Synthesis and evaluation of pyrazolo[3,4-b]pyridines and its structural analogues as TNF-α and IL-6 inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.06.042

In the present article, we have synthesized three different series of pyrazolo[3,4-b]pyridines and their structural analogues using novel synthetic strategy involving one-pot condensation of 5,6-dihydro-4H-pyran-3-carbaldehyde/2-formyl-3,4,6-tri-O-methyl-d-glucal /chromone-3-carbaldehyde with heteroaromatic amines. All synthesized compounds were evaluated for their anti-inflammatory activity against TNF-α and IL-6. Out of 28 compounds screened, 40, 51, 52 and 56 exhibited promising activity against IL-6 with 60-65% inhibition at 10 μM concentration. Amongst these, 51, 52 and 56 showed potent IL-6 inhibitory activity with IC₅₀'s of 0.2, 0.3 and 0.16 μM, respectively. Compound 56 was not cytotoxic in CCK-8 cells up to the concentration of >100 μM.

Full text of DOI:10.1016/j.bmc.2008.06.042

Bioorganic & Medicinal Chemistry 16 (2008) 7167–7176
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of pyrazolo[3,4-b]pyridines and its structural
analogues as TNF-a and IL-6 inhibitors
Sandip B. Bharate a, Tushar R. Mahajan a, Yogesh R. Gole a, Mahesh Nambiar b, T. T. Matan b,
Asha Kulkarni-Almeida b, Sarala Balachandran a, H. Junjappa a, Arun Balakrishnan b, Ram A. Vishwakarma a,
*
a Department of Medicinal Chemistry, Piramal Life Sciences Limited, 1, Nirlon Complex, Off Western Express Highway, Goregaon (E), Mumbai 400063, India
b Department of Screening and Biotechnology, Piramal Life Sciences Limited, 1, Nirlon complex, Off Western Express Highway, Goregaon (E), Mumbai 400063, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present article, we have synthesized three different series of pyrazolo[3,4-b]pyridines and their
structural analogues using novel synthetic strategy involving one-pot condensation of 5,6-dihydro-4H-
Received 14 May 2008
Revised 21 June 2008
Accepted 24 June 2008
Available online 26 June 2008
pyran-3-carbaldehyde/2-formyl-3,4,6-tri-O-methyl-
matic amines. All synthesized compounds were evaluated for their anti-inflammatory activity against
TNF- and IL-6. Out of 28 compounds screened, 40, 51, 52 and 56 exhibited promising activity against
IL-6 with 60–65% inhibition at 10 M concentration. Amongst these, 51, 52 and 56 showed potent IL-6
inhibitory activity with IC50’s of 0.2, 0.3 and 0.16
CCK-8 cells up to the concentration of >100 M.
D-glucal/chromone-3-carbaldehyde with heteroaro-
a
l
Keywords:
Pyrazolo[3,4-b]pyridines
Anti-inflammatory
lM, respectively. Compound 56 was not cytotoxic in
l
Ó 2008 Elsevier Ltd. All rights reserved.
TNF-a
IL-6
1. Introduction
shedding of TNF-a from the cell surface. Thus, TNF-a has received
a considerable amount of attention as a molecular target for the
Proinflammatory cytokines are involved in the pathogenesis of
a variety of autoimmune and inflammatory diseases. Interleukin-
6 (IL-6) and tumour necrosis factor-alpha (TNF-a) are two multi-
functional proinflammatory cytokines involved in the pathogene-
sis of cardiovascular, neurodegenerational diseases and cancer
through a series of cytokine signalling pathways.1,2 Inhibition of re-
lease of cytokines has become a major focus of current drug discov-
ery and development, and an important method for evaluating the
bioactivity of drugs. The inhibition of cytokines, in particular TNF-
treatment of diseases mentioned above.4
Amongst proinflammatory cytokines, interleukin-6 (IL-6) is
considered to contribute to the initiation and extension of the
inflammatory process. Interleukin-6 is a multifunctional cytokine
produced by wide range of cells, usually at sites of tissue inflam-
mation, and it regulates hepatic acute-phase response, the immune
response, inflammation and haematopoiesis. It appears to be the
central mediator in a range of inflammatory diseases, including
end-stage renal disease and rheumatoid arthritis.5 Inhibition of
IL-6 has not received desired attention in drug discovery.
a
, has been successful in several clinical trials for the treatment of
rheumatoid arthritis.3
Pyrazolo[3,4-b]pyridine class of compounds are reported to
possess diverse range of biological activities. Misra et al. identified
4-substituted 1H-pyrazolo[3,4-b]pyridine derivatives (1, SQ-
The inflammatory process is characterized by the production of
leukotrienes, histamine, bradykinin, and a variety of cytokines and
chemokines by tissues and migrating cells. Amongst many cyto-
67563: IC50 0.11 l
M) as a new class of cdk2 inhibitors.6 Lin et al.
kines responsible for regulating cellular physiology, TNF-
been proven to play a dominant role both in normal immune func-
tion and in disturbances leading to autoimmune disease. Overex-
pression of TNF-a can lead to a variety of pathological conditions
including rheumatoid arthritis (RA), multiple sclerosis, cachexia,
sepsis, ulcerative colitis, congestive heart failure and Crohn’s dis-
ease. To date, much research has been directed towards the inhibi-
a
has
identified substituted pyrazolo[3,4-b]pyridine analogue 2 as a po-
tent and selective cyclin-dependent kinase and cellular anti-prolif-
erative inhibitor (IC50 0.7 nM) against CDK1/cyclin B.7
Witherington et al. discovered 6-heteroaryl-pyrazolo[3,4-b]pyri-
dine 3 as a glycogen synthase kinase-3 (GSK-3) inhibitor (IC50
0.8 nM).8 Pyrazolo[3,4-b]pyridines are also reported to possess
anti-microbial (4, Escherichia coli: IC50 22
IC50 18
M),9,10 anti-chagasic (5, Trypanosoma cruzi: IC50 1.9
mL)11 and anti-leishmanial (6, Leishmania amazonensis: IC50
0.12
M)12 activities. It has also been reported that pyrazolo[3,4-
b]pyridine analogue, Y-25510 (7), stimulates production of IL-1
and IL-6 at the level of messenger RNA expression in cultured
lM; Candida albicans:
tion of TNF-
a
production, the antagonism of TNF-
a
and the
l
lg/
l
* Corresponding author. Tel.: +91 22 30818302; fax: +91 22 30818334.
E-mail addresses: ram.vishwakarma@piramal.com, ravboc@gmail.com (R.A.
Vishwakarma).
a
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.042

7168
S. B. Bharate et al. / Bioorg. Med. Chem. 16 (2008) 7167–7176
human monocytes as well as enhances in vivo production of IL-l
and IL-6 in mice.13,14 Compound 8 showed potent p38
MAP ki-
nase inhibitory activity (IC50 0.7 nM) and potent in vivo TNF-
a
discovery of new class of compounds against different therapeutic
areas, we have designed and synthesized different series of pyraz-
olo-pyridines and their structural analogues for evaluation of anti-
a
a
inhibition (97% inhibition of LPS-induced TNF-
a
release in mice
inflammatory activity against TNF-
Our dual interest in this work was to synthesize new series of
hybrid structures containing heterocycle and carbohydrate
-glucal) moiety as recognition element, and to evaluate these
against different therapeutic targets. Herein, we report synthesis
of three different series of pyrazolo[3,4-b]pyridines and other hy-
brid heterocycles via novel short and efficient synthetic route.
a and IL-6.
at 20 mg/kg po.).15
Pyrazolo[3,4-b]pyridines, 9 [IC50 15.7 nM (TNF-
IV)] and 10 [IC50 27 nM (TNF- ), >1 M (PDE-IV)], are reported to
and
a), >1
lM (PDE-
a
a
l
(D
exhibit anti-inflammatory activity by inhibition of TNF-
a
PDE-IV.16 Similarly biologically active pyrazolo[1,5-a]pyrimidines,
11 (non-steroidal anti-inflammatory drug)17 and 12 (COX-2 inhib-
itor)18, and isoxazolo[5,4-b]pyridine 13 (TACE inhibitor)19 are also
reported (Fig. 1).
