Imidazo[1,2- a]pyridine derivatives as aldehyde dehydrogenase inhibitors: Novel chemotypes to target glioblastoma stem cells

Article abstract of DOI:10.1021/acs.jmedchem.9b01910

Glioblastoma multiforme (GBM) is the deadliest form of brain tumor. It is known for its ability to escape the therapeutic options available to date thanks to the presence of a subset of cells endowed with stem-like properties and ability to resist to cyto

Full text of DOI:10.1021/acs.jmedchem.9b01910

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Article
Imidazo[1,2-a]pyridine Derivatives as Aldehyde Dehydrogenase
Inhibitors: Novel Chemotypes to Target Glioblastoma Stem Cells.
Luca Quattrini, Edoardo Luigi Maria Gelardi, Vito Coviello, Stefania Sartini, Davide
Ferraris, Mattia Mori, Ichiro Nakano, Silvia Garavaglia, and Concettina La Motta
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01910 • Publication Date (Web): 30 Mar 2020
Downloaded from pubs.acs.org on April 18, 2020
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Imidazo[1,2-a]pyridine Derivatives as Aldehyde Dehydrogenase Inhibitors:
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Novel Chemotypes to Target Glioblastoma Stem Cells.
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Luca Quattrini,§ç Edoardo Luigi Maria Gelardi,£ç Vito Coviello,§ Stefania Sartini,§ Davide Maria
Ferraris,£ Mattia Mori,# Ichiro Nakano,$ Silvia Garavaglia,£* Concettina la Motta.§*
§Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126, Pisa, Italy.
£Department of Drug Science, University of Piemonte Orientale, Via Bovio 6, 28100, Novara, Italy.
#Department of Biotechnology, Chemistry and Pharmacy “Department of Excellence 2018-2022”,
University of Siena, via Aldo Moro 2, Siena, Italy.
$Department of Neurosurgery, University of Alabama at Birmingham, WTI 401, 1824 6th Avenue
South, Birmingham, Alabama 35233.
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Abstract
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Glioblastoma multiforme (GBM) is the deadliest form of brain tumour. It is known for its ability to
escape the therapeutic options available to date, thanks to the presence of a subset of cells endowed
with stem-like properties and able to resist to cytotoxic treatments. As the cytosolic enzyme
aldehyde dehydrogenase 1A3 turns out to be overexpresses in this kind of cells, playing a key role
for their vitality, treatments targeting this enzyme may represent a successful strategy to fight GBM.
In this work we describe a novel class of imidazo[1,2-a]pyridine derivatives as aldehyde
dehydrogenase 1A3 inhibitors, reporting the evidence of their significance as novel drug candidates
for the treatment of GBM. Besides showing an interesting functional profile, in terms of activity
against the target enzyme and selectivity toward highly homologous isoenzymes, representative
examples of the series showed also a nanomolar to picomolar efficacy against patient-derived GBM
stem-like cells, thus proving the concept that targeting aldehyde dehydrogenase might represent a
novel and promising way to combat GBM by striking its ability to divide immortally.
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Introduction
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Glioblastoma multiforme (GBM) is the most aggressive and fast-growing astrocytic tumour,
accounting for about 15% of all brain tumours in adults. Although rare at presentation, with a yearly
incidence of about two to three per 100.000 adults in both North America and Europe, GBM
represents a critical health issue due to its unavoidably poor prognosis.1
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The treatment of choice for GMB relies in the maximal surgical resection of the tumour mass,
associated with external beam radiation therapy and the adjuvant administration of temozolomide
(Chart 1). However, the remarkable toxicity of the therapeutic intervention, combined with both its
marginal efficacy and the emergence of relapse, generally limit the median survival of the treated
patients to approximately 14 to 15 months from the diagnosis, with less than 5% surviving over 5
years.2 Accordingly, a ground-breaking therapeutic solution for people affected by GBM is urgently
needed.
Chart 1. Temozolomide and Aldehyde Dehydrogenase Inhibitors under Investigation
As a malignancy, GBM is made of a heterogeneous community of different cells governed by
cooperative interactions.3,4 A small percentage of them, bearing stem-like properties, plays more
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than most a key role in tumour perpetuating and progression, being endowed with indefinite self-
renewal ability and aberrant differentiation capacity.5,6
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Differently from tumour bulk cells, glioma stem-like cells (GSCs) are able to resist to cytotoxic
treatments commonly exploited to target rapidly dividing cells, as they are characterized by a slow
mitotic activity possibly associated with a quiescent G0/G1 state. Hence, rather than being
eradicated, GSCs turn out to be selected over the cancer bulk, being free to drive tumour relapse
and mediate metastasis. Therefore, they represent the reference sub-population that should be
brought into focus, as undermining self-renewal signaling pathways supporting their growth and
residence in the stem state may represent an effective strategy to wipe out efficiently the whole
tumour architecture. Indeed, experience gained in the management of different malignancies proof
the concept that targeting cancer stem cells improve patient outcomes by preventing tumours
recurrences,7 increasing chemotherapy efficacy8 and reversing chemotherapy resistance.9
To date, two distinct and mutually exclusive population of GSCs have been differentiated by
transcriptome array analyses, namely the proneural (PN-) and the mesenchymal (MES-) one, strictly
correlated to the clinically relevant proneural and mesenchymal subtypes of GBM.10
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While the PN-GSC subtype tends to adopt a MES identity in response to radiation treatment, MES-
GSCs display a well-characterized profile, turning out to be an aggressive phenotype markedly
resistant to radiation and showing high expression of genes associated with DNA repair. MES-
GSCs exhibit also high levels of the cytoplasmic enzyme aldehyde dehydrogenase, subtype 1A3
(ALDH1A3). Besides being acknowledged as the distinctive hallmark of this type of cancer cell
sub-population, ALDH1A3 plays also a crucial role in stem cells vitality, being involved in the
differentiation of progenitor cells into mature entities. Its overexpression endorses high grade
gliomas and is decisive for tumor invasion, thus having a prognostic value for poor clinical
outcomes.10,11 Accordingly, targeting this enzyme might represent a successful strategy to put in
place a keystone molecular treatment of GBM.
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By exploiting a rational approach, we recently succeeded in turning the natural ALDH inhibitor
daidzin into a novel effective derivative, 2,6-diphenylimidazo[1,2-a]pyridine, namely GA11 (Chart
1, compound 3a in Table 2), which proved to combine a significant in vitro inhibitory activity
against the target enzyme with a potent in vivo anti-tumor efficacy when administered in a murine
GSC-derived xenograft model of GBM.12 Taken together, these results were extremely encouraging
as they affirmed GA11 as a sound and viable candidate deserving of further development.
Therefore, guided by a preliminary crystallization study, performed to highlight GA11
accommodation into the active site of ALDH1A3, we embarked in the structural optimization of the
hit compound with the purpose to obtain a novel and effective class of ALDH1A3 inhibitors.
In this work, we present the synthesis of novel derivatives of GA11, whose phenyl rings in both
positions 2 and 6 of the heterocyclic core were suitably modified in their electronic and steric
features through the insertion suitable substituents. All the synthesized compounds were evaluated
in vitro for their activity against the target ALDH1A3 and selectivity towards the parent ALDH1A1
and ALDH1A2 subtypes, as well as for their anti-proliferative activity toward selected GBM cell
lines. Results obtained were rationalised through molecular docking reasoning.
