New quinoxaline derivatives as potential mt1 and mt2 receptor ligands

Article abstract of DOI:10.3390/molecules17077737

Ever since the idea arose that melatonin might promote sleep and resynchronize circadian rhythms, many research groups have centered their efforts on obtaining new melatonin receptor ligands whose pharmacophores include an aliphatic chain of variable length united to an N-alkylamide and a methoxy group (or a bioisostere), linked to a central ring. Substitution of the indole ring found in melatonin with a naphthalene or quinoline ring leads to compounds of similar affinity. The next step in this structural approximation is to introduce a quinoxaline ring (a bioisostere of the quinoline and naphthalene rings) as the central nucleus of future melatoninergic ligands.

Full text of DOI:10.3390/molecules17077737

Molecules 2012, 17, 7737-7757; doi:10.3390/molecules17077737
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
New Quinoxaline Derivatives as Potential MT1 and MT2
Receptor Ligands
Saioa Ancizu 1, Nerea Castrillo 1, Silvia Pérez-Silanes 1, Ignacio Aldana 1, Antonio Monge 1,
Philippe Delagrange 2, Daniel-Henry Caignard 2 and Silvia Galiano 1,*
1
Unidad en Investigación y Desarrollo de Medicamentos, Centro de Investigación en Farmacobiología
Aplicada (CIFA), Universidad de Navarra, C/Irunlarrea, 1, 31008 Pamplona, Spain;
E-Mails: sancizupere@alumni.unav.es (S.A.); ncastrillo@alumni.unav.es (N.C.);
sperez@unav.es (S.P.-S.); ialdana@unav.es (I.A.); amonge@unav.es (A.M.)
2
IdRServier, 125 Chemin de ronde, 78290 Croissy-sur-Seine, France;
E-Mails: philippe.delagrange@fr.netgrs.com (P.D.); daniel-henri.caignard@fr.netgrs.com (D.-H.C.)
* Author to whom correspondence should be addressed; E-Mail: sgaliano@unav.es;
Tel.: +34-948-425-653; Fax: +34-948-425-652.
Received: 17 April 2012; in revised form: 14 June 2012 / Accepted: 18 June 2012 /
Published: 25 June 2012
Abstract: Ever since the idea arose that melatonin might promote sleep and resynchronize
circadian rhythms, many research groups have centered their efforts on obtaining new
melatonin receptor ligands whose pharmacophores include an aliphatic chain of variable
length united to an N-alkylamide and a methoxy group (or a bioisostere), linked to a central
ring. Substitution of the indole ring found in melatonin with a naphthalene or quinoline
ring leads to compounds of similar affinity. The next step in this structural approximation
is to introduce a quinoxaline ring (a bioisostere of the quinoline and naphthalene rings) as
the central nucleus of future melatoninergic ligands.
Keywords: sleep disorders; melatonin; MT1/MT2 receptors; quinoxalinamide; quinoxalinurea
1. Introduction
The frequent pathology of sleep disorders has diverse origins. The clinical manifestations vary and
may include insomnia, hypersomnia, respiratory disorders, complex motor disorders, etc. These
disorders can appear alone or associated to each other, thereby making this a very complex pathology.

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Insomnia is the most frequent disorder within the general population [1]. The complaints from more
than 50% of primary care patients are related to insomnia and an estimated 9 to 18% of the adult
population suffers from chronic insomnia [2,3].
In the regulation of the awake-sleep system, a complex neural network is involved, in which
different areas of the brain are activated and inhibited, following a circadian rhythm that lasts 24 h.
The suprachiasmatic nucleus (SCN) is responsible for synchronizing the circadian rhythm as well as
for promoting sleep, by means of melatonin (MLT), the main hormone secreted by the pineal gland [4,5].
In fact, it has been observed that administration of MLT results in an increase of sleepiness.
Melatonin regulates sleep and the circadian rhythms by means of its receptors MT1 and MT2 which
in recent years have become one of the most interesting pharmacological targets [4,6].The diversity
and distribution of MT1 and MT2 in the different tissues indicate that each one of these receptors
possibly possesses a different physiological function, although this possibility must be more
thoroughly studied. Some studies suggest that the effect of promoting sleep is related to the
suppression of activity in the SCN neurons by means of activation of the receptors MT1. Other studies
report that the phase changes of the neuronal rhythm are related to the activation of the receptors MT2
at dawn [4,7].
Ever since the idea arose that MLT might promote sleep and resynchronize circadian rhythms, many
research groups have centered their efforts on obtaining new melatonin receptor ligands [4,6,8–11].
As can be observed in Figure 1, several series of MT2 ligands have been described as a competitive
agonists or antagonists with varying degrees of selectivity (IIK7 is a selective MT2 receptor agonist
and luzindole and 4P-PDOT are a selective MT2 receptor antagonists) as well as compounds with
affinity for both melatoninergic receptor subtypes (melatonin, ramelteon, agomelatine, LY156735 and
tasimelteon) [12–15].
Figure 1. Chemical structures of MT1/MT2 receptors agonist and MT2-selective ligands.

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Recently MT2 receptor subtype selectivity has registered some important advances that could result
in a safer compound with a more favorable pharmacological profile [16–19]. During the last two
decades, numerous research groups have sought to understand how melatonin interacts with its
receptors. A number of structure-activity relationships (SAR) have been studied proposing molecular
models of the melatonin binding site and 3D pharmacophore models [20–27]. These models have been
reviewed and completed in recent reviews [6,28].
A brief description of the general SAR concepts developed in these decades follows. The
pharmacophore structure that can be found in almost all MLT receptor agonists is composed by an
amide group connected by a linker chain of variable length to an aromatic nucleus (indole or a
bioisostere) carrying a methoxy group, or a bioisostere, such as bromine.
Since the indole ring of MLT is not essential for binding to the receptor, several potent ligands have
been designed by employing bioisosteric replacement of the indole moiety by other aromatic rings.
Substitution of the indole ring with the naphthalene ring system leads to agomelatine, which presents
higher affinity for MT1/MT2 than MLT. In addition, replacement of the naphthalene ring with a
quinoline scaffold leads to compounds of similar affinity [29,30]. The bioequivalency disclosed
between the indole moiety replaced by naphthalene and quinolone rings is a clear example of a
structural approach, in which the next logical step was to introduce the quinoxaline ring as shown in
Figure 2. The quinoxaline derivatives represent a privileged chemical structure because they possess
important biological properties with applications in diverse therapeutic fields as anticancerous [31],
anti-inflammatory/antioxidant [32], antibacterial [33] and sedative and anticonvulsant agents [34].
Thus, in this study, we decided to design and synthesize quinoxaline derivatives in order to verify
whether the quinoxaline is a valuable heterocyclic bioisostere of the melatonin indole nucleus.
Figure 2. Structural approach of the indole ring and the naphthalene ring to quinoline ring
and to quinoxaline ring.

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In addition, early SAR studies showed that both the 5-methoxy group and the N-acetylamino side
chain of melatonin are crucial for high receptor affinity and that the relative spatial distance between
these groups is also an important factor [35]. However, based on extended SAR data of a series of
tryptamines, it has been concluded that the 5-methoxy group is not an essential requirement for
biological activity, although it clearly shows major interactions with a specific binding site in the
melatonin receptor for a broad variety of ligands [36] and it is known to increase potency by forming a
hydrogen bond with His211 in the putative transmembrane domain 5 (TM5) [37,38].
Moreover, it has been reported that appropriate substituents ortho to the ethylamido side chain in
the naphthalenic melatonin analogues modulate the binding affinity to the melatonin receptor [39].
Thus, we were further interested in the effects of switching the methoxy group from position 6 of the
quinoxaline scaffold (analogous to position 5 in the melatonin indole ring) to position 2, contiguous to
the N-acylamido side chain. Herein, we report a preliminary design, synthesis and biological
evaluation of three novel series of quinoxaline derivatives as shown in Figure 2.
2. Results and Discussion
2.1. Chemistry
The synthetic route followed for the synthesis of series I is shown in Scheme 1. First, one of the
chlorine atoms of 2,3-dichloroquinoxaline was replaced by a methoxy group using sodium methoxide
in tetrahydrofuran (THF) leading to compound 1 [40]. According to the procedure described by
Herrman et al. [41,42] a nitrile group was then introduced into position 2 of the quinoxaline by
treatment with tetraethylammonium cyanide in the presence of acetonitrile at 50–60 °C to give
compound 2.
Scheme 1. Synthesis of series I.
Reagents and Conditions: (a) NaOMe, THF, 0 °C, 30 min, r.t., 1 h (86%); (b) Et4N+CN, AcCN,
50–60 °C, 2 h 30 min (20%); (c) (RCO)2O, THF, H2, Raney-Nickel, 50 psi, 50 °C, 7 h, (7–38%);
(d) RNCO, THF, Raney-Nickel, 50 psi, r.t., 7 h, (3–23%).
The amide derivatives 3a–e were obtained according to previously described methods [43,44].
Hydrogenation of the nitrile group of 2 over Raney-Nickel in THF and concomitant N-acylation with a
suitable anhydride gave the desired amide derivatives. In the case of the urea derivatives 3f–i, several
trials and strategies were carried out. At the beginning, a classical two-step strategy was designed in
which a catalytic hydrogenation of the nitrile group of 2 over Raney-Nickel in ammonia and ethanol

