Synthesis, antimicrobial and antioxidant activities of some new indole derivatives containing pyridopyrimidine and pyrazolopyridine moieties

Article abstract of DOI:10.1007/s00044-012-0066-2

New indole analogues containing pyrido[2,3-d]pyrimidin-4-(3H)-ones (3a-i), pyrazolo[3,4-b]pyridin-3-amines (4a-i) and pyrido[2,3-d]pyrimidin-4-amines (5a-i) were synthesized using appropriate synthetic routes. These newly synthesized compounds were screen

Full text of DOI:10.1007/s00044-012-0066-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:806–817
DOI 10.1007/s00044-012-0066-2
ORIGINAL RESEARCH
Synthesis, antimicrobial and antioxidant activities of some new
indole derivatives containing pyridopyrimidine
and pyrazolopyridine moieties
•
Anand R. Saundane Katkar Vijaykumar
•
•
A. V. Vaijinath Prabhaker Walmik
Received: 23 November 2011 / Accepted: 18 April 2012 / Published online: 11 May 2012
Ó Springer Science+Business Media, LLC 2012
Abstract New indole analogues containing pyrido[2,
3-d]pyrimidin-4-(3H)-ones (3a–i), pyrazolo[3,4-b]pyridin-
3-amines (4a–i) and pyrido[2,3-d]pyrimidin-4-amines (5a–
i) were synthesized using appropriate synthetic routes.
These newly synthesized compounds were screened for
their antimicrobial and antioxidant activities. Compounds
3a, 3g, 4c, 4h, 4i, 5a, 5c and 5f exhibited good antibacterial
and antifungal activities. The compounds 3b, 3c, 3g, 4a,
4h, 4i and 5a exhibited good radical scavenging activity.
Compounds 3a and 4d exhibited good metal chelating
activity compare with standards.
indole derivatives have been shown to exhibit antimicrobial
(Elisabete et al., 1996; Vijai Nath et al., 2008; Joseph et al.,
2008), anticancer (Rufine et al., 2009), antidiabetic (James
et al., 2005), anti-inflammatory (Eva-Maria et al., 2009), anti-
HIV (Silvestri et al., 1998), antitumor (Maria-Joao et al., 2008),
gastric ulcer (Fathi et al., 2011) activities. Pyridine is one of the
most popular N-heteroaromatic incorporated into the structure
of many pharmaceuticals. Many of the pyridine and pyrimidine
analogues have evaluated pharmacologically and found to
exhibit antioxidant activity, for example pyridopyrimidines 5
(Suresh et al., 2010), pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyr-
imidin-5-ones 6 (Thoraya et al., 2011), pyrazolo [4,3-c]pyri-
dines 7, pyrido[4,3-d]pyrimidin-2-thiones 8 (Mohamed et al.,
2005). Also anticancer (Ekhlass, 2010), antitumor (Harinadha
et al., 2004), urease inhibition (Abdul et al., 2010) and anti-
microbial (Aliye Altundas et al., 2011) activities have been
reported (Chart 1).
Keywords Indole analogues ꢀ Pyridine ꢀ
Pyridopyrimidine ꢀ Pyrazolopyridine ꢀ Antimicrobial ꢀ
Antioxidant
Introduction
In view of these findings and in continuation of our
ongoing search for new heterocycles (Saundane et al.,
2011a, b, c; Saundane and Veeresh Sharma, 2004) of
biological importance, we have synthesized the title com-
pounds and screened them for their antimicrobial and
antioxidant activities.
Nitrogen-containing heterocyclic compounds is one of the most
fruitful and extensively developing fields of heterocyclic
chemistry. These compounds exhibit various kinds of biologi-
cal activities. The biological activities of several analogues of
indole have been reported in the literature. For example,
6-hydroxy indole-3-acetamide 1 and 6-hydroxy indole-3-
carboxyaldehyde 2 (Won-gon et al., 1997), melatonin 3 (Deepa
et al., 2007), indolyl chromens 4 (Gnanamani et al., 2009) have
been reported to possess antioxidant activity. Apart from this
Results and discussion
The pathway for the synthesis of title compounds is illus-
trated in Scheme 1. The starting material 3-(50-substituted
20-phenyl-1H-indol-30-yl)-1-phenylprop-2-en-1-ones (1a–i)
were synthesized from indole-3-carboxyaldehydes (Hire-
math et al., 1982) and 4-substituted acetophenones in the
presence of piperidine. Compounds (1a–i) were subjected to
cyclocondensation with malononitrile and ammonium ace-
tate to afford 2-amino-3-cyano-4-(50-substituted 20-phenyl-
1H-indol-30-yl)-6-(4-substituted phenyl)pyridines (2a–i).
A. R. Saundane (&) ꢀ K. Vijaykumar ꢀ A. V. Vaijinath ꢀ
P. Walmik
Department of Post-Graduate Studies and Research in
Chemistry, Gulbarga University, Gulbarga 585 106,
Karnataka, India
e-mail: arsaundane@rediff.com
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Med Chem Res (2013) 22:806–817
807
Chart 1 Motivation for
synthesis of antioxidant active
compounds
R1
N
R3
R2
CH2CH2NHCOCH3
CHO
CONH2
H3CO
CN
N
N
N
HO
HO
O
4
NH2
H
H
H
2
3
1
HO
R1
HO
R1
N
N
N
R
H
N
S
N
N
5
N
NH
N
R2
R2
R
N
O
R'
N
R
N
R
N
N
OH
OH
Ar
6
7
8
Compound (2a) on heterocyclization with formic acid in
refluxing ethanol yielded 5-(50-chloro-20-phenyl-1H-indol-30-
yl)-7-(4-chlorophenyl)pyrido[2,3-d]pyrimidin-4-(3H)-one
(3a). The IR spectrum of compound 3a showed absorption
bands at 3,445 (indole-NH), 3,050 (NH), 1,705 (C=O) cm,-1
whereas absorption due to CN function was missing. In its 1H
NMR spectrum, the signals revealed at d, ppm 12.50 (s, 1H,
indole-NH), 10.00 (s, 1H, pyrimidine-NH), 8.80 (s, 1H,
pyrimidine-H), 8.20 (s, 1H, pyridine-H), 7.82 (s, 1H, indole-
H4), 7.75 (d, 1H, indole-H6), 7.60 (d, 1H, indole-H7),
7.30–7.55 (m, 9H, Ar–H). Its mass spectrum exhibited iso-
topic molecular ion peak at m/z 482 (M?) and 486 (M??4).
Compound (2a) on cyclocondensation with hydrazine
hydrate in alcohol at reflux temperature afforded 4-(50-
chloro-20-phenyl-1H-indol-30-yl)-6-(4-chlorophenyl)-1H-
pyrazolo[3,4-b]pyridin-3-amine (4a). The IR spectrum of 4a
exhibited absorption bands at 3,432 (indole-NH), 2,956
(NH2), 2,917 (NH) cm-1 and disappearance of absorption
whereas absorption due to CN function was missing. In its
1H NMR spectrum, signals due to different protons were
resonated at d, ppm 12.60 (s, 1H, indole-NH), 8.70 (s, 1H,
pyrimidine-H), 8.30 (s, 1H, pyridine-H), 7.85 (s, 1H,
indole-H4), 7.70 (d, 1H, indole-H6), 7.63 (d, 1H, indole-
H7), 7.40–7.60 (m, 9H, Ar–H), 6.00 (s, 2H, NH2). The mass
spectrum of 5a exhibited isotopic molecular ion peak at m/
z 481 (M?) and 485 (M??4).
