Antimicrobial, analgesic, DPPH scavenging activities and molecular docking study of some 1,3,5-triaryl-2-pyrazolines

Article abstract of DOI:10.1007/s00044-011-9735-9

A series of 1,3,5-triaryl-2-pyrazolines 2a-g were synthesized by the reaction of 4,40-disubstituted chalcone with phenyl hydrazine. All these compounds were characterized by NMR, IR and mass spectral and single crystal XRD data. All the synthesized produc

Full text of DOI:10.1007/s00044-011-9735-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2012–2022
DOI 10.1007/s00044-011-9735-9
ORIGINAL RESEARCH
Antimicrobial, analgesic, DPPH scavenging activities
and molecular docking study of some 1,3,5-triaryl-2-pyrazolines
•
Seranthimata Samshuddin Badiadka Narayana Balladka Kunhanna Sarojini
•
•
•
•
•
Mahmud Tareq Hassan Khan Hemmige S. Yathirajan Chenna Govindaraju Darshan Raj
Ramappa Raghavendra
Received: 8 March 2011 / Accepted: 27 June 2011 / Published online: 9 July 2011
Ó Springer Science+Business Media, LLC 2011
Abstract
A
series
of
1,3,5-triaryl-2-pyrazolines
Introduction
2a–g were synthesized by the reaction of 4,40-disubstituted
chalcone with phenyl hydrazine. All these compounds were
characterized by NMR, IR and mass spectral and single
crystal XRD data. All the synthesized products were
screened for their in vitro antimicrobial, analgesic and
antioxidant properties. The docking studies were carried
out for these compounds against the active site of
methionyl-tRNA synthetase (metRS). Some of the tested
compounds exhibited significant antimicrobial, analgesic,
DPPH scavenging activities and molecular binding.
Pyrazolines are well-known, and important nitrogen-con-
taining five-membered heterocyclic compounds and vari-
ous methods have been reported for their synthesis
(Fustero et al., 2009; Safaei-Ghomi et al., 2006; Rajendra
Prasad et al., 2005). Substituted pyrazolines are useful in
pharmaceutical and agrochemical research. They display
various biological activities such as antitumor, antibacte-
rial, antifungal, antiviral, antiparasitic, anti-tubercular and
insecticidal (Amir et al., 2008; Hes et al., 1978; Grosscurt
et al., 1979). Some of these compounds have also anti-
oxidant, anti-inflammatory and analgesic properties
(Sarojini et al., 2010; Amir and Kumar, 2005). Owing to
these interesting activities of diversely substituted pyraz-
olines as biological agents considerable attention has been
focused on this class. Several 1,3,5-triaryl-2-pyrazolines
were also used as scintillation solutes (Wiley et al., 1958).
Pyrazoline derivatives with a phenyl group at the
5-position have possessed good film-forming properties,
exhibit excellent characteristics of blue photolumines-
cence, fluorescence and electroluminescence (Zhang
et al., 2000). In addition, pyrazolines have played a cru-
cial part in the development of theory in heterocyclic
chemistry and also used extensively in organic synthesis
(Klimova et al., 1999).
Keywords Analgesic ꢀ Antimicrobial ꢀ
DPPH scavenging assay ꢀ 1,3,5-Triaryl-2-pyrazoline ꢀ
Molecular docking ꢀ metRS ꢀ Molecular recognition
S. Samshuddin ꢀ B. Narayana (&)
Department of Studies in Chemistry, Mangalore University,
Mangalagangotri 574 199, Karnataka, India
e-mail: nbadiadka@yahoo.co.uk
B. K. Sarojini ꢀ C. G. D. Raj
Research Department of Chemistry, P. A College
of Engineering, Nadupadavu, Mangalore 574153, Karnataka,
India
According to oxidative and nitrosylative damage
hypothesis, reactive oxygen species (ROS) and reactive
nitrogen species (RNS) play important roles in the initia-
tion and promotion of neurodegeneration in the brains of
patients with Alzheimer’s disease (AD). Some of these
free radicals are released during inflammatory reactions,
whereas others are formed during normal oxidative
metabolism and auto-oxidation of certain neurotransmit-
ters and by b-amyloid. Thus, the role of free radicals in
M. T. H. Khan
Holmboevegen 3B, 9010 Tromsø, Norway
H. S. Yathirajan
Department of Studies in Chemistry, University of Mysore,
Mysore 570 006, Karnataka, India
R. Raghavendra
Department of Postgraduate Studies and Research
in Biochemistry, Jnana Sahyadri, Kuvempu University,
Shankaraghatta, Shimoga, Karnataka, India
123

Med Chem Res (2012) 21:2012–2022
2013
the pathogenesis of AD should be considered, at least in
part, independent of inflammatory reactions. Clinical
studies showing the beneficial effects of high-dose anti-
oxidants such as vitamin E and nicotinamide adenine
dinucleotide (NADH) in the treatment of AD support
the role of free radicals in progressive degeneration of
neurons. Edaravone ‘‘(3-methyl-1-phenyl-2-pyrazolin-5-one)’’,
a strong novel free radical scavenger, was used for treat-
ment of patients with acute brain infarction. Antioxidant
actions of edaravone include enhancement of prostacyclin
production, inhibition of lipoxygenase metabolism of
arachidonic acid by trapping hydroxyl radicals, inhibition
of alloxan-induced lipid peroxidation, and quenching of
active oxygen, leading to protection of various cells, such
as endothelial cells, against damage by ROS (Kokura
et al., 2005).