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Results and Discussion
Crystallization study. The development of a compound able to target the human ALDH1A3 is a
difficult challenge, especially when a selectivity profile for the 1A1 and 1A2 isoenzymes is also
pursued. Therefore, an integrated approach that would use both structural studies and in silico drug
design is advisable. All the ALDH1A isoenzymes are able to catalyze the conversion of the natural
substrate retinal to retinoic acid, but they show preferences for a different isomer of retinal.
ALDH1A3, in particular, has a major affinity for all-trans retinal. It implies some diversity in the
features defining the catalytic site of the different isoforms, which should be taken into
consideration to get to novel active and selective derivatives.13 Indeed, the geometry of the substrate
access tunnel has a central role in ALDHs specificity.
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We started our research project co-crystallizing the reference hit GA11 (3a from here on out), with
the human recombinant ALDH1A3, to begin deciphering its binding mode to the target enzyme
(Figure 1, PDB code:6S6W). The obtained three-dimensional structure of human ALDH1A3 in
complex with 3a has been solved by molecular replacement using the human ALDH1A3 atomic
coordinates as a search model (PDB code: 5FHZ14), and refined at a resolution of 3.2 Å (Figure 1).
The final human ALDH1A3 model contains two identical chains per asymmetric unit, arranged as
dimer, and a total of 11 solvent molecules (Table 1).
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Table 1. Data collection and refinement statistics of the three-dimensional structure of human
ALDH1A3 in complex with compounds 3a (PDB code: 6S6W) and 3q (PDB code: 6TE5).
Data Collection
Compound 3aa
Compound 3bb
Space group
Cell dimensions
a, b, c (Å)
P21221
P21221
81.6 89.4 159.0
90, 90, 90
81.2 89.4 159.2
90, 90, 90
a, b, c (°)
Resolution (Å)
Rpim/Rmerge
48.02 - 3.2
48.02 - 3.2
0.064 (0.399)/0.128 (0.787)
9.8 (2.1)
0.076 (0.399)/0.168 (0.772)
8.9 (2.4)
Mean(I) / sd(I)
Completeness (%)
Redudancy
Refinement
Resolution (Å)
No. reflections
Rwork /Rfree
99.4 (99.3)
99.8 (99.3)
4.7 (4.6)
5.6 (5.9)
3.2
3.2
18833 (1840)
0.19 / 0.26
18833 (1823)
0.18 / 0.24
No. atoms
Protein
7446
11
7446
20
Water
Ligands
121
148
Mean B-factors
Protein (Å2)
77.6
56.7
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Water (Å2)
Ligands (Å2)
52.4
94.8
48.3
82.9
R.m.s. deviations
Bond lengths (Å)
Bond angles (°)
0.006
1.10
0.5
0.014
1.63
0.2
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Ramachandran outliers (%)
aPDB deposition name: GA11. bPDB deposition name: LQ43.
The structure analysis confirms that the obtained 3a-ALDH1A3 complex folds into 13 α-helices, 19
β-strands and the connecting loops, arranged into three functional domains. Both the NAD+ binding
domain and the catalytic domain are built on a topologically related βαβ type polypeptide fold
(Figure 1A). Our crystallographic data revealed that, in all monomers, 3a binds to the enzyme at the
entrance of the tunnel, which therefore turns out to be partially clogged by the compound. It is
important to highlight that 3a is a symmetric compound, and its phenyl rings at position 2 and 6 of
the imidazo[1,2-a]pyridine scaffold can binds the enzyme site in two overturned, almost equivalent
conformations Therefore, to fully unveil the binding mode of the compound, we decided to make
use of docking simulations. Molecular modelling was carried out by GOLD docking program15
towards the crystallographic structure of ALDH1A3 described above, and solved in complex with
3a. Results clearly show that two possible binding modes of 3a are possible, depicted in Figure 2.
They both share a comparable affinity, with a slightly higher theoretical affinity for the binding
mode described in Figure 2A, where the phenyl ring in position 6 of the imidazo[1,2-a]pyridine
scaffold is projected toward the binding site flanked by residues G136, L185, T189, L471, and
A473. It is worth noting that this is the same site occupied by the cyclohexene ring of the natural
ligand retinoic acid, such as recently highlighted by X-ray crystallography.14 By adopting this pose,
3a casts the 2-phenyl ring toward the side chain of the aromatic F308, thus allowing a π-π stacking
interaction which makes the compound binding more effective. No poses were found within the
hydrophobic pocket of the catalytic site, in proximity to the catalytic residues.
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Figure 1. Overall monomer structure of human ALDH1A3 in complex with 3a (PDB code: 6S6W,
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PDB deposition name GA11).
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A
B
NAD binding domain
N-ter
3a
NAD+
monA
C
GA11
3a
C-ter
catalytic domain
oligomerization domain
Panel A. Ribbon representation of the overall structure of the monomer of ALDH1A3. Each
monomer consists of three domains: the NAD+ binding-domain, the catalytic-domain and the
oligomerization domain. The ligands 3a and NAD+ are shown as yellow and grey sticks,
respectively. Panel B. Electron density map SA-omit (2mFo-DFc) of 3a, in grey, contoured at 1
sigma. Panel C. Superimposition between electron density map of 3a SA-omit (2mFo-DFc), in
grey, and 3a 2mFo-DFc, in blue.
Figure 2. Stick representations of the possible docking poses of compound 3a into the binding site
of ALDH1A3 (PDB code: 6S6W).
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The binding site of ALDH1A3 is represented as green ribbons. Residues relevant for the inhibitor
binding have been labelled, and their side chains are shown as green sticks.
Piecing together the inspection of the electron density map of the 3a-ALDH1A3 complex with the
evidences achieved by the molecular modeling study, we resolved to ascribe to 3a the binding pose
depicted in Figure 3. In this conformation, the N1 atom of the scaffold interacts with the carbonyl
oxygen of F131, while the whole heterocyclic core lies in a conserved hydrophobic pocket located
at the entrance of the tunnel that provides substrate access to and product release from, the catalytic
cysteine. Moreover, the phenyl ring in position 6 of the nucleus establishes contacts with the protein
backbone, including π-π stacking interaction with W189 (3.8 Å) and Van der Waals contacts with
G136 (3.6 Å), L185 (3.8 Å) and L471 (4.5 Å) (Figure 3).
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Figure 3. Representation of the main interactions between 3a and the ALDH1A3 binding site (PDB
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code: 6S6W).
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ALDH1A3_GA11 monA
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C314
GA11
F131
L185
E135
G136
W189
4
.
5
2
Ȧ
GA11
A473
L471
Y472
Buried surface areas of monomer A is colored in forest green and the ligand 3a is shown as yellow
stick. Side chains of key residues, involved in 3a binding, are shown in forest green sticks. E135,
F131, G136, L185, W189, A473, L471 and Y472 stabilize through π-π stacking and hydrophobic
interactions the 3a phenyl rings. The carbonyl oxygen of F131 make a hydrogen bond interaction
with N1 of 3a.
