.7, 71.7, 54.8, 50.4, 42.2, 37.1 (2C), 36.1, 33.7 (2C),
31.6, 31.2, 31.0, 20.8, 19.3, 15.1 ppm; MS-ESI: 399.40
[M+H]+, 381.28 [M+H-H2O]+.
3b-hydroxy-17-(2-chloropyridin-5-yl)androsta-
5,16-diene (4)
A 250-mL round-bottomed flask equipped with a magnetic
stirring bar was charged with DMF (60 mL) and flushed
with nitrogen. Palladium acetate (0.169 g, 0.75 mmol),
triphenylphosphine (0.395 g, 0.15 mmol), and potassium
carbonate (4.34 g, 18.83 mmol) were added, and the mix-
ture was stirred at 60 °C until the solution became red.
C
36H46N3O3: 568.3539, found: 568.3535.
622
Chem Biol Drug Des 2013; 82: 620–629

Abiraterone Analogues and Anticancer Activity
3b-hydroxy-17-(2-(4-[bis-(4-fluorophenyl)-methyl])
piperazin-1-yl)pyridin-5-yl)androsta-5,16-diene (5g)
Orange solid (372 mg, yield: 45% and average yield:
20%). mp: 152 °C; IR (KBr): υ = 3390 (C-alcohol), 3036
(CH aromatic), 2926–2849 (CH alkane), 1663 (C=N), 1600
(C=C aromatic) per cm; 1H NMR (400 MHz, CDCl3) :
d = 8.23 (s, 1H, H6pyr), 7.51–7.48 (dd, 1H, J = 8.7,
1.9 Hz, H4pyr), 7.40–7.36 (m, 4H, Har), 7.01–6.97 (m, 4H,
Har), 6.56 (d, 1H, J = 8.7 Hz, H3pyr), 5.80 (s, 1H, C16H),
5.39–5.38 (m, 1H, C6H), 4.90 (m, 1H, C3H), 4.25 (s, 1H,
-CH-piperazinyl), 3.51 (t, 4H, J = 4.8 Hz, 2 9 -CH2-piper-
azinyl), 2.48 (t, 4H, J = 4.8 Hz, 2 9 -CH2-piperazinyl),
2.31–2.25 (m, 2H, C4H), 1.87–1.82 (m, 2H, C7H), 1.06 (s,
3H, 19-Me), 1.01 (s, 3H, 18-Me); 13C NMR (100 MHz,
CDCl3) d = 163.0, 159.3 (2C, J1 = 257 Hz), 151.5, 145.3,
141.1, 137.9 (2C), 135.9, 129.2 (4C, J3 = 7 Hz), 125.5,
122.7, 121.4, 115.5 (4C, J2 = 21 Hz), 106.5, 74.5, 71.6,
51.5 (2C), 50.3, 47.0, 45.4 (2C), 42.2, 37.1, 36.6, 35.4,
31.5 (2C), 30.4, 30.1, 26.8, 20.9, 19.3, 16.4 ppm;
HRMS-ESI : m/z [M+H]+ calcd for C41H48N3OF2:
636.3765, found: 636.3782.
were expressed as difference between reference OD
(600 nm) and measured OD (450 nm).
Cell proliferation
Crystal violet test allows cell counting by staining adherent
cell (37). After WST-1 test, cells were washed with PBS
and socked at 4 °C until crystal violet analysis. One hun-
dred microlitre of crystal violet (1 g/L) were added to cells
and incubated for 30 min. After rinsing twofold with PBS/
Ca2+
, acetic acid 20% was added and allowed for
15 min with shaking. OD was measured with a spectro-
photometer at 600 nm, and cell number is correlated to
increasing OD.
Western blotting
The cell extract-associated proteins (20 lg) were resolved
on 8% polyacrylamide gel, using 1% SDS/Tris glycine
buffer. The proteins were then electrotransferred onto
polyvinylidene difluoride (PVDF) membrane (Millipore, Mols-
heim, France). Free protein-binding sites of the PVDF
membranes were blocked for 1 h 30 min at room temper-
ature in Tris buffer saline (TBST, 20 mM Tris, 137 mM
NaCl, 0.1% Tween 20) containing 10% non-fat dry milk,
and the membranes were incubated with an anticaspase-
3 (1:200 dilution), anti-Bax (1:200 dilution), anti-PARP
(1:1000 dilution), or anti-GAPDH (1:2000 dilution) antibod-
ies (Santa Cruz Biotechnology, Inc., Heidelberg, Germany).
