Discovery of imidazopyridines containing isoindoline-1,3-dione framework as a new class of BACE1 inhibitors: Design, synthesis and SAR analysis

Article abstract of DOI:10.1016/j.ejmech.2017.06.040

Alzheimer's disease is characterized by chronic neurodegeneration leading to dementia. The main cause of neurodegeneration is considered to be the accumulation of amyloid-β. Inhibiting BACE1 is a well-studied approach to lower the burden of amyloid-β aggregates. We designed a series of imidazopyridines-based compounds bearing phthalimide moieties as inhibitors of BACE1. The compounds 8a-o were synthesized by the Groebke–Blackburn–Bienaymé three-component reaction of heteroaromatic amidines, aldehydes and isocyanides. Evaluating the BACE1 inhibitory effects of the synthesized compounds revealed that introducing an aminocyclohexyl moiety in the imidazopyridine core resulted in a significant improvement in its BACE1 inhibitory potential. In this regard, compound 8e was the most potent against BACE1 with an IC₅₀ value of 2.84 (±0.95) μM. Molecular docking revealed that the nitrogen atom of imidazopyridines and the oxygen atom of the phenoxypropyl linker were involved in hydrogen bound interactions with Asp228 and Asp32 of BACE1 active site, respectively. The phthalimide moiety oriented toward the flap pocket and interacted with phe108, lle110, Trp115, Ile118 through van der Waal's and hydrophobic interactions. These findings demonstrate that imidazopyridines-based compounds bearing phthalimide moiety have the potential to decrease amyloid-β levels and ameliorate the symptoms of Alzheimer's disease.

Full text of DOI:10.1016/j.ejmech.2017.06.040

Accepted Manuscript
Discovery of imidazopyridines containing isoindoline-1,3-dione framework as a new
class of BACE1 inhibitors: Design, synthesis and SAR analysis
Sara Azimi, Afsaneh Zonouzi, Omidreza Firuzi, Aida Iraji, Mina Saeedi, Mohammad
Mahdavi, Najmeh Edraki
PII:
S0223-5234(17)30486-5
10.1016/j.ejmech.2017.06.040
EJMECH 9535
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 March 2017
Revised Date: 31 May 2017
Accepted Date: 22 June 2017
Please cite this article as: S. Azimi, A. Zonouzi, O. Firuzi, A. Iraji, M. Saeedi, M. Mahdavi, N. Edraki,
Discovery of imidazopyridines containing isoindoline-1,3-dione framework as a new class of BACE1
inhibitors: Design, synthesis and SAR analysis, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.06.040.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of Imidazopyridines Containing Isoindoline-1,3-dione Framework as a New
Class of BACE1 Inhibitors: Design, Synthesis and SAR Analysis
Sara Azimia, Afsaneh Zonouzia, Omidreza Firuzib, Aida Irajib, Mina Saeedic,d, Mohammad
Mahdavie*, Najmeh Edrakib*
Dedicated to Professor Abbas Shafiee (1937-2016) for his lifetime achievement in
pharmaceutical sciences research and education
a
School of Chemistry, College of Science, University of Tehran, PO Box 14155-6455,
Tehran, Iran
b Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran.
c
Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
d
Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences,
Tehran, Iran
e
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical
Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran.
Corresponding authors:
Najmeh Edraki, PhD
Medicinal and Natural Products Chemistry Research Center, Shiraz university of Medical
Sciences, Shiraz, Iran
Tel: +98 713 230 3872; Fax: +98 713 230 2225
E-mail: edrakin@sums.ac.ir, najmeh_edraki@yahoo.com
Mohammad Mahammad Mahdavi, PhD
Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical
Sciences, Tehran, Iran.
E-mail: mahdavi.chem@gmail.com
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Abstract
Alzheimer’s disease is characterized by chronic neurodegeneration leading to dementia. The
main cause of neurodegeneration is considered to be the accumulation of amyloid-β.
Inhibiting BACE1 is a well-studied approach to lower the burden of amyloid-β aggregates.
We designed a series of imidazopyridines-based compounds bearing phthalimide moieties as
inhibitors of BACE1. The compounds 8a-o were synthesized by the Groebke–Blackburn–
Bienaymé three-component reaction of heteroaromatic amidines, aldehydes and isocyanides.
Evaluating the BACE1 inhibitory effects of the synthesized compounds revealed that
introducing an aminocyclohexyl moiety in the imidazopyridine core resulted in a significant
improvement in its BACE1 inhibitory potential. In this regard, compound 8e was the most
potent against BACE1 with an IC50 value of 2.84 (±0.95) µM. Molecular docking revealed
that the nitrogen atom of imidazopyridines and the oxygen atom of the phenoxypropyl linker
were involved in hydrogen bound interactions with Asp228 and Asp32 of BACE1 active site,
respectively. The phthalimide moiety oriented toward the flap pocket and interacted with
phe108, lle110, Trp115, Ile118 through van der Waal’s and hydrophobic interactions. These
findings demonstrate that imidazopyridines-based compounds bearing phthalimide moiety
have the potential to decrease amyloid-β levels and ameliorate the symptoms of Alzheimer’s
disease.
Keywords: Alzheimer’s disease, β-secretase inhibitor, Groebke–Blackburn–Bienayme
reaction, imidazopyridines, phthalimide, molecular docking
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1. Introduction
Alzheimer’s disease is a neurodegenerative disorder and the most common cause of dementia
in the elderly [1]. The pathogenesis of the disease is often described by the “Amyloid
Cascade Hypothesis”(ACH) [2], which suggests that the deposition of amyloid beta (Aβ) is
the first pathological event that causes neuronal death and eventually leads to dementia. Two
proteases known as β- and γ-secretase endoproteolyze the amyloid precursor protein (APP) to
produce the Aβ peptide. β-Secretase is active in most tissues of the body [3, 4]; however, β-
site amyloid precursor protein cleaving enzyme-1 (BACE1) is the major β-secretase in the
CNS compared to BACE2 (close homolog of BACE1), which has a more widespread
expression pattern? [5]. BACE1 activity is increased in the brains of patients with sporadic
Alzheimer’s disease [6]. Consequently, BACE1 inhibitors have emerged as ideal candidates
for the treatment of Alzheimer’s disease by preventing Aβ accumulation and aggregation [7-
9]. The first generation of BACE1 inhibitors were designed based on peptide analogs of APP
[10]. Although they have shown high in vitro inhibitory activity, unfavorable in vivo
pharmacological properties were observed due to low blood-brain barrier (BBB) permeability
or oral bioavailability. Hence, developing non-peptidic BACE1 inhibitors is of particular
interest.
Among various non-peptidic scaffolds, amidine- or guanidine-containing heterocycles were
found to be suitable inhibitors due to the formation of a hydrogen-bond network with the
catalytic aspartyl dyad of BACE1 [11-13]. Recently, Merck has introduced a guanidine-based
drug for Alzheimer's disease known as Verubecestat (MK-8931) curently in phase II/III trials
[14]. Verubecestat is a potent inhibitor of BACE1 with an IC50 value of 0.4 nM [15]. The X-
ray cocrystal structure confirmed hydrogen-binding interactions between the amidine moiety
and the BACE1 catalytic dyad. High-affinity binding toward the relatively hydrophobic S1
and S3 subsites results from a diaryl amide substituent that occupies the above-mentioned
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subsites in BACE1. MK-8931 has favorable physicochemical properties including stability at
physiological pH, oral absorption, high cellular permeability and high BBB penetration [15].