Results and discussion
Chemistry
A series of 1,3,5-triaryl-2-pyrazolines 2a–g were synthe-
sized by the reaction of 4,40-disubstituted chalcone with
phenyl hydrazine (Scheme 1). The structures of title com-
pounds were confirmed by 1H and 13C NMR, IR, LCMS and
elemental analysis. The structure of compounds 2a, 2c, 2e
and 2f were characterized by single crystal XRD [2a:
˚
Monoclinic; P21/c; a = 12.2880 (3) A; b = 13.1678 (3) A;
˚
3;
˚
c = 11.3245 (3) A; V = 1690.91 (7) A
˚
Z = 4; 2c:
Orthorhombic; P212121;a = 10.5815 (3) A; b = 11.2119
˚
3
(3) A; c = 15.4569 (4) A; V = 1833.79 (9) A ; Z = 4; 2e:
˚
˚
˚
˚
Monoclinic, P21/n; a = 5.8113 (3) A; b = 10.6959
3;
(5) A;c = 28.4455 (13) A;V = 1761.41 (15) A Z = 4; 2f:
˚
˚
˚
˚ ˚
There is an urgent need to develop novel classes of
antibiotics to counter the inexorable rise of resistant
bacterial pathogens. Modern antibacterial drug discovery
is focused on the identification and validation of novel
protein targets that may have a suitable therapeutic
index. In combination with assays for function, the
advent of microbial genomics has been invaluable in
identifying novel antibacterial drug targets. The major
challenge in this field is the implementation of methods
that validate protein targets leading to the discovery of
new chemical entities. Ligand-directed drug discovery
has the distinct advantage of having a concurrent anal-
ysis of both the importance of a target in the disease
process and its amenability to functional modulation by
small molecules. VITATM is a process that enables a
target-based paradigm by using peptide ligands for direct
in vitro and in vivo validation of antibacterial targets and
the implementation of high-throughput assays to identify
novel inhibitory molecules. This process can establish
sufficient levels of confidence indicating that the target is
relevant to the disease process and inhibition of the
target will lead to effective disease treatment (Lapan
et al., 2002).
Monoclinic; P21/a; a = 9.4788 (5) A; b = 10.1893 (6) A;
3;
˚
˚
c = 19.9139 (10) A; V = 1921.79 (18) A Z = 4] (Fig. 1)
(Samshuddin et al., 2010; Jasinski et al., 2010a, b; Baktir
et al., 2011; Butcher et al., 2011). The IR spectra of all the
compounds showed –C=N– stretch at 1580–1595 cm-1
1
confirmed the formation of pyrazoline moiety. In the H
NMR spectra of pyrazoline, protons HA and HB are geminal
O
CHO
+
R
R
EtOH
KOH
O
1 a-g
R
R
As evident from the literature, in recent years a signif-
icant portion of research work in heterocyclic chemistry
has been devoted to 2-pyrazolines containing different aryl
groups as substituents. Among the methods employed in
synthesis of pyrazolines, condensation of substituted chal-
cones with hydrazine and its derivatives is commonly used
(Knorr, 1893; Thakare and Wadodkar, 1986; Ankhiwala
and Hathi, 1996; Azarifar and Ghasemnejad, 2003). In
view of the importance of 2-pyrazolines and in continua-
tion of our work on pyrazolines (Samshuddin et al., 2010;
Jasinski et al., 2010a, b; Fun et al., 2010; Baktir et al.,
2011), we report the synthesis and biological evaluation of
some 1,3,5-triaryl-2-pyrazolines.
