Design and synthesis of novel imidazo[1,2-a]quinoxalines as PDE4 inhibitors

Article abstract of DOI:10.1016/j.bmc.2003.11.034

New imidazo[1,2-a]quinoxaline derivatives have been synthesised by condensation of an appropriate α-aminoalcohol with a quinoxaline followed by intramolecular cyclisation and nucleophilic substitutions. Their phosphodiesterase inhibitory activities have b

Full text of DOI:10.1016/j.bmc.2003.11.034

Bioorganic & Medicinal Chemistry 12 (2004) 1129–1139
Design and synthesis of novel imidazo[1,2-a]quinoxalines as
PDE4 inhibitors
Carine Deleuze-Masquefa,a Gregori Gerebtzoff,a Guy Subra,a Jean-Roch Fabreguettes,a
Annabel Ovens,a Maelle Carraz,a Marie-Paule Strub,b Jacques Bompart,a Pascal Georgec
and Pierre-Antoine Bonneta,*
a
´
Pharmacochimie et Biomolecules, Laboratoire de Chimie Organique Pharmaceutique, Faculte de Pharmacie, 15, Av. Ch. Flahault,
´
BP 14 491, 34093 Montpellier Cedex 5, France
´
´ ´
Sanofi-Synthelabo Recherche, Departement de Recherche Systeme Nerveux Central, 31, Av. P.-V. Couturier,
b
Centre de Biochimie Structurale, Faculte de Pharmacie, Montpellier, France
`
c
92220 Bagneux, France
Received 8 August 2003; accepted 28 November 2003
Abstract—New imidazo[1,2-a]quinoxaline derivatives have been synthesised by condensation of an appropriate a-aminoalcohol
with a quinoxaline followed by intramolecular cyclisation and nucleophilic substitutions. Their phosphodiesterase inhibitory
activities have been assessed on a preparation of the PDE4 isoform purified from a human alveolar epithelial cell line (A549). These
studies showed potent inhibitory properties that emphasize the importance of a methyl amino group at position 4 and a weakly
hindered group at position 1.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
As part of our ongoing efforts to develop novel inflam-
matory lead molecules through their action on PDE,
and based on imidazo[1,2-a]quinoxalines as set out in a
preceding paper,9 we report here the design approach
and synthesis of derivatives substituted in positions 1
and 4, and their inhibitory profile on purified HSPDE4
isoenzyme obtained from a human alveolar epithelial
cell line (A549).
Cyclic AMP (cAMP) and cyclic GMP (cGMP) are
ubiquitous second messengers inducing numerous intra-
cellular responses by transduction of different inflam-
matory stimuli.1 An elevated level of cAMP has been
reported to lead to suppression of function in pro-
inflammatory and immunocompetent cells.1 Cyclic
nucleotide phosphodiesterases (PDE) are the only
enzymes which can hydrolyse cyclic nucleotides. Up to the
present time, 11 families of PDE have been identified,
sharing various properties (substrate specificities, enzy-
matic properties, responsiveness to inhibitors. . .).2ꢀ4
PDE4 is the only isoform found in most pro-inflamma-
tory and immunocompetent cells involved in airway
inflammation (e.g., lymphocytes, mast cells, eosinophils,
granulocytes),1,5 therefore being the preferred target to
control the cAMP intracellular level in diverse inflam-
matory disorders such as asthma or chronic obstructive
pulmonary disease (COPD).1,5ꢀ8
2. Chemistry
Compounds 4a and b were prepared following pre-
cedents for such transformations as described in ref 9.
Reaction of isovaleraldehyde or 3-phenylpropionalde-
hyde, for series a and b respectively, with sodium
cyanide led to 2-hydroxy-4-methylpentanenitrile 1a
and 2-hydroxy-4-phenylbutanenitrile 1b which, upon
reduction with lithium aluminium hydride, produced 1-
amino-4-methylpentan-2-ol 2a and 1-amino-4-phe-
nylbutan-2-ol 2b in 87% and 81% overall yields
(Scheme 1). Both compounds were condensed with 2,3-
dichloroquinoxaline in the presence of triethylamine in
dioxan to furnish the 1-[(3-chloroquinoxalin-2-yl)amino]-
4-methylpentan-2-ol 3a and 1-[(3-chloroquinoxalin-2-
Keywords: Phosphodiesterase 4; Imidazo[1,2-a]quinoxaline; Phospho-
diesterase inhibition.
* Corresponding author. Tel.: +33-4-67-54-86-43; fax: +33-4-67-54-
80-36; e-mail: pabonnet@univ-montp1.fr
0968-0896/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.11.034

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C. Deleuze-Masquefa et al. / Bioorg. Med. Chem. 12 (2004) 1129–1139
1130
various amines (Scheme 3). With the exception of com-
pounds 7a and b, a general protocol was used for all
compounds, albeit compounds 17a and b bearing an N-
Boc group required a deprotection step to give com-
pounds 18a and b respectively. Briefly the appropriate
amines were added to a solution of 5a or b dissolved in
Et2O, and the reaction mixtures were stirred either at
room temperature (3–40 h) or under reflux (8 to 24 h),
both with 80–90% yield for series a and 60–65% yield
for series b respectively.
Scheme 1. Synthesis of the a-aminoalcohol 2a and b.
yl)amino]-4-phenylbutan-2-ol 3b intermediates which
upon subsequent oxidation of their hydroxyl moiety, in
presence of sulphur trioxide trimethylamine complex,
gave rise to compounds 4a and b (Scheme 2) with yields
ranging from 50 to 60 for each intermediate step except
for the synthesis of 4b which yielded 90%.
3. Biology
The potential for the synthesised imidazo[1,2-a]quinox-
alines to inhibit PDE4 activity was tested on the HSPDE4
isoform. This isoform was isolated from the human
alveolar epithelial cell line A459 by sonication, ultra-
centrifugation and liquid chromatography purification
using a Q-Sepharose Fast-Flow column. The presence
of the PDE4 in the chromatography fractions was
revealed by SDS-PAGE gel electrophoresis as a single
band of ꢁ90 kDa. In addition to the predominant iso-
form HSPDE4, A459 cells also contain a low concen-
tration of PDE1 and a relatively large amount of
PDE3,6,12 whose active site has a similar structure to
that of PDE4.12 Since the characterisation of PDE4
inhibitors requires an enzymatic preparation containing
no detectable amount of PDE3, we investigated the
presumably pure PDE4 fractions for a possible con-
tamination by these isoforms (Fig. 1). To that effect, we
used rolipram, cilostamide and Ca2+/Calmodulin
(Ca2+/CaM) as these are a selective inhibitor of PDE4,
a selective inhibitor of PDE3 and a specific activator of
4-Chloro-1-isobutylimidazo[1,2-a]quinoxaline 5a and 4-
chloro-1-(2-phenylethyl)imidazo-[1,2-a]quinoxaline 5b,
the precursors of series a and b respectively, were
obtained by intramolecular cyclisation,10,11 a key step
in imidazo[1,2-a]quinoxalines skeletal construction, of
the corresponding 1-[(3-chloroquinoxalin-2-yl)amino]-4-
methylpentan-2-ol 4a and 1-[(3-chloroquinoxalin-2-
yl)amino]-4-phenylbutan-2-ol 4b (Scheme 2, step 3). In
this synthesis the problem of ring closure was effectively
controlled. Indeed, dehydration in trifluoroacetic anhy-
dride/trifluoroacetic acid led to the exclusive products
5a and 5b with yields of 84% and 66% respectively
(path A), whilst cyclising performed in the presence of
p-toluene sulfonic acid/xylene favoured an alternate
pathway generating lactams 6a and b (path B) with an
average yield of 85%. With compounds 5a and b in
hand, the next step in the synthesis plan required
nucleophilic substitution of the chlorine in position 1 by
Scheme 2. Synthesis of the imidazo[1,2-a]quinoxaline heterocycle.

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C. Deleuze-Masquefa et al. / Bioorg. Med. Chem. 12 (2004) 1129–1139
1131
Scheme 3. Nucleophilic substitution of chlorine by the appropriate amines.
