MED
gave the desired compound 2. If more than one free hydroxy
group is present on the benzopyran ring, silyl protection was
needed before the next reaction.
General procedure for the formation of enol-O-triflates (3): A so-
lution of compound 2 (1.0 equiv) and 2,6-di-tert-butyl-4-methylpyri-
dine (DTBMP, 1.3 equiv) in anhyd CH2Cl2 under N2 at 08C was treat-
ed with triflic anhydride (Tf2O, 1.2 equiv). The reaction was stirred
for 10 min at 08C. The reaction was filtered to remove any solid
and the filtrate was concentrated in vacuo. The residue was redis-
solved in EtOAc and washed with saturated aq NaHCO3 and brine.
The organic layer was dried (anhyd MgSO4), filtrated, and evaporat-
ed in vacuo. Purification by flash column chromatography (silica
gel) gave the desired compound 3.
Figure 4. Antagonistic activities of bicalutamide and compound 6 f in cell-
based reporter-gene assay in 293T cells co-transfected with ARE-luciferase
and WT AR (&) or AR mutant plasmids (W741L, &; T877A, &). RLU=relative
light units ÆSEM.
General procedure for compound 5 via Suzuki coupling fol-
lowed by Diels–Alder reaction: Compound 3 (1.0 equiv), boronic
acid (1.1 equiv), Pd(PPh3)4 (5 mol%) and Na2CO3 (3.0 equiv) were
suspended in EtOH/toluene/H2O (1:1:0.5) and the reaction mixture
was stirred at 708C for ~10 h. After completion (monitored by
TLC), the reaction was diluted with EtOAc and washed with brine.
The organic layer was dried (anhyd MgSO4), filtrated, and evaporat-
ed in vacuo. The resulting crude product 4 was used in the next re-
action without further purification. A solution of compound 4 and
maleimide derivative (1.1 equiv) in toluene was heated at reflux for
24 h. After completion (monitored by TLC), the reaction was con-
centrated in vacuo. Purification by flash column chromatography
(silica gel) gave the desired compound 5.
showed antagonistic activities against WT AR. However, bicalut-
amide acted as an agonist, not an antagonist, on the bicalut-
amide-resistant AR mutant (W741L) even without DHT treat-
ment, whereas compound 6 f retained its antagonistic activity
toward this AR mutant. In the case of T877A AR mutant, com-
pound 6 f effectively antagonized the AR activity. Surprisingly,
bicalutamide had little or no antagonistic effects on either of
the clinically relevant AR mutant cell lines. Therefore, com-
pound 6 f might be suitable for use as a potential therapeutic
agent in the treatment of hormone-refractory prostate cancer
through antagonizing antiandrogen-resistant mutant ARs. Un-
fortunately, we failed to demonstrate the in vivo efficacy of
compound 6 f, due to its poor solubility and oral bioavailability
(data not shown).
General procedure of allylic alcohol formation (6): A solution of
compound 5 (1.0 equiv) and DTBMP (2.0 equiv) in CH2Cl2 at 08C
was treated with m-chloroperoxybenzoic acid (mCPBA, 1.5 equiv)
and stirred for 1 h at the same temperature. After completion
(monitored by TLC), the reaction was diluted with EtOAc and
washed with saturated aq NH4Cl and brine. The organic layer was
dried (anhyd MgSO4), filtrated, and evaporated in vacuo. Purifica-
tion by flash column chromatography (silica gel) gave the desired
compound 6. If necessary, silyl deprotection was carried out.
In conclusion, we discovered a novel nonsteroidal AR antag-
onist using a cell-based reporter gene assay, along with our
small-molecule library constructed using a diversity-oriented
synthetic pathway. From 19 synthetic analogues containing a
novel benzopyran-fused tetracyclic core skeleton, compound
6 f was identified as having an excellent antagonistic activity
confirmed by western blot analysis, RT-PCR, and in vitro cellular
proliferation assay. We also demonstrated that compound 6 f is
active against not only WT AR but also mutant AR, such as
T877A or W741L (bicalutamide-resistant AR mutant). The poor
bioavailability/water solubility of compound 6 f may limit its
potential as a therapeutic agent; however, this new molecular
framework might provide valuable insight for the development
of therapeutics able to treat advanced prostate cancer. Studies
into the mode-of-action are currently underway.
1
Compound 6 f: Yellowish solid (59% from compound 5 f); H NMR
(500 MHz, [D6]DMSO): d=9.58 (s, 1H), 7.21–7.12 (m, 7H), 7.02–7.01
(d, J=6.5 Hz, 2H), 6.88–6.86 (m, 2H), 6.37 (dd, J=8.5, 2.0 Hz, 1H),
6.28 (d, J=2.0 Hz, 1H), 5.50 (d, J=6.0 Hz, 1H), 4.71 (dd, J=6.0,
1.5 Hz, 1H), 4.21 (d, J=7.5 Hz, 1H), 3.93–3.83 (AB q, JAB =15.2 Hz,
2H), 3.67–3.64 (m, 1H), 3.52 (dd, J=6.5, 1.5 Hz, 1H), 2.28–2.22 (m,
1H), 2.13–2.09 (m, 1H), 2.06–1.99 (m, 1H), 1.96–1.87 (m, 2H), 1.81–
1.68 ppm (m, 3H); 13C NMR (125 MHz, [D6]DMSO): d=178.0, 175.8,
159.1, 154.5, 138.1, 136.3, 131.5, 129.0, 128.2, 127.9, 127.8, 127.7,
125.7, 125.6, 123.8, 115.0, 109.1, 104.5, 104.4, 89.2, 64.4, 64.3, 47.8,
42.0, 41.5, 41.3, 41.2, 35.9, 32.8, 23.6, 22.9 ppm; HRMS-FAB: m/z
[M+H]+ calculated for C32H30NO5: 508.2124, found: 508.2126.
Cell-based ARE-luciferase reporter assay for androgen receptor antag-
onist: LNCaP stable cells (1ꢁ104) were seeded on 96-well white
plate coated with poly-d-lysine hydrobromide and cultured with
phenol red-free IMDM medium supplemented with 10% charcoal:
dextran stripped FBS and 1% (v/v) antibiotic/antimycotic solution.
After 24 h, cells were treated with 20 nm of dihydrotestosterone
(DHT), and 10 mm of test compound (about 2000 compounds) or
20 mm of bicalutamide, and incubate for a further 24 h. The cells
were washed with PBS, and the PBS was drained using Hydroflexꢂ.
After the injection of 50 mL of passive cell lysis buffer into individu-
al wells of the 96-well plate, the plate was incubated for 30 min at
room temperature. Finally, 50 mL of luciferase assay reagent solu-
tion was added to each well. The resulting luminescence was de-
tected by Synergy HT.
Experimental Section
General procedure for the cyclization of hydroxyacetophenones
(2): A solution of hydroxyacetophenone 1 (1.0 equiv) in EtOH was
treated with pyrrolidine (3.0 equiv) and cyclopentanone (3.0 equiv)
and then heated at reflux for ~24 h. After completion (monitored
by TLC), the reaction was concentrated in vacuo. The residue was
redissolved in EtOAc and washed with aq 1n HCl and brine. The
organic layer was dried (anhyd MgSO4), filtrated, and evaporated in
vacuo. Purification by flash column chromatography (silica gel)
532
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ChemMedChem 2010, 5, 529 – 533