Structure-activity relationships in 1,4-benzodioxan-related compounds. 10. Novel α1-adrenoreceptor antagonists related to openphendioxan: Synthesis, biological evaluation, and α1d computational study

Article abstract of DOI:10.1016/j.bmc.2010.08.002

A series of novel openphendioxan analogues were synthesized and tested at α₁-adrenoreceptor (AR) subtypes by binding and functional assays. The α_1d-AR binding profile was also examined by means of 2D, 3D-QSAR together with docking studies. Multiple regression analysis suggested the relevance of adequate number of heteroatoms in the whole molecule and of passive membrane diffusion to enhance α_1d-AR affinity. Docking simulations against a computational structural model of the biological target further proved this evidence and furnished support for chemiometric analysis, where polar, electrostatic, hydrophobic and shape effects of the ortho substituents in the phenoxy terminal, most likely governing ligand binding, helped the depiction of pharmacophore hypothesis for the examined ligands data set.

Full text of DOI:10.1016/j.bmc.2010.08.002

Bioorganic & Medicinal Chemistry 18 (2010) 7065–7077
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationships in 1,4-benzodioxan-related compounds. 10.
Novel
a1-adrenoreceptor antagonists related to openphendioxan: Synthesis,
biological evaluation, and a1d computational study q
b
b
b
b
Antonio Carrieri a, , Alessandro Piergentili , Fabio Del Bello , Mario Giannella , Maria Pigini ,
*
Amedeo Leonardi c, Francesca Fanelli d, Wilma Quaglia b,
*
a Dipartimento Farmaco-Chimico, Università di Bari, via Orabona 4, 70125 Bari, Italy
b Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino, via S. Agostino 1, 62032 Camerino, Italy
c Recordati S. p. A., Drug Discovery, via Civitali 1, 20148 Milano, Italy
d Istituto Dulbecco Telethon e Dipartimento di Chimica, Università di Modena & Reggio Emilia, via Campi 183, 41100 Modena, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel openphendioxan analogues were synthesized and tested at a1-adrenoreceptor (AR) sub-
Received 23 April 2010
Revised 28 July 2010
Accepted 2 August 2010
Available online 6 August 2010
types by binding and functional assays. The
3D-QSAR together with docking studies. Multiple regression analysis suggested the relevance of adequate
number of heteroatoms in the whole molecule and of passive membrane diffusion to enhance 1d-AR
a1d-AR binding profile was also examined by means of 2D,
a
affinity. Docking simulations against a computational structural model of the biological target further
proved this evidence and furnished support for chemiometric analysis, where polar, electrostatic, hydro-
phobic and shape effects of the ortho substituents in the phenoxy terminal, most likely governing ligand
binding, helped the depiction of pharmacophore hypothesis for the examined ligands data set.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
GPCRs
a1-Adrenoreceptor antagonists
Openphendioxan analogues
QSAR
Comparative modeling
Docking
1. Introduction
ery rate of novel compounds acting as binders to G-protein-cou-
pled receptors (GPCRs), the target of approximately half of the
Insights gained from target proteins and receptors three-dimen-
sional structure are pivotal for successful modern drug design. The
number of entries deposited at the Protein Data Bank2 (PDB) is
nowadays more than sixty thousand, with more than five thousand
new structures available each year. However, compared to these
valuable numbers, very few data refer to integral proteins (180 un-
ique structures).3 The paucity of robust information on structural
assembly of transmembrane (TM) receptors holds back the discov-
top 100 drugs currently on the market. Sensational breakdowns
were determined first in year 2000, when the X-ray structure of
bovine rhodopsin was solved by Palczewski et al.4 and later with
the achievement of human b1- and b2-adrenergic5,6 and A2A aden-
osine receptor crystallographic data.7 In the light of this mutated
scenario, the rational design of novel molecules interacting with
the adrenergic receptors turns to a more feasible effort.
The widespread expression in many human tissues and the
involvement in numerous physiological processes make adrenore-
ceptors (ARs) highly attractive pharmacological targets for the treat-
ment of numerous pathologies.8 Particularly, the three subtypes
Abbreviations: GPCR, G-protein-coupled receptor; AR, adrenoreceptor; TM,
transmembrane; LUTS, lower urinary tract symptoms; BPH, benign prostatic
hyperplasia; TM, transmembrane; SDM, site-directed mutagenesis; PDB, protein
data bank; CHO, chinese hamster ovary; QSARs, quantitative structure–activity
relationships; [3H]8-OH-DPAT, 8-hydroxy-2-(di-n-propylamino)tetralin; SAR,
structure–activity relationships; SAFIR, structure–affinity relationships; QPlogBB,
predicted blood–brain barrier partition coefficient; Ki, inhibition or dissociation
constant; Kb, dissociation constant; MRA, multiple regression analysis; PLS, partial
least squares; MIF, molecular interaction field.
belonging to the
tribution10 and modulate a large number of physiological functions
in cardiovascular and non cardiovascular tissues. The 1A-AR sub-
a1-AR family (a1A, a1B, and a
1D)9 have different dis-
a
type is considered to be the main system contributing to the dy-
namic (phasic) component of increased bladder outlet resistance11
and, together with the a1D subtype, mediating lower urinary tract
symptoms (LUTS) caused by benign prostatic hyperplasia (BPH).12
q
See Ref. 1.
* Corresponding authors. Tel.: +39 0805442638; fax: +39 0805442724 (A.C.); tel.:
+39 0737402237; fax: +39 0737637345 (W.Q.).
E-mail addresses: carrieri@farmchim.uniba.it (A. Carrieri), wilma.quaglia@uni
cam.it (W. Quaglia).
For this reason
AR subtypes with respect to the
the treatment of BPH,13 avoiding cardiovascular and orthostatic
a1-antagonists selective for
a1A- and/or a1A- + a1D-
a1B subtype are clinically useful in
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.002

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A. Carrieri et al. / Bioorg. Med. Chem. 18 (2010) 7065–7077
hypotensive side effects.14 The observation that the
a1B-AR subtype
were assessed by binding assays at human cloned a1-AR subtypes
controls locomotor and rewarding effects of psychostimulants and
opiates suggests the use of antagonists targeting this subtype in
and 5-HT1A receptors, expressed in Chinese hamster ovary (CHO)
and HeLa cell membranes, respectively, and by functional experi-
the treatment of drug abuse.15 Moreover, the role of
a1-ARs in the
ments in isolated rat vas deferens (
a1A), spleen (a1B), and aorta
mitogenic effect of catecholamines on prostate growth has been
(a
1D). Afterwards, computational quantitative structure–activity
demonstrated16 and recent studies have highlighted the efficacy of
relationships (QSARs), homology modeling, and docking studies
a
1D- and
proliferation.17 Unfortunately, the high homologyamong theTM do-
mains of the three 1-AR subtypes limits the availability of ligands
a1B-AR antagonists in the modulation of apoptosis and cell
were conducted to highlight the influence of the ortho substitution
in the terminal phenyl ring of the basic chain on
a1d-AR affinities.
a
The
a
1-AR antagonist WB410120 was included in this investigation
with adequate subtype selectivity. Indeed, site-directed mutagene-
sis (SDM) studies reported that two phenylalanines in TM7 domain
(Phe7.35 and Phe7.39 according to the Ballesteros and Weinstein
for useful comparison.
2. Chemistry
numbering)18 are highly conserved among all the three
a1-AR sub-
types19 and are involved in high-affinity binding for many
a1-AR
The novel compounds 2–14 were synthesized according to the
methods reported in Schemes 1 and 2. Alkylation of 2,6-dimeth-
oxybenzenethiol,22 2,6-diethoxyphenol,23 and 2-(benzyloxy)phe-
nol with 2-chloroacetamide afforded the intermediate amides
15–17, respectively, which were reduced with borane–dimethyl
sulfide complex in dry THF to give the corresponding amines
18–20. Amidation of 18, 19, and other proper substituted phen-
oxyethanamines, commercially available or prepared following
the procedure reported in the literature,24 with 2-[2-(benzyl-
oxy)phenoxy]acetic acid21 in the presence of Et3N and EtOCOCl
furnished amides 21–28, whose reduction with borane–dimethyl
sulfide complex in dry THF afforded the final compounds 2, 5, 7,
9–12, and 14. Oxidation of 2 with H2O2 in acetic acid gave com-
pound 4 (Scheme 1). Similarly, compounds 3, 6, 8, and 13 were
synthesized, according to Scheme 2, by amidation of 20 with the
proper disubstituted phenoxyacetic acid and subsequent reduction
of the amides 29–32 with borane–dimethyl sulfide complex in dry
THF. 3-(2,6-Dimethoxyphenyl)propanoic acid, 2-(2-methoxy-6-
nitrophenoxy)acetic acid, and 2-(2-methoxy-6-methylphen-
oxy)acetic acid were prepared according to the literature.25–27 In
the case of 2-(2,6-bis(methoxymethoxy)phenoxy)acetic acid (35),
the procedure reported in Scheme 3 was followed. Thus, lithiation
of 1,3-bis(methoxymethoxy)benzene28 with n-BuLi, followed by
boronation–oxidation with B(OMe3)3 and oxone in aqueous ace-
tone–NaHCO3, afforded the phenol 33, whose treatment with
methyl chloroacetate gave 34, which was hydrolyzed to the corre-
sponding acid 35.
antagonists, among which [2-(2,6-dimethoxyphenoxy)-ethyl][(2,3-
dihydro-1,4-benzodioxin-2-yl)methyl]-amine (WB4101) (Fig. 1).20
Some of us have previously demonstrated that the insertion of a
trans phenyl (phendioxan) or p-tolyl (mephendioxan) moiety in po-
sition 3 of WB4101 increased affinity and selectivity for
a1A-ARs,
whereas the replacement of the oxygen atom in position 4 with a
phenylmethine group afforded an a1D selective antagonist.20 a1D
selectivity was also obtained by opening the dioxane ring of phen-
dioxan affording openphendioxan (1)21 (Fig. 1). Moreover, we also
demonstrated that the introduction of substituents, having all the
possible combinations of
tionof 1, did notsignificantlyaffectitsalreadyhigh
r
and
p
parameters in the benzyloxy por-
a
1D-ARaffinity.17
However, since it is known that aromaticity/hydrophobicity
might play an important role in the antagonist receptor interaction
and subtype discrimination,19 in the present study, taken 1 as lead
compound, some chemical modifications were performed in its
2,6-dimethoxyphenoxy portion. The novel compounds were ob-
tained by replacing the oxygen atom of the bridge spacing the basic
centre and the 2,6-dimethoxyphenyl ring with its S or CH2 bioisos-
teres or an SO function (2–4), by removing one or both 2,6-dime-
thoxy substituents, or by replacing one or both of them with
groups of different physicochemical (electronic and steric) proper-
ties (5–14) (Fig. 1). The biological profiles of the novel compounds
O
O
H3CO
O
H
N
3. Results and discussion
OCH3
The affinity constants, expressed as pKi, of compounds 2–14
were evaluated by radio-receptor binding assays using 1 and
WB4101 as reference compounds. [3H]Prazosin was used to label
WB4101
cloned human
a
1-ARs expressed in CHO cells.29 Furthermore,
[3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) was
used to label cloned human 5-HT1A receptors expressed in HeLa
cells.30,31
O
O
R'
X
The pharmacological profile of compounds 2–14 was further
H
N
determined at
WB4101 as reference compounds.
ity, expressed as pKb, was assessed by antagonism of (ꢀ)-noradren-
a1-ARs on different isolated tissues using 1 and
a
1-AR subtypes blocking activ-
R
32
aline-induced contraction of rat prostatic vas deferens (a1A
)
or
thoracic aorta (a1D
)
33 and by antagonism of (ꢀ)-phenylephrine-in-
8: X=O, R=OCH3, R'=CH3
1: X=O, R=R'=OCH3
2: X=S, R=R'=OCH3
3: X=CH2, R=R'=OCH3
4: X=SO, R=R'=OCH3
5: X=O, R=R'=OC2H5
6: X=O, R=OCH3, R'=NO2
7: X=O, R=OCH3, R'=Cl
duced contraction of rat spleen (a
1B).34
9: X=O, R=OC2H5, R'=H
10: X=O, R=OCH3, R'=H
11: X=O, R=R'=Cl
12: X=O, R=R'=CH3
13: X=O, R=R'=OH
14: X=O, R=R'=H
From an analysis of the results reported in Table 1 interesting
considerations emerged. All the substitutions of the oxygen atom
in the bridge (2–4) induced the maintenance of high
and preferential 1d-AR subtype interaction with respect to a1a
and 1b-AR subtypes. However, while the bioisosteric sulfur
a1d-affinity
a
-
a
analogue 2 showed affinity values similar to those of 1, the replace-
ment of the oxygen atom with a methylene group (3) or the
oxidation of the sulfur atom to the sulfoxide function (4) did not
Figure 1. Chemical structures of WB4101, openphendioxan (1), and the novel
compounds 2–14.

