New quinoxaline derivatives as dual pim-1/2 kinase inhibitors: Design, synthesis and biological evaluation

Article abstract of DOI:10.3390/molecules26040867

Proviral integration site for Moloney murine leukemia virus (Pim)-1/2 kinase overexpression has been identified in a variety of hematologic (e.g., multiple myeloma or acute myeloid leukemia (AML)) and solid (e.g., colorectal carcinoma) tumors, playing a key role in cancer progression, metastasis, and drug resistance, and is linked to poor prognosis. These kinases are thus considered interesting targets in oncology. We report herein the design, synthesis, structure–activity relationships (SAR) and in vitro evaluations of new quinoxaline derivatives, acting as dual Pim1/2 inhibitors. Two lead compounds (5c and 5e) were then identified, as potent submicromolar Pim-1 and Pim-2 inhibitors. These molecules were also able to inhibit the growth of the two human cell lines, MV4-11 (AML) and HCT-116 (colorectal carcinoma), expressing high endogenous levels of Pim-1/2 kinases.

Full text of DOI:10.3390/molecules26040867

molecules
Article
New Quinoxaline Derivatives as Dual Pim-1/2 Kinase
Inhibitors: Design, Synthesis and Biological Evaluation
Bruno Oyallon 1, Marie Brachet-Botineau 2, Cédric Logé 3 , Thomas Robert 4,5, Stéphane Bach 4,5,6
,
Sajida Ibrahim 7 , William Raoul 7,8 , Cécile Croix 1, Pascal Berthelot 9, Jean Guillon 10 , Noël Pinaud 11
,
Fabrice Gouilleux 2 , Marie-Claude Viaud-Massuard 1 and Caroline Denevault-Sabourin 1,
*
1
EA GICC-ERL 7001 CNRS, Team IMT, University of Tours, F-37200 Tours, France;
bruno.oyallon@gmail.com (B.O.); cecile.croix@univ-tours.fr (C.C.);
marie-claude.viaud-massuard@univ-tours.fr (M.-C.V.-M.)
CNRS ERL7001 LNOx-EA GICC, University of Tours, F-37000 Tours, France;
marie.brachet.botineau@gmail.com (M.B.-B.); fabrice.gouilleux@univ-tours.fr (F.G.)
Department of Medicinal Chemistry, IICIMED-EA1155, IRS2, University of Nantes, F-44200 Nantes, France;
cedric.loge@univ-nantes.fr
Integrative Biology of Marine Models Laboratory (LBI2M), Sorbonne University, CNRS, UMR8227,
F-29680 Roscoff, France; trobert@sb-roscoff.fr (T.R.); bach@sb-roscoff.fr (S.B.)
Kinase Inhibitor Specialized Screening facility (KISSf), Sorbonne University, CNRS, FR2424,
F-29680 Roscoff, France
2
3
4
5
6
7
Centre of Excellence for Pharmaceutical Sciences, North-West University, Private Bag X6001,
Potchefstroom 2520, South Africa
EA GICC-ERL 7001 CNRS, Team PATCH, University of Tours, F-37200 Tours, France;
sajidaibrahim@hotmail.com (S.I.); william.raoul@univ-tours.fr (W.R.)
N2C, University of Tours, INSERM, UMR 1069, F-37032 Tours, France
UMR-S 1172-JPArc, University of Lille, INSERM, CHU Lille, F-59000 Lille, France;
pascal.berthelot@univ-lille2.fr
ARNA Laboratory, University of Bordeaux, INSERM U12132-UMR CNRS 5320, F-33076 Bordeaux, France;
jean.guillon@u-bordeaux.fr
ISM, University of Bordeaux, CNRS, UMR 5255, F-33405 Talence, France; noel.pinaud@u-bordeaux.fr
Correspondence: caroline.denevault@univ-tours.fr; Tel.: +33-2-47-36-72-31
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
8
9
Citation: Oyallon, B.;
Brachet-Botineau, M.; Logé, C.;
Robert, T.; Bach, S.; Ibrahim, S.; Raoul,
W.; Croix, C.; Berthelot, P.; Guillon, J.;
et al. New Quinoxaline Derivatives as
Dual Pim-1/2 Kinase Inhibitors:
Design, Synthesis and Biological
Evaluation. Molecules 2021, 26, 867.
https://doi.org/molecules26040867
10
11
*
Abstract: Proviral integration site for Moloney murine leukemia virus (Pim)-1/2 kinase overex-
pression has been identified in a variety of hematologic (e.g., multiple myeloma or acute myeloid
leukemia (AML)) and solid (e.g., colorectal carcinoma) tumors, playing a key role in cancer pro-
gression, metastasis, and drug resistance, and is linked to poor prognosis. These kinases are thus
considered interesting targets in oncology. We report herein the design, synthesis, structure–activity
relationships (SAR) and in vitro evaluations of new quinoxaline derivatives, acting as dual Pim1/2
inhibitors. Two lead compounds (5c and 5e) were then identified, as potent submicromolar Pim-1
and Pim-2 inhibitors. These molecules were also able to inhibit the growth of the two human cell
lines, MV4-11 (AML) and HCT-116 (colorectal carcinoma), expressing high endogenous levels of
Pim-1/2 kinases.
Academic Editors: William A. Denny
and Brullo Chiara
Received: 7 January 2021
Accepted: 3 February 2021
Published: 6 February 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Keywords: quinoxaline; Pim kinases; kinase inhibitor; anticancer targeted therapy
1. Introduction
Copyright:
© 2021 by the authors.
Proviral integration site for Moloney murine leukemia virus (Pim) kinases 1, 2 and 3
are homologous constitutively active proto-oncogenic serine/threonine protein kinases,
which share a high level of sequence homology, and subsequent functional redundan-
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 (https://
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4.0/).
cies [
are mainly expressed in hematopoietic cells, while Pim-3 is more abundant in breast, brain,
kidneys, and epithelia [ ]. These oncoproteins are positive regulators of cell cycle progres-
sion, and inhibit apoptosis, acting as oncogenic survival factors [ ]. They play a critical
1,2]. Nevertheless, they differ partially in their tissue distribution. Pim-1 and Pim-2
3
4,5
Molecules 2021, 26, 867. https://doi.org/10.3390/molecules26040867
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 867
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role in the control of cell proliferation, survival, differentiation, and migration [
6
,7]. Pim
kinases are aberrantly up-regulated in a variety of hematologic (e.g., multiple myeloma,
acute and chronic myeloid leukemias) and solid (e.g., prostate, breast, colon) tumors [ 12],
8–
contributing to malignant transformation, cancer progression, metastasis, drug resistance
and often poor prognosis. The Pim kinases overexpression observed in cancer cells results
from the abnormal activation of upstream kinases (e.g., BCR-Abl, Jak2) or receptor tyrosine
kinases (RTK) (e.g., fms-like tyrosine kinase 3 (FLT3)-ITD), responsible for the activation
of the signal transducer and activator of transcription (STAT) family transcription factors
(particularly STAT 3/5) [13].
Interestingly, it has been demonstrated that Pim kinases are involved in the expres-
sion, activation, and stabilization of drug efflux transporters, contributing to multidrug
resistance. More specifically, Pim-1 phosphorylates the P-glycoprotein (Pgp), resulting in
its protection from ubiquitination and proteasomal degradation. Pim-1 also mediates phos-
phorylation of the breast-cancer-resistant protein (BCRP), promoting its multimerization
and stable membrane expression [14,15]. Consequently, Pim kinases, by promoting survival
signals, and multiple drug resistance can reduce the efficacy of chemotherapeutic drugs.
All these findings, and the fact that pim genes triple knockout mice demonstrated
mild phenotypic changes [16], highlight the therapeutic potential of these kinases as targets
in oncology, especially in drug-resistant tumors, in order to restore chemosensitivity to
classical chemotherapies. Moreover, because of the redundant functions of the three Pim
isoforms, there is currently a great interest for the development of pan-Pim inhibitors.
A number of different chemical classes of Pim inhibitors have been reported to date, in-
cluding, for example, imidazo [1,2-b]pyridazines, thiazolidine-2,4-diones, benzo[d]imidazoles
or pyridines [17
,18] (Figure 1). For the most promising candidates, clinical trials were
carried out or are currently ongoing [18
–
21]. Thus, the pan-PIM inhibitors PIM447
(LGH447, Novartis) demonstrated antitumor activity in monotherapy in patients with
relapsed/refractory multiple myeloma with a good tolerance profile [21].
Crystallographic studies of Pim-1 and Pim-2 revealed unique structural features of
Pim kinases. Thus, in all three Pim kinases, the hinge region is characterized both by an
atypical conformation and by the lack of a hydrogen bond (H-bond) donor in the hinge.
Indeed, this region contains two proline residues (Pro123 and Pro125, in Pim-1), with no H-
bond donor property. Consequently, Pim kinases bound ATP, the natural substrate, via only
one hinge H-bond, involving the ATP adenine amino substituent and the hinge glutamate
(Glu121, in Pim-1) backbone carbonyl. Moreover, the lateral chain of the hinge prolines and
other hydrophobic amino acid residues in their environment, create a hydrophobic pocket
in the hinge region. Such differences between Pim kinases and other kinases provide an
opportunity for the design of selective pan-Pim inhibitors [22,23].
As part of our laboratory drug discovery program aimed at identifying new targeted
therapies for the treatment of hematologic malignancies, and, regarding the particular
potential of Pim-1 and Pim-2 as targets in leukemia [24
dual Pim-1/Pim-2 specific inhibitors.
,25], we decided to develop new
In a previous work, we identified the quinoxaline-2-carboxylic acid
1
as a new lead
compound (Figure 2). This molecule potently inhibits Pim-1 enzymatic activity at submi-
cromolar concentrations (IC50 of 74 nM) [26]. However, recent studies showed that this
compound exerted only a modest activity against Pim-2 with an IC50 of 2.10 µM (data not
published). In the light of these results, we planned to prepare analogues of compound
with an optimized activity on both Pim-1 and Pim-2 isoforms.
1,

