Discovery of non-LBD inhibitor for androgen receptor by structure-guide design

Article abstract of DOI:10.1016/j.bmcl.2013.04.065

In this study, we synthesized the BF-3 binding small molecules, a series of pyridazinone-based compounds, as a novel class of non-LBP antiandrogens for treating prostate cancer by inhibiting androgen receptor. The new class compound was discovered to inhibitor the viability of AR-dependent human prostate LNCap cells and AR activity combining with the computational method. It showed a good physicochemical and PK property.

Full text of DOI:10.1016/j.bmcl.2013.04.065

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3887–3890
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of non-LBD inhibitor for androgen receptor
by structure-guide design
Byung Jun Ryu c, Nakjeong Kim b, Jun Tae Kim a, Tae-Sung Koo a, Sung-Eun Yoo a, Seo Hee Jeong a,
Seong Hwan Kim c, Nam Sook Kang a,
⇑
a Graduate School of New Drug Discovery and Development, Chungnam National Univ., Daehakno 99, Yuseong-gu, Daejeon 305-764, Republic of Korea
b Metabolic Syndrome Therapeutics Research Center, Bio-Organic Science Division, Korea Research Institute of Chemical Technology, P.O. Box 107, Yuseong-gu,
Daejeon 305-600, Republic of Korea
c Laboratory of Translational Therapeutics, Pharmacology Research Center, Division of Drug Discovery Research, Korea Research Institute of Chemical Technology, P.O. Box 107,
Yuseong-gu, Daejeon 305-600, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we synthesized the BF-3 binding small molecules, a series of pyridazinone-based com-
pounds, as a novel class of non-LBP antiandrogens for treating prostate cancer by inhibiting androgen
receptor. The new class compound was discovered to inhibitor the viability of AR-dependent human
prostate LNCap cells and AR activity combining with the computational method. It showed a good phys-
icochemical and PK property.
Received 6 March 2013
Revised 11 April 2013
Accepted 25 April 2013
Available online 3 May 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Androgen receptor
BF-3 binding
LNCap cell
Androgen receptor (AR) that belongs to the nuclear receptor
superfamily of transcription factors is a major target for prostate
cancer.1,2 Prostate cancer (PCa) is the most popular cancer in
men and one of the major causes of cancer related death.3 AR
structure consists of a variable amino-terminal activation function
domain (AF-1), a highly conserved DNA-binding domain (DBD), a
hinge region including the nuclear localization signal, a C-terminal
ligand-binding domain (LBD) consisting of a 12 helical structure
that surrounds a ligand binding pocket (LBP), a second activation
function domain (AF-2) located at the carboxy terminal end of
the LBD,4 and a recently reported additional secondary binding
function domain (BF-3) located on the surface of the AR controlling
the allosteric modulation of the AF-2.5 Until now, all clinically ap-
proved AR-targeting inhibitors directly block the binding of andro-
gen to the ligand binding site of the receptor.6 But the targeting the
ligand binding pocket (LBP) has occurred several limitations such
as drug resistance.7 This is common problem for hormone nuclear
receptor antagonists including AR and estrogen receptor antago-
nists. To overcome these limitations, AF-2 or BF-3 blocking small
molecules without affecting natural ligand binding has been re-
cently demonstrated as a novel class of non-LBP antiandrogens.8
Here, we discovered the BF-3 binding small molecules, a series
of pyridazinone-based compounds, to inhibit the viability of LNCap
cells and AR activity combining with the computational method.
Since LNCap cells are the representative AR-positive human pros-
tate cancer cells lines, LNCap cells were used in this study. At early
stage, we screened in-house compounds in LNCap cell. The most
potent compound among the screened compounds was N-(3-(1-
(4-cyanophenyl)-1-methyl-6-oxo-1,6-dihydropyridazin-4-yl)phenyl)-
ethanesulfonamide (1) as shown in Figure 1.
In the viability of LNCap cells, compound 1 exhibited the IC50
value 39
the IC50 value 23
l
M when bicalutamide as a reference compound showed
l
M.9 Next, to improve the inhibitory activity of
compound 1 and its derivatives in LNCap cells, the docking exper-
iment to evaluate its binding mode was carried out. First, we tried
to dock at the LBP substituting androgen dihydrotestosterone
(DHT) using the known X-ray crystallography structure, but the
compound 1 was not fitted at the LBP of AR. We further explored
the known surface binding site, AF-2 and BF-3, of AR and then
found that the compound 1 can be well docked into BF-3
hydrophobic pocket of AR. Docking study was carried out from
the X-ray crystal structure having a testosterone and a BF-3 bind-
ing small molecule (pdb code: 2YLQ).8 The CDOCKER module10 in
Discovery Studio 3.5 with CHARMM force field was used. The eth-
ane sulfonamide group of hit compound 1 interacts with side chain
of N727 through hydrogen bonding and thus is expected to be good
for binding with AR, as shown in Figure 2a. But, 4-cyanide group of
phenyl ring of hit compound 1 is positioned nearby hydrophobic
pocket. The phenyl ring bonded 4-cyanide group formed
⇑
Corresponding author. Tel.: +82 01072925756.
