Oxazolo[4,5-b]pyridine-Based Piperazinamides as GSK-3β Inhibitors with Potential for Attenuating Inflammation and Suppression of Pro-Inflammatory Mediators

Article abstract of DOI:10.1002/ardp.201700022

Recent studies reveal that glycogen synthase kinase-3β (GSK-3β) acts as a pro-inflammatory enzyme, and by inhibiting this kinase, inflammation can be controlled. In this regard, a series of 17 piperazine-linked oxazolo[4,5-b]pyridine-based derivatives was

Full text of DOI:10.1002/ardp.201700022

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Full Paper
Oxazolo[4,5-b]pyridine-Based Piperazinamides as GSK-3b
Inhibitors with Potential for Attenuating Inflammation and
Suppression of Pro-Inflammatory Mediators
Mushtaq A. Tantray1, Imran Khan1, Hinna Hamid
and Ajaz Ahmad Ganai3
1, Mohammad Sarwar Alam1, Abhijeet Dhulap2,
1
Faculty of Science, Department of Chemistry, Jamia Hamdard (Hamdard University), New Delhi, India
CSIR ꢀ Unit for Research and Development of Information Products (URDIP), Pune, India
Molecular Virology Lab., Department of Biotechnology, Jamia Millia Islamia, New Delhi, India
2
3
Recent studies reveal that glycogen synthase kinase-3b (GSK-3b) acts as a pro-inflammatory enzyme,
and by inhibiting this kinase, inflammation can be controlled. In this regard, a series of 17
piperazine-linked oxazolo[4,5-b]pyridine-based derivatives was synthesized and evaluated for in vitro
GSK-3b inhibitory and in vivo anti-inflammatory activity. The compounds 7d, 7e, 7g, and 7c displayed
the best GSK-3b inhibitory activity among all the synthesized compounds, with corresponding IC50
values of 0.34, 0.39, 0.47, and 0.53 mM. Among the compounds 7d, 7e, 7g, and 7c examined for
in vivo anti-inflammatory activity in the rat paw edema model, compound 7d exhibited maximum
inhibition, reducing the paw volume by 62.79 and 65.91% at 3 and 5 h post-carrageenan
administration, respectively, in comparison to indomethacin (76.74% at 3 h and 79.54% at 5 h after
carrageenan administration). Furthermore, these compounds (7d, 7e, 7g, and 7c) were also found to
substantially inhibit pro-inflammatory mediators, i.e., TNF-a, IL-1b, and IL-6, ex vivo in comparison to
indomethacin and did not pose any gastric ulceration risk, indicating the potential of this
oxazolopyridine scaffold for the development of GSK-3b inhibitors and their application as anti-
inflammatory agents.
Keywords: Amide / Inflammation / Kinase inhibition / Oxazolopyridines
Received: January 20, 2017; Revised: April 28, 2017; Accepted: May 2, 2017
DOI 10.1002/ardp.201700022
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
proliferation, apoptosis, and embryonic development. This
multi-functional kinase, however, if deregulated, is reported
a culprit in various diseases, including diabetes, Alzheimer’s
Introduction
Glycogen synthase kinase-3 (GSK-3) is a serine/threonine
kinase prevalent in two major mammalian isoforms, GSK-3a
(MW 51 kDa) and GSK-3b (MW 47 kDa). GSK-3 modulates a
diversity of physiological processes such as insulin
signaling, glycogen metabolism, neuronal functions, cellular
disease (AD) and other CNS disorders, chronic inflammatory
disorders, malaria, and cancer [1, 2]. GSK-3 is thus targeted for
the development of therapeutic agents against these
diseases.
GSK-3b is also an important positive mediator of inflam-
mation possessing capacity to modulate the inflammatory
response in both innate and adaptive immune cells. GSK-3b
augments the production of various pro-inflammatory
cytokines, such as interleukin-6 (IL-6), interleukin-1b (IL-1b),
and tumor necrosis factor (TNF), while concurrently suppress-
ing the production of the anti-inflammatory cytokines such as
interleukin-10 (IL-10). Conversely, the application of GSK-3b
Correspondence: Dr. Hinna Hamid, Jamia Hamdard (Hamdard
University) Hamdard Nagar, New Delhi 110062, India.
E-mail: hhamid@jamiahamdard.ac.in
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inhibitors like lithium chloride, SB216763, TDZD8, and
CHIR99021 have been reported to reduce the effect by
inhibiting the production of pro-inflammatory cytokines and
increasing anti-inflammatory cytokine production (Fig. 1)
[3–7]. Toll-like receptor (TLR)-dependent activation of the
PI3K-Akt signaling pathway has been found to downregulate
GSK-3 activity [8, 9]. Inhibition of GSK-3b modulates the
inflammatory response by inducing down-regulation of
NF-kB-mediated transcription (without affecting IkB degra-
dation and nuclear translocation of NF-kB) [10], which is
responsible for gene expression and production of various
pro-inflammatory mediators like TNF-a, IL-1b, iNOS, IL-6,
etc. [3, 8, 11–13]. Attenuation of GSK-3 activity bolsters cAMP
response element-binding protein (CREB) to sequester
coactivator CREB-binding protein (CBP) from NF-kB, leading
to suppression of NF-kB/CBP-mediated activation of pro-
inflammatory cytokine gene transcription [3, 12]. Inhibition of
GSK-3b thus controls inflammation and protects the host
against inflammatory-mediated pathology and death in
animal models. Remarkably, the in vivo administration of
GSK-3b inhibitors has been reported to suppress inflamma-
tion in various animal models including TNF-a administration,
endotoxemia, experimental colitis, type II collagen-induced
arthritis, ovalbumin-induced asthma, carrageenan-induced
Figure 1. Some known GSK-3b inhibitors with reported anti-inflammatory potential.
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lung injury, and experimental autoimmune encephalomyeli-
tis [3, 5, 11–22]. This association between GSK-3b and
inflammation further increases the scope for development
of GSK-3b inhibitors for the treatment of inflammatory
disorders in a broad range of conditions.
been performed against GSK-3b to understand the molecular
interactions of these compounds with the GSK-3b site. Since
the anti-inflammatory compounds are known to pose a side
effect of gastric ulceration, the active compounds have also
been checked for their gastric ulceration risk.
