Exploration of antimicrobial potential of pyrazolo[3,4-b]pyridine scaffold bearing benzenesulfonamide and trifluoromethyl moieties

Article abstract of DOI:10.1007/s00044-013-0544-1

The synthesis and biological evaluation of a library of thirty differently substituted pyrazolo[3,4-b]pyridines bearing benzenesulfonamide moiety at position-1 and trifluoromethyl group at position-4 are reported. Fused heterocyclic system present in the target compounds (5a-j, 6a-j, and 7a-j) was constructed by refluxing various 5-aminopyrazoles (3a-c) with differently substituted trifluoromethyl-β-diketones (4a-j) in glacial acetic acid. All the target compounds (5-7) were evaluated for their in vitro antibacterial activity against four pathogenic bacterial strains namely, Staphylococcus aureus, Bacillus subtilis (Gram-positive), Escherichia coli, Pseudomonas aeruginosa (Gram-negative) and in vitro antifungal activity against two pathogenic fungal yeasts namely, Saccharomyces cerevisiae and Candida albicans. Graphical Abstract: Thirty differently substituted pyrazolo[3,4-b]pyridines bearing benzenesulfonamide moiety at position-1 and trifluoromethyl group at position-4 were synthesized and screened for antibacterial and antifungal activities.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-013-0544-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0544-1
ORIGINAL RESEARCH
Exploration of antimicrobial potential of pyrazolo[3,4-b]pyridine
scaffold bearing benzenesulfonamide and trifluoromethyl moieties
•
Navneet Chandak Satish Kumar Pawan Kumar
•
•
•
Chetan Sharma Kamal R. Aneja Pawan K. Sharma
•
Received: 6 December 2012 / Accepted: 15 February 2013
Ó Springer Science+Business Media New York 2013
Abstract The synthesis and biological evaluation of a
library of thirty differently substituted pyrazolo[3,4-
b]pyridines bearing benzenesulfonamide moiety at posi-
tion-1 and trifluoromethyl group at position-4 are reported.
Fused heterocyclic system present in the target compounds
(5a–j, 6a–j, and 7a–j) was constructed by refluxing various
5-aminopyrazoles (3a–c) with differently substituted tri-
fluoromethyl-b-diketones (4a–j) in glacial acetic acid. All
the target compounds (5–7) were evaluated for their
in vitro antibacterial activity against four pathogenic bac-
terial strains namely, Staphylococcus aureus, Bacillus
subtilis (Gram-positive), Escherichia coli, Pseudomonas
aeruginosa (Gram-negative) and in vitro antifungal activity
against two pathogenic fungal yeasts namely, Saccharo-
myces cerevisiae and Candida albicans.
quinoline in antimalarials, porphyrin in haemoglobin and
chlorophyll, indole in amino acid tryptophan and neuro-
transmitter serotonin, etc. (Blass, 2012; Chen et al., 2011).
Pyrazolo[3,4-b]pyridine ring system is an important mem-
ber of the family of fused heterocycles which has shown
promise in designing new pharmaceuticals with better
pharmacological profiles. This fused system is associated
with diverse biological activities such as antimicrobial
(Foks et al., 2005; Leal et al., 2008; El-Sayad, 2009; Parekh
and Maheria, 2012), anti-inflammatory (Bharate et al.,
2004), analgesic (Menegatti et al., 2006), antimalarial
(Menezes et al., 2002), antichagasic (Dias et al., 2007),
antileishmanial (Mello et al., 2004), anxiolytic (Bare et al.,
1989), antiviral (Bernardino et al., 2007), antidiabetic
(Hohn et al., 1973), antitumor (Lin et al., 2007; Hamama
et al., 2012), sedative/hypnotic (Menegatti et al., 2006), etc.
This nucleus is also known for better enzymatic inhibitory
activities such as cyclin-dependant kinases (CDK) (Misra
et al., 2003a, 2003b), glycogen synthase kinase-3 (GSK-3)
(Witherington et al., 2003a, b), phosphodiesterase 4 (PDE4)
(Hamblin et al., 2008), HIV-1 integrase (Wu et al., 2007),
etc. It is an established fact that the incorporation of fluorine
in many heterocyclic systems increases lipophilicity, thus
enhancing the rates of absorption as well as transportation
in vivo and hence making the fluoro compounds suitable for
drug formulation (Ojima, 2009). Trifluoromethyl (CF3)
group has been found to be one of the most lipophilic of all
the substituents. It is well known in the literature that in
general, increased lipophilicity makes the partition/perme-
ability of a bioactive molecule into the bacterial as well
fungal cell membrane much higher and thus tends to
increase the efficacy of a drug (Rezaee et al., 2009). Besides
increasing lipophilicity, introduction of a trifluoromethyl
moiety as well as its replacement with an existing functional
group can alter the physical properties as well as biological
Keywords Pyrazolo[3,4-b]pyridines Á
Benzenesulfonamide Á Trifluoromethyl-b-diketones Á
Antibacterial activity Á Antifungal activity
Introduction
Fused heterocycles have emerged as prime pharmaceutical
targets these days because of their abundant occurrence in
natural as well as synthetic bioactive molecules such as
b-lactam in antibiotics penicillins and cephalosporins,
N. Chandak Á S. Kumar Á P. Kumar Á P. K. Sharma (&)
Department of Chemistry, Kurukshetra University,
Kurukshetra 136119, India
e-mail: pksharma@kuk.ac.in
C. Sharma Á K. R. Aneja
Department of Microbiology, Kurukshetra University,
Kurukshetra 136119, India
123

Med Chem Res
activities of the parent compound in dramatic ways, e.g.
acidity, polarizability, enhanced hydrophobic binding, sta-
bility against metabolic oxidation, etc. (Schlosser, 2006;
Purser et al., 2008). All these findings make CF3 bearing
aromatics and heteroaromatics as increasingly attractive
targets in search of novel pharmaceuticals and thus enjoy a
special position in pharmaceutical as well as agrochemical
industries. Celecoxib (selective COX-2 inhibitor), prozac
(antidepressant), faslodex (estrogen receptor antagonist),
aprepitant (antiemetic), efavirenz (anti-HIV), casodex
(anticancer agent), desflurane (inhalation anesthetic), ind-
oxacarb (insecticide) are some of the drugs in the market
bearing a trifluoromethyl functionality.
subject matter of intense investigation in the recent past.
Formation of two different regioisomers of pyrazolo[3,4-
b]pyridine has been reported in the literature, under similar
reaction conditions, differing only in the position of CF3
which can be either at 4-position (Emelina et al., 2008) or
at 6-position (Singh et al., 2004b; Sharma et al., 2011a) of
the pyrazolopyridine nucleus. 19F NMR spectroscopy has
played a decisive role in establishing the structure of the
isomeric compound. It is reported in the literature (Numann
et al., 1985) that the CF3 group at ortho-position in pyri-
dine displays signal at higher negative value as compared
to that of para-position in 19F NMR. The relative differ-
ence in values at ortho- and para-positions may be attrib-
uted to the presence of nitrogen at adjacent position
attached via a double bond. A very recent report (Aggarwal
et al., 2012) has shown on the basis of 13C, 19F, and HMBC
experiments that solvent mediated stepwise synthesis of
pyrazolo[3,4-b]pyridine gave single regioisomer with CF3
at 4-position. Earlier reports presumed that the reaction of
5-aminopyrazoles with trifluoromethyl-b-diketones is ini-
tiated exclusively by the attack of amino group on the
carbonyl carbon adjoining the CF3 group (Singh et al.,
2004b). However, it is now believed that carbonyl carbon
adjacent to CF3 remains in hydrated form in protic solvent,
making attack of amino group forcibly on other carbonyl
carbon.
Benzenesulfonamide moiety in combination with vari-
ous heterocycles has been found to be associated with
a diverse range of bioactivities such as antimicrobial
(Basanagouda et al., 2010; Turkmen et al., 2011), anti-
inflammatory (Penning et al., 1997), anticancer (Ghorab
et al., 2010; Al-Said et al., 2011), anti-HIV (Iqbal et al.,
2006), etc. A perusal of literature reveals that the synthesis
of pyrazolo[3,4-b]pyridine ring system containing ben-
zenesulfonamide and trifluromethyl group for evaluation as
antimicrobial agents is still in its infancy (Sharma et al.,
2011a). Appreciation of these findings coupled with our
ongoing interest in the field of pyrazoles and other het-
erocyclic compounds of potential medicinal interest
(Sharma et al., 2010, 2011a, b, 2012a, b; Chandak et al.,
2012, 2013; Kumar et al., 2012a, b), we envisioned the
synthesis of a library of pyarazolo[3,4-b]pyridines bearing
benzenesulfonamide and trifluoromethyl moieties for their
evaluation as antibacterial and antifungal agents.
