Synthesis, study of 3D structures, and pharmacological activities of lipophilic nitroimidazolyl-1,4-dihydropyridines as calcium channel antagonist

Article abstract of DOI:10.1016/j.bmc.2006.03.016

QSAR studies indicated that the potency of nifedipine analogues was dependent upon lipophilicity, an electronic term and separated terms for each position on the DHP ring. Changes in the substitution pattern at the C₃, C₄, and C₅ positions of DHPs alter potency, tissue selectivity, and the conformation of the 1,4-DHP ring. In this project a group of alkyl ester analogues of new derivatives of nifedipine, in which the ortho-nitrophenyl group at position 4 is replaced by a 1-methyl-5-nitro-2-imidazolyl substituent, and the methyl group at position 6 is replaced by a phenyl substituent, were synthesized and evaluated as calcium channel antagonist using the high K⁺ contraction of guinea-pig ileal longitudinal smooth muscle. The results for asymmetrical esters showed that lengthening of the substituent in C₃ ester substituent increased activity. When increasing of the length is accompanied by increasing the hindrance, the activity decreased. The results demonstrate that all compounds were more active or similar in effect to that of the reference drug nifedipine.

Full text of DOI:10.1016/j.bmc.2006.03.016

Bioorganic & Medicinal Chemistry 14 (2006) 4842–4849
Synthesis, study of 3D structures, and pharmacological activities
of lipophilic nitroimidazolyl-1,4-dihydropyridines
as calcium channel antagonist
a,b
Ramin Miri,a,b, Katayoun Javidnia, Hasti Sarkarzadehb
*
and Bahram Hemmateenejada,c,
*
aMedicinal & Natural Products Chemistry Research Centre, Shiraz University of Medical Sciences, Shiraz, Iran
bDepartment of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Science, PO Box 71345-1149, Shiraz, Iran
cChemistry Department, Shiraz University, Shiraz 71454, Iran
Received 26 January 2006; revised 9 March 2006; accepted 13 March 2006
Available online 5 April 2006
Abstract—QSAR studies indicated that the potency of nifedipine analogues was dependent upon lipophilicity, an electronic term
and separated terms for each position on the DHP ring. Changes in the substitution pattern at the C3, C4, and C5 positions of DHPs
alter potency, tissue selectivity, and the conformation of the 1,4-DHP ring. In this project a group of alkyl ester analogues of new
derivatives of nifedipine, in which the ortho-nitrophenyl group at position 4 is replaced by a 1-methyl-5-nitro-2-imidazolyl substi-
tuent, and the methyl group at position 6 is replaced by a phenyl substituent, were synthesized and evaluated as calcium channel
antagonist using the high K+ contraction of guinea-pig ileal longitudinal smooth muscle. The results for asymmetrical esters showed
that lengthening of the substituent in C3 ester substituent increased activity. When increasing of the length is accompanied by
increasing the hindrance, the activity decreased. The results demonstrate that all compounds were more active or similar in effect
to that of the reference drug nifedipine.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
efficacy. It is also consistent with the recommendation
in the Sixth Report of the Joint National Committee
on Prevention, Evaluation, and Treatment of High
Blood Pressure that puts b-blockers and diuretics as
first-line agents for the treatment of uncomplicated
hypertension, with DHPs and ACE inhibitors second.8
Despite a rocky stretch, CCBs retain an important role
in hypertension therapy and their improvement remains
an important endeavor. Consequently, the search for
finding analogues with novel binding properties has
attracted renewed interest. Understanding the structure
and function of membrane bound ion channels remains
a challenge to chemists, and the design of molecules as
ligands to selectively modulate ion channels holds
significant therapeutic promise.
The discovery that the 4-aryl-1,4-dihydropyridine
(DHP) (Nifedipine, Nimodipine) class of calcium chan-
nel blockers (CCBs) inhibits Ca2+ influx represented a
major therapeutic advance in treatment of cardiovascu-
lar diseases such as hypertension, vasospastic angina,
and other spastic smooth muscle disorders.1–3 Recently,
controversy concerning side effects has emerged. The
first was an increased incidence of myocardial infarction
among patients on a regimen of ‘nifedipine’4,5 which led
the National Institute of Health to issue a warning to the
use of time release formulations. The second was a ret-
rospective correlation of increased incidence of cancer
among older patients taking calcium channel block-
ers.6,7 The current evidence on CCBs largely precludes
the adverse effects of the magnitude suggested by the
scare in the mid-1990 but attests to their safety and
QSAR studies indicated that the potency of nifedipine
analogues was dependent upon lipophilicity, an elec-
tronic term and separated terms for each position on
the aromatic ring.9 Changes in the substitution pattern
at the C3, C4, and C5 positions of DHPs alter potency,10
tissue selectivity,11 and the conformation of the
1,4-DHP ring.12 Natale et al.13 found that lipophilic
Keywords: Lipophilic-DHP; 1,4-Dihydropyridine; Calcium channel
antagonist; 5-Nitroimidazole, Three-dimensional structure.