A. Carrieri et al. / Bioorg. Med. Chem. 18 (2010) 7065–7077
7067
R'
XH
R
NH2
Cl
+
O
a or b
R'
O
X
R
15: X=S, R=R'=OCH3
NH2
16: X=O, R=R'=OC2H5
17: X=O, R=OCH2C6H5, R'=H
c
R'
X
R
18: X=S, R=R'=OCH3
NH2
19: X=O, R=R'=OC2H5
20: X=O, R=OCH2C6H5, R'=H
O
d
+
O
COOH
21: X=S, R=R'=OCH3
22: X=O, R=R'=OC2H5
23: X=O, R=OCH3, R'=Cl
24: X=O, R=OC2H5, R'=H
25: X=O, R=OCH3, R'=H
26:X =O, R=R'=Cl
O
O
R'
H
N
X
O
R
27: X=O, R=R'=CH3
28: X=O, R=R'=H
c
O
H3CO
O
O
R'
e
from 2
H
N
H
N
O
S
X
OCH3
R
O
2: X=S, R=R'=OCH3
5: X=O, R=R'=OC2H5
7: X=O, R=OCH3, R'=Cl
9: X=O, R=OC2H5, R'=H
10: X=O, R=OCH3, R'=H
11: X=O, R=R'=Cl
4
12: X=O, R=R'=CH3
14: X=O, R=R'=H
Scheme 1. Reagents: (a) Na, KI, EtOH; (b) K2CO3, acetone; (c) BH3ꢁMe2S, THF; (d) Et3N, EtOCOCl, CHCl3; (e) 30% H2O2, AcOH.
favourably contribute to the general
a
1-AR interaction. This result,
moiety enhanced the 5-HT1A-affinity of the lead. Removal of one or
both methoxy groups (10 and 14, respectively) as well as their
simultaneous replacement with smaller substituents such as
chloro, methyl and hydroxy groups (11–13) caused a sharp de-
which was more and more evident considering the functional
antagonist affinities, suggested that the good electronic density
provided by oxygen and sulfur atoms, and not associated with a
spatially defined orientation as in the case of the SO function,
determined more productive interactions. The pKb values observed
in functional experiments, comparable in most cases with the pKi
affinities derived from the binding assays, highlighted for 3 and,
crease in both binding and functional affinities for all the
a1-AR
subtypes and in 1d-AR subtype selectivity. Instead, such modifica-
a
tions did not significantly affect the 5-HT1A affinity. The 2,6-dieth-
oxy derivative 5 showed a biological affinity profile similar to that
of 1, suggesting that the small increase of the steric bulk was tol-
erated. In this case the removal of one ethoxy group (9) reduced
especially, for 4 the loss of the a1D selectivity. The discrepancy be-
tween functional and binding affinities meets a common justifica-
tion in the different arrangement of native or cloned receptor
populations.35 In contrast, the presence of a methylene or sulfoxide
the affinities for
a1b- and a1d-AR subtypes. The binding profile of
derivatives 5 and 9 was not confirmed by functional data, from

7068
A. Carrieri et al. / Bioorg. Med. Chem. 18 (2010) 7065–7077
R'
O
X
R
COOH
+
NH2
O
20
a
29: X=CH2, R=R'=OCH3
O
O
R'
30: X=O, R=OCH3, R'=NO2
31: X=O, R=OCH3, R'=CH3
32: X=O, R=R'=OCH2OCH3
H
N
X
O
b
R
3: X=CH2, R=R'=OCH3
6: X=O, R=OCH3, R'=NO2
8: X=O, R=OCH3, R'=CH3
13: X=O, R=R'=OH
O
O
R'
H
N
X
R
Scheme 2. Reagents: (a) Et3N, EtOCOCl, CHCl3; (b) BH3ꢁMe2S, THF.
O
OCH2OCH3
OCH2OCH3
OCH2OCH3
OCH2COOCH3
OCH2OCH3
OCH2COOH
OCH2OCH3
Cl
CH3
+
a
c
O
b
OH
OCH2OCH3
OCH2OCH3
OCH2OCH3
33
34
35
Scheme 3. Reagents: (a) n-BuLi, B(OMe)3, oxone, THF; (b) K2CO3, KI, acetone; (c) 2 N NaOH.
which compound 5 showed the highest antagonist affinity and a
preferential selectivity for 1B-AR subtype with respect to the
other subtypes. The removal of one ethoxy group (compound 9)
significantly affected only the potency at 1B-AR subtype. The
replacement of only one of the two methoxy groups of 1 with a ni-
tro, chloro, or methyl substituent (6–8) was compatible with a
preferential and efficient a1d interaction. The observation that
the derivatives 7 and 8 showed affinity values significantly higher
than those of the corresponding equally disubstituted derivatives
by the largeness of the substituents, as measured by QikProp vol-
ume (compound 1 was excluded most likely due to the data distri-
bution). This evidence is supported by the significant drop of the
same constants caused by the absence of any functional group in
the phenoxy moiety (compound 14). Moreover, our results agreed
with previous studies24,37 confirming the essential role of the ortho
substituents with certain steric hindrance.
Afterwards we gain better and further insights into the struc-
ture–affinity relationships (SAFIR) carrying out a statistical analy-
sis with STRIKE.38 This modeling tool, automatically, performs
multiple regression analysis to derive several QSAR equations
using different properties associated to ligand structures. Among
the whole set of fifty QikProp descriptors, the best and statistically
most significant relationship (Eq. 1) was achieved with two inde-
pendent variables, the predicted blood–brain barrier partition
coefficient QPlogBB and the number of nitrogen and oxygen atoms
present in the molecule #NandO. The aforementioned variables,
encoding passive membrane diffusion and polarity, scored with
positive coefficients, lipophilicity and hydrogen bonding capability
as major determinants for the antagonist binding. It could then be
suggested that molecular properties, mimicked by the parameters
present in (Eq. 1), should have a relevant weight on controlling and
affecting the largeness of the pKi value and, therefore, the whole
receptor binding process.
a
a
11 and 12 suggested that, for a high
a1d-affinity, at least one of
the two ortho substituents should be a methoxy group. Moreover,
interestingly, among the novel derivatives, analogously to lead 1,
compounds 2 and 5–7 proved to be endowed with a significant
a1d-AR/5-HT1A selectivity.
Classical QSARs were further applied on the highly congeneric
derivatives 1 and 5–14 to rationalize their a1d-AR affinity spectrum
using a large and fresh set of molecular descriptors providing a
complete level of information with different 1D and 2D indices, re-
lated not only to physicochemical, but also to topological, geomet-
rical, quantum chemical and pharmacokinetics features of the
ligand, included in the QikProp software package (see Table 2).36
In a first instance, taking into account affinities referred only to
compounds 5–14 the measured affinity constants, through plausi-
ble parabolic dependence (r2 >0.65), resulted to be nearly affected

