Imidazo[1,2-a]quinoxalines derivatives grafted with amino acids: Synthesis and evaluation on A375 melanoma cells

Article abstract of DOI:10.3390/molecules23112987

Imiqualines (imidazoquinoxaline derivatives) are anticancer compounds with high cytotoxic activities on melanoma cell lines. The first generation of imiqualines, with two lead compounds (EAPB0203 and EAPB0503), shows remarkable in vitro (IC₅₀ = 1 570 nM and IC₅₀ = 200 nM, respectively, on the A375 melanoma cell line) and in vivo activity on melanoma xenografts. The second generation derivatives, EAPB02302 and EAPB02303, are more active, with IC₅₀ = 60 nM and IC₅₀ = 10 nM, respectively, on A375 melanoma cell line. The aim of this study was to optimize the bioavailability of imiqualine derivatives, without losing their intrinsic activity. For that, we achieved chemical modulation on the second generation of imiqualines by conjugating amino acids on position 4. A new series of twenty-five compounds was efficiently synthesized by using microwave assistance and tested for its activity on the A375 cell line. In the new series, compounds 11a, 9d and 11b show cytotoxic activities less than second generation compounds, but similar to that of the first generation ones (IC₅₀ = 403 nM, IC₅₀ = 128 nM and IC₅₀ = 584 nM, respectively). The presence of an amino acid leads to significant enhancement of the water solubility for improved drugability.

Full text of DOI:10.3390/molecules23112987

molecules
Article
Imidazo[1,2-a]quinoxalines Derivatives Grafted with
Amino Acids: Synthesis and Evaluation on A375
Melanoma Cells
Adrien Chouchou 1 , Cindy Patinote 1,2,†, Pierre Cuq , Pierre-Antoine Bonnet *
,†
1
1,
and Carine Deleuze-Masquéfa 1
1
IBMM, Université de Montpellier, CNRS, ENSCM, 34000 Montpellier, France;
adrien.chouchou@umontpellier.fr (A.C.); cindy.patinote@umontpellier.fr (C.P.);
pierre.cuq@umontpellier.fr (P.C.); carine.masquefa@umontpellier.fr (C.D.-M.)
Société d’Accélération du Transfert de Technologies (SATT AxLR), CSU, 950 rue Saint Priest,
2
3
4090 Montpellier, France
*
†
Correspondence: pierre-antoine.bonnet@umontpellier.fr; Tel.: +33-4-11-75-95-41
These authors contributed equally to this work.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 30 October 2018; Accepted: 13 November 2018; Published: 15 November 2018
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Imiqualines (imidazoquinoxaline derivatives) are anticancer compounds with high
cytotoxic activities on melanoma cell lines. The first generation of imiqualines, with two
lead compounds (EAPB0203 and EAPB0503), shows remarkable in vitro (IC50 = 1 570 nM and
IC50 = 200 nM, respectively, on the A375 melanoma cell line) and in vivo activity on melanoma
xenografts. The second generation derivatives, EAPB02302 and EAPB02303, are more active, with
IC50 = 60 nM and IC50 = 10 nM, respectively, on A375 melanoma cell line. The aim of this study was to
optimize the bioavailability of imiqualine derivatives, without losing their intrinsic activity. For that,
we achieved chemical modulation on the second generation of imiqualines by conjugating amino
acids on position 4. A new series of twenty-five compounds was efficiently synthesized by using
microwave assistance and tested for its activity on the A375 cell line. In the new series, compounds
1
1a, 9d and 11b show cytotoxic activities less than second generation compounds, but similar to that of
the first generation ones (IC50 = 403 nM, IC50 = 128 nM and IC50 = 584 nM, respectively). The presence
of an amino acid leads to significant enhancement of the water solubility for improved drugability.
Keywords: imidazo[1,2-a]quinoxaline; melanoma; imiqualine; A375 structure–activity relationship
1
. Introduction
Cutaneous melanoma is a malignant tumour of melanocytes located in the basal epidermis. It is
the most aggressive and lethal form of skin cancer because of its fast-metastatic development [1,2]. Its
incidence has been increasing worldwide for several decades. While in its early stages remarkable
outcomes can be achieved with surgery alone, metastatic melanoma requires therapeutic treatment
intervention. Thanks to genomic studies, knowledge regarding the molecular biology of melanomas
has improved, leading to recent FDA-approved therapies [
disease are not equally treated [ ] and marked resistance mostly against kinase inhibitors rapidly
occurs [ ]. As the incidence among the worldwide fair-skinned population is increasing, this public
3–5]. However, all the subtypes of the
5
6,7
health concern remains challenging.
Our group is working on the development of the imidazo[1,2-a]quinoxaline derivatives presented
in Scheme 1, called imiqualines, as potential antitumoral agents, particularly for the treatment of
melanoma [8,9]. 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. The second
Molecules 2018, 23, 2987; doi:10.3390/molecules23112987
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2987
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generation is characterized by the presence of the 3,4-dihydroxyphenyl moiety on position 1 since the
presence of such catechol residue enhances global hydrophilicity. The chemical modulations of the
first hits, EAPB0203 and EAPB0503, afforded new leads EAPB02303 and its N-demethylated derivative
EAPB02302, with impressive in vitro activities in the nanomolar range on the A375 human melanoma
cancer cell line [10,11].
Scheme 1. General and lead compounds structures of the imiqualines.
EAPB02302 and EAPB02303 are considered as promising anticancer agents but exhibit high
lipophilicity (cLogP values estimated at 2.68 and 3.55, respectively), which might be critical for future
preclinical in vivo studies. Indeed, their low solubility in water might be a major drawback for further
development, especially in the case of intravenous use. In a preliminary study [12], the pharmacokinetic
parameters of EAPB02303 were determined in mice after a single intraperitoneal administration. For
this, the compound was solubilized in a mixture of DMSO, Tween 80 and sodium chloride solution 0.9%
(
10/10/80, v/v/v). The use of DMSO is recognized as toxic [13], in particular when used repeatedly as
would be the case in an efficacy study. In order to optimize the results of efficacy studies, we chose
to chemically modulate our lead compounds to obtain more soluble compounds with a moderate
impact on the cytotoxic activity. The result of the introduction of various amino acids on position 4 of
the heterocycle on both the physicochemical properties and biological activity was studied. Such an
approach to increase solubility, which remains a key factor for potential pharmaceutical development,
has already been described in the literature [14
showed to increase the water solubility as well as selective cytotoxicity [18
synthesis of new imidazo[1,2-a]quinoxalines decorated with a panel of natural
–
17]. The introduction of amino acid moieties has been
20]. We present herein the
-amino acids and their
–
α
in vitro preliminary evaluation on A375 cell line.
2
. Results and Discussion
2
.1. Synthesis of Imidazo[1,2-a]quinoxaline Derivatives
The synthetic pathways and the structures of imidazo[1,2-a]quinoxalines used in this study are
given in Schemes 2 and 3. Intermediates to were synthetized thanks to a route we previously
described [ 21]. Briefly, the carbonylimidazole dimer results from the condensation of the
1
5
9,
2

Molecules 2018, 23, 2987
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2
2
-imidazole carboxylic acid
gives the intermediate
1
in presence of thionyl chloride. Addition of O-fluoroaniline to the dimer
3
. Cyclisation is facilitated by using sodium hydride in dimethylacetamide.
Treatment of compound
4
with phosphorus oxychloride and N,N-diethylaniline gives the chlorinated
key intermediate 5.
Scheme 2. Synthesis of imidazo[1,2-a]quinoxaline derivatives grafted with the
α
-amine group of
◦
the amino acid. Reagents and Conditions: (
a
) SOCl , reflux, 18h; (
b
e
) NaHMDS, THF, 0 C to RT, 5 h;
) H-amino acid (PG)-OtBu, DIEA,
2
◦
(
c
) NaH, DMA, reflux, 48 h; (
d
) DEA, POCl , MW (130 C, 15 min); (
3
◦
DMF, MW (150 C, 30–60 min); (
f
) NBS, CHCl , reflux, 2 h; (
g) 3,4-dimethoxyphenylboronic acid,
3
◦
Pd(PPh ) , Na CO , DME, MW (140 C, 20 min); (
h
) BBr , CH Cl , RT, 1h–3h, (
i
◦
) BBr , CH Cl , RT,
3
4
2
3
3
2
2
3
2
2
1
h; (
j
) 3,4-dihydroxyphenylboronic acid 12, Pd(PPh ) , Na CO , DME, MW (140 C, 20 min); (
k) TFA/
3
4
2
3
CH Cl (1/1, v/v), RT, 1–2 h.
2
2
The chlorine of
5 can be substituted by an amino group in the presence of diisopropylethylamine
in DMF under microwave assistance. Among the commercially available amino acids, we chose to
study the effect of a short or long, substituted or not, aliphatic or aryl side chain with or without a
hydrophilic amino or phenolic group. The amino group could be either the α-amine of an amino acid
or the amine of the side chain for the ornithine residue. The introduction of HGlyOtBu, HAlaOtBu,
HValOtBu, HLeuOtBu, HLysOtBu, HOrnOtbu, HPheOtBu, HTyrOtBu is described in Scheme 2 and
the grafting of BocOrnOtBu is depicted in Scheme 3. The bromination of the intermediates
6 by

Molecules 2018, 23, 2987
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N-bromosuccinimide leads to compounds
7. The 3,4-dimethoxyphenyl group is introduced in position 1
via a Suzuki-Miyaura cross-coupling reaction to furnish compounds 8a
did not allow the cleavage of all the protections to give the targeted compounds 11a
supplementary equivalents of BBr were added at the beginning or during the reaction, or if the
–8f and 8i. Boron tribromide
–
11i, even if
3
reaction time was extended. Such an approach allows one to obtain compounds 10b–10d as the main
products. These compounds present remaining methoxy groups on the phenyl on position 1 without
the protecting groups on the amino acid. The by-products correspond to one remaining methoxy group
0
0
on the phenyl either at position 3 or 4 , but unfortunately, they could not be recovered separately.
Scheme 3. Synthesis of the ornithine-containing imidazo[1,2-a]quinoxaline derivative grafted by the
◦
side chain. Reagents and Conditions: (
e
) Boc-Orn-OtBu, HCl, DIEA, DMF, MW (150 C, 30–60 min);
(
(
f
) NBS, CHCl , reflux, 2 h; (
g
) 3,4-dimethoxyphenylboronic acid, Pd(PPh ) , Na CO , DME, MW
3
3 4
2
3
◦
140 C, 20 min); (
h
) BBr , CH Cl , RT, 1 h–3 h; (
i
) BBr , CH Cl , RT, 1 h; (
j) 3,4-dihydroxyphenylboronic
3
2
2
3
2
2
◦
acid 12, Pd(PPh ) , Na CO , DME, MW (140 C, 20 min); (k) TFA/ CH Cl (1/1, v/v), RT, 1–2 h.
3
4
2
3
2
2
As target compounds 11a
first the 3,4-dimethoxyphenylboronic acid using boron tribromide in order to obtain the
,4-dihydroxy-phenylboronic acid 12. This boronic acid readily reacts with intermediates under
microwave irradiation to furnish the hydroxylated derivatives 9a 9i. A final step of deprotection of
the amino acid moiety using TFA in CH Cl was used to obtain the final compounds 11a–11i.
–11f could not be obtained by this way, we decided to deprotect
3
7
–
2
2

Molecules 2018, 23, 2987
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2
.2. In Vitro Cytotoxic Activity on A375 Cell Line and Calculated ClogP
All new imidazo[1,2-a]quinoxaline derivatives 8b 8f 9a 9i
–d
,
,
–
,
10b
–d
and 11a
–
11i were tested
for their in vitro antiproliferative activities on the human melanoma cell line A375. Their IC50 values
concentration of the compound (nM) producing 50% cell growth inhibition after 96 h of drug exposure)
were determined using in vitro cytotoxicity assays and are displayed in Tables 1 and 2.
(
Table 1. Synthesis of imidazo[1,2-a]quinoxaline derivatives grafted with the α-amine of the amino acid
as described in Scheme 2: cLogP, theoretical water solubility (mg/mL) at pH 7.4 calculated values and
IC50 values against A375 (human melanoma cell line).
Theoretical
Water
IC50
Amino
Acids
–
R
–R’
Compounds ClogP a
Solubility
Values
(nM)
c
(
mg/mL) at
pH 7.4
b
−
−
4
3
ND d
1932
403
>10,000
6103
5947
584
>10,000
7180
8
a
5.25
4.99
2.59
5.61
5.34
3.21
2.94
6.47
6.21
4.07
3.81
5.88 × 10
1.79 × 10
47.18
3.80 × 10
1.15 × 10
6.01
Gly
Ala
9a
1a
1
−
−
4
3
8
b
9
b
1
1
0b
1b
19.92
−
−
4
4
8
9
c
c
1.35 × 10
4.07 × 10
1.91
Val
Leu
Lys
1
1
0c
1c
>10,000
7166
>10,000
6.5
−
−
5
4
8
d
6.98
7.43 × 10
9
d
6.72
4.58
4.32
2.23 × 10
128
>10,000
838
1
1
0d
1d
0.89
3.08
d
ND
5.70 × 10−
5
8
e
7.05
−
−
4
3
9
e
6.79
2.78
1.56 × 10
4575
673
1
1e
4.34 × 10
>
10,000
−
5
8
f
7.01
3.43 × 10
Orn
Phe
−
−
5
3
9
f
6.75
2.57
9.07 × 10
1111
3404
1
1f
5.82 × 10
1
999
−
4
9
g
6.82
3.71
1.06 × 10
1
1g
6.47
3.31 × 10−
10.17
951
4
117
5
9
h
7.99
3.05
Tyr
1
1h
2174
a,b
cLogP and theoretical water solubility (mg/mL) at pH 7.4 values are calculated using the ACDLabs®software;
c
IC50 values, concentration of the compound (nM) producing 50% cell growth inhibition after 96 h of drug exposure,
as determined by the MTT assay. Each experiment was performed in triplicate, and the results are presented as
average values. Coefficients of variation were less than 10%; d ND: Not determined.

Molecules 2018, 23, 2987
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Table 2. Synthesis of imidazo[1,2-a]quinoxaline derivatives grafted with the side chain amine of
ornithine as described in Scheme 3: ClogP, theoretical water solubility (mg/mL) at pH 7.4 calculated
values and IC50 values against A375 (human melanoma cell line).
Theoretical Water Solubility
Amino acid Compounds
ClogP a
IC50 Values c (nM)
(
mg/mL) at pH 7.4 b
−
−
−
4
4
3
8
i
6.12
5.86
2.99
1.62 × 10
4.50 × 10
3.05 × 10
>10,000
>10,000
5168
Orn
9i
1i
1
a,b
c
ClogP and theoretical water solubility (mg/mL) at pH 7.4 values are calculated using the ACDLabs®software.
IC50 values, concentration of the compound (nM) producing 50% cell growth inhibition after 96 h of drug exposure,
as determined by the MTT assay. Each experiment was performed in triplicate, and the results are presented as
average values. Coefficients of variation were less than 10%.
Lipophilicity, expressed as the logarithm of a compound’s octanol/water partition coefficient
ClogP), is a physicochemical property of drugs that affects many biological mechanisms, especially
(
drug absorption and distribution (absorption, plasma protein binding and membrane permeation) [22].
This parameter can also be correlated to solubility, metabolism and toxicity [23 24]. In a drug discovery
,
process, compounds must be sufficiently lipophilic to cross the membrane barriers and at the same time
be sufficiently water soluble to reach their targets. Therefore, a poor water solubility is a common cause
of rejection during development [25]. This is why we also estimated the lipophilicity and hydrophilicity
properties of all new compounds by predicting their ClogP and theoretical water solubility values
®
thanks to fragmentation methods available on the ACDLabs software. These calculated values are
purely theoretical but give a good estimate of what might be the solubility of the compounds in
blood circulation (pH 7.4). This approach is used in pharmaceutical industry for the screening of new
compounds [26,27]. Results of our new imidazo[1,2-a]quinoxaline derivatives were compared each
other as well as with the first and second generation imiqualine leads as shown in Table 3.
Table 3. First and second generation imiqualines leads: ClogP, theoretical water solubility (mg/mL) at
pH 7.4 calculated values and IC50 values against A375 (human melanoma cell line).
Theoretical Water Solubility
Compounds
ClogP a
IC50 Values c (nM)
(
mg/mL) at pH 7.4 b
−
−
−
−
3
3
2
2
EAPB0203
EAPB0503
EAPB02302
EAPB02303
4.6
3.46 × 10
2.60 × 10
4.28 × 10
1.74 × 10
1570
200
60
4.48
2.68
3.55
10
a,b
c
ClogP and theoretical water solubility (mg/mL) at pH 7.4 values are calculated using the ACDLabs®software.
IC50 values, concentration of the compound (nM) producing 50% cell growth inhibition after 96 h of drug exposure,
as determined by the MTT assay. Each experiment was performed in triplicate, and the results are presented as
average values. Coefficients of variation were less than 10%.
Compounds 8b, 8c, 8d and 8f have fully protected amino acid residues (side chain and carboxylic
function) with 3,4-dimethoxy substitution on the phenyl ring in position 1. They exhibit weak IC50
values higher than 10,000 nM. Compounds belonging to this chemical series are very lipophilic with
estimated ClogP values higher than 5 (5.25, 5.61, 6.47, 6.98, 7.05 and 7.01 for compounds 8a
8e and 8f, respectively). The theoretical water solubility of compounds at pH 7.4 is very low
10 mg/mL for compounds 8a and 8f, respectively.
, 8b, 8c,
8
d
,
8
−
4
−5
since the order varies from 5.88
Therefore, the presence of protective groups on the amino acids and the dimethoxy groups on the
phenyl appears not to be valuable, both in terms of water solubility and cytotoxic efficiency.
×
10 to 3.43
×
Compounds 9a–9i are only protected on the amino acids moieties. These compounds exhibit
various cytotoxic activities with IC50 values ranging from 128 to 7180 nM, for 9d and 9c, respectively.
These compounds show high lipophilicity since their ClogP values range from 4.99 to 7.99 for 9a
−
5
and 9h respectively. Similarly, the theoretical values of water solubility are between 3.31
×
10 and

