Imidazo[1,2-a]quinoxalines for melanoma treatment with original mechanism of action

Article abstract of DOI:10.1016/j.ejmech.2020.113031

The malignant transformation of melanocytes causes several thousand deaths each year, making melanoma an important public health concern. Melanoma is the most aggressive skin cancer, which incidence has regularly increased over the past decades. We described here the preparation of new compounds based on the 1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxaline structure. Different positions of the quinoxaline moiety were screened to introduce novel substituents in order to study their influence on the biological activity. Several alkylamino or alkyloxy groups were also considered to replace the methylamine of our first generation of Imiqualines. Imidazo[1,2-a]pyrazine derivatives were also designed as potential minimal structure. The investigation on A375 melanoma cells displayed interesting in vitro low nanomolar cytotoxic activity. Among them, 9d (EAPB02303) is particularly remarkable since it is 20 times more potent than vemurafenib, the reference clinical therapy used on BRAF mutant melanoma. Contrary to the first generation, EAPB02303 does not inhibit tubulin polymerization, as confirmed by an in vitro assay and a molecular modelisation study. The mechanism of action for EAPB02303 highlighted by a transcriptomic analysis is clearly different from a panel of 12 well-known anticancer drugs. In vivo EAPB02303 treatment reduced tumor size and weight of the A375 human melanoma xenografts in a dose-dependent manner, correlated with a low mitotic index but not with necrosis.

Full text of DOI:10.1016/j.ejmech.2020.113031

Journal Pre-proof
Imidazo[1,2-a]quinoxalines for melanoma treatment with original mechanism of action
Cindy Patinote, Carine Deleuze-Masquéfa, Kamel Hadj Kaddour, Laure-Anaïs
Vincent, Romain Larive, Zahraa Zghaib, Jean-François Guichou, Mona Diab Assaf,
Pierre Cuq, Pierre-Antoine Bonnet
PII:
S0223-5234(20)31003-5
DOI:
https://doi.org/10.1016/j.ejmech.2020.113031
EJMECH 113031
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 September 2020
Revised Date: 5 November 2020
Accepted Date: 14 November 2020
Please cite this article as: C. Patinote, C. Deleuze-Masquéfa, K.H. Kaddour, L.-A. Vincent, R. Larive,
Z. Zghaib, J.-F. Guichou, M.D. Assaf, P. Cuq, P.-A. Bonnet, Imidazo[1,2-a]quinoxalines for melanoma
treatment with original mechanism of action, European Journal of Medicinal Chemistry, https://
doi.org/10.1016/j.ejmech.2020.113031.
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Graphical Abstract
Novel imidazo[1,2-a]quinoxalines with original anticancer mechanism of action
EAPB02303 on A375 melanoma cells: IC50 = 3 nM

Imidazo[1,2-a]quinoxalines for melanoma treatment with original
mechanism of action.
Cindy Patinote1,2,a, Carine Deleuze-Masquéfa1,a,*, Kamel Hadj Kaddour1, Laure-Anaïs Vincent1, Romain
Larive1, Zahraa Zghaib1,3, Jean-François Guichou4, Mona Diab Assaf3, Pierre Cuq1, Pierre-Antoine
Bonnet1.
1. Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, CNRS, Université de Montpellier, Faculté de
Pharmacie, 15 avenue Charles Flahault, BP 14491, 34093 Montpellier Cedex 5, France.
2. Société d’Accélération du Transfert de Technologies (SATT AxLR), CSU, 950 rue Saint Priest, 34090
Montpellier, France.
3. Tumorigenèse et Pharmacologie Antitumorale, Lebanese University, EDST, BP 90656, Fanar Jdeideh,
Lebanon.
4. CNRS, UMR 5048, INSERM, U105, Université de Montpellier, Centre de Biochimie Structurale, 34090
Montpellier, France
a. These authors contribute equally to this work.
*
Corresponding author: carine.masquefa@umontpellier.fr
Abstract
The malignant transformation of melanocytes causes several thousand deaths each year, making
melanoma an important public health concern. Melanoma is the most aggressive skin cancer, which
incidence has regularly increased over the past decades. We described here the preparation of new
compounds based on the 1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxaline structure. Different
positions of the quinoxaline moiety were screened to introduce novel substituents in order to study
their influence on the biological activity. Several alkylamino or alkyloxy groups were also considered
to replace the methylamine of our first generation of Imiqualines. Imidazo[1,2-a]pyrazine derivatives
were also designed as potential minimal structure. The investigation on A375 melanoma cells
displayed interesting in vitro low nanomolar cytotoxic activity. Among them, 9d (EAPB02303) is
particularly remarkable since it is 20 times more potent than vemurafenib, the reference clinical
therapy used on BRAF mutant melanoma. Contrary to the first generation, EAPB02303 does not
inhibit tubulin polymerization, as confirmed by an in vitro assay and a molecular modelisation study.
The mechanism of action for EAPB02303 highlighted by a transcriptomic analysis is clearly different
from a panel of 12 well-known anticancer drugs. In vivo EAPB02303 treatment reduced tumor size
and weight of the A375 human melanoma xenografts in a dose-dependent manner, correlated with a
low mitotic index but not with necrosis.
R
R'
HO
R"
x
HO
N
N
N
N
N
N
R2
NHCH3
R3
Second Generation
EAPB02303
First Generation
EAPB0203
EAPB0503
1

Keywords
Imidazo[1,2-a]quinoxaline, Imidazo[1,2-a]pyrazine, Imiqualines, Melanoma, A375 cells.
1. Introduction
Melanoma, the sixth most frequently diagnosed cancer and the most dangerous and aggressive skin
cancer, corresponds to the degeneration of pigment-containing cells (melanocytes) into cancer cells.
This malignant cancer spreads quite early to others organs by metastases during the vertical growth
phase. The management of melanoma depends greatly on the location, depth and stage.8 While early
stages can achieve remarkable outcomes with surgery alone, metastatic melanoma requires
therapeutic treatment intervention, such as chemotherapeutic agents, targeted therapies
(Vemurafenib for BRAF mutation13, dabrafenib14, trametinib15 and cobimetinib16 for MEK inhibitors17)
or immunotherapies (prembrolizumab, nivolumab as PD-1 inhibitors21, ipilimumab as CTLA-4
inhibitors22, cytokines (IFN-α, IL-2)23). Although a steady improvement in survival among patients has
been reported over the last decades thanks to essentially early detection, therapy improvement and
diagnosis, the mortality rates from melanoma continue to rise in several European countries due to
an increasing incidence.5,6 There is always a need for the discovery of new therapeutic compounds.7
Originally developed as nucleoside analogue with antiviral properties, Imiquimod is a member of the
immune response modifier family (Scheme 1). 26 The interesting activity of this drug prompted us to
synthetize novel analogues in the pyrazoloquinoxaline and imidazoquinoxaline families. The first
generation of Imiqualines was essentially substituted on position 1 by multiple aromatic moieties
directly grafted to the main structure or via an alkyl linker.27–30 Our Imiqualines first generation were
characterized by significant anti-cancer activities with ICs50 in the µM range against a large variety of
cancer cell lines and the most active derivatives were found to interact with tubulin. Such interaction
is detrimental for further development since tubulin is present in all cancer as well as normal cells.
For further development of the series, it was important to study the possibility to disconnect the
anticancer activity from the tubulin interaction, which is a non-specific inhibition. In order to explore
this potential anti-cancer activity of our compounds, we first undertook a molecular modelling study
with the aim of finding a substituted phenyl to get rid off the interaction in the colchicin site of the
tubulin. These results allowed us to synthesize and develop a second generation of Imiqualines which
is characterized by the presence of the 3,4-dihydroxyphenyl moiety on position 1. We present herein
the design of new compounds belonging to the imidazo[1,2-a]quinoxaline and imidazo[1,2-
a]pyrazine series and their in vitro results. The mechanism of action of the best compound to date
EAPB02303 (9d) has been envisaged, as well as preliminary in vivo evaluation to treat melanoma.
2. Results and discussion
We previously synthesized a panel of imidazo[1,2-a]quinoxaline derivatives exhibiting remarkable
anti-proliferative effects on human melanoma cell line A375 (Scheme 1). Among this first series of
potent anti-cancer compounds, the most active compound EAPB0503 showed also potent inhibition
of tubulin polymerization.31,32 Molecular docking simulations revealed that EAPB0503 was able to
make a hydrogen bond with ASP682A and Van der Waals contacts with VAL174A, ASN689B,
ALA747B, ILE749B, LYS783B and ALA785B in the colchicine binding site of tubulin (Figure 1). The
quantitative analyses of the interaction estimate a binding free energy of -8.5 kcal/mol with a
contribution of -1.2 kcal/mol of the hydrogen bond, this result demonstrates a high contribution of
2

the Van der Waals contacts for the interaction of EAPB0503 with tubulin. The binding to tubulin was
experimentally determined in vitro (see 2.2.). Even if tubulin targeting agents have played a key role
in cancer treatment, colchicine binding site inhibitors have not yet reached the commercial phase.33
That is why focusing on the anti-cancer property while being free from any tubulin targeting property
could be the new insight into the design of new agents.
Figure 1: Analyses of the EAPB0503 interaction with the colchicine site.
2.1. Molecular docking: design of the novel Imiqualines generation.
In order to further explore the potential variability of Imiqualines to interact with the colchicine site
of tubulin, we underwent several molecular docking simulations based on the structure of our hit
EAPB0503 using analogues with a variety of substituents. These simulations exhibited high binding
discrepancies between imidazoquinoxalines bearing methoxy or hydroxyl groups on the aromatic
moiety fixed on position 1 of the tricyclic heterocycle. Actually, the presence of a catechol group on
position 1 was found to be highly detrimental for the virtual docking within the colchicine site. With
this dihydroxyphenyl moiety, we determined no binding in the tubulin colchicine site compared to
the results obtained with the first generation Imiqualines with one methoxy group such as our hit
EAPB0503 (Figure 2). This result can be explained since the driving forces of the EAPB0503
interaction with tubulin are Van der Waals contacts. The introduction of two hydroxyl groups induces
polarity which is highly unfavorable to such interaction.
3

Figure 2: Docking studies. Compounds in the colchicine binding pocket of tubulin chains a and b (PDB 4O2B):
Colchicine, EAPB0503, EAPB02303 (9d), EAPB02303 (9d) and colchicine overlay.
2.2. In vitro tubulin polymerization assay and first antiproliferative results
To investigate the results based on in silico molecular docking studies, we synthesized compound
EAPB02303 (9d) (see 2.3.) which is characterized by the presence of the 3,4-dihydroxyphenyl group
on position 1 of the 4-methylaminoimidazo[1,2-a]quinoxaline heterocycle platform instead of the 3-
methoxyphenyl group as in our first lead EAPB0503. As the mechanism of action of our hit EAPB0503
was previously linked to its capability to inhibit tubulin polymerization,31,32 we wanted to evaluate
the capability of the new derivative EAPB02303 for binding and inhibiting tubulin polymerization.
The results on tubulin polymerization are depicted on Figure 3. Tubulin was purified from pig brain
tissue according the protocol described in Experimental Section (see 4.3.). A well-known natural
tubulin inhibitor, colchicine, was used as reference. The previous reported data showed an important
role of EAPB0503 as antiproliferative and antimitotic agent, as EAPB0503 (at 5 µM) was highly
effective to inhibit tubulin assembly with 84% of inhibition, which is higher than colchicine (52% of
inhibition (P < 0.05)). This high effect on tubulin inhibition was clearly correlated with its potent
antiproliferative effect on human melanoma cell line. In the current study, it is clear that compound
EAPB02303 does not behave as our first hit EAPB0503 as the profile for the new compound is the
4

same as vehicle DMSO. By this way, it was clear that EAPB02303 was devoid of any inhibition on
tubulin polymerization.
Figure 3: Tubulin polymerization inhibition.
Based on these results, it was highly interesting to determine if such dramatic loss of tubulin
polymerization inhibition was detrimental to the anti-cancer activity of our Imiqualines series.
EAPB02303 was further tested on our A375 melanoma cell line model. Its IC50 found in the
nanomolar range (3 nM) is much lower than the best results obtained with compounds of the
Imiqualines first generation (e.g. 383 nM for EAPB0503). These results showed that the
antiproliferative activity was highly impacted by the loss of tubulin inhibition, but the observed
impact was in any case detrimental but, on the contrary, dramatically beneficial.
These very promising results prompted us to design derivatives of EAPB02303 with the catechol
moiety and to synthesize two new series of imidazo[1,2-a]quinoxalines and imidazo[1,2-a]pyrazine
derivatives with a catechol moiety directly linked to the imidazole ring.
R
R'
HO
R''
x
HO
1
N
N
N
N
N
N
N
N
8
7
R2
N
NH2
R1
R3
4
Imiquimod
First Generation
Second Generation
Scheme 1: Chemical structures of reference compound Imiquimod, first and second generations of
Imiqualines.
5

