Arylsulfonamide derivatives of (aryloxy)ethyl pyrrolidines and piperidines as α1-adrenergic receptor antagonist with uro-selective activity

Article abstract of DOI:10.1016/j.bmc.2016.09.017

A series of arylsulfonamide derivatives of (aryloxy)ethyl pyrrolidines and piperidines was synthesized to develop new α₁-adrenoceptor antagonists with uroselective profile. Biological evaluation for α₁- and α₂-adrenorecepo

Full text of DOI:10.1016/j.bmc.2016.09.017

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Arylsulfonamide derivatives of (aryloxy)ethyl pyrrolidines and
piperidines as a1-adrenergic receptor antagonist with uro-selective
activity
a
b
c
b
a
_
´
Aleksandra Rak , Vittorio Canale , Krzysztof Marciniec , Paweł Zmudzki , Magdalena Kotanska ,
Joanna Knutelska a, Agata Siwek e, Gabriela Stachowicz e, Marek Bednarski a, , Leszek Nowinski ,
⇑
d
´
Małgorzata Zygmunt a, Paweł Zajdel b, Jacek Sapa a
a Department of Pharmacological Screening, Faculty of Pharmacy, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Kraków, Poland
b Department of Medicinal Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Kraków, Poland
c Department of Organic Chemistry, Medical University of Silesia, 4 Jagiellonska Street, 41-200 Sosnowiec, Poland
d Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Kraków, Poland
e Department of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Kraków, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of arylsulfonamide derivatives of (aryloxy)ethyl pyrrolidines and piperidines was synthesized to
Received 23 June 2016
Revised 6 September 2016
Accepted 8 September 2016
Available online xxxx
develop new
a
a
with highest affinity and selectivity for
activity toward 1A- and
a
1-adrenoceptor antagonists with uroselective profile. Biological evaluation for
2-adrenorecepor showed that tested compounds 13–37 displayed high-to-moderate affinity for the
1-adrenoceptor (Ki = 34–348 nM) and moderate selectivity over 2-receptor subtype. Compounds
1-adrenoceptor were evaluated in vitro for their intrinsic
1B-adrenoceptor subtypes. All compounds behaved as antagonists at both
1-adrenoceptor subtypes, displaying 2- to 6-fold functional preference to 1A-subtype. Among them,
a1- and
a
a
a
a
Keywords:
a
a
LCAP flexible biomimetic
Arylsulfonamide derivatives of pyrrolidines
and piperidines
N-{1-[2-(2-methoxyphenoxy)ethyl]piperidin-4-yl}isoquinoline-4-sulfonamide (25) and 3-chloro-2-
fluoro-N-{[1-(2-(2-isopropoxyphenoxy)ethyl)piperidin-4-yl]methyl}benzene sulfonamide (34) displayed
the highest preference to a1A-adrenoceptor. Finally, compounds 25 and 34 (2–5 mg/kg, iv), in contrast to
a1-Adrenoceptors antagonists
tamsulosin (1–2 mg/kg, iv), did not significantly decrease systolic and diastolic blood pressure in
normotensive anesthetized rats to determine their influence on blood pressure.
Ó 2016 Elsevier Ltd. All rights reserved.
a1A/B receptor selectivity
Uroselective activity
Benign prostatic hyperplasia
1. Introduction
beneficial effects are not observed in all patients.7–10 According to
the current European Association of Urology guidelines,
Benign prostatic hyperplasia (BPH) is highly prevalent neo-
plasm in aging men, affecting up to 70% of patients over the age
of 80.1 Enlargement and elevated muscle tone of the prostate gland
lead to obstruction of the bladder outlet and manifests clinically
with lower urinary tract symptoms (LUTS).2,3 Although BPH is
rarely a life-threatening condition, the symptoms tend to be both-
ersome and result in significant impairment of quality of life.
Moreover BPH can be a progressive disease, especially if left
untreated. Therefore the aim of pharmacological therapy is to
relieve urinary symptoms, prevent progression and the develop-
ment of long term complications.4–6
alpha1-adrenoceptor blockers have an advantage over other groups
as they improve LUTS in a short period of time. In consequence,
they are considered the first-line drug treatment for BPH.11 Various
a1-adrenoceptor blockers are currently available for the treatment
of BPH with different selectivity for adrenoceptor subtypes, effi-
cacy and side-effect profile.
To date, four subtypes of
in human organs: 1A-,
Distribution studies have shown that
a
a
1-adrenoceptor have been identified
1B-, 1D-, and
1L-adrenoceptors.12
1A- and 1D-adrenoceptor
a
a
a
a
a
subtypes are three to nine fold greater expressed in the prostatic
gland in BPH comparing to healthy tissue.13 On the contrary,
There are several classes of medications available for the treat-
ment of BPH, but despite the different mechanisms of action, their
a
1B-adrenoceptors seem to be important for systematic blood pres-
sure control, as they are highly prevalent in vascular smooth mus-
cle.14 It has been demonstrated that blockade of
1A-adrenoceptors
relaxes prostate smooth muscle thus relieving obstructive outflow
symptoms, whereas 1D-adrenoceptor antagonists decrease
a
⇑
Corresponding author.
E-mail address: marek.bednarski@uj.edu.pl (M. Bednarski).
a
http://dx.doi.org/10.1016/j.bmc.2016.09.017
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Rak, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.017

2
A. Rak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
bladder over activity through direct influence on the bladder
detrusor muscle.12 The blockade of
1B-adrenoceptor may promote
O
O
a
O
N
N
O
O
O O
S
blood pressure-related side-effects, particularly orthostatic
S
hypotension, as it mediates vasodilation in blood vessels.15 The
N
N
H
H
F
most commonly used
line moiety, i.e. doxazosin, terazosin and alfuzosin, nonspecifically
interact with all
1-adrenoceptor subtypes.16 On the contrary, tam-
sulosin and the most recently introduced silodosin and naftopidil,
show specific affinity for 1A-adrenoceptor and 1D-adrenoceptor
subtypes.15–18 These new selective
1-adrenoceptor blockers
a1-adrenolitic drugs, derived from quinazo-
F
PZ-955
PZ-766
Ki (5-HT7) = 344 nM; Ki (α1) = 80 nM
Ki (5-HT7) = 0.3 nM; Ki (α1) = 630 nM
a
R
O
a
a
O
N
a
H
Ar
N
S
decrease risk for cardiovascular side effects comparing to the
n
m
O
O
non-selective representatives, due to the lower interaction with
13-37
a
1B-adrenoceptor subtype.19
In this context the search for
with stronger uroselective profile is still considered an important
approach in search for effective treatment of BPH. Among several
a1-adrenoreceptor antagonists
Figure 2. General structure of compounds PZ-766, PZ-955 and designed com-
pounds 13–37.
classes of
a1-adrenoceptors ligands, long-chain arylpiperazines
(LCAPs) represent one of the most privileged structure for develop-
(R and S 3-aminopyrrolidine, 4-aminopiperidine and 4-amino-
methylpiperidine) as well as diversification at the arylsulfonamide
moiety. Pharmacological tests were carried out to assess the affinity
for a1- and a2-adrenoceptors, to determine their intrinsic activity
using in vitro functional assay, and finally to evaluate an influence
ing derivatives with high affinity for a1A and
a1D-receptors and
high selectivity over the a1B subtypes20–22 (Fig. 1). Moreover, it
was found that a kind of substituent at the arylpiperazine moiety
as well as a kind of terminal fragment highly influenced interaction
with a
1-adrenoreceptors.23–28
of selected compounds on blood pressure in animal models.
Recently, Zajdel et al.29,30 have proposed a concept of biomimet-
ics of long-chain arylpiperazine derivatives. The idea was based on
the premise that arylpiperazine moiety might be replaced with
aryloxyalkyl alicyclic amines, e.g. piperidines and pyrrolidines.
Extended structure–activity relationship studies in a group of aryl-
sulfonamide derivatives of (aryloxy)ethyl piperidines revealed that
receptor affinity and selectivity, especially for serotonin 5-HT7 and
2. Chemistry
The synthesis of designed compounds 13–37 was performed
according to a multistep procedure reported in Scheme 1. In the first
step, commercially available phenols 1–2 were treated with
1,2-dibromoethane, in presence of potassium carbonate, in reflux-
ing acetone. Subsequently, the obtained alkylating agents 3–4
reacted with selected Boc-protected secondary amine (R and S-3-
amino-pyrrolidine, 4-amino-piperidine and 4-aminomethyl-
piperidine) yielding intermediates 5–12. After removal of Boc
group, final arylsulfonamides 13–37 were obtained upon treatment
of primary amines with the selected arylsulfonyl chlorides.
