Synthesis and biological evaluation of some new 1,4-dihydropyridines containing different ester substitute and diethyl carbamoyl group as anti-tubercular agents

Article abstract of DOI:10.1016/j.bmc.2008.12.070

Tuberculosis is a leading infectious cause of death worldwide. Because of the concern of the resistance to most of the commonly used drugs displayed by the considered mycobacteria, most efforts have been done to introduce new anti-tubercular agents. Recent studies showed that 1,4-dihydropyridine-3,5-dicarbamoyl derivatives with lipophilic groups have significant anti-tubercular activity. In this study, we synthesized new derivatives of 1,4-dihydropyridines in which different alkyl and aryl esters and diethyl carbamoyl are substituted in C-3 and C-5 of the DHP ring. In addition nitroimidazole ring is substitutes at C-4 position. These asymmetric analogues were synthesized by a modified Hantzsh reaction using procedure reported by Meyer. The in vitro anti-tubercular activity of compounds against Mycobacterium tuberculosis was evaluated. The results indicate that the compounds containing aromatic esters are more potent than alkyl ones. The most potent aromatic compound (R = 3-phenylpropyl) exhibits comparable anti-tubercular activity (MIC = 1 μmol/ml) with reference compound isoniazide (INH) (MIC = 1 μmol/ml). Conformational analysis, SAR studies of these compounds showed that increasing in lipophilicity and rotable bonds of these compounds resulted in increasing anti-tubercular activity.

Full text of DOI:10.1016/j.bmc.2008.12.070

Bioorganic & Medicinal Chemistry 17 (2009) 1579–1586
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of some new 1,4-dihydropyridines
containing different ester substitute and diethyl carbamoyl group
as anti-tubercular agents
Mehdi Khoshneviszadeh a,b, Najmeh Edraki a,b, Katayoun Javidnia a,b, Abdolvahab Alborzi c,
Bahman Pourabbas c, Jalal Mardaneh c, Ramin Miri a,b,
*
a Medicinal and Natural Products Chemistry Research Centre, Shiraz University of Medical Sciences, PO Box 3288-71345, Shiraz, Iran
b Department of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
c Prof. Alborzi Clinical Microbiology Research center, Shiraz University of Medical Sciences, Shiraz, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Tuberculosis is a leading infectious cause of death worldwide. Because of the concern of the resistance to
most of the commonly used drugs displayed by the considered mycobacteria, most efforts have been
done to introduce new anti-tubercular agents. Recent studies showed that 1,4-dihydropyridine-3,5-dic-
arbamoyl derivatives with lipophilic groups have significant anti-tubercular activity. In this study, we
synthesized new derivatives of 1,4-dihydropyridines in which different alkyl and aryl esters and diethyl
carbamoyl are substituted in C-3 and C-5 of the DHP ring. In addition nitroimidazole ring is substitutes at
C-4 position. These asymmetric analogues were synthesized by a modified Hantzsh reaction using proce-
dure reported by Meyer. The in vitro anti-tubercular activity of compounds against Mycobacterium tuber-
culosis was evaluated. The results indicate that the compounds containing aromatic esters are more
potent than alkyl ones. The most potent aromatic compound (R = 3-phenylpropyl) exhibits comparable
Received 15 September 2008
Revised 28 December 2008
Accepted 31 December 2008
Available online 6 January 2009
Keywords:
1,4-Dihydropyridines
Anti-tubercular activity
Conformational analysis
anti-tubercular activity (MIC = 1 lmol/ml) with reference compound isoniazide (INH) (MIC = 1 lmol/
ml). Conformational analysis, SAR studies of these compounds showed that increasing in lipophilicity
and rotable bonds of these compounds resulted in increasing anti-tubercular activity.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
in use to combat the emergence of the resistance. Therefore, the
search for new antimycobacterials is the subject of numerous recent
Tuberculosis is today amongst the worldwide health threats and
continues to be one of the most important global infectious causes
of morbidity and mortality.1,2 Mycobacterium tuberculosis (MTb) in-
fects approximately one-third of the world’s population, kills more
than 2 million people a year and is comparable only to human immu-
nodeficiency virus (HIV) as an infectious cause of death.1,3 Despite the
development of effective drug therapy for MTb over 50 years ago,
there remain several formidable challenges for controlling MTb infec-
tion. These obstacles include lengthy treatment regimens of 6–9
months duration, drug resistance, the lack of a highly efficacious vac-
cine, and an incomplete understanding of what controls infectivity
and progression of disease.1 The problem is being aggravated by
increasingresistantagainstfrontline drugsandsynergyofthisdisease
with HIV and mycotic infections in Immunocompromised patients.4
No new drug against tuberculosis has been developed in the last
30 years. Hence, there is urgent need for new chemotherapeutic
agents, preferably having a different mode of action than presently
studies.5–10 Recently, studies showed that 3,5-dicarbamoyl deriva-
tives of 1,4-dihydropyridine (DHP) with lipophilic groups have con-
siderable anti-tubercular activity against M. tuberculosis H37Rv.11 It
was also observed that esters or substituted isosters of pyridine and
pyrazinecarboxylic acids (such as tetrazoles) have been more active
than the parent acids specially against resistant strains. These esters
are presumably activated by an esterase to parent acid.12–15 Indeed,
esters of pyrazinoic acids have been shown to posses activity against
pyrazinamide-resistant isolates which has been attributed to a defi-
ciency of nicotinamidase.12–15 In addition, nitroimidazolyl moiety
(e.g., Metronidazole) and nitroimidazopyran exhibited significant
anti-tubercular activity.16 In view of this, it appeared of interest to
design and synthesize new derivatives of 1,4-dihydropyridines in
which different alkyl and aryl esters and diethyl carbamoyl are
substituted in C-3 and C-5 of the DHP ring, respectively. It seems that
such replacements could effectively overcome the resistant isolates
which have been attributed to a deficiency of amidase or esterase. In
addition, nitroimidazolyl moiety is substituted at C-4 position of
dihydropyridine ring. Theanti-mycobacterial activityof synthesized
compounds was evaluated against M. tuberculosis H37Rv strain.
