Synthesis, physicochemical properties, antimicrobial and antioxidant studies of pyrazoline derivatives bearing a pyridyl moiety

Article abstract of DOI:10.1007/s00044-013-0643-z

A series of new pyrazoline compounds bearing a pyridyl moiety (4a-i) were synthesized by condensing appropriate chalcones with hydrazine hydrate and tested for antimicrobial and antioxidant activities. According to in vitro antimicrobial activity against Bacillus subtilis, Staphylococcus epidermidis, Proteus vulgaris, Pseudomonas aeruginosa, Aspergillus niger and Penicillium chrysogenum and antioxidant activity by DPPH method, the compounds 4a, 4d, 4i and 4e, 4f, 4h showed maximum antimicrobial and antioxidant activities, respectively. Physiochemical properties and Lipinski's 'Rule of Five' analysis predicted higher intrinsic quality of the synthesized compounds and revealed that these compounds have good bioavailability and druglikeness properties.

Full text of DOI:10.1007/s00044-013-0643-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0643-z
ORIGINAL RESEARCH
Synthesis, physicochemical properties, antimicrobial
and antioxidant studies of pyrazoline derivatives bearing a pyridyl
moiety
•
Imtiyaz Hussain Lone Khaliquz Zaman Khan
•
Bharat Inder Fozdar
Received: 8 February 2013 / Accepted: 21 May 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of new pyrazoline compounds bearing
a pyridyl moiety (4a–i) were synthesized by condensing
appropriate chalcones with hydrazine hydrate and tested
for antimicrobial and antioxidant activities. According to
in vitro antimicrobial activity against Bacillus subtilis,
Staphylococcus epidermidis, Proteus vulgaris, Pseudomo-
nas aeruginosa, Aspergillus niger and Penicillium chrys-
ogenum and antioxidant activity by DPPH method, the
compounds 4a, 4d, 4i and 4e, 4f, 4h showed maximum
antimicrobial and antioxidant activities, respectively.
Physiochemical properties and Lipinski’s ‘Rule of Five’
analysis predicted higher intrinsic quality of the synthe-
sized compounds and revealed that these compounds have
good bioavailability and druglikeness properties.
compounds have medicinal importance. Owing to the
interesting activity of variously substituted pyrazolines as
biological agents, considerable attention has been focused on
this class (Mohamad and Payal, 2011). Amongst various
pyrazoline derivatives, 2-pyrazoline seems to be the most
frequently studied pyrazoline-type compounds. The phar-
maceutical importance of these compounds lies in the fact
that they can be effectively utilized as antibacterial, antide-
pressant, anticonvulsant, antihypotensive, psychoanaleptic,
antioxidant and tranquillizing agents (Sivakumar et al.,
2010; Ozdemir et al., 2007; Turan et al., 2000; Kumar et al.,
2009; Ji-Tai et al., 2007). In the development of drugs
intended for oral use, good drug absorption and appropriate
drug delivery are very important (Hou and Xu, 2004). About
30 % of oral drugs fail in development because of poor
pharmacokinetics (Waterbeemed and Gifford, 2003).
Among the pharmacokinetic properties, a low and highly
variable bioavailability is indeed the main reason for stop-
ping further development of the drug (Hou et al., 2007).
Moreover, the knowledge of ionization constants is an
important challenge for understanding various phenome-
nons like biological uptake, pharmacological activity or
in vivo studies. Hence, the discovery of new molecules
requires accurate determination of molecular properties.
