Synthesis and evaluation of imidazole-4,5-and pyrazine-2,3-dicarboxamides targeting dengue and yellow fever virus

Article abstract of DOI:10.1016/j.ejmech.2014.09.062

The results of a high-throughput screening assay using the dengue virus-2 replicon showed that the imidazole 4,5-dicarboxamide (I45DC) derivative (15a) has a high dengue virus inhibitory activity. Based on 15a as a lead compound, a novel class of both disubstituted I45DCs and the resembling pyrazine 2,3-dicarboxamides (P23DCs) were synthesized. Here, we report on their in vitro inhibitory activity against dengue virus (DENV) and yellow fever virus (YFV). Some of these first generation compounds have shown activity against both viruses in the micromolar range. Within this series, compound 15b was observed to display the highest antiviral potency against YFV with an EC₅₀ Combining double low line 1.85 μM. In addition, compounds 20a and 20b both potently inhibited replication of DENV (EC₅₀ Combining double low line 0.93 μM) in Vero cells.

Full text of DOI:10.1016/j.ejmech.2014.09.062

European Journal of Medicinal Chemistry 87 (2014) 529e539
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of imidazole-4,5- and pyrazine-2,3-
dicarboxamides targeting dengue and yellow fever virus*
Milind Saudi a, Joanna Zmurko b, Suzanne Kaptein b, Jef Rozenski a, Johan Neyts b,
,
*
Arthur Van Aerschot a
a Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium
b Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
The results of a high-throughput screening assay using the dengue virus-2 replicon showed that the
imidazole 4,5-dicarboxamide (I45DC) derivative (15a) has a high dengue virus inhibitory activity.
Based on 15a as a lead compound, a novel class of both disubstituted I45DCs and the resembling
pyrazine 2,3-dicarboxamides (P23DCs) were synthesized. Here, we report on their in vitro inhibitory
activity against dengue virus (DENV) and yellow fever virus (YFV). Some of these first generation
compounds have shown activity against both viruses in the micromolar range. Within this series,
compound 15b was observed to display the highest antiviral potency against YFV with an
Received 22 May 2014
Received in revised form
17 September 2014
Accepted 18 September 2014
Available online 22 September 2014
Keywords:
Flavivirus inhibitors
Dengue virus
Yellow fever virus
Imidazole dicarboxylic acid
Pyrazine dicarboxylic acid
EC50 ¼ 1.85
m
m
M. In addition, compounds 20a and 20b both potently inhibited replication of DENV
M) in Vero cells.
(EC50 ¼ 0.93
© 2014 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/3.0/).
1. Introduction
Unfortunately, none of these substances have entered into clinical
trials. Hence, further development of new chemical entities
Dengue is the most common arthropod-borne viral infection in
the world and is estimated to transmit 390 million new infections
every year [3]. Dengue virus (DENV) belongs to the Flavivirus genus
of the Flaviviridae. The genome of DENV is comprised of a 10.7 kb,
single, positive-stranded RNA with at least four different circulating
serotypes (DENV-1 to DENV-4) [4]. Reportedly, a fifth serotype now
complicates vaccine development [4]. With increased levels of
population growth, urbanization, and international travel, DENV
illness has increased 30-fold in the last 50 years [5]. Most of the
current research on dengue infections is focused on the treatment
of symptoms, which often can be a tedious and intensive process.
As there is neither a drug nor any vaccine available to combat DENV
infections till date [6], it is highly important to explore and uncover
small molecules that have potent anti-dengue activity. In the past
half-decade, a number of antiviral agents with such inhibitory
properties have been discovered. These include an adenosine
analog 1 [7], a N-sulfonylanthranilic acid derivate 2 [8], the
chlorophenyl-thiophene derivate 3 [9], and most recently some
endowed with dengue inhibitory properties is warranted. Our
previous efforts herein, focused on tritylated and alkylated nucle-
oside analogs, resulting in compounds endowed with strong anti-
flavivirus inhibitory properties. However, no clear SAR could be
determined [1,2]. Hence we now turned our attention to another
lead molecule.
The five-membered imidazole ring is a structural unit found in
many biologically active compounds. The strong therapeutic
properties of imidazole containing drugs have encouraged medic-
inal chemists to synthesize a large number of novel chemothera-
peutic agents comprising this entity. Amongst others, imidazole
core structures are found in different carboxypeptidase, hemeox-
ygenase and lactamase inhibitors, as well as among anti-
inflammatory, anticancer, antibacterial, antifungal, antitubercular,
antidiabetic and antiviral products, further highlighted with some
examples. Ramya et al. synthesized a series of novel 5-(nitro/
bromo)-styryl-2-benzimidazole derivatives (5) and tested them for
their antibacterial activity against Staphylococcus aureus, Escher-
ichia coli and likewise evaluated the antifungal activity against
Candida albicans and Aspergillus fumigates [11]. Biological activities
proved comparable to ciprofloxacin. Kavitha C.S. et al. synthesized a
series of 2-methylaminobenzimidazole derivatives in which com-
pound 6 showed analgesic and anti-inflammatory activity compa-
rable with the standard drug nimesulide [12]. Cenzo et al.
2,4-diaminoquinazoline derivatives
4
[10] (see Fig. 1).
*
This is part 3 in a series on Flavivirus inhibitors [1,2].
* Corresponding author.
E-mail address: Arthur.Vanaerschot@rega.kuleuven.be (A. Van Aerschot).
http://dx.doi.org/10.1016/j.ejmech.2014.09.062
0223-5234/© 2014 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).

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M. Saudi et al. / European Journal of Medicinal Chemistry 87 (2014) 529e539
Fig. 1. Structures of some recently described antiviral compounds being inhibitory for dengue virus.
synthesized a series of 1, 4-diarylimidazole-2(3H)-one derivatives 7
and their 2-thione analogs and found antitumor activity [13].
Finally, Sharma et al. synthesized imidazole derivatives for antiviral
screening and different [2-(substituted phenyl)-imidazol-1-yl]-
benzamides like 8 and 9 were selected as the most promising
antiviral agents [14] (see Fig. 2).
Imidazole-4,5-dicarboxylic acid (I45DC) and its derivatives such
as those bearing primary or secondary amides have previously
been reported to be active against HIV-1 protease [15]. Likewise,
some derivatives of this scaffold were uncovered as potential kinase
inhibitors with antiproliferative activity against HL-60 cells [16]. In
addition, inhibition of proteineprotein interaction between Hepa-
titis C glycoprotein E2 and CD81 has been reported previously [17].
The I45DCs are known as well to be a structural component of some
antibiotics [18] and also to affect memory [19]. Substitution on the
imidazole moiety herein has been reported, both in solution phase
as well as in a combinatorial fashion on solid support.
4,5-dicarboxylic acid 10 was allowed to reflux with SOCl2 in toluene
with catalytic DMF to afford pyrazinedione diacid dichloride 11 in
92% yield [20]. The literature route for amide formation suggests
hydrolysis of the acid chlorides to carboxylic acids by the addition
of water. Amines are then added to open both acyl imidazole bonds
affording two identical imidazole analogs. Baures et al., replaced
the water with phenol, thereby modulating the reactivity of the
acid chloride in comparison with an acyl imidazole bond for the
subsequent addition of two different amines [20]. The difficulty in
preparing such analogs is that their synthesis is highly dependent
upon the substituents to be introduced. Primary benzyl amines, for
example, are too reactive to provide selectivity in the reaction,
whereas anilines can be added in a proper stoichiometric ratio in
order to provide a pyrazine dione intermediate 12 which can often
be purified by crystallization. In the case of aniline derivatives, the
resulting pyrazine intermediates are often insoluble in the reaction
solvent and can be isolated simply by vacuum filtration. In parallel,
different bromoanilines or benzylamines were subsequently
coupled with commercially available heterocyclic boronic acids
through palladium catalyzed Suzuki reaction to give the desired
second aniline in 60e75% yield. Addition of this second aniline to
the pyrazine intermediates 12aec resulted in opening of the acyl
imidazole bond to obtain the expected product. Based on this
approach, a series of novel molecules was prepared within this
I45DC family containing various substituted phenyl rings. For some
of the final products 15, the reaction sequence was changed with
prior attachment of the aniline carrying a heterocycle.