The results suggest that the presence of pyridine and guanidine moieties results in satisfactory
curative effects. More recently, Al-Tel et al. introduced imidazopyridines with structure A
(Figure 1) as novel β-secretase inhibitors [16]. Three derivatives of compound A showed IC50
values of 5.51, 2.48, and 2.24 µM where X was replaced with hydrogen, fluorine, and
methoxy, respectively.
In addition, other studies reported isoindoline-1,3-dione (phthalimide) derivatives (compound
C, Figure 1) as cholinesterase and Aβ aggregation inhibitors with neuroprotective effects.
These compounds did not show cytotoxicity and had therapeutic potential for treating
Alzheimer’s disease [17]. Our previous study on phenyliminochromene carboxamide
derivatives bearing bromophenyl piperazine moieties (compound B, Figure 1) suggested that
the introduction of an isoindoline-1,3-dione moiety into the piperazine pendant results in a
significant improvement in BACE1 inhibitory activity (IC50= 0.098 µM) and suppression of
Aβ production in N2a-APPswe cells (% Inhibition of Aβ1–40 production= 39.4 at 10µM).
Phthalimide moiety could be considered as a non-peptidyl framework with low-molecular
weight involved in hydrophobic interaction with hydrophobic residue of the S2 sub-pocket of
active site and the network of hydrogen bonding interactions with Arg235, Thr23 and
Gly230. This observation might partially explain the significant inhibitory potential resulted
from the incorporation of phthalimide moiety into the BACE1 inhibitor scaffold [18].
As part of our ongoing research to design and synthesize novel anti-Alzheimer’s agents, [19-
21] we focused on an imidazopyridine core as a promising scaffold for inhibiting BACE1.
To this end, we employed molecular hybridization and bioisosterism replacement approaches
to identify novel non-peptidic inhibitors with aspartyl binding motifs as new entities for the
inhibition of this enzyme (Figure 1). Our design is based on bioisosteric replacement of the
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phenyl linker with a phenoxypropyl one (compound A, Figure 1) to increase the flexibility of
the linker and to allow the inhibitor to properly access and orient itself within the active site
of BACE1. Different secondary amines were incorporated into the structure to investigate the
importance of substituted groups at the 3-position of the imidazopyridines. Our pervious
results [18] prompted us to replace the benzimidazole group of compound A with a
phthalimide pendant to improve the anti-BACE1 effects and enhancing the accessibility to
the S2-sub pocket of the active site [18].
Figure 1. Chemical structure of previously reported BACE1 inhibitors; imidazopyridines A,
isoindoline-1,3-dione (phthalimide) containing derivative B, cholinesterase inhibitor with
neuroprotective effect C and our newly hybridized imidazopyridine-based derivatives as
potential novel anti-Alzheimer agents.
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2. Results and discussion
2.1. Chemistry
The synthetic procedure for the preparation of imidazopyridines bearing phthalimide moiety
8 is depicted in Scheme 1. Reaction of phthalimide 1 and 1,2-dibromopropane 2 in the
presence of K2CO3 in refluxing acetone gave 2-(2-bromopropyl)isoindoline-1,3-dione 3.
Next, the reaction of compound 3 and 4-hydroxyaldehyde derivative 4 in the presence of
K2CO3 in DMF at 80 °C gave the desired aldehyde 5.
The target compounds were prepared through the reaction of aldehyde 5, 2-aminopyridins 6,
and isocyanides 7 in refluxing toluene in the presence of ammonium chloride (NH4Cl
Scheme 1. Synthesis of imidazopyridines bearing phthalimide moiety 8a-o.
2.2. Determination of BACE1 inhibition
BACE1 inhibitory effects of the synthesized imidazopyridines bearing a phthalimide moiety
via the phenoxypropyl linker were determined using a FRET-based assay kit. The
concentration of compounds that produced 50% maximum inhibition of BACE1 activity
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(IC50) and enzyme inhibition percentages at 50 and 10 µM concentrations of the test
compounds were assessed and are summarized in Table 1. Experiments were repeated three
to four times for each derivative and mean percent of enzymatic inhibition at 50 and 10 µM
were used for comparing the potencies of the test compounds. Compounds 8e and 8c bearing
amino cyclohexyel pendents and a methyl substitute on imidazo pyridine core were the most
potent derivatives against BACE1 (IC50 = 2.84 µM and 5.93 µM; respectively). Investigation
of the structure activity relationship of synthesized derivatives resulted in the following
observations:
-
Assesment of aminoalkyl substitute (NHR2) at imidazopyridine core:
Aminocyclohexyl containing derivatives (R2=cyclohexyl) were more potent than their amino
t-butyl counterparts in most cases. Compound 8i bearing methoxy, amino cyclohexyl and 6-
chloro moieties at R1, R2 and R3 respectively, demonstrated a higher BACE1 inhibitory
potential (inhibition at 50 µM = 88.10%) over its amino t-butyl containing counterpart 8k
(inhibition at 50 µM = 37.90%). Similar results were observed in the case of the
aminocyclohexyl derivative 8e (R1=H and R3 =7-CH3, 100% BACE1 inhibition at 50 µM;
IC50= 2.84 µM) and its amino t-butyl counterpart (inhibition at 50 µM = 18.32%). These
findings indicate the important influence of the amino cyclohexyl moiety on BACE1
inhibition. Furthermore, the inappropriate orientation of t-butyl derivatives may hinder access
and prevent interaction between important functional groups of the ligand and the catalytic
residues of the enzyme’s active site.
-
Investigation of substituted moiety (R3) on amino cyclohexyl imidazopyridine
core.
The effect of type of substituted group R3 on the aminocyclohexyl imidazopyridine core
suggested that the introduction of methyl substitutes (R3=CH3) at different positions of
imidazopyridine ring increases the inhibitory potency against BACE1 and the preference
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order for the position of methyl substitute is 7 > 5 > 6,8. The most potent compound of this
series, 7-methylimidazopyridine derivative 8e (100% inhibition at 50 µM) demonstrated
superior potency over its 5-methylimidazopyridine (8c), 6-methylimidazopyridine (8d) and 8-
methylimidazopyridine (8g) counterparts with 87.94, 63.70 and 69.43 % BACE1 inhibition at
50 µM, respectively. Furthermore, the introduction of a small lipophilic electron-donating
moiety such as a methyl group into the 3-aminocyclohexyl imidazopyridine derivatives
increased the potency of compounds against BACE1. Finally, no significant difference in
potency was observed between bromine and chlorine derivatives.
-
Effect of substituted group R1 into the phenoxy propyl linker
Introduction of a methoxy substitute into the phenoxypropyl linker of the designed scaffold
did not significantly alter the potency of compounds against BACE1. The exception was in
the case of the amino cyclohexyl imidazopyridine bearing chlorine group at C6 position of
imidazopyridine ring and methoxy group in linker part (compound 8i); 8i was four times
more potent than its non-methoxylated counterpart in inhibiting BACE1 at 50µM (inhibition
was 88.10 and 19.6%, respectively). We attribute this to the structural and conformational
changes imposed by the spatial hindrance between the methoxy and cyclohexyl substitutes
resulting in proper orientation of functional groups into the key residues and pockets of
BACE1 active site.
