One-pot synthesis of 4,6-Diaryl-2-oxo(imino)-1,2-dihydropyridine-3- carbonitrile; A new scaffold for p38α MAP kinase inhibition

Article abstract of DOI:10.1021/cc1000488

Two series of new compounds with the general formula 4,6-diaryl-2-oxo-1,2- dihydropyridine-3-carbonitriles and their isosteric 2-imino derivatives were synthesized by the multicomponent reaction of the appropriate acetophenone, aromatic aldehyde, ammonium acetate, and malononitrile or ethyl cyanoacetate. The products were obtained with excellent yields. The prepared compounds were evaluated for their in vitro ability to inhibit p38 α-MAP kinase. Several compounds showed p38 MAP kinase inhibitory properties with IC₅₀ as low as 0.07 μM. This is the first time to report compounds with such scaffold as p38 α-MAP kinase inhibitors. This asserts the potentiality of multicomponent reactions in drug discovery.

Full text of DOI:10.1021/cc1000488

J. Comb. Chem. 2010, 12, 559–565
559
One-Pot Synthesis of 4,6-Diaryl-2-oxo(imino)-1,2-dihydropyridine-3-
carbonitrile; a New Scaffold for p38r MAP Kinase Inhibition
Aya M. Serry,† Sabine Luik,‡ Stefan Laufer,‡ and Ashraf H. Abadi*,†
Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German UniVersity
in Cairo, Cairo 11835, Egypt, and Department of Pharmaceutical and Medicinal Chemistry, Eberhard
Karls UniVersity Tu¨bingen, Auf der Morgenstelle 8, 72076 Tu¨bingen, Germany
ReceiVed March 22, 2010
Two series of new compounds with the general formula 4,6-diaryl-2-oxo-1,2-dihydropyridine-3-carbonitriles
and their isosteric 2-imino derivatives were synthesized by the multicomponent reaction of the appropriate
acetophenone, aromatic aldehyde, ammonium acetate, and malononitrile or ethyl cyanoacetate. The products
were obtained with excellent yields. The prepared compounds were evaluated for their in vitro ability to
inhibit p38 R-MAP kinase. Several compounds showed p38 MAP kinase inhibitory properties with IC50 as
low as 0.07 µM. This is the first time to report compounds with such scaffold as p38 R-MAP kinase inhibitors.
This asserts the potentiality of multicomponent reactions in drug discovery.
with cytochrome P450.8,9 Thus, herein we report the
syntheses of novel diarylheterocycle derivatives that are
devoid of the imidazole and the pyridine moiety; the core
heterocycles are cyanopyridone and cyanoiminopyridines
with the two aryls positioned meta to each other rather than
vicinal. This is accomplished by one-pot multicomponent
reaction of the appropriate acetophenone, appropriate aro-
matic aldehyde, ammonium acetate and malononitrile or ethyl
cyanoacetate. The newly synthesized compounds were
evaluated for their inhibitory activity toward p38R MAP
kinase in vitro.
Introduction
The p38 MAP kinase is a serine/threonine kinase that is
closely associated with the cytokine driven progression of
rheumatoid arthritis, autoimmune diseases, and cancer.
Almost all the pro-inflammatory, pro-oncogenic, and pro-
angiogenic mediators are activators of the p38R MAP kinase
cascade. Moreover, p38 MAP kinase is a key-player in the
biosynthesis of pro-inflammatory cytokines, such as IL-
1ꢀΓand TNF-R both at the translational and the transcrip-
tional level. Among the four identified p38 isoforms (p38R,
p38ꢀΓ, p38γ, and p38δ), the R-form is the fully studied.1–3
Thus, antagonists of this cascade may provide a multi-
modal strategy to counteract them. To date, p38R MAP
kinase inhibitors may be classified into six main classes:
diaryl heterocycles, particularly pyridinyl- and pyrimidinyl-
imidazole derivatives, (e.g., SB203580, SB202190: Figure
1, left and middle), bicyclic 6,6-heterocycles (pyridone
derivatives, Figure 1 right) and related structures, N,N′-
diarylureas and related structures, substituted benzamides,
diaryl ketones, and indole amides.4–7
Despite the efficient binding of p38R MAP kinase inhibi-
tors from the diaryl heterocycle class to their target, the
development of p38R MAP kinase inhibitors particularly
from the pyridinyl-imidazole class into anti-inflammatory
drugs was truncated by their severe liver toxicity, as the
pyridinyl imidazoles were found to interact with hepatic
cytochrome P450 enzymes through chelation of their iron
by the lone pair of electrons of the pyridine and/or imidazole
ring.8 It remains unclear whether this side effect is caused
by the pyridine or the imidazole ring.