* Corresponding author. Tel.: +98 711 2303872; fax: +98 711 2332225.
E-mail address: mirir@sums.ac.ir (R. Miri).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.070

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M. Khoshneviszadeh et al. / Bioorg. Med. Chem. 17 (2009) 1579–1586
Table 1
As the different studies have indicated, 4-aryl-1,4-dihydropyri-
Physical, and antimycobacterial data for compounds 7a–j
dines (DHPs) are the most studied class of organic calcium channel
antagonist. Therefore; as the synthesized compounds have 1,4-
DHP structure, they could potentially exhibit calcium channel
blocking (CCB) activity. As far as these compounds have been de-
signed as anti-tubercular agents, the CCB activity could be one of
the most important side effects of these compounds that cause
hypotension, bradycardia and other associated side effects. For this
reason; the in vitro CCB activity of synthesized compounds was
evaluated to know the structural requirement to decrease the side
effect and increase anti-tubercular activity.
O2N
N
N
H3C
ROOC
CON(C2H5)2
N
H3C
CH3
H
LogPa
2. Chemistry
Compound
R
Mp (°C)
Yield (%)
MIC (
lmol/lit)
7a
7b
7c
7d
7e
7f
7g
7h
CH3
180–184
193–196
185–190
205–209
207–212
185–189
166–169
139–143
124–129
113–118
10.45
12.2
15.8
13.52
16.2
11.43
11.8
13.72
12.32
16.7
>500
250
>500
À0.98
À0.64
À0.23
À0.17
0.22
0.79
1.04
1.44
1.84
2.23
—
The synthesis of the 1,4-dihydropyridine derivatives 7a–j was
achieved following the steps outlined in Figure 1. Reaction of alco-
hol 1d–e and 1g,i–j with 2,2,6-trimethyl-4H-1,3-dioxin-4-one 2
afforded the corresponding acetoacetic esters 3d–e and 3g,i–j
(70–85% yield).17 Reaction of acetoacetic esters 3 with ammonium
acetate afforded the corresponding alkyl 3-amminocrotonate 4. In
addition, reaction of 1-methyl-5-nitroimidazol-2-carboxaldehyde
5 with N,N-diethyl acetoacetamide afforded the corresponding
CH2CH3
(CH3)2CH2
(CH2)2CH3
(CH2)3CH3
CH2–C6H5
CH2)2C6H5)
CH2)3C6H5)
CH2)4C6H5)
CH2)5C6H5)
—
2
1
1
1
1
2
2
1
1
7i
7j
Isoniazide
Rifampine
intermediate,
N,N-diethyl-2-(1-methyl-5-nitro-1H-imidazole-2-
—
—
yl(methylen)3-oxobutan amide 6 (25% yield).18–20 The asymmetri-
cal analogues 7a–j were synthesized by a modified Hanstzch
reaction, using a procedure reported by Meyer et al. (10–17%
yield).21–23 These compounds were purified by preparative thin-
layer chromatography and recrystalization, then characterized by
mass spectroscopy, IR and 1H NMR. The yields and melting points
of final compounds are summarized in Table 1.
a
Calculated by Hyperchem software.
proportion method in Middlebrook 7H10 medium. The results of
the anti-mycocabacterial activity are listed in Table 1.
3.2. Pharmacology
3. Biology
As the earlier discuss, 4-aryl-1,4-dihydropyridines (DHPs) are
the most studied class of organic calcium channel modulators,
and since their introduction into clinical medicine in 1975, this
class has found widespread uses for the treatment of cardiovascu-
lar disease.24–26 Therefore; the calcium channel blocking (CCB)
activity could have been the potential side effect of the designed
3.1. Anti-mycobacterial activity
Minimum inhibitory concentration (MIC) of the compounds is
found against M. tuberculosis H37Rv strain ATCC 27294 by agar
O
-
+
4
O
O
ROOC
NH
O
CH COO
3
ROCCH2CCH3
ROH
+
Xylene
EtOH
O
CH3 NH2
1d-e/1g,i-j
2
3a-j
4a-j
O2N
O2N
O
O
N
H
N
H3C
CH3CCH2CN(C2H5)2
Piperidine, CH3COOH
N
N
H3C
CON(C2H5)2
CH3
CHO
O
5
6
O2N
N
N
H3C
ROOC
2-propanol
Reflux
CON(C2H5)2
CH3
4a-j
+
6
N
CH3
H
7a-j
Figure 1. Synthetic pathway for dihydropyridine derivatives used in this study.

