Design, synthesis, and pharmacological evaluation of new pyrazoline derivatives

Article abstract of DOI:10.1007/s00706-014-1371-x

In the present investigation, a new series of pyrazoline derivatives has been synthesized by the reaction of chalcones with hydrazine derivatives like hydrazine hydrate and phenyl hydrazines in the presence of different aliphatic acids. The chemical struc

Full text of DOI:10.1007/s00706-014-1371-x

Monatsh Chem
DOI 10.1007/s00706-014-1371-x
ORIGINAL PAPER
Design, synthesis, and pharmacological evaluation of new
pyrazoline derivatives
•
Majal Sapnakumari Badiadka Narayana
•
•
Purawarga Matada Gurubasavarajswamy
Balladka Kunhanna Sarojini
Received: 1 October 2014 / Accepted: 24 November 2014
Ó Springer-Verlag Wien 2014
Abstract In the present investigation, a new series of
pyrazoline derivatives has been synthesized by the reaction
of chalcones with hydrazine derivatives like hydrazine
hydrate and phenyl hydrazines in the presence of different
aliphatic acids. The chemical structure of the newly pre-
pared compounds was characterized by 1H NMR, 13C
NMR, FTIR, LC–MS and some of them by single crystal
XRD. The synthesized compounds were screened for their
antimicrobial, antitubercular, DPPH scavenging, and anti-
inflammatory properties. Some of the tested compounds
exhibited promising antimicrobial, antitubercular, DPPH
scavenging, and anti-inflammatory activities.
have been reported for their synthesis [1]. Pyrazolines
occupy a major place in medicinal and pesticide chemistry
due to their capability to exhibit a wide range of bioac-
tivities. 2-Pyrazolines seem to be the most frequently
studied pyrazoline-type compounds. Compounds with
pyrazoline moieties were known to possess antidepressant,
antitubercular, antipyretic, analgesic, tranquilizing, muscle
relaxant, psychoanaleptic, antiepileptic, antidepressant,
anti-inflammatory, insecticidal, antimicrobial, and antihy-
potensive activities [2, 3]. Their derivatives were also
found to exhibit cytotoxic activity, inhibitory activity of
platelet aggregation, herbicidal activity, and cannabinoid
CB1-receptor modulators. 1,3,5-Triaryl-2-pyrazolines were
also used as scintillation solutes. Pyrazoline derivatives
with a phenyl group at the 5-position found to possess good
film-forming properties, exhibit excellent characteristics of
blue photoluminescence, fluorescence, and electrolumi-
nescence [4]. Also, literature review revealed that phenyl
ring with halo substituent enhances the antimicrobial,
antitubercular, and anti-inflammatory activities [5–7].
Based on their utility in pharmaceutical and other fields, a
new series of pyrazoline derivatives was synthesized and
screened for their pharmacological activities.
Keywords Chalcone ꢀ Antimicrobial activity ꢀ
Anti-inflammatory activity ꢀ Antitubercular activity ꢀ
DPPH scavenging activity
Introduction
Pyrazolines are well-known nitrogen-containing five-
membered heterocyclic compounds and various methods
M. Sapnakumari ꢀ B. Narayana (&)
Department of Studies in Chemistry, Mangalore University,
Mangalagangotri 574 199, Karnataka, India
e-mail: nbadiadka@yahoo.co.uk
Results and discussion
The synthetic route for pyrazoline derivatives 2a–2j and
3a–3f is depicted in Scheme 1. Accordingly, the chalcones
1a–1c were reacted with hydrazine hydrate in the presence
of various aliphatic acids, namely formic acid, acetic acid,
propionic acid, butyric acid, and 2-chloropropionic acid to
yield N-alkyloxypyrazoline derivatives 2a–2j. Also, the
reaction of chalcones with phenyl hydrazine and 4-nitro-
phenyl hydrazine yielded 1,3,5-triaryl-2-pyrazolines 3a–3f.
P. M. Gurubasavarajswamy
Department of Pharmaceutical Chemistry, Acharya and B M
Reddy College of Pharmacy, Soledevanahalli,
Bangalore 560 090, Karnataka, India
B. K. Sarojini
Industrial Chemistry Section, Department of Studies in
Chemistry, Mangalore University, Mangalagangotri 574 199,
Karnataka, India
123

M. Sapnakumari et al.
The chalcones 1a–1c in turn were prepared by the Claisen–
Schmidt condensation of 4-fluoroacetophenone with dif-
ferent aldehydes. The structure of the newly synthesized
compounds was characterized by 1H NMR, 13C NMR,
FTIR, LC–MS, elemental analysis, and also by single
crystal XRD data. Characterization data of pyrazoline
derivatives are given in Table 1.
1,598–1,608 cm-1 confirmed the formation of pyrazolines.
Also, in the IR spectra of the newly synthesized N-al-
kyloxypyrazoline derivatives, appearance of a strong band
in the region 1,651–1,664 cm-1 along with C=N stretch-
ing, corresponds to the carbonyl group suggested the
formation of N-alkyloxy-substituted pyrazolines.
In the proton NMR spectra, the signals of the respective
protons of the synthesized compounds were verified on the
basis of their chemical shifts, multiplicities and coupling
constants. The proton NMR spectra of the newly prepared
compounds exhibited an ABX pattern of signals attributed
to methine (C-5) and methylene (C-4) protons. The protons
HA and HB on C-4 are geminal protons and appeared in the
region d = 3.02–3.29 and 3.43–3.98 ppm as doublet of
doublets. The proton HX at C-5 also appeared as a doublet
of doublet in the region 5.12–5.77 ppm due to the vicinal
coupling with two non-equivalent geminal protons of C-4
carbon. A similar pattern in spectrum for 2-pyrazolines was
reported in the literature [8, 9]. The presence of a singlet at
8.87 ppm in compound 2a corresponds to the aldehydic
proton. The protons of aliphatic CH3 and CH2 of com-
pounds 2d and 2e appeared as a triplet (1.10 and 1.11 ppm)
and quartet (2.70 and 2.23 ppm). Similarly, the compounds
2f and 2g exhibited a triplet and two multiplets corre-
sponding to the aliphatic protons. The appearance of a
doublet and multiplet in proton NMR spectrum of com-
pounds 2i and 2j was due to the protons of CH3 and CHCl
group, respectively. Similarly, the aromatic protons of new
triaryl pyrazolines appeared as multiplets in the aromatic
region (6.97–8.31 ppm). The proton NMR data confirmed
the formation of new derivatives.