The vital role played by small heterocyclic molecules in
drug design cannot be denied. These molecules act as highly
functionalized scaffolds. In this context, pyridyl ring, a
prominent scaffold present in numerous bioactive mole-
cules, has played a vital role in the development of different
medicinal agents (Jyh-Haur et al., 2004). On the other hand,
the interesting and versatile biological activities of pyrazo-
lines established them as important pharmacophore. A
variety of methods have been reported for the preparation of
this class of compounds. In the present article, we report the
reaction of 2-acetyl pyridine with different aromatic
Keywords Chalcones ꢀ Pyrazolines ꢀ
Physicochemical properties ꢀ Antimicrobials ꢀ Antioxidants
Introduction
The pyrazoline compounds are not known to occur in
nature. They are usually prepared by the reaction of
hydrazines with 1,3-diketones. Many synthetic pyrazoline
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0643-z) contains supplementary
material, which is available to authorized users.
I. H. Lone (&) ꢀ K. Z. Khan
Department of Chemistry, University of Kashmir,
Srinagar 190009, Jammu and Kashmir, India
e-mail: hussainjamia5@gmail.com
I. H. Lone ꢀ B. I. Fozdar
Department of Chemistry, IGNOU, New Delhi 110068, India
123

Med Chem Res
aldehydes to form pyrazolines bearing a pyridyl moiety and
studied their antimicrobial, antioxidant activities and phar-
macological properties.
compounds (4a–i) showed absorption bands in the region
of 1,600–1,700 cm-1 corresponding to C=N stretching.
Also, infrared spectra revealed N–H and C–H peaks at
2,900–3,250 cm-1.
In the 1H NMR of pyrazoline, the hydrogen atom attached to
N of pyrazoline ring appears at 5–6.5 ppm, and the hydrogen
atoms attached to C of pyrazoline ring appear at 2–3.5 ppm.
Aromatic and other protons were observed at expected ppm
values. In the 13C NMR spectra of these compounds (4a–i), the
chemical shift values of carbon atoms appeared: 147.8–151.2
(C-3), 33.5–47.6 (C-4) and 48.2–53.3 (C-5).
Results and discussion
Chemistry
The present study was undertaken to synthesize some novel
pyrazoline derivatives according to steps shown in
Scheme 1. In the initial step, chalcones (3a–i) were syn-
thesized by reacting 2-acetylpyridine with appropriate
aldehydes in the presence of base by conventional Claisen–
Schmidt condensation. The chalcones of the acetylpyridine
were reported earlier; however, their preliminary charac-
terizations like IR and NMR were carried out which con-
firmed them as the desired compounds (Prasad et al.,
2008). Reaction between the synthesized chalcones and
hydrazine hydrate in acetic acid led to the synthesis of
novel pyrazolines (4a–i). Most of the products were found
to be homogeneous, checked on pre-coated silica gel TLC
plates which were visualized by exposing them to iodine
vapours or UV light. Heterogeneous products were readily
purified by silica gel column chromatography using a
methanol/chloroform eluent.
Physiochemical properties
Physiochemical properties, mainly molecular size, flexi-
bility, hydrophobicity and the presence of various phar-
macophoric features, influence the behaviours of molecules
in a living organism, including bioavailability. A good
bioavailability can be achieved with an appropriate balance
between solubility and portioning properties. Thus, in order
to achieve good oral drugs, we have investigated a series of
pyrazoline derivatives bearing a pyridyl moiety (4a–i) for
the prediction of their molecular properties and Lipinski’s
‘Rule of Five’ (Lipinski et al., 1997).