M. Khoshneviszadeh et al. / Bioorg. Med. Chem. 17 (2009) 1579–1586
1581
compounds. Therefore, it is necessary to evaluate in vitro calcium
channel antagonist activity of the compounds in order to under-
stand the minimal structural requirements to decrease CCB activ-
ity. The calcium channel antagonist activity of compounds was
determined as the concentration needed to produce 50% inhibition
of the guinea-pig ileal longitudinal smooth muscle (GPILSM) con-
tractility is summarized in Table 2.
The results of CCB activity (Table 2) indicate that all compounds
7a–j are weak CCB agents in compared with reference drug nifedi-
pine (P < 0.05) (100–1000 times weaker than nifedipine). Struc-
ture–activity relationship study of 1,4-dihydropyridines as CCB
agents also indicate that the ester groups are important to the phar-
macological effect. Not only does variation of the ester groups lead to
quantitative changes in the activity, but replacement of an ester
groups by other groups can also produce dramatic qualitative
changes.25 Therefore, we can deduced that substitution of ester
group with amide type could significantly decrease CCB activity
and this result is favorable for design of DHPs as anti-tubercular
agents. Comparison of the activities of aryl ester compounds 7f–i
and alkyl ester series 7a–e indicates that aryl esters are more active
(in CCB effect) than alkyl compounds. As far as, aryl ester groups ex-
hibit much higher calcium channel blocking and anti-mycobacterial
activity in compared to alkyl ester group, it could be deduced that
lipophilicity and/or aromaticity of ester group have an increasing ef-
fect on both biological activities. However; the lipophilicity should
be optimal. As we can see in Tables 1 and 2; increasing in aliphatic
chain of aryl esters and hence increasing the lipophilicity (LogP) of
compound (compound 7j); resulted in decreased biological activities.
Comparison of conformational parameters of the compounds
(Table 3) with nifedipine indicates that the obvious conformational
feature of the designed compounds is significant deviation of DHP
ring from planarity (C2–C3–C5–C6 angle is deviated from nifedipine
angle); while, different X-ray structural investigation, computa-
tional and statistical calculationsconfirm a preference for a flattened
boat conformation of the DHP ring in nifedipine and its analogues as
CCB agents.25–28 In addition, different studies of 1,4-DHPs indicates
that cis/trans and cis/cis arrangements are preferred conformation of
ester groups for CCB activity.25–29 Conformational analysis of com-
pound 7a–j indicates that the ester and amide groups positioned at
C3 and C5 of DHP ring of designed compounds, specially in the most
potent anti-tubercular agents (7e–i) prefer the trans/trans orienta-
tion (C2–C3–C9–O10 and C5–C6–C11–O12 dihedral angles are between
+90 and +180); Figure 2. While cis/cis orientation is preferred in
nifedipine ester groups. Furthermore, the conformation of nitroimi-
dazolyl group is also deviated slightly from pseudoaxial form which
is predominant in nifedipine analogues.
4. Conformational analysis
The optimal 3D structure of the molecules obtained by semi-
emprical/AM1 calculations is represented in Figure 2 and some
important dihedral angles are listed in Table 3. The most important
conformational features considered in this study are:
–
cis or trans orientation of carbonyl groups bonded at C3 and C5;
C2–C3–C9–O10 and C5–C6–C11–O12 dihedral angles reflect it. If these
angles were between À90° and +90°, the orientation was consid-
ered cis, and the trans orientation was assumed when the dihedral
angels were between (+90°)–(+180°) or (À90°)–(À180°). The mol-
ecules were assigned as cis–cis, cis–trans and trans–trans based
on orientation of the individual carbonyl groups.
– The relative position of nitroimidazolyl ring with respect to DHP
ring as measured by C’2–C4–C3–C2 dihedral angle.
– The deviation from planarity of DHP ring as measured by C2–C3–
C5–C6 dihedral angle.
In addition numerical calculation of lipophilicity operated with
Hyperchem software, is listed in Table 1.
5. Results and discussion
Ten different alkyl and aryl ester derivatives of 5-(diethyl car-
bamoyl)2,6-dimethyl-4-(1-methyl-5-nitro-1H imidazole-2-yl)1,4-
dihydropyridine-3-carboxylate were synthesized and tested for
their inhibition activities against M. tuberculosis. The results indi-
cate that the overall anti-tubercular activity of aryl ester substitu-
ents is more than alkyl ones and could be comparable with INH
and rifampine. In aryl series, the compounds 7f–h (R = benzyl,
phenethyl, 3-phenyl propyl, respectively) show the most anti-
The most important conformational parameters for different
compounds specially the trans/trans conformation of carbamoyl
and ester substitutes, pseudoaxail orientation of nitroimidazolyl
group are represented in Figure 2.
It could be deduced that the conformational structure of de-
signed compounds deviated from CCB activity, makes them suit-
able anti-mycobacterial candidate.
mycobacterial activity (MIC = 1 lM). Moreover, increasing the ali-
phatic chain of aryl ester substituents (compounds 7i–j; R = 4-phe-
nyl butyl and 5-phenyl pentyl) resulted in decreased activity. In
alkyl series; increasing the aliphatic chain from methyl to n-butyl,
resulted in enhanced activity. But branching the aliphatic chain,
significantly abolish anti-mycobacterial activity (compound 7d;
R = n-propyl vs 7c; R = isopropyl). Therefore, 1,4-dihydropyridines
containing diethyl carbamoyl and aryl esters with optimal ali-
phatic chain could posses the suitable anti-mycobacterial activity.
