In vitro and in vivo anti-tumoral activities of imidazo[1,2-a]quinoxaline, imidazo[1,5-a]quinoxaline, and pyrazolo[1,5-a]quinoxaline derivatives

Article abstract of DOI:10.1016/j.bmc.2008.05.022

Imidazoquinoxaline and pyrazoloquinoxaline derivatives, analogues of imiquimod, were synthesized, and their in vitro cytotoxic and pharmacodynamic activities were evaluated. In vitro cytotoxicity studies were assessed against melanoma (A375, M4Be, RPMI-75

Full text of DOI:10.1016/j.bmc.2008.05.022

Bioorganic & Medicinal Chemistry 16 (2008) 6601–6610
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
In vitro and in vivo anti-tumoral activities of imidazo[1,2-a]quinoxaline,
imidazo[1,5-a]quinoxaline, and pyrazolo[1,5-a]quinoxaline derivatives
Georges Moarbess a, , Carine Deleuze-Masquefa a, , , Vanessa Bonnard a, Stéphanie Gayraud-Paniagua a,
*
Jean-Rémi Vidal c, Francßoise Bressolle b,c, Frédéric Pinguet c, Pierre-Antoine Bonnet a
a Laboratoire de Pharmacochimie et Biomolécules, EA 4215, Faculté de Pharmacie, Université Montpellier I, 15 av. Charles Flahault, BP 14491, 34 093 Montpellier Cedex 5, France
b Laboratoire de Pharmacocinétique Clinique, EA 4215, Faculté de Pharmacie, Université Montpellier I, 15 av. Charles Flahault, BP 14491, 34 093 Montpellier Cedex 5, France
c Centre Régional 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:
Received 5 February 2008
Revised 2 May 2008
Accepted 7 May 2008
Available online 10 May 2008
Imidazoquinoxaline and pyrazoloquinoxaline derivatives, analogues of imiquimod, were synthesized,
and their in vitro cytotoxic and pharmacodynamic activities were evaluated. In vitro cytotoxicity studies
were assessed against melanoma (A375, M4Be, RPMI-7591), colon (LS174T), breast (MCF7), and lym-
phoma (Raji) human cancer cell lines. In vivo studies were carried out in M4Be xenografted athymic mice.
EAPB0103, EAPB0201, EAPB0202, and EAPB0203 showed significant in vitro activities against A375
compared to fotemustine and imiquimod used as references. These compounds were 6–110 and 2–45
times more active than fotemustine and imiquimod, respectively. EAPB0203 bearing phenethyl as sub-
stituent at position 1 and methylamine at position 4 showed the highest activity. EAPB0203 has also a
more potent cytotoxic activity than imiquimod and fotemustine in M4Be and RPMI-7591 and interesting
cytotoxic activity in other tumor cell lines tested. In vivo, EAPB0203 treatment schedules caused a sig-
nificant decrease in tumor size compared to vehicle control and fotemustine treatments.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Imidazoquinoxalines
Pyrazoloquinoxaline
Imiquimod
Melanoma
In vitro activity
Pharmacodynamic evaluation
1. Introduction
requiring the activation of the ‘work horses’ of apoptosis, the casp-
ases’ family of proteases.
Imiquimod, an imidazoquinoline amine, is an immunomodulat-
ing agent approved in 1997 by the U.S Food and Drugs Administra-
tion for treating external genital and peri-anal warts.1–3 In murine
animal models, imiquimod has proven its efficacy in a variety of
transplantable tumors including colon carcinomas, melanomas,
lung sarcomas, mammary, and bladder carcinomas.2 It has shown
activities toward basal cell carcinomas (BCC),3,4 actinic keratos-
es,4,5 and some melanoma metastases,6–9, and its therapeutic spec-
trum was extended to cutaneous B-cell lymphomas.10
Extensive studies over the past years indicated that imiquimod
acts both: (i) indirectly, by activating the innate as well as the
adaptive immune system via binding to cell surface receptors such
as Toll-like receptors (TLR) 7 and 8, thereby inducing the activation
of transcription factors like nuclear factor NF-jB and resulting in
the secretion of pro-inflammatory cytokines predominantly inter-
feron (IFN)-a, tumor necrosis factor (TNF)-a and interleukin (IL)-
12,11,12 and (ii) directly, by inducing direct in vitro and in vivo
pro-apoptotic activities in a rather tumor selective manner,11,13,14
Responsible of several thousand deaths each year, melanoma is
the most frequent malignant tumor in the white human population
worldwide. Its frequency increases dramatically with age and
chronic sun exposure. INF-a, IL-2, some chemotherapeutic drugs,
and tumor-directed vaccination have been evaluated in clinical tri-
als. All of these approaches, unfortunately, met with rather limited
success. Given the limited effectiveness of a large variety of con-
ventional anti-cancer drugs in the treatment of melanoma such
as various chemo-and/or immunotherapies, novel approaches or
drugs selectively targeting melanoma cells are urgently needed.
NO
H
N
N
N
Cl
N
O
O
O
P
O
C2H5
N
NH2
C2H5
* Corresponding author. Tel: +33 4 67548665/8640; fax.: +33 4 67548036.
E-mail address: carine.masquefa@univ-montp1.fr (C. Deleuze-Masquefa).
These authors contributed equally to this work.
imiquimod
fotemustine
Figure 1. Chemical structures of imiquimod and fotemustine.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.022

6602
G. Moarbess et al. / Bioorg. Med. Chem. 16 (2008) 6601–6610
This study was related to the pharmacochemistry of nitrogen
with yields of 94% and 54%, respectively. Attempted reduction of
the nitro group by hydrogenation resulted in the formation of 4a
and 4b with yields of 45% and 94%, respectively. The formation
of the tricyclic compounds 5a and 5b was obtained after addition
of carbonyldiimidazole by intramolecular cyclization of 4a and
4b, respectively. The chlorination of 5a and 5b by phosphorus oxy-
chloride and substitution by an adapted amine formed the 7a and
7b compounds with yields of 53% and 86%, respectively.
heterocycles. The aim of this paper was to synthesize and to devel-
op three chemical series, analogues of imiquimod (Fig. 1): the imi-
dazo[1,2-a]quinoxalines, the imidazo[1,5-a]quinoxalines, and the
pyrazolo[1,5-a]quinoxalines, in order to evaluate their potential
anti-cancer properties, in vitro against a variety of human cancer
cell lines, and in vivo on melanoma by comparison with fotemus-
tine (Fig. 1), one of the main drug used in clinic to treat human
melanoma.
The synthesis of the 1H-pyrazolo[1,5-a]quinoxaline derivatives
is illustrated in Scheme 2.
Dimer 8 resulted from the bimolecular condensation of the 3-
isobutyl-1H-pyrazole-5-carboxylic acid, in presence of thionyl
chloride in 80% overall yield. Product 8 was coupled with orthoflu-
oroaniline to give the intermediate 9, with a yield of 46%. The tri-
cyclic compound 10 was obtained via an intramolecular cyclization
of 9 in the presence of sodium hydride and dimethylacetamide as
solvent with 80% yield. Treatment of compound 10 with phospho-
rus oxychloride yielded to 50% of compound 11 which was coupled
with methylamine (series a) and dimethylamine (series b) to form
12a and 12b with average yields of 75% and 85%, respectively. The
same strategy was applied to obtain the monosubstituted 1H-imi-
2. Results
2.1. Chemistry
2.1.1. Library synthesis
For the preparation of the 1H-imidazo[1,5-a]quinoxaline deriv-
atives, the procedure presented in Scheme 1 was used. In general,
treatment of ortho-fluoronitrobenzene with imidazole and an ex-
cess of potassium carbonate formed compound 1 in 95% overall
yield. After halogenation with N-bromosuccinimide, compounds
3a and 3b were obtained through Suzuki cross-coupling reactions
NO2
NO2
NO2
HN
N
N
N
NO2
F
N
N
N
N
N
c
R1
b
a
Br
1
2
a : R1= Me
3a, 3b
b :
R1= Ph
R1
R1
R1
R1
NH2
N
N
N
N
N
N
N
N
N
e
d
g
f
NHCH3
Cl
N
H
O
4a, 4b
5a, 5b
6a, 6b
7a, 7b
Scheme 1. Synthesis of 1H-imidazo[1,5-a]quinoxaline derivatives. Reagents and conditions: (a) MeCN, reflux; (b) NBS, MeCN, reflux; (c) R1-B(OH)2, Na2CO3, DME, Pd(PPh3);
(d) H2/Pd-C, EtOH; (e) Im2CO, PhCl2, reflux; (f) POCl3, reflux; (g) R0NH2, EtOH.