After incubation for 1 h 30 min at room temperature or
overnight incubation at 4 °C, the membranes were rinsed
and incubated for 1 h 30 min with a secondary antibody
(HRP-conjugated goat anti-rabbit antibody at 1:5000 dilu-
tion for Bax or 1:10 000 dilution for caspase-3 and PARP).
Protein signals were revealed using a Western blotting
Chemiluminescence Luminol Reagent (Tebu-Bio) and X-
ray films (Kodak, X-Omat, Perkins Elmers SAS, Courtab-
oeuf, France). GAPDH was used as control (housekeeping
gene).
3b-hydroxy-17-(2-(4-[2-(2,2′N-ditert-
butoxycarbonylamino)ethyl]piperazin-1-yl)pyridin-
5-yl)androsta-5,16-diene (5i)
Yellow amorphous solid (282 mg, yield: 32% and average
yield: 14%). IR (KBr): υ = 3432 (C-alcohol), 2971–2852
(CH alkane), 1702 (C=O ester), 1595 (C=C aromatic) per
cm; 1H NMR (400 MHz, CDCl3) : d = 8.36 (s, 1H, H6pyr),
7.61–7.58 (dd, 1H, J = 8.7, 2.9 Hz, H4pyr), 7.51 (d, 1H,
J = 8.7 Hz, H3pyr), 5.95 (s, 1H, C16H), 5.39–5.38 (m,
1H, C6H), 4.06 (m, 1H, C3H), 3.42 (t, 2H, J = 4.8 Hz,
-CH2CH2N(Boc)2), 3.35–3.30 (m, 4H, 2 9 -CH2-piperazi-
nyl), 2.62 (t, 2H, J = 6.8 Hz, -CH2CH2N(Boc)2), 2.46–
2.40 (m, 4H, 2 9 -CH2-piperazinyl), 2.25–2.18 (m, 2H,
C4H), 1.87–1.83 (m, 2H, C7H), 1.46–1.45 (m, 18H,
2 9 t-Bu), 1.07 (s, 3H, 19-Me), 1.03 (s, 3H, 18-Me)
ppm; 13C NMR (100 MHz, CDCl3) : d = 154.7 (2C),
154.0, 151.2, 145.1, 141.1, 134.8, 128.8, 128.3, 121.3,
119.1, 81.2, 79.6, 71.6, 57.5 (2C), 53.0 (2C), 52.7, 50.3,
47.2, 43.7, 42.2, 37.1, 36.7, 35.3, 31.6 (2C), 31.5, 30.4,
28.4 (6C), 28.3, 20.9, 19.3, 16.5 ppm; HRMS-ESI : m/z
[M+H]+ calcd for C40H61N4O5: 677.4642, found:
677.4630.
DNA ladder assay
DNA fragmentation by activated endonucleases is a well-
documented feature of cells undergoing apoptosis. We
used a modified rapid protocol reported by Herrmann et al.
(38) to detect apoptotic DNA fragment. After 48-h incuba-
tion of PC-3 and DU-145 cells in the presence or absence
of abiraterone and 5i (10 nM each), the monolayer and
floating cells (recovered after centrifugation at 800 9 g for
10 min) were incubated for 20 min at 4 °C in 100 mM Tris–
HCl lysis buffer, pH 5.8 containing 5 mM EDTA, 200 mM
NaCl and 0.2% SDS. After centrifugation at 10 000 9 g
for 15 min, supernatant was recovered and proteins were
extracted with phenol/chloroform and centrifuged again at
10 000 9 g for 15 min. Obtained supernatant was recov-
ered, and DNA was precipitated with 2 volumes of ethanol
overnight at À20 °C. After centrifugation at 10 000 9 g for
10 min, precipitated DNA was washed with 80% ethanol
Cell viability
Cell viability test was used according to the supplier
(Roche diagnostic). This test is based on the stable tetra-
zolium salt WST-1 reduction to a soluble formazan by a
complex cell mitochondrial mechanism (36). This bioreduc-
tion is largely dependent on the glycolytic production of
NAD(P)H in viable cells. Therefore, the amount of formazan
dye formed directly correlates to the number of metaboli-
cally active cells in the culture. One hundred ll of WST-1
was used at 1/40 dilution and incubated with cells for
45 min. Results, obtained by a spectrophotometric assay,
Chem Biol Drug Des 2013; 82: 620–629
623

Brossard et al.
and left to dry at room temperature. DNA was then dis-
solved in 25 lL of TE buffer (10 mM Tris–HCl, pH 8, 1 mM
EDTA) and incubated with RNase A (1 lg/lL) for 30 min at
room temperature. One lg of DNA was then loaded on a
1% agarose gel in a 1 9 TAE buffer (40 mM Tris–acetate,
2 mM EDTA) and visualized by GelRed.
