Thermal solvent-free synthesis of novel pyrazolyl chalcones and pyrazolines as potential antimicrobial agents

Article abstract of DOI:10.1016/j.bmcl.2011.03.080

A novel approach was adopted for the synthesis of series of new pyrazolyl chalcones (3a-c) by the reaction of 5-chloro-3-methyl-1-phenylpyrazole-4- carboxaldehyde (1) with different 5-acetylbarbituric acid derivatives (2a-c) under thermal solvent-free con

Full text of DOI:10.1016/j.bmcl.2011.03.080

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2860–2865
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Thermal solvent-free synthesis of novel pyrazolyl chalcones and pyrazolines
as potential antimicrobial agents
Zeba N. Siddiqui a, , T. N. Mohammed Musthafa a, Anis Ahmad b, Asad U. Khan b
⇑
a Department of Chemistry, Aligarh Muslim University, Aligarh 202 002, India
b Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202 002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel approach was adopted for the synthesis of series of new pyrazolyl chalcones (3a–c) by the reac-
tion of 5-chloro-3-methyl-1-phenylpyrazole-4-carboxaldehyde (1) with different 5-acetylbarbituric acid
derivatives (2a–c) under thermal solvent-free condition. The chalcones were then converted to the cor-
responding pyrazolines (4a–c) under the same condition in excellent yields. All the synthesized com-
pounds were characterized using elemental analysis and spectral data (IR, 1H NMR, and mass
spectrometry). The synthesized compounds were tested for their antimicrobial activity by disk diffusion
assay with slight modifications against Gram-positive, Gram-negative strains of bacteria as well as fungal
strains. The investigation of antimicrobial screening revealed that compounds (3a–4c) showed good anti-
bacterial and antifungal activities, respectively. Among the screened compounds, 3b showed more potent
Received 27 October 2010
Revised 4 March 2011
Accepted 23 March 2011
Available online 30 March 2011
Keywords:
Thermal solvent-free reactions
Chalcones
Pyrazolines
Antibacterial activity
Antifungal activity
inhibitory activity (MIC = 12.5
fluconazole.
lg/ml) nearly to that of standard antibiotics ciprofloxacin, griseofulvin and
Ó 2011 Elsevier Ltd. All rights reserved.
Development of new antimicrobial agents with novel structure
and mode of action remains the primary goal of scientists for the
solution of increasing bacterial resistance gained by microorgan-
ism to classical antimicrobial agents.1 Chalcones and their deriva-
tives are an attractive molecular scaffold for the search of new
biologically active molecules. Studies reveal that incorporation of
pyrazole moiety into various heterocyclic ring systems gives
worthwhile molecules from the biological point of view.2–4 Several
pharmaceutical drugs including celecoxib5 and rimonabant6 utilize
the pyrazole as their core molecular entity.7,8 Many pyrazole deriv-
atives are reported to have a broad spectrum of biological activi-
ties, such as anti-inflammatory,9 antifungal,10 herbicidal,11
antiviral,12 A3 adenosine receptor antagonists,13 etc. On the other
hand chalcones or 1,3-diaryl/heteroaryl-2-propene-1-ones, have
been recently subject of great interest due to their interesting
pharmacological activities including antioxidant,14 antibacterial,15
antileishmanial,16 anticancer,17 antiangiogenic,18 anti-infective,
anti-inflammatory,19 nitric oxide inhibition,20 antifungal,21 tyrosi-
nase inhibition,22 etc.
was perceived that the synergistic effect of heterocyclic moieties
in single nucleus might result in the formation of some worthwhile
molecules from the biological point of view.
One of the challenges facing chemists this century is to develop
new transformations that are not only efficient, selective, and high
yielding but also environmentally benign.33 During the last decade,
the topic of ‘green chemistry’ has received increasing attention.
‘Green chemistry’ aims the total elimination (or at least the mini-
mization) of waste, and the implementation of sustainable pro-
cesses. The utilization of non-toxic chemicals, renewable
materials and solvent-free conditions are the key issues of green
synthetic strategy.34 Thermal solvent-free reactions are gaining
importance in this context as it meets the requirement of
greenness.
To the best of our knowledge no reports have so far made in the
synthesis of pyrazolyl chalcones and pyrazolines under thermal
solvent-free ‘green’ condition. Thus, in accordance with the
increasing interest in the medicinal chemistry community in tech-
nologies and concepts that facilitate a more rapid environmentally
benign synthesis and consequently, the screening of novel chemi-
cal substances herein we report the novel synthesis of biologically
important pyrazolyl chalcones and pyrazolines under thermal sol-
vent-free condition in the absence of any acidic or basic catalyst.
Furthermore, the newly synthesized compounds were screened
for their in vitro antimicrobial activities against Gram-positive bac-
teria, Gram-negative bacteria and fungi.
