Affinity and activity profiling of unichiral 8-substituted 1,4-benzodioxane analogues of WB4101 reveals a potent and selective α1B- adrenoceptor antagonist

Article abstract of DOI:10.1016/j.ejmech.2012.09.049

Unichiral 8-substituted analogues of 2-[(2-(2,6-dimethoxyphenoxy)ethyl) aminomethyl]-1,4-benzodioxane (WB4101) were synthesized and tested for binding affinity at cloned human α_1a-, α_1b-and α_1d-adrenoreceptor (α_1a

Full text of DOI:10.1016/j.ejmech.2012.09.049

European Journal of Medicinal Chemistry 58 (2012) 184e191
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Original article
Affinity and activity profiling of unichiral 8-substituted 1,4-benzodioxane
analogues of WB4101 reveals a potent and selective a1B-adrenoceptor antagonist
Laura Fumagalli a, Marco Pallavicini a, Roberta Budriesi b, Marco Gobbi c, Valentina Straniero a,
Michael Zagami a, Giuseppe Chiodini a, Cristiano Bolchi a, Alberto Chiarini b, Matteo Micucci b,
,
Ermanno Valoti a
*
a Dipartimento di Scienze Farmaceutiche, Università di Milano, via Mangiagalli 25, I-20133 Milano, Italy
b Dipartimento di Scienze Farmaceutiche, Università di Bologna, via Belmeloro 6, I-40126 Bologna, Italy
c Istituto di Ricerche Farmacologiche ‘Mario Negri’, via La Masa 19, I-20156 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 June 2012
Received in revised form
27 September 2012
Accepted 28 September 2012
Available online 16 October 2012
Unichiral
8-substituted
analogues
of
2-[(2-(2,6-dimethoxyphenoxy)ethyl)aminomethyl]-1,4-
benzodioxane (WB4101) were synthesized and tested for binding affinity at cloned human a1a-, a1b-and
a1d-adrenoreceptor (a1a-, a1b-and a1d-AR) and at native rat 5-HT1A receptor and for antagonist affinity at
a1A-, a1B-and a1D-AR and at a2A/D-AR. Among the selected 8-substituents, namely fluorine, chlorine,
methoxyl and hydroxyl, only the last caused significant decrease of a1 binding affinity in comparison with
the lead compound. Functional tests on the S isomers confirmed the detrimental effect of OH positioned in
proximity to benzodioxane O(1). For the other three substituents (F, Cl, OMe), the a1A and the a1D antagonist
affinities were generally lower than the a1a and a1d binding affinities, but not the a1B antagonist affinity,
which was similar and sensibly higher compared to a1b binding affinity in the case of F and OMe respec-
tively. This trend confers significant a1B-AR selectivity, in particular, to the 8-methoxy analogue of (S)-
WB4101, a new potent (pA2 9.58) a1B-AR antagonist. The S enantiomers of all the tested compounds were
proved to act as a1-AR inverse agonists in a vascular model.
Keywords:
WB4101 analogues
8-Substituted 1,4-benzodioxane
a1-Adrenoreceptor
5-HT1A receptor
Binding affinity
Antagonist affinity
Inverse agonist
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
The S enantiomer of 2-(2,6-dimethoxyphenoxyethyl)amino-
methyl-1,4-benzodioxane (WB4101) is a potent a1-adrenoreceptor
The adrenergic receptors (ARs) have been extensively investi-
gated and their functions have been progressively elucidated. They
(a1-AR) antagonist, which exhibits, in binding experiments, high
and similar affinity at a1A-and a1D-AR and one order of magnitude
lower affinity for the a1B subtype [15]. In functional studies on
animal tissues, S-WB4101 shows subnanomolar antagonist affinity
at all the three a1-AR subtypes with a slight selectivity for the
a1A-AR [16]. In order to improve potency and subtype selectivity,
many a1-AR ligands structurally related to WB4101 have been
designed by making modifications at the benzodioxane nucleus, at
the amino function and at the (2,6-dimethoxyphenoxy)ethyl
moiety [7]. In particular, our research group has long been involved
in characterizing o-monosubstituted and o-disubstituted phenoxy
analogues as well as naphthoxy, naphthodioxane and tetrahy-
dronaphthodioxane analogues [15e18]. The most notable result of
such researches was the finding of two potent and selective a1D-AR
and a1A-AR antagonists, the former by replacement of the
2,6-dimethoxyphenoxy moiety of S-WB4101 with 2-methoxy-1-
naphthoxy and the latter by the same modification coupled with
substitution of benzodioxane by tetrahydronaphthodioxane [16]. In
are classified into a1, a2, b1, b2 and b3 [1e3]. The three a1 subtypes,
namely a1A a1B and a1D, exhibit distinct pharmacology and tissue
,
expression and subtype selective a1 antagonists have a therapeutic
potential in the treatment of several pathologies [4e7]. Indeed,
selective a1A and a1D antagonists are used in the therapy of diseases
such as hypertension and lower urinary tract symptoms (LUTS)
secondary to benign prostatic hyperplasia (BPH) [8e11], but also
selective a1B antagonists have been taken into account as thera-
peutic agents and pharmacological tools [12,13]. In particular, the
function of a1B-AR in CNS has been recently reinvestigated [13] and
this receptor subtype has been indicated as target for novel drugs
useful in the treatment of CNS disorders [14].
* Corresponding author. Tel.: þ39 2 50319334; fax: þ39 2 50319335.
E-mail address: ermanno.valoti@unimi.it (E. Valoti).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.049

L. Fumagalli et al. / European Journal of Medicinal Chemistry 58 (2012) 184e191
185
this class of a1-AR antagonists, however, modifications and
2. Chemistry
substitutions at the aromatic ring of WB4101 benzodioxane have
received little attention. In fact, while a large variety of WB4101
analogues modified or substituted at the dioxane have been
studied, only the 8-methylbenzodioxane and the three isomeric
naphthodioxane analogues have been reported by other research
groups and only as racemates [19,20]. Apart from the not ready
synthetic accessibility of the derivatives substituted at the aromatic
ring of benzodioxane, such a gap is rather surprising since a key role
has been attributed to benzodioxane in the interaction with a1-ARs
due to its overall steric and electronic features and its modification
has been postulated to be critical, in particular, for a1A-AR subtype
selectivity [21,22]. That’s why we decided to study the effects of the
insertion of a substituent at the aromatic ring of benzodioxane and
designed the WB4101 analogues 1e5 (Fig. 1). In particular, we
focused on the substitution at the 8-position of benzodioxane,
namely in proximity to dioxane O(1), which seems to exert a great
influence on the conformational properties of the ligand, due to
intramolecular interactions with ammonium protons [23], and to
play a peculiar and critical role in receptor binding, as suggested by
the fact that its replacement, in WB4101 or its analogues, with
a methylene, a methine or a hydroxymethine, but not with
a carbonyl, is deleterious [24e26]. As a first step, we considered
substitution with hydroxyl (1), a both HBA and HBD group, able to
interfere with the interaction potentiality of O(1) by giving rise to
hydrogen bond with it or to contribute itself to binding, if, as
reasonable, the counterpart of O(1) is a receptor polar pocket. The
8-methoxy analogue (2) was successively chosen as a necessary
term for comparison with the 8-hydroxy analogue 1. Besides the
8-methoxy substitution, for reasons discussed hereinafter, we
considered also the 5-methoxy substitution (5). The choice of
a substituent such as chlorine (3) was dictated by its lipophilic
character, opposite to OH, and by the steric and electronic effects
that it can exert on O(1) when positioned at benzodioxane C(8).