5-HT1A, dopamine D2 and also a1-adrenergic receptors, was highly
depended upon a kind of substituent in the aryloxy moiety. It was
found that, an introduction of small and electron donating sub-
stituents e.g. methoxy in a ortho-position at the (aryloxy)ethyl
fragment decreased the affinity for 5-HT7Rs (Ki >300 nM) when
compared with close analogs containing steric hindered sub-
stituents (i.e. isopropyl, t-butyl, phenyl, Ki <50 nM).30,31 At the
same time, this modification was favorable for interaction with
3. Pharmacology
a1-adrenoreceptors.
Inspired by these finding, we designed and synthesized a new
library of arylsulfonamide derivatives of (aryloxy)ethyl pyrrolidi-
3.1. In vitro radioligand binding and functional evaluation
nes and piperidines as a novel group of
nists (Fig. 2).
Structural modifications comprised an introduction of small or
sterically encumbered electron donor substituent in position-2 of
aryloxy fragment (methoxy and isopropoxy), different amine cores
a1A-/a1D-receptor antago-
The pharmacological profile of the new compounds was
evaluated by radioligand binding assays (the ability to displace
[3H]-Prazosin or [3H]-Clonidine from
a1- and a2-adrenoceptor,
respectively) using
a
tissue from the rat cerebral cortex.32
N
O
O
O
N
N
N
H
N
O
N
N
O
N
O
N
OH
B
N
O
N
HN
O
O
BMY-7378
A
O
K (α ) = 6.3 nM
K (α ) = 15.8 nM
i
K (α ) = 44.5 nM
i
1A
1A
i 1A
O
O
H
N
S
O
O
N
N
N
O
O
N
O
O
N
Cl
N
N
C
) = 1.2 nM
1A
D
K (α ) = 2 nM
E
α
K (
i
K (α ) = 0.72 nM
i
1A
i
1A
Figure 1. General structure of differently functionalized LCAPs as a1-adrenoreceptor antagonists.
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A. Rak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
R
R
R
The study showed that evaluated compounds displayed high-to-
O
O
O
moderate affinity for
a1-adrenoceptors (Ki = 34–348 nM), and low-
HO
O
O
Br
N
i
ii
to-moderate selectivity over
a
2-adrenoceptor subtypes (Table 1).
H
N
Structure–affinity relationship studies revealed that replace-
ment of the small methoxy substituent) in position-2 at the (ary-
loxy)ethyl fragment with the symmetric and sterically
Boc
n
m
1-2
3-4
5-12
iii, iv
encumbered isopropoxy one maintained high affinity for a1
-
R
O
adrenoceptors (25 vs 28 and 32 vs 37). Although it was difficult
to precisely determine the most favorable amine core, it was found
that 4-amino and 4-aminomethyl piperidine scaffolds were more
R = methyl, isopropyl; m= 0, 1 n = 0, 1;
Ar = 3-Cl-phenyl, 4-F-phenyl,
3-Cl,2-F-phenyl, 5-Cl,2-F-phenyl,
3,4-diMeO-phenyl,
O
N
H
N
Ar
S
preferable for interaction with
3-aminopyrrolidine (15 vs 22 and 30, and 18 vs 27 and 35). Inter-
estingly, compounds with 3-aminopyrrolidine core showed the
a1-adrenoceptor than the
n
m
8-quinolinyl, 4-isoquinolinyl
O
O
13-37
Scheme 1. Synthesis of compounds 13–37. Reagents and conditions: (i) 1,2-
dibromoethane, K2CO3, KI, (CH3)2CO, 60 °C, 48–72 h; (ii) alicyclic amine, K2CO3, KI,
(CH3)2CO, 60 °C, 48 h; (iii) TFA/CH2Cl2 (80:20; v/v), 2 h; (iv) ArSO2Cl, TEAÁCH2Cl2,
0 °C, 2–6 h.
highest selectivity over
18 vs 26 and 34). It was also found, that within this sub-series,
enantiomers S displayed a slight preference for
counterparts (13 vs 14 and 17 vs 18).
a2-adrenoceptors (16 vs 22 and 29, and
a1R over their R
To extend the structural modifications around this class of
derivatives, different substituents with electron-donating or elec-
tron-withdrawing properties at the arylsulfonamide fragments
were introduced. The selection was partially inspired by the work
of Chiu et al.25 who reported on a series of (phenylpiperidinyl)cyclo-
The inhibition constants (Ki) were calculated from the Cheng–
Prusoff equation.33
Selected compounds which showed high affinity for
ceptor Ki <100 nM, and displayed a1 a2 selectivity above 5 were
subsequently evaluated for their intrinsic activity at 1A- and
a1-adreno-
/
hexylsulfonamides as potent
a1R antagonists. In line with those
a
data, it seems that the type, the position as well as the number of
substituents at the sulfonamide did not influenced the affinity for
a1B-adrenoceptor test.
The intrinsic activity at
a1A-adrenoceptor was assessed by
a1Rs. Yet, an introduction of di-halogenated substituents generally
fluorescence detection of b-lactamase reporter gene using
a
increased the selectivity over the 2R subtypes (29 and 30 vs 31,
a
FRET-enabled substrate. The intrinsic activity at 1B-adrenoceptor
a
34 and 36 vs 33). Further structural modifications were based on
our previous findings demonstrating that localization of the nitro-
gen atom in the azine moiety significantly impacted the interaction
of azinesulfonamides of LCAPs and 3-(1,2,3,6-tetrahydropyridin-4-
yl)-1H-indoles with serotonin 5-HT1A, 5-HT6, 5-HT7Rs and dopamin-
ergic D2Rs.35–37 It was found that, among tested azinesulfonamides
was assessed by luminescence detection of calcium mobiliza-
tion using the recombinant expressed jellyfish photoprotein,
aequorin.
Finally, selected compounds with the highest functional selec-
tivity were further tested to determine their affinity for 5-HT1A
and 5-HT7Rs in screening radioligand binding studies using [3H]-
(with sulfonyl group in
with the sulfonamide localized at the pyridine ring (4-isoquinolinyl)
displayed higher affinity for 1Rs than their 8-quinolinyl analogs
a-position at azine fragment), compounds
8-Hydroxy-2-(dipropylamino)tetralin
([3H]-8-OH-DPAT)
and
[3H]-Lysergic acid diethylamide ([3H]-LSD), respectively. Experi-
ments were performed using membranes from CHO-K1 cells stably
transfected with the human 5-HT1A and 5-HT7Rs according to the
methods previously described.34
a
with sulfonamide at the phenyl moiety (24 vs 25 and 36 vs 37).
In the next step, selected compounds with the highest affinity
for
a1-adrenoceptors (Ki < 100 nM) and selectivity over a2-adreno-
ceptor subtypes (>5-fold) were in vitro tested for their intrinsic
3.2. In vivo pharmacology
activity behaving as potent antagonists at
tors (Table 2).
a1A- and a1B-adrenocep-
Knowing that blockade of
tate smooth muscle tone whereas a1B adrenoceptor antagonists
promote blood pressure related side effects, compounds 25 and
34, with the highest a1A/a1B selectivity ratio were chosen for fur-
ther in vivo evaluation. The hypotensive activity was determined
after one time iv administration to normotensive anaesthetized
rats at single doses of 2 and 5 mg/kg.
a1A-adrenoceptors reduce the pros-
It was found, that compounds containing benzene ring with
electron donating substituent as well as azine moieties at the sul-
fonamide displayed moderate selectivity for
a1ARs over the a1BRs
(31, 32 and 36). On the contrary, compounds 21, 29 and 30 with
halogen substituent at the sulfonamide revealed no preference
amongst tested receptors.
In the next step, selected compounds with the highest func-
tional selectivity were evaluated for their affinity for serotonin 5-
HT1A, and 5-HT7 receptors, displaying moderate affinity for the
evaluated receptors (Table 3).