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provided the desired amines which were converted to urea by reaction of the primary amine with the
corresponding isocyanate [45]. Due to the failure of this strategy, further investigations regarding these
reactions led to the development of a novel one-step procedure. Reduction of the nitrile group of 2 by
hydrogen over Raney-Nickel in THF and concomitant addition of an appropriate isocyanate gave the
desired urea derivatives mixed with the hydrolyzed isocyanates, as expected.
With the aim of obtaining the compounds of series II, a new synthetic route was designed, as shown
in Scheme 2. 2,3-Dichloro-6-nitroquinoxaline was first treated with sodium methoxide in THF to
give 4 [16]. Subsequently, compound 5 was obtained through the reduction of the nitrile group of 4
with hydrazinium hydroxide and palladium on carbon [46]. Finally, N-acylation of compound 5 using a
suitable anhydride and triethylamine in THF yielded the corresponding amide analogs 6a–e [47], while
treatment of 5 with different isocyanates in dichloromethane (DCM) afforded the urea analogs 6f–i [45].
Scheme 2. Synthesis of series II.
Reagents and Conditions: (a) NaOMe, THF, 0 °C, 30 min, r.t., 1 h (52%); (b) N2H4·OH, Pd-C,
MeOH, 0 °C, 30 min, r.t., 15 h (96%); (c) (RCO)2O, THF, Et3N, r.t., 1 h 30 min, (13–72%);
(d) RNCO, N2, dry DCM, 0 °C, 30 min, r.t., 3 h, (10–55%).
Compounds 10a–e of series III were synthesized as depicted in Scheme 3. A modified Beirut
reaction was adopted for the preparation of 7 by condensation of 6-methoxybenzofuroxane and
malononitrile.
Scheme 3. Synthesis of series III.
Reagents and Conditions: (a) CNCH2CN, N,N-DMF, Et3N, 0 °C-r.t., 6 h (86%); (b) (CH3)3CNO2,
N,N-DMF, N2, 65 °C, 1 h 30 min (31%); (c) Na2S2O4, H2O, MeOH, AcOEt, 65 °C, 10 min (39%);
(d) (RCO)2O, THF, H2, Raney-Nickel, 50 psi, 50 °C, 7 h, (6–69%).
Next, compound 7 was deaminated leading to compound 8 by means of the corresponding
diazonium salt intermediate, using tert-butyl nitrite in N,N-dimethylformamide (N,N-DMF) as previously

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described [48]. Treatment of compound 8 with sodium dithionite in water in the presence of methanol
(MeOH) and ethyl acetate (AcOEt) provided compound 9 [32,49]. Finally the amide derivatives, 10a–e,
were obtained by the hydrogenation of the nitrile group of 9 over Raney-Nickel in THF and
concomitant N-acylation with a suitable anhydride as previously described [43,44].
2.1. Pharmacology and Structure-Activity Relationship
The binding affinity of the newly synthesized quinoxalines for MT1 and MT2 receptors was tested
through competitive binding experiments using 2-[125I]Iodomelatonin as radioligand. Biological
evaluation was performed according to previously described methods [50] and the obtained results are
reported in Table 1.
Table 1. MT1 and MT2 binding affinities (µM) of new synthesized Quinoxalines.
Comp.
3a
R
CH3
CH2CH3
CH2CH2CH3
CH(CH3)2
Ph
NHCH2CH3
NHCH2CH2CH3
NHCH(CH3)2
NHPh
MT1 Ki (µM) ± SEM
2.98 ± 0.36
2.60 ± 0.23
0.75 ± 0.36
1.23 ± 0.32
>103
MT2 Ki (µM) ± SEM
0.88 ± 0.30
0.66 ± 0.01
1.10 ± 0.03
0.47 ± 0.02
>103
Quinoxalines
3b
3c
3d
3e
3f
3g
>103
0.40 ± 0.13
0.44 ± N.D.
>103
>103
3h
3i
>103
>103
>103
6a
6b
6c
6d
6e
CH3
20.00 ± 1.82
17.60 ± 7.81
11.50 ± 2.80
3.40 ± 1.37
>103
0.08 ± N.D.
4.36 ± 1.22
1.35 ± 0.31
10.50 ± 2.96
>103
CH2CH3
CH2CH2CH3
CH(CH3)2
Ph
6f
6g
6h
6i
10a
10b
10c
10d
10e
NHCH2CH3
NHCH2CH2CH3
NHCH(CH3)2
NHPh
3.41 ± 1.89
1.63 ± 0.44
>103
28.80 ± N.D
0.489 ± 0.07
>103
>103
>103
CH3
>103
>103
CH2CH3
CH2CH2CH3
CH(CH3)2
Ph
>103
0.34 ± 0.15
0.10 ± 0.01
0.16 ± 0.00
>103
0.21 ± 0.11
0.32 ± 0.04
>103
MLT
0.14·× 10−3 ± 0.03·× 10−3 0.41·× 10−3 ± 0.04·× 10−3
Binding affinities (μM) are expressed as mean Ki ± SEM of at least three independent experiments.
N.D. not determined.
As can be observed in Table 1, compounds 10c and 10d of Series III present the best affinity profile
towards MT1/MT2 receptors. Comparing these data with their analogues 3c and 3d of Series I, it can

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be concluded that the existence of the six-atom distance between the methoxy group and the first
nitrogen atom of the side chain is essential for obtaining a good melatoninergic MT1/MT2 binding
affinity. Derivative 10c exhibited the best melatoninergic binding affinity with both receptors and
therefore, this compound could be selected as a hit in the search for new and more active quinoxalines
acting as MT1/MT2 receptor ligands.
As shown by compounds of Series I and II, shifting the methoxy group from position C6 to C2 of
the quinoxaline moiety can modulate the affinity and selectivity of these ligands depending on the
distance between the methoxy group and the N-acylamino chain. The comparison of the obtained data
for compounds of both series and Series III reveals that switching the methoxy group to position 2 of
the quinoxaline ring has a much greater effect on binding affinity to the MT2 receptor subtype than it
does on affinity to MT1 receptor, displaying a significant selectivity for MT2 receptor in Series II,
where the relative spatial distance (six-atoms) between the two key pharmacophoric elements is also
the optimal.
Compound 6a showed the most promising profile in terms of affinity and selectivity for the MT2
receptor with MT2/MT1 selectivity ratio of 250 and the highest binding affinity (0.08 μM) for the MT2
receptor. These results indicate that compound 6a is a potent and selective MT2 ligand. Furthermore,
the functional activity of two of the best compounds of this series (6c and 6a) has been evaluated only
on the MT2 receptor subtype, due to the weak affinity for the MT1 subtype. The results are shown in
Table 2. Compound 6c showed full agonist profile with a EC50 = 1.3 μM and Emax = 84%. In the case
of compound 6a, due to the fact that this compounds exhibited an EC50 > 10 μM, the compound 6a
might considered as an antagonist.
Table 2. MT2 GTPγS (+) binding affinities of compounds 6a and 6c.
MT2
Compound
EC50 ± SEM (μM)
0.49 ×·10−3 ± 0.05·× 10−3
>10
Emax ± SEM (%)
MLT
6a
100
6c
1.3 ± 0.26
84 ± 9.5
Concentration-response curves were analysed by non-linear regression. Agonist potency was
expressed as EC50 ± SEM (μM) while the maximal efficacy, Emax ± SEM was expressed as a
Percentage of that observed with melatonin 1 μM (100%).
With regard to the amide/urea substitution, the results are inconclusive, therefore, it cannot be
determined which group (amide/urea) leads to better affinity for MT1/MT2 receptors. Comparing the
results obtained from Series I, it could be assumed that the amide derivatives 3a–e generally present
better affinity for MT1 receptor than their homologous urea derivatives 3f–i. However, the affinity of
urea derivatives for MT2 is higher than the affinity of amide derivatives for this same receptor,
provided that the aliphatic chain linked to the urea/amide group is a linear aliphatic chain. In contrast,
taking into account the results obtained for the compounds from Series II, it could be concluded that
the urea derivatives 6a–e present higher affinity for both receptors than their homologous amide
derivatives 6f–i, provided that the aliphatic chain linked to the urea/amide group is a linear aliphatic
chain. Thus, it can be said that the introduction of a urea function, instead of the amide function
existing in the MLT, does not negatively affect the affinity of the ligands for MT1 and MT2 receptors.

