Structure-based design of novel quinoxaline-2-carboxylic acids and analogues as Pim-1 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.04.056

We identified a new series of quinoxaline-2-carboxylic acid derivatives, targeting the human proviral integration site for Moloney murine leukemia virus-1 (HsPim-1) kinase. Seventeen analogues were synthesized providing useful insight into structure-activity relationships studied. Docking studies realized in the ATP pocket of HsPim-1 are consistent with an unclassical binding mode of these inhibitors. The lead compound 1 was able to block HsPim-1 enzymatic activity at nanomolar concentrations (IC₅₀ of 74 nM), with a good selectivity profile against a panel of mammalian protein kinases. In vitro studies on the human chronic myeloid leukemia cell line KU812 showed an antitumor activity at micromolar concentrations. As a result, compound 1 represents a promising lead for the design of novel anticancer targeted therapies.

Full text of DOI:10.1016/j.ejmech.2018.04.056

Accepted Manuscript
Structure-based design of novel quinoxaline-2-carboxylic acids and analogues as
Pim-1 inhibitors
Bruno Oyallon, Marie Brachet-Botineau, Cédric Logé, Pascal Bonnet, Mohamed
Souab, Thomas Robert, Sandrine Ruchaud, Stéphane Bach, Pascal Berthelot,
Fabrice Gouilleux, Marie-Claude Viaud-Massuard, Caroline Denevault-Sabourin
PII:
S0223-5234(18)30398-2
10.1016/j.ejmech.2018.04.056
EJMECH 10405
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 February 2018
Revised Date: 20 April 2018
Accepted Date: 28 April 2018
Please cite this article as: B. Oyallon, M. Brachet-Botineau, Cé. Logé, P. Bonnet, M. Souab, T. Robert,
S. Ruchaud, Sté. Bach, P. Berthelot, F. Gouilleux, M.-C. Viaud-Massuard, C. Denevault-Sabourin,
Structure-based design of novel quinoxaline-2-carboxylic acids and analogues as Pim-1 inhibitors,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.04.056.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Glu121
Phe187
Leu120
Lys67
Molecular
modeling
Hinge region
Pro123
DFG motif
Salt bridge
Asp186
IC50 (µM)
HsPim-1
KU812
1
0.074
38.9
ATP binding site pocket
PDB code: 3A99

ACCEPTED MANUSCRIPT
Structure-based design of novel quinoxaline-2-carboxylic acids and
analogues as Pim-1 inhibitors
1
2
3
4
Bruno Oyallon a, Marie Brachet-Botineau b,c, Cédric Logé d, Pascal Bonnet e,
Mohamed Souab f, Thomas Robert f, Sandrine Ruchaud f, Stéphane Bach f, Pascal
Berthelot g, Fabrice Gouilleux b, Marie-Claude Viaud-Massuard a, Caroline
Denevault-Sabourin a,*.
5
6
7
8
9
a EA GICC - ERL 7001 CNRS « Groupe Innovation et Ciblage Cellulaire », Team
Innovation Moléculaire et Thérapeutique, University of Tours, F-37200 Tours, France
b CNRS ERL7001 LNOx « Leukemic Niche and redOx metabolism » - EA GICC,
University of Tours, F-37000 Tours, France
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
c
CHRU de Tours, Service d’Hématologie Biologique, F-37044 Tours, France
d Université de Nantes, Nantes Atlantique Universités, Département de Chimie
Thérapeutique, Cibles et Médicaments des Infections et du Cancer, IICIMED-
EA1155, Institut de Recherche en Santé 2, F-44200 Nantes, France
e UMR University of Orléans-CNRS 7311, Institut de Chimie Organique et
Analytique (ICOA), University of Orléans, F-45067 Orléans, France
f Sorbonne Universités, USR3151 CNRS/UPMC, Plateforme de criblage KISSf
(Kinase Inhibitor Specialized Screening facility), Station Biologique, Place Georges
Teissier, F-29688 Roscoff, France
g UMR-S 1172 - JPArc - Centre de Recherche Jean-Pierre AUBERT Neurosciences et
Cancer, University of Lille, Inserm, CHU Lille, F-59000 Lille, France
* Corresponding author, Department of Innovation Moléculaire et Thérapeutique, EA
GICC - ERL 7001 CNRS, University of Tours, 31 avenue Monge, F-37200 Tours,
France. Tel.: +33 2 47367231.
E-mail address: caroline.sabourin@univ-tours.fr (C. Denevault-Sabourin).

ACCEPTED MANUSCRIPT
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
Abstract
We identified a new series of quinoxaline-2-carboxylic acid derivatives, targeting the
human proviral integration site for Moloney murine leukemia virus-1 (HsPim-1)
kinase. Seventeen analogues were synthesized providing useful insight into structure-
activity relationships studied. Docking studies realized in the ATP pocket of HsPim-1
are consistent with an unclassical binding mode of these inhibitors. The lead
compound 1 was able to block HsPim-1 enzymatic activity at nanomolar
concentrations (IC50 of 74 nM), with a good selectivity profile against a panel of
mammalian protein kinases. In vitro studies on the human chronic myeloid leukemia
cell line KU812 showed an antitumor activity at micromolar concentrations. As a
result, compound 1 represents a promising lead for the design of novel anticancer
targeted therapies.
Keywords
quinoxaline; Pim-1; kinase inhibitor; anticancer targeted therapy.
Abbreviations1
1
IC50, 50% inhibitory concentration; SAR, structure-activity relationships; Pim, proviral integration
site of Moloney murine leukemia virus; CML, chronic myeloid leukemia; 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.

ACCEPTED MANUSCRIPT
54
1. Introduction
55
56
Proviral integration site for Moloney murine leukemia virus (Pim) kinases belong to a
small family of constitutively activated proto-oncogenic serine/threonine protein
kinases, constituted of three isoforms: Pim-1, Pim-2 and Pim-3 [1]. These
oncoproteins control many cellular functions like cell cycle regulation, apoptosis, cell
survival, proliferation and differentiation [2,3], and are overexpressed in a large
number of human cancer types, such as hematopoietic malignancies [4,5] and solid
cancers (e. g. bladder [6], prostate [7], breast [8] or oral cancers [9]). These kinases
are positive regulators of cell cycle progression at G1/S and G2/M checkpoints, and
inhibit apoptosis, acting as oncogenic survival factors [10]. Interestingly, it has been
demonstrated that Pim1−/−2−/−3−/− triple knockout mice were viable and fertile, which
make these kinases very interesting for targeted cancer therapies [11].
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
Recently, Pim-1 has been shown to play a significant role in cancer stem cells growth,
and in resistance to chemotherapy drugs, promoting multiple drug resistance [12,13].
This kinase is thus considered as a relevant target for cancer therapy and a large
variety of small molecule inhibitors have been developed [14-18]. Many of these Pim-
1 kinase inhibitors demonstrated significant in vitro activity in cancer cell lines and in
different in vivo tumor xenograft models, and clinical trials are currently ongoing for
the most promising candidates [14,18].
A remarkable characteristic of Pim-1 active site in comparison to other protein
kinases is the presence of an original hinge region (region containing backbone
peptide atoms that forms hydrogen bond interactions (H-bonds) with the adenine
moiety of ATP). Indeed, this region contains a proline residue (Pro123), which has no
H-bond donor property and precludes the formation of one of the conserved H-bond
involving the hinge backbone and the ATP adenine ring, as it can be observed in other
kinases. Thus, Pim-1 bounds ATP via only one hinge H-bond between the ATP
adenine amino moiety and the backbone carbonyl of glutamate 121 (Glu121).
Moreover, the insertion in the hinge of a valine (Val126), absent in other kinases,
changes the hinge conformation, enlarging the catalytic pocket. This unique feature
can be exploited for the design of selective inhibitors [19].
The vast majority of Pim-1 inhibitors mainly act as ATP competitive inhibitors,
targeting the ATP-binding pocket. They can be classified into two categories: ATP-
mimetics, which bind to the Glu121 residue of the hinge region, and non-ATP

