Synthesis and antibacterial activity of some novel pyrazolopyridine derivatives

Article abstract of DOI:10.1007/s10593-011-0699-y

Eight pyrazolo[3,4-b]pyridine derivatives have been synthesized by Friedlaender condensation of 5-aminopyrazole-4-carbaldehyde with active methylene compounds in basic medium. These compounds have been screened for their antibacterial activity against two Gram-negative and two Gram-positive bacterium. Pyrazolopyridines having the carboxamide group at the 5-position showed moderate to good activity against P. aeruginosa, E. coli, S. pneumoniae, and B. cereus.

Full text of DOI:10.1007/s10593-011-0699-y

Chemistry of Heterocyclic Compounds, Vol. 46, No. 12, March, 2011 (Russian Original Vol. 46, No. 12, December, 2010)
SYNTHESIS AND ANTIBACTERIAL ACTIVITY OF
SOME NOVEL PYRAZOLOPYRIDINE DERIVATIVES
N. Panda1*, S. Karmakar, and A. K. Jena
Eight pyrazolo[3,4-b]pyridine derivatives have been synthesized by Friedländer condensation of
5-aminopyrazole-4-carbaldehyde with active methylene compounds in basic medium. These compounds
have been screened for their antibacterial activity against two Gram-negative and two Gram-positive
bacterium. Pyrazolopyridines having the carboxamide group at the 5-position showed moderate to good
activity against P. aeruginosa, E. coli, S. pneumoniae, and B. cereus.
Keywords: heterocycles, pyrazolo[3,4-b]pyridines, antimicrobial activity, disc diffusion method, Friedländer
condensation.
The evolution of antibacterial resistance in bacterial strains against the currently available antibacterial
agents is an increasing concern in recent years. For instance, Gram-positive bacterial pathogen such as
Staphylococcus aureus is resistant to Methicillin, and Streptococcus pneumoniae and Enterococci are resistant to
Penicillin and Vancomycin, respectively [1]. On the other hand, Gram-negative bacteria such as H. influenza and
M. catarrahalis are resistant to β-lactams, quinolones, and macrolides [2]. In order to overcome the threat of
widespread multidrug resistance in Gram-positive as well as Gram-negative bacterial strains, there is an ongoing
demand for new antibacterials.
Pyrazolo[3,4-b]pyridines as aza-analogs of indazoles [3] have long served as attractive targets in organic
synthesis due to their excellent antibacterial activity [4]. Moreover, pyrazolo[3,4-b]pyridines also exhibit other
promising biological activities, including anxiolytic (e.g., Tracazolate) [5], dopamine D3-receptor antagonist and
partial agonist [6], dopamine D4 antagonist [7], adenosine A1-receptor antagonist [8, 9], antiherpetic [10], anti-
inflammatory [11], and antiallergic [12] properties. The methods so far reported for the synthesis of
pyrazolopyridines include: i) the annelation of the pyrazole ring onto the suitably substituted pyridine ring [13-19],
and ii) the annelation of the pyridine ring onto the appropriately substituted pyrazole ring [13, 20-30]. As part of
our ongoing program [31] for the development of new antibacterials, we are particularly interested in the
synthesis of pyrazolo[3,4-b]pyridines by the latter method. Like others, our strategy involves the Friedländer
condensation of 5-aminopyrazoles with active methylene compounds as the key step. In this paper we report the
full details of our efforts on the synthesis of some novel pyrazolo[3,4-b]pyridine derivatives and their antimicrobial
profile against two Gram-positive and two Gram-negative bacterium species.
_______
* To whom correspondence should be addressed, e-mail: npanda@nitrkl.ac.in.
1 Department of Chemistry, National Institute of Technology, Rourkela-769008, India.
__________________________________________________________________________________________
Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 12, pp. 1857-1866, December, 2010.
Original article received June 10, 2010.
1500
0009-3122/11/4612-1500©2011 Springer Science+Business Media, Inc.