Biological results
Antimicrobial activity
All the synthesized compounds (3–5) were evaluated for
their antibacterial activity against Escherichia coli
(MTCC-723), Staphylococcus aureus (ATCC-29513),
Klebsiella penumoniae (NCTC-13368) and Pseudomonas
aeruginosa (MTCC-1688) and antifungal activity against
Aspergillus nizer (MTCC-281), Aspergillus oryzae
(MTCC-3567T), Aspergillus terrus (MTCC-1782) and
Aspergillus flavus (MTCC-1973) by cup-plate method at a
concentration 500 lg/mL. The zone of inhibition (in mm)
was compared with standards streptomycin and fluconazole
for antibacterial and antifungal activities, respectively. The
results are tabulated in Table 1.
1
due to CN functional group was noticed. In the H NMR
spectrum, the various signals were resonated at d, ppm 11.80
(s, 1H, indole NH), 8.70 (s, 1H, pyrazole-NH), 7.90 (s, 1H,
pyridine-H), 7.85 (s, 1H, indole-H4), 7.81 (d, 1H, indole-H6),
7.79 (d, 1H, indole-H7), 7.40–7.65 (m, 9H, Ar–H), 4.60 (s,
2H, NH2). The mass spectrum of 4a showed isotopic
molecular ions peak at m/z 469 (M?) and 473 (M??4).
Thus, confirmed the formation of compound 2a from 4a.
Also, compound (2a) on annulation with formamide
yielded, 5-(50-chloro-20-phenyl-1H-indol-30-yl)-7-(4-chlo-
rophenyl)pyrido[2,3-d]pyrimidin-4-amine (5a). Compound
5a in its IR spectrum exhibited characteristic absorption
The investigation of antibacterial screening revealed
that, compounds 3g and 4g exhibited maximum zone of
inhibition against K. pneumoniae. Compounds 3a and 5c
showed good zone of inhibition against P. aeruginosa. The
compounds 4c, 4g and 5a showed maximum zone of
bands at 3,438 (indole NH) and 2,921 (NH2) cm-1
,
123

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Med Chem Res (2013) 22:806–817
Scheme 1 Schematic pathway
for the synthesis of compounds
2–5
O
R
R'
N
H
1a-i
CH2(CN)2/
CH3COONH4
HN
R
CN
N
NH2
R'
2a-i
HCONH2
N2H4.H2O
HCOOH
R
HN
R
O
R
HN
HN
NH2
NH2
N
N
NH
N
H
N
N
N
N
N
R'
4a-i
3a-i
5a-i
R'
R'
Where
a
b
c
d
e
f
g
h
i
R = Cl, CH3, H, Cl, CH3, H, Cl, CH3 ,
H
R' = Cl, Cl, Cl, CH3, CH3, CH3, H, H, H,
inhibition against S. aureus. Compound 3g showed good
zone of inhibition against E. coli.
antioxidant reduces the risk for chronic diseases including
cancer and heart disease. The main characteristic of an anti-
oxidant is its ability to trap free radical. The scavenging of the
stable 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical model is
widely used method to evaluated antioxidant activities in rel-
atively short time compared with other methods. DPPH is
stable free radical that can accept an electron or hydrogen atom
and convert them into a stable diamagnetic molecule. DPPH
has an odd electron and so has strong absorption band at
517 nm. All compounds were tested for their interaction with
stable free radical DPPH. This interaction indicates their radical
scavenging activity (RSA). The RSA of synthesized com-
pounds was compared with the standards 2-tert-butyl-4-meth-
oxy phenol (butylated hydroxyl anisole, BHA), 2-(1,1-
dimethylethyl)-1,4-benzenediol (tertiary butylated hydroqui-
none, TBHQ) and ascorbic acid (AA). The results are shown in
Figs. 1, 2 and 3.
On the other hand, antifungal activity of test compounds
revealed that, compounds 3a, 3g, 4h and 4i exhibited good
zone of inhibition against A. oryzae. Compounds 3g, 4g
and 5a showed good zone of inhibition against A. nizer,
A. terrus and A. flavus, respectively. From the results of
antimicrobial activities, it could be assumed that, the
majority of synthesized compounds having chloro substi-
tution exhibited maximum growth inhibitory activity. The
electronegative nature of the chloro group may be
responsible to inhibit the growth of the microbes.
Antioxidant activities
Radical scavenging activity (RSA)
Antioxidant compounds in food play an important role
as health-protein factor. Scientific evidence suggests that
The analysis of results indicated that, compounds 3d, 3e and
5c exhibited the good RSA 61.55, 74.37 and 66.85 %,
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Med Chem Res (2013) 22:806–817
809
Table 1 Antimicrobial activity results of compounds 3–5
Comp.
Antibacterial activity (zone of inhibition in mm)
Antifungal activity (zone of inhibition in mm)
E. coli
S. aureus
K. penumoniae
P. aeruginosa
A. nizer
A. oryzae
A. terrus
A. flavus
3a
3b
3c
3d
3e
3f
11
08
10
10
05
10
12
05
10
10
07
10
06
10
10
08
08
10
11
06
08
09
05
10
09
08
05
13
–
11
09
10
08
08
08
05
06
02
10
10
14
10
11
11
12
10
08
12
08
10
10
10
11
10
10
10
13
–
11
06
08
11
12
11
14
09
10
11
10
10
09
05
09
13
09
10
10
10
05
09
06
11
05
06
10
13
–
13
08
02
05
10
10
08
10
10
06
08
08
09
06
09
05
05
11
11
11
02
09
05
10
08
05
09
13
–
10
11
10
11
10
06
12
09
08
06
10
10
08
10
05
05
02
10
08
10
10
08
10
10
10
08
10
–
14
08
10
10
08
06
12
08
10
10
08
10
10
03
10
10
14
12
08
08
02
05
08
10
10
06
09
–
08
09
11
09
10
10
10
10
08
10
02
08
11
09
02
14
05
08
08
05
10
04
05
08
05
10
05
–
03
08
11
08
05
05
09
10
11
08
11
11
05
11
08
10
06
06
12
11
08
10
05
08
08
11
10
–
3g
3h
3i
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
5g
5h
5i
Std1
Std2
14
14
14
14
Std1 streptomycin, Std2 flucanazole
respectively, at concentration 25 lg/mL. Compounds 3b, 4i
and 5g showed 72.70, 75.48 and 77.99 % radical scavenging
ability at concentration 50 lg/mL, respectively. Compound 3g
exhibited RSA 74.09 and 78.83 % at concentration 75 and
100 lg/mL, respectively. The compounds 4a and 4h exhibited
73.53and75.20 % RSA at 75 lg/mL, respectively, whereas 4h
showed 68.24 % RSA at concentration 100 lg/mL. The higher
activity of compound 3g may be due to presence of enolizable
amide group in pyrimidine ring, which leads to the aromatic
system. The probable mechanism for hydrogen donation to
DPPH free radical and stabilization of free radical formed is
shown in Schemes 2and 3for compound 3g. However, none of
the compounds exhibited RSA better than the standards.