The mentioned residues are conserved in each of the 1A1, 1A2 and 1A3 isozymes, being this
enzymatic area the one stabilizing retinoic acid binding in the whole members of the 1A subfamily,
as demonstrated in a previous work.14 ALDH1A3 shows a high homology degree compared to the
other members of the 1A subfamily. Actually, the 1A3 structure can be optimally superimposed
onto human ALDH1A1 (PDB: 4WB916), with a r.m.s.d. of 0.96 Å, and onto human ALDH1A2
(PDB: 6B5G17), with a r.m.s.d. of 0.92 Å, based on 485 equivalent Cpairs (Figure 4).
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Figure 4. Superposition of ALDH1A binding-sites conformations.
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ALDH1A1
ALDH1A2
ALDH1A3
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W189
L185
L471
C19
C18
C20
C17
G136
C21
C16
Y472
C03
C02
C04
C01
E135
A473
N05
C09
C06
N08
1
GA11
C07
sp|P47895|AL1A3_HUMAN
sp|P00352|AL1A1_HUMAN
sp|O94788|AL1A2_HUMAN
H
N
Q
C10
C15
C11
sp|P47895|AL1A3_HUMAN GT
sp|P00352|AL1A1_HUMAN
sp|O94788|AL1A2_HUMAN
F131
GT
GT
C14
C12
C13
Superposition ribbon representation of monomer overall structures of ALDH1A3 in forest green
(PDB code: 6S6W), ALDH1A2 in orange (PDB code: 6B5g), and ALDH1A1 in cyan (PDB code:
4WB9). The three structures maintain the same arrangement. The LigPlus representation of the
complex 3a-ALDH1A3 monomer A, highlights all the eight amino acids involved in 3a binding
site. The ALDH1A3 amino acid residues circled in green are not conserved in ALDH1A1 and
ALDH1A2.
In addition, ALDH1A3 shares 71% amino acid identity with human isozymes 1A1 and 72% amino
acid identity with 1A2. However, as detailed in Figure 5, a few differences in the amino acid
sequences of the three isozymes do exist. Indeed, the superposition of 1A3, 1A1 and 1A2 forms
highlights that three amino acids are not conserved. The hydrophilic F131 of ALDH1A3
corresponds to L120 in ALDH1A1 and Y137 in ALDH1A2, the E135 residue of ALDH1A3
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corresponds to A124 in ALDH1A1 and Q141 in ALDH1A2, while the Y472 residue of ALDH1A3
corresponds to S461 in ALDH1A1 and to N478 in ALDH1A2. The three mentioned residues affect
in particular the binding pocket hosting 3a, thus giving reason to the inhibitory selectivity displayed
by the compound against the three isozymes (Table 2).
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Figure 5. Superposition overview of different amino acids involved in 3a-ALDH1A3 binding.
E135
Q141
F131
A124
Y137
L120
S461
N478
Y472
GA11
130
140
sp|P47895|AL1A3_HUMAN
sp|P00352|AL1A1_HUMAN
sp|O94788|AL1A2_HUMAN
H
N
Q
AFFIDL
AYLNDL
AFYVDL
E
A
Q
GC
GC
GV
IRT
IKT
IKT
L
L
F
ALDH1A1
ALDH1A2
ALDH1A3
470
TVWINCY
TVWVNCY
TVWINCY
N
G
N
AL
VV
AL
Y
S
N
AQ
AQ
AQ
sp|P47895|AL1A3_HUMAN
sp|P00352|AL1A1_HUMAN
sp|O94788|AL1A2_HUMAN
Side chains of key residues involved in the binding (F131, E135, Y472, PDB code: 6S6W) are
shown in forest green sticks. The correspondent not conserved residues of ALDH1A2 (Y137, Q141,
N478, PDB code: 6B5G) and of ALDH1A1 (L120, A124, S461, PDB code: 4WB9) are represented
in orange sticks and cyan sticks, respectively. In the sequence overlapping of the three isoenzymes,
the not conserved residues are circled in green.
Overall, both crystallographic studies and molecular modeling investigations evidence that 3a is
able to bind the entrance of the catalytic site of ALDH1A3, without protruding inside the catalytic
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pocket. Clear docking sites for phenyl rings in positions 2- and 6- of the nucleus were identified,
thus offering interesting clues for the rational optimization of the hit. In particular, they lead to
speculate that the insertion of suitable substituents on the two pendant rings may help to fill in
further the binding pocket of the target proteins, thus getting to novel effective analogues. On the
basis of these premises, we synthesized a number of 6-substituted-imidazo[1,2-a]pyridines, 3a-v
(Schemes 1 and 2) as well as few examples of 7-substituted-imidazo[1,2-a]pyridines, 7a-i (Scheme
3), to shed light on both electronic and geometrical requirements of the imidazo[1,2-a]pyridine
class for the obtainment of ALDH1A3 inhibitors.
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Chemistry. The synthesis of the 6-substituted-imidazo[1,2-a]pyridines 3a-k,p-v was performed as
outlined in Scheme 1. The commercially available 2-amino-5-bromo pyridine 1 was treated with the
suitably substituted 2-bromo-1-phenylethan-1-one, in the presence of potassium carbonate, to
achieve the corresponding 2-phenyl-imidazo[1,2-a]pyridines 2a-e. Coupling the obtained
heterocycles with differently substituted phenylboronic acids, performed in the presence of
Pd(OAc)2 and PPh3 as the catalysts, provided the desired inhibitors. Compounds 3l,m were
synthesized following the alternative synthetic sequence described in Scheme 2, which allowed to
get to the imidazo[1,2-a]pyridine derivatives with comparable yields. Coupling 2-amino-5-
bromopyridine 1 with the appropriate phenylboronic acids, under the experimental conditions
outlined above, gave the intermediate 5-(4-chlorophenyl)pyridin-2-amine 4, which was cyclized to
the desired 2-phenyl-imidazo[1,2-a]pyrimidines 3l,m by reaction with 2-bromo-1-phenylethan-1-
one. 7-Substituted-imidazo[1,2-a]pyridines, 7a-i, represented in Scheme 3, were obtained following
similar reactions. The commercially available 2-amino-4-bromo pyridine 5 and the appropriate 2-
bromo-1-phenylethan-1-ones gave the key intermediates 7-bromo-2-phenylimidazo[1,2-a]pyridines
6a-c, which were converted into the target inhibitors 7a-i by reaction with different phenyl boronic
acids, under the customary coupling conditions in the presence of Pd(OAc)2 and PPh3 as the
catalysts.
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Functional evaluation and modeling studies. The novel 6-phenyl-substituted-imidazo[1,2-
a]pyridine derivatives were tested for both their inhibitory efficacy against the human recombinant
ALDH1A3 and selectivity towards the ALDH1A1 and ALDH1A2 isoforms. The kinetic parameters
were determined for all the compounds, following the experimental protocol described hereafter.
Regarding the reference hit 3a (GA11, IC50 4.7±1.7 M, Table 2), an in depth analysis of its
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functional activity revealed a Ki value of 0.54±0.11 M and a partially competitive mode of action
against the target enzyme ALDH1A3 (Figure 1-SI, Supporting Information). These results are
consistent with structural data, showing that the compound binds to the entrance of the catalytic
tunnel, hindering partially the substrate flow without coordinating directly the catalytic residue
C314. To provide atomistic insights into the mechanism of inhibition, the co-presence of 3a and the
substrate acetaldehyde in the catalytic cavity of ALDH1A3 was investigated by molecular docking
and molecular dynamic (MD) simulations (Figure 6). By referring to the crystallographic complex
between ALDH1A3 and 3a, two poses for acetaldehyde were found, i.e. in proximity of F308 and
close to C314 (Figure 6A). MD simulations on both docking poses clearly showed that, starting
from F308, acetaldehyde quickly leaves the catalytic site and moves to the bulk solvent (Figure 6B).
