Synthesis and structure–activity relationships of novel arylpiperazines as potent antagonists of α1-adrenoceptor

Article abstract of DOI:10.1016/j.ejmech.2016.06.052

Arylpiperazines 2–11 were synthesized, and their biological profiles at α1-adrenergic receptors (α1-ARs) assessed by binding assays in CHO cells expressing human cloned subtypes and by functional experiments in isolated rat vas deferens (α1A), spleen (α1B

Full text of DOI:10.1016/j.ejmech.2016.06.052

Accepted Manuscript
Synthesis and structure-activity relationships of novel arylpiperazines as potent
antagonists of α1-adrenoceptor
Renata Oliveira Silva, Andressa Souza de Oliveira, Laís Flávia Nunes Lemes,
Luciana de Camargo Nascente, Patrícia Coelho do Nascimento Nogueira, Edilberto
R. Silveira, Guilherme D. Brand, Giulio Vistoli, Antonio Cilia, Elena Poggesi, Michela
Buccioni, Gabriella Marucci, Maria Laura Bolognesi, Luiz Antonio Soares Romeiro
PII:
S0223-5234(16)30542-6
10.1016/j.ejmech.2016.06.052
EJMECH 8713
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 February 2016
Revised Date: 10 June 2016
Accepted Date: 28 June 2016
Please cite this article as: R.O. Silva, A.S. de Oliveira, L.F. Nunes Lemes, L. de Camargo Nascente, P.
Coelho do Nascimento Nogueira, E.R. Silveira, G.D. Brand, G. Vistoli, A. Cilia, E. Poggesi, M. Buccioni,
G. Marucci, M.L. Bolognesi, L.A. Soares Romeiro, Synthesis and structure-activity relationships of novel
arylpiperazines as potent antagonists of α1-adrenoceptor, European Journal of Medicinal Chemistry
(2016), doi: 10.1016/j.ejmech.2016.06.052.
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ACCEPTED MANUSCRIPT
Pharmacophoric
subunity
b
b
Y = H or OMe
Z = H or OMe
N
N
a
a
Y
N
OR
N
OMe
O
O
A
R = Et, iPr
LASSBio 772
2-11
Z

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Synthesis and Structure−Activity Relationships of Novel Arylpiperazines as Potent
Antagonists of α1-Adrenoceptor
Renata Oliveira Silva,a, b Andressa Souza de Oliveira,a, b Laís Flávia Nunes Lemes,b Luciana de
Camargo Nascente,b Patrícia Coelho do Nascimento Nogueira,c Edilberto R. Silveira,c Guilherme
D. Brand,d Giulio Vistoli,e Antonio Cilia,f Elena Poggesi,f Michela Buccioni,g Gabriella Marucci,g
Maria Laura Bolognesi,h,* Luiz Antonio Soares Romeiroa, b
*
a
Department of Pharmacy, Health Sciences Faculty, University of Brasília, Campus Universitário
Darcy Ribeiro, 70910-900, Brasília, DF, Brazil
b
LADETER, Catholic University of Brasília, QS 07, Lote 01, EPCT, Águas Claras, 71966-700,
Brasília, DF, Brazil
c
Departament of Organic and Inorganic Chemistry, Federal University of Ceará, 60021-970,
Fortaleza, CE, Brazil
d
Chemistry Institute, University of Brasília, Campus Universitário Darcy Ribeiro, 70910-900,
Brasília, DF, Brazil
e Department of Pharmaceutical Science, University of Milan, Via Mangiagalli 25, 20133, Milan,
Italy.
f Drug Discovery Department, Recordati S.p.A. Via Civitali 1, 20148 Milan, Italy
g School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, via S. Agostino, 1, I-
62032, Camerino, Italy
h
Department of Pharmacy and Biotechnology, University of Bologna, Via Belmeloro 6, 40126
Bologna, Italy
Key words: adrenergic receptors subtypes, BPH, α1-adrenergic antagonists, arylpiperazines
* Corresponding author: Phone +61 3107 1620; e-mail, luizromeiro@unb.br
* Corresponding author: Phone +39 051 2099717; e-mail, marialaura.bolognesi@unibo.it

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Abstract
Arylpiperazines 2-11 were synthesized, and their biological profiles at α1-adrenergic receptors (α1-
ARs) assessed by binding assays in CHO cells expressing human cloned subtypes and by functional
experiments in isolated rat vas deferens (α1A), spleen (α1B), and aorta (α1D). Modifications at the
1,3-benzodioxole and phenyl phamacophoric units resulted in the identification of a number of
potent compounds (moderately selective with respect to the α1b-AR), in binding experiments.
Notably, compound 7 (LDT451) showed a subnanomolar pKi of 9.41 towards α1a-AR. An
encouragingly lower α1B-potency was a general trend for all the series of compounds, which
showed α1A/D over α1B selectivity in functional assays. If adequately optimized, such peculiar
selectivity could have relevance for a potential LUTS/BPH therapeutic application.

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1. Introduction
α1-Adrenergic receptors (α1-ARsa), activated by norepinephrine (NE), play important roles in
cardiovascular and urogenital physiology.1 They also represent one of the most investigated
families of class A G protein coupled receptors (GPCRs).
The family encompasses at least three distinct subtypes (α1A, α1B, and α1D), which differs for
their biological structure, tissue distributions, pharmacological properties, and signal transduction.
In the past, studies aimed to assess the specific functional responses mediated by each α1-AR
subtype have been hindered by the lack of truly subtype-selective drugs.2 More recently, studies on
genetically modified mice lacking or overexpressing one or more α1-AR subtypes have shed some
light on the functional roles played by distinct receptors. However, our understanding on the
functional implications of α1-AR heterogeneity in physiological systems is still quite limited.3
In addition to their importance from a chemical biology perspective, α1-ARs are validated drug
targets for current drug discovery.2 Indeed, several studies confirmed that α1-ARs are critically
involved in benign prostatic hyperplasia (BPH), hypertension, prostate cancer, and other diseases
representing currently unmet medical needs.4
In particular, the alpha-blocker therapy for BPH dawned in the mid 1970s with the use of
nonselective agents, such as phenoxybenzamine. The therapeutic options have then expanded
significantly, giving rise to the receptor-specific alpha-blockers that encompass nowadays the first
line of therapy. They treat the dynamic component of BPH by blocking α1-receptor-mediated
sympathetic stimulation to relax the smooth muscle in the prostate. Tamsulosin (Fig. 1) is a
subtype-selective α1A- and α1D-adrenoceptor antagonist that demonstrated advantages over older
and less selective agents,5 in the management of patients with lower urinary tract symptoms (LUTS)
a Abbreviations: α1-ARs, α1-adrenergic receptors; BPH, benign prostatic hyperplasia; CHO,
Chinese hamster ovary; GPCRs, G protein coupled receptors; HBA, H-bond acceptor; IR, infrared;
LUTS, lower urinary tract symptoms; MW, microwave; NE, norepinephrine; SAR, structure-
activity relationships.

