New imidazo[1,2-a]quinoxaline derivatives: Synthesis and in vitro activity against human melanoma

Article abstract of DOI:10.1016/j.ejmech.2009.02.007

New imidazo[1,2-a]quinoxaline analogues have been synthesized in good yields via a bimolecular condensation of 2-imidazole carboxylic acid, followed by a coupling with ortho-fluoroaniline and subsequent substitution on the imidazole ring by Suzuki Cross-c

Full text of DOI:10.1016/j.ejmech.2009.02.007

European Journal of Medicinal Chemistry 44 (2009) 3406–3411
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New imidazo[1,2-a]quinoxaline derivatives: Synthesis
and in vitro activity against human melanoma
, ,
,
1
b
a
Carine Deleuze-Masquefa a 1, Georges Moarbess a , Sonia Khier , Nadege David , Stephanie Gayraud-
*
`
´
Paniagua a, Françoise Bressolle b , Frederic Pinguet , Pierre-Antoine Bonnet
,
c
c
a
´ ´
a EA 4215, Laboratoire de Chimie des Biomole´cules et de l’Environnement, Pharmacochimie et Biomole´cules, Universite´ Montpellier I,
Faculte´ de Pharmacie, 15 av. Charles Flahault, BP 14491, 34 093 Montpellier Cedex 5, France
b Pharmacocine´tique Clinique, Faculte´ de Pharmacie, Universite´ Montpellier I, 15 av. Charles Flahault, BP 14491, 34 093 Montpellier Cedex 5, France
c Centre R´egional de Lutte contre le Cancer, Val d’Aurelle, Rue de la Croix Verte, 34000 Montpellier, France
a r t i c l e i n f o
a b s t r a c t
Article history:
New imidazo[1,2-a]quinoxaline analogues have been synthesized in good yields via a bimolecular
condensation of 2-imidazole carboxylic acid, followed by a coupling with ortho-fluoroaniline and
subsequent substitution on the imidazole ring by Suzuki Cross-coupling reaction using microwave
assistance. Antitumor activities of these derivatives were evaluated by growth inhibition of A375 cells in
vitro. All compounds exhibited high activities compared to imiquimod and fotemustine used as
references.
Received 14 October 2008
Received in revised form
26 January 2009
Accepted 12 February 2009
Available online 21 February 2009
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Imidazoquinoxalines
Imiquimod
Melanoma
In vitro activity
1. Introduction
on melanoma in vitro and in vivo [13]. It was identified as a selective
inhibitor of I
k
B kinase that blocked NF-kB pathway [14].
In our previous studies [1–3], a general synthetic strategy has
been elaborated which can easily get access to three basic
heterocycles imidazo/pyrazoloquinoxalines. This method led us to
the intermediates which remain non-substituted on the
5-membered cycle with good yields. New imiquimod analogues in
the imidazo[1,2-a]quinoxaline, imidazo[1,5-a]quinoxaline and
pyrazolo[1,5-a]quinoxaline series have also been synthesised and
tested in vitro and in vivo for their anti-cancer activities.
Imiquimod, an imidazoquinoline amine, has been initially
approved for the topical treatment of external genital warts caused
by humanpapillomavirus [4–6]. It has shown activities toward basal
cell carcinomas (BCC), actinic keratoses, and some melanoma
metastases [7–10]. Fotemustine is a third generation nitrosourea
which is used both in melanoma and brain tumors [11]. However, its
clinical application is somewhat limited both by its toxicity and by
the resistance of melanoma cells [12]. BMS-345541, an imidazo[1,2-
a]quinoxaline derivative has shown important cytotoxic activities
EAPB0203,
a
new imidazoquinoxaline amine derivative
demonstrated significant in vitro activities against the human
melanoma cell line A375, 110 and 45 times more active than fote-
mustine and imiquimod, respectively [1,2]. Tested in vivo in female
Swiss Nude mice, EAPB0203 caused a significant decrease in tumor
(M4Be xenografts) growth compared to vehicle control and
fotemustine treatments [1,2].
Recently, EAPB0203 was evaluated in T-cell lymphomas and
HTLV-I associated adult T-cell leukaemia/lymphoma. EAPB0203
exhibits an important cytotoxicity in vitro on HTLV-I-infected CD4þ
T-cell lines HuT-102, MT-2, and C91-PL as well as HTLV-1-negative
malignant CD4þ T-cell lines CEM, Jurkat, HuT-78, and MOLT-4. The
imidazoquinoxaline derivative exhibits inhibition of cell prolifera-
tion, G2/M cell cycle arrest, and induction of apoptosis in HTLV-I
transformed and HTLV-I negative malignant T cells [15].
In continuation of our studies on the reactivity of nitrogen
heterocycles, we focused on the preparation of imidazo[1,2-a]qui-
noxaline derivatives using the new synthetic strategy described
previously [1,2]. To develop a convenient pathway in good agree-
ment with rapid pharmacomodulation, we were interested in using
the Suzuki Cross-coupling reaction. The application of this coupling
to these series was reported on the bromo derivatives [16]. In this
* Corresponding author. Tel.: þ33 4 67548665/8640; fax: þ33 4 67548036.