ACCEPTED MANUSCRIPT
88
89
mimetics, which interact with the ATP binding cleft in a different manner from ATP
[20].
90
In a continuing effort to develop new small molecule inhibitors with anticancer
properties, our laboratory has been recently focusing on the study of new inhibitors of
the signal transducer and activator of transcription 5 (STAT5) activation and
expression and their interest in chronic myeloid leukemia (CML) [21]. Indeed, the
STAT family transcription factors are commonly activated in cancer by upstream
mutations or cell surface signaling molecules. It has been demonstrated that the Pim
kinases are induced by the STAT family transcription factors (particularly STAT 3/5)
[14]. Regarding the potential of Pim-1 as target in cancer therapy and particularly in
leukemia [22,23], we decided to further explore the STAT signaling pathway, by
developing new Pim-1 kinase specific inhibitors. In this purpose, we first performed a
target-based approach, by realizing a focused in vitro screening of our chemical
library on a limited panel of kinases, comprising Homo sapiens Pim-1 (HsPim-1),
allowing the identification of the quinoxaline-2-carboxylic acid 1 as a new lead
compound (Fig. 1). This molecule was able to inhibit the in vitro enzymatic activity of
HsPim-1 with an IC50 of 74 nM.
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
Docking studies, using program GOLD (GOLD version 4.0; CCDC, Cambridge, UK),
were performed to understand the binding interactions between the lead compound 1
and the ATP pocket of HsPim-1 (PDB ID 3A99) (Fig. 2). Data analysis suggests that
the carboxylate group of this molecule can form a key salt bridge with the protonated
amino group side chain of catalytic Lys67, as it has already been described in other
Pim-1 inhibitors [24, 25], and shares also a H-bond interaction with the backbone NH
of Asp186 belonging to the DFG motif. Additionally, an H-bond interaction between
the 3-hydroxyphenyl moiety and the carboxylate group of residue Asp186 can be
observed. These studies suggested that compound 1 could act as an ATP competitive
inhibitor, with a non-ATP mimetic binding mode.
Sixteen new analogues were then synthesized, exploiting the unique sequence of
HsPim-1 ATP-binding cleft.
We report herein the design, synthesis, structure-activity relationships (SAR) and in
vitro evaluations of this new class of Pim-1 inhibitors.
2. Chemistry

ACCEPTED MANUSCRIPT
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
The preparation of quinoxaline-2-carboxylic acids 1, 5c-e, and 5h-i and potassium
carboxylate salts 5b, and 5g was performed as shown in Scheme 1 by amination of the
intermediate ethyl 3-chloroquinoxaline-2-carboxylate 3 with the appropriate amine
derivatives.
The synthesis of ethyl 3-chloroquinoxaline-2-carboxylate 3 was achieved in two steps
from commercial o-phenylenediamine according to literature procedures [26,27]
(Scheme 1). First, the o-phenylenediamine was condensed with diethyl 2-
oxomalonate in the presence of citric acid (3 mol%) at room temperature in ethanol to
give ester 2, which was then chlorinated using N,N-dimethylformamide (DMF) as a
catalyst in refluxing phosphorous oxychloride, affording the intermediate 3 in
quantitative yield.
Access to quinoxaline-2-carboxylic acids, and potassium carboxylate salts was then
performed using a two-step synthetic pathway. Thus, intermediate 3 undergoes initial
nucleophilic aromatic substitution with the appropriate amine in presence of p-TSA as
a catalyst in refluxing absolute ethanol to give esters 4a-f and 4h-i [28]. For
compound 4f, a supplementary step of tert-butyloxycarbonyl (Boc) group
deprotection, using trifluoroacetic acid in dichloromethane (DCM), was necessary to
obtain the derivative 4g. Then, hydrolysis of the intermediate ethyl esters 4a-c, 4e,
and 4g-i with potassium carbonate in refluxing 80% aqueous methanol was
performed. The potassium salts 5b and 5g were thus obtained without any treatment.
A subsequent acidification with a citric acid aqueous solution was realized to afford
acids 1, 5c-e, and 5h-i.
Ester 4d was saponified using a 10% aqueous sodium hydroxide solution in refluxing
ethanol, leading, after acidification with a citric acid aqueous solution, to the
corresponding carboxylic acid 5d.
Finally, synthesis of the carboxamide 6 was achieved from acid 5c, using N-
methylmorpholine and ethyl chloroformate in dichloromethane at 0 °C, followed by
the addition of a 28% ammonium hydroxide solution.
3. Results and discussion
3.1. Enzymatic Assays
3.1.1. Pim-1 enzymatic activity inhibition

ACCEPTED MANUSCRIPT
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
Compounds were first evaluated for their efficacy to inhibit the in vitro enzymatic
activity of HsPim-1, using a luminescence-based kinase assay [29]. Compounds that
displayed HsPim-1 IC50 > 10 µM were considered inactive.
To get closer insight into the potential binding mode of our compounds within the
ATP binding pocket of HsPim-1, we first decided to structurally vary the substitution
patterns of the quinoxaline scaffold of 1 in position 3 (Table 1). Docking analysis
revealed that the hydroxyl moiety of the phenyl ring in this position could be able to
form an H-bond interaction with the carboxylate side chain of residue Asp186 of the
HsPim-1 ATP binding pocket. Interestingly, it appears that position of the hydroxyl
group on the phenyl ring strongly modulates compound activity, since modification
from meta (1) to para (5e) or ortho (5i) position reduced significantly the inhibitory
potency (Table 1, entries 2, 11, and 17). Thus, compounds 5e and 5i maintained a
submicromolar activity on HsPim-1 (IC50 of 0.29 µM and 0.76 µM, respectively) but
were less potent (4-fold, 10-fold, respectively) than lead compound 1 (IC50 of 74 nM).
Surprisingly, the replacement of the hydroxyl moiety of compound 1 by an amino
group, able to form an H-bond with Asp186, led to a significant loss of potency (5b,
IC50 of 2.80 µM, 38-fold lower). However, as expected, the substitution by a
morpholino group, suppressing the formation of an H-bond, was not favorable for the
activity, as shown by derivative 5c (IC50 of 1.01 µM). Again, the para substitution on
the phenyl ring was deleterious for the activity, as shown by derivative 5g which was
> 3.5-fold less active than its “meta” analogue 5b, and by the inactive compound 5h
(Table 1, entries 4, 13 and 15).
Finally, replacement of the 3-hydroxyphenyl moiety of compound 1 by an 1H-indol-
5-yl group (5d) led to a drastic loss of potency (Table 1, entry 9).
Docking analysis also suggested that the carboxylate function in position 2 of the
quinoxaline scaffold of compound 1 was crucial for the activity, establishing, notably,
a key salt bridge with the catalytic Lys67. However, carboxylic acids are known to be
responsible for limited permeability across biological membranes, metabolic
instability, and potential adverse effects [30]. To circumvent these issues, and to
confirm the results of the modeling studies, we evaluated ethyl ester derivatives (4a-e
and 4g-i) of all synthesized acids and carboxylate salts, and we replaced the acid
group of compound 5c by a carboxamide isosteric moiety (6). Both ester and
carboxamide functions are not able to form a salt bridge like the carboxylic acid
group, resulting in a complete loss of HsPim-1 enzymatic activity inhibition (Table 1,

ACCEPTED MANUSCRIPT
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
entries 1, 3, 5, 7, 8, 10, 12, 14 and 16), highlighting the highly critical role of this type
of interaction in position 2 of the quinoxaline ring.
3.1.2. Selectivity over a panel of mammalian protein kinases
A selectivity profile of the most active HsPim-1 inhibitors was performed. In that
purpose, most promising candidates were further evaluated in an expanded panel of
mammalian protein kinases such as RnDYRK1A, HsCDK2/CyclinA,
HsCDK9/CyclinT, HsHaspin, MmCLK1, SscCK1δ/ε and SscGSK3α/β. Inhibition
values were determined using a luminescence-based kinase assay [29].
Similar inhibition trends were observed with 6 of the mammalian kinases tested
(HsCDK2/CyclinA, HsCDK9/CyclinT, HsHaspin, MmCLK1, SscCK1δ/ε and
SscGSK3α/β), with IC50 > 10 µM in every case, for each compound evaluated,
suggesting an interesting selectivity profile against these potential off-target kinases
(Table 2). Notably, we observed > 130-fold differences between IC50 values for
HsPim-1 over these mammalian kinases for our lead inhibitor 1.
In contrast, quinoxalines 1, 5b, 5e and 5i displayed a micromolar to submicromolar
inhibition of RnDYRK1A (table 2, entries 1, 2, 4 and 5). Lead 1 exhibited
nevertheless an IC50 value at least 3.5-fold higher for RnDYRK1A than for HsPim-1.
Interestingly, compound 5c, despite a less potent activity profile against HsPim-1, was
more than 10-fold selective with respect to this kinase (Table 2, entry 3).
3.2. In vitro cell-based assays
Most active HsPim-1 inhibitors were then tested in vitro on the human CML cell line
KU812, overexpressing Pim-1. Cytotoxic effects were evaluated using a MTT assay,
and living cells were also counted with the trypan blue dye exclusion method.
As expected, a same trend was observed between HsPim-1 enzymatic activity
inhibition and in vitro cytotoxic potency. Indeed, quinoxalines with a good level of
activity on HsPim1 (IC50 of 0.074 to 2.80 µM) also exhibited in vitro cytotoxic effects
on KU812 cell line with EC50 values ranging from 38.9 ± 3.4 µM to 177.5 ± 13.1 µM
(Table 2). Moreover, the best HsPim-1 inhibitor 1 (IC50 of 74 nM), was also the most
cytotoxic compound (EC50 of 38.9 ± 3.4 µM).