As far as the choice of solvent is concerned, the only
solvent which has been reported to work fine for pyrazol-
opyridine formation is glacial acetic acid (Joshi et al.,
1979; Singh et al., 2004b; Sharma et al., 2011a) which has
been further supported by a recently published paper (Hao
et al., 2012). However, in our hands, coupling of 3 with
various trifluoromethyl-b-diketones 4 in refluxing glacial
acetic acid gave two products, one desired isomer pyraz-
olo[3,4-b]pyridines (5–7) in major amount along with
acylated 5-aminopyrazoles (8a–c) in minor amount. The
formation of a side product like 8 in this reaction offers the
most probable reason for a moderate to low yield reported
in the literature for the synthesis of fused pyrazolopyridines
(Joshi et al., 1979; Singh et al., 2004b; Sharma et al.,
2011a; Aggarwal et al., 2012). The formation of 8 was
conclusively proved by refluxing 5-aminopyrazoles (3) in
glacial acetic acid in the absence of b-diketones where the
sole product obtained was acylated 5-aminopyrazoles (8a–
c) in high yield (86–92 %). It reveals that both the reac-
tions, i.e., (i) nucleophilic attack of amino group on the
carbonyl carbon, and (ii) amino acylation, go side by side
and compete with each other. We observed that substitution
of group R in 5-aminopyrazole plays an important role for
controlling yield ratio of both the products. Amount of
acylation follows the substituent order –CH3 (*10 %) \
–H (*15 %) \ –Cl (*20 %) revealing that acylation is
less when R group is electron releasing (3a, CH3) in nature
Results and discussion
Chemistry
The synthetic route used to synthesize the target pyrazol-
o[3,4-b]pyridines (5–7) is outlined in Scheme 1. The
starting materials, various a-cyanoacetophenones (2a–
c) were readily prepared by the reaction of appropriate
p-substituted phenacyl bromides with potassium cyanide in
aqueous ethanol at 50 °C following literature procedure
(Gakhar et al., 1971). 4-Hydrazinobenzenesulfonamide
hydrochloride (1) was prepared via diazotization of sulfa-
nilamide followed by reduction of the corresponding dia-
zonium salt with stannous chloride (Soliman, 1979).
Reaction of 1 with appropriate a-cyanoacetophenone (2) in
refluxing ethanol gave corresponding 4-(5-amino-3-aryl-
1H-pyrazol-1-yl)benzenesulfonamide (3a–c) following our
earlier adapted procedure (Sharma et al., 2011a). The
reaction of 5-aminopyrazoles with unsymmetrical 1,3-
diketones especially trifluoromethyl-b-diketones has been a
123

Med Chem Res
Scheme 1 Synthesis of
pyrazolo[3,4-b]pyridines (5–7)
H2NO2S
O
.
NHNH2 HCl
CN
N
N
EtOH
reflux, 6-7 h
+
H2N
H2NO2S
R
R
1
2a: R = H
b: R = CH3
c: R = Cl
3a: R = H
b: R = CH3
c: R = Cl
H2NO2S
H2NO2S
1
N
N2
OH
O
7
N
gl. AcOH
reflux, 12 h
N
N
3
3
+
+
R
F3C
R1
H3COCHN
6
R
R1
4
CF3
5
Major Product (48-80%)
Minor Product (6-25%)
8a: R = H
O
O
5, 6, 7
b: R = CH3
c: R = Cl
F3C
R1
4
R1 (4)
CF3
CH3
S
CH3
OCH3
F
Cl
Br
H
R (3)
5a
6a
7a
5b
6b
7b
5c
5d
6d
7d
5e
6e
7e
5f
6f
7f
5g
6g
7g
5h
6h
7h
5i
6i
7i
5j
6j
7j
H
CH3
6c
7c
Cl
and more when R is electron withdrawing (3c, Cl). Acyl-
ated compounds (8a–c) were easily separated from the
desired pyrazolopyridines (5a–j, 6a–j, and 7a–j) by frac-
tional crystallization from ethanol without using column
chromatography because of poor solubility of acylated
product in ethanol.
-59.01 which was in agreement with those reported
for similar structures in literature (Numann et al., 1985;
Aggarwal et al., 2012). 19F NMR signal of C6-CF3 reso-
nates in the narrow range d -65.45 to -65.47 as in the case
of 5b, 6b, and 7b. Acylated 5-aminopyrazoles, 8a–c were
characterized by the presence of an exchangeable singlet in
the narrow range d 10.18–10.20 due to NH proton and
another singlet at d 2.03 due to three protons of CH3 group
Spectral data (1H NMR, 13C NMR, 19F NMR, IR, and
mass) of the newly synthesized compounds were in full
agreement with the proposed structures. In general, 5-am-
inopyrazoles (3a–c) were characterized by the presence of
pyrazole C4-H proton resonating in the range d 5.93–5.99
appearing as a singlet and amino group resonating in the
narrow range d 5.65–5.71 appearing as an exchangeable
1
in H NMR.
Antimicrobial evaluation
All the thirty synthesized target compounds (5a–j, 6a–j, and
7a–j) were assayed for their in vitro antimicrobial activity
against Staphylococcus aureus (MTCC 96) and Bacillus
subtilis (MTCC 121) representing Gram-positive bacteria,
Escherichia coli (MTCC 1652) and Pseudomonas aeru-
ginosa (MTCC 741) representing Gram-negative bacteria,
and Saccharomyces cerevisiae (MTCC 170) and Candida
1
singlet in H NMR. Pyrazolo[3,4-b]pyridines (5a–j, 6a–j,
and 7a–j) were identified by the presence of a singlet for
pyridine C5-H proton resonating in the range d 7.75–7.79 in
5a, 6a, and 7a, d 8.99–9.00 in 5i, 6i and 7i and d 8.20–8.37
1
in others, sometimes merging with aromatic protons in H
NMR. Position of CF3 (C4-CF3) in range d -58.58 to
123

Med Chem Res
albicans (MTCC 227) representing fungal yeasts (Table 1)
by agar well diffusion method (Ahmad and Beg, 2001;
Andrews, 2001) using ciprofloxacin against bacteria and
amphotericin-B against fungi as the reference drugs. The
results were recorded for each tested compound as the
average diameter of inhibition zones of microbial growth
surrounding the well in mm. The minimum inhibitory
concentration (MIC) measurements were performed using a
modified agar well diffusion method (Okeke et al., 2001)
(Table 2).
Results revealed that in general, all the tested compounds
except 7c–7h possessed variable antibacterial activity against
both the Gram-positive bacteria (S. aureus, B. subtilis) and
some of the compounds (5a–f, 5h, 6i, 7b) possessed moderate
antifungal activity against both the yeasts (S. cerevisiae,
C. albicans). However, noneofthem wasfound tobeeffective
against any Gram-negative bacteria (E. coli, P. aeruginosa).
On the basis of zone of inhibition against the test bacterium,
two compounds (5b and 5d) were found to be the most
effective against S. aureus showing zone of inhibition 22.6
Table 1 In vitro antimicrobial activity of compounds 5–7 through agar well-diffusion method
Compoundsa
Diameter of growth of inhibition zone (mm)b
Staphylococcus
aureus
Bacillus
subtilis
Escherichia
coli
Pseudomonas
aeruginosa
Saccharomyces
cerevisiae
Candida
albicans
5a
19.3
22.6
18.6
21.3
19.3
17.6
15.0
18.3
13.6
14.6
15.3
15.6
18.3
15.3
14.6
13.6
15.6
14.3
15.6
15.3
18.6
16.3
–
20.3
21.6
17.0
20.3
21.6
19.6
16.3
17.6
14.6
15.6
16.3
16.6
17.6
16.3
17.3
15.6
16.3
15.6
15.6
16.3
17.6
15.6
–
–
–
15.3
14.6
16.3
15.6
14.6
15.3
–
16.3
15.6
15.3
16.3
15.6
14.6
–
5b
–
–
5c
–
–
5d
–
–
5e
–
–
5f
–
–
5g
–
–
5h
–
–
14.6
–
13.6
–
5i
–
–
5j
–
–
–
–
6a
–
–
–
–
6b
–
–
–
–
6c
–
–
–
–
6d
–
–
–
–
6e
–
–
–
–
6f
–
–
–
–
6g
–
–
–
–
6h
–
–
–
–
6i
–
–
15.6
–
18.3
–
6j
–
–
7a
–
–
–
–
7b
–
–
14.6
–
16.3
–
7c
–
–
7d
–
–
–
–
–
–
7e
–
–
–
–
–
–
7f
–
–
–
–
–
–
7g
–
–
–
–
–
–
7h
–
–
–
–
–
–
7i
15.3
14.6
26.6
Nt
16.3
15.6
24.0
Nt
–
–
–
–
7j
–
–
–
–
Ciprofloxacin
Amphotericin-B
25.0
Nt
22.0
Nt
Nt
19.3
Nt
16.6
–, No activity; Nt, not tested
a
Concentration 4.0 mg/mL
b
Values, including diameter of the well (8 mm), are means of three replicates
123

Med Chem Res
Table 2 Minimum inhibitory concentration (MIC) (in lg/mL) of compounds 5–7 by using modified agar well-diffusion method
Compound
Staphylococcus aureus
Bacillus subtilis
Saccharomyces cerevisiae
Candida albicans
5a
64
32
64
32
64
128
64
64
256
64
Nt
256
Nt
Nt
Nt
Nt
Nt
Nt
Nt
Nt
Nt
Nt
32
Nt
Nt
64
Nt
Nt
Nt
20
64
64
5b
5c
64
128
64
64
5d
32
64
5e
64
32
128
128
Nt
5f
128
128
64
64
5g
128
64
5h
[256
Nt
5i
256
256
128
128
64
256
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
5
5j
Nt
6a
Nt
6b
Nt
6c
Nt
6d
128
256
[256
128
256
128
128
64
Nt
6e
Nt
6f
Nt
6g
Nt
6h
Nt
6i
32
6j
Nt
7a
Nt
7b
128
128
128
5
64
7i
Nt
7j
Nt
Ciprofloxacin
Amphotericin-B
Nt
Nt
Nt
20
Nt not tested
and 21.3 mm, respectively, while four compounds showing
zone of inhibition 21.6 (5b and 5e) and 20.3 mm (5a and 5d)
against B. subtilis (Table 1) when compared with standard
drug ciprofloxacin which showed the zone of inhibition
26.6 mm against S. aureus and 24.0 mm against B. subtilis.