Molecules 2018, 23, 2987
7 of 26
−
3
1
.79
×
10 mg/mL for 9h and 9a, respectively. By comparing compounds
8
and
9
, we note that the
replacement of the methoxy groups by the hydroxy groups induces, for some residues, an important
increase of the biological activities. Nevertheless, such modification, with conservation of the amino
acid protection, does not improve the theoretical water solubility of the new synthesized compounds.
Compounds 10b
groups on the phenyl substitution at position 1. These compounds exhibit IC50 values higher than
000 nM (IC50 values at 5 947 nM for 10b and higher than 10,000 nM for 10c and 10d). However,
ClogP values are below 5 and theoretical water solubilities are higher than 0.8 mg/mL. The transition
from compounds to compounds 10 provides a significant decrease of lipophilicity as well as an
–10d are deprotected on the amino acid residues but still present dimethoxy
5
8
improvement of theoretical water solubility. The comparison of the compounds according to the
grafted residue permits to highlight this improvement. Actually, ClogP values decrease from 5.61
to 3.21, from 6.47 to 4.07 and from 6.98 to 4.58 for compounds 8b to 10b
, 8c to 10c and 8d to 10d,
−
4
respectively. Even more impressive, the theoretical water solubility values increase from 3.8
×
10 to
8c to 10c and 8d to
0d, respectively. The deprotection of the amino acid residues does not improve the cytotoxic activity
−
4
−5
10 to 0.89 mg/mL for compounds 8b to 10b,
6
1
.01, 1.35.10 to 1.91 and 7.43
×
but clearly improve the theoretical water solubility. The presence of dihydroxy groups on the phenyl
substitution at position 1 appears to be necessary for the conservation of the cytotoxic activity.
Compounds 11a
group at the position 1. Among these compounds, five have attractive IC50 values below 1000 nM: 403,
84, 673, 838 and 951 for 11a 11b 11e 11d and 11g, respectively. These compounds show ClogP values
ranged from 2.57 to 4.32 for 11f and 11d. Several compounds present very interesting theoretical water
solubility: compounds 11a 11b 11c 11d 11g and 11h exhibit values higher than 3 mg/mL. Very high
-11i are fully deprotected on the amino acid residues as well as on the catechol
5
,
,
,
,
,
,
,
values are obtained for compounds 11a and 11b at 47.18 and 19.92 mg/mL, respectively. The presence
of an alkyl or aryl moiety on the side chain of the amino acid residue does not decrease the water
solubility for these compounds. Nevertheless, the tendency appears to not be the same for compounds
−
2
1
1e
,
11f and 11i with theoretical water solubility values less than 1
×
10 mg/mL. These compounds
possess ornithine (grafted by the
α
-amine or by the amine of the side chain of the amino acid) or
lysine residues. These two residues are therefore not valuable for increasing the water solubility of the
compounds at pH 7.4.
Compounds 11a
, 11b, 11c and 11d present an alkyl chain at position 4 while compounds 11g and
1
1h display an aryl chain on this same position. Saturated alkyl groups appear to be most interesting
for our compounds. A trend that seems to stand out in these four compounds is that when the carbon
number of the alkyl chain increases, the biological activity and the theoretical solubility decrease
(
except for 11c which activity does not follow this tendency). Moreover, the presence of a phenyl group
causes an increase in lipophilicity and a decrease in biological activities, in particular with the presence
of a phenol (compound 11h) on the side chain of the amino acid moiety. The compounds with lysine
or ornithine residues did not show favorable results neither for biological activities or water solubility.
The transition of compounds 9 to compounds 11 is carried out by the cleavage of all the protecting
groups of the amino acid moieties. Such a deprotection step forms less lipophilic compounds (lower
ClogP) with similar or higher activity for all the amino acids tested, except for compounds 11d and 11f
with the leucine and ornithine residues grafted by the amine in the α-position, respectively. It should
be noted that the presence of an ornithine amino acid residue grafted by the α-amine or the amine of
the side chain of the amino acid does not result in any significant differences in the biological activity
(
IC50 values at 3404 nM for 11f and 5168 nM for 11i) or to the solubility (theoretical water solubility
−
3
−3
values at 5.82
1
×
10 and 3.05
×
10 mg/mL for 11f and 11i, respectively). Surprisingly, compound
1e which present a butan-1-amine substitution on the lateral chain of the amino acid show an higher
biological activity than compound 11f with a propan-1-amine substitution on its lateral chain (IC50
values at 673 nM for 11e and 3 404 nM for 11f) with equivalent theoretical water solubilities (values of
− −3
3
4
.34.10 and 5.82.10 mg/mL for 11e and 11f, respectively).

Molecules 2018, 23, 2987
8 of 26
Consequently, compounds 11a
,
11b
,
11d and 11g in particular hold our attention, both in terms of
improvement of the solubility and in terms of conservation of the biological activity. The activities
of these compounds are in the same potency order as the leads of the first generation of imiqualines
(IC50 values of 200 and 1570 nM for EAPB0503 and EAPB0203, respectively), presented in Table 3.
Moreover, these new compounds show highly improved water solubility. Indeed, the values for
−
3
−3
10 mg/mL for EAPB0203 and
the first generation compounds were only 2.60
×
10 and 3.46
×
EAPB0503, respectively. On the other hand, the second generation imiqualine compounds exhibit
higher activities than the new compounds, with IC50 values at 10 and 60 nM for EAPB02303 and
EAPB02302 respectively. However, the solubility values of these compounds are low, with values of
−
2
−2
1
.74 × 10 mg/mL for EAPB02303 and 4.28 × 10 mg/mL for EAPB02302.
All of these data and results allow us to put forward a preferential amino acid with a small alkyl
side chain in order to get a good compromise between maintaining the activity and increasing water
solubility. From the close analysis of Tables 1 and 2, it can be observed that compounds 11a 11b 9d
1d 11e and 11g are the most active members of this series against the tested melanoma A375 cell line
IC50 values less than 1000 nM). Surprisingly, compound 9d shows the lowest IC50 value among these
new synthesized compounds. Among these six attractive compounds, only 11a 11b 11d 11e and 11g
show a marked and considerable improvement of the theoretical water solubility.
,
,
,
1
,
(
,
,
,
3
3
3
. Experimental Section
.1. Chemistry
.1.1. General Information
All solvents and reagents were obtained from Sigma Aldrich Chemical Co. (Saint Louis, MO,
USA), Iris Biotech GmbH (Marktredwitz, Germany), Alfa Aesar Co. (Karlsruhe, Germany), VWR
Radnor, PA, USA) and FluoroChem UK (Hadfield, 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.). The progress of reaction was monitored by TLC exposure to UV light
254 nM and 366 nM). Thin layer chromatography plates (Kieselgel 60 F254) were purchased from
Merck (Darmstadt, Germany). Microwave assisted organic syntheses were performed on a Biotage
1
13
Initiator 2.0 microwave system (Uppsala, Sweden). H (400 MHz) and C-NMR (100 MHz) spectra
were obtained on a Brüker AC-400 spectrometer (Billerica, MA, USA). Chemical shifts are given as
parts per million (ppm) using residual dimethylsulfoxide signal for protons (
δ
= 2.46 ppm) and
DMSO
carbons (
δ
= 40.00 ppm). Coupling constants are reported in Hertz (Hz). Spectral splitting
DMSO
partners are designed as follow: singlet (s); doublet (d); triplet (t); quartet (q); multiplet (m). Mass
spectral data were obtained on a Waters Micromass Q-Tof (Milford, MA, USA) spectrometer equipped
with ESI source (Laboratoires de Mesures Physiques, Plateau technique de l’Institut des Biomolecules
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. The mass spectra
were recorded in positive mode, between 100 and 1500 Da. The capillary tension is 1000 V and the
◦
◦
cone tension is 30 V. The source and desolvation temperature are 120 C and 250 C, respectively. NMR
1
13
H and C spectra of all compounds are in Supplementary Materials.
3
.1.2. Amino Acids Grafted on 4-Chloroimidazo[1,2-a]quinoxaline
Tert-butyl 2-(imidazo[1,2-a]quinoxalin-4-ylamino)acetate (6a): Glycine tert-butyl ester hydrochloride (0.824 g,
.9 mmol), N,N-diisopropylethylamine (1.6 mL, 9.8 mmol) and 4-chloroimidazo[1,2-a]quinoxaline (0.5 g,
.5 mmol) were dissolved in dimethyl-formamide (10 mL) in a microwave adapted vial and sealed.
4
5
2
◦
The reaction mixture was irradiated at 150 C for 30 min. The solvent was removed under reduced

Molecules 2018, 23, 2987
9 of 26
pressure. The crude mixture was dissolved in ethyl acetate (100 mL) and successively washed with
saturated aqueous ammonium chloride, distilled water and finally brine. The organic phase was dried on
sodium sulfate, filtered and concentrated under reduced pressure. The product was purified by flash
chromatography eluted with cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as
1
a beige solid (42% yield). C H N O . M : 298.34 g/mol. H-NMR
δ
(ppm, DMSO-d ): 1.42 (s, 9H,
16
18
4
2
W
6
3
1
×
CH OtBu), 4.13 (d, 2H, J = 8 Hz, CH2
α
), 7.31–7.35 (m, 1H, CH 7), 7.40–7.44 (m, 1H, CH 8), 7.56 (dd,
H, J = 4 Hz, J = 8 Hz, CH 9), 7.66 (d, 1H, J = 4 Hz, CH 2), 7.96 (t, 1H, J = 8 Hz, NH), 8.13 (dd, 1H, J = 4 Hz,
J = 8 Hz, CH 6), 8.64 (d, 1H, J = 4 Hz, CH 1). 13C-NMR
(ppm, DMSO-d ): 28.23 (CH tBu), 43.30 (CH
), 80.85 (Cq tBu), 115.14 (CH 1), 115.91 (CH 6), 123.57 (CH 7), 124.93 (Cq 5a), 126.74 (CH 9), 126.88 (CH
3
δ
6
3
2
α
8
), 132.52 (CH 2), 132.71 (Cq 3a), 136.81 (Cq 9a), 147.49 (Cq 4), 169.79 (C=O). MS (ESI +, QTof, m/z): 299.0
+
[M + H] .
Tert-butyl 2-(imidazo[1,2-a]quinoxalin-4-ylamino)propanoate (6b): Same procedure used for the
synthesis of 6a was employed. L-Alanine tert-butyl ester hydrochloride (1.339 g, 7.4 mmol),
N,N-diisopropylethylamine (2.4 mL, 14.7 mmol) and 4-chloroimidazo[1,2-a]quinoxaline
5 (0.5 g,
2.5 mmol) were dissolved and reacted in dimethyl-formamide (10 mL). The product was purified
by flash chromatography eluted with cyclohexane/ethyl acetate 80/20 to 50/50. The compound is
1
obtained as a beige solid (48% yield). C H N O . M : 312.37 g/mol. H-NMR
δ
(ppm, DMSO-d6):
), 7.33–7.36 (m, 1H,
CH 7), 7.41–7.43 (m, 1H, CH 8), 7.56 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.66 (d, 1H, J = 4 Hz, CH 2), 7.75
17
20
4
2
W
1.41 (s, 9H, 3
×
CH OtBu), 1.51 (d, 3H, J = 8 Hz, CH3 β), 4.56–4.63 (m, 1H, CH α
3
(
(
(
(
d, 1H, J = 4 Hz, NH), 8.13 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 8.64 (d, 1H, J = 4 Hz, CH 1). 13C-NMR
ppm, DMSO-d ): 17.46 (CH3 ), 28.14 (CH tBu), 50.22 (CH ), 80.59 (Cq tBu), 115.18 (CH 1), 115.91
CH 6), 123.64 (CH 7), 124.93 (Cq 5a), 126.73 (CH 9), 126.88 (CH 8), 132.42 (CH 2), 132.58 (Cq 3a), 136.72
δ
β
α
6
3
+
Cq 9a), 147.03 (Cq 4), 172.84 (C=O). MS (ESI +, QTof, m/z): 313.2 [M + H] .
Tert-Butyl 2-(imidazo[1,2-a]quinoxalin-10-ylamino)-3-methylbutanoate (6c): Using the same procedure
as for the synthesis of 6a, L-valine tert-butyl ester hydrochloride (2.060 g, 9.8 mmol),
N,N-diisopropylethylamine (2.4 mL, 14.7 mmol) and 4-chloroimidazo[1,2-a]quinoxaline
5 (0.5 g,
2.5 mmol) were mixed in dimethylformamide (10 mL). The product was purified by flash
chromatography eluted with cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as
1
a beige solid (51% yield). C H N O . M : 340.42 g/mol. H-NMR
δ
(ppm, DMSO-d ): 1.03 (d, 3H,
19
24
4
2
W
6
J = 8 Hz, CH3
4
8
γ
), 1.06 (d, 3H, J = 8 Hz, CH3
.52 (t, 1H, J = 16 Hz, CH ), 7.15 (d, 1H, J = 8 Hz, NH), 7.33–7.37 (m, 1H, CH 7), 7.42–7.44 (m, 1H, CH
), 7.59 (dd, 1H J = 4 Hz, J = 8 Hz, CH 9), 7.68 (d, 1H, J = 4 Hz, CH 2), 8.14 (dd, 1H, J = 4 Hz, J = 8 Hz,
γ
’), 1.43 (s, 9H, 3
×
CH OtBu), 2.29–2.37 (m, 1H, CH β),
3
α
13
CH 6), 8.66 (d, 1H, J = 4 Hz, CH 1). C-NMR
tBu), 30.41 (CH ), 59.76 (CH ), 81.15 (Cq tBu), 115.37 (CH 1), 115.95 (CH 6), 123.88 (CH 7), 125.00 (Cq
a), 126.84 (CH 9), 126.95 (CH 8), 132.48 (CH 2), 132.58 (Cq 3a), 136.59 (Cq 9a), 147.29 (Cq 4), 171.59
δ (ppm, DMSO-d ): 19.42 (CH3 γ, CH3 γ’), 28.17 (CH3
6
β
α
5
+
(C=O). MS (ESI +, QTof, m/z): 341.0 [M + H] .
Tert-butyl 2-(imidazo[1,2-a]quinoxalin-4-ylamino)-4-methylpentanoate (6d): The same as for the synthesis
of 6a was used, employing L-leucine tert-butyl ester hydrochloride (2.198 g, 9.8 mmol),
N,N-diisopropylethylamine (2.4 mL, 14.7 mmol) and 4-chloro-imidazo[1,2-a]quinoxaline
5 (0.5 g,
2.5 mmol) in dimethylformamide (10 mL). The product was purified by flash chromatography eluted
with cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as a beige solid (60% yield).
1
C H N O . M : 354.45 g/mol. H-NMR
δ
(ppm, DMSO-d ): 0.93 (d, 3H, J = 8 Hz, CH3
δ
), 0.96 (d,
), 1.77–1.79 (m, 1H, CH ),
), 7.26–7.33 (m, 1H, CH 7), 7.40–7.44 (m, 1H, CH 8),
.57 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.64 (d, 1H, J = 4 Hz, NH), 7.66 (d, 1H, J = 4 Hz, CH 2), 8.13 (dd,
H, J = 4 Hz, J = 8 Hz, CH 6), 8.64 (d, 1H, J = 4 Hz, CH 1). 13C-NMR
(ppm, DMSO-d ): 22.06 (CH3 ),
3.31 (CH3 ’), 25.14 (CH ), 28.15 (CH tBu), 40.01 (CH2 ), 52.93 (CH ), 80.67 (Cq tBu), 115.21 (CH
20
26
4
2
W
6
3
H, J = 8 Hz, CH3
δ
’), 1.40 (s, 9H, 3
×
CH OtBu), 1.60–1.67 (m, 1H, CH2
β
γ
3
1
.93–1.95 (m, 1H, CH2
β
), 4.63 (t, 1H, J = 4 Hz, CH α
7
1
δ
δ
6
2
δ
γ
β
α
3
1
3
), 115.90 (CH 6), 123.64 (CH 7), 124.93 (Cq 5a), 126.76 (CH 8), 126.89 (CH 9), 132.41 (CH 2), 132.58 (Cq
a), 136.74 (Cq 9a), 147.40 (Cq 4), 172.75 (C=O). MS (ESI +, QTof, m/z): 355.0 [M + H] .
+