ACCEPTED MANUSCRIPT
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
4. Conclusion
In this study, we identified a new series of quinoxaline-2-carboxylic acids and
analogues, exhibiting a potent activity against the HsPim-1 oncoprotein. Among the 17
compounds synthesized, 5 significantly blocked HsPim-1 with IC50 values in the
submicromolar to low micromolar range. In particular, lead compound 1 showed the
best inhibitory effect against HsPim-1, with an IC50 value of 74 nM. SAR in positions 2
and 3 of the quinoxaline scaffold confirmed the molecular modeling studies,
highlighting the crucial role of the carboxylic acid function in position 2 for the HsPim-
1 inhibitory activity of these compounds. In vitro studies of the 5 most potent inhibitors
on the human CML cell line KU812, confirmed their interest, with antitumor activities
at micromolar concentrations.
This series of compounds, and particularly lead 1, could therefore represent new
attractive drug candidates for extending further pharmacomodulation studies in a way to
improve their potency and selectivity profile.
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
5. Experimental section
5.1. General remarks
All solvents were anhydrous reagents from commercial sources. Unless otherwise
noted, all chemicals and reagents were obtained commercially and used without
purification. 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 uncorrected. 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, m = multiplet and bs =
broad signal were used throughout. Known compounds were prepared according to

ACCEPTED MANUSCRIPT
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
literature procedures: tert-butyl (3-aminophenyl)carbamate, and tert-butyl (4-
aminophenyl)carbamate [31], 4-(1-methylpiperidin-4-yl)aniline [32].
5.2. Chemistry
5.2.1. Ethyl 3-oxo-3,4-dihydroquinoxaline-2-carboxylate (2)
A mixture of o-phenylenediamine (708 mg, 6.55 mmol), diethyl 2-oxomalonate (1.14 g,
6.55 mmol) and citric acid (41 mg, 0.20 mmol) in ethanol (13 mL) was stirred
magnetically at room temperature for 10 min. Ethanol was then evaporated under
reduced pressure, and the residue was stirred with crushed ice for 5 min, filtered and
dried under vacuum to give compound 2 (1.13 g, 79%) as a beige solid.
Mp 168.8 °C. 1H NMR (300 MHz, CDCl3) δ 12.85 (bs, 1H, NH), 7.97 (dd, 1H, J = 8.1,
1.2 Hz), 7.64 (ddd, 1H, J = 8.1, 7.2, 1.2 Hz), 7.48 (dd, 1H, J = 8.1, 1.2 Hz), 7.42 (ddd,
1H, J = 8.1, 7.2, 1.2 Hz), 4.56 (q, 2H, J = 7.2 Hz, CH2), 1.49 (t, 3H, J = 7.2 Hz, CH3).
13C NMR (75 MHz, CDCl3) δ 163.4, 154.6, 148.4, 132.8, 132.2, 132.1, 130.2, 125.0,
116.5, 62.6, 14.2.
5.2.2. Ethyl 3-chloroquinoxaline-2-carboxylate (3)
Into a dry three-neck round bottom flask was introduced compound 2 (218 mg, 1.00
mmol) in phosphorous oxychloride (2 mL) at ice bath temperature. Dimethylformamide
(0.1 mL) was then added at 0 °C and the reaction mixture was refluxed for 30 min.
After cooling, the resulting mixture was diluted with ethyl acetate and washed with a
10% sodium hydroxide solution (2 × 5 mL), and brine (2 × 10 mL). The combined
organic layers were dried over MgSO4, filtered, and evaporated under reduced pressure
to obtain derivative 3 (237 mg, 100%) as a beige solid.
1
Mp 46.4 °C. H NMR (300 MHz, CDCl3) δ 8.18 (m, 1H), 8.07 (m, 1H), 7.92-7.81 (m,
2H), 4.58 (q, 2H, J = 7.2 Hz, CH2), 1.49 (t, 3H, J = 7.2 Hz, CH3). 13C NMR (75 MHz,
CDCl3)δ 163.9, 144.7, 143.9, 142.2, 139.7, 132.6, 131.0, 129.6, 128.3, 63.0, 14.1.
5.2.3. Ethyl 3-((3-hydroxyphenyl)amino)quinoxaline-2-carboxylate (4a)
Method A: a solution of compound 3 (1.11 g, 4.70 mmol), 3-aminophenol (622 mg,
5.70 mmol) and p-TSA, as a catalyst, in absolute ethanol (40 mL) was refluxed for 110

ACCEPTED MANUSCRIPT
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
h. Ethanol was then evaporated under reduced pressure, and the resulting residue was
purified by silica column chromatography using cyclohexane with ethyl acetate gradient
(0-50%) as eluent to give the desired compound 4a (1.0 g, 69%) as a red powder.
Mp 233.3 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.08 (bs, 1H, NH), 9.52 (bs, 1H, OH),
7.99 (dd, 1H, J = 8.4, 0.6 Hz), 7.85-7.75 (m, 2H), 7.61-7.54 (m, 2H), 7.24-7.14 (m, 2H),
6.51 (ddd, 1H, J = 7.4, 2.4, 1.5 Hz), 4.48 (q, 2H, J = 7.2 Hz, CH2), 1.41 (t, 3H, J = 7.2
13
Hz, CH3). C NMR (75 MHz, DMSO-d6) δ 166.1, 158.3, 148.8, 142.4, 140.7, 136.0,
133.4, 132.7, 130.0 (2 × C), 126.7, 126.6, 111.1, 110.6, 107.3, 62.8, 14.5.
5.2.4. Ethyl 3-((3-aminophenyl)amino)quinoxaline-2-carboxylate (4b)
The title compound was synthesized according to the general method A from compound
3 (330 mg, 1.40 mmol) and tert-butyl (3-aminophenyl)carbamate (312 mg, 1.50 mmol)
in absolute ethanol (10 mL). The reaction mixture was refluxed for 110 h. Compound
4b was obtained (139 mg, 32%) as a red powder.
1
Mp 207.7 °C. H NMR (300 MHz, DMSO-d6) δ 10.00 (bs, 1H, NH), 7.97 (d, 1H, J =
8.1 Hz), 7.81-7.77 (m, 2H), 7.58-7.51 (m, 1H), 7.26 (bs, 1H), 7.09-6.97 (m, 2H), 6.32
(m, 1H), 5.17 (bs, 2H, NH2), 4.48 (q, 2H, J = 7.2 Hz, CH2), 1.41 (t, 3H, J = 7.2 Hz,
CH3). 13C NMR (75 MHz, DMSO-d6) δ 166.1, 149.7, 149.0, 142.6, 140.3, 135.9, 133.3,
132.5, 130.0, 129.7, 126.7, 126.4, 109.7, 108.2, 105.8, 62.8, 14.5.
5.2.5. Ethyl 3-((3-morpholinophenyl)amino)quinoxaline-2-carboxylate (4c)
The title compound was synthesized according to the general method A from compound
3 (361 mg, 1.53 mmol) and 3-morpholinoaniline (299 mg, 1.68 mmol) in absolute
ethanol (10 mL). The reaction mixture was refluxed for 110 h. Compound 4c was
obtained (439 mg, 76%) as an orange powder.
1
Mp 170.6 °C. H NMR (300 MHz, DMSO-d6) δ 10.09 (bs, 1H, NH), 7.98 (d, 1H, J =
8.1 Hz), 7.87-7.70 (m, 2H), 7.67-7.47 (m, 2H), 7.34 (d, 1H, J = 7.8 Hz), 7.24 (t, 1H, J =
8.1 Hz), 6.71 (d, 1H, J = 7.8 Hz), 4.48 (q, 2H, J = 6.9 Hz, CH2), 3.77 (m, 4H, 2 × CH2O
13
morpholine), 3.16 (m, 4H, 2 × CH2N, morpholine), 1.41 (t, 3H, J = 6.9 Hz, CH3). C
NMR (75 MHz, DMSO-d6) δ 166.0, 152.2, 148.9, 142.4, 140.4, 136.0, 133.5, 132.6,
130.0, 129.8, 126.7 (2 × C), 111.5, 110.7, 107.1, 66.6 (2 × C), 62.8, 48.9 (2 × C), 14.5.