2. Results and discussion
2.1 Chemistry
There are several reports on synthesis of pyrazolo-pyridines and
pyrazolo-pyrimidines using different synthetic routes. Lavecchia et
al. synthesized pyrazolo[3,4-b]pyridines via indirect iodination of
2-chloro-nicotinonitrile to yield 2-chloro-5-iodonicotinonitrile,
which was cyclized with methyl hydrazine leading to 3-amino-5-
iodopyrazolo[3,4-b]pyridine.20 Chebanov et al. synthesized
pyrazolo[3,4-b]pyridines via refluxing 5-amino-3-methyl-1-phen-
ylpyrazole with arylidenepyruvic acids.21 Jachak et al. synthesized
Our initial aim in this work was to devise a short and efficient
synthetic strategy for synthesis of hybrid structures containing
heterocycle and a carbohydrate component. Based on literature re-
ports on construction of heterocyclic skeletons using 3-alkoxyac-
roleins,29,30
we
designed
synthetic
strategy
involving
pyrazolo[3,4-b]pyridines
and
pyrazolo[40,30:5,6]pyrido[2,3-
condensation of 3-alkoxyacrolein analogues with heterocyclic
amines to get pyrazolo[3,4-b]pyridines via imine intermediate as
shown in Fig. 2.
Based on this strategy, we visualized 2-formyl glucal (2-formyl-
3,4,6-tri-O-protected-D-glucal) as a key starting material, which
d]pyrimidines via Friedlander condensation of 5-amino-pyrazole-
4-carbaldehyde with various active methylene compounds.22,23
Goda et al. in 2004 devised a novel route for synthesis of pyrazol-
o-pyridines starting from 1,3-diaryl chalcones and ketones to get
substituted pyrazolo-pyridines.9 Zheng et al. reported one-pot
conversion of 5-azidopyrazole-4-carboxaldehyde to pyrazolo[3,4-
b]pyridines via diazo-transfer and subsequent Friedlander reac-
tion.24 Apart from these reports, several other researchers
published synthesis of substituted 1H-pyrazolo[3,4-b] pyridines
and related skeletons.25–28
has 3-alkoxyacrolein moiety. In order to synthesize a key starting
material, we started with commercially available 3,4,6-tri-O-acet-
yl-D
-glucal (14). For conversion of 3,4,6-tri-O-acetyl-D-glucal (14)
to 3,4,6-tri-O-methyl-
D
-glucal (15), a number of reaction condi-
tions were attempted: (a) NaOH/tetrabutylammonium iodide
(TBAI), CH3I, THF; (b) K2CO3/MeOH; CH3I, NaH/DMF; (c) Na/MeOH;
CH3I, NaH/DMF. Desired product was obtained using all three
methods but TBAI/NaOH method was found to be better in terms
Based on the interesting biological activity profile of 1H-pyraz-
olo[3,4-b]pyridines and as a part of our continuing efforts towards
Figure 1. Some biologically active pyrazolo[3,4-b]pyridines, pyrazolo[1,5-a]pyrimidines and isoxazolo[5,4-b]pyridines.

S. B. Bharate et al. / Bioorg. Med. Chem. 16 (2008) 7167–7176
7169
Table 1
Conditions for condensation of 17 with heterocyclic amine 19
O
N
N
HO
+
N
CHO
N
H2N
N
17
20
19
Reagent/solvent
Condition
Yields (%)a
AcOH/EtOH
HCl/EtOH
pTSA/EtOH
AcOH
HCl/EtOH
pTSA/EtOH
RT, 5–10 h
RT, 5–10 h
RT, 5–10 h
120 °C, 2 h
90 °C, 2 h
90 °C, 2 h
0
0
0
60
40
45
Figure 2. Synthetic approach for pyrazolo[3,4-b]pyridines.
a
Isolated yields.
of yield and also it is one-pot conversion compared with the first
two methods. Treatment of 3,4,6-tri-O-acetyl-
solid NaOH and methyl iodide in THF in presence of TBAI resulted
in formation of 3,4,6-tri-O-methyl- -glucal (15) in 80% yield. Vils-
meier–Haack formylation31 of 15 using POCl3/DMF at room tem-
D-glucal (14) with
this interesting observation can be justified as due to presence of
phenyl moiety on N1 of pyrazole ring, which in conjugation with
D
pyridine ring makes the side chain
a-methoxy a better leaving
perature led to formation of 2-formyl-3,4,6-tri-O-methyl-
D-glucal
group and thus gets exchanged with comparatively stable ethoxy
moiety. This observation was supported by 1H, 13C NMR and MS
data.
(16) in 45% yield (Scheme 1).
After synthesis of 16, the key step involved in synthesis of target
compounds was condensation of formyl precursor 16 with hetero-
cyclic amine. As a model reaction, reaction of 5,6-dihydro-4H-pyr-
an-3-carbaldehyde (17, synthesized from 3,4-dihydropyran 18
using Vilsmeier–Haack formylation) with 1,3-dimethyl-1H-pyra-
zol-5-amine (19) was attempted using different reaction condi-
tions as depicted in Table 1.
As depicted in Table 1, desired hybrid compound 20 was ob-
tained in 40–60% yield using ethanol as a solvent and acetic acid,
HCl or pTSA as catalyst under reflux condition, while desired prod-
uct was not formed when reaction was kept at room temperature
even up to 10 h. The product 20 was characterized using 1H, 13C
NMR, MS and IR data.
After optimization of reaction conditions for condensation, de-
sired hybrid structures of glucal aldehyde 16 with different hetero-
cyclic amines were synthesized (Scheme 2). Yields were not
satisfactory (less than 15%) with the use of AcOH or HCl as a cata-
lyst but comparatively better yields (20–24%) were obtained with
pTSA, as well as very poor yields were obtained when only acetic
acid was used (without ethanol). Treatment of 2-formyl-3,4,6-tri-
In the case of reaction of 2-formyl-3,4,6-tri-O-methyl-D-glucal
(16) with 3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-5-amine
(22), hybrid structure 27b was obtained instead of desired com-
pound 27a (Fig. 3). 1H NMR spectrum of 27b showed (a) only three
methoxy signals (in region d3.15–4.00); (b) two extra protons in re-
gion d 4.00–3.00 ppm; (c) triplet at d 1.21 for three protons. This
observation was also supported by 13C NMR data where signal at
d 15.3 ppm (–CH2CH3) was observed. This observation along with
mass spectral data (478, M+1 peak) led to conclusion that one of
the methoxy groups of side chain gets replaced with ethoxy moi-
ety. DEPT experiments further confirmed presence of ethyl func-
tionality, as two CH2’s are seen in DEPT-135 spectrum of
compound 27b. Our assumption of ethanol (solvent used for reac-
tion) as a source of ethoxy group was confirmed by changing the
reaction solvent (methanol instead of ethanol). As expected, with
the use of methanol as a solvent, no exchange of methoxy group
was noticed, and compound 27a was formed as predicted from
1H NMR signals.
The location of ethyl functionality was further confirmed using
extensive 2-D NMR experiments viz. HSQC and HMBC. The com-
plete assignment of 1H NMR and 13C NMR signals (mentioned in
experimental section; Section 4.4.5) for compound 27b was done
based on DEPT, HSQC and HMBC experiments.
Further, we synthesized several analogues of compound 20 by
varying heterocycle component. Treatment of 5,6-dihydro-4H-pyr-
an-3-carbaldehyde (17) with different heterocyclic amines 19, 21–
23, 31–35 in presence of pTSA or acetic acid resulted in formation
of desired pyran-heterocycle hybrid structures 20, 36–43 in 50–
65% yield (Scheme 3). In the case of reaction of 17 with triazole
amine (31), pyrazole amine (32) and 2-amino benzimidazole (34)
using acetic acid, the terminal free hydroxyl of side chain under-
went acetylation (acyl moiety from acetic acid), and acetylated
products 38, 40 and 41 were obtained. Acetylated products (38
O-methyl-D-glucal (16) with heterocyclic amines 19, 21–23 in
presence of pTSA in EtOH under reflux for 2 h resulted in formation
of hybrid compounds 24–27 in 20–24% yield (Scheme 2). Hybrid
compounds 26–27 were further treated with BF3–DMS, which re-
sulted in formation of deprotected compounds 28–30 in 30% yield.