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associated with BPH.6 Silodosin (Fig. 1) is a recently approved α1A antagonist that exhibits an even
improved selectivity among the α1-adrenoceptor subtypes. Its exceptionally high in vitro selectivity
for α1A- versus α1B-adrenoceptors, likely accounts for the very favorable tolerability profile.7
Indeed, both preclinical and clinical studies support the assertion that silodosin markedly reduces
dynamic neutrally mediated smooth muscle relaxation in the lower urinary tract while minimizing
undesirable effects on blood pressure regulation.
All in all, the search for new α1-antagonists as both molecular probes8 and drug candidates is still
an active area of medicinal chemistry research.
Arylpiperazines represent one of the most studied classes of α1-AR antagonists. This class includes
a considerable variety of compounds that are characterized by a common arylpiperazine scaffold,
linked through a proper polymethylene spacer to various heteraromatic moieties.2 In previous
works, some of us reported that a series of 1-phenylpiperazines bind with high affinity to α1-ARs in
both binding and functional assay.9 In particular, the congener carrying a 2-methoxyphenyl moiety
on the piperazine ring and a 1,3-benzodioxole as a second pharmacophoric unit, named LASSBio-
772 (1 in Chart 1), was the most active of the series. It showed a remarkable pKi of 9.85 for the rat
α1A-AR, a value similar to that displayed by tamsulosin (pKi = 9.89).9 The measured pKi of 8.25
for the α1B-subtype gave raise to 40-fold higher affinity for α1A-AR. 1 also presented high affinity
(pKB = 10.60) for the α1D-AR subtype in the functional rat aorta assay, showing to be equipotent to
tamsulosin (pKB = 10.78).9
A similar high activity towards α1A-AR was found for another 1-(2-methoxyphenyl)piperazine
derivative developed by some of us, i.e. LDT66.10 In particular, this derivative simultaneously
blocked both the α1A- and α1D-adrenoceptors and the 5-HT1A receptors in vitro and in vivo, and
has therefore been proposed as a multi-target ligand for the treatment of BPH and LUTS.10
These recent findings prompted us to investigate whether derivatives of 1 have the capacity for
more selective discrimination among the different α1-AR subtypes. Thus, we thought of interest to
verify if modification in the 1-(2-methoxyphenyl)piperazine group and/or in the 1,3-benzodioxole

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moiety could lead to an improvement of the affinity profile for α1-adrenoceptors, and, hopefully, of
the therapeutic application. Particularly (Chart 1), the 2-methoxy substituent of 1 was replaced with
an ethoxy or isopropoxy one to increase at different extent the steric hindrance and the hydrophobic
properties of the parent compound. To note, the isopropoxy substituent has revealed a key feature of
some known potent and selective α1-adrenoceptor antagonists.11, 12 In addition, we evaluated the
effects of opening the 1,3-benzodioxole ring, by synthesizing the corresponding acyclic analogues,
with different H-bond acceptor (HBA) contribution patterns. Here we report the synthesis of
compounds 2-11 together with their affinity profiles assessed by binding assays in cells expressing
human cloned α1-AR subtypes, and by functional experiments in isolated rat vas deferens (α1A),
spleen (α1B), and aorta (α1D). Their putative binding mode at the three subtypes was also explored,
by molecular modeling simulations.
2. Chemistry
The novel series of derivatives (2-11) were prepared by straightforward SN2 methodologies,
exploiting phenethyl bromides (12-16) and 1-(2-alkoxyphenyl)piperazines (17-19) as starting
reagents (Scheme 1).
The required 5-(2-bromoethyl)-1,3-benzodioxole (12) was synthesized through the reduction of
(1,3-benzodioxol-5-yl)acetic acid (20) with lithium aluminum hydride in tetrahydrofuran, to afford
the alcohol 219 in 98% yield. Next, treatment of 21 with CBr4 and triphenylphosphine in acetonitrile
furnished the target bromide 12 in 76% yield (Scheme 2). Conversely, the unsubstituted-phenethyl
(13), 3-methoxyphenethyl (14), 4-methoxyphenethyl (15) and 3,4-dimethoxyphenethyl (16)
bromides were commercially available.
As for the 1-(2-alkoxyphenyl)piperazine starting fragments, 1-(2-methoxyphenyl)piperazine 17 and
1-(2-ethoxyphenyl)piperazine 18 were purchased by commercial vendors, whereas 1-(2-
isopropoxyphenyl)piperazine 19 was synthesized as previously reported.13

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Bromides 12-16 were then converted into final compounds 2-11 following nucleophilic
displacement with the proper 1-(2-alkoxyphenyl)piperazine (17-19) in acetonitrile, under irradiation
in a microwave (MW) oven (operating at 2.45 GHz, 450 W) for 4 minutes (Scheme 1).
All compounds described herein gave analytical and spectral data in agreement with the proposed
structures.
3. Biology
3.1 Binding Experiments. First, the affinity profile of compounds 2−11 was evaluated by
radioreceptor binding assays using 1 and tamsulosin as reference compounds. [3H]Prazosin was
used to label α1-adrenoceptor binding sites on membranes of Chinese hamster ovary (CHO) cells
expressing human α1a, α1b, and α1d-adrenoceptor subtypes. Binding affinities were expressed as
pKi values derived using the Cheng–Prusoff equation, as previously reported.14
3.2 Functional Studies. Receptor subtype selectivity of compounds 2−9 and 11 was also determined
at α1-ARs on different isolated rat tissues, using 1 and tamsulosin as reference standards. α1-AR
subtypes blocking activity was assessed by antagonism of (−)-NE-induced contraction of rat
prostatic vas deferens (α1A), rat spleen (α1B), or thoracic aorta (α1D). The potency was expressed
by the pKB value (with the exception of compound 7), according to van Rossum, and was calculated
at the lowest antagonist concentration giving a significant rightward shift of the agonist
concentration-response curve [log (concentration ratio −1) ≥ 0.5]. For compound 7 pA2 values at all
adrenoceptor subtypes were calculated from Schild plot.
4. Results and discussion
Our main goal in this study was to explore structure-activity relationships (SAR) around LASSBio-
772, a highly potent α1-AR antagonist. We thus sought to assess how modifying the
pharmacophoric features of 1, i.e. the 1,3-benzodioxole and the phenylpiperazine moieties, could
affect the antiadrenergic profile. To this end, the inhibitory affinities of 2-11 were first evaluated on

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human recombinant α1-AR. For a better comparison of the results, 1 and tamsulosin were used as
reference compounds (Table 1). To note, the inhibitory profile of 1 on human cloned receptors has
never been reported before. However, with human receptor clones available, collecting data from
single human proteins from the inception of a project would be undoubtedly more relevant from a
drug discovery point of view.
As matter of fact, we should remark that 1 showed a significant affinity profile even at human
subtypes, with Ki values in the single-digit nanomolar range. As regard as selectivity, 1 displayed a
slightly higher affinity for the α1d-adrenoceptor with respect to α1a and α1b subtypes (2.5- and 4-
fold, respectively). Next, the data obtained for compound 2 clearly highlight the role of the 1,3-
benzodioxole moiety of 1. Indeed, the presence of an unsubstituted phenyl ring in 2 and 8 resulted
in a general drop of affinity with respect to the benzodioxole derivative (compare 1 vs 2 and 7 vs 8).
This drop was more marked for the α1b-subtype, giving raise to a slight selectivity ratio.
An encouragingly lower α1b-affinity was a common trend for all the compounds, which, like 1,
showed a similar (albeit modest) α1a/d over α1b selectivity. This is a positive feature for the
potential application of these molecules in the treatment of BPH, as it leads to a reduced incidence
of cardiovascular side-effects (see below).
Conversely, the opening of the dioxole ring, affording 3-methoxy- (4, 9), 4-methoxhy- (5, 10) or
3,4-dimethoxy-phenyl derivatives (6, 11), only slightly affected inhibition. Intriguingly, the 3,4-
disubstituted phenyl turned out an optimal substituent for α1d recognition, with compound 6
resulting the most active of the series against this subtype. In details, it showed an excellent
subnanomolar affinity, with a pKi of 9.22, higher than that of 1 (pKi of 8.90).
To examine the influence of the 2-phenyl substituent on the affinity and selectivity of Lassbio-772
at α1-AR, 3 and 7 were synthesized. As expected, the sterically bulkier 2-isopropoxy moiety of 7
(adopted from RWJ 37914)11 was superior to the less bulky 2-ethoxy (3) or 2-methoxy (1) for both
α1a-AR binding affinity (pKi = 9.41 nM for 7 vs 8.95 for 3 or 8.46 for 1) and selectivity (α1a/α1b
32-fold versus 5-fold or 1.5-fold; α1a/α1d = 0.2-fold versus 0.5-fold or 2.5-fold, respectively).