Among the various derivatives of chalcones, synthesis of pyraz-
olines has engrossed substantial attention from organic23,24 and
medicinal chemists because of their pronounced biological activi-
ties such as tranquillizing, muscle relaxant, psychoanaleptic, anti-
depressant, antimicrobial, etc.25–32 Keeping in view the potential
biological activities of pyrazoles, chalcones and pyrazolines, it
Chalcones are usually synthesized by the Claisen–Schmidt
reaction in basic/acidic medium in polar solvents and involves
⇑
Corresponding author. Tel.: +91 9412653054.
E-mail address: siddiqui_zeba@yahoo.co.in (Z.N. Siddiqui).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.080

Z. N. Siddiqui et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2860–2865
2861
cumbersome purification process as the reaction often lead to a
complex mixture. Many of these reported synthetic procedures
require harsh reaction conditions, along with poor yields and low
selectivity.35 Due to exceptional reactivity of formyl group in
5-chloro-3-methyl-1-phenylpyrazole-4-carboxaldehyde 1 as well
as the versatile biological activities of barbituric acid derivatives,36
heteroaryl chalcones 3a–c were synthesized by the reaction of 1
with 5-acetyl-1,3-dimethylbarbituric acid (2a), 5-acetylbarbituric
acid (2b) and 5-acetylthiobarbituric acid (2c) in a variety of sol-
vents and in solvent-free condition (Scheme 1)43. The chalcones
3a–c synthesized using ethanol/methanol/acetic acid as solvents
in conventional method under the influence of basic catalyst, took
longer period for completion of reaction (8–20 h) with lower yields
(62–78%) (Table 1). In order to achieve the requirement of green-
ness, we then carried out reactions under thermal solvent-free
condition by simply heating the starting materials at 80 °C. To
our pleasant surprise, reactions were completed in shorter time
(10–20 min) with substantial increase in yield of products (82–
87%) even in the absence of any acidic or basic catalyst (Table 1).
Thus, the present work also describes the superiority of green syn-
thetic method over the conventional toxic solvent heating proce-
dures. The structures of the compounds isolated were
characterized by elemental and spectral analysis (IR, 1H NMR and
Mass spectrometry).
The infrared (IR) spectrum of 3a showed the carbonyl absorp-
tion band of barbituric moiety at 1715 cmÀ1. The carbonyl group
O
R
N
O
N
R
X
R = H or CH3
X= O or S
AcOH / POCl3
120 °C
H3C
O
Ha
O
H3C
H3C
Conventional
heating or
H
Q
Q
C
O
+
N
Thermal solvent-free
heating, 80 °C
N
Hb
N
Cl
N
Cl
C6H5
C6H5
2a-c
1
3a-c
Conventional heating
or
Thermal solvent-free
heating, 80 °C
NH 2NH 2
DMF / POCl3
70 °C
H3C
H
N
N
N
H3C
N
O
Q
He
Hd
Hc
N
C6H5
N
Cl
C6H5
4a-c
O
CH 3
N
3a, 4a =
Q=
O
N
O
CH3
O
H
N
Q=
3b, 4b =
O
O
N
O
H
O
H
N
Q=
3c, 4c =
N
H
S
Scheme 1. Synthetic route of pyrazolyl chalcones (3a–c) and pyrazolyl pyrazolines (4a–c).

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Z. N. Siddiqui et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2860–2865
Table 1
Synthesis of compounds 3a–4c under various reaction conditions
Entry
Product
Conventional heating methods
Methanol/piperidine, reflux
Thermal solvent-free method
Ethanol/pyridine, reflux
Acetic acid/sodium acetate, reflux
Timea
Yieldb(%)
Timea
Yieldb (%)
Timea
Yieldb (%)
Timea
Yieldb (%)
1
2
3
4
5
6
3a
3b
3c
4a
4b
4c
12 h
14 h
8 h
45 min
45 min
35 min
72
70
78
68
71
66
11.5
12 h
8 h
25 min
40 min
35 min
70
70
73
64
69
68
16 h
20 h
12 h
1 h
45 min
45 min
68
64
62
72
70
76
10 min
20 min
20 min
5 min
10 min
10 min
87
82
86
89
91
90
a
Reaction progress monitored by TLC.
All yields refer to recrystallized products.
b
of propenone moiety appeared as strong and sharp absorption
band at 1664 cmÀ1. Another sharp and strongly absorbed band at
spectrometer, Micromass Quattro II triple quadrupol mass spec-
trometer and Jeol-SX-102 (FAB) spectrometer. Elemental analyzes
(C, H, N) were conducted using Carlo Erba analyzer model 1108.