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Table 1. BACE1 inhibitory activity of synthesized compounds 8a-o.
N
N
R2
NH
O
R1
N
O
O
% Inhibition at
% Inhibition at
10 µM
Compounds
R1
R2
R3
50 µM
IC50 (µM)
8a
8b
8c
8d
8e
8f
H
OCH3
H
cyclohexyl
cyclohexyl
-
-
<10
<10
-
69.49(±7.52)
87.94(±6.66)
63.70(±5.26)
100
29.30(±6.12)
61.54 (±7.16)
37.61(±6.62)
61.32(±6.14)
24.24(±8.33)
44.42(±5.04)
11.50(±13.19)
26.86(±8.82)
17.67 (±11.01)
19.41(±5.85)
32.16(±10.05)
36.71(±2.71)
7.87(±0.02)
-
cyclohexyl 5-CH3
cyclohexyl 6-CH3
cyclohexyl 7-CH3
5.93 (±1.93)
29.42 (±8.98)
2.84 (±0.95)
-
H
H
H
t-Butyl
7-CH3
18.32(±10.89)
69.43(±8.71)
19.60(±6.86)
88.097(±7.53)
32.95(±11.30)
37.90(±1.38)
45.30(±5.83)
43.17(±1.18)
34.28(±9.20)
8g
8h
8i
H
cyclohexyl 8-CH3
H
cyclohexyl
cyclohexyl
t-Butyl
6-Cl
6-Cl
6-Cl
6-Cl
6-Br
6-Br
6-Br
-
OCH3
H
14.90(±8.98)
8j
8k
8l
OCH3
H
t-Butyl
-
-
-
-
cyclohexyl
cyclohexyl
t-Butyl
8m
8n
OCH3
H
8o
OCH3
t-Butyl
6-Br
48.72(±12.23)
33.19(±11.12)
-
OM99-2
-
-
-
-
-
0.014 (±0.0028)
Values represent mean (± standard error of mean (S.E.M.) of three or four independent experiments.
2.3. Molecular docking study
We performed molecular docking to better understand the Structure-activity relationship
(SAR) of all of the synthesized compounds. To validate and optimize our docking protocol,
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redocking of the co-crystallized conformation of a native ligand into the 4ACU active site
was performed [22]. The binding pose of the top rank cognate ligand was superimposed over
the X-ray crystallographic structure. The best-docked and the experimental conformations of
the inhibitor correlate well with an RMSD of 0.601 Å and binding free energies of −12.934
kcal/mol. This validated docking procedure was performed on all the compounds under
investigation. Estimated free binding energies (∆Gb) and Ki for synthesized molecules are
summarized in Table 2. All the ligand/receptor interactions were analyzed using Chimera
1.11 and Viewer lite 4.2 software and following results were obtained:
- In almost all docked structures containing an amino cyclohexyl pendent at R2 and a
different motif at R3 (compounds 8a, 8b, 8c, 8d, 8e and 8i) a key hydrogen bond
interaction between the nitrogen atom of imidazopyridine and Asp228 of the catalytic
dyad residues was observed. The Cyclohexyl moiety in these structures occupies the
P'2 pocket and phthalimide oriented toward flap pocket. The substituted
imidazopyridines was surrounded by Thr231, Thr232, and Asn233.
- As mentioned previously, replacing the cyclohexyl pendant with a t-butyl group
reduced the inhibitory activity of the compounds. To investigate this observation, we
performed computational analysis on of one of the most potent compounds, compound
8e, (aminocyclohexyl derivative) and its amino t-butyl counterpart 8f. As it is depicted
in Figure 2, 8e and 8f demonstrated different binding orientations in the active site of
BACE1. As it is shown, the phthalimide moiety of 8e was directed toward the flap
pocket of the active site surrounded by Phe108, Phe109 and Ileu118. Furthermore, the
methyl imidazopyridine core was oriented toward the P2 pocket and amino cyclohexyl
pendant occupied the P'2 pocket surrounded by Thr231, Thr232, Asn233 and Arg235.
Moreover, Asp32 and Asp228 are involved in H-bond interaction with the oxygen atom
of phenoxypropyl and the nitrogen atom of the imidazopyridine, respectively (Table 2).
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Surprisingly, the amino t-butyl derivative 8f, is inversely directed in the active site; the
phthalimide is oriented toward P'2 pocket, amino t-butyl pendant and imidazopyridine
are oriented toward P2 and flap pockets, respectively. Such an orientation partly
hindered H-bond interaction of 8c with key catalytic Asp32 and partly justifies its poor
BACE1 inhibitory potential. We conclude that the introduction of an amino t-butyl
pendant into the imidazopyridine core imposes an unfavorable binding mode that
hinders proper ligand-receptor interaction.
– The results indicate that the introduction of a methyl substitute (representing a small
electron donating group) into the imidazopyridine ring resulted in additional
hydrophobic interactions with the hydrophobic residues of P2 sub-pocket, therefore
increasing the BACE1 inhibitory activity as observed in the case of compounds 8c, 8d,
8e and 8g. Based on the above findings amino cyclohexyl derivatives 8e and 8c
containing 7-CH3 and 5-CH3 on imidazopyridine core, respectively, were the most
potent inhibitors against BACE1 and demonstrated a favorable orientation and H-bond
interactions with key residues of catalytic site. As depicted in Figure 3, 8e was involved
in hydrogen bond network with Asp32, Asp228 and Gln73 of catalytic site (∆Gb= -
11.90 Kcal/mol).
Table 2. Summary of molecular interactions, binding energy (∆Gb) and inhibition constant
(Ki) of synthesize compounds with BACE1 active site
Compounds
Mean binding free
energy (∆Gb) (kcal/mol)
Ki (nM)
Hydrogen bond interactions
Atom of the ligand Amino acid
NH (amino cyclohexyl)
-9.52
104.73
N of Thr 72
8a
N (imidazopyridin)
O of Asp 228
-11.67
2.77
N (imidazopyridin)
O of Asp 228
8b
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-12.09
1.37
NH (amino cyclohexyl)
O of Thr 72
O of Asp228
8c
N (imidazopyridin)
-11.68
-11.90
2.74
4.38
N (imidazopyridin)
O of Asp 228
8d
8e
O (phenoxyethyl)
N (imidazopyridin)
O of Asp 32
O of Asp 228
-9.61
-11.70
-11.37
-11.32
-10.24
20.13
2.62
CO (phthalimide)
N of Thr 71
8f
8g
8h
8i
NH (amino t-Butyl)
O of Thr 231
N (imidazopyridin)
O of Thr 72
O of Gly 34
NH (amino cyclohexyl)
4.59
CO (phthalimide)
CO (phthalimide)
O of Asp 32
O of Gly 230
5.03
CO (phthalimide)
N (imidazopyridin)
N of Thr 72
O of Asp 228
31.17
CO ( phthalimide)
OH of Tyr 198
O of Gly 230
8j
NH (amino t-Butyl)
-11.83
-11.02
-10.89
2.14
4.46 nM
10.33
CO (phthalimide)
N (imidazopyridin)
N and O of Thr 232
O of Thr 232
8k
8l
CO (phthalimide)
CO of phthalimide
O of Phe 108
O of Gly 230
NH (amino cyclohexyl)
CO (phthalimide)
N of Thr 72
O of Phe 108
O of Gly 230
O of Thr 231
8m
CO (phthalimide)
N (imidazopyridin)
-9.11
209.27
CO (phthalimide)
O of Gly 34
8n
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-11.18
14.67
CO (phthalimide)
O of Gln 34
O of Gln 73
8o
NH (amino t-Butyl)
Figure 2. Comparative binding orientation of compound 8e (displayed in blue) and 8f
(displayed in yellow) in the BACE1 active site.
