Synthetic utility of sydnones: Synthesis of pyrazolines derivatized with 1,2,4-triazoles as antihyperglymic, antioxidant agents and their DNA cleavage study

Article abstract of DOI:10.1007/s00044-011-9921-9

Ring transformation of sydnone (1a-i) to 1,3,4-oxadiazoline-2-one (2a-i) was carried out using bromine in acetic anhydride. The compounds (2a-i) on heating with hydrazine hydrate gave 1,2,4-triazole (3a-i) in good yields. The structure of these unknown co

Full text of DOI:10.1007/s00044-011-9921-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3709–3719
DOI 10.1007/s00044-011-9921-9
ORIGINAL RESEARCH
Synthetic utility of sydnones: synthesis of pyrazolines derivatized
with 1,2,4-triazoles as antihyperglymic, antioxidant agents
and their DNA cleavage study
•
Tasneem Taj Ravindra R. Kamble
•
•
Pramod P. Kattimani Bharati V. Badami
Received: 1 August 2011 / Accepted: 17 November 2011 / Published online: 2 December 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Ring transformation of sydnone (1a–i) to 1,3,4-
oxadiazoline-2-one (2a–i) was carried out using bromine in
acetic anhydride. The compounds (2a–i) on heating with
hydrazine hydrate gave 1,2,4-triazole (3a–i) in good yields.
The structure of these unknown compounds was confirmed
by IR, 1H NMR, MS and elemental analysis. Further, these
compounds were evaluated for the extent of penetration
into biological membranes (clogP) drug likeliness and
finally drug score was calculated. The title compounds
were also screened for their antihyperglycemic, DNA
cleavage and antioxidant activity.
1,3,4-Oxadiazole derivatives have a highlighted chemistry
(Bloom and Murray, 1992; Barnes etal., 1991; Fujiwara et al.,
1997). Several five-membered aromatic systems having three
hetero atoms at symmetrical position such as 1,3,4-oxadiaz-
oles have been synthesized with various substituted aromatic
acids in presence of POCl3 (Chudgar et al., 1989). Typically,
the synthesis was promoted by heat and reagents such as
thionyl chloride, phosphorous pentoxide, triphenylphosphine
and triflic anhydride (Al-Talib et al., 1990; Theocharis and
Alexandrou, 1990; Carlsen and Jorgensen, 1994; Brown etal.,
1997; Liras et al., 2000).
The common synthetic approaches to oxadiazoles
¨
(Loffler and Schobert, 1997) also involve cyclisation of
Keywords 1,3,4-Oxadiazoles ꢀ 1,2,4-Triazoles ꢀ
Drug score ꢀ Antihyperglycemic activity ꢀ DNA cleavage ꢀ
Antioxidant activity
diacylhydrazines.
The azole moiety is important and much attention has
been focused on 1H-1,2,4-triazole derivatives (Sweetman,
2004; Herbrecht et al., 2002) for their broad-spectrum
activities such as fungicidal, herbicidal, DNA cleavage,
antioxidant, anticonvulsant and also used as plant growth
regulators (Fig. 1) (Chai et al., 1989; Stankovsky et al.,
1993; Ferrer et al., 2004; Andreadou et al., 2002; Rage-
novic et al., 2001).
Introduction
Sydnones are representatives of mesoionic heterocyclic
compounds which possess a broad spectrum of pharmaco-
logicalactivitiesandhavebeenusedassynthonsin1,3-dipolar
cycloadditionstobetransformedtovariousamide derivatives,
1,3,4-oxadiazole, pyrazole, phenyl indazole, pyrazoline and
tetrazine (Taj et al., 2011, Ohta and Kato, 1969; Mallur and
Badami, 2000; Browne and Harrity, 2010; Wu et al., 2010).
Many synthetic studies have been focused on the design
of analogs of oxadiazoles and biological properties of
1,2,4-triazoles in our laboratory (Kattimani et al., 2011). In
view of this we have designed the title compounds bearing
1H-1,2,4-triazole in a molecular framework and the syn-
thesis involved the ring transformation of sydnone to 1,3,4-
oxadiazole followed by nitrogen insertion to get the title
compounds through the proposed reaction (Scheme 1).
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-011-9921-9) contains supplementary
material, which is available to authorized users.
Chemistry
T. Taj ꢀ R. R. Kamble (&) ꢀ P. P. Kattimani ꢀ B. V. Badami
PG Department of Studies in Chemistry, Karnatak University,
Dharwad 580 003, India
3-[4-(5-Aryl-4,5-dihydro-1H-pyrazolin-3-yl)-phenyl] syd-
none (1a–i) upon reaction with bromine in acetic anhydride
e-mail: kamchem9@gmail.com
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Med Chem Res (2012) 21:3709–3719
observed in the range 1,580–1,604 cm-1 (C=N) stretching
frequencies of pyrazoline and triazoline ring. In the com-
pounds 2i and 3i, a broad band around 3,400 cm-1
appeared indicating the presence of OH group.
F
N
N
N
N
H3C
N
N
N
N
N
N
N
OH
1
OH
F
The H NMR spectral analysis of all the compounds
F
(2a–i) and (3a–i) showed a singlet around 2.12–2.45 ppm
for three protons due to methyl protons of the N-acetyl
group. Whereas, the compounds (2a–i) have shown a sin-
glet for three protons in the range 2.38–2.55 ppm due to
protons of the methyl group attached at C5-carbon of the
1,3,4-oxadiazole ring. Similarly, the compounds (3a–
i) have shown a singlet in the range 2.31–2.55 ppm
attributed to the protons of the methyl group attached at the
C5-carbon of triazole ring.
F
F
Fluconazole
Voriconazole
Fig. 1 Structurally related bioactive compounds
gave 3-[4-(1-acetyl-5-aryl-4,5-dihydro-1H-pyrazol-3-yl)-
phenyl]-5-methyl-3H-[1,3,4]-oxadiazol-2-one (2a–i) and
further ring insertion with hydrazine hydrate yielded 2-[4-
(1-acetyl-5-aryl-4,5-dihydro-1H-pyrazol-3-yl)-phenyl]-4-
amino-5-methyl-2,4-dihydro-[1,2,4]-triazol-3-one (3a–i).
The mechanism for the conversion of the compounds
(1a–i) to the compounds (2a–i) and further ring insertion
with hydrazine hydrate to final product (3a–i) proposed in
(Scheme 2).