A. Carrieri et al. / Bioorg. Med. Chem. 18 (2010) 7065–7077
7069
Table 1
Affinity constants, expressed as pKi, of compounds 1–14 and WB4101 for human recombinant
a
1-AR subtypes and 5-HT1A receptora. Antagonist affinities, expressed as pKb values
b
at
a
1-ARs on isolated rat vas deferens (
a
1A), spleen (
a
1B), and thoracic aorta (a1D)
O
O
R'
X
H
N
R
b
Compd
R
R’
X
pKia human cloned receptors
pKb
a1a
a1b
a1d
5-HT1A
a1A
a1B
a1D
1
2
3
4
5
6
7
8
OCH3
OCH3
OCH3
OCH3
OC2H5
OCH3
OCH3
OCH3
OC2H5
OCH3
Cl
OCH3
OCH3
OCH3
OCH3
OC2H5
NO2
Cl
CH3
H
H
O
S
CH2
SO
O
O
O
O
O
O
O
O
O
9.33
8.50
8.01
7.70
8.50
8.95
8.60
8.60
8.70
8.50
8.20
8.10
8.30
8.0
9.27
9.30
8.16
7.80
9.10
8.30
8.10
8.30
7.90
7.70
7.10
7.0
10.17
9.75
8.92
8.56
9.41
9.29
9.13
9.04
8.43
8.40
8.15
7.85
7.82
7.75
9.29
7.93
7.80
8.60
8.50
7.50
7.80
7.50
8.40
8.30
8.60
7.50
7.80
7.90
8.10
8.68
8.39 0.18
8.74 0.10
7.80 0.04
7.95 0.05
7.92 0.04
8.41 0.04
8.16 0.05
8.35 0.02
8.03 0.04
7.95 0.07
6.57 0.13
7.33 0.14
7.44 0.07
7.65 0.10
9.51 0.06
8.30 0.15
8.57 0.09
7.34 0.18
7.65 0.08
8.57 0.03
8.19 0.05
7.84 0.07
7.91 0.09
7.94 0.04
7.89 0.14
7.75 0.03
7.78 0.10
7.02 0.01
7.74 0.11
8.16 0.09
9.37 0.15
9.47 0.11
8.12 0.05
7.49 0.03
8.03 0.17
8.92 0.09
8.74 0.02
8.36 0.13
8.35 0.07
8.08 0.06
8.02 0.02
7.59 0.03
7.44 0.03
7.32 0.03
8.80 0.12
9
10
11
12
13
14
WB4101
Cl
CH3
OH
CH3
OH
H
7.0
7.0
8.0
H
O
9.37
a
Equilibrium dissociation constants (Ki) were derived from IC50 values using the Cheng–Prusoff equation.48 The affinity estimates were derived from displacement of
[3H]prazosin and [3H]-8-hydroxy-2-(di-n-propylamino)tetralin binding for
a1-ARs and 5-HT1A receptor, respectively. Each experiment was performed in triplicate. Ki values
were from two to three experiments, which agreed within 20%.
b
pKb values were calculated according to van Rossum46 in the range 0.01–10
lM. Each concentration [B] of antagonist was tested four times.
Table 2
QikProp molecular descriptors of compounds 1 and 5–14
and hydrogen bonding acceptor groups might properly orient the
antagonist scaffold, favoring and stabilizing the different interac-
tions essential to increase affinity.
Compd
Va
QPlogBBb
#NandOc
1
5
6
7
8
146.61
273.91
139.21
107.57
161.62
147.54
75.24
77.67
102.65
37.75
0.135
0.015
ꢀ0.810
0.312
0.209
0.101
0.165
0.503
0.216
ꢀ0.819
0.227
6
6
8
5
5
5
5
4
4
6
4
This assumption can be perceived in details from Figure 2: li-
gands merged into the cleft comprising the TM helix bundle, with
the benzyloxy moiety deeply buried within the extracellular half of
the TM bundle. This ligand binding mode has already been ob-
served in other docking simulations carried out by some of us
9
using a rhodospin based
a
1d-AR homology model.39 According to
10
11
12
13
14
this docking mode, the charged nitrogen of the ligand anchors
the protein scaffold through an electrostatic interaction with
Asp176 (3.32), a fundamental receptor recognition point. This
interaction is shared by the docking poses selected for all the con-
sidered ligands. Furthermore, the presence of the same benzyloxy
aromatic moiety favors the stacking of binders towards some
essential residues, namely Trp361 (6.48), Phe364 (6.51) and
Phe365 (6.52), which are part of TM6. As assessed before, our past
0.00
a
Measured as difference between the solvent accessible volume in cubic Å of
substituted and unsubstituted compounds using a probe with a 1.4 Å radius.
b
Predicted blood–brain partitioning.
Number of nitrogen and oxygen atoms.
c
efforts17 aimed at exploring the
r and p effects did not result in
pKi ¼ 2:226ðꢂ0:360ÞQPlogBB
any gain of affinity constant. Therefore, it might be postulated that
the lonely steric hindrance, provided by this aromatic ring, could
probably hamper all the conformational changes of the same helix,
which are known to occur for activating the target.40–43
þ 1:033ðꢂ0:1307Þ#NandO þ 3:18ðꢂ0:701Þ
ð1Þ
n ¼ 11; r2 ¼ 0:887; s ¼ 0:296; F ¼ 31:30
To go further on this topic, WB4101 and derivatives 1–14 were
docked to a computational model of the 1d-AR, realized by com-
parative modeling by using the X-ray structure of the b2-AR as a
The most interesting observations might be done on the recep-
tor complex comprising the ligands disubstituted phenoxy ring,
the second extracellular loop (ECL2) and TM7. It has been sug-
gested that the triplet Gly247, Ile248 and Thr249 of the human
a
template. The aim was to sketch a more detailed framework of
a
1d-AR ligands binding process and to probe, at three-dimensional
a
1d-AR, highly conserved in the other two subtypes, might be the
level, the hypothesis formulated by the 2D-QSAR study about the
primary region of binding of antagonists, in agreement with SDM
role of ortho substituents as major molecular determinants for
experiments carried out on the equivalent position of the rat a1a
and hamster
a
1b-AR.44 Comfortably in our docking experiments
a1d-AR antagonism. Flexible docking furnished valuable indica-
tions on the binding topology characterizing openphendioxan ana-
logues and helped to rationalize the different experimentally
determined receptor affinities, suggesting that slightly hindered
one o-methoxy group, making significant van der Waals contacts
with the side chain of Ile248, can be observed. As a conse-
quence, this incidence might cause a proper orientation of the

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A. Carrieri et al. / Bioorg. Med. Chem. 18 (2010) 7065–7077
Gly247
Gly247
Thr249
Thr249
Asp176
Asp176
Ile248
Ile248
Tyr392
Tyr392
Phe388
Phe388
Phe384
Phe384
Phe364
Phe364
Trp361
Trp361
Phe365
Phe365
Figure 2. Receptor–ligand complex of
a
1d-AR and the high affine compound 1. The
Figure 3. Receptor–ligand complex of a1d-AR and the low affine compound 13. The
surface in magenta represents the void volume of the receptor binding cavity.
surface in magenta represents the void volume of the receptor binding cavity.
disubstituted phenyl ring which is afterward facing Phe384 (7.35)
and Phe388 (7.39) arising strong hydrophobic contact (i.e. face to
face and edge to face p–p stacking) with these residues side chains.
The GRIND descriptors were subsequently used in a Partial
Least Squares (PLS) analysis. From the data reported in Table 3 it
can be seen that good PLS statistics were obtained, with the per-
centage of explained variance being almost 95%. Similarly, the q2
cross-validated correlation coefficients in the Leave-One-Out
regression models is well above 0.3 which corresponds to a low
probability of chance correlation (i.e. p <5%).
Further insights into the 3D-QSAR results were gained consider-
ing signs and magnitudes of the PLS coefficients to determine the
chemical descriptors most responsible for antagonist affinity
(Fig. 4). This analysis indicates that the MIFs measured at certain
grid nodes and exact distances with the N1–TIP, O–TIP, and N1–
N1 probes have the greatest impact on antagonist affinity, while
variables related to DRY–DRY fields show negative control.
More specifically, variables N1–TIP 7, O–TIP 14 and 40, N1–N1
27 showed the largest coefficients and hence these pharmaco-
As further validation of docking poses, SDM experiments also evi-
denced the aforementioned aminoacids as crucial for ligand bind-
ing.19 All these evidences took place in the docking of 1 and 5–14,
in agreement with the importance of shape and polarity as came
out from 2D-QSAR. The increase of the steric bulk of the two ortho
substituents (5) or the replacement of only one methoxy group
with different substituents (6–8) do not dramatically affect the
binding mode. In the less affine compounds the mono-substitution
(9–10) or the total absence of o-methoxy groups (11–14) lock less
powerfully the ligand to the binding site. In particular, as shown in
Figure 3, the very low a1d-affinity value of the derivative 13 might
be due to the presence of the two polar o-hydroxy groups, which
might prevent productive interaction with Ile248. Moreover, an
additional hydrogen bond with Tyr392 might influence both ligand
conformation and ligand–receptor fit and affect the pKi value.
Also from the docking of compounds 2–4 some useful observa-
tions came out. In particular, the bioisosteric replacement of the
oxygen atom of 1 with a sulfur atom as in 2 preserves the same
electron rich hinge pointing towards the antagonists binding re-
gion of ECL2 (Fig. S1). Instead the methylene group of 3 or SO moi-
ety of 4, reducing the electron density on the disubstituted phenyl
ring, lowers the strength of the aromatic interaction. Moreover, in
the case of 4 the induced reduction of the torsional freedom of the
phoric moieties are likely to be present in the most affine
a1d-AR
antagonists and absent in the least affine ones, whereas variable
DRY–DRY 49, characterized by a large negative coefficient, should
be ascribable to the low affine antagonists (PLS variables in this
study were numbered according to the distances between interact-
ing nodes of a given type using a two-digit variable number equal
to Å distances by multiplying it by the grid spacing and smoothing
window, which were 0.5 and 0.8 Å, respectively).
These results suggested that certain, appropriately spaced,
structural elements such as convex molecular surfaces and/or polar
adjacent phenyl-ring also prevents the good fit and p–p stackings
with Phe384 (7.35) and Phe388 (7.39) obtained with compounds 1
and 2 (Figs. S2 and S3).
functional groups are pivotal for this class of
also that finding an optimal spacing between them might signifi-
a1d-AR binders, and
To infer the biological activity profile at three-dimensional level
and to produce a sound pharmacophore hypothesis, 3D-QSAR
study was performed. To accomplish this goal, docking was used
as support tool for chemometric analysis of the measured pKi. So
starting from the docked pose of each complex, a molecular align-
ment was generated, and affinity constant was related to GRID
independent descriptors (GRINDs)45 measured around the mole-
cules, producing statistical models capable to investigate the
molecular determinants most likely affecting the biological data
(see Section 4). GRINDs were obtained by a quick and automated
procedure involving: (i) computations of the molecular interaction
fields (MIFs) induced by selected probe atoms on a 3D grid, (ii) fil-
tering the MIFs in order to extract the most salient chemical infor-
mation regarding the receptor binding site, and (iii) encoding the
spatial relationship within the binding site nodes into new inde-
pendent 3D variables.
cantly enhance biological affinity. For instance, in the structural
model of
a1d-AR used for dockings Ile248 C and Asp178 (3.32)
c1
Od2 are spaced by 5.9 Å; this value seems to resemble field effects
measured around affine derivatives (i.e. 1) with a hydrogen bond
acceptor and a shape probe, as scored by variable N1–TIP 7, located
in the area surrounding the o-methoxy group and the amino func-
Table 3
Statistical results of PLS analysis for compounds 1–14 and WB4101
n
var
Probe
r2
q2
s
ONC
3
15
340
DRYa Ob N1c TIPd
0.944
0.633
0.168
a
b
c
Hydrophobic.
Negatively charged hydrogen bond acceptor.
Neutral hydrogen bond donor.
Shape.
d

A. Carrieri et al. / Bioorg. Med. Chem. 18 (2010) 7065–7077
7071
DRY-DRY
O-O
N1-N1
TIP-TIP
DRY-O
DRY-N1
DRY-TIP
O-N1
O-TIP
N1-TIP
0.6
0.5
0.4
0.3
0.2
0.1
0
7
14
27
40
-0.1
-0.2
-0.3
-0.4
-0.5
49
Figure 4. Plot of the PLS coefficients for data set (variables mentioned in the text are highlighted).
tion. Indeed, hydrophobic interactions (
p
–
p
stacking) occurring
Our docking experiments proved that the interaction of the phen-
oxy moiety of openphendioxan-related compounds is greatly fa-
vored by the ortho disubstitution. In particular, two methoxy
substituents allow an optimal interaction of the ligand to the bind-
ing sites, with one methoxy group forming a specific interaction
with the side chain of Ile248 and the other stabilizing a proper ori-
entation of the phenyl ring for strong hydrophobic contacts with
Phe384 (7.35) and Phe388 (7.39). Finally, the 3D-QSAR study sug-
gested that properly spaced structural elements, such as convex
molecular surface and/or polar functional groups, are pivotal for
over an intramolecular threshold distance might be detrimental
for ligand binding. In fact, the aromatic centroids of Phe365
(6.52) and Phe388 (7.39) are spaced by 11.3 Å that is well below
the distance values (19 Å) expressed by variable DRY–DRY 49. Fig-
ure 5 shows graphical representations of the above variables dis-
played for the high and low affine compounds
respectively.
It was supporting that fields effect emerged from chemometric
analysis (i.e. acceptor/donor hydrogen bond) might correspond, to
a great extent, to the molecular descriptors (i.e. #NandO) come out
as most relevant from the 2D-QSAR study.
1 and 13,
this class of a1d-AR binders.
In conclusion, comparative modeling and docking experiments,
4. Experimental section
4.1. Chemistry
together with QSAR, supported the understanding of
a1d-AR bind-
ing affinity, elucidating the SAFIR for a series of novel
a
1d-AR
antagonists related to openphendioxan. The pharmacophoric fea-
tures of the phenoxy terminal most likely capable of enhancing li-
gands affinity were interpreted with variables encoding the
lipopilicity (QPlogBB) and polarity (#NandO) of the substituents.
The suggestions of SAFIR regarding a possible lower of pKi arising
from the bioisosteric replacement of the oxygen atom of the bridge
spacing the basic centre and the 2,6-dimethoxyphenyl ring with
non polar (i.e. methylene) and spatially defined (i.e. SO) groups,
or the presence in the phenoxy ring of small substituents at one
or both ortho positions were validated by the computational study.
Melting points were taken in glass capillary tubes on a Büchi
SMP-20 apparatus and are uncorrected. IR and NMR spectra were
recorded on Perkin-Elmer 297 and Varian Gemini 200 instruments,
respectively. Chemical shifts are reported in parts per million
(ppm) relative to tetramethylsilane (TMS), and spin multiplicities
are given as s (singlet), d (doublet), dd (double 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
Figure 5. Grid filtered MIFs for the highest affine 1 (left) and low affine 13 (right). The most significant PLS variables 7, 14, 27, 40 and 49 are represented with magenta lines.

