Thiazolo[5,4-f]quinoxalines, Oxazolo[5,4-f]quinoxalines and Pyrazino[b,e]isatins: Synthesis from 6-Aminoquinoxalines and Properties

Article abstract of DOI:10.1002/ejoc.202100362

The regioselective iodination of different 2-mono-, 3-mono- and 2,3-disubstituted 6-aminoquinoxalines, which takes place at their 5-position, was rationalized on the basis of Hückel theory calculations. Oxazolo- and thiazolo[5,4-f]quinoxaline analogues of

Full text of DOI:10.1002/ejoc.202100362

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: Thiazolo[5,4-f]quinoxalines, oxazolo[5,4-f]quinoxalines and
pyrazino[b,e]isatins: synthesis from 6-aminoquinoxalines and
properties
Authors: Frederic LASSAGNE, Joshua M. Sims, William Erb, Olivier
Mongin, Nicolas Richy, Nour El Osmani, Ziad Fajloun, Laurent
Picot, Valerie Thiery, Thomas Robert, Stephane Bach,
Vincent Dorcet, Thierry Roisnel, and Florence Mongin
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202100362
Link to VoR: https://doi.org/10.1002/ejoc.202100362
0
1/2020

European Journal of Organic Chemistry
10.1002/ejoc.202100362
FULL PAPER
Thiazolo[5,4-f]quinoxalines, oxazolo[5,4-f]quinoxalines and
pyrazino[b,e]isatins: synthesis from 6-aminoquinoxalines and
properties
[
a]
[a,b]
[a]
[a]
[a]
Frédéric Lassagne,* Joshua M. Sims, William Erb, Olivier Mongin, Nicolas Richy,
[
c]
[c,d]
[e]
[e]
[f,g]
Nour El Osmani, Ziad Fajloun,* Laurent Picot,* Valérie Thiéry, Thomas Robert,
Stéphane Bach,*[f,g,h] Vincent Dorcet, Thierry Roisnel and Florence Mongin*
[a]
[a]
[a]
[
a]
F. Lassagne, J. M. Sims, Dr. W. Erb, Dr. O. Mongin, N. Richy, Dr. V. Dorcet, Dr. T. Roisnel, Prof. F. Mongin
Univ Rennes, CNRS, ISCR (Institut des Sciences Chimiques de Rennes) - UMR 6226, F-35000 Rennes, France
E-mails: frederic.lassagne@univ-rennes1.fr, florence.mongin@univ-rennes1.fr
J. M. Sims
Univ. Lyon, ENS de Lyon, CNRS UMR 5182, Université Claude Bernard Lyon 1, Laboratoire de Chimie, F-69342 Lyon, France
N. El Osmani
[
b]
c]
[
Laboratory of Applied Biotechnology (LBA3B), Azm Center for Research in Biotechnology and its Applications, EDST, Lebanese University, 1300 Tripoli,
Lebanon
[d]
[e]
[f]
Prof. Z. Fajloun
Faculty of Sciences 3, Lebanese University, Campus Michel Slayman 1352 Ras Maska - Tripoli, Lebanon
E-mail: ziad.fajloun@ul.edu.lb
Dr. L. Picot, Prof. V. Thiéry
La Rochelle Université, Laboratoire Littoral Environnement et Sociétés, UMRi CNRS 7266, 17042 La Rochelle, France
E-mail: laurent.picot@univ-lr.fr
T. Robert, Dr. S. Bach
Sorbonne Université, CNRS, FR2424, Plateforme de criblage KISSf (Kinase Inhibitor Specialized Screening facility), Station Biologique de Roscoff,
Place Georges Teissier, 29682 Roscoff, France
E-mail: bach@sb-roscoff.fr
[
g]
h]
T. Robert, Dr. S. Bach
Sorbonne Université, CNRS, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, CS 90074, 29688 Roscoff Cedex, France
Dr. S. Bach
[
Centre of Excellence for Pharmaceutical Sciences, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
Supporting information for this article is given via a link at the end of the document.
Abstract: The regioselective iodination of different 2-mono-, 3-
mono- and 2,3-disubstituted 6-aminoquinoxalines, which takes place
at their 5-position, was rationalized on the basis of Hückel theory
calculations. Oxazolo- and thiazolo[5,4-f]quinoxaline analogues of
reported disease-related protein kinases inhibitors were synthesized
from the obtained 6-amino-5-iodoquinoxalines by using as key steps
copper-catalyzed azole ring formation. Pyrazino[b,e]isatins were for
the first time obtained from the same substrates by recourse to
Sonogashira coupling, alkyne hydration and oxidative cyclization.
The absorption and emission properties of the most promising
compounds were recorded. In addition, most of the synthesized
polycycles were evaluated as protein kinase inhibitors and for their
antiproliferative activity towards cancer cells.
the topic. Pyrazino[b,e]isatins have, to our knowledge, never
been prepared.
