Synthesis and α1-adrenoceptor antagonist activity of tamsulosin analogues

Article abstract of DOI:10.1016/j.ejmech.2010.09.042

Tamsulosin (-)-1 is the most utilized α₁-adrenoceptor antagonist in the benign prostatic hyperplasia therapy owing to its uroselective antagonism and capability in relieving both obstructive and irritative lower urinary tract symptoms. Here we report the synthesis and pharmacological study of the homochiral (-)-1 analogues (-)-2-(-)-5, bearing definite modifications in the 2-substituted phenoxyethylamino group in order to evaluate their influence on the affinity profile for α₁-adrenoceptor subtypes. The benzyl analogue (-)-3, displaying a preferential antagonist profile for α_1A-than α_1D-and α_1B- adrenoceptors, and a 12-fold higher potency at α_1A- adrenoceptors with respect to the α_1B subtype, may have improved uroselectivity compared to (-)-1.

Full text of DOI:10.1016/j.ejmech.2010.09.042

European Journal of Medicinal Chemistry 45 (2010) 5800e5807
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and a1-adrenoceptor antagonist activity of tamsulosin analogues
Gianni Sagratini a, Piero Angeli a, Michela Buccioni a, Ugo Gulini a, Gabriella Marucci a, Carlo Melchiorre b,
,
Elena Poggesi c, Dario Giardinà a
*
a Scuola in Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino, Via S. Agostino 1, 62032 Camerino, Italy
b Dipartimento di Scienze Farmaceutiche, Università di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
c Drug Discovery Division, Recordati SpA, Via Civitali 1, 20148 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Tamsulosin (ꢀ)-1 is the most utilized a1-adrenoceptor antagonist in the benign prostatic hyperplasia
therapy owing to its uroselective antagonism and capability in relieving both obstructive and irritative
lower urinary tract symptoms. Here we report the synthesis and pharmacological study of the homo-
chiral (ꢀ)-1 analogues (ꢀ)-2e(ꢀ)-5, bearing definite modifications in the 2-substituted phenox-
yethylamino group in order to evaluate their influence on the affinity profile for a1-adrenoceptor
subtypes. The benzyl analogue (ꢀ)-3, displaying a preferential antagonist profile for a1A-than a1D-and
a1B-adrenoceptors, and a 12-fold higher potency at a1A-adrenoceptors with respect to the a1B subtype,
may have improved uroselectivity compared to (ꢀ)-1.
Received 11 May 2010
Received in revised form
16 September 2010
Accepted 17 September 2010
Available online 25 September 2010
Keywords:
a1-Adrenoceptor subtypes
a1-Adrenoceptor antagonists
a1A-Adrenoceptor selective antagonists
Tamsulosin analogues
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
obstructive symptoms to the urine flow [9,10]. At the same time, the
a1D-adrenoceptor is the most abundant subtype in human bladder
Three native adrenoceptors, a1A
,
a1B and a1D, have been phar-
detrusor [11] whose stimulation causes bladder instability and irri-
tability [10]. For these reasons, beside the non-selective antagonists,
some subtype selective antagonists are currently used as first line
medical treatment of lower urinary tract symptoms associated with
benign prostatic hyperplasia (BPH) [12,13].
macologically detected in animal and human tissues, correspond-
ing to the cloned a1a a1b, and a1d subtypes expressed in various cell
,
lines [1]. A fourth a1-adrenoceptor subtype, a1L, displaying low
affinity for prazosin [2] and conformationally related to the a1A
subtype [3] has also been reported.
Multiple a1-adrenoceptor subtypes are present in a variety of
organs, as brain, heart, blood vessels, liver, spleen, kidney, prostate,
where mediate, on activation by endogenous noradrenaline or
adrenaline, a range of physiological functions including neuronal
transmission, contraction of blood vessels and low urinary tract
smooth muscles, myocardial growth and inotropy [4].
(R)-(ꢀ)-5-[2-(2-(2-Ethoxyphenoxy)ethylamino)propyl]-2-
methoxybenzenesulfonamide ((ꢀ)-1, tamsulosin), a chiral sulfa-
moylphenethylamine derivative, is
a potent a1-adrenoceptor
antagonist [14]. Rather different results, with regard selectivity,
were obtained for (ꢀ)-1 in binding and functional assays. In binding
experiments, it showed high and similar affinity at a1A-and a1D
-
adrenoceptors and a slightly lower affinity for the a1B subtype
[15e17]. In functional studies on animal tissues, (ꢀ)-1 resulted
almost equipotent at a1D-and a1A-adrenoceptors while being
a weaker antagonist at the a1B subtype [18]. However, in other
studies, (ꢀ)-1 was significantly more potent at a1D-adrenoceptors
than at a1A- or a1B-adrenoceptors [16,17,19].
Pharmacological evidences indicate that the a1A-and the a1B
-
adrenoceptors are the subtypes predominantly expressed in human
blood vessels and involved in contraction of vasculatures responsible
of blood pressure inyoung men andin the elderly, respectively [5e8].
As a consequence, their specific antagonists display therapeutic
indications for the control of hypertension. Furthermore, the a1A
adrenoceptor is predominantly expressed, with respect to the a1D, in
human prostate gland where it mediates contractions producing
-
Tamsulosin is the most utilized a1-adrenoceptor antagonist in
the BPH therapy [20] owing to its uroselective a1-adrenoceptor
antagonism, with a superior profile in relieving both obstructive
and irritative lower urinary tract symptoms [21,22]. In this regard,
most of the pharmacological evidences indicate that a1-adreno-
ceptor antagonists with higher affinity for the a1A-adrenoceptor
* Corresponding author. Tel.: þ39 0737 402265; fax: þ39 0737 637345.
E-mail address: dario.giardina@unicam.it (D. Giardinà).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.042

G. Sagratini et al. / European Journal of Medicinal Chemistry 45 (2010) 5800e5807
5801
over the a1B subtype and, at the same time, with an important
affinity for the a1D-adrenoceptor, that is with a balanced a1A/D
selectivity, should find an optimal use in the BPH therapy [8,23,24].
Although endowed with minor side effects, such as orthostatic
hypotension and dizziness, in comparison to non-selective quina-
zoline antagonists, (ꢀ)-1 gave more frequently ejaculatory disor-
ders [8,25,26].
relative carbamate, obtained by reaction with (1R)-(ꢀ)-menthyl
chloroformate, in comparison with the mixture of menthylcarba-
mates of (ꢁ)-6. In the same conditions, the carbamates from (ꢁ)-6
showed two peaks with retention time of 36.19 and 36.53 min,
whereas that deriving from (ꢀ)-6 displayed, beside the principal
peak at 36.50 min, a significant and detectable small peak relative
to the less abundant diastereomer.
