Synthesis of a new series of 4-Aryl-1,4-dihydropyridines with calcium channel blocking and vasodilatory activity

Article abstract of DOI:10.1007/s00044-011-9600-x

Several new amide derivatives of 4-aryl-1, 4-dihydropyridine carboxylic congeners have been synthesized in this study to obtain therapeutically useful compounds. The changes in pharmacological properties of dihydropyridines by the presence of polar groups at different positions of 4-phenyl substituent and also by introduction of unsymmetrical ester groups in the synthesized symmetrical analogs have been studied. In vitro calcium channel blocking activity has been evaluated in cultures of neonatal rat cortical neurons by measuring the inhibitory response at L-type calcium channels activated by veratridine. The newly synthesized dihydropyridines displayed moderate calcium channel blockade with IC₅₀ values ranging from 2 to 10 μM in comparison to nifedipine (IC₅₀ = 57.7 nM). The vasodilatory activity was evaluated on isolated rat thoracic aortic rings precontracted by phenylephrine/KCl (30 mM). The symmetrically substituted dihydropyridine 8a exhibited maximum activity with IC₅₀ = 0.64 μMbut was found to be approximately 24 times less active in comparison to standard drug nifedipine with IC₅₀ = 27.5 nM. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9600-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:908–921
DOI 10.1007/s00044-011-9600-x
ORIGINAL RESEARCH
Synthesis of a New Series of 4-Aryl-1,4-Dihydropyridines
with Calcium Channel Blocking and Vasodilatory Activity
•
Ranju Bansal Priyanka Jain Carmen Calle
•
•
•
Rosalia Carron Karen Pemberton Alan L. Harvey
•
Received: 2 September 2010 / Accepted: 7 February 2011 / Published online: 20 February 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Several new amide derivatives of 4-aryl-1,
4-dihydropyridine carboxylic congeners have been synthe-
sized in this study to obtain therapeutically useful com-
pounds. The changes in pharmacological properties of
dihydropyridines by the presence of polar groups at different
positions of 4-phenyl substituent and also by introduction of
unsymmetrical ester groups in the synthesized symmetrical
analogs have been studied. In vitro calcium channel blocking
activity has been evaluated in cultures of neonatal rat cortical
neurons by measuring the inhibitory response at L-type
calcium channels activated by veratridine. The newly syn-
thesized dihydropyridines displayed moderate calcium
channel blockade with IC50 values ranging from 2 to 10 lM
in comparison to nifedipine (IC50 = 57.7 nM). The vasod-
ilatory activity was evaluated on isolated rat thoracic aortic
rings precontracted by phenylephrine/KCl (30 mM). The
symmetrically substituted dihydropyridine 8a exhibited
maximum activity with IC50 = 0.64 lM but was found to be
approximately 24 times less active in comparison to standard
drug nifedipine with IC50 = 27.5 nM.
Keywords Dihydropyridines Á
Calcium channel blockers Á Vasodilators Á
Unsymmetrical esters
Introduction
4-Aryl-1,4-dihydropyridines attract the interest of medicinal
chemists due to their high potency and selectivity of action as
calcium channel blockers (Mannhold, 1994; Triggle, 2003).
Many modifications of the prototypical structure of nifedipine
(Fig. 1) have been carried out leading to the formation of
several novel dihydropyridines (Steffen, 1999; Meredith,
1999). Structural variations of this lead molecule have been
focused mainly on ester functions and variations in phenyl
substitutions. The envisaged goal of these new developments
was an optimization of vascular selectivity. A new series of
4-aryl-1,4-dihydropyridines possessing vanilloid-based moie-
ties has attracted much attention lately (Yeh et al., 2000; Liang
et al., 2000; Liang et al., 2002). These vanilloid-based hybrid
molecules, e.g., vanidipinedilol and labedipinedilol (Fig. 1)
have produced potent vasodilatory effects with reduced sym-
pathetic activation-associated reflex tachycardia in the heart.
Aryl moieties bearing a basic side chain have been
considered as an essential active structural component in a
number of known calcium channel blockers such as
verapamil, diltiazem, prenylamine etc. (Janis and Triggle,
1983). Even in dihydropyridines, the presence of basic
moieties has led to the development of long acting, rela-
tively side effect free and potent calcium channel blockers
(Klotz, 2002). A series of 4-aryl-1,4-dihydropyridines
substituted with basic moieties possessing potent calcium
channel blocking and vasodilatory activity have also been
reported by us (Jain et al., 2006). This is attributed to
improve bioavailability of the molecules. Considering the
R. Bansal (&) Á P. Jain
University Institute of Pharmaceutical Sciences,
Panjab University, Chandigarh 160014, India
e-mail: ranju29in@yahoo.co.in
C. Calle Á R. Carron
´
´
Departamento de Fisiologıa y Farmacologıa,
Facultad de Farmacia, Campus ‘‘Miguel de Unamuno’’,
37007 Salamanca, Spain
K. Pemberton
Thistle Technologies, 11 Sycamore Way, Branford 06405, USA
A. L. Harvey
Strathclyde Institute for Drug Research and Department
of Physiology and Pharmacology, University of Strathclyde,
27 Taylor Street, Glasgow G4 0NR, UK
123

Med Chem Res (2012) 21:908–921
909
NO2
O
NO2
O
O
O
O
O
O
O
N
H
N
H
I
II
O
O
N
O
N
H
O
H
OH
O
O
OH
O
O
O
O
O
O
O
O
N
H
N
H
III
IV
Fig. 1 Structures of Nifedipine (I), Nitrendipine (II), Vanidipinedilol (III), and Labedipinedilol (IV)
substitution of hydroxy group present at different positions
of the 4-phenyl ring an interesting route to incorporate such
moieties, several new amide derivatives of 4-aryl-1,4-
dihydropyridine carboxylic congeners have been synthe-
sized in this study to obtain calcium channel blockers of
better therapeutic profile. The main objective of this study
is to study the effect of these weakly basic substituents on
pharmacological activity of dihydropyridines.
RX 1, FT-IR spectrophotometer model (Perkin-Elmer Ltd.,
Beaconsfield, Buckinghamshire, UK) using potassium
bromide pellets. Proton-NMR spectra were recorded on
Bruker AC-300F, 300 MHz (Bruker AG, Fallanden, Swit-
zerland) using deuterated-chloroform or deuterated dime-
thylsulfoxide-containing (Sigma-Aldrich, St. Louis, USA)
tetramethylsilane (Me4Si) as internal standard (chemical
shifts in d, ppm). The purity of compounds was established
by thin layer chromatography (TLC) (E. Merck, India) and
elemental analyses. Elemental analyses were carried out on
a Perkin-Elmer-2400 model CHN analyzer (Perkin-Elmer,
Norwalk, CT, USA). Heterocyclic amines morpholine from
E. Merck, India; pyrrolidine, piperidine, and 1-methylpi-
perazine from Himedia, India, and 3-pipecoline from
Sigma-Aldrich, Chemical Co. inc., Milwankee, wis. were
purchased. Tert-butylamine, methylchloroacetate, isopro-
panol, aldehydes, and methyl 3-aminocrotonate were from
E. Merck, India.
Further, the outstanding importance of the ester func-
tions with respect to the pharmacological properties of
dihydropyridines initiated the synthesis of innumerable
compounds with varied ester substitutions in 3- and
5-position of the dihydropyridine skeleton (Ramesh et al.,
1998). Dihydropyridines such as nitrendipine (Fig. 1) with
an unsymmetrical ester substitution are, in general, more
potent than symmetrically substituted derivatives due to a
gain in bulk at the port side ester, which presumably
interacts with a hydrophobic receptor domain (Mannhold,
1994). Therefore, it was also endeavored to introduce
asymmetrical ester groups in the synthesized symmetrical
analogs.
All solvents were distilled before use according to
standard procedures. Symmetric dihydropyridines 4–6
were prepared according to the reported procedure (Jain
et al., 2006).
Amide derivatives 7a–d, 8a–d and 9a–d and 9g were
synthesized by fusing the respective dihydropyridine car-
boxylate 7–9 with various heterocyclic amines like mor-
pholine, pyrrolidine, piperidine, 1-methylpiperazine, and
3-pipecoline at 80–90°C for 4 h. The 4-[(carbmethoxym-
ethyl)oxy]phenyl-dihydropyridines 7–9 in turn were pre-
pared by refluxing symmetrical dihydropyridines 4–6 (Jain
et al., 2006) with methylchloroacetate in ethyl methyl
Materials and Methods
Chemistry
Melting points were determined on a Veego melting point
apparatus (MP I, Veego, Mumbai, India) and are uncor-
rected. IR spectra were recorded on Perkin-Elmer spectrum
123

910
Med Chem Res (2012) 21:908–921
ketone using anhydrous potassium carbonate as shown in
Scheme 1. In case of amide derivatives, the prominent
carbonyl absorption band of tertiary amides appeared at a
lower wavenumber *1640 cm-1 than ‘‘normal’’ carbonyl
absorption due to the resonance effect. Methylenes of the
heterocyclic ring attached to nitrogen appeared downfield
R1
H2N
2 X
CH3
O
R2
+
CH3
O
Methyl 3-aminocrotonate
Trifluoroacetic acid
0 0C
CHO
( 1-3 )
R1
4
R2
5
6
3
2
O
O
1
(1,4)
(2,5)
R1 = OH, R2 = H
R1 = H, R2 = OH
R1 = OH, R2 = OCH3
H3C
CH3
4
1
O
O
3
5
(3,6)
2
6
H3C
N
CH3
H
( 4-6 )
(i) Methylchloroacetate
(ii) Amines
OCH2COR
OCH2COR
OMe
OCH2COR
O
O
O
O
O
O
H3C
CH3
H3C
CH3
H3C
CH3
O
O
O
O
O
O
H3C
N
H
CH3
H3C
N
CH3
H3C
N
H
CH3
H
7
(a-d)
8
(a-f )
9
(a-g)
(7-9) R=OCH3
Compd
a
b
c
d
e
f
g
No.
N
-NHC(CH3)3
O
-N
-N
-N
N
-N
-N
R
-N
Scheme 1 Synthetic route to symmetric dihydropyridines 7–9
123