E-mail address: carine.masquefa@univ-montp1.fr (C. Deleuze-Masquefa).
1
Carine Deleuze-Masquefa and Georges Moarbess contributed equally to this
work.
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.02.007

C. Deleuze-Masquefa et al. / European Journal of Medicinal Chemistry 44 (2009) 3406–3411
3407
work, we turned our interest to study the incorporation of different
arylboronic acids on the cytotoxic activity, in order to afford
a variety of new derivatives of our lead compound EAPB0203.
compounds 7b, 7c, 7e–7g are 7–9 times more potent against A375
than EAPB0203 (Table 1).
3. Discussion
2. Results
The procedure presented in Scheme 1 was used to prepare the
imidazo[1,2-a]quinoxaline derivatives. This method not only
reduced the number of purification steps but also led us to propose
a new common and efficient route for the preparation of the
unsubstituted imidazo[1,2-a]quinoxalines on the imidazole core
starting from the unsubstituted carboxylic acid. This new strategy
also permitted the introduction of R substituent at the last steps of
the procedure with different commercial Suzuki cross-coupling
reagents using microwave assistance.
The exact chemical structure of these imidazo[1,2-a]quinoxaline
derivatives and the assignments of the position of substitution on
the heterocycle using 1H and 13C NMR data were described
previously [17].
In order to confirm the bromine position on the imidazole ring,
we carried out a n.O.e study. The irradiation of H-9 at 9.10 ppm
showed a correlation with only one proton H-8 at 7.5 ppm and the
signal intensity of n.O.e effect was increased up to 15%. No
correlation was observed between H-9 and protons of the imid-
azole ring. The same results were obtained by irradiation of H-2 at
7.65 ppm. These results led us to confirm that the bromine was in
the position 1 of the imidazole ring.
2.1. Chemistry
2.1.1. Library synthesis
For the preparation of the 1H-imidazo[1,2-a]quinoxaline deriv-
atives, the procedure presented in Scheme 1 was used. Carbonyl-
imidazole dimer 1 resulted from the bimolecular condensation of
the 2-imidazole carboxylic acid, in presence of thionyl chloride in
95% overall yield. Product 1 was coupled with ortho-fluoroaniline to
give the intermediate 2, with a yield of 40%. The tricyclic compound
3 was obtained via an intramolecular cyclization of 2 accomplished
under strong basic conditions with 80% yield. Treatment of
compound 3 with phosphorus oxychloride and N,N-diethylaniline
yielded to 80% of compound 4 which was coupled with methyl-
amine to give compound 5 with average yields of 93% [1,3]. The
bromination of compound
5 by N-bromosuccinimide led to
compound 6. The position of the bromine on the imidazole ring was
confirmed by n.O.e study. Utilizing the appropriate arylbronic acid
allowed for introduction of various groups on the imidazole ring of
compound 6 via Suzuki reaction in the presence of palladium
catalyst, basic conditions and under microwave assistance to give
compounds 7a–7n in good yields.
We reported previously [1,2] that EAPB0203 bearing phenethyle
as substituent at position 1 was a lead compound with an activity
against A375 cell line 110 times higher than fotemustine
2.1.2. In vitro cytotoxic activity
(IC50 ¼ 173
mM) and 45 times higher than imiquimod (IC50 ¼ 70
mM).
All compounds were evaluated for their cytotoxic activity in
vitro against A375 human melanoma cell line by comparison with
imiquimod, fotemustine used as references and EAPB0203. Table 1
gives the IC50 values for all of the imidazo[1,2-a]quinoxaline
derivatives as determined by the microculture tetrazolium dye
assay. All the tested imidazo[1,2-a]quinoxalines caused a significant
inhibition of cell growth, their cytotoxicities were superior to that
of imiquimod and fotemustine. Compounds 7d, 7k–7n were
considerably 10–100 times less active than the present EAPB0203
known to be 110 and 45 times more active than fotemustine and
imiquimod on human melanoma cell line (A375), respectively [1,2].
Compounds 7h–7j showed a similar activity to EAPB0203. Five
In this paper, replacement of phenethyle (EAPB0203) by phenyle
(compound 7a) on the imidazole ring resulted in no change in
cytotoxicity. The presence of the carbon chain (EAPB0203) between
the phenyl and imidazole core appeared to be less interesting, the
activity was not influenced by the flexibility of the substituent on R
position. Introduction of an electron-donating group (compounds
7b, 7c, 7e, 7f) enhanced activity. This increase could be explained by
additional interaction type hydrogen bound with the biological
target. The substitution of an electron-withdrawing group on the
phenyl core (compounds 7k–7m) displayed decreased potency.