Here we report the design and synthesis of the homochiral (ꢀ)-1
analogues (ꢀ)-2, (ꢀ3), and (ꢀ)-4 [27], bearing definite modifica-
tions in the 2-substituted phenoxyethylamino group in order to
evaluate their influence on the affinity profile for a1-adrenoceptors,
which could, hopefully, lead to an improvement of the therapeutic
utilization. Specifically, the 2-ethoxy group of (ꢀ)-1 was replaced
with an i-propoxy ((ꢀ)-2), a benzyloxy ((ꢀ)-3) or a 2,2,2-tri-
fluoroethoxy moiety ((ꢀ)-4), to increase at different extent the
hindrance, the hydrophobic properties, or the polar character of the
progenitor. In addition, the i-propoxy [28] and the 2,2,2-tri-
fluoroethoxy [29] groups are structural component of some known
potent a1-adrenoceptor antagonists. In particular, some 2,2,2-tri-
fluoroethoxy-bearing compounds [13,30,31] displayed high affinity
and selectivity for the human cloned a1a-adrenoceptor together
with a marked uroselectivity. This finding suggested that the 2,2,2-
trifluoroethoxy moiety could be a key structural element for
prostate selectivity [32].
The alkylating agent 7 was synthesized by reaction of 1,2-
dibromoethane with 2-isopropoxyphenol in presence of KOH in
EtOH. The Swern oxidation [39] was used for the synthesis of
(ꢁ)-cis-3-methyl-2,3-dihydro-1,4-benzodioxine-2-carbaldehyde
(10) starting from (ꢁ)-[cis-3-methyl-2,3-dihydro-1,4-benzodioxin-
2-yl]methanol [33] and a mixture of oxalyl chloride and dime-
thylsulfoxide in dichloromethane at low temperature.
Compounds (ꢀ)-2e(ꢀ)-4, (ꢀ)-5a and (ꢀ)-5b were purified by
chromatography and characterized by elemental analysis, 1H NMR,
specific rotation and chromatographic parameters.
3. Pharmacology
The affinity profile of compounds (ꢀ)-2e(ꢀ)-4, (ꢀ)-5a, (ꢀ)-5b,
and (ꢀ)-1 as reference, was evaluated in radioreceptor binding
assays on human cloned a1-adrenoceptors. Competition experi-
ments were performed using [3H]prazosin to label a1-adrenoceptor
binding sites on membranes of Chinese hamster ovary (CHO) cells
expressing human a1a, a1b, and a1d-adrenoceptor subtypes [40].
Binding affinities were expressed as pKi values derived using the
ChengePrusoff equation [41].
In addition, to evaluate the effect of a specific conformational
constraint on affinity, the 2-(2-ethoxyphenoxy)ethanamine moiety
of (ꢀ)-1 was replaced with the 1-cis-(3-methyl-2,3-dihydro-1,4-
benzodioxin-2-yl)methanamino group affording the diastereomers
(ꢀ)-5a and (ꢀ)-5b. In fact, a similar structural modification on
All compounds were also studied on a1-adrenoceptor subtypes
of rat isolated tissues, using (ꢀ)-1 as reference compound. a1A-and
a1D-adrenoceptor blocking activities were evaluated by antagonism
of (ꢀ)-noradrenaline-induced contractions of rat prostatic vas
deferens [42] and aorta [43], respectively, whereas the a1B-adre-
noceptor antagonism was determined on the rat spleen tissue by
using phenylephrine as agonist [44]. The potency of (ꢀ)-4 and
(ꢀ)-5a at all a1 subtypes, of (ꢀ)-5b at a1A and a1D-adrenoceptors,
and of (ꢀ)-2 at the a1D subtype was expressed as the pA2 value
calculated by Schild plots at three different concentrations
according to Arunlakshana and Schild [45]. The potency of (ꢀ)-1
and (ꢀ)-3 at all a1 subtypes, of (ꢀ)-2 at a1A and a1B-adrenoceptors,
and of (ꢀ)-5b at the a1B subtype was expressed by the pKB value
according to van Rossum [46] because the slope of the Schild plot
was significantly different from unity. In this latter case, the pKB
value was calculated at the lowest antagonist concentration giving
a significant rightward shift of the agonist concentration-response
curve [log (concentration ratio ꢀ1) ꢂ 0.5].
a
WB4101-related compound resulted in
a
a1-adrenoceptor
antagonist potency and a1
/a2 selectivity higher than those
observed for the corresponding trans isomer [33] (Fig. 1).
2. Chemistry
Compounds (ꢀ)-2e(ꢀ)-4 were synthesized by reaction of (ꢀ)-5-
[(2R)-2-aminopropyl]-2-methoxybenzenesulfonamide ((ꢀ)-6) [34]
with the appropriate alkylating agent 1-(2-bromoethoxy)-2-iso-
propoxybenzene (7),1-(benzyloxy)-2-(2-bromoethoxy)benzene (8)
[35], or 2-[2-(2,2,2-trifluoroethoxy)-phenoxy]ethyl 4-methyl-
benzenesulfonate (9) [36] in refluxing i-AmOH. (ꢀ)-5a and (ꢀ)-5b
were synthesized as diastereomers by reductive alkylation of amine
(ꢀ)-6 with (ꢁ)-cis-3-methyl-2,3-dihydro-1,4-benzodioxine-2-car-
baldehyde (10) and sodium cyanoborohydride in MeOH (Scheme 1).
The intermediate amine (ꢀ)-6, which was included in a patent
[34], was obtained by resolution with (R)-(ꢀ)-mandelic acid of the
corresponding racemic amine (ꢁ)-6 [37], synthesized by reductive
amination of 2-methoxy-5-(2-oxopropyl)-1-benzenesulfonamide
[38] with ammonium acetate and sodium cyanoborohydride in
The experimental data were subjected to statistical analysis by
means of Student’s t-test. A p value < 0.05 was taken to indicate
a statistically significant difference.
MeOH. After repeated crystallizations from ethanol
a pure
(ꢀ)-mandelate was obtained. The treatment of an aqueous solution
of this salt with a K2CO3 solution gave (ꢀ)-6 with the same physical
characteristics of the reported compound [34]. Its optical purity
(99.3%) was determined by reversed phase HPLC analysis of the
4. Results and discussion
The results of radioligand binding assays and functional tests are
presented in Tables 1 and 2, respectively. In binding experiments,
O
O
H3C
H2N
O
CH3
(R)
H3C
H2N
O
CH3
(R)
H
H
O
O
O
O
S
N
S
N
O
H
O
H
R
H3C
(-)-5a (cis) , (-)-5b (cis)
(-)-1 R = CH3CH2; (-)-2 R = (CH3)2CH ;
(-)-3 R = C6H5CH2; (-)-4 R = CF3CH2
Fig. 1. Structure of compounds (ꢀ)-1 (tamsulosin) and analogues (ꢀ)-2e(ꢀ)-5.