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Indeed, 7 was the most active α1a-ligand, confirming that this substituent can optimally fit in a
hydrophobic subpocket, which is peculiar to the α1a-subtype. Conversely, the slightly reduced
potency of 7 at α1b-subtype could be related to the increased steric hindrance of the isopropoxy in
comparison with the methoxy moiety of 1, at this receptor site.
In summary, in binding assays the optimization at the X, Y, and R positions of 1 (Chart 1) resulted
in the identification of a number of equally or slightly more potent and more selective compounds
than 1 with respect to the α1b-AR.
Motivated by these interesting results, the synthesized compounds were also tested in functional
assays for their activity at α1-AR subtypes in isolated rat prostatic vas deferens (α1A-AR), spleen
(α1B) and thoracic aorta (α1AD-AR). Their pKB values, along with those of 1 and tamsulosin, are
listed in Table 2. We were pleased to observe that all compounds, in analogy with what reported
for 1,9 behaved as competitive antagonists. In fact, the concentration–response curves of reference
agonists after and before incubation with the tested compounds were parallel with no reduction of
the maximal effect, and the shift produced was proportional to the concentrations used.
However, we should highlight that in our hands 1 resulted significantly less potent in blocking NE-
induced contractions of rat thoracic aorta strips than what was reported before.9 One possible
explanation for the difference observed in the two studies could be attributed to the different tissue
preparation and the experimental conditions chosen in the assays. In particular, in the rat aorta strips
preparation all endothelium is preliminary removed, by rubbing the luminal surface, in order to
avoid the slight endothelium-dependent vasorelaxation through the stimulation of α1D-AR located
on endothelial cells.15 Moreover, the presence of cocaine, a known inhibitor of NE reuptake,
provides a higher endogenous agonist concentration at the receptor, influencing the dose-response
curve.
Encouragingly, 1 exhibited a functional profile very similar to that disclosed in binding
experiments. However, the results obtained in functional assays did not match the affinity profile
observed in binding studies for all the synthesized derivatives. It can be easily seen that pKB values

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derived from functional experiments of compounds 2-4 and 8, 9 and 11 are quantitatively in
disagreement with binding affinities. In particular, a general lower affinity with respect to that
found in binding occurred at the α1D-AR subtype. Conversely, functional profiles of 6 and 7 differ
from binding data both qualitatively and quantitatively. Specifically, 6, in functional assays, showed
a decrease in affinity at the α1D-AR together with a consistent concomitant increase at the α1A-
AR, resulting in an inversion of the selectivity profile. On the other hand, 7 showed a negative shift
in potency at α1A of 14-fold (pKi of 9.41 vs pKB of 8.27).
As previously proposed by Melchiorre et al.,16 the apparent discrepancy between binding and
functional data can be explained by considering the following: (i) these compounds act as inverse
agonists, and hence their affinity is system-dependent (just as does observed potency for positive
agonists)17; (ii) receptor form homo- and/or heterodimers, and hence native receptors in functional
tissues can be organized differently than cloned receptors (iii) species differences (human vs rat) ;
(iv) a different bioavailability of the compounds at the receptor level.
To rule out the first possibility, for compound 7, pA2 values at all adrenoceptor subtypes were
calculated from Schild plot.14, 18 Results showed that 7 behaves as competitive antagonist at α1A
adrenoceptor with a pA2 of 8.38 ± 0.11, but it is a not a competitive antagonist at the other subtypes.
These findings allow excluding that it acts as an inverse agonist since inverse agonist binds to the
same agonist receptor binding-site and the increase of agonist concentration restores the receptor
activity. On the other hand, a non-competitive antagonist may not interact with the same
neurotransmitter binding-site and it is impossible to have the full response of the receptor even in
the presence of high concentration of agonist. In conclusion, the different behavior of compound 7
in binding and functional assays could be due to its interaction with a binding site different from
that of NE on α1B, and α1D adrenoceptor subtypes.
To get further clues on these issues, we undertook docking simulations for molecules 2–11 by using
AR homology models. As further discussed in the supporting information (see also), we noticed
that the ligands can assume two predominant “symmetrical” binding poses, depending on the

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contact of the protonated nitrogen (ammonium head): the first is characterized by canonical
interaction with Asp106 and Tyr316, while the second involves a similar dyad of interacting
residues (Glu180 and Gln177); in both cases, the ligand can establish a key ion-pair reinforced by
H-bonds. Also the two lateral pockets accommodating the phenyl rings showed a certain degree of
symmetric similarity. As exemplified for 9 in Figure 2A, a first binding mode is stabilized by the
key ion-pair between the ammonium head and Asp106 reinforced, as mentioned above, by a H-
bond with Tyr316. The phenethyl group is inserted into a lateral subpocket where it can stabilize π-
π stacking interactions with Phe288 and Phe289 reinforced by hydrophobic contacts with Val107
and Ile178, while the methoxy substituent can elicit H-bonds with Ser188 and Ser192 plus apolar
interactions with the above cited alkyl side chains. Finally, the phenyl ring connected to the
piperazine is harbored within the second lateral subpocket where it is engaged in π-π stacking
interactions with Phe86 and Trp102, while the isopropoxy group elicits a H-bond with Gln177 plus
hydrophobic contacts with Phe308 and the carbon skeleton of Lys309. The second “symmetric”
binding mode observed for 9 is depicted in Figure 2B and is stabilized by the following set of
contacts: (1) the ion pair between the ammonium head and Glu180 reinforced by a H-bond with
Gln177; (2) the phenethyl group approaches Phe86 and Trp102 and the methoxy substituent elicits
a H-bond with Ser83 and (3) the phenyl ring connected to the piperazine contacts Phe288 and
Phe289 and the isopropoxy group stabilizes H-bonds with Ser188 and Ser192. A systematic
analysis of the best computed poses for all simulated ligands revealed that the two different
arrangements (and so the different contacts) of the ammonium head is the distinctive feature which
characterizes the monitored binding modes, while the specific pose of the two aromatic systems can
vary, depending on their steric hindrance and their capacity to stabilize H-bonds.
The possibility of two distinct arrangements with which the ammonium head interacts with the α1a
binding site is particularly relevant when considering that the role of Asp106, despite being the
most conserved negatively charged residue among the aminergic GPCRs, is largely debated. The
available mutational analyses reported contradictory results and, in particular, a recent study

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unambiguously demonstrated that mutations involving Asp106 have no significant effects on
antagonists’s affinity.19 The here reported existence of an alternative negatively charged residue
(Glu180) which can replace Asp106 might explain the still questioned role of Asp106, thus
suggesting that the two described binding modes can equally contribute in determining ligand
affinity. Clearly, the specific role of the two binding modes in influencing the ligand potency
remains to be clarified. To this end, a correlative analysis was performed, which suggest that both
binding modes contribute to ligand affinity (see the SI for experimental details) even though that
involving Asp106 seems to play a predominant role.
The correlations involving the potency values show different trends, since the relationship involving
the pose with Asp106 is clearly better than the corresponding correlation with affinity values, while
those involving the second binding mode or the average values perform largely worst. Taken
together, these results suggest that the potency is almost exclusively induced by the pose involving
Asp106 and, more importantly, the remarkable correlation involving the difference values
underlines that the other possible poses represent a side effect, the stability of which reduces the
measured potency. Overall, these results might explain the above mentioned contradictory results
coming from mutagenesis and suggest that, while the ligand affinity benefits of all binding modes a
ligand can assume, the ligand potency is strongly dependent by selected effective poses (such as
that involving Asp106). This also suggests that the ligand potency can be optimized by rendering
less favored the binding modes not involving Asp106. For example, the notable potency of 9 and 11
seems to indicate that an increased steric hindrance in both phenyl systems tends to disfavor the
ineffective pose involving Glu180. Such a symmetric architecture of the binding site, despite less
noticeable, is still present in α1b and α1d subtypes as evidenced by the corresponding docking
results which can be found in SI.
We also performed the in silico physico-chemical profiling (solubility, ionization, lipophilicity,
permeability) of the newly synthetized compounds to predict their pharmacokinetic properties (see
Table 2 in SI).