The methoxy group at position 2 on the phenyl ring (compound 7d)
has proved to be less favorable than the 3 and 4 positions
F
O
COOH
HN
N
F
N
N
c
NH2
a
N
NH
N
N
N
O
b
NH
2
O
1
N
N
N
N
N
f
e
d
N
NHCH3
N
H
O
N
Cl
5
4
3
R
Br
N
N
N
N
N
N
g
NHCH3
NHCH3
6
7a-7n
Scheme 1. Synthesis of 1H-imidazo[1,2-a]quinoxaline derivatives. Reagents and conditions: (a) SOCl2 reflux, 18 h; (b) NaHMDS, THF, 5 h; (c) NaH, DMA, reflux, 10 h; (d) POCl3,
reflux, 6 h; (e) EtOH, NHCH3, 20 h, rt; (f) NBS, CHCl3, reflux, 2 h; (g) arylboronic acid (R–B(OH)2), Pd(PPh3)4, Na2CO3, DME, MW (140 ꢀC, 20 min).

3408
C. Deleuze-Masquefa et al. / European Journal of Medicinal Chemistry 44 (2009) 3406–3411
Table 1
Imidazo[1,2-a]quinoxaline derivatives: formula, IC50 against A375 (human melanoma cell lines), pKa and log P calculated values.
R
N
N
N
imidazo[1,2-a]quinoxaline
NHCH3
a
Compounds
R1
Formula
IC50
(m
M)
pKa*
log P*
7a
C6H5–
C17H14N4
2.19 ꢂ 0.08
0.20 ꢂ 0.09
0.37 ꢂ 0.4
122 ꢂ 25
5.18 ꢂ 0.4
5.10 ꢂ 0.4
5.45 ꢂ 0.4
4.89 ꢂ 0.4
5.11 ꢂ 0.4
5.14 ꢂ 0.4
4.96 ꢂ 0.4
4.86 ꢂ 0.4
4.88 ꢂ 0.4
5.28 ꢂ 0.4
4.88 ꢂ 0.4
4.83 ꢂ 0.4
4.57 ꢂ 0.4
5.21 ꢂ 0.4
5.61 ꢂ 0.4
3.68 ꢂ 1.43
3.45 ꢂ 1.43
3.50 ꢂ 1.43
3.35 ꢂ 1.43
3.98 ꢂ 1.43
2.94 ꢂ 1.43
4.43 ꢂ 1.43
4.66 ꢂ 1.43
4.25 ꢂ 1.43
3.36 ꢂ 1.43
3.71 ꢂ 1.43
3.12 ꢂ 1.43
3.21 ꢂ 1.43
2.99 ꢂ 1.43
4.91 ꢂ 1.42
7b (EAPB0503)
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
3-OCH3–C6H4–
4-OCH3–C6H4–
2-OCH3–C6H4–
3-OC2H5–C6H4–
3-OH–C6H4–
3-Br–C6H4–
3-CF3–C6H4–
3-Cl–C6H4–
3-COOH–C6H4–
3-F–C6H4–
3-CN–C6H4–
3-NO2–C6H4–
3-(C4H3O)–
C18H16N4O
C18H16N4O
C18H16N4O
C19H18N4O
C17H14N4O
C17H13BrN4
C18H13F3N4
C17H13ClN4
C18H14N4O2
C17H13F N4
C18H13N5
0.30 ꢂ 0.01
0.56 ꢂ 0.12
0.65 ꢂ 0.02
1.28 ꢂ 0.18
1.78 ꢂ 0.35
3.47 ꢂ 0.55
24.9 ꢂ 0.27
27.0 ꢂ 2.2
40.0 ꢂ 3.9
74.1 ꢂ 3.1
C17H13N5O2
C15H12N4O
C19H18N4
7n
EAPB0203
Imiquimod
Fotemustine
C6H5–(CH2)2–
1.57 ꢂ 0.56
70.3 ꢂ 4.3
173 ꢂ 24
*pKa and log P values are calculated using the ACDlabs software.
a
IC50, concentration of the compound (mM) producing 50% cell growth inhibition after 96 h of drug exposure, as determined by the MTT assay. Each experiment was run at
least three times, and the results are presented as average values ꢂ standard deviation.
(compounds 7b, 7c). No significant differences were observed
between the pKa and log P calculated values (Table 1) of this series of
compounds which are all substituted by an amino group.
apparatus. pKa and log P values are calculated using the ACDLabs
software (Toronto, Canada).
In summary, a new series of imidazo[1,2-a]quinoxaline deriva-
tives were synthesized with short reaction times by using micro-
wave assistance and catalysts. These compounds were evaluated
for their antitumor activities against human melanoma. These new
analogues demonstrated interesting antitumor activities effect on
A375 cell lines more potent than imiquimod and fotemustine. We
found promising lead compounds (7b, 7c, 7e, 7f, 7g), EAPB0203
analogues, which exhibited high activities up to 9 times more
potent for compound 7b (EAPB0503) than EAPB0203 (lead
compound) on human melanoma cell lines. EAPB0503 can be
considered as a new lead compound of the imidazo[1,2-a]qui-
noxaline series. Further studies are now in progress for the opti-
mization of these compounds. On the other hand, in vivo studies
will be complemented and documented to depict the antitumor
activities of this new lead (EAPB0503) in female Swiss mice
xenografted with human melanoma cell lines.