C log of P
Structures of the synthesized compounds (4a–i) were
established on the basis of elemental analysis and spectral
data (IR, 1H NMR, 13C NMR and MS). IR spectra of
Poor solubility and permeability are amongst the main
causes for failure during drug development (Avdeef, 2001;
Scheme 1 Synthesis of
pyrazoline derivatives bearing
pyridyl moiety
O
O
O
CH3
a
H
+
R
N
N
2a- 2i
3a -3i
b
R
Compound
3a, 4a
3b, 4b
3c, 4c
3d, 4d
3e, 4e
3f, 4f
3g, 4g
3h, 4h
3i, 4i
H
R
3,4,5-trimethoxy
4-nitro
4
5
3
4-chloro
4-fluoro
NH
N
N
3-fluoro
1
2
4-methoxy
4- N' N-dimethyl
4-bromo
4a - 4i
a= Ethanol / KOH/rt
b= Acetic Acid/ Hydrazine Hydrate /reflux 24h
123

Med Chem Res
Oprea, 2002; Norinder and Bergstorm, 2006). The octanol/
water partition coefficient (P) (also referred as Kow) is a
measure of the propensity of a neutral compound to differ-
entiate dissolution in these immiscible phases. It is usually
referred to as the logarithmic ratio, log P, and serves as a
quantitative descriptor of lipophilicity. The octanol/water
partition coefficient C log P being a measure of hydro-
phobicity/lipophilicity was calculated using Marvin Sketch
tool (Balogh et al., 2009). Compounds with log P values \ 5
(Lipinski’s Rule of Five) mean that they can readily get past
ester/phosphate groups in skin membranes. The results
obtained are given in Table 1. The calculated values of log
P for the derivatives ranged from 2.556 to 4.223. The lipo-
philic aptitude of a compound increases with the increasing
log P. The C log P values of all the compounds show vari-
ation which can be attributed to the presence and position of
different substituents on the phenyl ring. The molar refrac-
tivity (MR—which represents size and polarizability of a
fragment of molecule) describing steric effects was calcu-
lated using Marvin Sketch Software (Table 1).
properties in formulating his ‘Rule of Five’. The rule states
that most molecules with good membrane permeability have
log P B 5, MW B 500, the number of hydrogen bond
acceptors B 10, and the number of hydrogen bond
donors B 5. The rule is widely used as a filter for drug-like
properties. A compound that fulfils at least three out of the
four criteria is said to adhere to ‘Lipinski’s Rule of Five’. A
poor permeation or absorption is more likely when there are
more than five H-bond donors and ten H-bond acceptors. The
series (4a–i) under investigation has not only the most of the
compounds possessing less number of hydrogen bond donors
(\5) but also does possess considerable number of acceptors
(\10). Table 1 lists the values of these properties for the new
scaffolds and suggest that the active compounds can be used
as templates for a drug discovery effort.
Disc diffusion assay
Table 2 summarizes the results obtained from antimicrobial
activity of pyrazoline derivatives with test compounds,
Kanamycin and Fluconazole. All the isolates Bacillus sub-
tilis, Staphylococcus epidermidis, Proteus vulgaris, Pseu-
domonas aeruginosa, Aspergillus niger and Penicillium
chrysogenum showed high degrees of sensitivity towards all
the test compounds (sharing a common structure) with slight
variation because of the presence of different substituents on
the phenyl ring. Compound ‘4d’ with a ‘chloro’ group at para
position of the phenyl ring showed higher antimicrobial
activity followed closely by compound 4i having a bromo
group at the same position. Compound ‘4f’ with a ‘fluoro’
group at meta position showed the least antimicrobial
activity. From the results, it can be inferred that the presence
and position of different groups on phenyl ring of the com-
pounds have marked effect on the antimicrobial efficiency of
the test compounds. Besides, an optimum electron density is
must for a compound to attain maximum activity. The most
important thing noticed is that the test compounds were
slightly more effective against bacteria as compared with
fungi. 1 % DMSO is used as a solvent (control), having no
effect on the tested organism. Hence, we can effectively
conclude here that whole of the antimicrobial effect is
because of the test compounds.
‘Rule of Five’ properties
High oral bioavailability is an important factor for the
development of bioactive molecules as therapeutic agents.
