From simple quinoxalines to potent oxazolo[5,4-: F] quinoxaline inhibitors of glycogen-synthase kinase 3 (GSK3)

Article abstract of DOI:10.1039/c9ob02002k

2,7-Disubstituted oxazolo[5,4-f]quinoxalines were synthesized from 6-amino-2-chloroquinoxaline in four steps (iodination at C5, substitution of the chloro group, amidation and copper-catalysed cyclization) affording 28 to 44% overall yields. 2,8-Disubstituted oxazolo[5,4-f]quinoxaline was similarly obtained from 6-amino-3-chloroquinoxaline (39% overall yield). For the synthesis of other oxazolo[5,4-f]quinoxalines, amidation was rather performed before substitution; moreover, time-consuming purification steps were avoided between the amines and the final products (38 to 54% overall yields). Finally, a more efficient method involving merging of the last two steps in a sequential process was developed to access more derivatives (37 to 65% overall yields). Most of the oxazolo[5,4-f]quinoxalines were evaluated for their activity on a panel of protein kinases, and a few 2,8-disubstituted derivatives proved to inhibit GSK3 kinase. While experiments showed an ATP-competitive inhibition on GSK3β, structure-activity relationships allowed us to identify 2-(3-pyridyl)-8-(thiomorpholino)oxazolo[5,4-f]quinoxaline as the most potent inhibitor with an IC₅₀ value of about 5 nM on GSK3α.

Full text of DOI:10.1039/c9ob02002k

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From simple quinoxalines to potent oxazolo[5,4-f]
quinoxaline inhibitors of glycogen-synthase kinase
3 (GSK3)†
Cite this: Org. Biomol. Chem., 2020,
18, 154
Frédéric Lassagne, *a Camille Duguépéroux,a Carlos Roca,b Concepcion Perez,c
Ana Martinez, b,d Blandine Baratte, e,f Thomas Robert,e,f Sandrine Ruchaud,e,f
a
Stéphane Bach,
*
e,f William Erb, a Thierry Roisnel a and Florence Mongin
*
2,7-Disubstituted oxazolo[5,4-f]quinoxalines were synthesized from 6-amino-2-chloroquinoxaline in four
steps (iodination at C5, substitution of the chloro group, amidation and copper-catalysed cyclization)
affording 28 to 44% overall yields. 2,8-Disubstituted oxazolo[5,4-f]quinoxaline was similarly obtained
from 6-amino-3-chloroquinoxaline (39% overall yield). For the synthesis of other oxazolo[5,4-f]quinoxa-
lines, amidation was rather performed before substitution; moreover, time-consuming purification steps
were avoided between the amines and the final products (38 to 54% overall yields). Finally, a more
efficient method involving merging of the last two steps in a sequential process was developed to access
more derivatives (37 to 65% overall yields). Most of the oxazolo[5,4-f]quinoxalines were evaluated for their
activity on a panel of protein kinases, and a few 2,8-disubstituted derivatives proved to inhibit GSK3
kinase. While experiments showed an ATP-competitive inhibition on GSK3β, structure–activity relation-
ships allowed us to identify 2-(3-pyridyl)-8-(thiomorpholino)oxazolo[5,4-f]quinoxaline as the most
potent inhibitor with an IC50 value of about 5 nM on GSK3α.
Received 12th September 2019,
Accepted 27th November 2019
DOI: 10.1039/c9ob02002k
rsc.li/obc
study their biological behaviour in order to find original
properties.
Introduction
Oxazolo[5,4-f]quinoxalines, to our knowledge, have never been
It has been shown in 2011 that 2-quinolinol can be functio-
synthesized before. Their structures can be seen either as a nalized either at its 6- or at its 7-position by nitration at room
pyrazine fused to a benzoxazole or as an oxazole fused to a temperature using potassium nitrate in sulfuric acid or nitric
quinoxaline1 (Fig. 1, top left). Oxazolo[4,5-h]quinolines, which acid in acetic acid, respectively.3 Subsequent conversion to the
are analogues of oxazolo[5,4-f]quinoxalines, lacking one nitro- corresponding chlorides (reflux with phosphorus oxychloride
gen (Fig. 1, top right), have shown anti-allergic activities.2 and phosphorus pentachloride) and nitro reduction3 can
Thus, it is of interest to access oxazolo[5,4-f]quinoxalines and easily furnish appropriate substrates (Fig. 1, bottom) for the
synthesis of various 2,7- and 2,8-disubstituted oxazolo[5,4-f]
quinoxalines.
