Regioselective synthesis and antibacterial evaluation of a new class of substituted pyrazolo[3,4-b]pyridines

Article abstract of DOI:10.3184/174751913X13735444714766

New derivatives of polyfunctional pyrazolo[3,4-b]pyridines were synthesised via a regioselective one-pot three-component reaction of 3- (cyanoacetyl)indole, 5-amino-3-methylpyrazole and arylaldehydes in excellent yields using Fe3+-montmorillonite as a reusable catalyst under conventional conditions and ultrasonic irradiation. In particular, valuable features of this method include high reaction rate, employment of a reusable catalyst, straightforward procedure, the easy work-up of the products and mild reaction conditions. The antibacterial activities of the synthesised products were examined which showed promising activities.

Full text of DOI:10.3184/174751913X13735444714766

494 RESEARCH PAPER
AUGUST, 494–498
JOURNAL OF CHEMICAL RESEARCH 2013
Regioselective synthesis and antibacterial evaluation of a new class of
substituted pyrazolo[3,4-b]pyridines
Manouchehr Mamaghania*, Khalil Tabatabaeiana, Mitra Bayata, Roghayeh Hossein Niaa and
Mehdi Rassab
aDepartment of Chemistry, Faculty of Sciences, University of Guilan, PO Box 41335-1914, Rasht, Iran
bDepartment of Biology, Faculty of Sciences, University of Guilan, PO Box 41335-1914, Rasht, Iran
New derivatives of polyfunctional pyrazolo[3,4-b]pyridines were synthesised via a regioselective one-pot three-com-
ponent reaction of 3- (cyanoacetyl)indole, 5-amino-3-methylpyrazole and arylaldehydes in excellent yields using
Fe3+-montmorillonite as a reusable catalyst under conventional conditions and ultrasonic irradiation. In particular,
valuable features of this method include high reaction rate, employment of a reusable catalyst, straightforward pro-
cedure, the easy work-up of the products and mild reaction conditions. The antibacterial activities of the synthesised
products were examined which showed promising activities.
Keywords: pyrazolo[3,4-b]pyridine, pyrazole, indole, Fe3+-montmorillonite, ultrasound, antibacterial activity
Aminopyrazoles belong to a very important class of heterocy-
cles due to their biological and pharmacological activities.1,2
Moreover, they are used as key starting materials for the
synthesis of heterocycles containing a pyrazole structural unit.
Many such compounds display a broad spectrum of pharmaco-
logical and biological activities, such as antimicrobial,3
antifungal4 and antitumour properties.5
Combination of the pyrazole moiety with various heterocy-
clic ring systems has resulted in interesting biological
and pharmacological properties.6–9 Pyrazolo[3,4-b]pyridine
skeletons possess diverse biological properties, including
antioxidant, antibacterial and antitubercular activities.10–14
Some derivatives exhibit potential antimalarial properties,15
fungicidal activities16 and also used as vasodilators.17
Following our continued interest in the development of
synthetic strategies to obtain functionalised heterocycles of
biological importance,18–23 also guided by the observation that
the presence of two or more different heterocyclic moieties in
a single molecule often enhances the biocidal profile remark-
ably,18 and considering the important biological properties
of indole containing heterocycles, we now describe a novel
and clean synthesis of indole-sustituted pyrazolopyridine
derivatives through a three-component condensation reaction
of 3-cyanoacetylindole (1), 5-amino-3-methylpyrazole (2) and
arylaldehydes (3) in the presence of Fe3+-montmorillonite
(Fe3+@Mont.) as a reusable catalyst in ethanol under reflux
conditions and ultrasonic irradiations (Scheme 1).
by the reaction of equimolar amounts of 3- (cyanoacetyl)indole
(1), 5-amino-3-methylpyrazole (2) and 2,4-dichlorobenzalde-
hyde (3a) in the presence of Fe3+@Mont. was selected as
model reaction. Different solvents and catalysts were screened
for the synthesis of 4a. The results are summarised in Tables 1
and 2. It is evident from the results that EtOH (Table 1, entry
5) and Fe3+@Mont. (Table 2, entry 2), are the most effective
solvent and catalyst respectively, resulting in the highest yield
and shortest reaction time among the solvents and catalysts
selected.
We also verified the amount of catalyst needed for the prep-
aration of 4a, and the best result was obtained using 0.05 g
Fe3+@Mont./1 mmol substrate in EtOH (5 mL) (Table 3).
