Synthesis of Novel Oxazolo[4,5-b]pyridine-2-one based 1,2,3-triazoles as Glycogen Synthase Kinase-3β Inhibitors with Anti-inflammatory Potential

Article abstract of DOI:10.1111/cbdd.12724

A novel series of oxazolo[4,5-b]pyridine-2-one based 1,2,3-triazoles has been synthesized by click chemistry approach and evaluated for in vitro GSK-3β inhibitory activity. Compound 4g showed maximum inhibition with IC₅₀ value of 0.19 μm. Keeping in view the effect of GSK-3β inhibition on inflammation, compounds 4g, 4d, 4f, 4i, 4n and 4q exhibiting significant GSK-3β inhibition were examined for in vivo anti-inflammatory activity in rat paw edema model. The compounds 4g, 4d, 4f and 4i showed pronounced in vivo anti-inflammatory activity (76.36, 74.54, 72.72 and 70.90%, respectively, after 5h post-carrageenan administration) and were further found to inhibit the pro-inflammatory mediators, viz. NO, TNF-α, IL-1β, and IL-6 substantially in comparison with indomethacin, an anti-inflammatory drug as well as SB216763, a GSK-3β inhibitor, reported to exert a similar effect. Histopathology studies confirmed the tolerance of gastric mucosa to these compounds.

Full text of DOI:10.1111/cbdd.12724

Chem Biol Drug Des 2016; 87: 918–926
Research Article
Synthesis of Novel Oxazolo[4,5-b]pyridine-2-one based
1,2,3-triazoles as Glycogen Synthase Kinase-3b
Inhibitors with Anti-inflammatory Potential
Mushtaq A. Tantray1, Imran Khan1,
virtue of regulating glycogen metabolism. GSK-3 exists as
two major isoforms in mammals, GSK-3a and GSK-3b.
Both are ubiquitously expressed in all tissues and share an
overall homology of 85%, with 98% homology within their
Hinna Hamid1,*, Mohammad Sarwar Alam1,
Sadiq Umar2, Yakub Ali1, Kalicharan Sharma3
and Firasat Hussain4
kinase domains (1). GSK-3b is the more conserved iso-
1Department of Chemistry, Faculty of Science, Hamdard
form in evolution with wide expression throughout the ani-
mal kingdom, while GSK-3a is only found in vertebrates
(2). GSK-3 has been recognized to modulate a diverse
array of key regulatory intra-cellular proteins such as meta-
bolic proteins (e.g. cyclin D1, APP, and presenilin), struc-
tural proteins (e.g. tau and other microtubule associated
proteins) and transcription factors (e.g. NF-jB, p53 and
Notch) (3,4). The plethora of substrates enable GSK-3 to
determine the output of various signaling pathways initi-
ated by diverse stimuli (5). Deregulation of GSK-3 activity
and its associated signaling pathways results in the devel-
opment of several human diseases as type II diabetes,
bipolar disorder, cancer, chronic inflammatory diseases,
and Alzheimer’s disease (AD) (6–9). Given its unique posi-
tion, GSK-3 has gained a spotlight status as an interesting
therapeutic target and a means to unravel the molecular
basis of its associated disorders (10). Inhibition of GSK-3,
thus, represents a plausible strategy to develop novel drug
therapy for the treatment of such pathologies.
University (Jamia Hamdard), New Delhi 110 062, India
2Department of Pharmaceutical Sciences, College of
Pharmacy, Washington State University, Spokane, WA
99202, USA
3Drug Design and Medicinal Chemistry Lab, Department
of Pharmaceutical Chemistry, Hamdard University (Jamia
Hamdard), New Delhi 110 062, India
4Department of Chemistry, University of Delhi, New Delhi
110 007, India
*Corresponding author: Hinna Hamid,
hhamid@jamiahamdard.ac.in
A novel series of oxazolo[4,5-b]pyridine-2-one based
1,2,3-triazoles has been synthesized by click chemistry
approach and evaluated for in vitro GSK-3b inhibitory
activity. Compound 4g showed maximum inhibition
with IC50 value of 0.19 lM. Keeping in view the effect
of GSK-3b inhibition on inflammation, compounds 4g,
4d, 4f, 4i, 4n and 4q exhibiting significant GSK-3b inhi-
bition were examined for in vivo anti-inflammatory
activity in rat paw edema model. The compounds 4g,
4d, 4f and 4i showed pronounced in vivo anti-inflam-
matory activity (76.36, 74.54, 72.72 and 70.90%,
respectively, after 5h post-carrageenan administration)
and were further found to inhibit the pro-inflammatory
mediators, viz. NO, TNF-a, IL-1b, and IL-6 substantially
in comparison with indomethacin, an anti-inflammatory
drug as well as SB216763, a GSK-3b inhibitor, reported
to exert a similar effect. Histopathology studies con-
firmed the tolerance of gastric mucosa to these com-
pounds.