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HO
H2N
N
N
F
F
F
N
N
N
N
N
N
N
H
H
N
H
N
OCF3
CF3
O
N
SGI-1776
TP-3654
Pim-1 Ki = 5 nm
Pim-2 Ki = 239 nM
Pim-3 Ki = 42 nM
LGH447 (PIM447)
Pim-1 IC50 = 7 nM
Pim-2 IC50 = 363 nM
Pim-3 IC50 = 69 nM
Pim-1 IC50 = 95 nM
Pim-2 IC50 = 522 nM
Pim-3 IC50 = 369 nM
Phase I clinical trial
Phase I clinical trial
Phase I/II clinical trial
Cardiotoxicity
Therapeutic efficacy
OH
NH2
NO2
H2N
Br
Br
F
F
F
N
N
O
HN
N
N
N
H
N
HN
O
S
O
N
HO
INCB053914
SEL24-B489
AZD1208
Pim-1 IC50 = 0.24 nM
Pim-2 IC50 = 30 nM
Pim-3 IC50 = 0.12 nM
Pim-1 Kd = 2 nM
Pim-2 Kd = 2 nM
Pim-3 Kd = 3 nM
Pim-1 IC50 = 0.4 nM
Pim-2 IC50 = 5 nM
Pim-3 IC50 = 1.9 nM
Phase I clinical trial
Phase I/II clinical trial
Phase I clinical trial
Lack of clinical responses
Figure 1. Selection of the most representative and/or promising proviral integration site for Moloney murine leukemia
virus (Pim) inhibitors.
Figure 2. Chemical structure of lead compound 1 [26].
Pim inhibitors of the literature can be classified into two classes: ATP mimetics, which
establish an H-bond with the hinge glutamate residue (Glu121, in Pim-1), and non-ATP
mimetics, which bind distant to the hinge or interact with the hinge through weak (mainly
hydrophobic) interactions, with multiple residues of the unique hydrophobic pocket in the
hinge environment. It has been shown that non-ATP mimetic Pim inhibitors could achieve
higher selectivity over other kinases [27].

Molecules 2021, 26, 867
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Consequently, in this work, we decided to probe the unique hydrophobic pocket of
the Pim kinases hinge environment, by adding halogenated substituents in positions 6 or
7 of the quinoxaline scaffold, oriented towards the hinge region of the ATP binding site,
and able to form weak interactions in this area. Eight new derivatives were then prepared
and tested for their Pim-1 and Pim-2 inhibitory activity. The selectivity profiles and anti-
proliferative activities of the most active compounds were further evaluated against a panel
of mammalian kinases and against four different cell lines, respectively.
2. Results and Discussion
2.1. Synthesis
The preparation of the 6- or 7-substituted-quinoxaline-2-carboxylic acids was per-
formed as described in Scheme 1, from commercial o-phenylenediamines according to
literature procedures [28,29].
Scheme 1. Reagents and conditions: (i) diethyl 2-oxomalonate (1 eq), citric acid (15 mol%), EtOH, rt, 2–24 h, 16–54%; (ii)
N,N-dim◦ethylformamide (DMF) (cat.), POCl3, 0 ◦C, and then reflux, 3 h, 51–100%; (iii) 3-aminophenol (1.1–3 eq), EtOH,
◦
MW 150 C, 3 h, 29–99%; (iv) 3-aminophenol (3 eq), p-toluenesulfonic acid (cat.), EtOH, MW 100 C, 8 h, 70%; (v) K2CO3
(3 eq), MeOH/H2O (4/1), reflux, 4 h, 94–100%.
First, the appropriate 4-substituted-1,2-phenylenediamine was condensed with di-
ethyl 2-oxomalonate in the presence of citric acid (15 mol%) at room temperature in
ethanol to give, after separation by column chromatography, ethyl 6-substituted-3-oxo-3,4-
dihydroquinoxaline-2-carboxylates 2a
2b 2d 2f, and 2h, respectively. The identification of each isomer was realized by compari-
son with literature spectroscopic data [30,31].
, 2c, 2e, and 2g, and their seven-substituted isomers
,
,

Molecules 2021, 26, 867
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Subsequent chlorination in position 3 of compounds 2a–h using N,N-dimethylformamide
(DMF) as a catalyst in refluxing phosphorous oxychloride afforded intermediates 3a–h
,
which were used directly for the next step because of a lack of stability.
Access to quinoxaline-2-carboxylic acids was then performed using a two-step syn-
thetic pathway. Intermediates 3a–h undergo initial nucleophilic aromatic substitution
with 3-aminophenol in ethanol under microwave irradiation, to yield esters 4a–h. For
compound 4g synthesis, a catalytic amount of p-toluenesulfonic acid (p-TSA) was used.
Then, hydrolysis of intermediate ethyl esters 4a–h with potassium carbonate in refluxing
80% aqueous methanol was performed, followed by an acidification with a 15% hydrochlo-
ric acid aqueous solution to afford acids 5a–h. The 3D structural determination of the
substituted-quinoxaline-2-carboxylic acids 5b
, 5d and 5e was established by X-ray crystal-
lography (Figure 3) [32] and confirmed the structure in the solid state.
Figure 3. Oak Ridge Thermal-Ellipsoid Plot Program (ORTEP) drawing of substituted-quinoxaline-2-carboxylic acids 5b
,
5d and 5e with thermal ellipsoids at 30% level [32].
2.2. Enzymatic Assays
2.2.1. Pim-1 and Pim-2 Enzymatic Activity Inhibition
Synthesized compounds were first evaluated for their ability to inhibit the in vitro en-
zymatic activity of Homo sapiens Pim-1 (HsPim-1) and HsPim-2, using a luminescence-based
kinase assay [33]. SGI-1776, a small-molecule pan-Pim protein kinase inhibitor [18], com-
mercially available, was used as a control for the in vitro studies. To increase our inhibitor
affinity on Pim kinases, we first decided to structurally vary the substitution patterns of the
quinoxaline scaffold of
1 in positions 6 and 7, introducing halogenated substituents, trying
to establish hydrophobic interactions with the unique hinge hydrophobic pocket (Table 1).

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Table 1. Enzymatic assays on HsPim-1 and HsPim-2 (ADP-Glo method at 10 µM ATP).
HsPim-1
HsPim-2
IC50 (µM) 1
Entry
Compound
X
IC50 (µM) 1
1
2
3
4
5
6
7
8
9
1
5a
5b
5c
5d
5e
5f
5g
5h
H
6-F
7-F
6-Cl
7-Cl
6-Br
7-Br
6-CF3
7-CF3
0.074
0.17
1.24
0.13
2.10
0.16
0.18
0.20
3.85
0.05
2.10
0.76
4.20
0.17
2.40
0.58
2.20
1.80
6.40
0.10
10
SGI-1776
1
IC50 on Pim-1/2 kinase activity were calculated from dose response curves. Each inhibitor concentration
−
was tested in duplicate. Inhibition curves for 5c and 5e are reported in supplementary data. Hs: Homo sapiens.
Values are a mean of n ≥ 3 independent experiments. Hs: Homo sapiens.
In position 7, it appears that the nature of the halogenated group has important
repercussions on compounds activity. Thus, introduction, in this position, of a bromine led
to the potent derivative 5f. This compound maintained a submicromolar activity on Pim-1
(IC50 of 180 nM) with approximately the same level of activity than lead compound 1 on
both Pim isoforms (Table 1, entries 1 and 7). In contrast, the 7-substitution by a fluorine or a
chlorine atom or by a trifluoromethyl group was not favorable for the activity, as evidenced
by derivatives 5b, 5d and 5h (IC50 > 1 µM on both Pim-1 and Pim-2 isoforms) (Table 1,
entries 3, 5 and 9).
In position 6, similar Pim-1 inhibition trends were observed with all halogenated
substituents whatever their size (Table 1, entries 2, 4, 6 and 8), with IC50 values in the range
of 130 to 200 nM, in every case, and nearly the same level of potency than lead compound 1
(Table 1, entry 1). Interestingly, the inhibition profile on Pim-2 isoform was significantly
improved for smaller 5a (6-F) and bulkier 5e (6-Br) derivatives, with IC50 values 3- and
4-fold lower than lead
1, respectively. Finally, the most active compound 5c (6-Cl) was
12-fold more active on Pim-2 isoform than lead
1
, and exhibited the same level of activity
than the reference drug SGI-1776 on both isoforms (Table 1, entry 10).
2.2.2. Selectivity over a Panel of Mammalian Protein Kinases
To compare the selectivity profile of our inhibitors, most active candidates were further
evaluated against a selected panel of mammalian protein kinases (comprising RnDYRK1A,
HsCDK5/p25, HsCDK9/CyclinT, HsHaspin, MmCLK1, HsCK1ε and HsGSK3β) using a
luminescence-based kinase assay [33].
As it can be observed with lead compound
1, derivatives 5c and 5e maintained an
interesting selectivity profile against the five potential off-target kinases HsCDK5/p25,
HsCDK9/CyclinT, HsHaspin, MmCLK1 and HsCK1ε with IC50 inhibition values > 10 µM
in every case (Table 2, entries 1, 2 and 3).

