Structure-activity relationships in 1,4-benzodioxan-related compounds. 8. {2-[2-(4-chlorobenzyloxy)phenoxy]ethyl}-[2-(2,6-dimethoxyphenoxy)ethyl]amine (clopenphendioxan) as a tool to highlight the involvement of α1D- and α1B-adrenore

Article abstract of DOI:10.1021/jm0580398

A series of new α₁-adrenoreceptor antagonists (5-18) was prepared by introducing various substituents (Topliss approach) into the ortho, meta, and para positions of the benzyloxy function of the phendioxan open analogue 4 ("openphendioxan"). Al

Full text of DOI:10.1021/jm0580398

7750
J. Med. Chem. 2005, 48, 7750-7763
Structure-Activity Relationships in 1,4-Benzodioxan-Related Compounds. 8.1
{2-[2-(4-Chlorobenzyloxy)phenoxy]ethyl}-[2-(2,6-dimethoxyphenoxy)ethyl]amine
(Clopenphendioxan) as a Tool to Highlight the Involvement of r1D- and
r1B-Adrenoreceptor Subtypes in the Regulation of Human PC-3 Prostate Cancer
Cell Apoptosis and Proliferation
Wilma Quaglia,§ Giorgio Santoni,‡ Maria Pigini,§ Alessandro Piergentili,§ Francesco Gentili,§ Michela Buccioni,§
Michela Mosca,‡ Roberta Lucciarini,‡ Consuelo Amantini,‡ Massimo Ivan Nabissi,‡ Patrizia Ballarini,|
Elena Poggesi, Amedeo Leonardi, and Mario Giannella*,§
Dipartimento di Scienze Chimiche, Universita` degli Studi di Camerino, via S. Agostino 1, 62032 Camerino, Italy,
Dipartimento di Medicina Sperimentale e Sanita` Pubblica, Universita` degli Studi di Camerino, via Scalzino, 62032 Camerino,
Italy, Dipartimento di Biologia Molecolare, Cellulare e Animale, Universita` degli Studi di Camerino, via Camerini, 62032
Camerino, Italy, and Divisione Ricerca e Sviluppo Farmaceutici, Recordati S. p. A., via Civitali 1, 20148 Milano, Italy
Received August 3, 2005
A series of new R1-adrenoreceptor antagonists (5-18) was prepared by introducing various
substituents (Topliss approach) into the ortho, meta, and para positions of the benzyloxy function
of the phendioxan open analogue 4 (“openphendioxan”). All the compounds synthesized were
potent antagonists and generally displayed, similarly to 4, the highest affinity values at R1D-
with respect to R1A- and R1B-AR subtypes and 5-HT1A subtype. By sulforhodamine B (SRB)
assay on human PC-3 prostate cancer cells, the new compounds showed antitumor activity
(estimated on the basis of three parameters GI50, TGI, LC50), at low micromolar concentration,
with 7 (“clopenphendioxan”) exhibiting the highest efficacy. Moreover, this study highlighted
for the first time R1D- and R1B-AR expression in PC3 cells and also demonstrated the involvement
of these subtypes in the modulation of apoptosis and cell proliferation. A significant reduction
of R1D- and R1B-AR expression in PC3 cells was associated with the apoptosis induced by 7.
This depletion was completely reversed by norepinephrine.
Introduction
carcinoma of the prostate (LNCap) cancer cells,7 while
no detectable R1A-AR mRNA expression has been found
in the androgen-independent human prostate PC-3 and
DU-145 cancer cells.8 No data concerning the expression
of R1B- and R1D-AR in prostate cancer cells have been
so far provided.
Some R1-AR antagonists are already in use for clinical
treatment of benign prostatic hyperplasia (BPH).9 Their
therapeutic effects may be attributed to not only reduced
prostatic smooth muscle tone but also inhibition of
nonepithelial (stromal and smooth muscle cells) cell
proliferation.
Recent studies have demonstrated that R1-AR an-
tagonists, such as doxazosin and terazosin, induce
apoptosis in primary human prostate cancer epithelial
cells, DU-145 and PC-3, and smooth muscle cells,
without affecting cell proliferation.8 The mechanisms of
apoptosis induction seem to be R1A-AR independent.8,10
Putative mechanisms underlying doxazosin-mediated
apoptosis include the up-regulation of transforming
growth factor beta (TGF-beta) signaling effectors11,12
and blocking of intracellular protein kinase B/Akt
activation.13 Terazosin-induced PC-3 and DU-145 cancer
cells death is p53- and Rb-independent and is associated
with G1 phase cell cycle arrest, up-regulation of p27Kip1
and Bax, and down-regulation of bcl-2,14-16 and I kappa
B alpha induction.12
The R1-adrenoreceptors (R1-ARs) belong to the super-
family of G-protein-coupled receptors and, on the basis
of pharmacological and binding studies, have been
subdivided into at least three subtypes, namely R1A (R1a),
R1B (R1b), and R1D (R1d), with upper and lower case
subscripts being used to designate the native or recom-
binant receptor, respectively.2 An additional R1-AR
subtype (R1L), characterized by low affinity for prazosin,
seems to be a functional phenotype of the R1A subtype.3
At the peripheral level, R1-ARs are expressed in a
wide variety of tissues, including liver, kidney, blood
vessels, heart, and prostate. The R1A-, R1B-, and R1D-AR
subtypes are differentially localized in human prostate,
with R1A-AR believed to be predominant in the fibro-
muscular stroma, but not in the glandular epithelium,4
while R1B-AR is mainly localized in the epithelium5 and
R
1D-AR principally detected in the stroma and blood
vessels.6
Few studies have been reported on the expression of
R1-AR subtypes on prostate cancer cells. R1A-AR expres-
sion, both at mRNA and protein levels, has been
detected in the human androgen-dependent lymph node
* To whom correspondence should be addressed. Phone: +39 0737
402257. Fax +39 0737 637345. E-mail: mario.giannella@unicam.it.
§ Dipartimento di Scienze Chimiche, Universita` degli Studi di
Camerino.
‡ Dipartimento di Medicina Sperimentale e Sanita` Pubblica, Uni-
versita` degli Studi di Camerino.
The role of R1-ARs in agonist-stimulated proliferation
has mainly been demonstrated for smooth muscle myo-
cytes, although there is evidence that these receptors
may participate in the promotion of other cell types
| Dipartimento di Biologia Molecolare, Cellulare e Animale, Uni-
versita` degli Studi di Camerino.
Recordati S.p.A.
10.1021/jm0580398 CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/02/2005

SAR of 1,4-Benzodioxan-Related Compounds
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7751
Chart 1
Scheme 1a
including prostate cells.17-19 In this respect, growing
evidence supports the role of R1-ARs in the direct
mitogenic effect of catecholamines on prostate growth.20
R1-Adrenergic stimulation of human prostate stromal
and vascular smooth muscle increases DNA synthesis
and cell proliferation via p44/42 (ERK1/2) MAPK acti-
vation.21,22 The identity of the R1-AR subtypes involved
in the norepinephrine (NE)-mediated stimulating effects
in prostatic nonepithelial cells are still unknown, whereas
the involvement of R1D-AR subtype has been sug-
gested.22 Finally, in human LNCap androgen-dependent
prostate cancer cells, epinephrine-induced prostate
cancer cell proliferation was inhibited by the competitive
R1-AR antagonists, prazosin and WB4101.7
In continued efforts to identify potent and selective
R1-AR antagonists, we previously reported the discovery
of compounds prepared by subsequent modifications of
WB 4101 (1):23 the insertion of a phenyl ring or a p-tolyl
moiety at the 3-position having a trans relationship with
the 2-side chain led to phendioxan (2)24 and mephen-
dioxan (3),25 the latter proving significantly selective for
R1A-AR subtype relative to both R1B and R1D subtypes.
Subsequently, the opening of the benzodioxan ring of
2 afforded N-[2-[2-benzyloxyphenoxy]ethyl]-N-[2-(2,6-
dimethoxyphenoxy)ethyl]amine (4), showing high po-
tency and significant selectivity for R1D subtype with
respect to the R1A and R1B subtypes.26 The name of
“openphendioxan” has now been given to compound 4.
In the present study, in an attempt to further improve
the R1D-AR selectivity of 4, while obviously maintaining
its high affinity, various substituents were introduced
into the ortho, meta, and para positions of the aromatic
ring of the benzyloxy function (compounds 5-18) (Chart
1), with the aim to recognize possible ancillary binding
sites. In fact, it is known that the aromatic areas play
a critical role in the interaction of R1-AR antagonists
with the corresponding receptors.27,28 The substituents
were chosen using the Topliss approach, which takes
into account the electronic, lipophilic, and steric factors
for substitution on a phenyl ring, using basic Hansch
principles in a noncomputerized manner.29,30
a Reagents: (a) NaH/DMF; (b) H2/Pd; (c) K2CO3/DMF; (d) 2-(2,6-
dimethoxyphenoxy)ethylamine,32 NaBH3CN/EtOH.
cell line used in this study. Finally, the ability of
compound 7 and the lead 4 to inhibit the R1-AR-
dependent growth of PC-3 cells by inducing apoptosis
and/or inhibiting cell proliferation was evaluated.
Chemistry. The new compounds 5-18 were synthe-
sized according to the methods reported in Schemes
1-3. Alkylation of 2-(benzyloxy)phenol with 2-chloro-
1,1-dimethoxyethane gave 1-(2,2-dimethoxyethoxy)-2-
[(benzyl)oxy]benzene (19). 2-(2,2-Dimethoxyethoxy)-
phenol (20) obtained by catalytic hydrogenation of 19
was then alkylated with the appropriate benzyl chlo-
Compounds 5-18 were evaluated for their affinity for
the R1-AR subtypes and their prostate antitumor activ-
ity with respect to unsubstituted lead 4, and doxazosin
was used as a reference.31 Furthermore, an in-depth
investigation was conducted to determine the R1-AR
subtypes expression in the human PC-3 prostate cancer

7752 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Quaglia et al.
Scheme 2a
a Reagents: (b) H2/Pd; (c) K2CO3/DMF; (e) MeOCOCl/Et3N/CHCl3; (f) DIAD/Ph3P/THF; (g) KOH/MeOH.
rides. Subsequent acidic hydrolysis of the substituted
benzyl ethers obtained (21a-i) yielded the correspond-
ing aldehydes (22a-i), which produced compounds
5-13 by reductive amination with 2-(2,6-dimethoxyphe-
noxy)ethylamine32 (Scheme 1). Since the acidic hydroly-
sis of p-alkoxy-substituted benzyl ethers afforded the
corresponding aldehydes with very low yields, ligands
14-17 were prepared according to Scheme 2, starting
from N-[2-[2-benzyloxyphenoxy]ethyl]-N-[2-(2,6-dimethox-
yphenoxy)ethyl]amine (4).26 Protection of the amino
group with methyl chloroformate (compound 23) and the
subsequent catalytic cleavage of the benzyl group
yielded the corresponding phenol (24), which was then
alkylated with the appropriate benzyl chlorides in DMF
and K2CO3 to obtain derivatives 25a-c, or else etheri-
fied with p-ethoxybenzyl alcohol in the presence of DIAD
and (C6H5)3P to give 26. The basic hydrolysis of the
carbamates 25a-c and 26 thus obtained yielded the
corresponding methoxy (14-16) and ethoxy (17) deriva-
tives. Finally, compound 18 was prepared as shown in
Scheme 3. (2-Hydroxyphenoxy)acetic acid methyl ester
was alkylated with (4-isopropoxyphenyl)-methanol to
the corresponding ester 27, which was transformed
into [2-(4-isopropoxybenzyloxy)phenoxy]acetic acid (28)
by subsequent basic hydrolysis. Compound 28 was
amidated in the presence of Et3N and EtOCOCl with
2-(2,6-dimethoxyphenoxy)ethylamine32 to the corre-
sponding amide 29, which was reduced with borane-
methyl sulfide complex in dry THF to give compound
18.
Results and Discussion
Binding and Functional Experiments. The phar-
macological profile of compounds 5-16 was evaluated
by radio-receptor binding assays and compared with 426
and doxazosin33 as reference compounds. [3H]Prazosin
was used to label cloned human R1-AR subtypes ex-
pressed in CHO cells.34 Furthermore, [3H]8-hydroxy-2-
(di-n-propylamino)tetralin was used to label cloned
human serotonin 5-HT1A receptors expressed in HeLa
cells.35 Affinity for the three R1-AR subtypes and for the
5-HT1A subtype was expressed as pKi values and is
shown in Table 1.
Receptor subtype selectivity of compounds 5-18 was
further determined on R1-ARs of different isolated
tissues and compared with 426 and doxazosin33 as
reference compounds. R1-AR subtypes blocking activity
was assessed by antagonism of (-)-NE-induced contrac-