The purity of all compounds was checked by TLC on glass plates
(20 Â 5 cm) coated with silica gel (E-Merck G254, 0.5 mm thickness).
The plates were run in chloroform–methanol (4:1) mixture and
were visualized by iodine vapors. 3-Methyl-1-phenylpyrazole-5-
one, barbituric acid, thiobarbituric acid and 1,3-dimethylbarbituric
acid were purchased from Sigma–Aldrich chemicals Pvt. Ltd. Other
chemicals were of commercial grade and used without further
purification. 5-Chloro-3-methyl-1-phenylpyrazole-4- carboxalde-
hyde,37 5-acetylbarbituric acid, 5-acetylthiobarbituric acid and 5-
acetyl-1,3-dimethylbarbituric acid38 were synthesized by using
DMF/POCl3 and AcOH/POCl3 according to the reported methods.
Antibacterial studies: The newly synthesized compounds were
screened for their antibacterial activity against Streptococcus pyog-
enes (clinical isolate), Methicillin resistant Staphylococcus aureus
(MRSA +Ve), Pseudomonas aeruginosa (ATCC-27853), Klebsiella
pneumoniae (clinical isolate) and Escherichia coli (ATCC-25922)
bacterial strains by disk diffusion method.39,40 A standard inocu-
lums (1–2 Â 107 c.f.u/ml 0.5 McFarland standards) was introduced
on to the surface of sterile agar plates, and a sterile glass spreader
was used for even distribution of the inoculums. The disks measur-
ing 6 mm in diameter were prepared from Whatman no. 1 filter pa-
per and sterilized by dry heat at 140 °C for 1 h. The sterile disks
previously soaked in a known concentration of the test compounds
were placed in nutrient agar medium. Solvent and growth controls
1618 cmÀ1 was assigned to carbon–carbon double bond of
a
,b-
unsaturated system. The 1H NMR spectrum showed trans olefinic
protons Ha and Hb as ortho coupled doublets at 8.00
d
(J = 16.2 Hz) and d 8.55 (J = 15.9 Hz), respectively. The value of
spin–spin coupling constant Jab in the range 15–16 Hz is indicative
of the E-configuration of chalcone. The aromatic protons of the N-
phenyl-pyrazole-moiety were present in the form of multiplet at d
7.42–7.58. The two N–CH3 group protons of barbituric acid moiety
were discernible as two sharp singlets at d 3.37 and 3.40 whereas
protons of CH3 group of pyrazole unit appeared as another sharp
singlet at d 2.59. Further confirmation of the structure was given
by mass spectra, which showed M+ at 400.12 as base peak. The
spectral data of other compounds followed similar pattern.
With the objective of synthesizing pyrazolines containing pyra-
zole moieties heterochalcones, (3a–c) were treated with hydrazine
hydrate in ethanol/methanol/acetic acid under basic medium in
conventional heating method (Scheme 1)44. The reactions as visu-
alized afforded the products (4a–c) in considerable yields (64–76%)
within 25 min–1 h (Table 1). The same reactions carried out under
solvent-free condition afforded the products (4a–c) with increase
in yields (89–91%) along with substantial reduction in reaction
time (5–10 min) (Table 1). All the reactions carried out in conven-
tional solvents were found to be incomplete in the absence of the
basic/acidic catalysts, however thermal solvent-free reactions pro-
ceeded smoothly in the absence of catalysts. Thus, our methodol-
ogy added a new efficient procedure for the generation of novel
pyrazolyl chalcones and pyrazolines in excellent yields, to the
existing conventional solvent heating techniques.
The IR spectrum of 4a showed a broad absorption band at
3200 cmÀ1 indicating the NH group of pyrazoline moiety. The car-
bonyl absorption band of barbituric acid unit was present at
1709 cmÀ1. The absorbtion bands at 1633,1363 cmÀ1 were as-
signed to C@N, C–N groups of pyrazoline moiety, respectively. In
1H NMR spectrum of 4a, the presence of the pyrazoline unit was
manifested by two doublets of doublets at d 3.58 (Hd), 4.05 (Hc)
and a triplet at 4.80 (He). The NH proton of pyrazoline unit ap-
peared as broad singlet at d 6.95. The five aromatic protons of N-
phenyl-pyrazole-moiety were discernible as multiplets in the
range of d 7.48–7.52. The six N–CH3 protons were present as sharp
singlet at d 3.34 whereas protons of CH3 group of pyrazole unit ap-
peared at d 2.24 as another sharp singlet. Further evidence of the
structure was given by mass spectrometry, which has given M+
ion peak at 414.15 as base peak. The spectral data of other pyraz-
olines as obtained are explained in references and notes.
were kept. Ciprofloxacin (30 lg) was used as positive control while
the disk poured in DMSO was used as negative control. The plates
were inverted and incubated for 24 h at 37 °C. The susceptibility
was assessed on the basis of diameter of zone of inhibition against
Gram-positive and Gram-negative strains of bacteria. Inhibition
zones were measured and compared with the controls. The bacte-
rial zones of inhibition values are given in Table 2.