Ph NH NH2
CH3COOH
N
N
H
HA
HB
2 a-g
R
R
Scheme 1 Synthesis of 1,3,5-triaryl-2-pyrazolines 2a–g
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Med Chem Res (2012) 21:2012–2022
Fig. 1 The molecular structure and numbering scheme for the compounds 2a, 2c, 2e, and 2f, with displacement ellipsoids drawn at the 50%
probability level for 2a, 2c, and 2e and 30% probability level for 2f
protons at C4 carbon, appeared in the region 3.00–3.14 ppm
(J = 6.3 Hz) and 3.80–3.98 ppm (J = 12.3 Hz) as doublet
of doublets for all newly synthesized compounds. The CH
proton at C5 also appeared as doublet of doublets in the
region of 5.37–5.57 ppm (J = 6.3 Hz), due to vicinal cou-
pling with two non-equivalent geminal protons of C4 car-
bon. LCMS and 13C-NMR spectral data supported the
formation of 2a–g. Elemental analysis also gave satisfactory
results for all the compounds.
2008; Saundane et al., 1998; Raghavendra and Neelagund,
2009). Further, their MIC values were determined against
these organisms by micro dilution method (Raghavendra
and Neelagund, 2009; Harish et al., 2007) using DMSO as a
solvent. Ciprofloxacin and Fluconazole were used as stan-
dard antibiotics. All the tested compounds were emerged as
active against all tested microorganisms.
The different substitutions on the pyrazoline moiety
almost equally contribute to the antimicrobial activity
comparable with that of standard drugs tested. However,
based on this promising observation, it is immature to
arrive at the conclusion on structure activity relationship
aspect of these molecules and further evaluation is needed
to use them for clinical use.
Biological evaluation
Antimicrobial studies
The synthesized 1,3,5-triaryl-2-pyrazolines 2a–g, were
assayed for their antimicrobial activities against four bac-
terial strains Gram positive, Bacillus subtilis, Streptococcus
haemolytius; Gram negative, Pseudomonas aeruginosa,
Klebsiella pneumoniae. The compounds were also tested
against two fungal strains Aspergillus niger, Candida
albicans using agar well diffusion method (Sharath et al.,
Analgesic activity
The analgesic activity of compounds 2a–g was performed
by the acetic acid-induced writhing test in mice (Vagdevi
et al., 2001; Bagavant et al., 1994; Satyanarayana and
Rao 1993). Among the tested compounds, 3,5-bis
(4-methoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazole 2f,
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Med Chem Res (2012) 21:2012–2022
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3,5-bis(4-methylphenyl)-1-phenyl-4,5-dihydro-1H-pyrazole
2e and 1,3,5-triphenyl-4,5-dihydro-1H-pyrazole 2d exhib-
ited good analgesic activity compared with acetyl salicylic
acid as a standard analgesic agent, whereas all other com-
pounds showed moderate activity. The enhanced activity of
these compounds might be attributed to methoxy phenyl,
methylphenyl and phenyl groups present in the pyrazoline
molecules.
newly synthesized compounds to methionyl-tRNA syn-
thetase enzyme was attempted. Many tRNA synthetases
can be considered good targets for antibacterial discovery
because they are broadly conserved, essential for growth
and distinct enough from their human orthologs to
anticipate the discovery of selective inhibitors (Hurdle
et al., 2005).
Putative molecular interactions with metRS
DPPH radical scavenging assay
The compounds 2a–g have been docked into the active site
of the active site of methionyl-tRNA synthetase (metRS)
(PDB ID: 1A8H).
A rapid, simple and inexpensive method to measure
antioxidant capacity of substances involves the use of the
free radical, 2,2-diphenyl-1-picrylhydrazyl (DPPH).
DPPH is widely used to test the ability of compounds to
act as free-radical scavengers or hydrogen donors. Anti-
oxidants tested on DPPH were also found extremely
effective in cell systems. This simple test further provides
information on the ability of a compound to donate
electrons during antioxidant action (Tiwari, 2004). The
radical scavenging mechanism is based on the transfer of
H-atom from the methylene group of 1,3,5-triaryl-2-pyr-
azolines to DPPH radical to form DPPH-H. Among the
Oxygen atom of the Glu138 formed a hydrogen bond
˚
(2.73 A) with F1 atom of compound 2a and N1 atom of the
compound formed the second hydrogen bond with the H21
˚
atom of Arg132 with a length of 2.5 A. All the phenyl rings
of the same compound formed hydrophobic interactions
and p–p stacking with different atoms and phenyl rings of
different amino acid residues like, Glu138, Tyr134, Thr55,
Ile146 and Pro145.
