Bioinspired imidazo[1,2-a:4,5-c’]dipyridines with dual antiproliferative and anti-migrative properties in human cancer cells: The SAR investigation

Article abstract of DOI:10.1016/j.ejmech.2021.113258

Herein, we report the design, synthesis and evaluation of novel bioinspired imidazo[1,2-a:4,5c’]dipyridines. The structural optimization identified four anti-proliferative compounds. Compounds 11, 18, 19 and 20 exhibited excellent anticancer activities in

Full text of DOI:10.1016/j.ejmech.2021.113258

European Journal of Medicinal Chemistry 218 (2021) 113258
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Bioinspired imidazo[1,2-a:4,5-c’]dipyridines with dual
antiproliferative and anti-migrative properties in human cancer cells:
The SAR investigation
,
b
c
a
a
Abdulrahim A. Alzain a , Lucie Brisson , Pierre-Olivier Delaye , Melanie Penichon ,
ꢀ
ꢀ
d
d
c
a
ꢀ
ꢀ
Stephanie Chadet , Pierre Besson , Stephan Chevalier , Hassan Allouchi ,
Magdi A. Mohamed e , Sebastien Roger
, Cecile Enguehard-Gueiffier
,
f
d
,
g
,
*
a, **
ꢀ
ꢀ
a University of Tours, Faculty of Pharmacy, EA 7502 SIMBA, 31 Avenue Monge, 37200, Tours, France
b University of Gezira, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, P.O box 20, Gezira, Sudan
c
ꢀ
University of Tours, INSERM, UMR 1069 N2C, 10 boulevard Tonnelle, 37032, Tours Cedex, France
d
ꢀ
University of Tours, EA 4245 T2I, 10 boulevard Tonnelle, 37032, Tours Cedex, France
e University of Khartoum, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Khartoum, Sudan
f Jouf University, College of Pharmacy, Department of Pharmaceutical Chemistry, Saudi Arabia
g Institut Universitaire de France, 75006, Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we report the design, synthesis and evaluation of novel bioinspired imidazo[1,2-a:4,5c’]dipyr-
idines. The structural optimization identified four anti-proliferative compounds. Compounds 11, 18, 19
and 20 exhibited excellent anticancer activities in vitro with IC50 of 0.4e5 mM against three human cancer
Received 10 November 2020
Received in revised form
28 January 2021
Accepted 30 January 2021
Available online 13 February 2021
cell lines (MDA-MB-468, MDA-MB-435s and MDA-MB-231). These four compounds induced apoptosis in
MDA-MB-231 cells in a dose-dependent manner, targeting different apoptotic proteins expression: 11
increased the expression of pro-apoptotic Bax protein while 18e20 reduced the level of anti-apoptotic
Bcl-2 protein. Compounds 18 and 19 also reduced MDA-MB-231 cells proliferation as measured by Ki-
67 staining.
Furthermore, compounds were also tested for the ability to inhibit cell migration in the highly
aggressive human MDA-MB-435s cell line. Six compounds of this series (8, 15, 18, 22, 23, 24) inhibited
cell migration by 41e50% while four compounds (20, 25, 27, 30) inhibited the migration by 53e62% in
wound-healing experiments. Interestingly, compound 20 presented both antiproliferative and anti-
migration activities and might be a promising anti-metastatic agent for cancer treatment.
© 2021 Elsevier Masson SAS. All rights reserved.
Keywords:
Imidazo[1,2-a:4,5-c’]dipyridines
Antiproliferative activity
Anti-migration activity
Harmine analogues
1. Introduction
development is a complex cascade that involves a first and neces-
sary step of invasion, with the acquisition of the ability by malig-
Breast cancer is the most commonly occurring cancer in women
worldwide and the second most common cancer overall. From the
World Health Organization, about 2.1 million new patients are
diagnosed with breast cancer each year and about 627,000 died
worldwide from the disease in 2018 [1]. According to the American
Cancer Society, the 5-year survival rate for a localized breast cancer
has improved significantly to 99%, while this rate drops to 26% if the
cancer is diagnosed at a distant metastasis stage [2]. Metastasis
nant cells to degrade and migrate through extracellular matrices to
distant tissues [3]. Inhibition of cancer cells migrative abilities
constitutes a highly interesting approach. Indeed the biggest hurdle
in controlling breast cancer associated mortality is the lack of
effective antimetastatic therapy. Thus, anti-invasive drugs are ur-
gently requested. They would be used in combination with other
anticancer agents, not only for the treatment of localized tumors,
but also in patients presenting with metastases at diagnosis.
ꢀ
* Corresponding author. University of Tours, EA 4245 T2I, 10 boulevard Tonnelle, 37032, Tours Cedex, France.
** Corresponding author.
E-mail addresses: sebastien.roger@univ-tours.fr (S. Roger), cecile.gueiffier@univ-tours.fr (C. Enguehard-Gueiffier).
https://doi.org/10.1016/j.ejmech.2021.113258
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

A.A. Alzain, L. Brisson, P.-O. Delaye et al.
European Journal of Medicinal Chemistry 218 (2021) 113258
[12]. However, imidazo[1,2-a:5,4-b0]dipyridines were lately pre-
sented as anticancer agents inhibiting human prostate cancer cell
DU-145 proliferation with an IC50 of 1.6
compound [13].
mM for the most active
Regarding the similarity between b-carboline and imidazo[1,2-
a:4,5-c’]dipyridine, we got interested in investigating the potential
anticancer activity of our compounds, drawing on all the relevant
literature. Indeed the b-carboline analogues were hugely studied
from decades in the field of oncology [14] and the structure-activity
relationships of this scaffold against cancer cells was described in
2007 by Cao et al. [8a]. Six sites of modulation were characterized
(Fig. 2).
Several harmine derivatives were developed on this model and
studied in order to promote antiproliferative properties. Despite a
high selectivity for cancer cells and an IC50 close to micromolar
concentrations, these derivatives are generally difficult to develop
as lead compounds for pre-clinical studies because of their poor
solubility [15].
Imidazo[1,2-a:4,5-c’]dipyridine structure present a supplemen-
tary nitrogen atom compared to harmine (Fig. 1). This difference
seems tenuous enough to conserve antitumoral properties, but
could critically affect the pharmacokinetic (specially improving the
aqueous solubility) and the reactivity of the scaffold to extend the
Fig. 1. Chemical structures of harmine, as representative
designed imidazo[1,2-a:4,5-c’]dipyridine core.
b-carboline derivative, and
Indeed, a growing body of evidence indicates that the burden put
on the primary tumor during radiotherapy or antiproliferative
treatment may promote further metastatic dissemination and
shorten survival time [4].
Natural product-oriented synthetic derivatives have played an
important role in drug discovery [5], and the majority of chemo-
therapeutic agents currently used in clinical settings are derived
from natural product scaffolds such as paclitaxel and vinblastine. In
recent years, our laboratories have described an efficient one-pot
sequence for the preparation of imidazo[1,2-a:4,5-c’]dipyridines
scope of the functionalization. Based on the SAR of the
scaffold (Fig. 2), we proposed in a first approach to explore posi-
tions 1, 2, 3, 7 and 10. Nevertheless, conversely to -carboline, we
b-carboline
b
rapidly noticed that position 2 of imidazo[1,2-a:4,5-c’]dipyridines
could not be studied. Indeed, N-2 was less nucleophilic than N-10 in
our series of compounds, and the benzylation did not occurred on
the 2 position but on the 10 position. Finally, we explored positions
1, 3, 7 and 10 (Fig. 3). As antiproliferative activities seemed to be
conserved with the preliminary screened 10-substituted com-
pounds (8 and 10, Table 1) we concluded that the presence but not
the position of this benzyl group is determinant. Moreover, we have
never been confronted to solubility limitation for our new imidazo
[1,2-a:4,5-c’]dipyridines.
[6], analogues of the
occurring alkaloids (Fig. 1) [7].
b
-carboline scaffold present in many naturally
-Carbolines alkaloids such as har-
b
mine and its derivatives display several pharmacological activities
[8], particularly antitumor effects. It is commonly reported that
both natural and synthetic b-carboline alkaloids elicit their anti-
In the present study, we explored the antiproliferative and anti-
migrative activities of this new series of compounds against breast
cancer cells as part of our on-going research, and attempted to
further elucidate the mechanisms of action, providing a basis for
future development of these compounds as human breast cancer
therapy.
cancer activities through multiple mechanisms such as DNA inter-
calation [9], topoisomerase I and II inhibition [10] or kinase
inhibition (CDK, PLKs, IKK, DYRK1A) [11].
Imidazo[1,2-a:4,5-c’]dipyridine scaffold was scarcely studied in
the literature, and to the best of our knowledge only one publica-
tion mentioned an antimalarial activity for such tricyclic system
Fig. 2. The structure-activity relationships of
b-carboline derivatives against tumor cells by Cao et al. [8a].
2

A.A. Alzain, L. Brisson, P.-O. Delaye et al.
European Journal of Medicinal Chemistry 218 (2021) 113258
achieved to avoid the reaction between ester and Grignard reagent.
The starting material 5b with a THP protected hydroxyl group was
first obtained and cyclized to 6b in the same conditions than
described above. After removal of the protecting group in HCl 6M at
room temperature for 1 h, the alcohol was oxidized to carboxylic
acid and esterified to 6i in non-optimized poor yield.
To extend the scope of the pharmacomodulation at position 1, a
new alternative protocol was developed using sodium methoxide
as nucleophile, in refluxing methanol overnight, in order to obtain
the 1-methoxy substituted compounds 6j-l.
The same synthetic route was then applied to the 6-brominated
starting materials 5e-f leading to compounds 6m-o via the
Grignard procedure and compound 6p after sodium methoxide
reaction (Scheme 3).
Finally, the 6-brominated imidazo[1,2-a:4,5-c’]dipyridines 6o-p
were involved in a Suzuki-Miyaura metal-catalyzed coupling re-
action leading to 7-methyl or (hetero)aryl-1-phenyl-3-tertiobuty-
limidazo[1,2-a:4,5-c’]dipyridines 7a-d or 1-methoxy-7-pyridin-4-
yl-3-tertiobutyl analogue 7e (Scheme 4).
All these compounds were converted into quaternary ammo-
nium salts 8e30, after reaction with benzyl bromide or ethyl iodide
in acetonitrile at 80 ꢀC for 24e48 h (Scheme 5, Table 1). The
structure of the salt 8 was confirmed by X-ray crystal structure
analysis (Fig. 4). This transformation not only aimed to improve the
solubility [16] but also to introduce substituent on the 10-position,
which has already been shown to be beneficial for antiproliferative
Fig. 3. Proposed SAR explorations around imidazo[1,2-a:4,5-c’]dipyridine core.
2. Results and discussion
2.1. Chemistry
The starting materials for the preparation of the attempted
imidazo[1,2-a:4,5-c’]dipyridines 6a-p are the 3-alkynyl-2-cyano-
imidazo[1,2-a]pyridines 5a-f.
The synthetic route to 5a-f is presented in Scheme 1 [6]. Esters
1a-b were transformed into amides 2a-b using aqueous solution of
ammonia (30%) at room temperature overnight. Amides 2a-b were
subjected to phosphorus oxychloride at reflux for 4 h to generate
nitriles 3a-b. Iodination at 3 position of 3a was performed as pre-
viously described [6a], when 3b was iodinated in the presence of
NIS overnight at reflux of acetonitrile. The Sonogashira coupling
reaction with various alkynes was then applied to 4 a-b in the
presence of Pd2dba3 and CuI in dioxane at room temperature for
3 h.
Scheme 2 displayed the preparation of the tricyclic 6a-h from
5a-d, by a Grignard reagent addition sequence to the nitrile group,
followed by a 6-endo-dig cyclization on the triple bond at room
temperature for 30 min to 2 h [6]. In order to introduce an ester
function in position 3 of the scaffold, an alternative protocol was
activity against cancer cell lines in the b-carboline series (Fig. 2)
[17]. During this salt formation, compound 6j underwent a deme-
thylation of the methoxy group leading to the 1-hydroxy derivative
22. To avoid this demethylation, the reaction time with benzyl
bromide was limited to 24 h leading to compound 18. Interestingly,
with a pyridine at the C7 position (7c and 7e) the salt did not form
selectively on N10 like for the other compounds but on pyridine
only (30) or on both pyridine and N10 (29), respectively.
Table 1
Compounds 8e30 and their anti-proliferative (cell culture over five days) and cytotoxic (over 24 h) activities (n ¼ 3 independent experiments).
Cpd
R1
R3
R7
R10
Antiproliferative activity (IC50
,
mM)
Cytotox. (IC50, mM)
MDA-MB-468
MDA-MB-435s
MDA-MB-231
4T1
MDA-MB-435s
8
9
Me
Me
Me
Me
Me
Et
Et
Ph
Ph
Ph
OMe
OMe
OMe
OMe
OH
Me
Et
Ph
Ph
Ph
Ph
t-Bu
CO2Et
c-Pr
Ph
t-Bu
t-Bu
c-Pr
t-Bu
c-Pr
c-Pr
t-Bu
c-Pr
Ph
c-Pr
t-Bu
t-Bu
c-Pr
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
Bn
Bn
Bn
Bn
Et
Bn
Et
Bn
Bn
Et
Bn
Bn
Bn
Et
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
H
18.4 6.1
>100
16.4 3.1
>100
n.d.
n.d.
n.d.
2.8 0.2
>100
n.d.
n.d.
n.d.
>100
n.d.
n.d.
n.d.
19.7 5.2
n.d.
n.d.
0.5 0.3
1.5 0.4
6.0 1.3
n.d.
n.d.
n.d.
74.4 28.0
>100
>100
>100
>100
35.4 14.6
>100
21.3 8.6
48.6 2.3
>100
11.9 1.7
16.3 3.3
16.9 2.4
>100
>100
59.6 14.8
>100
29.1 1.7
>100
37.2 5.0
30.8 6.5
98.7 7.9
>100
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Harmine
30.7 11.4
1.2 0.9
99.4 15.0
8.7 1.7
>100
13.3 2.3
31.1 16.1
>100
0.4 0.4
3.6 1.2
4.7 0.2
37.7 5.4
>100
11.0 9.2
47.1 4.7
14.1 11.0
n.d.
43.9 14.1
22.4 1.5
90.5 0.5
4.8 0.3
28.3 4.7
6.4 1.0
13.1 6.5
47.6 20.4
1.1 0.1
1.3 0.1
5.0 3.5
6.3 3.0
33.4 1.2
10.7 0.5
62.2 25.8
5.2 0.4
n.d.
10.7 2.6
>100
11.9 2.3
32.1 4.5
>100
1.7 0.3
3.1 0.4
3.0 0.4
24.8 3.7
>100
36.0 1.1
55.7 12.1
27.6 4.9
>100
Br
Br
Me
Ph
n.d.
31.8 8.3
>100
25.3 2.9
27.7 3.1
18.1 0,7
>100
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
17.3 5.7
62.9 0.4
14.3 2.0
>100
Thien-2-yl
1-benzyl pyridinium-4-yl
1-benzyl pyridinium-4-yl
OMe
Ph
n.d.
17,1 3,7
n.d.
>100
n.d. stands for not determinated.
3