E-mail address: nskang@cnu.ac.kr (N.S. Kang).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.065

3888
B. J. Ryu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3887–3890
pi-stacking interaction with the guanidium group of R840, but its
CN
O
surrounding residues made the shallow hydrophobic pocket
consisting of F673 and L674. To evaluate our result on the binding
of compound 1, we synthesized a series of compounds having
hydrophobic substituents instead of 4-CN group of compound 1.
According to the Scheme 1, we synthesized desired compounds,
as follows.
N
N
H
N
O
S
O
Figure 1. Primary hit compound (1).
The synthetic route of 2-aryl-5-(2-ethanesulfoneamido) pyrida-
zine-3(2H)-one analog is depicted in Scheme 1. Commercially
available 3-nitrophenylacetic acid 6 was reacted with 1-methylim-
idazole in acetic anhydride at room temperature to afford ketone
7.11 Alkylation of 7 with ethyl bromoacetate using lithium diiso-
propylamide (LDA) in THF followed by hydrolysis of ester to give
acid 8. Treatment of 8 in refluxing ethanol with hydrazine gave
the cyclized dihydropyridazinones, which was oxidized with
SeO2 in acetic acid to provide pyridazine-3(2H)-ones 9 in good
yield. Reduction of 9 with iron powder followed by sulfonylation
with ethanesulfonyl chloride in pyridine to afford key intermedi-
ates 10. Finally, desired compounds 1–512 were obtained by cou-
pling reaction of 10 with the appropriate aryl boronic acids or
pinacol aryl boronates13 using copper(II) acetate in moderate
yield.14
As we expected, the substitution of hydrophobic group instead
of 4-CN in the structure of compound 1 improved the inhibitory
potential to LNCap cell viability and AR transcriptional activity
(Table 1). Among them, compound 2 was the most active in the cell
viability assay and AR luciferase assay. The binding modes of com-
pound 1 and the most active compound 2 were shown in Fig. 2. The
di-Cl group was well fitted at the position of shallow hydrophobic
pocket. We further determined physicochemical properties,15,16
metabolic stability17 and CYP inhibition activity18 of compound
2. As shown in Table 2, compound 2 showed appropriate logP
(a)
N727
LBP
F673
R840
AF-2
(b)
N727
P723
F673
Table 1
Viability of LNCap cells and AR luciferase activity
Compound
X
IC50
(lM)
L674
LNCap cell viability AR luciferase activity
1
2
3
4
5
4-CN
39.02 5.74
11.57 2.67
22.93 2.06
26.65 1.62
6.26 0.35
2.65 0.01
13.19 0.03
5.07 0.81
9.76 0.27
R840
3,4-di-Cl
3,5-di-Cl
3-Cl, 4-F
Figure 2. The binding mode for the primary hit (a) and a compound 2 (b). The blue
lines represent hydrogen bond between AR residue and small molecules. The
magenta line in (b) represents hydrophobic interaction between guanidium group
of R840 residue and benzene ring of compounds.
2-Naphthalene 18.27 0.15
Bicalutamide
23.79 0.99
0.27 0.01
O
O
NH
N
OH
Me
O2N
OH
O2N
O2N
Me
c
a
b
e
O2N
O
O
Ac2O
O
H2N-NH2
6
d
7
8
9
O
O
X
NH
N
N
N
H
N
H
N
O
O
S
S
O
O
B(OH)2
X
10
1-5
Scheme 1. Synthesis of pyridazinone analogs, reagents and conditions: (a) 1-methylimidazole, Ac2O(5 equiv), rt, 56%; (b) (i) LDA, BrCH2COOEt, THF, ꢀ78 °C to 0 °C, 5 h, 80%;
(ii) 2N-NaOH, MeOH; (c) (i) H2NNH2, EtOH, reflux, 3 h, 84%; (ii) SeO2, AcOH, 100 °C, 3 h, 95%; (d) (i) Fe powder, c-HCl, EtOH, reflux, 2 h, 87%; (ii) EtSO2Cl, pyridine, 50 °C, 2 h,
74%; and (e) Aryl boronic acids or pinacol aryl boronate, Cu(OAc)2, Et3N, 4 Å molecular sieves, CH2Cl2, rt, 40–85%.