Compound 2b showed one hydrogen bonding between
the H atom of Tyr134 and N1 atom of the compound with a
˚
length of 2.46 A. Similar like compound 2a this one also
tested
compounds,
5-bis(4-flourophenyl)-1-phenyl-4,
5-dihydro-1H-pyrazole 2a showed good radical scaveng-
ing capacity whereas 5-bis(4-bromophenyl)-1-phenyl-4,
5-dihydro-1H-pyrazole 2c and 5-bis(4-methoxyphenyl)-1-
phenyl-4,5-dihydro-1H-pyrazole 2f exhibited moderate
radical scavenging capacity with concentration of 10 lg/ml
in comparison with the standard ascorbic acid. All other
compounds showed low activity. The variation exhibited
in DPPH scavenging capacity could be attributed to the
effect of different substitutions.
exhibited several hydrophobic interactions with Pro145,
Ile146, Thr55, Arg132 and Glu54.
Very similar sorts of interactions have been observed in
case of compound 2c like 2a and 2b. The same N1 atom of
compound 2c formed hydrogen bond between H-atom of
˚
Tyr134 with a length of 2.5 A and other phenyl rings
exhibited similar hydrophobic interactions and p–p
stackings.
Very similar interactions have been observed also by
compounds 2d, 2e and 2f. Compound 2g also showed
similar interaction, difference only is instead of Tyr134 the
N1 atom of the compounds formed hydrogen bonding with
HE2 atom of His147 residue and hydrophobic interactions
with Arg149, His147, Ser129, Ile146 and Thr55.
Docking calculations with ICMTM (Internal coordinate
mechanics) dock
Molecular docking and virtual screening based on
molecular docking have become an integral part of many
modern structure-based drug discovery efforts. The bind-
ing of small molecule ligands to large protein targets is
central to numerous biological processes. The accurate
prediction of the binding modes between the ligand and
protein (the docking problem) is of fundamental impor-
tance in modern structure-based drug design (Taylor
et al., 2002). Molecular docking for the screening of anti-
SARS drugs (Wei et al., 2006) and the docking studies of
antityrosinase activity of the Thai mango seed kernel
extract are just a few good examples of docking studies
that have shown a better results for further analysis
(Nithitanakool et al., 2009). So an in silico docking of the
All the molecules exhibited comparable binding energy
varying from -2.2 to -4.1 kcal/mol. But the docking
energy varied from -4.2 o -85.6 kcal/mol. Docking
energy gives an idea about the energy required to cover
the entire protein by a ligand molecule whereas the
binding energy gives the information about the putative
interaction of the molecules at the active site of the
enzyme. Lower the value of these energies, efficient will
be the molecule. So this study indicates that the com-
pound 2g is most active among the docked compounds.
However, both in vitro and in silico studies complement
each other. Further in vivo studies are needed to know the
exact nature of the compounds to recommend them as
possible drug candidates.
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3,5-Bis(4-flourophenyl)-1-phenyl-4,5-dihydro-1H-pyrazole
(2a)
Experimental section
Chemistry
1H NMR (DMSO, 400 MHz): d 3.08–3.14(dd, 1H, CH2-
HA, J = 6.36 Hz), 3.86–3.94(dd, 1H, CH2–HB, J = 12.24),
5.48–5.53(dd, 1H, Ar–CH, 6.32 Hz), 6.70–7.78(m, 13H,
Ar–H). LCMS: m/z 335.4 (M??1). IR (KBr, m cm-1):
3027 (Ar–H), 2926(CH2), 1587 (Pyrazoline C=N), 1498
(Ar–H).
Melting points were taken in open capillary tubes and are
uncorrected. The purity of the compounds confirmed by
thin layer chromatography using Merck silica gel 60 F254
coated aluminium plates. IR spectra were recorded on
Shimadzu-FTIR Infrared spectrometer in KBr (mmax in
1
cm-1). H(400 MHz) NMR spectra were recorded on a
3,5-Bis(4-chlorophenyl)-1-phenyl-4,5-dihydro-1H-
pyrazole (2b)
Bruker AMX 400 or Bruker DPX 300 spectrometer, with
5 mm PABBO BB -1H TUBES and 13C (100 MHz) NMR
spectra were recorded for approximately 0.03 M solutions
in DMSO-d6 at 75 MHz or 100 MHz with TMS as internal
standard. LCMS were obtained using Agilent 1200 series
LC and Micromass zQ spectrometer. Elemental analysis
was carried out by using VARIO EL-III (Elementar
Analysensysteme GmBH). All chemicals were purchased
from Sigma-Aldrich Co., U.S.A.
1H NMR (DMSO, 400 MHz): d 3.08–3.14(dd, 1H, CH2–
HA, J = 6.28 Hz), 3.86–3.94(dd, 1H, CH2–HB, J = 12.32),
5.52–5.57(dd,
1H,
pyrazoline
5C–H,
6.28 Hz),
6.72–7.74(m, 13H, Ar–H). LCMS: m/z 367.3 (M?), 365.3
(M?-2). IR (KBr, m cm-1): 3032 (Ar–H), 2920(CH2),
1592 (Pyrazoline C=N), 709 (C–Cl), 1503 (Ar–H).