Minimum inhibitory concentrations (MICs) were determined by
broth dilution technique. The nutrient broth, which contained log-
arithmic serially two fold diluted amount of test compound and
controls were inoculated with approximately 5 Â 105 c.f.u/ml of
actively dividing bacteria cells. The cultures were incubated for
24 h at 37 °C and the growth was monitored visually and spectro-
photometrically. The lowest concentration (highest dilution) re-
quired to arrest the growth of bacteria was regarded as
minimum inhibitory concentration (MIC). To obtain the minimum
bactericidal concentration (MBC), 0.1 ml volume was taken from
each tube and spread on agar plates. The number of c.f.u was
counted after 18–24 h of incubation at 35 °C. MBC was defined as
the lowest drug concentration at which 99.9% of the inoculums
were killed. The minimum inhibitory concentration and minimum
bactericidal concentration are given in Table 3.
Melting points were taken in Reichert Thermover instrument
and are uncorrected. The IR spectra were recorded on Perkin Elmer
RXI spectrometer in KBr, 1H NMR on Bruker DRX-300 and Bruker
Avance II-400 spectrometer using tetramethyl silane (TMS) as
the internal standard and DMSO-d6/CDCl3 as solvent. Mass
spectra were recorded on JEOL-Accu TOF JMS-T100LC DART-MS
The investigation of antibacterial screening data revealed that
all the tested compounds 3a–4c showed moderate to good
bacterial inhibition. All the compounds showed good inhibition

Z. N. Siddiqui et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2860–2865
2863
Table 2
Antibacterial activity of compounds 3a–4c
Compounds
Diameter of zone of inhibition (mm)
Gram-positive bacteria
S. pyogenes
MRSAa
20.1 0.4
22.2 0.2
18.4 0.4
17.1 0.2
16.8 0.1
15.2 0.1
24.0 0.3
—
Gram-negative bacteria
P. aeruginosa
K. pneumoniae
E. coli
3a
3b
3c
4a
4b
4c
Standard
DMSO
17.3 0.4
20.2 0.2
17.1 0.3
16.5 0.2
15.3 0.6
14.2 0.2
25.0 0.4
—
25.2 0.3
27.2 0.4
22.1 0.3
21.8 0.2
20.1 0.5
18.2 0.4
32.0 0.3
—
16.1 0.4
17.1 0.2
15.4 0.3
15.7 0.4
14.7 0.4
14.4 0.3
18.0 0.2
—
22.2 0.4
24.8 0.2
20.7 0.3
19.6 0.2
19.2 0.4
19.1 0.3
26.0 0.4
—
Positive control (standard): ciprofloxacin and negative control (DMSO) measured by the Halo Zone Test (Unit, mm).
a
Methicillin resistant Staphylococcus aureus (MRSA +Ve).
Table 3
MIC and MBC results of 3a–4c and positive control ciprofloxacin
Compounds
Gram-positive bacteria
S. pyogenes
MBC
Gram-negative bacteria
MRSA
P. aeruginosa
K. pneumoniae
E. coli
MIC
MIC
25
MBC
MIC
MBC
MIC
MBC
MIC
MBC
3a
3b
3c
4a
4b
4c
Standard
25
50
25
100
50
50
50
50
100
>100
>100
50
25
100
25
50
12.5
25
100
25
50
25
12.5
50
25
50
50
50
12.5
25
12.5
25
50
12.5
25
100
100
100
100
25
100
100
>100
100
50
25
50
50
50
50
50
50
50
50
12.5
100
>100
>100
50
100
100
50
100
100
12.5
50
12.5
12.5
12.5
MIC (lg/ml) = minimum inhibitory concentration, that is, the lowest concentration of the compound to inhibit the growth of bacteria completely; MBC (
lg/ml) = minimum
bactericidal concentration, that is, the lowest concentration of the compound for killing the bacteria completely.
Table 4
against S. pyogenes, Methicillin resistant S. aureus (MRSA +Ve), P.
Antifungal activity of compounds (3a–4c): Positive control (griseofulvin, fluconazole)
and negative control (DMSO) measured by the Halo Zone Test (unit, mm)
aeruginosa, K. pneumoniae and E. coli species. Compound 3b,
showed more potent antibacterial activity (MIC = 12.5 g/ml)
l
Compounds
Diameter of zone of inhibition (mm)
nearly equivalent to that of ciprofloxacin against all bacterial
strains tested. The MBC of compounds was found to be two, three
or four folds higher than the corresponding MIC results. A compar-
ative study also revealed that pyrazolyl chalcones 3a–c, are more
potent antimicrobial agents than pyrazolines 4a–c. The potential
bactericidal effects of chalcones can be related to the ability of
the a,b-unsaturated ketone system to undergo a conjugated addi-
tion to a nucleophilic group like a thiol group in an essential
protein.