In continuation of our search for GSK-3 inhibitors, we
focused our attention on oxazolo-pyridines. Oxazolo-
pyridines have been found to be relatively potent anti-
inflammatory, anti-analgesic, anti-pyretic, anti-leishmanial,
anti-cancerous, and anti-diabetic agents. They hold potential
value for a broad spectrum of conditions where one or more
of the symptoms of inflammation, fever, and pain are
manifested including rheumatoid arthritis, osteoarthritis,
gout, infectious arthritis, rheumatic fever, ultraviolet
erythema, atopic dermatitis, contact dermatitis, seborrheic
dermatitis, diaper rash, and inflammatory conditions of the
ocular system [23–28]. Oxazolo-pyridines have also been
reported as potent GSK-3b inhibitors [29, 30]. So far not much
work has been done on this scaffold and there is ample scope
for the development of more potent inhibitors from this
ligand. In view of the pharmacological potential of hitherto
relatively unexplored oxazolo[4,5-b]pyridine scaffold and the
privileged nature of piperazine linkage, we synthesized novel
piperazine-linked oxazolo[4,5-b]pyridine based derivatives as
GSK-3b inhibitors. A series of 17 compounds has been
synthesized, and assessed for in vitro GSK-3b inhibition.
Given the potential role of GSK-3b inhibition in controlling
inflammation, inhibitors of GSK-3b are thus believed to hold a
significant potential for the suppression of inflammatory
conditions. The synthesized compounds exhibiting significant
GSK-3b inhibition have been therefore evaluated for in vivo
anti-inflammatory activity by paw-edema method, and the
compounds showing prominent anti-inflammatory profile
have been examined for their effect on pro-inflammatory
mediators, like TNF-a, IL-1b, and IL-6 using ex vivo assays. The
molecular docking studies of the active compounds have also
Results and discussion
Chemistry
The compounds have been synthesized according to Scheme 1.
2-Amino-3-hydroxy pyridine 1 on refluxing with carbon
disulphide and potassium hydroxide in absolute ethanol
yielded 2-mercapto oxazolo[4,5-b]pyridine 2, which on
treatment with bromoacetic acid in the presence of potassium
carbonate in acetone formed oxazolo[4,5-b]pyridine-2-
mercaptoacetic acid 3. On the other hand, N-Boc piperazine
4 was coupled with various acids using EDC.HCl as a coupling
agent, to form various N-Boc piperazinamides 5 which when
deprotected with TFA and chloroform mixture gave the
requisite piperazinamides 6. Intermediates 3 and 6 were then
coupled by EDC.HCl in the presence of HOBt to afford the
target compounds 7a–q, as shown in Table 1. The target
compound structures have been established by 1H NMR,
13C NMR, IR and mass spectral techniques.
Biological activity
In vitro GSK-3b inhibitory activity
All the synthesized compounds were screened for in vitro
GSK-3b inhibition potential by Kinase-GloTM luminescent
method [31, 32]. The results as depicted in Table 1 indicate
that the compounds inhibited GSK-3b in the submicromolar
to micromolar range. Compounds 7d, 7e, 7g, and 7c were
most active among all the synthesized compounds with
corresponding IC50 values of 0.34, 0.39, 0.47, and 0.53 mM.
Staurosporine, taken as standard, exhibited an IC50 value of
Scheme 1. Synthetic approach for novel oxazolo[4,5-b]pyridine-based piperazinamides.
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Table 1. (Continued)
Table 1. Oxazolo[4,5-b]pyridine-based derivatives and
their GSK-3b inhibitory activity.
Compound
7o
Compound (R)
GSK-3b Inh. IC50
(mM) ꢁ SEMa)
19.31 ꢁ 1.03
Compound
Compound (R)
GSK-3b Inh. IC50
(mM) ꢁ SEMa)
7a
7b
5.79 ꢁ 0.17
7p
2.34 ꢁ 0.09
18.12 ꢁ 0.78
7q
18.90 ꢁ 0.87
0.04 ꢁ 0.01
7c
0.53 ꢁ 0.03
Staurosporine
aSEM, Standard error of the mean.
Numbers in bold indicate the activity of the most active
compounds.
7d
7e
0.34 ꢁ 0.02
0.39 ꢁ 0.03
7f
4.82 ꢁ 0.06
7g
7h
0.47 ꢁ 0.05
9.05 ꢁ 0.12
0.04 mM. Compound 7j also displayed moderate inhibition in
single-digit micromolar range while the remaining com-
pounds exhibited relatively weak inhibition.
From the in vitro GSK-3b inhibition results, it can be seen
that the benzyl derivatives demonstrated lower activity in
comparison to their phenyl counterparts. Furthermore,
among the phenyl derivatives, para-hydroxy substituent
exhibited better activity than para-methoxy substituent
which in turn imparted more activity than para-ethoxy
substituent, implying decrease in activity with increase in size.
7i
15.32 ꢁ 0.16
7j
0.72 ꢁ 0.08
In vivo anti-inflammatory activity
7k
7l
16.25 ꢁ 0.94
12.43 ꢁ 0.47
The synthesized compounds 7d, 7e, 7g, and 7c, displaying
potential GSK-3b inhibitory activity were also evaluated for
their in vivo anti-inflammatory activity by carrageenan-
induced hind-paw edema model. Indomethacin, an anti-
inflammatory drug, was taken as a standard. SB216763, a
known GSK-3b inhibitor, reported to exert anti-inflammatory
effect in various in vitro and in vivo models, was also used as
the reference. The results shown in Table 2 indicated that all
the tested compounds displayed a time-dependent increase in
anti-inflammatory activity after 3 and 5 h post-carrageenan
administration, by ameliorating inflamed rat paw edema. The
compounds 7d and 7e exhibited significantly controlled
inflammation, reducing the paw volume by 62.79 and
60.46% inhibition at 3 h and, 65.91 and 63.64% inhibition,
7m
7n
0.91 ꢁ 0.15
15.12 ꢁ 0.57
(Continued)
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Table 2. In vivo anti-inflammatory activity of oxazolo[4,5-b]pyridine-based derivatives.