4. Results and discussion
We have previously reported on the synthesis, in vitro and
in vivo pharmacological evaluation of a series of arylsulfonamide
derivatives of aryloxyalkyl piperidines and pyrrolidines.29–31 The
study confirmed, that the receptor binding profile, especially for
Compounds with the highest a1A/a1B selectivity ratio in in vitro
intrinsic activity studies—25 and 34 were chosen for further in vivo
evaluation to assess their influence on blood pressure parameters.
The hypotensive activity was determined after one time i.v. admin-
istration to normotensive anaesthetized rats at single doses 2.0 and
5.0 mg/kg. It was found that both compounds 25 and 34 did not
significantly decrease systolic (SBP) and diastolic blood pressure
(DBP) at dose range of 2–5 mg/kg i.v. in the whole period of obser-
vation (60 min). Compound 25 at dose of 5 mg/kg reduced SBP
from mean 124 to 110–116 mmHg (6–11%) and DBP from 92 to
85–90 mmHg (2–8%). At lower dose (2 mg/kg), the compound 25
reduced SBP from 133 to 120–127 mmHg (4–10%) and DBP from
97 to 89–93 mmHg (4–8%). Compound 34 at dose of 5 mg/kg
reduced the SBP from 126 to 120–124 mmHg (2–5%) and caused
5-HT1A, 5-HT7, and a1 receptors, was highly dependent upon a kind
of a substituent in position-2 at the aryloxy fragment. It was found
that an introduction of small methoxy group with strong electron
donating properties (PZ-955, Fig. 2) was preferred for an interac-
tion with adrenergic
the possibility towards exploration of the structural determinants
a
1Rs (Ki = 80 nM).31 These findings opened
which enable the development of a novel group of
tor antagonists.
a1A-/a1D-recep-
All newly synthesized compounds were tested in radioligand
binding assays to assess their affinity for 1- and 2-adrenoceptors.
a
a
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4
A. Rak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 1
The binding data of the synthesized compounds 13–37 for a1 and
a2Rs
R
O
O
N
H
N
Ar
S
n
m
O
O
13-37
Compound
Ar
Enant
n
m
R
Kia (nM) SEM
a1
a2
bS
8
>29
8
>35
2
>109
4
>41
10
4
22
1
6
1
3
3
14
24
8
8
5
a2/a1
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
3-Cl,2-F-phenyl
3-Cl,2-F-phenyl
5-Cl,2-F-phenyl
5-Cl,2-F-phenyl
3-Cl,2-F-phenyl
3-Cl,2-F-phenyl
5-Cl,2-F-phenyl
5-Cl,2-F-phenyl
3-Cl-phenyl
R
S
R
S
R
S
R
S
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Methyl
Methyl
Methyl
Methyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Methyl
Methyl
Methyl
Methyl
Methyl
Isopropyl
Isopropyl
Isopropyl
Methyl
Methyl
Methyl
241 14
348 36
289
214 22
176 13
92
106
242 16
46 12
34
99
93
1944 10
>10,000
2354 63
>10,000
378 12
>10,000
398 40
>10,000
439 18
129 15
2140 35
131 21
297 20
188 23
293 35
194 15
678 39
1345 55
644 70
695 53
416 51
1212 99
870 28
947 25
578 51
6
8
9
5-Cl,2-F-phenyl
3,4-DiOMe-phenyl
8-Quinolinyl
7
2
5
3
4-Isoquinolinyl
3-Cl,2-F-phenyl
5-Cl,2-F-phenyl
4-Isoquinolinyl
3-Cl,2-F-phenyl
5-Cl,2-F-phenyl
3,4-DiOMe-phenyl
4-Isoquinolinyl
4-F-phenyl
52
131
118 22
1
73
48
56
81
90
77
71
133 10
95
67
2
5
4
6
9
4
4
Methyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
3-Cl,2-F-phenyl
5-Cl,2-F-phenyl
8-Quinolinyl
17
7
10
9
8
2
4-Isoquinolinyl
a
Ki values based on two independent binding experiments.
Ratio of affinity for a2 and a1 adrenoreceptors.
b
Table 2
The intrinsic activity of selected compounds and references for a1A- and a1B-adrenoceptors
Compound
a1A
a1B
S1B/1A
EC50 (nM)
Profile
EC50 (nM)
Profile
21
23
25
29
30
31
32
34
120.6
191.6
99.7
ANTa
ANT
ANT
ANT
ANT
ANT
ANT
ANT
ANT
ANT
ANT
AGOa
15.5
461.1
608.4
14.15
12.65
598.6
514.8
42.1
ANT
ANT
ANT
ANT
ANT
ANT
ANT
ANT
ANT
ANT
ANT
AGO
0.13
2.40
6.10
0.17
0.08
3.73
3.68
3.80
3.07
1.93
0.03
0.01
84.2
148.2
160.7
140.1
11.1
34.1
0.07
36
104.8
0.13
1.73
Tamsulosin
Terazosin
Phenylephrine
51.89
55.99
0.86
a
AGO—agonist, ANT—antagonist. The means EC50 values were obtained from three experiments.
no DBP reduction. At lower dose (2 mg/kg), compound 34 reduced
Table 3
The binding data of selective compounds for
a1, 5-HT1A and 5-HT7 receptors
SBP from 132 to 123 mmHg (7%) whereas reduction in DBP has not
been observed as well. In comparison, the highly
a1A – selective
Compound
a1 Kia (nM)
5-HT1A
5-HT7
%inh @ 10À6
%inh @ 10À6/10À7
compound tamsulosin decreased SBP from 124 to 103–110 mmHg
(11–17%) and DBP from 94 to 77–84 mmHg (11–18%) already at a
dose of 2 mg/kg after i.v. administration (Fig. 3). Based on these
results we hypothesized that 25 and 34 reveal uroselective profile,
which may be slightly more potent than that of tamsulosin. In
order to confirm this expectances further pharmacological studies
will be undertaken.
25
31
32
34
36
52
81
90
71
95
3
6
9
4
8
69
72
78
59
61
87:41
52:16
89:54
90:45
91:51
a
Ki values based on two independent binding experiments.
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A. Rak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
b)
a)
1 4 0
1 4 0
1 2 0
1 0 0
8 0
1 2 0
*
*
*
1 0 0
8 0
6 0
6 0
0
2 0
4 0
6 0
0
2 0
4 0
6 0
T im e (m in )
T im e (m in )
d)
c)
1 4 0
1 2 0
1 0 0
8 0
1 4 0
1 2 0
1 0 0
8 0
6 0
6 0
0
2 0
4 0
6 0
0
2 0
4 0
6 0
T im e (m in )
T im e (m in )
e)
f)
1 4 0
1 2 0
1 0 0
8 0
1 4 0
1 2 0
1 0 0
8 0
6 0
6 0
0
2 0
4 0
6 0
0
2 0
4 0
6 0
T im e (m in )
T im e (m in )
Systolic blood pressure,
Diastolic blood pressure;
Figure 3. The hypotensive activity of compounds tamsulosin, 25 and 34 in anaesthetised rats after iv administration. (A) tamsulosin at the dose 1 mg/kg b.w., iv; (B)
tamsulosin at the dose 2 mg/kg b.w., iv; (C) compound 25 at the dose 2 mg/kg b.w., iv; (D) compound 25 at the dose 5 mg/kg b.w., iv; (E) compound 34 at the dose 2 mg/kg b.
w., iv; (F) compound 34 at the dose 5 mg/kg b.w., iv. Values are presented as the mean SEM of N = 6–8 experiments. The statistical significance was calculated using a one-
way ANOVA post-hoc Dunnett’s Multiple Comparison Test., Significant to time before compound administration (0 min): *p <0.05.
one time iv administration at a dose of 2 mg/kg, compounds 25 and
34 appear promising for further development as new uroselective
5. Conclusion
a
1-adrenoceptor antagonists.
In summary, the design, synthesis and pharmacological evalua-
tion of a series of new arylsulfonamide derivatives of (aryloxy)
ethyl pyrrolidines and piperidines was described. Study directed
6. Experimental
6.1. Chemistry
on identification of potent
a
1-adrenoceptor antagonists with selec-
1B-subtype allowed identification
tivity for 1A-adrenoceptor over
a
a
of compounds 25 and 34. Selected derivatives displayed no signif-
icant changes in the blood pressure parameters at doses 2 and
5 mg/kg (iv) in rats. Considering that tamsulosin, used as a refer-
ence drug, significantly decreased blood pressure parameters after
6.1.1. General chemical methods
Organic transformations were carried out at ambient tem-
perature, unless indicated otherwise. Organic solvents used in
Please cite this article in press as: Rak, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.017

6
A. Rak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
this study (Sigma–Aldrich, Chempur) were of reagent grade
and were used without purification. All other commercially
available reagents were of the highest purity (from Sigma–
Aldrich, Fluorochem). All workup and purification procedures
were carried out with reagent-grade solvents under ambient
atmosphere.
