Synthesis of diethyl 4-substituted-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylates as a new series of inhibitors against yeast α-glucosidase

Article abstract of DOI:10.1016/j.ejmech.2015.03.018

1,4-Dihydropyridine-3,5-dicarboxylate derivatives (1-25) were synthesized in high yields via Hantzsch reaction and evaluated for their α-glucosidase inhibitory activity. Compounds 1, 2, 6-8, 11, 13-15, and 23-25 showed a potent inhibitory activity against yeast α-glucosidase with IC₅₀ values in the range of 35.0-273.7 μM, when compared with the standard drug acarbose (IC₅₀ = 937 ± 1.60 μM). Their structures were characterized by different spectroscopic techniques. The kinetics, selectivity, and toxicity studies on these compounds were also carried out. The kinetic studies on most active compounds 14 and 25 determined their modes of inhibition and dissociation constants K_i. Compound 14 was found to be a non-competitive inhibitor with K_i = 25.0 ± 0.06, while compound 25 was identified as a competitive inhibitor with K_i = 66.0 ± 0.07 μM.

Full text of DOI:10.1016/j.ejmech.2015.03.018

Accepted Manuscript
Synthesis of diethyl 4-substituted-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylates
as a new series of inhibitors against yeast α-glucosidase
Huma Niaz, Hamdy Kashtoh, Jalaluddin A.J. Khan, Ajmal Khan, Atia tul-Wahab,
Muhammad Tanveer Alam, Khalid Mohammed Khan, Shahnaz Perveen, M. Iqbal
Choudhary
PII:
S0223-5234(15)00185-3
10.1016/j.ejmech.2015.03.018
EJMECH 7763
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 November 2014
Revised Date: 24 February 2015
Accepted Date: 9 March 2015
Please cite this article as: H. Niaz, H. Kashtoh, J.A.J. Khan, A. Khan, A. tul-Wahab, M.T. Alam,
K.M. Khan, S. Perveen, M.I. Choudhary, Synthesis of diethyl 4-substituted-2,6-dimethyl-1,4-
dihydropyridine-3,5-dicarboxylates as a new series of inhibitors against yeast α-glucosidase, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.03.018.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis of diethyl 4-substituted-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylates as a new series of inhibitors against yeast α-glucosidase
Huma Niaz,a Hamdy Kashtoh,b Jalaluddin A. J. Khan,b Ajmal Khan,a Atia-tul-Wahab,d
Muhammad Tanveer Alam,a Khalid Mohammed Khan,a, Shahnaz Perveen,c M. Iqbal
Choudharya,b,d,*
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
bDepartment of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah-21412,
Saudi Arabia
cPCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280,
Pakistan
dDr. Panjwani Center for Molecular Medicine and Drug Research, International Center for
Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
Abstract
1,4-Dihydropyridine-3,5-dicarboxylate derivatives (1-25) were synthesized in high yields via
Hantzsch reaction and evaluated for their α-glucosidase inhibitory activity. Compounds 1, 2, 6,
7, 8, 11, 13, 14, 15, 23, 24, and 25 showed a potent inhibitory activity against yeast α-
glucosidase with IC50 values in the range of 35.0-273.7 ꢀM, when compared with the standard
drug acarbose (IC50= 937 ± 1.60 ꢀM). Their structures were characterized by different
spectroscopic techniques. The kinetics, selectivity, and toxicity studies on these compounds were
also carried out. The kinetic studies on most active compounds 14 and 25 determined their
modes of inhibition and dissociation constants Ki. Compound 14 was found to be a non-
competitive inhibitor with Ki = 25.0 ± 0.06, while compound 25 was identified as a competitive
inhibitor with Ki = 66.0 ± 0.07 µM.
Keywords: 1,4-Dihydropyridine-3,5-dicarboxylates; Hantzsch reaction; α-glucosidase
inhibition; anti-hyperglycemic activity; type II diabetes
Corresponding authors: hassaan2@super.net.pk; khalid.khan@iccs.edu; iqbal.choudhary@iccs.edu; Tel.: 0092-21-
34824910; Fax: 0092-21-34819018; Tel.: (0092-21) 34824924-5; Fax: (0092-21) 4819018-9
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1. Introduction
The dihydropyridine derivatives exhibit a wide range of biological activities, such as
antianxiety, vasodilation, analgesic, neuroprotectant, bronchodilation, anti-inflammatory,
anticonvulsant, antidepressant, hypnotic, antitumor, and platelet anti-aggregatory activities [1].
4-Aryl-1,4-dihydropridines are used in the treatment of hypertension and cardiovascular diseases
functioning as calcium channels blockers. These compounds lower the blood pressure by
relaxing the smooth muscle wall of heart and arteries and reduce their external resistance [2].
Dialkyl-1,4-dihydro-2,6-dimethylpyridine-3,5-dicarboxylates are important drugs against
hypertension, and angina. Some of them, such as Nitrendipine,® Felodipine,® Isradipine,®
Nifedipine,® Lacidipine,® Nimodipine,® Nicardipine,® and Amlodipine® are in clinical use [3].
Hantzsch 1,4-dihydropyridine derivatives serve as reactive moieties in co-enzyme NADH
which is a redox reagent in biological system [4]. Based on the biological importance and diverse
therapeutic applications of 1,4-dihydropyridines, we evaluated their inhibitory potential against
several enzymes. Interestingly they showed a potent activity against yeast α-glucosidase in a
biochemical assay.
Diabetes mellitus is a metabolic syndrome, characterized by hyperglycemia [5, 6].
Epidemiological studies by International Diabetes Federation (IDF) showed that more than 375
million people worldwide have diabetes in 2012 [7]. Post-prandial high blood glucose levels,
associated with type-2 diabetes mellitus, play a crucial role in the development of
atherosclerosis, and cardiovascular disorders [8-9]. α-Glucosidase catalyzes the cleavage of
glucose from disaccharides, as only monosaccharides can be absorbed from the intestinal lumen
and transported into blood circulation in mammals. Thus, the inhibition of the action of this
enzyme in human can be an effective approach to control hyperglycemia in type-2 diabetes [10].
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α-Glucosidase inhibitors (AGIs), such as acarbose, miglitol, and voglibose, have sugar like
structures, and thus compete with the oligosaccharides for binding to the active site of the
enzyme. They effectively decrease the post-prandial glucose levels in type-2 diabetic patients
[11]. However, these classic AGIs cause various side effects, such as flatulence, diarrhea, and
abdominal discomfort [12]. In addition, all known AGIs have low efficacy with high IC50 values
against the enzyme. Due to the crucial role of this enzyme in hyperglycemia, and low tolerability
of the existing AGIs, there is a need to discover safe and efficient inhibitors for the effective
control of hyperglycemia in diabetes.
We describe here the synthesis and characterization of diethyl 4-substituted-2,6-dimethyl-
1,4-dihydropyridine-3,5-dicarboxylates (1-25). These compounds were first time evaluated for
their α-glucosidase inhibitory activity through a biochemical mechanism-based assay. Their
kinetics, selectivity, and toxicity were also evaluated. The kinetic studies on the most active
compounds 14 and 25 determined their modes of inhibition and dissociation constants Ki.
Compound 14 a non-competitive inhibitor showed a Ki = 25.0 ± 0.06, however, compound 25
was identified as a competitive inhibitor with a Ki = 66.0 ± 0.07 µM. The selectivity was checked
against phosphodiestrase-I, carbonic anhydrase-II, and β-glucuronidase, and no activity was
observed. The cytotoxicity was checked against 3T3 cell line and no toxicity was observed.
2. Results and discussion
2.1. Chemistry
Multi-component reactions (MCR) form an important sub-class of tandem reactions. They
are suitable for green syntheses as three or more components react efficiently to form a product
in good yield [13]. Due to their intrinsic atom economy, selectivity, simplicity, time and energy
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efficiency, as well as eco-friendliness, MCRs are gaining importance in research and
development [14].
Hantzsch MCR reaction has attracted much scientific attention in the synthesis of 1,4-
dihydropyridines (1,4-DHPs) [15]. The mechanism involves a Knoevenagel condensation of the
ethylacetoacetate with the aldehyde to form an α,β-unsaturated carbonyl compound, and
condensation of ammonia (produced in situ from (NH4)2CO3) with another equivalent of the
ethylacetoacetate to yield an enaminoester. The Michael addition of the enaminoester to α,β-
unsaturated carbonyl compound, and a finally intramolecular condensation results in the
formation of desired diethyl 4-aryl/alkyl-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylates.
Compounds 1-25 were synthesized by multi-component condensation reaction of ethyl
acetoacetate with different aliphatic, and aromatic aldehydes in water using ammonium
carbonate as a solid ammonia source (Scheme-1) [16].
O
R
O
O
O
13
10
H2O
14
H3C
9
4
(NH4)2CO3
R-CHO
12
11
+
+
O
O
CH3
5
6
3
2
80 oC
H3C