Med Chem Res (2012) 21:908–921
911
at *d 3.50 ppm for these derivatives due to the presence
of adjacent carbonyl group.
m.p. 215–216°C. Spectroscopic analysis: IR (KBr) mmax
/
cm-1: 3380, 3272, 2941, 1689, 1659, 1497, 1450, 1221,
1096, 1025, 952, 876, 816, 706; 1H NMR (CDCl3,
300 MHz, d, ppm): 1.22 (t, 3H, –COOCH2CH3,
J = 5.70 Hz), 2.29 (s, 6H, 2 9 CH3), 3.64 (s, 3H, –CO-
OCH3), 4.09 (m, 2H, –COOCH2CH3), 4.98 (s, 1H, 4-CH,
dihydropyridine), 5.46 (brs, 1H, –OH), 5.80 (t, 1H, NH,
J = 12.52 Hz), 6.60 (d, 1H, Jo = 8.20 Hz, 6-CH, aro-
matic), 6.76 (s, 1H, 2-CH, aromatic), 6.84 (d, 1H,
Jo = 7.43 Hz, 4-CH, aromatic), 7.05 (t, 1H, Jo = 7.82 Hz,
5-CH, aromatic); Anal. calcd. for C18H21NO5: C, 65.24; H,
6.39; N, 4.23; found: C, 65.05; H, 5.94; N, 4.12.
It was further endeavored to introduce a heteroaromatic
ring, e.g., Imidazole and also a primary amine such as
tert-butylamine, to observe the effect of such functional-
ities on the pharmacological profile since such type of
moieties form an integral part of many of the known
cardiovascular agents (Huber et al., 1991). The synthesis
of imidazolyl substituted dihydropyridines (8e, 9e)
required a longer time of 75 h and higher reaction tem-
perature of 90–110°C. tert-Butylamine substituted di-
hydopyridines 8f, 9f were prepared by thermal fusion of 8
and 9 with excess of tert-butylamine in dimethylform-
amide (DMF) for 70 h.
Ethyl methyl 4-(4-hydroxy-3-methoxyphenyl)-2,6-dime-
thyl-1,4-dihydropyridine-3,5-dicarboxylate (12). Yield:
65.21%; m.p. 100–101°C. Spectroscopic analysis: IR (KBr)
The synthesis of asymmetric dihydropyridines 10–12
was carried out by modified hantzsch condensation (Iwa-
nami et al., 1979) by continuously refluxing the equimolar
quantities of appropriate aldehyde, methyl 3-aminocroto-
nate and ethylacetoacetate in 2-propanol for 8–9 h to
generate the parent dihydropyridines (Scheme 2). Target
amide derivatives 13a–e, 14a–d, and 15a–e of asymmetric
4-phenyldihydropyridines were prepared by treating com-
pounds 10–12 with methyl chloroacetate to afford car-
boxylic congeners 13–15 and then further fusion with
various cyclic amines in a similar way as is followed for
symmetrical analogs.
m
max/cm-1: 3377, 2982, 1730, 1695, 1650, 1624, 1490,
1432, 1380, 1302, 1209, 1091, 1051, 1024, 861, 768, 607;
1H NMR (CDCl3, 300 MHz, d, ppm): 1.25 (m, 3H, –CO-
OCH2CH3), 2.34 (s, 6H, 2 9 CH3), 3.67 (s, 3H, –CO-
OCH3), 3.86 (s, 3H, –OCH3), 4.12 (m, 2H, –COOCH2CH3),
4.94 (s, 1H, 4-CH, dihydropyridine), 5.48 (s, 1H, –OH),
5.61 (t, 1H, –NH, J = 8.55 Hz), 6.74 (m, 2H, 5-CH and
6-CH, aromatic), 6.86 (s, 1H, 2-CH, aromatic); Anal. calcd.
for C19H23NO6: C, 63.15; H, 6.41; N, 3.88; found: C, 62.98;
H, 6.40; N, 3.53.
General Procedure for Alkylation of Parent
Dihydropyridines 4–6 and 10–12 (Formation of 7–9
and 13–15):
General Procedure for the Synthesis of Parent
Asymmetric Dihydropyridines 10–12
A solution of appropriate aldehydes 1–3 (8.19 mmol),
methyl 3-aminocrotonate (8.19 mmol) and ethyl acetoac-
etate (8.19 mmol) in isopropanol (10 mL) were refluxed
for 9 h with continuous stirring. The completion of the
reaction was monitored with TLC. The solvent was
removed under vacuum and the resultant solid was
repeatedly washed with water, filtered, dried, and crystal-
lized from ethyl acetate to obtain 10–12.
Methylchloroacetate (6.30 mmol) was added to a stirred
and refluxing suspension of respective dihydropyridine 4–6
and 10–12 (3.15 mmol) and anhydrous potassium carbonate
(2 g) in ethyl methyl ketone (35 ml). The reaction mixture
was further refluxed for 6 h. The resulting slurry was fil-
tered off, and the solvent was removed under reduced
pressure to obtain a solid product, which was crystallized
from a mixture of diethyl ether and ethyl acetate to afford
the corresponding dihydropyridine 7–9 and 13–15.
Ethyl methyl 4-(4-hydroxyphenyl)-2,6-dimethyl-1,4-
dihydropyridine-3,5-dicarboxylate (10). Yield: 81.54%;
Dimethyl 4-[4-(2-methoxy-2-oxoethoxy)phenyl]-2,6-di-
methyl-1,4-dihydropyridine-3,5-di-carboxylate (7). Yield:
40.98%; m.p. 139–141°C. Spectroscopic analysis: IR
(KBr) mmax/cm-1: 3339, 2946, 1741, 1699, 1643, 1499,
m.p. 205–206°C. Spectroscopic analysis: IR (KBr) mmax
/
cm-1: 3347, 3101, 2987, 2946, 1699, 1664, 1592, 1510,
1489, 1438, 1371, 1335, 1227, 1128, 1020, 843, 788, 760;
1H NMR (CDCl3, 300 MHz, d, ppm): 1.21 (t, 3H, –CO-
OCH2CH3, J = 6.36 Hz), 2.29 (s, 6H, 2 9 CH3), 3.61 (s,
3H, –COOCH3), 4.04 (m, 2H, –COOCH2CH3), 4.84 (s, 1H,
4-CH, dihydropyridine), 6.63 (d, 2H, Jo = 8.00 Hz, 3-CH
and 5-CH, aromatic), 7.04 (m, 2H, 2-CH and 6-CH, aro-
matic), 7.82 (t, 1H, –NH, J = 17.62 Hz); Anal. calcd. for
C18H21NO5: C, 65.24; H, 6.39; N, 4.23; found: C, 65.12; H,
6.11; N, 3.89.
1
1438, 1384, 1306, 1219, 1095, 849, 674, 603; H NMR
(CDCl3, 300 MHz, d, ppm): 2.28 (s, 6H, 2 9 CH3), 3.63
(s, 6H, 29 COOCH3), 3.79 (s, 3H, –OCH2COOCH3), 4.57
(s, 2H, –OCH2COOCH3), 4.94 (s, 1H, 4-CH, dihydropyr-
idine), 6.33 (s, 1H, –NH), 6.73 (dd, 2H, Jo = 6.74 Hz,
Jm = 1.97 Hz, 3-CH and 5-CH, aromatic), 7.18 (dd, 2H,
Jo = 6.70 Hz, Jm = 2.01 Hz, 2-CH and 6-CH, aromatic);
Anal. calcd. for C20H23NO7: C, 61.69; H, 5.95; N, 3.60;
found: C, 61.59; H, 5.64; N, 3.55.
Ethyl methyl 4-(3-hydroxyphenyl)-2,6-dimethyl-1,4-
dihydropyridine-3,5-dicarboxylate (11). Yield: 81.18%;
123

912
Med Chem Res (2012) 21:908–921
O
O
H2N
CH3
H3C
O
CH3
+
(1-3)
+
O
O
CH3
Ethylacetoacetae
Methyl 3-aminocrotonate
Reflux
2-Propanol
R1
(10)
(11)
(12)
R1 = OH, R2 = H
4
R2
R1 = H,
R2 = OH
5
6
3
R1 = OH, R2 = OCH3
2
O
O
1
5
H3C
3
2
CH2CH3
4
O
O
6
H3C
N1
H
CH3
(10-12)
(i)
Methylchloroacetate
Amines
(ii)
OCH2COR
OMe
OCH2COR
OCH2COR
O
O
O
O
O
O
H3C
CH2CH3
H3C
H3C
CH2CH3
CH2CH3
O
O
O
O
O
O
H3C
N
H
CH3
H3C
N
H
H3C
N
H
CH3
CH3
15
14
13
(a-e)
(a-e)
(a-d)
(13-15) R=OCH3
Compound
No.
a
b
c
D
e
O
-N
-N
-N
-N
N
R
-N
Scheme 2 Synthetic route to asymmetric dihydropyridines 13–15
123