Our studies began with the pyrazolones 2a,b, which were prepared by following a slight modificated
procedure reported earlier [32]. The standard procedure for such pyrazolones includes the reaction of
1,3-dicarbonyl compounds with phenylhydrazine under reflux condition in ethanol. However, this reaction suffers
from poor yield and long reaction time. In order to get a good yield of the product in a shorter reaction time, the
reaction was carried out under microwave irradiation. Thus, treatment of equimolar amounts of 1,3-dicarbonyl
compounds 1a,b with phenylhydrazine and catalytic amounts (1-2 drops) of concentrated sulfuric acid adsorbed
on silica gel G under microwave irradiation for 8 min gave pyrazolones 2a,b in excellent yield (Scheme 1). This
reaction procedure is remarkably simple and requires no solvent or inert atmosphere, and hence is an
environmentally friendly process. It was also observed that when the reaction was carried out under microwave
irradiation without silica gel, it gave a poor yield of the cyclic product.
Scheme 1
O
O
Silica gel G
R
O
+
PhNHNH2
N
N
OEt
H2SO4, MW,
8 min
R
Ph
1a,b
2a,b
1, 2 a R = Ph, b R = Me
The pyrazole derivatives 2a,b were regioselectively formylated at C-4 using Vilsmeier–Haack reaction
conditions (Scheme 2) to give compounds 3a,b in moderate to good yield [33].
Scheme 2
H
O
DMF, POCl3
reflux
R
Cl
2a,b
N
N
Ph
3a,b
3 a R = Ph, b R = Me
The chloro compounds 3a,b were turned into the corresponding azides 4a,b (Scheme 3) by the
nucleophilic displacement of chlorine atom by azide anion, catalyzed by the phase-transfer agent tetrabutyl-
ammonium bromide. The formation of 4a,b was evident from the spectral data. The IR spectra of compounds
4a,b show a strong absorbance at 2148 cm1 for the azide group. Chemoselective reduction of the azide group by
iron powder in the presence of ammonium chloride [33] furnished o-amino aldehydes 5a,b as pale-yellow
crystalline solids in excellent yield.
Scheme 3
O
H
O
H
Fe (powder)
NaN3, TBAB
R
NH2
R
N3
3a,b
N
N
N
N
EtOAc, NH4Cl
H2O, 4 h, rt
DMSO, 24 h, rt
Ph
Ph
5a,b
4a,b
TBAB  tetrabutylammonium bromide; 4, 5 a R = Ph, b R = Me
1501

The Friedländer condensation of o-amino aldehydes such as 5-aminopyrazole-4-carbaldehydes with
ketones is described to take place either with strong bases or acids as catalysts; in special cases the ring closure
can be observed without a catalyst at higher temperatures (e.g., under microwave irradiation) [34-37]. Having
5-aminopyrazolo-4-carbaldehyde in our hand, like others [4, 38–41] we have applied the Friedländer condensa-
tion reaction with active methylene compounds to get pyrazolo[3,4-b]pyridines. Initially we used piperidine [38]
as the base for the condensation reaction, which unfortunately gave poor yield (< 20%). Then we tried other bases
[29, 30, 42] like NaOH in EtOH, NaH in THF, and NaOMe in MeOH. We were pleased to find out that NaOMe
catalyzed the condensation reaction to give the best yield.
Scheme 4
R
R2
NaOMe, MeOH
R1CH2CN
5a,b
+
N
reflux
N
N
R3
6a–c
Ph
7a–f
6 a R1 = CONH2, b R1 = CN, c R1 = CO2Et; 7 a, d, f R = Me, b, c, e R = Ph,
a, b R2 = CONH2, R3 = NH2, c, d R2 = CN, R3 = NH2, e, f R2 = CONH2, R3 = OH
Thus compounds 5a,b on treatment with various active methylene compounds in the presence of an
excess of sodium methoxide under reflux in methanol gave the corresponding pyrazolopyridines 7a-f in good to
excellent yields (Scheme 4). The amino aldehydes 5a,b when reacting with active methylenes like ethyl
cyanoacetate 6c give amides 7e,f, but with malononitrile 6b the same reaction proceeds with the formation of
nitriles 7c,d. This may be due to the fact that the increased electronegativity of the oxygen atom in the OH group
makes the adjacent nitrile group more reactive for conversion to amide. Formation of compound 7 was evident
from spectral as well as analytical data. The reaction of compounds 5a,b with cyclohexanone in the presence of
KOH gave the corresponding annelated pyrazolopyridines 8a,b [43] in good yield (Scheme 5). In order to test the
biological activity of the synthesized pyrazolopyridines, compounds 7a,b and 8a,b were screened against two
pathogenic Gram-positive and two Gram-negative bacteria and the results are shown in Table 1.