Antifungal studies: Antifungal activity was also done by disk dif-
fusion method. For assaying antifungal activity Candida albicans,
Aspergillus fumigatus, Trichophyton mentagrophytes and Penicillium
marneffei were recultured in DMSO by agar diffusion method.41,42
Sabourauds agar media was prepared by dissolving peptone (1 g),
CA
AF
TM
PM
3a
3b
3c
4a
4b
4c
21.3 0.4
25.5 0.5
20.4 0.3
20.8 0.4
20.4 0.5
20.2 0.2
30.0 0.2
20.0 0.5
—
18.6 0.4
21.3 0.2
17.9 0.3
17.4 0.2
16.8 0.3
16.2 0.3
27.0 0.2
20.0 0.5
—
17.2 0.2
17.1 0.3
17.1 0.6
16.9 0.4
16.0 0.2
15.8 0.2
24.0 0.3
19.0 0.5
—
14.8 0.4
14.1 0.2
14.1 0.3
14.2 0.6
13.1 1.2
13.1 0.2
20.0 0.5
18.0 0.5
—
Griseofulvin
Fluconazole
DMSO
CA; Candida albicans, AF; Aspergillus fumigatus, TM; Trichophyton mentagrophytes,
PM; Penicillium marneffei.
D
-glucose (4 g) and agar (2 g) in distilled water (100 ml) and
at 35 °C and the growth was monitored. The lowest concentration
(highest dilution) required to arrest the growth of fungus was re-
garded as minimum inhibitory concentration (MIC). To obtain the
minimum fungicidal concentration (MFC), 0.1 ml volume was ta-
ken from each tube and spread on agar plates. The number of
c.f.u. was counted after 48 h of incubation at 35 °C. MFC was de-
fined as the lowest drug concentration at which 99.9% of the inocu-
lums were killed. The minimum inhibitory concentration and
minimum fungicidal concentration are given in Table 5.
The antifungal screening data of the compounds revealed good
to moderate activity. Compounds 3a, 3b, and 3c were showed a
good inhibitory activity against C. albicans and A. fumigatus. Com-
pounds 3a–c also showed moderate activity towards T. mentagro-
phytes and P. marneffei fungal strains. Compounds 4a, 4b, and 4c
were less active towards C. albicans, A. fumigatus, T. mentagrophytes
and P. marneffei fungal strains. Among the screened compounds, 3b
adjusting pH to 5.7. Normal saline was used to make a suspension
of spore of fungal strain for lawning. A loopful of particular fungal
strain was transferred to 3 ml saline to get a suspension of corre-
sponding species. Twenty milliliter of agar media was poured into
each petri dish. Excess of suspension was decanted and the plates
were dried by placing in an incubator at 37 °C for 1 h. Using an agar
punch, wells were made and each well was labeled. A control was
also prepared in triplicate and maintained at 37 °C for 3–4 days.
The antifungal activity of each compound was compared with gris-
eofulvin and fluconazole as standard drugs. Inhibition zones were
measured and compared with the controls. The fungal zones of
inhibition values are given in Table 4. The nutrient broth, which
contained logarithmic serially two fold diluted amount of test
compound and controls was inoculated with approximately
1.6 Â 104 À 6 Â 104 c.f.u/ml. The cultures were incubated for 48 h

2864
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MIC and MFC of compounds 3a–4c, positive control griseofulvin, fluconazole
Compounds
CA
AF
TM
PM
15. Avila, H. P.; Smania, E.; Monache, F. D.; Smania, A. Bioorg. Med. Chem. 2008, 16,
9790.
MIC
MFC
MIC MFC
MIC
MFC
MIC
MFC
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3a
3b
3c
4a
4b
4c
25
12.5
25
25
50
50
6.25
6.25
50
25
50
25
12.5
25
25
50
50
25
50
100
100
100
25
25
12.5
25
25
50
25
6.25
6.25
50
25
50
100
100
100
25
25
12.5
25
50
50
50
25
100
>100
100
100
100
100
12.5 12.5
12.5 6.5
25
>100 >100
12.5
Gris
Flu.
25
12.5
12.5
12.5 6.5
CA; Candida albicans, AF; Aspergillus fumigatus, TM; Trichophyton mentagrophytes,
PM; Penicillium marneffei. MIC ( g/ml) = minimum inhibitory concentration, that is,
the lowest concentration of the compound to inhibit the growth of fungus com-
pletely; MFC ( g/ml) = minimum fungicidal concentration, that is, the lowest
l
l
concentration of the compound for killing the fungus completely.