Good intestinal absorption, molecular flexibility (measured
by the number of rotatable bonds), low polar surface area or
total hydrogen bond count (sum of donors and acceptors), are
important predictors of good oral bioavailability (Veber
et al., 2002; Refsgaard et al., 2005). Molecular properties
such as membrane permeability and bioavailability are
always associated with some basic molecular descriptions
such as log P (partition coefficient), molecular weight (MW),
or hydrogen bond acceptor and donor count in a molecule
(Muegge, 2003). Lipinski et al. (1997) used these molecular
Table 1 The calculated molecular properties of compounds 4a–i and
Lipinski’s rule
Compounds Molecular MR C log P H-
weight
H-bond Rule
bond acceptors of Five
donors
criteria
4a
4b
4c
4d
4e
4f
221.25
311.33
266.25
255.70
239.24
239.24
251.28
264.32
300.15
66.49 3.345
88.24 2.556
78.55 3.125
71.10 4.073
66.90 3.503
66.38 3.503
73.74 3.322
81.67 3.578
74.18 4.223
1
1
1
1
1
1
1
1
1
3
6
7
3
3
3
4
4
3
Y
Y
Y
Y
Y
Y
Y
Y
Y
Antioxidant activity
The results summarized in Table 3 give the antioxidant data
in terms of mean % inhibition by DPPH method. All the
synthesized pyrazolines showed good antioxidant activity
with slight variation as expected because of the presence of
different substituents on phenyl ring. Compound 4h showed
maximum antioxidant activity as compared with other
derivatives, perhaps because of the availability of more
electron cloud on the core molecule. Inhibition due to control
4g
4h
4i
MR molar refractivity, Y yes, N no (Rule of Five criteria met or not)
123

Med Chem Res
Table 2 Antibacterial and antifungal screening data of compounds 4a–i
Compounds
Zone of inhibition in ‘mm’
Antibacterial activities
Antifungal activities
B. subtilis
(MTCC 619)
S. epidermidis
(MTCC 435)
P. vulgaris
(MTCC 426)
P. aeruginosa
(MTCC 424)
A. niger
(MTCC 1344)
P. chrysogenum
(MTCC 947)
4a
13
13
14
15
12
12
16
15
13
–
15
13
16
13
13
–
16
12
14
16
17
15
13
11
17
–
14
16
14
19
11
–
14
15
–
16
13
10
17
14
14
12
–
4b
4c
4d
14
–
4e
4f
12
10
12
14
–
4g
15
13
15
–
14
–
4h
4i
14
–
16
–
Control
Kanamycin
Fluconazole
24
–
24
–
22
–
17
–
–
18
14
–, no activity; P. vulgaris, Proteus vulgaris; B. subtilis, Bacillus subtilis; S. epidermidis, Staphylococcus epidermidis; P. aeruginosa, Pseudo-
monas aeruginosa; DMSO, negative control; well diameter/vol., 6 mm/50 ll, Kanamycin, standard for antibacterial activity; Fluconazole,
standard for anti fungal activity
(ethanol) was not seen during the test as shown in the table.
Ascorbic acid was used as a positive control.
on Bruker DPX200 instrument in CDCl3 with TMS as
internal standard for protons and solvent signals as internal
standard for carbon spectra. Chemical shift values are
mentioned in d (ppm). Mass spectra were recorded on
EIMS (Shimadzu) and ESI-esquire 3000 Bruker Daltonics
instrument. The progress of all reactions was monitored by
TLC on 2 9 5 cm2 pre-coated silica gel 60 F254 plates of
thickness of 0.25 mm (Merck). The chromatograms were
visualized under UV 254–366 nm and iodine vapours.
Conclusion
The present study has achieved the efficient synthesis of
pyrazoline bearing a pyridyl moiety and examined their pre-
liminary in vitro antimicrobial activity by disc diffusion
methodand antioxidant activity byDPPH method. From these
activities, it was found that the compounds 4a, 4d and 4i
exhibited maximum antimicrobial activity, while compounds
4e, 4f and 4h showed maximum antioxidant activity. The
molecular properties were predicted, which showed that these
active compounds can be used as templates for development
of new drugs. Also, it was found that all the compounds fol-
lowed Lipinski’s Rule of Five. Thus, the idea of appending the
pyridyl moiety to the pyrazoline nucleus so as to combine the
beneficial effects in a single structure proved to be successful.