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A. Carrieri et al. / Bioorg. Med. Chem. 18 (2010) 7065–7077
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. Mass spectra
were obtained using a Hewlett–Packard 1100 MSD instrument uti-
lizing electron-spray ionization (ESI) and a gas chromatograph/
mass spectrometer (GC–MS, EI-70 eV). Compounds were named
following IUPAC rules as applied by Beilstein-Institut AutoNom
4.1.5. 2-(2,6-Diethoxyphenoxy)ethanamine (19)
This was obtained following the procedure described for 18
starting from 16. The free base was transformed into the hydro-
chloride salt and recrystallized from 2-PrOH: 45% yield; mp 97–
98 °C. The hydrochloride salt was dissolved in 2 N NaOH and the
solution was extracted with CHCl3. The evaporation of the solvent
afforded the pure free base. 1H NMR (CDCl3): d 1.46 (t, 6, CH2CH3),
1.97 (br s, 2, NH2, exchangeable with D2O), 2.92 (t, 2, CH2N), 4.09
(m, 6, CH2CH3 and OCH2), 6.58 (d, 2, ArH), 6.95 (t, 1, ArH); MS
(ESI) m/z 226.1 ([M+H]+), 248.1 ([M+Na]+).
(version 2.1),
chemistry.
a software for systematic names in organic
4.1.6. 2-(2-(Benzyloxy)phenoxy)ethanamine (20)
This was obtained following the procedure described for 18
starting from 17. The evaporation of the solvent afforded an oil
which was used in the next step without further purification:
81% yield. 1H NMR (CDCl3): d 1.79 (br s, 2, NH2, exchangeable with
D2O), 3.07 (t, 2, CH2N), 4.07 (t, 2, OCH2), 5.12 (s, 2, OCH2Ar), 6.82–
7.52 (m, 9, ArH); MS (ESI) m/z 244.1 ([M+H]+), 266.1 ([M+Na]+).
4.1.1. 2-(2,6-Dimethoxyphenylthio)acetamide (15)
2,6-Dimethoxybenzenethiol22 (4.63 g, 27.2 mmol) was added to
a solution of Na (0.7 g, 30.4 mmol) in EtOH (120 mL) under
mechanical stirring. The mixture was refluxed for 30 min. Then
2-chloroacetamide (2.54 g, 27.2 mmol) and KI (0.5 g) were added
and heating was maintained for 5 h. After evaporation of the sol-
vent under vacuum, the residue was treated with 2 N NaOH and
extracted with CHCl3. The organic layer was dried over Na2SO4
and the solvent was evaporated under vacuum to give a solid:
5.5 g; 96% yield; mp 157–158 °C. 1H NMR (CDCl3): d 3.52 (s, 2,
SCH2), 3.88 (s, 6, OCH3), 5.49 and 7.62 (two br t, 2, NH2, exchange-
able with D2O), 6.55 (d, 2, ArH), 7.28 (t, 1, ArH); MS (ESI) m/z 228.0
([M+H]+), 250.0 ([M+Na]+).
4.1.7. 2-(2-(Benzyloxy)phenoxy)-N-(2-(2,6-dimethoxyphenyl
thio)ethyl)acetamide (21)
Et3N (0.81 mL, 5.81 mmol) and EtOCOCl (0.56 mL, 5.81 mmol)
were added to a solution of 2-(2-(benzyloxy)phenoxy)acetic acid21
(1.5 g, 5.81 mmol) in dry CHCl3 (40 mL) at 0 °C. After 30 min a solu-
tion of 18 (1.3 g, 5.81 mmol) in CHCl3 (20 mL) was added and the
reaction mixture was left at rt for 3 h. The solution was then
washed with 2 N HCl, 2 N NaOH, and the organic phase was dried
over Na2SO4. Removal of the solvent gave a residue, which was
purified by column chromatography. The residue was purified by
column chromatography. Eluting with cyclohexane/EtOAc (7:3)
afforded an oil: 2.3 g; 88% yield. 1H NMR (CDCl3): d 2.81 (t, 2,
CH2S), 3.30 (q, 2, NCH2), 3.81 (s, 6, OCH3), 4.50 (s, 2, CH2CO), 5.13
(s, 2, OCH2Ar), 6.51–7.48 (m, 12, ArH), 7.68 (br t, 1, NH, exchange-
able with D2O); MS (ESI) m/z 454.1 ([M+H]+), 476.1 ([M+Na]+).
4.1.2. 2-(2,6-Diethoxyphenoxy)acetamide (16)
A mixture of 2,6-diethoxyphenol23 (4.07 g, 22.34 mmol), 2-
chloroacetamide (2.09 g, 22.34 mmol), and K2CO3 (3.09 g,
22.34 mmol) in dry acetone (150 mL) was refluxed for 48 h. After
cooling, the solid was filtered and the solvent was evaporated.
The residue was dissolved in CHCl3 and washed with 2 N NaOH.
Removal of dried solvents gave a solid: 2.53 g; 47% yield; mp
156–157 °C. 1H NMR (CDCl3): d 1.46 (t, 6, CH2CH3), 4.09 (q, 4,
CH2CH3), 4.57 (s, 2, CH2CO), 5.72 and 8.01 (two br s, 2, NH2,
exchangeable with D2O), 6.55 (d, 2, ArH), 6.98 (t, 1, ArH); MS
(ESI) m/z 240.1 ([M+H]+), 262.1 ([M+Na]+).
4.1.8. 2-(2-(Benzyloxy)phenoxy)-N-(2-(2,6-diethoxyphenoxy)
ethyl)acetamide (22)
This was obtained following the procedure described for 21
starting from 19. The residue was purified by column chromatog-
raphy. Eluting with cyclohexane/EtOAc (8:2) afforded a solid:
68% yield; mp 75–76 °C. 1H NMR (CDCl3): d 1.33 (t, 6, CH2CH3),
3.60 (q, 2, NCH2), 4.02 (m, 6, CH2CH2O and CH2CH3), 4.58 (s, 2,
CH2CO), 5.09 (s, 2, OCH2Ar), 6.46–7.43 (m, 12, ArH), 7.80 (br t, 1,
NH, exchangeable with D2O); MS (ESI) m/z 466.2 ([M+H]+), 488.2
([M+Na]+).
4.1.3. 2-(2-(Benzyloxy)phenoxy)acetamide (17)
This was obtained following the procedure described for 16
starting from 2-(benzyloxy)phenol. The residue was treated with
petroleum ether to afford a solid: 65% yield; mp 158–159 °C. 1H
NMR (CDCl3): d 4.52 (s, 2, CH2CO), 5.08 (s, 2, OCH2Ar), 5.66 (br s,
2, NH2, exchangeable with D2O), 6.83–7.46 (m, 9, ArH); MS (ESI)
m/z 258.1 ([M+H]+), 280.1 ([M+Na]+).
4.1.9. 2-(2-(Benzyloxy)phenoxy)-N-(2-(2-chloro-6-methoxy
phenoxy)ethyl)acetamide (23)
4.1.4. 2-(2,6-Dimethoxyphenylthio)ethanamine (18)
A solution of 10 M BH3ꢁCH3SCH3 (3.0 mL) in dry THF (10 mL)
was added dropwise at rt to a stirred solution of 15 (2.0 g,
8.8 mmol) in dry THF (100 mL) under a stream of dry nitrogen with
exclusion of moisture. When the addition was completed, the reac-
tion mixture was heated at reflux temperature for 8 h. After the
mixture was cooled to 0 °C, excess borane was destroyed by cau-
tious dropwise addition of EtOH (15 mL). The resulting mixture
was left to stand overnight at rt, cooled to 0 °C, acidified with con-
centrated HCl, and then heated to 60 °C for 1 h. Removal of the sol-
vent under reduced pressure gave a residue, which was dissolved
in H2O. The aqueous solution was basified with 2 N NaOH and ex-
tracted with CHCl3. Removal of dried solvents gave a residue,
which was purified by column chromatography, eluting with
CHCl3/EtOH (9.9:0.1) to afford an oil: 1.75 g; 93% yield. 1H NMR
(CDCl3): d 1.62 (br s, 2, NH2, exchangeable with D2O), 2.65 and
2.85 (two t, 4, CH2CH2), 3.88 (s, 6, OCH3), 6.57 (d, 2, ArH), 7.27 (t,
1, ArH); MS (ESI) m/z 214.0 ([M+H]+), 236.0 ([M+Na]+).
This was obtained following the procedure described for 21
starting from 2-(2-chloro-6-methoxyphenoxy)ethanamine.24 The
residue was purified by column chromatography. Eluting with
cyclohexane/EtOAc (8:2) afforded an oil: 76% yield. 1H NMR
(CDCl3): d 3.62 (q, 2, NCH2), 3.72 (s, 3, OCH3), 4.08 (t, 2, CH2CH2O),
4.58 (s, 2, CH2CO), 5.11 (s, 2, OCH2Ar), 6.72–7.44 (m, 12, ArH), 7.78
(br t, 1, NH, exchangeable with D2O); MS (ESI) m/z 442.1 ([M+H]+),
464.1 ([M+Na]+).
4.1.10. 2-(2-(Benzyloxy)phenoxy)-N-(2-(2-ethoxyphenoxy)
ethyl)acetamide (24)
This was obtained following the procedure described for 21
starting from 2-(2-ethoxyphenoxy)ethanamine. The residue was
purified by column chromatography. Eluting with cyclohexane/
EtOAc (8:2) afforded a solid: 80% yield; mp 84–85 °C. 1H NMR
(CDCl3): d 1.33 (t, 3, CH2CH3), 3.63 (q, 2, NCH2), 4.02 (m, 4, CH2CH2O
and OCH2CH3), 4.58 (s, 2, CH2CO), 5.10 (s, 2, OCH2Ar), 6.82–7.44 (m,