IR spectra of all the compounds exhibited absorption
band due to C=N stretching vibrations in the range of
Scheme 1
13C
NMR
chemical
shift
values
at
Table 1 Characterization data of pyrazoline derivatives 2a–2j and
3a–3f
d = 53.41–59.41 ppm (C-5) and 38.98–42.89 ppm (C-4)
confirmed the formation of 2-pyrazolines. The presence of
a peak at 160.12 ppm in the 13C NMR spectrum of com-
pound 2a was accounted for the aldehydic carbon whereas
the other newly synthesized N-alkyloxypyrazoline deriva-
tives exhibited peaks in the range of 167.96–176.85 ppm,
accounted for the carbonyl carbons. The 13C NMR spec-
trum of the new compounds exhibited chemical shift values
in the range of 151.79–164.73 ppm corresponding to C=N
carbon, also confirmed the formation of 2-pyrazolines. LC–
MS data were also in agreement with the formation of
2-pyrazolines. Elemental analysis also gave satisfactory
results for all the compounds.
Comp. Molecular formula R1 R2
R3 Yield/ M.p./°C
%
2a
2b
2c
2d
2e
2f
C16H12ClFN2O
Cl
H
H
–
62
79
84
89
73
61
33
51
18
34
89
54
59
129–131
119–121
99–101
86–88
88–90
98–100
83–85
110–112
152–154
144–146
119–121
128
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
17H15FN2O
CH3
–
17H14BrFN2O
18H16ClFN2O
18H16BrFN2O
19H18ClFN2O
19H19FN2O
Br CH3
Cl C2H5
Br C2H5
Cl C3H7
–
–
–
–
2g
2h
2i
H
C3H7
–
19H18BrFN2O
18H15Cl2FNO
18H16ClFN2O
21H16ClFN2
21H17FN2
Br C3H7
–
Cl CHClCH3
–
The pyrazoline derivatives 2b, 2c, 2h, and 3c were
crystallized into neat single crystals by slow evaporation
technique and the structures of those compounds were
confirmed by single crystal XRD data (Fig. 1). The com-
pounds 2b and 2h were crystallized in an orthorhombic,
Pbca space group and triclinic P1 space group, respec-
tively, while the compounds 2c and 3c were crystallized in
a monoclinic P21/c space group [10–13].
2j
H
CHClCH3
–
3a
3b
3c
3d
3e
3f
Cl
H
–
–
H
H
H
21H16BrFN2
21H15ClFN3O2
21H16FN3O2
21H15BrFN3O2
Br –
124–126
110
Cl
H
–
–
NO2 46
NO2 47
NO2 39
143
Br –
134
123

A new series of pyrazoline derivatives
Fig. 1 The molecular structure and numbering scheme for the compounds 2b, 2c, 2h, and 3c, with displacement ellipsoids drawn at the 50 %
probability level
Antibacterial activity
compounds, compound 2j exhibited the maximum zone of
inhibition of 8 mm at MIC 6.25 lg/cm3 against all the
tested bacterial strains except E. coli while the compound
3a against S. aureus and E. coli. The compounds 3-(4-
fluorophenyl)-1,5-diphenyl-4,5-dihydro-1H-pyrazole (3b)
and 5-(4-bromophenyl)-3-(4-fluorophenyl)-1-phenyl-4,5-
dihydro-1H-pyrazole (3c) inhibited all the tested bacterial
strains except Klebsiella pneumoniae. The compounds 2d
(with 4-chlorophenyl and propyl group), 2g (with butyl
group), and 2h (with 4-bromophenyl and butyl group) also
exhibited the zone of inhibition of 8 mm at MIC of
6.25 lg/cm3 against S. aureus. The results of the antibac-
terial activity revealed that the majority of the synthesized
pyrazoline derivatives showed varying degree of inhibition
against the tested microorganisms.
The in vitro antibacterial activities of pyrazoline deriva-
tives 2a–2j and 3a–3f were carried out using Gram positive
Staphylococcus aureus (ATTC-25923), Gram negative
Escherichia coli (ATTC-25922), Pseudomonas aeruginosa
(ATCC-27853), and Klebsiella pneumoniae (recultured)
bacterial strains by serial plate dilution method. Activity of
each compound was compared with ciprofloxacin as stan-
dard. The results are summarized in Table 2. Among the
newly synthesized compounds, 2-chloro-1-[3-(4-fluoro-
phenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl]propan-1-
one (2j) and 5-(4-chlorophenyl)-3-(4-fluorophenyl)-1-phe-
nyl-4,5-dihydro-1H-pyrazole (3a) were found to be active
against all the tested microorganisms. Among these two
123

M. Sapnakumari et al.
Table 2 Antibacterial activity
of the synthesized compounds:
MIC/lg cm-3 (zone of
inhibition/mm)
Compound
Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa Klebsiella pneumonia
2a
2b
2c
2d
2e
2f
6.25 (6)
R
6.25 (7)
R
R
R
R
R
R
R
R
R
6.25 (8)
R
R
R
R
R
R
R
12.5 (6)
6.25 (8)
6.25 (8)
6.25 (6)
6.25 (8)
6.25 (8)
12.5 (6)
12.5 (6)
R
12.5 (6)
R
R
R
2g
2 h
2i
R
R
R
R
R
12.5 (6)
6.25 (7)
6.25 (8)
12.5 (6)
12.5 (6)
R
R
R
2j
6.25 (8)
12.5 (7)
12.5 (7)
12.5 (6)
R
6.25 (8)
3a
3b
3c
3d
3e
3f
12.5 (6)
R
R
R
6.25 (7)
12.5 (6)
12.5 (6)
R
R
R
R
R
R indicates bacterial strains are
resistant to the compounds
[100 lg/cm3
Ciprofloxacin 6.25 (20)
6.25 (19)
6.25 (17)
6.25 (18)