Figure 3. Binding orientation of compound 8e (displayed in violet) in BACE1 active site. H-
bonds are shown as green dash lines..
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Conclusion
In this study, we synthesized and evaluated the anti-BACE1 activity of fifteen hybrid
imidazopyridines containing phthalimide moieties The goal was to discover novel small
.
compounds with potentially improved BACE1 inhibitory properties. Our preliminary BACE1
inhibitory results suggested that a cyclohexyl substitution at R2 was a key structure for
improving the inhibitory activity. In the case of aminocyclohexyl containing derivatives,
introducing a methyl substitute at 6 or 7 positions of the imidazopyridine core (as in 8d and
8e) resulted in considerable improvement of BACE1 inhibitory potential. Besides, molecular
docking simulation indicated that the amino t-butyl derivatives have different binding
orientations in the active site of BACE1 in comparison with aminocyclohexyl derivatives.
Moreover, all aminocyclohexyl derivatives showed favorable binding interactions with P2,
P'2 and stacking pockets; imidazopyridines and phenoxypropyl linkers were involved in
hydrogen bond interaction with Asp228 and Asp32 of BACE1 active site, respectively. The
Phthalimide portion was oriented toward the flap pocket and demonstrated van der Waal’s
and hydrophobic interactions with phe108, lle110, Trp115, Ile118. The results of the present
study could guide the rational design of more potent BACE1 blocking agents.
3. Experimental Section
3.1. Chemistry
All chemicals were purchased from commercial sources (Merck and Aldrich) and used
without further purification.
Melting points were taken on a Kofler hot stage apparatus and were uncorrected. 1H- and 13C-
NMR spectra were recorded on Bruker FT-500 (Germany), using TMS as an internal
standard. The IR spectra were obtained on a Nicolet MagnaFTIR 550 spectrometer (KBr
disks). The elemental analysis was performed with an Elementar Analysensystem GmbH
VarioEL CHNS mode (Germany).
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Synthesis of aldehyde 5: A mixture of phthalimide 1 (1 mmol), 1,2-dibromopropane 2 (1
mmol), and K2CO3 (1.3 mmol) in acetone (15 mL) was heated at reflux for 8 h. After
completion of the reaction (checked by TLC), the mixture was poured into crushed ice and
the precipitate was filtered of and dried to give 2-(3-bromopropyl)isoindoline-1,3-dione 3.
Next, mixture of compound 3 (1 mmol), 4-hydroxyaldehyde derivative 4 (1 mmol), and
K2CO3 (1.3 mmol) in DMF (10 mL) was heated at 80 °C for 8 h. Upon completion of the
following reaction, the mixture was poured into crushed ice and the precipitate was filtered of
and dried affording aldehyde 5.
Synthesis of imidazopyridine derivatives 8: A mixture of aldehyde 5 (1 mmol), 2-
aminopyridins 6 (1 mmol), isocyanides 7 (1.2 mmol), NH4Cl (1 mmol) and toluene (10 mL)
was heated under reflux conditions for 8-12 h. After completion of the reaction (checked by
TLC), the solvent was evaporated under vacuum and the residue was recrystallized from
ethanol to afford pure imidazopyridines 8.
2-(3-(4-(3-(Cyclohexylamino)imidazo[1,2-a]pyridin-2-yl)phenoxy)propyl)isoindoline-
1,3-dione (8a). Yield: 79%, Mp: 182-184 °C, IR (KBr): 3205, 2917, 2850, 1771, 1705, 1610,
1523 cm-1; MS: m/z (%) 495 (M+, 100), 412(20), 385(24), 264(10), 225(7), 188(100), 170(6),
1
160(55), 142(23), 130(12), 104 (4), 90(11), 79(26), 55(13); H NMR (CDCl3, 500 MHz):
δH(ppm)= 8.50-8.46 (m, 3H, H5, H7, H8), 8.02 (d, J= 8.5 Hz, 2H, H3´, H5´), 7.86-7.85 (m,
2H, H1″, H4″), 7.74-7.72 (m, 2H, H2″, H3″), 6.87-6.85 (m, 3H, H2´, H6´, H6), 4.08 (t, J =
6.5 Hz, 2H, CH2), 3.94 (t, J = 6.5 Hz, 2H, CH2), 3.51 (bs, 1H, NH), 2.96-2.94 (m, 1H, CH),
2.22 (q, J= 6.5 Hz, 2H, CH2), 1.79-1.14 (m, 10H, H(cyclohexyl)); 13C NMR (CDCl3, 125
MHz): δc(ppm) = 168.5, 159.1, 134.0, 132.1, 130.8, 129.1, 128.8, 127.3, 127.2, 123.3, 123.2,
122.1, 119.1, 117.9, 114.5, 65.6, 56.8, 35.4, 34.0, 28.3, 25.6, 24.7. Anal. Calcd for
C29H28N4O3: C, 72.48; H, 5.87; N, 11.66. Found: C, 72.69; H, 5.64; N, 11.47.
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2-(3-(4-(3-(Cyclohexylamino)imidazo[1,2-a]pyridin-2-yl)-2-
methoxyphenoxy)propyl)isoindoline-1,3-dione (8b). Yield: 71%, Mp: 181-183 °C, IR
(KBr): 3228, 2923, 2849, 1773, 1708, 1611, 1503 cm-1; MS: m/z (%) 525 (M+, 30), 415 (4),
248(5), 228(7), 188(100), 160(45), 130(15), 104(8), 79(15), 57(10);
1H NMR (CDCl3, 500
MHz): δH(ppm)= 8.47-8.44 (m, 3H, H5, H7, H8), 7.86-7.84 (m 2H, H1″, H4″), 7.76 (s, 1H,
H3´), 7.73-7.72 (m, 2H, H2″, H3″), 7.59 (d, J= 8.0 Hz, 1H, H5′), 6.90 (m, 2H, H6 , H6´),
4.14 (t, J = 5.0 Hz, 2H, CH2), 3.95 (t, J = 5.0 Hz, 2H, CH2), 3.79 (s, 3H, OCH3), 3.48 (bs, 1H,
NH), 3.00-2.99 (m, 1H, CH), 2.28 (q, J= 6.5 Hz, 2H, CH2), 1.81-1.16 (m, 10H,
H(cyclohexyl)); 13C NMR (CDCl3, 125 MHz): δc(ppm) = 168.4, 165.7, 160.1, 150.0, 149.5,
133.8, 132.2, 131.6, 130.4, 127.5, 123.1, 122.8, 119.5, 117.8, 115.9, 112.7, 111.1, 66.9, 56.8,
56.0, 35.6, 34.1, 28.4, 25.6, 24.7. Anal. Calcd for C30H30N4O4: C, 70.57; H, 5.92; N, 10.97.