Besides 5b and 5d, six compounds (5a, 5c, 5e, 5h, 6c, and 7a)
showed moderate antibacterial activity against S. aureus with
zone of inhibition [18.0 mm while compound 5f showed
moderate antibacterial activity against B. subtilis with zone of
inhibition 19.6 mm. Rest of the compounds did not show any
significant antibacterial activity against any of the Gram-
positive bacteria. However, in terms of MIC, none of the
compounds was found to possess appreciable antibacterial
activity. Amongst all the active compounds, the MIC ranged
between 32 and C256 lg/mL against Gram-positive bacteria
as compared to standard drug ciprofloxacin having MIC of
5 lg/mL (Table 2). Compound 5b was found to be the most
potent member in the whole series in terms of zone of inhi-
bition as well as MIC.
compound 5c was found to be the most effective against S.
cervisiae showing zone of inhibition 16.3 mm and four
compounds against C. albicans producing zone of inhibi-
tion 18.3 mm (6i) and 16.3 mm (5a, 5d, and 7b) when
compared with standard drug amphotericin-B producing
zone of inhibition 19.3 mm against S. cerevisiae and
16.6 mm against C. albicans. Rest of the compounds
showed moderate antifungal activity against S. cervisiae as
well as C. albicans producing zone of inhibition[14.0 and
[13.0 mm, respectively (Table 1). However, none of the
compounds was found to be close to the standard drug in
terms of MIC. Amongst the all active compounds, the MIC
ranged between 32 and C256 lg/mL against fungal yeasts
as compared to standard drug amphotericin-B having MIC
of 20 lg/mL (Table 2). Out of all the compounds synthe-
sized, compound 5c was found to be the most potent
against S. cerevisiae and 6i against C. albicans in terms of
zone of inhibition as well as MIC.
Analysis of the results in terms of structure–activity rela-
tionshipindicatesthatingeneral, pyrazolopyridines5, bearing
a phenyl group at C-3 of pyrazole, exhibited better
In case of fungal yeasts, only nine compounds (5a–f, 5h,
6i, and 7b) were found to be active. Amongst them,
123

Med Chem Res
¨
antibacterial as well as antifungal activity as compared to
analogues 6 and 7, bearing a p-toluyl and p-chlorophenyl
group, respectively, in terms of zone of inhibition as well as
MIC pointing that the presence of either an electron releasing
(6) or electron withdrawing R group (7) results in decrease of
both types of activities. No correlation could be drawn within
individual homologues of 5, 6, and 7 with respect to group R1.
et al., 2006; Ahlstrom et al., 2007) as well as a-cynaoace-
tophenones (2a–c) (Gakhar et al., 1971) were prepared using
established literature procedures and confirmed by their
available spectral data and melting points.
General procedure for the preparation
of 5-aminopyrazoles (3a–c)
Appropriate a-cyanoacetophenone (2, 24 mmol) was dis-
solved in ethanol (100 mL) by warming at 50 °C followed
by the addition of 4-hydrazinobenzenesulfonamide hydro-
chloride (1, 20 mmol) along with four to five drops of
acetic acid and the reaction mixture was refluxed for 5–6 h.
After completion of the reaction, solution was reduced to
1/3rd of its volume and cooled to room temperature.
Crystalline solid separated out which was filtered, washed
with water (100 mL) followed by cold ethanol (20 mL),
dried and recrystallized from aqueous ethanol to afford
target 5-aminopyrazoles 3.
Conclusion
The objective of this study was to synthesize and investigate
antimicrobial activity of a library of novel pyrazolo [3,4-
b]pyridines bearing benzenesulfonamide and trifluoromethyl
moieties with electron releasing or electron withdrawing
groups (R and R1). Pyrazolopyridines 5 having a phenyl
substituent were found to exhibit better antimicrobial activity
as compared to analogues 6 and 7. Out of the tested com-
pounds, 5b was found to be the most potent analogue exhib-
iting moderate antibacterial activity against Gram-positive
bacteria while 5c and 6i exhibiting moderate antifungal
activity against S. cerevisiae and C. albicans, respectively.
However, none of the newly synthesized compounds was
found to be superior over the reference drugs.
4-(5-Amino-3-phenyl-1H-pyrazol-1-yl)benzenesulfonamide
(3a)
Yield: 64 %; m.p. 183–185 °C (Sharma et al. 2011a); IR
(KBr) cm-1: 3356, 3302 and 3263 (m, N–H stretch), 1628
(s, C=N stretch), 1597 (s, C=N stretch), 1512 (m, N–H
bend), 1327 and 1165 (s, SO2 stretch); 1H NMR (300 MHz,
DMSO-d6): d 7.88–7.95 (m, 4H, Ar), 7.78 (d, 2H,
J = 7.2 Hz, Ar), 7.43 (s, ex, 2H, SO2NH2), 7.40 (d, 2H,
J = 7.2 Hz, Ar), 7.32 (t, 1H, J = 7.2 Hz, Ar), 5.97 (s, 1H,
pyrazole C4-H), 5.68 (s, ex, 2H, NH2); 13C NMR
(75.5 MHz, DMSO-d6): d 151.4, 149.3, 142.3, 141.6,
133.6, 129.0, 128.3, 127.3, 125.6, 123.5, 122.7, 88.6.
Experimental protocols
All reactions were carried out under atmospheric pressure.
Melting points were determined in open glass capillaries in an
electrical melting point apparatus and are uncorrected. The
infrared (IR) spectra were recorded on ABB MB 3000 DTGS
FT-IR Spectrophotometer using the KBr pellet technique. 1H
NMR and 13C NMR spectra were recorded either in pure
DMSO-d6 or in CDCl3/DMSO-d6 mixture on Bruker NMR
spectrometers at 300/400 and 75.5/100 MHz, respectively,
using tetramethylsilane (TMS) as internal standard, whereas
19F NMR spectra were recorded at 376.4 MHz in DMSO-d6
using fluorinated chloromethane, CFCl3 as an internal stan-
dard. Chemical shifts are expressed in d, ppm. Mass spectra
(DART-MS) were recorded on a JEOL-AccuTOF JMS-
T100LC Mass spectrometer having a DART (direct analysis
in real time) source in ES? mode. The purity of the com-
pounds was checked by 1H NMR and thin layer chromatog-
raphy (TLC) on silica gel plates using a mixture of petroleum
ether and ethyl acetate as eluent. Iodine or UV lamp was used
as a visualizing agent. Abbreviations ‘‘s’’ for singlet, ‘‘d’’ for
doublet, ‘‘m’’ for multiplet, ‘‘ex’’ for exchangeable proton are
used for NMR assignments and ‘‘s’’ for strong, ‘‘m’’ for
medium for IR assignments. ‘‘d’’ stands for decomposition in
melting point data. Trifluoromethyl-b-diketones, 4a–c and 4j
were purchased from ACROS-ORGANICS, New Jersey,
USA and remaining diketones (Singh et al., 2004a; Sloop
4-[5-Amino-3-(4-methylphenyl)-1H-pyrazol-1-
yl]benzenesulfonamide (3b)
Yield 70 %; m.p. 242–244 °C; IR (KBr) cm-1: 3356, 3302,
and 3263 (m, N–H stretch), 1628 (s, C=N stretch), 1597 (s,
C=N stretch), 1512 (m, N–H bend), 1327 and 1165 (s, SO2
1
stretch); H NMR (300 MHz, DMSO-d6): d 7.93 (d, 2H,
J = 9.0 Hz, Ar), 7.90 (d, 2H, J = 9.0 Hz, Ar), 7.67 (d, 2H,
J = 8.1 Hz, Ar), 7.44 (s, ex, 2H, SO2NH2), 7.21 (d, 2H,
J = 8.1 Hz, Ar), 5.93 (s, 1H, pyrazole C4-H), 5.65 (s, ex,
2H, NH2), 2.32 (CH3); 13C NMR (75.5 MHz, DMSO-d6): d
151.4, 149.2, 142.3, 141.4, 137.6, 130.9, 129.5, 127.2,
125.6, 122.5, 88.4, 21.3 (CH3).