Scheme 5
R
O
5a,b
N
KOH, EtOH,
reflux, 2 h
N
N
Ph
8a,b
8 a R = Ph, b R = Me
Antimicrobial Evaluation. The antimicrobial properties of the synthesized compounds were tested in
vitro by the disc diffusion method [44, 45]. The antimicrobial properties are tested against two Gram-positive
(e.g., S. pneumoniae, MTCC 1936, and B. cereus, MTCC 1305) and two Gram-negative (e.g., P. aeruginosa,
NCIM 2074, and E. coli, NCIM 5051) pathogenic bacterial strains. The preliminary screening results indicated
that compounds 7a,b having the amino group at position 6 and the amide group at position 5 of the pyridine ring
show pronounced biological activity. In compounds 7c,d, although the amino group is present at position 6, the
presence of the cyano group at position 5 diminishes the antibacterial activity. The presence of the OH group at
1502

TABLE 1. Screening of Compounds Against Pathogenic Bacteria
Diameter of inhibition zone, mm / Concentration of compound, µg/disk
Compound
P. aeruginosa
60 80 100
E. coli
80 100
S. pneumoniae
B. cereus
80 100
60
60
80 100
60
8
9
10
11
—
—
9
14
16
—
—
15
14
11
11
7
—
—
—
7
9
11
—
—
—
12
—
7
7
—
—
—
10
9
10
—
—
—
12
11
6
14
—
—
—
16
16
8
8
12
—
—
9
12
14
—
—
11
12
7
16
15
—
—
15
16
7
7a
7b
7c
7d
7e
7f
—
—
—
7
—
—
10
—
7
8
10
10
10
18
—
6
10
5
8
6
8a
8b
8
6
7
7
6
6
8
7
8
10
Tetracycline
(30 µg)
14
18
18
position 6 along with the amide group at position 5 also confers similar inhibitory activity. Furthermore,
compounds 8a,b having no amide group at position 5 show somewhat lower antibacterial activity than
compounds 7a,b,e,f. From this observation it may be concluded that the presence of the cyano group at position 5
in the pyrazolo[3,4-b]pyridine ring is responsible for the loss of biological activity.
Conclusion. We have successfully synthesized a series of pyrazolo[3,4-b]pyridine via Friedländer
condensation of 5-aminopyrazole-4-carbaldehyde with active methylene compounds. Compounds 7a,b,e,f show
significant antibacterial activity against four pathogenic bacterium species. The presence of the carboxamide
group at position 5 of pyrazolo[3,4-b]pyridine may be responsible for pronounced antibacterial activity which, in
turn, is suppressed by the cyano group in the same position.
EXPERIMENTAL
Reactions are generally performed under nitrogen atmosphere in flame-dried round bottom flasks.
Reagents were purchased from suppliers like SRL, Spectrochem, Loba, s.d. Fine etc., and were used without
further purification unless otherwise stated. Products were purified by column chromatography on silica gel
(60120 mesh, Rankem) or by recrystallization from distilled solvents. IR spectra were recorded on Perkin Elmer
spectrophotometer on KBr disks. 1H and 13C NMR spectra were recorded on Bruker Avance Digital (400 and 100
MHz respectively) spectrometers using TMS as an internal standard in CDCl3 (compounds 2a,b, 3a,b, 4a,b, 5a,b,
7c,d, 8a,b) and DMSO-d6 (compounds 7a,b,e,f). All 13C spectra were proton decoupled; the "multiplicity" of the
signals was determined using the DEPT technique. Mass spectra were taken by the EI (70 eV) technique on the
Shimadzu QP 2010 PLUS GC-MS system. Elemental analyses were carried out with a Vario EL Elemental
Analyzer. Melting points were uncorrected.