OC2H5
8
1
7
H3C
N
H
CH3
1-25
Scheme-1: Synthesis of 1,4-dihydropyridine-3,5-dicarboxylate derivatives 1-25
Aromatic aldehydes, containing either electron withdrawing or electron donating
substituents, aliphatic, and heteroaryl aldehydes, were reacted efficiently with ethyl acetoacetate
in the presence of ammonium carbonate. Desired products were obtained in generally good
1
yields. The structures of synthetic compounds were confirmed by H-NMR, EI and HREI-MS
spectroscopy and CHN analyses. Compounds 1-25 have been previously reported [17-30].
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2.2. Biological activity
Compounds 1-25 were evaluated for their inhibitory potential against the
enzyme (EC 3.2.1.20, Saccharomyces cerevisiae (Table-1) and showed significant activity with
IC50 values in the range 35.0 ± 1.01 to 273.7 ± 1.04 M (Table-1). The 4-substituted-2,6-
α-glucosidase
)
µ
dimethyl-1,4-dihydropyridine-3,5-dicarboxylates 1, 2, 6, 7, 8, 11, 13, 14, 15, 23, 24, and 25 were
found to be active, while rest of the compounds showed less than 50% inhibition and were not
screened for their IC50 values. Compounds 14 and 25 were about 26- and 18-folds more active
with IC50 values of 35.0 ± 0.17 and 51 ± 0.98 µM, respectively, as compared to the standard drug
acarbose (IC50 = 937 ± 1.60
Insert Table-1 here
µM).
Insert Figure-1 here
Limited SAR suggests that the activity mainly depends upon the presence of R (aliphatic or
aromatic) group and in case of aromatic, nature and position of substituents on it (Figure-1).
Compounds 14 (IC50 = 35.0 ± 0.17 µM) and 25 (IC50 = 51 ± 0.98 µM) containing nitro group at
phenyl ring showed a potent activity. The enhanced activity of compound 14 may be due to
presence of an electron-withdrawing NO2 group at meta position which may be responsible for
its binding with enzyme. In comparision relatively decreased activity of compound 25 may be
due to change in the position of NO2 from meta to para. Compounds 11 (IC50 = 60.2 ± 1.01
µM)
having small fluorine group at ortho position and 1 (IC50 = 72.4 ± 1.03 M) having a relatively
µ
large chlorine residues at para position are the determing factor for difference in activities. The
presence of bulky carboxyl moiety at ortho position of phenyl ring made the derivative 18
inactive against the enzyme. Compounds containing para substituted phenyl moiety were also
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found to be active. In addition, compound 5 containing unsubstituted phenyl ring, was found to
be inactive.
When meta position is substituted with an electron-donating hydroxyl group, the resulting
compound 17 showed no activity, however, derivative 23 have both meta and para hydroxyl
substitution showed a weak inhibition (IC50 = 273.7 ± 1.04
compounds. Same pattern was observed in compounds 8, 13, and 16
a methoxy/ethoxy group at para position, showed good activities (IC50 = 65.1 ± 0.92 and IC50 =
124.3 ± 0.97 M, respectively), but when another methoxy was introduced at meta position, the
µ
M) as compare to other active
.
Compounds 13 and 8 with
µ
resulting compound 16 became inactive.
When R group was changed to aliphatic moieties as in analogues 4, 19-22, the resulting
compounds displayed no activity against the enzyme. However, in case of compound 24 where
ethyl benzene moiety was present as R made the derivative potent inhibitor of the enzyme (IC50 =
112 ± 0.64 µM). This difference in activitity suggested that remote phenyl ring may be helful for
its binding with active site of the enzyme.
Heterocyclic residues containing analogues 3, 9, 10 and 12 were found to be inactive, except
sulphur containing compounds 2 (IC50 = 74.3 ± 0.76 µM) and 6 (IC50 = 97.4 ± 0.32 µM) showed
good activity.
From aforementioned discussion it can easily be extracted that the yeast α-glucosidase inhibitory
activity of diethyl 4-substituted-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylates (1-25) is
depending upon the nature of R (aliphatic or aromatic) group and in case of aromatic, position
and effect of substituents on it.
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2.3. Kinetic studies
To study the mechanism of inhibition, the kinetic studies of the most active compounds 14
and 25 were performed. In the kinetic studies, different concentrations of test compounds and
substrates were used. The result indicated that compound 14 is a non-competitive inhibitor with
Ki value 25 ± 0.06
M.
µM, while compound 25 is a competitive inhibitor with Ki value 68 ± 0.07
µ
The type of inhibition was determined by Lineweaver-Burk plots, the reciprocal of the rate
of the reaction were plotted against the reciprocal of substrate concentrations to monitor the
effect of inhibitor on both, Km and Vmax. Figure-2A showed that Vmax of enzyme decreased
without affecting the Km of enzyme which indicated that compound 14 is a non-competitive
inhibitor of α-glucosidase.
The secondary re-plots of Lineweaver-Burk plots were plotted to determine the Ki values
(Figure-2B). The Ki value was calculated by plotting the slope of each line in the Lineweaver-
Burk plots against the different concentrations of compound 14. The Ki value was confirmed
from Dixon plot by plotting the reciprocal of the rate of reaction against different concentrations
of compound 14 (Figure-2C).
Insert Figure-2 here
The kinetic studies showed that the compound 25 is a competitive inhibitor of α-glucosidase
(Figure-3). Figure-3A showed that the Vmax of enzyme was not affected with varying
concentrations of compound 25, while the Km increased. This indicated a pure competitive-type
of inhibition.
Insert Figure-3 here
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2.4. Selectivity
In order to asses the selectivity of the compounds for
2,13,14, and 25 were evaluated for their inhibitory activity against the phosphodiestrase-I [31],
α-glucosidase, the most potent compounds
carbonic anhydrase-II [32], and
(Table-2).
β-glucuronidase enzymes [33], and no activity was observed
Insert Table-2 here
2.5. Cytotoxicity
The cytotoxicity of compounds 1, 2, 6-8, 11, 13, 14, and 23-25 was evaluated against 3T3
cell line [34]. Most of these compounds exhibited a non-toxic behavior, except compounds 1, 23,
and 24 which showed low cytotoxicity with IC50 values of 27.857 ± 0.25, 28.634 ± 0.24, and
26.726 ± 0.52, respectively (Table-3).
Insert Table-3 here
3. Conclusion
Compounds 1, 2, 6-8, 11, 13-15, and 23-25 showed a potent inhibitory activity against yeast
α-glucosidase with IC50 values in the range of 35 ± 0.17 to 273.7 ± 1.04
µM, when compared
with the standard drug acarbose (IC50 = 937 ± 1.60 M). Limited SAR studies indicated that
µ
meta position in aromatic aldehydes is important for activity. Introduction of electron
withdrawing groups at meta position enhanced the activity, while introduction of electron
donating groups at meta position, resulted in either decrease or loss of activity. Compounds
containing nitro group, halogens or sulphur expressed a strong inhibitory potential against the
yeast
α-glucosidase enzyme, therefore future development or structural modifications of these
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moieties are requird to explore inhibitory mechanism. However, these molecules needs to be
further evaluated through animal model, i.e. in vivo studies to establish their therapeutic potential
to overcome the pathologies in diabetes.
4. Materials and methods
NMR experiments were performed on Avance Bruker AM 300, 400, 500, and 600 MHz
instruments, Rheinstetten, Germany. CHN analyses were carried on a Carlo Erba Strumentazione-
Mod-1106, Italy, apparatus, and electron impact mass spectra (EI-MS) on a Finnigan MAT-311A,
instrument, Bremen, Germany. Thin-layer chromatography (TLC) was performed on pre-coated
silica gel glass plates (Kieselgel 60, 254, E. Merck, Darmstadt, Germany). Chromatograms were
visualized by either UV at 254 or 365 nm. Melting points (uncorrected) were recorded on a Büchi
Labortechnik AG, Switzerland, melting point apparatus model B-540.
4.1. General procedure for the synthesis of 1,4-dihydropyridine-3,5-dicarboxylate derivatives 1-
25
A mixture of aldehyde (1 mmol), ethyl acetoacetate (2 mmol) and ammonium carbonate (2
mmol) was stirred in water at 80 °C for 3 to 5 h. The progress of reaction was monitored through
the TLC analysis. After completion of the reaction as indicated by TLC, the mixture was diluted
with cold H2O, precipitated product was filtered, washed with distilled water, and crystallized
from absolute ethanol.
4.1.1. Diethyl 4-(4-chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (1)
º
º
Yield: 85%; M.p.: 145-147 C (Lit. M.p.24 147-150 C); Rf: 0.67 (
n
-hexane/ethyl acetate,
8:2); 1H-NMR: (300 MHz, DMSO-d6):
δ
8.84 (s, 1H, NH), 7.24 (d, 2H, J2′,3′ = J6′,5′ = 8.4 Hz, H-
2
′
/H-6
′
), 7.13 (d, 2H, J3′,2′
=
J5′,6′ =8.4 Hz, 2H, H-3
′/H-5
′), 4.82 (s, 1H, H-4), 3.98 (m, 4H, 2CH2),
9

