Rational design, synthesis, biological evaluation, homology and docking studies of coumarin derivatives as α1-adrenoceptor antagonists

Article abstract of DOI:10.1002/cbdv.201000135

According to a three-point pharmacophore for some uro-selective α₁-adrenoceptor (AR) antagonists, a novel class of coumarin (=2H-1-benzopyran-2-one) derivatives have been successfully designed and synthesized with high efficacies for α₁

Full text of DOI:10.1002/cbdv.201000135

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Rational Design, Synthesis, Biological Evaluation, Homology and Docking
Studies of Coumarin Derivatives as a1-Adrenoceptor Antagonists
by Xiang Zhoua), Ya-Dong Chenb), Tao Wangc), Xiao-Bing Wanga), and Ling-Yi Kong*a)
a) Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang,
210009 Nanjing, P. R. China (phone: þ86-25-85391289; fax: þ86-25-85301528;
e-mail: cpu_lykong@126.com)
b) Department of Inorganic Chemistry, China Pharmaceutical University, 210009 Nanjing, P. R. China
c) Jiangsu Center for Drug Screening, China Pharmaceutical University, 210009 Nanjing, P. R. China
According to
a three-point pharmacophore for some uro-selective a1-adrenoceptor (AR)
antagonists, a novel class of coumarin (¼2H-1-benzopyran-2-one) derivatives have been successfully
designed and synthesized with high efficacies for a1-AR. These synthesized coumarin derivatives
exhibited high efficacies towards a1-AR in in vitro pharmacological assays. Compared with prazosin (pKi
value of 8.77), among those coumarins, tolylpiperazine-substituted derivatives, 7 and 8, have comparable
pKi values of 8.81 and 8.77, respectively. The trend in efficacies of these coumarin derivatives towards a1A
-
adrenoceptor was further rationalized by intensive molecular docking. Our work demonstrated that the
designed coumarin derivatives can inhibit a1-AR in vitro. These findings will provide a guide for further
studies of the medical therapy of benign prostatic hyperplasia (BPH).
Introduction. – The a1-adrenoceptors (a1-ARs) regulate several functions of the
sympathetic nervous system, whose divergent antagonists have primarily been
developed for the treatment of cardiovascular diseases and benign prostatic hyper-
plasia (BPH) [1–5]. So far, drug therapy for BPH has been classified into two
categories: 5a-reductase inhibitors acting by reducing the size of the prostate; a1-AR
antagonists acting by relaxing prostate muscle. The a1-AR antagonists have an
advantage over 5a-reductase inhibitors, since they can provide effective relief of
symptoms in a short time. Analysis of a number of chemical structures of various newly
synthesized a1-AR antagonists [6–13] indicates that a large group of active compounds
contain arylpiperazine moieties, such as prazosin [14][15], 5-methylurapidil, BMY-
7378, REC-1512739, and RA36 (Fig. 1) [6]. So, to further develop a1-AR antagonists
for treating BPH, a rational strategy on the basis of prazosin was applied as depicted in
Scheme 1 with double objectives: a) to further clarify the structureꢀactivity relation-
ship with respect to the heterocyclic moiety presented in the upper part of the molecule
and to explore the nature of the a1-AR binding pocket, and b) to introduce a naturally
occurring heterocycle, the coumarin backbone, which was perceived as a critical
physicochemical characteristic to enhance the efficacy towards a1-AR.
At the same time, the construction of a three-dimensional pharmacophore model of
a1-AR antagonists have been studied [11][12], which shared some characteristics: an
aromatic ring (A); a positive ionizable center (P); and a H-bond donor (HBD); and
the distances of AꢀP, AꢀHBD, and PꢀHBD are 5.296–5.477, 5.429–6.823, and 3.000 ꢀ,
ꢁ 2011 Verlag Helvetica Chimica Acta AG, Zꢂrich

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1053
Fig. 1. Structures of potent a1-adrenoceptor antagonists
respectively. Considering those factors, a series of new compounds on the basis of the
piperazinyl-quinazoline molecular scaffold were designed and synthesized. The
piperazinyl-quinazoline moiety in prazosin was replaced by a coumarin ring, a
naturally occurring phytochemical with a wide range of biological activities, such as
anti-inflammatory, antitumor [16–18], anti-allergic, and anti-HIV-1 properties [19][20].
Furthermore, in the designed coumarin derivatives, the following chemical modifica-
tions were also considered: i) some groups such as Cl, MeO, or Me were chosen as the
substituents on the Ph ring linked to the piperazine nucleus; ii) the alkanediyl chain
acting as a spacer contained 2–4 C-atoms. The effect of substitutions in the o-, m-, and
p-positions of the Ph group bound to the piperazine ring was also considered. The
target coumarin derivatives 3–20 and 24–31 were synthesized as depicted in Scheme 1.
Here, the design, preparation, and in vitro pharmacological characterization of a
series of coumarin derivatives are reported. Moreover, recent studies had demon-
strated that the a1A-subtype was the predominantly expressed a1-AR in human prostate
and in the lower urinary tract (LUT). So, to further study the a1-ARs antagonists to
treat BPH, the docking behavior of the synthesized compounds has been studied based
on a1A-AR. Owing to the unavailable structure of the human a1A-AR, the homology

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Scheme 1. Design Strategy for the Target Compounds
models of a1A-AR was constructed. Based on these models, the intensive molecular
docking was performed. The interaction mode of the designed coumarin derivatives
with a1A-AR was also investigated.
Results and Discussion. – 1. Chemistry. Several possible chemical variations were
considered on the basis of piperazine derivatives. The synthesis of the target
compounds is shown in Schemes 2 and 3 (and Tables 1 and 2). The key intermediate,
compound 1, was prepared starting from resorcinol and ethyl acetoacetate with
concentrated H2SO4 as the catalyst and 1,4-dioxane as solvent. Then, 1 was reacted with
Br(CH2)nBr to give the key intermediates 2. Compounds 2 were reacted with
correspondingly substituted phenylpiperazines to give the target compounds 3–20. The
target compounds could be transformed with HCl in MeOH into the hydrochlorides
(Scheme 2).
The second series of compounds were prepared by the reaction of resorcinol with
malonic acid to give the intermediate compound 21, which was methylated with MeOH
and concentrated H2SO4 to afford 22. Compound 22 was reacted with Br(CH2)nBr and
K2CO3 to give the key intermediates 23. Then, compounds 23 were reacted with
correspondingly substituted phenylpiperazines to give the target compounds 24–31.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1055
Scheme 2. Synthesis of Compounds 3–201)
a) H2SO4, 608. b) Br(CH2)nBr, K2CO3, reflux. c) RH, K2CO3, reflux. d) MeOH, HCl, reflux.