Antifungal activity
against Mycobacterium tuberculosis using the microplate
Alamar Blue assay (MABA) method. Minimum inhibitory
concentration (MIC) was determined and the results
obtained were compared with standard drugs pyrazinamide
and streptomycin (Table 4). The compounds 1-[5-(4-
bromophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-
1-yl]butan-1-one (2h) and 1,3,5-triaryl-2-pyrazolines, 5-(4-
chlorophenyl)-3-(4-fluorophenyl)-1-phenyl-4,5-dihydro-
1H-pyrazole (3a), 3-(4-fluorophenyl)-1,5-diphenyl-4,5-
dihydro-1H-pyrazole (3b), 5-(4-bromophenyl)-3-(4-fluoro-
phenyl)-1-phenyl-4,5-dihydro-1H-pyrazole (3c) inhibited
M. tuberculosis at a concentration of 0.8 lg/cm3.
The in vitro antifungal activity of the synthesized com-
pounds was examined against Aspergillus fumigatus
(NCIM No. 902), Penicillium marneffei (recultured),
Trichophyton mentagrophytes (recultured), and Aspergillus
flavus (NCIM No. 524) by serial plate dilution method.
Activity of each compound was compared with itraconaz-
ole as standard. The results are summarized in Table 3. The
compounds 1-[3-(4-fluorophenyl)-5-phenyl-4,5-dihydro-
1H-pyrazol-1-yl]ethanone (2b), 1-[5-(4-bromophenyl)-3-
(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl]butan-1-one
(2h), and 3-(4-fluorophenyl)-1,5-diphenyl-4,5-dihydro-1H-
pyrazole (3b) were found to be active against all the tested
fungal strains. The compound 2g with butyl substituent was
active against all tested microorganisms except A. Fumig-
atus, whereas compounds 3a with 4-chlorophenyl group
and 3d with 4-chlorophenyl and 4-nitrophenyl group were
active against only three fungal stains T. mentagrophytes,
A. flavus, and A. fumigatus. Among the tested compounds,
the maximum zone of inhibition of 8 mm at MIC 6.25 lg/
cm3 was observed for the compound 2g against P. mar-
neffei, 2h and 3a against A. flavus, 3b and 3d against T.
mentagrophytes, and compound 3b against A. fumigatus. In
general, the antifungal activity of the newly synthesized
pyrazoline derivatives depends on different substituents
present in the molecule.
DPPH radical scavenging assay
A rapid, simple, accurate, and inexpensive method to
measure antioxidant capacity of substances involves the
use of organic nitrogen radical DPPH. It is widely used to
test the ability of compounds to act as free radical scav-
engers or hydrogen donors. Further, this simple test
provides information on the ability of a compound to
contribute electrons during antioxidant action [14]. The
odd electron in the DPPH free radical gives strong
absorption maximum at 517 nm and is purple in color. The
color changes from purple to yellow as the molar absorp-
tivity of the DPPH radical at 517 nm reduces upon the
transfer of acidic H-atom from the compound to DPPH
radical to form DPPH-H. The resulting decolorization is
stoichiometric with respect to number of electrons cap-
tured. The results are summarized in Table 5.
Antitubercular activity
All the newly synthesized pyrazoline derivatives 2a–2j and
3a–3f were assessed for their in vitro antitubercular activity
Among the tested compounds, compounds 5-(phenyl/4-
chlorophenyl/4-bromophenyl)-3-(4-fluorophenyl)-1-phenyl-
123

A new series of pyrazoline derivatives
Table 3 Antifungal activity of
Compound
Penicillium
marneffei
Trichophyton
mentagrophytes
Aspergillus
flavus
Aspergillus
fumigatus
the synthesized compounds:
MIC/lg cm-3 (zone of
inhibition/mm)
2a
12.5 (6)
6.25 (7)
R
R
R
R
2b
12.5 (6)
12.5 (6)
6.25 (6)
2c
R
R
R
2d
R
R
R
R
2e
R
R
R
R
2f
12.5 (6)
6.25 (8)
12.5 (6)
R
R
R
R
2g
12.5 (6)
12.5 (6)
R
6.25 (7)
6.25 (8)
R
R
2h
6.25 (6)
2i
R
2j
R
R
R
R
3a
R
12.5 (6)
6.25 (8)
R
6.25 (8)
12.5 (6)
R
12.5 (6)
6.25 (8)
R
3b
6.25 (7)
12.5 (6)
R
3c
3d
6.25 (8)
12.5 (6)
R
12.5 (6)
R
6.25 (7)
R
3e
6.25 (7)
12.5 (6)
6.25 (17)
R indicates fungal strains are
resistant to the compounds
[100 lg/cm3
3f
R
R
Itraconazole
6.25 (19)
6.25 (18)
6.25 (17)
Table 4 Antitubercular activity
of synthesized compounds
Anti-inflammatory activity
Compound
MIC/lg cm-3
2a
3.125
–
The anti-inflammatory activity of the test compounds was
assessed by carrageenan induced rat paw edema method.
The tested compounds exhibited anti-inflammatory activity
ranging from 27 to 59 %, whereas the standard diclofe-
nac showed 65 % inhibition after 24 h. Among the
tested compounds, compound 1-[5-(4-bromophenyl)-3-(4-
fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl]ethanone (2b)
showed 71 % inhibition of paw edema at 4th hour and this
inhibition is of significant when compared to the standard
diclofenac. The compounds, 1-[5-(4-bromophenyl)-3-(4-
fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl]ethanone (2c)
and 1-[5-(4-chlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-
1H-pyrazol-1-yl]propan-1-one (2d) exhibited 58 % inhi-
bition of paw edema at 4th hour whereas 1-[5-(4-
bromophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-
1-yl]propan-1-one (2e) showed 62 % inhibition of paw
edema at 5th hour. The results are tabulated in Tables 6
and 7.
2b
2c
–
2d
50
50
50
–
2e
2f
2g
2h
0.8
–
2i
2j
–
3a
0.8
0.8
0.8
6.25
–
3b
3c
3d
3e
3f
–
Pyrazinamide
Streptomycin
3.125
6.25
4,5-dihydro-1H-pyrazole 3a–3c and 5-(phenyl/4-chlor-
ophenyl/4-bromophenyl)-3-(4-fluorophenyl)-1-(4-nitro-
phenyl)-4,5-dihydro-1H-pyrazole 3d–3f showed moderate
radical scavenging capacity with concentration of 1 mg/cm3
in comparison with the standard glutathione. The radical
scavenging capacity of the compounds 3a–3c was due to the
presence of phenyl ring in the pyrazoline. The scavenging
capacity of compounds 3d–3f is decreased slightly because
of the electron withdrawing nitro group. All other pyrazoline
derivatives exhibited low scavenging capacity.