All synthesized compounds were characterized by 1H, 13C NMR, MS
and IR data.
Interestingly, it was noticed that during condensation of 2-for-
myl-3,4,6-tri-O-methyl-D-glucal (16) with 1-phenyl pyrazole
amines (21–22), a side-chain methoxy located at
a-position to pyr-
idine ring gets replaced with ethoxy moiety, which appears to
come from ethanol, a solvent used for reaction. But this ethyl ex-
change was not observed in the case of reaction of 16 with 1-
methyl pyrazole amine (19). The probable mechanistic reason for
Scheme 1. Reagents and conditions: (a). CH3l, solid NaOH, TBAI, THF, RT, 6 h, 80%; (b). POCl3, DMF, rt, 4 h, 45%.

7170
S. B. Bharate et al. / Bioorg. Med. Chem. 16 (2008) 7167–7176
Scheme 2. Reagents and conditions: (a) pTSA, EtOH, 90 °C, 2 h, 20–24%; (b) BF3–DMS, rt, 4 h, 25–30%.
bonded phenolic OH group. This was confirmed by deuterium ex-
change experiment.
2.2. Biological evaluation
All synthesized compounds were evaluated for anti-inflamma-
tory activity against TNF-
a and IL-6. Results are shown in Table
2. Dexamethasone was used as a reference standard for assay. Glu-
cal-heterocycle hybrids, 24–27, exhibited 15–20% and 30–35%
inhibition of TNF-
a
at 10 and 30
lM, respectively, while none of
the compounds showed significant TNF-
a
inhibitory activity.
Several compounds showed promising activity against IL-6.
Glucal-heterocycle hybrid structures, 26 and 27, showed 44% and
35% inhibition of IL-6 at 10
hybrid compounds pyrazolo-pyrimidine 40 showed promising
activity against IL-6 with 60% inhibition at 10 M. Compounds
37, 43 and 44 showed 35–45% inhibition of IL-6 at 10 M. From
lM while amongst pyran-heterocycle
l
l
chromone-heterocycle hybrid structures, pyrazolo-pyridines, 51–
52, and isoxazolo-pyridines, 56–57, showed 54–63% inhibition of
Figure 3. Condensation of 2-formyl-3,4,6-tri-O-methyl-
phenyl-3-(4-methoxyphenyl)-5-pyrazole amine (22).
D-glucal (16) with 1-
IL-6 at 10
nificant toxicity at 10
ity at 10 M = 37–41% inhibition).
lM (Table 2). Most of the compounds did not show sig-
lM except compounds 51, 52 and 58 (toxic-
l
Most promising compounds 51, 52 and 56 were further tested,
and IC50 was determined. These showed promising IC50 against IL-
and 41) further on treatment with catalytic amounts of potassium
carbonate in methanol led to formation of desired products, 44–45,
in 85–90% yield. All synthesized compounds were characterized by
1H, 13C NMR, MS and IR spectroscopic data.
6 inhibition with 0.2, 0.3 and 0.16
tent IL-6 inhibitory compound 56 did not show cytotoxicity (IC50
>100 M).
lM, respectively (Table 3). A po-
l
From the present work, chromone-heterocycle hybrid com-
pounds, isoxazolo-pyridine 56, showed promising IL-6 inhibition
with IC50 of 0.16 lM. Thus, isoxazolo-pyridine class of compounds
have a potential to emerge as a new class of anti-inflammatory
Based on structural similarity, we further selected 3-formyl chr-
omones, 46–48, as 3-alkoxyacrolein part and synthesized several
hybrid compounds by varying heterocycle part. Treatment of 3-for-
myl chromones, 46–48, with different heterocyclic amines 19, 21–
23, 34–35, 49–50 in presence of pTSA or acetic acid resulted in for-
mation of desired chromone-heterocycle hybrid structures 51–60,
in 70–85% yield (Scheme 4). All synthesized compounds were char-
acterized by 1H, 13C NMR, MS and IR spectroscopic data. The 1H
NMR spectrum of chromone-heterocycle hybrid structures, 51–
60 showed presence of signals at d 11.50–11.70 ppm for hydrogen
agents with specific IL-6 inhibitory activity.
3. Conclusion
In conclusion, three different series of pyrazolo[3,4-b]pyridines
and other related structural analogues were synthesized using

S. B. Bharate et al. / Bioorg. Med. Chem. 16 (2008) 7167–7176
7171
Scheme 3. Reagents and conditions: (a) EtOH/pTSA, or AcOH, 90 °C, 2–4 h, 50-65%; (b) K2CO3, MeOH, 30 min, rt, 85–90%.
new, short and efficient synthetic strategy involving condensation
of 3-alkoxyacroleins with different heterocyclic amines. From the
activity results, we could conclude that only three analogues, 51,
were Kromasil C18 (150 ꢀ 4.6 mm, 3.5
l) column; mobile phase:
ACN (A), 0.01 M NH4OAc + 0.5% TEA, pH 5.0, with AcOH (Time/
A%: 0/10, 20/90, 22/10, 30/10); flow rate: 1 mL/min. Elemental
analysis was carried out using elemental analyzer (Flash 1112 ser-
ies EA). All chromatographic purifications were performed with sil-
ica gel (60–120 mesh), whereas all TLC (silica gel) developments
were performed on silica gel coated (Merck Kiesel 60F254, 0.2 mm
thickness) sheets. All reagents and solvents were of commercial
quality and were used as supplied unless otherwise stated. Yields
reported are isolated yields of the materials.
52 and 56 (IC50 0.2, 0.3 and 0.16
promising compounds. Moreover, since compound 56 did not
show cytotoxicity even up to 100 M concentration, we have
lM, respectively), are the most
l
selected it as a potential lead for further optimization as an IL-6
inhibitor.
4. Experimental
4.1. General
4.2. Synthesis of 3,4,6-tri-O-methyl-
D
-glucal (15)
To a solution of 3,4,6-tri-O-acetyl-
D
-glucal (14, 1 mmol) in THF
Melting points were recorded on Labindia visual melting point
apparatus. 1H NMR spectra were recorded on 300 MHz Bruker
FT-NMR (Avance DPX300) spectrometer using tetramethylsilane
as internal standard, and chemical shifts are reported in d units.
Mass spectra were recorded on either GCMS (Focus GC with TSQ
II mass analyzer and thermoelectron) with auto sampler/direct
injection (EI/CI) or LCMS (APCI/ ESI; Bruker Daltonics MicroTOF
Q). HPLC purity was checked using Waters Alliance or Dionex Ulti-
ma 3000 HPLC system. HPLC conditions used for checking purity
were added powdered NaOH (1 equiv), TBAI (0.5 equiv) and methyl
iodide (5 equiv) successively. Reaction mixture was further al-
lowed to stir for 6 h at room temperature. The reaction mixture
was poured into water and extracted with EtOAc (3ꢀ 150 mL).
The organic layer was washed with water, dried over sodium sul-
fate and concentrated to dryness. Crude reaction mixture was puri-
fied by column chromatography over silica gel (100–200 mesh)
using 20% EtOAc/petroleum ether as eluent to get yellow viscous

7172
S. B. Bharate et al. / Bioorg. Med. Chem. 16 (2008) 7167–7176
Scheme 4. Reagents and conditions: (a) EtOH/pTSA, 90 °C, 2–4 h, 70–85%.
oil (yield: 60%). It was characterized by comparison of spectral data
with literature values.31
was refluxed for 2–4 h. In the case of reactions of pyran aldehyde
or glucal aldehyde with different amines, reaction mixture was
concentrated under vacuum and purified by column chromatogra-
phy over silica gel (100–200 mesh) using 50% EtOAc/petroleum
ether as eluent. In the case of reaction of chromone aldehyde with
different amines, the reaction mixture was filtered and residue was
washed with cold ethanol and finally recrystallized from ethanol.