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Table 2. Kinase selectivity profile of the most active quinoxalines (ADP-Glo method at 10 µM ATP).
Kinase Enzymatic IC50 (µM) 1
Entry
Compound
X
Pim-1
Pim-2
DYRK1A CDK5/p25 CDK9/CyclinT Haspin
CLK1
CK1ε
GSK3β
1
2
3
4
1
1
5c
5e
H
6-Cl
6-Br
0.07
0.13
0.16
0.05
2.10
0.17
0.58
0.10
0.27
2.58
1.98
3.80
>10
>10
>10
9.53
>10
>10
>10
1.08
>10
>10
>10
0.05
>10
>10
>10
0.43
>10
>10
>10
6.54
>10
2.80
3.22
>10
SGI-1776
IC50 on disease-related kinase activity were calculated from dose response curves. Each inhibitor concentration was tested in duplicate.
−
Inhibition curves for 5c and 5e are reported in supplementary data. All protein kinases used here are human with the exception of
DYRK1A (Rattus norvegicus) and CLK1 (Mus musculus). DYRK1A: dual specificity tyrosine phosphorylation regulated kinase 1A, CDK:
cyclin-dependent kinase, Haspin: haploid germ cell-specific nuclear protein kinase, CLK1: CDC2-like kinase 1, CK1: casein kinase 1, GSK3:
glycogen synthase kinase 3.
The new quinoxalines tested have limited activity against HsGSK3
β, displaying
micromolar inhibition, in contrast to lead (IC50 > 10 M) (Table 2, entries 1, 2 and 3). Our
1
µ
best inhibitor 5c exhibited nevertheless an IC50 value at least 21- and 16-fold higher for
HsGSK3β than for HsPim-1 and HsPim-2, respectively.
In contrast, while compound
1 potently inhibits RnDYRK1A (IC50 of 0.27 µM), the
most potent quinoxalines, 5c and 5e, displayed only micromolar inhibition of RnDYRK1A
(Table 2, entries 1, 2 and 3).
Importantly, the general selectivity profile of our best inhibitors (5c and 5e) was
significantly improved in comparison to the reference drug SGI-1776, which inhibited
eight of the nine mammalian kinases tested, with IC50 values in the submicromolar to low
micromolar range (0.05–9.53 µM) (Table 2, entry 4).
2.3. Docking Studies
As already hypothesized for compound 1, docking analysis of compound 5c (6-Cl)
into the ATP pocket of Pim-1 (Figure 4a) showed a key salt bridge between the carboxylate
function in position 2 of the quinoxaline scaffold and the ammonium side chain of the
catalytically essential Lys67. As expected, the 6-chloro substituent is oriented towards
the hydrophobic pocket formed by Leu44 (P-loop residue), Ala65, the alkyl side chain of
Arg122, Val126 and Leu174 and contributes to enhance van der Waals interactions. This
hypothesis can be extrapolated for the other 6-halogenated substituents (5a (6-F), 5e (6-Br))
and for the 6-trifluoromethyl group (5g). Isomerization of the bromine atom from the 6- to
the 7-position (5f) does not modify the IC50 values in Pim-1, while it impacts ones with a
fluorine atom (5b), chlorine atom (5d) or a trifluoromethyl group (5h). The docking results
of compound 5f (Figure 4b) into the ATP pocket of Pim-1 revealed a similar salt bridge
interaction with Lys67, even if the 7-bromo substituent can influence both conformation and
binding in this sterically restricted area of the hinge region, probably to promote a halogen-
bond interaction with the carbonyl group of Glu121. This hypothesis is consistent with
the halogen-bonding theory where heavier halogens bound to aryl groups show a partial
positive charge opposite the C-X bond (with the exception of fluorine atoms due to their
high electronegativity) that allows for interactions with classical H-bond acceptors [34].

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(a)
(b)
Figure 4. (a) Illustration of the possible binding mode within the ATP pocket of Pim-1 of compound 5c (b) Illustration of the
possible binding mode within the ATP pocket of Pim-1 of compound 5f. Hydrogen bonds are indicated as yellow lines.
Pim-1 PDB ID: 3A99.

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2.4. In Vitro Cell-Based Assays
Most active Pim inhibitors were then tested in vitro on the human acute myeloid
leukemia (AML) cell line MV4-11, harboring the FLT3-ITD mutation, and overexpressing
Pim kinases. Indeed, Pim 1 and Pim-2 kinases have been shown to be key downstream
effectors of FLT3-induced signaling [35,36]. Moreover, the FLT3-ITD mutation confers poor
prognosis in patients with AML [36]. Cytotoxic effects were evaluated using an MTT assay,
and living cells were also counted with the trypan blue dye exclusion method (Table 3).
Table 3. Cell-based assays of the most active quinoxalines.
EC50 (µM) 1
Entry
Compound
X
MV4-11 2
HCT-116 2
HS-27a 2
MSC 2
1
2
3
4
1
5c
5e
H
6-Cl
6-Br
61.2 ± 3.9
35.5 ± 1.1
32.9 ± 9.6
0.03 ± 0.003
2
32.9 ± 10.2
45.3 ± 1.0
40.7 ± 0.1
4.4 ± 1.7
84.8 ± 4.0
86.3 ± 3.3
>100
11.3 ± 4.2
43.6 ± 11.5
63.2 ± 13.1
>100
4.0 ± 1.1
SGI-1776
1
Values are a mean of n
100 nM to 100
≥
3 independent experiments. Cells were treated with concentrations ranging from
µ
M for 48 h. Cell viability was then determined by MTT assays, and EC50 values were calculated
using Graphpad PRISM 7 software (n = 3 in triplicate; data are the mean ± SEM).
The most potent Pim1/2 inhibitors, 5c and 5e, exhibited in vitro cytotoxic effects
on the MV4-11 cell line with EC50 values in the micromolar range (35.5
±
1.1
1 (EC50 of
µ
m and
32.9 ± 9.6 µM, respectively), and a better activity than the lead compound
61.2 ± 3.9 µM) (Table 3, entries1, 2 and 3).
To determine if the growth inhibitory activity of these compounds was restricted
to leukemic cells, we analyzed the effects of these quinoxalines on normal bone marrow
stromal and mesenchymal stem cells, major cellular components of the leukemic and
hematopoietic microenvironment. To this purpose, we used bone marrow mesenchymal
stem cells (MSCs), freshly isolated from brain-dead donors, and the human bone marrow
stromal cell line HS-27a, sharing similar biological properties with MSCs.
The results show that, as can be observed with lead compound
demonstrated a moderate inhibitory activity on these cells’ growth with EC50 values of
63.2 ± 13.1 µM and 86.3 3.3 M, on MSCs and HS-27a, respectively (Table 3, entries 1
and 2). Interestingly, quinoxaline 5e did not significantly inhibit HS-27a and MSC cell
growth (EC50 values > 100 M) (Table 3, entry 3), suggesting a selective effect of this
1, quinoxaline 5c
±
µ
µ
compound on myeloid leukemia cell growth, sparing normal bone marrow stromal cells of
the leukemic niche.
The best candidates were finally evaluated on the human colorectal carcinoma cell
line HCT-116, a solid tumor cell line, highly expressing Pim-1 and Pim-2 kinases [12
,37],
using an MTT assay. Compounds 5c, and 5e exhibited the same level of activity on this
1,
cell line with EC50 values ranging from 32.9
2 and 3).
±
10.2
µM to 45.3
±
1.0 µM (Table 3, entries 1,
It is interesting to notice that, even if the reference drug SGI-1776 demonstrated higher
activity on leukemic and colorectal carcinoma cells’ growth than our derivatives, this
compound also potently inhibited MSC and HS-27a cells’ growth (Table 3, entry 4), which
is consistent with its lack of selectivity on the selected panel of mammalian protein kinases
studied (Table 2).

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3. Materials and Methods
3.1. General Remarks
All solvents were anhydrous reagents from commercial sources. Unless otherwise
noted, all chemicals and reagents were obtained commercially and used without purifi-
cation. Microwave heating was carried out with a single-mode Initiator Alstra (Biotage)
unit. Melting points (Mp) were determined on a Stuart capillary apparatus and are uncor-
rected. High-resolution mass spectra (HRMS) were performed in positive mode with an
ESI source on a Q-TOF mass spectrometer (Bruker maXis) with an accuracy tolerance of
2 ppm. NMR spectra were recorded at 300 MHz (1H) or 75 MHz (13C) on a Bruker Avance
(300 MHz) spectrometer. The chemical shifts are reported in parts per million (ppm, δ)
relative to residual deuterated solvent peaks. The abbreviations s = singlet, d = doublet,
t = triplet, q = quadruplet, qt = quintuplet, m = multiplet and bs = broad signal were used
throughout. In the 1H-NMR spectra of quinoxalines 5a
–
5h, the carboxylic acid proton was
not observed. The presence of the carbonyl peak has been confirmed by 13C-NMR and
the compound structure by HRMS. The identification of compounds 2b 2d 2f, and 2g
was realized by comparison with literature spectroscopic data [30 31]. The reference of the
,
,
,
literature-relevant spectroscopic data is given below the characterization of each concerned
compound.
3.2. Chemistry
Ethyl 6-fluoro-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (2a) and ethyl 7-fluoro-3-oxo-3,4-
dihydroquinoxaline-2-carboxylate (2b), Method A: a mixture of 4-fluoro-1,2-phenylenediamine
(366.8 mg, 2.91 mmol), diethyl 2-oxomalonate (506.5 mg, 2.91 mmol) and citric acid (83.8 mg
,
0.44 mmol) in ethanol (15 mL) was stirred magnetically at room temperature overnight.
Ethanol was then evaporated under reduced pressure, and the resulting residue was
purified by silica column chromatography using cyclohexane with ethyl acetate gradient
(0–60%) as eluents to give compounds 2a (190.6 mg, 28%) and 2b (372.0 mg, 54%) as
yellow powders.
◦
1
Compound 2a: Mp 186.8 C. H NMR (300 MHz, DMSO-d6)
δ 12.96 (bs, 1H, NH),
7.91 (dd, 1H, J = 9.0, 5.7 Hz), 7.24 (ddd, 1H, J = 9.0, 2.7 Hz), 7.07 (dd, 1H, J = 9.0,
2.7 Hz), 4.36 (q, 2H, J = 7.1 Hz, CH2), 1.32 (t, 3H, J = 7.1 Hz, CH3). 13C NMR (75 MHz,
DMSO-d6)
δ 164.0 (d, J = 248.9 Hz), 163.9, 152.7, 149.8, 135.0 (d, J = 13.0 Hz), 132.4 (d,
J = 11.0 Hz), 128.3 (d, J = 1.5 Hz), 112.7 (d, J = 24.2 Hz), 102.2 (d, J = 26.7 Hz), 62.3, 14.4. 19F
NMR (282 MHz, DMSO-d6) δ −106.0. HRMS (ESI) m/z: [M+H]+ calcd for C11H10FN2O3,
237.0670; found: 237.0668.
Compound 2b: Mp 207.6 ◦C. 1H NMR (300 MHz, DMSO-d6)
δ
12.97 (bs, 1H, NH), 7.73
(dd, 1H, J = 9.0, 2.9 Hz), 7.59 (ddd, 1H, J = 9.0, 2.9 Hz), 7.38 (dd, 1H, J = 9.0, 5.1 Hz), 4.38 (q,
2H, J = 7.1 Hz, CH2), 1.32 (t, 3H, J = 7.1 Hz, CH3). 13C NMR (75 MHz, DMSO-d6)
163.9,
δ
158.4 (d, J = 239.3 Hz), 152.5, 152.3, 131.4 (d, J = 11.6 Hz), 130.1, 120.9 (d, J = 24.6 Hz), 117.8
(d, J = 9.2 Hz), 114.7 (d, J = 22.7 Hz), 62.4, 14.4. 19F NMR (282 MHz, DMSO-d6) δ −118.4.
HRMS (ESI) m/z: [M+H]+ calcd for C11H10FN2O3, 237.0670; found: 237.0667.
Literature spectroscopic data Reference [30].
Ethyl 6-chloro-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (2c) and ethyl 7-chloro-3-oxo-3,4-
dihydroquinoxaline-2-carboxylate (2d): The title compound was synthesized according to the
general method A from 4-chloro-1,2-phenylenediamine (285.2 mg, 2.00 mmol), diethyl 2-
oxomalonate (348.3 mg, 2.00 mmol) and citric acid (57.7 mg, 0.30 mmol) in ethanol (10 mL).
The reaction mixture was stirred magnetically at room temperature for 2 h. The solvent
was then removed under reduced pressure, and the resulting residue was purified by silica
column chromatography using cyclohexane with ethyl acetate gradient (0–40%) as eluent
to give compounds 2c (161.7 mg, 32%) and 2d (274.2 mg, 54%) as yellow powders.
◦
1
Compound 2c: Mp 236.0 C. H NMR (300 MHz, DMSO-d6)
7.85 (d, 1H, J = 8.7 Hz), 7.40 (dd, 1H, J = 8.7, 2.2 Hz), 7.34 (d, 1H, J = 2.2 Hz), 4.37 (q, 2H,
J = 7.1 Hz, CH2), 1.32 (t, 3H, J = 7.1 Hz, CH3). 13C NMR (75 MHz, DMSO-d6)
163.8, 152.6,
δ 12.94 (bs, 1H, NH),
δ