*
Corresponding authors. Tel.: +98 711 230 3872; fax: +98 711
2307594; e-mail addresses: mirir@sums.ac.ir; hemmatb@sums.ac.ir
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.03.016

R. Miri et al. / Bioorg. Med. Chem. 14 (2006) 4842–4849
4843
4-isoxazolyl-1,4-dihydropyridines that have a aryl group
2. Chemistry
on isoxazole ring exhibit high binding affinity. They pro-
posed a model for DHP binding and found a highly
lipophilic pocket in the receptor’s active site. Examina-
tion of their model indicated that aryl substituent could
be properly oriented to interact with the lipophilic pock-
et of their hypothesized channel. Natale also employed
Striessnig’s model suggesting that potassium channel is
a usable working mimic of the L-type calcium channel
and observed that lipophilic substituent on isoxazolyl
ring would easily be accommodated within the lipophilic
channel as oriented and could be formed as a result of a
p–p interaction between Tyr-1463 and the aryl moiety.14
Other studies suggested that C4 heterocycle substituents
gave active compounds as calcium channel antago-
nists.15–17 Parallel to this finding, we were able to prove
that the bioisosteric replacement of the 4-aryl moiety
with a 4-imidazolyl group yields 4-imidazolyl-1,4-dihy-
dropyridines which retain potent calcium antagonist
activity.18–23
The synthesis of the 1,4-dihydropyridine derivatives 8a–
f was achieved following the steps outlined in Figure 1.
Reaction of alcohol 1 with 2,2,6-trimethyl-4H-1,3-diox-
in-4-one 2 afforded the corresponding acetoacetic esters
3c–f (84–91% yield).23 Reaction of acetoacetic esters
3c–f with ammonium acetate afforded the corresponding
alkyl 3-aminocrotonate 4a–f. Also, reaction of 1-methyl-
5-nitroimidazol-2-carboxaldehyde 5 with ethylbenzoy-
lacetate 6 afforded the corresponding ethyl-2-benzoyl-
3-(1-methyl-5-nitroimidazolyl)-2-propionate
7
(52%
yield).24,25 The asymmetrical analogues 8a–f were
synthesized by a modified Hantzsch reaction, using a
procedure reported by Meyer et al. (8–52% yield).26–29
The symmetrical analogue 9 was prepared (5% yield)
by the classical Hantzsch condensation.30 The yield
and melting point of final compounds are summarized
in Table 1.
It was therefore of interest to determine the Ca2+ chan-
nel antagonist activity of the hitherto unknown 1,4-
dihydropyridine compounds that have 1-methyl-5-ni-
tro-2-imidazolyl and a phenyl substituents at position
4 and 6 of DHP ring, respectively.
3. Pharmacology
The calcium channel antagonist activity of compounds
was determined as the concentration needed to produce
50% inhibition of the guinea-pig ileal longitudinal
O
O
O
R1O2C
O
+
-
R1OH
1c-f
+
R1OCCH2CCH3
CH3CO2 NH4
+
O
H3C
4a-f
NH2
3a-f
2
NO2
NO2
N
N
CH3
N
O
O
CH3
CO2CH2CH3
CH3CH2OCCH2CC6H5
+
N
COH
CHO
O
7
6
5
NO2
NO2
N
CH3
CO2CH2CH3
N
N
CH3
N
R1O2C
R1O2C
CO2CH2CH3
+
H3C
NH2
H3C
N
H
O
8a-f
4a-f
7
NO2
N
CH3
N
CH3CH2O2C
CO2CH2CH3
+
NH3
6
5
+
N
H
9
Figure 1. Synthetic pathway for the DHP derivatives used in this study.

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R. Miri et al. / Bioorg. Med. Chem. 14 (2006) 4842–4849
Table 1. Physical and calcium channel modulation data for compounds 8a–f and 9
NO2
CH3
N
N
R1O2C
CO2CH2CH3
N
H
R2
Compound
R1
R2
Mp (ꢁC)
Yield (%)
Calcium channel antagonist activity IC50 SEMa (n = 5)
8a
8b
8c
8d
8e
8f
9
Methyl
Ethyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Phenyl
200–202
167–169
186–188
157–159
168–171
168–169
183–185
8
10
21
29
20
52
5
8.24 0.41 · 10ꢀ8
1.37 0.78 · 10ꢀ8
3.33 0.37 · 10ꢀ9
3.47 0.22 · 10ꢀ8
2.21 0.42 · 10ꢀ10
1.54 0.25 · 10ꢀ9
1.14 0.73 · 10ꢀ7
1.12 0.36 · 10ꢀ8
n-Propyl
iso-Propyl
n-Butyl
iso-Butyl
Ethyl
Nifedipine
a The molar concentration of antagonist test compound causing a 50% in the tonic contractile response (IC50 SEM) in guinea-pig ileum smooth
muscle by KCl (80 mmol/L) was determined graphically from dose–response curve. The number of experiments was 5 for all compounds.
smooth muscle (GPILSM) contractility is summarized
in Table 1. Comparison of the activities of asymmetrical
esters in alkyl ester series (Table 1, 8a–f) indicates that
increasing of size of substituents in C-3 ester position
increases activity. When increasing of the size is accom-
panied by increasing the hindrance, the activity decreas-
es. Moreover, the results show that compounds 8b and
8d are similar in effect to the reference drug nifedipine.
The compounds 8a and 9 represent lower calcium chan-
nel antagonist activity, while compounds 8c, 8e and 8f
exhibit much higher activity relative to the reference
drug nifedipine. Comparison of the activities of symmet-
rical compound (9) and asymmetrical esters in alkyl
ester series (8a–f) indicates that asymmetrical esters are
more active than symmetrical compound.