Med Chem Res (2012) 21:908–921
913
Dimethyl
dimethyl-1,4-dihydropyridine-3,5-di-carboxylate
4-[3-(2-methoxy-2-oxoethoxy)phenyl]-2,6-
(8).
4-CH, aromatic), 7.14 (t, 1H, Jo = 7.69 Hz, 5-CH, aromatic);
Anal. calcd. for C21H25NO7:C, 62.52;H, 6.25; N, 3.47;found:
C, 61.88; H, 5.95; N, 3.44.
Yield: 81.96%; m.p. 170–172°C. Spectroscopic analysis:
IR (KBr) mmax/cm-1: 3370, 2950, 1770, 1700, 1650, 1480,
1430, 1300, 1210, 1100, 1010, 790, 710, 570; H NMR
Ethyl methyl 4-[3-methoxy-4-(2-methoxy-2-oxoethoxy)
phenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
(15). Yield: 92.43%; m.p. 105–107°C. Spectroscopic anal-
ysis: IR (KBr) mmax/cm-1: 3373, 2981, 1730, 1694, 1497,
1376, 1296, 1208, 1095, 1024, 860, 768, 670, 610; 1H NMR
(CDCl3, 300 MHz, d, ppm): 1.23 (t, 3H, –COOCH2CH3,
J = 7.27 Hz), 2.31 (s, 6H, 2 9 CH3), 3.65 (s, 3H, –CO-
OCH3), 3.77 (s, 3H, –OCH2COOCH3), 3.82 (s, 3H,
–OCH3), 4.09 (m, 2H, –COOCH2CH3), 4.63 (s, 2H,
–OCH2COOCH3), 4.95 (s, 1H, 4-CH, dihydropyridine),
5.88 (t, 1H, –NH, J = 12.60 Hz), 6.66 (d, 1H, Jo =
8.24 Hz, 6-CH, aromatic), 6.73 (m, 1H, 5-CH, aromatic),
6.89 (d, 1H, Jm = 1.84 Hz, 2-CH, aromatic); Anal. calcd.
for C22H27NO8: C, 60.96; H, 6.28; N, 3.23; found: C, 60.90;
H, 6.18; N, 3.15.
1
(CDCl3, 300 MHz, d, ppm): 2.32 (s, 6H, 2 9 CH3), 3.64 (s,
6H, 29 COOCH3), 3.79 (s, 3H, –OCH2COOCH3), 4.58 (s,
2H, –OCH2COOCH3), 4.99 (s, 1H, 4-CH, dihydropyri-
dine), 5.81 (s, 1H, –NH), 6.65 (dd, 1H, Jo = 8.13 Hz,
Jm = 2.50 Hz, 6-CH, aromatic), 6.84 (m, 1H, 2-CH, aro-
matic), 6.92 (d, 1H, Jo = 7.93 Hz, 4-CH, aromatic), 7.13
(t, 1H, Jo = 7.82 Hz, 5-CH, aromatic); Anal. calcd. for
C20H23NO7: C, 61.69; H, 5.95; N, 3.60; found: C, 61.42; H,
5.74; N, 3.49.
Dimethyl 4-[3-methoxy-4-(2-methoxy-2-oxoethoxy)phe-
nyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (9).
Yield: 66.66%; m.p. 178–180°C. Spectroscopic analysis: IR
(KBr) mmax/cm-1: 3370, 2920, 1770, 1690, 1660, 1510, 1470,
1210, 1120, 1020, 810, 750, 690;1H NMR (CDCl3, 300 MHz,
d, ppm): 2.32 (s, 6H, 2 9 CH3), 3.65 (s, 6H, 29 COOCH3),
3.77 (s, 3H, –OCH2COOCH3), 3.83 (s, 3H, –OCH3), 4.63 (s,
2H, –OCH2COOCH3), 4.96 (s, 1H, 4-CH, dihydropyridine),
5.78 (s, 1H, –NH, D2O exchangeable), 6.66 (d, 1H, Jo =
8.45 Hz, 5-CH, aromatic), 6.72 (dd, 1H, Jo = 8.24 Hz,
Jm = 1.97 Hz, 6-CH, aromatic), 6.88 (d, 1H, Jm = 1.62 Hz,
2-CH, aromatic); Anal. calcd. for C21H25NO8: C, 60.13; H,
6.01; N, 3.34; found: C, 59.85; H, 5.96; N, 3.16.
General Procedure for the Synthesis of Amide
Derivatives 7a–d, 8a–f, and 9a–g/13a–e, 14a–d,
and 15a–e
A mixture of 7–9 (2.57 mmol) and requisite amine a–d and
g (5.14 mmol) was thermally fused at 80–90°C for 7 h.
After the completion of the reaction, which was monitored
by TLC, ice-cold water was added to remove excess of
amines. The solid product thus obtained was filtered, dried,
and crystallized from a mixture of diethyl ether and ethyl
acetate to obtain 7a–d, 8a–d, and 9a–d and 9g. The syn-
thesis of imidazolyl substituted dihydropyridines 8e, 9e
required a longer time of 75 h and higher reaction tem-
perature 90–110°C. Tert-butylamine substituted dihydo-
pyridines 8f, 9f were prepared by thermal fusion of 8 and 9
with excess of tert-butylamine in DMF for 70 h.
Ethyl methyl 4-[4-(2-methoxy-2-oxoethoxy)phenyl]-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (13). Yield:
74.38%; m.p. 165–166°C. Spectroscopic analysis: IR (KBr)
m
max/cm-1: 3340, 3112, 2948, 1744, 1695, 1640, 1504, 1283,
1214, 1094, 850, 742; 1H NMR (CDCl3, 300 MHz, d, ppm):
1.21 (t, 3H, –COOCH2CH3, J = 7.07 Hz), 2.31 (s, 6H,
2 9 CH3), 3.63 (s, 3H, –COOCH3), 3.79 (s, 3H, –OCH2-
COOCH3), 4.07 (m, 2H, –COOCH2CH3), 4.58 (s, 2H,
–OCH2COOCH3), 4.93 (s, 1H, 4-CH, dihydropyridine), 5.85
(s, 1H, –NH), 6.75 (d, 2H, Jo = 7.59 Hz, 3-CH and 5-CH,
aromatic), 7.18 (m, 2H, 2-CH and 6-CH, aromatic); Anal.
calcd. for C21H25NO7: C, 62.52; H, 6.25; N, 3.47; found: C,
62.35; H, 6.11; N, 3.37.
Thermal fusion of 13–15 (12.48 mmol) and amines a–e
(4.96 mmol) at 80–90°C for 4 h afforded corresponding
amide derivatives 13a–e, 14a–d, and 15a–e.
Dimethyl 2,6-dimethyl-4-[4-(2-morpholin-4-yl-2-oxo-
ethoxy)phenyl]-1,4-dihydropyridine
-3,5-dicarboxylate
Ethyl methyl 4-[3-(2-methoxy-2-oxoethoxy)phenyl]-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (14). Yield:
78.51%; m.p. 134–135°C. Spectroscopic analysis: IR (KBr)
(7a). Yield: 74.56%; m.p.: 183–185°C. Spectroscopic
analysis: IR (KBr) mmax/cm-1: 3274, 3217, 3093, 2943,
2868, 1695, 1651, 1505, 1437, 1377, 1306, 1277, 1215,
1
m
max/cm-1: 3340, 3094, 2949, 1742, 1698, 1645, 1598, 1441,
1097, 1029, 849, 746.4; H NMR (CDCl3, 300 MHz, d,
1382, 1283, 1207, 1093, 861, 797; 1H NMR (CDCl3,
300 MHz, d, ppm): 1.22 (t, 3H, –COOCH2CH3, J =
6.01 Hz), 2.31 (s, 6H, 2 9 CH3), 3.64 (s, 3H, –COOCH3),
3.79 (s, 3H, –OCH2COOCH3), 4.09 (m, 2H, –COOCH2CH3),
4.58 (s, 2H, –OCH2COOCH3), 4.98 (s, 1H, 4-CH, dihydro-
pyridine), 6.06 (t, 1H, –NH, J = 12.87 Hz), 6.64 (dd, 1H,
Jo = 8.06 Hz, Jm = 1.55 Hz, 6-CH, aromatic), 6.85 (d, 1H,
Jm = 1.60 Hz, 2-CH, aromatic), 6.94 (d, 1H, Jo = 7.77 Hz,
ppm): 2.32 (s, 6H, 2 9 CH3), 3.61 (m, 8H, –N(CH2)2– and
O(CH2)2– morpholine and s (merged), 6H, 2 9 COOCH3),
4.62 (s, 2H, –OCH2CO–), 4.94 (s, 1H, 4-CH, dihydropyr-
idine), 5.72 (s, 1H, –NH), 6.77 (dd, 2H, Jo = 6.74 Hz,
Jm = 1.94 Hz, 3-CH and 5-CH, aromatic), 7.17 (dd, 2H,
Jo = 6.70 Hz, Jm = 2.00 Hz, 2-CH and 6-CH aromatic);
Anal. calcd. for C23H28N2O7: C, 62.15; H, 6.35; N, 6.30;
found: C, 62.01; H, 5.96; N, 5.83.
123