Conclusion
A new series of 2-pyrazoline derivatives was synthesized
1
from chalcones and characterized by H NMR, 13C NMR,
IR, mass spectral data, and some of them by single crystal
XRD. The synthesized compounds were screened for their
antimicrobial, antitubercular, DPPH scavenging, and anti-
inflammatory properties. Some of the tested compounds
exhibited promising antimicrobial, antitubercular, DPPH
scavenging, and anti-inflammatory activities.
123

M. Sapnakumari et al.
General procedure for the synthesis of pyrazoline
Experimental
derivatives 2a–2j
Melting points were taken in open capillary tubes. The
purity of the compounds was confirmed by thin layer
chromatography using Merck silica gel 60 F254-coated
aluminum plates using ethyl acetate: n-hexane (1:3 v/v) as
solvent system. IR spectra were recorded on Shimadzu-
A mixture of chalcone (0.01 mol) and 0.5 cm3 hydrazine
hydrate (0.01 mol) in 20 cm3 formic acid/acetic acid/pro-
pionic acid/2-chloropropionic acid/butyric acid was
refluxed for 8 h. The reaction mixture was cooled and
poured into the 50 cm3 ice-cold water. The precipitate
formed was filtered and purified by recrystallization from
ethanol.
1
FTIR Infrared spectrometer in KBr. H NMR spectra were
recorded on a Bruker AMX 400 spectrometer (400 MHz)
with 5 mm PABBO BB-1H TUBES and 13C NMR spectra
were recorded for approximately 0.03 M solutions in
DMSO-d6 at 75 MHz or 100 MHz with TMS as internal
standard. LC–MS were obtained using Shimadzu LCMS-
8030/Agilent 1200 series LC and Micromass zQ spec-
trometer. Elemental analysis was carried out using VARIO
EL-III (Elementar Analysensysteme GmBH).
5-(4-Chlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-
pyrazole-1-carbaldehyde (2a, C16H12ClFN2O)
IR (KBr): v = 1,662 (CHO), 1,606 (pyrazoline C=N),
;
2,922 (CH), 1,219 (C–F) cm-1 1H NMR (400 MHz,
DMSO-d6): d = 3.19 (dd, 1H, CH2-HA, JAB = 18 Hz,
JAX = 5.2 Hz), 3.86 (dd, 1H, CH2-HB, JBA = 18.2 Hz,
JBX = 11.8 Hz), 5.51 (dd, 1H, pyrazoline 5C–H,
JXA = 5.2 Hz, JXB = 11.6 Hz), 7.25–8.87 (m, 8H, Ar–
H), 8.87 (s, 1H, CHO) ppm; 13C NMR (100 MHz, DMSO-
d6): d = 42.89 (aliphatic CH2, C-4), 59.10 (aliphatic CH,
C-5), 116.28, 122.35, 126.18, 127.97, 129.21, 131.98,
141.67, 144.67, 155.72 (C=N of pyrazoline ring), 160.12
(C=O, C-6) ppm; LC–MS: m/z = 303.6 ([M ? 1]?).
Table 5 DPPH scavenging
Compound DPPH scavenging/
assay of synthesized compounds
%
2a
2b
2c
2d
2e
2f
19.23 0.22
14.93 0.33
32.41 0.24
23.11 0.31
15.15 0.42
16.12 0.23
17.34 0.21
22.12 0.35
15.98 0.16
27.36 0.13
52.43 0.32
49.35 0.14
53.40 0.41
43.83 0.23
42.54 0.41
45.32 0.18
1-[3-(4-Fluorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-
1-yl]ethanone (2b)
M.p.: 119–121 °C (Ref. [10] 119–121 °C).
2g
2h
2i
1-[5-(4-Bromophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-
pyrazol-1-yl]ethanone (2c)
2j
M.p.: 99–101 °C (Ref. [11] 99–101 °C).
3a
3b
3c
3d
3e
3f
1-[5-(4-Chlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-
pyrazol-1-yl]propan-1-one (2d, C18H16ClFN2O)
IR (KBr): v = 1,664 (C=O), 1,598 (pyrazoline C=N),
;
2,981 (CH), 1,224 (C–F) cm-1 1H NMR (400 MHz,
DMSO-d6): d = 1.10 (t, 3H, CH3, J = 7.6 Hz), 2.70 (q,
2H, CH2), 3.10 (dd, 1H, CH2-HA, JAB = 18 Hz,
JAX = 4.8 Hz), 3.79 (dd, 1H, CH2-HB, JBA = 18 Hz,
Glutathione 92.02 0.34
Table 6 Anti-inflammatory activity data of the compounds 2a–2f
Compounds
Paw edema volume/cm3 (mean SEM)
0 h
1 h
2 h
3 h
4 h
5 h
24 h
Control
0.11 0.03
0.1 0.01
0.11 0.04
0.1 0.04
0.11 0.04
0.1 0.04
0.11 0.04
0.11 0.04
0.216 0.03
0.2 0.01
0.333 0.01
0.191 0.02
0.216 0.01
0.133 0.02
0.15 0.02
0.216 0.02
0.15 0.02
0.233 0.02
0.333 0.02
0.183 0.02
0.216 0.02
0.133 0.03
0.240 0.03
0.183 0.02
0.191 0.03
0.266 0.02
0.516 0.02
0.2 0.01
0.433 0.02
0.2 0.01
0.283 0.02
0.1 0.02
Diclofenac
2a
2b
2c
2d
2e
2f
0.216 0.02
0.133 0.01
0.158 0.02
0.158 0.01
0.158 0.02
0.133 0.02
0.35 0.01
0.15 0.02
0.262 0.02
0.216 0.01
0.216 0.02
0.281 0.02
0.30 0.01
0.25 0.02
0.316 0.02
0.215 0.02
0.2 0.02
0.2 0.02
0.128 0.02
0.271 0.02
0.116 0.02
0.141 0.02
0.208 0.02
0.333 0.02
SEM standard error of mean
123

A new series of pyrazoline derivatives
141.67, 161.23, 151.83 (C=N of pyrazoline ring), 172.31
(C=O, C-6) ppm; LC–MS: m/z = 344.7 (M?).
