Discovery of colon tumor cell growth inhibitory agents through a combinatorial approach

Article abstract of DOI:10.1016/j.ejmech.2009.09.029

Two series with the general formula of 4,6-diaryl-2-oxo-1,2 dihydropyridine-3-carbonitriles and their isosteric 4,6-diaryl-2-imino-1,2-dihydropyridine-3-carbonitrile were synthesized through one pot reaction of the appropriate acetophenone, aldehyde, ammonium acetate with ethyl cyanoacetate or malononitrile, respectively. The synthesized compounds were evaluated for their tumor cell growth inhibitory activity against the human HT-29 colon tumor cell line, as well as their PDE3 inhibitory activity. Compound 4-(2-Ethoxyphenyl)-2-oxo-6-thiophen-3-yl-1,2-dihydropyridine-3 carbonitrile (21) showed tumor cell growth inhibitory activity with an IC₅₀ value of 1.25 μM. Meanwhile, 4-(4-Ethoxyphenyl)-2-imino-6-(thiophen-3-yl)-1,2-dihydropyridine-3-carbonitrile (26) showed inhibitory effect upon PDE3 using cAMP or cGMP as substrate. No correlation exists between PDE3 inhibition and the tumor cell growth inhibitory activity. Docking compound 21 to other possible molecular targets showed the potential to bind PIM1 Kinase.

Full text of DOI:10.1016/j.ejmech.2009.09.029

European Journal of Medicinal Chemistry 45 (2010) 90–97
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of colon tumor cell growth inhibitory agents
through a combinatorial approach
,
b
b
c,d
Ashraf H. Abadi a , Dalal A. Abouel-Ella , Jochen Lehmann , Heather N. Tinsley
,
*
Bernard D. Gary c, Gary A. Piazza c d, Mohammed A.O. Abdel-Fattah a
,
a Department of Parmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo 11835, Egypt
b Institute of Pharmacy, Department of Pharmaceutical/Medicinal Chemistry, Friedrich-Schiller University, Jena, Philosophenweg 14, D-07743, Germany
c Division of Drug Discovery, Department of Biochemistry and Molecular Biology, Southern Research Institute, Birmingham, AL 35205, USA
d Department of Pharmacology and Toxicology, The University of Alabama at Birmingham, 2000 Ninth Avenue South, Birmingham, AL 35205, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series with the general formula of 4,6-diaryl-2-oxo-1,2 dihydropyridine-3-carbonitriles and their
isosteric 4,6-diaryl-2-imino-1,2-dihydropyridine-3-carbonitrile were synthesized through one pot
reaction of the appropriate acetophenone, aldehyde, ammonium acetate with ethyl cyanoacetate or
malononitrile, respectively. The synthesized compounds were evaluated for their tumor cell growth
inhibitory activity against the human HT-29 colon tumor cell line, as well as their PDE3 inhibitory
activity. Compound 4-(2-Ethoxyphenyl)-2-oxo-6-thiophen-3-yl-1,2-dihydropyridine-3 carbonitrile (21)
Received 18 May 2009
Received in revised form
17 July 2009
Accepted 17 September 2009
Available online 20 September 2009
showed tumor cell growth inhibitory activity with an IC50 value of 1.25 mM. Meanwhile, 4-(4-Ethoxyphenyl)-
Keywords:
Multi-component reaction
Growth inhibition
2-imino-6-(thiophen-3-yl)-1,2-dihydropyridine-3-carbonitrile (26) showed inhibitory effect upon PDE3
using cAMP or cGMP as substrate. No correlation exists between PDE3 inhibition and the tumor cell
growth inhibitory activity. Docking compound 21 to other possible molecular targets showed the
potential to bind PIM1 Kinase.
Phosphodiesterase inhibition
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
carcinoma in head and neck, malignant melanoma, osteosarcoma
(HOSM-1), and also in malignant salivary gland cells (HSG) [5,6].
3-Cyano-2-Pyridones and 3-cyano-2-iminopyridines are known
to have diverse biological and pharmacological activity, particularly
antimicrobial [1], antidepressant [2], cardiotonic [3], and anticancer
activity [4]. There is much interest in the anticancer activity of these
compounds owing to different types of biological targets they
might interfere with for this effect to occur e.g. PDE3, PIM1 Kinase,
and Survivin protein.
PDE3 is a member of the phosphodiesterase super family of
isozymes and is characterized by having high affinity for both
cAMP and cGMP as substrates. Specific inhibition of PDE3s in
different tissues leads to various biological actions such as stim-
ulation of myocardial contractility, inhibition of platelet aggrega-
tion, and the relaxation of vascular smooth muscle, most probably
due to the elevation of cyclic adenosine monophosphate levels
(cAMP). Moreover, inhibition of PDE3 results in blocking both the
anti-lipolytic and the induced lipogenesis actions of insulin, most
probably through inhibiting PDE3B [4]. PDE3 is recently shown to
be over-expressed in some malignant cells such as squamous cell
The inhibition of PDE3 was able to inhibit the growth and prolif-
eration of the squamous cell carcinoma cell line HeLa, and in HSG
cells and further studies revealed that the pyridone derivative,
cilostamide– a selective PDE3 inhibitor– has synergism action to
the anti-apoptotic action of PDE4 inhibitors in leukemia cells [5–
7]. This effect may be explained by the elevation of cAMP levels
caused by PDE3 inhibition that leads to anti-angiogenic effect, and
interfering with the activation of EGRF to inhibit DNA synthesis
and cell proliferation [8,9]. However, most of the studies showed
that cross inhibition of different PDEs rather than a specific
isozyme is associated anti-angiogenesis or tumor cell growth
inhibitory activities.
PIM1 kinase is a member of a family of proteins containing
homologues PIM-2 and PIM-3, and is transcriptionally regulated by
cytokines, mitogens, and numerous growth factors. PIM1 Kinase is
responsible for the phosphorylation, and activation of a large
number of endogenous substrates that are responsible for cell
division and proliferation [10–12].