Fig. 1 Antioxidant activity of compounds (3a–i)
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Med Chem Res (2013) 22:806–817
Iron metal ions chelating activity
agents, followed by clinical experience, with regard to
monitoring metal excretion and status of tissue damage
(Flora et al., 2008). Metal chelating capacity was signifi-
cant, since it reduces the concentration of the catalyzing
transition metal in lipid peroxidation. It was reported that
chelating agents, which form r-bonds with a metal, are
effective as secondary antioxidants because they reduce
the redox potential therapy stabilizing the oxidized form of
metal ion (Gulcin et al., 2007). Transition metals (iron,
Extensive experience demonstrates that acute and chronic
human intoxications with a wide range of metals can be
treated with considerable efficiency by the administration
of a relevant chelating agent. Development of effective
chelating agent is based on combinations of chemical
consideration and whole animal experimentation on the
toxicokinetics and toxicodynamics of metal and chelating
Fig. 2 Antioxidant activity of compounds (4a–i)
Scheme 2 Probable
Fig. 3 Antioxidant activity of compounds (5a–i)
Cl
Cl
mechanism for H-donation to
HN
HN
DPPH radical and stabilization
of free radical formed from
pyrimidine systems
O
OH
H
N
N
N
N
N
N
3g
DPPH
Cl
Cl
HN
HN
O
N
O
N
DPPH-H
N
etc
N
N
N
Cl
HN
O
N
N
N
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Med Chem Res (2013) 22:806–817
811
Scheme 3 Probable
N
HN
mechanism for H-donation to
DPPH radical and stabilization
of free radical formed from
indole-NH
N
HN
N
O
N
Cl
O
Cl
DPPH
DPPH-H
N
N
H
3g
N
HN
N
HN
N
O
N
O
Cl
Cl
etc
N
N
N
HN
N
O
Cl
N
H
Fig. 4 Reducing activity of compounds (3a–i)
Fig. 5 Reducing activity of compounds (4a–i)
Fe2þ + H2O2 ! Fe3þ + OHꢁ + OH
Fe3? ion also produces radical from peroxides, although
the rate is 10-fold less than that of Fe?2 ion, which is the
most powerful pro-oxidant among the various types of
metal ions (C¸ alis¸ et al., 1993).
copper, chromium, cobalt, vanadium, cadmium and nickel)
can also be mediators in the formation of free radicals
(Marijana et al., 2009). Among the transition metal ions,
iron is known as the most important lipid oxidation pro-
oxidant due to its high reactivity. Iron can stimulate lipid
peroxidation by the Fenton reaction and also accelerates
peroxidation by decomposing lipid hydroperoxides into
peroxy and alkoxy radicals that can themselves abstract
hydrogen and perpetuate the chain reaction of lipid perox-
idation (Gulcin et al., 2005; Halliwell, 1991).
Ferric ions (Fe3?) reducing antioxidant power (FRAP)
The ferric ion (Fe3?) is the relatively biological inactive
form of iron. However, it can be reduced to the active Fe2?
,
depending on condition, particularly pH (Strlic et al., 2002)
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Med Chem Res (2013) 22:806–817
Fig. 6 Reducing activity of compounds (5a–i)
Fig. 9 Metal chelating activity of compounds (5a–i)
increasing concentration of test compounds (25–100 lg/
mL) and increased absorbance at 700 nm indicates
increased in reducing ability. The results are shown in
Figs. 4, 5 and 6. The results indicated that, only the com-
pounds 4d and 4h exhibited moderate reducing power
activity. This activity may be due to chloro and methyl
group attached to fused pyridine system.
Ferrous ions (Fe2?) chelating activity The chelating
effect of ferrous ions (Fe2?) towards the test compounds
(3–5) and standards was determined by following reported
method (Dinis et al., 1994). Ferrozine can make complexes
with ferrous ions. In the presence of chelating agents,
complex (red coloured) formation is interrupted and as a
result, the red colour of the complex is decreased. Thus, the
chelating effect of the coexisting chelator can be deter-
mined by measuring the rate of colour reduction. The
chelating activity results of test compounds are shown in
the Figs. 7, 8 and 9. The compounds 3a, 3i and 4c exhib-
ited good metal chelating activity, whereas other com-
pounds exhibited either moderate or poor chelating activity
Fig. 7 Metal chelating activity of compounds (3a–i)
Conclusion
Fig. 8 Metal chelating activity of compounds (4a–i)
This study revealed that fused pyridine system containing
the chloro substitutions are founds to exhibit good anti-
microbial and antioxidant activities compared to pyridine
moiety.
and oxidized back through Fenton type reaction with pro-
duction of hydroxyl radical or Haber–Weiss reaction with
superoxide anions. Reducing power is to measure the
reductive ability of antioxidant and it is evaluated by the
transformation of Fe3? to Fe2? by donation of an electron,
in the presence of test compounds. Therefore, the Fe2? can
be monitored by measuring the formation of Perl’s Prussian
blue at 700 nm.
Experimental procedures
Chemistry
Materials and methods
The FRAP of synthesized compounds (3–5) was deter-
mined at concentrations (25, 50, 75 and 100 lg/mL) at
pH 6.6 using literature method (Oyaizu, 1986). Thus,
the reducing power of test compounds increases with
All the reagents were obtained commercially and used by
further purification. Melting points were determined by an
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Med Chem Res (2013) 22:806–817
813
open capillary method and are uncorrected. Purity of the
compounds was checked by TLC using silica gel-G coated
aluminum plates (Merck) and spots were visualized by
exposing the dry plates in iodine vapours. The IR (KBr)
spectra were recorded with a Perkin-Elmer spectrum one
FT-IR spectrometer. The 1H NMR (DMSO-d6) spectra
recorded with on Bruker NMR (500 MHz) and the chem-
ical shifts were expressed in ppm (d scale) downfield from
TMS. Mass spectra were recorded with a JEOL GCMATE
II GC–MS mass spectrometer. Elemental analysis carried
out using Flash EA 1112 series elemental analyzer.
5-(20-Phenyl-1H-indol-30-yl)-7-(4-chlorophenyl)pyrido[2,
3-d]pyrimidin-4(3H)-one (3c): Yield: 62 %, mp 203–204
°C, Rf 0.42 ethyl acetate: benzene (9:1) mixture, FTIR (KBr)
1
cm-1: 3,423 (indole NH); 3,100 (NH); 1,700 (C=O); H
NMR (DMSO-d6, d ppm): 12.01 (s, 1H, indole-NH); 10.00
(s, 1H, pyrimidine-NH); 9.00 (s, 1H, pyrimidine-H); 7.85 (s,
1H, pyridine-H); 7.41–7.60 (m, 13H, Ar–H); Anal. %
C27H17N4ClO: C, 72.24; H, 3.82; N, 12.48. Found: C, 72.35;
H, 3.98; N, 12.36.
5-(50-Chloro-20-phenyl-1H-indol-30-yl)-7-(4-methylphenyl)
pyrido[2,3-d]pyrimidin-4(3H)-one (3d): Yield: 64 %, mp 248–
249 °C, Rf 0.60 ethyl acetate: benzene (9:1) mixture, FTIR
(KBr) cm-1: 3,400 (indole-NH); 3152 (NH); 1,693 (C=O); 1H
NMR (DMSO-d6, d ppm): 12.53 (s, 1H, indole-NH); 10.11
(s, 1H, pyrimidine-NH); 8.89 (s, 1H, pyrimidine-H); 8.10
(s, 1H, pyridine-H); 7.82 (s, 1H, indole-H4); 7.71 (d, 1H,
indole-H6); 7.62 (d, 1H, indole-H7); 7.40–7.63 (m, 9H, Ar–H);
2.26 (s, 3H, CH3); Anal. % C28H19N4ClO: C, 72.65; H, 4.14; N,
12.10. Found: C, 72.78; H, 4.39; N, 11.96.
5-(50-Methyl-20-phenyl-1H-indol-30-yl)-7-(4-methylphenyl)
pyrido[2,3-d]pyrimidin-4(3H)-one (3e): Yield: 68 %, mp 222–
223 °C, Rf 0.42 ethyl acetate: benzene (9:1) mixture, FTIR
(KBr) cm-1: 3,444 (indole-NH); 3,211 (NH); 1,699 (C=O); 1H
NMR (DMSO-d6, d ppm): 12.12 (s, 1H, indole-NH); 10.01 (s,
1H, pyrimidine-NH); 9.00 (s, 1H, pyrimidine-H); 7.83 (s, 1H,
pyridine-H); 7.45–7.61 (m, 9H, Ar–H); 7.33 (s, 1H, indole-H4);
7.15 (d, 1H, indole-H6); 7.00 (d, 1H, indole-H7); 2.31 (s, 3H,
CH3); 2.26 (s, 3H, CH3); Anal. % C29H22N4O: C, 78.71; H,
5.01; N, 12.66. Found: C, 78.62; H, 5.32; N, 12.52.