Lastly, we considered the introduction of fluorine (4), which has
a large electronic effect due to high electronegativity and a weak
HBA character, but with low additional steric demand in
comparison with hydrogen.
(S)-and (R)-WB4101, necessary in binding and functional tests as
reference compounds, are accessible, as previously reported, from
different unichiral synthons, such as (S)-isopropylidene glycerol
[23], (R)-and (S)-1,4-benzodioxane-2-carboxylic acid [28e30] and
the respective methyl esters [28,31]. The enantiomers of compound
1 were prepared from (R)-and (S)-2-tosyloxymethyl-8-hydroxy-
1,4-benzodioxane, whose synthesis we had previously described
[27,32], by conversion into iodomethyl derivatives and amination
with 2-(2,6-dimethoxyphenoxy)ethylamine. The enantiomers of
compound 3 were synthesized by the same strategy, namely
reacting 2-(2,6-dimethoxyphenoxy)ethylamine with enantiopure
2-mesyloxymethyl-8-chloro-1,4-benzodioxane
15,
in
turn
prepared by using (S)- or (R)-glycerol acetonide and 2-benzyloxy-
3-chlorophenol 10 as building blocks. The synthesis of (S)-3 is
illustrated in Scheme 1. The 2,3-disubstituted phenol 10 was
obtained from 2-chlorophenol by (a) esterification with acetyl
chloride, (b) Fries rearrangement, (c) benzylation of the resulting
o-acetylphenol 7, (d) Baeyer-Villiger oxidation to 2-benzyloxy-3-
chlorophenyl acetate 9 and (e) methanolysis of the acetate 9.
Displacement of mesylate from the mesyl ester of (S)-glycerol
acetonide by 2-benzyloxy-3-chlorophenoxide and subsequent
hydrolysis of the cyclic ketal provided the phenoxypropanediol
(R)-12, which was dimesylated and then debenzylated. Intra-
molecular nucleophilic substitution of the mesylate at C(2) of the
glycerol skeleton by the ortho-phenoxy moiety formed the
1,4-dioxane ring giving (R)-2-mesyloxymethyl-8-chloro-1,4-
benzodioxane (R)-15, which was reacted with 2-(2,6-
dimethoxyphenoxy)ethylamine to yield (S)-3. The R enantiomer
of 3 was synthesized by the same route as illustrated in Scheme 1
but starting from the mesyl ester of (R)-glycerol acetonide.
3. Results and discussion
Table 1 reports the affinities, expressed as pKi values (ꢀlogKi, M),
at the three cloned human a1-AR subtypes and native 5-HT1A
serotoninergic receptor from rat hippocampus of the enantiomeric
pairs of compounds 1e5 and, as comparison terms, of (S)-and
(R)-WB4101.
The affinities of the S enantiomers are always higher than those
of their antipodes, the eudismic index being generally higher than 1
and, only in the case of some modest affinities of the enantiomers
of 5, significantly lower than 1. Compared to (S)-WB4101, the S
enantiomers of the 8-OMe, 8-Cl and 8-F analogues have quite
similar affinity profiles showing a1a and a1d affinities close to those
of (S)-WB4101 (pKi 9.39 and 9.29, respectively). For these three
compounds as for (S)-WB4101, the a1a and a1d affinities are always
higher than the a1b affinity, but the difference is less pronounced.
We have previously reported the synthesis of 2, 4 and 5 in both
the enantiomeric forms [27]. Herein, we describe the synthesis of
the S and R stereoisomers of the 8-hydroxy and 8-chloro benzo-
dioxane analogues,1 and 3 respectively, and the biological profile of
all the five enantiomeric pairs 1e5 in binding tests at a1a-, a1b-and
a1d-ARs and the 5-HT1A receptor and, for the S enantiomers, in
functional experiments at a1A-, a1B-and a1D-and a2-ARs. The SAFIR
and SAR data of 1e5 are discussed as well as their nature of inverse
agonists, which was studied and semi-quantitatively determined
by means of a vascular model.
Therefore, they are a little less a1a/a1b and a1d/a1b selective than
X
OMe
(S)-WB4101. A different behaviour is shown by (S)-1, the 8-hydroxy
analogue, for which significant affinity decreases at all the three a1
subtypes were registered in comparison with (S)-WB4101 and with
the other 8-substituted derivatives. Concerning the 5-HT1A
receptor, the S enantiomers of all the four 8-substituted analogues
display affinity in the nanomolar range, slightly higher than
(S)-WB4101. The 5-methoxy analogue, (S)-5, radically differs from
the 8-substituted derivatives, its affinities at the a1-AR subtypes
and 5-HT1A serotoninergic receptor being from one to two orders of
magnitude lower.
The functional assays were carried out only for the S enantio-
mers. Their antagonist affinities at a1-AR subtypes and a2-AR,
expressed as pA2, are listed in Table 2.
a1-AR subtypes and a2-AR blocking activity was determined on
different rat tissues. In particular, a1-AR subtypes blocking activity
+
O
O
O
*
N
H H
Cl
MeO
-
Y
WB4101 X = H
Y = H
1
2
3
4
5
OH
OMe
Cl
F
H
H
H
H
H
OMe
Fig. 1. 8-and 5-substituted analogues of WB4101.

186
L. Fumagalli et al. / European Journal of Medicinal Chemistry 58 (2012) 184e191
O
OH
O
OBn O
OH
O
Cl
Cl
Cl
Cl
Cl
b
d
a
c
6
7
8
OBn
OBn
OBn
O
O
O
O
Cl
Cl
OH
Cl
O
f
e
g
9
10
(S)-11
OH
OMs
OBn
OH
OBn
OMs
OMs
OH
OMs
Cl
O
Cl
O
O
h
j
i
(R)-12
(S)-13
(S)-14
Cl
Cl
OMe
+
O
O
O
O
O
k
OMs
N
H
H
MeO
Cl-
(S)-3
(R)-15
Scheme 1. Reagents: (a) Acetyl chloride, pyridine, dichloromethane. (b) Aluminium chloride, dichlorobenzene. (c) Benzyl bromide, tetrabutylammonium hydroxide, 10% aqueous
NaOH, dichloromethane. (d) 3-Chloroperbenzoic acid, dichloromethane. (e) 2.5 N NaOH, methanol. (f) (R)-1-Mesiloxy-2,3-propanediol acetonide, potassium tert-butoxide, DMF. (g)
1 N HCl, ethanol. (h) Mesyl chloride, triethylamine, dichloromethane. (i) H2ePd/C, MeOH. (j) K2CO3, acetone. (k) 2-(2,6-Dimethoxyphenoxy)ethylamine, isobutanol; HCl/EtOH.