Survivin belongs to a family of proteins known as Inhibitors of
Apoptosis (IAP) and several studies have shown that survivin is
highly expressed in many common human cancers. Moreover,
survivin has a very significant role in mitotic cell division where it
* Corresponding author. Tel.: þ202 27590716; fax: þ202 27581041.
E-mail address: ashraf.abadi@guc.edu.eg (A.H. Abadi).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.09.029

A.H. Abadi et al. / European Journal of Medicinal Chemistry 45 (2010) 90–97
91
displays cell cycle-dependent expression, with a pronounced
accumulation at mitosis. Survinin over expression in tumor cells is
often associated with poor prognosis and shorter survival rates of
the patient. In addition, survivin is one of the chromosomal
passenger proteins; it complexes and localizes to Aurora B kinase in
the mitotic machinery [13–16].
stabilization of I with the hinge The role of the Br atom on the
phenyl at position 6 of the pyridone ring is less well defined from
a SAR perspective although its importance is exemplified by the
fact that replacement of the Br by a methyl group leads to a 7-
fold loss of inhibition [11].
Cl
The lead compound 6-(5-bromo-2-hydroxyphenyl)-2-oxo-4-
phenyl-1,2-dihydropyridine-3-carbonitrile I has been reported
both as PIM1 kinase inhibitor and survivin inhibitor with IC50 of
Cl
50 nm and KD of 5.7 mM, respectively. The latter compound was
N
N
successfully co-crystallized with PIM-1 kinase (PDB ID code
2OBJ). Compound I bound convincingly within the ATP-binding
site of PIM-1 suggesting an ATP-competitive inhibitory mecha-
nism. Perhaps the most significant finding from this complex
structure was the prominent interaction of the carbonyl group on
the pyridone ring with both the Lys67 side chain (interacts with
phosphates from ATP) of PIM-1 together with a hydrogen bond to
a water molecule that appeared to play an integral part of
a larger H-bonding network in this region. The apparent H-
bonding network further consisted of a second conserved water
molecule that interacted with both the PIM-1 kinase catalytic
residue Phe187. Interestingly, the only potential interaction of
Compound I with the hinge region involves a weak H-bond
between the main carbonyl of Glu 121 and an aromatic hydrogen,
potential H-bonding between the same carbonyl of Glu 121 and
the aromatic H at the C-4-position may also contribute to
OH
Br
OH
Br
N
H
O
N
H
O
I
II
Nuclear overhauser effect experiments have shown also that this
2-pyridone derivative is also able to inhibit survivin protein through
multiple interactions. Moreover, the affinity of I to survivin was
enhanced by improving the liophilicity through the introduction of
2 halogen atoms to the phenyl at position 4 of the pyridone ring
e.g., compound II with KD of 0.8
m
M. This indicates the potentiality of
O
O
+
O
NH4
O-
+
+
R
R`
O
N
N
N
O
R
N
R
N
NH
N
H
O
N
H
R`
R`
1-5, 11, 13
6-10, 12, 14
Compound
R
R`
1, 6
2,7
2-hydroxyl
2-chloro
4-bromo
4-bromo
4-bromo
4-bromo
4-bromo
3-bromo
2-bromo
3, 8
3-chloro
4, 9
2,5-dimethoxy
2,4-dimethoxy
2-hydroxyl
2-hydroxyl
5, 10
11, 12
13, 14
Scheme 1.

92
A.H. Abadi et al. / European Journal of Medicinal Chemistry 45 (2010) 90–97
O
+
O
O
NH4
O-
+
+
R
H
O
N
N
N
O
R
R
N
N
HN
HN
NH
O
15, 16
17, 18
R
Compound
15, 17
3-chlorophenyl
3-thiophene
16, 18
Scheme 2.
4,6-diaryl-3-cyano-2-pyridone skeleton as a template for further
optimization and with multimodal anticancer molecular targets.
Herein, we report upon novel derivatives with the 3-cyano-2-
pyridone skeleton and their isosteric 2-imino derivatives, their
tumor cell growth inhibitory against the human HT-29 colon
adenocarcinoma tumor cell line and the human PDE3A isozyme.
These studies provide insight to defining the scope and limitations
of this class of compounds as potential anticancer agents.
O
O
+
O
NH4
O-
+
+
S
R
O
N
N
N
O
N
R
N
R
N
H
NH
N
H
O
S
S
25-30
19-24
Compound
19, 25
R
2-hydroxy
4-ethoxy
2-ethoxy
4-methoxy
2-methoxy
4-chloro
20, 26
21, 27
22, 28
23, 29
24, 30
Scheme 3.

A.H. Abadi et al. / European Journal of Medicinal Chemistry 45 (2010) 90–97
93
Table 1
2. Chemistry
Inhibitory effect of the synthesized compounds upon HT29 cells and PDE3.
The general synthesis of the 3-cyano-2-pyridone and the
3-cyano-2-iminopyridine derivatives is illustrated in Schemes 1–3.
We utilized the in-solution one-pot synthesis. Whereby, the
respective aromatic ketone, the respective aromatic aldehyde,
ammonium acetate and ethyl cyanoacetate or malononitrile were
refluxed in ethanol [3,16]. The desired compounds were purified by
re-crystallization from a mixture of DMF-Ethanol in different ratios
or by column chromatography.
Cpd # % Growth Inhibition Growth inhibition % PDE3 PDE3
at 50
mM
IC50 m
M
Inhibition at inhibition
50 IC50 m
m
M
M
cAMP cGMP cAMP cGMP
1
2
3
4
5
6
7
8
94
5
7
33
0
23
ꢀ16
7
21
10
18
8
8
2
22
36
10
20
25
20
18
34
20
10
7
28
36
21
17
ꢀ8
27
47
13
20
70
45
36
50
40
24
9
20
0
37
0
20
0
8
ND*
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
13.3
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
4
ND
ND
ND
ND
ND
2.1
ND
ND
ND
46
ND
1.25
ND
ND
ND
ND
ND
6
All the synthesized compounds are diarylpyridine derivatives.