5-(20-Phenyl-1H-indol-30-yl)-7-(4-methylphenyl)pyrido[2,
3-d]pyrimidin-4(3H)-one (3f): Yield: 74 %, mp 215–216 °C,
Rf 0.56 ethyl acetate: benzene (9:1) mixture, FTIR (KBr)
cm-1: 3433 (indole-NH); 3211 (NH); 1700 (C=O); 1H NMR
(DMSO-d6, d ppm): 12.10 (s, 1H, indole-NH); 10.00 (s, 1H,
pyrimidine-NH); 8.74 (s, 1H, pyrimidine-H); 8.10 (s, 1H,
pyridine-H); 7.35–7.80 (m, 13H, Ar–H); 2.30 (s, 3H, CH3);
Anal. % C28H20N4O: C, 78.49; H, 4.70; N, 13.08. Found: C,
78.58; H, 4.78; N, 13.15.
General procedure for the synthesis of 3-(50-substituted 20-
phenyl-1H-indol-30-yl)-1-phenyl prop-2-en-1-ones (1a–i)
These compounds were prepared by reported method
(Tsukarman et al., 1969).
General procedure for the synthesis of 2-amino-3-cyano-4-
(50-substituted 20-phenyl-1H-indol-30-yl)-6-(4-substituted
phenyl)pyridines (2a–i) These compounds were prepared
by reported method (Saundane et al., 2011b).
General procedure for the synthesis of 5-(50-substituted 20-
phenyl-1H-indol-30-yl)-7-(4-substituted phenyl)pyrido[2,3-d]
pyrimidin-4(3H)-ones (3a–i) A solution of 2-amino-3-cy
ano-4-(50-substituted 20-phenyl-1H-indol-30-yl) -6-(4-sub
stituted phenyl)pyridines (2a–i) (0.01 mol) in formic acid
(8 mL) was heated under reflux for 6 h. The excess of for-
mic acid was removed under reduced pressure. After cooling
the product separated was filtered, washed with cold etha-
nol, dried, and crystallized from ethanol.
5-(50-Chloro-20-phenyl-1H-indol-30-yl)-7-(4-chlorophenyl)
pyrido[2,3-d]pyrimidin-4(3H)-one (3a): Yield: 66 %, mp
174–175 °C, Rf 0.76 ethyl acetate: benzene (9:1) mixture,
FTIR (KBr) cm-1: 3445 (indole-NH); 3050 (NH); 1705
1
(C=O); H NMR (DMSO-d6, d ppm): 12.50 (s, 1H, indole-
NH); 10.00 (s, 1H, pyrimidine-NH); 8.80 (s, 1H, pyrimidine-
H); 8.20 (s, 1H, pyridine-H); 7.82 (s, 1H, indole-H4); 7.75 (d,
1H, indole-H6); 7.60 (d, 1H, indole-H7); 7.30–7.55 (m, 9H, Ar–
H); MS (EI) m/z 482 (M?); 486 (M??4). Anal. %
C27H16N4OCl2: C, 67.09; H, 3.34; N, 11.59. Found: C, 67.23;
H, 3.42; N, 11.75.
5-(50-Chloro-20-phenyl-1H-indol-30-yl)-7-phenylpyrido[2,
3-d]pyrimidin-4(3H)-one (3g): Yield: 80 %, mp 162–163 °C,
Rf 0.71 ethyl acetate: benzene (9:1) mixture, FTIR (KBr)
cm-1: 3441 (indole NH); 3211 (NH); 1701 (C=O); 1H NMR
(DMSO-d6, d ppm): 12.00 (s, 1H, indole-NH); 10.03 (s, 1H,
pyrimidine-NH); 8.83 (s, 1H, pyrimidine-H); 7.85 (s, 1H,
pyridine-H); 7.78 (s, 1H, indole-H4); 7.69 (d, 1H, indole-H6);
7.60 (d, 1H, indole-H7); 7.33–7.58 (m, 10H, Ar–H); Anal. %
C27H17N4OCl: C, 72.24; H, 3.82; N, 12.48. Found: C, 72.38;
H, 3.94; N, 12.55.
5-(50-Methyl-20-phenyl-1H-indol-30-yl)-7-(4-chlorophenyl)
pyrido[2,3-d]pyrimidin-4(3H)-one (3b): Yield: 75 %, mp
172–173 °C, Rf 0.51 ethyl acetate: benzene (9:1) mixture,
FTIR (KBr) cm-1: 3435 (indole-NH); 3010 (NH); 1705
1
(C=O); H NMR (DMSO-d6, d ppm): 12.00 (s, 1H, indole-
5-(50-Methyl-20-phenyl-1H-indol-30-yl)-7-phenylpyrido[2,
3-d]pyrimidin-4(3H)-one (3h): Yield: 56 %, mp 151–152
°C, Rf 0.65 ethyl acetate: benzene (9:1) mixture, FTIR (KBr)
cm-1: 3,389 (indole-NH); 3,200 (NH); 1700 (C=O); 1H
NMR (DMSO-d6, d ppm): 12.24 (s, 1H, indole-NH); 10.00
NH); 9.78 (s, 1H, pyrimidine-NH); 8.75 (s, 1H, pyrimidine-H);
8.18 (s, 1H, pyridine-H); 7.65–8.15 (m, 9H, Ar–H); 7.41 (s,
1H, indole-H4); 7.30 (d, 1H, indole-H6); 7.11 (d, 1H, indole-
H7); 2.25 (s, 3H, CH3); Anal. % C28H19N4ClO: C, 72.65; H,
4.14; N, 12.10. Found: C, 72.78; H, 4.23; N, 12.26.
123

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Med Chem Res (2013) 22:806–817
(s, 1H, pyrimidine-NH); 8.81 (s, 1H, pyrimidine-H); 8.00
(s, 1H, pyridine-H); 7.80–7.94 (m, 10H, Ar–H); 7.31 (s, 1H,
indole-H4); 7.20 (d, 1H, indole-H6); 7.08 (d, 1H, indole-H7);
2.21(s, 3H, CH3); Anal. % C28H20N4O: C, 78.49; H, 4.70; N,
13.08. Found: C, 78.56; H, 4.57; N, 12.82.
4-(50-Chloro-20-phenyl-1H-indol-30-yl)-6-(4-methylphenyl)-
1H-pyrazolo[3,4-b]pyridin-3-amine (4d): Yield: 80 %, mp
169–170 °C, Rf 0.68 chloroform: ethanol (5:1) mixture, FTIR
(KBr) cm-1: 3,400 (indole-NH); 3,254 (NH2); 3,200 (NH); 1H
NMR (DMSO-d6, d ppm): 12.13 (s, 1H, indole-NH); 8.95 (s,
1H, pyrazole-NH); 7.93 (s, 1H, pyridine-H); 7.85 (s, 1H, indole-
H4); 7.75 (d, 1H, indole-H6); 7.71 (d, 1H, indole-H7); 7.41–7.63
(m, 9H, Ar–H); 5.10 (s, 2H, NH2); 2.30 (s, 3H, CH3); Anal. %
C27H20N5Cl: C, 72.07; H, 4.48; N, 15.57. Found: C, 72.28; H,
4.51; N, 15.35.