6. Conclusion
The results indicate that replacement of ester substitute with
amide type in Nifedipine analogues significantly reduces muscle
relaxing activity and increase anti-mycobacterial effect. Therefore;
1,4-dihydropiridines with N,N-diethyl carbamoyl and aryl esters
with optimal lipophilicity could be a suitable candidates against M.
tuberculosis in vivo anti-mycobacterial evaluation is needed for fur-
ther optimization. In addition, in vitro evaluation of designed com-
pounds against resistant strain of M. tuberculosis could be valuable.
Table 2
Calcium channel blocking data for compounds 7a–j
Compound
R
Calcium channel antagonist activity IC50 SEM (n = 3)
7a
7b
7c
7d
7e
7f
7g
7h
CH3
CH2CH3
3.22 0.011 Â 10À5
2.97 0.008 Â 10À5
1.88 0.008 Â 10À5
1.20 0.007 Â 10À5
8.57 0.015 Â 10À6
4.2 0.118 Â 10À6
2.4 0.209 Â 10À6
2.7 0.064 Â 10À6
3.5 0.246 Â 10À6
2.4 0.110 Â 10À5
1.07 0.170 Â 10À8
(CH3)2CH2
(CH2)2CH3
(CH2)3CH3
CH2–C6H5
CH2)2C6H5)
CH2)3C6H5)
CH2)4C6H5)
CH2)5C6H5)
—
7. Experimental
Melting points were determined on an Electrothermal 9200
apparatus (England) and are uncorrected. 1H NMR spectra were ob-
tained with a Bruker-Avance DPX-250 MHz spectrometer (Ger-
many) in chloroform-d1 and tetramethylsilane (TMS) was used as
an internal standard. The mass spectra were measured with a
7i
7j
Nifedipine

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M. Khoshneviszadeh et al. / Bioorg. Med. Chem. 17 (2009) 1579–1586
Figure 2. The optimized 3D structures of the compounds obtained by semiemprical/AM1 calculation.
Hewlett Packard (HP) 6890 (Germany) at 70 eV. The IR spectra
were obtained by using a Perkin Elmer (KBr disks) (England). The
results of elemental analyses (C, H and N) were within 0.4% of
the calculated values. Silica gel HT-254 (E. Merck, Darmstadt, Ger-
many) was used for thin-layer chromatography. All spectra were
consistent with the assigned structures. Methyl, ethyl, isopropyl,
benzyl and 3-phenyl propyl acetoacetates 1a–c, 1f and 1h, 2,2,6-
trimethyl-4H-1,3-dioxin-4-one 2 and N,N-diethyl acetoecetamide
purchased from the Aldrich Chemical Co. (Sigma–Aldrich Chemie
GmbH, Deisenhofon, Germany).
7.1.1. n-Propyl acetoacetate (3d)
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).
IR (KBr):
m
1743 (C@O, ester), 1714 cmÀ1 (C@O, ketone).
7.1.2. n-Butyl acetoacetate (3e)
1H 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).
IR (KBr):
m
1740 (C@O, ester), 1717 cmÀ1 (C@O, ketone).
7.1.3. Phenethyl acetoacetate (3g)
7.1. General procedure for the synthesis of acetoacetic esters
(3d–e and 3g,i–j)
1H NMR (CDCl3): d 7.32 (m, 5H, Phenyl), 4.18 (t, J = 6.5 Hz, 2H,
CO2CH2), 3.39 (s, 2H, COCH2CO2), 2.69 (t, J = 6.5, 2H, CH2Phenyl),
2.46 (s, 3H, CH3CO).
A solution of alcohol 1d–e and 1g,i–j (50 mmol) and 2,2,6-tri-
methyl-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 preheated to 150 °C, and the solution
was vigorously stirred. Heating was continued for a total 30 min.
The reaction mixture was cooled. The xylene was removed. Distil-
lation of the mixture afforded 3d–e and 3g,i–j which were used
immediately in subsequent reactions.
IR (KBr):
m
1747 (C@O, ester), 1715 cmÀ1 (C@O, ketone).
7.1.4. 4-Phenyl butyl acetoacetate (3i)
1H NMR (CDCl3): d 7.23 (m, 5H, Phenyl), 4.19 (t, J = 6.2 Hz, 2H,
CO2CH2), 3.26 (s, 2H, COCH2CO2), 2.69 (t, J = 6.7, 2H, CH2Phenyl),
2.27 (s, 3H, CH3CO), 1.83 (m, 4H, CH2–CH2).
IR (KBr):
m
1736 (C@O, ester), 1716 cmÀ1 (C@O, ketone).