Results and Discussion
Chemistry. The general synthesis of the target 4,6-
diaryl-2-imino-1,2-dihydropyridine-3-carbonitrile 1-7,
15-17 and the isosteric 2-oxopyridine derivatives 8-14,
18-20 is illustrated in Schemes 1 and 2. Briefly, a one-
pot synthetic approach was utilized, whereby the respec-
tive acetophenone, the respective aromatic aldehyde,
ammonium acetate and malononitrile or ethyl cyanoacetate
were refluxed in ethanol for 10-14 h. Previously reported
methods to this class of compounds include sequential
reaction of the aldehyde with the acetophenone with the
aldehyde to give the corresponding chalcone, followed by
reaction with ammonium acetate and ethyl cyanoacetate
or malononitrile.10,11 In our methodology we have all the
advantages of one-pot reaction over stepwise ones in
addition to operational simplicity.12
In most of cases, the successful formation of the end
product is indicated by the formation of a fine solid
throughout the refluxing process, which was collected by
filtration followed by crystallization from N,N-dimethyl-
formamide (DMF)/ethanol in high yields. A variety of
aromatic heteroaryl, aryl aldehydes both with electron
withdrawing and electron donating substituents were
employed, and they yielded the desired products under
It remains a medicinal chemistry challenge to design novel
ligands to dissect the inhibition of p38R from interference
* To whom correspondence should be addressed. E-mail: ashraf.abadi@
guc.edu.eg. Phone: +202-27590716. Fax: +202-27581041.
† German University in Cairo.
‡ Eberhard Karls University Tu¨bingen.
10.1021/cc1000488  2010 American Chemical Society
Published on Web 06/02/2010

560 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4
Serry et al.
Figure 1. Structures of p38R MAP kinase inhibitors SB203580 (left), SB202190 (middle) and the 2-pyridone derivative Pamapimod (right)
that are structurally relevant to our synthesized compounds.
Scheme 1
shift ranging around 6.90 ppm. All compounds with 5-methyl-
furan-2-yl substitution were indicated by a singlet at about
2.50 ppm indicating the deshielding effect of the neighboring
oxygen atom. Meanwhile, compounds having trimethox-
yphenyl substituent at position 4 of the 3-cyano-2-imino
(oxo) pyridine ring showed two peaks at chemical shifts
around 3.86 for 6 protons and at around 3.75 equivalent to
3 protons. Electron impact (EI) mass spectrometry of all
products derived from 4-bromoacetophenone showed mo-
lecular ion peaks at M+ and M++2 as the element bromine
is composed of two isotopes with different masses. In
addition, the molecular ion peaks were also the base peaks
signifying their stable characters. The infrared spectra (IR)
of the 4,6-diaryl-2 imino-1,2-dihydropyridine-3-carbonitrile
derivatives showed bands at a frequency around 3300 cm-1
corresponding to the NH and a band at around 2200 cm-1
corresponding to the CN function. On the other hand, the
pyridone derivatives showed a band at around 1700 cm-1
for the lactam carbonyl. The correct elemental analysis of
all compounds and the GC/MS of particular compounds, for
example, compound 2 showed purity more than 95% (Figure
2).
Biology. All the final compounds were tested for their in
vitro ability to inhibit p38R MAP Kinase inhibitory proper-
ties. First, the percentage inhibition at a screening dose of
10 µM was performed in triplicate, then the IC50 was
determined for the compounds displaying a percentage of
inhibition >60% by testing a range of 4 concentrations with
3 replicates per concentration. Results are summarized in
Table 1. The nine active compounds as p38R MAP kinase
inhibitors were of 4-bromophenyl substituent at position 6
and 2-oxo or 2-imino 3-cyano substituent on the pyridine
ring. Derivatives with a 4-bromobiphenyl substituent at
position 6 of the pyridine 15-20 were inactive; thus a bulky
substituent at this position is deleterious for activity.
Scheme 2
Compounds 1, 2, 3, 6, and 7 bear an imino group at
position 2 of the pyridine ring, while compounds 8, 11, and
14 were of oxo substituent at the same position; compounds
8 and 11 with 2-oxo substituent were more active than their
2-imino congeners 1 and 4, meanwhile, the 2-oxo analogue
14 was less active relative to its imino analogue 7; this
indicates that there is no preference for a carbonyl or an
imino at position 2 of the pyridine ring for the MAP kinase
inhibition. It should be borne in mind that the NH and O
are isosteric functions.
Position 4 of the pyridine has been substituted by 3,4,5-
trimethoxyphenyl,5-methyl-2-furanyl,3-pyridinyl,3,4-dichloro-
phenyl, 2,4-dichlorophenyl, 3-indolyl, and 4-bromophenyl
moieties; almost all derivatives except those with 2,4-
dichlorophenyl substitution were active. This finding indi-
cates that a wide variety of aryls and with electron
similar circumstances. This indicates the applicability of
the procedure for the preparation of large libraries as well.