A. Carrieri et al. / Bioorg. Med. Chem. 18 (2010) 7065–7077
7073
13, ArH), 7.52 (br t, 1, NH, exchangeable with D2O); MS (ESI) m/z
(br t, 1, NH, exchangeable with D2O); MS (ESI) m/z 453.1 ([M+H]+),
422.1 ([M+H]+), 444.1 ([M+Na]+).
475.1 ([M+Na]+).
4.1.11. 2-(2-(Benzyloxy)phenoxy)-N-(2-(2-methoxyphenoxy)
ethyl)acetamide (25)
4.1.17. N-(2-(2-(Benzyloxy)phenoxy)ethyl)-2-(2-methoxy-6-
methylphenoxy)acetamide (31)
This was obtained following the procedure described for 21
starting from 2-(2-methoxyphenoxy)ethanamine. The residue
was purified by column chromatography. Eluting with cyclohex-
ane/EtOAc (7:3) afforded a solid: 76% yield; 75–76 °C. 1H NMR
(CDCl3): d 3.62 (q, 2, NCH2), 3.71 (s, 3, OCH3), 4.0 (t, 2, CH2CH2O),
4.52 (s, 2, CH2CO), 5.07 (s, 2, OCH2Ar), 6.79–7.42 (m, 14, ArH and
NH, exchangeable with D2O); MS (ESI) m/z 408.1 ([M+H]+), 430.1
([M+Na]+).
This was obtained following the procedure described for 21
starting from 20 and 2-(2-methoxy-6-methylphenoxy)acetic
acid.27 The residue was purified by column chromatography. Elut-
ing with cyclohexane/EtOAc (7:3) afforded an oil: 76% yield. 1H
NMR (CDCl3): d 2.22 (s, 3, CH3Ar), 3.72 (s, 3, OCH3), 3.82 (q, 2,
NCH2), 4.20 (t, 2, OCH2CH2), 4.42 (s, 2, COCH2), 5.09 (s, 2, OCH2Ar),
6.64–7.40 (m, 12, ArH), 7.87 (br t, 1, NH, exchangeable with D2O);
MS (ESI) m/z 422.1 ([M+H]+), 444.1 ([M+Na]+).
4.1.12. 2-(2-(Benzyloxy)phenoxy)-N-(2-(2,6-dichlorophenoxy)
ethyl)acetamide (26)
4.1.18. N-(2-(2-(Benzyloxy)phenoxy)ethyl)-2-(2,6-bis(methoxy
methoxy)phenoxy)acetamide (32)
This was obtained following the procedure described for 21
starting from 2-(2,6-dichlorophenoxy)ethanamine.24 The residue
was purified by column chromatography. Eluting with cyclohex-
ane/EtOAc (8.5:1.5) afforded a solid which was crystallized from
cyclohexane: 60% yield; mp 112–113 °C. 1H NMR (CDCl3): d 3.68
(q, 2, NCH2), 4.05 (t, 2, CH2CH2O), 4.60 (s, 2, CH2CO), 5.12 (s, 2,
OCH2Ar), 6.92–7.46 (m, 12, ArH), 7.69 (br t, 1, NH, exchangeable
with D2O); MS (ESI) m/z 446.0 ([M+H]+), 468.0 ([M+Na]+).
This was obtained following the procedure described for 21
starting from 20 and 35. The residue was purified by column chro-
matography. Eluting with cyclohexane/EtOAc (7:3) afforded an oil:
58% yield. 1H NMR (CDCl3): d 3.41 (s, 6, OCH3), 3.80 (q, 2, CH2N),
4.18 (t, 2, OCH2CH2), 4.58 (s, 2, COCH2), 5.10 (s, 2, OCH2Ar), 5.13
(s, 4, OCH2O), 6.78–7.43 (m, 12, ArH), 8.31 (br t, 1 NH, exchange-
able with D2O); MS (ESI) m/z 498.2 ([M+H]+), 520.2 ([M+Na]+).
4.1.19. 2-(2-(Benzyloxy)phenoxy)-N-(2-(2,6-dimethoxyphenyl-
thio)ethyl)ethanamine (2)
4.1.13. 2-(2-(Benzyloxy)phenoxy)-N-(2-(2,6-dimethylphenoxy)
ethyl)acetamide (27)
This was obtained following the procedure described for 18
starting from 21. The residue was purified by column chromatog-
raphy. Eluting with CHCl3/EtOH (9.9:0.1) afforded the free base
as an oil: 52% yield. 1H NMR (CDCl3): d 2.02 (br s, 1, NH, exchange-
able with D2O), 2.70 (t, 2, CH2S), 2.95 and 2.99 (two t, 4, CH2NCH2),
3.85 (s, 6, OCH3), 4.12 (t, 2, OCH2CH2), 5.12 (s, 2, OCH2Ar), 6.52–
7.49 (m, 12, ArH); MS (ESI) m/z 440.1 ([M+H]+), 462.1 ([M+Na]+.
The free base was transformed into the oxalate salt and recrystal-
lized from EtOH; mp 161–162 °C. Anal. Calcd for C25H29NO4Sꢁ
C2H2O4: C, 61.23; H, 5.90; N, 2.64; S, 6.05. Found: C, 61.03; H,
6.19; N, 2.88; S, 5.86.
This was obtained following the procedure described for 21
starting from 2-(2,6-dimethylphenoxy)ethanamine. Removal of
the solvent gave a residue, which was purified by column chroma-
tography. Eluting with cyclohexane/EtOAc (8:2) afforded a solid:
47% yield; mp 95–96 °C. 1H NMR (CDCl3): d 2.18 (s, 6, CH3), 3.65
(q, 2, NCH2), 3.76 (t, 2, CH2CH2O), 4.60 (s, 2, CH2CO), 5.09 (s, 2,
OCH2Ar), 6.84–7.43 (m, 12, ArH), 7.63 (br t, 1, NH, exchangeable
with D2O); MS (ESI) m/z 406.1 ([M+H]+), 428.1 ([M+Na]+).
4.1.14. 2-(2-(Benzyloxy)phenoxy)-N-(2-phenoxyethyl)acetamide
(28)
This was obtained following the procedure described for 21
starting from 2-phenoxyethanamine. The residue was purified by
column chromatography. Eluting with cyclohexane/EtOAc (7:3)
afforded a solid: 71% yield; 103–104 °C. 1H NMR (CDCl3): d 3.60
(q, 2, NCH2), 3.92 (t, 2, CH2CH2O), 4.52 (s, 2, CH2CO), 5.08 (s, 2,
OCH2Ar), 6.72–7.52 (m, 15, ArH and NH, exchangeable with
D2O); MS (ESI) m/z 378.1 ([M+H]+), 400.1 ([M+Na]+).
4.1.20. 2-(2-(Benzyloxy)phenoxy)-N-(2-(2,6-dimethoxyphenyl
sulfinyl)ethyl)ethanamine (4)
A solution of 2 (0.36 g, 0.82 mmol), glacial AcOH (0.3 mL) and
30% H2O2 (0.4 mL) was left at rt for 9 h. The solution was basified
with 2 N NaOH, extracted with CHCl3 and the organic solvents
were dried over Na2SO4. After evaporation of the solvent, the resi-
due was purified by column chromatography. Eluting with CHCl3/
EtOH (9.9:0.1) afforded the free base as an oil: 35% yield. 1H NMR
(CDCl3): d 1.79 (br s, 1, NH, exchangeable with D2O), 3.0–3.26 (m, 6,
CH2NCH2CH2S), 3.86 (s, 6, OCH3), 4.12 (t, 2, OCH2CH2), 5.09 (s, 2,
OCH2Ar), 6.52–7.49 (m, 12, ArH); MS (ESI) m/z 456.1 ([M+H]+),
478.1 ([M+Na]+). The free base was transformed into the oxalate
salt and recrystallized from 2-PrOH; mp 146–147 °C. Anal. Calcd
for C25H29NO5SꢁC2H2O4ꢁ0.5H2O: C, 58.47; H, 5.82; N, 2.53; S, 5.78.
Found: C, 58.60; H, 5.93; N, 2.52; S, 6.12.
4.1.15. N-(2-(2-(Benzyloxy)phenoxy)ethyl)-3-(2,6-dimethoxy
phenyl)propanamide (29)
This was obtained following the procedure described for 21
starting from 20 and 3-(2,6-dimethoxyphenyl)propanoic acid.25
The residue was purified by column chromatography. Eluting with
cyclohexane/EtOAc (7:3) afforded a solid: 45% yield; mp 114–
115 °C. 1H NMR (CDCl3): d 2.38 (t, 2, COCH2), 2.91 (t, 2, COCH2CH2),
3.63 (q, 2, NCH2), 3.75 (s, 6, OCH3), 4.06 (t, 2, OCH2CH2), 5.08 (s, 2,
OCH2Ar), 6.38 (br t, 1, NH, exchangeable with D2O), 6.43–7.44 (m,
12, ArH); MS (ESI) m/z 436.2 ([M+H]+), 458.2 ([M+Na]+).
4.1.21. N-(2-(2-(Benzyloxy)phenoxy)ethyl)-3-(2,6-dimethoxy
phenyl)propan-1-amine (3)
This was obtained following the procedure described for 18
starting from 29. The residue was purified by column chromatog-
raphy. Eluting with CHCl3/EtOH (9.9:0.1) afforded the free base
4.1.16. N-(2-(2-(Benzyloxy)phenoxy)ethyl)-2-(2-methoxy-6-
nitrophenoxy)acetamide (30)
This was obtained following the procedure described for 21
starting from 20 and 2-(2-methoxy-6-nitrophenoxy)acetic acid.26
The residue was purified by column chromatography. Eluting with
cyclohexane/EtOAc (5:5) afforded an oil: 65% yield. 1H NMR
(CDCl3): d 3.78 (s, 3, OCH3), 3.80 (q, 2, NCH2), 4.20 (t, 2, OCH2CH2),
4.61 (s, 2, COCH2), 5.08 (s, 2, OCH2Ar), 6.90–7.43 (m, 12, ArH), 7.56
as an oil: 63% yield. 1H NMR (CDCl3):
d 1.75 (quintet, 2,
CH2CH2CH2), 1.91 (br s, 1, NH, exchangeable with D2O), 2.67 (m,
4, CH2NCH2), 3.02 (t, 2, CH2Ar), 3.79 (s, 6, OCH3), 4.15 (t, 2,
OCH2CH2), 5.12 (s, 2, OCH2Ar), 6.48–7.49 (m, 12, ArH); MS (ESI)
m/z 422.2 ([M+H]+), 444.2 ([M+Na]+). The free base was trans-
formed into the oxalate salt and recrystallized from 2-PrOH; mp