The protons of pyrazolin ring, i.e. a methine proton and the
diastereotopic methylene protons show a characteristic fea-
ture. ThemethyleneprotonscanbeassignedasHA, HB andthe
methine proton as HX. The HA proton appears as doublet of
doublet in the range d 3.48–3.73 ppm. The HB also appears as
doublet of doublet at 3.08–3.47 ppm. The Hx always
appears as four line spectrum in the range d 4.32–
4.92 ppm. Aromatic protons appear around 7.0–8.0 ppm. The
compounds (3a–i) show a singlet (D-2O exchanged) due to
NH group in the range 6.80–8.10. The compounds 2i and 3i
show a broad singlet (D2O exchanged) at around 8.0 ppm due
to OH proton.
Results and discussion
The structures of the compounds (2a–i) and title com-
1
pounds (3a–i) were confirmed by IR, H NMR and mass
Molecular osiris property explorer
spectral and elemental analysis. All the compounds (2a–
i) have shown a common strong absorption band around
1,775–1,781 cm-1 and a medium intensity sharp band
around 1,650–1,660 cm-1 due to carbonyl of acetyl group,
sharp common band around 1,580–1,604 cm-1 appeared
for C=N stretching frequencies of pyrazoline ring.
A new molecule to qualify as a drug molecule, it has to be
analysed for the parameters set by Lipinski’s rule of five
(Lipinski, 2004; http://www.organic-chemistry.org/prog/peo/;
Emami et al., 2011; Sangamwar et al., 2007). This is a thumb
rule to evaluate drug likeliness to determine whether a com-
pound with a certain pharmacological activity has properties
that make it a likely orally active drug in humans. It is
important for drug development where pharmacologically
active lead structure is optimised step wise for increased
activity, selectivity, aswellasdrug-likeproperties. Also, these
The final compounds (3a–i) have shown common strong
absorption bands around 3,310–3,320 cm-1 (NH2 group),
1,697–1,710 cm-1 (carbonyl of triazoline ring) and a
medium intensity sharp band around 1,650–1,660 cm-1
(carbonyl of N-acetyl group). Another sharp band is
Scheme 1 Ring transformation
of sydnone (1a–i) to
O
H
O
O
N
i,ii
HN
N
1,3,4-oxadiazolin-2one
(2a–i) followed by ring
insertion of hydrazine hydrate to
1,2,4-triazole (3a–i). Reagents
and conditions: (i) Br2, Ac2O
0–5°C, (ii) 60°C, (iii)
NH2NH2ꢀH2O, 6 h reflux.
a Phenyl, b o-chlorophenyl,
c m-chlorophenyl,
H3C
N
O
N
N
O
N
R
R
H
N
CH3
HB
(2a-i)
X HA
(1a-i)
iii
O
O
d p-chlorophenyl,
N
H3C
N
NNH2
e p-nitrophenyl, f p-tolyl,
g p-hydroxyphenyl, h p-anisyl,
i 3,4-dimethoxy
N
R
H
N
HB
CH3
X HA
(3a-i)
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Med Chem Res (2012) 21:3709–3719
3711
Br
Br
H
O
O
O
CO
(CH3CO)2O
(CH3CO)2O
0 °C, Br2
R'
N
O
R'
N
O-COCH3
CH3
R'
N
O
O
60 °C
-HBr
N
N
N
(1a-i)
Br
N
Br
O-COCH3
O
R"
N
O
CH3COO-
CH3
COCH3
-Br-
NH2NH2
O
N
-CO2
-CH3COO
R'
R'
N
O
N
N
CH3
CH3
(2a-i)
O
O
O
R"
R''
H
R"
N
N
N
N
N
NHNH2
OH
NHNH2
-H2O
(3a-i)
N
NH2
OH
O
N
CH3
H3C
CH3
Ac
H
R
N
N
N
N
H
R' =
R" =
R
HX
HB
HB
XHA
HA
Scheme 2 Proposed mechanism for the formation of compounds (2a–i) and (3a–i)
properties are important for a drug pharmacokinetics in
human body, including its absorption, distribution, metabo-
lism and excretion (ADME). The modification of the molec-
ular structure often leads to drugs with higher molecular
weight, more rings, more rotatable bonds and higher lipo-
philicity. The rule states that in general an orally active drug
has no more than 5 hydrogen bond donors, and not more than
10 hydrogen acceptors. A molecular weight under 500 and
partitioncoefficient (clogP)lessthan5donot qualify asadrug
molecule. The title compounds donot violate the Lipinski rule
and they fall well in the range. All the title compounds showed
clogP well within the range and also molecular weights less
than 500. The drug likeliness ranged from 3 to 7 where as the
drug score ranged from 0.12 to 0.35. The chloro substituent in
compounds 2b, 2c, 2d, 3b, 3c, 3d was shifted to different
positions (ortho, para, meta) to study the change on the
positional effect of substituent. Interestingly, these com-
pounds showed almost the same drug score which indicating
that there is no positional effect on these compounds. The
compounds 2e and 3e which have electron withdrawing nitro
group showed drug likeliness in a negative value and also the
drug score was less. The compounds 2f, 2h, 3f and 3h which
haveelectrondonatinggroupsshowalmostsamedrugscoreas
depicted in (Table 1). The compounds 2g and 3g have also
shown diversified effects, and 2i, 3i with two electron
donating methoxy groups based on drug likeliness and drug
score, but clogP values are well within the range as mentioned
by the rule.
Amylase inhibition assay
The basic heterocyclic rings present in the various medic-
inal agents are 1,2,3-triazole and 1,2,4-triazole. A large
volume of research has been carried out on triazole and
their derivatives, which has proved the pharmacological
importance of this heterocyclic nucleus (Rajeev et al.,
2011). This study is an attempt to study the antihypergly-
cemic activities of triazole (3a–i) derivatives (Fig. 2).
In this study, the compounds 3a (phenyl), 3d (p-chloro)
and 3e (p-nitro) have shown strong inhibitory activity. 3b
(o-chloro), 3g, 3h, 3i (hydroxyl, –OCH3, 3,4-dimethoxy)
have exhibited good inhibitory activity against a-amylase
compared with the control enzyme inhibitor (Fig. 3). Some
of the derivatives like 3c (m-chloro) and 3f (p-tolyl) have
shown moderate activity. The bar graph representation of
comparative activities of the title compounds (3a–i) is
shown in (Table 2).