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G. Sagratini et al. / European Journal of Medicinal Chemistry 45 (2010) 5800e5807
O
X
R
O
O
H3C
H2N
O
CH3
(R)
H
7- 9
O
O
S
N
O
H
a, b
R
O
S
H3C
H2N
O
CH3
(R)
(-)-2 R = (CH3)2CH ; (-)-3 R = C6H5CH2;
(-)-4 R = CF3CH2
H
NH2
O
(-)-6
c
O
H3C
H2N
O
CH3
(R)
H
O
O
O
CHO
CH3
S
N
O
H
H3C
O
10 (cis)
(-)-5a (cis) , (-)-5b (cis)
Scheme 1.. Reagents and conditions: (a) X ¼ Br with R ¼ (CH3)2CH (7) and C6H5CH2 (8); X ¼ TsO with R ¼ CF3CH2 (9); (b) i-AmOH, reflux; (c) NaBH3CN, MeOH, Hþ, r.t.
Table 1
Binding affinity constants, expressed as pKi, of compounds (ꢀ)-2e(ꢀ)-4, (ꢀ)-5a, (ꢀ)-5b, and (ꢀ)-1 as reference, at cloned human a1-adrenoceptor subtypes expressed in CHO
cells.
O
O
S
H3C
H2N
O
CH3
(R)
H3C
H2N
O
CH3
H
(R)
O
O
H
O
O
S
N
N
O
H
O
H
R
H3C
(-)-1 - (-)-4
(-)-5a (cis) , (-)-5b (cis)
Compound
pKia
Selectivity ratiob
a1a
a1b
a1d
a1a
/a1b
a1d/a1b
a1a/a1d
(ꢀ)-1
(ꢀ)-2
(ꢀ)-3
(ꢀ)-4
(ꢀ)-5a
(ꢀ)-5b
10.30 ꢁ 0.04
9.47 ꢁ 0.02
9.46 ꢁ 0.06
9.22 ꢁ 0.02
7.72 ꢁ 0.03
7.94 ꢁ 0.01
9.20 ꢁ 0.08
8.72 ꢁ 0.04
8.68 ꢁ 0.01
8.26 ꢁ 0.03
7.17 ꢁ 0.07
7.50 ꢁ 0.09
10.00 ꢁ 0.04
9.61 ꢁ 0.01
9.49 ꢁ 0.04
9.02 ꢁ 0.07
7.76 ꢁ 0.01
7.81 ꢁ 0.02
13
5
6
9
4
6
8
6
6
4
2
2
0.7
0.9
2
0.9
1
3
Equilibrium dissociation constants (Ki) were calculated from IC50 values using the ChengePrusoff equation. The affinity estimates, derived from displacement of [3H]
prazosin binding from a1-adrenoceptors and expressed as mean values of pKi ꢁ SEM, were from two to three experiments performed in triplicate, which agreed within ꢁ20%.
a
b
Calculated by the antilog of the difference between pKi values at different a1-adrenoceptor subtypes.
compounds (ꢀ)-2e(ꢀ)-4 displayed a slightly lower affinity than
(ꢀ)-1 at all the three a1-adrenoceptor subtypes, which was more
pronounced (9- to 12-fold) for the fluoro derivative (ꢀ)-4. In
addition, all compounds, like (ꢀ)-1, showed similar and low a1a,d
over a1b selectivity. In contrast, the cyclic analogues (ꢀ)-5a and
(ꢀ)-5b were markedly less potent than non-cyclic analogues and
(ꢀ)-1 at all three a1-adrenoceptor subtypes.
The slightly reduced potency of (ꢀ)-2 at a1D-, and (ꢀ)-3 at a1B
-
and a1D-adrenoceptors, relatively to (ꢀ)-1, could be related to an
increased steric hindrance of the i-propoxy and benzyloxy groups
in comparison with the ethoxy moiety of (ꢀ)-1. Differently, the
electronic properties of the 2,2,2-trifluoroethoxy group might be
responsible for the reduced potency of (ꢀ)-4 at a1D-adrenoceptor.
On 2002, a molecular modeling study [47] has shown that (ꢀ)-1
binds at the a1-adrenoceptor subtypes through the interaction of its
secondary amine function and methoxy group with three aspartate
residues in the third transmembrane (TM3) domain and with three
serine residues in TM5 domain, respectively. An additional inter-
action would take place between the nitrogen atom of the sul-
phonamide moiety of (ꢀ)-1 and a glutamate residue located into
the TM4 domain of a1D-adrenoceptor. Furthermore, previous
mutagenesis studies [48] had identified two phenylalanine resi-
dues, which are conserved in TM7 domains of all a1-adrenoceptor
subtypes, as binding sites for a non-selective interaction with many
a1-antagonists. As consequence, we hypothesize that the phenyl
ring of the 2-ethoxyphenoxy moiety of (ꢀ)-1 could interact with
these phenylalanine residues, as advanced for the related 2,6-
dimethoxyphenoxy fragment of the a1A-adrenoceptor antagonist
In rat tissues, (ꢀ)-1 displayed a slightly higher potency for the
a1D-adrenoceptor with respect to a1A and a1B subtypes (3- and 5-
fold, respectively) as previously reported [17,18]. Compounds
(ꢀ)-2e(ꢀ)-4 were almost equipotent with (ꢀ)-1 at a1A and a1B
subtypes, and slightly less potent (5e10-fold) at the a1D subtype,
whereas (ꢀ)-5a and (ꢀ)-5b, in agreement with the binding results,
were much weaker antagonists at all a1-adrenoceptors (from 35 to
600 times less potent than (ꢀ)-1). Compound (ꢀ)-3 displayed the
best antagonist profile, being as potent as (ꢀ)-1 at the a1A-adre-
noceptor (pKB, 9.66 vs. 9.46) and 5e6-fold less potent at the a1B and
a1D subtypes (pKB, 8.57 and 9.20 vs. 9.30 and 10.00, respectively).
Thus, it was 12-fold more selective for the a1A-adrenoceptor over
the a1B subtype while maintaining a relevant blocking activity at
the a1D-adrenoceptor.

G. Sagratini et al. / European Journal of Medicinal Chemistry 45 (2010) 5800e5807
5803
Table 2
Functional antagonist affinities, expressed as pA2 or pKB, of compounds (ꢀ)-2 e (ꢀ)-4, (ꢀ)-5a, (ꢀ)-5b, and (ꢀ)-1 as reference, at a1-adrenoceptor subtypes of isolated rat
prostatic vas deferens (a1A), spleen (a1B) and thoracic aorta (a1D).
