Synthesis, antimicrobial, and antioxidant activities of some new indole analogues containing pyrimidine and fused pyrimidine systems

Article abstract of DOI:10.1007/s00044-011-9915-7

To examine new leads with potential antimicrobial and antioxidant activities, a new series of tetrahydropyrimidines (2a-c, 3a-c and 4a-c), pyrazolo[3, 4-d]pyrimidines (5a-c), and ditetrazolo[1,5-A;10,50-c] pyrimidines (6a-c) were synthesized in this study using appropriate synthetic routes. The newly synthesized compounds have been tested for their antimicrobial and antioxidant activities against DPPH stable free radical. In the case of antibacterial activity, compounds 2a, 6a, and 6c exhibited the maximum zone of inhibition against Staphylococcus aureus; compound 6c exhibited maximum zone of inhibition against Pseudomonas aeruginosa; and compound 2a showed maximum inhibitory growth against Klebsiella pneumonia. While in the case of antifungal activities, compound 5a showed good zone of inhibition against Aspergillus oryzae, compounds 2b and 6a exhibited maximum zone of inhibition against Aspergillus niger. In case of antioxidant activities, compound 2a showed the highest DPPH radical scavenging activity. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9915-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3809–3817
DOI 10.1007/s00044-011-9915-7
ORIGINAL RESEARCH
Synthesis, antimicrobial, and antioxidant activities of some new
indole analogues containing pyrimidine and fused pyrimidine
systems
•
Saundane Anand Raghunath Yarlakatti Manjunatha
•
Kalpana Rayappa
Received: 11 August 2011 / Accepted: 16 November 2011 / Published online: 2 December 2011
Ó Springer Science+Business Media, LLC 2011
Abstract To examine new leads with potential anti-
microbial and antioxidant activities, a new series of tetra-
hydropyrimidines (2a–c, 3a–c and 4a–c), pyrazolo[3,
4-d]pyrimidines (5a–c), and ditetrazolo[1,5-a;10,50-c]
pyrimidines (6a–c) were synthesized in this study using
appropriate synthetic routes. The newly synthesized com-
pounds have been tested for their antimicrobial and anti-
oxidant activities against DPPH stable free radical. In the
case of antibacterial activity, compounds 2a, 6a, and 6c
exhibited the maximum zone of inhibition against Staph-
ylococcus aureus; compound 6c exhibited maximum zone
of inhibition against Pseudomonas aeruginosa; and com-
pound 2a showed maximum inhibitory growth against
Klebsiella pneumonia. While in the case of antifungal
activities, compound 5a showed good zone of inhibition
against Aspergillus oryzae, compounds 2b and 6a exhibited
maximum zone of inhibition against Aspergillus niger. In
case of antioxidant activities, compound 2a showed the
highest DPPH radical scavenging activity.
Introduction
Food oxidation by atmospheric oxygen and free radicals is
a destructive process, causing the loss of nutritional values
and changes in chemical composition. The formation of
reactive oxygen species (ROS) is a natural consequence of
aerobic metabolism and is an integral part of tissue oxygen
homeostasis maintenance (Castro and Freeman, 2001).
When present in high concentrations, these compounds can
damage cellular proteins, lipids, and form carcinogenic
DNA adducts. Although ROS play crucial roles in normal
physiological processes, such as the apoptotic elimination
of damaged cells, aberrant production or regulation of ROS
activity has been demonstrated to contribute to the devel-
opment of some prevalent diseases, including cancer and
cardiovascular (Seifried et al., 2007). The purpose of
antioxidants is to prevent ROS concentrations from react-
ing harmful intracellular levels and degenerative processes
by various mechanisms including scavenging of free
radicals.
Keywords Indole ꢀ Pyrazolopyrimidine ꢀ
Indole is an important substance used in organic syn-
thesis (Bajtos et al., 2007), and its physiological activities
attracted a lot of scientific attention as well (Bittner et al.,
2007). The antioxidant effect of indole derivatives, such as
melatonin, tryptophan, and serotonin on cisplatin-induced
ROS have been reported (Fukutomi et al., 2006; Matuszak
et al., 1997). In vitro radical scavenging activity (RSA) of
several N-substituted indole-2-carboxylic acid esters (1)
was studied (Kruk et al., 2007). This property of indole
attracted researchers’ attention because of the correlation
of serious diseases with the oxidative damage of the
membrane, DNA- and RNA-induced radicals (Ratnam
et al., 2006). Several indole derivatives are well known for
their manifold uses because of their potential application in
medicinal chemistry as antitumor (Sunjoo et al., 2011;
Tetrazolopyrimidines ꢀ Antimicrobial ꢀ Antioxidant
Presented in the 47th Annual Convention of Chemists and
International Conference on Recent Advances in Chemical Sciences
2010 held at School of Studies in Chemistry, Pt. Ravishankar Shukla
University, Raipur (Chhattisgarh, India) during December 23–27,
2010.
S. A. Raghunath (&) ꢀ Y. Manjunatha ꢀ K. Rayappa
Department of Post-Graduate Studies and Research
in Chemistry, Gulbarga University, Gulbarga,
Karnataka 585 106, India
e-mail: arsaundane@rediff.com
123

3810
Med Chem Res (2012) 21:3809–3817
Mohamed and Mahmoud, 2007) antivascular (Nancy et al.,
2008), antituberculosis (Basavarajaiah and Mruthyunja-
yaswamy, 2009), and anti-inflammatory (Mohamed et al.,
2007) activities.