2.3. Chemistry: synthesis of the novel Imiqualines generation
2.3.1. Imidazo[1,2-a]quinoxaline derivatives
Structural modifications on our imidazo[1,2-a]quinoxaline derivatives have been consequently done.
Further to first impressive results obtained with compound EAPB02303, the second generation we
designed is based on the 3,4-dihydroxyphenyl group on position 1. This substituent will not change
during this study for the target imidazo[1,2-a]quinoxalines and imidazo[1,2-a]pyrazines. We present
here a series of novel compounds with modifications on the one hand on the quinoxaline ring as
methyl group was until today the only considered substituent, and on the other hand on position 4
by introducing small alkylamino or hydroxy groups.34,35 Firstly, we worked on the screening of new
substituents of the quinoxaline ring. The smallest alkylamine group on position 4, that is to say
methylamine, remained. The trifluoromethyl and trifluoromethoxy derivatives were chosen among
the commercially available o-fluoroanilines substituted on positions 7 or 8 to finally afford
compounds 8a-8c and 9a-9c. Secondly, we modified the methylamine group on position 4 by
introducing different kinds of alkyldiamine or alkoxy (8j, 9j) moieties, remaining the quinoxaline
devoided of any ring substituent. Among the commercially available monoprotected diamine, we
chose the ethyl (8e, 9e), propyl (8f, 9f), hexyl (8g, 9g) and piperazine (8h, 9h) derivatives. The
dimethylamine derivatives were obtained by using the corresponding nucleophilic amine (8i, 9i).
From a chemical strategy point of view, the synthetic pathways of the imidazo[1,2-a]quinoxaline
derivatives used in this study are given in schemes 2 and 3. Compounds were synthesized according
to a route we previously partially described.27,31,34,36 The carbonylimidazole dimer 2 results from the
condensation of the 2-imidazole carboxylic acid 1 in presence of thionyl chloride.
Diversification of the quinoxaline ring was until today few explored by our team. We only considered
mono- or dimethyl groups (R2 substitution). Among the commercially available o-fluoroanilines, we
chose the cyano, trifluoromethyl and trifluoromethoxy derivatives in position 4 or 5. Addition of o-
fluoroaniline on the dimer 2 gives the intermediates 3a-3d. The others commercially available
reagents (methylcarboxylic acid and tert-butycarbamate) do not allow us to complete the synthetic
pathway due to their sensitivity to acid and basic conditions used during the synthesis, leading to
ester saponification and amino-deprotection, respectively. Moreover, the intermediates
corresponding to the methylsulfonyl substituted o-fluoroaniline were never detected by mass
spectrometry. Unfortunately, the o-fluoroanilines substituted in position 3 weren’t suitable reagents,
their lack of reactivity was probably due to their inductive and mesomeric effects on the
fluoroaniline. The o-fluoroanilines substituted in position 6 were not investigated.
Cyclisation of 3a-3d is allowed by using sodium hydride in dimethylacetamide to afford 4a-4d.
Treatment of compounds 4a-4d with phosphorus oxychloride and N,N-diethylaniline gives the
chlorinated compounds with a satisfactory yield for 5d, lower for 5c and very low for 5a and 5b. The
use of phosphorus pentachloride as chlorinated agent and solvent37 leads globally to lower yields
(data not shown).
The chlorine of 5a-5d is substituted by diverse nucleophilic groups under microwave assistance with
good to excellent yields. To obtain the compounds 6a-6d, methylamine is used in ethanol. The
compounds 6e-6h are obtained by addition of the corresponding monoprotected diamine in
6

presence of diisopropylethylamine in acetonitrile. Compound 6i is obtained with the same procedure
using dimethylamine as nucleophile. The preparation of compound 6j required an adjustment. The
use of sodium methanolate in methanol in the same microwave conditions used for the others lead
exclusively to the hydroxy derivative, whereas the methoxy derivative could be furnished under
reflux during 2 hours followed by a stirring overnight at room temperature.38 Cherng and al. describe
nucleophilic substitutions of halides on quinoline or isoquinoline structures under microwave
irradiations.39 The use of sodium methanolate in methanol under microwave irradiations at 70°C
during 2 minutes leads to the desired compound 6j with an excellent yield. Compound 6k is obtained
by using aqueous ammonia under microwave assistance at 130°C during 2 hours.
The bromination of intermediates 6a-6k by N-bromosuccinimide in chloroform leads to compounds
7a-7k quantitatively. The 3,4-dimethoxyphenyl moiety is introduced in position 1 of the imidazo[1,2-
a]quinoxaline via a Suzuki-Miyaura cross-coupling reaction using the corresponding boronic acid to
furnish compounds 8a-8k. Optimal conditions correspond to the use of tetrakis(triphenylphosphine)
palladium (0) with sodium carbonate in dimethoxyethane/water (2/1) under microwave irradiations
at 140°C during 20 minutes.
Intermediates 8a-8k are submitted to deprotection to afford the targeted compounds 9a-9k. We
previously used iodocyclohexane in dimethylformamide under microwave irradiations at 200°C
during 1h20. Those conditions do not allow the cleavage of all the protections engaged in this
synthetic pathway and afford generally low yields. Greene and al. described many methods for the
cleavage of methyl ethers of phenols.40 The more common conditions are based on bore derivatives
as demethylating agents. Spadoni and al. use boron tribromide in dichloromethane.41 These most
described conditions in literature give good yields and are compatible with different kind of
structures and chemical functions. Perspicace and al. use the complex BF3-SMe2 in presence or not of
triethylamine in dichloromethane.42,43 All these conditions were tested with some of our derivatives.
Only boron tribromide in dichloromethane leads readily, with completion and cleanly to the targeted
compounds 9a-9k. Moreover, with these conditions, we observed that the Boc protective group is
removed faster than the methyl protecting groups.
7

Scheme 2: General synthetic pathway to synthetize the targeted imidazo[1,2-a]quinoxalines 9a to 9k.
Reagents and conditions : (a) SOCl2, reflux, 24h; (b) corresponding o-fluoroaniline, NaHMDS, THF, 1h at 0°C
then 3h at RT; (c) NaH, DMA, reflux, 48h; (d) N,N-DEA, POCl3, MW (2 x 15 min, 130°C); (e) NH2CH3, EtOH, MW
(20 min, 150°C); (e’) RNH2 (R ≠ CH3) or NH(CH3)2, DIEA, ACN, MW (20 min, 150°C); (e’’) MeONa, MeOH, MW (2
min, 70°C); (e’’’) NH4OH, ACN, MW (2 h, 140°C); (f) NBS, CHCl3, reflux, 1h30; (g) 3,4-dimethoxyphenylboronic
acid, Pd(PPh3)4, Na2CO3, DME/H2O (2/1), MW (20 min, 140°C); (h) BBr3, CH2Cl2, 0°C to RT, 3h.
8

Two of our target compounds 9e and 9g were converted in ammonium chloride salts 10e and 10g by
using hydrochloric acid in dioxane (4M) (Scheme 3).
Scheme 3: Synthetic pathway for ammonium chloride salts preparation. Reagents and conditions: (a)
HCl/dioxane (4M), 2h, RT.
2.3.2. Imidazo[1,2-a]pyrazine derivatives
In parallel of the synthesis of the imidazo[1,2-a]quinoxaline derivatives, we also designed a series of
imidazo[1,2-a]pyrazines to evaluate the potency of this minimal structure. Several synthetic
pathways to obtain imidazo[1,2-a]pyrazine derivatives have been described in the literature.44–46 We
chose to focus on the following one we previously described (Scheme 4)34, because of the diverse
substituents we wanted to graft on positions 1 and 4.47 Starting material 3-bromo-8-
chloroimidazo[1,2-a]pyrazine is commercially available. Methylamine or ammoniac is first introduced
in position 4 under microwave assistance without shifting the bromine to obtain compounds 11a or
11b, respectively. The 3,4-dimethoxyphenyl group is then introduced by the Suzuki-Miyaura cross-
coupling reaction with the appropriate aryl boronic acid in presence of carbonate salt and palladium
catalyst under microwave irradiations to furnish compounds 12a and 12b. A deprotecting step is
engaged with boron tribromide in dichloromethane to regenerate the hydroxyl groups on the aryl
moiety to afford 13a and 13b.
Scheme 4: Synthesis of imidazo[1,2-a]pyrazine derivatives. Reagents and conditions: (a) NH4OH, ACN, MW
(2h, 130°C); (a’) CH3NH2, EtOH, MW (20 min, 140°C) ; (b) 3,4-dimethoxyphenylboronic acid, Na2CO3,
Pd(PPh3)4, DME/H2O (1/1), MW (20 min, 140°C) ; (c) BBr3, CH2Cl2, 0°C to RT, 2h.
2.4. In vitro cell growth inhibition assay
We have evaluated the cellular activity of compounds 6d, 7d, 8b-8j, 9a-9k, 10e, 12a-12b, 13a-
13b to investigate their efficacy on the A375 human melanoma cell line proliferation, which
9

possesses the BRAF mutation. Results are reported in Table 1 and compared to the positive
control vemurafenib, used as targeted therapy of BRAF mutant melanoma.
The analysis of the structure-activity relationship confirms the interest of small alkylamino groups
(such as methylamine, dimethylamine or amine) in position 4 as depicted by compounds 8d, 8i, 9d, 9i
and 9k, compared to vemurafenib. Nor longer chains as ethyl (8e and 9e), propyl (8f and 9f), hexyl
(8g and 9g) or cyclic alkylamine chain as piperazine (8h and 9h), nor the hydrochlorine salt of the
ethyl derivative 10e improve the cytotoxic activities. Even if compound 8j bearing a methoxy group in
position 4 displays a micromolar activity, its demethylated analogue 9j completely loses the cytotoxic
activity. Moreover, substitutions of the quinoxaline ring by the trifluoromethyl or trifluoromethoxy
group are tolerated on position 7 (8b, 9a, 9b) , which is not the case in position 8 (8c and 9c). Their
synthetic intermediates 6d and 7d do not display better in vitro activity, confirming the interest of
the catechol moiety. The in vitro results highlight the necessity of the entire aromatic structure for
antiproliferative activity, that is to say the hetero-tricyclic nucleus of the imidazo[1,2-a]quinoxaline,
since the IC50 values of the four prepared imidazo[1,2-a]pyrazines 12a-b and 13a-b are above 10 µM.
These results allowed us to highlight compounds EAPB02303 (9d) and its N-demethylated derivative
EAPB02302 (9k) as very active leads of the second Imiqualine generation, with ICs50 of 3 and 60 nM
respectively. Additional modifications did not contribute to improve the anti-proliferative activity,
since the compounds show micromolar activities similar to those of our first Imiqualine series.
Table 1 : IC50 values on human melanoma A375 cell line.
IC50 (nM)
Compounds
vemurafenib
139
>10000
6d
7d
8b
8c
8d
8e
8f
2354
3493
EAPB02203-7b
EAPB02203-8a
EAPB02203
EAPB02211
EAPB02214
EAPB02212
EAPB02213
EAPB02204
EAPB02215
EAPB02303-7a
EAPB02303-7b
EAPB02303-8a
EAPB02303
EAPB02306
EAPB02309
EAPB02307
EAPB02308
EAPB02304
EAPB02300
EAPB02302
>10000
193
>10000
>10000
>10000
>10000
414
8g
8h
8i
2929
8j
6909
9a
9b
9c
9d
9e
9f
199
>10000
3
1284
5588
5694
>10000
30
9g
9h
9i
> 10 000
60
9j
9k
10

1704
10e
12a
12b
13a
13b
EAPB02306s
EAPB32203
EAPB32202
EAPB32303
EAPB32302
> 10 000
> 10 000
> 10 000
> 10 000
Considering the astonishing activity of EAPB02303, we evaluated its potential on different melanoma
cell lines (A375, ME WO, A2058, IPC 298), compared to our first generation hit EAPB0503 and
vemurafenib (Figure 3 and Table 2). Whereas vemurafenib selectively inhibits BRAF v600e melanoma
cell lines, our compound EAPB02303 is able at a nanomolar range to inhibit growth of all melanoma
cell lines tested in this assay. It is interesting to note that EAPB02303 does not exhibit any
cytotoxicity on freshly isolated hPBMCs at concentrations as high as 0.2 mM. Those in vitro data
prompt us to only considered EAPB02303 for the rest of the entire study to start an in-depth
research of the biological target(s) and/or mechanism of action.
Figure 4: Chemical structures of first and second Imiqualines generations
Table 2: IC50 values of EAPB02303 and EAPB0503 on different melanoma cell lines compared to vemurafenib
IC50 (nM)
Cell lines
BRAF mutation
EAPB02303 EAPB0503 vemurafenib
A375
ME WO
A2058
IPC 298
3
3
4
383
125
185
469
139
28350
v600e
-
v600e
-
42550
40
1300051
2.5. Transcriptomic data
The mode of action of treatments on cancer cells can be correlated with the modification of their
transcriptomes. To precise the mechanism of action of our Imiqualines family, we designed a
transcriptomic study to carry out a comparison with different therapies used in human cancer
treatment. Thus, EAPB0203, EAPB0503 and EAPB02303 were compared to a panel of 12 drugs,
which are representative of the diversity of anticancer mechanisms of action. Among them, 11
conventional chemotherapies belonging to different therapeutic classes were selected. The alkylating
agents were represented by dacarbazine (reference molecule for Multiple Myeloma (MM) treatment
11

until 2011) and fotemustine (also used in MM treatment in case of brain metastases). The anti-
metabolites were represented by 5-fluorouracil (5-FU, antipyrimidine) and methotrexate (folic acid
antagonist). SN38 (the active metabolite of irinotecan) and Doxorubicin were chosen as
topoisomerase inhibitors (topoisomerases I and II respectively). Finally, we included vemurafenib
(BRAF inhibitor) as targeted therapy. Moreover, as the first generations of Imiqualines (EAPB0203
and EAPB0503) conserved the anti-microtubule activity of imiquimod31, imiquimod and a panel of
molecules with anti-tubulin activities were also included in the study. These drugs fix different
“binding sites” of tubulin: vinca-alkaloid binding site (vinorelbine), taxane binding site (paclitaxel),
colchicine binding site (colchicine), and maytansine binding site (maytansine). EAPB02303 was
compared to all these mentioned drugs.
From an experimental point of view, A375 cells were treated for 6 hours by each drug at a
concentration of 10 µM. Then, the transcriptome of treated or non-treated cells (DMSO was used as
negative control) was established after hybridization on Affymetrix HG U133 + PM genechip. The
experiment aimed to characterize the genes whose level of transcription was disturbed following the
application of the different studied drugs. The analysis of transcription profiles allowed the
calculation of a distance between all the drugs taken two by two. These distances were converted
into a dendrogram which is represented here by the synthetic form of a tree (Figure 5).
Figure 5: Dendrogram of transcriptomic profiles. Comparison of gene expression profiles of A375 cells after a
treatment with Imiqualines derivatives (EAPB0203, EPAB0503, EAPB02303), or a panel of anticancer drugs
(6h, 10 µM). The distances between each pair of cell lines were calculated as described in materials and
methods and represented as a dendrogram of distances.
In this Figure 5, we can observe that molecules sharing a common mechanism of action are grouped,
which give a good confidence in the obtained results. Two of the three tested Imiqualines
derivatives, EAPB0203 and EAPB0503 (Figure 1), are included among anti-tubulin agents. This set is
itself split into two groups, one of these consisting in EAPB0203, EAPB0503 and colchicine. This
notable result confirms both the anti-tubulin component of their mechanism of action, and the
probably binding on the colchicine site.31 In addition, EAPB02303 is clearly set apart from all anti-
tubulin agents, confirming the loss of the anti-tubulin component in its mechanism of action.
Moreover, and very interestingly, EAPB02303 is not associated with any group. Thus, its mechanism
of action appears to significantly differ from other first generation Imiqualines derivatives, but also
from those of the other tested drugs. A finer and targeted analysis of these transcriptomic data could
allow to better understand the original mechanism of action of EAPB02303 in highlighting cellular or
12