Change in paw edema volume (mL) after drug
% Inhibition
treatment (ꢁSEM)
Group
3 h
5 h
3 h
5 h
Control
Indomethacin
SB216763
7c
7d
7e
7g
1.016 ꢁ 0.101
1.033 ꢁ 0.088
–
–
0.467 ꢁ 0.056ꢂꢂꢂ
0.567 ꢁ 0.076ꢂꢂ
0.650 ꢁ 0.043ꢂꢂ
0.583 ꢁ 0.048ꢂꢂ
0.617 ꢁ 0.060ꢂꢂ
0.600 ꢁ 0.073ꢂꢂ
0.483 ꢁ 0.048ꢂꢂꢂ
0.550 ꢁ 0.084ꢂꢂ
0.633 ꢁ 0.076ꢂꢂ
0.567 ꢁ 0.099ꢂꢂ
0.600 ꢁ 0.073ꢂꢂ
0.583 ꢁ 0.079ꢂꢂ
76.74
67.44
51.16
62.79
60.46
58.14
79.54
70.45
54.55
65.91
63.64
61.36
Data are analyzed by one-way ANOVA followed by Dunnett’s t-test and expressed as mean ꢁ SEM from six observations;
ꢂ
ꢂꢂꢂp < 0.001, ꢂꢂp < 0.01, and p < 0.05.
respectively, at 5 h post-carrageenan administration relative
to indomethacin (76.74% at 3 h and 79.54% at 5 h after
carrageenan administration). Compounds 7g and 7c also
showed a fairly good activity, reducing the paw volume by
58.14 and 51.16% after 3 h interval and 61.36 and 54.55%,
respectively, after 5 h interval. SB216763 exhibited inhibition
of 67.44% at 3 h and 70.45% at 5 h post-carrageenan
treatment in comparison to indomethacin.
On comparison of these results with GSK-3b inhibitory data,
it was found that the anti-inflammatory profile of the tested
compounds 7d, 7e, 7g, and 7c was observed to be in
agreement with their observed GSK-3b inhibition activities.
The compounds, therefore, exhibited good correlation
between GSK-3b inhibition and anti-inflammatory property,
suggesting the role GSK-3b inhibitors can play against
inflammation and inflammatory associated conditions.
carrageenan group, with 7d possessing the highest potential
(Fig. 2).
Ulcerogenic risk evaluation
The compounds possessing potential anti-inflammatory
activity were also studied for their gastric ulcerogenic risk.
As compared to indomethacin, SB216763 and the active
compounds 7d, 7e, 7g, and 7c did not cause any gastric
ulceration at even three times higher dose than used for anti-
inflammatory activity (Fig. 3).
Molecular docking studies
In order to understand binding interactions of the synthesized
compounds, docking studies of the active compounds (7d, 7e,
7g, and 7c) were performed against GSK-3b protein (PDB:
1Q3D). Staurosporine was taken as the reference. As depicted
in Table 3, compound 7d shows the highest glide score of
ꢀ6.29 in comparison to staurosporine with glide score of
ꢀ10.01. It forms H-bond with LYS-85 and water residue
(Fig. 4). These interactions might account for its observed GSK-
3b inhibitory activity in vitro.
Effects of compounds 7d, 7e, 7g, and 7c on pro-inflammatory
mediator levels
The inflammatory process is characterized by the generation
of large volumes of the pro-inflammatory mediators like TNF-
a, IL-1b, and IL-6, which have
a central role in the
perpetuation of chronic inflammation and tissue damage
during progression of inflammatory disorder. GSK-3b upre-
gulates the production of these pro-inflammatory mediators,
thereby positively modulates the inflammation. The suppres-
sion of these molecules has been reported to result in a
reduction of disease severity [33]. The compounds showing
potent activity against both GSK-3b as well as in vivo
inflammation were therefore further evaluated to study
their effect on pro-inflammatory mediators, TNF-a, IL-1b, and
Conclusion
A series of seventeen piperazine-linked oxazolo[4,5-b]pyri-
dine-based derivatives has been synthesized and screened for
in vitro GSK-3b inhibitory activity. The compounds 7d, 7e, 7g,
and 7c displayed highest activity among all the synthesized
compounds with corresponding IC50 values of 0.34, 0.39, 0.47,
and 0.53 mM. Out of compounds 7d, 7e, 7g, and 7c examined
for in vivo anti-inflammatory activity in rat-paw edema
model, compound 7d exhibited maximum inhibition of
65.91% at 5 h post-carrageenan administration in comparison
to indomethacin (79.54%). Furthermore, these compounds
(7d, 7e, 7g, and 7c) were also found to inhibit the pro-
inflammatory mediators, viz., TNF-a, IL-1b, and IL-6 ex vivo
substantially in comparison to indomethacin, and did not
pose any gastric ulceration risk, indicating the potential of this
IL-6.
A significant increase in the level of these pro-
inflammatory markers was observed in carrageenan-induced
inflammation group in comparison to control, as evident
from a higher concentration of TNF-a, IL-1b, and IL-6 in
carrageenan-injected rat paws relative to indomethacin and
SB216763-treated groups. The treatment with selected
conjugates 7d, 7e, 7g, and 7c suppressed the elevation in
the level of IL-1b, and TNF-a significantly when compared to
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Figure 2. Effects of test compounds, indomethacin and SB216763 on inflammatory mediators was measured in rat paw homogenate.
(a) TNF-a, (b) IL-1b, and (c) IL-6. Data are expressed as mean ꢁ SEM of six rats. ꢂꢂp < 0.01, ꢂꢂꢂp < 0.001 when compared with the
carrageenan group.
oxazolopyridine scaffold for the development of GSK-3b
inhibitors and their application as anti-inflammatory agents.
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting
Information.
Synthesis of 2-mercapto-1,3-oxazolopyridine (2)
Experimental
A mixture of 1.8 g (32.7 mmol) of KOH, 2.1 mL of CS2 and 50 mL
of dry alcohol was taken in 100 mL R.B. flask and refluxed for
30 min. Then 3 g (27.2 mmol) of 2-amino-3-hydroxy-pyridine
(1) was added to the reaction mixture and refluxing was
continued for 10 h. The progress of reaction was monitored
continuously by TLC. The reaction mixture was then concen-
trated, cooled, poured in to a beaker containing crushed ice
and stirred for few minutes and then filtered. A yellow-
colored solid was formed which was dried. It was then
dissolved in ethyl acetate and filtered to remove insoluble
impurities. The ethyl acetate fraction was concentrated to
obtain the pure compound.