In H NMR spectra the aromatic proton at position 5 of
1
the dihydropyridine ring appeared as a singlet at a chemical

4,6-Diaryl-2-oxo(imino)-1,2-dihydropyridine-3-carbonitrile
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4 561
Figure 2. GC/MS of compound 2 showing purity of >95%, base peak as the molecular ion peak, and isotopic peak due to the bromine
content of the molecule.
Table 1. Inhibitory Effects of the Synthesized Compounds on
p38R MAP Kinase Enzyme
with the NH of the backbone Gly110. In addition, the
4-bromophenyl substituent at position 6 fills in a hydrophobic
pocket circumferenced by hydrophobic amino acids, namely,
Leu104, Ala51, Leu75, and Val38, Figures 4 and 5. Compar-
ing our proposed interaction with the known inhibitor
SB203580 (PDB 1A9U) it seems that the 2-imino-3-cyano
part interacts with MAP kinase in a way similar to the
pyridine of SB203580; the bromophenyl occupies the lipo-
philic pocket occupied by the corresponding fluorophenyl.
The methylfuran is directed toward a pocket surrounded by
Gly31, Ser32, and Ala34 with no specific interaction.
It is worth to mention that our compounds are devoid of
the pyridine and the imidazole rings of known p38R MAP
kinase inhibitors; thus, they are expected to be of lower iron
chelating properties, interaction with CYP enzymes, and
hence lower hepatotoxicity.13
% inhibition
% inhibition
cpd. no.
(10 µM)
IC50 (µM) cpd. no.
(10 µM)
IC50 (µM)
1
2
3
4
5
6
7
8
93
87
86
64
39
87
83.5
79
58
8
2
0.07
3.7
11
12
13
14
15
16
17
18
19
20
91
49
17
70
18
17
35
44
29
27
0.12
ND
ND
4.4
ND
ND
ND
ND
ND
ND
3.68
ND
2.65
0.76
1.22
ND
ND
0.035
9
10
SB203580
90
withdrawing and electron donating substituent can be ac-
commodated at position 4 and still retaining activity. The
most active compound 2 is of the smallest substituent at
position 4, and its energy minimized form showed planarity
of its 2-methylfuranoyl substituent with the dihydropyridine
ring; thus, steric and conformational aspects of the substituent
at position 4 may be issues as well, Figure 3.
A docking experiment of compound 2 to p38R MAP
kinase enzyme showed the nitrogen of the 2-imino substituent
forms a crucial hydrogen bond with the NH of the backbone
residue Met109; meanwhile its hydrogen forms hydrogen
bonding with the carbonyl oxygen of the backbone residue
His107. The cyano function nitrogen forms a hydrogen bond
3. Experimental Section
3.1. Chemistry. All reactions were performed with com-
mercially available reagents, and they were used without
further purification. Solvents were dried by standard methods
and stored over molecular sieves. All reactions were moni-
tored by thin-layer chromatography (TLC) carried out on
precoated silica gel plates (ALUGRAM SILG/UV254), and
detection of the components was made by short and long
UV light. Melting points were determined in open capillaries

562 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4
Serry et al.
Figure 3. Energy minimized form of compound 2 (left) and compound 5 (right), showing coplanarity and non-coplanarity of the substituent
at position 4 relative to the 2-iminopyridine ring, respectively.
(m, 4H, aromatic), 7.07 (s, 2H, C2 and C6 trimethoxyphenyl),
6.90 (s, 1H, C5 cyanopyridine), 3.83 (s, 6H, 2 × -OCH3),
3.76 (s, 3H, -OCH3); Anal. Calcd. for C21H18BrN3O3: C,
57.29; H, 4.12; N, 9.54. Found: C, 57.31; H, 4.13; N 9.54.
3.2.2. 6-(4-Bromophenyl)-2-imino-4-(5-methylfuran-2-
yl)-1,2-dihydropyridine-3-carbonitrile (2). Yield 75%; mp
337-339 °C; IR (cm-1): 3355, 2217; MS (EI): m/z 355
(M++2), 353 (M+; 100%); 1H NMR (DMSO-d6): 8.20-7.71
(m, 4H, aromatic), 7.43-7.42 (d, 1H, C4 furan), 7.02 (s,
1H, C5 cyanopyridine), 6.36-6.35 (d, 1H, C3 furan), 2.42
(s, 3H, CH3); Anal. Calcd. for C17H12BrN3O: C, 57.65; H,
3.41; N, 11.86. Found: C, 57.68; H, 3.55; N, 11.53.