Molecules 2018, 23, 2987
10 of 26
Tert-butyl 6-((tert-butoxycarbonyl)amino)-2-(imidazo[1,2-a]quinoxalin-4-ylamino)hexanoate (6e): The same
procedure used for the synthesis of 6a was employed with N- -tert-butyloxycarbonyl-L-lysine tert-butyl
ester hydrochloride (2.565 g, 7.6 mmol), N,N-diisopropyl-ethylamine (2.4 mL, 14.7 mmol) and
-chloroimidazo[1,2-a]quinoxaline (0.5 g, 2.5 mmol) in dimethylformamide (10 mL). The product
ε
4
5
was purified by flash chromatography eluted with cyclohexane/ethyl acetate 80/20 to 50/50. The
1
compound is obtained as a beige solid (32% yield). C H N O . M : 469.58 g/mol. H-NMR
δ
(ppm,
, CH2
25
35
5
4
W
DMSO-d ): 1.35 (s, 9H, 3
δ
×
CH OtBu), 1.41 (s, 9H, 3
×
CH OtBu), 1.43–1.49 (m, 4H, CH2
γ
6
3
3
), 1.86–1.92 (m, 2H, CH2
NH-CH2 ), 7.26–7.35 (m, 1H, CH 7), 7.39–7.42 (m, 1H, CH 8), 7.52 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9),
.64 (d, 1H, J = 4 Hz, NH-CH ), 7.66 (d, 1H, J = 4 Hz, CH 2), 8.12 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6),
.64 (d, 1H, J = 4 Hz, CH 1). 13C-NMR
(ppm, DMSO-d ): 23.52 (CH2 ), 28.19 (CH tBu), 28.74 (CH
), 30.92 (CH2 ), 40.35 (CH2
CH 1), 115.93 (CH 6), 123.70 (CH 7), 124.98 (Cq 5a), 126.81 (CH 9), 126.92 (CH 8), 132.44 (CH 2), 132.60
β), 2.91–2.93 (m, 2H, CH2 ε), 4.52–4.56 (m, 1H, CH α), 6.79 (t, 1H, J = 4 Hz,
ε
7
8
α
δ
γ
6
3
3
tBu), 29.67 (CH2
δ
β
ε), 54.64 (CH α), 79.68 (Cq tBu), 80.80 (Cq tBu), 115.25
(
(
+
Cq 3a), 136.75 (Cq 9a), 147.35 (Cq 4), 172.41 (C=O). MS (ESI +, QTof, m/z): 470.0 [M + H] .
Tert-butyl 5-(((benzyloxy)carbonyl)amino)-2-(imidazo[1,2-a]quinoxalin-4-ylamino)-pentanoate (6f): Using
the same procedure used for the synthesis of 6a with N- -carbobenzoxy-L-ornithine -tert-butyl
ester hydrochloride (3.702 g, 10.3 mmol), N,N-diisopropylethylamine (2.4 mL, 14.7 mmol) and
-chloroimidazo[1,2-a]quinoxaline (0.5 g, 2.5 mmol) in dimethylformamide (10 mL). The product
δ
α
4
5
was purified by flash chromatography eluted with cyclohexane/ethyl acetate 80/20 to 50/50. The
1
compound is obtained as a beige solid (38% yield). C H N O . M : 489.57 g/mol. H-NMR
δ
27
31
5
4
W
1
(
2
ppm, 400 MHz, DMSO-d ): H-NMR
δ
(ppm, DMSO-d ): 1.29 (s, 9H, 3
×
CH OtBu), 1.46–1.50 (m,
6
6
3
H, CH2
γ
), 1.79–1.84 (m, 2H, CH2
β
), 2.94–2.98 (m, 2H, CH2
), 7.24–7.27 (m, 5H, CH Phenyl), 7.29–7.33
m, 1H, CH 8), 7.46 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.52 (d, 1H, J = 4 Hz, NH-CH ), 7.56 (d, 1H,
J = 4 Hz, CH 2), 8.02 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 8.53 (d, 1H, J = 4 Hz, CH 1). 13C-NMR
δ), 4.44–4.46 (m, 1H, CH α), 4.90 (s, 2H,
CH –Phenyl), 7.17–7.21 (m, 1H, CH 7), 7.23 (s, 1H, NH-CH2
δ
2
(
α
δ
(
ppm, DMSO-d ): 26.63 (CH2
γ
), 28.13 (CH tBu), 28.66 (CH2
β), 40.71 (CH2
δ), 54.50 (CH α), 65.67
6
3
(
CH -Phenyl), 80.91 (Cq tBu), 115.30 (CH 1), 115.99 (CH 6), 123.77 (CH 7), 125.03 (Cq 5a), 126.84 (CH 9),
2
1
26.97 (CH 8), 128.86 (CH Phenyl), 132.50 (CH 2), 132.65 (Cq 3a), 136.77 (Cq 9a), 147.36 (Cq 4), 156.65
+
(Cq Phenyl), 172.28 (C=O). MS (ESI +, QTof, m/z): 490.0 [M + H] .
Tert-Butyl 2-(imidazo[1,2-a]quinoxalin-4-ylamino)-3-phenylpropanoate (6g):The same procedure as for
the synthesis of 6a was used with L-phenylalanine tert-butyl ester hydrochloride (1.9 g, 7.4 mmol),
N,N-diisopropylethylamine (2.4 mL, 14.7 mmol) and 4-chloroimidazo[1,2-a]quinoxaline
5 (0.5 g,
2.5 mmol) in dimethylformamide (10 mL). The product was purified by flash chromatography eluted
with cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as a beige solid (37% yield).
1
C H N O . M : 388.46 g/mol. H-NMR δ (ppm, DMSO-d ): 1.33 (s, 9H, 3 × CH OtBu), 3.19–3.23
2
3
24
4
2
W
6
3
(
m, 1H, CH
β
), 3.32–3.36 (m, 1H, CH2
β
), 4.82–4.84 (m, 1H, CH
α
), 7.21 (t, 1H, J = 8 Hz, CH Phenyl),
2
7
.29 (t, 2H, J = 8 Hz, 2
.57 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.61 (d, 1H, J = 8 Hz, NH), 7.66 (d, 1H, J = 4 Hz, CH 2), 8.13
(ppm, DMSO-d ): 26.97 (CH tBu),
×
CH Phenyl), 7.32–7.34 (m, 3H, CH 7, 2
×
CH Phenyl), 7.41–7.44 (m, 1H, CH 8),
7
(
dd, 1H, J = 8, 4 Hz, CH 6), 8.64 (d, 1H, J = 4 Hz, CH 1). 13C-NMR
δ
6
3
3
5.91 (CH2 β), 54.93 (CH α), 79.88 (Cq tBu), 114.18 (CH 1), 114.85 (CH 6), 122.74 (CH 7), 123.89 (Cq 5a),
1
2
25.74 (CH 9), 125.84 (CH Phenyl), 125.86 (CH 8), 127.59 (CH Phenyl), 128.69 (CH Phenyl), 131.41 (CH
), 135.54 (Cq 3a), 137.05 (Cq 9a), 146.02 (Cq 4), 170.53 (C=O). MS (ESI +, QTof, m/z): 389.0 [M + H] .
+
Tert-butyl 3-(4-(tert-butoxy)phenyl)-2-(imidazo[1,2-a]quinoxalin-4-ylamino)propanoate (6h): The same
procedure as for the synthesis of 6a was used with O-tert-butyl-L-tyrosine tert-butyl ester
hydrochloride (1.6 g, 4.9 mmol), N,N-diisopropylethylamine (2.4 mL, 14.7 mmol) and
4-chloroimidazo[1,2-a]quinoxaline 5 (0.5 g, 2.5 mmol) in dimethylformamide (10 mL). The product
was purified by flash chromatography eluted with cyclohexane/ethyl acetate 80/20 to 50/50. The
1
compound is obtained as a beige solid (26% yield). C H N O . M : 460.57 g/mol. H-NMR
δ
(ppm,
27
32
4
3
W
DMSO-d ): 1.23 (s, 9H, 3
×
CH OtBu), 1.31 (s, 9H, 3
×
CH OtBu), 3.11–3.17 (m, 1H, CH2
β), 3.25–3.31
6
3
3

Molecules 2018, 23, 2987
11 of 26
(m, 1H, CH2
β), 4.78–4.83 (m, 1H, CH
α
), 6.86 (dd, 2H, J = 4 Hz, J = 8 Hz, CH Phenyl), 7.22 (dd, 2H,
J = 4 Hz, J = 8 Hz, CH Phenyl), 7.32–7.36 (m, 1H, CH 7), 7.40–7.44 (m, 1H, CH 8), 7.57 (dd, 1H, J = 4 Hz,
J = 8 Hz, CH 9), 7.61 (d, 1H, J = 4 Hz, NH), 7.66 (d, 1H, J = 4 Hz, CH 2), 8.13 (dd, 1H, J = 4 Hz, J = 8 Hz,
CH 6), 8.64 (d, 1H, J = 4 Hz, CH 1). 13C-NMR
6.63 (CH2 ), 56.09 (CH
23.98 (2 CH Phenyl), 124.96 (Cq 5a), 126.79 (CH 9), 126.90 (CH 8), 130.31 (2
δ (ppm, DMSO-d ): 28.02 (CH tBu), 28.93 (CH tBu),
6 3 3
3
1
β
α
), 78.11 (Cq tBu), 80.87 (Cq tBu), 115.22 (CH 1), 115.90 (CH 6), 123.80 (CH 7),
CH Phenyl), 132.47
×
×
(
CH 2), 132.61 (Cq 3a), 136.60 (Cq 9a), 147.04 (Cq 4), 154.03 (2 × Cq Phenyl), 171.72 (C=O). MS (ESI +,
+
QTof, m/z): 461.2 [M + H] .
Tert-butyl 6-((tert-butoxycarbonyl)amino)-2-(imidazo[1,2-a]quinoxalin-4-ylamino)hexanoate (6i): Same
procedure used for the synthesis of 6a was employed with N-
tert-butyl ester hydrochloride (1.596 g, 4.9 mmol), N,N-diisopropyl-ethylamine (1.6 mL, 9.8 mmol) and
-chloroimidazo[1,2-a]quinoxaline (0.5 g, 2.5 mmol) in dimethylformamide (10 mL). The product
α-tert-butyloxycarbonyl-L-ornithine
4
5
was purified by flash chromatography eluted with cyclohexane/ethyl acetate 80/20 to 50/50. The
1
compound is obtained as a beige solid (48% yield). C H N O . M : 455.56 g/mol. H-NMR
δ
(ppm,
), 2.91–2.95
), 6.79 (d, 1H, J = 4 Hz, NH-CH ),
.32–7.35 (m, 1H, CH 7), 7.39–7.42 (m, 1H, CH 8), 7.56 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.60 (d, 1H,
J = 4 Hz, NH-CH2 ), 7.66 (s, 1H, CH 2), 8.13 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 8.64 (d, 1H, CH 1).
(ppm, DMSO-d ): 25.87 (CH2 ), 28.04 (CH tBu), 28.67 (CH tBu), 28.43 (CH ), 39.79
), 78.45 (Cq tBu), 80.53 (Cq tBu), 115.02 (CH 1), 115.79 (CH 6), 122.99 (CH 7), 124.65
24
33
5
4
W
DMSO-d ): 1.35 (s, 9H, CH-COOtBu), 1.41 (s, 9H, NH-COOtBu), 1.85–1.96 (m, 2H, CH2
β
6
(
m, 2H, CH2
γ
), 3.35–3.39 (m, 2H, CH2
δ
), 4.51–4.56 (m, 1H, CH
α
α
7
δ
13
C-NMR
δ
β
γ
2
6
3
3
(
(
(
CH2
δ
), 54.75 (CH α
Cq 5a), 126.50 (CH 9), 126.73 (CH 8), 132.20 (CH 2), 132.87 (Cq 3a), 147.78 (Cq 4), 156.00 (Cq 9a), 172.33
+
C=O). MS (ESI +, QTof, m/z): 456.0.1 [M + H] .
3
.1.3. Bromination
Tert-butyl 2-((1-bromoimidazo[1,2-a]quinoxalin-4-yl)amino)acetate (7a): A solution of 6a (0.26 g, 0.87 mmol)
and N-bromosuccinimide (0.19 g, 1.0 mmol) in CHCl (50 mL) was heated under reflux for 2 h. The solvent
3
was removed under reduced pressure. The crude mixture was dissolved in ethyl acetate (50 mL) and
successively washed with saturated aqueous ammonium chloride, saturated aqueous sodium bicarbonate,
distilled water and finally brine (50 mL). The organic phase was dried on sodium sulfate, filtered and
concentrated under reduced pressure. The compound was obtained as a beige oil (97% yield) and used
1
without purification. C H BrN O . M : 377.24 g/mol. H-NMR
δ (ppm, DMSO-d ): 1.42 (s, 9H,
6
16
17
4
2
W
3
1
×
CH OtBu), 4.12 (d, 2H, J = 8 Hz, CH2
α
), 7.34–7.37 (m, 1H, CH 7), 7.45–7.50 (m, 1H, CH 8), 7.59 (dd,
H, J = 4 Hz, J = 8 Hz, CH 9), 7.76 (s, 1H, CH 2), 8.02 (t, 1H, J = 8 Hz, NH), 8.96 (dd, 1H, J = 4 Hz, J = 8 Hz,
CH 6). 13C-NMR δ (ppm, DMSO-d ): 28.22 (CH tBu), 43.26 (CH α), 80.93 (Cq tBu), 99.61 (Cq 1), 115.12
3
6
3
2
(
CH 6), 123.08 (CH 7), 126.08 (Cq 5a), 127.35 (CH 9), 127.78 (CH 8), 134.01 (Cq 3a), 134.79 (CH 2), 137.63
+
(Cq 9a), 147.05 (Cq 4), 169.64 (C=O). MS (ESI +, QTof, m/z): 377.0 [M + H] .
Tert-butyl 2-((1-bromoimidazo[1,2-a]quinoxalin-4-yl)amino)propanoate (7b): A solution of 6b (0.3 g,
.96 mmol) and N-bromosuccinimide (0.2 g, 1.1 mmol) in CHCl (50 mL) was heated under reflux
0
3
for 2 h. The solvent was removed under reduced pressure. The crude mixture was dissolved in ethyl
acetate (50 mL) and successively washed with saturated aqueous ammonium chloride, saturated
aqueous sodium bicarbonate, distilled water and finally brine. The organic phase was dried on sodium
sulfate, filtered and concentrated under reduced pressure. The compound was obtained as a beige
1
oil (98% yield) and used without purification. C H BrN O . M : 391.26 g/mol. H-NMR
δ
(ppm,
17
19
4
2
W
DMSO-d ): 1.41 (s, 9H, 3
×
CH OtBu), 1.51 (d, 3H, J = 8 Hz, CH3
β
), 4.55–4.59 (m, 1H, CH
α
), 7.34–7.38
6
3
(
(
m, 1H, CH 7), 7.46–7.48 (m, 1H, CH 8), 7.60 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.76 (s, 1H, CH 2), 7.82
d, 1H, J = 4 Hz, NH), 8.97 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6). C-NMR
13
δ (ppm, DMSO-d ): 17.40 (CH
6 3
β
), 28.03 (CH tBu), 50.24 (CH ), 80.70 (Cq tBu), 99.68 (CH 1), 115.06 (CH 6), 123.17 (CH 7), 126.06 (Cq
α
3
5
a), 127.30 (CH 9), 127.45 (CH 8), 133.87 (Cq 3a), 134.73 (CH 2), 137.47 (Cq 9a), 146.58 (Cq 4), 172.66
+
(C=O). MS (ESI +, QTof, m/z): 391.1 [M + H] .