4.1.1. Diimidazo[1,2-a]piperazine-5,10-dione (1)
The imidazole-2-carboxylic acid (5 g, 44.6 mmol) in suspension
in thionyl chloride (75 mL) was heated to reflux, under agitation for
18 h. The reaction mixture was cooled, then filtered, washed with
toluene, and dried under high vacuum to obtain a yellow solid
(yield, 90–95%). M.p.: 164 ꢀC. 1H NMR (300 MHz, DMSO-d6)
d
: 8.85
(s, 2H); 8.2 (s, 2H). 13C NMR (300 MHz, DMSO-d6)
d: 145.85, 144.95,
125.20, 120.15. Anal. calcd for C8H4N4O2: C, 51.07; H, 2.14; N, 29.78.
Found: C, 51.15; H, 2.21; N, 29.46.
4.1.2. (2-Fluoroaniline)-1H-imidazole-4-carboxamide (2)
To 2-fluoroaniline (1.91 ml, 19.77 mmol) in THF (13 ml), cooled
in a ꢁ10 ꢀC bath, (45.2 ml, 45,2 mmol, 1.0 M) sodium bis-(trime-
thylsilyl)amide in THF was added. The mixture was stirred for 1 h,
and a suspension of 1 (1.77 g, 9.4 mmol) in THF (20 mL) was added
and allowed to warm to room temperature. The mixture was stirred
for 2 h, and acetic acid was added to pH 7. The reaction mixture was
concentrated in vacuum followed by the addition of water and
saturated NaHCO3. The solid was collected by filtration, washed
with water and cyclohexane, and dried under high vacuum to give 2
as a beige solid (yield, 40%). M.p.: 250 ꢀC. 1H NMR (300 MHz,
4. Experimental
4.1. Chemistry
DMSO-d6)
d
: 8.12 (dd,1H), 7.92 (dd,1H), 6.92 (t,1H), 7.13 (t,1H), 7.07
All solvents and reagents were obtained from commercial
sources and used without further purification unless indicated
otherwise. 1H and 13C NMR spectra were recorded using a Bruker
AC 300 spectrometer. Chemical shifts are reported in parts per
million (ppm) from the tetramethylsilane resonance in the
indicated solvent. Coupling constants are reported in Hertz (Hz),
spectral splitting partners are designed as follow: singlet (s);
doublet (d); triplet (t); multiplet (m). Column chromatography
was performed on Merck silica gel 60 (200–400 mesh).
Elemental analysis was carried out at the Microanalytical
Central Department (Montpellier, France). The microwave organic
synthesis was assisted by the Biotage initiator (France). Melting
points of solid compounds were measured by the Biocote
(s, 2H). 19F NMR: 126. 13C NMR (300 MHz, DMSO-d6)
d: 151.69,
150.92, 150.52, 130.14, 128.79, 126.96, 123.97, 121.03, 119.53, 115.03.
Anal. calcd for C10H8FN3O: C, 58.54; H, 3.93; N, 20.48C, 64,37; H,
6.13; N, 16.09. Found: C, 58.33; H, 3.55; N, 20.14.
4.1.3. Imidazo[1,2-a]quinoxaline-6-(5H)-one (3)
A mixture of 2 (2 g, 9.75 mmol) and sodium hydride (0.3 g,
1.17 mmol) in dimethylacetamide (80 mL) was heated to reflux for
6 h. The reaction mixture was then concentrated in vacuum fol-
lowed by the addition of water and saturated ammonium chloride.
The solid was collected by filtration, washed with water, and dried
to give 3 (yield, 80%). M.p.: 322 ꢀC. 1H NMR (300 MHz, DMSO-d6)

C. Deleuze-Masquefa et al. / European Journal of Medicinal Chemistry 44 (2009) 3406–3411
3409
d
: 9.4 (s, 1H), 8.2 (d, 1H), 7.8 (d, 1H), 7.31 (d, 1H), 7.1–7.3 (m, 3H).
4.2.1. N-Methyl-1-phenylimidazo[1,2-a]quinoxalin-4-amine (7a)
13C NMR (300 MHz, DMSO-d6)
d
: 149.26, 139.11, 137.89, 132.43,
Phenylboronic acid (260 mg, 2.13 mmol). White solid (90%).
126.43, 123.94, 123.02, 119.40, 118.17, 115.42. Anal. calcd for
C10H7N3O: C, 64.86; H, 3.81; N, 22.69. Found: C, 64.44; H, 4.03; N,
22.98.
M.p.: 186 ꢀC. 1H NMR (300 MHz, CDCl3)
d
: 8.15 (s, 1H), 7.99 (dd, 1H),
7.52 (m, 8H), 5.7(s, 1H), 3.01 (d, 3H). 13C NMR (300 MHz, CDCl3)
d
:
142.86, 139.37, 131.13, 130.74, 129.67, 129.02, 126.77, 125.75, 124.90,
124.78, 124.47, 121.12, 114.96, 29.20. Anal. calcd for C17H14N4: C,
74.43; H, 5.14; N, 20.42. Found: C, 74.15; H, 5.46; N, 20.09.