F
O
N
N
F
NH2
HN
O
N
N
NH
a
b
N
N
N
H
COOH
O
9
8
N
N
N
N
N
N
c
e
d
N
H
O
N
Cl
N
NR'R''
11
12a,12b
10
a : R'=CH3, R''=H
b : R'=R''=CH3
Scheme 2. Synthesis of 1H-pyrazolo[1,5-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, R0R00NH, 20 h, rt.

G. Moarbess et al. / Bioorg. Med. Chem. 16 (2008) 6601–6610
6603
dazo[1,5-a]quinoxalines 17a and 17b starting from imidazole-4-
carboxylic acid (Scheme 3).
All synthesized compounds with their corresponding pKa and
logP calculated values are presented in Table 1. The 1H-imi-
N
F
O
N
HN
F
NH2
N
N
N
N
NH
N
c
O
NH
14
b
a
HOOC
O
13
N
N
N
N
N
N
e
N
d
NR'R''
N
H
O
Cl
a : R' = CH3, R'' = H
b : R' = R'' = CH3
17a, 17b
15
16
Scheme 3. New synthesis of the monosubstituted 1H-imidazo[1,5-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, R0R00NH, 20 h, rt.
Table 1
1H-Imidazo[1,5-a]quinoxaline, 1H-imidazo[1,2-a]quinoxaline and 1H-pyrazolo[1,5-a]quinoxaline: formulas and in vitro cytotoxic activities against A375 human melanoma cell
line
R1
R1
R1
N
N
N
N
N
N
N
N
R4
N
R4
R4
Pyrazolo[1,5-a]quinoxaline
Imidazo[1,5-a]quinoxaline
Imidazo[1,2-a]quinoxaline
IC50 (lM)a against A375 cell line
pKa
LogP*
*
Compounds
R1
R4
Formula
Imidazo[1,2-a]quinoxalines
EAPB0103
(CH3)2–CH–CH2–
C6H5–(CH2)2–
CH3–NH–
CH3–NH–
NH2–
C15H18N4
C19H18N4
C18H16N4
C18H14ClN3
C16H20N4
C20H20N4
C19H17N3O
31.6 2.0
1.57 0.56
2.35 0.15
24.0 0.5
66.3 7.5
80.1 7.0
47.8 5.0
5.61 0.4
5.61 0.4
5.13 0.4
ꢀ1.57 0.4
5.73 0.4
5.73 0.4
0.85 0.4
3.91 1.42
4.91 1.42
4.97 1.42
4.53 1.27
3.33 1.42
4.32 1.42
4.56 1.42
EAPB0203
EAPB0202
EAPB0201
EAPB0104
EAPB0204
EAPB0206
C6H5–(CH2)2–
C6H5–(CH2)2–
(CH3)2–CH–CH2–
C6H5–(CH2)2–
C6H5–(CH2)2–
Cl–
(CH3)2–N–
(CH3)2–N–
OCH3
N
EAPB0105
(CH3)2–CH–CH2–
C21H21ClN4
>400
2.25 0.4
4.56 1.42
7.25 1.43
Cl
N
EAPB0205
C6H5–(CH2)2–
C25H21ClN4
329 63
2.25 0.4
Cl
Imidazo[1,5-a]quinoxalines
17a
17 b
7a
H
H
C6H5–
CH3–
CH3–NH–
(CH3)2–N–
CH3–NH–
CH3–NH–
C11H10N4
C12H12N4
C17H14N4
C12H12N4
85.3 7.0
121 17
>400
6.50 0.4
6.62 0.4
6.45 0.4
7.33 0.4
0.93 1.07
0.95 1.08
3.38 1.09
1.19 1.08
7b
100
9
Pyrazolo[1,5-a]quinoxalines
12a
12b
(CH3)2–CH–CH2–
(CH3)2–CH–CH2–
CH3–NH–
(CH3)2–N–
C15H18N4
C16H20N4
78.6 7.8
101
6.20 0.4
6.32 0.4
3.38 1.06
3.40 1.06
5
Imiquimod
Fotemustine
70.3 4.3
173 24
a
IC50, concentration of the compound (lM) 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 standard deviation.
*
pKa and logP values are calculated using the ACDlabs software.

6604
G. Moarbess et al. / Bioorg. Med. Chem. 16 (2008) 6601–6610
dazo[1,2-a]quinoxaline substituted derivatives were obtained as
activity higher than fotemustine used as reference. To further eval-
uate the in vivo induction of melanoma death by EAPB0203 we
proceeded as described in materials and methods. Tumor size is
the most common parameter used to assess effective anti-cancer
treatment in preclinical models. The EAPB0203 treatments were
well tolerated without apparent side-effects or weight losses. As
expected, M4Be xenografts grew progressively using the vehicle
control. In contrast, in mice treated with EAPB0203 or fotemustine,
a highly significant delay in tumor growth was observed (Fig. 2). At
highest doses (20 mg/kg), from day 18 to day 55, the simple con-
trast test showed a high significant difference in tumor growth be-
tween the EAPB0203 and the control groups (P values ranging
from 0.0026 to 0.00004). Lower significant differences were ob-
served between the fotemustine and the control groups (not signif-
icant at days 18 through 24, 34, 37, and 55; and P: 0.014 to 0.0024
at days 27, 31, 41 through 51). Most interesting, EAPB0203 treat-
ment produced a statistically significant decrease in tumor growth
at days 21, 24, 41, and 44 compared to the fotemustine treatment.
Results are presented in Figure 2. At low doses (5 mg/kg), the vari-
ations in tumor growth according to the treatment group have the
same profile than at high doses (data not shown) but lower signif-
icant differences were observed. Between fotemustine and
EAPB0203, significant difference occurred on day 34.
Efficacy of treatment is dose-related. In mice receiving
EAPB0203, the decrease in tumor growth was statistically higher
on days 18 (P = 0.026), 21 (P = 0.0058), 24 (P = 0.0073), 31
(P = 0.0033), and 34 (P = 0.016) at the 20 mg/kg dose than at the
5 mg/kg dose. In animals treated by fotemustine, significant differ-
ences occurred at days 24 (P = 0.017), 27 (P = 0.019), 31
(P = 0.0046), and 34 (P = 0.0052).
In the control group, all mice were sacrificed on day 55. In the
fotemustine group (20 mg/kg), all mice were killed on day 76, since
tumor volumes were superior to 2 cm3, while in the EAPB0203
group (20 mg/kg), one mouse (1/6) was still alive with a tumor vol-
ume less than 2 cm3 (the limit fixed by the protocol).
previously described.15
2.1.2. In vitro biological activity
All compounds were firstly evaluated for their cytotoxic activity
in vitro against the A375 human melanoma cell line by comparison
with imiquimod and fotemustine used as references. Concentra-
tions of the compounds which produced 50% cell growth inhibition
(IC50 values) are summarized in Table 1 for the imidazo- and
pyrazoloquinoxaline compounds, and for fotemustine and imiqui-
mod. Among the compounds tested, EAPB0203 (IC50 = 1.57 lM)
and EAPB0202 (IC50 = 2.35 lM) exhibited the highest cytotoxic
activities. EAPB0203 containing phenethyle as substituent at posi-
tion 1 and methylamine at position 4 was the ‘lead’ compound
with an activity against A375 cell line 110 times higher than fote-
mustine (IC50 = 173 lM) and 45 times higher than imiquimod
(IC50 = 70 lM). The EAPB0202 (R1 = phenethyl and R4 = amine)
had an activity very close to that of EAPB0203 (almost 2 times
more active than EAPB0202) with an IC50 value 70 times lower
than fotemustine and 30 times lower than imiquimod. The
EAPB0103 compound having isobutyle at position 1 and methyl-
amine at position 4 exhibited an interesting activity; this com-
pound is 7 times more active than fotemustine and 3 times more
active than imiquimod. The EAPB0201 (R1 = phenethyl and
R4 = chloro) also presents a good cytotoxic activity, this compound
is 2 and 6 times more active than imiquimod and fotemustine,
respectively. The other compounds, substituted or unsubstituted
at position 1 and bearing dimethylamine or chloromethylaniline
as substituent at position 4, are less potent and have IC50 values be-
tween 20 and 300 lM. The cytotoxic activities of 7a and EAPB0105
have not been evaluated due to the very low solubility of these
compounds in the culture medium. The pyrazolo[1,5-a]quinoxa-
line analogue 12a showed similar activity than imiquimod.