Found: C, 70.34; H, 6.18; N, 11.23.
2-(3-(4-(3-(Cyclohexylamino)-5-methylimidazo[1,2-a]pyridin-2-
yl)phenoxy)propyl)isoindoline-1,3-dione (8c). Yield: 86%, Mp:204-206 °C, IR (KBr):
3325, 3050, 2926, 2851, 1770, 1712, 1609, 1562 cm-1; MS: m/z (%) 508 (M+, 88), 425(100),
398(25), 264(7), 238(10), 211(12), 188(62), 160(37.5), 130(14), 92(50), 65(14). 1H NMR
(CDCl3, 500 MHz): δH(ppm)= 7.83-7.85 (m, 4H, H3´, H5´, H1″, H4″), 7.73-7.71 (m, 2H,
H3″, H2″), 7.46 (d, J= 7.0 Hz, 1H, H6), 7.03 (t, J = 7.0 Hz, 1H, H7), 6.84 (d, J= 8.5 Hz, 2H,
H2´, H6´), 6.45 (d, J= 7.0 Hz, 1H, H8), 4.07 (t, J = 6.5 Hz, 2H, CH2), 3.94 (t, J = 6.5 Hz, 2H,
CH2), 3.25 (s, 1H, NH), 2.94 (s, 3H, CH3), 2.76-2.74 (m, 1H, CH), 2.21 (q, J= 6.5 Hz, 2H,
CH2), 1.65-1.04 (m, 10H, H(cyclohexyl)); 13C NMR (CDCl3, 125 MHz): δc(ppm) =168.3,
161.1, 158.2, 149.3, 142.3, 136.6, 133.9, 132.1, 128.8, 125.9, 124.9, 123.2, 114.8, 114.2,
113.9, 65.5, 58.4, 35.5, 32.1, 28.3, 25.7, 24.8, 20.1. Anal. Calcd for C30H30N4O3: C, 72.85; H,
6.11; N, 11.33. Found: C, 73.10; H, 6.31; N, 11.56.
16

ACCEPTED MANUSCRIPT
2-(3-(4-(3-(Cyclohexylamino)-6-methylimidazo[1,2-a]pyridin-2-
yl)phenoxy)propyl)isoindoline-1,3-dione (8d). Yield:81%, Mp:175-177 °C, IR (KBr):
3462, 2931, 2853, 1771, 1708 1600, 1561 cm-1; MS: m/z (%) 508 (M+, 1), 482(14), 374(10),
1
212(7), 188(100), 160(40), 130(12), 104(3), 83(4), 55(14). H NMR (CDCl3, 500 MHz):
δH(ppm)= 8.57 (d, J= 9.0 Hz, 2H, H3´, H5´), 7.97 (s, 1H, H5), 7.86-7.84 (m, 2H, H1″, H4″),
7.74-7.72 (m, 2H, H3″, H2″), 7.27-7.26 (m, 2H, H6, H7), 6.87 (d, J= 9.0 Hz, 2H, H2´, H6´),
4.61 (s, 1H, NH), 4.13 (t, J = 6.5 Hz, 2H, CH2), 3.93 (t, J = 6.5 Hz, 2H, CH2), 2.27 (q, J= 6.5
Hz, 2H, CH2), 2.24 (s, 3H, CH3), 2.26-2.22 (m, 1H, CH), 1.91-1.25 (m, 10H, H(cyclohexyl));
13C NMR (CDCl3, 125 MHz): δc(ppm) = 170.7, 169.3, 165.8, 163.4, 157.0, 151.9, 140.1,
138.2, 134.0, 133.0, 132.0, 123.4, 133.3, 117.8, 114.7, 65.9, 60.6, 35.2, 33.0, 28.4, 28.1, 25.5,
23.9. Anal. Calcd for C30H30N4O3: C, 72.85; H, 6.11; N, 11.33. Found: C, 73.11; H, 6.21; N,
11.12.
2-(3-(4-(3-(Cyclohexylamino)-7-methylimidazo[1,2-a]pyridin-2-
yl)phenoxy)propyl)isoindoline-1,3-dione (8e). Yield: 67%, Mp:193-195 °C, IR (KBr):
3224, 2926, 2851, 1769, 1712, 1656, 1607 cm-1; MS: m/z (%) 508 (M+, 85), 425 (100),
398(20), 258(5), 237(5), 211(9), 188(83), 160(50), 130(10), 92(40), 65(10). 1H NMR
(CDCl3, 500 MHz): δH(ppm)= 8.25 (d, J = 7.0 Hz, 1H, H5), 7.97 (d, J= 8.5 Hz, 2H, H3´,
H5´), 7.88-7.86 (m, 2H, H1″, H4″), 7.77-7.75 (m, 2H, H2″, H3″), 7.68 (s, 1H, H8), 6.86 (d,
J= 7.0 Hz, 1H, H6), 6.72 (d, J= 8.5 Hz, 2H, H2´, H6´), 4.84 (bs, 1H, NH), 4.02 (t, J = 6.5 Hz,
2H, CH2), 3.94 (t, J = 6.5 Hz, 2H, CH2), 2.86-2.82 (m, 1H, CH), 2.37 (s, 3H, CH3), 2.22 (q,
J= 6.5 Hz, 2H, CH2), 1.74-1.10 (m, 10H, H(cyclohexyl)); 13C NMR (CDCl3, 125 MHz):
δc(ppm) =168.4, 159.2, 143.7, 136.6, 134.1, 132.0, 128.2, 126.8, 124.0, 123.5, 123.3, 118.5,
118.2, 114.5, 111.2, 65.6, 56.2, 35.3, 33.7, 28.3, 25.5, 24.6, 21.4. Anal. Calcd for
C30H30N4O3: C, 72.85; H, 6.11; N, 11.33. Found: C, 72.64; H, 6.31; N, 11.18.
17

ACCEPTED MANUSCRIPT
2-(3-(4-(3-(tert-butylamino)-7-methylimidazo[1,2-a]pyridin-2-
yl)phenoxy)propyl)isoindoline-1,3-dione (8f). Yield: 79%, Mp:201-203°C, IR (KBr): 3297,
3058, 2927, 2852, 1773, 1712, 1606, 1584 cm-1; MS: m/z (%) 482 (M+, 20), 425 (100), 398
(15), 237(3), 211(3), 188(50), 160(30), 130(7), 92(25), 65 (10). 1H NMR (CDCl3, 500 MHz):
δH(ppm)= 8.07 (d, J= 7.0 Hz, 1H, H5), 7.85-7.83 (m, 2H, H1″, H4″), 7.79 (d, J= 8.5 Hz, 2H,
H3´, H5´), 7.72-7.70 (m, 2H, H2″, H3″), 7.28 (s, 1H, H8), 6.58 (d, J= 8.5 Hz, 2H, H2´, H6´),
6.58 (d, J = 7.0 Hz, 1H, H6), 4.07 (t, J = 6.5 Hz, 2H, CH2), 3.93 (t, J = 6.5 Hz, 2H, CH2),
3.05 (s, 1H, NH), 2.28 (q, J= 6.5 Hz, 2H, CH2), 2.37 (s, 3H, CH3), 1.02 (s, 9H, H(t-Bu)); 13C
NMR (CDCl3, 125 MHz): δc(ppm) = 168.3, 157.9, 142.1, 138.6, 134.7, 133.8, 132.1, 129.1,
127.8, 123.1, 122.6, 122.3, 115.3, 114.0, 113.8, 65.6, 56.2, 35.5, 30.2, 28.4, 21.2. Anal. Calcd
for C28H28N4O3: C, 71.78; H, 6.02; N, 11.96. Found: C, 71.59; H, 5.87; N, 12.22.
