Design, synthesis and biological evaluation of 6-(benzyloxy)-4-methylquinolin-2(1H)-one derivatives as PDE3 inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.11.044

Selective PDE3 (phosphodiesterase 3) inhibitors improve cardiac contractility and may be used in congestive heart failure. However, their proarrhythmic potential is the most important side effect. In this work ten new synthetic compounds (3-[(4-methyl-2-oxo-1,2-dihydro-6-quinolinyl)oxy]methylbenzamide analogs: 4a-j) were designed, synthesized and tested for the inhibitory activity against human PDE3A and PDE3B. The strategy of the design was based on the structure of vesnarinone (a selective PDE3 inhibitor) and its docking analysis results. The synthetic compounds showed better PDE3 inhibitory activity in comparison with vesnarinone. Using docking analysis, a common binding model of each compound toward PDE3 was suggested. In the next step the potential cardiotonic activity of the best PDE3A inhibitors (4b, IC₅₀ = 0.43 ± 0.04 μM) was evaluated by using the spontaneously beating atria model. In the experiment, atrium of reserpine-treated rat was isolated and the contractile and chronotropic effects of the synthetic compound were assessed. That was carried out in comparison with vesnarinone. The best pharmacological profile was obtained for the compound 4b, which displayed selectivity for increasing the force of contraction (46 ± 3% change over the control) rather than the frequency rate (16 ± 4% change over the control) at 100 μM.

Full text of DOI:10.1016/j.bmc.2009.11.044

Bioorganic & Medicinal Chemistry 18 (2010) 855–862
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of
6-(benzyloxy)-4-methylquinolin-2(1H)-one derivatives as PDE3 inhibitors
c
d
Mohsen Nikpour a, Hamid Sadeghian b, , Mohammad Reza Saberi , Reza Shafiee Nick ,
*
Seyed Mohammad Seyedi e, Azar Hosseini d, Heydar Parsaee d, Alireza Taghian Dasht Bozorg a
a Department of Chemistry, School of Sciences, Islamic Azad University, Ahvaz Branch, Ahvaz, 61349-68875, Islamic Republic of Iran
b Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences, Mashhad, Islamic Republic of Iran
c School of Pharmacy, Pharmaceutical Research Center, Mashhad University of Medical Sciences, BuAli Square, Mashhad 91857-63788, Islamic Republic of Iran
d Department of Pharmacology, School of Medicine, Pharmacological Research Center of Medicinal Plants, Mashhad University of Medical Sciences, Mashhad, Islamic Republic of Iran
e Department of Chemistry, School of Sciences, Ferdowsi University of Mashhad, Mashhad, Islamic Republic of Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Selective PDE3 (phosphodiesterase 3) inhibitors improve cardiac contractility and may be used in conges-
tive heart failure. However, their proarrhythmic potential is the most important side effect. In this work
ten new synthetic compounds (3-[(4-methyl-2-oxo-1,2-dihydro-6-quinolinyl)oxy]methylbenzamide
analogs: 4a–j) were designed, synthesized and tested for the inhibitory activity against human PDE3A
and PDE3B. The strategy of the design was based on the structure of vesnarinone (a selective PDE3 inhib-
itor) and its docking analysis results. The synthetic compounds showed better PDE3 inhibitory activity in
comparison with vesnarinone. Using docking analysis, a common binding model of each compound
toward PDE3 was suggested. In the next step the potential cardiotonic activity of the best PDE3A inhib-
Received 22 October 2009
Revised 19 November 2009
Accepted 20 November 2009
Available online 26 November 2009
Keywords:
Inotropic
Vesnarinone
Chronotropic
Rat atria
itors (4b, IC50 = 0.43 0.04 lM) was evaluated by using the spontaneously beating atria model. In the
experiment, atrium of reserpine-treated rat was isolated and the contractile and chronotropic effects
of the synthetic compound were assessed. That was carried out in comparison with vesnarinone. The best
pharmacological profile was obtained for the compound 4b, which displayed selectivity for increasing the
force of contraction (46 3% change over the control) rather than the frequency rate (16 4% change over
Docking
the control) at 100 lM.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
out the family, and an N-terminal regulatory domain unique for
each isozyme. They are expressed in tissue and cell-specific distri-
bution patterns, and they show different substrate affinities and
inhibitor sensitivities.1 Most cell types express one or more PDE
isozymes, each regulating intracellular cAMP and/or cGMP concen-
trations in different cellular compartments and in different man-
ners. In cardiovascular tissues, PDE3 and PDE4 are well
established as the dominant cAMP-hydrolysis isozymes.2 PDE1,
PDE3, PDE4, and PDE5 are expressed in aortic smooth muscle
cells;3,4 PDE1, PDE2, PDE3, and PDE4 are expressed in the heart,5
whereas PDE2, PDE3, and PDE5 are found in platelets.2,6,7 PDE3 is
Congestive heart failure (CHF) is a major cause of death in pa-
tients with heart disease. Digitalis glycosides have been used for
the treatment of CHF for many years. However, application of these
agents is limited because of their narrow therapeutic window and
their propensity that cause life-threatening arrhythmias (arrhyth-
mogenic lability). The literature survey for orally active ‘non-glyco-
side’ cardiotonic drugs displays
a greater safety profile and
improved efficacy on patient survival that resulted in establishing
the selective inhibitors of cyclic nucleotide phosphodiesterase
(PDE) enzymes as a new class of cardiotonic agents.
thus
a unique cAMP-regulating isozyme expressed in all of
PDE enzymes specifically hydrolyze cAMP (cyclic adenosine
monophosphate) and cGMP (cyclic guanosine monophosphate) in
the cells. On the basis of their amino-acid sequence homology, bio-
chemical properties and inhibitor profiles, 11 PDE families have
been recognized in mammalian tissues, PDE1, PDE2 to PDE11. Each
PDE isozyme has a C-terminal catalytic domain conserved through-
mentioned tissues. These tissues contribute significantly to the
pathogenesis of arteriosclerosis obliterans and restenosis after
angioplasty. It was shown that the inhibition of PDE3 activity in
cardiovascular tissues resulted in increasing the levels of cAMP
with consequent reduction in platelet aggregation and smooth
muscle cell proliferation in vitro, and induction of a cardiotonic
effect.7,8
The PDE3 subfamily consists of two closely related subtypes:
PDE3A and PDE3B. PDE3A is mostly expressed in cardiac tissue,
* Corresponding author. Tel.: +98 05117610111; fax: +98 05117628088.