Molecules 2018, 23, 2987
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Tert-butyl 2-((1-bromoimidazo[1,2-a]quinoxalin-4-yl)amino)-3-methylbutanoate (7c): A solution of 6c
0.38 g, 1.1 mmol) and N-bromosuccinimide (0.24 g, 1.4 mmol) in CHCl (50 mL) was heated under
(
3
reflux for 2 h. The solvent was removed under reduced pressure. The crude mixture was dissolved in
ethyl acetate (50 mL) and successively washed with saturated aqueous ammonium chloride, saturated
aqueous sodium bicarbonate, distilled water and finally brine. The organic phase was dried on sodium
sulfate, filtered and concentrated under reduced pressure. The compound was obtained as a beige
1
oil (96% yield) and used without purification. C H BrN O . M : 419.31 g/mol. H-NMR
δ
(ppm,
19
23
4
2
W
DMSO-d ): 1.02 (d, 3H, J = 8 Hz, CH3
γ
), 1.05 (d, 3H, J = 8 Hz, CH3
), 4.49 (t, 1H, J = 16 Hz, CH ), 7.20 (d, 1H, J = 8 Hz, NH), 7.35–7.40 (m, 1H, CH
), 7.47–7.49 (m, 1H, CH 8), 7.62 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.76 (s, 1H, CH 2), 8.97 (dd, 1H, J = 4
γ
’), 1.43 (s, 9H, 3
×
CH OtBu),
6
3
2
7
.31–2.36 (m, 1H, CH
β
α
1
3
Hz, J = 8 Hz, CH 6). C-NMR
δ (ppm, DMSO-d ): 19.42 (CH3 γ, CH3 γ’), 28.17 (CH tBu), 30.40 (CH
6 3
β
), 59.76 (CH ), 81.25 (Cq tBu), 99.86 (Cq 1), 115.08 (CH 6), 123.38 (CH 7), 126.16 (Cq 5a), 127.44 (CH
α
9
), 127.47 (CH 8), 133.81 (Cq 3a), 134.74 (CH 2), 137.40 (Cq 9a), 146.84 (Cq 4), 171.44 (C=O). MS (ESI +,
+
QTof, m/z): 419.1 [M + H] .
Tert-butyl 2-((1-bromoimidazo[1,2-a]quinoxalin-4-yl)amino)-4-methylpentanoate (7d): A solution of 6d
(
0.35 g, 1.0 mmol) and N-bromosuccinimide (0.2 g, 1.2 mmol) in CHCl (50 mL) was heated under
3
reflux for 2 h. The solvent was removed under reduced pressure. The crude mixture was dissolved in
ethyl acetate (50 mL) and successively washed with saturated aqueous ammonium chloride, saturated
aqueous sodium bicarbonate, distilled water and finally brine. The organic phase was dried on sodium
sulfate, filtered and concentrated under reduced pressure. The compound was obtained as a beige
1
oil (95% yield) and used without purification. C H BrN O . M : 433.34 g/mol. H-NMR
δ
(ppm,
20
25
4
2
W
DMSO-d ): 0.91 (d, 3H, J = 8 Hz, CH3
δ
), 0.95 (d, 3H, J = 8 Hz, CH3
), 1.77–1.79 (m, 1H, CH ), 1.94–1.96 (m, 1H, CH2
), 7.35–7.38 (m, 1H, CH 7), 7.46–7.50 (m, 1H, CH 8), 7.60 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.71 (d, 1H,
(ppm, DMSO-d6):
), 52.95 (CH ), 80.76 (Cq
δ
’), 1.40 (s, 9H, 3
×
CH OtBu),
6
3
1.60–1.62 (m, 1H, CH2
β
γ
β), 4.60 (t, 1H, J = 4 Hz, CH
α
J = 4 Hz, NH), 7.76 (s, 1H, CH 2), 8.97 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6). 13C-NMR
δ
2
2.03 (CH3 ), 23.31 (CH3 ’), 25.12 (CH ), 28.14 (CH tBu), 40.10 (CH2
δ
δ
γ
β
α
3
tBu), 99.21 (Cq 1), 115.07 (CH 6), 123.17 (CH 7), 126.08 (Cq 5a), 127.37 (CH 9), 127.46 (CH 8), 133.88 (Cq
+
3
a), 134.71 (CH 2), 137.55 (Cq 9a), 146.97 (Cq 4), 172.59 (C=O). MS (ESI +, QTof, m/z): 433.1 [M + H] .
Tert-butyl 2-((1-bromoimidazo[1,2-a]quinoxalin-4-yl)amino)-6-((Tert-butoxycarbonyl)amino)-hexanoate (7e):
A solution of 6e (1.2 g, 2.5 mmol) and N-bromosuccinimide (0.54 g, 3.1 mmol) in CHCl (50 mL) was
3
heated under reflux for 2 h. The solvent was removed under reduced pressure. The crude mixture
was dissolved in ethyl acetate (50 mL) and successively washed with saturated aqueous ammonium
chloride, saturated aqueous sodium bicarbonate, distilled water and finally brine. The organic phase
was dried on sodium sulfate, filtered and concentrated under reduced pressure. The compound was
obtained as a beige oil (78% yield) and used without purification. C H BrN O . M : 548.47 g/mol.
2
5
34
5
4
W
1
H-NMR
OtBu), 1.54–1.56 (m, 2H, CH2
CH ), 6.78 (t, 1H, J = 4 Hz, NH-CH2
J = 4 Hz, J = 8 Hz, CH 9), 8.03 (s, 1H, CH 2), 8.34 (d, 1H, J = 4 Hz, NH-CH
J = 8 Hz, CH 6). 13C-NMR
(ppm, DMSO-d ): 22.90 (CH2 ), 28.15 (CH tBu), 28.73 (CH tBu), 29.46
δ
(ppm, DMSO-d ): 1.22–1.24 (m, 2H, CH2
γ
), 1.34 (s, 9H, 3
), 1.89–1.91 (m, 2H, CH2 ), 2.83–2.89 (m, 2H, CH2
), 7.34–7.39 (m, 1H, CH 7), 7.46–7.48 (m, 1H, CH 8), 7.65 (dd, 1H,
), 8.97 (dd, 1H, J = 4 Hz,
×
CH OtBu), 1.40 (s, 9H, 3
×
CH3
6
3
β
δ
ε), 4.13–4.19 (m, 1H,
α
ε
α
δ
γ
6
3
3
(
(
(
CH2 δ), 31.34 (CH2 β), 39.36 (CH2 ε), 54.61 (CH α), 80.85 (Cq tBu), 81.14 (Cq tBu), 99.70 (Cq 1), 115.06
CH 6), 123.19 (CH 7), 126.09 (Cq 5a), 127.39 (CH 9), 127.46 (CH 8), 133.87 (CH 2), 134.70 (Cq 3a), 137.54
+
Cq 9a), 146.88 (Cq 4), 171.39 (C=O), 172.21 (C=O). MS (ESI +, QTof, m/z): 548.1 [M + H] .
Tert-butyl 5-(((benzyloxy)carbonyl)amino)-2-((1-bromoimidazo[1,2-a]quinoxalin-4-yl)-amino)pentanoate (7f):
A solution of 6f (0.45 g, 0.9 mmol) and N-bromosuccinimide (0.20 g, 1.1 mmol) in CHCl (50 mL) was
3
heated under reflux for 2 h. The solvent was removed under reduced pressure. The crude mixture
was dissolved in ethyl acetate (50 mL) and successively washed with saturated aqueous ammonium
chloride, saturated aqueous sodium bicarbonate, distilled water and finally brine. The organic phase
was dried on sodium sulfate, filtered and concentrated under reduced pressure. The compound was

Molecules 2018, 23, 2987
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obtained as a beige oil (94% yield) and used without purification. C H BrN O . M : 568.46 g/mol.
2
7
30
5
4
W
1
H-NMR
H, CH2
NH-CH2
H, J = 4 Hz, J = 8 Hz, CH 9), 7.67 (d, 1H, J = 4 Hz, NH-CH
J = 8 Hz, CH 6). 13C-NMR
(ppm, DMSO-d ): 26.53 (CH2
CH2 ), 54.43 (CH ), 65.59 (CH -Phenyl), 80.92 (Cq tBu), 99.69 (Cq 1), 115.07 (CH 6), 123.20 (CH 7),
26.10 (Cq 5a), 127.39 (CH 9), 127.45 (CH 8), 128.18 (CH Phenyl), 128.78 (CH Phenyl), 133.87 (Cq 3a),
δ
(ppm, DMSO-d ): 1.40 (s, 9H, 3
×
CH OtBu), 1.51–1.59 (m, 2H, CH2
γ
), 1.89–1.92 (m,
6
3
2
β
), 3.03–3.08 (m, 2H, CH2
), 7.31–7.35 (m, 5H, CH Phenyl), 7.36–7.39 (m, 1H, CH 7), 7.46–7.50 (m, 1H, CH 8), 7.59 (dd,
), 7.76 (s, 1H, CH 2), 8.99 (dd, 1H, J = 4 Hz,
), 28.13 (CH tBu), 28.50 (CH2 ), 40.42
δ), 4.52–4.54 (m, 1H, CH α), 5.00 (s, 2H, CH -Phenyl), 7.26 (s, 1H,
2
δ
1
α
δ
γ
β
6
3
(
1
1
δ
α
2
34.70 (CH 2), 137.72 (Cq 9a), 146.85 (Cq 4), 156.56 (Cq Phenyl), 172.05 (C=O). MS (ESI +, QTof, m/z):
+
5
68.3 [M + H] .
Tert-butyl 2-((1-bromoimidazo[1,2-a]quinoxalin-4-yl)amino)-3-phenylpropanoate (7g): A solution of 6g (0.34 g,
.9 mmol) and N-bromosuccinimide (0.18 g, 1.1 mmol) in CHCl (50 mL) was heated under reflux for 2
0
3
h. The solvent was removed under reduced pressure. The crude mixture was dissolved in ethyl acetate
50 mL) and successively washed with saturated aqueous ammonium chloride, saturated aqueous
(
sodium bicarbonate, distilled water and finally brine. The organic phase was dried on sodium sulfate,
filtered and concentrated under reduced pressure. The compound was obtained as a beige oil (83%
1
yield) and used without purification. C H BrN O . M : 467.36 g/mol. H-NMR
δ
(ppm, DMSO-d6):
23
23
4
2
W
1
.33 (s, 9H, 3
CH
Phenyl), 7.35–7.38 (m, 1H, CH 7), 7.46–7.49 (m, 1H, CH 8), 7.60 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.65
×
CH OtBu), 3.18–3.22 (m, 1H, CH2
), 7.21 (t, 1H, J = 8 Hz, CH Phenyl), 7.27–7.30 (m, 2H, 2
β
), 3.31–3.34 (m, 1H, CH2
β), 4.79–4.81 (m, 1H,
3
α
×
CH Phenyl), 7.32–7.34 (m, 2H, 2xCH
13
(d, 1H, J = 8 Hz, NH), 7.75 (s, 1H, CH 2), 8.97 (dd, 1H, J = 2 Hz, J = 8 Hz, CH 6). C-NMR
δ (ppm,
DMSO-d ): 28.04 (CH tBu), 36.91 (CH ), 56.01 (CH ), 81.06 (Cq tBu), 99.76 (Cq 1), 115.07 (CH 6),
β
2
α
6
3
1
1
1
23.33 (CH 7), 126.13 (Cq 5a), 126.96 (CH Phenyl), 127.45 (CH 9), 127.47 (CH 8), 128.68 (CH Phenyl),
29.77 (CH Phenyl), 133.80 (Cq Phenyl), 134.76 (CH 2), 137.45 (Cq 3a), 138.05 (Cq 9a), 146.66 (Cq 4),
+
71.45 (C=O). MS (ESI +, QTof, m/z): 467.1 [M + H] .
Tert-butyl 2-((1-bromoimidazo[1,2-a]quinoxalin-4-yl)amino)-3-(4-(Tert-butoxy)phenyl)-propanoate
(7h):
A solution of 6h (0.34 g, 0.9 mmol) and N-bromosuccinimide (0.18 g, 1.1 mmol) in CHCl (50 mL) was
3
heated under reflux for 2 h. The solvent was removed under reduced pressure. The crude mixture
was dissolved in ethyl acetate (50 mL) and successively washed with saturated aqueous ammonium
chloride, saturated aqueous sodium bicarbonate, distilled water and finally brine. The organic phase
was dried on sodium sulfate, filtered and concentrated under reduced pressure. The compound was
obtained as a beige oil (91% yield) and used without purification. C H BrN O . M : 467.36 g/mol.
2
3
23
4
2
W
1
H-NMR
H, CH2
Phenyl), 7.20 (dd, 2H, J = 4 Hz, J = 8 Hz, CH Phenyl), 7.34–7.38 (m, 1H, CH 7), 7.46–7.50 (m, 1H, CH 8),
.59 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.65 (d, 1H, J = 8 Hz, NH), 7.75 (d, 1H, J = 4 Hz, CH 2), 8.98 (dd,
H, J = 4 Hz, J = 8 Hz, CH 6). 13C-NMR
(ppm, DMSO-d ): 28.04 (CH tBu), 28.93 (CH tBu), 36.61
CH2 ), 56.03 (CH ), 78.10 (Cq tBu), 81.01 (Cq tBu), 99.71 (Cq 1), 115.08 (CH 6), 123.32 (CH 7), 123.96
CH Phenyl), 126.13 (Cq 5a), 127.42 (CH 9), 127.47 (CH 8), 130.33 (2 CH Phenyl), 132.54 (Cq 3a),
Cq Phenyl), 171.53 (C=O). MS (ESI +, QTof,
δ
(ppm, DMSO-d ): 1.22 (s, 9H, 3
×
CH OtBu), 1.31 (s, 9H, 3
×
CH OtBu), 3.11–3.16 (m,
6
3
3
1
β), 3.24–3.26 (m, 1H, CH2 β), 4.76–4.81 (m, 1H, CH α), 6.85 (dd, 2H, J = 4 Hz, J = 8 Hz, CH
7
1
δ
6
3
3
(
(
β
α
2
×
×
1
34.78 (CH 2), 137.43 (Cq 9a), 146.61 (Cq 4), 154.06 (2
×
+
m/z): 467.1 [M + H] .
Tert-butyl 5-((1-bromoimidazo[1,2-a]quinoxalin-4-yl)amino)-2-((Tert-butoxycarbonyl)amino)-pentanoate (7i):
A solution of 6i (0.49 g, 1.1 mmol) and N-bromosuccinimide (0.23 g, 1.3 mmol) in CHCl (50 mL) was
3
heated under reflux for 2 h. The solvent was removed under reduced pressure. The crude mixture
was dissolved in ethyl acetate (50 mL) and successively washed with saturated aqueous ammonium
chloride, saturated aqueous sodium bicarbonate, distilled water and finally brine. The organic phase
was dried on sodium sulfate, filtered and concentrated under reduced pressure. The compound was
obtained as a beige oil (92% yield) and used without purification. C H BrN O . M : 534.45 g/mol.
2
4
32
5
4
W
1
H-NMR δ (ppm, DMSO-d ): 1.44 (s, 9H, CH-COOtBu), 1.46 (s, 9H, NH-COOtBu), 1.79–1.83 (m, 2H,
6

Molecules 2018, 23, 2987
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CH2
β), 1.91–1.94 (m, 2H, CH2
γ
), 3.73–3.78 (m, 2H, CH2
δ
), 4.25–4.29 (m, 1H, CH
α
), 5.39 (d, 1H, J = 4
Hz, NH-CH
α
), 6.33 (d, 1H, J = 4 Hz, NH-CH2 δ), 7.27–7.31 (m, 1H, CH 7), 7.41–7.45 (m, 1H, CH 8),
7
.48 (s, 1H, CH 2), 7.81 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 8.98 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6).
1
3
C-NMR ), 40.42
δ
(ppm, DMSO-d ): 25.74 (CH2
β
), 28.07 (CH tBu), 28.45 (CH tBu), 30.17 (CH γ
6
3
3
2
(
(
(
CH2 ), 54.00 (CH α), 79.75 (Cq tBu), 81.98 (Cq tBu), 99.05 (Cq 1), 115.02 (CH 6), 122.77 (CH 7), 124.54
δ
Cq 5a), 127.12 (CH 8), 127.98 (CH 9), 134.22 (CH 2), 135.98 (Cq 3a), 146.86 (Cq 4), 155.59 (Cq 9a), 171.92
+
C=O). MS (ESI +, QTof, m/z): 534.1 [M + H] .
3
.1.4. Suzuki-Miyaura Cross-Coupling Reactions
Tert-butyl 2-((1-(3,4-dimethoxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)acetate (8a): To a mixture of
(0.270 g, 0.71 mmol) in DME/H O (2/1, 15 mL) were added 3,4-dimethoxyphenylboronic acid
7
a
2
(0.143 g, 0.79 mmol), tetrakis(triphenylphosphine) palladium (0.042 g, 0.036 mmol) and sodium
carbonate (0.152 g, 1.43 mmol) in a microwave-adapted vial. The reaction was submitted to microwave
◦
irradiations during 20 min at 150 C and then filtered on a Celite pad. The filtrate was concentrated
under reduced pressure and successively washed with saturated aqueous ammonium chloride,
saturated aqueous sodium bicarbonate, distilled water and finally brine. The organic phase was
dried on sodium sulfate, filtered and concentrated under reduced pressure. The product was purified
by flash chromatography eluted with cyclohexane/ethyl acetate 80/20 to 50/50. The compound is
1
obtained as a white solid (19% yield). C H N O . MW: 434.49 g/mol. H-NMR
δ
(ppm, DMSO-d6):
24
26
4
4
1
.44 (s, 9H, 3
CH2 ), 7.01–7.05 (m, 1H, CH 7), 7.11 (d, 1H, J = 4 Hz, CH Phenyl), 7.14 (s, 1H, J = 4 Hz, CH Phenyl),
.18 (d, 1H, J = 4 Hz, CH Phenyl), 7.31 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.33–7.35 (m, 1H, CH 8), 7.54
×
CH OtBu), 3.74 (s, 3H, OCH Phenyl), 3.87 (s, 3H, OCH Phenyl), 4.14 (d, 2H, J = 8 Hz,
3 3 3
α
7
13
(
s, 1H, CH 2), 7.56 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.96 (t, 1H, J = 8 Hz, NH). C-NMR
DMSO-d ): 28.26 (CH tBu), 43.33 (CH ), 80.86 (Cq tBu), 112.31 (CH Phenyl), 114.25 (CH Phenyl),
15.93 (CH 6), 122.75 (CH 7), 123.30 (CH Phenyl), 126.03 (Cq 5a), 126.58 (CH 8), 127.20 (CH 9), 132.66
δ (ppm,
α
2
6
3
1
(
(
CH 2), 133.141 (Cq 3a), 130.93 (Cq 1), 137.71 (Cq 9a), 149.27 (Cq Phenyl), 150.14 (Cq Phenyl), 147.74
Cq 4), 169.85 (C=O). MS (ESI +, QTof, m/z): 435.1 [M + H] .
+
Tert-butyl 2-((1-(3,4-dimethoxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)propanoate (8b): Following
the same procedure used for the synthesis of 8a, to a mixture of 7b (0.440 g, 1.12 mmol) in
DME/H O (2/1, 15 mL) were added 3,4-dimethoxyphenylboronic acid (0.225 g, 1.24 mmol),
2
tetrakis(triphenylphosphine) palladium (0.065 g, 0.056 mmol) and sodium carbonate (0.237 g,
2.24 mmol) in a microwave-adapted vial. The product was purified by flash chromatography eluted
with cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as a white solid (42% yield).
1
C H N O . MW: 448.51 g/mol. H-NMR
δ
(ppm, DMSO-d ): 1.32 (s, 9H, 3
×
CH OtBu), 1.42 (d,
3
25
28
4
4
6
3
H, J = 8 Hz, CH3
β), 3.64 (s, 3H, OCH Phenyl), 3.76 (s, 3H, OCH Phenyl), 4.48–4.52 (m, 1H, CH α),
3 3
6
.90–6.95 (m, 1H, CH 7), 7.01 (d, 1H, J = 4 Hz, CH Phenyl), 7.03 (s, 1H, CH Phenyl), 7.08 (d, 1H, J = 4
Hz, CH Phenyl), 7.20 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.22–7.25 (m, 1H, CH 8), 7.42 (s, 1H, CH 2),
.46 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.82 (d, 1H, J = 4 Hz, NH). 13C-NMR
(ppm, DMSO-d ): 17.59
CH3 ), 28.25 (CH tBu), 50.30 (CH ), 80.72 (Cq tBu), 112.40 (CH Phenyl), 114.33 (CH Phenyl), 116.00
CH 6), 122.77 (CH 7), 122.90 (Cq 1), 123.37 (CH Phenyl), 126.10 (CH 8), 126.65 (Cq 5a), 127.28 (CH 9),
7
δ
6
(
(
β
α
3
1
31.05 (Cq 3a), 132.63 (CH 2), 137.68 (Cq 9a), 147.33 (Cq 4), 149.36 (Cq Phenyl), 150.22 (Cq Phenyl),
+
172.96 (C=O). MS (ESI +, QTof, m/z): 449.2 [M + H] . HRMS calculated for C H N O 449.2189,
25
29
4
4
found 449.2186.
Tert-butyl
2-((1-(3,4-dimethoxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)-3-methylbutanoate
(8c):
Followigng the same procedure used for the synthesis of 8a, to a mixture of 7c (0.370 g, 0.88 mmol)
in DME/H O (2/1, 15 mL) were added 3,4-dimethoxyphenylboronic acid (0.176 g, 0.97 mmol),
2
tetrakis(triphenylphosphine) palladium (0.051 g, 0.044 mmol) and sodium carbonate (0.186 g,
1.76 mmol) in a microwave-adapted vial. The product was purified by flash chromatography eluted
with cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as a white solid (69%