4. Results and discussion
PDE1, respectively. Their activities on an ultra-
centrifuged crude extract and chromatography-purified
PDE4 were compared (Fig. 1). The high inhibition
activity of 100 mM rolipram on crude extract as well as
on purified PDE confirmed a high prevalence of PDE4
in A549 cells. Cilostamide (2.5 mM) partially inhibited
the crude extract, thus signalling the possible presence
of PDE3 in A549 cells. After chromatography, results of
inhibition activity are in favour of the elimination of
significant PDE3 activity in the purified preparation. As
an increase of enzymatic activity was observed with 0.1
mM Ca2+/150 mM CaM between the crude extract and
the purified PDE, a co-purification of PDE1 and PDE4
could not be excluded. Fortunately, PDE1, which is
present at low concentration in A549 cells, is strongly
Based on an early report from this laboratory,9 we
designed novel imidazo[1,2-a]quinoxalines with improved
pharmacological profiles. SAR studies performed at
that time clearly demonstrated that a substitution in
position 1 and/or 2 has a strong influence on the tar-
geted PDE isoform; a substitution in position 2 unam-
biguously led to a preferential inhibition of PDE3 whilst
an imidazo[1,2-a]quinoxaline substitution in R1 exhib-
ited selective PDE4 inactivation. Furthermore it was
shown that the most potent PDE4 inhibitor possessed a
hydrophobic side chain at R1. In the present work, we
further explored the tolerance for steric hindrance in
position 1 and synthesised two series of imidazo[1,2-
a]quinoxaline of twelve and ten analogues each, in
which R1 was either an iso-butyl (series a) or a 2-phe-
nylethyl moiety (series b). In position 4, different groups
were placed which had different electronic, hydrophobic
and steric properties such as the chloro, methylamino,
pyrolidyl, (2-methoxyethyl)amino, phenethylamino or
piperazinyl functions which gave rise to compounds 5
through 18 presented in Table 1.
inhibited in absence of Ca2+ 13
Since our activity mea-
.
surements of imidazo[1,2-a]quinoxalines on PDE4 were
performed at a low concentration of Ca2+ (50 mM), the
possible presence of small amounts of PDE1 in the
purified preparation should not significantly influence
the characterisation of the synthesised compounds as
PDE4 inhibitors. Besides, the experimental IC50 value
of rolipram (6.03ꢂ0.84 mM) determined from the pur-
ified PDE4 preparation was similar to the published
value14 which would not be the case if PDE1 levels were
present.
All derivatives were assessed for their inhibitory activity
on HSPDE4 from human alveolar epithelial cell line
A459. The residual enzyme activity in presence of 100
mM for each inhibitor is shown in Figure 2 for series a.
In Table 1 are reported the residual enzyme activity in
presence of 100 mM for all compounds and the concen-
tration of compounds that inhibited 50% of the PDE
activity (IC50), only for compounds that showed a resi-
dual enzyme activity less than 50% at 100 mM (except
for compound 11a which showed ꢁ52% of PDE activ-
ity at 100 mM).
A rapid comparison of the residual PDE4 activity
obtained for series a and b did not show any significant
variation which could be correlated to the properties of
the moiety in position R1. Of particular notice are
compounds 18a and b, for which less than 5% activity is
retained. Nevertheless, for compounds 8 and 9, and to a
lesser extend compounds 5 and 7, the isobutyl substitu-
tion appears to be more favorable than the phenylethyl
group.
Figure 1. Characterization of the PDE preparation. aControl: Positive
control, cAMP without inhibitor. Rolipram: cAMP+100 mM roli-
pram (PDE4 inhibitor). Cilostamide: cAMP+2.5 mM cilostamide
(PDE3 inhibitor). Ca/CaM: cAMP+0.1 mM Ca2++150 mM Calmo-
dulin (PDE1 activator).

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1132
The different substitutions at R4 within the series a
affected quite differently the PDE4 activity (Fig. 2).
Whilst compounds 5a, 6a, 7a and 8a with respectively a
chloro, a hydroxyl, a primary amino and an amino-
methyl moiety group with a mesomer donor effect
exhibited a potent PDE4 inhibitory activity (over 80%
of inhibition) as expected,9,15 substitution on the amino
group led to a significant drop in activity. This decrease
is remarkable not only because it closely parallels the
increase of the length of the lipophylic side chain (com-
pound 8a and 15a) but also the decrease of the length of
aliphatic spacer between the amino and the phenyl
moieties (compound 13a, 14a and 12a). A similar profile
was observed for compound 9a, 10a and 11a when R4
was an aminodimethyl, a pyrolidyl and a piperidyl
group, respectively; however substitution with a piper-
azyl group (compound 18a) dramatically reduced PDE4
activity. These preliminary results suggested that it is
not total steric volume per se which hampers PDE4
inhibition but the deficiency in accessibility of the amino
group.
Further analysis of the most potent inhibitors (i.e.,
those producing more than 50% inhibition) in both
series showed that the majority of the compounds tested
have an IC50 of the same order of magnitude as the
reference drug rolipram with the exception of com-
pounds 6a, 9a and 13a which are 3 to 5 times less active.
These differences might be related to the possible keto/
enol form of compound 6a on one hand and to the poor
Table 1. Imidazo[1,2-a]quinoxalines: formulas and phosphodiesterase 4 inhibitory activities
Compd
R1
R4
mp (ꢃC)
Formula
PDE4 residual
activity (%)a
IC50 (mM)b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
(CH3)2–CH–CH2–
C6H5-(CH2)2–
(CH3)2–CH–CH2–
C6H5-(CH2)2–
(CH3)2–CH–CH2–
C6H5-(CH2)2–
(CH3)2–CH–CH2–
C6H5-(CH2)2–
Cl–
Cl–
HO
HO
118
122–124
– >250
–>250
>250
186
126–127
148–149
118–119
140
C14H14N3Cl
C18H14N3Cl
C14H14N3O13.87
C18H14N3O97.40
C14H16N4
C18H16N4
C15H18N4
C19H18N4
C16H20N4
C20H20N4
9.64ꢂ1.44
24.00ꢂ4.07
ꢂ1.33
4.00ꢂ0.92
3.00ꢂ0.63
20.00ꢂ4.63
—
2.00ꢂ0.18
10.00ꢂ1.00
0.20ꢂ0.05
—
ꢂ7.43
NH2–
NH2–
CH3-NH–
CH3-NH–
(CH3)2-N–
(CH3)2-N–
6.79ꢂ1.45
28.05ꢂ2.40
6.10ꢂ3.00
68.28ꢂ6.80
21.56ꢂ3.86
61.76ꢂ9.45
(CH3)2–CH–CH2–
C6H5-(CH2)2–
20.00ꢂ7.00
—
10a
10b
11a
11b
(CH3)2–CH–CH2–
C6H5-(CH2)2–
112–113
128
C18H22N4
C22H22N4
C19H24N4
C23H24N4
62.04ꢂ7.58
62.01ꢂ10.78
47.79ꢂ3.63
53.34ꢂ2.51
—
—
—
—
(CH3)2–CH–CH2–
C6H5-(CH2)2–
60–61
112–113
12a
12b
13a
13b
14a
15a
(CH3)2–CH–CH2–
C6H5-(CH2)2–
(CH3)2–CH–CH2–
C6H5-(CH2)2–
(CH3)2–CH–CH2–
(CH3)2–CH–CH2–
C6H5-NH–
C6H5-NH–
C6H5-(CH2)2-NH–
C6H5-(CH2)2-NH–
C6H5–CH2-NH–
CH3O-(CH2)2-NH–
119–120
126–128
105–106
120
136–137
98–99
C20H20N4
C24H20N4
C22H24N4
C26H24N4
C21H22N4
C17H22N4O18.09
53.13ꢂ10.85
52.98ꢂ1.56
41.02ꢂ8.73
63.48ꢂ5.96
50.90ꢂ11.72
ꢂ1.58
—
—
30.00ꢂ5.00
—
—
—
18a
18b
(CH3)2–CH–CH2–
C6H5-(CH2)2–
>250
>250
C18H23N5
C22H23N5
3.36ꢂ1.42
3.00ꢂ0.84
5.00ꢂ1.00
2.87ꢂ0.40
a,bThe results presented are meansꢂSEM of at least three experiments.
a All compounds were tested at 100 mM.
b IC50 values were determined using a nonlinear regression curve fitting program for compounds which inhibited 50% or more of the PDE activity at
100 mM. Rolipram IC50 value: 6.03ꢂ0.84 mM.