E-mail addresses: sadeghianh@mums.ac.ir, hd_sn@yahoo.com (H. Sadeghian).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.044

856
M. Nikpour et al. / Bioorg. Med. Chem. 18 (2010) 855–862
platelets, and vascular smooth muscle cells, while PDE3B is preva-
lently expressed in hepatocytes and adipose tissue.9,10
position 4 of carbostiryl moiety, the PDE3 inhibitory activity of
the compounds increases. In order to obtain new vesnarinone ana-
logs with a better PDE3 inhibitory activity, we planned to substi-
tute 4-methyl group on 6-hydroxycarbostiryl and to replace
piperazine with benzamide moiety (Fig. 3). In the later modifica-
Vesnarinon (3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-pip-
erazinyl]-2(1H)-quinolinone) (Fig. 1) was documented as an effec-
tive inotropic agent for treating congestive heart failure.11,12 This
compound increases contractility by inhibiting phosphodiesterase
III activity and prolonging the potential action duration via increas-
ing inward Ca2+ currents and inhibiting repolarizing K+ currents.13
Also vesnarinone has demonstrated a unique spectrum of intrigu-
ing biologic effects, including tumor cell differentiating and
proapoptotic properties.14 Clinical application of vesnarinone is re-
stricted because of the severe side effect of agranulocytosis.15
Despite considerable research efforts towards providing in-
sights into PDE3–ligand interactions,16–19 the progress in this area
still relies heavily on information about structure–activity relation-
ships (SAR) collected from identification of new ligands that were
discovered by trial and error. Using computerized study of PDE3–
ligand interaction, we designed and synthesized new series of
cardiotonic agents by modifying the structure of vesnarinone.
Afterward, the inhibitory activities of the synthetic compounds
were measured against PDE3A and PDE3B and the potential cardio-
tonic activity of the best PDE3A inhibitors was assessed.
tion, the hydrophobic interaction with side chain of Leu895, Ile938
,
Ile955 and Phe991 increases. Among the mentioned residues
,
Phe991 is the most critical amino acid over all species so that in
PDE3A, Phe1004?Ala mutant (Phe1004 is the homolog of Phe991 in
PDE3B27) decreased the catalytic efficiency (kcat/Km) by 286-fold
from wild type enzyme.22 On the basis of the mentioned hypothe-
sis (modifying the structure of vesnarinone), compounds 4a and
4a0 was designed, synthesized and their PDE3 (types A and B)
inhibitory activities was assessed in comparison with vesnarinone.
Among the three compounds, 4a was the most potent inhibitors.
The IC50 value of 4a was lower than vesnarinone by six and four-
fold for PDE3A and PDE3B, respectively (Table 2). The results of Ta-
ble
2 indicate that the substitution of 4-methyl on 6-
hydroxycarbostiryl moiety will lead to an increase in inhibitory
activity.
Considering the inhibitory activity of 4a, other analogs were de-
signed. Binding affinity of designed molecular structures (com-
pounds 4a–j) toward PDE3A and PDE3B was studied. 100 Docked
conformers of 4a–j were generated in ADT software. A detailed
assessment of each conformer (all of the 100 docked models) re-
vealed that more than 30% of docking results had nearly identical
orientations towards suggested inhibitory model of docked ves-
narinone. The most mimic conformer with vesnarinone at lowest
Ki was adopted from each 100 docked models as the ‘consensus’
structure and then used for further analysis (Fig. 4). The calculated
Ki for the consensus structure of the mentioned compounds was in
2. Results and discussions
In order to give further proof to the mechanism of action of de-
signed inhibitors, molecular models of the complex enzyme–inhib-
itor were generated for both ligands and vesnarinone, using PDE3B
co-crystalized with MERCK119 and the site-directed mutagenesis
data available for PDE3A.17,20–23
There is reasonable homology between the PDE3A and PDE3B
(identities: 48%, positives: 67%, extracted from NCBI-BLAST24–26).
This homology increases (ꢀ95%) within 15 Å in the active site
pocket.27
The lactamic portion of vesnarinone was supposed to be the pri-
mary binding moiety and as common anchor point. A similar case
was confirmed by Scapin et al. for cilostamide, cilostazol and amri-
none (Fig. 1).19
a good range of 10e-8 to 10e-10 lM (Table 1). Therefore other ana-
logs of 4a (compounds 4b–j) were synthesized and their inhibitory
activity against human PDE3A and PDE3B was tested.
The synthetic compounds 4a–j showed a short range of inhibi-
tion activity against the two enzymes (IC50 = 0.43–1.76
lM for
PDE3A and IC50 = 0.91–3.50 M for PDE3B; Table 2). The synthetic
l
compounds showed a better inhibitory activity against PDE3A in
comparison with PDE3B while 4b having a 4-methylpiperidine
substituent was the most potent inhibitor of PDE3A at 0.43 lM.
We generated 100 docked conformers of vesnarinone in ADT
software. A detailed assessment of each conformers (all of the
100 docked models) revealed that more than 50% of docking re-
sults had nearly identical orientations in which: (1) quinolinone
group of inhibitors oriented toward Glu988, (2) piperazine moiety
Considering the IC50 values in Table 2, there is no satisfactory rela-
tionship between IC50 values of PDE3B and Ki of proposed bonding
conformation of docking study. To earn the best bonding confor-
mation for these series of compounds, further analysis on docking
results was tried.