The catalyst was recycled and reused in the preparation of
4a as model compound. After each run, the catalyst was simply
filtered, washed and activated at 120 °C. The result showed
that after three successive runs, catalytic activity of the catalyst
was retained without significant loss of activity (Table 4).
The plausible mechanism for the formation of 4a–i is out-
lined in Scheme 2. The formation of these products can be
visualised by initial Knoevenagel condensation of aldehyde
(3) and 3-cyanoacetylindoles (2). The Fe3+@Mont. activated
arylidene intermediate, seems to be a good acceptor for the
Michael addition of 2 via attack of the nucleophilic C-4 of the
pyrazole, followed by cyclisation and loss of H2O to furnish
the desired pyrazolopyridines 4a–i.
The regioselectivity observed in this reaction may be ratio-
nalised by hard–soft–acid–base interaction in the reaction
intermediate, in which the nitrogen group prefer carbonyl
group functionality rather than the CN group. The result of
the reaction between 3- (cyanoacetyl)indole (1), 3-amino-
5-methylpyrazole (2), and various aryl aldehydes (3) in the
Result and discussion
Synthesis of 4-(2,4-dichlorophenyl)-6-(1H-indol-3-yl)-3-methyl-
4,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (4a),
Scheme 1 Synthesis of indolylpyrazolo[3,4-b]pyridine derivatives 4a–i.
* Correspondent. E-mail: m-chem41@guilan.ac.ir;
mchem41@gmail.com

JOURNAL OF CHEMICAL RESEARCH 2013 495
Table 1 Synthesis of 4a in the presence of Fe3+@Mont. (0.05 g
/L mmol substrate) in several solvents
Table 3 Optimisation of the amount of catalyst in the synthesis
of 4a
Entry
Solvent
Time/min
Yield/%a
Entry
Amount of catalyst/g
Time/min
Yield/%a
1
2
3
4
5
6
7
8
9
Ethylenglycol
MeOH
DMF
60
70
75
68
62
55
89
45
40
35
30
1
2
3
0.02
0.05
0.1
35
25
25
80
89
85
90
THF
100
25
150
180
210
240
a Isolated yield.
EtOH
AcOH
CH3CN
CH2Cl2
H2O
Table 4 Reusability of Fe3+@Mont. in the preparation of 4a
under ultrasonic irradiation
a Isolated yield.
Entry
No. of runs
Time/min
Yield/%a
1
2
3
4
5
First
Second
Third
Fourth
Fifth
4
4
4
4
4
98
95
89
73
64
Table 2 Effect of various catalysts in the synthesis of 4aa
Entry
Catalyst
Time/min
Yield/%b
a Isolated yield.
1
2
3
4
5
6
7
8
K10
45
25
40
45
50
50
55
60
75
89
80
70
80
75
75
75
Fe3+@Mont.
p-TSA
KSF
L-proline
Cellulose–sulfuric acid
presence of Fe3+@Mont. in refluxing ethanol under optimised
conditions has been summarised in Table 5. The aromatic alde-
hydes bearing electron-donating groups as well as electron
withdrawing groups underwent reactions successfully.
Recently, organic chemists have tried to develop green and
environmentally benign routes for the preparation of organic
AcOH
–
a EtOH as a solvent.
b Isolated yield.
Scheme 2 Plausible mechanism of synthesis of indolylpyrazolo[3,4-b]pyridines 4a–i.

496 JOURNAL OF CHEMICAL RESEARCH 2013
Table 5 Synthesis of products 4a–i using Fe3+@Mont. in refluxing EtOH and under ultrasonic irradiations at 60 °C
Entry
Product
R1
Ar
Classical method
Sonochemical method
Time /min
Yield/%a
Time /min
Yield/%a
1
2
3
4
5
6
7
8
9
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
H
2,4-Cl2C6H3
3-HOC6H4
25
30
30
20
40
25
25
25
25
89
90
80
85
80
90
90
90
90
4
5
5
3
7
3
3
4
3
98
98
90
95
85
98
98
98
98
H
3-MeOC6H4
3-O2NC6H4
4-CH3C6H4
2,6-ClFC6H3
2,4-Cl2C6H3
3-HOC6H4
H
H
H
Me
Me
Me
2,6-ClFC6H3
a Isolated yield.