CYP17A1 and CYP19, the two key enzymes involved in
hormone-dependent cancers such as prostate and breast
cancers (20,42–47). Screening assay procedures were
used to assess the potential inhibitory effects of abirater-
one analogues towards CYP17A1 and CYP19. Abiraterone
(17-(3-pyridyl)androsta-5,16-dien-3b-ol) was used as refer-
ence for CYP17A1 inhibition, whereas anastrozole and
4-hydroxy-androst-4-ene-3,17-dione (4OHA) were chosen
for CYP19 (aromatase) inhibition.
Automated ligand docking
The automated ligand docking protocol was adapted
from Haider et al. (39). Ligands 5d, 5g, abiraterone, and
androstenedione were built, minimized, and charges
assigned using the PRODRG server (40). The structures
of CYP17A1 (PDB id 3SWZ) and CYP19 (PDB id 3EQM)
were downloaded from the protein data bank (www.pdb.
org). Docking of abiraterone and androstenedione was
used as controls to identify the orientation that is consis-
tent with being positioned above haem. The haem moiety
was treated as an integral part of the protein and remains
fixed during the entire docking process. The parameters
for haem were extrapolated from Haider et al. (39). Auto-
mated ligand docking was carried out using the Autodock
v4.2 (41). The models for both CYP17A1 and CYP19
contain ligands that have been cocrystallized in the bind-
ing sites. The docking grid boxes of size 50A 9
The results presented in Table 1 show that compound 5d
expressed inhibitory effect only towards CYP17A1
(IC50 = 5.09 lM), while the aromatase enzyme was
affected by compound 5g (IC50 = 7.40 lM). Compound 5i
inhibits similarly both enzymes with an IC50 value of about
10 lM (see Figures S1 and S2). We then made an attempt
dock compounds 5d and 5g, presenting the best and
moderate inhibitory potency for CYP17A1 and CYP19 to
rationalize the structural influence of different piperazinyl
substituents. The coordinate bond that is required for the
inhibition of the enzyme does not form between the het-
eroaryl atom and haem iron, and as a result, the inhibitory
effect of these compounds with bulky groups was dramat-
ically reduced (see Figure S7). Our results suggest that the
inhibitor designed to bind cytochrome P450 haem iron is a
necessary condition but not the only rationale to explain
inhibitory activity.
66A 9 90A were generated with
a grid spacing of
0.375A. The grid spacing was identified to be optimal to
accommodate all ligands. Lamarckian genetic algorithm
was employed as the search method with 250 runs. The
number of population in each set was set to 150; the
energy evaluation limit was limited to 2 500 000, and
the maximum number of generations was set to 27 000.
The top docking orientations were fixed to 1; rate of gene
mutation was kept at 0.02 and that of crossover was kept
at 0.8. The docking permits the ligand to be totally flexi-
ble, while only a limited side chains on the protein can be
flexible. The side chains on the protein, which were
assigned to be flexible, were carefully selected based on
their contributions to the active site. From the top 50
solutions, the final docked conformation was chosen
based on its orientation and the interaction energies
within the active sites.
On the other hand, we investigated the effect of these
hybrid heterocycle steroids (5a-i) on viability and growth of
hormone-dependent prostate and breast cancer cell lines
LNCaP (androgen sensitive) and MCF-7 (oestrogen sensi-
tive). We also determined the effect of these abiraterone
analogues on the viability and growth of two human
androgen-independent prostate cancer cell lines, PC-3
and DU-145.
When two cells lines PC-3 and DU-145 were incubated in
the presence of abiraterone and its analogues (5a-i) with
concentrations ranging from 0.001 to 1 lM, the results of
WST-1 viability test show that the DU-145 cell line is more
Table 1: IC50 values (in lM) of tested compounds for CYP17A1
and CYP19
Results and Discussion
Compound
CYP17A1
CYP19
A series of nine novel 17-(piperazin-1-yl)pyridin-5-yl)steroid
derivatives was prepared as shown in Scheme 1.
Abiraterone
Anastrazole
4OHA
5a
0.84
–
–
–
0.078
0.358
>50
>50
>50
>50
>50
>50
7.40
>50
10.60
The key steps of this synthesis involved a convenient
method for the synthesis of 3b-hydroxy-17-(2-chloropyri-
din-5-yl)androsta-5,16-diene, a key intermediate, which
was used as a building block for the synthesis of piperazi-
nyl abiraterone derivatives 5a-i using Buchwald–Hartwig
cross-coupling reaction.