In conclusion, the present study showed that synthesized
compounds can be used as template for future development
through modification and derivatization.
Chemical synthesis
General procedure for the preparation
of chalcones (3a–i)
To the stirring solution of 2-acetyl pyridine 1 (0.01 mol)
and aryl aldehyde 2 (0.01 mol) in ethanol (30 ml) was
added an aqueous solution of KOH (40 %, 15 ml). The
mixture was kept overnight at room temperature. It was
poured into crushed ice and acidified with concentrated
HCl. The product obtained was filtered, washed with water
and crystallized from ethanol.
Experimental section
General procedure for the preparation
of pyrazolines (4a–i)
General methods
The appropriate chalcones (0.001 mol) and hydrazine
hydrate (0.001) were immediately dissolved in acetic acid
(5 ml) and refluxed for 24 h. After completion of reaction,
the reflux condenser was removed, and the reaction mixture
Melting points were recorded on Buchi melting point
apparatus D-545; IR spectra (KBr discs) were recorded on
Bruker Vector 22 instrument. NMR spectra were recorded
123

Med Chem Res
2-[5-(4-Nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl]pyridine
(4c) The compound 4c was prepared by reacting (2E)-3-
(4-nitrophenyl)-1-(pyridin-3-yl)prop-2-en-1-one (3c) with
hydrazine hydrate in acetic acid. The product was obtained
Table 3 Antioxidant activity (DPPH assay data) of compounds 4a–i
Compounds % inhibition
25 lg/ml 50 lg/ml 75 lg/ml 100 lg/ml 125 lg/ml
as coloured powder. The yield was 45 %. IR (KBr) cm-1
:
4a
8.23
12.33
20.34
18.18
25.45
23.78
26.56
17.87
27.23
16.22
–
15.77
22.43
29.34
36.65
31.11
35.44
24.11
38.38
21.33
–
22.67
28.59
43.47
44.89
45.56
46.44
33.78
49.27
32.12
–
26.23
34.51
54.19
52.56
59.87
59.39
40.65
60.19
39.26
–
3,125 (N–H Str.), 3,100 (C–H Str.), 1,630 (C=N Str.),
1,500 (C=C Str.),1H NMR (d ppm) (CDCl3): d 3.1 (d, 2H,
CH2), d 6.0 (s, 1H, N–H), d 7.38–7.99 (m, Ar–H), d 8.2 (d,
1H, N–C–H); 13C NMR (500 MHz, CDCl3): d 44.3 (C-4),
d 53.1 (C-5,), d 121.3–137.2 (C–Ar), d 147.1 (C-3); ESI-
MS: 267 (M?H); Anal. Calcd. for C14H10N4O2: C, 63.15;
H, 3.7; N, 21.06; O, 12.03; Found C, 63.27; H, 3.23.; N,
21.46; O, 12.20.
4b
12.81
10.29
11.56
14.11
13.56
10.22
15.81
10.55
–
4c
4d
4e
4f
4g
4h
4i
Control
Standard
2-[5-(4-Chlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl]pyridine
(4d) The compound 4d was prepared by reacting (2E)-3-
25.13
45.33
55.42
80.10
96.67
5 lg/ml 10 lg/ml 15 lg/ml 20 lg/ml 25 lg/ml
(4-chlorophenyl)-1-(pyridin-3-yl)prop-2-en-1-one
(3d)
Values represent the mean standard error mean (SEM) of three
experiments
with hydrazine hydrate in acetic acid. The product was
obtained as coloured powder. The yield was 41 %. IR
(KBr) cm-1: 3,105 (N–H Str.), 3,090 (C–H Str.), 1,617
(C=H Str.), 1,513 (C=C Str.); 1H NMR (d ppm) (CDCl3): d
2.9 (d, 2H, CH2), d 6.12 (s, 1H, N–H), d 7.18–7.8 (m, Ar–
H), d 8.52 (d, 1H, N–C–H); 13C NMR (500 MHz, CDCl3):
d 46.13 (C-4,), d 56.13 (C-5,), d 118.3–137.32 (C–Ar), d
151.1 (C-3); ESI-MS: 256 (M?H); Anal. Calcd. for
C14H10ClN3: C, 65.7; H, 3.9; N, 16.42; Found C, 65.63; H,
3.83.; N, 16.56;
–, no inhibition; Standard, ascorbic acid
was washed, first with cold water and then with excess
sodium bicarbonate. It was filtered and crystallized to give
pure compound. However, impure compounds were readily
purified by silica gel column chromatography using a
methanol/chloroform eluent.
