Synthesis, structure-activity relationship and biological evaluation of novel arylpiperzines as α1A/1D-AR subselective antagonists for BPH

Article abstract of DOI:10.1016/j.bmc.2015.11.020

A series of novel arylpiperazine derivatives as α_1A/1D-adrenergic receptors (AR) subtype selective antagonists were designed, synthesized and evaluated for their antagonistic activities towards α₁-ARs (α_1A, α_1B,

Full text of DOI:10.1016/j.bmc.2015.11.020

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, structure–activity relationship and biological evaluation
of novel arylpiperzines as a1A/1D-AR subselective antagonists for BPH
Fang Xu a, , Hong Chen , Jingyi Xu , Xue Liang , Xuelan He , Binhao Shao , Xianqiang Sun , Bing Li ,
⇑
,y
b,y
c
c
c
c
d
e
e
c,
⇑
Xiaoliang Deng , Mu Yuan
a
Institute of Tumor Pharmacology, College of Pharmacy, Jinan University, 601 West Huangpu Blvd, Guangzhou 510632, People’s Republic of China
College of Chemistry and Chemical Engineering, Luoyang Normal University, Luoyang, Henan 471022, People’s Republic of China
Pharmaceutical Research Center, Guangzhou Medical University, 195# Dongfengxi Road, Guangzhou 510182, People’s Republic of China
Division of Theoretical Chemistry and Biology, School of Biotechnology, KTH Royal Institute of Technology, S-106 91 Stockholm, Sweden
Experimental Medical Research Center, Guangzhou Medical University, Guangzhou 510182, People’s Republic of China
b
c
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel arylpiperazine derivatives as a1A/1D-adrenergic receptors (AR) subtype selective antag-
Received 14 September 2015
Revised 2 November 2015
Accepted 19 November 2015
Available online xxxx
onists were designed, synthesized and evaluated for their antagonistic activities towards
and 1D). Compounds 9, 12, 13, 15, 17, 18, 21, 22, 25 and 26 exerted strong antagonistic effects on
and/or 1D subtypes over 1B in vitro. SAR analysis indicated that chloride at the ortho-phenyl position
for compound 17 was beneficial for the highest 1A/D-AR sub-selectivity. Moreover, molecular docking
study of compound 17 with the homology-modeled -ARs ( 1B, and 1D) structures exhibited dif-
ferences of key amino resides in the docking pocket which may influence the subtype selectivity. ILE 193
of 1A was validated as the key residues for binding ligand. This work provides useful information for
finding more new potential drugs in clinic in treating benign prostatic hyperplasia (BPH).
Ó 2015 Elsevier Ltd. All rights reserved.
1
a -ARs (
a
1A
,
a
1B
,
a
a
1A
a
a
a
a
1
a
1A
,
a
a
Keywords:
a -ARs
1
Subtype selectivity
Arylpiperazine derivatives
Antagonic activity
a
Homology modeling
Structure–activity relationship
1
. Introduction
-Adrenergic receptors (
their inability to differentiate between
the prostate and those involved in maintaining vascular tone.
Tamsulosin (V, Fig. 2), the first 1A-AR ‘selective’ antagonist, has
demonstrated 15-fold selectivity for 1A-AR over 1B-AR and
1
a -AR subtypes present in
8
a
1
a1A-,
a
1B-, and
a
1D-ARs), which belong
a
to the G-protein coupled receptor family, play significant roles in
lower urinary tract symptoms (LUTS) associated with benign
prostatic hyperplasia (BPH). These receptors are widely expressed
in many human tissues and involved in numerous physiological
processes. Therefore,
targets for treatment of several pathologies.
a
a
almost no selectivity for clinical trials on BPH patients. However,
9
tamsulosin still shows side effects. Naftopidil (VI, Fig. 2), an
arylpiperazine compound, is a specific
antagonist,
a
1D-adrenergic receptor
and is one of the most widely used -adrenergic
receptor antagonists in Japan for the treatment of benign prostatic
1
0,11
a
1
-ARs are highly attractive pharmacological
a
1
1
1
a -AR antagonists
1
2,13
14–20
can relax the prostatic smooth muscle and are used as first-line
medical treatment for patients with LUTS associated with BPH.
hyperplasia (BPH).
Other studies
have demonstrated that
2
compounds with an open-chain linker between arylpiperazinyl
Many efforts have been devoted to the development of
a
1
-AR
and isoindole-1,3-dione-2-yl groups (e.g., NAN-190, VII, Fig. 2)
antagonists, which resulted in discovery of first-generation antag-
bind to
1
a -ARs with high affinity but demonstrate poor subtype
3
4
onists against
a
1
-ARs, such as prazosin (I, Fig. 1), terazosin (II),
selectivity. We hypothesized that the compounds with ethylbenzyl
linker instead of n-butyl linker, such as NAN-190, will retain bind-
doxazosin (III), and alfuzosin (IV),6 which are used clinically for
5
the treatment of BPH by relaxing the smooth muscle of the
prostate. However, these agents also exhibit side effects, including
ing affinity and improve selectivity among the three
1
a -AR sub-
7
types ( 1B, and 1D). In the current study, we designed and
a
1A
,
a
a
orthostatic hypotension, dizziness, decreased blood pressure, nasal
congestion, and impotence, which may be partially attributed to
synthesized (phenylpiperazinyl)ethylbenzylphthalimides derived
from isoindole-1,3-diones and evaluated their antagonistic
activities in the three
models of -ARs were built using the crystal structure of
-adrenergic receptor (2RH1) as template and analized the key
amino residues in each binding pocket. Molecular docking was
1
a -AR subtypes. Furthermore, the homology
a
1
⇑
Corresponding authors. Tel./fax: +86 20 85220799.
E-mail addresses: xufang@jnu.edu.cn (F. Xu), mryuanmu@aliyun.com (M. Yuan).
Contributed equally to this work.
b
2
y
http://dx.doi.org/10.1016/j.bmc.2015.11.020
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
0
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2
F. Xu et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
O
O
O
SAR analysis revealed the following results: (1) Phenylpiperazine
derivative 5 exhibited better 1A subtype selectivity over
1A ratio = 5.8). However, benzylpiperazine derivative 6 showed
O
O
N
N
a
1B 1B
a (a /
O
O
N
N
O
O
N
N
a
N
N
better
1B 1B
a1D subtype selectivity over a (a /a1D ratio = 8.7). (2)
(I)
(II)
NH2
NH2
Although compounds 7, 19, and 24 demonstrated strong antagonis-
tic effects on 1A and 1D subtypes, these compounds also exhibited
strong activities in 1B subtype and weak 1A and/or 1D subtype
selectivities. (3) Compared with o-methyl-substituted phenyl group
derivative 8 (IC50 = 227.70 nM and 1A ratio = 4.2), p-methyl-
substituted phenyl group derivative 9 ( 1A ratio = 14.2) exhib-
ited strong antagonistic effects on 1A subtype and better 1A sub-
type selectivity over 1B. However, compound 9 also displayed
strong activity on 1B subtype (IC50 = 28.91 nM). (4) Compounds
containing an o-methoxyl-substituted phenyl group showed better
activity on 1A subtype and better 1A subtype selectivity over
than did the p-methoxyl substituted group, as exemplified by com-
pounds 10 (IC50 = 30.12 nM and a1B a1A ratio = 3.3) and 11
(IC50 = 884.70 nM and a1B a1A ratio = 0.5). Similar result was
observed in compound 13 versus 14 (IC50 = 229.40 nM and
1A ratio = 3.0). (5) Compound 12 ( 1A ratio = 13.4) exhibited
better 1A subtype selectivity over 1B than compound 10
1A ratio = 3.3). These results suggest that the introduction of
a
a
a
a
a
O
O
N
O
H
N
O
O
N
N
O
N
N
1B
a /a
N
N
O
(III)
O
(
IV)
1B
a /a
NH2
NH2
a
a
a
Figure 1.