In contrast, starting from C314, acetaldehyde persists within the catalytic tunnel for around 150 ns
(Figure 6C) before moving to the bulk solvent, being 3a firmly anchored at the entrance of the site.
Overall, the structural analysis suggests that both acetaldehyde and 3a can occupy simultaneously
the ALDH1A3 catalytic site, thus giving rise to a partially competitive mechanism of action.
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Figure 6. Predicted binding mode and MD simulations of acetaldehyde in the 3a-ALDH1A3
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crystallographic complex (PDB code: 6S6W).
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B
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A
C
Panel A: obtained docking poses of acetaldehyde, represented as sticks and either cyan or yellow
balls. Panels B and C: plots of the distances between acetaldehyde and the catalytic C314 along MD
trajectories starting from the docking poses, represented in cyan and yellow respectively, indicated
by black arrows. MD simulations on the cyan pose were stopped after around 150 ns, as the small
molecule detaches from the ALDH1A3 catalytic site and moves to the bulk solvent.
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As for the novel imidazo[1,2-a]pyridine derivatives, most of them proved to inhibit the target
ALDH1A3 isoform when tested at 25 M concentration, displaying different degrees of efficacy
depending on the substitution patterns on the pendant phenyl rings (Figure 2-SI, Supporting
Information). Compounds showing good inhibitory activity at 25 M were investigated further, to
determine their IC50 values (Table 2). Results obtained enable us to draft structure-activity
relationships for the imidazo[1,2-a]pyridine class.
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Table 2. ALDH Inhibitory activity of Selected Imidazo[1,2-a]pyridine Derivatives 3a-v.
IC50 (Ma) or % Inhibitionb
ALDH1A1 ALDH1A2 ALDH1A3
N
R1
R2
R3
H
H
H
19.6±1.6
n.a.c
54.6±1.9
n.a.c
n.a.c
n.a.c
n.a.c
n.a.c
n.a.c
n.a.c
n.a.c
n.a.c
n.a.c
n.a.c
n.a.c
n.a.c
n.a.c
n.a.c
4.7±1.7
22.8±1.6
45.8±1.7
27.1±1.5
17.8±1.5
27.4±1.6
5.3±1.5
5.2±1.7
11.3±1.5
n.a.c
3a
H
H
H
H
4-F
4-Cl
3b
3c
3d
3e
3f
68.8±1.5
n.a.c
H
H
4-SCH3
4-OCH3
4-COOCH3
3-CN
H
H
n.a.c
H
H
96.9±2.2
11.3%
15.9%
22.4%
17.3±1.6
22.9%
17.5%
197.7±3.0
n.a.c
H
H
3h
3i
H
H
4-CN
CH3
Br
H
H
3j
H
4-Cl
3m
3o
3p
3q
3r
3u
3v
OCH3
OCH3
OCH3
CH3
H
H
4-OCH3
3,4-diOCH3
3,5-diOCH3
4-F
11.4±1.6
8.3±1.2
3.5±1.2
92.9±1.8
21.2±1.7
6.4±1.3
H
H
H
OCH3
4-F
n.a.c
n.a.c
H
OCH3
4-Cl
aIC50 values represent the concentration required to produce 50% enzyme inhibition..bPercentage of enzyme
c
inhibition. Standard errors of the means (SEMs)  10 %. Not active. No significant enzyme inhibition was
observed at 25 M.
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In particular, starting from the unsubstituted hit 3a (GA11, IC50 4.7±1.7 M), modifications of the
6-phenyl area alone, through the insertion of increasingly bulky substituents as in compounds 3b-i,
were well tolerated, regardless the electronic properties of the added groups. The best results were
achieved with compounds 3h,i, bearing the electro-withdrawing nitrile group in position meta and
para of the ring, respectively, which turned out to be as potent as the parent hit 3a (3h, IC50 5.3±1.5
M; 3i, IC50 5.2±1.7 M). The inhibitory efficacy of the hit was kept unaltered also by inserting a
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methyl group in position para of the 2-phenyl ring, as in derivative 3j (IC50 11.3±1.5 M).
Double substitution patterns on both the pendant phenyl rings of the hit were explored as well,
pursuing different combinations of both electron-withdrawing and electron-donating substituents. In
this regard, the best results in terms of inhibitory efficacy were obtained by using the electron-
donating methoxy group. Actually, by merging the 2-(4-methoxy)phenyl residue with a mono- or
di-methoxy substitution on the 6-phenyl ring, a relevant activity was obtained and the resulting
compounds, 3o-q, displayed IC50 values in the low micromolar range.
Co-crystallizing 3q (IC50 3.5±1.2 M) with the human ALDH1A3 enzyme (PDB code: 6TE5, PDB
deposition name LQ43, Figure 7), it has been possible to verify that the oxygen atom in position
para on the 2-phenyl ring is involved in a hydrogen bond with Q304. This interaction is
complemented by a further hydrogen bond between the methoxy group in position meta of the 6-
phenyl ring (O2) and the NE1 residue of W189 (3.5 Å) and OG1 of T140 (3.39 Å), which ties
firmly the compound into the ALDH1A3 binding site. The 6-phenyl ring of the compound
establishes also hydrophobic contacts with the protein backbone, including - stacking interaction
with E135 and Y472, crucial residues of ALDH1A3 not conserved in the parent isoenzymes.
Actually, as highlighted in Figure 5, the key Y472 is replaced by S461 and N478 in the 1A1 and
1A2 isoenzymes, respectively, thus accounting for the inhibitory selectivity displayed by 3q (Table
2).
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Figure 7. Overall monomer structure of human ALDH1A3 in complex with 3q (PDB code: 6TE5,
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6
PDB deposition name LQ43).
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NAD binding domain
A
B
N-ter
E135
T140
NAD+
monB
3.
50
Ȧ
W189
3b
LQ43
LQ43
Y472
C
D
catalytic domain
C-ter
oligomerization domain
Ala473
A473
Tyr472
Y472
3b
L471
Leu471
C04
C20
O02
C21
T140
Thr140
C19
O01
C18
C01
C17
C16
Gly136
G136
C06
C07
C05
C08
W189
Trp189
E135
Glu135
N04
LQ43
C09
C03
Lq
N01
C02
C10
C11
C15
C12
3b
C14
C13
O03
C25
Panel A. Ribbon representation of the overall structure of the monomer of ALDH1A3. The LigPlus
representation of the complex 3q-ALDH1A3 monomer B highlights all the amino acids involved in
the binding. The ALDH1A3 amino acid residues circled in green are not conserved in ALDH1A1
and ALDH1A2. Panel B. Compound 3q into the enzyme binding site. The ligand is shown as cyan
stick and the side chains of key residues, involved in the binding, are shown in forest green sticks.
The electron density map (2mFo-dFc) countered at 1.0 sigma, used to determine the presence of 3q
into the enzyme binding site, is in blue. Panel C. Electron density map SA-omit (2mFo-dFc) of 3q,
in grey, contoured at 1 sigma. Panel D. Superimposition between electron density map of 3q SA-
omit (2mFo-dFc), in grey, and 3q 2mFo-dFc, in blue.