Recently, our random screening of original polyaromatic
compounds revealed inhibitors of protein kinases.4 Following
aUniv Rennes, CNRS, ISCR (Institut des Sciences Chimiques de Rennes) – UMR 6226,
F-35000 Rennes, France. E-mail: florence.mongin@univ-rennes1.fr
bCentro de Investigaciones Biologicas-CSIC, Ramiro de Maeztu 9, 28040 Madrid,
Spain. E-mail: ana.martinez@csic.es
cInstituto de Química Médica-CSIC, Juan de la Cierva 3, 28006 Madrid, Spain
dCentro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas
(CIBERNED), Instituto Carlos III, 28031 Madrid, Spain
eSorbonne Université, CNRS, FR2424, Plateforme de criblage KISSf (Kinase Inhibitor
Specialized Screening facility), Station Biologique de Roscoff, CS 90074,
29682 Roscoff Cedex, France. E-mail: bach@sb-roscoff.fr
fSorbonne Université, CNRS, UMR 8227, Integrative Biology of Marine Models,
Station Biologique de Roscoff, CS 90074, 29688 Roscoff Cedex, France
†Electronic supplementary information (ESI) available: General information,
experimental procedures and analyses of the compounds and NMR spectra.
CCDC 1947178 (10ba) and 1964834 (10ci). For ESI and crystallographic data in
CIF or other electronic format see DOI: 10.1039/c9ob02002k
Fig. 1 Top: Structures of oxazolo[5,4-f]quinoxaline (left) and oxazolo
[4,5-h]quinoline (right). Bottom: Planned quinoxaline substrates.
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these studies, we report in the present paper the synthesis of assium carbonate, and selected dimethylsulfoxide as the
original oxazolo[5,4-f]quinoxalines and evaluation of their solvent in order to avoid an additional ligand. As expected,
activity on a panel of protein kinases. Docking studies are after 12 h at 110 °C, 2,7-disubstituted oxazolo[5,4-f]quinoxa-
carried out, leading to a potent inhibitor of GSK3.
lines 6aa and 6ab were isolated in good yields.
similar four-step synthesis, employing isonicotinoyl
A
chloride instead of benzoyl chloride, was used to obtain 2,8-di-
substituted oxazolo[5,4-f]quinoxaline 10a (Scheme 2).
Results and discussion
For the purpose of moving towards a more convergent
strategy, we reversed the order of the steps and performed
amidation before substitution of the chloro group
(Scheme 3). A further benefit of this approach would be the
possibility to carry out substitution under milder conditions;
indeed, the reaction should be favoured with a less electron-
donating group such as aroylamino at C6 than the previous
amino group. In a preliminary attempt, iodide 3 was treated
with nicotinoyl chloride; to solve the solubility issue some-
times encountered with toluene, acetonitrile containing 5
equivalents of pyridine was rather employed. Next, chlorocar-
boxamide 11b was reacted with morpholine in dimethyl-
sulfoxide, a solvent also suitable for the formation of benzox-
azole, and an excess of potassium carbonate was introduced
right from the beginning of the reaction. After the required
time to achieve nucleophilic substitution at room tempera-
ture, catalytic copper(I) iodide was introduced and the
mixture containing 5ba was heated to 110 °C in order to
allow cyclization to take place. Under these conditions, 2,7-di-
substituted oxazolo[5,4-f]quinoxaline 6ba was isolated after
6-Amino-2-chloroquinoxaline (1) was converted into 2,7-di-
substituted
oxazolo[5,4-f]quinoxalines
in
four
steps
(Scheme 1). An approach to obtain benzoxazole is by construct-
ing a ring from 2-haloanilide.5 Therefore, inspired by the
reported bromination of 6-aminoquinoxaline at C5,6 we
treated a solution of 1 in 4 : 1 dioxane–water with an excess of
iodine and sodium bicarbonate at room temperature for 4 h.