ACCEPTED MANUSCRIPT
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
5.2.6. Ethyl 3-((1H-indol-5-yl)amino)quinoxaline-2-carboxylate (4d)
The title compound was synthesized according to the general method A from compound
3 (237 mg, 1.00 mmol) and 5-aminoindole (397 mg, 3.00 mmol) in absolute ethanol (10
mL). The reaction mixture was refluxed for 36 h. Compound 4d was obtained (245 mg,
73%) as a red powder.
1
Mp 204.7 °C. H NMR (300 MHz, CDCl3) δ 10.24 (bs, 1H, NH), 8.80 (bs, 1H, NH),
8.26 (s, 1H, indolyl), 8.00 (dd, 1H, J = 8.4, 0.9 Hz), 7.77 (dd, 1H, J = 8.4, 0.9 Hz), 7.66
(ddd, 1H, J = 8.4, 6.9, 0.9 Hz), 7.50-7.38 (m, 3H), 7.23 (t, 1H, J = 2.4 Hz, indolyl),
6.56 (bs, 1H, indolyl), 4.60 (q, 2H, J = 7.2 Hz, CH2), 1.53 (t, 3H, J = 7.2 Hz, CH3). 13C
NMR (75 MHz, CDCl3) δ 166.4, 150.0, 143.6, 136.2, 133.0, 132.8, 131.5, 130.6, 130.1,
128.1, 126.6, 125.4, 125.1, 117.2, 112.8, 111.2, 102.6, 62.9, 14.3.
5.2.7. Ethyl 3-((4-hydroxyphenyl)amino)quinoxaline-2-carboxylate (4e)
The title compound was synthesized according to the general method A from compound
3 (302 mg, 1.28 mmol) and 4-aminophenol (418 mg, 3.83 mmol) in absolute ethanol
(16 mL). The reaction mixture was refluxed for 20 h. Compound 4e was obtained (155
mg, 39%) as a red powder.
Mp 231.8 °C. 1H NMR (300 MHz, DMSO-d6) δ 9.84 (bs, 1H, NH), 9.30 (bs, 1H, OH),
7.94 (d, 1H, J = 8.1 Hz), 7.80-7.60 (m, 4H), 7.51 (m, 1H), 6.80 (d, 2H, J = 8.7 Hz),
4.47 (q, 2H, J = 7.2 Hz, CH2), 1.41 (t, 3H, J = 7.2 Hz, CH3). 13C NMR (75 MHz,
DMSO-d6) δ 166.0, 154.0, 149.2, 142.8, 135.9, 133.3, 132.5, 131.0, 130.0, 126.5,
126.1, 122.7 (2 × C), 115.8 (2 × C), 62.7, 14.5.
5.2.8. Ethyl 3-((4-((tert-butoxycarbonyl)amino)phenyl)amino)quinoxaline-2-carboxylate
(4f)
The title compound was synthesized according to the general method A from compound
3 (95 mg, 0.40 mmol), tert-butyl (4-aminophenyl)carbamate (250 mg, 1.20 mmol) in
absolute ethanol (6.5 mL). The reaction mixture was refluxed for 64 h in a sealed tube.
After purification by silica column chromatography using CH2Cl2 with MeOH gradient
(0-2%) as eluent, compound 4f was obtained (152 mg, 93%) as an orange powder.

ACCEPTED MANUSCRIPT
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
Mp 198.1 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.00 (bs, 1H, NH), 9.35 (bs, 1H, NH),
8.00-7.40 (m, 8H), 4.48 (q, 2H, J = 7.2 Hz, CH2), 1.49 (s, 9H, 3 × CH3), 1.41 (t, 3H, J =
7.2 Hz, CH3CH2O).
5.2.9. Ethyl 3-((4-aminophenyl)amino)quinoxaline-2-carboxylate (4g)
To a solution of compound 4f (37 mg, 0.09 mmol) in CH2Cl2 (5 mL) was added
dropwise trifluoroacetic acid (1 ml, 13.06 mmol). The mixture was stirred at room
temperature for 6 h. The resulting mixture was made alkaline with a saturated sodium
carbonate solution and extracted with CH2Cl2. The combined organic layers were dried
over MgSO4, filtered, and evaporated under reduced pressure to give the desired
derivative 4g (27 mg, 96%) as a red powder.
Mp 203.3 °C. 1H NMR (300 MHz, CDCl3) δ 10.06 (bs, 1H, NH), 8.01 (dd, 1H, J = 8.4,
1.2 Hz), 7.80-7.64 (m, 4H), 7.44 (ddd, 1H, J = 8.1, 6.6, 1.2 Hz), 6.77 (d, 2H, J = 8.7
Hz), 4.61 (q, 2H, J = 7.2 Hz, CH2), 3.70 (bs, 2H, NH2), 1.55 (t, 3H, J = 7.2 Hz, CH3).
13C NMR (75 MHz, CDCl3) δ 166.4, 149.7, 143.6, 142.6, 136.2, 132.8, 130.6, 130.5,
130.2, 126.6, 125.4, 122.4 (2 × C), 115.5 (2 × C), 62.9, 14.3.
5.2.10. Ethyl 3-((4-(1-methylpiperidin-4-yl)phenyl)amino)quinoxaline-2-carboxylate
(4h)
The title compound was synthesized according to the general method A from compound
3 (45 mg, 0.19 mmol) and 4-(1-methylpiperidin-4-yl)aniline (40 mg, 0.21 mmol) in
absolute ethanol (1.5 mL). The reaction mixture was refluxed for 112 h. After
purification by silica column chromatography using CH2Cl2 with MeOH gradient (0-
10%) as eluent, compound 4h was obtained (47 mg, 63%) as an orange powder.
1
Mp 127 °C. H NMR (300 MHz, CDCl3) δ 10.28 (bs, 1H, NH), 8.02 (dd, 1H, J = 8.1,
1.2 Hz), 7.84 (d, 2H, J = 8.4 Hz), 7.77 (dd, 1H, J = 8.1, 1.2 Hz), 7.69 (ddd, 1H, J = 8.1,
6.6, 1.2 Hz), 7.47 (ddd, 1H, J = 8.1, 6.6, 1.2 Hz), 7.26 (d, 2H, J = 8.4 Hz), 4.59 (q, 2H,
J = 7.2 Hz, CH2), 3.30 (d, 2H, J = 11.7 Hz), 2.70-2.45 (m, 6H), 2.24-2.09 (m, 2H), 1.96
(d, 2H, J = 11.7 Hz), 1.53 (t, 3H, J = 7.2 Hz, CH3). 13C NMR (75 MHz, CDCl3) δ
166.4, 149.3, 143.2, 139.4, 137.7, 136.4, 132.9, 130.5, 130.2, 127.3 (2 × C), 126.7,
125.9, 120.5 (2 × C), 63.0, 55.7 (2 × C), 45.1, 40.4, 31.9 (2 × C), 14.3.

