Investigations on substituted (2-aminothiazol-5-yl)(imidazo[1,2-a]pyridin-3-yl)methanones for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2021.116091

Alzheimer's disease (AD) is a neurodegenerative disease majorly affecting old age populations. Various factors that affect the progression of the disease include, amyloid plaque formation, neurofibrillary tangles, inflammation, oxidative stress, etc. Here

Full text of DOI:10.1016/j.bmc.2021.116091

Bioorg. Med. Chem. 36 (2021) 116091
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Investigations on substituted (2-aminothiazol-5-yl)(imidazo[1,2-a]
pyridin-3-yl)methanones for the treatment of Alzheimer’s disease
,
c
,
,
Sneha R. Sagara 1, Devendra Pratap Singhb 2, Rajesh D. Dasa, Nirupa B. Panchala,
Vasudevan Sudarsanama, Manish Nivsarkarb, Kamala K. Vasua
,
*
a Department of Medicinal Chemistry, B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, S. G. Highway, Thaltej, Ahmedabad 380054,
Gujarat, India
b Department of Pharmacology and Toxicology, B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, S. G. Highway, Thaltej, Ahmedabad
380054, Gujarat, India
c Institute of Pharmacy, NIRMA University, S. G. Highway, Chandlodia, Gota, Ahmedabad, Gujarat 382481, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Alzheimer’s disease (AD) is a neurodegenerative disease majorly affecting old age populations. Various factors
that affect the progression of the disease include, amyloid plaque formation, neurofibrillary tangles, inflam-
mation, oxidative stress, etc. Herein we report of a new series of substituted (2-aminothiazol-5-yl)(imidazo[1,2-
a]pyridin-3-yl)methanones. The designed compounds were synthesized and characterized by spectral data. In
vivo anti-inflammatory activity was carried out for screening of anti-inflammatory potential of synthesized
compounds. All the compounds were tested for acute inflammatory activity by using carrageenan induced acute
inflammation model. Compounds 10b, 10c, and 10o had shown promising acute anti-inflammatory activity and
they were further tested for formalin induced chronic inflammation model. Compound 10c showed both acute
and chronic anti-inflammatory activity. Compound 10c also showed promising results in AlCl3 induced AD
model. Studies on various behavioral parameters suggested improved amnesic performance of compound 10c
treated rats. Compound 10c treated rats also exhibited excellent antioxidant and neuroprotective effect with
inherent gastrointestinal safety.
Imidazo[1,2-a]pyridines
Thiazoles
Neuronal inflammation
Alzheimer’s disease
1. Introduction
aggregation, cholinergic neurotransmitter imbalance, oxidative stress
and neuronal inflammation can be largely attributed as its hallmarks.3
Alzheimer’s disease (AD) is a progressive and unremitting neurode-
generative disorder of old age which affects wide areas of cerebral cortex
and hippocampus. The malformations in AD are initially detected in
brain tissue that involves the frontal and temporal lobes and then slowly
propagates to other regions of the neocortex at different rates in
different individuals.1 The peculiar characteristics are irrevocable
memory impairment, decline in cognitive ability and higher intellectual
functions.1 In 2020, World Health Organization (WHO) reported 50
million people were living with dementia globally and this figure is set
to increase to 152 million by 2050.2 Amidst various factors governing
In recent years, a retrospection into underlying inflammatory re-
sponses in context of AD mechanism, proposes neuronal inflammation to
be a critical factor for pathogenesis of AD. Neuronal inflammation is
largely manifested by the activation of microglia and astrocytes. Under
normal physiological condition, microglia cells are in a state of rest.4
However, in neurodegenerative diseases, they are constantly activated
and trigger the release of inflammatory cytokines eventually leading to
chronic neuroinflammation. Studies show that these inflammatory cy-
tokines aggravate oxidative stress in brain and stimulate the generation,
aggregation and accumulation of insoluble Aβ plaques in extracellular
spaces, as well as in walls of blood vessels.5 The accumulated insoluble
the pathophysiology of AD, β-amyloid (Aβ) deposition,
τ-protein
Abbreviations: AD, Alzheimer’s disease; COXs, cyclooxygenases; RT, room temperature; NC, normal control; DC, disease control; PC, positive control; LPO, Lipid
peroxidation; SOD, Superoxide dismutase.
* Corresponding author.
E-mail address: kamkva@gmail.com (K.K. Vasu).
1
Present address: L. J. Institute of Pharmacy, S. G. Highway, Sanand Circle, Sarkhej, Ahmedabad, Gujarat 382210, India.
2
Present address: Manager R&D, (FDD-NDDS), Sun Pharmaceutical Industries Limited, Tandalija, Vadodara, Gujarat, India.
https://doi.org/10.1016/j.bmc.2021.116091
Received 3 January 2021; Received in revised form 15 February 2021; Accepted 16 February 2021
Available online 27 February 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

S.R. Sagar et al.
Bioorganic & Medicinal Chemistry 36 (2021) 116091
Aβ plaques induce cycloxygenase-2 (COX-2) expression. In addition, the
activation of microglia occurs before neurodegenerative symptoms
appear, and this can be regarded as an early event related to patholog-
ical changes in AD. Thus, anti-inflammatory agents are considered to be
potential candidates for Alzheimer’s disease. Researchers showed that
anti-inflammatory drugs could even control the release of inflammatory
factors and oxygen free radicals caused by the deposition of Aβ, and
reduce the amount and area of Aβ deposition.6 However, the available
literature reported controversial role of anti-inflammatory agents in the
progression of AD. The reports suggested that anti-inflammatory bene-
fits were observed only in low doses during AD pathology and some of
the NSAIDs were failed to show clinical efficacy. The reason behind
lower activity could be due to some factors like brain penetration of
drug, pharmacological properties of drugs, dosing schedule and selec-
tion of patients.7
In present study, imidazo[1,2-a]pyridines were clubbed with thiazoles
to get the beneficial effects of both the pharmacophores. Thus, we hy-
pothesize that taking these two pharmacophores in a single molecule
may increase anti-inflammatory potential of the compounds as depicted
in Fig. 1.
2. Results & discussion
2.1. Chemistry
The designed compounds were synthesized as depicted in Scheme 1.
Substituted pyridin-2-amines reacted with 1,1-dimethoxy-N,N-dime-
◦
thylmethanamine in methanol at 70 C to yield N,N-dimethyl-N′-(pyr-
idin-2-yl)formimidamide derivatives 2a-d.8 Substituted 2-chloro-1-
(imidazo[1,2-a]pyridin-3-yl)ethanone 3a-d were synthesized from 2a-
d using 1,3-dichloropropan-2-one in acetonitrile at room temperature as
described in Scheme 1.8,14 Reaction of imidamide derivatives 4a-c in
presence of (isothiocyanatomethanetrityl)tribenzene in THF at 50 ◦C for
3–4 h afforded substituted N′-(tritylcarbamothioyl)formimidamides 5a-
c.15 Thiourea 6 reacted with 1,1-dimethoxy-N,N-dimethylmethanamine
to afford N′-carbamothioyl-N,N-dimethylformimidamide 7. 7 was reac-
ted in presence of (isothiocyanatomethanetrityl) tribenzene in THF at
50 ◦C for 3–4 h to yield N,N-dimethyl-N′-(tritylcarbamothioyl) for-
mimidamide 8.16 Substituted 2-chloro-1-(imidazo[1,2-a]pyridin-3-yl)
ethanones 3a-d were reacted with substituted N′-(tritylcarbamothioyl)
formimidamides 5a-c and 8 to yield 9a-p. 9a-p were further reacted for
the deprotection of trityl group by trifluoro acetic acid to get the final
compounds, substituted (2-aminothiazol-5-yl)(imidazo[1,2-a]pyridin-3-
yl)methanones 10a-p (refer Table 1 for specific substitutions). All the
synthesized compounds were purified by column chromatography and
purity of the compounds was checked by HPLC chromatogram. Further,
compounds were characterized by 1H NMR and 13C NMR spectra (See
supplementary material for spectral data).
