Download Expedient synthesis of 1,2,4-triazolin-3

Indian Journal of Chemistry
Vol. 52B, June 2013, pp 794-801
Expedient synthesis of 1,2,4-triazolin-3-one derivatives as
DNA cleavage and antioxidant agents
Tasneem Taja, Ravindra R Kamble*a, Pramod P Kattimania & Sudha S Belgurb
a
Department of Studies in Chemistry, Karnatak University Dharwad 580 003, India
b
Department of Chemistry, Yuvaraja’s College, University of Mysore, Mysore 590 005, India
E-mail: [email protected]
Received 24 January 2012; accepted (revised) 5 April 2013
The present study involves the synthesis of fluoren-9-ylidene 5a-e and isoindolin-1,3-dione derivatives 5f-j appended
to 1,2,4-triazolin-2-one under solvent and neat conditions. The title compounds have been evaluated for the extent of cell
penetration through biological membranes (clogP) and drug score has been calculated and further screened for DNA
cleavage and antioxidant activities.
Keywords: Sydnone, fluorenone, isoindolin-1,3-dione, DNA cleavage, antioxidant
Sydnones are extensively studied mesoionic
compounds which have gained importance due to
their use as synthons for the ring transformations into
various heterocyclic systems1-4. Recent years have
witnessed a great deal of interest in the synthesis and
characterization of Schiff bases5. Several reports have
indicated that the presence of a lone pair of electrons
in sp2 hybridized orbital of nitrogen atom of the
azomethine group has considerable chemical and
biological importance6-12. Benzo[b] fluorenone
skeleton characterized by an uncommon diazo subunit belongs to a family of complex natural products
of kinamycins13-16. The lomaiviticins are glycosylated
dimeric analogues of the kinamycins and were
reported to have potent anti-cancer/antibiotic activity.
Lomaiviticin A was reported to cleave DNA under
reducing conditions17. Many research programmes
have been directed towards understanding the
mechanism by which the kinamycins/lomaiviticins
cleave DNA and subsequently harnessing this
information toward the design of simple analogues for
the
development
of
potential
anti-cancer
compounds18,19.
Several 4-substituted-isoindolinones were linked
through various spacers to adenosine and are reported
in literature as weak inhibitor of PARP activity20.
Interaction of transition metal complexes with DNA
has long been intensively investigated for applications
in molecular biology, biotechnology, and medicine21, 22.
Since DNA is the intracellular target in treating a
wide range of anticancer cells, binding of small
molecules with DNA is extremely useful in
understanding drug–DNA interactions. Several 2-(2hydroxybenzylideneamino)-isoindoline-1,3-dione transition metal complexes have been reported as DNA
cleavage agents23 and pthalidomide (2-(2,6-dioxo-3piperidyl-isoindoline-1,3-dione, Contergan)
was
developed as a sedative24 and is also effective in
cancer treatment25-27. Molecules containing a
dithiolane moiety are widely investigated due to their
antioxidant properties. The archetypal representative
of this class of compounds is lipoic acid and indeed
the lipoic acid, dihydrolipoic acid couple is part of the
antioxidant defense system of the cell28. The
deleterious effects of an imbalance between reactive
oxygen species (ROS) production and the available
antioxidant defense capacity, termed oxidative stress,
as well as its role in the aggravation of a plethora of
pathological conditions are also documented in the
literature29. A variable sensitivity to this excessive
oxidative state is observed between tissues and
organs, the brain being the most vulnerable due to its
high oxygen consumption, high content of unsaturated
membrane fatty acids and low level of antioxidant
enzymes or vitamins30. In the last decade much effort
has been directed towards finding molecules capable
of opposing the oxidative stress challenge31.