ACCEPTED MANUSCRIPT
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
5.2.11 Ethyl 3-((2-hydroxyphenyl)amino)quinoxaline-2-carboxylate (4i)
The title compound was synthesized according to the general method A from compound
3 (239 mg, 1.01 mmol) and 2-aminophenol (121 mg, 1.11 mmol) in absolute ethanol
(10 mL). The reaction mixture was refluxed for 110 h. Compound 4i was obtained (156
mg, 50%) as an orange powder.
1
Mp 166.6 °C. H NMR (300 MHz, DMSO-d6) δ 10.66 (bs, 1H, NH), 10.16 (bs, 1H,
OH), 8.89 (m, 1H), 7.99 (d, 1H, J = 8.1 Hz), 7.80 (m, 2H), 7.56 (m, 1H), 7.00-6.82 (m,
3H), 4.49 (q, 2H, J = 7.2 Hz, CH2), 1.43 (t, 3H, J = 7.2 Hz, CH3). 13C NMR (75 MHz,
DMSO-d6) δ 165.8, 149.0, 147.0, 142.6, 135.9, 133.5, 132.3, 130.1, 128.4, 126.6,
126.5, 123.1, 119.7, 119.6, 114.7, 62.7, 14.6.
5.2.12. 3-((3-Hydroxyphenyl)amino)quinoxaline-2-carboxylic acid (1)
Method B: to ester 4a (72 mg, 0.23 mmol) in aqueous methanol (80%, 10 mL), was
added potassium carbonate (97 mg, 0.70 mmol) 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 saturated citric acid aqueous solution, and extracted with
ethyl acetate. The combined organic layers were dried over MgSO4, filtered, and
evaporated under reduced pressure to yield the acid 1 (33 mg, 50%) as a red powder.
Mp 199.6 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.52 (bs, 1H, NH), 9.51 (bs, 1H, OH),
7.98 (d, 1H, J = 8.4 Hz), 7.84-7.76 (m, 2H), 7.60-7.54 (m, 2H), 7.25-7.14 (m, 2H), 6.50
(d, 1H, J = 7.8 Hz). 13C NMR (75 MHz, DMSO-d6) δ 168.1, 158.3, 149.3, 142.5, 140.7,
136.0, 133.2, 132.9, 130.1, 130.0, 126.6, 126.5, 111.0, 110.5, 107.2. HRMS (ESI) m/z:
[M+H]+ calcd for C15H12N3O3, 282.2742; found, 282.0873.
5.2.13. Potassium 3-((3-aminophenyl)amino)quinoxaline-2-carboxylate (5b)
Method C: to ester 4b (82 mg, 0.26 mmol) in aqueous methanol (80%, 10 mL) was
added potassium carbonate (37 mg, 0.26 mmol), and the reaction mixture was refluxed
for 4 h. After cooling, the solvent was removed under reduced pressure, and freeze-
dried to obtain compound 5b (84 mg, 100%) as an orange powder.
Mp > 375 °C. 1H NMR (300 MHz, DMSO-d6) δ 13.15 (bs, 1H, NH), 7.82 (dd, 1H, J =
8.4, 1.5 Hz), 7.65 (dd, 1H, J = 7.2, 1.8 Hz), 7.58 (td, 1H, J = 7.2, 1.5 Hz), 7.38 (ddd,
1H, J = 8.4, 7.2, 1.8 Hz), 7.21 (t, 1H, J = 1.8 Hz), 7.14 (dd, 1H, J = 7.8, 1.8 Hz), 6.97

ACCEPTED MANUSCRIPT
13
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
(t, 1H, J = 7.8 Hz), 6.22 (dd, 1H, J = 7.8, 1.8 Hz), 5.04 (bs, 2H, NH2). C NMR (75
MHz, DMSO-d6) δ 166.7, 150.4, 149.6, 143.2, 141.6, 141.5, 136.6, 130.2, 129.5 (2 ×
C), 125.9, 124.4, 108.5, 107.5, 104.9. HRMS (ESI) m/z: [M+H]+ calcd for C15H13N4O2,
281.2895; found, 281.1032.
5.2.14. 3-((3-Morpholinophenyl)amino)quinoxaline-2-carboxylic acid (5c)
The title compound was synthesized according to the general method B from compound
4c (150 mg, 0.40 mmol) and potassium carbonate (218 mg, 1.58 mmol) in aqueous
methanol (80%, 5 mL). The reaction mixture was refluxed for 4 h. Compound 5c was
obtained (139 mg, 100%) as an orange powder.
1
Mp 186.6 °C. H NMR (300 MHz, DMSO-d6) δ 10.49 (bs, 1H, NH), 7.97 (m, 1H),
7.82-7.74 (m, 2H), 7.67 (s, 1H), 7.56 (ddd, 1H, J = 8.4, 6.3, 2.1 Hz) 7.32 (d, 1H, J = 8.7
Hz), 7.23 (t, 1H, J = 8.1 Hz), 6.69 (dd, 1H, J = 8.1, 1.5 Hz), 3.77 (m, 4H, 2 × CH2O
13
morpholine), 3.16 (m, 4H, 2 × CH2N, morpholine). C NMR (75 MHz, DMSO-d6) δ
168.0, 152.2, 149.4, 142.5, 140.5, 135.9, 133.2, 132.9, 129.9, 129.7, 126.7, 126.4,
111.4, 110.5, 106.9, 66.6 (2 × C), 49.0 (2 × C). HRMS (ESI) m/z: [M+H]+ calcd for
C19H19N4O3, 351.3793; found, 351.1453.
5.2.15. 3-((1H-Indol-5-yl)amino)quinoxaline-2-carboxylic acid (5d)
To ester 4d (81 mg, 0.24 mmol) in ethanol (5 mL), was added a 10% sodium hydroxide
solution (2 mL) and the reaction mixture was refluxed for 18 h. Ethanol was then
evaporated under reduced pressure, and the resulting residue was acidified to pH 2 with
a 15% citric acid solution and extracted with ethyl acetate. The organic layers were then
washed with water and brine, dried over MgSO4, filtered, and evaporated under reduced
pressure. The residue was finally purified by silica column chromatography using
CH2Cl2 with MeOH gradient (0-20%) as eluent to give the acid 5d (30 mg, 41%) as a
red powder.
1
Mp 244.4 °C. H NMR (300 MHz, DMSO-d6) δ 12.45 (bs, 1H, NH), 11.07 (bs, 1H,
NH), 8.70-6.80 (m, 8H), 6.44 (bs, 1H, indolyl). 13C NMR (75 MHz, DMSO-d6) δ
168.6, 151.4, 133.6, 133.1, 132.5, 129.9, 129.4, 129.1 (3 × C), 127.1, 126.9, 125.7,
116.6, 112.9, 111.6, 102.5. HRMS (ESI) m/z: [M+H]+ calcd for C17H13N4O2, 305.3109;
found, 305.1034.

ACCEPTED MANUSCRIPT
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
5.2.16. 3-((4-Hydroxyphenyl)amino)quinoxaline-2-carboxylic acid (5e)
The title compound was synthesized according to the general method B from compound
4e (90 mg, 0.29 mmol) and potassium carbonate (80 mg, 0.58 mmol) in aqueous
methanol (80%, 10 mL). The reaction mixture was refluxed for 4 h. Compound 5e was
obtained (79 mg, 97%) as an orange powder.
Mp 197.4 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.22 (bs, 1H, NH), 9.31 (bs, 1H, OH),
7.93 (dd, 1H, J = 8.1, 0.9 Hz), 7.78-7.63 (m, 4H), 7.51 (ddd, 1H, J = 8.1, 6.6, 1.5 Hz),
13
6.80 (d, 2H, J = 8.7 Hz). C NMR (75 MHz, DMSO-d6) δ 168.1, 153.9, 149.6, 142.9,
135.8, 133.2, 132.7, 131.1, 129.9, 126.4, 125.9, 122.5 (2 × C), 115.8 (2 × C). HRMS
(ESI) m/z: [M+H]+ calcd for C15H12N3O3, 282.2742; found, 282.0873.
5.2.17. Potassium 3-((4-aminophenyl)amino)quinoxaline-2-carboxylate (5g)
The title compound was synthesized according to the general method C from compound
4g (16 mg, 0.05 mmol) and potassium carbonate (7 mg, 0.05 mmol) in aqueous
methanol (80%, 5 mL). The reaction mixture was refluxed for 4 h. Compound 5g was
obtained (14 mg, 87%) as a red powder.
1
Mp > 375 °C. H NMR (300 MHz, DMSO-d6) δ 12.80 (bs, 1H, NH), 7.84 (d, 1H, J =
7.8 Hz), 7.60-7.50 (m, 4H), 7.32 (m, 1H), 6.60 (d, 2H, J = 8.4 Hz), 4.84 (bs, 2H NH2).
13C NMR (75 MHz, DMSO-d6) δ 167.0, 150.4, 144.2, 143.0, 142.0, 136.4, 130.4,
130.2, 129.5, 125.6, 123.8, 120.9 (2 × C), 114.7 (2 × C). HRMS (ESI) m/z: [M+H]+
calcd for C15H13N4O2, 281.2895; found, 281.1032.
5.2.18. 3-((4-(1-Methylpiperidin-4-yl)phenyl)amino)quinoxaline-2-carboxylic acid (5h)
The title compound was synthesized according to the general method B from compound
4h (42 mg, 0.11 mmol) and potassium carbonate (45 mg, 0.32 mmol) in aqueous
methanol (80%, 5 mL). The reaction mixture was refluxed for 4 h. Compound 5h was
obtained (19 mg, 49%) as a yellow powder.
Mp 321 °C. 1H NMR (300 MHz, DMSO-d6) δ 13.33 (bs, 1H, NH), 8.00 (d, 1H, J = 7.8
Hz), 7.85 (d, 2H, J = 8.4 Hz), 7.68-7.58 (m, 2H), 7.54-7.31 (m, 1H), 7.23 (d, 2H, J =
8.4 Hz), 2.86 (d, 2H, J = 11.4 Hz), 2.46-2.36 (m, 1H), 2.19 (s, 3H, CH3), 1.95 (td, 2H, J
= 11.4, 1.8 Hz), 1.77-1.62 (m, 4H). 13C NMR (75 MHz, DMSO-d6) δ 166.9, 150.4,