>50
>50
33.42
5.09
>50
>50
>50
>50
10.03
5b
5c
5d
5e
5f
5g
5h
5i
First, we aimed to compare the in vitro inhibitory activity of
new abiraterone analogues (5a-i) on cytochromes P450
624
Chem Biol Drug Des 2013; 82: 620–629

Abiraterone Analogues and Anticancer Activity
A
B
C
D
Figure 1: Effect of abiraterone and its analogues 5a-i on cell viability (A and C) and proliferation (B and D) of human hormone-
independent prostate cancer cell lines PC-3 (A and B) and DU-145 (C and D)a. aFollowing treatment of these cell lines with abiraterone
and analogues for 48 h, the effect on cell viability was measured using WST-1 assay. Growth inhibition of cell lines was determined with
N-hexamethylpararosaniline (crystal violet = CV). The results are expressed as mean Æ SEM of 3–5 samples and representative of three
separate experiments showing the same profile. Statistical significance of difference between treated cells and control (Ctl, 0.1% DMSO)
or between abiraterone and its derivatives was determined using Student’s t-test. “a”: p < 0.01 versus Ctl; “b”: p < 0.001 versus Ctl; “c”:
p < 0.01 versus 0.001 lM; “d”: p < 0.001 versus 0.001lM; “e”: p < 0.01 versus abiraterone; “f”: p < 0.001 versus abiraterone.
sensitive to treatment with these molecules than PC-3 cell
line and that the viability of DU-145 cell line is significantly
reduced (80% decrease from 0.1% DMSO control) even in
the presence of low concentration of 0.001 lM abiraterone
and its analogues, this inhibition is maintained in the pres-
ence of other higher concentrations (Figure 1A,C).
reference molecule abiraterone on the inhibition of the via-
bility and proliferation of DU-145 cell line (Figure 1C,D).
This screening has also allowed us to identify two similar
analogues 5 h and 5i, which showed very interesting
results with respect to the PC-3 cell line against which
they exhibit better inhibition compared with the reference
molecule abiraterone namely at 0.01 lM.
For PC-3 cell line, low dose of 0.001 lM led to a decrease
in the viability in the order of 20 to 40% for all molecules
except 5 h and 5i. Indeed, this cell line is more sensitive
only to the dose of 0.01 lM of 5 h and 5i which abolished
the viability to 80%, but this significant decrease was not
confirmed with the higher doses of these molecules nor
for any of the molecules tested (Figure 1A). The results of
the proliferation test with crystal violet (Figure 1B,D) were
confirmed, with few exceptions, the same decrease profile
previously observed with the WST-1 test. Taken together,
these results show that 5c and 5e-i are as effective as the
These same molecules have little or no effect on hor-
mone-dependent prostate cancer cell line LNCaP and
hormone-dependent breast cancer cell line MCF-7 even
with concentrations up to 50 times of those used against
hormone-independent cell lines PC-3 and DU-145. Even
if a drop in proliferation of LNCap up to 30% is observed
with certain doses and structures (abiraterone, 5a, 5d
and 5c), for these same doses and structures, the viability
of this cell line remains unchanged (see Figures S3–S6).
Taken together, these results show that in general, with
Chem Biol Drug Des 2013; 82: 620–629
625

Brossard et al.
some exceptions, the profile of cell viability follows that of
cell proliferation for both cell lines PC-3 and DU-145, and
the reduced viability observed is connected to decrease
in cell proliferation. The differential effects observed in cell
proliferation and viability for some analogues and doses
clearly showed that these two phenomena are sometimes
dissociated and a decrease in cell viability still does not
reflect a decrease in cell proliferation. Both hormone-inde-
pendent prostate cancer cell lines are more sensitive to
treatment with abiraterone and its analogues than the
hormone-dependent LNCaP and MCF-7 cell lines (see
supporting information). DU-145 cell line has more sensi-
tivity than PC-3 cell line. Analogues 5 h and 5i are two
potential candidates against the hormone-independent
prostate cancer cell lines. However, our results did not
allow us to establish the correlation curves between
doses and cytotoxic/antiproliferative effects, and therefore,
it was impossible to calculate the IC50. The absence of
this correlation may be attributed to the stimulation or
inhibition of different cell signalling pathways in response
to the different doses leading to the lack of a uniform cell
toxicity/proliferation.