Many studies have been focused on the design of
analogues of fluorenones and isoindoline-1,3-dione
which possess DNA cleavage ability of pathogens and
TAJ et al.: SYNTHESIS OF 1,2,4-TRIAZOLIN-3-ONE
795
O
O
O
CH3 N N
N
HB
HX H
A
R
N
N
N
CH3
6a-e
X
O
R
O
N N
HX
O
O
CH3
NNH2
N
N
HB
HA
N
CH3 N N
N
3
CH3
HX HA
R
N
HB
N
CH3
2a-j
5a-e
4
O
O
CH3
R = H, p-OH, o-OH, p-NO2, p-OCH3
O
O
N N
HX
R
HB
HA
N N
N
N
CH3
3=
O
4=
O
O
5f-j
O
Scheme I — Synthesis of Schiff bases of fluorenone 5a-e and isoindolin-1,3-dione derivatives 5f-j in solvent
CH3
6a-e
X
5a-e
O
3
O
N N
NH2NH2.H2O
R
HX
O
N
HB
N
4
CH3
NH2NH2.H2O
5f-j
HA
1a-j
Scheme II — Synthesis of Schiff bases of fluorenone 5a-e and isoindolin-1,3-dione derivatives 5f-5j under neat condition
also which act as anti-oxidants. Owing to the above
literature survey and our zeal to explore newer
fluorenone-9-ylidene 5a-e and isoindoline-1,3-diones
5f-j, we herein report the synthesis, in vitro DNA
cleavage, antioxidant evaluations and drug score
values.
Results and Discussion
Starting materials viz., 3-(4-(1-acetyl-4,5-dihydro5-aryl-1H-pyrazol-3-yl)phenyl)-5-methyl-1,3,4-oxadiazol-2-(3H)-one 1a-j and 2-[4-(1-acetyl-5-aryl-4,5dihydro-1H-pyrazol-3-yl)-phenyl]-4-amino-5-methyl2,4-dihydro-[1,2,4]triazol-3-one 2a-j were prepared
according to reported method32. The title compounds
5a-j were synthesized by two methods. First method
involved the reaction of 2-[4-(1-acetyl-5-aryl-4,5dihydro-1H-pyrazol-3-yl)-phenyl]-4-amino-5-methyl2,4-dihydro-[1,2,4]-triazol-3-one 2a-j with fluorenone
3 and/or phthalic anhydride 4 in solvents viz., ethanol
and dry butanol respectively to yield the title
compounds 5a-j (Scheme I). By this method
moderate yield of 62-70% was observed.
In an alternative method, under neat condition 3-(4(1-acetyl-4,5-dihydro-5-aryl-1H-pyrazol-3-yl)phenyl)5-methyl-1,3,4-oxadiazol-2(3H)-one 1a-j was reacted
with hydrazine hydrate, fluorenone 3 and/or phthalic
anhydride 4 (as the case may be) in a single flask
operation to get directly the final compounds
5a-j (Scheme II). Interestingly, an appreciable yield
of 73-81% was observed. Both the methods differ in
INDIAN J. CHEM., SEC B, JUNE 2013
796
Table I — Characterization data of compounds 5a-j
Compd
Mol. formula
m.p. (°C)
Yield (g)
(%)
Method 1*
Yield (g)
(%)
Method 2#
5a
C33H26N6O2
103-104
0.530
65
0.577
78
5b
C33H26N6O3
123-24
0.539
67
0.583
79
5c
C33H26N6O3
143-44
0.504
62
0.549
75
5d
C33H25N7O4
135-36
0.480
70
0.515
72
5e
C34H28N6O3
155-56
0.510
68
0.535
74
5f
C28H22N6O4
123-25
0.482
70
0.511
73
5g
C28H22N6O5
132-33
0.481
63
0.519
75
5h
C28H22N6O5
143-44
0.495
65
0.526
76
5i
C28H21N7O6
125-27
0.513
69
0.515
81
5j
C29H24N6O5
138-39
0.468
70
0.500
73
Elemental analysis
Found % (Calcd)
C
H
N
73.59
(73.56
71.47
(71.45
71.47
(71.44
67.92
(67.89
71.82
(71.80
66.40
(66.38
64.36
(64.34
64.36
(64.35
60.98
(60.95
64.92
(64.89
4.87
4.85
4.73
4.71
15.60
15.62)
15.15
15.12)
4.73
4.74
4.32
4.30
4.96
4.94
15.15
15.17)
16.80
16.78)
14.78
14.75)
4.38
4.35
4.24
4.22
4.24
4.23
16.59
16.62)
16.08
16.05)
16.08
16.07)
3.84
3.82
4.51
4.53
17.78
17.75)
15.66
15.63)
*Triazole, #Oxadiazole
the reaction condition and reaction time. The yields
observed in both methods were given in Table I. It
was observed that the neat condition exhibited
excellent yields. However, attempts to synthesize
azetidinone derivatives from Schiff bases 5a-e were
failed which is due to steric hindrance of bulkier
fluorenone ring.