Imidazopyridine ring scaffold has been proved to be efficacious
against neuroinflammation by regulating various pro-inflammatory
mediators and COXs enzymes.8–11 Based on this mechanism, Imidazo
[1,2-a]pyridin-3-yl(7-methylimidazo[1,2-a]pyridin-3-yl)methanone (1)
was designed and it exhibited 67% inhibition against acute inflamma-
tion and 61% inhibition of chronic inflammation in rat paw edema
model.8 1′,3′-dihydro-2H-spiro[imidazo[1,2-a]pyridine-3,2′-inden]-2-
one (2) was found to be a potent complex cognitive enhancer as it is able
to influence the processes in neuroinflammation pathways.9 Moreover,
our previous work on Imidazo[1,2-a]pyridines linked with thiazoles/
thiophene motif through keto spacer, indicated the importance of Imi-
dazopyridines against the progression of various inflammatory events
and NF-ĸB and AP-1 mediated transcriptional processes. Thiazole moi-
ety has important structural features of having H-bond acceptor and H-
bond donor.12,13 Methyl 3-(4-(dimethylamino)-2-(methylamino)thiazol-
5-yl)-2-(methoxyimino)-3-oxopropanoate (3) was reported as an anti-
inflammatory agent with 83% inhibition of rat paw edema in acute
inflammation and 66% inhibition of rat paw edema in chronic inflam-
mation.12 N,N′-(5,5′-oxalylbis(4-methylthiazole-5,2-diyl)dibenzamide
(4) showed 73% inhibition of edema in acute inflammation rat model.12
Fig. 1. Design of thiazolyl-imidazo[1,2-a]pyridines.
2

S.R. Sagar et al.
Bioorganic & Medicinal Chemistry 36 (2021) 116091
Scheme 1. Synthesis of thiazolyl-imidazo[1,2-a]pyridine derivatives.
2.2. In vivo acute inflammation study
chronic inflammation model. On 5th day of chronic model, % inhibition
of edema was obtained as 54% and 63% for 10b and 10c, respectively.
Celecoxib (selective COX-2 inhibitor) was used as a standard drug
because COX-2 is a major contributor to prostanoid synthesis in chronic
phase of inflammation.17–19 Celecoxib was found to inhibit 68% of rat
paw edema as given in Table 2.
The anti-inflammatory potential of the compounds 10a-p were
tested by studying acute and chronic inflammation model. All the
compounds were examined using carrageenan induced acute inflam-
mation rat model. Carrageenan was given subcutaneously to the left
hind paw of each rat. This causes metabolism of arachidonic acid and
further produces plasma and leukocyte extravasations, plasma protein
exudation along with neutrophil extravasations and increased tissue
water. This inflammatory event was diminished by our compounds at
50 mg⋅kgꢀ 1 dose. % decrease in the size of edema was calculated and
reported as % edema inhibition in Table 1. During carrageenan induced
acute inflammation, COX-1 and COX-2 both the enzymes are involved in
the inflammatory event. Therefore, non-selective COX inhibitor, Diclo-
fenac was used as a standard drug.17–19 Data expressed as mean ± SEM.
Test compounds were compared with the standard drug diclofenac (dose
of 7.5 mg⋅kgꢀ 1). Compounds 10b, 10c, 10h, 10i, 10k, 10o and 10p
were found to inhibit inflamed paw with % inhibition of 81%, 85%,
77%, 53%, 66%, 77%, and 53%, respectively. Standard drug, diclofenac
was found to inhibit rat paw edema with 72%.
Compound 10c had shown both acute as well as chronic anti-
inflammatory activities, so it was taken further to evaluate its activity
in AlCl3 induced Alzheimer’s disease model. During the 28 days of
Alzheimer’s disease model, various behavioral parameters, biochemical
parameters and haematological parameters were assessed. Meloxicam
was taken as a standard drug for AlCl3 induced (AD) model because it
protects neurons by reducing metal ion imbalance induced oxidative
stress.20
2.4. AlCl3 induced Alzheimer’s disease (AD) model
2.4.1. Estimation of behavioral parameters
2.4.1.1. Evaluation of transfer latency by elevated plus maze test. Spatial
working memory of the rats was evaluated using elevated plus maze test.
It is a natural tendency of rats to escape from the elevated open space
environment and to be in the close and safe environment. Transfer la-
tency (TL) measures this behavior of rats. Time required for each rat to
reach to the closed arm from an open arm was measured (in seconds).21
Treatment with compound 10c improved the spatial working memory of
rats when compared to DC rats as shown in Fig. 2A. Memory enhancing
2.3. In vivo chronic inflammation study
Active compounds from the acute inflammation study, i.e. 10b, 10c
and 10o were taken further to evaluate their anti-inflammatory activity
using formalin induced chronic inflammation model. Compounds 10b
and 10c were found to show anti-inflammatory activity during 5 days of
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S.R. Sagar et al.
Bioorganic & Medicinal Chemistry 36 (2021) 116091
Table 1
List of synthesized compounds with % inhibition of edema in acute inflamma-
tion model.
Code
R1
R2
% inhibition of edema [mean ± SEM]a
10a
H
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
C6H5
C6H5
C6H5
C6H5
CH3
32 ± 1.0
81 ± 1.5
85 0.9
23 ± 2.1
40 ± 3.5
20 ± 2.5
26 ± 3.9
77 1.5
53 1.2
45 ± 1.8
66 1.1
33 ± 2.4
33 ± 2.6
31 ± 2.4
77 0.8
53 1.0
72 1.3
10b
10c
10d
10e
3-CH3
4-CH3
5-CH3
H
10f
3-CH3
4-CH3
5-CH3
H
10g
10h
10i
10j
3-CH3
4-CH3
5-CH3
H
CH3
10k
10l
CH3
CH3
10m
10n
10o
10p
Diclofenac
H
3-CH3
4-CH3
5-CH3
—
H
H
H
—
a
Data expressed as the % inhibition in edema size relative to the DC group.
Statistical analysis was performed by One-way ANOVA followed by Tukey’s
multiple comparison test. Mean ± SEM (n = 6 rats/group) *p < 0.05.
Table 2
% edema inhibition against formalin induced chronic inflammation.
Compound
% Inhibition of edema in chronic inflammation [mean ± SEM]a
Day 1
Day 2
Day 3
Day 4
Day 5
10b
55 3.3
58 3.2
30 ± 1.6
67 ± 1.8
56 2.0
67 3.0
24 ± 2.3
74 ± 1.1
56 1.0
65 3.9
36 ± 4.7
70 ± 4.4
54 3.7
60 4.7
23 ± 1.0
70 ± 2.9
54 3.9
63 3.8
18 ± 4.9
68 ± 1.0
10c
10o
Celecoxib
a
Data expressed as the % inhibition in edema size relative to the DC group.
Statistical analysis was performed by One-way ANOVA followed by Tukey’s
multiple comparison test. Mean ± SEM (n = 6 rats/group) *p < 0.05.
effect of compound 10c was evaluated during 7th, 14th, 21st and 28th
day of AlCl3 induced AD model.
2.4.1.2. Evaluation of conditioned avoidance response using pole climbing
apparatus. Conditioned avoidance response (CAR) was evaluated using
pole climbing apparatus. This test demonstrates learning memory of
rats.22 During acquisition session, rats showed low CAR values as they
learned to escape from the foot shock. Whereas during test session, DC
rats showed significantly high CAR values on 7th, 14th, 21st and 28th
day of AlCl3 model. Learning memory of rats were improved when
treated with the compound 10c when compared with DC group rats (#p
< 0.05) as depicted in Fig. 2B.
Fig. 2. Behavioral parameters estimation. (A) Results of transfer latency using
elevated plus maze test. (B) Results of conditioned avoidance response (CAR)
using pole climbing apparatus (C) Results of Y maze test expressed as %
alteration. Data expressed as mean ± SEM. Asterisk denotes statistical signifi-
cance (*p < 0.05) when compared to NC group and (#p < 0.05) when compared
to DC group, using two-way ANOVA followed by Tukey’s multiple compari-
son test.
2.4.2. Haematological parameters estimation
2.4.1.3. Evaluation of simultaneous alteration performance using Y maze
apparatus. Simultaneous alteration performance was assessed by using
Y maze apparatus. This test uses the natural tendency of rats to explore
new environment, so rats were preferred to visit different maze then
previously visited maze.23 Immediate working memory of rats was
monitored by calculating % alteration of all the groups as shown in
Fig. 2C. Treatment with 10c significantly improves the working memory
when compared with the DC group rats (#p < 0.05).
On 29th day of AlCl3 model, rats were sacrificed and various hae-
matological parameters, biochemical parameters and anti-oxidant
properties were measured using brain tissues. Haemoglobin (HGB)
and haematocrit (HCT) level were estimated on the 29th day of AlCl3
model. HGB and HCT level were significantly reduced in the PC animals
compared to NC animals. Whereas, 10c treated rats showed no differ-
ence in the HGB and HCT level in blood samples as shown in Fig. 3A and
3B. Reduction in HGB and HCT level in PC animals is due to the gastric
injury and bleeding, which was further confirmed in gastrointestinal
4

S.R. Sagar et al.
