Synthesis and evaluation of pharmacological activities of 3,5-dialkyl 1,4-dihydro-2,6-dimethyl-4-nitroimidazole-3,5-pyridine dicarboxylates

Article abstract of DOI:10.1002/ardp.200300762

New analogues of nifedipine, in which the 2-nitrophenyl group at position 4 is replaced by a 1 -methyl-5-nitro-2-imidazolyl substituent, were synthesized. The symmetrical dialkyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-2-imidazolyl)-3, 5-pyridinedicarboxylates were prepared by a classical Hantzsch condensation. The asymmetrical analogues were synthesized using a procedure reported by Iwanami that involved the condensation of alkylacetoacetate with methyl-, ethyl- or isopropyl-3-aminocrotonate and 1 -methyl-5-nitroimidazole-2-carboxaldehyde. Calcium channel antagonist activities were determined in vitro using a guinea pig ileum longitudinal smooth muscle (GPILSM) assay. Many compounds exhibited superior, or equipotent, calcium antagonist activity (IC₅₀ = 10- ¹⁰ to 10-¹³ M range) relative to the reference drug nifedipine (IC₅₀ = 1.09? 0.12 × 10-¹¹ M). Antinociceptive effects of some compounds were evaluated by the mouse tail-flick assay in vivo. Results demonstrate that some of the compounds were active as an antinociceptive.

Full text of DOI:10.1002/ardp.200300762

4
22 Miri et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 422–428
R. Miria,
K. Javidnia ,
Synthesis and Evaluation of Pharmacological
Activities of 3,5-Dialkyl
a
b
A. Kebriaie-Zadeh ,
c
H. Niknahad ,
1,4-Dihydro-2,6-Dimethyl-4-Nitroimidazole-
c
N. Shaygani ,
d
3,5-Pyridine Dicarboxylates
S. Semnanian ,
A. Shafieee
New analogues of nifedipine, in which the 2-nitrophenyl group at position 4 is re-
placed by a 1-methyl-5-nitro-2-imidazolyl substituent, were synthesized. The sym-
metrical dialkyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-2-imidazolyl)-3,5-pyr-
idinedicarboxylates were prepared by a classical Hantzsch condensation. The
asymmetrical analogues were synthesized using a procedure reported by Iwanami
that involved the condensation of alkylacetoacetate with methyl-, ethyl- or isopropyl-
a
Medicinal & Natural
Products Chemistry
Research Center, Shiraz
University of Medical
Science, Shiraz, Iran
3
-aminocrotonate and 1-methyl-5-nitroimidazole-2-carboxaldehyde.Calcium chan-
b
Department of
nel antagonist activities were determined in vitro using a guinea pig ileum longitudi-
Pharmacology & Toxicology,
Faculty of Pharmacy, Tehran
University of Medical
nal smooth muscle (GPILSM) assay. Many compounds exhibited superior, or equi-
–
10
–13
potent, calcium antagonist activity (IC50 = 10 to 10 M range) relative to the refer-
–
11
ence drug nifedipine (IC50 = 1.09 ± 0.12 × 10 M). Antinociceptive effects of some
compounds were evaluated by the mouse tail-flick assay in vivo. Results demon-
strate that some of the compounds were active as an antinociceptive.
Science, Tehran, Iran
Department of
c
Pharmacology & Toxicology,
Faculty of Pharmacy, Shiraz
University of Medical
Keywords: Dihydropyridine; Ca+2 channel antagonist; Antinociceptive; Nitroimid-
azole
Science, Shiraz, Iran
Department of Physiology,
Tarbiat Modarres University,
Tehran, Iran
Department of Chemistry,
Received: January 7, 2003; Accepted: April 22, 2003 [FP762]
DOI 10.1002/ardp.200300762
d
e
Faculty of Pharmacy,
Tehran University of Medical
Science, Tehran, Iran
Changes in substitution pattern at the C-3, C-4, and C-5
positions of nifedipine alter activity and tissue selectivity
Introduction
The influx of extracellular Ca2+ through potential depend-
ent calcium channels is responsible for the regulation of
many physiological functions, including smooth and car-
diac muscle contraction [1–3]. The discovery that the
[11–13].It was our interest to determine the effects of dif-
ferent C-3 alkyl substituents, in conjugation with C-4
nitroimidazolyl substituents, on the calcium channel
antagonist activity.We now report the synthesis, calcium
channel antagonist and antinociceptive activities of
dialkyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-2-
imidazolyl)-3,5-pyridinedicacboxylate.
1
,4-dihydropyridine (Nifedipine) class of calcium chan-
2
+
nel antagonists inhibits this Ca influx represented a
major therapeutic advance in treatment of cardiovascu-
lar diseases such as hypertension, angina pectoris, and
other spastic smooth muscle disorders [4–7].In addition,
the antinociceptive activities of some calcium channel
blockers such as, nifedipine, nimodipine, verapamil, and
diltiazem had been reported previously. These finding
suggest a pharmacological role of Ca channel blockers
in the modulation of antinociception under acute condi-
tion [8–10].