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The same order of efficacy was also obtained with compounds 3u (IC50 21.1±1.7 M) and 3v (IC50
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7
8
6.4±1.3 M), bearing a 2-(3-methoxy)phenyl fragment combined with a fluoro and a chloro atom,
respectively, in the para position of the 6-phenyl ring (Table 2).
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Tested against the related dehydrogenase isoforms ALDH1A1 and ALDH1A2, none of the
synthesized compounds but the unsubstituted 3a showed any relevant inhibitory efficacy (Figure 2-
SI, Supporting Information), thus proving that suitably amending the aromatic tails of the
imidazo[1,2-a]pyridine core a complete selectivity between similar enzymes can be achieved.
However, it is worth mentioning derivative 3m, 2-(4-bromophenyl)-6-(4-chlorophenyl)imidazo[1,2-
a]pyridine, whose double substitutions carried out with a 4-bromo and a 4-chloro on the 2- and 6-
phenyl ring, respectively, turned upside down the activity profile of the imidazopyridine class
proving to be fully selective for the ALDH1A1 subtype (IC50 17.3±1.6 M).
To verify that the 2,6-diphenyl motif is the structural geometry that best comply with the structural
requirements of the ALDH1A3 binding site, the series of 7a-i analogues was synthesized and tested
as well. Shifting the phenyl ring to position 7 of the imidazopyridine nucleus modified significantly
the inhibitory activity of the resulting compounds, which was substantially downsized against the
ALDH1A3 isoform and even nullified in the case of ALDH1A1 and ALDH1A2 enzymes (Figure 3-
SI, Supporting Information).
Anti-proliferative activity. Representative examples of the imidazopyridine derivatives, 3b-c,f,v,
chosen among those possessing the best profile of inhibitory activity and selectivity, were tested for
their anti-proliferative efficacy against three different patient-derived glioma sphere samples, both
proneural (PN-157) and mesenchymal (MES-267 and MES-374) ones, already characterized for
their ALDH1 expression patterns.11,12 The 72-hour exposure to the test compounds significantly
inhibited the growth of glioma spheres. All the tested imidazopyridines displayed IC50 values in the
nanomolar/sub-nanomolar range against the different cell subtypes, showing a more marked activity
against the MES-374 line (Table 3). The 6-(4-fluoro)phenyl derivative 3b, combining the best
functional profile in terms of activity and selectivity against the ALDH1 proteins (Table 2)
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confirmed its efficacy also in the cell-based test, showing IC50 values of 25.2 nM, 63.4 nM, and
2.58 pM against the PN-157, MES-267, and MES-374 cell lines, respectively.
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Table 3. Anti-proliferative Activity of Selected Imidazo[1,2-a]pyridine Derivatives 3a-c,e,f,u,v
against 157-, 267-, and 374-GSC lines.
GSC Lines
157 (IC50, nMa)
267 (IC50, nMa)
374 (IC50, nMa)
N
151.0
25.2
46.9
63.4
21.5
660
2.74
0.00258
0.0196
n.t.b
n.tb
n.t.b
3a
3b
3c
3e
3f
53.1
2660
822.0
1510
29.6
823.0
350
3u
3v
71.2
13.5
aIC50 values represent the concentration of drug that decreased cell count by 50%. Standard errors
of the means (SEMs)  10%. bN.t.: not tested.
Concluding Remarks
In this work we described a novel series of 2,6-diphenyl-imidazo[1,2-a]pyridine derivatives as
aldehyde dehydrogenase inhibitors, proving their significance not only as pharmacological tools but
also as promising functional leads. Moving from the previously described hit GA1112 and taking
advantage of both crystallographic studies and molecular modeling investigations, we succeeded in
customizing the selectivity profile of the compound by varying its substitution pattern on both the
pendant phenyl rings. A 2-(4-bromophenyl)-6-(4-chlorophenyl) decoration, as in derivative 3m,
gave a compound fully selective for the ALDH1A1 isoenzyme, while most of the other explored
substituents led to compounds able to inhibit the key ALDH1A3 subtype, accountable for GBM
stem cells vitality being involved in the differentiation of progenitor cells into mature entities. In
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this respect, derivative 3b turned out to be the most promising one. Besides showing the most
efficient combination of inhibitory activity against the target 1A3 isoenzyme and selectivity towards
the parent 1A1 and 1A2, 3b displayed a relevant anti-proliferative efficacy when tested against
well-characterized patient-derived glioma sphere samples, showing IC50 values in the
nanomolar/picomolar range. This compound represents therefore an original example of ALDH
inhibitor whose functional profile may provide, in principle, a novel and viable chance to treat
GBM by targeting stem-like cells. Clearly, thorough investigations in animal models of GBM are
now necessary, to prove its robustness as a novel prototype of drug candidate.
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Experimental Section
Chemistry. Melting points were determined using a Reichert Köfler hot-stage apparatus and are
1
uncorrected. Routine H-NMR and 13C-NMR spectra were recorded in DMSO-d6 solution on a
Bruker 400 spectrometer operating at 400 MHz. Evaporation was performed in vacuo (rotary
evaporator). Analytical TLCs were carried out on Merck 0.2 mm precoated silica gel aluminium
sheets (60 F-254). Purity of the target inhibitors was determined by HPLC analysis, using a Merck
Hitachi D-7000 liquid chromatograph (PDA, 250-500 nm) and a Luna® C18 column (250 mm x
4.6 mm, 5 μm, Phenomenex), with a gradient of 30% water and 70% acetonitrile and a flow rate of
1.0 mL/min. All the compounds showed percent purity values ≥95% (Figures 4-SI to 18-SI,
Supporting Information). 4-Bromopyridin-2-amine, 5-bromopyridin-2-amine, the suitably
substituted 2-bromo-1-phenylethan-1-one, and the appropriate boronic acids, used to obtain the
target inhibitors, were from Alfa Aesar, Aldrich and Fluka. 6-Bromo-2-phenylimidazo[1,2-
a]pyridine
2a,18
6-bromo-2-(p-tolyl)imidazo[1,2-a]pyridine
2b,18
6-bromo-2-(4-
methoxyphenyl)imidazo[1,2-a]pyridine 2c,18 5-(4-chlorophenyl)pyridin-2-amine 4,19 were
synthesized following reported procedures.
General Procedure for the Synthesis of 6-bromo-2-(substituted)phenylimidazo[1,2-
a]pyridines, 2d,e. A solution of 5-bromopyridin-2-amine 1 (1.00 mmol) in ethanol was added with
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the suitably substituted 2-bromo-1-phenylethan-1-one (1.00 mmol) and potassium carbonate (138
mg, 1.00 mmol), and the resulting mixture was refluxed under stirring until the disappearance of the
starting material (TLC analysis). After cooling, the crude obtained was evaporated to dryness, then
water was added to the residue and the desired heterocycle, 2d,e, separated as a white solid, was
collected by filtration, purified through recrystallization from the suitable solvent and characterized
with physio-chemical and spectroscopic data.
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6-Bromo-2-(4-nitrophenyl)imidazo[1,2-a]pyridine, 2d. White solid. M.p. 258-260 °C. Cryst.