Since EAPB0203 exhibited the highest activity against the A375
melanoma cell line, we further evaluated its cytotoxic activity
against the following human cell lines: M4Be and RPMI-7591 (mel-
anoma), LS174T (colon cancer), MCF7 (breast cancer), and Raji (B
lymphoma). The IC50 values are illustrated in Table 2. The most
interesting results were obtained on M4Be and RMPI-7591.
EAPB0203 exhibited cytotoxic activity 30-130 times higher than
fotemustine, with IC50 values of 2.58 and 4.23 lM, respectively.
In addition EAPB0203 showed a cytotoxic activity (3 times less po-
tent) similar to irinotecan on LS174T cell line.
3. Discussion
Imiquimod is a low-molecular-weight immune response modi-
fier that can induce the synthesis of interferon-a and other cyto-
kines in a variety of cell types. Imiquimod has potent anti-viral
and anti-tumor properties. Several mechanisms by which this
compound exerts its activity have been mentioned. Many stud-
ies11,16,17 show that imiquimod activates immune cells via the
Toll-like receptor 7 (TLR7)-MyD88-dependent signalling pathways.
Based on some reports14,18 of apoptosis in skin cancer, imiquimod
exerts a direct pro-apoptotic effect against human melanoma. It is
able to induce apoptosis in malignant melanoma cells both in vitro
and in vivo in a rather tumor-selective manner. Imiquimod-in-
duced apoptosis in tumor cells could not be antagonized by func-
tional blockage of various membrane-bound death receptors,
2.1.3. In vivo activity
The 50% lethal dose (LD50) of EAPB0203 was determined, in
Swiss mice, after intraperitoneal administrations at doses ranging
from 30 to 300 mg/kg. At the highest doses, EAPB0203 appeared
to be non toxic.
Previous studies have shown that EAPB0203 is a potent in vitro
growth inhibitor for human melanoma cell lines with a cytotoxic
Table 2
IC50 values for imiquimod, fotemustine, methotrexate, irinotecan, doxorubicin, and EAPB0203 against A375, M4Be, and RMPI-7591 (melanoma cell lines), LS174T (colon cancer),
MCF7 (breast cancer), and Raji (B lymphoma) human cell lines
Compounds
IC50 (lM)a
A375
M4Be
RPMI-7591
125 49
MCF7
LS174T
Raji
Fotemustine
Doxorubicin
Irinotecan
173 24
326 34
0.13 0.01
1.16 0.07
Methotrexate
Imiquimod
EAPB0203
0.04 0.005
139 12
6.23 0.06
70.3 4.3
1.57 0.56
33.5 7.7
2.58 0.40
53.7 9.8
4.23 0.48
145
1.08 0.46
7
34.4 9.7
4.12 0.67
a
IC50, concentration of the compounds (lM) 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 standard deviation.

G. Moarbess et al. / Bioorg. Med. Chem. 16 (2008) 6601–6610
6605
Figure 2. Effects of fotemustine (20 mg/kg once a week for 3 weeks) and EAPB0203 (20 mg/kg twice a week for 3 weeks) on tumor growth (ab2) in xenografted nude mice by
M4Be melanoma cell lines. ", fotemustine and EAPB0203 administrations; , EAPB0203 administrations N, control mice; j, EAPB0203; ꢁ, fotemustine. Data are the mean of
one experiment carried out on six mice. Error bars represent SEM. Significant difference between the 3 treatment groups: , P < 0.05; , 0.002 < P < 0.009; , P 6 0.001.
***
*
**
including Fas/APO-1 (CD95), TRAIL, and TNF receptors, which sug-
gests that imiquimod-inducing apoptosis is independent from
apoptosis mediated by membrane-bound death receptors. Its tu-
mor-selective pro-apoptotic activity is correlated with transloca-
tion of cytochrome c from the mitochondria to the cytosol by
shifting the mitochondria-associated Bcl-2/Bax ratio, and activat-
ing caspases 9 and 3. Imidazo[1,2-a]quinoxalines are imiquimod
analogues that inhibit cyclic nucleotide phosphodiesterase en-
zymes 4.15 Furthermore, in studies on murine fibroblast L929 cells,
we recently showed that these imidazo[1,2-a]quinoxalines acti-
vate the p38 MAPK pathway, and inhibit the PI3K pathway.19 Fur-
thermore, we recently demonstrated that one of these compounds
(EAPB0203) induced inhibition of cell proliferation, G2/M cell cycle
arrest, and apoptosis in HTLV-I transformed and HTLV-I-negative
malignant T cells as well in fresh adult T-cell leukaemia (ATL) cells,
while normal resting or activated T lymphocytes were resistant.
EAPB0203 initiated the apoptotic process via the intrinsic cell
death pathway converging at the mitochondria.21
In the present study, we report the synthesis and preliminary
pharmacological evaluation of a new series of imiquimod’s ana-
logues, the imidazo/pyrazoloquinoxaline derivatives. Imidazoquin-
oxalines were prepared via two different methods, the first one
previously described15 and the second one is presented in Scheme
1. The first method15 was used to synthesize the imidazo[1,2-
a]quinoxaline compounds which were obtained in seven steps in
good yields. The second method outlined in Scheme 1 was em-
ployed for the synthesis of the imidazo[1,5-a]quinoxaline com-
pounds. Using these two methods, a limited number of analogues
were obtained. Actually, the alkyl or aryl substitution at the R1 po-
sition must be done at the beginning steps of the synthesis leading
to a low variability in the moieties on the imidazole cycle. Like-
wise, the purifications after each step caused further limitations
of the synthetic process. Our framework was to optimize the meth-
ods of synthesis (i) to obtain a common strategy for the three
chemical series, (ii) to reduce the number of purification steps,
and (iii) to increase the structural diversity of these series essential
for the SAR studies. The procedure presented in Scheme 2 was used
to prepare the pyrazolo[1,5-a]quinoxaline derivatives.20 This
method not only reduced the number of purification steps but also
led us to propose a new common and efficient route for the prep-
aration of the unsubstituted imidazo[1,5-a]quinoxalines on the
imidazole core (Table 1) starting from the unsubstituted carboxylic
acid (Scheme 3). This new strategy also permitted the introduction
the R1 substituent at the last steps of the procedure with different
commercial Suzuki cross coupling reagents (syntheses under
study, data not shown).
All compounds were screened in vitro against the human mel-
anoma cell line A375. First, we assessed the influence of the sub-
stituent at the R4 position by the introduction of four types of
amines with increasing steric hindrance EAPB0202, EAPB0203,
EAPB0204, and EAPB0205 (Table 1). At R4 position, the methyl-
amino group (EAPB0203) appears to be essential for the potency.
Indeed, accessibility at the electronic doublet of the nitrogen on
this position of the heterocycle might be critical for activity,
thanks to the low steric bulk of this secondary amine. No signif-
icant differences were observed between the pKa and logP calcu-
lated values (Table 1) of this series of compounds which are all
substituted by an amino group at the R4 position. We next fo-
cused our attention on the substitution on the imidazole core
with the objective to improve in vitro potency against A375 cells.