1
a -AR antagonists clinically used for BPH treatment. prazosin (I),
terazosin (II), doxazosin (III), and alfuzosin (IV).
a
a
a
a
1B
applied to analyze the binding mode of the representive compound
-ARs and validate its subtype selectivity. The SAR was fur-
ther discussed on the basis of the obtained experimental data.
with
a
1
/
/
1B
a /
2
2
. Results and discussion
a
1B
a /a
a
a
.1. Chemistry
1B
(a /a
an ethoxyl moiety at the o-position on the phenyl group was
beneficial for improving subtype selectivity. (6) Compared with
compounds 13 and 15, compound 14 displayed decreased potency
Arylpiperazine derivatives were designed and synthesized in
order to find novel
a1A- and/or a1D-selective ligands for the treat-
ment of BPH, from commercially available 2-[4-(bromomethyl)
phenyl]acetic acid 1 (Scheme 1). First, compound 1 was reduced
to alcohol 2 in the presence of borane–methyl sulfide complex
2 M in tetrahydrofuran) at 0 °C for 1 h, then at room temperature
for 10 h. The intermediate 2 was directly used without further
on a1A subtype and lower a1A subtype selectivity over a1B. The
obtained results suggested that a fluoro group in the p-position on
the phenyl group was unfavorable for antagonistic activity and sub-
type selectivity. (7) o-Chloro-substituted phenyl group derivative
(
17 showed strong activities in
1A and 1D subtypes selectivity over
substituted phenyl group derivative 18 showed strong activities in
1D subtype and excellent 1D subtype selectivity over 1B. (8)
a
1A and
a1D subtypes and excellent
purification. The nucleophilic substitution reaction of compound
a
a
a
1B. However, p-chloro-
2
with potassium phthalimide in the presence of potassium car-
bonate (K CO ) yielded compound 3 (70% yield from compound
) after 16 h at reflux. Compound 3 was treated with 4-toluene-
a
a
a
2
3
1
Compounds with a cyano group at the o- or p-position or a methyl-
sulfonyl chloride in the presence of triethylamine and a catalytic
amount of 4-dimethylaminopyridine at 0 °C for 16 h to generate
compound 4 (95% yield). Finally, the reactions of compound 4 with
sulfonyl group at the o-position on the phenyl group also displayed
strong activities in
types selectivity over
25. (9) Compound 16 with an acetyl group at the p-position on the
phenyl group displayed potent activity in 1A subtype and excellent
1A subtype selectivity over
a1A and
a1D subtypes and better a1A and a1D sub-
a1B, as exemplified by compounds 21, 22, and
2 3
various arylpiperazines in the presence of K CO at reflux for 16 h
yielded arylpiperazine derivatives 5–26 (45–95% yield; Scheme 1).
a
1
13
The structures of the compounds were confirmed by H NMR,
C
a
1B
a .
NMR, and HRMS (ESI).
2
.3. Homology model building of
1
a -ARs and molecular docking
2
1
.2. Antagonistic activity in a -ARs
studies
Antagonistic activities of 5–26 towards
using dual-luciferase reporter assays21 (Table 1). Compounds 9,
2, 13, 15, and 26 exhibited antagonistic effects on 1A subtype
IC50 = 2.03, 32.43, 96.01, 89.94, 14.25 nM, respectively) and better
1A subtype selectivity over 1A ratio = 8.6–19.5). Com-
pound 18 (IC50 = 35.16 nM) displayed soundly antagonistic activity
on 1D subtype and marked 1D subtype selectivity over
1D ratio = 22.6). Compounds 17, 21, 22, and 25 also showed strong
antagonistic activities both on 1A (IC50 = 15.16, 36.14, 89.24,
1D (IC50 = 36.62, 16.73, 63.54,
a
1
-ARs were tested
The structures of
the template structure of b
(Fig. 3). For the crystal structure of b
2
a
1
-ARs were generated from the alignment of
-AR (PDB ID 2RH1) using Schrodinger
-AR, the intracellular loop
2
1
(
a
a
region 3 (IL3) was replaced by T4 lysozyme to increase the solubil-
ity and stability of the receptor, and the active site of the GPCRs
was located in the extracellular region near loop 2 (EL2). IL3 loop
was removed in the modeling process of the three targets since this
loop was unlikely to play a major role in ligand binding. The highly
conserved disulfide bond, DRY region in loop 3, NPxxY in loop 7,
and CWxP in loop 6 were retained in the models. Ramachandran
plot (Supplementary data in Fig. S2) of the three protein models
showed that residues located in the most favored region were
1B 1B
a (a /a
a
a
1B 1B
a (a /
a
a
4
6.44 nM, respectively) and
0.07 nM, respectively), with
a
1
a
1B 1B 1D
/a1A ratio = 4.3–51.5 and a /a
ratio = 10.8–21.3, respectively.
9
0.8%, 91.7%, and 90.9%, respectively; residues located in the addi-
tionally allowed region were 7.9%, 7%, and 5.8%, respectively; resi-
dues located in the generously allowed region were 0.4%, 0.8%, and
1.7%, respectively; and residues located in the disallowed region
were 0.8%, 0.4%, and 1.7%, respectively. Meanwhile, profile 3D of
the three models indicated that the verified scores of the amino
acid residues were more than 0.2, which was predicted to be reli-
able (Supplementary data in Fig. S3). The models of the three sub-
type proteins were considered satisfactory based on the above
O
N
O
H
N
O
S
OH
N
O
H2N
O
OEt
O
(
V)
(VI)
O
O
N
N
N
(VII)
O
Figure 2. Structures of Tamsulosin (V), Naftopidil (VI) and NAN-190 (VII).
examples. The possible binding pockets of
1
a -ARs were predicted
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F. Xu et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
O
OH
OH
a
OH
b
Br
Br
N
O
1
2
O
3
O
O
OTs
d
N
N
Ar
O
c
N
N
5
-26
4
O
O
O
F
F
S
O
5, Ar =
10, Ar =
11, Ar =
15, Ar =
16, Ar =
F
20, Ar =
Br 25, Ar =
26, Ar =
CN
NC
O
6, Ar =
O
CN 21, Ar =
O
F
Cl
N
7, Ar =
8, Ar =
9, Ar =
12, Ar =
13, Ar =
14, Ar =
17, Ar =
18, Ar =
19, Ar =
22, Ar =
Cl 23, Ar =
24, Ar =
3
F C
F
CF
3
Cl
Scheme 1. Reagents and conditions: (a) BH
3
ꢀS(CH
3
)
2
, THF, 0 °C for 1 h, and then room temperature for 10 h; (b) phthalimide potassium salt, K
CO , CH CN, reflux, 16 h.