GSK-3b is also a crucial mediator of inflammation. The
paramount importance of GSK-3b in controlling inflamma-
tion is evident by its capacity to modulate the inflamma-
tory response in both innate and adaptive immune cells
(1). Numerous transcription factors such as NF-jB neces-
sary for controlling acute inflammation act as substrates
of GSK-3b and are responsible for production of various
pro-inflammatory mediators such as TNF-a, IL-1b, iNOS,
and IL-6 (11,12). Inhibition of GSK-3b controls inflamma-
tion and protects the host against inflammatory-mediated
pathology and death in animal models. GSK-3b inhibitors
have been reported to suppress inflammation in various
animal models against TNF-a administration, endotox-
emia, experimental colitis, type II collagen-induced arthri-
tis, ovalbumin-induced asthma, carrageenan induced lung
injury, and experimental autoimmune encephalomyelitis
(11,13–22). The development of GSK-3b inhibitors may
thus provide an important therapeutic approach for the
treatment of inflammatory diseases. Also, as various other
pathologies such as diabetes, cancer, and AD linked to
abnormal functioning of GSK-3 are associated with
Key words: click chemistry, glycogen synthase kinase-3, in-
flammation, oxazolo[4,5-b]pyridine-2-one, pro-inflammatory
mediators
Received 23 October 2015, revised 6 January 2016 and
accepted for publication 16 January 2016
Glycogen synthase kinase-3 (GSK-3) is a ubiquitous multi-
functional serine/threonine kinase, originally identified by
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ª 2016 John Wiley & Sons A/S. doi: 10.1111/cbdd.12724

Oxazolopyridinone-triazoles as GSK-3b Inhibitors
inflammation, the inhibitors of GSK-3b may provide thera-
peutic anti-inflammatory effects in a broad range of con-
ditions (23).
Corporation (New Delhi, India). Kinase-Glo Luminescent
Kinase Assay (catalog no. V6713) was obtained from Pro-
mega Corporation (Madison, WI, USA). Indomethacin, Car-
rageenan, Carboxymethylcellulose, Lipopolysaccharide
(LPS), Lithium chloride, and Staurosporine were purchased
from Sigma-Aldrich Chemicals Pvt. Limited, Bangalore,
India.
In continuation to the work performed by our group on
benzoxazolinone
and
2-mercaptobenzoxazole-based
1,2,3-triazole conjugates as anti-inflammatory compounds
(24–26), we extended our studies to oxazolo[4,5-b]pyri-
dine-2-one moiety, the bio-isostere of benzoxazolinone
and 2-mercaptobenzoxazole. Oxazolopyridinones have
been reported to be pharmaceutically important as anti-
nociceptive, anti-inflammatory, anti-malarial, and Met
kinase inhibitors (27–33). Recently, oxazolo-pyridines have
also been reported as potent GSK-3b inhibitors (34). So
far not much work has been performed on this scaffold
and there is ample scope for the development of more
potent inhibitors from this ligand. Similarly 1,2,3-triazoles
and their derivatives have received considerable attention
due to their role in diverse biological activities such as
antimicrobial, anti-inflammatory, local anesthetic, anticon-
vulsant, anti-neoplastic, antimalarial, and antiviral activities
(35,36). 1,2,3-triazole moiety has also been reported as a
novel class of selective GSK-3b inhibitors (37). We there-
fore focused our attention on the development of novel
oxazolo[4,5-b]pyridine-2-one based GSK-3 inhibitors by
conjugating them to 1,2,3-triazoles through a click chem-
istry approach. The aim of the work was to develop
molecules having GSK-3b inhibitory activity with anti-
inflammatory properties. A library of 21 compounds has
been synthesized and evaluated for in vitro GSK-3b inhibi-
tion and in vivo anti-inflammatory activity. The compounds
exhibiting significant GSK-3b inhibition and anti-inflamma-
tory profile have been further evaluated for their effect on
pro-inflammatory mediators such as NO, TNF-a, IL-1b,
and IL-6, using ex vivo assays. In silico molecular docking
studies of the active compounds were also carried out
against GSK-3b to get an insight into the molecular inter-
actions of these compounds with its active site. Further-
more, as the anti-inflammatory compounds are known to
cause gastric ulceration as side effect, the active com-
pounds have been assessed for their gastric ulceration
risk.