While a few thiazolo[5,4-f]quinoxalines have been claimed
6
in patents, the first oxazolo[5,4-f]quinoxalines were prepared by
7
our group in 2020, and notably the 8-piperidino-2-(3-pyridyl) Fl-
290 and the 2-(3-pyridyl)-8-thiomorpholino CD-07 derivatives
identified as potent inhibitors of glycogen-synthase kinase 3
8
(GSK3) with respective IC50 values of about 5 and 11 nM on the
isoform GSK3α (Figure 1, bottom).7
9
Introduction
Oxazolo[5,4-f]quinoxalines,
thiazolo[5,4-f]quinoxalines
and
pyrazino[b,e]isatins (Figure 1, top) share similar structures in
which
a
pyrazine ring is fused to
a
(hetero)aromatic
1
2
3
(
benzoxazole, benzothiazole or isatin, respectively) of
biological interest. These compounds can also be seen as a
quinoxaline ring fused to
a
five-membered heterocycle
4
4
5
(
oxazole, thiazole or 2,3-pyrrolidinedione, respectively) found
in numerous bioactive molecules. However, in spite of the
potential of these structures in fields such as medicinal
chemistry and materials, very few results have been reported on
Figure 1. Top: Structures of oxazolo[5,4-f]quinoxaline (left), thiazolo[5,4-
f]quinoxaline (middle) and pyrazino[b,e]isatin (right). Bottom: Recently
discovered kinase inhibitors based on the oxazolo[5,4-f]quinoxaline skeleton.
1
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European Journal of Organic Chemistry
10.1002/ejoc.202100362
FULL PAPER
Encouraged by these promising results, our goal in the
present paper is to show that 6-amino-5-iodoquinoxalines can
be used as common substrates to access these three families,
to evaluate the photochemical properties of these compounds
and to screen their biological activities.
Table 1. HOMO orbital coefficients on the different C(-H) atoms of the
substituted 6-aminoquinoxalines 2, and the 6- and 7-aminoquinolines.[
a]
Results and Discussion
1
2
Synthesis
Entry
2 (R , R )
C2
C3
C5
C7
C8
We previously showed that 2- and 3-chloro-6-aminoquinoxalines
could be regioselectively iodinated at C5-position by using iodine
1
2
3
4
5
6
7
8
9
2a (H, H)
0.29
0.00
0.54
-0.38
-0.44
-0.26
0.49
-0.55
-0.55
0.43
0.47
0.50
-0.54
-0.55
-0.54
C4:
0.07
-0.15
-0.10
-0.11
0.10
-0.07
-0.07
0.14
0.12
0.09
-0.07
-0.06
-0.07
0.04
-0.32
0.26
0.29
0.23
-0.31
0.32
0.32
-0.29
-0.30
-0.32
0.32
0.32
0.32
-0.34
2b (Ph, Ph)
2c (Ph, H)
2d (2-thienyl, H)
2e (Cl, H)
-
-
(
2.5 equiv) and sodium hydrogenocarbonate (2.5 equiv) in a 4:1
dioxane-water mixture at room temperature.7 As part of our
ongoing research, we were eager to see if this interesting result
could be extended to various 2-mono-, 3-mono- and 2,3-
-
-0.09
-
-0.17
disubstituted
substrates
easily
synthesized
from
4-
-
0.07
nitrophenylenediamine and various 1,2-dicarbonyl compounds
under mild conditions (Scheme 1).10
2f (H, Cl)
-0.29
-
2g (H, Ph)
2h (OMe, OMe)
2i (Cl, Cl)
0.29
-
-
-
-
-
10
11
12
13
14
2j (Me, H)
2k (H, Me)
-
0.06
-
-0.29
-
2
2l (NO , H)
-0.28
-
2m (H, NO
2
)
-0.29
0.25
-0.12
-0.32
C5:
0.52
Scheme 1. Regioselective synthesis of various 2-mono-, 3-mono- and 2,3-
disubstituted 6-aminoquinoxalines 2.
15
C1:
0.02
-0.55
0.00
0.34
-0.22
C2:
In order to firstly rationalize the regioselective iodination at
C5 of 6-aminoquinoxalines and evaluate its scope with various
substituents at C2 or/and C3, we used the HuLiS calculator11 to
get both the amplitudes of the highest occupied molecular orbital
-0.34
[a] For representations of the amplitudes and complete results of the
calculations, see Supporting information. Because we used a numbering with
the amino group fixed at the 6-position for all calculated substrates, the
numbering for 7-aminoquinoline is not consistent with its name.
(
HOMO) coefficients (Table 1) and the charges (Table 2) on the
C atoms of various substituted 6-aminoquinoxalines series 2 and
of quinoline analogues of 2a.