5-(20-Phenyl-1H-indol-30-yl)-7-phenylpyrido[2,3-d]pyrim
idin-4(3H)-one (3i): Yield: 58 %, mp 183–184 °C, Rf 0.50
ethyl acetate: benzene (9:1) mixture, FTIR (KBr) cm-1
:
1
3,441 (indole-NH); 3,148 (NH); 1,685 (C=O); H NMR
(DMSO-d6, d ppm): 12.11 (s, 1H, indole-NH); 10.00 (s, 1H,
pyrimidine-NH); 8.95 (s, 1H, pyrimidine-H); 8.19 (s, 1H,
pyridine-H); 7.60–8.00 (m, 14H, Ar–H); Anal. %
C27H18N4O: C, 78.24; H, 4.38; N, 13.52. Found: C, 78.48;
H, 4.46; N, 13.36.
4-(50-Methyl-20-phenyl-1H-indol-30-yl)-6-(4-methylphenyl)
-1H-pyrazolo[3,4-b]pyridin-3-amine (4e): Yield: 65 %, mp
182–183 °C, Rf 0.70 chloroform: ethanol (5:1) mixture, FTIR
1
(KBr) cm-1: 3418 (indole NH); 3219 (NH2); 3153 (NH); H
NMR (DMSO-d6, d ppm): 12.00 (s, 1H, indole-NH); 9.00 (s,
1H, pyrazole-NH); 8.15 (s, 1H, pyridine-H); 7.89–8.03 (m, 9H,
Ar–H); 7.53 (s, 1H, indole-H4); 7.35 (d, 1H, indole-H6); 7.30
(d, 1H, indole-H7); 5.32 (s, 2H, NH2); 2.31 (s, 3H, CH3); 2.19 (s,
3H, CH3); Anal. % C28H23N5: C, 78.30; H, 5.40; N, 16.31.
Found: C, 78.17; H, 5.62; N, 16.23.
4-(20-Phenyl-1H-indol-30-yl)-6-(4-methylphenyl)-1H-pyr-
azolo[3,4-b]pyridin-3-amine (4f): Yield: 60 %, mp 193–194
°C, Rf 0.70 chloroform: ethanol (5:1) mixture, FTIR (KBr)
cm-1: 3,418 (indole NH); 3,200 (NH2); 3,189 (NH); 1H NMR
(DMSO-d6, d ppm): 12.32 (s, 1H, indole-NH); 9.00 (s, 1H,
pyrazole-NH); 8.03 (s, 1H, pyridine-H); 6.20–7.89 (m, 13H,
Ar–H); 5.11 (s, 2H, NH2); 2.18 (s, 3H, CH3); Anal. %
C27H21N5: C, 78.05; H, 5.09; N, 16.86. Found: C, 78.19; H,
5.27; N, 16.64.
4-(50Chloro-20-phenyl-1H-indol-30-yl)-6-phenyl-1H-pyraz
olo[3,4-b]pyridin-3-amine (4g): Yield: 78 %, mp 201–202
°C, Rf 0.68 chloroform: ethanol (5:1) mixture, FTIR (KBr)
cm-1: 3431 (indole-NH); 3298 (NH2); 3200 (NH); 1H NMR
(DMSO-d6, d ppm): 12.15 (s, 1H, indole-NH); 8.78 (s, 1H,
pyrazole-NH); 7.98 (s, 1H, pyridine-H); 7.80 (s, 1H, indole-
H4); 7.75 (d, 1H, indole-H6); 7.72 (d, 1H, indole-H7); 7.40–
7.65 (m, 10H, Ar–H); 5.38 (s, 2H, NH2); Anal. % C26H18
N5Cl: C, 71.64; H, 4.16; N, 16.07. Found: C, 71.43; H, 4.39;
N, 16.29.
General procedure for the synthesis of 4-(50-substituted
20-phenyl-1H-indol-30-yl)-6-(4-substituted phenyl)-1H-pyr-
azolo[3,4-b]pyridin-3-amines (4a–i). A solution of 2-amino
-3-cyano-4-(50substituted 20-phenyl-1H-indol-30-yl)-6-(4-
substituted phenyl)pyridines (2a–i) (0.01 mol) and hydra-
zine hydrate (99 %, 0.03 mol) in ethanol (5 mL) was heated
under reflux for 3 h. After cooling the product separated was
filtered, washed with cold ethanol and crystallized from
ethanol.
4-(50-Chloro-20-phenyl-1H-indol-30-yl)-6-(4-chlorophenyl)
-1H-pyrazolo[3,4-b]pyridin-3-amine (4a): Yield: 75 %, mp
195–196 °C, Rf 0.61 chloroform: ethanol (5:1) mixture, FTIR
(KBr) cm-1: 3432 (indole-NH); 2956 (NH2); 2917 (NH); 1H
NMR (DMSO-d6, d ppm): 11.80 (s, 1H, indole-NH); 8.70 (s,
1H, pyrazole-NH); 7.90 (s, 1H, pyridine-H); 7.85 (s, 1H,
indole-H4); 7.81 (d, 1H, indole-H6); 7.79 (d, 1H, indole-H7);
7.40–7.65 (m, 9H, Ar–H); 4.60 (s, 2H, NH2); MS (EI) m/z 469
(M?); 473 (M??4). Anal. % C26H17N5Cl2: C, 66.39; H, 3.64;
N, 14.89. Found: C, 66.47; H, 3.86; N, 14.73.
4-(50-Methyl-20-phenyl-1H-indol-30-yl)-6-(4-chlorophenyl)
-1H-pyrazolo[3,4-b]pyridin-3-amine (4b): Yield: 63 %, mp
197–198 °C, Rf 0.72 chloroform: ethanol (5:1) mixture, FTIR
(KBr) cm-1: 3419 (indole-NH); 3238 (NH2); 3,100 (NH); 1H
NMR (DMSO-d6, d ppm): 12.00 (s, 1H, indole-NH); 8.95 (s,
1H, pyrazole-NH); 8.19 (s, 1H, pyridine-H); 7.54–7.85 (m, 9H,
Ar–H); 7.45 (s, 1H, indole-H4); 7.30 (d, 1H, indole-H6); 7.20
(d, 1H, indole-H7); 5.00 (s, 2H, NH2); 2.28 (s, 3H, CH3);
Anal. % C27H20N5Cl: C, 72.07; H, 4.48; N, 15.57. Found: C,
72.23; H, 4.55; N, 15.38.
4-(50-Methyl-20-phenyl-1H-indol-30-yl)-6-phenyl-1H-
pyrazolo[3,4-b]pyridin-3-amine (4h): Yield: 48 %, mp
133–134 °C, Rf 0.53 chloroform: ethanol (5:1) mixture,
FTIR (KBr) cm-1: 3,401 (indole-NH); 3,300 (NH2); 3210
1
(NH); H NMR (DMSO-d6, d ppm): 12.00 (s, 1H, indole-
4-(20-Phenyl-1H-indol-30-yl)-6-(4-chlorophenyl)-1H-pyraz
NH); 9.00 (s, 1H, pyrazole-NH); 8.00 (s, 1H, pyridine-H);
7.60–7.95 (m, 10H, Ar–H); 7.43 (s, 1H, indole-H4); 7.35
(d, 1H, indole-H6); 7.18 (d, 1H, indole-H7); 5.38 (s, 2H,
NH2); 2.13 (s, 3H, CH3); Anal. % C27H21N5: C, 78.05; H,
5.09; N, 16.86. Found: C, 78.23; H, 5.36; N, 16.69.