We also explored the effect of the incorporation of a phenethyle
substituent at the R1 position. EAPB0203, bearing phenethyle on
R1, significantly enhanced the activity (IC50 = 1.57 lM) by creation
of an P interaction necessary for its binding. The improved bioac-
tivity of the EAPB0203 compound may be attributed to the addi-
tional effect and the synergy between the methylamino and
phenethyle groups in addition to the basic pharmacophore that
might be defined by the sequence of the three intracyclic nitro-
gens of the imidazo[1,2-a]quinoxaline series. Overall, the other
analogues appeared to be less potent. In a general way, it is no-
ticed that the imidazo[1,2-a]quinoxaline derivatives have a better
activity than the two other series of compounds, but this still re-
mains to be proven by comparison with a larger variety of imi-
dazo-/pyrazolo[1,5-a]quinoxaline derivatives.
Two drugs are approved in the European Union for treatment of
advanced or metastatic melanoma: dacarbazine and fotemustine.22

6606
G. Moarbess et al. / Bioorg. Med. Chem. 16 (2008) 6601–6610
Furthermore, imiquimod was recently approved for treatment of
different cutaneous cancers.
was heated to reflux for 0.5 h. The reaction mixture was cooled to
room temperature and the 4-chloro-1-isobutylimidazo[1,2-a]quin-
oxaline15 (150 mg, 0.5 mmol) in THF (10 mL) was added. The mix-
ture was again heated to reflux for 0.5 h and then cooled to room
temperature and stopped with acetic acid until pH 7. The reaction
mixture was concentrated in vacuum followed by addition of
water and saturated sodium bicarbonate. The solid was collected
by filtration, washed with water and hexane, and dried under high
vacuum to give EAPB0105 (110 mg, 60%); 1H NMR (300 MHz,
CDCl3-d6): d 8.01 (t, 1H), 7.6 (m, 3H), 7.5 (d, 1H), 7.38 (d, 1H),
7.06 (s, 1H), 6.67 (t, 1H), 3.00 (t, 2H), 2.2 (m, 5H), 0.81 (t, 6H).
13C NMR (300 MHz, CDCl3): d 142.61, 140.68, 139.55, 131.44,
130.32, 129.71, 129.19, 128.07, 126.78, 126.27, 125.55, 123.44,
122.79, 114.36, 33.68, 32.75, 21.99, 18.10. Anal. calcd for
EAPB0203 displayed potent cellular cytotoxic activity with
micromolar IC50 values against different human cancer cell lines
and was compared to fotemustine and imiquimod (Table 2). Upon
such encouraging results, EAPB0203 was selected for detailed in
vivo studies. In human melanoma (M4Be) xenografted athymic
mice, EAPB0203, administered twice a week at 20 mg/kg, has ap-
proached a significant delay in tumor growth and also a decrease
in tumor size compared to that of control and fotemustine groups
(Fig. 2). Interestingly, a stabilization and even an arrest in tumor
growth were observed after each treatment cycle. The efficacy of
treatment is dose-related. The EAPB0203 treatment was well tol-
erated without side-effects.
C21H21ClN4: C, 69.13; H, 5.80; N, 15.35. Found: C, 69.33; H, 5.42;
N, 15.65.
4. Conclusion
5.1.2. 2-Chloro-6-methylphenyl-N-methyl-1-phenethylimidazo
[1,2-a]quinoxalin-4-amine (EAPB0205)
In this paper, we have reported the synthesis of imidazoquinox-
aline and pyrazoloquinoxaline derivatives, and their in vitro cyto-
toxic activity against human melanoma cell lines and the in vivo
anti-proliferative activity of the EAPB0203 derivative on mela-
noma. The synthesis was based on a bimolecular condensation of
a carboxylic acid, followed by a coupling with orthofluoroaniline,
an intramolecular cyclisation and a substitution with appropriate
amines. This strategy will be the starting point for the development
of other compounds. The incorporation of phenethyle as moiety at
the R1 position and methylamine at the R4 position led to the
EAPB0203 compound with enhanced cytotoxic activity against
A375 human melanoma cell line. Its IC50 (1.57 lM) was 110 times
lower than fotemustine (IC50 = 173 lM), and EAPB0203 was 45
times more potent than imiquimod (IC50 = 70 lM). Interestingly,
compared to imiquimod and fotemustine, this compound also
had lower IC50 values (30–130-fold) against the two other mela-
noma cell lines tested. In vivo, EAPB0203 had potent anti-tumor
activity on human melanoma (M4Be) xenografted nude mice.
Our results support a potential therapeutic role of EAPB0203 in
the treatment of malignant melanoma and prompt us to modulate
the chemical structure of this lead molecule to optimize its effect.
Further studies are now in progress to study the mode of action
and should lead to an interesting class of new anti-cancer drugs
with innovating mechanism of action.
EAPB0205 was prepared with the same procedure as the one
for the preparation of EAPB0105; using 4-chloro-1-phenethylimi-
dazo[1,2-a]quinoxaline15 (200 mg, 0.65 mmol) in 10 ml of THF.
EAPB0205 was obtained as a white solid (120 mg, 45%); 1H NMR
(300 MHz, CDCl3-d6): d 7.93 (d, 1H), 7.49 (d, 1H), 7,56 (m, 3H),
7.39 (d, 1H), 7.24 (m, 5H), 6.73 (t, 1H), 3.3 (t, 4H), 2.23 (s, 1H).
13C NMR (300 MHz, CDCl3): d 143.12, 142.57, 140.69, 139.77,
137.67, 131.44, 130.42, 130.12, 129.71, 129.19, 128.81, 126.78,
126.44, 126.27, 125.55, 124.38, 124.3, 114.22, 35.17, 23.83, 18.10.
Anal. calcd for C25H21ClN4: C, 72.72; H, 5.13; N, 13.57. Found: C,
72.41; H, 5.38; N, 13.39.
5.1.3. 1-(2-Nitrophenyl)-1H-imidazole (1)
A mixture of 2-nitrofluorobenzene (14.85 mL, 0.142 mol), imid-
azole (14.9 g, 0.128 mol), and 40 g of K2CO3 in 200 ml of CH3CN
was heated to reflux for 24 h. The solvent was removed under vac-
uum, and the residue was slurred in 200 mL of CH2CL2, washed
twice with 200 mL of water, dried over Mg2SO4, and the solvent
was removed under vacuum. We obtained 95% of 1 as a yellow so-
lid. 1H NMR (300 MHz, CDCl3): d 9.12 (s, 1H), 7.58 (d, 1H), 7.42 (d,
1H), 7.37 (d, 1H), 7.26 (d, 1H), 7.10 (m, 3H, ArH). Anal. calcd for
C9H7N3O: C, 57.14; H, 3.70; N, 22.22. Found: C, 56.99; H, 3.9; N,
22.06.
5. Experimental
5.1. Chemistry
5.1.4. 2-Bromo-1-(2-nitrophenyl)-1H-imidazole (2)
To a solution of 1 (5 g, 0.27 mmol) in dry acetonitrile (250 mL),
N-bromosuccinimide (5.65 g, 0.032 mmol) was added. The reaction
mixture was stirred to reflux for 3 h. The solvent was removed un-
der vacuum and the residue was slurred in 250 mL of CH2CL2,
washed twice with a solution of 5% NaHCO3, dried over MgSO4,
and purified by column chromatography (CH2Cl2-MeOH, 98:2, v/
v) to give 50% of 2. 1H NMR (300 MHz, CDCl3): d 8.03 (d, 1H),
7.70 (d, 1H), 7.47 (t, 1H), 7.33 (d, 1H), 7.09 (m, 2H). Anal. calcd
for C9H6N3O2Br: C, 40.30; H, 2.24; N, 15.42. Found: C, 40.65; H,
2.01; N, 15.52.
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 mil-
lion (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 car-
ried out at the Microanalytical Central Department (Montpellier,
France). All melting points are determined using köfler hot plate
melting point apparatus and are not corrected. pKa and logP values
are calculated using the ACDLabs software (Toronto, Canada).