6-(20,50-disubstituted 1H-indol-30-yl)-4-oxo-1,2,3,4-tetra-
hydro-2-thioxo pyrimidin-5-carbonitriles (2.a–2.c) was
conveniently prepared by the reaction of 2,5-disubstituted
indole-3-carboxaldehydes (1.a–1.c) (Hiremath et al., 1982)
with ethyl acetoacetate, thiourea, and anhydrous potassium
carbonate in dry ethanol under reflux conditions (Kambe
et al., 1979; Ram et al., 1988). The IR spectrum of com-
pound 2.a showed absorption bands at 3259 (NH), 3056
(indole NH), 2210 (CN), 1693 (C=O) cm-1 functions, and
Pyrimidine and purine compounds play an essential role
in several biological processes and have very important
chemical and pharmacological properties. Some of the
pyrimidine analogues (2) (Agarwal et al., 2002), and (3)
(Polak and Scholer, 1975) have been reported as potential
antimicrobial agents. Also, pyrazolopyrimidine systems, as
they are structurally related to purine, considered as typical
examples of purine analogues (4) (Aly and Gad El Karim,
2011) and (5) (Wang et al., 2008), have been reported as
antibacterial agents, inhibitors for the syntheses of DNA
and RNA in cells of some kind of cancers (Seela et al.,
2000), and viruses (Tomonaga et al., 1990). The literature
survey has revealed that, several tetrazolopyrimidines have
been reported as antimicrobial agents (6) (El-Assiery and
El-Haiza, 1998) and (7) (Dave and Shah, 2002). Tetrazol-
opyrimidines are also used in the treatment of obesity,
atherosclerosis, glaucoma (Aspens et al., 2002), hyperten-
sion (Takaya et al., 1988; Uehata et al., 2001), coronary
heart disease (Fujii et al., 2005), and diabetic retinotherapy
(Takayama et al., 2006).
1
its H NMR spectrum revealed the signals at d ppm 11.71
(s, 1H, indole NH), 9.92 (s, 1H, NH), 7.78 (s, 1H, Ar–H),
7.70 (d, 1H, Ar–H), 7.65 (d, 1H, Ar–H), 7.59 (t, 1H, Ar–H),
7.30 (t, 2H, Ar–H), 7.01 (dd, 2H, Ar–H), and 5.13 (s, 1H,
SH). Its mass spectrum exhibited isotopic molecular ion
peak at m/z 378 and 380. Compounds (2.a–2.c) on reaction
with phosphorus pentasulfide in boiling pyridine gave 2.4-
dithiopyrimidines (3.a–3.c). Compound 3.a in its IR
spectrum exhibited absorption bands at 3289, 3057, 2210,
and 1239 cm-1 due to NH, indole NH, CN, and C=S
functions, respectively. Further, its 1H NMR spectrum
showed signals at d ppm 12.24 (s, 1H, indole NH), 10.01 (s,
1H, NH), 8.11 (s, 1H, Ar–H), 8.02 (d, 1H, Ar–H), 7.83 (d,
1H, Ar–H), 7.45 (t, 2H, Ar–H), 7.38 (t, 1H, Ar–H), 7.09
(dd, 2H, Ar–H), and 5.23 (s, 1H, SH). Its mass spectrum
showed isotopic molecular ion peak at m/z 394 and 396,
which confirms the formation of 3.a from 2.a. Compounds
(3.a–3.c) upon refluxing with hydrazine hydrate in ethanol
afforded 6-(20,50-disubstituted 1H-indole-30-yl)-2,4-dihy-
drazinylpyrimidin-5-carbonitriles (4.a–4.c). Compound 4.a
in its IR spectrum exhibited absorption bands at 3434,
3322, and 3150 cm-1 due to indole NH, NH, and NH2
NH
H
N
O
Cl
NH2
CN
Ar
N
N
N
R2
R1
N
F
N
R
COOCHCH2
N
N
N
NH2
RS
N
Ar
R1
(4)
(3)
(1)
O
(2)
CH3
CN
N
N
RS
R
N
NH2
N
1
N
N
functions, respectively. While in its H NMR spectrum,
N
N
N
N
N
N
N
N
NHAr
signals due to various protons appeared at d ppm 11.72 (s,
1H, indole NH), 8.91 (bs, 2H, NH), 8.45 (s, 1H, Ar–H),
8.03 (d, 1H, Ar–H), 7.74 (d, 1H, Ar–H), 7.53 (t, 2H, Ar–H),
7.32 (t, 1H, Ar–H), 7.08 (dd, 2H, Ar–H), and 6.83 (s, 2H,
NH2). In mass spectrum, isotopic molecular ion peaks at
m/z 390 and 392 were observed which confirmed the for-
mation of 4.a from 3.a.
N
N
N
Ph
N
R1
N
(6)
(7)
(5)
Literature Compounds 1-7
As a result, encouraged by these pharmacological
properties of indole, pyrozolopyrimidine, and tetrazolo-
pyrimidines and guided by the observation that many times
the combination of one or more heterocyclic nuclei in a
molecule enhances the biological profile manifold, and in
continuation of our interest in the synthesis of biologically
active indole analogues (Saundane et al., 2009; Saundane
and Manjunatha, 2011), we have synthesized the title
compounds and screened them for their antimicrobial and
antioxidant activities.
Then, compounds (4.a–4.c) were invested in the syn-
thesis of pyrazolopyramidine and tetrazolopyrimidine sys-
tems. Thus, compounds (4.a–4.c) when subjected to
thermal cyclization in 1-butanol under reflux conditions for
5 h, yielded pyrazolopyrimidines (5.a–5.c). The structure
of compound (5.a) was confirmed by its spectral data.