3,5-Bis(4-bromophenyl)-1-phenyl-4,5-dihydro-1H-
pyrazole (2c)
General procedure for synthesis of 1,3,5-triaryl-2-
pyrazolines (2a–g)
1H NMR (DMSO, 400 MHz): d 3.08–3.14(dd, 1H, CH2–
HA, J = 6.28 Hz), 3.86–3.94(dd, 1H, CH2–HB, J = 12.32),
A series of 4,40-disubstituted chalcones 1a–g were prepared
by stirring a mixture of p-substituted benzaldehyde
(0.02 mol) and p-substituted acetophenone (0.02 mol) in
ethanolic potassium hydroxide solution (50 ml) for several
hours at 5–10°C. The precipitate thus formed was collected
by filtration and purified by recrystallization from ethanol.
Further, a solution of each of these chalcones 1a–
g (0.01 mol) and phenyl hydrazine (0.01 mol) in 50 ml
glacial acetic acid was refluxed for 6 h. The reaction
mixture was cooled and poured onto 50 ml ice-cold water.
The precipitate was collected by filtration, dried and re-
crystallized from ethanol or acetonitrile to obtain the pure
1,3,5-triaryl-2-pyrazolines 2a–g. Characterization data are
given in Table 1.
5.50–5.55(dd,
1H,
pyrazoline
5C–H,
6.28 Hz),
6.72–7.69(m, 13H, Ar–H). LCMS: m/z 456.3 (M?). IR
(KBr,m cm-1): 3058 (Ar–H), 2922(CH2), 1580 (Pyrazoline
C=N), 528 (C–Br), 1500 (Ar–H).
1,3,5-Triphenyl-4,5-dihydro-1H-pyrazole (2d)
1H NMR (DMSO, 400 MHz): d 3.08–3.14(dd, 1H, CH2–
HA, J = 6.4 Hz), 3.88–3.96(dd, 1H, CH2–HB, J = 12.28),
5.45–5.50(dd, 1H, pyrazoline 5C–H, 6.4 Hz), 6.69–7.74(m,
15H, Ar–H). 13C NMR (100 MHz, DMSO): d 43.05, 63.21,
113.00, 118.67, 125.75, 125.91, 127.46, 128.70, 128.75,
128.92, 129.09, 132.34, 142.62, 144.30, 147.24. LCMS:
Table 1 Characterization data of 1,3,5-triaryl-2-pyrazolines 2a–g
Compounds
Molecular formula
R
Yield (%)
Melting point (°C)
Elemental analysis %, found (calculated)
C
H
N
2a
2b
2c
2d
2e
2f
C21H16F2N2
–F
84
77
86
79
78
76
83
112–114
120–122
206–208
134–135
141–142
139–141
136–138
75.40 (75.43)
68.59 (68.68)
55.21 (55.29)
84.46 (84.53)
84.56 (84.63)
77.01 (77.07)
87.91 (87.97)
4.77 (4.82)
4.32 (4.39)
3.48 (3.54)
6.03 (6.08)
6.71 (6.79)
6.10 (6.19)
5.77 (5.82)
8.31 (8.38)
7.55 (7.63)
6.10 (6.14)
9.34 (9.36)
8.52 (8.58)
7.79 (7.82)
6.16 (6.22)
C
C
C
C
C
C
21H16Cl2N2
21H16Br2 N2
21H18N2
–Cl
–Br
–H
23H22N2
–CH3
–OCH3
–Ph
23H22N2O2
33H26N2
2g
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Med Chem Res (2012) 21:2012–2022
2017
m/z 297.4 (M?-1). IR (KBr,m cm-1): 3020 (Ar–H),
2921(CH2), 1595 (Pyrazoline C=N), 1490 (Ar–H).