4-[5-Amino-3-(4-chlorophenyl)-1H-pyrazol-1-
yl]benzenesulfonamide (3c)
Yield 65 %; m.p. 220–221 °C; IR (KBr) cm-1: 3371, 3271
& 3202 (m, N–H stretch), 1628 (s, C=N stretch), 1597 (s,
123

Med Chem Res
4-[3-Phenyl-4,6-bis(trifluoromethyl)-1H-pyrazolo[3,4-
b]pyridin-1-yl]benzenesulfonamide (5b)
C=N stretch), 1512 (m, N–H bend), 1327 and 1157 (s, SO2
1
stretch); H NMR (300 MHz, DMSO-d6): d 7.95 (d, 2H,
J = 9.0 Hz, Ar), 7.90 (d, 2H, J = 9.0 Hz, Ar), 7.81 (d, 2H,
J = 8.4 Hz, Ar), 7.45–7.47 (m, 4H, SO2NH2, Ar), 5.99 (s,
1H, pyrazole C4-H), 5.71 (s, ex, 2H, NH2); 13C NMR
(75.5 MHz, DMSO-d6): d 150.2, 149.5, 142.2, 141.7,
132.7, 132.5, 129.0, 127.3, 127.2, 124.9, 122.7, 88.4.
Yield 71 %; m.p. 240–242 °C (Sharma et al., 2011a); IR
(KBr) cm-1: 3356 and 3271 (m, N–H stretch), 1597 (s,
C=N stretch), 1504 (m, N–H bend), 1335 and 1142 (s, SO2
1
stretch); H NMR (300 MHz, DMSO-d6): d 8.41 (d, 2H,
J = 8.7 Hz, Ar), 8.27 (s, 1H, pyridine C5-H), 8.09 (d, 2H,
J = 8.7 Hz, Ar), 7.56-7.64 (m, 5H, Ar), 7.51 (s, ex, 2H,
SO2NH2); 13C NMR (75.5 MHz, DMSO-d6): d 150.5,
146.9, 146.5, 146.0, 143.0, 140.4, 133.4, 132.1, 130.1,
129.9, 128.5, 127.6, 122.3, 113.5, 112.5; 19F NMR
(376.4 MHz, DMSO-d6): d –58.64 (C4-CF3), –65.46 (C6-
CF3); DART-MS: m/z 487.10 (M?H)?, C20H12F6N4O2
SH? calcd. 487.05.
General procedure for the preparation of pyrazolo[3,4-
b]pyridines (5a–j, 6a–j, and 7a–j)
A solution of appropriate 5-aminopyrazole (3, 1.5 mmol)
and appropriate trifluoromethyl-b-diketone (4, 1.8 mmol)
in glacial acetic acid (8 mL) was refluxed for 10–12 h.
After completion of the reaction, the contents were poured
into ice cold water and stirred vigorously whereupon solid
separated out which was filtered, washed with excess of
cold water and dried to afford a crude mixture which upon
fractional crystallization from ethanol yielded two prod-
ucts: pyrazolo[3,4-b]pyridines (5, 6, or 7) in major amount
and 5-acetamidopyrazoles (8a–c) in minor amount.
(8a) Yield 11 %.
4-[3,6-Diphenyl-4-(trifluoromethyl)-1H-pyrazolo[3,4-
b]pyridin-1-yl]benzenesulfonamide (5c)
Yield 58 %; m.p. 234–236 °C; IR (KBr) cm-1: 3286 and
3217 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1335 and 1157 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.57 (d, 2H, J = 8.7 Hz, Ar),
8.34–8.38 (m, 3H, pyridine C5-H, Ar), 8.10 (d, 2H,
J = 8.7 Hz, Ar), 7.56–7.63 (m, 4H, Ar), 7.45–7.50 (m, 4H,
SO2NH2, Ar), 7.40–7.41 (m, 2H, Ar); 13C NMR
(75.5 MHz, DMSO-d6): d 157.7, 151.3, 145.8, 142.2,
141.1, 137.1, 131.3, 130.0, 129.6, 128.4, 128.2, 127.5,
121.8, 114.4, 109.2; 19F NMR (376.4 MHz, DMSO-d6): d
–58.75 (C4-CF3); DART-MS: m/z 495.15 (M?H)?,
C25H17F3N4O2SH? calcd. 495.10.
4-[6-Methyl-3-phenyl-4-(trifluoromethyl)-1H-
pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (5a)
Yield 60 %; m.p. 248–250 °C; IR (KBr) cm-1: 3348 &
3240 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1335 and 1149 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.49 (d, 2H, J = 8.7 Hz, Ar),
8.04 (d, 2H, J = 8.7 Hz, Ar), 7.75 (s, 1H, pyridine C5-H),
7.51–7.55 (m, 5H, Ar), 7.48 (s, ex, 2H, SO2NH2), 2.80 (s,
3H, CH3); 13C NMR (75.5 MHz, DMSO-d6): d 161.2,
151.6, 145.6, 142.1, 141.2, 132.9 131.3, 130.8, 130.0,
129.5, 128.4, 127.4, 124.5, 121.7, 120.9, 116.7, 116.6,
108.8, 25.1 (CH3); 19F NMR (376.4 MHz, DMSO-d6): d –
59.01 (C4-CF3); DART-MS: m/z 433.12 (M?H)?,
C20H15F3N4O2SH? calcd. 433.08.
(8a) Yield 17 %.
4-[6-(4-Methylphenyl)-3-phenyl-4-(trifluoromethyl)-1H-
pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (5d)
Yield 59 %; m.p. 260–262 °C; IR (KBr) cm-1: 3340 &
3232 (m, N–H stretch), 1597 (s, C=N stretch), 1497 (m, N–
H bend), 1358 and 1142 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.57 (d, 2H, J = 8.7 Hz, Ar),
8.28-8.30 (m, 3H, pyridine C5-H, Ar), 8.10 (d, 2H,
J = 8.7 Hz, Ar), 7.54–7.62 (m, 5H, Ar), 7.48 (s, ex, 2H,
SO2NH2), 7.43 (d, 2H, J = 8.1 Hz, Ar), 2.42 (s, 3H, CH3);
13C NMR (75.5 MHz, DMSO-d6): d 157.7, 151.8, 145.8,
142.1, 141.3, 141.1, 134.4, 132.8, 130.2, 130.0, 129.5,
128.4, 128.1, 127.5, 121.7, 115.0, 112.8, 109.1, 21.4 (CH3);
19F NMR (376.4 MHz, DMSO-d6): d –58.75 (C4-CF3);
DART-MS: m/z 509.20 (M?H)?, C26H19F3N4O2SH?
calcd. 509.11.
N-{1-[4-(Aminosulfonyl)phenyl]-3-phenyl-1H-pyrazol-5-
yl}acetamide (8a)
Yield 13 %; m.p. 254–256 °C; IR (KBr) cm-1: 3271 (m,
N–H stretch), 1666 (s, C=O stretch), 1597 (s, C=N stretch),
1327 and 1157 (s, SO2 stretch); 1H NMR (300 MHz,
DMSO-d6): d 10.19 (s, ex, 1H, NHCO), 7.96 (d, 2H,
J = 8.4 Hz, Ar), 7.87 (d, 2H, J = 8.1 Hz, Ar), 7.80 (d, 2H,
J = 8.4 Hz, Ar), 7.47 (s, ex, 2H, SO2NH2), 7.40–7.42 (m,
3H, Ar), 6.92 (s, 1H, pyrazole C4-H), 2.03 (s, 3H,
NHCOCH3); 13C NMR (75.5 MHz, DMSO-d6): d 169.5
(NHCO), 151.3, 142.9, 141.5, 138.2, 132.9, 129.2, 128.7,
127.3, 125.7, 123.8, 100.8, 23.3 (NHCOCH3).
(8a) Yield 15 %.
123

Med Chem Res
4-[6-(4-Methoxyphenyl)-3-phenyl-4-(trifluoromethyl)-1H-
pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (5e)
4-[6-(4-Bromophenyl)-3-phenyl-4-(trifluoromethyl)-1H-
pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (5h)
Yield 59 %; m.p. 270–271 °C; IR (KBr) cm-1: 3302 &
3240 (m, N–H stretch), 1597 (s, C=N stretch), 1512 (m, N–
H bend), 1358 and 1142 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.57 (d, 2H, J = 8.7 Hz, Ar),
8.36 (d, 2H, J = 8.4 Hz, Ar), 8.26 (s, 1H, pyridine C5-H),
8.10 (d, 2H, J = 8.4 Hz, Ar), 7.49–7.60 (m, 7H, SO2NH2,
Yield 62 %; m.p. 278–280 °C; IR (KBr) cm-1: 3379 and
3286 (m, N–H stretch), 1589 (s, C=N stretch), 1504 (m, N–
H bend), 1342 and 1134 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.55 (d, 2H, J = 8.7 Hz, Ar),
8.32–8.34 (m, 3H, pyridine C5-H, Ar), 8.09 (d, 2H,
J = 8.7 Hz, Ar), 7.80 (d, 2H, J = 8.7 Hz, Ar), 7.56–7.60
(m, 5H, Ar), 7.50 (s, ex, 2H, SO2NH2); 13C NMR
(75.5 MHz, DMSO-d6): d 156.5, 151.7, 145.8, 142.2,
141.0, 136.3, 132.7, 132.5, 130.2, 130.0, 129.6, 128.4,
127.6, 125.1, 113.4, 110.0; 19F NMR (376.4 MHz, DMSO-
d6): d -58.77 (C4-CF3); DART-MS: m/z 573.10 (M?H)?,
C25H16BrF3N4O2SH? calcd. 573.01.