Method 2: The mixture of equimolar amounts of the appropriate
aldehyde (1 mmol) and heterocyclic amine (1 mmol) in glacial ace-
tic acid (20 mL) was refluxed for 2–4 h. The reaction mixture was
neutralized with saturated NaHCO3 solution and extracted with
EtOAc (3ꢀ 150 mL). Organic layer was concentrated under reduced
pressure and purified by column chromatography over silica gel
(100–200 mesh) using 50% EtOAc/petroleum ether as eluent.
4.3. Vilsmeier–Haack formylation: synthesis of 16–17
To a cooled solution of 15 or 18 (1 mmol) in N,N-dimethylform-
amide (1.1 equiv), phosphorus oxychloride (1.1 equiv) was added
dropwise. Reaction mixture was allowed to stir for 4–6 h at room
temperature. The reaction mixture was neutralized with saturated
NaHCO3 solution and extracted with EtOAc (3ꢀ 150 mL). Organic
layer was washed 7–8 times with cold water followed by brine
in order to remove DMF completely. After concentrating, crude
reaction mixture was purified by column chromatography over sil-
ica gel (100–200 mesh) using 20% EtOAc/petroleum ether as elu-
ent. Compounds 16 and 17 were characterized by comparison of
spectral data with literature values.31,32
4.4.1. 5-Propyl-3-ol-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridine
(20)
4.4. Synthesis of pyrazolo/triazolo[3,4-b]pyridines/pyrimidines
Synthesized by method 1; brown sticky oil; yield: 71%; HPLC:
97.02%; IR (neat): 3326, 2926, 1624, 1508, 1053 cmꢁ1 1H NMR
;
Method 1: To a mixture of equimolar amounts of appropriate
aldehyde (1 mmol) and heterocyclic amine (1 mmol) in ethanol
(20 mL), pTSA (catalytic amount) was added and resulting mixture
(300 MHz, CD3OD): d 8.41 (s, 1H), 8.01 (s, 1H), 4.00 (s, 3H), 3.59
(d, J = 6.3 Hz, 2H), 2.84 (d, J = 7.8 Hz, 2H), 2.52 (s, 3H), 1.95–1.84
(m, 2H); 13C NMR (75 MHz, CD3OD): d 149.8, 149.6, 140.2, 129.9,

S. B. Bharate et al. / Bioorg. Med. Chem. 16 (2008) 7167–7176
7173
(APCI): m/z 310 [M+1]+; 332 [M+Na]+; analysis for C15H23N3O4
(309.2) calcd, C, 58.24; H, 7.49; N, 13.58; found, C, 58.12; H,
7.30; N, 13.42.
Table 2
Anti-inflammatory activity against TNF-
a and IL-6
Entry
% Inhibition at 10
IL-6
lM
TNF-a
Toxicity
4.4.4. 5-(1-Ethoxy-2,4-dimethoxylbutyl-3-ol)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-b]pyridine (26)
20
24
25
26
27
28
29
30
36
37
38
39
6
18
20
19
15
0
3
2
8
12
7
27
34
35
44
35
40
44
43
19
36
45
0
7
3
5
4
6
5
9
8
Synthesized by method 1; brown sticky solid; yield: 22%; HPLC:
98.82%; IR (neat): 3433, 2867, 1532, 1548, 1052 cmꢁ1 1H NMR
;
(300 MHz, CD3OD): d 8.68 (s, 1H), 8.26-8.18 (m, 3H), 7.56–7.52
(m, 2H), 7.35–7.31 (m, 1H), 4.62 (s, 1H), 3.91-3.85 (m, 1H), 3.54–
3.46 (m, 4H), 3.40–3.38 (m, 1H), 3.36 (s, 3H), 3.08 (s, 3H), 2.66 (s,
3H), 1.22 (t, J = 7.5 Hz, 3H); MS (APCI): m/z 386.2 [M+1]+, 408.2
[M+Na]+; analysis for C21H27N3O4 (385.2) calcd, C, 65.44; H, 7.06;
N, 10.90; found, C, 65.31; H, 6.96; N, 10.62.
16
10
0
0
9
40
41
42
43
44
45
51
52
53
54
55
56
57
58
59
60
13
0
0
0
4
0
0
0
0
0
0
5
0
5
0
6
72
66
16
18
37
43
45
60
54
49
34
44
58
63
32
9
0
13
9
12
0
8
41
39
27
19
14
16
25
37
5
4.4.5. 5-(1-Ethoxy-2,4-dimethoxylbutyl-3-ol)-3-(4-
methoxyphenyl)-1-phenyl-1H-pyrazolo[3,4-b]pyridine (27)
For numbering see Fig. 3; synthesized by method 1; brown
sticky solid; yield: 24%; HPLC: 97.04%; 1H NMR (300 MHz, CDCl3):
d 8.70 (d, J = 1.2 Hz, 1H, ArH-C6), 8.39 (d, J = 1.2 Hz, 1H, ArCH-C4),
8.35 (d, J = 8.1 Hz, 2H, ArH-C2’’’, C6’’’), 8.02 (d, J = 9 Hz, 2H, ArH-C2’’,
6’’), 7.57-7.55 (m, 2H, ArH-C3’’’, 5’’’), 7.32 (t, J = 6.0 Hz, 1H, ArH-
C4’’’), 7.11 (d, J = 6 Hz, 2H, ArH-C3’’, 5’’), 4.72 (d, J = 4.6 Hz, 1H, CH-
C1’), 3.91 (s, 3H, OCH3-C7’’), 3.70–3.64 (m, 1H, CH2-C3’), 3.57–3.54
(m, 2H, CH2-C4’), 3.49 (t, J = 6.9 Hz, 1H, CH-C2’), 3.44 (q, J = 3.2,
7.8 Hz, 2H, OCH2-C7’), 3.40 (s, 3H, OCH3-C5’), 3.25 (s, 3H, OCH3-
C6’), 1.22 (t, J = 6.9 Hz, 3H, CH3-C8’); 13C NMR (75 MHz, CDCl3): d
160.1 (C-C4’’), 151.3 (C-C8), 149.5 (CH-C6), 144.0 (C-C3), 139.5 (C-
39
94
0
0
Dexamethasone (1
l
M)
C1’’’), 130.0 (CH-C4), 129.1 (CH-C3’’’,5’’’), 128.8 (CH-C5), 128.6 (CH-
C2’’, 6’’), 125.9 (CH-C4’’’), 125.4 (C-C1’’), 121.3 (CH-C2’’’, 6’’’), 115.0
(C-C9), 114.5 (CH-C3’’, 5’’), 83.4 (CH-C2’), 81.7 (CH-C1’), 73.3 (CH2-
C4’), 71.7 (CH-C3’), 64.7 (CH2-C7’), 60.5 (OCH3-C6’), 59.1 (OCH3-
C5’), 55.4 (OCH3-C7’’), 15.3 (CH3-C8’); MS (APCI): m/z 478.2
[M+1]+; analysis for C27H31N3O5 (477.2) calcd, C, 67.91; H, 6.54;
N, 8.80; found, C, 67.43; H, 6.32; N, 8.44.
Table 3
IC50’s of compounds 51, 52 and 56
Entry
IC50 (l
M)a
TNF-a
IL-6
Toxicity
51
52
56
>100
0.2
0.3
0.16
0.007
2.6
10
>100
>100
4.4.6. 5-Propyl-3-ol-3-methyl-1-phenyl-1H-pyrazolo[3,4-
b]pyridine (36)
>100
>100
0.05
Dexamethasone
Synthesized by method 1; white amorphous solid; yield: 72%;
mp 105–107 °C; HPLC: 97.02%; IR (KBr): 3403, 3018, 2862, 1593,
a
IC50: the concentration that affords 50% inhibition of TNF-a or IL-6.