Biological evaluation
Antimicrobial activity
3,5-Bis(4-methylphenyl)-1-phenyl-4,5-dihydro-1H-
pyrazole (2e)
The antimicrobial activity of synthesized compounds 2a–
g was carried out using agar well-diffusion method. The
bacterial strains were collected from different infectious
status of patients who had not administered any antibac-
terial drugs for at least 2 weeks with the suggestions of an
authorized physician, in Kiran diagnostic health centre of
Chitradurga, Karnataka state, India. Fungal strains were
procured from the culture maintained at National College
of Pharmacy Shimoga. The in vitro antimicrobial activity
was carried out against 24 h culture of four bacterial strains
Gram positive Bacillus subtilis, Streptococcus haemolytius
Gram negative, Pseudomonas aeruginosa, Klebsiella
pneumoniae. Two fungal strains were Aspergillus niger
and Candida albicans. The compounds were tested at
40 lg/ml concentration against both bacterial and fungal
strains. DMSO was used as a vehicle. Ciprofloxacin and
Fluconazole were used as standard drugs for comparison of
antibacterial and antifungal activities, respectively. The
zone of inhibition was compared with standard drug after
24 h of incubation at 37°C for antibacterial activity and
72 h at 25°C for antifungal activity. The results are
recorded in Table 2.
1H NMR (DMSO, 400 MHz): d 2.07(s, 3H, –CH3), 2.24(s,
3H, –CH3), 3.00–3.07 (dd, 1H, CH2–HA, J = 6.28 Hz),
3.83–3.90(dd, 1H, CH2–HB, J = 12.16), 5.37–5.42 (dd,
1H, pyrazoline 5C–H, 6.28 Hz), 6.67–7.64(m, 13H, Ar–H).
13C NMR (100 MHz, DMSO): d 20.69, 21.00, 43.13,
62.93, 112.94, 118.43, 125.71, 125.83, 128.86, 129.28,
129.57, 129.65, 136.56, 138.33, 139.70, 144.44, 147.33.
LCMS: m/z 325.3 (M?-1). IR (KBr,m cm-1): 3030 (Ar–
H), 2900(CH3), 1595 (Pyrazoline C=N), 1490 (Ar–H).
3,5-Bis(4-methoxyphenyl)-1-phenyl-4,5-dihydro-1H-
pyrazole (2f)
1H NMR (DMSO, 400 MHz): d 3.00–3.07(dd, 1H, CH2–
HA, J = 6.28 Hz), 3.70(s, 3H, OCH3–), 3.79(s, 3H, OCH3–
), 3.80–3.98(dd, 1H, CH2–HB, J = 12.32), 5.52–5.57(dd,
1H, pyrazoline 5C–H, 6.28 Hz), 6.72–7.74(m, 13H, Ar–H).
LCMS: m/z 359.4 (M?-1). IR (KBr,m cm-1): 3000 (Ar–
H), 2950(CH2), 2825 (OCH3–),1585 (Pyrazoline C=N),
1490 (Ar–H).
The MIC of all synthesized compounds 2a–g was
determined by a micro dilution method. The respective
clinical strain was spread separately on the medium. The
wells were created using a stainless steel sterilized cork
borer under aseptic conditions. The synthesized com-
pounds at different concentrations viz. 10, 20, 30, 40 and
50 lg was dissolved, respectively, in 25, 50, 75, 100 and
125 ll of DMSO and later loaded into corresponding wells.
The standard drug Ciprofloxacin (40 lg in100 ll and
Fluconazole (40 lg in100 ll) were used as standard drugs
3,5-Di(biphenyl-4-yl)-1-phenyl-4,5-dihydro-1H-pyrazole
(2g)
1H NMR (DMSO, 400 MHz): d 3.04–3.10(dd, 1H, CH2–
HA, J = 6.28 Hz), 3.84–3.92(dd, 1H, CH2–HB, J =
12.32), 5.42–5.47(dd, 1H, pyrazoline 5C–H, 6.28 Hz),
6.60–7.92(m, 23H, Ar–H). LCMS: m/z 450.0 (M?). IR
(KBr,m cm-1): 3027 (Ar–H), 2925(CH2), 1584 (Pyrazoline
C=N), 1501 (Ar–H).
Table 2 Anti-microbial
Zone of inhibition in (mm)
activity of synthesized
compounds 2a–g
Compounds
Antibacterial strains
Antifungal strains
B. subtilis Streptococcus Pseudomonas Klebsiella
haemolytius
A. niger C. albicans
aeruginosa
pneumoniae
2a
21
22
23
23
20
21
23
24
–
23
24
21
23
22
23
21
24
–
20
21
22
22
23
24
22
24
–
22
20
21
23
24
22
23
24
–
20
22
20
23
24
21
22
–
20
21
20
21
22
20
21
–
2b
2c
2d
2e
2f
2g
Ciprofloxacin
Fluconazole
Control (DMSO)
24
0
23
0
0
0
0
0
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Med Chem Res (2012) 21:2012–2022
Antifungal Strains
Table 3 Minimum inhibitory
concentration (MIC) of
synthesized compounds 2a–g
MIC (lg/ll)
Compounds
10–50 (lg)
Antibacterial strains
B. subtilis Streptococcus Pseudomonas Klebsiella
haemolytius
A. niger C. albicans
aeruginosa
pneumoniae
2a
30
30
40
40
30
40
40
0
30
30
40
40
20
40
40
0
40
30
30
30
40
30
30
0
30
30
40
40
30
30
30
0
40
30
40
30
40
30
40
0
40
30
20
30
30
40
40
0
2b
2c
2d
2e
2f
2g
Control DMSO
for comparison of antibacterial and antifungal activities,
respectively. The zone of inhibition was compared with
standard drug after 24 h of incubation at 37°C for anti-
bacterial activity and 72 h at 25°C for antifungal activity.