1,3-Diphenyl-1H-pyrazol-5(4H)-one (2a). To a mixture of ethyl benzoylacetate 1a (0.45 ml, 2.6 mmol)
and phenylhydrazine (0.25 ml, 2.6 mmol) a drop of concentrated sulfuric acid was added, and the resulting
mixture along with 2 g of silica gel G was ground thoroughly. The mixture was heated in a domestic microwave
oven (900 W) for 8 min. Then it was washed with CH2Cl2 repeatedly, the combined CH2Cl2 extracts were dried
over Na2SO4, and the solvent was removed under reduced pressure yielding compound 2a as a yellow crystalline
solid, yield 92%; mp 110-128°C. IR spectrum, ν, cm–1: 1709 (C=O). 1H NMR spectrum, , ppm: 8.01-7.22 (10H,
m, two C6H5); 3.85 (2H, s, CH2). 13C NMR spectrum, δ, ppm: 170.26 (s); 154.67 (s); 138.13 (s); 130.87 (s, C=O);
130.74 (d); 128.94 (d); 128.89 (d); 126.00 (d); 125.33 (d); 119.09 (d); 39.67 (t, CH2). Found, %: C 76.11; H 5.08;
N 11.80; O 6.60. C15H12N2O. Calculated, %: C 76.25; H 5.12; N 11.86; O 6.77.
1503

3-Methyl-1-phenyl-1H-pyrazol-5(4H)-one (2b). Ethyl acetoacetate (0.49 ml, 3.84 mmol) and phenyl-
hydrazine (0.38 ml, 3.84 mmol) were taken, and compound 2b was synthesized, following the same procedure as
1
reported for 2a, as a yellow solid, yield 90%; mp 112-122°C. IR spectrum, ν, cm–1: 1598 (C=N). H NMR
spectrum, , ppm: 7.88-7.15 (5H, m, C6H5); 3.44 (2H, s, CH2); 2.21 (3H, s, 3-CH3). 13C NMR spectrum, δ, ppm:
170.57 (s, C=O); 156.27 (s); 138.04 (s); 128.83 (d); 125.05 (d); 118.89 (d); 43.11 (t, CH2); 17.03 (q, 3-CH3).
Found, %: C 68.91; H 5.77; N 15.98; O 9.10. C10H10N2O. Calculated, %: C 68.95; H 5.79; N 16.08; O 9.18.
5-Chloro-1,3-diphenyl-1H-pyrazole-4-carbaldehyde (3a). Compound 2a (5.5 g, 23.5 mmol) was
refluxed for 6 h with a mixture of POCl3 (8.79 ml, 94.06 mmol) and N,N-dimethylformamide (7.27 ml, 94.06
mmol) and allowed to cool to room temperature. Then, cold water (80 ml) was added followed by neutralization
with concentrated aqueous sodium hydroxide. The mixture was extracted with dichloromethane, and the organic
layers were dried with anhydrous sodium sulfate and concentrated under reduced pressure. The crude solid was
purified by column chromatography, yielding compound 3a as white crystalline solid, yield 83%; mp 75°C. IR
spectrum, ν, cm–1: 1683 (C=O). 1H NMR spectrum, , ppm (J, Hz): 10.01 (1H, s, CHO); 7.91-7.71 (2H, m, C6H5),
7.65-7.50 (2H, m, C6H5) 7.48–7.32 (6H, m, C6H5). 13C NMR spectrum, δ, ppm: 183.94 (CHO); 154.30 (s); 136.99
(s); 133.26 (s); 130.79 (s); 129.61 (d); 129.49 (d); 129.33 (d); 128.97 (d); 128.61 (d); 125.50 (d); 116.46 (s).
5-Chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde (3b). A mixture of compound 2b (5 g,
28.73 mmol), DMF (7.11 ml, 91.95 mmol), and POCl3 (18.79 ml, 201.11 mmol) was refluxed for 90 min. The
reaction mixture was cooled to room temperature; cold water was added to it, followed by neutralization with
aqueous sodium hydroxide. The mixture was extracted with dichloromethane, and the organic layer was dried
over Na2SO4, and concentrated under reduced pressure. The residue was purified by flash-chromatography on
silica gel to give compound 3b as a white crystalline solid, yield 65%; mp 120°C. IR spectrum, ν, cm–1: 1676
(C=O). 1H NMR spectrum, , ppm: 10.00 (1H, s, CHO); 7.65-7.42 (5H, m, C6H5); 2.56 (3H, s, 3-CH3). 13C NMR
spectrum, δ, ppm: 183.90 (d, CHO); 151.79 (s); 136.96 (s); 133.48 (s); 129.29 (d); 129.21 (d); 125.20 (d); 117.42
(s); 13.85 (q, 3-CH3).