Compound (5.a) in its IR spectrum showed the absence of
CN at 2210 cm-1 function, and fresh appearances of the
absorption bands at 3308, 3286, 3186, 3114, and
3056 cm-1 due to NH2, NH2, NH, NH, and indole NH
1
Results and discussion
functions, respectively were noticed. Also, the H NMR
spectrum showed the presence of signals at d ppm 12.02 (s,
1H, indole NH). 9.04 (s, 1H, pyrazole NH), 8.41 (1H, NH,
and hydrazinyl NH), 8.12 (s, 1H, Ar–H), 7.97 (d, 1H,
The reaction sequence employed for the synthesis of title
compounds is shown in Scheme 1. Starting material
123

Med Chem Res (2012) 21:3809–3817
3811
Scheme 1 Pathway for
synthesis of compounds 2–6
S
NH
O
CHO
R
HN
K2CO3
EtOH
NH2CSNH2
NCCH2COOC2H5
R
N
H
(1 a-c)
CN
N
H
(2 a-c)
H2NHN
N
NH
N
N
R
P2S5
NH2
S
N
NHNH2
N
H
NH
S
HN
N
(5 a-c)
NHNH2
R
n-butanol
R
2 NH2NH2 H2O
CN
CN
N
N
H
H
NaNO2/HCl
(3 a-c)
(4 a-c)
N
N
N
N
N
N
N
N
a
b
c
H
R
Where R= Cl CH3
CN
N
H
(6 a-c)
Ar–H), 7.84 (d, 1H, Ar–H), 7.75 (t, 2H, Ar–H), 7.67 (t, 1H,
Ar–H), 7.54 (dd, 2H, Ar–H), 7.32 (s, 2H, NH2), and 7.11 (s,
2H, NH2). The structure of this pyrazolopyrimidine was
further confirmed by recording on mass spectrum, which
showed that isotopic molecular ion peaks at m/z 390 and 392
confirmed the formation of 5.a from 4.a. Again, synthons
(4.a–4.c) on diazotization with sodium nitrite at 0–5°C
afforded ditetrazolo[1,5-a;10,50-c]pyrimidines (6.a–6.c). In
the IR spectrum of compound 6.a, the bands due to indole
NH and CN, which are present in all studied compounds,
were observed at about 3053 and 2210 cm-1 functions,
respectively. The bands at 1240 and 1012 cm-1 were char-
Pseudomonas aeruginosa, and Klebsiella pneumonia,
whereas antifungal activities against Aspergillus niger,
A. terrus, and A. oryzae were evaluated by cup plate
method at a concentration of 1 mg/ml following the liter-
ature procedure (Indian pharmacopoeia, 1985). The zone of
inhibition (in mm) was compared with the standard gen-
tamycin and fluconazole for antibacterial and antifungal
activities, respectively. The results are tabulated in
Table 1. In case of antibacterial activities, compounds 2.a
and 6.a showed maximum zone of inhibition against
S. aureus and K. Pneumonia, whereas, compounds 5.a and
6.c exhibited maximum zone of inhibition against S. aureus
and P. aeruginosa. Compound 5.c showed maximum
inhibitory growth against S. aureus and K. pneumonia.
Compound 4.a showed maximum zone of inhibition
against P. aeruginosa, whereas compounds 2.c, 4.b, and
5.b exhibited good zone of inhibition against K.
pneumonia.
1
acteristic of the tetrazole ring. In H NMR spectrum, dif-
ferent protons were resonated at d ppm: 12.12 (s, 1H, indole
NH) and 8.02 (s, 1H, Ar–H), 7.95 (d, 1H, Ar–H), 7.83 (d, 1H,
Ar–H), 7.65 (t, 2H, Ar–H), 7.59 (t, 1H, Ar–H), 7.24 (dd, 2H,
Ar–H), and the absence of signals due to NH/NH2 functions
confirmed the formation of 6.a from 4.a. Further, formation
of 6.a was confirmed by its mass spectrum, which showed
isotopic molecular ion peaks at m/z 412 and 414.
On the other hand, antifungal activities of test com-
pounds revealed that, compound 2.b showed maximum
fungal growth inhibition against all the three fungi. Com-
pounds 2.a, 5.a, and 5.c exhibited maximum inhibitory
growth against A. oryzae. Compound 6.b exhibited maxi-
mum inhibition against A. terrus, and compound 6.a pos-
sessed good zone of inhibition against A. niger.
Biological results
Antimicrobial activity
From the results of antimicrobial activities, it is revealed
that, the majority of the synthesized compounds having
chloro substitution exhibited the maximum growth
All the synthesized compounds (2–6) were evaluated for
their antibacterial activities against Staphylococcus aureus,
123

3812
Med Chem Res (2012) 21:3809–3817
Table 1 Antimicrobial activity results of compounds 2–6
Comp. no.
Antibacterial activity (zone of inhibition in mm)
Antifungal activity (zone of inhibition in mm)
S. aureus
P. aeruginosa
K. pneumonia
A. oryzae
A. terrus
A. niger
2.a
14
12
11
12
09
08
11
10
08
13
11
12
14
10
14
15
–
10
10
11
10
10
07
13
10
10
13
12
11
12
10
14
16
–
14
11
12
09
08
08
10
12
11
09
13
12
13
11
12
15
–
12
11
10
11
09
10
10
09
10
13
09
12
10
09
07
–
09
11
10
10
10
11
10
11
11
10
10
10
11
12
10
–
10
13
10
11
09
11
08
11
10
09
10
12
13
09
10
–
2.b
2.c
3.a
3.b
3.c
4.a
4.b
4.c
5.a
5.b
5.c
6.a
6.b
6.c
Gentamycin
Fluconazole
14
15
14
inhibitory activity. In general, it is worth mentioning that
compounds 2.a, 3.a, 4.a, 5.a, and 6.a contain chloro sub-
stitution at position-5 of the indole nucleus. The electro-
negative nature of the chloro group may be responsible for
inhibiting the growth of the microbes. Antimicrobial
activities of compounds (5) and (6) may be due to the
presence of pyrazolopyrimidine and diterazolopyrimidine
systems, respectively. However, none of the compounds
exhibited zone of inhibition more than that of the
standards.
Radical scavenging activity (RSA)
Free radical scavenging is one of the best known mecha-
nisms by which antioxidants inhibit lipid oxidation. 1,1-
diphenyl-2-picrylhydrazyl (DPPH) RSA evaluation is
standard assay in antioxidant activity studies and offers a
rapid technique for screening the RSA of specific com-
pounds. The RSA of synthesized compounds (2–6) were
carried out using methanolic solution of the stable free
radical DPPH, and the results are shown in Figs. 1 and 2.
The freshly prepared DPPH solution exhibits a deep purple
color with absorption maxima at 517 nm. The purple color
generally fades/disappears when an antioxidant is present
in the medium. Thus, antioxidant molecule can quench
DPPH free radical (i.e., by providing hydrogen atoms or by
electron donation, conceivably via a free radical attack on
DPPH molecule) and convert them to a colorless/bleached
Fig. 1 Antioxidant activities of compounds 2–4
product, resulting in the absorbance at 517 nm. Therefore,
the more rapidly the absorbance decreases, the more potent
the Antioxidant activities of the compounds. Hence, this
method is based on the reduction of alcoholic DPPH
solution in the presence of a hydrogen-donating antioxidant
due to the formation of the non-radical form DPPH-H by
the reaction.