25. Batulin, Y. M. Farmakol. Toksikol. 1968, 31, 533.
26. Parmar, S. S.; Pandey, B. R.; Dwivedi, C.; Harbinson, R. D. J. Pharm. Sci. 1974, 63,
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showed more potent inhibition against all the fungal strains
(MIC = 12.5 lg/ml). The MFC of compounds were found to be two
or three or four folds higher than the corresponding MIC results.
Thus, overall the data revealed that the compounds 3a–4c have
produced the marked enhancement in the potency of these analogs
as antibacterial and antifungal agents. A comparative study with
known chalcones and pyrazolines revealed that the newly synthe-
sized compounds are equally potent as most of the reported com-
pounds. However, the mode of action of these compounds and
their effect on mammalian cell still needs to be evaluated.
The fact that readily synthesized starting materials, available
reagents along with short reaction time, no additives and simple
work-up and isolation of the products under green condition make
the current method a feasible and attractive protocol for genera-
tion of series of pyrazole derivatives in good yields. Further,
in vitro antibacterial and antifungal evaluation of compounds has
proved them as potent antimicrobial agents. The importance of
such kind of work lies in the possibility that the new compounds
might be more effective against microbes for which a thorough
study regarding the structure–activity relationship, toxicity and
their biological effects would be helpful in designing more potent
antimicrobial agents.
28. Turan-Zitouni, G.; Chevallet, P.; Kilic, F. S.; Erol, K. Eur. J. Med. Chem. 2000, 35,
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43. General method for the preparation of pyrazolyl chalcones (3a–c) under
conventional heating conditions.
Acknowledgments
Financial assistance in the form of major research project [F.No.
37-15/2009 (SR)] from UGC, New Delhi, is gratefully acknowl-
edged. The authors thank SAIF, CDRI, Lucknow and SAIF, Punjab
University, Chandigarh for spectral data.
To a well stirred solution of 5-acetyl-1,3-dimethylbarbituric acid (4.52 mmol)/
5-acetylbarbituric acid (4.52 mmol)/5-acetylthiobarbituric acid (4.52 mmol) in
ethanol (15 ml)/methanol (15 ml)/acetic acid (12 ml), containing pyridine
(1 ml)/piperidine (0.5 ml)/sodium acetate (4 mmol), 5-chloro-3-methyl-1-
phenylpyrazole-4-carboxaldehyde (4.52 mmol) was added in portion. The
reaction mixture was then refluxed in a heating mantle for specified time
(Table 1) and cooled at room temperature. The yellow solid, thus, obtained was
filtered, washed with water, alcohol and dried to afford 3a–c. Further
purification was made by recrystallization from chloroform-methanol (4:1v/
v) mixture.
References and notes
1. Moneer, A. A.; Abouzid, K. A. M.; Said, M. M. Az. J. Pharm. Sci. 2002, 30, 150.
2. Tandon, V. K.; Yadav, D. B.; Chaturvedi, A. K.; Shukla, P. K. Bioorg. Med. Chem.
Lett. 2005, 15, 3288.
3. Akbas, E.; Berber, I. Eur. J. Med. Chem. 2005, 40, 401.
4. Bekhit, A. A.; Ashour, H. M. A.; Ghany, Y. S. A.; Bekhit, A. E. A.; Baraka, A. Eur. J.
Med. Chem. 2008, 43, 456.
5. Penning, T. D.; Talley, J. J.; Bertenshaw, S. R.; Carter, J. S.; Collins, P. W.; Docter,
S.; Graneto, M. J.; Lee, L. F.; Malecha, J. W.; Miyashiro, J. M.; Rogers, R. S.; Rogier,
D. J.; Yu, S. S.; Anderson, G. D.; Burton, E. G.; Cogburn, J. N.; Gregory, S. A.;
Koboldt, C. M.; Perkins, W. E.; Seibert, K.; Veenhuizen, A. W.; Zhang, Y. Y.;
Isakson, P. C. J. Med. Chem. 1997, 40, 1347.
General method for the preparation of pyrazolyl chalcones (3a–c) under thermal
heating conditions.
A
mixture
of
5-chloro-3-methyl-1-phenylpyrazole-4-carboxaldehyde
(4.52 mmol) and 5-acetyl-1,3-dimethylbarbituric acid (4.52 mmol)/5-
acetylbarbituric acid (4.52 mmol)/5-acetyl thiobarbituric acid (4.52 mmol)
was mixed well using a mortar and pestle. The reaction mixture was then
transferred into a 50 ml R.B flask and heated at 80 °C in a heating mantle for
specified time (Table 1). The progress of reaction was monitored by TLC. On
completion, reaction mixture was cooled and 25 ml of ice cold water added.