2-(5-Phenyl-4,5-dihydro-1H-pyrazol-3-yl)pyridine (4a) The
compound 4a was prepared by reacting (2E)-3-phenyl-1-
(pyridin-3-yl)prop-2-en-1-one (3a) with hydrazine hydrate
in acetic acid. The product was obtained as coloured
powder. The yield was 45 %. IR (KBr) cm-1: 3,104 (N–H
Str.), 3,050 (C–H Str.), 1,637 (C=N Str.), 1,403 (C=C Str.);
1H NMR (d ppm) (CDCl3): d 2.4 (d, 2H, CH2,), d 5.4 (s,
1H, N–H), d 7.12–7.9 (m, Ar–H), d 8.02 (d, 1H, N–C–H,);
13C NMR (500 MHz, CDCl3): d 44.6 (C-4), d 55.6 (C-5), d
124.69–150.6 (Ar–C), d 147.6 (C-3); ESI–MS: 222
(M?H); Anal. Calcd. for C17H21N3O3: C, 76.01; H, 4.91;
Found C, 76.37; H, 4.83.
2-[5-(4-Fluorophenyl)-4,5-dihydro-1H-pyrazol-3-yl]pyridine
(4e) The compound 4e was prepared by reacting (2E)-3-
(4-fluorophenyl)-1-(pyridin-3-yl)prop-2-en-1-one (3e) with
hydrazine hydrate in acetic acid. The product was obtained
as coloured powder. The yield was 45 %. IR (KBr) cm-1
:
3,115 (N–H Str.), 3,080 (C–H Str.), 1,605 (C=H Str.),
1,505 (C=C Str.); 1H NMR (d ppm) (CDCl3): d 2.6 (d, 2H,
CH2), d 6.42 (s, 1H, N–H), d 7.2–7.8 (m, Ar–H), d 8.49 (d,
1H, N–C–H); 13C NMR (500 MHz, CDCl3): d 41.23 (C-4),
d 46.53 (C-5,), d 116.2–137.3 (C–Ar), d 150.4 (C-3); ESI-
MS: 240 (M?H); Anal. Calcd. for C14H10FN3: C, 70.22; H,
4.17; N, 17.55; Found C, 70.16; H, 4.0; N, 17.46;
2-[5-(3,4,5-Trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl]
pyridine (4b) The compound 4b was prepared by reacting
(2E)-3-(3,4,5-trimethoxyphenyl)-1-(pyridin-3-yl)prop-2-en-
1-one (3b) with hydrazine hydrate in acetic acid. The
product was obtained as coloured powder. The yield was
40 %. IR (KBr) cm-1: 3,114 (N–H Str.), 3,012 (C–H Str.),
2-[5-(3-Fluorophenyl)-4,5-dihydro-1H-pyrazol-3-yl]pyridine
(4f) The compound 4f was prepared by reacting (2E)-3-
(3-fluorophenyl)-1-(pyridin-3-yl)prop-2-en-1-one (3f) with
hydrazine hydrate in acetic acid. The product was obtained
as coloured powder. The yield was 40 %. IR (KBr) cm-1
:
1
1,627 (C=N Str.), 1,403 (C=C Str.); H NMR (CDCl3): d
3,125 (N–H Str.), 3,085 (C–H Str.), 1,600 (C=N Str.),
1,495 (C=C Str.); 1H NMR (d ppm) (CDCl3): d 3.1 (d, 2H,
CH2), d 6.0 (s, 1H, N–H), d 6.9–7.8 (m, Ar–H), d 8.6 (d,
1H, N–C–H); 13C NMR (500 MHz, CDCl3): d 45.1 (C-4),
d 52.7 (C-5), d 120.19–147.8 (Ar–C), d 150.6 (C-3); ESI-
MS: 240 (M?H); Anal. Calcd. for C14H10FN3: C, 70.22; H,
4.17; N, 17.55; Found C, 70.26; H, 4.1; N, 17.51;
2.5 (d, 2H, CH2), d 2.67 (s, 9H, OCH3), d 6.0 (s, 1H, N–H),
d 7.3–7.9 (m, Ar–H), d 8.2 (d, 1H, N–C–H); 13C NMR
(500 MHz, CDCl3): d 47.3 (C-4), d 56.3 (C-5), d 120–139
(C–Ar), d 149 (C-3); ESI-MS: 312 (M?H); Anal. Calcd.