Chemistry
General
The chemicals and reagents employed in this study were
purchased from Sigma–Aldrich, Merck (India), and Spectro-
chem. Melting points were measured using Electro-thermal IA
9100 apparatus (Shimadzu, Japan) and are uncorrected; IR
spectra were recorded on a Bruker spectrophotometer (USA),
NMR spectra were determined on a Bruker (300 and 400 MHz)
spectrometer in DMSO-d6 and the chemical shifts are
expressed as ppm against TMS as internal reference. Mass
spectra were recorded at 70 eV (EI Ms-QP 1000EX, Shimadzu,
Japan) or by a Waters UPLC-TQD (ESI-MS). Column chroma-
tography was performed on Merck silica gel 60 (particle size
0.06–0.20 mm). Elemental analysis was carried out using
Elemental Vario EL III elemental analyzer and data are
reported in % standard. All the compounds synthesized in the
current study are novel and were confirmed from spectral
data.
Synthesis
of
1,3-oxazolopyridine-2-mercapto-acetic
acid (3)
A mixture of 2 g (13.07 mmol) of 2-mercapto-1,3-oxazolopyr-
idine (3), 2.6 mL (19.6 mmol) of TEA and around 40–50 mL THF
was taken in a 100 mL R.B. flask and refluxed for half an hour.
Then 2.92 g (19.6 mmol) of bromoacetic acid was added and
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Figure 3. Histopathology report of ulcerogenic risk potential of active compounds. (a) Control, 10ꢃ; (b) SB216763, 10ꢃ; (c)
indomethacin, 10ꢃ; (d) 7c, 10ꢃ; (e) 7d, 10ꢃ; (f) 7e, 10ꢃ; (g) 7g, 10ꢃ.
refluxing was continued for 8 h. The progress of the reaction
mixture was monitored continuously by TLC. On completion
of the reaction, the reaction mixture was concentrated,
poured on crushed ice and 2 drops of conc. H2SO4 were added,
white-colored precipitate was formed which was filtered,
dried, and used as such for the next step.
temperature followed by addition of N-Boc piperazine (4)
(1.2eq). Thereactionmixturewasallowedtostirfor8–10 hwith
regular monitoring by TLC. After completion of the reaction,
the reaction mixture was poured onto crushed ice and the solid
obtained was filtered and air dried. The compounds were then
purified in ethyl acetate to afford the desired intermediates.
Synthesis of the N-Boc piperazinamides (5)
Synthesis of the piperazinamides (6)
Appropriate aryl acids or arylalkyl acids (1 eq) were dissolved in
dry DMF. EDC.HCl (1.5 eq) and HOBt (catalytic amount) were
added and stirring was continued for 30min at room
The N-Boc piperazinamides (5) were deprotected by adding to
it a mixture of TFA and chloroform (2:3) and stirred in a
magnetic stirrer for 8 h. On completion of the reaction, the
reaction mixture was concentrated to near dryness. The crude
residue was packed onto a silica gel column and the desired
intermediates (6) were eluted with methanol/chloroform
(3:7).
Table 3. In silico docking scores with respect to GSK-3b
protein target (PDBID: 1Q3D).
Compound code
Docking score
Synthesis of the target compounds 7a–q
1,3-Oxazolopyridine-2-mercapto-acetic acid (3) (1 eq) was
dissolved in dry DMF. EDC.HCl (1.5 eq) and HOBt (catalytic
amount) were added and stirring was continued for 30 min at
room temperature followed by addition of piperazinamides (6)
(1.2eq). Thereactionmixturewasallowedtostirfor8–10 hwith
regular monitoring by TLC. After completion of the reaction,
7d
7e
7g
7c
ꢀ6.29
ꢀ6.25
ꢀ6.24
ꢀ6.24
ꢀ10.01
Standard (staurosporine)
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Figure 4. Docking poses and interaction of
compound 7d with respect to the GSK-3b
protein.
the reaction mixture was poured onto crushed ice and the solid
obtained was filtered and air dried. The compounds were then
purified in methanol to afford the target compounds (7a–q).
4.47 (s, 2H), 7.12 (dd, 1H, J ¼ 8.0 and 5.2 Hz), 7.50 (d, 2H,
J ¼ 8.4 Hz), 7.86 (d, 1H, J ¼ 8.0 Hz), 8.07–8.09 (m, 2H), 8.20–8.22
(m, 1H); 13C NMR (100 MHz, DMSO-d6): d 37.03, 42.11, 45.71,
118.49, 120.06, 124.25, 128.88, 142.42, 144.02, 146.30, 148.38,
155.73, 165.34, 167.85, 168.85; MS (ESI) m/z: 428 (Mþ1)þ. Anal.
calcd. for C19H17N5O5S: C, 53.39; H, 4.01; N, 16.38; O, 18.72; S,
7.50%. Found: C, 53.35; H, 4.03; N, 16.36; S, 7.48%.
1-[4-(4-Methoxybenzoyl)piperazin-1-yl]-2-{[1,3]-
oxazolo[4,5-b]pyridin-2-ylsulfanyl}ethan-1-one (7a)
Creamish powder; yield: 82%; m.p. 179–180°C; IR (KBr): n
(cmꢀ1) 3418, 3396, 3013, 2850, 1697, 1681, 1600, 1493, 1296,
1247, 1164, 1116, 1010; 1H NMR (400 MHz, DMSO-d6): d
3.44–3.58 (m, 8H), 3.74 (s, 3H), 4.58 (s, 2H); 6.94 (d, 2H,
J ¼ 8.8 Hz), 7.29 (dd, 1H, J ¼ 8.0 and 4.8 Hz), 7.35 (d, 2H,
J ¼ 8.4 Hz), 8.02 (dd, 1H, J ¼ 8.0 and 1.2 Hz), 8.37 (dd, 1H, J ¼ 4.8
and 1.2 Hz); 13C NMR (100 MHz, DMSO-d6): d 36.66, 41.71,
45.31, 55.24, 113.65, 118.02, 119.56, 127.44, 129.16, 143.51,
145.80, 155.24, 160.30, 164.77, 168.39, 169.20; MS (ESI) m/z:
413 (Mþ1)þ. Anal. calcd. for C20H20N4O4S: C, 58.24; H, 4.89; N,
13.58; O, 15.52; S, 7.77%. Found: C, 58.29; H, 4.86; N, 13.55; S,
7.78%. The 13C NMR, 1H NMR, and ESI-MS spectra of 7a are
provided as Supporting Information.