2
CO
3
, CH
3
CN, reflux, 16 h; (c)
TsCl, Et N and 4-dimethylaminopyridine, CH
3
2
Cl , 0 °C, 16 h; (d) arylpiperazines, K
2
2
3
3
Table 1
versus Ile193, Met293 versus Leu293 versus Leu293, and Lys302
versus Leu302 versus Leu302, and so on to 1B and 1D. More-
over, the key amino residues around the binding pocket of the
Antagonistic activities (IC50) on
a
1
-ARs (
a
1A
,
a
1B, and
a
1D) of 5–26
Selectivity ratio
a
1A
,
a
a
a
Compd
IC50 (nM)
three subtypes were differentiated (Fig. 4). The conformations of
a
1A
a
1B
a
1D
a
1B
/
a
1A
1B 1D
a /a
Ile193 in
that there was conformation difference of the same binding ligand.
Ile193 in 1A, which was closer to the binding pocket, exhibited
hydrogen bonding interaction with the ligand; this phenomenon
was unlikely to occur in 1B. The different conformations of the
a1A and Val193 in a1B were entirely different indicating
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
151.30
1200.00
49.61
227.70
2.03
875.30
1007.00
12.57
948.80
28.91
100.90
406.20
433.30
1876.00
684.00
787.50
34.76
781.50
794.40
31.62
1003.00
180.00
769.40
250.40
19.69
199.00
121.90
634
1758.00
115.50
11.31
5.8
0.8
0.3
4.2
14.2
3.3
0.5
8.7
1.1
0.01
0.08
0.08
0.2
1.8
0.2
5.6
1.6
a
80550.00
343.60
1235.00
1862.00
239.90
10630.00
122.00
493.30
957.20
36.62
35.16
31.28
7426.00
16.73
63.54
a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
30.12
884.70
32.43
96.01
229.40
89.94
128.00
15.16
551.90
34.47
3421.00
36.14
89.24
384.20
66.23
46.44
14.25
555
same amino residues at the same region of the three subtypes
can induce change in the active pocket conformation. Cys191
0.5
13.4
19.5
3.0
8.8
0.3
51.5
1.4
0.9
0.3
5.0
8.6
0.7
0.3
4.3
8.6
11.53
was the typical residue in that its stereo conformation in
pletely differed from those in 1A and
a1B com-
a
1D
a .
Considering compound 17 was confirmed with high affinity for
0.04
21.3
22.6
1.0
a1A/1D-AR subtypes, approximately 51.5- and 21.3-fold higher
potency was observed for
a1A and 1D than for
a1B, respectively.
The docking-binding mode of a1A/1D-AR and 17 were performed
0.1
using Surflex-Dock (SYBYL) (Fig. 5). Compound 17 with a carbonyl
group promoted intermolecular hydrogen bonding with ILE193
10.8
12.1
1.5
1.5
19.8
0.2
163.70
12.87
10.07
866.40
55.2
Naftopidil
114.8
a
IC50 values are taken as means ± standard deviation from three experiments.
based on the common binding region of GPCRs. We found that Glu
residue, the key amino acid residue on the pocket, was the residue
responsible for ligand binding. The binding pockets of the three
subtypes were similar, such as Phe289 from the
p–p bond and
Gln195 from the hydrogen bonding with ligands from their
sequence alignment of the binding region. However, they still
had their unique amino acids, respectively: Ile193 versus Val193
Figure 3. The models of a1-ARs (A: 1A, B: 1B, and C: 1D).
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4
F. Xu et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Figure 4. The key amino residues surrounded the
1
a -ARs’s pockets (3 Å, A: 1A, B: 1B, and C: 1D).
Figure 5. Molecular docking models of compound 17 inside active site (3 Å) of
a1A-AR (A) and a1D-AR (B).
amino residue in
perdition of binding sites of
a
1A-AR model, which was consistent with the
1A-AR. VAL114 was also reasonable
for the binding mode of 17 and 1D-AR by charge–charge interac-
tions. The conformation of 17 was highly folded by charge–charge
intramolecular interactions to match the pocket of 1D-AR.
identified the important ILE193 residue to
study showed that hydrophobic interactions played an important
role in the 1A/1D-AR ligand selectivity. This results provided by
the binding-mode map can help elucidate the subtype selectivity
and aid the design of high 1A and/or 1D subtype selective
arylpiperazine derivatives in the future.
a1A-AR. The docking
a
a
a
a
a
a
3
. Conclusion
4
. Experimental section
This study reported the synthesis and biological evaluation of a
novel class of arylpiperazine derivatives. Some compounds exhib-
ited strong antagonistic effects on 1A and/or 1D subtypes and
better 1A and/or 1D subtype selectivity over 1B. Compounds
with a chloro (17) group at the o-position on the phenyl group or
cyano (22) group at the p-position demonstrated strong
antagonistic effects on 1A and 1D subtypes, and better 1A and
1D subtypes selectivity over 1B. The docking study of -ARs with
the most potent ligands (17) exhibited a good docking score and
4.1. Chemistry
a
a
a
a
a
Reagents and solvents were commercially available. Solvents
were dried and purified using standard procedures prior to use.
Melting points were determined on a Fisher Johns hot-stage
apparatus and are uncorrected. NMR spectra were determined on
Bruker AV-400 NB spectrometer in CDCl
standard, and coupling constants (J) are in Hz. EI mass spectra were
a
a
a
a
a
a
a
1
3
using TMS as internal
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F. Xu et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
recorded on a DSQ mass spectrometer, and HRMS spectra were
recorded on LTQ Orbitrap LC–MS (Thermo, Rockford, IL, USA). All
derivatives tested for biological activity showed >95% putity by
HPLC analysis (detection at 254 nm). Flash column chromatogra-
phy was performed with silica gel (Qing Dao Ocean Chemical Fac-
tory, 300–400 mesh) eluted with petroleum ether–ethyl acetate.
filtration the filtrate was concentrated in vacuo and the residue
was purified by silica gel column chromatography using ethyl acet-
ate/petroleum ether (1:4, v/v) as eluent to afford the corresponding
products, and all compounds were recrystallized from trichloro-
methane and n-hexane.
4
.1.5. 2-(4-(2-(4-Phenylpiperazin-1-yl)ethyl)benzyl)isoindoline-
1,3-dione (5)
White solid; mp 130.2–131.0 °C; yield, 95%; H NMR (400 MHz,
CDCl ) d in ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.70 (dd, J = 5.5,
4
.1.1. 2-(4-(Bromomethyl)phenyl)ethanol (2)
To a cooled (0 °C) solution of carboxylic acid 1 (5 g, 0.021 mol)
1
in dry tetrahydrofuran (THF, 100 mL) borane–dimethyl sulfide
complex (21.9 mL, 0.042 mol, 2 M in THF) was added dropwise.
The reaction mixture was stirred at 0 °C for 1 h and at room tem-
perature for 10 h. Water (20 mL) was added slowly and extracted
with ethyl acetate (3 ꢁ 100 mL). The combined organic phase
was successively washed with water, brine, dried over anhydrous
magnesium sulfate, and concentrated in vacuo. The resulting resi-
due was directly used without further purification in the following
step.