The Hückel theory calculations12 were inspired by a study
of Begunov and co-workers in which the authors attributed the
Table 2. Charges on the different C(-H) atoms of the substituted 6-
aminoquinoxalines 2, and the 6- and 7-aminoquinolines.
E
high regioselectivity in aromatic electrophilic substitution (S Ar)
of condensed imidazole derivatives to orbital control.13 In our
case, according to Fukui’s concept (reaction of a compound with
an electrophile at its carbon with the highest HOMO
1
2
Entry
2 (R , R )
C2
C3
C5
C7
C8
1
2
3
4
5
6
2a (H, H)
0.08
0.10
-
-0.09
-0.10
-0.09
-0.09
-0.09
-0.10
-0.02
-0.03
-0.02
-0.02
-0.02
-0.03
0.00
0.00
0.00
0.00
0.00
0.01
14
E
coefficient), the reaction centre of the S Ar first step is the C5
atom, whatever of the nature of the substituents (when present)
at C2 or/and C3 (Table 1). However, that the iodination is under
orbital control cannot be inferred from these calculations. Indeed,
by considering the charges on the different C atoms, the same
result can be deduced, with the highest negative charge always
localized at C5 (Table 2). Experimentally, all the performed
reactions took place at C5 as expected, and proceeded in high
yields ranging from 75 to 93% (Table 3; Figure 2).
2b (Ph, Ph)
2c (Ph, H)
2d (2-thienyl, H)
2e (Cl, H)
-
-
0.11
0.10
0.08
-
-
-
2f (H, Cl)
0.06
2
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European Journal of Organic Chemistry
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7
8
9
1
1
1
1
1
2g (H, Ph)
2h (OMe, OMe)
2i (Cl, Cl)
0.09
-
-0.10
-0.10
-0.10
-0.09
-0.10
-0.09
-0.09
C4:
-0.03
-0.03
-0.03
-0.02
-0.03
-0.02
-0.01
-0.02
0.00
0.00
0.00
0.00
0.01
0.01
0.00
-0.01
-
-
-
-
0
1
2
3
4
2j (Me, H)
-
0.08
-
2k (H, Me)
0.06
-
2
2l (NO , H)
0.12
-
2m (H, NO
2
)
0.10
0.09
Scheme 2. Benzoylation of the different 6-amino-5-iodoquinoxalines 3.
-0.01
0.05
C5:
0.08
-
15
C1:
0.10
-0.10
-0.04
0.02
0.07
C2:
0.02
-
Table 3. Regioselective iodination of the different 2-mono-, 3-mono- and 2,3-
disubstituted 6-aminoquinoxalines 2.
1
2
Yield (%)[a]
Entry
2 (R , R )
Product 3
1
2a (H, H)
3a
3b
3c
3d
3e
3f
83
93
93
85
88
88
75
86
83
2
2b (Ph, Ph)
2c (Ph, H)
2d (2-thienyl, H)
2e (Cl, H)
3
Figure 2. ORTEP diagrams (thermal ellipsoids shown at the 30% probability
level) of the compounds 3c (top left), 3g (top right) and 3k (middle, see
Scheme 5), and H-bond (2.169 Å) in 3g (bottom).
4
5[b]
6[b]
7
2f (H, Cl)
2g (H, Ph)
2h (OMe, OMe)
2i (Cl, Cl)
3g
3h
3i
8
9
[
a] After purification (see experimental part). [b] Results previously obtained.7
In order to progress towards the new oxazolo[5,4-
f]quinoxalines 5a-d,g,h, the 6-amino-5-iodoquinoxalines 3a-
d,g,h were N-benzoylated (Scheme 2), and the obtained
carboxamides 4a-d,g,h were cyclized to the corresponding
7
oxazoles as previously reported (Scheme 3).
Scheme 3. Cyclization of compounds 4 to oxazolo[5,4-f]quinoxalines.
3
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European Journal of Organic Chemistry
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The synthesis of the oxazolo[5,4-f]quinoxalines 7a-c,
which are C8-alkoxy analogues of the GSK3 kinase inhibitors Fl-
equiv).16 In our case, we first prepared the 6-(N’-
benzoyl)thioureido-5-iodoquinoxaline 9b from 3k, by reaction
with benzoyl isothiocyanate at the reflux of acetonitrile. The
cyclization of 9b, which is less soluble than 3k, to the 2-
(benzoylamino)thiazolo[5,4-f]quinoxaline 9c had to be performed
at 90 °C in DMSO containing triethylamine (2 equiv) to reach a
56% yield. Thus, the original thiazolo[5,4-f]quinoxalines 9a and
9c were synthesized from 6-amino-3-chloro-5-iodoquinoxaline
290 and CD-07, was also achieved from 6-amino-3-chloro-5-
iodoquinoxaline (3f), either by ‘substitution of chlorine-
a
aroylation of amine-cyclization to oxazole’ sequence (Scheme 4,
top and right), or changing the step order with amine aroylation
followed by one pot chlorine substitution/oxazole ring formation
(
Scheme 4, left).