molecular signaling pathways, that are recruited or inhibited. A screening over 450 kinases could not
allow us to put forward any enzyme inhibition. Furthermore, chemical biology studies including SILAC
method will allow us to refine and precise biological targets.
2.6. In vivo study
To test the efficacy of the EAPB02303 to inhibit tumor growth in a heterotopic xenograft mouse
model of human melanoma, we inoculated subcutaneously A375 cells to immunodeficient mice and
treated the animals by intraperitoneal injection of the compound as soon as the tumors reached a
minimal volume.
At the maximum tolerated dose (75 mg/kg - 2 times a week), EAPB02303 treatment induced a
significant reduction in the tumor size, around 80% smaller as compared to the vehicle group (Figure
6A). However severe adverse side effects were observed such as significant reduction in body weight,
morbidity and mortality (Figure 6B and data not shown). Gross pathology findings at study
termination revealed pathological findings mainly on the liver, spleen, gut and pancreas.
(Supplementary Table S1).
Interestingly, by giving the same dose level per week (150 mg/kg) but by changing the dose regimen
(5 times a week), the compound had the same efficacy on tumor size without apparent toxicity. No
significant impact on body weight, neither side effects were observed (Figures 6A and 6B). This
clinical observation was supported by gross pathology analysis at study termination (data not shown).
For treatment at 25 mg/kg – 3 times a week, the reduction of tumor size was found to be 50%, which
suggest a dose-dependent response (Figure 6A). Finally, for all regimen, tumor weight measurements
at necropsy showed a similar pattern to the calculated tumor size (Figures 6C and 6D).
Histologic analyses, as displayed in supplementary Table S2, show EAPB02303 did not induce changes
regarding tumor stroma, inflammation and vascularization. Tumors blood flow or indicative
angiogenic parameters were not modified in comparison to the vehicle group as indicated by Laser
Doppler measurements (data not shown). It was observed a marked trend toward a lower invasion in
group 30 mg/kg – 5 times a week, a moderately low trend to invade in group 25 mg/kg – 3 times a
week, intermediate invasion in group 75 mg/kg – 2 times a week and a higher trend toward invasion
in vehicle group.
EAPB02303 also induced a trend toward a decreased curve of the mitotic index. In the groups 75
mg/kg – 2 times a week and 25 mg/kg – 3 times a week, the number of mitosis was lower than in the
vehicle group (Figures 6E and 6F). Moreover, the mitotic index was even below in group 30 mg/kg – 5
times a week.
To summarize, EAPB02303 exerted in vivo an anti-cancer effect that was highly correlated with a low
mitotic index and a lower invasion capacity correlate but not with an anti-angiogenic nor necrotic
activity. Therefore, the efficacy of this compound is probably linked to an effect involving mitosis.
EAPB02303 compound, at the highest dose and when administered 2 times a week, was coupled to a
significant toxicity. By changing the dose regimen with the same dose level per week (150 mg/kg),
same efficacy was achieved without apparent toxicity indicating that EAPB02303 toxicity might relate
to Cmax but not its efficacy.
13

Figure 6: Tumorigenesis experiment with subcutaneously inoculated A375 melanoma cells in animals treated
with indicated regimen of EAPB02303. EAPB02303 treatment inhibits tumor growth in heterotopic xenograft
mouse model. A: Growth kinetics of tumors in animals treated with the indicated regimen (number of
independent transplants is indicated in the figure). B: Averages of normalized body weight of tumor-bearing
animals during treatment with the indicated regimen. C: Representative macroscopic pictures of tumors
after mid-sagittal section from indicated mice. D: Weight of tumors after necroscopy of the indicated mice. E:
Representative hematoxylin/eosin/saffron-stained samples from tumors of the indicated mice (scale bar, 40
µm). F: Quantification of mitosis from the indicated mice. n = 4 mice for each group. Data are presented as
mean ± s.e.m. * p<0.05; ** p<0.01; *** p<0.001 relative to values obtained by vehicle-treated animals.
14

3. Conclusion
Imiqualines first generation exhibited in vitro antiproliferative effects on melanoma cell lines and high
tubulin inhibitory effects. As tubulin inhibition is not specific of cancer cells, molecular docking
studies drove us to the synthesis of new compounds with a catechol moiety directly linked to the
imidazole ring of the tricyclic heterocycle scaffold. Structural modifications of the first hits, EAPB0203
and EAPB0503, afforded two new leads, EAPB02303 (9d) and its N-demethylated derivative
EAPB02302 (9k), with utmost impressive in vitro activities on A375 human melanoma cancer cell line
in the nanomolar range with a loss of tubulin polymerization for EAPB02303. The transcriptomic
analysis further demonstrated that the mechanism of action for EAPB02303 is clearly different from
a panel of well-known anticancer drugs, attesting the novelty and originality of its mechanism of
action. In vivo EAPB02303 treatment reduced tumor size and weight of the A375 human melanoma
xenografts in a dose-dependent manner, correlated with a low mitotic index but not with necrosis.
The patented compound EAPB02303 is today under preclinical studies and further on-going studies
are implemented to determine the molecular target(s) and to precise the mechanism of action of
Imiqualines second generation.
4. Materials and Methods
4.1. Chemistry
4.1.1. General
All solvents and reagents were obtained from Sigma Aldrich Chemical Co., Iris Biotech GmbH, Alfa
Aesar Co, VWR and FluoroChem UK and used without further purification unless indicated otherwise.
Silica gel chromatography was conducted with 230-400 mesh 60 Å silica gel (Sigma Aldrich Chemical
Co.). Thin layer chromatography plates (Kieselgel 60 F254) were purchased from Merck. The progress
of reaction was monitored by TLC exposure to UV light (254 nm and 366 nm). Microwave assisted
1
organic syntheses were performed on the Biotage Initiator 2.0 Microwave. H and 13C NMR spectra
were obtained on Brüker AC-400 spectrometer (Laboratoires de Mesures Physiques, Plateau
technique de l'Institut des Biomolécules Max Mousseron, Université de Montpellier, Montpellier,
France). Chemical shifts are given as parts per million (ppm) using residual dimethylsulfoxide signal
for protons (δDMSO = 2.46 ppm) and carbons (δDMSO = 40.00 ppm). Abbreviations used for signal
patterns are the following: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Coupling
constants are reported in Hertz (Hz). Mass spectral data were obtained on a Micromass Q-Tof
(Waters) spectrometer equipped with ESI source (Laboratoires de Mesures Physiques, Plateau
technique de l'Institut des Biomolécules Max Mousseron, Université de Montpellier, Montpellier,
France). Mass spectra were recorded in positive mode between 50 and 1500 Da, capillary and cone
tension were 3000 and 20 V, respectively. The High Resolution Mass Spectroscopy (HRMS) analyses
are carried out by direct introduction on a Synapt G2-S mass spectrometer (Waters, SN: UEB205)
equipped with ESI source (Laboratoires de Mesures Physiques, Plateau technique de l'Institut des
Biomolécules Max Mousseron, Université de Montpellier, Montpellier, France). The mass spectra
were recorded in positive mode, between 100 and 1500 Da. The capillary tension is 3000 V and the
cone tension is 30 V. The source and desolvation temperature are 120°C and 250°C, respectively.
15

Compounds 2, 3d, imidazo[1,2-a]quinoxalines 4d, 5d, 6d, 7d, and imidazo[1,2-a]pyrazines 11a-b,
12a-b and 13a-b were prepared as previously described in literature.27,31,34,36
4.1.2. General procedure for the addition of the fluoroaniline.
To 2-fluoroaniline (56.5 mmol) in THF (28 mL) cooled in a -10oC bath, sodium bis(trimethylsilyl)amide
(141 mmol, 1.0 M in THF) was added. After stirring for 1 hour, a suspension of 2 (25.6 mmol) in THF
(30 mL) was added. The mixture was allowed to warm to room temperature and stirred for 3 hours.
Acetic acid was carefully added to obtain pH 7. The reaction mixture was concentrated under
vacuum, followed by the addition of water and saturated aqueous NaHCO3 solution (v/v, 1/1, 100
mL). The solid was collected by filtration, washed with distilled water and cyclohexane until red-color
disappearance, and dried under vacuum. The compound 3 is obtained as a solid and used without
further purification.
4.1.3. General procedure for the cyclisation.
To 3 (20 mmol) in dimethylacetamide (160 mL) was carefully added and sodium hydride (30 mmol).
The reaction mixture was heated to reflux for 24 hours. After being cooled, a second portion of
sodium hydride (30 mmol) was added and the reaction mixture was heated to reflux during 24 hours.
The sodium hydride was neutralized with saturated aqueous NH4Cl solution (40 mL) and the reaction
mixture was then concentrated under vacuum. The residue was suspended in water, filtered, washed
with water. The precipitate 4 was dried under vacuum and used without further purification.
4.1.4. General procedure for the chlorination.
Phosphorus oxychloride (60.2 mmol) and N,N-diethylaniline (5.72 mmol) were added to 4 (2.86
mmol) in a microwave-adapted vial and sealed. The reaction was submitted to microwave
irradiations at 130oC for 15 min. The reaction mixture was carefully poured dropwise in a saturated
aqueous Na2CO3 solution (50 mL) to be neutralized. When effervescence stopped, the solution was
extracted with dichloromethane (3x50 mL). The organic layer was dried over MgSO4, filtered and
concentrated under reduced pressure. The crude mixture was purified by flash chromatography
eluted with cyclohexane/ethyl acetate to afford compound 5.
4-chloro-7-(trifluoromethyl)imidazo[1,2-a]quinoxaline (5a). Yield: 7%. C11H5ClF3N3. Mw: 271.63 g/mol.
Mp: 139°C. 1H-NMR δ (ppm, 400 MHz, DMSO d6) 7.97 (s, 1H, CH 2), 8.15 (d, 1H, CH 8, J=8Hz), 8.39 (s,
1H, CH 6), 8.67 (d, 1H, CH 9, J=8Hz), 9.08 (s, 1H, CH 1). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 117.97
(CH 1), 118.32 (CH 9), 126.03 (CH 8), 126.77 (CH 6), 127.63 and 127.96 (Cq 7, CF3), 130.23 (Cq 5a),
134.40 (Cq 3a), 135.37 (CH 2), 136.12 (Cq 9a), 144.42 (Cq 4). 19F-RMN δ (ppm, 376.5 MHz, DMSO d6) -
60.47. MS (ESI +, QTof, m/z): 272.10 [M+H]+
4-chloro-7-(trifluoromethoxy)imidazo[1,2-a]quinoxaline (5b). Yield: 34%. C11H5ClF3N3O. Mw: 287.63
g/mol. Mp: 176°C. 1H-NMR δ (ppm, 400 MHz, DMSO d6) 7.84 (d, 1H, CH 8, J=8Hz), 7.93 (d, 1H, CH 2,
J=4Hz), 8.02 (s, 1H, CH 6), 8.56 (d, 1H, CH 9, J=8Hz), 9.01 (d, 1H, CH 1, J=4Hz). 13C-NMR δ (ppm, 100
MHz, DMSO d6) 117.75 (CH 1), 118.69 (CH 9), 121.26 (CH 6), 122.98 (CH 8), 126.75 (Cq 5a), 135.19
(CH 2), 135.29 (Cq 3a), 135.75 (Cq 9a), 144.30 (Cq 4), 146.60 and 146.62 (Cq 7, OCF3). 19F-RMN δ
(ppm, 376.5 MHz, DMSO d6) -57.47. MS (ESI +, QTof, m/z): 287.90 [M+H]+
16