High throughput screening of a compound library led to the
identification of N5-(4-fluorophenyl)-N4-(4-methyl-2-(3-methyl-
1H-pyrazol-5-yl)phenyl)-1H-imidazole-4,5-dicarboxamide
15a
(see Fig. 3) with an EC50 of 2.50 M and 3.47 M against DENV and
m
m
YFV, respectively. In search for new compounds with potential for
clinical use as antiviral agents, a series of compounds based on this
I45DC scaffold were synthesized with regiospecific attachment of
the substituents and these analogs were investigated for their
inhibitory properties against DENV and YFV. In addition, the
imidazole core was substituted for a pyrazine ring leading to a se-
ries of pyrazine-2,3-dicarboxylic acids (P23DC). The intended sub-
stitution of the central imidazole ring would result in slightly
different orientation of the attached amide groups, and in a change
of the hydrogen bonding pattern exchanging the combination of a
hydrogen donating and a hydrogen accepting nitrogen into two
hydrogen accepting nitrogen atoms. In this communication, we
report on the synthesis and biological activity of both new series of
dissymmetric I45DCs and P23DCs, which we studied for their
inhibitory properties against YFV and DENV.
Pyrazines are important pharmacophores present in a number
of biologically active compounds such as antimycobacterial, anti-
bacterial, antidiabetic, and hypnotic agents. To further explore
previous structureeactivity relationship, we planned to substitute
the five-membered imidazole ring with a pyrazine ring. Hereto,
regioselective functionalization of the pyrazine started from com-
mercial pyrazine-2,3-dicarboxylic acid (P23DA) and a four-step
synthesis of the target compounds has been described in Scheme
2. P23DA was converted to its corresponding anhydride 17 by
treatment with acetic anhydride at room temperature.
Reaction with p-fluoroaniline or p-toluidine resulted in opening
of the five-membered ring with concomitant formation of the first
amide bond (18a,b). Reaction with trifluoroacetic anhydride at 0 ꢀC
afforded the anhydric activated product 19a or 19b allowing
introduction of the second amide. Hereto, the intermediate was
reacted with preformed anilines or benzyl amines 14aem to give
2. Results and discussion
2.1. Synthetic aspects
The general procedure employed for the synthesis of dissym-
metrically disubstituted I45DCs is shown in Scheme 1. Imidazole-

M. Saudi et al. / European Journal of Medicinal Chemistry 87 (2014) 529e539
531
Fig. 2. Some examples of imidazole containing structures endowed with pronounced biological activity.
Fig. 3. Lead compound 15a and envisaged modifications of different parts of the antiviral lead for optimization of the structure.
the target compounds 20aen in 60e80% overall yields. All final
products were purified by silica gel chromatography and charac-
terized by 1H NMR, 13C NMR and HRMS before evaluation of their
inhibitory properties against DENV and YFV.
heterocyclic moiety as shown in Table 1. Using p-fluoroaniline (ring
A) and the I45DC core as a fixed fragment, substitution of the five-
membered pyrazole moiety (ring D) with a thienyl or furanyl ring
mostly led to considerable loss of activity (i.e., compare 15h, 15i, 15j
versus 15a). However, activity remarkably was restored upon sub-
stitution of a methyl group for the para-fluorine in ring A (15k, 15l).
The benzylamine substitution for ring A likewise annihilated the
inhibitory properties (15m), while in contrast a p-fluoro-phenyl-
ethylamine again displayed nice activity (15n). Exchanging aniline
ring C for a benzylamine preserved the activity when the supple-
mentary heterocycle D was attached at the ortho position (15c,
15d), while para substitution led to considerable loss of activity
(15eeg) within this context. A single attempt with an amino-
pyridine ring at site C proved inactive (15o). Overall, the com-
2.2. Assessment of antiviral activity
As shown in Fig. 3 and Schemes 1 and 2, four main structural
domains were chosen for optimization of hit 15a: the aromatic
moiety (ring A), the central pyrazine/imidazole core (ring B), the
substituted aromatic moiety (ring C) and the heterocyclic group
(ring D). These efforts have provided a few compounds that
exhibited an improved biological profile with respect to the initial
lead compound 15a. Overall, improvement or reduction of the
inhibitory activities for either DENV or YFV is mostly running in
parallel.
The overall structure of 15a comprises an imidazole core car-
rying two aniline moieties coupled via an amide bond, with one of
the aniline rings substituted with an additional heterocyclic ring
(ring D). Therefore, our efforts to understand the structureeactivity
relationship of the initial lead started with modifying the
pounds 15b (EC50 ¼ 1.85
m
M, YFV), 15c (EC50 ¼ 1.93
m
M, DENV), 15d
(EC50 ¼ 2.61
m
M, YFV) and 15k (EC50 ¼ 2.02
mM, DENV) are
endowed with the best inhibitory properties within this imidazole
series.
With these results in mind, we hoped to further improve the
potency via replacement of the central five-membered imidazole
(ring B) with a pyrazine scaffold, both rings containing two nitrogen

532
M. Saudi et al. / European Journal of Medicinal Chemistry 87 (2014) 529e539
Scheme 1. General reaction scheme for synthesis of the I45DC analogs. Conditions: a) SOCl2, cat. DMF, Toluene, 85 ꢀC; b) DIPEA, substituted aniline, DCM, ꢁ10 ꢀC; c) acetoni-
trile:water (1:1), K2CO3, PdCl2TPP2, boronic acid; d) DIPEA, substituted aniline or benzyl amine, DCM, room temperature (rt).
Scheme 2. General scheme for assembly of different P23DC analogs. Conditions: a) acetic anhydride; b) acetonitrile:water (1:1), substituted aniline, dodecyl hydrogen sulfate
sodium salt, rt; c) trifluoroacetic anhydride, TEA, THF; d) substituted aniline or benzyl amine, THF, rt.
atoms but providing a slightly different orientation for the aniline
substituents. Focusing on compound 15c as most potent repre-
proved deleterious for the biological activity (see 15eeg). In
contrast with these results, the para-pyrimidin-4-yl substituent on
ring C proved advantageous within the pyrazine series and led to
compound 20a displaying a 2-fold increase in anti-DENV potency
sentative for inhibition of DENV (EC50 ¼ 1.92
mM), we synthesized a
series of pyrazine analogs with a pyridine substituent as the het-
erocyclic moiety D, attached at different positions of ring C. The
inhibitory potency of these compounds was likewise examined
against DENV and YFV and is summarized in Table 2 (compounds
20aꢁ20n).
with EC50 of 0.93 mM. A distinction however can be made having
either a benzylamine (n ¼ 1) or an aniline (n ¼ 0) aromatic ring C.
Within the benzylamine series, the heterocycle is preferred at the
para position with preference for concomitant presence of fluo-
roaniline at the A-site (20a, 20c) over toluidine (20h, 20j). However,
ortho attachment of the heterocycle on the benzylamine ring
seriously reduced the activity (20f, 20m). Having an aniline ring at
site C the results were less clear with mostly intermediate inhibi-
Unfortunately, the compound 20f corresponding to 15c,
exhibited 20-fold decrease in activity against DENV
(EC50 ¼ 40.70
m
M) and 6-fold against YFV (EC50 ¼ 21.29
mM).
Concentrating on anti-DENV activity, we noticed within the imid-
azole series that the para attachment of the heterocycle to ring C
tory properties (5e25
mM), except for the strongly inhibiting

M. Saudi et al. / European Journal of Medicinal Chemistry 87 (2014) 529e539
533
Table 1
Inhibitory properties for yellow fever virus and dengue virus of the imidazole analogs 15ae15o.