GSK-3b inhibitory activity
The synthesized compounds were screened for their GSK-
3b inhibition as per the Kinase-Glo assay method of Baki
et al. (38). The sensitivity of the assay is comparable to the
radioactive method. This assay determines the kinase
activity by quantifying the amount of ATP remaining in
solution following a kinase reaction. In a typical assay,
10 lL of test compound of different concentrations (dis-
solved in dimethyl sulfoxide [DMSO] and diluted with assay
buffer) and 10 lL (20 ng) of enzyme were added to each
well followed by 20 lL of assay buffer containing substrate
and ATP to get a concentration of 25 lM substrate and
1 lM ATP per well. The final DMSO concentration in the
reaction mixture was <1%. After incubation at 30 °C for
30 min, the enzymatic reaction was quenched with 40 lL
of Kinase-Glo reagent. Luminescence was recorded after
10 min using Infinite F200ꢀ PRO multimode reader (Tecan
€
Group Ltd., Mannedorf, Switzerland). The activity is pro-
portional to the difference of the total and consumed ATP.
Maximum activity was found in the absence of inhibitor
and was used to calculate the inhibitory activities of test
compounds. Staurosporine was used as a standard GSK-
3b inhibitor.
Animals
Wistar rats of either sex (150–200 g) were obtained from
Central Animal House, Hamdard University, New Delhi.
They 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 had free access to standard rodent pellet diet and
water ad libitum. Fourteen hours prior to the start of the
experiment, the animals were sent to laboratory and fed
only with water ad libitum. The experimental study was
conducted in accordance with the Institutional Animal
Ethics Committee of the University, Jamia Hamdard, New
Delhi, India.
Experimental
Chemistry
The synthetic procedure and the characterization data of
target compounds (4a–4u) has been provided in the
Appendix S1.
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 injection of 0.1 mL of 1% freshly prepared
saline solution (39) of carrageenan subcutaneously into the
sub-plantar region of the right hind paw of rats. The stan-
dard anti-inflammatory drug, indomethacin (20 mg/kg
Pharmacology
Chemicals
Human recombinant glycogen synthase kinase-3b and
pre-phosphorylated polypeptide substrate glycogen
synthase-2 (GS-2) were purchased from Merck-Millipore
Chem Biol Drug Des 2016; 87: 918–926
919

Tantray et al.
BW), was given orally as a positive control. GSK-3b inhibi-
tor, SB216763 (20 mg/kg BW), was also administered to
one group orally. The control group was only administered
with 0.1 mL of 0.9% saline solution orally. The test groups
were given the synthesized compounds orally at a dosage
of 20 mg/kg BW, 1 h preceding the carrageenan adminis-
tration. The paw volumes were measured at an interval of
3 and 5 h using plethysmometer (40).
prepared for docking using ligprep application. The protein
for docking study was taken from Protein Data Bank (PDB
ID: 4IQ6) and prepared by removing solvent, adding
hydrogen, and further minimization in the presence of
bound ligand (IQ6) using protein preparation wizard. Grids
for molecular docking were generated with bound co-crys-
tallized ligand. For the validation of docking parameters,
the co-crystal ligand (IQ6) was re-docked at the catalytic
site of protein and the RMSD between co-crystal and re-
ꢀ
docked pose was found to be 0.255 A. All compounds
Lipopolysaccharide (LPS)-induction into rat paw
Wistar albino rats weighing 150–200 g were used. Rats
were fasted for 18 h before i.p. dosing with the test com-
pound. The standard drug indomethacin (10 lM) and
GSK-3b inhibitor SB216763 (10 lM) 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 com-
pounds at 20 lM, 2 h before an intra-planter injection of
LPS into rat paw. Following dosing of drugs, rats were
injected with 50 lL of saline containing 10 lg/paw LPS.
Two hours later, inflamed rat paw were collected from the
inflamed animals (41,42).
were docked using Glide extra-precision (XP) mode, with
up to three poses saved per molecule.