Mass spectra were recorded on a UPLC-MS/MS system consisted
of a Waters ACQUITYÒ UPLCÒ (Waters Corporation, Milford, MA,
USA) coupled to a Waters TQD mass spectrometer (electrospray
ionization mode ESI-tandem quadrupole). Chromatographic sepa-
rations were carried out using the Acquity UPLC BEH (bridged ethyl
6.1.5. General procedure for preparation of final compounds
(13–37)
A mixture of the appropriate deprotected amine (0.38 mmol) in
CH2Cl2 (3 mL), and TEA (1.14 mmol) was cooled down (ice bath),
and arylsulfonyl chloride (1.37 mmol) was added at 0 °C in one
portion. The reaction mixture was stirred for 2–6 h under cooling.
Then, the solvent was evaporated and the sulfonamides were puri-
fied using silica gel column with CH2Cl2/MeOH (9:0.7 v/v) as an
eluting system. Compound 24, which evaluated in in vivo testes
was further converted into the hydrochloride salts by treatment
of their solution in anhydrous ethanol with 1.25 M HCl in MeOH.
hybrid) C18 column; 2.1 Â 100 mm, and 1.7
equipped with Acquity UPLC BEH C18 VanGuard pre-column;
2.1 Â 5 mm, and 1.7 m particle size. The column was maintained
lm particle size,
6.1.6. Characterization of final compounds
l
6.1.6.1.
(R)-3-Chloro-2-fluoro-N-{1-[2-(2-methoxyphenoxy)
Yellow oil,
at 40 °C, and eluted under gradient conditions from 95% to 0% of
eluent A over 10 min, at a flow rate of 0.3 mL minÀ1. Eluent A:
water/formic acid (0.1%, v/v); eluent B: acetonitrile/formic acid
(0.1%, v/v). Chromatograms were made using Waters ek PDA detec-
tor. Spectra were analyzed in 200–700 nm range with 1.2 nm res-
olution and sampling rate 20 points/s. MS detection settings of
Waters TQD mass spectrometer were as follows: source tempera-
ture 150 °C, desolvation temperature 350 °C, desolvation gas flow
ethyl]pyrrolidin-3-yl}benzenesulfonamide (13).
110 mg (78% yield) following chromatographic purification over
silica gel with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 99%,
tR = 4.31, C19H22ClFN2O4S, MW 428.90, Monoisotopic Mass
428.10, [M+H]+ 429.3.1H NMR (300 MHz, CDCl3) d 1.61–1.73 (m,
2H), 2.29–2.39 (m, 2H), 2.71 (dd, J = 9.9, 2.2 Hz, 2H), 2.79–3.03
(m, 3H), 3.84 (s, 3H), 3.98 (br s, 1H), 4.07 (t, J = 5.7 Hz, 2H), 6.84–
6.97 (m, 4H), 7.19 (td, J = 8.0, 1.1 Hz, 1H), 7.57 (ddd, J = 8.1, 6.6,
1.7 Hz, 1H), 7.79 (ddd, J = 7.9, 6.3, 1.6 Hz, 1H).
rate 600 L hÀ1
, , capillary potential
cone gas flow 100 L hÀ1
3.00 kV, cone potential 40 V. Nitrogen was used for both nebulizing
and drying gas. The data were obtained in a scan mode ranging
from 50 to 1000 m/z in time 0.5 s intervals. Data acquisition soft-
ware was MassLynx V 4.1 (Waters). The UPLC/MS purity of all
the final compounds was confirmed to be 95% or higher.
1H NMR and 13C NMR spectra were obtained in Varian BB 200
spectrometer using TMS (0.00 ppm) as an internal standard in d6-
DMSO, and were recorded at 300 and 75 MHz, respectively. The J
values are reported in Hertz (Hz), and the splitting patterns are
designated as follows: s (singlet), br s (broad singlet), d (doublet),
t (triplet), q (quartet), dd (doublet of doublets), m (multiplet).
Elemental analyses for C, H, N and S were carried out using the
elemental Vario EL III Elemental Analyser (Hanau, Germany). All
values are given as percentages, and were within 0.4% of the cal-
culated values.
6.1.6.2.
(S)-3-Chloro-2-fluoro-N-{1-[2-(2-methoxyphenoxy)
Yellow oil,
ethyl]pyrrolidin-3-yl}benzenesulfonamide (14).
120 mg (80% yield) following chromatographic purification over
silica gel with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 96%,
tR = 4.26, C19H22ClFN2O4S, MW 428.90, Monoisotopic Mass
428.10, [M+H]+ 429.3.1H NMR (300 MHz, CDCl3) d 1.61–1.73 (m,
2H), 2.29–2.39 (m, 2H), 2.74 (dd, J = 10.0, 2.7 Hz, 2H), 2.81–3.03
(m, 3H), 3.85 (s, 3H), 4.01 (br s, 1H), 4.08 (t, J = 5.8 Hz, 2H), 6.84–
6.95 (m, 4H), 7.19 (td, J = 7.9, 1.1 Hz, 1H), 7.57 (ddd, J = 8.1, 6.6,
1.7 Hz, 1H), 7.79 (ddd, J = 7.9, 6.3, 1.7 Hz, 1H).
6.1.6.3.
(R)-5-Chloro-2-fluoro-N-{1-[2-(2-methoxyphenoxy)
Yellow oil,
ethyl]pyrrolidin-3-yl}benzenesulfonamide (15).
90 mg (72% yield) following chromatographic purification over sil-
Melting points (mp) were determined with a Büchi apparatus
and are uncorrected.
ica gel with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 95%, tR = 4.55,
C
19H22ClFN2O4S, MW 428.90, Monoisotopic Mass 428.10, [M+H]+
429.3.1H NMR (300 MHz, CDCl3) d 1.61–1.73 (m, 2H), 2.29–2.39
(m, 2H), 2.71 (dd, J = 9.9, 2.2 Hz, 2H), 2.79–3.03 (m, 3H), 3.84 (s,
3H), 3.98 (br s, 1H), 4.07 (t, J = 5.7 Hz, 2H), 6.84–6.96 (m, 4H),
7.02 (t, J = 9.0 Hz, 1H), 7.43 (ddd, J = 8.7, 4.2, 2.7 Hz, 1H), 7.87
(dd, J = 6.1, 2.6 Hz, 1H).
6.1.2. General procedure for the alkylation of phenols (3–4)
Commercial phenols 1–2 (0.05 mol) was dissolved in acetone
(30 ml). Then K2CO3 (0.15 mol) and catalytic amount of KI were
added followed by dropwise addition of 1,2-dibromoethane
(0.2 mol). The reaction was refluxed for 48–72 h. Inorganic resi-
dues were filtered off and organic mixture was concentrated under
reduced pressure. The obtained crude product was purified using
silica gel with AcOEt/hexane (1:9 v/v) as an eluting system.
6.1.6.4.
(S)-5-Chloro-2-fluoro-N-{1-[2-(2-methoxyphenoxy)
Yellow oil,
ethyl]pyrrolidin-3-yl}benzenesulfonamide (16).
100 mg (75% yield) following chromatographic purification over
silica gel with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 95%,
tR = 4.63, C19H22ClFN2O4S, MW 428.90, Monoisotopic Mass
428.10, [M+H]+ 429.3.1H NMR (300 MHz, CDCl3) d 1.61–1.73 (m,
2H), 2.29–2.39 (m, 2H), 2.71 (dd, J = 9.9, 2.2 Hz, 2H), 2.79–3.03
(m, 3H), 3.84 (s, 3H), 3.98 (br s, 1H), 4.07–4.12 (m, 2H), 6.84–
6.96 (m, 4H), 7.03 (t, J = 9.0 Hz, 1H), 7.43 (ddd, J = 8.7, 4.2, 2.7 Hz,
1H), 7.86 (dd, J = 6.1, 2.6 Hz, 1H).