5.1.5. 2-Methyl-1-(2-nitrophenyl)-1H-imidazole (3a)
To a mixture of 2 (2 g, 7.46 mmol) and Pd(PPh3)4 (0.8 g,
0.7 mmol) in 1,4-dioxane (34 mL), 1.4 mL of the trimethylboroxine
was added followed by Na2CO3 (2.37 g, 22.38 mmol) in 5 mL of
water. The reaction mixture was heated to 75 °C with vigorous stir-
ring under nitrogen atmosphere. After 24 h, a second portion of
trimethylboroxine was added. After that, the mixture was poured
into water and extracted with dichloromethane. The combined or-
ganic extracts were washed with water, dried over calcium chlo-
ride, and concentrated to dryness under vacuum. The crude
products were purified by column chromatography (silica gel, elu-
5.1.1. 2-Chloro-1-isobutyl-6-methylphenyl-N-methylimidazo
[1,2-a]quinoxalin-4-amine (EAPB0105)
To a solution of 6-chloro-3-methylaniline (89 mg, 0.63 mmol)
in THF (12 ml), was added a solution of sodium bis(trimethyl-
silyl)amide (4 mL, 4 mmol, 1.0 M in THF), and the reaction mixture

G. Moarbess et al. / Bioorg. Med. Chem. 16 (2008) 6601–6610
6607
tion with CH2Cl2–MeOH, 97:7, v/v) to give 3a (yield, 94%). 1H NMR
(300 MHz, CDCl3): d 7.40 (m, 2H), 7.22 (d, 1H), 7.11 (d, 1H), 7.01
(m, 2H), 2.3 (s, 3H). Anal. calcd for C10H9N3O2: C, 59.11; H, 4.43;
N, 20.69. Found: C, 59.32; H, 4.65; N, 20.51.
5.1.12. 4-Chloro-1-phenylimidazo[1,5-a]quinoxaline (6b)
6b was prepared from 5b following the protocol described for
6a (i.e., from 5b (0.57 g, 2.18 mmol) and POCl3 (5 mL)). A yellow
solid was obtained after column chromatography purification as
specified for 6a (yield, 61%). 1H NMR (300 MHz, CDCl3): d 8.1 (s,
1H), 7.9 (dd, 1H), 7.5 (m, 5H), 7.4 (m, 2H), 7.32 (t, 1H). Anal. calcd
for C16H10N3Cl: C, 68.69; H, 3.58; N, 15.03. Found: C, 68.51; H, 3.39;
N, 15.38.
5.1.6. 1-(2-Nitrophenyl)-2-phenyl-1H-imidazole (3b)
3b was prepared from 2 following the protocol described for 3a
(i.e., from 2 (2 g, 7.46 mmol), Pd(PPh3)4 (0.5 g, 0.4 mmol) in DME
(85 mL), 2 ꢂ 1 g (16.4 mmol) of phenylboronic acid and Na2CO3
(2.37 g, 22.38 mmol) in 5 mL of water). The crude products were
purified by column chromatography (silica gel, elution with
CH2Cl2–MeOH, 97:7, v/v) to give 3b (yield, 88%). 1H NMR
(300 MHz, CDCl3): d 8.45 (dd, 2H), 7.60 (t, 1H), 7.57 (m, 2H), 7.40
(m, 5H), 7.02 (d, 1H). Anal. calcd for C15H11N3O2: C, 67.92; H,
4.15; N, 15.85. Found: C, 67.75; H, 4.47; N, 15.70.
5.1.13. N-Methyl-1-methylimidazo[1,5-a]quinoxalin-4-amine
(7a)
Methylamine (0.5 mL of a 40% (w/v) aqueous solution, 4 mmol)
was added dropwise to a stirred solution of 6a (0.3 g, 1.33 mmol)
in absolute EtOH (7 mL) at room temperature. After 40 h, another
portion of methylamine (0.5 mL of a 40% (w/v) aqueous solution,
4 mmol) was added and stirring was maintained for additional
3 h. The solvent was removed under reduced pressure and the res-
idue was dissolved in CH2Cl2 (10 mL). The organic fraction was suc-
cessively washed with 5% NaHCO3 (15 mL) and water (15 mL),
dried (Na2SO4), and concentrated under reduced pressure. The
product was purified by column chromatography on silica gel elut-
ing with CH2Cl2–MeOH (98:2, v/v) to yield a solid cream of 7a
(yield, 53%). 1H NMR (300 MHz, CDCl3): d 8.01 (d, 1H), 7.7 (d,
1H), 7.5 (s, 1H), 7.3 (t, 1H), 7.21 (t, 1H), 3.22 (d, 3H), 2.95 (s, 3H).
13C NMR (300 MHz, CDCl3): d 149.30, 142.34, 126.96, 126.33,
124.9, 122.85, 120.85, 119.78, 115.20, 30.84, 27.69. Anal. calcd
for C12H12N4: C, 67.92; H, 5.66; N, 26.41. Found: C, 67.52; H,
5.95; N, 26.36.
5.1.7. 2-(2-Methylimidazol-1-yl)phenylamine (4a)
Compound 3a (0.3 g, 1.48 mmol) was dissolved in 20 mL of eth-
anol with 0.3 g of 10% palladium on carbon and hydrogenated for
2 h. The catalyst was removed by filtration, and the residue con-
centrated under vacuum and purified by column chromatography
on silica gel using CH2Cl2–MeOH (97:3) as eluent to yield 43% of
4a. 1H NMR (300 MHz, DMSO-d6): d 7.36 (m, 2H), 7.03 (d, 1H),
6.90 (m, 2H), 6.79 (d, 1H), 2.1 (s, 3H). Anal. calcd for C10H11N3: C,
69.36; H, 6.36; N, 24.28. Found: C, 69.5; H, 6.11; N, 24.12.
5.1.8. 2-(2-Phenylimidazol-1-yl)phenylamine (4b)
This compound was prepared as described for 4a to obtain 4b
(yield, 93%) (i.e., from 3b (1.31 g, 5 mmol), EtOH (60 mL), and
0.3 g of 10% palladium on carbon). 1H NMR (300 MHz, DMSO-d6):
d 7.36 (m, 2H), 7.25 (m, 5H), 7.12 (t, 1H), 6.8 (m, 2H), 3.67 (s, 2H,
–NH2). Anal. calcd for C15H13N3: C, 76.59; H, 5.53; N, 17.97. Found:
C, 76.72; H, 5.34; N, 18.02.
5.1.14. N-Methyl-1-phenylimidazo[1,5-a]quinoxalin-4-amine
(7b)
7b was prepared from 6b following the protocol described for
7a, that is, from 6b (0.4 g, 1.4 mmol) and methylamine (0.12 g of
40% (w/v), 4 mmol). A yellow solid was obtained after column
chromatography purification as specified for 7a (yield, 86%). 1H
NMR (300 MHz, CDCl3): d 7.6 (m, 4H), 7.56 (m, 3H), 7.2 (m, 2H),
6.93 (t, 1H), 3.22 (d, 3H). 13C NMR (300 MHz, CDCl3): d 149.31,
144.42, 138.31, 132.53, 129.76, 129.71, 129.00, 128.77, 126.96,
126.55, 124.11, 122.37, 122.31, 120.07, 116.25, 27.73. Anal. calcd
for C17H14N4: C, 74.45; H, 5.11; N, 20.40. Found: C, 74.10; H,
5.22; N, 20.57.
5.1.9. 1-Methyl-5H-imidazo[1,5-a]quinoxalin-4-one (5a)
Compound 4a (1.38 g, 8 mmol) and carbonyldiimidazole
(1.55 g, 9.57 mmol) were dissolved in 83 mL of 1,2-dichloroben-
zene and heated to reflux under N2 for 2 h. After cooling to room
temperature, the residue was slurred with CH2Cl2, washed with
water, and dried over MgSO4. The product was purified by chro-
matography on silica gel using CH2Cl2–MeOH (97:3, v/v) as elu-
ent to give 62% of 5a. 1H NMR (300 MHz, DMSO-d6): d 8.1 (d,
1H), 7.7 (s, 1H), 7.37 (m, 2H), 7.25 (t, 1H), 2.5 (s, 3H). Anal. calcd
for C11H9N3O: C, 68.38; H, 4.66; N, 16.09. Found: C, 68.49; H,
4.41; N, 16.20.