Molecules 2018, 23, 2987
15 of 26
1
yield). C H N O . MW: 476.57 g/mol. H-NMR
δ
(ppm, DMSO-d ): 0.95 (d, 3H, J = 8 Hz, CH3
γ
),
), 3.63 (s, 3H, OCH3
), 6.92–6.97 (m, 1H, CH 7), 7.00 (s, 1H,
27
32
4
4
6
0.98 (d, 3H, J = 8 Hz, CH3
γ
’), 1.34 (s, 9H, 3
×
CH OtBu), 2.20–2.28 (m, 1H, CH β
3
Phenyl), 3.77 (s, 3H, OCH Phenyl), 4.44 (t, 1H, J = 16 Hz, CH
α
3
CH Phenyl), 7.02 (d, 1H, J = 4 Hz, NH), 7.06 (d, 1H, J = 4 Hz, CH Phenyl), 7.08 (d, 1H, J = 4 Hz, CH
Phenyl), 7.22 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.23–7.26 (m, 1H, CH 8), 7.42 (s, 1H, CH 2), 7.49 (dd,
1
3
1
H, J = 4 Hz, J = 8 Hz, CH 9). C-NMR
δ
(ppm, DMSO-d ): 19.43 (CH3
γ
’), 19.56 (CH3
), 81.31 (Cq tBu), 112.41
CH Phenyl), 114.35 (CH Phenyl), 116.02 (CH 6), 122.67 (Cq 1), 123.14 (CH 7), 123.39 (CH Phenyl),
γ), 28.28 (CH3
6
tBu), 30.57 (CH ), 56.15 (OCH Phenyl), 56.24 (OCH Phenyl), 59.73 (CH
β
α
3
3
(
1
26.19 (Cq 5a), 126.71 (CH 8), 127.39 (CH 9), 131.25 (Cq Phenyl), 132.68 (CH 2), 133.00 (Cq 3a), 137.55
(
4
Cq 9a), 147.56 (Cq 4), 149.36 (Cq Phenyl), 150.26 (Cq Phenyl), 171.70 (C=O). MS (ESI +, QTof, m/z):
+
77.1 [M + H] . HRMS calculated for C H N O 477.2502, found 477.2505.
27
33
4
4
Tert-butyl 2-((1-(3,4-dimethoxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)-4-methyl-pentanoate:
(8d):
Following the same procedure used for the synthesis of 8a, to a mixture of 7d (0.360 g, 0.83 mmol)
in DME/H O (2/1, 15 mL) were added 3,4-dimethoxyphenylboronic acid (0.166 g, 0.91 mmol),
2
tetrakis(triphenylphosphine) palladium (0.048 g, 0.041 mmol) and sodium carbonate (0.176 g, 1.66 mmol)
in a microwave-adapted vial. The product was purified by flash chromatography eluted with
cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as a white solid (61% yield).
1
C H N O . MW: 490.59 g/mol. H-NMR
δ
(ppm, DMSO-d ): 0.83 (d, 3H, J = 8 Hz, CH3
δ
), 0.86 (d,
), 1.67–1.70 (m, 1H, CH ),
), 3.63 (s, 3H, OCH Phenyl), 3.76 (s, 3H, OCH Phenyl), 4.55 (t, 1H, J = 4 Hz, CH
28
34
4
4
6
3H, J = 8 Hz, CH3
δ
’), 1.31 (s, 9H, 3
×
CH OtBu), 1.55–1.58 (m, 1H, CH2
β
γ
3
1.84–1.88 (m, 1H, CH2
β
3
3
α), 6.90–6.94 (m, 1H, CH 7), 7.01 (d, 1H, J = 4 Hz, CH Phenyl), 7.05 (s, 1H, CH Phenyl), 7.08 (d, 1H, J = 4
Hz, CH Phenyl), 7.20 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.22–7.24 (m, 1H, CH 8), 7.41 (s, 1H, CH 2), 7.45
dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.50 (d, 1H, J = 4 Hz, NH). 13C-NMR
(ppm, DMSO-d ): 22.14 (CH3 ),
3.41 (CH3 ’), 25.25 (CH ), 28.25 (CH tBu), 40.29 (CH2 ), 52.96 (CH ), 56.14 (OCH Phenyl), 56.23
OCH Phenyl), 80.79 (Cq tBu), 112.40 (CH Phenyl), 114.34 (CH Phenyl), 115.99 (CH 6), 122.78 (Cq 1),
(
δ
δ
6
2
δ
γ
β
α
3
3
(
3
122.88 (CH 7), 123.36 (CH Phenyl), 126.11 (Cq 5a), 126.69 (CH 8), 127.29 (CH 9), 131.09 (Cq 3a), 132.61 (CH
2
), 137.70 (Cq 9a), 147.66 (Cq 4), 149.36 (Cq Phenyl), 150.23 (Cq Phenyl), 172.86 (C=O). MS (ESI +, QTof,
+
m/z): 491.0 [M + H] . HRMS calculated for C H N O 491.2658, found 491.2654.
28
35
4
4
Tert-butyl 6-((Tert-butoxycarbonyl)amino)-2-((1-(3,4-dimethoxyphenyl)imidazo[1,2-a]-quinoxalin-4-yl)amino)
hexanoate (8e): Following the same procedure used for the synthesis of 8a, to a mixture of 7e (1.090 g,
1
.99 mmol) in DME/H O (2/1, 15 mL) were added 3,4-dimethoxyphenylboronic acid (0.397 g,
2
2.18 mmol), tetrakis(triphenylphosphine) palladium (0.114 g, 0.099 mmol) and sodium carbonate
(0.421 g, 3.97 mmol) in a microwave-adapted vial. The product was purified by flash chromatography
eluted with cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as a white solid (7%
1
yield). C H N O . MW: 605.72 g/mol. H-NMR
δ
(ppm, DMSO-d ): 1.36 (s, 9H, 3
×
CH OtBu),
3
33
43
5
6
6
1
3
.41–1.46 (m, 13H, CH2
γ
, 3
×
CH OtBu, CH2
δ), 1.88–1.91 (m, 2H, CH2
β
), 2.93–2.95 (m, 2H, CH2
), 6.80 (t, 1H, J = 4 Hz,
ε), 7.01–7.05 (m, 1H, CH 7), 7.11 (d, 1H, J = 4 Hz, CH Phenyl), 7.15 (s, 1H, CH Phenyl), 7.18
ε),
3
.73 (s, 3H, OCH Phenyl), 3.87 (s, 3H, OCH Phenyl), 4.54–4.58 (m, 1H, CH
α
3
3
NH-CH2
(
1
d, 1H, J = 4 Hz, CH Phenyl), 7.31 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.31–7.35 (m, 1H, CH 8), 7.52 (s,
13
H, CH 2), 7.55 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.59 (d, 1H, J = 4 Hz, NH-CH
α
). C-NMR
), 40.40 (CH2
α), 56.05 (OCH Phenyl), 56.14 (OCH Phenyl), 77.78 (Cq tBu), 80.80 (Cq tBu), 112.30 (CH
δ (ppm,
DMSO-d ): 23.49 (CH2 ), 28.18 (CH tBu), 28.72 (CH tBu), 29.64 (CH ), 30.94 (CH2 β
ε
γ
δ
2
6
3
3
), 54.56 (CH
3
3
Phenyl), 114.22 (CH Phenyl), 115.91 (CH 6), 122.67 (CH 7), 123.28 (CH Phenyl), 126.03 (Cq 5a), 126.56
(
CH 8), 127.23 (CH 9), 131.01 (Cq 1), 132.53 (CH 2), 132.98 (Cq 3a), 137.61 (Cq 9a), 147.51 (Cq 4), 149.27
(
Cq Phenyl), 150.14 (Cq Phenyl), 156.04 (Cq Phenyl), 172.41 (C=O). MS (ESI +, QTof, m/z): 606.2 [M +
+
H] . HRMS calculated for C H N O 606.3292, found 606.3291.
33
44
5
6
Tert-butyl 5-(((benzyloxy)carbonyl)amino)-2-((1-(3,4-dimethoxyphenyl)imidazo[1,2-a]-quinoxalin-4-yl)amino)
pentanoate (8f): Following the same procedure used for the synthesis of 8a, to a mixture of 7f (0.470 g,
0
.89 mmol) in DME/H O (2/1, 15 mL) were added 3,4-dimethoxyphenylboronic acid (0.165 g,
2

Molecules 2018, 23, 2987
16 of 26
0
(
.91 mmol), tetrakis(triphenylphosphine) palladium (0.048 g, 0.041 mmol) and sodium carbonate
0.175 g, 1.65 mmol) in a microwave-adapted vial. The product was purified by flash chromatography
eluted with cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as a white solid
1
1
(42% yield). C H N O . MW: 625.71 g/mol. H-NMR
δ
(ppm, DMSO-d ): H-NMR
δ
(ppm, 400
), 1.99–2.03 (m, 1H, CH2 ),
δ), 3.34–3.39 (m, 2H, CH2 β), 3.88 (s, 3H, OCH Phenyl), 4.02 (s, 3H, OCH
35
39
5
6
6
MHz, DMSO-d ): 1.52 (s, 9H, 3
×
CH OtBu), 1.77–1.81 (m, 2H, CH2
γ
δ
6
3
2.10–2.13 (m, 1H, CH2
3
3
Phenyl), 5.12 (s, 2H, CH -Phenyl), 5.37–5.39 (m, 1H, CH
1
α
), 6.99 (d, 2H, J = 4 Hz, CH Phenyl), 7.03 (s,
H, CH Phenyl), 7.10–7.12 (m, 1H, CH 7), 7.28 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.32–7.35 (m, 7H, CH
, CH2, CH Phenyl), 7.36 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.47 (d, 1H, J = 4 Hz, NH-CH ), 7.76 (dd,
2
8
δ
2
1
3
1
H, J = 4 Hz, J = 8 Hz, NH-CH
CH2 ), 40.61 (CH2 ), 54.42 (CH
CH Phenyl), 114.69 (Cq 1), 115.99 (CH 6), 123.16 (CH 7), 126.40 (Cq 5a), 126.82 (CH 9), 126.87 (CH 8),
28.01 (CH Phenyl), 128.08 (CH Phenyl), 129.49 (CH Phenyl), 131.85 (CH 2), 132.87 (Cq 3a), 136.68 (Cq
α
). C-NMR
δ (ppm, DMSO-d ): 25.62 (CH2 γ), 28.09 (CH tBu), 30.04
6
3
(
(
1
β
δ
α
), 66.58 (CH -Phenyl), 82.42 (Cq tBu), 111.30 (CH Phenyl), 113.24
2
9
a), 149.16 (Cq 4), 150.12 (Cq Phenyl), 152.37 (Cq Phenyl), 156.51 (Cq Phenyl), 171.59 (C=O). MS (ESI +,
+
QTof, m/z): 626.0 [M + H] . HRMS calculated for C H N O 626.2979, found 626.2982.
35
40
5
6
Tert-butyl 2-((Tert-butoxycarbonyl)amino)-5-((1-(3,4-dimethoxyphenyl)imidazo[1,2-a]-quinoxalin-4-yl)amino)
pentanoate (8i): Following the same procedure used for the synthesis of 8a, to a mixture of 7i (0.515 g,
0
.96 mmol) in DME/H O (2/1, 15 mL) were added 3,4-dimethoxyphenylboronic acid (0.193 g,
2
1.06 mmol), tetrakis(triphenylphosphine) palladium (0.056 g, 0.048 mmol) and sodium carbonate
(0.204 g, 1.93 mmol) in a microwave-adapted vial. The product was purified by flash chromatography
eluted with cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as a white solid
1
(
45% yield). C H N O . MW: 591.70 g/mol. H-NMR
δ
(ppm, DMSO-d ): 1.34 (s, 9H, CH-COOtBu),
32
41
5
6
6
1
δ
.39 (s, 9H, NH-COOtBu), 1.73–1.74 (m, 2H, CH2
β
), 1.77–1.78 (m, 2H, CH2
γ
), 3.56–3.59 (m, 2H, CH2
), 6.95–7.00 (m, 1H,
CH 7), 7.09 (dd, 1H, J = 4 Hz, J = 8 Hz, CH Phenyl), 7.14 (s, 1H, CH Phenyl), 7.15 (dd, 1H, J = 4 Hz, J = 8
Hz, CH Phenyl), 7.19 (d, 1H, J = 4 Hz, NH-CH ), 7.28 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.30–7.32 (m,
H, CH 8), 7.47 (s, 1H, CH 2), 7.58 (dd, 1H, J = 4 Hz, J = 8 Hz CH 9), 7.72 (t, 1H, J = 4 Hz, NH-CH2 ).
), 39.79
), 56.05 (OCH Phenyl), 56.15 (OCH Phenyl), 78.46 (Cq tBu), 80.54 (Cq tBu),
), 3.74 (s, 3H, OCH Phenyl), 3.87 (s, 3H, OCH Phenyl), 3.89–3.92 (m, 1H, CH
α
3
3
α
1
δ
13
C-NMR
δ (ppm, DMSO-d ): 25.97 (CH2 β), 28.06 (CH tBu), 28.25 (CH tBu), 28.67 (CH γ
6 3 3 2
(CH2 ), 54.77 (CH α
δ
3
3
112.32 (CH Phenyl), 114.22 (CH Phenyl), 115.83 (CH 6), 122.12 (CH 7), 122.82 (CH Phenyl), 123.26 (Cq
1
), 125.75 (Cq 5a), 126.41 (CH 8), 127.07 (CH 9), 132.32 (CH 2), 138.24 (Cq 3a), 148.04 (Cq 4), 149.26
+
(Cq Phenyl), 150.10 (Cq Phenyl), 156.01 (Cq 9a), 172.36 (C=O). MS (ESI +, QTof, m/z): 592.1 [M + H] .
HRMS calculated for C H N O 592.3135, found 592.3137.
32
42
5
6
Tert-butyl 2-((1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)acetate (9a): To a mixture of 7a
0.320 g, 0.85 mmol) in DME/H O (2/1, 15 mL) were added compound 12 (0.392 g, 2.54 mmol),
(
2
tetrakis(triphenylphosphine) palladium (0.049 g, 0.040 mmol) and sodium carbonate (0.179 g,
1
.70 mmol) in a microwave-adapted vial. The reaction was submitted to microwave irradiations
◦
during 20 min at 150 C and then filtered on a Celite pad. The filtrate was concentrated under reduced
pressure and successively washed with saturated aqueous ammonium chloride, saturated aqueous
sodium bicarbonate, distilled water and finally brine. The organic phase was dried on sodium sulfate,
filtered and concentrated under reduced pressure. The product was purified by flash chromatography
eluted with cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as a white solid (16%
1
yield). C H N O . MW: 406.43 g/mol. 1H-NMR
δ
(ppm, DMSO-d ): H-NMR
δ
(ppm, DMSO-d6):
22
22
4
4
6
1
6
1
.42 (s, 9H, 3
×
CH OtBu), 4.15 (d, 2H, J = 8 Hz, CH2
α
), 6.82 (dd, 1H, J = 4 Hz, J = 8 Hz, CH Phenyl),
.89 (dd, 1H, J = 4 Hz, J = 8 Hz, CH Phenyl), 6.93 (s, 1H, CH Phenyl), 7.02–7.06 (m, 1H, CH 7), 7.33 (dd,
H, J = 4 Hz, J = 8 Hz, CH 6), 7.35–7.38 (m, 1H, CH 8), 7.46 (s, 1H, CH 2), 7.54 (dd, 1H, J = 4 Hz, J = 8 Hz,
CH 9), 7.96–8.00 (m, 1H, NH), 9.32 (s, 1H, C-OH Phenyl), 9.41 (s, 1H, C-OH Phenyl). 13C-NMR
(ppm,
DMSO-d ): 28.23 (CH tBu), 43.30 (CH ), 80.85 (Cq tBu), 115.37 (Cq 1), 115.92 (CH 6), 116.48 (CH
), 117.89 (CH Phenyl), 121.13 (CH Phenyl), 122.06 (CH 8), 122.81 (CH Phenyl), 125.98 (Cq 5a), 126.54
3
δ
α
2
6
3
7