Table 1. Structures of Compounds 3–20
Compound
n
R
Compound
n
R
3
4
5
6
7
8
9
10
11
2
2
2
2
2
2
2
2
2
Ph
12
13
14
15
16
17
18
19
20
2
2
4
4
4
4
3
3
3
4-MeOꢀC6H4ꢀCO
2,5-Cl2ꢀC6H3
4-MeꢀC6H4
2-MeOꢀC6H4
4-MeOꢀC6H4
4-ClꢀC6H4
2-MeꢀC6H4
4-MeꢀC6H4
4-MeOꢀC6H4
2-MeOꢀC6H4
4-MeꢀC6H4
2-MeꢀC6H4
3-MeOꢀC6H4
4,6-Dimethoxypyrimidin-2-yl
4-MeꢀC6H4ꢀCO
2-MeꢀC6H4
4-MeOꢀC6H4
The target compounds could be transformed with HCl in MeOH into the hydro-
chlorides (Scheme 3).
The structures of all the new compounds were characterized by analytical and
spectroscopic methods (1H- and 13C-NMR; cf. the Exper. Part). The molecular weights
of all compounds synthesized were confirmed by ESI-MS, and their purity was also
determined by HPLC analysis.
2. In vitro Activities. Antagonist efficacies were expressed as apparent pKi values
calculated from the following equation: pKi ¼ ꢀlog [B]þlog (rꢀ1), where [B] is the
molar concentration of antagonists, and r is the ratio of agonist EC50 determined in the
presence and absence of antagonist [21]. The a1-AR antagonist efficacies of
(arylpiperazinyl)alkyl-coumarin derivatives 3–20 and 24–31, expressed as pKi values,
1
)
For the structure of compounds 3–20, see Table 1.

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Scheme 3. Synthesis of Compounds 24–312)
a) ZnCl2, POCl3. b) MeOH, H2SO4. c) Br(CH2)nBr, K2CO3, reflux. d) RH, K2CO3, reflux. e) MeOH,
HCl, reflux.
Table 2. Structures of Compounds 24–31
Compound
n
R
Compound
n
R
24
25
26
27
2
2
2
2
Ph
28
29
30
31
2
2
3
4
2-MeOꢀC6H4
4-ClꢀC6H4
4-ClꢀC6H4
4-ClꢀC6H4
4-MeꢀC6H4
2-MeꢀC6H4
4-MeOꢀC6H4
have been determined in vitro and compiled in Table 3. All these newly designed
coumarin derivatives exhibit high efficacies (Table 3) towards a1-AR compared with
prazosin. Different substituents on the phenyl group linked to the piperazine nucleus
led to diverse binding potencies. For example, 2,5-dichlorophenyl-substituted deriva-
tive 13, and monochlorophenyl-substituted analogs 6, 29, 30, and 31 exhibited moderate
efficacies compared to the Me- and MeO-substituted ones. The efficacies of the
compounds that bear propane-1,3-diyl or butane-1,4-diyl spacers were weaker than
those with a ethane-1,2-diyl spacer (compare 14 and 18 to 7, 15 and 19 to 8, and 20 and
16 to 5), indicating that the increase in the length of the alkanediyl chain lowered the
activity. Moreover, the p-substituents on the phenylpiperazine moiety were found to be
more relevant for activity. Particularly, in our study, compounds 7 and 8 with Me groups
on the benzene ring are the most potent examples of this series with pKi values of 8.81
and 8.77, respectively.
2
)
For the structure of compounds 24–31, see Table 2.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1057
Table 3. Apparent pKi Values of the Synthesized Compoundsa)
Compounds
N
pKi
Compounds
N
pKi
3
4
5
6
7
8
9
10
11
12
13
14
3
3
3
3
3
3
3
3
3
3
3
3
3
4
7.88ꢁ0.12
6.86ꢁ0.21
8.34ꢁ0.11
7.14ꢁ0.17
8.81ꢁ0.13
8.77ꢁ0.09
7.39ꢁ0.15
7.47ꢁ0.18
7.47ꢁ0.20
7.06ꢁ0.14
6.34ꢁ0.13
7.78ꢁ0.18
7.35ꢁ0.16
8.79ꢁ0.07
16
17
18
19
20
24
25
26
27
28
29
30
31
3
3
3
3
3
3
3
3
3
3
3
3
3
6.61ꢁ0.11
8.14ꢁ0.15
6.76ꢁ0.19
7.31ꢁ0.11
6.65ꢁ0.11
6.91ꢁ0.14
6.78ꢁ0.15
7.88ꢁ0.13
7.12ꢁ0.17
7.78ꢁ0.09
7.33ꢁ0.15
6.88ꢁ0.17
7.30ꢁ0.18
15
Prazosinb)
a) Data are meansꢁstandard deviation of N independent experiments. b) Prazosin was used as positive
control.
3. The Homology and Docking Studies. By using the homology models of a1A-
AR built, a pharmacophore (Fig. 2,a) was generated with LIGANDSCOUT 2.0
(Inte:ligand, Vienna), based on the hypothetic binding mode between the receptor and
compound 7. This pharmacophore displayed that compound 7 mainly had five features
including four hydrophobic regions and one H-bond interaction. The C¼O group on the
coumarin ring acted as an H-bond acceptor, which occurred also in other potent
compounds (such as 5, 8, and 17) and interacted with NꢀH group on the backbone of
Phe289. The residues Phe308, Leu290, Val291, Ile178, and Val185 shaped a hydro-
phobic pocket which can accommodate the 4-Me group and the benzo moiety of the
coumarin moiety. The 4-MeO-coumarin derivatives exhibited limited reduction in
receptor-binding potency compared with the 4-Me-coumarin derivatives, revealing that
small substituents at C(4) of coumarin ring might favor more potent binding capacity.
This is in agreement with the result that the 4-Me group fits the hydrophobic feature
better than the 4-MeO group (compare 26 to 8, 25 to 7, and 28 to 4). Fig. 2,b, revealed
that the spatial resistance between the MeO group and residues Val291 and Leu290
could be the reason for the lower efficacy of the 4-MeO-substituted derivatives (i.e.,
24–31). For the phenylpiperazine moiety, the phenyl group and the 4-Me group
occupied the narrow hydrophobic groove formed by the residues Val282, Leu197,
Phe193, and Phe281 (Fig. 2,b). Decreases in efficacies (compare 5 and 6 to 7, 16 to 15,
and 20 to 18) were attributed to the Cl and MeO substituents. Whereas the spatial
resistance resulted in low efficacies for MeO substituents, the presence of the Cl
substituents weakened the efficacies due to its source of binding energy: a) lipophilic
interactions; b) a positive electrostatic potential at the tip of the Cl-atom, which was not
suitable here, although it had fairly similar size and hydrophobicity as the Me group.
The p-substituents were sensitive to the length of the alkanediyl chain (compare 7 to 14,
8 to 15), and p-substitution turned out to be crucial for the efficacy. The piperazine N-

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Fig. 2. a) The pharmacophore based on the hypothetic binding mode of compound 7 with five features:
one H-bond acceptor (deep red arrow) and four hydrophobic groups (yellow). b) The surface model for 7
bound to the active site of a1A-AR, of which the active site fragment are colored according to the
electrostatic potentials, and the active-site amino acid residues (green) of compound 7 (gray) shown as
stick models.
atom, which was considered as a positive ion center, displayed a weak H-bond
interaction with Ser192.