Table 7 Percentage inhibition of in vivo anti-inflammatory activity
of the compounds 2a–2f
Compounds
Inhibition/%
1-[3-(4-Fluorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-
1-yl]butan-1-one (2g, C19H19FN2O)
IR (KBr): v = 1,653 (C=O), 1,602 (pyrazoline C=N),
1 h
2 h
3 h
4 h
5 h
24 h
Control
–
–
–
–
–
–
2,935 (CH), 1,222 (C–F) cm-1 1H NMR (400 MHz,
;
Diclofenac
10**
0**
43**
35**
60**
55**
35**
55**
33**
45**
35**
60**
43**
45**
43**
20**
62**
32**
71**
58**
58**
58**
52**
62**
31**
52**
52**
58**
62**
45**
65**
30**
27**
50**
59**
50**
27**
DMSO-d6): d = 0.94 (t, 3H, CH3, J = 7.6 Hz), 1.58 (m,
2H, CH2), 2.66 (m, 2H, CH2), 3.21 (dd, 1H, CH2-HA,
JAB = 18 Hz, JAX = 4.8 Hz), 3.82 (dd, 1H, CH2-HB,
JBA = 18 Hz, JBX = 12 Hz), 5.54 (dd, 1H, pyrazoline
5C–H, JXA = 4.8 Hz, JXB = 12 Hz), 7.19–7.88 (m, 8H,
Ar–H) ppm; 13C NMR (100 MHz, DMSO-d6): d = 14.21
(aliphatic CH3), 19.48 (aliphatic CH2), 37.91 (aliphatic
CH2), 39.96 (aliphatic CH2, C-4), 59.41 (aliphatic CH,
C-5), 115.62, 128.31, 128.68, 129.51, 130.77, 132.51,
141.80, 160.34, 151.79 (C=N of pyrazoline ring), 172.34
(C=O, C-6) ppm; LC–MS: m/z = 310.2 (M?).
2a
2b
2c
2d
2e
2f
38**
27**
27**
27**
38**
(ANOVA) followed by Dunnett’s t test for multiple comparisons
* P \ 0.05 and ** P \ 0.01 were taken as significant
JBX = 12 Hz), 5.50 (dd, 1H, pyrazoline 5C–H,
JXA = 4.8 Hz, JXB = 12 Hz), 7.19–7.83 (m, 8H, Ar–H)
ppm; 13C NMR (100 MHz, DMSO-d6): d = 10.58 (ali-
phatic CH3), 29.43 (aliphatic CH2), 39.13 (aliphatic CH2,
C-4), 57.18 (aliphatic CH, C-5), 115.61, 128.07, 128.71,
129.66, 130.43, 132.45, 141.62, 159.54, 164.73 (C=N of
pyrazoline ring), 176.85 (C=O, C-6) ppm; LC–MS: m/
z = 331.6 ([M ? 1]?).
1-[5-(4-Bromophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-
pyrazol-1-yl]butan-1-one (2h)
M.p.: 110–112 °C (Ref. [12] 110–112 °C).
2-Chloro-1-[5-(4-chlorophenyl)-3-(4-fluorophenyl)-4,5-
dihydro-1H-pyrazol-1-yl]propan-1-one
(2i, C18H15Cl2FN2O)
IR (KBr): v = 1,658 (C=O), 1,600 (pyrazoline C=N),
;
2,804 (CH), 1,220 (C–F) cm-1 1H NMR (400 MHz,
1-[5-(4-Bromophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-
pyrazol-1-yl]propan-1-one (2e, C18H16BrFN2O)
DMSO-d6): d = 1.50 (d, 3H, CH3, J = 6.8 Hz), 4.59 (q,
1H, CH–Cl), 3.10 (dd, 1H, CH2-HA, JAB = 18.2 Hz,
JAX = 4.8 Hz), 3.79 (dd, 1H, CH2-HB, JBA = 18 Hz,
JBX = 12 Hz), 5.47 (dd, 1H, pyrazoline 5C–H,
JXA = 4.8 Hz, JXB = 12 Hz), 7.11–7.94 (m, 9H, Ar–H)
ppm; 13C NMR (100 MHz, DMSO-d6): d = 22.70 (ali-
phatic CH3), 39.91 (aliphatic CH2, C-4), 58.26 (aliphatic
CH), 58.78 (aliphatic CH, C-5), 115.61, 128.45, 128.76,
129.64, 130.68, 132.34, 141.65, 165.20, 152.61 (C=N of
pyrazoline ring), 168.09 (C=O, C-6) ppm; LC–MS: m/z
= 366.4 ([M ? 1]?).
IR (KBr): v = 1,662 (C=O), 1,602 (pyrazoline C=N), 2,989
(CH), 1,226 (C–F) cm-1; 1H NMR (400 MHz, DMSO-d6):
d = 1.11 (t, 3H, CH3, J = 7.8 Hz), 2.23(q, 2H, CH2), 3.02
(dd, 1H, CH2-HA, JAB = 18.2 Hz, JAX = 4.8 Hz), 3.43
(dd, 1H, CH2-HB, JBA = 18 Hz, JBX = 12 Hz), 5.12 (dd,
1H, pyrazoline 5C–H, JXA = 4.8 Hz, JXB = 11.8 Hz),
7.21–7.85 (m, 8H, Ar–H) ppm; 13C NMR (100 MHz,
DMSO-d6): d = 9.41 (aliphatic CH3), 24.63 (aliphatic
CH2), 39.60 (aliphatic CH2, C-4), 55.16 (aliphatic CH,
C-5), 115.28, 127.94, 128.85, 129.82, 130.29, 131.98,
141.91, 158.98, 160.34 (C=N of pyrazoline ring), 176.81
(C=O, C-6) ppm; LC–MS: m/z = 375.9 ([M ? 1]?).