Figure 4. Schematic presentation of the interaction between the
3.2.3. 6-(4-Bromophenyl)-2-imino-4-(pyridin-3-yl)-1,2-
inhibitor 2 and human p38R MAP kinase enzyme. The bromophenyl
dihydropyridine-3-carbonitrile (3). Yield 85%; mp 309-310
is located in the hydrophobic pocket which is between Thr106 and
Lys53; possible hydrogen bonding is shown in dashed lines.
°C; IR (cm-1): 3359, 2210; MS (EI): m/z 352 (M++2), 350
1
(M+; 100%); H NMR (DMSO-d6): 8.83 (s, 1H, C2 pyri-
dine), 8.82-7.54 (m, 7H, aromatic), 6.90 (s, 1H, C5
using a Buchi Melting Point B-540 apparatus and are
uncorrected. FTIR spectra were recorded on a Nicolet Avatar
cyanopyridine); Anal. Calcd. for C17H11BrN4: C, 58.14; H,
380 spectrometer, 1H NMR spectra were recorded on Varian
3.16; N, 15.95. Found: C, 57.93; H, 3.61; N, 15.66.
Mercury VX-300 MHz spectrometer using DMSO-d6 as a
solvent; chemical shifts (δ) were reported in parts per million
3.2.4. 6-(4-Bromophenyl)-4-(3,4-dichlorophenyl)-2-imino-
1,2-dihydropyridine-3-carbonitrile (4). Yield 75%; mp
369-370 °C; IR (cm-1): 3365, 2216; MS (EI): m/z 419
(M++2), 417 (M+; 100%); 1H NMR (DMSO-d6): 8.11-7.59
(m, 6H, aromatic), 7.36 (s, 1H, C2 dichlorophenyl), 6.87 (s,
1H, C5 cyanopyridine); Anal. Calcd. for C18H10BrCl2N3: C,
51.58; H, 2.40; N, 10.03. Found: C, 51.16; H, 2.43; N, 10.14.
3.2.5. 6-(4-Bromophenyl)-4-(2,4-dichlorophenyl)-2-imino-
1,2-dihydropyridine-3-carbonitrile (5). Yield 70%; mp
235-237 °C; IR (cm-1): 3281, 2160; MS (EI): m/z
419(M++2), 417 (M+; 100%); 1H NMR (DMSO-d6):
8.22-7.62 (m, 8H, aromatic), 7.59 (s, 1H, C3 dichlorophe-
nyl), 6.92 (s, 1H, C5 cyanopyridine); Anal. Calcd. for C18H10-
BrCl2N3: C, 51.58; H, 2.40; N, 10.03. Found: C, 51.70; H,
2.67; N, 10.41.
3.2.6. 6-(4-Bromophenyl)-2-imino-4-(1H-indol-3-yl)-1,2-
dihydropyridine-3-carbonitrile (6). Yield 70%; mp 228-230
°C; IR (cm-1): 3310, 2212; MS (EI): m/z 390 (M++2), 388
(M+; 100%); 1H NMR (DMSO-d6): 8.56 (s, 1H, C2 indole),
8.55-7.20 (m, 9H, aromatic), 6.95 (s, 1H, C5 cyanopyri-
dine); Anal. Calcd. for C20H13BrN4: C, 61.71; H, 3.37; N,
14.39. Found: C, 62.02; H, 3.77; N, 14.09.
(ppm) downfield from TMS; multiplicities are abbreviated
as follows: s, singlet; d, doublet; q, quartet; m, multiplet;
dd, doublet of doublets; brs, broad. Mass spectra were made
on Hewlett-Packard GC-MS, model 5890, series II at an
ionization potential of 70 eV. Elemental analyses were
performed by the Microanalytical Unit, Faculty of Science,
Cairo University; the values found were within (0.4% of
the theoretical ones, unless otherwise indicated.
3.2. General Procedure for the Preparation of 4-Aryl-
6-(4-bromophenyl)-2-imino-1,2-dihydropyridine-3-carbo-
nitrile (1-7). 4-Bromoacetophenone (0.5 g, 2.5 mmol)
together with the respective aromatic aldehyde (2.5 mmol),
malononitrile (0.16 g, 2.5 mmol) and ammonium acetate
(1.50 g, 20 mmol) were refluxed for 10-14 h in ethyl alcohol
(30 mL). The precipitate formed was filtered, washed with
ethyl alcohol and recrystallized from DMF-ethyl alcohol (1:
10).