3
3.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.28–7.24 (m, 2H), 7.18 (d,
J = 8.0 Hz, 2H), 6.93 (d, J = 8.0 Hz, 2H), 6.85 (t, J = 7.3 Hz, 1H), 4.82
(s, 2H), 3.22 (t, J = 5.0 Hz, 4H), 2.81 (dd, J = 9.8, 6.3 Hz, 2H), 2.67
13
(t, J = 5.0 Hz, 4H), 2.62 (dd, J = 9.8, 6.3 Hz, 2H);
(101 MHz, CDCl ) d in ppm: 168.1, 151.4, 140.0, 134.3, 134.0,
132.3, 129.2, 129.1, 128.9, 123.4, 119.8, 116.2, 60.4, 53.3, 49.3,
C NMR
3
+
41.4, 33.3; HRMS (ESI) m/z [M+1] : calcd for
27 28 2 3
C H O N ,
426.2176, found, 426.2169.
4
.1.2. 2-(4-(2-Hydroxyethyl)benzyl)isoindoline-1,3-dione (3)
To a solution of compound 2 (4 g, 18.7 mmol) in acetone
4.1.6. 2-(4-(2-(4-Benzylpiperazin-1-yl)ethyl)benzyl)isoindoline-
1,3-dione (6)
1
(
100 mL) potassium phthalimide (3.46 g, 18.7 mmol) and potas-
White solid; mp 122.8–123.4 °C; yield, 80%; H NMR (400 MHz,
sium carbonate (2.58 g, 18.7 mmol) were added, and the reaction
mixture was stirred at reflux for16 h. After cooling to ambient tem-
perature, the reaction mixture was filtered through a Buchner fun-
nel. After filtration the filtrate was concentrated in vacuo and the
residue was purified by silica gel column chromatography using
ethyl acetate/petroleum ether (1:8, v/v) as eluent to afford 4.31 g
3
CDCl ) d in ppm: 7.83 (dd, J = 5.5, 3.0 Hz, 2H), 7.69 (dd, J = 5.5,
3.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 7.31–7.28 (m, 5H), 7.14 (d,
J = 8.0 Hz, 2H), 4.81 (s, 2H), 3.51 (s, 2H), 2.75 (dd, J = 9.8, 6.3 Hz,
1
3
3
2H), 2.66–2.38 (m, 10H); C NMR (101 MHz, CDCl ) d in ppm:
168.1, 140.2, 138.3, 134.2, 134.0, 132.3, 129.3, 129.1, 128.8,
128.3, 127.1, 123.4, 63.2, 60.5, 53.3, 53.2, 41.4, 33.4; HRMS (ESI)
+
of compound 3. White solid; mp 101.2–101.8 °C; yield, 70% (from
m/z [M+1] : calcd for C28
30
H O
2
N
3
, 440.2332, found, 440.2327.
1
compound 1); H NMR (400 MHz, CDCl
3
) d in ppm: 7.84 (dd,
J = 5.5, 3.0 Hz, 2H), 7.70 (dd, J = 5.5, 3.0 Hz, 2H), 7.38 (d, J = 8.0 Hz,
4
.1.7. 2-(4-(2-(4-(Pyridin-2-yl)piperazin-1-yl)ethyl)benzyl)
2
2
H), 7.17 (d, J = 8.0 Hz, 2H), 4.82 (s, 2H), 3.82 (t, J = 6.5 Hz, 2H),
isoindoline-1,3-dione (7)
.83 (t, J = 6.5 Hz, 2H), 1.44 (s, 1H); 13C NMR (101 MHz, CDCl
3
) d
White solid; mp 123.0–123.4 °C; yield, 86.8%; 1H NMR
400 MHz, CDCl ) d in ppm: 8.18 (dd, J = 5.0, 2.0 Hz, 1H), 7.83
dd, J = 5.5, 3.0 Hz, 2H), 7.69 (dd, J = 5.5, 3.0 Hz, 2H), 7.46 (m, 1H),
in ppm: 168.5, 138.6, 135.0, 134.4, 132.6, 129.7, 129.3, 123.7,
(
(
3
+
6
4.0, 41.7, 39.3; MS (EI) m/z: 281 (M ), 251 (100%), 232, 204,
1
92, 178, 160.
7
1
4
.36 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 6.63 (d, J = 8.8,
H), 6.60 (dd, J = 7.2, 5.0 Hz, 1H), 4.82 (s, 2H), 3.57 (t, J = 5.0 Hz,
H), 2.81 (dd, J = 9.8, 6.3 Hz, 2H), 2.64–2.59 (m, 6H); C NMR
4
.1.3. 2-(4-((1,3-Dioxoisoindolin-2-yl)methyl)phenyl)ethyl-4-
13
methylbenzenesulfonate (4)
(
101 MHz, CDCl
3
) d in ppm: 168.4, 160.0, 148.4, 140.3, 137.8,
To a solution of compound 3 (4 g, 14.2 mmol), triethylamine
1
4
4
34.6, 134.4, 132.6, 129.4, 129.2, 123.7, 113.7, 107.5, 60.8, 53.4,
5.6, 41.8, 33.6; HRMS (ESI) m/z [M+1] : calcd for C26H O N ,
27 2 4
27.2128, found, 427.2130.
(
1
5.75 g, 56.8 mmol) and 4-dimethylaminopyridine (0.17 g,
.42 mmol) in dry dichloromethane (CH Cl , 100 mL) at 0 °C was
added a solution of 4-toluene sulfonyl chloride (4.06 g, 21.3 mmol)
in CH Cl (10 mL) dropwise. The reaction mixture was stirred at
+
2
2
2
2
4
1
.1.8. 2-(4-(2-(4-o-Tolylpiperazin-1-yl)ethyl)benzyl)isoindoline-
,3-dione (8)
0
°C for 16 h.
2 2
Water (20 mL) was added slowly and extracted with CH Cl
1
White solid; mp 120.8–121.6 °C; yield, 87%; H NMR (400 MHz,
(
3 ꢁ 100 mL). The combined organic phase was successively
CDCl
.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.19–7.14 (m, 4H), 7.06–6.95
m, 2H), 4.83 (s, 2H), 2.97 (t, J = 5.0 Hz, 4H), 2.82 (dd, J = 9.8,
.3 Hz, 2H), 2.68 (br t, 4H), 2.64 (dd, J = 9.8, 6.3 Hz, 2H), 2.30 (s,
3
) d in ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.70 (dd, J = 5.5,
washed with water, brine, dried over anhydrous magnesium sul-
fate, and concentrated in vacuo. The residue was purified by silica
gel column chromatography using ethyl acetate/petroleum ether
3
(
6
(
1:10, v/v) as eluent to afford 5.88 g of compound 4. White solid;
1
3
1
3H); C NMR (101 MHz, CDCl
3
) d in ppm: 168.1, 151.6, 140.1,
34.3, 134.0, 132.7, 132.3, 131.1, 129.1, 128.9, 126.7, 123.4,
23.2, 119.1, 60.6, 53.8, 51.8, 41.5, 33.4, 18.0; HRMS (ESI) m/z [M
mp 108.2–108.9 °C; yield, 95%; H NMR (400 MHz, CDCl
3
) d in
1
1
ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.71 (dd, J = 5.5, 3.0 Hz, 2H),
7
2
2
.66 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.3 Hz,
+
30 2 3
+1] : calcd for C28H O N , 440.2333, found, 440.2326.