Bioorganic & Medicinal Chemistry 36 (2021) 116091
Fig. 3. Estimation of haematological parameters. (A) HGB level was
significantly decreased in the PC animals. Data presented as mean ± SEM (*p <
0.05). (B) HCT level estimation in all the groups indicated that there was a
significant reduction in the HCT level in PC group when compared to all the
groups. Data expressed as mean ± SEM (*p < 0.05) by using 1 way ANOVA
followed by Tukey’s multiple comparison test.
Fig. 4. Estimation of biochemical parameters. (A) Serum albumin level
estimation. Data presented as mean ± SEM (*p < 0.05). (B) Total protein
estimation. Data expressed as mean ± SEM (*p < 0.05) by using 1 way ANOVA
followed by Tukey’s multiple comparison test.
of all the groups of rats were observed under 100X magnified micro-
scope. Hippocampal neuronal degeneration is observed in the DC rats.
Notably, NC, PC and 10c treated animals had shown intact neuronal
cells. 10c treated rats had shown the neuro-protective effects compa-
rable with that of PC rats (Fig. 6).
safety studies.
2.4.3. Biochemical parameters estimation
Serum biochemical parameters viz. serum albumin and total protein
were measured in all groups of animals. There was no significant dif-
ference in the serum albumin level of all the groups of animals. How-
ever, the PC group showed significant decrease in the total protein level
as compared to NC animals (*p < 0.05) (Fig. 4).
2.4.6. Anti-amyloid properties of compound 10c
Determination of amyloid plaque formation was done by congo red
stain. Effect of compound 10c was observed against amyloid plaques.
DC group rats had amyloid plaques in their brain and treatment with 10c
reduces the plaque formation in the cortex region of brain as shown in
Fig. 7.
2.4.4. Antioxidant activity
AlCl3 is responsible for the cell damage via free radial generation.
This causes increase in the oxidative stress in brain. These free radicals
mediate increase in the peroxidase level, which was evaluated by lipid
peroxidation assay (LPO).8 Antioxidant property of the 10c was evalu-
ated by superoxide dismutase assay (SOD) of brain tissues. Fig. 5 shows
LPO and SOD activity of brain tissues in different groups of rats. LPO
activity was measured as formation of MDA level. LPO activity was
expressed as [(nmol/gm fresh weight) × 10ꢀ 5]. AlCl3 treated group
significantly increased MDA level when compared to NC animals (*p <
0.05). Treatment with 10c reduces the LPO activity and showed com-
parable results with PC rats as shown in Fig. 5A. In case of SOD, sig-
nificant change (#p < 0.05) was observed in PC animals and 10c treated
rats when compared with DC rats. Significant change in SOD values were
also observed between NC and DC animals (*p < 0.05). Normal defense
mechanism of body causes slight increase in the SOD level in DC group
animals as shown in Fig. 5B.
2.5. Determination of gastrointestinal damage
Macroscopic evaluation of gastrointestinal tract was carried out on
the 29th day of AlCl3 model. Damage of gastrointestinal tract was
measured by using Vernier calipers. Stomach and intestines were
observed for lesion indices. PC group rats showed damage in both
stomach and intestines. Lesion index in stomach was found to be 12.19
± 0.8 mm and 29.15 ± 1.6 mm for intestines as shown in Fig. 8. Com-
pound 10c treated animals had not shown any lesions in the stomach
and intestines. Despite being an anti-inflammatory compound, 10c had
no gastrointestinal damaging effect when compared with other
NSAIDS.24,25
2.5.1. Histopathological evaluation of gastrointestinal damage
Damage of gastrointestinal track was further checked by microscopic
evaluation of stomach and intestinal sections. Sections were stained
with HE stain and gastric injuries were confirmed in PC treated animals.
2.4.5. Neuroprotective effects of compound 10c
Brain sections were stained with HE stain and hippocampal regions
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S.R. Sagar et al.
Bioorganic & Medicinal Chemistry 36 (2021) 116091
4. Experimental section
4.1. General methods
Unless stated otherwise solvents and reagents were purchased from
commercial suppliers and used without further purification. Determi-
nation of melting point was done by using scientific melting point
apparatus (Veego; Model VMP-DS) and uncorrected. FTIR spectra were
recorded using Shimadzu’s IR Affinity 1 apparatus. 1H NMR and 13C
NMR spectra were recorded using Bruker Avance II NMR spectropho-
tometer at frequencies 400 MHz & 100 MHz, respectively. Chemical
shift values are represented in parts per million. Signals are described as
s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and dd
(doublet of doublets). LC-ESI-MS spectral data were obtained using
Perkin Elmer mass spectrometer. Q-TOF micromass (ESI-MS) was used
for HRMS spectral analysis. Pre-coated TLC plates were used for reaction
monitoring (Merck). Visualization of TLC plates was done using UV
chamber. SHIMADZU-LC-2010 HPLC was used for determination of
purity of the compounds. Paw volumes were recorded using manual
mercury plethysmometer. Samples were homogenized using Polytron
homogenizer (Kinematica, Switzerland). Sorvall Legend X1R centrifuge
(Thermo Scientific) was used for Centrifugation. Samples were vortexed
in Vortex Mixer (Eltek, VM301). Shimadzu UV/Visible Spectropho-
tometer UV-1800 was used for absorbance of samples. Serum albumin
and total protein were determined using fully automated random access
clinical chemistry analyser Transasia EM 360. VetScan HM-5 was used
for Haemoglobin and haematocrit level estimation. Histopathological
slides were observed under optical microscope ProgRes C3 OLYMPUS,
U-TV1 X,Japan.
4.2. Chemistry
Fig. 5. Anti-oxidative effect of compound 10c. (A) Lipid peroxidation (LPO)
expressed as MDA level in rat brain homogenate (B) Super oxide dismutase
activity in rat brain homogenate. Data shown as mean ± SEM (n = 6 rats/
group) *p < 0.05 when compared with NC group and #p < 0.05 when
compared to DC group using 1 way ANOVA followed by Tukey’s multiple
comparison test.
4.2.1. Synthesis and characterization of substituted N,N-dimethyl-N′-
(pyridin-2-yl)formimidamide (2a-d)
4.2.1.1. N,N-dimethyl-N′-(pyridin-2-yl)formimidamide (2a). To the so-
lution of pyridin-2-amine (1.0 eq.) in methanol, 1,1-dimethoxy-N,N-
dimethylmethanamine (2.0 eq.) was added drop wise. Reaction was
allowed to proceed at 70 ◦C for 15 h. After completion of reaction,
methanol was evaporated in vacuo and concentrated reaction mixture
was poured into ice cold water. The product was extracted by
dichloromethane (3 × 50 mL). The organic layers were combined, dried
over sodium sulfate and evaporated in vacuo to get yellow liquid N,N-
dimethyl-N′-(pyridin-2-yl)formimidamide (2a).8,14 Yield: 81%; Boiling
point: 101–103 ◦C; LC-ESI-MS (m/z): 150.09 [M+H]+.
PC treated rats had focal erosions present in the stomach and epithelium
damage was seen in the intestinal sections. Compound 10c treated an-
imals revealed no erosions in stomach and intestine as shown in Fig. 9
and Fig. 10.
3. Conclusion
There are several factors that affect the AD aetiology. One of the
major factors that influences AD progression are neuronal cell inflam-
mation. Damaged neurons and amyloid plaques release several inflam-
4.2.1.2. N,N-dimethyl-N′-(3-methylpyridin-2-yl)formimidamide (2b). In-
termediate 2b was prepared by the procedure described in 2a using 3-
methylpyridin-2-amine. Yield: 75%; Yellow solid; Melting point:
50–52 ◦C; LC-ESI-MS (m/z): 164.1 [M+H]+.
matory cytokines namely TNF-α, IL-1β and COXs. This inflammatory
event lead to neuronal cell degeneration and cell death. Hence to combat
AD, treatment with anti-inflammatory leads can serve as a therapeutic
weapon. Imidazo[1,2-a]pyridines and thiazoles were fused in a single
molecule to make thiazolyl-imidazo[1,2-a]pyridines in order to enhance
the anti-inflammatory potency of this fused scaffold. A series of 16
compounds were synthesized and characterized by spectral data.
Further, these molecules were tested for their anti-inflammatory bene-
fits using in vivo acute and chronic inflammation model. Overall, com-
pound 10c exhibited promising anti-inflammatory activity. Compound
10c was further evaluated for anti-Alzheimer’s activity using in vivo
AlCl3 induced model. In this study, 10c showed promising results by
exhibiting anti-amnesic, neuroprotective and anti-amyloid activities.