A.A. Alzain, L. Brisson, P.-O. Delaye et al.
European Journal of Medicinal Chemistry 218 (2021) 113258
Scheme 1. Synthetic routes to the compounds 5a-f (THP ¼ tetrahydropyrane, c-propyl ¼ cyclopropyl, n.d. ¼ not determined).
Scheme 2. Synthetic routes to the compounds 6a-l. Reactions conditions for 6i: i: HCl 6M, CH2Cl2, r.t., 1 h, 93%; ii: a) KMnO4, acetone, 40 ꢀC, overnight; b) EtOH, H2SO4, reflux, 4 h,
7%.
Scheme 3. Synthetic route to the brominated compounds 6m-p.
4

A.A. Alzain, L. Brisson, P.-O. Delaye et al.
European Journal of Medicinal Chemistry 218 (2021) 113258
Scheme 4. Synthetic route to the 7-substituted imidazo[1,2-a:4,5-c’]dipyridines 7a-e.
Scheme 5. Formation of quaternary ammonium salts 8e30.
(control) of compounds 8e30 or harmine, at concentrations from
10 nM to 100 M. For each compound, the concentration required
m
to reduce the global growth by 50% (IC50) was determined on each
cell line (Table 1). Dimethylsulfoxyde (DMSO) was used in the
control condition.
Compounds 8e10 from our initial screening, were preliminary
tested for their cancer cell growth inhibitory activity on MDA-MB-
468 and MDA-MB-435s cells (Table 1). Compound 8 exhibited the
best activity with IC50 of 18.4 and 16.4
mM, respectively, compare to
9 (completely inactive) and 10 (30.7 and 43.9
mM). These first
encouraging results gave a trend for the C3 position: t-butyl > c-
propyl [ CO2Et. All the next newly synthetized compounds 11e25
were then tested on the three human cancer cell lines. To complete
the study focusing on the C3 position, the phenyl analogue 11
(R1 ¼ Methyl and R3 ¼ Phenyl) was prepared and exhibited the best
antiproliferative activity against MDA-MB-468. The superiority of
the phenyl group at the C3 position was not recovered with com-
pound 20 (R1 ¼ OMe, R3 ¼ Phenyl) compare to analogues 18
(R1 ¼ OMe, R3 ¼ t-Butyl) and 19 (R1 ¼ OMe, R3 ¼ c-Propyl). In the
methoxylated series, the best compound 18 present a tertio-butyl in
position 3.
Changing the benzyl group (compound 8) to an ethyl group
(compound 12) at the N-10 position resulted in a complete loss of
activity. This observation (benzyl [ Ethyl) was confirmed for
compounds 17 and 21, compare to 16 and 19 respectively, and for
compound 14, regardless of the nature of substituents in other
positions.
Fig. 4. ORTEP diagram for 8 [18].
2.2. Biological evaluation
2.2.1. In vitro antiproliferative activity against MDA-MB-468, MDA-
MB-435, MDA-MB-231 human cancer cells
Determination of in vitro growth inhibitory activity was pre-
liminary performed on three highly aggressive human cancer cell
lines (MDA-MB-468, MDA-MB-435s, MDA-MB-231) using a color-
imetric MTT assay. The best compounds were then tested against
4T1 murine mammary carcinoma in vitro to anticipate the evalua-
tion of our compounds in the experimental mouse model. Cells
were cultured during five days in presence (test) or absence
We then explored the C1 position with compounds 13
(R1 ¼ Ethyl) and 15 (R1 ¼ Phenyl) compare to 8 (R1 ¼ Methyl). These
three compounds with a t-butyl group at the C3 position gave
5

A.A. Alzain, L. Brisson, P.-O. Delaye et al.
European Journal of Medicinal Chemistry 218 (2021) 113258
similar results, with a slightly higher activity of the ethyl group. We
then moved to 1-methoxy substituted compounds 18e21. Besides
compound 21 (R10 ¼ Ethyl), they all exhibited strong anti-
that the relative levels of pro-apoptotic Bax expression was
increased in the presence of 11, while 18e20 reduced the levels of
anti-apoptotic Bcl-2 expression. Furthermore, the four tested
compounds resulted in more significant cleavage of PARP than the
control group. Taken together, these results confirm that 11, 18e20
induced apoptosis in MDA-MB-231 cells. Nevertheless, a different
signaling pathway seems to be impacted by 11 compare to 18e20.
proliferative effect with IC50 from 0.4 to 6.0 mM regardless of the
cancer cell line, whereas harmine presented an IC50 of 17.1 mM
against MDA-MB-435s cells. Compound 18 (R3 ¼ t-Butyl) proved to
be the most active derivative with the lowest IC50 on the three cell
lines. On the opposite, introduction of a hydroxyl group at the C1
position led to a loss of activity (compound 22).
2.2.3. Effect on MDA-MB-231 cell proliferation using Ki-67 staining
In addition to the induction of apoptosis by compounds 11,
18e20, the reduction of cell number could be attributed to a
reduced cell proliferation. Cell proliferation was evaluated using Ki-
Finally, we introduced structural modifications at the C7 posi-
tion. A bromine atom was well tolerated in this position if we
compare the 7-brominated compounds 23 (R1 ¼ Methyl) and 25
(R1 ¼ Phenyl) to the corresponding analogues 8 (R1 ¼ Methyl) and
15 (R1 ¼ Phenyl) with almost no effect on the IC50 values. The
following compounds 26e30 were then tested only on MDA-MB-
231 and 4T1 murine mammary carcinoma. Introduction of a
methyl or thienyl moiety drastically affected the antiproliferative
activity (compounds 26 and 28) while a phenyl or benzylpyr-
idinium group seems to be tolerated in this position (IC50 around
67 staining in MDA-MB-231 cells treated with 30 mM of compounds
11, 18e20 for 48 h. A decrease in nuclear Ki-67 staining indicates a
blockage in cell cycle and a decrease in cell proliferation. All com-
pounds induced a decrease in nuclear Ki-67 expression with
compounds 18 and 19 being the most effective (Fig. 7).
Altogether, compounds 11, 18e20 lead to cancer cell growth
inhibition with slightly different mechanisms. Compounds 18, 19
and 20 are the most potent to induce apoptosis and compounds 18
and 19 also reduced cell proliferation.
15 mM respectively for 27 and 29 on MDA-MB-231). We could again
confirm the critical importance of the benzyl substituent in position
10, with the inactive compound 30.
From the in vitro growth inhibitory activity evaluation, 18
appeared as the most promising antiproliferative agent against the
three highly aggressive human cancer cell lines tested (MDA-MB-
468, MDA-MB-435s, MDA-MB-231) as well as against 4T1 murine
2.2.4. Effects on MDA-MB-435s cells migration in vitro
Cell invasion and migration are critical processes in tumor
metastasis. The inhibitory effect of compounds 8e30 and harmine
on migration of the highly metastatic human cancer cell lines MDA-
MB-435s was investigated using time-lapse videomicroscopy of
mammary carcinoma cells. As in the SAR investigation of the b-
carboline series (Fig. 2), we can observe that the nature of the
substituent in position 1 deeply influences the antiproliferative
efficacy when position 3 is less decisive.
in vitro wound healing. These experiments were conducted at 1 mM,
concentration for which no cytotoxicity at 24 h can be observed
(Fig. 8).
We also measured the cytotoxic activity of these compounds by
assessing cell survival after one-day treatment to exclude any
compound with acute toxic effect that could induce high toxicity
in vivo. Among the best compounds, 11, 18e20 were chosen for
further evaluation because of their low IC50 for antiproliferative
activity and higher IC50 for cytotoxicity test.
In this assay, six compounds inhibited the migration of MDA-
MB-435s cells by 41e50% (compounds 8, 15, 18, 22e24) while
four compounds inhibited the migration by 53e62% (20, 25, 27, 30).
From these ten compounds, eight present a tertio-butyl group in
position 3 and a structural diversity in position 1, which seems to be
less decisive for the anti-migrative activity than for anti-
proliferative activity.
Three closely related compounds 25, 27, and 30 deeply inhibited
2.2.2. Effect on apoptosis in MDA-MB-231 cells
MDA-MB-435s cell migration at 1 mM by 62, 53 and 62%, respec-
A double staining flow cytometric assay using annexin-V fluo-
rescein isothiocyanate (FITC) and propidium iodide (PI) was carried
out to evaluate the potential of compounds 11 and 18e20 to induce
apoptosis in MDA-MB-231 cancer cells. MDA-MB-231 cells were
treated with compounds 11 and 18e20 for 48 h at two different
tively. Their structures present a phenyl group in position 1 and a
tertio-butyl group in position 3. Diversity occurs in position 7 with
either a bromine atom for compound 25, a phenyl group for 27 and
a benzylpyridinium for 30. As the 10-benzyl part is absent in
compound 30, this substituent did not appear to be essential for the
anti-migration activity. Interestingly, compound 20 presented dual
antiproliferative and anti-migration activity with an average IC50 of
concentrations, 3 and 30 mM corresponding to about 1xIC50 and
10xIC50, respectively (Fig. 5). Dimethylsulfoxyde (DMSO) 0.1%, used
as a vehicle for these compounds, was used in the control condition.
The different parts of the graph represented the percentage of
living, apoptotic, and necrotic cell populations. Compared to the
control group with a total apoptosis rate of 2.38%, almost no effect
3.2
MDA-MB-435s cell proliferation, and 43% inhibition in the MDA-
MB-435s migration assay at 1 M (Fig. 6). Conversely, harmine
mM for the inhibition of the MDA-MB-231, MDA-MB-468 and
m
had almost no effect on migration of MDA-MB-435s cells (MDA-
MB-435s cells migration of 93%).
was observed at 3
for compounds 11 and 18e20, respectively. Nevertheless, at 30
mM with apoptosis of 3.30, 3.34, 3.50 and 4.11%
mM
the total apoptosis rate strongly increased for compounds 11,18e20
to 7.78, 6.72, 12.5 and 6.03% respectively. Late apoptotic and
3. Conclusions
necrotic cell population also dramatically increased with 30 mM of
11 (from 2.94% in control group to 13.4%) and 18e20 (from 2.94% to
around 30%).
To further investigate the induction of apoptosis by our com-
pounds, we examined the expression of pro-apoptotic proteins Bax
and Bak, anti-apoptotic Bcl-2 and Bcl-XL, and the cleavage state of
PARP in MDA-MB-231 cells in response to compounds 11, 18e20 at
In conclusion, a series of bioinspired imidazo[1,2-a:4,5-c’]
dipyridines, diversely substituted on positions 1, 3, 7 and 10, were
synthetized and tested for their in vitro antitumor activity against
three strains of highly aggressive human cancer cell lines (MDA-
MB-468, MDA-MB-435s and MDA-MB-231). Among them, com-
pounds 18e20 exhibited excellent cancer inhibitory activity in vitro
the single dose of 30
without (control condition with 0.1% DMSO) the tested compounds
for 48 h and then lysed and analyzed by western blotting. -Actin
expression was used as an internal control (Fig. 6). It was revealed
m
M. MDA-MB-231 cells were treated with or
with IC50 of 0.4e5
harmine activity against MDA-MB-435s. Compound 11 was also
selected for further exploration (IC50 of 1.2 and 2.8
MDA-MB-468 and 231 respectively).
mM against the three cell lines, higher than
b
mM against
6

A.A. Alzain, L. Brisson, P.-O. Delaye et al.
European Journal of Medicinal Chemistry 218 (2021) 113258
Fig. 5. Annexin V/PI staining for detection of apoptosis of MDA-MB-231 cells treated with compounds 11, 18e20 at 3 and 30
experiments).
mM for 48 h (Representative of n ¼ 3 independent
SAR analysis showed that the 10-benzyl group played a critical
role in the antitumor activity, as well as the 1-methoxy group that
deeply improved the antiproliferative effect. On the opposite, the
presence of an ester group on the 3 position or the substitution of
the 7 position seems detrimental.
Furthermore, wound-healing experiments showed that this
series of compounds present also very interesting anti-migration
activity (conversely to harmine that proved to be inactive) with
six compounds inhibiting MDA-MB-435s cell migration by 41e50%
while four compounds (20, 25, 27, 30) inhibited the migration by
53e62%. SAR analysis showed that the nature of the substitution in
position 1 and the presence of a 10-benzyl group are less
discriminant for anti-migration activity. The 7 position appeared to
be important to explore further.
Flow cytometry assay showed that compounds 11 and 18e20
induce apoptosis in MDA-MB-231 cells in
a dose-dependent
manner, compounds 18e20 being more potent than 11. Moreover,
a different signaling pathway seems to be impacted by 11 compare
to 1-methoxylated 18e20: 11 increased the expression of pro-
apoptotic Bax while 18e20 reduced the level of anti-apoptotic
Bcl-2 expression. The effect of these compounds on MDA-MB-
231 cell proliferation using Ki-67 staining revealed that 18 and 19
also reduced cell proliferation.
Interestingly, compound 20 may constitute a promising drug
candidate with dual antiproliferative and anti-migration activity on
aggressive human breast cancer cell lines.
7