3.2.1. 6-(4-Bromophenyl)-2-imino-4-(3,4,5-trimethoxy-
phenyl)-1,2-dihydropyridine-3-carbonitrile (1). Yield 83%;
mp 300-302 °C; IR (cm-1): 3194, 2206; MS (EI): m/z 441
(M++2), 439 (M+; 100%); 1H NMR (DMSO-d6): 7.73-7.20

4,6-Diaryl-2-oxo(imino)-1,2-dihydropyridine-3-carbonitrile
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4 563
Figure 5. 3D interaction of compound 2 with human p38R MAP kinase; the NH interacts by hydrogen bonding with the backbone residues
His107 and Met109, meanwhile CN interacts by hydrogen bonding with Gly110 residue.
3.2.7. 4,6-Bis-(4-bromophenyl)-2-imino-1,2-dihydropyr-
idine-3-carbonitrile (7). Yield 80%; mp 284-285 °C; IR
(cm-1): 3355, 2217; MS (EI): m/z 429 (M++2), 427 (M+;
100%); 1H NMR (DMSO-d6): 7.77-7.56 (m, 8H, aromatic),
6.79 (s, 1H, C5 cyanopyridine); Anal. Calcd. for C18H11Br2N:
C, 50.38; H, 2.58; N, 9.79. Found: C, 50.29; H, 2.19; N,
10.13.
3.3. General Procedure for the Preparation of 4-Aryl-
6-(4-bromophenyl)-2-oxo-1,2-dihydropyridine-3 Carbo-
nitrile Derivatives (8-14). 4-Bomoacetophenone (0.5 g, 2.5
mmol) together with the aromatic aldehyde (2.5 mmol), ethyl
cyanoacetate (0.28 g, 2.5 mmol) and ammonium acetate (1.50
g, 20 mmol) were refluxed for 10-14 h in ethyl alcohol (30
mL). The precipitate formed was filtered, washed with ethyl
alcohol, dried and recrystallized fom DMF-ethyl alcohol (1:
10).
3.3.1. 6-(4-Bromophenyl)-2-oxo-4-(3,4,5-trimethoxyphe-
nyl)-1,2-dihydropyridine-3-carbonitrile (8). Yield 80%; mp
320-322 °C; IR (cm-1): 3265, 2248, 1641; MS (EI): m/z
442 (M++2), 440 (M+; 100%); 1H NMR (DMSO-d6): 12.78
(s, 1H, NH), 7.87-7.73 (m, 4H, aromatic),7.06 (s, 2H, C2
and C6 trimethoxyphenyl), 6.94 (s, 1H, C5 pyridone), 3.86
(s, 6H, 2 × -OCH3), 3.75 (s, 3H, -OCH3); Anal. Calcd. for
C21H17BrN2O4: C, 57.16; H, 3.88; N, 6.35. Found: C, 57.44;
H, 3.55; N, 6.00.
3.3.2. 6-(4-Bromophenyl)-4-(5-methylfuran-2-yl)-2-oxo-
1,2-dihydropyridine-3-carbonitrile (9). Yield 80%; mp
335-337 °C; IR (cm-1): 3378, 2221, 1657; MS (EI): m/z
356 (M++2), 354 (M+; 100%); 1H NMR (DMSO-d6):
7.82-7.76 (m, 4H, aromatic), 7.73-7.61 (d, 1H, C4 furan),
7.00 (s, 1H, C5 pyridone), 6.50-6.49 (d, 1H, C3 furan), 3.34
(s, 3H, CH3); Anal. Calcd. for C17H11BrN2O2: C, 57.49; H,
3.12; N, 7.89. Found: C, 57.77; H, 3.32; N, 7.56.
3.3.3. 6-(4-Bromophenyl)-2-oxo-4-(pyridin-3-yl)-1,2-di-
hydropyridine-3-carbonitrile (10). Yield 70%; mp 330-332
°C; IR (cm-1): 3200, 2222, 1666; MS (EI): m/z 353 (M++2),
351 (M+; 100%); 1H NMR (DMSO-d6): 12.94 (s, 1H, NH),
8.92 (s, 1H, C2 pyridine), 8.90-7.59 (m, 7H, aromatic), 7.03
(s, 1H, C5 pyridone); Anal. Calcd. for C17H10BrN3O: C,
57.98; H, 2.86; N, 11.93. Found: C, 57.66; H, 3.12; N, 11.64.
3.3.4. 6-(4-Bromophenyl)-4-(3,4-dichlorophenyl)-2-oxo-
1,2-dihydropyridine-3-carbonitrile (11). Yield 70%; mp
289-290 °C; IR (cm-1): 3310, 2211, 1692; MS (EI): m/z
420 (M++2), 418 (M+; 100%); 1H NMR (DMSO-d6):
8.04-7.55 (m, 6H, aromatic), 7.41 (s, 1H, C2 dichlorophe-
nyl), 7.02 (s, 1H, C5 pyridone); Anal. Calcd. for C18H9-
BrCl2N2O: C, 51.46; H, 2.16; N, 6.67. Found: C, 51.75; H,
2.46; N, 6.50.