Sr. No.
n1
n2
R1
F
R2
Heterocycle (positiona)
Dengue
EC50 M)
Yellow fever
EC50 M)
(
m
CC50
(
m
M)
SI
(
m
CC50
(
m
M)
SI
15a
0
0
p-Methyl
3-Methyl-1H-pyrazol-5-yl (ortho)
Initial Lead
2.50
>120
48
3.47
>120
34.5
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
15m
15n
15o
0
0
0
0
0
0
0
0
0
0
0
1
2
0
0
1
1
1
1
1
0
0
0
0
0
0
0
0
F
F
F
F
F
F
F
F
H
CH3
CH3
H
F
H
H
H
H
H
H
3-Methyl-1H-pyrazol-5-yl (ortho)
Pyridin-4-yl (ortho)
Thien-3-yl (ortho)
Pyridin-3-yl (para)
Thien-3-yl (para)
Pyridin-4-yl (para)
Thien-3-yl (ortho)
Furan-2-yl (ortho)
Thien-3-yl (ortho)
Thien-3-yl (ortho)
Furan-2-yl (ortho)
Furan-2-yl (ortho)
Furan-2-yl (ortho)
See structure above
6.03
1.93
8.88
21.7
50.1
17.32
>120
20.21
>120
>117
>124
>124
>50
>125
>125
>116.5
26.40
3.6
26
1.95
2.85
0.60
1
2.4
1
1
24.5
36.2
1
30.9
0.22
1.85
3.90
2.61
>121
>120
34.5
>118
>119
>120
>119
e
67
31
13.2
10
4.13
42.10
33.69
>120
48.75
>123.6
>124
2.02
3.47
>125
3.77
11.81
28.77
>120
1
1
p-Methyl
p-Methyl
p-Methyl
p-Methyl
p-Methyl
p-Methyl
p-Methyl
p-Methyl
>119
ND
e
9.20
>120
ND
>120
>120
e
13
1
e
e
e
ND
ND
e
e
H
>120
>120
>119
0.97
a
Indicates the attachment point of the heterocycle on the phenyl ring.
compound 20b and the non-active analog 20e. Within this pyrazine
series (20aen) the YFV inhibitory properties in general were
slightly weaker compared to DENV inhibition by these compounds.
spectra. Exact mass measurements were performed with a quad-
rupole/orthogonal acceleration time-of-flight tandem mass spec-
trometer (qTOF2, Micromass, Manchester, UK) fitted with
a
standard electrospray ionization (ESI) interface. All solvents were
carefully dried or bought as such.
3. Conclusion
A series of tetracyclic inhibitors were prepared using I45CD and
P23DC scaffolds and which further were part of a structur-
eꢁactivity investigation. The SAR results of both series, I45DCs and
P23DCs derivatives, as potent anti-dengue and anti-yellow fever
virus agents are described above. Our investigation led to the dis-
covery of 15b, which is the most potent inhibitor of the series
4.1. Chemistry
4.1.1. 5,10-Dioxo-5,10-dihydrodiimidazo[1,5-a:10,50-d]pyrazine-1,6-
dicarbonyl dichloride (11)
To a dry round-bottom flask was added 1.0 g of imidazole-4,5-
dicarboxylic acid (10, 1 mmol) and 10 mL of toluene. To this stir-
red suspension were added 3.0 mL of thionyl chloride (6.5 mmol)
and 0.250 mL of DMF (catalytic). The resulting mixture was refluxed
for 16 h. After the mixture was cooled in ice bath, the solid product
was collected by vacuum filtration, washed with two 20 mL por-
tions of toluene, and dried under vacuum to yield 0.95 g of crude 11
(Yield: 95%) as a yellow solid. This product was used further
without characterization.
(EC50 ¼ 1.85
mM) against YFV, but followed closely in potency by the
quite deviating structure 15d. The structural analog 15c on the
other hand proved most inhibitory for dengue virus (see Fig. 4). In
addition, within the pyrazine series both compounds 20a and 20b
were found to have potent DENV inhibitory properties
(EC50 ¼ 0.93
mM) uncovered to date. Key features of these inhibitors
include the central bis-amide arrangement which is found to be a
metal-chelating component in many drugs. In summary, determi-
nation of the antiviral activities shows that I45DC and P23DC are
attractive candidate scaffolds for further studies and the interesting
activities observed so far, warrant an in depth study of their
mechanism of action.
4.1.2. General procedure for 12aee
To a suspension of 11 (1.0 mmol) in 10 mL of dichloromethane
was added N,N-diethylaniline (2 mmol), and corresponding
substituted aniline (1.7 mmol) at ꢁ10 ꢀC. The reaction mixture was
stirred at room temperature for 3e5 h before collecting highly
insoluble colored solid 12aee by vacuum filtration. The product
was washed with portions of 10 mL DCM, cold water, and acetone,
respectively. Note: Addition of excess of aniline results in opening of
acyl imidazole bonds affording unwanted products.
4. Materials and methods
All other chemicals were provided by Aldrich or ACROS and
were of the highest quality. 1H and 13C NMR spectra were deter-
mined with a 300 MHz Varian Gemini apparatus with tetrame-
thylsilane as internal standard for the 1H NMR spectra (s ¼ singlet,
d ¼ doublet, dd ¼ double doublet, t ¼ triplet, br. s ¼ broad signal, br.
d ¼ broad doublet, m ¼ multiplet) and the solvent signal e DMSO-
4.1.2.1. 5,10-Dioxo-N1,N6-diphenyl-5,10-dihydrodiimidazo[1,5-a:10,50-
d]pyrazine-1,6-dicarboxamide (12a). Yield: 85%; Orange solid; 1H
NMR (300 MHz, CDCl3):
d 10.74 (s, 2H), 9.09 (s, 2H), 7.80 (d, 4H,
d6 (
d
¼ 39.6 ppm) or CDCl3 (
d
¼ 76.9 ppm) e for the 13C NMR
J ¼ 7.8 Hz), 7.41 (t, 4H, J ¼ 8.1 Hz), 7.17 (t, 2H, J ¼ 7.5 Hz). 13C NMR

534
M. Saudi et al. / European Journal of Medicinal Chemistry 87 (2014) 529e539
Table 2
Inhibitory properties for yellow fever virus and dengue virus of the pyrazine containing compounds 20ae20n.
Sr. No.
n
R1
R2
Heterocycle (positiona)
Dengue
EC50 M)
Yellow fever
EC50 M)
(
m
CC50
(m
M)
SI
(
m
CC50
(m
M)
SI
20a
20b
20c
20d
20e
20f
20g
20h
20i
20j
20k
20l
20m
20n
1
0
1
0
0
1
0
1
0
1
0
0
1
0
F
F
F
F
F
F
F
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
Pyridin-4-yl (para)
Pyridin-3-yl (ortho)
Pyridin-3-yl (para)
Pyridin-4-yl (ortho)
Pyridin-3-yl (ortho)
Pyridin-4-yl (ortho)
Pyridin-4-yl (ortho)
Pyridin-4-yl (para)
Pyridin-3-yl (ortho)
Pyridin-3-yl (para)
Pyridin-4-yl (ortho)
Pyridin-3-yl (ortho)
Pyridin-4-yl (ortho)
Pyridin-4-yl (ortho)
0.94
0.94
2.99
11.71
23.19
40.70
>117.5
>117.5
>115.1
>117.1
>120.5
>118.0
>117
74.7
47.5
>116.1
>122.5
>128.13
>118
>125
>125
38.5
7.49
13.10
8.90
>120
>118
>116
>123.3
>119
>127
>125
>110.5
75.5
>124
>125
>122
>120
>117.5
16
9
13
15
2.5
6
2.6
3
4.1
1.7
10
1
p-Methyl
H
H
H
10.4
5.2
2.9
1
4.5
4.5
17.2
26
8.22
47.53
21.29
48.43
36.83
18.41
73.08
12.46
>122
70.61
30.93
H
p-Methyl
H
p-Methyl
H
H
H
H
>117
16.60
10.55
6.75
4.71
14.56
57.86
11.90
8.8
2.04
10
1.7
3.8
p-Methyl
>119
a
Indicates the attachment point of the heterocycle on the phenyl ring.
Fig. 4. Overview of all new compounds most inhibitory to either DENV or YFV as reported in this communication.
(75 MHz, CDCl3):
d
162.3, 158.1, 149.2, 145.2, 138.7, 138.2, 129.0,
found: 427.1151. HRMS calcd for C22H13F2N6O4 [MþH]þ: 463.0961;
124.5, 120.1. HRMS calcd for C22H15N6O4 [MþH]þ: 427.1149; found:
found: 463.0961.
427.1151.