compounds of synthetic and biological values. In this area, the
use of ultrasound has gained popularity compared to conven-
tional heating.24 We have used ultrasonic irradiation in the
synthesis of potent compounds with medicinal values18–20,25,26
which has encouraged us to explore the use of this methodol-
ogy in the synthesis of pyrazolopyridinones. We therefore
investigated the effect of ultrasonic irradiation on the synthesis
of novel fused pyrazolopyrines (4a–i). An equimolar mixture
of reactants in the presence of Fe3+@Mont. (0.05 g) in EtOH
(5 mL) were placed in a pyrex-glass open vessel and irradiated
at 60 °C by ultrasonic irradiation (40 kHz) to furnish the
desired products (4a–i) in very short reaction times (3–7 min)
and excellent yields (85–98%) (Table 5).
Experimental
Melting points were measured on an electrothermal 9100 apparatus.
IR spectra were determined on a Shimadzo IR-470 spectrometer. H
1
NMR and 13C NMR spectra were recorded on a 400 MHz Bruker
DRX-400 in DMSO-d6 as solvent and TMS as an internal standard.
Chemical shifts on 1H and 13C NMR were expressed in ppm downfield
from tetramethylsilane. Sonication was performed in Elmasonic S
40H ultrasonic cleaning unit (40 kHz). Elemental analyses were done
on a Carlo-Erba EA1110CNNO-S analyser and agreed with the calcu-
lated values. All the chemicals were purchased from Merck and used
without further purification. All solvents used were dried and distilled
according to standard procedures.
Synthesis of (4a–j); general procedure
A mixture of 3-amino-5-methyl pyrazole (1 mmol), 3- (cyanoacetyl)-
indole27 (1) (1 mmol), arylaldehyde (1 mmol) and Fe3+@ Mont. (0.05 g)
in EtOH (5 mL) was heated under reflux or by ultrasonic irradiation
using Elmasonic S 40H ultrasonic cleaning unit (40 kHz) at 60 °C.
The progress of the reaction was monitored by TLC (EtOAc/petro-
leum ether 8:4). After completion of the reaction, the catalyst was
removed by filtration, and the filtrate was evaporated under reduced
pressure to remove the solvent. The residue was purified by column
chromatography (EtOAc/petroleum ether 8:4) to provide the pure
desired products.
To extend the synthetic potential of this protocol to other
cyanoacetyl substrates, the synthesis of pyrrole substituted
pyrazolo[3,4-b]pyridine derivatives was studied. We investi-
gated the reaction between 2- (cyanoacetyl)-N-methylpyrrole,
5-amino-3-methylpyrazole (2) and arylaldehyde (3j), in the
presence of Fe3+@Mont., using the optimised conditions
described above and obtained 1-methyl-1H-pyrrol-2-yl)-4,7-
dihydro-1H-pyrazolo[3,4-b]pyridine (4j) (Fig. 1) in 120 min
and 70% yield under classic conditions. Under ultrasonic
irradiation, the reaction was completed in 20 min with 73%
yield.
Synthesis of Fe3+@Mont.28
A total of 1% suspension of montmorillonite (K10) in a 1.5 mole dm−3
solution of FeCl3.6H2O was stirred overnight. On settling, the super-
natant solution was discarded and the hhexchange process repeated
three times. The ion-exchanged material was filtered and washed free
of chloride ion (checked by 0.1M AgNO3) with deionised water and
dried in air.
The structures of all the products were fully characterised
1
by spectroscopic (IR, H NMR, 13C NMR) and elemental
analyses.
The antibacterial activity of synthesised compounds 4a–j
was examined using Escherichia coli (EC), Bacillus subtilis
(BS), Pseudomonas Aeruginosa (A), Salmonella enterica (SE)
and Staphylococcus aureus (SA). For comparison, two rou-
tinely used antibiotics, erythromycin and nalidixic acid, were
also used. The results revealed that most of the products (4a–i)
exhibit strong activities towards both Gram-negative and
Gram-positive bacteria, as revealed by the diameters of their
inhibition zones. Among the tested compounds, 4j shows very
strong inhibitory effects on both types of bacterium. The results
of this study are listed in Table 6.
(2,4-Dichlorophenyl)-6-(1H-indol-3-yl)-3-methyl-4,7-dihydro-1H-
pyrazolo[3,4-b]pyridine-5-carbonitrile (4a): Cream powder; m.p.