A.A. Alzain, L. Brisson, P.-O. Delaye et al.
European Journal of Medicinal Chemistry 218 (2021) 113258
4.1.1. Ethyl 6-bromoimidazo[1,2-a]pyridine-2-carboxylate (1b)
To a solution of 2-amino-5-bromopyridine (8.5 g, 49 mmol,1 eq)
in DME (300 mL) was added dropwise ethyl bromopyruvate (11 mL,
73.7 mmol, 1.5 eq). After stirring for 5 h, the solid was filtered and
washed with DME. The solid was then transfered in a second flask
and heated in refluxing ethanol (500 mL) overnight. The ethanol
was evaporated to dryness and the resulting solid was partitioned
between water and CH2Cl2 (300 mL/300 mL). The aqueous phase
was alkalinized with a saturated solution of Na2CO3 and extracted
with twice 300 mL of CH2Cl2. The combined organic phases were
dried over MgSO4 and evaporated to yield 11.5 g (42,7 mmol, 87%)
of crude product, as a beige solid. 1b was used directly for the next
step without further purification.
Fig. 6. Immunoblot analyses of levels of pro-apoptotic proteins Bax and Bak, anti-
apoptotic Bcl-2 and Bcl-XL, and the cleavage state of PARP in MDA-MB-231 cells in
m.p.: 122e126 ꢀC. 1H NMR (300 MHz, CDCl3)
d: 8.31 (d,
response to compounds 11, 18e20 treatment at 30
independent experiments).
m
M for 48 h (Representative of n ¼ 3
3
4J ¼ 1.0 Hz, 1H, H-5), 8.14 (s, 1H, H-3), 7.61 (d, J ¼ 9.6 Hz, 1H, H-8),
7.32 (dd, 3J ¼ 9.6 Hz, 4J ¼ 1.7 Hz, 1H, H-7), 4.46 (q, 3J ¼ 7.1 Hz, 2H,
OCH2CH3), 1.44 (t, 3J ¼ 7.1 Hz, 3H, OCH2CH3). 13C NMR (75 MHz,
4. Experimental section
CDCl3) d: 162.8 (CO), 143.7 (C-8a), 137.5 (C-2), 130.2 (C-7), 126.3 (C-
5), 119.8 (C-3), 116.9 (C-8), 109.1 (C-6), 61.6 (OCH2CH3), 14.5
4.1. General methods of chemistry
(OCH2CH3). HRMS (ESI): m/z calc. for C10H799BrN2O2 [MþI]þ:
81
268.99202, found: 268.99118; m/z calc. for C
H
BrN2O2 [MþI]þ:
10
9
All reagents were used directly as obtained commercially. Har-
mine was purchased from Sigma-Aldrich (St Louis, MO, USA). Thin-
layer chromatographies (TLC) were performed using Merck® silica
gel 60F254 plates. Column chromatographies were performed using
270.98997, found: 270.98902.
4.1.2. 6-Bromoimidazo[1,2-a]pyridine-2-carboxamide (2b)
1b (11.5 g, 42.7 mmol) was stirred at room temperature over-
night in a mixture of 65 mL of THF and 350 mL of an aqueous so-
lution of ammonia (30%). Solvents were then evaporated to
dryness. The obtained solid was washed with water and CH2Cl2 to
afford 9.35 g of a beige solid (38.9 mmol, 91%). 2b was used directly
for the next step without further purification.
Merck Geduran® Si 60 (40e63 mm) silica. Melting points were
determined on a capillary apparatus (Stuart, Staffordshire, United
Kingdom) and are uncorrected. Microwave heating was performed
using CEM® Explorer SP 12 S class apparatus (maw power 300 W).
NMR experiments were performed at 300 MHz (1H) and 75 MHz
13C) on a Bruker-Avance 300 MHz spectrometer. Assignment of
m.p.: 253e257 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
d: 8.94 (d,
(
4J ¼ 1.0 Hz, 1H, H-5), 8.29 (s, 1H, H-3), 7.76 (bs, 1H, NH), 7.58 (d,
carbons noted C* may be interchanged. Mass spectra were deter-
mined on a Hewlett Packard 5988A spectrometer or on a Shimadzu
QP 2010 spectrometer by direct inlet at 70 eV. Compounds 1a, 2a,
3a, 4a, 5a, 5c, 5d, 6a, 6c, 6d, 6f and 6h were prepared according to
literature [6].
3J ¼ 9.6 Hz, 1H, H-8), 7.46e7.42 (m, 2H, H-7 & NH). 13C NMR
(75 MHz, DMSO‑d6) d: 163.8 (CO), 142.3 (C-8a*), 140.5 (C-2*), 129.1
(C-7), 127.7 (C-5), 118.4 (C-3), 115.1 (C-8), 106.8 (C-6). HRMS (ESI):
m/z calc. for C8H769BrN3O [MþI]þ: 239.97670, found: 239.97592; m/z
Fig. 7. Ki-67 staining for detection of cell proliferation of MDA-MB-231 cells treated with compounds 11, 18e20 at 30
positive cells is indicated (Representative of n ¼ 3 independent experiments).
mM for 48 h. Total nuclei are stained with Dapi. % of Ki-67
8

A.A. Alzain, L. Brisson, P.-O. Delaye et al.
European Journal of Medicinal Chemistry 218 (2021) 113258
4.1.4. 6-Bromo-3-iodoimidazo[1,2-a]pyridine-2-carbonitrile (4b)
To a solution of 3b (8.4 g, 37.8 mmol) in CH3CN (170 mL) was
added NIS (11.1 g, 49.2 mmol, 1.3 eq) in one portion. The reaction
mixture was heated at reflux overnight. The CH3CN was then
evaporated to dryness and the solid was washed several times with
water, petroleum ether and diethyl ether. The crude mixture was
purified by flash chromatography on alumina (CH2Cl2) to afford
10.5 g (30 mmol, 80%) of 4b as a white solid.
m.p.: 275e279 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
d: 8.56 (dd,
4J ¼ 1.7 Hz, 5J ¼ 1.0 Hz, 1H, H-5), 7.67 (dd, 3J ¼ 9.6 Hz, 5J ¼ 0.9 Hz, 1H,
H-8), 7.61 (dd, 3J ¼ 9.6 Hz, 4J ¼ 1.7 Hz, 1H, H-7). 13C NMR (75 MHz,
DMSO‑d6) d: 145.8 (C-8a), 131.6 (C-7), 127.8 (C-5), 124.0 (C-2), 118.9
(C-8), 115.0 (CN), 110.0 (C-6), 78.3 (C-3). HRMS (ESI): m/z calc. for
C8H739BrIN3 [MþI]þ: 347.86278, found: 347.86179; m/z calc. for
C8H831BrIN3 [MþI]þ: 349.86073, found: 349.85975.
4.1.5. General method for Sonogashira cross-coupling (5b, 5e, 5f)
4 (5 mmol), Pd2dba3 (0.25 mmol, 5 mol%) and CuI (0.5 mmol,
10 mol%) were introduced in a sealed tube. Vacuum/refilling with
argon cycles were repeated thrice and dioxane (5.5 mL), Et3N
(5.5 mL) and finally alkyne (1.2 eq) were added. The reaction
mixture was stirred for 3 h at room temperature. The reaction
mixture was then filtered on celite® with CH2Cl2. Organic phase
was washed with a saturated solution of NH4Cl, dried over MgSO4
and evaporated. The crude mixture was purified by flash chroma-
tography with silica or alumina with appropriate eluent.
4.1.6. 3-(3-((tetrahydro-2H-pyran-2-yl)oxy)prop-1-yn-1-yl)
imidazo[1,2-a]pyridine-2-carbonitrile (5b)
Compound 5b was obtained after purification as an inseparable
mixture (80/20) with de-iodinated side-product. It was used like
this for the cyclization.
4.1.7. 6-Bromo-3-(3,3-dimethylbut-1-yn-1-yl)imidazo[1,2-a]
pyridine-2-carbonitrile (5e)
Silica, PE/Et2O 80/20 / 50/50, yellow solid, m.p.: 137e141 ꢀC,
3.26 g. Yield: 94%. 1H NMR (300 MHz, CDCl3)
d
: 8.26 (dd, 4J ¼ 1.8 Hz,
5J ¼ 0.9 Hz, 1H, H-5), 7.53 (dd, 3J ¼ 9.6 Hz, 5J ¼ 0.8 Hz, 1H, H-8), 7.42
Fig. 8. (A) Effect of compound 20 on MDA-MB-435s cell migration in a wound healing
assay using time-lapse video microscopy. Representative photographs taken at 0 h
(T0), 12 h (T12) and 24 h (T24) in the control condition (DMSO) or under the treatment
with compound 20 (1 mM). (B) Effect of the most active compounds on MDA-MB-
435 cells migration for 24 h in wound healing assay. Results are presented as
means SEM of three independent experiments.
(dd, 3J ¼ 9.6 Hz, 4J ¼ 1.8 Hz, 1H, H-7), 1.42 (s, 9H, t-Bu). 13C NMR
(75 MHz, CDCl3) d: 143.1 (C-8a), 131.6 (C-7), 125.4 (C-5), 121.1 (C-2),
119.3 (C-8), 114.7 (C-3*), 113.9 (C-6*), 110.1 (CN), 63.5 (-C^C-t-Bu),
30.7 (CH3 t-Bu), 29.0 (Cq t-Bu). One carbon is missing. HRMS (ESI):
79
m/z calc. for C
H
BrN3 [MþI]þ: 302.02874, found: 302.02784; m/z
14 12
81
calc. for C
H
14 12
BrN3 [MþI]þ: 304.02669, found: 304.02566.
calc. for C8H681BrN3O [MþI]þ: 241.97465, found: 241.97374.
4.1.8. 6-Bromo-3-(cyclopropylethynyl)imidazo[1,2-a]pyridine-2-
carbonitrile (5f)
4.1.3. 6-Bromoimidazo[1,2-a]pyridine-2-carbonitrile (3b)
2b (8.47 g, 35 mmol) was charged in a 250 mL round bottom
flask, followed by POCl3 (66 mL, 705 mmol, 20 eq). The reaction
mixture was heated at reflux for 4 h. POCl3 was then directly
evaporated from the flask, using vacuum pump equipped with ice-
cooled trap. The resulting solid was then suspended in a mixture of
water and CH2Cl2 (200 mL/200 mL), followed by alkalization with
aqueous saturated solution of Na2CO3. The aqueous phase was then
extracted with CH2Cl2 (3 ꢁ 200 mL). The combined organic phases
were dried over MgSO4 and evaporated to yield 6.5 g (29 mmol,
83%) of crude product, as a brown solid. 3b was used directly for the
next step without further purification.
Alumina, CH2Cl2, yellow solid, m.p.: 161e165 ꢀC, 1.05 g. Yield:
74%. 1H NMR (300 MHz, CDCl3)
d
: 8.33 (dd, 4J ¼ 1.8 Hz, 5J ¼ 0.9 Hz,
1H, H-5), 7.52 (dd, 3J ¼ 9.6 Hz, 5J ¼ 0.9 Hz, 1H, H-8), 7.41 (dd,
3J ¼ 9.6 Hz, 4J ¼ 1.8 Hz, 1H, H-7), 1.72e1.58 (m, 1H, CH c-Pr),
1.13e0.95 (m, 4H 2 CH2 c-Pr). 13C NMR (75 MHz, CDCl3)
d: 143.0 (C-
8a),131.6 (C-7),125.5 (C-5),121.3 (C-2),119.3 (C-8),113.9 (C-3), 110.1
(CN*), 110.1 (C-6*), 59.6 (-C^C-c-Pr), 9.9 (CH2 c-Pr), 0.7 (CH c-Pr).
79
One carbon is missing. HRMS (ESI): m/z calc. for C
285.99744, found: 285.99663; m/z calc. for C
287.99539, found: 287.99444.
H
H
BrN3 [MþI]þ:
13
8
BrN3 [MþI]þ:
81
13
8
m.p.: 223e227 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
d
: 8.97 (dd,
4.1.9. General method for cyclization with Grignard reagent
(compounds 6e, 6g, 6i, 6m, 6n and 6o)
4J ¼ 1.8 Hz, 5J ¼ 0.9 Hz,1H, H-5), 8.69 (s,1H, H-3), 7.66 (d, 3J ¼ 9.7 Hz,
1H, H-8), 7.57 (dd, 3J ¼ 9.7 Hz, 4J ¼ 1.9 Hz, 1H, H-7). 13C NMR
To a solution of 3-alkynylimidazo[1,2-a]pyridine-2-carbonitriles
(e.g. 1 mmol) in CPME (10 mL) were added dropwise Grignard re-
agent (2 eq) at room temperature. After completion of the reaction
(followed by TLC, ca. 30min-2h), 10 mL of NH4Cl aqueous saturated
solution were added. After 30 min of hydrolysis the phases are
(75 MHz, DMSO‑d6) d: 143.3 (C-8a), 130.9 (C-7), 127.7 (C-5), 121.5
(C-3), 118.4 (C-8),116.5 (C-2),115.0 (CN),108.3 (C-6). HRMS (ESI): m/
z calc. for C8H749BrN3 [MþI]þ: 221.96614, found: 221.96547; m/z calc.
for C8H481BrN3 [MþI]þ: 223.96409, found: 223.96336.
9