A
B
Moreover, our results of enzyme inhibition show that while
compounds 5e, 5 h and 5i inhibited weakly or moderately
P450 CYP17A1 and CYP19 activities (IC50 values of
>50 lM for 5e/5 h and 10.03 lM for 5i), they exhibited a
strong antiproliferative effect on hormone-independent
cancer cell lines PC-3 and DU-145 at 10 nM. In contrast,
compound 5d presenting a moderate CYP17A1 inhibitory
effect (IC50 = 5.09 lM) showed no antiproliferative activities
on the same cell lines. For these reasons and those of cell
viability and proliferation, we have therefore maintained the
abiraterone and 5i analogue for further analysis of Western
blotting and DNA ladder assay (see Figure 2A,B).
Figure 2: Western blotting of the pro-apoptotic markers (A) and
DNA ladder assay (B) in the presence of 10 nM of abiraterone and
5i analogue with PC-3 and DU-145 cells.
realized Western blotting of pro-apoptotic markers such as
Bax, a protein of 23 kDa and a member of the Bcl-2
family, Casp-3 (35 kDa) and its substrate, the poly (ADP–
ribose) polymerase (PARP, 116 kDa), an enzyme impli-
cated in the DNA reparation and antiapoptotic process
(50–53). Results are presented in Figure 2A.
As observed from our data, the best CYP17A1 and/or
CYP19 inhibitors are not the best antiproliferative com-
pounds, and this divergence might be explained by either
bioavailability of compound or its possible metabolism into
more active metabolite in a cellular context. Finally, the two
approaches used in this work, that is, enzymatic activity
evaluation and cell viability/proliferation determination, may
not be associated, and the lead compounds showing
strong cytotoxic and antiproliferative activities in cell model
may not even involved in the CYP17A1 and CYP19 inhibi-
tion in microsomal preparation. Because the expression of
steroidogenic enzymes such as CYP17A1 in prostatic cell
lines or tumours is absent (48) or extremely low (49), a cor-
relation between these approaches may be searched using
unique test model such as the overexpression of CYP17A1
and CYP19 in prostatic cell lines for better understanding
of mechanistic effect of such a compound synthesized and
undergoing bioevaluation in a screening protocol.
As illustrated in Figure 2A, abiraterone and 5i (10 nM each)
induced Casp-3 cleavage (35 kDa) and its activation
(17 kDa) allowing therefore PARP inactivation (85 kDa) in
PC-3 cells but not in DU-145 cells. It is likely that the acti-
vation of Casp-3 in PC-3 cells takes place under the effect
and a prior activation of the Bax. Abiraterone and 5i
analogue-induced apoptosis in PC-3 cells were then
confirmed by DNA ladder test (Figure 2B). As showed in
Figure 2B, DNA fragmentation that is a feature of the cells
undergoing apoptosis (54) occurs only in the presence of
abiraterone and 5i (10 nM each) in PC-3 cell line.
Altogether, these results suggest that cell death mecha-
nism induced by abiraterone and its analogues (especially
5i) is different in both PC-3 and DU-145 cell lines. We
confirm here the involvement of Bax/caspase/PARP path-
way in the induction of apoptosis at least in PC-3 prostate
cell line. It is likely that this mechanism is not Casp-3-
dependent in DU-145 cells and that other pathways may
Finally, to search for the involvement of the apoptotic path-
way in the mechanism of cytotoxicity and cell growth inhi-
bition observed with WST-1 and crystal violet tests, we
626
Chem Biol Drug Des 2013; 82: 620–629

Abiraterone Analogues and Anticancer Activity
be involved in the death of these cells observed with crys-
tal violet test under abiraterone and analogue treatment.
on antileishmanial steroidal alkaloids from Sarcococca
hookeriana. Nat Prod Res;21:292–297.
7. Devkota K.P., Lenta B.N., Choudhary M.I., Naz Q.,
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esterase inhibiting and antiplasmodial steroidal alkaloids
Conclusion and Future Directions
from
Sarcococca
hookeriana.
Chem
Pharm
In conclusion, these experiments validate the approach of
coupling nitrogen heterocycles to steroids. We have intro-
duced novel compounds as abiraterone analogues, which
have been shown to be highly antiproliferative agents on
prostate hormone-independent cancer cell lines DU-145
and PC-3, and they appear to be extremely good lead
compounds in design and synthesis of potential drugs. It
can also be considered to study the in vivo cytotoxicity of
these compounds.
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This work was financially supported by ‘Conseil Regional
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€
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Chem Biol Drug Des 2013; 82: 620–629
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