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In addition, since it is already well known that urea derivatives usually present better metabolic
stability than amide derivatives, the urea derivatives are preferred.
Finally, the introduction of a benzene ring, replacing the aliphatic chain substituted over the
amide/urea group, results in a total loss of affinity. By contrast, substitution with an aliphatic linear
chain in general, and particularly with a propyl group, appeared to be the best option in order to obtain
high affinity compounds.
With these results, we were not able to state that the quinoxaline scaffold can act as a useful
bioisostere of indole in the search for melatonin receptor ligands. In addition, The SAR study
suggested that a six-atom length is the best distance between the methoxy group and the first nitrogen
atom of the side chain in order to obtain high MT1/MT2 affinity. Moreover, new bibliographic review
has revealed that the optimum distance should be obtained through a two-methylene linker in order to
allow compounds to adopt a MLT-like configuration [51]. Thus, the structural requirements initially
described have been redefined and the synthesis of a new series of quinoxalines has been proposed
in which the aforementioned distance is obtained through a two-methylene linker (Figure 3). These
conclusions open a new line of research in order to determine if the quinoxaline ring is suitable as
central scaffold of melatoninergic ligands.
Figure 3. Structural requirements of future series of quinoxalines as MT1/MT2 receptor ligands.
3. Experimental
3.1. Chemical Synthesis
3.1.1. General Remarks
All of the synthesized compounds were chemically characterized by thin layer chromatography
(TLC), infrared (IR), proton nuclear magnetic resonance (1H-NMR) and elemental microanalyses
(CHN). Alugram SIL G/UV254 (Layer: 0.2 mm) (Macherey-Nagel GmbH & Co. KG., Düren,
Germany) was used for TLC, and Silica gel 60 (0.040–0.063 mm, Merck, Darmstadt, Germany) was
1
used for flash column chromatography. The H-NMR spectra were recorded on a Bruker 400
Ultrashield instrument (Bruker, Rheinstetten, Germany) 400 MHz), using TMS as internal standard
and with DMSO-d6 or CDCl3 as solvents; the chemical shifts are reported in ppm (δ) and coupling
constant (J) values are given in Hertz (Hz). Signal multiplicities are represented by: s (singlet), bs
(broad singlet), d (doublet), dd (doublet of doublets), ddd (doublet of doublet of doublets), t (triplet), dt
(doublet of triplets), tt (triplet of triplets); q (quartet); dq (doublet of quartets) and m (multiplet). The
IR spectra were recorded on a Nicolet Nexus FTIR (Thermo, Madison, WI, USA) in KBr pellets.
Elemental microanalyses were obtained on a CHN 900 Elemental Analyzer (Leco, Tres Cantos, Spain)
from vacuum-dried samples. The analytical results for C, H and N, were within ±0.4 of the theoretical

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values. Chemicals were purchased from Panreac Química S.A. (Montcada i Reixac, Spain),
Sigma-Aldrich-Fluka Química S.A. (Alcobendas, Spain), Alfa Aesar-Avocado GmbH & Co. KG
(Karlsruhe, Germany), and E. Merck (Darmstadt, Germany).
3.1.2. Synthesis of 2-Chloro-3-methoxyquinoxaline (1)
2,3-Dichloroquinoxaline (90%, 2.30 g, 10.04 mmol) and THF (20.00 mL) were placed in a flask.
The mixture was cooled to 0 °C. A solution of sodium methoxide was obtained from the reaction
between Na (0.50 g) and methanol (5.00 mL). NaOMe (2.34 mL, 10.04 mmol) was added dropwise to
the other suspension. The color of the mixture changed from purple to yellow. The mixture was stirred
during 30 min at 0 °C and during 1 h at room temperature. DCM (80.00 mL) was added and the
mixture was quenched with brine. The DCM solution was dried over Na2SO4 and filtered. The solvent
was removed under reduced pressure to give compound 1 as a yellow solid. Yield: 86%. IR (cm−1):
1
3032, 2987, 2936. H-NMR (CDCl3) δ: 7.96 (ddd, 1H, H8, J8-7 = 8.3 Hz, J8-6 = 1.5 Hz, J8-5 = 0.5 Hz);
7.88 (ddd, 1H, H5, J5-6 = 8.3 Hz, J5-7 = 1.4 Hz, J5-8 = 0.5 Hz); 7.71 (ddd, 1H, H6, J6-5 = 8.4 Hz,
J6-7 = 7.1 Hz, J6-8 = 1.5 Hz); 7.60 (ddd, 1H, H7, J7-8 = 8.4 Hz, J7-6 = 7.1 Hz, J7-5 = 1.5 Hz); 4.20 (s, 3H,
OCH3) ppm. Anal. Calcd for C9H7N2OCl: C: 55.53%; H: 3.60%; N: 14.40%; Found: C: 55.23%; H:
3.34%; N: 14.41%.
3.1.3. Synthesis of 3-Methoxyquinoxaline-2-carbonitrile (2)
Compound 1 (1.59 g, 8.17 mmol) and tetraethylammonium cyanide (2.55 g, 16.32 mmol) were
dissolved in acetonitrile (60.00 mL). The mixture was heated between 50–60 °C during 2 h 30 min.
The solution was poured onto ice and a black precipitate was formed which was purified by silica gel
column chromatography using DCM as mobile phase. The solvent was removed under reduced
pressure to yield compound 2. Yield: 20%. IR (cm−1): 3026, 2943, 2232. 1H-NMR (CDCl3) δ: 8.08 (ddd,
1H, H8, J8-7 = 8.4 Hz, J8-6 = 1.5 Hz, J8-5 = 0.5 Hz); 7.88 (ddd, 1H, H5, J5-6 = 8.3 Hz, J5-7 = 1.4 Hz,
J5-8 = 0.5 Hz); 7.71 (ddd, 1H, H6, J6-5 = 8.4 Hz, J6-7 = 6.9 Hz, J6-8 = 1.4 Hz); 7.60 (ddd, 1H, H7,
J7-8 = 8.4 Hz, J7-6 = 6.9 Hz, J7-5 = 1.5 Hz); 4.20 (s, 3H, OCH3) ppm. Anal. Calcd for C10H7N3O: C:
64.86%; H: 3.78%; N: 22.70%; Found: C: 65.23%; H: 3.61%; N: 22.70%.
3.1.4. General Procedure for Synthesis of N-(3-Methoxyquinoxalin-2-ylmethyl)-alkylamidea 3a–e
The previously obtained compound 2, THF and the corresponding anhydride in proportion 1:2 were
placed in a hydrogenation flask. A spatula of Raney-Nickel was added and the mixture was
hydrogenated during 7 h at 50 °C and 50 psi. Next, DCM was added and the mixture was decanted off.
Next, the mixture was quenched with water, dried over Na2SO4 and filtered. The solvent was removed
under reduced pressure to obtain brown oil. Finally, n-hexane was added to the oil and a white solid
precipitated, which was filtered and washed with diethyl ether and water. If necessary, it was purified
by silica gel column chromatography using DCM/MeOH as mobile phase. The solvent was removed
under reduced pressure and the desired amides 3a–e were thus obtained.
N-(3-Methoxyquinoxalin-2-ylmethyl)-acetamide (3a). Yield: 7%. IR (cm−1): 3314, 3084, 2937, 1648.
1H-NMR (CDCl3) δ: 8.11 (dd, 1H, H8, J8-7 = 8.1 Hz, J8-6 = 1.1 Hz); 7.91 (ddd, 1H, H5, J5-6 = 8.3 Hz,