Docking procedure was carried on several times in order to find
the best fitness between experimental and docking results. Some-
times, we had to increase the generation number to more than 250
to get the proper conformer of ligand. This is aligned with other
synthetic compounds in the same direction up to keeping the min-
imal accepted Ki value. The result of this strategy was so satisfac-
tory that there was at least one conformer in proper orientation
and proper Ki value for each ligand (Fig. 4).
was flanked by the hydrophobic portions of the Leu895, Ile938
,
Ile955, and Phe991 side chain and (3) dimethoxybenzamide moiety
was surrounded by His737, His825 and Thr829 side chains. Due to
the orientation mentioned, the docked conformers displayed
hydrogen bond with the amide group of Gln988 side chain in PDE3B
while the 4-(3,4-dimethoxybenzoyl)-1-piperazinyl moiety, as the
second bonding part of vesnarinone, interact with pocket formed
by residues: Tyr736, His737, His825, Thr829, Leu895, Ile938, Ile955
,
Phe959, Leu987 and Phe991 (Fig. 2). The importance of the mentioned
amino acids in catalytic activity of the PDE3 has been proved by
site direct mutagenesis17,21,22 and multiple alignment technique.27
In previous work we found that by placing methyl group in
Figures 2 and 4 show that the inhibitors could interact with
the active site in quite opposite direction than that reported by
O
O
N
O
N
O
NH2
O
O
N
N
N
N
O
N
N
H
O
N
N
H
N
H
O
N
H
O
vesnarinone
cilostamide
cilostazol
amrinone
Figure 1. Chemical structure of some PDE inhibitors.

M. Nikpour et al. / Bioorg. Med. Chem. 18 (2010) 855–862
857
Figure 2. Stick (left) and solvent surface (right) view of the amino acids interacted with vesnarinone (A), 4b in traditional orientation (B) and 4b in new proposed orientation
(C). The residues which have intermolecular interactions with amide and piperazine moiety of vesnarinone were distinguished by yellow and blue color, respectively. In this
figure hydrogen bonds of Gln988 with the docked structures are shown in red line.
Scapin et al.19 while the Ki is still in a proper range. In this orien-
tation consensus structures of the docked compounds show
hydrophobic interaction with Phe991 via their phenyl portion
and hydrogen bond with highly conserved His825 and Gln988 via
quinolinone and benzamide portions, respectively (Fig. 4). It is
notable that mutant His840?Ala in PDE3A lead to no catalytic
activity even at high concentration of the mutated enzyme.20
Considering the high identity (ꢀ95%) between PDE3A and PDE3B,
the catalytic role of His840 in PDE3A can be extended to its homo-
log His825 in PDE3B. Both residues are conserved among all
PDEs.27 In PDE3B, His825 directly bind to the magnesium ion
which is involved in hydrolysis.19 The direct linear relation be-
tween ꢁlog Ki and ꢁlog IC50 of PDE3B proved the proposed direc-
tion for the inhibitors in the active site of the enzyme (Fig. 5). It
became more clear when two series of orientations were com-
pared in which, graph A showed the Ki for aforementioned pro-
posed orientation while graph B represented the Ki for our
proposed orientation against IC50 (Fig. 5). There was not any
acceptable line for graph A while there was a suitable line for
graph B with 0.96 regressions.

858
M. Nikpour et al. / Bioorg. Med. Chem. 18 (2010) 855–862
N
N
O
O
N
H
O
4a'
X
O
O
N
H
O
Compd.
4a
Compd.
X
X
N
N
N
N
N
N
N
HO
N
N
4f
4b
4g
4c
4h
N
N
N
HO
4d
O
4i
4j
O
O
4e
N
HO
Figure 3. The structure of amide moiety of compounds 4a–j and 4a0.
Table 1
Docking analysis data of consensus conformers
*
*
Compd
Ki*
D
Gb
Ed
Ki
D
Gb
Ed
4a
4b
4c
4d
4e
4f
4g
4h
4i
5.05eꢁ9
1.43eꢁ9
5.68eꢁ10
4.50eꢁ8
3.50eꢁ8
2.37eꢁ8
3.72eꢁ8
8.64eꢁ9
5.24eꢁ8
7.64eꢁ8
ꢁ11.32
ꢁ12.07
ꢁ12.61
ꢁ10.04
ꢁ10.17
ꢁ10.40
ꢁ10.14
ꢁ11.00
ꢁ9.93
ꢁ13.01
ꢁ13.92
ꢁ14.61
ꢁ11.90
ꢁ12.09
ꢁ11.30
ꢁ11.69
ꢁ12.62
ꢁ11.73
ꢁ11.01
3.55eꢁ8
1.37eꢁ8
2.01eꢁ8
1.37eꢁ8
8.94eꢁ9
6.37eꢁ9
1.36eꢁ8
1.23eꢁ8
1.04eꢁ8
5.02eꢁ8
ꢁ10.16
ꢁ10.73
ꢁ10.50
ꢁ10.73
ꢁ10.98
ꢁ11.18
ꢁ10.73
ꢁ10.79
ꢁ10.89
ꢁ9.96
ꢁ11.41
ꢁ12.06
ꢁ12.08
ꢁ12.05
ꢁ11.77
ꢁ12.41
ꢁ11.35
ꢁ12.27
ꢁ12.08
ꢁ11.07
4j
ꢁ9.71
D
Gb: estimated free energy of bonding, Ed: final docking energy, Ki: estimated
*
inhibition constant. The docking parameters distinguished with related to tradi-
tional orientation.
We also investigated other possible orientations which might
be candidate for interaction with the active site of PDE3B. There
was no relation between the Ki and IC50 values.
In light of PDE3A as mostly expressed in cardiac tissue versus
PDE3B, compound 4b which had showed the best PDE3A inhibitory
activity was tested for its effect on the force of contraction and fre-
quency rate of rat spontaneously beating atria in comparison with
vesnarinone. To avoid bias of catecholamine release, we used prep-
arations obtained from reserpine-pretreated animals.
Figure 4. Superimposition of the consensus bonding conformations of compounds
4a–j in the PDE3B active site. The above figure represents the traditional orientation
and the below one represents the new proposed orientation.