In the 1H-NMR spectra all compounds showed aromatic protons as
multiplet peaks at
d 6.90–8.50 ppm. The aromatic proton at posi-
9
95
2
tion 5 of the pyridine ring appeared as singlet peak at
6.90 ppm.
d about
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
ꢀ5
30
22
28
80
ꢀ10
20
25
79
ꢀ1
80
ꢀ20
20
5
44
ꢀ10
92
75
78
65
Mass Spectroscopy of the synthesized compounds showed
molecular ion peak [Mþ] corresponding to either the exact mass or
the molecular weight of the target compounds. The molecular ion
peaks were also the base peaks indicating the stable nature of these
compounds. Bromine containing derivatives showed molecular ion
peak at [Mþ] and [Mþ þ 2] in a ratio of almost1:1 due to the isotopic
nature of the Bromine atom. Meanwhile, the chlorine containing
derivatives showed molecular ion peak [Mþ] and [Mþ þ 2] in a ratio
of 3:1 indicating the isotopic nature of the chlorine atom.
Infrared spectra of all compounds showed bands at z3100–
3480 cmꢀ1 (NeH) stretching and a band at z2200 cmꢀ1 (CN)
stretching. On the other hand, the 3-cyano 2-pyridone derivatives
showed an extra band at 1640–1753 cmꢀ1 for the pyridone
carbonyl amide stretching. This indicates that the ionization status
of the carbonyl is variable and the potentiality for amide-iminol
equilibrium.
15
4
21
7
5
8
10
5
14
26
80
ꢀ10
30
ꢀ95
45
21
55
37
10
47
28
13.5
2.7
35
ND
*ND ¼ Not determined.
3. Biological results and discussion
For compounds with potent tumor cell growth inhibitory
activity that contained a carbonyl function, such as Compounds 1,
15, 19 and 21 we noticed the activity is relatively higher when the
All compounds were tested for their in vitro tumor cell growth
inhibitory activity using the human HT-29 colon tumor cell line,
which is known to express different PDEs, including PDE3. Most of
compounds were evaluated in 2 steps, first, the percentage inhi-
infrared
y
cmꢀ1 of the carbonyl is lower, specifically 1753, 1654,
1738 and 1641 cmꢀ1 versus. 21, 2.1, 46 and 1.25
m
M, respectively.
bition at a screening dose of 50 mM was performed in triplicate,
This indicates that more single bond character to the eC]O
(enolization) may be beneficial for tumor cell growth inhibitory
then for compounds displaying a percentage of inhibition >50%
were evaluated by testing a range of 10 concentrations with at least
two replicates per concentration to calculate an IC50 value.
The results are summarized in Table 1.
Only compound 26, showed significant PDE3 inhibition (>50%)
at the screening dose using either cAMP or cGMP as substrates with
almost equal IC50s but no tumor cell growth inhibitory activity.
This reveals no direct correlation between inhibiting PDE3 and
tumor cell growth inhibitory activity.
The energy minimized form of 26 (Fig. 1) showed a dihedral
angle of 91ꢁ between the phenyl and pyridone, and a dihedral
angle of 138ꢁ between the thiophen and pyridone, this non-
coplanarity seems essential for proper interaction with the
receptor and indicates the topology of the area of PDE3 with
which 26 interacts.
A series of compounds, however, displayed tumor cell growth
inhibitory activity including Compounds 1, 9, 15, 19, 21, 27, 28, and
29. Analysis of SAR reveals the following conclusions: For the
2-pyridone function versus the isosteric imino, there is no prefer-
entiality in this regard, compounds 21 and 27 are the exact isostere
of each other; both were active with IC50 ¼ 1.25 and 6
mM, respec-
tively. In addition, four of the active compounds 1, 15, 19 and 21
were 2-pyridone derivatives, while compounds 9, 27, 28 and 29
were 2-iminopyridine derivatives. Thus, the tumor cell growth
inhibitory activity of this group of derivatives appears to be
modulated by the aryl substituents at position 4 and 6.
Fig. 1. The energy minimized form of compound 26, showing non-coplanarity of the
pyridone and phenyl ring at position 4 with dihedral angle 91ꢁ and the thiophene ring
to pyridone ring, dihedral angle 138ꢁ.

94
A.H. Abadi et al. / European Journal of Medicinal Chemistry 45 (2010) 90–97
that alkoxy substituent on the phenyl and the carbonyl of the
pyridone will create negative electrostatic potential that seems
essential for activity as shown in Fig. 2. Moreover, this substituent
at the ortho position will lead to non coplanarity of the pyridone
and phenyl, a feature that appears to be essential for tumor cell
growth inhibitory activity. Comparing the activity of compound
21 relative to 20 verifies the importance of the substituent
positioning.
Position 6 of the pyridone was substituted with 4-bromophenyl,
3-bromophenyl, 2-bromophenyl, phenyl and thiophene, significant
activity is obtained with those with 4-bromophenyl, phenyl and
thiophene substitution.
AS the anticancer activity is not correlated with PDE3 inhibi-
tion, thus other targets such as survivin protein or PIM1 kinase
may be involved. As a predictive tool, docking the most active
candidate 21 to PIM1 (PDB ID code 2OBJ). Fig. 3 shows hydrogen
bonding interaction of the pyridine N and carbonyl with Lys67
which may also interact with the CN at position 3. Also, Ser 189
interacts via hydrogen bonding with the CN group. It is worthy to
mention that compound I interacts similarly with the same
protein. Interestingly, the ethoxy group of compound 21 is
involved in extra hydrogen bonding through water molecule with
Asp 167, Asp 186, and Ser 189. This may explain the high potency
of compound 21.