The structures of compounds 5a-i were confirmed
by elemental analysis, IR, 1H and 13C NMR and mass
spectra. In case of IR spectral analysis, the title
compounds 5a-j showed weak bands for CH stretching
around 2924-2853, 2930-2855 cm-1, C=N stretching for
the pyrazoline ring in the range 1630-1646 cm-1.
Compounds 5b, 5c, 5g, and 5h showed two broad
medium intensity bands in the range 3400 cm-1 due to
OH group. The 1H NMR spectra of the pyrazoline ring
(methine and methylene protons) have shown a
characteristic feature and form an ABX pattern. The
methylene protons can be assigned as HA, HB and the
methine proton as Hx. HA and HB are diastereotopic
and also anisochronous as they differ in chemical shifts
and since this difference is not large as they are
identified as AB protons. The methine proton on
adjacent carbon with larger shift downfield is the Hx
proton and hence all together form ABX pattern. The
HA and HB protons appear as doublet due to geminal
and vicinal coupling. These HA and HB differ in
coupling with the Hx and hence they are also
anisogamous. The HA proton appears as doublet of
doublet in the range δ 3.29-3.65 with two coupling
constants, JAB = 17.6 Hz and JAx = 4.5Hz. HB also
appears as doublet of doublet at δ 3.83-4.25 where JBA
is 17.6 Hz and JBX = 11.8 Hz. The Hx always appeared
as four line spectrum with JXA = 4.49 Hz and JXB =
11.84 in the range δ 4.25-5.50. A broad singlet for OH
in the compounds 5b, 5c, 5e and 5g appeared at δ 5.385.49 (D2O exchangeable). The aromatic protons appear
around δ 6.85-8.99. The compounds 5e and 5j showed
a singlet at δ 3.72-3.77 due to methoxy protons. The
13
C NMR spectral analysis of the title compounds
showed the number of signals consistent with number
of carbons which are magnetically different and the
mass spectra of the title compounds exhibited
molecular ion peaks at their respective molecular
weights which confirmed their formation.
Pharmacological Evaluation
Molecular Osiris property
For a new molecule to qualify as a drug candidate,
it has to be analyzed for the parameters set by
Lipinski’s rule of five using Osiris property explorer33-35.
TAJ et al.: SYNTHESIS OF 1,2,4-TRIAZOLIN-3-ONE
Lipinski’s rule of five is thumb rule to evaluate drug
score or determine if a new molecule with a certain
pharmacological or biological activity has properties
that would make it a likely orally active drug in
humans. The molecular properties are important for a
drug pharmacokinetics in human body, including their
absorption, distribution, metabolism and excretion
(ADME). The rule is important for drug development
where pharmacologically active lead structure is
optimized step wise for increased activity and
selectivity, as well as drug like properties as described
by Lipinski rule. The modification of the molecular
structure often leads to drugs with higher molecular
weight, more rings, more rotatable bonds and higher
lipophilicity. 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 partition co-efficient clogP less
than 5. Interestingly, it was observed that the title
compounds do not violate the Lipinski rule and they
fall well in the range as mentioned when evaluated by
the Osiris property explorer, and also the drug
Table II — Pharmacological parameters for bioavailability
Compd
clogP
Drug Score
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
6.96
6.66
6.66
6.83
6.85
3.18
2.88
2.88
3.05
3.08
0.06
0.06
0.06
0.06
0.06
0.13
0.13
0.13
0.10
0.12
797
likeliness in the range -12.36 to -0.15 and drug score
0.03-0.39 which will surely lead us to evaluate the
compounds experimentally Table II. Promising results
were observed for compounds 5f, 5g, 5h and 5j with
the drug score of 0.13. All the title compounds were
shown as safe drugs without any mutagenic,
tumourigenic, irritability and reproductive effects as
evaluated in Osiris property explorer.
DNA cleavage activity
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 the title compounds. The relative
efficacy of the drugs to stimulate DNA cleavage
varies considerably from one congener to another. A
detailed comparison between the compounds tested
allows us to make some significant observations.