Ar), 7.15 (d, 2H, J = 8.7 Hz, Ar), 3.87 (s, 3H, OCH3); 13
C
NMR (75.5 MHz, DMSO-d6): d 162.0, 157.3, 151.8, 145.8,
142.1, 141.2, 132.9, 131.8, 130.0, 129.8, 129.5, 128.4,
127.5, 121.6, 115.0, 112.7, 109.1, 55.8 (OCH3); 19F NMR
(376.4 MHz, DMSO-d6): d –58.85 (C4-CF3); DART-MS:
m/z 525.19 (M?H)?, C26H19F3N4O3SH? calcd. 525.11.
(8a) Yield 15 %.
(8a) Yield 13 %.
4-[6-(4-Fluorophenyl)-3-phenyl-4-(trifluoromethyl)-1H-
pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (5f)
4-[6-(2-Naphthyl)-3-phenyl-4-(trifluoromethyl)-1H-
pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (5i)
Yield 57 %; m.p. 230–232 °C; IR (KBr) cm-1: 3302 and
3271 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1342 and 1149 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.55 (d, 2H, J = 8.7 Hz, Ar),
8.46 (dd, 2H, JHF = 5.4 Hz, JHH = 8.4 Hz, Ar), 8.33 (s,
1H, pyridine C5-H), 8.09 (d, 2H, J = 8.7 Hz, Ar),
7.54–7.56 (m, 3H, Ar), 7.49 (s, 4H, SO2NH2, Ar), 7.44 (t,
2H, J = 8.7 Hz, Ar); 13C NMR (100 MHz, CDCl3/DMSO-
d6): d 156.1, 151.3, 145.3, 141.3, 140.8, 137.4, 133.1,
132.3, 132.1, 129.69, 129.60, 126.8, 126.7, 125.0, 123.1,
120.7, 115.9, 115.7; 19F NMR (376.4 MHz, DMSO-d6): d
–58.70 (C4-CF3); DART-MS: m/z 513.15 (M?H)?,
C25H16F4N4O2SH? calcd. 513.09.
Yield 55 %; m.p. 278–280 °C; IR (KBr) cm-1: 3371 and
3286 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1342 and 1126 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 9.00 (s, 1H, pyridine C5-H), 8.62
(d, 2H, J = 8.7 Hz, Ar), 8.50–8.53 (m, 2H, Ar), 8.12–8.18
(m, 4H, Ar), 8.00–8.03 (m, 1H, Ar), 7.61–7.64 (m, 4H, Ar),
7.56–7.57 (m, 3H, Ar), 7.50 (s, ex, 2H, SO2NH2); 13C
NMR (75.5 MHz, DMSO-d6): d 157.5, 151.8, 145.9, 142.2,
141.1, 134.4, 134.3, 133.4, 132.8, 130.0, 129.6, 129.5,
129.2, 128.6, 128.4, 128.1, 128.0, 127.6, 127.2, 124.9,
121.8, 114.4, 109.7; 19F NMR (376.4 MHz, DMSO-d6): d
-58.73 (C4-CF3); DART-MS: m/z 545.16 (M?H)?,
C29H19F3N4O2SH? calcd. 545.11.
4
3
(8a) Yield 17 %.
(8a) Yield 17 %.
4-[6-(4-Chlorophenyl)-3-phenyl-4-(trifluoromethyl)-1H-
pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (5g)
4-[3-Phenyl)-4-(2-thienyl)-6-(trifluoromethyl)-1H-
pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (5j)
Yield 60 %; m.p. 287–288 °C; IR (KBr) cm-1: 3379 and
3279 (m, N–H stretch), 1589 (s, C=N stretch), 1497 (m, N–
H bend), 1342 and 1134 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.55 (d, 2H, J = 8.7 Hz, Ar),
8.42 (d, 2H, J = 8.4 Hz, Ar), 8.35 (s, 1H, pyridine C5-H),
8.09 (d, 2H, J = 8.7 Hz, Ar), 7.67 (d, 2H, J = 8.7 Hz, Ar),
7.61–7.63 (m, 2H, Ar), 7.55–7.56 (m, 3H, Ar), 7.50 (s, ex,
2H, SO2NH2); 13C NMR (75.5 MHz, DMSO-d6): d 156.4,
151.7, 145.8, 142.2, 141.0, 136.2, 135.9, 132.7, 132.6,
130.0, 129.6, 128.4, 127.6, 125.0, 121.8, 113.3, 109.9; 19F
NMR (376.4 MHz, DMSO-d6): d –58.76 (C4-CF3); DART-
MS: m/z 529.11 (M?H)?, C25H16ClF3N4O2SH? calcd.
529.06.
Yield 57 %; m.p. 288–290 °C; IR (KBr) cm-1: 3371 and
3271 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1366 and 1134 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.52 (d, 2H, J = 8.7 Hz, Ar),
8.28–8.29 (m, 2H, pyridine C5-H, thienyl), 8.07 (d, 2H,
J = 8.7 Hz, Ar), 7.87 (d, 1H, J = 5.1 Hz, thienyl),
7.51–7.61 (m, 5H, Ar), 7.49 (s, ex, 2H, SO2NH2), 7.28 (t,
1H, J = 4.5 Hz, thienyl); 13C NMR (100 MHz, DMSO-
d6): d 153.0, 151.3, 146.0, 142.9, 142.1, 141.1, 132.7,
131.9, 130.0, 129.6, 128.4, 127.4, 124.4, 121.4, 112.2,
109.4; 19F NMR (376.4 MHz, DMSO-d6): d –58.84 (C4-
CF3); DART-MS: m/z 501.15 (M?H)?, C23H15F3N4O2S2H?
calcd. 501.05.
(8a) Yield 13 %.
(8a) Yield 14 %.
123

Med Chem Res
bend), 1327 and 1134 (s, SO2 stretch); 1H NMR (300 MHz,
DMSO-d6): d 8.56 (d, 2H, J = 8.7 Hz, Ar), 8.34–8.37 (m,
2H, Ar), 8.29 (s, 1H, pyridine C5-H), 8.10 (d, 2H,
J = 8.7 Hz, Ar), 7.59–7.62 (m, 3H, Ar), 7.49–7.51 (m, 4H,
SO2NH2, Ar), 7.34 (d, 2H, J = 8.1 Hz, Ar), 2.42 (s, 3H,
CH3); 13C NMR (75.5 MHz, DMSO-d6): d 157.6, 151.8,
145.9, 142.1, 141.1, 139.0, 137.1, 132.0, 131.2, 129.9, 129.6,
129.0, 128.2, 127.5, 121.8, 113.3, 109.8, 21.4 (CH3); 19F
NMR (376.4 MHz, DMSO-d6): d –58.73 (C4-CF3); DART-
MS: m/z 509.19 (M?H)?, C26H19F3N4O2SH? calcd.
509.11.
4-[6-Methyl-3-(4-methylphenyl)-4-(trifluoromethyl)-1H-
pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (6a)
Yield 73 %; m.p. 244–246 °C; IR (KBr) cm-1: 3348 and
3263 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1319 and 1149 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.48 (d, 2H, J = 8.7 Hz, Ar),
8.03 (d, 2H, J = 8.7 Hz, Ar), 7.75 (s, 1H, pyridine C5-H),
7.45–7.47 (m, 4H, SO2NH2, Ar), 7.33 (d, 2H, J = 7.8 Hz,
Ar), 2.80 (s, 3H, CH3), 2.41 (s, 3H, CH3); 13C NMR
(75.5 MHz, DMSO-d6): d 161.1, 151.6, 145.7, 142.1,
141.2, 138.9, 131.2, 130.8, 129.9, 129.0, 127.4, 121.7,
116.8, 108.9, 25.1 (CH3), 21.4 (CH3); 19F NMR
(376.4 MHz, DMSO-d6): d –58.94 (C4-CF3); DART-MS:
m/z 447.16 (M ? H)?, C21H17F3N4O2SH? calcd. 447.10.
(8b) Yield 10 %.
4-[3,6-bis(4-Methylphenyl)-4-(trifluoromethyl)-1H-
pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (6d)
N-[1-[4-(Aminosulfonyl)phenyl]-3-(4-methylphenyl)-1H-
pyrazol-5-yl]acetamide (8b)
Yield 62 %; m.p. 250–252 °C; IR (KBr) cm-1: 3340 and
3232 (m, N–H stretch), 1597 (s, C=N stretch), 1497 (m, N–
H bend), 1358 & 1142 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.56 (d, 2H, J = 8.7 Hz, Ar),
8.26–8.29 (m, 3H, pyridine C5-H, Ar), 8.09 (d, 2H,
J = 8.7 Hz, Ar), 7.50 (d, 4H, J = 8.4 Hz, SO2NH2, Ar),
7.41 (d, 2H, J = 8.1 Hz, Ar), 7.35 (d, 2H, J = 7.8 Hz, Ar),
2.43 (s, 3H, CH3), 2.42 (s, 3H, CH3); 13C NMR (75.5 MHz,
DMSO-d6): d 157.6, 151.8, 145.9, 142.1, 141.3, 141.2,
138.9, 134.4, 130.2, 129.9, 129.0, 128.1, 127.5, 121.7,
113.1, 109.2, 21.4 (CH3); 19F NMR (376.4 MHz, DMSO-
d6): d –58.76 (C4-CF3); DART-MS: m/z 523.21 (M?H)?,
C27H21F3N4O2SH? calcd. 523.13.