Under these conditions, the 5-iodo derivative 3, formed regio-
selectively, was isolated in 88% yield.
In order to progress towards compounds of biological inter-
est, the chloro group was successfully replaced by morpholino
and 4-methylpiperazino at the reflux temperature of toluene,
affording 4a,b. Their reaction with benzoyl chloride in the
presence of calcium carbonate under similar conditions led to
benzamides 5aa and 5ab.
Next, we sought simple and efficient conditions under
which we could attempt the synthesis of benzoxazole.5,7
Convinced with the interest in copper-catalysed reactions, we
decided to use catalytic copper(I) iodide in the presence of pot-
Scheme 1 Synthesis of 2,7-disubstituted oxazolo[5,4-f]quinoxalines 6aa and 6ab.
Scheme 2 Synthesis of 2,8-disubstituted oxazolo[5,4-f]quinoxaline 10a.
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Scheme 3 Synthesis of 2,7-disubstituted oxazolo[5,4-f]quinoxalines 6ba and 2,8-disubstituted oxazolo[5,4-f]quinoxalines 10aa, 10ab, 10ba and 10bb.
purification with an overall yield of 51% in this three-step from 7 as, after a suitable work-up, they can be directly involved
process (Scheme 3, top).
in the substitution reaction and benzoxazole formation.
Protein kinases can be deregulated in diseases such as
In a similar way, iodide 7 was readily converted into carbox-
amide 12a,b using either benzoyl chloride or nicotinoyl chlor- cancers and neurodegenerative disorders.8 Since the end of
ide. Further replacement of the chloro group by morpholino or the nineties, this class of signalling biomolecules has become
4-methylpiperazino benefited from the higher reactivity of 12a, indispensable drug targets. As a result, many candidates are at
b towards nucleophiles when compared with 11b. From the present undergoing clinical evaluation and 50 FDA-approved
compounds 9aa, 9ab, 9ba and 9bb, here only isolated from kinase inhibitors are already in the market.9
their reaction mixture by a simple work-up to ascertain their
In continuation of our ongoing research on novel protein
formation by NMR, cyclization was performed, leading to the kinase inhibitors,4 most of the synthesized oxazolo[5,4-f]quin-
new 2,8-disubstituted oxazolo[5,4-f]quinoxalines 10aa, 10ab, oxalines as well as a few precursors were tested against a short
10ba (unambiguously identified by X-ray diffraction; the corres- panel of disease-related protein kinases: cyclin-dependent
ponding ORTEP diagram is given in Fig. 2) and 10bb kinases 2 (CDK2/CyclinA), 5 (CDK5/p25) and 9 (CDK9/CyclinT),
(Scheme 3, bottom). It is worth noting that there is no need to proto-oncogene kinase PIM1, CDC2-like kinase 1 (CLK1), dual
purify the intermediates of this three-step synthesis process specificity tyrosine phosphorylation regulated kinase 1A
(DYRK1A), glycogen synthase kinase 3 (GSK3; isoform β, and
possibly α or/and α/β), casein kinase 1 (CK1; isoform ε, and
possibly δ/ε) and mitotic kinase Haspin (Table 1).
Interestingly, some of the oxazolo[5,4-f]quinoxalines and, to
a lesser extent, their non-cyclized precursors proved to signifi-
cantly inhibit CDK9/CyclinT, DYRK1A and GSK3 at 10 μM. In
addition, even at a lower concentration of 1 μM, compounds
10ba and 10bb bearing a 3-pyridyl ring at C2 still exhibit good
activity against GSK3 protein kinase. IC50 values were calcu-
lated for the most promising compounds; while micromolar
values for GSK3 inhibition were found in the case of 10aa and
10ab, encouraging submicromolar values were recorded for
10ba and 10bb (Table 2).