Molecules 2018, 23, 2987
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(
(
CH 9), 127.77 (Cq Phenyl), 131.43 (Cq 3a), 132.36 (CH 2), 132.89 (Cq 9a), 146.04 (Cq Phenyl), 147.13
Cq Phenyl), 147.65 (Cq 4), 169.76 (C=O). MS (ESI +, QTof, m/z): 407.2 [M + H] . HRMS calculated for
+
C H N O 407.1706, found 407.1710.
22
23
4
4
Tert-butyl 2-((1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)propanoate (9b): Following the
same procedure used for the synthesis of 9a (see 4.1.4.8.), to a mixture of 7b (0.290 g, 0.74 mmol) in
DME/H O (2/1, 15 mL) were added compound 12 (0.342 g, 2.22 mmol), tetrakis- (triphenylphosphine)
2
palladium (0.043 g, 0.037 mmol) and sodium carbonate (0.157 g, 1.48 mmol) in a microwave-adapted
vial. The product was purified by flash chromatography eluted with cyclohexane/ethyl acetate 80/20
to 50/50. The compound is obtained as a white solid (6% yield). C H N O . MW: 420.46 g/mol.
2
3
24
4
4
1
H-NMR
H, CH
.01–7.05 (m, 1H, CH 7), 7.30 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.33–7.37 (m, 1H, CH 8), 7.45 (s, 1H,
CH 2), 7.53 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.66 (d, 1H, J = 4 Hz, NH), 9.36 (s, 2H, C-OH Phenyl).
δ
(ppm, DMSO-d ): 1.42 (s, 9H, 3
×
CH OtBu), 1.51 (d, 3H, J = 8 Hz, CH3
β), 4.59–4.63 (m,
6
3
1
7
α), 6.81 (d, 1H, J = 4 Hz, CH Phenyl), 6.90 (d, 1H, J = 4 Hz, CH Phenyl), 6.93 (s, 1H, CH Phenyl),
13
C-NMR
δ (ppm, DMSO-d ): 17.57 (CH3 β), 28.16 (CH tBu), 50.17 (CH α), 80.66 (Cq tBu), 115.87 (CH
6 3
8
), 116.47 (CH Phenyl), 117.88 (CH Phenyl), 121.16 (Cq 1), 122.05 (CH Phenyl), 122.78 (CH 7), 126.02
(
1
Cq 5a), 127.16 (CH 9), 131.35 (Cq 3a), 132.19 (CH 2), 132.80 (Cq 9a), 137.57 (Cq 4), 146.03 (Cq Phenyl),
47.10 (Cq Phenyl), 147.23 (Cq Phenyl), 172.87 (C=O). MS (ESI +, QTof, m/z): 421.2 [M + H] . HRMS
+
calculated for C H N O 421.1876, found 421.1875.
23
25
4
4
Tert-butyl 2-((1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)-3-methyl-butanoate
Following the same procedure used for the synthesis of 9a (see 4.1.4.8.), to a mixture of 7c (0.380 g,
.91 mmol) in DME/H O (2/1, 15 mL) were added compound 12 (0.340 g, 2.72 mmol), tetrakis-
(9c):
0
2
(triphenylphosphine) palladium (0.052 g, 0.045 mmol) and sodium carbonate (0.192 g, 1.81 mmol)
in a microwave-adapted vial. The product was purified by flash chromatography eluted with
cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as a white solid (32% yield).
1
C H N O . MW: 448.51 g/mol. H-NMR
δ
(ppm, DMSO-d ): 1.03 (d, 3H, J = 8 Hz, CH3
γ
), 1.06 (d,
), 4.53 (t, 1H, J = 8 Hz, CH ),
.81 (dd, 1H, J = 4 Hz, J = 8 Hz, CH Phenyl), 6.90 (d, 1H, J = 4 Hz, J = 8 Hz, CH Phenyl), 6.93 (d, 1H,
25
28
4
4
6
3
H, J = 8 Hz, CH3
γ
’), 1.44 (s, 9H, 3
×
CH OtBu), 2.31–2.35 (m, 1H, CH
β
α
3
6
J = 4 Hz, J = 8 Hz, CH Phenyl), 7.03–7.05 (m, 1H, CH 7), 7.07 (d, 1H, J = 4 Hz, NH), 7.34–7.35 (m, 1H,
CH 8), 7.37 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.46 (s, 1H, CH 2), 7.56 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9),
9
.32 (s, 1H, C-OH Phenyl), 9.41 (s, 1H, C-OH Phenyl). 13C-NMR
δ
(ppm, DMSO-d ): 19.30 (CH3
γ
’),
), 81.25 (Cq tBu), 115.90 (CH 6), 116.47 (CH
Phenyl), 117.89 (CH Phenyl), 121.05 (Cq 1), 122.07 (CH Phenyl), 123.07 (CH 7), 126.11 (Cq 5a), 126.56
6
19.47 (CH3 γ), 28.19 (CH tBu), 30.54 (CH β), 59.60 (CH α
3
(
1
CH 8), 127.28 (CH 9), 131.55 (Cq Phenyl), 132.24 (CH 2), 132.72 (Cq 3a), 137.43 (Cq 9a), 146.03 (Cq 4),
47.14 (Cq Phenyl), 147.47 (Cq Phenyl), 171.62 (C=O). MS (ESI +, QTof, m/z): 449.3 [M + H] . HRMS
+
calculated for C H N O 449.2189, found 449.2188.
25
29
4
4
Tert-butyl 2-((1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)-4-methyl-pentanoate
Following the same procedure used for the synthesis of 9a (see 4.1.4.8.), to a mixture of 7d (0.460 g,
.06 mmol) in DME/H O (2/1, 15 mL) were added compound 12 (0.400 g, 2.6 mmol), tetrakis-
(9d):
1
2
(triphenylphosphine) palladium (0.062 g, 0.053 mmol) and sodium carbonate (0.224 g, 2.11 mmol)
in a microwave-adapted vial. The product was purified by flash chromatography eluted with
cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as a white solid (16% yield).
1
C H N O . MW: 462.54 g/mol. H-NMR
δ
(ppm, DMSO-d ): 0.93 (d, 3H, J = 8 Hz, CH3
δ
), 0.97 (d,
), 1.79–1.83 (m, 1H, CH ),
), 6.81 (d, 1H, J = 4 Hz, CH Phenyl), 6.86 (s, 1H,
CH Phenyl), 6.90 (d, 1H, J = 4 Hz, CH Phenyl), 7.01–7.05 (m, 1H, CH 7), 7.28–7.32 (m, 1H, CH 8), 7.34
dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.46 (s, 1H, CH 2), 7.55 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.59 (d, 1H,
J = 4 Hz, NH), 9.38 (s, 2H, C-OH Phenyl). 13C-NMR
(ppm, DMSO-d ): 22.09 (CH3 ), 23.32 (CH3
’), 25.17 (CH ), 28.18 (CH tBu), 39.60 (CH2 ), 52.88 (CH ), 80.76 (Cq tBu), 115.87 (CH 6), 116.48
CH Phenyl), 117.90 (CH Phenyl), 121.15 (Cq 1), 122.18 (CH Phenyl), 122.79 (CH 7), 126.11 (Cq 5a),
26
30
4
4
6
3
H, J = 8 Hz, CH3
δ
’), 1.42 (s, 9H, 3
×
CH OtBu), 1.56–1.60 (m, 1H, CH2
β
γ
3
1.91–1.95 (m, 1H, CH2
β
), 4.65 (t, 1H, J = 4 Hz, CH α
(
δ
δ
6
δ
γ
β
α
3
(

Molecules 2018, 23, 2987
18 of 26
1
25.80 (CH 8), 126.04 (CH 9), 131.42 (Cq 3a), 132.18 (CH 2), 137.58 (Cq 9a), 146.06 (Cq 4), 147.14 (Cq
+
Phenyl), 147.57 (Cq Phenyl), 172.77 (C=O). MS (ESI +, QTof, m/z): 463.2 [M + H] . HRMS calculated for
C H N O 463.2345, found 463.2343.
26
31
4
4
Tert-butyl 6-((Tert-butoxycarbonyl)amino)-2-((1-(3,4-dihydroxyphenyl)imidazo[1,2-a]-quinoxalin-4-yl)amino)
hexanoate (9e): Following the same procedure used for the synthesis of 9a (see 4.1.4.8.), to a mixture
of 7e (0.242 g, 0.44 mmol) in DME/H O (2/1, 15 mL) were added compound 12 (0.170 g, 1.10 mmol),
2
tetrakis- (triphenylphosphine) palladium (0.025 g, 0.022 mmol) and sodium carbonate (0.093 g,
0.88 mmol) in a microwave-adapted vial. The product was purified by flash chromatography eluted
with cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as a white solid (40%
1
yield). C H N O . MW: 577.67 g/mol. H-NMR
δ
(ppm, DMSO-d ): 1.35 (s, 9H, 3
×
CH OtBu),
3
31
39
5
6
6
1
β
.40–1.42 (m, 2H, CH2
), 2.92–2.94 (m, 2H, CH2
J = 4 Hz, CH Phenyl), 6.89 (s, 1H, CH Phenyl), 6.93 (d, 1H, J = 4 Hz, CH Phenyl), 7.02–7.05 (m, 1H, CH
), 7.31–7.33 (m, 1H, CH 8), 7.36 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.45 (s, 1H, CH 2), 7.54 (dd, 1H, J = 4
Hz, J = 8 Hz, CH 9), 7.57 (d, 1H, J = 4 Hz, NH-CH ), 9.33 (s, 1H, C-OH Phenyl), 9.41 (s, 1H, C-OH
Phenyl). 13C-NMR
(ppm, DMSO-d ): 22.40 (CH2 ), 27.11 (CH tBu), 27.65 (CH tBu), 28.56 (CH ),
9.91 (CH2 ), 39.25 (CH2
Phenyl), 116.80 (CH Phenyl), 120.07 (CH Phenyl), 121.73 (CH 7), 124.97 (Cq 5a), 125.42 (CH 8), 126.12
γ
), 1.43 (s, 9H, 3
×
CH OtBu), 1.45–1.47 (m, 2H, CH2 δ), 1.88–2.00 (m, 2H, CH
3
2
ε
), 4.55–4.56 (m, 1H, CH
α), 6.79 (t, 1H, J = 4 Hz, NH-CH2 ε), 6.83 (d, 1H,
7
α
δ
γ
δ
2
6
3
3
2
β
ε), 53.44 (CH α), 76.70 (Cq tBu), 79.75 (Cq tBu), 114.79 (CH 6), 115.40 (CH
(
1
CH 9), 130.32 (Cq 1), 131.09 (CH 2), 131.70 (Cq 3a), 136.49 (Cq 9a), 144.96 (Cq 4), 146.04 (Cq Phenyl),
46.43 (Cq Phenyl), 154.96 (Cq Phenyl), 172.41 (C=O). MS (ESI +, QTof, m/z): 578.3 [M + H] . HRMS
+
calculated for C H N O 578.2979, found 578.2980.
31
40
5
6
Tert-butyl 5-(((benzyloxy)carbonyl)amino)-2-((1-(3,4-dihydroxyphenyl)imidazo[1,2-a]-quinoxalin-4-yl)amino)
pentanoate (9f): Following the same procedure used for the synthesis of 9a (see 4.1.4.8.), to a mixture
of 7f (0.260 g, 0.46 mmol) in DME/H O (2/1, 15 mL) were added compound 12 (0.211 g, 1.37 mmol),
2
tetrakis- (triphenylphosphine) palladium (0.026 g, 0.023 mmol) and sodium carbonate (0.097 g,
0.91 mmol) in a microwave-adapted vial. The product was purified by flash chromatography eluted
with cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as a white solid (26%
1
1
yield). C H N O . MW: 597.66 g/mol. H-NMR
δ
(ppm, DMSO-d ): H-NMR
δ
(ppm, 400 MHz,
), 1.91–1.95 (m, 2H, CH2 ), 3.05–3.08
α), 5.01 (s, 2H, CH -Phenyl), 6.81 (d, 1H, J = 4 Hz, CH Phenyl),
33
35
5
6
6
DMSO-d ): 1.40 (s, 9H, 3
×
CH OtBu), 1.58–1.60 (m, 2H, CH2
γ
β
6
3
(m, 2H, CH2
δ
), 4.55–4.59 (m, 1H, CH
2
6
.89 (s, 1H, CH Phenyl), 6.93 (d, 1H, J = 4 Hz, CH Phenyl), 7.01–7.06 (m, 1H, CH 7), 7.27 (d, 1H, J = 4
Hz, NH-CH2
δ
), 7.31–7.34 (m, 5H, CH Phenyl), 7.37 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.45 (s, 1H, CH
), 7.56–7.60 (m, 1H, CH 8), 7.63 (dd, 1H, J = J = 4 Hz, J = 8 Hz, CH 9).
(ppm, DMSO-d ): 26.54 (CH2 ), 28.09 (CH2 ), 28.17 (CH tBu), 40.45 (CH2 ), 54.34 (CH
), 65.60 (CH -Phenyl), 80.92 (Cq tBu), 115.88 (CH 6), 116.48 (CH Phenyl), 117.90 (CH Phenyl), 121.16
2
), 7.54 (d, 1H, J = 4 Hz, NH-CH α
1
3
C-NMR
δ
γ
β
δ
6
3
α
2
(Cq 1), 122.84 (CH 7), 126.06 (Cq 5a), 126.50 (CH 9), 127.20 (CH 8), 128.18 (CH Phenyl), 128.79 (CH
Phenyl), 129.16 (CH Phenyl), 131.90 (CH 2), 132.79 (Cq 3a), 137.55 (Cq 9a), 146.04 (Cq 4), 147.12 (Cq
+
Phenyl), 147.47 (Cq Phenyl), 156.59 (Cq Phenyl), 172.23 (C=O). MS (ESI +, QTof, m/z): 598.3 [M + H] .
HRMS calculated for C H N O 598.2666, found 598.2670.
33
36
5
6
Tert-butyl 2-((1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)-3-phenyl-propanoate
Following the same procedure used for the synthesis of 9a (see 4.1.4.8.), to a mixture of 7g (0.360 g,
.77 mmol) in DME/H O (2/1, 15 mL) were added compound 12 (0.356 g, 2.31 mmol), tetrakis-
(9g):
0
2
(triphenylphosphine) palladium (0.044 g, 0.038 mmol) and sodium carbonate (0.163 g, 1.54 mmol)
in a microwave-adapted vial. The product was purified by flash chromatography eluted with
cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as a white solid (16% yield).
1
C H N O . MW: 496.56 g/mol. H-NMR
(
δ
(ppm, DMSO-d ): 1.36 (s, 9H, 3
×
CH OtBu), 3.23–3.27
3
29
28
4
4
6
m, 1H, CH2 β), 3.33–3.38 (m, 1H, CH2 β), 4.91–4.93 (m, 1H, CH α), 6.81 (dd, 1H, J = 4 Hz, J = 8 Hz, CH
Phenyl), 6.89 (d, 1H, J = 4 Hz, CH Phenyl), 6.92 (d, 1H, J = 8 Hz, CH Phenyl), 7.08 (t, 1H, J = 8 Hz, CH
Phenyl), 7.20 (t, 1H, J = 8 Hz, CH Phenyl), 7.29 (t, 2H, J = 8 Hz, CH Phenyl), 7.34 (d, 2H, J = 8 Hz, CH 6,