1-(4-(4-Chlorobenzoyl)piperazin-1-yl)-2-(oxazolo[4,5-b]-
pyridin-2-ylthio)ethanone (7d)
White powder; yield: 83%; m.p. 161–162°C; IR (KBr): n (cmꢀ1
)
3410, 3379, 3011, 2844, 1696, 1681, 1592, 1485, 1289, 1244,
1161, 1109; 1H NMR (400 MHz, DMSO-d6): d 3.35–3.69 (m, 8H),
4.47 (s, 2H), 6.87 (dd, 1H, J ¼ 8.0 and 5.2 Hz), 7.16–7.20 (m, 1H),
7.29–7.31 (m, 1H), 7.38–7.40 (m, 1H), 7.48 (d, 1H, J ¼ 8.4 Hz),
7.75 (d, 1H, J ¼ 8.4 Hz), 7.80 (dd, 1H, J ¼ 5.6 and 1.2 Hz);
13C NMR (100 MHz, DMSO-d6): d 37.01, 42.24, 45.72, 113.67,
117.65, 122.57, 127.82, 128.56, 135.85, 136.76, 145.83, 148.52,
163.58, 168.38, 169.41; MS (ESI) m/z: 417 (Mþ1)þ. Anal. calcd.
for C19H17ClN4O3S: C, 54.74; H, 4.11; Cl, 8.50; N, 13.44; O, 11.51;
S, 7.69%. Found: C, 54.71; H, 4.06; N, 13.42; S, 7.68%.
1-[4-(2-Methoxybenzoyl)piperazin-1-yl]-2-{[1,3]-oxazolo
[4,5-b]-pyridin-2-ylsulfanyl}ethan-1-one (7b)
Creamish powder;yield: 77%; m.p. 161–163°C; IR (KBr): n (cmꢀ1
)
1-(4-(2-Methylbenzoyl)piperazin-1-yl)-2-(oxazolo[4,5-b]-
3430, 3386, 3012, 2848, 1696, 1683, 1598, 1495, 1293, 1251,
1161, 1118, 1010; 1H NMR (400MHz, DMSO-d6): d 3.42–3.55 (m,
8H),3.74(s,3H),4.46(s,2H),7.01–7.07(m,2H),7.21–7.25(m,1H),
7.29(dd, 1H, J ¼ 8.0 and 4.8 Hz), 7.42–7.45(m, 1H), 7.74–7.76(m,
1H), 8.32 (dd, 1H, J ¼ 4.8 and 1.2 Hz); 13C NMR (100 MHz, DMSO-
d6): d 36.74, 42.26, 45.76, 55.05, 113.71, 118.01, 120.56, 122.59,
126.45, 129.96, 130.73, 143.68, 145.80, 155.34, 160.30, 164.75,
166.59, 168.21; MS (ESI) m/z: 413 (Mþ1)þ. Anal. calcd. for
pyridin-2-ylthio)ethanone (7e)
White powder; yield: 71%; m.p. 141–143°C; IR (KBr): n (cmꢀ1
)
3415, 3391, 3009, 2847, 1694, 1683, 1597, 1491, 1292, 1249, 1157,
1103; 1H NMR (400 MHz, DMSO-d6): d 2.38 (s, 3H), 3.38–3.59 (m,
8H), 4.58(s, 2H), 7.27–7.31(m, 3H), 7.72–7.75(m,2H),7.76(dd,1H,
J¼ 5.2 and 1.2 Hz), 8.02 (dd, 1H, J¼ 8.0 and 1.2Hz); 13C NMR
(100 MHz, DMSO-d6): d 17.69, 37.05, 41.06, 46.14, 117.65, 122.56,
126.71, 128.60, 129.86, 130.71, 136.92, 138.34, 145.81, 148.52,
160.27, 164. 58, 166.55, 168.24; MS (ESI) m/z: 397 (Mþ1)þ. Anal.
calcd. for C20H20N4O3S: C, 60.59; H, 5.08; N, 14.13; O, 12.11; S,
8.09%; S, 7.69%. Found: C, 60.63; H, 5.05; N, 14.15; S, 8.06%.
C
20H20N4O4S: C, 58.24; H, 4.89; N, 13.58; O, 15.52; S, 7.77%.
Found: C, 58.26; H, 4.84; N, 13.56; S, 7.76%.
1-(4-(4-Nitrobenzoyl)piperazin-1-yl)-2-(oxazolo[4,5-b]-
pyridin-2-ylthio)ethanone (7c)
1-(4-(3-Methylbenzoyl)piperazin-1-yl)-2-(oxazolo[4,5-b]-
White powder; yield: 83%; m.p. 182–184°C; IR (KBr): n (cmꢀ1
)
pyridin-2-ylthio)ethanone (7f)
3420, 3377, 3017, 2855, 1701, 1688, 1601, 1493, 1294, 1246,
White powder; yield: 74%; m.p. 152–154°C; IR (KBr): n (cmꢀ1
)
1164, 1130; 1H NMR (400 MHz, DMSO-d6): d 3.19–3.55 (m, 8H),
3440, 3375, 3014, 2865, 1697, 1691, 1593, 1495, 1296, 1247,
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1151, 1110; 1H NMR (400 MHz, DMSO-d6): d 2.37 (s, 3H),
3.32–3.63 (m, 8H), 4.56 (s, 2H), 7.26–7.31 (m, 3H), 7.71–7.74 (m,
2H), 7.76 (dd, 1H, J ¼ 5.2 and 1.2 Hz), 8.03 (dd, 1H, J ¼ 8.0 and
1.2 Hz); 13C NMR (100 MHz, DMSO-d6): d 18.82, 37.01, 42.24,
45.74, 118.07, 122.06, 125.67, 128.60, 129.82, 130.11, 135.89,
138.33, 145.82, 148.46, 160.74, 164.57, 166.91, 168.22; MS (ESI)
m/z: 397 (Mþ )þ. Anal. calcd. for C20H20N4O3S: C, 60.59; H, 5.08;
N, 14.13; O, 12.11; S, 8.09%; S, 7.69%. Found: C, 60.63; H, 5.10;
N, 14.11; S, 8.10%.