O
H3C
H2N
O
CH3
(R)
O
S
H3C
H2N
O
CH3
H
O
O
(R)
H
S
N
O
O
O
H
N
O
H
H3C
R
(-)-1 - (-)-4
(-)-5a (cis) , (-)-5b (cis)
Selectivity ratioc
a
b
Compound
R
pA2 or pKB
a1A
a1B
a1D
a1A
/a1B
a1A
/a1D
a1D/a1B
(ꢀ)-1
(ꢀ)-2
(ꢀ)-3
(ꢀ)-4
(ꢀ)-5a
(ꢀ)-5b
CH3CH2
9.46 ꢁ 0.13b
9.66 ꢁ 0.08b
9.66 ꢁ 0.01b
9.58 ꢁ 0.08a
7.52 ꢁ 0.06a
7.79 ꢁ 0.04a
9.30 ꢁ 0.08b
8.81 ꢁ 0.10b
8.57 ꢁ 0.02b
8.96 ꢁ 0.02a
7.51 ꢁ 0.04a
7.76 ꢁ 0.08b
10.00 ꢁ 0.10b
9.30 ꢁ 0.04a
9.20 ꢁ 0.04b
9.02 ꢁ 0.01a
7.22 ꢁ 0.03a
7.67 ꢁ 0.03a
1.5
7
12
4
1
1
0.3
2
3
4
2
5
3
4
1
0.5
0.8
(CH3)2CH
C6H5CH2
CF3CH2
1
a
pA2 values, expressed as means ꢁ SEM of three different concentrations, each tested at least four times.
pKB values (ꢁSEM) calculated according to van Rossum.
b
c
Calculated by the antilog of the difference between pA2 or pKB values at different a1-adrenoceptor subtypes.
WB4101 [49], whereas the 2-ethoxy function could bind the OH
fragment of (ꢀ)-1 into the cis-3-methyl-[2,3-dihydro-1,4-benzo-
dioxin-2-yl]methylamino moiety. This structural modification
could negatively affect one or both of above supposed interactions
with TM6 and TM7 domains with a possible and important
reduction of the overall binding (Fig. 2).
serine residue present into the TM6 domain of all three a1-adre-
noceptors [47]. Thus, it is possible to speculate that specific alkoxy
or arylalkoxy groups, in place of the ethoxy moiety of (ꢀ)-1, could
disrupt these interactions by steric or electronic effects established
with some aminoacidic residues into the a1-adrenoceptor TM
domains (Fig. 2). This may explain the small reduction of antagonist
activity at a1B-and a1D-adrenoceptors, with respect to (ꢀ)-1, of
compounds (ꢀ)-2e(ꢀ)-4, bearing an i-propoxy, benzyloxy, and
2,2,2-trifluoroethoxy groups, respectively.
5. Conclusion
The benzyl analogue (ꢀ)-3 of (ꢀ)-1 is the most interesting
compound of the present study, owing to its antagonist profile at
On the other hand, the marked drop in affinity observed for
(ꢀ)-5a and (ꢀ)-5b at all the a1-adrenoceptor subtypes could be due
to the reduced flexibility and the conformational constraint
induced by cyclization of the 2-ethoxyphenoxyethylamino
a1A-and a1D-adrenoceptors (a1A
> a1D > a1B) in functional tests,
with a 12-fold higher potency at a1A-adrenoceptor with respect to
the a1B subtype. These properties could have relevance in BPH
therapy because compounds like (ꢀ)-3 may have improved
Fig. 2. Hypothetical interaction model of tamsulosin analogue compounds (ꢀ)-2 e (ꢀ)-4 and (ꢀ)-5a and (ꢀ)-5b with the aminoacidic residues of seven transmembrane domains of
a1-adrenoceptors. The model derives from that suggested by Ishiguro for tamsulosin [47], involving the binding of some chemical functions with the TM3, TM4, and TM5 domains,
which has been here adapted with new supposed interactions. It is conceivable that compounds (ꢀ)-2e(ꢀ)-4, with R ¼ (CH3)2CH, C6H5CH2, and CF3CH2, would bind also to the
serine and phenylalanine residues of TM6 and TM7 domains, respectively (left); on the contrary, the same interactions would be missing for compounds (ꢀ)-5a and (ꢀ)-5b (right).

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G. Sagratini et al. / European Journal of Medicinal Chemistry 45 (2010) 5800e5807
uroselectivity compared to (ꢀ)-1. This is because of the potential
major beneficial clinical effects deriving from the preferential
antagonism of the a1A-adrenoceptor, which relieves the voiding
symptoms due to the bladder outlet obstruction mediated by
prostate smooth muscle contraction, and from an effective antag-
onism towards the a1D subtype, which alleviates the symptoms of
bladder filling. At the same time, the reduced blocking activity of
the a1B-adrenoceptor subserves the uroselectivity character of
(ꢀ)-3 because of minimization of the hypotensive side effect,
especially in the elderly.
CH2Ar, partly overlapped to solvent), 2.98 (sextet, J ¼ 6.2 Hz, 1H,
CHCH3), 3.85 (s, 3H, OCH3), 7.10 (m, 1H, AreH3), 7.32e7.45 (m, 1H,
AreH4), 7.53 (m, 1H, AreH6). The NH2 and SO2NH2 protons are
unapparent on the spectrum; they are expected under the waters’
low and flattened hump observed in the range 3.20e3.80 d. Anal.
Calc. for C10H16N2O3S$0.25H2O: C, 48.28; H, 6.68; N,11.25. Found: C,
48.27; H, 6.98; N, 11.16%.
6.1.1.1. Resolution of (ꢁ)-6. A solution of (ꢁ)-6 (20 g, 81.86 mmol)
and (R)-(ꢀ)-mandelic acid (12.46 g, 81.86 mmol) in MeOH
(180 mL) was evaporated to dryness to give a residue that was
6. Experimental protocols
crystallized seven times from EtOH, affording the less soluble
20
diastereomeric mandelate: 1.1 g, mp 205e206 ꢄC, [
a]
¼ ꢀ44.6
D
6.1. Chemistry
(c ¼ 1, MeOH); 1H NMR (DMSO-d6):
d
1.03 (d, J ¼ 6.2 Hz, 3H,
CH3CH), 2.46e2.68 (m, 1H, CH2CH, partly overlapped to solvent),
2.80e2.96 (m, 1H, CH2CH), 3.41 (sextet, J ¼ 6.2 Hz, 1H, CHCH3),
3.84 (s, 3H, OCH3), 4.50 (s, 1H, CHOH), 6.80e7.42 (m, 13H, AreH,
AreH3, AreH4, SO2NH2, NH3þ, OH, partly exchangeable with D2O),
7.55 (m, 1H, AreH6). Anal. Calc. for C18H24N2O6S$1.5H2O$0.1C2H5-
OH: C, 51.06; H, 6.49; N, 6.47. Found: C, 50.77; H, 6.21; N, 6.17%.
Potassium carbonate (3.5 g) was added to a solution of the above
salt (0.7 g) dissolved in H2O (11.2 mL). Following 3 h stirring at
room temperature, the precipitate was collected by filtration to give
Melting points were taken in glass capillary tubes on a Büchi SMP-
20 apparatus and are uncorrected. IR and NMR spectra were recorded
on PerkineElmer 297 and Varian VXR 300 instruments, respectively.