Fig. 2. Electrostatic potential map of compound 21 on Connolly surface (red ¼ negative);
(blue ¼ positive); (white ¼ neutral), hydrogens removed for clarification. (For interpre-
tation of the references to colour in this figure legend, the reader is referred to the web
version of this article).
activity. Compound 21 is of 2- ethoxy (electron donating)
substituent on the phenyl at position 4; thus in a mesomeric
fashion may increase the electron density of carbonyl oxygen
(H bond acceptor).
For compounds with potent tumor cell growth inhibitory
activity that contained an imino function, such as Compounds
29, 27, 9 and 28, the tumor cell growth inhibitory activity is directly
On the other hand, docking of 21 to the binding pocket of
survivin (PDB ID code 1F3H) showed no specific interaction with
the residues of the binding pocket (data not shown). From these
data we conclude that PIM1 kinase is the most appropriate
molecular target for the tumor cell growth inhibitory activity of this
series of derivatives. It should be taken into consideration that the
survivin inhibitor II is of highly lipophilic substituent at position
4 and an oxygenated aryl at position 6; meanwhile our most active
anticancer compound 21 is of an oxygenated aryl at position 4 and
a lipophilic aryl (thiophene) at position 6. This retro order of
substituents hydrophobicity nature may explain the bias towards
PIM1 kinase in our case.
proportional to the
y
cmꢀ1 of the ]NH, specifically 3102, 3242,
3360 and 3477 cmꢀ1 Versus 35, 6, 4 and 2.7
mM, respectively. This
confirms the critical role of the NH acidic protons upon activity,
although other substituents on the pyridone ring should be
considered.
Six out of the 8 active compounds, viz 1, 9,19, 27, 28 and 29 were
substituted with an alkoxy substituent particularly at the ortho
position of the phenyl at Position 4 of the pyridone, hydrogen bond
formation from the receptor may be involved. It should be noted
Gly
Asp
203
167
Asp
202
Phe
187
Asp
His
186
165
H O
2
Ser
189
O
N
S
N
H
Gly
O
188
Ser
Phe
46
49
H O
2
Lys
67
Glu
89
Fig. 3. 2D interaction of compound 21 with PIM1 kinase.

A.H. Abadi et al. / European Journal of Medicinal Chemistry 45 (2010) 90–97
95
4. Experimental
3278, 2246,1753; 1H-NMR (DMSO-d6): 7.37-8.55 (m, 9H, aromatic),
12.35 (s, 1H, NH); MS (EI): m/z 369 (Mþ þ 2), 367 (Mþ); Anal.
(C18H11BrN2O2) C, H, N.
4.1. Chemistry
All reactions were performed with commercially available
reagents and they were used without further purification. Solvents
were dried by standard methods and stored over molecular sieves.
All reactions were monitored by thin-layer chromatography (TLC)
carried on fluorescent precoated plates and detection of the
components was made by short UV light. Melting points were
determined in open capillaries using MEL-TEMP II and Buchi B-540
Melting Point apparatus and are uncorrected. FTIR spectra were
recorded on Nicolet Avatar 380 spectrometer. 1H-NMR spectra were
recorded on Varian Mercury VX-300 MHz and spectrometer. Mass
spectra were obtained with Hewlett Packard GC-MS, model 5890,
series II at an ionization potential of 70 ev.
4.1.1.7. 6-(2-Bromophenyl)-4-(2-hydroxyphenyl)-2-oxo-1,2-dihydropy-
ridine-3-carbonitrile (13). Yield 87%; mp 295–297 ꢁC; IR (cmꢀ1):
3394, 3278, 2246, 1753; 1H-NMR (DMSO-d6): 7.17-8.33 (m, 10H,
aromatic and OH), 12.5 (s, 1H, NH); MS (EI): m/z 369 (Mþ þ 2), 367
(Mþ); Anal. (C18H11BrN2O2) C, H, N.
4.1.1.8. 4-(3-Chlorophenyl)-2-oxo-6-phenyl-1,2-dihydropyridine-3-car-
bonitrile (15). Yield 83%; mp 244–246 ꢁC; IR (cmꢀ1): 3325, 2219,
1654; 1H-NMR (DMSO-d6): 6.87-7.89 (m, 10H, aromatic), 12.87 (s, 1H,
NH); MS (EI): m/z 307 (Mþþ1), 329 (Mþþ Naþ); Anal. (C18H11ClN2O)
C, H, N.
Elemental analyses were performed by Institute of Organic
Chemistry, Jena University and the Microanalytical Unit, Faculty of
Science, Cairo University; found values were within ꢂ0.4% of the
theoretical ones, unless otherwise indicated. Yields are not optimized.
4.1.1.9. 2-Oxo-6-phenyl-4-thiophen-3-yl-1,2-dihydropyridine-3-carbo-
nitrile (16). Yield 82%; mp 300–302 ꢁC; IR (cmꢀ1): 3438, 2213,
1637; 1H-NMR (DMSO-d6): 6.92 (s, 1H, aromatic), 7.52–7.89 (m,
7H aromatic), 8.35 (s, 1H, aromatic), 12.69 (s, 1H, NH); Anal
(C16H10N2OS) C, H, N.
4.1.1. General procedure for the preparation of 4,6-diaryl-2-oxo-1,2
dihydropyridine-3-carbonitriles
4.1.1.10. 4-(2-Hydroxyphenyl)-2-oxo-6-thiophen-3-yl-1,2-dihydropyr-
idine-3-carbonitrile (19). Yield 89%; mp 295–296 ꢁC; IR (cmꢀ1):
3441, 3330, 2224, 1738; 1H-NMR (DMSO-d6): 7.34-8.50 (m, 8H,
aromatic and OH), 8.65 (s, 1H, aromatic), 12.10 (s, 1H, NH); MS (EI):
m/z 294 (Mþ); Anal. (C16H10N2O2S): C, H, N.