2-(3-(4-(3-(Cyclohexylamino)-8-methylimidazo[1,2-a]pyridin-2-
yl)phenoxy)propyl)isoindoline-1,3-dione (8g). Yield: 94%, Mp:185-187 °C, IR (KBr):
3292, 2929, 2851, 1771, 1716, 1610, 1559 cm-1; MS: m/z (%) 508 (M+, 88), 425(100),
398(37), 325(40), 264(5), 238(6), 211(8), 188(47), 160(37), 130(9), 92(45), 65(12); 1H NMR
(CDCl3, 500 MHz): δH(ppm)= 7.96 (d, J= 6.5 Hz, 1H, H5), 7.90 (d, J= 9.0 Hz, 2H, H3´, H5´),
7.86-7.83 (m, 2H, H1″, H4″), 7.74-7.71 (m, 2H, H2″, H3″), 6.91 (d, J= 6.5 Hz, 1H, H7), 6.87
(d, J= 9.0 Hz, 2H, H2´, H6´), 6.69 (t, J = 6.5 Hz, 1H, H6), 4.08(t, J = 6.5 Hz, 2H, CH2), 3.93
(t, J = 6.5 Hz, 2H, CH2), 3.12 (bs, 1H, NH), 2.92-2.88 (m, 1H, CH), 2.62 (s, 3H, CH3), 2.21
13
(q, J= 6.5 Hz, 2H, CH2), 1.80-1.13 (m, 10H, H(cyclohexyl)); C NMR (CDCl3, 125 MHz):
δc(ppm) =168.3, 158.0, 144.6, 142.0, 133.9, 132.1, 128.4, 126.8, 124.5, 123.2, 122.6, 120.5,
118.8, 114.4, 111.5, 65.6, 56.8, 35.5, 34.1, 28.3, 25.7, 24.7, 16.6. Anal. Calcd for
C30H30N4O3: C, 72.85; H, 6.11; N, 11.33. Found: C, 73.10; H, 5.87; N, 11.15.
2-(3-(4-(6-Chloro-3-(cyclohexylamino)imidazo[1,2-a]pyridin-2-
yl)phenoxy)propyl)isoindoline-1,3-dione (8h). Yield: 69%, Mp:255-257 °C, IR (KBr):
18

ACCEPTED MANUSCRIPT
3250, 3049, 2927, 2852, 1770, 1711, 1608, 1561 cm-1; MS: m/z (%) 528 (M+, 90), 445 (60),
1
418(20), 258(5), 258(7), 231(14), 188(100), 160(65), 130(15), 112(25), 76(10), 57(15); H
NMR (CDCl3, 500 MHz): δH(ppm)= 8.15 (s, 1H, H5), 7.90 (d, J= 9.0 Hz, 2H, H5´, H3´),
7.86-7.85 (m, 2H, H1″, H4′′), 7.74-7.72 (m, 2H, H2″, H3′′), 7.52 (d, J= 9.5 Hz, 1H, H8), 7.11
(d, J= 9.5 Hz, 1H, H7), 6.87 (d, J= 9.0 Hz, 2H, H2´, H6´), 4.08 (t, J = 6.5 Hz, 2H, CH2), 3.94
(t, J = 6.5 Hz, 2H, CH2), 3.28 (s, 1H, NH), 2.94-2.93 (m, 1H, CH), 2.27 (q, J= 6.5 Hz, 2H,
CH2), 1.79-1.15 (m, 10H, H(cyclohexyl)); 13C NMR (CDCl3, 125 MHz): δc(ppm) = 168.4,
158.5, 141.5, 133.9, 132.1, 131.0, 129.0, 128.2, 126.2, 125.9, 124.4, 123.2, 120.7, 116.9,
114.5, 65.6, 56.7, 35.4, 34.0, 28.4, 25.6, 24.7. Anal. Calcd for C29H27ClN4O3: C, 67.63; H,
5.28; N, 10.88. Found: C, 67.45; H, 5.41; N, 10.63.
2-(3-(4-(6-Chloro-3-(cyclohexylamino)imidazo[1,2-a]pyridin-2-yl)-2-
methoxyphenoxy)propyl) isoindoline-1,3-dione (8i). Yield: 82%, Mp: 189-191 °C, IR
(KBr): 3295, 3058, 2926, 2853, 1774, 1712, 1605, 1584 cm-1; MS: m/z (%) 558 (M+, 55),
475(14), 448 (5), 261(7), 188(100), 160(50), 130(10), 112(15), 76(5), 57(8); 1H NMR
(CDCl3, 500 MHz): δH(ppm)= 8.16 (s, 1H, H5), 7.86-7.84 (m, 2H, H1″, H4″), 7.73-7.71 (m,
2H, H2″, H3″), 7.60 (d, J= 1.5 Hz, 1H, H3´), 7.51 (d, J = 9.0 Hz, 1H, H8), 7.47 (dd, J= 8.0,
1.5 Hz, 1H, H5´), 7.13 (d, J= 9.0 Hz, 1H, H7), 6.88 (d, J= 8.0 Hz, 1H, H6´), 4.13 (t, J = 6.5
Hz, 2H, CH2), 3.94 (t, J = 6.5 Hz, 2H, CH2), 3.81 (s, 3H, OCH3), 3.46 (bs, 1H, NH), 2.98-
2.97 (m, 1H, CH), 2.22 (q, J= 6.5 Hz, 2H, CH2), 1.80-1.15 (m, 10H, H(cyclohexyl)); 13C
NMR (CDCl3, 125 MHz): δc(ppm) = 168.4, 164.5, 155.8, 150.0, 149.4, 148.1, 137.7, 133.8,
132.2, 131.1, 124.6, 123.2, 120.7, 119.2, 116.6, 112.8, 110.6, 66.8, 56.6, 55.9, 35.5, 34.0,
28.3, 25.6, 24.7. Anal. Calcd for C30H29ClN4O4: C, 66.11; H, 5.36; N, 10.28. Found: C,
65.86; H, 5.58; N, 10.41.