3.3.5. 6-(4-Bromophenyl)-4-(2,4-dichlorophenyl)-2-oxo-
1,2-dihydropyridine-3-carbonitrile (12). Yield 75%; mp
368-370 °C; IR (cm-1): 3276, 2223, 1640; MS (EI): m/z
420 (M++2), 418 (M+; 100%); 1H NMR (DMSO-d6): 12.90
(s, 1H, NH), 7.87-7.58 (m, 6H, aromatic), 7.55 (s, 1H, C3
dichlorophenyl), 6.90 (s, 1H, C5 pyridone); Anal. Calcd. for
C18H9BrCl2N2O: C, 51.46; H, 2.16; N, 6.67. Found: C, 51.67;
H, 2.22; N, 6.36.
3.3.6. 6-(4-Bromophenyl)-4-(1H-indol-3-yl)-2-oxo-1,2-
dihydropyridine-3-carbonitrile (13). Yield 85%; mp 260-

564 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4
Serry et al.
261 °C; IR (cm-1): 3268, 2218, 1670; MS (EI): m/z
391(M++2), 389 (M+; 100%); 1H NMR (DMSO-d6): 12.71
(s, 1H, NH), 8.68 (s, 1H, C2 indole), 8.51-7.22 (m, 8H,
aromatic), 7.02 (s, 1H, C5 pyridone); Anal. Calcd. for
C20H12BrN3O: C, 61.56; H, 3.10; N, 10.77. Found: C, 61.13;
H, 3.12; N, 10.67.
(EI): m/z 518 (M++2), 516 (M+; 100%); 1H NMR (DMSO-
d6): 8.04-7.40 (m, 8H, aromatic), 7.15 (s, 2H, C2 and C6
trimethoxyphenyl), 6.90 (s, 1H, C5 pyridone), 3.87 (s, 6H,
2 × -OCH3), 3.75 (s, 3H, -OCH3); Anal. Calcd. for
C27H21BrN2O4: C, 62.68; H, 4.09; N, 5.41. Found: C, 62.98;
H, 4.08; N, 5.21.
3.3.7. 4,6-Bis(4-Bromophenyl)-2-oxo-1,2-dihydropyri-
dine-3-carbonitrile (14). Yield 90%; mp 320-322 °C; IR
(cm-1): 3277, 2219, 1639; MS (EI): m/z 430 (M++2), 428
3.5.2. 6-(4′-Bromobiphenyl-4-yl)-4-(5-methylfuran-2-
yl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (19). Yield
80%; mp 320-322 °C; IR (cm-1): 3332, 2208, 1750; MS
(EI): m/z 432 (M++2), 430 (M+; 100%); 1H NMR (DMSO-
d6): 8.05-7.56 (m, 8H, aromatic), 7.47-7.36 (d, 1H, C4
furan), 7.03 (s, 1H, C5 pyridone), 6.43-6.41 (d, 1H C3
furan), 2.61 (s, 3H, CH3); Anal. Calcd. for C23H15BrN2O2:
C, 64.05; H, 3.51; N, 6.50. Found: C, 63.80; H, 3.69; N,
6.0.
1
(M+; 100%); H NMR (DMSO-d6): 12.90 (s, 1H, NH),
7.87-7.65 (m, 8H, aromatic), 6.90 (s, 1H, pyridone); Anal.
Calcd. for C18H10Br2N2O: C, 50.27; H, 2.34; N, 6.51. Found:
C, 50.51; H, 2.60; N, 6.30.
3.4. General Procedure for the Preparation of 4-Aryl-
6-(4′-bromo-biphenyl-4-yl)-2-imino-1,2-dihydropyridine-
3-carbonitrile Derivatives (15-17). 4′-(4-Bromophenyl)ac-
etophenone (0.7 g, 2.5 mmol) together with the respective
aromatic aldehyde (2.5 mmol), malononitrile (0.160 g, 2.5
mmol), and ammonium acetate (1.5 g, 20 mmol) were
refluxed for 10-14 h in ethyl alcohol (30 mL). The
precipitate formed was filtered, washed with ethyl alcohol,
and recrystallized fom DMF-ethyl alcohol (1:10).
3.5.3. 6-(4′-Bromobiphenyl-4-yl)-4-(3,4-dichlorophenyl)-
2-oxo-1,2-dihydropyridine-3-carbonitrile (20). Yield 85%;
mp 300-304 °C; IR (cm-1): 3220, 2202, 1722; MS (EI):
1
m/z 496 (M++2), 494 (M+; 100%); H NMR (DMSO-d6):
8.12-7.42 (m, 11H, aromatic), 6.87 (s, 1H, C5 pyridone);
Anal. Calcd. for C24H13BrCl2N2O: C, 58.09; H, 2.64; N, 5.65.