Conclusions. – A novel class of coumarin derivatives with high efficacies for a1-AR
have been successfully designed and synthesized. These coumarin derivatives inhibited
a1-AR in vitro. All of these compounds shifted the concentrationꢀresponse curves for
phenylephrine in parallel without diminishing the maximum contraction, which
suggested reversible antagonistic effects on a1-AR, particularly when the substituent
of phenyl ring was in o-position. Therefore, it can be concluded that coumarin with a
phenylpiparazinyl substitution may show favorable anti-a1-receptor activities. At the
same time, the interaction mode of the designed coumarin derivatives with the a1A-AR
was investigated.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1059
This work was supported by the Program for Changjiang Scholars (L.-Y. K.), Ministry of Education
of the P. R. of China.
Experimental Part
General. Chemicals: Shanghai Chemical Reagent Company. Column chromatography (CC): silica gel
60 (SiO2, 200–300 mesh; Qingdao Ocean Chemical Company, P. R. China). TLC: 60 F254 silica-gel
plates (250 mm, Qingdao Ocean Chemical Company, P. R. China). HPLC: Agilent 1100 LC. M.p.: RY-1
melting-point tester; in cap. tube; uncorrected. IR Spectra: Shimadzu FTIR-8400S spectrometer; n˜ in
cmꢀ1. 1H- and 13C-NMR spectra: Bruker ACF-300 and ꢀ500 NMR instruments; d in ppm, J in Hz. LR-
MS: Hewlett-Packard 1100 LC/MSD spectrometer.
7-Hydroxy-4-methyl-2H-chromen-2-one (1). Conc. H2SO4 (2 ml) was dropped slowly into 0.05 mol
of resorcinol dissolved in dioxane (10 ml). The mixture was kept under 258. After the addition of H2SO4,
ethyl acetoacetate (¼ethyl 3-oxobutanoate; 7 ml) was added dropwise within 20 min. Then, the mixture
was heated to 608 and refluxed for 4 h. The mixture was then poured into H2O (300 ml), and the
precipitate was filtered off. The residue was purified by CC (cyclohexane/AcOEt 7:3) to give 1 (2.2 g,
70%).
7-(2-Bromoethoxy)-4-methyl-2H-chromen-2-one (2a). A mixture of 1 (0.005 mol), BrCH2CH2Br
(0.04 mol) and K2CO3 (1.0 g, 0.01 mol) in dry EtOH was refluxed for 24 h. The mixture was filtered, and
the org. phase was evaporated under reduced pressure. The residue was purified by CC (cyclohexane/
AcOEt 8 :2) to give 2a (55%). White crystals.
7-(3-Bromopropoxy)-4-methyl-2H-chromen-2-one (2b) and 7-(4-bromobutoxy)-4-methyl-2H-chro-
men-2-one (2c) were prepared according to the same method using 1 and 1,3-dibromopropane or 1,4-
dibromobutane, resp.
General Procedure for the Preparation of Compounds 3–20. To a mixture of acetone and EtOH
(15 ml each) were added 0.01 mol of 2, 0.01 mol of substituted piperazine derivative, and 0.02 mol
K2CO3. The mixture was refluxed for 48 h and then evaporated. The flash chromatography (FC) was
performed to afford the target compound.
4-Methyl-7-[2-(4-phenylpiperazin-1-yl)ethoxy]-2H-chromen-2-one (3). Yield 41%. Colorless pow-
der. M.p. 154–1568. HPLC 96.7% (MeOH/H2O 75 :25 (v/v); tR 13.78 min). IR (KBr): 3546, 1699, 1618,
1386, 1299, 1147, 1070, 692. 1H-NMR (CDCl3, 300 MHz): 7.51 (d, J¼8.8, HꢀC(5)); 6.90–6.98 (m, 5 arom.
H); 7.27–7.30 (m, HꢀC(6), HꢀC(8)); 6.15 (s, HꢀC(3)); 4.27–4.29 (m, CH2O); 3.26–3.29 (m,
2 CH2N(4)); 2.78–2.80 (m, 2 CH2N(1)); 2.94–2.97 (m, N(1)ꢀCH2CH2O); 2.38 (s, MeꢀC(4)). ESI-MS:
365.0 ([MþH]þ ).
7-{2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethoxy}-4-methyl-2H-chromen-2-one (4). Yield 45%. Col-
orless powder. M.p. 156–1578. HPLC 96.3% (MeOH/H2O 75 :25 (v/v); tR 14.58 min). IR (KBr): 3529,
1697, 1618, 1510, 1386, 1070, 823. 1H-NMR (CDCl3, 300 MHz): 7.51 (d, J¼8.8, HꢀC(5)); 6.84–7.26 (m, 4
arom. H, HꢀC(6), HꢀC(8)); 6.14 (s, HꢀC(3)); 4.24–4.28 (m, CH2O); 3.16–3.19 (m, 2 CH2N(4)); 2.85–
2.88 (m, 2 CH2N(1)); 2.96–2.99 (m, N(1)ꢀCH2CH2O); 2.38 (s, Me); 3.87 (s, MeO). 13C-NMR (CDCl3,
125 MHz): 161.8; 161.3; 155.3; 152.5; 152.3; 141.2; 125.5; 123.0; 121.0; 118.2; 113.7; 112.7; 112.0; 111.3;
101.6; 66.5; 56.9; 55.3; 53.9; 50.5; 18.6. ESI-MS: 395.3 ([MþH]þ ).
7-{2-[4-(4-Methoxyphenyl)piperazin-1-yl]ethoxy}-4-methyl-2H-chromen-2-one (5). Yield 45%. Col-
orless powder. M.p. 156–1578. HPLC 98.7% (MeOH/H2O 80 :20 (v/v); tR 6.35 min). IR (KBr): 2943,
1
1719, 1608, 1511, 1450, 1389, 1269, 1030, 710. H-NMR (CDCl3, 500 MHz): 7.51 (d, J¼8.8, HꢀC(5));
6.84–6.92 (m, 4 arom. H, HꢀC(6), HꢀC(8)); 6.14 (s, HꢀC(3)); 4.19–4.22 (m, CH2O); 3.11–3.15 (m,
2 CH2N(4)); 2.75–2.78 (m, 2 CH2N(1)); 2.90–2.93 (m, N(1)ꢀCH2CH2O); 2.39 (s, Me); 3.76 (s, MeO).
13C-NMR (CDCl3, 125 MHz): 161.8; 161.2; 155.3; 153.9; 152.5; 125.5; 118.3; 145.6; 114.5; 113.7; 112.7;
112.1; 101.6; 66.5; 56.9; 55.5; 53.8; 50.5; 18.6. ESI-MS: 395.2 ([MþH]þ ).