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151.1, 136.8, 134.3, 131.4, 130.0, 124.5, 115.6, 62.3, 14.4. HRMS (ESI) m/z: [M+H]+ calcd for
C11H10ClN2O3: 253.0374; found: 253.0374.
◦
1
Compound 2d: Mp 213.2 C. H NMR (300 MHz, DMSO-d6)
δ 13.00 (bs, 1H, NH),
7.93 (d, 1H, J = 2.3 Hz), 7.69 (dd, 1H, J = 8.8, 2.3 Hz), 7.36 (d, 1H, J = 8.8 Hz), 4.38 (q, 2H,
J = 7.1 Hz, CH2), 1.32 (t, 3H, J = 7.1 Hz, CH3). 13C NMR (75 MHz, DMSO-d6)
δ 163.8, 152.6,
152.2, 132.6, 132.2, 131.7, 128.6, 128.0, 118.0, 62.4, 14.4. HRMS (ESI) m/z: [M+H]+ calcd for
C11H10ClN2O3: 253.0374; found: 253.0373.
Literature spectroscopic data Reference [30].
Ethyl 6-bromo-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (2e) and ethyl 7-bromo-3-oxo-3,4-
dihydroquinoxaline-2-carboxylate (2f): The title compounds were synthesized according to
the general method A from 4-bromo-1,2-phenylenediamine (748.2 mg, 4.00 mmol), diethyl
2-oxomalonate (696.6 mg, 4.00 mmol) and citric acid (115.3 mg, 0.60 mmol) in ethanol
(
15 mL). The reaction mixture was stirred magnetically at room temperature for 24 h.
The solvent was then removed under reduced pressure, and the resulting residue was
purified by silica column chromatography using cyclohexane with ethyl acetate gradient
(40–70%) as eluent to give compound 2e (393.5 mg, 33%) as a beige powder and compound
2f (304.5 mg, 26%) as an orange powder.
Compound 2e: Mp 234.8◦C. 1H NMR (300 MHz, DMSO-d6)
δ
12.93 (bs, 1H, NH), 7.77
(d, 1H, J = 8.6 Hz), 7.52 (dd, 1H, J = 8.6, 2.0 Hz), 7.49 (d, 1H, J = 2.0 Hz), 4.36 (q, 2H,
J = 7.1 Hz, CH2), 1.31 (t, 3H, J = 7.1 Hz, CH3). 13C NMR (75 MHz, DMSO-d6)
δ 163.9, 152.6,
151.3, 134.5, 131.5, 130.2, 127.3, 125.6, 118.6, 62.3, 14.4. HRMS (ESI) m/z: [M+H]+ calcd for
C11H10BrN2O3: 296.9869; found: 296.9869.
Compound 2f: Mp 234.8◦C. 1H NMR (300 MHz, DMSO-d6)
δ
13.00 (bs, 1H, NH), 8.06
(d, 1H, J = 2.2 Hz), 7.80 (dd, 1H, J = 8.8, 2.2 Hz), 7.29 (d, 1H, J = 8.8 Hz), 4.37 (q, 2H,
J = 7.1 Hz, CH2), 1.32 (t, 3H, J = 7.1 Hz, CH3). 13C NMR (75 MHz, DMSO-d6)
δ 163.8, 152.6,
152.1, 135.2, 132.6, 132.1, 131.6, 118.2, 115.6, 62.4, 14.4. HRMS (ESI) m/z: [M+H]+ calcd for
C11H10BrN2O3: 296.9869; found: 296.9868.
Literature spectroscopic data Reference [30].
Ethyl 3-oxo-6-(trifluoromethyl)-3,4-dihydroquinoxaline-2-carboxylate (2g) and ethyl 3-oxo-
7-(trifluoromethyl)-3,4-dihydroquinoxaline-2-carboxylate (2h): The title compounds were syn-
thesized according to the general method A from 4-trifluoromethyl-1,2-phenylenediamine
(387.5 mg, 2.20 mmol), diethyl 2-oxomalonate (383.1 mg, 2.20 mmol) and citric acid (63.4 mg
,
0.33 mmol) in ethanol (15 mL). The reaction mixture was stirred magnetically at room
temperature overnight. The solvent was then removed under reduced pressure, and the
resulting residue was purified by silica column chromatography using dichloromethane
with ethyl acetate gradient (0–20%) as eluent to give the desired compounds 2g (325.8 mg,
52%) and 2h (98.9 mg, 16%) as◦beige powders.
1
Compound 2g: Mp 204.1 C. H NMR (300 MHz, DMSO-d6)
δ 13.11 (bs, 1H, NH),
8.06 (d, 1H, J = 8.4 Hz), 7.67 (dd, 1H, J = 8.4, 1.8 Hz), 7.63 (d, 1H, J = 1.8 Hz), 4.40 (q, 2H,
J = 7.2 Hz, CH2), 1.33 (t, 3H, J = 7.2 Hz, CH3). 13C NMR (75 MHz, DMSO-d6)
δ
163.7, 153.6,
152.6, 133.4, 133.0, 131.7 (q, J = 32.3 Hz), 131.1, 124.0 (q, J = 271.0 Hz), 120.3 (q, J = 3.5 Hz),
113.5 (q, J = 3.8 Hz), 62.5, 14.4. 19F RMN (282 MHz, DMSO-d6) δ −61.4. HRMS (ESI) m/z:
[M+H]+ calcd for C12H10F3N2O3: 287.0638; found: 287.0637.
Literature spectroscopic data Reference [31].
◦
1
Compound 2h: Mp 161.2 C. H NMR (300 MHz, DMSO-d6)
8.20 (d, 1H, J = 1.5 Hz), 7.96 (dd, 1H, J = 8.7, 1.5 Hz), 7.51 (d, 1H, J = 8.7 Hz), 4.39 (q, 2H,
J = 7.2 Hz, CH2), 1.33 (t, 3H, J = 7.2 Hz, CH3). 13C NMR (75 MHz, DMSO-d6)
163.7, 152.9,
δ 13.18 (bs, 1H, NH),
δ
152.6, 136.1, 130.4, 128.7 (q, J = 3.3 Hz), 127.0 (q, J = 4.1 Hz), 124.6 (q, J = 32.7 Hz), 124.3 (q,
J = 270.2 Hz), 117.7, 62.5, 14.4. 19F RMN (282 MHz, DMSO-d6) δ −60.4. HRMS (ESI) m/z:
[M+H]+ calcd for C12H10F3N2O3: 287.0638; found: 287.0635.
Ethyl 3-chloro-6-fluoroquinoxaline-2-carboxylate (3a), Method B: into a dry three-neck
round bottom flask was introduced compound 2a (141.3 mg, 0.60 mmol) in phosphorous
oxychloride (1.4 mL) at ice bath temperature. N,N-dimethylformamide (140
µL) was then
added at 0 ◦C and the reaction mixture was refluxed for 3 h. After cooling, the resulting