7074
A. Carrieri et al. / Bioorg. Med. Chem. 18 (2010) 7065–7077
159–160 °C. Anal. Calcd for C26H31NO4ꢁC2H2O4: C, 65.74; H, 6.50; N,
([M+Na]+). The free base was transformed into the oxalate salt
and recrystallized from EtOH; mp 164–165 °C. Anal. Calcd for
2.74. Found: C, 65.92; H, 6.82; N, 2.88.
C
25H29NO4ꢁC2H2O4: C, 65.18; H, 6.28; N, 2.82. Found: C, 64.95; H,
4.1.22. 2-(2-(Benzyloxy)phenoxy)-N-(2-(2,6-diethoxyphenoxy)
ethyl)ethanamine (5)
6.57; N, 2.89.
This was obtained following the procedure described for 18
starting from 22. The residue was purified by column chromatog-
raphy. Eluting with CHCl3/EtOH (9.9:0.1) afforded the free base
as an oil: 83% yield. 1H NMR (CDCl3): d 1.41 (t, 6, CH2CH3), 2.38
(br s, 1, NH, exchangeable with D2O), 3.01 and 3.13 (two t, 4,
CH2NCH2), 4.03 (q, 4, CH2CH3), 4.18 (m, 4, OCH2 and CH2O), 5.12
(s, 2, OCH2Ar), 6.50–7.44 (m, 12, ArH); MS (ESI) m/z 452.2
([M+H]+), 474.2 ([M+Na]+). The free base was transformed into
the oxalate salt and recrystallized from 2-PrOH; mp 109–110 °C.
Anal. Calcd for C27H33NO5ꢁC2H2O4: C, 64.31; H, 6.51; N, 2.59.
Found: C, 63.97; H, 6.82; N, 2.67.
4.1.27. 2-(2-(Benzyloxy)phenoxy)-N-(2-(2-methoxyphenoxy)
ethyl)ethanamine (10)
This was obtained following the procedure described for 18
starting from 25. The residue was purified by column chromatog-
raphy. Eluting with CHCl3/EtOH (9.9:0.1) afforded the free base
as an oil: 72% yield. 1H NMR (CDCl3): d 2.42 (br s, 1, NH, exchange-
able with D2O), 3.10 (m, 4, CH2NCH2), 3.77 (s, 3, OCH3), 4.08 and
4.18 (two t, 4, OCH2 and CH2O), 5.09 (s, 2, OCH2Ar), 6.79–7.43
(m, 13, ArH); MS (ESI) m/z 394.1 ([M+H]+), 416.1 ([M+Na]+). The
free base was transformed into the oxalate salt and recrystallized
from EtOH; mp 199–200 °C. Anal. Calcd for
C24H27
NO4ꢁC2H2O4ꢁ0.25H2O: C, 63.99; H, 6.09; N, 2.87. Found: C, 63.96;
4.1.23. 2-(2-(Benzyloxy)phenoxy)-N-(2-(2-methoxy-6-nitrophen
oxy)ethyl)ethanamine (6)
H, 6.38; N, 2.70.
This was obtained following the procedure described for 18
starting from 30. The residue was purified by column chromatog-
raphy. Eluting with CHCl3/EtOH (9.7:0.3) afforded the free base
as an oil: 60% yield. 1H NMR (CDCl3): d 2.81 (br s, 1, NH, exchange-
able with D2O), 3.25 and 3.32 (two t, 4, CH2NCH2), 3.84 (s, 3, OCH3),
4.35 (m, 4, OCH2 and CH2O), 5.08 (s, 2, OCH2Ar), 6.88–7.47 (m, 12,
ArH); MS (ESI) m/z 439.1 ([M+H]+), 461.1 ([M+Na]+). The free base
was transformed into the oxalate salt and recrystallized from
EtOH; mp 161–162 °C. Anal. Calcd for C24H26N2O6ꢁC2H2O4: C,
59.09; H, 5.34; N, 5.30. Found: C, 59.09; H, 5.53; N, 5.34.
4.1.28. 2-(2-(Benzyloxy)phenoxy)-N-(2-(2,6-dichlorophenoxy)
ethyl)ethanamine (11)
This was obtained following the procedure described for 18
starting from 26. The residue was purified by column chromatog-
raphy. Eluting with CHCl3/EtOH (9.9:0.1) afforded the free base
as a solid: 56% yield; mp 66–67 °C. 1H NMR (CDCl3): d 2.60 (br s,
1, NH, exchangeable with D2O), 3.20 and 3.27 (two t, 4, CH2NCH2),
4.21 and 4.30 (two t, 4, OCH2 and CH2O), 5.12 (s, 2, OCH2Ar), 6.88–
7.46 (m, 12, ArH); MS (ESI) m/z 432.1 ([M+H]+), 454.1 ([M+Na]+).
The free base was transformed into the oxalate salt and recrystal-
lized from EtOH; mp 157–158 °C. Anal. Calcd for
C23H23Cl2
4.1.24. 2-(2-(Benzyloxy)phenoxy)-N-(2-(2-chloro-6-methoxy
phenoxy)ethyl)ethanamine (7)
NO3ꢁC2H2O4: C, 57.48; H, 4.82; N, 2.68. Found: C, 57.61; H, 5.15;
N, 2.73.
This was obtained following the procedure described for 18
starting from 23. The residue was purified by column chromatog-
raphy. Eluting with CHCl3/EtOH (9.9:0.1) afforded the free base
as an oil: 80% yield. 1H NMR (CDCl3): d 2.14 (br s, 1, NH, exchange-
able with D2O), 3.07 and 3.13 (two t, 4, CH2NCH2), 3.78 (s, 3, OCH3),
4.18 (m, 4, OCH2 and CH2O), 5.12 (s, 2, OCH2Ar), 6.72–7.47 (m, 12,
ArH); MS (ESI) m/z 428.1 ([M+H]+), 450.1 ([M+Na]+). The free base
was transformed into the oxalate salt and recrystallized from
EtOH; mp 151–152 °C. Anal. Calcd for C24H26ClNO4ꢁC2H2O4: C,
60.29; H, 5.45; N, 2.70. Found: C, 59.97; H, 5.71; N, 2.75.
4.1.29. 2-(2-(Benzyloxy)phenoxy)-N-(2-(2,6-dimethylphenoxy)
ethyl)ethanamine (12)
This was obtained following the procedure described for 18
starting from 27. The residue was purified by column chromatog-
raphy. Removal of dried solvents gave a residue, which was puri-
fied by column chromatography, eluting with CHCl3/EtOH
(9.9:0.1) to afford an oil: 0.5 g; 79% yield. 1H NMR (CDCl3): d 2.26
(s, 6, CH3), 2.52 (br s, 1, NH, exchangeable with D2O), 3.12 and
3.20 (two t, 4, CH2NCH2), 3.88 and 4.24 (two t, 4, OCH2 and
CH2O), 5.11 (s, 2, OCH2Ar), 6.88–7.45 (m, 12, ArH); MS (ESI) m/z
392.2 ([M+H]+), 414.1 ([M+Na]+). The free base was transformed
into the oxalate salt and recrystallized from MeOH; mp 180–
181 °C. Anal. Calcd for C25H29NO3ꢁC2H2O4: C, 67.34; H, 6.49; N,
2.91. Found: C, 67.10; H, 6.68; N, 2.96.
4.1.25. 2-(2-(Benzyloxy)phenoxy)-N-(2-(2-methoxy-6-methyl
phenoxy)ethyl)ethanamine (8)
This was obtained following the procedure described for 18
starting from 31. The residue was purified by column chromatog-
raphy. Eluting with CHCl3/EtOH (9.9:0.1) afforded the free base
as an oil: 74% yield. 1H NMR (CDCl3): d 2.15 (s, 3, CH3), 2.72 (br
s, 1, NH, exchangeable with D2O), 3.05 and 3.13 (two t, 4,
CH2NCH2), 3.78 (s, 3, OCH3), 4.02 and 4.20 (two t, 4, OCH2 and
CH2O), 5.08 (s, 2, OCH2Ar), 6.67–7.42 (m, 12, ArH); MS (ESI) m/z
408.2 ([M+H]+), 430.2 ([M+Na]+). The free base was transformed
into the oxalate salt and recrystallized from EtOH; mp 164–
165 °C. Anal. Calcd for C25H29NO4ꢁC2H2O4: C, 65.18; H, 6.28; N,
2.82. Found: C, 64.94; H, 6.65; N, 2.88.
4.1.30. 2-(2-(2-(2-(Benzyloxy)phenoxy)ethylamino)ethoxy)
benzene-1,3-diol (13)
This was obtained following the procedure described for 18
starting from 32. The residue was purified by column chromatog-
raphy. Eluting with CHCl3/EtOH (8.5:1.5) afforded the free base
as a solid: 60% yield; mp 118–119 °C. 1H NMR (CDCl3): d 2.88
and 3.08 (two t, 4, CH2NCH2), 3.91 and 4.20 (two t, 4, OCH2 and
CH2O), 5.07 (s, 2, OCH2Ar), 5.20 (br s, 1 NH, exchangeable with
D2O), 6.42–7.48 (m, 14, OH and ArH); MS (ESI) m/z 396.1
([M+H]+), 418.1 ([M+Na]+). The free base was transformed into
the oxalate salt and recrystallized from 2-PrOH; mp 113–114 °C.
Anal. Calcd for C23H25NO5ꢁC2H2O4: C, 61.85; H, 5.61; N, 2.89.
Found: C, 61.58; H, 5.33; N, 2.93.
4.1.26. 2-(2-(Benzyloxy)phenoxy)-N-(2-(2-ethoxyphenoxy)
ethyl)ethanamine (9)
This was obtained following the procedure described for 18
starting from 24. The residue was purified by column chromatog-
raphy. Eluting with CHCl3/EtOH (9.9:0.1) afforded the free base
as an oil: 67% yield. 1H NMR (CDCl3): d 1.40 (t, 3, CH2CH3), 1.92
(br s, 1, NH, exchangeable with D2O), 3.11 (m, 4, CH2NCH2), 4.03
(q, 2, CH2CH3), 4.13 (m, 4, OCH2 and CH2O), 5.11 (s, 2, OCH2Ar),
6.81–7.48 (m, 13, ArH); MS (ESI) m/z 408.2 ([M+H]+), 430.2
4.1.31. 2-(2-(Benzyloxy)phenoxy)-N-(2-phenoxyethyl)ethan
amine (14)
This was obtained following the procedure described for 18 start-
ing from 28. The residue was purified by column chromatography.