2-Chloro-1-[3-(4-fluorophenyl)-5-phenyl-4,5-dihydro-1H-
pyrazol-1-yl]propan-1-one (2j, C18H16ClFN2O)
IR (KBr): v = 1,662 (C=O), 1,608 (pyrazoline C=N),
;
2,823 (CH), 1,218 (C–F) cm-1 1H NMR (400 MHz,
1-[5-(4-Chlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-
pyrazol-1-yl]butan-1-one (2f, C19H18ClFN2O)
IR (KBr): v = 1,651 (C=O), 1,602 (pyrazoline C=N),
;
2,870 (CH), 1,217 (C–F) cm-1 1H NMR (400 MHz,
DMSO-d6): d = 1.57 (d, 3H, CH3, J = 6.8 Hz), 5.48 (q,
1H, CH–Cl), 3.17 (dd, 1H, CH2-HA, JAB = 18 Hz,
JAX = 4.8 Hz), 3.88 (dd, 1H, CH2-HB, JBA = 18.2 Hz,
JBX = 11.8 Hz), 5.56 (dd, 1H, pyrazoline 5C–H,
JXA = 4.8 Hz, JXB = 11.6 Hz), 7.14–7.90 (m, 9H, Ar–H)
ppm; 13C NMR (100 MHz, DMSO-d6): d = 22.64 (ali-
phatic CH3), 38.98 (aliphatic CH2, C-4), 58.22 (aliphatic
CH), 58.71 (aliphatic CH, C-5), 115.64, 128.41, 128.72,
129.60, 130.71, 131.94, 141.55, 165.23, 152.63 (C=N of
pyrazoline ring), 167.96 (C=O, C-6) ppm; LC–MS:
m/z = 331.6 ([M ? 1]?).
DMSO-d6): d = 0.92 (t, 3H, CH3, J = 7.4 Hz), 1.53 (m,
2H, CH2), 2.61 (m, 2H, CH2), 3.09 (dd, 1H, CH2-HA,
JAB = 18 Hz, JAX = 4.8 Hz), 3.79 (dd, 1H, CH2-HB,
JBA = 18 Hz, JBX = 12 Hz), 5.51 (dd, 1H, pyrazoline
5C–H, JXA = 4.8 Hz, JXB = 12 Hz), 7.18–7.87 (m, 8H,
Ar–H) ppm; 13C NMR (100 MHz, DMSO-d6): d = 14.06
(aliphatic CH3), 19.63 (aliphatic CH2), 38.80 (aliphatic
CH2), 39.92 (aliphatic CH2, C-4), 59.26 (aliphatic CH,
C-5), 115.81, 128.46, 128.70, 129.60, 130.82, 132.35,
123

M. Sapnakumari et al.
General procedure for the synthesis of pyrazoline
3-(4-Fluorophenyl)-1-(4-nitrophenyl)-5-phenyl-4,5-
derivatives 3a–3f
dihydro-1H-pyrazole (3e, C21H16FN3O2)
IR (KBr): v = 1,598 (pyrazoline C=N), 2,928 (CH), 1,496
1
A mixture of chalcone (0.01 mol) and phenyl hydrazine or
4-nitrophenyl hydrazine (0.01 mol) in 20 cm3 glacial ace-
tic acid was refluxed for 8 h. The reaction mixture was
cooled and poured into the 50 cm3 ice-cold water. The
precipitate formed was filtered and purified by recrystalli-
zation from ethanol.
(Ar–H), 1,218 (C–F) cm-1; H NMR (400 MHz, DMSO-
d6): d = 3.21 (dd, 1H, CH2-HA, JAB = 18.2 Hz,
JAX = 4.6 Hz), 3.91 (dd, 1H, CH2-HB, JBA = 17.8 Hz,
JBX = 12 Hz), 5.77 (dd, 1H, pyrazoline 5C–H,
JXA = 4.6 Hz, JXB = 11.8 Hz), 7.14–8.24 (m, 12H, Ar–
H) ppm; 13C NMR (100 MHz, DMSO-d6): d = 40.03
(aliphatic CH2, C-4), 53.41 (aliphatic CH, C-5), 114.48,
115.58, 121.89, 128.28, 128.82, 129.72, 130.81, 132.43,
136.86, 141.54, 149.94, 165.72, 152.47 (C=N of pyrazoline
ring) ppm; LC–MS: m/z = 362.1 ([M ? 1]?).
5-(4-Chlorophenyl)-3-(4-fluorophenyl)-1-phenyl-4,5-
dihydro-1H-pyrazole (3a, C21H16ClFN2)
IR (KBr): v = 1,604 (pyrazoline C=N), 2,916 (CH), 1,496
(Ar–H), 1,218 (C–F) cm-1; H NMR (400 MHz, DMSO-
1
d6): d = 3.07 (dd, 1H, CH2-HA, JAB = 17.4 Hz,
JAX = 6.6 Hz), 3.87 (dd, 1H, CH2-HB, JBA = 17.6 Hz,
JBX = 12.4 Hz), 5.43 (dd, 1H, pyrazoline 5C–H,
JXA = 6.6 Hz, JXB = 12.2 Hz), 6.97–7.80 (m, 13H, Ar–
H) ppm; 13C NMR (100 MHz, DMSO-d6): d = 40.01
(aliphatic CH2, C-4), 53.47 (aliphatic CH, C-5), 113.51,
115.64, 117.24, 128.45, 128.79, 129.63,129.67, 130.82,
132.32, 141.65, 143.83, 165.28, 151.82 (C=N of pyrazoline
ring) ppm; LC–MS: m/z = 351.9 ([M ? 1]?).
5-(4-Bromophenyl)-3-(4-fluorophenyl)-1-(4-nitrophenyl)-
4,5-dihydro-1H-pyrazole (3f, C21H15BrFN3O2)
IR (KBr): v = 1,597 (pyrazoline C=N), 2,924 (CH), 1,500
1
(Ar–H), 1,219 (C–F) cm-1; H NMR (400 MHz, DMSO-
d6): d = 3.25 (dd, 1H, CH2-HA, JAB = 18 Hz,
JAX = 4.4 Hz), 3.98 (dd, 1H, CH2-HB, JBA = 17.8 Hz,
JBX = 11.8 Hz), 5.75 (dd, 1H, pyrazoline 5C–H,
JXA = 4.6 Hz, JXB = 11.8 Hz), 7.06–8.27 (m, 12H, Ar–
H) ppm; 13C NMR (100 MHz, DMSO-d6): d = 40.01
(aliphatic CH2, C-4), 53.47 (aliphatic CH, C-5), 114.46,
115.63, 121.92, 128.43, 128.80, 129.66, 130.85, 132.37,
136.82, 141.60, 149.92, 165.29, 152.43 (C=N of pyrazoline
ring) ppm; LC–MS: m/z = 441.1 ([M ? 1]?).