Molecules 2012, 17
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J5-7 = 1.3 Hz); 7.71 (ddd, 1H, H6, J6-5 = 8.3 Hz, J6-7 = 7.1 Hz, J6-8 = 1.3 Hz); 7.62 (ddd, 1H, H7,
J7-8 = 8.3 Hz, J7-6 = 7.1 Hz, J7-5 = 1.4 Hz); 4.76 (d, 2H, CH2, JCH2-NH = 4.3 Hz); 4.17 (s, 3H, OCH3);
1
2.21 (s, 3H, CH3) ppm. Anal. Calcd for C12H13N3O2 · /4 H2O: C: 61.14%; H: 5.73%; N: 17.83%;
Found: C: 61.26%; H: 5.47%; N: 17.65%. M.p.: 231–235 °C.
N-(3-Methoxyquinoxalin-2-ylmethyl)-propionamide (3b). Yield: 27%. IR (cm−1): 3308, 3078, 2969,
2943, 1638.1H-NMR (DMSO-d6) δ: 8.27 (t, 1H, NH, JNH-CH2 = 5.3 Hz); 7.96 (dd, 1H, H8, J8-7 = 8.2 Hz,
J8-6 = 1.2 Hz); 7.85 (dd, 1H, H5, J5-6 = 8.3 Hz, J5-7 = 1.0 Hz); 7.72 (ddd, 1H, H6, J6-5 = 8.3 Hz,
J6-7 = 7.0 Hz, J6-8 = 1.5 Hz); 7.63 (ddd, 1H, H7, J7-8 = 8.3 Hz, J7-6 = 7.0 Hz, J7-5 = 1.5 Hz); 4.53 (d, 2H,
CH2NH, JCH2-NH = 5.6 Hz); 4.07 (s, 3H, OCH3); 2.23 (q, 2H, CH2CH3, JCH2-CH3 = 7.6 Hz); 1.06 (t, 3H,
1
CH2CH3, JCH3-CH2 = 7.6 Hz) ppm. Anal. Calcd for C13H15N3O2 · /10 H2O: C: 63.21%; H: 6.16%; N:
17.02%; Found: C: 63.44%; H: 6.08%; N: 16.65%. M.p.: 164 °C.
N-(3-Methoxyquinoxalin-2-ylmethyl)-butyramide (3c). Yield: 9%. IR (cm−1): 3309, 3065, 2951, 1641.
1H-NMR (DMSO-d6) δ: 8.30 (bs, 1H, NH); 7.93 (d, 1H, H8, J8-7 = 7.9 Hz); 7.84 (d, 1H, H5, J5-6 = 8.2 Hz);
7.70 (dd, 1H, H6, J6-5 = 8.2 Hz, J6-7 = 6.7 Hz); 7.61 (dd, 1H, H7, J7-8 = 8.1 Hz, J7-6 = 6.9 Hz); 4.52 (d,
2H, CH2NH, JCH2-NH = 5.4 Hz); 4.07 (s, 3H, OCH3); 2.18 (t, 2H, CH2CH2CH3, JCH2-CH2 = 7.2 Hz); 1.56
(dd, 2H, CH2CH2CH3, JCH2-CH2 = 7.2 Hz, JCH2-CH3 = 7.2 Hz); 0.91 (t, 3H, CH3, JCH3-CH2 = 7.3 Hz) ppm.
Anal. Calcd for C14H17N3O2·1/8 H2O: C: 64.31%; H: 6.60%; N: 16.08%; Found: C: 64.34%; H: 6.65%;
N: 15.93%. M.p.: 259 °C.
N-(3-Methoxyquinoxalin-2-ylmethyl)-isobutyramide (3d). Yield: 28%. IR (cm−1): 3312, 3065, 2968,
2937, 1644. 1H-NMR (DMSO-d6) δ: 8.23 (t, 1H, NH, JNH-CH2 = 5.6 Hz); 7.93 (dd, 1H, H8, J8-7 = 8.2 Hz,
J8-6 = 1.0 Hz); 7.85 (dd, 1H, H5, J5-6 = 8.2 Hz, J5-7 = 0.9 Hz); 7.72 (ddd, 1H, H6, J6-5 = 8.3 Hz, J6-7 = 7.1 Hz,
J6-8 = 1.5 Hz); 7.62 (ddd, 1H, H7, J7-8 = 8.4 Hz, J7-6 = 7.1 Hz, J7-5 = 1.5 Hz); 4.51 (d, 2H, CH2NH,
JCH2-NH = 5.6 Hz); 4.07 (s, 3H, OCH3); 2.55 (q, 1H, CH(CH3)2, JCH-(CH3)2 = 6.8 Hz); 1.08 (d, 6H,
CH(CH3)2, J(CH3)2-CH = 6.8 Hz) ppm. Anal. Calcd for C14H17N3O2: C: 64.86%; H: 6.56%; N: 16.22%;
Found: C: 64.55%; H: 6.79%; N: 15.91%. M.p.: 142–144 °C.
N-(3-Methoxy-quinoxalin-2-ylmethyl)-benzamide (3e). Yield: 38%. IR (cm−1): 3391, 3071, 3026, 2943,
1
2904, 1659. H-NMR (DMSO-d6) δ: 9.00 (t, 1H, NH, JNH-CH2 = 5.4 Hz); 7.94–7.92 (m, 3H,
H8+H2'+H6'); 7.86 (d, 1H, H5, J5-6 = 8.3 Hz); 7.71 (dd, 1H, H6, J6-5 = 8.1 Hz, J6-7 = 7.2 Hz); 7.63–7.47 (m,
4H, H7+H3'+H4'+H5'); 4.74 (d, 2H, CH2, JCH2-NH = 5.4 Hz); 4.10 (s, 3H, OCH3) ppm. Anal. Calcd for
1
C17H15N3O2 · /8 H2O: C: 69.09%; H: 5.17%; N: 14.23%; Found: C: 69.13%; H: 5.14%; N: 13.99%.
M.p.: 137 °C.
3.1.5. General Procedure for Synthesis of 1-Alkyl-3-(3-methoxyquinoxalin-2-ylmethyl)ureas 3f–i
Compound 2 (1 eq.), THF and the corresponding isocyanate (2 eq.) were placed in a hydrogenation
tube. A spatula of Raney-Nickel was added and the reaction was carried out during 7 h at room
temperature and 50 psi. Next, DCM was added and the mixture was decanted off with the aim of
leaving behind the catalyst. Next, the mixture was quenched with water, dried over Na2SO4 and
filtered. The solvent was removed under reduced pressure and the orange oil obtained was purified by