The gel electrophoresis was used for the analysis of
5a-j and after treatment, it was observed that the DNA
treated with 5i and 5j (isoindolin-1,3-dione derivatives)
have cleaved the DNA completely. In both 5i and 5j
lane, with p-NO2 and p-OCH3 of the phenyl group,
whole band of DNA was disappeared indicating the
complete cleavage of DNA. DNA treated with 5j (with
p–OCH3 group) showed significant diminishing of the
band with a prominent streak indicating DNA cleavage
activity of compound. Only partial degradation activity
was found with any other sample in this gel with the
compounds 5e-h. Among the compounds 5a-d in
another gel also showed partial degradation of DNA as
shown in the Figure 1.
Figure 1 — DNA cleavage activity of compounds 5a-j
Note: In the photograph M- Standard DNA molecular weight marker (λ DNAEcoRI digest,
Banglore Genei, Bangalore) C- Control DNA (untreated sample)
INDIAN J. CHEM., SEC B, JUNE 2013
798
Figure 2 — Comparison of ferric reducing antioxidant power of 5a-j with butylated hydroxy anisole
Anti-oxidant activity
The FRAP assay was carried out to determine the
ferric reducing ability of biological fluids and aqueous
solutions of pure compounds. Butylated hydroxy
anisole (BHA) was used as a standard. Although the
final dilution of the sample in the reaction mixture
was high (100 µg), for any possible influence of the
solvent on the reaction was checked. Therefore,
aqueous solutions of the FeSO4.7H2O standard used
for calibration were prepared and analyzed with the
FRAP reagent. Ferrous sulfate dissolved well in both
solvents and there was no precipitation problem. The
values of absorbance at 700 nm compared to BHA
with concentrations of 10, 50, 100 µg are represented
in Figure 2. The compounds 5c, 5g and 5j
respectively with groups like o-OH and p-OCH3, have
shown very good activity compared to BHA, at all the
concentrations tested. 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, complexers of prooxidant metal ions and quenchers of singlet-oxygen
formation. Compounds 5a, 5e, 5f with phenyl and -OCH3
(isoindolin-1,3-dione) substituents have shown
moderate activity compared to the standard.
Compounds 5b, 5d, 5h and 5i have shown least
activity compared to standard BHA.
Experimental Section
Melting points of the synthesized compounds were
determined in open capillaries. The TLC was
performed on alumina silica gel 60 F254 (Merck).
The mobile phase was hexane and ethyl acetate (1:1)
and detection was made using UV light. IR spectra
were recorded using KBr pellets on Nicolet 5700 FTIR instrument. The 1H and 13C NMR were recorded
on Bruker Avance-300 (300 MHz) model spectrophotometer in CDCl3 and DMSO-d6 as solvents and
TMS as internal standard with 1H resonance
frequency of 300 MHz. The chemical shifts were
measured in ppm downfield from internal standard
TMS at δ = 0. Mass spectra were recorded on
Shimadzu Japan QP2010 S model spectrometer and
elemental analyses were carried out using Heraus
CHN rapid analyzer. The resulting compounds were
purified by recrystallization with either methanol or
ethanol. All the compounds gave C, H and N analysis
within +/- 0.5% of the theoretical values. DNA
cleavage and antioxidant evaluations were carried out
at Biogenics, Research and training centre in
Biotechnology, Hubli, Karnataka, India. The clog p,
drug likeliness and drug scores have been evaluated in
Osiris property explorer software for the structural
analogues of the synthesized compounds and are
uncorrected.
General procedure
Preparation of 4-(9H-fluoren-9-ylideneamino)-2(4-(1-acetyl-4,5-dihydro-5-aryl-1H-pyrazol-3-yl)phenyl)-5-methyl-2H-1,2,4-triazol-3(4H)-one, 5a-e
Method 1: Compound 2a (0.5 g, 1.38 mmol),
fluorenone 3 (0.237 g, 1.32 mmol) and ethanol (5 mL)
were refluxed at 98-100°C for about 6 hr on a water
bath. The reaction mixture after cooling was poured
into ice cold water and the solid mass separated was
recrystallized in ethanol to get pale yellow needles of
compound 5a. (Scheme I)
Method 2: Compound 1a (0.5 g, 1.32 mmol),
fluorenone 3 (0.234 g, 1.38 mmol) and hydrazine
TAJ et al.: SYNTHESIS OF 1,2,4-TRIAZOLIN-3-ONE
hydrate (5 mL) were refluxed at 130-35°C for 5 hr.