5-Azido-1,3-diphenyl-1H-pyrazole-4-carbaldehyde (4a). A mixture of compound 3a (2.70 g, 9.82 mmol),
sodium azide (0.77 g, 11.79 mmol), and tetrabutylammonium bromide (0.38 g, 1.17 mmol) was dissolved in
DMSO (22 ml) and stirred at room temperature for 24 h. The reaction mixture was poured onto ice, and the solid
precipitate was extracted with dichloromethane. The organic layer was washed repeatedly with water and dried
over Na2SO4. The organic layer was concentrated under reduced pressure to give compound 4a as a cream-
1
colored solid, yield 84%; mp 102–104°C. IR spectrum, ν, cm–1: 2148 (N3), 1666 (C=O). H NMR spectrum, ,
ppm: 9.99 (1H, s, CHO); 7.82–7.62 (5H, m, C6H5); 7.58–7.42 (5H, m, C6H5). 13C NMR spectrum, δ, ppm: 185.51
(d, CHO); 155.07 (s); 141.07 (s); 137.14 (s); 131.01 (d); 129.51 (d); 129.12 (d); 129.08 (d); 128.90 (d); 128.73
(d); 124.53 (s); 111.69 (s). Found, %: C 66.20; H 3.84; N 23.88; O 5.54. C16H11N5O. Calculated, %: C 66.43;
H 3.83; N 24.21; O 5.53.
5-Azido-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde (4b). Compound 3b (3.00 g, 13.61 mmol)
was taken, and, following the same procedure as for compound 4a, compound 4b was synthesized in 95% yield;
mp 42°C. IR spectrum, ν, cm–1: 2144 (N3), 1673 (C=O). 1H NMR spectrum, , ppm: 9.93 (1H, s, CHO); 8.10-7.01
(5H, m, C6H5); 2.51(3H, s, 3-CH3). 13C NMR spectrum, δ, ppm: 183.62 (d, CHO); 152.07 (s); 137.02 (s); 129.11
(d); 128.56 (d); 124.28 (d); 112.33 (s); 12.72 (q, 3-CH3). Found, %: C 57.90; H 4.06; N 30.55; O 7.09. C11H9N5O.
Calculated, %: C 58.14; H 3.99; N 30.82; O 7.04.
5-Amino-1,3-diphenyl-1H-pyrazole-4-carbaldehyde (5a). To a mixture of compound 4a (2.76 g, 9.5 mmol),
iron powder (1.72 g, 30.6 mmol), and ethyl acetate (10 ml), a solution of ammonium chloride (2.72 g, 50.9 mmol) in
water (10 ml) was added, and the resulting emulsion was stirred at room temperature for 4 h. The mixture was
filtered and the residue washed with ethyl acetate (210 ml). The organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The crude solid was purified by column chromatography to give compound
5a as a cream-coloured crystalline solid, yield 92%; mp 135-137°C. IR spectrum, ν, cm–1: 3424, 3307 (NH2),
1
1642 (C=O). H NMR spectrum, , ppm: 9.90 (1H, s, CHO); 7.80-7.70 (2H, m, C6H5); 7.66-7.42 (8H, m,
1504

13
C6H5); 6.00 (2H, br. s, NH2). C NMR spectrum, δ, ppm: 185.49 (d, CHO); 153.47 (s); 149.94 (s); 136.9 (s);
131.57 (s); 129.95 (d); 129.13 (d); 128.78 (d); 128.61 (d); 128.49 (d); 124.01 (d); 104.73 (s). Mass spectrum, m/z,
(I, %): 263 [M]+ (36), 77 [M–C10H8N3O]+ (100). Found, %: C 72.51; H 4.88; N 15.45; O 5.98. C16H13N3O.
Calculated, %: C 72.99; H 4.98; N 15.96; O 6.08.