H), 7.05 (d, J = 8.0 Hz, 2H), 4.81 (s, 2H), 4.16 (t, J = 7.0 Hz, 2H),
.91 (t, J = 7.0 Hz, 2H), 2.42 (s, 3H); 13C NMR (101 MHz, CDCl
) d
3
in ppm: 168.4, 145.1, 136.3, 135.5, 134.4, 133.4, 132.6, 130.2,
29.6, 129.2, 128.3, 123.8, 70.8, 41.6, 35.4, 22.0; MS (EI) m/z: 435
4.1.9. 2-(4-(2-(4-p-Tolylpiperazin-1-yl)ethyl)benzyl)isoindoline-
1,3-dione (9)
1
+
Light yellow solid; mp 150.4–151.1 °C; yield, 45%; 1H NMR
(
M ), 363, 250 (100%), 235, 204, 178, 148.
(
3
400 MHz, CDCl ) d in ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.70
4
.1.4. General procedure for the synthesis of compounds 5–26
To a solution of 4 (100 mg, 0.23 mmol) in acetonitrile (CH CN,
0 mL) was added the corresponding arylpiperazines (1.2 equiv)
(dd, J = 5.5, 3.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.0 Hz,
2H), 7.07 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 4.82 (s, 2H),
3.17 (t, J = 5.0 Hz, 4H), 2.81 (dd, J = 9.8, 6.3 Hz, 2H), 2.67 (t,
J = 5.0 Hz, 4H), 2.62 (dd, J = 9.8, 6.3 Hz, 2H), 2.27 (s, 3H); 13C NMR
3
1
and potassium carbonate (6.0 equiv). The reaction mixture was
stirred at reflux for 16 h. After cooling to ambient temperature,
the reaction mixture was filtered through a Buchner funnel. After
3
(101 MHz, CDCl ) d in ppm: 169.4, 150.6, 141.3, 135.5, 135.3,
133.5, 130.9, 130.5, 130.3, 130.1, 124.6, 117.7, 61.7, 54.6, 51.0,
Please cite this article in press as: Xu, F.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.11.020

6
F. Xu et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
+
4
4
2.7, 34.6, 21.7; HRMS (ESI) m/z [M+1] : calcd for C28
40.2332, found, 440.2324.
H
30
O
2
N
3
,
4.1.15. 2-(4-(2-(4-(2,4-Difluorophenyl)piperazin-1-yl)ethyl)
benzyl)isoindoline-1,3-dione (15)
White solid; mp 122.4–123.0 °C; yield, 75.5%; 1H NMR
(
(
400 MHz, CDCl
3
) d in ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.70
4
.1.10. 2-(4-(2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethyl)
dd, J = 5.5, 3.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.0 Hz,
benzyl)isoindoline-1,3-dione (10)
White solid; mp 132.8–133.6 °C; yield, 83%; H NMR (400 MHz,
CDCl ) d in ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.70 (dd, J = 5.5,
1
2H), 6.93–6.86 (m, 1H), 6.83–6.76 (m, 2H), 4.82 (s, 2H), 3.07 (t,
J = 5.0 Hz, 4H), 2.80 (dd, J = 9.8, 6.3 Hz, 2H), 2.69 (t, J = 5.0 Hz,
4
3
+
H), 2.63 (dd, J = 9.8, 6.3 Hz, 2H); HRMS (ESI) m/z [M+1] : calcd
3
6
.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 7.02–
.85 (m, 4H), 4.82 (s, 2H), 3.86 (s, 3H), 3.12 (br t, 4H), 2.82 (dd,
26 2 3 2
for C27H O N F , 462.1988, found, 462.1982.
J = 9.8, 6.3 Hz, 2H), 2.73 (br t, 4H), 2.64 (dd, J = 9.8, 6.3 Hz, 2H);
C NMR (101 MHz, CDCl ) d in ppm: 168.1, 152.4, 141.5, 140.1,
3
1
3
4.1.16. 4-(4-(2-(4-((1,3-Dioxoisoindolin-2-yl)methyl)phenyl)
ethyl)piperazin-1-yl)-3-fluorobenzonitrile (16)
1
1
34.3, 134.0, 132.3, 129.1, 128.9, 123.4, 123.0, 121.1, 118.3,
11.4, 60.5, 55.5, 53.5, 50.7, 41.5, 33.3; HRMS (ESI) m/z [M+1] :
White solid; mp 170.3–171.3 °C; yield, 46.5%; 1H NMR
+
(
3
400 MHz, CDCl ) d in ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.70
calcd for C28
30 3 3
H O N , 456.2282, found, 456.2273.
(
dd, J = 5.5, 3.0 Hz, 2H), 7.37–7.33 (m, 3H), 7.24 (dd, J = 2.0,
2
2
.0 Hz, 1H), 7.16 (d, J = 8.0 Hz, 2H), 6.90 (t, J = 8.4 Hz, 1H), 4.82 (s,
H), 3.24 (t, J = 5.0 Hz, 4H), 2.80 (dd, J = 9.8, 6.3 Hz, 2H), 2.67 (t,
4
.1.11. 2-(4-(2-(4-(4-Methoxyphenyl)piperazin-1-yl)ethyl)
benzyl)isoindoline-1,3-dione (11)
1
3
Light yellow solid; mp 142.6–143.6 °C; yield, 71.7%; 1H NMR
J = 5.0 Hz, 4H), 2.63 (dd, J = 9.8, 6.3 Hz, 2H); C NMR (101 MHz,
CDCl ) d in ppm: 169.3, 156.5, 154.1, 145.5, 145.4, 141.0, 135.6,
3
(
400 MHz, CDCl
dd, J = 5.5, 3.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.0 Hz,
H), 6.92–6.81 (m, 4H), 4.82 (s, 2H), 3.76 (s, 3H), 3.11 (t,
J = 5.0 Hz, 4H), 2.81 (dd, J = 9.8, 6.3 Hz, 2H), 2.67 (t, J = 5.0 Hz,
H), 2.62 (dd, J = 9.8, 6.3 Hz, 2H); 13C NMR (101 MHz, CDCl
d in ppm: 168.1, 153.9, 145.9, 140.1, 134.3, 134.0, 132.3, 129.1,
28.9, 123.4, 118.3, 114.6, 60.4, 55.7, 53.4, 50.7, 41.4, 33.4;
3
) d in ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.69
1
1
35.3, 133.5, 130.7, 130.3, 130.1, 124.6, 121.1, 120.9, 120.0,
20.0, 119.7, 104.9, 104.8, 61.4, 54.2, 51.0, 50.9, 42.6, 34.5; HRMS
(
2
+
(
ESI) m/z [M+1] : calcd for
C
28
26
H O
2
N
4
F, 469.2034, found,
469.2025.