Yield 8 %; m.p. 214–216 °C (d); IR (KBr) cm-1: 3271 (m,
N–H stretch), 1666 (s, C=O stretch), 1597 (s, C=N stretch),
1327 and 1157 (s, SO2 stretch); 1H NMR (300 MHz, DMSO-
d6): d 10.18 (s, ex, 1H, NHCO), 7.95 (d, 2H, J = 8.1 Hz, Ar),
7.79 (d, 2H, J = 8.1 Hz, Ar), 7.75 (d, 2H, J = 7.8 Hz, Ar),
7.48 (s, ex, 2H, SO2NH2), 7.24 (d, 2H, J = 7.8 Hz, Ar), 6.87
(s, 1H, pyrazole C4-H), 2.33 (s, 3H, CH3), 2.03 (s, 3H,
NHCOCH3); 13C NMR (75.5 MHz, DMSO-d6): d 169.5
(NHCO), 151.4, 142.8, 141.6, 138.1, 130.9, 129.8, 127.3,
123.7, 100.7, 23.3 (NHCOCH3), 21.3 (CH3).
4-[3-(4-Methylphenyl-4,6-bis(trifluoromethyl)-1H-
pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (6b)
(8b) Yield 12 %.
Yield 80 %; m.p. 234–236 °C; IR (KBr) cm-1: 3356 and
3263 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1327 and 1165 (s, SO2 stretch); 1H NMR
(400 MHz, DMSO-d6): d 8.40 (d, 2H, J = 8.4 Hz, Ar),
8.22 (s, 1H, pyridine C5-H), 8.08 (d, 2H, J = 8.4 Hz, Ar),
7.49–7.51 (m, 4H, SO2NH2, Ar), 7.35 (d, 2H, J = 7.6 Hz,
Ar), 2.42 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): d
150.0, 146.3, 146.0, 145.5, 142.4, 139.9, 138.9, 133.3,
132.9, 129.4, 128.7, 128.6, 127.1, 122.2, 121.7, 113.1,
112.0, 20.9 (CH3); 19F NMR (376.4 MHz, DMSO-d6): d –
58.58 (C4-CF3), –65.47 (C6-CF3); DART-MS: m/z 501.11
(M?H)?, C21H14F6N4O2SH? calcd. 501.07.
4-[6-(4-Methoxyphenyl)-3-(4-methylphenyl)-4-
(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridin-1-
yl]benzenesulfonamide (6e)
Yield 59 %; m.p. 274–276 °C; IR (KBr) cm-1: 3348 and
3263 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1366 and 1149 (s, SO2 stretch); 1H NMR
(400 MHz, DMSO-d6): d 8.56 (d, 2H, J = 8.8 Hz, Ar),
8.35 (d, 2H, J = 8.8 Hz, Ar), 8.24 (s, 1H, pyridine C5-H),
8.09 (d, 2H, J = 8.4 Hz, Ar), 7.50 (d, 2H, J = 8.0 Hz, Ar),
7.48 (s, ex, 2H, SO2NH2), 7.34 (d, 2H, J = 8.0 Hz, Ar),
7.14 (d, 2H, J = 9.2 Hz, Ar), 3.87 (s, 3H, OCH3), 2.43 (s,
3H, CH3); 13C NMR (100 MHz, DMSO-d6): d 161.5,
156.9, 156.8, 151.4, 145.4, 141.5, 140.7, 138.4, 133.9,
129.5, 129.4, 129.0, 128.5, 127.0, 121.2, 114.5, 112.2,
108.6, 104.1, 55.4 (OCH3), 20.9 (CH3); 19F NMR
(376.4 MHz, DMSO-d6): d –58.78 (C4-CF3); DART-MS:
m/z 539.15 (M ? H)?, C27H21F3N4O3SH? calcd. 539.12.
(8b) Yield 12 %.
(8b) Yield 6 %.
4-[3-(4-Methylphenyl)-6-phenyl-4-(trifluoromethyl)-1H-
pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (6c)
Yield 67 %; m.p. 256–258 °C; IR (KBr) cm-1: 3348 and
3263 (m, N–H stretch), 1597 (s, C=N stretch), 1497 (m, N–H
123

Med Chem Res
DART-MS: m/z 587.12 (M?H)?, C26H18BrF3N4O2SH?
calcd. 587.03.
4-[6-(4-Fluorophenyl)-3-(4-methylphenyl)-4-
(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridin-1-
yl]benzenesulfonamide (6f)
(8b) Yield 11 %.
Yield 60 %; m.p. 214–216 °C; IR (KBr) cm-1: 3302 and
3248 (m, N–H stretch), 1597 (s, C=N stretch), 1512 (m, N–
H bend), 1358 and 1173 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.55 (d, 2H, J = 8.7 Hz, Ar),
8.44–8.47 (m, 2H, Ar), 8.32 (s, 1H, pyridine C5-H), 8.09 (d,
2H, J = 8.7 Hz, Ar), 7.50 (d, 4H, J = 7.5 Hz, SO2NH2,
Ar), 7.42 (d, 2H, J = 8.7 Hz, Ar), 7.35 (d, 2H, J = 7.5 Hz,
Ar), 2.43 (s, 3H, CH3); 13C NMR (75.5 MHz, DMSO-d6):
d 156.6, 151.9, 145.9, 142.2, 141.1, 139.0, 136.3, 134.0,
130.8, 130.7, 129.9, 129.0, 127.6, 121.9, 116.7, 113.3, 21.4
(CH3); 19F NMR (376.4 MHz, DMSO-d6): d –58.71 (C4-
CF3); DART-MS: m/z 527.19 (M?H)?, C26H18F4N4O2
SH? calcd. 527.10.
4-[3-(4-Methylphenyl)-6-(2-naphthyl)-4-(trifluoromethyl)-
1H-pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (6i)
Yield 60 %; m.p. 298–300 °C; IR (KBr) cm-1: 3379 &
3286 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1342 and 1157 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.99 (s, 1H, pyridine C5-H), 8.61
(d, 2H, J = 8.7 Hz, Ar), 8.50-8.53 (m, 2H, Ar), 8.11–8.18
(m, 4H, Ar), 8.00-8.03 (m, 1H, Ar), 7.61-7.64 (m, 2H, Ar),
7.52 (d, 2H, J = 8.1 Hz, Ar), 7.49 (s, ex, 2H, SO2NH2),
7.36 (d, 2H, J = 8.1 Hz, Ar), 2.44 (s, 3H, CH3); 13C NMR
(75.5 MHz, DMSO-d6): d 157.4, 151.9, 145.9, 142.1,
141.2, 139.0, 134.4, 134.3, 133.3, 129.9, 129.5, 129.2,
129.0, 128.5, 128.0, 127.6, 127.2, 124.9, 121.7, 113.5,
109.7, 21.4 (CH3); 19F NMR (376.4 MHz, DMSO-d6): d –
58.81 (C4-CF3); DART-MS: m/z 559.22 (M?H)?,
C30H21F3N4O2SH? calcd. 559.13.
(8b) Yield 10 %.
4-[6-(4-Chlorophenyl)-3-(4-methylphenyl)-4-
(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridin-1-
yl]benzenesulfonamide (6g)
(8b) Yield: 12 %.
Yield 60 %; m.p. 240–242 °C; IR (KBr) cm-1: 3333 and
3240 (m, N–H stretch), 1597 (s, C=N stretch), 1497 (m, N–
H bend), 1358 and 1149 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.54 (d, 2H, J = 8.7 Hz, Ar),
8.40 (d, 2H, J = 8.4 Hz, Ar), 8.32 (s, 1H, pyridine C5-H),
8.09 (d, 2H, J = 8.7 Hz, Ar), 7.65 (d, 2H, J = 8.4 Hz, Ar),
7.48-7.50 (m, 4H, SO2NH2, Ar), 7.34 (d, 2H, J = 8.1 Hz,
Ar), 2.42 (s, 3H, CH3); 13C NMR (75.5 MHz, DMSO-d6):
d 156.3, 151.7, 145.9, 142.1, 141.0, 139.0, 136.2, 135.9,
130.0, 129.9, 129.6, 129.0, 127.5, 121.8, 113.6, 109.9, 21.4
(CH3); 19F NMR (376.4 MHz, DMSO-d6): d –58.70 (C4-
CF3); DART-MS: m/z 543.16 (M ? H)?, C26H18ClF3N4O2
SH? calcd. 543.07.
4-[3-(4-Methylphenyl)-6-(2-thienyl)-4-(trifluoromethyl)-
1H-pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (6j)
Yield 60 %; m.p. 276–278 °C; IR (KBr) cm-1: 3371 and
3271 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1319 and 1134 (s, SO2 stretch); 1H NMR
(400 MHz, DMSO-d6): d 8.51 (d, 2H, J = 9.2 Hz, Ar),
8.25-8.26 (m, 2H, pyridine C5-H, thienyl), 8.07 (d, 2H,
J = 9.2 Hz, Ar), 7.86 (d, 1H, J = 4.8 Hz, thienyl), 7.47 (d,
4H, J = 9.2 Hz, SO2NH2, Ar), 7.33 (d, 2H, J = 8.0 Hz,
Ar), 7.26 (t, 1H, J = 4.4 Hz, thienyl), 2.41 (s, 3H, CH3);
13C NMR (100 MHz, DMSO-d6): d 152.5, 150.9, 145.6,
142.5, 141.6, 140.6, 138.5, 131.4, 129.5, 129.4, 129.1,
128.5, 126.9, 120.9, 111.7, 109.0, 20.9 (CH3); 19F NMR
(376.4 MHz, DMSO-d6): d -58.82 (C4-CF3); DART-MS:
m/z 515.15 (M?H)?, C24H17F3N4O2S2H? calcd. 515.07.