4.1.2.2. N1,N6-bis(4-fluorophenyl)-5,10-dioxo-5,10-dihydrodiimidazo
[1,5-a:10,50-d]pyrazine-1,6-dicarboxamide (12b). Yield: 87%; Yellow
4.1.2.3. 5,10-Dioxo-N1,N6-di-p-tolyl-5,10-dihydrodiimidazo[1,5-
a:10,50-d]pyrazine-1,6-dicarboxamide (12c). Yield: 89%; Red Solid;
Solid; 1H NMR (300 MHz, CDCl3):
d
10.76 (s, 2H), 9.09 (s, 2H),
1H NMR (600 MHz, DMSO):
d 10.62 (s, 2H), 9.07 (s, 2H), 7.67 (d, 4H,
7.85e7.81 (m, 4H), 7.25 (m, 4H, J ¼ 9.0 Hz). 13C NMR (75 MHz,
J ¼ 8.4 Hz), 7.20 (d, 4H, J ¼ 7.8 Hz), 2.30 (s, 6H). 13C NMR (125 MHz,
CDCl3):
d
160.3, 158.1, 157.2, 149.0, 145.0, 138.6, 134.8, 134.8, 122.1,
DMSO): d157.8, 149.2, 145.2, 138.6, 135.9, 133.5, 129.4, 120.4, 120.0,
122.0, 120.7, 115.5. HRMS calcd for C22H15N6O4 [MþH]þ: 427.1149;
20.6. HRMS calcd for
C
22H15N6O4 [MþH]þ: 427.1149; found:

M. Saudi et al. / European Journal of Medicinal Chemistry 87 (2014) 529e539
535
427.1151. HRMS calcd for C24H19N6O4 [MþH]þ: 455.1462; found:
(m, 1H), 7.91e7.87 (m, 1H), 7.60e7.57 (m, 2H), 7.47e7.40 (m, 2H),
7.38e7.36 (m, 1H), 3.96 (s, 2H, CH2), 1.62 (bs, 2H, NH2). 13C NMR
455.1461.
(75 MHz, CDCl3):
C
d 149.9, 127.5, 126.8, 121.1, 45.7. HRMS calcd for
4.1.2.4. N1,N6-bis(4-methyl-2-(thiophen-2-yl)phenyl)-5,10-dioxo-
5,10-dihydrodiimidazo [1,5-a:10,50-d]pyrazine-1,6-dicarboxamide
(12d). Yield: 74%; Red Solid; 1H NMR (300 MHz, DMSO): 10.01 (s,
2H), 8.97 (s, 2H), 7.79e7.63 (m, 6H), 7.35e7.23 (m, 6H), 2.37 (s, 6H,
2CH3). HRMS calcd for C32H23N6O4S2 [MþH]þ: 619.1237; found:
619.1238. In view of insufficient solubility 13C NMR could not be
determined.
12H13N2 [MþH]þ: 185.1073; found: 185.1072.
4.1.3.7. (4-(Pyridin-4-yl)phenyl)methanamine (14g) [22]. Yield:
59%; White solid; 1H NMR (300 MHz, CDCl3):
8.64e8.62 (m, 2H),
7.61e7.41 (m, 6H), 3.92 (s, 2H, CH2), 1.77 (bs, 2H, NH2). 13C NMR
(75 MHz, CDCl3): 149.2, 126.9, 126.2, 120.5, 45.1.
d
d
4.1.3.8. (2-(Pyridin-4-yl)phenyl)methanamine (14h) [23]. Yield:
65%; White solid; 1H NMR (300 MHz, CDCl3):
8.76e8.74 (m, 1H),
7.56e7.10 (m, 8H), 3.92 (s, 2H, CH2). 13C NMR (75 MHz, CDCl3):
149.1,132.0,131.3,131.1,131.1,128.2,127.7,127.6,127.5,126.9,122.7,
4.1.2.5. N1,N6-bis(2-(furan-2-yl)-4-methylphenyl)-5,10-dioxo-5,10-
d
dihydrodiimidazo[1,5-a:10,50-d]pyrazine-1,6-dicarboxamide
(12e).
Yield: 72%; Yellow Solid; 1H NMR (300 MHz, DMSO): 10.46 (s, 2H),
9.07 (s, 2H), 7.81e7.61 (m, 6H), 7.25e6.63 (m, 6H), 2.38 (s, 6H,
2CH3). HRMS calcd for C32H23N6O6 [MþH]þ: 587.1673; found:
587.1672. In view of insufficient solubility 13C NMR could not be
determined.
d
120.5, 46.0.
4.1.3.9. 4-Methyl-2-(thiophen-3-yl)aniline (14i). Yield: 62%; Off-
white solid; 1H NMR (300 MHz, CDCl3):
d 7.47e7.40 (m, 2H),
7.33e7.31 (m, 1H), 7.09 (s, 1H), 7.03e7.00 (m, 1H), 6.75e6.72 (d, 1H,
4.1.3. General procedure for 14aem
J ¼ 8.1 Hz), 3.73 (bs, 2H, NH2), 2.33 (s, 3H, CH3). 13C NMR (75 MHz,
To a solution of corresponding aniline/benzyl amine (1.0 mmol)
in 10 mL dioxane:water (1:1), was added anhydrous K2CO3
(1.5 mmol), aryl boronic acids (1.2 mmol) and Pd(TPP)2Cl2
(0.025 mmol) in a seal tube. The mixture was purged with argon for
30 min at rt and heated at 100 ꢀC for 1 h in microwave. All reactions
and manipulations were run under argon atmosphere. After
completion, the solvent was evaporated under reduced pressure
and the residue was purified by column chromatography on a silica
gel to give the desired product.
CDCl3): d 141.5, 140.1, 130.7, 129.1, 128.5, 127.8, 126.0, 122.6, 122.5,
116.0, 20.5. HRMS calcd for C11H12N1S1 [MþH]þ: 190.0685; found:
190.0686.
4.1.3.10. (4-(Thiophen-3-yl)phenyl)methanamine
(14j)
[24].
Yield: 58%; Off-white solid; 1H NMR (300 MHz, CDCl3):
d
7.61e7.58
(m, 2H), 7.47e7.28 (m, 6H), 3.92 (s, 2H, CH2). 13C NMR (75 MHz,
CDCl3):
d 127.7, 126.8, 126.5, 126.3, 120.2, 46.3.
4.1.3.11. 2-(Thiophen-3-yl)phenyl)methanamine
(14k)
7.58e7.12 (m,
6H), 3.91 (s, 2H, CH2). 13C NMR (150 MHz, CDCl3):
d 162.1, 160.8,
[25].
4.1.3.1. 2-(5-Methyl-1H-pyrazol-3-yl)aniline (14a) [21]. Yield: 52%;
Yield: 52%; White solid; 1H NMR (300 MHz, CDCl3):
d
White solid; 1H NMR (300 MHz, CDCl3):
d
7.50 (d, 1H, J ¼ 7.2 Hz),
7.13 (t, 1H, J ¼ 7.5 Hz), 6.79e6.75 (m, 2H), 6.39 (s, 1H), 2.37 (s, 3H,
160.5, 142.0, 141.1, 135.8, 132.7, 130.1, 129.8, 129.3, 129.0, 128.8,
128.6,128.4,128.2,127.7,127.5,127.4,127.1,127.0,126.8,125.5,125.0,
123.4, 122.7, 46.8, 44.2.
CH3).
4.1.3.2. 4-Methyl-2-(pyridin-3-yl) aniline (14b). Yield: 62%; Yellow
Oil; 1H NMR (300 MHz, CDCl3):
d
8.70 (s, 1H), 8.61e8.59 (m, 1H),
4.1.3.12. 2-(Furan-2-yl)-4-methylaniline (14l). Yield: 63%; White
7.83e7.80 (m, 1H),7.40e7.35 (m, 1H), 7.05e7.01 (m, 1H), 6.94 (m,
1H), 6.73e6.71 (d, 1H, J ¼ 8.1 Hz), 3.60 (bs, 2H, NH2), 2.30 (s, 3H,
solid; 1H NMR (300 MHz, CDCl3):
d
7.52 (s, 1H), 7.32 (s, 1H),
6.97e6.94 (m, 1H), 6.71e6.52 (m, 3H), 2.30 (s, 3H, CH3). 13C NMR
CH3). 13C NMR (75 MHz, CDCl3):
d
149.2, 147.4, 140.3, 135.7, 130.0,
(75 MHz, CDCl3): d 152.5, 141.2, 140.8, 129.5, 127.8, 127.6, 116.9,
129.0, 127.3, 122.6, 115.2, 19.5. HRMS calcd for C12H13N2 [MþH]þ:
116.2, 111.3, 106.3, 20.4. HRMS calcd for C11H12N1O1 [MþH]þ:
185.1073; found: 185.1078.
174.0913; found: 174.0910.