4. Three-dimensional structure
The optimized 3D structure of the molecules obtained
by DFT calculations at the level of 6–31G* is represent-
ed in Figure 2 and some geometrical properties includ-
ing angles, dihedrals, and atomic distances are listed in
Table 2. As one can see from Figure 2, the studied
DHP derivatives adapted to relatively the same geome-
try similar to that we found in our previous studies for
other DHP derivatives.12 The DHP ring exhibited a
semi-boat conformer and the methyl-nitroimidazolyl
substituent at C-4 position of the DHP ring positioned
at the axial coordinate. Interestingly, in all of the deriv-
atives the methyl group of methyl-nitroimidazolyl ori-
ented in the anti-preplanar with respect to the DHP
ring. In addition, the nitroimidazolyl ring made a dihe-
dral angle about 30ꢁ with the DHP ring. The phenyl
ring, attached to the C-6 position of DHP ring, made
a dihedral angle about 58ꢁ with the DHP ring for all
derivatives except for 8e. The dihedral angle for this
compound was found to be 25ꢁ. It should be noted that
the optimization was performed many times with differ-
ent starting geometries and in all trials similar final
geometries were found for 8e. The major difference be-
tween the geometry of the studied molecules upon vari-
ation of the substituent pattern on the C-3 position of
the DHP ring (R1) is the orientation of the CO double
bond of the C3-COOEt with respect to the DHP ring.
As seen, for compounds 8a and 9 a cis orientation result-
ed and the rest adapted to the trans conformation.
The structure–activity data indicate that the 1-methyl-
5-nitro-2-imidazolyl moiety is a bioisoester of ortho-
nitrophenyl group. As lipophilic aromatic substituents
attached to the C-6 position of DHP ring are supposed
to improve penetration into organ, these compounds
are more active in comparison of similar compounds
that contain methyl moiety in C-6 position of DHP
ring. On the other hand, the activity data listed in
Table 1 indicate that one-order decrease in activity is
obtained when methyl at C-2 position is replaced by
a phenyl substituent (compounds 8b and 9, respective-
ly). This observation that is in the reverse order of the
effect of increasing lipophilicity may be due to the
increase in steric hindrance by phenyl group since the
binding of DHP derivatives to their receptor is sand-
wich-type and there is a challenge between lipophilicity
and steric for the calcium channel antagonist activity of
the DHP derivatives.31 Therefore, replacement of meth-
yl by phenyl in one site of DHP ring (i.e., C-6) increas-
es the activity because of higher lipophilicity but
replacement of both methyls at C-2 and C-6 positions
of DHP ring decreasing the activity due to increase
in steric hindrance.
It is reported from the crystal structures of DHP-based
derivatives that the 3D geometry of the molecules plays
an important role in their activity.12 As it is observed
from Figure 2 and Table 2, the methyl of the nitroimi-
dazolyl ring adapted to the anti-periplanar configura-
tion with respect to the DHP ring and this is required
for calcium channel antagonist activity. The syn-prepla-
nar orientation leads to agonist activity. Another 3D

R. Miri et al. / Bioorg. Med. Chem. 14 (2006) 4842–4849
4845
Figure 2. The optimized three-dimensional structural representation of the lipophilic DHP derivatives used in this study.
Table 2. Some geometrical features of the optimized DHP derivative
atom C-5 of DHP ring the cis conformer of the CO
makes the OCH2CH3 to be formed from the phenyl ring.
In contrast, a decreased distance between the methyl-
ene’s hydrogens and phenyl carbons is obtained for
the trans conformer of CO. The inter-atomic distance
between the carbon of methylene and C-20 carbon of
phenyl ring, listed in Table 2 (footnote d), supports this
postulate. For compounds 8a and 9 this distance is high-
e
Compound Splittinga U1b (ꢁ) U2c (ꢁ) U3d (ꢁ) d (A)
˚
8a
8b
8c
8d
8e
8f
9
q
58.4
58.8
32.6
29.1
28.9
28.9
31.2
28.9
32.1
cis
5.36
4.03
4.02
4.06
6.23
4.07
5.33
m
m
m
m
m
q
trans
trans
trans
trans
trans
cis
57.5
59.1
ꢀ25.1
59.3
56.8
˚
er than 5.3 A and for 8b, 8c, 8d, and 8f the distance is
˚
about 4.0 A. Here also the distance for 8e is an
expectation.
a q, quartet and m, multiplet.
b Dihedral angle between phenyl and DHP rings.
c Dihedral angle between nitroimidazolyl and DHP rings.
d Dihedral orientation between C-5-CO and DHP ring.
e The interatomic distance between methylene carbon and C-20 carbon
of phenyl.
5. Experimental
5.1. Chemistry
geometry–activity relationship is observed for the cis/
trans orientation of the CO double bond of the C3-
COOEt with respect to the DHP ring. Compounds 8a
and 9 that exhibited lower activity preferred the cis ori-
entation and the rest adapted to the trans orientation.
Melting points were determined on a Kofler hot stage
apparatus (Reichert, Vienna, Austria) and are uncorrect-
ed. 1H NMR spectra were obtained with a Bruker
80 MHz spectrometer (Bruker Analytische Messetech-
nik, Rheinstetten, Germany) in d1-chloroform or
d6-DMSO and tetramethylsilane (TMS) was used as an
internal standard. The mass spectra were measured with
a Finnigan TSQ-70 spectrometer(Finnigan Mat, Bremen,
Germany) at 70 eV. The IR spectra were obtained by
using a Nicolet 50X-FT spectrometer (KBr disks) (Nico-
let, Madison, WI, USA). The results of elemental analyses
(C, H, and N) were within 0.4% of the calculated values.