Compound 4b which was the most active PDE3A inhibitor in-
creased the atrial contractility nearly equal to vesnarinone
sus PDE3B (0.43 lM vs 1.61 lM) and more inhibitory activity in
(46 3% vs 41 3% increases at 100
l
M, respectively) (Table 3).
comparison with vesnarinone by 25 and 8-fold for PDE3A and
PDE3B, respectively. Compound 4b could have an advantage for
its clinical application that produces inotropic effect without any
obvious change in heart rate. This prevents unnecessary increase
in heart oxygen consumption that is an important determinant
to exacerbate the heart failure syndrome.
Washing out the myocardial preparations, the contractility to the
pre-drug state was completely retained. This enables us to suppose
the effects of our compound as reversible. During the experiment,
4b did not cause any arrhythmias. Vesnarinone and 4b increased
heart rate by 14 5% and 16 4% at 100 lM, respectively.
Despite the equal inotropic activity of compounds 4b and ves-
narinone, we observed a better selectivity of 4b toward PDE3A ver-
In summary we have designed and synthesized 3-[(4-methyl-2-
oxo-1,2-dihydro-6-quinolinyl)oxy]methylbenzamides 4a–j as

M. Nikpour et al. / Bioorg. Med. Chem. 18 (2010) 855–862
859
Table 2
with oxygenated Krebs–Henseleit solution. The enzymes were ob-
tained from BPS BioScience Inc. All chemicals purchased from
Merck and Fluka Chemical Co.
Enzyme inhibitory assessment data of compounds 4a–j, 4a0 and vesnarinone
Compd
IC50 (lM)
Melting points were recorded on an Electrothermal type 9100
melting point apparatus. 1H NMR (500 MHz) was obtained by
using a Bruker Avance DRX-500 Fourier transformer spectrometer.
Chemical shifts are reported in ppm (d) downfield from tetrameth-
ylsilane (TMS). Electron impact (EI) mass spectra were recorded on
a Varian Match 7A spectrometer. Elemental analysis was obtained
on a Thermo Finnigan Flash EA microanalyzer. The developed ten-
sion was recorded isometrically by means of a high-sensitivity
force transducer (type E. Zimmermann, Eipzig-Berlin) connected
to powerlab 8/30 (model 870).
PDE3A
PDE3B
4a0
4a
4b
4c
4d
4e
4f
4g
2.84 0.05
1.76 0.03
1.44 0.04
1.65 0.02
1.26 0.06
0.84 0.06
1.35 0.05
0.66 0.03
0.43 0.04
1.34 0.03
0.64 0.05
10.7 0.09
5.67 0.09
3.51 0.11
1.93 0.15
2.80 0.08
1.14 0.02
1.28 0.05
0.91 0.06
1.73 0.07
1.61 0.10
1.54 0.09
1.20 0.09
13.2 0.13
4h
4i
4j
Vesnarinone
3.2. Chemistry
potent PDE3 inhibitors and inotropic agents. As a conclusion
compound 4b with high inhibitory potency for PDE3A may be a
good candidate for treatment of CHF since; in spite of a high effi-
cacy, it may introduce less proarrhythmic side effects.
The synthesis of 6-hydroxy-4-methylquinolin-2(1H)-one (1)
and 6-hydroxyquinolin-2(1H)-one (10) was reported in last litera-
tures.29,30 Condensation of
1
and 10 with methyl-3-(bromo-
methyl)benzoate in the presence of 1,8-diazabicyclo[5,4,0]undec-
7-ene (DBU) afforded the corresponding esters 2 and 20.14 Hydroly-
sis of 2 and 20 with HCl 20% gave the corresponding carboxylic
acids 3 and 30. Various amide derivatives 4a0 and 4a–j were pre-
pared from various secondary amines and an intermediate acid
chloride which was formed from reacting of acids 3 and 30 with iso-
butyl chloroformate in the presence of DBU25 (Scheme 1). Vesnari-
none were prepared according to the method reported in
previously published work.31
3. Materials and methods
3.1. Animals, drugs, chemicals and instruments
Adult male Wistar rats (250–350 g), obtained from animal
house of Mashhad medical school, were kept in controlled environ-
mental conditions (temperature: 23 2 °C; light–dark cycle: 7 am
to 7 pm). Animals had free access to a standard laboratory diet and
water. In order to obtain atrial preparation, depleted in endoge-
nous catecholamine, the animals were treated intraperitoneally
with reserpine (5 mg/kg b.wt) 24 h before euthanasia.28 In each
experiment, the animals were anaesthetized with intraperitoneal
injection of thiopental 80 mg/kg, and after midline thoracotomy,
the heart was rapidly excised and placed in a dissection dish filled
3.3. Molecular modeling, docking and SAR study
3.3.1. Structure optimization
Structures 4a–j and vesnarinone were simulated in chem3D
professional; Cambridge software; using MM2 method (RMS gradi-
ent = 0.05 kcal/mol).32 Output files were minimized under semi-
Figure 5. Log IC50 versus ꢁlog Ki for PDE3B. Diagram A represents the traditional orientation and diagram B represents the new proposed orientation.
Table 3
Effects of the compound 4b upon force of contractility and frequency rate of whole atria from reserpine-treated rats in comparison with vesnarinone
Compd
Base
1 ꢂ 10ꢁ6
1 ꢂ 10ꢁ5
1 ꢂ 10ꢁ4
4b
100
100
3
4
101
102
2
4
123
121
2
3
146
141
3
3
Contractile force
Frequency rate
Vesnarinone
4b
100
100
2
3
106
103
1
7
108
109
5
6
116
114
4
5
Vesnarinone
The effect of each concentration (mol Lꢁ1) of a compound was defined by the difference between the contraction and frequency before and after its addition to the bathing
fluid, and was expressed as a percent variation in contraction and frequency in respect to the controls. Results are means SEM from six atria.