5-Amino-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde (5b). Compound 4b (2.9 g, 12.9 mmol) was
taken, and following the same procedure as for 5a, compound 5b was obtained as a yellow solid, yield 91%; mp
1
96°C. IR spectrum, ν, cm–1: 3399, 3287 (NH2), 1645 (C=O). H NMR spectrum, , ppm (J, Hz): 9.64 (1H, s,
13
CHO); 7.49-7.30 (5H, m, C6H5); 5.77 (2H, br. s, NH2); 2.33 (3H, s, 3-CH3). C NMR spectrum, δ, ppm: 183.94
(d, CHO); 150.78 (s); 149.2 (s); 136.9 (s); 129.87 (d); 128.19 (d); 123.69 (d); 105.74 (s); 11.17 (q, 3-CH3).
Found, %: C 65.79; H 5.46; N 20.80; O 7.95. C11H11N3O. Calculated, %: C 65.66; H 5.51; N 20.88; O 7.95.
6-Amino-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (7a). To a solution of
NaOMe (0.134 g, 2.487 mmol) in methanol, cyanoacetamide (0.049 g, 0.497 mmol) was added dropwise under an
inert atmosphere. The resulting mixture was stirred for 0.5 h. A solution of compound 5b (0.10 g, 0.49 mmol)
dissolved in methanol was injected into the stirred mixture, which was then refluxed for 48 h, cooled to room
temperature, neutralized by adding diluted HCl, and extracted with ethyl acetate. The organic layer was washed
repeatedly with water, dried over Na2SO4, and concentrated under reduced pressure. The crude residue was
purified by column chromatography using methanol and diethylamine as eluent. Compound 7a was obtained in
1
91% yield; mp 228°C. IR spectrum, ν, cm–1: 3410, 3319 (NH2), 1695 (C=O). H NMR spectrum, , ppm: 8.52
(1H, s, H-4); 8.31-8.22 (2H, m, C6H5); 8.07 (1H, br. s, CONH2); 7.78 (1H, br. s, CONH2); 7.59-7.44 (2H, m,
13
C6H5); 7.40 (2H, br. s, NH2); 7.25-7.19 (1H, m, C6H5); 2.51 (3H, s, 3-CH3). C NMR spectrum, δ, ppm: 169.87
(s, CONH2); 159.13 (s); 151.05 (s); 144.10 (s); 139.49 (s); 132.34 (d); 128.87 (d); 124.68 (d); 119.36 (d); 108.77
(s); 106.66 (s); 12.07 (q, 3-CH3). Found, %: C 62.82; H 4.76; N 26.01; O 5.43. C14H13N5O. Calculated, %:
C 62.91; H 4.90; N 26.20; O 5.99.
6-Amino-1,3-diphenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (7b) was obtained by Friedländer
condensation of compound 5a (0.1 g, 0.38 mmol) with cyanoacetamide in 65% yield; mp 234°C. IR spectrum, ν,
1
cm–1: 3412, 3330 (NH2), 1665 (C=O). H NMR spectrum, , ppm: 8.74 (1H, s, H-4); 8.41-8.28 (3H, m, C6H5);
8.14-8.08 (2H, m, C6H5); 7.85-7.75 (2H, br. s, CONH2); 7.62-7.49 (6H, m, C6H5 + NH2); 7.31 (1H, m, C6H5). 13C
NMR spectrum, δ, ppm: 169.82 (s, CONH2); 158.90 (s); 151.60 (s); 144.86 (s); 139.27 (s); 132.86 (d); 132.11 (s);
128.96 (d); 128.92 (d); 128.84 (d); 127.12 (d); 125.44 (d); 120.29 (d); 108.20 (s); 106.49 (s). Mass spectrum, m/z
(I, %): 329 [M]+ (65), 311 [M-NH4]+ (46), 77 [M-C13H10N5O]+ (100). Found, %: C 69.07; H 4.33; N 21.22;
O 4.74. C19H15N5O. Calculated, %: C 69.29; H 4.59; N 21.26; O 4.86.
6-Amino-1,3-diphenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (7c). To a solution of sodium (0.8 g,
3.8 mmol) in dry methanol, malononitrile (0.04 ml, 0.76 mmol) was added dropwise under an inert atmosphere.
The resulting mixture was stirred for 30 min. Compound 5b (0.2 g, 0.76 mmol) dissolved in methanol was
injected to the stirred mixture and the reaction mixture was refluxed for 8 h. The mixture was cooled to room
temperature and neutralized by adding diluted HCl. The compound was extracted with chloroform, and the
organic layer was washed with water several times. The organic layer was dried over Na2SO4 and concentrated.