Molecules 2021, 26, 867
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mixture was poured over ice, stirred magnetically at room temperature overnight, and
extracted with dichloromethane. The combined organic layers were dried over MgSO4,
filtered, and evaporated under reduced pressure to obtain derivative 3a (112.9 mg, 74%)
1
as a brown oil. H NMR (300 MHz, DMSO-d6)
δ 8.32 (dd, 1H, J = 9.2, 5.8 Hz), 8.02–7.90
(m, 2H), 4.49 (q, 2H, J = 7.1 Hz, CH2), 1.38 (t, 3H, J = 7.1 Hz, CH3). 19F RMN (282 MHz,
DMSO-d6) δ −103.6.
Ethyl 3-chloro-7-fluoroquinoxaline-2-carboxylate (3b): The title compound was synthe-
sized according to the general method B from compound 2b (236.2mg, 1.00 mmol), phos-
phorous oxychloride (2.3 mL) and DMF (230
100%) as a brown oil. H NMR (300 MHz, DMSO-d6)
µ
L). Compound 3b was obtained (254.6 mg,
1
δ
8.23 (dd, 1H, J = 9.2, 5.6 Hz, 1H),
8.09 (dd, 1H, J = 9.2, 2.8 Hz), 8.00 (ddd, 1H, J = 9.2, 8.4, 2.8 Hz), 4.50 (t, 2H, J = 7.1 Hz, CH2),
1.39 (t, 3H, J = 7.1 Hz, CH3). 19F RMN (282 MHz, DMSO-d6) δ −105.8.
Ethyl 3,6-dichloroquinoxaline-2-carboxylate (3c): The title compound was synthesized
according to the general method B from compound 2c (50.0 mg, 0.20 mmol), phosphorous
oxychloride (461
brown oil. H NMR (300 MHz, DMSO-d6)
µ
L) and DMF (46
µ
L). Compound 3c was obtained (53.7 mg, 100%) as a
1
δ
8.29 (d, 1H, J = 2.3 Hz), 8.26 (d, 1H, J = 9.0 Hz),
8.03 (dd, 1H, J = 9.0, 2.3 Hz), 4.50 (q, 2H, J = 7.1 Hz, CH2), 1.39 (t, 3H, J = 7.1 Hz, CH3).
Ethyl 3,7-dichloroquinoxaline-2-carboxylate (3d): The title compound was synthesized
according to the general method B from compound 2d (200.0 mg, 0.79 mmol), phosphorous
oxychloride (1.9 mL) and DMF (185
brown oil. H NMR (300 MHz, DMSO-d6)
µ
L). Compound 3d was obtained (202.0 mg, 94%) as a
1
δ
8.37 (d, 1H, J = 2.2 Hz), 8.17 (d, 1H, J = 9.0 Hz),
8.07 (dd, 1H, J = 9.0, 2.2 Hz), 4.51 (q, 1H, J = 7.1 Hz, CH2), 1.39 (t, 1H, J = 7.1 Hz, CH3).
Ethyl 6-bromo-3-chloroquinoxaline-2-carboxylate (3e): The title compound was synthe-
sized according to the general method B from compound 2e (200.0 mg, 0.67 mmol), phos-
phorous oxychloride (1.6 mL) and DMF (157
µ
L). Compound 3e was obtained (212.4 mg,
100%) as a brown oil. 1H NMR (300 MHz, DMSO-d6)
δ
8.39 (dd, 1H, J = 1.9, 0.8 Hz),
8.16–8.08 (m, 2H), 4.49 (q, 2H, J = 7.1 Hz, CH2), 1.38 (t, 3H, J = 7.1 Hz, CH3).
Ethyl 7-bromo-3-chloroquinoxaline-2-carboxylate (3f): The title compound was synthe-
sized according to the general method B from compound 2f (200.0 mg, 0.67 mmol), phos-
phorous oxychloride (1.6 mL) and DMF (157
100%) as a brown oil. H NMR (300 MHz, DMSO-d6)
µ
L). Compound 3f was obtained (212.2 mg,
1
δ
8.52 (dd, 1H J = 2.1, 0.5 Hz), 8.18
¸
(dd, 1H¸ J = 9.0, 2.1 Hz), 8.08 (dd, 1H, J = 9.0, 0.5 Hz), 4.50 (q, 2H, J = 7.1 Hz, CH2), 1.38 (t,
3H, J = 7.1 Hz, CH3).
Ethyl 3-chloro-6-(trifluoromethyl)quinoxaline-2-carboxylate (3g): The title compound was
synthesized according to the general method B from compound 2g (278.0 mg, 0.97 mmol),
phosphorous oxychloride (2.3 mL) and DMF (230
100%) as a yellow oil. H NMR (300 MHz, DMSO-d6)
µ
L). Compound 3g was obtained (295.8 mg
,
1
δ
8.57 (d, 1H, J = 2.0 Hz), 8.44 (d, 1H,
J = 8.8 Hz), 8.25 (dd, 1H, J = 8.8, 2.0 Hz), 4.52 (q, 2H, J = 7.1 Hz, CH2), 1.40 (t, 3H, J = 7.1 Hz,
CH3). 19F RMN (282 MHz, DMSO-d6) δ −61.3.
Ethyl 3-chloro-7-(trifluoromethyl)quinoxaline-2-carboxylate (3h): The title compound was
synthesized according to the general method B from compound 2h (50.0 mg, 0.18 mmol),
phosphorous oxychloride (0.4 mL) and DMF (40
µ
L). Compound 3h was obtained (27.0 mg
,
1
51%) as a yellow oil. H NMR (300 MHz, DMSO-d6) δ 8.64 (d, 1H, J = 0.8 Hz), 8.33 (d, 1H,
J = 8.9 Hz), 8.28 (dd, 1H, J = 8.9, 0.8 Hz), 4.52 (q, 2H, J = 7.1 Hz, CH2), 1.39 (t, 3H, J = 7.1 Hz,
CH3). 19F RMN (282 MHz, DMSO-d6) δ −61.3 (s).
Ethyl 6-fluoro-3-((3-hydroxyphenyl)amino)quinoxaline-2-carboxylate (4a), Method C1: a
solution of compound 3a (84.6 mg, 0.33 mmol) and 3-aminophenol (39.8 mg, 0.365 mmol) in
◦
absolute ethanol (2 mL) was stirred under microwave irradiation for 3 h at 150 C. Ethanol
was then evaporated under reduced pressure, and the resulting residue was purified by
silica column chromatography using cyclohexane with ethyl acetate gradient (0–30%) as
eluent to give compound 4a (88.9 mg, 82%) as an orange powder. Mp 199.8 ◦C. 1H NMR
(300 MHz, DMSO-d6)
δ 10.15 (bs, 1H), 9.49 (bs, 1H), 8.06 (ddd, 1H, J = 9.1, 6.1, 0.5 Hz), 7.55
(t, 1H, J = 2.0 Hz), 7.53–7.42 (m, 2H), 7.24–7.14 (m, 2H), 6.53 (ddd, 1H, J = 7.7, 2.0, 1.3 Hz),
4.48 (q, 2H, J = 7.1 Hz, CH2), 1.41 (t, 3H, J = 7.1 Hz, CH3). 13C NMR (75 MHz, DMSO-d6)

