Pyrazolopyridines II: Synthesis and antibacterial screening of 6-Aryl-3-methyl-1-phenyl-1H-pyrazolo[3,4-B]pyridine-4-carboxylic Acids

Article abstract of DOI:10.14233/ajchem.2014.15918

5-Amino-3-methyl-1-phenyl-1 H-pyrazole was prepared to react with various aromatic aldehydes and pyruvic acid to afford 6-aryl-3-methyl-1-phenyl-1 H-pyrazolo[3,4-b]pyridine-4-carbxylic acids. The prepared compounds were characterized by Mass, IR, ¹H, ¹³C NMR spectra and CHN analysis data. Various 6-aryl-3-methyl-1-phenyl-1 H-pyrazolo[3,4-b]pyridine-4- carbxylic acids synthesized during this work were also evaluated for their antibacterial activities. Some of these compounds were found to be good antibacterial agents.

Full text of DOI:10.14233/ajchem.2014.15918

Asian Journal of Chemistry; Vol. 26, No. 10 (2014), 2870-2872
ASIAN JOURNAL OF CHEMISTRY
http://dx.doi.org/10.14233/ajchem.2014.15918
Pyrazolopyridines II: Synthesis and Antibacterial Screening of
6-Aryl-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylicAcids
1
2
3,*
4
1,2
TAHIR MAQBOOL , AREESHA NAZEER , MUHAMMAD NAEEM KHAN , MARK C. ELLIOTT , MISBAHUL AIN KHAN ,
5
1
5
2
MUHAMMAD ASHRAF , MUHAMMAD NASRULLAH , SHAFIA ARSHAD and MUNAWAR ALI MUNAWAR
1Department of Chemistry, The Islamia University of Bahawalpur, Bahawalpur, Pakistan
2Institute of Chemistry, University of the Punjab, Lahore, Pakistan
3Applied Chemistry Research Centre, PCSIR Laboratories Complex, Lahore, Pakistan
4School of Chemistry, Cardiff University, Cardiff, UK
5Department of Biochemistry and Biotechnology, The Islamia University of Bahawalpur, Bahawalpur, Pakistan
*Corresponding author: Email: changwani_1@yahoo.com
Received: 15 June 2013;
Accepted: 26 December 2013;
Published online: 10 May 2014;
AJC-15137
5-Amino-3-methyl-1-phenyl-1H-pyrazole was prepared to react with various aromatic aldehydes and pyruvic acid to afford 6-aryl-3-
methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbxylic acids. The prepared compounds were characterized by Mass, IR, 1H, 13C NMR
spectra and CHN analysis data. Various 6-aryl-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbxylic acids synthesized during this
work were also evaluated for their antibacterial activities. Some of these compounds were found to be good antibacterial agents.
Keywords: Pyrazolopyridines, Doebner Reaction, Schiff's bases, Pyruvic acid.
TABLE-1
6-ARYL-3-METHYL-1-PHENYL-1H-PYRAZOLO[3,4-
b]PYRIDINE-4-CARBOXYLIC ACIDS (2a-2h)
INTRODUCTION
Pyrazolopyridines are well known for their biological
activities. These are found to be good antiviral agents, potent
P-38 kinase and HIV reverse transcriptases1-4. A number of
pyrazolopyridines have also been reported as good vasodila-
ting, anti inflammatory, analgesics, antipyretic, antimalarial
and antibacterial compounds5-11. We have already presented
the synthesis of some novel ethyl 1,6-diaryl-1H-pyrazolo[3,4-
b]pyridine-4-carboxylates12 which may be considered as ester
analogs of cincophen. In continuation of our work, we would
now like to report our success with their corresponding 4-
carboxylic acids and their antibacterial screening.
Compound
X
Yield (%)
m.p. (°C)
272-274∗
214-216
294∗∗
2a
2b
2c
2d
2e
2f
H
35
46
47
38
34
29
46
40
2-Cl
4-Cl
3-CH3
4-OC2H5
3-NO2
4-F
176-178
192-194
210-212
266-268
260-262
2g
4-OH
2h
∗lit.11 mp:285-287; ∗∗ lit.11 m.p.:280-282
EXPERIMENTAL
aldehydes (2 mmol) and one drop of HCl was heated without
any solvent at 100 °C on an oil bath. It was followed by
successive addition of an equimolar amount of pyruvic acid
and 3 mL of glacial acetic acid. The mixture was further heated
under reflux for 2 h, allowed to cool to room temperature,
filtered and dried. The crude compounds were recrystallized
from ethanol.