The respective aromatic ketone (1 mmol), together with the
respective aromatic aldehyde (1 mmol), ethyl cyanoacetate
(1 mmol), and ammonium acetate (8 mmol) were dissolved in ethyl
alcohol (30 ml) and put under reflux for 10 to 12 h. The precipitate
obtained was filtered, washed with ethyl alcohol and dried. For the
purification purpose, the precipitate was subjected either to
re-crystallization from a mixture of DMF-Ethanol (1:10), or to
column chromatography on silica gel, eluting with chloroform.
4.1.1.11. 4-(4-Ethoxyphenyl)-2-oxo-6-thiophen-3-yl-1,2-dihydropyrid-
ine-3-carbonitrile (20). Yield 85%; mp 295–297 ꢁC; IR (cmꢀ1): 3449,
2212, 1637; 1H-NMR (DMSO-d6): 1.32-1.38 (t, 3H, -CH3), 4.07–4.15
(q, 2H, -OCH2), 6.90-8.51 (m, 8H, aromatic), 12.56 (s, 1H, NH); Anal.
(C18H14N2O2S) C, H, N.
4.1.1.1. 6-(4-Bromophenyl)-4-(2-hydroxyphenyl)-2-oxo-1,2-dihydropy-
ridine-3-carbonitrile (1). Yield 92%; mp 294–296 ꢁC; IR (cmꢀ1): 3394,
3278, 2246, 1753; 1H-NMR (DMSO-d6): 6.90 (s, 1H, aromatic), 7.36–
8.27 (m, 9H, aromatic and NH); MS (EI): m/z 369 (Mþ þ 2), 367 (Mþ);
Anal (C18H11BrN2O2) C, H, N.
4.1.1.12. 4-(2-Ethoxyphenyl)-2-oxo-6-thiophen-3-yl-1,2-dihydropyr-
idine-3 carbonitrile (21). Yield 78%; mp 280–281 ꢁC; IR (cmꢀ1):
3418, 2216, 1641; 1H-NMR (DMSO-d6): 1.24–1.30 (t, 3H, eCH3),
4.05–4.14 (q, 2H, eOCH2), 6.84–8.49 (m, 8H, aromatic), 12.60 (s,
1H, NH); MS (EI): m/z 322 (Mþ), 345 (Mþ þ Naþ). Anal.
(C18H14N2O2S) C, H, N.
4.1.1.2. 6-(4-Bromophenyl)-4-(2-chlorophenyl)-2-oxo-1,2-dihydropyr-
idine-3-carbonitrile (2). Yield 87%; mp 293–295 ꢁC; IR (cmꢀ1): 3300,
2223, 1640; 1H-NMR (DMSO-d6): 6.90 (s, 1H, aromatic), 7.49–7.86
(m, 9H, aromatic and NH); MS (EI): m/z 388 (Mþ þ 2), 386 (Mþ);
Anal. (C18H10BrClN2O) C, H, N.
4.1.1.13. 4-(4-Methoxyphenyl)-2-oxo-6-thiophen-3-yl-1,2-dihydropyr-
idine-3-carbonitrile (22). Yield 88%; mp 338–340 ꢁC; IR (cmꢀ1):
3453, 2221,1650; 1H-NMR (DMSO-d6): 3.83 (s, 3H, -OCH3), 6.90–8.51
(m, 8H, aromatic), 12.60 (s, 1H, NH); MS (EI): m/z 308 (Mþ), 331
(Mþ þ Naþ, 100%). Anal. (C17H12N2O2S) C, H, N.
4.1.1.3. 6-(4-Bromophenyl)-4-(3-chlorophenyl)-2-oxo-1,2-dihydropyr-
idine-3-carbonitrile (3). Yield 85%; mp 294–296 ꢁC; IR (cmꢀ1): 3300,
2221,1657; 1H-NMR (DMSO-d6): 6.90 (s,1H, aromatic), 7.57–7.90 (m,
9H, aromatic and NH); MS (EI): m/z 388 (Mþ þ 2), 386 (Mþ); Anal.
(C18H10BrClN2O) C, H, N.
4.1.1.14. 4-(2-Methoxyphenyl)-2-oxo-6-thiophen-3-yl-1,2-dihydropyr-
idine-3-carbonitrile (23). Yield 82%; mp 311–313 ꢁC; IR (cmꢀ1): 3356,
2220, 1648; 1H-NMR (DMSO-d6): 3.80 (s, 3H, eOCH3), 6.83–8.48 (m,
8H, aromatic), 12.62 (s, 1H, NH); MS (EI): m/z 308 (Mþ), 331
(Mþ þ Naþ). Anal. (C17H12N2O2S) C, H, N.
4.1.1.4. 6-(4-Bromophenyl)-4-(2,5-dimethoxyphenyl)-2-oxo-1,2-dihy-
dropyridine-3-carbonitrile (4). Yield 65%; mp 296–297 ꢁC; IR
(cmꢀ1): 3476, 2218, 1683; 1H-NMR (DMSO-d6): 3.70 (s, 3H, -OCH3),
3.86 (s, 3H, -OCH3), 6.99 (s, 1H, aromatic) 7.01–7.15 (m, 4H, aromatic
and NH), 7.71–7.74 (d, 2H, aromatic), 7.81–7.84 (d, 2H, aromatic); MS
(EI): m/z 412 (Mþ þ 2), 410 (Mþ); Anal. (C20H15BrN2O3) C, H, N.
4.1.1.15. 4-(4-Chlorophenyl)-2-oxo-6-thiophen-3-yl-1,2-dihydropyr-
idine-3-carbonitrile (24). Yield 78%; mp 310–312 ꢁC; IR (cmꢀ1):
3446, 2220, 1644; 1H-NMR (DMSO-d6): 6.94 (s, 1H, aromatic),
7.62–7.83 (m, 6H, aromatic), 8.52 (s, 1H, aromatic), 12.72 (s, 1H,
NH); Anal. (C16H9Cl N2OS) C, H, N.