4.1.3.3. 4-Methyl-2-(pyridin-4-yl)aniline (14c). Yield: 59%; White
4.1.3.13. 5-Methyl-3-(5-methylthiophen-2-yl)pyridin-2-amine
solid; 1H NMR (300 MHz, CDCl3):
d
8.69e8.67 (m, 2H), 7.44e7.42
(14m). Yield: 57%; White solid; 1H NMR (300 MHz, CDCl3):
d 7.84
(m, 2H), 7.06e6.96 (m, 2H), 6.72 (d, 1H, J ¼ 8.1 Hz), 3.69 (bs, 2H,
(s, 1H), 7.28 (s, 1H), 6.99 (d, J ¼ 3.3 Hz, 1H), 6.73e6.72 (m, 1H), 4.96
NH), 2.30 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d
150.4, 130.6,
(bs, 2H, NH2), 2.49 (s, 3H, CH3), 2.18 (s, 3H, CH3). 13C NMR (75 MHz,
130.5, 124.0, 116.4, 20.5. HRMS calcd for C12H13N2 [MþH]þ:
CDCl3):
d 153.6, 146.5, 139.8, 138.5, 137.1, 125.6, 125.5, 122.7, 114.5,
185.1073; found: 185.1074.
17.0, 14.9.
4.1.3.4. 2-(Pyridin-3-yl)aniline (14d). Yield: 72%; Yellow solid; 1H
4.1.4. General procedure for 15beo
NMR (300 MHz, CDCl3):
(m, 1H), 7.41e7.36 (m, 1H), 7.24e7.11 (m, 2H), 6.89e6.79 (m, 2H),
3.75 (bs, 2H, NH2). 13C NMR (75 MHz, CDCl3):
150.2, 148.6, 143.9,
d
8.73 (s, 1H), 8.62e8.60 (m, 1H), 7.84e7.80
To a stirred solution of 12aee (1.0 mmol) in 10 mL DCM, was
added DIPEA (3.0 mmol), and 14aem (3.0 mmol) under argon at-
mosphere at room temperature. The mixture was allowed to reflux
for 16e48 h. Two alternative workup procedures were followed
depending on whether the product precipitated out of solution or
not.
Workup 1 (for soluble products): The DCM was evaporated using
a rotavapor. The product was purified by flash chromatography on
silica gel.
d
136.6, 135.4, 130.7, 129.5, 123.6, 119.0, 116.0. HRMS calcd for
C
11H11N2 [MþH]þ: 171.0917; found: 171.0921.
4.1.3.5. 2-(Pyridin-4-yl)aniline (14e). Yield: 69%; White solid; 1H
NMR (300 MHz, CDCl3): 8.69e8.67 (m, 2H), 7.44e7.28 (m, 2H),
7.25e7.13 (m, 2H), 6.89e6.78 (m, 2H), 3.82 (bs, 2H, NH2). 13C NMR
d
(75 MHz, CDCl3):
d
149.4, 129.2, 128.8, 123.0, 118.0, 115.2. HRMS
Workup 2 (for insoluble products): The precipitate was collected
by filtration using a Buchner funnel and washed on the frit with
DCM (20e25 mL) until the yellow impurities had disappeared. The
product obtained from this workup procedure did not need chro-
matographic purification.
calcd for C11H11N2 [MþH]þ: 171.0917; found: 171.0925.
4.1.3.6. (4-(Pyridin-3-yl)phenyl)methanamine (14f). Yield: 55%;
White solid; 1H NMR (300 MHz, CDCl3):
d 8.86 (m, 2H), 8.61e8.59

536
M. Saudi et al. / European Journal of Medicinal Chemistry 87 (2014) 529e539
4.1.4.1. N5-(4-fluorophenyl)-N4-(2-(3-methyl-1H-pyrazol-5-yl)
phenyl)-1H-imidazole-4,5-dicarboxamide (15b). Yield: 86%; 1H
(300 MHz, DMSO): d 13.69 (bs, 1H, NH), 13.20 (bs, 1H, NH), 10.60 (s,
1H, NH), 8.05e7.55 (m, 6H), 7.27e6.56 (m, 5H), 2.38 (s, 3H, CH3).
NMR (500 MHz, DMSO):
d 13.69 (bs, 1H, NH), 13.30 (bs, 1H, NH),
HRMS calcd for C22H18F1N4O3 [MþH]þ: 405.1357; found: 405.1353.
13.05 (bs, 1H, NH), 12.78 (bs, 1H, NH), 8.65 (d, 1H, J ¼ 8.0 Hz), 7.96 (s,
1H), 7.78e7.23 (m, 9H), 6.54 (s, 1H), 2.31 (s, 3H, CH3). 13C NMR
4.1.4.9. N4-(4-methyl-2-(thiophen-3-yl)phenyl)-N5-phenyl-1H-imid-
(125 MHz, DMSO):
d 163.3, 159.6, 158.0, 156.4, 150.3, 140.2, 137.3,
azole-4,5-dicarboxamide (15j). Yield: 81%; 1H NMR (600 MHz,
135.2, 135.0, 133.9, 130.0, 128.7, 124.8, 123.2, 121.9, 121.6, 116.4,
116.2, 103.1, 10.8. HRMS calcd for C21H16F1N6O2 [MꢁH]þ: 403.1324;
found: 403.1323.
DMSO):
12.47 (s, 0.3H, NH), 10.34 (s, 0.3H, NH), 10.12 (s, 0.7H, NH), 7.96e7.12
(m, 12H), 2.36 (s, 3H, CH3). 13C NMR (125 MHz, DMSO):
162.4,
d 13.63 (s, 0.7H, NH), 13.56 (s, 0.3H, NH), 13.18 (s, 0.7H, NH),
d
156.0, 138.5, 138.3, 137.1, 135.2, 132.8, 131.5, 130.6, 129.9, 129.3,
128.6, 128.4, 127.0, 124.2, 124.0, 120.9, 119.4, 20.6. HRMS calcd for
C
4.1.4.2. N5-(4-fluorophenyl)-N4-(2-(pyridin-4-yl)benzyl)-1H-imid-
azole-4,5-dicarboxami-de (15c). Yield: 83%; 1H NMR (600 MHz,
22H17N4O2 S1 [MꢁH]þ: 401.1078; found: 401.1078.
DMSO):
d
8.76 (d, 0.5H, J ¼ 6.6 Hz), 7.96e7.17 (m, 10H), 4.57 (d, 2H,
J ¼ 6.0 Hz, CH2). 13C NMR (150 MHz, DMSO):
d 159.5, 157.9, 150.0,
137.4, 127.1, 134.8, 132.7, 132.4, 131.8, 131.7, 129.3, 129.1, 129.0, 128.7,
128.1, 122.4, 122.0, 116.0, 115.8, 43.1. HRMS calcd for C23H18F1N5O2
[MꢁH]þ: 414.1372; found: 414.1372.
4.1.4.10. N4-(4-methyl-2-(thiophen-3-yl)phenyl)-N5-(p-tolyl)-1H-
imidazole-4,5-dicarboxamide (15k). Yield: 79%; 1H NMR (600 MHz,
DMSO): d 13.59 (s, 0.7H, NH),13.54 (s, 0.3H, NH), 13.09 (s, 0.3H, NH),
12.52 (s, 0.7H, NH), 10.26 (s, 0.3H, NH), 10.10 (s, 0.7H, NH), 7.98e7.18
4.1.4.3. N5-(4-fluorophenyl)-N4-(2-(thiophen-3-yl)benzyl)-1H-imid-
azole-4,5-dicarbox amide (15d). Yield: 86%; 1H NMR (600 MHz,
(m, 10H), 2.36 (s, 3H, CH3), 2.28 (s, 3H, CH3). 13C NMR (125 MHz,
DMSO):
d 162.4, 155.8, 138.3, 137.0, 136.0, 135.1, 133.1, 132.7, 131.5,
DMSO):
NH), 9.23 (bs, 0.5H, NH), 7.99e7.18 (m, 12H), 4.58 (d, 2H, J ¼ 6.6 Hz,
CH2). 13C NMR (150 MHz, DMSO):
164.3, 156.1, 140.5, 137.3, 137.0,
132.7, 132.5, 129.9, 129.0, 128.0, 127.6, 127.1, 126.3, 123.7, 121.1, 115.9,
115.7, 42.9. HRMS calcd for C22H16F1N4O2S1 [MꢁH]þ: 419.0973;
found: 419.0967.
