Anti-androgens with full antagonistic activity toward human prostate tumor LNCaP cells with mutated androgen receptor

Article abstract of DOI:10.1016/S0960-894X(03)00575-4

Anti-androgens were designed based on the principle of inhibiting the folding of helix 12 of the nuclear androgen receptor. The prepared anti-androgens exhibited full antagonistic activity toward human prostate tumor LNCaP cells with T877A point-mutated nuclear androgen receptor, as far as examined, towards which other known anti-androgens, including hydroxyflutamide, are inactive or act as androgen agonists.

Full text of DOI:10.1016/S0960-894X(03)00575-4

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2655–2658
Anti-Androgens with Full Antagonistic Activity Toward Human
Prostate Tumor LNCaP Cells with Mutated Androgen Receptor
Toshiyasu Ishioka, Aya Tanatani, Kazuo Nagasawa and Yuichi Hashimoto*
Institute of Molecular & Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
Received 28 April 2003; accepted 31 May 2003
Abstract—Anti-androgens were designed based on the principle of inhibiting the folding of helix 12 of the nuclear androgen
receptor. The prepared anti-androgens exhibited full antagonistic activity toward human prostate tumor LNCaP cells with T877A
point-mutated nuclear androgen receptor, as far as examined, towards which other known anti-androgens, including hydroxy-
flutamide, are inactive or act as androgen agonists.
#
2003 Elsevier Ltd. All rights reserved.
Androgens, typically testosterone, are endogenous
ligands for the nuclear androgen receptor (AR) which is
a member of the steroid/thyroid/retinoid/vitamin D3
nuclear receptor superfamily, and play diverse physio-
which induce misfolding of helix 12 (denoted as mis-
folding inducers in this paper), and the other is AAs
which inhibit the folding of helix 12 (denoted as folding
inhibitors in this paper) (Fig. 1). Ourcompute r- assisted
docking studies using the X-ray structure of the LBD of
AR suggested that all currently known AAs, including
clinically useful flutamide and its active form, hydroxy-
flutamide, can be categorized as misfolding inducers.
1
,2
logical and pathophysiological roles. AR, like other
nuclear receptors, consists of three main functional
domains including the ligand-binding domain (LBD),
DNA-binding domain (DBD) and amino-terminal
3À5
domain (A/B region).
The general structure of the
LBD has been elucidated to be composed of 12 a helices
and a small b sheet by X-ray crystallography. Among
the substructures, the helix H12 plays a critical role in
the ligand-dependent activation of the receptor, that is,
it functions as a lid covering the ligand binding pocket,
being in the closed conformation when an agonist
occupies the binding pocket and in the open conforma-
tion without the agonist orwhen an antagonist occupies
The major obstacle in the treatment of prostate tumor
with AAs is the sudden appearance of AA-resistant
cells. These cells have generally lost androgen-depen-
dency in their growth and, further, AAs promote their
growth, whereas AAs had suppressed their growth
before the cells acquired AA-resistance. One major
molecularmechanism of AA- er sistance is point muta-
tion of AR. Some point mutations, including T877A
8,9
6
,7
the pocket.
and T874H, are clinically established.
ARs which
possess such a point mutation are considered to take a
helix 12-folded conformation, and are constitutively
active even in the absence of the cognate ligand, andro-
gen (Fig. 1). Of course, these mutated ARs can bind
androgens and misfolding inducer-type AAs, which sta-
bilize the active conformation of the AR, leading to
super-activation of the mutated ARs. Therefore, to
overcome the problem of AA-resistance based on AR
mutation, othertypes of AAs, which bind to the muta-
ted ARs and induce unfolding (orinhibit the folding of)
helix 12, would be useful (Fig. 1).