4.1.4. 6-Chloroimidazo[1,2-a]quinoxaline (4)
To 3 (1.42 g, 7.7 mmol), phosphorus oxychloride (10 mL) and
N,N-diethylaniline (1.6 mL) were added and the mixture was
heated to reflux for 2 h. The reaction mixture was concentrated
under vacuum, and the residue was cooled in an ice bath. Water
was added to the residue and neutralized with saturated NaHCO3.
The solid was collected by filtration and purified by recrystallisation
from methanol to yield 4 as a white solid (yield, 80%). M.p.: 188 ꢀC.
4.2.2. 1-(3-Methoxyphenyl)-N-methylimidazo[1,2-a]quinoxalin-4-
amine (7b) (EAPB0503)
3-Methoxyphenylboronic acid (329 mg, 2.16 mmol). White solid
(90%). M.p.: 168 ꢀC. 1H NMR (300 MHz, CDCl3)
d
: 9.04 (s, 1H), 8 (dd,
1H), 7.82 (m, 2H), 7.4 (m, 3H), 6.99 (d, 1H), 6.86 (s, 1H), 5.7 (s, 1H),
3.83 (s, 3 H), 2.88 (d, 3H). 13C NMR (300 MHz, CDCl3)
: 160.98,
d
1H NMR (300 MHz, DMSO-d6)
7.35 (m, 1H), 7.25 (m, 2H). 13C NMR (300 MHz, DMSO-d6)
d
: 8.5 (s, 1H), 8.06 (d, 1H), 7.56 (s, 1H),
: 135.82,
142.86, 139.57, 131.13, 130.56, 129.5, 129.02, 128.4, 127.39, 124.9,
124.40, 122.10, 118.02, 114.96, 111.87, 108.08, 55.30, 29.20. Anal.
calcd for C18H16N4O: C, 71.04; H, 5.30; N, 18.41. Found: C, 71.40; H,
5.66; N, 18.09.
d
132.19, 131.75, 130.4, 129.95, 127.12, 125.03, 115.67, 113.4. Anal. calcd
for C10H6N3Cl: C, 58.98; H, 2.97; N, 20.64. Found: C, 59.12; H, 2.76;
N, 20.45.
4.2.3. 1-(4-Methoxyphenyl)-N-methylimidazo[1,2-a]quinoxalin-4-
amine (7c)
4.1.5. N-Methylimidazo[1,2-a]quinoxalin-4-amine (5)
Methylamine of a 40% (w/v) aqueous solution, was added
dropwise to a stirred solution of 4 (0.110 g, 0.54 mmol) in absolute
EtOH (10 mL) at room temperature. After 40 h, another portion of
methylamine (0.15 ml, 1.74 mmol) of a 40% (w/v) aqueous solution
was added and stirring was maintained for additional 3 h. The
solvent was removed under reduced pressure, and the residue was
dissolved in CH2Cl2 (10 mL). The organic fraction was successively
washed with 5% NaHCO3 (15 mL) and water (15 mL), dried
(Na2SO4), and concentrated under reduced pressure. The product
4-Methoxyphenylboronic acid (329 mg, 2.16 mmol). White solid
(95%). M.p.: 198 ꢀC. 1H NMR (300 MHz, CDCl3)
d
: 8.14 (s, 1H), 8.00
(d, 1H), 7.67 (m, 3H), 7.5 (m, 2H), 7.05 (m, 2H), 5.77 (s, 1H), 3.82 (s,
3H), 2.99 (s, 3H). 13C NMR (300 MHz, CDCl3)
: 158.05, 142.86,
d
139.57, 131.13, 129.61, 129.02, 125.96, 125.68, 124.47, 124.02, 121.12,
114.96, 113.35, 57.83, 29.20. Anal. calcd for C18H16N4O: C, 71.04; H,
5.30; N, 18.41. Found: C, 71.18; H, 5.58; N, 18.13.
4.2.4. 1-(2-Methoxyphenyl)-N-methylimidazo[1,2-a]quinoxalin-4-
amine (7d)
was purified by column chromatography on silica gel with C6H12
EtOAc (70:30, v/v) as eluent to yield a white solid (yield, 93%). M.p.:
180 ꢀC. 1H NMR (300 MHz, DMSO-d6)
: 7.92 (s, 1H), 7.75 (dd, 1H),
7,65 (dd, 1H), 7.52 (s, 1H), 7.4 (t,1H), 7.25 (t, 1H), 6.15 (s, 1H), 3.25 (d,
3H). 13C NMR (300 MHz, DMSO-d6)
: 142.25, 139.17, 131.99, 129.4,
–
2-Methoxyphenylboronic acid (329 mg, 2.16 mmol). Beige solid
d
(94%). M.p.: 180 ꢀC. 1H NMR (300 MHz, CDCl3)
d
: 8.1 (d, 1H), 7.60 (m,
3H), 7.48 (t, 1H), 7.30 (m, 4H), 5.74 (s, 1H), 3.85 (s, 3H), 3.00 (s, 3H).