>300 °C. IR (KBr) (νmax/cm−1): 3396, 3050, 2926, 2858, 2189, 1649,
1
1591, 1514, 1384, 1099, 802, 744. H NMR (DMSO-d6, 400 MHz):
δ 1.83 (3H, s, CH3), 5.45 (1H, s, CH), 7.17–7.09 (2H, m, ArH), 7.54–
7.41 (4H, m, ArH), 7.63 (1H, s, ArH), 7.73 (1H, s, ArH), 9.83 (1H, br.,
s, NH), 11.76 (1H, br., s, NH), 12.08 (1H, br., s, NH) ppm. 13C NMR
(DMSO-d6, 100 MHz): δ 9.8, 29.5, 77.1, 100.1, 109.4, 112.4, 120.0,
120.6, 122.2, 122.6, 125.6, 127.6, 128.6, 129.2, 132.4, 132.8, 133.1,
135.6, 136.3, 142.4, 147.2, 156.6 ppm. Anal. Calcd for C22H15Cl2N5
(420.29): C, 62.87; H, 3.60; N, 16.66. Found: C, 62.75; H, 3.49; N,
16.50%.
4-(3-Hydroxyphenyl)-6-(1H-indol-3-yl)-3-methyl-4,7-dihydro-1H-
pyrazolo[3,4-b]pyridine-5-carbonitrile (4b): Cream powder; m.p.
260–263 °C. IR (KBr) (νmax/cm−1): 3353, 3188, 3068, 2955, 2199,
1650, 1602, 1539, 1504, 1383, 1271, 876, 741. 1H NMR (DMSO-d6,
400 MHz): δ 1.87 (3H, s, CH3), 4.82 (1H, s, CH), 6.64 (1H, ddd,
J = 7.2, 2.4, 0.8 Hz, ArH), 6.72 (1H, t, J = 1.8 Hz, ArH), 6.76 (1H, d,
J = 7.6 Hz, ArH), 7.08–7.19 (3H, m, ArH), 7.46 (1H, d, J = 8.4 Hz,
ArH), 7.54 (1H, d, J = 7.6 Hz, ArH), 7.72 (1H, s, ArH), 9.38 (1H, br.
s, OH), 9.60 (1H, br., s, NH), 11.62 (1H, br. s, NH), 11.89 (1H, br. s,
NH) ppm. 13C NMR (DMSO-d6, 100 MHz): δ 10.0, 33.2, 79.2, 101.4,
109.7, 112.4, 114.1, 114.9, 118.7, 120.0, 120.6, 122.2, 123.3, 125.7,
127.4, 129.7, 136.3, 138.6, 146.2, 148.2, 156.8, 158.0 ppm. Anal.
Calcd for C22H17N5O (367.40): C, 71.92; H, 4.66; N, 19.06. Found: C,
71.75; H, 4.60; N, 19.00%.
Fig. 1 Structures of 3j and 4j.

JOURNAL OF CHEMICAL RESEARCH 2013 497
Table 6 Antimicrobial activity of the compounds 4a–j
Compound
Conc. of compound
µg/well
Antimicrobial activity (Zone of inhibition in mm)
Staphylococcus
aureus
Salmonella
Pseudomonas
aeruginosa
Bacillus subtilis Escherichia coli
4a
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
15
17 (16)a (17)b
16 (17)a
18 (17)a (17)b
16 (16)a
17 (17)a
15
14
15
4b
15
16 (16)a
17 (16)a
14
4c
14
15
12
18 (19)a
4d
13
14
16 (17)a
15
4e
15
16 (16)a
16 (15)a
14
14
17 (18)a
4f
13
15
15
14
4g
16 (17)a
14
16 (15)a (16)b
16 (17)a
14
13
15
4h
14
16 (16)a
13
16 (17)a
23 (23)a (21)b
17
15
4i
15
13
15
4j
20 (21)a (20)b
25 (24)a (26)b
23 (22)a (24)b
25
24 (23)a (24)b
Erythromycin
Nalidixic acid
21
30
25
27
18
20
30
20
19
a Data of duplicated experiments.
b Data of triplicated experiments.
6-(1H-Indol-3-yl)-4-(3-methoxyphenyl)-3-methyl-4,7-dihydro-1H-
pyrazolo[3,4-b]pyridine-5-carbonitrile (4c): Cream powder; m.p.
252–254 °C. (KBr) (νmax/cm−1): 3350, 3050, 2920, 2190, 1645, 1600,
for C22H15ClFN5 (403.84): C, 65.43; H, 3.74; N, 17.34. Found: C,
65.36; H, 3.61; N, 17.25%.