M. Khoshneviszadeh et al. / Bioorg. Med. Chem. 17 (2009) 1579–1586
1583
Table 3
Dihedral angles for the most stable conformers of the studied DHP derivatives, obtained by AM1 calculations
O2N
H3C
NO2
10
N
N
1'
12
O
O
2'
2'
O10
O
12
4
9
4
1
11
Me O
OMe
ORO
5
6
N(Et)2
3
2
11
9
3
5
6
2
1
H3C
CH3
N
H3C
N
CH3
H
H
Nifedipine
7a-7j
Compound
R
C2–C3–C5–C6
C2–C3–C9–O10
C6–C5–C11–O12
C’2–C4–C3–C2
7a
7b
7c
7d
7e
7f
7g
7h
CH3
CH2CH3
0.21
0.11
0.82
0.72
0.79
0.27
0.90
0.31
0.37
0.80
0.004
96.31
102.53
128.13
103.22
159.09
175.64
176.61
177.57
163.64
87.89
65.09
92.86
87.89
106.89
102.26
100.32
98.37
(CH3)2CH2
(CH2)2CH3
(CH2)3CH3
CH2–C6H5
CH2)2C6H5)
CH2)3C6H5)
CH2)4C6H5)
CH2)5C6H5)
—
81.10
124.22
105.24
107.98
102.7
110.24
128.13
8.76
94.63
102.08
105.99
115.95
115.09
119.77
92.91
7i
7j
Nifedipine
52.90
7.1.5. 5-Phenyl pentyl acetoacetate (3j)
7.2.7. Phenethyl 3-aminocrotonate (4g)
IR (KBr):
3460 and 3341 (NH2), 1709 cmÀ1 (C@O, ester).
1H NMR (CDCl3): d 7.25 (m, 5H, Phenyl), 4.21 (t, J = 6.6 Hz, 2H,
CO2CH2), 3.53 (s, 2H, COCH2CO2), 2.71 (t, J = 6.2, 2H, CH2Phenyl),
2.25 (s, 3H, CH3CO), 1.91 (m, 6H, CH2–CH2–CH2).
m
7.2.8. 3-Phenyl propyl 3-aminocrotonate (4h)
IR (KBr):
3461 and 3334 (NH2), 1717 cmÀ1 (C@O, ester).
IR (KBr):
m
1737 (C@O, ester), 1713 cmÀ1 (C@O, ketone).
m
7.2.9. 4-Phenyl butyl 3-aminocrotonate (4i)
IR (KBr):
3454 and 3329 (NH2), 1711 cmÀ1 (C@O, ester).
7.2. General procedure for the synthesis of alkyl 3-
aminocrotonate (4a–j)
m
7.2.10. 5-Phenyl pentyl 3-aminocrotonate (4j)
IR (KBr):
3446 and 3341 (NH2), 1709 cmÀ1 (C@O, ester).
A solution of alkyl acetoacetic esters 3a–j (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 preheated to 90 °C, and the solution was vigor-
ously stirred. Heating was continued for a total 24 h. The reac-
tion mixture was cooled. The ethanol was removed and IR
spectra of the compounds were recorded to confirm the struc-
ture of 4a–j. Then, the compounds were immediately used in
subsequent reactions.
m
7.3. General procedure for the synthesis of N,N-diethyl-2-(1-
methyl-5-nitro-1H-imidazole-2-yl (methylen)3-oxobutan
amide (6)
A solution of 1-methyl-5-nitroimidazol-2-carboxaldehyde 5
(300 mg, 2 mmol), N,N-diethyl acetoacetamide (314 mg, 2 mmol),
glacial acetic acid (0.5 ml), piperidine (0.75 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 re-
moved and the residue was purified by chromatography on silica
gel with chloroform–methanol–ethylacetate (92/4/4), to give pure
compound 6 (24% yield) as solid (mp 115–118 °C).
7.2.1. Methyl 3-aminocrotonate (4a)
IR (KBr):
m
3449 and 3370 (NH2), 1709 cmÀ1 (C@O, ester).
7.2.2. Ethyl 3-aminocrotonate (4b)
IR (KBr):
3456and 3400 (NH2), 1712 cmÀ1 (C@O, ester).
MS: m/z(%) 294(M+, 54), 222(35), 206(13), 194(100), 180(70),
167(35), 154(17), 134(29), 106(20), 97(19), 83(16), 72(66),
58(51), 43(26).
Elemental Anal. Calcd for C13H18N4O4: C, 53.05; H, 6.16; N,
19.04.
m
7.2.3. Isopropyl 3-aminocrotonate (4c)
IR (KBr):
3454 and 3338 (NH2), 1712 cmÀ1 (C@O, ester).
m
7.2.4. n-Propyl 3-aminocrotonate (4d)
IR (KBr):
3454 and 3330 (NH2), 1710 cmÀ1 (C@O, ester).
7.4. General procedure for the synthesis of alkyl/aryl 5-(diethyl
carbamoyl)2,6-dimethyl-4-(1-methyl-5-nitro-1H imidazole-2-
yl)1,4 dihydropyridine-3-carboxylate (7a–j)
m
7.2.5. n-Butyl 3-aminocrotonate (4e)
IR (KBr):
3443 and 3329 (NH2), 1708 cmÀ1 (C@O, ester).
m
A solution of compounds 4a–j (1.2 mmol) and N-diethyl-2-(1-
methyl-5-nitro-1H-imidazole-2-yl (methylen)3-oxobutan amide
6 (353 mg, 1.2 mmol) in 2-propanol (5 ml) was protected from
light and refluxed for 24–30 h. After cooling, the solution was
7.2.6. Benzyl 3-aminocrotonate (4f)
IR (KBr):
3412 and 3341 (NH2), 1714 cmÀ1 (C@O, ester).
m

1584
M. Khoshneviszadeh et al. / Bioorg. Med. Chem. 17 (2009) 1579–1586
concentrated under reduced pressure and purified by thin-layer
chromatography on silica gel with chloroform–methanol (94–6%).