13C NMR (300 MHz, CDCl3)
: 153.81, 142.74, 139.57, 131.64, 131.13,
d
d
128.17, 127.2, 125.14, 124.34, 114.14, 113.89, 29.20. Anal. calcd for
C11H10N4 C, 66.65; H, 5.08; N, 28.26. Found: C, 66.26; H, 5.53; N,
28.23.
129.02, 128.44, 127.58, 124.45, 124.28, 121.37, 118.02, 114.86, 11.98,
55.32, 29.20. Anal. calcd for C18H16N4O: C, 71.04; H, 5.30; N, 18.41.
Found: C, 70.98; H, 5.12; N, 18.22.
4.1.6. 1-Bromo-N-methylimidazo[1,2-a]quinoxalin-4-amine (6)
A solution of 5 (1.5 g, 7.5 mmol) and N-bromosuccinimide (1.5 g,
7.5 mmol) in CHCl3 (200 mL) was heated under reflux for 2 h. The
resulting solution was cooled, washed with 5% sodium hydrogen
carbonate (50 mL), dried and concentrated. The residue was puri-
fied by recristallisation from chloroform to a white solid (1.18 g,
4.2.5. 1-(3-Ethoxyphenyl)- N-methylimidazo[1,2-a]quinoxalin-4-
amine (7e)
3-Ethoxyphenylboronic acid (360 mg, 2.17 mmol). White solid
(52%). M.p.: 162 ꢀC. 1H NMR (300 MHz, CDCl3)
d: 8.34 (s, 1H), 8.02
(d,1H), 7.83 (d,1H), 7.67 (t,1H), 7.5 (m, 2H), 6.96 (d,1H), 6.85 (s,1H),
5.74 (s, 1H), 3.89 (m, 2H), 2.89 (s, 3H), 1.38 (t, 3H). 13C NMR
67%). M.p.: 202 ꢀC. 1H NMR (300 MHz, DMSO-d6)
(dd, 1H), 7,65 (dd, 1H), 7.4 (t,1H), 7.25 (t, 1H), 6.15 (s, 1H), 3.25 (d,
3H). 13C NMR (300 MHz, DMSO-d6)
: 141.97, 140.54, 138.85, 128.96,
d: 9.10 (s, 1H), 7.75
(300 MHz, CDCl3) d: 161.36, 142.86, 139.57, 131.13, 130.22, 129.5,
129.02, 128.41, 127.05, 124.9, 122.1, 118.02, 114.96, 113.77, 108.12,
63.8, 29.2, 15.2. Anal. calcd for C19H18N4O: C, 71.68; H, 5.70; N,17.60.
Found: C, 71.43; H, 5.56; N, 17.39.
d
127.78, 127.09, 126.03, 125.13, 113.86, 101.4, 29.20. Anal. calcd for
C11H9N4Br: C, 47.68; H, 3.27; N, 20.22. Found: C, 47.33; H, 3.53; N,
20.53.
4.2.6. 1-(3-Hydroxyphenyl)-N-methylimidazo[1,2-a]quinoxalin-4-
amine (7f)
4.2. General procedure for the Suzuki reaction
3-Hydroxyphenylboronic acid (329 mg, 2.16 mmol). White solid
(85%). M.p.: 278 ꢀC. 1H NMR (300 MHz, CDCl3)
d
: 8.33 (s, 1H), 7.99
(d, 1H), 7.79 (d, 1H), 7.70 (m, 2H), 7.67 (t, 1H), 7.50 (m, 3H), 6.857 (m,
2H), 3.02 (s, 3H). 13C NMR (300 MHz, CDCl3)
: 160.07, 142.86,
To a mixture of 6 (300 mg, 1.08 mmol) and Pd(PPh3) (63 mg,
0.05 mmol) in DME (15 mL) was added the corresponding aryl
boronic acid followed by the addition of sodium carbonate
(234 mg) in water (5 mL). The reaction was irradiated in a sealed
tube at 140 ꢀC for 20 min using a Biotage Initiator microwave
synthesizer. The reaction mixture was poured on water and
then extracted with dichloromethane (2 ꢃ 40 mL). The combined
organic extracts were washed with water (40 mL), dried and
concentrated to dryness under vacuo. The crude product was
purified by column chromatography (silica gel eluting with
dichloromethane).
d
139.57, 131.46, 131.13, 130.35, 129.63, 129.02, 127.64, 126.78, 125.39,
124.9, 124.40, 122.83, 120.71, 114.86, 114.54, 29.02. Anal. calcd for
C17H14N4O: C, 70.33; H, 4.86; N, 19.30. Found: C, 70.49; H, 5.19; N,
19.09.