(m, 8H), 4.42 (s, 2H), 6.58 (d, 2H, J ¼ 8.8 Hz), 7.06–7.14 (m, 3H),
7.86 (dd, 1H, J ¼ 8.0 and 1.2 Hz), 8.20 (dd, 1H, J ¼ 4.8 and
1.2 Hz), 9.65 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 36.04,
42.26, 45.34, 115.41, 118.50, 120.06, 126.22, 129.83, 144.03,
146.30, 155.74, 159.36, 165.29, 168.87, 170.05; MS (ESI) m/z:
399 (Mþ1)þ. Anal. calcd. for C19H18N4O4S: C, 57.27; H, 4.55; N,
14.06; O, 16.06; S, 8.05%. Found: C, 57.23; H, 4.56; N, 14.04; S,
8.08%.
1-(4-(4-Ethoxybenzoyl)piperazin-1-yl)-2-(oxazolo[4,5-b]-
1-(4-(4-Methylbenzoyl)piperazin-1-yl)-2-(oxazolo[4,5-b]-
pyridin-2-ylthio)ethanone (7k)
pyridin-2-ylthio)ethanone (7g)
White powder; yield: 81%; m.p. 163–165°C; IR (KBr): n (cmꢀ1
)
White powder; yield: 74%; m.p. 157–159°C; IR (KBr): n (cmꢀ1
)
3432, 3393, 3035, 2846, 1697, 1683, 1595, 1493, 1291, 1244,
1147, 1118, 1030; 1H NMR (400 MHz, DMSO-d6): d 1.11 (t, 3H,
J ¼ 6.8 Hz), 3.27–3.41 (m, 8H), 3.84 (q, 2H, J ¼ 6.8 Hz), 4.42 (s,
2H), 6.75 (d, 2H, J ¼ 8.4 Hz), 7.10–7.18 (m, 3H), 7.86 (dd, 1H,
J ¼ 8.0 and 1.2 Hz), 8.20 (dd, 1H, J ¼ 4.8 and 1.2 Hz); 13C NMR
(100 MHz, DMSO-d6): d 15.02, 37.06, 42.23, 45.81, 63.71,
114.57, 118.48, 120.04, 127.80, 129.65, 144.02, 146.29, 155.75,
160.11, 165.29, 168.86, 169.74; MS (ESI) m/z: 427 (Mþ1)þ. Anal.
calcd. for C21H22N4O4S: C, 59.14; H, 5.20; N, 13.14; O, 15.01; S,
7.52%. Found: C, 59.11; H, 5.17; N, 13.15; S, 7.52%.
3430, 3386, 3015, 2847, 1697, 1681, 1596, 1487, 1288, 1246,
1161, 1105; 1H NMR (400 MHz, DMSO-d6): d 2.37 (s, 3H),
3.51–3.61 (m, 8H), 4.65 (s, 2H), 7.30–7.33 (m, 3H), 7.75–7.79 (m,
3H), 8.38 (dd, 1H, J ¼ 8.0 and 1.2 Hz); 13C NMR (100 MHz,
DMSO-d6): d 18.79, 36.87, 41.04, 46.76, 118.48, 121.97, 126.59,
127.10, 129.63, 131.02, 136.37, 137.98, 145.82, 148.43, 160.84,
163.95, 166.76, 168.17; MS (ESI) m/z: 397 (Mþ1)þ. Anal. calcd.
for C20H20N4O3S: C, 60.59; H, 5.08; N, 14.13; O, 12.11; S, 8.09%;
S, 7.69%. Found: C, 60.59; H, 5.08; N, 14.13; S, 8.09%.
1-(4-Benzoylpiperazin-1-yl)-2-(oxazolo[4,5-b]-pyridin-2-
1-(4-(4-Bromobenzoyl)piperazin-1-yl)-2-(oxazolo[4,5-b]-
ylthio)ethanone (7h)
pyridin-2-ylthio)ethanone (7l)
White powder; yield: 73%; m.p. 145–147°C; IR (KBr): n (cmꢀ1
)
White powder; yield: 72%; m.p. 160–162°C; IR (KBr): n (cmꢀ1
)
3430, 3386, 3015, 2851, 1696, 1674, 1589, 1499, 1283, 1247,
1154, 1103; 1H NMR (400 MHz, DMSO-d6): d 3.52–3.61 (m, 8H),
4.65 (s, 2H), 7.32–7.35 (m, 1H), 7.37–7.40 (m, 2H), 7.43–7.49 (m,
1H), 7.82 (dd, 1H, J ¼ 8.0 and 1.2 Hz), 7.86–7.91 (m, 2H), 8.38
(dd, 1H, J ¼ 4.8 and 1.2 Hz); 13C NMR (100 MHz, DMSO-d6): d
36.79, 42.25, 45.83, 116.64, 121.56, 128.17, 129.76, 130.21,
135.82, 144.97, 148.14, 160.13, 163.71, 165.10, 168.36; MS (ESI)
m/z: 383 (Mþ1)þ. Anal. calcd. for C19H18N4O3S: C, 59.67; H,
4.74; N, 14.65; O, 12.55; S, 8.38%. Found: C, 59.71; H, 4.72; N,
14.66; S, 8.39%.
3445, 3391, 3030, 2847, 1699, 1681, 1593, 1503, 1291, 1247,
1139, 1119; 1H NMR (400 MHz, DMSO-d6): d 3.58–3.67 (m, 8H),
4.66 (s, 2H), 7.33–7.37 (m, 1H), 7.48–7.55 (m, 4H), 8.08 (d, 1H,
J ¼ 7.8 Hz), 8.43–8.45 (m, 1H); 13C NMR (100 MHz, DMSO-d6): d
37.08, 42.10, 45.73, 115.51, 118.47, 120.04, 129.01, 129.54,
134.86, 144.02, 146.28, 155.74, 165.30, 168.72, 168.86; MS (ESI)
m/z: 461 (Mþ1)þ. Anal. calcd. for C19H17BrN4O3S: C, 49.47; H,
3.71; Br, 17.32; N, 12.14; O, 10.40; S, 6.95%. Found: C, 49.43; H,
3.72; N, 12.15; S, 6.96%.