Furthermore, compound 10c did not exhibit GI toxicity like other
NSAIDS. In future, compound 10c may emerge as a promising lead
candidate for the treatment of AD.
4.2.1.3. N,N-dimethyl-N′-(4-methylpyridin-2-yl)formimidamide (2c). In-
termediate 2c was prepared by the procedure described in 2a using 4-
methylpyridin-2-amine. Yield: 84%; Yellow solid; Melting point:
55–57 ◦C; LC-ESI-MS (m/z): 164.2 [M+H]+.
4.2.1.4. N,N-dimethyl-N′-(5-methylpyridin-2-yl)formimidamide (2d). In-
termediate 2d was prepared by the procedure described in 2a using 5-
methylpyridin-2-amine. Yield: 77%; Yellow solid; Melting point:
53–55 ◦C; LC-ESI-MS (m/z): 164.1 [M+H]+.
4.2.2. Synthesis and characterization of substituted 2-chloro-1-(imidazo
[1,2-a]pyridin-3-yl)ethanone (3a-d)
4.2.2.1. 2-chloro-1-(imidazo[1,2-a]pyridin-3-yl)ethanone (3a). To the
6

S.R. Sagar et al.
Bioorganic & Medicinal Chemistry 36 (2021) 116091
Fig. 6. Typical microscopic HE stained sections (under 100X magnification) of brain hippocampus (A) NC animals had shown normal and healthy hippocampal
neurons (B) DC animals had shown degenerated neuronal cells in hippocampus (C) PC animals showed healthy neurons in hippocampus, (D) 10c treated rats showed
healthy neuronal cells.
Fig. 7. Congo red stained sections (under 100X magnification) of brain cortex in all the groups. (A) NC animal’s brain cortex without amyloid plaques (B) Amyloid
plaques were present in the cortex of DC rats (stained red) indicated by an arrow, (C) PC animals without plaques (D) Reduction in the amyloid plaque by treatment
with compound 10c as indicated by an arrow.
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S.R. Sagar et al.
Bioorganic & Medicinal Chemistry 36 (2021) 116091
N-dimethyl-N′-(3-methylpyridin-2-yl)formimidamide.
Yield:
68%;
Colorless solid; Melting point: 135–137 ◦C; LC-ESI-MS (m/z): 209.5
[M+H]+.
4.2.2.3. 2-chloro-1-(7-methylimidazo[1,2-a]pyridin-3-yl)ethanone (3c).
Intermediate 3c was prepared by the procedure described in 3a using N,
N-dimethyl-N′-(4-methylpyridin-2-yl)formimidamide. Yield: 69%; Pale
yellow solid, Melting point: 134–136 ◦C; LC-ESI-MS (m/z): 209.5
[M+H]+.
4.2.2.4. 2-chloro-1-(6-methylimidazo[1,2-a]pyridin-3-yl)ethanone (3d).
Intermediate 3d was prepared by the procedure described in 3a using N,
N-dimethyl-N′-(5-methylpyridin-2-yl)formimidamide.
Yield:
64%;
Colorless solid, Melting point: 135–137 ◦C; LC-ESI-MS (m/z): 209.5
[M+H]+.
4.2.3. Synthesis and characterization of substituted N′-
(tritylcarbamothioyl) formimidamides (5a-c)
Fig. 8. Determination of lesion index: PC group animals showed significantly
higher lesion index when compared to NC, DC and 10c treated groups. Data
expressed as mean ± SEM (*p < 0.05) by using 1 way ANOVA followed by
Tukey’s multiple comparison test.
4.2.3.1. N,N-diethyl-N′-(tritylcarbamothioyl)benzimidamide (5a). To the
solution of N,N-diethylbenzimidamide 4a (1.0 eq.) in THF, addition of
(isothiocyanatomethanetrityl)tribenzene (1.0 eq.) was done. Reaction
mixture was allowed to stir at room temperature for 3–4 h. After
completion of reaction, THF was evaporated in vacuo to get colorless
solid of 5a.15 The crude was purified by column chromatography (1:9 =
Ethyl acetate: Hexane). Yield: 82%; colorless solid; Melting point:
195–197 ◦C; LC-ESI-MS (m/z): 478.1 [M+H]+.
solution of 1,3-dichloropropan-2-one (1.5 eq.) in acetonitrile, N,N-
dimethyl-N′-(pyridin-2-yl)formimidamide (1.0 eq.) was added drop
wise. Reaction mixture was allowed to stir at room temperature for 7 h.
After completion of reaction, reaction mixture was poured into ice cold
water to obtain crude brown solid of 3a. Resulting crude was crystallized
in methanol to get pure 2-chloro-1-(imidazo[1,2-a]pyridin-3-yl)etha-
none (3a).14 Yield: 62%; White solid; Melting point: 138–140 ◦C; LC-ESI-
MS (m/z): 195.5 [M+H]+.
4.2.3.2. N,N-diethyl-N′-(tritylcarbamothioyl)acetimidamide (5b). Inter-
mediate 5b was prepared by the procedure described in 5a using N,N-
diethylacetimidamide. Yield: 70%; Melting point: 188–190 ◦C; LC-ESI-
MS (m/z): 416.2 [M+H]+.
4.2.2.2. 2-chloro-1-(8-methylimidazo[1,2-a]pyridin-3-yl)ethanone (3b).
Intermediate 3b was prepared by the procedure described in 3a using N,
Fig. 9. Typical microscopic HE stained sections (under 100X magnification) of stomach in all the groups. (A) NC animals without stomach erosions, (B) DC animals
without erosions, (C) PC animals with focal erosions indicated by an arrow, (D) Absence of ulceration and erosions in 10c treated animals.
8

S.R. Sagar et al.
Bioorganic & Medicinal Chemistry 36 (2021) 116091
Fig. 10. Typical microscopic HE stained sections (under 100X magnification) of intestine in all the groups. (A) NC animals did not show any ulceration (B) DC
animals without any focal erosions, (C) PC animals showed focal erosions of the superficial epithelium and epithelial stratification indicated by an arrow, (D) No
erosions or epithelium stratification was observed in 10c treated animals.
4.2.3.3. 1-(bis(dimethylamino)methylene)-3-tritylthiourea (5c). Interme-
diate 5c was prepared by the procedure described in 5a using 1,1,3,3-
tetramethylguanidine. Yield: 86%; Melting point: 182–184 ◦C; LC-ESI-
MS (m/z): 417.2 [M+H]+.
Methanol = 9.5:0.5). SHIMADZU-LC-2010 was used to determine the
purity of each compound [Mobile phase (10 mM):- KH2PO4 buffer:
Acetonitrile (50:50); Column: Reverse phase; pH: 7]. Pale yellow solid;
◦
Yield: 76%; Purity: 99.1%; Melting point: 180–182 C; Molecular for-
mula: C13H13N5OS; LC-ESI-MS (m/z): 288.1 [M+H]+; IR (KBr, cmꢀ 1):
3391.23, 3305.25, 3056.21, 1643.52; 1H NMR (400 MHz, DMSO‑d6) δ:
3.05 (s, 6H), 7.045 (t, J = 5.6 Hz, 2H), 7.352 (s, 1H), 7.90 (d, J = 6.5 Hz,
2H), 8.2114 (s, D2O exchangeable, 2H); 13C NMR (100 MHz, DMSO‑d6)
δ: 40.1 (2C), 115.50, 117.28, 127.76, 128.03, 128.28, 129.03, 130.40,
139.87, 140.81, 151.24, 164.12.
4.2.4. Synthesis and characterization of N,N-dimethyl-N′-
(tritylcarbamothioyl)formimidamide (8)
Thiourea 6 (1.0 eq) in methanol was reacted with 1,1-dimethoxy-N,
N-dimethylmethanamine (2.0 eq) at room temperature for 3 h to get N′-
carbamothioyl-N,N-dimethylformimidamide 7 (yield 82%). 7 (1.0 eq)
was further reacted with (isothiocyanatomethanetrityl)tribenzene in
◦
THF, at 50 C for 3–4 h to yield intermediate 8. After completion of
4.2.5.2. (2-amino-4-(dimethylamino)thiazol-5-yl)(8-methylimidazo[1,2-
a]pyridin-3-yl)methanone (10b). Final compound 10b was synthesized
as per the procedure described in 10a using starting materials 2-chloro-
1-(8-methylimidazo[1,2-a]pyridin-3-yl)ethanone 3b and 1-(bis(dime-
thylamino)methylene)-3-tritylthiourea 5c. 10b was purified by column
chromatography (Chloroform: Methanol = 9.7:0.3). Pale yellow solid;
reaction, THF was evaporated in vacuo to get compound 8.16 8 was
purified by column chromatography (1:9 = Ethyl acetate: Hexane).