(
3f) in respectively two and three steps and overall yields of 64.5
and 41% (Scheme 5, top and left).
Scheme 5. Synthesis of the thiazolo[5,4-f]quinoxalines 9a (sulfur analogue of
the kinase inhibitor Fl-260) and 9c from 3f.
Scheme 4. Synthesis of alkoxy analogues of the GSK3 kinases inhibitors Fl-
7
290 and CD-07.
Very few thiazolo[5,4-f]quinoxalines have been obtained
up to now by following lengthy syntheses.6 We planned to
investigate if this skeleton could be reached from 6-amino-5-
iodoquinoxalines, and targeted the synthesis of the thiazolo[5,4-
f]quinoxaline 9a. The latter is a sulfur analogue of 8-(N’-
methylpiperazino)-2-phenyloxazolo[5,4-f]quinoxaline (Fl-260), an
inhibitor of the disease-related protein kinases CDK9/CyclinT
(
cyclin-dependent kinase 9; IC50 = 3.1 μM), GSK3α (IC50 = 1.6
μM) and GSK3β (IC50 = 3.1 μM). Inspired by the recent study of
Wu and Jiang who used 2-iodoanilines, elemental sulfur (1.2
equiv) and terminal alkynes (1.5 equiv) in the presence of
tripotassium phosphate (2 equiv) copper(I) iodide (0.1 equiv),
1
,10-phenanthroline (0.2 equiv) under oxygen (1 atm) to prepare
benzothiazole derivatives in dimethylsulfoxide (DMSO) at
10 °C,15 the reaction of the 6-amino-5-iodoquinoxaline 3k
coming from 3f) with phenylacetylene under similar reaction
1
(
conditions led to the expected product 9a in 75% yield without
further optimization (Scheme 5, top and right).
Scheme 6. Towards pyrazino-fused isatins from 3.
To progress towards thiazolo[5,4-f]quinoxalines having an
additional substituent at C2, we were interested by the
methodology reported by Verma and co-workers. Indeed, these
authors showed in 2019 the possible synthesis of 2-
As pyrazino[b,e]isatins have, to our knowledge, never
been synthesized before, we were interested in the development
of an approach towards these compounds. By starting from the
(
aroylamino)benzothiazoles from 2-iodoanilines and aroyl
6-amino-5-iodoquinoxalines 3b and 3h, the required two-carbon
isothiocyanate at 90 °C in water containing triethylamine (2
4
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European Journal of Organic Chemistry
10.1002/ejoc.202100362
FULL PAPER
chain was introduced by Sonogashira coupling17 with
trimethylsilylacetylene under conditions reported by Mukai and
18
co-workers.
Thus, in the presence of diisopropylamine,
catalytic palladium(II) chloride bis(triphenylphosphine) and
copper(I) iodide in tetrahydrofuran (THF) at room temperature,
the reactions afforded 10b and 10h in correct yields (Scheme 6,
top). The alkynes were next hydrated by using sulfuric acid in a
mixture of solvents to provide the 5-acetyl-6-amino derivatives
11b and 11h in 82 and 75% yield, respectively (Scheme 6, right).
Oxidative cyclization was finally applied by using selenium oxide
in refluxing pyridine19 to furnish the pyrazino-fused isatins 12b
and 12h in 60 and 17% yield, respectively (Scheme 6, bottom).
Figure 3. Absorption (solid line) and emission (dotted line) spectra of 5d, 5g
and Fl-290 in acetonitrile.
Photophysical properties
A preliminary study of the photophysical properties of selected
compounds has been performed in order to identify potential
fluorescent candidates combining protein kinase inhibition and
fluorescence imaging or sensing properties. Such kind of
compounds would be highly desirable tools to perform imaging
studies of in vitro and/or in vivo protein kinase inhibition.20 The
UV-visible absorption and emission properties of 5d, 5g and Fl-
Bioactivity
Most of the synthesized compounds were evaluated in biological
assays, first by looking at their antioxidant properties. As shown
in Table 5, while the in vitro antioxidant activity of the derivatives
does not exceed 60% of the DPPH radical scavenger activity
(RSA), 5h (64%) and above all 8a (88%) can be considered as
potent antioxidant compounds.
290 were investigated in acetonitrile, and the results are
gathered in Table 4.
Table 5. Antioxidant activity (at t = 30 min) of some of the synthesized
compounds.