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Med Chem Res (2012) 21:3709–3719
Table 1 Pharmacological parameters for bioavailability of com-
pounds (2a–i) and (3a–i)
0.015
0.01
0.005
0
Entry no.
clogP
Mol wt
Druglikeness
Drug score
2a
2b
2c
2d
2e
2f
3.43
4.04
4.04
4.04
3.30
3.74
3.13
3.32
3.22
1.94
2.55
2.55
2.55
1.81
2.55
3.16
1.83
1.73
362
396
396
396
407
376
378
392
422
376
410
410
410
421
390
392
406
436
-5.22
-5.53
-6.02
-4.33
-16.08
-7.33
-5.66
-5.81
-5.63
3.51
0.35
0.28
0.28
0.28
0.31
0.31
0.36
0.34
0.33
0.27
0.23
0.22
0.23
0.12
0.23
0.27
0.26
0.25
2g
2h
2i
Fig. 3 Antihyperglycemic activity of the compounds (3a–i)
Table 2 Antihyperglycemic activity of compounds (3a–i)
3a
3b
3c
3d
3e
3f
3.19
Sample
OD
540 nm
C (lg)
Activity
(lmoles/ml/min)
% Activity
2.72
4.43
3a
0.50
0.19
0.40
0.49
0.41
0.40
0.19
0.40
0.14
0.40
40
24
30
39
32
30
24
30
20
31
0.0111
0.0066
0.0083
0.0108
0.0088
0.0083
0.0066
0.0083
0.0055
0.0086
129.07
76.74
96.51
125.58
102.33
96.51
76.74
96.51
63.95
100.00
-7.32
1.41
3b
3c
3g
3h
3i
3.06
3d
2.89
3e
3.08
3f
3g
3h
It is apparent that the C5 position of the pyrazoline ring
is sterically sensitive and this overides any acidity contri-
butions (e.g., from phenyl, 4-nitrophenyl). The lipophilicity
range of active compounds is quite narrow and this
parameter too can overide acidity through hindrance of oral
absorption. It is also possible that a specific tautomeric
form of the heterocycle is required for in vivo activity
(Kees et al., 1995), tailed significance and hence the
compounds 3a (phenyl) and 3e (nitro phenyl) groups show
strong inhibitory activity. Electron-withdrawing character,
associated with a chloro or bromo group appears to be
optimal for antihyperglycemic activity (Ellingboe et al.,
1993). Hence, compound 3d with p-chloro phenyl moiety
exhibits strong antihyperglycemic activity.
3i
Control
C concentration of maltose liberated
T test analysis for amylase inhibition assay
T test analysis is carried out to check the level of confi-
dence compared to the control drug (Landau and Everitt,
2004; SPSS, 20081) which is done using the ‘‘Statistical
Package of Programs’’ (SPSS). It is a package of programs
for manipulating, analysing and presenting data; the
package is widely used in the social and behavioural sci-
ences. There are several forms of SPSS. The core program
is called SPSS Base and there are a number of add-on
modules that extend the range of data entry, statistical, or
reporting capabilities. According to this statistical analysis,
amylase inhibition assay indicated the two-tailed signifi-
cance which lies at 0.595 and is well within the permissible
value for the 95% confidence interval of the difference as
compared to the control. Hence, it can be inferred that
1
SPSS is a computer programme used for survey authoring and
deployment (IBM data Collection), data mining (IBM SPSS Modeler),
text analytics, statistical analysis and collaboration and deployment
(batch and automated scoring services). The t test is used to test the null
hypothesis that the means of two populations are the same, H0: l1 = l2,
when a sample of observations from each population is available. The
observations made on the sample members must all be independent of
each other. One sample t tests can be used to determine if the mean of a
sample is different from a particular value.
Fig. 2 Standard maltose curve
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Med Chem Res (2012) 21:3709–3719
3713
antihyperglycemic activity is excellent for the compounds
Table 3 Percentage of free radical scavenging activity
(3a–i) (given in Supplementary information).
Entry no.
Test sample (lg)
10
50
100
Antioxidant activity (DPPH method)
3a
3b
3c
24.70
20.00
15.25
21.66
00.40
23.00
03.08
00.60
00.48
21.64
30.16
67.83
63.76
69.87
01.09
70.10
09.03
02.28
01.18
69.86
56.15
78.38
73.79
81.48
01.49
82.45
16.87
02.40
01.67
81.46
Free radical scavenging is one of the best known mecha-
nisms by which antioxidants inhibit lipid oxidation. The
1,1-diphenyl-2-picryl-hydrazyl (DPPH) method assay was
developed to determine the reducing ability of biological
fluids and aqueous solutions of pure compounds. Butylated
hydroxyl anisole (BHA) was used as a standard. Although,
the final dilution of the sample in the reaction mixture was
high (100 lg), any influence of the solvent on the reaction
was also checked. The values of absorbance at 700 nm
compared to BHA with concentrations of 10, 50, 100 lg
are given in Table 3 and are represented in Fig. 4.
3d
3e
3f
3g
3h
3i
BHA
The compounds 3d and 3f (p-chloro and p-tolyl) have
shown very good activity compared to BHA at all the
concentrations tested (Fig. 3). Compounds 3a, 3b and 3c
(phenyl, o and m-chloro) have shown moderate activity
compared to the standard. Compound 3g (hydroxyl) has
shown less activity compared to the standard. Compounds
3e, 3h and 3i (p-NO2, p-OCH3, 3,4-dimethoxy) have shown
least activity compared to standard BHA. The antioxidant
activity of these compounds may be related to their redox
properties which allow them to act as reducing agents or
hydrogen-atom donors, their ability to chelate metals,
inhibit lipoxygenase and scavenge free radicals. Such
antioxidants function as free-radical scavengers and chain
breakers, complexes of pro-oxidant metal ions and
quenchers of singlet-oxygen formation (Scheme 3).
100
80
60
40
20
0
10 µg
50 µg
100 µg
Fig. 4 Antioxidant activity of the compounds (3a–i)
significant observations. Studies show that 1,2,4-triazoles
can be induced to undergo photoextrusion under conditions
required for DNA cleavage, producing intermediates
capable of hydrogen abstraction (Wender et al., 1996).
After treatment with DNA samples 3g and 3h have
cleaved the DNA completely. In 3g lane (hydroxyl and
methoxy substituent), whole band of DNA is missing
which indicated the complete cleavage of DNA. DNA
treated with remaining samples has shown partial cleavage
(Fig. 5).