ACCEPTED MANUSCRIPT
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
142.1, 141.7, 140.1, 138.8, 136.4, 130.7, 129.8, 127.5 (2 × C), 125.9, 124.7, 119.3 (2 ×
C), 56.4 (2 × C), 46.7, 41.2, 33.7 (2 × C). HRMS (ESI) m/z: an abundant fragment ion
has been observed at m/z 213.1022 that have been attributed to the loss of the 1-
methylpiperidin-4-yl)phenyl)amino moiety from the quinoxaline ring to form the ion
[C9H6N2O2 +K]+ (m/z calcd for C9H6KN2O2, 213.2545; found, 213.1022).
5.2.19. 3-((2-Hydroxyphenyl)amino)quinoxaline-2-carboxylic acid (5i)
The title compound was synthesized according to the general method B from compound
4i (101 mg, 0.33 mmol) and potassium carbonate (135 mg, 0.98 mmol) in aqueous
methanol (80%, 10 mL). The reaction mixture was refluxed for 4 h. Compound 5i was
obtained (60 mg, 65%) as a red powder.
Mp 191.1 °C. 1H NMR (300 MHz, DMSO-d6) δ 14.00 (bs, 1H, COOH), 10.84 (bs, 1H,
NH), 10.11 (bs, 1H, OH), 8.88 (m, 1H), 7.97 (d, 1H, J = 8.1 Hz), 7.80 (m, 2H), 7.56 (m,
13
1H), 7.00-6.68 (m, 3H). C NMR (75 MHz, DMSO-d6) δ 167.1, 148.7, 146.5, 142.2,
135.3, 132.7, 132.4, 129.4, 127.9, 126.1, 125.8, 122.5, 119.2, 119.0, 114.2. HRMS
(ESI) m/z: [M+H]+ calcd for C15H12N3O3, 282.2742; found, 282.0871.
5.2.20. 3-((3-Morpholinophenyl)amino)quinoxaline-2-carboxamide (6)
To a solution of compound 5c (50 mg, 0.14 mmol) and N-methylmorpholine (31 µL,
0.29 mmol) in dichloromethane (5 mL) at 0 °C, was added ethyl chloroformate (21 µL,
0.21 mmol). The reaction mixture was stirred magnetically at 0 °C for 1 h, and a 28%
solution of ammonium hydroxide (5 mL) was added. The reaction was stirred overnight
at room temperature and extracted with dichloromethane. The organic layer was then
washed with water and brine, dried over MgSO4, filtered, and evaporated under reduced
pressure to give the carboxamide 6 (50 mg, 100%) as a red powder.
1
Mp 203.9 °C. H NMR (300 MHz, DMSO-d6) δ 11.52 (bs, 1H, NH), 8.75 (bs, 1H,
NH2), 8.26 (bs, 1H, NH2), 7.93 (d, 1H, J = 8.1 Hz), 7.82-7.50 (m, 2H), 7.69 (bs, 1H),
7.56 (m, 1H), 7.32-7.20 (m, 2H), 6.69 (d, 1H, J = 8.1 Hz), 3.78 (m, 4H, 2 × CH2O
13
morpholine), 3.17 (m, 4H, 2 × CH2N, morpholine). C NMR (75 MHz, DMSO-d6) δ
168.5, 152.2, 149.4, 142.7, 140.6, 135.3, 133.1, 132.8, 129.8, 129.6, 126.7, 126.3,
111.1, 110.3, 106.6, 66.6 (2 × C), 49.0 (2 × C). HRMS (ESI) m/z: [M+H]+ calcd for
C19H20N5O2, 350.3946; found, 350.1613.

ACCEPTED MANUSCRIPT
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
5.3. Molecular Modeling
Molecular modeling studies were performed using SYBYL-X 1.3 software [33] running
on a Dell precision T3400 workstation. The three-dimensional structure of compound 1
(under its carboxylate form to imitate physiological conditions) was built from a
standard fragments library and optimized using the Tripos force field [34] 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 AMP-PNP at 1.6 Å resolution (PDB ID 3A99) [35] 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 compound 1 into ATP-binding site was performed using GOLD
software [36]. The most stable docking model was selected according to the best scored
conformation predicted by the GoldScore scoring function. Finally, the complexe was
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.
5.4. Biology
5.4.1. Mammalian protein kinase assays
Kinase enzymatic activities were assayed in 384-well plates using the ADP-GloTM assay
kit (Promega, Madison, WI) according to the recommendations of the manufacturer.
This assay is a luminescent ADP detection assay that provides a homogeneous and
high-throughput screening method to measure kinase activity by quantifying the amount
of ADP produced during a kinase reaction. Briefly, the reactions were carried out in a
final volume of 5 µL for 30 min at 30 °C in ADP-Glo buffer and 10 µM ATP (40 mM
Tris pH 7.5, 20 mM MgCl2 and 0.1 mg/mL of BSA). After that, 5 µL of ADP-GloTM
Kinase Reagent was added to stop the kinase reaction. After an incubation time of 50
min at room temperature (rt), 10 µL of Kinase Detection Reagent was added for one

ACCEPTED MANUSCRIPT
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
hour at rt. The transmitted signal was measured using the Envision (PerkinElmer,
Waltham, MA) microplate luminometer and expressed in Relative Light Unit (RLU). In
order to determine the half maximal inhibitory concentration (IC50), the assays were
performed in triplicate in the absence or presence of increasing doses of the tested
compounds. 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 (human proto-
oncogene, recombinant, expressed in bacteria) was assayed with 0.8 µg/µL of histone
H1 (Sigma #H5505) as substrate; 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 µg/µL of
the following peptide: KKISGRLSPIMTEQ as substrate; HsCDK2/CyclinA (cyclin-
dependent kinase-2, human, kindly provided by Dr. A. Echalier-Glazer, Leicester, UK)
was assayed with 0.8 µg/µL of histone H1 as substrate; HsCDK9/CyclinT (human,
recombinant, expressed by baculovirus in Sf9 insect cells) was assayed with 0.27 µg/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
µg/µL of
Histone
H3
(1-21)
peptide
(ARTKQTARKSTGGKAPRKQLA) as substrate; MmCLK1 (from Mus musculus,
recombinant, expressed in bacteria) was assayed with 0.027 µg/µL of the following
peptide: GRSRSRSRSRSR as substrate; SscCK1δ/ε (casein kinase 1δ/ε, porcine brain,
native, affinity purified) was assayed with 0.022 µg/µL of the following peptide:
RRKHAAIGSpAYSITA (“Sp” stands for phosphorylated serine) as CK1-specific
substrate; SscGSK-3α/β (glycogen synthase kinase-3, porcine brain, native, affinity
purified) isoforms were assayed with 0.010 µg/µL of GS-1 peptide, a GSK-3-selective
substrate (YRRAAVPPSPSLSRHSSPHQSpEDEEE). To validate the kinase assay,
model inhibitors were used for each tested enzyme: Staurosporine from Streptomyces
sp. (#S5921, purity ≥95%, Sigma-Aldrich) for SscCK1δ/ε; Indirubin-3’-oxime (#I0404,
purity ≥98%, Sigma-Aldrich) for SscGSK-3α/β, HsPim-1, human Cyclin-dependent
kinases, RnDYRK1A and MmCLK1; CHR-6494 (#SML0648, purity ≥98%, Sigma-
Aldrich) for Haspin.