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Med Chem Res (2012) 21:908–921
Dimethyl 2,6-dimethyl-4-[4-(2-oxo-2-pyrrolidin-1-yleth-
oxy)phenyl]-1,4-dihydropyridine-3,5-dicarboxylate (7b).
4-CH, dihydropyridine), 5.91 (s, 1H, –NH), 6.69 (dd, 1H,
Jo = 7.99 Hz, Jm = 3.02 Hz, 6-CH, aromatic), 6.87 (m, 1H,
2-CH, aromatic), 6.93 (d, 1H, Jo = 7.54 Hz, 4-CH, aro-
matic), 7.13 (t, 1H, Jo = 7.84 Hz, 5-CH, aromatic); Anal.
calcd. for C23H28N2O7: C, 62.15; H, 6.35; N, 6.30; found: C,
61.77; H, 6.51; N, 5.88.
Yield: 45.45%; m.p.: 223–225°C. Spectroscopic analysis: IR
(KBr) mmax/cm-1: 3276, 3214, 3092, 2950, 1697, 1647,
1506, 1444, 1383, 1306, 1277, 1212, 1189, 1136, 1098,
1
1024, 851, 756, 686; H NMR (CDCl3, 300 MHz, d, ppm:
1.87 (p, 2H, –CH2– pyr), 1.95 (p, 2H, –CH2–, pyr), 2.31 (s,
6H, 2 9 CH3), 3.51 (t, 4H, –N(CH2)2– pyr, J = 3.25 Hz),
3.63 (s, 6H, 2 9 COOCH3), 4.55 (s, 2H, –OCH2CO–), 4.94
(s, 1H, 4-CH, dihydropyridine), 6.26 (s, 1H, –NH), 6.77 (dd,
2H, Jo = 6.68 Hz, Jm = 2.01 Hz, 3-CH and 5-CH, aro-
matic) and 7.16 (dd, 2H, Jo = 6.73 Hz, Jm = 2.01 Hz,
2-CH and 6-CH aromatic); Anal. calcd. for C23H28N2O6:
C, 64.47; H, 6.59; N, 6.54; found: C, 63.96; H, 6.55; N,
5.98.
Dimethyl 2,6-dimethyl-4-[3-(2-oxo-2-pyrrolidin-1-yleth-
oxy)phenyl]-1,4-dihydropyridine-3,5-dicarboxylate (8b).
Yield: 76.45%; m.p.: 157–160°C; Spectroscopic analysis:
IR (KBr) mmax/cm-1: 3330, 3083, 2960, 1695, 1651, 1479,
1379, 1346, 1305, 1206, 1095, 1019, 858, 783, 714, 588;
1H NMR (CDCl3, 300 MHz, d, ppm): 1.84 (p, 2H, –CH2–,
pyr), 1.95 (p, 2H, –CH2–, pyr), 2.32 (s, 6H, 2 9 CH3), 3.50
(t, 4H, –N(CH2)2–, pyr, J = 6.81 Hz), 3.64 (s, 6H,
2 9 COOCH3), 4.55 (s, 2H, –OCH2CO–), 4.98 (s, 1H,
4-CH, dihydropyridine), 6.44 (s, 1H, –NH), 6.68 (dd, 1H,
Jo = 7.92 Hz, Jm = 2.88 Hz, 6-CH, aromatic), 6.88 (m,
1H, 2-CH aromatic), 6.92 (d, 1H, Jo = 7.79 Hz, 4-CH,
aromatic), 7.11 (t, 1H, Jo = 7.95 Hz, 5-CH, aromatic);
Anal. calcd. for C23H28N2O6: C, 64.47; H, 6.59; N, 6.54;
found: C, 64.09; H, 6.25; N, 6.16.
Dimethyl 2,6-dimethyl-4-[4-(2-oxo-2-piperidin-1-yleth-
oxy)phenyl]- 1,4-dihydropyridine-3,5-dicarboxylate (7c).
Yield: 41.59%; m.p.: 201–203°C. Spectroscopic analysis:
IR (KBr) mmax/cm-1: 3275, 3214, 3094, 2937, 2857, 1696,
1644, 1505, 1444, 1381, 1305, 1228, 1098, 1017, 850, 781,
1
756; H NMR (CDCl3, 300 MHz, d, ppm): 1.63 (m, 6H,
3-CH2– pip), 2.30 (s, 6H, 2 9 CH3), 3.46 (brt, 2H,
–NCH2–, pip, J = 5.17 Hz), 3.55 (brt, 2H, –NCH2–, pip,
J = 5.15 Hz), 3.63 (s, 6H, 2 9 COOCH3), 4.61 (s, 2H,
–OCH2CO–), 4.93 (s, 1H, 4-CH, dihydropyridine), 6.17 (s,
1H, –NH), 6.77 (dd, 2H, Jo = 6.80 Hz, Jm = 1.90 Hz,
3-CH and 5-CH, aromatic) and 7.16 (dd, 2H, Jo = 6.79 Hz,
Jm = 1.91 Hz, 2-CH and 6-CH, aromatic); Anal. calcd. for
C24H30N2O6: C, 65.14; H, 6.83; N, 6.33; found: C, 64.79;
H, 6.46; N, 6.55.
Dimethyl 2,6-dimethyl-4-[3-(2-oxo-2-piperidin-1-yleth-
oxy)phenyl]- 1,4-dihydropyridine-3,5-dicarboxylate (8c).
Yield: 58.40%; m.p. 158–161°C. Spectroscopic analysis: IR
(KBr) mmax/cm-1: 3288, 3225, 3094, 2939, 1696, 1645,
1492, 1437, 1379, 1308, 1214, 1096, 1019, 858, 784, 715;
1H NMR (CDCl3, 300 MHz, d, ppm): 1.53 (brt, 4H, –CH2–,
pip, J = 5.06 Hz), 1.60 (brt, 2H, –CH2–, pip, J = 4.96 Hz),
2.33 (s, 6H, 2 9 CH3), 3.48 (t, 2H, –NCH2–, pip,
J = 5.18 Hz), 3.53 (t, 2H, –NCH2– pip, J = 5.39 Hz), 3.64
(s, 6H, 2 9 COOCH3), 4.61 (s, 2H, –OCH2CO–), 4.97 (s,
1H, 4-CH, dihydropyridine), 5.97 (s, 1H, –NH), 6.69 (dd,
1H, Jo = 8.04 Hz, Jm = 1.97 Hz, 6-CH, aromatic), 6.89 (d,
1H, Jm = 2.74 Hz, 2-CH, aromatic), 6.92 (s, 1H, 4-CH,
aromatic), 7.11 (t, 1H, Jo = 7.80 Hz, 5-CH, aromatic);
Anal. calcd. for C24H30N2O6: C, 65.14; H, 6.83; N, 6.33;
found: C, 64.88; H, 6.81; N, 5.95.
Dimethyl 2,6-dimethyl-4-{4-[2-(4-methylpiperazin-1-yl)-
2-oxoethoxy]- phenyl}-1,4-dihydropyridine-3,5-dicarboxyl-
ate (7d). Yield: 48.80%; m.p. 164–165°C. Spectroscopic
analysis: IR (KBr) mmax/cm-1: 3278, 3217, 3095, 2948,
2847, 2801, 1700, 1649, 1506, 1444, 1380, 1230, 1095,
1036, 866, 752, 685; 1H NMR (CDCl3, 300 MHz, d, ppm):
2.30 (s, 8H, 2 9 CH3 and [ N–CH3), 2.38 (brs, 4H, CH3–
N(CH2)2–, piperazine), 3.56 (brt, 2H, –NCH2–, piperazine,
J = 4.84 Hz), 3.63 (s, 8H, 2 9 COOCH3 and –NCH2–,
piperazine), 4.62 (s, 2H, –OCH2CO–), 4.94 (s, 1H, 4-CH,
dihydropyridine), 6.17 (brs, 1H, –NH), 6.77 (dd, 2H,
Jo = 8.63 Hz, Jm = 2.81 Hz, 3-CH and 5-CH, aromatic),
7.18 (dd, 2H, Jo = 8.57 Hz, Jm = 2.85 Hz, 2-CH and
6-CH, aromatic); Anal. calcd. for C24H31N3O6: C, 63.00; H,
6.83; N, 9.18; found: C, 63.00; H, 6.59; N, 9.45.
Dimethyl
2,6-dimethyl-4-{3-[2-(4-methylpiperazin-1-
yl)-2-oxoethoxy]-phenyl}-1,4-dihydropyridine-3,5-dicarbo-
xylate (8d). Yield: 57.69; m.p. 173–175°C. Spectroscopic
analysis: IR (KBr) mmax/cm-1: 3299, 3233, 3097, 2944,
1703, 1650, 1595, 1494, 1433, 1307, 1212, 1118, 1093,
1
1023, 857, 812, 795, 713; H NMR (CDCl3, 300 MHz, d,
ppm): 2.27 (s, 3H, [ N–CH3), 2.35 (s, 6H, 2 9 CH3 and t
(merged), 4H, CH3–N(CH2)2– piperazine, J = 2.33 Hz),
3.63 (t, 4H, –N(CH2)2– piperazine, J = 3.64 Hz and s
(merged), 6H, 2 9 COOCH3), 4.62 (s, 2H, –OCH2CO–),
4.98 (s, 1H, 4-CH, dihydropyridine), 5.90 (s, 1H, –NH),
6.69 (dd, 1H, JoJo = 8.00 Hz, Jm = 1.90 Hz, 6-CH, aro-
matic), 6.87 (m, 1H, 2-CH, aromatic), 6.91 (d, 1H,
Jo = 7.52 Hz, 4-CH, aromatic), 7.12 (t, 1H, Jo = 7.77 Hz,
5-CH, aromatic); Anal. calcd. for C24H31N3O6: C, 63.00;
H, 6.83; N, 9.18; found: C, 63.01; H, 6.11; N, 8.97.
Dimethyl 2,6-dimethyl-4-[3-(2-morpholin-4-yl-2-oxoeth-
oxy)phenyl]- 1,4-dihydropyridine-3,5-dicarboxylate (8a).
Yield: 47.37%; m.p. 165–168°C. Spectroscopic analysis: IR
(KBr) mmax/cm-1: 3346, 3092, 2954, 2865, 1700, 1653,
1
1488, 1437, 1348, 1298, 1220, 1121, 1019, 794, 686; H
NMR (CDCl3, 300 MHz, d, ppm): 2.33 (s, 6H, 2 9 CH3),
3.60 (s, 8H, –N(CH2)2– and O(CH2)2–, morpholine), 3.64 (s,
6H, 2 9 COOCH3), 4.63 (s, 2H, –OCH2CO–), 4.98 (s, 1H,
123