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Finally, to get preliminary clues on the cellular profile of the current series, the effect of 7 on
prostate cell growth was evaluated in vitro. Indeed, previous studies reported that treatment of PC-3
prostate cancer cells with α1-antagonists doxazosin and terazosin results in a significant loss of cell
viability, whereas tamsulosin had no effect.20 In agreement with these findings, we did not observe
any antiproliferative effect for compound 7 in a range of concentration from 0.01 to 10 ꢀM. These
data seem to confirm that the described capability of inhibiting prostate cell growth is independent
from the α1-antagonist properties, and it likely resides in the quinazoline chemotype common to
doxazosin and terazosin.
4. Conclusions
In the current work, we have described a series of novel 1-phenylpiperazines related to hit
compound 1, which show high affinity and potency towards α1-adrenoceptors. The added SAR
trends help to define the structural features involved in optimal recognition at the different receptor
subtypes for this class of molecules. The investigation of the binding mode for 2-11 provides an
explanation for these results, and the molecular insights of receptor-ligand interactions gained from
the performed docking study can be exploited for the development of novel derivatives. Of note, the
α1a/d over α1b selectivity disclosed for most of the compounds, albeit modest, could have
relevance for a potential LUTS/BPH therapeutic application. This is because of the proposed more
beneficial treatment effects deriving from the preferential antagonism of the α1A-adrenoceptor,
which relieves the voiding symptoms due to the bladder outlet obstruction mediated by prostate
smooth muscle contraction, and from a concomitant effective antagonism towards the α1D subtype,
which alleviates the symptoms of bladder filling. At the same time, the lower activity at the α1B-
adrenoceptor of the arterial vessels should minimize the blood pressure-related adverse effects, such
as orthostatic hypotension.5
Indeed, compound 3 (LDT8) has been selected for a more detailed pharmacological characterization
aimed to disclose its therapeutic potential in the treatment of BPH.21 Notably, it showed a low

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affinity (micromolar range) for receptors unrelated to BPH such as α2A-adrenoceptors, muscarinic
and 5-HT2A receptors, which is a desirable profile in terms of putative side effects.21
Based on the considerations above and also considering the simpler and achiral structures of the
current phenylpiperazines with respect to those marketed for BPH (i.e. tamsulosin and silodosin), 2-
11 provide a good starting point in the design of novel analogues for BPH.
5. Experimental
5.1. Chemistry. General information
Melting points were determined on a Quimis MQAPF 302 apparatus and are uncorrected. Infrared
1
(IR) spectra were obtained on a Perkin Elmer – Spectrum BX infrared spectrophotometer. H and
13C NMR spectra were recorded on Bruker Avance DRX500 or DRX300 instruments. Chemical
shifts (δ) are expressed in parts per million relative to internal tetramethylsilane; coupling constants
(J) are in Hertz. Microanalyses were obtained with Thermofinnigan EA1112 analyzer, using a
Metler MX5 electronic balance. Reactions were routinely monitored by thin-layer chromatography
(TLC) in silica gel (F254 Silicycle plates) and the products visualized with iodine or ultraviolet
lamp (254 and 365 nm). For normal pressure and flash column chromatography purifications,
Silicycle silica gel type 60 (size 70-230 and 230-400 mesh, respectively) was used. Unless stated
otherwise, starting materials used were high-grade commercial products. The purity of the final
compounds was assessed by a Biotage Isolera One System and was found > 95%. HRMS spectra
were acquired on a TripleTof 5600+ (Sciex, Ontario, Canada) by flow injection analysis using a
liquid chromtographer (Eksigent UltraLC 100, Sciex) set to a flow rate of 0.3mL/min. A DuoSpray
Ion Source (EsI) source was used and MS spectra were acquired in positive mode in a 100 – 1000
Da mass range using external calibration. Acquisition parameters were: TEM 450, GS1 45, CUR
25, GS2 50, ISVF 5500 and DP 80. Product ion scans were acquired with CE 45 and CES 20. Data
were analyzed using the PeakView v2.1 software.

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5.2. General procedures of synthesis and spectral data
5.2.1 Synthesis of 2-(1,3-Benzodioxol-5-yl)ethanol (18)9
To a suspension of lithium aluminum hydride (0.114 g, 3.00 mmol) in anhydrous THF (20 mL) a
solution of the acid 20 (3.00 mmol) in anhydrous THF (10 mL) was added dropwise over 15
minutes. After 4 hours, the excess of reductive agent was quenched with methanol (1 mL) and
10% aqueous NaOH solution (2 mL) until formation of aluminum hydroxide, which was
neutralized with 10% aqueous hydrochloric acid solution (ca. 5 mL). The obtained mixture was
extracted with ethyl acetate (3 x 15 mL) and the combined organic extracts were washed with
brine and concentrated at reduced pressure after drying over anhydrous sodium sulfate. The
residue was purified by chromatography (silica gel, CHCl3/EtOH 100:1) to give the
corresponding primary alcohol 21 as a yellow oil; 0.489 g (98%); Rf = 0.5 (CHCl3/EtOH 20:1);
1
IR (film, cm-1) 3351, 2883, 1607, 1503, 1489, 1442, 1247, 1040; H NMR (CDCl3): δ 1.87 (br;
1H, ArCH2CH2OH); 2.76 (t, J = 6.5 Hz, 2H, ArCH2CH2OH); 3.78 (t, J = 6.5 Hz, 2H,
ArCH2CH2OH); 5.91 (s, 2H, OCH2O); 6.65 (dd, J = 7.8 Hz, J = 1.6 Hz, 1 H, 4’); 6.71-6.70 (m,
1H, 2’); 6.75 (d, J = 7.8 Hz, 1H, 5’). 13C NMR (CDCl3): δ 38.6 (ArCH2CH2OH); 63.5
(ArCH2CH2OH); 100.6 (OCH2O); 108.1 (CH, 2’); 109,1 (CH, 5’); 121,7 (CH, 6’); 132,0 (C, 1’);
145,9 (CH, 4’); 147,5 (CH, 3’);
5.2.2 Synthesis of 5-(2-Bromoethyl)-1,3-benzodioxole (12)
To a solution of alcohol 21 (1.00 mmol) in anhydrous acetonitrile (2 mL) triphenylphosphine (0.26
g, 1.00 mmol) was added. The reaction was maintained at 0°C, while tetrabromomethane (0.33 g,
1.00 mmol) was slowly added, protected against the light, and followed by warming to room
temperature, under vigorous stirring and nitrogen atmosphere for 24 hours. The solvent was then
evaporated and the residue was purified by chromatography
(silica gel,
hexane→dichloromethane→chloroform) to give 12 as a yellow oil; 0.174 g (76%); Rf = 0.62
(CHCl3/EtOH 20:1); IR (film, cm-1) 2943, 2895, 1500, 1490, 1444, 1040; 1H NMR (CDCl3): δ 3.07