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1133
relative to TMS as internal standard. Coupling con-
stants are reported in hertz (Hz), spectral splitting pat-
terns are designed as follows: s, singlet; br, broad; d,
doublet; t, triplet; m, multiplet. All melting points are
uncorrected and were determined using a Kofler hot
plate melting point apparatus. All reagents and solvents
were of reagent grade and used without further pur-
ification unless indicated otherwise. Column chromato-
graphy was performed on Merck silica gel 60 (200–400
mesh). Elemental analysis was carried out at the
Microanalytical Central Department, Vernaison, France.
Its results were not corrected.
All reactions were monitored by TLC on 20ꢄ60 mm
plastic sheets precoated with silica gel (HT-254 Merck)
to a thickness of 0.25 mm and viewed at 254 nm
UV-light.
Figure 2. PDE4 activity without (Control) and with different
imidazo[1,2-a]quinoxalines (100 mM). aControl: Positive control,
cAMP without inhibitor. 5a–18a: cAMP with 100 mM imidazo[1,2-a]-
quinoxalines.
6.1.2. 2-Hydroxy-4-methylpentanenitrile (1a). Isovaler-
aldehyde (10 g, 116 mmol) was added over 2–3 min to a
stirred solution of 37% aqueous NaHSO3 (25 mL, 116
mmol), at 0 ꢃC. A white bisulphite precipitate was
formed almost immediately. A solution of NaCN (5.7 g,
116 mmol) in H2O(30 mL) was then added dropwise,
over 45 min. Stirring was maintained for 18 h at room
temperature, during which time the precipitate was
redissolved and two immiscible layers were formed. The
mixture was extracted with Et2O(30 mL). The com-
bined dried (Na2SO4) organic layers were evaporated to
give a yellow oil which was used without further pur-
ification (12.21 g, 93%); 1H NMR (100 MHz, CDCl3) d:
0.88 (d, J=6 Hz, 6H), 1.51–1.98 (m, 3H, H-3+H-4),
4.20 (s, br, OH), 4.45 (t, J=7 Hz, 1H); 13C NMR
(25 MHz, DMSO-d6) d: 21.6, 22.1, 23.8, 43.1, 58.3,
121.3. Anal. calcd for C6H11NO: C, 63.68; H, 9.80; N,
12.38. Found: C, 63.56; H, 9.75; N, 12.42.
exposure of the amino group in the case of compounds
9a and 13a. Interestingly, compounds 5a and b, which
are the precursors of both series, show an IC50 half of
that of rolipram but no effect related to the steric or
the electronic differences in R1 could be established,
whereas a slight difference was noted for compounds 18
and 7. Indeed in both cases, the b series was less active
(40% for compound 18 and 20% for compound 7) than
the a series. It is also worth noticing that although
compound 7a was 3 times more potent than the refer-
ence compound, one should really consider compound
8a which was 30 times more active than rolipram. Thus
an amino group in R1 is essential for PDE4 inactivation
and a single substitution with a methyl group is optimal.
Whilst this confirms results reported previously9,15 it
would be interesting to further probe the role of the
linker in compounds based on compound 18a.
6.1.3. 2-Hydroxy-4-phenylbutanenitrile (1b). 1b was pre-
pared from 3-phenylpropionaldehyde following the
protocol described for 1a; 3-phenylpropionaldehyde
(11.14 g, 83.2 mmol), NaHSO3 (17.7 mL of 37% aqu-
eous solution, 83 mmol), NaCN (4.08 g, 83.2 mmol) in
H2O(18 mL). The product, a yellow oil, was used
without further purification (12.4 g, 93%); 1H NMR
(100 MHz, CDCl3) d: 2.02–2.25 (m, 2H), 2.76–2.91 (m,
2H), 3.98 (s, 1H), 4.39 (t, J=8 Hz, 1H), 7.26 (d, J=3
Hz, 5H); 13C NMR (25 MHz, CDCl3) d: 30.49, 36.37,
59.92, 120.04, 126.28, 128.28, 128.49, 139.60. Anal.
calcd for C10H11NO: C, 74.51; H, 6.88; N, 8.69. Found:
C, 74.38; H, 6.97; N, 8.42.
5. Conclusion
In the present paper, we describe the synthesis of
imidazo[1,2-a]quinoxaline derivatives and their PDE4
inhibitory activities. The synthesis was based on intra-
molecular cyclisation of quinoxaline substituted by an
aminoketone group, followed by nucleophilic substitu-
tion with appropriate amines. Their inhibitory activities
were assayed on PDE4 extracted from a human alveolar
epithelial cell line (A549). Substitution in position 1 was
previously shown to be essential.9 Comparative results
obtained with either an alkyl or a phenylethyl group on
this position show that the nature of the groups may in
certain cases have a substantial effect on PDE4 inhibi-
tory activity. Substitution in position 4 clearly shows the
need of a weakly hindered amino group.
6.1.4. 1-Amino-4-methylpentan-2-ol (2a). A solution of
1a (12.13 g, 107 mmol) dissolved in Et2O(50 mL), was
added dropwise, over 45 min, under gentle reflux to a
stirred slurry of LiAlH4 (8.12 g, 214 mmol) in Et2O(160
mL). The mixture was further refluxed for 90 min. After
cooling to 0–5 ꢃC, the excess of LiAlH4 was quenched
by dropwise addition of H2O(8 mL), 15% aqueous
NaOH (8 mL) and H2O(40 mL). The mixture was stir-
red until all LiAlH4 was consumed and a white pre-
cipitate was formed. The mixture was filtered and the
precipitate was washed with Et2O. The organic fraction
was dried (KOH pellets) and the solvent was removed
6. Experimental
6.1. Chemistry
6.1.1. General procedures. 1H and 13C NMR spectra
were recorded using a Bruker AC 100 spectrometer.
Chemical shifts are reported in parts per million (ppm)

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1134
under reduced pressure to yield an orange oil which was
used without further purification (11.78 g, 94%); 1H
NMR (100 MHz, CDCl3) d: 0.74 (d, J=7 Hz, 6H), 1.04
(m, 2H, H-3), 1.56 (m, J=7 Hz, 1H, H-4), 2.41 (m, 5H,
H-1, OH, NH2), 3.40 (m, J=4 Hz, 1H, H-2); 13C NMR
(25 MHz, DMSO-d6) d: 21.90, 23.20, 24.30, 43.80, 47.80,
69.90. Anal. calcd for C6H15NO: C, 61.49; H, 12.90; N,
11.95. Found: C, 61.70; H, 12.53; N, 11.67.
61%); 1H NMR (100 MHz, CDCl3) d: 0.93 (s, 3H), 1.00
(s, 3H), 2.34 (m, 1H), 2.40 (s, 2H), 4.38 (d, 2H), 6.39 (t,
1H), 7.59 (m, 4H); 13C NMR (25 MHz, CDCl3) d: 22.56,
25.01, 49.28, 51.55, 125.34, 125.87, 127.98, 130.14,
136.65, 137.92, 140.99, 147.30, 205.01. Anal. calcd for
C14H16N3OCl: C, 60.54; H, 5.81; N, 15.13. Found: C,
60.32; H, 6.06; N, 14.95.
6.1.9. 1-[(3-Chloroquinoxalin-2-yl)amino]-4-phenylbutan-
2-one (4b). 4b was prepared from 3b following the pro-
tocol described for 4a; 3b (4.7 g, 14.4 mmol), 14.4 mL
DMSO, 14.4 mL Et3N, and Me3N.SO3 (4 g, 28.8
mmol). The product was purified by column chromato-
graphy on silica gel eluting with C6H12/Et2O(80:20) to
6.1.5. 1-Amino-4-phenylbutan-2-ol (2b). 2b was prepared
from 1b following the protocol described for 2a; LiAlH4
(6.2 g, 163 mmol), Et2O(120 mL), 1b (12.4 g, 77 mmol),
H2O(6.2 mL), 15% aqueous NaOH (6.2 mL) and H 2O
(18 mL). The product, an orange oil, was used without
1
1
further purification (11 g, 87%); H NMR (100 MHz,
give a yellow solid (4.51 g, 96%); H NMR (100 MHz,
CDCl3) d: 1.80 (m, 3H), 2.65 (m, 4H), 3.62 (m, 3H), 7.21
(s, 5H); 13C NMR (25 MHz, CDCl3) d: 33.98, 36.25,
70.92, 125.46, 128.04, 141.74. Anal. calcd for
C10H15NO: C, 72.69; H, 9.15; N, 8.48. Found: C, 72.45;
H, 9.33; N, 8.24.