4-(20-Phenyl-1H-indol-30-yl)-6-phenyl-1H-pyrazolo[3,4-
b]pyridin-3-amine (4i): Yield: 48 %, mp 159–160 °C, Rf
olo[3,4-b]pyridin-3-amine (4c):Yield: 59 %, mp 185–186 °C,
Rf 0.66 chloroform: ethanol (5:1) mixture, FTIR (KBr) cm-1
:
3,455 (indole NH); 3,300 (NH2); 3,215 (NH); 1H NMR
(DMSO-d6, d ppm): 12.08 (s, 1H, indole-NH); 9.00 (s, 1H,
pyrazole-NH); 8.10 (s, 1H, pyridine-H); 7.60–8.00 (m, 13H,
Ar–H); 5.13 (s, 2H, NH2); Anal. % C26H18N5Cl: C, 71.64; H,
4.16; N, 16.07. Found: C, 71.45; H, 4.25; N, 15.98.
0.68 chloroform: ethanol (5:1) mixture, FTIR (KBr) cm-1
:
123

Med Chem Res (2013) 22:806–817
815
1
3,400 (indole-NH); 3,300 (NH2); 3,235 (NH); H NMR
(DMSO-d6, d ppm): 12.12 (s, 1H, indole-NH); 9.08 (s, 1H,
pyrazole-NH); 8.18 (s, 1H, pyridine-H); 7.72–8.12 (m,
14H, Ar–H); 5.43 (s, 2H, NH2); Anal. % C26H19N5: C,
77.79; H, 4.77; N, 17.44. Found: C, 78.05; H, 4.95; N, 17.67.
5-(50-Methyl-20-phenyl-1H-indol-30-yl)-7-(4-methylphenyl)
pyrido[2,3-d]pyrimidin-4-amine (5e): Yield: 79 %, mp
214–215 °C, Rf 0.68 benzene: methanol (2:1) mixture, FTIR
(KBr) cm-1: 3430 (indole-NH); 3218 (NH2); 1H NMR
(DMSO-d6, d ppm): 12.38 (s, 1H, indole-NH); 8.83 (s, 1H,
pyrimidine-H); 8.25 (s, 1H, pyridine-H); 7.81–8.00 (m, 9H, Ar–
H); 7.61 (s, 1H, indole-H4); 7.40 (d, 1H, indole-H6); 7.30 (d, 1H,
indole-H7); 6.03 (s, 2H, NH2); 2.18 (s, 3H, CH3); 2.10 (s, 3H,
CH3); Anal. % C29H23N5: C, 78.89; H, 5.25; N, 15.86. Found:
C, 79.15; H, 5.52; N, 16.03.
General procedure for the synthesis of 5-(50-substituted 20-
phenyl-1H-indol-30-yl)-7-(4-substituted phenyl)pyrido[2,
3-d]pyrimidin-4-amines (5a–i) A solution of 2-amino-3-
cyano-4-(50-substituted 20-phenyl-1H-indol-30-yl)-6-(4-sub
stituted phenyl)pyridines (2a–i) (0.01 mol) in formamide
(15 mL) was heated under reflux for 6 h. The excess of
formamide was removed under reduced pressure and resi-
due was decomposed in crushed ice, solid obtained was
filtered washed with cold water and crystallized from
ethanol.
5-(50-Chloro-20-phenyl-1H-indol-30-yl)-7-(4-chlorophenyl)
pyrido[2,3-d]pyrimidin-4-amine (5a): Yield: 62 %, mp 143–
144 °C, Rf 0.73 benzene: methanol (2:1) mixture, FTIR (KBr)
cm-1: 3,438 (indole-NH); 2,921 (NH2); 1H NMR (DMSO-d6,
d ppm): 12.60 (s, 1H, indole-NH); 8.70 (s, 1H, pyrimidine-H);
8.30 (s, 1H, pyridine-H); 7.85 (s, 1H, indole-H4); 7.70 (d, 1H,
indole-H6); 7.63 (d, 1H, indole-H7); 7.40–7.60 (m, 9H, Ar–H);
6.00 (s, 2H, NH2); MS (EI) m/z 481 (M?); 485 (M??4).
Anal. % C27H17N5Cl2: C, 67.23; H, 3.55; N, 14.52. Found: C,
67.49; H, 3.72; N, 14.68.
5-(50-Methyl-20-phenyl-1H-indol-30-yl)-7-(4-chlorophenyl)
pyrido[2,3-d]pyrimidin-4-amine (5b): Yield: 66 %, mp 162–
163 °C, Rf 0.54 benzene: methanol (2:1) mixture, FTIR (KBr)
cm-1: 3444 (indole-NH); 3293 (NH2); 1H NMR (DMSO-d6, d
ppm): 12.18 (s, 1H, indole-NH); 9.00 (s, 1H, pyrimidine-H);
8.21 (s, 1H, pyridine-H); 7.70–8.00 (m, 9H, Ar–H); 7.43 (s,
1H, indole-H4); 7.35 (d, 1H, indole-H6); 7.20 (d, 1H, indole-
H7); 6.01 (s, 2H, NH2); 2.35 (s, 3H, CH3); Anal. %
C28H20N5Cl: C, 72.80; H, 4.36; N, 15.16. Found: C, 72.96; H,
4.58; N, 15.36.
5-(20-Phenyl-1H-indol-30-yl)-7-(4-methylphenyl)pyrido[2,
3-d]pyrimidin-4-amine (5f): Yield: 65 %, mp 210–211 °C,
Rf 0.50 benzene: methanol (2:1) mixture, FTIR (KBr) cm-1
:
1
3,444 (indole-NH); 3,300 (NH2); H NMR (DMSO-d6, d
ppm): 12.30 (s, 1H, indole-NH); 8.93 (s, 1H, pyrimidine-H);
8.00 (s, 1H, pyridine-H); 7.60–7.85 (m, 13H, Ar–H); 6.13 (s,
2H, NH2); 2.43 (s, 3H, CH3); Anal. % C28H21N5: C, 78.67;
H, 4.95; N, 16.38. Found: C, 78.86; H, 5.22; N, 16.28.
5-(50-Chloro-20-phenyl-1H-indol-30-yl)-7-phenylpyrido[2,
3-d]pyrimidin-4-amine(5g):Yield:72 %,mp142–143 °C, Rf
0.68 benzene: methanol (2:1) mixture, FTIR (KBr) cm-1
:
1
3,430 (indole-NH); 3,300 (NH2); H NMR (DMSO-d6, d
ppm): 12.11 (s, 1H, indole-NH); 9.18 (s, 1H, pyrimidine-H);
7.93 (s, 1H, pyridine-H); 7.88 (s, 1H, indole-H4); 7.80 (d, 1H
indole-H6); 7.72 (d, 1H, indole-H7); 7.38–7.62 (m, 10H, Ar–
H); 6.00 (s, 2H, NH2); Anal. % C27H18N5Cl: C, 72.40; H,
4.05; N, 15.64. Found: C, 72.68; H, 4.35; N, 15.88.
5-(50-Methyl-20-phenyl-1H-indol-30-yl)-7-phenylpyrido[2,
3-d]pyrimidin-4-amine(5h): Yield:66 %, mp 144–145 °C, Rf
0.53 benzene: methanol (2:1) mixture, FTIR (KBr) cm-1
:
1
3,444 (indole-NH); 3,342 (NH2); H NMR (DMSO-d6, d
ppm): 12.09 (s, 1H, indole-NH); 8.83 (s, 1H, pyrimidine-H);
8.13 (s, 1H, pyridine-H); 7.73–8.00 (m, 10H, Ar–H); 7.48 (s,
1H, indole-H4); 7.30 (d, 1H, indole-H6); 7.10 (d, 1H, indole-
H7); 6.05 (s, 2H, NH2); 2.53 (s, 3H, CH3); Anal. % C28H21N5:
C, 78.67;H, 4.95;N, 16.38. Found:C, 78.93;H, 5.18;N, 16.52.