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2.24 (s, 6H, 2CH3), 1.11 (t, 6H,
11 and C-12, C=O), 147.0 (C-1
(C-2 and C-6 , C-Ar), 127.7 (C-3
J
= 7.2 Hz, 2CH3); 13C-NMR (100 MHz, DMSO-d6):
, C-Ar), 145.5 (C-2 and C-6, C=C), 130.3 (C-4 , C-Ar), 129.1
and C-5 , C-Ar), 101.4 (C-3 and C-5, C=C), 59.0 (C-10 and
δ 166.7 (C-
′
′
′
′
′
′
C-13, OCH2), 38.4 (C-4, CH), 18.1 (C-7 and C-8, CH3), 14.1 (C-9 and C-14, OCH2CH3); EI-MS:
m/z (rel. abund. %), 365 (M+ +2, 8.3), 364 (M++1, 9.1), 363 (M+, 23.6), 252 (100), 224 (66), 196
(71), 150 (21); HREI-MS: m/z Calcd for C19H22ClNO4: 363.1237; Found: 363.1202; Anal. Calcd
for C19H22ClNO4 (363.84): C, 62.72; H, 6.09; Cl, 9.74; N, 3.85; O, 17.59
6.04; N, 3.82.
; Found:C, 62.77; H,
4.1.2. Diethyl 2,6-dimethyl-4-(2-thienyl)-1,4-dihydropyridine-3,5-dicarboxylate (2)
º
º
Yield: 87%; M.p.: 154-156 C (Lit. M.p.20 153-155 C); Rf: 0.59 (
n-hexane/ethyl acetate,
1
8:2); H-NMR: (300 MHz, DMSO-d6):
δ
8.99 (s, 1H, NH), 7.17 (dd, 1H, J5′, 3′ = 0.9 Hz, J5′, 4′
), 6.81 (dd, 1H, J4′, 3′ = 3.3 Hz, J4′, 5′ = 4.8 Hz, H-4 ), 6.64 (d, 1H, J3′,4′ = 3.3 Hz, H-3
5.16 (s, 1H, H-4), 4.05 (m, 4H, 2CH2), 2.25 (s, 6H, 2CH3), 1.11 (t, 6H,
NMR (75 MHz, DMSO-d6): 166.6 (C-11 and C-12, C=O), 151.8 (C-2
C-6, C=C), 126.3 (C-4 , C-Ar), 123.3 (C-3 , C-Ar), 122.4 (C-5 , C-Ar), 101.3 (C-3 and C-5,
=
4.8 Hz, H-5
′
′
′
),
J
= 7.2 Hz, 2CH3); 13C-
δ
′, C-Ar), 145.8 (C-2 and
′
′
′
C=C), 59.1 (C-10 and C-13, OCH2), 33.7 (C-4, CH), 18.1 (C-7 and C-8, CH3), 14.2 (C-9 and C-
14, OCH2CH3); EI-MS: m/z (rel. abund. %), 337 (M+ +2, 1.6), 335 (M+, 20), 137 (100), 109 (52),
65 (12); HREI-MS: m/z Calcd for C17H21NO4S: 335.1191; Found: 335.1199; Anal. Calcd for
C17H21NO4S (335.42): C, 60.87; H, 6.31; N, 4.18; O, 19.08; S; 9.56; Found:C, 60.83; H, 6.28; N,
4.21.
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4.1.3. Diethyl 2,6-dimethyl-4-(3-pyridinyl)-1,4-dihydropyridine-3,5-dicarboxylate (3)
º
º
Yield: 80%; M.p.: 191-192 C (Lit. M.p.16 190-192 C); Rf: 0.21 (
n
-hexane/ethyl acetate,
J2′,6′ = 1.5 Hz,
H-2′), 8.29 (br. d, 1H, J4′,5′ = 3.9 Hz, H-4′), 7.48 (br. d, 1H, J6′,5′ = 7.8 Hz, H-6′), 7.23 (dd, 1H, J5′,
8:2); 1H-NMR: (300 MHz, DMSO-d6):
δ
8.91 (s, 1H, NH), 8.35 (br. d, 1H, J2′,4′ =
4′ = 4.8 Hz, J5′, 6′ = 7.8 Hz, H-5′), 4.82 (s, 1H, H-4), 3.97 (m, 4H, 2CH2), 2.25 (s, 6H, 2CH3), 1.10
(t, 6H,
(C-6
Ar), 123.4 (C-5
J
= 7.2 Hz, 2CH3); 13C-NMR (75 MHz, DMSO-d6):
δ
166.5 (C-11 and C-12, C=O), 148.7
, C-Ar), 134.8 (C-4 , C-
, C-Ar), 101.0 (C-3 and C-5, C=C), 59.0 (C-10 and C-13, OCH2), 36.9 (C-4,
′
, C-Ar), 147.1 (C-2
′
, C-Ar), 146.0 (C-2 and C-6, C=C), 143.2 (C-3
′
′
′
CH), 18.1 (C-7 and C-8, CH3), 14.1 (C-9 and C-14, OCH2CH3); EI-MS: m/z (rel. abund. %), 330
(M+, 6.3), 252 (100), 224 (28), 196 (50), 150 (7.6); HREI-MS: m/z Calcd for C18H22N2O4 :
330.1580; Found: 330.1571; Anal. Calcd for C18H22N2O4 (330.37): C, 65.44; H, 6.71; N, 8.48;
O, 19.37; Found: C, 65.39; H, 6.68; N, 8.51.
4.1.4. Diethyl 4-ethyl-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (4)
Yield: 78%; M.p.: 111-112 ºC (Lit. M.p.28 112 ºC); Rf: 0.67 ( -hexane/ethyl acetate, 8:2); 1H-
NMR: (300 MHz, DMSO-d6): 8.58 (s, 1H, NH), 4.04 (m, 4H, 2CH2), 3.73 (t, 1H, = 5.4 Hz,
n
δ
J
H-4), 2.18 (s, 6H, 2CH3), 1.18 (t, 6H,
J = 7.2 Hz, 2CH3), 1.21 (m, 2H, aliphatic-CH2), (t, 3H, J =
7.5 Hz, aliphatic- CH3); 13C-NMR (100 MHz, DMSO-d6):
δ
167.2 (C-11 and C-12, C=O), 146.1
(C-2 and C-6, C=C), 100.2 (C-3 and C-5, C=C), 58.7 (C-10 and C-13, OCH2), 33.3 (C-4, CH),
29.0 (aliphatic-CH2CH3), 18.1 (C-7 and C-8, CH3), 14.3 (C-9 and C-14, OCH2CH3), 8.9
(aliphatic-CH2CH3); EI-MS: m/z (rel. abund. %), 281 (M+), 252 (100), 236 (42), 224 (72); HREI-
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MS: m/z Calcd for C15H23NO4: 281.1627; Found: 281.1633; Anal. Calcd for C15H23NO4
(281.34): C, 64.03; H, 8.24; N, 4.98; O, 22.75; Found:C, 64.06; H, 8.19; N, 5.01.
4.1.5. Diethyl 2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (5)
º
Yield: 85%; M.p.: 159-160 C (Lit. M.p.24 158-160); Rf: 0.59 (
n
-hexane/ethylacetate, 8:2);
1H-NMR: (300 MHz, DMSO-d6):
δ
8.78 (s, 1H, NH), 7.14 (m, 5H, aromatic), 4.84 (s, 1H, H-4),
13
3.99 (m, 4H, 2CH2), 2.24 (s, 6H, 2CH3), 1.11 (t, 6H,
DMSO-d6): 166.9 (C-11 and C-12, C=O), 148.1 (C-1
(C-3 and C-5 , C-Ar), 127.2 (C-2 and C-6 , C-Ar), 125.8 (C-4′
J
= 7.2 Hz, 2CH3); C-NMR (75 MHz,
δ
′
, C-Ar), 145.2 (C-2 and C-6, C=C), 127.7
, C-Ar), 101.8 (C-3 and C-5,
′
′
′
′
C=C), 58.9 (C-10 and C-13, OCH2), 38.8 (C-4, CH), 18.1 (C-7 and C-8, CH3), 14.1 (C-9 and C-
14, OCH2CH3); EI-MS: m/z (rel. abund. %), 330 (M++1, 3.8), 329 (M+, 15.2), 252 (100), 224
(43), 196 (55), 150 (9.2); HREI-MS: m/z Calcd for C19H23NO4: 329.1627; Found: 329.1639;
Anal. Calcd for C19H23NO4 (329.39): C, 69.28; H, 7.04; N, 4.25; O, 19.43; Found: C, 69.30; H,
7.01; N, 4.22.
4.1.6. Diethyl 2,6-dimethyl-4-[4-(methylsulfanyl)phenyl]-1,4-dihydropyridine-3,5-dicarboxylate
(6)
º
º
Yield: 92%; M.p.: 140-142 C (Lit. M.p.24 138-141 C); Rf: 0.69 (
n
-hexane/ethyl acetate,
8:2); 1H-NMR: (300 MHz, DMSO-d6):
δ
8.78 (s, 1H, NH), 7.10 (d, 2H, J2′,3′ = J6′,5′ = 8.7 Hz, H-
2
′
/H-6
2.39 (s, 3H, -SCH3), 2.23 (s, 6H, 2CH3), 1.12 (t, 6H,
DMSO-d6):
(C-1 , C-Ar), 127.9 (C-2
′
), 7.04 (d, 2H, J3′,2′
=
J5′,6′ = 8.7 Hz, 2H, H-3
′
/H-5
′
), 4.79 (s, 1H, H-4), 3.95 (m, 4H, 2CH2),
J
= 7.2 Hz, 2CH3); 13C-NMR (100 MHz,
δ
166.8 (C-11 and C-12, C=O), 145.2 (C-2 and C-6, C=C), 145.0 (C-4
′, C-Ar), 135.0
′
′
and C-6 , C-Ar), 125.7 (C-3 and C-5 , C-Ar), 101.7 (C-3 and C-5,
′
′
′
C=C), 58.9 (C-10 and C-13, OCH2), 38.3 (C-4, CH), 18.1 (C-7 and C-8, CH3), 14.8 (-SCH3),
12