Molecules 2018, 23, 2987
19 of 26
CH Phenyl), 7.34–7.37 (m, 1H, CH 7), 7.38–7.40 (m, 1H, CH 8), 7.52 (s, 1H, CH 2), 7.60 (d, 1H, J = 8
Hz, CH 9), 7.92–7.93 (m, 1H, NH), 9.38 (s, 2H, C-OH Phenyl). 13C-NMR
(ppm, DMSO-d ): 26.98
CH tBu), 35.93 (CH2 ), 55.04 (CH ), 80.23 (Cq tBu), 114.96 (CH 6), 115.42 (CH Phenyl), 116.75 (CH
Phenyl), 119.67 (Cq 1), 120.96 (CH Phenyl), 122.32 (CH 7), 124.69 (Cq 5a), 125.73 (CH Phenyl, CH 9),
25.94 (CH Phenyl), 127.90 (CH Phenyl), 128.71 (CH 8), 130.76 (Cq Phenyl), 131.11 (CH 2), 131.21 (Cq
δ
6
(
β
α
3
1
Phenyl), 136.87 (Cq 3a), 138.12 (Cq 9a), 146.17 (Cq 4), 170.13 (C=O). MS (ESI +, QTof, m/z): 497.1 [M +
+
H] . HRMS calculated for C H N O 497.2189, found 497.2198.
29
29
4
4
Tert-butyl
propanoate (9h): Following the same procedure used for the synthesis of 9a (see 4.1.4.8.), to a mixture of
(0.260 g, 0.57 mmol) in DME/H O (2/1, 15 mL) were added compound 12 (0.170 g, 1.72 mmol),
3-(4-(Tert-butoxy)phenyl)-2-((1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)
7
h
2
tetrakis- (triphenylphosphine) palladium (0.028 g, 0.028 mmol) and sodium carbonate (0.101 g,
.15 mmol) in a microwave-adapted vial. The product was purified by flash chromatography eluted
1
with cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as a white solid (22%
1
yield). C H N O . MW: 568.66 g/mol. H-NMR
δ
(ppm, DMSO-d ): 1.24 (s, 9H, 3
×
CH OtBu),
3
33
36
4
5
6
1.33 (s, 9H, 3
×
CH OtBu), 3.12–3.18 (m, 1H, CH2
CH α), 6.80 (d, 1H, J = 8 Hz, CH Phenyl), 6.88 (s, 1H, CH Phenyl), 6.90 (dd, 2H, J = 4 Hz, J = 8 Hz,
β
), 3.26–3.32 (m, 1H, CH2
β), 4.80–4.84 (m, 1H,
3
CH Phenyl), 6.92 (d, 1H, J = 8 Hz, CH Phenyl), 7.01–7.05 (m, 1H, CH 7), 7.25 (d, 2H, J = 8 Hz, CH
Phenyl), 7.30–7.32 (m, 1H, CH 8), 7.34 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.45 (s, 1H, CH 2), 7.54 (dd,
1
H, J = 4 Hz, J = 8 Hz, CH 9), 7.57 (d, 1H, J = 8 Hz, NH), 9.31 (s, 1H, C-OH Phenyl), 9.40 (s, 1H, C-OH
Phenyl). 13C-NMR
(ppm, DMSO-d ): 28.05 (CH tBu), 28.95 (CH tBu), 36.64 (CH ), 55.87 (CH ),
8.12 (Cq tBu), 80.91 (Cq tBu), 115.90 (CH 6), 116.46 (CH Phenyl), 117.87 (CH Phenyl), 121.08 (Cq 1),
22.05 (CH Phenyl), 122.91 (CH 7), 124.00 (2 CH Phenyl), 126.07 (Cq 5a), 126.49 (CH 8), 127.21 (CH
), 130.33 (2 CH Phenyl), 132.26 (CH 2), 132.62 (Cq 3a), 137.46 (Cq 9a), 146.02 (Cq 4), 147.11 (Cq
δ
β
2
α
6
3
3
7
1
9
×
×
Phenyl), 147.22 (Cq Phenyl), 154.05 (2
H] . HRMS calculated for C H N O 569.2764, found 569.2773.
×
Cq Phenyl), 171.75 (C=O). MS (ESI +, QTof, m/z): 569.3 [M +
+
33
37
4
5
Tert-butyl 2-((Tert-butoxycarbonyl)amino)-5-((1-(3,4-dihydroxyphenyl)imidazo[1,2-a]-quinoxalin-4-yl)amino)
pentanoate (9i): Following the same procedure used for the synthesis of 9a (see 4.1.4.8.), to a mixture
of 7i (0.460 g, 0.86 mmol) in DME/H O (2/1, 15 mL) were added compound 12 (0.330 g, 2.15 mmol),
2
tetrakis- (triphenylphosphine) palladium (0.050 g, 0.043 mmol) and sodium carbonate (0.182 g,
1.72 mmol) in a microwave-adapted vial. The product was purified by flash chromatography eluted
with cyclohexane/ethyl acetate 80/20 to 50/50. The compound is obtained as a white solid (30% yield).
1
C H N O . MW: 563.64 g/mol. H-NMR
δ
(ppm, DMSO-d ): 1.33 (s, 9H, CH-COOtBu), 1.39 (s, 9H,
30
37
5
6
6
NH-COOtBu), 1.66–1.72 (m, 4H, CH2
β, CH2 γ), 3.56–3.57 (m, 2H, CH2 δ), 3.89–3.93 (m, 1H, CH α),
6
.79 (dd, 1H, J = 4 Hz, J = 8 Hz, CH Phenyl), 6.87 (d, 1H, J = 4 Hz, CH Phenyl), 6.90 (d, 1H, J = 8 Hz,
CH Phenyl), 6.95–7.00 (m, 1H, CH 7), 7.17 (d, 1H, J = 8 Hz, NH-CH ), 7.27–7.29 (m, 1H, CH 8), 7.31
dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.40 (s, 1H, CH 2), 7.56 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.69 (t, 1H,
J = 4 Hz, NH-CH2 (ppm, DMSO-d ): 26.80 (CH2 ), 28.04
), 9.35 (s, 2H, C-OH Phenyl). 13C-NMR
CH tBu), 28.67 (CH tBu), 30.42 (CH γ), 39.79 (CH δ), 54.78 (CH α), 78.46 (Cq tBu), 80.53 (Cq tBu),
α
(
δ
δ
β
6
(
3
3
2
2
115.79 (CH 6), 116.45 (CH Phenyl), 117.86 (CH Phenyl), 121.29 (Cq 1), 122.01 (CH Phenyl), 122.06 (CH
7
), 125.76 (Cq 5a), 126.29 (CH 8), 127.03 (CH 9), 131.91 (CH 2), 138.20 (Cq 3a), 146.02 (Cq 4), 147.05
+
(Cq Phenyl), 148.03 (Cq Phenyl), 156.02 (Cq 9a), 172.34 (C=O). MS (ESI +, QTof, m/z): 564.3 [M + H] .
HRMS calculated for C H N O 564.2822, found 564.2827.
30
38
5
6
3
.1.5. Cleavage of the Protective Groups
◦
2
-((1-(3,4-Dimethoxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)propanoic acid (10b): To a cooled (0 C)
solution of 8b (0.170 g, 0.36 mmol) in anhydrous CH Cl (20 mL) was added boron tribromide (2.1 mL,
2
2
2
.1 mmol). The resulting solution was allowed to warm to room temperature and stirred until complete
consumption of the starting material (1–3 h, monitored by TLC). The solution was neutralized by
addition of saturated aqueous sodium bicarbonate (20 mL). The crude mixture was extracted with

Molecules 2018, 23, 2987
20 of 26
CH Cl (3
×
20 mL). The organic phase was dried on sodium sulfate, filtered and concentrated under
2
2
reduced pressure. The product was purified by flash chromatography eluted with cyclohexane/ethyl
acetate 90/20 to 40/50. The compound is obtained as a white solid (71% yield). C H N O . MW:
2
1
20
4
4
1
3
92.41 g/mol. H-NMR
δ
(ppm, DMSO-d ): 1.24 (s, 1H, COOH), 1.53 (d, 3H, J = 8 Hz, CH3
β
), 3.75 (s,
6
3
H, OCH Phenyl), 3.87 (s, 3H, OCH Phenyl), 4.61–4.63 (m, 1H, CH
α
), 7.00–7.04 (m, 1H, CH 7), 7.11
3
3
(d, 1H, J = 4 Hz, CH Phenyl), 7.13 (s, 1H, CH Phenyl), 7.17 (d, 1H, J = 4 Hz, CH Phenyl), 7.31 (dd, 1H,
J = 4 Hz, J = 8 Hz, CH 6), 7.33–7.35 (m, 1H, CH 8), 7.50 (s, 1H, CH 2), 7.52 (d, 1H, J = 4 Hz, NH), 7.60
(
(
(
dd, 1H, J = 4 Hz, J = 8 Hz, CH 9). 13C-NMR
OCH Phenyl), 56.16 (OCH Phenyl), 112.31 (CH Phenyl), 114.22 (CH Phenyl), 115.92 (CH 6), 122.54
CH 7), 122.70 (Cq 1), 123.28 (CH Phenyl), 126.54 (CH 8), 125.90 (Cq 5a), 127.21 (CH 9), 130.95 (Cq 3a),
δ (ppm, DMSO-d ): 18.71 (CH3 β), 50.30 (CH α), 56.07
6
3
3
1
32.63 (CH 2), 137.99 (Cq 9a), 147.05 (Cq 4), 149.26 (Cq Phenyl), 150.132 (Cq Phenyl), 172.96 (C=O). MS
+
(ESI +, QTof, m/z): 393.0 [M + H] . HRMS calculated for C H N O 393.1563, found 393.1558.
21
21
4
4
2
-((1-(3,4-Dimethoxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)-3-methylbutanoic acid (10c): Following
the same procedure used for the synthesis of 10b, to a cooled solution of 8c (0.290 g, 0.61 mmol) in
anhydrous CH Cl (20 mL) was added boron tribromide (3.6 mL, 3.6 mmol). The product was purified
2
2
by flash chromatography eluted with cyclohexane/ethyl acetate 90/20 to 40/50. The compound is
1
obtained as a white solid (80% yield). C H N O . MW: 420.46 g/mol. H-NMR
δ
(ppm, DMSO-d6):
23
24
4
4
0
β
.99 (d, 3H, J = 8 Hz, CH3
γ
), 1.01 (d, 3H, J = 8 Hz, CH3
γ
’), 1.23 (s, 1H, COOH), 2.36–2.41 (m, 1H, CH
), 6.97–7.01 (m,
H, CH 7), 7.10 (dd, 1H, J = 4 Hz, J = 8 Hz, CH Phenyl), 7.14 (s, 1H, CH Phenyl), 7.16 (dd, 1H, J = 4 Hz,
J = 8 Hz, CH Phenyl), 7.19 (d, 1H, NH-CH ), 7.28 (d, 1H, J = 4 Hz, CH 6), 7.29–7.32 (m, 1H, CH 8),
.48 (s, 1H, CH 2), 7.54 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9). 13C-NMR
(ppm, DMSO-d ): 19.02 (CH
’), 19.96 (CH3 ), 31.19 (CH ), 56.06 (OCH Phenyl), 56.16 (OCH Phenyl), 59.54 (CH
Phenyl), 114.26 (CH Phenyl), 115.87 (CH 6), 122.35 (CH 7), 122.75 (Cq 1), 123.29 (CH Phenyl), 125.89
Cq 5a), 126.49 (CH 8), 127.18 (CH 9), 130.95 (Cq Phenyl), 132.44 (CH 2), 133.40 (Cq 3a), 138.10 (Cq 9a),
), 3.74 (s, 3H, OCH Phenyl), 3.87 (s, 3H, OCH Phenyl), 4.55 (t, 1H, J = 16 Hz, CH
α
3
3
1
α
7
γ
δ
6
3
γ
β
α), 112.31 (CH
3
3
(
1
47.60 (Cq 4), 149.25 (Cq Phenyl), 150.12 (Cq Phenyl), 170.00 (C=O). MS (ESI +, QTof, m/z): 420.9 [M +
+
H] . HRMS calculated for C H N O 421.1876, found 421.1873.
23
25
4
4
2
-((1-(3,4-Dimethoxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)-4-methylpentanoic acid (10d): Following
the same procedure used for the synthesis of 10b, to a cooled solution of 8d (0.210 g, 0.43 mmol)
in anhydrous CH Cl (20 mL) was added boron tribromide (2.5 mL, 2.53.6 mmol). The product
2
2
was purified by flash chromatography eluted with cyclohexane/ethyl acetate 90/20 to 40/50. The
1
compound is obtained as a white solid (76% yield). C H N O . MW: 434.49 g/mol. H-NMR
δ
24
26
4
4
(
ppm, DMSO-d ): 0.95 (d, 3H, J = 8 Hz, CH3
δ
), 0.98 (d, 3H, J = 8 Hz, CH3
δ
’), 1.76–1.80 (m, 3H, CH2
), 6.98–7.02 (m,
H, CH 7), 7.11 (d, 1H, J = 4 Hz, CH Phenyl), 7.13 (s, 1H, CH Phenyl), 7.17 (d, 1H, J = 4 Hz, CH Phenyl),
.29 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.31–7.33 (m, 1H, CH 8), 7.44 (d, 1H, J = 4 Hz, NH), 7.49 (s,
H, CH 2), 7.56 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9). 13C-NMR
(ppm, DMSO-d ): 22.68 (CH3 ), 23.69
CH3 ’), 25.24 (CH ), 40.42 (CH2 ), 53.00 (CH ), 56.05 (OCH Phenyl), 56.15 (OCH Phenyl), 112.30
CH Phenyl), 114.24 (CH Phenyl), 115.88 (CH 6), 122.29 (CH 7), 122.77 (Cq 1), 123.27 (CH Phenyl),
β,
6
CH
1
7
1
(
(
γ
), 3.74 (s, 3H, OCH Phenyl), 3.87 (s, 3H, OCH Phenyl), 4.66 (t, 1H, J = 4 Hz, CH α
3
3
δ
δ
6
δ
γ
β
α
3
3
1
1
25.86 (Cq 5a), 126.49 (CH 8), 127.13 (CH 9), 130.90 (Cq 3a), 132.42 (CH 2), 138.12 (Cq 9a), 147.44 (Cq 4),
49.25 (Cq Phenyl), 150.10 (Cq Phenyl), 172.31 (C=O). MS (ESI +, QTof, m/z): 434.9 [M + H] . HRMS
+
calculated for C H N O 435.2032, found 435.2032.
24
27
4
4
◦
2
-((1-(3,4-Dihydroxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)acetic acid (11a): To a cooled (0 C)
solution of 9a (0.055, 0.14 mmol) in anhydrous CH Cl (10 mL) was added TFA (5 mL). The resulting
2
2
solution was allowed to warm to room temperature and stirred until complete consumption of the
starting material (1–2 h, monitored by TLC). The solvent was removed under reduced pressure. The
product was purified by flash chromatography eluted with cyclohexane/ethyl acetate 90/20 to 40/50.
1
The compound is obtained as a white solid (92% yield). C H N O . MW: 350.33 g/mol. H-NMR
δ
18
14
4
4
(
ppm, DMSO-d ): 1.23 (s, 1H, COOH), 4.24 (d, 2H, J = 8 Hz, CH2 α), 6.82 (dd, 1H, J = 4 Hz, J = 8 Hz,
6

Molecules 2018, 23, 2987
21 of 26
CH Phenyl), 6.90 (dd, 1H, J = 4 Hz, J = 8 Hz, CH Phenyl), 6.93 (s, 1H, CH Phenyl), 7.03–7.07 (m, 1H,
CH 7), 7.31–7.34 (m, 1H, CH 8), 7.35 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.48 (s, 1H, CH 2), 7.58 (dd, 1H,
J = 4 Hz, J = 8 Hz, CH 9), 8.02–8.05 (m, 1H, NH), 9.39–9.43 (m, 2H, C-OH Phenyl). 13C-NMR
δ
(ppm,
DMSO-d ): 42.37 (CH2
α), 115.97 (CH 6), 116.49 (CH Phenyl), 117.86 (CH Phenyl), 120.69 (Cq 1), 122.06
6
(
1
CH Phenyl), 122.96 (CH 7), 126.13 (Cq 5a), 126.51 (CH 8), 126.62 (CH 9), 132.36 (CH 2), 146.01 (Cq 3a),
47.17 (Cq 9a), 147.56 (Cq 4), 152.74 (Cq Phenyl), 172.09 (C=O). MS (ESI +, QTof, m/z): 351.2 [M + H] .
+
HRMS calculated for C H N O 351.1093, found 351.1093.
18
15
4
4
2
-((1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)propanoic acid (11b): Following the same
◦
procedure for the synthesis of 11a, to a cooled (0 C) solution of 9b (0.040, 0.09 mmol) in anhydrous
CH Cl (10 mL) was added TFA (5 mL). The product was purified by flash chromatography eluted
2
2
with cyclohexane/ethyl acetate 90/20 to 40/50. The compound is obtained as a white solid (88% yield).
1
C H N O . MW: 364.35 g/mol. H-NMR
δ
(ppm, DMSO-d ): 1.23 (s, 1H, COOH), 1.56 (d, 3H, J = 8
19
16
4
4
6
Hz, CH3
β), 4.82–4.84 (m, 1H, CH α), 6.82 (d, 1H, J = 4 Hz, CH Phenyl), 6.90 (d, 1H, J = 4 Hz, CH
Phenyl), 6.93 (s, 1H, CH Phenyl), 7.05–7.08 (m, 1H, CH 7), 7.33 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6),
.35–7.37 (m, 1H, CH 8), 7.50 (s, 1H, CH 2), 7.59 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.95 (d, 1H, J = 4 Hz,
7
13
NH), 9.37 (s, 2H, C-OH Phenyl). C-NMR
δ
(ppm, DMSO-d ): 17.79 (CH3
β), 49.27 (CH
α), 116.01
6
(CH 6), 116.51 (CH Phenyl), 117.87 (CH Phenyl), 120.42 (Cq 1), 120.95 (Cq 5a), 122.06 (CH Phenyl),
123.17 (CH 7), 125.80 (CH 9), 126.69 (CH 8), 131.82 (Cq 3a), 132.42 (CH 2), 132.64 (Cq 9a), 137.42 (Cq 4),
1
46.06 (Cq Phenyl), 146.95 (Cq Phenyl), 147.22 (Cq Phenyl), 174.64 (C=O). MS (ESI +, QTof, m/z): 365.1
+
[M + H] . HRMS calculated for C H N O 365.1250, found 365.1240.
19
17
4
4
2
-((1-(3,4-Dihydroxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)-3-methylbutanoic acid (11c): Following
◦
the same procedure for the synthesis of 11a, to a cooled (0 C) solution of 9c (0.035, 0.08 mmol) in
anhydrous CH Cl (10 mL) was added TFA (5 mL). The product was purified by flash chromatography
2
2
eluted with cyclohexane/ethyl acetate 90/20 to 40/50. The compound is obtained as a white solid
1
(
91% yield). C H N O . MW: 392.41 g/mol. H-NMR
δ
(ppm, DMSO-d ): 1.04 (d, 3H, J = 8 Hz, CH
3
21
20
4
4
6
γ
), 1.07 (d, 3H, J = 8 Hz, CH3
γ’), 1.18 (s, 1H, COOH), 2.36–2.41 (m, 1H, CH β), 4.74–4.76 (m, 1H, CH
α
), 6.82 (dd, 1H, J = 4 Hz, J = 8 Hz, CH Phenyl), 6.90 (d, 1H, J = 4 Hz, J = 8 Hz, CH Phenyl), 6.94 (d, 1H,
J = 4 Hz, J = 8 Hz, CH Phenyl), 7.06–7.10 (m, 1H, CH 7), 7.17 (d, 1H, J = 4 Hz, NH), 7.33–7.36 (m, 1H,
CH 8), 7.38 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.51 (s, 1H, CH 2), 7.60 (dd, 1H, J = 4 Hz, J = 8 Hz, CH
9
β
), 9.40 (s, 2H, C-OH Phenyl). 13C-NMR
), 58.70 (CH
Phenyl), 123.29 (CH 7), 126.05 (Cq 5a), 126.72 (CH 8), 126.94 (CH 9), 132.12 (CH 2), 132.52 (Cq Phenyl),
δ
(ppm, DMSO-d ): 18.86 (CH3
γ
’), 19.63 (CH3
γ), 30.50 (CH
6
α), 115.98 (CH 6), 116.49 (CH Phenyl), 117.86 (CH Phenyl), 120.83 (Cq 1), 122.07 (CH
1
32.82 (Cq 3a), 137.42 (Cq 9a), 146.06 (Cq 4), 147.23 (Cq Phenyl), 147.47 (Cq Phenyl), 171.62 (C=O). MS
+
(ESI +, QTof, m/z): 393.2 [M + H] . HRMS calculated for C H N O 393.1563, found 393.1561.
21
21
4
4
2
-((1-(3,4-Dihydroxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)-4-methylpentanoic acid (11d): Following
◦
the same procedure for the synthesis of 11a, to a cooled (0 C) solution of 9d (0.050, 0.11 mmol) in
anhydrous CH Cl (10 mL) was added TFA (5 mL). The product was purified by flash chromatography
2
2
eluted with cyclohexane/ethyl acetate 90/20 to 40/50. The compound is obtained as a white solid (78%
1
yield). C H N O . MW: 406.43 g/mol. H-NMR
δ
(ppm, DMSO-d ): 0.93 (d, 3H, J = 8 Hz, CH3
δ
),
),
22
22
4
4
6
0
.98 (d, 3H, J = 8 Hz, CH3
.99–2.02 (m, 1H, CH2 ), 4.85 (t, 1H, J = 4 Hz, CH
Phenyl), 6.93 (d, 1H, J = 4 Hz, CH Phenyl), 7.05–7.08 (m, 1H, CH 7), 7.32–7.35 (m, 1H, CH 8), 7.36 (dd,
H, J = 4 Hz, J = 8 Hz, CH 6), 7.50 (s, 1H, CH 2), 7.59 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.90 (d, 1H, J = 4
Hz, NH), 9.38 (s, 2H, C-OH Phenyl). 13C-NMR
(ppm, DMSO-d ): 20.74 (CH3 ), 22.28 (CH3 ’), 24.07
), 114.92 (CH 6), 115.42 (CH Phenyl), 116.77 (CH Phenyl), 119.82
Cq 1), 120.98 (CH Phenyl), 122.10 (CH 7), 124.67 (Cq 5a), 125.64 (CH 8), 125.72 (CH 9), 130.79 (Cq 3a),
δ
’), 1.42 (s, 1H, COOH), 1.69–1.72 (m, 1H, CH2
β), 1.76–1.79 (m, 1H, CH
γ
1
β
α), 6.82 (d, 1H, J = 4 Hz, CH Phenyl), 6.90 (s, 1H, CH
1
δ
δ
δ
6
(
(
CH γ), 39.08 (CH2 β), 50.92 (CH α
1
31.28 (CH 2), 144.99 (Cq 9a), 146.14 (Cq 4), 146.28 (Cq Phenyl), 157.42 (Cq Phenyl), 173.51 (C=O). MS
+
(ESI +, QTof, m/z): 407.1 [M + H] . HRMS calculated for C H N O 407.1719, found 407.1712.
22
23
4
4