2
+
Chemistry
Alkyl acetoacetates 10–14 were synthesized by the re-
action of 2,2,6-trimethyl-4H-1,3-dioxine-4-one 7 with al-
cohols 1–5 in 43–57 % overall yield. The symmetrical
analogues 18–25 were prepared by the classical
Hantzsch condensation [14] in which 1-methyl-5-
nitroimidazole-2-carboxaldehyde 15 was reacted with
the acetoacetic esters 7–14 and ammonium hydroxide in
49-81 % yield [15–16] (Scheme 1).
Correspondence: Ramin Miri, Medicinal & Natural Products
Chemistry Research Center Shiraz University of Medical
Science, Zand Street, 71345 Shiraz, Iran. Phone:
+
98 711 235-7282, Fax: +98 711 230-3872, e-mail:
mirir@sums.ac.ir.
©
2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 422–428
Pharmacological Activities of a Dicarboxylate 423
Scheme 1. Synthesis of symmetrical and asymmetrical analogues of nifedipine.
The unsymmetrical analogues 26–38 were synthesized
Results and discussion
by a modified Hantsch reaction using a procedure re-
ported by Iwanami [17]. Thus, condensation of alkyl-3-
aminocrotonates 16–17, acetoacetic esters 7–14, and
The in vitro calcium channel antagonist activities (IC50
)
of compounds 18–36 were determined as the molar con-
centration of the test compounds required to produce
50 % inhibition of the muscarinic receptor-mediated
1
-methyl-5-nitroimidazole-2-carboxaldehyde 15 afford-
ed the required products in 48–76 % yield [18]
Scheme 1).
–
7
+2
(
(carbacol, 1.67 × 10 M) Ca -dependent contraction
©
2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

4
24 Miri et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 422–428
Table 1. Physical properties and calcium channel antagonist activity of symmetrical analogues 18–25.
Compound
R
1
Mp (°C)
Yield
%)
Calcium channel antagonist
activity IC50 ± SEM, (n)
(
–
12
18
19
20
21
22
23
24
25
Me
Et
nPro
isoPro
nBut
isoBut
tBut
211–213
205–207
221–223
229–231
196–198
208–210
236–237
140–143
54
49
59
81
53
69
61
58
9.03 ± 0.34 × 10 (6)*
–
12
1.55 ± 0.24 × 10 (4)*
–
11
8.78 ± 0.40 × 10 (5)*
–
11
7.76 ± 0.93 × 10 (4)
–
11
5.11 ± 0.83 × 10 (7)
–
10
4.38 ± 1.01 × 10 (4)
–
10
8.68 ± 0.89 × 10 (4)
–
10
nPen
Nifedipine
9.53 ± 0.67 × 10 (5)
–
11
1.09 ± 0.12 × 10 (3)*
*
Single asterisk indicates P < 0.05 compared to nifedipine in that experiment using Student’s t-test.
Table 2. Physical properties and calcium channel antagonist activity of asymmetrical analogues 26–38.
Compound
R
1
R
2
Mp (°C)
Yield
%)
Calcium channel antagonist activity
IC50 ± SEM, (n)
(
12
26
27
28
29
30
31
32
33
34
35
36
37
38
Me
nPro
nPro
isoPro
isoPro
nBut
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
221–224
200–202
215–217
218–220
241–243
171–173
187–189
232–234
241–243
241–244
238–240
180–183
161–163
65
76
64
59
72
57
52
63
56
73
58
48
53
7.39 ± 0.43 × 10– (5)*
–
12
4.78 ± 1.02 × 10 (4)
–
12
1.72 ± 0.51 × 10 (6)*
–
13
9.49 ± 1.19 × 10 (4)*
–
12
5.16 ± 1.05 × 10 (3)*
–
12
0.33 ± 0.22 × 10 (6)*
–
12
nBut
3.25 ± 0.61 × 10 (4)*
–
12
isoBut
isoBut
tBut
0.91 ± 0.85 × 10 (3)*
–
11
1.15 ± 0.29 × 10 (5)
–
12
1.77 ± 0.71 × 10 (3)*
–
11
tBut
4.91 ± 1.17 × 10 (4)
–
13
nPen
nPen
Nifedipine
4.66 ± 0.42 × 10 (6)*
–
12
7.34 ± 0.04 × 10 (4)*
–
11
1.09 ± 0.12 × 10 (3)*
*
Single asterisk indicates P < 0.05 compared to nifedipine in that experiment using Student’s t-test.
2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
©

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 422–428
Pharmacological Activities of a Dicarboxylate 425
Figure 1. Comparing the effects
of compounds 21, 24, and 29 on
tail-flick latency time, Pre-drug,
and post-drug latency time. n =
4
, control was distilled water.
Compound administrated 50
mg/kg (i.p.), standard was nifed-
ipine.
(
tonic response) of guinea pig ileal longitudinal smooth
with the report of Akula et al.[19] indicated that 1,4-dihy-
dropyridine compounds with nitroimidazole substituted
on C-4 position of the ring have more activity than similar
compounds with a phenyl or pyridine substitute. In addi-
tion, antinociceptive activity of the compounds 18, 21,
muscle (GPILSM), and those values are presented inTa-
bles 1 and 2.