Among the pathophysiological effects elicited by
androgens, a role as endogenous tumor promoters,
especially forp ro state tumo r, is well known. Because of
this, anti-androgens (AA), which antagonize endogen-
ous androgens by competitively binding to AR, are
expected to be effective for treatment of androgen-
dependent tumors, especially for prostate tumor. On
the basis of a mechanistic consideration of AR activa-
tion, we can considertwo types of AAs. One is AAs
2
In this paper, we describe the design and biological
activities of AAs with an isoxazolone moiety (1–4, Fig. 2)
*
8
Corresponding author. Tel.: +81-3-5841-7847; fax: +81-3-5841-
495; e-mail: hashimot@iam.u-tokyo.ac.jp
0
960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00575-4

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T. Ishioka et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2655–2658
consideration, as well as our previous studies on nuclear
4
,5,7,10À12
receptor ligands,
azolone derivatives 1–4 (Fig. 2).
led us to design 3-phenylisox-
Compounds 1–4 were prepared by usual synthetic
methods as illustrated in Figure 3, and the structures
were confirmed by NMR and mass spectrometry, and
1
3À16
by appropriate analytical values.
Biological Activities of Compounds 1–4
Preliminary evaluation of androgenic and anti-andro-
genic activities, that is, activities toward normal ARs, of
the compounds was done by using a combination of two
assay systems, that is, receptor-binding assay and
growth promotion/inhibition assay using androgen-
1
2,17À19
dependent Shionogi carcinoma cells SC-3.
The
ability of the compounds to be ligands forAR is indi-
cated by the former assay, while the latter assay deter-
mines whether the compounds are androgen agonists or
antagonists.
For receptor-binding assay, we adopted competitive
3
binding assay using [ H]testosterone and recombinant
1
Figure 1. Schematic illustration of putative mechanisms of activation
and inactivation of ARs by point mutation and with AAs.
2,19
human AR as described previously. The recombi-
nant AR was prepared from cytosol of Escherichia coli
transformed with a human AR LBD expression vector
(GST-hARLBD), which codes amino acids 627–919 of
hAR fused with GST protein under the lac pro-
1
2,19
moter.
thus prepared toward [ H]testosterone, that is, the K
The binding activity of the GST-hARLBD
3
d
(dissociation constant) value, was determined to be 1.4
nM by Scatchard analysis, and this value is close to that
of AR prepared from intact SC-3 cells. The binding
activity of a test compound was determined by incu-
3
bation of GST-hARLBD with [ H]testosterone in the
presence of various concentrations of the test com-
pound. The concentration of a test compound that
3
inhibits [ H]testosterone-binding by 50% (IC5 value)
0
was determined after log–logit transformation, and the
3
K value was calculated based on the K of [ H]testos-
d
terone. The K value of flutamide thus obtained was 1.3
i
mM. The values of relative receptor-binding activity of
i
Figure 2. Structures of folding inhibitor-type AAs (1–4) and known
AAs [hydroxyflutamide: HF (5) and RU (6)].
the test compounds, that is, (K value of flutamide)/(Ki
i
value of test compound), were in the range of 100–150
(details will be published elsewhere). In the growth pro-
motion/inhibition assay using androgen-dependent SC-
3 cells, none of the compounds prepared showed
growth-promoting activity, suggesting that none was an
androgen agonist. All the compounds prepared showed
dose-dependent growth-inhibitory activity on SC-3 cells
incubated in the presence of 10 nM testosterone, sug-
gesting that all the compounds act as AAs, at least
toward normal AR.
which act as possibly full antagonists in human prostate
tumorLNCaP cells with T877A mutation in the AR,
towards which currently known AAs act as androgen
agonists.
Design of Folding Inhibitor-type AAs
Recently, we reported novel structural types of AAs
1
0À12
with an isoxazolone skeleton.