860
M. Nikpour et al. / Bioorg. Med. Chem. 18 (2010) 855–862
O
R
R
HO
Br
O
O
O
O
N
H
O
N
H
O
DBU
1': R = H, 1: R = Me
2 and 2'
HCl 20%
R
R
O
R
O
O
O
Isobutylchloroformate
DBU / RRNH
N
OH
R
N
H
O
N
H
O
4a-j and 4a'
3 and 3'
4a-j: R = Me, 4a': R = H and RRNH = 4-methylpiperazine
Scheme 1. General procedure for the synthesis of compounds 4a–j and 4a0.
empirical AM1 method in the second optimization (Convergence
limit = 0.01; Iteration limit = 50; RMS gradient = 0.05 kcal/mol;
Fletcher–Reeves optimizer algorithm) in HyperChem7.5.33,34
Crystal structure of human PDE3B complex with MERCK1 was
retrieved from RCSB Protein Data Bank (PDB entry: 1ISO).
amine was verified by lack of any positive inotropic effect induced
by tyramine (1.5
M).18 Experiments were performed only in prep-
arationsthatdid notresponseto tyramine. Thetestcompoundswere
added cumulatively (1–100 M) and the responses of each concen-
l
l
tration were recorded up to the maximum.
3.3.2. Molecular docking
3.5. Assessment of PDE3A and PDE3B inhibitory activity
Automated docking simulation was implemented to dock 4a–j
into the active site of PDE3B with AutoDockTools 4.0 (ADT4) ver-
sion 1.5.235 using Lamarckian genetic algorithm.46 This method
has been previously shown to produce binding models similar to
the experimentally observed models.16,36,37 The torsion angles of
the ligands were identified, hydrogens were added to the macro-
molecule, bond distances were edited and solvent parameters were
added to the enzyme 3D structure. Partial atomic charges were
then assigned to the macromolecule as well as ligands (Gasteiger
for the ligands and Kollman for the protein).
Assays were conducted following the instruction from the man-
ufacture of the assay kit. The enzymatic reactions were conducted
at room temperature for 60 min in a 50 ll mixture containing
IMAP Reaction Buffer, 100 nM FAM-cAMP, 1 ng PDE3A or PDE3B
and the test compound.
The binding reactions were performed in the present of Binding
Reagent (1:600 dilution in a reagent binding buffer containing 85%
Binding Buffer A and 15% Binding Buffer B) at room temperature
for 60 min.
The regions of interest of the enzyme were defined by consider-
ing Cartesian chart 58.4, 2.4 and 10.0 as the central of a grid size of
40, 50 and 40 points in X, Y and Z axises. The docking parameter
files were generated using Genetic Algorithm and Local Search
Parameters (GALS) while number of generations was set to 100–
250. The docked complexes were clustered with a root-mean-
square deviation tolerance of 0.2 Å. Autodock generated 100–250
docked conformers of 4a–j-corresponding to the lowest-energy
structures. After docking procedure in AD4, docking results were
submitted to Accelrys DS Visualizer v2.0.1.734738 and Swiss-Pdb-
Viewer v3.7b2 (spdbv)39 for further evaluations. The results of
Fluorescence intensity was measured at an excitation of 485 nm
and an emission of 528 nm using a BioTek Synergy™ 2 microplate
reader.
PDE3 activity assays were performed in duplicates at each con-
centration. Fluorescence intensity is converted to fluorescence
polarization using the Gen5 software. The fluorescence polariza-
tion data were analyzed using the computer software, Graphpad
Prism. The highest value of fluorescence polarization (FPt) in each
data set was defined as 100% activity. In the absence of PDE3, the
value of fluorescent polarization (FPb) in each data set was defined
as 0% activity. The percent activity in the presence of the com-
pound was calculated according to the following equation: %bind-
ing = (FP–FPb)/(FPt–FPb) ꢂ 100, where FP = the fluorescence
polarization in the presence of the compound, FPb = the fluorescent
polarization in the absence of PDE3, and FPt = the highest fluores-
cent polarization in each data set.
docking processing (DGb: estimated free energy of bonding, Ed:
final docked energy and Ki: estimated inhibition constant) are out-
lined in Table 1.
3.4. Assessment of inotropic and chronotropic activities
The IC50 values were determined from the % activity of the en-
The whole atrium was separated from ventricle (for each com-
pounds six atrium was used) and mounted vertically in a 50 ml
organ-bath containing Krebs–Henseleit solution constantly gassed
by 95% O2 and 5% CO2, 35–37 °C, pH of 7.35–7.45. The bathing solu-
tion was made up (in mM): NaCl 118, KCl 4.5, CaCl2 1.36, MgSO4 1.21,
NaH2PO4 1.22, NaHCO3 25, and glucose 11.18 Resting tension was ad-
justed to about 0.5 g in the whole atria24 and the initial equilibration
period was 40–50 min for each preparation. Since the atria were iso-
lated from reserpine-pretreated animals, depletion of catechol-
zymes at 0.01, 0.1, 1, 10 and 100 lM concentrations of synthetic
inhibitors using sigmoidal dose–response curve in Graphpad Prism.
3.6. Methyl 3-[(4-methyl-2-oxo-1,2-dihydro-6-quinolinyl)-
oxy]methylbenzoate 2
To a solution of 5 g (0.029 mol) of 6-hydroxy-4-methylquinolin-
2(1H)-one 1 and 6 g of DBU in 80 ml of isopropyl alcohol was
added methyl-3-(bromomethyl)benzoate (6.5 g, 0.033 mol) drop-

M. Nikpour et al. / Bioorg. Med. Chem. 18 (2010) 855–862
861
wise. The mixture was refluxed for 5 h, the solvent was evaporated
3.8.3. 6-[(3-{[4-(2-Hydroxyethyl)piperazino]carbonyl}-
benzyl)oxy]-4-methyl-1,2-dihydro-2-quinolinone 4c
off, and the residue was extracted with chloroform. After removal
of the solvent, the residue was recrystallized from methanol to give
2 as colorless needles (6.8 g, 74%).
