Hantzsch reaction: Synthesis and characterization of some new 1,4-dihydropyridine derivatives as potent antimicrobial and antioxidant agents

Article abstract of DOI:10.1016/j.ejmech.2011.09.026

In the present study two new series of Hantzsch 1,4-dihydropyridine derivatives (1,4-DHPs) containing substituted pyrazole moiety (4a-f and 5a-f) were synthesized by the reaction of 3-aryl-1H-pyrazole-4-carbaldehydes with 1,3-dicarbonylcompounds (ethylace

Full text of DOI:10.1016/j.ejmech.2011.09.026

European Journal of Medicinal Chemistry 46 (2011) 5591e5597
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Hantzsch reaction: Synthesis and characterization of some new
1,4-dihydropyridine derivatives as potent antimicrobial and antioxidant agents
,
,
c
d
a
e
A.M. Vijesha b, Arun M. Isloorb , S.K. Peethambar , K.N. Shivananda , T. Arulmoli , Nishitha A. Isloor
*
a SeQuent Scientific Ltd., No: 120 A & B, Industrial Area, Baikampady, New Mangalore, Karnataka 575 011, India
b Medicinal Chemistry Division, Department of Chemistry, National Institute of Technology-Karnataka, Surathkal, Mangalore 575 025, India
c Department of Bio-Chemistry, Jnanasahyadri, Kuvempu University, Shankaraghatta, Karnataka 577 451, India
d Department of Chemistry, Technion Israel Institute of Technology, Haifa 32000, Israel
e Biotechnology Division, Chemical Engg. Department, National Institute of Technology-Karnataka, Surathkal, Mangalore 575 025, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 June 2011
Accepted 15 September 2011
Available online 22 September 2011
In the present study two new series of Hantzsch 1,4-dihydropyridine derivatives (1,4-DHPs) containing
substituted pyrazole moiety (4aef and 5aef) were synthesized by the reaction of 3-aryl-1H-pyrazole-4-
carbaldehydes with 1,3-dicarbonylcompounds (ethylacetoacetate and methylacetoacetate) and ammo-
nium acetate. The newly synthesized compounds were characterized by IR, NMR, mass spectral study
and also by C, H, N analyses. New compounds were screened for their antimicrobial activity by well plate
method (zone of inhibition). Antioxidant studies of the synthesized compounds were also performed by
measuring the DPPH radical scavenging assay. Compounds 4c, 4e and 4f were found to be potent
antibacterial and antioxidant agents. The acute oral toxicity study for the compounds 4c, 4e and 4f were
carried out and the experimental studies revealed that compounds 4c and 4e is safe up to 3000 mg/kg
and no death of animals were recorded. However in compound 4f, we found mortality above 2000 mg
and also significant behavioral changes in experimental animals.
Keywords:
1,4-Dihydropyridine
Pyrazoles
1,3-Dicarbonylcompounds
Antimicrobial studies
Antioxidant studies
Toxicity study
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
agents [8,9]. Oxidative aromatization reactions of DHPs are taking
place in biological systems in presence of certain enzymes. The
In recent decades, multicomponent reactions (MCR’s) have
gained wide applicability in the field of synthetic organic chemistry
as they increase the efficiency of the reaction and decrease the
number of laboratory operations along with quantities of solvent
and chemicals used. These methods also considerably reduce the
reaction time and facilitate the yield of products than the normal
multiple step methods. One-pot, four-component synthesis of
symmetrically substituted 1,4-dihydropyridines were first reported
by Arthur Hantzsch in 1882 [1]. Hantzsch 1,4-dihydropyridines
(1,4-DHPs) and their derivatives are an important class of bioac-
tive molecules in the pharmaceutical field [2]. They possess anti-
inflammatory, anti-microbial [3], anti-oxidant, antiulcer activities
[4]. DHPs are commercially used as calcium channel blockers for
the treatment of cardiovascular diseases, including hypertension
[5]. Recently, the synthesis of DHPs with respect to Multidrug
Resistance (MDR) reversal in tumor cell gave a new dimension to
their applications [6,7]. In addition, 1,4-DHP class of compounds are
excellent starting synthons for development of antitubercular
nitrogen heterocycles thus prepared by Hantzsch method are of
great importance because of their role in biological systems. They
have been served as model compounds for the NAD-NAPH bio-
logical redox systems [10e12].
Recently, antibiotic-resistant microbes are making their inexo-
rable march and medicinal chemists have now realized that the
discovery of more powerful antibiotics is not the only answer to this
threat. But, a real need exists in searching a novel antimicrobial that
expresses antimicrobial properties, possibly acting through mech-
anisms different from those of existing drugs. In this context, it is
very essential to successfully develop novel, efficient antimicrobial
agents with clinically unexploited mode of action.
Further, pyrazole derivatives have showed significant biological
activities, such as anti-microbial [13], analgesic [14], anti-
inflammatory [15] and, anticancer [16]. This gave a great impetus
to the search for potential pharmacologically active drugs carrying
pyrazole substituents. Keeping in view of this and in continuation
of our search on biologically potent molecules [17e21], we hereby
report the synthesis of some new 1,4-dihydropyridine derivatives
containing pyrazole nucleus. These compounds were evaluated for
their antimicrobial and antioxidant properties.
* Corresponding author. Tel.: þ91 824 2474000; fax: þ91 824 2474033.