4.2.7. 1-(3-Bromophenyl)- N-methylimidazo[1,2-a]quinoxalin-4-
amine (7g)
3-Bromophenylboronic acid (435 mg, 2.16 mmol). Yellow solid
(78%). M.p.: 144 ꢀC. 1H NMR (300 MHz, CDCl3)
d: 8.18 (s, 1H), 7.90

3410
C. Deleuze-Masquefa et al. / European Journal of Medicinal Chemistry 44 (2009) 3406–3411
(m, 2H), 7.67 (t, 1H), 7.50 (m, 2H), 7.45 (m, 3H), 5.74 (s, 1H), 2.98 (s,
3H). 13C NMR (300 MHz, CDCl3)
: 142.86, 139.57, 132.86, 131.13,
139.57, 131.19, 131.13, 129.02, 127.32, 127.16, 126.99, 126.43, 124.90,
124.66, 124.40, 122.75, 120.76, 114.96, 29.20. Anal. calcd for
d
130.53, 129.63, 129.03, 127.64, 126.78, 125.39, 124.90, 124.30,
122.93, 120.71, 114.96, 29.20. Anal. calcd for C17H13 Br N4: C, 57.81;
H, 3.71; N, 15.86. Found: C, 58.05; H, 3.55; N, 15.99.
C17H13N5O2: C, 63.94; H, 4.10; N, 21.93. Found: C, 64.13; H, 3.89; N,
21.66.
4.2.14. 1-(3-Furan-N-methylimidazo[1,2-a]quinoxalin-4-amine
(7n)
4.2.8. 1-(3-Trifluoromethylphenyl)- N-methylimidazo[1,2-
a]quinoxalin-4-amine (7h)
3-Furanboronic acid (243 mg, 2.16 mmol). White solid (96).
3-Trifluoromethylphenylboronic acid (411 mg, 2.16 mmol).
M.p.: 190 ꢀC. 1H NMR (300 MHz, CDCl3)
d
: 8.24 (s, 1H), 7.90 (m, 3H),
7.83 (s, 1H), 7.62 (t, 1H), 7.50 (m, 2H), 7.12 (m, 2H), 6.98 (s, 1H), 5.74
(s, 1H), 4.85 (d, 1H), 2.85 (s, 3H). 13C NMR (300 MHz, CDCl3)
White solid (98%). M.p.: 210 ꢀC. 1H NMR (300 MHz, CDCl3)
d
: 8.14 (s,
1H), 8.10 (s, 1H), 8.02 (m, 2H), 7.85 (d, 1H), 7.60 (m, 2H), 7.48 (t, 1H),
5.74 (s, 1H), 2.96 (s, 3H). 13C NMR (300 MHz, CDCl3)
: 142.86,
d
:
d
141.50, 137.73, 135.60, 130.13, 128.79, 128.02, 124.24, 123.70, 117.85,
116.09, 113.61, 107.72, 94.65, 29.20. Anal. calcd for C15H12N4O: C,
68.17; H, 4.58 N, 21.20. Found : C, 68.20 ; H, 4.56 ; N, 21.17.
139.57, 132.62, 132.30, 132.02, 131.60, 131.13, 129.02, 128.86, 124.90,
124.47, 122.51, 121.59, 120.40, 114.86, 114.54, 29.02. Anal. calcd for
C18H13N4F3: C, 63.16; H, 3.83; N, 16.65. Found: C, 63.03; H, 3.76; N,
16.74.
4.3. Cell lines and culture techniques
4.2.9. 1-(3-Chlorophenyl)-N-methylimidazo[1,2-a]quinoxalin-4-
amine (7i)
Melanoma (A375) human cancer cell lines were obtained from
American Type Culture Collection (Rockville, Md., USA). Cells were
cultured in RPMI medium containing RPMI-1640 (Gibco Laborato-
ries, France), 10% heat-inactived (56 ꢀC) foetal bovine serum (FBS)
3-Chlorophenylboronic acid (339 mg, 2.16 mmol). Yellow solid
(63%). M.p.: 188 ꢀC. 1H NMR (300 MHz, CDCl3)
d
: 8.48 (s, 1H), 8.02
(d, 1H), 7.67 (t, 1H), 7.58 (m, 2H), 7.49 (m, 3H), 7.10 (s, 1H), 5.74 (s,
1H), 2.81 (s, 3H). 13C NMR (300 MHz, CDCl3)
: 142.86, 139.57,
d
(Polylabo, Paris, France), 2 mM
L-glutamine, 100 IU/ml penicillin G
135.86, 131.13, 130.50, 130.16, 129.76, 129.36, 129.02, 126.68, 124.9,
124.46, 123.39, 122.96, 121.74, 114.96, 29.15. Anal. calcd for
C17H13ClN4: C, 66.13; H, 4.24; N, 11.48. Found: C, 66.35; H, 4.01; N,
11.25.
sodium, 100 g/ml streptomycin sulphate, and 0.25 m
tericin B. Cells were maintained in a humidified atmosphere of 5%
CO2 in air at 37 ꢀC.