In the case of 6-(20,50-disubstituted 1H-indol-30-yl)-4-
oxo-1,2,3,4-tetrahydro-2-thioxopyrimidin-5-carbonitriles
(2a–c), derivative 2a showed the highest DPPH scavenging
activity with percent inhibition of 79.7 at concentration of
100 lg/ml (IC50 16.43 lg/ml), when compared with the
123

Med Chem Res (2012) 21:3809–3817
3813
Experimental procedures
Chemistry
Materials and methods
All the reagents were obtained commercially and used by
further purification. Melting points were determined by an
open capillary method and are uncorrected. The IR (KBr)
spectra were recorded with a Perkin-Elmer spectrum one
FT-IR spectrometer. The 1H NMR (DMSO-d6) spectra
were recorded using Bruker NMR (500 MHz), and the
chemical shifts were expressed in ppm (d scale) downfield
from TMS. Mass spectra were recorded with a JEOL
GCMATE II GC–MS mass spectrometer. Elemental anal-
ysis carried out using Flash EA 1112 series elemental
analyzer. The progress of the reaction was monitored by
TLC, and the purities of the synthesized compounds were
also checked by TLC.
Fig. 2 Antioxidant activities of compounds 5 and 6
other compounds. The higher activity of this compound
may be due to the presence of enolizable thio and oxo
groups. These groups may be responsible for stabilization
of free radicals formed after donating hydrogen or electron
to the stable DPPH radical as shown in Scheme 2. How-
ever, none of the compounds showed better RSA activity
than standards BHA (IC50 14.35 lg/ml) and TBHQ (IC50
14.46 lg/ml).
General procedure for the synthesis of 6-(20,50-
disubstituted 1H-indol-30-yl)-4-oxo-1,2,3,4-tetrahydro-2-
thioxopyrimidin-5-carbonitriles (2.a–2.c)
A mixture of 2,5-disubstitutedindole-3-carboxaldehyde
(1.a–1.c) (Hiremath et al., 1982) (0.01 mol), ethyl cya-
noacetate (0.01 mol), thiourea (0.01 mol), and potassium
carbonate (0.01 mol) in absolute ethanol (30 ml) was
refluxed for 20 h. The reaction mixture was cooled to room
temperature and acidified with dil. HCl. The precipitate
formed was filtered off, washed with water, dried, and
crystallized from ethanol to give compound (2.a–2.c).
Conclusion
In general, it was found that the compounds having chloro
substitution along with the pyrazolopyrimidine and ditet-
razolopyrimidine systems exhibited good antioxidant and
antimicrobial activities.
S
SH
CN
Scheme 2 DPPH RSA:
probable mechanism for
hydrogen donation from
compound 2a to DPPH radical
and stabilization of free radical
formed
S
H
H
O
N
N
N
N
N
N
Cl
Cl
Cl
OH
OH
DPPH
CN
CN
N
N
H
N
H
H
2.a
DPPH
SH
SH
CN
N
N
N
N
Cl
Cl
O
O
etc
DPPH-H
CN
N
H
N
H
SH
CN
N
N
Cl
OH
N
H
123

3814
Med Chem Res (2012) 21:3809–3817
6-(50-Chloro-20-phenyl-1H-indol-30-yl)-4-oxo-1,2,3,4-
(indole NH), 2215 (CN), 1283 (C=S). 1HNMR (DMSO-d6,
d ppm): 12.12 (s, 1H, indole NH); 10.13 (s, 1H, NH); 8.25
(s, 1H, Ar–H), 8.05 (d, 1H, Ar–H), 7.83 (d, 1H, Ar–H),
7.52 (t, 2H, Ar–H), 7.39 (t, 1H, Ar–H), 7.11 (dd, 2H,
Ar–H); 5.27 (s, 1H, SH); 2.26 (s, 3H, CH3); Anal. %
C20H14N4S2: C, 64.17; H, 3.74; N, 14.97. Found: C, 65.35;
H, 3.97; N, 15.15.
6-(20-Phenyl-1H-indol-30-yl)-2-4-dithioxo-1,2,3,4,-tetra-
hydropyrimidin-5-carbonitrile (3.c): Yield 65%, mp
249–250°C. FTIR (KBr) cm-1: 3287 (NH); 3059 (indole
NH); 2219 (CN); 1285 (C=S); 1HNMR (DMSO-d6, d
ppm): 12.19 (s, 1H, indole NH); 10.02 (s, 1H, NH); 8.28 (d,
1H, Ar–H), 8.10 (t, 1H, Ar–H), 7.87 (t, 1H, Ar–H), 7.55 (d,
1H, Ar–H), 7.41 (t, 2H, Ar–H), 7.25 (t, 1H, Ar–H), 7.14
(dd, 2H, Ar–H); 5.17 (s, 1H, SH); Anal. % C19H12N4S2: C,
63.33; H, 3.33; N, 15.56. Found: C, 63.57; H, 3.51; N,
15.78.
tetrahydro-2-thioxopyrimidin-5-carbonitrile (2.a): Yield:
71%, mp 265–266°C. FTIR (KBr) cm-1: 3259 (NH); 3056
(indole NH); 2210 (CN); 1693 (C=O); 1HNMR (DMSO-d6,
d ppm) 11.71 (s, 1H, indole NH); 9.92 (s, 1H, NH); 7.78 (s,
1H, Ar–H), 7.70 (d, 1H, Ar–H), 7.65 (d, 1H, Ar–H), 7.59 (t,
1H, Ar–H), 7.30 (t, 2H, Ar–H), 7.01 (dd, 2H, Ar–H); 5.13
(s, 1H, SH); MS (EI) m/z 378 (M?); 380 (M??2). Anal. %
C19H11N4OSCl: C, 60.32; H, 2.91; N, 14.81. Found: C,
60.54; H, 2.72; N, 14.71.