E-mail address: isloor@yahoo.com (A.M. Isloor).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.026

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A.M. Vijesh et al. / European Journal of Medicinal Chemistry 46 (2011) 5591e5597
2. Results and discussion
NH protons of dihydropyridine appeared as a singlet at
d 8.56. A
singlet appeared at 12.58 is due to pyrazole-NH further confirmed
d
2.1. Chemistry
the structure. The mass spectrum of 4a showed molecular ion peak
at m/z ¼ 424.1 (mꢀ1), which is in agreement with the molecular
formula C23H27N3O5. Similarly the spectral values for all the
compounds and C, H, N analyses are given in the experimental part
and the characterization is provided in Tables 1 and 2.
The schematic representation of the new 1,4-dihydropyridine
derivatives (4aef and 5aef) has been presented in Scheme 1. 3-
Substituted-1H-pyrazole-4-carbaldehydes (3aef) were synthe-
sized by the Vilsmayer Haack reaction of semicarbazones (2aef)
[22]. Refluxing 3-substituted-1H-pyrazole-4-carbaldehydes (3aef),
2.2. Antimicrobial studies
1,3-dicarbonylcompounds
(ethylacetoacetate
and
methyl-
acetoacetate) and ammonium acetate in ethanol resulted in the
target compounds (4aef and 5aef) via one-pot multicomponent
reaction. The proposed mechanism for the formation of dihy-
dropyridine derivatives has been presented in Scheme 2.
2.2.1. Antibacterial studies
The in vitro antibacterial activity of newly synthesized
compounds 4aef and 5aef were determined by well plate method
in Mullere Hinton Agar [23,24]. In this work, Escherichia coli ATCC
25922 (Gram-negative), Staphylococcus aureus ATCC 25923 (Gram-
positive) and Pseudomonas aeruginosa ATCC 27853 (Gram-nega-
tive) were selected due to their infectious nature. The test
compounds were dissolved in dimethyl sulfoxide (DMSO) at
concentrations of 1, 0.5 and 0.25 mg/mL.
The antibacterial screening revealed that, some of the tested
compounds showed good inhibition against various tested micro-
bial strains. The result indicated that among the tested compounds,
4c and 4f showed excellent activity against all the tested microbial
strains E. coli, S. aureus and P. aeruginosa at concentrations of 1, 0.5
and 0.25 mg/mL compared to standard drug streptomycin.
Compound 4d showed good antibacterial activity against E. coli and
P. aeruginosa as compared to the standard drug. 4e showed
Structures of the synthesized compounds 4aef and 5aef were
confirmed by recording their IR, NMR, mass spectra and C, H, N
elemental analyses. All compounds were characterized after
recrystallization from appropriate solvents. IR spectrum of
compound 4a showed absorption at 3243 cmꢀ1 which is due to the
NH stretching. Band at 1667 cmꢀ1 is due to C]O stretch. Similarly,
band at 1608 cmꢀ1 is due to C]N group confirmed the structure.
The 1H NMR spectrum of 4a showed a triplet at
protons of CH2eCH3 group. A singlet at 2.28 is due to CH3 protons.
A quartet appeared at 3.87 which is due to CH2 protons of
OeCH2eCH3 group. Pyridine-4H appeared as a singlet at 5.04.
Two doublets at 7.02 and 7.76 are due to aromatic protons of p-
d 0.96 is due to CH3
d
d
d
d
d
anisyl moiety. Pyrazole-5H proton appeared as a singlet at
d
7.25.
O
NH2
Ar
O
CHO
N
NH
Semicarbazide.HCl
DMF
Ar
N
Ethanol
POCl3
Ar
CH3
N
H
CH3
Sodiumacetate
3a-f
1a-f
2a-f
EAA
CH3COONH4
Ethanol
MAA
CH3COONH4
Methanol
H
N
H
N
H3C
CH3
H3C
CH3
O
O
O
H3C
O
CH3
H3C
CH3
O
O
O
O
Ar
Ar
N
N
H
N
N
H
5a-f
4a-f
Where Ar = 2,4-Dichlorophenyl, 4-Thioanisyl, 2,5-Dichlorothiophene, Biphenyl,
4-Anisyl, 4-Chlorophenyl
Scheme 1. Synthetic route for the compounds 4aef and 5aef.

A.M. Vijesh et al. / European Journal of Medicinal Chemistry 46 (2011) 5591e5597
5593
Step-1: The reaction can be visualized as proceeding through a Knoevenagel Condensation
product as a key intermediate
O
O
H
O
O
+
-H2O
R'
R'
Pyz
O
R
O
R
O
Pyz
Step-2: Formation of enamine-type intermediate
O
O
NH2
O
NH3
R'
R'
R
O
R
O
-H2O
Step-3: Condensation between the two fragments gives the dihydropyridine derivative.
R'
Pyz
R'
Pyz
O
Pyz
O
R'OOC
Tautomerism
COOR'
R
R'OOC
O
COOR'
R
O
O
O
H2N
R
H2N
R
R
H2N
R
O
Pyz
Pyz
H
R'OOC
R
COOR'
R
R'OOC
COOR'
R
-H2O
R
N
H
N
H
OH
4a-f / 5a-f
Substituted pyrazoles, R = CH3, R1 =
CH3 or C2H5
Pyz =
Scheme 2. Proposed reaction mechanism for the formation of substituted dihydropyridines.
excellent activity as that of standard, against S. aureus and
P. aeruginosa at all concentrations. Compounds 4a and 4b showed
moderately good anti-microbial activity against all the tested
microbial strains. The remaining compounds have showed less
activity against all of the three tested bacterial strains compared to
standard, streptomycin. The antibacterial results were summarized
in Table 3.
Compound 4c has thioanisyl moiety on pyrazole ring, which is
accounted for the enhanced antibacterial activity. Compound 4f has
4-chlorophenyl substituent, 4d has 2,5-dichlorothiophene and 4e
has biphenyl respectively on pyrazole ring, which is accounted for
the enhanced activity of the compounds. Similarly compounds 4a
and 4b have anisyl and 2,4-dichlorophenyl substituents, which is
responsible for their biological activity.