Molecules 2012, 17
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flash chromatography using DCM/MeOH as mobile phase. The obtained solid was dissolved in
water/diethyl ether with vigorous stirring and then quenched. The organic layer was dried over Na2SO4
and filtered. The solvent was removed under reduced pressure and the desired urea derivatives 3f–i,
were obtained.
Ethyl-3-(3-methoxyquinoxalin-2-ylmethyl)urea (3f). Yield: 3%. IR (cm−1): 3334, 3061, 2978, 2939,
1
1629. H-NMR (DMSO-d6) δ: 7.96 (dd, 1H, H8, J8-7 = 8.2 Hz, J8-6 = 1.2 Hz); 7.85 (dd, 1H, H5,
J
5-6 = 8.2 Hz, J5-7 = 1.2 Hz); 7.71 (ddd, 1H, H6, J6-5 = 8.4 Hz, J6-7 = 7.0 Hz, J6-8 = 1.4 Hz); 7.63 (ddd,
1H, H7, J7-8 = 8.4 Hz, J7-6 = 7.1 Hz, J7-5 = 1.4 Hz); 6.41 (t, 1H, ArCH2NH, JNH-CH2 = 5.0 Hz); 6.33 (t,
1H, NHCH2CH3, JNH-CH2 = 5.3 Hz); 4.50 (d, 2H, ArCH2NH, JCH2-NH = 5.4 Hz); 4.07 (s, 3H, OCH3);
3.05 (dq, 2H, CH2CH3, JCH2-CH3 = 7.2 Hz, JCH2-NH = 5.8 Hz); 1.02 (dt, 3H, CH3, JCH3-CH2 = 7.2 Hz,
JCH3-NH = 1.3 Hz) ppm. Anal. Calcd for C13H16N4O2: C: 60.00%; H: 6.15%; N: 21.54%; Found: C:
59.91%; H: 6.12%; N: 21.14%. M.p.: 160.5–160.8 °C.
1-(3-Methoxyquinoxalin-2-ylmethyl)-3-propylurea (3g). Yield: 9%. IR (cm−1): 3330, 3061, 2960, 1633.
1H-NMR (DMSO-d6) δ: 7.95 (d, 1H, H8, J8-7 = 8.1 Hz); 7.85 (d, 1H, H5, J5-6 = 8.2 Hz); 7.71 (dd, 1H, H6,
J6-5 = 8.0 Hz, J6-7 = 7.3 Hz); 7.63 (dd, 1H, H7, J7-8 = 8.0 Hz, J7-6 = 7.1 Hz); 6.41 (t, 1H, ArCH2NH,
JNH-CH2 = 5.5 Hz); 6.37 (t, 1H, NHCH2CH2CH3, JNH-CH2 = 6.0 Hz); 4.50 (d, 2H, ArCH2NH,
JCH2-NH = 5.2 Hz); 4.07 (s, 3H, OCH3); 2.98 (dd, 2H, NHCH2CH2CH3, JCH2-NH = 6.4 Hz,
JCH2-CH2 = 6.7 Hz); 1.40 (tq, 2H, NHCH2CH2CH3, JCH2-CH2 = 6.8 Hz, JCH2-CH3 = 7.1 Hz); 0.86 (t, 3H,
CH3, JCH3-CH2 = 7.4 Hz) ppm. Anal. Calcd for C14H18N4O2: C: 61.31%; H: 6.57%; N: 20.44%; Found:
C: 60.99%; H: 6.45%; N: 20.31%. M.p.: 115.3–116 °C.
1-Isopropyl-3-(3-methoxyquinoxalin-2-ylmethyl)urea (3h). Yield: 23%. IR (cm−1): 3363, 3283, 3100,
1
2971, 1620. H-NMR (DMSO-d6) δ: 7.95 (dd, 1H, H8, J8-7 = 8.1 Hz, J8-6 = 1.1 Hz); 7.85 (dd, 1H, H5,
J5-6 = 8.2 Hz, J5-7 = 0.9 Hz); 7.71 (ddd, 1H, H6, J6-5 = 8.2 Hz, J6-7 = 7.7 Hz, J6-8 = 1.2 Hz); 7.63 (ddd,
1H, H7, J7-8 = 8.0 Hz, J7-6 = 7.4 Hz, J7-5 = 1.1 Hz); 6.34 (t, 1H, ArCH2NH, JNH-CH2 = 5.1 Hz); 6.26 (d, 1H,
NHCH(CH3)2, JNH-CH = 7.6 Hz); 4.49 (d, 2H, ArCH2NH, JCH2-NH = 5.3 Hz); 4.07 (s, 3H, OCH3);
3.71–3.67 (m, 1H, CH(CH3)2); 1.06 (d, 6H, CH-(CH3)2, J(CH3)2-CH = 6.5 Hz) ppm. Anal. Calcd for
C14H18N4O2: C: 61.31%; H: 6.57%; N: 20.44%; Found: C: 61.33%; H: 6.58%; N: 20.43%. M.p.: 171.2 °C.
1-(3-Methoxyquinoxalin-2-ylmethyl)-3-phenyl-urea (3i). Yield: 4%. IR (cm−1): 3310, 3177, 3023, 2952,
1
2907, 1640. H-NMR (DMSO-d6) δ: 9.01 (s, 1H, CONH-Ph); 7.99 (dd, 1H, H8, J8-7 = 8.2 Hz,
J8-6 = 1.2 Hz); 7.87 (dd, 1H, H5, J5-6 = 8.3 Hz, J5-7 = 1.1 Hz); 7.73 (ddd, 1H, H6, J6-5 = 8.4 Hz, J6-7 = 7.1 Hz,
J6-8 = 1.5 Hz); 7.64 (ddd, 1H, H7, J7-8 = 8.4 Hz, J7-6 = 7.1 Hz, J7-5 = 1.5 Hz); 7.43 (dd, 2H, H2'+H6',
J2'-3' = J6'-5' = 8.6 Hz, J2'-4' = J6'-4' = 1.1 Hz); 7.24 (dd, 2H, H3'+H5', J3'-2' = J5'-6' = 8.4 Hz, J3'-4' = J5'-4'
=
7.5 Hz); 6.91 (tt, 1H, H4', J4'-3' = J4'-5' = 7.5 Hz, J4'-2' = J4'-6' = 1.1Hz); 6.83 (t, 1H, ArCH2NH,
JNH-CH2 = 5.2 Hz); 4.61 (d, 2H, ArCH2NH, JCH2-NH = 5.2 Hz); 4.10 (s, 3H, OCH3) ppm. Anal. Calcd
for C17H16N4O2 · 1/8 H2O: C: 65.75%; H: 5.24%; N: 18.05%; Found: C: 65.76%; H: 5.32%; N: 18.12%.
M.p.: 201–201.5 °C.

Molecules 2012, 17
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3.1.6. Synthesis of 2,3-Dimethoxy-6-nitroquinoxaline (4)
2,3-Dichloro-6-nitroquinoxaline (5.12 g, 20.35 mmol) and THF (100.00 mL) were placed in a round
bottomed flask. The mixture was cooled to 0 °C. NaOMe (9.6 mL, 41.00 mmol) prepared
in situ by means of a reaction between Na (1.20 g) and MeOH (12.00 mL) was added dropwise to the
other suspension. The color of the mixture changed from white-yellow to orange. The mixture was
stirred during 30 min at 0 °C and during 1 h at room temperature. Then, DCM was added and the
mixture was quenched with brine. The DCM solution was dried over Na2SO4 and filtered. The solvent
was removed under reduced pressure in order to obtain compound 4 as a yellow solid. Yield: 52%. IR
1
(cm−1): 3116, 3014, 2956. H-NMR (DMSO-d6) δ: 8.50 (d, 1H, H5, J5-7 = 2.6 Hz); 8.30 (dd, 1H, H7,
J7-8 = 9.0 Hz, J7-5 = 2.6 Hz); 7.93 (d, 1H, H8, J8-7 = 9.0 Hz); 4.11 (s, 3H, OCH3-C2); 4.10 (s, 3H, OCH3-C3)
ppm. Anal. Calcd for C10H9N3O4: C: 51.06%; H: 3.83%; N: 17.87%; Found: C: 50.99%; H: 3.79%; N:
17.40%.
3.1.7. Synthesis of 6-Amino-2,3-dimethoxyquinoxaline (5)
Compound 4 (2.50 g, 10.64 mmol) was disolved in MeOH (150.00 mL) and the mixture was cooled
to 0 °C. Then a tip of spatula of palladium on carbon and a great excess of hydrazine hydroxide were
added. After stirring 30 min at 0 °C the mixture was stirred at room temperature for 15 h. The mixture
was filtered to eliminate the catalyst and the solvent was removed under reduced pressure. Then, DCM
was added and the mixture was quenched with brine. The DCM solution was dried over Na2SO4 and
filtered. The solvent was removed under reduced pressure yielding to compound 5. Yield: 96%. IR
(cm−1): 3442, 3347, 3219, 2937. 1H-NMR (DMSO-d6) δ: 7.42 (d, 1H, H8, J8-7 = 8.6 Hz); 6.87 (dd, 1H,
H7, J7-8 = 8.6 Hz, J7-5 = 2.4 Hz); 6.78 (d, 1H, H5, J5-7 = 2.5 Hz); 5.43 (s, 2H, NH2); 3.97 (s, 3H, OCH3-C2);
3.94 (s, 3H, OCH3-C3) ppm. Anal. Calcd for C10H11N3O2: C: 58.54%; H: 5.37%; N: 20.49%; Found: C:
58.08%; H: 5.37%; N: 20.41%.
3.1.8. General Procedure for Synthesis of N-(2,3-Dimethoxyquinoxalin-6-yl)alkylamides 6a–e
Compound 5 (1 eq.) was placed in suspension in THF. Next, the corresponding anhydride (3 eq.)
and a catalytic amount of triethylamine were added dropwise. The reaction was carried out during 1 h
30 min at room temperature. THF was removed under reduced pressure. The obtained residue was
treated with n-hexane and the obtained solid was then filtered. The solid was washed with water to
yield the desired compounds 6a–e.
N-(2,3-Dimethoxyquinoxalin-6-yl)acetamide (6a). Yield: 13%. IR (cm−1): 3276, 3116, 2943, 1664.
1H-NMR (DMSO-d6) δ: 10.19 (s, 1H, NH); 8.21 (d, 1H, H5, J5-7 = 2.2 Hz); 7.67 (d, 1H, H8,
J8-7 = 8.8 Hz); 7.57 (dd, 1H, H7, J7-8 = 8.9 Hz, J7-5 = 2.2 Hz); 4.03 (s, 3H, OCH3-C2); 4.01 (s, 3H,
1
OCH3-C3); 2.10 (s, 3H, CH3) ppm. Anal. Calcd for C12H13N3O3 · /2 H2O: C: 56.25%; H: 5.47%; N:
16.41%; Found: C: 56.23%; H: 5.42%; N: 16.04%. M.p.: 290 °C.
N-(2,3-Dimethoxyquinoxalin-6-yl)propionamide (6b). Yield: 68%. IR (cm−1): 3284, 3175, 3105, 2987,
2943, 1663. 1H-NMR (DMSO-d6) δ: 10.11 (s, 1H, NH); 8.21 (s, 1H, H5); 7.67 (dd, 1H, H8, J8-7 = 8.8 Hz,
J8-5 = 1.2 Hz); 7.60 (ddd, 1H, H7, J7-8 = 8.9 Hz, J7-5 = 2.2 Hz, JH7-NH = 1.5 Hz); 4.03 (d, 3H, OCH3-C2
,