4
3
)
4
.1.17. 2-(4-(2-(4-(2-Chlorophenyl)piperazin-1-yl)ethyl)benzyl)
1
+
isoindoline-1,3-dione (17)
30 3 3
HRMS (ESI) m/z [M+1] : calcd for C28H O N , 456.2281, found,
1
White solid; mp 147.7–148.1 °C; yield, 86%; H NMR (400 MHz,
4
56.2273.
CDCl
3
) d in ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.70 (dd, J = 5.5,
3
.0 Hz, 2H), 7.38–7.34 (m, 3H), 7.24–7.16 (m, 3H), 7.05 (dd,
4
.1.12. 2-(4-(2-(4-(2-Ethoxyphenyl)piperazin-1-yl)ethyl)benzyl)
J = 8.0, 1.5 Hz, 1H), 6.96 (td, J = 8.0, 1.5 Hz, 1H), 4.82 (s, 2H), 3.10
(br t, 4H), 2.81 (dd, J = 9.8, 6.3 Hz, 2H), 2.71 (br t, 4H), 2.64 (dd,
isoindoline-1,3-dione (12)
White solid; mp 122.4–123.1 °C; yield, 86%; H NMR (400 MHz,
CDCl ) d in ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.69 (dd, J = 5.5,
1
1
3
J = 9.8, 6.3 Hz, 2H); C NMR (101 MHz, CDCl
3
) d in ppm: 169.4,
50.6, 141.3, 135.5, 135.3, 133.5, 132.0, 130.3, 130.1, 130.1,
28.9, 125.0, 124.6, 121.7, 61.7, 54.7, 52.5, 42.7, 34.6; HRMS (ESI)
3
1
1
3
6
2
6
.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 6.98–
.83 (m, 4H), 4.82 (s, 2H), 4.07 (q, J = 7.0 Hz, 2H), 3.15 (br t, 4H),
.82 (dd, J = 9.8, 6.3 Hz, 2H), 2.72 (br t, 4H), 2.64 (dd, J = 9.8,
+
27 2 3
m/z [M+1] : calcd for C27H O N Cl, 460.1786, found, 460.1782.
.3 Hz, 2H), 1.45 (t, J = 7.0 Hz, 3H); 13C NMR (101 MHz, CDCl
) d
3
4
.1.18. 2-(4-(2-(4-(4-Chlorophenyl)piperazin-1-yl)ethyl)benzyl)
isoindoline-1,3-dione (18)
Light yellow solid; mp 160.0–160.6 °C; yield, 73.9%; 1H NMR
400 MHz, CDCl ) d in ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.70
dd, J = 5.5, 3.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 7.19 (dd, J = 7.2,
in ppm: 168.4, 152.0, 141.8, 140.4, 134.6, 134.3, 132.6, 129.4,
1
4
4
29.2, 123.7, 123.1, 121.4, 118.6, 113.0, 64.0, 60.9, 53.8, 50.9,
1.8, 33.6, 15.4; HRMS (ESI) m/z [M+1] : calcd for C29H O N ,
32 3 3
70.2438, found, 470.2432.
+
(
(
3
2
4
2
.4 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 6.83 (dd, J = 7.2, 2.4 Hz, 2H),
.82 (s, 2H), 3.18 (t, J = 5.0 Hz, 4H), 2.80 (dd, J = 9.8, 6.3 Hz, 2H),
.65 (t, J = 5.0 Hz, 4H), 2.62 (dd, J = 9.8, 6.3 Hz, 2H); C NMR
4
.1.13. 2-(4-(2-(4-(2-Fluorophenyl)piperazin-1-yl)ethyl)benzyl)
isoindoline-1,3-dione (13)
White solid; mp 133.6–134.1 °C; yield, 82%; H NMR (400 MHz,
CDCl ) d in ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.69 (dd, J = 5.5,
13
1
(
101 MHz, CDCl
3
) d in ppm: 169.4, 151.3, 141.2, 135.6, 135.3,
3
1
4
4
33.5, 130.3, 130.3, 130.1, 125.8, 124.6, 118.5, 61.5, 54.4, 50.5,
2.7, 34.5; HRMS (ESI) m/z [M+1] : calcd for C27H O N Cl,
27 2 3
60.1786, found, 460.1782.
3
6
6
.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 7.08–
.89 (m, 4H), 4.82 (s, 2H), 3.14 (t, J = 5.0 Hz, 4H), 2.81 (dd, J = 9.8,
.3 Hz, 2H), 2.70 (t, J = 5.0 Hz, 4H), 2.63 (dd, J = 9.8, 6.3 Hz, 2H);
+
1
3
3
C NMR (101 MHz, CDCl ) d in ppm: 168.1, 157.1, 154.6, 140.3,
4
.1.19. 2-(4-(2-(4-(5-Chloro-2-methylphenyl)piperazin-1-yl)
1
1
4
4
40.2, 140.0, 134.3, 134.0, 132.3, 129.1, 128.9, 124.6, 124.5,
23.4, 122.5, 122.5, 119.0, 119.0, 116.3, 116.1, 60.4, 53.4, 50.6,
1.4, 33.3; HRMS (ESI) m/z [M+1] : calcd for
44.2082, found, 444.2075.
ethyl)benzyl)isoindoline-1,3-dione (19)
White solid; mp 183.8–184.7 °C; yield, 80%; H NMR (400 MHz,
CDCl ) d in ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.70 (dd, J = 5.5,
.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 7.07
d, J = 8.0 Hz, 1H), 6.97 (d, J = 2.0 Hz, 1H), 6.94 (dd, J = 8.0, 2.0 Hz,
1H), 4.82 (s, 2H), 2.94 (t, J = 5.0 Hz, 4H), 2.81 (dd, J = 9.8, 6.3 Hz,
2H), 2.66 (br t, 4H), 2.64 (dd, J = 9.8, 6.3 Hz, 2H), 2.24 (s, 3H); 13
1
+
27 27 2 3
C H O N F,
3
3
(
4
.1.14. 2-(4-(2-(4-(4-Fluorophenyl)piperazin-1-yl)ethyl)benzyl)
isoindoline-1,3-dione (14)
C
Light yellow solid; mp 152.0–152.6 °C; yield, 59%; 1H NMR
NMR (101 MHz, CDCl ) d in ppm: 169.4, 153.9, 141.2, 135.5,
3
(
(
2
(
6
1
1
400 MHz, CDCl
dd, J = 5.5, 3.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.0 Hz,
H), 6.98–6.85 (m, 4H), 4.82 (s, 2H), 3.14 (t, J = 5.0 Hz, 4H), 2.80
dd, J = 9.8, 6.3 Hz, 2H), 2.66 (t, J = 5.0 Hz, 4H), 2.62 (dd, J = 9.8,
3
) d in ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.70
135.3, 133.5, 133.2, 133.1, 132.1, 130.3, 130.1, 124.6, 124.3,
120.8, 61.6, 54.8, 52.8, 42.7, 34.5, 18.8; HRMS (ESI) m/z [M+1] :
+
calcd for C28H29O N Cl, 474.1942, found, 474.1938.