The results are recorded in Table 3.
acetic acid. After 5 min of acetic acid injection, mice were
observed for the number of writhes for the duration of
20 min. The mean value for each group was calculated and
compared with control. Acetyl salicylic acid was used as a
standard for comparison of analgesic activity. Results are
recorded in Table 4. Percent protection was calculated using
following formula: (1 - Vc/Vt) 9 100; where Vt = Mean
number of writhing in test animals and Vc = Mean number of
writhing in control.
Analgesic activity
The analgesic activity was performed by the acetic acid-
induced writhing test in mice and approved by the animal
ethics committee (Institutional Animal Ethical Committee,
IAEC) of the National College of Pharmacy, Harapanah-
alli. Five groups of six male Swiss albino mice, each
25–35 g of body weight, were used. 0.6% acetic acid
(dose = 10 ml/kg of body weight) was injected intraperi-
toneally. The numbers of writhes were counted for 20 min,
after 5 min of injection of acetic acid into each mice. This
reading was taken as a control. Next day, same groups of
mice were used for evaluating analgesic activity. Each
group was administered orally with the suspension of test
compound in 0.1% Tween-80 solution at a dose of 100 mg/kg
body weight of animal was given 1 h before injection of
DPPH radical scavenging assay
The DPPH assay was based on the reported method
(Kokura et al., (2005). In brief, the DMSO sample of
compounds at 10 lg/ml and it was diluted to 4 ml using
distilled water. To this 1 ml of 1,1-diphenyl-2-picryl-
hydrazyl (DPPH) solution in methanol was added. The
mixed solution was incubated at room temperature for
30 min. The absorbance of stable DPPH was read at
517 nm using UV–visible spectrophotometer and the
remaining DPPH was calculated. Ascorbic acid was taken
as standard. The free radical scavenging activity was
expressed as follows:
Table 4 Analgesic activity of synthesized compounds 2a–g
½Ac ꢁ Asꢂ
DPPH scavenging activity (%) ¼
ꢃ 100
Compounds Dose
(mg/kg)
Mean no. of writhing
Before drug After drug
%
Protection
½Ac ꢁ Abꢂ
where Ac was the absorbance of the control, As for the
sample and Ab for the blank (MeOH). Each sample was
assayed at 10 lg/ml and all experiments were carried out in
triplicate and the % RSC is shown in Table 5.
2a
2b
2c
2d
2e
2f
100
31.1 0.40 15.66 0.85 49.65
100
100
100
100
100
100
100
31.0 6.73
32.0 0.25
31.5 0.44
32.5 0.57
31.5 0.44
31.1 0.48
32.33 0.57
15.5 0.44 50.0
14.8 0.31 53.75
13.3 0.63 57.8
13.6 0.57 58.16
13.0 0.36 58.74
14.6 0.51 53.1
13.5 0.68 58.25
Docking calculations with ICMTM (internal coordinate
mechanics) dock
2g
Standard
(Aspirin)
All the docking calculations of compounds in this article
were performed using the ICMTM docking module with the
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Med Chem Res (2012) 21:2012–2022
2019
Table 5 DPPH radical scavenging assay of synthesized compounds
2a–g
DG ¼ DGH þ DGEL þ DGS þ C
DGEL ¼ DGCOUL þ DGDESOLV
ð1Þ
ð2Þ
Compounds
%DPPH scavenging
Here DGH is the hydrophobic or cavity term, which
accounts for the variation of water/non-water interface
area. DGEL is the electrostatic term composed of coulombic
(DGCOUL) interactions and desolvation (DGDESOLV) of
partial charges transferred from an aqueous medium to a
protein core environment. DGS is the entropic term which
results from the decrease in the conformational freedom of
functional groups buried upon complexation; and finally
the C is a constant accounts for the change of entropy of
the system due to the decrease of free molecules concen-
tration (cratic factor), and loss of rotational/translational
degrees of freedom (Schapira et al., 1999). The calculated
docking and binding (DG) energies (in Kcal/mol) of the
compounds are shown in Table 6.