A. Carrieri et al. / Bioorg. Med. Chem. 18 (2010) 7065–7077
7075
Eluting with CHCl3/EtOH (9.8:0.2) afforded the free base as a solid:
80% yield; mp 40–41 °C. 1H NMR (CDCl3): d 1.31 (br s, 1, NH,
exchangeable with D2O), 3.08 (m, 4, CH2NCH2), 4.02 and 4.15
(two t, 4, OCH2 and CH2O), 5.09 (s, 2, OCH2Ar), 6.89–7.45 (m, 14,
ArH); MS (ESI) m/z 364.1 ([M+H]+), 386.1 ([M+Na]+). The free base
was transformed into the oxalate salt and recrystallized from
EtOH; mp 195–196 °C. Anal. Calcd for C23H25NO3ꢁC2H2O4ꢁ0.5H2O:
C, 64.92; H, 6.10; N, 3.03. Found: C, 65.17; H, 6.47; N, 2.93.
4.2.1.1. Rat vas deferens prostatic portion. This tissue was used
to assess
1A-adrenergic antagonism.32 Prostatic portions of 2 cm
length were mounted under 0.5 g of tension in Tyrode solution of
the following composition (mM): NaCl, 130.0; KCl, 2.0; CaCl2,
1.8; MgCl2, 0.89; NaHCO3, 25.0; NaH2PO4, 0.42; glucose, 5.6. Co-
a
caine hydrochloride (0.1 lM) was added to the Tyrode to prevent
the neuronal uptake of (ꢀ)-noradrenaline. The preparations were
equilibrated for 60 min with washing every 15 min. After the
equilibration period, tissues were primed two times by addition
4.1.32. 2,6-Bis(methoxymethoxy)phenol (33)
of 10
l
M (ꢀ)-noradrenaline. After another washing and equilibra-
1.6 M n-Butillitium (14 mL) was added to an ice-cooled solution
of 1,3-bis(methoxymethoxy)benzene28 (3.16 g, 15.21 mmol) in dry
THF (50 mL) under nitrogen. After stirring for 1 h at rt, the solution
was cooled to 0 °C and trimethyl borate (1.52 g, 25.51 mmol) was
added. After 30 min under stirring the solution was concentrated
under vacuum and the crude residue was dissolved in 20% aqueous
acetone (54 mL) containing NaHCO3 (15.14 g). Oxone (3.8 g) was
added and stirring continued. After 5 min NaHSO3 (2.09 g) was
added. The reaction mixture was extracted with EtOAc and dried
over Na2SO4. Evaporation of the solvent gave an oil, which was
purified by column chromatography. Eluting with cyclohexane/
EtOAc (9:1) afforded 33 as an oil: 1.11 g; 33% yield. 1H NMR
(CDCl3): d 3.52 (s, 6, OCH3), 5.20 (s, 4, OCH2), 6.63–6.84 (m, 4, OH
and ArH); MS (ESI) m/z 213.0 ([MꢀH]ꢀ).
tion period of 60 min, a (ꢀ)-noradrenaline concentration-response
curve was constructed (basal response). The antagonist was al-
lowed to equilibrate with the tissue for 30 min, followed by
30 min of washing. Then, a new concentration-response curve to
the agonist was obtained. (ꢀ)-Noradrenaline solutions contained
0.05% Na2S2O5 to prevent oxidation.
4.2.1.2. Rat spleen. This tissue was used to assess a1B-adrenergic
antagonism.34 The spleen was removed and bisected longitudinally
into two strips, which were suspended in tissue baths containing
Krebs solution of the following composition (mM): NaCl, 120.0;
KCl, 4.7; CaCl2, 2.5; MgSO4, 1.5; NaHCO3, 20.0; KH2PO4, 1.2; glu-
cose, 11.0; K2ethylenediaminetetraacetic acid (EDTA), 0.01. ( )-
Propranolol hydrochloride (4.0 lM) was added to block b-ARs.
The spleen strips were placed under 1 g of resting tension and
equilibrated for 2 h. The cumulative concentration-response curves
to (ꢀ)-phenylephrine were measured isometrically and obtained at
30 min intervals; the first one was discarded, and the second one
was taken as the control. The antagonist was allowed to equilibrate
with the tissue for 30 min, followed by 30 min of washing. Then, a
new concentration-response curve to the agonist was constructed.
4.1.33. Methyl 2-(2,6-bis(methoxymethoxy)phenoxy)acetate
(34)
A mixture of 33 (1.11 g, 5.18 mmol) methyl 2-chloroacetate
(0.53 mL, 6.06 mmol), K2CO3 (0.84 g, 6.06 mmol) and KI (0.106 g)
in dry acetone (40 mL) was refluxed for 13 h. After cooling the solid
was filtered and the filtrate was evaporated. The residue was dis-
solved in EtOAc, was washed with 2 N NaOH and the organic layer
was dried over Na2SO4. After evaporation of the solvent the residue
was purified by column chromatography. Eluting with cyclohex-
ane/EtOAc (9:1) afforded an oil: 1.1 g; yield = 74%. 1H NMR (CDCl3):
d 3.52 (s, 6, OCH3), 3.80 (s, 3, COOCH3), 4.66 (s, 2, CH2CO), 5.20 (s, 4,
OCH2O), 6.81–7.02 (m, 3, ArH); MS (EI) m/z 286 [M+], 254, 227, 213,
121, 62, 45 (100), 39.
4.2.1.3. Rat aorta. This tissue was used to assess a1D-adrenergic
antagonism.33 Thoracic aorta was cleaned from extraneous con-
nective tissue and placed in Krebs solution of the following compo-
sition (mM): NaCl, 118.4; KCl, 4.7; CaCl2, 1.9; MgSO4, 1.2; NaHCO3,
25.0; NaH2PO4, 1.2; glucose, 11.7. Cocaine hydrochloride (0.1
lM)
and ( )-propranolol hydrochloride (4.0 M) were added to prevent
l
the neuronal uptake of (ꢀ)-noradrenaline and to block b-ARs,
respectively. Two helicoidal strips (15 ꢃ 3 mm) were cut from each
aorta beginning from the end most proximal to the heart. The
endothelium was removed by rubbing with filter paper; the ab-
4.1.34. 2-(2,6-Bis(methoxymethoxy)phenoxy)acetic acid (35)
A mixture of 34 (1.0 g; 3.49 mmol) and 2 N NaOH (40 mL) was
stirred at 70 °C for 3 h. After cooling to 0 °C, 2 N HCl was added till
pH 7 followed by NH4Cl saturated solution till pH 4–5. Extraction
with CHCl3 followed by washing, drying, and evaporation of the ex-
tracts gave an oil: 0.85 g; 81% yield. 1H NMR (CDCl3): d 3.52 (s, 6,
OCH3), 4.66 (s, 2, CH2CO), 5.25 (s, 4, OCH2O), 6.87 (d, 2, ArH),
7.02 (t, 1, ArH), 7.65 (br s, 1, COOH, exchangeable with D2O); MS
(ESI) m/z 271.0 ([MꢀH]ꢀ).
sence of acetylcholine (100
tions contracted with (ꢀ)-noradrenaline (1
l
M)-induced relaxation to prepara-
M) was taken as an
l
indicator that the vessel was denuded successfully. Vascular strips
were then tied with surgical thread and suspended in a jacketed
tissue bath containing Tyrode solution. Strips were secured at
one end to Plexiglas hooks and connected to a transducer for mon-
itoring changes in isometric contraction. After at least 2 h equili-
bration period under an optimal tension of 2 g, cumulative (ꢀ)-
noradrenaline concentration-response curves were recorded at
1 h intervals; the first two were discarded, and the third one was
taken as the control. The antagonist was allowed to equilibrate
with the tissue for 30 min before the generation of the fourth
cumulative concentration-response curve to (ꢀ)-noradrenaline.
(ꢀ)-Noradrenaline solutions contained 0.05% K2EDTA and 0.9%
NaCl to prevent oxidation.
4.2. Pharmacology
4.2.1. Functional antagonism in isolated tissues
Male Wistar rats (275–300 g) were killed by cervical disloca-
tion, and the organs required were isolated, freed from adhering
connective tissue, and set up rapidly under a suitable resting ten-
sion in 20 mL organ baths containing physiological salt solution
kept at 37 °C and aerated with 5% CO2:95% O2 at pH 7.4. Concentra-
tion-response curves were constructed by cumulative addition of
agonist. The concentration of agonist in the organ bath was in-
creased approximately threefold at each step, with each addition
being made only after the response to the previous addition had at-
tained a maximal level and remained steady. Contractions were re-
corded by means of a force displacement transducer connected to
the MacLab system PowerLab/800. In addition, parallel experi-
ments in which tissues did not receive any antagonist were run
in order to check any variation in sensitivity.
4.2.2. Binding studies
4.2.2.1. Radioligand binding assays. Binding to cloned human
a
1-AR subtypes was performed in membranes from CHO cells
transfected by electroporation with DNA expressing the gene
encoding each 1-AR subtype. Cloning and stable expression of
the human
1-AR gene was performed as previously described.29
CHO cell membranes (30 g proteins) were incubated in 50 mM
a
a
l