was assessed by antagonism of (ꢀ)-noradrenaline (NA)-induced
contraction of vas deferens prostatic portion (a1A) [33] and of
thoracic aorta (a1D) [34] and by antagonism to (ꢀ)phenylephrine-
induced contraction of spleen (a1B) [34], while a2-adrenoceptor
blocking activity was determined by antagonism to clonidine-
inhibited twitch responses of the field-stimulated prostatic
portion of vas deferens. Furthermore, considering the demon-
strated a1A-AR involvement in maintaining prostate smooth muscle
tone and the consequent therapeutic potential of agents reducing
the latter for LUTS, the antagonist affinity was also evaluated in rat
prostate [35]. Inspection of the results reported in Table 2 reveals
trends, which can be summarized as follows: (a) all the compounds
these three compounds, the 8-F and, even more markedly, the
8-OMe analogue show significant a1B selectivity, opposite to
(S)-WB4101. It was this last observation that induced us to consider
the introduction of methoxy substituent also at the 5 position of
benzodioxane and to include the 5-methoxy analogue in the
present investigation otherwise devoted only to 8-substituted
WB4101 analogues.
The analysis of the functional affinities suggests that the intro-
duction of a substituent at the 8-position of (S)-WB4101 benzo-
dioxane, unlike the introduction of a 5-substituent, is well tolerated
and relatively different steric and electronic effects, such as those of
fluorine, chlorine and methoxyl, are allowed to be exerted by the
8-substituent if not interfering, as in the case of hydroxyl, with the
interaction potential of O(1) or with its influence on the ligand
conformation. Such an explanation is substantiated by the high
antagonist affinities of the 8-OMe analogue (S)-2 compared to the
exhibit high a1/a2 selectivity; (b) the 8-OH and the 5-OMe
analogues are significantly less potent a1 antagonists than the
8-OMe, 8-Cl and 8-F analogues, which display from high to very
high antagonist affinities at all the three a1-AR subtypes; (c) among
Table 1
Experimental affinity constants, expressed as pKi (ꢀlogKi, M) ꢁ SE, of the enantiomers of compounds 1e5 and of WB4101 for cloned human a1-adrenoceptor
subtypes and 5-HT1A receptor.
Benzodioxane
substituent
Compound
pKi
a1a
a1b
a1d
5-HT1A
e
(S)-WB4101a
(R)-WB4101a
(S)-1
(R)-1
(S)-2
(R)-2
(S)-3
(R)-3
(S)-4
9.39 (ꢁ0.06)
7.95 (ꢁ0.04)
8.66 (ꢁ0.04)
7.18 (ꢁ0.04)
9.04 (ꢁ0.07)
7.78 (ꢁ0.04)
9.05 (ꢁ0.11)
8.25 (ꢁ0.02)
9.19 (ꢁ0.05)
7.74 (ꢁ0.04)
7.62 (ꢁ0.04)
6.09 (ꢁ0.07)
8.24 (ꢁ0.04)
7.14 (ꢁ0.06)
7.51 (ꢁ0.07)
6.40 (ꢁ0.05)
8.56 (ꢁ0.06)
7.10 (ꢁ0.08)
8.66 (ꢁ0.09)
7.47 (ꢁ0.05)
8.62 (ꢁ0.05
6.99 (ꢁ0.04)
6.35 (ꢁ0.05)
6.19 (ꢁ0.02)
9.29 (ꢁ0.11)
7.98 (ꢁ0.08)
8.05 (ꢁ0.06)
6.49 (ꢁ0.03)
9.43 (ꢁ0.09)
7.78 (ꢁ0.06)
9.15 (ꢁ0.17)
8.40 (ꢁ0.04)
9.31 (ꢁ0.05)
8.13 (ꢁ0.05)
7.10 (ꢁ0.09)
6.58 (ꢁ0.07)
8.61 (ꢁ0.04)
7.39 (ꢁ0.03)
9.00 (ꢁ0.03)
7.76 (ꢁ0.03)
9.07 (ꢁ0.03)
7.89 (ꢁ0.04)
8.77 (ꢁ0.07)
7.76 (ꢁ0.04)
8.85 (ꢁ0.02)
7.50 (ꢁ0.03)
7.54 (ꢁ0.05)
6.91 (ꢁ0.06)
8-OH
8-OMe
8-Cl
8-F
(R)-4
(S)-5
(R)-5
5-OMe
a
Data taken from Ref. [15].

L. Fumagalli et al. / European Journal of Medicinal Chemistry 58 (2012) 184e191
187
Table 2
S Enantiomers of WB4101 and of compounds 1e5: Antagonist affinities, expressed as pA2, at a1A-, a1B-, a1D-and a2-AR on isolated rat tissues and inverse agonism expressed as
magnitude of inhibition of calcium-induced increase in the resting tension (IRT) of calcium depleted guinea pig thoracic aorta.
a
Benzodioxane
substituent
Compd
pA2
Affinity ratiosb
Inhibition of
Caþþ induced
IRT (%)c
a1A
a1B
a1D
a2
a1A
/a1B
a1A
/a1D
a1D/a1B
Prostate
Prostatic vas
deferens
Spleen
Thoracic
aorta
Prostatic vas
deferens
e
(S)-WB4101
(S)-1
(S)-2
(S)-3
(S)-4
9.49 (ꢁ0.05)
7.30 (ꢁ0.01)
8.55 (ꢁ0.02)
9.04 (ꢁ0.05)
8.92 (ꢁ0.03)
7.40 (ꢁ0.02)
9.98 (ꢁ0.01)
6.78 (ꢁ0.07)
8.77 (ꢁ0.03)
9.56 (ꢁ0.01)
7.54 (ꢁ0.03)
7.27 (ꢁ0.02)
9.17 (ꢁ0.01)
7.80 (ꢁ0.02)
9.58 (ꢁ0.04)
8.05 (ꢁ0.04)
8.56 (ꢁ0.02)
7.78 (ꢁ0.04)
9.20 (ꢁ0.06)
7.29 (ꢁ0.09)
7.93 (ꢁ0.03)
8.52 (ꢁ0.02)
8.03 (ꢁ0.03)
8.24 (ꢁ0.02)
6.92 (ꢁ0.02)
<5
6.23 (ꢁ0.01)
6.02 (ꢁ0.01)
6.26 (ꢁ0.02)
5.67 (ꢁ0.01)
6.5
6.0
0.31
6.9
11.0
0.32
0.11
1.1
84
100
60
80
100
79
8-OH
8-OMe
8-Cl
8-F
5-OMe
0.10
0.15
32.4
0.10
0.31
0.31
0.02
3.0
0.30
2.9
(S)-5
a
pA2 values ꢁ SE were calculated from Schild plots [38], constrained to a slope of ꢀ1.0, unless otherwise specified [39]. pA2 is the positive value of the intercept of line
derived by plotting log(DR-1) vs log[antagonist]. The log(DR-1) was calculated at least at three different antagonist concentrations, and each was tested from three to five
times. Dose-ratio (DR) values represent the ratio of the potency of the agonist (EC50) in the presence of the antagonist and in its absence. Parallelism of dose-response curves
was checked by linear regression, and the slopes were tested for significance (p < 0.05).
b
Antilog of
D
pA2.