5-(20-Phenyl-1H-indol-30-yl)-7-phenylpyrido[2,3-d]pyr-
imidin-4-amine (5i): Yield: 77 %, mp 234–235 °C, Rf 0.69
benzene: methanol (2:1) mixture, FTIR (KBr) cm-1: 3413
5-(20-Phenyl-1H-indol-30-yl)-7-(4-chlorophenyl)pyrido[2,
3-d]pyrimidin-4-amine (5c): Yield: 62 %, mp 101–102 °C,
Rf 0.73 benzene: methanol (2:1) mixture, FTIR (KBr) cm-1
:
3401 (indole-NH); 3293 (NH2); 1H NMR (DMSO-d6, d
ppm): 12.00 (s, 1H, indole-NH); 9.02 (s, 1H, pyrimidine-H);
8.12 (s, 1H, pyridine-H); 7.50–7.95 (m, 13H, Ar–H); 5.92 (s,
2H, NH2); Anal. % C27H18N5Cl: C, 72.40; H, 4.05; N, 15.64.
Found: C, 72.28; H, 4.23; N, 15.83.
(indole-NH); 3300 (NH2); H NMR (DMSO-d6, d ppm):
1
12.10 (s, 1H, indole-NH); 8.94 (s, 1H, pyrimidine-H); 8.00
(s, 1H, pyridine-H); 7.52–7.98 (m, 14H, Ar–H); 6.00 (s,
2H, NH2); Anal. % C27H19N5: C, 78.43; H, 4.63; N, 16.94.
Found: C, 78.69; H, 4.43; N, 16.73.
5-(50-Chloro-20-phenyl-1H-indol-30-yl)-7-(4-methylphenyl)
pyrido[2,3-d]pyrimidin-4-amine (5d): Yield: 58 %, mp 232–
233 °C, Rf 0.70 benzene: methanol (2:1) mixture, FTIR (KBr)
cm-1: 3,418 (indole NH); 3,249 (NH2); 1H NMR (DMSO-d6, d
ppm): 12.18 (s, 1H, indole NH); 8.83 (s, 1H, pyrimidine-H);
7.89 (s, 1H, pyridine-H); 7.80 (s, 1H, indole-H4); 7.76 (d, 1H,
indole-H6); 7.70 (d, 1H, indole-H7); 7.45–7.63 (m, 9H, Ar–H);
6.01 (s, 2H, NH2); 2.31 (s, 3H, CH3); Anal. % C28H20N5Cl: C,
72.80; H, 4.36; N, 15.16. Found: C, 72.53; H, 4.19; N, 14.95.
Biological assay
Antimicrobial activities
The in vitro biological screening of the synthesized com-
pounds (3–5) was carried out against bacteria species,
namely E. coli, S. aureus, K. penumoniae and P. aerginosa
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Med Chem Res (2013) 22:806–817
and fungal species, namely, A. nizer, A. oryzae, A. terrus
and A. flaves by cup-plate method (Indian pharmacopoeia,
Government of India, 1985) using nutrient agar as medium.
The holes of 6 mm diameter were punched carefully using
a sterile cork borer and these were filled with test solution
(500 lg/mL in DMF), standards (500 lg/mL in DMF) and
DMF as control. The plates were incubated at 37 °C for 24
and 72 h in case antibacterial and antifungal activity,
respectively. The diameter of the zone of inhibition for all
the test compounds was measured (in mm) and the results
were compared with the standard drug streptomycin for
antibacterial activity and fluconazole for antifungal activity.
and standards was estimated by following reported method
(Dinis et al., 1994). The test samples (25, 50, 75 and
100 lg/mL) in ethanolic solution (0.4 mL) were added to a
solution of FeCl2 (0.05 mL, 2 mM). The reaction was
initiated by the addition of ferrozine (0.2 mL, 5 mM) and
the total volume was adjusted to 4 mL with ethanol. Fer-
rozine reacted with the divalent iron to form stable
magenta complex species that were very soluble in water.
The mixture was shaken vigorously and kept at room
temperature for 10 min. Then the absorbance of the solu-
tion was measured spectrophotometrically at 562 nm. All
test analyses were run in triplicate and averaged. The
percentage of inhibition of the ferrozine Fe2? complex
formations was calculated using the formula
Antioxidant activities
Ferrous ion chelating effect ð%Þ
Absorbance of control ꢁ Absorbance of test sample
Radical scavenging activity RSA
¼
Absorbance of control
ꢂ 100
The RSA of test compounds (3–5) in methanolic solution at
concentrations 25, 50, 75, 100 lg/mL containing freshly
prepared DPPH solution (0.004 % w/v) was carried out and
compared with those of standards BHA, TBHQ and AA by
using reported method (Hatano et al., 1988). All the test
analyses were performed on three replicates were averaged.
The results in percentage are expressed as the ratio of
absorption decrease in the presence of test compounds and
absorption of DPPH solution in the absence of test com-
pounds at 517 nm on ELICO SL171 mini spec spectrom-
eter. The percentage scavenging activity of the DPPH free
radical was determined using the following equation.
Acknowledgments The authors are thankful to the Chairman,
Department of Chemistry, Gulbarga University, Gulbarga, for pro-
viding laboratory facilities and Chairman, Department of Microbiol-
ogy, Gulbarga University, Gulbarga for providing facilities to carry
out antimicrobial activity. We are also thankful to Director, Indian
Institute of Technology, Madras, Chennai for providing spectral data.
One of us (V.K.) is thankful to University Grants Commission, New
Delhi, India for providing financial assistance through Research
Fellowship in Science Meritorious Students (RFSMS).
References
% DPPH radical scavenging
Abdul R, Sumaira L, Ashfaq MQ, Muhammad Y, Aziz UR,
Mahmood UH, Zahid HC, Faiz UHN, Ben Hadda T (2010)
Synthesis, characterization and urease inhibition of 5-substi-
tuted-8-methyl-2H-pyrido[1,2-a]pyrimidine-2,4(3H)-diones.
Med Chem Res. doi:10.1007/s00044-010-9491-2
Altundas Aliye, Selcuk A, Elif L (2011) Synthesis and evaluation of
series of amino cyanopyridines as antimicrobial agents. Med
Chem Res 20:1–8
C¸ alis¸ I, Hosny M, Khalifa T, Nishibe S (1993) Secoiridoids from
Fraxinus angustifolia. Phytochemistry 33:1453–1456
Deepa SM, Beverley DG, Santy D (2007) Melatonin: new places in
therapy. Biosci Rep 27:299–320
Dinis TCP, Madeira VMC, Almeida LM (1994) Action of phenolic
derivatives (acetoaminophenol, salicylate and 5-amino salicy-
late) as inhibitors of membrane lipid peroxidation and as peroxyl
radical scavengers. Arch Biochem Biophys 315:161–169
Ekhlass N (2010) Synthesis, (in vitro) antitumor and antimicrobial
activity of some pyrazoline, pyridine and pyrimidine derivatives
linked to indole moiety. J Am Sci 6(8):463–471
Elisabete RP, Martine S, Jean-Jacques T, Michelle P, Anne-Marie M,
Mousset Guy, Maryse R (1996) Synthesis and antimicrobial
activities of five-membered ring heterocycles coupled to indole
moieties. J Antibiot 49(4):380–385
Eva-Maria K, Susann L, Carlo P, Ulrike B, Antonietta R, Hinnak N,
Lidia S, Reinhard T, Oliver W (2009) Structural optimization
and biological evaluation of 2-substituted 5-hydroxy indole-3-
carboxylates as potent inhibitors of human 5-lipoxygenase.