5.1.15. 5,5-Diisobutyldipyrazolo[1,5-a]piperazine-5,10-dione
(8)
The
3-isobutyl-1H-pyrazole-5-carboxylic
acid
(0.9 g,
5.35 mmol) in suspension in thionyl chloride (7 mL) was heated
to reflux, under agitation for 18 h. The reaction mixture was
cooled, then filtered and washed with toluene, and dried under
high vacuum to obtain a white solid (yield, 80 %). 1H NMR
(300 MHz, DMSO-d6): d 0.9 (d, 12H), 1.9 (m, 1H), 2.5 (d, 2H), 7.4
(s, 2H). Anal. calcd for C16H20N4O2: C, 64.00; H, 6.67; N, 18.67.
Found: C, 64.31; H, 6.33; N, 18.56.
5.1.10. 1-Phenyl-5H-imidazo[1,5-a]quinoxalin-4-one (5b)
This compound was prepared as described for 5a, that is, from
4b (1.08 g, 4.6 mmol) and carbonyldiimidazole (0.9 g, 5.51 mmol)
in 56 mL of 1,2-dichlorobenzene. After cooling to room tempera-
ture, the solid was filtered and washed with diethylether to give
5b (yield, 42%). 1H NMR (300 MHz, DMSO-d6): d 7.9 (s, 1H), 7.72
(m, 5H), 7.35 (m, 2H), 7.01 (d, 1H), 6.9 (t, 1H). Anal. calcd for
C
16H11N3O: C, 73.31; H, 4.21; N, 16.09. Found: C, 73.42; H, 4.32;
5.1.16. 2-Fluoroaniline-3-isobutyl-1H-pyrazole-5-carboxamide
(9)
N, 16.21.
To 2-fluoroaniline (0.734 mL, 7.6 mmol) in THF (8 mL), cooled in
a ꢀ10 °C bath, 18.3 mL (18.30 mmol, 1.0 M) sodium bis-(trimethyl-
silyl)amide in THF was added. The mixture was stirred for 1 h, and
a suspension of 8 (1.14 g, 3.8 mmol) in THF (15 mL) was added and
let warm to room temperature. The mixture was stirred for 2 h,
and acetic acid was added to pH 7. The reaction mixture was con-
centrated in vacuum followed by the addition of water and satu-
rated NaHCO3. The solid was collected by filtration, washed with
water and cyclohexane, and dried under high vacuum to give 9
as a beige solid (yield, 46%).1H NMR (300 MHz, DMSO-d6): d 7.85
(m, 1H), 7.25 (m, 1H), 7.15 (m, 2H), 6.5 (s, 1H), 2.46 (d, 2H), 1.88
5.1.11. 4-Chloro-1-methylimidazo[1,5-a]quinoxaline (6a)
A mixture of 5a (0.9 g, 4.5 mmol) and 10 mL of phosphorus oxy-
chloride was heated at reflux under N2 for 5 h. The excess of POCl3
was removed under vacuum, and the residue dissolved in 20 ml
CH2Cl2, washed with 5% Na2CO3, dried over MgSO4, and purified
by chromatography on silica gel using CH2Cl2–MeOH (95:5, v/v).
Concentration of the pure fractions provided 30% of 6a. 1H NMR
(300 MHz, CDCl3): d 8.29 (dd, 1H), 7.82 (dd, 1H), 7.7 (s, 1H), 7.54
(m, 2H), 3.04 (s, 3H). Anal. calcd for C11H8N3Cl: C, 60.69; H, 3.68;
N, 19.31. Found: C, 60.78; H, 3.92; N, 19.06.

6608
G. Moarbess et al. / Bioorg. Med. Chem. 16 (2008) 6601–6610
(m, 1H), 0.85 (d, 6H). 19F NMR: 126. Anal. calcd for C14H16N3OF: C,
64,37; H, 6.13; N, 16.09. Found: C, 64.09; H, 6.01; N, 16.25.
scribed for 8. We obtained 4.7 g of a white solid (yield, 90–95%).1H
NMR (300 MHz, DMSO-d6): d 8.85 (s, 2H); 8.2 (s, 2H). Anal. calcd
for C8H4N4O2: C, 51.33; H, 2.13; N, 29.79. Found: C, 51.23; H, 2.01;
N, 29.56.
5.1.17. 3-Isobutylpyrazolo[1,5-a]quinoxaline-pyrazine-6-(5H)-
one (10)
A mixture of 9 (0.88 g, 3.37 mmol) and sodium hydrure (0.97 g,
4.04 mmol) in dimethylacetamide (25 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 10 (yield, 80%). 1H NMR (300 MHz, DMSO-d6): d 0.9 (d, 6H),
2 (m, 1H), 3.3 (s, 2H), 6.9 (s,1H), 7.3 (m, 4H), 8.05 (s, 1H). Anal.
calcd for C14H15N3O: C, 69.71; H, 6.22; N, 17.43. Found: C, 69.98;
H, 6.51; N, 17.38.
5.1.22. (2-Fluoroaniline)-1H-imidazole-4-carboxamide (14)
14 was prepared from 13 following the protocol described for 9,
that is, from 2-fluoroaniline (5.9 g, 53 mmol) in THF (26 ml), so-
dium bis-(trimethylsilyl)amide (133.0 mmol, 1.0 M in THF) and
13 (4.7 g, 24.0 mmol) in THF (30 mL). We obtained 7.50 g of 14
as a light beige solid (yield, 42%). 1H NMR (300 MHz, DMSO-d6):
d 9.45 (s, 2H), 8.13 (s, 1H), 8.1 (s, 1H), 8.03 (s, 1H), 7.8 (s, 1H), 7–
7.25 (m, 2H) 19F NMR: 128. Anal. calcd for C10H8N3F: C, 63.49; H,
4.23; N, 22.22. Found: C, 63.28; H, 4.51; N, 22.09.
5.1.18. 6-Chloro-3-isobutylpyrazolo[1,5-a]quinoxaline (11)
To 10 (1.04 g, 4.3 mmol), 12 mL of phosphorus oxychloride was
added and the mixture was heated to reflux for 6 h. The reaction
mixture was concentrated under vacuum, and the residue was
cooled in an ice bath. Water was added to the residue and neutral-
ized with saturated NaHCO3. The solid was collected by filtration
and purified using column chromatography on silica gel to yield
11 as a yellow solid (yield, 50%). 1H NMR (300 MHz, DMSO-d6): d
0.9 (d, 6H), 2 (m, 1H), 3.3 (s, 2H), 6.9 (s,1H), 7.3 (m, 4H), 8.05 (s,
1H). Anal. calcd for C14H14N3OCl: C, 64.74; H, 5.39; N, 16.18.
Found: C, 64.52; H, 5.24; N, 16.33.
5.1.23. Imidazo[1,5-a]quinoxaline-pyrazine-6-(5H)-one (15)
A mixture of 14 (7.50 g, 36 mmol) and sodium hydrure (1.1 g,
43 mmol) in dimethylacetamide (280 mL) was heated to reflux
for 6 h. The reaction mixture was then concentrated under vacuum
followed by the addition of water and saturated ammonium chlo-
ride. The solid was collected by filtration, washed with water, and
dried to give 4 g of 15 (yield, 80%). 1H NMR (300 MHz, DMSO-d6): d
9.4 (s, 1H), 8.2 (d, 1H), 7.8 (d, 1H), 7.31 (d, 1H), 7.1-7.3 (m, 3H).
Anal. calcd for C10H7N3O: C, 64.86; H, 3.78; N, 22.70. Found: C,
65.11; H, 3.87; N, 22.93.
5.1.24. 6-Chloroimidazo[1,5-a]quinoxaline (16)
5.1.19. 3-Isobutyl-N-methylpyrazolo[1,5-a]quinoxalin-4-amine
(12a)
16 was prepared from 15 following the protocol described for 11,
that is, from 15 (4 g, 21.6 mmol) and phosphorus oxychloride (44
ml). The solid was collected by filtration and purified using column
chromatography on silica gel to yield 16 as a yellow solid (yield,
20%). 1H NMR (300 MHz, DMSO-d6): d 9.4 (s, 1H), 8.2 (d, 1H), 7.8
(d, 1H), 7.31 (d, 1H), 7.1-7.3 (m, 3H). Anal. calcd for C10H6N3Cl: C,
58.97; H, 2.95; N, 20.64. Found: C, 58.77; H, 2.72; N, 20.88.