Computer-assisted
docking studies suggested that (Z)-4-(4-N,N-diethyl-
aminophenylmethylene)-3-methyl-5(4H)-isoxazolone can
be a ligand forAR, with the 3-methyl moiety inte ra cting
with helix 12 of AR. Based on this result, we considered
that by the use of the isoxazolone skeleton as a scaffold,
folding inhibitor-types AAs might be prepared by
replacing the 3-methyl group with a bulky group. This
Activity of compounds 1–4 toward mutated, con-
stitutively active ARs was analyzed by the use of
LNCaP and 22Rv1 cells, which express T877A and
T874H point-mutated ARs, respectively. All of the
compounds prepared showed growth-inhibitory activity
on these cells, while known classical AAs, hydroxy-
flutamide (HF: 5, Fig. 2) and RU (6, Fig. 2) showed
8
,9

T. Ishioka et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2655–2658
2657
growth-promoting activity (data for LNCaP cells were
shown in Fig. 4a, bottom). The result suggests that
compounds 1–4 do not elicit androgenic activity (do not
super-activate the mutated ARs) and act as AAs on
LNCaP and 22Rv1 cells, while HF (5) and RU (6) act as
androgen agonists on these cells.
The amount of PSA was measured by the use of an
ELISA PSA Assay Kit (Cayman Co. Ltd) according to
the protocol recommended by the supplier. The experi-
ment was performed in triplicate and repeated at least
two times. Though the values deviated from experiment
to experiment, the results (order of potency) were
basically reproducible, and a typical set of data is shown
in Figure 4.
Quantitative assessment of androgenic and anti-andro-
genic activities of the compounds was performed by
measurement of the amount of prostate-specific antigen
As shown in Figure 4, LNCaP cells incubated underthe
normal conditions produced a small amount of PSA
(the amount was defined as 1-fold in Fig. 4a), and
addition of 10 nM testosterone (TS) markedly enhanced
the production (the amount was defined as 100% in Fig.
4b). Classical AAs, HF (5) and RU (6), also enhanced
the production to 4–6 times (Fig. 4a), suggesting that
these AAs act as androgen agonists toward LNCaP
cells, which is in accordance with the cell growth pro-
motion/inhibition assay data forLNCaP and 22Rv1
described above. Compounds 1–4 did not enhance the
PSA production at all in the concentration range of 10–
(
marker molecule of prostate tumor malignancy and its
PSA) produced by LNCaP cells (Fig. 4). PSA is a
2
0,21
production is known to be induced by androgens.
Therefore, androgenic activity can be detected in terms
of enhanced production of PSA (Fig. 4a), and anti-
androgenic activity can be assessed in terms of inhibi-
tion of PSA production induced by androgens (Fig. 4b).
1
000 nM, suggesting that these compounds possess no
androgenic activity in the concentration range examined
Fig. 4a). In the inhibition assay of testosterone-induced
(
PSA production (Fig. 4b), HF (5) was almost com-
pletely inactive (98%), and RU (6) showed moderate
inhibitory activity (58%). The results suggest that HF
3
Figure 3. Synthetic procedures of compounds 1–4: (a) POCl , DMF,
8
ꢀ
.
2
0 C, 2 h; (b) NH OH HCl, phosphoric acid, rt, overnight.
(
5) and RU (6) act as a full agonist and a partial ago-
nist/antagonist toward LNCaP cells in these assay sys-
tems, respectively. In contrast, all of compounds 1–4
showed inhibitory activity on testosterone-induced PSA
production (Fig. 4b). This result, combined with the
results in Fig. 4a, suggests that the compounds act as
possibly full AAs even on the LNCaP cells, which con-
stitutively express active mutated AR. Among the com-
pounds prepared, the 4-hydroxyl analogue (4) showed
the most potent inhibitory activity on PSA production. No
substituent effect at the 3-phenyl moiety of the isoxazolone
derivatives on the activity was apparent, as far as com-
pared among compounds 1–4. Further investigation is in
progress.
Conclusion
We designed AAs 1–4 as candidate inhibitors of the
folding of helix 12 of AR, with the aime of obtaining
full antagonistic activity toward constitutively active
point-mutated ARs. The prepared compounds 1–4 were
indeed found to act as full AAs on human prostate
tumorLNCaP cells exp er ssing T877A mutated AR,
as faras examined, while classical and clinically used
AAs act as full androgen agonists or partial androgen
agonists/antagonists on these cells.