4-chloro-8-(trifluoromethyl)imidazo[1,2-a]quinoxaline (5c). Yield: 12%. C11H5ClF3N3. Mw: 271.63
g/mol. Mp: 155°C. 1H-NMR δ (ppm, 400 MHz, DMSO d6) 7.96 (s, 1H, CH 2), 8.00 (d, 1H, CH 7, J=8Hz),
8.19 (d, 1H, CH 6, J=8Hz), 8.94 (s, 1H, CH 9), 9.18 (s, 1H, CH 1). 13C-NMR δ (ppm, 100 MHz, DMSO
d6) 114.92 (CH 9), 118.20 (CH 1), 123.68 (CH 7), 125.01 (CF3), 127.86 (CH 6), 129.16 (Cq 8), 129.87 (Cq
5a), 130.69 (CH 2), 135.94 (Cq 3a), 136.93 (Cq 9a), 145.22 (Cq 4). 19F-RMN δ (ppm, 376.5 MHz, DMSO
d6) -60.32. MS (ESI +, QTof, m/z): 271.80 [M+H]+
4.1.5. General procedure for the introduction of the methylamino group.
A 33% (w/v) ethanol solution of methylamine (49.0 mmol) was added to a solution of 5 (2.45 mmol)
in ethanol (7 mL) in a microwave-adapted vial. The reaction was submitted to microwave irradiations
during 20 minutes at 150°C. The solvent was removed under reduced pressure. The crude mixture
was dissolved in ethyl acetate and successively washed with saturated aqueous chloride ammonium,
distilled water and finally brine. The organic layer was dried on sodium sulphate, filtered and
concentrated under reduced pressure. The crude mixture was purified by flash chromatography
eluted with cyclohexane/ethyl acetate to afford compounds 6a-6d as solids.
N-methyl-7-(trifluoromethyl)imidazo[1,2-a]quinoxalin-4-amine (6a). Yield: 94%. C12H9F3N4. Mw:
266.22 g/mol. Mp: 225°C. 1H-NMR δ (ppm, 400 MHz, DMSO d6) 3.04 (d, 3H, CH3NH, J=8Hz), 7.60 (d,
1H, CH 8, J=8Hz), 7.67 (d, 1H, CH 2, J=4Hz), 7.85 (s, 1H, CH 6), 8.06 (d, 1H, NHCH3, J=4Hz), 8.31 (d, 1H,
CH 9, J=8Hz), 8.69 (d, 1H, CH 1, J=4Hz). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 27.73 (CH3NH), 115.71
(CH 1), 117.15 (CH 9), 118.92 (CH 8), 123.22 (CH 6), 127.10 to 127.42 (CF3, Cq 7, Cq 5a), 132.76 (CH 2),
132.87 (Cq 3a), 137.63 (Cq 9a), 149.11 (Cq 4). 19F-RMN δ (ppm, 376.5 MHz, DMSO d6) -60.42. MS (ESI
+, QTof, m/z): 267.10 [M+H]+
N-methyl-7-(trifluoromethoxy)imidazo[1,2-a]quinoxalin-4-amine (6b). Yield: 70%. C12H9F3N4O. Mw:
282.22 g/mol. Mp: 189°C. 1H-NMR δ (ppm, 400 MHz, DMSO d6) 3.04 (d, 3H, CH3NH, J=8Hz), 7.28 (d,
1H, CH 8, J=8Hz), 7.49 (s, 1H, CH 6), 7.64 (d, 1H, CH 2, J=4Hz), 8.02 (d, 1H, NHCH3, J=4Hz), 8.21 (d, 1H,
CH 9, J=8Hz), 8.62 (d, 1H, CH 1, J=4Hz). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 27.73 (CH3NH), 115.51
(CH 1, CH 8), 117.49 (CH 9), 117.99 (CH 6), 123.79 (Cq 5a), 132.51 (CH 2), 132.84 (Cq 3a), 138.75 (Cq
9a), 146.69 and 146.71 (OCF3, Cq 7), 149.06 (Cq 4). 19F-RMN δ (ppm, 376.5 MHz, DMSO d6) -56.81
(OCF3). MS (ESI +, QTof, m/z): 282.9 [M+H]+
N-methyl-8-(trifluoromethyl)imidazo[1,2-a]quinoxalin-4-amine (6c). Yield: 82%. C12H9F3N4. Mw:
1
266.22 g/mol. Mp: 196°C. H-NMR δ (ppm, 400 MHz, DMSO d6) 3.06 (d, 3H, CH3NH, J=4Hz), 7.65 (d,
1H, CH 2, 4Hz), 7.67 (d, 1H, CH 7, J=8Hz), 7.72 (d, 1H, CH 6, J=8Hz), 8.15 (m, 1H, NH), 8.55 (s, 1H, CH
9), 8.81 (s, 1H, CH 1). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 27.75 (CH3NH), 113.72 (CH 9), 115.88 (CH
1), 123.06 and 123.09 (CF3, CH 7), 124.64 (Cq 5a), 126.28 (Cq 8), 127.08 (CH 6), 132.57 (CH 2), 132.95
(Cq 3a), 140.53 (Cq 9a), 149.49 (Cq 4). 19F-RMN δ (ppm, 376.5 MHz, DMSO d6) -59.36 (CF3). MS (ESI +,
QTof, m/z): 267.00 [M+H]+
4.1.6. General procedure for the introduction of an alkyldiamino group.
N-Boc-diaminoalkyl (3.69 mmol) and DIEA (4.9 mmol) were added to 5 (2.45 mmol) in acetonitrile (10
mL) in a microwave-adapted vial and sealed. The reaction was submitted to microwave irradiations
at 150oC for 20 min and then evaporated. The residue was dissolved in ethyl acetate, washed with
saturated ammonium chloride solution, water and brine. The organic layer was dried over MgSO4,
17

filtered and concentrated under reduced pressure. Compound 6i is obtained as a solid and used
without further purification. The crudes mixtures were purified by flash chromatography eluted with
cyclohexane/ethyl acetate to afford compounds 6e, 6f, 6g, 6h.
tert-butyl (2-(imidazo[1,2-a]quinoxalin-4-ylamino)ethyl)carbamate (6e). Yield: 81%. C17H21N5O2. Mw:
1
327.38 g/mol. Mp: 135°C. H-NMR δ (ppm, 400 MHz, DMSO d6) 1.36 (s, 9H, 3 x CH3 tBu), 3.26 (qd,
2H, CH2NHBoc, J=4Hz), 3.61 (qd, 2H, CH2NH, J=4Hz), 7.01 (t, 1H, NHBoc, J=4Hz), 7.28 (t, 1H, CH 7,
J=8Hz), 7.30 (t, 1H, CH 8, J=8Hz), 7.58 (d, 1H, CH 9, J=8Hz), 7.62 (d, 1H, CH 2, J=4Hz), 7.68 (t, 1H, NH,
J=4Hz), 8.11 (d, 1H, CH 6, J=8Hz), 8.60 (d, 1H, CH 1, J=4Hz). 13C-NMR δ (ppm, 100 MHz, DMSO
d6) 28.68 (CH3 tBu), 40.00 (CH2NHBoc, CH2NH), 78.07 (Cq tBu), 115.03 (CH 1), 115.83 (CH 6), 123.17
(CH 7), 124.77 (Cq 5a), 126.62 (CH 9), 126.78 (CH 8), 132.26 (CH 2), 132.45 (Cq 3a), 137.17 (Cq 9a),
147.79 (Cq 4), 156.23 (C=O carbamate). MS (ESI +, QTof, m/z): 328.18 [M+H]+
tert-butyl (3-(imidazo[1,2-a]quinoxalin-4-ylamino)propyl)carbamate (6f). Yield: 93%. C18H23N5O2. Mw:
341.41 g/mol. Mp: 152°C. 1H-NMR δ (ppm, 400 MHz, DMSO d6) 1.37 (s, 9H, 3 x CH3 tBu), 1.74 (m, 2H,
CH2CH2NHBoc), 3.02 (qd, 2H, CH2NHBoc, J=4Hz), 3.56 (qd, 2H, CH2NH, J=4Hz), 6.93 (t, 1H, NHBoc,
J=4Hz), 7.26 (t, 1H, CH 7, J=8Hz), 7.37 (t, 1H, CH 8, J=8Hz), 7.58 (d, 1H, CH 9, J=8Hz), 7.60 (s, 1H, CH 2,
J=4Hz), 7.70 (t, 1H, NH, J=4Hz), 8.08 (d, 1H, CH 6, J=8Hz), 8.59 (s, 1H, CH 1, J=4Hz). 13C-NMR δ (ppm,
100 MHz, DMSO d6) 28.72 (CH3 tBu), 29.77 (CH2CH2NHBoc), 37.76 (CH2NH), 37.94 (CH2NHBoc), 77.92
(Cq tBu), 115.04 (CH 1), 115.83 (CH 6), 123.03 (CH 7), 124.70 (Cq 5a), 126.50 (CH 9), 126.76 (CH 8),
132.23 (CH 2), 132.53 (Cq 3a), 137.26 (Cq 9a), 147.79 (Cq 4), 156.11 (C=O carbamate). MS (ESI +,
QTof, m/z): 342.19 [M+H]+
tert-butyl (6-(imidazo[1,2-a]quinoxalin-4-ylamino)hexyl)carbamate (6g). Yield: 89%. C21H29N5O2. Mw:
1
383.49 g/mol. Mp: 118°C. H-NMR δ (ppm, 400 MHz, DMSO d6) 1.29 to 1.40 (m, 15H, 3 x CH3 tBu,
CH2CH2CH2NH, CH2CH2CH2NHBoc, CH2CH2NHBoc), 1.64 (qt, 2H, CH2CH2NH, J=8Hz), 2.91 (qd, 2H,
CH2NHBoc, J=4Hz), 3.56 (qd, 2H, CH2NH, J=4Hz), 6.76 (t, 1H, NHBoc, J=4Hz), 7.27 (t, 1H, CH 7, J=8Hz),
7.37 (t, 1H, CH 8, J=8Hz), 7.57 (d, 1H, CH 9, J=8Hz), 7.61 (d, 1H, CH 2, J=4Hz), 7.68 (t, 1H, NH, J=4Hz),
8.07 (d, 1H, CH 6, J=8Hz), 8.58 (d, 1H, CH 1, J=4Hz). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 26.55 and
26.73 (CH2CH2CH2NH, CH2CH2CH2NHBoc), 28.73 (CH3 tBu), 29.24 (CH2CH2NH), 29.95 (CH2CH2NHBoc),
40.00 (CH2NHBoc, CH2NH), 77.73 (Cq tBu), 114.96 (CH 1), 115.78 (CH 6), 122.88 (CH 7), 124.66 (Cq
5a), 126.59 (CH 9), 126.74 (CH 8), 132.15 (CH 2), 132.93 (Cq 3a), 137.42 (Cq 9a), 147.74 (Cq 4), 156.04
(C=O carbamate). MS (ESI +, QTof, m/z): 384.24 [M+H]+
tert-butyl 4-(imidazo[1,2-a]quinoxalin-4-yl)piperazine-1-carboxylate (6h). Yield: 63%. C19H23N5O2. Mw:
353.42 g/mol. Mp: 154°C. 1H-NMR δ (ppm, 400 MHz, DMSO d6) 1.44 (s, 9H, 3 x CH3 tBu), 3.51 (m, 4H,
2 x CH2NBoc), 4.30 (m, 4H, 2 x CH2NC=N), 7.33 (t, 1H, CH 7, J=8Hz), 7.44 (t, 1H, CH 8, J=8Hz), 7.60 (d,
1H, CH 9, J=8Hz), 7.69 (d, 1H, CH 2, J=4Hz), 8.14 (d, 1H, CH 6, J=8Hz), 8.68 (d, 1H, CH 1, J=4Hz). 13C-
NMR δ (ppm, 100 MHz, DMSO d6) 28.54 (CH3 tBu), 44.5 (CH2NBoc), 46.06 (CH2NC=N), 79.52 (Cq tBu),
114.82 (CH 1), 115.67 (CH 6), 124.01 (CH 7), 124.98 (Cq 5a), 126.97 (CH 9, CH 8), 136.03 (CH 2),
132.52 (Cq 3a), 136.03 (Cq 9a), 147.53 (Cq 4), 154.44 (C=O carbamate). MS (ESI +, QTof, m/z): 354.19
[M+H]+
4.1.7. Procedure for the introduction of the methoxy group.
Sodium methanolate (0.59 mmol) was added to 5 (0.1 mmol) in methanol (5 mL) in a microwave-
adapted vial and sealed. The reaction was submitted to microwave irradiations at 70oC for 2 min and
18

then evaporated. The residue was dissolved in dichloromethane and washed with brine. The organic
layer was dried over MgSO4, filtered and concentrated under reduced pressure to afford 6j.
1
4-methoxyimidazo[1,2-a]quinoxaline (6j). Yield: 89%. C11H9N3O. Mw: 199.21 g/mol. Mp: 169°C. H-
NMR δ (ppm, 400 MHz, DMSO d6) 4.14 (s, 3H, OCH3), 7.56 (m, 2H, CH 7, CH 8), 7.73 (s, 1H, CH 2), 7.81
(s, 1H, CH 9), 8.29 (s, 1H, CH 6), 8.76 (s, 1H, CH 1). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 54.19
(OCH3), 115.78 (CH 1), 116.22 (CH 6), 126.37 (Cq 5a), 126.50 (CH 8), 127.15 (CH 7), 128.01 (CH 9),
132.36 (Cq 3a), 133.59 (CH 2), 134.40 (Cq 9a), 153.25 (Cq 4). MS (ESI +, QTof, m/z): 200.2 [M+H]+
4.1.8. Procedure for the introduction of the amino group.
A 30% (w/v) ammoniac aqueous solution (78.6 mmol) was added to a solution of 5 (9.8 mmol) in
acetonitrile (10 mL) in a microwave-adapted vial. The reaction was submitted to microwave
irradiations during 2 hours at 140°C. The solvent was removed under reduced pressure. The crude
mixture was purified by flash chromatography eluted with cyclohexane/ethyl acetate to afford
compound 6k as a white solid.
Imidazo[1,2-a]quinoxalin-4-amine (6k). Yield: 84%. C10H8N4. Mw: 184.19 g/mol. Mp: 183°C. 1H-NMR δ
(ppm, 400 MHz, DMSO d6) 7.24 (s, 2H, NH2), ), 7.32 (t, 1H, CH 7, J=8Hz), 7.40 (t, 1H, CH 8, J=8Hz), 7.54
(d, 1H, CH 9, J=8Hz), 7.64 (s, 1H, CH 2, J=4Hz), 8.09 (d, 1H, CH 6, J=8Hz), 8.61 (s, 1H, CH 1, J=4Hz). 13C-
NMR δ (ppm, 100 MHz, DMSO d6) 115.15 (CH 1), 115.88 (CH 6), 123.14 (CH 7), 124.99 (Cq 5a), 126.04
(CH 9), 126.78 (CH 8), 132.48 (CH 2), 132.72 (Cq 3a), 137.21 (Cq 9a), 149.26 (Cq 4). MS (ESI +, QTof,
m/z): 185.10 [M+H]+
4.1.9. General procedure for bromination.
A solution of 6 (3.5 mmol) and N-bromosuccinimide (3.85 mmol) in chloroform (100 mL) was heated
under reflux for 2 hours. The solvent was evaporated under vacuum and the residue was dissolved in
ethyl acetate, washed twice with a saturated aqueous NH4Cl solution, saturated aqueous NaHCO3
solution, distillated water, brine, then dried over Na2SO4, filtered and concentrated under vacuum.
Compounds 7 are used without further purification as the reaction is considered as complete.
1-bromo-N-methyl-7-(trifluoromethyl)imidazo[1,2-a]quinoxalin-4-amine (7a). C12H8BrF3N4. Mw:
345.12 g/mol. Mp: 187°C. 1H-NMR δ (ppm, 400 MHz, DMSO d6) 3.04 (d, 3H, CH3NH, J=8Hz), 7.65 (m,
2H, CH 8), 7.78 (s, 1H, CH 2), 7.88 (s, 1H, CH 6), 8.11 (m, 1H, NHCH3), 9.12 (d, 1H, CH 9, J=8Hz). 19F-
RMN δ (ppm, 376.5 MHz, DMSO d6) -60.42. 13C-NMR δ (ppm, 100 MHz, DMSO d6) 27.71 (CH3NH),
100.03 (Cq 1), 117.19 (CH 9), 118.43 (CH 8), 123.62 (CF3), 127.74 (Cq 5a), 128.30 (CH 6), 132.72 (Cq
7), 134.29 (Cq 3a), 135.00 (CH 2), 138.56 (Cq 9a), 148.68 (Cq 4). MS (ESI +, QTof, m/z): 344.90 [M+H]+
1-bromo-N-methyl-7-(trifluoromethoxy)imidazo[1,2-a]quinoxalin-4-amine (7b). C12H8BrF3N4O. Mw:
1
361.12 g/mol. Mp: 181°C. H-NMR δ (ppm, 400 MHz, DMSO d6) 3.02 (d, 3H, CH3NH, J=8Hz), 7.30 (d,
1H, CH 8, J=8Hz), 7.51 (s, 1H, CH 6), 7.73 (s, 1H, CH 2), 8.05 (m, 1H, NHCH3), 9.02 (d, 1H, CH 9, J=8Hz).
13C-NMR δ (ppm, 100 MHz, DMSO d6) 27.70 (CH3NH), 99.85 (Cq 1), 114.79 (CH 8), 116.88 (CH 9),
119.34 (CH 6), 121.89 (CF3), 124.80 (Cq 5a), 134.19 (CH 2), 134.71 (Cq 3a), 139.55 (Cq 9a), 146.82 (Cq
7), 148.03 (Cq 4). 19F-RMN δ (ppm, 376.5 MHz, DMSO d6) -56.77. MS (ESI +, QTof, m/z): 360.90
[M+H]+
1-bromo-N-methyl-8-(trifluoromethyl)imidazo[1,2-a]quinoxalin-4-amine (7c). C12H8BrF3N4. Mw:
345.12 g/mol. Mp: 194°C. 1H-NMR δ (ppm, 400 MHz, DMSO d6) 3.04 (d, 3H, CH3NH, J=8Hz), 7.75 (m,
19