CDCl3) d: 2.82–3.01 (m, 4H), 4.38 (d, J=5 Hz, 2H), 6.4
(m, 1H), 7.23–7.84 (m, 9H). Anal. calcd for
C18H16N3OCl: C, 66.36; H, 4.95; N, 12.90. Found: C,
66.53; H, 5.23; N, 12.78.
6.1.10. 4-Chloro-1-isobutylimidazo[1,2-a]quinoxaline (5a).
4a (16.39 g, 23 mmol) was dissolved in a mixture of tri-
fluoroacetic anhydride (100 mL) and trifluoroacetic acid
(1 mL) and stirred (under N2) for 24 h at room temper-
ature. The solvent was then removed under reduced
pressure, and the crude concentrate was dissolved in
CH2Cl2 (300 mL). The organic fraction was successively
washed with 5% NaHCO3 (75 mL) and water, dried
(Na2SO4) and concentrated under vacuum to yield a
dark orange oil which was purified by column chroma-
tography on silica gel eluting with C6H12/Et2O(95:5) to
6.1.6. 1-[(3-Chloroquinoxalin-2-yl)amino]-4-methylpentan-
2-ol (3a). 2,3-dichloroquinoxaline (18.05 g, 90.7 mmol)
was added to a solution of crude 2a (11.7 g, 99.8 mmol)
and Et3N (19 mL, 13.8 g, 136 mmol) in dioxan (210
mL). The resulting solution was refluxed (under N2) for
6 h, whereupon it was cooled to room temperature.
Et3N, HCl was removed by filtration and the filtrate
was concentred under reduced pressure. The dark
orange residue was purified by column chromatography
on silica gel eluting with CH2Cl2/MeOH (100:0!99:1)
to yield a yellow solid (12.14 g, 48%); 1H NMR
(100 MHz, CDCl3) d: 0.72 (d, 3H), 0.78 (d, 3H), 1.60 (m,
3H), 3.68 (m, 4H), 5.82 (t, 1H), 7.58 (m, 4H); 13C NMR
(25 MHz, CDCl3) d: 22.17, 23.35, 24.60, 44.32, 48.62,
69.67, 125.30, 125.62, 127.91, 130.36, 136.52, 137.92,
140.59, 148.62. Anal. calcd for C14H18N3OCl: C, 60.10;
H, 6.49; N, 15.02. Found: C, 59.87; H, 6.62; N, 15.25.
1
give a cream solid, (5.04 g, 84%); H NMR (100 MHz,
CDCl3) d: 1.07 (d, J=6–7 Hz, 6H), 2.16 (m, 1H), 3.11
(d, J=6–7 Hz, 2H), 7.53–7.64 (m, 3H), 7.96–8.16 (m,
2H); 13C NMR (25 MHz, CDCl3) d: 22.41, 26.88, 36.66,
115.39, 126.50, 128.41, 128.94, 130.04, 132.02, 134.37,
135.61, 136.60, 143.66. Anal. calcd for C14H14N3Cl: C,
64.74; H, 5.43; N, 16.18. Found: C, 64.66; H, 5.55; N,
15.86.
6.1.7. 1-[(3-Chloroquinoxalin-2-yl)amino]-4-phenylbutan-
2-ol (3b). 3b was prepared from 2b following the proto-
col described for 3a; 2,3-dichloroquinoxaline (9.95 g, 50
mmol), 2b (9.02 g, 55 mmol) in dioxan (250 mL) with
Et3N (7.57 g, 75 mmol). The crude product was purified
by column chromatography on silica gel eluting with
CH2Cl2/MeOH (98:2) to yield a yellow solid (10 g,
61%); 1H NMR (100 MHz, CDCl3) d: 1.76–1.97 (m,
2H), 2.70–2.90 (m, 2H), 3.53–4.05 (m, 4H), 5.96 (t, J=5
Hz, 1H), 7.23–7.82 (m, 9H). Anal. calcd for
C18H18N3OCl: C, 65.95; H, 5.53; N, 12.82. Found: C,
66.16; H, 5.43; N, 12.58.
6.1.11. 4-Chloro-1-(2-phenylethyl)imidazo[1,2-a]quinoxa-
line (5b). 5b was prepared from 4b following the proto-
col described for 5a; 4b (4 g, 12.2 mmol), trifluoroacetic
anhydride (100 mL), trifluoroacetic acid (4 mL). The
product was purified by column chromatography on
silica gel eluting with CH2Cl2/MeOH (98:2) to give a
1
yellow solid (2.5 g, 66%); H NMR (100 MHz, CDCl3)
d: 3.20–3.31 (m, 2H), 3.53–3.68 (m, 2H), 7.32 (s, 5H),
7.54–7.63 (m, 3H), 7.99–8.31 (m, 2H); 13C NMR
(25 MHz, CDCl3) d: 30.05, 33.80, 115.34, 126.54,
126.66, 128.19, 128.46, 128.70, 129.94, 129.20, 133.00,
139.72. Anal. calcd for C18H14N3Cl: C, 70.24; H, 4.58;
N, 13.65. Found: C, 70.04; H, 4.96; N, 13.82.
6.1.8. 1-[(3-Chloroquinoxalin-2-yl)amino]-4-methylpentan-
2-one (4a). A mixture of 3a (6.96 g, 24.8 mmol), 28.4
mL Et3N and sulphur trioxide trimethylamine complex
(7.9 g, 56.8 mmol) in 28.4 mL DMSO, was stirred
overnight (under N2) at room temperature, whereupon
ice-cold water (50 mL) was added. The aqueous layer
was extracted with CH2Cl2 (3ꢄ30 mL). The combined
organic phases were dried (CaCl2) and the solvents
removed under reduced pressure. The residues were
purified by column chromatography on silica gel eluting
with C6H12/Et2O(85:15) to give a beige solid (4.18 g,
6.1.12. 1-Isobutylimidazo[1,2-a]quinoxaline-4(5H)-one (6a).
p-Toluene sulfonic acid (30 mg, 0.15 mmol) was added,
to a stirred solution of 4a (0.5 g, 1.8 mmol) under reflux
in xylene (300 mL). Refluxing in a Dean-Stark was
maintained for 24 h. The solvent was eliminated under
reduced pressure and the residue was dissolved in
CH2Cl2 (500 mL). The organic fraction was then suc-
cessively washed with 5% Na2CO3 (5 mL) and water (5
mL) prior to be dried (Na2SO4) and concentred to give

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1135
the crude product which was purified by column chro-
matography on silica gel eluting with CH2Cl2/MeOH
(90:10) to yield a yellow solid (0.35 g, 82%); H NMR
(100 MHz, CDCl3) d: 1.07 (d, 6H), 2.16 (m, 1H), 3.11 (d,
2H), 7.53–7.64 (m, 3H), 7.96–8.16 (m, 2H); 13C NMR
(25 MHz, CDCl3) d: 22.41, 26.88, 36.66, 115.39, 126.50,
128.41, 128.94, 130.04, 132.02, 134.37, 135.61, 136.60,
143.66. Anal. calcd for C14H14N3O: C, 69.69; H, 6.27;
N, 17.415. Found: C, 70.01; H, 6.01; N, 17.70.
dried (Na2SO4) and concentred under reduced pressure.