T test for free radical scavenging activity
One sample t test is used for DDPH activity for individual
concentrations of 10, 50 and 100 lg. The two tailed sig-
nificance lies at 0.29, 0.12 and 0.17 respectively. But the
values are low as compared to the control BHA for the 95%
confidence interval of the difference. Hence, it can be
concluded that the compounds (3a–i) possess moderate to
low activity (given in supplementary information).
DNA cleavage activity
Experimental
A number of studies have shown that the clinical efficacies
of many drugs correlate with their abilities to induce
enzyme mediated DNA cleavage. The inhibitory potency
of the test compounds was assessed by comparing the
cleavage of DNA with the control used and presence of
title compounds. The relative efficacy of the drugs to
stimulate DNA cleavage varies considerably from one
congener to another. A detailed comparison between the
nine compounds (3a–i) tested allows us to make some
Melting points were determined in open capillaries and are
uncorrected. The IR spectra were recorded on Nicolet
Impact 5200 USA FT IR spectrophotometer using KBr
pellets. 1H NMR spectra were recorded on FT NMR
Spectrometer model: Avance 300 MHz Bruker supercon-
ducting with TMS as an internal standard. Mass spectra
were recorded on Shimadzu Japan QP2010 S model
spectrometer and elemental analyses were carried out using
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Med Chem Res (2012) 21:3709–3719
Scheme 3 Schematic
representation of quenching of
DPPH radical
N
N
N
NH
RH
R
O2N
NO2
O2N
NO2
NO2
NO2
Fig. 5 DNA cleavage activity
of the compounds (3a–i)
3-[4-(1-Acetyl-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-
phenyl]-5-methyl-3H-[1,3,4]-oxadiazol-2-one (2a)
Heraus CHN rapid analyzer. The purity of the compounds
was checked by thin layer chromatography (TLC) on silica
gel plate using benzene and ethyl acetate as eluent. The
Osiris property explorer was used to evaluate clogP values,
drug likeliness and drug score of the synthesised mole-
cules. Antihyperglycemic, DNA cleavage and antioxidant
activities of the synthesised molecules were carried out at
Biogenics, Hubli, Karnataka, India.
Brown amorphous solid (ethanol). Yield; 71%; Rf 0.87;
mp; (°C); 95–96. IR (m max in cm-1); 1776, 1659, 1603. 1H
NMR (300 MHz, d ppm, CDCl3-d6): 2.43 (s, 3H, CH3
–C5), 2.54 (s, 3H, CH3 of NCOCH3), 3.1 (dd, 1H, [HA]
CH2), 3.6 (dd, 1H, [HB] CH2, J = 4.65 Hz), 4.56 (m, 1H,
[HX] CH2,), 7.27–7.86 (m, 9H, Ar H). GCMS (m/z); 362
[M], 337 (12), 265 (10), 251 (60), 235 (30), 212 (40), 209
(30), 194 (35), 176 (35), 159 (55), 150 (30), 134 (100), 119
(75), 106 (40), 91(60), 74 (10), 65 (25), 54 (5), 49 (10).
CHN analysis; calculated for C20H18N4O3: C, 66.25; H,
4.90; N, 15.42. Found: C, 66.29; H, 4.97; N, 15.46.
3-[4-(5-Aryl-4,5-dihydro-1H-pyrazolin-3-yl)-phenyl]-
sydnone (1a–i)
The compounds (1a–i) was prepared according to the lit-
erature method (Dambal et al., 1984).
3-{4-[1-Acetyl-5-(3-chlorophenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenyl}-5-methyl-3H-[1,3,4]-oxadiazol-2-
one (2b)
General procedure for the preparation of 3-[4-(1-acetyl-
5-aryl-4,5-dihydro-1H-pyrazol-3-yl)-phenyl]-5-methyl-
3H-[1,3,4]-oxadiazol-2-one (2a–i)
Brown amorphous solid (methanol).Yield; 87%; Rf 0.90;
mp; (°C); 154–155. IR (m max in cm-1); 1778, 1660, 1600.
1H NMR (300 MHz, d ppm, CDCl3-d6): 2.34 (s, 3H, CH3–
C5), 2.41 (s, 3H, CH3 of NCOCH3), 3.11 (dd, 1H, [HA]
CH2), 3.55 (dd, 1H, [HB] CH2, J = 4.79 Hz), 4.59 (m, 1H,
[HX] CH2), 7.14–8.10 (m, 8H, Ar H). GCMS (m/z); 398
[M ? 2, 12], 396 [M, 74], 362 (30), 337 (12), 265 (10) 251
(60), 235(30), 212 (40), 209 (30), 194 (35), 176 (35), 159
(55), 150 (30), 134 (80), 119 (75), 106 (40), 91(60), 74
The compound (1a–i) (0.01 mol) was suspended in acetic
anhydride (5 ml) and bromine (0.5 ml) in acetic anhydride
(5 ml) was added at 0–5°C within a period of 15 min. The
reaction mixture was then stirred at room temperature
(30 min), and warmed on water bath (60°C) till the evo-
lution of CO2 ceased and poured to ice cold water. Filtered
and recrystallized using ethanol to yield the compound
(2a–i) (yield 80–85%).
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Med Chem Res (2012) 21:3709–3719
3715
(10), 65 (25), 54 (5), 49 (100). CHN analysis; calculated
for C20H17N4O3Cl C, 60.48; H, 4.21; N, 14.07. Found: C,
60.52; H, 4.28; N, 14.12.
54 (5), 49 (10). CHN analysis; calculated for C21H20N4O4: C,
64.28; H, 5.14; N, 14.28. Found: C, 64.27; H, 5.13; N, 14.30.
3-{4-[1-Acetyl-5-(4-chlorophenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenyl}-5-methyl-3H- [1,3,4]-oxadiazol-2-
one (2f)
3-{4-[1-Acetyl-5-(2-chlorophenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenyl}-5-methyl-3H [1,3,4]-oxadiazol-2-
one (2c)
Brown amorphous solid (methanol). Yield; 79%; Rf 0.89,
mp; (°C); 202–203. IR (m max in cm-1); 1780, 1659, 1604.