Data are the percent decreases of the Ca2þ (1.8 mM)-induced IRT in the presence of the antagonist at 10 nM concentration, calculated assuming as a reference IRT the Ca2þ
(1.8 mM)-induced IRT in the absence of any agent. Data represent the mean ꢁ SE from 4 to 7 experiments.
c
modest ones of (S)-1, whose 8-OH can give rise to hydrogen bond
with O(1).
other compounds not producing the maximum inhibition under the
adopted experimental conditions.
Matching of binding with functional tests indicates that only the
overall lower potencies of (S)-1 and (S)-5, compared to (S)-2-(S)-4,
were correctly predicted by the binding assays. Indeed, (S)-1 and
(S)-5 are both less affinitive and less potent than (S)-2, (S)-3 and
(S)-4. Apart from such a parallelism, the a1A-, a1B-and a1D-antag-
onist affinities of compounds (S)-1ꢀ(S)-5 significantly diverge, for
the most part, from the binding affinities at the corresponding
cloned AR subtypes being almost always lower at a1A-and a1D-AR
and higher at a1B-AR. Such a trend is particularly evident for the
8-OMe analogue (S)-2, whose subnanomolar a1B antagonist affinity
is one order of magnitude greater than the a1b binding affinity,
while its a1A and a1D antagonist affinities are significantly lower
than the a1a and a1d binding affinities.
As previously for WB4101 and some of its derivatives [16], the
discrepancy between binding and functional data prompted us to
verify if these antagonists behave as inverse agonists thus showing
affinity values not system-independent, but different according to
the relative proportion of receptor in the resting state and receptor
in the active state typical of the system employed for the determi-
nation. Inhibition of calcium induced increase in the resting tension
(IRT) of calcium depleted guinea pig thoracic aorta was assumed to
indicate inverse agonist behaviour [36], in particular at the a1A-AR
since the a1-AR subtype involved in the contraction of guinea pig
thoracic aorta is pharmacologically similar to a1A-AR of rat vas def-
erens. In Table 2, the inhibition of the IRT produced by 1.8 mM Caþþ
administered after incubation with 10 nM antagonist is expressed in
comparison to the IRT produced by the same concentration of Caþþ
in the absence of the antagonist. For instance, (S)-3 shows a 80%
inhibition, which means that 1.8 mM Caþþ induced IRT is, after
incubationwith 10 nM (S)-3, the 20% of the 1.8 mM Caþþ induced IRT
in the absence of the antagonist. In the case of (S)-1 and (S)-4 (100%
inhibition), 1.8 mM Caþþ doesn’t induce any IRT after incubation
with the antagonist at 10 nM. This semi-quantitative test indicates
that all the five compounds would be a1A-AR inverse agonists.
Ranking their potencies is difficult, because, at the tested concen-
tration (10 nM), four compounds, namely (S)-WB4101, (S)-2, (S)-3
and (S)-5, exert percentual IRT inhibitions, which are similar and
lower than 100%, while (S)-1 and (S)-4 give the maximum response
(100%). This notwithstanding, it is worthy of note that these two
maximum 100% inhibitions are associated with the two largest
negative divergences of a1A pA2 (vas deferens) from a1a pKi (ꢀ1.88
and ꢀ1.65 for (S)-1 and (S)-4 respectively), while the a1A pA2ꢀa1a pKi
differences are more modest and either negative or positive for the
4. Conclusion
The present investigation focused on substitution of the hydrogen
at the 8-position of 1,4-benzodioxane of WB4101, a quite neglected
modification of this lead compound of a1-AR selective antagonists.
Fluorine, chlorine, hydroxyl and methoxyl were selected as
8-substituents; methoxyl was introduced also at the 5 position.
Binding assays at the three a1-AR subtypes indicated that F, Cl and
OMe at the 8 position don’t substantially modify the affinity profile of
(S)-WB4101, whereas OH at the same position and, more markedly,
OMe at the 5 position are invariably deleterious. Interferencewith the
role played by benzodioxane O(1) and steric hindrance might be
respectively invoked to justify the negative effects of these two
substitutions. The antagonist affinities at the three a1-AR subtypes,
which confirm the 8-hydroxy and the 5-methoxy analogues as the
least potent derivatives in this set of compounds, diverge from the
corresponding binding affinities and such a difference might be
explained by inverse agonist nature, demonstrated and quantified at
the a1A-AR fortheSenantiomersofallthe fiveanalogues. Forthemost
part, the a1A and a1D antagonist affinities are lower than the a1a and
a1d binding affinities, while the opposite behaviour was observed for
the a1B subtype. This trend results in a new potent and significantly
a1B-AR selective antagonist, the (S)-WB4101 analogue 8-methoxy
substituted at the benzodioxane nucleus.
5. Experimental protocols
5.1. Chemistry
Melting points were measured on Buchi melting point appa-
ratus and are uncorrected. 1H NMR spectra were recorded oper-
ating at 300 MHz and 13C NMR at 75 MHz. Chemical shifts are
reported in ppm relative to residual solvent (CHCl3 or DMSO) as
internal standard. Signal multiplicity is designed according to the
following abbreviations: s ¼ singlet, d ¼ doublet, dd ¼ doublet of
doublets, t ¼ triplet, m ¼ multiplet, br s ¼ broad singlet, br
t ¼ broad triplet. Coupling constants are reported in Hertz (Hz).
Optical rotations were determined by a PerkineElmer 241 Polar-
imeter at 25 ꢂC. Elemental analyses (C, H, N, S, Cl, I) of the new
substances are within 0.40% of theoretical values. Purifications
were performed by flash chromatography using silica gel (particle
size 40e63 mm, Merck).

188
L. Fumagalli et al. / European Journal of Medicinal Chemistry 58 (2012) 184e191
5.1.1. (R)-2-Iodomethyl-8-hydroxy-1,4-benzodioxane
mixture of (R)-2-tosyloxymethyl-8-hydroxybenzodioxane
NMR (CDCl3)
2.36 (s, 3H).
d 7.44 (dd, 1H), 7.29 (dd, 1H), 7.19 (dt, 1H), 7.13 (dt, 1H),
A
(4.0 g, 11.9 mmol) and NaI (5.35 g, 35.7 mmol) in acetone (100 mL)
was refluxed for 3 h. The solvent was evaporated and the residue
treated with 10% aqueous HCl (100 mL) and ethyl acetate. The
organic phase was separated, washed with a saturated aqueous
solution of Na2S2O5, dried and concentrated. The residue was puri-
fied by chromatography on silica gel (eluent: cyclohexane/ethyl
acetate 80:20) yielding 3 g (86%) of (R)-2-iodomethyl-8-hydroxy-
5.1.6. 2-Hydroxy-3-chloroacetophenone (7)
At room temperature, 6 (152.5 g, 893.7 mmol) was added
dropwise to a solution of aluminium chloride (119.0 g, 893.7 mmol)
in dichlorobenzene (120 mL). The reaction mixture was stirred at
100 ꢂC for 5 h. After cooling to room temperature, dichloromethane
was added and the resulting mixture was poured into H2SO4 2 N at
1,4-benzodioxane as a white solid: m.p. 82 ꢂC; ½a D25
ꢃ
¼ ꢀ13.3 (c 1,
0
ꢂC. Precipitated 3-chloro-4-hydroxyacetophenone was removed
CHCl3); 1H NMR (CDCl3)
d
6.71e6.80 (t,1H), 6.59 (d,1H), 6.46 (d,1H),
by filtering the mixture. The aqueous layer was separated and
extracted with dichloromethane. The organic phases were
combined, rinsed with water, dried and concentrated. The residue
was crystallized from cyclohexane, yielding additional undesired
3-chloro-4-hydroxyacetophenone (23.6 g) as a yellowish solid and,
by concentration of the crystallization mother liquor, 47.3 g (31.0%)
5.44 (s, 1H, exchangeable with D2O), 4.29e4.41 (m, 2H), 4.11e4.19
(m, 1H), 3.36 (d, 2H). Anal. Calcd for C9H9IO3 (292.07).