SAR of 1,4-Benzodioxan-Related Compounds
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7753
Scheme 3a
a Reagents: (f) DIAD/Ph3P/THF; (h) NaOH; (i) EtOCOCl/Et3N; 2-(2,6-dimethoxyphenoxy)ethylamine32/CHCl3; (l) BH3‚Me2S/THF.
tion of rat prostatic vas deferens (R1A
)
36 or thoracic aorta
not significantly different from that of the unsubsti-
tuted-phenyl 4, we decided to explore both branches of
Topliss tree. So we also synthesized the p-methoxyphe-
nyl derivative (16), whose R1D-AR affinity was compa-
rable to those of compounds 4 and 7. Since the rank
order of the substituents (p-Cl and p-OCH3), with
respect to activity, did not correspond completely to any
parameter dependency, with the two substituents hav-
ing opposite σ and π effects (p-OCH3: -π, -σ; p-Cl: +π,
+σ), synthesis proceeded under the branch of p-chlo-
rophenyl 7 with the preparation of the p-methyl ana-
logue 10, whose substituent is a +π, -σ type. The
equiactivity of 10 with respect to 7 led us to suppose
that the expected increase of potency was hindered by
steric reasons of the para substitution; thus, the next
compound synthesized was the m-chloro derivative 6,
which represented the descending term both of p-
chlorophenyl 7 in the central branch, and of p-methox-
yphenyl 16 in the left branch of the Topliss operational
scheme. Since also compound 6 showed an affinity value
similar to that of lead compounds of both branches, the
sequence was continued with the m-methyl derivative
(compound 9) and, subsequently, with the o-chloro-,
o-methyl-, and o-methoxy-derivatives (compounds 5, 8,
and 14, respectively). Also these compounds did not
significantly modify affinity for the R1D-AR subtype.
Therefore, the p-nitro analogue 13 was synthesized on
the grounds that a +σ effect was operating, but that
lower lipophilicity was optimal. Finally, compound 13
proved significantly more active than the preceding
derivatives, even though it was equiactive with the
unsubstituted lead compound 4.
(R1D)
37 and by antagonism of (-)-phenylephrine-induced
contraction of rat spleen (R1B).38 The antagonist potency
of the compounds, expressed as pKB values,39 is reported
in Table 1.
The agonist efficacy of compounds 4-8, 12, 13, and
16 toward 5-HT1A receptors was assessed by determin-
ing the induced stimulation of [35S]GTPγS binding in
cell membranes from HeLa cells transfected with hu-
man cloned 5-HT1A receptors,40 and was expressed as
pD2 (-log ED50) values (Table 1). ED50 represents the
molar concentration of agonist, which produces 50% of
the maximum possible response for that agonist.
The analysis of the results shows that all the com-
pounds synthesized were potent R1-AR antagonists,
whose pKB values observed in the functional experi-
ments were comparable, in most cases, with the pKi
affinity derived from the binding assays; the latter were
in any case higher than the former by 0.5 to 1.5
logarithmic units. Similarly to lead 4,26 all the new
compounds generally displayed the highest affinity
values at the R1D subtype with respect to the other R1-
AR subtypes and to 5-HT1A, at which all tested mol-
ecules behaved as partial agonists.
Attempts to improve the R1D-affinity of 4 were made
by inserting, into the ortho, meta, and para positions
of the aromatic ring of the benzyloxy function, various
substituents having different electronic, lipophilic, and
steric contributions, and chosen using the Topliss ap-
proach.29,30
The first compound prepared was the p-chlorophenyl
derivative (7). Since its potency was slightly lower but

7754 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Quaglia et al.
Table 1. Affinity Constants Expressed as pKi (-log Ki) for Human Recombinant R1-AR Subtypes and 5-HT1A Receptors,a Affinity
Constants Expressed as pKB (-log KB) at R1-AR Subtypes on Isolated Tissues,bAgonist Efficacy ([35S]GTPγS Binding) Expressed as
pD2 (-log ED50) on 5-HT1A Serotoninergic Receptors,a and Cytotoxic Activityc of Compounds 4-18, in Comparison with Doxazosin
pKi cloned receptors
(human brain)
cloned 5-HT1A receptors
(human brain)
cytotoxic activity
(human PC-3 prostate cancer cells)
pKB isolated tissues
compd
R1
R1a
R1b
R1d
R1A
R1B
R1D
pKi
pD2
% max
GI50 µM
TGI µM
LC50 µM
4
5
6
7
8
9
H
9.33d 9.27d 10.17d 8.39 ( 0.18d 8.30 ( 0.15 9.37 ( 0.15d 7.93
7.11
45.7
26.9 ( 1.2 76.9 ( 3.8 193.1 ( 9.3
5.4 ( 0.2 17.8 ( 0.8 58.9 ( 2.9
5.9 ( 0.3 20.4 ( 1.0 58.9 ( 3.4
2.4 ( 0.1 14.5 ( 0.8 52.5 ( 2.9
o-Cl
8.85 9.25
9.60 8.56 ( 0.13 7.52 ( 0.09 8.59 ( 0.05
8.32
8.18
7.81
7.95
7.74
7.65
7.62
7.68
7.93
7.63
7.51
7.76
f
7.31
74.4
m-Cl
p-Cl
o-CH3
9.60 9.80 10.3
9.60 9.00 10.5
8.12 ( 0.21 7.93 ( 0.22 8.72 ( 0.15
8.17 ( 0.13 8.06 ( 0.06 9.11 ( 0.20
7.15
67.1
6.98
60.0
8.80 9.10
9.90 7.81 ( 0.02 7.67 ( 0.18 8.64 ( 0.20
9.70 8.30 ( 0.16 8.20 ( 0.01 8.85 ( 0.10
6.96
75.6
11.5 ( 0.4 33.9 ( 1.3
g
m-CH3 9.40 9.60
p-CH3 8.70 9.60 10.20 8.23 ( 0.06 8.43 ( 0.18 9.12 ( 0.23
o-NO2 8.10 8.60 9.04 8.20 ( 0.06 8.56 ( 0.22 8.95 ( 0.01
m-NO2 9.50 9.10 10.20 8.85 ( 0.03 8.74 ( 0.18 9.28 ( 0.22
p-NO2 8.90 9.40 10.30 8.58 ( 0.09 9.02 ( 0.12 9.41 ( 0.06
o-OMe 8.70 8.20
m-OMe 8.70 8.70
p-OMe 8.70 9.40 10.00 8.54 ( 0.07 9.51 ( 0.01 9.21 ( 0.19
p-OEt
p-OiPr
-
f
f
12.0 ( 0.5 32.3 ( 1.4 87.1 ( 6.3
7.8 ( 0.5 27.5 ( 1.1 61.7 ( 3.0
13.2 ( 0.7 34.7 ( 1.3 95.5 ( 5.6
10
f
f
11
f
f
12
7.16
44.4
13.2 ( 0.6 38.9 ( 1.5
12.0 ( 0.6 43.6 ( 1.9
g
g
13
6.15
79.1
14
8.70 8.53 ( 0.14 8.13 ( 0.17 8.86 ( 0.13
9.50 8.30 ( 0.05 8.31 ( 0.11 9.12 ( 0.21
f
f
f
f
8.9 ( 0.4 30.2 ( 1.5 100.0 ( 4.9
2.5 ( 0.1 15.1 ( 1.0 61.7 ( 3.4
15
16
7.92
21.6
8.5 ( 0.2 34.7 ( 1.2
g
17
f
f
f
f
f
f
8.50 ( 0.18 8.23 ( 0.19 9.11 ( 0.13
8.17 ( 0.19 8.62 ( 0.11 9.14 ( 0.11
f
f
f
f
f
f
5.2 ( 0.3 18.2 ( 1.0 51.3 ( 2.7
2.0 ( 0.1 17.8 ( 0.9 63.1 ( 3.0
26.9 ( 1.3 49.0 ( 2.5 75.8 ( 3.6
18
f
f
Doxazosin
9.27e 9.09e
9.09e 8.69 ( 0.70e 9.51 ( 0.41e 8.97 ( 0.23e
a Equilibrium dissociation constants (Ki) were derived from IC50 values using the Cheng-Prusoff equation.51 The affinity estimates
were derived from displacement of [3H]prazosin for R1-ARs, and [3H]8-hydroxy-2-(di-n-propylamino)tetralin and [35S]GTPγS binding for
5-HT1A receptors (antagonism and agonism, respectively). Each experiment was performed in triplicate. Ki values were from two to three
experiments, which agreed within (20%. b R1-AR subtypes blocking activity was assessed by antagonism of (-)-NE-induced contractions
on isolated rat prostatic vas deferens (R1A) )
36 or thoracic aorta (R1D 37 and by antagonism of (-)-phenylephrine-induced contractions on rat
spleen (R1B).38 pKB values were calculated according to van Rossum39 in the range of 0.01-1 µM. Each concentration [B] of antagonist
was tested four times. c In vitro cytotoxic activity on human PC-3 prostate cancer cells was carried out by sulforhodamine B (SRB) assay,
according to the National Cancer Institute protocol.43 Growth Inhibition 50 (GI50) represents the drug concentration (µM) required to
inhibit 50% net of cell growth. Total growth inhibition (TGI) represents the drug concentration (µM) required to inhibit 100% of cell
growth. Lethal concentration 50 (LC50) represents the drug concentration required to kill 50% of the initial cell number. Each quoted
value represents the mean of quadruplicate determinations ( standard error (n ) 5). d Data from ref 26. e Data from ref 33. f Not
determined. g Cytotoxicity activity < 50% net of cell growth.
Up to this point, the SAR studies at the benzyloxy
moiety led to several compounds, which, even though
modified with substituents having all the possible
combinations of σ and π parameters, did not display any
increase in the already high R1D-affinity of lead com-
pound 4. The reason for this behavior can be found in
an examination of the molecular complex that the
antagonist 4 forms with the R1D-AR subtype, as was
recently described.41 In fact, in this type of interaction,
because of the high flexibility of the ligand, the benzyl-
oxy function is buried in the core of the receptor helices
bundle of the transmembrane domain, inside an area
sterically accessible to the various substituent groups
inserted in the aromatic ring. However, none of them
is able to recognize additional binding sites: therefore,
the main contribution to the binding energy, both in the
case of 4 and for the whole series of derivatives 5-18,
is afforded by the strong hydrophobic interaction be-
tween the benzyloxy function of the ligand and the
corresponding receptor site.
Neither did the R1A-affinity potency draw any notice-
able advantage from the introduction of substituents
into the various positions of the aromatic ring; only the
m-nitro derivative 12 showed slight improvement of
affinity with a pKB value of 8.85 compared with that of
8.39 of the reference compound 4.
Instead, in the case of the R1B-AR subtype, an
interesting modulation of affinity was obtained: this
drew advantage from the substitution of the nitro group
and, in particular, when in the para position (compound
13) (pKB o-nitro derivative 8.56; m-nitro derivative 8.74;
p-nitro derivative 9.02) and with the methoxy group
exclusively in the para position (compound 16) (pKB
p-methoxy derivative 9.51 against a value of 8.30 for
prototype 4). The increase of the R1B-affinity, which
cannot be attributed to electronic effect, in that the
two groups mentioned above possess parameter values
of opposite sign (σpOMe ) -0.27, σpNO2 ) +0.78), is
favored by the hydrophilic character (πpOMe ) -0.02,
πpNO2 ) -0.28) and by a definite steric hindrance
(MRpOMe ) 7.87, MRpNO2 ) 7.36). Lipophilic (Cl, CH3,
OC2H5, OCHMe2) and more sterically hindered (OC2H5,
OCHMe2) substituents seem to negatively affect the R1B
-
antagonist potency.
In Vitro Cytotoxic Activity. In vitro cytotoxic
activity on human PC-3 prostate cancer cells of com-
pounds 4-18 and doxazosin42 for useful comparison,
was carried out by sulforhodamine B (SRB) assay,
according to the National Cancer Institute protocol.43
The antitumor activity was estimated on the basis of
measurements of three parameters: GI50, the molar
concentration of the compound that inhibits 50% net of
cell growth; TGI, the molar concentration of the com-
pound that causes total inhibition; and LC50, the molar
concentration of the compound causing 50% net of cell
death. The new compounds 5-18 were active at low
micromolar concentration and more effective than lead
4 in suppressing cell growth of PC-3 cells (Table 1).