3-(4-Fluorophenyl)-1,5-diphenyl-4,5-dihydro-1H-pyrazole
(3b, C21H17FN2)
IR (KBr): v = 1,598 (pyrazoline C=N), 2,908 (CH), 1,498
1
(Ar–H), 1,222 (C–F) cm-1; H NMR (400 MHz, DMSO-
d6): d = 3.04 (dd, 1H, CH2-HA, JAB = 18 Hz,
JAX = 6.2 Hz), 3.79 (dd, 1H, CH2-HB, JBA = 18.2 Hz,
JBX = 12 Hz), 5.46 (dd, 1H, pyrazoline 5C–H,
JXA = 6.4 Hz, JXB = 12.2 Hz), 7.03–7.84 (m, 13H, Ar–
H) ppm; 13C NMR (100 MHz, DMSO-d6): d = 40.09
(aliphatic CH2, C-4), 53.41 (aliphatic CH, C-5), 113.52,
115.60, 117.32, 128.53, 128.81, 129.77,129.89, 130.83,
132.51, 141.76, 143.63, 165.31, 152.80 (C=N of pyrazoline
ring) ppm; LC–MS: m/z = 317.7 ([M ? 1]?).
Antibacterial activity
The antibacterial efficacy of the newly synthesized pyraz-
oline derivatives was tested against Gram positive S.
aureus (ATTC-25923), Gram negative E. coli (ATTC-
25922), P. aeruginosa (ATCC-27853), and K. pneumoniae
(recultured) bacterial strains by serial plate dilution
method. Serial dilutions of the drug in Mueller–Hinton
broth were taken in tubes and their pH was adjusted to 5.0
using phosphate buffer. A standardized suspension of the
test bacterium was inoculated and incubated for 16–18 h at
37 °C. The minimum inhibitory concentration (MIC) was
noted by seeing the lowest concentration of the drug at
which there was no visible growth. A number of antimi-
crobial discs were placed on the agar for the sole purpose
of producing zones of inhibition in the bacterial lawn.
About 20 cm3 of agar media was poured into each petri
dish. Excess of suspension was decanted and plates were
dried by placing in an incubator at 37 °C for 1 h. Using a
punch, wells were made on these seeded agar plates and
minimum inhibitory concentrations of the test compounds
in dimethylsulfoxide (DMSO) were added into each
labeled well. A control was also prepared for the plates in
the same way using solvent DMSO. The petri dishes were
prepared in triplicate and maintained at 37 °C for 3–4 days
5-(4-Bromophenyl)-3-(4-fluorophenyl)-1-phenyl-4,5-
dihydro-1H-pyrazole (3c)
M.p.: 124–126 °C (Ref. [13] 124–126 °C).
5-(4-Chlorophenyl)-3-(4-fluorophenyl)-1-(4-nitrophenyl)-
4,5-dihydro-1H-pyrazole (3d, C21H15ClFN3O2)
IR (KBr): v = 1,600 (pyrazoline C=N), 2,923 (CH), 1,502
1
(Ar–H), 1,220 (C–F) cm-1; H NMR (400 MHz, DMSO-
d6): d = 3.29 (dd, 1H, CH2-HA, JAB = 18 Hz,
JAX = 4.6 Hz), 3.95 (dd, 1H, CH2-HB, JBA = 18 Hz,
JBX = 11.8 Hz), 5.72 (dd, 1H, pyrazoline 5C–H,
JXA = 4.6 Hz, JXB = 12 Hz), 7.13–8.31 (m, 12H, Ar–H)
ppm; 13C NMR (100 MHz, DMSO-d6): d = 40.04 (ali-
phatic CH2, C-4), 53.63 (aliphatic CH, C-5), 114.38,
115.47, 121.94, 128.52, 128.84, 129.72, 130.88, 132.34,
136.78, 141.64, 149.98, 165.33, 152.45 (C=N of pyrazoline
ring) ppm; LC–MS: m/z = 395.9 ([M ? 1]?).
123

A new series of pyrazoline derivatives
DPPH radical scavenging assay
[15]. Antibacterial activity was determined by measuring
the diameter of inhibition zone. Activity of each compound
was compared with ciprofloxacin as standard.
Free radical scavenging activity of compounds was mea-
sured by DPPH using the reported method [19]. Briefly,
1 mM solution of DPPH in ethanol was prepared, and
1 cm3 of this solution was added to sample solutions 1 mg/
cm3 of distilled water. The mixture was shaken vigorously
and allowed to stand at room temperature for 20 min.
Then, reaction mixture indicated higher free radical scav-
enging activity. The capability to scavenge the DPPH
radical was calculated using the following equation:
Antifungal activity
Antifungal studies of newly synthesized pyrazoline deriv-
atives were determined by serial plate dilution method
against A. fumigates (NCIM No. 902), P. marneffei (re-
cultured), T. mentagrophytes (recultured), and A. flavus
(NCIM No. 524). Sabouraud agar media were prepared by
dissolving 4 g D-glucose, 1 g peptone, and 2 g agar in
100 cm3 distilled water and adjusting the pH to 5.7. Nor-
mal saline was used to make a suspension of spore of
fungal strains for lawning. A loopful of particular fungal
strain was transferred to 3 cm3 saline to get a suspension of
corresponding species. Agar media (20 cm3) was poured
into each petri dish. Excess of suspension was decanted and
plates were dried by placing in incubator at 37 °C for 1 h.
Using a punch, wells were made on these seeded agar
plates. Minimum inhibitory concentrations of the test
compounds in DMSO were added into each labeled well. A
control was also prepared for the plates in the same way
using solvent DMSO. The Petri dishes were prepared in
triplicate and maintained at 37 °C for 3–4 days [16, 17].