4-(2,4-Dichlorophenyl)-3-methyl-6-(2-methyl-1H-indol-3-yl)-4,7-
dihydro-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (4g): Cream
powder; m.p. >300 °C. (KBr) (νmax/cm−1): 3350, 3300, 3050, 2900,
1
1540, 1510, 1485, 1455, 1420, 1380, 1258, 1040, 875, 740, 698. H
NMR (DMSO-d6, 400 MHz): δ 1.88 (3H, s, CH3), 3.75 (3H, s, OMe),
4.91 (1H, s, CH), 6.83 (1H, dd, J = 7.8, 2.2 Hz, ArH), 6.88 (1H, s,
ArH), 6.90 (1H, d, J = 7.6 Hz, ArH), 7.05–7.21 (3H, m, ArH), 7.47
(1H, d, J = 8.0 Hz, ArH), 7.53 (1H, d, J = 8.0 Hz, ArH) 7.73 (1H, s,
ArH), 9.64 (1H, br. s, NH), 11.65 (1H, br. s, NH), 11.92 (1H, br. s,
NH) ppm. 13C NMR (DMSO-d6, 100 MHz): δ 10.0, 31.7, 55.8 (OMe),
77.6, 101.6, 111.4, 111.9, 112.4, 114.9, 117.7, 120.0, 120.6, 121.0,
121.3, 122.4, 125.0, 127.4, 129.4, 139.3, 139.4, 148.4, 158.9, 161.9
ppm. Anal. Calcd for C23H19N5O (381.43): C, 72.42; H, 5.02; N, 18.36.
Found: C, 72.30; H, 4.89; N, 18.24%.
1
2200, 1645, 1590, 1514, 1460, 1340, 1100, 830, 800, 740. H NMR
(DMSO-d6, 400 MHz): δ 1.83 (3H, s, CH3-pyrazole), 2.37 (3H, s,
CH3-indole), 5.45 (1H, s, CH), 7.32–7.35 (2H, m, ArH), 7.40–7.53
(4H, m, ArH), 7.64 (1H, s, ArH), 9.79 (1H, br. s, NH), 11.42 (1H, br.
s, NH), 11.96 (1H, br., s, NH) ppm. 13C NMR (DMSO-d6, 100 MHz):
δ 9.7, 13.1, 29.4, 76.2, 100.2, 106.9, 111.1, 119.7, 119.8, 121.4, 121.5,
125.3, 127.2, 129.0, 130.3, 133.0, 133.1, 136.2, 136.4, 142.2, 147.2,
157.1 ppm. Anal. Calcd for C23H17Cl2N5 434.32): C, 63.60; H, 3.95;
(
N, 16.12. Found: C, 63.51; H, 3.84; N, 16.01%.
4-(3-Hydroxyphenyl)-3-methyl-6-(2-methyl-1H-indol-3-yl)-4,7-
dihydro-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (4h): Cream
powder; m.p. 242–245 °C. (KBr) (νmax/cm−1): 3355, 3250, 3050,
2930, 2910, 2198, 1650, 1600, 1558, 1510, 1485, 1455, 1380, 1260,
875, 740, 690. 1H NMR (DMSO-d6, 400 MHz): δ 1.87 (3H, s,
CH3-pyrazole), 2.37 (3H, s, CH3-indole), 4.83 (1H, s, CH), 6.63 (1H,
ddd, J = 7.20, 2.4, 0.8 Hz, ArH), 6.72 (1H, s, ArH), 6.77 (1H, d,
J = 8.0 Hz, ArH), 7.01–7.16 (3H, m, ArH), 7.34 (1H, d, J = 8.0 Hz,
ArH), 7.43 (1H, d, J = 8.0 Hz, ArH), 9.53 (1H, br. s, OH), 9.61 (1H,
br. s, NH), 11.39 (1H, br. s, NH), 11.88 (1H, br. s, NH) ppm. 13C
NMR (DMSO-d6, 100 MHz): δ 9.9, 13.1, 32.7, 78.9, 101.4, 107.0,
111.3,114.0, 114.8, 114.9, 118.6, 119.7, 121.3, 122.7, 122.9, 127.2,
127.3, 129.7,135.3, 136.3, 146.1, 148.3, 155.7, 157.9 ppm. Anal.