Med Chem Res (2012) 21:908–921
915
Dimethyl 4-{3-[2-(1H-imidazol-1-yl)-2-oxoethoxy]phenyl}-
2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (8e).
Yield: 22.93; m.p. 175–177°C. Spectroscopic analysis: IR
(mmax): 3345, 3147, 2957, 1694, 1650, 1567, 1486, 1439,
1307, 1226, 1107, 1057, 870, 830, 779, 715, 630; 1H NMR
(CDCl3 ? DMSO-d6, 300 MHz, d, ppm): 2.30 (s, 6H,
2 9 CH3), 3.64 (s, 6H, 2 9 COOCH3), 4.59 (s, 2H,
–OCH2CO–), 4.99 (s, 1H, 4-CH, dihydropyridine), 6.10 (s,
1H, –NH), 6.67 (dd, 1H, Jo = 7.97 Hz, Jm = 2.02 Hz,
6-CH, aromatic), 6.81 (s, 1H, 2-CH, aromatic), 6.92 (d, 1H,
Jo = 8.00 Hz, 4-CH, aromatic), 7.12 (t, 1H, Jo = 7.85 Hz,
5-CH, aromatic), 7.17 (s, 2H, 4-CH and 5-CH, imidazole),
8.41 (s, 1H, 2-CH, imidazole); Anal. calcd. for
C22H23N3O6: C, 62.11; H, 5.45; N, 9.88; found: C, 62.01;
H, 4.98; N, 9.31.
2H, –OCH2CO–), 4.88 (s, 1H, 4-CH, dihydropyridine), 6.62
(dd, 1H, Jo = 8.28 Hz, Jm = 1.66 Hz, 6-CH, aromatic),
6.70 (d, 1H, Jo = 8.22 Hz, 5-CH, aromatic), 6.84 (d, 1H,
Jm = 1.89 Hz, 2-CH, aromatic), 7.25 (s, 1H, –NH, D2O
exchangeable); Anal. calcd. for C24H30N2O7: C, 62.87; H,
6.59; N, 6.11; found: C, 62.41; H, 6.61; N, 6.23.
Dimethyl 4-[3-methoxy-4-(2-oxo-2-piperidin-1-ylethoxy)
phenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
(9c). Yield: 56.25%; m.p. 210–214°C. Spectroscopic anal-
ysis: IR (KBr) mmax/cm-1: 3350, 2920, 1700, 1640, 1500,
1270, 1210, 1110, 1020, 790, 700, 630; 1H NMR
(CCl4 ? CDCl3, 300 MHz, d, ppm): 1.61 (m, 6H, 3-CH2–
pip), 2.27 (s, 6H, 2 9 CH3), 3.53 (t, 4H, –N(CH2)2–, pip,
J = 5.20 Hz), 3.64 (s, 6H, 2 9 COOCH3), 3.77 (s, 3H,
–OCH3), 4.62 (s, 2H, –OCH2CO–), 4.89 (s, 1H, 4-CH,
dihydropyridine), 6.64 (dd, 1H, Jo = 8.35 Hz, Jm =
1.82 Hz, 6-CH, aromatic), 6.74 (d, 1H, Jo = 8.27 Hz, 5-CH,
aromatic), 6.84 (d, 1H, Jm = 2.00 Hz, 2-CH, aromatic), 6.98
(s, 1H, –NH, D2O exchangeable); Anal. calcd. for
C25H32N2O7: C, 63.54; H, 6.83; N, 5.93; found: C, 63.11; H,
6.59; N, 5.51.
Dimethyl 4-{3-[2-(tert-butylamino)-2-oxoethoxy]phenyl}-
2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (8f).
Yield: 12.96%; m.p. 233–235°C. Spectroscopic analysis:
FT-IR (KBr) mmax/cm-1: 3380, 3275, 2984, 1689, 1659,
1596, 1496, 1450, 1310, 1255, 1221, 1122, 1096, 1026,
1
875, 782, 610; H NMR (CDCl3 ? DMSO-d6, 300 MHz,
d, ppm): 1.29 (s, 9H, –NHC(CH3)3), 2.33 (s, 6H,
2 9 CH3), 3.63 (s, 6H, 2 9 COOCH3), 4.17 (s, 2H,
–OCH2CO–), 4.91 (s, 1H, 4-CH, dihydropyridine), 6.59 (d,
1H, Jo = 8.10 Hz, 6-CH, aromatic), 6.66 (s, 1H, 2-CH,
aromatic), 6.71 (d, 1H, Jo = 7.14 Hz, 4-CH, aromatic),
7.11 (t, 1H, Jo = 7.80 Hz, 5-CH, aromatic), 8.40 (brs, 1H,
–NH), 9.01 (s, 1H, –NH); Anal. calcd. for C23H30N2O6: C,
64.17; H, 7.02; N, 6.51; found: C, 63.95; H, 6.75; N, 6.26.
Dimethyl 4-[3-methoxy-4-(2-morpholin-4-yl-2-oxoethoxy)
phenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
(9a). Yield: 56.64%; m.p.: 190–193°C. Spectroscopic
analysis: IR (KBr) mmax/cm-1: 3350, 2950, 1700, 1650,
1490, 1450, 1220, 1110, 1010, 850, 800, 650, 600; 1H
NMR (CDCl3, 300 MHz, d, ppm): 2.33 (s, 6H, 2 9 CH3),
3.65 (s, 6H, 2 9 COOCH3 and t (merged), 8H, –N(CH2)2–
and O(CH2)2– morph, J = 3.65 Hz), 3.81 (s, 3H, –OCH3),
4.67 (s, 2H, –OCH2CO–), 4.95 (s, 1H, 4-CH, dihydropyr-
idine), 5.75 (s, 1H, -NH, D2O exchangeable), 6.73 (dd, 1H,
Jo = 8.33 Hz, Jm = 1.70 Hz, 6-CH, aromatic), 6.79 (d,
1H, Jo = 8.22 Hz, 5-CH, aromatic), 6.88 (d, 1H,
Jm = 1.87 Hz, 2-CH, aromatic); Anal. calcd. for
C24H30N2O8: C, 60.75; H, 6.37; N, 5.90; found: C, 60.32;
H, 6.49; N, 6.19.
Dimethyl 4-{3-methoxy-4-[2-(4-methylpiperazin-1-yl)-
2-oxoethoxy]-phenyl}-2,6-dimethyl-1,4-dihydropyridine-3,
5-dicarboxylate (9d). Yield: 43.10%; m.p. 202–204°C.
Spectroscopic analysis: IR (KBr) mmax/cm-1: 3314, 3088,
2948, 2800, 1694, 1634, 1489, 1380, 1256, 1215, 1095,
1
1031, 866, 764, 706, 629; H NMR (CDCl3, 300 MHz, d,
ppm): 2.30 (s, 3H, [ N–CH3), 2.31 (s, 6H, 2 9 CH3), 2.37
(t, 4H, –CH3–N(CH2)2–, piperazine, J = 4.79 Hz), 3.65
(s, 10H, 2 9 COOCH3 and –N(CH2)2–, piperazine), 3.80
(s, 3H, –OCH3), 4.66 (s, 2H, –OCH2CO–), 4.95 (s, 1H,
4-CH, dihydropyridine), 6.05 (s, 1H, –NH, D2O
exchangeable), 6.71 (dd, 2H, Jo = 8.33 Hz, Jm = 1.79 Hz,
6-CH, aromatic), 6.78 (d, 1H, Jo = 8.25 Hz, 5-CH, aro-
matic), 6.88 (d, 1H, Jm = 1.61 Hz, 2-CH, aromatic); Anal.
calcd. for C25H33N3O7: C, 61.59; H, 6.82; N, 8.62; found:
C, 61.49; H, 6.59; N, 8.31.
Dimethyl 4-{4-[2-(1 h-imidazol-1-yl)-2-oxoethoxy]-3-meth-
oxyphenyl}-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarbox-
ylate (9e). Yield: 37.96%; m.p. 216–220°C. Spectroscopic
analysis: IR (KBr) mmax/cm-1: 3357, 3146, 2947, 1680,
1655, 1491, 1212, 1117, 1023, 865, 611; 1H NMR
(CDCl3 ? DMSO-d6, 300 MHz, d, ppm): 2.29 (s, 6H,
2 9 CH3), 3.61 (s, 6H, 2 9 COOCH3), 3.79 (s, 3H,
–OCH3), 4.52 (s, 2H, –OCH2CO–), 4.87 (s, 1H, 4-CH,
dihydropyridine), 6.66 (s, 2H, 5-CH and 6-CH, aromatic),
6.83 (s, 1H, 2-CH, aromatic), 7.04 (s, 2H, 4-CH and 5-CH,
imidazole), 7.74 (s, 1H, 2-CH, imidazole), 8.50 (s, 1H, –NH,
D2O exchangeable); Anal. calcd. for C23H25N3O7: C, 60.65;
H, 5.53; N, 9.23; found: C, 59.99; H, 4.95; N, 9.30.
Dimethyl 4-[3-methoxy-4-(2-oxo-2-pyrrolidin-1-ylethoxy)
phenyl]-2,6-dimethyl-1,4,-dihydropyridine-3,5-dicarboxylate
(9b). Yield: 55.96%; m.p. 192–194°C. Spectroscopic anal-
ysis: IR (KBr) mmax/cm-1: 3350, 2960, 1700, 1650, 1500,
1
1430, 1320, 1210, 1110, 1010, 750, 650; H NMR (CDCl3,
300 MHz, d, ppm): 1.85 (m, 2H, –CH2–, pyr), 1.95 (m, 2H,
–CH2–, pyr), 2.26 (s, 6H, 2 9 CH3), 3.48 (t, 2H, –NCH2–,
pyr, J = 6.83 Hz), 3.55 (t, 2H, –NCH2–, pyr, J = 6.64 Hz),
3.64 (s, 6H, 2 9 COOCH3), 3.75 (s, 3H, –OCH3), 4.56 (s,
Dimethyl 4-{4-[2-(tert-butylamino)-2-oxoethoxy]-3-meth-
oxyphenyl}-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarbox-
ylate (9f). Yield: 71.55%; m.p.[ 270°C. Spectroscopic
123

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Med Chem Res (2012) 21:908–921
analysis: IR (KBr) mmax/cm-1: 3150, 2897, 1660, 1492, 1301,
1259, 1079, 787, 680, 623; H-NMR (CDCl3, 300 MHz, d,
–COOCH2CH3), 4.56 (s, 2H, –OCH2CO-), 4.93 (s, 1H,
4-CH, dihydropyridine), 6.53 (s, 1H, –NH), 6.76 (d, 2H,
Jo = 8.52 Hz, 3-CH and 5-CH, aromatic), 7.19 (m, 2H,
2-CH and 6-CH aromatic); Anal. calcd. for C24H30N2O6: C,
65.14; H, 6.83; N, 6.33; found: C, 65.02; H, 6.52; N, 6.03.
Ethyl methyl 2,6-dimethyl-4-[4-(2-oxo-2-piperidin-1-
ylethoxy)- phenyl]-1,4-dihydropyridine-3,5-dicarboxylate
(13c). Yield: 56.63%; m.p. 187–188°C. Spectroscopic
analysis: IR (KBr) mmax/cm-1: 3278, 3221, 3097, 2939,
2859, 1695, 1643, 1505, 1445, 1382, 1304, 1274, 1227,
1096, 1018, 855, 781, 688; 1H NMR (CDCl3, 300 MHz, d,
ppm): 1.21 (t, 3H, –COOCH2CH3, J = 5.89 Hz), 1.62 (m,
6H, 3-CH2–, pip), 2.31 (s, 6H, 2 9 CH3), 3.45 (t, 2H,
–NCH2–, pip, J = 4.92 Hz), 3.55 (t, 2H, –NCH2–, pip,
J = 4.75 Hz), 3.63 (s, 3H, –COOCH3), 4.08 (m, 2H,
–COOCH2CH3), 4.62 (s, 2H, –OCH2CO–), 4.93 (s, 1H,
4-CH, dihydropyridine), 6.00 (brs, 1H, –NH), 6.77 (d, 2H,
Jo = 8.52 Hz, 3-CH and 5-CH, aromatic), 7.17 (m, 2H,
2-CH and 6-CH, aromatic); Anal. calcd. for C25H32N2O6:
C, 65.77; H, 7.07; N, 6.14; found: C, 65.70; H, 6.56; N,
6.01.
1
ppm): 2.17 (s, 9H, –NH(CH3)3), 2.34 (s, 6H, 2 9 CH3), 3.66
(s, 6H, 2 9 COOCH3), 3.87 (s, 3H, –OCH3), 4.59 (s, 2H,
–OCH2CO–), 4.98 (s, 1H, 4-CH, dihydropyridine), 5.64 (s,
1H, –NH, D2O exchangeable), 6.80 (m, 2H, 5-CH and 6-CH
aromatic), 6.92 (d, 1H, Jm = 1.16 Hz, 2-CH, aromatic), 7.26
(s, 1H, –NH(CH3)3); Anal. calcd. for C24H32N2O7: C, 62.59;
H, 7.00; N, 6.08; found: C, 62.11; H, 7.02; N, 5.86.
Dimethyl 4-{3-methoxy-4-[2-(3-methylpiperidin-1-yl)-2-
oxoethoxy]- phenyl}-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (9g). Yield: 72.17%; m.p. 148–150°C.
Spectroscopic analysis: IR (KBr) mmax/cm-1: 3306, 2948,
1700, 1631, 1479, 1214, 1029, 767; 1H NMR (CDCl3,
300 MHz, d, ppm): 0.90 (t, 3H, –C(CH3)H–, pipec,
J = 4.54 Hz), 1.16 (m, 1H, –C(CH3)H–, pipec), 1.54 (m,
1H, –CH(H)–, pipec), 1.68 (m, 1H, –CH(H)–, pipec), 1.79
(m, 1H, –CH(H)–, pipec), 1.98 (m, 1H, –CH(H)–, pipec),
2.32 (s, 6H, 2 9 CH3), 2.64 (m, 1H, –NCH(H)–, pipec),
3.00 (q, 1H, –NCH(H)– pipec), 3.65 (s, 6H, 2 9 CO-
OCH3), 3.81 (s, 3H, –OCH3), 3.92 (t, 1H, –NCH(H)– pi-
pec, J = 9.45 Hz), 4.34 (t, 1H, –NCH(H)– pipec,
J = 10.72 Hz), 4.66 (s, 2H, –OCH2CO–), 4.95 (s, 1H,
4-CH, dihydropyridine), 6.08 (s, 1H, –NH, D2O
exchangeable), 6.71 (dd, 1H, Jo = 8.30 Hz, Jm = 1.92 Hz,
6-CH aromatic), 6.78 (d, 1H, Jo = 8.29 Hz, 5-CH aro-
matic), 6.88 (d, 1H, Jm = 1.63 Hz, 2-CH aromatic); Anal.
calcd. for C26H34N2O7: C, 64.18; H, 7.04; N, 5.76; found:
C, 63.95; H, 6.51; N, 5.91.
Ethyl methyl 2,6-dimethyl-4-{4-[2-(4-methylpiperazin-1-
yl)-2-oxo- ethoxy]phenyl}-1,4-dihydropyridine-3,5-dicar-
boxylate (13d). Yield: 44.82%; m.p. 176–178°C. Spectro-
scopic analysis: IR (KBr) mmax/cm-1: 3280, 3222, 3096,
2942, 2801, 1694, 1649, 1503, 1444, 1377, 1273, 1225, 1095,
1039, 862, 826, 754; 1H NMR (CDCl3, 300 MHz, d, ppm):
1.21 (t, 3H, –COOCH2CH3, J = 6.12 Hz), 2.30 (s,
9H, [ N–CH3 and 2 9 CH3), 2.39 (brs, 4H, –N(CH2)2–
piperazine), 3.61 (m, 7H, –COOCH3 and –N(CH2)2– piper-
azine), 4.08 (m, 2H, –COOCH2CH3), 4.61 (s, 2H,
–OCH2CO–), 4.93 (s, 1H, 4-CH, dihydropyridine), 5.90 (t,
1H, –NH, J = 15.83 Hz), 6.76 (d, 2H, Jo = 8.59 Hz, 3-CH
and 5-CH, aromatic), 7.18 (m, 2H, 2-CH and 6-CH, aro-
matic). Anal. calcd. for C25H33N3O6: C, 63.68; H, 7.05; N,
8.91; found: C, 62.86; H, 6.75; N, 8.46.
Ethyl methyl 2,6-dimethyl-4-[4-(2-morpholin-4-yl-2-
oxoethoxy)-phenyl]-1,4-dihydropyridine-3,5-dicarboxylate
(13a). Yield: 59.29%; m.p.: 165–167°C. Spectroscopic
analysis: IR (KBr) mmax/cm-1: 3281, 3223, 3097, 2952,
1694, 1646, 1504, 1445, 1375, 1275, 1217, 1106, 1028,
1
853, 747; H NMR (CDCl3, 300 MHz, d, ppm): 1.22 (t,
3H, –COOCH2CH3, J = 5.83 Hz), 2.27 (t, 4H, –N(CH2)2–,
morph, J = 14.39 Hz and s (merged), 6H, 2 3 CH3), 3.63
(m, 4H, O(CH2)2–, morph and s (merged), 3H, –COOCH3),
4.08 (m, 2H, –COOCH2CH3), 4.62 (s, 2H, –OCH2CO–),
4.92 (s, 1H, 4-CH, dihydropyridine), 5.81 (s, 1H, –NH),
6.77 (d, 2H, Jo = 8.43 Hz, 3-CH and 5-CH, aromatic),
7.18 (m, 2H, 2-CH and 6-CH, aromatic); Anal. calcd. for
C24H30N2O7: C, 62.87; H, 6.59; N, 6.11; found: C, 62.55;
H, 6.16; N, 6.10.
Ethyl methyl 4-(4-{2-[(3-methylpiperidin-1-yl)oxy]-2-
oxoethoxy}- phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (13e). Yield: 65.51%; m.p. 166–167°C.
Spectroscopic analysis: IR (KBr) mmax/cm-1: 3281, 3218,
3095, 2939, 1692, 1645, 1505, 1444, 1382, 1304, 1207,
1095, 1051, 1022, 854, 825, 781, 752, 687; 1H NMR
(CDCl3, 300 MHz, d, ppm): 0.89 (t, 3H, –C(CH3)H–, pi-
pec, J = 5.71 Hz), 1.15 (m, 1H, –C(CH3)H–, pipec), 1.21
(t, 3H, –COOCH2CH3, J = 6.22 Hz), 1.52 (m, 2H, –CH2–,
pipec), 1.69 (m, 1H, –CH(H)–, pipec), 1.81 (m, 1H,
–CH(H)–, pipec), 2.31 (s, 6H, 2 9 CH3), 2.66 (m, 1H,
–NCH(H)–, pipec), 2.99 (m, 1H, –NCH(H)–, pipec), 3.63
(s, 3H, –COOCH3), 3.80 (m, 1H, –NCH(H)–, pipec), 4.08
(m, 2H, –COOCH2CH3), 4.34 (m, 1H, –NCH(H)–, pipec),
4.59 (s, 2H, –OCH2CO–), 4.93 (s, 1H, 4-CH, dihydropyr-
idine), 5.95 (t, 1H, –NH, J = 18.73 Hz), 6.76 (d, 2H,
Jo = 8.50 Hz, 3-CH and 5-CH aromatic), 7.18 (m, 2H,
Ethyl methyl 2,6-dimethyl-4-[4-(2-oxo-2-pyrrolidin-1-
ylethoxy)-phenyl]-1,4-dihydropyridine-3,5-dicarboxylate
(13b). Yield: 64.22%; m.p. 189–190°C. Spectroscopic
analysis: IR (KBr) mmax/cm-1: 3280, 3219, 3095, 2980, 1694,
1646, 1504, 1382, 1304, 1275, 1210, 1098, 1026, 856, 756;
1H NMR (CDCl3, 300 MHz, d, ppm): 1.21 (t, 3H, –CO-
OCH2CH3, J = 5.39 Hz), 1.88 (m, 2H, –CH2–, pyr), 1.97
(m, 2H, –CH2–, pyr), 2.30 (s, 6H, 2 3 CH3), 3.50 (m, 4H,
–N(CH2)2–, pyr), 3.63 (s, 3H, –COOCH3), 4.08 (m, 2H,
123