Ulcerogenic activity
The compounds exhibiting significant anti-inflammatory
activity were assessed further for their ulcerogenic risk
evaluation (44). This was performed at 3-fold higher dose
as compared to that used for anti-inflammatory activity.
Each group constituted of three animals and were later
sacrificed 5 h after oral drug administration. In comparison
with indomethacin, the tested compounds were not found
to cause any gastric ulceration and disruption of gastric
epithelial cells at the given dose. SB216763 was also not
found to cause any gastric ulceration. The gastric toler-
ance of these compounds toward the gastric mucosa was
thus better than that of standard drug indomethacin.
Measurement of Nitric oxide (NO) level
After the experiment, animals were sacrificed and the hind
paw tissues were washed with PBS (pH 7.4) and placed
on ice by the method described earlier (43). Briefly a
50 lL sample was added to 100 lL of Griess reagent,
and the reaction mixture was incubated for about
5–10 min at room temperature and protected from light.
The optical density was measured at 540 nm in microplate
reader as per the reagent manufacturer’s protocol. Calcu-
lations were performed after generating a standard curve
from sodium nitrite in the same buffer as used for prepara-
tion of homogenate.
Statistical analysis
Results are expressed as the mean ꢀ SEM, and different
groups were compared using one-way analysis of variance
(ANOVA) followed by Tukey Kramer test for multiple compar-
isons.
Results and Discussion
Chemistry
Measurement of TNF-a IL-1b, and IL-6
2-Amino-3-hydroxypyridine 1 was refluxed with 1,1-carbo-
nyldiimidazole in dry THF for 5–6 h under nitrogen atmo-
Levels of inflammatory cytokines TNF-a IL-1b, and IL-6 in
the serum were determined using commercially available
cytokine ELISA kits (R&D and Cayman, Ann Arbor, MI,
USA). Supernatants were removed and assayed in dupli-
cate according to the manufacturer’s guidelines. Cytokine
concentrations were expressed as picograms of antigen
per milliliter.
sphere to yield oxazolo[4,5-b]pyridin-2(3H)-one
2
as
depicted in Scheme 1. It was then refluxed with propargyl
bromide in presence of potassium carbonate in dry acetone
for 4–5 h to obtain 3-ethynyloxazolo[4,5-b]pyridin-2(3H)-one
3. The propargylated oxazolopyridinone 3 was subsequently
treated with various aromatic azides under click chemistry
conditions to afford the target compounds, oxazolo[4,5-b]
pyridin-2(3H)-one based 1,2,3-triazoles (4a–4u) in quantita-
tive yields (Table S1). The formation of propargylated inter-
mediate 3 was validated by the appearance of singlets at d
4.65 and d 2.27 in the 1H-NMR spectrum, corresponding
to methylene protons and terminal alkyne proton, respec-
tively. Presence of a signal in the region of d 8.58–9.10 due
to H-C(5) of triazole ring confirmed the synthesis of tria-
zoles. The 13C NMR spectra further confirmed the struc-
tural assignments, showing the C-atom signals
corresponding to triazole derivatives. The final structure veri-
Docking studies
For better understanding of binding mode of substituted
oxazolo pyridine-2-one derivatives at the molecular level,
we carried out molecular docking simulations of synthe-
sized molecules (4d, 4f, 4g, 4i) at the GSK-3b catalytic
ligand binding site. The docking simulations of synthesized
compounds were performed using MAESTRO, version 9.4
implemented from SCHRODINGER software suite. The ligands
were sketched in 3D format using build panel and were
920
Chem Biol Drug Des 2016; 87: 918–926

Oxazolopyridinone-triazoles as GSK-3b Inhibitors
OH
O
O
O
N
(a)
(b)
(c)
O
O
O
N
N
N
H
N
N
N
NH2
R
N
N
N
(4a-4t)
1
2
3
Scheme 1: Synthetic approach for novel oxazolo[4,5-b]pyridin-2(3H)-one based 1,2,3-triazoles. (a) 1,10-Carbonyldiimidazole, THF, Reflux;
(b) Propargyl bromide, K2CO3, Acetone, Reflux; (c) Azides (R-N3), CuSO4.5H2O, Sodium ascorbate, tBuOH: H2O (1:1), r.t.
fication was performed by ESI-MS analysis which showed
ity against both GSK-3b as well as in vivo induced inflam-
mation. Moreover, the anti-inflammatory activity of the
tested compounds was observed to be in conformity with
their GSK-3b inhibition profile. The compounds, therefore,
displayed good coordination between GSK-3b inhibition
and anti-inflammatory property, that is, compounds inhibit-
ing GSK-3b also reduced inflammation, indicating the role
GSK-3b inhibitors can play against inflammation and
inflammatory associated conditions.
the presence of [M]+ or [M + 1]+ or [M + 2]+ ion peaks.