3H, CH 2, CH 6, CH 7), 8.21 (m, 1H, NH), 9.24 (s, 1H, CH 9). 13C-NMR δ (ppm, 100 MHz, DMSO
d6) 27.73 (CH3NH), 100.32 (Cq 1), 112.26 (CH 9), 123.60 and 123.63 (CF3, CH 7), 125.58 (Cq 5a),
126.18 (Cq 8), 127.81 (CH 6), 134.25 (Cq 3a), 134.83 (CH 2), 141.44 (Cq 9a), 149.02 (Cq 4). 19F-RMN δ
(ppm, 376.5 MHz, DMSO d6) -59.89 (CF3). MS (ESI +, QTof, m/z): 344.90 [M+H]+
tert-butyl (2-((1-bromoimidazo[1,2-a]quinoxalin-4-yl)amino)ethyl)carbamate (7e). C17H20BrN5O2. Mw:
406.28 g/mol. Mp: 137°C. 1H-NMR δ (ppm, 400 MHz, DMSO d6) 1.36 (s, 9H, 3 x CH3 tBu), 3.26 (m, 2H,
CH2NHBoc), 3.59 (m, 2H, CH2NH), 6.98 (m, 1H, NHBoc), 7.30 (t, 1H, CH 7, J=8Hz), 7.35 (t, 1H, CH 8,
J=8Hz), 7.61 (d, 1H, CH 9, J=8Hz), 7.72 (m, 2H, CH 2, NH), 8.96 (d, 1H, CH 6, J=8Hz). 13C-NMR δ (ppm,
100 MHz, DMSO d6) 28.68 (CH3 tBu), 40.00 (CH2NHBoc, CH2NH), 78.09 (Cq tBu), 99.35 (Cq 1), 114.98
(CH 6), 122.66 (CH 7), 125.91 (Cq 5a), 127.34 (CH 8, CH 9), 134.20 (CH 2), 134.35 (Cq 3a), 138.03 (Cq
9a), 147.35 (Cq 4), 156.24 (C=O carbamate). MS (ESI +, QTof, m/z): 406.08 [M+H]+.
tert-butyl (3-((1-bromoimidazo[1,2-a]quinoxalin-4-yl)amino)propyl)carbamate (7f). C18H22BrN5O2.
1
Mw: 420.30 g/mol. Mp: 148°C. H-NMR δ (ppm, 400 MHz, DMSO d6) 1.38 (s, 9H, 3 x CH3 tBu), 1.74
(m, 2H, CH2CH2NHBoc), 3.00 (qd, 2H, CH2NHBoc, J=4Hz), 3.56 (qd, 2H, CH2NH, J=4Hz), 6.90 (m, 1H,
NHBoc), 7.54 (m, 3H, CH 7, CH 8, CH 9), 7.73 (s, 1H, CH 2, J=4Hz), 7.97 (m, 1H, NH), 9.04 (d, 1H, CH 6,
J=8Hz). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 28.71 (CH3 tBu), 29.59 (CH2CH2NHBoc), 37.93 (CH2NH,
CH2NHBoc), 77.95 (Cq tBu), 100.04 (Cq 1), 117.42 (CH 6), 126.66 (CH 7), 127.42 (Cq 5a), 128.59 (CH
8), 130.02 (CH 9), 134.03 (Cq 3a), 134.73 (CH 2), 137.24 (Cq 9a), 147.45 (Cq 4), 156.11 (C=O
carbamate). MS (ESI +, QTof, m/z): 420.10 [M+H]+
tert-butyl (6-((1-bromoimidazo[1,2-a]quinoxalin-4-yl)amino)hexyl)carbamate (7g). C21H28BrN5O2. Mw:
1
462.38 g/mol. Mp: 113°C. H-NMR δ (ppm, 400 MHz, DMSO d6) 1.29 to 1.40 (m, 15H, 3 x CH3 tBu,
CH2CH2CH2NH, CH2CH2CH2NHBoc, CH2CH2NHBoc), 1.63 (m, 2H, CH2CH2NH), 2.87 (qd, 2H, CH2NHBoc,
J=4Hz), 3.51 (qd, 2H, CH2NH, J=4Hz), 6.76 (t, 1H, NHBoc, J=4Hz), 7.28 (t, 1H, CH 7, J=8Hz), 7.42 (t, 1H,
CH 8, J=8Hz), 7.58 (d, 1H, CH 9, J=8Hz), 7.63 (s, 1H, CH 2), 7.74 (m, 1H, NH), 8.94 (d, 1H, CH 6, J=8Hz).
13C-NMR δ (ppm, 100 MHz, DMSO d6) 26.53 and 26.69 (CH2CH2CH2NH, CH2CH2CH2NHBoc), 28.73 (CH3
tBu), 29.14 (CH2CH2NH), 29.93 (CH2CH2NHBoc), 40.00 (CH2NHBoc, CH2NH), 77.73 (Cq tBu), 99.39 (Cq
1), 114.96 (CH 6), 122.45 (CH 7), 125.73 (Cq 5a), 127.31 (CH 8, CH 9), 134.20 (Cq 3a), 134.50 (CH 2),
134.64 (Cq 9a), 149.14 (Cq 4), 156.03 (C=O carbamate). MS (ESI +, QTof, m/z): 462.15 [M+H]+
tert-butyl 4-(1-bromoimidazo[1,2-a]quinoxalin-4-yl)piperazine-1-carboxylate (7h). C19H22BrN5O2. Mw:
432.31 g/mol. Mp: 139°C. 1H-NMR δ (ppm, 400 MHz, DMSO d6) 1.38 (s, 9H, 3 x CH3 tBu), 3.45 (m, 4H,
2 x CH2NBoc), 4.15 (m, 4H, 2 x CH2NC=N), 7.28 (t, 1H, CH 7, J=8Hz), 7.44 (t, 1H, CH 8, J=8Hz), 7.54 (d,
1H, CH 9, J=8Hz), 7.70 (d, 1H, CH 2, J=4Hz), 8.93 (d, 1H, CH 6, J=8Hz). 13C-NMR δ (ppm, 100 MHz,
DMSO d6) 28.53 (CH3 tBu), 42.5 (CH2NBoc), 46.42 (CH2NC=N), 79.54 (Cq tBu), 99.64 (Cq 1), 114.97 (CH
6), 123.49 (CH 7), 126.11 (Cq 5a), 127.47 (CH 9, CH 8), 134.29 (CH 2), 134.66 (Cq 3a), 136.85 (Cq 9a),
147.33 (Cq 4), 154.43 (C=O carbamate). MS (ESI +, QTof, m/z): 432.10 [M+H]+
1-bromo-N,N-dimethylimidazo[1,2-a]quinoxalin-4-amine (7i). C12H11BrN4. Mw: 291.14 g/mol. Mp:
129°C. 1H-NMR δ (ppm, 400 MHz, DMSO d6) 3.50 (s, 6H, 2 x CH3), 7.27 (td, 1H, CH 7, J=8Hz, J=1.4Hz),
7.43 (td, 1H, CH 8, J=8Hz, J=1.4Hz), 7.58 (dd, 1H, CH 9, J=8Hz, J=1.4Hz), 7.71 (s, 1H, CH 2), 8.96 (dd,
1H, CH 6, J=8Hz, J=1.4Hz). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 40.00 (2 x CH3), 99.43 (Cq 1), 114.88
(CH 6), 122.41 (CH 7), 125.77 (Cq 5a), 127.06 (CH 9), 127.30 (CH 8), 134.38 (CH 2), 134.90 (Cq 3a),
135.51 (Cq 9a), 147.98 (Cq 4). MS (ESI +, QTof, m/z): 290.9 [M+H]+.
20

1
1-bromo-4-methoxyimidazo[1,2-a]quinoxaline (7j). C11H8BrN3O. Mw: 278.10 g/mol. Mp: 148°C. H-
NMR δ (ppm, 400 MHz, DMSO d6) 4.19 (s, 3H, OCH3), 7.67 (m, 2H, CH 7, CH 8), 7.74 (s, 1H, CH 2), 7.92
(m, 1H, CH 9), 9.16 (m, 1H, CH 6). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 54.33 (OCH3), 100.29 (Cq 1),
115.29 (CH 6), 125.95 (CH 7, Cq 5a), 127.66 (CH 8), 128.60 (CH 9), 133.86 (Cq 3a), 135.25 (Cq 9a),
135.89 (CH 2), 152.74 (Cq 4). MS (ESI +, QTof, m/z): 277.8 [M+H]+.
1
1-bromoimidazo[1,2-a]quinoxalin-4-amine (7k). C10H7BrN4. Mw: 263.09 g/mol. Mp: 134°C. H-NMR δ
(ppm, 400 MHz, DMSO d6) 7.24 (s, 2H, NH2), 7.30 (td, 1H, CH 7, J=8Hz, J=1.4Hz), 7.40 (td, 1H, CH 8,
J=8Hz, J=1.4Hz), 7.59 (dd, 1H, CH 9, J=8Hz, J=1.4Hz), 7.72 (s, 1H, CH 2), 8.97 (dd, 1H, CH 6, J=8Hz,
J=1.4Hz). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 99.41 (Cq 1), 115.00 (CH 6), 122.54 (CH 7), 126.20 (Cq
5a), 126.79 (CH 9), 127.29 (CH 8), 134.06 (Cq 3a), 134.72 (CH 2), 138.25 (Cq 9a), 148.93 (Cq 4). MS
(ESI +, QTof, m/z): 263.00 [M+H]+.
4.1.11. General procedure for the Suzuki-Miyaura cross-coupling.
To a mixture of 7 (1.53 mmol) in DME/H2O (v/v, 1/1, 10 mL) were added 3,4-dimethoxyphenylboronic
acid (1.84 mmol), tetrakis(triphenylphosphine) palladium (0.077 mmol) and sodium carbonate (3.06
mmol) in a microwave-adapted vial and sealed. The reaction was submitted to microwave
irradiations during 20 minutes at 150°C and then filtered on a Celite pad. The filtrate was
concentrated under reduced pressure and purified by flash chromatography eluted with
cyclohexane/ethyl acetate to ethyl acetate/methanol to afford 8 as solid.
EAPB02203-7a:
1-(3,4-dimethoxyphenyl)-N-methyl-7-(trifluoromethyl)imidazo[1,2-a]quinoxalin-4-
1
amine (8a). Yield: 13%. C20H17F3N4O2. Mw: 402.37 g/mol. Mp: 165°C. H-NMR δ (ppm, 400 MHz,
DMSO d6) 3.06 (d, 3H, CH3NH, J=8Hz), 3.75 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 7.15 (m, 2H, CH 5’, CH
6’), 7.20 (s, 1H, CH 2’), 7.33 (d, 1H, CH 8, J=8Hz), 7.45 (d, 1H, CH 9, J=8Hz), 7.54 (s, 1H, CH 2), 7.85 (s,
1H, CH 6), 8.05 (m, 1H, NHCH3). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 27.72 (CH3NH), 56.03 (OCH3),
56.16 (OCH3), 112.37 (CH 5’), 114.01 (CH 2’), 117.09 (CH 9), 118.97 (CH 8), 122.18 (Cq 1’), 122.88 (CH
6, CH 6’), 125.85 (CF3), 126.34 (Cq 5a), 131.29 (Cq 1), 132.82 (CH 2), 133.01 (Cq 3a), 137.53 (Cq 9a),
138.47 (Cq 7), 149.27 (Cq 3’, Cq 4’), 150.22 (Cq 4). 19F-RMN δ (ppm, 400 MHz, DMSO d6) -60.42. MS
(ESI +, QTof, m/z): 403.00 [M+H]+
EAPB02203-7b: 1-(3,4-dimethoxyphenyl)-N-methyl-7-(trifluoromethoxy)imidazo[1,2-a]quinoxalin-4-
1
amine (8b). Yield: 67%. C20H17F3N4O3. Mw: 418.37 g/mol. Mp: 169°C. H-NMR δ (ppm, 400 MHz,
DMSO d6) 3.08 (d, 3H, CH3NH, J=8Hz), 3.77 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 7.01 (d, 1H, CH 8, J=8Hz),
7.12 (d, 1H, CH 6’, J=8Hz), 7.17 (d, 1H, CH 5’, J=8Hz), 7.20 (s, 1H, CH 2’), 7.36 (d, 1H, CH 9), 7.48 (s, 1H,
CH 6), 7.51 (d, 1H, CH 2, J=4Hz), 8.02 (d, 1H, NHCH3, J=4Hz). 13C-NMR δ (ppm, 100 MHz, DMSO
d6) 27.75 (CH3NH), 56.01 (OCH3), 56.14 (OCH3), 112.34 (CH 5’), 114.14 (CH 2’), 114.59 (CH 8), 117.23
(CH 9), 118.12 (CH 6), 122.35 (Cq 1’), 123.29 (CH 6’), 124.01 (Cq 5a), 124.40 (CF3), 131.01 (Cq 1),
132.56 (CH 2), 133.24 (Cq 3a), 139.82 (Cq 9a), 146.29 (Cq 7), 149.27 and 149.36 (Cq 4’, Cq 3’), 150.20
(Cq 4). 19F-RMN δ (ppm, 376.5 MHz, DMSO d6) -56.86 (OCF3). MS (ESI +, QTof, m/z): 419.20 [M+H]+.
HRMS calculated for C20H18F3N4O3 419.1326, found 419.1329.
EAPB02203-8a:
1-(3,4-dimethoxyphenyl)-N-methyl-8-(trifluoromethyl)imidazo[1,2-a]quinoxalin-4-
1
amine (8c). Yield: 21%. C20H17F3N4O2. Mw: 402.37 g/mol. Mp: 179°C. H-NMR δ (ppm, 400 MHz,
DMSO d6) 2.94 (d, 3H, CH3NH), 3.75 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 7.16 (m, 2H, CH 5’, CH 6’), 7.23
(s, 1H, CH 2’), 7.55 (s, 1H, CH 2), 7.59 (m, 2H, CH 7, CH 9), 7.72 (d, 1H, CH 6, J=8Hz), 8.16 (m, 1H, NH).
21