These results indicate that compounds 18–36 exhibit su-
perior or equipotent calcium channel antagonist activity
2
4, 26, and 29 have been investigated by the tail-flick
method. As shown in Figure1, compounds 21, 24, and
9 (at 50 mg/kg) had antinociceptive effects on the tail-
–
11
–13
(10 –10 M) relative to the reference drug nifedipine
–
11
(IC50 = 1.09 ± 0.12 × 10 M).Comparison of the activi-
2
ties of symmetrical esters 18–25 on GPILSM indicates
that the potency order was based on the substitutions
ethyl>methyl>iso-butyl>iso-propyl>n-pentyl. A compari-
son of the activities of the asymmetrical series of esters
flick test (P < 0.05), but the negative control (distilled wa-
ter) did not have any significant effect on the nociception.
2
6–38 indicates compounds possessing methyl substit-
Acknowledgement
uent (R = Me), the R C-3 ester substituent was a deter-
minant of calcium channel antagonist activity where the
potency order was: n-pentyl>n-butyl>t-butyl>isopropyl.
2
1
This work was supported by a grant from the Research
Council of the Medical Sciences University of Shiraz.
Also in compounds with ethyl substituent (R
2
= Et), the
R
1
C-3 ester substituent was a determinant and the
Experimental
potency order was: n-propyl>n-butyl>n-pentyl>t-butyl.
Chemistry
These results show that in symmetrical esters 18–25 an
increase in the steric size of the R
decreases antagonist action ethyl>n-propyl>n-butyl>n-
pentyl, and in asymmetrical series of esters 26–38 that
Melting points were determined on a Kofler hot stage appara-
tus (Reichert, Vienna, Austria) and are uncorrected. H-NMR
1
C-3 ester substituent,
1
spectra were run at a Varian Unity Plus 400 MHz spectrometer
(Varian, Darmstadt, Germany). Chemical shifts are reported in
parts per million (δ) relative toTMS as an internal standard.The
mass spectra were measured with a Finnigan TSQ-70 spec-
trometer (Finnigan, Bremen, Germany) at 70 eV. The IR spec-
tra were obtained by using a Nicolet 50X-FT spectrometer (KBr
disks) (Nicolet, Madison, WI, USA). Microanalyses were within
± 0.40 % of theoretical values for C, H, and N. All spectra were
consistent with the assigned structures. 2,2,6-trimethyl-4H-
compounds possessing one small R
Me or Et) an increase in the steric size or lipophilic prop-
erties of R C-3 ester substituent increases the activity.
The comparison of the activities of the compounds
8–36 with the compounds reported by Shafiee et al.
15] reveals that the presence of an aryl or cycloalkyl
2 2
substituent (R =
1
1
[
1
7
,3-dioxine-4-one 6, methyl (ethyl or t-butyl) acetoacetates
–9, and methyl (ethyl) 3-aminocrotontes 16–17 were pur-
group substituted on the C-3 or C-5 position of the 1,4-di-
hydropyridine ring increases the smooth muscle relax-
ant activity. Also, comparison of the results of this study
chased from the Aldrich Chemical Co. (Sigma-Aldrich Chemie
GmbH, Deisenhofen, Germany).
©
2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

4
26 Miri et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 422–428
General procedure for the synthesis of acetoacetic esters 10–
3,5-Diethyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-2-im-
idazolyl)-3,5-pyridinedicarboxylate 19
1
4
A solution of alcohol 1–5 (50 mmol) and 2,2,6-trimethyl-4H-1,3-
dioxine-4-one 6 (7.1 g, 50 mmol) in 10 mL of xylene was placed
in a 50 mL Erlenmeyer flask. The flask was immersed in an oil
bath that had been preheated to 150 °C, and the solution was
vigorously stirred. The evolution of acetone became apparent
within several minutes, heating was continued for a total
1H NMR (CDCl
H-4), 5.09 (s, 1 H, C
(s, 3 H, N-CH ), 2.97 (s, 6 H, C
7.1 Hz, 6 H, CH ).
3
): δ 9.01 (br s, 1 H, NH), 7.78 (s, 1 H, imidazole
-H), 4.87 (q, J = 7.1 Hz, 4 H, CO CH ), 4.09
-CH & C -CH ), 1.67 (t, J =
4
2
2
3
2
3
6
3
3
–
1
IR (KBr):ν 3426 (NH), 1698 (C=O), 1522 and 1385 cm (NO
2
).
3
0 min. The reaction was cooled. The xylene was removed.
3
,5-Di-npropyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-2-
Distillation of the mixture afforded 10–14 which were used im-
mediately in subsequent reactions.
imidazolyl)-3,5-pyridinedicarboxylate 20
1H NMR (CDCl
): δ 9.16 (br s, 1 H, NH), 7.95 (s, 1 H, imidazole
3
nPropyl acetoacetate (10)
H-4), 5.17 (s, 1 H, C -H), 4.24 (s, 3 H, N-CH ), 4.04 (t, J =
6.8 Hz, 4 H, CO CH ), 2.21 (s, 6 H, C -CH & C
H, CH ), 0.88 (t, J = 7.1 Hz, 6 H, CH ).