2-(4-Chlorophenyl)-1-(4-(2-(oxazolo[4,5-b]-pyridin-2-
1-(4-(2-Nitrobenzoyl)piperazin-1-yl)-2-(oxazolo[4,5-b]-
ylthio)acetyl)piperazin-1-yl)ethanone (7m)
pyridin-2-ylthio)ethanone (7i)
White powder; yield: 76%; m.p. 138–139°C; IR (KBr): n (cmꢀ1
)
White powder; yield: 77%; m.p. 135–137°C; IR (KBr): n (cmꢀ1
)
3435, 3390, 3029, 2833, 1697, 1683, 1593, 1496, 1281, 1246,
1151, 1118; 1H NMR (400 MHz, DMSO-d6): d 3.49–3.58 (m, 8H),
3.72 (m, 2H), 4.65 (s, 2H), 7.27–7.31 (m, 1H), 7.52–7.57 (m, 4H),
8.03 (d, 1H, J ¼ 8.0 Hz), 8.21–8.24 (m, 1H); 13C NMR (100 MHz,
DMSO-d6): d 37.07, 42.21, 43.65, 45.76, 117.58, 121.93, 129.61,
130.15, 133.90, 135.34, 144.64, 146.71, 159.45, 163.14, 168.27,
169.12; MS (ESI) m/z: 431 (Mþ1)þ. Anal. calcd. for
3426, 3391, 3012, 2847, 1693, 1681, 1589, 1497, 1281, 1251,
1147, 1110; 1H NMR (400 MHz, DMSO-d6): d 3.43–3.55 (m, 8H),
4.64 (s, 2H), 7.26 (dd, 1H, J ¼ 8.0 and 5.2 Hz), 7.76–7.77 (m, 1H),
7.80–7.86 (m, 2H), 8.07–8.09 (m, 1H), 8.25–8.28 (m, 1H),
8.42–8.46 (m, 1H); 13C NMR (100 MHz, DMSO-d6): d 37.02,
42.12, 45.71, 118.49, 120.46, 123.58, 126.37, 131.08, 133.03,
137.20, 144.32, 146.72, 148.56, 155.03, 165.13, 166.71, 169.12;
MS (ESI) m/z: 428 (Mþ1)þ. Anal. calcd. for C19H17N5O5S: C,
53.39; H, 4.01; N, 16.38; O, 18.72; S, 7.50%. Found: C, 53.43; H,
4.02; N, 16.36; S, 7.48%.
C
20H19ClN4O3S: C, 55.75; H, 4.44; Cl, 8.23; N, 13.00; O, 11.14;
S, 7.44%. Found: C, 55.78; H, 4.45; N, 13.01; S, 7.43%.
2-(4-Methoxyphenyl)-1-(4-(2-(oxazolo[4,5-b]-pyridin-2-
ylthio)acetyl)piperazin-1-yl)ethanone (7n)
1-(4-(4-Hydroxybenzoyl)piperazin-1-yl)-2-(oxazolo[4,5-b]-
White powder; yield: 76%; m.p. 182–184°C; IR (KBr): n (cmꢀ1
)
pyridin-2-ylthio)ethanone (7j)
3433, 3373, 3040, 2841, 1693, 1681, 1595, 1503, 1291, 1253,
1146, 1119, 1037; 1H NMR (400 MHz, DMSO-d6): d 3.46–3.62
(m, 8H), 3.73 (s, 2H), 3.76 (s, 3H), 4.64 (s, 2H), 7.03 (d, 2H,
J ¼ 8.4 Hz), 7.32 (dd, 1H, J ¼ 8.0 and 4.8 Hz), 7.36 (d, 2H,
White powder; yield: 80%; m.p. 165–167°C; IR (KBr): n (cmꢀ1
)
3521, 3430, 3373, 3011, 2847, 1693, 1681, 1586, 1503, 1279,
1246, 1145, 1106; 1H NMR (400 MHz, DMSO-d6): d 3.27–3.40
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Archiv der Pharmazie
J ¼ 8.4 Hz), 8.04 (dd, 1H, J ¼ 8.0 and 1.2 Hz), 8.32 (dd, 1H, J ¼ 4.8
and 1.2 Hz); 13C NMR (100 MHz, DMSO-d6): d 37.07, 42.21,
43.65, 45.76, 55.81, 113.79, 118.22, 122.43, 127.64, 129.96,
143.78, 145.89, 155.65, 160.13, 163.79, 168.25, 169.36; MS (ESI)
m/z: 427 (Mþ1)þ. Anal. calcd. for C21H22N4O4S: C, 59.14; H,
5.20; N, 13.14; O, 15.01; S, 7.52%. Found: C, 59.17; H, 5.21; N,
13.13; S, 7.51%.
Promega Corporation (Madison, WI). Indomethacin, carra-
geenan, carboxymethylcellulose, SB216763, and staurospor-
ine were purchased from Sigma–Aldrich Chemicals Pvt.
Limited, Bangalore, India.
GSK-3b inhibitory activity
The compound library was assessed for their GSK-3b inhibi-
tion as per the Kinase-Glo assay method of Baki et al. [31]. This
assay determines the activity of the kinase by quantifying the
amount of ATP which remains in solution following a kinase
reaction. In a typical assay, 10 mL of test compound of
different concentrations (dissolved in dimethyl sulfoxide
(DMSO) and diluted with assay buffer) and 10 mL (20 ng) of
enzyme were added to each well followed by 20 mL of assay
buffer containing substrate and ATP to obtain a concentra-
tion of 25 mM substrate and 1 mM ATP per well. The final
DMSO concentration in the reaction mixture was kept less
than 1%. After incubation at 30°C for 30 min, the enzymatic
reaction was quenched with 40 mL of Kinase-Glo reagent.
Luminescence was recorded after 10 min using an Infinite
F2001PRO multimode reader (Tecan). The activity is propor-
tional to the difference of the total and consumed ATP. The
activity was maximum in the absence of inhibitor and was
used to calculate the inhibitory activities of test compounds.
2-(4-Nitrophenyl)-1-(4-(2-(oxazolo[4,5-b]-pyridin-2-ylthio)
acetyl)piperazin-1-yl)ethanone (7o)
White powder; yield: 75%; m.p. 178–180°C; IR (KBr): n (cmꢀ1
)
3434, 3391, 3018, 2846, 1693, 1681, 1593, 1493, 1301, 1248,
1145, 1122; 1H NMR (400 MHz, DMSO-d6): d 3.54–3.63 (m, 8H),
3.75 (s, 2H), 4.58 (s, 2H), 7.13 (dd, 1H, J ¼ 8.0 and 5.2 Hz), 7.47
(d, 2H, J ¼ 8.4 Hz), 7.80 (d, 1H, J ¼ 8.0 Hz), 8.02–8.05 (m, 2H),
8.21–8.24 (m, 1H); 13C NMR (100 MHz, DMSO-d6): d 37.11,
42.31, 43.56, 45.69, 118.54, 120.35, 124.71, 129.02, 141.86,
144.32, 146.38, 148.17, 155.76, 164.82, 168.57, 169.64; MS (ESI)
m/z: 442 (Mþ1)þ. Anal. calcd. for C20H19N5O5S: C, 54.41; H,
4.34; N, 15.86; O, 18.12; S, 7.26%. Found: C, 54.38; H, 4.32; N,
15.86; S, 7.23%.