Found: C, 58.01; H, 2.68; N, 5.39.
3.6. Biology. 3.6.1. p38 MAP Kinase Assay.14 Microtiter
plates were coated with 50 µL/well of the p38R MAPK
substrate ATF-2 (10 µg/mL in TBS) for 1.5 h at 37 °C. After
washing three times with bidistilled water, the remaining
open binding sites were blocked with blocking buffer (BB;
0.05% Tween 20 (Bio-Rad), 0.25% BSA, 0.02% NaN3 in
TBS) for 30 min at room temperature. Plates were washed
again, 50 µL of the respective test solution was filled into
the wells, and the plates were incubated for 1 h at 37 °C.
Test solutions containing 12 ng/well p38R MAPK were
diluted in kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, 10 mM ꢀ-Glycerophosphate, 100 µg/mL BSA, 1 mM
Dithiothreitol, 0.1 mM Na3VO4, 100 µM rATP) with or
without test substance (10-4-10-8 M).
Test substances were dissolved in DMSO to form stock
solutions of 10-2 M; all further dilution steps were carried
out in kinase buffer. After subsequent washing, plates were
blocked again with BB for 15 min followed by a fourth
washing step. Wells were filled with 50 µL of the primary
AB; Phospho-ATF-2 (Thr69/71)-Antibody (1:500 in BB) and
incubated for 1 h at 37°L of the secondary AB; Antirabbit
IgG-AP-Antibody (alkaline phosphatase conjugated) (1:4000
in BB). Then 100 µL of 4-NPP (Nitrophenylphosphate) was
pipetted in each well after a final washing step, and the color
development was measured 1.5-2 h later with an enzyme-
linked immunosorbent assay reader linked equipped with
SOFTmax PRO software at 405 nm.14
3.4.1. 6-(4′-Bromobiphenyl-4-yl)-2-imino-4-(3,4,5-tri-
methoxyphenyl)-1,2-dihydropyridine-3-carbonitrile (15).
Yield 90%; mp 307-309 °C; IR (cm-1): 3200, 2207; MS
(EI): m/z 517 (M++2), 515 (M+; 100%); 1H NMR (DMSO-
d6): 7.84-7.23 (m, 8H, aromatic), 7.10 (s, 2H, C2 and C6
trimethoxyphenyl), 7.06 (s, 1H, C5 cyanopyridine), 3.84 (s,
6H, 2 × -OCH3), 3.75 (s, 3H, -OCH3); Anal. Calcd. for
C27H22BrN3O3: C, 62.80; H, 4.29; N, 8.14. Found: C, 63.09;
H, 4.42; N, 8.55.
3.4.2. 6-(4′-Bromobiphenyl-4-yl)-2-imino-4-(5-methyl-
furan-2-yl)-1,2-dihydropyridine-3-carbonitrile (16). Yield
80%; mp 300-301 °C; IR (cm-1): 3311, 2208; MS (EI):
1
m/z 431 (M++2), 429 (M+; 100%); H NMR (DMSO-d6):
8.22-7.24 (m, 8H, aromatic), 7.17-7.04 (d, 1H, C4 furan),
6.78 (s, 1H, C5 cyanopyridine), 6.39-6.38 (d, 1H, C3 furan),
2.50 (s, 3H, CH3); Anal. Calcd. for C23H16BrN3O: C, 64.20;
H, 3.75; N, 9.77. Found: C, 64.31; H, 3.49; N, 10.14.
3.4.3. 6-(4′-Bromobiphenyl-4-yl)-4-(3,4-dichlorophenyl)-
2-imino-1,2-dihydropyridine-3-carbonitrile (17). Yield
90%; mp 298-300 °C; IR (cm-1): 3359, 2210; MS (EI):
1
m/z 495 (M++2), 493 (M+; 100%); H NMR (DMSO-d6):
8.04-7.64 (m, 10H, aromatic), 7.04 (s, 1H, C2 dichlorophe-
nyl), 6.90 (s, 1H, C5 cyanopyridine); Anal. Calcd. for
C24H14BrCl2N3: C, 58.21; H, 2.85; N, 8.49. Found: C, 58.49;
H, 3.09; N, 8.19.