ACCEPTED MANUSCRIPT
14.1 (C-9 and C-14, OCH2CH3); EI-MS: m/z (rel. abund. %), 376 (M++1, 7.4), 375 (M+, 35), 373
(62), 252 (100), 224 (32), 196 (43), 150 (10), 83 (27); HREI-MS: m/z Calcd for C20H25NO4S:
375.1504; Found: 375.1509; Anal. Calcd for C20H25NO4S 375.48): C, 63.97; H, 6.71; N, 3.73; O,
17.04; S, 8.54; Found:C, 63.92; H, 6.75; N, 3.70.
4.1.7. Diethyl 2,6-dimethyl-4-(4-methylphenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7)
º
º
Yield: 83%; M.p.: 132-133 C (Lit. M.p.29 130-132 C); Rf: 0.66 (
n
-hexane/ethyl acetate,
8:2); 1H-NMR: (300 MHz, DMSO-d6):
δ
8.73 (s, 1H, NH), 7.01 (d, 2H, J2′,3′ = J6′,5′ = 8.4 Hz, H-
2
′
/H-6
′
), 6.97 (d, 2H, J3′,2′
=
J5′,6′ = 8.4 Hz, H-3
′
/H-5
′
), 4.79 (s, 1H, H-4), 3.95 (m, 4H, 2CH2),
= 7.2 Hz, 2CH3); 13C-NMR (125
2.22 (s, 6H, 2CH3), 2.19 (s, 3H, aromatic-CH3), 1.11 (t, 6H,
J
MHz, DMSO-d6):
δ
166.9 (C-11 and C-12, C=O), 145.2 (C-1
and C-6 , C-Ar), 127.1 (C-3
′
, C-Ar), 145.0 (C-2 and C-6, C=C),
and C-5 , C-Ar), 101.9 (C-3 and C-
134.7 (C-4 , C-Ar), 128.3 (C-2
′
′
′
′
′
5, C=C), 58.9 (C-10 and C-13, OCH2), 38.3 (C-4, CH), 20.5 (Ar-CH3), 18.1 (C-7 and C-8, CH3),
14.1 (C-9 and C-14, OCH2CH3); EI-MS: m/z (rel. abund. %), 344 (M++1, 2), 343 (M+, 9), 252
(100), 224 (56), 196 (62), 150 (20), 91 (12); HREI-MS: m/z Calcd for C20H25NO4: 343.1784;
Found: 343.1785; Anal. Calcd for C20H25NO4 (343.42): C, 69.95; H, 7.34; N, 4.08; O, 18.64
;
Found: C, 69.98; H, 7.32; N, 4.03.
4.1.8. Diethyl 4-(4-ethoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (8)
º
º
Yield: 77%; M.p.: 104-106 C (Lit. M.p.25 104-106 C); Rf: 0.57 (
n
-hexane/ethyl acetate,
8:2); 1H-NMR: (300 MHz, DMSO-d6):
δ
8.73 (s, 1H, NH), 7.01 (d, 2H, J2′,3′ = J6′,5′ = 8.7 Hz, H-
2
΄/H-6΄), 6.72 (d, 2H, J3′,2′
=
J5′,6′ = 8.7 Hz, H-3′/H-5′), 4.77 (s, 1H, H-4), 4.01 (m, 6H, 3CH2),
2.22 (s, 6H, 2CH3), 1.26 (t, 3H,
J
= 6.0, CH3), 1.11 (t, 6H, J
= 7.2 Hz, 2CH3); 13C-NMR (100
13

ACCEPTED MANUSCRIPT
MHz, DMSO-d6):
δ
166.9 (C-11 and C-12, C=O), 156.6 (C-4
′
, C-Ar), 144.8 (C-2 and C-6, C=C),
140.3 (C-1 , C-Ar), 128.2 (C-2
′
′
and C-6 , C-Ar), 113.6 (C-3
′
′
and C-5 , C-Ar), 102.0 (C-3 and C-
′
5, C=C), 62.7 (Ar-OCH2), 58.8 (C-10 and C-13, OCH2), 37.8 (C-4, CH), 18.1 (C-7 and C-8,
CH3), 14.6 (Ar-OCH2CH3), 14.1 (C-9 and C-14, OCH2CH3); EI-MS: m/z (rel. abund. %), 374
(M++1, 7), 373 (M+, 27), 344 (60), 300 (64), 252 (100), 224 (62), 196 (67), 150 (22) ; HREI-MS:
m/z Calcd for C21H27NO5: 373.1889; Found: 373.1882; Anal. Calcd for C21H27NO5 (373.44): C,
67.54; H, 7.29; N, 3.75; O, 21.42; Found: C, 67.51; H, 7.32; N, 3.72.
4.1.9. Diethyl 2,6-dimethyl-4-(1H-pyrrol-2-yl)-1,4-dihydropyridine-3,5-dicarboxylate (9)
º
1
Yield: 88%; M.p.: 214-216 C; Rf: 0.50 (
DMSO-d6): 9.73 (s, 1H, NH), 8.74 (s, 1H, NH), 6.42 (d, 1H, J3′,4′ = 1.8 Hz
J4′,3′ = 2.4 Hz
2CH3), 1.15 (t, 6H,
n
-hexane/ethyl acetate, 8:2); H-NMR: (300 MHz,
δ
,
H-3 ), 5.76 (d, 1H,
′
,
H-4
′
), 5.52 (br. s, 1H, H-5
′), 4.87 (s, 1H, H-4), 4.05 (m, 4H, 2CH2), 2.21 (s, 6H,
J
= 7.2 Hz, 2CH3); 13C-NMR (125 MHz, DMSO-d6):
δ
167.1 (C-11 and C-
, C-Ar), 106.7 (C-4 , C-Ar),
, C-Ar), 100.4 (C-3 and C-5, C=C), 58.9 (C-10 and C-13, OCH2), 31.8 (C-4, CH),
12, C=O), 145.2 (C-2 and C-6, C=C), 137.1 (C-2
103.6 (C-3
′
, C-Ar), 115.9 (C-5
′
′
′
18.3 (C-7 and C-8, CH3), 14.2 (C-9 and C-14, OCH2CH3); EI-MS:m/z (rel. abund. %), 319
(M++1, 6), 318 (M+, 27 ), 252 (13), 245 (22), 206 (100), 195 (20), 178 (34), 150 (16); HREI-MS:
m/z Calcd for C17H22N2O4: 318.1580; Found: 318.1566; Anal. Calcd for C17H22N2O4 (318.37):
C, 64.13; H, 6.97; N, 8.80; O, 20.10; Found:C, 64.10; H, 6.99; N, 8.77.
4.1.10. Diethyl 4-(2-furyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (10)
º
º
Yield: 92%; M.p.: 160-162 C (Lit. M.p.24 159-161 C); Rf: 0.57 (
n
-hexane/ethyl acetate,
8:2); 1H-NMR: (300 MHz, DMSO-d6):
δ
8.89 (s, 1H, NH), 7.37 (s, 1H, H-3′), 6.23 (dd, 1H, J4′, 3′
14