Silica gel HT-254 (E.Merck, Darmstadt, Germany) was
used for thin-layer chromatography. All spectra were
consistent with the assigned structures. Methyl(ethyl)
acetoacetate 1a–b, 2,2,6-trimethyl-4H-1,3-dioxin-4-one
2, and methyl(ethyl) 3-aminocrotonate 4a–b were
In the NMR investigation of the synthesized DHP, we
found that the multiplicity of the HNMR of methylene’s
hydrogens of the C5-COOCH2CH3 is different for com-
pounds 8a and 9 with the other compounds. Ideally,
these hydrogens must produce a quartet signal due to
the presence of methyl’s protons. However, all com-
pounds exhibited multiplet signal except for 8a and 9
that produced quartet signal. This can be attributed to
the long-range coupling between the protons of methy-
lene and those of phenyl ring, especially one attached
to the C-20 of the phenyl. A close insight into the 3D
structures represented in Figure 2 reveals that at the

4846
R. Miri et al. / Bioorg. Med. Chem. 14 (2006) 4842–4849
purchased from the Aldrich Chemical Co. (Sigma–
Aldrich Chemie GmbH, Deisenhofon, Germany).
5.1.2.4. Iso-butyl 3-aminocrotonate (4f). IR (KBr): m
3453 and 3336 (NH2), 1716 cmꢀ1 (C@O, ester).
5.1.1. General procedure for the synthesis of acetoacetic
esters (3c–f). A solution of alcohol 1c–f (50 mmol) and
2,2,6-trimethyl-4H-1,3-dioxine-4-one 2 (7.1 g, 50 mmol)
in 10 ml xylene was placed in a 50 ml Erlenmeyer flask.
The flask was immersed in an oil bath that had been pre-
heated to 150 ꢁC, and the solution was vigorously stir-
red. The evolution of acetone became apparent within
several minutes, heating was continued for a total
30 min. The reaction mixture was cooled. The xylene
was removed. Distillation of the mixture afforded 3c–f
which were used immediately in subsequent reactions.
5.1.3. Procedure for the synthesis of ethyl-2-benzoyl-3-(1-
methyl-5-nitroimidazolyl)-2-propionate (7). A solution of
1-methyl-5-nitroimidazol-2-carboxaldehyde 5 (460 mg,
3 mmol), ethylbenzoylacetate 6 (580 mg, 3 mmol), gla-
cial acetic acid (0.1 ml), piperidine (0.05 ml), and dry
benzene (5 ml) was refluxed for 7 h, during which the
resultant water was removed via a Dean–Stark trap.
After cooling, the benzene was removed and the residue
was purified by chromatography on silica gel with chlo-
roform–methanol (20:1 v/v), to give pure compound 7
(52% yield) as solid (mp 135–137 ꢁC).
5.1.1.1. n-Propyl acetoacetate (3c). 1H NMR (CDCl3):
d 4.10 (t, J = 6.5 Hz, 2H, CO2CH2), 3.45 (s, 2H,
COCH2CO2), 2.27 (s, 3H, CH3CO), 1.63 (m, 2H, CH2),
0.94 (t, J = 6.5 Hz, 3H, CH3).
1H NMR (CDCl3): d 7.82 (s, 1H, imidazol H-4), 7.70
(s, 1H, @C–H), 7.41–7.33 (m, 5H, C6H5), 4.26
(q, J = 7.2 Hz, 2H, CO2CH2), 4.10 (s, 3H, N–CH3),
1.18 (t, J = 7.2 Hz, 3H, CH3).
IR (KBr): m 1747 (C@O, ester), 1719 cmꢀ1 (C@O, ketone).
IR (KBr): m 1739 (C@O, ester), 1716 (C@O, ketone),
1627 (C@C), 1520, 1370 cmꢀ1 (NO2).
5.1.1.2. Iso-propyl acetoacetate (3d). 1H NMR
(CDCl3): d 5.06 (septet, J = 6.1 Hz, 1H, CO2CH), 3.41
(s, 2H, COCH2CO2), 2.26 (s, 3H, CH3CO), 1.26
(d, J = 6.1 Hz, 6H, CH(CH3)2).
MS: m/z (%) 392 (M+, 9), 300 (61), 272 (14), 242 (10), 228
(17), 192 (10), 155 (18), 125 (28), 105 (100) and 77 (37).
5.1.4. General procedure for the synthesis of alkyl ethyl
1,4-dihydro-2-methyl-6-phenyl-4-(1-methyl-5-nitro-2-im-
idazolyl)-3,5-pyridinedicarboxylates (8a–f). A solution of
compounds 4a–f (1.5 mmol) and ethyl-2-benzoyl-3-(1-
IR (KBr): m 1742 (C@O, ester), 1727 cmꢀ1 (C@O, ketone).