B. J. Ryu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3887–3890
3889
Table 2
Furthermore, we want to seriously optimize for our compound 2
to discovery a new prostate cancer treatment.
Physicochemical properties, metabolic stability, CYP inhibition of thiazole derivatives
Compound Physicochemical properties
CYPa
MS
Log P
PAMPA
permeability
3A4
(%)
2D6
(%)
Rat,
in vitro
References and notes
1. Brinkmann, A. O.; Blok, L. J.; de Ruiter, P. E.; Doesburg, P.; Steketee, K.;
Berrevoets, C. A.; Trapman, J. J. Steroid Biochem. Mol. Biol. 1999, 69, 307.
2. Quigley, C. A.; De Bellis, A.; Marschke, K. B.; El-Awady, M. K.; Wilson, E. M.;
French, F. S. Endocr. Rev. 1995, 16, 271.
2
3.82 0.03 ꢀ5.86 0.06
6.7
0
>99%
a
Inhibition at 10 M CYP concentration (%).
l
3. Greenlee, R. T.; Taylor, M.; Bolden, S.; Wingo, P. A. CA Cancer J. Clin. 2000, 50, 7.
4. Estebanez-Perpiña, E.; Moore, J. M.; Mar, E.; Delgado-Rodrigues, E.; Nguyen, P.;
Baxter, J. D.; Buehrer, B. M.; Webb, P.; Fletternick, R. J.; Guy, R. K. J. Biol. Chem.
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5. Buzon, V.; Carbó, L. R.; Estruch, S. B.; Fletterick, R. J.; Estebanez-Perpiña, E. Mol.
Cell. Endocrinol. 2012, 348, 394.
6. Ryan, C. J.; Tindall, D. J. J. Clin. Oncol. 2011, 29, 3651.
7. Estebanez-Perpiña, E.; Arnold, A. A.; Nguyen, P.; Rodrigues, E. D.; Mar, E.;
Bateman, R.; Pallai, P.; Shokat, K. M.; Baxter, J. D.; Guy, R. K.; Webb, P.;
Fletternick, R. J. Proc. Natl. Acad. Sci. USA 2007, 104, 16074.
8. Lack, N. A.; Axerio-Cilies, P.; Tavassoli, P.; Han, F. Q.; Chan, K. H.; Feau, C.;
LeBlanc, E.; Guns, E. T.; Guy, R. K.; Rennie, P. S.; Cherkasov, A. J. Med. Chem.
2011, 54, 8563.
9. (a) Cell culture: Human prostate cancer LNCap cells cells were cultured in
RPMI1640 (Hyclone, UT) supplemented with 10% fetal bovine serum (FBS,
Hyclone), 100 U/ml of penicillin and 100 lg/ml of streptomycin in humidified
atmosphere of 5% CO2 at 37 °C. The culture medium was changed every 3 days.
(b) Cell viability assay: Cells were seeded in a 96-well plate at 4 ꢁ 103 cells/well,
cultured for 24 h and then incubated with compounds for 2 days. Then, cell
viability was measured in triplicates by the Cell Counting Kit-8 (Dojindo
Molecular Technologies, ML) according to the manufacturer’s protocol.
Absorbance was measured by using Wallac EnVision microplate reader
(PerkinElmer, Finland). (c) AR reporter luciferase assay: LNCap cells were
seeded in a 12-well plate at 8 ꢁ 104 cells/well. After 24 h, cells were transduced
by SureENTRY transduction reagent with Cignal Lenti AR reporter (5 ꢁ 105 TU;
SABioscience, MD) in RPMI1640 with 10% FBS according to the manufacturer’s
protocol. After 2 days, medium was changed to RPMI1640 with 10% FBS, 1%
antibiotics and 1% NEAA, and then transduced cells were selected in culture
Figure 3. Plasma concentration-time plots after an intravenous (h) and oral (s)
administration of compound 2 in SD male rats (n = 3).
medium with 30 lg/ml of puromycin (Sigma, MO). After puromycin selection,
selected cells (1.5 ꢁ 104 cells/well) were incubated in a 96-well plate for 1 day
and treated with compounds for 1 day. Then, AR activation was evaluated by
luciferase reporter assay (Promega, WI).
10. Wu, G.; Robertson, D. H.; Brooks, C. L., III; Vieth, M. J. Comput. Chem. 2003, 24,
1549.