6-(50-Methyl-20-phenyl-1H-indol-30-yl)-4-oxo-1,2,3,4-
tetrahydro-2-thioxopyrimidin-5-carbonitrile (2.b): Yield:
69%, mp 269–270°C. FTIR (KBr) cm-1: 3255 (NH); 3059
(indole NH); 2210 (CN); 1693 (C=O); 1HNMR (DMSO-d6,
d ppm): 11.82 (s, 1H, indole NH); 9.91 (s, 1H, NH); 7.83
(s, 1H, Ar–H), 7.75 (d, 1H, Ar–H), 7.68 (d, 1H, Ar–H),
7.58 (t, 2H, Ar–H), 7.35 (t, 1H, Ar–H),7.04 (dd, 2H,
Ar–H); 5.11 (s, 1H, SH); 2.15 (s, 3H, CH3); Anal. %
C20H14N4OS: C, 67.03; H, 3.91; N, 15.64. Found: C, 67.32;
H, 4.12; N, 15.59.
6-(20-Phenyl-1H-indol-30-yl)-4-oxo-1,2,3,4-tetrahydro-
2-thioxopyrimidin-5-carbonitrile (2.c): Yield 69%, mp
258–259°C. FTIR (KBr) cm-1: 3257 (NH); 3059 (indole
NH); 2216 (CN); 1690 (C=O); 1HNMR (DMSO-d6, d
ppm): 11.84 (s, 1H, indole NH); 9.83 (s, 1H, NH); 7.82 (d,
1H, Ar–H), 7.73 (t, 1H, Ar–H), 7.70 (t, 1H, Ar–H), 7.62 (d,
1H, Ar–H), 7.54 (t, 2H, Ar–H), 7.30 (t, 1H, Ar–H),7.10
(dd, 2H, Ar–H); 5.23 (s, 1H, SH); Anal. % C19H12N4OS: C,
66.28; H, 3.49; N, 16.28. Found: C, 66.52; H, 3.71; N,
16.53.
General procedure for the synthesis of 6-(20,50-
disubstituted-1H-indol-30-yl)-2,4-dihydrazinylpyrimidin-5-
carbonitriles (4.a–4.c)
An equimolar mixture of compounds (3.a–3.c) (0.01 mol)
and hydrazine hydrate (0.01 mol, 99–100%) in ethanol
(15 ml) was refluxed for 4 h. After cooling, the resulting
solid was collected by filtration, washed with cold ethanol,
and recrystallized from ethanol to give (4.a–4.c).
6-(50-Chloro-20-phenyl-1H-indol-30-yl)-2,4-dihydrazinylpyr-
imidin-5-carbonitrile (4.a): Yield 69%, mp 210–211°C.
FTIR (KBr) cm-1: 3434 (NH2), 3322 (NH) and 3150
(indole NH); 2195 (CN); 1HNMR (DMSO-d6, d ppm):
11.72 (s, 1H, indole NH); 8.91 (bs, 2H, 2 NH); 8.45 (s, 1H,
Ar–H), 8.03 (d, 1H, Ar–H), 7.74 (d, 1H, Ar–H), 7.53 (t, 2H,
Ar–H), 7.32 (t, 1H, Ar–H), 7.08 (dd, 2H, Ar–H), 6.83 (s,
2H,NH2); MS (EI) m/z 390 (M?), 392 (M??2). Anal. %
C19H15N8Cl: C, 58.46; H, 3.85; N, 28.72. Found: C, 58.71;
H, 3.99; N, 28.93.
General procedure for the synthesis of 6-(20,50-
disubstituted 1H-indol-30-yl)-2-4-dithioxo-1,2,3,4,-
tetrahydropyrimidin-5-carbonitriles (3.a–3.c)
A solution of (2.a–2.c) (0.01 mol) in pyridine (10 ml) and
P2S5 (0.01 mol) was refluxed for 5 h, then cooled, poured
to crushed ice, filtered, washed with water, dried, and re-
crystallized from ethyl acetate to obtain (3.a–3.c).
6-(50-Methyl-20-phenyl-1H-indole-30-yl)-2,4-dihydrazinylpyr-
imidin-5-carbonitrile (4.b): Yield 65%, mp 215–216°C.
FTIR (KBr) cm-1: 3435 (NH2), 3320 (NH) 3125 (indole
6-(50-Chloro-20-phenyl-1H-indol-30-yl)-2-4-dithioxo-
1,2,3,4,-tetrahydropyrimidin-5-carbonitrile (3.a): Yield
70%, mp 235–236°C. FTIR (KBr) cm-1: 3289 (NH); 3057
(indole NH); 2210 (CN); 1293 (C=S); 1HNMR (DMSO-d6,
d ppm): 12.24 (s, 1H, indole NH); 10.01 (s, 1H, NH); 8.11
(s, 1H, Ar–H), 8.02 (d, 1H, Ar–H), 7.83 (d, 1H, Ar–H),
7.45 (t, 2H, Ar–H), 7.38 (t, 1H, Ar–H), 7.09 (dd, 2H, Ar–
H); 5.23 (s, 1H, SH); MS (EI) m/z 394 (M?), 396 (M??2).
Anal. % C19H11N4S2Cl: C, 57.87; H, 2.79; N, 14.21.
Found: C, 57.79; H, 2.57; N, 14.53.