The product was recrystallized from methanol to give pure com-
pounds 7a–j.
O–CH2–CH2–CH2–CH3), 1–1.2 (m, 5H, O–(CH2)2–CH2–CH3), 0.08 (t,
J = 7.2, 6H, N(CH2–CH3)2).
IR (NaCl):
m 3238 (NH), 1693 (C@O, ester), 1622 (C@O, amide),
1525 and 1373cmÀ1 (NO2).
MS: m/z(%) 433(M+ 53), 416(19), 360(100), 345(43), 333(37),
307(45), 259(100), 234(32), 213(33), 178(49), 150(24), 100(37),
72(39), 42(11).
7.4.1. Methyl-5-(diethyl carbamoyl)2,6-dimethyl-4-(1-methyl-
5-nitro-1H imidazole-2-yl)1,4 dihydropyridine-3-carboxylate
(7a)
Elemental Anal. Calcd for C21H31N5O5: C, 58.18; H, 7.21; N,
16.16.
1H NMR (CDCl3): d 8.45 (br s, 1H, DHP NH), 8 (s, 1H, Imidazole
H-4), 5.2 (s, 1H, C4–H), 3.9 (s, 3H, N–CH3), 3.7 (q, J = 7.2 Hz, 4H,
((CH2CH3)2), 3.68 (s, 3H, CO2CH3), 2.3 (s, 3H, C6–CH3), 1.79 (s, 3H,
C2–CH3), 1.2 (t, J = 7.2 Hz, 6H, N(CH2–CH3).
7.4.6. Benzyl- 5-(diethyl carbamoyl)2,6-dimethyl-4-(1-methyl-
5-nitro-1H imidazole-2-yl)1,4 dihydropyridine-3-carboxylate
(7e)
IR (KBr):
m 3244 (NH), 1701 (C@O, ester), 1608 (C@O, amide),
1518, 1373 cmÀ1 (NO2).
1H NMR (CDCl3): d 9.45 (br s, 1H, NH-DHP), 8.32 (s, 1H, H-Imid-
azole), 7.15–7.22 (m, 5H-Phenyl), 5.91 (s, 1H, C4H), 4.93 (3H, N–
CH3), 3.9–3.97 (m, 4H, N(CH2CH3)2, 3.16 (s, 2H, CO2CH2), 1.70 (s,
6H, C2–CH3 and C6–CH3), 0.70–0.80 (m,6H N(CH2CH3)2.
MS: m/z(%) 391(M+, 26), 374(18), 318(100), 303(32), 291(8),
259(46), 243(12) 213(20), 192(57), 100(20), 72(37), 42(31).
Elemental Anal. Calcd for C18H25N5O5: C, 55.23; H, 6.44; N, 17.89.
IR (KBr):
m 3317 (NH), 3008 (CH, aromatic), 1695 (C@O, ester),
7.4.2. Ethyl- 5-(diethyl carbamoyl)2,6-dimethyl-4-(1-methyl-5-
nitro-1H imidazole-2-yl)1,4 dihydropyridine-3-carboxylate (7b)
1H NMR (CDCl3): d 9.2 (br s, 1H, DHP NH), 7.9 (s, 1H, Imidazole
H-4), 4.5 (s, 1H, C4–H), 3.9 (m, 7H, N–CH3 and N((CH2CH3)2), 3.7 (q,
J = 7.2 Hz, 2H, OCH2–CH3, 2.4 (s, 3H, C6–CH3), 1.9 (s, 3H,C2–CH3), 1.2
(t, J = 7.2 Hz, 3H, OCH2–CH3), 0.09 (t, J = 7.2, 6H, N((CH2CH3)2).
1611 (C@O, amide), 1522 and 1371cmÀ1 (NO2).
MS: m/z(%) 467(M+ 62), 450(16), 394(99), 367(30), 341(48),
259(100), 91(99), 72(28).
Elemental Anal. Calcd for C24H29N5O5: C, 61.66; H, 6.25; N,
14.98.
IR (KBr):
m
3243 (NH), 1697 (C@O, ester), 1616 (C@O, amide),
7.4.7. Phenethyl-5-(diethyl carbamoyl)2,6-dimethyl-4-(1-
methyl-5-nitro-1H imidazole-2-yl)1,4 dihydropyridine-3-
carboxylate (7f)
1529, 1373 cmÀ1 (NO2).
MS: m/z(%) 405(M+, 8), 388(4), 332(26), 317(11), 305(57),
279(10), 259(100) 206(16), 178(16), 71(29), 57(38), 43(24).
Elemental Anal. Calcd for C19H27N5O5: C, 56.28; H, 6.71; N, 17.27.
1H NMR (CDCl3): d 9.14 (br s, 1H, NH-DHP), 7.86 (s, 1H, H-Imid-
azole), 7.02–7.20 (m, 5H-Phenyl), 5.35 (s, 1H, C4H), 4.20 (3H, N–
CH3), 3.35–3.55 (m, 4H, N(CH2CH3)2, 2.79 (t, 2H, CO2CH2), 2.19 (s,
6H, C2–CH3 and C6–CH3), 1.16 (m, 6H, N(CH2CH3)2, 0.75–0.93 (t,
CO2CH2CH2).