2.2.2. Antifungal studies
Newly synthesized compounds 4aef and 5aef were also
screened for their antifungal activity against Aspergillus flavus
MTCC 3306, Chrysosporium keratinophilum MTCC 2827 and Candida
albicans MTCC 3017, because of their infectious nature. The
compounds were dissolved in DMSO and antimicrobial activity was
determined by well plate method [25,26] at concentrations of 1, 0.5
and 0.25 mg/mL.
Table 2
Characterization data of the compounds 4aef and 5aef.
Comp. Ar
No.
Molecular Formula
(Mol. wt.)
Yield M.p.
(%) (ꢁC)
Color
4a
4b
4c
4d
4-Anisyl
C
23H27N3O5 (425.47)
76
180e182 Off white
184e186 Off white
162e164 Off white
169e171 Off white
2,4-Dichlorophenyl C22H23Cl2N3O4 (464.34) 73
4-Thioanisyl
2,5-Dichloro
thiophene
C23H27N3O4S (441.54)
C
82
Table 1
20H21Cl2N3O4S (470.36) 69
Characterization data of the compounds 3aef.
Comp. Ar
No.
Molecular Formula
(Mol. wt.)
Yield M.p.
(%)
(ꢁC)
Color
4e
4f
Biphenyl
4-Chlorophenyl
C
28H29N3O4 (471.57)
74
192e194 Yellow
186e188 Off white
202e204 Off white
194e196 Yellow
214e216 Yellow
C22H24ClN3O4 (429.89) 67
5a
5b
5c
2,4-Dichlorophenyl C20H19Cl2N3O4 (436.28) 77
3a
3b
3c
2,4-Dichlorophenyl
C
10H6Cl2N2O (241.07) 87
114e116 Off white
148e150 Off white
138e140 Off white
4-Thioanisyl
2,5-Dichloro
thiophene
C21H23N3O4S (413.49)
C18H17Cl2N3O4S (442.31) 80
84
4-Thioanisyl
2,5-Dichloro
thiophene
C11H10N2OS (218.27) 91
C8H4Cl2N2OS (247.10) 90
5d
5e
5f
Biphenyl
4-Anisyl
4-Chlorophenyl
C
26H25N3O4 (443.49)
78
81
246e248 Off white
162e164 Off white
252e254 Off white
3d
3e
3f
Biphenyl
4-Anisyl
4-Chlorophenyl
C
16H12N2O (248.27)
88
178e180 Yellow
162e164 Off white
142e144 Off white
C21H23N3O5 (397.42)
C20H20ClN3O4 (401.84) 75
C11H10N2O2 (202.20) 86
C10H7ClN2O (206.62) 87

5594
A.M. Vijesh et al. / European Journal of Medicinal Chemistry 46 (2011) 5591e5597
Table 3
Antibacterial activity of the compounds 4aef and 5aef (Zone of inhibition in mm.).
Comp. No.
Escherichia coli
Staphylococcus aureus
Pseudomonas aeruginosa
Concn. (mg/mL)
1
0.5
0.25
8 ꢂ 0.02
1
0.5
0.25
9 ꢂ 0.02
1
0.5
0.25
Standard Streptomycin
16 ꢂ 0.01
12 ꢂ 0.01
06 ꢂ 0.02
17 ꢂ 0.02
16 ꢂ 0.02
13 ꢂ 0.02
17 ꢂ 0.01
06 ꢂ 0.01
04 ꢂ 0.01
10 ꢂ 0.01
13 ꢂ 0.02
07 ꢂ 0.02
10 ꢂ 0.01
10 ꢂ 0.02
08 ꢂ 0.02
04 ꢂ 0.01
12 ꢂ 0.01
10 ꢂ 0.01
10 ꢂ 0.01
12 ꢂ 0.01
03 ꢂ 1
15 ꢂ 0.02
13 ꢂ 0.02
11 ꢂ 0.01
16 ꢂ 0.01
13 ꢂ 0.01
16 ꢂ 0.01
15 ꢂ 0.01
06 ꢂ 0.01
06 ꢂ 0.02
10 ꢂ 0.02
09 ꢂ 0.01
06 ꢂ 0.02
08 ꢂ 0.01
10 ꢂ 0.01
08 ꢂ 0.01
07 ꢂ 0.02
12 ꢂ 0.02
09 ꢂ 0.02
12 ꢂ 0.01
10 ꢂ 0.02
03 ꢂ 0.01
03 ꢂ 0.02
08 ꢂ 0.02
07 ꢂ 0.02
04 ꢂ 0.02
06 ꢂ 0.02
16 ꢂ 9.02
10 ꢂ 0.01
12 ꢂ 0.01
14 ꢂ 0.01
15 ꢂ 0.02
17 ꢂ 0.02
16 ꢂ 0.01
08 ꢂ 0.02
06 ꢂ 0.02
07 ꢂ 0.01
10 ꢂ 0.02
07 ꢂ 0.01
09 ꢂ 0.01
13 ꢂ 0.01
07 ꢂ 0.02
09 ꢂ 0.02
11 ꢂ 0.01
12 ꢂ 0.01
13 ꢂ 0.01
13 ꢂ 0.01
04 ꢂ 0.01
04 ꢂ 0.02
03 ꢂ 0.02
08 ꢂ 0.02
04 ꢂ 0.01
07 ꢂ 0.02
11 ꢂ 0.02
05 ꢂ 0.01
07 ꢂ 0.01
08 ꢂ 0.01
10 ꢂ 0.01
11 ꢂ 0.02
10 ꢂ 0.02
02 ꢂ 0.01
03 ꢂ 0.01
01 ꢂ 0.01
07 ꢂ 0.01
02 ꢂ 0.02
04 ꢂ 0.01
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
05 ꢂ 0.01
02 ꢂ 0.02
09 ꢂ 0.01
08 ꢂ 0.01
07 ꢂ 0.02
10 ꢂ 0.02
02 ꢂ 0.01
01 ꢂ 0.01
05 ꢂ 0.01
08 ꢂ 0.02
02 ꢂ 0.01
05 ꢂ 0.02
06 ꢂ 0.02
05 ꢂ 0.01
10 ꢂ 0.01
08 ꢂ 0.01
10 ꢂ 0.02
08 ꢂ 0.01
02 ꢂ 0.01
02 ꢂ 0.01
04 ꢂ 0.01
05 ꢂ 0.01
02 ꢂ 0.02
03 ꢂ 0.01
02 ꢂ 0.01
07 ꢂ 0.02
10 ꢂ 0.01
04 ꢂ 0.02
08 ꢂ 0.01
All the compounds (4aef and 5aef) showed less antifungal
activity against all the tested micro organisms compared to stan-
dard drug, fluconazole. Results of antifungal studies have been
presented in Table 4.