J Med Chem 52:3474–3483
Absorbanceof control ꢁ Absorbanceof testsample
¼
ꢂ 100
Absorbance of control
Iron metal ions chelating activity
Ferric ions (Fe3?) reducing antioxidant power (FRAP)
The reducing power of the synthesized compounds (3–5)
was determined according to the literature method (Oyaizu,
1986). Different concentration of samples (25, 50, 75 and
100 lg/mL) in DMSO (1 mL) were mixed with phosphate
buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide
(2.5 mL, 1 %). The mixture was incubated at 50 °C for
20 min. After which a portion of trichloroacetic acid
(2.5 mL, 10 %) was added to the mixture and centrifuged
for 10 min at 1,0009g. The upper layer of solution
(2.5 mL) mixed with distilled water (2.5 mL) and ferric
chloride (0.5 mL, 0.1 %). Then the absorbance at 700 nm
was measured in spectrophotometer. Higher absorbance of
the reaction mixture indicated greater reducing power.
Ferrous ions (Fe2?) chelating activity The chelating
activity of ferrous ions by synthesized compounds (3–5)
123

Med Chem Res (2013) 22:806–817
817
Fathi FM, Harita H, Mahmood A, Hamid K, Hapipah MA (2011)
Cytoprotective effect of benzyl N0-(indol-30-yl-methylidene)-
hydrazinecarbodithioate against ethanol induced gastric mucosal
injury in rats. Afr J Pure Appl Chem 5(3):34–42
Rufine AG, Eric D, Yoan F, Laurent M, Michelle P, Fabrice A,
Pascale M (2009) Synthesis and biological activities of amin-
opyrimidyl indoles structurally related to meridianins. Bioorg
Med Chem 17:4420–4424
Flora SJS, Mittal M, Mehta A (2008) Heavy metal induced oxidative
stress and its possible reversal by chelation therapy. Indian J
Med Res 128:501–523
Gnanamani S, Paramasivan TP, Urmila R, Praveen Kumar S (2009)
Synthesis and antioxidant activity of indolyl chromens. Indian J
Chem 48B:1319–1323
Gulcin I, Alici AH, Cesur M (2005) Determination of in vitro
antioxidant and radical scavenging activities of Propofol. Chem
Pharm Bull 53(3):281–285
Saundane AR, Veeresh Sharma PM (2004) Synthesis and antimicro-
bial activity of 2-(20,50-substituted indolidineamino-30-yl)-4,
6-diaryl pyrimidines and 2-[(20, 50-substituted indol-30-yl)pheny-
lazomethyleneimino]pyrimidines. Indian J Heterocycl Chem
13:275–276
Saundane AR, Manjunath Y, Kalpana R (2011a) Synthesis, antimi-
crobial and antioxidant activities of some new indole analogous
containing pyrimidine and fused pyrimidine systems. Med Chem
Res. doi:10.1007/s00044-011-9915-7
Gulcin I, Elmastas M, Aboul-Enein HY (2007) Determination of
antioxidant and radical scavenging activity of Basil (Ocimum
basilicum L. family Lamiaceae) assayed by different methodol-
ogies. Phytother Res 21:354–361
Halliwell B (1991) Reactive oxygen species in living systems: source,
biochemistry and role in human disease. Am J Med 91:14–22
Harinadha BV, Senthilkumar P, Srinivasan KK, Vardaraja Bhat G
(2004) Synthesis, antitumour and antibacterial activities of
certain substituted pyrimidine bearing benzofuran. Indian J
Pharm Sci 66(5):647–652
Hatano T, Kagawa H, Yasuhara T, Okuda T (1988) Two new
flavonoids and other constituents in licorice root: their relative
astringency and radical scavenging effects. Chem Pharm Bull
36(6):2090–2097
Hiremath SP, Biradar JS, Pourohit MG (1982) A new route to
indolo[3,2-b]isoquinolines. Indian J Chem 21B:249–251
Indian Pharmacopoeia, Government of India (1985) Appendix IV, 90,
3rd edn. Government of India, New Delhi
Saundane AR, Vijaykumar K, Yarlakatti M, Prabhaker W, Vaijinath
AV (2011b) Synthesis and biological activities of some indole
analogues containing pyridine, pyridopyrimidine and pyra-
nonapthyridine systems. Heterocycl Lett 1(4):339–350
Saundane AR, Vijaykumar K, Yarlakatti M, Vaijinath AV, Prabhaker
W (2011c) Synthesis and biological activities of some-5-
substituted 2-phenyl-3-(6-aryl-3-cyano-2-substituted pyridine-4-
yl)indoles. Indian J Heterocycl Chem 20:321–324
Silvestri R, Artico M, Bruno B, Massa S, Novellino E, Greco G,
Marongiu ME, Pani A, De Montis A, La Colla P (1998)
Synthesis and biological evaluation of 5H-indolo[3,2-b][1,5]ben-
zothiazepine derivatives, designed as conformationally con-
strained analogous of the human immunodeficiency virus type I
reverse transcriptase inhibitors L-737, 126. Antiviral Chem
Chemother 9:139–148
Strlic M, Radovic T, Kolar J, Pihlar B (2002) Anti- and prooxidative
properties of gallic acid in fenton-type systems. J Agri Food
Chem 50:6313–6317
James FD, Taro A, Monica E, Bahanu H, Tom D, Joel PB, Peter T,
Meinke GuoQS (2005) Synthesis and biological activities of
novel aryl indole-2-carboxylic acid analogous as PPARgamma,
partial agonist. Bioorg Med Chem Lett 15:5035–5038
Joseph IA, Michael JK, John BB, Anthony RB, Gabriel C, Lewis K
(2008) Structure–activity relationship of 2-aryl-1H-indole inhib-
itors of the NorA efflux pump in Staphylococcus aureus. Bioorg
Med Chem Lett 18:4294–4297
Maria-Joao RPQ, Ana SA, Solange MDC, Paula MTF, Nair N, Sao-
jose MN (2008) Synthesis of new heteroaryl and heterannulated
indoles from dehydrophenylalanines: antitumor evaluation. Bio-
org Med Chem 16:5584–5589
Marijana ZK, Raji Z, Petri N, Branka Z (2009) Antioxidant activity of
NSAID hydroxamic acids. Acta Pharm 59:235–242
Mohamed AA, Khairia MY, Magda AE, Sit Albanat AA, Hussein IE
(2005) Synthesis and in vitro antioxidant activity of some new
fused pyridine analogous. Arch Pharm Chem Life Sci 338:
175–180
Suresh M, Lavanya P, Vasu K, Sudhakar D, Venkata RC (2010)
Synthesis and bioassay studies of 7-substituted pyrido[2,3-
d]pyrimidines. J Chem Pharm Res 2(2):82–89
Thoraya AF, Ikhlas MA, Mohamed MA, Raghda OAM (2011)
Synthesis and pharmacological activities of fused pyrimidinones.
World J Chem 6(1):8–18
Tsukarman SV, Nikitchenko VM, Bugai AI, Lavruchin VF (1969)
Synthesis of analogs of chalcones and derivatives of D2-
pyrazoline based on 3-formyl indole. Khim Geterotsikl Soedin
Chem 2:268. Chem Abstr 71:22060 (1969)
Vijai Nath P, Gupta R, Gupta N (2008) Synthesis and biological
evaluation of some new 4,5-dihydro-3-(2-aryl-indol-3-yl)5-(4-chlo-
rophenyl)-N1-phenylpyrazoles. Indian J Chem 47B:1303–1307
Won-Gon K, In-Kyoung L, Jong-Pyung K, In-Ja R, Hiroyuki K,
Ick-dong Y (1997) New indole derivatives with free radical
scavenging activity from Agrocybe cylindracea. J Nat Prod
60:721–723
Oyaizu M (1986) Studies on product of browning reaction prepared
from glucose amine. Jpn J Nutr 44:307–316
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