Molecules 2018, 23, 2987
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6
-Amino-2-((1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)hexanoic acid (11e): Following
◦
the same procedure for the synthesis of 11a. To a cooled (0 C) solution of 9e (0.035, 0.06 mmol) in
anhydrous CH Cl (10 mL) was added TFA (5 mL). The product was purified by flash chromatography
2
2
eluted with cyclohexane/ethyl acetate 90/20 to 40/50. The compound is obtained as a white solid (84%
1
yield). C H N O . MW: 421.45 g/mol. H-NMR
δ
(ppm, DMSO-d ): 1.23 (s, 1H, COOH), 1.47–1.51
22
23
5
4
6
(
(
m, 2H, CH2
m, 1H, CH
γ
), 1.58–1.64 (m, 2H, CH2 δ), 1.98–2.04 (m, 2H, CH2 β), 2.79–2.83 (m, 2H, CH2 ε), 4.76–4.78
α
), 6.81 (d, 1H, J = 4 Hz, CH Phenyl), 6.90 (s, 1H, CH Phenyl), 6.93 (d, 1H, J = 4 Hz, CH
Phenyl), 7.03–7.07 (m, 1H, CH 7), 7.31–7.35 (m, 1H, CH 8), 7.37 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.47
(
s, 1H, CH 2), 7.58 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.64 (d, 1H, J = 4 Hz, NH-CH
α
), 7.68–7.70 (m, 2H,
), 26.08 (CH2 ),
13
NH ), 9.40–9.46 (m, 2H, C-OH Phenyl). C-NMR
δ
(ppm, DMSO-d ): 22.04 (CH2
γ
δ
2
6
29.65 (CH2
β), 38.04 (CH2 ε), 52.17 (CH α), 114.84 (CH 6), 115.40 (CH Phenyl), 116.79 (CH Phenyl),
1
20.96 (CH Phenyl), 121.87 (CH 7), 124.88 (Cq 5a), 125.49 (CH 8), 126.01 (CH 9), 130.49 (Cq 1), 131.14
(
CH 2), 131.69 (Cq 3a), 136.24 (Cq 9a), 145.00 (Cq 4), 146.10 (Cq Phenyl), 146.51 (Cq Phenyl), 157.59
+
(
Cq Phenyl), 173.29 (C=O). MS (ESI +, QTof, m/z): 422.1 [M + H] . HRMS calculated for C H N O
22
24
5
4
4
22.1828, found 422.1834.
5
-Amino-2-((1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)pentanoic acid (11f): Following
◦
the same procedure for the synthesis of 11a, to a cooled (0 C) solution of 9f (0.055, 0.09 mmol) in
anhydrous CH Cl (10 mL) was added TFA (5 mL). The product was purified by flash chromatography
2
2
eluted with cyclohexane/ethyl acetate 90/20 to 40/50. The compound is obtained as a white solid (10%
1
1
yield). C H N O . MW: 407.42 g/mol. H-NMR
δ
(ppm, DMSO-d ): H-NMR
δ
(ppm, 400 MHz,
), 2.66–2.70 (m,
), 6.69 (d, 1H, J = 8 Hz, CH Phenyl), 6.80
21
21
5
4
6
DMSO-d ): 1.70–1.74 (m, 2H, CH2
γ
), 1.89–1.93 (m, 1H, CH2
H, CH2 δ), 2.78–2.82 (m, 1H, CH2 δ), 4.33–4.34 (m, 1H, CH
β
), 2.07–2.11 (m, 1H, CH2 β
6
1
α
(
d, 1H, J = 8 Hz, CH Phenyl), 6.89 (d, 1H, J = 4 Hz, CH Phenyl), 6.97–7.01 (m, 1H, CH 7), 7.29-.33 (m, 1H,
CH 8), 7.36 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.40 (s, 1H, J = 4 Hz, CH 2), 7.46 (d, 1H, J = 4 Hz, NH-CH
), 7.57 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 8.69 (s, 2H, C-OH Phenyl). 13C-NMR
(ppm, DMSO-d6):
3.61 (CH2 ), 29.41 (CH2 ), 40.41 (CH2 ), 54.57 (CH ), 115.88 (CH 6), 116.50 (CH Phenyl), 117.95
α
δ
2
γ
β
δ
α
(
(
CH Phenyl), 121.23 (Cq 1), 122.04 (CH 7), 122.18 (CH Phenyl), 125.85 (Cq 5a), 126.39 (CH 8), 127.17
CH 9), 132.08 (CH 2), 133.25 (Cq 3a), 138.31 (Cq 9a), 146.07 (Cq 4), 146.84 (Cq Phenyl), 147.12 (Cq
+
Phenyl), 174.19 (C=O). MS (ESI +, QTof, m/z): 408.2 [M + H] . HRMS calculated for C H N O
21
22
5
4
4
08.1672, found 408.1668.
2
-((1-(3,4-Dihydroxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)-3-phenylpropanoic acid (11g): Following
◦
the same procedure for the synthesis of 11a, to a cooled (0 C) solution of 9g (0.045, 0.09 mmol) in
anhydrous CH Cl (10 mL) was added TFA (5 mL). The product was purified by flash chromatography
2
2
eluted with cyclohexane/ethyl acetate 90/20 to 40/50. The compound is obtained as a white solid (87%
1
yield). C H N O . MW: 440.45 g/mol. H-NMR
δ
(ppm, DMSO-d ): 1.23 (s, 1H, COOH), 3.33–3.39
25
20
4
4
6
(
m, 2H, CH2
β), 5.04–5.07 (m, 1H, CH α), 6.81 (d, 1H, J = 8 Hz, CH Phenyl), 6.89 (d, 1H, J = 4 Hz, CH
Phenyl), 6.91 (d, 1H, J = 8 Hz, CH Phenyl), 7.05–7.08 (m, 1H, CH 7), 7.17 (t, 1H, J = 8 Hz, CH Phenyl),
7
7
.26 (t, 2H, J = 8 Hz, CH Phenyl), 7.31–7.333 (m, 1H, CH 8), 7.34 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6),
.36–7.37 (m, 2H, CH Phenyl), 7.48 (s, 1H, CH 2), 7.61 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9), 7.76–7.77
(
m, 1H, NH), 9.39 (s, 2H, C-OH Phenyl). 13C-NMR
δ
(ppm, DMSO-d ): 35.61 (CH2
β
), 5.31 (CH
14.91 (CH 6), 115.40 (CH Phenyl), 116.75 (CH Phenyl), 119.75 (Cq 1), 120.96 (CH Phenyl), 122.23 (CH
), 124.71 (Cq 5a), 125.64 (CH 9), 125.88 (CH 8), 127.67 (CH Phenyl), 128.58 (CH Phenyl), 130.78 (Cq
α),
6
1
7
Phenyl), 131.35 (CH 2), 137.26 (Cq 3a), 138.42 (Cq 9a), 144.98 (Cq Phenyl), 146.14 (Cq 4), 157.48 (Cq
+
Phenyl), 157.77 (Cq Phenyl), 172.35 (C=O). MS (ESI +, QTof, m/z): 441.2 [M + H] . HRMS calculated for
C H N O 441.1563, found 441.1569.
25
21
4
4
2
-((1-(3,4-Dihydroxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)-3-(4-hydroxyphenyl)-propanoic acid (11h):
◦
Following the same procedure for the synthesis of 11a, to a cooled (0 C) solution of 9h (0.030,
0
.06 mmol) in anhydrous CH Cl (10 mL) was added TFA (5 mL). The product was purified by flash
2 2
chromatography eluted with cyclohexane/ethyl acetate 90/20 to 40/50. The compound is obtained as

Molecules 2018, 23, 2987
23 of 26
1
a white solid (89% yield). C H N O . MW: 456.45 g/mol. H-NMR δ (ppm, DMSO-d ): 1.29 (s, 1H,
25
20
4
5
6
COOH), 3.21–3.23 (m, 2H, CH2
β), 4.94–4.98 (m, 1H, CH
α
), 6.71 (d, 2H, J = 8 Hz, CH Phenyl), 6.86
(
7
dd, 1H, J = 4 Hz, J = 8 Hz, CH Phenyl), 6.94 (d, 1H, CH Phenyl), 6.98 (d, 1H, J = 8 Hz, CH Phenyl),
.04–7.08 (m, 1H, CH 7), 7.12 (d, 2H, J = 8 Hz, CH Phenyl), 7.31–7.32 (m, 1H, CH 8), 7.35 (dd, 1H, J = 4
Hz, J = 8 Hz, CH 6), 7.47 (s, 1H, CH 2), 7.59–7.60 (m, 1H, NH), 7.64 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 9),
13
9
.32–9.35 (m, 2H, C-OH Phenyl). C-NMR
CH Phenyl), 115.96 (CH 6), 116.47 (CH Phenyl), 117.82 (CH Phenyl), 120.86 (Cq 1), 122.05 (CH
Phenyl), 123.19 (CH 7), 125.81 (Cq 5a), 126.68 (CH 9), 126.70 (CH 8), 130.59 (2 CH Phenyl), 132.35
δ (ppm, DMSO-d ): 36.01 (CH2 β), 55.24 (CH α), 115.56
6
(2
×
×
(Cq 3a), 132.48 (CH 2), 137.32 (Cq 9a), 146.03 (Cq 4), 147.05 (Cq Phenyl), 147.19 (Cq Phenyl), 156.44
+
(
Cq Phenyl), 173.55 (C=O). MS (ESI +, QTof, m/z): 457.1 [M + H] . HRMS calculated for C H N O
25
21
4
5
4
57.1512, found 457.1514.
2
-Amino-5-((1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)amino)pentanoic acid (11i): Following
◦
the same procedure for the synthesis of 11a, to a cooled (0 C) solution of 9i in anhydrous CH Cl
2
2
(10 mL) was added TFA (5 mL). The product was purified by flash chromatography eluted with
cyclohexane/ethyl acetate 90/20 to 40/50. The compound is obtained as a white solid (79% yield).
1
C H N O . MW: 407.42 g/mol. H-NMR
δ
(ppm, DMSO-d ): 1.23 (s, 1H, COOH), 1.85–1.89 (m, 4H,
21
21
5
4
6
CH2
Phenyl), 6.89 (d, 1H, J = 4 Hz, CH Phenyl), 6.92 (d, 1H, J = 8 Hz, CH Phenyl), 7.07–7.11 (m, 1H, CH 7),
.35 (dd, 1H, J = 4 Hz, J = 8 Hz, CH 6), 7.37–7.39 (m, 1H, CH 8), 7.51 (s, 1H, CH 2), 7.55 (dd, 1H, J = 4
Hz, J = 8 Hz, CH 9), 7.66 (t, 1H, J = 4 Hz, NH-CH2 ), 8.24 (s, 2H, NH ), 9.40 (s, 2H, C-OH Phenyl).
γ, CH2 β), 3.64–3.66 (m, 2H, CH2 δ), 3.99–4.01 (m, 1H, CH α), 6.80 (dd, 1H, J = 4 Hz, J = 8 Hz, CH
7
δ
2
13
C-NMR
δ (ppm, DMSO-d ): 24.80 (CH2 γ), 28.04 (CH2 β), 39.99 (CH2 δ), 52.30 (CH α), 116.12 (CH 6),
6
1
16.51 (CH Phenyl), 117.77 (CH Phenyl), 120.74 (Cq 1), 121.96 (CH Phenyl), 123.27 (CH 7), 125.76 (Cq
5
a), 126.83 (CH 8), 129.28 (CH 9), 132.68 (CH 2), 138.42 (Cq 3a), 146.12 (Cq 4), 147.29 (Cq Phenyl), 158.41
+
(
Cq Phenyl), 158.75 (Cq 9a), 171.54 (C=O). MS (ESI +, QTof, m/z): 408.2 [M + H] . HRMS calculated for
C H N O 408.1672, found 408.1668.
21
22
5
4
3
.1.6. 3,4-Dihydroxyphenylboronic Acid (12)
◦
To a cooled (0 C) solution of 3,4-dimethoxyphenylboronic acid (0.800 g, 4.39 mmol) in anhydrous
CH Cl (50 mL) was added boron tribromide (10 mL, 10 mmol). The resulting solution was allowed
2
2
to warm to room temperature and stirred until complete consumption of the starting material (1–2h,
monitored by TLC). The solution was neutralized by addition of methanol (50 mL). The crude mixture
was concentrated under reduced pressure. The compound was obtained as a white solid (84% yield)
1
and used without purification. C H BO . MW: 153.93 g/mol. H-NMR
δ
(ppm, DMSO-d ): 2.08 (s, 2H,
6
7
4
6
B-OH), 6.47 (d, 1H, CH Phenyl), 6.60 (d, 1H, CH Phenyl), 6.71 (d, 1H, CH Phenyl), 8.00 (s, 2H, C-OH
Phenyl). 13C-NMR
δ
(ppm, DMSO-d ): 108.10 (CH Phenyl), 116.14 (CH Phenyl), 119.73(CH Phenyl),
6
−
1
45.73 (C-OH). MS (ESI +, QTof, m/z): 153.2 [M
53.0358.
−
H] . HRMS calculated for C H O B 153.0359, found
6
6
4
1
3
.2. Cell Line and Culture Techniques
The melanoma (A375) human cancer cell line is obtained from American Type Culture Collection
(
Rockville, MD, USA). Cells were cultured in RPMI Gibco medium containing RPMI-1640 (Waltham,
◦
MA, USA), 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% CO in air at 37 C.
2
3
.3. 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

Molecules 2018, 23, 2987
24 of 26
2
4 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 EAPB02303, EAPB02302 and the synthesized
−
5
−9
compounds at concentrations of 10 –3.2
medium (v/v). After 96 h of incubation, cell supernatant was removed and 100
3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide) solution in fresh medium was added
×
10 M dissolved in a mixture 0.15% DMSO/culture
µL of a MTT
(
◦
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 isopropyl alcohol and 1 M hydrochloric acid (96/4, v/v) was added to
each well. After 10 min of incubation and vigorous shaking to solubilize formazan crystals, the optical
density was measured at 570 nM in a microculture plate reader (Zaragoza, Spain). For each assay, at
least three experiments were performed in triplicate. The individual cell line growth curves confirmed
that all A375 line 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
. Conclusions
The synthesis and study of the amino acid groups grafted on position 4 within the imiqualine
series highlight the fact that the nature of the substituent on position 4 is not essential for the biological
activity. Indeed, large modifications between EAPB02302, which only has a primary amine, and the
new compounds with a complete amino acid residue do not significantly modify the activity, which
remains similar to that of our first imiqualine generation. However, these modulations allow one to
significantly increase the theoretical water solubility. The presence of dihydroxy groups on the phenyl
appears to be necessary for the conservation of the cytotoxic activity on the melanoma cell line tested.
These encouraging results obtained on the representative A375 melanoma cell line will prompt us to
study further in vivo evaluation on xenografted mice.
Supplementary Materials: The following are available online. NMR H and 13C spectra of all compounds
1
evaluated on A375 melanoma cells.
Author Contributions: Conceptualization, A.C., C.P., P.-A.B. and C.D.-M.; Investigation, A.C.; Methodology, A.C.,
C.P. and C.D.-M.; Supervision, P.C., P.-A.B. and C.D.-M.; Writing—original draft, A.C. and C.P.; Writing—review
&
editing, P.-A.B. and C.D.-M.
Funding: This research was funded by the Société d’Accélération du Transfert de Technologies (SATT AxLr).
Acknowledgments: Authors thank the Société d’Accélération du Transfert de Technologies (SATT AxLr) for
financial support to Cindy Patinote, Stephanie Paniagua and Amandine Dejean for their technical help, AGV
Discovery for the biological study, and the French department of Biotage for lending us a HPFC system.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).