Yield: 79%; Pale yellow solid; Melting point: 210–212 ◦C; LC-ESI-MS (m/
z): 374.1 [M+H]+.
◦
4.2.5. Synthesis and characterization of final compounds: Substituted (2-
Yield: 71%; Purity: 99.6%; Melting point: 170–172 C; Molecular for-
mula: C14H15N5OS; LC-ESI-MS (m/z): 302.09 [M+H]+; HRMS (TOF) m/
z calcd for C14H15N5OS [M+H]+ 302.3668, found: 302.3623; IR (KBr,
cmꢀ 1): 3471.87, 3313.71, 3084, 1681.93; 1H NMR (400 MHz, DMSO‑d6)
δ: 2.51 (s, 3H), 3.02 (s, 6H), 6.99 (t, J = 6.8 Hz, 1H), 7.27 (d, J = 6.7 Hz,
1H), 7.95 (s, D2O exchangeable, 2H), 8.08 (s, 1H), 9.17 (d, J = 6.6 Hz,
1H); 13C NMR (100 MHz, DMSO‑d6) δ: 16.43, 112.44, 113.21, 125.57,
125.88, 126.59, 137.92, 138.43, 140.86, 164.31, 169.96, 170.04,
175.37.
aminothiazol-5-yl)(imidazo[1,2-a]pyridin-3-yl)methanones (10a-p)
4.2.5.1. (2-amino-4-(dimethylamino)thiazol-5-yl)(imidazo[1,2-a]pyridin-
3-yl)methanone (10a). To a solution of 2-chloro-1-(imidazo[1,2-a]pyr-
idin-3-yl)ethanone (1.0 eq.) 3a in acetonitrile, K2CO3 (3.0 eq.) was
added. Reaction mixture was allowed to stir and addition of 1-(bis
(dimethylamino)methylene)-3-tritylthiourea (1.0 eq.) 5c was done
slowly. Reaction was allowed to stir at room temperature for 5 h. After
completion of reaction, reaction mixture was poured into ice cold water
to get the precipitates of tritylated 9a. Deprotection of tritylated 9a was
done using trifluoro acetic acid (50.0 eq.) in DCM for 3 h. After
completion of reaction, reaction mixture was poured into ice cold water.
Solution was neutralized with sodium bicarbonate to get the product
10a. 10a was purified by column chromatography (Chloroform:
4.2.5.3. (2-amino-4-(dimethylamino)thiazol-5-yl)(7-methylimidazo[1,2-
a]pyridin-3-yl)methanone (10c). Final compound 10c was synthesized
as per the procedure described in 10a using starting materials 2-chloro-
1-(7-methylimidazo[1,2-a]pyridin-3-yl)ethanone 3c and 1-(bis(dime-
thylamino)methylene)-3-tritylthiourea 5c. 10c was purified by column
9

S.R. Sagar et al.
Bioorganic & Medicinal Chemistry 36 (2021) 116091
chromatography (Chloroform: Methanol = 9.7:0.3). Yellow solid; Yield:
68%; Purity: 99.9%; Melting point: 173–175 ◦C; Molecular formula:
C14H15N5OS; LC-ESI-MS (m/z): 302.09 [M+H]+; HRMS (TOF) m/z calcd
for C14H15N5OS [M+H]+ 302.3668, found: 302.3654; IR (KBr, cmꢀ 1):
3471.87, 3313.71, 3064.89, 1681.93; 1H NMR (400 MHz, DMSO‑d6) δ:
2.33 (s, 3H), 3.02 (s, 6H), 6.96 (d, J = 6.8 Hz, 1H), 7.7 (s, 1H), 7.92 (s,
D2O exchangeable, 2H), 8.02 (s, 1H), 9.2 (d, J = 7.2 Hz, 1H); 13C NMR
(100 MHz, DMSO‑d6) δ: 20.75, 41.72 (2C), 115.57, 116.14, 126.60,
127.03, 127.50, 138.22, 138.60, 147.06, 147.73, 164.19, 169.88.
13C NMR (100 MHz, DMSO‑d6) δ: 20.98, 117.39, 117.98, 118.12,
120.90, 122.49, 127.22, 127.27, 127.66, 128.24, 129.13, 135.19,
140.66, 141.35, 144.37, 148.97, 171.31, 180.27.
4.2.5.8. (2-amino-4-phenylthiazol-5-yl)(6-methylimidazo[1,2-a]pyridin-
3-yl)methanone (10h). Final compound 10h was synthesized as per the
procedure described in 10a using starting materials 2-chloro-1-(6-
methylimidazo[1,2-a]pyridin-3-yl)ethanone 3d and N,N-diethyl-N′-
(tritylcarbamothioyl)benzimidamide 5a. 10h was purified by column
chromatography (Hexane: Ethyl acetate = 1:1). Yellow solid; Yield:
52%; Purity: 99.9%; Melting point: 175–177 ◦C; Molecular formula:
C18H14N4OS; LC-ESI-MS (m/z): 335.3 [M+H]+; IR (KBr, cmꢀ 1):
3362.71, 3287.25, 3078.15, 1646.12; 1H NMR (400 MHz, DMSO‑d6) δ:
2.401 (s, 3H), 7.254–7.312 (m, 5H), 7.47 (d, J = 8.8 Hz, 1H), 7.74 (d, J
= 7.2 Hz, 1H), 8.108 (s, 1H), 8.112 (s, D2O exchangeable, 2H), 9.301 (s,
1H); 13C NMR (100 MHz, DMSO‑d6) δ: 18.12, 113.62, 115.89, 122.01,
124.50, 125.41, 126.75, 127.03, 127.61, 128.14, 130.92, 140.99,
146.73, 147.52, 148.02, 173.26, 173.42, 178.79.
4.2.5.4. (2-amino-4-(dimethylamino)thiazol-5-yl)(6-methylimidazo[1,2-
a]pyridin-3-yl)methanone (10d). Final compound 10d was synthesized
as per the procedure described in 10a using starting materials 2-chloro-
1-(6-methylimidazo[1,2-a]pyridin-3-yl)ethanone 3d and 1-(bis(dime-
thylamino)methylene)-3-tritylthiourea 5c. 10d was purified by column
chromatography (Chloroform: Methanol = 9.7:0.3). Yellow solid; Yield:
79%; Purity: 99.9%; Melting point: 172–174 ◦C; Molecular formula:
C14H15N5OS; LC-ESI-MS (m/z): 302.1 [M+H]+; IR (KBr, cmꢀ 1):
3442.94, 3282.84, 3178.69, 1633.71; 1H NMR (400 MHz, DMSO‑d6) δ:
2.34 (s, 3H), 3.02 (s, 6H), 7.352 (d, J = 1.6 Hz, 1H), 7.6285 (d, J = 9.2
Hz, 1H), 7.931 (s, D2O exchangeable, 2H), 8.033 (s, 1H), 9.14 (d, J = 7.0
Hz, 1H); 13C NMR (100 MHz, DMSO‑d6) δ: 17.78, 41.72 (2C), 92.69,
116.5, 123.04, 125.07, 125.40, 130.12, 138.31, 145.61, 164.32, 169.93,
170.00.
4.2.5.9. (2-amino-4-methylthiazol-5-yl)(imidazo[1,2-a]pyridin-3-yl)
methanone (10i). Final compound 10i was synthesized as per the pro-
cedure described in 10a using starting materials 2-chloro-1-(imidazo
[1,2-a]pyridin-3-yl)ethanone 3a and N,N-diethyl-N′-(tritylcarbamo-
thioyl)acetimidamide 5b. 10i was purified by column chromatography
(Hexane: Ethyl acetate = 2.5:7.5). Colorless solid; Yield: 63%; Purity:
99.4%; Melting point: 183–185 ◦C; Molecular formula: C12H10N4OS; LC-
ESI-MS (m/z): 259.2 [M+H]+; IR (KBr, cmꢀ 1): 3483.44, 3238.48,
3091.89, 1654.92; 1H NMR (400 MHz, DMSO‑d6) δ: 2.469 (s, 3H),
7.220–7.297 (m, 2H), 7.599 (s, D2O exchangeable, 2H), 7.8255 (d, J =
7.6 Hz, 1H), 8.362 (s, 1H), 9.4535 (d, J = 8.4 Hz, 1H); 13C NMR (100
MHz, DMSO‑d6) δ: 18.60, 114.79 (2C), 117.37, 126.60, 127.49, 127.73,
128.24, 128.97, 141.27, 147.73, 159.22.