In vitro GSK-3b inhibitory activity
All the synthesized oxazolopyridinone triazoles were evalu-
ated for inhibition of GSK-3b by Kinase-GloTM luminescent
technique (38,45). The results as illustrated in Table S1
shows that the compounds inhibited GSK-3b in the submi-
cromolar to micromolar range. Among these, compounds
4g, 4d, 4f, and 4i were found to be the most active with
IC50 values of 0.19, 0.23, 0.31 and 0.37 lM, respectively.
Compounds 4n, 4q, 4l, and 4b displayed moderate inhibi-
tion in single-digit micromolar range while as the rest of the
compounds exhibited relatively weak inhibition.
Effect of compounds 4g, 4d, 4f, and 4i on pro-
inflammatory mediator levels
Pro-inflammatory cytokines such as TNF-a, IL-1b as well
as IL-6 have a central role in the perpetuation of chronic
inflammation and tissue damage during progression of
inflammatory disorders. Blockade of these molecules result
in a reduction of disease severity (46). As GSK-3b inhibi-
tors such as lithium chloride, SB216763, TDZD8, and
CHIR99021 have been reported to suppress inflammation
by inhibiting the production of pro-inflammatory cytokines
and increasing anti-inflammatory cytokine production,
compounds 4g, 4d, 4f, and 4i exhibiting significant in vivo
anti-inflammatory potential were, therefore, examined for
their effect on concentration of various pro-inflammatory
mediators.
In vivo anti-inflammatory activity
In view of the nexus between GSK-3b inhibition and allevi-
ation of inflammatory disorders, the reported anti-inflam-
matory potential of oxazolopyridinones and 1,2,3-triazoles,
the synthesized compounds exhibiting significant GSK-3b
inhibitory activity were also examined for their in vivo anti-
inflammatory activity by carrageenan-induced hind paw
edema model. Indomethacin, an anti-inflammatory drug
was taken as standard. SB216763, a well-known GSK-3b
inhibitor, reported to exert anti-inflammatory effect in vari-
ous in vitro and in vivo models, was also used as the ref-
erence. As illustrated in Table S2 indicated that all the
tested compounds showed a time-dependent increase in
the inhibition of inflammation after 3 h and 5 h post-carra-
geenan administration. The compounds 4g and 4d exhib-
ited significant anti-inflammatory potential (73.58 and
71.69% inhibition at 3 h and 76.36 and 74.54% inhibition,
respectively, at 5 h post-carrageenan administration) com-
parable to that of indomethacin (79.24% at 3 h and
83.63% at 5 h after carrageenan administration). Com-
pounds 4f and 4i also showed a fairly good profile, reduc-
ing the paw volume by 69.81% and 67.92% after 3 h
interval and 72.72% and 70.90%, respectively, after 5 h
interval, in comparison with indomethacin. However, com-
pounds 4n and 4q displayed moderate activity. SB216763
was found to exhibit 75.47% inhibition at 3 h and 78.18%
inhibition at 5 h post-carrageenan treatment in comparison
with indomethacin.
Nitric oxide
Nitric oxide (NO) is an important signaling molecule, pro-
duced as part of the inflammatory response from activated
cells and macrophages (47,48). Huang et al. (19) have
demonstrated that GSK-3 inhibition can block NO and
iNOS production in response to LPS in primary microglia.
In this study, increased NO levels have been detected in
the LPS group similar with those previously reported in the
literature (49). Analysis of nitrite estimation is summarized
in Figure S1. A significant increase in nitrite was observed
in LPS-induced inflammation group as compared to con-
trol. The treatment with synthesized compounds 4g, 4d,
4f, and 4i led to a decline in the nitrite level as compared
to the inflammation group.