1
NH); 2196 (CN); HNMR (DMSO-d6, d ppm): 11.84 (s,
1H, indole NH); 8.88 (bs, 2H, 2 NH); 8.35 (s, 1H, Ar–H),
8.05 (d, 1H, Ar–H), 7.71 (d, 1H, Ar–H), 7.49 (t, 2H, Ar–H),
7.30 (t, 1H, Ar–H), 7.15 (dd, 2H, Ar–H), 6.82 (s, 2H,NH2);
2.11 (s, 3H, CH3); Anal. % C20H18N8: C, 64.86; H, 4.86; N,
30.27. Found: C, 64.95; H, 4.95; N, 30.55.
6-(20-Phenyl-1H-indol-30-yl)-2,4-dihydrazinylpyrimidin-5-
carbonitrile (4.c): Yield 67%, mp 201–203°C. FTIR
(KBr) cm-1: 3430 (NH), 3315 (NH) 3125 (indole NH);
2198 (CN); 1HNMR (DMSO-d6, d ppm): 11.71 (s, 1H,
indole NH); 8.95 (bs, 2H, 2 NH); 8.35 (d, 1H, Ar–H), 8.06
(t, 1H, Ar–H),8.01 (t, 1H, Ar–H), 7.89 (d, 1H, Ar–H), 7.54
6-(50-Methyl-20-phenyl-1H-indol-3’-yl)-2-4-dithioxo-1,2,
3,4,-tetrahydropyrimidin-5-carbonitrile (3.b): Yield 68%,
mp 239–240°C. FTIR (KBr) cm-1: 3291 (NH), 3060
123

Med Chem Res (2012) 21:3809–3817
3815
(t, 2H, Ar–H), 7.43 (t, 1H, Ar–H), 7.31 (dd, 2H, Ar–H),
6.72 (s, 2H,NH2); Anal. % C19H16N8: C, 64.04; H, 4.49; N,
31.46.Found: C, 64.35; H, 4.67; N, 31.71.
0–5°C. The mixture was then allowed to react at same
temperature for 3 h. The formed solid was filtered, washed
with water, dried, and recrystallized from alcohol to obtain
(6.a–6.c).
5-(50-Chloro-20-phenyl-1H-indol-30-yl)ditetrazolo[1,5-a;
10,50-c]pyrimidin-6-carbonitrile (6.a): Yield 61%, mp
278–279°C. FTIR (KBr) cm-1: 3053 (indole NH); 2210
General procedure for the synthesis of 4-(20,50-
disubstituted-1H-indol-30-yl)-6-hydrazino-1H-
pyrazolo[3,4-d]pyrimidin-3-amines (5.a–5.c)
1
(CN); HNMR (DMSO-d6, d ppm): 12.12 (s, 1H, indole
NH), 8.02 (s, 1H, Ar–H), 7.95 (d, 1H, Ar–H), 7.83 (d, 1H,
Ar–H), 7.65 (t, 2H, Ar–H), 7.59 (t, 1H, Ar–H), 7.24 (dd,
2H, Ar–H); MS (EI) m/z 412 (M?), 414 (M??2); Anal. %
C19H9N10Cl: C, 55.34; H, 2.18; N, 33.98. Found: C, 55.59;
H, 2.39; N, 33.71.
5-(50-Methyl-20-phenyl-1H-indol-30-yl)ditetrazolo[1,5-a;
10,50-c]pyrimidin-6-carbonitrile (6.b): Yield 64%, mp
282–283°C. FTIR (KBr) cm-1: 3050 (indole NH), 2214
(CN). 1HNMR (DMSO-d6, d ppm): 12.0 (s, 1H, indole
NH); 8.05 (s, 1H, Ar–H), 7.93 (d, 1H, Ar–H), 7.85 (d, 1H,
Ar–H), 7.61 (t, 2H, Ar–H), 7.57 (t, 1H, Ar–H), 7.14 (dd,
2H, Ar–H); 2.12 (s, 3H, CH3); Anal. % C20H12N10: C,
61.22; H, 3.06; N, 35.71. Found: C, 61.15; H, 3.31; N,
35.97.
5-(20-Phenyl-1H-indol-30-yl)ditetrazolo[1,5-a;10,50-c]
pyrimidin-6-carbonitrile (6.c): Yield 60%, mp 275–276°C.
IR: m/cm-1: 3062 (indole NH), 2214 (CN). 1HNMR
(DMSO-d6, d ppm): 12.11 (s, 1H, indole NH); 8.25 (d, 1H,
Ar–H), 8.02 (t, 1H, Ar–H), 7.78 (t, 1H, Ar–H), 7.85 (d, 1H,
Ar–H), 7.77 (t, 2H, Ar–H), 7.53 (t, 1H, Ar–H),7.15 (dd,
2H, Ar–H); MS, 378 (M?); Anal. % C19H10N10: C, 60.32;
H, 2.64; N, 37.04. Found: C, 60.55; H, 2.81; N, 36.95.
A solution of compounds (4.a–4.c) (0.001 mol) in 1-buta-
nol (20 ml) was refluxed for 7 h. The excess solvent was
removed under reduced pressure, and the residue was re-
crystallized form ethanol to give compound (5.a–5.c).
4-(50-Chloro-20-phenyl-1H-indol-30-yl)-6-hydrazino-1H-
pyrazolo[3,4-d]pyrimidin-3-amine (5.a): Yield 63%, mp
186–187°C. FTIR (KBr) cm-1: 3308 (NH2); 3286 (NH2);
3186 (NH); 3114 (NH); 3056 (indole NH); 1HNMR
(DMSO-d6, d ppm): 12.02 (s, 1H, indole NH); 9.04 (s, 1H,
pyrazole NH); 8.91 (s, 1H, hydrazinyl NH); 8.12 (s, 1H,
Ar–H), 7.97 (d, 1H, Ar–H), 7.84 (d, 1H, Ar–H), 7.75 (t, 2H,
Ar–H), 7.67 (t, 1H, Ar–H), 7.54 (dd, 2H, Ar–H), 7.32 (s,
2H, NH2), 7.11 (s, 2H, NH2); MS (EI) m/z 390 (M?), 392
(M??2); Anal. % C19H15N8Cl: C, 58.46; H, 3.85; N,
28.72. Found: C, 58.69; H, 3.99; N, 28.93.
4-(50-Methyl-20-phenyl-1H-indol-30-yl)-6-hydrazino-
1H-pyrazolo[3,4-d]pyrimidin-3-amine (5.b): Yield 65%,
mp 190–191°C. FTIR (KBr) cm-1: 3310 (NH2); 3289
(NH2); 3189 (NH); 3116 (NH); 3060 (indole NH); 1HNMR
(DMSO-d6, d ppm): 11.91 (s, 1H, indole NH); 8.92 (s, 1H,
pyrazole NH); 8.81 (s, 1H, hydrazinyl NH); 8.16 (s, 1H,
Ar–H), 7.95 (d, 1H, Ar–H), 7.82 (d, 1H, Ar–H), 7.71 (t, 2H,
Ar–H), 7.65 (t, 1H, Ar–H), 7.52 (dd, 2H, Ar–H), 7.30 (s,
2H, NH2), 7.05 (s, 2H, NH2); 2.21 (s, 3H, CH3); Anal. %
C20H18N8: C, 64.86; H, 4.86; N, 30.27. Found: C, 64.97; H,
5.01; N, 30.53.