SAR of 1,4-Benzodioxan-Related Compounds
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7755
Figure 1. Expression of R1D- and R1B-AR subtypes in PC-3 cells. (A) R1A-, R1B-, and R1D-AR subtypes mRNA expression in PC-3
cells and PBMC were evaluated by RT-PCR. PCR products were analyzed by 1.7% ethidium bromide-stained agarose gel and
acquired using a Chemidoc (Bio-Rad). (B) The expression of R1B- and R1D-AR subtypes in PC-3 cells was evaluated by
immunofluorescence and FACS analysis using a goat anti-R1B-AR and a rabbit anti-R1D-AR polyclonal Abs, respectively. FITC-
conjugated RAG and FITC-conjugated GARB were used as secondary Abs. The data are expressed as mean fluorescence intensity
(MFI); RAG ) 2.94; GARB-FITC ) 2.36. The white area indicates negative control; the red area indicates R1B- or R1D-AR positive
cells. (C) Immunocytochemical localization of R1D- and R1B-AR subtypes in PC-3 cells was evaluated by confocal microscopy. Goat
anti-R1B-AR and rabbit anti-R1D-AR polyclonal Abs were used as primary Abs; FITC-conjugated RAG and FITC-conjugated GARB
were used as secondary Abs. Each image is the sum of one of three single optical sections taken at 1-µM intervals. Bars ) 5 µM.
(D) Lysates from PC-3 cells and human PBMC were separated on 8% SDS-PAGE and probed with a goat anti-R1B-AR and a
rabbit anti-R1D-AR polyclonal Abs. Sizes are shown in kDa; the arrowhead indicates the band corresponding to R1B- and R1D-AR
subtypes. All data shown are representative of three separate experiments.
Thus, the presence of a substituent in the benzyloxy
moiety of these compounds seems to favor PC-3 cell
growth inhibition, with 7 exhibiting potency (GI50 ) 2.4
( 0.1; TGI ) 14.5 ( 0.08) significant higher than those
of lead 4 (GI50 ) 26.9 ( 1.2; TGI ) 76.9 ( 3.8) and
doxazosin (GI50 ) 26.9 ( 1.3; TGI ) 49 ( 2.5). Moreover,
para substitution seems to improve growth inhibition
activity. Concerning cytotoxic activity, 7 showed an
effect higher (LC50 ) 52.5 ( 2.9) than those of lead 4
and doxazosin (LC50 ) 193.1 ( 9.3 and 75.8 ( 3.6,
respectively). Compounds 8, 12, 13, and 16 were devoid
of cytotoxic activity. Finally, since serotonin has been
reported to show an enhancing effect on human prostate
cancer cells growth,13 and 4-8, 12, 13, and 16 are

7756 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Quaglia et al.
endowed with a 5-HT1A partial agonist activity, the
effect of these compounds on PC-3 cell growth also in
the presence of the 5-HT1A antagonist, (S)-WAY 100135,
was evaluated. In these experiments, (S)-WAY 100135,
neither alone nor in combination with 4, 7, or doxazosin
reversed the inhibitory effects induced by compounds
4, 7, or doxazosin on PC-3 cell growth (data not shown).
Expression of r1-AR Subtypes in PC-3 Cells. To
directly demonstrate the R1-AR subtypes mRNA expres-
sion in PC-3 (p53-/-) human androgen-nonresponsive
prostate cancer cells, a qualitative PCR analysis on PC-3
cells and human peripheral blood mononuclear cells
(PBMC), used as positive control, was performed. Our
results indicated that R1B- and R1D-AR subtypes mRNAs
were expressed in PC-3 cells and PBMC, as shown by
the finding of PCR products of expected size, [637 bp
(R1B-AR) and 247 bp (R1D-AR), respectively]. As previ-
ously reported,8 no R1A-AR mRNA expression was
detected in PC-3 cells (Figure 1A). The expression of R1B
-
AR and R1D-AR subtypes in PC-3 cells was also assessed
at protein level. Immunofluorescence and FACS analy-
sis showed that these cells expressed high levels of R1D
-
AR protein (MFI ) 80.9), whereas lower expression
(MFI ) 16.1) was observed for the R1B-AR protein
(Figure 1B). Confocal microscopy analysis showed that
R
1B-AR subtype was localized near the plasma mem-
brane of PC-3 cells, whereas a broader expression of R1D
-
AR on the plasma membrane, cytosol, and nucleus was
found (Figure 1C).
Figure 2. R1-AR antagonists affect cell viability and induce
PS exposure in PC-3 cells. (A) Cell viability of PC-3 cells,
treated with different doses (10 and 50 µM) of 7, lead 4 and
doxazosin for 24 h at 37 °C, was evaluated by PI staining and
cytofluorimetric analysis. Data are representative of one of
three separate experiments. (B) The expression of Annexin V
on PC-3 cells, treated with 50 µM of 7, lead 4, and doxazosin,
was evaluated by immunofluorescence and FACS analysis.
Data are the mean ( SD of three separate experiments.
Statistical analysis was determined by comparing the MFI
from untreated with 4-, 7-, and doxazosin (10 and 50 µM)-
treated PC-3 cells. *P < 0.01 determined by Student’s t-test.
ns ) Not significant comparing the MFI from untreated with
doxazosin (10 µM)-treated PC-3 cells.
Moreover, Western blot analysis of cell lysates re-
vealed a band with an apparent MW of about 56 kDa,
and a band of 70 kDa (Figure 1D), likely corresponding
to the R1B- and R1D-AR subtypes, respectively; similar
bands were also evident in the lysates from human
PBMC used as positive control. No reactivity was
observed with normal goat serum and with normal
rabbit serum used as negative control (data not shown).
Taken together, these results demonstrate for the first
time the expression of the R1B- and R1D-AR subtypes on
human PC-3 cells, both at mRNA and protein levels.
Apoptotic Activity. A characteristic feature of apo-
ptotic cell death is the loss of phospholipid asymmetry
and expression of phosphatidylserine (PS) on the outer
layer of the plasma membrane.44 Thus, we analyzed
whether the treatment of PC-3 cells with the new
antagonist 7, selected for its interesting biological
profile, and the lead compound 4 induced externaliza-
tion of PS residues from the inner to the outer leaflet
of the plasma membrane in these cells and consequently
increased the Annexin V binding. Since doxazosin has
been proved to induce apoptosis of PC-3 cells,8,10 it was
used as positive control. Exposure of PC-3 cells to
compounds 7 and 4 resulted in a time- and dose-
dependent reduction of cell viability and induction of
apoptotic cell death. Compound 7 showed the highest
potency to affect PC-3 cell viability and to induce
apoptosis (Figure 2A-B). Although PC-3 cells were
susceptible to compound 4- or doxazosin-induced apo-
ptosis at 50 µM, very low and no appreciable apoptotic
death was evaluated at 10 µM with compound 4 and
doxazosin, respectively.
with 7 and 4, resulted in a dose-dependent modulation
of R1B- and R1D-AR expression on PC-3 cells. As shown
in Figure 3A, treatment of PC-3 cells with 7 and 4
induced a marked reduction of R1D-AR expression, with
antagonist 7 showing the highest inhibitory effect (MFI
from 80.9 to 21.5 at 50 µM concentration). Doxazosin
used at 50 µM showed the lowest ability to affect R1D
-
AR expression. Compounds 7 and, partially, 4 affect,
mainly at higher dose, the R1B-AR expression on PC-3
cells, whereas doxazosin did not show any significant
effect at either of the dose used (Figure 3B).
Overall, these results indicate that the apoptosis
triggered by the R1-AR antagonists 7 and 4 is associated
with a significant reduction of R1D-AR and R1B-AR
subtypes in PC-3 positive cells, suggesting a role for
these AR subtypes in the modulation of apoptosis.
NE Protects PC-3 Cells from r1-AR Antagonist-
Induced Apoptosis. NE has been shown to promote
survival in different cell types by acting as endogenous
modulator of cell death.45-47 Thus, the ability of the
endogenous agonist NE, used at different concentrations
(10 and 50 µM), to reverse the R1-AR antagonist (50 µM)-
induced inhibitory effects on PC-3 cells has been evalu-
Finally, once the expression of R1B- and R1D-AR
subtypes on PC-3 cells was observed, we analyzed
whether the proapoptotic effect induced by treatment

SAR of 1,4-Benzodioxan-Related Compounds
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7757
Figure 4. NE protects PC-3 cells from R1-AR antagonist-
induced apoptosis. (A) Cell viability of PC-3 cells treated with
50 µM of 7, lead 4, and doxazosin for 24 h at 37 °C, alone or
in combination with different doses (10 and 50 µM) of NE, was
evaluated by PI staining and cytofluorimetric analysis. Data
are the mean ( SD of three separate experiments. Statistical
analysis was determined by comparing the percentage of PI+
cells from 4-, 7-, and doxazosin-treated with NE (10 and 50
µM) plus 4, 7, and doxazosin coadministered PC-3 cells. *P <
0.01 determined by Student’s t-test. (B) The expression of
Annexin V on PC-3 cells treated with 7, lead 4, and doxazosin
(50 µM), alone or in combination with different doses of NE
(10 and 50 µM), was evaluated by immunofluorescence and
FACS analysis. Data are the mean ( SD of three separate
experiments. Statistical analysis was determined by comparing
the percentage of cell viability or Annexin V+ cells from 4-, 7-,
and doxazosin-treated with NE-treated (10 and 50 µM) plus
4, 7, and doxazosin coadministered PC-3 cells. *P < 0.01
determined by Student’s t-test. ns ) Not significant comparing
the percent of cell viability from 4- (10 µM)-treated with NE-
treated (10 µM) plus 4, and the percentage of cell viability or
Annexin V+ cells from doxazosin-treated with NE-treated (10
and 50 µM) plus doxazosin coadministered PC-3 cells.
Figure 3. R1-AR antagonists treatment modulates R1D- and
R1B-AR expression in PC-3 cells. The expression of R1D- (panel
A) and R1B-AR (panel B) subtypes in PC-3 cells, treated with
different concentrations (10 and 50 µM) of 7, lead 4, and
doxazosin, was evaluated by immunofluorescence and FACS
analysis as described above. The data shown represent the
mean ( SD of three separate experiments and are expressed
as mean MFI. Statistical analysis was determined by compar-
ing the MFI from untreated with 4-, 7-, and doxazosin (10 and
50 µM)-treated PC-3 cells. *P < 0.01 determined by Student’s
t-test. ns ) Not significant comparing the MFI from untreated
with 4- (10 µM)- and doxazosin (10 and 50 µM)-treated PC-3
cells.
ated. NE markedly protected PC-3 cells from R1-AR
antagonist-dependent apoptosis and completely reversed
in a dose-dependent manner the reduction of PC-3 cell
viability (Figure 4A) and apoptosis (Figure 4B) induced
by 7, and, to a lesser extent, by exposure to 4. On the
other hand, treatment with NE did not affect the ability
of doxazosin to inhibit cell viability and to induce
apoptosis of PC-3 cells.
to stimulate PC-3 cell proliferation was evaluated by
BrdU incorporation into the DNA of proliferating cells
using a colorimetric ELISA kit. NE markedly enhanced
in a dose-dependent manner (10 µM ) 33% and 50 µM
) 65%) PC-3 cell proliferation. Moreover, the new
antagonist 7 (0.5 µM), and, to a lesser extent, the lead
4 (1.0 µM), but not doxazosin (1.0 µM), completely
inhibited the NE-induced increase of PC-3 cell prolifera-
tion (Figure 6).
As reported in Figure 5A,B, NE (10 and 50 µM)
significantly increased in a dose-dependent manner the
expression of R1D-AR and, at 50 µM, the expression of
R
1B-AR on PC-3 cells. Moreover, treatment with NE
completely reversed the reduction of R1D- and R1B-AR
expression induced by 7 on PC-3 cells; it also reversed
that of R1D-AR expression induced by exposure to 4,
whereas it did not affect that of R1B-AR subtype expres-
sion. Finally, it did not reverse the inhibitory effect of
doxazosin on the R1D-AR subtype expression in PC-3
cells.
Conclusions
The present study, for the first time, highlights the
expression of R1D- and R1B-AR subtypes in human
androgen nonresponsive PC-3 prostate cancer cells.
Interestingly, R1D-AR seems to be predominantly located
in intracellular vescicles, which are widespread in the
cytosol and nucleus, while the majority of the R1B-AR
is clustered around the plasma membrane of PC-3 cells.
The R1D-AR subtype is a phosphoprotein whose function
NE Stimulates PC-3 Cell Proliferation. Given the
growing evidence on the role of R1-ARs in the direct
mitogenic effect of catecholamines on prostate growth,7,20
we examined whether the protective effects, induced by
NE on R1-AR positive PC-3 cells, were partially related
to its ability to induce PC-3 cell proliferation. Thus, the
effect of NE, at different doses (0, 0.1, 1, 10, and 50 µM),