A.A. Alzain, L. Brisson, P.-O. Delaye et al.
European Journal of Medicinal Chemistry 218 (2021) 113258
separated and aqueous phase was extracted with thrice 15 mL of
EtOAc. The crude mixture was purified by flash chromatography on
silica gel (petroleum ether/diethyl ether mixture).
between 10 mL of a saturated aqueous solution of Na2CO3 and
10 mL of CHCl3. The aqueous solution was extracted two more times
with 10 mL of CHCl3. The combined organic phases were dried over
MgSO4 and evaporated. The crude mixture was purified on alumina
column with a mixture CH2Cl2/AcOEt (9/1) as eluent. 17 mg
(0.07 mmol, 7%) of 6i were obtained. Due to the small amount
obtained, 6i was entirely engaged in the salt formation and only the
4.1.10. 3-(tert-Butyl)-1-ethylimidazo[1,2-a:4,5-c']dipyridine (6e)
PE/Et2O: 50/50, yellow solid, m.p.: 121e125 ꢀC, 45 mg. Yield:
40%. 1H NMR (300 MHz, CDCl3)
d
: 8.41 (d, 3J ¼ 6.9 Hz, 1H, H-6), 7.73
(d, 3J ¼ 9.4 Hz, 1H, H-9), 7.60 (s, 1H, H-4), 7.42 (ddd, 3J ¼ 9.3 Hz,
proton NMR was recorded. 1H NMR (300 MHz, CDCl3)
d: 8.63 (s,1H),
6.6 Hz, 4J ¼ 1.1 Hz, 1H, H-8), 6.84 (td, 3J ¼ 6.7 Hz, J ¼ 0.9 Hz, 1H, H-
8.53 (d, 3J ¼ 6.9 Hz, 1H), 7.81 (d, 3J ¼ 9.4 Hz, 1H), 7.58 (ddd, 3J ¼ 9.3,
4
7), 3.39 (q, 3J ¼ 7.5 Hz, 2H, CH2CH3), 1.51e1.46 (m, 12H, t-Bu &
6.7 Hz, 4J ¼ 1.2 Hz, 1H), 7.01 (td, 3J ¼ 6.8 Hz, 4J ¼ 0.9 Hz, 1H), 4.53 (q,
CH2CH3). 13C NMR (75 MHz, CDCl3)
d
: 160.0 (C-3), 155.7 (C-1), 148.2
3J ¼ 7.1 Hz, 2H), 3.08 (s, 3H), 1.48 (t, J ¼ 7.1 Hz, 3H).
3
(C-9a), 137.1 (C-10a), 133.4 (C-4a), 129.9 (C-8), 125.6 (C-6), 119.0 (C-
9), 110.9 (C-7), 97.6 (C-4), 37.8 (Cq t-Bu), 30.8 (CH3, t-Bu), 27.2
(CH2CH3), 13.0 (CH2CH3). HRMS (ESI): m/z calc. for C16H19N3
[MþI]þ: 254.16517, found: 254.16426.
4.1.13. 7-Bromo-3-(tert-butyl)-1-methylimidazo[1,2-a:4,5-c']
dipyridine (6m)
PE/Et2O: 10/90, yellow solid, m.p.: 226e230 ꢀC, 90 mg. Yield:
42%. 1H NMR (300 MHz, CDCl3)
d: 8.54 (s, 1H, H-6), 7.62 (d,
4.1.11. 3-(tert-Butyl)-1-phenylimidazo[1,2-a:4,5-c']dipyridine (6g)
PE/Et2O: 50/50, yellow solid, m.p.: 128e132 ꢀC, 105 mg. Yield:
3J ¼ 9.8 Hz, 1H, H-9), 7.57 (s, 1H, H-4), 7.44 (d, 3J ¼ 9.8 Hz, 1H, H-8),
2.97 (s, 3H, CH3),1.46 (s, 9H, t-Bu). 13C NMR (75 MHz, CDCl3)
d: 160.8
77%. 1H NMR (300 MHz, CDCl3)
d: 8.93e8.91 (m, 2H, Ph-2,6), 8.42
(C-3), 151.8 (C-1), 146.4 (C-9a), 137.9 (C-10a), 133.3 (C-8), 132.9 (C-
(d, 3J ¼ 6.9 Hz, 1H, H-6), 7.76 (d, 3J ¼ 9.4 Hz, 1H, H-9), 7.72 (s, 1H, H-
4), 7.60e7.55 (m, 2H, Ph-3,5), 7.47e7.39 (m, 2H, Ph-4 & H-8), 6.83
(td, 3J ¼ 6.8 Hz, 4J ¼ 0.7 Hz, 1H, H-7), 1.56 (s, 9H, t-Bu). 13C NMR
4a), 125.8 (C-6), 119.7 (C-9), 105.3 (C-7), 97.8 (C-4), 37.7 (Cq t-Bu),
79
30.7 (CH3 t-Bu), 20.7 (Me). HRMS (ESI): m/z calc. for C15
[MþI]þ: 318.06004, found: 318.05912; m/z calc. for C15
[MþI]þ: 320.05799, found: 320.05698.
H
H
BrN3
BrN3
16
81
16
(75 MHz, CDCl3) d: 159.8 (C-3), 148.7 (C-9a), 147.7 (C-1), 138.8 (Ph-
1), 137.0 (C-10a*), 135.2 (C-4a*), 130.2 (C-8), 129.5 (Ph-2,6), 128.9
(Ph-4),128.5 (Ph-3,5),125.5 (C-6),119.1 (C-9),111.0 (C-7), 98.8 (C-4),
38.0 (Cq t-Bu), 30.9 (CH3 t-Bu). HRMS (ESI): m/z calc. for C20H19N3
[MþI]þ: 302.16517, found: 302.16415.
4.1.14. 7-Bromo-3-cyclopropyl-1-ethylimidazo[1,2-a:4,5-c']
dipyridine (6n)
CH2Cl2/EtOAc: 70/30 / 60/40, tan solid, m.p.: 121e125 ꢀC,
4.1.12. Ethyl 1-methylimidazo[1,2-a:4,5-c']dipyridine-3-carboxylate
(6i)
Note: The sequence leading to compound 6i is an un-optimized
sequence which was performed only once.
164 mg. Yield: 74%. 1H NMR (300 MHz, CDCl3)
d: 8.46 (dd,
4J ¼ 1.5 Hz, 5J ¼ 0.6 Hz, 1H, H-6), 7.60 (dd, 3J ¼ 9.6 Hz, 5J ¼ 0.6 Hz, 1H,
H-9), 7.43 (dd, 3J ¼ 9.6 Hz, 4J ¼ 1.8 Hz,1H, H-8), 7.36 (s,1H, H-4), 3.30
(q, 3J ¼ 7.5 Hz, 2H, CH2CH3), 2.25e2.16 (m, 1H, CH c-Pr), 1.43 (t,
3J ¼ 7.5 Hz, 3H, CH2CH3), 1.11e0.99 (m, 4H, 2 CH2 c-Pr). 13C NMR
Cyclization was performed on 5b (981 mg: mixture with de-
iodinated side-product, 80/20) following general procedure.
529 mg of 1-methyl-3-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)
imidazo[1,2-a:4,5-c']dipyridine (6b) were obtained. 1H NMR
(75 MHz, CDCl3) d: 157.2 (C-1), 153.9 (C-3), 146.1 (C-9a), 137.6 (C-
10a),133.3 (C-8),125.7 (C-6),119.8 (C-9),105.2 (C-7), 99.2 (C-4), 27.4
(CH2CH3), 17.9 (CH c-Pr), 13.1 (CH2CH3), 9.8 (CH2 c-Pr). One carbon
79
(300 MHz, CDCl3)
d
: 8.41 (d, 3J ¼ 6.9 Hz, 1H), 7.82 (s, 1H), 7.72 (d,
is missing. HRMS (ESI): m/z calc. for C
found: 316.04351; m/z calc. for C
found: 318.04135.
H
H
BrN3 [MþI]þ: 316.04439,
15 14
3
81
3J ¼ 9.4 Hz, 1H), 7.45 (ddd, J ¼ 9.4, 6.6 Hz, 4J ¼ 1.2 Hz, 1H), 6.88 (td,
3J ¼ 6.9 Hz, 4J ¼ 0.9 Hz, 1H), 5.09 (d, 2J ¼ 13.3 Hz, 1H), 4.86e4.81 (m,
2H), 3.98e3.91 (m, 1H), 3.60e3.54 (m, 1H), 3.00 (s, 3H), 1.94e1.55
BrN3 [MþI]þ: 318.04234,
15 14
(m, 6H). 13C NMR (75 MHz, CDCl3)
d: 151.8, 148.5, 148.3, 138.7, 133.1,
4.1.15. 7-Bromo-3-(tert-butyl)-1-phenylimidazo[1,2-a:4,5-c']
dipyridine (6o)
130.3, 125.7, 118.7, 111.2, 101.1, 98.6, 70.2, 62.5, 30.6, 25.4, 20.3, 19.6.
Deprotection of the THP was performed by adding 10 mL of HCl
(6M) to a solution of 6b (529 mg, 1.78 mmol) in CH2Cl2 (40 mL). The
reaction mixture was stirred at room temperature for 1 h. The
aqueous phase was alkalinized with Na2CO3 aqueous saturated
solution and extracted with CH2Cl2 (twice 40 mL). The organic
phase was dried over MgSO4 and then evaporated to give 141 mg of
a yellow solid. The aqueous phase was cooled in an ice-bath for 2 h.
After filtration, 214 mg more of yellow solid was obtained. All the
solids were combined to yield 355 mg of the free alcohol (1-
methylimidazo[1,2-a:4,5-c']dipyridin-3-yl)methanol (1.66 mmol,
PE/Et2O: 10/90, yellow solid, m.p.: 216e220 ꢀC, 230 mg. Yield:
91%. 1H NMR (300 MHz, CDCl3)
d: 8.85e8.83 (m, 2H, Ph-2,6), 8.63 (s,
1H, H-6), 7.76 (d, 3J ¼ 9.7 Hz, 1H, H-9), 7.71 (s, 1H, H-4), 7.60e7.43
(m, 4H, H-8 & Ph-3,4,5), 1.55 (s, 9H, t-Bu). 13C NMR (75 MHz, CDCl3)
d
: 160.7 (C-3), 148.3 (C-1), 146.8 (C-9a), 138.5 (Ph-1), 136.9 (C-4a*),
134.9 (C-10a*), 133.7 (C-8), 129.6 (Ph-2,6), 129.2 (Ph-4), 128.5 (Ph-
3,5), 125.7 (C-6), 119.9 (C-9), 105.4 (C-7), 98.7 (C-4), 38.1 (Cq t-Bu),
79
30.8 (CH3 t-Bu). HRMS (ESI): m/z calc. for C20
380.07569, found: 380.07470; m/z calc. for C
382.07364, found: 382.07253.
H
H
BrN3 [MþI]þ:
18
81
BrN3 [MþI]þ:
20 18
93%). 1H NMR (300 MHz, DMSO‑d6)
d
: 8.84 (d, 3J ¼ 7.0 Hz, 1H), 8.08
(s, 1H), 7.70e7.62 (m, H), 7.08e7.03 (m, 1H), 4.92 (s, 2H), 2.89 (s,
3H). 13C NMR (75 MHz, DMSO‑d6)
d: 152.9, 151.8, 150.3, 138.8, 135.2,
4.1.16. General method for cyclization with sodium methoxide
(compounds 6j-l and 6p)
133.4, 128.2, 118.4, 113.3, 102.5, 65.7, 19.7.
218 mg (1.02 mmol) of (1-methylimidazo[1,2-a:4,5-c']dipyridin-
3-yl)methanol was dissolved in 22 mL of acetone and 403 mg
(2.55 mmol, 2.5 eq) of KMnO4 were added in one portion. The re-
action mixture was heated at 40 ꢀC overnight. The mixture was
then filtrated, the solid washed with water and the filtrate was
evaporated to dryness. The obtained residue was directly used for
the next step and dissolved in 10 mL of EtOH. 250 mL of H2SO4 were
added and the reaction mixture was heated to reflux for 4 h. The
solvent was evaporated to dryness and the residue was partitioned
3-Alkynylimidazo[1,2-a]pyridine-2-carbonitriles (e.g. 1 mmol)
were added in one portion to a 0.4 M solution of MeONa in MeOH
(12 mL). The reaction mixture was refluxed overnight. Methanol
was then evaporated and crude solid was partitioned between
25 mL of water and 25 mL of CH2Cl2. Aqueous phase was extracted
several time with 20 mL of CH2Cl2. The combined organic phases
were dried over MgSO4, filtered and evaporated to dryness. The
crude mixture was purified by flash chromatography on silica gel
with appropriate eluent.
10