d
13.53 (bs, 0.5H, NH), 13.45 (bs, 0.5H, NH), 9.40 (bs, 0.5H,
130.6, 129.9, 129.6, 129.4, 129.0, 128.6, 128.5, 128.4, 128.4, 127.0,
124.2, 124.0, 120.9, 119.4, 20.6. HRMS calcd for C23H21N4O2S1
[MþH]þ: 417.1380; found: 417.1377.
d
4.1.4.11. N4-(2-(furan-2-yl)-4-methylphenyl)-N5-(p-tolyl)-1H-imid-
azole-4,5-dicarboxamide (15l). Yield: 76%; 13.58 (s, 0.7H, NH), 13.52
(s, 0.3H, NH), 13.07 (s, 0.3H, NH), 12.50 (s, 0.7H, NH), 10.23 (s, 0.3H,
NH), 10.08 (s, 0.7H, NH), 7.97e7.93 (m, 2H), 7.71e7.56 (m, 4H),
7.33e7.11 (m, 5H), 2.35 (s, 3H, CH3), 2.28 (s, 3H, CH3). 13C NMR
4.1.4.4. N5-(4-fluorophenyl)-N4-(4-(pyridin-3-yl)benzyl)-1H-imid-
azole-4,5-dicarboxamide (15e). Yield: 76%; 1H NMR (600 MHz,
DMSO): d 13.55 (s, 1H, NH), 13.49 (bs, 1H, NH), 10.49 (s, 1H), 10.06 (s,
(125 MHz, DMSO):
d 162.4, 155.8, 138.3, 136.9, 136.0, 135.1, 133.1,
1H), 9.47 (s, 1H), 8.85e7.20 (m, 9H), 4.59 (d, 2H, J ¼ 6.6 Hz, CH2). 13
C
132.7, 131.5, 130.6, 129.8, 129.6, 129.4, 129.0, 128.6, 128.5, 128.4,
128.4, 127.0, 124.2, 124.0, 120.9, 119.4, 20.6, 20.6. HRMS calcd for
NMR (150 MHz, DMSO):
d 164.2, 156.4, 148.6, 147.7, 139.3, 137.2,
136.0, 135.6, 135.1, 134.4, 133.0, 128.9, 128.4, 128.3, 127.3, 127.2,
124.2, 121.4, 121.4, 116.1, 116.9, 42.3. HRMS calcd for C23H17F1N5O2
[MꢁH]þ: 414.1372; found: 414.1372.
C
23H21N4O3 [MþH]þ: 401.1608; found: 401.1602.
4.1.4.12. N5-benzyl-N4-(2-(furan-2-yl)-4-methylphenyl)-1H-imid-
4.1.4.5. N5-(4-fluorophenyl)-N4-(4-(thiophen-3-yl)benzyl)-1H-imid-
azole-4,5-dicarboxamide (15f). Yield: 78%; 1H NMR (300 MHz,
azole-4,5-dicarboxamide (15m). Yield: 83%; 1H NMR (300 MHz,
CDCl3):
d 11.84 (bs, 1H, NH), 11.57 (bs, 1H, NH), 10.32 (bs, 1H, NH),
DMSO):
d
9.84 (bs, 1H, NH), 7.97 (s, 1H), 7.87e7.19 (m, 15H), 4.57 (d,
8.16 (d, 1H, J ¼ 8.4 Hz), 7.61e7.18 (m, 10H), 6.74 (d, 1H, J ¼ 3.3 Hz),
2H, J ¼ 6.3 Hz, CH2). 13C NMR (75 MHz, DMSO):
d
160.1, 157.0, 141.4,
6.57e6.55 (m, 1H), 4.70 (d, 2H, J ¼ 6.0 Hz, CH2), 2.40 (s, 3H, CH3). 13
C
141.4, 138.0,137.0, 134.9,134.0, 128.1, 128.0, 127.6, 127.1, 126.3, 126.0,
121.7, 121.6, 120.9, 120.7, 115.9, 115.6, 42.2. HRMS calcd for
NMR (75 MHz, CDCl3): d 161.7, 159.0,142.4,134.9,134.8,130.5,129.1,
128.6, 127.9, 127.4, 127.2, 123.0, 111.7, 108.5, 43.5, 21.0. HRMS calcd
C
22H16F1N5O2 S1 [MꢁH]þ: 419.0983; found: 419.0993.
for C23H21N4O3 [MþH]þ: 401.1608; found: 401.1590.
4.1.4.6. N5-(4-fluorophenyl)-N4-(4-(pyridin-4-yl)benzyl)-1H-imid-
azole-4,5-dicarboxamide (15g). Yield: 81%; 1H NMR (600 MHz,
4.1.4.13. N5 -(4-fluorophenethyl)-N4 -(2-(furan-2-yl)-4-
methylphenyl)-1H-imidazole-4,5-dicarboxamide (15n). Yield: 75%;
DMSO)
d 13.56 (s, 1H, NH), 13.47 (bs, 1H, NH), 9.50 (s, 1H, NH),
1H NMR (300 MHz, CDCl3):
d 11.13 (bs, 1H, NH), 10.32 (bs, 1H, NH),
8.61e7.20 (m, 13H), 4.59 (d, 2H, J ¼ 6.0 Hz, CH2). 13C NMR (150 MHz,
8.18 (d, 1H, J ¼ 8.7 Hz), 7.67e6.55 (m, 10H), 3.73 (m, 2H, CH2), 3.00
DMSO):
d 164.2, 156.2, 150.5, 150.3, 146.9, 140.4, 137.1, 135.9, 135.0,
(t, 2H, J ¼ 7.5 Hz CH2), 2.42 (s, 3H, CH3). 13C NMR (150 MHz, DMSO):
133.0, 128.8, 128.3, 128.2, 127.1, 127.0, 121.3, 121.2, 116.0, 115.9, 42.2.
d
161.5, 157.9, 157.1, 150.6, 150.3, 143.1, 142.8, 142.3, 142.2, 138.4,
HRMS calcd for C23H17F1N5O2 [MꢁH]þ: 414.1372; found: 414.1369.
137.4, 136.8, 136.5, 135.7, 135.0, 133.3, 132.6, 130.4, 130.2, 129.0,
128.7, 128.3, 127.6, 127.3, 127.1, 125.5, 125.0, 124.5, 123.8, 118.0, 115.7,
115.4,113.1,112.8,112.1,109.1,108.9, 77.1, 60.9, 34.81. HRMS calcd for
4.1.4.7. N5-(4-fluorophenyl)-N4-(4-methyl-2-(thiophen-3-yl)
phenyl)-1H-imidazole-4,5-dicarboxamide (15h). Yield: 89%; 1H
C
24H22FN4O3 [MþH]þ: 433.1670; found: 433.1668.
NMR (500 MHz, DMSO):
d 13.64 (s, 0.7H, NH), 13.56 (s, 0.3H, NH),
13.22 (s, 0.7H, NH), 12.44 (s, 0.3H, NH), 10.46 (s, 0.3H, NH), 10.12 (s,
0.7H, NH), 7.99e7.15 (m, 13H), 2.36 (s, 3H, CH3). 13C NMR (125 MHz,
4.1.4.14. N5-(5-methyl-3-(5-methylthiophen-2-yl)pyridin-2-yl)-N4-
DMSO):
d
162.5, 156.0, 138.3, 137.1, 136.7, 135.2, 134.9, 132.9, 131.5,
phenyl-1H-imidazole-4,5-dicarboxamide (15o). Yield: 76%; 1H NMR
130.7, 130.6, 130.0, 129.2, 128.6, 128.6, 128.4, 128.4, 127.0, 126.2,
124.9, 124.2, 124.0, 122.9, 199.9, 121.2, 121.2, 116.0, 115.8, 115.2, 115.1,
20.6. HRMS calcd for C22H18F1N5O2 S1 [MꢁH]þ: 421.1128; found:
421.1117.
(600 MHz, CDCl3):
(bs, 0.3H, NH), 10.70 (bs, 0.7H, NH), 10.54 (bs, 0.3H, NH), 8.31e7.08
(m, 8H), 6.79e6.76 (m, 1H), 2.39 (s, 3H, CH3), 2.36 (s, 3H, CH3). 13
NMR (150 MHz, CDCl3): 184.6, 149.5, 147.4, 144.6, 143.7, 141.2,
138.3,138.2,137.4,137.3,135.6,132.9,129.3,129.2,129.1,128.5,127.1,
d 13.63 (bs, 1H, NH), 13.08 (bs, 0.7H, NH), 12.92
C
d
4.1.4.8. N5-(4-fluorophenyl)-N4-(2-(furan-2-yl)-4-methylphenyl)-
1H-imidazole-4,5-dicarboxamide (15i). Yield: 77%; 1H NMR
126.8, 126.3, 128.5, 17.5, 15.1. HRMS calcd for C22H19N5O2S1
[MꢁH]þ: 416.1187; found: 416.1187.