4
3
1H NMR (CDCl
2
0
2
2
2
3
6
-CH
3
), 1.61 (m,
3
): δ 4.10 (t, J = 6.5 Hz, 2 H, CO
), 2.27 (s, 3 H, CH CO), 1.63 (m, 2 H, CH
.94 (t, J = 6.4 Hz , 3 H, CH ).
2
CH
2
), 3.45 (s,
4
2
3
H, COCH
2
CO
2
3
2
),
–1
3
2
IR (KBr):ν 3426 (NH), 1698 (C=O), 1522 and 1385 cm (NO ).
–
1
IR (KBr): ν 1747 (C=O, ester), 1719 cm (C=O, ketone).
isoPropyl acetoacetate (11)
3
2
,5-Di-isopropyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-
-imidazolyl)-3,5-pyridinedicarboxylate 21
1H NMR (CDCl
): δ 8.95 (br s, 1 H, NH), 7.96 (s, 1 H, imidazole
H-4), 5.12 (s, 1 H, C -H), 5.03 (m, 2 H, CH(CH ), 4.26 (s, 3 H,
N-CH ), 2.23 (s, 6 H, C ), 1.24 and 1.19 (dd, J =
.4 Hz, 6 H each, CH(CH
1H NMR (CDCl
3
3
): δ 5.06 (septet, J = 6.1 Hz, 1 H, CO
s, 2 H, COCH CO ), 2.26 (s, 3 H, CH CO), 1.26 (d, J = 6.1 Hz,
3 2
H, CH(CH ) ).
2
CH), 3.41
4
3 2
)
(
6
2
2
3
3
2
-CH
3
)
3
2
& C
).
6 3
-CH
6
–
1
IR (KBr): ν 1742 (C=O, ester), 1727 cm (C=O, ketone).
nButyl acetoacetate (12)
–
1
IR (KBr):ν 3426 (NH), 1698 (C=O), 1522 and 1385 cm (NO
2
).
3
,5-Di-nbutyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-2-
1H NMR (CDCl
2
1
): δ 4.15 (t, J = 6.5 Hz, 2 H, CO
), 2.16 (s, 3 H, CH CO), 1.41 (m, 4 H, CH
.01 (t, J = 6.4 Hz, 3 H, CH ).
CH
), 3.44 (s,
imidazolyl)-3,5-pyridinedicarboxylate 22
3
2
2
H, COCH
2
CO
2
3
2
CH ),
2
1H NMR (CDCl
): δ 8.81 (br s, 1 H, NH), 7.94 (s, 1 H, imidazole
3
3
H-4), 5.15 (s, 1 H, C -H), 4.28 (s, 3 H, N-CH ), 4.08 (t, J = 6.9
Hz, 4 H, CO CH ), 2.19 (s, 6 H, C -CH & C -CH ), 1.61 & 1.26
two m, each 4 H, CH CH ), 0.89 (t, J = 6.8 Hz, 6 H, CH ).
4
3
–
1
IR (KBr): ν 1757 (C=O, ester), 1729 cm (C=O, ketone).
isoButyl acetoacetate (13)
2
2
2
3
6
3
(
2
2
3
–
1
2
IR (KBr):ν 3387 (NH), 1716 (C=O), 1519 and 1363 cm (NO ).
1H NMR (CDCl
H, COCH CO
d, J = 6.5 Hz, 6 H, CH(CH
): δ 4.48 (d, J = 6.6 Hz, 2 H, CO
), 2.26 (s, 3 H, CH CO), 2.46 (m, 1 H, CH), 1.48
).
3
2 2
CH ), 3.46 (s,
2
(
2
2
3
3,5-Di-isobutyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-2-
imidazolyl)-3,5-pyridinedicarboxylate 23
3
)
2
–
1
1H NMR (CDCl
H-4), 5.17 (s, 1 H, C
H, CO CH ), 2.16 (s, 6 H, C
CH(CH ) ), 1.23 and 1.19 (d, J = 6.8 Hz, 12 H, CH(CH ) ).
IR (KBr): ν 1756 (C=O, ester), 1727 cm (C=O, ketone).
nPentyl acetoacetate (14)
3
): δ 9.32 (br s, 1 H, NH), 7.81 (s, 1 H, imidazole
-H), 4.24 (s, 3 H, N-CH ), 3.89 (d, J = 6 Hz,
-CH & C -CH ), 1.19 (m, 2 H,
4
3
4
2
2
2
3
6
3
1H NMR (CDCl
): δ 4.13 (t, J = 6.4 Hz, 2 H, CO
), 2.26 (s, 3 H, CH CO), 1.52 (m, 6 H,
), 0.91 (t, J = 6.1 Hz, 3 H, CH ).
2
CH
2
), 3.43 (s,
3
2
3 2
3
2
H, COCH
2
CO
2
3
–1
IR (KBr):ν 3416 (NH), 1718 (C=O), 1522 and 1371 cm (NO
2
).