1
5.1.1.3. n-Butyl acetoacetate (3e). H NMR (CDCl3):
d 4.15 (t, J = 6.4 Hz, 2H, CO2CH2), 3.44 (s, 2H,
COCH2CO2), 2.16 (s, 3H, CH3CO), 1.41 (m, 4H,
CH2CH2), 1.01 (t, J = 6.4 Hz, 3H,CH3).
methyl-5-nitroimidazolyl)-2-propionate
7
(493 mg,
1.5 mmol) in ethanol (5 ml) was protected from light
and refluxed for 24–30 h. After cooling, the solution
was concentrated under reduced pressure and purified
by thin-layer chromatography on silica gel with chloro-
form–methanol (50:1 v/v). The product was recrystal-
lized from chloroform to give pure compounds 8a–f.
IR (KBr): m 1757 (C@O, ester), 1729 cmꢀ1 (C@O, ketone).
5.1.1.4. Iso-butyl acetoacetate (3f). 1H NMR (CDCl3):
d 4.48 (d, J = 6.6 Hz, 2H, CO2CH2), 3.46 (s, 2H,
COCH2CO2), 2.26 (s, 3H, CH3CO), 2.46 (m, 1H, CH),
1.48 (d, J = 6.6 Hz, 6H, CH(CH3)2).
5.1.4.1. 5-Ethyl-3-methyl-1,4-dihydro-2-methyl-6-phen-
yl-4-(1-methyl-5-nitro-2-imidazolyl)-3,5-pyridinedicarb-
oxylate (8a). 1H NMR (CDCl3): d 8.01 (s, 1H, imidazole
H-4), 7.41–7.27 (m, 5H, C6H5), 6.22 (br s, 1H, NH),
5.20 (s, 1H, C4-H), 4.23 (s, 3H, N–CH3), 3.81 (q,
J = 7.2 Hz, 2H, CO2CH2), 3.71 (s, 3H, CO2CH3), 2.38
(s, 3H, C2–CH3), 0.75 (t, J = 7.2 Hz, 3H, CH3).
IR (KBr): m 1756 (C@O, ester), 1727 cmꢀ1 (C@O, ketone).
5.1.2. General procedure for the synthesis of alkyl 3-
aminocrotonate (4c–f). A solution of alkyl acetoacetic es-
ters 3a–f (4 mmol) and ammonium acetate (6 mmol) in
5 ml ethanol was placed in a 10 ml Erlenmeyer flask.
The flask was immersed in an oil bath that had been pre-
heated to 90 ꢁC, and the solution was vigorously stirred.
Heating was continued for a total 24 h. The reaction
mixture was cooled. The ethanol was removed and IR
spectra of the compounds were recorded to confirm
the structure of 4a–f. Then, the compounds were imme-
diately used in subsequent reactions.
IR (KBr): m 3421 (NH), 1702 (C@O), 1526, 1373 cmꢀ1
(NO2).
MS: m/z (%) 426 (M+, 51), 380 (8), 367 (15), 353 (36),
321 (100), 300 (31), 272 (13) and 252 (7).
Elemental analysis calculated for C21H22N4O6: C, 59.15;
H, 5.20; N, 13.14. Found: C, 59.34; H, 5.18; N, 13.08.
5.1.2.1. n-Propyl 3-aminocrotonate (4c). IR (KBr): m
3449 and 3342 (NH2), 1715 cmꢀ1 (C@O, ester).
5.1.4.2. 3,5-Diethyl-1,4-dihydro-2-methyl-6-phenyl-4-(1-
methyl-5-nitro-2-imidazolyl)-3,5-pyridinedicarboxylate (8b).
1H NMR (CDCl3): d 7.99 (s, 1H, imidazole H-4), 7.40–
7.31 (m, 5H, C6H5), 6.35 (br s, 1H, NH), 5.20 (s, 1H,
C4-H), 4.24 (s, 3H, N–CH3), 4.17 (q, J = 7.2 Hz, 2H,
C3–CO2CH2), 3.82–3.78 (m, 2H, C5–CO2CH2), 2.38 (s,
3H, C2–CH3), 1.27 (t, J = 7.2 Hz, 3H, C3–CO2CH2CH3),
0.75 (t, J = 7.1 Hz, 3H, C5–CO2CH2CH3).
5.1.2.2. Iso-propyl 3-aminocrotonate (4d). IR (KBr): m
3453 and 3336 (NH2), 1717 cmꢀ1 (C@O, ester).
5.1.2.3. n-Butyl 3-aminocrotonate (4e). IR (KBr): m
3449 and 3334 (NH2), 1713 cmꢀ1 (C@O, ester).

R. Miri et al. / Bioorg. Med. Chem. 14 (2006) 4842–4849
4847
IR (KBr): m 3193 (NH), 1673 (C@O), 1525, 1370 cmꢀ1
(NO2).
Elemental analysis calculated for C24H28N4O6: C, 61.53;
H, 6.02; N, 11.96. Found: C, 61.72; H, 5.99; N, 12.01.
MS: m/z (%) 440 (M+, 39), 394 (6), 367 (44), 321 (100),
314 (22), 286 (6), 258 (11) and 240 (10).