for C17H17N3O3: C, 65.30; H, 5.41; N, 13.5; O, 15.4; Found
C, 64.87; H, 5.63; N, 13.46; O, 15.00.
123

Med Chem Res
2-[5-(4-Methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl]pyri-
dine (4g) The compound 4g was prepared by reacting
(2E)-3-(4-methoxyphenyl)-1-(pyridin-3-yl)prop-2-en-1-one
(3g) with hydrazine hydrate in acetic acid. The product was
obtained as coloured powder. The yield was 40 %. IR
(KBr) cm-1: 3,110 (N–H Str.), 3,085 (C–H Str.), 1,665
(C=N Str.), 1,625 (C=C Str.); 1H NMR (d ppm) (CDCl3): d
3.12 (s, 3H, OCH3), d 2.9 (d, 2H, CH2), d 5.1 (s, 1H, N–H),
d 6.1–7.6 (m, Ar–H), d 8.6 (d, 1H, N–C–H); 13C NMR
(500 MHz, CDCl3): d 43.1 (C-4), d 53.97 (C-5), d
105.19–137.8 (Ar–C), d 148.1 (C-3); ESI-MS: 252 (M?H);
Anal. Calcd. for C15H13N3O: C, 71.70; H, 5.17; N, 16.13;
Found C, 71.79; H, 5.13; N,16.23.
Growth conditions
Stock cultures of all the bacterial and fungal strains were
maintained on agar slants at 4 °C to initiate growth; one
loopful of cells from the agar culture was inoculated into
their respective broth media and incubated at 35 2 °C
for 24–28 h up to the stationary phase (primary culture).
The cells from the primary culture (108 cells ml) were re-
inoculated into 100-ml fresh media and grown for 8 2 h,
i.e. up to mid-log phase (106 cells/ml).
Disc diffusion assay
The target compounds (4a–i) were evaluated for antimicrobial
activity against the test strains by agar disc diffusion method
(Stefan et al., 2010). Strains were inoculated into liquid media
and kept overnight for growth at 35 2 °C. The cells were
then pelleted, and washed three times with distilled water.
Approximately, 105 cells/ml were inoculated in molten agar
media at 40 °C and poured into 100-mm-diameter petriplates.
4-mm filter discs were impregnated on solid agar, and test
compounds (1,000 lg/ml dissolved in 1 % DMSO) were
spotted on the filter discs. Solvent control (1 % DMSO) was
pipetted onto filter disc to serve as positive control. The
diameter of zone of inhibition was recorded in millimetres
after 48 h and was compared to that of control. This experi-
mentwas independently repeated thrice, and valuesare shown
in terms of mean standard error of mean (SEM).