1508, 1063 cmꢁ1 1H NMR (300 MHz, CDCl3): d 8.43 (s, 1H), 8.19
;
(d, J = 8.1 Hz, 2H), 7.81 (s, 1H), 7.46 (t, J = 8.1 Hz, 2H), 7.22 (t,
J = 7.2 Hz, 1H), 3.68 (t, J = 6 Hz, 2H), 2.84 (t, J = 7.8 Hz, 2H), 2.59
(s, 3H), 1.98-1.89 (m, 2H); 13C NMR (75 MHz, CDCl3): d 150.1,
149.9, 142.1, 139.6, 130.2, 129.1, 128.0, 125.4, 120.7, 117.0, 61.7,
34.5, 29.1, 12.4; MS (APCI): m/z 268 [M+1]+; analysis for
129.0, 115.0, 60.4, 34.3, 32.2, 28.5, 10.6; MS (APCI): m/z 206
[M+1]+; analysis for C11H15N3O (205.1) calcd, C, 64.37; H, 7.37; N,
20.47; found, C, 64.07; H, 7.23; N, 20.13.
4.4.2 6-(1,2,4-Trimethoxybutyl-3-ol)-2-p-tolylpyrazolo[1,5-
a]pyrimidine (24)
C16H17N3O2 (267.1) calcd, C 71.89; H 6.41; N 15.72; found, C
70.31, H 6.54, N 14.96.
Synthesized by method 1; brown sticky solid; yield: 24%; HPLC:
96.06%; IR (Neat): 3323, 2924, 1717, 1622, 1521, 1447, 1322, 1123,
1003 cmꢁ1; 1H NMR (300 MHz CDCl3): d 8.80 (s, 1H), 8.52 (d, J = 1.8
Hz, 1H), 7.90 (d, J = 9.0 Hz, 2H), 7.30 (d, J = 8.2 Hz, 2H), 6.97 (s, 1H),
4.65 (d, J = 3.0 Hz 1H), 4.02 (m, 1H), 3.62–3.56 (m, 2H), 3.39 (s, 3H),
3.32 (s, 3H), 3.32 (m, 1H), 3.18 (s, 3H), 2.42 (s, 3H); MS (APCI): m/z
372.2 [M+1]+; 394.2 [M+Na]+; analysis for C20H25N3O4 (371.2)
calcd, C, 64.67; H, 6.78; N, 11.31; found, C, 64.38; H, 6.54; N, 11.10.
4.4.7. 5-Propyl-3-ol-3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazolo[3,4-b]pyridine (37)
Synthesized by method 1; brown viscous oil; yield: 65%; HPLC:
99.01%; IR (KBr): 3373, 3040, 2927, 1598, 1500, 1032, 1249 cmꢁ1
;
1H NMR (300 MHz, CD3OD + DMSO-d6 - 4:1): d 8.49 (d, J = 1.0 Hz,
1 H), 8.31 (d, J = 1.2 Hz, 1H), 8.27 (d, J = 4.8 Hz, 2H), 7.97 (d,
J = 5.4 Hz, 2H), 7.50 (t, J = 4.8 Hz, 2H), 7.30 (t, J = 4.5 Hz, 1H), 7.08
(d, J = 5.1 Hz, 2H), 3.85 (s, 3 H), 3.59 (t, J = 3.6 Hz, 2 H), 2.88 (t,
J = 4.8 Hz, 2H), 1.94-1.89 (m, 2H); 13C NMR (75 MHz, CD3OD): d
158.5, 147.9, 141.9, 137.5, 130.1, 128.1, 126.4, 123.8, 123.1,
119.2, 113.2, 112.2, 58.6, 52.6, 32.4, 26.9; MS (APCI): m/z 360
[M+1]+; analysis for C22H21N3O2 (359.2) calcd, C, 73.52; H, 5.89;
N, 11.69; found, C, 73.29; H, 5.59; N, 11.72.
4.4.3. 5-(1,2,4-Trimethoxylbutyl-3-ol)-1,3-dimethyl-1H-
pyrazolo[3,4-b]pyridine (25)
Synthesized by method 1; brown sticky solid; yield: 20%; HPLC:
98.65%; IR (Neat): 3020, 2855, 1610, 1516, 1216, 1093, 1032 cmꢁ1
;
1H NMR (300 MHz CDCl3,): d 8.56 (s, 1H), 8.07 (s, 1H), 4.74 (d,
J = 3.2 Hz, 1H), 4.11 (s, 3H), 4.01–3.97 (m, 1H), 3.63-3.55 (m, 3H),
3.41 (s, 3H), 3.34 (s, 3H), 3.31-3.27 (m, 1H), 3.12 (s, 3H), 2.59 (s,
3H); 13C NMR (75 MHz, CDCl3): d 149.3, 146.9, 138.6, 126.6,
124.5, 113.1, 82.5, 78.7, 71.5, 67.9, 58.9, 57.2, 55.7, 31.8, 27.9; MS
4.4.8. 6-(3-Acetoxypropyl)-[1,2,4]triazolo[1,5-a]pyrimidine (38)
Synthesized by method 2; brown sticky solid; yield: 62%; HPLC:
99.11%; IR (KBr): 3322, 2920, 1710, 1623, 1455, 1332, 1135 cmꢁ1
;

7174
S. B. Bharate et al. / Bioorg. Med. Chem. 16 (2008) 7167–7176
1H NMR (300 MHz, CDCl3): d 8.74 (s, 1H), 8.70 (s, 1H), 8.48 (s, 1H),
4.16 (t, J = 6 Hz, 2H), 2.86 (t, J = 8.1 Hz, 2H), 2.11-2.04 (m, 2H), 2.08
(s, 3H); 13C NMR (75 MHz, CD3OD): d 171.3, 157.4, 154.8, 153.8,
134.9, 125.2, 63.0, 29.1, 25.6, 19.3; MS (APCI): m/z 221 [M+1]+;
analysis for C10H12N4O2 (220.1) calcd, C, 54.54; H, 5.49; N, 25.44;
found, C, 54.37; H, 5.41; N, 25.29.
4.4.14. 5-(2-Hydroxy-5-methyl-benzoyl)-1,3-dimethyl-1H-
pyrazolo[3,4-b]pyridine (51)
Synthesized by method 1; yellow solid; yield: 70%; mp 173–
175 °C; HPLC: 99.92%; IR (KBr): 3432, 2980, 2922, 2376, 1624,
1617, 1591, 1508, 1480, 1408, 1373, 1359, 1327, 1296, 1244,
1215, 1056 cmꢁ1 1H NMR (300 MHz, CDCl3): d 11.65 (s, 1H),
;
8.86 (d, J = 1.2 Hz, 1H), 8.39 (d, J = 1.2 Hz, 1H), 7.36–7.35 (m,
4.4.9. 6-Propyl-3-ol-2-p-tolyl-pyrazolo[1,5-a]pyrimidine (39)
Synthesized by method 1; brown sticky solid; yield: 62%;
HPLC: 98.08%; IR (KBr): 3326, 2926, 1711, 1624, 1528, 1457,
2 H), 7.02 (m, 1H), 4.14 (s, 3H), 2.62 (s, 3H), 2.27 (s, 3H); 13C
NMR (75 MHz, CDCl3):
d 197.3, 159.2, 149.8, 148.0, 140.7,
135.7, 130.9, 129.9, 126.3, 124.6, 117.3, 116.6, 112.6, 32.0,
18.7, 10.6; MS (APCI): m/z 282.2 [M+1]+, 304.1 [M+Na]+; analysis
for C16H15N3O2 (281.1) calcd, C, 68.31; H, 5.37; N, 14.94; found,
C, 68.20; H, 5.28; N, 14.88.
1342, 1140, 1053 cmꢁ1 1H NMR (300 MHz, CDCl3): d 8.47 (s,
;
1H), 8.39 (s, 1H), 6.87 (s, 1H), 7.82 (d, J = 8.1 Hz, 2H), 7.24 (d,
J = 7.2 Hz, 2H), 3.70 (t, J = 6 Hz, 2H), 2.74 (t, J = 7.8 Hz, 2H), 2.36
(s, 3H), 1.94–1.85 (m, 2H); 13C NMR (75 MHz, CDCl3): d 154.2,
149.3, 146.8, 137.1, 130.8, 128.1, 127.7, 124.5, 119.5, 91.1, 59.3,
31.2, 24.3, 19.5; MS (APCI): m/z 268 [M+1]+; analysis for
C16H17N3O (267.1) calcd, C, 71.89; H, 6.41; N, 15.72; found, C,
71.65; H, 6.50; N, 15.40.