2-(3-(4-(3-(tert-butylamino)-6-chloroimidazo[1,2-a]pyridin-2-
yl)phenoxy)propyl)isoindoline-1,3-dione (8j). Yield: 74%, Mp: 229-231 °C, IR (KBr):
19

ACCEPTED MANUSCRIPT
3289, 2967, 2877, 1770, 1714, 1612, 1557 cm-1; MS: m/z (%) 502 (M+, 29), 445 (70),
418(15), 258(5), 231(7), 188(100), 160(60), 130(10), 112(20), 76(7), 57(8); 1H NMR (CDCl3,
500 MHz): δH(ppm)= 8.27 (s, 1H, H5), 7.86-7.84 (m, 2H, H1″, H4″), 7.78 (d, J= 8.5 Hz, 2H,
H3´, H5´), 7.73-7.72 (m, 2H, H2″, H3″), 7.55 (d, J= 9.5 Hz, 1H, H8), 7.14 (d, J= 9.5 Hz, 1H,
H7), 6.85 (d, J= 8.5 Hz, 2H, H2´, H6´), 4.07 (t, J = 6.5 Hz, 2H, CH2), 3.94 (t, J = 6.5 Hz, 2H,
CH2), 3.23 (bs, 1H, NH), 2.28 (q, J= 6.5 Hz, 2H, CH2), 1.04 (s, 9H, H(t-Bu)); 13C NMR
(CDCl3, 125 MHz): δc(ppm) = 168.4, 158.6, 137.9, 133.9, 132.1, 129.3, 128.1, 126.1, 123.3,
123.2, 122.6, 121.4, 120.5, 116.9, 114.3, 65.6, 56.5, 35.4, 30.3, 28.4. Anal. Calcd for
C27H25ClN4O3: C, 66.32; H, 5.15; N, 11.46. Found: C, 66.54; H, 5.34; N, 11.67.
2-(3-(4-(3-(tert-butylamino)-6-chloroimidazo[1,2-a]pyridin-2-yl)-2-
methoxyphenoxy)propyl)isoindoline-1,3-dione (8k). Yield: 67%, Mp:267-269 °C, IR
(KBr): 3290, 3079, 2929, 2851, 1772, 1711, 1610, 1559 cm-1; MS: m/z (%) 604 ((M+2)+, 20),
602 (M+
, 20), 520 (34), 522(34), 506(7), 504(7), 399(68), 360(56), 325(45), 291(100),
259(10), 261(), 188(100), 160(44), 151(20), 125(75), 104(4), 97(18), 57(12)
.
1H NMR (CDCl3, 500 MHz): δH(ppm)= 8.28 (s, 1H, H5), 7.85-7.84 (m, 2H, H1″, H4″), 7.72-
7.71 (m, 2H, H2″, H3″), 7.54 (d, J= 9.0 Hz, 1H, H8), 7.47 (d, J = 1.5 Hz, 1H, H3´), 7.38 (d,
J= 8.0 Hz, 1H, H5´), 7.15 (d, J= 9.0 Hz, 1H, H7), 6.87 (d, J= 8.0. Hz, 1H, H6´), 4.12 (t, J =
6.5 Hz, 2H, CH2), 3.94 (t, J = 6.5 Hz, 2H, CH2), 3.80 (s, 3H, OCH3), 3.34 (bs, 1H, NH), 2.22
13
(q, J= 6.5 Hz, 2H, CH2), 1.05 (s, 9H, H(t-Bu)); C NMR (CDCl3, 125MHz): δc(ppm) =
168.3, 149.5, 148.0, 141.9, 133.8, 132.2, 131.4, 126.8, 125.4, 123.5, 123.2, 122.7, 121.5,
120.5, 116.6, 112.7, 111.7, 66.9, 56.0, 55.9, 35.5, 30.3, 28.2. Anal. Calcd for C28H27ClN4O4:
C, 64.80; H, 5.24; N, 10.80. Found: C, 64.59; H, 5.51; N, 10.71.
2-(3-(4-(6-Bromo-3-(cyclohexylamino)imidazo[1,2-a]pyridin-2-
yl)phenoxy)propyl)isoindoline-1,3-dione (8l).Yield: 85%, Mp: 263-265°C, IR (KBr): 3248,
3045, 2928, 2852, 1770, 1711, 1608, 1561 cm-1; MS: m/z (%) 574 ((M+2)+, 40), 574 (M+,
20

ACCEPTED MANUSCRIPT
40), 493(25), 491(25), 466(10), 464(8), 277(5), 275(5), 188(100), 160(60), 130(20), 104(5),
77(7), 55(10). 1H NMR (CDCl3, 400 MHz): δH (ppm) = 8.28 (s, 1H, H5), 7.91 (d, J = 8.5 Hz,
2H, H3′, H5′), 7.87-8.50 (m, 2H, H1″, H4′′), 7.74-7.72 (m, 2H, H2′′, H3′′), 7.54 (d, J = 8.0
Hz, 1H, H8), 7.17 (d, J = 8.0 Hz, 1H, H7), 6.85 (d, J = 8.5 Hz, 2H, H2´, H6´), 4.08 (t, J = 6.5
Hz, 2H, CH2), 3.94 (t, J = 6.5 Hz, 2H, CH2), 3.52 (bs, 1H, NH), 3.00-2.80 (m, 1H, CH), 2.20
(q, J= 6.5 Hz, 2H, CH2), 1.78- 1.15 (m, 10H, H (cyclohexyl)); 13C NMR (CDCl3, 100 MHz):
δc (ppm) =168.4, 158.6, 157.2, 144.8, 143.2, 136.3, 135.1, 133.9, 132.1, 128.3, 127.0, 124.3,
123.2, 123.1, 114.6, 65.6, 56.6, 35.4, 34.0, 28.3, 25.5, 24.7.Anal. Calcd for C29H27BrN4O3: C,
62.26; H, 4.86; N, 10.01. Found: C, 62.41; H, 4.62; N, 9.86.
2-(3-(4-(6-Bromo-3-(cyclohexylamino)imidazo[1,2-a]pyridin-2-yl)-2-
methoxyphenoxy)propyl) isoindoline-1,3-dione (8m).Yield: 83%, Mp:196-198°C, IR
(KBr): 3294, 3047, 2992, 2926, 2852, 1773, 1710, 1606, 1583 cm-1; MS: m/z (%) 604
((M+2)+, 20), 602 (M+
, 20), 520 (34), 522(34), 506(7), 504(7), 399(68), 360(56), 325(45),
291(100), 259(10), 261(), 188(100), 160(44), 151(20), 125(75), 104(4), 97(18), 57(12)
.