7-{2-[4-(4-Chlorophenyl)piperazin-1-yl]ethoxy}-4-methyl-2H-chromen-2-one (6). Yield 35%. Color-
less powder. M.p. 125–1278. HPLC 98.3% (MeOH/H2O 80 :20 (v/v); tR 5.65 min). IR (KBr): 3452, 1726,
1
1498, 1392, 1284, 1234, 1141, 673. H-NMR (CDCl3, 500 MHz): 7.50 (d, J¼8.8, HꢀC(5)); 7.20–7.23 (m,
HꢀC(6), HꢀC(8)); 6.82–6.90 (m, 4 arom. H); 6.13 (s, HꢀC(3)); 4.21 (t, J¼5.7, CH2O); 3.17–3.20 (m,

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
2 CH2N(4)); 2.74–2.77 (m, 2 CH2N(1)); 2.91 (t, J¼5.7, N(1)ꢀCH2CH2O); 2.39 (s, Me). 13C-NMR
(CDCl3, 125 MHz): 161.3; 155.4; 152.5; 141.2; 125.7; 125.5; 18.7; 117.5; 114.7; 112.6; 112.3; 101.8; 66.5;
56.9; 53.5; 48.9. ESI-MS: 399.2 ([MþH]þ ).
4-Methyl-7-{2-[4-(4-methylphenyl)piperazin-1-yl]ethoxy}-2H-chromen-2-one (7). Yield 42%. Color-
less powder. M.p. 141–1438. HPLC 97.6% (MeOH/H2O 80 :20 (v/v); tR 4.75 min). IR (KBr): 3530, 1708,
1620, 1514, 1451, 1387, 1304, 1008, 529. 1H-NMR (CDCl3, 500 MHz): 7.49 (d, J¼8.8, HꢀC(5)); 7.06–7.08
(m, HꢀC(6), HꢀC(8)); 6.83–6.89 (m, 4 arom. H); 6.13 (s, HꢀC(3)); 4.20 (t, CH2O, J ¼ 5.7); 3.16–3.19
(m, 2 CH2N(4)); 2.74–2.77 (m, 2 CH2N(1)); 2.91 (t, J¼5.7, N(1)ꢀCH2CH2O); 2.39 (s, Me); 2.67 (s, Me).
13C-NMR (CDCl3, 125 MHz): 161.8; 161.3; 155.3; 152.5; 149.2; 129.7; 125.5; 116.5; 113.7; 112.7; 112.1;
101.6; 66.6; 56.9; 53.7; 49.6; 20.4; 18.6. ESI-MS: 379.2 ([MþH]þ ).
4-Methyl-7-{2-[4-(2-methylphenyl)piperazin-1-yl]ethoxy}-2H-chromen-2-one (8). Yield 45%. Color-
less powder. M.p. 112–1138. HPLC 97.5% (MeOH/H2O 80 :20 (v/v); tR 5.36 min). IR (KBr): 2927, 1710,
1
1617, 1492, 1393, 1289, 1223, 835. H-NMR (CDCl3, 300 MHz): 7.49 (d, J¼8.8, HꢀC(5)); 7.13–7.16 (m,
HꢀC(6), HꢀC(8)); 6.84–7.04 (m, 4 arom. H); 6.13 (s, HꢀC(3)); 4.19–4.21 (m, CH2O); 2.97–3.00 (m,
3 CH2N(1)); 2.77–2.81 (m, 2 CH2N(4)); 2.39 (s, Me); 2.31 (s, Me). 13C-NMR (CDCl3, 125 MHz): 161.3;
155.3; 152.5; 147.3; 132.7; 131.1; 126.6; 125.6; 123.3; 112.7; 112.1; 101.7; 66.6; 56.9; 54.2; 51.5; 18.6; 17.8.
ESI-MS: 379.2 ([MþH]þ ).
7-{2-[4-(3-Methoxyphenyl)piperazin-1-yl]ethoxy}-4-methyl-2H-chromen-2-one (9). Yield 37%. Col-
orless powder. M.p. 134–1368. HPLC 96.9% (MeOH/H2O 80 :20 (v/v); tR 4.56 min). IR (KBr): 2928,
1715, 1612, 1501, 1389, 1362, 1258, 1065, 1020, 749. 1H-NMR (CDCl3, 300 MHz): 7.51 (d, J¼8.8,
HꢀC(5)); 6.84–7.26 (m, 4 arom. H, HꢀC(6), HꢀC(8)); 6.14 (s, HꢀC(3)); 4.24–4.27 (m, CH2O); 3.14–
3.18 (m, 2 CH2N(4)); 2.85–2.89 (m, 2 CH2N(1)); 2.97–2.99 (m, N(1)ꢀCH2CH2O); 2.38 (s, Me); 3.87 (s,
MeO). 13C-NMR (CDCl3, 125 MHz): 161.8; 161.2; 160.6; 155.3; 152.6; 152.4; 129.8; 125.5; 113.7; 112.6;
112.1; 108.9; 104.5; 101.6; 66.5; 56.9; 55.2; 53.6; 48.9; 18.6. ESI-MS: 395.3 ([MþH]þ ).
7-{2-[4-(4,6-Dimethoxypyrimidin-2-yl)piperazin-1-yl]ethoxy}-4-methyl-2H-chromen-2-one (10).
Yield 34%. Colorless powder. M.p. 112–1148. HPLC 96.8% (MeOH/H2O 80 :20 (v/v); tR 5.77 min).
IR (KBr): 2943, 1718, 1603, 1579, 1529, 1361, 1282, 1194, 1157, 1008. 1H-NMR (CDCl3, 500 MHz): 7.51 (d,
J¼8.8, HꢀC(5)); 6.83–6.89 (m, HꢀC(6), HꢀC(8)); 6.14 (br. s, 1 H, pyrimidine); 5.37 (s, HꢀC(5)); 6.14 (s,
HꢀC(3)); 4.20–4.23 (m, CH2O); 3.83–3.89 (m, 2 MeO, 2 CH2N(4)); 2.85–2.88 (m, CH2N(1)); 2.63–2.66
(m, N(1)ꢀCH2CH2O)); 2.39 (s, Me); 2.88–2.91 (m, CH2N(1)). ESI-MS: 427.1 ([MþH]þ ).
4-Methyl-7-{2-[4-(4-methylbenzoyl)piperazin-1-yl]ethoxy}-2H-chromen-2-one (11). Yield 36%. Col-
orless powder. M.p. 135–1378. HPLC 98.8% (MeOH/H2O 80 :20 (v/v); tR 5.37 min). IR (KBr): 1720,
1616, 1429, 1391, 1156, 1070, 750. 1H-NMR (CDCl3, 500 MHz): 7.49 (d, J¼8.8, HꢀC(5)); 6.81 (d, J¼2.5,
HꢀC(8)); 6.81 (dd, J¼2.5, 8.8, HꢀC(6)); 7.19–7.31 (m, 4 arom. H); 6.13 (s, HꢀC(3)); 4.16 (t, J¼5.6,
CH2O); 3.76–3.81 (m, CH2N(4)); 3.48–3.51 (m, CH2N(4)); 2.98 (t, J¼5.6, N(1)ꢀCH2CH2O); 2.48–2.52
(m, 2 CH2N(1)); 2.39 (s, Me); 2.37 (s, Me). ESI-MS: 407.2 ([MþH]þ ).
7-{2-[4-(4-Methoxybenzoyl)piperazin-1-yl]ethoxy}-4-methyl-2H-chromen-2-one (12). Yield 35%.