7758 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Quaglia et al.
Figure 5. NE increases the expression of R1D- and R1B-AR subtypes in untreated, NE- and R1-AR antagonists-treated PC-3 cells.
The expression of R1D- and R1B-AR subtypes in PC-3 cells, untreated, treated with different doses of NE (10 and 50 µM), or with
7, lead 4, and doxazosin, alone or in combination with different doses (10 and 50 µM) of NE, was evaluated by immunofluorescence
and FACS analysis as described above. The data shown represent the mean ( SD of three separate experiments and are expressed
as MFI. Statistical analysis was determined by comparing the MFI of R1D- and R1B-AR subtypes positive cells, from untreated
and NE-treated (10 and 50 µM) (**P < 0.01) and 4-, 7-, and doxazosin-treated with that of NE (10 and 50 µM) plus 4, 7, and
doxazosin coadministered PC-3 cells. *P < 0.01 determined by Student’s t-test.
significant reduction of R1D- and R1B-AR expression in
PC-3 cells. NE markedly protects, in a dose-dependent
manner, PC-3 cells from apoptosis induced by these R1-
AR antagonists and completely reverses the depletion
of R1D-AR and R1B-AR PC-3 positive cells induced by
treatment with 7. Moreover, our study provides evidence
that NE promotes the proliferation of PC-3 cells. The
specificity of NE-induced proliferation is proven by its
complete suppression by 7 and, at higher concentration,
by 4. Compound 7 also shows higher potency, with
respect to 4, to induce apoptosis. The high R1-AR
antagonist potency, the more significant R1D-selectivity
and the higher lipophilic character with respect to
doxazosin might be the factors responsible for the effects
evoked by 7. Moreover, it is known that R1D-AR forms
heterodimers with the R1B-AR subtype, and that such
association may have consequences in their pharmaco-
logical properties and their function/regulation.50 Thus,
these effects may be related to the ability of 7 and, to a
lesser extent, of 4, to affect both R1D-AR and R1B-AR
PC-3 cell populations.
In conclusion, clopenphendioxan (7) may represent a
valid pharmacological tool and promising lead to design
new ligands useful for prostate cancer treatment. Fur-
ther studies have already been planned with the aim
to obtain structurally optimized antagonists. The dif-
ference between interaction of the novel antagonists and
doxazosin with NE is interesting and may warrant
further investigation with other agonists and antago-
nists.
Figure 6. Lead 4 and 7 (1 and 0.5 µM), but not doxazosin (1
µM), treatment inhibits NE (50 µM)-induced increase of PC-3
cell proliferation, as evaluated by immunoassay using cell
proliferation ELISA BrdU. Data are the mean ( SD of three
separate experiments. Statistical analysis was determined by
comparing the percentage of growth from untreated with NE-
treated, and NE with NE plus 4, 7, and doxazosin (1, 0.5, and
1 µM, respectively) coadministered PC-3 cells. *P < 0.01
determined by Student’s t-test. ns ) Not significant comparing
the percentage of growth from NE with NE plus doxazosin (1
µM) coadministered PC-3 cells.
is regulated through phosphorylation, so that its intra-
cellular localization could be the result of its intrinsic
activity, which could trigger phosphorylation and in-
ternalization. On the other hand, the presence of R1B
-
AR near the plasma membrane may support its ability
to act as a chaperon for the proper insertion of R1D-AR
in the plasma membrane.48,49
We also demonstrate the involvement of these recep-
tors in the regulation of apoptosis and cell proliferation.
Thus, 7- and 4-induced apoptosis is associated with a

SAR of 1,4-Benzodioxan-Related Compounds
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7759
4.06 (d, 2, OCH2), 4.75 (t, 1, CH), 5.09 (s, 2, CH2Ar), 6.83-
7.43 (m, 8, ArH).
Experimental Protocols
Chemistry. Melting points were taken in glass capillary
tubes on a Bu¨chi SMP-20 apparatus and are uncorrected. IR
and NMR spectra were recorded on Perkin-Elmer 297 and
Varian EM-390 instruments, respectively. Chemical shifts are
reported in parts per million (ppm) relative to tetramethylsi-
lane (TMS), and spin multiplicities are given as s (singlet), d
(doublet), t (triplet), q (quartet), or m (multiplet). IR spectral
data (not shown because of the lack of unusual features) were
obtained for all compounds reported and are consistent with
the assigned structures. The microanalyses were performed
by the Microanalytical Laboratory of our department. The
elemental composition of the compounds agreed to within
(0.4% of the calculated value. Chromatographic separations
were performed on silica gel columns (Kieselgel 40, 0.040-
0.063 mm, Merck) by flash chromatography. The term “dried”
refers to the use of anhydrous sodium sulfate. Compounds
were named following IUPAC rules as applied by Beilstein-
Institut AutoNom (version 2.1), a software for systematic
names in organic chemistry.
(2-Benzyloxyphenoxy)acetaldehyde Dimethyl Acetale
(19). A 60% NaH dispersion in mineral oil (0.22 g, 5.5 mmol)
was washed with hexane under nitrogen, suspended in dim-
ethylformamide (DMF; 0.7 mL), and then, after 30 min, added
with a solution of 2-benzyloxyphenol (1.0 g; 4.99 mmol) in dry
DMF (0.5 mL). The mixture was stirred at room temperature
for 1.5 h and cooled to 0 °C. A solution of 2-chloro-1,1-
dimethoxyethane (0.65 mL; 5.7 mmol) in DMF (8.0 mL) was
added, and the reaction mixture was heated at 120 °C for 8 h.
After cooling, the mixture was poured in 2 N NaOH (15 mL)
and extracted with Et2O. Removal of dried solvents gave a
residue, which was purified by column chromatography. Elut-
ing with cyclohexane/AcOEt (98:2) gave an oil; 0.85 g (59%
yield). 1H NMR (CDCl3) δ 3.43 (s, 6, OCH3), 4.07 (d, 2, OCH2),
4.73 (t, 1, CH), 5.11 (s, 2, CH2Ar), 6.88-7.48 (m, 9, ArH).
2-(2,2-Dimethoxyethoxy)phenol (20). A solution of 19
(0.5 g; 1.73 mmol) in AcOEt/AcOH (9:1; 4.3 mL) was hydro-
genated over 10% Pd on charcoal (0.05 g) for 4 h at 50 psi of
pressure. Following catalyst removal, the solution was diluted
with Et2O, and the extracts were washed with NaHCO3 and
ice. Removal of dried solvent gave an oil, which was purified
by column chromatography. Eluting with cyclohexane/AcOEt
(8:2) gave an oil; 0.3 g (87% yield). 1H NMR (CDCl3) δ 3.42 (s,
6, OCH3), 4.02 (d, 2, OCH2), 4.68 (t, 1, CH), 6.19 (br s, 1, OH),
6.76-6.94 (m, 4, ArH).
1-(2,2-Dimethoxyethoxy)-2-[(4-methylbenzyl)oxy]ben-
zene (21f). Eluting solvent: cyclohexane/AcOEt (9.5:0.5); 82%
1
yield. H NMR (CDCl3) δ 2.38 (s, 3, CH3), 3.47 (s, 6, OCH3),
4.09 (d, 2, OCH2), 4.77 (t, 1, CH), 5.10 (s, 2, CH2Ar), 6.88-
7.40 (m, 8, ArH).
1-(2,2-Dimethoxyethoxy)-2-[(2-nitrobenzyl)oxy]ben-
zene (21g). Eluting solvent: cyclohexane/AcOEt (9.5:0.5); 37%
yield. 1H NMR (CDCl3) δ 3.44 (s, 6, OCH3), 4.10 (d, 2, OCH2),
4.80 (t, 1, CH), 5.55 (s, 2, CH2Ar), 6.95-8.22 (m, 8, ArH).
1-(2,2-Dimethoxyethoxy)-2-[(3-nitrobenzyl)oxy]ben-
zene (21h). Eluting solvent: cyclohexane/AcOEt (9:1); 60%
yield; crystallized from AcOEt/petroleum ether, mp 86-87 °C.
1H NMR (CDCl3) δ 3.50 (s, 6, OCH3), 4.10 (d, 2, OCH2), 4.82
(t, 1, CH), 5.22 (s, 2, CH2Ar), 6.90-8.42 (m, 8, ArH).
1-(2,2-Dimethoxyethoxy)-2-[(4-nitrobenzyl)oxy]ben-
zene (21i). Eluting solvent: cyclohexane/AcOEt (9:1); 30%
yield; crystallized from AcOEt/petroleum ether, mp 106-107
°C. 1H NMR (CDCl3) δ 3.48 (s, 6, OCH3), 4.10 (d, 2, OCH2),
4.79 (t, 1, CH), 5.23 (s, 2, CH2Ar), 6.90-8.26 (m, 8, ArH).
[2-(2-Chlorobenzyloxy)phenoxy]acetaldehyde (22a).
Compound 21a (0.78 g; 2.42 mmol) was added to a solution of
2 N HCl (3.9 mL) in acetone (6.7 mL), and the resulting
solution was refluxed for 1.5 h with stirring. After cooling to
0 °C, Et2O (25 mL) and H2O (10 mL) were added. The ether
layer was separated and washed with 5% Na2CO3 (1 × 25 mL)
and H2O (2 × 25 mL). Removal of dried solvent gave a residue,
which was purified by column chromatography. Eluting with
cyclohexane/AcOEt (8:2) gave a solid; 0.57 g (85% yield); mp
59-60 °C. 1H NMR (CDCl3) δ 4.63 (d, 2, OCH2), 5.26 (s, 2,
CH2Ar), 6.83-7.63 (m, 8, ArH), 9.92 (t, 1, CHO).
Similarly, compounds 22b-i were obtained via the proce-
dure described for 22a.
[2-(3-Chlorobenzyloxy)phenoxy]acetaldehyde (22b).
Eluting solvent: cyclohexane/AcOEt (8:2); 67% yield. 1H NMR
(CDCl3) δ 4.61 (d, 2, OCH2), 5.10 (s, 2, CH2Ar), 6.58-7.24 (m,
8, ArH), 9.84 (t, 1, CHO).
[2-(4-Chlorobenzyloxy)phenoxy]acetaldehyde (22c). 72%
1
Yield. H NMR (CDCl3) δ 4.60 (d, 2, OCH2), 5.09 (s, 2, CH2-
Ar), 6.81-7.38 (m, 8, ArH), 9.84 (t, 1, CHO). This compound
was used in the next step without further purification.
[2-(2-Methylbenzyloxy)phenoxy]acetaldehyde (22d).
71% Yield. 1H NMR (CDCl3) δ 2.42 (s, 3, CH3), 4.60 (d, 2,
OCH2), 5.14 (s, 2, CH2Ar), 6.61-7.44 (m, 8, ArH), 9.88 (t, 1,
CHO). This compound was used in the next step without
further purification.
1-(2,2-Dimethoxyethoxy)-2-[(2-chlorobenzyl)oxy]ben-
zene (21a). A mixture of 20 (0.6 g; 3.01 mmol), 2-chlorobenzyl
chloride (0.57 mL; 4.51 mmol), and K2CO3 (0.42 g; 3.01 mmol)
in DMF (6 mL) was refluxed for 45 min at 125 °C. After cooling
to 0 °C, a solution of 5% NaOH was added and the solution
was extracted with Et2O.The extracts were washed with 5%
NaOH and H2O. Removal of dried solvent gave a residue,
which was purified by column chromatography. Eluting with
cyclohexane/AcOEt (9:1) gave an oil; 0.78 g (80% yield). 1H
NMR (CDCl3) δ 3.48 (s, 6, OCH3), 4.10 (d, 2, OCH2), 4.78 (t, 1,
CH), 5.23 (s, 2, CH2Ar), 6.91-7.72 (m, 8, ArH).
[2-(3-Methylbenzyloxy)phenoxy]acetaldehyde (22e).
Eluting solvent: cyclohexane/AcOEt (8:2); 72% yield. 1H NMR
(CDCl3) δ 2.40 (s, 3, CH3), 4.62 (d, 2, OCH2), 5.12 (s, 2, CH2-
Ar), 6.79-7.34 (m, 8, ArH), 9.90 (t, 1, CHO).
[2-(4-Methylbenzyloxy)phenoxy]acetaldehyde (22f).
Eluting solvent: cyclohexane/AcOEt (8:2); 96% yield. 1H NMR
(CDCl3) δ 2.39 (s, 3, CH3), 4.59 (d, 2, OCH2), 5.13 (s, 2, CH2-
Ar), 6.70-7.38 (m, 8, ArH), 9.85 (t, 1, CHO).
[2-(2-Nitrobenzyloxy)phenoxy]acetaldehyde (22g). Elut-
ing solvent: cyclohexane/AcOEt (8:2); 85% yield. 1H NMR
(CDCl3) δ 4.15 (d, 2, OCH2), 5.53 (s, 2, CH2Ar), 6.82-8.22 (m,
8, ArH), 9.93 (t, 1, CHO).
[2-(3-Nitrobenzyloxy)phenoxy]acetaldehyde (22h). Elut-
ing solvent: cyclohexane/AcOEt (8:2); 42% yield. 1H NMR
(CDCl3) δ 4.10 (d, 2, OCH2), 5.21 (s, 2, CH2Ar), 6.84-8.25 (m,
8, ArH), 9.85 (t, 1, CHO).
[2-(4-Nitrobenzyloxy)phenoxy]acetaldehyde (22i). Elut-
ing solvent: cyclohexane/AcOEt (8:2); 75% yield; mp 81-82 °C.
1H NMR (CDCl3) δ 4.12 (d, 2, OCH2), 5.23 (s, 2, CH2Ar), 6.84-
8.24 (m, 8, ArH), 9.92 (t, 1, CHO).
{2-[2-(2-Chlorobenzyloxy)phenoxy]ethyl}-[2-(2,6-
dimethoxyphenoxy)ethyl]amine (5). A 2 M solution of HCl
gas in EtOH (1.55 mL) was added to a solution of 2-(2,6-
dimethoxyphenoxy)ethylamine32 (1.82 g; 9.23 mmol) and 22a
(0.43 g; 1.55 mmol) in EtOH (12.0 mL), followed by the addition
of NaBH3CN (0.085 g; 1.35 mmol) and molecular sieves (4 Å).
The mixture was stirred at room temperature for 1 h, then
Similarly, compounds 21b-i were obtained via the proce-
dure described for 21a.
1-(2,2-Dimethoxyethoxy)-2-[(3-chlorobenzyl)oxy]ben-
zene (21b). Eluting solvent: cyclohexane/AcOEt (9:1); 73%
yield. 1H NMR (CDCl3) δ 3.44 (s, 6, OCH3), 4.05 (d, 2, OCH2),
4.68 (t, 1, CH), 5.09 (s, 2, CH2Ar), 6.81-7.62 (m, 8, ArH).
1-(2,2-Dimethoxyethoxy)-2-[(4-chlorobenzyl)oxy]ben-
zene (21c). Eluting solvent: cyclohexane/AcOEt (9.8:0.2); 53%
yield. 1H NMR (CDCl3) δ 3.42 (s, 6, OCH3), 4.02 (d, 2, OCH2),
4.73 (t, 1, CH), 5.08 (s, 2, CH2Ar), 6.88-7.42 (m, 8, ArH).
1-(2,2-Dimethoxyethoxy)-2-[(2-methylbenzyl)oxy]ben-
zene (21d). Eluting solvent: cyclohexane/AcOEt (9.5:0.5); 63%
1
yield. H NMR (CDCl3) δ 2.42 (s, 3, CH3), 3.42 (s, 6, OCH3),
4.07 (d, 2, OCH2), 4.73 (t, 1, CH), 5.12 (s, 2, CH2Ar), 6.93-
7.52 (m, 8, ArH).
1-(2,2-Dimethoxyethoxy)-2-[(3-methylbenzyl)oxy]ben-
zene (21e). Eluting solvent: cyclohexane/AcOEt (9:1); 77%
1
yield. H NMR (CDCl3) δ 2.37 (s, 3, CH3), 3.46 (s, 6, OCH3),