5.1.2. (S)-2-[((2-(2,6-Dimethoxyphenoxy)ethyl)amino)methyl]-8-
hydroxy-1,4-benzodioxane hydrochloride [(S)-1]
of the desired product 7 as a yellow oil: 1H NMR (CDCl3)
d 12.84 (br
A solution of (R)-2-iodomethyl-8-hydroxy-1,4-benzodioxane
(1.3 g, 4.45 mmol) and 2-(2,6-dimethoxyphenoxy)ethylamine
(1.71 g, 8.9 mmol) in 2-propanol (5 mL) was refluxed for 20 h. After
cooling, dichloromethane (10 mL) and 1 M aqueous NaOH (10 mL)
were added. The organic phase was separated, washed with water,
dried and concentrated. The residue was purified by chromatog-
raphy on silica gel (eluent CH2Cl2/CH3OH/Et3N 95:5:1) yielding
700 mg (44%) of (S)-2-[((2-(2,6-Dimethoxyphenoxy)ethyl)amino)
methyl]-8-hydroxy-1,4-benzodioxane as a solid: m.p. 122e124 ꢂC;
s, 1H, exchangeable with D2O), 7.67 (dd, 1H), 7.57 (dd, 1H), 6.87 (dd,
1H), 2.66 (s, 3H). 3-Chloro-4-hydroxyacetophenone: m.p. 109 ꢂC;
1H NMR (CDCl3)
d 7.98 (d, 1H), 7.81 (m, 1H), 7.08 (m, 1H), 6.30 (br s,
1H, exchangeable with D2O), 2.56 (s, 3H).
5.1.7. 2-Benzyloxy-3-chloroacetophenone (8)
Tetrabutylammonium bromide (8.94 g, 27.7 mmol) and 10%
aqueous NaOH (222 mL) were added to a solution of 7 (47.3 g,
277.3 mmol) in dichloromethane (500 mL). Benzyl bromide
(36.5 mL, 305.1 mmol) was added dropwise to the mixture while
vigorously stirring. After 5 h at room temperature, the organic
phase was separated, treated with 10% HCl (450 mL), separated
again, washed with water, dried and concentrated. Column
chromatography on silica gel (eluent: cyclohexane/ethyl acetate
95:5) of the resulting residue allowed 48.5 g (67.1%) of 8 to be
½
a 2D5
ꢃ
¼ ꢀ43.3 (c 1, CHCl3); 1H NMR (CDCl3)
d 6.96e7.05 (t, 1H),
6.67e6.75 (t, 1H), 6.41e6.61 (m, 4H), 3.89e4.40 (m, 5H), 3.84 (s,
6H), 2.94e2.97 (m, 4H). The free amine (500 mg, 1.39 mmol) was
dissolved in ethanol (6 mL) and added with 2 M ethanolic HCl
(1.5 mL). Recovery of the precipitated solid by filtration yielded
300 mg of (S)-1 as a white solid: m.p. 211e212 ꢂC; ½a D25
¼ ꢀ53.9 (c 1,
ꢃ
CH3OH); 1H NMR (CDCl3)
d 9.40 (br s, 2H, exchangeable with D2O),
isolated as a yellow oil: 1H NMR (CDCl3)
d 7.56 (dd, 1H), 7.48 (m,
9.06 (s, 1H, exchangeable with D2O), 7.06e7.14 (t, 1H), 6.39e6.77
3H), 7.38 (m, 3H), 7.15 (t, 1H), 5.02 (s, 2H), 2.55 (s, 3H).
(m, 5H), 4.65 (m, 1H), 4.45 (dd, 1H), 4.22 (m, 2H), 4.05 (dd, 4H),
3.82 (s, 6H), 3.39 (m, 4H).13C NMR (d6-DMSO)
d 47.3, 47.4, 56.6, 65.6,
5.1.8. 2-Benzyloxy-3-chlorophenyl acetate (9)
68.4, 69.6, 106.1, 108.1, 109.4, 121.5, 125.2, 131.3, 136.1, 144.1, 147.2,
153.7. Anal. Calcd for C19H24ClNO6 (397.86).
3-Chloroperbenzoic acid (55%) (64.24 g, 372.3 mmol) was added
in small portions to a solution of 8 (48.53 g, 186.1 mmol) in
dichloromethane at 0 ꢂC. The reaction mixture was stirred for 7
days at room temperature and then poured into a saturated
NaHCO3 solution. The aqueous layer was separated and extracted
with dichloromethane. The organic phases were combined, washed
with water, dried and concentrated to give 51.50 g (100%) of 9 as
5.1.3. (S)-2-Iodomethyl-8-hydroxy-1,4-benzodioxane
Prepared in 87.3% yield from (S)-2-tosyloxymethyl-8-
hydroxybenzodioxane as described for the
R enantiomer:
½
a 2D5
ꢃ
¼ þ2.7 (c 1, CHCl3); m.p. and 1H NMR identical to (R)-2-
iodomethyl-8-hydroxy-1,4-benzodioxane. Anal. Calcd for C9H9IO3
(292.07).
a dark oil: 1H NMR (CDCl3)
d 7.40 (m, 5H), 7.29 (dd, 1H), 7.07 (t, 1H),
7.00 (dd, 1H), 5.03 (s, 2H), 2.17 (s, 3H).
5.1.4. (R)-2-[((2-(2,6-Dimethoxyphenoxy)ethyl)amino)methyl]-8-
hydroxy-1,4-benzodioxane hydrochloride [(R)-1]
5.1.9. 2-Benzyloxy-3-chlorophenol (10)
A solution of 9 (51.5 g, 186 mmol) in methanol (520 mL) was
added to 2.5 N aqueous NaOH 2.5 N (83 mL). After stirring for 2 h,
the reaction mixture was concentrated under vacuum. The residue
was dissolved in dichloromethane and water. The aqueous layer
was separated, acidified with HCl (pH 1) and extracted with
dichloromethane. The organic phases were combined, dried and
concentrated to give 35.7 g (81.7%) of 10 as a dark oil: 1H NMR
Prepared
from
(S)-2-iodomethyl-8-hydroxy-1,4-benzodi-
oxane as described for the S enantiomer. (R)-2-[((2-(2,6-Dimet
hoxyphenoxy)ethyl)amino)methyl]-8-hydroxy-1,4-benzodioxane:
½
a 2D5
ꢃ
¼ þ39.6 (c 1, CHCl3); m.p. and 1H NMR identical to (S)-2-[((2-
(2,6-Dimethoxyphenoxy)ethyl)amino)methyl]-8-hydroxy-1,4-ben-
zodioxane. (R)-1: m.p. 210e211 ꢂC; ½a D25
ꢃ
¼ þ52.9 (c 1, CH3OH); 1H
NMR and 13C NMR identical to (S)-1. Anal. Calcd for C19H24ClNO6
(CDCl3)
d 7.41 (m, 5H), 6.97 (m, 2H), 6.85 (m, 1H), 5.62 (br s, 1H,
(397.86).
exchangeable with D2O), 5.10 (s, 2H).