The IR spectra, not included, were consistent with all the assigned
structures. The elemental analyses of compounds, performed on
a Fisons instrument mod. EA1108CHNS-O, agreed with the calculated
values within the range ꢁ0.4%. Electron impact ionization (EI) mass
spectra were obtained with a HewlettePackard instrument, consist-
ing of model 5890 A for the separation section and model 5971 A for
the mass section. Optical rotations were measured on a PerkineElmer
241 MC polarimeter. Analytical HPLC was performed on a Hew-
lettePackard 1090 apparatus, series II, with UV detector, equipped
enantiomer (ꢀ)-6 as amorphous white solid: 0.45 g; mp
20
165e166 ꢄC; [
a]
¼ ꢀ16.8 (c ¼ 1, MeOH) (ref. 27, mp 166e167 ꢄC,
D
23
[a
]
¼ ꢀ17.3 (c ¼ 1.07, MeOH)). 1H NMR (DMSO-d6):
d 0.92 (d,
D
J ¼ 6.2 Hz, 3H, CH3CH), 2.40e2.70 (m, 2H, CH2CH, partly overlapped
to solvent), 2.95 (sextet, J ¼ 6.2 Hz, 1H, CHCH3), 3.85 (s, 3H, OCH3),
7.10 (m, 1H, AreH3), 7.30e7.40 (m, 1H, AreH4), 7.51 (m, 1H, AreH6).
The NH2 and SO2NH2 protons are unapparent on the spectrum;
they are expected under the waters’ low and flattened hump
with a Luna C18 column (250 mm ꢃ 4.6 mm i.d., 5
mm) from Phe-
nomenex (Chesire, UK). Chromatographic separations were per-
formed on silica gel columns (Kieselgel 40, 0.040e0.063 mm, Merck)
by flash (when non specified) or gravity chromatography. Rf values
were determined with silica gel TLC plates (Kieselgel 60 F254, layer
thickness 0.25 mm, Merck). The composition and volumetric ratio of
eluting mixtures were: A, chloroformeethyl acetateemethanole28%
ammonia (4:4:2:0.1); B, ethyl acetateecyclohexane (5:5); C, chlor-
oformeethyl acetateemethanole28% ammonia (4:4:1:0.1); D,
chloroformeethyl acetateemethanole28% ammonia (4:4:0.5:0.05);
E, ethyl acetateepetroleum ether (2:8); F, chloroformeethyl
acetateemethanole28% ammonia (4:4:0.2:0.05); G, petroleum
etheremethanol (9:1). Petroleum ether refers to the fraction with
a boiling point of 40e60 ꢄC. The term “dried” refers to the use of
anhydrous sodium sulphate. Compounds were named following
IUPAC rules as applied by ACD/Name software, version 7.0 (Advanced
Chemistry Development, Inc., Toronto, Canada). Yields of purified
products were not optimized. Chemicals and reagents were
purchased from SigmaeAldrich Srl (Milano, Italy) or Lancaster
research chemicals (Chiminord, Srl, Cusano Milanino, Milano, Italy).
observed in the range 3.20e3.80
d.
Anal. Calc. for
C10H16N2O3S$0.25H2O: C, 48.28; H, 6.68; N, 11.25. Found: C, 48.01;
H, 6.77; N, 11.44%.
6.1.1.2. Determination of theoptical purityof (ꢀ)-6. (1R)-(ꢀ)-Menthyl
chloroformate (0.13 g, 0.61 mmol) and 2 N NaOH (0.15 mL,
0.31 mmol) were simultaneously added dropwise to a cooled (0 ꢄC)
and stirred solution of (ꢁ)-6 (0.15 g, 0.61 mmol) in 2 N NaOH
(0.15 mL, 0.31 mmol). After stirring 2 h at 0 ꢄC and 2 h at room
temperature, the reaction mixture was acidified with 2 N HCl and
extracted with ethyl acetate. Removal of the dried solvent gave
a crude residue that was purified by column chromatography
(mixture B) to give 0.1 g of a mixture of the two diastereomers as
waxy solid: MS (EI) m/z ¼ 426 [Mþ]. 1H NMR (DMSO-d6),
d:
0.75e2.08 (m, 21H, CH3CHN and menthyl), 2.60e2.92 (m, 2H,
CH2CHN), 4.02 (s, 3H, OCH3), 4.32e4.60 (m, 2H, CH3CHN and OCH
menthyl), 5.06 (br s, 3H, CONH and SO2NH2, exchangeable with
D2O), 6.98 (m, 1H, AreH3), 7.32e7.44 (m, 1H, AreH4), 7.73 (m, 1H,
AreH6).
6.1.1. (ꢁ)-5-(2-Aminopropyl)-2-methoxybenzenesulfonamide
[(ꢁ)-6]
Amonium acetate (8.41 g, 109 mmol) and sodium cyanobor-
ohydride (0.69 g, 10.9 mmol) were added consecutively to
Similarly, the diastereomer of (ꢀ)-6 with (1R)-(ꢀ)-menthyl
chloroformate was prepared following the above procedure: MS
a
suspension of 2-methoxy-5-(2-oxopropyl)-1-benzenesulfona-
(EI) m/z ¼ 426 [Mþ]. 1H NMR (DMSO-d6),
d: 0.68e2.07 (m, 21H,
mide (2.5 g, 10.9 mmol) in MeOH (75 mL), then the mixture was
stirred at room temperature for 15 h. After removal of inorganic
salts by filtration through a short silica column and quick elution
with mixture A, evaporation of the solvent left an oily residue that
was transformed into the hydrochloride salt, which was crystal-
lized from MeOH. The corresponding free base was obtained
treating an aqueous solution (11 mL) with K2CO3 (7.46 g). After 2 h
stirring at room temperature, the crude precipitate was collected
and purified by column chromatography eluting with mixture A to
give the racemic amine (ꢁ)-6 as amorphous solid: 0.9 g (35%); mp
166e168 ꢄC (ref. 30, 166e167 ꢄC); Rf ¼ 0.30 (mixture A); 1H NMR
CH3CHN and menthyl), 2.60e2.93 (m, 2H, CH2CHN), 4.00 (s, 3H,
OCH3), 4.30e4.60 (m, 2H, CH3CHN and OCH menthyl), 5.12 (br s, 3H,
CONH and SO2NH2, exchangeable with D2O), 6.99 (m, 1H, AreH3),
7.30e7.44 (m, 1H, AreH4), 7.53 (m, 1H, AreH6).
The HPLC analysis of the diastereomer obtained from (ꢀ)-6 and
of the mixture of the two diastereomers mixture obtained from
(ꢁ)-6 was performed with a reversed phase Luna C18 column
(250 mm ꢃ 4.6 mm i.d., 5
mm) fluxed (1 mL/min) 20 min with
a mixture 70:30 (v/v) of H2O/CH3CN, then changed to reach in
15 min a 30:70 ratio. The injection of a sample solution (5 L, c
m
2.5 mg/mL) of the mixture obtained from (ꢁ)-6, detectable at
(DMSO-d6):
d
0.93 (d, J ¼ 6.2 Hz, 3H, CH3CH), 2.42e2.66 (m, 2H,
275 nm, displayed two peaks with retention time of 36.19 and

G. Sagratini et al. / European Journal of Medicinal Chemistry 45 (2010) 5800e5807
5805
36.53 min, whereas the diastereomer obtained from (ꢀ)-6 gave
a principal peak at a retention time of 36.50 min with a 99.3% of
enantiomeric purity.
d
1.30 (d, J ¼ 6.6 Hz, 3H, CH3), 4.19 (dq, J ¼ 2.8, 6.6 Hz, 1H, H3), 4.30
(d, J ¼ 2.8 Hz, 1H, H2), 6.80e7.00 (m, 4H, AreH), 9.63 (s, 1H, CHO).