Because of its association with key biological processes
(metabolism, cell signalling, cellular transport, apoptosis, pro-
liferation, intracellular communication, etc.), the serine-threo-
nine protein kinase GSK3 is implicated in various diseases
(type 2 diabetes, bipolar disorder, schizophrenia, Alzheimer’s
disease, Parkinson’s disease, developmental disorders and
cancer), and has become an important target for drug develop-
ment.10 Thus, our preliminary results prompted us to identify
Fig. 2 ORTEP diagrams (30% probability) of 10ba (top) and 10ci
(bottom).
both the binding site and the binding mode of our best inhibi-
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Table 2 Selected IC50 values (nM) for compounds in Table 1
Entry
Product
CDK9/CyclinT
GSK3α
GSK3β
SscGSK3α/β
1
2
3
4
5
9aa
2500
>10000
3100
—
2200
610
1600
15
6800
980
3100
25
8800
1100
1900
—
10aa
10ab
10ba
10bb
—
24
55
30
tors 10ba and 10bb through (i) ATP-competition experiments
and (ii) docking studies in order to optimize their structure.
(i) The kinetic mechanism of GSK3β inhibition was experi-
mentally investigated for one of the most potent derivatives
synthesized, quinoxaline 10bb, by varying the concentrations
of both ATP and the tested inhibitor in the kinase reaction.
The Lineweaver–Burk or double-reciprocal plots of 1/v versus 1/
[ATP] at fixed concentrations of the new inhibitor 10bb showed
an intersecting pattern consistent with competitive inhibition
as all lines converge at or near the y-axis (Fig. 3). These results
clearly show that this new family of quinoxaline derivatives
and specifically compound 10bb are ATP-competitive GSK3β
inhibitors because an increase in the ATP concentration (from
1 to 100 µM) does not interfere with enzyme inhibition.
(ii) In order to develop structure–activity relationships and
propose novel substituents able to increase kinase inhibition
potency, we performed docking studies in the ATP-binding
cavity of GSK3β for compounds 10ab and 10bb. Both com-
pounds are able to bind to the cavity through an H-bond inter-
action between the N6 atom of the oxazolo quinoxaline ring
(see Scheme 3 for numbering) and the backbone amine group
of Val135 (Fig. 4), orienting the substituent at C2 to the cata-
lytic residue Lys85 and the substituent at C8 to the solvent.
The main difference between both compounds is the substitu-
ent at C2, a phenyl ring (compound 10ab) and a pyridine ring
(compound 10bb). The pyridine ring is able to interact through
Fig. 3 Kinetic data determined for derivative 10bb. ATP concentrations
in the reaction mixture varied from 1 to 100 µM. Compound concen-
trations used are depicted in the plot, and the concentration of the sub-
strate was kept constant at 12.5 µM. Each point is the mean of two
different experiments, each one analysed in duplicate.
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We first decided to keep the pyridine ring at C2 and replace
the morpholine or 4-methylpiperazine present at C8 with
various cyclic amines (Table 3, top scheme). To access the
planned structures, we applied the procedure involving the
merging of substitution and cyclization to obtain benzoxazole
in a sequential process (Scheme 3, top). Indeed, we observed
in the course of synthesis of 6ba that there was no need to
isolate intermediate 5ba and to purify precursor 11b. Thus,
under these conditions, 2,8-disubstituted oxazolo[5,4-f]quinox-
alines 10bc–bi were prepared from the dihalogenated carboxa-
mide 12b and isolated after purification in yields ranging from
44 to 86% (Table 3, entries 1–7).
Fig. 4 Proposed binding mode for compounds 10ab (left) and 10bb
(right) in the ATP binding pocket of GSK3β.
Then, we prepared 10ci, an analogue of 10bi in which the
3-pyridyl group at C2 is replaced by 1-ethyl-5-imidazolyl
(Table 3, bottom scheme). The idea was to verify whether the
interaction between Lys85 and the nitrogen of the ring at C2
could be improved. Compound 10ci was prepared similarly
(Table 3, entry 8), and its structure was identified unambigu-
ously by X-ray diffraction (see the ORTEP diagram in Fig. 2).