5.1.4.6. 5-Ethyl-3-iso-butyl-1,4-dihydro-2-methyl-6-phen-
yl-4-(1-methyl-5-nitro-2-imidazolyl)-3,5-pyridinedicar-
boxylate (8f). 1H NMR (CDCl3): d 7.99 (s, 1H, imidazole
H-4), 7.40–7.29 (m, 5H, C6H5), 6.41 (br s, 1H, NH), 5.20
(s, 1H, C4-H), 4.24 (s, 3H, N–CH3), 4.00–3.81 (m, 2H, C3–
CO2CH2), 3.80–3.77 (m, 2H, C5–CO2CH2), 2.38 (s, 3H,
C2–CH3), 1.97 (m, 1H, CH2CH(CH3)2), 0.90 (d,
J = 7.0 Hz, 6H, CH(CH3)2), 0.75 (t, J = 7.2 Hz, 3H,
CO2CH2CH3).
Elemental analysis calculated for C22H24N4O6: C, 59.99;
H, 5.49; N, 12.72. Found: C, 59.87; H, 5.51; N, 12.77.
5.1.4.3. 5-Ethyl-3-n-propyl-1,4-dihydro-2-methyl-6-phen-
yl-4-(1-methyl-5-nitro-2-imidazolyl)-3,5-pyridinedicar-
boxylate (8c). 1H NMR (CDCl3): d 8.01 (s, 1H, imidazole
H-4), 7.40–7.26 (m, 5H, C6H5), 6.11 (br s, 1H, NH), 5.20
(s, 1H, C4-H), 4.24 (s, 3H, N–CH3), 4.09–4.06 (m, 2H, C3–
CO2CH2), 3.82–3.78 (m, 2H, C5–CO2CH2), 2.39 (s, 3H,
C2–CH3), 1.66 (m, 3H, C3–CO2CH2CH2CH3), 0.92 (t,
IR (KBr): m 3415 (NH), 1677 (C@O), 1532, 1376 cmꢀ1
(NO2).
J = 7.2 Hz,
(t, J = 7.1 Hz, 3H, C5–CO2CH2CH3).
3H,
C3–CO2
CH2CH2CH3),
0.75
MS: m/z (%) 468 (M+, 39), 395 (22), 367 (39), 342 (16),
333 (10), 321 (100), 286 (9) and 240 (10).
IR (KBr):
m
3421 (NH), 1676 (C@O), 1527,
Elemental analysis calculated for C24H28N4O6: C 61.53;
H, 6.02; N, 11.96. Found: C, 61.44; H, 6.05; N, 11.91.
1372 cmꢀ1(NO2).
MS: m/z (%) 454 (M+, 68), 440 (15), 395 (6), 367 (52),
321 (100), 276 (7), 258 (9) and 240 (10).
5.1.5. Procedure for the synthesis of 3,5-diethyl-1,4-
dihydro-2,6-diphenyl-4-(1-methyl-5-nitro-2-imidazolyl)-
3,5-pyridinedicarboxylate (9). A solution of ammonium
hydroxide (25%, 0.75 ml) was added to a stirring solu-
tion of 1-methyl-5-nitro-imidazole-2-carboxaldehyde 5
(0.78 g, 5 mmol) ethylbenzoylacetate 6 (1.92 g, 10 mmol)
in absolute ethanol (5 ml). The mixture was heated un-
der reflux for 20 h. After that, the reaction mixture
was cooled, solvent removed under vacuum. The residue
was purified by thin-layer chromatography on silica gel
using chloroform–methanol (98:2 v/v), to give pure com-
pound 9 (5% yield) as solid (mp 183–185 ꢁC).
Elemental analysis calculated for C23H26N4O6: C, 60.78;
H, 5.77; N, 12.33. Found: C, 60.56; H, 5.79; N, 12.37.
5.1.4.4. 5-Ethyl-3-iso-propyl-1,4-dihydro-2-methyl-6-
phenyl-4-(1-methyl-5-nitro-2-imidazolyl)-3,5-pyridine
dicarboxylate (8d). 1H NMR (CDCl3): d 7.99 (s, 1H, imid-
azole H-4), 7.39–7.27 (m, 5H, C6H5), 6.31 (br s, 1H, NH),
5.18 (s, 1H, C4-H), 5.05 (m, 1H, CO2CH), 4.25 (s, 3H, N–
CH3), 3.82–3.79 (m, 2H, CO2CH2), 2.37 (s, 3H, C2–CH3),
1.27 and 1.20 (two d, J = 6.0 Hz, 3H each, CH(CH3)2),
0.74 (t, J = 7.1 Hz, 3H, C5–CO2CH2CH3).
1H NMR (CDCl3): d 8.07 (s, 1H, imidazole H-4), 7.42–
7.37 (m, 10H, C6H5), 6.19 (br s, 1H, NH), 5.29 (s, 1H,
C4-H), 4.31 (s, 3H, N–CH3), 3.86 (q, J = 7.2 Hz, 4H,
C3 and C5–CO2CH2), 0.81 (t, J = 7.2 Hz, 6H, C3 and
C5–CO2CH2CH3).
IR (KBr): m 3419 (NH), 1677 (C@O), 1528, 1372 cmꢀ1
(NO2).
MS: m/z (%) 454 (M+, 27), 381 (9), 367 (47), 339 (10),
321 (100), 307 (6) and 286 (14).
IR (KBr): m 3181 (NH), 1676 (C@O), 1532, 1381 cmꢀ1
(NO2).
Elemental analysis calculated for C23H26N4O6: C, 60.78;
H, 5.77; N, 12.33. Found: C, 60.61; H, 5.74; N, 12.28.
MS: m/z (%) 502 (M+, 58), 456 (16), 429 (54), 383 (100),
376 (32) and 230 (11).