The product was purified by column chromatography
on silica gel eluting with C6H12/EtOAc (70:30) to yield
a cream solid (0.49 g, 83%); 1H NMR (100 MHz,
CDCl3) d: 1.04 (d, J=6.2 Hz, 6H), 1.90–2.35 (m, 1H),
3.03 (d, J=7.0 Hz, 2H), 3.20 (d, J=4.9 Hz, 3H), 6.10–
6.40 (m, 1H), 7.10–7.55 (m, 3H), 7.60–8.00 (m, 2H); 13C
NMR (25 MHz, CDCl3) d: 22.43, 26.93, 27.37, 36.65,
115.15, 122.49, 125.93, 126.63, 127.38, 130.43, 131.32,
134.01, 138.23, 148.41. Anal. calcd for C15H18N4: C,
70.84; H, 7.13; N, 22.03. Found: C, 71.12; H, 7.44; N,
22.21.
1
6.1.13. 1-(2-Phenylethyl)-imidazo[1,2-a]quinoxaline-4(5H)-
one (6b). 6b was prepared from 4b following the proto-
col described for 6a; 4b (0.5 g, 1.5 mmol). The product
was purified by column chromatography as specified for
4a to give a yellow solid (0.38 g, 88%); 1H NMR
(100 MHz, DMSO-d6) d: 3.04–3.19 (m, 2H), 3.46–3.61
(m, 2H), 7.13–7.39 (m, 9H), 8.10 (d, J=7.6 Hz, 1H),
11.87 (s, 1H); 13C NMR (25 MHz, DMSO-d6) d: 28.53,
52.93, 116.25, 116.53, 122.45, 123.00, 134.90, 135.19,
128.12, 128.74, 131.21, 131.91, 140.21, 152.25. Anal.
calcd for C18H14N3O: C, 74.72; H, 5.23; N, 14.52.
Found: C, 74.65; H, 5.14; N, 14.75.
6.1.17. N-Methyl-1-(2-phenylethyl)imidazo[1,2-a]quinoxal-
ine-4-amine (8b). 8b was prepared from 5b following
the protocol described for 8a; methylamine (0.260 mL
of a 40% (w/v) aqueous solution, 3 mmol), 5b (0.307 g,
1 mmol). The crude product was purified by column
chromatography eluting with CH2Cl2/MeOH (90:10) to
1
yield a yellow solid (0.2 g, 66%); H NMR (100 MHz,
CDCl3) d: 3.07–3.22 (m, 5H), 3.42–3.58 (m, 2H), 6.56
(d, J=5.6 Hz, 1H), 7.12–7.45 (m, 7H), 7.73 (d, J=9.1
Hz, 1H), 7.98 (d, J=7.9 Hz, 1H); 13C NMR (25 MHz,
CDCl3) d: 27.27, 29.76, 33.96, 114.93, 122.28, 125.79,
126.33, 127.05, 129.16, 129.49, 129.79, 130.51, 133.72,
133.97, 140.24, 149.14. Anal. calcd for C19H18N4: C,
75.47; H, 6.00; N, 18.43. Found: C, 75.68; H, 6.15; N,
18.51.
6.1.14. 1-Isobutylimidazo[1,2-a]quinoxaline-4-amine (7a).
5a (1 g, 3.85 mmol) was heated to 120 ꢃC for 4 h in the
presence of ammonia (60 mL of a 30% (w/v) aqueous
solution, 0.5 mmol). The reaction mixture was allowed
to cool to room temperature and filtered. The precipitate
was washed with H2O(10 mL), dissolved in CH Cl2 (25
2
mL), and the organic fraction was dried (Na2SO4) and
concentred under reduced pressure. The crude product
was purified by column chromatography on silica gel
eluting with CH2Cl2/MeOH (90:10) to give a yellow
6.1.18. 1-Isobutyl-N,N-dimethylimidazo[1,2-a]quinoxaline-
4-amine (9a). 9a was prepared from 5a following the
protocol described for 8a; dimethylamine (1.3 mL of a
40% (w/v) aqueous solution, 11.55 mmol), 5a (1.03 g,
3.85 mmol) in absolute EtOH (10 mL). The crude pro-
duct was purified by column chromatography on silica
gel eluting with CH2Cl2/MeOH (97:3) to give a white
1
solid (0.75 g, 81%); H NMR (100 MHz, DMSO-d6 ) d:
2.07 (m, J=6-7 Hz, 1H), 3.09 (d, J=6-7 Hz, 2H), 7.08–
8.05 (m, 7H). Anal. calcd for C14H16N4: C, 69.97; H,
6.71; N, 23.32. Found: C, 70.13; H, 6.97; N, 23.07.
1
solid (0.8 g, 78%); H NMR (100 MHz, CDCl3) d: 1.03
(d, J=6-7 Hz, 6H), 2.16 (m, J=6–7 Hz, 1H), 3.03 (d,
J=6–7 Hz, 2H), 3.58 (s, 6H), 7.15–7.41 (m, 3H), 7.62–
7.72 (m, 1H), 7.86–7.95 (m, 9H); 13C NMR (25 MHz,
CDCl3) d: 22.42, 26.75, 36.89, 40.00, 114.86, 122.05,
125.80, 126.50, 126.80, 126.98, 129.51, 131.08, 134.74,
137.74, 149.15. Anal. calcd for C16H20N4: C, 71.61; H,
7.51; N, 20.88. Found: C, 71.77; H, 7.23; N, 20.64.
6.1.15. 4-Chloro-1-(2-phenylethyl)imidazo[1,2-a]quinoxa-
line (7b). 7b was prepared from 5b following the proto-
col described for 7a. 5b (0.8 g, 2.6 mmol); ammonia (48
mL of a 30% (w/v) aqueous solution, 0.4 mmol). A
yellow solid was obtained after column chromatography
purification as specified for 7a (0.465 g, 62%); 1H NMR
(100 MHz, CDCl3) d: 3.10–3.24 (m, 2H), 3.48–3.63 (m,
2H), 5.74 (s, 2H), 7.20–7.48 (m, 8H), 7.69 (d, J=7.2 Hz,
1H), 8.05 (d, J=7.9 Hz, 1H); 13C NMR (25 MHz,
CDCl3) d: 29.75, 34.00, 115.01, 123.09, 125.94, 126.33,
126.78, 128.08, 128.46, 130.51, 130.89, 137.50, 140.04,
148.44. Anal. calcd for C18H16N4: C, 74.98; H, 5.59; N,
19.43. Found: C, 74.86; H, 5.75; N, 19.37.
6.1.19. N,N-Dimethyl-1-(2-phenylethyl)imidazo[1,2-a]qui-
noxaline-4-amine (9b). 9b was prepared from 5b follow-
ing the protocol described for 9a; 5b (0.32 g, 1 mmol),
dimethylamine (0.44 mL of a 40% (w/v) aqueous solu-
tion, 3.90 mmol). The product was purified by column
chromatography as specified for 9a and yield a beige
solid (0.2 g, 65%); 1H NMR (100 MHz, CDCl3) d: 3.09–
3.26 (m, 2H), 3.42–3.54 (m, 2H), 3.59 (s, 6H), 7.14–7.42
(m, 8H), 7.70 (d, J=6.5 Hz, 1H), 8.00 (d, J=6.9 Hz,
1H); 13C NMR (25 MHz, CDCl3) d: 30.41, 34.30, 40.12,
114.99, 125.25, 126.02, 126.58, 127.07, 128.43, 128.75,
129.89, 130.02, 134.84, 137.82, 140.57, 149.21. Anal.
calcd for C20H20N4: C, 75.92; H, 6.37; N, 17.71. Found:
C, 75.82; H, 6.15; N, 17.57.
6.1.16. 1-Isobutyl-N-methyl-imidazo[1,2-a]quinoxaline-4-
amine (8a). Methylamine (0.6 mL of a 40% (w/v) aqu-
eous solution, 6.93 mmol) was added dropwise to a
stirred solution of 5a (0.6 g, 2.31 mmol) in absolute
EtOH (15 mL) at room temperature. After 40 h,
another portion of methylamine (0.6 mL of a 40% (w/v)
aqueous solution, 6.93 mmol) was added and stirring was
maintained for an additional 3 h. The solvent was removed
under reduced pressure and the residue was dissolved in
CH2Cl2 (50 mL). The organic fraction was successively
washed with 5% NaHCO3 (30 mL) and H2O(30 mL),
6.1.20. (1-Isobutyl-4-pyrrolidin-1-yl)imidazo[1,2-a]quinoxa-
line (10a). Pyrrolidine (0.58 mL, 0.49 g, 6.93 mmol) was
added dropwise to a stirred solution of 5a (0.6 g, 2.31

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1136
mmol) in absolute EtOH (15 mL) at room temperature.