7760 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Quaglia et al.
acidified at pH 1 with 2 N HCl, filtered, and evaporated. The
residue was taken up with water and basified with 6 N NaOH,
and the mixture was extracted with Et2O. Removal of dried
solvent gave a residue, which was purified by column chro-
matography. Eluting with CHCl3/EtOH (9.8:0.2) gave 5 as the
free base; 0.27 g (38% yield). 1H NMR (CDCl3) δ 1.88 (br s, 1,
NH, exchangeable with D2O), 2.99 and 3.12 (two t, 4, CH2-
NCH2), 3.81 (s, 6, OCH3), 4.18 (m, 4, OCH2 and CH2O), 5.22
(s, 2, CH2Ar), 6.53-7.64 (m, 11, ArH).
The free base was transformed into the oxalate salt by
treating an ether solution with oxalic acid; the solid was
crystallized from EtOH; mp 164-165 °C. Anal. (C25H28ClNO5‚
H2C2O4‚0.5H2O) C, H, N.
(CDCl3) δ 2.15 (br s, 1, NH, exchangeable with D2O), 3.01 and
3.14 (two t, 4, CH2NCH2), 3.81 (s, 6, OCH3), 4.20 (m, 4, OCH2
and CH2O), 5.20 (s, 2, CH2Ar), 6.50-8.12 (m, 11, ArH). The
oxalate salt was crystallized from EtOH; mp 167-168 °C. Anal.
(C25H28N2O7‚H2C2O4‚0.25H2O) C, H, N.
[2-(2-Benzyloxyphenoxy)ethyl]-[2-(2,6-dimethoxyphe-
noxy)ethyl]carbamic Acid Methyl Ester (23). Methyl
chloroformate (0.45 mL; 5.82 mmol) was added dropwise to a
stirred solution of N-[2-[2-(benzyloxy)phenoxy]ethyl]-N-[2-(2,6-
dimethoxyphenoxy)ethyl]amine (4)26 (1,95 g; 4.6 mmol) and
Et3N (0.65 mL; 4.6 mmol) in CHCl3 (30 mL). Removal of
solvent gave a residue, which was purified by column chro-
matography. Eluting with cyclohexane/AcOEt (8:2) gave an oil;
1
1.7 g (77% yield). H NMR (CDCl3) δ 3.62-4.0 (m, 13, OCH3
Similarly, compounds 6-13 were obtained via the procedure
described for 5.
and CH2NCH2), 4.02-4.30 (m, 4, OCH2 and CH2O), 5.09 (s, 2,
CH2Ar), 6.52-7.53 (m, 12, ArH).
{2-[2-(3-Chlorobenzyloxy)phenoxy]ethyl}-[2-(2,6-
dimethoxyphenoxy)ethyl]amine (6). Eluting with CHCl3/
[2-(2,6-Dimethoxyphenoxy)ethyl]-[2-(2-hydroxyphe-
noxy)ethyl]carbamic Acid Methyl Ester (24). A solution
of 23 (1.1 g; 2.28 mmol) in EtOAc/AcOH (9:1; 5.7 mL) was
hydrogenated over 10% Pd on charcoal (0.07 g) for 27 h at 50
psi of pressure. Following catalyst removal, the solution was
diluted with Et2O, and the extracts were washed with NaHCO3
and ice. Removal of dried solvent gave an oil, which was
purified by column chromatography. Eluting with cyclohexane/
1
EtOH (9.5:0.5) gave 6 as the free base; (45% yield). H NMR
(CDCl3) δ 2.47 (br s, 1, NH, exchangeable with D2O), 2.94 and
3.10 (two t, 4, CH2NCH2), 3.78 (s, 6, OCH3), 4.13 (m, 4, OCH2
and CH2O), 5.06 (s, 2, CH2Ar), 6.60-7.41 (m, 11, ArH). The
oxalate salt was crystallized from EtOH; mp 135-136 °C. Anal.
(C25H28ClNO5‚H2C2O4‚0.5H2O) C, H, N.
{2-[2-(4-Chlorobenzyloxy)phenoxy]ethyl}-[2-(2,6-
dimethoxyphenoxy)ethyl]amine (7). Eluting with AcOEt/
EtOH (9:1) gave 7 as the free base; (28% yield). 1H NMR
(CDCl3) δ 2.15 (br s, 1, NH, exchangeable with D2O), 2.95 and
3.08 (two t, 4, CH2NCH2), 3.77 (s, 6, OCH3), 4.13 (m, 4, OCH2
and CH2O), 5.03 (s, 2, CH2Ar), 6.49-7.34 (m, 11, ArH). The
oxalate salt was crystallized from EtOH; mp 161-162 °C. Anal.
(C25H28ClNO5‚H2C2O4‚0.5H2O) C, H, N.
1
AcOEt (8:2) gave an oil; 0.58 g (65% yield). H NMR (CDCl3)
δ 3.62-4.0 (m, 13, OCH3 and CH2NCH2), 4.03-4.32 (m, 4,
OCH2 and CH2O), 6.52-7.04 (m, 7, ArH).
[2-(2,6-Dimethoxyphenoxy)ethyl]-{2-[2-(2-methoxy-
benzyloxy)phenoxy]ethyl}carbamic Acid Methyl Ester
(25a). A mixture of 24 (0.36 g; 0.92 mmol), 2-methoxybenzyl
chloride (0.2 mL; 1.44 mmol), and K2CO3 (0.13 g; 0.94 mmol)
in DMF (1.82 mL) was refluxed for 4 h at 125 °C. After cooling
to 0 °C, a solution of 5% NaOH was added and the solution
was extracted with Et2O.The extracts were washed with 5%
NaOH and H2O. Removal of dried solvent gave a residue,
which was purified by column chromatography. Eluting with
petroleum ether/Et2O (5:5) gave an oil; 0.43 g (91% yield). 1H
NMR (CDCl3) δ 3.66-3.98 (m, 16, OCH3 and CH2NCH2), 4.03-
4.30 (m, 4, OCH2 and CH2O), 5.12 (s, 2, CH2Ar), 6.52-7.53
(m, 11, ArH).
[2-(2,6-Dimethoxyphenoxy)ethyl]-{2-[2-(2-methyl-
benzyloxy)phenoxy]ethyl}amine (8). Eluting with CHCl3/
1
EtOH (9.9:0.1) gave 8 as the free base; (28% yield). H NMR
(CDCl3) δ 1.63 (br s, 1, NH, exchangeable with D2O), 2.38 (s,
3, CH3), 2.97 and 3.12 (two t, 4, CH2NCH2), 3.80 (s, 6, OCH3),
4.16 (m, 4, OCH2 and CH2O), 5.09 (s, 2, CH2Ar), 6.53-7.50
(m, 11, ArH). The oxalate salt was crystallized from EtOH;
mp 155-156 °C. Anal. (C26H31NO5‚H2C2O4‚0.5H2O) C, H, N.
[2-(2,6-Dimethoxyphenoxy)ethyl]-{2-[2-(3-methyl-
benzyloxy)phenoxy]ethyl}amine (9). Eluting with CHCl3/
Similarly, compounds 25b-c were obtained via the proce-
dure described for 25a.
1
EtOH (9.9:0.1) gave 9 as the free base; (41% yield). H NMR
(CDCl3) δ 1.78 (br s, 1, NH, exchangeable with D2O), 2.32 (s,
3, CH3), 2.99 and 3.12 (two t, 4, CH2NCH2), 3.82 (s, 6, OCH3),
4.18 (m, 4, OCH2 and CH2O), 5.08 (s, 2, CH2Ar), 6.53-7.28
(m, 11, ArH). The oxalate salt was crystallized from EtOH;
mp 126-127 °C. Anal. (C26H31NO5‚H2C2O4‚0.25H2O) C, H, N.
[2-(2,6-Dimethoxyphenoxy)ethyl]-{2-[2-(4-methyl-
benzyloxy)phenoxy]ethyl}amine (10). Eluting with CHCl3/
EtOH (9.8:0.2) gave 10 as the free base; (42% yield). 1H NMR
(CDCl3) δ 2.20 (br s, 1, NH, exchangeable with D2O), 2.28 (s,
3, CH3), 2.95 and 3.08 (two t, 4, CH2NCH2), 3.78 (s, 6, OCH3),
4.12 (m, 4, OCH2 and CH2O), 5.05 (s, 2, CH2Ar), 6.50-7.32
(m, 11, ArH). The oxalate salt was crystallized from EtOH;
mp 145-146 °C. Anal. (C26H31NO5‚H2C2O4‚0.5H2O) C, H, N.
[2-(2,6-Dimethoxyphenoxy)ethyl]-{2-[2-(2-nitro-
benzyloxy)phenoxy]ethyl}amine (11). Eluting with CHCl3/
EtOH (9.9:0.1) gave 11 as the free base; (49% yield). 1H NMR
(CDCl3) δ 1.93 (br s, 1, NH, exchangeable with D2O), 3.0 and
3.14 (two t, 4, CH2NCH2), 3.80 (s, 6, OCH3), 4.19 (m, 4, OCH2
and CH2O), 5.52 (s, 2, CH2Ar), 6.53-8.17 (m, 11, ArH). The
oxalate salt was crystallized from MeOH; mp 171-172 °C.
Anal. (C25H28N2O7‚H2C2O4‚0.25H2O) C, H, N.
[2-(2,6-Dimethoxyphenoxy)ethyl]-{2-[2-(3-methoxy-
benzyloxy)phenoxy]ethyl}carbamic Acid Methyl Ester
(25b). 91% Yield. 1H NMR (CDCl3) δ 3.67-3.98 (m, 16, OCH3
and CH2NCH2), 4.03-4.30 (m, 4, OCH2 and CH2O), 5.08 (s, 2,
CH2Ar), 6.52-7.32 (m, 11, ArH).
[2-(2,6-Dimethoxyphenoxy)ethyl]-{2-[2-(4-methoxy-
benzyloxy)phenoxy]ethyl}carbamic Acid Methyl Ester
(25c). 73% Yield; mp 85-87 °C. 1H NMR (CDCl3) δ 3.67-3.94
(m, 16, OCH3 and CH2NCH2), 4.01-4.25 (m, 4, OCH2 and
CH2O), 5.02 (s, 2, CH2Ar), 6.52-7.39 (m, 11, ArH).
[2-(2,6-Dimethoxyphenoxy)ethyl]-{2-[2-(4-ethoxy-
benzyloxy)phenoxy]ethyl}carbamic Acid Methyl Ester
(26). A solution of DIAD (0.2 mL; 0.984 mmol) in dry THF
(1.5 mL) was added dropwise to a solution of 24 (0.35; 0.894
mmol), 4-ethoxybenzyl alcohol (0.14 g; 0.894 mmol), and
(C6H5)3P (0.235 g; 0.894 mmol) in dry THF (2 mL) with stirring
under a stream of dry nitrogen. When the addition was
completed, the reaction mixture was left for 2 h at room
temperature. Removal of solvent gave a residue, which was
purified by column chromatography. Eluting with cyclohexane/
1
Et2O (7:3) gave an oil; 0.25 g (52% yield). H NMR (CDCl3) δ
1.40 (t, 3, CH2CH3), 3.60-4.28 (m, 19, OCH3, OCH2CH3, CH2-
NCH2, OCH2, and CH2O), 5.0 (s, 2, CH2Ar), 6.52-7.38 (m, 11,
ArH).
[2-(2,6-Dimethoxyphenoxy)ethyl]-{2-[2-(3-nitro-
benzyloxy)phenoxy]ethyl}amine (12). Eluting with CHCl3/
EtOH (9.9:0.1) gave 12 as the free base; (45% yield). 1H NMR
(CDCl3) δ 1.93 (br s, 1, NH, exchangeable with D2O), 2.99 and
3.12 (two t, 4, CH2NCH2), 3.80 (s, 6, OCH3), 4.19 (m, 4, OCH2
and CH2O), 5.21 (s, 2, CH2Ar), 6.51-8.31 (m, 11, ArH). The
oxalate salt was crystallized from EtOH; mp 137-138 °C. Anal.
(C25H28N2O7‚H2C2O4‚0.25H2O) C, H, N.
[2-(4-Isopropoxybenzyloxy)phenoxy]acetic Acid Meth-
yl Ester (27). Compound 27 was obtained via the procedure
described for 26 starting from (2-hydroxyphenoxy)acetic acid
methyl ester (1.74; 9.56 mmol) and 4-isopropoxybenzyl alcohol
(1.59 g; 9.56 mmol). Eluting solvent: cyclohexane/AcOEt (9.5:
1
[2-(2,6-Dimethoxyphenoxy)ethyl]-{2-[2-(4-nitro-
benzyloxy)phenoxy]ethyl}amine (13). Eluting with CHCl3/
EtOH (9.9:0.1) gave 13 as the free base; (24% yield). 1H NMR
0.5); 41% yield; mp 75-76 °C. H NMR (CDCl3) δ 1.32 (d, 6,
CH(CH3)2), 3.78 (s, 3, OCH3), 4.55 (m, 1, CH(CH3)2), 4.70 (s,
2, OCH2CO), 5.04 (s, 2, CH2Ar), 6.80-7.40 (m, 8, ArH).