(8b) Yield 12 %.
(8b) Yield 12 %.
4-[6-(4-Bromophenyl)-3-(4-methylphenyl)-4-
(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridin-1-
yl]benzenesulfonamide (6h)
4-[3-(4-Chlorophenyl)-6-methyl-4-(trifluoromethyl)-1H-
pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (7a)
Yield 59 %; m.p. 259–261 °C; IR (KBr) cm-1: 3387 and
3294 (m, N–H stretch), 1589 (s, C=N stretch), 1497 (m, N–
H bend), 1350 and 1157 (s, SO2 stretch); 1H NMR
(400 MHz, DMSO-d6): d 8.53 (d, 2H, J = 8.4 Hz, Ar),
8.28 (d, 3H, J = 8.4 Hz, pyridine C5-H, Ar), 8.09 (d, 2H,
J = 8.0 Hz, Ar), 7.75 (d, 2H, J = 7.6 Hz, Ar), 7.47–7.49
(m, 4H, SO2NH2, Ar), 7.33 (d, 2H, J = 7.2 Hz, Ar), 2.41
(s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): d 155.8,
151.2, 145.4, 141.7, 140.6, 138.5, 135.8, 132.0, 129.7,
129.4, 128.5, 127.1, 124.6, 121.2, 112.7, 109.5, 20.9 (CH3);
19F NMR (376.4 MHz, DMSO-d6): d –58.69 (C4-CF3);
Yield 60 %; m.p. 252–253 °C; IR (KBr) cm-1: 3364 and
3271 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1319 & 1126 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.48 (d, 2H, J = 8.7 Hz, Ar),
8.04 (d, 2H, J = 8.7 Hz, Ar), 7.79 (s, 1H, pyridine C5-H),
7.60 (d, 4H, J = 8.4 Hz, Ar), 7.47 (s, ex, 2H, SO2NH2),
2.81 (CH3); 13C NMR (75.5 MHz, DMSO-d6): d 161.4,
151.6, 144.4, 142.3, 141.1, 134.5, 131.9, 128.5, 127.4,
121.9, 116.9, 108.9, 25.1 (CH3); 19F NMR (376.4 MHz,
123

Med Chem Res
4-[3-(4-Chlorophenyl)-6-(4-methylphenyl)-4-
(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridin-1-
yl]benzenesulfonamide (7d)
DMSO-d6): d –58.91 (C4-CF3); DART-MS: m/z 467.11
(M?H)?, C20H14ClF3N4O2SH? calcd. 467.04.
N-[1-[4-(Aminosulfonyl)phenyl]-3-(4-chlorophenyl)-1H-
pyrazol-5-yl]acetamide (8c)
Yield 58 %; m.p. 258–260 °C; IR (KBr) cm-1: 3350 and
3271 (m, N–H stretch), 1597 (s, C=N stretch), 1497 (m, N–
H bend), 1327 and 1157 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.54 (d, 2H, J = 8.7 Hz, Ar),
8.32-8.36 (m, 3H, pyridine C5-H, Ar), 8.09 (d, 2H,
J = 8.7 Hz, Ar), 7.80 (d, 2H, J = 8.7 Hz, Ar), 7.64 (s, 4H,
Ar), 7.51 (s, ex, 2H, SO2NH2), 2.50 (s, 3H, CH3); 13C
NMR (75.5 MHz, DMSO-d6): d 157.8, 156.6. 151.8, 144.6,
142.3, 142.2, 141.4, 141.0, 140.9, 134.5, 134.3, 132.5,
131.9, 131.7, 130.2, 128.6, 128.1, 127.5, 125.2, 121.9,
121.8, 113.4, 109.4, 21.4 (CH3); 19F NMR (376.4 MHz,
DMSO-d6): d -58.80 (C4-CF3); DART-MS: m/z 543.16
(M?H)?, C26H18ClF3N4O2SH? calcd. 543.07.
Yield 17 %; m.p. 270–271 °C; IR (KBr) cm-1: 3271 (m,
N–H stretch), 1666 (s, C=O stretch), 1597 (s, C=N stretch),
1327 and 1157 (s, SO2 stretch); 1H NMR (300 MHz,
DMSO-d6): d 10.20 (s, ex, 1H, NHCO), 7.96 (d, 2H,
J = 8.4 Hz, Ar), 7.90 (d, 2H, J = 8.4 Hz, Ar), 7.80 (d, 2H,
J = 8.4 Hz, Ar), 7.48–7.50 (m, 4H, SO2NH2, Ar), 6.96 (s,
1H, pyrazole C4-H), 2.03 (s, 3H, NHCOCH3); 13C NMR
(75.5 MHz, DMSO-d6): d 169.5 (NHCO), 150.2, 143.1,
141.4, 138.4, 133.2, 131.8, 129.2, 127.4, 127.3, 123.9,
100.9, 23.3 (NHCOCH3).
(8c) Yield 20 %.
4-[3-(4-Chlorophenyl-4,6-bis(trifluoromethyl)-1H-
pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (7b)
4-[3-(4-Chlorophenyl)-6-(4-methoxyphenyl)-4-
(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridin-1-
yl]benzenesulfonamide (7e)
Yield 70 %; m.p. 230–231 °C; IR (KBr) cm-1: 3387 and
3279 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1335 and 1149 (s, SO2 stretch); 1H NMR
(400 MHz, DMSO-d6): d 8.41 (d, 2H, J = 8.4 Hz, Ar),
8.28 (s, 1H, pyridine C5-H), 8.09 (d, 2H, J = 8.4 Hz, Ar),
7.65 (s, 4H, Ar), 7.50 (s, ex, 2H, SO2NH2); 13C NMR
(75.5 MHz, DMSO-d6): d 150.0, 146.2, 145.8, 144.3,
142.6, 139.8, 134.4, 133.0, 132.8, 131.5, 130.5, 128.2,
127.1, 123.4, 122.2, 121.8, 113.0, 112.2; 19F NMR
(376.4 MHz, DMSO-d6): d –58.59 (C4-CF3), –65.45 (C6-
CF3); DART-MS: m/z 521.10 (M?H)?, C20H11ClF6N4O2
SH? calcd. 521.01.
Yield 53 %; m.p. 262–264 °C; IR (KBr) cm-1: 3340 and
3271 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1358 and 1134 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.55 (d, 2H, J = 8.7 Hz, Ar),
8.35 (d, 2H, J = 8.4 Hz, Ar), 8.26 (s, 1H, pyridine C5-H),
8.09 (d, 2H, J = 8.7 Hz, Ar), 7.62–7.64 (m, 4H, Ar), 7.50
(s, ex, 2H, SO2NH2), 7.14 (d, 2H, J = 8.7 Hz, Ar), 3.86
(OCH3); 13C NMR (100 MHz, CDCl3/DMSO-d6): d 162.0,
156.9, 149.8, 144.0, 142.4, 140.9, 137.6, 133.0, 131.1,
128.3, 126.7, 126.5, 123.2, 114.8, 112.2, 108.6, 104.2, 55.2
(OCH3); 19F NMR (376.4 MHz, DMSO-d6): d -58.77 (C4-
CF3); DART-MS: m/z 559.12 (M?H)?, C26H18ClF3N4O3SH?
calcd. 559.07.
(8c) Yield 12 %.
4-[3-(4-Chlorophenyl)-6-phenyl-4-(trifluoromethyl)-1H-
pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (7c)
(8c) Yield 22 %.
Yield 58 %; m.p. 238–240 °C; IR (KBr) cm-1: 3333 and
3194 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1335 and 1165 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.56 (d, 2H, J = 8.7 Hz, Ar),
8.33–8.36 (m, 3H, pyridine C5-H, Ar), 8.10 (d, 2H,
J = 8.7 Hz, Ar), 7.80 (d, 2H, J = 8.4 Hz, Ar), 7.62–7.65
(m, 3H, Ar), 7.48–7.50 (m, 4H, SO2NH2, Ar); 13C NMR
(100 MHz, CDCl3/DMSO-d6): d 157.3, 151.8, 144.0,
141.6, 140.7, 136.7, 134.3, 131.0, 130.5, 128.9, 128.4,
127.9, 127.4, 126.9, 126.7, 126.6, 123.2, 120.8, 113.2,
109.1; 19F NMR (376.4 MHz, DMSO-d6): d –58.76 (C4-
CF3); DART-MS: m/z 529.16 (M?H)?, C25H16ClF3N4O2
SH? calcd. 529.06.