Effect on IL-1b, TNF-a, and IL-6
During inflammatory processes, large amounts of the pro-
inflammatory mediators are generated, which affect the
immune system by suppressing the proliferation of T and
B cells, as well as cytokine synthesis (50). IL-1b and TNF-
To get a better picture, these results were compared with
GSK-3b inhibitory data of these compounds. Compounds
4g, 4d, 4f, and 4i were found to possess significant activ-
Chem Biol Drug Des 2016; 87: 918–926
921

Tantray et al.
a stimulation upregulates the expression of pro-inflamma-
tory genes, such as cyclooxygenase 2 (COX-2) and nitric
oxide synthase (NOS). Takada et al. (21), showed LPS
induced the production of these cytokines through glyco-
gen synthase kinase-3b. Our results as depicted in Fig-
ure S2 showed that there is a significant increase in the
level of TNF-a, IL-1b, and IL-6 in LPS-induced inflamma-
tion in experimental rats in comparison with control.
Administration of selected compounds 4g, 4i, 4d, and 4f
suppressed the increase in the level of TNF-a, IL-1b, and
IL-6 significantly when compared to the LPS group.
Ulcerogenic risk evaluation
The compounds showing potential anti-inflammatory activ-
ity were tested for their gastric ulcerogenic effect. As com-
pared to indomethacin, SB216763 and the active
compounds 4g, 4d, 4f, and 4i did not show any gastric
ulceration and disruption of gastric epithelial cells at even
three times higher dose than used for anti-inflammatory
activity (Figure S4).
Structure activity relationship
Based on the results, a preliminary structure–activity rela-
tionship was determined to correlate the effect of different
structural features of synthesized compounds on the effi-
cacy of their GSK-3b inhibition and anti-inflammatory prop-
erties.
Molecular docking studies
To analyze the binding pattern of the active compounds
(4g, 4d, 4f, and 4i), molecular docking studies were per-
formed against GSK-3b. The docking scores are shown
in Table S3. Compound 4g exhibited a docking score of
ꢁ6.34 in comparison with that of the reference ligand IQ6
(6-chloro-N-cyclohexyl-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyri-
din-2-amine), which showed a docking score of ꢁ9.86.
The binding pose of compound 4g and its comparison
with the standard co-crystal ligand (IQ6) bonded to GSK-
3b protein (PDBID: 4IQ6) is shown in Figure S3A–D. The
oxazolopyridine-2-one moiety plays an important role in
the binding, as the carbonyl oxygen atom is involved in
hydrogen bonding interaction with backbone atom of the
amino acid residue Val-135 in hinge region. The Val-135
residue has been known to contribute to equivalent
hydrogen bonds in most of the GSK-3b inhibitor classes
known including paullones, indirubin-30-oxime, stau-
rosporine, ARA-014418, maleimides, benzimidazoles, oxa-
diazoles, and pyrazolopyridine (51–53). Interaction with
hinge region residues such as Val-135, Asp-133, and/or
Tyr-134 is thus important for exhibiting potent GSK-3b
inhibition (54–56). In addition, the phenyl ring of the tria-
zole moiety exhibits p–p stacking interaction with the phe-
nyl ring (backbone) of the amino acid residue Phe-67 in
the DFG motif region and hydrophobic interactions with
GSK-3b protein Leu-188, Ileu-62, Val-70, and Ala-83
amino acid residues, interactions which are reported to
determine selectivity as well potency for GSK-3b inhibition
(54). These interactions probably explain the in vitro GSK-
3b inhibitory activity exhibited by oxazolopyridine-2-one
based 1,2,3-triazoles. We also compared the binding
interactions of the most active compound 4g with the co-
crystallized ligand (IQ6) against the target site as shown in
Figure S3E. Standard ligand IQ6 exhibits high docking
score as it shows binding interaction with both Val 135
and Asp 133 in hinge region. This standard ligand also
shows the binding interaction with Asp 200 in the DFG
motif region. Some part of the ligand 4g is partially super-
imposed with standard IQ6 in hinge region and shows
the common interaction with Val 135. The difference in
the GSK-3b inhibitory activity of the docked synthesized
compounds (4g, 4d, 4f, and 4i), may be mainly due to
the lack of interaction with Asp-133 in the hinge region
when compared to the standard.
ꢂ
Compounds with substitution at ortho-position on the
phenyl ring were found to be more active against both
GSK-3b as well as induced inflammation in comparison
with their corresponding para-substituted derivatives.