CH CH CH
2
2
2
3
3
,5-Di-tbutyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-2-
–
1
IR (KBr): ν 1752 (C=O, ester), 1721 cm (C=O, ketone).
imidazolyl)-3,5-pyridinedicarboxylate 24
1H NMR (CDCl
): δ 7.95 (s, 1 H, imidazole H-4), 7.81 (br s, 1 H,
3
General procedure for the synthesis of symmetrical esters of
NH), 5.07 (s, 1 H, C -H), 5.03 (m, 2 H, CH(CH ), 4.24 (s, 3 H,
), 2.20 (s, 6 H, C -CH & C -CH ), 1.43 (m, 18 H each,
).
4
3 2
)
1
3
,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-2-imidazolyl)-
,5-pyridinedicarboxylates 18–25
N-CH
C(CH
3
3
2
3
6
3
)
3
A solution of ammonium hydroxide (25 %, 0.75 mL) was added
to a stirring solution of 1-methyl-5-nitro-imidazole-2-carboxal-
dehyde 15 (0.78 g, 5 mmol) or the respective acetoacetic esters
–
1
2
IR (KBr):ν 3407 (NH), 1709 (C=O), 1521 and 1378 cm (NO ).
3,5-Di-npentyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-2-
7
–14 (11 mmol) in absolute ethanol (5 mL). The mixture was
imidazolyl)-3,5-pyridinedicarboxylate 25
heated under reflux overnight.The reaction mixture was cooled
and poured into ice-water (10 mL). The sticky solid which pre-
cipitated was removed by filtration and recrystallized from etha-
nol.
1H NMR (CDCl
H-4), 5.16 (s, 1 H, C
H, CO CH ), 2.26 (s, 6 H, C
CH CH CH ), 0.91 (t, J = 6.1 Hz, 6 H, CH
3
): δ 9.26 (br s, 1 H, NH), 7.96 (s, 1 H, imidazole
-H), 4.25 (s, 3 H, N-CH ), 4.12 (t, J = 6 Hz,
-CH & C -CH ), 1.29 (m, 12 H,
).
3
4
3
4
2
2
2
3
6
3
2
2
2
–
1
2
IR (KBr):ν 3411 (NH), 1706 (C=O), 1516 and 1362 cm (NO ).
3
,5-Dimethyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-2-
imidazolyl)-3,5-pyridinedicarboxylate 18
General procedure for the synthesis of unsymmetrical esters
of 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-2-imidazolyl)-
3,5-pyridinedicarboxylates 26–38
1H NMR (CDCl
): δ 8.92 (br s, 1 H, NH), 7.71 (s, 1 H, imidazole
3
H-4), 5.21 (s, 1 H, C4-H), 4.61 (s, 6 H, CO CH ), 4.18 (s, 3 H, N-
CH ), 2.31 (s, 6 H, C -CH & C -CH ).
2
3
3
2
3
6
3
A mixture of the respective acetoacetic esters 7–14 (5.0 mmol),
1-methyl-5-nitro-imidazole-2-carboxaldehyde 15 (0.78 g,
–
1
2
IR (KBr):ν 3374 (NH), 1747 (C=O), 1543 and 1333 cm (NO ).
©
2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 422–428
mmol), and the respective alkyl 3-aminocrotonates (5.0
mmol) 16–18 in absolute ethanol (25 mL) was refluxed for 16 h
with stirring. After cooling, the precipitated product was filtered
off, washed with cold ethanol, and then dried in vacuo. Recrys-
tallization from methanol gave 26–36 (27–53 %) as yellow or
white crystals.3-Ethyl, 5-methyl 1,4-dihydro-2,6-dimethyl-4-(1-
methyl-5-nitro-2-imidazolyl)-3,5-pyridinedicarboxylate 26
Pharmacological Activities of a Dicarboxylate 427
5
CO
2
CH
2
), 2.23 (s, 6 H, C
), 1.24 (t, J = 7.1 Hz, 3 H, CH
2
-CH
3
& C
6 3
-CH ), 1.34 (m, 4 H,
CH
CH
2
CH
).
2
3
), 0.90 (t, J = 7.1 Hz, 3 H,
3
–
1
IR (KBr): ν 3418 (NH), 1728 (C=O), 1548,1369 cm (NO
2
).
3
-isoButyl, 5-methyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-
nitro-2-imidazolyl)-3,5-pyridinedicarboxylate 33
1H NMR (CDCl
H-4), 5.15 (s, 1 H, C
Hz, 2 H, CO CH ), 3.77 (s, 3 H, CO
-CH ), 1.67 (t, J = 6.8 Hz, 3 H, CH
3
): δ 8.94 (br s, 1 H, NH), 7.81 (s, 1 H, imidazole
-H), 4.27 (s, 3 H, N-CH ), 4.26 (t, J = 6.8
CH ), 2.22 (s, 6 H, C -CH
).