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δ
165.9, 164.9 (d, J = 247.5 Hz), 158.3, 149.2, 143.7 (d, J = 14.6 Hz), 140.3, 133.3, 132.7 (d,
J = 11.0 Hz), 132.2, 130.1, 116.5 (d, J = 25.6 Hz), 111.4, 110.9, 110.4 (d, J = 21.8 Hz), 107.6,
60.2, 14.5. 19F RMN (282 MHz, DMSO-d6) δ −105.3. HRMS (ESI) m/z: [M+H]+ calcd for
C17H15FN3O3: 328.1092; found: 328.1094.
Ethyl 7-fluoro-3-((3-hydroxyphenyl)amino)quinoxaline-2-carboxylate (4b): The title com-
pound was synthesized according to the general method C1 from compound 3b
(254.6 mg,
1.00 mmol) and 3-aminophenol (120.0 mg, 1.10 mmol) in absolute ethanol (5 mL). Com-
◦
pound 4b was obtained (138.6 mg, 42%) as a red powder. Mp 223.4 C. 1H NMR (300 MHz,
DMSO-d6) 10.01 (bs, 1H), 9.47 (bs, 1H), 7.85–7.70 (m, 3H), 7.55 (t, 1H, J = 1.8 Hz), 7.22–7.13
(m, 2H), 6.51 (ddd, 1H, J = 7.3, 1.8 Hz), 4.48 (q, 2H, J = 7.1 Hz, CH2), 1.41 (t, 3H, J = 7.1 Hz,
CH3). 13C NMR (75 MHz, DMSO-d6)
165.8, 160.1 (d, J = 243.1 Hz), 158.3, 148.5, 140.6,
δ
δ
139.6, 136.0 (d, J = 12.8 Hz), 133.6, 130.0, 128.7 (d, J = 9.6 Hz), 123.1 (d, J = 26.7 Hz), 113.5 (d,
J = 21.4 Hz), 111.2, 110.6, 107.3, 62.9, 14.5. 19F RMN (282 MHz, DMSO-d6)
(ESI) m/z: [M+H]+ calcd for C17H15FN3O3: 328.1092; found: 328.1092.
δ -114.4. HRMS
Ethyl 6-chloro-3-((3-hydroxyphenyl)amino)quinoxaline-2-carboxylate (4c): The title com-
pound was synthesized according to the general method C1 from compound 3c (203.1 mg
,
0.75 mmol) and 3-aminophenol (245.2 mg, 2.25 mmol) in absolute ethanol (5 mL). After
purification by silica column chromatography using cyclohexane with ethyl acetate gradi-
ent (0–40%) as eluent, compound 4c was obtained (253.9 mg, 99%) as an orange powder.
Mp 226.8 ◦C. 1H NMR (300 MHz, DMSO-d6)
δ 10.14 (bs, 1H), 9.48 (bs, 1H), 8.01 (d, 1H,
J = 8.9 Hz), 7.82 (dd, 1H, J = 2.3, 1.0), 7.63 (m, 1H), 7.57 (dd, 1H, J = 8.9, 2.2 Hz), 7.22–7.14 (m,
2H), 6.56–6.50 (m, 1H), 4.48 (q, 2H, J = 7.1 Hz, CH2), 1.40 (t, 3H, J = 7.1 Hz, CH3). 13C NMR
(75 MHz, DMSO-d6)
δ 165.8, 158.3, 149.2, 142.9, 140.3, 137.8, 134.6, 133.3, 131.8, 130.1, 127.0,
125.3, 111.3, 110.9, 107.5, 62.9, 14.5. HRMS (ESI) m/z: [M+H]+ calcd for C17H15ClN3O3:
344.0796; found: 344.0794.
Ethyl 7-chloro-3-((3-hydroxyphenyl)amino)quinoxaline-2-carboxylate (4d): The title com-
pound was synthesized according to the general method C1 from compound 3d (167 mg,
0.62 mmol) and 3-aminophenol (74 mg, 0.68 mmol) in absolute ethanol (5 mL). After purifi-
cation by silica column chromatography using dichloromethane with ethyl acetate gradient
(0–10%) as eluent, compound 4d was obtained (192 mg, 91%) as a red powder. Mp 216.9 ◦C
.
1H NMR (300 MHz, DMSO-d6)
(dd, 1H, J = 8.8, 2.2 Hz), 7.77 (d, 1H, J = 8.8 Hz), 7.56 (t, 1H, J = 2.1 Hz), 7.22–7.14 (m,
2H), 6.52 (ddd, 1H, J = 7.0, 2.1 Hz), 4.48 (q, 2H, J = 7.1 Hz, CH2), 1.40 (t, 3H, J = 7.1 Hz
δ 10.07 (bs, 1H), 9.49 (bs, 1H), 8.06 (d, 1H, J = 2.2 Hz), 7.82
,
CH3). 13C NMR (75 MHz, DMSO-d6)
δ 165.7, 158.3, 148.8, 141.1, 140.4, 136.1, 133.9, 133.6,
130.4, 130.0, 128.5, 128.3, 111.3, 110.8, 107.5, 62.9, 14.5. HRMS (ESI) m/z: [M+H]+ calcd for
C17H15ClN3O3: 344.0796; found: 344.0799.
Ethyl 6-bromo-3-((3-hydroxyphenyl)amino)quinoxaline-2-carboxylate (4e): The title com-
pound was synthesized according to the general method C1 from compound 3e (203.2 mg,
0.64 mmol) and 3-aminophenol (210.8 mg, 1.93 mmol) in absolute ethanol (5 mL). After
cooling, the reaction mixture was filtered, giving compound 4e (72.4 mg, 29%) as a red
powder. Mp 230.6 ◦C. 1H NMR (300 MHz, DMSO-d6)
δ 10.14 (bs, 1H), 9.48 (bs, 1H), 7.98
(d, 1H, J = 2.1 Hz), 7.91 (d, 1H, J = 8.8 Hz), 7.70–7.64 (m, 2H), 7.21–7.13 (m, 2H), 6.55–6.49
(m, 1H), 4.47 (q, 2H, J = 7.1 Hz, CH2), 1.40 (t, 2H, J = 7.1 Hz, CH3). 13C NMR (75 MHz,
DMSO-d6)
δ 165.8, 158.3, 149.1, 143.1, 140.3, 134.8, 133.3, 131.8, 130.1, 129.6, 128.6, 126.7,
111.3, 110.8, 107.5, 62.9, 14.5. HRMS (ESI) m/z: [M+H]+ calcd for C17H15BrN3O3: 388.0291;
found: 388.0291.
Ethyl 7-bromo-3-((3-hydroxyphenyl)amino)quinoxaline-2-carboxylate (4f): The title com-
pound was synthesized according to the general method C1 from compound 3f (210.9 mg,
0.67 mmol) and 3-aminophenol (218.7 mg, 2.00 mmol) in absolute ethanol (5 mL). Af-
ter purification by silica column chromatography using cyclohexane with ethyl acetate
gradient (0–50%) as eluent, compound 4f was obtained (88.4 mg, 34%) as a red powder.
1
Mp 244.1 ◦C. H NMR (300 MHz, DMSO-d6)
δ 10.08 (bs, 1H), 9.50 (bs, 1H), 8.20 (d, 1H,
J = 2.2 Hz), 7.91 (dd, 1H, J = 8.9, 2.2 Hz), 7.70 (d, 1H, J = 8.9 Hz), 7.58–7.56 (m, 1H), 7.22–7.14
(m, 2H), 6.52 (ddd, 1H, J = 7.1, 2.1 Hz), 4.48 (q, 2H, J = 7.1 Hz, CH2), 1.40 (t, 3H, J = 7.1 Hz,

Molecules 2021, 26, 867
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CH3). 13C NMR (75 MHz, DMSO-d6)
δ
165.7, 158.3, 148.9, 141.4, 140.4, 136.6, 136.1, 133.8,
131.7, 130.0, 128.5, 118.5, 111.3, 110.8, 107.5, 62.9, 14.5. HRMS (ESI) m/z: [M+H]+ calcd for
C17H15BrN3O3: 388.0291; found: 388.0290.
Ethyl 3-((3-hydroxyphenyl)amino)-6-(trifluoromethyl)quinoxaline-2-carboxylate (4g), Method
C2: a solution of compound 3g (256.9 mg, 0.84 mmol), 3-aminophenol (276.0 mg, 2.53 mmol
)
and p-TSA, as a catalyst, in absolute ethanol (1.5 mL) was stirred under microwave irra-
diation for 8 h at 100 ◦C. Ethanol was then evaporated under reduced pressure, and the
resulting residue was purified by silica column chromatography using cyclohexane with
ethyl acetate gradient (0–30%) as eluent to give the desired compound 4g (222.7 mg, 70%)
as a red powder. Mp 184.7 ◦C. 1H NMR (300 MHz, DMSO-d6)
δ 10.16 (bs, 1H), 9.52 (bs,
1H), 8.20 (d, 1H, J = 8.6 Hz), 8.10 (d, 1H, J = 1.8 Hz), 7.79 (dd, 1H, J = 8.6, 1.8 Hz), 7.67 (m,
1H), 7.24–7.16 (m, 2H), 6.54 (ddd, 1H, J = 6.9, 2.4 Hz), 4.50 (q, 2H, J = 7.2 Hz, CH2), 1.42
(t, 3H, J = 7.2 Hz, CH3). 13C NMR (75 MHz, DMSO-d6)
δ 165.5, 158.3, 149.2, 141.7, 140.2,
137.2, 135.6, 132.7 (q, J = 31.9 Hz), 131.6, 130.1, 124.2 (q, J = 271.1 Hz), 124.1 (q, J = 3.5 Hz),
121.6 (q, J = 4.0 Hz), 111.4, 111.0, 107.6, 63.1, 14.4. 19F RMN (282 MHz, DMSO-d6) δ −61.4.
HRMS (ESI) m/z: [M+H]+ calcd for C18H15F3N3O3: 378.1060; found: 378.1059.
Ethyl 3-((3-hydroxyphenyl)amino)-7-(trifluoromethyl)quinoxaline-2-carboxylate (4h): The
title compound was synthesized according to the general method C1 from compound 3h
(86.2 mg, 0.28 mmol) and 3-aminophenol (33.9 mg, 0.31 mmol) in absolute ethanol (2 mL).
After purification by silica column chromatography using cyclohexane with ethyl acetate
gradient (0–20%) as eluent, compound 4h was obtained (38.8 mg, 36%) as a yellow powder.
Mp 178.5 ◦C. 1H NMR (300 MHz, DMSO-d6)
δ 10.22 (bs, 1H), 9.56 (bs, 1H), 8.34 (d, 1H,
J = 1.8 Hz), 8.05 (dd, 1H, J = 8.7, 1.8 Hz), 7.93 (d, 1H, J = 8.7 Hz), 7.60 (m, 1H), 7.23–7.17
(m, 2H), 6.56 (ddd, 1H, J = 7.2, 1.8 Hz), 4,50 (q, 2H, J = 7.1 Hz, CH2), 1.42 (t, 3H, J = 7.1 Hz,
CH3). 13C NMR (75 MHz, DMSO-d6)
δ 165.5, 158.3, 149.7, 144.2, 140.1, 134.8, 134.7, 130.1,
128.4 (q, J = 3.1 Hz), 128.1, 127.7 (q, J = 4.4 Hz), 126.3 (q, J = 32.3 Hz), 124.4 (q, J = 270.0 Hz),
111.6, 111.2, 107.8, 63.0, 14.4. 19F RMN (282 MHz, DMSO-d6) δ −60.6 (t, J = 2.9 Hz). HRMS
(ESI) m/z: [M+H]+ calcd for C18H15F3N3O3: 378.1060; found: 378.1059.
6-Fluoro-3-((3-hydroxyphenyl)amino)quinoxaline-2-carboxylic acid (5a), Method D: to ester
4a (43.2 mg, 0.13 mmol) in aqueous methanol (80%, 15 mL), potassium carbonate (54.7 mg,
0.40 mmol) was added and the reaction mixture was refluxed for 4 h. After cooling, the
solvent was removed under reduced pressure. Then, the residue was acidified with a 15%
hydrochloric acid aqueous solution, and extracted with dichloromethane. The combined
organic layers were washed with brine, dried over MgSO4, filtered, and evaporated under
reduced pressure to yield the acid 5a (39.5 mg, 100%) as a dark red powder. Mp 200.4 ◦C
1H NMR (300 MHz, DMSO-d6)
10.59 (bs, 1H), 9.47 (bs, 1H), 8.04 (m, 1H), 7.56 (t, 1H
J = 2.0 Hz), 7.52–7.40 (m, 2H), 7.24 (ddd, 1H, J = 7.9, 2.0, 1.0 Hz), 7.17 (t, 1H, J = 7.9 Hz), 6.52
(ddd, 1H, J = 7.9, 2.0, 1.0 Hz). 13C NMR (75 MHz, DMSO-d6)
168.0, 164.8 (d, J = 249.7 Hz),
.
δ
δ
158.3, 149.7, 144.0, 143.8, 140.4, 133.2, 132.5 (d, J = 11.6 Hz), 130.1, 116.2 (d, J = 25.9 Hz),
111.2, 110.8, 110.3 (d, J = 21.8 Hz), 107.4. 19F RMN (282 MHz, DMSO-d6) δ −105.6. HRMS
(ESI) m/z: [M+H]+ calcd for C15H11FN3O3: 300.0779; found: 300.0779.
7-Fluoro-3-((3-hydroxyphenyl)amino)quinoxaline-2-carboxylic acid (5b): The title com-
pound was synthesized according to the general method D from compound 4b (107.3 mg,
0.33 mmol) and potassium carbonate (135.9 mg, 0.98 mmol) in aqueous methanol (80%,
1
15 mL). Compound 5b was obtained (97.1 mg, 99%) as a red powder. Mp 180.1 ◦C. H
NMR (300 MHz, DMSO-d6)
1H, J = 2.0 Hz), 7.22 (ddd, 1H, J = 7.7, 2.0, 1.2 Hz), 7.16 (t, 1H, J = 7.7 Hz), 6.50 (ddd,
1H, J = 7.7, 2.0, 1.2 Hz). 13C NMR (75 MHz, DMSO-d6)
167.9, 160.0 (d, J = 243.1 Hz),
δ 10.44 (bs, 1H), 9.47 (bs, 1H), 7.85–7.69 (m, 3H), 7.56 (t,
δ
158.3, 149.0, 140.7, 139.7, 136.0 (d, J = 12.2 Hz), 133.9, 130.0, 128.6 (d, J = 9.5 Hz), 122.9 (d,
J = 25.7 Hz), 113.4 (d, J = 21.2 Hz), 111.0, 110.5, 107.2. 19F RMN (282 MHz, DMSO-d6)
−114.7. HRMS (ESI) m/z: [M+H]+ calcd for C15H11FN3O3: 300.0779; found: 300.0779.
δ
6-Chloro-3-((3-hydroxyphenyl)amino)quinoxaline-2-carboxylic acid (5c): The title com-
pound was synthesized according to the general method D from compound 4c (29.7 mg,
0.09 mmol) and potassium carbonate (35.7 mg, 0.26 mmol) in aqueous methanol (80%,