4.4.15. 5-(2-Hydroxy-5-methyl-benzoyl)-3-methyl-1-phenyl-1H
-pyrazolo[3,4-b]pyridine (52)
Synthesized by method 1; yellow solid; yield: 73.23%; mp 128–
130 °C; HPLC: 99.90%; IR (KBr): 3431, 3050, 2964, 2924, 1632,
1590, 1509, 1482, 1442 1338, 1299, 1226, 1143, 1116,
4.4.10. 6-(3-Acetoxy)propyl-pyrazolo[1,5-a]pyrimidine (40)
1017 cmꢁ1 1H NMR (300 MHz, CDCl3) d: 11.69 (s, 1H), 8.95 (d,
;
Synthesized by method 2; sticky brown solid; yield: 52%; HPLC:
J = 2.1 Hz, 1H), 8.47 (d, J = 2.1 Hz, 1H), 8.26 (d, J = 7.8 Hz, 2H) 7.55
(t, J = 7.8 Hz, 2H), 7.39 (m, 3H), 7.05 (d, J = 8.7 Hz, 1H), 2.72 (s,
3H), 2.17 (s, 3H); 13C NMR (75 MHz, CDCl3): d 197.0, 159.3,
149.9, 148.2, 142.5, 137.1, 135.9, 130.9, 129.9, 127.4, 126.4,
125.7, 124.5, 119.5, 117.2, 116.7, 114.6, 18.7, 10.7; MS (APCI): m/
z 344.1 [M+1]+; analysis for C21H17N3O2 (343.1) calcd, C, 73.45;
H, 4.99; N, 12.24; found, C, 73.34; H, 5.01; N, 12.32.
95.42%; IR (KBr): 3131, 1736, 1629, 1559, 1239, 1123 cmꢁ1 1H
;
NMR (300 MHz, CDCl3): d 8.51 (s, 1H), 8.39 (s, 1H), 8.07 (s, 1H),
6.68 (s, 1H), 4.14 (t, J = 6.3 Hz, 2H), 2.75 (t, J = 8.1 Hz, 2H), 2.07 (s,
3H), 2.07-2.01 (m, 2H,); 13C NMR (75 MHz, CD3OD): d 169.6,
149.7, 145.2, 142.3, 126.7, 120.2, 93.7, 61.3, 27.3, 23.9, 17.5; MS
(APCI): m/z 220 [M+1]+; analysis for C11H13N3O2 (219.1) calcd, C,
60.26; H, 5.98; N, 19.17; found, C, 60.26; H, 5.98; N, 19.17.
4.4.16. 5-(2-Hydroxy-5-methyl-benzoyl)-3-(4-methoxy
phenyl)-1-phenyl-1H-pyrazolo[3,4-b]pyridine (53)
4.4.11. 5-(3-Acetoxy)propyl-1-ethyl-1H-pyrazolo[3,4-b]pyridine
(41)
Synthesized by method 1; yellow solid; yield: 95.81%; mp 161–
163 °C; HPLC: 99.70%; IR (KBr): 3029, 2967, 2930, 2837, 2558,
1938, 1685, 1632, 1608, 1587, 1479.4, 1438, 1278, 1256, 1185,
Synthesized by method 2; brown sticky oil; yield: 85%; HPLC:
99.05%; IR (neat): 3020, 1734, 1612, 1567, 1513, 1508, 1368,
1216 cmꢁ1 1H NMR (300 MHz, CDCl3): d 8.41 (s, 1H), 7.96 (s,
;
1137, 1022 cmꢁ1 1H NMR (300 MHz, CDCl3): d 11.71 (s, 1H),
;
1H), 7.87 (s, 1H), 4.60 (q, J = 7.2, 14.4 Hz, 2H), 4.36 (t, J = 6.6 Hz,
2H), 2.85 (t, J = 8.1 Hz, 2H), 2.08 (s, 3H), 2.05-1.98 (m, 2H),
1.55 (d, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3): d 170.6,
149.2, 148.4, 130.7, 128.8, 128.3, 115.2, 62.9, 41.7, 30.1, 28.9,
20.5, 14.5; MS (APCI): m/z 248 [M+1]+; analysis for C13H17N3O2
(247.1) calcd, C, 63.14; H, 6.93; N, 16.99; found, C, 62.97; H,
7.03; N, 16.69.
8.97 (s, 1H), 8.76 (s, 1H), 8.37 (d, J = 8.1 Hz, 2H), 8.02 (d,
J = 8.7 Hz, 2H), 7.59 (t, J = 7.5 Hz, 2H), 7.42-7.36 (m, 3H), 7.10-
7.03 (m, 3H), 3.91 (s, 3H), 2.46 (s, 3H); 13C NMR (75 MHz, CDCl3):
d 197.0, 159.3, 158.7, 149.8, 148.0, 143.8, 137.1, 136.0, 131.0,
130.9, 127.4, 126.9, 126.5, 126.4, 124.8, 122.7, 119.8, 117.2,
116.7, 113.0, 112.8, 53.6, 18.7; MS (ESI): m/z 434.2 [Mꢁ1]+; analy-
sis for C27H21N3O3 (435.1) calcd, C 74.74; H 4.86; N 9.65; found, C
74.37, H 4.70, N 9.91.
4.4.12. 3-Propyl-3-ol-benzo[4,5]imidazo[1,2-a]pyrimidine (42)
Synthesized by method 1; green amorphous solid; yield: 87%;
mp 120–121 °C; HPLC: 98.06%; IR (KBr): 3061, 2896, 1736, 1612,
4.4.17. 5-(2-Hydroxy-5-fluoro-benzoyl)-1,3-dimethyl-1H-
pyrazolo[3,4-b]pyridine (54)
1497, 1238, 1043 cmꢁ1 1H NMR (300 MHz, CD3OD): d 9.22 (s,
;
Synthesized by method 1; yellow solid; yield: 54%; mp 158–
160 °C; HPLC: 99.76%; IR (KBr): 3407, 3026, 2982, 2930, 2371,
1895, 1617, 1596, 1474, 1373, 1360, 1312, 1286, 1209, 1035,
1H), 8.81 (s, 1H), 8.20 (d, J = 8.1 Hz, 1H), 7.86 (d, J = 8.1 Hz, 1H),
7.59 (t, J = 7.8 Hz, 1H), 7.49 (t, J = 7.8 Hz, 1H), 4.19 (t, J = 6.3 Hz,
2H), 2.89 (t, J = 8.1 Hz, 2H), 2.15–2.07 (m, 2H), 2.03 (s, 3H); 13C
NMR (75 MHz, CD3OD): d 169.6, 156.6, 148.0, 141.1, 130.8, 125.1,
120.0, 119.1, 116.7, 109.7, 61.3, 27.4, 24.0, 17.5; MS (ESI): m/z
255 [Mꢁ1]+; analysis for C15H15N3O2 (269.1) calcd, C 66.90; H
5.61; N 15.60; found, C 67.28, H 5.85, N 15.30.
1000 cmꢁ1 1H NMR (300 MHz, CDCl3): d 11.56 (s, 1H), 8.89 (s,
;
1H), 8.39 (s, 1H), 7.33–7.30 (m, 2H), 7.11-7.07 (m, 1H), 4.15 (s,
3H), 2.63 (s, 3H); 13C NMR (75 MHz, CDCl3): d 196.4, 167.9,
157.4, 149.9, 147.9, 140.9, 130.0, 123.9, 122.4, 122.1, 118.3,
116.1, 115.8, 32.0, 10.7; MS (ESI): m/z 284.2 [Mꢁ1]+; analysis for
C15H12FN3O2 (285.1) calcd, C, 63.15; H, 4.24; N, 14.73; found, C,
4.4.13. 6-(3-Hydroxypropyl)pyrido[2,3-d]pyrimidine-2,4-
(1H,3H)-dione (43)
63.00; H, 4.17; N, 14.49.