Methylamine (0.15 mL of
a 40% (w/v) aqueous solution,
1.74 mmol) was added dropwise to a stirred solution of 11 (0.15 g,
0.57 mmol) in absolute EtOH (10 mL) at room temperature. After
40 h, another portion of methylamine (0.25 mL of a 40% (w/v) aque-
ous solution, 2.7 mmol) was added and stirring was maintained for
additional 3 h. The solvent was removed under reduced pressure,
andtheresiduewasdissolvedinCH2Cl2 (10 mL). Theorganicfraction
was successively washed with 5% NaHCO3 (15 mL) and water
(15 mL), dried (Na2SO4), and concentrated under reduced pressure.
The product was purified by column chromatography on silica gel
with C6H12–EtOAc (70:30, v/v) as eluent to yield a cream solid (yield,
75%). Mp 145–150°C. 1H NMR (300 MHz, DMSO-d6): d 8.25 (dd,
J1 = 1.37 Hz, J2 = 7.8, 1H), 7.7 (dd, J1 = 1.46, J2 = 8.11,1H), 7.3 (m, 2H),
6.37 (s, 1H), 4.95 (s, 1H), 3.25 (d, 3H), 3.75 (d, 2H), 2.08 (m, 1H),
0.95 (d, 6H).13C NMR (300 MHz, DMSO-d6): d 152.43, 150.30,
139.7, 125.4, 125.16, 124.6, 123.7, 122.21, 112.21, 108.25, 35.10,
29.61, 28.57, 23.14. Anal. calcd for C15H18N4: C, 70.84; H, 7.13; N,
22.03. Found: C, 70.37; H, 7.44; N, 21.99.
5.1.25. N-Methylimidazo[1,5-a]quinoxalin-4-amine (17a)
17a was prepared from 16 following the protocol described for
12a, that is, from methylamine 2 ꢂ (0.25 mL of a 40% (w/v) aqueous
solution, 2.7 mmol), 16 (0.11 g, 0.54 mmol), and EtOH (8 mL). The
product was purified by column chromatography on silica gel with
C6H12–EtOAc (70:30, v/v) as eluent to yield a cream solid (yield,
80–85%). Mp 255–258 °C. 1H NMR (300 MHz, DMSO-d6): d 8.54 (s,
1H), 7.75 (dd, J1 = 1.33, J2 = 8.07, 1H), 7,65 (dd, J1 = 1.3, J2 = 8.11,
1H), 7.59 (s, 1H), 7.35 (s, 1H), 7.25 (s, 1H), 3.24 (d, 3H).13C NMR
(300 MHz, DMSO-d6): d 147.50, 142.34, 135.54, 130.67, 129.16,
125.14, 124.34, 114.14, 111.24, 26.98. Anal. calcd for C11H10N4: C,
66.65; H, 5.08; N, 28.26. Found: C, 66.54; H, 4.98; N, 28.15.
5.1.20. 3-Isobutyl-N,N-dimethylpyrazolo[1,5-a]quinoxalin-4-
amine (12b)
5.1.26. N,N-Dimethylimidazo[1,5-a]quinoxalin-4-amine (17b)
17b was prepared from 16 following the protocol described for
17a, that is, from dimethylamine (0.15 mL of a 40% (w/v) aqueous
solution, 1.19 mmol), 16 (0.11 g, 0.54 mmol), and EtOH (10 mL). The
product was purified by column chromatography on silica gel with
C6H12–EtOAc (70:30, v/v) as eluent to give a white solid (yield, 96%).
Mp 160–165°C. 1H NMR (300 MHz, DMSO-d6): d 8.55 (s, 1H), 7.78 (s,
1H), 7.71 (dd, J1 = 1.25, J2 = 8.07, 1H), 7.6 (dd, J1 = 1.21, J2 = 8.12, 1H),
7.35 (t, 1H), 7.15 (t, 1H), 3.43 (s, 6H). 13C NMR (300 MHz, DMSO-d6):
d 146.93, 143.22, 135.50, 129.56, 129.08, 128.29, 125.48, 124.75,
114.49, 111.16, 37.89. Anal. Calcd for C12H12N4: C, 67.91; H, 5.70; N,
26.40. Found: C, 67.64; H, 5.48; N, 26.53.
12b was prepared from 11 following the protocol described for
12a, that is, from dimethylamine (0.22 mL of a 40% (w/v) aqueous
solution, 1.73 mmol) and 11 (0.15 g, 0.57 mmol) in absolute EtOH
(10 mL). The product was purified by column chromatography on
silica gel with C6H12–EtOAc (70:30, v/v) as eluent to give a white
solid (yield, 83%). Mp 160–165°C. 1H NMR (300 MHz, DMSO-d6):
d 8.25 (dd, J1 = 1.32, J2 = 7.9, 1H), 7.65 (dd, J1 = 1.24, J2 = 9.12, 1H),
7.3 (m, 2H), 6.6 (s, 1H), 3.3 (s, 6H), 2.75 (d, 2H), 2.09 (m, 1H), 1
(d, 6H). 13C NMR (300 MHz, DMSO-d6): d 149.79, 145.9, 140.85,
125.56, 125.42, 124.98, 124.00, 122.63, 114.26, 112.60, 39.48,
35.10, 29.61, 23.01. Anal. calcd: for C16H20N4: C, 71.61; H, 7.51;
N, 20.88. Found: C, 71.37; H, 7.62; N, 21.09.
5.2. In vitro cytotoxic activity
5.1.21. Diimidazo[1,5-a]piperazine-5,10-dione (13)
5.2.1. Materials and reagents
13 was prepared from the imidazole-4-carboxylic acid (5 g,
44.6 mmol) and thionyl chloride (75 mL) following the protocol de-
Imiquimod (Molekula, Wessex House, Shaftesbury, Dorset, UK),
doxorubicin hydrochloride (Teva laboratory, Courbevoie, France),

G. Moarbess et al. / Bioorg. Med. Chem. 16 (2008) 6601–6610
6609
fotemustine (Servier, Orléans, France), irinotecan (Aventis Pharma
laboratories, Vitry-sur-Seine, France), methotrexate (Teva labora-
tory) were used in this study. 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), isopropyl alcohol, and hydro-
chloric acid were obtained from Merck (Darmstadt, Germany). All
other reagents were of analytical grade and were obtained from
commercial sources.
chased from Charles River (L’Arbresle Cedex, France) and were kept
under sterile conditions. They received sterile food and water. Tu-
mor growth was monitored twice a week. Two perpendicular
diameters (a and b) were measured with a Vernier caliper, and tu-
mor volumes (V) were calculated using the following formula:
V = 0.5ab2 . At a tumor size of ꢃ0.7 cm3, athymic mice were ran-
domized into three groups (6 mice per group): (i) the vehicle con-
trol group (weekly ip administration of DMSO/intralipid (50:50, v/
v)), (ii) the EAPB0203 group (two protocols: protocol 1, weekly ip
administration of 5 mg/kg for 3 weeks and protocol 2, administra-
tion of 20 mg/kg twice a week (monday and thursday) for 3 weeks,
this cycle was repeated after 15 days), and (iii) the fotemustine
group (two protocols: protocol 1, weekly ip administration of
5 mg/kg for 3 weeks and protocol 2, weekly ip administration of
20 mg/kg for 3 weeks, this cycle was repeated after 15 days).
EAPB0203 being poorly soluble in water was dissolved in a mix-
ture intralipid/DMSO (50:50, v/v). Fotemustine was dissolved in
isotonic glucose. The dosing regimen of fotemustine is near to
the fotemustine dose of 100 mg/m2 used clinically. Animals were
euthanized with CO2 when the tumor size reached 2 cm3.
5.2.2. Cell lines and culture techniques
Melanoma (A375, M4Be and RPMI-7951), breath (MCF7), colon
(LS174T), and B lymphoma (Raji) human cancer cell lines were ob-
tained from American Type Culture Collection (Rockville, Md.,
USA). Cells were cultured in RPMI medium containing RPMI-1640
(Gibco Laboratories, France), with the exception of the M4Be line
that required DMEM medium supplemented with 10% heat-inac-
tived (56 °C) foetal bovine serum (FBS) (Polylabo, Paris, France),
2 mM L-glutamine, 100 IU/ml penicillin G sodium, 100 lg/ml strep-
tomycin sulphate, and 0.25 lg/ml amphotericin B. Cells were main-
tained in a humidified atmosphere of 5% CO2 in air at 37 °C.