Molecules 2012, 17
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JOCH3-OCH3 = 1.4 Hz); 4.01 (d, 3H, OCH3-C3, JOCH3-OCH3 = 1.4 Hz); 2.38 (dq, 2H, CH2, JCH2-CH3 = 7.5 Hz,
JCH2-NH = 1.3 Hz); 2.12 (dt, 3H, CH3, JCH3-CH2 = 7.5 Hz, JCH3-NH = 1.4 Hz) ppm. Anal. Calcd for
1
C13H15N3O3 · /2 H2O: C: 57.78%; H: 5.93%; N: 15.56%; Found: C: 58.15%; H: 5.92%; N: 15.61%.
M.p.: 182–183 °C.
N-(2,3-Dimethoxyquinoxalin-6-yl)butyramide (6c). Yield: 26%. IR (cm−1): 3289, 2949, 1656.
1H-NMR (DMSO-d6) δ: 10.11 (s, 1H, NH); 8.21 (s, 1H, H5); 7.66 (dd, 1H, H7, J7-8 = 8.4 Hz, J7-5 = 2.9 Hz);
7.59 (d, 1H, H8, J8-7 = 8.7 Hz); 4.03 (d, 3H, OCH3-C2, JOCH3-OCH3 = 2.6 Hz); 4.01 (d, 3H, OCH3-C3
,
JOCH3-OCH3 = 2.6 Hz); 2.36–2.32 (m, 2H, COCH2CH2CH3); 1.66–1.62 (m, 2H, COCH2CH2CH3);
0.96–0.92 (m, 3H, CH3) ppm. Anal. Calcd for C14H17N3O3: C: 61.09%; H: 6.18%; N: 15.27%; Found:
C: 60.78%; H: 6.32%; N: 15.59%. M.p.: 136–140 °C.
N-(2,3-Dimethoxyquinoxalin-6-yl)isobutyramide (6d). Yield: 72%. IR (cm−1): 3253, 3026, 2969, 1653.
1H-NMR (DMSO-d6) δ: 10.07 (s, 1H, NH); 8.21 (d, 1H, H5, J5-7 = 2.2 Hz); 7.67 (d, 1H, H8, J8-7 = 8.9 Hz);
7.62 (dd, 1H, H7, J7-8 = 8.9 Hz, J7-5 = 2.3 Hz); 4.03 (d, 3H, OCH3-C2, JOCH3-OCH3 = 1.2 Hz); 4.01 (d, 3H,
OCH3-C3, JOCH3-OCH3 = 1.3 Hz); 2.64 (dq, 1H, CH(CH3)2, JCH-(CH3)2 = 6.7 Hz); 1.14 (d, 6H, CH(CH3)2,
1
J(CH3)2-CH = 6.8 Hz) ppm. Anal. Calcd for C14H17N3O3 · /8 H2O: C: 60.60%; H: 6.22%; N: 15.15%;
Found: C: 60.60%; H: 6.07%; N: 15.12%. M.p.: 201–203 °C.
N-(2,3-Dimethoxyquinoxalin-6-yl)benzamide (6e). Yield: 42%. IR (cm−1): 3285, 3061, 2984, 2939,
1
1651. H-NMR (DMSO-d6) δ: 10.49 (s, 1H, NH); 8.37 (d, 1H, H5, J5-7 = 2.2 Hz); 8.00–7.98 (m, 2H,
H2'+H6'); 7.87 (dd, 1H, H7, J7-8 = 8.9 Hz, J7-5 = 2.4 Hz); 7.73 (d, 1H, H8, J8-7 = 8.8 Hz); 7.65–7.53 (m, 3H,
H3'+H4'+H5'); 4.05 (s, 3H, OCH3-C2, JOCH3-OCH3 = 0.5 Hz); 4.03 (s, 3H, OCH3-C3, JOCH3-OCH3 = 0.5 Hz)
ppm. Anal. Calcd for C17H15N3O3 · 1/8 H2O: C: 65.54%; H: 4.90%; N: 13.49%; Found: C: 65.52%; H:
4.99%; N: 13.40%. M.p.: 165–170 °C.
3.1.9. General Procedure for Synthesis of 1-(2,3-Dimethoxyquinoxaline-6-yl)-3-alkylureas 6f–i
Compound 5 (1 eq.) was dissolved in dry DCM (15 mL) and placed under N2 atmosphere. The
mixture was cooled to 0 °C and the corresponding isocyanate (2 eq.) was added. The reaction was
carried out during 30 min at 0 °C and 3 h at room temperature. Next, the obtained precipitate was
filtered to achieve the corresponding compounds 6f–i.
1-(2,3-Dimethoxyquinoxalin-6-yl)-3-ethylurea (6f). Yield: 24%. IR (cm−1): 3303, 3103, 2981, 1633.
1H-NMR (DMSO-d6) δ: 8.69 (s, 1H, CONHAr); 7.97 (d, 1H, H5, J5-7 = 2.2 Hz); 7.60 (d, 1H, H8,
J8-7 = 8.8 Hz); 7.41 (dd, 1H, H7, J7-8 = 8.8 Hz, J7-5 = 2.5 Hz); 6.20 (t, 1H, CONHCH2, JNH-CH2 = 5.4 Hz);
4.02 (d, 3H, OCH3-C2, JOCH3-OCH3 = 0.9 Hz); 3.99 (s, 3H, OCH3-C3, JOCH3-OCH3 = 0.9 Hz); 3.14 (dq, 2H,
CH2, JCH2-NH = 5.9 Hz, JCH2-CH3 = 7.2 Hz); 1.08 (t, 3H, CH3, JCH3-CH2 = 7.3 Hz) ppm. Anal. Calcd for
1
C13H16N4O3 · /4 H2O C: 55.61%; H: 5.88%; N: 19.96%; Found: C: 55.57%; H: 5.66%; N: 19.94%.
M.p.: >300 °C.
1-(2,3-Dimethoxyquinoxalin-6-yl)-3-propylurea (6g). Yield: 29%. IR (cm−1): 3289, 2949, 1656.
1H-NMR (DMSO-d6) δ: 8.67 (s, 1H, CONHAr); 7.96 (dd, 1H, H5, J5-7 = 2.2 Hz, J5-8 = 0.6 Hz); 7.60 (dd,
1H, H8, J8-7 = 8.8 Hz, J8-5 = 0.6 Hz); 7.40 (ddd, 1H, H7, J7-8 = 8.9 Hz, J7-5 = 2.4 Hz, JH7-NH = 0.8 Hz); 6.24

Molecules 2012, 17
7750
(t, 1H, CONHCH2, JNH-CH2 = 5.8 Hz); 4.02 (s, 3H, OCH3-C2, JOCH3-OCH3 = 0.9 Hz); 3.99 (s, 3H, OCH3-C3
,
JOCH3-OCH3 = 0.9 Hz); 3.08 (dd, 2H, NHCH2CH2CH3, JCH2-CH2 = 6.4 Hz, JCH2-NH = 6.3 Hz); 1.47 (tq, 2H,
NHCH2CH2CH3, JCH2-CH3 = 7.3 Hz, JCH2-CH2 = 7.2 Hz); 0.89 (t, 3H, CH3, JCH3-CH2 = 7.4 Hz) ppm.
Anal. Calcd for C14H18N4O3: C: 57.93%; H: 6.21%; N: 19.31%; Found: C: 57.78%; H: 6.11%; N:
19.31%. M.p.: 122–123 °C.
1-(2,3-Dimethoxyquinoxalin-6-yl)-3-isopropylurea (6h). Yield: 10%. IR (cm−1): 3334, 3297, 3097, 2962,
1
1637. H-NMR (DMSO-d6) δ: 8.55 (s, 1H, CONHAr); 7.95 (dd, 1H, H5, J5-7 = 2.3 Hz, J5-8 = 1.2 Hz);
7.60 (dd, 1H, H8, J8-7 = 8.9 Hz, J8-5 = 1.0 Hz); 7.39 (ddd, 1H, H7, J7-8 = 8.9 Hz, J7-5 = 2.4 Hz,
JH7-NH = 1.1 Hz); 6.10 (d, 1H, CONHCH(CH3)2, JNH-CH = 7.7 Hz); 4.02 (s, 3H, OCH3-C2
,
J
OCH3-OCH3 = 1.3 Hz); 3.99 (s, 3H, OCH3-C3, JOCH3-OCH3 = 1.3 Hz); 3.84–3.75 (m, 1H, CH); 1.12 (dd, 6H,
1
CH(CH3)2, J(CH3)2-CH = 6.5 Hz, J(CH3)2-NH = 1.2 Hz) ppm. Anal. Calcd for C14H18N4O3 · /4 H2O: C:
57.05%; H: 6.28%; N: 19.02%; Found: C: 57.12%; H: 6.24%; N: 18.88%. M.p.: 250 °C.
1-(2,3-Dimethoxyquinoxalin-6-yl)-3-phenylurea (6i). Yield: 55%. IR (cm−1): 3275; 3068, 3030, 2997,
2939; 1639. 1H-NMR (DMSO-d6) δ: 8.94 (s, 1H, NH); 8.77 (s, 1H, NH); 8.02 (d, 1H, H5, J5-7 = 2.1 Hz);
7.66 (d, 1H, H8, J8-7 = 8.8 Hz); 7.54–7.43 (m, 3H, H7+H2'+H6'); 7.30 (dd, 2H, H3'+H5', J3'-2' = J5'-6'
=
8.1 Hz, J3'-4' = J5'-4' = 7.7 Hz); 6.99 (t, 1H, H4’, J4'-3' = J4'-5' = 7.4 Hz); 4.03 (s, 3H, OCH3-C2); 4.00
(s, 3H, OCH3-C3) ppm. Anal. Calcd for C17H16N4O3: C: 62.96%; H: 4.94%; N: 17.28%; Found: C:
63.30%; H: 5.23%; N: 17.56%. M.p.: 260 °C.
3.1.10. Synthesis of 3-Amino-7-methoxy-1,4-di-N-oxidequinoxaline-2-carbonitrile (7)
5-Methoxybenzofuroxan (2.50 g, 15.06 mmol), malononitrile (1.80 mL, 18.00 mmol) and
N,N-DMF (5.00 mL) were placed in a flask. Next, the mixture was cooled to 0 °C and triethylamine
(1.50 mL, 10.77 mmol) was added dropwise. The reaction was carried out during 6 h at room
temperature. Diethyl ether and methanol were added and the mixture was filtered. The obtained
compound was used without further purification. This compound has already been described by
Monge et al. [24].
3.1.11. Synthesis of 7-Methoxy-1,4-di-N-oxidequinoxaline-2-carbonitrile (8)
Compound 7 (1.60 g, 6.88 mmol) and N,N-DMF (30.00 mL) were placed in a flask under a N2
atmosphere. Next, the mixture was heated to 65 °C and tert-butyl nitrite (2.80 mL, 23.57 mmol) was
added dropwise. Effervescence could be observed. After stirring 10 min at 65 °C, another portion of
tert-butyl nitrite (2.10 mL, 17.68 mmol) was added and the mixture was stirred for 1 h 30 min. The
solvent was removed under reduced pressure. Finally, the solid was purified by traditional column
chromatography using DCM as mobile phase to yield compound 8. Yield: 31%. IR (cm−1): 3090, 2975,
2238, 1367. 1H-NMR (DMSO-d6) δ: 9.19 (s, 1H, H3); 8.38 (d, 1H, H5, J5-6 = 9.4 Hz); 7.74 (d, 1H, H8,
J
8-6 = 2.7 Hz); 7.69 (dd, 1H, H6, J6-5 = 9.4 Hz, J6-8 = 2.8 Hz); 4.02 (s, 3H, OCH3) ppm. Anal. Calcd for
C10H7N3O3 · 1/8 H2O: C: 54.73%; H: 3.31%; N: 19.16%; Found: C: 54.86%; H: 3.30%; N: 18.85%.

Molecules 2012, 17
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3.1.12. Synthesis of 7-Methoxyquinoxaline-2-carbonitrile (9)
Compound 8 (0.91 g, 0.97 mmol) and AcOEt/MeOH (1:1, 40.00 mL) were placed in a flask and
heated to 65 °C. Next, a solution of sodium dithionite in water was prepared in situ (1.01 g of Na2O4S2
in 20.00 mL of water) and added over the mixture. The reaction was carried out during 10 min at 65 °C.
Next, the mixture was filtered with the aim of removing the sodium dithionite, and the solvent was
removed under reduced pressure. Finally, the crude residue was purified by flash column
1
chromatography to yield compound 9. Yield: 39%. IR (cm−1): 3090, 3039, 2975, 2226. H-NMR
(DMSO-d6) δ: 9.22 (s, 1H, H3); 8.13 (d, 1H, H5, J5-6 = 9.3 Hz); 7.73 (dd, 1H, H6, J6-5 = 9.3 Hz,
J6-8 = 2.8 Hz); 7.58 (d, 1H, H8, J8-6 = 2.8 Hz); 4.00 (s, 3H, OCH3) ppm. Anal. Calcd for
C10H7N3O · 1/8 H2O: C: 64.09%; H: 3.87%; N: 22.43%; Found: C: 64.36%; H: 3.73%; N: 22.29%.
3.1.13. General Procedure for Synthesis of N-[(7-Methoxyquinoxalin-2-yl)methyl]alkylamides 10a–e
Compound 9 (1 eq.), THF (20 mL) and the corresponding anhydride (2 eq.) were placed in a
hydrogenation flask. A spatula of Raney-Nickel was added and the mixture was hydrogenated during
7 h at 50 °C and 50 psi. Next, DCM was added and the mixture was decanted off with the aim of
leaving off the catalyst. Next, the mixture was quenched with water, dried over Na2SO4 and filtered.
The solvent was removed under reduced pressure and the orange oil obtained was purified through
flash chromatography in order to obtain the amide derivatives 10a–e.
N-[(7-Methoxyquinoxalin-2-yl)methyl]acetamide (10a). Yield: 6%. IR (cm−1): 3289, 3068, 2965, 2920,
1
1648. H-NMR (DMSO-d6) δ: 8.71 (s, 1H, H3); 8.62 (t, 1H, NH, JNH-CH2 = 5.3 Hz); 7.97 (d, 1H, H5,
J5-6 = 9.1 Hz); 7.62 (dd, 1H, H6, J6-5 = 9.1 Hz, J6-8 = 2.8 Hz); 7.40 (d, 1H, H8, J8-6 = 2.6 Hz); 4.56 (d,
2H, CH2NH, JCH2-NH = 5.8 Hz); 3.95 (s, 3H, OCH3); 1.93 (s, 3H, CH3) ppm. Anal. Calcd for
1
C12H13N3O2 · /8 H2O: C: 61.74%; H: 5.68%; N: 18.01%; Found: C: 61.99%; H: 5.69%; N: 17.67%.
M.p.: 123.9–126.9 °C.
N-[(7-Methoxyquinoxalin-2-yl)methyl]propionamide (10b). Yield: 24%. IR (cm−1): 3280, 3068, 2971,
2913, 1643. 1H-NMR (DMSO-d6) δ: 8.70 (s, 1H, H3); 8.54 (t, 1H, NH, JCH2-NH = 5.6 Hz); 7.97 (d, 1H, H5,
J5-6 = 9.1 Hz); 7.46 (dd, 1H, H6, J6-5 = 9.1 Hz, J6-8 = 2.8 Hz); 7.40 (d, 1H, H8, J8-6 = 2.8 Hz); 4.56
(d, 2H, CH2NH, JCH2-NH = 5.8 Hz); 3.95 (s, 1H, OCH3); 2.21 (q, 2H, CH2CH3, JCH2-CH3 = 7.6 Hz); 1.04
(t, 3H, CH3, JCH3-CH2 = 7.6 Hz) ppm. Anal. Calcd for C13H15N3O2 · 1/4 H2O: C: 62.53%; H: 6.21%; N:
16.83%; Found: C: 62.92%; H: 6.23%; N: 16.85%. M.p.: 143–145 °C.
N-[(7-Methoxyquinoxalin-2-yl)methyl]butyramide (10c). Yield: 24%. IR (cm−1): 3282, 3081, 2965,
1643. 1H-NMR (DMSO-d6) δ: 8.69 (s, 1H, H3); 8.58 (s, 1H, NH); 7.96 (d, 1H, H5, J5-6 = 8.7 Hz); 7.45
(d, 1H, H6, J6-5 = 8.4 Hz); 7.38 (s, 1H, H8); 4.56 (d, 2H, CH2NH, JCH2-NH = 4.8 Hz); 3.94 (s, 3H, OCH3);
2.17 (t, 2H CH2CH2CH3, JCH2-CH2 = 6.5 Hz); 1.60–1.50 (m, 2H, CH2CH2CH3); 0.88 (t, 3H, CH3,
1
JCH3-CH2 = 6.6 Hz) ppm. Anal. Calcd for C14H17N3O2 · /2 H2O: C: 62.69%; H: 6.72%; N: 15.67%;
Found: C: 62.49%; H: 6.55%; N: 15.53%. M.p.: 128–129 °C.
N-[(7-Methoxyquinoxalin-2-yl)methyl]isobutyramide (10d). Yield: 62%. IR (cm−1): 3280, 3074, 2971,
2926, 1648. 1H-NMR (DMSO-d6) δ: 8.67 (s, 1H, H3); 8.53 (t, 1H, NH, JNH-CH2 = 5.3 Hz); 7.97 (d, 1H, H5,

Molecules 2012, 17
7752
J5-6 = 9.2 Hz); 7.46 (dd, 1H, H6, J6-5 = 9.2 Hz, J6-8 = 2.8 Hz); 7.39 (d, 1H, H8, J8-6 = 2.7 Hz); 4.55 (d,
2H, CH2NH, JCH2-NH = 5.8 Hz); 3.95 (s, 3H, OCH3); 2.52–2.48 (m, 1H, CH); 1.07 (d, 6H, CH(CH3)2,
J(CH3)2-CH = 6.9 Hz) ppm. Anal. Calcd for C14H17N3O2: C: 64.86%; H: 6.56%; N: 16.22%; Found: C:
64.76%; H: 6.67%; N: 16.27%. M.p.: 133–133.5 °C.
N-[(7-Methoxyquinoxalin-2-yl)methyl]benzamide (10e). Yield: 29%. IR (cm−1): 3299, 3061, 3010,
1
2965, 2920, 1640. H-NMR (DMSO-d6) δ: 9.28 (t, 1H, NH, JNH-CH2 = 5.4 Hz); 8.79 (s, 1H, H3); 7.98
(d, 1H, H5, J5-6 = 9.1 Hz); 7.94 (dd, 2H, H2'+H6', J2'-3' = J6'-5' = 7.1 Hz, J2'-4' = J6'-4' = 1.2 Hz); 7.59–7.44
(m, 4H, H6+H3'+H4'+H5'); 7.41 (d, 1H, H8, J8-6 = 2.7 Hz); 4.79 (d, 2H, CH2NH, JCH2-NH = 5.7 Hz);
1
3.94 (s, 3H, OCH3) ppm. Anal. Calcd for C17H15N3O2 · /4 H2O: C: 68.57%; H: 5.21%; N: 14.12%;
Found: C: 68.66%; H: 5.35%; N: 13.95%. M.p.: 142–143.9 °C.
3.2. Pharmacology
3.2.1. Reagents and Chemicals
2-[125I]Iodomelatonin (2200 Ci/mmol) was purchased from NEN (Boston, MA, USA). Other drugs
and chemicals were purchased from Sigma-Aldrich (Saint Quentin, France).
3.2.2. Cell Culture
HEK (provided by A.D. Strosberg, Paris, France) and CHO cell lines stably expressing the human
melatonin MT1 or MT2 receptors were grown in DMEM medium supplemented with 10% fetal calf
serum, 2 mM glutamine, 100 IU/mL penicillin and 100 µg/mL streptomycin. Grown at confluence at
37 °C (95% O2/5% CO2), they were harvested in PBS containing EDTA 2 mM and centrifuged at
1,000 g for 5 min (4 °C). The resulting pellet was suspended in Tris 5 mM (pH 7.5), containing EDTA
2 mM and homogenized using a Kinematica polytron. The homogenate was then centrifuged (95,000 g,
30 min, 4 °C) and the resulting pellet suspended in 75 mM Tris (pH 7.5), 12.5 mM MgCl2 and 2 mM
EDTA. Aliquots of membrane preparations were stored at −80 °C until use.
3.2.3. Binding Assays
2-[125I]Iodomelatonin binding assay conditions are essentially as previously described [50]. Briefly,
binding is initiated by the addition of membrane preparations from stable transfected HEK or CHO
cells diluted in binding buffer (50 mM Tris-HCl buffer, pH 7.4, containing 5 mM MgCl2) to
2-[125I]Iodomelatonin (25 or 200 pM for MT1 and MT2 receptors, respectively, expressed in HEK cells
or 20 pM for MT1 and MT2 receptors expressed in CHO cells) and the tested drug. Non-specific
binding was defined in the presence of 1 µM melatonin. After 120 min of incubation at 37 °C, the
reaction was stopped by rapid filtration through GF/B filters presoaked in 0.5% (v/v) polyethylenimine.
Filters were washed three times with 1 mL of ice-cold 50 mM Tris-HCl buffer, pH 7.4. Data from
dose-response curves (seven concentrations in duplicate) were analyzed using the program PRISM
(Graph Pad Software Inc., San Diego, CA, USA) to yield IC50 (inhibitory concentration 50). Results
were expressed as Ki using the Cheng and Prusoff equation: Ki = IC50/[1 + (L/KD)], where [L] is the

Molecules 2012, 17
7753
concentration of radioligand used in the assay and KD, the dissociation constant of the radioligand
characterizing the membrane preparation.
The equilibrium binding constant (KD) for the 2-iodomelatonin at hMT1 and hMT2 receptors
expressed in CHO and HEK cell membranes were 21 ± 3 pM and 107 ± 11 pM (n = 4), for CHO-hMT1
and CHO-hMT2 membranes, respectively and 10 ± 1 pM and 83 ± 19 pM (n = 4) for HEK-hMT1 and
HEK-hMT2, respectively.
[35S]GTPγS binding assay was performed according to published methodology [50]. Briefly,
membranes from transfected CHO cells expressing MT1 and MT2 receptor subtypes and compounds
were diluted in binding buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 3 μM GDP, 3 mM MgCl2, and
20 μg/mL saponin). Incubation was started by the addition of 0.2 nM [35S]GTPγS to membranes
(20 μg/mL) and drugs, and further followed for 1h at room temperature.
Usual levels of [35S]GTPγS binding (expressed in dpm) were for CHO-MT2 membranes: 2,000 for
basal activity, 8,000 in the presence of melatonin 1 μM and 180 in the presence of GTPγS 10 μM
which defined the non-specific binding. Data from the dose-response curves (seven concentrations in
duplicate) were analyzed by using the program PRISM (Graph Pad Software Inc., San Diego, CA,
USA) to yield EC50 (effective concentration 50%) and Emax (maximal effect) for agonists.
4. Conclusions
In conclusion, we were not able to assert whether the quinoxaline moiety can replace the melatonin
indole nucleus as a useful bioisostere in the search of new melatoninergic ligands. Series III showed
the best binding affinities to MT1/MT2 receptors due to the fact that the distance between the methoxy
group and the nitrogen atom of the amide/urea group is comparable to the distance in the MLT
molecule, a six-atom distance. Compound 10c showed the best binding affinity to both receptors. We
found a large selectivity on the MT2 receptor subtype by switching the methoxy group to position 2
and establishing a six-atom distance between this group and N-acylamido chain (Series II), displaying
a very selective MT2 ligand with a MT2/MT1 ratio of 250 and the higher binding affinity to MT2
(0.08 μM) of this study. This may provide a good preliminary starting point taking into consideration
the new structural characteristics were set up as essential requirements for obtaining new quinoxalines
as potent MT1 and MT2 receptor ligands.
Acknowledgments
Saioa Ancizu is indebted to the Navarre Government for a grant. We would also like to express our
gratitude to ADA of the University of Navarra for the grant given to Nerea Castrillo. We want to thank
Carmen Elizalde for her help in the identification assays of this study and to Laura Stokes for having
edited the English language in this manuscript.
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