A.A. Alzain, L. Brisson, P.-O. Delaye et al.
European Journal of Medicinal Chemistry 218 (2021) 113258
4.1.17. 3-(tert-Butyl)-1-methoxyimidazo[1,2-a:4,5-c']dipyridine
(6j)
4.1.22. 3-(tert-Butyl)-7-methyl-1-phenylimidazo[1,2-a:4,5-c']
dipyridine (7a)
PE/Et2O: 10/90, beige solid, m.p.: 80e84 ꢀC, 45 mg. Yield: 39%.
CH2Cl2, pale yellow solid, m.p.: 190e194 ꢀC, 74 mg. Yield: 89%.
1H NMR (300 MHz, CDCl3)
d
: 8.40 (d, 3J ¼ 6.5 Hz, 1H, H-6), 7.81 (d,
1H NMR (300 MHz, CDCl3)
d: 8.91e8.88 (m, 2H, Ph-2,6), 8.23 (s, 1H,
3J ¼ 9.2 Hz, 1H, H-9), 7.44 (dd, 3J ¼ 8.8 Hz, 4J ¼ 6.8 Hz, 1H, H-8), 7.34
H-6), 7.71e7.68 (m, 2H, H-4 & H-9), 7.59e7.54 (m, 2H, Ph-3,5),
(s, 1H, H-4), 6.92 (t, 3J ¼ 6.4 Hz, 1H, H-7), 4.19 (s, 3H, OCH3), 1.43 (s,
7.46e7.40 (m, 1H, Ph-4), 7.31 (dd, 3J ¼ 9.5 Hz, 4J ¼ 1.7 Hz, 1H, H-8),
9H, t-Bu). 13C NMR (75 MHz, CDCl3)
d: 159.2 (C-3), 154.7 (C-1), 147.2
2.41 (s, 3H, Me), 1.55 (s, 9H, t-Bu). 13C NMR (75 MHz, CDCl3)
d: 159.6
(C-9a), 134.8 (C-4a*), 129.9 (C-8), 126.3 (C-10a*), 125.4 (C-6), 118.8
(C-9), 111.9 (C-7), 94.4 (C-4), 53.6 (OCH3), 37.8 (Cq t-Bu), 30.4 (CH3 t-
Bu). HRMS (ESI): m/z calc. for C15H17N3O [MþI]þ: 256.14444, found:
256.14351.
(C-3), 148.1 (C-9a), 147.7 (C-1), 138.9 (Ph-1), 137.2 (C-10a*), 135.1 (C-
4a*), 133.7 (C-8), 129.5 (Ph-2,6), 128.9 (Ph-4), 128.5 (Ph-3,5), 122.8
(C-6), 120.7 (C-7), 118.5 (C-9), 98.8 (C-4), 38.0 (Cq t-Bu), 30.9 (CH3 t-
Bu), 18.3 (Me). HRMS (ESI): m/z calc. for C21H21N3 [MþI]þ:
316.18082, found: 316.17996.
4.1.18. 3-Cyclopropyl-1-methoxyimidazo[1,2-a:4,5-c']dipyridine
(6k)
4.1.23. 3-(tert-Butyl)-1-phenyl-7-(thien-2-yl)imidazo[1,2-a:4,5-c']
dipyridine (7b)
PE/Et2O: 10/90, yellow solid, m.p.: 188e192 ꢀC, 88 mg. Yield:
CH2Cl2, pale yellow solid, m.p.: 229e233 ꢀC, 90 mg. Yield: 89%.
57%. 1H NMR (300 MHz, CDCl3)
d
: 8.26 (dt, 3J ¼ 6.9 Hz, 4J ¼ 1.0 Hz,
1H NMR (300 MHz, CDCl3)
d: 8.91e8.88 (m, 2H, Ph-2,6), 8.60 (s, 1H,
1H, H-6), 7.69 (d, 3J ¼ 9.3 Hz, 1H, H-9), 7.36 (ddd, 3J ¼ 9.3 Hz, 6.6 Hz,
4J ¼ 1.2 Hz, 1H, H-8), 7.23 (s, 1H, H-4), 6.83 (td, 3J ¼ 6.8 Hz,
4J ¼ 1.0 Hz, 1H, H-7), 4.11 (s, 3H, OCH3), 2.12e2.04 (m, 1H, CH c-Pr),
1.14e1.09 (m, 2H, 2 CHaHb c-Pr), 0.97e0.91 (m, 2H, 2 CHaHb c-Pr).
H-6), 7.80 (dd, 3J ¼ 9.6 Hz, 5J ¼ 0.8 Hz,1H, H-9), 7.75 (s,1H, H-4), 7.71
(dd, 3J ¼ 9.6 Hz, 4J ¼ 1.8 Hz, 1H, H-8), 7.60e7.55 (m, 2H, Ph-3,5),
7.47e7.42 (m, 1H, Ph-4), 7.37e7.35 (m, 2H, thienyl-3,5), 7.15 (dd,
3J ¼ 4.9 Hz, 3.8 Hz, 1H, thienyl-4), 1.57 (s, 9H, t-Bu). 13C NMR
13C NMR (75 MHz, CDCl3)
d: 155.9 (C-1), 151.5 (C-3), 147.3 (C-9a),
(75 MHz, CDCl3)
d: 160.3 (C-3), 148.0 (C-1), 147.8 (C-9a), 139.4
135.0 (C-10a*), 129.2 (C-8), 127.5 (C-4a*), 125.3 (C-6), 119.1 (C-9),
111.4 (C-7), 96.6 (C-4), 53.4 (OCH3), 17.5 (CH c-Pr), 9.3 (CH2 c-Pr).
HRMS (ESI): m/z calc. for C14H13N3O [MþI]þ: 240.11314, found:
240.11230.
(thienyl-2), 138.7 (Ph-1), 137.3 (C-4a), 135.5 (C-10a), 130.4 (C-8),
129.5 (Ph-2,6), 129.0 (Ph-4), 128.5 (Ph-3,5 & thienyl-4), 125.6
(thienyl-5*), 124.4 (thienyl-3*), 121.1 (C-6), 119.6 (C-7), 119.1 (C-9),
98.9 (C-4), 38.1 (Cq t-Bu), 30.9 (CH3 t-Bu). HRMS (ESI): m/z calc. for
C
24H21N3S [MþI]þ: 384.15289, found: 384.15177.
4.1.19. 1-Methoxy-3-phenylimidazo[1,2-a:4,5-c']dipyridine (6l)
PE/EtOAc: 50/50 / 30/70, beige solid, m.p.: 248e252 ꢀC,
4.1.24. 3-(tert-Butyl)-1-phenyl-7-(pyridin-4-yl)imidazo[1,2-a:4,5-
c']dipyridine (7c)
100 mg. Yield: 44%. 1H NMR (300 MHz, CDCl3)
d
: 8.37 (d, 3J ¼ 6.9 Hz,
CH2Cl2/CH2Cl2/MeOH 99/1, pale yellow solid, m.p.:
1H, H-6), 8.15e8.12 (m, 2H, Ph-2,6), 7.80 (s, 1H, H-4), 7.72 (d,
254e258 ꢀC, 72 mg. Yield: 73%. 1H NMR (300 MHz, CDCl3)
d:
3J ¼ 9.4 Hz, 1H, H-9), 7.50e7.35 (m, 4H, H-8 & Ph-3,4,5), 6.89 (t,
8.89e8.86 (m, 2H, Ph-2,6), 8.72 (bs, 2H, pyr-2,6), 8.67 (s, 1H, H-6),
3J ¼ 6.8 Hz, 1H, H-7), 4.30 (s, 3H, OCH3). 13C NMR (75 MHz, CDCl3)
d:
3
7.82 (d, J ¼ 9.6 Hz, 1H, H-9), 7.77 (s, 1H, H-4), 7.67 (dd, 3J ¼ 9.6 Hz,
155.9 (C-1), 148.3 (C-9a), 146.0 (C-3), 139.6 (Ph-1), 135.4 (C-4a),
129.6 (C-8*), 129.4 (C-10a), 128.8 (Ph-3,5), 128.3 (Ph-4*), 126.7 (Ph-
2,6), 125.4 (C-6), 119.3 (C-9), 111.7 (C-7), 96.3 (C-4), 53.8 (OCH3).
HRMS (ESI): m/z calc. for C17H13N3O [MþI]þ: 276.11314, found:
276.11235.
4J ¼ 1.5 Hz, 1H, H-8), 7.56e7.51 (m, 4H, Ph-3,5 & pyr-3,5), 7.45e7.40
(m, 1H, Ph-4), 1.56 (s, 9H, t-Bu). 13C NMR (75 MHz, CDCl3)
d: 160.5
(C-3), 150.7 (pyr-2,6), 148.1 (C-1*), 147.9 (C-9a*), 144.1 (pyr-4), 138.5
(Ph-1), 137.3 (C-4a), 135.5 (C-10a), 129.5 (C-8), 129.5 (Ph-2,6), 129.1
(Ph-4), 128.4 (Ph-3,5), 123.5 (C-6), 122.5 (C-7), 121.2 (pyr-3,5), 119.5
(C-9), 98.9 (C-4), 38.1 (Cq t-Bu), 30.8 (CH3 t-Bu). HRMS (ESI): m/z
calc. for C25H22N4 [MþI]þ: 379.19172, found: 379.19058.
4.1.20. 7-Bromo-3-(tert-butyl)-1-methoxyimidazo[1,2-a:4,5-c']
dipyridine (6p)
CH2Cl2, beige solid, m.p.: 207e211 ꢀC, 143 mg. Yield: 43%. 1H
4.1.25. 3-(tert-Butyl)-1,7-diphenylimidazo[1,2-a:4,5-c']dipyridine
(7d)
NMR (300 MHz, CDCl3)
d
: 8.51 (d, 4J ¼ 1.5 Hz, 1H, H-6), 7.66 (d,
CH2Cl2, orange solid, m.p.: 267e271 ꢀC, 92 mg. Yield: 93%. 1H
3J ¼ 9.7 Hz, 1H, H-9), 7.44 (dd, 3J ¼ 9.7 Hz, 4J ¼ 1.8 Hz, 1H, H-8), 7.30
(s, 1H, H-4), 4.19 (s, 3H, OCH3), 1.43 (s, 9H, t-Bu). 13C NMR (75 MHz,
NMR (300 MHz, CDCl3) d: 8.93e8.89 (m, 2H, 1-Ph-2,6), 8.60 (m, 1H,
H-6), 7.86 (dd, 3J ¼ 9.6 Hz, 5J ¼ 0.8 Hz,1H, H-9), 7.78 (s,1H, H-4), 7.75
(dd, 3J ¼ 9.6 Hz, 4J ¼ 1.8 Hz, 1H, H-8), 7.68e7.67 (m, 2H, 7-Ph-2,6),
7.60e7.41 (m, 6H, 1-Ph-3,4,5 & 7-Ph-3,4,5), 1.56 (s, 9H, t-Bu). 13C
CDCl3) d: 159.7 (C-3), 155.1 (C-1), 145.7 (C-9a), 134.7 (C-10a), 132.7
(C-8), 127.6 (C-4a), 125.5 (C-6), 119.8 (C-9), 105.9 (C-7), 94.2 (C-4),
53.6 (OCH3), 37.8 (Cq t-Bu), 30.4 (CH3 t-Bu). HRMS (ESI): m/z calc.
79
BrN3O [MþI]þ: 334.05495, found: 334.05389; m/z calc.
NMR (75 MHz, CDCl3) d: 160.1 (C-3), 148.1 (C-1*), 148.0 (C-9a*),
for C
for C
H
H
15 16
81
BrN3O [MþI]þ: 336.05290, found: 336.05169.
138.8 (1-Ph-1), 137.4 (7-Ph-1), 136.9 (C-10a), 135.6 (C-4a), 131.3 (C-
8), 129.6 (1-Ph-2,6), 129.4 (1-Ph-3,5*), 129.0 (1-Ph-4*), 128.5 (7-Ph-
3,5*), 128.3 (7-Ph-4*), 127.0 (7-Ph-2,6), 125.6 (C-7), 122.5 (C-6),
119.1 (C-9), 98.9 (C-4), 38.1 (Cq t-Bu), 30.1 (CH3 t-Bu). HRMS (ESI):
m/z calc. for C26H23N3 [MþI]þ: 378.19647, found: 378.19548.
15 16
4.1.21. General method for Suzuki-Miyaura cross-coupling
(compounds 7a-e)
7-Bromo-imidazo[1,2-a:4,5-c']dipyridines (6n-o) (e.g. 100 mg),
Pd(PPh3)4 (10 mol%), Na2CO3 (2 eq) and boronic acid (1.1 eq) were
introduced in a microwave reaction vial. DME (1 mL) and water
4.1.26. 3-(tert-Butyl)-1-methoxy-7-(pyridin-4-yl)imidazo[1,2-
a:4,5-c']dipyridine (7e)
(500
m
L) were then added. The mixture was heated using micro-
CH2Cl2/MeOH 99/1/CH2Cl2/MeOH 96/4, brown solid, m.p.:
wave oven until completion (ca. 1e3 h). The crude mixture was
partitioned between 20 mL of water and 20 mL of CH2Cl2. Aqueous
phase was then extracted with twice 20 mL of CH2Cl2. The com-
bined organic phases were dried over MgSO4, filtered and evapo-
rated to dryness. The crude mixture was purified by flash
chromatography on silica gel with appropriate eluent.
190e194 ꢀC, 106 mg. Yield: 99%. 1H NMR (300 MHz, CDCl3)
d:
4
5
8.75e8.73 (m, 2H, pyr-2,6), 8.62 (dd, J ¼ 1.5 Hz, J ¼ 0.9 Hz, 1H, H-
6), 7.81 (dd, 3J ¼ 9.6 Hz, 5J ¼ 0.7 Hz, 1H, H-9), 7.65 (dd, 3J ¼ 9.6 Hz,
4J ¼ 1.7 Hz, 1H, H-8), 7.59e7.56 (m, 2H, pyr-3,5), 7.39 (s, 1H, H-4),
4.19 (s, 3H, OCH3), 1.45 (s, 9H, t-Bu). 13C NMR (75 MHz, CDCl3)
d:
159.4 (C-3), 155.2 (C-1), 150.7 (pyr-2,6), 147.0 (C-9a), 144.5 (pyr-4),
11