ACCEPTED MANUSCRIPT
= 2.1 Hz, J4′, 5′ = 3.0 Hz, H-4
′
), 5.81 (d, 1H, J5′,4′ = 3.0 Hz, H-5′), 5.03 (s, 1H, H-4), 4.04 (m, 4H,
2CH2), 2.23 (s, 6H, 2CH3), 1.15 (t, 6H,
J
= 7.2 Hz, 2CH3); 13C-NMR (100 MHz, DMSO-d6):
δ
166.7 (C-11 and C-12, C=O), 158.7 (C-2
110.1 (C-4 , C-Ar), 103.9 (C-3 , C-Ar), 98.4 (C-3 and C-5, C=C), 59.0 (C-10 and C-13, OCH2),
ʹ, C-Ar), 146.3 (C-2 and C-6, C=C), 141.0 (C-5
′, C-Ar),
′
′
32.7 (C-4, CH), 18.1 (C-7 and C-8, CH3), 14.2 (C-9 and C-14, OCH2CH3); EI-MS: m/z (rel.
abund. %), 321 (M+ +2, 6), 319 (M++1, 37), 319 (M+, 73), 290 (72), 274 (69), 262 (68), 252 (68),
246 (100), 218 (70), 196 (66), 150 (50); HREI-MS: m/z Calcd for C17H21NO5: 319.1420; Found:
319.1394; Anal. Calcd for C17H21NO5(319.35): C, 63.94; H, 6.63; N, 4.39; O, 25.05; Found:C,
63.91; H, 6.61; N, 4.40.
4.1.11. Diethyl 4-(2-fluorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (11)
º
º
Yield: 75%; M.p.: 147-149 C (Lit. M.p.20 148-152 C); Rf: 0.60 (
n-hexane/ethyl acetate,
1
8:2); H-NMR: (300 MHz, DMSO-d6):
δ
8.79 (s, 1H, NH), 7.20 (td, 1H, J5′,3′ = 1.8 Hz, J5′,4′
=
J5′,6′ = 7.5 Hz, H-5
′), 7.11 (m, 1H, H-4
′), 7.03 (dd, 1H, J3′,5′ = 1.8 Hz, J3′,4′ = 7.2 Hz, H-3
′
), 6.96
(br. d, 1H, J6′,5′ = 8.4 Hz
,
H-6
′
), 3.94 (m, 4H, 2CH2), 2.22 (s, 6H, 2CH3), 1.07 (t, 6H,
J
= 7.2 Hz,
= 247.5 Hz,
= 3.0 Hz,
= 24.0 Hz, C-
2CH3); 13C-NMR (150 MHz, DMSO-d6):
δ
166.7 (C-11 and C-12, C=O), 159.4 (d,
= 15.0 Hz, C-1 , C-Ar), 130.6 (d,
, C-Ar), 124.0 (C-5 , C-Ar), 114.6 (d, C-3
J
C-2
′
′
, C-Ar), 145.7 (C-2 and C-6, C=C), 135.5 (d,
, C-Ar), 127.7 (d, = 7.5 Hz, C-4
J
′
J
C-6
J
′
′
′, J
Ar), 101.0 (C-3 and C-5, C=C), 58.9 (C-10 and C-13, OCH2), 33.1 (C-4, CH), 18.1 (C-7 and C-
8, CH3), 13.9 (C-9 and C-14, OCH2CH3); EI-MS: m/z (rel. abund. %), 347 (M+, 6), 274 (22), 252
(100), 224 (36), 196 (50), 150 (13); HREI-MS: m/z Calcd for C19H22NO4F: 347.1533 Found:
347.1513; Anal. Calcd for C19H22NO4F (347.38): C, 65.69; H, 6.38; F, 5.47; N, 4.03; O, 18.42;
Found:C, 65.72; H, 6.35; N, 4.01.
15

ACCEPTED MANUSCRIPT
4.1.12. Diethyl 2,6-dimethyl-4-(5-methyl-2-furyl)-1,4-dihydropyridine-3,5-dicarboxylate (12)
º
1
Yield: 93%; M.p.: 132-134 C; Rf: 0.58 (
n
-hexane/ethyl acetate, 8:2); H-NMR: (300 MHz,
DMSO-d6): 8.83 (s, 1H, NH), 5.81 (d, 1H, J3′,4′ = 2.7 Hz
δ
,
H-3 ), 5.64 (d, 1H, J4′,3′ = 2.7 Hz H-
′
,
4
′
), 4.95 (s, 1H, H-4), 4.06 (m, 4H, 2CH2), 2.22 (s, 6H, 2CH3), 2.11 (s, 3H, CH3), 1.16 (t, 6H,
J
=
7.2 Hz, 2CH3); 13C-NMR (100 MHz, DMSO-d6):
δ
166.7 (C-11 and C-12, C=O), 157.2 (C-2
′
, C-
Ar), 149.3 (C-5′, C-Ar), 146.1 (C-2 and C-6, C=C), 106.0 (C-3′, C-Ar), 104.6 (C-4′, C-Ar), 98.6
(C-3 and C-5, C=C), 58.9 (C-10 and C-13, OCH2), 32.7 (C-4, CH), 18.1 (C-7 and C-8, CH3),
14.2 (C-9 and C-14, OCH2CH3), 13.4 (Ar-CH3); EI-MS: m/z (rel. abund. %), 335 (M+ +2, 2), 334
(M++1, 10), 333 (M+, 54), 304 (39), 260 (100), 252 (11), 232 (14), 214 (14), 196 (7) ; HREI-MS:
m/z Calcd for C18H23NO5: 333.1576; Found: 333.1581; Anal. Calcd for C18H23NO5 (333.38): C,
64.85; H, 6.95; N, 4.20; O, 24.00; Found:C, 64.80; H, 6.92; N, 4.25.
4.1.13. Diethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (13)
Yield: 85%; M.p. 160-161 ºC (Lit. M.p.24 158-161 ºC); Rf: 0.56 (
1H-NMR: (300 MHz, DMSO-d6):
n
-hexane/ethyl acetate, 8:2);
J6′,5′ = 8.4 Hz, H-2 /H-
δ
8.73 (s, 1H, NH), 7.03 (d, 2H, J2′,3′
=
′
6
′
), 6.74 (d, J3′,2′
3H, -OCH3), 2.23 (s, 6H, 2CH3), 1.12 (t, 6H,
d6):
C-Ar), 128.2 (C-2
=
J5′,6′ = 8.4 Hz, 2H, H-3
′
/H-5
′
), 4.78 (s, 1H, H-4), 3.97 (m, 4H, 2CH2), 3.66 (s,
= 7.2 Hz, 2CH3); 13C-NMR (100 MHz, DMSO-
J
δ
166.9 (C-11 and C-12, C=O), 157.4 (C-4
′
, C-Ar), 144.9 (C-2 and C-6, C=C), 140.4 (C-1
′,
′
and C-6 , C-Ar), 113.1 (C-3
′
′
and C-5 , C-Ar), 102.1 (C-3 and C-5, C=C), 58.9
′
(C-10 and C-13, OCH2), 54.8 (Ar-OCH3), 37.9 (C-4, CH), 18.1 (C-7 and C-8, CH3), 14.1 (C-9
and C-14, OCH2CH3); EI-MS: m/z (rel. abund. %), 361 (M++2, 1.3), 360 (M++1, 6.5), 359 (M+,
35), 330 (46), 286 (82), 252 (100), 224 (45), 196 (51), 150 (13), 43 (9); HREI-MS: m/z Calcd for
16

ACCEPTED MANUSCRIPT
C20H25NO5: 359.1733; Found: 359.1755; Anal. Calcd for C20H25NO5 (359.42): C, 66.83; H, 7.01;
N, 3.90; O, 22.26; Found: C, 66.80; H, 6.98; N, 3.94.
4.1.14. Diethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (14)
º
º
Yield: 90%; M.p.: 163-164 C (Lit. M.p.24 162-165 C); Rf: 0.53 (
n-hexane/ethyl acetate,
8:2); 1H-NMR: (300 MHz, DMSO-d6):
δ
8.99 (s, 1H, NH), 7.98 (dd, 1H, J2′,4′ = 0.9 Hz, J2′,6′ = 2.1
Hz, H-2
′
), 7.55 (m, 3H, H-4
′
/5′
/6′
), 4.94 (s, 1H, H-4), 4.94 (s, 1H, H-4), 3.97 (m, 4H, 2CH2),
2.27 (s, 6H, 2CH3), 1.10 (t, 6H,
11 and C-12, C=O), 150.2 (C-3
J
= 7.2 Hz, 2CH3); 13C-NMR (75 MHz, DMSO-d6):
δ 166.4 (C-
′
, C-Ar), 147.3 (C-1
′
, C-Ar), 146.3 (C-2 and C-6, C=C), 134.1
, C-Ar), 101.0 (C-3 and C-5,
(C-2
′
, C-Ar), 129.5 (C-4
′
, C-Ar), 121.8 (C-6
′
, C-Ar), 121.0 (C-5′
C=C), 59.1 (C-10 and C-13, OCH2), 39.2 (C-4, CH), 18.1 (C-7 and C-8, CH3), 14.0 (C-9 and C-
14, OCH2CH3); EI-MS: m/z (rel. abund. %), 374 (M+, 4), 252 (100), 224 (21), 196 (33), 176
(14), 150 (10), 43 (25); HREI-MS: m/z Calcd for C19H22N2O6: 374.1478; Found: 374.1483; Anal.
Calcd for C19H22N2O6 (374.15): C, 60.95; H, 5.92; N, 7.48; O, 25.64; Found: C, 60.92; H, 5.96;
N, 7.43.
4.1.15. Diethyl 4-butyl-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (15)
Yield: 85%; M.p.: 94-95 ºC (Lit. M.p.24 92-94 ºC); Rf: 0.75 (
n
-hexane/ethyl acetate, 8:2); 1H-
NMR: (300 MHz, DMSO- d6): 8.61 (s, 1H, NH), 4.02 (m, 4H, 2CH2), 3.75 (t, 1H,
δ
J
= 5.7, H-
4), 2.18 (s, 6H, 2CH3), 1.18 (t, 6H,
J = 7.2 Hz, 2CH3), 1.12 (m, 6H, aliphatic-3CH2), 0.78 (t, 3H,
J
= 7.2 Hz, aliphatic-CH3); 13C-NMR (100 MHz, DMSO-d6):
δ 167.2 (C-11 and C-12, C=O),
145.8 (C-2 and C-6, C=C), 100.9 (C-3 and C-5, C=C), 58.7 (C-10 and C-13, OCH2), 36.4 (-
CH2CH2CH2CH3), 31.9 (C-4, CH), 26.5 (-CH2CH2CH2CH3), 22.2 (-CH2CH2CH2CH3), 18.1 (C-7
and C-8, CH3), 14.3 (C-9 and C-14, OCH2CH3), 14.0 (-CH2CH2CH2CH3); EI-MS:m/z (rel.
17