Med Chem Res (2012) 21:908–921
917
2-CH and 6-CH, aromatic); Anal. calcd. for C26H34N2O6:
C, 66.36; H, 7.28; N, 5.95; found: C, 66.33; H, 6.65; N,
5.36.
ylate (14d). Yield: 45.51%; m.p. 135–136°C. Spectroscopic
analysis: IR (KBr) mmax/cm-1: 3301, 3234, 3099, 2943,
1700, 1650, 1493, 1306, 1212, 1168, 1095, 1027, 780, 711;
1H NMR (CDCl3, 300 MHz, d, ppm): 1.22 (t, 3H, –CO-
OCH2CH3, J = 6.61 Hz), 2.33 (s, 9H, 2 9 CH3 and [ N–
CH3), 2.46 (brt, 4H, –N(CH2)2–, piperazine, J = 11.59 Hz),
3.64 (s, 3H, –COOCH3), 3.66 (brs, 4H, –N(CH2)2–, piper-
azine), 4.08 (m, 2H, –COOCH2CH3), 4.63 (s, 2H,
–OCH2CO–), 4.97 (s, 1H, 4-CH, dihydropyridine), 5.87 (t,
1H, –NH, J = 11.18 Hz), 6.70 (dd, 2H, Jo = 6.62 Hz,
Jm = 1.39 Hz, 6-CH, aromatic), 6.87 (d, 1H, Jm =
1.59 Hz, 2-CH, aromatic), 6.93 (m, 1H, 4-CH, aromatic),
7.11 (t, 1H, Jo = 7.66 Hz, 5-CH aromatic); Anal. calcd. for
C25H33N3O6: C, 63.68; H, 7.05; N, 8.91; found: C, 63.23;
H, 6.68; N, 8.52.
Ethyl methyl 2,6-dimethyl-4-[3-(2-morpholin-4-yl-2-
oxoethoxy)- phenyl]-1,4-dihydropyridine-3,5-dicarboxylate
(14a). Yield: 54.36%; m.p. 137–139°C. Spectroscopic
analysis: IR (KBr) mmax/cm-1: 3301, 3231, 3098, 2944,
1701, 1650, 1493, 1434, 1307, 1211, 1167, 1094, 1002,
1
778, 756, 711; H NMR (CDCl3, 300 MHz, d, ppm): 1.22
(t, 3H, –COOCH2CH3, J = 6.04 Hz), 2.33 (brs, 10H,
2 9 CH3 and –N(CH2)2–, morph), 3.58 (t, 4H, O(CH2)2–,
morph, J = 4.51 Hz), 3.64 (s, 3H, –COOCH3), 4.09 (m,
2H, –COOCH2CH3), 4.61 (s, 2H, –OCH2CO–), 4.97 (s,
1H, 4-CH, dihydropyridine), 5.89 (t, 1H, –NH, J =
11.17 Hz), 6.68 (dd, 1H, Jo = 7.99 Hz, Jm = 1.63 Hz,
6-CH, aromatic), 6.88 (d, 1H, Jm = 2.05 Hz, 2-CH, aro-
matic), 6.92 (m, 1H, 4-CH aromatic), 7.10 (t, 1H, Jo =
7.93 Hz, 5-CH, aromatic); Anal. calcd. for C24H30N2O7: C,
62.87; H, 6.59; N, 6.11; found: C, 62.58; H, 6.36; N, 5.83.
Ethyl methyl 2,6-dimethyl-4-[3-(2-oxo-2-pyrrolidin-1-
ylethoxy)-phenyl]-1,4-dihydropyridine-3,5-dicarboxylate
(14 b). Yield: 67.27%; m.p. 87–89°C. Spectroscopic anal-
ysis: IR (KBr) mmax/cm-1: 3273, 3211, 3086, 2981, 2891,
1692, 1648, 1596, 1493, 1441, 1304, 1271, 1205, 1094,
Ethyl methyl 4-{3-methoxy-4-[2-(morpholin-4-yloxy)-2-
oxoethoxy]- phenyl}-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (15a). Yield: 85.71%; m.p.: 157–158°C;
Spectroscopic analysis: IR (KBr) mmax/cm-1: 3328, 2966,
1692, 1642, 1509, 1330, 1219, 1118, 1027, 794, 757, 696;
1H NMR (CDCl3, 300 MHz, d, ppm): 1.24 (t, 3H, –CO-
OCH2CH3, J = 7.65 Hz), 2.32 (s, 6H, 2 9 CH3), 3.65 (s,
11H, –COOCH3 and –N–(CH2)2–, O–(CH2)2–, morph),
3.81 (s, 3H, –OCH3), 4.10 (m, 2H, –COOCH2CH3), 4.67
(s, 2H, –OCH2CO–), 4.95 (s, 1H, 4-CH, dihydropyridine),
6.05 (brs,1H, –NH), 6.74 (m, 2H, 5-CH and 6-CH, aro-
matic), 6.89 (d, 2H, Jm = 1.27 Hz, 2-CH, aromatic); Anal.
calcd. for C25H32N2O8: C, 61.46; H, 6.60; N, 5.73; found:
C, 61.00; H, 6.49; N, 5.49.
1
1021, 863, 760, 719; H NMR (CDCl3): d 1.22 (t, 3H,
–COOCH2CH3, J = 7.20 Hz), 1.84 (p, 2H, –CH2–, pyr),
1.95 (p, 2H, –CH2–, pyr), 2.32 (s, 6H, 2 9 CH3), 3.50 (t,
4H, –N(CH2)2–, pyr, J = 6.74 Hz), 3.64 (s, 3H, –CO-
OCH3), 4.08 (m, 2H, –COOCH2CH3), 4.55 (s, 2H,
–OCH2CO–), 4.97 (s, 1H, 4-CH, dihydropyridine), 6.44 (t,
1H, –NH, J = 17.62 Hz), 6.67 (dd, 1H, Jo = 7.94 Hz,
Jm = 1.46 Hz, 6-CH, aromatic), 6.92 (m, 2H, 2-CH and
4-CH, aromatic), 7.11 (t, 1H, Jo = 7.81 Hz, 5-CH, aro-
matic). Anal. calcd. for C24H30N2O6: C, 65.14; H, 6.83; N,
6.33. Found: C, 64.84; H, 6.54; N, 6.12.
Ethyl methyl 4-{3-methoxy-4-[2-oxo-2-(pyrrolidin-1-
yloxy)ethoxy]- phenyl}-2,6-dimethyl-1,4-dihydropyridine-
3,5-dicarboxylate (15b). Yield: 94.49%; m.p. 176–177°C.
Spectroscopic analysis: IR (KBr) mmax/cm-1: 3283, 3087,
2944, 2881, 1692, 1649, 1508, 1307, 1259, 1211, 1092,
1
1036, 863, 765.6; H NMR (CDCl3, 300 MHz, d, ppm):
Ethyl methyl 2,6-dimethyl-4-[3-(2-oxo-2-piperidin-1-
ylethoxy)-phenyl]-1,4-dihydropyridine-3,5-dicarboxylate
(14c). Yield: 78.31%; m.p. 157–158°C. Spectroscopic
analysis: IR (KBr) mmax/cm-1: 3289, 3220, 3095, 2938,
1701, 1645, 1495, 1435, 1307, 1210, 1115, 1041, 811, 781
and 712. 1H NMR (CDCl3, 300 MHz, d, ppm): 1.22 (t, 3H,
–COOCH2CH3, J = 5.95 Hz), 1.52 (brs, 4H, 2-CH2– pip),
1.61 (m, 2H, –CH2– pip), 2.33 (s, 6H, 2 9 CH3), 3.51 (m,
4H, –N(CH2)2– pip), 3.64 (s, 3H, –COOCH3), 4.08 (m, 2H,
–COOCH2CH3), 4.63 (s, 2H, –OCH2CO–), 4.97 (s, 1H,
4-CH, dihydropyridine), 6.04 (t, 1H, –NH, J = 13.09 Hz),
6.69 (dd, 2H, Jo = 8.00 Hz, Jm = 1.61 Hz, 6-CH, aro-
matic), 6.91 (m, 2H, 2-CH and 4-CH, aromatic), 7.11 (t,
1H, Jo = 7.84 Hz, 5-CH, aromatic); Anal. calcd. for
C25H32N2O6: C, 65.77; H, 7.07; N, 6.14; found: C, 65.65;
H, 7.00; N, 6.13.
1.24 (t, 3H, –COOCH2CH3, J = 7.61 Hz), 1.88 (m, 4H,
2-CH2– pyr), 2.31 (s, 6H, 2 9 CH3), 3.52 (p, 4H,
–N(CH2)2– pyr), 3.65 (s, 3H, –COOCH3), 3.81 (s, 3H,
–OCH3), 4.10 (m, 2H, –COOCH2CH3), 4.62 (s, 2H,
–OCH2CO–), 4.94 (s, 1H, 4-CH, dihydropyridine), 6.25 (t,
1H, –NH, J = 20.89 Hz), 6.74 (m, 2H, 5-CH and 6-CH
aromatic), 6.89 (s, 1H, 2-CH, aromatic); Anal. calcd. for
C25H32N2O7: C, 63.54; H, 6.83; N, 5.93; found: C, 62.96;
H, 7.01; N, 5.43.
Ethyl methyl 4-{3-methoxy-4-[2-oxo-2-(piperidin-1-
yloxy)ethoxy]- phenyl}-2,6-dimethyl-1,4-dihydropyridine-
3,5-dicarboxylate (15c). Yield: 60.71%; m.p. 159–160°C.
Spectroscopic analysis: IR (KBr) mmax/cm-1: 3295, 3223,
3095, 2942, 1695, 1641, 1507, 1442, 1264, 1212, 1090,
1
1031, 857 and 789; H NMR (CDCl3, 300 MHz, d, ppm):
1.23 (t, 3H, –COOCH2CH3, J = 7.11 Hz), 1.59 (m, 6H,
3-CH2–, pip), 2.31 (s, 6H, 2 9 CH3), 3.53 (m, 4H,
–N(CH2)2–, pip), 3.65 (s, 3H, –COOCH3), 3.80 (s, 3H,
Ethyl methyl 2,6-dimethyl-4-{3-[2-(4-methylpiperazin-1-
yl)-2-oxoethoxy]phenyl}-1,4-dihydropyridine-3,5-dicarbox-
123