were observed in experimental animals. But in compound 4f, we
found mortality in above 2000 mg and also significant behavioral
changes were observed in experimental animals.
3. Conclusion
2.3. Antioxidant studies: DPPH radical scavenging assay
Two series of new substituted Hantzsch 1,4-dihydropyridine
derivatives (1,4-DHPs) containing substituted pyrazole moiety
(4aef and 5aef) were synthesized in reasonably good yields. They
were characterized by spectral studies and elemental analyses. All
the newly synthesized compounds were screened for antimicrobial
and antioxidant activity.
The free radical scavenging activity of test samples 4aef and
5aef was measured by DPPH according to Brand-Williams et al.
[27]. All compounds have exhibited free radical scavenging capacity
by comparison with the standard Butylated Hydroxytoulene (BHT).
DPPH assay were carried out for compounds 4aef and 5aef at
100
mM concentration. The antioxidant activity may be, one
As regards the relationships between the structure of the
heterocyclic scaffold and the detected antibacterial properties, it
showed varied biological activity. Compounds 4c and 4f have
showed excellent antibacterial activity. Among the two series, 4aef
which contains ethyl ester moiety on dihydropyridine ring showed
good activity as compared to the second series, 5aef which consists
of a methyl ester group. Antifungal studies reveal that all the tested
compounds were less active compared to the standard drug. From
the antimicrobial results we can conclude that, synthesized
compounds are specific antibacterial agents. A combination of two
different heterocyclic systems namely pyrazole and 1,4-
dihydropyridine has enhanced the pharmacological effect and
hence they are ideally suited for further modifications to obtain
more efficacious antibacterial compounds.
possible mechanism responsible for the organ protective effects of
1,4-dihydropyridine calcium channel blockers [28,29]. Nifedipine is
a well known 1,4-dihydropyridine derivative [30].
Among the tested compounds (4aef and 5aef), compounds 4c,
4e and 4f showed significant amount of DPPH activity (>60%).
Remaining were non significant compared to the standard BHT. The
variation exhibited in DPPH scavenging capacity could be attrib-
uted to the effect of different substitutions and results are pre-
sented in Table 5.
2.4. Acute toxicity and behavioral studies
The acute oral toxicity study for the test compounds
(compounds 4c, 4e and 4f) was carried out by following the OECD
guidelines [31,32]. The experimental studies revealed that
compounds 4c and 4e are safe up to 3000 mg/kg and no deaths of
animals were recorded. Further, no significant behavioral changes
The free radical scavenging activity of test samples 4aef and
5aef was measured by DPPH. The data reported herein indicates
that compounds 4c, 4e and 4f showed significant DPPH activity
(>60%) and the remaining compounds were non significant
Table 4
Antifungal activity of the compounds 4aef and 5aef (Zone of inhibition in mm.).
Comp. No.
Aspergilus Flavus
Chrysosporium Keratinophilum
Candida Albicans
Concn. (mg/mL)
1
0.5
0.25
1
0.5
0.25
1
0.5
0.25
Standard Flucanazole
13 ꢂ 0.01
02 ꢂ 0.01
03 ꢂ 0.01
00
04 ꢂ 0.02
05 ꢂ 0.01
02 ꢂ 0.01
03 ꢂ 0.01
00
03 ꢂ 0.01
05 ꢂ 0.02
00
06 ꢂ 0.02
11 ꢂ 0.02
01 ꢂ 0.01
02 ꢂ 0.01
00
03 ꢂ 0.01
02 ꢂ 0.01
00
10 ꢂ 0.01
00
17 ꢂ 0.01
03 ꢂ 0.01
04 ꢂ 0.01
00
05 ꢂ 0.01
03 ꢂ 0.01
03 ꢂ 0.01
05 ꢂ 0.01
00
04 ꢂ 0.02
03 ꢂ 0.01
00
05 ꢂ 0.02
15 ꢂ 0.02
02 ꢂ 0.01
02 ꢂ 0.01
00
02 ꢂ 0.01
00 ꢂ 0.01
00
14 ꢂ 0.01
22 ꢂ 0.01
03 ꢂ 0.01
04 ꢂ 0.01
00
05 ꢂ 0.01
06 ꢂ 0.02
04 ꢂ 0.01
03 ꢂ 0.01
00
21 ꢂ 0.02
01 ꢂ 0.01
01 ꢂ 0.01
00
02 ꢂ 0.01
04 ꢂ 0.01
02 ꢂ 0.01
00
20 ꢂ 0.02
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
00
02 ꢂ 0.01
02 ꢂ 0.01
01 ꢂ 0.01
01 ꢂ 0.01
00
00
00
03 ꢂ 0.01
01 ꢂ 0.01
00
00
00
02 ꢂ 0.01
02 ꢂ 0.01
00
02 ꢂ 0.01
00
00
00
00
00
00
00
0
02 ꢂ 0.01
01 ꢂ 0.01
00
00
00
02 ꢂ 0.01
04 ꢂ 0.01
00
03 ꢂ 0.01
00
02 ꢂ 0.01
02 ꢂ 0.01
00
04 ꢂ 0.01
01 ꢂ 0.01
03 ꢂ 0.01
02 ꢂ 0.01
02 ꢂ 0.01
00
00
00
00
00
02 ꢂ 0.01
00
03 ꢂ 0.01
00
01 ꢂ 0.01
00
00
01 ꢂ 0.01

A.M. Vijesh et al. / European Journal of Medicinal Chemistry 46 (2011) 5591e5597
5595
Table 5
4.3.1. 3-(2,4-Dichlorophenyl)-1H-pyrazole-4-carbaldehyde (3a)
1H NMR (400 MHz, DMSO-d6):
7.49e7.54 (m, 2H, AreH), 7.76
(s, 1H, AreH), 8.47 (s, 1H, pyrazole-5H), 9.71 (s, 1H, eCHO), 13.74 (s,
1H, pyrazole-NH); 13C NMR:
184.40, 147.59, 133.89, 133.29, 129.04,
DPPH radical scavenging activity of synthesized compounds
4aef and 5aef.
d
Compound No.
DPPH Assay in %
d
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
30.3
29.2
61.1
32.0
65.3
62.3
35.2
32.2
30.3
25.3
30.2
29.4
72.42
127.28, 120.95; MS: m/z ¼ 242.0 (M þ 1); Anal. calcd. for
C10H6Cl2N2O: C, 49.82; H, 2.51; N, 11.62; Found: C, 49.80; H, 2.48; N,
11.59%.
4.3.2. 3-[4-(Methylsulfanyl)phenyl]-1H-pyrazole-4-carbaldehyde
(3b)
1H NMR (400 MHz, DMSO-d6):
d
3.29 (s, 3H, SCH3), 7.35 (m, 2H,
AreH), 7.79 (m, 2H, AreH), 8.56 (s, 1H, pyrazole-5H), 9.87 (s, 1H,
eCHO), 13.64 (s, 1H, pyrazole-NH); 13C NMR:
184.65, 141.54,
d
130.68, 128.84, 125.60, 14.35; MS: m/z ¼ 219.2 (M þ 1); Anal. calcd.
for C11H10N2OS: C, 60.53; H, 4.62; N, 12.83; Found: C, 60.50; H, 4.61;
N, 12.81%.
BHT
compared to the standard BHT. The acute oral toxicity study for the
compounds 4c, 4e and 4f were carried out and the experimental
studies revealed that compounds 4c and 4e is safe up to 3000 mg/
kg and no death of animals were recorded. But in compound 4f we
found mortality in above 2000 mg and also significant behavioral
changes were observed in experimental animals.
4.3.3. 3-(2,5-Dichlorothiophen-3-yl)-1H-pyrazole-4-carbaldehyde
(3c)
1H NMR (400 MHz, DMSO-d6):
d
7.35 (s, 1H, 2,5-
dichlorothiophene), 8.49 (s, 1H, pyrazole-5H), 9.80 (s, 1H, eCHO),
13.69 (s, 1H, pyrazole-NH); 13C NMR:
184.43, 161.15, 128.87,
d
120.69; MS: m/z ¼ 248.1 (M þ 1); Anal. calcd. for C8H4Cl2N2OS: C,
4. Experimental
38.89; H, 1.63; N, 11.34; Found: C, 38.86; H, 1.61; N, 11.31%.
4.1. Chemistry
4.4. General procedure for the synthesis of diethyl-4-(3-aryl-1H-
pyrazol-4-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
(4aef)
Melting points were determined by open capillary method and
were uncorrected. The IR spectra (in KBr pellets) were recorded on
a JASCO FT/IR-4100 spectrophotometer. 1H NMR spectra were
recorded (DMSO-d6) on a Bruker (400 MHz, 300 MHz) using TMS as
3-(4-Substituted)-1H-pyrazole-4-carbaldehyde (1.0 mmol),
ethylacetoacetate (2.0 mmol) and ammonium acetate (1.12 mmol)
in ethanol (20 mL) were refluxed for 8 h in an oil bath. After the
reaction completion, reaction mixture was concentrated and
poured in to crushed ice. The precipitated product was filtered,
washed with water. The resulting solid was recrystallized from hot
ethanol.
internal standard. Chemical shift values are given in d (ppm) scales.
The mass spectra were recorded on a JEOL JMS-D 300 spectrometer
operating at 70 eV. Elemental analyses were performed on a Flash
EA 1112 series CHNS-O Analyzer. The completion of the reaction
was checked by thin layer chromatography (TLC) on silica gel
coated aluminum sheets (silica gel 60 F254) obtained from Merck.
Commercial grade solvents and reagents were used without further
purification.
4.4.1. Diethyl-4-[3-(4-methoxyphenyl)-1H-pyrazol-4-y]-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (4a)
IR (KBr, nmax cmꢀ1): 3243 (NeH-str), 3066, 2975 (CeH-str), 1667
(C]O), 1608 (C]N); 1H NMR (400 MHz, DMSO-d6):
d 0.86 (t, 6H,
4.2. General procedure for the synthesis of (2E)-2-(1-
arylethylidene)hydrazinecarboxamide (2aef)
CH2eCH3), 2.16 (s, 6H, CH3), 3.74 (s, 3H, OCH3), 3.97 (m, 4H, CH2),
4.99 (s, 1H, pyridine-4H), 6.98 (d, 2H, J ¼ 8.17 Hz, AreH), 7.18 (s, 1H,
pyrazole-5H), 7.68 (d, 2H, J ¼ 8.28, AreH), 8.77 (s, 1H, pyridine-NH),
A solution of semicarbazide hydrochloride (1.07 mmol) in 20 mL
of water was added drop wise to a round bottom flask containing
mixture of substituted carbonyl compounds 1aef (1.0 mmol),
sodium acetate (1.3 mmol) and ethanol (20 mL). The reaction
mixture was stirred at 80 ꢁC for 8 h. After the reaction completion,
the separated solids were filtered, washed with water and dried.
The crude products (2aef) as such taken for next stage preparation
without further purification.
12.46 (s, 1H, pyrazole-NH); 13C NMR:
d 167.61, 159.19, 145.20,
129.42, 114.05, 103.17, 59.27, 55.66, 28.66, 18.65, 14.48; MS: m/
z ¼ 424.1 (M ꢀ 1); Anal. calcd. for C23H27N3O5: C, 64.93; H, 6.40; N,
9.88; Found: C, 64.90; H, 6.36; N, 9.85%.
4.4.2. Diethyl-4-[3-(2,4-dichlorophenyl)-1H-pyrazol-4-y]-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (4b)
IR (KBr, nmax cmꢀ1): 3286 (NeH-str), 3061, 2982 (CeH-str), 1678
4.3. General procedure for the synthesis of 3-substituted-1H-
(C]O), 1651 (C]N); 1H NMR (400 MHz, DMSO-d6):
d 1.08 (t, 6H,
pyrazole-4-carbaldehydes (3aef)
CH2eCH3), 2.52 (s, 6H, CH3), 3.90 (m, 4H, CH2), 5.04 (s, 1H, pyridine-
4H), 7.24e7.65 (m, 4H, AreH, pyrazole-5H), 8.47 (s, 1H, pyridine-
3-Substituted-1H-pyrazole-4-carbaldehydes
(3aef)
were
NH), 12.59 (s, 1H, pyrazole-NH); 13C NMR:
d 167.40, 145.25,
synthesized by VilsmayereHaack reaction. To an ice cold solution of
(2E)-2-(1-arylethylidene)hydrazinecarboxamide (2aef) (0.1 mmol)
in DMF (20 mL), POCl3 (8 mL) was added drop wise. After the
addition, the reaction mixture was stirred for 30 min at ambient
temperature and then stirred at 60e65 ꢁC for 6 h. The reaction
mixture was quenched into ice cold water and pH adjusted to 7
using 25% sodium hydroxide solution. The solids thus precipitated
were filtered and dried. Crude product was recrystallized from
ethyl acetate.
135.46, 133.96, 101.39, 59.26, 28.96, 18.43, 14.64; MS: m/z ¼ 465.3
(M þ 1); Anal. calcd. for C22H23Cl2N3O4: C, 59.91; H, 4.99; N, 9.05;
Found: C, 59.89; H, 4.96; N, 9.02%.
4.4.3. Diethyl-4-{3-[4-(methylsulfanyl)phenyl]-1H-pyrazol-4-yl}-
2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (4c)
IR (KBr, nmax cmꢀ1): 3213 (NeH-str), 3066, 2978 (CeH-str), 1680
(C]O), 1623 (C]N); 1H NMR (300 MHz, DMSO-d6):
d
0.91 (t, 6H,
CH2eCH3), 2.22 (s, 6H, CH3), 2.51 (s, 3H, SCH3), 3.96 (m, 4H, CH2),

5596
A.M. Vijesh et al. / European Journal of Medicinal Chemistry 46 (2011) 5591e5597
4.88 (s, 1H, pyridine-4H), 7.22(s, 1H, pyrazole-5H), 7.35 (m, 2H,
AreH), 7.74 (m, 2H, AreH), 8.83 (s, 1H, pyridine-NH), 12.60 (s, 1H,
NH), 12.49 (s, 1H, pyrazole-NH); MS: m/z ¼ 414.4 (M þ 1); Anal.
calcd. for C21H23N3O4S: C, 61.00; H, 5.61; N, 10.16; Found: C, 60.98;
H, 5.58; N, 10.14%.
pyrazole-NH); MS: m/z
C23H27N3O4S: C, 62.56; H, 6.16; N, 9.52; Found: C, 62.54; H, 6.13; N,
9.50%.
¼
442.5 (M
þ
1); Anal. calcd. for
4.5.3. Dimethyl-4-[3-(2,5-dichlorothiophen-3-yl)-1H-pyrazol-4-
yl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (5c)
IR (KBr, nmax cmꢀ1): 3269 (NeH-str), 3064, 3006 (CeH-str), 1682
4.4.4. Diethyl-4-[3-(2,5-dichlorothiophen-3-yl)-1H-pyrazol-4-y]-
2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (4d)
(C]O), 1648 (C]N); 1H NMR (300 MHz, DMSO-d6):
d 2.17 (s, 6H,
IR (KBr, nmax cmꢀ1): 3240 (NeH-str), 3052, 2966 (CeH-str), 1668
CH3), 3.32 (s, 6H, OeCH3), 4.79 (s, 1H, pyridine-4H), 7.19e7.36 (m,
2H, 2,5-dichlorothiophene, pyrazole-5H), 8.73 (s, 1H, pyridine-NH),
12.73 (s,1H, pyrazole-NH); MS: m/z ¼ 443.3 (M þ 1); Anal. calcd. for
C18H17Cl2N3O4S: C, 48.88; H, 3.87; N, 9.50; Found: C, 48.85; H, 3.84;
N, 9.48%.
(C]O), 1614 (C]N); 1H NMR (300 MHz, DMSO-d6):
d 1.10 (t, 6H,
CH2eCH3), 2.50 (s, 6H, CH3), 3.92 (m, 4H, CH2), 4.84 (s, 1H, pyridine-
4H), 7.22e7.36 (m, 2H, 2,5-dichlorothiophene, pyrazole-5H), 8.63
(s, 1H, pyridine-NH), 12.72 (s, 1H, pyrazole-NH); MS: m/z ¼ 471.3
(M þ 1); Anal. calcd. for C20H21Cl2N3O4S: C, 51.07; H, 4.50; N, 8.93;
Found: C, 51.05; H, 4.47; N, 8.90%.
4.5.4. Dimethyl-4-[3-(biphenyl-4-yl)-1H-pyrazol-4-yl]-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (5d)
4.4.5. Diethyl-4-[3-(biphenyl-4-yl)-1H-pyrazol-4-yl]-2,6-dimethyl-
1,4-dihydropyridine-3,5-dicarboxylate (4e)
IR (KBr, nmax cmꢀ1): 3230 (NeH-str), 3037, 2956 (CeH-str), 1667
(C]O), 1609 (C]N); 1H NMR (300 MHz, DMSO-d6):
d 2.21 (s, 6H,
IR (KBr, nmax cmꢀ1): 3256 (NeH-str), 3064, 2927 (CeH-str), 1682
CH3), 3.19 (s, 6H, OeCH3), 5.09 (s, 1H, pyridine-4H), 7.26 (s, 1H,
pyrazole-5H), 7.34e7.81 (m, 9H, AreH), 8.88 (s, 1H, pyridine-NH),
(C]O), 1625 (C]N); 1H NMR (300 MHz, DMSO-d6):
d 1.09 (t, 6H,
CH2eCH3), 2.24 (s, 6H, CH3), 3.91 (m, 4H, CH2), 5.02 (s, 1H, pyridine-
4H), 7.25e7.79 (m, 10H, AreH, pyrazole-5H), 8.83 (s, 1H, pyridine-
NH), 12.69 (s, 1H, pyrazole-NH); MS: m/z ¼ 472.5 (M þ 1); Anal.
calcd. for C28H29N3O4: C, 71.32; H, 6.20; N, 8.91; Found: C, 71.30; H,
6.17; N, 8.87%.
12.71 (s, 1H, pyrazole-NH); 13C NMR:
d 167.86, 145.58, 140.29,
139.29, 129.45, 128.73, 127.91, 127.01, 126.81, 126.07, 102.71, 50.56,
28.74, 18.45; MS: m/z ¼ 444.4 (M þ 1); Anal. calcd. for C26H25N3O4:
C, 70.41; H, 5.68; N, 9.47; Found: C, 70.40; H, 5.66; N, 9.45%.
4.5.5. Dimethyl-4-[3-(4-methoxyphenyl)-1H-pyrazol-4-y]-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (5e)
4.4.6. Diethyl-4-[3-(4-chlorophenyl)-1H-pyrazol-4-yl]-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (4f)
IR (KBr, nmax cmꢀ1): 3202 (NeH-str), 3085, 2952 (CeH-str), 1687
IR (KBr, nmax cmꢀ1): 3235 (NeH-str), 3072, 2937 (CeH-str), 1671
(C]O), 1649 (C]N); 1H NMR (400 MHz, DMSO-d6):
d 2.14 (s, 6H,
(C]O), 1635 (C]N); 1H NMR (300 MHz, DMSO-d6):
d
0.91 (t, 6H,
CH3), 3.74 (s, 3H, OCH3), 3.87 (m, 6H, COOCH3), 5.07 (s,1H, pyridine-
4H), 7.02e7.68 (m, 5H, AreH, pyrazole-5H), 8.66 (s, 1H, pyridine-
NH), 12.53 (s, 1H, pyrazole-NH); MS: m/z ¼ 398.4 (M þ 1); Anal.
calcd. for C21H23N3O5: C, 63.46; H, 5.83; N, 10.57; Found: C, 63.44;
H, 5.81; N, 10.55%.
CH2eCH3), 2.21 (s, 6H, CH3), 3.90 (m, 4H, CH2), 5.03 (s, 1H, pyridine-
4H), 7.29 (s, 1H, pyrazole-5H), 7.52 (m, 2H, AreH), 7.82 (m, 2H,
AreH), 8.83 (s,1H, pyridine-NH),12.67 (s, 1H, pyrazole-NH); MS: m/
z ¼ 430.8 (M þ 1); Anal. calcd. for C22H24ClN3O4: C, 61.46; H, 5.63;
N, 9.77; Found: C, 61.42; H, 5.61; N, 9.74%.