Table 4. Absorption and emission properties of 5d, 5g and Fl-290 in CH
3
CN.
a
max (M-1 cm )
b
-1
c
d
RSA (%)[a]
RSA (%)[a]
Compound

abs (nm)


em (nm)

F
Compound
Compound
5
d
g
312, 377
298, 351
300, 385
33300
38700
31400
431
419
455
0.09
0.03
0.08
5a
5b
5c
57
35
56
31
49
5h
64
62
56
56
88
5
Fl-290
7a
Fl-290
5
d
g
7c
[
[
a] Absorption maxima. [b] Molar extinction coefficient at absorption maximum.
c] Emission maximum. [d] Fluorescence quantum yield using as a standard
quinine bisulfate in 0.5 M H
maximum.
5
8a
2 4
SO , upon excitation at lowest energy absorption
[
a] 100 g tested at a concentration of 50 g/mL in DMSO at room
temperature.
The three oxazoloquinoxalines 5d, 5g and Fl-290 exhibit
two main absorption bands in the near UV, one of higher energy
with a maximum near 300 nm and a large molar extinction
coefficient (> 30000 M-1 cm ), and the other one of lower energy
-1
with
a maximum between 350 and 400 nm and lower
absorptivity (Figure 3). These compounds are fluorescent and
their emission band is situated in the violet-blue part of the
visible. Compound 5d, bearing two phenyl groups, is the less
emissive, with a fluorescence quantum of 3%. Replacement of
these phenyl groups with an electron-donating piperidinyl group
at the 8-position of the oxazoloquinoxaline and an electron-
withdrawing pyridyl group on the other side leads to an increase
of the quantum yield of Fl-290 up to 8%. A slightly higher
fluorescence efficiency (9%) is obtained with 5g, substituted with
an electron-rich thienyl ring at the 7-position of the
oxazoloquinoxaline. Moreover, the absorption and emission
bands of 5d and Fl-290 are red-shifted in comparison with 5g,
presumably in relation with an intramolecular charge transfer
Figure 4. Cytotoxicity assay representing the antiproliferative effects (%
viability) of some of the synthesized compounds on HCT116 (human
colorectal) cancer cells. 100 g tested at a concentration of 1 g/L in DMSO at
room temperature. Data are collected from
n = 1 experiment, and are
expressed in % of maximal viability (DMSO control).
A study was also performed in order to investigate the
antiproliferative activity of some of the prepared derivatives
towards cancer cell lines. First, HCT116 (human colorectal)
cancer cells were chosen to evaluate the cytotoxic potential of
(
ICT) process.
5
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European Journal of Organic Chemistry
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the compounds 5a-d, 5g,h, 7a,c, 8a and Fl-290. Pleasingly,
except in the case of 2,7,8-triphenyloxazolo[5,4-f]quinoxaline
μM), its thiazolo[5,4-f]quinoxaline analogue 9a is also less
efficient. In addition, while a weak activity was noticed for most
of the tested derivatives, compound 9c was found active against
CK1 at both 10 M and 1 M. Therefore, to clearly establish its
potency, its IC50 values of inhibition against both CK1ε (0.31 M)
and CK1δ/ε (1.58 M) were determined, thus establishing 9c as
a potent CK1 kinase inhibitor.
(
5b),
a
remarkable antiproliferative activity (>80%) was
evidenced for all the tested compounds (Figure 4).
We also evaluated the antiproliferative activity of the
compounds 5b,d,h, 7a,b,c, 9c, 11b,h, 12b,h, Fl-290, Fl-291 and
CD-07 against the A2058 melanoma cell line, considered as
highly resistant to anticancer drugs. Molecules at 10-5 M induced
a growth inhibition at 72 h ranging from 0.3% to 66% (Figure 5).
The original thiazolo[5,4-f]quinoxaline 9c showed a promising
antiproliferative activity against the selected melanoma cells.
Because they are often deregulated in diseases such as
cancers and neurodegenerative disorders, protein kinases have
become a major class of drug targets.21 As part of our ongoing
studies on the discovery of new protein kinase inhibitors,19,22
most of the compounds here synthesized were tested7 against
several disease-related protein kinases: cyclin-dependent
kinases 2 (CDK2/Cyclin A), 5 (CDK5/p25) and 9 (CDK9/Cyclin
T), proto-oncogene kinase PIM1, CDC2-like kinase 1 (CLK1),
dual specificity tyrosine phosphorylation regulated kinase 1A
(
DYRK1A), glycogen-synthase kinase-3 (GSK-3 isoforms /),
casein kinase 1 (CK1 isoforms δ/ε), mitotic kinase Haspin and
vascular endothelial growth factor receptor 2 (VEGFR2).
As shown in Table 5, the compounds 7a-c were found less
efficient than the potent kinase inhibitors Fl-290, Fl-291 and CD-
Figure 5. Antiproliferative activity (% growth inhibition) of compounds here
-
5
synthesized at 10 M after 72 h in 2000 A2058 human melanoma cells
DMSO control).