ACCEPTED MANUSCRIPT
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
5.4.2. Cell Cultures and Reagents
KU812 cell lines were obtained from the American Type Culture Collection (ATCC)
and maintained according to the supplier’s recommendations. Cell lines were cultured
in RPMI, with 10% fetal bovine serum, 1% glutamine, and 1% penicillin/streptomycin
at 37 °C and 5% CO2.
5.4.3. Cell Proliferation Assays
Cell viability and proliferation were studied using a MTT cell proliferation assay.
Briefly, 0.2 × 105 cells were incubated in 100 µL of X-Vivo red phenol free medium
(Lonza, Basel, Switzerland) in 96 well plates. In initial screening assays, cells were
incubated with 10 µM of each compound (quinoxalines stock solution at 50 mM in
DMSO) for 24, 48, and 72 h. Imatinib mesylate (Selleckchem, stock solution at 10 mM
in DMSO) was used as reference. To determine the concentration-effect of the
molecules, cells were treated with concentrations ranging from 100 nM to 50 µM for 24
or 48 h. Cells were incubated with 10 µL of MTT working solution (5 g/L of
methylthiazolyldiphenyl-tetrazolium bromide) during 4 h. Cells were then lysed
overnight at 37 °C with 100 µL of 10% SDS and 0.003% HCl. Optical density (OD) at
570 nm was measured using a spectrophotometer (Dynex, Chantilly, United States).
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 ± standard errors of the
mean.
Acknowledgments
This work was supported 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 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. The

ACCEPTED MANUSCRIPT
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
authors also thank the Cancéropôle Grand Ouest (axis: Natural sea products in cancer
treatment), GIS IBiSA (Infrastructures en Biologie Santé et Agronomie) and
Biogenouest (Western France life science and environment core facility network) for
supporting KISSf screening facility. The authors wish to thank the Région Centre Val de
Loire for financial support.
Supplementary materials
Electronic Supplementary Information (ESI) available: spectroscopic data for selected
compounds.
References
[1] H. T. Cuypers, G. Selten, W. Quint, M. Zijlstra, E. R. Maandag, W. Boelens, P.
Van Wezenbeek, C. Melief, A. Berns, Murine leukemia virus-induced T-cell
lymphomagenesis: integration of proviruses in a distinct chromosomal region,
Cell. 37 (1984) 141-150.
[2] T. Mochizuki, C. Kitanaka, K. Noguchi, T. Muramatsu, A. Asai, Y. J. Kuchino,
Physical and functional interactions between Pim-1 kinase and Cdc25A
phosphatase. Implications for the Pim-1-mediated activation of the c-Myc
signaling pathway, J. Biol. Chem. 274 (1999) 18659-18666.
[3] M. Bachmann, C. Kosan, P. X. Xing, M. Montenarh, I. Hoffmann, T. Möröy, The
oncogenic serine/threonine kinase Pim-1 directly phosphorylates and activates the
G2/M specific phosphatase Cdc25C, Int. J. Biochem. Cell Biol. 38 (2006) 430-
443.
[4] R. Amson, F. Sigaux, S. Przedborski, G. Flandrin, D. Givoland, A. Telerman, The
human protooncogene product p33pim is expressed during fetal hematopoiesis
and in diverse leukemias, Proc. Natl. Acad. Sci. U. S. A. 86 (1989) 8857-8861.

ACCEPTED MANUSCRIPT
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
[5] E. D. Hsi, S. H. Jung, R. Lai, J. L. Johnson, J. R. Cook, D. Jones, S. Devos, B. D.
Cheson, L. E. Damon, J. Said, Ki67 and PIM1 expression predict outcome in
mantle cell lymphoma treated with high dose therapy, stem cell transplantation
and rituximab: a Cancer and Leukemia Group B 59909 correlative science study,
Leuk. Lymphoma. 49 (2008) 2081-2090.
[6] S. J. Guo, X. P. Mao, J. X. Chen, B. Huang, C. Jin, Z. B. Xu, S. P. Qiu,
Overexpression of Pim-1 in bladder cancer, J. Exp. Clin. Cancer Res. 29 (2010)
161.
[7] S. M. Dhanasekaran, T. R. Barrette, D. Ghosh, R. Shah, S. Varambally, K.
Kurachi, K. J. Pienta, M. A. Rubin, A. M. Chinnaiyan, Delineation of prognostic
biomarkers in prostate cancer, Nature. 412 (2001) 822-826.
[8] F. Brasó-Maristany, S. Filosto, S. Catchpole, R. Marlow, J. Quist, E. Francesch-
Domenech, D. A. Plumb, L. Zakka, P. Gazinska, G. Liccardi, P. Meier, A. Gris-
Oliver, M. C. Cheang, A. Perdrix-Rosell, M. Shafat, E. Noël, N. Patel, K.
McEachern, M. Scaltriti, P. Castel, F. Noor, R. Buus, S. Mathew, J. Watkins, V.
Serra, P. Marra, A. Grigoriadis, A. N. Tutt, PIM1 kinase regulates cell death,
tumor growth and chemotherapy response in triple-negative breast cancer, Nat.
Med. 22 (2016) 1303-1313.
[9] B. Yan, E. X. Yau, S. Samanta, C. W. Ong, K. J. Yong, L. K. Ng, B.
Bhattacharya, K. H. Lim, R. Soong, K. G. Yeoh, N. Deng, P. Tan, Y. L. Lam, M.
Salto-Tellez, Clinical and therapeutic relevance of PIM1 kinase in gastric cancer,
Gastric Cancer. 15 (2012) 188-197.
[10] Y. Tursynbay, J. Zhang, Z. Li, T. Tokay, Z. Zhumadilov, D. Wu, Y. Xie, Pim-1
kinase as cancer drug target: An update, Biomed. Rep. 4 (2016) 140-146.

ACCEPTED MANUSCRIPT
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
[11] H. Mikkers, M. Nawijn, J. Allen, C. Brouwers, E. Verhoeven, J. Jonkers, A.
Berns, Mice Deficient for All PIM Kinases Display Reduced Body Size and
Impaired Responses to Hematopoietic Growth Factors, Mol. Cell Biol. 24 (2004)
6104-6115.
[12] R. A. Darby, A. Unsworth, S. Knapp, I. D. Kerr, R. Callaghan, Overcoming
ABCG2-mediated drug resistance with imidazo-[1,2-b]-pyridazine-based Pim1
kinase inhibitors, Cancer Chemother. Pharmacol. 76 (2015) 853-864.
[13] Y. Xie, S. Bayakhmetov, PIM1 kinase as a promise of targeted therapy in prostate
cancer stem cells, Mol. Clin. Oncol. 4 (2016) 13-17.
[14] C. Liang, Y. Y. Li, Use of regulators and inhibitors of Pim-1, a serine/threonine
kinase, for tumour therapy (review), Mol. Med. Rep. 9 (2014) 2051-2060.
[15] F. Anizon, A. A. Shtil, V. N. Danilenko, P. Moreau, Fighting tumor cell survival:
advances in the design and evaluation of Pim inhibitors, Curr. Med. Chem. 17
(2010) 4114-4133.
[16] Y. Xu, B. G. Brenning, S. G. Kultgen, J. M. Foulks, A. Clifford, S. Lai, A. Chan,
S. Merx, M. V. McCullar, S. B. Kanner, K.-K. Ho, Synthesis and biological
evaluation of pyrazolo[1,5-a]pyrimidine compounds as potent and selective Pim-1
inhibitors, ACS Med. Chem. Lett. 6 (2015) 63-67.
[17] H.-B. Sun, X.-Y. Wang, G.-B. Li, L.-D. Zhang, J. Liu, L.-F. Zhao, Design,
synthesis and biological evaluation of novel C3-functionalized oxindoles as
potential Pim-1 kinase inhibitors, RSC Adv. 5 (2015) 29456-29466.
[18] C. Blanco-Aparicio, A. Carnero, Pim kinases in cancer: diagnostic, prognostic and
treatment opportunities, Biochem. Pharmacol. 85 (2013) 629-643.