The reaction mixture was extracted with ether (3×5
mL), washed with brine solution and water. The
ethereal layer was dried over anhyd. Na2SO4, ether
was evaporated and the separated solid obtained was
recrystallized using ethanol to get the compound 5a.
(Scheme II). Similarly, the compounds 5b-e were
prepared according to this procedure.
4-(9H-Fluoren-9-ylideneamino)-2-(4-(1-acetyl4,5-dihydro-5-phenyl-1H-pyrazol-3-yl)phenyl)-5methyl-2H-1,2,4-triazol-3(4H)-one, 5a: Pale yellow
solid; IR (KBr): 2961, 2923, 2848 (CH), 1712, 1653
(C=O), 1603 (C=N) cm-1; 1H NMR (CDCl3): δ 2.35
(s, 3H, CH3), 3.06 (s, 3H, CH3), 3.62 (dd, 1H, CH2),
4.25 (dd, 1H, CH2), 5.50 (m, 1H, CH), 6.87-8.94 (m,
17H, Ar-H); 13C NMR (CDCl3): δ 22.80 (CH3), 23.54
(CH3), 39.90 (CH2), 58.68 (CH), 118.18, 118.35,
120.71, 121.34, 124.72, 125.93, 128.05, 129.29,
129.47, 129.73, 134.54 (Ar-C), 145.10 (C=N),
144.82, 194.34 (C=O); MS: m/z 538 (M+).
4-(9H-Fluoren-9-ylideneamino)-2-(4-(1-acetyl-4,
5-dihydro-5-(4-hydroxyphenyl)-1H-pyrazol-3-yl)phenyl)-5-methyl-2H-1,2,4-triazol-3(4H)-one, 5b:
Pale brown solid; IR (KBr): 2965, 2925, 2850 (CH),
1715, 1654 (C=O), 1605 (C=N) cm-1; 1H NMR
(CDCl3): δ 2.40 (s, 3H, CH3), 3.10 (s, 3H, CH3), 3.65
(dd, 1H, CH2), 4.20 (dd, 1H, CH2), 5.23 (m, 1H, CH),
5.45 (s, 1H, -OH), 7.01-8.84 (m, 16H, Ar-H); 13C
NMR (CDCl3): δ 23.84 (CH3), 24.55 (CH3), 38.90
(CH2), 56.68 (CH), 119.18, 119.32, 120.73, 121.46,
124.87, 125.83, 128.15, 129.30, 129.50, 129.78 (ArC), 134.60 (C-OH), 146.10 (C=N), 147.82, 195.34
(C=O); MS: m/z 554 (M+).
4-(9H-Fluoren-9-ylideneamino)-2-(4-(1-acetyl-4,
5-dihydro-5-(2-hydroxyphenyl)-1H-pyrazol-3-yl)phenyl)-5-methyl-2H-1,2,4-triazol-3(4H)-one, 5c:
Brown solid; IR (KBr): 2968, 2925, 2853 (CH), 1712,
1655 (C=O), 1601 (C=N) cm-1; 1H NMR (CDCl3): δ
2.44 (s, 3H, CH3), 3.12 (s, 3H, CH3), 3.63 (dd, 1H,
CH2), 4.15 (dd, 1H, CH2), 5.25 (m, 1H, CH), 5.50 (s,
1H, OH), 6.95-8.45 (m, 16H, Ar-H); 13C NMR
(CDCl3): δ 22.80 (CH3), 23.54 (CH3), 39.90 (CH2),
58.68 (CH), 118.18, 118.35, 120.71, 121.34, 124.72,
125.93, 128.05, 129.29, 129.47, 129.73, 134.54 (ArC), 137.60 (C-OH), 146.10 (C=N), 147.82, 198.34
(C=O); MS: m/z 554 (M+).