5.1.5. 2-Chlorophenyl acetate (6)
5.1.10. (S)-3-(2-Benzyloxy-3-chlorophenoxy)-1,2-propanediol
acetonide [(S)-11]
Acetyl chloride (66.6 mL, 933 mmol) was added drop-wise to
a solution of 2-chlorophenol (120 g, 933 mmol) and pyridine
(85.3 mL) in dichloromethane (600 mL) at 25 ꢂC. The mixture was
stirred for 4 h at room temperature and then washed with water
(480 mL). The organic phase was washed with 10% HCl (180 mL)
and then with water (120 mL), dried and concentrated to give 159 g
(quantitative yield) of the desired product as a colourless oil: 1H
(R)-1-Mesiloxy-2,3-propanediol acetonide (5.91 g, 28.1 mmol) in
DMF was added dropwise to a solution of 10 (6.6 g, 28.1 mmol) and
potassium tert-butoxide (3.31 g, 29.5 mmol) in DMF (100 mL). After
4 h the solvent was removed under vacuum; the residue was dis-
solved in dichloromethaneand treated with 2.5 Naqueous NaOH. The
aqueous layer was separated and extracted with dichloromethane.

L. Fumagalli et al. / European Journal of Medicinal Chemistry 58 (2012) 184e191
189
The organic phases were combined, washed with HCl 10% and then
(5 mL) was refluxed for 24 h. The solvent was evaporated and the
residue treated with 10% aqueous NaOH (30 mL) and dichloro-
methane. The organic phase was separated, washed with water,
dried and concentrated. Purification of the residue by chromatog-
raphy on silica gel (eluent: cyclohexane/ethyl acetate 1:1) gave
690 mg of (S)-2-[((2-(2,6-Dimethoxyphenoxy)ethyl)amino)
with water, dried and concentrated to give 7.64 g (77.8%) of (S)-11 as
a dark oil: ½a 2D5
ꢃ
¼ þ9.1 (c 0.5, EtOH); 1H NMR (CDCl3)
d 7.58 (m, 2H),
7.37 (m, 3H), 7.00 (m, 2H), 6.85 (m,1H), 5.05 (s, 2H), 4.50 (m,1H), 4.15
(m, 2H), 4.10 (m, 1H), 3.93 (m, 1H), 1.43 (s, 3H),1.38 (s, 3H).
5.1.11. (R)-3-(2-Benzyloxy-3-chlorophenoxy)-1,2-propanediol [(R)-
12]
methyl]-8-chloro-1,4-benzodioxane as an oil: ½a D25
¼ ꢀ21.0 (c 1,
ꢃ
CHCl3); 1H NMR (CDCl3)
d 6.92 (t,1H), 6.85 (m,1H), 6.70 (m, 2H), 6.49
1 N HCl (40 mL) was added to a solution of (S)-11 (7.64 g,
21.9 mmol) in ethanol (20 mL) and the mixture was heated at 75 ꢂC
for 4 h. After cooling, ethanol was removed under vacuum and the
residual aqueous phase was extracted with dichloromethane. The
organic extract was washed with water, dried and concentrated to
give an oily residue which was chromatographed on silica gel.
Elution with dichloromethane/methanol (95:5) yielded 2.66 g
(d, 2H), 4.27 (m, 2H), 4.04 (m, 3H), 3.77 (s, 6H), 3.01 (dd,1H), 2.90 (m,
3H), 2.01 (br s, 1H). The free amine was dissolved in ethanol (4 mL)
and 2.6 m ethanolic HCl (1.1 mL) was added while stirring. After
10 min, ethanol was evaporated and ethyl acetate (10 mL) was added
to the residue. Ethanol (2 mL) was added to the mixture while
refluxing. After cooling to 0 ꢂC, the suspension was filtered to give
293 mg (24.4%) of (S)-3 as a white solid: m.p. 161e162 ꢂC;
(39.2%) of (R)-12 as a light brown solid: m.p. 86e87 ꢂC; ½a D25
ꢃ
¼ ꢀ5.2
½
a 2D5
ꢃ
¼ ꢀ41.7 (c 1, CH3OH); 1H NMR (DMSO-d6)
d 9.63 (br s, 1H),
9.36 (br s,1 H), 7.05 (m, 2H), 6.88 (m, 2H), 6.68 (m, 2H), 4.85 (m,1H),
(c 0.5, EtOH); 1H NMR (DMSO-d6)
d
7.50 (m, 2H), 7.38 (m, 3H), 7.01
(m, 3H), 5.01 (m, 2H þ 1H exchangeable with D2O), 4.71 (t, 1H,
exchangeable with D2O), 4.07 (m, 1H), 3.96 (m, 1H), 3.86 (m, 1H),
3.48 (m, 2H). Anal. Calcd for C16H17ClO4 (308.76).
4.45 (br d,1H), 4.16 (m, 3H), 3.76 (s, 6H), 3.28e3.58 (m, 4H).13C NMR
(DMSO-d6)
121.9, 122.5, 124.8, 136.6, 138.7, 144.2, 153.2. Anal. Calcd for
19H23Cl2NO5 (416.30).
d 46.3, 47.4, 56.1, 56.2, 65.2, 67.9, 70.7, 105.6, 116.4, 121.1,
C
5.1.12. (S)-3-(2-Benzyloxy-3-chlorophenoxy)-1,2-
dimesyloxypropane [(S)-13]
5.1.16. (R)-3-(2-Benzyloxy-3-chlorophenoxy)-1,2-propanediol
acetonide [(R)-11]
Mesyl chloride (1.33 mL, 17.2 mmol) was added dropwise to
a solution of (R)-12 (2.66 g, 8.6 mmol) and triethylamine (2.4 mL,
17.2 mmol) in dichloromethane (50 mL) at 0e5 ꢂC. The mixture was
stirred for 2 h and then diluted with dichloromethane (50 mL) and
treated with a saturated aqueous solution of NaHCO3 (30 mL). The
organic phase was separated, washed with water, dried and
concentrated under vacuum to give 4.6 g of crude product, which
was triturated in diisopropyl ether (25 mL) yielding 2.23 g (55.6%)
Prepared in quantitative yield from (S)-1-mesyloxy-2,3-
propanediol acetonide as described for the
S enantiomer:
½
a 2D5
ꢃ
¼ ꢀ9.0 (c 0.5, EtOH); 1H NMR identical to (S)-11.