6.1.3. General procedure for the alkylation with the ((aryloxy)
ethyl)bromides of Boc-protected-amines (5–12)
Commercial Boc-protected amines (0.015 mol) was dissolved in
acetone (15 ml). Then K2CO3 (0.045 mol) and catalytic amount of
KI were added followed by dropwise addition of (aryloxy)ethyl-
bromide (0.018 mol). The reaction was refluxed for 48 h. Inorganic
residues were filtered off and organic mixture was concentrated
under reduced pressure. The obtained crude product was purified
using silica gel with CH2Cl2/MeOH (9:0.7 v/v) as an eluting system.
6.1.6.5. (R)-3-Chloro-2-fluoro-N-{1-[2-(2-isopropoxyphenoxy)
ethyl]pyrrolidin-3-yl}benzenesulfonamide (17).
Yellow oil,
90 mg (71% yield) following chromatographic purification over sil-
ica gel with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 97%, tR = 4.82,
21H26ClFN2O4S, MW 456.96, Monoisotopic Mass 456.13, [M+H]+
6.1.4. Boc-deprotection protocol
C
457.3.1H NMR (300 MHz, CDCl3) d 1.32 (d, J = 6.0 Hz, 6H), 1.65–
1.71 (m 2H), 2.19–2.29 (m, 1H), 2.30–2.39 (m, 1H), 2.63 (dd,
J = 10.2, 6.1 Hz, 1H), 2.71–2.76 (m, 1H), 2.78–3.01 (m, 3H), 3.93
(br s, 1H), 4.06 (t, J = 5.6 Hz, 2H), 4.46 (spt, J = 6.1 Hz, 1H),
TFA (4 ml) was added to the solution of Boc-protected
intermediates (5–12) in CH2Cl2 (2 ml) and stirred for 2 h at room
temperature. The excess of reagent and solvent were removed
under reduced pressure and left under vacuum overnight.
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A. Rak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
6.83–6.91 (m, 4H) 7.17 (td, J = 8.0, 1.1 Hz, 1H), 7.56 (ddd, J = 8.1,
6.6, 1.6 Hz, 1H), 7.78 (ddd, J = 7.9, 6.3, 1.7 Hz, 1H).
130 mg (79% yield) following chromatographic purification over
silica gel with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 100%,
tR = 3.89, C22H30N2O6S, MW 450.55, Monoisotopic Mass 450.18,
[M+H]+ 451.3.1H NMR (300 MHz, CDCl3) d 1.45–1.61 (m, 2H),
1.76–1.80 (m, 2H), 2.16–2.22 (m, 2H), 2.80 (t, J = 6.1 Hz, 2H),
2.83–2.93 (m, 2H), 3.11–3.20 (m, 1H), 3.83 (s, 3H), 3.91 (s, 3H),
3.93 (s, 3H), 4.05–4.12 (m, 2H), 4.92 (br s, 1H), 6.83–6.94 (m,
5H), 7.36 (d, J = 1.7 Hz, 1H), 7.49 (dd, J = 8.5, 2.0 Hz, 1H). 13C NMR
(75 MHz, CDCl3) d 32.84, 50.52, 52.33, 55.85, 56.13, 56.26, 56.86,
66.68, 109.44, 110.48, 111.87, 113.55, 120.79, 120.85, 121.40,
132.88, 148.11, 149.12, 149.46, 152.39.
6.1.6.6. (S)-3-Chloro-2-fluoro-N-{1-[2-(2-isopropoxyphenoxy)
ethyl]pyrrolidin-3-yl}benzenesulfonamide (18).
Yellow oil,
100 mg (75% yield) following chromatographic purification over
silica gel with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 97%,
tR = 4.84, C21H26ClFN2O4S, MW 456.96, Monoisotopic Mass
456.13, [M+H]+ 457.3.1H NMR (300 MHz, CDCl3)
d 1.33 (d,
J = 6.0 Hz, 6H), 1.61–1.73 (m, 2H), 2.19–2.29 (m, 1H), 2.30–2.39
(m, 1H), 2.68–2.72 (m, 1H), 2.75–2.77 (m, 1H), 2.80–3.02 (m,
3H), 3.98 (br s, 1H), 4.05 (t, J = 5.6 Hz, 2H), 4.46 (spt, J = 6.0 Hz,
1H), 6.84–6.92 (m, 4H), 7.18 (td, J = 8.0, 1.1 Hz, 1H), 7.57 (ddd,
J = 8.1, 6.6, 1.6 Hz, 1H), 7.79 (ddd, J = 7.9, 6.2, 1.6 Hz, 1H).
6.1.6.12.
N-(1-(2-(2-Methoxyphenoxy)ethyl)piperidin-4-yl)
Yellow oil, 120 mg (85%
quinoline-8-sulfonamide (24).
yield) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9:0.7), UPLC/MS purity 100%, tR = 3.96, C23H27N3O4-
S, MW 441.54, Monoisotopic Mass 441.17, [M+H]+ 442.2.1H NMR
(300 MHz, CDCl3) d 1.25–1.47 (m, 3H), 1.65–1.71 (m, 2H), 2.08–
2.16 (m, 2H), 2.71–2.77 (m, 4H), 3.80 (s, 3H), 4.01–4.05 (m, 2H),
6.36–6.39 (m, 1H), 6.80–7.25 (m, 4H), 7.53–7.57 (m, 1H), 7.62–
7.67 (m, 1H), 8.04 (dd, J = 7.1, 1.3 Hz, 1H), 8.27 (dd, J = 7.1, 1.7 Hz,
1H), 8.42 (dd, J = 7.1, 1.5 Hz, 1H), 8.99–9.02 (m, 1H).
6.1.6.7. (R)-5-Chloro-2-fluoro-N-{1-[2-(2-isopropoxyphenoxy)
ethyl]pyrrolidin-3-yl}benzenesulfonamide (19).
Yellow oil,
100 mg (74% yield) following chromatographic purification over
silica gel with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 97%,
tR = 5.01, C21H26ClFN2O4S, MW 456.96, Monoisotopic Mass
456.13, [M+H]+ 457.4.1H NMR (300 MHz, CDCl3)
d 1.31 (d,
J = 6.0 Hz, 6H), 1.67–1.81 (m, 2H), 2.19–2.27 (m, 1H), 2.39–2.51
(m, 1H), 2.64 (dd, J = 10.2, 6.1 Hz, 1H), 2.82–2.89 (m, 1H), 2.91–
3.04 (m, 2H), 3.12 (t, J = 9.0, 4.2 Hz, 1H), 4.04 (br s, 1H), 4.05 (t,
J = 5.5 Hz, 2H), 4.45 (spt, J = 6.0 Hz, 1H), 6.83–6.91 (m, 4H), 7.03
(t, J = 9.0 Hz, 1H), 7.44 (ddd, J = 8.7, 4.2, 2.7 Hz, 1H), 7.86 (dd,
J = 6.1, 2.6 Hz, 1H).
6.1.6.13. N-{1-[2-(2-Methoxyphenoxy)ethyl]piperidin-4-yl}iso-
quinoline-4-sulfonamide (25).
Yellow oil, 90 (74% yield) fol-
lowing chromatographic purification over silica gel with CH2Cl2/
MeOH (9:0.7), UPLC/MS purity 100%, tR = 3.76, C23H27N3O4S, MW
441.54, Monoisotopic Mass 441.17, [M+H]+ 442.3. 1H NMR
(300 MHz, CDCl3) d 1.46–1.55 (m, 2H), 1.69–1.72 (m, 2H), 2.08–
2.16 (m, 2H), 2.73–2.77 (m, 4H), 2.79–2.84 (m, 1H), 3.84 (s, 3H),
4.01–4.03 (m, 2H), 6.85–6.97 (m, 4H), 7.71–7.76 (m, 1H), 7.83–
7.89 (m, 1H), 8.08–8.11 (m, 1H), 8.57–8.59 (m, 1H), 9.15 (s, 1H),
9.41 (s, 1H).13C NMR (75 MHz, CDCl3) d 29.85, 52.21, 55.57,
55.85, 63.93, 66.90, 111.91, 115.02, 115.20, 120.96, 123.06,
124.98, 128.06, 131.45, 131.85, 133.30, 133.69, 135.67, 138.53,
146.43, 149.48, 151.49.