Halo-substituted ortho derivatives were found to exhibit
better activity in comparison with compounds having either
alkyl or alkoxy substituents at ortho-position.
ꢂ
Among the halo-substituted derivatives, chloro-substitu-
tion resulted in maximum activity followed by bromo- and
fluoro-substitution at ortho-position. However, the order of
activity at para-position was Br>Cl>F.
ꢂ
Compounds possessing chloro group at either ortho- or
para-position in the phenyl ring showed better activity than
corresponding meta-substituted and di-substituted com-
pounds.
ꢂ
Substitution of alkyl groups on aromatic ring led to
increase in activity than the substitution of corresponding
alkoxy groups.
ꢂ
Substitution of ethoxy group at para-position of aromatic
ring reduced GSK-3b as well as in vivo inflammatory activ-
ity in comparison with substitution of methoxy group at the
same position.
ꢂ
Presence of bulky ter-butyl group at para-position of
aromatic ring decreased the activity as compared to ethyl
group at the corresponding position.
ꢂ
Mono-alkyl substituted phenyl derivatives resulted in
higher activity than dialkyl-substituted derivative.
Crystallographic study
Intensity data were collected at 293(2) K an Oxford Xcal-
ibur Sapphire
Enhance X-ray
3
diffractometer (a single wavelength
source with MoKa radiation,
ꢀ
k = 0.71073 A) (57). The selected suitable single crystals
922
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Oxazolopyridinone-triazoles as GSK-3b Inhibitors
were mounted using paratone oil on the top of a glass
fiber fixed on a goniometer head and immediately trans-
ferred to the diffractometer. Pre-experiment, data collec-
tion, data reduction, and analytical absorption corrections
(58) were performed with the Oxford program suite CrysA-
lisPro (59). The crystal structures were solved with
OLEX2.Solve (60) using direct methods. The structure
refinements were performed by full-matrix least-squares on
F2 with SHELXL-97 (61,62).
Acknowledgments
The authors wish to express their thanks to Dr. G. N. Qazi,
Vice-Chancellor, Jamia Hamdard for providing necessary
facilities to carry out this work. The authors thank DST-
SERB, Govt. of India, for awarding research project to HH
and fellowship to IK. The author MAT is thankful to HNF
for providing research assistance.
Conflict of Interest
The chemical formula and ring labeling system are shown in Fig-
ure S5. Crystal data for compound 4q: C16H13N5O3, Mr,
323.31; system, monoclinic; space group, P 21/n; unit cell
The authors report no conflict of interests.
ꢀ
ꢀ
ꢀ
dimensions, a = 11.555(8) A; b = 9.048(4) A; c = 14.588(9) A;
ꢀ3
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b = 97.89(6)°; V = 1510.7(16) A ; Z = 4; T = 293 K; Rint,
0.0375; R(all), 0.0943; Gof = 1.065; Dqmax = 0.153 e A ;
ꢀ3
ꢀ3
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a riding model, with C-
ꢀ
H = 0.93 A + Uiso(H) = 1.2Ueq(C) for aromatic H atoms, with
ꢀ
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Figure S3 (a) The binding mode of 4g (Grey) is shown in
the GSK-3b active site and important residues are high-
lighted with Silver stick. (b) Ligplot of 4g with GSK-3b
active site (c) The binding mode of IQ6 (Grey) is shown in
the GSK-3b active site and important residues are high-
lighted with Silver stick. (d) Ligplot of IQ6 with GSK-3b
active site (e) Comparison of 4g (Cyan) and co-crystal
ligand (Dark green) binding mode in the GSK-3b active
site.
Figure S4 Histopathology report of ulcerogenic risk
potential of active compounds.
Supporting Information
Figure S5 Crystal structure of 4q.
Additional Supporting Information may be found in the
online version of this article:
Table S1 Oxazolo[4,5-b]pyridin-2-one based 1,2,3-tria-
zoles and their GSK-3b inhibitory activity.
Appendix S1 Experimental.
Table S2 In vivo Anti-inflammatory activity of oxazolo[4,5-
b]pyridin-2-one based 1,2,3-triazoles.
Figure S1 Effects of test compounds, indomethacin and
SB216763 on nitric oxide as measured in rat paw homo-
genate.
Table S3 In silico docking scores with respect to GSK-3b
protein target (PDBID:4IQ6).
Figure S2 Effects of test compounds, indomethacin and
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Chem Biol Drug Des 2016; 87: 918–926