White crystal, mp: 212–213 °C; 1H NMR (CDCl3/DMSO-d6): d
2.38 (s, 1H, –CH3), 2.52–2.58 (m, 6H, NCH2CH2OH & –CH2NCH2–
(piperazine)), 2.98 (s, 1H, –OH), 3.36 (m, 2H, –CH2NCOCH2– (piper-
azine)), 3.61 (m, 2H, NCH2CH2OH), 3.77 (m, 2H, –CH2NCOCH2–
(piperazine)), 5.10 (s, 2H, –CH2O–), 6.46 (s, 1H, H-3), 7.10–7.13
White crystal, mp: 190–191 °C; 1H NMR (CDCl3): d 2.51 (s, 3H, –
CH3), 3.98 (s, 3H, CH3O–), 5.20 (s, 2H, –CH2O–), 6.40 (s, 1H, H-3),
7.21 (d, J = 1.8 Hz, 1H, H-5), 7.27 (dd, J = 8.9 Hz, 1.8 Hz, 1H, H-7),
7.43 (d, J = 8.9 Hz, 1H, H-8), 7.52 (t, J = 7.7 Hz, 1H, H-5 (benzoate)),
7.71 (d, J = 7.5, 1H, H-4 (benzoate)), 8.06 (d, J = 7.7, 1H, H-6 (benzo-
ate)), 8.19 (s, 1H, H-2 (benzoate)), 12.33 (s, 1H, NHCO); MS m/z:
323 (M+), 321 (100%).
(m, 2H, H-5
& H-7), 7.23 (d, J = 8.7 Hz, 1H, H-8), 7.32 (d,
J = 7.6 Hz, 1H, H-4 (benzoate)), 7.41 (t, J = 7.6 Hz, 1H, H-5 (benzo-
ate)), 7.45–7.48 (m, 2H, H-2 (benzoate) & H-6 (benzoate)), 10.91
(s, 1H, NHCO); MS m/z: 421 (M+), 247 (100%); C24H27N3O4 re-
quires: C, 68.39; H, 6.46; N, 9.97. Found: C, 68.49; H, 6.39; N, 9.88.
3.7. 3-[(4-Methyl-2-oxo-1,2-dihydro-6-quinolinyl)oxy]meth-
ylbenzoic acid 3
3.8.4. 4-Methyl-6-(3-[(4-oxopiperidino)carbonyl]benzyloxy)-
1,2-dihydro-2-quinolinone 4d
White crystal, mp: 216–217 °C; 1H NMR (DMSO-d6): d 2.35 (m,
2H, –CH2COCH2– (piperidone)), 2.41 (s, 3H, –CH3), 2.43 (m, 2H, –
CH2COCH2– (piperidine)), 3.75 (m, 4H, –CH2NCOCH2– (piperi-
done)), 5.14 (s, 2H, –CH2O–), 6.37 (s, 1H, H-3), 7.24–7.26 (m, 3H,
1H, H-5 & H-7), 7.33 (d, J = 7.6 Hz, 1H, H-4 (benzoate)), 7.43–7.47
(m, 2H, H-2 & H-5 (benzoate)), 7.56 (m, d, J = 7.6 Hz, 1H, H-6 (ben-
zoate)), 11.59 (s, 1H, NHCO); IR cmꢁ1: MS m/z: 390 (M+), 216
(100%); C23H22N2O4 requires: C, 70.75; H, 5.68; N, 7.17. Found: C,
71.00; H, 5.71; N, 7.15.
A suspension of 3 (6 g, 0.019 mol) in 60 ml of 20% HCl was stir-
red at 85–90 °C for 2 h, and then cooled down to room tempera-
ture. The precipitated crystals were collected, and washed with
water. The crystals were recrystallized from DMF–water, oven
dried gave 3 (5.4 g, 94%).
White crystal, mp: 223–225 °C; 1H NMR (DMSO-d6): d 2.42 (s,
3H, –CH3), 4.70 (br, COOH & H2O), 5.25 (s, 2H, –CH2O–), 6.46 (s,
1H, H-3), 7.21 (d, J = 1.8 Hz, 1H, H-5), 7.27 (dd, J = 8.9 Hz, 1.8 Hz,
1H, H-7), 7.00–7.29 (m, 3H, H-5, H-7, H-8), 7.45–8.15 (m, 4H, (ben-
zoate)), 11.80 (s, 1H, NHCO); MS m/z: 309 (M+), 174 (100%).
3.8.5. 1-(3-{[(4-Methyl-2-oxo-1,2-dihydro-6-quinolinyl)oxy]-
methyl}benzoyl)-4-piperidinecarboxylic acid 4e
3.8. General procedure for the synthesis of 4a–j
White crystal, mp: 236–237 °C; 1H NMR (DMSO-d6): d 1.50 (m,
2H, –CH2CHCH2– (piperidine)), 1.70–2.00 (m, 2H, –CH2CHCH2–
(piperidine)), 2.39 (s, 3H, –CH3), 2.54 (m, 1H, –CH– (piperidine)),
2.90–3.20 (m, 2H, –CH2NCOCH2– (piperidine)), 3.51 (m, 1H, –
CH2NCOCH2– (piperidine)), 4.35 (m, 1H, –CH2NCOCH2– (piperi-
dine)), 5.22 (s, 2H, –CH2O–), 6.39 (s, 1H, H-3), 7.25–7.27 (m, 3H,
1H, H-5 & H-7), 7.33 (d, J = 7.6 Hz, 1H, H-4 (benzoate)), 7.46–7.49
(m, 2H, H-2 & H-5 (benzoate)), 7.55 (m, d, J = 7.6 Hz, 1H, H-6 (ben-
zoate)), 11.48 (s, 1H, NHCO), 12.35 (s, 1H, -COOH); IR cm-1: MS m/z:
420 (M+), 246 (100%); C24H24N2O5 requires: C, 68.56; H, 5.75; N,
6.66. Found: C, 68.37; H, 5.71; N, 6.72.
Isobuthyl chloroformate (1.5 g, 0.011 mol) was added dropwise
to a solution of 3 (0.010 mol) and 1.7 g of DBU in 50 ml chloroform
while stirring in ice-water. After removing the ice-bath, the reac-
tion mixture was stirred at room temperature for 1 h. the required
amine (0.010 mol) was then added dropwise and stirring contin-
ued for 3 h. The resulting solution was washed with 0.5 N NaOH
(2 ꢂ 50 ml), dilute HCl (2 ꢂ 50 ml) and water (2 ꢂ 50 ml). The or-
ganic layer was dried over sodium sulfate. After removing of the
solvent under reduced pressure, the residue was recrystallized
from acetone to give 4a–j.