6.1.6.8. (S)-5-Chloro-2-fluoro-N-{1-[2-(2-isopropoxyphenoxy)
ethyl]pyrrolidin-3-yl}benzenesulfonamide (20).
Yellow oil,
110 mg (76% yield) following chromatographic purification over
silica gel with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 97%,
tR = 5.01, C21H26ClFN2O4S, MW 456.96, Monoisotopic Mass
456.13, [M+H]+ 457.3.1H NMR (300 MHz, CDCl3)
d 1.34 (d,
J = 6.0 Hz, 6H), 1.67–1.81 (m, 2H), 2.19 (dtd, J = 13.3,, 8.8, 4.3 Hz,
1H), 2.39–2.51 (m, 1H), 2.64 (dd, J = 10.2, 6.1 Hz, 1H), 2.82–2.89
(m, 1H), 2.91–3.04 (m, 2H), 3.12 (t, J = 9.0, 4.2 Hz, 1H), 4.04 (br s,
1H), 4.05 (t, J = 5.4 Hz, 2H), 4.46 (spt, J = 6.0 Hz, 1H), 6.82–6.93
(m, 4H), 7.03 (t, J = 9.0 Hz, 1H), 7.44 (ddd, J = 8.7, 4.2, 2.6 Hz, 1H),
7.86 (dd, J = 6.1, 2.6 Hz, 1H).13C NMR (75 MHz, CDCl3) d 21.80,
52.43, 64.53, 115.40, 118.35, 118.66, 122.88, 129.78, 129.79,
134.69, 134.80, 147.51, 147.58, 147.60, 155.33, 158.70.
Anal. calcd for C23H27N3O4SÁ2HCl: C: 53.70, H: 5.68, N: 8.17, S:
6.23; Found C: 53.47, H: 5.99, N: 8.78, S: 6.45. Mp for C23H27N3O4-
SÁ2HCl 140.5–141.2 °C.
6.1.6.14.
3-Chloro-2-fluoro-N-{1-[2-(2-isopropoxyphenoxy)
Yellow oil,
ethyl]piperidin-4-yl}benzenesulfonamide (26).
120 mg (77% yield) following chromatographic purification over
silica gel with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 97%,
tR = 5.07, C22H28ClFN2O4S, MW 470.99, Monoisotopic Mass
6.1.6.9. 3-Chloro-N-{[1-(2-(2-methoxyphenoxy)ethyl)piperidin-
4-yl]methyl}benzenesulfonamide (21).
(82% yield) following chromatographic purification over silica gel
with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 97%, tR = 4.50, C21H27
Yellow oil, 120 mg
470.14, [M+H]+ 471.3.1H NMR (300 MHz, CDCl3)
d 1.30 (d,
-
J = 6.0 Hz, 6H), 1.49–1.67 (m, 2H), 1.76–1.85 (m, 2H), 2.26 (t,
J = 10.5 Hz, 2H), 2.81 (t, J = 5.5 Hz, 2H), 2.96 (d, J = 12.0 Hz, 2H),
3.30–3.33 (m, 1H), 4.07 (t, J = 5.5 Hz, 2H), 4.46 (spt, J = 6.0 Hz,
1H), 6.85–6.90 (m, 4H), 7.21 (td, J = 8.0, 1.0 Hz, 1H), 7.60 (ddd,
J = 8.1, 6.7, 1.6 Hz, 1H), 7.80 (ddd, J = 7.9, 6.3, 1.6 Hz, 1H).
ClN2O4S, MW 438.97, Monoisotopic Mass 438.14, [M+H]+
439.3.1H NMR (300 MHz, CDCl3) d 1.24–1.39 (m, 2H), 1.49–1.54
(m, 1H), 1.67–1.72 (m, 2H), 2.12–2.19 (m, 2H), 2.86–2.91 (m,
4H), 3.05–3.09 (m, 2H), 3.83 (s, 3H), 4.12–4.16 (m, 2H), 6.86–6.94
(m, 4H), 7.41–7.46 (m, 1H), 7.51–7.54 (m, 1H), 7.72–7.75 (m,
1H), 7.83–7.84 (m, 1H).
6.1.6.15.
5-Chloro-2-fluoro-N-{1-[2-(2-isopropoxyphenoxy)
Yellow oil,
ethyl]piperidin-4-yl}benzenesulfonamide (27).
6.1.6.10. 5-Chloro-2-fluoro-N-{1-[2-(2-methoxyphenoxy)ethyl]
110 mg (78% yield) following chromatographic purification over
silica gel with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 97%,
tR = 5.11, C22H28ClFN2O4S, MW 470.99, Monoisotopic Mass
piperidin-4-yl}benzenesulfonamide
(22).
Yellow
oil,
100 mg (76% yield) following chromatographic purification over
silica gel with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 100%,
tR = 4.57, C20H24ClFN2O4S, MW 442.93, Monoisotopic Mass
442.11, [M+H]+ 443.2.1H NMR (300 MHz, CDCl3) d 1.53–1.62 (m,
2H), 1.80–1.83 (m, 2H), 2.20–2.27 (m, 2H), 2.81–2.92 (m, 4H),
3.28–3.31 (m, 1H), 3.84 (s, 3H), 4.07–4.11 (m, 2H), 6.86–6.94 (m,
4H), 7.12–7.18 (m, 1H), 7.48–7.54 (m, 1H), 7.87–7.9 (m, 1H).
470.14, [M+H]+ 471.2.1H NMR (300 MHz, CDCl3)
d 1.30 (d,
J = 6.0 Hz, 6H), 1.47–1.61 (m, 2H), 1.80 (dd, J = 12.7, 3.6 Hz, 2H),
2.18–2.29 (m, 2H), 2.79 (t, J = 5.6 Hz, 2H), 2.91 (d, J = 12.2 Hz,
2H), 3.11–3.25 (m, 1H), 4.06 (t, J = 5.6 Hz, 2H), 4.46 (spt,
J = 6.0 Hz, 1H), 6.85–6.91 (m, 4H), 7.15 (t, J = 9.0 Hz, 1H), 7.51
(ddd, J = 8.7, 4.2, 2.7 Hz, 1H), 7.89 (dd, J = 6.1, 2.6 Hz, 1H).
6.1.6.11.
3,4-Dimethoxy-N-{1-[2-(2-methoxyphenoxy)ethyl]
(23). Yellow oil,
6.1.6.16.
quinoline-8-sulfonamide (28).
N-{1-[2-(2-Isopropoxyphenoxy)ethyl]piperidin-4-yl}
Yellow oil, 100 mg (75%
piperidin-4-yl}benzenesulfonamide
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8
A. Rak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
yield) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9:0.7), UPLC/MS purity 100%, tR = 4.54, C25H31N3O4-
S, MW 469.60, Monoisotopic Mass 469.20, [M+H]+ 470.4.1H NMR
(300 MHz, CDCl3) d 1.24 (d, J = 1.0 Hz, 6H), 1.31–1.45 (m, 2H),
1.65 (dd, J = 12.8, 3.8 Hz, 2H), 2.11 (t, J = 10.3 Hz, 2H), 2.70 (t,
J = 5.7 Hz, 2H), 2.75–2.80 (m, 1H), 3.28 (d, J = 7.2 Hz, 2H), 3.99 (t,
J = 5.7 Hz, 2H), 4.41 (spt, J = 6.1 Hz, 1H), 6.38 (d, J = 7.3 Hz, 1H),
6.81–6.84 (m, 1H), 6.84–6.87 (m, 2H), 7.54 (dd, J = 8.3, 4.3 Hz,
1H), 7.64 (dd, J = 8.1, 7.3 Hz, 1H), 8.04 (dd, J = 8.2, 1.3 Hz, 1H),
8.27 (dd, J = 8.3, 1.7 Hz, 1H), 8.42 (dd, J = 7.2, 1.4 Hz, 1H), 9.00
(dd, J = 4.2, 1.7 Hz, 1H).
116.17, 116.47, 121.13, 122.62, 129.89, 130.01, 135.54, 135.58,
147.88, 148.08, 163.27, 166.64.