2a
51.22 1.36
31.76 1.33
40.28 2.04
23.13 2.11
22.37 1.38
39.61 1.68
18.22 1.53
69.22 1.67
2b
2c
2d
2e
2f
2g
Std (Ascorbic acid)
default setup as earlier mentioned (Abagyan et al., 1994;
Khan et al., 2009).
Preparations of the inhibitors and target molecules
Interpretations of intermolecular interactions
The 2D structures of the compounds (in mol file formats)
have been converted to 3D and energy minimized at the 3D
space of ICM environment. The atom types using local
chemical environment, Merck molecular force field
(MMFF) (Halgren, 1996a, b, c, d, 1999a, b; Halgren and
Nachbar1996) formal charges and 3D topology were
assigned. The lowest energy conformers of the compounds
were then docked into the 3D space of the targets active
site [methionyl-tRNA synthetase (metRS) (PDB ID:
1A8H)] (Sugiura et al., 2000).
To study the intermolecular interactions between the tar-
gets and the compounds LigPlot (Wallace et al., 1995)
were used to plot the interactions from 3D to 2D. Beside
LigPlot, ICM (http://www.molsoft.com) and Discovery
Studio Visualizer (http://www.accelrys.com) also been
used to analyze the interactions in 3D space. The molecular
interactions between the compounds and the active site
residues of metRS at 3D space are shown in Fig. 2, dif-
ferent compounds in different panels, accordingly.
Docking process
Conclusion
All the docking calculations were performed using the
‘interactive docking’ menu at the ICM environment. After
docking the stack of docking poses were checked visually.
Multiple stack conformations were selected based on their
docking energies, rmsd values (compared between the
docked model and X-ray conformation) and similarities to
closely related X-ray crystal structures from PDB. Then the
best conformations for each of the compounds were finally
chosen, and then their binding energies were calculated
using ICM script (briefly described in the following ‘Cal-
culations of free energies of binding’ section).
A series of 1,3,5-triaryl-2-pyrazolines 2a–g were synthe-
sized by the reaction of 4,40-disubstituted chalcones with
phenyl hydrazine and screened for their antimicrobial,
analgesic and antioxidant properties. All the tested com-
pounds were emerged as active against all tested
Table 6 Calculated docking and binding (DG) energies of the com-
pounds 2a–g against metRS
Compounds
Calculated energies (in Kcal/mol)
The correlation between the activity profiles and the
binding energies (Cal. DG) are presented in ‘Results and
discussion’ section.
Docking
Binding (DG)
2a
2b
2c
2d
2e
2f
-14.0
-6.1
-2.3
-3.3
-3.2
-2.2
-4.1
-3.7
-3.7
Calculations of free energies of binding
-6.1
-4.2
For each of the individual docked complexes the free
energies of binding (Cal. DG) between the protein and
ligand was calculated using ICM script utilizing the Eqs. 1
and 2 (Schapira et al., 1999).
-7.2
-12.3
-85.6
2g
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2020
Med Chem Res (2012) 21:2012–2022
Fig. 2 Molecular interactions between the compounds 2a–g and the methionyl-tRNA synthetase (metRS) (PDB ID: 1A8H)
microorganisms. Among them, 3,5-bis(4-methoxyphenyl)-
1-phenyl-4,5-dihydro-1H-pyrazole 2f, 3,5-bis(4-methyl-
phenyl)-1-phenyl-4,5-dihydro-1H-pyrazole 2e and 1,3,
5-triphenyl-4,5-dihydro-1H-pyrazole 2d have exhibited
good analgesic activity. The compound 5-bis(4-flourophe-
nyl)-1-phenyl-4,5-dihydro-1H-pyrazole 2a has shown good
123

Med Chem Res (2012) 21:2012–2022
2021
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DPPH scavenging activity where as compounds 5-bis
(4-bromophenyl)-1-phenyl-4,5-dihydro-1H-pyrazole 2c and
5-bis(4-methoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazole
2f have shown moderate DPPH scavenging activity. The
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the active site of methionyl-tRNA synthetase (metRS). Some
of the tested compounds exhibited good molecular binding.
Hence this study has widened the scope of developing these
1,3,5-triaryl-2-pyrazoline derivatives as promising antimi-
crobial, analgesic and antioxidant agents.
Acknowledgments The authors are thankful to Mangalore Uni-
versity and the UGC-SAP for financial assistance for the purchase of
chemicals. MTHK is grateful to Prof. Ingebrigt Sylte, Medical
Pharmacology and Toxicology, Department of Medical Biology,
Faculty of Health Science, University of Tromsø, N-9037 Tromsø,
Norway, for the access of the license of ICM-Pro version.
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