Molecules 2021, 26, 867
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◦
5 mL). Compound 5c was obtained (27.2 mg, 100%) as a dark red powder. Mp 194.6 C. 1H
NMR (300 MHz, DMSO-d6) 10.60 (bs, 1H), 9.48 (bs, 1H), 7.98 (d, 1H, J = 8.8 Hz), 7.82 (d,
1H, J = 2.3 Hz), 7.64 (t, 1H, J = 1.9 Hz), 7.57 (dd, 1H, J = 8.8, 2.3 Hz), 7.23–7.13 (m, 2H), 6.51
δ
(ddd, 1H, J = 7.2, 1.9 Hz). 13C NMR (75 MHz, DMSO-d6)
δ 167.9, 158.3, 149.6, 143.1, 140.4,
137.6, 134.6, 133.5, 131.7, 130.1, 126.9, 125.3, 111.2, 110.7, 107.4. HRMS (ESI) m/z: [M+H]+
calcd for C15H11ClN3O3: 316.0483; found: 316.0484.
7-Chloro-3-((3-hydroxyphenyl)amino)quinoxaline-2-carboxylic acid (5d): The title com-
pound was synthesized according to the general method D from compound 4d (128 mg,
0.37 mmol) and potassium carbonate (154 mg, 1.12 mmol) in aqueous methanol (80%,
15 mL). Compound 5d was obtained (110 mg, 94%) as a dark red powder. Mp 173–174 ◦C
.
1H NMR (300 MHz, DMSO-d6)
δ 10.48 (bs, 1H), 9.49 (bs, 1H), 8.03 (d, 1H, J = 2.2 Hz),
7.84–7.76 (m, 2H), 7.57 (t, 1H, J = 1.9 Hz), 7.23–7.14 (m, 2H), 6.53–6.49 (m, 1H). 13C NMR
(75 MHz, DMSO-d6)
δ 167.3, 157.8, 148.8, 140.8, 140.0, 135.7, 135.5, 133.0, 129.7, 129.6, 128.0,
127.8, 110.7, 110.2, 106.9. HRMS (ESI) m/z: [M+H]+ calcd for C15H11ClN3O3: 316.04855;
found: 316.04778.
6-Bromo-3-((3-hydroxyphenyl)amino)quinoxaline-2-carboxylic acid (5e): The title com-
pound was synthesized according to the general method D from compound 4e (39.0 mg,
0.10 mmol) and potassium carbonate (41.5 mg, 0.30 mmol) in aqueous methanol (80%,
5 mL). Compound 5e was obtained (36.0 mg, 100%) as a red powder. Mp 199.0 ◦C. 1H
NMR (300 MHz, DMSO-d6)
δ 10.54 (bs, 1H), 9.47 (bs, 1H), 7.98 (d, 1H, J = 2.2 Hz), 7.90
(d, 1H, J = 8.8 Hz), 7.70–7.64 (m, 2H), 7.22–7.13 (m, 2H), 6.51 (ddd, 1H, J = 6.6, 2.4 Hz).
13C NMR (75 MHz, DMSO-d6)
δ 167.9, 158.3, 149.5, 143.3, 140.4, 134.7, 133.4, 131.7, 130.1,
129.5, 128.6, 126.6, 111.2, 110.7, 107.4. HRMS (ESI) m/z: [M+H]+ calcd for C15H11BrN3O3:
359.9978; found: 359.9977.
7-Bromo-3-((3-hydroxyphenyl)amino)quinoxaline-2-carboxylic acid (5f): The title com-
pound was synthesized according to the general method D from compound 4f (40.7 mg,
0.11 mmol) and potassium carbonate (43.5 mg, 0.32 mmol) in aqueous methanol (80%, 5
mL). Compound 5f was obtained (37.8 mg, 100%) as an orange powder. Mp 193.1 ◦C. 1H
NMR (300 MHz, DMSO-d6)
δ 10.48 (bs, 1H), 9.53 (bs, 1H), 8.16 (d, 1H, J = 2.3 Hz), 7.91 (dd,
1H, J = 8.9, 2.3 Hz), 7.71 (d, 1H, J = 8.9 Hz), 7.58 (t, 1H, J = 1.9 Hz), 7.23–7.13 (m, 2H), 6.52
(dd, 1H, J = 7.5, 1.9 Hz). 13C NMR (75 MHz, DMSO-d6)
δ 167.8, 158.3, 149.3, 141.5, 140.4,
136.6, 136.0, 133.9, 131.7, 130.1, 128.5, 118.3, 111.1, 110.7, 107.4. HRMS (ESI) m/z: [M+H]+
calcd for C15H11BrN3O3: 359.9978; found: 359.9979.
3-((3-Hydroxyphenyl)amino)-6-(trifluoromethyl)quinoxaline-2-carboxylic acid (5g): The title
compound was synthesized according to the general method D from ester 4g (30.8 mg,
0.08 mmol) and potassium carbonate (33.6 mg, 0.24 mmol) in aqueous methanol (80%,
15 mL). After cooling, the solvent was removed under reduced pressure. Then, the residue
was acidified with a 15% hydrochloric acid aqueous solution, and extracted with diethyl
ether. The combined organic layers were washed with brine, dried over MgSO4, filtered
and evaporated under reduced pressure to yield the acid 5g (28.6 mg, 100%) as a dark red
◦
powder. Mp 196.2 C. 1H NMR (300 MHz, DMSO-d6)
1H, J = 8.4 Hz), 8.09 (d, 1H, J = 1.8 Hz), 7.79 (dd, 1H, J = 8.4, 1.8 Hz), 7.68 (m, 1H), 7.24–7.15
(m, 2H), 6.54 (ddd, 1H, J = 7.2, 1.8 Hz). 13C NMR (75 MHz, DMSO-d6)
167.7, 158.3, 149.7,
δ 10.56 (bs, 1H), 9.51 (bs, 1H), 8.17 (d,
δ
141.8, 140.3, 137.2, 135.7, 132.4 (q, J = 37.5 Hz), 131.6, 130.1, 124.3 (q, J = 270.0 Hz), 124.1 (q,
J = 3.0 Hz), 121.4 (q, J = 2.3 Hz), 111.3, 110.9, 107.4. 19F RMN (282 MHz, DMSO-d6) δ −61.4.
HRMS (ESI) m/z: [M+H]+ calcd for C16H11F3N3O3: 350.0747; found: 350.0749.
3-((3-Hydroxyphenyl)amino)-7-(trifluoromethyl)quinoxaline-2-carboxylic acid (5h): The title
compound was synthesized according to the general method D from ester 4h (30.8 mg, 0.08
mmol) and potassium carbonate (33.6 mg, 0.25 mmol) in aqueous methanol (80%, 15 mL).
After cooling, the solvent was removed under reduced pressure. Then, the residue was
acidified with a 15% hydrochloric acid aqueous solution, and extracted with diethyl ether.
The combined organic layers were washed with brine, dried over MgSO4, filtered and
evaporated under◦reduced pressure to yield the acid 5h (33.2 mg, 100%) as a red ocher
powder. Mp 178.7 C. 1H NMR (300 MHz, DMSO-d6)
δ 10.90 (bs, 1H), 9.52 (bs, 1H), 8.29 (s,