918
Med Chem Res (2012) 21:908–921
–OCH3), 4.09 (m, 2H, –COOCH2CH3), 4.67 (s, 2H,
–OCH2CO–), 4.94 (s, 1H, 4-CH, dihydropyridine), 6.42 (t,
1H, –NH, J = 21.30 Hz), 6.73 (m, 2H, 5-CH and 6-CH,
aromatic), 6.89 (d, 1H, Jm = 1.26 Hz, 2-CH aromatic);
Anal. calcd. for C26H34N2O7: C, 64.18; H, 7.04; N, 5.76;
found: C, 64.08; H, 7.00; N, 5.45.
with approximately 25,000 cells. The plates were placed
into a humidified 5% CO2 incubator at 37°C and kept for at
least 4 days before fluorescence screening. Before the
beginning of the experiments, the cells were washed thor-
oughly with saline solution using Tomtec liquid handling
robot. The fluorescence screening was done using fluo-3-
ester calcium dye (2 lM). It was loaded into cells with
pluronic acid (20%). Once in the cytoplasm, esterases
cleave the ester from the fluo-3 dye effectively trapping the
dye within the cell. The cells were incubated for 45 min at
37°C. The plates were washed thoroughly before carrying
out the experiment. Initial readings were taken with a
Cytofluor 4000 HT fluorimeter (Perceptives) set at 36°C to
assess the background fluorescence in each of the plates.
The excitation/emission settings were 483/530 nm.
Ethyl methyl 4-(3-methoxy-4-{2-[(4-methylpiperazin-1-
yl)oxy]-2-oxoethoxy}phenyl)-2,6-dimethyl-1,4-dihydropyri-
dine-3,5-dicarboxylate (15d). Yield: 73.04%; m.p.
153–155°C. Spectroscopic analysis: IR (KBr) mmax/cm-1
:
3321, 2941, 1695, 1631, 1510, 1260, 1214, 1094, 1033,
1
798, 760; H NMR (CDCl3, 300 MHz, d, ppm): 1.24 (t,
3H, –COOCH2CH3, J = 6.96 Hz), 2.32 (m, 4H,
–N(CH2)2– piperazine and s (merged), 9H, 2 9 CH3
and [ N–CH3), 3.65 (brs, 7H, –COOCH3 and –N(CH2)2–,
piperazine), 3.81 (s, 3H, –OCH3), 4.10 (m, 2H,
–COOCH2CH3), 4.67 (s, 2H, –OCH2CO–), 4.94 (s, 1H,
4-CH, dihydropyridine), 6.00 (t, 1H, –NH, J = 15.27 Hz),
6.76 (m, 2H, 5-CH and 6-CH, aromatic), 6.90 (s, 1H, 2-CH,
aromatic); Anal. calcd. for C26H35N3O7: C, 62.26; H, 7.03;
N, 8.38; found: C, 61.96; H, 6.59; N, 7.95.
Cells were exposed to veratridine for 2 min before
readings were taken. For those experiments that require
pretreatment, cells were pre-treated for 30 s with the
compound before application of veratridine. In each plate,
a saline and a veratridine lane were included. The data
were analyzed using Microsoft Excel. The initial back-
ground readings were subtracted from the treatment read-
ings. The saline control was then subtracted, and the results
were expressed as % of the control veratridine response.
Increase in intracellular calcium measured with the fluo-3
dye is reflected as rises in fluorescence and decreases in
fluorescence represent a drop in intracellular calcium. All
compounds were brought up to 10 mM stocks in 100%
DMSO and then serially diluted. All the results are
expressed as means the standard error of the mean
(SEM), and statistical analysis was performed with Stu-
dent’s t-test. Dose–response curves were analyzed by a
sigmoidal curve-fitting analysis to give the IC50 values.
Ethyl methyl 4-(3-methoxy-4-{2-[(3-methylpiperidin-1-
yl)oxy]-2-oxoethoxy}phenyl) 2,6-dimethyl-1,4-dihydropyri-
dine-3,5-dicarboxylate (15e). Yield: 85.21%; m.p.
155–157°C. Spectroscopic analysis: IR (KBr) mmax/cm-1
:
3380, 3272, 2942, 1687, 1659, 1596, 1497, 1310, 1221,
1123, 1026, 875, 703; 1H NMR (CDCl3, 300 MHz, d,
ppm): 0.88 (t, 3H, –C(CH3)H–, pipec, J = 5.56 Hz), 1.13
(m, 1H, –C(CH3)H–, pipec), 1.23 (t, 3H, –COOCH2CH3,
J = 7.11 Hz), 1.46 (m, 2H, –CH2–, pipec), 1.69 (m, 2H,
–CH2–, pipec), 2.32 (s, 6H, 2 9 CH3), 2.64 (m, 1H,
–NCH(H)–, pipec), 2.99 (m, 1H, –NCH(H)–, pipec), 3.65
(s, 3H, –COOCH3), 3.81 (s, 3H, –OCH3), 3.89 (m, 1H,
–NCH(H)–, pipec), 4.10 (m, 2H, –COOCH2CH3), 4.35 (m,
1H, –NCH(H)–, pipec), 4.67 (s, 2H, –OCH2CO–), 4.93 (s,
1H, 4-CH, dihydropyridine), 5.92 (t, 1H, –NH, J =
18.50 Hz), 6.71 (m, 1H, 6-CH, aromatic), 6.79 (d, 1H, Jo =
8.28 Hz, 5-CH, aromatic), 6.88 (d, 1H, Jm = 1.12 Hz,
2-CH, aromatic); Anal. calcd. for C27H36N2O7: C, 64.78;
H, 7.25; N, 5.60; found: C, 64.66; H, 6.91; N, 5.12.
Vasodilatory Activity Using KCl (30 mM)
(a) and using Phenylephrine (b)
(a) Wistar rats of either sex weighing 300–400 g were
killed by a blow on the head. The descending thoracic aorta
was rapidly dissected and placed in a physiological saline
solution (PSS) of the composition (mM): NaCl 118,
NaHCO3 25, MgSO4 1.2, CaCl2 2.5, KH2PO4 1.2, KCl
4.75, and glucose 11. After excess of fat and connective
tissue was removed, the aorta were cut into rings (4–5 mm
in length), mounted under basal tension of 2 g in 5 ml
organ baths containing PSS and attached to force–dis-
placement transducers to measure isometric contractile
force. The tissue bath was maintained at 37°C and bubbled
with 95% O2–5% CO2 gas mixture. The preparation was
allowed to equilibrate for at least 60 min before initiation
of experimental procedures, and during this period the
incubation media was changed every 30 min.
Biological Methods
Calcium Channel Blocking Activity
The calcium channel blocking activity of newly synthesized
compounds was assessed using 96-well plate fluorimetery.
Primary cultures of cortex were prepared from neonatal rat
pups. The isolated cells were placed onto poly-D-lysine
coated 96-well plates (Becton-Dickenson) using a fluid-
handling robot (Quadra 96, Tomtec). Each well was loaded
123