Synthesized by method 1; CH2Cl2: MeOH as mobile phase for
column chromatography; greenish sticky semisolid; yield: 52%;
HPLC: 98.09%; IR (KBr): 3754, 3438, 2856, 1701, 1438, 1388,
4.4.18 5-(2-Hydroxy-3,5-dimethyl-benzoyl)-3-methyl-1-phenyl-
1H-pyrazolo[3,4-b]pyridine (55)
1290 cmꢁ1
; d 11.57 (s, 1H),
1H NMR (300 MHz, DMSO-d6):
Synthesized by method 1; yellow solid; yield: 72%, mp 162–
164 °C; HPLC: 99.91%; IR (KBr): 3050, 2620, 1654, 1619, 1509,
11.39 (s, 1H), 8.45 (s, 1H), 8.06 (s, 1H), 4.50 (t, J = 4.8 Hz, 2H),
2.66 (t, J = 7.8 Hz, 2H), 1.74-1.65 (m, 2 H); 13C NMR (75 MHz,
DMSO-d6): d 161.1, 153.3, 149.3, 149.0, 134.0, 131.3, 108.0,
58.2, 32.4, 26.4; MS (ESI): m/z 220 [Mꢁ1]+; analysis for
1466, 1441, 1427 cmꢁ1 1H NMR (300 MHz, CDCl3): d 11.95 (s,
;
1H), 8.90 (s, 1H), 8.43 (s, 1H), 8.23 (d, J = 9 Hz, 2H), 7.52 (t,
J = 9 Hz, 2H), 7.31 (t, J = 9 Hz, 1H), 7.12 (s, 1H), 2.68 (s, 3H), 2.29
(s, 3H), 2.21 (s, 3H); 13C NMR (75 MHz, CDCl3): d 197.3, 157.7,
149.4, 148.2, 142.4, 137.1, 136.9, 129.8, 128.4, 127.4, 125.9,
C10H11N3O3 (221.1) calcd, C, 54.29; H, 5.01; N, 19.00; found, C,
53.89; H, 5.27; N, 18.78.

S. B. Bharate et al. / Bioorg. Med. Chem. 16 (2008) 7167–7176
7175
125.6, 124.5, 119.4, 116.5, 114.6, 18.7, 13.8, 10.7; MS (APCI): m/z
358.2 [M+1]+; analysis for C22H14N3O2 (357.2) calcd, C 73.93; H
5.63; N 11.76; found, C 73.99, H 5.24, N 12.09.
DMSO-d6): d 12.09 (s, 1H), 11.64 (s, 1H), 10.08 (s, 1H), 8.87 (d,
J = 3 Hz, 1H), 8.35 (d, J = 3.2 Hz, 1H), 7.28 (d, J = 8.4 Hz, 1H), 7.19
(s, 1H), 6.90 (d, J = 8.1 Hz, 1H), 2.24 (s, 3H); 13C NMR (75 MHz,
DMSO-d6): d 192.2, 160.6, 153.9, 153.2, 152.7, 148.9, 136.1,
132.9, 128.8, 126.9, 122.9, 115.3, 108.0, 18.5; MS (ESI): m/z 296.1
[Mꢁ1]+; analysis for C15H11N3O4 (297.1) calcd, C 60.61; H 3.73; N
14.14; found, C 60.13, H 3.75, N 14.38.
4.4.19. 5-(2-Hydroxy-5-methyl-benzoyl)-3-methyl
isoxazolo[5,4-b]pyridine (56)
Synthesized by method 1; yellow solid; yield: 69.12%; mp 175–
177 °C; HPLC: 99.44%; IR (KBr): 3046, 2974, 2925, 2590, 2370,
1911, 1632, 1588, 1603, 1522, 1507, 1432, 1379, 1356, 1319,
4.5. Demethylation: synthesis of 28-30
1289, 1250, 1213, 1137, 1080 cmꢁ1 1H NMR (300 MHz, CDCl3): d
;
11.53 (s, 1H), 8.93 (s, 1H), 8.40 (s, 1H), 7.41 (d, J = 8.4 Hz, 1H),
7.28 (s, 1 H), 7.05 (d, J = 8.4 Hz, 1H), 2.67 (s, 3H), 2.27 (s, 3H); 13C
NMR (75 MHz, CDCl3): d 196.1, 168.7, 159.4, 154.6, 149.7, 136.6,
131.1, 130.6, 128.6, 126.7, 116.9, 111.9, 18.7, 9.1; MS (APCI): m/z
268 [M+1]+; analysis for C15H12N2O3 (268.2) calcd, C 67.16, H
4.51, N 10.44; found, C 67.37, H 3.93, N 10.39.
To a stirred solution of 26 or 27 in dry CH2Cl2 at ꢁ48 °C, BF3–
DMS (3 equiv) was added dropwise and reaction mixture was stir-
red further at room temperature for 8–10 h. The reaction mixture
was neutralized by NaHCO3 and extracted with CH2Cl2. Solvent
was evaporated on vacuo rotavapor and crude residue was purified
by silica gel (100–200 mesh) column chromatography using
CH2Cl2: MeOH (99:1).
4.4.20. 5-(2-Hydroxy-5-methyl-benzoyl)-3-phenyl
isoxazolo[5,4-b]pyridine (57)
4.5.1. 5-(1-Ethoxybutyl-2,3,4-triol)-3-(4-hydroxy phenyl)-1-
phenyl-1H-pyrazolo[3,4-b]pyridine (28)
Synthesized by method 1; yellow solid; yield: 70.6%; mp 179–
182 °C; HPLC: 96.37%; IR (KBr): 3423, 3059, 2966, 2918, 1924,
1598, 1508, 1481.8, 1458, 1388, 1337, 1313, 1296, 1243, 1222,
Yellowish brown viscous liquid; yield: 30%; HPLC: 98.53%; 1H
NMR (300 MHz, CDCl3):
d 8.70 (d, J = 1.8 Hz, 1H), 8.53 (d,
1154, 1070, 1046, 1001 cmꢁ1 1H NMR (300 MHz, CDCl3): d 11.57
;
J = 1.8 Hz, 1H), 8.30 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 6.9 Hz, 2H),
7.55 (t, J = 7.5 Hz, 2H), 7.34 (t, J = 7.2 Hz, 1H), 6.99 (d, J = 8.7 Hz,
2H), 4.84 (d, J = 4.2 Hz, 1H), 3.99 (dd, J = 4.5, 7.8 Hz, 1H), 3.73 (dd,
J = 3.3, 11.4 Hz, 1H), 3.68–3.59 (m, 1H), 3.56–3.43 (m, 2H), 1.22
(t, J = 7.2 Hz, 3H); MS (APCI): m/z 336 [M+1]+; analysis for
(s, 1H), 8.99 (d, J = 1.8 Hz, 1H), 8.69 (d, J = 1.8 Hz, 1H), 7.99 (m,
2H), 7.61 (m, 3H), 7.42 (d, J = 8.4 Hz, 1H), 7.30 (s, 1H), 7.06 (d,
J = 8.7 Hz, 1H), 2.28 (s, 1H); 13C NMR (75 MHz, CDCl3): d 195.5,
158.4, 156.0, 149.7, 136.1, 132.2, 130.4, 129.1, 127.6, 127.4,
126.8, 125.8, 123.6, 117.4, 116.5, 95.9, 62.4, 18.5; MS (ESI): m/z
329 [Mꢁ1]+; analysis for C20H14N2O3 (330.1) calcd, C 72.72; H
4.27; N 8.48; found, C 72.99, H 4.43, N 8.41.
C24H25N3O5 (435.12) calcd, C, 66.19; H, 5.79; N, 9.65; found, C,
66.12; H, 5.64; N, 9.71.
4.5.2. 5-(1-Ethoxybutyl-2,3,4-triol)-3-methyl-1-phenyl-1H-
pyrazolo[3,4-b]pyridine (29)
4.4.21. 3-(2-Hydroxy5-methyl-benzoyl)-benzimidazo[1,2-
a]pyrimidine (58)
Yellow oil; yield: 32%; HPLC: 97.03%; 1H NMR (300 MHz,
CDCl3): d 8.59 (d, J = 3 Hz, 1H), 8.24 (d, J = 8.9 Hz, 2H) 8.07 (d,
J = 3 Hz, 1H), 7.51 (t, J = 6 Hz, 3H), 7.31-7.26 (m, 2H), 4.73 (d,
J = 3 Hz, 1H), 3.81 (s, 1H), 3.52-3.45 (m, 3H), 2.67 (s, 3H), 1.22 (t,
J = 7.2 Hz, 3H); MS (APCI): m/z 358.2 [M+1]+; 380.1 [M+Na]+; anal-
ysis for C19H23N3O4 (357.16) calcd, C, 63.85; H, 6.49; N, 11.76;
found, C, 63.69; H, 6.38; N, 11.59.