The chemicals used were commercially available from
Merck or Fluka and were used as such. However when needed
were purified using normal techniques. The solvents used were
distilled and dried. FTIR spectra were recorded on Bruker
Tensor-27. Melting points were taken on a GallenKamp melting
point apparatus and are uncorrected. The H and 13C NMR
1
spectra were taken on Bruker DPX instrument at 400 MHz
and 100 MHz, respectively.
In the case of solid aldehydes (products 2c and 2f), the
Schiff bases were prepared by heating the mixture under reflux
in ethanol (5 mL). Ethanol was evaporated and followed by
the same general procedure as described above.
Preparation of 6-Aryl-1H-pyrazolo[3,4-b]pyridine-4-car-
boxylic acids (2a-2h) (Table-1)
1,6-Diphenyl-3-methyl-1H-pyrazolo[3,4-b]pyridine-4-
carboxylic acid. (2a): Yield: 35 %; m.p.: 270-274 °C. lit.11
General procedure: A mixture of equimolar amounts of
respective 5-aminopyrazole (2.0 mmol), appropriate aromatic

Vol. 26, No. 10 (2014)
Pyrazolopyridines II: Synthesis and Antibacterial Screening 2871
285-287 °C; IR (KBr, νmax, cm-1): 3170 (O-H); 3060-2950
(C-H); 1714 (C=O); 1H NMR (CDCl3): δ 2.81 (s, 3H, CH3),
7.35-8.36 (m, 11H, ArH), 13.81 (br.s, 1H, COOH); 13C NMR
(CDCl3): δ 15.89 (CH3), 112.00, 115.46, 121.48, 126.57,
127.43, 128.82, 128.93, 129.64, 134.90, 138.32, 139.08,
142.92, 152.02, 156.85, 167.41 (C=O); MS: m/z (%) = Calcd.
for (C20H15N3O2) [M+]: 329.12; Found: 329.0 Anal. Calcd. for
C20H15N3O2: C, 72.94; H, 4.59; N, 12.76. Found: C, 72.54; H,
5.09; N, 13.07.
NMR (DMSO): δ 15.52 (CH3), 112.22, 114.70, 120.70,
120.97, 121.76, 124.5, 126.11, 129.04, 129.19, 130.70, 133.59,
138.59, 139.10, 142.38 148.49, 151.11, 153.59, 166.34 (C=O);
MS: m/z (%) = Calcd. for (C20H14N4O4) [M+]: 374.10; Found:
374.0; HRMS (EI): Calcd. for (C20H14N4O4) [M+]: 374.1671 ;
Found: 374.1626.
6-(4'-Fluorophenyl)-3-methyl-1-phenyl-1H-pyrazolo
[3,4-b]pyridine-4-carboxylic acid. (2g): Yield: 46 %; m.p.:
266-268 °C; IR (KBr, νmax, cm-1): 3160 (O-H); 3060-2950
(C-H); 1710 (C=O); 1593 (C=C); 1H NMR (DMSO): δ 2.70
(s, 3H, CH3), 7.30-8.35 (m, 9H, ArH), 8.15 (s, 1H, H-5); 13C
NMR (DMSO): δ 15.58 (CH3), 111.48, 114.31, 115.86,
116.08, 120.75, 125.84, 129.18, 129.60, 129.69, 134.05,
136.18, 138.79, 142.27, 151.27, 155.04, 162.19, 164.66,
166.49 (C=O); MS: m/z (%) = Calcd. for (C20H14N3O2F) [M+]:
347.11; Found: 347.1; HRMS (EI): Calcd. for (C20H14N3O2F)
[M+]: 347.1072 ; Found: 347.1097.