4.2.5.5. (2-amino-4-phenylthiazol-5-yl)(imidazo[1,2-a]pyridin-3-yl)meth-
anone (10e). Final compound 10e was synthesized as per the procedure
described in 10a using starting materials 2-chloro-1-(imidazo[1,2-a]
pyridin-3-yl)ethanone 3a and N,N-diethyl-N′-(tritylcarbamothioyl)ben-
zimidamide 5a. 10e was purified by column chromatography (Hexane:
Ethyl acetate = 4.5:5.5). Yellow solid; Yield: 67%; Purity: 98.9%;
Melting point: 187–189 ◦C; Molecular formula: C17H12N4OS; LC-ESI-MS
(m/z): 321.1 [M+H]+; IR (KBr, cmꢀ 1):3371.57, 3278.99, 3128.54,
1612.49; 1H NMR (400 MHz, DMSO‑d6) δ: 7.234 (t, J = 7.2 Hz, 4H), 7.47
(d, J = 6.8 Hz, 2H), 7.583 9 (t, J = 8 Hz, 1H), 7.71 (d, J = 8.8 Hz, 1H),
7.781 (s, 1H), 7.925 (s, D2O exchangeable, 2H), 9.37 (d, J = 6.8 Hz, 2H);
13C NMR (100 MHz, DMSO‑d6) δ: 113.21, 124.32, 126.61 (2C), 127.34,
128.12, 128.93 (2C), 131.30, 132.75, 134.92 (2C), 151.43 (2C), 152.15,
168.63, 174.54.
4.2.5.10. (2-amino-4-methylthiazol-5-yl)(8-methylimidazo[1,2-a]pyridin-
3-yl)methanone (10j). Final compound 10j was synthesized as per the
procedure described in 10a using starting materials 2-chloro-1-(8-
methylimidazo[1,2-a]pyridin-3-yl)ethanone 3b and N,N-diethyl-N′-
(tritylcarbamothioyl)acetimidamide 5b. 10j was purified by column
chromatography (Hexane: Ethyl acetate = 3:7). Colorless solid; Yield:
68%; Purity: 99.9%; Melting point: 179–181 ◦C; Molecular formula:
C13H12N4OS; LC-ESI-MS (m/z): 273.3 [M+H]+; HRMS (TOF) m/z calcd
for C13H12N4OS [M+H]+ 273.3256, found: 273.3248; IR (KBr, cmꢀ 1):
3284.77, 3046.28, 1691.57; 1H NMR (400 MHz, DMSO‑d6): δ 2.335 (s,
3H); δ 2.671 (s, 3H); δ 7.145 (t, J = 5.6 Hz, 1H); δ 7. 432 (d, J = 6.0 Hz,
1H); δ 7.906 (s, D2O exchangeable, 2H); δ 8.338 (s, 1H) δ 9.3 (d, J = 6.4
Hz, 1H); 13C NMR (100 MHz, DMSO‑d6) δ: 16.39, 18.60, 114.71, 116.16,
124.54, 125.90, 126.87, 127.54, 140.72, 147.05, 159.13, 170.46,
174.08.
4.2.5.6. (2-amino-4-phenylthiazol-5-yl)(8-methylimidazo[1,2-a]pyridin-
3-yl)methanone (10f). Final compound 10f was synthesized as per the
procedure described in 10a using starting materials 2-chloro-1-(8-
methylimidazo[1,2-a]pyridin-3-yl)ethanone 3b and N,N-diethyl-N′-
(tritylcarbamothioyl)benzimidamide 5a. 10e was purified by column
chromatography (Hexane: Ethyl acetate = 1:1). Pale Yellow solid; Yield:
64%; Purity: 99.4%; Melting point: 176–178 ◦C; Molecular formula:
C18H14N4OS; LC-ESI-MS (m/z): 335.3 [M+H]+; IR (KBr, cmꢀ 1):
3378.25, 3262.45, 3048.13, 1618.92; 1H NMR (400 MHz, DMSO‑d6) δ:
2.401 (s, 3H), 7.144–7.485 (m, 8H), 7.910 (d, J = 5.6 Hz, 2H), 9.255 (d,
J = 5.2 Hz, 1H), 13C NMR (100 MHz, DMSO‑d6) δ: 20.22, 113.32,
114.12, 124.42, 126.88, 127.45, 128.14, 129.07, 131.36, 132.46,
135.01, 151.41, 152.36, 156.53, 158.69, 159.15, 171.23, 177.54.
4.2.5.11. (2-amino-4-methylthiazol-5-yl)(7-methylimidazo[1,2-a]pyridin-
3-yl)methanone (10k). Final compound 10k was synthesized as per the
procedure described in 10a using starting materials 2-chloro-1-(7-
methylimidazo[1,2-a]pyridin-3-yl)ethanone 3c and N,N-diethyl-N′-(tri-
tylcarbamothioyl)acetimidamide 5b. 10k was purified by column
chromatography (Hexane: Ethyl acetate = 3:7). Pale yellow solid; Yield:
78%; Purity: 99.6%; Melting point: 176–178 ◦C; Molecular formula:
C13H12N4OS; LC-ESI-MS (m/z): 273.3 [M+H]+; IR (KBr, cmꢀ 1):
3477.66, 3265.49, 3082.25, 1691.57; 1H NMR (400 MHz, DMSO‑d6): δ
2.452 (s, 3H), δ 3.318 (s, 3H), δ 7.287 (d, J = 6.4 Hz, 1H), δ 7.593 (s, D2O
exchangeable, 2H), δ 7.847 (s, 1H), δ 8.288 (s, 1H), δ 9.3315 (d, J = 6.8
Hz, 1H); 13C NMR (100 MHz, DMSO‑d6) δ: 16.51, 19.42, 115.52, 117.33,
131.34, 132.48, 133.12, 135.91, 140.12, 150.52, 156.42, 170.52,
179.61.
4.2.5.7. (2-amino-4-phenylthiazol-5-yl)(7-methylimidazo[1,2-a]pyridin-
3-yl)methanone (10g). Final compound 10g was synthesized as per the
procedure described in 10a using starting materials 2-chloro-1-(7-
methylimidazo[1,2-a]pyridin-3-yl)ethanone 3c and N,N-diethyl-N′-(tri-
tylcarbamothioyl)benzimidamide 5a. 10g was purified by column
chromatography (Hexane: Ethyl acetate = 1:1). Yellow solid; Yield:
55%; Purity: 99.7%; Melting point: 171–173 ◦C; Molecular formula:
C
18H14N4OS; LC-ESI-MS (m/z): 335.3 [M+H]+; IR (KBr, cmꢀ 1):
3460.30, 3242.55, 3086.11, 1645.28; 1H NMR (400 MHz, DMSO‑d6) δ:
2.349 (s, 3H), 6.422 (d, J = 5.2 Hz, 1H), 7.231–7.560 (m, 5H), 7.921 (s,
D2O exchangeable, 2H), 8.342 (d, J = Hz, 2H), 9.37 (d, J = 6.8 Hz, 1H);
10

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Bioorganic & Medicinal Chemistry 36 (2021) 116091
4.2.5.12. (2-amino-4-methylthiazol-5-yl)(6-methylimidazo[1,2-a]pyridin-
3-yl)methanone (10l). Final compound 10l was synthesized as per the
procedure described in 10a using starting materials 2-chloro-1-(6-
methylimidazo[1,2-a]pyridin-3-yl)ethanone 3d and N,N-diethyl-N′-
(tritylcarbamothioyl)acetimidamide 5b. 10l was purified by column
chromatography (Hexane: Ethyl acetate = 3:7). Pale yellow solid; Yield:
59%; Purity: 99.8%; Melting point: 170–172 ◦C; Molecular formula:
C12H10N4OS; LC-ESI-MS (m/z): 259.2 [M+H]+; IR (KBr, cmꢀ 1):
3383.14, 3302.13, 3089.96, 1627.92; 1H NMR (400 MHz, DMSO‑d6): δ
2.378 (s, 3H); δ 7.468 (d, J = 8.8 Hz, 1H); δ 7.7385 (d, J = 9.2 Hz, 1H); δ
8.045(s, 1H), 8.075 (s, D2O exchangeable, 2H); δ 8.505 (s, 1H) δ 9.262
(s,1H); 13C NMR (100 MHz, DMSO‑d6) δ: 17.78, 124.53, 125.57, 126.68,
127.06, 127.53, 127.66, 131.84, 146.70, 147.46, 147.56, 173.52.