2-(Oxazolo[4,5-b]pyridin-2-ylthio)-1-(4-(2-(o-tolyl)acetyl)-
piperazin-1-yl)ethanone (7p)
White powder; yield: 75%; m.p. 153–155°C; IR (KBr): n (cmꢀ1
)
3441, 3392, 3018, 2845, 1693, 1669, 1593, 1493, 1279, 1239,
1151, 1123; 1H NMR (400 MHz, DMSO-d6): d 2.21 (s, 3H),
3.52–3.62 (m, 8H), 3.73 (s, 2H), 4.65 (s, 2H), 7.10–7.15 (m, 4H),
7.35 (dd, 1H, J ¼ 8.1 and 4.8 Hz), 8.08 (dd, 1H, J ¼ 8.1 and
0.9 Hz), 8.44 (dd, 1H, J ¼ 4.8 and 1.2 Hz); 13C NMR (100 MHz,
DMSO-d6): d 36.05, 117.06, 118.36, 121.52, 122.25, 126.68,
131.63, 137.81, 141.27, 143.32, 147.15, 153.26; MS (ESI) m/z:
411 (Mþ1)þ. Anal. calcd. for C21H22N4O3S: C, 61.44; H, 5.40; N,
13.65; O, 11.69; S, 7.81%. Found: C, 61.47; H, 5.38; N, 13.66; S,
7.80%.
Animals
Wistar rats of either sex (150–200 g) were acquired from
Central Animal House, Hamdard University, New Delhi. Rats
were kept in the Central Animal House of Hamdard University
in colony cages at an ambient temperature of 25 ꢁ 2°C and
relative humidity 45–55% with 12 h light/dark cycles after
initial acclimatization for about 1 week. They were provided
free access to standard rodent pellet diet and water ad
libitum. Fourteen hours preceding the start of the experi-
ment, the animals were sent to lab and fed only with water ad
libitum. The experimental study was conducted in accordance
with guidelines of the Institutional Animal Ethics Committee
of the University, Jamia Hamdard (Hamdard University), New
Delhi, India.
2-(Oxazolo[4,5-b]pyridin-2-ylthio)-1-(4-(2-phenylacetyl)-
piperazin-1-yl)ethanone (7q)
Creamish white powder; yield: 77%; m.p. 133–135°C; IR (KBr):
n (cmꢀ1) 3430, 3386, 3015, 2851, 1696, 1674, 1589, 1499, 1283,
1247, 1154, 1103; 1H NMR (400 MHz, DMSO-d6): d 3.49–3.57
(m, 8H), 3.74 (s, 2H), 4.67 (s, 2H), 7.08–7.11 (m, 1H), 7.22–7.29
(m, 3H), 7.37–7.40 (m, 2H), 7.80 (dd, 1H, J ¼ 8.1 and 0.9 Hz),
8.36 (dd, 1H, J ¼ 4.8 and 1.2 Hz); 13C NMR (100 MHz, DMSO-d6):
d 37.15, 42.47, 43.32, 46.24, 118.12, 120.97, 127.67, 128.45,
129.83, 135.62, 144.74, 147.97, 159.23, 163.52, 165.61, 168.49;
MS (ESI) m/z: 397 (Mþ1)þ. Anal. calcd. for C20H20N4O3S: C,
60.59; H, 5.08; N, 14.13; O, 12.11; S, 8.09%. Found: C, 60.65; H,
5.04; N, 14.12; S, 8.10%.
Anti-inflammatory activity
The synthesized compounds were evaluated for their in vivo
anti-inflammatory activity using carrageenan-induced hind
paw edema method. The rat paw edema was induced by
subplantar injection of 0.1 mL of 1% freshly prepared
carrageenan suspension in normal saline into the right hind
paw of rats. The standard anti-inflammatory drug, indometh-
acin (0.05 mmol/kg) was taken as positive control while as
negative control was given as equivalent volume of 0.9%
saline solution. GSK-3b inhibitor SB216763 (0.05 mmol/kg)
was also administered to one group orally. The test groups
were administered synthesized compounds at a dosage
equimolar to the positive control. The positive control,
negative control, SB216763, and test compounds were
administered orally to respective groups 1 h preceding the
carrageenan treatment. The paw volumes were measured in
Pharmacology
Chemicals
Human recombinant GSK-3b and pre-phosphorylated poly-
peptide substrate glycogen synthase-2 (GS-2) were purchased
from Merck-Millipore Corporation (India). Kinase-Glo Lumi-
nescent Kinase Assay (catalog no. V6713) was obtained from
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two time points at an interval of 3 and 5 h post-carrageenan
treatment by using plethysmometer [34].
The authors have declared no conflicts of interest.
Effects of active compounds on production of
References
carrageenan-induced pro-inflammatory mediators
Wistar albino rats weighing 150–200 g were used. Rats were
fasted for 18 h before i.p. dosing with the test compound. The
standard drug indomethacin (10 mM) and GSK-3b inhibitor
SB216763 (10 mM) were given orally as a positive control. The
control group was administered orally with 0.9% of 0.1 mL of
saline solution only. The test groups were administered orally
with the synthesized compounds at equimolar dosage of the
standard drug, 2 h before an intra-planter injection of
carrageenan into rat paw. Following dosing of drugs, rats
were injected with 0.2 mL of 1% freshly prepared carra-
geenan in normal saline. Five hours later, the blood was
collected from the tail vein. Blood was centrifuged for 10 min
at 3000 rpm to isolate the serum. Serum levels of TNF-a, IL-1b,
IL-6, and COX-2 in control and experimental rats were
measured by ELISA using a commercially available diagnostic
kit (Invitrogen, Carlsbad, CA). The results were expressed as
pg/mL. COX-2 levels were also measured by ELISA. The results
were expressed as IU/dL.
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