6.1.4. (ꢀ)-2-Methoxy-5-((2R)-2-cis-{[(3-methyl-2,3-dihydro-1,4-
benzodioxin-2-yl)methyl]-amino} propyl)benzenesulfonamide
[(ꢀ)-5a and (ꢀ)-5b]
6.1.2. General procedure for the synthesis of (ꢀ)-2e(ꢀ)-4
A mixture of (ꢀ)-6 (0.25 g, 1.23 mmol) and the appropriate
alkylating agent 7, 8, or 9 (0.51 mmol) in i-AmOH (10 mL) was
refluxed 3 h. After filtration, the solvent was distilled at reduced
pressure and the residue was purified by gravity column
chromatography.
A solution of 10 (0.09 g, 0.51 mmol) in MeOH (5 mL), NaBH3CN
ꢀ
(0.032 g, 0.51 mmol), and an excess of molecular sieves 3 A were
consecutively added to a solution of (ꢀ)-6 (0.25 g, 1.23 mmol) in
MeOH (10 mL) acidified to pH 6 with 2 N HCl in MeOH. After 72 h
stirring at room temperature, the molecular sieves were removed
by filtration and the solvent evaporated. The residue was purified
by gravity column chromatography (mixture F) to give diastereo-
mers (ꢀ)-5a and (ꢀ)-5b as white spongy solids.
6.1.2.1. (ꢀ)-5-((2R)-2-{[2-(2-Isopropoxyphenoxy)ethyl]amino}
propyl)-2-methoxybenzenesulfonamide (ꢀ)-2. 0.08 g (37%); mp
20
108e109 ꢄC; Rf ¼ 0.32 (mixture C); [
a
]
D
¼ ꢀ15 (c ¼ 0.5, MeOH). 1H
NMR (DMSO-d6):
d
0.91 (d, J ¼ 6.0 Hz, 3H, CH3CH), 1.20 (d,
J ¼ 6.1 Hz, 6H, (CH3)2CH), 1.62 (br s, 1H, NH, exchangeable with
D2O), 2.37e2.51 (m, 1H, CH2CH, partly overlapped to solvent),
2.66e2.98 (m, 4H, CH2CH, CH3CH, and NCH2), 3.84 (s, 3H, OCH3),
3.97 (t, J ¼ 5.6 Hz, 2H, CH2CH2O), 4.43 (septet, J ¼ 6.1 Hz, 1H,
(CH3)2CH), 6.82e7.15 (m, 7H, AreH, AreH3, SO2NH2, partly
exchangeable with D2O), 7.33e7.40 (m, 1H, AreH4), 7.54 (m, 1H,
AreH6). Anal. Calc. for C21H30N2O5S$0.25H2O: C, 59.06; H, 7.20; N,
6.56. Found: C, 59.24; H, 7.59; N, 6.67%.
6.1.4.1. (ꢀ)-5a. 0.011 g; mp 106e108 ꢄC; Rf ¼ 0.48 (mixture F);
20
[a
]
¼ ꢀ23.6 (c ¼ 0.5, MeOH). 1H NMR (DMSO-d6):
d 0.93 (d,
D
J ¼ 5.8 Hz, 3H, CH3CHN), 1.27 (d, 3H, CH3CHO), 1.68 (br s, 1H, NH,
exchangeable with D2O), 2.35e2.60 (m, 1H, CH2CH, partly over-
lapped to solvent), 2.64e3.05 (m, 4H, CH2CH, CH3CHN, NCH2),
3.75e3.95 (m, 4H OCH3, H2 benzodioxin), 4.00e4.20 (m, 1H, H3
benzodioxin), 6.75e6.90 (m, 4H, AreH benzodioxin), 6.95e7.15 (m,
3H, H3, SO2NH2, partly exchangeable with D2O), 7.35e7.43 (m, 1H,
H4), 7.55 (m, 1H, H6). Anal. Calc. for C20H26N2O5S$0.75H2O: C, 57.19;
H, 6.59; N, 6.66. Found: C, 57.46; H, 6.39; N, 6.59%.
6.1.2.2. (ꢀ)-5-[(2R)-2-({2-[2-(Benzyloxy)phenoxy]ethyl}amino)
propyl]-2-methoxybenzenesulfonamide (ꢀ)-3. 0.06 g (25%); mp
20
126e127 ꢄC; Rf ¼ 0.44 (mixture D); [
a
]
¼ ꢀ11.2 (c ¼ 0.5, MeOH).
6.1.4.2. (ꢀ)-5b. 0.018 g; mp 106e108 ꢄC; Rf ¼ 0.40 (mixture F);
D
20
1H NMR (DMSO-d6):
d
0.87 (d, J ¼ 5.8 Hz, 3H, CH3CH), 1.75 (br s,
[a
]
¼ ꢀ19.6 (c ¼ 0.5, MeOH). 1H NMR (DMSO-d6):
d 0.91 (d,
D
1H, NH, exchangeable with D2O), 2.33e2.52 (m, 1H, CH2CH,
partly overlapped to solvent), 2.63e3.00 (m, 4H, CH2CH, CH3CH,
and NCH2), 3.83 (s, 3H, OCH3), 4.01 (t, J ¼ 5.6 Hz, 2H, CH2CH2O),
5.08 (s, 2H, C6H5CH2), 6.82e7.10 (m, 7H, AreH, AreH3, SO2NH2,
partly exchangeable with D2O), 7.28e7.46 (m, 6H, C6H5CH2,
AreH4), 7.53 (m, 1H, AreH6). Anal. Calc. for C25H30N2O5S$0.5H2O:
C, 62.61; H, 6.51; N, 5.84. Found: C, 62.88; H, 6.86; N, 5.51%.
J ¼ 5.8 Hz, 3H, CH3CHN), 1.18 (d, 3H, CH3CHO), 1.72 (br s, 1H, NH,
exchangeable with D2O), 2.35e2.55 (m, 1H, CH2CHN, partly over-
lapped to solvent), 2.62e2.98 (m, 4H, CH2CHN, CH3CHN, NCH2),
3.85 (s, 3H, OCH3), 4.05e4.20 (m, 1H, H2 benzodioxin), 4.35e4.48
(m, 1H, H3 benzodioxin), 6.75e6.87 (m, 4H, AreH benzodioxin),
6.95e7.16 (m, 3H, H3, SO2NH2, partly exchangeable with D2O),
7.30e7.42 (m, 1H, H4), 7.54 (m, 1H, H6). Anal. Calc. for
C20H26N2O5S$0.25H2O: C, 58.45; H, 6.50; N, 6.81. Found: C, 58.30;
H, 6.82; N, 6.58%.