SAR of 1,4-Benzodioxan-Related Compounds
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7761
[2-(2,6-Dimethoxyphenoxy)ethyl]-{2-[2-(2-methoxy-
benzyloxy)phenoxy]ethyl}amine (14). A saturated solution
of KOH (5 mL) was added to a solution of 25a (0.43 g; 0.84
mmol) in MeOH (15 mL). The reaction mixture was refluxed
for 48 h. Removal of solvent gave a residue, which was
dissolved in H2O and extracted with CHCl3. Removal of dried
solvent gave an oil as the free base; 0.15 g (39% yield). 1H NMR
(CDCl3) δ 1.98 (br s, 1, NH, exchangeable with D2O), 3.0 and
3.14 (two t, 4, CH2NCH2), 3.82 and 3.85 (two s, 9, OCH3), 4.17
(m, 4, OCH2 and CH2O), 5.18 (s, 2, CH2Ar), 6.52-7.52 (m, 11,
ArH). The oxalate salt was crystallized from EtOH; mp 141-
142 °C. Anal. (C26H31NO6‚H2C2O4‚0.5H2O) C, H, N.
in H2O (10 mL) and ice. The aqueous solution was extracted
with Et2O. Removal of dried solvent gave a residue, which was
purified by column chromatography. Eluting with cyclohexane/
Et2O/EtOH (5:5:1.5) afforded 18 as the free base: 0.33 g (62%
1
yield). H NMR (CDCl3) δ 1.32 [d, 6, CH(CH3)2], 2.13 (br s, 1,
NH, exchangeable with D2O), 2.97 and 3.10 (two t, 4, CH2-
NCH2), 3.80 (s, 6, OCH3), 4.14 (m, 4, OCH2 and CH2O), 4.47
(m, 1, CH(CH3)2), 5.02 (s, 2, CH2Ar), 6.52-7.37 (m, 11, ArH).
The oxalate salt was crystallized from EtOH/Et2O; mp 152-
153 °C. Anal. (C28H35NO6‚H2C2O4) C, H, N.
Biology. Functional antagonism in isolated tissues and
radioligand binding assays were performed according to the
protocols reported in ref 41. Agonist efficacy, tested with [35S]-
GTPγS binding on cells transfected with human cloned 5-HT1A
serotoninergic receptors, was evaluated according to the
protocol reported in ref 26.
Cell Culture. PC-3 (p53-/-) human androgen-nonrespon-
sive prostate cancer cells were purchased from the American
Type Tissue Collection (Manassas, VA). Cells were cultured
in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, BRL,
Life Technology, Milan) supplemented with 10% fetal bovine
serum, 2 mM L-glutamine (Euroclone, Devon) at 37 °C in a
humidified incubator containing 5% CO2.
Immunofluorescence and FACS Analysis. To determine
the expression of R1B- and R1D-AR subtypes on the PC-3 line,
1 × 106 cells were fixed and permeabilized using CytoFix/
CytoPerm Plus (BD Biosciences, Milano) before the addition
of anti-R1B-AR and anti- R1D-AR polyclonal Abs (1:25 dilution).
Normal goat serum and rabbit normal serum were used as
negative controls. After 30 min at 4 °C, cells were washed twice
and labeled with FITC-conjugated RAG and with FITC-
conjugated GARB (1:20 dilution), respectively. In some experi-
ments, PC-3 cells were exposed to the test agents, norepine-
phrine (NE) at the indicated concentration, alone or in
combination, for 24 h, and stained as described above. The
percentage of positive cells, determined over 10 000 events,
was analyzed on a FACScan cytofluorimeter (Becton Dickin-
son, San Jose´, CA). Fluorescent intensity is expressed in
arbitrary units on a logarithmic scale.
Cell viability and apoptotic cell death were detected by flow
cytometry using propidium iodide (PI) exclusion and Annexin
V staining. PC-3 cells exposed to the test agents, NE at the
indicated concentration, alone or in combination, for different
times, were trypsinized and washed in PBS. Cells were then
resuspended in PBS containing 40 µM of PI (Molecolar Probes,
Leiden) for 30 min, and PI uptake was analyzed by cytofluo-
rimetric and FACS analysis. Viability was defined as cells
excluding PI and maintaining a high forward scatter.
Apoptotic cell death was evaluated by phosphatidylserine
(PS) exposure on PC-3 cells by Annexin V staining.44 Briefly,
1 × 106 PC-3 cells exposed to the test agents (50 µM) alone or
in combination with NE (10 and 50 µM), for 24 h, were
resuspended in 0.2 mL of binding buffer (10 mM Hepes/NaOH,
pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2)
in the presence of 5 µL of FITC-Annexin V (Bender MedSys-
tem, Vienna) for 10 min at room temperature in the dark. The
percentage of positive cells determined over 10 000 events was
analyzed on a FACScan cytofluorimeter. Fluorescence inten-
sity is expressed in arbitrary units on a logarithmic scale.
Similarly, compounds 15-17 were obtained via the proce-
dure described for 14.
[2-(2,6-Dimethoxyphenoxy)ethyl]-{2-[2-(3-methoxy-
benzyloxy)phenoxy]ethyl}amine (15). 48% Yield. 1H NMR
(CDCl3) δ 1.84 (br s, 1, NH, exchangeable with D2O), 3.0 and
3.12 (two t, 4, CH2NCH2), 3.79 and 3.81 (two s, 9, OCH3), 4.18
(m, 4, OCH2 and CH2O), 5.11 (s, 2, CH2Ar), 6.52-7.29 (m, 11,
ArH). The oxalate salt was crystallized from EtOH; mp 117-
118 °C. Anal. (C26H31NO6‚H2C2O4) C, H, N.
[2-(2,6-Dimethoxyphenoxy)ethyl]-{2-[2-(4-methoxy-
benzyloxy)phenoxy]ethyl}amine (16). 49% Yield. 1H NMR
(CDCl3) δ 1.88 (br s, 1, NH, exchangeable with D2O), 2.98 and
3.12 (two t, 4, CH2NCH2), 3.75 and 3.81 (two s, 9, OCH3), 4.18
(m, 4, OCH2 and CH2O), 5.05 (s, 2, CH2Ar), 6.53-7.40 (m, 11,
ArH). The oxalate salt was crystallized from EtOH; mp 157-
158 °C. Anal. (C26H31NO6‚H2C2O4‚0.5H2O) C, H, N.
[2-(2,6-Dimethoxyphenoxy)ethyl]-{2-[2-(4-ethoxy-
benzyloxy)phenoxy]ethyl}amine (17). The free base was
purified by column chromatography eluting with CHCl3/EtOH
1
(9.8:0.2); 50% yield. H NMR (CDCl3) δ 1.19 (t, 3, CH2CH3),
2.17 (br s, 1, NH, exchangeable with D2O), 2.97 and 3.09 (two
t, 4, CH2NCH2), 3.80 (s, 6, OCH3), 3.97 (q, 2, CH2CH3), 4.15
(m, 4, OCH2 and CH2O), 5.02 (s, 2, CH2Ar), 6.52-7.38 (m, 11,
ArH). The oxalate salt was crystallized from EtOH/Et2O; mp
136-137 °C. Anal. (C27H33NO6‚H2C2O4‚H2O) C, H, N.
[2-(4-Isopropoxybenzyloxy)phenoxy]acetic Acid (28).
A mixture of 27 (1.0 g; 3.03 mmol) and 2 N NaOH (25.0 mL)
was stirred at 70 °C for 1.5 h. The mixture was extracted with
CHCl3, and the aqueous layer was acidified with concentrated
HCl. Extraction with CHCl3, followed by washing, drying, and
evaporation of the extracts gave a solid (0.8 g; 83% yield),
which was crystallized from cyclohexane; mp 55-56 °C. 1H
NMR (CDCl3) δ 1.32 (d, 6, CH(CH3)2), 4.55 (m, 1, CH(CH3)2),
4.72 (s, 2, OCH2CO), 5.17 (s, 2, CH2Ar), 6.12 (br s, 1, COOH),
6.72-7.32 (m, 8, ArH).
N-[2-(2,6-Dimethoxyphenoxy)ethyl]-2-[2-(4-isopropoxy-
benzyloxy)phenoxy]acetamide (29). Ethyl chloroformate
(0.28 g; 2.53 mmol) was added dropwise to a stirred and cooled
(0 °C) solution of 28 (0.8 g; 2.53 mmol) and Et3N (0.26 g; 2.53
mmol) in CHCl3 (50 mL), followed after 30 min by the addition
of a solution of 2-(2,6-dimethoxyphenoxy)ethylamine32 (0.5 g;
2.53 mmol) in CHCl3 (10 mL). The resulting reaction mixture
was stirred for 3 h at room temperature and then washed with
2 N HCl, 2 N NaOH, and finally water. Removal of dried
solvent gave an oil, which was purified by column chromatog-
raphy. Eluting with cyclohexane/AcOEt (6:4) gave an oil; 0.67
1
g (54% yield). H NMR (CDCl3) δ 1.32 (d, 6, CH(CH3)2), 3.57
Confocal Laser Scanning Microscopy Analysis. 2 × 105/
mL PC-3 cells, grown for 24 h at 37 °C and 5% CO2 in poly-
L-lysine-coated slides, were permeabilized using 2% of paraform-
aldehyde with 0.5% of Triton X-100 in PBS and fixed by 4% of
paraformaldehyde in PBS. After three washes in PBS, cells
were incubated with 3% of BSA and 0.1% of Tween-20 in PBS
for 1 h at room temperature and then with a goat anti-R1B-AR
or a rabbit anti-R1D-AR polyclonal Abs (1:50) at 4 °C overnight.
Samples were finally washed with 0.3% of Triton X-100 in PBS
three times, incubated with FITC-conjugated RAG Ab or with
FITC-conjugated GARB Ab (1:100) for 1 h at 4 °C, mounted,
and analyzed with a MRC600 confocal laser scanning micro-
scope (BioRad, Hercules, CA) equipped with a Nikon (Diaphot-
TMD) inverted microscope. Fluorochrome was excited with the
600 line of an argon-krypton laser and imaged using a 488
(q, 2, NCH2), 3.75 (s, 6, OCH3) 4.08 (t, 2, CH2O), 4.48 (m, 1,
CH(CH3)2), 4.58 (s, 2, OCH2CO), 5.04 (s, 2, CH2Ar), 6.52-7.34
(m, 11, ArH), 7.92 (br t, 1, NH, exchangeable with D2O).
[2-(2,6-Dimethoxyphenoxy)ethyl]-{2-[2-(4-isopro-
poxybenzyloxy)phenoxy]ethyl}amine (18). A solution of
BH3‚Me2S (0.3 mL) in dry THF (2 mL) was added dropwise at
room temperature to a solution of 28 (0.67 g; 1.36 mmol) in
dry THF (60 mL) with stirring under a stream of dry nitrogen
with exclusion of moisture. When the addition was completed,
the reaction mixture was heated at 70 °C for 6 h. After cooling
to 0 °C, excess borane was destroyed by cautious dropwise
addition of EtOH (1 mL). After standing overnight at room
temperature, 2 N NaOH (3.3 mL) was added, and the resulting
mixture was heated at 60 °C for 3 h. Removal of the solvent
under reduced pressure gave a residue, which was dissolved