1H NMR (CDCl
3
): δ 9.02 (br s, 1 H, NH), 7.95 (s, 1 H, imidazole
-H), 4.23 (s, 3 H, N-CH ), 3.88 (t, J =
), 3.76 (s, 3 H, CO CH ), 2.23 (s, 6 H, C
), 0.87 (d, J = 6.6 Hz,
3
4
3
H-4), 5.17 (s, 1 H, C
.8 Hz, 2 H, CO CH
CH & C -CH
6 H, CH(CH
4
3
2
2
2
3
2
3
&
6
2
-
2
2
2
3
C
6
3
3
6
3
), 1.90 (m, 1 H, CH(CH
3 2
) ).
3 2
)
–
1
IR (KBr): ν 3462 (NH), 1698 (C=O), 1597,1365 cm (NO
2
).
–
1
IR (KBr): ν 3401 (NH), 1715 (C=O), 1561, 1368 cm (NO
2
).
3
-nPropyl, 5-methyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-
nitro-2-imidazolyl)-3,5-pyridinedicarboxylate 27
3
-isoButyl, 5-ethyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-ni-
1H NMR (CDCl
tro-2-imidazolyl)-3,5-pyridinedicarboxylate 34
3
): δ 9.23 (br s, 1 H, NH), 7.94 (s, 1 H, imidazole
-H), 4.22 (s, 3 H, N-CH ), 4.05 (t, J = 6.7
), 3.71 (s, 3 H, CO CH ), 2.19 (s, 6 H, C
), 1.62 (m, 2 H, CH ), 0.87 (t, J = 6.7 Hz, 3 H, CH
H-4), 5.17 (s, 1 H, C
Hz, 2 H, CO CH
-CH
4
3
1H NMR (CDCl
3
): δ 8.78 (br s, 1 H, NH), 7.94 (s, 1 H, imidazole
-H), 4.23 (s, 3 H, N-CH ), 4.14 (q, J =
), 3.89 (d, J = 6.6 Hz, 2 H, CO CH ), 2.23
-CH ), 1.90 (m, 1 H, CH(CH ), 1.23 (t, J =
), 0.87 (d, J = 6.4 Hz, 6 H, CH(CH
2
2
2
3
2
-CH
3
&
H-4), 5.16 (s, 1 H, C
.8 Hz, 2 H, CO CH
(s, 6 H, C -CH & C
.8 Hz, 3 H, CH
4
3
C
6
3
2
3
).
6
2
2
2
2
–
1
2
3
6
3
3 2
)
IR (KBr): ν 3341 (NH), 1718 (C=O), 1504,1368 cm (NO
2
).
6
3
3
)
2
).
3
-nPropyl, 5-ethyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-
–
1
IR (KBr): ν 3411 (NH), 1731 (C=O), 1547, 1381 cm (NO
2
).
nitro-2-imidazolyl)-3,5-pyridinedicarboxylate 28
1H NMR (CDCl
3-tButyl, 5-methyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-ni-
tro-2-imidazolyl)-3,5-pyridinedicarboxylate 35
3
): δ 8.57 (br s, 1 H, NH), 7.95 (s, 1 H, imidazole
-H), 4.22 (s, 3 H, N-CH ), 4.14 (q, J =
), 4.04 (t, J = 6.2 Hz, 2 H, CO CH ), 2.24
-CH ), 1.62 (m, 2 H, CH ), 1.24 (t, J =
), 0.89 (t, J = 6.2 Hz, 3 H, CH ).
H-4), 5.15 (s, 1 H, C
.2 Hz, 2 H, CO CH
s, 6 H, C -CH & C
.2 Hz, 3 H, CO CH
4
3
7
2
2
2
2
1H NMR (CDCl
3
): δ 8.21 (br s, 1 H, NH), 7.95 (s, 1 H, imidazole
-H), 4.23 (s, 3 H, N-CH ), 3.68 (s, 3 H,
), 2.46 & 2.19 (two s, 3 H each, C -CH & C -CH ), 1.42
(s, 9 H, C(CH ).
(
2
3
6
3
2
H-4), 5.19 (s, 1 H, C
CO CH
4
3
7
2
3
3
2
3
2
3
6
3
–
1
3 3
)
IR (KBr): ν 3421 (NH), 1723 (C=O), 1528, 1365 cm (NO
2
).
–
1
IR (KBr): ν 3414 (NH), 1728 (C=O), 1549, 1367 cm (NO
2
).
3
-isoPropyl, 5-methyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-
nitro-2-imidazolyl)-3,5-pyridinedicarboxylate 29
3
-tButyl, 5-ethyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-
1H NMR (CDCl
2-imidazolyl)-3,5-pyridinedicarboxylate 36
3
): δ 8.63 (br s, 1 H, NH), 7.96 (s, 1 H, imidazole
-H), 5.02 (m, 1 H, CO CH(CH ), 4.23 (s,
), 3.68 (s, 3 H, CO CH ), 2.22 (two s, 3 H each, C
), 1.23 and 1.16 (two d, J = 6.0 Hz, 3 H each,
H-4), 5.14 (s, 1 H, C
H, N-CH
4
2
3
)
2
1H NMR (CDCl
3
): δ 8.31 (br s, 1 H, NH), 7.95 (s, 1 H, imidazole
-H), 4.24 (s, 3 H, N-CH ), 4.13 (q, J =
), 2.25 & 2.19 (two s, 3 H each, C -CH
), 1.98 (s, 9 H, C(CH ), 1.23 (t, J = 5.6 Hz, 3 H, CH
3
CH
CH(CH
3
2
3
2
-
H-4), 5.12 (s, 1 H, C
.6 Hz, 2 H, CO CH
-CH
4
3
3
& C
6
-CH
).