3.8.6. 6-({3-[(4-Hydroxypiperidino)carbonyl]benzyl}oxy)-4-
methyl-1,2-dihydro-2-quinolinone 4f
3.8.1. 4-Methyl-6-({3-[(4-methylpiperazino)carbonyl]-
benzyl}oxy)-1,2-dihydro-2-quinolinone 4a
White crystal, mp: 251–252 °C; 1H NMR (DMSO-d6): d 1.20–
1.50 (m, 2H, –CH2CHCH2– (piperidine)), 1.60–1.90 (m, 2H, –
CH2CHCH2– (piperidine)), 2.39 (s, 3H, –CH3), 3.00–3.30 (m, 2H, -
CH2NCOCH2- (piperidine)), 3.44 (m, 1H, –CH2NCOCH2– (piperi-
dine)), 3.73 (m, 1H, –CH– (piperidine)), 4.01 (m, 1H, –CH2NCOCH2–
(piperidine)), 4.78 (d, J = 4.0 Hz, 1H, –OH (piperidine)), 5.22 (s, 2H,
–CH2O–), 6.39 (s, 1H, H-3), 7.25–7.27 (m, 3H, 1H, H-5 & H-7), 7.33
(d, J = 7.6 Hz, 1H, H-4 (benzoate)), 7.45–7.48 (m, 2H, H-2 & H-5
(benzoate)), 7.55 (m, d, J = 7.6 Hz, 1H, H-6 (benzoate)), 11.49 (s,
1H, NHCO); MS m/z: 392 (M+), 218 (100%); C23H24N2O4 requires:
C, 70.39; H, 6.16; N, 7.14. Found: C, 70.29; H, 6.11; N, 7.18.
White crystal, mp: 217–218 °C; 1H NMR (CDCl3): d 2.35 (m, 5H,
NCH3 & –CH2NCH2– (piperazine)), 2.51 (m, 5H, –CH3 & –CH2NCH2–
(piperazine)), 3.45 (m, 2H, –CH2NCOCH2– (piperazine)), 3.84 (m,
2H, –CH2NCOCH2– (piperazine)), 5.18 (s, 2H, –CH2O–), 6.63 (s,
1H, H-3), 7.20 (d, J = 2.6 Hz, 1H, H-5), 7.24 (dd, J = 8.9 Hz, 2.6 Hz,
1H, H-7), 7.40 (d, J = 7.6 Hz, 1H, H-4 (benzoate)), 7.43 (d,
J = 8.9 Hz, 1H, H-8), 7.48 (t, J = 7.7 Hz, 1H, H-5 (benzoate)), 7.54–
756 (m, 2H, H-2 (benzoate) & H-6 (benzoate)), 12.45 (s, 1H, NHCO);
MS m/z: 391 (M+), 217 (100%); C23H25N3O3 requires: C, 70.57; H,
6.44; N, 10.73. Found: C, 70.39; H, 6.41; N, 10.87.
3.8.2. 4-Methyl-6-({3-[(4-ethylpiperazino)carbonyl]-
benzyl}oxy)-1,2-dihydro-2-quinolinone 4b
3.8.7. 4-Methyl-6-{[3-(piperidinocarbonyl)benzyl]oxy}-1,2-
dihydro-2-quinolinone 4g
White crystal, mp: 193–194 °C; 1H NMR (CDCl3): d 1.12 (t,
J = 7.2 Hz, 3H, CH3CH2N–), 2.38 (m, 2H, –CH2NCH2– (piperazine)),
2.47 (q, J = 7.2 Hz, 2H, CH3CH2N–), 2.51 (m, 3H, –CH3), 2.56 (m,
2H, –CH2NCH2– (piperazine)), 3.45 (m, 2H, –CH2NCOCH2– (pipera-
zine)), 3.97 (m, 2H, –CH2NCOCH2– (piperazine)), 5.19 (s, 2H, –
CH2O–), 6.63 (s, 1H, H-3), 7.20 (d, J = 2.6 Hz, 1H, H-5), 7.24 (dd,
J = 8.9 Hz, 2.6 Hz, 1H, H-7), 7.39–74.2 (m, 2H, H-8 & H-4 (benzo-
ate)), 7.48 (t, J = 7.7 Hz, 1H, H-5 (benzoate)), 7.54–7.56 (m, 2H, H-
2 (benzoate) & H-6 (benzoate)), 12.00 (br, 1H, NHCO); MS m/z:
405 (M+), 231 (100%); C24H27N3O3 requires: C, 71.09; H, 6.71; N,
10.36. Found: C, 71.21; H, 6.81; N, 10.46.
White crystal, mp: 200–201 °C; 1H NMR (CDCl3/DMSO-d6): d
1.63 (m, 1H, –CH2CH2CH2– (piperidine)), 1.71 (m, 4H,
–
CH2CH2CH2– (piperidine)), 1.88 (m, 1H, –CH2CH2CH2– (piperi-
dine)), 2.50 (s, 3H, –CH3), 3.28 (t, J = 5.1 Hz, 2H, –CH2NCOCH2–
(piperidine)), 3.36 ((t, J = 5.1 Hz, 2H, –CH2NCOCH2– (piperidine)),
5.18 (s, 2H, –CH2O–), 6.62 (s, 1H, H-3), 7.19 (d, J = 2.5 Hz, 1H, H-
5), 7.24 (dd, J = 8.7 Hz, 2.5 Hz, 1H, H-7), 7.37–741 (m, 2H, H-8 &
H-4 (benzoate)), 7.46 (t, J = 7.5 Hz, 1H, H-5 (benzoate)), 7.52–7.54
(m, 2H, H-2 (benzoate) & H-6 (benzoate)), 11.33 (s, 1H, NHCO);
MS m/z: 376 (M+), 202 (100%); C23H24N2O3 requires: C, 73.38; H,
6.43; N, 7.44. Found: C, 72.89; H, 6.41; N, 7.51.