Calcd for C23H19N5O (381.43): C, 72.42; H, 5.02; N, 18.36. Found: C,
72.31; H, 4.89; N, 18.45%.
6-(1H-Indol-3-yl)-3-methyl-4-(3-nitrophenyl)-4,7-dihydro-1H-pyra-
zolo[3,4-b]pyridine-5-carbonitrile (4d): Yellow powder; m.p. 266–
268 °C. (KBr) (νmax/cm−1): 3360, 3250, 3050, 2910, 2198, 1650, 1600,
1
1505, 1470, 1455, 1520, 1345, 1380, 800, 740, 725, 700. H NMR
(DMSO-d6, 400 MHz): δ 1.87 (3H, s, CH3), 5.23 (1H, s, CH), 7.10
(1H, td, J = 7.46, 0.8 Hz, ArH), 7.18 (1H, td, J = 7.73, 0.8 Hz, ArH),
7.47 (1H, d, J = 8.0, ArH), 7.53 (1H, d, J = 8.00 Hz, ArH), 7.71 (1H,
t, J = 8.0 Hz, ArH), 7.76 (1H, s, ArH), 7.83 (1H, dt, J = 7.60, 1.2 Hz,
ArH), 8.15 (1H, ddd, J = 8.0, 2.4, 0.8 Hz, ArH), 8.17 (1H, s, ArH),
9.82 (1H, br. s, NH), 11.68 (1H, br. s, NH), 12.04 (1H, br. s, NH) ppm.
13C NMR (DMSO-d6, 100 MHz): δ 9.9, 28.4, 78.0, 100.4, 109.4,
112.4, 113.3, 120.1, 120.5, 122.2, 124.4, 125.0, 125.6, 127.7, 130.7,
135.7, 136.2, 136.3, 147.0, 148.5, 148.8, 155.5 ppm. Anal. Calcd for
C22H16N6O2 (396.40): C, 66.66; H, 4.07; N, 21.20. Found: C, 66.51; H,
3.92; N, 21.25%.
6-(1H-Indol-3-yl)-3-methyl-4-(p-tolyl)-4,7-dihydro-1H-pyra-
zolo[3,4-b]pyridine-5-carbonitrile (4e): Cream powder; m.p. >300 °C.
(KBr) (νmax/cm−1): 3350, 3100, 3060, 2900, 2850, 2190, 1650, 1558,
1540, 1505, 1430, 1380, 820, 785, 740. 1H NMR (DMSO-d6,
400 MHz): δ 1.84 (3H, s, CH3), 2.30 (3H, s, CH3), 4.88 (1H, s, CH),
7.07–7.24 (4H, m, ArH), 7.46 (2H, t, J = 8.0 Hz, ArH), 7.50–7.53 (2H,
m, ArH), 7.71 (1H, s, ArH), 9.63 (1H, br. s, NH), 11.74 (1H, br. s,
NH), 12.02 (1H, br. s, NH) ppm. 13C NMR (DMSO-d6, 100 MHz):
δ 10.0, 21.5, 29.5, 79.4, 98.4, 101.4, 119.9, 120.5, 121.0, 122.1, 125.7,
127.9, 129.1, 129.4, 129.5, 136.0, 136.3, 136.8, 142.7, 152.7, 155.5
4-(2-Chloro-6-fluorophenyl)-4,7-dihydro-3-methyl-6-(2-methyl-
1H-indol-3-yl)-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (4i): Cream
powder; m.p. 234–236 °C. (KBr) (νmax/cm−1): 3350, 3280, 3065, 2920,
2195, 1600, 1560, 1570, 1520, 1450, 1380, 1135, 1080, 895, 780,
1
740. H NMR (DMSO-d6, 400 MHz): δ 1.87 (3H, s, CH3-pyrazole),
2.36 (3H, s, CH3-indole), 5.66 (1H, s, CH), 6.99–7.12 (2H, m, ArH),
7.22–7.46 (5H, m, ArH), 9.74 (1H, br. s, NH), 11.39 (1H, br.s, NH),
11.89 (1H, br. s, NH) ppm. Anal. Calcd for C23H17ClFN5 417.87): C,
(
66.11; H, 4.10; N, 16.76. Found: C, 66.01; H, 4.03; N, 16.65%
4-[1-(4-Chlorophenyl)-3-phenyl-1H-pyrazol-4-yl]-3-methyl-6-(1-
methyl-1H-pyrrol-2-yl)-4,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-
carbonitrile (4j): White powder; m.p. 245–247 °C. (KBr) (νmax/cm−1):
3300, 3050, 2920, 2200, 1635, 1537, 1520, 1500, 1420, 1385, 1085,
ppm. Anal. Calcd for C23H19N5 365.43): C, 75.59; H, 5.24; N, 19.16.