15
ACCEPTED MANUSCRIPT
(t, J = 6.0 Hz, 2H, ArCH2CH2Br), 3.51 (t, J = 6.0 Hz, 2H, ArCH2CH2Br), 5.94 (s, 2H, OCH2O),
6.67-6.76 (m, 3H, 2’, 5’ e 6’); 13C NMR (CDCl3): δ 33.3 (ArCH2CH2Br), 39.1 (ArCH2CH2Br),
101.0 (OCH2O), 108.4 (CH, 2’), 109.0 (CH, 5’), 121.7 (CH, 6’), 132.7 (C, 1’), 146.4 (C, 4’), 147.7
(C, 3’);
5.2.3 Synthesis of 1-(2-(1,3-Benzodioxol-5-yl)ethyl)-4-(2-methoxyphenyl)piperazine (1)
1 was synthesized according to published procedure.9 Rf = 0.55 (CHCl3/EtOH 20:1), mp 90-92 °C;
1
IR (KBr, cm-1) 3022, 2951, 2804, 1586, 1494, 1459, 1445, 1314, 1245, 1135, 1038, 752; H NMR
(CDCl3): δ 2.59-2.83 (m, 8H, ArCH2CH2N (4H) e NCH2CH2NAr (4H)); 3.11-3.16 (m, 4H,
NCH2CH2NAr); 3.75 (s, 3H, OCH3); 5.91 (s, 2H, OCH2O); 6.65-6.69 (m, 1H, 6’); 6.73 (m, 1H, 2’);
6.75 (d, J = 7.7 Hz, 1H, 5’); 6.85-6.88 (m, 1H, 6); 6.91-7.05 (m, 3H, 3, 4 e 5); 13C NMR (CDCl3): δ
33.1 (ArCH2CH2N); 50.4 (NCH2CH2N); 53.1 (NCH2CH2N); 55.2 (ArOCH3); 60.6 (ArCH2CH2N);
100.6 (OCH2O); 108.0 (CH, 2’); 109.0 (CH, 5’); 111.0 (CH, 3); 118.0 (CH, 6); 120.8 (CH, 5);
121.2 (CH, 6’); 122.7 (CH, 4); 133.9 (C, 1’); 141.1 (C, 1); 145.6 (C, 4’); 147.3 (C, 3’); 152.1 (Ar-
C, 2). HRMS (ESI) calcd for C20H24N2O3 [M + H]+ 341.1867, found 341.1860.
5.2.4 General procedure for the synthesis of 1-(2-alkoxyphenyl)piperazine derivatives (2-11)
In an Ace pressure tube, the corresponding bromide (12-16) (1.00 mmol), 1-phenylpiperazine
derivatives (17-19) (1.25 mmol), triethylamine (1.25 mmol), and acetonitrile (0.5 mL) were added.
The mixture was then irradiated in the microwave oven (operating at 2.45 GHz, 450 W) for 4
minutes (4 x 1’). At the end of this time the solution was concentrated in a rotatory evaporator,
solubilized in dichloromethane and mixed with silica gel. The material was purified by
chromatography (silica gel, dichloromethane→chloroform) to give the desired 1-(2-
alkoxyphenyl)piperazine derivative.
5.2.4.1 1-(2-Methoxyphenyl)-4-phenethylpiperazine (2,LDT2)

16
ACCEPTED MANUSCRIPT
Prepared by nucleophilic substitution of bromide (13) with 1-(2-methoxyphenyl)piperazine (17), as
a oil; 0.275 g (93%); Rf = 0.38 (CHCl3/EtOH 20:1); IR (film, cm-1) 2942, 2808, 1594, 1500, 1309,
1240, 1026; 1H NMR (CDCl3): δ 2.72-2.78 (m, 2H, ArCH2CH2N), 2.83 (br, 4H, NCH2CH2N), 2.90-
2.95 (m, 2H, ArCH2CH2N), 3.20 (br, 4H, NCH2CH2N), 3.88 (s, 3H, OCH3), 6.87-6.89 (m, 1H, 6);
6.92-7.05 (m, 3H, 3, 4 e 5); 7.20-7.34 (m, 5H, 2’, 3’, 4’, 5’, 6’); 13C NMR (CDCl3): δ 33.4
(ArCH2CH2N), 50.5 (NCH2CH2NAr), 53.5 (NCH2CH2NAr), 60.6 (ArCH2CH2N), 111.5 (CH, 3),
118.5 (CH, 6), 121.2 (CH, 5), 123.2 (CH, 4), 126.3 (C, 4’), 128.6 (2CH, 3’, 5’), 128.9 (2CH, 2’, 6’),
140.1 (C, 1’), 141.3 (C, 1), 152.5 (C, 2); HRMS (ESI) calcd for C19H24N2O [M + H]+ 297.1969,
found 297.1961.
5.2.4.2. 1-(2-(1,3-Benzodioxol-5-yl)ethyl)-4-(2-ethoxyphenyl)piperazine (3, LDT8)
Prepared by nucleophilic substitution of bromide (12) with 1-(2-ethoxyphenyl)piperazine (18), as a
oil; 0.330 g (93%); Rf = 0.40 (CHCl3/EtOH 20:1); IR (film, cm-1) 3022, 2951, 2804, 1586, 1494,
1
1459, 1445, 1314, 1245, 1135, 1038, 752; H NMR (CDCl3): δ 1.46 (t, J = 6.0 Hz, 3H,
ArOCH2CH3), 2.61-2.66 (m, 2H, ArCH2CH2N), 2.76-2.81 (m, 4H, NCH2CH2N e 2H,
ArCH2CH2N), 3.16 (br, 4H, NCH2CH2N), 4.07 (q, J = 6.0 Hz, 2H, ArOCH2CH3), 5.92 (s, 2H,
OCH2O), 6.66-6.76 (m, 2H, 6’, 2’), 6.78-6.99 (m, 5H, 5’, 3, 4, 5, 6); 13C NMR (CDCl3): δ
HRMS (ESI) calcd for C21H26N2O3 [M + H]+ 355.2023, found 355.2016.
5.2.4.3. 1-(3-Methoxyphenethyl)-4-(2-ethoxyphenyl)piperazine (4, LDT243)
Prepared by nucleophilic substitution of bromide (14) with 1-(2-ethoxyphenyl)piperazine (18), as a
white solid; 0.231 g (68%); Rf = 0.40 (CHCl3/EtOH 20:1); mp 58-59 °C; IR (KBr, cm-1) 2943,
1
2813, 1592, 1495, 1452, 1238, 1026; H NMR (CDCl3): δ 1.47 (t, J = 6.9 Hz, 3H, ArOCH2CH3),
2.73-2.94 (m, 8H, ArCH2CH2N, NCH2CH2N, ArCH2CH2N), 3.22 (br, 4H, NCH2CH2N), 3.81 (s,