1H NMR (300 MHz, d ppm, CDCl3-d6): 2.39 (s, 3H, CH3–
C5), 2.47 (s, 3H, CH3 of NCOCH3), 3.23 (dd, 1H, [HA]
CH2), 3.56 (dd, 1H, [HB] CH2, J = 4.58 Hz), 4.55 (m, 1H,
[HX] CH2), 7.22–8.14 (m, 8H, Ar H). GCMS (m/z); 398
[M ? 2], 396 [M, 69], 362 (30), 337 (10), 265 (12), 251
(60), 235(30), 212 (40), 209 (30), 194 (35), 176 (100), 159
(55), 150 (30), 134 (60), 119 (75), 106 (40), 91(60), 74
(10), 65 (25), 54 (5), 49 (10). CHN Analysis; Calculated for
C20H17ClN4O3: C, 60.53; H, 4.32; N, 14.12. Found: C,
60.50; H, 4.30; N, 14.10.
Brown amorphous solid (methanol). Yield; 80%; Rf 0.90;
mp; (°C); 169–170. IR (m max in cm-1); 1780, 1660, 1600.
1H NMR (300 MHz, d ppm, CDCl3-d6): 2.23 (s, 3H, CH3–
C5), 2.49 (s, 3H, CH3 of NCOCH3), 3.22 (dd, 1H, [HA]
CH2), 3.59 (dd, 1H, [HB] CH2, J = 4.60 Hz) 4.47 (m, 1H,
[HX] CH2), 7.20–7.91 (m, 8H, Ar H). GCMS (m/z); 398
[M ? 2, 10], 396 [80], 362 (30), 338 (10), 267 (12), 251
(60), 235 (30), 209 (40), 205 (30), 194 (35), 176 (35), 159
(55), 150 (30), 134 (80), 109 (75), 106 (40), 91 (60), 74
(10), 65 (25), 54 (100), 49 (10). CHN analysis; calculated
for C20H17N4O3Cl: C, 60.48; H, 4.21; N, 14.07. Found: C,
60.51; H, 4.27; N, 14.11.
3-{4-[1-Acetyl-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-
3-yl]-phenyl}-5-methyl-3H- [1,3,4]-oxadiazol-2-one (2g)
3-[4-(1-Acetyl-5-p-tolyl-4,5-dihydro-1H-pyrazol-3-yl)-
phenyl]-5-methyl-3H-[1,3,4]-oxadiazol-2-one (2d)
Brown amorphous solid (methanol). Yield; 86%; Rf 0.93,
mp; (°C); 216–217. IR (m max in cm-1); 1781, 1659, 1604.
1H NMR (300 MHz, d ppm, CDCl3-d6): 2.44 (s, 3H, CH3–
C5), 2.50 (s, 3H, CH3 of NCOCH3), 3.13 (dd, 1H, [HA]
CH2), 3.56 (dd, 1H, [HB] CH2), J = 4.70 Hz), 4.70 (m, 1H,
[HX] CH2), 7.00–7.95 (m, 8H, Ar H). GCMS (m/z); 407
[M], 397 [M], 362 (30), 336 (10), 265 (12), 251 (60), 230
(30), 212 (40), 209 (30), 194 (35), 176 (30), 159 (55), 150
(30), 134 (100), 119 (75), 106 (40), 90 (60), 74 (10), 65
(25), 50 (5), 49 (10). CHN analysis; calculated for
C20H17N5O5: C, 58.97; H, 4.21; N, 17.19. Found: C, 58.95;
H, 4.20; N, 17.20.
Brown amorphous solid (methanol). Yield; 76%; Rf 0.80,
mp; (°C); 218–219. IR (m max in cm-1); 1775, 1665, 1600.
1H NMR (300 MHz, d ppm, CDCl3-d6): 2.25 (s, 3H, CH3),
2.52 (s, 3H, CH3–C5), 2.55 (s, 3H, CH3 of NCOCH3), 3.20
(dd, 1H, [HA] CH2), 3.65 (dd, 1H, [HB] CH2, HB,
J = 4.60 Hz), 4.68 (m, 1H, [HX] CH2), 7.0–8.0 (m, 8H, Ar
H). GCMS (m/z); 376 [M], 337 (50), 335 (60), 265 (25),
251 (60), 235 (30), 212 (40), 209 (30), 194 (35), 176 (30),
159 (55), 150 (30), 134 (70), 119 (75), 106 (40), 91(80), 74
(10), 65 (25), 54 (5), 49 (100). CHN analysis; calculated
for C21H20N4O3: C, 67.01; H, 5.36; N, 14.88. Found: C,
67.00; H, 5.37; N, 14.88.
3-{4-[1-Acetyl-5-(2,4-dimethoxyphenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenyl}-5-methyl-3H [1,3,4]-oxadiazol-2-
one (2h)
3-{4-[1-Acetyl-5-(4-methoxyphenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenyl}-5-methyl-3H-[1,3,4]-oxadiazol-2-
one (2e)
Brown amorphous solid (methanol). Yield; 75%; Rf 0.93,
mp; (°C); 160–161. IR (m max in cm-1); 1779, 1659, 1604.
1H NMR (300 MHz, d ppm, CDCl3- d6): 2.20 (s, 3H, CH3–
C5), 2.40 (s, 3H, CH3 of NCOCH3), 3.26 (dd, 1H, [HA]
CH2), 3.54 (dd, 1H, [HB] CH2, J = 4.58 Hz), 3.71 (s, 6H,
CH3 of OCH3), 4.79 (m, 1H, [HX] CH2), 7.2–8.0 (m, 8H,
Ar H). GCMS (m/z); 422 [M], 407 (40), 397 (30), 362 (30),
337 (10), 265 (12), 251 (60), 235(30), 212 (40), 209 (30),
194 (35), 176 (35), 159 (55), 150 (100), 134 (80), 119 (75),
106 (40), 91(60), 74 (10), 65 (25), 54 (100), 49 (10). CHN
analysis; calculated for C22H23N5O3: C, 58.95; H, 4.13; N,
17.15. Found: C, 58.96; H, 4.17; N, 17.19.
Brown amorphous solid (ethanol). Yield; 75%; Rf 0.80, mp;
(°C); 224–225. IR (mmax in cm-1); 1767, 1660, 1602. 1HNMR
(300 MHz, d ppm, CDCl3-d6): 2.35 (s, 3H, CH3), 2.56 (s, 3H,
CH3–C5), 2.60 (s, 3H, CH3 of NCOCH3), 3.21 (dd, 1H, [HA]
CH2), 3.55 (dd, 1H, [HB] CH2, J = 4.80 Hz), 3.71 (s, 3H, CH3
of OCH3), 4.62 (m, 1H, [HX] CH2), 7.24–7.79 (m, 8H, ArH).