1
solvent: Acetonitrile. Yield: 55%. H-NMR (δ, ppm): 8.953 (d, 1H, J=1.20 Hz), 8.603 (s, 1H),
8.321 (d, 2H, J = 8.88 Hz), 8.242 (d, 2H, J = 8.88 Hz), 7.638 (d, 1H, J = 9.60 Hz), 7.445 (dd, 1H, J
= 9.60 Hz, J= 1.71 Hz).
6-Bromo-2-(3-methoxyphenyl)imidazo[1,2-a]pyridine, 2e. White solid. M.p. 232-234 °C. Cryst.
solvent: EtOH. Yield: 76%. 1H-NMR (δ, ppm): 9.189 (s, 1H), 8.678 (s, 1H), 7.928 (d, 1H, J = 9.28
Hz), 7.853 (d, 1H, J = 9.44 Hz), 7.557 (s, 1H), 7.550 (d, 1H, J = 7.95 Hz), 7.499 (t, J = 8.09 Hz,
1H), 7.091 (d, 1H, J = 7.92 Hz), 3.867 (s, 3H).
General Procedure for the Synthesis of 2,6-(substituted)diphenylimidazo[1,2-a]pyridines, 3a-
k,n-v. A solution of the appropriate imidazo[1,2-a]pyridine derivative 2a-e (1.00 mmol), Pd(OAc)2
(0.10 mmol) and PPh3 (0.20 mmol) in toluene was added with the suitable phenyl boronic acid
(1.50 mmol), dissolved in ethanol, and 2 mL of Na2CO3 2M. The resulting mixture was refluxed
under stirring until the disappearance of the starting material (TLC analysis). After cooling, the
crude obtained was evaporated to dryness and the residue was purified by column chromatography
(silica gel, ethyl acetate/petroleum ether). The pure product was then recrystallized from the
suitable solvent and characterized by physio-chemical and spectroscopic data.
2,6-Diphenylimidazo[1,2-a]pyridine, 3a. White solid. M.p. 179-181 °C. Cryst. solvent: MeOH.
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Yield: 78%. H-NMR (δ, ppm): 8.899 (s, 1H), 8.420 (s, 1H), 7.999 (d, 2H, J = 7.10 Hz), 7.755 (d,
2H, J = 7.20 Hz), 7.679 (d, 1H, J = 9.40 Hz), 7.615 (dd, 1H, J = 9.40 Hz, J = 1.80 Hz), 7.524 (t, 2H,
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J = 7.40 Hz), 7.466 (t, 3H, J = 7.44 Hz), 7.441 (t, 1H, J = 7.32 Hz), 7.343 (t, 1H, J = 7.30 Hz). C-
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NMR (, ppm): 145.45, 144.66, 137.14, 134.32, 129.57, 129.20, 128.24, 128.18, 126.99, 126.07,
125.70, 125.41, 124.47, 117.12, 110.02.
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6-(4-Fluorophenyl)-2-phenylimidazo[1,2-a]pyridine, 3b. White solid. M.p. 173-175 °C. Cryst.
solvent: AcOEt. Yield: 58%. 1H-NMR (δ, ppm): 8.879 (s, 1H), 8.405 (s, 1H), 8.002 (d, 2H, J = 7.70
Hz), 7.786 (dd, 2H, J = 7.10 Hz, J = 5.36 Hz), 7.679 (d, 1H, J = 9.16 Hz), 7.595 (dd, 1H, J = 9.38
Hz, J = 1.86 Hz), 7.463 (t, 2H, J = 6.78 Hz), 7.358 (t, 3H, J = 8.33 Hz). 13C-NMR (, ppm): 163.64,
161.21, 145.40, 144.55, 136.92, 134.17, 133.60, 133.57, 129.22, 129.09, 129.01, 128.31, 126.05,
125.46, 124.83, 124.45, 117.06, 116.51, 116.29, 114.84, 110.05.
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6-(4-Chlorophenyl)-2-phenylimidazo[1,2-a]pyridine, 3c. White solid. M.p. 192-194 °C. Cryst.
solvent: MeOH. Yield: 56%. 1H-NMR (δ, ppm): 8.934 (s, 1H), 8.413 (s, 1H), 8.004 (d, 2H, J = 7.08
Hz), 7.779 (d, 2H, J = 8.60 Hz), 7.690 (d, 1H, J = 9.36 Hz), 7.614 (dd, 1H, J = 9.44 Hz, J = 1.88
Hz), 7.582 (d, 2H, J = 8.61 Hz), 7.466 (t, 2H, J = 7.62 Hz), 7.345 (t, 1H, J = 7.37 Hz). 13C-NMR (,
ppm): 145.54, 144.62, 136.03, 134.23, 129.52, 129.21, 128.74, 128.29, 126.08, 125.13, 124.73,
124.44, 117.19, 110.08.
6-(4-(Methylthio)phenyl)-2-phenylimidazo[1,2-a]pyridine, 3d. White solid. M.p. 158-160 °C.
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Cryst. solvent: Acetonitrile. Yield: %. H-NMR (δ, ppm): 8.873 (s, 1H), 8.394 (s, 1H), 7.990 (d,
2H, J = 7.32 Hz), 7.678 (d, 2H, J = 8.30), 7.590 (d, 2H, J = 7.60 Hz), 7.493-7.329 (m, 5H), 3. 603
(s, 3H). 13C-NMR (, ppm): 145.39, 144.58, 138.37, 134.30, 133.56, 129.20, 128.24, 127.37,
126.95, 126.05, 125.18, 125.11, 124.09, 117.12, 109.99, 15.13.
6-(4-Methoxyphenyl)-2-phenylimidazo[1,2-a]pyridine, 3e. White solid. M.p. 173-175 °C. Cryst.
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solvent: EtOH. Yield: %. H-NMR (δ, ppm): 8.765 (s, 1H), 8.369 (s, 1H), 7.944 (d, 2H, J = 7.81
Hz), 7.631 (d, 4H, J = 8.78), 7.444 (t, 2H, J = 6.84 Hz), 7.327 (t, 1H, 6.35), 7.047 (d, 2H, J = 7.32
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Hz), 3. 809 (s, 3H). C-NMR (, ppm): 159.56, 145.14, 144.42, 134.27, 129.37, 129.20, 128.15,
126.03, 125.52, 123.63, 116.96, 115.03, 109.92, 55.71.
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Methyl 4-(2-phenylimidazo[1,2-a]pyridin-6-yl)benzoate, 3f. White solid. M.p. 165-168 °C.
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Cryst. solvent: EtOH. Yield: %. H-NMR (δ, ppm): 9.014 (s, 1H), 8.413 (s, 1H), 8.063 (d, 2H, J =
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8.30 Hz), 7.994 (d, 2H, J = 8.31), 7.889 (d, 2H, J = 8.30 Hz), 7.454 (t, 2H, 6.83), 7.328 (t, 1H, J =
7.32 Hz), 3.875 (s, 3H). 13C-NMR (, ppm): 166.52, 166.40, 145.16, 143.74, 139.29, 130.35,
130.31, 130.27. 130.23, 129.74, 129.57, 128.91, 128.88, 127.81, 127.78, 127.45, 127.43, 61.33,
52.71, 52.62, 14.64.