4-(9H-Fluoren-9-ylideneamino)-2-(4-(1-acetyl-4,
5-dihydro-5-(4-nitrophenyl)-1H-pyrazol-3-yl)phenyl)-5-methyl-2H-1,2,4-triazol-3(4H)-one, 5d: Pale
yellow solid; IR (KBr): 2963, 2935, 2855 (CH), 1717,
1657 (C=O), 1610 (C=N) cm-1; 1H NMR (CDCl3): δ
799
2.38 (s, 3H, CH3), 3.15 (s, 3H, CH3), 3.68 (dd, 1H,
CH2), 4.22 (dd, 1H, CH2), 5.27 (m, 1H, CH), 7.118.99 (m, 16H, Ar-H); 13C NMR (CDCl3): δ 24.84
(CH3), 25.55 (CH3), 39.90 (CH2), 57.68 (CH), 120.18,
120.32, 121.73, 122.46, 125.87, 126.83, 129.15,
130.30, 130.50, 130.78 (Ar-C), 136.60 (C-OH),
147.10 (C=N), 148.82, 197.34 (C=O); MS: m/z 583
(M+).
4-(9H-Fluoren-9-ylideneamino)-2-(4-(1-acetyl-4,
5-dihydro-5-(4-methoxyphenyl)-1H-pyrazol-3-yl)phenyl)-5-methyl-2H-1,2,4-triazol-3(4H)-one, 5e:
Yellow solid; IR (KBr): 2961, 2921, 2851, 1711,
1651 (C=O), 1601 (C=N) cm-1; 1H NMR (CDCl3): δ
2.38 (s, 3H, CH3), 3.09 (s, 3H, CH3), 3.64 (dd, 1H,
CH2), 3.72 (s, 3H, -OCH3), 4.18 (dd, 1H, CH2), 5.10
(m, 1H, CH), 7.05-8.85 (m, 16H, Ar-H); 13C NMR
(CDCl3): δ 22.85 (CH3), 23.57 (CH3), 37.89 (CH2),
57.69 (CH), 118.18, 119.32, 121.73, 122.46, 124.87,
126.83, 127.15, 128.30, 129.50, 129.83 (Ar-C),
133.62 (C-OH), 147.15 (C=N), 149.82, 197.38
(C=O); MS: m/z 568 (M+).
Preparation of 2-(1-(4-(1-acetyl-4,5-dihydro-5aryl-1H-pyrazol-3-yl)phenyl)-3-methyl-5-oxo-1H1,2,4-triazol-4(5H)-yl)isoindoline-1,3-dione, 5f-j
Method 1: Compound 1a (0.5 g, 1.38 mmol),
phthalic anhydride 4 (0.195 g, 1.32 mmol) and dry
butanol (5 mL) were refluxed at 120-21°C for 6hr.
The reaction mixture after cooling was poured to ice
cold water and the separated solid was recrystallized
using ethanol to yield the compound 5f (Scheme I).
Method 2: A mixture of compound 2a (0.5 g, 1.32
mmol), phthalic anhydride 4 (0.192 g, 1.38 mmol)
and hydrazine hydrate (5 mL) were refluxed at 13840°C for 5hr. The reaction mixture after cooling was
extracted with DCM (3×5 mL), washed with brine
solution and water. The organic layer was dried over
anhyd. Na2SO4, DCM was evaporated and the
separated solid was recrystallized using ethanol to
yield the compound 5f (Scheme II). Similarly, the
compounds 5b-e were prepared according to this
procedure.
2-(1-(4-(1-Acetyl-4,5-dihydro-5-phenyl-1H-pyrazol-3-yl)phenyl)-3-methyl-5-oxo-1H-1,2,4-triazol-4(5H)-yl)isoindoline-1,3-dione, 5f: Pale yellow solid;
IR (KBr): 2961, 2932, 2871, 1809 (CH), 1722, 1710,
1640 (C=O), 1606 (C=N) cm-1; 1H NMR (DMSO): δ
2.36 (s, 3H, CH3), 2.51 (s, 3H, CH3), 3.29 (dd, 1H,
CH2), 3.64 (dd, 1H, CH2), 4.25 (m, 1H, CH), 7.64-8.14
(m, 13H, Ar-H); 13C NMR (DMSO): δ 22.3 (CH3),
23.4 (CH3), 39.9 (CH2), 58.9 (CH), 121.7, 122.0, 126.8,
800
INDIAN J. CHEM., SEC B, JUNE 2013
127.1, 127.4, 127.6, 127.8, 128.6, 128.9, 129.4, 129.6,
130.0, 132.1, 132.5, 132.3, 132.7, 140.1, 143.5 (Ar-C),
151.0 (C=N), 154.0 (C=O), 155.0 (C=N), 164.8,
164.09, 168.3 (C=O); MS: m/z 506 (M+).