GCMS (m/z); 392 [M], 387 (90), 364 (60), 361(10) 340 (80),
324 (65), 309 (15), 299 (10), 282 (70), 265 (87), 266 (25), 251
(60), 235 (40), 212 (40), 209 (30), 194 (35), 170 (35), 159 (55),
150 (30), 133 (75), 119 (75), 106 (40), 91(60), 75 (100), 65 (25),
123

3716
Med Chem Res (2012) 21:3709–3719
3-{4-[1-Acetyl-5-(4-hydroxyphenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenyl}-5-methyl-3H-[1,3,4] oxadiazol-2-
one (2i)
4.67 (m, 1H, [HX] CH2), 7.22–7.90 (m, 8H, Ar H), 8.2 (s,
2H, NH2). GCMS (m/z); 412 [M ? 2, 16] 410 [M, 84], 392
(50), 387 (90), 377, 376, 362 (30), 330 (10), 265 (12), 251
(60), 235(30), 212 (40), 210 (30), 195 (35), 176 (30), 159
(55), 150 (30), 134 (60), 119 (75), 106 (40), 91(60), 74
(10), 65 (25), 54 (5), 49 (100). CHN analysis; calculated
for C20H19 ClN6O2: C, 60.53; H, 4.66; N, 20.45. Found: C,
60.50; H, 4.64; N, 20.43.
Brown amorphous solid (ethanol). Yield; 70%; Rf 0.95,
mp; (°C); 155–156. IR (m max in cm-1); 3400, 1780, 1659,
1604. 1H NMR (300 MHz, d ppm, CDCl3-d6): 2.31 (s, 3H,
CH3 of NCOCH3), 2.38 (s, 3H, CH3–C5), 3.15 (dd, 1H,
[HA] CH2), 3.62 (dd, 1H, [HB] CH2, J = 4.65 Hz) 3.71 (s,
3H, CH3 of OCH3), 4.81 (m, 1H, [HX] CH2), 7.0–8.1 (m,
8H Ar H), 8.4 (bs, 1H, OH). GCMS (m/z); 378 [M], 362
(30), 337 (10), 260 (12), 251 (60), 235 (35), 210 (40), 209
(30), 194 (35), 176 (30), 159 (55), 151 (30), 134 (90), 119
(75), 106 (40), 91(60), 74 (10), 65 (25), 54 (5), 49 (100).
CHN analysis; calculated for C20H18N4O4: C, 63.48; H,
4.79; N, 14.81. Found: C, 63.45; H, 4.80; N, 14.80.
2-{4-[1-Acetyl-5-(2-chlorophenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenyl}-4-amino-5-methyl-2,4-dihydro-
[1,2,4]-triazolin-3-one (3c)
Pale brown solid (ethanol). Yield; 71%; Rf 0.90, mp; (°C);
1
212–213. IR (m max in cm-1); 3319, 1709, 1649, 1605. H
NMR (300 MHz, d ppm, CDCl3-d6): 2.35 (s, 3H, CH3–C5),
2.42 (s, 3H, CH3 of NCOCH3), 3.10 (dd, 1H, [HA] CH2),
3.40 (dd, 1H, [HB] CH2, J = 4.70 Hz), 4.60 (m, 1H, [HX]
CH2), 7.23–7.88 (m, 8H, Ar H), 8.2 (s, 2H, NH2). GCMS
(m/z); 412 [M ? 2, 12], 410 [M, 79], 392 [M], 387 (90),
377 (30), 370 (45), 362 (30), 337 (10), 265 (12), 251 (60),
235(30), 212 (40), 210 (30), 190 (35), 175 (35), 159 (55),
150 (30), 134 (90), 119 (75), 106 (40), 91(60), 74 (10), 65
(25), 54 (100), 49 (10). CHN analysis; calculated for
C20H19ClN6O2: C, 60.53; H, 4.66; N, 20.45. Found: C,
60.51; H, 4.63; N, 20.42.
General procedure for the preparation of 2-[4-(1-acetyl-
5-aryl-4,5-dihydro-1H-pyrazol-3-yl)-phenyl]-4-amino-
5-methyl-2,4-dihydro-[1,2,4]-triazolin-3-one (3a–i)
The compound (2a–i) (0.01 mol) was refluxed with hydrazine
hydrate (0.01 mol) in ethanol (25 ml) for 6 h. The contents of
the flask were poured in water to yield the crude product (3a–
i) which was thenfiltered. Recrystallization fromethanol gave
the yellow needles (yield 75–84%).
2-[4-(1-Acetyl-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-
phenyl]-4-amino-5-methyl-2,4-dihydro-[1,2,4]-triazolin-3-
one (3a)
2-[4-(1-Acetyl-5-p-tolyl-4,5-dihydro-1H-pyrazol-3-yl)-
phenyl]-4-amino-5-methyl-2,4-dihydro-[1,2,4]-triazolin-3-
one (3d)
Pale yellow amorphous solid (ethanol). Yield; 65%; Rf
0.95, mp; (°C); 12–121. IR (m max in cm-1); 3321, 1712,
1649, 1605. H NMR (300 MHz, d ppm, CDCl3-d6): 2.18
Brown amorphous solid (ethanol). Yield; 76%; Rf 0.90, mp;
(°C); 218–219. IR (m max in cm-1); 3325, 1712, 1649, 1605.
1H NMR (300 MHz, d ppm, CDCl3- d6): 2.27 (s, 3H, CH3),
2.34(s, 3H, CH3–C5),2.50(s, 3H, CH3 ofNCOCH3),3.19(dd,
1H, [HA] CH2), 3.56 (dd, 1H, [HB] CH2, J = 4.58 Hz) 4.62
(m, 1H, [HX] CH2), 7.24–7.85 (m, 8H, Ar H), 8.25 (s, 2H,
NH2). GCMS(m/z);390[M], 387(90), 377(40), 376(10), 362
(30), 337 (10), 265 (12), 251 (60), 240 (30), 212 (40), 209 (30),
194 (35), 176 (35), 159 (55), 150 (30), 134 (86), 119 (75), 106
(40), 91(60), 74 (10), 65 (25), 54 (5), 49 (100). CHN Analysis;
Calculated for C21H22N6O2: C, 64.60; H, 5.68; N, 21.52.
Found: C, 64.59; H, 5.67; N, 21.50.