Figure 4. Effects of prepared AAs (1–4) and known AAs [hydroxy-
flutamide: HF (5) and RU (6)] on PSA production by LNCaP cells.
(
A) Amount of PSA produced in the absence or presence of testoster-
one (TS: 10 nM) ortest compounds (100 nM). The amount of PSA
produced by non-treated cells was defined as 1-fold. Values below the
panel indicate cell growth promoting activity (%) of the compounds
when the activity of TS was defined as 100%. (B) Effect of test com-
pounds (300 nM) on the amount of PSA produced in the presence of
Acknowledgements
The work described in this paper was partially sup-
ported by Grants-in-Aid for Scientific Research from
The Ministry of Education, Science, Sports and Culture,
Japan.
10 nM testosterone (TS). The amount of PSA produced in the pres-
ence of 10 nM testosterone (TS) alone was defined as 100%.

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T. Ishioka et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2655–2658
7.53 (d, J=9.0 Hz, 1H), 7.31 (d, J=7.5 Hz, 1H), 7.23 (d,
References and Notes
J=9.0 Hz, 1H), 7.14 (d, J=2.0 Hz, 1H), 6.65 (d, J=9.0 Hz,
2H), 3.47 (tt, J=3.5, 3.5 Hz, 4H). 2.08 (tt, J=3.5, 3.5 Hz, 4H).
Anal. (%): calcd forC 20H N O F; C, 71.42; H, 5.09; N, 8.33.
17 2 2
Found; C, 71.46; H, 5.26; N, 8.12.
1. Mooradian, A. D.; Morley, J. E.; Korenman, S. G. Endocr.
Rev. 1987, 8, 1.
2
3
3
. Zhi, L.; Martinborough, E. Ann. Rep. Med. Chem. 2001,
6, 169.
. MacLean, I. J.; Warne, G. L.; Zajac, J. D. J. Steroid Bio-
15. (Z)-4-(4-Pyrrolidinophenylmethylene)-3-(4-fluoro-phenyl)-
5(4H)-isoxazolone (ISOP-4F: 3): purple crystals [hexane/
ꢀ
+
1
chem. Mol. Biol. 1997, 62, 233.
EtOAc]; mp 217 C; MS(FAB) m/z 337 (MH ); H NMR
(500 MHz, CDCl ) d: 8.40 (brs, 2H), 7.58 (ddd, J 8.5, 5.5, 2.0
Hz, 2H), 7.31 (s, 1H), 7.23 (td, J=8.5, 2.0 Hz, 2H), 6.60 (d,
4
5
6
. Hashimoto, Y.; Shudo, K. Cell Biol. Rev. 1991, 25, 209.
. Hashimoto, Y. Cell Struct. Funct. 1991, 16, 113.
. Wurtz, J.; Bourguet, W.; Renaud, J.; Vivat, V.; Chambon,
3
=
J=9.0 Hz, 2H), 3.48 (tt, J=3.5, 3.5 Hz, 4H), 2.09 (tt, J=3.5,
P.; Moras, D.; Gronemeyer, H. Nat. Struct. Biol. 1996, 3, 87.
. Eyrolles, L.; Kawachi, E.; Matsushima, Y.; Nakajima, O.;
Kagechika, H.; Hashimoto, Y.; Shudo, K. Med. Chem. Res.
17 2 2
3.5 Hz, 4H). Anal. (%). calcd forC 20H N O F; C, 71.42; H,
7
5.09;N, 8.33. Found; C, 71.51; H, 5.29; N, 8.21.
16. (Z) - 4 - (4 - Pyrrolidinophenylmethylene) - 3 - (4 - hydroxy -
phenyl)-5(4H)-isoxazolone (ISOP-4H: 4): red crystals
1
992, 2, 361.