11. Tran, K. V.; Bickar, D. J. Org. Chem. 2006, 71, 6640.
values and medium permeability against artificial permeable
membrane. In the metabolic stability assay, compound 2 remained
99% after 1 h incubation with rat liver microsomes. Compound 2
did not show the strong CYP inhibition activity. In in vitro assay,
compound 2 exhibited an acceptable in the permeability, meta-
bolic stability and CYP inhibition, but good in vitro activity may
not reflect good in vivo efficacy. Therefore, we further investigated
the in vivo pharmacokinetic profile of compound 2. As shown in
Figure 3 and Table 3, after oral administration of a 2.5 mg/kg dose
of compound 2 to rats, the peal plasma concentration (Cmax) of
12. General experimental methods and analytical data for the compounds: 1H NMR
spectra were recorded on a Bruker Fourier AG-300 spectrometer. Chemical
shifts are expressed as d values in parts per million (ppm), relative to TMS.
Coupling constant (J) was reported in Hertz unit (Hz). High-resolution mass
spectra (HRMS) was measured on a JMS-700 mass spectrometer (JEOL, Japan)
using electron impact (EI) method. N-(3-(1-(4-cyanophenyl)-3-methyl-6-oxo-
1,6-dihydropyridazin-4-yl)phenyl)ethanesulfonamide (1): 1H NMR (300 MHz,
CDCl3) d 7.95 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.46 (dd, J = 7.7, 7.6 Hz,
1H), 7.31 (d, J = 7.7 Hz, 1H), 7.30 (s, 1H), 7.14 (d, J = 7.6 Hz, 1H), 6.92 (s, 1H),
3.20 (q, 2H), 2.32 (s, 3H), 1.41 (t, 3H). HRMS (EI, M+): calcd for C20H18N4O3S:
394.1100, found: 394.1102; N-(3-(1-(3,4-dichlorophenyl)-3-methyl-6-oxo-
1,6-dihydropyridazin-4-yl)phenyl)ethanesulfonamide (2): 1H NMR (300 MHz,
CDCl3) d 7.88 (d, J = 2.3 Hz, 1H), 7.76 (br s, NH), 7.65 (dd, J = 8.7, 2.3 Hz, 1H),
7.55 (d, J = 8.7 Hz, 1H), 7.45 (dd, J = 7.7, 7.6 Hz, 1H), 7.32 (d, J = 7.7 Hz, 1H), 7.30
(s, 1H), 7.13 (d, J = 7.6 Hz, 1H), 6.93 (s, 1H), 3.19 (q, 2H), 2.30 (s, 3H), 1.39 (t,
3H). HRMS (EI, M+): calcd for C19H17Cl2N3O3S: 437.0368, found: 437.0364; N-
(3-(1-(3,5-dichlorophenyl)-3-methyl-6-oxo-1,6-dihydropyridazin-4-yl)-
phenyl)ethanesulfonamide (3): 1H NMR, (300 MHz, CDCl3) d 7.63 (s, 2H), 7.41
(dd, J = 7.7, 7.6 Hz, 1H), 7.31 (s, 1H), 7.22 (d, J = 7.7 Hz, 1H), 7.19 (s, 1H), 7.09 (d,
J = 7.6 Hz, 1H), 6.83 (s, 1H), 6.64 (br s, NH), 3.14 (q, 2H), 2.24 (s, 3H), 1.35 (t,
3H). HRMS (EI, M+): calcd for C19H17Cl2N3O3S: 437.0368, found: 437.0364; N-
(3-(1-(3-chloro-4-fluorophenyl)-3-methyl-6-oxo-1,6-dihydropyridazin-4-yl)-
phenyl)ethanesulfonamide (4): 1H NMR (300 MHz, CDCl3) d 7.81 (dd, J = 7.6
2.2 Hz, 1H), 7.63 (m, 2H), 7.47 (dd, J = 7.7, 7.6 Hz, 1H), 7.28 (d, J = 7.7 Hz, 1H),
7.27 (s, 1H), 7.15 (d, J = 7.6 Hz, 1H), 6.89 (s, 1H), 6.69 (br s, NH), 3.20 (q, 2H),
2.30 (s, 3H), 1.41 (t, 3H). HRMS (EI, M+): calcd for C19H17ClFN3O3S: 421.0663,
found: 421.0662; N-(3-(3-methyl-1-(naphthalen-2-yl)-6-oxo-1,6-dihydro-
pyridazin-4-yl)phenyl)ethanesulfonamide (5): 1H NMR (300 MHz, CDCl3) d
8.17 (d, J = 2.1 Hz, 1H), 7.95 (d, J = 8.7 Hz, 1H), 7.89 (m, 2H), 7.76 (dd, J = 2.1,
8.7 Hz, 1H), 7.52 (m, 2H), 7.45 (dd, J = 7.7, 7.6 Hz, 1H), 7.20 (br s, NH), 7.29 (m,
2H), 7.16 (d, J = 7.6 Hz, 1H), 6.96 (s, 1H), 3.18 (q, J = 7.4, 14.8 Hz, 2H), 2.33 (s,
3H), 1.40 (t, J = 7.4 Hz, 3H). HRMS (EI, M+): calcd for C23H21N3O3S: 419.1304,
found: 419.1307.