ACCEPTED MANUSCRIPT
abund. %), 309 (M+), 264 (33), 252 (100), 224 (40), 196 (49), 179 (12), 150 (8); HREI-MS: m/z
Calcd for C17H27NO4: 309.1940; Found: 309.1943; Anal. Calcd for C17H27NO4 (309.40): C,
65.99; H, 8.80; N, 4.53; O, 20.68; Found:C, 66.01; H, 8.78; N, 4.56.
4.1.16. Diethyl 4-(3,4-dimethoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
(16)
Yield: 84%; M.p.: 145-147 ºC (Lit. M.p.22 147 ºC); Rf: 0.32 (
NMR: (300 MHz, DMSO-d6): 8.75 (s, 1H, NH), 6.77 (d, 1H, J5′,6′ = 8.4 Hz, H-5
J2′,6′ = 1.5 Hz, H-2 ′), 4.77 (s, 1H, H-4), 3.99
n
-hexane/ethyl acetate, 8:2); 1H-
δ
′
), 6.72 (d, 1H,
′
), 6.61 (dd, 1H, J6′,5′ = 8.4 Hz, J6′,2′ = 1.5 Hz, H-6
= 7.2 Hz, 2CH3); 13C-NMR
(m, 4H, 2CH2), 3.66 (s, 6H, 2CH3), 2.23 (s, 6H, 2CH3), 1.13 (t, 6H,
J
(100 MHz, DMSO-d6):
δ
167.0 (C-11 and C-12, C=O), 147.8 (C-4
′
, C-Ar), 147.0 (C-3
′
, C-Ar),
145.0 (C-2 and C-6, C=C), 140.9 (C-1
′
, C-Ar), 119.1 (C-6
′
, C-Ar), 111.7 (C-5
′
, C-Ar), 111.4 (C-
2′, C-Ar), 101.9 (C-3 and C-5, C=C), 58.9 (C-10 and C-13, OCH2), 55.4 (Ar-OCH3), 55.2 (Ar-
OCH3), 38.2 (C-4, CH), 18.1 (C-7 and C-8, CH3), 14.2 (C-9 and C-14, OCH2CH3); EI-MS: m/z
(rel. abund. %), 390 (M++1, 7.8), 389 (M+, 30), 360 (42), 316 (56), 252 (100), 224 (57), 196
(86), 150 (18), 138 (14), 77 (12); HREI-MS: m/z Calcd for C21H27NO6: 389.1838; Found:
389.1840; Anal. Calcd for C21H27NO6 (389.44): C, 64.77; H, 6.99; N, 3.60; O, 24.65; Found: C,
64.73; H, 6.95; N, 3.58.
4.1.17. Diethyl 4-(3-hydroxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (17)
º
º
Yield: 78%; M.p.: 181-183 C (Lit. M.p.16 180-182 C); Rf: 0.27 (
n-hexane/ethyl acetate,
8:2); 1H-NMR: (300 MHz, DMSO-d6):
δ
9.08 (s, 1H, OH), 8.73 (s, 1H, NH), 6.95 (t, 1H, J5′/4′,6′ =
7.8 Hz, H-5′), 6.57 (br. d, 2H, J4′,6′/5′ = 7.2 Hz, H-4′/H-6′), 6.47 (dd, 1H, J2′,4′ = 1.0, J2′,6′ = 1.8 Hz,
H-2 ), 4.79 (s, 1H, H-4), 3.99 (m, 4H, 2CH2), 2.23 (s, 6H, 2CH3), 1.13 (t, 6H,
′
J
= 7.2 Hz, 2CH3);
18

ACCEPTED MANUSCRIPT
13C-NMR (100 MHz, DMSO-d6):
, C-Ar), 145.0 (C-2 and C-6, C=C), 128.5 (C-5
112.7 (C-2 , C-Ar), 101.7 (C-3 and C-5, C=C), 58.9 (C-10 and C-13, OCH2), 38.5 (C-4, CH),
δ
167.0 (C-11 and C-12, C=O), 156.9 (C-3
′
, C-Ar), 149.3 (C-
1
′
′
, C-Ar), 117.9 (C-6 , C-Ar), 114.3 (C-4
′
′
, C-Ar),
′
18.2 (C-7 and C-8, CH3), 14.1 (C-9 and C-14, OCH2CH3); EI-MS: m/z (rel. abund. %), 346
(M++1, 4.7), 345 (M+, 20), 272 (26), 252 (100), 224 (49), 196 (68), 150 (10); HREI-MS: m/z
Calcd for C19H23NO5: 345.1576; Found: 345.1582; Anal. Calcd for C19H23NO5 (345.39): C,
66.07; H, 6.71; N, 4.06; O, 23.16; Found: C, 66.04; H, 6.69; N, 4.09.
4.1.18. 2-[3,5-Bis(ethoxycarbonyl)-2,6-dimethyl-1,4-dihydro-4-pyridinyl]benzoic acid (18)
º
1
Yield: 90%; M.p.: 208-209 C; Rf: 0.21 (
DMSO-d6): 12.50 (s, 1H, COOH), 8.72 (s, 1H, NH), 7.55 (d, 1H, J3′,4′ = 7.5 Hz, H-3
2H, H-4 /H-6 = J5′,6′ = 7.8 Hz, H-5′), 5.72 (s, 1H, H-4), 4.0 (m,
n
-hexane/ethyl acetate, 8:2); H-NMR: (300 MHz,
δ
′
), 7.37 (m,
′
′
), 7.15 (td, 1H, J5′,3′ = 1.8 Hz, J5′,4′
= 7.2 Hz, 2CH3); 13C-NMR (100 MHz, DMSO-d6):
4H, 2CH2), 2.19 (s, 6H, 2CH3), 1.04 (t, 6H,
J
δ
168.7 (-COOH), 167.3 (C-11 and C-12, C=O), 148.9 (C-2
′
, C-Ar), 145.0 (C-2 and C-6, C=C),
, C-Ar), 125.6 (C-3 , C-
131.5 (C-4 , C-Ar), 130.3 (C-1 , C-Ar), 129.6 (C-6 , C-Ar), 128.8 (C-5
′
′
′
′
′
Ar), 103.0 (C-3 and C-5, C=C), 58.9 (C-10 and C-13, OCH2), 34.9 (C-4, CH), 18.2 (C-7 and C-
8, CH3), 14.0 (C-9 and C-14, OCH2CH3); EI-MS: m/z (rel. abund. %), 373 (M+), 344 (69), 300
(99), 252 (50), 224 (14), 196 (43), 105 (100), 77 (51); HREI-MS: m/z Calcd for C20H23NO6:
373.1525; Found: 373.1532; Anal. Calcd for C20H23NO6 (373.40): C, 64.33; H, 6.21; N, 3.75; O,
25.71; Found: C, 64.28; H, 6.25; N, 3.72.
4.1.19. Diethyl 2,4,6-trimethyl-1,4-dihydropyridine-3,5-dicarboxylate (19)
Yield: 92%; M.p.: 130-131 ºC (Lit. M.p.22 130 ºC); Rf: 0.64 ( -hexane/ethyl acetate, 8:2); 1H-
n
NMR: (300 MHz, DMSO-d6):
δ
8.62 (s, 1H, NH), 4.04 (m, 4H, 2CH2), 3.65 (q, 1H, H-4), 2.17
19