6.1.2.3. (ꢀ)-2-Methoxy-5-[(2R)-2-({2-[2-(2,2,2-trifluoroethoxy)phe-
noxy]ethyl}amino)propyl]benzenesulfonamide (ꢀ)-4. 0.01 g (4%);
20
mp 117e118 ꢄC; Rf ¼ 0.42 (mixture C);
[
a
]
¼ ꢀ12 (c ¼ 0.5,
6.1.5. 1-(2-Bromoethoxy)-2-isopropoxybenzene (7)
D
MeOH). 1H NMR (DMSO-d6):
d
0.92 (d, J ¼ 6.2 Hz, 3H, CH3CH),
A mixture of 2-isopropoxyphenol (3 g, 19 mmol), 1,2-dibromo-
ethane (29.62 g, 150 mmol), and KOH (1.06 g, 19 mmol) in EtOH
(50 mL) was refluxed for 75 h. After distillation of solvent and 1,2-
dibromoethane excess at reduced pressure, the residue was washed
with 2 N NaOH and extracted with CHCl3. Removal of the dried
solvent gave a residue that was purified by column chromatog-
raphy (mixture G), affording 7 as an oil: 2.2 g (45%); Rf ¼ 0.56
1.70 (br s, 1H, NH, exchangeable with D2O), 2.36e2.52 (m, 1H,
CH2CH, partly overlapped to solvent), 2.68e3.06 (m, 4H, CH2CH,
CH3CH, and NCH2), 3.84 (s, 3H, OCH3), 4.04 (t, J ¼ 5.6 Hz, 2H,
CH2CH2O), 4.68 (quartet, J ¼ 9.0, 2H, CH2CF3), 6.88e7.16 (m, 7H,
AreH, AreH3, SO2NH2, partly exchangeable with D2O), 7.32e7.43
(m, 1H, AreH4), 7.55 (m, 1H, AreH6). Anal. Calc. for C20H25F3N2O5-
S$0.5H2O: C, 50.94; H, 5.55; N, 5.94. Found: C, 50.65; H, 5.36; N, 6.30%.
(mixture G); MS (EI) m/z ¼ 258 [Mþ]. 1H NMR (CDCl3):
d 1.37 (d,
J ¼ 6.2 Hz, 6H, (CH3)2CH), 3.66 (t, J ¼ 6.6 Hz, 2H, CH2Br), 4.33 (t,
J ¼ 6.6 Hz, 2H, OCH2), 4.56 (septet, J ¼ 6.2 Hz, 1H, (CH3)2CH),
6.82e7.12 (m, 4H, AreH).
6.1.3. (ꢁ)-cis-3-Methyl-2,3-dihydro-1,4-benzodioxine-2-
carbaldehyde (10)
A solution of DMSO (0.4 g, 5.28 mmol) in dry CH2Cl2 (0.5 mL)
was added dropwise to a stirred and cooled at ꢀ 60 ꢄC solution of
(COCl)2 (0.31 g, 2.4 mmol) in dry CH2Cl2 (12 mL). After 2 min, the
temperature was allowed to raise until ꢀ 30 ꢄC, then a solution of
[cis-3-methyl-2,3-dihydro-1,4-benzodioxin-2-yl]methanol (0.2 g,
1.10 mmol) in dry CH2Cl2 (2 mL) was added to reaction mixture.
After stirring for additional 15 min, triethylamine (0.56 g,
5.5 mmol) was dropped inwards at the same temperature, which
was left to increase to room temperature. The resulting mixture
was stirred for further 2 h, treated with H2O/CH2Cl2 and then
extracted several time with CHCl3. After washing with 1% HCl, 5%
Na2CO3 and H2O, the organic solvents were dried and then evap-
orated. The residue was purified by gravity column chromatog-
raphy (mixture E) to give the aldheyde 10 as an oil: 0.11 g (61%);
Rf ¼ 0.45 (mixture E); MS (EI) m/z ¼ 178 [Mþ]; 1H NMR (DMSO-d6):
6.2. Pharmacology
6.2.1. Binding assays
Competition binding assays to cloned human a1a, a1b, and a1d-
adrenoceptor subtypes were performed in membrane preparations
from CHO (Chinese Hamster Ovary) cell lines transfected by elec-
troporation with DNA expressing the gene encoding each
a1-adrenoceptor. Cloning and stable expression of the human a1
adrenoceptor gene was performed as previously described [40].
-
Briefly, CHO cells membranes (30 mg proteins) were incubated in
50 mM TriseHCl buffer, pH 7.4, with 0.1e0.4 nM [3H]prazosin, in
a final volume of 1.02 mL for 30 min at 25 ꢄC, in the absence or
presence of competing drugs (1 pM-10
was determined in the presence of 10
m
M). Non-specific binding
M phentolamine. The
m

5806
G. Sagratini et al. / European Journal of Medicinal Chemistry 45 (2010) 5800e5807
incubation was stopped by addition of ice-cold TriseHCl buffer and
rapid filtration through 0.2% poly(ethylenimine)-pretreated
Whatman GF/B or Schleicher & Schuell GF52 filters.
strips (15 mm ꢃ 3 mm) were cut helically from rat thoracic aorta
beginning from the end most proximal to the heart. The endothelium
was removed by rubbing with filter paper: the absence of 100
acetylcholine-induced relaxation to preparations contracted with
M noradrenaline was taken as an indicator that the vessel was
mM
6.2.2. Functional experiments
1 m
Tissues for experiments were taken from male Wistar rats
(275e300 g; Charles River, Como, Italy). All animal testing was
carried out according to the European Community Council
Directive of 24 November 1986 (86/609/EEC). Animals were
sacrificed by cervical dislocation and the required organs were
isolated. Vas deferens prostatic portion, spleen and aorta
were freed from adhering connective tissue and set up rapidly,
under a suitable tension, in 20-mL organ baths. The bath medium,
containing physiological salt solution (pH 7.4), was kept at 37 ꢄC
and aerated with 5% CO2: 95% O2. Concentration-response curves
were constructed by cumulative addition of agonist. The agonist
concentration in the bath was increased approximately 3-fold at
each step, with each addition being made only after the response
of the previous addition had attained a maximal level and
remained steady. Contractions were recorded by means of a force
displacement transducer connected to the MacLab System Pow-
erLab/800.
denuded successfully. The strips were then tied with surgical thread
and suspended in an organ bath containing Krebs solution of the
following composition (mM): NaCl,118.4; KCl, 4.7; CaCl2,1.9; MgSO4,
1.2; NaH2PO4, 1.2; NaHCO3, 25; glucose, 11.7. Cocaine hydrochloride
(10
hydrochloride (1
extraneuronaluptakeoftheagonistnoradrenalineand toblock the
m
M), normetanephrine hydrochloride (1
mM), and propranolol
mM) were added to prevent the neuronal and
b-
adrenoceptors, respectively. In the absence of these inhibitors the
noradrenaline concentration-response curve was significantly dis-
placed to the right (data not shown).