M. Saudi et al. / European Journal of Medicinal Chemistry 87 (2014) 529e539
537
4.1.5. General procedure for 18aeb
121.2, 115.7, 115.4, 42.2. HRMS calcd for C24H19F1N5O2 [MþH]þ:
Sodium dodecyl sulfate (SDS, 6 mmol) was added to a stirred
heterogeneous suspension of amine (5 mmol) in water (20 mL)
until a homogeneous solution was formed, (in case of turbidity, the
mixture was warmed to obtain a clear solution). Anhydride 17
(5 mmol) dissolved in acetonitrile (5 mL) was added to this solution
in one lot. After stirring for 1 h at room temperature, the acetoni-
trile was evaporated and the product precipitated from the aqueous
layer. To the aqueous solution containing precipitate, solid sodium
hydrogen carbonate was added pinch-wise until the effervescence
ceased and the pH was 6.0. The remaining precipitated product was
filtered, washed with water (20 mL), dried in a vacuum desiccator.
In cases where the product did not precipitate, the reaction mixture
was extracted with ethyl acetate (2 ꢂ 25 mL). The combined
organic extracts were dried with anhydrous Na2SO4 and the solvent
was removed in a rotary evaporator under reduced pressure to
yield the pure product.
428.1517; found: 428.1520.
4.1.7.2. N2-(4-fluorophenyl)-N3-(4-methyl-2-(pyridin-3-yl) phenyl)
pyrazine-2,3-dicarboxamide (20b). Yield: 74%; 1H NMR (300 MHz,
CDCl3):
d
9.02 (bs, 1H, NH), 8.69e8.54 (m, 5H), 8.27 (d, J ¼ 8.4 Hz,
1H), 7.84 (d, J ¼ 8.1 Hz, 1H), 7.70e7.66 (m, 2H), 7.36e7.26 (m, 2H),
7.11e7.03 (m, 3H), 2.39 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d
161.8, 161.5, 150.1, 149.1, 144.3, 137.3, 135.7, 134.2, 131.8, 131.0,
130.1, 123.7, 123.2, 122.2, 122.1, 116.1, 115.8, 21.1. HRMS calcd for
C
24H19F1N5O2 [MþH]þ: 428.1517; found: 428.1514.
4.1.7.3. N2-(4-fluorophenyl)-N3-(4-(pyridin-3-yl) benzyl) pyrazine-
2,3-dicarboxamide (20c). Yield: 72%; 1H NMR (300 MHz, CDCl3):
d
9.33 (bs, 1H, NH), 8.76 (bs, 1H, NH), 8.63 (s, 2H), 8.54e8.52 (d,
J ¼ 4.8 Hz, 1H), 7.86e7.27 (m, 9H), 7.05 (m, J ¼ 8.7 Hz, 2H), 4.76 (d,
J ¼ 6.0 Hz, 2H, CH2). 13C NMR (75 MHz, CDCl3):
d164.9, 161.6, 161.5,
148.6, 148.3, 147.6, 146.1, 144.6, 144.0, 137.8, 137.3, 136.3, 134.4,
133.6, 128.8, 127.6, 123.7, 122.1, 122.0, 116.0, 115.7, 43.7. HRMS calcd
for C24H19F1N5O2 [MþH]þ: 428.1517; found: 428.1517.
4.1.5.1. 3-((4-Fluorophenyl)carbamoyl)pyrazine-2-carboxylic
(18a) [26]. Yield: 72%; White solid; 1H NMR (300 MHz, DMSO):
10.86 (bs, 1H), 8.90 (s, 2H), 7.83e7.78 (m, 2H), 7.22 (t, 2H,
acid
d
J ¼ 9.0 Hz).
4.1.7.4. N2-(4-fluorophenyl)-N3-(2-(pyridin-4-yl) phenyl) pyrazine-
2,3-dicarboxamide (20d). Yield: 66%; 1H NMR (300 MHz, CDCl3):
4.1.5.2. 3-(p-Tolylcarbamoyl)pyrazine-2-carboxylic acid (18b) [27].
d
10.72 (bs, 1H, NH), 10.27 (bs, 1H, NH), 8.90e8.86 (m, 2H),
Yield: 65%; White solid; 1H NMR (300 MHz, DMSO):
d
10.68 (bs,
1H), 8.88 (s, 2H), 7.67 (d, J ¼ 8.4 Hz, 2H), 7.17 (t, J ¼ 8.4 Hz, 2H), 2.29
(s, 3H, CH3). 13C NMR (75 MHz, DMSO):
166.5, 162.3, 146.5, 145.8,
8.58e8.56 (m, 2H), 7.85e7.76 (m, 3H), 7.55e7.37 (m, 5H), 7.22 (t,
J ¼ 9.0 Hz, 2H). 13C NMR (75 MHz, CDCl3):
d 163.2,163.1, 149.8,147.0,
d
146.2, 145.1, 144.9, 135.2, 134.4, 133.6, 130.3, 129.3, 126.6, 126.2,
124.0, 121.9, 121.8, 115.7, 115.4, 79.3. HRMS calcd for C23H17F1N5O2
[MþH]þ: 414.1361; found: 414.1363.
145.6, 144.6, 136.0, 133.4, 129.3, 120.2, 20.7.
4.1.6. General procedure for 19aeb
Trifluoroacetic anhydride (1.5 equiv) was added dropwise to a
stirring solution of acid 18aeb (1 equiv) and Et3N (TEA, 3 equiv) in
1,4-dioxane (20 mL) that was kept at 0 ꢀC with an ice bath. After
15 min the yellow solution was allowed to warm to room tem-
perature and was stirred for 30 min, then it was poured in cold H2O
(100 mL). A precipitate formed, which was collected by filtration
using a Buchner funnel and washed with H2O. The product was
dried overnight under high vacuum.
4.1.7.5. N2-(4-fluorophenyl)-N3-(2-(pyridin-3-yl) phenyl) pyrazine-
2,3-dicarboxamide (20e). Yield: 76%; 1H NMR (300 MHz, CDCl3):
d
9.04 (bs, 1H, NH), 8.78 (bs, 1H, NH), 8.70e8.56 (m, 4H), 8.45 (d,
J ¼ 8.1 Hz, 1H), 7.87 (d, J ¼ 7.8 Hz, 1H), 7.72e7.69 (m, 2H),
7.52e7.7.28 (m, 4H), 7.07 (t, J ¼ 8.7 Hz, 2H). 13C NMR (75 MHz,
CDCl3):
d 161.7, 161.5, 150.1, 149.2, 144.3, 137.3, 134.4, 134.0, 133.5,
130.5, 129.9, 129.5, 125.8, 123.7, 123.1, 122.2, 122.1, 116.1, 115.8.
HRMS calcd for C23H17F1N5O2 [MþH]þ: 414.1361; found: 414.1365.
4.1.6.1. (Z)-7-((4-fluorophenyl)imino)furo[3,4-b]pyrazin-5(7H)-one
(19a). Yield: 70%; Off-white solid; 1H NMR (300 MHz, CDCl3):
4.1.7.6. N2-(4-fluorophenyl)-N3-(2-(pyridin-4-yl) benzyl) pyrazine-
2,3-dicarboxamide (20f). Yield: 68%; 1H NMR (600 MHz, DMSO):
d
9.06 (d, 2H, J ¼ 5.1 Hz), 7.73e7.69 (m, 2H), 7.15 (t, J ¼ 8.4 Hz, 2H).
13C NMR (75 MHz, CDCl3):
d
150.9, 149.5, 128.7, 128.6, 116.2, 116.0.
d
10.67 (bs, 1H, NH), 9.41 (t, J ¼ 6.0 Hz, 1H), 8.91e8.89 (m, 2H),
7.75e7.19 (m, 8H), 4.51 (d, J ¼ 6.0 Hz, 2H). 13C NMR (125 MHz,
DMSO):
d 164.3, 163.6, 159.2, 157.6, 150.6, 149.0, 148.1, 145.3, 144.5,
4.1.6.2. (Z)-7-(p-tolylimino)furo[3,4-b]pyrazin-5(7H)-one
Yield: 66%; White solid; 1H NMR (300 MHz, CDCl3):
9.07e9.03 (m,
2H), 7.69e7.28 (m, 4H), 2.43 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3):
150.5, 149.0, 129.5, 126.4, 21.0.