Colorless powder. M.p. 113–1158. HPLC 99.1% (MeOH/H2O 80 :20 (v/v); tR 4.87 min). IR (KBr):
1
3436, 1702, 1618, 1511, 1453, 1423, 1393, 1249, 1004, 841. H-NMR (CDCl3, 500 MHz): 7.49 (d, J¼8.8,
HꢀC(5)); 6.81 (d, J¼2.4, HꢀC(8)); 6.86 (dd, J¼2.4, 8.8, HꢀC(6)); 7.37–7.40 (m, 2 arom. H); 6.90–6.93
(m, 2 arom. H); 6.13 (s, HꢀC(3)); 4.16 (t, J¼5.5, CH2O); 2.85–2.89 (m, N(1)ꢀCH2CH2O); 3.64–3.68 (m,
2 CH2N(4)); 2.48–2.53 (m, 2 CH2N(1)); 2.39 (s, Me); 3.87 (s, MeO). ESI-MS: 423.2 ([MþH]þ ).
7-{2-[4-(2,5-Dichlorophenyl)piperazin-1-yl]ethoxy}-4-methyl-2H-chromen-2-one (13). Yield 36%.
Colorless powder. M.p. 134–1358. HPLC 98.1% (MeOH/H2O 80 :20 (v/v); tR 4.07 min). IR (KBr): 2946,
1713, 1615, 1475, 1389, 1158, 1071, 808. 1H-NMR (CDCl3, 500 MHz): 7.50 (d, J¼8.8, HꢀC(5)); 6.84 (d,
J ¼ 2.5, HꢀC(8)); 6.89 (dd, HꢀC(6), J¼2.5, 8.8); 7.28 (s, 1 arom. H); 6.95–6.97 (m, 2 arom. H); 6.14 (s,
HꢀC(3)); 4.21–4.23 (m, CH2O); 3.10–3.13 (m, 2 CH2N(4)); 2.92–2.96 (m, N(1)ꢀCH2CH2O); 2.77–2.81
(m, 2 CH2N(1)); 2.39 (s, Me). 13C-NMR (CDCl3, 125 MHz): 161.3; 155.4; 152.5; 133.2; 131.5; 127.0; 125.7;
120.9; 112.7; 112.2; 101.7; 66.5; 56.9; 53.7; 50.9; 18.7. ESI-MS: 433.1 ([MþH]þ ).
4-Methyl-7-{4-[4-(4-methylphenyl)piperazin-1-yl]butoxy}-2H-chromen-2-one (14). Yield 37%. Col-
orless powder. M.p. 141–1428. HPLC 98.4% (MeOH/H2O 80 :20 (v/v); tR 5.13 min). IR (KBr): 3412,
1715, 1614, 1491, 1445, 1386, 1146, 1124, 996, 557. 1H-NMR (CDCl3, 500 MHz): 7.49 (d, J¼8.8, HꢀC(5));
6.82 (d, J ¼ 2.5, HꢀC(8)); 6.86 (d, HꢀC(6), J¼2.5, 8.8); 7.15–7.18 (m, 2 arom. H); 6.98–7.01 (m, 2 arom.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1061
H); 6.13 (s, HꢀC(3)); 4.14–4.17 (m, CH2O); 2.94–2.98 (m, 2 CH2N(4)); 2.63–2.66 (m, 2 CH2N(1));
2.49–2.52 (m, N(1)ꢀCH2(CH2)2CH2O)); 2.39 (s, Me); 2.31 (s, Me); 1.87–1.90 (m, CH2); 1.73–1.75 (m,
CH2). 13C-NMR (CDCl3, 125 MHz): 162.2; 161.3; 152.5; 146.6; 132.6; 131.1; 126.6; 125.5; 123.1; 119.1;
113.5; 112.7; 111.9; 101.4; 68.3; 58.2; 53.8; 51.7; 27.0; 23.3; 18.6; 17.8. ESI-MS: 407.3 ([MþH]þ ).
4-Methyl-7-{4-[4-(2-methylphenyl)piperazin-1-yl]butoxy}-2H-chromen-2-one (15). Yield 39%. Col-
orless powder. M.p. 101–1038. HPLC 96.8% (MeOH/H2O 80 :20 (v/v); tR 6.03 min). IR (KBr): 3425,
1716, 1614, 1552, 1366, 1294, 1146, 749. 1H-NMR (CDCl3, 500 MHz): 7.48 (d, J¼8.8, HꢀC(5)); 7.05–7.07
(m, 2 arom. H); 6.81–6.86 (m, 4 arom. H, HꢀC(6), HꢀC(8)); 6.13 (s, HꢀC(3)); 4.05–4.07 (m, CH2O);
3.15–3.19 (m, 2 CH2N(4)); 2.62–2.66 (m, 2 CH2N(1)); 2.47–2.50 (m, N(1)ꢀCH2(CH2)2CH2O); 2.39 (s,
Me); 2.68 (s, Me); 1.88–1.91 (m, CH2); 1.74–1.76 (m, CH2). 13C-NMR (CDCl3, 125 MHz): 162.2; 161.3;
152.5; 149.3; 129.6; 129.3; 125.5; 116.5; 113.5; 112.7; 111.9; 101.4; 68.3; 58.1; 53.3; 49.7; 27.0; 23.3; 20.3;
18.6. ESI-MS: 405.3 ([MþH]þ ).
7-{4-[4-(4-Methoxyphenyl)piperazin-1-yl]butoxy}-4-methyl-2H-chromen-2-one (16). Yield 40%.
Colorless powder. M.p. 108–1098. HPLC 96.5% (MeOH/H2O 80 :20 (v/v); tR 7.11 min). IR (KBr):
1
3420, 1729, 1608, 1511, 1451, 1386, 1155, 1135, 874, 819. H-NMR (CDCl3, 500 MHz): 7.48 (d, J¼8.8,
HꢀC(5)); 6.81–6.91 (m, 4 arom. H, HꢀC(6), HꢀC(8)); 6.12 (s, HꢀC(3)); 4.05–4.07 (m, CH2O); 3.76 (s,
MeO); 3.09–3.13 (m, 2 CH2N(4)); 2.62–2.65 (m, 2 CH2N(1)); 2.46–2.49 (m, N(1)ꢀCH2(CH2)2CH2O);
2.39 (s, Me); 2.68 (s, Me); 1.87–1.90 (m, CH2); 1.72–1.75 (m, CH2). 13C-NMR (CDCl3, 125 MHz): 162.2;
161.3; 155.4; 153.9; 152.5; 145.7; 125.5; 118.2; 114.5; 113.5; 112.6; 111.9; 101.4; 68.3; 58.1; 55.6; 53.4; 50.6;
27.0; 23.3; 18.6. ESI-MS: 423.3 ([MþH]þ ).
7-{4-[4-(2-Methoxyphenyl)piperazin-1-yl]butoxy}-4-methyl-2H-chromen-2-one (17). Yield 48%.