7762 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Quaglia et al.
(FITC)-nm band-pass filter. Serial optical sections were taken
at 1-µm intervals through the cells. Images were processed
using Jacs Paint Shop Pro (Jacs Sotfware Inc).
drug treatment, as compared to that at the beginning (LC50).
The percentage of growth inhibition was calculated as [(Ti -
Tz)/(C - Tz)] × 100 for concentrations for which Ti g Tz and
[(Ti - Tz)/Tz] × 100 for those for which Ti < Tz, where Tz )
absorbance time zero, C ) absorbance in the presence of
vehicle, and Ti ) absorbance in the presence of drug at
different concentrations. GI50, TGI and LC50 were obtained by
interpolating % of growth versus Log(M) in a graph. Each
quoted value represents the mean of quadruplicate determina-
tions.
Western Blot Analysis. Lysates obtained from PC-3 cells
and human lymphocytes, used as positive control, were
resuspended in 0.2 mL of RIPA (0.1% Nonidet-P40, 1 mM
CaCl2, 1 mM MgCl2, 0.1% sodium azide, 1 mM PMSF, 0.03
mg/mL aprotinin, 1 mM NaVO4). Samples were separated on
7.5% SDS-polyacrylamide gel, transferred onto Immobilon-P
membranes (Millipore, Bedford, MA), and blotted with goat
anti-R1B-AR and a rabbit anti- R1D-AR polyclonal Abs (1:400)
followed by the incubation with HRP-conjugated RAG (1:
20000) and HPR-conjugated donkey anti-rabbit (1:10000) Abs,
respectively. Immunoreactivity was detected using the En-
hanced Chemiluminescence (ECL, Amersham) and the Chemi-
doc (Bio-Rad) apparatus.
RNA Isolation, Reverse Transcription and RT-PCR
Analysis. Messenger RNA was extracted from PC-3 cells or
human lymphocytes as positive control, using the Rnasy Mini
Kit (Qiagen Sciences, MA). The mRNA samples resuspended
in diethylpyrocarbonate (DEPC) water, and their concentration
and purity were evaluated by A260 measurement. RNA samples
(4 µg) were subjected to reverse transcription using the High
Capacity cDNA Archive Kit protocol (RT buffer, dNTP mixture,
Random Primers, MultiScribe RT, RNase inhibitor, Applied
Biosystems, Monza). Two microliters of resulting cDNA prod-
ucts was used as template for Reverse Transcriptase (RT)-
PCR.
Proliferation Assay. A total of 7 × 104 PC-3 cells,
dispersed for 24 h into tissue cultured-treated plastic 96-well
flat-bottomed microtiter plates at 37 °C, 5% CO2 humidified
air atmosphere, were incubated with different doses of NE (0,
0.1, 1, 10, and 50 µM) alone or in combination with compound
4 and doxazosin (1 µM) or compound 7 (0.5 µM), for 16 h in a
total volume of 200 µL of culture medium. During the last 4
h, all samples received the addition of the pyrimidine analogue
BrdU, that, after incorporation into the DNA of proliferating
cells, was detected by immunoassay using a Cell Proliferation
ELISA BrdU (colorimetric) kit (Boehringer Mannheim, Roche).
Statistical Analysis. Statistical significance was deter-
mined by using analysis of variance (ANOVA) followed by post
hoc Newman-Keuls multiple comparison at P < 0.01 and by
Student’s t-test at P < 0.01.
Acknowledgment. We thank the MIUR (Rome) and
the University of Camerino for financial support.
RT-PCR was performed using a GeneAmp 5700 Sequence
Detection system (Applied Biosystems, Monza). The reaction
mixture contained the HotStarTaq Master Mix (Qiagen Sci-
ences, Maryland) and primers.
Supporting Information Available: Elemental analysis.
This material is available free of charge via the Internet at
http://pubs.acs.org.
R1-AR subtypes primer sequences (forward and reverse)
References
(1) For part 7, see ref 41.
(2) (a) Calzada, B. C.; De Artinano A. A. R-Adrenoceptors Subtypes.
Pharmacol. Res. 2001, 44, 195-208. (b) Piascik, M. T.; Perez,
D. M. R1-Adrenergic Receptors: New Insights and Directions.
J. Pharmacol. Exp. Ther. 2001, 298, 403-410.
were as follows:
R1A
CAGTAGGTT-3′
1B: 5′-AGATGACTCCTGCCAG-3′; 5′-ACTGAGCAGCGC-
CAAGAT-3′
: 5′-AATGATACGGAACAGCATT-3′; 5′-GTGGCTGT-
R
(3) Daniels, D. V.; Gever, J. R.; Jasper, J. R.; Kava, M. S.; Lesnick,
J. D.; Meloy, T. D.; Stepan, G.; Williams, T. J.; Clarke, D. E.;
Chang, D. J.; Ford, A. P. Human Cloned R1A-Adrenoceptor
Isoforms Display R1L-Adrenoceptor Pharmacology in Functional
Studies. Eur. J. Pharmacol. 1999, 370, 337-343.
R
1D: 5′-CTACGAATTGGCCGACT-3′; 5′-GGATGGGGCAGT-
GTTTC-3′
Each PCR amplification consisted of heat activation for 15
min at 95 °C followed by 30 cycles of 94 °C for 30 s, 54 °C for
30 s and 72 °C for 30 s, by 30 cycles of 94 °C for 30 s, 52 °C for
30 s and 72 °C for 30 s and by 30 cycles of 94 °C for 30 s, 54
°C for 30 s and 72 °C for 30 s for R1A-, R1B- and R1D-subtypes,
respectively. PCR products were analyzed by electrophoresis
on 1.5% ethidium bromide-stained agarose gel, visualized by
UV transilluminator and acquired by a Chemi Doc (BioRad).
In Vitro Cytotoxicity Assay. In vitro cytotoxicity was
evaluated by SRB assay.43 Cells were maintained as stocks in
DMEM (Gibco) supplemented with 10% fetal bovine serum
(Gibco), 2 mM L-glutamine (Gibco). Cell cultures were passaged
twice weekly using trypsin-EDTA to detach the cells from their
culture flasks. The rapidly growing cells were harvested,
counted, and incubated under the appropriate concentrations
(7 × 105 cells/well) in 96-well microtiter plates. After incuba-
tion for 24 h, target and test agents dissolved in culture
medium were applied to the culture wells in quadruplicate and
incubated for 48 h at 37 °C in a 5% CO2 atmosphere and 95%
relative humidity. At the same time, a plate was tested to
evaluate the cell population before addition of the test agents
addition (Tz). In some experiments, an SRB assay was
performed using the test agents in combination with 0.1 µM
of the 5-HT1A antagonist, (S)-WAY 100135. Culture, fixed with
cold trichloroacetic acid (TCA), was stained by 0.4% SRB
dissolved in 1% acetic acid. Bound stains were subsequently
solubilized with 10 mM Trizma, and the absorbance was read
on the microplate reader Dynatech Model MR 700 at a
wavelength of 520 nm. The cytotoxic activity was evaluated
by measuring the drug concentration resulting in a 50%
reduction in the net protein increase (as measured by SRB
staining) in control cells during the drug incubation (GI50), that
resulting in total growth inhibition (TGI), and that resulting
in a 50% reduction in the measured protein at the end of the
(4) Price, D. T.; Schwinn, D. A.; Lomasney, J. W.; Allen, L. F.; Caron,
M. G.; Lefkowitz, R. J. Identification, Quantification, and
Localization of mRNA for Three Distinct R1-Adrenergic Receptor
Subtypes in Human Prostate. J. Urol. 1993, 150, 546-551.
(5) Walden, P. D.; Gerardi, C.; Lepor, H. Localization and Expres-
sion of the R1A-, R1B- and R1D-Adrenoceptors in Hyperplastic and
Non-Hyperplastic Human Prostate. J. Urol. 1999, 161, 635-
640.
(6) (a) Tseng-Crank, J.; Kost, T.; Goetz, A.; Hazum, S.; Roberson,
K. M.; Haizlip, J.; Godinot, N.; Robertson, C. N.; Saussy, D. The
R1C-Adrenoceptor in Human Prostate: Cloning, Functional
Expression, and Localization to Specific Prostate Cell Types. Br.
J. Pharmacol. 1995, 115, 1475-1485. (b) Moriyama, N.; Kurim-
oto, S.; Horie, S.; Nasu, K.; Tanaka, T.; Yano, K.; Hirano, H.;
Tsujimoto, G.; Kawabe, K. Detection of R1-Adrenoceptor Sub-
types in Human Hypertrophied Prostate by in Situ Hybridiza-
tion. Histochem. J. 1996, 28, 283-288.
(7) Thebault, S.; Roudbaraki, M.; Sydorenko, V.; Shuba, Y.; Lem-
onnier, L.; Slomianny, C.; Dewailly, E.; Bonnal, J. L.; Mauroy,
B.; Skryma, R.; Prevarskaya, N. R1-Adrenergic Receptors Acti-
vate Ca(2+)-Permeable Cationic Channels in Prostate Cancer
Epithelial Cells. J. Clin. Invest. 2003, 111, 1691-1701.
(8) Kyprianou, N.; Benning, C. M. Suppression of Human Prostate
Cancer Cell Growth by R1-Adrenoceptor Antagonists Doxazosin
and Terazosin via Induction of Apoptosis. Cancer Res. 2000, 60,
4550-4555.
(9) (a) Caine, M. R-Adrenergic Blockers for the Treatment of Benign
Prostatic Hyperplasia. Urol. Clin. North Am. 1990, 17, 641-
649. (b) Thorpe, A.; Neal, D. Benign Prostatic Hyperplasia.
Lancet 2003, 361, 1359-1367.
(10) Benning, C. M.; Kyprianou, N. Quinazoline-Derived R1-Adreno-
ceptor Antagonists Induce Prostate Cancer Apoptosis via an R1-
Adrenoceptor-Independent Action. Cancer Res. 2002, 62, 597-
602.
(11) Yang, G.; Timme, T. L.; Park, S. H.; Wu, X.; Wyllie, M. G.;
Thompson, T. C. Transforming Growth Factor beta 1 Transduced
Mouse Prostate Reconstitutions: II. Induction of Apoptosis by
Doxazosin. Prostate 1997, 33, 157-163.