The bright yellow solid, thus, obtained was filtered, washed with water, alcohol
and dried to afford 3a–c. Further purification was made by recrystallization
from chloroform–methanol (4:1v/v) mixture.
6. Deng, X.; Mani, N. S. Org. Lett. 2008, 10, 1307.
7. Katritzky, A. R.; Wang, M.; Zhang, S.; Voronkov, M. V. J. Org. Chem. 2001, 66,
6787.
(2E)-3-(5-chloro-3-methyl-1-phenylpyrazol-4-yl)-1-(1,3-dimethyl-2,4,6-
8. Deng, X.; Mani, N. S. Org. Lett. 2006, 8, 3505.
pyrimidinetrione-5-yl)-2-propen-1-one (3a)
9. Smith, S. R.; Denhardt, G.; Terminelli, C. Eur. J. Pharmacol. 2001, 432, 107.
10. Prakash, O.; Kumar, R.; Parkash, V. Eur. J. Med. Chem. 2008, 43, 435.
11. Mahajan, R. N.; Havaldar, F. H.; Fernandes, P. S. J. Indian Chem. Soc. 1991, 68,
245.
12. Baraldi, P. G.; Manfredini, S.; Romagnoli, R.; Stevanato, L.; Zaid, A. N.;
Manservigi, R. Nucleosides, Nucleotides Nucleic Acids 1998, 17, 2165.
Yellow crystals; yield 87%, mp 263–266 °C. IR (m
max, cmÀ1, KBr): 1618 (C@C),
1664 (C@O), 1715 (C@O). 1H NMR (300 MHz, CDCl3, d, ppm): 2.59 (3H, s, CH3),
3.37 (3H, s, N–CH3), 3.40 (3H, s, N–CH3), 7.42–7.58 (5H, m, Ar-H), 8.00 (1H, d,
J = 16.2 Hz, Ha), 8.55 (1H, d, J = 15.9 Hz, Hb). ESI-MS m/z: 400.12 (M+). Anal. Calcd
for C19H17N4O4Cl: C, 56.93; H, 4.27; N, 13.97. Found: C, 56.76; H, 4.38; N, 13.91.

Z. N. Siddiqui et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2860–2865
2865
(2E)-3-(5-chloro-3-methyl-1-phenylpyrazol-4-yl)-1-(2,4,6-pyrimidinetrione-5-
white solid, thus, obtained was filtered, washed with water, acetone and dried
to afford 4a–c. Further purification was made by recrystallization from
chloroform–methanol (3:2) mixture.
yl)-2-propen-1-one (3b)
White powder; Yield 82%, mp >300 °C. IR (m
max, cmÀ1, KBr): 1601 (C@C), 1657
(C@O), 1698 (C@O), 3193(NH). 1H NMR(400 MHz, DMSO-d6, d, ppm):2.62 (3H, s,
CH3), 7.47–7.85 (5H, m, Ar-H), 8.32(1H, d, J = 15.8 Hz, Ha), 9.01 (1H, d, J = 15.8 Hz,
Hb), 11.45 (1H, s, NH), 11.60 (1H, s, NH), 16.89 (1H, s, OH, D2O exchangable). FAB-
MS m/z (%): 372 (M+, 75), 371 (55), 336 (10), 261 (20), 260 (10), 232 (40), 214 (15),
157 (100). Anal. Calcd for C17H13N4O4Cl: C, 54.77; H, 3.51; N, 15.02. Found: C,
54.86; H, 3.64; N, 15.04.
5-(5-Chloro-3-methyl-1-phenylpyrazol-4-yl)-3-(1,3-dimethyl-2,4,6-
pyrimidinetrione-5-yl)pyrazoline (4a)
White crystals; yield 89%, mp >300 °C. IR (m
max, cmÀ1, KBr): 1363 (C–N), 1633
(C@N), 1709 (C@O), 3200 (NH). 1H NMR (300 MHz, DMSO-d6, d, ppm): 2.24
(3H, s, CH3), 3.34 (6H, s, 2 N–CH3), 3.58 (1H, dd, J = 18.6 Hz, 9.3 Hz, Hd), 4.05
(1H, dd, J = 18.9 Hz, 10.5 Hz, Hc), 4.80 (1H, t, J = 18.3 Hz He), 6.95 (1H, s, NH),
7.48–7.52 (5H, m, Ar-H). ESI-MS m/z: 414.15 (M+), 413.15, 412.15, 277.15.
Anal. Calcd for C19H19N6O3Cl: C, 55.01; H, 4.61; N, 20.25. Found: C, 55.13; H,
4.78; N, 20.13.