7076
A. Carrieri et al. / Bioorg. Med. Chem. 18 (2010) 7065–7077
Tris–HCl, pH 7.4, with 0.1–0.4 nM [3H]prazosin, in a final volume of
1.02 mL for 30 min at 25 °C, in absence or presence of competing
membrane/water model.51 With respect to the physical parame-
ters representing the membrane, the surface tension coefficient
(representing the non-polar solvation energy) was set to
0.03 kcal/(mol.A2). The membrane thickness centered at Z = 0
was set to 30.0 Å with a membrane smoothing length of 5.0 Å
(wm = 2.5 Å). Minimizations were carried out by using 1500 steps
of steepest descent followed by Adopted Basis Newton-Raphson
(ABNR) minimization, until the root mean square gradient was less
than 0.001 kcal/mol Å. Docking was first carried out with Flo+ suite
drugs (1 pM–10
lM). Nonspecific binding was determined in the
presence of 10
l
M phentolamine. The incubation was stopped by
addition of ice-cold Tris–HCl buffer and rapid filtration through
0.2% polyethyleneimine-pretreated Whatman GF/B or Schleicher
& Schuell GF52 filters. Genomic clone G-21 coding for the human
5-HT1A receptor was stably transfected in a human cell line
(HeLa).30 HeLa cells were grown as monolayers in Dulbecco’s mod-
ified Eagle’s medium (DMEM), supplemented with 10% fetal calf
of programs.49 The
a1d-AR and WB 4101 receptor complex was first
serum and gentamicin (100
l
g/mL), and 5% CO2 at 37 °C. Cells were
submitted to 100 steps of flexible dynamic docking (DYNDOCK),
with each step 300 ps long. The residues within 15 Å from the neg-
atively charged oxygen atom of Asp176 were restrained in their
original position, and a harmonic distance constraint between
the same oxygen and the positively charged nitrogen atom of WB
4101 was imposed. In each run the temperature was cooled down
from 600 to 300 K and the receptor structure then quenched. The
unique conformations, according to a RMSD P0.5 calculated on
detached from the growth flask at 95% confluence by a cell scraper
and were lysed in ice-cold Tris 5 mM and EDTA 5 mM buffer (pH
7.4). Homogenates were centrifuged at 40,000g for 20 min, and
pellets were resuspended in a small volume of ice-cold Tris
5 mM and EDTA 5 mM buffer (pH 7.4) and immediately frozen
and stored at ꢀ70 °C until use. On the experimental day, cell mem-
branes were resuspended in binding buffer: 50 mM Tris–HCl (pH
7.4), 2.5 mM MgCl2, and 10
l
M pargiline.31 Membranes were incu-
the Ca atom trace, were stored and the best energy solution se-
bated in a final volume of 1 mL for 30 min at 30 °C with 0.7–1.4 nM
lected. Compounds 1–14 were further docked into the binding site
carrying out fifty thousand runs of stochastic Monte Carlo docking
(MCDOCK) with the whole binding kept fixed. The energy window
for the selected agonists poses did not exceeded 3.3 kcal/mol above
the best energy solution. GRINDs were calculated in GRID using the
DRY, O, N1, and TIP atom probes to give four auto-correlograms
and six cross-correlograms. PCA and PLS analyses were performed
using the ALMOND software (version 3.3.0) using default settings.
No initial scaling was applied to the original X-variable matrix
which was subsequently filtered by three Fractional Factorial De-
sign runs. Cross-validation was performed using the leave one
out (LOO) procedure to select the optimal number of components
in the PLS model equal to a significant increase of the cross-valida-
tion coefficient q2 (at least 5%).
[3H]8-OH-DPAT, in the absence or presence of competing drugs.
Nonspecific binding was determined in the presence of 10 lM 5-
HT. The incubation was stopped by the addition of ice-cold Tris–
HCl buffer and rapid filtration through 0.2% polyethyleneimine-
pretreated Whatman GF/B or Schleicher & Schuell GF52 filters.
4.2.3. Data analysis
In functional studies, responses were expressed as percentage
of the maximal contraction observed in the agonist concentra-
tion-response curve taken as a control. The agonist concentra-
tion-response curves were analyzed by pharmacological
computer programs. pKb values were calculated from only one con-
centration. Compounds 2–14 were tested at only one concentra-
tion, in the range of 0.01–10 lM, when determining a1-AR
blocking activity. pKb values were calculated according to van
Acknowledgments
Rossum.46
Binding data were analyzed using the nonlinear curve-fitting
program Allfit.47 Scatchard plots were linear in all preparations.
All pseudo-Hill coefficients (nH) were not significantly different
from unity (p >0.05). Equilibrium inhibition constants (Ki) were de-
rived from the Cheng–Prusoff48 Equation: Ki = IC50/(1 + L/Kd),
where L and Kd are the concentration and the equilibrium dissoci-
ation constant of the radioligand. pKi values are the mean SE of
2–3 separate experiments performed in triplicate.
We thank the MIUR (Rome) and the University of Bari and Cam-
erino for financial support. This study was also supported by Tele-
thon-Italy (S00068TELU to F.F.).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.08.002.
4.3. Modeling study
References and notes
In the 2D-QSAR statistical analysis was performed relating pKi
with the fifty QikProp molecular descriptors by multiple linear
regression, automatically selecting the optimal subset of variables
and removing any outlier. A ratio of one variable per eight com-
pounds was applied. Molecular structures of the protonated com-
pounds 1–14 were built using the Flo+ suite of software49 with
standard bond distances and valence angles, while for WB 4101
the X-ray structure of the (S)-enantiomer was used in calculations
1. For part 9: Quaglia, W.; Piergentili, A.; Del Bello, F.; Farande, Y.; Giannella, M.;
Pigini, M.; Rafaiani, G.; Carrieri, A.; Amantini, C.; Lucciarini, R.; Santoni, G.;
Poggesi, E.; Leonardi, A. J. Med. Chem. 2008, 51, 6359.
2. http://www.pdb.org.
3. White, S. H. Nature 2009, 459, 344.
4. Palczewski, K.; Kumasaka, T.; Hori, T.; Behnke, C. A.; Motoshima, H.; Fox, B. A.;
Le Trong, I.; Teller, D. C.; Okada, T.; Stenkamp, R. E.; Yamamoto, M.; Miyano, M.
Science 2000, 289, 739.
5. Warne, T.; Serrano-Vega, M. J.; Baker, J. G.; Moukhametzianov, R.; Edwards, P.
C.; Henderson, R.; Leslie, A. G. W.; Tate, C. G.; Schertler, G. F. X. Nature 2008,
454, 486.
6. Cherezov, V.; Rosenbaum, D. M.; Hanson, M. A.; Rasmussen, S. G. F.; Thian, F. S.;
Kobilka, T. S.; Choi, H.-J.; Kuhn, P.; Weis, W. I.; Kobilka, B. K.; Stevens, R. C.
Science 2007, 318, 1258.
7. Jaakola, V.-P.; Griffith, M. T.; Hanson, M. A.; Cherezov, V.; Chien, E. Y. T.; Lane, J.
R.; Ijzerman, A. P.; Stevens, R. C. Science 2008, 322, 1211.
8. Fanelli, F.; De Benedetti, P. In Antitargets; Vaz, R. J., Klabunde, T., Eds.; Wiley-
VCH, 2008; p 155.
9. Zhong, H.; Minneman, K. P. Eur. J. Pharmacol. 1999, 375, 261.
10. Price, D. T.; Lefkowitz, R. J.; Caron, M. G.; Berkowitz, D.; Schwinn, D. A. Mol.
Pharmacol. 1994, 45, 171.
11. Michel, M. C. Eur. Urol. Suppl. 2002, 1, 5.
12. Roehrborn, C. G.; Schwinn, D. A. J. Urol. 2004, 171, 1029.
13. Romics, I. Neurochem. Int. 2007, 51, 328.
(CSD code: BAXBEH). The a1d-AR model was built by comparative
modeling (by means of MODELLER 7v7)50 by using the crystal
structure of b2-AR as a template. All the receptor domains but N-
term, third intracellular loop and C-term were modeled (i.e. the
92–419 sequence). A modified b2-AR template was used lacking
the amino acid segments 176–190 and 263–265. External a-helical
restraints were imposed to the 415–419 sequence. Moreover, a
disulphide bridge patch was employed between C169 (3.25) and
C244. The best a1d-AR model was subjected to rotations of the ami-
no acid side chains when in non allowed conformations and then
subjected to energy minimization, by using the GBSW implicit

A. Carrieri et al. / Bioorg. Med. Chem. 18 (2010) 7065–7077
7077
14. Chiu, G.; Li, S.; Connolly, P. J.; Pulito, V.; Liu, J.; Middleton, S. A. Bioorg. Med.
Chem. Lett. 2007, 17, 3930.
31. Fargin, A.; Raymond, J. R.; Lohse, M. J.; Kobilka, B. K.; Caron, M. G.; Lefkowitz, R.
J. Nature 1988, 335, 358.
15. Drouin, C.; Darracq, L.; Trovero, F.; Blanc, G.; Glowinski, J.; Cotecchia, S.; Tassin,
J. P. J. Neurosci. 2002, 22, 2873.
32. Eltze, M.; Boer, R.; Sanders, K. H.; Kolassa, N. Eur. J. Pharmacol. 1991, 202, 33.
33. Ko, F. N.; Guh, J. H.; Yu, S. M.; Hou, Y. S.; Wu, Y. C.; Teng, C. M. Br. J. Pharmacol.
1994, 112, 1174.
34. Buckner, S. A.; Oheim, K. W.; Morse, P. A.; Knepper, S. M.; Hancock, A. A. Eur. J.
Pharmacol. 1996, 297, 241.
35. Scapecchi, S.; Nesi, M.; Matucci, R.; Bellucci, C.; Buccioni, M.; Dei, S.;
Guandalini, L.; Manetti, D.; Martini, E.; Marucci, G.; Romanelli, M. N.;
Teodori, E.; Cirilli, R. J. Med. Chem. 2008, 51, 3905, and references therein.
36. QikProp, version 3.3, Schrödinger, LLC, New York, NY, 2010.
37. Fumagalli, L.; Bolchi, C.; Colleoni, S.; Gobbi, M.; Moroni, B.; Pallavicini, M.;
Pedretti, A.; Villa, L.; Vistoli, G.; Valoti, E. Bioorg. Med. Chem. 2005, 13, 2547.
38. Strike, version 1.9, Schrödinger, LLC, New York, NY, 2010.
39. Quaglia, W.; Pigini, M.; Piergentili, A.; Giannella, M.; Gentili, F.; Marucci, G.;
Carrieri, A.; Carotti, A.; Poggesi, E.; Leonardi, A.; Melchiorre, C. J. Med. Chem.
2002, 45, 1633.
16. McVary, K. T.; McKenna, K. E.; Lee, C. Prostate Suppl. 1998, 8, 2.
17. Quaglia, W.; Santoni, G.; Pigini, M.; Piergentili, A.; Gentili, F.; Buccioni, M.;
Mosca, M.; Lucciarini, R.; Amantini, C.; Nabissi, M. I.; Ballarini, P.; Poggesi, E.;
Leonardi, A.; Giannella, M. J. Med. Chem. 2005, 48, 7750.
18. Ballesteros, A.; Weinstein, H. Methods Neurosci. 1995, 25, 366.
19. Waugh, D. J. J.; Gaivin, R. J.; Zuscik, M. J.; Gonzalez-Cabrera, P.; Ross, S. A.; Yun,
J.; Perez, D. M. J. Biol. Chem. 2001, 276, 25366.
20. Rosini, M.; Bolognesi, M. L.; Giardinà, D.; Minarini, A.; Tumiatti, V.; Melchiorre,
C. Curr. Top. Med. Chem. 2007, 7, 147.
21. Quaglia, W.; Pigini, M.; Piergentili, A.; Giannella, M.; Marucci, G.; Poggesi, E.;
Leonardi, A.; Melchiorre, C. J. Med. Chem. 1999, 42, 2961.
22. Wada, M.; Natsume, S.; Suzuki, S.; Uo, A.; Nakamura, M.; Hayase, S.; Erabi, T. J.
Organomet. Chem. 1997, 548, 223.
23. Gardner, P. D.; Horton, W. J.; Pincock, R. E. J. Am. Chem. Soc. 1956, 78, 2541, and
reference therein.
40. Ballesteros, J. A.; Jensen, A. D.; Liapakis, G.; Rasmussen, S. G. F.; Shi, L.; Gether,
U.; Javitch, J. A. J. Biol. Chem. 2001, 276, 29171.
24. Pallavicini, M.; Fumagalli, L.; Gobbi, M.; Bolchi, C.; Colleoni, S.; Moroni, B.;
Pedretti, A.; Rusconi, C.; Vistoli, G.; Valoti, E. Eur. J. Med. Chem. 2006, 41,
1025.
25. Bestmann, H. J.; Schulz, H.; Kunstmann, R.; Rostock, K. Chem. Ber. 1966, 99,
1906.
26. Berti, G.; Saettone, M. F. Il Farmaco Ed Sci. 1960, 15, 431.
27. Chen, X.; Green, B. E.; Kempf, D. J.; Li, L.; Norbeck, D. W.; Sham, H. L. Patent US
5905068 A, 1999; Chem. Abstr. 1999, 130, 352501.
41. Shi, L.; Liapakis, G.; Xu, R.; Guarnieri, F.; Ballesteros, J. A.; Javitch, J. A. J. Biol.
Chem. 2002, 277, 40989.
42. Greasley, P. J.; Fanelli, F.; Rossier, O.; Abuin, L.; Cotecchia, S. Mol. Pharmacol. 2002,
61, 1025; reviewed in Fanelli, F.; De Benedetti, P. G. Chem. Rev. 2005, 105, 3297.
43. Topiol, S.; Sabio, M. Biochem. Pharmacol. 2009, 78, 11, and references therein.
44. Zhao, M. M.; Hwa, J.; Perez, D. M. Mol. Pharmacol. 1996, 50, 1118.
45. Pastor, M.; Cruciani, G.; McLay, I.; Pickett, S.; Clementi, S. J. Med. Chem. 2000,
43, 3233.
28. Donnelly, A. C.; Mays, J. R.; Burlison, J. A.; Nelson, J. T.; Vielhauer, G.;
Holzbeierlein, J.; Blagg, B. S. J. J. Org. Chem. 2008, 73, 8901.
29. 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. Pharmacol. Commun. 1995, 6, 79.
46. van Rossum, J. M. Arch. Int. Pharmacodyn. Ther. 1963, 143, 299.
47. De Lean, A.; Munson, P. J.; Rodbard, D. Am. J. Physiol. 1978, 235, E97.
48. Cheng, Y. C.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
49. McMartin, C.; Bohacek, R. S. J. Comput. Aided Mol. Des. 1997, 11, 333.
50. Sali, A.; Blundell, T. L. J. Mol. Biol. 1993, 234, 779.
30. Fargin, A.; Raymond, J. R.; Regan, J. W.; Cotecchia, S.; Lefkowitz, R. J.; Caron, M.
G. J. Biol. Chem. 1989, 264, 14848.
51. Im, W.; Feig, M.; Brooks, C. L., III Biophys. J. 2003, 85, 2900.