1H NMR (CDCl3, 500 MHz): δH (ppm)= 8.24 (s, 1H, H5), 7.86-7.83 (m, 2H, H1″, H4′′),
7.73-7.71 (m, 2H, H2″, H3′′), 7.60 (d, J = 2.0 Hz, 1H, H3′), 7.49 (dd, J = 8.5, 2.0 Hz, 1H,
H5´), 7.44 (d, J = 9.0 Hz, 1H, H8), 7.20 (d, J = 9.0 Hz, 1H, H7), 6.89 (d, J = 8.5 Hz, 1H,
H6´), 4.14 (t, J = 6.5 Hz, 2H, CH2), 3.94 (t, J= 6.5 Hz, 2H, CH2), 3.81 (s, 3H, OCH3), 3.28
(bs, 1H, NH), 3.10-2.98 (m, 1H, CH), 2.24 (q, J= 6.5 Hz, 2H, CH2), 1.80-1.15 (m, 10H,
13
H(cyclohexyl)); C NMR (CDCl3, 125 MHz): δc(ppm) =168.3, 155.8, 149.4, 148.0, 145.1,
143.5, 139.8, 133.8, 132.1, 128.0, 124.4, 123.1, 122.8, 119.2, 117.2, 112.8, 110.62, 66.8,
56.7, 55.9, 35.5, 34.0, 28.1, 25.6, 24.7.Anal. Calcd for C30H29BrN4O4: C, 61.13; H, 4.96; N,
9.50. Found: C, 61.37; H, 5.27; N, 9.74.
2-(2-(4-(6-Bromo-3-(tert-butylamino)imidazo[1,2-a]pyridin-2-
yl)phenoxy)propyl)isoindoline-1,3-dione (8n).Yield: 78%, Mp: 233-235°C, IR (KBr):
21

ACCEPTED MANUSCRIPT
3231, 3066, 2926, 2852, 1769, 1714, 1657, 1606, 1572 cm-1; MS: m/z (%) 548 ((M+2)+, 25),
546 (M+, 25), 491(30), 489(30), 464(12), 462(12), 411(7), 341(5),188(100), 160(80),
130(15), 85 (20), 77(23), 55(40); 1H NMR (CDCl3, 500 MHz): δH(ppm)= 8.35 (s, 1H, H5),
7.86-7.84 (m, 2H, H1″, H4′′), 7.78 (d, J= 8.0 Hz, 2H, H3´, H5´), 7.73-7.72 (m, 2H, H2″,
H3′′), 7.48 (d, J= 9.0 Hz, 1H, H8), 7.21 (d, J=9.0 Hz, 1H, H7), 6.85 (d, J= 8.0 Hz, 2H, H2´,
H6´), 4.07 (t, J = 6.5 Hz, 2H, CH2), 3.93 (t, J = 6.5 Hz, 2H, CH2), 3.21 (bs, 1H, NH), 2.22 (q,
J= 6.5 Hz, 2H, CH2), 1.04 (s, 9H, H (t-Bu)); 13C NMR (CDCl3, 125 MHz): δc(ppm) = 168.3,
158.5, 150.6, 143.1, 139.2, 136.1, 133.9, 132.1, 129.3, 128.2, 123.7, 123.2, 117.2, 115.9,
114.3, 65.6, 56.5, 35.5, 30.3, 28.3. Anal. Calcd for C27H25BrN4O3: C, 60.79; H, 4.72; N,
10.50. Found: C, 60.58; H, 4.58; N, 10.74.
2-(3-(4-(6-Bromo-3-(tert-butylamino)imidazo[1,2-a]pyridin-2-yl)-2-
methoxyphenoxy)propyl) isoindoline-1,3-dione (8o). Yield: 71%, Mp:165-167°C, IR
(KBr): 3295, 3048, 2992, 2926, 2852, 1774, 1710, 1606, 1583 cm-1; MS: m/z (%) 578
((M+2)+, 10), 576 (M+, 10), 521(20), 519 (18), 441(4), 188(100), 160(40), 130(7), 104(4),
77(7), 55(9);
1H NMR (CDCl3, 500 MHz): δH(ppm)= 8.35 (s, 1H, H5), 7.85-7.83 (m, 2H,
H1″, H4′′), 7.72-7.71 (m, 2H, H2″, H3′′), 7.47-7.45 (m, 2H, H8, H3´), 7.36 (d, J= 8.0 Hz, 1H,
H5´), 7.21 (d, J= 9.5Hz, 1H, H7), 6.89 (d, J= 8.0 Hz, 1H, H6´), 4.13 (t, J= 6.5 Hz, 2H, CH2),
3.94 (t, J= 6.5 Hz, 2H, CH2), 3.80 (s, 3H, OCH3), 3.21 (bs, 1H, NH), 2.21 (q, J= 6.5 Hz, 2H,
CH2), 1.05 (s, 9H, H(t-Bu)); 13C NMR (CDCl3, 125 MHz): δc(ppm) = 168.3, 161.2, 149.4,
148.1, 139.5, 135.9, 133.8, 132.2, 127.9, 123.6, 123.3, 123.1, 120.5, 117.3, 114.8, 112.7,
111.7, 66.9, 56.5, 55.9, 35.5, 30.3, 28.3. Anal. Calcd for C28H27BrN4O4: C, 59.69; H, 4.83; N,
9.94. Found: C, 59.27; H, 4.62; N, 10.24.
3.2. BACE1 enzymatic assay
Enzyme inhibition assay was carried out using BACE1 (β-secretase) FRET assay kit, from
Invitrogen (former Pan Vera, Madison, WI) according to the manufacturer instructions [16,
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23]. The analysis was carried out according to the manufacturer’s instructions with small
modifications. Stock solutions of all tested compounds were prepared in DMSO and further
diluted with the assay buffer (50 mM sodium acetate; pH 4.5). Ten µl of BACE1 substrate
(Rh-EVNLDAEFK-Quencher) was mixed with 10 µL of each tested compound and then 10
µL of enzyme (1 U/mL) was added to start the reaction. After around 90 min of incubation at
room temperature, 10 µL of the stop solution (2.5 M sodium acetate) was applied to stop the
reaction. The fluorescence was monitored at 545 nm (Ex) and 585 nm (Em) [19, 27]. The
assays were performed in triplicate for each concentration in a 96-well polystyrene black
plates and OM99 was used as a reference inhibitor agent.
3.3. Molecular docking
Flexible-ligand docking studies were carried out using AutoDock version (1.5.4). X-ray
crystallographic holo structures of BACE1 were retrieved from the Brookhaven Protein Data
Bank (4acu; http://www.rcsb.org/). Water molecules and cognate ligand were removed from
the 4acu and hydrogen atoms were added to the receptor PDB. Next, non-polar hydrogens
were merged into related carbon atoms of the receptor and Kollman charges were also
assigned. Structures of the desired ligands were constructed using HyperChem software
version 8.0.10 and energetically minimized using MM+ force field. The active sites were
defined based on the center and radius of cognate ligand which are mentioned in X-ray
crystal structure. Docking computations were performed using the Lamarkian genetic
algorithm with grid sizes 60× 60×60 (grid spacing 0.375 Å), the maximum number of
evaluations were set to 2500000, the number of GA runs were 150 and the maximum number
of generations were set as 27000 and all other options were set as default. The lowest energy
conformation of the highest populated cluster was selected for analysis. Schematic 2D
representations of the ligand–receptor interactions were all generated using Chimera 1.11.
Acknowledgement
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The authors gratefully acknowledge financial support from Tehran University of Medical
Sciences. The authors also acknowledge the financial support of Shiraz University of Medical
Sciences, Vice Chancellor of Research (Grant: 12-12200). We thank Alireza Edraki and
Samantha J. Nelson from Umass Medical School, MA, USA for thorough editing of this manuscript
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Highlights
•
A novel class of imidazopyridine- phthalimide hybrids was rationally designed and
synthesized as novel anti-Alzheimer agents.
•
•
All compounds were examined for their BACE1 inhibitory potential.
Compound 8e was the most potent against BACE1 with an IC50 value of 2.84 (±0.95)
µM.
•
Molecular docking revealed the key binding interactions of 8e in BACE1 active site