Stirring was maintained for 3 h. The solvent was
removed under reduced pressure and the residue
was dissolved in CH2Cl2 (50 mL). The organic phase was
successively washed with 5% Na2CO3 (30 mL) and H2O
(30 mL), dried (Na2SO4) and concentrated under
reduced pressure. The crude product was purified by
column chromatography on silica gel eluting with
C6H12/EtOAc (85:15) to yield a cream solid (0.6 g,
6.1.24. 1-Isobutyl-N-phenylimidazo[1,2-a]quinoxaline-4-
amine (12a). Aniline (0.63 mL, 0.645 g, 6.93 mmol) was
added dropwise to a stirred solution of 5a (0.6 g, 2.31
mmol) in absolute EtOH (15 mL) at room temperature.
Stirring was maintained for 67 h. The solvent was removed
under reduced pressure and the residue was dissolved in
CH2Cl2 (50 mL). The organic fraction was washed suc-
cessively with 5% Na2CO3 (30 mL) and H2O(30 mL),
dried (Na2SO4) and concentrated under reduced pres-
sure. The crude product was purified by column chro-
matography on silica gel eluting with C6H12/EtOAc
1
88%); H NMR (100 MHz, CDCl3) d: 1.03 (d, J=6.5
Hz, 6H), 1.85–2.30 (m, 5H), 3.04 (d, J=6.7 Hz, 2H),
4.00–4.25 (m, 4H), 7.05–7.45 (m, 3H), 7.60–7.95 (m,
2H); 13C NMR (25 MHz, CDCl3) d: 22.38, 25.41, 26.73,
36.83, 49.24, 114.84, 121.36, 125.74, 126.22, 126.26,
129.53, 131.34, 134.90, 138.50, 147.33. Anal. calcd for
C18H22N4: C, 73.44; H, 7.53; N, 19.03. Found: C, 73.24;
H, 7.68; N, 19.31.
1
(80:20) to yield a white solid (0.41 g, 56%); H NMR
(100 MHz, CDCl3) d: 1.07 (d, J=6.8 Hz, 6H), 1.95–2.45
(m, 1H), 3.07 (d, J=7.0 Hz, 2H), 6.95–7.55 (m, 5H),
7.70–8.30 (m, 5H); 13C NMR (25 MHz, CDCl3) d:
22.456, 26.95, 36.67, 115.11, 119.33, 122.73, 123.82,
126.06, 127.09, 128.38, 128.96, 131.04, 131.44, 133.60,
137.31, 139.49, 144.65. Anal. calcd for C20H20N4: C,
75.92; H, 6.37; N, 17.71. Found: C, 75.89; H, 6.03; N,
17.95.
6.1.21. [1-(2-Phenylethyl)-4-pyrrolidin-1-yl]imidazo[1,2-
a]quinoxaline (10b). 10b was prepared from 5b follow-
ing the protocol described for 10a; pyrrolidine (0.37 mL,
0.32 g, 4.5 mmol), 5b (0.46 g, 1.5 mmol). The product
was purified by column chromatography as specified for
6.1.25. N-Phenyl-1-(2-phenylethyl)imidazo[1,2-a]quinoxa-
line-4-amine (12b). 12b was prepared from 5b following
the protocol described for 10a; aniline (0.63 mL, 0.645
g, 6.93 mmol), 5b (0.7 g, 2.3 mmol). The product was
purified by column chromatography as specified for 12a
to yield a yellow solid (0.376 g, 45%); 1H NMR
(100 MHz, CDCl3) d: 3.13–3.30 (m, 2H), 3.50–3.64 (m,
2H), 6.66–6.82 (m, 1H), 7.10–7.50 (m, 10H), 7.85–9.23
(m, 5H); 13C NMR (25 MHz, CDCl3) d: 29.99, 34.12,
115.06, 119.46, 119.23, 122.70, 123.94, 126.00, 126.55,
127.00, 128.30, 128.69, 128.93, 129.21, 130.16, 131.28,
133.66, 137.21, 139.42, 140.26, 144.53. Anal. calcd for
C24H20N4: C, 79.10; H, 5.53; N, 15.37. Found: C, 79.35;
H, 5.36; N, 15.69.
1
10a and yield a yellow solid (0.32 g, 62%); H NMR
(100 MHz, CDCl3) d: 1.93–2.06 (m, 4H), 3.08–3.26 (m,
2H), 3.40–3.60 (m, 2H), 4.05–4.17 (m, 4H), 7.11–7.41
(m, 8H), 7.67 (d, J=6.8 Hz, 1H), 7.98 (d, J=8.2 Hz,
1H); 13C NMR (25 MHz, CDCl3) d: 25.55, 30.36, 34.35,
49.11, 115.30, 121.62, 126.01, 126.56, 126.81, 128.43,
128.75, 130.19, 134.80, 138.65, 140.61, 147.48. Anal.
calcd for C22H22N4: C, 77.16; H, 6.48; N, 16.36. Found:
C, 77.48; H, 6.96; N, 16.22.
6.1.22. (1-Isobutyl-4-piperidin-1-yl)imidazo[1,2-a]quinoxa-
line (11a). 11a was prepared from 5a following the pro-
tocol described for 10a; piperidine (0.7 mL, 0.59 g, 6.93
mmol); 5a (0.6 g, 2.31 mmol) in absolute EtOH (15 mL).
The product was purified by column chromatography
on silica gel eluting with C6H12/Et2O(90:10) to yield a
6.1.26. 1-Isobutyl-N-(2-phenylethyl)imidazo[1,2-a]quinoxa-
line-4-amine (13a). 2-Phenylethyl-amine (0.8 mL, 0.84
g, 6.93 mmol) was added dropwise to a stirred solution
of 5a (0.6 g, 2.31 mmol) in absolute EtOH (15 mL) at
room temperature. Stirring was maintained for 23 h and
then the reaction mixture was refluxed for an additional
24 h. The solvent was removed under reduced pressure
and the residue was dissolved in CH2Cl2 (50 mL). The
organic fraction was successively washed with 5%
Na2CO3 (30 mL) and H2O(30 mL), dried (Na 2SO4) and
concentrated under reduced pressure. The crude pro-
duct was purified by column chromatography on silica
gel eluting with C6H12/EtOAc (85:15) to yield a cream
1
white solid (0.68 g, 96%); H NMR (100 MHz, CDCl3)
d: 1.04 (d, J=6.2 Hz, 6H), 1.60–1.90 (m, 6H), 1.90–2.40
(m, 1H), 3.04 (d, J=6.6 Hz, 2H), 4.10–4.40 (m, 4H),
7.10–7.45 (m, 2H), 7.60–8.05 (m, 3H); 13C NMR
(25 MHz, CDCl3) d: 22.41, 25.02, 26.22, 26.78, 36.92,
47.90, 114.82, 122.33, 125.72, 126.67, 127.21, 129.40,
131.01, 134.70, 137.57, 148.79. Anal. calcd for C19H24N4:
C, 73.99; H, 7.84; N, 18.17. Found: C, 73.61; H, 7.72; N,
18.43.