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2-(2-Phenylimidazo[1,2-a]pyridin-6-yl)benzonitrile, 3g. White solid. M.p. 213-215 °C. Cryst.
solvent: EtOH. Yield: 26%. 1H-NMR (δ, ppm): 8.857 (s, 1H), 8.855 (s, 1H), 8.852 (d, 3H, J = 7.76
Hz), 7.879 (t, 1H, J = 7.64), 7.775-7.740 (m, 2H), 7.655 (t, 1H, J = 7.68 Hz), 7.475 (t, 3H, J = 7.72
Hz), 7.355 (t, 1H, J = 7.37 Hz). 13C-NMR (, ppm): 145.85, 144.56, 141.31, 134.36, 134.15,
134.09, 130.81, 129.24, 129.16, 128.43, 126.88, 126.55, 126.16, 123.44, 118.80, 116.94, 111.19,
110.24.
3-(2-Phenylimidazo[1,2-a]pyridin-6-yl)benzonitrile, 3h. White solid. M.p. 170-171 °C. Cryst.
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solvent: EtOH. Yield: 30%. H-NMR (δ, ppm): 9.043 (s, 1H), 8.415 (s, 1H), 8.272 (s, 1H), 8.110
(d, 1H, J = 8.00 Hz), 8.022 (d, 2H, J = 7.30 Hz), 7.881 (d, 1H, J = 7.80), 7.737 (d, 2H, J = 7.77),
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7.707 (s, 1H), 7.470 (t, 2H, J = 7.52), 7.352 (t, 1H, J = 7.28). C-NMR (, ppm): 145.71, 144.68,
138.36, 134.12, 131.69, 130.72, 130.54, 129.20, 128.36, 126.14, 125.44, 125.00, 123.67, 119.18,
117.23, 112.69, 110.12.
4-(2-Phenylimidazo[1,2-a]pyridin-6-yl)benzonitrile, 3i. White solid. M.p. 209-210 °C. Cryst.
solvent: Acetonitrile. Yield: 39%. 1H-NMR (δ, ppm): 9.066 (s, 1H), 8.436 (s, 1H), 8.017 (d, 2H, J =
7.08), 7.979 (d, 4H, J = 10.20 Hz), 7.729 (d, 1H, J = 9.37 Hz), 7.690 (dd, 1H, J = 10.16, Hz, J =
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1.60 Hz), 7.472 (t, 2H, J = 7.60 Hz), 7.354 (t, 1H, J = 7.40). C-NMR (, ppm): 145.81, 144.75,
141.83, 134.10, 133.46, 129.23, 128.40, 127.76, 125.84, 124.91, 119.26, 117.33, 110.67, 110.25.
6-Phenyl-2-(p-tolyl)imidazo[1,2-a]pyridine, 3j. White solid. M.p. 165-167 °C. Cryst. solvent:
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Acetonitrile. Yield: 36%. H-NMR (δ, ppm): 8.879 (s, 1H), 8.359 (s, 1H), 7.887 (d, 2H, J = 8.01
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Hz), 7.739 (d, 2H, J = 7.33 Hz), 7.663 (d, 1H, J = 9.29 Hz), 7.603 (dd, 1H, J = 9.36 Hz, J = 1.76
Hz), 7.520 (t, 2H, J = 7.58 Hz), 7.415 (t, 1H, J = 7.37 Hz), 7.272 (d, 2H, J = 8.09 Hz), 2.353 (s, 3H).
13C-NMR (, ppm): 159.62, 145.38, 144.49, 137.19, 134.52, 130.44, 129.57, 128.14, 127.79,
127.41, 126.97, 126.82, 125.58, 125.21, 124.34, 116.79, 114.67, 108.97, 55.64.
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2-(4-Methoxyphenyl)-6-phenylimidazo[1,2-a]pyridine, 3k. White solid. M.p. 176-179 °C. Cryst.
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solvent: EtOH. Yield: %. H-NMR (δ, ppm): 8.879 (s, 1H), 8.359 (s, 1H), 7.887 (d, 2H, J = 8.01
Hz), 7.739 (d, 2H, J = 7.33 Hz), 7.662 (d, 1H, J = 9.29 Hz), 7.603 (dd, 1H, J = 9.36, Hz, J = 1.76
Hz), 7.521 (t, 2H, J = 7.40 Hz), 7.415 (t, 1H, J = 7.37 Hz), 7.272 (d, 2H, J = 8.09 Hz). 13C-NMR (,
ppm): 145.57, 144.57, 137.54, 137.17, 131.54, 129.78, 129.56, 128.15, 126.96, 126.00, 125.58,
125.23, 124.38, 117.00, 109.55, 21.34.
6-(4-Chlorophenyl)-2-(4-nitrophenyl)imidazo[1,2-a]pyridine, 3n. White solid. M.p. 198-200 °C.
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Cryst. solvent: EtOH. Yield: 28%. H-NMR (δ, ppm): 8.957 (s, 1H), 8.644 (s, 1H), 8.324 (d, 2H, J
= 8.88 Hz), 8.270 (d, 2H, J = 8.89 Hz), 7.789 (d, 2H, J= 8.52 Hz), 7.736 (d, 1H, J = 9.37 Hz), 7.677
(dd, 1H, J = 9.40 Hz, J= 1.56 Hz), 7.588 (d, 2H, J = 8.48 Hz). 13C-NMR (, ppm): 146.92, 144.96,
143.08, 140.72, 135.69, 133.17, 129.48, 128.74, 126.77, 126.03, 124.94, 124.54, 117.47, 112.54.
2,6-Bis(4-methoxyphenyl)imidazo[1,2-a]pyridine, 3o. White solid. M.p. 202-203 °C. Cryst.
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solvent: EtOH. Yield: %. H-NMR (δ, ppm): 8.767 (s, 1H), 8.257 (s, 1H), 7.899 (d, 2H, J = 6.84
Hz), 7.644 (d, 2H, J = 6.84 Hz), 7.566 (d, 2H, J = 6.84 Hz), 7.052 (d, 2H, J = 7.08 Hz), 7,006 (d,
2H, J = 7.08 Hz), 3.794 (s, 6H). 13C-NMR (, ppm): 159.54, 145.19, 144.29, 133.76, 132.48,
131.91, 129.28, 128.13, 126.82, 125.24, 123.51, 116.66, 115.03, 114.66, 108.87, 55.72, 55.63.
6-(3,4-Dimethoxyphenyl)-2-(4-methoxyphenyl)imidazo[1,2-a]pyridine, 3p. White solid. M.p.
245-248 °C. Cryst. solvent: EtOH. Yield: %. 1H-NMR (δ, ppm): 8.805 (s, 1H), 8.259 (s, 1H), 7.806
(d, 2H, J = 8.79 Hz), 7.580 (s, 1H) 7.567 (d, 1H, J = 7.37 Hz), 7.20 (d, 2H, J = 8.06 Hz), 7.039 (d,
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1H, J = 7.56 Hz), 6.998 (d, 2H, J = 8.55 Hz), 3.865 (s, 3H), 3.796 (s, 6H). C-NMR (, ppm):
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159.52, 149.67, 149.12, 145.37, 144.45, 129.83, 127.34, 126.99, 125.53, 125.26, 123.63, 119.16,
116.64, 114.62, 112.74, 110.74, 108.75, 56.12, 56.06, 55.60.