5.1.17. (S)-3-(2-benzyloxy-3-chlorophenoxy)-1,2-propanediol [(S)-
12]
Prepared in 28.0% yield from (R)-11 as described for (R)-12:
of (S)-13 as a light brown solid: m.p. 85 ꢂC; ½a D25
ꢃ
¼ ꢀ6.2 (c 1, CHCl3);
½
a 2D5
ꢃ
¼ þ5.7 (c 0.5, EtOH); m.p. and 1H NMR identical to (R)-12.
1H NMR (DMSO-d6)
d
7.50 (m, 2H), 7.35 (m, 3H), 7.08 (m, 3H), 5.27
Anal. Calcd for C16H17ClO4 (308.76).
(m, 1H), 5.00 (dd, 2H), 4.60 (m, 1H), 4.50 (m, 1H), 4.37 (m, 2H), 3.28
(s, 6H). Anal. Calcd for C18H21ClO8S2 (464.93).
5.1.18. (R)-3-(2-Benzyloxy-3-chlorophenoxy)-1,2-
dimesyloxypropane [(R)-13]
5.1.13. (S)-3-(2-Hydroxy-3-chlorophenoxy)-1,2-dimesyloxypropane
[(S)-14]
Prepared in 51.4% yield from (S)-12 as described for (S)-13:
½
a 2D5
ꢃ
¼ þ7.1 (c 1, CHCl3); m.p. and 1H NMR identical to (S)-13. Anal.
A solution of (S)-13 (2.23 g, 4.8 mmol) in methanol (56 mL) was
added with 10% Pd/C (0.2 g) and vigorously shaken under hydrogen
at room temperature until hydrogen uptake ceased. The catalyst
was removed by filtration and the filtrate concentrated to give
1.77 g of crude product. Purification by chromatography on silica
gel (eluent: cyclohexane/ethyl acetate 1:1) yielded 1.16 g (64.4%) of
Calcd for C18H21ClO8S2 (464.93).
5.1.19. (R)-3-(2-Hydroxy-3-chlorophenoxy)-1,2-
dimesyloxypropane [(R)-14]
Prepared in 45.3% yield from (R)-13 as described for (S)-14:
½
a 2D5
ꢃ
¼ þ11.8 (c 1, CHCl3); 1H NMR identical to (S)-14.
(S)-14 as an orange oil: ½a D25
ꢃ
¼ ꢀ12.2 (c 1, CHCl3); 1H NMR (DMSO-
d6)
d
9.25 (br s, 1H, exchangeable with D2O), 6.98 (m, 2H), 6.78 (m,
5.1.20. (S)-2-Mesyloxymethyl-8-chloro-1,4-benzodioxane [(S)-15]
Prepared in 53.3% yield from (R)-14 as described for (R)-15:
1H), 5.20 (m, 1H), 4.75 (m, 1H), 4.60 (m, 1H), 4.25 (m, 2H), 3.30 (s,
3H), 2.25 (s, 3H).
½
a 2D5
ꢃ
¼ ꢀ6.4 (c 1, CHCl3); m.p. and 1H NMR identical to (R)-15. Anal.
Calcd for C10H11ClO5S (278.71).
5.1.14. (R)-2-Mesyloxymethyl-8-chloro-1,4-benzodioxane [(R)-15]
Potassium carbonate (0.43 g) was added to a solution of (S)-14
(1.16 g, 3.1 mmol) in acetone (12 mL) and the mixture was refluxed
for 24 h. After evaporating the solvent, the resultant residue was
treated with ethyl acetate (80 mL) and aqueous 10% HCl (70 mL).
The organic phase was separated, washed with water, dried and
concentrated to give 0.99 g of crude product, which was crystallized
from diisopropyl ether yielding 0.44 g (50.9%) of (R)-15 as a white
5.1.21. (R)-2-[((2-(2,6-Dimethoxyphenoxy)ethyl)amino)methyl]-8-
chloro-1,4-benzodioxane hydrochloride [(R)-3]
Prepared from (S)-15 as described for (S)-3: ½a D25
¼ þ43.9 (c 1,
ꢃ
CH3OH); m.p. and 1H NMR and 13C NMR identical to (S)-3.Anal.
Calcd for C19H23Cl2NO5 (416.30).
5.2. Biology
solid: m.p. 76e77 ꢂC; ½a D25
ꢃ
¼ þ7.5 (c 1, CHCl3); 1H NMR (CDCl3)
d
6.90 (m, 1H), 6.75 (m, 3H), 4.50 (m, 1H), 4.48 (m, 3H), 4.26 (dd,
5.2.1. Binding assays
1H), 4.07 (dd, 1H), 3.08 (s, 3H). Anal. Calcd for C10H11ClO5S (278.71).
The pharmacological profile of both the S and R enantiomers of
compounds 1e5 was assessed by measuring their affinities for a1a
a1b a1d AR-subtypes and 5-HT1A serotoninergic receptor with
in vitro binding studies.
,
5.1.15. (S)-2-[((2-(2,6-Dimethoxyphenoxy)ethyl)amino)methyl]-8-
chloro-1,4-benzodioxane hydrochloride [(S)-3]
,
A
solution of (R)-15 (850 mg, 3.05 mmol) and 2-(2,6-
Briefly, membranes derived from Chinese Hamster Ovary (CHO)
cells expressing a1-AR subtypes [37] were resuspended in Tris HCl,
dimethoxyphenoxy)ethylamine (1.2 g, 6.1 mmol) in isobutanol

190
L. Fumagalli et al. / European Journal of Medicinal Chemistry 58 (2012) 184e191
50 mM, pH ¼ 7.7 containing 10
m
M pargyline and 0.1% ascorbic acid,
5.2.2.2. Vas deferens prostatic portion. This tissue (from rats of
and incubated for 30 min at 25 ꢂC with 0.5 nM [3H]-Prazosin (NEN,
80.5 Ci/mmol) in the absence or presence of different concentrations
of the tested compounds (from 0.3 to 1000 nM depending on the
200e250 g) was used to assess a1A-adrenoceptor antagonist
activity [33]. Prostatic portions of 2 cm length were set up in Tyrode
solution of the following composition (mM): NaCl, 130.0; KCl, 2.0;
CaCl2$2H2O, 1.8; MgCl2 0.89; NaHCO3, 25.0; NaH2PO4$2H2O, 0.42;
affinity). Prazosin 1 mM was used to determine non-specific binding.
Binding studies at 5-HT1A receptor were carried out using crude
membrane preparations from rat hippocampus, which were
glucose, 5.6; desipramine hydrochloride (0.01 mM) was added to
prevent the neuronal uptake of (ꢀ)-noradrenaline. The medium
was maintained at 37 ꢂC The preparations were equilibrated for 1 h
under a resting tension of 0.35 g. The preparations were equili-
brated for 45e60 min and during this time the bathing solution was
changed every 10 min. Contraction-response curves for isotonic
contractions in response to (ꢀ)-noradrenaline were recorded at
30 min intervals; the first one being discarded and the second one
taken as control. After the incubation with antagonist concentra-
tion for 30 min, a third dose-response curve was obtained.
resuspended in Tris HCl 50 mM (pH ¼ 7.7, 10
mM pargyline and
4 mM CaCl2) and incubated for 30 min at 25 ꢂC with 1 nM [3H]-8-
OH-DPAT, in the absence or presence of different concentrations of
the tested compounds. 5-HT 1
mM was used to determine non-
specific binding.