(2E)-3-(5-chloro-3-methyl-1-phenylpyrazol-4-yl)-1-(2-mercapto-4,6-
pyrimidinedione-5-yl)-2-propen-1-one (3c)
Yellow powder; yield 86%, mp >300 °C. IR (m
max, cmÀ1, KBr): 1057 (C@S), 1608
(C@C), 1647 (C@O), 1746 (C@O), 3188 (NH). 1H NMR (400 MHz, DMSO-d6, d,
ppm): 2.56 (3H, s, CH3), 7.47–7.87 (5H, m, Ar-H), 7.87 (1H, d, J = 15.7 Hz, Ha), 9.04
(1H, d, J = 15.8 Hz, Hb), 12.19 (1H, s, NH), 12.73 (1H, s, NH). ESI-MS m/z: 388.6
(M+), 361.6, 360.6, 310.6, 274.6, 202.6. Anal. Calcd for C17H13N4O3SCl: C, 52.51; H,
3.36; N, 14.40. Found: C, 52.57; H, 3.39; N, 14.32.
5-(5-Chloro-3-methyl-1-phenylpyrazol-4-yl)-3-(2,4,6-pyrimidinetrione-5-yl)
pyrazoline (4b)
White powder; yield 91%, mp >300 °C. IR (m
max, cmÀ1, KBr): 1294 (C–N), 1616
(C@N), 1727 (C@O), 3169 (NH), 3215 (NH). 1H NMR (400 MHz, DMSO-d6, d,
ppm): 2.32 (3H, s, CH3), 3.74 (1H, dd, J = 18.9 Hz, 8.7 Hz, Hd), 4.07 (1H, dd,
J = 19.0 Hz, 10.1 Hz, Hc), 4.93 (1H, t, J = 18.7 Hz, He), 7.33 (1H, s, NH), 7.41–7.66
(5H, m, Ar-H), 10.23 (1H, s, NH), 11.12 (1H, s, NH). 16.09 (1H, s, OH, D2O
exchangable). ESI-MS m/z: 386.6 (M+), 376.6, 362.6, 361.6, 360.6, 358.6, 332.6,
318.6, 301.5, 274.6, 202.6. Anal. Calcd for C17H15N6O3Cl: C, 52.79; H, 3.90; N,
21.72. Found: C, 52.86; H, 3.98; N, 21.64.
44. General method for the preparation (4a–c) under conventional heating
conditions.
A mixture of 3a–c (2.52 mmol) and hydrazine hydrate (2.52 mmol) in ethanol
(10 ml)/pyridine (0.6 ml) or methanol (10 ml)/piperidine (0.4 ml) or acetic acid
(8 ml)/sodium acetate (2 mmol) was refluxed in a heating mantle for specified
time (Table 1). After completion of the reaction (checked by TLC), the reaction
mixture was cooled and the solid precipitated out was filtered, washed with
water, acetone and dried to afford 4a–c. Further purification was made by
recrystallization from chloroform–methanol (3:2) mixture.
General method for the preparation of pyrazolines (4a–c) under thermal heating
conditions
The mixture of 3a–c (2.49 mmol) and hydrazine hydrate (2.49 mmol) was
5-(5-Chloro-3-methyl-1-phenylpyrazol-4-yl)-3-(2-mercapto-4,6-
pyrimidinedione-5-yl)pyrazoline (4c)
White powder; Yield 90%, mp >300 °C. IR (m
max, cmÀ1, KBr): 1011 (C@S), 1313
(C–N), 1633 (C@N), 1688 (C@O), 3126 (NH), 3234 (NH). 1H NMR (400 MHz,
DMSO-d6, d, ppm): 2.42 (3H, s, CH3), 3.69 (1H, dd, J = 18.6 Hz, 8.5 Hz, Hd), 4.09
(1H, dd, J = 18.9 Hz, 10.1 Hz, Hc), 4.93 (1H, t, J = 18.7 Hz, He), 7.35 (1H, s, NH),
7.47–7.61 (5H, m, Ar-H), 11.51 (1H, s, NH), 11.92 (1H, s, NH). ESI-MS m/z: 402.6
(M+), 388.6, 362.6, 361.6, 360.6, 358.6, 356.6, 332.6, 302.5, 301.5, 274.6, 202.6,
158.5. Anal. Calcd for C17H15N6O2SCl: C, 50.68; H, 3.74; N, 20.86. Found: C,
50.73; H, 3.85; N, 20.77.
mixed well using
a mortar and pestle. The reaction mixture was then
transferred in to a 50 ml R.B flask and heated at 80 °C over a heating mantle
for specified time (Table 1). After completion of the reaction (checked by TLC),
the reaction mixture was cooled and 20 ml of ice cold water was added. The