07. When compared with Fl-260 which inhibits CDK9/CyclinT
(
(
IC50 = 3.1 μM), GSK3α (IC50 = 1.6 μM) and GSK3β (IC50 = 3.1
Table 5. Inhibitory activities of some of the synthesized compounds against a short panel of disease-related protein kinases. The table displays the remaining
kinase activities detected after treatment with 10 and 1 µM of the tested compounds. The values obtained after treatment with 1 µM are given in brackets. Results
are expressed in % of maximal activity, i.e. measured in the absence of inhibitor but with an equivalent dose of DMSO (solvent of the tested compounds). ATP
concentration used in the kinase assays was 10 µmol/L (values are means, n = 2). Kinases are from human origin unless specified: Mm, Mus musculus; Rn,
Rattus norvegicus; Ssc, Sus scrofa domesticus.
Compd. CDK2/
CDK5/
p25
CDK9/
CyclinT
PIM1
MmCLK1 RnDYRK1A GSK3α
GSK3β
SscGSK3 CK1ε
α/β
SscCK1δ/ε Haspin
VEGFR2
CyclinA
[
a]
[a]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[a]
[a]
5
5
5
5
5
5
7
7
7
a
b
c
d
g
h
a
b
c
67 (84)
66 (96)
53 (93)
92 (96)
83 (91)
91 (82)
84 (≥100) 93 (97)
[
a]
[a]
[a]
[a]
≥100 (99)
96 (99)
≥100 (94) 99 (93)
[a]
[
a]
[a]
[a]
90 (≥100)
98 (64)
[
b]
[a]
92 (≥100) ≥100 (93) 100 (78) 94 (≥100)
≥100 (73) 98 (≥100) ≥100 (97)
[
a]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[a]
≥100 (94)
96 (≥100)
[
[
[
87 (99)
63 (71)
61 (76)
66 (68)
92 (80)
11 (40)
27 (45)
4 (69)
97 (90)
[a]
94 (79)
[a]
94 (73)
≥100 (79)
34 (69)
34 (64)
38 (69)
1 (5)
85 (77)
[
a]
[a]
[a]
[a]
[a]
b]
b]
b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
39 (98)
90 (≥100)
44 (79)
13 (≥100)
23 (43)
14 (31)
15 (49)
19 (43)
4 (21)
28 (45)
32 (41)
26 (50)
11 (13)
9 (9)
33 (61)
43 (60)
18 (57)
21 (58)
12 (23)
8 (63)
75 (≥100)
83 (93)
65 (75)
44 (86)
72 (≥100)
[b]
7
[b]
[b]
[b]
Fl-290
58 (97)
66 (98)
53 (99)
Fl-2917
3 (11)
2 (2)
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
CD-077
18 (≥100)
98 (≥100)
75 (89)
5 (19)
[b]
-1 (-3)
57 (99)
-7 (54)
21 (14)
55 (99)
12 (58)
[
a]
9a
86 (≥100)
57 (64)
4 (61)
58 (≥100) 90 (≥100) 86 (≥100) 61 (69)
Fl-2607 52 (98)
[b]
[b]
[a]
[a]
[a]
[b]
[b]
85 (81)
14 (69)
85 (92)
40 (94)
[
a]
[c]
[d]
9
1
1
1
c
≥100 (91) 73 (≥100) 88 (91)
86 (≥100) 90 (99)
24 (67)
[b]
9 (39)
[a]
89 (90)
[a]
[
[
[
b]
b]
b]
[b]
[b]
[b]
[b]
[b]
[b]
1b
2b
2h
99 (100)
≥100 (96)
93 (92)
≥100 (96) 42 (98)
89 (≥100)
[a]
≥100 (93)
[a]
[b]
[b]
[a]
[a]
[a]
73 (98)
89 (94)
76 (98)
[a]
[a]
[a]
95 (≥100)
[
a] ≥100 (≥100). [b] Not determined. [c] IC50 = 0.31 M. [d] IC50 = 1.58 M.
isatin. It is worth noting that the Hückel theory calculations
similarly predict a regioselective halogenation at the 5-position
for 6-aminoquinoline,23 and at the 8-position for 7-
aminoquinoline.24
Conclusion
An early photophysical evaluation of some of the compounds
was performed and their biological properties were finally
investigated. By this way, we not only managed to access new
GSK3 kinase inhibitors, but also identified a new CK1 kinase
selective inhibitor, therefore establishing these original
In the present study, we have first rationalized the regioselective
iodination at their 5-position of 2-mono-, 3-mono- and 2,3-
disubstituted 6-aminoquinoxalines, and used the reaction to
access original derivatives in which a pyrazine ring is fused to a
benzoxazole, a benzothiazole and, for the first time, even an
6
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European Journal of Organic Chemistry
10.1002/ejoc.202100362
FULL PAPER
heterocycle-fused quinoxalines as promising scaffolds for drug
development.