6-(3'-Chlorophenyl)-3-methyl-1-phenyl-1H-pyrazolo-
[3,4-b]pyridine-4-carboxylic acid. (2b): Yield: 45 %; m.p.:
214-216 °C; IR (KBr, νmax, cm-1): 3152 (O-H); 3030-2920 (C-
H); 1705 (C=O); 1585 (C=C); 1H NMR (DMSO): δ 2.70 (s,
3H, CH3), 7.3-8.3 (m, 10H, ArH); 13C NMR (DMSO): δ 15.96
(CH3), 115.31, 121.47, 125.55, 125.93, 127.49, 128.96, 129.66,
130.06, 134.88, 135.01, 139.01, 140.09, 142.90, 155.15,
167.16 (C=O); MS: m/z (%) = Calcd. for (C20H14N3O2Cl) [M+]:
363.08 & [M + 2]: 365.08; Found: 363.0 & 365.1;Anal. Calcd.
for C20H14N3O2Cl: C, 66.03; H, 3.88; N, 11.55. Found: C, 65.74;
H, 3.78; N, 11.34.
6-(4'-Hydroxyphenyl)-3-methyl-1-phenyl-1H-
pyrazolo[3,4-b]pyridine-4-carboxylic acid. (2h): Yield: 40
%; m.p.: 260-262 °C; IR (KBr, νmax, cm-1): 3298 (O-H); 3025-
2940 (C-H); 1706 (C=O); 1594 (C=C); 3298 (OH); 1H NMR
(DMSO): δ 2.70 (s, 3H, CH3), 6.95-8.35 (m, 9H, ArH), 8.05
(s, 1H, H-5), 9.98 (br. s, 1H, OH); 13C NMR (DMSO): δ 15.58
(CH3), 110.77, 113.66, 115.86, 120.59, 125.56, 128.39,
128.90, 129.15, 135.93, 138.97, 142.21, 151.47, 156.25,
159.57, 166.68 (C=O); MS: m/z (%) = Calcd. for (C20H15N3O3)
[M+]: 345.11; Found: 345.11;Anal. Calcd. for C20H15N3O3: C,
69.56; H, 4.38; N, 12.17. Found: C, 69.29; H, 4.59; N, 12.08.
6-(4'-Chlorophenyl)-3-methyl-1-phenyl-1H-pyrazolo-
[3,4-b]pyridine-4-carboxylic acid. (2c): Yield: 47 %; m.p.:
294 °C lit.11 280-282 °C; IR (KBr, νmax, cm-1): 3148 (O-H);
1
3055-2940 (C-H); 1702 (C=O); 1582 (C=C); H NMR
(DMSO); δ 2.70 (s, 3H, CH3), 7.30-8.4 (m, 9H, ArH), 8.18 (s,
1H, H-5); 13C NMR (DMSO): δ 15.56 (CH3), 111.71, 114.37,
120.77, 125.86, 129.05, 129.17, 134.98, 136.19, 136.34,
138.73,142.28, 151.24, 154.78, 166.41 (C=O); MS: m/z (%)
= Calcd. for (C20H14N3O2Cl) [M+]: 363.08 & [M + 2]: 365.08;
Found: 363.1 & 365.1; Anal. Calcd. for C20H14N3O2Cl: C,
66.03; H, 3.88; N, 11.55. Found: C, 66.03; H, 3.74; N, 11.60.
3-Methyl-1-phenyl-6-m-tolyl-1H-pyrazolo[3,4-b]-
pyridine-4-carboxylic acid. (2d) : Yield: 38 %; m.p.: 176-
178 °C; IR (KBr, νmax, cm-1): 3120 (O-H); 3030-2920 (C-H);
1715 (C=O); 1H NMR (CDCl3): δ 2.46 (s, 3H, PhCH3), 2.82
(s, 3H, CH3), 7.26-8.35 (m, 9H, ArH), 8.22 (s, 1H, H-5); 13C
NMR (CDCl3): δ 16.43 (CH3), 21.63 (ArCH3), 111.97, 116.62,
124.65, 125.87, 126.84, 128.91, 129.01, 129.85, 130.79,
132.88, 138.16, 138.60, 139.30, 142.64, 152.21, 156.99,
169.45 (C=O); MS: m/z (%) = Calcd. for (C21H17N3O2) [M+]:
343.13; Found: 343.2; Anal. Calcd. for C21H17N3O2: C, 73.45;
H, 4.99; N, 12.24. Found: C, 73.69; H, 5.24; N, 11.78.