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6-(3,5-Dimethoxyphenyl)-2-(4-methoxyphenyl)imidazo[1,2-a]pyridine, 3q. White solid. M.p.
213-215 °C. Cryst. solvent: EtOH. Yield: %. 1H-NMR (δ, ppm): 9.176 (s, 1H), 8.553 (s, 1H), 8.136
(d, 1H, J = 9.45 Hz), 7.934 (d, 2H, J = 8.84 Hz), 7.903 (d, 1H, J = 9.56 Hz), 7.171 (d, 2H, J = 8.76
Hz), 6.945 (s, 1H), 6.939 (s, 1H), 6.637 (s, 1H), 3.858 (s, 9H). 13C-NMR (, ppm): 161.47, 159.57,
145.52, 144.66, 139.28, 127.38, 126.89, 125.38, 125.21, 124.59, 116.65, 114.64, 109.87, 105.11,
100.05, 55.84, 55.62.
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6-(4-Fluorophenyl)-2-(p-tolyl)imidazo[1,2-a]pyridine, 3r. White solid. M.p. 156-158 °C. Cryst.
solvent: Acetonitrile. Yield: 39%. 1H-NMR (δ, ppm): 8.862 (s, 1H), 8.348 (s, 1H), 7.888 (d, 2H, J =
8.09 Hz), 7.781 (dd, 2H, J = 7.76 Hz, J = 4.36 Hz), 7.656 (d, 1H, J = 9.41 Hz), 7.580 (dd, 1H, J =
9.37 Hz, J = 1.80 Hz), 7.356 (t, 2H, J = 8.87 Hz), 7.270 (d, 2H, J = 7.43 Hz), 2.351 (s, 3H). 13C-
NMR (, ppm): 163.62, 161.19, 145.60, 144.48, 137.54, 133.67, 133.64, 131.51, 129.76, 129.04,
128.96, 126.00, 125.18, 124.65, 124.36, 116.98, 116.46, 116.25, 109.54, 21.32.
6-(4-Chlorophenyl)-2-(p-tolyl)imidazo[1,2-a]pyridine, 3s. White solid. M.p. 188-190 °C. Cryst.
solvent: Acetonitrile. Yield: 36%. 1H-NMR (δ, ppm): 8.914 (s, 1H), 8.353 (s, 1H), 7.889 (d, 2H, J =
8.09 Hz), 7.775 (d, 2H, J = 8.56 Hz), 7.660 (d, 1H, J = 9.32 Hz), 7.597 (dd, 1H, J = 9.12 Hz, J =
1.80 Hz), 7.577 (d, 2H, J = 8.53 Hz), 7.271 (d, 2H, J = 8.01 Hz), 2.352 (s, 3H). 13C-NMR (, ppm):
145.67, 144.54, 137.60, 136.06, 132.97, 131.46, 129.78, 129.50, 128.72, 126.02, 124.95, 124.63,
124.32, 117.06, 109.61, 21.34.
2-(3-Methoxyphenyl)-6-phenylimidazo[1,2-a]pyridine, 3t. White solid. M.p. 140-141 °C. Cryst.
solvent: Acetonitrile. Yield: 39%. 1H-NMR (δ, ppm): 8.877 (s, 1H), 8.433 (s, 1H), 7.741 (d, 2H, J =
7.24 Hz), 7.690 (d, 1H, J = 9.36 Hz), 7.618 (dd, 1H, J = 9.36 Hz, J = 1.76 Hz), 7.574 (s, 1H), 7.560
(d, 1H, J = 7.42), 7.522 (t, 2H, J = 7.44 Hz), 7.418 (t, 1H, J = 7.37), 7.370 (t, 1H, J = 8.20 Hz),
6.912 (dd, 1H, J = 7.91 Hz, J = 1.76 Hz), 3.845 (s, 3H). 13C-NMR (, ppm): 160.13, 145.38, 144.52,
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136.02, 135.64, 133.02, 130.29, 129.52, 128.77, 125.18, 124.70, 124.48, 118.49, 117.20, 114.15,
111.15, 110.38, 55.57.
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6-(4-Fluorophenyl)-2-(3-methoxyphenyl)imidazo[1,2-a]pyridine, 3u. White solid. M.p. 147-148
°C. Cryst. solvent: Acetonitrile. Yield: 27%. 1H-NMR (δ, ppm): 8.865 (s, 1H), 8.424 (s, 1H), 7.785
(dd, 2H, J = 9.00 Hz, J = 5.78 Hz), 7.686 (d, 1H, J = 9.24 Hz), 7.587-7.564 (m, 3H), 7.391-7.337
(m, 3H), 6.916 (d, 1H, J = 9.40 Hz), 3.845 (s, 3H). 13C-NMR (, ppm): 163.65, 161.21, 160.13,
145.30, 144.45, 135.69, 133.63, 133.60, 130.27, 129.10, 129.01, 125.41, 124.80, 124.43, 118.47,
117.11, 116.49, 116.28, 114.10, 111.14, 110.31, 55.55.
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6-(4-Chlorophenyl)-2-(3-methoxyphenyl)imidazo[1,2-a]pyridine, 3v. White solid. M.p. 161-163
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°C. Cryst. solvent: MeOH. Yield: 24%. H-NMR (δ, ppm): 8.911 (s, 1H), 8.426 (s, 1H), 7.777 (d,
2H, J = 6.64 Hz), 7.693 (d, 1H, J = 9.36 Hz), 7.610 (dd, 1H, J = 9.44 Hz, J = 1.76 Hz), 7.371 (t, 1H,
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J = 8.10 Hz), 7.915 (dd, 1H, J = 6.00 Hz, J = 0.40 Hz), 3.844 (s, 3H). C-NMR (, ppm): 160.14,
145.39, 144.52, 136.03, 135.65, 133.02, 130.29, 129.52, 128.76, 125.18, 124.70, 124.49, 118.50,
117.20, 114.15, 111.17, 110.37, 55.58.
Synthesis of 2,6-bis(4-chlorophenyl)imidazo[1,2-a]pyridine, 3l, and 2-(4-bromophenyl)-6-(4-
chlorophenyl)imidazo[1,2-a]pyridine, 3m. A solution of 5-(4-chlorophenyl)pyridin-2-amine19
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(1.00 mmol) in ethanol was added with the suitably substituted 2-bromo-1-phenylethan-1-one (1.00
mmol) and potassium carbonate (138 mg, 1.00 mmol), and the resulting mixture was refluxed under
stirring until the disappearance of the starting material (TLC analysis). After cooling, the crude
obtained was evaporated to dryness, then water was added to the residue and the desired heterocycle
3l,m, separated as a white solid, was collected by filtration, purified through recrystallization from
the suitable solvent and characterized with physio-chemical and spectroscopic data.
2,6-Bis(4-chlorophenyl)imidazo[1,2-a]pyridine, 3l. White solid. M.p. 278-280 °C. Cryst. solvent:
EtOH. Yield: 81%. 1H-NMR (δ, ppm): 9.192 (s, 1H), 8.692 (s, 1H), 8.102 (s, 1H), 8.031 (d, 2H, J =
8.60 Hz), 7.945 (d, 1H, J = 8.90 Hz), 7.842 (d, 2H, J = 8.50 Hz), 7.686-7.638 (m, 4H). 13C-NMR (,
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