4.1.1.5. 6-(4-Bromophenyl)-4-(2,4-dimethoxyphenyl)-2-oxo-1,2-dihy-
dropyridine-3-carbonitrile (5). Yield 62%; mp 274–276 ꢁC; IR (cmꢀ1):
3436, 2215, 1710; 1H-NMR (DMSO-d6): 3.80 (s, 3H, eOCH3), 3.83 (s,
3H, eOCH3), 6.65-7.77 (m, 9H, aromatic and NH); Anal.
(C20H15BrN2O3) C, H, N.
4.1.2. General procedure for the preparation of 4,6-diaryl-2-imino-
1,2-dihydropyridine-3-carbonitrile
The respective aromatic keton (1 mmol), together with the
respective aromatic aldehyde (1 mmol), malononitrile (1 mmol),
and ammonium acetate (8 mmol) were dissolved in ethyl alcohol
(30 ml) and put under reflux for 10–12 h. The precipitate obtained
4.1.1.6. 6-(3-Bromophenyl)-4-(2-hydroxyphenyl)-2-oxo-1,2-dihydropy-
ridine-3-carbonitrile (11). Yield 88%; mp 294–296 ꢁC; IR (cmꢀ1): 3394,

96
A.H. Abadi et al. / European Journal of Medicinal Chemistry 45 (2010) 90–97
was filtered, washed with ethyl alcohol and dried. For the purifi-
cation purpose, the precipitate was subjected either to re-crystal-
lization from a mixture of DMF-Ethanol (1:10), or to column
chromatography on silica gel, eluting with chloroform and/or
methylene chloride.
4.1.2.12. 4-(2-Ethoxyphenyl)-2-imino-6-(thiophen-3-yl)-1,2-dihydro-
pyridine-3-carbonitrile (27). Yield 65%; mp 242–244 ꢁC; IR (cmꢀ1):
3243, 2199; 1H-NMR (DMSO-d6): 1.38–1.43 (t, 3H, -CH3), 4.19e4.26
(q, 2H, -OCH2), 7.09e8.31 (m, 10H, aromatic and NHs); Anal.
(C18H15N3OS) C, H, N.
4.1.2.1. 6-(4-Bromophenyl)-4-(2-hydroxyphenyl)-2-imino-1,2-dihydro-
pyridine-3-carbonitrile (6). >Yield 73%; mp 294–296 ꢁC; IR (cmꢀ1):
3430, 3138, 2195; 1H-NMR (DMSO-d6): 6.87–7.72 (m, 11H, aromatic
and NHs), 9.86 (s, 1H, OH); MS (EI): m/z 368 (Mþ þ 2), 366 (Mþ). Anal.
(C18H12BrN3O) C, H, N.
4.1.2.13. 2-Imino-4-(4-methoxyphenyl)-6-(thiophen-3-yl)-1,2-dihydro-
pyridine-3-carbonitrile (28). Yield 65%; mp 270–272 ꢁC; IR (cmꢀ1):
3477, 2203; 1H-NMR (DMSO-d6): 3.82 (s, 3H, eOCH3), 6.83–8.48 (m,
10H, aromatic and NHs); Anal. (C17H13N3OS) C, H, N.
4.1.2.14. 2-Imino-4-(2-methoxyphenyl)-6-(thiophen-3-yl)-1,2-dihydro-
pyridine-3-carbonitrile (29). Yield 60%; mp 288–290 ꢁC; IR (cmꢀ1):
3102, 2205; 1H-NMR (DMSO-d6): 3.94 (s, 3H, eOCH3), 7.11–8.25 (m,10H,
aromatic and NHs); MS (EI): m/z 307 (Mþ); Anal. (C17H13N3OS) C, H, N.
4.1.2.2. 6-(4-Bromophenyl)-4-(2-chlorophenyl)-2-imino-1,2-dihydro-
pyridine-3-carbonitrile (7) [17]. Yield 75%; mp 255–257 ꢁC; IR
(cmꢀ1): 3446, 2220.
4.1.2.3. 6-(4-Bromophenyl)-4-(3-chlorophenyl)-2-imino-1,2-dihydro-
pyridine-3-carbonitrile (8) [18]. Yield 80%; mp 265–277 ꢁC; IR
(cmꢀ1): (eNH), (eCN); 1H-NMR (DMSO-d6): 6.84–8.33 (m, 11H,
aromatic and NHs); MS (EI): m/z 387 (Mþ þ 2), 385 (Mþ).
4.1.2.15. 4-(4-Chlorophenyl)-2-imino-6-(thiophen-3-yl)-1,2-dihydrop-
yridine-3-carbonitrile (30). Yield 65%; mp 270–272 ꢁC; IR (cmꢀ1):
3355, 2219; 1H-NMR (DMSO-d6): 6.79–8.30 (m, 10H, aromatic and
NHs); MS (EI): m/z 313 (Mþ þ 2), m/z 311 (Mþ); Anal. (C16H10ClN3S)
C, H, N.
4.1.2.4. 6-(4-Bromophenyl)-4-(2,5-dimethoxyphenyl)-2-imino1,2-dihy-
dropyridine3-carbonitrile (9). Yield 68%; mp 284–286 ꢁC; IR (cmꢀ1):
3360, 2217; 1H-NMR (DMSO-d6): 3.60 (s, 3H, eOCH3), 3.86 (s, 3H,
eOCH3), 7.09–7.75 (m, 10H, aromatic and NHs); MS (EI): m/z 411
(Mþ þ 2), 409 (Mþ); Anal. (C20H16BrN3O2) C, H, N.