2-(1-(4-(1-Acetyl-4,5-dihydro-5-(4-hydroxyphenyl)-1H-pyrazol-3-yl)phenyl)-3-methyl-5-oxo-1H-1,
2,4-triazol-4(5H)-yl)isoindoline-1,3-dione,
5g:
Yellow solid; IR (KBr): 3447, 2961, 2924, 2848
(CH), 1800, 1742, 1640 (C=O), 1605 (C=N) cm-1; 1H
NMR (CDCl3): δ 2.14 (s, 3H, CH3), 2.55 (s, 3H, CH3),
3.49 (dd, 1H, CH2), 4.23 (dd, 1H, CH2), 4.28 (m, 1H,
CH), 5.38 (s, 1H, OH), 7.20-7.82 (m, 12H, Ar-H); 13C
NMR (CDCl3): δ 23.2, 25.3 (CH3), 40.9 (CH2), 57.9
(CH), 115.9, 121.8, 127.6, 132.1, 132.2, 136.1, 140.3
(Ar-C), 151.9 (C=N), 154.4 (C=O), 155.4 (C=O),
156.5 (C-OH), 164.8, 165.1, 168.9 (C=O); MS:
m/z 523 (M+).
2-(1-(4-(1-Acetyl-4,5-dihydro-5-(2-hydroxyphenyl)-1H-pyrazol-3-yl)phenyl)-3-methyl-5-oxo-1H-1,
2,4-triazol-4(5H)-yl)isoindoline-1,3-dione, 5h: Pale
yellow solid; IR (KBr): 3440, 2967, 2927, 2846 (CH),
1807, 1740, 1642 (C=O), 1600 (C=N) cm-1; 1H NMR
(CDCl3 d6): δ 2.20 (s, 3H, CH3), 2.56 (s, 3H, CH3),
3.50 (dd, 1H, CH2), 4.25 (dd, 1H, CH2), 4.32 (m, 1H,
CH), 5.49 (s, 1H, OH), 7.25-7.92 (m, 12H, Ar-H); 13C
NMR (CDCl3): δ 23.5, 25.5 (CH3), 41.9 (CH2), 58.9
(CH), 115.5, 121.9, 127.4, 132.4, 132.8, 136.3, 140.8
(Ar-C), 152.1 (C=N), 154.6 (C=O), 155.7 (C=O),
156.9 (C-OH), 164.9, 165.3, 169.0 (C=O); MS:
m/z 523 (M+).
2-(1-(4-(1-Acetyl-4,5-dihydro-5-(4-nitrophenyl)1H-pyrazol-3-yl)phenyl)-3-methyl-5-oxo-1H-1,2,4triazol-4(5H)-yl)isoindoline-1,3-dione, 5i: Pale
brown solid; IR (KBr): 2963.8 (CH), 1747, 1715,
1646 (C=O), 1607 (C=N) cm-1; 1H NMR (DMSO): δ
2.09 (s, 3H, CH3), 2.51 (s, 3H, CH3), 3.37 (dd, 1H,
CH2), 3.83 (dd, 1H, CH2), 4.24 (m, 1H, CH), 7.218.24 (m, 12H, Ar-H); 13C NMR (DMSO): δ 38.63,
38.84 (CH3), 39.05 (CH2), 39.88 (CH), 106.17,
114.63, 125.54, 125.61, 129.23, 129.89, 129.98 (ArC), 130.23 (C=N), 132.35, 133.07 (C=O), 133.24 (CNO2), 139.84, 148.85, 163.85 (C=O); MS: m/z 551
(M+).