5.2.3. Cytotoxicity assay
Tumor response was assessed by comparing the tumor volume
in each mouse group (control, fotemustine, and EAPB0203).
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, M4Be, RPMI 7950, LS147T, MCF7, and
Raji 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) to doxorubicin, irinotecan, fotemustine, methotrexate,
and imiquimod at concentrations of 10ꢀ4 to 10ꢀ10 lM, diluted in
the culture medium, and (iii) to the synthesized compounds
(10ꢀ4–10ꢀ10 lM) dissolved in a mixture 1% DMSO/culture medium
(v/v). After 96 h of incubation, 10 ll of 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 metabolically unimpaired tu-
mor-cell targets to reduce MTT to a blue formazan product. At
the end of the incubation period, the supernatant was carefully
aspirated, then, 100 ll 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 micro-
culture plate reader (Dynatech MR 5000, France). For each assay,
at least three experiments were performed in triplicate.
5.2.5. Statistical analysis
In vivo results are presented as mean standard error of the
mean (SEM). A one way ANOVA for repeated measurements was
performed to compare (i) the efficacy of treatment according to
the administered dose (5 and 20 mg/kg), both for fotemustine
and EAPB0203; (ii) the variations of tumor volume between the
three treatment groups (control, fotemustine, and EAPB0203) both
at low and high administered doses. A simple contrast test was
used to compare 2-by-2 each group; in this case, the level of signif-
icance was corrected for multiple comparisons and fixed at 0.02.
This analysis was performed using the PK-fit software. Probability
values of less than 0.05 were considered statistically significant.
Acknowledgments
We thank Dr. Bompart Jacques for providing assistance in the
chemistry part, Margout Delphine, Heintz Geneviève, Brissac Mi-
chel, and Imed Ait-Arsa for their precious technical support in
the biological studies, and La Ligue contre le Cancer for its financial
support.
Supplementary data
The individual cell line growth curves confirmed that all tumor
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.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.05.022.
References and notes
1. Rudy, S. J. Dermatol. Nurs. 2002, 14, 268–270.
2. Sidky, Y. A.; Borden, E. C.; Weeks, C. E.; Reiter, M. J.; Hatcher, J. F.; Bryan, G. T.
Cancer Res. 1992, 52, 2528–2533.
5.2.4. Xenografts and in vivo study protocol
3. Sauder, D. N. Br. J. Dermatol. 2003, 149, 5–8.
This research adhered to the ‘Principles of Laboratory Animal
Care’ (NIH publication #85-23, revised 1985). The animal study
was approved by the local Animal Use Committee. Before to start
4. Reiter, M. J.; Testerman, T. L.; Miller, R. L.; Weeks, C. E.; Tomai, M. A. J. Leukoc.
Biol. 1994, 55, 234–240.
5. Megyeri, K.; Au, W. C.; Rosztoczy, I.; Raj, N. B.; Miller, R. L.; Tomai, M. A.; Piha, P.
M. Mol. Cell. Biol. 1995, 15, 2207–2218.
6. Steinmann, A.; Funk, J. O.; Schuler, G.; Von den Driech, P. J. Am. Acad. Dermatol.
2000, 43, 555–556.
7. Bong, A. B.; Bonnekoh, B.; Franke, I.; Schon, M. P.; Ulrich, J.; Gollnick, H. J.
Dermatol. 2002, 205, 135–138.
8. Ugurel, S.; Wagner, A.; Phohler, C.; Tilgen, W.; Reinhold, U. Br. J. Dermatol. 2002,
147, 621–624.
9. Hengge, U. R.; Cusini, M. Br. J. Dermatol. 2003, 149, 15–19.
10. Spanner, D. E.; Miller, R. L.; Mena, J.; Crossman, L.; Sorrenti, V.; Ski, Y. Leuk.
Lymphoma 2005, 46, 935–939.
pharmacodynamic studies in mouse, the 50% lethal dose (LD50
)
of the EAPB0203 compound was evaluated in healthy animal after
intraperitoneal (ip) administration. The LD50 was higher than
300 mg/kg.
M4Be tumor cells at logarithmic growth in vitro were harvested
and washed twice with PBS. Tumor cells (3 ꢂ 106) suspended in
150 ll of DMEM medium without FBS were inoculated subcutane-
ously into the flank area of female Swiss Nude mice (7 weeks old,
weighing 20–22 g) through a 23-gauge needle. Mice were pur-
11. Hemmi, H.; Kaisho, T.; Takeuchi, O.; Sato, S.; Sanjo, H.; Hoshimo, K.; Horiuchi,
T.; Tomizawa, H.; Takeda, K.; Akira, S. Nat. Immunol. 2002, 3, 196–200.

6610
G. Moarbess et al. / Bioorg. Med. Chem. 16 (2008) 6601–6610
12. Schon, M.; Bong, A. B.; Drewniok, C.; Herz, J.; Geilen, C. C.; Reifenberger, J.;
Beinninghoff, B.; Slade, H. B.; Gollnick, H.; Schon, M. P. J. Natl. Cancer Inst. 2003,
95, 1138–1149.
18. Schön, M.; Bong, A. B.; Drewniok, C.; Herz, J.; Geilen, C. C.; Reifenberger, J.;
Benninghoff, B.; Slade, H. B.; Gollnick, H.; Shön, M. P. J. Natl. Cancer Inst. 2003,
95, 1138–1149.
13. Stockfeth, E.; Trefzer, U.; Gracia-Bartels, C.; Wegner, T.; Schmook, T.; Sterry, W.
Br. J. Dermatol. 2003, 149, 53–56.
19. Morjarial, S.; Deleuze-Masquefa, C.; Lafont, V.; Gayraud, S.; Bompart, J.; Bonnet,
P. A.; Dornand, J. J. Immunopathol. Pharmacol. 2006, 19, 525–538.
20. Chen, P.; Doweyko, A. M.; Norris, D.; Gu, H. H.; Spergel, S. H.; Das, J.; Moquin, M.
V.; Lin, L.; Wityak, J.; Iwanowics, E. J. J. Med. Chem. 2004, 47, 4517–4529.
21. Moarbess, G.; El-Hajj, H.; Kfoury, Y.; El-Sabban, M. E.; Lepelletier, Y.; Hermine,
O.; Deleuze-Masquéfa, C.; Bonnet, P. A.; Bazarbachi, A. Blood 2008, 111, 3770–
3777.
14. Schön, M. P.; Wienrich, B. G.; Drewniok, C.; Bong, A. B.; Eberle, J.; Geilen, C. C.;
Gollnick, H.; Schön, M. J. Invest. Dermatol. 2004, 122, 1266–1276.
15. Deleuze-Masquefa, C.; Gerebtzoff, G.; Subra, G.; Fabreguettes, J. R.; Ovens, A.;
Carraz, M.; Strub, M. P.; Bompart, J.; George, P.; Bonnet, P. A. Bioorg. Med. Chem.
2004, 12, 1129–1139.
16. Wagner, T. L.; Ahonen, C. L.; Couture, A. M.; Gibson, S. J.; Miller, R. L.; Smith, R.
M. Cell. Immunol. 1999, 191, 10–19.
17. Gibson, S. J.; Imbertson, L. M.; Wagner, T. L. J. Interferon Cytokine Res. 1995, 15,
2207–2218.
22. Avril, M. F.; Aamdal, S.; Grob, J. J.; Hauschild, A.; Mohr, P.; Bonerandi, J. J.;
Weichenthal, M.; Neuber, K.; Bieber, T.; Gilde, K.; Guillem Porta, V.; Fra, J.;
Bonneterre, J.; Sa¨ıag, P.; Kamanabrou, D.; Pehamberger, H.; Sufliarsky, J.;
Gonzalez Larriba, J. L.; Scherrer, A.; Menu, Y. J. Clin. Oncol. 2004, 18, 1118–1125.