3.5. General Procedure for the Preparation of 4-Aryl-
6-(4′-bromobiphenyl-4-yl)-2-oxo-1,2-dihydropyridine-3-
carbonitrile Derivatives (18-20). 4′-(4-Bromophenyl)aceto-
phenone (0.7 g, 2.5 mmol) together with the aromatic
aldehyde (2.5 mmol), ethyl cyanoacetate (0.28 g, 2.5 mmol),
and ammonium acetate (1.50 g, 20 mmol) were refluxed for
10-14 h in ethyl alcohol (30 mL). The precipitate formed
was filtered, washed with ethyl alcohol, and recrystallized
fom DMF-ethyl alcohol (1:10).
3.6.2. Molecular Modeling. 3.6.2.1. Energy Minimiza-
tion. The compounds were drawn on ChemSketch 11
software and saved as mol file; the latter were subjected to
energy minimization using the AM1 procedure. The drawn
compounds were first subjected to MM2 followed by the
semiempirical AM1 in the MOPAC module of Chem3D
Ultra 9.0; the methods were repeated consecutively until the
rms gradient <0.01 and <0.1, respectively.
3.5.1. 6-(4′-Bromobiphenyl-4-yl)-2-oxo-4-(3,4,5-trimeth-
oxyphenyl)-1,2-dihydropyridine-3-carbonitrile (18). Yield
85%; mp 320-322 °C; IR (cm-1): 3350, 2217, 1640; MS
3.6.2.2. Docking Procedure. The X-ray structures of p38R
MAP kinase enzyme (PDB ID code: 1WBW) for docking

4,6-Diaryl-2-oxo(imino)-1,2-dihydropyridine-3-carbonitrile
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4 565
(4) Liverton, N. J.; Butcher, J. W.; Claiborne, C. F.; Claremon,
D. A.; Libby, B. E.; Nguyen, K. T.; Pitzenberger, S. M.;
Selnick, H. G.; Smith, G. R.; Tebben, A.; Vacca, J. P.; Varga,
S. L.; Agarwal, L.; Dancheck, K.; Forsyth, A. J.; Fletcher,
D. S.; Frantz, B.; Hanlon, W. A.; Harper, C. F.; Hofsess, S. J.;
Kostura, M.; Lin, J.; Luell, S.; O’Neill, E. A.; O’Keefe, S. J.
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(5) Laufer, S. A.; Ahrens, G. M.; Karcher, S. C.; Hering, J. S.;
Niess, R. J. Med. Chem. 2006, 49, 7912–7915.
(6) Pargellis, C.; Tong, L.; Churchill, L.; Cirillo, F. P.; Gilmore,
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were downloaded from the official pdb database and prepared
with the Protein Preparation Wizard using default settings.
This tool is available in Maestro, the GUI of the Schro¨dinger
Software package. The co-crystallized ligand and the binding
site with its residues were identified. The old ligand was
removed and redocked to the protein to reveal the different
types of interaction as a validation for the coming docking
procedure. Compound 2 was prepared with the tool LigPrep
from the Schro¨dinger software package, using the OPLS_2005
force field. The docking was done by using the Induced Fit
Docking protocol with the XP scoring function. The func-
tional form of the OPLS force field is very similar to the
AMBER force field. The lowest energy conformation was
selected as the best.15
Acknowledgment. The authors are indebted to Ms. Verena
Schattel for her help with the docking part.
(11) Drabu, S.; Archna Singh, S.; Munirajam, S.; Kumar, N. Indian
J. Heterocycl. Chem. 2007, 16, 411–412.
(12) Padwa, A.; Bur, S. K. Tetrahedron 2007, 63, 5341–5378.
(13) Adams, J. L.; Boehm, J. C.; Gorycki, P. D.; Webb, E. F.;
Hall, R.; Sorenson, M.; Lee, J. C.; Ayrton, A.; Griswold, D. E.;
Gallagher, T. F. Bioorg. Med. Chem. Lett. 1998, 22, 3111–
3116.
(14) Laufer, S.; Thuma, S.; Peifer, C.; Greim, C.; Herweh, Y.;
Albrecht, A.; Dehner, F. Anal. Biochem. 2005, 344, 135–137.
(15) Schroedinger Suite 2008, Induced Fit Docking Protocol; Glide
Version 5.0; Schroedinger, LLC: New York, 2005.
References and Notes
(1) Cheeseright, T. J.; Holm, M.; Lehmann, F.; Luik, S.; Gottert,
M.; Melville, J. L.; Laufer, S. J. Med. Chem. 2009, 52, 4200–
420.
(2) Chen, Z.; Gibson, T. B.; Robinson, F.; Silvestro, L.; Pearson,
G.; Xu, B.; Wright, A.; Vanderbilt, C.; Cobb, M. H. Chem
ReV 2001, 101, 2449–2476.
(3) Mudgett, J. S.; Ding, J.; Guh-Siesel, L.; Chartrain, N. A.;
Yang, L.; Gopal, S.; Shen, M. M. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 10454–10459.
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