General procedure 6 for the conversion of 6-(3-pyridoylamino)-3-
chloro-5-iodoquinoxaline (6) by nucleophilic substitution followed
by cyclization. To 6-(3-pyridoylamino)-3-chloro-5-iodoquinoxaline (6; 51
mg, 0.125 mmol) under argon were added anhydrous DMSO (0.5 mL)
and the required sodium ethanolate (prepared by heating under argon
the 2-substituted ethanol in the presence of 60% NaH (1 equiv) in dry
toluene at 50 °C for 1 h before removal of the solvent, addition of dry
Experimental Section
General information, analyses of the compounds and NMR spectra are
given in Supporting information.
Et
50 °C for 6 h. CuI (2.6 mg, 12.5 μmol) was next added, followed by
NaHCO (13 mg, 0.15 mmol), and the reaction mixture was heated at
110 °C for 12 h. The reaction was monitored by TLC using CH Cl -MeOH
0:10 as eluent. When the reaction was finished, the solvent was
removed under reduced pressure. The residue was dissolved in CHCl
washed with brine. Drying over Na SO and removal of the solvent under
reduced pressure led to the crude product which was purified by
preparative TLC plate on silica gel (the eluent is given in the product
description).
2
O and filtration; 0.15 mmol). The red reaction mixture was stirred at
3
General procedure 1 for the cyclocondensation.10 To the required
2
2
9
1,2-dicarbonyl compound (10 mmol) in methanol (10 mL) were added
3
,
saccharin (92 mg, 0.50 mmol) and 4-nitrophenylenediamine (10 mmol).
The reaction mixture was stirred at room temperature for 24 h and
poured onto water (10 mL). The solid was collected by filtration and dried
under reduced pressure to afford the title product which was used in the
next step without further purification.
2
4
General procedure 7 for the Sonogashira coupling.18 Anhydrous
tetrahydrofuran (4 mL) was degassed 5 min with argon and introduced
into a Schlenk tube containing the iodoquinoxaline (1.5 mmol). To the
solution obtained after stirring, were added CuI (28.5 mg, 0.15 mmol) and
General procedure 2 for the reduction of the nitro compounds.25 To
a stirred suspension of the nitro compound (10 mmol) in EtOH (25 mL)
were added Pd/C (0.30 g) and hydrazine hydrate (5.0 mL). After 30 min,
the reaction mixture was heated under reflux for 2 h. After cooling to
room temperature, filtration over Celite® and rinsing with AcOEt, the
solvents were evaporated under reduced pressure. The solid was
dissolved in AcOEt and the organic phase washed with brine. Drying
PdCl
2
(PPh
3
)
2
(52.5 mg, 75 mol). The mixture was stirred under argon
NH (1.3 mL, 9.6
for 5 min before addition of degassed anhydrous iPr
2
mmol) and (trimethylsilyl)acetylene (0.80 mL, 5.7 mmol). After 5 h stirring
at room temperature, the reaction mixture was partitioned between
2 4
over Na SO and removal of the solvent under reduced pressure led to
AcOEt and H
2
O. The organic layer was washed with brine and dried over
the expected product which was employed in the next step without
purification.
Na SO . Removal of the solvent under reduced pressure led to the crude
2
4
product which was purified by column chromatography over silica gel
(eluent given in the product description).
7
General procedure 3 for the iodination of the amino compounds. To
a solution of the amine (17 mmol) in 4:1 dioxane-water (80 mL) at 0 °C
General procedure 8 for the conversion of 10 into 11. A solution of 10
were successively added NaHCO
mmol). The solution was stirred at room temperature for 4 h and poured
onto a saturated aqueous Na solution (40 mL). The product was
extracted with CH Cl (3 x 20 mL); the organic layer was washed with
water (20 mL) and dried over Na SO . After evaporation of the solvent
3 2
(3.5 g, 42 mmol) and I (11 g, 42
(0.20 g, 0.5 mmol) in tetrahydrofuran (2 mL) was added to a solution of
H
1
2
SO
h and evaporated. The residue was dissolved in AcOEt and washed
solution and then brine. The organic
. Removal of the solvent under reduced
4
(83 L, 1.5 mmol) in methanol (3 mL). The mixture was stirred for
2 2 3
S O
2
2
with a saturated aqueous NaHCO
phase was dried over Na SO
3
2
4
under reduced pressure, the iodide was purified as indicated in the
product description (see an example below).
2
4
pressure led to the crude product which was purified by column
chromatography over silica gel (eluent given in the product description).