(
Found: C, 75.43; H, 5.10; N, 19.25%.
1
4-(2-Chloro-6-fluorophenyl)-6-(1H-indol-3-yl)-3-methyl-4,7-dihy-
dro-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (4f): Cream powder;
m.p. 260–263 °C. (KBr) (νmax/cm−1): 3350, 3280, 3065, 2920, 2195,
1647, 1600, 1560, 1570, 1520, 1450, 1380, 1135, 1080, 895, 780, 740;
1H NMR (DMSO-d6, 400 MHz): δ 1.86 (1H, s, CH3), 5.66 (1H, s,
CH), 7.09 (1H, td, J = 7.2, 0.8 Hz, ArH), 7.16 (1H, td, J = 7.40, 1.2 Hz,
ArH), 7.37 (2H, t, J = 6.40 Hz, ArH), 7.46 (2H, d, J = 8.0 Hz, ArH),
7.53–7.57 (1H, m, ArH), 7.70 (1H, s, ArH), 9.75 (1H, br. s, NH), 11.67
(1H, br. s, NH), 11.94 (1H, br. s, NH) ppm. 13C NMR (DMSO-d6,
100 MHz): δ 9.5, 24.4, 98.8, 109.6, 110.0, 112.4, 112.6, 113.0, 118.4,
120.0, 121.2, 122.1, 122.7, 123.1, 126.0, 126.4,127.2, 129.3, 136.3,
137.0, 143.0, 147.1, 156.0, 161.1 (d, 1JC-F = 240 Hz) ppm. Anal. Calcd
800, 757, 730, 690. H NMR (DMSO-d6, 400 MHz): δ 2.10 (3H, s,
CH3-pyrazole), 3.92 (3H, s, CH3-pyrrole), 5.23 (1H, s, CH), 6.18 (1H,
dd, J = 4.0, 2.8 Hz, ArH), 6.31 (1H, dd, J = 4.8, 2.0 Hz, ArH), 6.81
(1H, dd, J = 3.6, 1.6 Hz, ArH), 7.22 (1H, t, J = 7.6 Hz, ArH), 7.41 (2H,
d, J = 8.4 Hz, ArH), 7.48 (2H, d, J = 8.4 Hz, ArH), 7.62 (2H, t, J =
7.6 Hz, ArH), 7.80 (2H, d, J = 8.0 Hz, ArH), 8.06 (1H, s, ArH), 9.12
(1H, br. s, N–H), 13.84 (1H, br. s, N–H) ppm. 13C NMR (DMSO-d6,
100 MHz): δ 9.9, 21.2, 36.6, 82.2, 101.2, 108.0, 111.1, 114.7, 115.2,
118.5, 119.0, 127.7, 128.5, 128.9, 129.4, 130.3, 130.9, 131.4, 133.6,
139.4, 143.1, 144.0, 149.3, 152.3, 152.4 ppm. Anal. Calcd for
C28H22ClN7 (491.97): C, 68.36; H, 4.51; N, 19.93. Found: C, 68.22; H,
4.39; N, 19.78%.

498 JOURNAL OF CHEMICAL RESEARCH 2013
Determination of antimicrobial activity: The antibacterial activity
of the synthesised compounds (4a–j) was examined using EC, BS, PS,
Salmonella and SA. All media were prepared according to manufac-
turers’ instructions. A colony of each test organism was subcultured
from nutrient agar plates into nutrient broth and incubated at 37 °C for
18 h. Mueller–Hinton agar plates were prepared and four plates were
inoculated with bacteria from nutrient broth cultures. The antibacte-
rial activity was performed by well diffusion technique. A concentra-
tion of 100 µg mL−1 of sample was prepared in DMSO. A sterilised
glass tube (5 mm diameter) was used to aseptically scoop out the solid
medium from the plate to create wells and 40 µL of the sample solu-
tion (6.25 µg/well) was aseptically added. The plates were incubated
at 37 °C for 24 h. After incubation, zone of inhibition was measured.
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Received 2 May 2013; accepted 3 June 2013
Paper1301922 doi: 10.3184/174751913X13735444714766
Published online: 9 August 2013