4.2. Biology
4.2.1. Cell cultures
HT-29 tumor cells were obtained from ATCC and were grown
under standard cell culture conditions at 37 ꢁC in a humidified
atmosphere with 5% CO2. Cells were grown in RPMI 1640 con-
taining 5% fetal bovine serum. Cell count and viability was deter-
mined by Trypan blue staining followed by hemocytometry. Only
cultures displaying >95% cell viability were used for experiments.
4.1.2.5. 6-(4-Bromophenyl)-4-(2,4-dimethoxyphenyl)-2-imino-1,2-dih-
ydropyridine3-carbonitrile (10). Yield 76%; mp 225–227 ꢁC; IR
(cmꢀ1): 3448, 2215; 1H-NMR (DMSO-d6): 3.82 (s, 3H, eOCH3), 3.83
(s, 3H, eOC H3), 6.60–7.81 (m, 10H, aromatic and NHs); MS (EI): m/z
432 (Mþ þ Naþ), m/z 411 (Mþ þ 2), m/z 409 (Mþ);
Anal.(C20H16BrN3O2) C, H, N.
4.2.2. Growth assays
Tissue culture treated microtiter 96-well plates were seeded at
a density of 5000 cells/well. The plates were incubated for 18–24 h
prior to any treatment. Cell viability was measured 72 h after
treatment by the Cell Titer Glo Assay (Promega), which is a lumi-
nescent assay that is an indicator of live cells as a function of
metabolic activity and ATP content. The assay was performed
according to manufacturer’s specifications [19].
4.1.2.6. 6-(3-Bromophenyl)-4-(2-hydroxyphenyl)-2-imino-1,2-dihydro-
pyridine3-carbonitrile (12). Yield 85%; mp 291–293 ꢁC; IR (cmꢀ1):
3415, 3316, 2195; 1H-NMR (DMSO-d6): 6.85–8.96 (m, 11H, aromatic
and NHs); MS (EI): m/z 368 (Mþ þ 2), 366 (Mþ); Anal.
(C18H12BrN3O) C, H, N.
4.1.2.7. 6-(2-Bromophenyl)-4-(2-hydroxyphenyl)-2-imino-1,2-dihydro-
pyridine3-carbonitrile (14). Yield 84%; mp 284–286 ꢁC; IR (cmꢀ1):
3412, 3309, 2205; 1H-NMR (DMSO-d6): 6.88-8.98 (m, 11H, aromatic
and NHs); MS (EI): m/z 368 (Mþ þ 2), 366 (Mþ); Anal.
(C18H12BrN3O) C, H, N.
4.2.3. Phosphodiesterase assay
PDE activity was measured using an adaptation of the IMAPÒ
fluorescence polarization phosphodiesterase assay (Molecular
Devices). PDE hydrolysis of the fluorescent-labeled substrate allows
it to bind the IMAPÒ reagent, which increases fluorescence polari-
zation (FP). The assay used fluorescein (Fl)-cAMP and tetrame-
thylrhodamine (TAMRA)-cGMP as substrates. The different
excitation and emission spectra of the substrates (485–530 nm for Fl
and 530–590 nm for TAMRA) allowed for simultaneous measure-
ment of cAMP and cGMP hydrolysis in the same well. The assays
were performed in 96-well microtiter plates using a reaction buffer
containing 10 mM Tris-HCl (pH 7.2) 10 mM MgCl2, 0.05% NaN3 and
4.1.2.8. 4-(3-Chlorophenyl)-2-imino-6-phenyl-1,2-dihydropyridine3-
carbonitrile (17) [18]. Yield 70%; mp 214–216 ꢁC; IR (cmꢀ1): 3354,
2217.
4.1.2.9. 2-Imino-6-phenyl-4-(thiophen-3-yl)-1,2-dihydropyridine-3-
carbonitrile (18). Yield 62%; mp 150–152 ꢁC; IR (cmꢀ1): 3476,
2204; 1H-NMR (DMSO-d6): 6.96–8.17 (m, 11H, aromatic and NHs);
Anal. (C16H11N3S) C, H, N.
0.1% phosphate-free BSA as the carrier. Each well contained 20
recombinant enzyme (BPS Biosciences, San Diego, CA) and 10
inhibitor. The reaction was initiated by the addition of 10 l of
a substrate solution containing 50 nM Fl–cAMP and/or TAMRA–
cGMP. After incubating at room temperature for 60 minutes, the
ml of
ml
4.1.2.10. 2-Imino-4-phenyl-6-(thiophen-3-yl)-1,2-dihydropyridine-3-
carbonitrile (25). Yield 84%; mp 290–292 ꢁC; IR (cmꢀ1): 3408, 3108,
2195; 1H-NMR (DMSO-d6):7.39–8.51 (m, 10H, aromatic and NHs);
MS (EI): m/z 293 (Mþ); Anal. (C16H11N3OS) C, H, N.
m
reactionwas terminated byadding 120
measured with a BioTek Synergy 4.
ml of binding solution. FP was
4.1.2.11. 4-(4-Ethoxyphenyl)-2-imino-6-(thiophen-3-yl)-1,2-dihydropy-
ridine-3-carbonitrile (26). Yield 70%; mp 220–222 ꢁC, IR (cmꢀ1):
3200, 2198; 1H-NMR (DMSO-d6): 1.33–1.37 (t, 3H, eCH3), 4.06–4.13
(q, 2H, eOCH2), 6.99–8.13 (m, 10H, aromatic and NHs); Anal.
(C18H15N3OS) C, H, N.
4.2.4. Experimental design and data analysis
Compound effects on tumor cell growth and PDE activity were
measured and potency was expressed by an IC50 value (50%
inhibitory concentration). For growth assays, the IC50 value was

A.H. Abadi et al. / European Journal of Medicinal Chemistry 45 (2010) 90–97
97
determined by testing a range of 8 concentrations with at least four
replicates per concentration. For enzyme assays, the IC50 value was
determined by testing a range of 10 concentrations with at least
two replicates per concentration. Dose response curves were
analyzed using PrismÔ 4 software (GraphPad) to calculate IC50
values using a four parameter logistic equation.
favorable binding configurations between the ligand and the
macromolecular target. The lowest energy conformation was
selected as the best.