RESULTS AND DISCUSSION
6-Aryl-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-
4-carboxylic acids (2a-2h) were prepared according to
Scheme-I, from 5-amino-3-methyl-1-phenyl-1H-pyrazole13. 5-
Aminopyrazoles were prepared to react with various aromatic
aldehydes to obtain respective Schiff's bases which were, without
isolation, subjected to condensation with pyruvic acid to give
3-methyl-1-phenyl-6-aryl-1H-pyrazolo[3,4-b]pyridine-4-
carboxylic acids (2a-2h). The yields of the recrystallized
products are in the range of 29 to 47 %; the crude yields are
although much higher. No attempts, at this stage, were made
to optimize them. All the products obtained as fine crystals,
are stable compounds at room temperature and were charac-
terized by IR, 1H, 13C NMR spectra.
6-(4'-Ethoxyphenyl)-3-methyl-1-phenyl-1H-pyrazolo-
[3,4-b]pyridine-4-carboxylic acid. (2e): Yield: 34 %;
m.p.:192-194 °C; IR (KBr, νmax, cm-1): 3220 (O-H); 3040-2930
(C-H); 1713 (C=O); 1572 (C=C); 1H NMR (DMSO): δ 1.45
(t, 3H, OCH2CH3, J = 6.8), 2.80 (s, 3H, CH3), 4.11 (q, 2H,
OCH2CH3, J = 6.8), 7.0-8.35 (m, 10H, ArH); 13C NMR
(DMSO): δ 14.38 (OCH2CH3), 15.58 (CH3), 63.45 (OCH2),
111.22, 114.57, 121.56, 124.93, 125.93, 128.93, 129.82,
130.50, 134.69, 138.95, 142.79, 151.89, 156.52, 160.38,
167.32 (C=O); MS: m/z (%) = Calcd. for (C22H19N3O3) [M+]:
373.14; Found: 373.1; Anal. Calcd. for C22H19N3O3: C, 70.76;
H, 5.13; N, 11.25. Found: C, 70.31; H, 5.52; N, 11.16.
COOH
H3C
H3C
(i) XC H CHO,C H OH,HCl
4
6
2 5
N
N
(ii) Pyruvic acid, acetic acid,Reflux
NH2
N
N
N
C6H5
C6H5
X
(2a- 2h)
(1)
Scheme-I
Spectral characterization of synthesized compounds:
In all these compounds C=O group of acid functionality at
4th position showed an absorption band in the IR spectral range
3-Methyl-6-(3'-nitrophenyl)-1-phenyl-1H-pyrazolo-
[3,4-b]pyridine-4-carboxylic acid. (2f): Yield: 29 %; m.p.:
210-212 °C; IR (KBr, νmax, cm-1): 3130 (O-H); 3050-2930
(C-H); 1700 (C=O); 1596 (C=C), 1525, 1345 (NO2); 1H NMR
(DMSO): δ 2.71 (s, 3H, CH3), 7.3-9.0 (m, 10H, ArH); 13C
1
of 1715-1700 cm-1. In the H NMR spectra a singlet in the
range of 2.70 to 2.80 ppm confirmed the presence of three
proton of methyl group present at position 3 of pyrazole ring.

2872 Maqbool et al.
Asian J. Chem.