SAR of 1,4-Benzodioxan-Related Compounds
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7763
(12) Partin, J. V.; Anglin, I. E.; Kyprianou, N. Quinazoline-Based R1-
Adrenoceptor Antagonists Induce Prostate Cancer Cell Apoptosis
via TGF-beta Signalling and I kappa B alpha Induction. Br. J.
Cancer 2003, 88, 1615-1621.
(13) Dizeyi, N.; Bjartell, A.; Nilsson, E.; Hansson, J.; Gadaleanu, V.;
Cross, N.; Abrahamsson, P. A. Expression of Serotonin Receptors
and Role of Serotonin in Human Prostate Cancer Tissue and
Cell Lines. Prostate 2004, 59, 328-336.
(14) Keledjian, K.; Kyprianou, N. Anoikis Induction by Quinazoline
Based R1-Adrenoceptor Antagonists in Prostate Cancer Cells:
Antagonistic Effect of bcl-2. J. Urol. 2003, 169, 1150-1156.
(15) Xu, K.; Wang, X.; Ling, P. M.; Tsao, S. W.; Wong, Y. C. The R1-
Adrenoceptor Antagonist Terazosin Induces Prostate Cancer Cell
Death through a p53 and Rb Independent Pathway. Oncol. Rep.
2003, 10, 1555-1560.
(16) Zeng, L.; Rowland, R. G.; Lele, S. M.; Kyprianou, N. Apoptosis
Incidence and Protein Expression of p53, TGF-beta Receptor II,
p27Kip1, and Smad4 in Benign, Premalignant, and Malignant
Human Prostate. Hum. Pathol. 2004, 35, 290-297.
(17) Noveral, J. P.; Grunstein, M. M. Adrenergic Receptor-Mediated
Regulation of Cultured Rabbit Airway Smooth Muscle Cell
Proliferation. Am. J. Physiol. 1994, 267, 291-299.
(18) Mimura, Y.; Kobayashi, S.; Notoya, K.; Okabe, M.; Kimura, I.;
Horikoshi, I.; Kimura, M. Activation by R1-Adrenergic Agonists
of the Progression Phase in the Proliferation of Primary Cultures
of Smooth Muscle Cells in Mouse and Rat Aorta. Biol. Pharm.
Bull. 1995, 18, 1373-1376.
(19) Gao, B. B.; Lei, B. L.; Zhang, Y. Y.; Han, Q. D. Cell Proliferation
and Ca(2+)-Calmodulin Dependent Protein Kinase Activation
Mediated by R1A-and R1B-Adrenergic Receptors in HEK293 Cells.
Acta Pharmacol. Sin. 2000, 21, 55-59.
(20) McVary, K. T.; McKenna, K.E.; Lee, C. Prostate Innervation.
Prostate Suppl. 1998, 8, 2-13.
(21) Xin, X.; Yang, N.; Eckhart, A. D.; Faber, J. E. R1D-Adrenergic
Receptors and Mitogen-Activated Protein Kinase Mediate In-
creased Protein Synthesis by Arterial Smooth Muscle. Mol.
Pharmacol. 1997, 51, 764-775.
(22) Kanagawa, K.; Sugimura, K.; Kuratsukuri, K.; Ikemoto, S.;
Kishimoto, T.; Nakatani, T. Norepinephrine Activates P44 and
P42 MAPK in Human Prostate Stromal and Smooth Muscle
Cells but not in Epithelial Cells. Prostate 2003, 56, 313-318.
(23) Mottram, D. R.; Kapur, H. The R-Adrenoceptor Blocking Effects
of a New Benzodioxane. J. Pharm. Pharmacol. 1975, 27, 295-
296.
(24) Quaglia, W.; Pigini, M.; Giannella M.; Melchiorre, C. 3-Phenyl
Analogues of 2-([(2-(2,6-Dimethoxyphenoxy)ethyl]amino]-meth-
yl)-1,4-benzodioxan (WB 4101) as Highly Selective Rl-Adreno-
receptor Antagonists. J. Med. Chem. 1990, 33, 2946-2948.
(25) Quaglia, W.; Pigini, M.; Tayebati, S. K.; Piergentili, A.; Giannella,
M.; Marucci, G.; Melchiorre, C. Structure-Activity Relationships
in 1,4-Benzodioxan-Related Compounds. 4. Effect of Aryl and
Alkyl Substituents at Position 3 on R-Adrenoreceptor Blocking
Activity. J. Med. Chem. 1993, 36, 1520-1528.
(26) Quaglia, W.; Pigini, M.; Piergentili, A.; Giannella, M.; Marucci,
G.; Poggesi, E.; Leonardi, A.; Melchiorre, C. Structure-Activity
Relationships in 1,4-Benzodioxan-Related Compounds. 6. Role
of the Dioxane Unit on Selectivity for R1-Adrenoreceptor Sub-
types. J. Med. Chem. 1999, 42, 2961-2968.
(27) Zhao, M. M.; Hwa, J.; Perez, D. M. Identification of Critical
Extracellular Loop Residues Involved in R1-Adrenergic Receptor
Subtype-Selective Antagonist Binding. Mol. Pharmacol. 1996,
50, 1118-1126.
(28) Hamaguchi, N.; True, T. A.; Saussy, D. L., Jr.; Jeffs, P. W.
Phenylalanine in the Second Membrane-Spanning Domain of
(34) Testa, R.; Taddei, C.; Poggesi, E.; Destefani, C.; Cotecchia, S.;
Hieble, J. P.; Sulpizio, A. C.; Naselsky, D.; Bergsma, D.; Ellis,
S.; Swift, A.; Ganguly, S.; Ruffolo, R. R.; Leonardi, A. Rec 15/
2739 (SB 216469): a Novel Prostate Selective R1-Adrenoceptor
Antagonist. Pharmacol Commun. 1995, 6, 79-86.
(35) (a) Fargin, A.; Raymond, J. R.; Lohse, M. J.; Kobilka, B. K.;
Caron, M. G.; Lefkowitz, R. J. The Genomic Clone G-21 Which
Resembles
a â-Adrenergic Receptor Sequence Encodes the
5-HT1A Receptor. Nature 1988, 335, 358-360. (b) Fargin, A.;
Raymond, J. R.; Regan, J. W.; Cotecchia, S.; Lefkowitz, R. J.;
Caron, M. G. Effector Coupling Mechanisms of the Cloned
5-HT1A Receptor. J. Biol. Chem. 1989, 284, 14848-14852.
(36) Eltze, M.; Boer, R.; Sanders, K. H.; Kolossa, N. Vasodilatation
Elicited by 5-HT1A Receptor Agonists in Constant-Pressure-
Perfused Rat Kidney Is Mediated by Blockade of R1A-Adrenore-
ceptors. Eur. J. Pharmacol. 1991, 202, 33-44.
(37) Ko, F. N.; Guh, J. H.; Yu, S. M.; Hou, Y. S.; Wu, Y. C.; Teng, C.
M. (-)-Discretamine, a Selective R1D-Adrenoreceptor Antagonist,
Isolated from Fissistigma glaucescens. Br. J. Pharmacol. 1994,
112, 1174-1180.
(38) Buckner, S. A.; Oheim, K. W.; Morse, P. A.; Knepper, S. M.;
Hancock, A. A. R1-Adrenoceptor-Induced Contractility in Rat
Aorta Is Mediated by the R1D Subtype. Eur. J. Pharmacol. 1996,
297, 241-248.
(39) van Rossum, J. M. Cumulative Dose-Response Curves. II.
Technique for the Making of Dose-Response Curves in Isolated
Organs and the Evaluation of Drug Parameters. Arch. Int.
Pharmacodyn. Ther. 1963, 143, 299-330.
(40) Stanton, J. A.; Beer, M. S. Characterization of a Cloned Human
5-HT1A Receptor Cell Line Using [35S]GTPγS Binding. Eur. J.
Pharmacol. 1997, 320, 267-275.
(41) Quaglia, W.; Pigini, M.; Piergentili, A.; Giannella, M.; Gentili,
F.; Marucci, G.; Carrieri, A.; Carotti, A.; Poggesi E.; Leonardi,
A.; Melchiorre C. Structure-Activity Relationships in 1,4-
Benzodioxan-Related Compounds. 7. Selectivity of 4-Phenyl-
chroman Analogues for R1-Adrenoreceptor Subtypes. J. Med.
Chem. 2002, 45, 1633-1643.
(42) Cal, C.; Uslu, R.; Gunaydin, G.; Ozyurt, C.; Omay, S. B.
Doxazosin: a New Cytotoxic Agent for Prostate Cancer? BJU
Int. 2000, 85, 672-675.
(43) Grever, M. R.; Shepartz, S. A.; Chabner, B. A. The National
Cancer Institute: Cancer Drug Discovery and Development
Program. Semin. Oncol. 1992, 19, 622-638.
(44) Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutelingsperger,
C. A Novel Assay for Apoptosis. Flow Cytometric Detection of
Phosphatidylserine Expression on Early Apoptotic Cells Using
Fluorescein Labelled Annexin V. J. Immunol. Methods 1995,
184, 39-51.
(45) Lindquist, J. M.; Rehnmark, S. Ambient Temperature Regula-
tion of Apoptosis in Brown Adipose Tissue. Erk1/2 Promotes
Norepinephrine-Dependent Cell Survival. J. Biol. Chem. 1998,
273, 30147-30156.
(46) Noh, J. S.; Kim, E. Y.; Kang, J. S.; Kim, H. R.; Oh, Y. J.; Gwag,
B. J. Neurotoxic and Neuroprotective Actions of Catecholamines
in Cortical Neurons. Exp. Neurol. 1999, 159, 217-224.
(47) Popovik, E.; Haynes, L. W. Survival and Mitogenesis of Neu-
roepithelial Cells Are Influenced by Noradrenergic but not
Cholinergic Innervation in Culture Embryonic Rat Neopallium.
Brain Res. 2000, 853, 227-235.
R
1A-Adrenergic Receptor Determines Subtype Selectivity of
Dihydropyridine Antagonists. Biochemistry 1996, 35, 14312-
14317.
(48) Garcia-Sainz, J. A.; Villalobos-Molina, R. The Elusive R1D
-
(29) Topliss J. G. Utilization of Operational Schemes for Analogue
Synthesis in Drug Design. J. Med. Chem. 1972, 15, 1006-1011.
(30) Topliss J. G. A Manual Method for Applying the Hansch
Approach to Drug Design. J. Med. Chem. 1977, 20, 463-469.
(31) Keledjian, K.; Borkowski, A.; Kim, G.; Isaacs, J. T.; Jacobs S.
C.; Kyprianou, N. Reduction of Human Prostate Tumor Vascu-
larity by the R1-Adrenoceptor Antagonist Terazosin. Prostate
2001, 48, 71-78.
Adrenoceptor: Molecular and Cellular Characteristics and
Integrative Roles. Eur. J. Pharmacol. 2004, 500, 113-120.
(49) Garcia-Sainz, J. A.; Rodriguez-Perez, C. E.; Romero-Avila, M.
T. Human R1D-Adrenoceptor Phosphorylation and Desensitiza-
tion. Biochem. Pharmacol. 2004, 67, 1853-1858.
(50) Hague, C.; Uberti, M. A.; Chen, Z.; Hall, R.A.; Minneman, K. P.
Cell Surface Expression of R1D-Adrenergic Receptors Is Con-
trolled by Heterodimerization with R1B-Adrenergic Receptors. J.
Biol. Chem. 2004, 279, 15541-15549.
(51) Cheng, Y.; Prusoff, W. H. Relationship Between the Inhibition
Constant (Ki) and the Concentration of Inhibitor Which Causes
50% Inhibition (I50) of an Enzymatic Reaction. Biochem. Phar-
macol. 1973, 22, 3099-3108.
(32) Woolley, D. W. Probable Evolutionary Relations of Serotonin and
Indole-Acetic Acid, and Some Practical Consequences Therefrom.
Nature (London) 1957, 180, 630-633.
(33) Hancock, A. A.; Buckner, S. A.; Brune, M. E.; Katwala, S.; Milicic,
I.; Ireland, L. M.; Morse, P. A.; Knepper, S. M.; Meyer, M. D.;
Chapple, C. R.; Chess-Williams, R.; Noble, A. J.; Williams, M.;
Kerwin, J. F., Jr. Pharmacological Characterization of A-131701,
a Novel R1-Adrenoceptor Antagonist Selective for R1A- and R1D
-
Compared to R1B-Adrenoceptors. Drug Dev. Res. 1998, 44, 140-
162.
JM0580398