ACCEPTED MANUSCRIPT
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
[19] A. Kumar, V. Mandiyan, Y. Suzuki, C. Zhang, J. Rice, J. Tsai, D. R. Artis, P.
Ibrahim, R. Bremer, Crystal structures of proto-oncogene kinase Pim1: a target of
aberrant somatic hypermutations in diffuse large cell lymphoma, J. Mol. Biol. 348
(2005) 183-193.
[20] B. T. Le, M. Kumarasiri, J. R. Adams, M. Yu, R. Milne, M. J. Sykes, S. Wang,
Targeting Pim kinases for cancer treatment: opportunities and challenges, Future
Med. Chem. 7 (2015) 35-53.
[21] L. Juen, M. Brachet-Botineau, C. Parmenon, J. Bourgeais, O. Hérault, F.
Gouilleux, M. C. Viaud-Massuard, G. Prié, New Inhibitor Targeting Signal
Transducer and Activator of Transcription 5 (STAT5) Signaling in Myeloid
Leukemias, J. Med. Chem. 60 (2017) 6119-6136.
[22] Z. Guo, A. Wang, W. Zhang, M. Levit, Q. Gao, C. Barberis, M. Tabart, J. Zhang,
D. Hoffmann, D. Wiederschain, J. Rocnik, F. Sun, J. Murtie, C. Lengauer, S.
Gross, B. Zhang, H. Cheng, V. Patel, L. Schio, F. Adrian, M. Dorsch, C. Garcia-
Echeverria, S. M. Huang, PIM inhibitors target CD25-positive AML cells through
concomitant suppression of STAT5 activation and degradation of MYC oncogene,
Blood. 124 (2014) 1777-1789.
[23] L. S. Chen, S. Redkar, D. Bearss, W. G. Wierda, V. Gandhi, Pim kinase inhibitor,
SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells, Blood. 114
(2009) 4150-4157.
[24] Y. Xiang, B. Hirth, G. Asmussen, H. P. Biemann, K. A. Bishop, A. Good, M.
Fitzgerald, T. Gladysheva, A. Jain, K. Jancsics, J. Liu, M. Metz, A. Papoulis, R.
Skerlj, J.D. Stepp, R.R. Wei, The discovery of novel benzofuran-2-carboxylic
acids as potent Pim-1 inhibitors, Bioorg. Med. Chem. Lett. 21 (2011) 3050-3056.

ACCEPTED MANUSCRIPT
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
[25] J. Bogusz, K. Zrubek, K. P. Rembacz, P. Grudnik, P. Golik, M. Romanowska, B.
Wladyka, G. Dubin, Crystal structure of PIM1 kinase in complex with small-
molecule inhibitor, Sci. Rep. 7 (2017) 13399-13399.
[26] R. Mahesh, A. K. Dhar, T. Sasank T. V. N. V., S. Thirunavukkarasu, T.
Devadoss, Citric acid: An efficient and green catalyst for rapid one pot synthesis
of quinoxaline derivatives at room temperature, Chinese Chemical Letters. 22
(2011) 389-392.
[27] R. Mahesh, T. Devadoss, A. K. Dhar, S. M. Venkatesh, S. Mundra, D. K. Pandey,
S. Bhatt, A. K. Jindal, Ligand-based design, synthesis, and pharmacological
evaluation of 3-methoxyquinoxalin-2-carboxamides as structurally novel
serotonin type-3 receptor antagonists, Arch. Pharm. Chem. Life Sci. 345 (2012)
687-694.
[28] A. Kumar, K. Srivastava, S. R. Kumar, M. I. Siddiqi, S. K. Puri, J. K. Sexana, P.
M. Chauhan, 4-Anilinoquinoline triazines: a novel class of hybrid antimalarial
agents, Eur. J. Med. Chem. 46 (2011) 676-690.
[29] H. Zegzouti, M. Zdanovskaia, K. Hsiao, S. A. Goueli, ADP-Glo: A
Bioluminescent and homogeneous ADP monitoring assay for kinases, Assay Drug
Dev. Technol. 7 (2009) 560-572.
[30] P. Lassalas, B. Gay, C. Lasfargeas, M. J. James, V. Tran, K. G. Vijayendran, K.
R. Brunden, M. C. Kozlowski, C. J. Thomas, A. B. Smith, III, D. M. Huryn, C.
Ballatore, Structure property relationships of carboxylic acid isosteres, J. Med.
Chem. 59 (2016) 3183-3203.
[31] F. Jahani, M. Tajbakhsh, H. Golchoubian, S. Khaksar, Guanidine hydrochloride as
an organocatalyst for N-Boc protection of amino groups, Tetrahedron Lett. 52
(2011) 1260-1264.

ACCEPTED MANUSCRIPT
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
[32] R. Wurz, A. Tasker, S. Tadesse, L. H. Pettus, T. T. Nguyen, F.-T. Hong, B. J.
Herberich, E. Harrington, J. J. Chen, J. Brown, Substituted 7-oxo-pyrido[2,3-d]
pyrimidines and their use for the treatment of EGFR/ErbB2 related disorders,
From PCT Int. Appl., 2014. WO2014134308.
[33] SYBYL-X 1.3, Tripos Associates, Inc. 1699 South Hanley Road, St. Louis, MO
63144, U.S.A.
[34] M. Clarck, R. D. Cramer III, N. Van Opdenbosch, Validation of the general
purpose tripos 5.2 force field, J. Comput. Chem. 10 (1989), 982-1012.
[35] D. Morishita, M. Takami, S. Yoshikawa, R. Katayama, S. Sato, N. Kukimoto-
Niino, T. Umehara, M. Shirouzu, K. Sekimizu, S. Yokoyama, N. Fujita, Cell-
permeable carboxyl-terminal p27Kip1 peptide exhibits anti-tumor activity by
inhibiting Pim-1 kinase, J. Biol. Chem. 286 (2011) 2681-2688.
[36] G. Jones, P. Willet, R. C. Glen, Development and validation of a genetic
algorithm for flexible docking, J. Mol. Biol. 267 (1997) 727-748.

ACCEPTED MANUSCRIPT
781
782
Fig. 1. Chemical structure of compound 1.
783
784

ACCEPTED MANUSCRIPT
Glu121
Phe187
Leu120
Hinge region
Pro123
DFG motif
Lys67
Salt bridge
Asp186
ATP binding site pocket
785
786
Fig. 2. Binding pose found by the docking program GOLD for compound 1 into the
787
788
ATP pocket of Pim-1 (PDB ID 3A99). Hydrogen bonds are indicated as yellow lines.

ACCEPTED MANUSCRIPT
NH2
NH2
N
N
COOEt
Cl
N
COOEt
O
i
ii
N
H
2
3
N
COOEt
Cl
N
N
COOEt
NH
N
N
COOH
NH
v
iii
N
3
Lead 1
4a
OH
OH
N
N
COOEt
N
N
COR2
v or vi
iii
NH
R1
NH
R1
5b-e, 5g-i
4b-f, 4h-i
NHBoc
4b-5b R1 =
4f R1 =
4g R1 =
5c-e, 5h-i R2 = OH
5b, 5g R2 = OK
5c R2 = OH
iv
NH2
N
NH2
NH2
4c-5c, 6 R1 =
5g R1 =
viii
O
6 R2 = NH2
4h-5h R1 =
N
NH
OH
4d-5d R1 =
4e-5e R1 =
4i-5i R1 =
HO
789
790
791
792
793
794
795
796
Scheme 1. Reagents and conditions: (i) diethyl 2-oxomalonate (1 eq), citric acid (3
mol%), EtOH, rt, 10 min, 79%; (ii) DMF (cat.), POCl3, 0°C, and then reflux, 30 min,
100%; (iii) amine (1.1-3 eq), p-toluenesulfonic acid (cat.), EtOH, reflux, 20-112 h, 32-
93%; (iv) TFA (20%), DCM, rt, 6 h, 96%; (v) K2CO3 (1-4 eq), MeOH/H2O (4/1), reflux,
4 h, 49-100%; (vi) NaOH (10%), EtOH, reflux, 18 h, 41%; (vii) ClCOOEt (1.5 eq),
NMM (2 eq), DCM, 0°C, 1 h, and NH4OH, rt, overnight, 100%.