Colorless powder. M.p. 98–1008. HPLC 98.2% (MeOH/H2O 80 :20 (v/v); tR 6.71 min). IR (KBr):
3426, 1716, 1612, 1389, 1145, 1070, 754. 1H-NMR (CDCl3, 500 MHz): 7.48 (d, J¼8.8, HꢀC(5)); 6.81–6.94
(m, 4 arom. H, HꢀC(6), HꢀC(8)); 6.12 (s, HꢀC(3)); 4.05–4.08 (m, CH2O); 3.86 (s, MeO); 3.09–3.13 (m,
2 CH2N(4)); 2.66–2.70 (m, 2 CH2N(1)); 2.50–2.53 (m, N(1)ꢀCH2(CH2)2CH2O); 2.39 (s, Me); 1.88–1.91
(m, CH2); 1.72–1.75 (m, CH2). 13C-NMR (CDCl3, 125 MHz): 162.2; 161.3; 155.4; 152.4; 141.4; 125.5;
122.9; 121.0; 118.3; 113.5; 112.7; 111.9; 111.3; 101.4; 68.3; 58.2; 55.3; 53.5; 50.6; 27.0; 23.3; 18.6. ESI-MS:
423.3 ([MþH]þ ).
4-Methyl-7-{3-[4-(4-methylphenyl)piperazin-1-yl]propoxy}-2H-chromen-2-one (18). Yield 38%.
Colorless powder. M.p. 146–1478. HPLC 97.6% (MeOH/H2O 80 :20 (v/v); tR 5.67 min). IR (KBr):
3409, 1711, 1611, 1513, 1388, 1201, 1139, 927, 813. 1H-NMR (CDCl3, 500 MHz): 7.48 (d, J¼8.8, HꢀC(5));
6.83–7.08 (m, 4 arom. H, HꢀC(6), HꢀC(8)); 6.12 (s, HꢀC(3)); 4.10–4.12 (m, CH2O); 3.14–3.17 (m,
2 CH2N(4)); 2.64–2.66 (m, 2 CH2N(1)); 2.57–2.61 (m, N(1)ꢀCH2CH2CH2O); 2.39 (s, Me); 2.27 (s, Me);
2.02–2.05 (m, CH2). 13C-NMR (CDCl3, 125 MHz): 162.1; 161.3; 155.4; 152.5; 149.2; 129.7; 129.6; 129.3;
125.5; 116.4; 113.6; 112.6; 111.9; 101.5; 66.8; 54.9; 53.3; 49.7; 26.5; 20.3; 18.6. ESI-MS: 393.3 ([MþH]þ ).
4-Methyl-7-{3-[4-(2-methylphenyl)piperazin-1-yl]propoxy}-2H-chromen-2-one (19). Yield 40%.
Colorless powder. M.p. 108–1108. HPLC 97.8% (MeOH/H2O 80 :20 (v/v); tR 5.65 min). IR (KBr):
3428, 1713, 1618, 1555, 1293, 1013, 770. 1H-NMR (CDCl3, 500 MHz): 7.49 (d, J¼8.8, HꢀC(5)); 6.84 (d,
J ¼ 2.5, HꢀC(8)); 6.87 (d, J¼2.5, 8.8, HꢀC(6)); 7.16–7.18 (m, 2 arom. H); 6.96–6.99 (m, 2 arom. H); 6.12
(s, HꢀC(3)); 4.11–4.13 (m, CH2O); 2.95–2.98 (m, 2 CH2N(4)); 2.63–2.66 (m, 3 CH2N(1)); 2.39 (s, Me);
2.31 (s, Me); 2.04–2.07 (m, CH2). 13C-NMR (CDCl3, 125 MHz): 162.2; 161.3; 155.4; 152.5; 151.5; 132.6;
131.1; 126.6; 125.5; 123.2; 119.1; 113.6; 112.6; 111.9; 101.5; 66.8; 55.0; 53.8; 51.6; 26.5; 18.6. ESI-MS: 393.3
([MþH]þ ).
7-{3-[4-(4-Methoxyphenyl)piperazin-1-yl]propoxy}-4-methyl-2H-chromen-2-one (20). Yield 37%.
Colorless powder. M.p. 144–1458. HPLC 96.8% (MeOH/H2O 80 :20 (v/v); tR 5.34 min). IR (KBr): 3439,
1
3079, 1709, 1610, 1510, 1461, 1202, 1140, 840. H-NMR (CDCl3, 500 MHz): 7.48 (d, J¼8.8, HꢀC(5));
6.83–6.92 (m, 4 arom. H, HꢀC(6), HꢀC(8)); 6.12 (s, HꢀC(3)); 4.10–4.13 (m, CH2O); 3.11–3.14 (m,
2 CH2N(4)); 2.64–2.67 (m, 2 CH2N(1)); 2.57–2.61 (m, N(1)ꢀCH2CH2CH2O); 2.39 (s, Me); 3.87 (s,
MeO); 2.04–2.06 (m, CH2). 13C-NMR (CDCl3, 125 MHz); 162.2; 161.3; 155.4; 152.6; 152.5; 145.8; 125.6;
118.3; 114.6; 113.6; 112.7; 112.6; 112.1; 112.0; 101.6; 66.8; 55.6; 54.7; 53.5; 50.7; 26.6; 18.6. ESI-MS: 409.2
([MþH]þ ).
4,7-Dihydroxy-2H-1-benzopyran-2-one (21). Resorcin (0.07 mol), malonic acid (0.07 mol), and
ZnCl2 (30 g) were dissolved in 20 ml of POCl3. The mixture was kept for 24 h at 608. Then, the mixture

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
was poured into 250 ml of ice-H2O and deposited overnight. The precipitate was filtered and then
recrystallized with 5% EtOH to give 21 5.91 g (48%).
7-Hydroxy-4-methoxy-2H-chromen-2-one (22). Compound 21 (1.25 g) and 7 ml of oil of vitriol was
dissolved in 70 ml of MeOH. The mixture was refluxed for 1.5 h and then cooled to r.t. The crystals
formed were filtered to give 22 (1.19 g, 88%).
7-(2-Bromoethoxy)-4-methoxy-2H-chromen-2-one (23). A mixture of 22 (0.005 mol), BrCH2CH2Br
(0.04 mol), and K2CO3 (1.0 g, 0.01 mol) in dry EtOH was refluxed for 24 h. The mixture was filtered, and
the org. phase was evaporated. The residue was purified by CC (SiO2; cyclohexane/AcOEt 8 :2) to give
23 (55%) as white crystals. The 7-(3-bromopropoxy)-4-methoxy-2H-chromen-2-one and 7-(4-bromobu-
toxy)-4-methoxy-2H-chromen-2-one were prepared by the same method using 1 and corresponding 1,3-
dibromopropane or 1,4-dibromobutane.
General Procedure for the Preparation of Compounds 24–31. To 15 ml of acetone and 15 ml of
alcohol were added 0.01 mol 23, 0.01 mol of substituted piperazine derivative, and 0.02 mol of K2CO3.
The mixture was refluxed for 48 h and then evaporated. The FC provided the target compounds.