Incubations were stopped by rapid filtration, through GF/B fibre
filters, which were then washed, dried and counted in an LK1214
rack
b Liquid scintillation Spectrometer.
At least three different experiments, in triplicate, were carried
out for each compound and usually each compound was tested
simultaneously on the different a1-AR subtypes. Prazosin or 5-HT
was always tested in parallel, as reference drugs. The % inhibitory
effects obtained in the different experiments were pooled together
and the inhibition curves were analyzed using the “one-site
competition” equation built into GraphPad Prism 4.0 (GraphPAD
Software, San Diego, CA). This analysis gives the IC50 (i.e. the drug
concentration inhibiting specific binding by 50%), calculated with
the relative standard error. Ki values were then calculated by IC50
using the Cheng and Prusoff equation in which the Kd of [3H]-
a2-Adrenoreceptor antagonist activity was determined also on
prostatic portions of 1.5e2 cm length which were set up in organ
bath containing Krebs solution of the following composition (mM):
NaCl, 118.4; KCl, 4.7; CaCl2$2H2O, 2.52; MgSO4, 0.6; KH2PO4, 1.2;
NaHCO3, 25.0; glucose, 11.1. Propranolol hydrochloride (1
desipramine hydrochloride (0.01 M) were present in the above
described Krebs solutions throughout the experiments to block
-adrenoreceptors and to prevent the neuronal uptake of
mM) and
m
b
(ꢀ)-noradrenaline respectively. The physiological salt solution was
kept at 37 ꢂC. Field stimulation of the tissues was carried out by
means of two platinum electrodes, connected to a Grass S88
stimulator, placed near the top and bottom of the vas deferens at
0.1 Hz, using square pulses of 3 ms duration at voltage of 20e40 V.
A 1 h equilibration period under a resting tension of 0.35 g was
allowed. A first clonidine concentration-response curve, taken as
control, was obtained cumulatively. The antagonist concentration
was allowed to equilibrate with the tissue for 30 min before
obtaining a second dose-response curve.
Prazosin for a1a, a1b, a1d AR-subtypes were 0.4, 0.4 and 0.7 nM,
respectively, whereas the Kd of [3H]-8-OH-DPAT for 5-HT1A recep-
tors was 1.2 nM.
5.2.2. Functional antagonism in isolated rat tissues
Male Sprague-Dawley rats (Charles River, Italy) were killed by
cervical dislocation under ketamine anaesthesia and the organ
required were isolated, freed from adhering connective tissue, and
set up rapidly under resting tension in organ bath (15 mL) con-
taining physiological salt solution kept at appropriate concentra-
tion (see below) and gassed with 95%O2 and 5%CO2 at pH 7.4.
Concentration-response curves were constructed by cumulative
addition of agonist. The concentration of agonist in the organ bath
was increase approximately 5-fold at each step, with each addition
being made only after the response to the previous addition had
attained a maximal level and remained steady.
All experimental data were recorded by means of isometrically
or isotonically FT.03 Grass force transducers using Power LabÒ
software (AD-Instruments Pty Ltd, Castle Hill, Australia). In addition
parallel experiments in which tissues did not receive any antagonist
were run in order to check any variation in sensitivity.
5.2.2.3. Spleen. This tissue (from rats of 250e300 g) was used to
assess a1B-adrenoceptor antagonist activity [34]. The spleens were
bisected transversally into two strips and were suspended in organ
baths maintained at 37 ꢂC and containing Krebs solution of the
following composition (mM): NaCl, 118.4; KCl, 4.7; CaCl2, 1.9;
MgSO4 1.2; NaHCO3, 25.0; NaH2PO4$2H2O, 1.2; glucose, 11.7; desi-
pramine hydrochloride (0.01
ride (1 M) were added to prevent the neuronal uptake of
(ꢀ)-phenylephrine and to block -adrenoreceptors, respectively.
mM) and (ꢁ)-propranolol hydrochlo-
m
b
The spleen strips were placed under 1 g of resting tension and
equilibrated for 1 h. The cumulative concentration-response curves
to phenylephrine were measured isometrically and obtained at
30 min intervals, the first one being discarded and the second one
taken as control. The antagonist was allowed to equilibrate with the
tissue for 30 min, and then a new concentration-response curve to
the agonist was constructed.
5.2.2.1. Prostate. This tissue (from rats of 200e250 g) was used to
assess a1A-adrenoceptor antagonist activity [35]. Prostatic strips
measuring 8e10 mm in length and 1e2 mm in width were placed
under a resting tension of 2 g in modified Krebs solution of the
following composition (mM): NaCl, 118.0; KCl, 4.7; CaCl2$2H2O, 2.5;
MgSO4∙7H2O 1.18; NaHCO3, 25.0; KH2PO4, 1.18; glucose, 5.5. The
preparations were equilibrated for 60 min; during this time the
bathing solution was changed every 20 min. Before the
concentration-curves were started, tissues were exposed to
5.2.2.4. Thoracic aorta. This tissue (from rats of 250e300 g) was
used to assess a1D-adrenoceptor antagonist activity [34]. The
thoracic portion of aorta was cleaned from extraneous connective
tissue and placed in organ bath containing Krebs solution main-
tained at 37 ꢂC of the following composition (mM): NaCl, 118.4; KCl,
4.7; CaCl2, 1.9; MgSO4 1.2; NaHCO3, 25.0; NaH2PO4, 1.2; glucose,
(ꢀ)-noradrenaline at a concentration of 1.0
m
M. A minimum
11.7; desipramine hydrochloride (0.01
hydrochloride (1 M) were added to prevent the neuronal uptake of
(ꢀ)-noradrenaline and to block -adrenoreceptors, respectively.
m
M) and (ꢁ)-propranolol
response of 0.5 g of tension was required for the tissue to be used
for concentration-response curves. After a 90 min time period,
m
b
a
cumulative response-curve to (ꢀ)-noradrenaline was con-
Two helicoids strips were cut in strips from each aorta of about
1.5 cm length. The endothelium was removed by rubbing with filter
paper: the functional loss of endothelial cells was confirmed by the
absence of the relaxing response to acetylcholine. After at least 1 h
equilibration period under an optimal tension of 1 g, cumulative
structed. After completion of the concentration-response curve, the
tissue was washed for 90 min, and the antagonist was added and
incubated for 30 min before a second cumulative concentration-
response curve was obtained.

L. Fumagalli et al. / European Journal of Medicinal Chemistry 58 (2012) 184e191
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a1-antagonist as inverse agonist [16,36]. Aortic strips were isolated
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containing the Krebs solution maintained at 37 ꢂC of the following
composition (mM): NaCl, 118; KCl, 4.75; CaCl2, 1.8; MgCl2, 1.2;
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Acknowledgements
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Appendix A. Supplementary data
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