13C-NMR δ (ppm, 100 MHz, DMSO d6) 29.42 (CH3NH), 56.18 (OCH3), 56.24 (OCH3), 112.55 (CH 5’),
113.31 (CH 9), 114.22 (CH 2’), 122.12 (Cq 1’), 122.765 (CH 7), 123.46 (CH 6’), 125.40 (Cq 5a),127.53
(CH 6), 129.18 (CF3), 131.21 (Cq 1), 132.53 (CH 2), 133.36 (Cq 3a), 141.46 (Cq 9a), 142.19 (Cq 8),
149.55 (Cq 3’, Cq 4’), 150.47 (Cq 4). 19F-RMN δ (ppm, 376.5 MHz, DMSO d6) -60.28. MS (ESI +, QTof,
m/z): 403.00 [M+H]+. HRMS calculated for C20H18F3N4O2 403.1376, found 403.1385.
EAPB02203: 1-(3,4-dimethoxyphenyl)-N-methylimidazo[1,2-a]quinoxalin-4-amine (8d). Yield: 47%.
C19H18N4O2. Mw: 334.37 g/mol. Mp: 168°C. 1H-NMR δ (ppm, 400 MHz, DMSO d6) 3.09 (s, 3H, NHCH3),
3.69 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 7.03 (t, 1H, CH 7, J=8Hz), 7.13 (m, 3H, CH 2’, CH 5’, CH 6’), 7.26
(d, 1H, CH 6, J=8Hz), 7.36 (t, 1H, CH 8, J=8Hz), 7.51 (s, 1H, CH 2), 7.69 (d, 1H, CH 9, J=8Hz). 13C-NMR δ
(ppm, 100 MHz, DMSO d6) 27.26 (NHCH3), 54.87 (OCH3), 54.93 (OCH3), 111.10 (CH 6’), 112.47 (CH 2’),
115.03 (CH 6), 120.87 (Cq 1’), 122.32 and 122.40 (CH 7, CH 5’), 123.57 (Cq 5a), 123.92 (CH 8), 126.26
(CH 9), 130.70 (Cq 1), 131.68 and 131.89 (CH 2, Cq 3a), 133.58 (Cq 9a), 146.62 (Cq 3’), 147.90 (Cq 4’),
148.93 (Cq 4). MS (ESI +, QTof, m/z): 335.20 [M+H]+. HRMS calculated for C19H19N4O2 335.1518, found
335.1503.
EAPB02211:
tert-butyl
(2-((1-(3,4-dimethoxyphenyl)imidazo[1,2-a]quinoxalin-4-
1
yl)amino)ethyl)carbamate (8e). Yield: 57%. C25H29N5O4. Mw: 463.53 g/mol. Mp: 168°C. H-NMR δ
(ppm, 400 MHz, DMSO d6) 1.38 (s, 9H, 3 x CH3 tBu), 3.27 (qd, 2H, CH2NHBoc, J=8Hz), 3.63 (qd, 2H,
CH2NH, J=8Hz), 3.74 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 6.98 (m, 2H, NHBoc, CH 7), 7.16 (m, 3H, CH 2’,
CH 5’, CH 6’), 7.30 (m, 2H, CH 8, CH 6), 7.49 (s, 1H, CH 2), 7.58 (d, 2H, CH 9, J=8Hz), 7.71 (t, 1H, NH,
J=4Hz). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 28.70 (CH3 tBu), 40.00 (CH2NHBoc, CH2NH), 56.05
(OCH3), 56.15 (OCH3), 78.10 (Cq tBu), 112.30 (CH 2’), 114.19 (CH 5’), 115.88 (CH 6), 122.35 (Cq 1’),
122.76 (CH 7), 123.26 (CH 6’), 125.84 (Cq 5a), 126.49 (CH 8), 127.09 (CH 9), 130.85 (Cq 1), 132.42 (CH
2), 133.30 (Cq 3a), 138.06 (Cq 9a), 148.00 (Cq 3’), 149.25 (Cq 4’), 150.10 (Cq 4), 156.25 (C=O
carbamate). MS (ESI +, QTof, m/z): 464.23 [M+H]+. HRMS calculated for C25H30N5O4 464.2292, found
464.2294.
EAPB02214 :
tert-butyl
(3-((1-(3,4-dimethoxyphenyl)imidazo[1,2-a]quinoxalin-4-
1
yl)amino)propyl)carbamate (8f). Yield: 69%. C26H31N5O4. Mw: 477.56 g/mol. Mp: 171°C. H-NMR δ
(ppm, 400 MHz, DMSO d6) 1.36 (s, 9H, 3 x CH3 tBu), 1.77 (m, 1H, CH2CH2NHBoc), 3.02 (qd, 2H,
CH2NHBoc, J=4Hz), 3.56 (qd, 2H, CH2NH, J=4Hz), 3.74 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 6.97 (m, 2H,
CH 7, NHBoc), 7.17 (m, 3H, CH 6’, CH 2’, CH 5’), 7.29 (m, 2H, CH 6, CH 8), 7.49 (s, 1H, CH 2), 7.61 (m,
1H, CH 9). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 28.73 (CH3 tBu), 29.80 (CH2CH2NHBoc), 37.91
(CH2NHBoc, CH2NH), 56.05 (OCH3), 56.15 (OCH3), 77.95 (Cq tBu), 112.30 (CH 5’), 114.22 (CH 2’),
115.92 (CH 6), 122.27 (Cq 1’), 123.28 (CH 7, CH 6’), 125.73 to 127.93 (CH 8, CH 9, Cq 5a), 130.85 (Cq
1), 132.43 (CH 2), 133.13 (Cq 3a), 139.56 (Cq 9a), 147.95 (Cq 3’), 149.25 (Cq 4’), 150.11 (Cq 4), 156.12
(C=O carbamate). MS (ESI +, QTof, m/z): 478.25 [M+H]+. HRMS calculated for C26H32N5O4 478.2454,
found 478.2456.
EAPB02212:
tert-butyl
(6-((1-(3,4-dimethoxyphenyl)imidazo[1,2-a]quinoxalin-4-
1
yl)amino)hexyl)carbamate (8g). Yield: 61%. C29H37N5O4. Mw: 519.64 g/mol. Mp: 133°C. H-NMR δ
(ppm, 400 MHz, DMSO d6) 1.31 to 1.40 (m, 15H, 3 x CH3 tBu, CH2CH2CH2NH, CH2CH2CH2NHBoc,
CH2CH2NHBoc), 1.66 (qt, 2H, CH2CH2NH, J=8Hz), 2.91 (qd, 2H, CH2NHBoc, J=4Hz), 3.53 (qd, 2H, CH2NH,
J=4Hz), 3.74 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 6.77 (t, 1H, NHBoc, J=4Hz), 6.95 (t, 1H, CH 7, J=8Hz),
7.10 (m, 3H, CH 2’, CH 5’, CH 6’), 7.28 (m, 2H, CH 6, CH 8), 7.47 (s, 1H, CH 2), 7.57 (d, 1H, CH 9,
J=8Hz), 7.67 (t, 1H, NH, J=4Hz). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 26.5 and 26.72 (CH2CH2CH2NH,
22

CH2CH2CH2NHBoc), 28.74 (CH3 tBu), 29.30 (CH2CH2NH), 29.97 (CH2CH2NHBoc), 40.00 (CH2NHBoc,
CH2NH), 56.04 (OCH3), 56.14 (OCH3) 77.74 (Cq tBu), 112.28 (CH 2’), 114.23 (CH 5’), 115.85 (CH 6),
122.05 (CH 7), 122.84 (Cq 1’), 123.27 (CH 6’), 125.73 (Cq 5a), 126.44 (CH 8), 127.05 (CH 9), 130.79 (Cq
1), 132.31 (CH 2), 133.35 (Cq 3a), 138.31 (Cq 9a), 147.96 (Cq 3’), 149.24 (Cq 4’), 150.08 (Cq 4), 156.05
(C=O carbamate). MS (ESI +, QTof, m/z): 520.29 [M+H]+. HRMS calculated for C29H38N5O4 520.2918,
found 520.2925.
EAPB02213:
tert-butyl
4-(1-(3,4-dimethoxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)piperazine-1-
1
carboxylate (8h). Yield: 85%. C27H31N5O4. Mw: 489.57 g/mol. Mp: 170°C. H-NMR δ (ppm, 400 MHz,
DMSO d6) 1.45 (s, 9H, 3 x CH3 tBu), 3.53 (m, 4H, 2 x CH2NBoc), 3.73 (s, 3H, OCH3), 3.87 (s, 3H, OCH3),
4.29 (m, 4H, 2 x CH2NC=N), 7.05 (t, 1H, CH 7, J=8Hz), 7.10 (d, 1H, CH 6’), 7.15 (m, 2H, CH 2’, CH 5’),
7.30 (d, 1H, CH 6, J=8Hz), 7.35 (t, 1H, CH 8, J=8Hz), 7.56 (s, 1H, CH 2), 7.63 (d, 1H, CH 9, J=8Hz). 13C-
NMR δ (ppm, 100 MHz, DMSO d6) 28.56 (CH3 tBu), 43.00 (CH2NBoc), 46.40 (CH2NC=N), 56.07 (OCH3),
56.16 (OCH3), 79.54 (Cq tBu), 112.36 (CH 5’), 114.12 (CH 2’), 115.81 (CH 6), 122.79 (CH 7), 123.23 (CH
6’, Cq 1’), 125.73 (Cq 5a), 126.44 (CH 8), 127.35 (CH 9), 130.36 (Cq 1), 132.51 (CH 2), 133.35 (Cq 3a),
136.91 (Cq 9a), 147.94 (Cq 3’), 149.33 (Cq 4’), 150.18 (Cq 4), 154.46 (C=O carbamate). MS (ESI +,
QTof, m/z): 490.25 [M+H]+. HRMS calculated for C27H32N5O4 490.2449, found 490.2451.
EAPB02204: 1-(3,4-dimethoxyphenyl)-N,N-dimethylimidazo[1,2-a]quinoxalin-4-amine (8i). Yield: 28%.
1
C20H20N4O2. Mw: 348.40 g/mol. Mp: 185°C. H-NMR δ (ppm, 400 MHz, DMSO d6) 3.74 (s, 3H, CH3),
3.78 (s, 3H, CH3), 3.84 (s, 3H, CH3), 3.87 (s, 3H, CH3), 7.02 (m, 2H, CH 7, CH 6’), 7.15 (m, 3H, CH 2, CH
2’, CH 5’), 7.26 (d, 1H, CH 6, J = 8Hz), 7.33 (t, 1H, CH 8, J = 8Hz), 7.64 (m, 1H, CH 9). 13C-NMR δ (ppm,
100 MHz, DMSO d6) 56.07 (4 x CH3), 110.89 (CH 5’), 112.37 (CH 2’), 112.63 (CH 6’), 115.94 (CH 6),
118.99 (CH 2), 122.21 (CQ 1’, Cq 5’), 122.64 (CH 7), 125.44 (Cq 3’), 126.78 (CH 8), 132.73 (Cq 1),
132.73 (Cq 1), 133.59 (Cq 9’), 133.72 (CH 9), 148.54 (Cq 3’), 149.45 (Cq 4’), 150.23 (Cq 4). MS (ESI +,
QTof, m/z): 349.20 [M+H]+. HRMS calculated for C20H21N4O2 349.1663, found 349.1665.
EAPB02215: 1-(3,4-dimethoxyphenyl)-4-methoxyimidazo[1,2-a]quinoxaline (8j). Yield: 32%.
1
C19H17N3O3. Mw: 335.36 g/mol. Mp: 178°C. H-NMR δ (ppm, 400 MHz, DMSO d6) 3.75 (s, 3H, OCH3),
3.88 (s, 3H, OCH3), 4.16 (s, 3H, OCH3), 7.18 (m, 3H, CH 2’, CH 5’, CH 6’), 7.28 (t, 1H, CH 8, J=8Hz), 7.42
(m, 2H, CH 7, CH 9), 7.56 (s, 1H, CH 2), 7.80 (d, 1H, CH 6, J=8Hz). 13C-NMR δ (ppm, 100 MHz, DMSO
d6) 54.22 (OCH3), 56.07 and 56.17 (2 x OCH3), 112.34 (CH 5’), 114.16 (CH 2’), 116.10 (CH 9), 122.35
(Cq 1’), 123.29 (CH 6’), 125.61 (CH 8), 127.45 (CH 7), 127.98 (Cq 9a), 128.70 (CH 6), 131.32 (Cq 1),
132.76 (Cq 3a), 133.87 (CH 2), 135.24 (Cq 5), 149.32 (Cq 4’), 150.25 (Cq 3a), 153.41 (Cq 4). MS (ESI +,
QTof, m/z): 335.9 [M+H]+. HRMS calculated for C19H18N3O3 336.1343, found 336.1348.
EAPB02202: 1-(3,4-dimethoxyphenyl)-4-methoxyimidazo[1,2-a]quinoxaline (8k). Yield: 70%.
1
C18H16N4O2. Mw: 320.34 g/mol. Mp: 178°C. H-NMR δ (ppm, 400 MHz, DMSO d6) 3.74 (s, 3H, OCH3),
3.87 (s, 3H, OCH3), 6.99 (t, 1H, CH 7, J=8Hz), 7.13 (m, 3H, CH 2’, CH 5’, CH 6’), 7.22 (m, 2H, CH 6, CH 8),
7.50 (s, 1H, CH 2), 7.56 (m, 2H, NH2), 7.63 (d, 1H, CH 9, J=8Hz). 13C-NMR δ (ppm, 100 MHz, DMSO
d6) 54.94 (OCH3), 55.04 (OCH3), 111.13 (CH 6’), 113.07 (CH 2’), 114.80 (CH 6), 121.15 (CH 7), 121.70
(Cq 1’), 122.19 (CH 5’), 125.01 (Cq 5a), 125.47 (CH 8), 128.11 (CH 9), 130.83 (Cq 1), 131.55 (CH 2),
132.04 (Cq 3a), 137.16 (Cq 9a), 148.12 (Cq 3’), 148.42 (Cq 4’), 148.96 (Cq 4). MS (ESI +, QTof, m/z):
321.10 [M+H]+. HRMS calculated for C18H17N4O2 336.1343, found 336.1348.
4.1.12. General procedure for the cleavage of all protections.
23