Med Chem Res (2012) 21:908–921
919
After equilibration, aortic rings were contracted by
single concentration of 30 mM KCl. When the contractions
were stable, compound was added in progressively
increasing cumulative concentrations (10-8 to 10-5 M) at
30-min intervals. The results were expressed as a per-
centage of maximal control KCl-induced responses, and
statistical analysis was performed with Student’s t-test.
Differences between control and experimental values were
considered significant when P \ 0.05. Dose–response
slopes were analyzed to give the concentration of com-
pounds producing a 50% inhibition of the maximal con-
tractile response (IC50) by linear regression analysis
(method of least squares). The compounds were dissolved
in dimethyl sulfoxide (DMSO). DMSO had no effect on
KCl-contractile response.
Table 1 IC50 values derived from logistical fits to the calcium
inhibitory data of various dihydropyridines
Compound No. (Code)
IC50 SEM (lM)
7 (DPJ-RG-1212)
8 (DPJ-RG-1126)
8a (DPJ-RG-1161)
8b (DPJ-RG-1162)
8c (DPJ-RG-1163)
8d (DPJ-RG-1164)
9b (DPJ-RG-1144)
9c (DPJ-RG-1145)
9d (DPJ-RG-1160)
9e (DPJ-RG-1201)
9f (DPJ-RG-1203)
9g (DPJ-RG-1159)
Nifedipine
2.1 4.2
10.0 3.0
9.9 10.0
3.6 3.0
10.0 5.7
10.0 2.9
5.6 1.2
10.0 5.7
9.1 2.1
10.1 7.1
2.5 1.3
10.0 5.8
57.7 48.6 nM
(b) Vasodilatory activity was carried out using
descending thoracic aortic rings of Wistar rats. The aortic
rings were contracted by phenylephrine (10-6 M). After
stabilization, compounds were added in progressively
increasing cumulative concentrations (10-8 to 10-5 M) at
30-min intervals. All the results were expressed as a per-
centage of the maximal control phenylephrine-induced
responses, and statistical analysis was performed with
Student’s t-test.
P \ 0.05 versus DMSO
Vasodilatory Activity
The compounds 8, 8a, 8b, 8d, 9a, 9b, 9d, 9f, and 9g have
been screened for vasodilatory activity (Carron et al., 1991;
Perez-Vizcaino et al., 1993) using rat thoracic aortic rings,
precontracted with phenylephrine (10-6 M). The results are
expressed as relaxation as a percentage of the maximal
control phenylephrine-induced responses. The compounds
produced a concentration-dependent inhibition of the con-
tractile response of phenylephrine (Table 2). Relaxation (%
of control) and pD2 (-log IC50) of various compounds to
inhibit the contractions induced by phenylephrine (10-6 M)
are given in Table 3. Compounds 14c, 15, and 15c were
also tested for their vasorelaxant action using KCl
(30 mM) as the contractile agent. The relaxation (% of
control) and pD2 (-log IC50) values of various compounds
to inhibit the contractions produced by KCl (30 mM) are
depicted in Table 3. IC50 values of the various compounds
showing potential vasodilatory activity using phenyleph-
rine (10-6 M) and KCl (30 mM) have been calculated and
are given in Tables 2 and 3, respectively.
Results
Calcium Channel Blocking Activity
The calcium channel blocking activity of newly synthe-
sized compounds 7–9, 7a–d, 8a–f, 9a–g, 10–12, 13–15,
13a–e, 14 a–f, and 15a–e was assessed using 96-well plate
fluorimetery at L-type calcium channels activated by
veratridine in neonatal rat cortical neurons maintained in
cell culture. Veratridine (10 lM) was used as the depo-
larizing agent for all the experiments. Initially, the com-
pounds were screened for calcium channel blocking
activity at a single concentration of 10 lM on NG108-15
cells (Neuroblastoma X Glioma). Veratridine-induced
increase (5 min read) in intracellular calcium was inhibited
with 10 lM nifedipine (IC50 = 57.7 48.6 nM) indicat-
ing a large component of the veratridine response occurring
through L-type channels. Percentage veratridine response
produced by various compounds was observed. The com-
pounds that showed more than 50% inhibition of the
veratridine response at 10 lM have been selected for fur-
ther detailed testing to calculate their IC50 values. IC50
values derived from logistical fits to the calcium flux
inhibitory data from various dihydropyridines are shown in
Table 1.
Discussion
Although the carboxylates 7 and 8 substituted at para and
meta positions of the 4-aryl group of symmetrical dihy-
dropyridines displayed medium calcium channel blocking
activity, the vanilloid-based derivative 9 with an ortho-
methoxy group did not exhibit significant activity at
10 lM. The compound 7 displayed the most potent cal-
cium channel inhibiting activity with IC50 = 2.1 lM.
123

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Med Chem Res (2012) 21:908–921
Table 2 Relaxation (% of control), pD2 (-log IC50), and IC50 (lM) values to inhibit the contractions induced by phenylephrine (10-6 M)
Compound No. (Code)
10-9
M
10-8
0
M
10-7
M
10-6
M
10-5
M
pD2
IC50 SEM (lM)
8 (DPJ-RG-1126)
8a (DPJ-RG-1161)
8b (DPJ-RG-1162)
8d (DPJ-RG-1164)
9a * (DPJ-RG-1143)
9b (DPJ-RG-1144)
9d (DPJ-RG-1160)
9f * (DPJ-RG-1203)
9g (DPJ-RG-1159)
Nifedipine
0
0
0
0
0
0
0
0
0
0
3.1 4.9
26.6 3.2
0.0 0.0
4.1 4.1
36.1 6.8
30.6 3.1
0.2 0.2
0.9 0.6
0.4 0.4
55.7 7.7
47.6 4.4
55.7 2.7
36.2 4.6
26.9 4.2
56.5 6.3
54.8 5.2
37.5 3.2
4.3 1.4
81.6 1.3
81.5 5.1
60.3 3.0
52.5 6.4
5.25 0.25
76.1 8.6
51.3 5.6
8.8 2.4
5.84 0.01
6.19 0.11
5.34 0.10
5.02 0.19
–
1.44 0.01
0.64 0.11
4.57 0.10
9.54 0.19
–
5.6 3.5
0
0
0.4 1.7
8.1 3.3
6.08 0.20
5.16 0.18
–
0.83 0.20
6.91 0.18
–
0
0
0
36.7 4.3
68.0 8.5
83.1 2.6
78.3 4.7
5.76 0.04
7.56 0.63
1.73 0.04
27.5 nM
39.5 8.7
Values are expressed as mean SEM, n = 5
P \ 0.05 versus DMSO
*These compounds did not produce 50% relaxation up to 10-5
M
Table 3 Relaxation (% of control), pD2 (-Log IC50), and IC50 (lM) values to inhibit the contractions induced by KCl 30 mM on rat aortic rings
Compound
No. (Code)
10-8
M
10-7
M
10-6
M
10-5
M
3 9 10-5
M
10-4
M
pD2
IC50 SEM
(lM) (K-30)
14c (DPJ-RG-1335)
15 (DPJ-RG-1263)
15c (DPJ-RG-1267)
0
0
0
11.42 7.79 44.92 8.97 92.12 2.43
5.53 2.57 12.28 4.28 39.96 7.52
99.50 0.27
100 0.0
5.90 0.10 1.25 0.10
64.31 7.77 80.38 6.59 4.89 0.15 12.8 0.15
4.27 1.47 23.99 6.43 56.30 10.84 76.18 8.76 85.92 7.82 5.41 0.17 3.89 0.17
10-11
M
10-10
M
10-9
M
10-8
M
10-7
M
pD2
IC50 SEM (lM) (K-30)
Nifedipine
0
8.71 3.17
28.88 3.53
83.63 3.70
99.47 0.29
8.59 0.07
2 nM
Values are expressed as means SEM, n = 5–8
P \ 0.05 versus DMSO
Formation of amides of carboxylates resulted in com-
pounds with moderate calcium channel blocking activity
with IC50 values ranging from 2 to 10 lM; however, there
was loss of potency in comparison to nifedipine
(IC50 = 57.7 nM). Even though introduction of unsym-
metrical ester groups in the dihydropyridines has been
reported to impart more potency (Mannhold, 1994), the
introduction of asymmetry in compounds 13a–e, 14a–d,
and 15a–e did not even produce 50% inhibition of verat-
ridine response at 10 lM in this study. In general, the
newly synthesized dihydropyridines displayed only mar-
ginal activity in comparison to nifedipine; however, these
compounds are nitro group free and represent a suitable
lead to be structurally modified to get therapeutically useful
dihydropyridines.
dihydropyridines, rather decreased it in comparison to
nifedipine. The symmetrical pyrrolidinyl substituted deriv-
ative 8a displayed maximum potency of 0.64 lM but was
found to be approximately 24 times less active in comparison
to standard drug nifedipine with IC50 = 27.5 nM. It seems
that conformational and electronic changes caused by the
substitution at different sites of the phenyl ring might affect
the interaction of the concerned molecule with drug recep-
tors and thereby influence their potency.
Overall, it was observed that incorporation of an ester/
amide group on 4-phenyl ring in all the three series of
compounds resulted in decreased calcium channel blocking
and vasodilatory effects of dihydropyridines in comparison
to nifedipine.
As vasodilators, the compounds are producing significant
relaxation of KCl as well as phenylephrine induced con-
tractions suggesting that compounds may be acting at vari-
ous sites other than calcium channels. Substitution of esters,
heterocyclic ring, heteroaromatic, or acyclic amine did not
improve upon the vasodilatory potential of the
Conclusions
Amide derivatives of symmetrical and unsymmetrical
4-aryl-1,4-dihydropyridine carboxylic congeners have been
synthesized in this study. The newly synthesized nitro
123

Med Chem Res (2012) 21:908–921
921
Liang JC, Yeh JL, Chiang LC, Yang YC, Sheu SH, Lai WT, Chen IJ
(2000) Labedipinedilol-A: a vanilloid-based a/b-adrenoceptor
blocker with calcium entry blocking and long-lasting antihyper-
tensive properties. Drug Dev Res 49:94–108
Liang JC, Yeh JL, Wang CS, Liou SF, Tsai CH, Chen IJ (2002) The
new generation dihydropyridine type calcium blockers, bearing
4-phenyl oxypropanolamine, display alpha-/beta-adrenoceptor
antagonist and long-acting antihypertensive activities. Bioorg
Med Chem 10:719–730
group free dihydropyridines exhibited moderate calcium
channel blocking and vasodilatory activity. Further struc-
tural modifications might lead to a new series of thera-
peutically useful dihydropyridines.
Acknowledgments The authors are thankful to University Grants
Commission, India for providing financial support.
Mannhold R (1994) Medicinal chemistry of DHP-like calcium
antagonist. Drugs Today 30:103–122
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