5.1.4.5. 5-Ethyl-3-n-butyl-1,4-dihydro-2-methyl-6-
phenyl-4-(1-methyl-5-nitro-2-imidazolyl)-3,5-pyridine
dicarboxylate (8e). H NMR (CDCl3): d 8.00 (s, 1H,
Elemental analysis calculated for C27H26N4O6: C, 64.53;
H, 5.21; N, 11.15. Found: C, 64.80; H, 5.20; N, 11.14.
1
imidazole H-4), 7.41–7.26 (m, 5H, C6H5), 6.09 (br s,
1H, NH), 5.20 (s, 1H, C4-H), 4.24 (s, 3H, N–CH3),
4.18–4.10 (m, 2H, C3–CO2CH2), 3.83–3.78 (m, 2H,
C5–CO2CH2), 2.38 (s, 3H, C2–CH3), 1.64–1.58
(m, 3H, C3–CO2CH2CH2CH2), 1.37–1.0 (m, 3H, C3–
CO2CH2CH2CH2CH3), 0.92 (t, J = 7.0 Hz, 3H,
C3–CO2 CH2CH2CH2CH3), 0.75 (t, J = 7.1 Hz, 3H,
C5–CO2CH2CH3).
5.2. Pharmacology
5.2.1. Determination of calcium channel antagonist activ-
ity. Male albino guinea pigs (300–450 g) were purchased
from SUMS animal house department. They had free
access to standard rodent chow and tap water at all
times. The animals were housed in a room maintained
at 23 2 ꢁC temperature, 55 10 % humidity, and on
a 12 h light/dark cycle. The feeding was disrupted 1
day before starting in vitro tests. The animals were sac-
rificed by a blow to the head. The intestine was removed
above the ileocecal junction and longitudinal smooth
muscle segments of 2 cm length were mounted under a
IR (KBr): m 3420 (NH), 1677 (C@O), 1529,1373 cmꢀ1
(NO2).
MS: m/z (%) 468 (M+, 29), 440 (7), 395 (16), 367 (38),
342 (13), 321 (100), 276 (7), 258 (8) and 240 (9).

4848
R. Miri et al. / Bioorg. Med. Chem. 14 (2006) 4842–4849
5. Psaty, B. M.; Heckbert, S.; Koepsell, T.; Siscovick, D. S.;
Raghunathan, T. E.; Weiss, N. S.; Rosendal, F. R.;
Lemaitre, R. N.; Smith, N. L.; Wahl, P. W.; Wagner, E.
H.; Furberg, C. D. J. Am. Med. Assoc. 1995, 274, 620.
6. Pahor, M.; Guralnik, J. M.; Ferruci, L.; Corti, M. C.;
Salive, M. E.; Cerhan, J. R.; Wallace, R. B.; Havlik, R. J.
Lancet 1996, 348, 493.
7. Fitzpatrick, A. L.; Daling, J. R.; Furberg, C. D.; Kron-
mal, R. A.; Weissfeld, J. L. Cancer 1997, 80, 1438.
8. Joint National Committee. The Sixth Report of the Joint
National Committee on Prevention, Detection, Evalua-
tion, and Treatment of high Blood Pressure (JNC-VI).
Arch. Intern. Med. 1997, 157, 2413.
9. Coburn, R. A.; Wierzba, M.; Suto, M. J.; Solo, A. J.;
Triggle, A. M.; Triggle, D. J. J. Med. Chem. 1988, 31, 2103.
10. Janis, R. A.; Triggle, D. J. J. Med. Chem 1983, 26, 775.
11. Schramm, M.; Thomas, G.; Towart, R.; Frankowiak, G.
Nature 1983, 303, 535.
12. Hemmateenejad, B.; Miri, R.; Safarpour, M. A.; Khoshn-
eviszadeh, M.; Edraki, N. J. Mol. Struct. (Theochem.)
2005, 717, 139.
resting tension of 0.5 g. The segments were maintained
at 37 ꢁC in a 20 ml jacketed organ bath containing oxy-
genated physiological saline solution of the following
(mmol) composition: NaCl, 137; CaCl2, 1.8; KCl, 2.7;
MgSO4, 1.1; NaH2PO4, 0.4; NaHCO3, 12 and glucose
5. The muscle was equilibrated for 1 h with a solution
changing every 15 min. The contractions were recorded
with a force displacement transducer (F-50) on a Narco
Physiograph (Narco Biosystems, Houston, TX, USA).
Test agents were prepared as 10–2 mol/L stock solutions
in DMSO and stored protected from light. Dilutions
were made with DMSO. The contractile response was
taken as the 100% value for the tonic (slow) component
of the response. The contraction was elicited with
80 mmol/L KCl. Test compounds were cumulatively
added and compound-induced relaxation of contracted
muscle was expressed as percent of control. The IC50
values (concentration needed to produce 50% relaxation
on contracted ileal smooth muscle) were graphically
determined from the concentration–response curves.32,33
Nifedipine was used as reference compound.
13. Natale, N. R.; Rogers, M. E.; Staples, R. J.; Triggle, D. J.;
Rutledge, A. J. Med. Chem 1999, 42, 3087.
14. Zamponi, G. W.; Stotz, S. C.; Staples, R. J.; Andro, T. M.;
Nelson, J. K.; Hulubei, V.; Blumenfeld, A.; Natale, N. R.