Biology
Antioxidant activity: 1,1-diphenyl-2-picryl hydrazyl
(DPPH) RSA
4-(20-Phenyl-1H-indol-30-yl)-6-hydrazino-1H-pyrazolo
[3,4-d]pyrimidin-3-amine (5.c): Yield 63%, mp 196–197°C.
FTIR (KBr) cm-1: 3305 (NH2); 3284 (NH2); 3180 (NH);
3119 (NH); 3050 (indole NH); 1HNMR (DMSO-d6, d ppm):
12.11 (s, 1H, indole NH); 9.15 (s, 1H, pyrazole NH); 8.89 (s,
1H, hydrazinyl NH); 8.11 (d, 1H, Ar–H), 7.96 (t, 1H, Ar–H),
7.83 (t, 1H, Ar–H), 7.79 (d, 1H, Ar–H), 7.67 (t, 2H, Ar–H),
7.63 (t, 1H, Ar–H), 7.56 (dd, 2H, Ar–H), 7.35 (s, 2H, NH2),
7.04 (s, 1H, NH2); Anal. % C19H16N8: C, 64.04; H, 4.50; N,
31.46. Found: C, 64.35; H, 4.73; N, 31.78.
The free RSAs of compounds 2–6 were carried out in the
presence of the stable free radical DPPH following the
literature procedure. (Hatano et al., 1988) using 2-tert-
butyl-4-methoxyphenol (butylated hydroxyl anisole, BHA)
and 2-(1,1-dimethylethyl)-1,4-benzenediol (2-tert. butyl
hydroquinone, TBHQ) as standards. The RSAs for meth-
anolic solutions of compounds 2–6 at concentrations 25,
50, 75, and 100 lg/ml containing freshly prepared DPPH
solution (0.004% w/v) were carried out and compared to
those of standards, BHA and TBHQ. All the test analyses
were performed on three replicates, and the results are
averaged. The results in percentage are expressed as the
ratio of the decrease in the absorption of DPPH in the
presence of test compounds and the absorption of DPPH in
the absence of test compounds at 517 nm using ELICO SL
171 Mini Spec spectrophotometer. The percentage
General procedure for the synthesis of 5-(20,50-
disubstituted 1H-indol-30-yl)ditetrazolo[1,5-a;10,50-
c]pyrimidin-6-carbonitriles (6.a–6.c)
A solution of sodium nitrite (0.001 mol) in HCl (8 ml) was
added to the stirred solution of compounds (4.a–4.c)
(0.001 mol) in 20% aqueous hydrochloric acid (10 ml) at
123

3816
Med Chem Res (2012) 21:3809–3817
Dave CG, Shah RD (2002) Annelation of triazole and tetrazole
systems onto pyrrolo[2,3-d] pyrimidines: synthesis of tetrazol-
o[1,5-c]pyrrolo[2,3-e]pyrimidines and triazolo[1,5-c] pyrrol-
o[3,2-e]pyrimidines as potential antibacterial agents. Molecules
7:554–565
El-Assiery SA, El-Haiza MA (1998) Synthesis of some new
pyrimidine and fused pyrimidine derivatives. J King Saud Univ
10:101–117
Fujii A, Tanaka H, Otsuki M, Kawaguchi T, Oshita K (2005)
Antitumor effect potentiators. US Patent No. 6930115
Fukutomi J, Fukuda A, Fukuda S, Hara M, Terada A, Yoshida M
(2006) Scavenging activity of indole compounds against cis-
platin-induced reactive oxygen species. Life Sci 80:254–257
Hatano T, Kanawa 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
36:2090–2097
Hiremath SP, Biradar JS, Purohit MG (1982) A new route to
indolo[3,2-b]isoquinolines. Indian J Chem 21B:249
Indian Pharmacopoeia, Government of India (1985) 3rd edn, New
Delhi Appendix IV, 90
Kambe S, Saito K, Kishi H, Sakurai A, Midorikawa H (1979) A one-
step synthesis of 4-oxo-2-thioxopyrimidine derivatives by the
ternary condensation of ethyl cyanoacetate, aldehyde and
thiourea. Synthesis 287–289
Kruk I, Aboul-Enein HY, Michalska T, Lichszteld K, Kubasik-
Kladna K, Olgen S (2007) In vitro scavenging activity for
reactive oxygen species by N-substituted indole-2-carboxylic
acid esters. Luminescence 22:379–386
scavenging activity of the DPPH free radical was measured
using the following equation
%DPPH raddical scavenging
Absorbance of control - Absorbance of test sample
¼
Absorbance of control
ꢁ 100
The results are shown in Figs. 1 and 2.
Antimicrobial activities
The antimicrobial activities (Indian pharmacopoeia, 1985)
of the synthesized compounds were evaluated against
P. aeruginosa, S. aureus (Gram negative bacteria),
K. pneumoniae (gram negative bacteria), A. oryzae, and
A. niger (fungi). An aliquot 0.1 ml of each bacterial strain
was spread on nutrient agar, while 0.1 ml of the fungal
spore suspension was spread on potato dextrose agar
(PDA). An agar-well diffusion test was performed in each
case. In these tests, 6 mm wells were produced using a
sterile cork borer, and each well was then inoculated with
100 ll of each key substance in DMF. Nutrient agar plates
were incubated at 37°C for 24 h, while the PDA plates
incubated at 25°C for 72 h. The zone of inhibition around
the well was determined. Gentamycin and fluconazole were
used as the reference standards for antibacterial and anti-
fungal activity, respectively.