17
ACCEPTED MANUSCRIPT
3H, OCH3), 4.08 (q, J = 6.9 Hz, 2H, ArOCH2CH3), 6.77 (dd, J = 8.1 Hz, J = 2.4 Hz, 1H, 4'), 6.81-
6.87 (m, 3H, 2’, 6’, 6), 6.91-6.97 (m, 3H, 3, 4, 5), 7.23 (t, J = 7.8 Hz, 1H, 5’); 13C NMR (CDCl3): δ
15.1 (ArOCH2CH3), 33.3 (ArCH2CH2N), 50.3 (NCH2CH2N), 53.5 (NCH2CH2N), 55.3 (OCH3, 3’),
60.5 (ArCH2CH2N), 63.7 (ArOCH2CH3), 111.7 (CH, 4’), 112.7 (CH, 3), 114.7 (CH, 2’), 118.3 (CH,
6), 121.2 (CH, 5), 121.3 (CH, 6’), 123.0 (CH, 4), 129.6 (CH, 5’), 141.2 (C, 1’), 141.5 (C, 1), 151.7
(C, 2), 159.9 (C, 3’); HRMS (ESI) calcd for C21H28N2O2 [M + H]+ 341.2231, found 341.2224.
5.2.4.4 1-(4-Methoxyphenethyl)-4-(2-ethoxyphenyl)piperazine (5, LDT244)
Prepared by nucleophilic substitution of bromide (15) with 1-(2-ethoxyphenyl)piperazine (18), as a
white solid; 0.286 g (84%); Rf = 0.40 (CHCl3/EtOH 20:1); mp 48-49 °C; IR (KBr, cm-1) 2973,
1
2947, 2814, 1609, 1589, 1307, 1245, 1044; H NMR (CDCl3): δ 1.47 (t, J = 6.8 Hz, 3H,
ArOCH2CH3), 2.67-2.70 (m, 2H, ArCH2CH2N), 2.79 (br, 4H, NCH2CH2N), 2.83-2.86 (m, 2H,
ArCH2CH2N), 3.20 (br, 4H, NCH2CH2N), 3.80 (s, 3H, OCH3), 4.08 (q, J = 6.8 Hz, 2H,
ArOCH2CH3), 6.85-6.86 (m, 3H, 3’, 5’, 6), 6.91-6.99 (m, 3H, 3, 4, 5), 7.16 (d, J = 8.2 Hz, 2H, 2’,
6’). 13C NMR (CDCl3): δ 15.1 (ArOCH2CH3), 32.6 (ArCH2CH2N), 50.5 (NCH2CH2N), 53.5
(NCH2CH2N), 55.4 (OCH3, 4’), 60.9 (ArCH2CH2N), 63.7 (ArOCH2CH3), 112.6 (CH, 3), 114.0
(2CH, 3’, 5’), 118.3 (CH, 6), 121.1 (CH, 5), 122.9 (CH, 4), 129.8 (2CH, 2’, 6’), 132.3 (C, 1’), 141.4
(C, 1), 151.7 (C, 2), 158.1 (C, 4’); HRMS (ESI) calcd for C21H28N2O2 [M + H]+ 341.2231, found
341.2224.
5.2.4.5 1-(3,4-Dimethoxyphenethyl)-4-(2-ethoxyphenyl)piperazine (6, LDT245)
Prepared by nucleophilic substitution of bromide (16) with 1-(2-ethoxyphenyl)piperazine (18), as
an oil; 0.256 g (69%); Rf = 0.40 (CHCl3/EtOH 20:1); IR (film, cm-1) 2927, 2827, 1609, 1582, 1304,
1
1241, 1028; H NMR (CDCl3): δ 1.48 (t, 3H, J = 6.93 Hz, ArOCH2CH3), 2.68-2.73 (m, 2H,
ArCH2CH2N), 2.77 (br, 4H, NCH2CH2N), 2.82-2.89 (m, 2H, ArCH2CH2N), 3.19 (br, 4H,
NCH2CH2N), 3.81 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 4.09 (q, J = 6.90 Hz, 2H, ArOCH2CH3),

18
ACCEPTED MANUSCRIPT
6.75-6.83 (m, 3H, 3’, 5’, 6), 6.83-6.92 (d, J = 6.0 Hz, 1H, 6’), 6.92-6.99 (m, 3H, 3, 4, 5); 13C NMR
(CDCl3): δ 15.0 (ArOCH2CH3), 33.7 (ArCH2CH2N), 50.6 (NCH2CH2N), 53.6 (NCH2CH2N), 55.2
(OCH3, 3’), 55.2 (OCH3, 4’), 60.5 (ArCH2CH2N), 63.7 (ArOCH2CH3), 111.4 (CH, 3), 112.7 (CH,
5’), 114.6 (CH, 2’), 118.3 (CH, 6), 120.1 (CH, 6’), 121.2 (CH, 5), 122.8 (CH, 4), 129.5 (C, 1),
141.5 (C, 1), 142.0 (C, 4’), 151.7 (C, 3’), 159.8 (C, 2); HRMS (ESI) calcd for C22H30N2O3 [M + H]+
371.2336, found 371.2329.
5.2.4.6 1-(2-(1,3-Benzodioxol-5-yl)ethyl)-4-(2-isopropoxyphenyl)piperazine (7, LDT451)
Prepared by nucleophilic substitution of bromide (12) with 1-(2-isopropoxyphenyl)piperazine (19),
as a white solid; 0.295 g (80%); Rf = 0.43 (CHCl3/EtOH 20:1); mp 59-60 °C; IR (KBr, cm-1) 2974,
1
2941, 2816, 1595, 1492, 1382, 1317, 1311, 1122, 1008; H NMR (CDCl3): δ 1.37 (d, J = 6.0 Hz,
6H, ArOCH(CH3)2), 2.62-2.68 (m, 2H, ArCH2CH2N, 4H, NCH2CH2N e 2H, ArCH2CH2N), 3.17
(br, 4H, NCH2CH2N), 4.61 (hp, J = 6,0 Hz, 1H, ArOCH(CH3)2), 5.92 (s, 2H, OCH2O), 6.67-6.70
13
(m, 1H, 6’), 6.67-6.77 (m, 2H, 2’, 5’), 6.84-6.91 (m, 1H, 6), 6.92-6.98 (m, 3H, 3, 4, 5); C NMR
(CDCl3): δ 22.5 (ArOCH(CH3)2), 33.4 (ArCH2CH2N), 50.5 (NCH2CH2N), 53.7 (NCH2CH2N),
60.9 (ArCH2CH2N), 70.4 (ArOCH(CH3)2), 100.9 (OCH2O), 108.2 (CH, 2’), 109.3 (CH, 5’), 116.4
(CH, 3), 118.7 (CH, 6), 121.6 (2CH, 5, 6’), 122.6 (CH, 4), 134.0 (C, 1’), 142.9 (C, 1), 145.9 (C, 4’),
147.7 (C, 3’), 150.6 (C, 2); HRMS (ESI) calcd for C22H28N2O3 [M + H]+ 369.2180, found
369.2173.
5.2.4.7 1-(2-Isopropoxyphenyl)-4-phenethylpiperazine (8, LDT452)
Prepared by nucleophilic substitution of bromide (13) with 1-(2-isopropoxyphenyl)piperazine (19),
as a white solid; 0.266 g (82%); Rf = 0.38 (CHCl3/EtOH 20:1); mp 67-68 °C; IR (KBr, cm-1) 2975,

19
ACCEPTED MANUSCRIPT
1
2943, 2811, 1592, 1495, 1382, 1371, 1237, 1128, 1040; H NMR (CDCl3): δ 1.38 (d, J = 10.0 Hz,
6H, ArOCH(CH3)2), 2.68-2.75 (m, 2H, ArCH2CH2N e 4H, NCH2CH2N), 2.85-2.98 (m, 2H,
ArCH2CH2N), 3.19 (br, 4H, NCH2CH2N), 4.62 (hp, J = 10,0 Hz, 1H, ArOCH(CH3)2), 6.88-6.90 (m,
13
1H, 6), 6.89-6.98 (m, 3H, 3, 4, 5), 7.20-7.34 (m, 5H, 2’, 3’, 4’, 5’, 6’); C NMR (CDCl3): δ 22.4
(ArOCH(CH3)2), 33.7 (ArCH2CH2N), 50.5 (NCH2CH2NAr), 53.7 (NCH2CH2NAr), 60.7
(ArCH2CH2N), 70.4 (ArOCH(CH3)2), 116.3 (CH, 3), 118.6 (CH, 6), 121.6 (CH, 5), 122.7 (CH, 4),
126.2 (C, 4’), 128.6 (2CH, 3’, 5’), 128.9 (2CH, 2’, 6’), 140.0 (C, 1’), 142.3 (C, 1), 150.5 (C, 2);
HRMS (ESI) calcd for C21H28N2O [M + H]+ 325.2282, found 326.2274.
5.2.4.8 1-(3-Methoxyphenethyl)-4-(2-isopropoxyphenyl)piperazine (9, LDT453)
Prepared by nucleophilic substitution of bromide (14) with 1-(2-isopropoxyphenyl)piperazine (19),
as a white solid; 0.142 g (40%); Rf = 0.42 (CHCl3/EtOH 20:1); mp 57-58 °C; IR (KBr, cm-1) 2944,
1
2809, 1583, 1492, 1444, 1370, 1311, 1236, 1125, 1054; H NMR (CDCl3): δ 1.37 (d, J = 6.0 Hz,
6H, ArOCH(CH3)2), 2.71-2.75 (m, 2H, ArCH2CH2N), 2.78 (br, 4H, NCH2CH2N), 2.87-2.91 (m,
2H, ArCH2CH2N), 3.20 (br, 4H, NCH2CH2N), 3.81 (s, 3H, OCH3), 4.61 (hp, J = 6.0 Hz, 1H,
ArOCH(CH3)2), 6.77 (dd, J = 8.1 Hz, J = 2.0 Hz, 1H, 4'), 6.81 (br, 1H, 2’), 6.84 (d, J = 7.5 Hz, 1H,
13
6’), 6.88 (d, J = 7.2 Hz, 1H, 6), 6.91-6.97 (m, 3H, 3, 4, 5), 7.23 (t, J = 7.8 Hz, 1H, 5’); C NMR
(CDCl3): δ 22.5 (ArOCH(CH3)2), 33.5 (ArCH2CH2N), 50.3 (NCH2CH2N), 53.6 (NCH2CH2N), 55.3
(OCH3, 3’), 60.5 (ArCH2CH2N), 70.4 (ArOCH(CH3)2), 111.6 (CH, 4’), 114.7 (CH, 2’), 116.1 (CH,
3), 118.7 (CH, 6), 121.3 (CH, 5), 121.6 (CH, 6’), 122.7 (CH, 4), 129.6 (CH, 5’), 142.0 (C, 1’),
143.0 (C, 1), 150.5 (C, 2), 159.9 (C, 3’); HRMS (ESI) calcd for C22H30N2O2 [M + H]+ 355.2387,
found 355.2380.
5.2.4.9 1-(4-Methoxyphenethyl)-4-(2-isopropoxyphenyl)piperazine (10, LDT454)
Prepared by nucleophilic substitution of bromide (15) with 1-(2-isopropoxyphenyl)piperazine (19),
as an oil; 0.184 g (52%); Rf = 0.42 (CHCl3/EtOH 20:1); IR (film, cm-1) 2973, 2936, 2812, 1610,
1
1595, 1512, 1496, 1372, 1300, 1239, 1134, 1039; H NMR (CDCl3): δ 1.37 (d, J = 6.0 Hz, 6H,