J. Med. Chem. 2003, 46, 87.
15. Ramesh, M.; Matowe, W. C.; Wolowyk, M. W.; Knaus,
E. E. Drug Des. Deliv. 1987, 2, 79.
5.2.2. Statistical analysis. Data are expressed as means
SEM. The one-way analysis of variance (ANOVA)
followed by Tukey–Kramer multiple comparisons was
used to analyze the data. A value of P < 0.05 was con-
sidered as the significance level between the groups.
16. Akula, M. R.; Matowe, W. C.; Wolowyk, M. W.; Knaus,
E. E. Drug Des. Deliv. 1989, 5, 117.
17. Ramesh, M.; Matowe, W. C.; Wolowyk, M. W.; Knaus,
E. E. Pharm. Res. 1990, 7, 919.
5.3. Computation details
18. Shafiee, A.; Miri, R.; Dehpour, A. R. Pharm. Sci. 1996, 2,
541.
19. Pourmorad, F.; Hadizadeh, F.; Shafiee, A. Pharm. Sci.
1997, 3, 165.
20. Shafiee, A.; Dehpour, A. R.; Hadizadeh, F. Pharm. Acta
Helv. 1998, 73, 75.
21. Amini, M.; Golabchifar, A. A.; Dehpour, A. R.; Pirali, H.
M.; Shafiee, A. Arzneim-Forsch./Drug Res. 2002, 52, 21.
22. Shafiee, A.; Rastkary, N.; Jorjani, M. Arzneim-Forsch./
Drug Res. 2002, 52, 537.
23. Clemens, R. J.; Hyatt, H. A. J. Org. Chem. 1985, 50, 2431.
24. Shafiee, A.; Miri, R.; Dehpour, A. R.; Soleymani, F.
Pharm. Sci. 1996, 2, 541.
25. Rovnyak, G. C.; Atwal, K. S.; Hedberg, A.; Kimball, S.
D.; Moreland, S.; Gougoutas, J. Z.; O’Reilly, Z. B. C.;
Schwartz, J.; Malley, M. F. J. Med. Chem. 1992, 35, 3236.
26. Meyer, H.; Bossert, F.; Wehinger, E.; Stoepel, K.; Vater,
W. Arzneim-Forsch./Drug Res. 1981, 31, 407.
27. Iwanami, M.; Shibanuma, T.; Fujimoto, M.; Kawai, R.;
Tamazawa, K.; Takenaka, T.; Takahashi, K.; Murakami,
M. Chem. Pharm. Bull. 1979, 27, 1426.
28. Miri, R.; Dehpour, A. R.; Azimi, M.; Shafiee, A. Daru
2001, 9, 40.
Chemical structure of each molecule was built by
Hyperchem software (Version 7, Hypercube Inc.) for
the structural chemistry. Gaussian98 program34 was
operated to optimize the molecular structure. The struc-
tures were optimized by the B3LYP method of density
functional theory (DFT) at the level of 6-31G*. No
molecular symmetry constraint was applied; rather full
optimization of all bond lengths and angles was carried
out. The root mean square of 0.1 kcal molꢀ1 was used
as ending criteria in geometry optimization. Then, the
molecules were reloaded to Hyperchem and some an-
gles, dihedrals, and distances were calculated in this
software.
Acknowledgments
This research was supported by a grant from the re-
search council of Shiraz University of Medical Sciences.
The quantum chemical calculation by Mrs. U. Nirou-
mand is acknowledged.
29. Miri, R.; McEwen, C. A.; Knaus, E. E. Drug Dev. Res.
2000, 51, 225.
30. Hantzsch, A. Justus Liebigs Ann. Chem. 1882, 215, 1.
31. Hemmateenejad, B.; Miri, R.; Akhond, M.; Shamsipur,
M. Arch. Pharm. Pharm. Med. Chem. 2002, 10, 472.
32. Triggle, C. R.; Swamy, V.; Triggle, D. J. Can. J. Physiol.
Pharmacol. 1979, 57, 804.
References and notes
1. Luscher, T. F.; Consentino, F. Drugs 1998, 55, 509.
2. Navidpour, L.; Miri, R.; Shafiee, A. Arzeneinihel Fors-
chung Drug Res. 2004, 54, 499.
3. Striessnig, J. Cell Physiol. Biochem. 1999, 9, 242.
4. New analyses regarding the safety of calcium channel
blockers: A statement for health professionals from the
National Hearth, Lung, and Blood Institute, Septem-
ber 1, 1995. http://www.nhlbi.nih.gov/new/press/cutlrccb.
txt.
33. Miri, R.; Niknahad, H.; Vesal, Gh.; Shafiee, A. IL
Farmaco 2002, 57, 123.
34. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G.
E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.;
Montgomery, J. A.; Jr., Stratmann, R. E.; Burant, J. C.;
Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.;
Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi,

R. Miri et al. / Bioorg. Med. Chem. 14 (2006) 4842–4849
4849
M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.;
Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.;
Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.;
Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz,
J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.;
Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.;
Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara,
A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.;
Johnson, B.; Chen, W.; Wong, M. W.; Andres, J. L.;
Gonzalez, C.; Head-Gordon, M.; Replogle, E. S.; Pople, J.
A. GAUSSIAN 98 (Revision A.7), Gaussian Inc., Pitts-
burgh PA, 1998.