. Beer, H.-D.; Florence, C.; Dammeier, J.; McGuire, L.;
ꢀ
[CH Cl ]; mp 234–235 C; MS(FAB) m/z 335 (MH ); H
2 2
+
1
8
Werner, S.; Duan, D. R. Oncogene 1997, 15, 2211.
. Sramkoski, R. M.; Pretlow, T. G.; Giaconia, J. M.; Pre-
tlow, T. P.; Schwartz, S.; Sy, M. S.; Marengo, S. R.; Rhim,
J. S.; Zhang, D. S.; Jacobberger, J. W. In Vitro Cell. Dev. Biol.
Anim 1999, 35, 403.
NMR (500 MHz, DMSO-d ) d: 9.98 (s, 1H), 8.43 (brd,
J=9.0 Hz, 2H), 7.45 (s, 1H), 7.44 (d, J=8.0 Hz, 2H), 6.93
(d, J=8.0 Hz, 2H), 6.70 (d, J=9.0 Hz, 2H), 3.46 (td,
6
9
J=3.5, 3.5 Hz, 4H), 1.98 (tt, J=3.5, 3.5 Hz, 4H), HRMS
+
(FAB, MH )
335.1432.
20 19 2 3
C H N O , calcd for335.1396, found
10. Hashimoto, Y. Bioorg. Med. Chem. 2002, 10, 461.
11. Hashimoto, Y. Mini. Rev. Med. Chem. 2002, 2, 543.
12. Ishioka, T.; Kubo, A.; Koiso, Y.; Nagasawa, K.; Itai, A.;
17. Hiraoka, D.; Nakamura, N.; Nishizawa, Y.; Uchida, N.;
Noguchi, S.; Matsumoto, K.; Sato, B. Cancer Res. 1987, 47,
6560.
18. Miyachi, H.; Azuma, A.; Kitamoto, T.; Hayashi, K.;
Kato, S.; Koga, M.; Sato, B.; Hashimoto, Y. Bioorg. Med.
Chem. Lett. 1997, 7, 1483.
19. Takahashi, H.; Ishioka, T.; Koiso, Y.; Sodeoka, M.;
Hashimoto, Y. Biol. Pharm. Bull. 2000, 23, 1387.
20. Lee, C.; Sutkowski, D. M.; Sensibar, J. A.; Zelner, D.;
Kim, I.; Amsel, I.; Shaw, N.; Prins, G. S.; Kozlowski, J. M.
Endocrinology 1995, 136, 796.
21. Riegman, P. H. J.; Vlietstra, R. J.; van der Korput,
H. A. G. M.; Brinkmann, A. O.; Trapman, J. Mol. Endocrinol.
1991, 5, 1921.
Hashimoto, Y. Bioorg. Med. Chem. 2002, 10, 1555.
3. (Z)-4-(4-Pyrrolidinophenylmethylene)-3-phenyl-5(4H)-iso-
xazolone (ISOP-00: 1): red crystals [CH Cl /EtOAc]; mp 172–
73 C; MS(FAB) m/z 319 (MH ), 318 (M ); H NMR
500MHz, CDCl ) d: 8.42 (brs, 2H), 7.50–7.60 (m, 5H), 7.370
s, 1H), 6.59 (d, J=9.0 Hz, 2H), 3.47 (tt, J=3.0, 3.0 Hz, 4H),
.08 (tt, J=3.0, 3.0 Hz, 4H). Anal. (%): calcd forC 20H N O ;
1
2
2
ꢀ
+
+
1
1
(
(
3
2
18
2
2
C, 75.45; H, 5.70; N, 8.80. Found; C, 75.35; H, 5.82; N, 8.69.
1
5
4. (Z)-4-(4-Pyrrolidinophenylmethylene)-3-(2-fluoro-phenyl)-
(4H)-isoxazolone (ISOP-2F: 2): red needles [hexane/EtOAc];
ꢀ
+
1
mp 200–201 C; MS(FAB) m/z 337 (MH ); H NMR
500 MHz, CDCl ) d: 8.41 (brs, 2H), 7.55 (d, J=7.5 Hz, 1H),
(
3