C
13H12N4OS; LC-ESI-MS (m/z): 273.3 [M+H]+; IR (KBr, cmꢀ 1):
3382.15, 3254.91, 3045.22, 1646.88; 1H NMR (400 MHz, DMSO‑d6): δ
2.384 (s, 3H), δ 2.454 (s, 3H), δ 7.4675 (d, J = 9.2 Hz, 1H), δ 7.7165 (d, J
= 9.2 Hz, 1H), δ 7.877 (s, D2O exchangeable, 2H), δ 8.299 (s, 1H), δ
9.276 (s, 1H); 13C NMR (100 MHz, DMSO‑d6) δ: 16.42, 18.46, 115.22,
117.34, 123.96, 126.01, 126.92, 127.46, 141.05, 148.42, 160.11,
170.52, 174.16.
4.3. Animals
In vivo work was carried out using Sprague-Dawley rats (either sex,
150–200 g) (3 animals per cage). Animals were kept in animal house of
B. V. Patel PERD Centre under standard conditions (Relative humidity:
60 ± 5%, Temperature: 25 ± 3 ◦C, 10% air exhaust conditioning unit, 12
h light/dark cycle). Good Laboratory Practice (GLP) mentioned in
CPCSEA guidelines were followed for housing and handling of animals.
All the experiments were performed after the approval of Institutional
Animal Ethics Committee (Approval No: PERD/IAEC/2016/049 &
PERD/IAEC/2016/050).
4.2.5.13. (2-aminothiazol-5-yl)(imidazo[1,2-a]pyridin-3-yl)methanone
(10m). Final compound 10m was synthesized as per the procedure
described in 10a using starting materials 2-chloro-1-(imidazo[1,2-a]
pyridin-3-yl)ethanone 3a and N,N-dimethyl-N′-(tritylcarbamothioyl)
formimidamide 8. 10m was purified by column chromatography
(Hexane: Ethyl acetate = 1.5:8.5). Pale yellow solid; Yield: 62%; Purity:
99.1%; Melting point: 190–192 ◦C; Molecular formula: C11H8N4OS; LC-
ESI-MS (m/z): 245.2 [M+H]+; IR (KBr, cmꢀ 1): 3477.66, 3383.14,
4.4. Anti-inflammatory activity
1
3082.25, 1687.71; H NMR (400 MHz, DMSO‑d6): δ 7.222–7.303 (m,
4.4.1. Carrageenan induced rat paw edema model for acute inflammation
A series of 16 compounds were tested for their acute anti-
inflammatory activity using carrageenan induced rat paw edema
model. Animals were randomly grouped in to 19 groups (n = 6). These
groups were denoted as normal control (NC), disease control (DC),
positive control (PC) and 16 test groups (10a-10p). Test group (50 mg/
kg body weight) and PC group (Diclofenac: 7.5 mg/kg body weight)
animals were orally pre-dosed using a suspension of 0.2% agar. NC and
DC group animals were received 0.2% agar only. Rat paw edema was
induced by 0.1 mL of 1% carrageenan, prepared in normal saline (sub-
cutaneously). The injection was given after 1 h of oral dosing into the left
hind paw (sub plantar region) of each rat. Paw volumes were measured
using mercury plethesmometer before and after 3 h of carrageenan
injection.26–28 The reduction in volume of paw edema was calculated as
percentage (%) edema inhibition using following equation.
4H); δ 7.598 (s, 1H); δ 8.093 (d, J = 12.4 Hz, 1H); δ 8.571 (s, 1H); δ
9.4325 (d, J = 5.6 Hz, 1H); 13C NMR (100 MHz, DMSO‑d6) δ: 117.37,
122.21, 126.60, 127.01, 127.49, 127.73, 128.01, 128.87, 141.28,
147.73, 173.71.
4.2.5.14. (2-aminothiazol-5-yl)(8-methylimidazo[1,2-a]pyridin-3-yl)
methanone (10n). Final compound 10n was synthesized as per the
procedure described in 10a using starting materials 2-chloro-1-(8-
methylimidazo[1,2-a]pyridin-3-yl)ethanone 3b and N,N-dimethyl-N′-
(tritylcarbamothioyl)formimidamide 8. 10n was purified by column
chromatography (Hexane: Ethyl acetate = 2:8). Pale yellow solid; Yield:
60%; Purity: 99.5%; Melting point: 194–196 ◦C; Molecular formula:
C
12H10N4OS; LC-ESI-MS (m/z): 259.2 [M+H]+; IR (KBr, cmꢀ 1):
3493.09, 3373.50, 3064.80, 1664.57; 1H NMR (400 MHz, DMSO‑d6): δ
2.580 (s, 3H); δ 7.128 (t, J = 6.8 Hz, 1H); δ 7.4205 (d, J = 6.8 Hz, 1H); δ
8.054 (s, 1H), 8.090 (s, D2O exchangeable, 2H); δ 8.526 (s, 1H) δ 9.2735
(d, J = 6.8 Hz, 1H); 13C NMR (100 MHz, DMSO‑d6) δ: 16.40, 114.74,
122.68, 125.68, 126.89, 127.07, 127.45, 140.71, 147.64, 147.87,
173.41, 173.66.
% edem a inhibition = (Vt/V0)DC – (Vt/V0)treated/(Vt/V0)DC × 100
Vt = rat paw volum e at 3rd hour
V0 = rat paw volum e at 0th hour
The results of % edema inhibition are presented as mean ± SEM (n =
6 animals/group). The statistical analysis was performed by one-way
ANOVA followed by Tukey’s multiple comparison test (*p < 0.05).
4.2.5.15. (2-aminothiazol-5-yl)(7-methylimidazo[1,2-a]pyridin-3-yl)
methanone (10o). Final compound 10o was synthesized as per the
procedure described in 10a using starting materials 2-chloro-1-(7-
methylimidazo[1,2-a]pyridin-3-yl)ethanone 3c and N,N-dimethyl-N′-
(tritylcarbamothioyl)formimidamide 8. 10o was purified by column
chromatography (Hexane: Ethyl acetate = 2:8). Colorless solid; Yield:
77%; Purity: 99.9%; Melting point: 196–198 ◦C; Molecular formula:
4.4.2. Formalin induced rat paw edema model for chronic inflammation
Chronic anti-inflammatory study was carried out using six groups
NC, DC, PC and 3 test groups (n = 6). Animals were pre dosed with test
compounds (50 mg/kg body weight), celecoxib (positive control, 40
mg/kg body weight) and vehicle (normal control) prepared in 0.2%
agar. After 1 h of oral dosing, 0.1 mL of 2% formalin, prepared in normal
saline was injected in the sub plantar region of rat’s left hind paw
(subcutaneously) for consecutive 5 days. After 5 h, the changes in paw
volume were recorded using mercury plethesmometer for each day.26–28
% edema reduction was calculated using following equation.
C
12H10N4OS; LC-ESI-MS (m/z): 259.2 [M+H]+; IR (KBr, cmꢀ 1):
3432.51, 3342.87, 3085.44, 1664.75; 1H NMR (400 MHz, DMSO‑d6): δ
2.472 (s, 3H), δ 7.136 (d, J = 7.2 Hz, 1H), δ 7.481 (s, 1H), δ 7.614 (s, 1H),
δ 8.038 (s, D2O exchangeable, 2H), 8.575 (s, 1H), δ 9.313 (d, J = 6.4 Hz,
1H); 13C NMR (100 MHz, DMSO‑d6) δ: 18.42, 115.01, 122.74, 124.97,
125.78, 127.12, 127.93, 141.02, 148.22, 149.31, 171.23, 178.58.
% edem a inhibition = (Vt/V0)DC – (Vt/V0)treated/(Vt/V0)DC × 100
Vt = rat paw volum e at 5th hour
4.2.5.16. (2-aminothiazol-5-yl)(6-methylimidazo[1,2-a]pyridin-3-yl)
methanone (10p). Final compound 10p was synthesized as per the
procedure described in 10a using starting materials 2-chloro-1-(6-
methylimidazo[1,2-a]pyridin-3-yl)ethanone 3d and N,N-dimethyl-N′-
(tritylcarbamothioyl)formimidamide 8. 10p was purified by column
chromatography (Hexane: Ethyl acetate = 2:8). Colorless solid; Yield:
67%; Purity: 99.9%; Melting point: 202–204 ◦C; Molecular formula:
V0 = rat paw volum e at 0th hour
The results of % edema inhibition are presented as mean ± SEM (n =
6 animals/group). The statistical analysis was performed by one-way
ANOVA followed by Tukey’s multiple comparison test (*p < 0.05).