Synthesized by method 1; yellow solid; yield: 51%; mp 260–
262 °C; HPLC: 99.52%; IR (KBr): 3448, 3152, 3026, 2808, 2712,
1678.6, 1654, 1613, 1582, 1506, 1485, 1458, 1419, 1373, 1336,
1275, 1256, 1221, 1198, 1138, 1115, 1008 cmꢁ1
;
1H NMR
(300 MHz, DMSO-d6): d 11.65 (s, 1H), 7.70-7.62 (m, 3H), 7.54-
7.42 (m, 2H), 7.30 (s, 1H), 7.27-7.22 (m, 2H), 7.11 (d, J = 8.4 Hz,
1H), 2.32 (s, 3H); 13C NMR (75 MHz, DMSO-d6): d 178.1, 152.8,
142.6, 140.3, 135.3, 132.5, 130.5, 130.3, 125.0, 121.9, 121.7,
120.2, 116.8, 116.0, 109.7, 101.8, 79.6, 18.8; MS (ESI): m/z 304.1
[Mꢁ1]+; analysis for C18H13N3O2 (305.1) calcd, C 71.28, H 4.32, N
13.85; found C 71.00, H 3.66, N 13.48.
4.5.3. 5-(4-Methoxy-1-ethoxybutyl-2,3-diol)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-b]pyridine (30)
Yellowish brown viscous liquid; yield: 30%; HPLC: 98.21%; 1H
NMR (300 MHz, CDCl3,): d 8.60 (s, 1H), 8.25 (d, J = 8.8 Hz, 2H),
8.09 (s, 1H), 7.52 (t, J = 6.2 Hz, 2H), 7.28 (t, J = 6.4 Hz, 1H), 4.88
(d, J = 3 Hz, 1H), 3.80–3.71 (m, 2H), 3.59-3.33 (m, 2H), 3.39 (s,
3H), 2.67 (s, 3H), 1.27 (t, J = 6 Hz, 3H); MS (APCI): m/z 372.2
[M+1]+; 394.2 [M+Na]+; analysis for C20H25N3O4 (371.18) calcd, C,
64.67; H, 6.78; N, 11.31; found, C, 64.54; H, 6.65; N, 11.19.
4.4.22. 6-(2-Hydroxy-5-methyl-benzoyl)-2-p-tolyl
pyrazolo[1,5-a]pyrimidine (59)
Synthesized by method 1; yellow solid; yield: 69.23%; mp
208–210 °C; HPLC: 99.96%; IR (KBr): 3422, 3134, 3078, 2917,
2370, 1631, 1597, 1476, 1460, 1372, 1337, 1308, 1295, 1248,
1200, 1165, 1064, 1001 cmꢁ1
; d
1H NMR (300 MHz, CDCl3):
4.6. Deacetylation: synthesis of 44–45
11.37 (s, 1H), 9.07 (s, 1H), 8.82 (s, 1H), 7.92 (d, J = 7.8 Hz, 2H),
7.45 (s, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.33 (d, J = 7.8 Hz, 2H),
7.08 (s, 1H), 7.05 (d, J = 8.4 Hz, 1H), 2.43 (s, 3H), 2.32 (s, 3H);
13C NMR (75 MHz, CDCl3): d 193.0, 159.1, 157.8, 147.9, 147.4,
138.2, 136.4, 135.5, 129.7, 127.9, 127.2, 126.8, 124.8, 116.9,
116.7, 93.1, 19.6, 18.7; MS (ESI): m/z 344.1 [M-1]+; analysis for
To the solution of 38 or 41 in methanol, potassium carbonate
(0.5 equiv) was added and reaction mixture was stirred at room
temperature for 30 min. Water was added to reaction mixture
and extracted with EtOAc (3ꢀ 150 mL). Solvent was evaporated
under vacuum and purified by column chromatography over silica
gel (100–200 mesh) using 50% EtOAc/petroleum ether as eluent.
C21H17N3O2 (343.4) calcd, C 73.45, H 4.99, N 12.24; found, C
73.54, H 5.26, N 12.12.
4.6.1. 6-Propyl-3-ol-[1,2,4]triazolo[1,5-a]pyrimidine (44)
4.4.23. 6-(2-Hydroxy-5-methylbenzoyl) pyrido[2,3-
d]pyrimidine-2,4-(1H,3H)-dione (60)
Brownish sticky solid; yield: 65%; HPLC: 97.05%; IR (KBr):
3366, 2929, 1628, 1450, 1322, 1102 cmꢁ1 1H NMR (300 MHz,
;
Synthesized by method 1; yellow solid; yield: 62%; mp 300–
302 °C (degrad.); HPLC: 96.73%; IR (KBr): 3448, 3195, 3063, 2834,
1727, 1686, 1610, 1590, 1474, 1443, 1363, 1340, 1296, 1250,
CD3OD): d 9.09 (s, 1 H), 8.86 (s, 1H), 8.53 (s, 1H), 3.65 (t,
J = 6.3 Hz, 2H), 2.91 (t, J = 8.1 Hz, 2H), 2.05–1.88 (m, 2H); 13C
NMR (75 MHz, CDCl3 + DMSO-d6 = 4:1): d 155.0, 153.9, 132.2,
1239, 1210, 1178, 1144, 1042, 1024 cmꢁ1 1H NMR (300 MHz,
;

7176
S. B. Bharate et al. / Bioorg. Med. Chem. 16 (2008) 7167–7176
123.0, 58.4, 31.2, 24.1; MS (APCI): m/z 179.2 [M+1]+; analysis for
C8H10N4O (178.1) calcd, C 53.92; H 5.66; N 31.44; found, C
52.02, H 5.38, N 33.05.
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4.6.2. 5-Propyl-3-ol-1-ethyl-1H-pyrazolo[3,4-b] pyridine (45)
Greenish viscous liquid; yield: 60%; HPLC: 99.08%; IR (neat):
3584, 3384, 1612, 1569, 1508, 1457, 1414, 1299, 1260,
1057 cmꢁ1 1H NMR (300 MHz, CD3OD): d8.43 (d, J = 1.2 Hz, 1H),
;
8.01 (d, J = 1.2 Hz, 1H), 8.04 (s, 1H), 4.54 (q, J = 7.2, 14.4, 2H),
3.60 (t, J = 6.3 Hz, 2H), 2.85 (m, 2H), 1.93–1.86 (m, 2H), 1.47 (t,
J = 7.5 Hz, 3H); 13C NMR (75 MHz, CDCl3): d 147.9, 146.6, 129.3,
128.1, 127.0, 113.8, 59.8, 40.3, 32.7, 27.3, 13.2; MS (APCI): m/z
206 [M+1]+; analysis for C11H15N3O (205.1) calcd, C, 64.37; H,
7.37; N, 20.47; found, C, 63.97; H, 7.27; N, 20.03.
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Proinflammatory cytokine production by lipopolysaccharide
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in RPMI 1640 culture medium (Gibco BRL, Pasley, UK) containing
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(FBS, JRH). Cells were differentiated with phorbol myristate acetate
(PMA, Sigma). Following cell plating, the test compounds or vehicle
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30 min at 37 °C. Finally, LPS (Escherichia coli 0127:B8, Sigma Chem-
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ical Co., St. Louis, MO) was added, at a final concentration of 1
Plates were incubated at 37 °C for 24 h, 5% CO2. Supernatants were
harvested, and assayed for TNF- and IL-6 by ELISA as described
lg/mL.
a
by the manufacturer (BD Biosciences). The cells were simulta-
neously evaluated for cytotoxicity using CCK-8 from Dojindo Labo-
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control was calculated. The 50% inhibitory concentration (IC50) val-
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