The inhibitory activities of these new compounds were eval-
uated for different kinases as mentioned previously and com-
pared with those of 10ba and 10bb (Table 4). In addition, the
dose-dependent effects of the most promising compounds
were tested on GSK3β and GSK3α; the corresponding IC50
values are given in Table 5. Most of the derivatives lost their
activities against GSK3, except compounds 10bg and 10bi
an H-bond interaction with Lys85, increasing the affinity and
improving the inhibition of the target. Thus, in order to access
more potent inhibitors, we could maintain the pyridine ring or
replace it with a five-membered heterocycle that allows similar
interaction. The substituent at C8 in both cases is a piperazine
ring that is oriented to the solvent-exposed region. This orien-
tation allows the introduction of different substituents in order
to grow the ligand in this region. Moreover, substituents at this
position with aromatic rings could be interesting to allow inter-
action with Arg141 through a π–cation interaction. Furthermore,
H-bond acceptor groups at this position would also be able to
interact with Arg141.
Table 3 Synthesis of 2,8-disubstituted oxazolo[5,4-f]quinoxalines 10bc-bi and 10ci
Entry
Product
X
Yield (%)
1
2
10bc
10bd
76
86
3
10be
10bf
76
44
4a
5
6
10bg
10bh
CH2
78
49
7
8
10bi
10ci
S
—
83
67
a 10bf was prepared from 10be (see the ESI†).
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which behaved similar to 10ba against GSK3β, but showed
potency towards GSK3α. It is worth noting that in spite of
similar ring sizes, increasing activities are noticed from 10ba
to 10bg and 10bi, suggesting that either conformational
changes or additional interactions might occur with these
latter compounds. Finally, the dose-dependent effect of 10ba,
10bg, 10bi and 10ci was determined on DYRK1A, which
showed a rather good preference of these inhibitors for GSK3
when compared with DYRK1A.
Conclusions
These results allowed us to identify the oxazolo[5,4-f]quinoxa-
lines 10ba, 10bg and 10bi as strong ATP-competitive inhibitors
of GSK3. The most potent GSK3 inhibitors reported in the lit-
erature are pyrimidine CHIR 9801414 and bis(aryl)maleimide
LY2090314,15 which have subnanomolar or nanomolar IC50
values (Fig. 5, left; Table 5, entries 14 and 15); however, while
CHIR 98014 selectively inhibits GSK3β, LY2090314 binds
almost equally to GSK3α and GSK3β. In contrast, while inhibi-
tors with high isoform-selectivity for GSK3α exist (e.g.
BRD070516 and G28_1417 with IC50(GSK3β)/IC50(GSK3α) ratios
of 7.8 and 6.6, respectively), they are less potent (Fig. 5, right;
Table 5, entries 16 and 17). Our best inhibitor, the sulphur-
containing molecule 10bi is potent and it binds GSK3α more
easily than GSK3β (ratio of 4.6). Thus, it will be a subject of
further studies aimed at identifying potent and selective
inhibitors of GSK3α. It is indeed of interest to identify
isoform-selective kinase inhibitors for the development of new
therapies.17 Efforts devoted to cellular testing in order to ident-
ify and develop applications for this new family of heterocycles
are also ongoing in the lab.