Matuszak Z, Reszka KJ, Chignell CF (1997) Reaction of melatonin
and related indoles with hydroxyl radicals: EPR and spin
trapping investigations. Free Radic Biol Med 23:367–372
Mohamed AAR, Mahmoud ES (2007) Synthesis and antitumor
activity of indolylpyrimidines: marine natural product meridia-
nin-D analogues. Bioorg Med Chem 15:1206–1211
Mohamed AAR, Eman AR, Nermien MS, Siham MES (2007)
Synthesis and biological evaluation of new 3-substituted indole
derivatives as potential anti-inflammatory and analgesic agents.
Bioorg Med Chem 15:3832–3841
Nancy Ty, Gregory D, Guy GC, Johanne S, Francois T, Daniel S,
Sylvie M, Xavier C (2008) Synthesis and biological evaluation
of new disubstituted analogues of 6-methoxy-3-(30,40,50-trim-
ethoxybenzoyl)-1H-indole (BPROL075), as potential antivascu-
lar agents. Bioorg Med Chem 16:7494–7503
Acknowledgments The authors are thankful to UGC, New Delhi
for providing financial support and to the Chairman, Department of
Chemistry, Gulbarga University, Gulbarga, for providing laboratory
facilities. The authors are also thankful to the Director, Indian Insti-
tute of Technology, Madras, Chennai, for providing spectral data. One
of the authors, Miss K R as a project fellow, is thankful to the UGC,
New Delhi for providing the financial assistance fellow.
Polak A, Scholer HJ (1975) Mode of action of 5-fluorocytosine and
mechanisms of resistance. Chemotherapy 21:113–130
Ram VJ, Vanden Berghe DA, Vlietinck AJ (1988) Pyrido[2,3-
d]pyrimidines and pyrido[2,3-d; 5-d0]dipyrimidines as potential
chemotherapeutic agents. VIII. J Heterocycl Chem 25: 217–219
Ratnam DV, Ankola DD, Bhardwaj V, Sahana DK, Kumar MNVR
(2006) Role of antioxidants in prophylaxis and therapy: a
pharmaceutical perspective. J Control Release 113:189–207
Saundane AR, Manjunatha Y (2011) Synthesis, antimicrobial and
antioxidant activities of 2-oxo-6-phenyl-2-yl-4-(20-phenyl-50-substi-
tuted 1H-indol-30-yl)-1,2-dihydropyridin-3-carbonitriles and their
derivatives. Arab J Chem. doi:10.1016/j.arabjc.2011.06.011
Saundane AR, Manjunatha Y, Prabhakar W (2009) Synthesis and
antimicrobial activities of 5-(20,50-disubstituted 1H-indole-30-yl)-
5H-thiazolo[4,3-b]1,3,4-oxadiazole-1,3,4-thiadiazole-1,2,4-triazole
and their derivatives. Heterocycl Commun 15:303–310
Seela F, Changfu W, Georg B, Matthias Z, Peter L (2000) The
influence of modified purine bases on the stability of parallel
DNA. Bioorg Med Chem Lett 10:289–292
References
Agarwal N, Srivastava P, Raghuwanshi SK, Upadhyay DN, Sinha S,
Shukla PK, Ram VJ (2002) Chloropyrimidines as a new class of
antimicrobial agents. Bioorg Med Chem 10:869–874
Aly AA, Gad El Karim IA (2011) Facile synthesis of new
pyrazolopyrimidine derivatives of potential biosignificant inter-
est. J Korean Chem Soc 55:781–786
Aspens GE, Yuan C, Chiang P (2002) Tetrazole compounds as
thyroid receptor ligands. US Patent No. 6441015
Bajtos B, Yu M, Zhao H, Pagenkopf BL (2007) C-2/C-3 Annulation
and C-2 alkylation of indoles with 2-alkoxycyclopropanoate
esters. J Am Chem Soc 129:9631–9634
Basavarajaiah SM, Mruthyunjayaswamy BHM (2009) Synthesis and
antimicrobial activity of some 5-substituted-3-phenyl-Nb-(sub-
stituted-2-oxo-2H-pyrano[2,3-b]quinoline-3-carbonyl)-1H-indole-
2-carboxyhydrazide. Chem Pharm Bull 57:557–560
Bittner S, Scherzer R, Harlev E (2007) The 5-bromotryptophans.
Amino Acids 33:19–42
Seifried HE, Anderson DE, Fisher EI, Milner JA (2007) A review of
the interaction among dietary antioxidants and reactive oxygen
species. J Nutr Biochem 18(9):567–579
Castro L, Freeman B (2001) Reactive oxygen species in human health
and disease. Nutrition 17:161–165
123

Med Chem Res (2012) 21:3809–3817
3817
Sunjoo A, Dong JH, Christina MB, Jun Y, Charles BD (2011) A novel
bis-indole destabilizes microtubules and displays potent in vitro
and in vivo antitumor activity in prostate cancer. Cancer
Chemother Pharmacol 67:293–304
Tomonaga Y, Hakowa Y, Hera M, Hosomi A (1990) Synthesis of
pyrazolo[3,4-d]pyrimidine derivatives using ketene dithioace-
tals. J Heterocycl Chem 27:775–779
Uehata M, Ono T, Satoh H, Yamagami K, Kawahara T (2001)
Medicines comprising Rho kinase inhibitor. US Patent No.
6218410
Wang HQ, Wei-Pimg Z, Yu-Yuan W, Can-Rong L (2008) Synthesis
and antifungal activities of novel 5-amino-6-arylamino-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one derivatives. J Agric Food
Chem 56:7321–7325
Takaya T, Murata M, Ito K (1988) Pyrimidine compounds having
activity as
a cardiotonic anti-hypertensive cerebrovascular
vasodilator and anti-platelet aggregation agent. US Patent No.
4725600. Application No. 06/751867
Takayama Y, Yoshida Y, Uehata M, (2006) Visual function disorder
improving agents, US Patent No. 7109208. Application No.
10474369
123