3
5
C
3
&
2
2
2
3
)
2
)
).
6
3
3
3
3
–
1
IR (KBr): ν 3383 (NH), 1737 (C=O), 1517, 1362 cm (NO
2
).
–1
IR (KBr): ν 3371 (NH), 1738 (C=O), 1561,1388 cm (NO
2
).
3
-isoPropyl, 5-ethyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-
3
-nPentyl, 5-methyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-ni-
nitro-2-imidazolyl)-3,5-pyridinedicarboxylate 30
tro-2-imidazolyl)-3,5-pyridinedicarboxylate 37
1H NMR (CDCl
): δ 8.25 (br s, 1 H, NH), 7.97 (s, 1 H, imidazole
1H NMR (CDCl
3
): δ 8.87 (br s, 1 H, NH), 7.95 (s, 1 H, imidazole
-H), 4.21 (s, 3 H, N-CH ), 4.07 (t, J =
), 3.76 (s, 3 H, CO CH ), 2.22 & 2.17 (two
), 1.29 (m, 6 H, CH CH CH ), 0.89
3
H-4), 5.13 (s, 1 H, C -H), 5.06 (m, 1 H, CH(CH ), 4.23 (s, 3 H,
N-CH ), 4.17 (q, J = 7.2 Hz, 2 H, CO CH ), 2.19 (s, 6 H, C -CH
-CH ), 1.58 (t, J = 7.2 Hz, 3 H, CH ) 1.21 and 1.19 (two d,
J = 6.0 Hz, 3 H each, CH(CH ).
4
3 2
)
H-4), 5.15 (s, 1 H, C
.0 Hz, 2 H, CO CH
s, 3 H each, C -CH & C
t, J = 7.2 Hz, 3 H, CH
4
3
3
2
2
2
3
6
2
2
2
3
&
C
6
3
3
2
3
6
-CH
).
3
2
2
2
3 2
)
(
3
–
1
IR (KBr): ν 3411 (NH), 1728 (C=O), 1542,1364 cm (NO
2
).
–1
IR (KBr): ν 3361 (NH), 1717 (C=O), 1549, 1367 cm (NO
2
).
3
-nButyl, 5-methyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-ni-
3
-nPentyl, 5-ethyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-ni-
tro-2-imidazolyl)-3,5-pyridinedicarboxylate 31
tro-2-imidazolyl)-3,5-pyridinedicarboxylate 38
1H NMR (CDCl
): δ 9.01 (br s, 1 H, NH), 7.99 (s, 1 H, imidazole
1H NMR (CDCl
3
): δ 9.08 (br s, 1 H, NH), 7.95 (s, 1 H, imidazole
-H), 4.23 (s, 3 H, N-CH ), 4.12 (m, 2 H,
), 2.21 (s, 6 H, C -CH & C -CH ), 1.59 (t, J = 6.8 Hz,
). 1.27 (m, 6 H, CH CH CH ), 0.86 (t, J = 6.7 Hz, 3 H,
3
H-4), 5.08 (s, 1 H, C -H), 4.19 (s, 3 H, N-CH ), 4.02 (t, J =
.6 Hz, 2 H, CO CH ), 3.76 (s, 3 H, CO CH ), 2.21 (s, 6 H, C
), 1.51 (m, 4 H, CH CH ), 0.91 (t, J = 6.7 Hz, 3 H,
4
3
H-4), 5.16 (s, 1 H, C
CO CH
H, CH
CH ).
4
3
6
CH
CH
2
2
2
3
2
-
2
2
2
3
6
3
3
3
& C
).
6
-CH
3
2
2
3
3
2
2
2
3
–
1
IR (KBr): ν 3317 (NH), 1728 (C=O), 1542, 1391 cm (NO
2
).
–1
2
IR (KBr): ν 3421 (NH), 1731 (C=O), 1518, 1351 cm (NO ).
Pharmacology
3
2
-nButyl, 5-ethyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-
-imidazolyl)-3,5-pyridinedicarboxylate 32
Investigations on isolated ileum of guinea pigs
1H NMR (CDCl
): δ 8.67 (br s, 1 H, NH), 7.95 (s, 1 H, imidazole
H-4), 5.17 (s, 1 H, C -H), 4.23 (s, 3 H, N-CH ), 4.11 (m, 4 H,
Male albino guinea pig (300–450 g) was killed by a blow to the
head.The intestine was removed above the ileocecal junction.