Fig. 5 Left: Potent inhibitors of GSK3α/β CHIR 9801414 and LY2090314.15
Right: Selective inhibitors of GSK3α vs. GSK3β BRD070516 and G28_14.17
Experimental section
Procedure used to convert 12b into 10bi
To a suspension of iodinated amide 12b (0.41 g, 1.0 mmol) in
dry DMSO (4 mL) were added thiomorpholine (0.11 mL,
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Table 5 IC50 values (nM) for the most promising inhibitors of GSK3a
b
Entry
Product
X
GSK3α
GSK3β
RnDYRK1A
1
2
3
4
5
6
7
8
10ba
10bb
10bc
10bd
10be
10bf
10bg
10bh
O
N-Me
15
24
40
88
57
24
11
22
25
55
102
127
97
68
33
35
22
1.7
2.3
2.55
1.4
1.7
2.8
3.0
1.6
4.6
1.6
—
—
—
—
—
—
—
200
—
—
—
—
—
390
—
130
447
N-Benzyl
N-CH2-5-(1,3-benzodioxolyl)
N-Boc
N–H
CH2
CH-morpholino
S
—
9
10bi
10ci
4.8
31
10
11
12
13
1414
1515
1616
1717
50
Indirubine-3′-monoxime
Hymenialdisine
Alsterpaullone
CHIR 98014
LY2090314
BRD0705
G28_14
72
55 (22)c,11
10 (10)c,12
22 (4)c,13
0.6
0.76
2.8
0.73
0.09
0.60
7.8
6.6
3.6
30 (4)c,13
7.0
1.5
66
0.9
515
218
33
a Three known inhibitors (indirubine-3′-monoxime, hymenialdisine and alsterpaullone) were also measured as controls. Values obtained from
the literature for known potent or selective inhibitors were also added for comparison. b IC50(GSK3β)/IC50(GSK3α) as an evaluation of the isoform
selectivity. c Values obtained from the literature (radioactive method using 15 µM ATP).
1.1 mmol) and potassium carbonate (0.29 g, 2.2 mmol). After cules, and protonating the residues at pH = 7.2. To develop the
stirring at room temperature for 1 h, copper(I) iodide (19 mg, docking studies, we used Glide22 to perform the docking calcu-
0.10 mmol) was added. The tube was sealed, heated at 110 °C lations using the Extra Precision Glide mode. The innerbox
for 12 h and then cooled. The resulting dark mixture was size of the grid was 16 × 16 × 16 Å and the outerbox size was
poured into cold water (20 mL); the precipitate was filtered, 33.5 × 33.5 × 33.5 Å. The grid box was centred on coordinates
washed with cold water (5 mL) and dissolved in chloroform. 0.432, 9.255, 31.434, that correlate with the centre of the ATP-
The organic layer was dried over sodium sulphate and the binding site. 10 poses per ligand were calculated and classified
solvent was removed under reduced pressure. The product was by the binding energy. To validate the docking protocol, we
purified by chromatography over silica gel (eluent: CHCl3– employed some GSK3β inhibitors with a known binding mode
MeOH 97 : 3).
to the ATP-binding site of the enzyme.16,22,23
Kinetic studies
Conflicts of interest
Kinetic experiments were performed by varying the concen-
trations of ATP (from 1 to 100 µM) and inhibitor 10bb (0.05
and 0.02 µM), while the concentration of the substrate GS2
was kept constant (12.5 µM). The ADP-Glo kinase assay18 was
used. Double-reciprocal plotting of the data is depicted in
Fig. 3.
There are no conflicts to declare.
Acknowledgements
We thank the Université de Rennes 1 and the Centre National
de la Recherche Scientifique for supporting this research. We
Docking studies
Before the docking studies, ligands and targets need to be pre- acknowledge the support from the Fonds Européen de
processed. Ligands were prepared for docking using the Développement Régional (FEDER; D8 VENTURE Bruker AXS
LigPrep tool,19 using the OPLS2005 force field, and by calculat- diffractometer). A. M. acknowledges financial support from
ing the protonation state at pH = 7.2 0.2. For target prepro- Spanish Ministry of Innovation, Science and Universities
cessing, the Maestro Protein Preparation Wizard20 was used. (MICU, grant no. SAF2016-76693-R) and ISCiii (CIBERNED,
The crystallographic structure of GSK3β in a complex with a grant no. CB18/05/00040). S. B. and S. R. also thank the
known inhibitor (PDBID 4ACD)21 was preprocessed by adding Cancéropôle Grand Ouest (axis: Natural Sea Products in
hydrogen atoms, removing the counterions and water mole- Cancer Treatment), IBiSA (French Infrastructures en sciences du
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vivant: biologie, santé et agronomie) and Biogenouest (Western
France Life Science and Environment Core Facility Network)
for supporting KISSf screening facility. We also thank Aurélie
Assicot for her contribution to the study and Ludovic Paquin
for the generous gift of secondary amines.
S. R. Depaolini, A. Feuchtinger, Z. Wu, T. Schmidt,
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