(19b).
137.4, 135.3, 132.4, 129.0, 128.5, 127.8, 122.1, 121.5, 115.5, 115.4, 42.9.
d
HRMS calcd for C24H19F1N5O2 [MþH]þ: 428.1517; found: 428.1520.
d
4.1.7.7. N2-(4-fluorophenyl)-N3-(4-methyl-2-(pyridin-4-yl)phenyl)
pyrazine-2,3-dicarbox amide (20g). Yield: 76%; 1H NMR (300 MHz,
4.1.7. General procedure for 20aen
CDCl3):
d
8.95 (bs, 1H, NH), 8.66e8.58 (m, 5H), 8.32 (d, J ¼ 8.4 Hz,
Phthalisoimide 19aeb (1 equiv) was added to stirring solution of
the aniline/benzylamine 14aem (1.2 equiv) in THF. The mixture
was stirred overnight at room temperature. The THF was evapo-
rated on a rotavapor. The mixture was diluted with EtOAc (triple the
volume of THF) and washed three times with 1 M HCl. The organic
phase was dried with Na2SO4 and evaporated. The product was
purified by flash chromatography on silica gel.
1H), 7.72e7.67 (m, 2H), 7.43 (d, J ¼ 8.4 Hz, 2H), 7.30 (d, J ¼ 8.4 Hz,
2H), 7.13e7.7.04 (m, 3H), 2.40 (s, 3H, CH3). 13C NMR (75 MHz,
CDCl3):
d 150.0, 145.9, 144.0, 143.8, 130.9, 130.0, 129.9, 124.0, 122.6,
121.7, 121.6, 115.6, 115.3, 20.6. HRMS calcd for C24H19F1N5O2
[MþH]þ: 428.1517; found: 428.1518.
4.1.7.8. N2-(4-(pyridin-4-yl) benzyl)-N3-(p-tolyl) pyrazine-2,3-
4.1.7.1. N2-(4-fluorophenyl)-N3-(4-(pyridin-4-yl) benzyl) pyrazine-
2,3-dicarboxamide (20a). Yield: 76%; 1H NMR (300 MHz, DMSO):
dicarboxamide (20h). Yield: 78%; 1H NMR (300 MHz, CDCl3):
d
9.19 (bs, 1H, NH), 8.66e8.62 (m, 4H), 7.64e7.47 (m, 8H), 7.17 (d,
d
10.65 (bs, 1H, NH), 9.45 (t, J ¼ 6.6 Hz, 1H, NH), 8.88 (q, 2H,
J ¼ 8.1 Hz, 3H), 4.78 (d, J ¼ 6.0 Hz, 2H, CH2), 2.36 (s, 3H, CH3). 13
C
J ¼ 2.4 Hz), 8.64e8.61 (m, 2H), 7.78e7.69 (m, 6H), 7.50 (d, J ¼ 8.4 Hz,
NMR (75 MHz, CDCl3): d 165.1, 161.2, 150.4, 148.1, 148.0, 145.8, 144.8,
2H), 7.21 (t, J ¼ 9.0 Hz, 2H), 2.39 (d, J ¼ 6.3 Hz, 2H, CH2). 13C NMR
143.8, 139.0, 137.6, 134.9, 134.8, 129.7, 128.8, 127.5, 121.7, 120.3, 43.7,
(75 MHz, DMSO):
d
164.0, 163.7, 160.1, 156.9, 150.3, 148.3, 146.9,
21.1. HRMS calcd for
424.1765.
C
25H22N5O2 [MþH]þ: 424.1768; found:
145.4, 145.3, 144.5, 140.5, 135.8, 135.5, 128.3, 126.9, 121.7, 121.6,

538
M. Saudi et al. / European Journal of Medicinal Chemistry 87 (2014) 529e539
4.1.7.9. N2-(4-methyl-2-(pyridin-3-yl) phenyl)-N3-(p-tolyl) pyrazine-
infectious doses (i.e., CCID50) of virus. After 7 days of incubation, 2%
FCS culture medium was discarded and cells were fixed with
ethanol and stained with 1% methylene blue, and EC50 and CC50
were determinate visually. The 50% effective concentration (EC50),
which is defined as the compound concentration that is required to
inhibit the virus-induced cytopathogenic effect (CPE) by 50%, and
50% cytotoxic concentration (CC50), which is defined as the com-
pound concentration that is required to inhibit the cell growth by
50%.
2,3-dicarboxamide (20i). Yield: 76%; 1H NMR (300 MHz, CDCl3):
d
8.95 (bs, 1H, NH), 8.72e8.56 (m, 5H), 8.33 (d, J ¼ 8.1 Hz, 1H), 7.89
(d, J ¼ 7.5 Hz, 1H), 7.62 (d, J ¼ 8.1 Hz, 2H), 7.38e7.14 (m, 4H), 2.42 (s,
3H, CH3), 2.37 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3):
150.2, 149.2,
d
144.5,144.1, 137.3, 135.5, 134.8,134.2,131.9, 131.0, 130.1,123.6,123.3,
120.3, 21.1. HRMS calcd for C25H22N5O2 [MþH]þ: 424.1768; found:
424.1767.
4.1.7.10. N2-(4-(pyridin-3-yl) benzyl)-N3-(p-tolyl) pyrazine-2,3-
dicarboxamide (20j). Yield: 71%; 1H NMR (300 MHz, CDCl3):
Acknowledgments
d
9.24 (bs, 1H, NH), 8.78e8.54 (m, 4H), 7.88e7.84 (m, 1H), 7.63 (d,
J ¼ 8.4 Hz, 2H), 7.56e7.17 (m, 8H), 4.79 (d, J ¼ 6.0 Hz, 2H, CH2), 2.36
(s, 3H, CH3). 13C NMR (75 MHz, CDCl3):
165.2, 161.2, 148.6, 148.3,
Milind Saudi holds a scholarship of the Erasmus Mundus
Cooperation Window. Mass spectrometry was made possible by the
support of the Hercules Foundation of the Flemish Government
(grant 20100225e7). The antiviral work was supported by the
Wellcome Trust (grant 089328), EU FP7 project SILVER (contract no
HEALTH-F3-2010-260644) and the Marie Curie Initial Training
Network “EUVIRNA”, while the chemistry part was supported by
the Rega Foundation. We are indebted to C. Biernaux for final
typesetting.
d
148.1, 145.7, 144.6,143.8,137.9,137.2, 136.4,134.9,134.7,134.4,129.7,
128.9, 127.6, 123.7, 120.3, 43.7, 21.1. HRMS calcd for C25H22N5O2
[MþH]þ: 424.1768; found: 424.1769.
4.1.7.11. N2-(2-(pyridin-4-yl) phenyl)-N3-(p-tolyl) pyrazine-2,3-
dicarboxamide (20k). Yield: 72%; 1H NMR (300 MHz, CDCl3):
d
8.95 (bs, 1H, NH), 8.68e8.51 (m, 6H), 7.63 (d, J ¼ 8.4 Hz, 2H),
7.50e7.19 (m, 7H), 2.37 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d
150.5, 146.2, 144.6, 144.1, 134.9, 134.1, 129.9, 129.8, 125.7, 124.5,
Appendix A. Supplementary data
123.0, 120.3, 21.1. HRMS calcd for C24H20N5O2 [MþH]þ: 410.1611;
found: 410.1608.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.09.062.These data include MOL
files and InChiKeys of the most important compounds described in
this article.
4.1.7.12. N2-(2-(pyridin-3-yl) phenyl)-N3-(p-tolyl) pyrazine-2,3-
dicarboxamide (20l). Yield: 68%; 1H NMR (300 MHz, CDCl3):
d
8.70 (bs, 1H, NH), 8.70e8.47 (m, 6H), 7.90 (d, J ¼ 7.8 Hz, 2H),
7.63e7.18 (m, 8H), 2.36 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3):
162.0, 160.1, 149.6, 148.5, 144.0, 143.7, 137.0, 123.2, 134.0, 133.6,
References
d
120.0, 129.2, 129.1, 125.2, 123.2, 122.8, 119.8, 20.6. HRMS calcd for
C
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