The crude compound was purified by column chromatography to give the target compound 7c in 67% yield; mp
1
305°C. IR spectrum, ν, cm–1: 2210 (CN), 3334, 3470 (NH2). H NMR spectrum, , ppm: 8.46 (1H, s, H-4);
8.28-8.22 (2H, m, C6H5); 7.98-7.90 (2H, m, C6H5); 7.60-7.45 (5H, m, C6H5); 7.40-7.32 (1H, m, C6H5); 5.45 (2H,
13
br. s, NH2). C NMR spectrum, δ, ppm: 157.69 (s); 151.42 (s); 145.69 (s); 138.80 (s); 137.71 (d); 131.83 (s);
129.32 (d); 129.11 (d); 129.02 (d); 127.32 (d); 126.50 (d); 121.62 (d); 117.07 (s); 108.91 (s); 88.94 (s). Found, %:
C 73.15; H 4.05; N 22.33. C19H13N5. Calculated, %: C 73.30; H 4.21; N 22.49.
6-Amino-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (7d) was obtained by
Friedländer condensation of compound 5b (0.1 g, 0.497 mmol) with malononitrile in 63% yield; mp 225-226°C.
1
IR spectrum, ν, cm–1: 3469, 3333 (NH2), 2209 (CN). H NMR spectrum, , ppm: 8.10-7.95 (3H, m, H-4 and
1505

13
C6H5); 7.45-7.30 (2H, m, C6H5); 7.25-7.12 (1H, m, C6H5); 5.31 (2H, br. s, NH2); 2.46 (3H, s, 3-CH3). C NMR
spectrum, δ, ppm: 157.93 (s); 150.95 (s); 144.08 (s); 138.80 (s); 136.49 (d); 128.99 (d); 126.10 (d); 121.15 (d);
117.20 (s); 110.66 (s); 87.72 (s); 12.35 (q, 3-CH3). Found, %: C 67.34; H 4.38; N 28.03. C14H11N5. Calculated, %:
C 67.46; H 4.45; N 28.10.
6-Hydroxy-1,3-diphenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (7e) was synthesized by
Friedländer condensation of compound 5a (0.1 g, 0.38 mmol) with ethyl cyanoacetate (0.04 ml, 0.38 mmol).
Compound 7e was purified by recrystallization from methanol, yield 95%; mp 280-282°C. IR spectrum, ν, cm–1:
1
3457, 3354 (NH2), 1688 (C=O). H NMR spectrum, , ppm: 8.81 (1H, s, H-4); 8.38–8.30 (2H, m, C6H5);
8.05-7.98 (2H, m, C6H5); 7.80 (2H, br. s, CONH2); 7.60-7.28 (5H, m, C6H5); 7.25-7.12 (1H, m, C6H5). 13C NMR
spectrum, δ, ppm: 168.40 (s, CONH2); 159.17 (s); 152.36 (s); 145.21 (s); 139.09 (s); 136.17 (d); 131.89 (d);
129.13 (d); 129.04 (d); 128.99 (d); 126.96 (d); 125.64 (d); 120.44 (s); 107.24 (s); 104.82 (s). Mass spectrum, m/z
(I, %): 330 [M]+ (78), 77 [M-C13H9N4O2]+ (100). Found, %: C 68.97; H 4.11; N 16.71; O 9.74. C19H14N4O2.
Calculated, %: C 69.08; H 4.27; N 16.96; O 9.69.
6-Hydroxy-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (7f) was synthesized by
Friedländer condensation of compound 5b (0.05 ml, 0.497 mmol) with ethyl cyanoacetate (0.05 ml, 0.49 mmol),
1
yield 63%; mp 270-276°C. IR spectrum, ν, cm–1: 3410, 3313 (NH2), 1698 (C=O). H NMR spectrum, , ppm:
8.60 (1H, s, H-4); 8.30-8.20 (2H, m, C6H5); 7.77 (2H, br. s, CONH2); 7.52-7.44 (2H, m, C6H5); 7.28-7.20 (1H, m,
C6H5); 2.4 (3H, s, CH3). 13C NMR spectrum, δ, ppm: 168.57 (s, CONH2); 159.39 (s); 151.83 (s); 144.75 (s);
139.32 (s); 135.82 (d); 128.88 (d); 124.86 (d); 119.54 (d); 109.50 (s); 103.38 (s); 12.04 (q, 3-CH3). Mass
spectrum, m/z (I, %): 268 [M]+ (100), 250 [M–H2O]+ (35), 77 [M-C8H7N4O2]+. Found, %: C 62.53; H 4.40;
N 20.54; O 11.78. C14H12N4O2. Calculated, %: C 62.68; H 4.51; N 20.88; O 11.93.