1
6.1.23. [1-(2-Phenylethyl)-4-piperidin-1-yl]imidazo[1,2-
a]quinoxaline (11b). 11b was prepared from 5b follow-
ing the protocol described for 10a; piperidine (0.7 mL,
0.59 g, 6.93 mmol), 5b (0.7 g, 2.3 mmol). The product
was purified by column chromatography as specified for
11a to yield a yellow solid (0.62 g, 75%); 1H NMR
(100 MHz, CDCl3) d: 1.76 (s, 6H), 3.11–3.30 (m, 2H),
3.47–3.65 (m, 2H), 4.28 (s, 4H), 7.14–7.46 (m, 8H), 7.72
(d, J=8.4 Hz, 1H), 8.05 (d, J=8.7 Hz, 1H); 13C NMR
(25 MHz, CDCl3) d: 25.11, 26.36, 30.30, 34.17, 47.83,
53.79, 115.81, 122.29, 125.78, 126.41, 126.97, 128.40,
128.61, 129.62, 129.97, 137.63, 140.74, 148.90. Anal.
calcd for C23H24N4: C, 77.50; H, 6.79; N, 15.72. Found:
C, 77.33; H, 6.91; N, 15.48.
solid (0.7 g, 88%); H NMR (100 MHz, CDCl3) d: 1.05
(d, J=6.5 Hz, 6H), 1.90–2.30 (m, 1H), 2.95–3.15 (m,
4H), 3.80–4.05 (m, 2H), 6.26 (t, J=5.7 Hz, 1H), 7.10–
7.50 (m, 8H), 7.70–8.00 (m, 2H); 13C NMR (25 MHz,
CDCl3) d: 22.45, 26.95, 35.81, 36.67, 41.87, 115.15,
122.59, 125.94, 126.38, 126.70, 127.50, 128.57, 128.81,
130.49, 131.36, 137.91, 139.26, 147.59. Anal. calcd for
C22H24N4: C, 76.71; H, 7.52; N, 16.27. Found: C, 77.05;
H, 7.47; N, 16.21.
6.1.27. N,1-bis(2-Phenylethyl)imidazo[1,2-a]quinoxaline-
4-amine (13b). 13b was prepared from 5b following the
protocol described for 13a; 2-Phenylethylamine (0.87
mL, 0.84 g, 6.93 mmol), 5b (0.7 g, 2.3 mmol). The crude

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1137
product was purified by column chromatography as
specified for 13a and yield a yellow solid (0.56 g,
62%); H NMR (100 MHz, CDCl3) d: 3.02 (m, 4H),
was added to a stirred solution of 5a (0.60 g, 2.31 mmol)
in absolute EtOH (15 mL). After 22 h of reflux, the
solvent was removed under reduced pressure. The resi-
due was dissolved in CH2Cl2 (50 mL). The organic
fraction was successively washed with 5% NaHCO3 (10
mL) and H2O(10 mL), dried (Na 2SO4) and con-
centrated under reduced pressure. The crude product
was purified by column chromatography on silica gel
eluting with C6H12/EtOAc (85:15) to yield a white solid
1
3.48 (m, 2H), 3.87 (m, 2H), 6.21 (m, 1H), 7.27 (s, 13H),
7.23 (d, J=7.1 Hz, 1H), 7.99 (d, J=7.8 Hz, 1H); 13C
NMR (25 MHz, CDCl3) d: 29.83, 34.04, 35.59, 41.73,
114.94, 122.39, 125.80, 126.19, 126.39, 127.27, 129.19,
129.39, 129.53, 129.65, 129.85, 130.59, 133.62, 137.99,
139.12, 140.29, 147.39. Anal. calcd for C26H24N4: C,
79.56; H, 6.16; N, 14.27. Found: C, 79.91; H, 6.22; N,
14.33.
1
(0.88 g, 93%); H NMR (100 MHz, CDCl3) d: 1.05 (d,
J=6.9 Hz, 6H), 1.47 (s, 9H), 2.00–2.40 (m, 1H), 3.06 (d,
J=7.0 Hz, 2H), 3.50–3.70 (m, 4H), 4.20–4.40 (m, 4H),
7.15–7.50 (m, 3H), 7.65–8.05 (m, 2H); 13C NMR
(25 MHz, CDCl3) d: 22.46, 26.82, 28.43, 36.90, 43.78,
46.59, 79.82, 114.95, 123.18, 125.94, 126.88, 127.56,
129.73, 131.23, 134.42, 137.07, 148.44, 154.90.
6.1.28. 1-Isobutyl-N-benzylimidazo[1,2-a]quinoxaline-4-
amine (14a). Benzylamine (0.76 mL, 0.743 g, 6.93
mmol) was added dropwise to a stirred solution of 5a
(0.6 g, 2.31 mmol) in absolute EtOH (15 mL) at room
temperature. Stirring was continued for 19.5 h and the
reaction mixture was then refluxed for an additional 8 h.
The solvent was removed under reduced pressure and
the residue was dissolved in CH2Cl2 (50 mL). The
organic fraction was successively washed with 5%
6.1.32. t-Butyl-4-[1-(2-phenylethyl)imidazo[1,2-a]quinox-
alin-4-yl]piperazine-1-carboxylate (17b). 17b was pre-
pared from 5b following the protocol described for 17a;
16 (0.74 g, 3 mmol), 5b (0.62 g, 2 mmol), absolute EtOH
(15 mL). The crude product was purified by column
chromatography as specified for 17a to give a white
solid (0.54 g, 59%); 1H NMR (100 MHz, CDCl3) d: 1.43
(s, 9H), 3.04–3.22 (m, 2H), 3.43–3.61 (m, 6H), 4.19–4.28
(t, 2H), 7.11–7.34 (m, 3H+5H), 7.64–7.71 (m, 1H),
7.96–8.03 (m, 1H).
NaHCO3 (10 mL) and H2O(10 mL), dried (Na SO4)
2
and concentrated under reduced pressure. The crude
product was purified by column chromatography as
1
specified for 13a to yield a white solid (0.5 g, 66%); H
NMR (100 MHz, CDCl3) d: 1.05 (d, J=6.6 Hz, 6H),
1.90–2.45 (m, 1H), 3.03 (d, J=6.8 Hz, 2H), 4.87 (d,
J=5.7 Hz, 2H), 6.35–6.65 (m, 1H), 7.05–7.55 (m, 8H),
7.65–8.05 (m, 2H); 13C NMR (25 MHz, CDCl3) d:
22.43, 26.91, 36.63, 44.51, 115.12, 122.67, 125.93,
126.79, 127.29, 127.53, 127.99, 128.53, 130.53, 131.39,
133.80, 138.06, 137.78, 147.47. Anal. calcd for
C21H22N4: C, 76.33; H, 6.71; N, 16.96. Found: C, 76.23;
H, 6.99; N, 16.90.
6.1.33. (1-Isobutyl-4-piperazin-1-yl)imidazo[1,2-a]quinoxa-
line (18a). A solution of 17a (0.82 g, 2 mmol) in TFA/
CH2Cl2 (1:1) (50 mL) was stirred at room temperature
for 30 min during which time a yellow solution resulted.
The volatiles were removed under reduced pressure to
leave a yellow oil which was dissolved in CH2Cl2 (30
mL), washed with 5% NaHCO3 (30 mL) and then
extracted with 1 M HCl (2ꢄ20 mL). The pH of the
combined aqueous layers was adjusted to 10 with 1 M
Na2CO3. The precipitate formed was collected by fil-
tration, washed with a small amount of ice-cold water
and dried to constant weight in a drying pistol to yield a
white solid which was not further purified (0.46 g, 74%);
1H NMR (100 MHz, CDCl3) d: 1.04 (d, J=6.7 Hz, 6H),
1.95–2.35 (m, 1H), 3.06 (d, J=6.9 Hz, 2H), 3.25–3.60
(m, 4H), 4.50–4.85 (m, 4H), 6.10 (s, br, 1H), 7.15–7.50
(m, 3H), 7.60–8.10 (m, 2H); 13C NMR (25 MHz,
CDCl3) d: 22.45, 26.80, 36.82, 43.48, 43.71, 114.99,
124.05, 126.07, 127.10, 127.94, 130.03, 131.43, 133.88,
136.46, 147.40. Anal. calcd for C18H23N5: C, 69.87; H,
7.49; N, 22.63. Found: C, 70.15; H, 7.13; N, 23.01.