3
4
3
©
2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

4
28 Miri et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 422–428
Smooth muscle segments of about 1 cm length were mounted
under a resting tension of 500 mg and were maintained at 37 °C
in a 20 mL jacketed organ bath containing oxygenated (95 %
[3] N. Morel, V. Buryi, O. Feron, J. P. Gomez, M. O. Christen, T.
Godfraind, Br. J. Pharmacol. 1998, 125, 1005–1012.
[
4] D. J. Triggle, Can. J. Physio. Pharmacol. 1990, 68, 1474–
481.
O
2
, 5 % CO
molar compositions: NaCl 137; CaCl
NaH PO 0.4; NaHCO 12, and glucose 5. The muscle was
2
) physiologic saline solution of the following milli-
1
2
1.8; KCl 2.7; MgSO 1.1;
4
2
4
3
[
[
5] T. F. Luscher, F. Cosentino, Drugs, 1998, 55, 509–516.
6] L. Lacinova, F. Hofmann, Receptors Channels, 1998, 5,
equilibrated for 1 h with a solution changing every 15 min.The
contractions were recorded with a forced displacement trans-
ducer (FTO3C) on a GRASS physiograph (GRASS Instru-
ments, Quincy, MA, USA). All compounds were dissolved in
DMSO and the same volume of solvent was used as the con-
trol.The contractile response was taken as the 100 % value for
the tonic (slow) component of the response. Test compounds
were added in accumulative increment after the dose response
1
53–164.
[
7] S. Yusuf, Circulation, 1995, 92, 1079–1082.
[8] H. F. Miranda, D. Bustamante, V. Kramer, T. Pelissier, H.
Saavedra, C. Paeile, E. Fernandez, G. Pinardi, Eur. J.
Pharmacol. 1992, 217(2–3), 137–141.
–
7
for carbacol (1.67 × 10 M).Test compound-induced relaxation
of contracted muscle was expressed as the percent of the con-
trol [20, 21].The IC50 values were graphically determined from
the contraction-response curve.
[
9] W.A.Prado, Braz.J.Med.Biol.Res.2001, 34(4), 449–461.
[10] M. Murakami, O. Nakagawasai, S. Fujii, K. Kameyama, S.
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isawa, K. Tanno, T. Tadano, K. Kitamura, K. Kisara, Eur. J.
Pharmacol. 2001, 419(2–3), 175–181.
Tail-flick test
All experiments were preformed on mice (albino males, in-
house bred, 25–35 g). 12 animals were kept in one cage on
straw bedding in an animal holding room at 24 ± 2 °C with a
[
11] A. C. Gaudio, A. Korolkovas, Y. Takahata, J. Pharm. Sci.
994, 83, 1110–1115.
1
1
2 h light-dark cycle. Balanced diet pellets and tap water were
[12] B. Hemmateenejad, R. Miri, M. Akhond, M. Shamsipur,
Chemometrics and Intelligent Laboratory Systems, 2002,
64, 91–99.
continuously available.Changes in pain perception due to drug
treatments were determined in a quiet laboratory with ambient
illumination and temperature close to those of the holding
room. Mice were allowed to acclimate to the testing area for 1 h
before the experiments began. Nociceptive response was as-
sessed with a tail flick apparatus (HSE, March-Hugstetten,
Germany) using a method initially described by D’Amour and
Smith [22]. Animal responded to a focused heat-stimulus by
flicking or removing their inflicted tail. The base-line latency
time (reaction time, in seconds) was obtained with three meas-
urements. The mean of these measurements was considered
as the pre-drug latency time.The drug was administered intra-
peritoneal (50 mg/kg) immediately after the third pre-drug
measure (n = 4).Another set of three measurements was taken
[
13] B. Hemmateenejad, R. Miri, M. Akhond, M. Shamsipur,
Arch. Pharm. Pharm. Med. Chem. 2002, 10, 472–480.
[
14] A. Hantzsch, Justus Liebigs Ann. Chem. 1882, 215, 1–82.
[15] A. Shafiee, R. Miri, A. R. Dehpour, F. Soleymani, Pharm.
Sci. 1996, 2, 541–543.
[
16] R. J. Clemans, H. A. Hyatt, J. Org. Chem. 1985, 50, 2431–
435.
2
[17] M. Iwanami, T. Shibanuma, M. Fujimoto, R. Kawai, K.
Tamazawa, T. Takenaka, K. Takahashi, M. Murakami,
Chem. Pharm. Bull. 1979, 27, 1426–1440.
2
5 min afterwards for intraperitoneal (i.p.) administrations, and
their mean was considered as post-drug latency time. A cut-off
time of 10 s was used to prevent tissue damage [23, 24].
[
[
[
[
[
18] A. Shafiee, B. Pirouzzadeh, F. Ghasemian, K. Parang, J.
Heterocyclic Chem, 1992, 29, 1021–1023.
Statistical analysis
19] R. Akula, W. C.Matowe, M.W.Wolowyk, E. E.Kanus, Drug
Design and Delivery 1990, 7, 11–17.
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considered significant.
20] C. R. Triggle, V. Swamy, D. J. Triggle, Can. J. Physiol.
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©
2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim