Synthesis and evaluation of new chalcones, derived pyrazoline and cyclohexenone derivatives as potent antimicrobial, antitubercular and antileishmanial agents

Article abstract of DOI:10.1007/s00044-013-0803-1

A series of chalcones (1-8) were prepared by Claisen-Schmidt condensation of 3-nitroacetophenone with various aldehydes. These chalcones on cyclization with hydrazine hydrate in the presence of glacial acetic acid, hydrazine hydrate in absolute ethanol an

Full text of DOI:10.1007/s00044-013-0803-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:2019–2032
DOI 10.1007/s00044-013-0803-1
ORIGINAL RESEARCH
Synthesis and evaluation of new chalcones, derived pyrazoline
and cyclohexenone derivatives as potent antimicrobial,
antitubercular and antileishmanial agents
•
Vikramdeep Monga Kamya Goyal Mario Steindel Manav Malhotra
•
•
•
•
Dhanji P. Rajani Smita D. Rajani
Received: 1 May 2013 / Accepted: 21 September 2013 / Published online: 1 October 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of chalcones (1–8) were prepared by
Claisen–Schmidt condensation of 3-nitroacetophenone
with various aldehydes. These chalcones on cyclization
with hydrazine hydrate in the presence of glacial acetic
acid, hydrazine hydrate in absolute ethanol and ethyl ace-
toacetate in the presence of barium hydroxide gave corre-
sponding N-acetyl pyrazolines (9–16), N-unsubstituted
pyrazolines (17–19) and cyclohexenone derivatives (20–
22). All the synthesized compounds were evaluated for
their in vitro antibacterial and antifungal activity by using
broth microdilution method, and many compounds showed
promising activity against various tested bacteria and fungi.
Among various tested compounds, 16 and 19 exhibited
strongest activities against Streptococcus pyogenes and
Pseudomonas aeruginosa; both have MIC value of 25 lg/
mL, which is fourfold greater than the standard drug. Many
compounds showed good activity against Candida albican.
Analogs 11, 12, 15–17 and 19 displayed two- to fivefold
greater activity against C. albican as compared to the
standard drug. Results of antitubercular evaluation revealed
that compounds 4 and 19 displayed strong antimycobac-
terial activity against H37Rv having MIC of 25 and
62.5 lg/mL, respectively. All analogs were found to be
inactive against Leishmania braziliensis except analogs 4
and 5 which exhibited strong leishmanicidal activity
against Leishmania promastigotes with IC50 values of 9.3
and 8.9 lg/mL, respectively.
Keywords Chalcones ꢀ Pyrazolines ꢀ Cyclohexenones ꢀ
Antimicrobial ꢀ Antitubercular ꢀ Antileishmanial
Introduction
Diseases caused by microbial infection are a serious
menace to the health of human beings and is a major cause
of death in developed and developing countries (Sharma
et al., 2009). In recent decades, problems of drug-resistant
microorganism have reached alarming level in many
countries around the world (Bogatcheva et al., 2006;
Goldstein, 2007). A number of recent clinical reports
describe the increasing occurrence of methicillin-resistant
Staphylococcus aureus and other antibiotic-resistant human
pathogenic microorganisms in United States and European
countries (Moreillon, 2008; Boucher et al., 2009). The high
mortality rate associated with multidrug-resistant gram-
negative enteric bacteria is a very serious problem (Arias,
et al., 2010). Mycobacterium tuberculosis (M. tuberculo-
sis), the main causative agent for tuberculosis (TB)
infecting around 32 % of the world population, is the
leading cause of mortality claiming about 2 million lives
annually. Further, one of the major factors contributing
V. Monga (&)
Department of Pharmaceutical Chemistry, Rajendra Institute of
Technology and Sciences, 4th Milestone, Hisar Road,
Sirsa 125055, Haryana, India
e-mail: vikramdeepmonga@gmail.com
K. Goyal
Laureate Institute of Pharmacy, VPO Kathog, Teh. Dehra,
Kangra, Himachal Pardesh, India
M. Steindel
Departamento de Microbiologia e Parasitologia, Universidade
´
Federal de Santa Catarina (UFSC), Florianopolis, SC, Brazil
M. Malhotra
Department of Pharmaceutical Chemistry, I.S.F. College of
Pharmacy, Moga, Punjab, India
D. P. Rajani ꢀ S. D. Rajani
Microcare Laboratory, Surat, Gujrat, India
123

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Med Chem Res (2014) 23:2019–2032
toward the increasing incidence of tuberculosis is the
occurrence of multidrug-resistant strains of M. tuberculosis
(MDR-TB) (Pai et al., 2006; Lawn and Zumla, 2011).
Moreover, a high number of infections with non-tubercu-
lous mycobacteria, especially in immune-compromised
patients such as those infected with the human immuno-
deficiency virus (HIV), render the treatment even more
difficult. Leishmaniasis is a group of parasitic diseases,
widespread in many parts of the world, and therapy of
patients with leishmaniasis still poses a serious problem
due to the side effects and toxicity associated with the
clinically used drugs. Besides, treatment regimen is lengthy
and requires long-term parenteral administration, and
resistance to these drugs has been observed in many cases
(Le Pape, 2008; Kobets et al., 2012). To overcome this
problem of rapid development of drug resistance, one of
the major challenges in drug discovery is the development
of new chemical entities which should preferably consist of
chemical characteristic that clearly differs from those of
existing drugs.
Result and discussion
Chemistry
The synthetic route followed for the preparation of the
titled compounds is illustrated in Scheme 1. The conden-
sation of 3-nitroacetophenone with various aromatic alde-
hydes afforded the 1,3-diaryl-2-propen-1-one (chalcone)
derivatives (1–8). The cyclization of chalcones with
hydrazine hydrate in the presence of glacial acetic acid
provided N-acetyl pyrazolines 9–16, whereas cyclization
with hydrazine hydrate in absolute ethanol resulted in
pyrazoline derivatives 17–19. The reaction of chalcones
with ethyl acetoacetate in the presence of barium hydroxide
produces cyclohexenone derivatives 20–22. The purity of
the compounds was checked by thin-layer chromatography
(TLC) and elemental analyses and characterized by spec-
tral data. The physical data of the compounds are shown in
Table 1. The structures of various synthesized compounds
were established by FT-IR, H-NMR, 13C-NMR and ele-
1
Chalcones consist of open-chain flavonoids and are
well-known intermediates for synthesizing various hetero-
cyclic compounds, which are associated with impressive
array of biological activities, including anti-inflammatory,
antibacterial, antioxidant, antimalarial, anticancer, antimi-
crobial and antileishmanial activities (Go et al., 2005;
Nowakowska, 2007; Romagnoli et al., 2009; Kumar et al.,
2010; Zheng et al., 2011). On the other hand, pyrazoline
derivatives have attracted considerable interest due to their
diverse biological properties and therapeutic applications
(Palaska et al., 2001; Ali et al., 2007; Amir et al., 2008;
Kaplancikli et al., 2009; Shaharyar et al., 2010; Ozdemir
et al., 2010; Senturk et al., 2012; Sharshira and Hamada
2012), namely antifungal, anti-inflammatory, antidepres-
sant, antimicrobial, anticancer, antinociceptive, antituber-
cular activity and inhibitors of mammalian monoamine
oxidase. Further, cyclohexenone derivatives represent
another class of cyclic compounds with interesting bio-
logical activities, among which its antimicrobial activity is
well documented (Mayekar et al., 2010; Kanagarajan et al.,
2011). Prompted by these observations and in continuation
of our efforts in search of new effective antimicrobial
agents, it was envisaged to synthesize a series of nitro-
chalcones (1–8) that were further cyclized to N-acetyl-
substituted and unsubstituted pyrazolines (9–19) and
cyclohexenone (20–22) derivatives. All the compounds
were structurally elucidated and screened for in vitro
antimicrobial activity against two gram-positive bacteria,
two gram-negative bacteria and three fungi. The promising
results obtained prompted us to further screen them for
antitubercular and antileishmanial activity. The results of
antimicrobial evaluation of all the synthesized compounds
are discussed in this paper.
mental analysis. All derivatives showed typical absorption
bands at 1,315–1,532 cm-1 due to their NO2 stretching
vibrations. The IR spectrum of chalcones (1–8) showed
absorption band at 1,643–1,666 cm-1 due to C=O
stretching. The formation of compounds 1–8 is observed in
1H NMR by the disappearance of the aldehydic proton
signal and the appearance of two doublets due to the ole-
finic protons at d 8.16–7.72 ppm and d 7.63–7.06 ppm,
respectively, with coupling constant between them of
J = 15.6–15.9 Hz, which agrees with a trans configuration.
The IR spectra of pyrazolines (9–19) showed the
expected absorption bands at 1,592–1,617 cm-1 corre-
sponding to C=N stretching bands because of ring closure,
at 1,658–1,671 cm-1 due to C=O group in 9–16 and at
3,327–3,349 cm-1 due to NH functionality in 17–19. In the
1H NMR spectra of these compounds, the CH2 protons of
the pyrazoline ring resonated as a pair of doublets of
doublets at d 3.03–3.26 ppm (Ha) and d 3.47–3.85 ppm
(Hb), with JAB = 16.5–17.7 Hz. The –CH(Hx) proton
appeared as a doublet of doublets at d 4.93–6.21 ppm due
to vicinal coupling with the two magnetically non-equiv-
alent protons of the methylene group at position 4 of the
pyrazoline ring with JAX = 4.5–5.1 Hz for 9–16,
JAX = 8.7–8.9 Hz for 17–19 and JBX = 11.1–12.3 Hz for
9–19. The FT-IR spectrum of cyclohexenone derivatives
(20–22) shows two strong characteristic absorptions in the
range of 1,726–1,733 cm-1 and 1,655–1,675 cm-1 due to
the ester carbonyl and ketone functional groups, respec-
tively. In the 1H NMR spectra, the protons of the ethyl ester
group resonated as a triplet and quartet in the range of d
1.08–1.12 ppm and 4.07–4.12 ppm, respectively, with
J = 7.2 Hz. The signal for =CH–CO– of cyclohexenone
ring appeared as singlet in the range of d 6.60–6.62 ppm,
123

Med Chem Res (2014) 23:2019–2032
2021
O
O2N
CH3
Ar-CHO
+
CHO
5% Aq. NaOH
EtOH
O
O2N
5
α
8
O
2'
5'
O2N
4'
Ar
α
NH2NH2. H2O, AcOH
1'
6'
1-7
4
5
O2N
CH3COCH2COOC2H5/Ba(OH)2
NH2NH2. H2O
1
N N
NH2NH2. H2O, AcOH
2
O
O
16
O
3
O C2H5
4
1
Ha
Hb
3
5 Ar
O2N
O2N
6
Ar
O2N
4
5
Ar
Hx
N NH
1
N
N
20-22
17-19
O
1, 9, 17, 20: Ar = 4-OCH3C6H4
2, 10, 18, 21: Ar = C6H3OCH2O
3,11:
Ar = 3,4-(OCH3)2C6H3
4, 12, 22:
5, 13:
6, 14:
Ar = 3,4,5-(OCH3)3C6H2
Ar = 2, 3,4-(OCH3)3C6H2
Ar = 3,4,5-(OCH3)3C6H2
Ar = 2-napthyl
7, 15, 19:
Scheme 1 Synthetic pathway for the compounds 1–22
whereas multiplets due to the protons of two CH groups
and CH2 group of the ring resonated at d 3.74–3.79 and
3.03–3.07 ppm. The protons belonging to the aromatic ring
and phenyl substituents were observed at expected chem-
ical shift and integral values. The spectral analyses of all
the compounds are listed in the ‘‘Experimental’’ section
and found to be in accordance with the assigned structures.
14, 16, 18 and 19 were found to be active against both
gram-positive and gram-negative bacteria with MIC values
in the range of 25–125 lg/mL. Among the various tested
analogs, 16 was found to be most active against S. pyog-
enes and 19 was found to be most potent against P. aeru-
ginosa, both having MIC value of 25 lg/mL, which is
fourfold greater than the standard drug used. Compounds
11 and 15 exhibited very good activity against gram-
positive bacteria and moderate activity against gram-neg-
ative bacteria, whereas compounds 6 and 13 displayed
good antibacterial activity against gram-negative bacteria
as compared to their activity against gram-positive bacte-
ria. Analogs 2, 5, 9, 10, 12 and 22 displayed moderate
activity against various tested strains of bacteria. All other
derivatives exhibited moderate to weak antibacterial
activity.
Antimicrobial screening
Antibacterial activity
All the synthesized compounds (1–22) were tested for their
antibacterial activity in vitro against gram-positive bacteria
(S. aureus MTCC-96, Streptococcus pyogenes MTCC-443)
and gram-negative bacteria (Escherichia coli MTCC-442,
Pseudomonas aeruginosa MTCC-2488). Ampicillin was
used as the standard drug. The minimum inhibitory con-
centration (MIC) values in lg/mL are given in Table 2. A
close survey of the antimicrobial data revealed that most of
the tested compounds exhibited good to moderate anti-
bacterial activity against all the tested bacterial strains with
MIC values in the range of 25–200 lg/mL. Analogs 7, 8,
As regards the relationships between the structures of
the various synthesized scaffolds and the detected anti-
bacterial properties, it showed varied biological activity.
However, conversion of chalcones to pyrazoles in some
cases (as in case of chalcone 7 and 8 and derived pyrazo-
lines 16 and 19) not only improves the potency but also the
spectrum of activity. Moreover, the presence of different
substituents causes
a
certain change of activity.
123

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Med Chem Res (2014) 23:2019–2032
Table 1 Physical data of
synthesized compounds
Compound no.
Molecular formula
Molecular weight
Yield (%)
Mp (°C)
Rfa
1
C16H13NO4
C16H11NO5
C17H15NO5
C18H17NO6
C18H17NO6
C18H17NO6
C19H13NO3
C17H13NO3
C18H17N3O4
C18H15N3O5
C19H19N3O5
C20H21N3O6
C20H21N3O6
C20H21N3O6
C21H17N3O3
C19H17N3O3
C16H15N3O3
C16H13N3O4
C19H15N3O2
C22H21NO6
C22H19NO7
C24H25NO8
283.28
297.26
313.3
86
75
62
77
71
69
68
79
65
57
45
70
49
61
69
82
60
54
62
47
54
60
132–134
127–129
130–132
128–129
115–117
132–133
139–141
114–116
135–137
186–188
138–140
125–127
110–111
124–126
131–133
141–143
101–102
101–102
98–99
0.78a1
0.75a1
0.52a1
0.59a1
0.60a1
0.38a1
0.75a1
0.62a1
0.32a2
0.24a2
0.20a2
0.18a2
0.28a2
0.17a2
0.34a2
0.45a2
0.50a1
0.70a1
0.42a1
0.52a1
0.54a1
0.55a1
2
3
4
343.33
343.33
343.33
303.31
279.29
339.35
353.33
369.37
399.4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
399.4
399.4
359.38
335.36
297.31
311.29
317.34
395.4
a1
Solvent system—hexane/
ethyl acetate (7:3)
a2
106–107
130–131
137–139
409.39
455.46
solvent system—toluene/
ethyl acetate (9.5:0.5)
Substitution with an electron-donating methoxy group at
para position of the phenyl ring in chalcone 1, derived
pyrazoline 9 and cyclohexenone derivative 20 imparts
weak to moderate activity; in case of pyrazoline 17, it
exhibits strong activity against gram-negative bacteria and
weak activity against gram-positive bacteria. Analogs 2, 10
and 21 containing methylenedioxy-substituted phenyl ring
exhibited moderate antibacterial activity, whereas analog
18 displayed good antibacterial activity against various
tested strains. Further, di- or tri-substitution with methoxy
group improves the activity of compounds against various
tested strains. Compound 4 possessing 3,4.5-trimethoxy
substituent showed fivefold and compound 5 carrying
2,3,4-trimethoxy substituent showed twofold increase in
potency against gram-positive bacteria as compared to
chalcone 1. Replacement of phenyl ring with napthyl ring
as in case of analogs 7 and 19 and increased separation
between two aromatic rings by additional double bond as in
case of analogs 8 and 16 imparts good activity to the
compounds. Chalcone 7 containing napthyl ring displayed
twofold increase in potency against S. aureus and E. coli,
whereas derived pyrazoline 19 showed two- and fourfold
increased activity against S. aureus and P. aeruginosa as
compared to the standard drug. Similarly, cinnamaldehyde-
derived chalcone 8 exhibited almost similar pattern of
activity as that of standard drug, while the derived pyraz-
oline derivative 16 found to posses five- and fourfold
increase in activity against S. pyogenes and E. coli,
respectively. The synthesized cyclohexenone derivatives
20 and 21 also showed improved antibacterial activity
against both gram-positive and gram-negative bacteria as
compared to the parent chalcones.
Antifungal activity
All the synthesized compounds (1–22) were tested for their
antifungal activity in vitro against three fungal strains (C.
albican MTCC-227, Aspergillus niger MTCC-282 and
Aspergillus clavatus MTCC-1323). Griseofulvin was used
as the standard drug. The minimum inhibitory concentra-
tion (MIC) values in lg/mL are given in Table 2. A close
survey of the antifungal data revealed that some of the
tested compounds exhibited good activity (100–250 lg/
mL) against C. albican and very few compounds were
found to display good activity (50–200 lg/mL) against A.
niger and A. clavatus. Compounds 5, 6 and 9 were found to
have no antifungal activity against C. albican, A. niger and
A. clavatus, respectively, even at a high concentration of
1,000 lg/mL. Chalcone derivatives 2, 4 and 8 were found
to exhibit twofold activity, whereas analogs 3 and 7 were
found to exhibit similar MIC values activity against C.
albican as that of standard. All chalcone derivatives were
found to exhibit very weak or no antifungal activity against
A. niger and A. clavatus except 1 which showed good
123

Med Chem Res (2014) 23:2019–2032
2023
Table 2 In vitro antibacterial and antifungal activity (MIC) values of the tested compounds
No.
MIC (lg/mL)
Gram-positive bacteria
Gram-negative bacteria
Fungi
S. aureus
MTCC-96
S. pyogenes
MTCC-443
E. coli
MTCC-442
P. aeruginosa
MTCC-2488
C. albican
MTCC-227
A. niger
MTCC-282
A. clavatus
MTCC-1323
1
500
250
200
100
250
500
100
125
250
250
125
250
250
100
100
250
500
100
50
500
250
200
100
250
500
125
125
250
250
125
250
250
125
100
25
200
250
100
250
200
125
62.5
200
250
250
500
250
100
125
250
62.5
62.5
100
250
100
125
200
100
–
200
250
200
250
200
125
200
100
250
200
250
200
100
125
250
100
100
200
25
1,000
250
500
250
1,000
1,000
500
250
1,000
500
200
250
1,000
1,000
250
500
250
250
100
500
500
100
–
200
200
2
1,000
[1,000
[1,000
[1,000
1,000
1,000
1,000
1,000
500
1,000
[1,000
[1,000
[1,000
1,000
1,000
1,000
1,000
500
3
4
5
6
7
8
9
10
11
500
500
12
500
500
13
250
250
14
250
250
15
[1,000
[1,000
1,000
1,000
100
[1,000
[1,000
1,000
1,000
250
16
17
500
125
125
500
200
200
100
–
18
19
20
250
250
200
250
–
200
200
200
100
–
[1,000
1,000
[1,000
–
[1,000
1,000
[1,000
–
21
22
Ampicillin
Griseofulvin
500
100
100
activity against both of them with a MIC value of 200 lg/
mL. Napthyl-substituted pyrazoline 19 showed fivefold
increased activity against C. albican and similar activity
against A. niger (100 lg/mL) as compared to standard
drug. Twofold increased activity was noted for 11, 12, 15,
17 and 18 against C. albican, whereas all other pyrazoline
derivatives displayed moderate to weak activity against
various fungal strains. Among various tested cyclohexe-
none derivatives, only 22 was found to exhibit better
activity (100 lg/mL) against C. albican and weak anti-
fungal activity against other tested fungal strains.
derivative 19 possessing napthyl ring attached to C-5 of the
pyrazoline ring showed better antitubercular activity, hav-
ing MIC values of 25 and 62.5 lg/mL, respectively. The
trimethoxy-substituted N-acetyl pyrazoline derivative 12
turns out to be the most potent compound which exhibited
a MIC value of 12.5 lg/mL and showed twofold increase
in antitubercular activity as compared to the parent chal-
cone 4. All other synthesized analogs displayed moderate
to weak activity.
Antileishmanial activity
Antitubercular activity
All the synthesized analogs (1–22) were evaluated for their
inhibitory activity against Leishmania braziliensis promas-
tigote forms. Amphotericin B was used as the standard drug,
and the results of antileishmanial evaluation are reproduced
in Table 4. Among the various tested analogs, compound 4
possessing 3,4,5-trimethoxy-substituted phenyl ring and
compound 5 having 2,3,4-trimethoxy-substituted phenyl
ring displayed potential inhibitory activity with IC50 values
The encouraging results from the antibacterial studies
impelled us to go for preliminary screening of synthesized
title compounds against M. tuberculosis. The results of
preliminary antimycobacterial screening of synthesized
compounds are tabulated in Table 3. Compound 4 con-
taining 3,4,5-trimethoxy-substituted phenyl ring and
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Med Chem Res (2014) 23:2019–2032
Table 3 In vitro effect of the synthesized compounds on the growth
of M. tuberculosis (MICs, lg/mL)
Table 4 In vitro effect of the synthesized compounds on the growth
of L. braziliensis and cytotoxicity on mouse macrophages
No.
MIC values (lg/mL)
M. tuberculosis H37Rv
% Inhibition
No.
IC50 values (lM)
L. braziliensis strain H3 (lM) cytotoxicity
on J774-A1
CC50 values
SIa
1
1,000
250
500
25
98
99
98
99
98
99
98
97
99
99
99
99
99
98
98
98
99
98
99
98
99
99
98
1
NA
NA
[100
9.30
8.90
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
[100
NA
NA
NA
NA
NA
–
–
2
2
–
–
3
3
–
–
4
4
48.95
5.26
5
500
250
500
1,000
250
250
500
12.5
250
500
1,000
250
250
500
62.5
500
250
250
40
5
89.49
10.06
6
6
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
7
7
8
8
9
9
10
10
11
12
13
14
15
16
17
18
19
20
21
22
11
12
13
14
15
16
17
18
19
20
21
22
Rifampicin
Amphotericin B 0.3
of 9.3 and 8.9 lg/mL, respectively, whereas dimethoxy-
phenyl-ring-substituted chalcone derivative 3 and pyrazo-
line derivative 17 displayed weak activity. All other syn-
thesized chalcones, derived pyrazoline and cyclohexenone
derivatives were found to be inactive. Thus, the nature of ring
may not have any effects on activity, and cyclization of
chalcones resulted in the loss of activity as in case of chal-
cone derivatives 4 and 5. In order to determine the toxicity/
activity index, the most active compounds against prom-
astigotes were also tested for their cytotoxicity toward mouse
J774-A1 macrophages. The selectivity index (SI) was
defined as the ratio of the CC50 values of compounds against
macrophages relative to IC50 values obtained against L.
braziliensis promastigotes. It has been found that compounds
4 and 5 displayed moderately good SI values of 5.26 and
10.06, respectively. Although the mechanisms of antileish-
manial activity displayed by chalcones were not addressed in
this work, depending upon literature reports, it can be pre-
dicted that chalcones could potentially inhibit the activity
of nicotinamide adenine dinucleotide-reduced fumarate
reductase (NADH-FRD), succinate dehydrogenase, NADH
dehydrogenase, or succinate- and NADH-cytochrome c
reductase in the parasite mitochondria (Lunardi et al., 2003;
Nowakowska, 2007).
NA no activity
a
Assay performed at Departamento de Microbiologia e Parasitolo-
gia, UFSC, Brazil; SI = CC50macrophages/IC50L. Braziliensis promastigotes
Conclusions
In conclusion, we have presented the synthesis, character-
ization and biological evaluation of series of chalcone,
pyrazoline and cyclohexenone derivatives. Their antimi-
crobial activities were evaluated against S. aureus, S. py-
ogenes, E. coli, P. aeruginosa, C. albican, A. niger,
A. clavatus as well as M. tuberculosis. Of all the analogs
screened for activity, some of the compounds were asso-
ciated with higher antibacterial and antifungal activity than
the standard drugs used (ampicillin and griseofulvin).
Compounds 16 and 19 displayed strongest activities against
tested bacteria S. pyogenes and P. aeruginosa with MIC
value of 25 lg/mL, whereas 11, 12, 15–17 and 19 exhib-
ited greater activity against Candida albican as compared
to the standard drugs. Results of the antitubercular evalu-
ation showed that compounds 4 and 19 exhibited potent
activity against M. tuberculosis with MIC values of
25–62.5 lg/mL, whereas pyrazoline derivative 12
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Med Chem Res (2014) 23:2019–2032
2025
substituted with electron-donating methoxy groups at the
meta/para position of the phenyl rings displayed highest
activity against M. tuberculosis with MIC value of 12.5 lg/
mL. Among all the tested analogs, only trimethoxy-
substituted chalcone derivatives 4 and 5 were found to be
active against L. braziliensis with IC50 values of 9.3 and
8.9 lg/mL, respectively, and exhibited good therapeutic
index. These results showed that cyclization of chalcones
leads to improvement in their antibacterial and antifungal
activity and loss of antiparasitic activity. Based on close
examination of substitutions, it may be concluded that role
of electron-donating groups (OCH3) on the phenyl ring and
the replacement of phenyl with napthyl ring has great
influence on antimicrobial activity. The present study has
generated leads with broad-spectrum activity, and further
SAR studies on identified bioactive lead molecules for
enhancing the antimicrobial activity are in progress.
allowed to stir at room temperature for appropriate time
until the starting reactants were disappeared. After com-
pletion of the reaction, the reaction mixture was quenched
on ice-cold water and neutralized with dilute hydrochloric
acid (5 %). The precipitated solid was filtered, washed with
distilled water and dried to give the crude product that was
recrystallized from ethanol. The spectral data of known
compounds 2 and 7 were found to be identical with those
reported in literature (Chiaradia et al., 2008). The charac-
terization data for all the compounds are given below.
(2E)-3-(4-methoxyphenyl)-1-(30-nitrophenyl)-2-propen-1-
one (1)
Light orange solid, Yield 86 %; m.p.: 132–134 °C; IR
(KBr) m (cm-1): 3,041 (=C–H, aromatic), 1,655 (C=O),
1
1,589 (C=C), 1,526 & 1,343 (–NO2); H NMR (300 MHz,
CDCl3) d ppm: 8.83 (s, 1H, H-20), 8.44 (d, 1H, J = 8.1 Hz,
H-40), 8.35 (d, 1H, J = 7.5 Hz, H-60), 7.88 (d, 1H,
J = 15.6 Hz, H-b), 7.70 (m, 3H, H-2, 6 and 50), 7.42 (d,
1H, J = 15.6 Hz, H-a), 6.97 (d, 2H, J = 8.7 Hz, H-3 and
5), 3.88 (s, 3H, OCH3); d ppm: 189.74 (C=O), 159.09 (C-
4), 148.59 (C-30), 145.98 (C-b), (133.43, 132.76, 130.84,
128.21, 127.16, 126.13, 124.62, 114.27, phenyl), 121.45
(C-a), 55.29 (OCH3); MS (APCI) m/z = 284 (M ? 1);
Anal. Calcd. for C16H13NO4 (283.28): C, 67.84; H, 4.63;
N, 4.94, Found: C, 67.93; H, 4.61; N, 4.72.
Experimental
Chemistry
Solvents and organic reagents were purchased from Sigma-
Aldrich, Merck and Loba Chemie (India) and were used
without further purification. All the evaporations were
carried out under reduced pressure on Buchi-rotary evap-
orator. Reactions were monitored by thin-layer chroma-
tography (TLC) using commercially available precoated
plates (Merck Kieselgel 60 F254 silica) with UV light
(256 nm), iodine and vanillin-sulfuric acid as developing
agents. Melting points were determined in open capillaries
and are uncorrected. IR spectra (KBr disks) were recorded
(2E)-1-(30-nitrophenyl)-3-(1,3-benzodioxol-5-yl)-2-
propen-1-one (2)
Yellowish brown solid, Yield 75 %; m.p.: 143–145 °C; IR
(KBr) m (cm-1): 3,067 (=C–H, aromatic), 1,662 (C=O),
1,566 (C=C), 1,505 & 1,347 (–NO2), 1,248 (C–O); 1H
NMR (300 MHz, CDCl3) d ppm: 8.82 (s, 1H, H-20), 8.48
(d, 1H, J = 8.1 Hz, H-40), 8.34 (d, 1H, J = 7.9 Hz, H-60),
7.82 (d, 1H, J = 15.6 Hz, H-b), 7.71 (m, 1H, H-50), 7.36
(d, 1H, J = 15.6 Hz, H-a), 7.23 (s, 1H, H-2), 7.16 (d, 1H,
J = 8.0 Hz, H-6), 6.87 (d, 2H, J = 8.0 Hz, H-5), 6.05 (s,
2H, –OCH2O–); 13C NMR (75 MHz, CDCl3) d ppm:
187.85 (C=O), 148.57 (C-30), 147.24–148.16 (C-3, C-4),
146.78 (C-b), (139.55, 134.28, 131.12, 129.03, 127.19,
126.20, 118.83, 109.03, 107.02, phenyl), 123.37 (C-a),
102.1 (–OCH2O–); MS (APCI) m/z = 298 (M ? 1); Anal.
Calcd. for C16H13NO4 (297.26): C, 67.84; H, 4.63; N, 4.94,
Found: C, 67.93; H, 4.61; N, 4.72.
1
on a NICOLET-380 FT-IR spectrophotometer. H NMR
and 13C NMR spectra were recorded on Bruker AM 300
(1H NMR at 300 MHz and 13C NMR at 75 MHz) spec-
trometer (Bruker Biosciences, USA) using CDCl3 as sol-
vent with TMS (tetramethylsilane) as the internal standard.
Splitting patterns are designated as follows: s, singlet; d,
doublet; dd, doublet of doublets; t, triplet; and m, multiplet.
Chemical shift values (d) are given in ppm. Mass spectra
were recorded on Finnigan Mat LCQ spectrometer. The
results of elemental analyses (% C, H, N) for the synthe-
sized compounds were in agreement with calculated values
within 0.4 % range.
General procedure used for the synthesis of 1,3-diaryl-
2-propen-1-ones (1–7) and 1,5-diaryl-2,4-pentadien-1-
one (8)
(2E)-3-(3,4-dimethoxyphenyl)-1-(30-nitrophenyl)-2-
propen-1-one (3)
To a solution of substituted aromatic aldehyde (1 mmol)
and m-nitroacetophenone (1 mmol) in ethanol (20 mL),
10 % aqueous NaOH was added. The reaction mixture was
Light green solid, Yield 62 %; m.p.: 130–132 °C; IR (KBr)
m (cm-1): 3,067 (=C–H, aromatic), 1,657 (C=O), 1,579
(C=C), 1,518 & 1,315 (–NO2); 1H NMR (300 MHz,
123

2026
Med Chem Res (2014) 23:2019–2032
CDCl3) d ppm: 8.83 (s, 1H, H-20), 8.40 (m, 2H, H-40 and
H-60), 7.86 (d, 1H, J = 15.59 Hz, H-b), 7.72 (m, 1H, H-50),
7.42 (d, 1H, J = 15.59 Hz, H-a), 7.28 (m, 2H, H-2 and 6),
6.93 (d, 1H, J = 8.2 Hz, H-5), 3.97 (s, 6H, 2 9 OCH3);
13C NMR (75 MHz, CDCl3) d ppm: 187.61(C=O), 155.21
(C-3), 153.93 (C-4), 148.58 (C-30), 143.57 (C-b), (140.50,
139.78, 134.91, 127.30, 118.26, 114.51, 111.76, phenyl),
122.96 (C-a), 56.29 (OCH3); MS (APCI) m/z = 314
(M ? 1); Anal. Calcd. for C17H15NO5 (313.3): C, 65.17;
H, 4.83; N, 4.47, Found: C, 65.12; H, 4.72; N, 4.41.
1,522 & 1,351 (NO2); 1H NMR (300 MHz, CDCl3) d ppm:
8.82 (s, 1H, H-20), 8.43 (m, 1H, H-40), 8.33 (d, 1H,
J = 7.8 Hz, H-60), 8.16 (d, 1H, J = 15.6 Hz, H-b), 7.69 (t,
1H, J = 8.1 Hz, H-50), 7.45 (d, 1H, J = 15.6 Hz, H-a), 7.13
(s, 1H, H-5), 6.51 (s, 1H, H-3), 3.96 (s, 3H, OCH3), 3.93 (s,
6H, 2 9 OCH3); 13C NMR (75 MHz, CDCl3) d ppm: 189.82
(C=O), 153.67 (C-2), 153.68 (C-4), 148.67 (C-30), 145.23
(C-b), 142.37 (C-5), (139.67, 133.43, 131.88, 129.55,
124.61, 118.52, 109.93, 101.02 phenyl ring), 122.86 (C-a),
56.43–56.28 (3 9 OCH3); MS (APCI) m/z = 344 (M ? 1);
Anal. Calcd. for C18H17NO6 (343.33): C, 62.97; H, 4.99; N,
4.08, Found: C, 62.88; H, 4.79; N, 4.01.
(2E)-1-(30-nitrophenyl)-3-(3,4,5-trimethoxyphenyl)-2-
propen-1-one (4)
(2E)-1-(30-nitrophenyl)-3-(2-naphthyl)-2-propen-1-one (7)
Yellow solid, Yield 77 %; m.p.: 128–129 °C; IR (KBr) m
(cm-1): 3,092 (=C–H, aromatic), 1,666 (C=O), 1,572
(C=C), 1,514 & 1,358 (NO2); 1H NMR (300 MHz, CDCl3)
d ppm: 8.84 (s, 1H, H-20), 8.46 (d, 1H, J = 8.1 Hz, H-40),
8.36 (d, 1H, J = 7.8 Hz, H-60), 7.82 (d, 1H, J = 15.6 Hz,
H-b), 7.73 (m, 1H, H-50), 7.40 (d, 1H, J = 15.6 Hz, H-a),
6.90 (s, 2H, H-2 and H-6), 3.94 (s, 6H, 2 9 OCH3), 3.92 (s,
3H, OCH3); 13C NMR (75 MHz, CDCl3) d ppm: 189.35
(C=O), 153.66 (C-3 and C-5), 148.69 (C30), 143.78 (C-b),
140.51 (C-4), (138.21, 133.57, 131.86, 129.96, 124.73,
121.21, 102.37, phenyl), 123.23 (C-a), 60.78 (m-OCH3),
56.15 (p-OCH3); MS (APCI) m/z = 344 (M ? 1); Anal.
Calcd. for C18H17NO6 (343.33): C, 62.97; H, 4.99; N, 4.08,
Found: C, 62.99; H, 4.92; N, 3.98.
Yellow solid, m.p.: 139–141 °C; Yield 62 %; IR (KBr) m
(cm-1): 3,094 (=C–H, aromatic), 1,659 (C=O), 1,563
(C=C), 1,522 & 1,351 (NO2); 1H NMR (300 MHz, CDCl3)
d ppm: 8.87 (s, 1H, H-20), 8.45 (d, 1H, J = 8.1 Hz, H-40),
8.38 (d, 1H, J = 7.8 Hz, H-60), 8.07 (d, 1H, J = 15.6 Hz,
H-b), 8.03 (s, 1H, H-1), 7.89 (d, 2H, J = 8.4 Hz, H-5 and
8), 7.84–7.81 (m, 2H, H-3 and 4), 7.72 (t, 1H, J = 8.1 Hz,
H-50), 7.63 (d, 1H, J = 15.6 Hz, H-a), 7.56–7.54 (m, 2H,
H-6 and 7); 13C NMR (75 MHz, CDCl3) d ppm: 187.97
(C=O), 148.69 (C-30), 145.93 (C-b), 139.21 (C-10),
(134.38, 133.98, 133.21, 132.48, 131. 09, 131.12, 129.37,
129.18, 128.22, 128.01, 127.56, 125.07, 122. 76, phenyl
and naphthyl), 123.43 (C-a); MS (APCI) m/z = 304
(M ? 1); Anal. Calcd. for C19H13NO3 (303.31): C, 75.24;
H, 4.32; N, 4.62, Found: C, 75.08; H, 4.39; N, 4.86.
(2E)-3-(2,3,4-trimethoxyphenyl)-1-(3-nitrophenyl)-2-
propen-1-one (5)
(2E, 4E)-1-(3-nitrophenyl)-5-phenyl-2,4-pentadien-1-one
Light brown solid, m.p.: 115–117 °C; Yield 71 %; IR (KBr)
m (cm-1): 3,109 (=C–H, aromatic), 1,646 (C=O), 1,568
(C=C), 1,533 & 1,356 (NO2); 1H NMR (300 MHz, CDCl3) d
ppm: 8.82 (s, 1H, H-20), 8.42 (d, 1H, J = 8.1 Hz, H-40), 8.34
(d, 1H, J = 7.8 Hz, H-60), 8.07 (d, 1H, J = 15.9 Hz, H-b),
7.72 (t, 1H, J = 7.8 Hz, H-50), 7.55 (d, 1H, J = 15.9 Hz,
H-a), 7.42 (d, 1H, J = 8.7 Hz, H-5), 6.74 (d, 1H,
J = 8.7 Hz, H-6), 3.97 (s, 3H, OCH3), 3.93 (s, 3H, OCH3),
3.90 (s, 3H, OCH3); 13C NMR (75 MHz, CDCl3) d ppm:
187.88 (C=O), 156.67 (C-4), 153.78 (C-2), 148.64 (C-30),
142.18 (C-b), 140.40 (C-3), 139.50 (C-10), (134.99, 131.02,
127.58, 124.27, 121.13, 120.17, 108.94, phenyl), 123.14 (C-
a), 62.05 (OCH3), 60.93 (OCH3), 56.58 (OCH3); MS (APCI)
m/z = 344 (M ? 1); Anal. Calcd. for C18H17NO6 (343.33):
C, 62.97; H, 4.99; N, 4.08, Found: C, 63.12; H, 5.05; N, 3.97.
(8)
Yellow crystals; Yield 79 %; m.p.: 1,14–1,16 °C; IR (KBr) m
(cm-1): 3,092 (=C–H, aromatic), 1,657 (C=O), 1,586 (C=C),
1,521 & 1,347 (NO2); 1H NMR (300 MHz, CDCl3) d ppm:
8.81 (s, 1H, H-20), 8.42 (d, 1H, J = 7.2 Hz, H-40), 8.31 (d,
1H, J = 7.8 Hz, H-60), 7.72–7.64 (m, 2H, H-b and H-50),
7.54–7.51 (m, 2H, H-2 and 6 of ArH), 7.42–7.35 (m, 3H, H-3,
4 and 5 of ArH), 7.13–7.06 (m, 3H, H-a, H-3 and 5 of
pentadiene); 13C NMR (75 MHz, CDCl3) d ppm: 187.91
(C=O), 148.52 (C-30), 146.31(C-b or C-2), 143.1 (C-3),
139.27 (C-5), (136.31, 134.68, 131.06, 129.87(C-a), 129.38,
127.83, 127.56, 125.14, phenyl), 122.98 (C-4); MS (APCI)
m/z = 280 (M ? 1); Anal. Calcd. for C17H13NO3 (279.29):
C, 73.11; H, 4.69; N, 5.02, Found: C, 73.03; H, 4.63; N, 4.88.
(2E)-1-(30-nitrophenyl)-3-(2,4,5-trimethoxyphenyl)-2-
General procedure for the synthesis of 1-acetyl-3,5-
propen-1-one (6)
diaryl-4,5-dihydro(1H)pyrazoles (9–16)
Yellow solid, m.p.: 132–133 °C; Yield 69 %; IR (KBr) m
To a solution of substituted chalcone (0.5 g) in glacial
acetic acid (10 mL), hydrazine hydrate (0.5 mL) was
(cm-1): 3,094 (=C–H, aromatic), 1,643 (C=O), 1,597 (C=C),
123

Med Chem Res (2014) 23:2019–2032
2027
1-Acetyl-5-(3,4-dimethoxyphenyl)-3-(30-nitrophenyl)-4,5-
added, and the mixture was refluxed for appropriate time
until the chalcone disappeared. After cooling, the mixture
was poured in ice-cold water. Precipitated solid was fil-
tered, dried and recrystallized using ethanol. The spectral
data of known compounds 9–11 were found to be identical
with those reported in literature (Nepali et al., 2011). The
characterization data for all the compounds are provided
below.
dihydro(1H)pyrazole (11)
Light orange solid; Yield 45 %; m.p.: 156–158 °C; IR
(KBr) m (cm-1): 3,080 (=C–H, aromatic), 1,666 (C=O),
1,593 (C=N), 1,530 & 1,349 (NO2), 1,258 (C–N); 1H
NMR- (300 MHz, CDCl3) d ppm: 8.55 (s, 1H, H-20), 8.28
(d, 1H, J = 8.1 Hz, H-40), 8.09 (d, 1H, J = 7.8 Hz, H-60),
7.63 (dd, 1H, J = 7.8, 8.1 Hz, H-50), 6.83–6.75 (m, 3H,
H-2, 5 and 6), 5.61 [dd, 1H, J = 4.8, 11.7 Hz, Hx (pyraz-
oline ring)], 3.86–3.74 (m, 7H, 2 9 OCH3 and Hb), 3.21
[dd, 1H, J = 4.8, 17.7 Hz, Ha (pyrazoline ring)], 2.45 (s,
3H, COCH3); 13C NMR (75 MHz, CDCl3) d ppm: 169.09
(C=O), 151.47 (C-3, pyrazoline ring), (149.39, 148.75,
148.64, 134.03, 133.33, 131.98, 129.84, 124.55, 121.35,
117.47, 111.65, 109.15, phenyl ring), 60.24 (C-5, pyrazo-
line ring), 55.98 (2 9 OCH3), 42.22 (C-4, pyrazoline ring),
22.01 (CH3); MS (APCI) m/z = 370 (M ? 1); Anal. Calcd.
for C19H19N3O5 (369.37): C, 61.78; H, 5.18; N, 11.38,
Found: C, 61.61; H, 5.26; N, 11.47.
1-Acetyl-5-(4-methoxyphenyl)-3-(30-nitrophenyl)-4,5-
dihydro(1H)pyrazole (9)
Pale brown solid; Yield 70 %; m.p.: 117–119 °C; IR (KBr)
m (cm-1): 3,093 (=C–H, aromatic), 1,664 (C=O), 1,610
(C=N), 1,522 & 1,348 (NO2), 1,252 (C–N); 1H NMR-
(300 MHz, CDCl3) d ppm: 8.53 (s, 1H, H-20), 8.27 (d, 1H,
J = 8.1 Hz, H-40), 8.08 (d, 1H, J = 7.8 Hz, H-60), 7.62
(dd, 1H, J = 7.8, 8.1 Hz, H-50), 7.15 (d, 1H, J = 8.7 Hz,
H-2 and 6), 6.85 (d, 1H, J = 8.7 Hz, H-3 and 5), 5.62 [dd,
1H, J = 4.8, 11.7 Hz, Hx (pyrazoline ring)], 3.83–3.73 (m,
4H, OCH3 and Hb), 3.20 [dd, 1H, J = 4.6, 17.7 Hz, Ha
(pyrazoline ring)], 2.43 (s, 3H, C OCH3); 13C NMR
(75 MHz, CDCl3) d ppm: 168.96 (C=O), 159.17 (C-OMe),
151.38 (C-3, pyrazoline ring), 148.58 (C–NO2), (133.55,
133.36, 131.95, 129.78, 126.80, 124.46, 121.28, 114.32,
phenyl ring), 59.91 (C-5, pyrazoline ring), 55.26 (OCH3),
42.08 (C-4, pyrazoline ring), 21.98 (CH3); Anal. Calcd. for
C18H17N3O4: C, 63.71; H, 5.05; N, 12.38, Found: C, 63.66;
H, 5.28; N, 10.44; MS (APCI) m/z = 340 (M ? 1); Anal.
Calcd. for C18H17N3O4 (339.35): C, 63.71; H, 5.05; N,
12.38, Found: C, 63.66; H, 5.28; N, 10.44.
1-Acetyl-5-(3,4,5-trimethoxyphenyl)-3-(30-nitrophenyl)-
4,5-dihydro(1H)pyrazole (12)
Light brown solid; Yield 70 %; m.p.: 125–127 °C; IR
(KBr) m (cm-1): 3,093 (=C–H, aromatic), 1,670 (C=O),
1,595 (C=N), 1,522 & 1,348 (NO2), 1,232 (C–N); 1H
NMR- (300 MHz, CDCl3) d ppm: 8.55 (s, 1H, H-20), 8.28
(d, 1H, J = 8.1 Hz, H-40), 8.08 (d, 1H, J = 7.5 Hz, H-60),
7.63 (dd, 1H, J = 7.5, 8.1 Hz, H-50), 6.41 (s, 2H, H-2 and
6), 5.59 [dd, 1H, J = 4.8, 11.7 Hz, Hx (pyrazoline ring)],
3.83 (s, 6H, 2 9 OCH3), 3.79 (s, 3H, OCH3), 3.76 [dd, 1H,
J = 11.7 & 17.7 Hz, Hb (pyrazoline ring)], 3.20 [dd, 1H,
J = 4.5, 17.7 Hz, Ha (pyrazoline ring)], 2.48 (s, 3H,
COCH3); 13C NMR (75 MHz, CDCl3) d ppm: 169.18
(C=O), 153.77 (2 9 C-OMe), 151.59 (C-3, pyrazoline
ring), 148.64 (C–NO2), (137.54, 133.19, 131.99, 129.87,
124.62, 121.37, 102.38, phenyl ring), 60.74 (C-5, pyrazo-
line ring), 60.63 (OCH3), 56.19 (2 9 OCH3), 42.37 (C-4,
pyrazoline ring), 21.98 (CH3); MS (APCI) m/z = 400
(M ? 1); Anal. Calcd. for C20H21N3O6 (399.4): C, 60.14;
H, 5.30; N, 10.52, Found: C, 60.03; H, 5.28; N, 10.44.
1-(5-benzo[d][1,3]dioxol-5-yl)-4,5dihydro-3-(30-
nitrophenyl)pyrazol-1-yl)ethanone (10)
Orange solid; Yield 57 %; m.p.: 196–198 °C; IR (KBr) m
(cm-1): 3,083 (=C–H, aromatic), 1,660 (C=O), 1,599
(C=N), 1,529 & 1,348 (NO2), 1,239 (C–N); 1H NMR-
(300 MHz, CDCl3) d ppm: 8.52 (s, 1H, H-20), 8.26 (d, 1H,
J = 8.1 Hz, H-40), 8.08 (d, 1H, J = 7.6 Hz, H-60), 7.62
(dd, 1H, J = 7.6, 8.1 Hz, H-50), 6.74–6.66 (m, 3H, H-2, 5
and 6), 5.92 (s, 2H, –OCH2O–), 5.57 [dd, 1H, J = 4.6,
11.9 Hz, Hx (pyrazoline ring)], 3.77 (dd, 1H, J = 11.9,
17.7 Hz, Hb), 3.17 [dd, 1H, J = 4.6, 17.7 Hz, Ha (pyraz-
oline ring)], 2.44 (s, 3H, C OCH3); 13C NMR (75 MHz,
CDCl3) d ppm: 168.99 (C=O), 151.35 (C-3, pyrazoline
ring), 148.58 (C–NO2), (148.17, 147.20, 135.36, 133.25,
131.94, 129.79, 124.52, 121.29, 119.02, 108.55, phenyl
ring), 101.15 (–OCH2O–), 60.13 (C-5, pyrazoline ring),
42.19 (C-4, pyrazoline ring), 21.97 (CH3); MS (APCI) m/
z = 354 (M ? 1); Anal. Calcd. for C18H15N3O5 (353.33):
C, 61.19; H, 4.28; N, 11.89, Found: C, 61.29; H, 4.39; N,
11.78.
1-Acetyl-5-(2,3,4-trimethoxyphenyl)-3-(30-nitrophenyl)-
4,5-dihydro(1H)pyrazole (13)
Brown solid; Yield 49 %; m.p.: 110–111 °C; IR (KBr) m
(cm-1): 3,064 (=C–H, aromatic), 1,658 (C=O), 1,592
(C=N), 1,532 & 1,349 (NO2), 1,266 (C–N); 1H NMR
(300 MHz, CDCl3) d ppm: 8.54 (s, 1H, H-20), 8.26 (d, 1H,
J = 8.1 Hz, H-40), 8.07 (d, 1H, J = 7.8 Hz, H-60), 7.61
(dd, 1H, J = 7.8, 8.1 Hz, H-50), 6.80 (d, 1H, J = 8.7 Hz,
H-5), 6.59 (d, 1H, J = 8.7 Hz, H-6), 5.72 [dd, 1H, J = 5.1,
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Med Chem Res (2014) 23:2019–2032
12.3 Hz, Hx (pyrazoline ring)], 3.90 (s, 3H, OCH3), 3.86 (s,
3H, OCH3), 3.82 (s, 3H, OCH3), 3.73 [dd, 1H, J = 12.3 &
17.7 Hz, Hb (pyrazoline ring)], 3.16 [dd, 1H, J = 5.1 &
17.7 Hz, Ha (pyrazoline ring)], 2.44 (s, 3H, COCH3); 13C
NMR (75 MHz, CDCl3) d ppm: 169.09 (C=O), 153.57 (C-
3, pyrazoline ring), (152.37, 150.79, 148.61, 142.44,
133.55, 132.00, 129.77, 127.00, 124.41, 121.36, 107.31,
phenyl ring), 60.73 (C-5, pyrazoline ring), 56.91 (OCH3),
56.91 (OCH3), 56.01 (OCH3), 41.58 (C-4, pyrazoline ring),
22.02 (CH3); MS (APCI) m/z = 400 (M ? 1); Anal. Calcd.
for C20H21N3O6 (399.4): C, 60.14; H, 5.30; N, 10.52,
Found: C, 60.18; H, 5.25; N, 10.43.
pyrazoline ring), 42.22 (C-4, pyrazoline ring), 20.03 (CH3);
MS (APCI) m/z = 360 (M ? 1); Anal. Calcd. for
C21H17N3O3 (359.38): C, 70.18; H, 4.77; N, 11.69, Found:
C, 70.27; H, 4.71; N, 11.56.
(E)-1-(3-(30-nitrophenyl)-5-styryl-4,5-dihydropyrazol-1-
yl)ethanone (16)
Yellow solid, Yield 82 %; m.p.: 141–143 °C; IR (KBr) m
(cm-1): 3,058 (=C–H, aromatic), 1,659 (C=O), 1,602
(C=N), 1,528 & 1,347 (NO2), 1,248 (C–N); 1H NMR
(300 MHz, CDCl3) d ppm: 8.53 (s, 1H, H-20), 8.27 (d, 1H,
J = 8.1 Hz, H-40), 8.08 (d, 1H, J = 7.8 Hz, H-60), 7.62
(dd, 1H, J = 7.8, 8.1 Hz, H-50), 7.36 (d, 2H, J = 7.2 Hz,
H-2 and 6), 7.31–7.20 (m, 3H, H-3, 4 and 5), 6.62 (d, 1H,
J = 15.9 Hz, –CH=CH–Ph), 6.21 (d, 1H, J = 15.9 Hz,
–CH=CH–Ph), 5.39–5.3 [m, 1H, Hx (pyrazoline ring)],
3.60 [dd, 1H, J = 11.9, 17.4 Hz, Hb (pyrazoline ring)],
3.17 [dd, 1H, J = 4.5, 17.4 Hz, Ha (pyrazoline ring)], 2.44
(s, 3H, COCH3); 13C NMR (75 MHz, CDCl3) d ppm:
169.29 (C=O), 151.70 (C-3, pyrazoline ring), 148.62 (C–
NO2), (136.00, 133.40, 131.95, 129.81, 128.54, 128.02,
126.65, 126.55, 124.53, 121.30, phenyl ring and styryl),
58.47 (C-5, pyrazoline ring), 39.20 (C-4, pyrazoline ring),
22.09 (CH3); MS (APCI) m/z = 336 (M ? 1); Anal. Calcd.
for C19H17N3O3 (335.36): C, 68.05; H, 5.11; N, 12.53,
Found: C, 67.02; H, 5.16; N, 12.41.
1-Acetyl-5-(2,4,5-trimethoxyphenyl)-3-(30-nitrophenyl)-
4,5-dihydro(1H)pyrazole (14)
Brown solid; Yield 61 %; m.p.: 124–126 °C; IR (KBr) m
(cm-1): 3,019 (=C–H, aromatic), 1,671 (C=O), 1,611
(C=N), 1,525 & 1,348 (NO2), 1,264 (C–N); 1H NMR
(300 MHz, CDCl3) d ppm: 8.54 (s, 1H, H-20), 8.26 (d, 1H,
J = 8.1 Hz, H-40), 8.08 (d, 1H, J = 7.8 Hz, H-60), 7.61
(dd, 1H, J = 7.8, 8.1 Hz, H-50), 6.61 (s, 1H, H-3), 6.53 (s,
1H, H-6), 5.77 [dd, 1H, J = 4.8, 11.7 Hz, Hx (pyrazoline
ring)], 3.86 (s, 3H, OCH3), 3.79 (s, 6H, 2 9 OCH3), 3.71
[dd, 1H, J = 12.0, 17.7 Hz, Hb (pyrazoline ring)], 3.12 [dd,
1H, J = 5.1, 17.7 Hz, Ha (pyrazoline ring)], 2.46 (s, 3H,
COCH3); 13C NMR (75 MHz, CDCl3) d ppm: 169.10
(C=O), 152.47 (C-3, pyrazoline ring), (150.67, 149.51,
148.59, 143.18, 133.60, 131.98, 129.75, 124.39, 121.29,
120.57, 111.31, 98.26, phenyl ring), 57.07 (C-5, pyrazoline
ring), 56.61(OCH3), 56.30 (OCH3), 56.15 (OCH3), 41.24
(C-4, pyrazoline ring), 21.95 (CH3); MS (APCI) m/z = 400
(M ? 1); Anal. Calcd. for C20H21N3O6 (399.4): C, 60.14;
H, 5.30; N, 10.52, Found: C, 60.21; H, 5.34; N, 10.46.
General procedure for the synthesis of 3,5-diaryl-4,5-
dihydro-1H-pyrazole (17–19)
A solution of the appropriate chalcone (0.03 mol) and
hydrazine hydrate (0.06 mol) in ethanol (30 mL) was
refluxed for the appropriate time until the chalcone disap-
peared as indicated by the TLC. After completion, the
reaction mixture was cooled and poured into ice-cold water
and then neutralized with glacial acetic acid. The solid
separated was filtered, washed with water, dried and re-
crystallized from ethanol to afford the titled compounds
good yield and purity.
1-(5-(naphthalen-2-yl)-3-(30-nitrophenyl)-4,5-
dihydropyrazol-1-yl)ethanone (15)
Brown solid; Yield 69 %; m.p.: 176–178 °C; IR (KBr) m
(cm-1): 3,052 (=C–H, aromatic), 1,665 (C=O), 1,595
(C=N), 1,530 & 1,348 (NO2), 1,270 (C–N); 1H NMR
(300 MHz, CDCl3) d ppm: 8.55 (s, 1H, H-20), 8.26 (d, 1H,
J = 8.1 Hz, H-40), 8.08 (d, 1H, J = 7.8 Hz, H-60),
7.83–7.72 (m, 3H, H-4, 5 and 8), 7.68 (s, 1H, H-1), 7.61
(dd, 1H, J = 7.8, 8.1 Hz, H-50), 7.46–7.41 (m, 2H, H-6 and
7), 7.31–7.28 (m, 1H, H-3), 5.82 [dd, 1H, J = 4.8, 11.7 Hz,
Hx (pyrazoline ring)], 3.85 [dd, 1H, J = 11.7 & 17.7 Hz,
Hb (pyrazoline ring)], 3.26 [dd, 1H, J = 4.8, 17.7 Hz, Ha
(pyrazoline ring)], 2.48 (s, 3H, COCH3); 13C NMR
(75 MHz, CDCl3) d ppm: 164.14 (C=O), 151.50 (C-3,
pyrazoline ring), 148.63 (C–NO2), (138.53, 133.35, 133.00,
132.03, 129.85, 129.22, 127.98, 127.69, 126.43, 126.13,
124.58, 123.19, 121.38, phenyl and naphthyl), 60.64 (C-5,
4,5-dihydro-5-(4-methoxyphenyl)-3-(30-nitrophenyl)-1H-
pyrazole (17)
Yellow solid; Yield 60 %; m.p.: 103–104 °C; IR (KBr) m
(cm-1): 3,327 (N–H), 3,016 (=C–H, aromatic), 1,607
(C=N), 1,536 & 1,344 (NO2), 1,247 (C–N); 1H NMR
(300 MHz, CDCl3) d ppm: 8.42 (s, 1H, H-20), 8.17 (d, 1H,
J = 8.1 Hz, H-40), 8.09 (d, 1H, J = 7.8 Hz, H-60), 7.54
(dd, 1H, J = 7.8, 8.1 Hz, H-50), 7.27 (d, 2H, J = 8.7 Hz,
H-2 and 6), 6.88 (d, 2H, J = 8.7 Hz, H-3 and 5), 6.19 (bs,
1H, NH), 4.98 [dd, 1H, J = 8.7, 11.6 Hz, Hx (pyrazoline
ring)], 3.80 (s, 3H, OCH3), 3.48 [dd, 1H, J = 11.1,
123

Med Chem Res (2014) 23:2019–2032
2029
General method for the synthesis of ethyl 6-(substituted
16.5 Hz, Hb (pyrazoline ring)], 3.06 (dd, 1H, J = 8.7,
16.5 Hz, Ha (pyrazoline ring)]; 13C NMR (75 MHz,
CDCl3) d ppm: 159.07 (C-OMe), 149.78 (C-3, pyrazoline
ring), 148.56 (C–NO2), (133.78, 133.57, 132.21, 129.97,
126.85, 124.98, 121.72, 114.62, phenyl ring), 64.69 (C-5,
pyrazoline ring), 55.26 (OCH3), 41.07 (C-4, pyrazoline
ring); MS (APCI) m/z = 298 (M ? 1); Anal. Calcd. for
C16H15N3O3 (297.31): C, 64.64; H, 5.09; N, 14.13, Found:
C, 64.51; H, 5.17; N, 14.07.
aryl)-4-(3-nitrophenyl)-2-oxocyclohex-3-
enecarboxylate (20–22)
To a solution of chalcone (0.01 mol) in the ethanol
(30 mL), barium hydroxide (0.001 mol) was added fol-
lowed by the addition of ethyl acetoacetate (0.01 mol). The
mixture was refluxed for the appropriate time until the
chalcone disappeared. After completion of reaction as
indicated by the TLC, the solvent was removed under
vacuum and the crude residue was extracted with chloro-
form (2 9 25 mL). The organic layers were combined and
dried over sodium sulfate, and the chloroform layer was
concentrated to get the crude product that was then re-
crystallized from ethanol to get pure product.
5-(benzo[d][1,3]dioxol-5-yl)-4,5-dihydro-3-(30-
nitrophenyl)-1H-pyrazole (18)
Yellow solid, Yield 54 %; m.p.: 101–102 °C; IR (KBr) m
(cm-1): 3,338 (N–H), 3,024 (=C–H, aromatic),
1,611(C=N), 1,529 & 1,349 (NO2), 1,245 (C–N); 1H NMR
(300 MHz, CDCl3) d ppm: 8.41 (s, 1H, H-20), 8.16 (d, 1H,
J = 8.1 Hz, H-40), 8.02 (d, 1H, J = 7.8 Hz, H-60), 7.54
(dd, 1H, J = 7.8, 8.1 Hz, H-50), 6.86 (s, 1H, H-2),
6.81–6.75 (m, 2H, H-5 and 6), 6.23 (bs, 1H, NH), 5.95 (s,
2H, –OCH2O–), 4.93 [m, 1H, Hx (pyrazoline ring)], 3.47
[dd, 1H, J = 11.1, 16.5 Hz, Hb (pyrazoline ring)], 3.03 [dd,
1H, J = 8.7, 16.5 Hz, Ha (pyrazoline ring)]; 13C NMR
(75 MHz, CDCl3) d ppm: 150.22 (C-3, pyrazoline ring),
148.57 (C–NO2), (148.49, 148.21, 147.38, 136.06, 134.75,
131.34, 129.46, 123.01, 120.63, 119.66, 108.41, 106.48,
101.19), 64.61 (C-5, pyrazoline ring), 41.02 (C-4, pyrazo-
line ring); MS (APCI) m/z = 312 (M ? 1); Anal. Calcd.
for C16H13N3O4 (311.29): C, 61.73; H, 4.21; N, 13.50,
Found: C, 61.54; H, 4.23; N, 13.53.
Ethyl 6-(4-methoxyphenyl)-4-(3-nitrophenyl)-2-
oxocyclohex-3-enecarboxylate (20)
Yellow solid, Yield 47 %; m.p.: 106–107 °C; IR (KBr) m
(cm-1): 3,076 (=C–H, aromatic), 1,731 (C=O), 1,655
(C=O), 1,609 (C=C), 1,529 & 1,349 (NO2), 1,249 (C–O);
1H NMR (300 MHz, CDCl3) d ppm: 8.38 (s, 1H, H-20),
8.28 (d, 1H, J = 8.1 Hz, H-40), 7.87 (d, 1H, J = 7.8 Hz,
H-60), 7.63 (dd, 1H, J = 7.8, 8.1 Hz, H-50), 7.25 (d, 2H,
J = 8.7 Hz, Ar–H), 6.89 (d, 2H, J = 8.7 Hz, Ar–H), 6.62
(s, 1H, H-3 of cyclohexenone), 4.02 (q, 2H, J = 7.2 Hz,
–CH2CH3), 3.81 (s, 3H, OCH3), 3.78–3.74 (m, 2H, H-1 and
6 of cyclohexenone), 3.06–3.02 (m, 2H, H-5 of cyclo-
hexenone), 1.08 (t, 3H, J = 7.2 Hz, CH3); 13C NMR
(75 MHz, CDCl3) d ppm: 193.72 (C-2 of cyclohexenone),
168.91 (C=O at C-1 of cyclohexenone), 159.04 (C-OMe),
155.67 (C-4 of cyclohexenone), 148.64 (C–NO2), (139.60,
132.50, 131.82, 130.01, 128.31, 127.89, 125.90, 124.79,
121.08, 114.29, phenyl ring and C-3 of cyclohexenone),
61.08 (C-1 of cyclohexenone), 59.82 (–O–CH2CH3), 55.30
(OCH3), 43.26 (C-6 of cyclohexenone), 36.22 (C-5 of
cyclohexenone), 13.98 (CH3); MS (APCI) m/z = 396
(M ? 1); Anal. Calcd. for C22H21NO6 (395.41): C, 66.83;
H, 5.35; N, 3.54, Found: C, 66.92; H, 5.42; N, 3.68.