1,3-Diphenyl-5,6,7,8-tetrahydro-1H-pyrazolo[3,4-b]quinoline (8a). A solution of compound 5a (0.1 g,
0.38 mmol) and cyclohexanone (0.04 ml, 0.38 mmol) was refluxed in 2% KOH in ethanol for 2 h. Then ethanol
was evaporated and to the residue water was added. After neutralization with dilute HCl the compound was
extracted with dichloromethane. Recrystallization from ethanol gave the pure compound 8a as yellow crystals,
1
yield 86%; mp 138-142°C. IR spectrum, ν, cm–1: 3040 (C–H), 2934, 2862 (CH2). H NMR spectrum, , ppm:
8.46-8.42 (2H, m, H-4, C6H5); 8.31-8.00 (3H, m, C6H5); 7.81-7.42 (5H, m, C6H5); 7.34-7.21 (1H, m, C6H5);
3.30-2.90 (4H, m, 5,8-CH2); 2.22-1.80 (4H, m, 6,7-CH2). 13C NMR spectrum, δ, ppm: 158.42 (s); 150.07 (s);
143.48 (s); 139.88 (s); 133.17 (s); 130.07 (d); 128.97 (d); 128.89 (d); 128.48 (d); 127.27 (d); 127.16 (s); 125.48
(d); 120.95 (d); 114.18 (s); 33.60 (t, CH2); 29.38 (t, CH2); 23.09 (t, 6,7-CH2). Found, %: C 81.07; H 5.69; N 12.66.
C22H19N3. Calculated, %: C 81.20; H 5.89; N 12.91.
3-Methyl-1-phenyl-5,6,7,8-tetrahydro-1H-pyrazolo[3,4-b]quinoline (8b) was synthesized by Friedländer
condensation of compound 5b (0.1 g, 0.49 mmol) with cyclohexanone (0.05 ml, 0.49 ml) as white crystals in 82%
yield; mp 72-82°C. IR spectrum, ν, cm–1: 3063 (C–H), 2935, 2862 (CH2). 1H NMR spectrum, , ppm: 8.50–8.21 (2H,
m, H-4, C6H5); 7.88-7.68 (1H, m, C6H5); 7.65-7.42 (2H, m, C6H5); 7.40-7.12 (1H, m, C6H5); 3.35–2.82 (4H, m,
6,7-CH2); 2.61 (3H, s, 3-CH3); 2.26-1.80 (4H, m, 5,8-CH2). 13C NMR spectrum, δ, ppm: 158.22 (s); 149.29 (s); 129.27
(d); 128.97 (d); 126.05 (s); 125.09 (d); 120.51 (d); 115.93 (s); 33.51 (t, CH2); 29.22 (t, CH2); 23.07 (t, 6,7-CH2); 12.46
(q, 3-CH3). Found, %: C 77.43; H 6.32; N 15.65. C17H17N3. Calculated, %: C 77.54; H 6.51; N 15.96.
Antimicrobial Screening. The antibacterial activity of the compounds was determined by the disc
diffusion method [44, 45]. The bacteria were cultured in nutrient broth medium (from HiMedia) and used as
inocula for the study. Bacterial cells were swabbed into nutrient agar medium in Petri dishes. The test solutions
were prepared in DMSO to a concentration of 5 mg/ml, and then 12, 16, and 20 µl (60, 80, and 100 µg,
respectively) of this solution were applied to filter paper discs (Whatmann No. 4, 5 mm dia), which were placed
upon already seeded plates. These plates were incubated at 35±2°C for 24 h. Tetracycline was used as a standard
positive control.
The authors are thankful to DST (Ref No. SR/FTP/CS-101/2006), India, for financial support.
1506

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