6.1.6.22.
ethyl)piperidin-4-yl]methyl}benzenesulfonamide
(34). Yellow oil, 120 mg (79% yield) following chromato-
3-Chloro-2-fluoro-N-{[1-(2-(2-isopropoxyphenoxy)
graphic purification over silica gel with CH2Cl2/MeOH (9:0.7),
UPLC/MS purity 98%, tR = 5.16, C23H30ClFN2O4S, MW 485.01,
Monoisotopic Mass 484.16, [M+H]+ 485.8.1H NMR (300 MHz,
CDCl3) d 1.23–1.26 (m, 2H), 1.31 (d, J = 1.0 Hz, 6H), 1.41–1.56 (m,
2H), 1.70 (d, J = 12.8 Hz, 2H), 2.11 (td, J = 11.7, 1.9 Hz, 2H), 2.83
(t, J = 5.7 Hz, 2H), 2.88–2.90 (m, 1H), 3.06 (d, J = 11.8 Hz, 2H),
4.11 (t, J = 5.7 Hz, 2H), 4.47 (spt, J = 6.0 Hz, 1H), 6.86–6.92 (m,
4H), 7.22 (td, J = 8.0, 1.1 Hz, 1H), 7.62 (ddd, J = 8.1, 6.7, 1.7 Hz,
1H), 7.79 (ddd, J = 7.9, 6.3, 1.7 Hz, 1H). 13C NMR (75 MHz, CDCl3)
d 22.21, 29.52, 35.95, 48.75, 53.56, 57.31, 67.04, 71.81, 114.58,
117.32, 121.40, 121.62, 124.72, 124.79, 128.61, 129.60, 135.19,
147.82, 149.86, 152.67, 156.05.
6.1.6.17. 3-Chloro-2-fluoro-N-{[1-(2-(2-methoxyphenoxy)ethyl)
piperidin-4-yl]methyl}benzenesulfonamide
(29).
Yellow
oil, 80 mg (65% yield) following chromatographic purification over
silica gel with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 96%, tR = 4.50,
C21H26ClFN2O4S, MW 456.96, Monoisotopic Mass 456.13, [M+H]+
457.3.1H NMR (300 MHz, CDCl3) d 1.24–1.36 (m, 2H), 1.48–1.53
(m, 1H), 1.71–1.74 (m, 2H), 2.12–2.17 (m, 2H), 2.84–2.91 (m,
4H), 3.03–3.07 (m, 2H), 3.86 (s, 3H), 4.12–4.17 (m, 2H), 6.86–6.94
(m, 4H), 7.19–7.25 (m, 1H), 7.59–7.64 (m, 1H), 7.76–7.81 (m, 1H).
Anal. calcd for C23H30ClFN2O4SÁHCl: C: 52.97, H: 5.99, N: 5.37, S:
6.15; Found C: 52.72, H: 6.35, N: 5.18, S: 6.39. Mp for C23H30ClFN2-
O4SÁHCl: 133.5–134.2 °C.
6.1.6.18. 5-Chloro-2-fluoro-N-{[1-(2-(2-methoxyphenoxy)ethyl)
6.1.6.23.
ethyl)piperidin-4-yl]methyl}benzenesulfonamide
(35). Yellow oil, 110 mg (78% yield) following chromato-
5-Chloro-2-fluoro-N-{[1-(2-(2-isopropoxyphenoxy)
piperidin-4-yl]methyl}benzenesulfonamide
(30).
Yellow
oil, 90 mg (72% yield) following chromatographic purification over
silica gel with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 95%, tR = 4.73,
graphic purification over silica gel with CH2Cl2/MeOH (9:0.7),
UPLC/MS purity 95%, tR = 5.24, C23H30ClFN2O4S, MW 485.01,
Monoisotopic Mass 484.16, [M+H]+ 485.5. 1H NMR (300 MHz,
CDCl3) d 1.23–1.28 (m, 2H), 1.30 (d, J = 5.90 Hz, 6H), 1.84–2.00
(m, 4H), 2.73–2.82 (m, 1H), 3.10–3.24 (m, 2H), 3.55–3.64 (m,
2H), 3.91 (d, J = 9.75 Hz, 4H), 4.38–4.49 (m, 1H), 6.74–6.92 (m,
4H), 7.15 (t, J = 9.09 Hz, 2H), 7.89–8.02 (m, 1H).
C
21H26ClFN2O4S, MW 456.96, Monoisotopic Mass 456.13, [M+H]+
457.3.1H NMR (300 MHz, CDCl3) d 1.24–1.37 (m, 2H), 1.38–1.53
(m, 1H), 1.71–1.74 (m, 2H), 2.12–2.21 (m, 2H), 2.86–2.91 (m,
4H), 3.05–3.09 (m, 2H), 3.84 (s, 3H), 4.13–4.17 (m, 2H), 6.86–6.94
(m, 4H), 7.12–7.18 (m, 1H), 7.48–7.53 (m, 1H), 7.84–7.87 (m, 1H).
6.1.6.19.
3,4-Dimethoxy-N-{[1-(2-(2-methoxyphenoxy)ethyl)
(31). Yellow
piperidin-4-yl]methyl}benzenesulfonamide
6.1.6.24. N-{[1-(2-(2-Isopropoxyphenoxy)ethyl)piperidin-4-yl]
oil, 100 mg (74% yield) following chromatographic purification
over silica gel with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 99%,
tR = 3.96, C23H32N2O6S, MW 464.57, Monoisotopic Mass 464.2, [M
+H]+ 465.3.1H NMR (CDCl3) d 1.24–1.32 (m, 2H), 1.37–1.51 (m,
2H), 1.68–1.72 (m, 2H), 2.13–2.21 (m, 2H), 2.78–2.91 (m, 4H),
3.05–3.09 (m, 2H), 3.83 (s, 3H), 3.91 (s, 3H), 3.93 (s, 3H), 4.13–
4.17 (m, 2H), 6.86–6.94 (m, 4H), 7.32–7.33 (m, 1H), 7.44–7.48
(m, 1H).
methyl}quinoline-8-sulfonamide (36).
Yellow oil, 100 mg
(73% yield) following chromatographic purification over silica gel
with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 94%, tR = 4.82, C26H33N3-
O4S, MW 483.62, Monoisotopic Mass 483.22, [M+H]+ 484.3.1H NMR
(300 MHz, CDCl3) d 1.23–1.29 (m, 2H), 1.31 (d, J = 1.0 Hz, 6H), 1.61–
1.73 (m, 2H), 2.28–2.35 (m, 2H), 2.69 (t, J = 6.7 Hz, 2H), 2.79–2.85 (m,
1H), 3.00 (d, J = 11.2 Hz, 2H), 3.98 (dd, J = 5.8, 1.4 Hz, 2H), 4.08 (t,
J = 5.8 Hz, 2H), 4.39–4.53 (m, 1H), 6.36–6.43 (m, 1H), 6.85–6.91
(m, 3H), 7.57 (dd, J = 8.3, 4.3 Hz, 1H), 7.67 (dd, J = 8.1, 7.3 Hz, 1H),
8.06 (dd, J = 8.2, 1.4 Hz, 1H), 8.29 (dd, J = 8.3, 1.7 Hz, 1H), 8.44 (dd,
J = 7.2, 1.4 Hz, 1H), 9.02 (dd, J = 4.3, 1.7 Hz, 1H).
6.1.6.20.
methyl}isoquinoline-4-sulfonamide (32).
(70% yield) following chromatographic purification over silica gel
with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 96%, tR = 3.99, C24H29
N-{[1-(2-(2-Methoxyphenoxy)ethyl)piperidin-4-yl]
Yellow oil, 90 mg
-
6.1.6.25. N-{1-[2-(2-Isopropoxyphenoxy)ethyl]pyrrolidin-3-yl}
N3O4S, MW 455.77, Monoisotopic Mass 455.19, [M+H]+ 456.3.1H
NMR (300 MHz, CDCl3) d 1.30–1.34 (m, 2H), 1.35–1.49 (m, 2H),
1.89–1.93 (m, 4H), 2.87–3.01 (m, 2H), 3.43–3.45 (m, 4H), 3.84 (s,
3H), 4.32–4.34 (m, 2H), 6.84–6.88 (m, 4H), 7.69–7.74 (m, 1H),
7.82–7.87 (m, 1H), 8.06–8.08 (m, 1H), 8.60–8.63 (m, 1H), 9.07 (s,
1H), 9.38 (s, 1H).
isoquinoline-4-sulfonamide (37).