6
-Amino-5-iodoquinoxaline (3a).26 The general procedure 3 from 6-
aminoquinoxaline (2a; 2.5 g) gave, after purification by chromatography
over silica gel (eluent: CH Cl ; R = 0.59), 3a in 83% yield (3.8 g) as a
dark red solid: mp 180-182 °C; IR (ATR): 507, 861, 950, 1042, 1419,
General procedure 9 for the conversion of 11 into isatins.19 To the
ketone (11; 0.50 mmol) in pyridine (1 mL) was added SeO (115 mg, 1.0
2
mmol). The mixture was heated under reflux for 2 h. After cooling to room
temperature, an aqueous 1 M solution of HCl (2 mL) was added before
2
2
f
494, 1534, 1618, 3186, 3297, 3444 cm ; 1H NMR (CDCl
-
1
1
3
)  4.87 (br s,
), 7.26 (d, 1H, J = 9.0 Hz, H7), 7.85 (d, 1H, J = 9.0 Hz, H8), 8.54
) 
3.0 (C, C5, C-I), 120.8 (CH, C7), 130.5 (CH, C8), 138.8 (C), 141.5 (CH,
C2 or C3), 144.4 (C), 145.6 (CH, C2 or C3), 149.5 (C).
filtration and washing with H
the isatin.
2
O (2 x 5 mL). Drying under vacuum afforded
2
(
H, NH
2
d, 1H, J = 1.9 Hz, H2), 8.75 (d, 1H, J = 1.9 Hz, H3); 13C NMR (CDCl
3
8
Cellular viability assay. HCT116 cells were obtained from the American
Type Culture Collection (ATCC). The cell line was cultured in DMEM
(
Gibco Dulbecco's Modified Eagle Medium, Sigma Eldrish) at 37 °C in a
humidified atmosphere 5% CO and 95% air. Media was amplified with
% Penicillin Streptomycin (100 U·mL−1) and 10% heat-inactivated fetal
bovine serum (FBS). The cellular viability assay was carried out by MTT
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) test. MTT
General procedure
4 for the conversion of the amines into
7
2
benzamides. To a suspension of the amine (2.0 mmol) and calcium
carbonate (0.22 g, 2.2 mmol) in toluene (10 mL) was added benzoyl
chloride (0.31 g, 2.2 mmol). The mixture was heated under reflux for 15 h.
After filtration of the hot solution, the filtrate was concentrated under
reduced pressure to give the expected product which was used in the
next step without further purification.
1
(
assay works on the mitochondria of the cell by the enzyme mitochondrial
reductase.27 It converts the yellow dye of MTT to purple formazan. Cells
were seeded in 96 well plates at a density of 10 . At 70% confluency, the
4
7
cells are ready to be treated. The treatment was for 24 h. After 24 h, the
media was discarded. 100 µl of MTT solution was added to each well.
The absorbance was measured by ELISA READER at 570 nm. The
antiproliferative activity in A2058 melanoma cells (LGC Promochem,
France) was evaluated as previously reported by our team.19
General procedure 5 for the cyclization of the iodinated amides. To
a suspension of the iodinated amide (0.125 mmol) in dry DMSO (0.5 mL)
under argon was added K
2.5 μmol). The reaction mixture was heated at 110 °C for 12 h and
cooled. The resulting dark mixture was poured onto a cold saturated
aqueous NaHCO solution (5 mL); the precipitate was filtered, washed
with cold water and dissolved in CHCl . The organic layer was dried over
Na SO and the solvent was removed under reduced pressure. The
product was purified as specified below.
2 3
CO (20 mg, 0.15 mmol) and CuI (2.4 mg,
1
3
Biological evaluation: protein kinase assays. Kinase activities were
assayed in 384-well plates using the ADP-GloTM assay kit following the
recommendations of the manufacturer (Promega, Madison, WI). The
experimental conditions used to detect the enzymatic activity of each
protein kinase tested in this study are reported.28 Controls were
3
2
4
7
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European Journal of Organic Chemistry
10.1002/ejoc.202100362
FULL PAPER
performed in appropriate dilutions of dimethyl sulfoxide (DMSO). IC50
values were determined from the dose response curves using Prism-
GraphPad (GraphPad Software, San Diego, CA, USA).
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Acknowledgements
We thank the “Comité d’Ille et Vilaine 35” de la Ligue Nationale
Contre le Cancer, the Université de Rennes 1 and the Centre
National de la Recherche Scientifique for supporting this
research. We acknowledge the Fonds Européen de
Développement Régional (FEDER; D8 VENTURE Bruker AXS
diffractometer). V. T. and L. P. also thank the “Comité 17 de la
Ligue Nationale Contre le Cancer for financial support. T. R. and
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4
7
8
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European Journal of Organic Chemistry
10.1002/ejoc.202100362
FULL PAPER
Entry for the Table of Contents
Oxazolo[5,4-f]quinoxalines, thiazolo[5,4-f]quinoxalines and pyrazino[b,e]isatins were all obtained from 5-iodo-6-aminoquinoxalines.
While the first two families were synthesized by nitrogen functionalization and subsequent copper-catalyzed cyclization, the latter one
was obtained by Sonogashira coupling, alkyne hydration and oxidative cyclization. Most of the synthesized polycycles were
evaluated in biological tests.
Institute and/or researcher Twitter usernames:
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9
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