A.A. Alzain, L. Brisson, P.-O. Delaye et al.
European Journal of Medicinal Chemistry 218 (2021) 113258
135.4 (C-4a*), 128.4 (C-10a*), 128.3 (C-8), 123.4 (C-6), 122.8 (C-7),
121.3 (pyr-3,5), 119.7 (C-9), 94.3 (C-4), 53.5 (OCH3), 37.9 (Cq t-Bu),
30.5 (CH3 t-Bu). HRMS (ESI): m/z calc. for C20H20N4O [MþI]þ:
333.17099, found: 333.16997.
117.4 (C-7), 111.4 (C-9), 103.2 (C-4), 48.3 (CH2Ph), 22.4 (Me). HRMS
(ESI): m/z calc. for C24H20N3 [M]þ: 350.16517, found: 350.16432.
4.1.32. 3-(tert-Butyl)-10-ethyl-1-methylimidazo[1,2-a:4,5-c']
dipyridin-10-ium iodide (12)
4.1.27. General method for salt formation (compounds 8e30)
To a solution of 6a, 6b-p or 7a-e (e.g. 0.5 mmol) in CH3CN (5 mL)
was added benzyl bromide or ethyl iodide (8 eq for 6a, 6b-i, 6m-o
and 7a-d or 1.5 eq for 6j-l, 6p and 7e). The tube was sealed and the
reaction heated to 80 ꢀC for ca. 24e72 h. After cooling in an ice bath
the solid was filtered and washed with EtOAc.
Brown solid, m.p: 244e248 ꢀC, 52 mg. Yield: 63%. 1H NMR
(300 MHz, DMSO‑d6)
d
: 9.72 (d, 3J ¼ 6.9 Hz, 1H, H-6), 8.61 (s, 1H, H-
4), 8.50e8.40 (m, 2H, H-9 & H-8), 7.76 (td, 3J ¼ 6.7 Hz, 4J ¼ 1.2 Hz,
3
1H, H-7), 4.88 (q, J ¼ 7.1 Hz, 2H, CH2CH3), 3.08 (s, 3H, Me), 1.49 (t,
3J ¼ 7.2 Hz, 3H, CH2CH3), 1.44 (s, 9H, t-Bu). 13C NMR (75 MHz,
DMSO‑d6) d: 162.9 (C-3), 143.6 (C-1*), 142.8 (C-9a*), 140.3 (C-8),
133.4 (C-4a), 129.9 (C-6), 125.2 (C-10a), 116.6 (C-7), 111.4 (C-9), 101.8
(C-4), 40.8 (CH2CH3), 37.6 (Cq t-Bu), 30.2 (CH3 t-Bu), 22.7 (Me), 15.2
(CH2CH3). HRMS (ESI): m/z calc. for C17H22N3 [M]þ: 268.18082,
found: 268.17990.
4.1.28. 10-Benzyl-3-(tert-butyl)-1-methylimidazo[1,2-a:4,5-c']
dipyridin-10-ium bromide (8)
Beige solid, m.p.: 138e142 ꢀC, 156 mg. Yield: 69%. 1H NMR
(300 MHz, DMSO‑d6)
d
: 9.84 (d, 3J ¼ 6.6 Hz, 1H, H-6), 8.70 (s, 1H, H-
4), 8.50e8.42 (m, 2H, H-8 & H-9), 7.82 (m, 1H, H-7), 7.40e7.36 (m,
3H, Ph-3,4,5), 7.25e7.23 (m, 2H, Ph-2,6), 6.18 (s, 2H, CH2Ph), 2.76 (s,
4.1.33. 10-Benzyl-3-(tert-butyl)-1-ethylimidazo[1,2-a:4,5-c']
dipyridin-10-ium bromide (13)
3H, CH3), 1.43 (s, 9H, t-Bu). 13C NMR (75 MHz, DMSO‑d6)
d: 163.3 (C-
Beige solid, m.p: 158e162 ꢀC, 30 mg. Yield: 40%. 1H NMR
3), 144.0 (C-1*), 143.6 (C-9a*), 141.0 (C-8), 135.7 (Ph-1),133.9 (C-4a),
130.3 (C-6), 129.1 (Ph-3,5), 128.0 (Ph-4), 125.7 (Ph-2,6), 125.5 (C-
10a), 117.0 (C-7), 111.2 (C-9), 102.0 (C-4), 48.2 (CH2Ph), 37.7 (Cq t-
Bu), 30.2 (CH3 t-Bu), 22.4 (Me). HRMS (ESI): m/z calc. for C22H24N3
[M]þ: 330.19647, found: 330.19572.
(300 MHz, CD3OD)
d
: 9.68 (d, 3J ¼ 6.8 Hz, 1H, H-6), 8.52 (s, 1H, H-4),
8.42 (ddd, 3J ¼ 9.3 Hz, 6.9 Hz, 4J ¼ 0.9 Hz, 1H, H-8), 8.29 (d,
3J ¼ 9.3 Hz, 1H, H-9), 7.78 (t, 3J ¼ 6.9 Hz, 1H, H-7), 7.43e7.21 (m, 5H,
Ph), 6.18 (s, 2H, CH2Ph), 3.23 (q, 3J ¼ 7.4 Hz, 2H, CH2CH3), 1.52 (s, 9H,
t-Bu), 1.30 (t, 3J ¼ 7.4 Hz, 3H, CH2CH3). 13C NMR (75 MHz, CD3OD)
d:
165.8 (C-3), 150.1 (C-1), 145.5 (C-9a), 142.2 (C-8), 136.0 (Ph-1*),
135.4 (C-4a*), 131.1 (C-6), 130.5 (Ph-3,5), 129.5 (Ph-4), 126.7 (Ph-
2,6),126.5 (C-10a),118.5 (C-7),112.3 (C-9),102.5 (C-4), 50.4 (CH2Ph),
39.2 (Cq t-Bu), 30.7 (CH3 t-Bu), 28.7 (CH2CH3), 12.6 (CH2CH3). HRMS
(ESI): m/z calc. for C23H26N3 [M]þ: 344.21212, found: 344.21126.
4.1.29. 10-Benzyl-3-(ethoxycarbonyl)-1-methylimidazo[1,2-a:4,5-
c']dipyridin-10-ium bromide (9)
Beige solid, m.p.: 256e260 ꢀC, 5 mg. Yield: 15%. 1H NMR
(300 MHz, CD3OD)
d
: 9.73 (d, 3J ¼ 6.9 Hz, 1H, H-6), 9.28 (s, 1H, H-4),
8.52 (ddd, 3J ¼ 9.3 Hz, 7.2 Hz, 4J ¼ 0.9 Hz, 1H, H-8), 8.37 (d,
3J ¼ 9.3 Hz, 1H, H-9), 7.87 (t, 3J ¼ 6.9 Hz, 1H, H-7), 7.44e7.37 (m, 3H,
Ph-3,4,5), 7.26e7.23 (m, 2H, Ph-2,6), 6.24 (s, 2H, CH2Ph), 4.55 (q,
3J ¼ 7.1 Hz, 2H, OCH2CH3), 2.99 (s, 3H, CH3), 1.49 (t, 3J ¼ 7.1 Hz, 3H,
4.1.34. 3-Cyclopropyl-1,10-diethylimidazo[1,2-a:4,5-c']dipyridin-
10-ium iodide (14)
Pale yellow solid, m.p.: 256e260 ꢀC, 30 mg. Yield: 15%. 1H NMR
OCH2CH3). 13C NMR (75 MHz, CD3OD)
d
: 165.3 (C-3), 147.8 (C-1),
(300 MHz, DMSO‑d6)
d
: 9.52 (d, 3J ¼ 6.9 Hz, 1H, H-6), 8.47e8.38 (m,
146.6 (C-9a), 143.53 (C-4a), 143.47 (C-8), 135.9 (C-10a), 135.2 (Ph-1),
131.7 (C-6), 130.6 (Ph-3,5), 129.7 (Ph-4), 126.7 (Ph-2,6), 119.4 (C-7),
112.6 (C-9), 110.4 (C-4), 63.5 (OCH2CH3), 50.3 (CH2Ph), 22.4 (Me),
14.6 (OCH2CH3). CO is missing. HRMS (ESI): m/z calc. for C21H20N3O2
[M]þ: 346.15500, found: 346.15400.
3H, H-4, H-8 & H-9), 7.75 (td, 3J ¼ 6.6 Hz, 4J ¼ 1.5 Hz, 1H, H-7), 4.83
(q, 3J ¼ 7.1 Hz, 2H, CH2CH3), 3.32 (q, 3J ¼ 7.5 Hz, 2H, CH2CH3),
2.35e2.27 (m, 1H, CH c-Pr), 1.47 (t, 3J ¼ 7.1 Hz, 3H, CH2CH3), 1.38 (t,
3J ¼ 7.3 Hz, 3H, CH2CH3), 1.13e1.02 (m, 4H, 2 CH2 c-Pr). 13C NMR
(75 MHz, DMSO‑d6) d: 156.2 (C-3), 148.7 (C-1), 142.7 (C-9a), 140.2
(C-8), 133.5 (C-4a), 129.6 (C-6), 124.5 (C-10a), 116.7 (C-7), 111.4 (C-
9), 102.7 (C-4), 41.1 (CH2CH3), 27.1 (CH2CH3), 17.2 (CH c-Pr), 15.0
(CH2CH3), 12.2 (CH2CH3), 10.2 (CH2 c-Pr). HRMS (ESI): m/z calc. for
4.1.30. 10-Benzyl-3-cyclopropyl-1-methylimidazo[1,2-a:4,5-c']
dipyridin-10-ium bromide (10)
Brown solid, m.p.: 158e162 ꢀC, 7 mg. Yield: 39%. 1H NMR
C
17H20N3 [M]þ: 266.16517, found: 266.16450.
(300 MHz, CD3OD)
d
: 9.54 (d, 3J ¼ 6.8 Hz, 1H, H-6), 8.49e8.43 (m,
2H, H-8 & H-4), 8.31 (d, 3J ¼ 9.3 Hz, 1H, H-9), 7.81 (td, 3J ¼ 7.0 Hz,
4J ¼ 0.8 Hz, 1H, H-7), 7.44e7.36 (m, 3H, Ph-3,4,5), 7.25e7.22 (m, 2H,
Ph-2,6), 6.17 (s, 2H, CH2Ph), 2.90 (s, 3H, CH3), 2.47e2.39 (m, 1H, CH
4.1.35. 10-Benzyl-3-(tert-butyl)-1-phenylimidazo[1,2-a:4,5-c']
dipyridin-10-ium bromide (15)
Yellow solid, m.p: 208e212 ꢀC, 54 mg. Yield: 34%. 1H NMR
c-Pr), 1.26e1.20 (m, 4H, 2 CH2 c-Pr). 13C NMR (75 MHz, CD3OD)
d
:
(300 MHz, DMSO‑d6)
d
: 9.94 (d, 3J ¼ 6.6 Hz, 1H, H-6), 8.92 (s, 1H, H-
158.6 (C-3), 146.9 (C-1*), 146.0 (C-9a*), 143.7 (C-8), 137.2 (Ph-1),
135.7 (C-4a), 131.5 (C-6), 130.6 (Ph-3,5), 129.7 (Ph-4), 127.9 (C-10a),
126.8 (Ph-2,6), 119.2 (C-7), 112.5 (C-9), 105.1 (C-4), 50.3 (CH2Ph),
20.6 (Me), 16.8 (CH c-Pr), 11.6 (CH2 c-Pr). HRMS (ESI): m/z calc. for
4), 8.49 (t, 3J ¼ 8.7 Hz, 1H, H-8), 8.42 (d, 3J ¼ 9.0 Hz, 1H, H-9), 7.88 (t,
3J ¼ 6.6 Hz, 1H, H-7), 7.52e7.41 (m, 5H, 1-Ph), 7.20e7.13 (m, 3H,
CH2Ph-3,4,5), 6.56 (d, 3J ¼ 7.2 Hz, 2H, CH2Ph-2,6), 5.64 (s, 2H,
CH2Ph), 1.46 (s, 9H, t-Bu). 13C NMR (75 MHz, DMSO‑d6)
d: 163.3 (C-
C
21H20N3 [M]þ: 314.16517, found: 314.16443.
3), 145.2 (C-1), 144.3 (C-9a),141.5 (C-8), 136.8 (1-Ph-1), 134.9 (C-4a),
134.0 (CH2Ph-1), 130.6 (C-6), 129.4 (1-Ph-4), 129.2 (1-Ph-2,6), 128.5
(1-Ph-3,5*), 128.3 (CH2Ph-3,5*), 127.8 (CH2Ph-4), 125.8 (CH2Ph-
2,6), 124.5 (C-10a), 117.3 (C-7), 111.5 (C-9), 103.3 (C-4), 48.5 (CH2Ph),
37.9 (Cq t-Bu), 30.2 (CH3 t-Bu). HRMS (ESI): m/z calc. for C27H26N3
[M]þ: 392.21212, found: 392.21099.
4.1.31. 10-Benzyl-1-methyl-3-phenylimidazo[1,2-a:4,5-c']dipyridin-
10-ium bromide (11)
Brown solid, m.p: 238e242 ꢀC, 35 mg. Yield: 30%. 1H NMR
(300 MHz, DMSO‑d6)
d
: 9.85 (d, 3J ¼ 6.9 Hz, 1H, H-6), 9.37 (s, 1H, H-
4), 8.54e8.47 (m, 2H, H-8 & H-9), 8.26 (d, 3J ¼ 7.2 Hz, 2H, Ph-2,6),
7.91 (td, 3J ¼ 6.9 Hz, 4J ¼ 0.9 Hz, 1H, H-7), 7.61e7.47 (m, 3H, Ph-
3,4,5), 7.42e7.26 (m, 5H, CH2Ph), 6.23 (s, 2H, CH2Ph); 2.84 (s, 3H,
4.1.36. 10-Benzyl-3-cyclopropyl-1-phenylimidazo[1,2-a:4,5-c']
dipyridin-10-ium bromide (16)
CH3). 13C NMR (75 MHz, DMSO‑d6)
d
: 150.4 (C-3), 144.8 (C-1), 144.2
Beige solid, m.p.: 252e256 ꢀC, 133 mg. Yield: 55%. 1H NMR
(C-9a), 141.5 (C-8), 137.5 (3-Ph-1), 135.7 (CH2Ph-1*), 134.7 (C-4a*),
130.4 (C-6), 129.5 (3-Ph-4), 129.1 (CH2Ph-3,5*), 129.0 (3-Ph-3,5*),
128.1 (CH2Ph-4), 126.8 (C-10a), 126.7 (3-Ph-2,6), 125.7 (CH2Ph-2,6),
(300 MHz, CD3OD)
d
: 9.57 (d, 3J ¼ 6.8 Hz, 1H, H-6), 8.46 (s, 1H, H-4),
8.41 (ddd, 3J ¼ 9.1 Hz, 7.1 Hz, 4J ¼ 1.1 Hz, 1H, H-8), 8.18 (d, 3J ¼ 9.3 Hz,
1H, H-9), 7.79 (td, 3J ¼ 7.0 Hz, 4J ¼ 0.9 Hz,1H, H-7), 7.56e7.49 (m, 1H,
12