Acknowledgment
The first author is grateful to the Alexander von Humboldt foun-
dation Germany, for providing him with a research stay in Germany.
The second author is grateful for the Faculty of graduate studies,
German University in Cairo for partial financing of the project.
4.3. Molecular modeling
4.3.1. Molecular modeling and conformational search
ThecompoundsweredrawnonChemoffice2002andsavedasmol
file, thelatter were subjected to energy minimization using MMFF94x
and systemic conformational search using MOE software [20]. Dihe-
dral angles of the most favorable conformer were then measured.
References
[1] A. Abdel-Aziz, H.I. El-Subbagh, T. Kunieda, Bioorg. Med. Chem. 13 (2005)
4929–4935.
[2] N. Abdel-Latif, Sci. Pharm. 73 (2005) 195–216.
[3] A.A. Bekhit, A.M. Baraka, Boll. Chim. Farma. 145 (2005) 133–138.
[4] P. Thompson, V.C. Manganiello, E. Degerman, Curr. Top. Med. Chem. 7 (2007)
421–436.
4.3.2. Electrostatic potential map
[5] K. Shimizu, T. Murata, K. Okumura, V.C. Manganiello, T.T. Tagawa, Anticancer
Drugs 13 (2002) 875–880.
[6] T. Murata, T. Sugatani, K. Shimizu, V.C. Manganiello, T. Tagawa, Anticancer
Drugs 12 (2001) 79–83.
[7] E. Moon, R. Lee, R. Near, L. Weintraub, S. Wolda, A. Lerner, Clin. Cancer Res. 8
(2002) 589–595.
[8] N. Stuart, M. Donald, Mol. Pharmacol. 67 (2005) 263–272.
[9] S.H. Shafer, S.H. Phelps, C.L. Williams, Biochem. Pharmacol. 56 (1998)
1229–1236.
The relevant energy minimized compounds were opened on
MOE software, their partial charges were calculated, and their EPS
were obtained on Connolly surface.
4.3.3. Docking
For docking on the PIM1 Kinase, the crystal structure of Human
PIM1 Kinase complexed with the inhibitor was downloaded from
the Protein data bank (PDB ID code 2obj) and opened with MOE
software. The Molecular Operating Environment of docking was
used to calculate the docking energies between ligand and the
enzyme catalytic site as given in the software manual. MOE-Dock is
used to search for favorable binding configurations between the
ligand and the macromolecular target. The lowest energy confor-
mation was selected as the best.
For docking on Survivin, the crystal structure of Human Survivin
was downloaded from the Protein data bank (PDB ID code 1f3 h)
and opened with MOE software. The protein was then edited
through removing zinc and sulphate molecules, then the reported
amino acid pocket namely; L 102, F 93, L 96, L 98, L 104, V 89, K 15,
and F 86 was selected and the residue around the pocket was
extended, and the partial charges were calculated. The Molecular
Operating Environment of docking was used to calculate the
docking energies between ligand and the protein catalytic site as
given in the software manual. MOE-Dock is used to search for
[10] I.W. Cheney, S. Yan, T. Appleby, H. Walker, T. Vo, N. Yao, R. Hamatake, Z. Hong,
J.Z. Wu, Bioorg. Med. Chem. Lett. 17 (2007) 1679–1683.
[11] M.D. Wendt, C. Sun, A. Kunzer, D. Sauer, K. Sarris, E. Hoff, L. Yu,
D.G. Nettesheim, J. Chen, S. Jin, K.M. Comess, Y. Fan, S.N. Anderson, B. Isaac,
E.T. Olejniczak, P.J. Hajduk, S.H. Rosenberg, S.W. Elmore, Bioorg. Med. Chem.
Lett. 17 (2007) 3122–3129.
[12] Y. Tong, K.D. Stewart, S. Thomas, M. Przytulinska, E.F. Johnson, V. Klinghofer,
J. Leverson, O. McCall, N.B. Soni, Y. Luo, N.H. Lin, T.J. Sowin, V.L. Giranda,
T.D. Penning, Bioorg. Med. Chem. Lett. 18 (2008) 5206–5208.
[13] L. Zhonghong, L. Lianjie, Z. Changqing, H. Ying, J. Yu, L. Yan, J. Exp .Clin. Cancer
Res. 27 (2008) 20.
[14] W.V. Rankin, C.J. Henry, S.E. Turnquist, J.R. Turk, M.E. Beissenherz, J.W. Tyler,
E.B. Rucker, D.W. Knapp, C.O. Rodriguez, J.A. Green, Vet. Comp. Oncol. 6 (2008)
141–150.
[15] S. Sejeong, S. Byung-Je, C. Yong-Soon, K. Hyun-Ju, H. Nam-Chul, H. Jong-Ik,
C. Chul-Woong, J. Yong-Keun, O. Byung-Ha, Biochemistry 40 (2001) 1117–1123.
[16] A.A. Abadi, O. Al-Deeb, A. Al-Afify, H. El-Kashef, Farmaco 54 (1999) 195–201.
[17] S. Khatoon, A.K. Yadav, Phosph. Sulf. Silicon 179 (2004) 345–352.
[18] H. Popat, V. Kachadia, K. Nimavat, H.S. Joshi, J. Ind. Chem. Soc. 81 (2004)
157–159.
[19] C.M. Whitehead, K.A. Earle, J. Fetter, S. Xu, T. Hartman, D.C. Chan, T.L. Zhao,
G.A. Piazza, A.J. Klein-Szanto, R. Pamukcu, H. Alila, P.A. Bunn Jr.,
W.J. Thompson, Mol. Cancer Ther. 2 (2003) 479–488.
[20] MOE, Chemical Computing Group Inc.: Montreal, http://www.chemcomp/com.