4,5-dihydro-5-(naphthalen-2-yl)-3-(30-nitrophenyl)-1H-
pyrazole (19)
Yellow solid, Yield 62 %; m.p.: 98–99 °C; IR (KBr) m
(cm-1): 3,349 (N–H), 3,013 (=C–H, aromatic), 1,617
(C=N), 1,523 & 1,348 (NO2), 1,254 (C–N); 1H NMR
(300 MHz, CDCl3) d ppm: 8.45 (s, 1H, H-20), 8.17 (d, 1H,
J = 7.2 Hz, H-40), 8.04 (d, 1H, J = 7.8 Hz, H-60),
7.86–7.79 (m, 4H, H-1 4, 5, and 8), 7.55 (dd, 1H, J = 7.8,
8.1 Hz, H-50), 7.49–7.47 (m, 3H, H-3, 6 and 7), 6.12 (bs,
1H, NH), 5.22–5.11 [m, 1H, Hx (pyrazoline ring)], 3.57
[dd, 1H, J = 11.1, 16.5 Hz, Hb (pyrazoline ring)], 3.16 [dd,
1H, J = 8.9, 16.5 Hz, Ha (pyrazoline ring)]; 13C NMR
(75 MHz, CDCl3) d ppm: 150.02 (C-3, pyrazoline ring),
148.58 (C–NO2), (138.96, 133.74, 133.52, 133.26, 132.37,
129.94, 129.26, 128.03, 127.72, 126.61, 126.19, 124.75,
123.21, 121.35, phenyl and naphthyl), 64.67 (C-5, pyraz-
oline ring), 41.04 (C-4, pyrazoline ring); MS (APCI) m/
z = 318 (M ? 1); Anal. Calcd. for C19H15N3O2 (317.34):
C, 71.91; H, 4.76; N, 13.24, Found: C, 71.83; H, 4.87; N,
13.28.
Ethyl 6-(benzo[d][1,3]dioxol-5-yl)-4-(3-nitrophenyl)-2-
oxocyclohex-3-enecarboxylate (21)
White solid, Yield 54 %; m.p.: 152–153 °C; IR (KBr) m
(cm-1): 3,091 (=C–H, aromatic), 1,726 (C=O), 1,666
(C=O), 1,597 (C=C), 1,533 & 1,347 (NO2), 1,243 (C–O);
1H NMR (300 MHz, CDCl3) d ppm: 8.37 (s, 1H, H-20),
8.29 (d, 1H, J = 8.4 Hz, H-40), 7.86 (d, 1H, J = 7.8 Hz,
H-60), 7.62 (dd, 1H, J = 7.8, 8.1 Hz, H-50), 6.81-6.78 (m,
3H, ArH), 6.60 (s, 1H, H-3 of cyclohexenone), 5.96 (s, 2H,
–OCH2O–), 4.10 (q, 2H, J = 7.2 Hz, –CH2CH3),
3.77–3.70 (m, 2H, H-1 and 6 of cyclohexenone), 3.04 (m,
123

2030
Med Chem Res (2014) 23:2019–2032
2H, H-5 of cyclohexenone), 1.12 (t, 3H, J = 7.2 Hz, CH3);
13C NMR (75 MHz, CDCl3) d ppm: 193.58 (C-2 of
cyclohexenone), 168.84 (C=O at C-1 of cyclohexenone),
155.59 (C-4 of cyclohexenone), 148.65 (C–NO2), (148.03,
147.06, 139.52, 134.29, 131.86, 130.07, 125.89, 124.88,
121.11, 120.63, 108.61, 107.50 phenyl ring and C-3 of
cyclohexenone), 101.23 (OCH2O), 61.20 (C-1 of cyclo-
hexenone), 59.77 (–O–CH2CH3), 43.73 (C-6 of cyclo-
hexenone), 36.24 (C-5 of cyclohexenone), 14.06 (CH3);
MS (APCI) m/z = 410 (M ? 1); Anal. Calcd. for
C22H19NO7 (409.39): C, 64.54; H, 4.68; N, 3.42, Found: C,
64.65; H, 4.77; N, 3.51.
and griseofulvin was used as a standard antifungal agent.
All MTCC cultures were collected from Institute of
Microbial Technology, Chandigarh, and tested against the
above-mentioned known drugs.
Mueller–Hinton broth and Sabouraud’s broth were used
as nutrient medium to grow bacteria and fungus, respec-
tively. Inoculum size for the test strain was adjusted to 106
colony-forming unit (CFU) per milliliter by comparing the
turbidity.
Serial dilutions were prepared in primary and secondary
screening. The control tube containing no antibiotic was
immediately subcultured (before inoculation) by spreading
a loopful evenly over a quarter of plate of medium suitable
for the growth of test organism and incubated at 37 °C for
bacteria and 22 °C for fungi overnight. The MIC of the
control organism was read to check the accuracy of the
drug concentrations. The lowest concentration inhibiting
growth of the organism was recorded as the MIC. Each test
compound was diluted, obtaining 2,000 lg/mL concentra-
tion, as a stock solution. In primary screening, 500, 250 and
125 lg/mL concentrations of the test compounds were
taken. The active synthesized compounds found in this
primary screening were further tested in a second set of
dilution against all organisms. The drugs found active in
primary screening were similarly diluted to obtain 100, 50,
25, 12.5, 6.250, 3.125 and 1.5625 lg/mL concentrations.
The highest dilution showing at least 99 % inhibition is
taken as MIC. The antimicrobial screening data are shown
in Table 2.
Ethyl 6-(3,4,5-methoxyphenyl)-4-(3-nitrophenyl)-2-
oxocyclohex-3-enecarboxylate (22)
White solid, Yield 60 %; m.p.: 162–163 °C; IR (KBr) m
(cm-1): 3,084 (=C–H, aromatic), 1,733 (C=O), 1,675
(C=O), 1,591 (C=C), 1,539 & 1,349 (NO2), 1,253 (C–O);
1H NMR (300 MHz, CDCl3) d ppm: 8.39 (s, 1H, H-20),
8.30 (d, 1H, J = 8.1 Hz, H-40), 7.88 (d, 1H, J = 7.8 Hz,
H-60), 7.64 (dd, 1H, J = 7.8, 8.1 Hz, H-50), 6.62 (s, 1H,
H-3 of cyclohexenone), 6.53 (s, 2H, ArH), 4.12 (q, 2H,
J = 7.2 Hz, –CH2CH3), 3.87 (s, 6H, 2 9 OCH3), 3.84 (s,
3H, OCH3), 3.81–3.77 (m, 2H, H-1 and 6 of cyclohexe-
none), 3.09–3.04 (m, 2H, H-5 of cyclohexenone), 1.11 (t,
3H, J = 7.2 Hz, CH3); 13C NMR (75 MHz, CDCl3) d ppm:
193.48 (C-2 of cyclohexenone), 168.86 (C=O at C-1 of
cyclohexenone), 155.53 (C-4 of cyclohexenone), 148.66
(C–NO2), (153.55, 139.53, 136.15, 141.83, 125.92, 124.89,
121.11, 104.40, phenyl ring and C-3 of cyclohexenone),
61.19 (C-1 of cyclohexenone), 60.86 (OCH3), 59.42 (-O-
CH2CH3), 56.24 (2 9 OCH3), 44.25 (C-6 of cyclohexe-
none), 36.30 (C-5 of cyclohexenone), 14.07 (CH3); MS
(APCI) m/z = 456 (M ? 1); Anal. Calcd. for C24H25NO8
(455.46): C, 63.29; H, 5.53; N, 3.08, Found: C, 63.42; H,
5.61; N, 3.14.
Antitubercular activity
MIC of the test compounds against M. tuberculosis H37Rv
was determined by L.J. agar (MIC) method (Desai et al.,
1984; Shah et al., 1985; Anargyros et al., 1990) where
primary 1,000, 500 and 250 lg/mL and secondary 200,
100, 50, 25, 12.5, 6.250 and 3.125 lg/mL dilutions of each
test compound were added to liquid L.J medium and then
media were sterilized by inspissation method. A culture of
M. tuberculosis H37Rv growing on L.J. medium was har-
vested in 0.85 % saline in bijou bottles. For all test com-
pounds, first a stock solution of 2,000 lg/mL concentration
was prepared in DMSO. These tubes were then incubated
at 37 °C for 24 h followed by streaking of M. tuberculosis
H37Rv (5 9 104 bacilli per mL). These tubes were then
incubated at 37 1 °C. Growth of bacilli was seen after
12, 22 and finally 28 days of incubation. Tubes having the
compounds were compared with control tubes where
medium alone was incubated with M. tuberculosis H37Rv.
The concentration at which no development of colonies
occurred or less than 20 colonies was taken as MIC of test
compound. The standard strain M. tuberculosis H37Rv was
tested with known drug rifampicin.
Microbiology
Antibacterial and antifungal activity
The MICs of synthesized compounds were carried out by
broth microdilution method as described by Rattan (Rattan,
2000). DMSO was used as diluents to get desired con-
centration of drugs to test upon standard bacterial strains.
Antibacterial activity was screened against two gram-
positive (S. aureus MTCC 96 and S. pyogenes MTCC 443)
and two gram-negative (E. coli MTCC 442 and P. aeru-
ginosa MTCC 2488) bacteria by using ampicillin as a
standard antibacterial agent. Antifungal activity was
screened against three fungal species (C. albican MTCC
227, A. niger MTCC 282 and A. clavatus MTCC 1323),
123

Med Chem Res (2014) 23:2019–2032
2031
carrying out the antileishmanial screening of the compounds. The
author dedicated this paper to his mentor and guide Prof. (Dr.) Rahul
Jain.
In vitro antileishmanial activity against promastigotes
Drugs All derivatives (1–22) were added into the cultures
as a dimethyl sulfoxide (DMSO) solution (50 lM). The
final solvent (DMSO) concentrations never exceeded 1 %
(v/v) and had no effect on the parasites’ proliferation or
morphology.
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