6.1.29. 1-Isobutyl-N-(2-methoxyethyl)imidazo[1,2-a]qui-
noxaline-4-amine (15a). 2-Methoxyethyl-amine (0.603
mL, 0.521 g, 6.93 mmol) was added dropwise to a stir-
red solution of 5a (0.60 g, 2.31 mmol) in absolute EtOH
(15 mL) at room temperature. Stirring was maintained
for 22 h and then the reaction mixture was refluxed for
24 h. The solvent was removed under reduced pressure
and the residue was dissolved in CH2Cl2 (50 mL). The
organic fraction was successively washed with 5%
NaHCO3 (10 mL) and H2O(10 mL), dried (Na SO4)
2
and concentrated under reduced pressure. The crude
product was purified by column chromatography on
silica gel eluting with C6H12/EtOAc (70:30) to yield a
cream solid (0.61 g, 89%); 1H NMR (100 MHz, CDCl3)
d: 1.03 (d, J=6.7 Hz, 6H), 1.95–2.35 (m, 1H), 3.02 (d,
J=6.9 Hz, 2H), 3.39 (s, 3H), 3.65 (t, J=4.8 Hz, 2H),
3.75–4.00 (m, 2H), 6.35–6.60 (m, 1H), 7.05–7.50 (m,
3H), 7.60–8.00 (m, 2H); 13C NMR (25 MHz, CDCl3) d:
2.37, 26.87, 36.59, 40.06, 58.76, 71.13, 115.08, 122.51,
125.84, 126.66, 127.32, 130.41, 131.40; 133.81, 138.02,
147.63, 175.15. Anal. calcd for C17H22N4O: C, 68.43; H,
7.43; N, 18.78. Found: C, 68.71; H, 7.24; N, 18.83.
6.1.34. [1-(2-phenylethyl)-4-piperazin-1-yl]imidazo[1,2-a]-
quinoxaline (18b). 18b was prepared from 17b follow-
ing the protocol described for 18a; 17a (0.54 g, 1.18
mmol), TFA/CH2Cl2 (1:1) (25 mL). The product
obtained, a white solid, was not further purified (0.2 g,
1
47%); H NMR (100 MHz, MeOD) d: 3.16–3.61 (m,
6H), 4.40–4.54 (m, 4H), 7.22–7.51 (m, 8H), 7.70–7.78
(m, 1H), 8.18–8.25 (m, 1H); 13C NMR (25 MHz,
MeOD) d: 30.75, 35.14, 45.08, 44.66, 116.73, 125.85,
127.49, 128.42, 129.00, 129.52, 129.67, 131.69, 132.35,
137.69, 141.81, 149.10. Anal. calcd for C22H23N5: C,
73.92; H, 6.49; N, 19.59. Found: C, 74.29; H, 6.11; N,
19.36.
6.1.30. t-Butyl-4-piperazine (16). Compound 16 was
prepared as described in the litterature.16
6.1.31. t-Butyl-4-(1-isobutylimidazo[1,2-a]quinoxalin-4-yl)-
piperazine-1-carboxylate (17a). 16 (0.86 g, 4.62 mmol)

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C. Deleuze-Masquefa et al. / Bioorg. Med. Chem. 12 (2004) 1129–1139
1138
6.2. Biochemistry: measurement of PDE4 inhibitory
activity
were filled with 0.5 mL AG-50 Wx8 mesh resuspended
in water. Prior to use, the columns were washed with 1
mL of 1 N NaOH, two times 1 mL of 1 N HCl, and four
times 1 mL of H2O.
6.2.1. Materials. A549 cells were from the American
Type Culture Collection, Rockville, MD, USA. T75 cul-
ture flasks were obtained from Costar. Dulbecco’s mod-
ified Eagle’s Medium (DMEM)-F12 came from Gibco-
BRL, and l-glutamine, penicillin, streptomycin and
phosphate-buffered saline (PBS) from BioMedia. Foetal
calf serum (FCS) was supplied by Bio Whittaker Europe.
Dimethylsulfoxyde (DMSO) was provided by ICN Bio-
medicals. Tris-2-amino-2-(hydroxymethyl)-1,3-propane-
diol pH 7.5 was from Q-Biogene. AG-50 Wx8 (200–400
mesh) and Poly-Prep (0.8ꢄ4 cm) columns were from Bio-
Rad. [3,8-3H]cAMP and Q-Sepharose Fast-Flow were
from Amersham Biosciences. All other chemicals were
from Sigma-Aldrich except when otherwise stated. Ultima
Gold MV was supplied by Packard. Radioactivity was
measured in a Beckman LS-3801 liquid scintillation b-
counter. The A549 cells were counted in a Casy cell-coun-
ter. Protein concentration was determined with a com-
mercial kit (Coomassie Plus Protein Assay Reagent1,
Pierce), using bovine serum albumin (BSA) as a standard.
The reaction mixture (100 mL) routinely contained 40
mM Tris–HCl, pH 8.0, 0.05 mM CaCl2, 3 mM MgCl2,
0.1 mg/mL BSA and 1% DMSO. The reaction, carried
out at 37 ꢃC, was started by addition of [3H]cAMP (15
000 to 25 000 cpm, 0.1ꢄ10ꢀ6 and 1ꢄ10ꢀ3 M) and stop-
ped with 0.5 mL of 5% TCA containing 1 mM AMP
and 1 mM cAMP. The reaction mixtures were applied
on the columns which were washed three times with 0.4
mL and four times with 1 mL H2O , 3[H]AMP was
eluted with five successive additions of 0.6 mL 0.4 M
sodium citrate pH 7.5, collected directly in scintillation
vials and immediately measured by scintillation spec-
troscopy after addition of 12 mL of Ultima Gold MV.
The purity of the PDE4 isoenzyme prepared was asses-
sed by (i) its sensitivity to 100 mM rolipram, a selective
PDE4 inhibitor, (ii) the effect of 2.5 mM cilostamide, a
PDE3 selective inhibitor and (iii) the response to Ca2+
/
Calmodulin (0.1 mM/150 mM) stimulation, a PDE1
specific activator.
6.2.2. A549 cells culture and HSPDE4 extraction. A549
cells were cultured to confluence in T75 culture flasks, at
37 ꢃC in 5% CO2 in DMEM-F12 medium supplemented
with 2 mM l-glutamine, 100 U/mL penicillin/strepto-
mycin and 10% heat-inactivated FCS whereupon they
were trypsinated for 4 min, collected by centrifugation
at room temperature at 2000 g for 5 min and washed
twice with PBS without calcium and magnesium and
stored at ꢀ80 ꢃC.
Stock solutions for the inhibitors were prepared in
100% DMSO. The final concentration of DMSO in the
enzyme assay was 1%.
Acknowledgements
We wish to thank Dr. P.W. Stemmer, Wayne State
University, Detroit, MI, USA, for kindly providing the
Calmodulin sample used in this work. We also thank
Dr. J.J. Bourguignon, Laboratoire de Pharmacochimie
de la Communication Cellulaire, UMR 7081, Faculte de
Pharmacie, Illkirch, France, for supplying a generous
sample of cilostamide. We are grateful to Prof. L. Vian
and Prof. A. Muller for the use of their culture room
and hot lab, respectively, and to Dr. C. Roumestan and
Pr. A. Michel for the cellular line and the help during
the start-off of the cell cultures. GG was the recipient of
DEA stipend from the Ministere de la Jeunesse, de
l’Education Nationale et de la Recherche.
The pellets were thawed on ice and resuspended to a
density of 107 cells/mL in 10 mL ice-cold lysis buffer (10
mM Tris–HCl pH 7.5, 2 mM MgCl2, 5 mM b-mercapto-
ethanol, 10 mM antipain, 10 mM leupeptin, 10 mM pep-
statin A, 200 mM PMSF, 1 mM EDTA, 0.2 mM EGTA,
10 mM NaF, and 10 mM TLCK) and disrupted by
sonication (ten pulses, 30 seconds each, on ice, at 60 W).
The lysat was clarified by centrifugation at 100 000 g for
1 h at 4 ꢃC and the supernatant was applied on a
Q-Sepharose Fast-Flow (1.6ꢄ10 cm) column pre-equili-
brated with 10 mM Tris–HCl pH 7.5, 2 mM MgCl2 and
5 mM b-mercaptoethanol. The column was developed
at a flow rate of 1 mL/min, with a non-linear sodium ace-
tate gradient (100 min at 0 M, 50 min from 0.5 to 0.6 M,
60 min from 0.6 to 0.9 M, 25 min at 1 M, and 20 min at
0 M), 1 mL fractions were collected. The protein contain-
ing fractions were determined by 12.5% SDS–PAGE
and the presence of PDE4 by enzymatic activity measure-
ments, with or without rolipram, as described below. The
active fractions were pooled and either kept on ice for at
most one month or stored frozen at ꢀ80 ꢃC after addition
of 20% v/v glycerol. All subsequent enzymatic determi-
nations were performed using this batch of PDE4.
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