A.A. Alzain, L. Brisson, P.-O. Delaye et al.
European Journal of Medicinal Chemistry 218 (2021) 113258
Ph-4), 7.44e7.40 (m, 4H, Ph-2,3,5,6), 7.25e7.14 (m, 3H, Bn-3,4,5),
6.69e6.67 (m, 2H, Bn-2,6), 5.60 (s, 2H, CH2Ph), 2.48e2.39 (m, 1H,
4.1.41. 3-Cyclopropyl-10-ethyl-1-methoxyimidazo[1,2-a:4,5-c']
dipyridin-10-ium iodide (21)
CH c-Pr), 1.16e1.14 (m, 4H, CH2 c-Pr). 13C NMR (75 MHz, CD3OD)
d
:
Yellow solid, m.p.: >260 C, 69 mg. Yield: 47%. 1H NMR (300 MHz,
160.1 (C-3), 148.4 (C-1), 145.9 (C-9a), 142.5 (C-8), 138.0 (1-Ph-1),
136.2 (C-4a), 135.0 (CH2Ph-1), 131.2 (C-6), 130.9 (1-Ph-4), 130.4 (1-
Ph-3,5*), 130.0 (1-Ph-2,6*), 129.5 (CH2Ph-3,5*), 129.2 (CH2Ph-4),
126.8 (CH2Ph-2,6), 126.2 (C-10a), 118.6 (C-7), 112.7 (C-9), 104.7 (C-
4), 50.2 (CH2Ph), 18.3 (CH c-Pr), 11.3 (CH2 c-Pr). HRMS (ESI): m/z
calc. for C26H22N3 [M]þ: 376.18082, found: 376.18001.
DMSO‑d6)
d
: 9.46 (d, 3J ¼ 6.8 Hz, 1H, H-6), 8.45 (d, 3J ¼ 9.3 Hz, 1H, H-
9), 8.36 (ddd, 3J ¼ 9.0 Hz, 6.9 Hz, 4J ¼ 1.2 Hz, 1H, H-8), 8.17 (s, 1H, H-
4), 7.73 (td, 3J ¼ 6.8 Hz, 4J ¼ 1.0 Hz, 1H, H-7), 4.77 (q, 3J ¼ 7.1 Hz, 2H,
CH2CH3), 4.13 (s, 3H, OCH3), 2.28e2.20 (m, 1H, CH c-Pr), 1.45 (t,
3J ¼ 7.1 Hz, 3H, CH2CH3), 1.08e1.06 (m, 4H, 2 CH2 c-Pr). 13C NMR
(75 MHz, DMSO‑d6) d: 155.1 (C-3), 150.8 (C-1), 142.0 (C-9a), 139.2
(C-8), 134.8 (C-4a), 129.4 (C-6), 116.7 (C-7), 114.9 (C-10a),111.6 (C-9),
99.0 (C-4), 54.2 (OCH3), 41.5 (CH2CH3), 17.1 (CH c-Pr), 15.1 (CH2CH3),
9.9 (CH2 c-Pr). HRMS (ESI): m/z calc. for C16H18N3O [M]þ:
268.14444, found: 268.14368.
4.1.37. 3-Cyclopropyl-10-ethyl-1-phenylimidazo[1,2-a:4,5-c']
dipyridin-10-ium iodide (17)
Pale yellow solid, m.p.: 237e241 ꢀC, 103 mg. Yield: 44%. 1H NMR
(300 MHz, DMSO‑d6)
d
: 9.62 (d, 3J ¼ 6.9 Hz, 1H, H-6), 8.63 (s, 1H, H-
4.1.42. 10-Benzyl-3-(tert-butyl)-1-hydroxyimidazo[1,2-a:4,5-c']
dipyridin-10-ium bromide (22)
4), 8.48e8.43 (m, 2H, H-8 & H-9), 7.81 (td, 3J ¼ 6.6 Hz, 4J ¼ 1.5 Hz,
1H, H-7), 7.68e7.60 (m, 5H, Ph), 4.29 (q, 3J ¼ 7.1 Hz, 2H, CH2CH3),
2.45e2.35 (m, 1H, CH c-Pr), 1.16e1.02 (m, 4H, 2 CH2 c-Pr), 0.95 (t,
Yellow solid, m.p.: >260 ꢀC, 31 mg. Yield: 80%. 1H NMR (300 MHz,
CD3OD)
d
: 9.31 (dt, 3J ¼ 6.9 Hz, 4J ¼ 5J ¼ 0.9 Hz, 1H, H-6), 8.24e8.22
3J ¼ 7.1 Hz, 3H, CH2CH3). 13C NMR (75 MHz, DMSO‑d6)
d: 156.8 (C-3),
(m, 2H, H-8 & H-9), 7.69 (m, 1H, H-7), 7.51e7.48 (m, 2H, Ph-2,6),
7.36e7.33 (m, 4H, Ph-3,4,5 & H-4), 6.20 (s, 2H, CH2Ph), 1.48 (s, 9H,
145.9 (C-1), 143.4 (C-9a), 140.8 (C-8), 136.9 (Ph-1), 134.4 (C-4a),
129.8 (C-6*), 129.7 (Ph-4*), 129.1 (Ph-2,6*), 128.5 (Ph-3,5*), 124.4
(C-10a), 117.0 (C-7), 111.7 (C-9), 103.9 (C-4), 40.7 (CH2CH3), 17.3 (CH
c-Pr), 13.5 (CH2CH3), 10.5 (CH2 c-Pr). HRMS (ESI): m/z calc. for
t-Bu). 13C NMR (75 MHz, CD3OD)
d: 156.9 (C-3*), 156.8 (C-1*), 143.4
(C-9a), 139.3 (C-8), 136.1 (Ph-1), 134.9 (C-4a), 130.4 (C-6), 130.2 (Ph-
3,5), 129.8 (Ph-4), 128.8 (Ph-2,6), 120.2 (C-10a), 118.9 (C-7), 112.7 (C-
9), 89.6 (C-4), 49.8 (CH2Ph), 36.8 (Cq t-Bu), 29.3 (CH3 t-Bu). HRMS
(ESI): m/z calc. for C21H22N3O [M]þ: 332.17574, found: 332.17473.
C
21H20N3 [M]þ: 314.16517, found: 314.16421.
4.1.38. 10-Benzyl-3-(tert-butyl)-1-methoxyimidazo[1,2-a:4,5-c']
dipyridin-10-ium bromide (18)
4.1.43. 10-Benzyl-7-bromo-3-(tert-butyl)-1-methylimidazo[1,2-
a:4,5-c']dipyridin-10-ium bromide (23)
White solid, m.p: 188e192 ꢀC, 38 mg. Yield: 50%. 1H NMR
(300 MHz, DMSO‑d6)
d
: 9.71 (d, 3J ¼ 6.6 Hz, 1H, H-6), 8.49e8.38 (m,
Pale yellow solid, m.p.: 220e224 ꢀC, 145 mg. Yield: 99%. 1H NMR
2H, H-8 & H-9), 8.34 (s, 1H, H-4), 7.79 (t, 3J ¼ 6.6 Hz, 1H, H-7),
7.43e7.33 (m, 5H, Ph), 6.03 (s, 2H, CH2Ph), 4.11 (s, 3H, OCH3), 1.43 (s,
(300 MHz, DMSO‑d6)
d
: 10.23 (d, 4J ¼ 1.2 Hz, 1H, H-6), 8.70 (s, 1H, H-
4), 8.66 (dd, 3J ¼ 9.7 Hz, 4J ¼ 1.7 Hz, 1H, H-8), 8.49 (d, 3J ¼ 9.8 Hz, 1H,
H-9), 7.41e7.31 (m, 3H, Ph-3,4,5), 7.24e7.21 (m, 2H, Ph-2,6), 6.19 (s,
2H, CH2Ph), 2.74 (s, 3H, Me), 1.45 (s, 9H, t-Bu). 13C NMR (75 MHz,
9H, t-Bu). 13C NMR (75 MHz, DMSO‑d6)
d: 161.9 (C-3); 149.8 (C-1),
142.8 (C-9a), 140.0 (C-8), 135.3 (C-4a*), 135.1 (Ph-1*), 130.1 (C-6),
128.8 (Ph-3,5), 128.2 (Ph-4), 127.2 (Ph-2,6), 117.0 (C-7), 115.2 (C-
10a), 111.5 (C-9), 98.0 (C-4), 54.0 (OCH3), 48.9 (CH2Ph), 37.8 (Cq t-
Bu), 29.9 (CH3 t-Bu). HRMS (ESI): m/z calc. for C22H24N3O [M]þ:
346.19139, found: 346.19042.
DMSO‑d6) d: 163.6 (C-3), 143.9 (C-1), 143.3 (C-9a), 142.9 (C-8), 135.6
(C-4a), 133.6 (Ph-1), 130.6 (C-6), 129.1 (Ph-3,5), 128.1 (Ph-4), 125.7
(Ph-2,6), 125.5 (C-10a), 112.5 (C-9), 110.7 (C-7), 102.2 (C-4), 48.4
(CH2Ph), 37.7 (Cq t-Bu), 30.1 (CH3 t-Bu), 22.4 (Me). HRMS (ESI): m/z
79
calc. for C
H
BrN3 [M]þ: 408.10699, found: 408.10601; m/z calc.
22 23
81
for C H
22 23
BrN3 [M]þ: 410.10494, found: 410.10392.
4.1.39. 10-Benzyl-3-cyclopropyl-1-methoxyimidazo[1,2-a:4,5-c']
dipyridin-10-ium bromide (19)
4.1.44. 10-Benzyl-7-bromo-3-cyclopropyl-1-ethylimidazo[1,2-
a:4,5-c']dipyridin-10-ium bromide (24)
Off-white solid, m.p: 218e222 ꢀC, 88 mg. Yield: 43%. 1H NMR
(300 MHz, CDCl3)
d
: 10.08 (d, 3J ¼ 6.8 Hz, 1H, H-6), 8.53 (s, 1H, H-4),
Pale yellow solid, m.p.: 232e236 ꢀC, 31 mg. Yield: 20%. 1H NMR
8.35 (d, 3J ¼ 9.3 Hz,1H, H-9), 8.14 (m, 1H, H-8), 7.54 (t, 3J ¼ 6.9 Hz, H-
7), 7.37e7.29 (m, 5H, Ph), 6.21 (s, 2H, CH2Ph), 4.14 (s, 3H, OCH3),
2.34e2.26 (m, 1H, CH c-Pr), 1.17e1.01 (m, 4H, 2 CH2 c-Pr). 13C NMR
(300 MHz, DMSO‑d6)
d
: 10.09 (s, 1H, H-6), 8.65 (dd, 3J ¼ 9.7 Hz,
4J ¼ 1.2 Hz, 1H, H-8), 8.51e8.47 (m, 2H, H-4 & H-9), 7.36e7.34 (m,
3H, Ph-3,4,5), 7.22e7.19 (m, 2H, Ph-2,6), 6.16 (s, 2H, CH2Ph), 3.01 (q,
3J ¼ 7.3 Hz, 2H, CH2CH3), 2.34e2.24 (m, 1H, CH c-Pr), 1.11e1.02 (m,
(75 MHz, CDCl3) d: 158.1 (C-3), 151.0 (C-1), 141.9 (C-9a), 139.5 (C-8),
134.6 (C-4a*), 134.2 (Ph-1*), 131.2 (C-6), 129.5 (Ph-3,5), 129.1 (Ph-
4), 127.5 (Ph-2,6), 117.7 (C-7), 115.0 (C-10a), 112.5 (C-9), 100.3 (C-4),
54.4 (OCH3), 50.5 (CH2Ph), 17.8 (CH c-Pr), 10.8 (CH2 c-Pr). HRMS
(ESI): m/z calc. for C21H20N3O [M]þ: 330.16009, found: 330.15921.
7H, 2CH2 c-Pr & CH2CH3). 13C NMR (75 MHz, DMSO‑d6)
d: 157.1 (C-
3), 149.0 (C-1), 143.3 (C-8), 142.8 (C-9a), 135.2 (Ph-1), 133.7 (C-4a),
130.3 (C-6), 129.0 (Ph-3,5), 128.1 (Ph-4), 125.7 (Ph-2,6), 124.7 (C-
10a), 112.5 (C-9), 110.6 (C-7), 102.8 (C-4), 48.7 (CH2Ph), 26.8
(CH2CH3), 17.3 (CH c-Pr), 12.1 (CH2CH3), 10.4 (2 CH2 c-Pr). HRMS
79
4.1.40. 10-Benzyl-1-methoxy-3-phenylimidazo[1,2-a:4,5-c']
dipyridin-10-ium bromide (20)
(ESI): m/z calc. for C H
BrN3 [M]þ: 406.09134, found: 406.09033;
22 21
81
m/z calc. for C
H
22 21
BrN3 [M]þ: 408.08929, found: 408.08815.
Off-white solid, m.p: 190e194 ꢀC, 48 mg. Yield: 49%. 1H NMR
(300 MHz, DMSO‑d6)
d
: 9.71 (d, 3J ¼ 6.6 Hz, 1H, H-6), 9.06 (s, 1H, H-
4.1.45. 10-Benzyl-7-bromo-3-(tert-butyl)-1-phenylimidazo[1,2-
a:4,5-c']dipyridin-10-ium bromide (25)
4), 8.53e8.42 (m, 2H, H-8 & H-9), 8.26 (d, 3J ¼ 7.5 Hz, 2H, 3-Ph-2,6),
7.86 (t, 3J ¼ 6.6 Hz, 1H, H-7), 7.60e7.31 (m, 8H, 3-Ph-3,4,5 & CH2Ph),
6.08 (s, 2H, CH2Ph), 4.23 (s, 3H, OCH3). 13C NMR (75 MHz, DMSO‑d6)
Yellow solid, m.p.: 156e160 ꢀC, 112 mg. Yield: 33%. 1H NMR
(300 MHz, CDCl3)
d
: 10.67 (d, 4J ¼ 0.9 Hz, 1H, H-6), 9.05 (s, 1H, H-4),
d
: 150.6 (C-1), 148.6 (C-3), 143.0 (C-9a), 140.4 (C-8), 137.1 (C-4a),
8.50 (d, 3J ¼ 9.7 Hz, 1H, H-9), 8.15 (dd, 3J ¼ 9.6 Hz, 4J ¼ 1.1 Hz, 1H, H-
8), 7.57e7.41 (m, 5H, 1-Ph), 7.18e7.10 (m, 3H, CH2Ph-3,4,5),
6.75e6.72 (m, 2H, CH2Ph-2,6), 5.92 (s, 2H, CH2Ph), 1.48 (s, 9H, t-Bu).
135.7 (3-Ph-1), 135.2 (CH2Ph-1), 130.2 (C-6), 129.6 (3-Ph-4), 129.0
(3-Ph-3,5*), 128.8 (CH2Ph-3,5*), 128.2 (CH2Ph-4), 127.2 (CH2Ph-
2,6), 126.5 (3-Ph-2,6), 117.4 (C-7), 116.4 (C-10a), 111.7 (C-9), 99.2 (C-
4), 54.4 (OCH3), 49.0 (CH2Ph). HRMS (ESI): m/z calc. for C24H20N3O
[M]þ: 366.16009, found: 366.15899.
13C NMR (75 MHz, CDCl3)
d: 166.3 (C-3), 146.3 (C-1), 143.2 (C-8),
142.5 (C-9a), 136.7 (1-Ph-1), 133.9 (CH2Ph-1*), 133.4 (C-4a*), 130.7
(C-6), 130.1 (1-Ph-4), 129.7 (1-Ph-2,6), 129.2 (1-Ph-3,5*), 128.8
13