862
M. Nikpour et al. / Bioorg. Med. Chem. 18 (2010) 855–862
3.8.8. 4-Methyl-6-({3-[(4-methylpiperidino)carbonyl]-
benzyl}oxy)-1,2-dihydro-2-quinolinone 4h
Acknowledgments
White crystal, mp: 230–231 °C; 1H NMR (CDCl3): d 0.84 (d,
J = 6.4, 3H, –CH3 (piperidine)), 0.91 (m, 1H, –CH2CHCH2– (piperi-
dine)), 1.11 (m, 1H, –CH2CHCH2– (piperidine)), 1.44 (m, 1H, –
CH2CHCH2– (piperidine)), 1.55 (m, 1H, –CH2CHCH2– (piperidine)),
1.67 (m, 1H, –CH2CHCH2– (piperidine)), 2.34 (s, 3H, –CH3), 2.67
(m, 1H, –CH2NCOCH2– (piperidine)), 2.87 (m, 1H, –CH2NCOCH2–
(piperidine)), 3.56 (m, 1H, –CH2NCOCH2– (piperidine)), 4.52 ((m,
1H, –CH2NCOCH2– (piperidine)), 5.05 (s, 2H, –CH2O–), 6.41 (s,
1H, H-3), 7.05 (d, J = 2.5 Hz, 1H, H-5), 7.07 (dd, J = 8.7 Hz, 2.5 Hz,
1H, H-7), 7.19–7.25 (m, 2H, H-8 & H-4 (benzoate)), 7.31 (t,
J = 7.6 Hz, 1H, H-5 (benzoate)), 7.37–741 (m, 2H, H-2 (benzoate)
& H-6 (benzoate)), 11.52 (s, 1H, NHCO); MS m/z: 390 (M+), 216
(100%); C24H26N2O3 requires: C, 73.82; H, 6.71; N, 7.17. Found: C,
73.57; H, 6.64; N, 7.09.
We express our sincere gratitude to Dr. Hossein Orafaie and
Mohammad Naser Shafiee for reviewing the manuscript. We are
also grateful to BPS Bioscience Inc. for enzyme assays.
References and notes
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3.8.9. 4-Methyl-6-{[3-(morpholinocarbonyl)benzyl]oxy}-1,2-
dihydro-2-quinolinone 4i
White crystal, mp: 211–212 °C; 1H NMR (CDCl3): d 2.51 (s, 3H, –
CH3), 3.48–3.81 (m, 8H, (morpholin)), 5.18 (s, 2H, –CH2O–), 6.41 (s,
1H, H-3), 7.21 (d, J = 2.6 Hz, 1H, H-5), 7.25 (dd, J = 8.7 Hz, 2.6 Hz,
1H, H-7), 7.40–7.43 (m, 2H, H-8 & H-4 (benzoate)), 7.31 (t,
J = 7.7 Hz, 1H, H-5 (benzoate)), 7.56–758 (m, 2H, H-2 (benzoate)
& H-6 (benzoate)), 12.32 (s, 1H, NHCO); MS m/z: 378 (M+), 204
(100%); C22H22N2O4 requires: C, 69.83; H, 5.86; N, 7.40. Found: C,
69.77; H, 5.84; N, 7.51.
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3.8.10. 4-Methyl-6-{[3-(tetrahydro-1H-1-pyrrolylcarbonyl)-
benzyl]oxy}-1,2-dihydro-2-quinolinone 4j
White crystal, mp: 193–194 °C; 1H NMR (CDCl3): d 1.90 (m, 2H,
–CH2CH2– (pyrrolydine)), 2.00 (m, 2H, –CH2CH2– (pyrrolydine)),
2.51 (s, 3H, CH3–), 3.43 (t, J = 6.6 Hz, 2H, –CH2NCOCH2– (pyrroly-
dine)), 3.69 t, J = 6.6 Hz, 2H, –CH2NCOCH2– (pyrrolydine)), 5.18
(s, 2H, –CH2O–), 6.63 (s, 1H, H-3), 7.20 (d, J = 2.6 Hz, 1H, H-5),
7.25 (dd, J = 8.9 Hz, 2.6 Hz, 1H, H-7), 7.41 (d, J = 8.9 Hz, 1H, H-8),
7.48 (d, J = 7.5 Hz, 1H, H-4 (benzoate)), 7.52–7.55 m, 2H, H-5 (ben-
zoate) & H-6 (benzoate)), 767 (s, 1H, H-2 (benzoate)), 12.19 (s, 1H,
NHCO); MS m/z: 362 (M+), 188 (100%); C22H22N2O3 requires: C,
72.91; H, 6.12; N, 7.73. Found: C, 72.77; H, 6.10; N, 7.81.
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from 10, is same as which was reported for 4a–j.
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3.9.1. 4-Methyl-6-({3-[(4-methylpiperazino)carbonyl]-
benzyl}oxy)-1,2-dihydro-2-quinolinone 4a0
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White crystal, mp: 227–228 °C; 1H NMR (CDCl3): d 2.38 (m, 5H,
NCH3 & –CH2NCH2– (piperazine)), 2.55 (m, 2H, –CH2NCH2– (piper-
azine)), 3.49 (m, 2H, –CH2NCOCH2– (piperazine)), 3.87 (m, 2H, –
CH2NCOCH2– (piperazine)), 5.15 (s, 2H, –CH2O–), 6.62 (d, J = 8.8,
1H, H-3), 7.20 (d, J = 2.6 Hz, 1H, H-5), 7.24 (dd, J = 8.9 Hz, 2.6 Hz,
1H, H-7), 7.41 (d, J = 7.7 Hz, 1H, H-4 (benzoate)), 7.41 (d,
J = 8.8 Hz, 1H, H-8), 7.44 (t, J = 7.6 Hz, 1H, H-5 (benzoate)), 7.48–
754 (m, 2H, H-2 (benzoate) & H-6 (benzoate)), 7.66 (d, J = 8.8 Hz,
1H, H-4), 12.45 (s, 1H, NHCO); MS m/z: 377 (M+), 217 (100%);
C23H25N3O3 requires: C, 70.01; H, 6.14; N, 11.13. Found: C, 70.22;
H, 6.21; N, 10.99.
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