7.4.3. Isopropyl-5-(diethyl carbamoyl)2,6-dimethyl-4-(1-
methyl-5-nitro-1H imidazole-2-yl)1,4 dihydropyridine-3-
carboxylate (7c)
IR (KBr):
m 3252 (NH), 3086 (CH, aromatic), 1696 (C@O, ester),
1H NMR (CDCl3): d 9.1 (br s, 1H, DHP NH), 7.9 (s, 1H, Imidazole
C4–H), 5.1 (s, 1H, C4–H), 3.9 (m, 7H, N–CH3 and N(CH2–CH3)2), 3.2
(m, 1H, O–CH (CH3)2), 2.2 (s, 3H, C6–CH3), 1.7 (s, 3H, C2–CH3), 1.1
(two d, J = 6.2 Hz, 3H each, CH(CH3)2,), 0.7(t, 6H, N(CH2–CH3)2).
1654 (C@O, amide), 1550 and 1373cmÀ1 (NO2).
MS: m/z(%) 481(M+ 40), 464(20), 408(100), 393(21), 381(24),
355(32), 259(77), 178(39),105(91),72(37).
Elemental Anal. Calcd for C25H31N5O5: C, 62.36; H, 6.49; N,
14.54.
IR (KBr):
m 3241 (NH), 1697 (C@O, ester), 1618 (C@O, amide),
1529 and 1372 cmÀ1 (NO2).
MS: m/z(%) 419(M+ 40), 402(11), 360(8), 346(63), 332(16),
319(38), 304(53), 259(100), 213(20), 178(45), 150(18), 100(17),
72(16), 43(16).
7.4.8. 3-Phenyl propyl-5-(diethyl carbamoyl)2,6-dimethyl-4-(1-
methyl-5-nitro-1H imidazole-2-yl)1,4 dihydropyridine-3-
carboxylate (7g)
Elemental Anal. Calculd for C20H29N5O5: C, 57.27; H, 6.97; N,
16.7.
1H NMR (CDCl3): d 8.5 (br s, 1H, NH-DHP), 7.922 (s, 1H, H-Imid-
azole), 7.07–7.19 (m, 5H-Phenyl), 4.70 (s, 1H, C4H), 3.9–4.0 (m, 7H,
N–CH3 and N(CH2CH3)2), 2.49 (t, CO2CH2), 2.28 (s, 6H, C2CH3 and
C6CH3), 1.03 (m, 4H, CO2–CH2–(CH2)2–), 0.80–1.18 (m, 6H,
N(CH2CH3)2.
7.4.4. n-Propyl-5-(diethyl carbamoyl)2,6-dimethyl-4-(1-
methyl-5-nitro-1H imidazole-2-yl)1,4 dihydropyridine-3-
carboxylate (7d)
IR (KBr):
m 3302 (NH), 3112 (CH, aromatic), 1694 (C@O, ester),
1H NMR (CDCl3): d 9.4 (br s, 1H, DHP N–H), 7.9 (s, 1H, Imidazole
C4–H), 5.2 (s, 1H, C4–H), 3.8–3.9 (m, 9H, N–CH3 and N(CH2–CH3)2
and O–CH2–CH2–CH3), 2.2 (s, 3H, C6–CH3), 1.6 (s, 3H, C2–CH3),
1.5 (m, 2H, O–CH2–CH2–CH3), 1.1 (t, 3H, O–CH2–CH2–CH3), 0.7 (t,
J = 7.2, 6H, N(CH2–CH3)2).
1609 (C@O, amide), 1518 and 1371cmÀ1 (NO2).
MS: m/z(%) 495(M+ 42), 478(18), 422(100), 395(22), 259(59),
213(17), 178(22.95), 91(37.70), 72(16.39).
Elemental Anal. Calcd for C26H33N5O5: C, 63.01; H, 6.71; N,
14.13.
IR (NaCl):
m 3251 (NH), 1699 (C@O, ester), 1609 (C@O, amide),
1518 and 1372cmÀ1 (NO2).
7.4.9. 4-Phenyl butyl-5-(diethyl carbamoyl)2,6-dimethyl-4-(1-
methyl-5-nitro-1H imidazole-2-yl)1,4 dihydropyridine-3-
carboxylate (7h)
MS: m/z(%) 419(M+ 33), 402(10), 346(100), 331(32), 319(19),
303(27), 293(28), 259(71), 243(20), 213(24), 178(24), 150(17),
100(23), 72(21), 43(12).
1H NMR (CDCl3): d 9.24 (br s, 1H, NH-DHP), 7.87 (s, 1H, H-Imid-
azole), 7.05–7.21 (m, 5H-Phenyl), 5.03 (s, 1H, C4H), 4.27 (s. 3H, N–
CH3), 3.97–4.04 (m, 4H, N(CH2CH3)2, 2.50 (t, CO2CH2), 2.10 (s, 6H,
C2CH3 and C6CH3), 1.49–1.59 (m, 6H, CO2–CH2–(CH2)3–), 1.18–
1.27 (m, 6H, N(CH2CH3)2.
Elemental Anal. Calcd for C20H29N5O5: C, 57.27; H, 6.97; N, 16.7.