2-(1-(4-(1-Acetyl-4,5-dihydro-5-(4-methoxyphenyl)-1H-pyrazol-3-yl)phenyl)-3-methyl-5-oxo-1H1,2,4-triazol-4(5H)-yl)isoindoline-1,3-dione,
5j:
Brown solid; IR (KBr): 2966, 2923, 2854, 2300 (CH),
1739, 1725, 1644 (C=O), 1606 (C=N) cm-1; 1H NMR
(CDCl3): δ 2.30 (s, CH3), 2.42 (s, CH3), 3.39 (dd,
CH2), 3.77 (s, -OCH3), 3.86 (dd, CH2), 4.32 (m, CH),
6.85-8.05 (m, Ar-H); 13C NMR (CDCl3): δ 23.20,
25.35 (CH3), 41.9 (CH2), 55.90 (-OCH3), 58.9 (CH),
114.93, 122.82, 126.64, 131.14, 131.25, 135.10,
141.30 (Ar-C), 150.9 (C=N), 155.40 (C=O), 154.40
(C=O), 156.52 (C-OH), 164.91, 165.16, 168.94
(C=O); MS: m/z 536 (M+).
Biological evaluation assays
DNA Cleavage analysis36
Culture media
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 inoculated with the seed
culture and incubated at 37°C for 24 hr.
Isolation of DNA
DNA was isolated using the procedure mentioned
below.
The bacterial culture (1.5 mL) was centrifuged to
obtain the pellet. The pellet was then dissolved in
lysis buffer (0.5 mL, 100 mM tris pH 8.0, 50 mM
EDTA, 50 mM lysozyme) followed by addition of
saturated phenol (0.50 mL) and incubated at 55°C for
10 min. This mixture was centrifuged at 10,000 rpm
for 10 min and to the supernatant liquid equal volume
of chloroform: isoamyl alcohol (24:1) and 1/20th
volume of 3M sodium acetate (pH 4.8) were added.
Then the mixture was centrifuged at 10,000 rpm for
10 min and to the supernatant liquid three volumes of
chilled absolute alcohol was added. The precipitated
DNA was separated by centrifugation. The pellet was
dried and dissolved in Tris buffer (10 mM tris pH 8.0)
and stored under cold condition.
Treatment of DNA with the samples
The synthesized compounds (100 µg) were added
separately to the DNA sample. The sample mixtures
were incubated at 37°C for 2 hr.
Agarose gel electrophoresis
Following the treatment of DNA samples, the
electrophoresis of the samples was done according to
the following procedure.
Agarose (200 mg) was dissolved in TAE buffer (25
mL) (4.84 g Tris base, pH 8.0, 0.5 M EDTA/1 L) by
boiling. When the gel attained ≈ 55°C, it was poured
into the gel cassette fitted with comb. The gel was
then allowed to solidify. The comb was carefully
TAJ et al.: SYNTHESIS OF 1,2,4-TRIAZOLIN-3-ONE
removed and the gel was placed in the electrophoresis
chamber flooded with TAE buffer. DNA sample (20
µL, mixed with bromophenol blue dye at 1:1 ratio),
was loaded carefully into the wells, along with
standard DNA marker and constant 50 V of electricity
was passed for around 45 min. The gel was removed
and carefully stained with ETBR solution (10 µg/mL)
for 10-15 min and the bands were observed under UV
transilluminator.
Anti-oxidant analysis37
Various concentrations of samples (10µg, 50µg,
100µg) were mixed with 200 mM sodium phosphate
buffer (2.5 mL, pH 6.6) and potassium ferricyanide
(1%, 2.5 mL). The mixture was incubated at 50ºC for
20 min. Trichloroacetic acid. (10%, 2.5 mL) (w/v)
were added; above solution (5.0 mL) was mixed with
distilled water (5.0 ml) and ferric chloride (0.1%, 1
mL).
The
absorbance
was
measured
spectrophotometrically at 700 nm. BHA was used as
standard antioxidant.
Conclusions
A series of novel fluoren-9-ylidene 5a-e and isoindolin-3,4-dione 5a-j with ease under neat conditions
were prepared and analysed for their drug likeliness,
drug score values. Further, these results were
substantiated by DNA cleavage and antioxidant
property. In case of DNA cleavage activity, the
compounds 5i and 5j cleaved DNA completely.
Antioxidant activity revealed the potent activity of the
compounds 5c, 5g and 5j as compared to BHA.
Acknowledgements
The authors are thankful to the University
Scientific Instrumentation Centre (USIC), K U
Dharwad, for providing spectral as well as analytical
data and to Prof. (Mrs) Bharathi V. Badami for the
encouragement. One of the authors (TT) thanks UGC,
New Delhi for the award of RFSMS fellowship to
carry out the present work.
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