2-[5-(4-N,N-dimethylphenyl)-4,5-dihydro-1H-pyrazol-3-yl]
pyridine (4h) The compound 4h was prepared by reacting
(2E)-3-(4-N,N-dimethylphenyl)-1-(pyridin-3-yl)prop-2-en-
1-one (3h) with hydrazine hydrate in acetic acid. The
product was obtained as coloured powder. The yield was
43 %. IR (KBr) cm-1: 3,105 (N–H Str.), 2,900 (C–H Str.),
1,680 (C=N Str.), 1,490 (C=C Str.); 1H NMR (d ppm)
(CDCl3): d 2.52 (s, 6H, N(CH3)2), d 2.7 (d, 2H, CH2), d
5.91 (s, 1H, N–H), d 6.2–7.9 (m, Ar–H), d 8.5 (d, 1H, N–
C–H); 13C NMR (500 MHz, CDCl3): d 44.1 (C-4), d 52.27
(C-5), d 114.6–137 (Ar–C), d 147.21 (C-3); ESI-MS: 265
(M?H); Anal. Calcd. for C16H16N4: C, 64.36; H, 6.0; N;
21.2; Found C, 64.17; H, 5.83; N, 20.89.
2-[5-(4-Bromophenyl)-4,5-dihydro-1H-pyrazol-3-yl]pyridine
(4i) The compound 4i was prepared by reacting (2E)-3-
(4-bromophenyl)-1-(pyridin-3-yl)prop-2-en-1-one (3i) with
hydrazine hydrate in acetic acid. The product was obtained
Antioxidant activity
General free radical scavenging-DPPH assay
as coloured powder. The yield was 40 %. IR (KBr) cm-1
:
IR (KBr) cm-1: 3,125 (N–H Str.), 3,038 (C–H Str.), 1,650
(C=N Str.), 1,412 (C=C Str.); 1H NMR (d ppm) (CDCl3): d
2.4 (d, 2H, CH2), d 5.86 (s, 1H, N–H), d 7.2–7.8 (m, Ar–
H), d 8.3 (d, 1H, N–C–H); 13C NMR (500 MHz, CDCl3): d
42.8 (C-4), d 50.97 (C-5), d 120.5–147.5 (Ar–C), d 149.21
(C-3); ESI-MS: 301 (M?H); Anal. Calcd. for C14H10BrN3:
C,56; H,3.3; N,14.2; Br, 26.2; Found C, 56.37; H, 3.13; N,
14.6; Br, 26.5;
The antioxidant potential of any compound can be deter-
mined on the basis of its capacity to trap the stable 1,1-
diphenyl-2-picryl hydrazyl (DPPH) free radical (Sherwin,
1978; Brand-Willams et al., 1995; Espin et al., 2000). The
antioxidant activity by this method is measured as the
decrease in the absorbance of DPPH at 517 nm resulting
from the colour change from purple to yellow. The
decrease in absorbance is because of formation of stable
molecule of DPPH on reaction with an antioxidant through
donation of hydrogen or electron by an antioxidant. The
free electron on DPPH radical is responsible for giving
absorbance peak at 517 nm and appears purple in colour.
The antioxidant agents pair up through donation of electron
or release of hydrogen with the free electron on DPPH
radical and form stable molecule of DPPH-H. The change
of colour from purple to yellow is attributed to the decrease
of molar absorptivity of DPPH radical when the odd
electron of DPPH pairs up with the antioxidant agent. The
resulting decrease in colour is also stoichiometric with the
number of electrons captured.
Antimicrobial activity
Strain and media
The bacterial and fungal strains used in the study were B.
subtilis (MTCC 619), S. epidermidis (MTCC 435), P. vul-
garis (MTCC 426), P. aeruginosa (MTCC 424), A. niger
(MTCC 1344), and P. chrysogenum (MTCC 947). All the
bacterial and fungal strains were maintained on Mueller
Hinton agar (MHA) and potato dextrose agar (PDA),
respectively.
123

Med Chem Res
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% Inhibition ¼ ðBlank ODꢁSample OD=Blank ODÞꢂ100
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