TABLE-2
ANTIBACTERIAL ACTIVITY OF 6-ARYL-3-METHYL-1-PHENYL-1H-PYRAZOLO[3,4-b]PYRIDINE-4-CARBOXYLIC ACIDS
B. subtilis (+)
MIC50
S.aureus (+)
MIC50
S.typhi (-)
MIC50
P.aureginosa (-)
MIC50
E.coli (-)
S. sonnei (-)
MIC50
Compound
MIC50
8.41 0.18
18.17 0.17
19.04 0.51
13.56 0.50
-
16.78 0.22
15.42 0.15
-
13.04 0.33
11.05 0.32
12.27 0.35
12.88 0.13
14.38 0.00
15.33 0.23
13.26 0.41
-
12.99 0.24
11.73 0.28
13.07 0.12
14.31 0.22
12.02 0.29
14.55 0.01
12.01 0.21
-
16.85 0.00
7.65 0.11
7.39 0.77
16.03 0.11
13.91 0.17
10.91 0.32
13.72 0.41
10.98 0.23
-
2a
-
2b
-
2c
18.62 0.29
19.21 0.28
13.58 0.21
7.65 0.21
-
16.03 0.46
2d
-
2e
2f
14.69 0.33
-
-
18.17 0.50
-
2g
-
2h
Ampicilin
Gentamycin
Ciprofloxacin
11.66 0.14
10.36 0.13
8.36 0.12
10.69 0.06
8.42 0.12
9.42 0.11
10.85 0.16
11.21 0.31
7.59 0.11
12.33 0.15
10.89 0.11
11.03 0.10
11.32 0.13
8.21 0.02
8.21 0.02
11.98 0.13
9.31 0.18
7.31 0.08
The H-5 of the pyridine ring appeared as a singlet in the range
of 8.15 to 8.22 ppm.The carbonyl carbon in all 13C NMR spectra
gave a discrete signal in the range of 166.34 to 167.45 ppm.
CHN analysis and HRMS also confirmed the structure of the
prepared compounds.
value against P. aureginosa (-) (MIC50 = 12.01 0.21) than
ampicilin (MIC50 = 12.33 0.15). Moreover it is found to be
more active against S. sonnei (-) (MIC50 = 10.98 0.23) than
ampicilin (MIC50 = 11.98 0.13). So we can assume that this
series of compound showed significant antibacterial activity.
Antibacterial studies
ACKNOWLEDGEMENTS
Procedure: The antibacterial activity was performed in
sterile 96-wells microplates under aseptic environments14,15
The authors (TM,AN and MN) are indebted to the Higher
Education Commission, Government of Pakistan for Indig-
enous Ph.D. Scholarships and IRSIP fellowships. One of the
authors, MAK are thankful to the HEC for the Instrumental
Analysis Support programme.
.
The test samples with suitable solvents and dilutions were
pipetted into wells (20 µg/well). Overnight maintained fresh
bacterial culture after suitable dilution with fresh nutrient broth
was poured into wells (180 µL). The initial absorbance of the
culture was maintained between 0.12-0.19 at 540 nm. The total
volume in each well was kept to 200 µL. The incubation was
done at 37 °C for 16-24 h with lid on the microplate. The
absorbance was measured at 540 nm using microplate reader,
before and after incubation and the difference was noted as an
index of bacterial growth. Ciprofloxacin, gentamycin and
ampicilin were taken as standard. Minimum inhibitory concen-
tration (MIC) was measured with suitable dilutions (5-30 µg/
well) and results were calculated using EZ-Fit5 Perrella
Scientific Inc. Amherst USA software and data expressed as
MIC50 (µg/mL). Results are mean of triplicate (n = 3, sem).
Table-2 shows the results of MIC50 studies of 6-aryl-3-
methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbxylic
acids (2a-2h) against six strains. Compound 2a showed compa-
rable activity against the B. subtilis (+) and S. sonnei (-) to the
standard ciprofloxacin. Compound 2b was found to show
lower MIC50 value (MIC50 = 11.05 0.32) against S. typhi (-)
than the gentamycin (MIC50 = 10.85 0.16). It is also found
to be more active against P. aureginosa (-) (MIC50 = 11.73
0.28) than ampicilin (MIC50 = 12.33 0.15). However it has a
comparable MIC50bvalue (MIC50 = 7.39 0.77) to the cipro-
floxacin (MIC50 = 7.31 0.08) against S. sonnei (-). Compound
2e is a little more active (MIC50 = 12.02 0.29) than ampicilin
(MIC50 = 12.33 0.15) against P. aureginosa (-). Compound
2g showed some significant results. It is found to be more
active against S. aureus (+) (MIC50 = 7.65 0.21) than
gentamycin (MIC50 = 8.42 0.12). It also showed lower MIC50
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