4-Methoxy-7-[2-(4-phenylpiperazin-1-yl)ethoxy]-2H-chromen-2-one (24). Yield 42%. Colorless
powder. M.p. 115–1178. HPLC 96.5% (MeOH/H2O 80 :20 (v/v); tR 7.35 min). IR (KBr): 2948, 1746,
1
1716, 1618, 1492, 1147, 1001. H-NMR (CDCl3, 500 MHz): 7.70 (d, J¼8.8, HꢀC(5)); 6.81–7.25 (m, 5
arom. H, HꢀC(6), HꢀC(8)); 5.57 (s, HꢀC(3)); 4.20–4.23 (m, CH2O); 3.22–3.25 (m, 2 CH2N(4)); 2.74–
2.77 (m, 2 CH2N(1)); 2.91–2.93 (m, N(1)ꢀCH2CH2O); 3.97 (s, MeO). 13C-NMR (CDCl3, 125 MHz):
166.8; 163.4; 162.4; 155.1; 151.3; 129.1; 119.8; 116.2; 112.6; 109.1; 101.2; 87.7; 66.6; 56.9; 56.2; 53.7; 49.1.
ESI-MS: 381.3 ([MþH]þ ).
4-Methoxy-7-{2-[4-(4-methylphenyl)piperazin-1-yl]ethoxy}-2H-chromen-2-one (25). Yield 36%.
Colorless powder. M.p. 162–1648. HPLC 97.8% (MeOH/H2O 80 :20 (v/v); tR 8.15 min). IR (KBr):
2948, 1746, 1716, 1618, 1492, 1241, 1147, 766. 1H-NMR (CDCl3, 500 MHz): 7.70 (d, J¼8.8, HꢀC(5)); 7.06–
7.08 (m, HꢀC(6), HꢀC(8)); 6.81–6.87 (m, 4 arom. H); 5.57 (s, HꢀC(3)); 4.20–4.22 (m, CH2O); 3.17–
3.21 (m, 2 CH2N(4)); 2.89–2.92 (m, N(1)ꢀCH2CH2O); 2.75–2.78 (m, 2 CH2N(1)); 3.97 (s, MeO); 2.67 (s,
Me). 13C-NMR (CDCl3, 125 MHz): 166.8; 163.4; 162.4; 155.1; 149.2; 129.6; 129.4; 124.1; 116.5; 112.6;
109.1; 101.2; 87.7; 66.5; 56.9; 56.2; 53.7; 49.6; 20.3. ESI-MS: 395.2 ([MþH]þ ).
4-Methoxy-7-{2-[4-(2-methylphenyl)piperazin-1-yl]ethoxy}-2H-chromen-2-one (26). Yield 36%.
Colorless powder. M.p. 122–1248. HPLC 98.1% (MeOH/H2O 80 :20 (v/v); tR 8.64 min). IR (KBr):
2931, 1718, 1633, 1392, 1295, 917. 1H-NMR (CDCl3, 500 MHz): 7.70 (d, J¼8.8, HꢀC(5)); 6.81 (d, J¼2.4,
HꢀC(8)); 6.87 (dd, J¼8.8, 2.4, HꢀC(6)); 6.98–7.18 (m, 4 arom. H); 5.57 (s, HꢀC(3)); 4.21–4.24 (m,
CH2O); 2.95–2.98 (m, 2 CH2N(4), N(1)ꢀCH2CH2O); 2.76–2.79 (m, 2 CH2N(1)); 3.97 (s, MeO); 2.67 (s,
Me). 13C-NMR (CDCl3, 125 MHz): 166.8; 163.4; 162.4; 155.1; 151.4; 132.6; 131.1; 124.1; 119.1; 112.6;
109.0; 101.2; 87.7; 66.6; 56.9; 56.2; 54.2; 51.6; 17.8. ESI-MS: 395.2 ([MþH]þ ).
4-Methoxy-7-{2-[4-(4-methoxyphenyl)piperazin-1-yl]ethoxy}-2H-chromen-2-one (27). Yield 37%.
Colorless powder. M.p. 141–1438. HPLC 98.4% (MeOH/H2O 80 :20 (v/v); tR 8.19 min). IR (KBr): 2951,
1754, 1716, 1617, 1392, 1254, 831. 1H-NMR (CDCl3, 500 MHz): 7.70 (d, J¼8.8, HꢀC(5)); 6.83–6.94 (m, 4
arom. H, HꢀC(6), HꢀC(8)); 5.59 (s, HꢀC(3)); 4.23–4.25 (m, CH2O); 3.14–3.17 (m, 2 CH2N(4)); 2.79–
2.83 (m, 2 CH2N(1)); 2.92–2.95 (m, N(1)ꢀCH2CH2O); 3.97 (s, MeO); 3.99 (s, MeO). 13C-NMR (CDCl3,
125 MHz): 166.8; 163.4; 162.4; 155.1; 153.9; 145.6; 124.1; 118.3; 114.6; 114.5; 112.6; 109.1; 101.2; 87.7;
66.6; 56.9; 56.2; 53.8; 50.5. ESI-MS: 411.2 ([MþH]þ ).
4-Methoxy-7-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethoxy}-2H-chromen-2-one (28). Yield 36%.
Colorless powder. M.p. 228–2298. HPLC 97.3% (MeOH/H2O 80 :20 (v/v); tR 7.35 min). IR (KBr): 2929,
1720, 1636, 1613, 1394, 1296, 810. 1H-NMR (CDCl3, 500 MHz): 7.70 (d, J¼8.8, HꢀC(5)); 6.81 (d, J¼2.4,
HꢀC(8)); 6.85 (br. s, HꢀC(6)); 6.87–7.02 (m, 4 arom. H); 5.57 (s, HꢀC(3)); 4.19–4.21 (m, CH2O); 3.12–
3.15 (m, 2 CH2N(4)); 2.78–2.82 (m, 2 CH2N(1)); 2.90–2.93 (m, N(1)ꢀCH2CH2O); 3.97 (s, MeO); 3.87 (s,
MeO). 13C-NMR (CDCl3, 125 MHz): 166.8; 163.4; 162.4; 155.1; 152.3; 141.3; 124.0; 122.9; 121.0; 118.2;
112.6; 111.3; 109.0; 101.2; 87.7; 66.6; 56.9; 56.2; 53.9; 50.6. ESI-MS: 411.3 ([MþH]þ ).
7-{2-[4-(4-Chlorophenyl)piperazin-1-yl]ethoxy}-4-methoxy-2H-chromen-2-one (29). Yield 39%.
Colorless powder. M.p. 168–1708. HPLC 96.7% (MeOH/H2O 80 :20 (v/v); tR 7.33 min). IR (KBr):
2950, 1718, 1624, 1497, 1295, 1244, 1027, 811. 1H-NMR (CDCl3, 500 MHz): 7.73 (d, J¼8.8, HꢀC(5)); 6.83–
7.24 (m, 4 arom. H, HꢀC(6), HꢀC(8)); 5.59 (s, HꢀC(3)); 4.23–4.25 (m, CH2O); 3.22–3.25 (m,

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
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2 CH2N(4)); 2.88–2.93 (m, 3 CH2N(1)); 3.99 (s, MeO). 13C-NMR (CDCl3, 125 MHz): 166.8; 163.4;
162.3; 155.1; 149.8; 141.9; 129.0; 128.9; 124.7; 124.1; 117.3; 112.6; 109.1; 101.2; 87.7; 66.6; 56.8; 56.2; 53.5;
49.1. ESI-MS: 415.2 ([MþH]þ ).
7-{3-[4-(4-Chlorophenyl)piperazin-1-yl]propoxy}-4-methoxy-2H-chromen-2-one (30). Yield 33%.