Yellow oil, 90 mg (70%
yield) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9:0.7), UPLC/MS purity 99%, tR = 4.46, C25H31N3O4S,
MW 469.59, Monoisotopic Mass 469.20, [M+H]+ 470.2.1H NMR
(300 MHz, CDCl3) d 1.25 (d, J = 1.0 Hz, 6H), 1.41–1.56 (m, 2H),
1.71–1.81 (m, 2H), 2.16–2.28 (m, 2H), 2.78 (t, J = 5.6 Hz, 2H), 2.90
(d, J = 12.3 Hz, 2H) 3.11–3.25 (m, 1H), 4.18 (t, J = 5.6 Hz, 2H), 4.44
(spt, J = 6.0 Hz, 1H), 6.80–6.93 (m, 4H), 7.67–7.75 (m, 1H), 7.90 (t,
J = 7.5 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 8.65 (d, J = 8.6 Hz, 1H),
9.15 (s, 1H), 9.39 (s, 1H).
6.1.6.21. 4-Fluoro-N-{[1-(2-(2-isopropoxyphenoxy)ethyl)piperi-
din-4-yl]methyl}benzenesulfonamide
(33).
Yellow
oil,
100 mg (76% yield) following chromatographic purification over
silica gel with CH2Cl2/MeOH (9:0.7), UPLC/MS purity 95%,
tR = 4.85, C23H31FN2O4S, MW 450.57, Monoisotopic Mass 450.20,
[M+H]+ 451.3.1H NMR (300 MHz, CDCl3) d 1.23–1.28 (m, 2H),
1.30 (d, J = 5.9 Hz, 6H), 1.84–2.00 (m, 4H), 2.73–2.82 (m, 1H),
3.10–3.24 (m, 2H), 3.55–3.64 (m, 2H), 3.91 (d, J = 9.7 Hz, 2H),
4.38–4.49 (m, 3H), 6.74–6.92 (m, 4H), 7.12 (t, J = 8.1 Hz, 2H),
7.89–8.02 (m, 2H). 13C NMR (75 MHz, CDCl3) d 22.21, 27.30,
33.85, 47.44, 53.06, 56.43, 64.90, 70.95, 76.68, 115.48, 115.65,
6.2. In vitro pharmacology
6.2.1. Determination of the affinity of the tested compounds at
the a1-adrenoreceptors and a2-adrenoreceptors
The affinity of the obtained compounds was evaluated by radi-
oligand binding assays (the ability to displace [3H]-Prazosin and
[3H]-Clonidine from
a1- and a2-adrenoceptor, respectively) on rat
Please cite this article in press as: Rak, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.017

A. Rak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
cerebral cortex. The brains is homogenized in 20 volumes of an ice-
cold 50 mM Tris–HCl buffer (pH 7.6) and is centrifuged at 20,000 g
for 20 min (0–4 °C). The cell pellet is resuspended in the Tris–HCl
buffer and centrifuged again. Radioligand binding assays are per-
formed in plates (MultiScreen/Millipore).The final incubation mix-
well), diluted in Assay Buffer, were prepared in ½ white polystyr-
ene area plates, and the cell suspension was dispensed in 50 l vol-
ume on the ligands using the injector. The light emitted was record
l
for 20 s. Cells with antagonist were incubate for 15 min at room
temperature. Therefore 50
tion) was injected onto the mix of cells and antagonist and record
l
l of agonist (3 Â EC80 final concentra-
ture (final volume 300
suspension, 30
l of [3H]-Prazosin (0.2 nM) or [3H]-Clonidine
(2 nM) solution and 30 l of the buffer containing seven to eight
concentrations (10À11 to 10À4 M) of the tested compounds. For
measuring the unspecific binding, phentolamine, 10 M (in the
case of [3H]-Prazosin) and clonidine, 10 M (in the case of [3H]-
ll) consisted of 240
l
l of the membrane
l
the light emitted for 20 s.
l
6.3. In vivo pharmacology
l
l
6.3.1. Determination of the effect of the test compounds on
blood pressure after a single administration in rats
Clonidine) are applied. The incubation is terminated by rapid filtra-
tion over glass fiber filters (Whatman GF/C)using a vacuum mani-
fold (Millipore). The filters are then washed twice with the assay
buffer and placed in scintillation vials with a liquid scintillation
cocktail. Radioactivity was measured in a WALLAC 1409 DSA liquid
scintillation counter. All the assays were made in duplicate.
The normotensive rats were anesthetized with thiopental
(70 mg/kg) by ip injection. The left carotid artery was cannulated
with polyethylene tubing filled with heparin solution in saline to
facilitate pressure measurements using PowerLab Apparatus
(ADInstruments). Blood pressure was measured: before adminis-
tration of the compounds—time 0 min (control pressure) and
60 min thereafter. For each compound, studies were performed
in three doses: 2 mg/kg, 5 mg/kg and 10 mg/kg b.w. Compounds
were dissolved in water and administered intravenously.
6.2.2. Determination of the affinity of the tested compounds at
the 5-HT1A and 5-HT7 receptors
Binding experiments were conducted in 96-well microplates in
a total volume of 250
the assay buffers was as follows: 50 mM Tris–HCl, 0.1 mM EDTA,
10 mM MgCl2. Reaction mix included 50 l solution of test com-
pound, 50 l of radioligand and 150 l of diluted membranes. All
assays were incubated for 1 h (5-HT1ARs) or 2 h (5-HT7Rs) at
37 °C. Radioactivity was counted in MicroBeta2 scintillation coun-
ll of appropriate buffers. The composition of
6.3.2. Animals
l
The experiments were carried out on male Wistar rats (body
weight 200–250 g). The animals were housed in pairs in plastic
cages in constant temperature facilities exposed to 12:12 h light/-
dark cycle, water and food were available ad libitum. Experimental
groups consisted of six animals each. All experiments were con-
ducted according to the guidelines of the Animal Use and Care
Committee of the Jagiellonian University (2012, Poland).
l
l
ter (PerkinElmer). Non-specific binding is defined with 10
HT and 10 M of methiothepine in 5-HT1AR and 5-HT7R binding
experiments, respectively. Each compound was tested in screening
assay at two final concentrations of 10 M and 1 M. Results were
expressed as percent inhibition of specific binding.
lM of 5-
l
l
l
6.3.3. Statistical analysis
Statistical calculations were carried out with the GraphPadPr-
ism 6 program. Results are given as the arithmetic means with
standard error of the mean (SEM). The statistical significance was
calculated using a one-way ANOVA post-hoc Dunnett’s Multiple
Comparison Test. Differences were considered statistically signifi-
cant at: *p 60.05, **p 60.01, ***p 60.001. Differences were consid-
6.2.3. Determination of the intrinsic activity of the a1A
adrenoreceptors
Intrinsic activity assay was performed according to the manu-
facturer of the assay kit (Invitrogen, Life Technologies). The cells
were harvested and suspended in Assay Medium to a density of
-
*
ered statistically significant at: p 0.05, **p 0.01, ***p 0.001.
312,500 cells/ml. 32 ll per well of the cell suspension was added
to the Test Compound wells, the Unstimulated Control wells, and
Stimulated Control wells and incubated per 16–24 h. To perform
Acknowledgement
an agonist assay 8 concentrations of 8 ll of the tested compound
(10À4–10À11 M), e.g. in 5 fold higher concentration in comparison
to the final tested concentration in the well, was added to the cells.
The studies were financially supported by the National Science
Centre in Poland within the Grant No. 2011/03/B/NZ7/00724.
To perform an antagonist assay 8 concentrations of 4 ll of the
tested compound (10À4–10À11 M), e.g. in 10 fold higher concentra-
tion in comparison to the final tested concentration in the well,
A. Supplementary data
was added to the cells. Then, after 30 min 4 ll of standard agonist
MS, 1H NMR and 13C NMR spectra for representative final com-
pounds. This material is available free of charge via the Internet.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.09.017.
in EC80 (10 fold higher concentration in comparison to the EC80 in
the well), in Assay Medium, was added to the cells. Then both the
agonist and the antagonist plate were incubated in a humidified
37 °C/5% CO2 incubator for 5 h. After the incubation 8 ll of Live-
BLAzerTM-FRET B/G Substrate Mixture (CCF4-AM) was loaded cells
in the absence of direct strong lighting, covered and incubated at
room temperature for 2 h.
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10
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Please cite this article in press as: Rak, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.017