20
ACCEPTED MANUSCRIPT
ArOCH(CH3)2), 2.66-2.69 (m, 2H, ArCH2CH2N), 2.76 (br, 4H, NCH2CH2N), 2.82-2.86 (m, 2H,
ArCH2CH2N), 3.19 (br, 4H, NCH2CH2N), 3.80 (s, 3H, OCH3), 4.62 (hp, J = 6.0 Hz, 1H,
ArOCH(CH3)2), 6.85-6.89 (m, 3H, J = 8.4 Hz, 3’, 5’, 6), 6.94-6.95 (m, 3H, 3, 4, 5), 7.16 (d, J = 8.4
Hz, 2H, 2’, 6’); 13C NMR (CDCl3): δ 22.5 (ArOCH(CH3)2), 33.7 (ArCH2CH2N), 50.5
(NCH2CH2N), 53.7 (NCH2CH2N), 55.4 (OCH3, 4’), 60.9 (ArCH2CH2N), 70.5 (ArOCH(CH3)2),
114.1 (2CH, 3’, 5’), 116.4 (CH, 3), 118.7 (CH, 6), 121.7 (CH, 5), 122.7 (CH, 4), 129.8 (2CH, 2’,
6’), 132.4 (C, 1’), 142.9 (C, 1), 150.6 (C, 2), 158.2 (C, 4’); HRMS (ESI) calcd for C22H30N2O2
[M + H]+ 355.2387, found 355.2380.
5.2.4.10 1-(3,4-Dimethoxyphenethyl)-4-(2-isopropoxyphenyl)piperazine (11, LDT455)
Prepared by nucleophilic substitution of bromide (16) with 1-(2-isopropoxyphenyl)piperazine (19),
as an oil; 0.212 g (55%); Rf = 0.45 (CHCl3/EtOH 20:1); IR (film, cm-1) 2972, 2936, 2812, 1592,
1
1516, 1497, 1354, 1372, 1237, 1141, 1030; H NMR (CDCl3): δ 1.36 (d, J = 6.0 Hz, 6H,
ArOCH(CH3)2), 2.68-2.71 (m, 2H, ArCH2CH2N), 2.76 (br, 4H, NCH2CH2N), 2.82-2.87 (m, 2H,
ArCH2CH2N), 3.19 (br, 4H, NCH2CH2N), 3.86 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.60 (hp, J =
6.0 Hz, 1H, ArOCH(CH3)2), 6.77-6.82 (m, 3H, 2’, 5’, 6), 6.86-6.88 (m, 1H, 6’), 6.93-6.95 (m, 3H,
13
3, 4, 5); C NMR (CDCl3): δ 22.5 (ArOCH(CH3)2), 33.2 (ArCH2CH2N), 50.4 (NCH2CH2N), 53.7
(NCH2CH2N), 56.0 (OCH3, 4’), 56.1 (OCH3, 3’), 60.9 (ArCH2CH2N), 70.5 (ArOCH(CH3)2), 111.6
(CH, 5’), 112.4 (CH, 2’), 116.4 (CH, 3), 118.7 (CH, 6), 120.7 (CH, 6’), 121.7 (CH, 5), 122.7 (CH,
4), 132.9 (C, 1’), 142.9 (C, 1), 147.5 (C, 4’), 149.0 (C, 3), 151.7 (C, 2); HRMS (ESI) calcd for
C23H32N2O3 [M + H]+ 385.2493, found 385.2486.
5.3. Biology
5.3.1. Binding assays
Competition binding assays to cloned human α1a, α1b, and α1d-adrenoceptor subtypes were
performed in membrane preparations from CHO (Chinese Hamster Ovary) cell lines transfected by
electroporation with DNA expressing the gene encoding each α1-adrenoceptor. Cloning and stable

21
ACCEPTED MANUSCRIPT
expression of the human α1-adrenoceptor gene was performed as previously described.22 Briefly,
CHO cells membranes (30 ꢀg proteins) were incubated in 50 mM Tris–HCl buffer, 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 the absence or
presence of competing drugs (1 pM-10 ꢀM). Non-specific binding was determined in the presence
of 10 ꢀM phentolamine. The incubation was stopped by addition of ice-cold Tris–HCl buffer and
rapid filtration through 0.2% poly(ethylenimine)-pretreated Whatman GF/B or Schleicher &
Schuell GF52 filters.
5.3.2 Functional experiments
Male Wistar rats (275-300 g) were killed by cervical dislocation and the required organs were
isolated, freed from adhering connective tissue, and set up rapidly under a suitable resting tension in
20 mL organ baths containing physiological salt solution kept at 37 °C and aerated with 5%
CO2:95% O2 at pH 7.4. Concentration-response curves were constructed by cumulative addition of
reference agonist. The concentration of agonist in the organ bath was increased approximately 3-
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. Contractions were recorded by means of a transducer
connected to the MacLAb system PowerLab/800. At first the compounds under study were added in
the organ bath in order to construct a concentration response curve such as that for the reference
agonist, but no response was obtained; after this, the compounds were treated as antagonists. In
particular, after construction of concentration-response curves of the reference agonist following 30
min of washing, tissues were incubated with the compound under study for 30 min and a new
concentration-response curve to the agonist was recorded. In all cases, parallel experiments in
which tissues received only the reference agonist were run in order to check any variation in
sensitivity.
All animal testing was carried out according to European Communities Council Directive of 24
November 1986 (86/609/EEC).