1
(s, 3H, CH3–C5), 2.31 (s, 3H, CH3 of NCOCH3), 3.10 (dd,
1H, [HA] CH2), 3.8 (dd, 1H, [HB] CH2 J = 4.65 Hz), 4.52
(m, 1H, [HX] CH2), 7.22–7.95 (m, 9H, Ar H), 8.22 (s, 2H,
NH2). GCMS (m/z); 377 [M ? 1, 6], 376 [M], 362 (30),
337 (10), 265 (12), 251 (60), 235(30), 212 (40), 209 (30),
194 (35), 176 (35), 159 (55), 150 (30), 134 (100), 119 (75),
106 (40), 91(60), 74 (10), 65 (25), 54 (5), 49 (10). CHN
analysis; calculated for C20H20N6O2: C, 63.82; H, 5.36; N,
22.33. Found: C, 63.80; H, 5.35; N, 22.30.
2-{4-[1-Acetyl-5-(3-chlorophenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenyl}-4-amino-5-methyl-2,4-dihydro-
[1,2,4]triazolin-3-one (3b)
2-{4-[1-Acetyl-5-(4-methoxyphenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenyl}-4-amino-5-methyl-2,4-dihydro-
[1,2,4]-triazolin-3-one (3e)
Pale brown amorphous solid (ethanol). Yield; 70%; Rf
0.90, mp; (°C); 210–211. IR (m max in cm-1); 3321, 1712,
1645, 1605. H NMR (300 MHz, d ppm, CDCl3-d6): 2.33
(s, 3H, CH3–C5–), 2.40 (s, 3H, CH3 of NCOCH3), 3.20 (dd,
1H, [HA] CH2), 3.35 (dd, 1H, [HB] CH2, J = 4.70 Hz),
Pale green amorphous solid (ethanol). Yield; 80%; Rf 0.91,
mp; (°C); 235-236. IR (m max in cm-1); 3318, 1710, 1649,
1605. 1H NMR (300 MHz, d ppm, CDCl3-d6): 2.21 (s, 3H,
CH3–C5), 2.41 (s, 3H, CH3 of NCOCH3), 3.15 (dd, 1H,
[HA] CH2), 3.3 (dd, 1H, [HB] CH2, J = 4.65 Hz), 3.72 (s,
1
123

Med Chem Res (2012) 21:3709–3719
3717
3H, CH3 of OCH3), 4.73 (m, 1H, [HX] CH2), 6.80–8.08 (m,
9H, Ar H), 9.27 (s, 2H, NH). GCMS (m/z); 408 (30), 405
(10), 390 (40), 385 (90), 364 (70), 360 (12), 340 (80), 324
(65), 309 (15), 300 (10), 282 (70), 265 (87), 266 (25), 251
(60), 235 (40), 212 (40), 209 (30), 194 (35), 176 (35), 159
(55), 150 (30), 134 (70), 119 (75), 106 (40), 91(60), 74
(10), 65 (25), 54 (100), 49 (10). CHN analysis; calculated
for C21H22N6O3: C, 62.06; H, 5.46; N, 20.68. Found: C,
62.07; H, 5.43; N, 20.65.
[HA] CH2), 3.57 (dd, 1H, [HB] CH2, J = 4.65 Hz), 3.75 (s,
6H, CH3 of OCH3), 4.60 (m, 1H, [HX] CH2), 7.26–7.92
(m,7H, Ar H), 8.84 (s, 2H, NH). GCMS (m/z); 436, [M],
427 (10), 423(15), 421 (20), 403 (10), 385 (10), 361 (10),
345 (15), 329 (15), 316 (40), 305 (20), 291 (15), 273 (10),
251 (10), 237 (10), 219 (20), 206 (10), 191 (35), 175 (26),
159 (15), 147 (50), 131 (15), 119 (20), 105 (20), 91 (25), 78
(60), 57 (80), 44 (50), 41 (100). CHN analysis; calculated
for C22H24N6O4: C, 60.54; H, 5.54; N, 19.25. Found: C,
60.52; H, 5.51; N, 19.19.
2-{4-[1-Acetyl-5-(4-chlorophenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenyl}-4-amino-5-methyl-2,4-dihydro-
[1,2,4]-triazolin-3-one (3f)
2-{4-[1-Acetyl-5-(4-hydroxyphenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenyl}-4-amino-5-methyl-2,4-dihydro-
[1,2,4]-triazolin-3-one (3i)
Pale brown amorphous solid (ethanol). Yield; 80%; Rf 0.90,
mp; (°C); 210–211. IR (m max in cm-1); 3318, 1710, 1649,
1605. H NMR (300 MHz, d ppm, CDCl3-d6): 2.45 (s, 3H,
Brown amorphous solid (ethanol). Yield; 81%; Rf 0.89,
mp; (°C); 210–211. IR (m max in cm-1); 3438, 3328, 1700,
1658, 1606. H NMR (300 MHz, d ppm, CDCl3- d6): 2.12
1
1
CH3–C5), 2.55 (s, 3H, CH3 of NCOCH3), 3.22 (dd, 1H, [HA]
CH2), 3.45 (dd, 1H, [HB]CH2, J = 4.70 Hz), 4.65 (m, 1H, [HX]
CH2), 6.15 (s, 2H, NH), 7.02–8.10 (m, 8H, Ar H). GCMS (m/z);
412 [M ? 2, 18], 410 [M, 96], 403 (10), 385 (10), 361 (10), 345
(15), 329 (15), 316 (40), 305 (20), 291 (15), 273 (10), 251 (10),
237 (10), 219 (20), 206 (10), 191 (35), 175 (26), 159 (15), 147
(55), 131 (15), 119 (20), 105 (20), 91 (25), 78 (60), 57 (80), 44
(100), 41 (30). CHN analysis; calculated for C20H19 ClN6O3:C,
60.53; H, 4.66; N, 20.45. Found: C, 60.50; H, 4.64; N, 20.43.
(s, 3H, C5–CH3), 2.33 (s, 3H, CH3 of NCOCH3), 3.33 (dd,
1H, [HA] CH2), 3.66 (dd, 1H, [HB] CH2, J = 4.70 Hz),
4.55 (m, 1H, [HX] CH2), 5.5 (s, 2H, NH), 6.98–7.46 (m,
7H, Ar H), 8.0 (s, 1H, OH). GCMS (m/z); 392 [M], 383
(10), 369 (10), 334 (10), 319 (10), 290 (10), 278 (10), 261
(10), 249 (10), 236 (10), 211 (10), 196 (15), 183 (15), 168
(15), 155 (15), 137 (20), 125 (30), 111 (40), 100 (45), 85
(60), 71 (70), 57 (90), 47 (100), 41 (50). CHN analysis;
calculated for C20H20N6O3: C, 61.21; H, 5.14; N, 21.42.