0.767 lg/mL was observed at 10.3 h. The elimination half-life for
compound 2 following oral administration was 21.6 h in rats. Com-
pound 2 showed perfect oral bioavailability (F = 102%) and low
blood clearance (CL = 84.4 mL/kg/h) in rats.
In this study, we synthesized the BF-3 binding small molecules
without affecting natural ligand binding, a series of pyridazinone-
based compounds, to inhibit the viability of AR-dependent human
prostate LNCap cells and AR activity combining with the computa-
tional method, having a good physicochemical and PK property.
Table 3
Pharmacokinetic parameters after an intravenous and oral administration of
compound 2 in SD male rats (n = 3)
Parameters
I.v. (2.5 mg/kg)
P.o. (2.5 mg/kg)
Tmax (h)
–
–
10.3 2.9
0.767 0.055
21.6 2.9
26.8 3.4
30.5 4.9
–
Cmax
(
lg/mL)
Tk1/2 (h)
AUC0–72 h
AUC0–1 (lg h/mL)
CL (mL/kg/h)
Vss (mL/kg)
MRT (h)
19.2 4.5
27.5 2.4
29.8 3.0
84.4 7.9
2250 259
26.9 4.2
–
13. (a) Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60, 7508; (b)
Gerbino, D. C.; Mandolesi, S. D.; Schmalz, H. G.; Podesta, J. C. Eur. J. Org. Chem.
2009, 3964.
14. (a) Patent WO2008/16239.; (b) Asano, T.; Yamazaki, H.; Kasahara, C.; Kubota,
H.; Kontani, T.; Harayama, Y.; Ohno, K.; Mizuhara, H.; Yokomoto, M.; Misumi,
K.; Kinoshita, T.; Ohta, M.; Tkeuchi, M. J. Med. Chem. 2012, 55,
7772.
(l
g h/mL)
–
34.9 6.3
102.5 16.4
F (%)

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B. J. Ryu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3887–3890
15. Log P: Log P is measured by a pH-metric method, based on a two-phase acid–
base titration in a mixture of water and octanol using GLpKa system by Sirius.
16. Parallel artificial membrane permeability (PAMPA) permeability: Donor solution
plate following the manufacturer’s instruction with some modification. The
compounds including ketoconazole known as CYP3A4 inhibitor were prepared
in acetonitrile to give final concentrations of 10 mM. NADP generating solution
(1.0 mM NADPþ, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl2ꢂ6H2O, and 0.4 U/
mL glucose-6-phosphate dehydrogenase in 10 mM KPO4, pH 8.0) was added to
each well of the microtiter plate followed by the vehicle acetonitrile (control)
and the test samples. The plate was covered and then incubated at 37 °C for
20 min. Enzyme reaction was initiated by the addition of enzyme/substrate (E/
S) mixture (0.5 pmol recombinant human CYP3A4 enzyme and 5 mM
dibenzylfluorescein, DBF). The plate was further incubated for 20 min,
followed by the addition of the stop solution to terminate the enzyme
activity. Background reading was measured in a similar manner except for the
E/S mixture which was added after the enzyme reaction was terminated. The
fluorescence of DBF metabolite fluorescein was measured on a fluorescence
plate reader with an excitation wavelength of 485 nm and an emission
wavelength of 530 nm.
(500
using the diluted system buffer (pH adjusted to 7.4). Five microliters lipid
solution was applied to each filter membrane. Donor solution (150 L) was
lM) was prepared by diluting 1 mM DMSO compound stock solution
l
added to each well of the filter plate. The filter plate was then put onto a
receiver plate with preloaded acceptor sink buffer. The sandwich was
incubated at room temperature for 16 h. Samples were taken from both
receiver and donor sides at the end of the incubation and analyzed using UV
spectrometer. Donor solutions were also analyzed for initial concentration.
17. Metabolic stability: Using rat liver microsomes, the amount of parent
compound remaining after 1 h incubation. The concentration of the used
compound is 5 mM and protein concentration is 1 mg/mL.
18. CYP3A4 enzyme assay: The assay was carried out using fluorometric enzyme
assays with Vivid CYP3A4 assay kit (PanVera, USA, CA) in a 96-well microtiter