After an equilibration period of at least 2 h under an optimal
tension of 1 g, cumulative noradrenaline concentration-response
curves were recorded isometrically at 1 h intervals, the first being
discarded and the second one taken as control. After inspection of
vehicle activity, the antagonist was allowed to equilibrate with the
tissue for 30 min before generation of the third cumulative
concentration-response curve to the agonist. Noradrenaline solu-
tions contained 0.05% K2EDTA in 0.9% NaCl to prevent oxidation.
In all experiments a control agonist concentration-response
curve (vehicle) was constructed in the presence of the maximum
DMSO concentration (0.5%) contained in the bathing solutions being
the solvent used for dissolution of tested antagonists on preparing
the initial stock solution. These curves were not different from the
previous one indicating no interference of solvent in the agonist
effect. The agonist-elicited concentration-response curves obtained
in the presence of the tested concentrations of antagonist were
related to the vehicle control curve, taking the maximal response as
100%. Parallel experiments in which tissues did not receive any
antagonist were run in order to check any variation in sensitivity.
The experimental conditions used for the investigation at a1-adre-
noceptor subtypes are procedures taken from quoted literatures.
All pharmacological graphics were drawn by a Prism 4.0
computer program (GraphPad Software, Inc., San Diego, CA, USA).
Chemicals, (ꢀ)-noradrenaline bitartrate, (ꢀ)-phenylephrine
hydrochloride, cocaine hydrochloride, normetanephrine hydro-
chloride and (ꢁ)-propranolol hydrochloride were purchased from
SigmaeAldrich Srl (Milano, Italy).
6.2.2.3. Spleen. Affinity at rat spleen a1B-adrenoceptor was evalu-
ated according to a reported procedure [44]. The spleen was
removed and bisected longitudinally in two strips, which were
suspended in tissue baths containing Krebs solution of the following
composition (mM): NaCl, 120; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.5;
KH2PO4, 1.2; NaHCO3, 20; glucose, 11; EDTA, 0.01. Propranolol
hydrochloride (4 mM) was added to block b-adrenoceptors.
Following the reported procedure the spleen strips were placed
under 1 g resting tension and equilibrated for 2 h. A first cumulative
concentration-response curve to the agonist phenylephrine was fast
taken isometrically, followed by 30 min washing. Subsequently,
a second cumulative curve was constructed followed by 30 min
washing. Each tissue was then incubated for 30 min either with
vehicle or different antagonist concentrations before constructing
the new phenylephrine concentration-response curve (third curve).
6.2.3. Data analysis
Data from binding assays were analysed using a non-linear
curve-fitting program Allfit [50]. Scatchard plots were linear in all
preparations and the pseudo-Hill coefficients non significantly
different from the unity (p > 0.05). The inhibition of the radioligand
specific binding by tested compounds allowed the estimation of
IC50 values that were converted to affinity constants (Ki) by the
ChengePrusoff equation [41]: Ki ¼ IC50/(1 þ L/Kd), where L and Kd
are the concentration and the equilibrium dissociation constant of
the radioligand. Results were expressed as pKi values.
In functional studies, responses were expressed as percentage of
the maximal contraction observed in the agonist concentration-
response curve taken as control. Each response was plotted graph-
icallyas a mean fromat least four separateexperiments. Curves were
fitted to all the data by a non-linear regression using the Prism 3.0
program to calculate pEC50 values. In all cases, 50% of the maximum
for each concentration-response curve was used to evaluate the
EC50. This value, calculated in presence and in absence of antagonist
in a single tissue, was used to determine the concentration ratio.
Schild plots were constructed to estimate the pA2 values and the
slope of the regression line using experimental series obtained
from at least three different concentrations [45]. The Schild
diagrams were constructed by plotting the log (concentration
ratio ꢀ1) against the log [antagonist] and deriving it from a linear
regression using the Prism 3.0 program. When the Schild plot slope
6.2.2.1. Prostatic rat vas deferens. Affinity at a1A-adrenoceptor was
evaluated on prostatic rat vas deferens according to a reported
procedure [42]. Prostatic portions of 2 cm length were mounted
under 0.35 g tension at 37 ꢄC in Tyrode solution of the following
composition (mM): NaCl, 130; KCl, 2; CaCl2, 1.8; MgCl2, 0.89;
NaH2PO4, 0.42; NaHCO3, 25; glucose, 5.6. To prevent the neuronal
uptake of the agonist noradrenaline, cocaine hydrochloride (10 mM)
was added to the Tyrode solution 20 min before the agonist
cumulative concentration-response curve. Vasa deferentia were
equilibrated for 45 min, with washing every 15 min. After the
equilibration period, tissues were primed twice by addition of
10 mM noradrenaline in order to obtain a constant response. After
another washing and equilibration period of 45 min, a cumulative
isotonic noradrenaline concentration-response curve was con-
structed to determine the relationship between agonist concen-
trations and contractile response. When measuring the effect of the
antagonist, it was allowed to equilibrate with the tissue for 30 min
before constructing a new concentration-response curve to the
agonist. The noradrenaline solution contained 0.05% Na2S2O5 to
prevent oxidation.
6.2.2.2. Aorta. Affinity at rat aorta a1D-adrenoceptor was evaluated
using a procedure adapted from that already reported [43]. Two

G. Sagratini et al. / European Journal of Medicinal Chemistry 45 (2010) 5800e5807
5807
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was not significantly different from unity (p > 0.05), the regression
was recalculated with a constrained slope of 1 and the result given
as a pA2 value. In a number of cases, Schild analysis could not be
performed due to the nonparallel slopes of concentration-response
curves. As consequence, pKB values were calculated, according to
van Rossum [46], at the lowest antagonist concentration giving
a significant rightward shift of the agonist concentration-response
curve [log (concentration ratio ꢀ1) ꢂ 0.5]. Thus, the potency of
(ꢀ)-4 and (ꢀ)-5a at all a1 subtypes, of (ꢀ)-5b at a1A and a1D
-
adrenoceptors, and of (ꢀ)-2 at the a1D subtype was expressed as the
pA2 value, whereas the potency of (ꢀ)-1 and (ꢀ)-3 at all a1
subtypes, of (ꢀ)-2 at a1A and a1B-adrenoceptors, and of (ꢀ)-5b at
the a1B subtype was expressed by the pKB value.
All data were compared by Student’s t-test and presented as
means ꢁ SEM of 2e3 experiments, performed in triplicate, in
binging assays, and of 4e6 experiments in functional tests. A p val-
ue < 0.05 was taken to indicate a statistically significant difference.
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