Found: C, 61.20; H, 5.10; N, 21.40.
2-{4-[1-Acetyl-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-
3-yl]-phenyl}-4-amino-5-methyl-2,4-dihydro-[1,2,4]-
triazolin-3-one (3g)
Pharmacological evaluation
Pale yellow amorphous solid (ethanol). Yield; 81%; Rf
0.92, mp; (°C); 220–221. IR (m max in cm-1); 3330, 1697,
1646, 1610. H NMR (300 MHz, d ppm, CDCl3- d6): 2.25
The title compounds were analysed for drug likeliness and
drug score by Osiris molecular property explorer and sub-
jected to antihyperglycemic, DNA cleavage and antioxidant
assay.
1
(s, 3H, CH3–C5), 2.46 (s, 3H, CH3 of NCOCH3), 3.29 (dd,
1H, [HA] CH2), 3.49 (dd, 1H, [HB] CH2, J = 4.60 Hz),
4.80 (m, 1H, [HX] CH2), 6.83–8.05 (m, 8H, Ar H), 9.7 (s,
2H, NH). GCMS (m/z); 421 [M], 403 (10), 385 (10), 361
(10), 345 (15), 329 (15), 316 (40), 305 (20), 291 (15), 273
(10), 251 (10), 237 (10), 219 (20), 206 (10), 191 (35), 175
(26), 159 (15), 147 (55), 131 (15), 119 (20), 105 (20), 91
(25), 78 (60), 57 (80), 44 (100), 41 (30). CHN analysis;
calculated for C20H19 ClN7O4: C, 57.00; H, 4.54; N, 23.27.
Found: C, 57.01; H, 4.53; N, 23.25.
Amylase inhibition assay (Sadasivam and Manickam
1996)
The antihyperglycemic assay (Himedia, Mumbai) was carried
out with different concentrations of samples. Sodium phos-
phate buffer (control) (1.4 ml, 50 mM, pH 7.0-7.3) was added
to the test tubes. Different volumes of solvent, starch (0.5 ml
in buffer) and enzyme (0.1 ml) were also added to the sample
in buffer (1 mg/ml). The tubes were incubated at 37°C for
10 min followed by addition of dinitrosalicylic acid (DNS;
1 ml). The blank (control) tube was added with DNS (1 ml)
before the addition of enzyme. The tubes were then incubated
in boiling water bath for 10 min, cooled and recorded the
absorbance at 540 nm against blank. The maltose liberated
was determined with the help of standard maltose curve and
activities were calculated according to the following for-
mula.Activity was expressed as lmoles/ml/min.
2-{4-[1-Acetyl-5-(2,4-dimethoxyphenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenyl}-4-amino-5-methyl-2,4-dihydro-
[1,2,4]-triazolin-3-one (3h)
Brown amorphous solid (ethanol). Yield; 81%; Rf 0.90,
mp; (°C); 200–201. IR (m max in cm-1); 3330, 1700, 1646,
1610. 1H NMR (300 MHz, d ppm, CDCl3-d6): 2.34 (s, 3H,
CH3–C5), 2.40 (s, 3H, CH3 of NCOCH3), 3.25 (dd, 1H,
123

3718
Med Chem Res (2012) 21:3709–3719
Conc: of maltose liberated ꢁ Vol of enzyme used ðmlÞ ꢁ Dilution factor
Mol wt of maltose ꢁ Incubation time ðminÞ
Activity ¼
The inhibitory/induction property shown by the sample
was compared with that of control and expressed as percent
induction/inhibition. This was calculated according to the
following formula.
control. This test is applied to antihyperglycemic and
antioxidant assay.
Conclusions
Activity in presence of compound
% Inhibition ¼
ꢁ 100
Control activity
We have successfully ring transformed sydnone to 1,3,4-
oxadiazolin-3-one and further to triazolin-3-one moiety.
Ring transformation and ring insertion with ease and pro-
cedural simplicity are the key aspects of the synthesis. The
result of the antihyperglycemic screening reveals that
among all the title compounds, the compounds 3b, 3g, 3h
and 3i showed strong inhibition while the other compounds
3c, 3e, 3f, 3a and 3d displayed moderate to low inhibition.
T test analysis for amylase Inhibition Assay lies at 0.595
and hence the antihyperglycemic activity is excellent for
the title compounds. Antioxidant activity revealed three
compounds 3d and 3f have exhibited good activity. T test
for free radical scavenging activity lies at 0.29, 0.12 and
0.17, respectively, and the values are low as compared to
the control BHA and the title compounds show moderate to
low activity. In case of DNA cleavage activity it was
observed that compounds 3g and 3h exhibited complete
cleavage of DNA and other compounds have shown partial
cleavage.
Antioxidant activity [free radical scavenging activity
by 1,1-diphenyl-2-picryl hydrazyl (DPPH method;
Singh et al., 2002)]
Different concentrations (10, 50 and 100 lg) of sample in
DMSO and butylated hydroxy anisole (BHA), a synthetic
antioxidant were taken in different test tubes. The volume
was adjusted to 500 ll by adding methanol. Methanolic
solution (0.1 mM, 5 ml) of DPPH was then added to tubes
and were shaken vigorously. A control without the test
compound was maintained. The tubes were allowed to
stand at room temperature for 20 min. The absorbance of
the samples was measured at 517 nm. Radical scavenging
activity was calculated using the following formula
% Radical scavenging activity
ðControl absorbance ꢂ Sample absorbanceÞ
¼
ꢁ 100
Control absorbance
DNA cleavage activity (Sambrook et al., 1989)
Acknowledgements The authors are thankful to the University
Scientific Instrumentation Centre, Karnatak University Dharwad,
India for providing spectral as well as analytical data. One of the
authors (TT) thanks UGC, New Delhi for the award of RFSMS fel-
lowship to carry out the present work.
Nutrient broth was used for the growth of the organism.
The media (50 ml) was prepared, autoclaved for 15 min at
121°C, 15 lb pressure. The autoclaved media were inocu-
lated with the seed culture and incubated at 37°C for 24 h.
Isolation of DNA
References
DNA was isolated using the procedure as reported (Singh
et al., 2002). The synthesised compounds (100 lg) were
added separately to the DNA sample. The sample mixtures
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