4.5.6. Dimethyl 4-[3-(4-chlorophenyl)-1H-pyrazol-4-yl]-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (5f)
4.5. General procedure for the synthesis of dimethyl 4-(3-aryl-1H-
pyrazol-4-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
(5aef)
IR (KBr, nmax cmꢀ1): 3235 (N-H-str), 3042, 2936 (CeH-str), 1664
(C]O), 1622 (C]N); 1H NMR (300 MHz, DMSO-d6):
d 2.22 (s, 6H,
CH3), 3.18 (s, 6H, CH2), 4.99 (s, 1H, pyridine-4H), 7.33(s, 1H,
pyrazole-5H), 7.51 (m, 2H, AreH), 7.74 (m, 2H, AreH), 8.88 (s, 1H,
pyridine-NH), 12.64 (s, 1H, pyrazole-NH); MS: m/z ¼ 402.8 (M þ 1);
Anal. calcd. for C20H20ClN3O4: C, 59.78; H, 5.02; N, 10.46; Found: C,
59.75; H, 4.99; N, 10.43%.
3-(4-Aryl)-1H-pyrazole-4-carbaldehyde (1.0 mmol), methyl-
acetoacetate (2.0 mmol) and ammonium acetate (1.12 mmol) in
ethanol (20 mL) were refluxed for 8 h in an oil bath. After the
reaction completion, reaction mixture was concentrated and
poured in to crushed ice. The precipitated product was filtered,
washed with water. The resulting solid was recrystallized from hot
ethanol.
4.6. Antibacterial studies
The antibacterial activity of newly synthesized compounds 4aef
and 5aef were determined by well plate method in Mueller-Hinton
Agar. The in vitro antibacterial activity was carried out against 24 h
old cultures of bacterial strains. In this work, E. coli, S. aureus and P.
aeruginosa were used to investigate the activity. The test
compounds were dissolved in dimethyl sulfoxide (DMSO) at
concentration of 1 and 0.5 mg/mL. Twenty milliliters of sterilized
agar media was poured into each pre-sterilized Petri dish. Excess of
suspension was decanted and plates were dried by placing in an
4.5.1. Dimethyl 4-[3-(2,4-dichlorophenyl)-1H-pyrazol-4-yl]-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (5a)
IR (KBr, nmax cmꢀ1): 3262 (NeH-str), 3057, 2946 (CeH-str), 1692
(C]O), 1632 (C]N); 1H NMR (400 MHz, DMSO-d6):
d 2.32 (s, 6H,
CH3), 3.48 (s, 6H, OeCH3), 4.97 (s, 1H, pyridine-4H), 7.22e7.64 (m,
4H, AreH, pyrazole-5H), 8.56 (s, 1H, pyridine-NH), 12.63 (s, 1H,
pyrazole-NH); 13C NMR:
d 166.78, 145.23, 136.58, 132.91, 101.39,
52.24, 28.37, 18.22; MS: m/z ¼ 437.0 (M þ 1); Anal. calcd. for
C20H19Cl2N3O4: C, 55.06; H, 4.39; N, 9.63; Found: C, 55.03; H, 4.37;
N, 9.60%.
incubator at 37 ꢁC for an hour. About 60
mL of 24 h old culture
suspension were poured and neatly swabbed with the pre-
sterilized cotton swabs. Six millimeter diameter well were then
4.5.2. Dmiethyl-4-{3-[4-(methylsulfanyl)phenyl]-1H-pyrazol-4-yl}-
2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (5b)
punched carefully using a sterile cork borer and 30 mL of test
solutions of different concentrations were added into each labeled
well. The plates were incubated for 24 h at 37 ꢁC. The inhibition
zone that appeared after 24 h, around the well in each plate were
measured as zone of inhibition in mm. Experiments were in trip-
licates and standard deviation was calculated.
IR (KBr, nmax cmꢀ1): 3252 (NeH-str), 3078, 2946 (CeH-str), 1676
(C]O), 1618 (C]N); 1H NMR (300 MHz, DMSO-d6):
d 2.12 (s, 6H,
CH3), 2.49 (s, 3H, SCH3), 3.72 (m, 6H, OeCH3), 4.92 (s, 1H, pyridine-
4H), 7.25e7.71 (m, 5H, AreH, pyrazole-5H), 8.52 (s, 1H, pyridine-

A.M. Vijesh et al. / European Journal of Medicinal Chemistry 46 (2011) 5591e5597
5597
4.7. Antifungal studies
onset of toxic symptoms and behavioral changes were also
recorded.
Antifungal studies of newly synthesized compounds 4aef and
5aef were carried out against Aspergillus flavus, Chrysosporium
Keratinophilum and Candida albicans. Sabourands agar media was
Acknowledgments
prepared by dissolving peptone (10 g), D-glucose (40 g) and agar
AMI thanks Department of atomic energy, Board for research in
Nuclear Sciences, Government of India for ‘Young Scientist’ award.
AMV is thankful to the management, SEQUENT SCIENTIFIC LTD,
New Mangalore, India for their invaluable support and allocation
of resources for this work. Authors are also thankful to Prof.
K. Kaliyappan, Chemistry Dept. of Indian Institute of Technology-
Bombay, India for the technical support.
(20 g) in distilled water (1000 mL) and adjusting the pH to 5.7.
Normal saline was used to make a suspension of spore of fungal
strains for lawning. A loopful of particular fungal strain was trans-
ferred to 3 mL saline to get a suspension of corresponding species.
Twenty milliliters of agar media was poured into each petri dish.
Excess of suspension was decanted and plates were dried by placing
in incubator at 37 ꢁC for 1 h. Using sterile cork borer punched
carefully, wells were made on these seeded agar plates different
concentrations of the test compounds in DMSO were added into
each labeled well. A control was also prepared for the plates in the
same way using solvent DMSO. The Petri dishes were prepared in
triplicate and maintained at 25 ꢁC for 72 h. Antifungal activity was
determined by measuring the diameter of inhibition zone. Activity
of each compound was compared with fluconazole as standard.
Zones of inhibition were determined for compounds 4aef and 5aef.
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