2
3
1
3
3
.3 Hz, 2H); C NMR (101 MHz, CDCl ) d in ppm: 169.4, 159.7,
4.1.20. 2-(4-(2-(4-(4-Bromophenyl)piperazin-1-yl)ethyl)benzyl)
isoindoline-1,3-dione (20)
57.3, 149.3, 141.2, 135.5, 135.3, 133.5, 130.3, 130.1, 124.6,
Light yellow solid; mp 167.4–167.9 °C; yield, 70%; 1H NMR
19.2, 119.1, 116.9, 116.7, 61.6, 54.5, 51.5, 42.7, 34.6; HRMS (ESI)
+
m/z [M+1] : calcd for C27
H
27
O
2
N
3
F, 444.2081, found, 444.2075.
(400 MHz, CDCl ) d in ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.70
3
Please cite this article in press as: Xu, F.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.11.020

F. Xu et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
(
dd, J = 5.5, 3.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 7.33 (dd, J = 6.8,
.0 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 6.78 (dd, J = 6.8, 2.0 Hz, 2H),
2H), 3.33 (s, 3H), 3.12 (br t, 4H), 2.81 (dd, J = 9.8, 6.3 Hz, 2H),
1
3
2
4
2
2.72 (br t, 4H), 2.65 (dd, J = 9.8, 6.3 Hz, 2H); C NMR (101 MHz,
CDCl ) d in ppm: 168.4, 152.9, 140.2, 137.5, 135.1, 134.7, 134.4,
132.6, 130.3, 129.4, 129.2, 126.0, 124.3, 123.7, 60.7, 54.1, 54.0,
.82 (s, 2H), 3.17 (t, J = 5.0 Hz, 4H), 2.79 (dd, J = 9.8, 6.3 Hz, 2H),
3
1
3
.64 (t, J = 5.0 Hz, 4H), 2.61 (dd, J = 9.8, 6.3 Hz, 2H); C NMR
) d in ppm: 168.1, 150.4, 140.0, 134.3, 134.1,
32.3, 132.0, 129.1, 128.9, 123.4, 117.7, 111.9, 60.3, 53.1, 49.1,
+
(
101 MHz, CDCl
3
30 4 3
43.2, 41.7, 33.6; HRMS (ESI) m/z [M+1] : calcd for C28H O N S,
1
4
5
504.1951, found, 504.1941.
+
27 2 3
1.4, 33.3; HRMS (ESI) m/z [M+1] : calcd for C27H O N Br,
04.1281, found, 504.1271.
4.1.26. 2-(4-(2-(4-(4-Acetylphenyl)piperazin-1-yl)ethyl)benzyl)
isoindoline-1,3-dione (26)
4
.1.21. 2-(4-(2-(4-(2-Isocyanophenyl)piperazin-1-yl)ethyl)
Light yellow solid; mp 157.4–157.8 °C; yield, 55.9%; 1H NMR
benzyl)isoindoline-1,3-dione (21)
White solid; mp 141.7–142.2 °C; yield, 82.8%; 1H NMR
400 MHz, CDCl ) d in ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.70
dd, J = 5.5, 3.0 Hz, 2H), 7.55 (dd, J = 8.0, 1.3 Hz, 1H), 7.47 (m, 1H),
3
(400 MHz, CDCl ) d in ppm: 7.87 (d, J = 9.0 Hz, 2H), 7.84 (dd,
J = 5.5, 3.0 Hz, 2H), 7.70 (dd, J = 5.5, 3.0 Hz, 2H), 7.36 (d, J = 8.0 Hz,
2H), 7.17 (d, J = 8.0 Hz, 2H), 6.86 (d, J = 9.0 Hz, 2H), 4.82 (s, 2H),
3.38 (t, J = 5.0 Hz, 4H), 2.81 (dd, J = 9.8, 6.3 Hz, 2H), 2.65–2.61 (m,
(
(
3
1
3
7
4
(
.37 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 7.01–6.98 (m, 2H),
.82 (s, 2H), 3.26 (t, J = 5.0, 4H), 2.81 (dd, J = 9.8, 6.3 Hz, 2H), 2.74
3
6H), 2.51 (s, 3H); C NMR (101 MHz, CDCl ) d in ppm: 197.8,
169.4, 155.4, 135.6, 135.3, 133.5, 131.7, 130.3, 130.1, 129.0,
t, J = 5.0, 4H), 2.65 (dd, J = 9.8, 6.3 Hz, 2H); 13C NMR (101 MHz,
124.6, 114.7, 61.5, 54.1, 48.6, 42.6, 34.5, 27.4; HRMS (ESI) m/z [M
+
CDCl
3
) d in ppm: 168.1, 155.8, 139.9, 134.5, 134.3, 134.0, 133.9,
+1] : calcd for C29
H
30
O
3
N
3
, 468.2281, found, 468.2273.
1
5
4
32.3, 129.1, 128.9, 123.4, 121.8, 118.8, 118.5, 106.1, 60.2, 53.2,
+
1.6, 41.4, 33.3; HRMS (ESI) m/z [M+1] : calcd for C28
H
27
O
2
N
4
,
4.2. Dual-luciferase reporter gene assay
51.2128, found, 451.2122.
Firefly and Renilla luciferase activities, which are indicated as
RLUs, were determined using Dual-Glo luciferase assay kits (Pro-
mega) according to the manufacturer’s instructions. RLUs were
measured using a luminometer (GloMaxTM 96-Microplate Lumi-
nometer, Promega) and are reported as the mean ± SEM of three
4
.1.22. 4-(4-(2-(4-((1,3-Dioxoisoindolin-2-yl)methyl)phenyl)
ethyl)piperazin-1-yl)benzonitrile (22)
Light yellow solid; mp 150.0–150.8 °C; yield, 48.4%; 1H NMR
400 MHz, CDCl ) d in ppm: 7.83 (dd, J = 5.5, 3.0 Hz, 2H), 7.70
dd, J = 5.5, 3.0 Hz, 2H), 7.48 (d, J = 9.0 Hz, 2H), 7.36 (d, J = 8.0 Hz,
(
(
3
individual experiments. For agonists, fold of induction = LUinduced
/
2
3
6
1
1
H), 7.16 (d, J = 8.0 Hz, 2H), 6.84 (d, J = 9.0 Hz, 2H), 4.82 (s, 2H),
RLUuninduced. For antagonists, % of control = 100 ꢁ RLU (agonist
+ antagonist)/RLU (agonist alone). All RLUs were normalized
against firefly RLUs/Renilla RLUs. Data are expressed as EC50/IC50
values in lM, and the IC50 of phenylephrine (lM) was calculated
by plotting the data using nonlinear regression analysis in Graph-
.33 (t, J = 5.0 Hz, 4H), 2.79 (dd, J = 9.8, 6.3 Hz, 2H), 2.64–2.59 (m,
H); 1 C NMR (101 MHz, CDCl
3
) d in ppm: 169.3, 154.7, 141.0,
3
35.6, 135.3, 134.9, 134.8, 133.5, 130.3, 130.1, 124.6, 121.3,
+
15.5, 101.6, 61.4, 54.0, 48.5, 42.6, 34.5; HRMS (ESI) m/z [M+1] :
calcd for C28
27 2 4
H O N , 451.2128, found, 451.2127.
Pad Prism 5 software.