A.A. Alzain, L. Brisson, P.-O. Delaye et al.
European Journal of Medicinal Chemistry 218 (2021) 113258
(CH2Ph-3,5*), 128.7 (CH2Ph-4), 126.4 (CH2Ph-2,6), 124.7 (C10a),
(pyr-CH2Ph-1), 132.3 (C-6), 131.1 (10-CH2Ph-4*), 130.8 (10-CH2Ph-
3,5*), 130.32 (pyr-CH2Ph-3,5*), 130.26 (10-CH2Ph-2,6*), 129.8 (pyr-
CH2Ph-4*), 128.4 (pyr-CH2Ph-2,6*), 127.4 (pyr-3,5), 125.9 (C-7),
117.3 (C-10a), 113.5 (C-9), 99.2 (C-4), 65.4 (pyr-CH2Ph), 54.9 (OCH3),
51.3 (10-CH2Ph), 39.4 (Cq t-Bu), 30.6 (CH3 t-Bu). HRMS (ESI): m/z
calc. for C34H34N4O [M]2þ: 257.13608, found: 257.13525.
114.6 (C-9), 112.1 (C-7), 104.1 (C-4), 50.4 (CH2Ph), 38.8 (Cq t-Bu),
79
30.7 (CH3 t-Bu). HRMS (ESI): m/z calc. for C27
H
BrN3 [M]þ:
25
81
470.12264, found: 470.12126; m/z calc. for C
H
BrN3 [M]þ:
27 25
472.12059, found: 472.11915.
4.1.46. 10-Benzyl-3-(tert-butyl)-7-methyl-1-phenylimidazo[1,2-
a:4,5-c']dipyridin-10-ium bromide (26)
White solid, m.p.: 237e241 ꢀC, 57 mg. Yield: 53%. 1H NMR
4.1.50. 1-Benzyl-4-(3-(tert-butyl)-1-phenylimidazo[1,2-a:4,5-c']
dipyridin-7-yl)pyridin-1-ium bromide (30)
(300 MHz, CD3OD) d: 9.56 (s, 1H, H-6), 8.62 (s, 1H, H-4), 8.30 (dd,
3J ¼ 9.4 Hz, 4J ¼ 1.5 Hz, 1H, H-8), 8.10 (d, 3J ¼ 9.4 Hz, 1H, H-9),
7.57e7.40 (m, 5H, 1-Ph), 7.25e7.13 (m, 3H, CH2Ph-3,4,5), 6.69e6.66
(m, 2H, CH2Ph-2,6), 5.61 (s, 2H, CH2Ph), 2.65 (s, 3H, Me), 1.53 (s, 9H,
Orange solid, m.p.: 324e328 ꢀC, 103 mg. Yield: 97%. 1H NMR
(300 MHz, DMSO‑d6)
d
: 10.09 (s, 1H, H-6), 9.34 (d, 3J ¼ 7.0 Hz, 2H,
pyr-2,6), 8.92e8.84 (m, 2H, 1-Ph-2,6), 8.69 (d, 3J ¼ 7.0 Hz, 2H, pyr-
3,5), 8.53 (s, 1H, H-4), 8.21 (dd, 3J ¼ 9.9 Hz, 4J ¼ 1.8 Hz, 1H, H-8),
7.94 (d, 3J ¼ 9.7 Hz, 1H, H-9), 7.64e7.61 (m, 2H, CH2Ph-2,6),
7.56e7.40 (m, 6H, 1-Ph-3,4,5 & CH2Ph-3,4,5), 5.88 (s, 2H, CH2Ph),
t-Bu). 13C NMR (75 MHz, CD3OD)
d: 166.1 (C-3),147.6 (C-1),144.9 (C-
8), 144.8 (C-9a), 138.5 (1-Ph-1), 135.9 (C-4a), 135.1 (CH2Ph-1), 130.8
(1-Ph-4), 130.6 (1-Ph-2,6), 130.0 (1-Ph-3,5*), 129.8 (C-7), 129.5
(CH2Ph-3,5*),129.2 (CH2Ph-4),129.0 (C-6), 126.9 (CH2Ph-2,6),126.2
(C-10a), 111.9 (C-9), 103.5 (C-4), 50.1 (CH2Ph), 39.2 (Cq t-Bu), 30.7
(CH3 t-Bu), 17.9 (Me). HRMS (ESI): m/z calc. for C28H28N3 [M]þ:
406.22777, found: 406.22663.
1.54 (s, 9H, t-Bu). 13C NMR (75 MHz, DMSO‑d6)
d: 159.7 (C-3), 151.8
(pyr-4), 147.6 (C-9a), 145.7 (C-1), 144.6 (pyr-2,6), 137.8 (1-Ph-1),
136.7 (C-4a), 136.2 (C-10a), 134.6 (CH2Ph-1), 130.2 (C-6), 129.4 (C-
8), 129.3 (1-Ph-4*), 129.2 (1-Ph-3,5*), 129.1 (CH2Ph-4*), 128.8 (1-
Ph-2,6), 128.6 (CH2Ph-2,6), 128.3 (CH2Ph-3,5*), 124.1 (pyr-3,5),
118.2 (C-7),117.7 (C-9),101.7 (C-4), 62.4 (CH2Ph), 37.7 (Cq t-Bu), 30.6
(CH3 t-Bu). HRMS (ESI): m/z calc. for C32H29N4 [M]þ: 469.23867,
found: 469.23716.
4.1.47. 10-Benzyl-3-(tert-butyl)-1,7-diphenylimidazo[1,2-a:4,5-c']
dipyridin-10-ium bromide (27)
White solid, m.p.: 244e248 ꢀC, 103 mg. Yield: 97%. 1H NMR
(300 MHz, CD3OD) d: 9.99 (s, 1H, H-6), 8.86 (s, 1H, H-4), 8.74 (dd,
3J ¼ 9.6 Hz, 4J ¼ 1.7 Hz, 1H, H-8), 8.27 (d, 3J ¼ 9.6 Hz, 1H, H-9),
7.97e7.94 (m, 2H, 7-Ph-2,6), 7.72e7.39 (m, 8H, 7-Ph-3,4,5 & 1-Ph),
7.30e7.12 (m, 3H, CH2Ph-3,4,5), 6.74e6.72 (m, 2H, CH2Ph-2,6), 5.67
4.2. Cell culture
(s, 2H, CH2Ph), 1.54 (s, 9H, t-Bu). 13C NMR (75 MHz, CD3OD)
d
: 166.4
Human cancer MDA-MB-435s, MDA-MB-231, MDA-MB-
468 cells and murine mammary cancer 4T1 lines were obtained
from the American Type Culture Collection (LGC Promochem,
France). MDA-MB-435s, MDA-MB-231 and MDA-MB-468 cell lines
were cultivated in plastic culture flasks in Dulbecco's Modified
Eagle's medium (DMEM) supplemented with 5% fetal bovine serum
(FBS). 4T1 cells were grown in RPMI supplemented with 10% FBS.
All cells were cultured at 37 ꢀC under a humidified atmosphere
containing 5% CO2.
(C-3), 147.7 (C-1), 145.1 (C-9a), 142.1 (C-8), 138.4 (1-Ph-1), 136.5 (C-
4a), 135.5 (7-Ph-1), 135.1 (CH2Ph-1), 133.1 (C-7), 130.8 (7-Ph-4),
130.7 (1-Ph-3,4,5), 130.6 (7-Ph-3,5), 130.0 (CH2Ph-3,5*), 129.6 (1-
Ph-2,6*), 129.3 (CH2Ph-4), 128.6 (7-Ph-2,6), 128.3 (C-6), 126.9
(CH2Ph-2,6), 126.4 (C-10a), 112.6 (C-9), 104.0 (C-4), 50.3 (CH2Ph),
39.3 (Cq t-Bu), 30.7 (CH3 t-Bu). HRMS (ESI): m/z calc. for C33H30N3
[M]þ: 468.24342, found: 468.24191.
4.1.48. 10-Benzyl-3-(tert-butyl)-1-phenyl-7-(thien-2-yl)imidazo
Test compounds were dissolved in DMSO at a stock solution of
[1,2-a:4,5-c']dipyridin-10-ium bromide (28)
100 mM and further diluted to 100
mM, 10 mM, 1 mM, 0.1 mM and
Off-white solid, m.p.: 251e255 ꢀC, 119 mg. Yield: 90%. 1H NMR
0.01 M in culture medium with a final concentration of DMSO of
m
5
(300 MHz, CD3OD)
d
: 10.0 (q, 4J ¼ 1.8 Hz, J ¼ 0.9 Hz, 1H, H-6), 8.87
0.1% including the control condition.
(s, 1H, H-4), 8.70 (dd, 3J ¼ 9.6 Hz, 4J ¼ 1.8 Hz, 1H, H-8), 8.23 (dd,
3J ¼ 9.6 Hz, 5J ¼ 0.7 Hz, 1H, H-9), 7.85 (dd, 3J ¼ 3.7 Hz, 4J ¼ 1.1 Hz, 1H,
thienyl-3), 7.71 (dd, 3J ¼ 5.1 Hz, 4J ¼ 1.1 Hz, 1H, thienyl-5), 7.59e7.41
(m, 5H, 1-Ph), 7.29 (dd, 3J ¼ 5.1 Hz, 3.7 Hz, 1H, thienyl-4), 7.24e7.15
(m, 3H, CH2Ph-3,4,5), 6.72e6.70 (m, 2H, CH2Ph-2,6), 5.65 (s, 2H,
4.3. Cell viability and toxicity assay
Cell viability and toxicity were evaluated in vitro using the MTT
assay. This assay is based on that the yellow tetrazolium MTTcan be
reduced by living cells to colored formazan dye. The absorbance of
the dye was measured and is proportional to the cell viability. Cells
were seeded in 24 well plates at a density of 5 ꢁ 103 - 30 ꢁ 103 cells/
CH2Ph), 1.55 (s, 9H, t-Bu). 13C NMR (75 MHz, CD3OD)
d: 166.4 (C-3),
147.7 (C-1), 144.8 (C-9a), 140.9 (C-8), 138.4 (1-Ph-1), 137.6 (thienyl-
2), 136.4 (C-4a), 135.0 (CH2Ph-1), 130.8 (1-Ph-4), 130.6 (1-Ph-2,6),
130.03 (1-Ph-3,5*), 130.00 (thienyl-4), 129.6 (CH2Ph-3,5*), 129.34
(CH2Ph-4*), 129.32 (thienyl-5*), 128.2 (thienyl-3), 127.4 (C-7), 126.9
(CH2Ph-2,6), 126.4 (C-6), 126.3 (C-10a), 112.9 (C-9), 104.0 (C-4), 50.3
(CH2Ph), 39.3 (Cq t-Bu), 30.7 (CH3 t-Bu). HRMS (ESI): m/z calc. for
well in 500 mL of medium containing FBS and allowed to attach
overnight at 37 ꢀC and 5% CO2. The growth medium in each well
was then replaced with culture medium containing compounds to
be tested at 5 different concentrations and incubated for 1 day for
toxicity or for 5 days for viability assay. After incubation, the me-
dium was replaced by MTT reagent (5 mg/mL prepared in
DMEMþ5%FCS) and incubated for 30e40 min in the incubator at
37 ꢀC, 5% CO2. The formazan crystals were dissolved in DMSO and
the absorbance was measured using a microplate spectrophotom-
eter (Epoch, BioTek Instruments Inc, USA) at 570 nm. The obtained
data were normalized to the DMSO treated cells and IC50, corre-
sponding to the concentration of the test compound giving 50%
inhibition of growth, were calculated by using nonlinear regression
with variable slope using graph GraphPad Prism™ v.5 (GraphPad
Software, Incorporation, California, USA).
C
31H28N3S [M]þ: 474.20039, found: 474.19820.
4.1.49. 10-Benzyl-7-(1-benzylpyridin-1-ium-4-yl)-3-(tert-butyl)-1-
methoxyimidazo[1,2-a:4,5-c'] dipyridin-10-ium dibromide (29)
Red solid, m.p.: 208e212 ꢀC, 65 mg. Yield: 32%. 1H NMR
(300 MHz, CD3OD)
d
: 10.38 (s, 1H, H-6), 9.28 (d, 3J ¼ 6.7 Hz, 2H, pyr-
2,6), 8.89 (dd, 3J ¼ 9.7 Hz, 4J ¼ 1.5 Hz, 1H, H-8), 8.79 (d, 3J ¼ 6.7 Hz,
2H, pyr-3,5), 8.52 (d, 3J ¼ 9.7 Hz, 1H, H-9), 8.45 (s, 1H, H-4),
7.67e7.29 (m, 10H, Ph), 6.16 (s, 2H, 10-CH2Ph), 5.95 (s, 2H, pyr-
CH2Ph), 4.24 (s, 3H, OCH3), 1.52 (s, 9H, t-Bu). 13C NMR (75 MHz,
CD3OD)
d: 165.8 (C-3), 152.3 (C-1*), 152.0 (pyr-4*), 146.5 (pyr-2,6),
144.4 (C-9a), 139.2 (C-8), 137.1 (C-4a), 135.9 (10-CH2Ph-1), 134.6
14

A.A. Alzain, L. Brisson, P.-O. Delaye et al.
European Journal of Medicinal Chemistry 218 (2021) 113258
4.4. Apoptosis assay
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Acknowledgements
We gratefully acknowledge the generous support provided by
the Ligue Nationale Contre le Cancer e Interregion Grand-Ouest.
Abdulrahim Alzain research was supported by a grant from the
Ministry of Higher Education and University of Gezira, Sudan.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113258.
15

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16