7.4.5. n-Butyl-5-(diethyl carbamoyl)2,6-dimethyl-4-(1-methyl-
5-nitro-1H imidazole-2-yl)1,4 dihydropyridine-3-carboxylate
(7e)
IR (KBr):
m 3337 (NH), 3015 (CH, aromatic), 1711 (C@O, ester),
1H NMR (CDCl3): d 8.7(br s, 1H, DHP NH), 7.9 (s, 1H, Imidazole
C4–H), 5 (s, 1H, C4–H), 3.9 (m, 9H, N–CH3 and O–CH2(CH3)3 and
N(CH2CH3)2), 2.2 (s, 3H, C6–CH3), 1.6 (s, 3H, C2–CH3), 1.4 (m, 2H,
1656 (C@O, amide), 1585 and 1359cmÀ1 (NO2).
MS: m/z(%) 509(M+, 8), 492(46), 382(37), 367(22), 259(12),
104(27), 91(100), 72(24), 42(22).

M. Khoshneviszadeh et al. / Bioorg. Med. Chem. 17 (2009) 1579–1586
1585
Elemental Anal. Calcd for C27H35N5O5: C, 63.64; H, 6.92; N,
13.74.
contraction) was considered as control. Then, tissues were prein-
cubated with one certain concentration of each compound (for
15 min) and then the effect of KCl (40 mM) was assessed once
again. Each segment was treated with only one compound. From
concentration–response curves, the pIC50 (ÀlogIC50) value of each
compound was calculated. nifedipine was used as reference com-
pound.30–32
7.4.10. 5-Phenyl pentyl-5-(diethyl carbamoyl)2,6-dimethyl-4-
(1-methyl-5-nitro-1H imidazole-2-yl)1,4 dihydropyridine-3-
carboxylate (7i)
1H NMR (CDCl3): d 9.43 (br s, 1H, NH-DHP), 7.86 (s, 1H, H-Imid-
azole), 7.04–7.17 (m, 5H-Phenyl), 5.07 (s, 1H, C4H), 4.15 (s. 3H, N–
CH3), 3.96–4.03 (m, 4H, N(CH2CH3)2, 2.46 (t, CO2CH2), 2.08 (s, 6H,
C2CH3 and C6CH3), 1.50–1.59 (m, 6H, CO2–CH2–(CH2)4–), 1.20–
1.29 (m, 6H, N(CH2CH3)2.
7.6.2. Statistical methods
Results are expressed as mean SEM The one-way analysis of
variance (ANOVA) followed by Tukey–Kramer multiple compari-
sons was used to analyze the data. A value of P < 0.05 was consid-
ered as the significance level between the groups.
IR (KBr):
m 3277(NH), 3021 (CH, aromatic), 1693 (C@O, ester),
1663 (C@O, amide), 1531 and 1369cmÀ1 (NO2).
MS: m/z(%) 523(M+ 4), 506(12), 423(28), 324(16), 259(53),
196(16), 146(36), 91(100), 43(18).
7.7. Computational details
Elemental Anal. Calcd for C28H37N5O5: C, 64.23; H, 7.12; N,
13.37.
All the computations were run on a Pentium IV personal com-
puter (CPU at 2.6 GH) with the windows XP operating system. Chem-
ical structure of each molecule was built by Hyperchem software
(Version 7, Hypercube Inc.) for the structural chemistry. The Z-
matrices of the structures were constructed by the software and
transferred to the GAUSSIAN 98 program.33 Complete geometryoptimi-
zation was performed taking the most extended conformations as
starting geometries. Semiempirical molecular orbital calculations
(AM1) of the structures were performed using the GAUSSIAN 98 pro-
gram. 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 molÀ1 was used as ending criteria in geom-
etryoptimization. Then, themoleculeswerereloadedtoHyperchem,
some dihedrals angles and LogP were calculated in this software.
7.5. Determination of anti-mycobacterial activity
In vitro antimicrobial activity of the compounds was elevated
against M. tuberculosis H37Rv strain ATCC 27294 that susceptible
to Rifampin and Isoniazid. Minimum inhibitory concentration
(MIC) was determined by using agar proportion method. The com-
pounds in dimethylsulfoxide (DMSO) solutions were adding to the
Middlebrook 7H10 (Quelab Co. UK) medium with glycerol and en-
riched with OADC. The following concentrations were used: 1, 2, 4,
8, 16, 32, 62, 125, 250 and 500 lmol/l. A culture of M. tuberculosis
H37Rv cultivated in 7H9 broth (Quelab Co. UK) at 37 °C for a period
of 4–7 days, was adjusted, using the same medium, to the optical
density of McFarland standard no. 1. Two dilutions of this suspen-
sion, 10À2 and 10À4, were used as an inoculum, 0.1 ml per each
tubes. MIC values were determined after incubation at 37°C in
the presence of 5 to 7% CO2 for a period of 21 days. MIC shows
the lowest substance concentration indicating the inhibitory effect
on the Mycobacterium growth. The colonies on each tube were
counted, and the numbers of colonies on drug-containing tubes
were compared with negative control. The present results were ob-
tained from two independent measurements. Isoniazid and rifam-
picin (Sigma Chemical Co.) were used as a positive control.
Acknowledgments
Financial supports of this project by research council of Shiraz
University of Medical Sciences are acknowledged (Grant No.
3583). Also, authors are grateful of Pasteur Institute of Iran for kind
providing of microorganism.
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