After solubilisation of compound 8 (0.411 mmol) in dichloromethane, boron tribromide (2
eq/protecting group) was slowly added at 0°C. The reaction was allowed to warm up to room
temperature and stirred for 2 hours. Methanol was added to quench the reaction which was then
concentrated under reduced pressure. The crude mixture was purified by flash chromatography
eluted with ethyl acetate/methanol to afford 9 as solid.
EAPB02303-7a: 4-(4-(methylamino)-7-(trifluoromethyl)imidazo[1,2-a]quinoxalin-1-yl)benzene-1,2-diol
1
(9a). Yield: 28%. C18H13F3N4O2. Mw: 374.32 g/mol. Mp: 248°C. H-NMR δ (ppm, 400 MHz, DMSO d6)
3.06 (d, 3, CH3NH, J=8Hz), 6.81 (d, 1H, CH 6’, J=8Hz), 6.89 (s, 1H, CH 2’), 6.92 (d, 1H, CH 5’, J=8Hz),
7.34 (d, 1H, CH 8, J=8Hz), 7.47 (s, 1H, CH 2), 7.51 (d, 1H, CH 9, J=8Hz), 7.82 (s, 1H, CH 6), 8.03 (m, 1H,
NHCH3). 19F-RMN δ (ppm, 376.5 MHz, DMSO d6) -60.72 (CF3). 13C-NMR not available due to weak
product amount. MS (ESI +, QTof, m/z): 375.10 [M+H]+. HRMS calculated for C18H14F3N4O2 375.1063,
found 375.1072.
EAPB02303-7b: 4-(4-(methylamino)-7-(trifluoromethoxy)imidazo[1,2-a]quinoxalin-1-yl)benzene-1,2-
1
diol (9b). Yield: 67%. C18H13F3N4O3. Mw: 390.32 g/mol. Mp: 255°C. H-NMR δ (ppm, 400 MHz, DMSO
d6) 3.08 (d, 3H, CH3NH, J=8Hz), 6.84 (d, 1H, CH 6’, J=8Hz), 6.94 (s, 1H, CH 2’), 6.97 (d, 1H, CH 5’,
J=8Hz), 7.03 (d, 1H, CH 8, J=8Hz), 7.40 (d, 1H, CH 9, J=8Hz), 7.42 (s, 1H, CH 2), 7.45 (s, 1H, CH 6), 8.01
(d, 1H, NHCH3, J=4Hz). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 27.74 (CH3NH), 114.58 (CH8), 116.56 (CH
5’), 117.15 (CH 9), 117.72 (CH 2’), 118.12 (CH 6), 120.80 (Cq 1’), 121.99 (CH 6’), 124.76 (Cq 5a),
131.38 (Cq 1), 132.20 (CH 2), 133.04 (Cq 3a), 139.79 (Cq 9a), 146.12 (Cq 3’), 146.25 (Cq 7), 147.18 (Cq
4’), 149.27 (Cq 4). 19F-RMN δ (ppm, 376.5 MHz, DMSO d6) -56.83 (OCF3). MS (ESI +, QTof, m/z):
391.10 [M+H]+. HRMS calculated for C18H14F3N4O3 391.1013, found 391.1018.
EAPB02303-8a: 4-(4-(methylamino)-8-(trifluoromethyl)imidazo[1,2-a]quinoxalin-1-yl)benzene-1,2-diol
1
(9c). Yield: 19%. C18H13F3N4O2. Mw: 374.32 g/mol. Mp: 259°C. H-NMR δ (ppm, 400 MHz, DMSO
d6) 3.12 (d, 3H, CH3NH, J=8Hz), 6.83 (d, 1H, CH 6’, J=8Hz), 6.91 (s, 1H, CH 2’), 6.96 (d, 1H, CH 5’), 7.55
(s, 1H, CH 2), 7.60 (s, 1H, CH 9), 7.67 (d, 1H, CH 7, J=8Hz), 7.82 (d, 1H, CH 6). 13C-NMR δ (ppm, 100
MHz, DMSO d6) 28.38 (CH3NH), 113.45 (CH 9), 116.52 (CH 5’), 118.50 (CH 2’), 120.08 (Cq 1’), 121.93
(CH 1’), 123.11 (CH 7), 125.08 (Cq 5a), 132.68 to 132.75 (CH 2, Cq 1, Cq 3a, Cq 9a), 146.36 (Cq 3’, Cq
4’), 147.49 (Cq 4). 19F-RMN δ (ppm, 376.5 MHz, DMSO d6) -60.44 (CF3). MS (ESI +, QTof, m/z): 375.20
[M+H]+. HRMS calculated for C18H14F3N4O2 375.1063, found 375.1070.
EAPB02303: 4-(4-(methylamino)imidazo[1,2-a]quinoxalin-1-yl)benzene-1,2-diol (9d). Yield: 96 %.
C17H14N4O2. Mw: 306.31 g/mol. Mp: 267°C. 1H-NMR δ (ppm, 400 MHz, DMSO d6) 3.04 (d, 3H, NHCH3,
J=4Hz), 6.80 (dd, 1H, CH 6’, J=4Hz, J=8Hz), 6.88 (d, 1H, CH 2’, J=4Hz), 6.89 (d, 1H, CH 5’, J=8Hz), 6.97
(td, 1H, CH 7, J=8Hz, J=4Hz), 7.31 (m, 2H, CH 6, CH 8), 7.39 (s, 1H, CH 2), 7.58 (dd, 1H, CH 9, J=4Hz,
J=8Hz), 7.69 (qd, 1H, NH, J=4Hz). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 27.25 (NHCH3), 115.86 (CH
6), 116.45 (CH 5’), 117.87 (CH 2’), 121.27 (Cq 1’), 122.00 (CH 6’), 122.08 (CH 7), 125.78 (Cq 5’), 126.38
(CH 8), 126.99 (CH 9), 131.12 (Cq 1), 131.98 (CH 2), 133.27 (Cq 3’ quinoxaline), 138.28 (Cq 9’), 146.04
(Cq 3’ phenyl), 147.07 (Cq 4’ phenyl), 148.55 (Cq 4). MS (ESI +, QTof, m/z) : 307.12 [M+H]+. HRMS
calculated for C17H15N4O2 307.1195, found 307.1194.
EAPB02306: 4-(4-((2-aminoethyl)amino)imidazo[1,2-a]quinoxalin-1-yl)benzene-1,2-diol (9e). Yield:
1
94%. C18H17N5O2. Mw: 335.36 g/mol. Mp: 263°C. H-NMR δ (ppm, 400 MHz, DMSO d6) 3.14 (m, 2H,
CH2NH2), 3.81 (m, 2H, CH2NH), 6.79 (d, 1H, CH 6’, J=8Hz), 6.90 (s, 1H, CH 2’), 6.94 (d, 1H, CH 5’,
J=8Hz), 7.01 (t, 1H, CH 7, J=8Hz), 7.31 (m, 2H, CH 8, CH 6), 7.43 (s, 1H, CH 2), 7.60 (d, 1H, CH 9, J=8Hz),
24

7.87 (t, 1H, NH, J=4Hz). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 40.00 (CH2NH2, CH2NH), 115.83 (CH 6),
116.52 (CH 5’), 117.86 (CH 2’), 121.05 (Cq 1’), 121.96 (CH 6’), 122.71 (CH 7), 125.95 (Cq 5a), 126.52
(CH 8), 127.13 (CH 9), 131.34 (Cq 1), 132.13 (CH 2), 133.02 (Cq 3a), 137.65 (Cq 9a), 146.04 (Cq 3’),
147.10 (Cq 4’), 148.13 (Cq 4). MS (ESI +, QTof, m/z): 336.15 [M+H]+. HRMS calculated for
C18H18N5O2 336.1455, found 336.1460.
EAPB02309: 4-(4-((3-aminopropyl)amino)imidazo[1,2-a]quinoxalin-1-yl)benzene-1,2-diol (9f). Yield:
1
80%. C19H19N5O2. Mw: 349.39 g/mol. Mp: 258°C. H-NMR δ (ppm, 400 MHz, DMSO d6) 2.00 (t, 2H,
CH2CH2NH2, J=8Hz), 2.90 (m, 2H, CH2NH), 3.64 (m, 2H, CH2NH2), 6.80 (d, 1H, CH 6’, J=8Hz), 6.94 (s, 1H,
CH 2’), 6.96 (d, 1H, CH 5’, J=8Hz), 7.01 (t, 1H, CH 7, J=8Hz), 7.30 (m, 2H, CH 6, CH 8), 7.46 (s, 1H, CH 2),
7.59 (d, 1H, CH 9, J=8Hz), 7.90 (m, 2H, NH2). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 27.54
(CH2CH2NH2), 37.17 and 37.26 (CH2NH2, CH2NH), 115.86 (CH 6), 116.48 (CH 5’), 117.88 (CH 2’), 121.14
(Cq 1’), 122.01 (CH 6’), 122.80 (CH 7), 124.80 (Cq 5a), 125.82 (CH 8), 126.46 (CH 9), 133.31 (Cq 1),
132.08 (CH 2), 133.01 (Cq 3a), 137.84 (Cq 9a), 146.00 (Cq 3’), 147.06 (Cq 4’), 148.08 (Cq 4). MS (ESI +,
QTof, m/z): 350.16 [M+H]+. HRMS calculated for C19H20N5O2 350.1612, found 350.1615.
EAPB02307: 4-(4-((6-aminohexyl)amino)imidazo[1,2-a]quinoxalin-1-yl)benzene-1,2-diol (9g). Yield:
1
81%. C22H25N5O2. Mw: 391.47 g/mol. Mp: 251°C. H-NMR δ (ppm, 400 MHz, DMSO d6) 1.39 (m, 4H,
CH2CH2CH2NH, CH2CH2CH2NH2), 1.54 (m, 2H, CH2CH2NH2), 1.69 (m, 2H, CH2CH2NH), 2.76 (m, 2H,
CH2NH2), 3.57 (m, 2H, CH2NH), 6.79 (d, 1H, CH 6’, J=8Hz), 6.88 (s, 1H, CH 2’), 6.93 (d, 1H, CH 5’,
J=8Hz), 6.97 (t, 1H, CH 7), 7.29 (m, 2H, CH 6, CH8), 7.39 (s, 1H, CH 2), 7.54 (d, 1H, CH 9, J=8Hz), 7.64
(t, 1H, NH, J=4Hz). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 26.08 and 26.53 (CH2CH2CH2NH,
CH2CH2CH2NH2), 27.47 (CH2CH2NH2), 29.16 (CH2CH2NH), 40.00 (CH2NH2, CH2NH), 115.85 (CH 6),
116.46 (CH 5’), 117.89 (CH 2’), 121.26 (Cq 1’), 122.09 (CH 7, CH 6’), 125.75 (Cq 5a), 126.41 (CH 8),
126.99 (CH 9), 131.22 (Cq 1), 131.96 (CH 2), 133.14 (Cq 3a), 138.24 (Cq 9a), 146.02 (Cq 3’), 147.05 (Cq
4’), 147.97 (Cq 4). MS (ESI +, QTof, m/z): 392.21 [M+H]+. HRMS calculated for C22H26N5O2 392.2081,
found 392.2085.
EAPB02308: 4-(4-(piperazin-1-yl)imidazo[1,2-a]quinoxalin-1-yl)benzene-1,2-diol (9h). Yield: 54%.
1
C20H19N5O2. Mw: 361.40 g/mol. Mp: 257°C. H-NMR δ (ppm, 400 MHz, DMSO d6) 3.09 (m, 4H, 2 x
CH2NH), 4.38 (m, 2H, 2 x CH2NC=N), 6.79 (d, 1H, CH 6’, J=8Hz), 6.88 (s, 1H, CH 2’), 6.92 (d, 1H, CH 5’,
J=8Hz), 7.04 (t, 1H, CH 7, J=8Hz), 7.34 (m, 3H, CH 6, CH 8, NH), 7.48 (s, 1H, CH 2), 7.60 (d, 1H, CH 9,
J=8Hz), 7.95 (s, 1H, NH). 13C-NMR not available due to weak product amount. MS (ESI +, QTof, m/z):
362.16 [M+H]+. HRMS calculated for C20H20N5O2 362.1612, found 362.1616.
EAPB02304: 4-(4-(dimethylamino)imidazo[1,2-a]quinoxalin-1-yl)benzene-1,2-diol (9i). Yield: 61%.
1
C18H16N4O2. Mw: 320.34 g/mol. Mp: 247°C. H-NMR δ (ppm, 400 MHz, DMSO d6) 3.04 (2s, 6H, 2 x
CH3), 6.81 (dd, 1H, CH 6’, J=8Hz, J=1.5Hz), 6.87 (d, 1H, CH 2’, J=1.5Hz), 6.90 (d, 1H, CH 5’, J=8Hz), 6.98
(td, 1H, CH 7, J=8Hz, J=1.5Hz), 7.27 (td, 1H, CH 8, J=8Hz, J=1.5Hz), 7.30 (d, 1H, CH 6, J=8Hz), 7.34 (s,
1H, CH 2), 7.60 (d, 1H, CH 9, J=8Hz). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 115.38 (CH 6), 115.96 (CH
5’), 117.39 (CH 2’), 120.79 (Cq 1’), 121.52 (CH 6’, CH 7), 125.30 (Cq 5a), 125.89 (CH 8), 126.50 (CH 9),
130.64 (Cq 1), 131.49 (CH 2), 132.79 (Cq 3a), 137.80 (Cq 9a), 145.55 (Cq 3’), 146.59 (Cq 4), 148.07 (Cq
4’). MS (ESI +, QTof, m/z): 321.03 [M+H]+. HRMS calculated for C18H17N4O2 321.1346, found 321.1352.
EAPB02300: 4-(4-hydroxyimidazo[1,2-a]quinoxalin-1-yl)benzene-1,2-diol (9j). Yield: 43%.C16H11N3O3.
Mw: 293.28 g/mol. Mp: 261°C. 1H-NMR δ (ppm, 400 MHz, DMSO d6) 3.37 (m, 3H, OH), 6.79 (d, 1H, CH
6’, J=8Hz), 6.87 (s, 1H, CH 2), 6.92 (d, 1H, CH 5), 7.00 (t, 1H, CH 8, J=8Hz), 7.23 (d, 1H, CH 9, J=8Hz),
7.31 (t, 1H, CH 7, J=8Hz), 7.37 (d, 1H, CH 6, J=8Hz), 7.40 (s, 1H, CH 2). 13C-NMR δ (ppm, 100 MHz,
25