4
.1.23. 2-(4-(2-(4-(2-(Trifluoromethyl)phenyl)piperazin-1-yl)
4.3. Homology model building of
studies
1
a -ARs and molecular docking
ethyl)benzyl)isoindoline-1,3-dione (23)
White solid; mp 141.3–142.3 °C; yield, 79.4%; 1H NMR
(
(
400 MHz, CDCl
3
) d in ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.70
The structure of
X-ray diffraction structural data. The sequence of the three
subtypes (SwissProt 1A-AR: P35348; 1B-AR: P35368; and
P25100) were based to predict the membrane protein and TM
1
a -ARs is difficult to elucidate because of lack of
2
2
dd, J = 5.5, 3.0 Hz, 2H), 7.61 (dd, J = 7.8, 0.8 Hz, 1H), 7.51 (t,
1
a -AR
J = 7.8 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H),
.22 (d, J = 7.8 Hz, 1H), 7.17 (d, J = 8.0 Hz, 2H), 4.82 (s, 2H), 2.98
t, J = 5.0 Hz, 4H), 2.81 (dd, J = 10.0, 6.2 Hz, 2H), 2.68–2.62 (m,
a
a
a1D-AR:
7
(
6
1
1
2
3
region within the SOSUI program. Protein blast was used to
search the template protein. b -Adrenergic G protein-coupled
receptor (Protein code: 2RH1) was the best match to the
H); 1 C NMR (101 MHz, CDCl
3
) d in ppm: 168.1, 152.7, 140.1,
3
2
34.3, 134.0, 132.8, 132.3, 129.1, 128.9, 127.3, 127.3, 124.8,
24.1, 123.4, 60.5, 53.6, 53.5, 41.5, 33.4; HRMS (ESI) m/z [M+1] :
a1A-,
+
a
1B
-
,
and
a
a
1D-AR subtypes; these sequence identity obtained for
calcd for C28
H
27
O
2
N
3
F
3
, 494.2049, found, 494.2041.
the three
1
-AR subtypes were 36%, 35% and 38%, respectively24
-ARs
were built using homology modeling protocol (Prime, Schrodinger);
the diffusible ligand carazolol in the crystal structure of b -adrener-
gic receptor (b -AR) was chosen for the study of receptor–ligand
(Supplementary data in Fig. S1). The models of the three a
1
4
.1.24. 2-(4-(2-(4-(4-(Trifluoromethyl)phenyl)piperazin-1-yl)
ethyl)benzyl)isoindoline-1,3-dione (24)
2
Light yellow solid; mp 164.3–164.9 °C; yield, 48.5%; 1H NMR
2
5,26
2
(
400 MHz, CDCl
3
) d in ppm: 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.70
interactions.
The three models were refined using energy min-
(
dd, J = 5.5, 3.0 Hz, 2H), 7.47 (d, J = 8.7 Hz, 2H), 7.37 (d, J = 8.0 Hz,
H), 7.17 (d, J = 8.0 Hz, 2H), 6.91 (d, J = 8.7 Hz, 2H), 4.82 (s, 2H),
imization with macromodel (Schrodinger) by (a) fixing the main
chain and refining the side chains by 500 steps; (b) fixing the side
chains and refining the main chain by 500 steps; and (c) refining
the whole body by 500 steps in a simulated octanol environment.
3D Ranachandran plots and profiles were obtained to evaluate the
2
3
6
1
5
.33 (t, J = 5.0 Hz, 4H), 2.80 (dd, J = 9.8, 6.3 Hz, 2H), 2.66–2.60 (m,
H); 1 C NMR (101 MHz, CDCl
3
) d in ppm: 168.5, 153.7, 140.2,
3
34.7, 134.4, 132.6, 129.4, 129.2, 126.8, 126.8, 123.7, 114.9, 60.6,
+
3.3, 48.4, 41.8, 33.6; HRMS (ESI) m/z [M+1] : calcd for C28
, 494.2049, found, 494.2040.
H
27
O
2
-
2
model structures. For the crystal structure of b -AR, the intracellu-
N
F
3 3
lar loop region 3 (IL3) was replaced by T4 lysozyme to increase the
solubility and stability of the receptor, and the active site of the
GPCRs was located in the extracellular region near loop 2 (EL2).
IL3 loop was removed in the modeling process of the three targets
since this loop was unlikely to play a major role in ligand binding.
The highly conserved disulfide bond, DRY region in loop 3, NPxxY
in loop 7, and CWxP in loop 6 were retained in the models.
4
.1.25. 2-(4-(2-(4-(2-(Methylsulfonyl)phenyl)piperazin-1-yl)
ethyl)benzyl)isoindoline-1,3-dione (25)
White solid; mp 228.5–229.4 °C; yield, 73.5%; 1H NMR
400 MHz, CDCl ) d in ppm: 8.07 (dd, J = 8.0, 1.6 Hz, 1H), 7.84
dd, J = 5.5, 3.0 Hz, 2H), 7.70 (dd, J = 5.5, 3.0 Hz, 2H), 7.61 (td,
(
(
3
J = 8.0, 1.6 Hz, 1H), 7.41 (dd, J = 8.0, 0.8 Hz, 1H), 7.37 (d, J = 8.0 Hz,
H), 7.32 (td, J = 8.0, 0.8 Hz, 1H), 7.17 (d, J = 8.0 Hz, 2H), 4.82 (s,
Molecular docking was performed using Surflex-Dock
(SYBYL 8.1 molecular modeling software). The diffusible ligand
Ò
2
Please cite this article in press as: Xu, F.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.11.020

8
F. Xu et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5. Bendix-Holme, J.; Christensen, M. M.; Rasmussen, P. C.; Jacobsen, F.; Nielsen, J.;
carazolol in the crystal structure of
2
b -adrenergic receptor
Norgaard, J. P.; Olesen, S.; Noer, L.; Wolf, H.; Elkjaer-Husted, S. Scand. J. Urol.
Nephrol. 1994, 28, 77.
Jardin, A.; Bensadoun, H.; Delauche-Cavallier, M. C.; Stalla-Bourdillon, A.; Attali,
P. Br. J. Urol. 1984, 74, 579.
Wong, W. C.; Chiu, G.; Wetzel, J. M.; Marzabadi, M. R.; Nagarathnam, D.; Wang,
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2
-AR) was retained as reference ligand in order to study the
-ARs–ligand interactions. Molecule was sketched and minimized
a
1
6
.
.
using Powell optimization in the presence of the Tripos force
field with a convergence criterion of 0.001 kcal/mol Å and then
assigned with the Gasteiger–Hückel charges. Automatic docking
was employed. Other parameters were established by default in
software.
7
8. Lepor, H. Urology 1995, 45, 406.
Djavan, B.; Marberger, M. Eur. Urol. 1999, 36, 1.
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1
1
0. Dellabella, M.; Milanese, G.; Muzzonigro, G. J. Urol. 2003, 170, 2202.
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006, 97, 747.
2
1
3. Kojima, Y.; Sasaki, S.; Kubota, Y.; Hayase, M.; Hayashi, Y.; Shinoura, H.;
This part of research were funded by The National Science
Foundation of Guangdong Province (Grant 2015A030313464 to F.
X., S2013040014088 to H.C.), and Science Foundation of Zhejiang
Province, China (Grant LY12B02008 to F.X.). We acknowledge the
technical assistance of Experimental Medical Research Center,
Guangzhou Medical University.
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Supplementary data
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