ACCEPTED MANUSCRIPT
(s, 6H, 2CH3), 1.18 (t, 6H,
J = 7.2 Hz, 2CH3), 0.81 (d, 3H, J
= 6.6 Hz, CH3); 13C-NMR (75 MHz,
DMSO-d6): 166.9 (C-11 and C-12, C=O), 145.6 (C-2 and C-6, C=C), 102.3 (C-3 and C-5,
δ
C=C), 58.8 (C-10 and C-13, OCH2), 27.8 (C-4, CH), 22.5 (Aliphatic-CH3), 18.1 (C-7 and C-8,
CH3), 14.3 (C-9 and C-14, OCH2CH3); EI-MS: m/z (rel. abund. %), 267 (M+, 2.7), 252 (100),
224 (31), 83 (16); HREI-MS: m/z Calcd for C14H21NO4: 267.1471; Found: 267.1458; Anal.
Calcd for C14H21NO4 (267.32): C, 62.90; H, 7.92; N, 5.24; O, 23.94; Found:C, 62.89; H, 7.94; N,
5.27.
4.1.20. Diethyl 2,6-dimethyl-4-propyl-1,4-dihydropyridine-3,5-dicarboxylate (20)
Yield: 75%; M.p.: 122-123 ºC (Lit. M.p.19 122 ºC); Rf: 0.67 (
n
-hexane/ethyl acetate, 8:2); 1H-
NMR: (300 MHz, DMSO-d6): 8.61 (s, 1H, NH), 4.04 (m, 4H, 2CH2), 3.75 (t, 1H,
δ
J
= 5.1 Hz,
H-4), 2.18 (s, 6H, 2CH3), 1.18 (t, 6H,
J = 7.2 Hz, 2CH3), 1.12 (m, 4H, aliphatic-2CH2), 0.77 (t,
3H,
J
= 6.6, CH3); 13C-NMR (100 MHz, DMSO-d6):
δ 167.2 (C-11 and C-12, C=O), 145.9 (C-2
and C-6, C=C), 100.9 (C-3 and C-5, C=C), 58.8 (C-10 and C-13, OCH2), 39.1 (-CH2CH2CH3),
31.8 (C-4, CH), 18.1 (C-7 and C-8, CH3), 17.4 (-CH2CH2CH3), 14.3 (C-9 and C-14, OCH2CH3),
14.1 (-CH2CH2CH3); EI-MS: m/z (rel. abund. %), 295 (M+), 252 (100), 224 (37), 206 (58), 196
(48), 178 (24), 150 (69); HREI-MS: m/z Calcd for C16H25NO4: 295.1784 Found: 295.1764; Anal.
Calcd for C16H25NO4 (295.37): C, 65.06; H, 8.53; N, 4.74; O, 21.67; Found: C, 65.02; H, 8.48;
N, 4.77.
4.1.21. Diethyl 4-isopropyl-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (21)
Yield: 75%; M.p.: 94-96 ºC (Lit. M.p.21 95-98 ºC); Rf: 0.72 (
NMR: (300 MHz, DMSO- d6): 8.62 (s, 1H, NH), 4.02 (m, 4H, 2CH2), 3.73 (d, 1H,
H-4), 2.0 (s, 6H, 2CH3), 1.42 (m, 1H, CH), 1.18 (t, 6H, = 7.2 Hz, 2CH3), 0.64 (d, 6H,
n
-hexane/ethyl acetate, 8:2); 1H-
δ
J
= 5.1 Hz,
J
J
= 6.9
20

ACCEPTED MANUSCRIPT
Hz, 2CH3); 13C-NMR (100 MHz, DMSO-d6):
δ
167.8 (C-11 and C-12, C=O), 145.6 (C-2 and C-
6, C=C), 99.4 (C-3 and C-5, C=C), 58.7 (C-10 and C-13, OCH2), 38.0 (C-4, CH), 35.0 (-
CH(CH3)2), 18.2 (-CH(CH3)2), 18.1 (C-7 and C-8, CH3), 14.3 (C-9 and C-14, OCH2CH3); EI-
MS: m/z (rel. abund. %), 295 (M+), 252 (100), 224 (68), 196 (94), 179 (23), 150 (20); HREI-MS:
m/z Calcd for C16H25NO4: 295.1784; Found: 295.1778; Anal. Calcd for C16H25NO4 (295.37): C,
65.06; H, 8.53; N, 4.74; O, 21.67; Found: C, 65.03; H, 8.51; N, 4.78.
4.1.22. Diethyl 4-isobutyl-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (22)
º
1
Yield: 72%; M.p.: 82-84 C; Rf: 0.75 (
n
-hexane/ethyl acetate, 8:2); H-NMR: (300 MHz,
= 6.6 Hz, H-4), 2.18 (s, 6H,
= 6.6 Hz, CH2), 0.80 (d,
167.2 (C-11 and C-12, C=O), 145.7
DMSO-d6): 8.70 (s, 1H, NH), 4.02 (m, 4H, 2CH2), 3.78 (t, 1H,
δ
J
2CH3), 1.39 (m, 1H, CH), 1.18 (t, 6H,
J = 7.2 Hz, 2CH3), 0.97 (t, 2H, J
6H,
J
= 6.6 Hz, 2CH3); 13C-NMR (100 MHz, DMSO-d6):
δ
(C-2 and C-6, C=C), 101.8 (C-3 and C-5, C=C), 58.8 (C-10 and C-13, OCH2), 47.1 (-
CH2CH(CH3)2), 30.0 (C-4, CH), 23.1 (-CH2CH(CH3)2), 22.9 (-CH2CH(CH3)2), 18.1 (C-7 and C-
8, CH3), 14.2 (C-9 and C-14, OCH2CH3); EI-MS: m/z (rel. abund. %), 309 (M+), 262 (35), 252
(84), 206 (100), 195 (24), 178 (51), 150 (22), 106 (16); HREI-MS: m/z Calcd for C17H27NO4:
309.1940; Found: 309.1923; Anal. Calcd for C17H27NO4 (309.40): C, 65.99; H, 8.80; N, 4.53; O,
20.68; Found: C, 65.95; H, 8.77; N, 4.51.
4.1.23. Diethyl 4-(3,4-dihydroxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (23)
Yield: 78%; M.p.:149-150 ºC (Lit. M.p.24 147-151 ºC); Rf: 0.21 (
1H-NMR: (300 MHz, DMSO-d6):
8.66 (s, 1H, 3′-OH), 8.58 (s, 1H, 4′
n
-hexane/ethyl acetate, 8:2);
-OH), 8.49 (s, 1H, NH),
), 6.39 (dd, 1H, J6′, 2′ = 2.0 Hz,
), 4.68 (s, 1H, H-4), 3.97 (m, 4H, 2CH2), 2.21 (s, 6H, 2CH3), 1.13 (t, 6H, J =
δ
6.54 (d, 1H, J2′,6′ = 2.0 Hz, H-2
′), 6.50 (d, 1H, J5′,6′ = 8.0 Hz, H-5′
J6′, 5′ = 8.0 Hz, H-6
′
21

ACCEPTED MANUSCRIPT
7.0 Hz, 2CH3); 13C-NMR (75 MHz, DMSO-d6):
δ
167.1 (C-11 and C-12, C=O), 144.5 (C-2 and
C-6, C=C), 144.4 (C-4
′
, C-Ar), 143.3 (C-3
′
, C-Ar), 139.3 (C-1′, C-Ar), 118.0 (C-6′, C-Ar), 114.9
(C-5 , C-Ar), 114.8 (C-2′, C-Ar), 102.2 (C-3 and C-5, C=C), 58.8 (C-10 and C-13, OCH2), 37.8
′
(C-4, CH), 18.1 (C-7 and C-8, CH3), 14.2 (C-9 and C-14, OCH2CH3); EI-MS: m/z (rel. abund.
%), 362 (M++1, 2.2), 361 (M+, 10.7), 332 (15), 288 (27), 252 (100), 224 (19), 196 (32), 150 (7);
HREI-MS: m/z Calcd for C19H23NO6: 361.1525; Found: 361.1520; Anal. Calcd for C19H23NO6
(361.39): C, 63.15; H, 6.41; N, 3.88; O, 26.56; Found: C, 63.12; H, 6.39; N, 3.83.
4.1.24. Diethyl 2,6-dimethyl-4-phenethyl-1,4-dihydropyridine-3,5-dicarboxylate (24)
º
1
Yield: 84%; M.p.: 104-105 C; Rf: 0.70 (
DMSO-d6): 8.71 (s, 1H, NH), 7.20 (t, 2H, J3′/2′,4′ = J5′/4′,6′ = 7.5 Hz, H-3
7.5 Hz, H-4 ), 7.06 (br. d, 2H, J2′,3′ J6′,5′ = 7.5 Hz, H-2 /H-6 ), 4.06 (m, 4H, 2CH2), 3.87 (t, 1H,
n
-hexane/ethyl acetate, 8:2); H-NMR: (300 MHz,
δ
′/5′), 7.10 (t, 1H, J4′/3′,5′ =
′
=
′
′
J
= 6.0 Hz, H-4), 2.40 (m, 2H, CH2), 2.20 (s, 6H, 2CH3), 1.48 (m, 2H, CH2), 1.17 (t, 6H,
Hz, 2CH3); 13C-NMR (100 MHz, DMSO-d6):
J
= 7.5
δ
167.1 (C-11 and C-12, C=O), 146.3 (C-2 and C-
6, C=C), 142.5 (C-1
, C-Ar), 100.5 (C-3 and C-5, C=C), 58.8 (C-10 and C-13, OCH2), 38.2 (-CH2CH2Ph), 32.4 (C-
′, C-Ar), 128.1 (C-3′ and C-5′, C-Ar), 127.9 (C-2′ and C-6′, C-Ar), 125.3 (C-
4
′
4, CH), 30.5 (-CH2CH2Ph), 18.2 (C-7 and C-8, CH3), 14.3 (C-9 and C-14, OCH2CH3); EI-MS:
m/z (rel. abund. %), 357 (M+), 312 (16), 252 (100), 224 (39), 196 (55), 150 (11), 105 (12),
91(13); HREI-MS: m/z Calcd for C21H27NO4:357.1940; Found: 357.1953; Anal. Calcd for
C21H27NO4 (357.44): C, 70.56; H, 7.61; N, 3.92; O, 17.90; Found: C, 70.61; H, 7.59; N, 3.88.
4.1.25. Diethyl 2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (25)
Yield: 95%; M.p.: 131-133 ºC (Lit. M.p.24 128-132 ºC); Rf: 0.51 (
n
-hexane/ethyl acetate, 8:2); 1H-
J5′,6′ = 8.5 Hz, H-3 /H-5 ),
22
NMR: (300 MHz, DMSO-d6): 8.96 (s, 1H, NH), 8.09 (d, 2H, J3′,2′
δ
=
′
′