4-[3-(4-Chlorophenyl)-6-(4-fluorophenyl)-4-
(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridin-1-
yl]benzenesulfonamide (7f)
Yield 50 %; m.p. 220–222 °C; IR (KBr) cm-1: 3302 & 3240
(m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–H bend),
1311 and 1165 (s, SO2 stretch); 1H NMR (300 MHz, DMSO-
d6): d 8.55 (d, 2H, J = 8.7 Hz, Ar), 8.48 (dd, 2H,
4JHF = 5.4 Hz, 3JHH = 8.7 Hz, Ar), 8.37 (s, 1H, pyridine C5-
H), 8.10 (d, 2H, J = 8.7 Hz, Ar), 7.67 (d, 2H, J = 8.7 Hz,
Ar), 7.63 (d, 2H, J = 8.7 Hz, Ar), 7.51 (s, ex, 2H, SO2NH2),
7.45 (t, 2H, J = 8.7 Hz, Ar); 13C NMR (100 MHz, CDCl3/
DMSO-d6): d 156.2, 151.0, 145.2, 142.4, 137.7, 132.9, 131.1,
(8c) Yield 21 %.
123

Med Chem Res
129.8, 128.3, 126.7, 126.6, 125.0, 123.2, 120.6, 116.2, 115.8;
19F NMR (376.4 MHz, DMSO-d6): d -58.75 (C4-CF3);
DART-MS: m/z 547.15 (M ? H)?, C25H15ClF4N4O2SH?
calcd. 547.05.
142.4, 141.5, 137.7, 134.3, 134.0, 133.8, 132.9, 130.9,
128.6, 128.3, 126.9, 126.7, 126.57, 126.51, 123.2, 120.7,
114.5, 109.9; 19F NMR (376.4 MHz, DMSO-d6): d -58.77
(C4-CF3); DART-MS: m/z 579.15 (M?H)?, C29H18ClF3N4
O2SH? calcd. 579.07.
(8c) Yield 24 %.
(8c) Yield 25 %.
4-[3,6-bis(4-Chlorophenyl)-4-(trifluoromethyl)-1H-
pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (7g)
4-[3-(4-Chlorophenyl)-6-(2-thienyl)-4-(trifluoromethyl)-
1H-pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (7j)
Yield 56 %; m.p. 230–232 °C; IR (KBr) cm-1: 3340 &
3271 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1327 and 1157 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.54 (d, 2H, J = 8.7 Hz, Ar),
8.42 (d, 2H, J = 8.7 Hz, Ar), 8.36 (s, 1H, pyridine C5-H),
8.09 (d, 2H, J = 8.7 Hz, Ar), 7.67 (d, 2H, J = 8.7 Hz, Ar),
7.61–7.64 (m, 4H, Ar), 7.52 (s, ex, 2H, SO2NH2); 13C
NMR (75.5 MHz, DMSO-d6): d 156.5, 151.7, 144.6, 142.3,
140.9, 136.3, 135.8, 134.6, 131.9, 131.6, 130.0, 129.6,
128.6, 127.6, 121.9, 113.6, 109.8; 19F NMR (376.4 MHz,
DMSO-d6): d –58.73 (C4-CF3); DART-MS: m/z 563.12
(M?H)?, C25H15Cl2F3N4O2SH? calcd. 563.02.
Yield 54 %; m.p. 268–270 °C; IR (KBr) cm-1: 3340 and
3217 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1335 and 1157 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.51 (d, 2H, J = 8.7 Hz, Ar),
8.32 (s, 1H, pyridine C5-H), 8.30 (d, 1H, J = 3.9 Hz, thi-
enyl), 8.07 (d, 2H, J = 8.7 Hz, Ar), 7.88 (d, 1H,
J = 5.1 Hz, thienyl), 7.62-7.64 (m, 4H, Ar), 7.50 (s, ex,
2H, SO2NH2), 7.27-7.30 (m, 1H, thienyl); 13C NMR
(75.5 MHz, DMSO-d6): d 153.1, 151.3, 144.8, 142.8,
142.0, 141.0, 134.5, 131.9, 131.8, 131.5, 130.0, 129.6,
128.5, 127.4, 121.4, 113.0, 109.3; 19F NMR (376.4 MHz,
DMSO-d6): d –58.86 (C4-CF3); DART-MS: m/z 535.15
(M?H)?, C23H14ClF3N4O2S2H? calcd. 535.01.
(8c) Yield 20 %.
4-[6-(4-Bromophenyl)-3-(4-chlorophenyl)-4-
(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridin-1-
yl]benzenesulfonamide (7h)
(8c) Yield 22 %.
Antimicrobial assays
Yield 53 %; m.p. 270–272 °C; IR (KBr) cm-1: 3350 and
3271 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1335 and 1134 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 8.54 (d, 2H, J = 8.7 Hz, Ar),
8.37 (s, 1H, pyridine C5-H), 8.35 (d, 2H, J = 8.7 Hz, Ar),
8.09 (d, 2H, J = 8.7 Hz, Ar), 7.81 (d, 2H, J = 8.7 Hz, Ar),
7.63-7.66 (m, 4H, Ar), 7.51 (s, ex, 2H, SO2NH2); 13C NMR
(75.5 MHz, DMSO-d6): d 156.6, 151.7, 144.6, 142.3,
140.9, 136.3, 134.6, 132.6, 131.9, 131.6, 130.3, 128.6,
127.6, 125.2, 121.9, 113.7, 109.9; 19F NMR (376.4 MHz,
DMSO-d6): d –58.73 (C4-CF3); DART-MS: m/z 607.05
(M?H)?, C25H15BrClF3N4O2SH? calcd. 606.97.
Determination of antimicrobial activity (bacteria
and yeasts)
The antimicrobial activity of thirty synthesized pyrazol-
o[3,4-b]pyridines was evaluated in vitro by agar well dif-
fusion method (Ahmad and Beg, 2001; Andrews, 2001).
All the microbial cultures were adjusted to 0.5 McFarland
standard, which is visually comparable to a microbial
suspension of approximately 1.5 9 108 cfu/mL. Twenty
milliliter of Mueller–Hinton agar medium was poured into
each petri plate and the agar plates were swabbed with
100 lL inocula of each test bacterium and kept for 15 min
for adsorption. Using sterile cork borer of 8 mm diameter,
wells were bored into seeded agar plates and these were
loaded with a 100 lL volume with concentration of
4.0 mg/mL of each compound reconstituted in dimethyl-
sulphoxide (DMSO). All the plates were incubated at
37 °C for 24 h. Antimicrobial activity of each compound
was evaluated by measuring the zone of growth inhibition
against the test microorganisms with zone reader (HiAn-
tibiotic zone scale). DMSO was used as a negative control
whereas ciprofloxacin was used as positive control for
bacteria and amphotericin-B for fungal yeasts. This pro-
cedure was performed in three replicate plates for each
organism.
(8c) Yield 24 %.
4-[3-(4-Chlorophenyl)-6-(2-naphthyl)-4-(trifluoromethyl)-
1H-pyrazolo[3,4-b]pyridin-1-yl]benzenesulfonamide (7i)
Yield 48 %; m.p. 262–265 °C; IR (KBr) cm-1: 3333 and
3186 (m, N–H stretch), 1597 (s, C=N stretch), 1504 (m, N–
H bend), 1335 and 1165 (s, SO2 stretch); 1H NMR
(300 MHz, DMSO-d6): d 9.00 (s, 1H, pyridine C5-H), 8.61
(d, 2H, J = 8.7 Hz, Ar), 8.51 (d, 2H, J = 8.7 Hz, Ar),
8.11–8.16 (m, 4H, Ar), 7.98–8.02 (m, 1H, Ar), 7.59–7.69
(m, 6H, Ar), 7.51 (s, ex, 2H, SO2NH2); 13C NMR
(100 MHz, CDCl3/DMSO-d6): d 157.1, 151.4, 144.1,
123

Med Chem Res
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Determination of minimum inhibitory concentration (MIC)
MIC is the lowest concentration of an antimicrobial com-
pound that will inhibit the visible growth of a microor-
ganism after overnight incubation. MIC of newly
synthesized pyrazolopyridines against bacterial and yeast
strains was tested through a modified agar well-diffusion
method (Okeke et al., 2001). In this method, a twofold
serial dilution of each tested compound was prepared by
first reconstituting the compound in DMSO followed by
dilution in sterile distilled water to achieve a decreasing
concentration range of 256–0.5 lg/mL. A 100 lL volume
of each dilution was introduced into wells (in triplicate) in
the agar plates already seeded with 100 lL of standardized
inoculum (106 cfu/mL) of the test microbial strain. All test
plates were incubated aerobically at 37 °C for 24 h and
observed for the inhibition zones. MIC, shown by a clear
zone of inhibition, was recorded for each test organism.
Ciprofloxacin (bacteria) and amphotericin-B (yeasts) were
used as positive control while DMSO as negative control.
Chandak N, Bhardwaj JK, Sharma RK, Sharma PK (2013) Inhibitors
of apoptosis in testicular germ cells: synthesis and biological
evaluation of some novel IBTs bearing sulfonamide moiety. Eur
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thermochromic properties. Org Lett 13:1658–1661
Acknowledgments Defence Research and Development Organiza-
tion (DRDO), New Delhi is thankfully acknowledged for financial
support in the form of a research project. Authors (NC and SK) are
grateful to the Council of Scientific and Industrial Research (CSIR),
New Delhi and University Grants Commission (UGC), New Delhi,
respectively, for the award of senior research fellowships. The authors
are thankful to Sophisticated Analytical Instrument Facility, Central
Drug Research Institute, Lucknow for Mass spectra.
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and pyrazolo[3,4-d]pyrimidine derivatives bearing 6,6-diphenyl-
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Conflict of interest The authors declare that they have no conflict
of interest.
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