Molecules 2021, 26, 867
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1H), 8.03 (dd, 1H, J = 8.8, 1.7 Hz), 7.92 (d, 1H, J = 8.8 Hz), 7.61 (t, 1H, J = 2.0 Hz), 7.26–7.16
(m, 2H), 6.54 (ddd, 1H, J = 7.7, 1.3 Hz). 13C NMR (75 MHz, DMSO-d6)
δ
167.4, 158.3, 150.4,
144.5, 140.3, 136.0, 134.6, 130.1, 128.0 (2
×
C), 127.4, 125.9 (q, J = 30 Hz), 124.7 (q, J = 270 Hz),
111.3, 111.0, 107.5. 19F RMN (282 MHz, DMSO-d6) —60.5. HRMS (ESI) m/z: [M+H]+ calcd
δ
for C16H11F3N3O3: 350.0747; found: 350.0747.
3.3. Docking Studies
Molecular modeling studies were performed using SYBYL-X 1.3 software [38] running
on a Dell precision T3400 workstation. The three-dimensional structure of compounds 5c
and 5f (under their carboxylate forms to imitate physiological conditions) were built from
a standard fragments library and optimized using the Tripos force field [39] including the
electrostatic term calculated from Gasteiger and Hückel atomic charges. Powell’s method
available in Maximin2 procedure was used for energy minimization until the gradient
value was smaller than 0.001 kcal/(mol*Å). The crystal structure of Pim-1 in complex with
0
5 -adenylyl-
β,γ
-imidodiphosphate (AMP-PNP) at 1.6 Å resolution (PDB ID 3A99) [40] was
used as template for docking. Water molecules were removed from the coordinates set
since no information about conserved water molecules is known for this chemical series in
Pim-1. Flexible docking of compounds 5c and 5f into the ATP-binding site was performed
using GOLD software [41]. The most stable docking models were selected according to the
best scored conformation predicted by the Chemscore scoring function implemented in
GOLD. Finally, the complexes were energy-minimized using Powell’s method available in
Maximin2 procedure with the Tripos force field and a dielectric constant of 4.0, until the
gradient value reached 0.1 kcal/mol.Å. Biovia Discovery Studio Visualizer [42] was used
for graphical display.
3.4. Biology
3.4.1. Mammalian Protein Kinase Assays
Kinase enzymatic activities were assayed with 10 µM ATP in 384-well plates using
the luminescent ADP-GloTM assay (Promega, Madison, WI, USA) according to the rec-
ommendations of the manufacturer (see [32] for details on this method). The transmitted
signal was measured using the Envision (PerkinElmer, Waltham, MA, USA) microplate
luminometer and expressed in Relative Light Unit (RLU). In order to determine the half
maximal inhibitory concentration (IC50), the assays were performed in duplicate in the ab-
sence or presence of increasing doses of the tested compounds. GraphPad Prism6 software
(GraphPad Software, San Diego, CA, USA) was used to fit dose–response curves and to
determine the IC50 values. Kinase activities are expressed in % of maximal activity, i.e.,
measured in the absence of inhibitor. Peptide substrates were obtained from Proteogenix
(Schiltigheim, France).
The following kinases were analyzed during this study: HsPim-1 and HsPim-2 (human
proto-oncogene, recombinant, expressed in bacteria) were assayed with 0.20 µg/µL of
consensus peptide substrate: ARKRRRHPSGPPTA; RnDYRK1A-kd (Rattus norvegicus,
amino acids 1 to 499 including the kinase domain, recombinant, expressed in bacteria,
DNA vector kindly provided by Dr. W. Becker, Aachen, Germany) was assayed with
0.033
(human cyclin-dependent kinase-2, kindly provided by Dr. A. Echalier-Glazer, Leicester,
UK) was assayed with 0.8 g/ L of histone H1 as substrate; HsCDK9/CyclinT (human,
recombinant, expressed by baculovirus in Sf9 insect cells) was assayed with 0.20 g/
µg/µL of the following peptide: KKISGRLSPIMTEQ as substrate; HsCDK2/CyclinA
µ
µ
µ
µL
of the following peptide: YSPTSPSYSPTSPSYSPTSPSKKKK, as substrate; HsHaspin-kd
(human, kinase domain, amino acids 470 to 798, recombinant, expressed in bacteria) was
assayed with 0.007
as substrate; MmCLK1 (from Mus musculus, recombinant, expressed in bacteria) was
assayed with 0.027 g/ L of the following peptide: GRSRSRSRSRSR as substrate; HsCK1
(human casein kinase 1 , recombinant, expressed by baculovirus in Sf9 insect cells) was
assayed with 0.02 g/ L of the following peptide: RRKHAAIGSpAYSITA (“Sp” stands for
µg/µL of Histone H3 (1–21) peptide (ARTKQTARKSTGGKAPRKQLA)
µ
µ
ε
ε
µ
µ

Molecules 2021, 26, 867
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phosphorylated serine) as CK1-specific substrate; HsGSK-3
β
(human glycogen synthase
kinase-3) was assayed with 0.010 g/µL of GS-1 peptide, a GSK-3-selective substrate
µ
(YRRAAVPPSPSLSRHSSPHQSpEDEEE). For kinases expressed in bacteria, Escherichia coli
BL21 DE3 pLysS strain (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) was
transformed with pGEX-2T-1 (Sigma-Aldrich, St. Louis, MO, USA) containing the coding
region of the corresponding kinase. For kinases expressed in Sf9 insect cells, the coding
region of the corresponding kinase was cloned in pFastBacTM vector. Purification of the
recombinant kinases were performed following the protocol “Bac-to-Bac® Baculovirus
Expression System” provided by the manufacturer (Invitrogen, ThermoFisher Scientific,
Waltham, MA, USA).
To validate the kinase assay, model inhibitors were used for each tested enzyme:
Staurosporine from Streptomyces sp. (#S5921, purity
≥
95%, Sigma-Aldrich) for HsCK1
ε;
Indirubin-30-oxime (#I0404, purity
≥
98%, Sigma-Aldrich) for HsGSK-3
β, HsPim-1 and
2, human cyclin-dependent kinases, RnDYRK1A and MmCLK1; CHR-6494 (#SML0648,
purity ≥ 98%, Sigma-Aldrich) for Haspin.
3.4.2. Cell Cultures and Reagents
MV4-11 cell lines were obtained from the Deutshe Sammlung von Mikroorganismens
und Zellkulturen (DSMZ). HS-27a cell lines were obtained from the American Type Culture
Collection (ATCC). All cell lines were cultured in Roswell Park Memorial Institute medium
(RPMI), with 10% fetal bovine serum, 1% glutamine, and 1% penicillin/streptomycin at
37 ◦C and 5% CO2. Bone marrow (BM) MSCs from brain-dead donors were isolated and
cultured as described [43]. Informed consent was obtained before BM samples were taken.
The HCT-116 cell line (colorectal carcinoma) was obtained from ATCC and cultured in
McCoy’s medium with 10% fetal bovine serum without antibiotics at 37 ◦C and 5% CO2.
3.4.3. In Vitro Cell-Based Assays
Cell viability was studied using an MTT cell proliferation assay. To determine the
concentration effect of the molecules, 0.2
cells were incubated in 100 L of RPMI red phenol-free medium (ThermoFisher Scientific),
0.035 L of alphaMEM medium (Gibco) and 0.05
105 MSCs were incubated in 100 105
HCT-116 cells were incubated in 100 L of McCoy’s medium, in 96-well plates for 48 h
×
105 MV4-11 leukemic cells or 0.1
×
105 HS-27a
µ
×
µ
×
µ
and then treated with quinoxalines (stock solution at 50 mM in DMSO) or SGI-1776 (SAB
Signalway antibody, stock solution at 50 mM in DMSO), as reference, with concentrations
ranging from 100 nM to 100 µM for 48 h.
Cells were incubated with 10 µL of MTT working solution (5 g/L of methylthiazolyl
diphenyl-tetrazolium bromide from Sigma Aldrich, Lyon, France) during 4 h. Cells were
◦
then lysed overnight at 37 C with 100
µL of 10% sodium dodecyl sulfate (SDS) and
0.003% HCl. Optical density (OD) at 570 nm was measured using a spectrophotometer
CLARIOstar® (BMG Labtech, Offenburg, Germany) or Mithras Multimode Microplate
Reader LB940 (Berthold, Versailles, France). Living cells were also counted with the trypan
blue dye exclusion method. When a dose-dependent activity was observed, EC50 values
were calculated using Graphpad PRISM 7 software (n = 3 in triplicate). Data were collected
from at least three independent experiments and the values reported are means
errors of the mean (SEM).
±
standard
4. Conclusions
In summary, using a structure-based design approach, we have developed a new
promising quinoxaline-2-carboxylic acid series of dual Pim-1/2 inhibitors. Starting from
the lead compound 1, moderately active on Pim-2, we significantly improved the inhibition
profile on Pim-2 isoform by adding halogenated substituents in position 6. Docking studies
demonstrated that this 6-halogenated group was oriented towards the unique hydrophobic
pocket of the Pim kinases hinge environment, contributing to increase van der Waals
interactions in this area. Two lead compounds, 5c (6-Cl) and 5e (6-Br), were then identified,

Molecules 2021, 26, 867
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exhibiting submicromolar potency on both Pim-1 and Pim-2 isoforms, with an interesting
selectivity profile against the panel of mammalian kinases studied. In vitro cell-based
assays on human hematologic (AML) and solid tumor (colorectal carcinoma) cell lines
overexpressing Pim-1/2 kinases were then realized, showing growth inhibitory activities
at micromolar concentrations. These encouraging results make them promising new leads
for further pharmacomodulation studies.
Supplementary Materials: The following are available online, spectroscopic data for final com-
pounds, and Figure S1: Inhibition curves of compound 5c on HsPim1 (A), HsPim2 (B), RnDYRK1A
(C), and HsGSK3β (D), Figure S2: Inhibition curves of compound 5e on HsPim1 (A), HsPim2 (B),
RnDYRK1A (C), and HsGSK3β (D).
Author Contributions: Conceptualization, C.D.-S. and C.L.; methodology, C.D.-S. and M.-C.V.-M.; in-
vestigation, B.O., M.B.-B., C.L., C.D.-S., F.G., S.I., W.R., C.C., J.G., N.P., T.R. and S.B.; writing—original
draft preparation, C.D.-S. and C.L.; writing—review and editing, C.D.-S. and C.L.; supervision, C.D.-S.
and M.-C.V.-M.; project administration, C.D.-S. and M.-C.V.-M.; funding acquisition, M.-C.V.-M., P.B.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by a grant from the « Association pour le Développement de
la Recherche et de l’Innovation dans le NORD PAS DE CALAIS » (ADRINORD) (to B.O.). The
authors also thank the Cancéropôle Grand Ouest (3MC network—Marine Molecules, Metabolism
and Cancer), GIS IBiSA (Infrastructures en Biologie Santé et Agronomie) and Biogenouest (Western
France life science and environment core facility network) for supporting the KISSf screening facility.
The authors wish to thank the Région Centre Val de Loire and LabEx SynOrg (ANR-11-LABX-0029)
for financial support.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments: The authors thank the « Plateforme Scientifique et Technique Analyses des
Systèmes Biologiques » (PST-ASB), Tours (France), for NMR spectrometry and the « Fédération de
Recherche » ICOA/CBM (FR2708) platform for HRMS analyses.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
Sample Availability: Samples of the compounds are available from the authors.
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