ACCEPTED MANUSCRIPT
7.39 (d, 2H, J2′,3′
=
J6′,5′ =8.5 Hz, 2H, H-2
′
/H-6′), 4.95 (s, 1H, H-4), 3.97 (m, 4H, 2CH2), 2.26 (s,
6H, 2CH3), 1.11 (t, 6H,
C-12, C=O), 155.4 (C-4
J
= 7.5 Hz, 2CH3); 13C-NMR (100 MHz, DMSO-d6):
δ
166.5 (C-11 and
, C-Ar), 128.5 (C-3 and
′, C-Ar), 100.8 (C-3 and C-5, C=C), 59.2 (C-10 and C-13,
′
, C-Ar), 146.2 (C-2 and C-6, C=C), 145.8 (C-1
′
′
C-5
′
, C-Ar), 123.2 (C-2
′
and C-6
OCH2), 39.4 (C-4, CH), 18.2 (C-7 and C-8, CH3), 14.1 (C-9 and C-14, OCH2CH3); EI-MS: m/z
(rel. abund. %), 375 (M++1, 4.6), 374 (M+, 20), 345 (42), 329 (42), 301 (43), 252 (100), 224
(52), 196 (78), 150 (14); HREI-MS: m/z Calcd for C19H22N2O6:374.1478; Found: 374.1497;
Anal. Calcd for C19H22N2O6 (374.39): C, 60.95; H, 5.92; N, 7.48; O, 25.64; Found: C, 60.91; H,
5.89; N, 7.51.
4.2. In vitro α-glucosidase inhibition assay
α-Glucosidase (EC 3.2.1.20, Saccharomyces cerevisiae, Sigma catalog # G0660) inhibitory
°
activity was assayed by using 0.1 M phosphate buffer (pH 6.8) at 37 C [35]. The enzyme (0.2
U/mL) in phosphate buffer saline was incubated with various concentrations of test compounds
°
at 37 C for 15 min, test compounds were dissolved in DMSO (final concentration 7%). The
substrate and p-nitrophenyl α-D-glucopyranoside (0.7 mM, final) were added and change in
absorbance at 400 nm was monitored up to 30 min. Test compound was replaced by DMSO (7%
final) as control. Acarbose was used as the standard inhibitor. The percent inhibition was
calculated by using the following formula:
% Inhibition = 100−(OD test well/OD control) × 100
23

ACCEPTED MANUSCRIPT
4.3. Cytotoxicity evaluation of compounds on rat fibroblast 3T3 cell lines
The experiment was performed according to the method described by Dimas et al. [34]. Rat
fibroblast 3T3 cells were used in this assay. Briefly the 3T3-adherent cells (2×105 cells/mL)
°
were cultured in a 96-well plate overnight in a CO2 environment at 37 C. Supernatant was
removed and 50
medium (DMEM supplemented with 5% (v/v) fetal bovine serum, penicillin (100 units/mL) and
streptomycin (100 g/mL) were added to each well. After the incubation, the culture medium
was aspirated carefully and 50 L of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium
bromide (MTT) solution (2 mg/mL in PBS) was added to each well and further incubated for 4 h.
After this, MTT solution was aspirated and cells were PBS-washed once, and 100 L of DMSO
ꢀL of serially diluted compounds (100 - 12.5 ꢀg/mL) and 150 ꢀL complete
ꢀ
ꢀ
ꢀ
was added to dissolve the blue insoluble MTT formazan produced by the action of mitochondrial
dehydrogenase. The plate was agitated at room temperature for 15 minutes and then read at 540
nm by using microplate readers (SpectraMax Plus-384). The percentage of viable cells was
calculated as the relative ratio of optical densities.
4.4. In vitro carbonic anhydrase-II inhibition assay
In this assay, 4-nitrophenyl acetate (4-NPA), which is colorless, is hydrolyzed to 4-
nitrophenol and CO2 and reaction is followed by measuring the formation of 4-nitrophenol, a
yellow colored compound. The experiment was performed in buffer containing HEPES and Tris
at a total concentration of 20 mM and pH was 7.4. For each sample the reaction tube contained
140
µ
L of the HEPES-Tris solution, 20
µ
L of freshly prepared aqueous solution of purified
L of test compound
bovine erythrocyte CA-II (0.1 mg/mL of deionized water for 96-well), 20
µ
dissolve in DMSO (10% final concentration), 20
µ
L of substrate 4-PNA at concentration of 0.7
24

ACCEPTED MANUSCRIPT
mM diluted in ethanol. The reaction was initiated by addition of 4-PNA after 15 min. incubation
of test compound. The compounds were tested for 3-times at different concentration. In this
assay the reaction was performed in 96-well plate. SPECTRA max 340 spectrophotometer,
Molecular devices (USA) was used to monitor the reaction and the amount of product formed
was monitored at 1 min interval for 30min at 400 nm [36].
4.5. In vitro phosphodiesterase-I inhibition assay
Activity against snake venom was assayed by taking 33 mM Tris–HC1 buffer pH 8.8, 30
mM Mg-acetate with 0.000742 U/well final concentration of enzyme using a microtiter plate
assay and 0.33 mM bis-(p-nitropheny1) phosphate (Sigma N-3002) as substrate. From Merck
Cystein and EDTA were used as positive controls (1C50 = 748
respectively).
µM ± 0.015, 274 µM ± 0.007,
After 30 min pre-incubation of the enzyme with the test samples, enzyme activity was monitored
spectrophotometrically at 37 °C on a microtitre plate reader (SpectraMax, Molecular Devices) by
following the rate (change in O.D/min) of release of p-nitrophenol from p-nitrophenyl phosphate
at 410 nm. All assays were conducted in triplicate [37].
4.6. In vitro β-Glucuronidase inhibition assay
β
-Glucuronidase activity was determined by means of the spectrophotometer by measuring
the absorbance at 405 nm of -nitrophenol produced from the substrate. The entire reaction
volume was 250
p
µ
L. The reaction mixture contained 185
µL of 0.1 M acetate buffer, 5 µL of test
compound solution, 10
µ
L of (1U) enzyme solution was incubated at 37 ºC for 30 min. The test
compound was dissolve in DMSO, from which 5
µ
L was added to per well (, which become 2%
25

ACCEPTED MANUSCRIPT
in the ultimate assay and the similar conditions were used for standard (D-saccharic acid 1,4-
lactone). The plates were read on a multiplate reader (SpectraMax plus 384) after the addition of
50 µL of 0.4 mM p-nitrophenyl-β-D-glucuronide. All assays were run in triplicate [38].
4.7. Statistical analysis
The EZ-Fit Enzyme Kinetics program (Perrella Scientific Inc., Amherst, USA) were
employed to calculate the IC50 values. All graphs were plotted by using GraFit program (1999).
Values of the correlation coefficients, intercepts, slopes, and their standard errors were calculated
by the linear regression analysis by using the same program. Each point in the constructed graphs
represents the mean of the three experiments [39].
Acknowledgements: This work was economically sustained by the Higher Education
Commission (HEC), Pakistan, Project No. 20-1910 under the National Research Program for
Universities. The authors are grateful to the King Abdulaziz University, Jeddah, for supporting
the study visit of one of the authors (Hamdy Kashtoh) to the International Center for Chemical
and Biological Sciences, University of Karachi, Pakistan.
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