m
g/ml ampho-
4.2.10. 1-(3-Carboxyphenyl)- N-methylimidazo[1,2-a]quinoxalin-4-
amine (7j)
4.4. In vitro cytotoxicity assay
3-Carboxyphenylboronic acid (357 mg, 2.16 mmol). Orange
Previously to the experiments, the number of cells by well, the
doubling time and the MTT concentration have been optimized. In
all the experiments, A375 cells were seeded at a final concentration
of 5000 cells/well in 96-well microtiter plates and allowed to attach
overnight. After 20–24 h incubation, the medium was aspirated
carefully from the plates using a sterile Pasteur pipette, and cells
were exposed (i) to vehicle controls (1% DMSO/culture medium and
culture medium alone), (ii) fotemustine and imiquimod at concen-
trations of 10ꢁ4–10ꢁ10 mM, diluted in the culture medium, and (iii) to
the synthesized compounds (10ꢁ4–10ꢁ10 mM) dissolved in a mixture
solid (87%). M.p.: 130 ꢀC. 1H NMR (300 MHz, CDCl3)
d
: 8.41 (d, 1H),
8.26 (s,1H), 8.01 (m, 2H), 7.80 (m, 4H), 7.58 (m, 2H), 7.47 (t,1H), 2.95
(s, 3H). 13C NMR (300 MHz, CDCl3)
: 167.35, 142.86, 139.54, 131.88,
d
131.13, 130.34, 129.02, 128.76, 127.1, 126.72, 124.9, 124.47, 122.51,
122.38, 114.96, 29.20. Anal. calcd for C18H14N4O2: C, 67.92; H, 4.43;
N, 17.60. Found: C, 67.72; H, 4.35; N, 17.63.
4.2.11. 1-(3-Fluorophenyl)- N-methylimidazo[1,2-a]quinoxalin-4-
amine (7k)
3-Fluorophenylboronic acid (303 mg, 2.16 mmol). Yellow solid
1% DMSO/culture medium (v/v). After 96 h of incubation, 10 ml of
(66%). M.p.: 220 ꢀC. 1H NMR (300 MHz, CDCl3)
d
: 8.22 (s, 1H), 7.98
(d, 1H), 7.59 (m, 3H), 7.48 (m, 2H), 7.22 (s, 1H), 7.03 (d, 1H), 5.74 (s,
1H), 2.96 (s, 1H). 13C NMR (300 MHz, CDCl3)
: 163.95, 142.86,
MTT solution in PBS (5 mg/ml, phosphate-buffer saline pH 7.3) was
added to each well, and the wells were incubated at 37 ꢀC for 4 h.
This colorimetric assay is based on the ability of live and metaboli-
cally unimpaired tumor-cell targets to reduce MTT to a blue for-
mazan product. At the end of the incubation period, the supernatant
d
139.57, 132.57, 132.15, 131.13, 129.02, 128.63, 126.77, 124.90,
124.47, 121.59, 115.29, 114.96, 111.90, 111.36, 29.20. Anal. calcd for
C17H13 F N4: C, 69.85; H, 4.48; N, 19.17. Found: C, 69.79; H, 4.48; N,
18.97.
was carefullyaspirated, then,100 ml of a mixture of isopropyl alcohol
and 1 M hydrochloric acid (96/4, v/v) was added to each well. After
10 min of incubation and vigorous shaking to solubilize formazan
crystals, the optical density was measured at 570 nm in a microcul-
ture plate reader (Dynatech MR 5000, France). Foreachassay, at least
three experiments were performed in triplicate.
The individual cell line growth curves confirmed that all A375
lines in control medium remained in the log phase of cell growth
96 h after plating. Cell survival was expressed as percent of vehicle
control. The IC50 values defined as the concentrations of drugs
which produced 50% cell growth inhibition; 50% reduction of
absorbance, were estimated from the sigmoidal dose–response
curves.
4.2.12. 1-(3-Cyanophenyl)- N-methylimidazo[1,2-a]quinoxalin-4-
amine (7l)
3-Cyanophenylboronic acid (351 mg, 2.16 mmol). Beige solid
(84%). M.p.: 249 ꢀC. 1H NMR (300 MHz, CDCl3)
d
: 8.41 (s,1H), 8.13 (d,
1H), 8.00 (d, 1H), 7.77 (d, 1H), 7.64 (m, 3H), 7.51 (m, 2H), 5.77 (s, 1H),
2.76 (s, 3H). 13C NMR (300 MHz, CDCl3)
: 142.86, 139.57, 132.25,
d
131.64, 131.13, 130.04, 129.02, 128.70, 128.67, 127.96, 127.41, 124.90,
124.47, 119.70, 114.96, 113.80, 29.20. Anal. calcd for C18H13N5: C,
72.23; H, 4.38; N, 23.40. Found: C, 72.01; H, 4.71; N, 23.25.
4.2.13. 1-(3-Nitrophenyl)- N-methylimidazo[1,2-a]quinoxalin-4-
amine (7m)
3-Nitrophenylboronic acid (399 mg, 2.16 mmol). Yellow solid
Acknowledgements
(95%). M.p.: 236 ꢀC. 1H NMR (300 MHz, CDCl3)
d
: 8.21 (s, 1H), 8.01
(d, 1H), 7.70 (m, 2H), 7.58 (d, 1H), 7.40 (m, 2H), 7.17 (m, 2H), 5.17 (s,
1H), 3.01 (s, 3H). 13C NMR (300 MHz, CDCl3)
: 147.62, 142.86,
Special thanks to the Ligue contre le Cancer for its financial
support.
d

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3411
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