22
ACCEPTED MANUSCRIPT
The antagonist potency was expressed by pKB at a single concentration23 or pA2 when indicated.14,
18
pKB values were calculated from the equation pKB = log(DR - 1) - log[B], where DR is the ratio
of ED50 values of agonist after and before treatment with one or two antagonist concentrations [B].
pA2 was calculated from Schild plot constraining the slope to -1. Data are presented as the mean ±
SE of 4-5 experiments. Differences between mean values were tested for significance by Student's
t-test.
5.3.2.1 Rat vas deferens prostatic portion
This tissue was used to assess α1A-AR antagonism.24 Prostatic portions of 2-cm length were
mounted under 0.35 g tension at 37 °C in a 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. Cocaine
hydrochloride (10 mM) was added to the Tyrode to prevent the neuronal uptake of (−)-NE. After
the equilibration period, tissues were primed twice by addition of 10 µM of the agonist (−)-NE in
order to obtain a constant response. After another washing and equilibration period of 45 min, a
cumulative (−)-NE concentration-response curve was constructed isotonically to determine the
relationship between agonist concentrations and the contractile response (basal response). When
measuring the effect of the antagonist, it was allowed to equilibrate with the tissue for 60 min
before constructing a new concentration-response curve to the agonist. (−)-NE solution contained
0.05% Na2S2O5 to prevent oxidation.
5.3.2.2 Rat spleen
This tissue was used to assess α1B-AR antagonism.25 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; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.5; KH2PO4, 1.2;
NaHCO3, 20; glucose, 11; K2EDTA, 0.01. (±)-Propranolol hydrochloride (4 µM) was added to

23
ACCEPTED MANUSCRIPT
block β-adrenoceptors. The spleen strips were placed under 1 g 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 being discarded and the second one taken as control.
The antagonist was allowed to equilibrate with the tissue for 30 min; then a new concentration-
response curve to the agonist was constructed.
5.3.2.3 Rat aorta
This tissue was used to assess α1D-AR antagonism.14 Thoracic aorta was cleaned from extraneous
connective tissue and placed in 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 µM), normetanephrine hydrochloride (1 µM) and (±)-propranolol hydrochloride
(1 µM) were added to prevent the neuronal and extraneuronal uptake of (−)-NE and to block β-
adrenoceptors, respectively. Two helicoidal strips (15 x 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 absence of acetylcholine (100 µM)-induced relaxation to preparations contracted with
(−)-NE (1 µM) was taken as an indicator that vessel was denuded successfully. Vascular strips were
then tied with surgical thread and suspended in a jacketed tissue bath containing Krebs solution.
Strip contractions were measured isometrically. After at least a 2 h equilibration period under an
optimal tension of 1 g, cumulative (−)-NE concentration-response curves were recorded at 1 h
intervals, the first two being discarded and the third one taken as a control. The antagonist was
allowed to equilibrate with the tissue for 60 min before the generation of the fourth cumulative
concentration-response curve to (−)-NE. (−)-NE solutions contained 0.05% Na2S2O4 to prevent
oxidation.
5.3.3 Cellular studies

24
ACCEPTED MANUSCRIPT
Caucasian prostate adenocarcinoma PC-3 cell line was used to study compound 7’s antiproliferative
activity. This cell line was grown adherently and maintained in minimum essential medium
supplemented with 100 U/mL penicillin, 100 ꢀg/mL streptomycin, and 10% fetal bovine serum
(FBS) and cultured at 37∘ C and aerated with 5% CO2 : 95% O2.
Tested compound was dissolved in methanol (MeOH) at a concentration of 10.000 ꢀM and diluted
with specific cells medium prior to use. Ten thousand cells were suspended in 98 ꢀL of specific
medium and incubated in a 96-well plate for overnight. After the incubation, 2 ꢀL of understudy
compound was added to the well with the final concentrations of 0.01 to 10 ꢀM . After 24, 48, and
72 h incubation at 37 °C, viability of the cells was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)- 2-(4-sulfenyl)-2H-tetrazolium (MTS) assay using Cell Titer 96 Aqueous
One Solution Cell Proliferation Assay (Promega Italia Srl).26 After the addiction of MTS, in
combination with the electron coupling agent phenazine methosulfate, the cells were allowed to
incubate for 1 h and absorbance was measured at 492 nm in a microplate reader, GeniosPro. Cell
viability was calculated as a percentage using the formula: (mean OD of treated cells/mean OD of
control cells) × 100. Results are expressed as percent of control cells which are not treated. The
growth control (GC) and growth control with MeOH (GCM) were run for each set of cell line. All
experiments were done in triplicate.
5.4. Docking studies
The primary sequences of the three α1-AR subtypes were retrieved from UniProt (ID: α1a =
P35348, ADA1A_HUMAN; α1b
=
P35368, ADA1B_HUMAN; α1d
=
P25100,
27
ADA1D_HUMAN) and the homology models were generated by using Modeller 9.15 based on
the resolved structure of human β2-adrenergic receptor (PDB Id: 2RH1). Among the 10 produced
models, the best structure was chosen by considering the DOPE and GA341 scoring functions as
well as some well-known structural parameters such as the percentages of residues falling in the
allowed regions of the Ramachandran plot and the chi-space. For all considered subtypes, the

25
ACCEPTED MANUSCRIPT
selected models did not include the large intracellular C-terminal domains, which indeed is not
involved in the binding pocket (excluded residues: 330-446 for a1a, 352-520 for a1b and 406-572
for a1d). After a set of geometrical checks to avoid unphysical occurrences such as unpredicted
gaps, incorrect D-residues, cis peptide bonds, colliding side-chains or unsuitable bond lengths, the
selected models were optimized by energy minimization keeping fixed the backbone atoms to
preserve the predicted folding.
The ligands were simulated in their protonated state since this is involved in receptor binding. Their
conformational profile was explored by quenched MonteCarlo simulations (as implemented in the
VEGA program,28 which produced 1000 minimized conformations by randomly rotating the
rotatable bonds and the so computed lowest energy conformers underwent docking simulations.
Docking analyses were carried out by using Plants, which calculates reliable ligand poses by ant
colony organization algorithms.29 For all receptor models, the search was focused into a 8 Å radius
sphere around the key aspartate residue, namely Asp106 or a1a, Asp125 for a1b and Asp176 for
a1d. 10 poses were generated for each ligand and scored by the Plp95 function with a speed equal to
1. The obtained best complexes were finally minimized by keeping fixed all atoms outside a 8 Å
radius sphere around the bound ligand and the so optimized complexes were utilized to recalculate
the docking scores.
Acknowledgments
ERS and LASR are senior fellow of the National Counsel of Technological and Scientific
Development (CNPq #310509/2011-4, Brazil). This work was supported by CNPq #473053/2004-7
grant to LASR. MLB is supported by a grant of Visiting Professor (PVE – CNPq#401864/2013-8)
from the programme “Ciência sem Fronteiras”, Brazil.
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Table 1. Binding affinity constants of target compounds 2-11 and reference compounds 1 and
tamsulosin, at cloned human α1-AR subtypes expressed in CHO cells.
a
Compounds
pKi,
Selectivityb
No
Code
α1a
α1b
α1d
α1d/α1a α1d/α1b α1a/α1b
LASSBio 772 8.46 8.28 8.90
2.5
2.0
0.5
1.0
1.0
3.5
0.2
1.0
2.0
0.5
1.5
0.5
4.0
5.5
3.5
9.5
7.5
26
7.5
17
10
1.5
3.0
5.0
8.5
9.0
7.5
32
15
5
6.5
5.0
13
1
2
3
4
5
6
7
8
LDT2
7.50 7.04 7.78
8.95 8.27 8.82
8.53 7.60 8.59
8.95 7.99 8.87
8.68 7.80 9.22
9.41 7.89 8.76
8.30 7.11 8.34
8.56 7.86 8.88
8.65 7.83 8.51
8.70 7.98 8.87
10.30c 9.20c 10.00c
LDT8
LDT243
LDT244
LDT245
LDT451
LDT452
LDT453
LDT454
LDT455
9
10
11
4.5
7.5
6.0
Tamsulosin
a
Log equilibrium dissociation constants (pKi) were calculated from IC50 values using the Cheng–
Prusoff equation. The affinity estimates, derived from displacement of [3H]prazosin binding from
α1-adrenoceptors and expressed as mean values, were from two to three experiments performed in
triplicate, which agreed within ±20%.
b
Calculated by the antilog of the difference between pKi values at different α1-adrenoceptor
subtypes.
c Data from ref 14.