Colorless powder. M.p. 164–1668. HPLC 97.3% (MeOH/H2O 80 :20 (v/v); tR 6.75 min). IR (KBr): 2944,
1
1712, 1611, 1497, 1139, 1070. H-NMR (CDCl3, 500 MHz): 7.48 (d, J¼8.8, HꢀC(5)); 6.83–7.21 (m, 4
arom. H, HꢀC(6), HꢀC(8)); 6.13 (s, HꢀC(3)); 4.10–4.13 (m, CH2O); 3.18–3.22 (m, 2 CH2N(4)); 2.61–
2.64 (m, 3 CH2N(1)); 3.97 (s, MeO); 2.04–2.06 (m, CH2). ESI-MS: 430.2 ([MþH]þ ).
7-{4-[4-(4-Chlorophenyl)piperazin-1-yl]butoxy}-4-methoxy-2H-chromen-2-one (31). Yield 41%.
Colorless powder. M.p. 114–1168. HPLC 97.7% (MeOH/H2O 80 :20 (v/v); tR 7.32 min). IR (KBr):
1
2935, 1738, 1610, 1497, 1241, 1199, 1153, 1070. H-NMR (CDCl3, 500 MHz): 7.50 (d, J¼8.8, HꢀC(5));
7.17–7.27 (m, HꢀC(6), HꢀC(8)); 6.83–6.88 (m, 4 arom. H); 6.14 (s, HꢀC(3)); 4.07–4.09 (m, CH2O);
3.16–3.19 (m, 2 CH2N(4)); 2.62–2.65 (m, 2 CH2N(1)), 2.46–2.49 (m, N(1)ꢀCH2(CH2)2CH2O); 3.97 (s,
MeO); 1.90–1.92 (m, CH2); 1.72–1.75 (m, CH2). 13C-NMR (CDCl3, 125 MHz): 162.1; 161.3; 155.3;
152.5; 149.9; 126.9; 125.5; 124.5; 117.2; 113.5; 112.6; 111.9; 101.4; 58.0; 53.1; 48.1; 26.9; 23.3; 18.6. ESI-
MS: 427.2 ([MþH]þ ).
Biological Studies [22]. The Ethical Committee for Conduct of Animal Studies at China
Pharmaceutical University (CPU) approved the exper. protocol, and all animals were taken care of in
accordance with the Act to Administer the Care and Use of Experimental Animals of the Jiangsu
Province of China. Male SpragueꢀDawley rats (Shanghai Sipper-BK Lab Animal Co., LTD., Shanghai,
P. R. China), eight weeks of age and weighing 180ꢁ20 g each, were used. The anococcygeus muscles
were isolated after cervical dislocation under anesthesia with Et2O. Preparations were suspended in an
organ bath (Model 832, HSE Co., Ltd., Germany) filled with KrebsꢀHenseleit soln. (20 ml) of the
following composition [mmol/l]: NaCl, 118.4; KCl, 4.7; CaCl2, 2.52; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3,
25; and glucose, 11.1. Each bath was maintained at 378 and continuously bubbled with a gas mixture
consisting of 95% O2 and 5% CO2. The responses of the preparations were isometrically recorded with
an automatic magnus system (IM-400C, Japan Tobacco, Tokyo, Japan) under a resting tension of 1.0 g.
The preparations were equilibrated for ca. 1 h and contracted twice by 3ꢂ10ꢀ5 m of phenylephrine, and
the maximum contraction of the second time was taken as 100%. Cumulative concentrations of
phenylephrine (3ꢂ10ꢀ9 –10ꢀ4 m) were then added to the organ bath as a a1-AR agonist, and
concentrationꢀresponse curves obtained to determine the relationship between agonist concentrations
and contractile responses. After a successive concentrationꢀresponse curve for the agonist had been
obtained, the antagonist prazosin (10ꢀ8 –10ꢀ7 m) or the compound to be tested (10ꢀ6 –10ꢀ7 m) was added
to the bath. Responses for the agonist in the presence of the antagonists were calculated as the
percentage of the maximal response. Schild plots were constructed, and pKi values were determined from
the intercept on the abscissa scale [23].
a1A-ARs Homology Model and Molecular Docking. As known, G-protein-coupled receptors
(GPCRs) share similar transmembrane (TM) boundaries and overall topology. Moreover, the homology
models have been used in the studies of adrenergic receptors in many cases, which have proved to be
considerably effective. Here, we chose the human b2-adrenergic receptorꢃs crystal structure (PDB ID:
2RH1, R¼2.4 ꢀ) as a template to construct a1A-AR homology model by Discovery Studio 2.0 (Accelrys
software Inc, San Diego) on the basis of the assumption that a1A-AR may be more homologous with b2-
AR also (identity 25%) from human sources than bovine rhodopsin (identity 21%), which was used for
the modeling of GPCRs as the unique template of this sort of receptors, before the crystal structure of b2-
AR was reported. The modeling process was divided into three steps: first, the C(a) backbones of the
conserved 7TM domains were built with loop modeling using the DOPE energy function which
represents an improved energy function from potentials extracted from a library of non-redundant high-
resolution crystal structures, and has been shown to provide higher quality models. Second, the model
was verified using the Profile-3D method, evaluating the likelihood that an amino acid should be present
within its current environment, and the verify scores used for further refinement were obtained for all
amino acid residues. Third, we performed the CHARMm-based structural refinement of loops and side
chains with LOOPER and ChiRotor algorithm, resp. In particular, the side chains of all residues were
refined first, followed by the residues around the active site of the receptor identified, using the binding-

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
site searching tool according to the viewpoint that the active site could be influenced by the rest residues.
Finally, the minimization was carried out by 1000 steps of steepest descent and then conjugate gradient
minimization until, the rms gradient of potential energy was less than 0.1 kcal molꢀ1 ꢀꢀ1 using
minimization protocol.
With the homology model of a1A-AR, molecular docking was carried out by GOLD 3.1.1 (CCDC,
Cambridge). We constructed the ligands using the SYBYL 7.1 molecular-modeling package (Tripos,
Missouri), followed by minimization with MMFF94s force field applied. The ligands including prazosin
and the newly designed coumarin derivatives 3–31 were docked into the active sites of a1A-AR, and then
we obtained the putative binding modes of a1A-AR and the ligands. The GOLDScore fitness was used to
rank top ten conformations for each compound, taking into account H-bond and hydrophobic
interactions. The docking process was terminated, while the three top-ranked answers obtained were
within 1.5 ꢀ rms deviation from another, according to the suggestion that these three poses represented
the most probable docking conformations. We analyzed docking results based on the scoring functions of
the software or a visual inspection of the docked poses of each compound.
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Received May 13, 2010