DMSO d6) 116.53 (CH 5’), 116.73 (CH 9), 117.37 (CH 6), 117.64 (CH 2’), 120.74 (Cq 1’), 122.45 (CH 6’),
123.01 (CH 8), 124.30 (Cq 9a), 127.00 (CH 7), 129.63 (Cq 5), 132.35 (Cq 1), 133.22 (CH 2), 137.39 (Cq
3a), 146.12 (Cq 4’), 147.24 (Cq 3a), 153.21 (Cq 4). MS (ESI +, QTof, m/z): 294.1 [M+H]+. HRMS
calculated for C16H12N3O3 294.0873, found 294.0877.
EAPB 02302: 4-(4-aminoimidazo[1,2-a]quinoxalin-1-yl)benzene-1,2-diol (9k). Yield: 74%.
1
C16H12N4O2.Mw: 292.29 g/mol. Mp: 264°C. H-NMR δ (ppm, 400 MHz, DMSO d6) 6.80 (dd, 1H, CH 6’,
J=4Hz, J=8Hz), 6.88 (d, 1H, CH 2’, J=4Hz), 6.90 (d, 1H, CH 5’, J=8Hz), 6.99 (td, 1H, CH 7, J=8Hz, J=4Hz),
7.13 (s, 2H, NH2), 7.32 (m, 2H, CH 6, CH 8), 7.42 (s, 1H, CH 2), 7.51 (dd, 1H, CH 9, J=4Hz, J=8Hz). 13C-
NMR δ (ppm, 100 MHz, DMSO d6) 115.88 (CH 6), 116.46 (CH 5’), 117.87 (CH 2’), 121.24 (Cq 1’),
122.02 (CH 6’), 122.28 (CH 7), 126.08 (CH 9), 126.41 (CH 8, Cq 5’), 131.35 (Cq 1), 132.32 (CH 2),
132.92 (Cq 3’ quinoxaline), 137.95 (Cq 9’), 146.02 (Cq 3’ phenyl), 147.06 (Cq 4’ phenyl), 149.48 (Cq 4).
MS (ESI +, QTof, m/z) : 293.10 [M+H]+. HRMS calculated for C16H13N4O2 293.1039, found 293.1043.
4.1.13. General procedure for the preparation of ammonium chloride salts.
Compound 9 was solubilized in HCl/dioxane (4N) and stirred for 2 hours at room temperature. The
reaction mixture was concentrated under reduced pressure, then dissolved in dichloromethane and
concentrated again. The precipitate was crushed with diethyl ether and filtered to afford desired salt
10.
EAPB
02306s:
2-((1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)ethanaminium
1
chloride (10e). C18H18ClN5O2. Mw: 371.82 g/mol. Mp: 221°C. H-NMR δ (ppm, 400 MHz, DMSO
d6) 3.18 (m, 2H, CH2NH3), 4.05 (m, 2H, CH2NH), 6.80 (d, 1H, CH 6’, J=8Hz), 6.94 (s, 1H, CH 2’), 6.99 (d,
1H, CH 5’, J=8Hz), 7.14 (t, 1H, CH 7, J=8Hz), 7.33 (m, 1H, CH 6), 7.39 (m, 1H, CH 8), 7.52 (s, 1H, CH 2),
7.60 (d, 1H, CH 9, J=8Hz), 8.05 (m, 1H, CH 9), 8.27 (m, 3H, NH3). 13C-NMR δ (ppm, 100 MHz, DMSO d6)
40.00 (CH2NH3, CH2NH), 116.22 (CH 6), 116.69 (CH 5’), 117.73 (CH 2’), 120.06 (Cq 1’), 122.03 (CH 6’),
124.34 (CH 7), 125.75 (Cq 5a), 127.19 (CH 8, CH 9), 132.23 (CH 2), 132.93 to 133.22 (Cq 1, Cq 3a, Cq
9a), 146.18 (Cq 3’), 146.68 (Cq 4’), 147.46 (Cq 4). MS (ESI +, QTof, m/z): 336.15 [M+H]+.
EAPB02307s:
6-((1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)hexan-1-aminium
1
chloride (10g). C22H26ClN5O2. Mw: 427.93 g/mol. Mp: 214°C. H-NMR δ (ppm, 400 MHz, DMSO
d6) 1.39 (m, 4H, CH2CH2CH2NH, CH2CH2CH2NH2), 1.59 (m, 2H, CH2CH2NH2), 1.73 (m, 2H, CH2CH2NH),
2.77 (m, 2H, CH2NH2), 3.85 (m, 2H, CH2NH), 6.81 (d, 1H, CH 6’, J=8Hz), 6.95 (s, 1H, CH 2’), 6.99 (d, 1H,
CH 5’, J=8Hz), 7.22 (t, 1H, CH 7, J=8Hz), 7.34 (d, 1H, CH 6, J=8Hz), 7.45 (t, 1H, CH 8 , J=8Hz), 7.68 (s,
1H, CH 2), 7.97 (m, 3H, NH3), 8.35 (m, 1H, CH 9). 13C-NMR δ (ppm, 100 MHz, DMSO d6) 25.95 and
26.03 (CH2CH2CH2NH, CH2CH2CH2NH2), 27.23 (CH2CH2NH2), 28.44 (CH2CH2NH), 40.00 (CH2NH2,
CH2NH), 116.57 (CH 6), 116.71 (CH 5’), 117.61 (CH 2’), 119.60 (CH 9), 120.16 (Cq 1’), 121.82 (CH 6’),
124.19 (CH 7), 125.21 (Cq 5a), 127.52 (CH 8), 131.73 (Cq 1), 134.10 to 134.20 (CH 2, Cq 3a, Cq 9a),
144.88 (Cq 3’), 146.27 (Cq 4’), 147.69 (Cq 4). MS (ESI +, QTof, m/z): 392.21 [M+H]+.
4.2. Cell lines and culture techniques
4.2.1. General
26

Melanoma (A375) human cancer cell lines were obtained from American Type Culture Collection
(Rockville, Md., USA). Cells were cultured in RPMI medium containing RPMI-1640 (Gibco
Laboratories, France), 10% heat-inactived (56°C) foetal bovine serum (FBS) (Polylabo, Paris, France), 2
mM L-glutamine, 100 IU/mL penicillin G sodium, 100 mg/mL streptomycin sulfate, and 0.25 mg/mL
amphotericin B. Cells were maintained in a humidified atmosphere of 5% CO2 in air at 37°C.
4.2.2. In vitro cytotoxicity assay
Previously to the experiments, the number of cells by well, the doubling time and the MTT
concentration have been optimized. In all the experiments, A375 cells were seeded at a final
concentration of 5000 cells/well in 96-well microtiter plates and allowed to attach overnight. After
24 h incubation, the medium (phosphate-buffer saline pH 7.3) was aspirated carefully from the plates
using a sterile Pasteur pipette, and cells were exposed (i) to vehicle controls (0.15% DMSO/culture
medium (v/v) and culture medium alone), (ii) to the synthesized compounds at concentrations of 10-
5–3.2.10-9 M dissolved in a mixture 0.15% DMSO/culture medium (v/v). After 96 h of incubation, cell
supernatant was removed and 100 μL of a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide) solution in fresh medium was added per well (MTT final concentration of 0.5 mg/ml) and
incubated for 4 h at 37°C. This colorimetric assay is based on the ability of live and metabolically
unimpaired tumor-cell targets to reduce MTT to a blue formazan product. At the end of the
incubation period, the supernatant was carefully aspirated, then, 100 µL of a mixture of SDS 10% and
0.01 M hydrochloric acid was added to each well. After 2 hours at 37°C of incubation and vigorous
shaking to solubilize formazan crystals, the optical density was measured at 570 nm in a microculture
plate reader (Dynatech MR 5000, France). For each assay, at least three experiments were performed
in triplicate. The individual cell line growth curves confirmed that all A375 lines in control medium
remained in the log phase of cell growth 96 h after plating. Cell survival was expressed as percent of
vehicle control. The IC50 values defined as the concentrations of drugs which produced 50% cell
growth inhibition; 50% reduction of absorbance, were estimated from the sigmoidal dose–response
curves.
4.3. In vitro tubulin polymerization analysis
Tubulin was prepared from pig brain according to the purification procedure described by Williams
and Lee.52,53 To evaluate the effect of the compounds on tubulin assembly in vitro, tubulin
polymerization was monitored turbidimetrically at 350 nm with a MC2 spectrophotometer (Safas,
Monaco) equipped with a thermal-jacketed cuvette holder. The reaction mixture was prepared at
0°C, and contained PEM (Pipes 0.1 M, EGTA 2 mM, MgSO4 1 mM pH 6.9) buffer, 25% glycerol (v/v), 1
mM GTP, MgSO4 5 mM, and 12 µM tubulin. GTP and tubulin were added at the very last minute.
EAPB0503, EAPB02303 and colchicine stock solutions were diluted in DMSO to the desired
concentration, and 2 µL of the compound solution was added to the medium (final concentration: 5
µM). The same volume of DMSO alone was used for negative control. The final volume of the sample
was 200 µL. The reaction was started by placing the cuvette in the spectrophotometer cell
compartment thermostated at 37°C. Ice was added 45 min later to initiate depolymerization to check
for signal specificity.
4.4. Molecular docking assay in the colchicine site
The molecular modeling studies were performed with GOLD.54 The crystal structure of tubulin
complexed with colchicine (PDB 4O2B)55 was retrieved from the RCSB Protein Data Bank. The binding
27

sphere with a radius of 25.0 Å was defined with residue Leu255 as the binding site. All compounds
were drawn with PRODRG and protonated using BABEL.56 Finally, they were docked into the binding
site using the GOLD protocol with the default settings. The pictures were generated by the program
PYMOL.57
The docking pose of EAPB0503 was analyzed with two servers (https://projects.biotec.tu-
dresden.de/plip-web/plip/index (refX) and https://dockthor.lncc.br/v2/ (refY)) in order to determine
a quantitative analysis.
Ref x: PLIP: fully automated protein–ligand interaction profiler
Sebastian Salentin, Sven Schreiber, V. Joachim Haupt, Melissa F. Adasme, Michael Schroeder
Nucleic Acids Res. 2015 Jul 1; 43(Web Server issue): W443–W447. Published online 2015 Apr 14.
doi: 10.1093/nar/gkv315
Ref y: K. B. dos Santos, I. A. Guedes, A. L. M. Karl, and L. Dardenne. Highly Flexible Ligand Docking:
Benchmarking of the DockThor Program on the LEADS-PEP Protein-peptide Dataset, J. Chem. Inf.
Model., Jan. 2020, doi: 10.1021/acs.jcim.9b00905.
4.5. In vitro transcriptomic study
4.5.1. Experimental design and drug treatments
To explore the mechanism of action of EAPB02303, a transcriptomic study was performed. At first,
the question of the originality of the mechanism of action was addressed. For this, the A375 cell line
was treated without (negative control, drug vehicle: DMSO) or with different Imiqualines derivatives,
chemotherapies and targeted therapies used in human clinics at the single concentration of 10 µM
for 6 hours in DMEM. Fifteen molecules were used: vemurafenib, dacarbazine, SN38, doxorubicin,
fotemustine, 5-FU, methotrexate, vinorelbine, paclitaxel, colchicine, maytansine, Imiquimod,
EAPB02303, EAPB0203, EAPB0503. The goal was to compare the mechanisms of action of these
different treatments through there associated expression profiles. Then, the expression alterations
associated specifically with the EAPB02303 treatment were further analyzed for mechanistic
hypotheses.
4.5.2. RNA isolation and Affymetrix GeneChip processing
Total RNA was extracted from each cell line using RNeasy Mini Kit (Qiagen) according to the
manufacturer’s protocol. Then, the quality of purified RNA sample was checked with an Agilent
BioAnalyzer (Agilent Technologies). Total RNA from each cell line was therefore prepared for
hybridization with Affymetrix HG-U133+PM GeneChip according to the manufacturer’s protocol in
the platform “TRANSCRIPTOME” of Montpellier (IRMB).
4.5.3. Microarray data processing
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