Antifungal activity was determined by measuring the
diameter of inhibition zone. Activity of each compound
was compared with itraconazole as standard.
DPPH scavenging effect ð% Þ ¼ ðA0 ꢁ A1 = A0Þ ꢂ 100
where A0 is the absorbance of the control reaction and A1 is
the absorbance in the presence of the samples or standards.
Each sample was assayed at 1 mg/cm3 and all experiments
were carried out in triplicate.
Anti-inflammatory activity
The anti-inflammatory activity of the test compounds was
assessed by carrageenan induced rat paw edema method
[20–22]. The wistar albino rats weighing between 150 and
250 g were selected and divided into different groups each
containing six rats. The first group of rats was treated with
normal saline (control), second group was administered
with a dose of 20 mg/kg of diclofenac (standard) and the
other groups were treated with 20 mg/kg of the suspension
of test compounds. After 1 h, 0.1 cm3 1 % carrageenan
suspension in isosaline solution was injected into the sub-
plantar tissue of the right hind paw. The paw volume of
legs was noted for 0, 1, 2, 3, 4, 5, and 24 h after carra-
geenan challenge using plethysmometer. The mean paw
edema value for the test group is compared with its mean
value for the control group. Anti-inflammatory activity was
measured as the percentage reduction in edema level when
drug was present, relative to control and was calculated by
the formula
Antitubercular activity
The antitubercular activity of newly synthesized com-
pounds was assessed against M. tuberculosis using
Microplate Alamar Blue assay (MABA). This methodol-
ogy is non-toxic, uses a thermally stable reagent and
shows good correlation with proportional and BACTEC
radiometric method. Sterile deionized water (0.2 cm3)
was added to all outer perimeter wells of sterile 96 wells
plate to minimize evaporation of medium in the test wells
during incubation. The 96 plates received 0.1 cm3 of the
Middlebrook 7H9 broth and serial dilution of compounds
was made directly on the plate. The final drug concen-
trations tested were 100–0.2 lg/cm3. Plates were covered
and sealed with parafilm and incubated at 37 °C for
5 days. After this, 0.025 cm3 of freshly prepared 1:1
mixture of Alamar Blue reagent and 10 % tween 80 were
added to the plate and incubated for 24 h [18]. A blue
color in the well was interpreted as no bacterial growth
and pink color was scored as growth. The minimum
inhibition concentration (MIC) was defined as lowest drug
concentration which prevented the color change from blue
to pink.
% Inhibition ¼ ð1 ꢁ Vt = VcÞ ꢂ 100
where Vt is edema volume in drug-treated group and Vc is
edema volume in the control group.
Statistical analysis: the results were expressed in
mean SEM. The data from experiments were analyzed
separately using one-way ANOVA followed by Dunnett’s
t test to determine significant difference between the
groups and P \ 0.05 was considered significant.
Acknowledgments The authors are thankful to the Director, IISc,
Bangalore for NMR data and DST-PURSE for LC–MS data. Authors
are also thankful to Nalilu Suchetha Kumari, Department of Bio-
chemistry, K. S. Hegde Medical Academy, Deralakatte for the
microbial screening. BN thanks the UGC for financial assistance
through BSR one time grant for the purchase of chemicals. MS thanks
123

M. Sapnakumari et al.
DST for providing financial help for the research work through
INSPIRE Fellowship.
12. Fun HK, Loh WS, Sapnakumari M, Narayana B, Sarojini BK
(2012) Acta Cryst E68:o2655
13. Fun HK, Ooi CW, Sapnakumari M, Narayana B, Sarojini BK
(2012) Acta Cryst E68:o2634
14. Tiwari AK (2004) Current Sci 86:1092
15. Barry AL (1991) Procedure for testing antimicrobial agents in
agar media. In: Corian VL (ed) Antibiotics in laboratory medi-
cine. Williams and Wilkins, Baltimore
16. Arthington-Skaggs BA, Motley M, Warnock DW, Morrison CJ
(2000) J Clin Microbiol 38:2254
References
1. Fustero S, Simon-Fuentes A, Sanz-Cervera JF (2009) Org Prep
Proced Int 41:253
2. Li JT, Zhang XH, Lin ZP (2007) Beilstein J Org Chem 3:13
3. Rahman MA, Siddiqui AA (2010) Int J Pharm Sci Drug Res
2:165
17. Fenlon CH, Cynamon MH (1986) Antimicrob Agents Chemother
29:386
4. Sarojini BK, Vidyagayatri M, Darshanraj CG, Bharath BR,
Manjunatha H (2010) Lett Drug Des Disc 7:214
5. Alam S, Panda R, Kachroo M (2014) Indian J Chem 53B:440
6. Giles D, Roopa K, Sheeba FR, Gurubasavarajaswamy PM, Di-
vakar G, Vidhya T (2012) Eur J Med Chem 58C:478
7. Kumar C, Reddy V, Fasiulla (2013) Int J Sci Res Publications 3:1
8. Levai A, Jeko J, Brahmbhatt DI (2005) J Heterocycl Chem
42:1231
9. Samshuddin S, Narayana B, Sarojini BK, Khan MTH, Yathirajan
HS, Raj CGD, Raghavendra R (2012) Med Chem Res 21:2012
10. Fun HK, Chia TS, Sapnakumari M, Narayana B, Sarojini BK
(2012) Acta Cryst E68:o2680
18. Maria CSL, Marcus VND, Alessandra CP, de Marcelle LF,
Rasnisb BG, Thais Cristina MN, Monica AP (2007) Arkivoc
15:181
19. Blois MS (1958) Nature 181:1199
20. Alagarsamy V, Meena S, Ramseshu KV, Solomon VR, Thiru-
murugan K, Dhanabal K, Murugan M (2006) Eur J Med Chem
41:1293
21. Giles D, Roopa K, Sheeba FR, Venkatesh DP, Gurubasavara-
jaswamy PM (2011) J Pharm Sci Res 3:1253
22. Winter CA, Risley EA, Nuss CW (1962) Proc Soc Exp Biol Med
111:544
11. Fun HK, Loh WS, Sapnakumari M, Narayana B, Sarojini BK
(2012) Acta Cryst E68:o2586
123