11

S.R. Sagar et al.
Bioorganic & Medicinal Chemistry 36 (2021) 116091
4.5. AlCl3 induced Alzheimer’s disease model
calculated as SAP. SAP was measured as total number of entries minus
two. % alteration was measured as (actual correct alteration
÷
Animals were divided in to 4 groups NC, DC, PC and test group (n =
6). Animals were predosed with normal saline (for NC and DC),
Meloxicam (5 mg/kg body weight, for PC) and test compound (10c, 50
mg/kg body weight) using 0.2% agar suspension for 28 days. DC, PC and
test group animals were injected with aluminium chloride (4.2 mg/kg, i.
p.) for 28 days, after oral dosing. NC group received vehicle only.31,32
Various studies were performed during this 28 days as described in
following procedures.
maximum alteration) × 100.23 Rats were trained for consecutive 5 days
and % alteration was calculated at 7th, 14th, 21st and 28th day of AlCl3
model.27,28 Data are given as mean % alteration ± SEM and statistical
analysis was performed by two-way ANOVA test followed by multiple
comparisons of Tukey’s test. Statistical significance was measured as *p
< 0.05 compared to NC and as #p < 0.05 compared to DC.
4.5.2. Haematological parameters estimation
After 28 days of AlCl3 treatment, blood samples were collected from
the retro orbital sinus of rats on 29th day. For haematological studies,
rats were anaesthetized by isoflurane and blood samples were collected
in 1 mL heparinized micro centrifuge tubes. Haemoglobin and haema-
tocrit level were determined using automated haematology analyser
(VetScan HM-5; Abaxis Inc., Union City, CA, USA).29
4.5.1. Behavioral study
Training was given to each animal prior to the AlCl3 dosing for 5
days. Afterwards, behavioral parameters were observed during 28 days
of AlCl3 induced AD model using elevated plus maze, pole climbing
apparatus and Y maze.
4.5.3. Serum biochemical assays
4.5.1.1. Elevated plus maze test. Spatial working memory of rats can be
assessed by elevated plus maze test (EPM). It is a natural tendency of rats
to be in the safe closed environment rather than an open heighted
environment. In this test, rat’s ability to remember closed arm location
was determined. EPM was made up of two open arms (29 × 5 cm), two
closed arms (29 × 5 × 15 cm) and a central platform (5 × 5 cm). This
apparatus is placed at the height of 40 cm from the floor. Training ses-
sion was given for 5 days. During this session, each rat was placed in an
open maze away from the central platform and allowed to explore the
maze environment for 5 min. The time required for rat to move from an
open arm to close arm was recorded as transfer latency.21 Same pro-
cedure was employed during retention session at 7th, 14th, 21st and
28th day of AlCl3 model. Transfer latency (in seconds) was recorded for
each rat.27,28 Data are given as mean transfer latency (sec) ± SEM and
statistical analysis was performed by two-way ANOVA test followed by
multiple comparisons of Tukey’s test. Statistical significance was
measured as *p < 0.05 compared to NC and as #p < 0.05 compared to
DC.
Rats were anaesthetized using isoflurane and blood samples were
collected in 1 mL non-heparinized micro centrifuge tubes from retro
orbital sinus. Serum was collected from the blood samples. Biochemical
parameters, % albumin and total protein was determined by automated
analyser (Transasia EM 360).29
4.5.4. Anti-oxidant activity
After 28 days of AlCl3 model, on 29th day all the rats were eutha-
nized and intact brain was removed. Brains were washed with saline to
remove traces of blood. Estimation of lipid peroxidase and superoxide
dismutase assays were performed using brain tissues.
4.5.4.1. Lipid peroxidase (LPO) assay. Brain tissues obtained from each
group were taken into 5 mL of Hank’s balanced salt solution (HBSS, pH
7.4). These tissues were homogenized at 5000 rpm of 3 cycles (30 sec of
each cycle) using Polytron homogenizer (Kinematica, Switzerland).
Further, homogenates were centrifuged at 35,000 rpm for 10 min using
sorvall, legend X1R centrifuge. After that, supernatant was discarded
from each sample and pellet was re-suspended in 0.1 mL of HBSS for
further use. LPO activity was determined based on the reaction of
malonaldehyde (MDA): thiobarbituric acid (TBA). The previously pro-
cessed tissue homogenate was taken to initiate the reaction. The assay
reaction also contained 2 mL of 8.1% of sodium dodecyl sulfate, 1.5 mL
of 20% acetic acid (pH 3.5 was adjusted using 1 M NaOH), and TBA (1.5
mL of 0.8% aqueous solution). The final assay volume was made up to 4
mL by addition of 0.7 mL double distilled water. Then, reaction mixture
was heated for 1 h at 95 ◦C in a water bath. After heating, double
distilled water (1 mL) and 15:1 v/v n-butanol: pyridine mixture (5 mL)
was added and vortexed for 5 min. After that, reaction mixture was
centrifuged at 3000 rpm for 7 min and supernatant (organic) was taken
and processed further for MDA calculation. MDA formed in reaction
mixture was measured using an Ultra violet/Visible Spectrophotometer
(Shimadzu UV-1800) at 532 nm. LPO was calculated using MDA
extinction coefficient (1.45 × 10ꢀ 5/min/cm).8,29
4.5.1.2. Conditioned avoidance response (CAR) rat model. Cognitive
function of rats was determined using Cook’s pole climbing apparatus. A
wooden sound proof apparatus with 25 × 25 × 25 cm dimensions was
used for the study. The chamber has metallic grid floor that conduct
electric shock. A wooden pole of diameter 2.5 cm was placed on the
center of the apparatus. Initially, an animal was placed and allowed to
explore the environment of the apparatus for 45 sec. Conditioned stim-
ulus (buzzer signal) and unconditioned stimulus (electric shock) was
given through grid floor for 45 sec. All the animals were trained before
the actual experiment.22 Acquisition session was given to each animal
for 3 days (1st day 5 trials, 2nd day 3 trials and 3rd day 1 trial). During
the training session, animals learned to escape from the electric shock by
climbing to wooden pole after buzzer signal. Afterwards, 1 trial of CAR
was noted for each animal during 7th, 14th, 21st and 28th day of AlCl3
model.27,28 Data are represented as mean ± SEM and statistical analysis
was performed by two-way ANOVA test followed by multiple compar-
isons of Tukey’s test. Statistical significance was measured as *p < 0.05
compared to NC and #p < 0.05 compared to DC.
LPO was calculated using following formula:
LPO = [(Sam ple-Blank)*145]*10ꢀ 5/weight of organ (gram s)
4.5.1.3. Y maze test. Y-maze test was used to assess immediate working
memory of rats. It is a natural tendency of rodent’s to explore novel
environment. This test emphasis on this tendency of rodents and
calculated in terms of ‘simultaneous alteration performance’ (SAP). In
this test, Y-maze apparatus was used which was made up of 3 identical
wooden arms (40 × 9 × 16 cm) denoted as ‘A’, ‘B’, and ‘C’. These arms
were placed at an angle of 120◦ with respect to each other. A rat was
placed in one of the arm and allowed it to explore the environment of
maze for 5 min. Rats generally chose to visit unexplored arm over pre-
viously visited arm. Arm entries visited by each rat were recorded and
4.5.4.2. Superoxide dismutase (SOD) assay. SOD activity was measured
using brain tissues. Tissues were taken in 2 mL of chilled 50 mM Tris
buffer (pH 8.2 was adjusted by 2 mM EDTA). Afterwards, tissues were
homogenized using 3 cycles of 30 s. Tissue homogenate was treated with
1 mL of 0.1% Triton X 100 (v/v) at 4 ◦C for 30 min. Further, reaction
◦
mixture was centrifuged at 5600 rpm for 30 min (temperature: 4 C).
Supernatant was taken and divided into two parts. One part of super-
natant was stored at 4–8 ◦C and other part was heated at 95 ◦C for 1 h.
12

S.R. Sagar et al.
Bioorganic & Medicinal Chemistry 36 (2021) 116091
Pyrogallol was added to each reaction to determine SOD activity.
Absorbance was recorded on 0th and 9th min of pyrogallol addition at
420 nm. All the calculations were made in terms of per gram fresh tissue
weight.30,32 SOD enzyme activity was calculated by following equation:
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14