Design, synthesis and biological evaluation of novel pyridine derivatives as anticancer agents and phosphodiesterase 3 inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.06.063

Two series of 4,6-diaryl-2-imino-1,2-dihydropyridine-3-carbonitriles and their isosteric 4,6-diaryl-2-oxo-1,2-dihydropyridine-3-carbonitriles were synthesized through a combinatorial approach. The prepared analogues were evaluated for their in vitro capac

Full text of DOI:10.1016/j.bmc.2009.06.063

Bioorganic & Medicinal Chemistry 17 (2009) 5974–5982
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of novel pyridine derivatives
as anticancer agents and phosphodiesterase 3 inhibitors
a
b
c
d
Ashraf H. Abadi a, , Tamer M. Ibrahim , Khaled M. Abouzid , Jochen Lehmann , Heather N. Tinsley ,
*
Bernard D. Gary d, Gary A. Piazza d
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo 11835, Egypt
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo, Egypt
c Institute of Pharmacy, Department of Pharmaceutical/Medicinal Chemistry, Friedrich-Schiller University, Jena, Philosophenweg 14, D-07743, Germany
d Division of Drug Discovery, Department of Biochemistry and Molecular Biology, Southern Research Institute, 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 of 4,6-diaryl-2-imino-1,2-dihydropyridine-3-carbonitriles and their isosteric 4,6-diaryl-2-
oxo-1,2-dihydropyridine-3-carbonitriles were synthesized through a combinatorial approach. The pre-
pared analogues were evaluated for their in vitro capacity to inhibit PDE3A and the growth of the human
HT-29 colon adenocarcinoma tumor cell line. Compound 6-(4-bromophenyl)-4-(2-ethoxyphenyl)-2-
imino-1,2-dihydropyridine-3-carbonitrile (Id) exhibited the strongest PDE3 inhibition when cGMP but
Received 11 March 2009
Revised 19 June 2009
Accepted 28 June 2009
Available online 3 July 2009
not cAMP is the substrate with a IC50of 27 lM, which indicates a highly selective mechanism of enzyme
inhibition. On the other hand, compound 6-(1,3-benzodioxol-5-yl)-4-(2-ethoxyphenyl)-2-imino-1,2-
dihydropyridine-3-carbonitrile (Ii) was the most active in inhibiting colon tumor cell growth with a
IC50 of 3 lM. The electronic effects, steric effects and conformational aspects of Id seem to be the most
crucial for the PDE3 inhibition. Meanwhile, steric factors and the H-bonding capability seem to be the
most important factors for tumor cell growth inhibitory activity. Conversely, there is no direct correlation
between PDE3 inhibition and anticancer activity for the prepared compounds. An in silico docking exper-
iment indicates the potential involvement of other potential molecular targets such as PIM-1 kinase to
explain its tumor cell growth inhibitory activity.
Keywords:
Combinatorial chemistry
3-Cyano-2-pyridone
3-Cyano-2-iminopyridine
PDE3 inhibitor
Growth inhibition
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
inhibition, leading to high levels of cAMP and consequent inotropic
effect. Recent studies showed that PDE3, PDE4 and PDE5 are over
Cheney et al., reported novel 4, 6-diaryl-2-oxo-1,2-dihydropyr-
idine-3-carbonitriles (e.g., Fig. 1, compound 1), as inhibitors of the
oncogenic serine/threonine kinase PIM-1, which plays a role in
cancer cell survival, differentiation and proliferation. PIM-1 kinase
has been shown to be over expressed in a variety of cancer cell
lines.1 Wendt et al., showed that several compounds with the same
general formula as above but with higher lipophilic properties (e.g.,
Fig. 1, compound 2) can inhibit survivin which is a member of the
inhibitor of apoptosis family (IAP).2 The level of expression of sur-
vivin in tumor cells is often associated with poor prognosis and
shorter patient survival rates. Survivin is highly expressed in most
human tumors and fetal tissue but undetectable in most terminally
differentiated adult tissues. This fact therefore makes survivin an
ideal target for cancer therapy.3,4
expressed in cancerous cells compared with normal cells. In addi-
tion, cross inhibition of PDE3 together with other PDEs may lead to
inhibition of tumor cell growth and angiogenesis.5–9
In this work, we report upon the synthesis of novel derivatives of
the formula 4,6-diaryl-2-oxo-1,2-dihydropyridine-3-carbonitriles
and their 2-imino isosteres. The final compounds were evaluated
for their PDE3A inhibitory effect, in addition to their tumor cell
growth inhibitory using the human HT-29 colon adenocarcinoma
tumor cell line. Structure–activity relationships were studied.
2. Results and discussion
2.1. Chemistry
Milrinone (Fig. 1, compound 3) is a 3-cyano-2-oxopyridine
derivative that has been introduced to the clinic for the treatment
of congestive heart failure. Its mechanism of action involves PDE3
The general synthesis of 4,6-diaryl-2-imino-1,2-dihydropyri-
dine-3-carbonitriles (Ia–j) and 4,6-diaryl-2-oxo-1,2-dihydropyri-
dine-3-carbonitriles (IIa–j) is illustrated in Schemes 1 and 2. A
combinatorial approach was adopted utilizing in-solution phase
MCRs. Briefly, 4-bromoacetophenone or 1-(1,3-benzodioxol-5-yl)
* Corresponding author. Tel.: +20 2 27590716; fax: +20 2 27581041.
E-mail addresses: ashraf.abadi@guc.edu.eg, ahabadi@yahoo.com (A.H. Abadi).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.063

A. H. Abadi et al. / Bioorg. Med. Chem. 17 (2009) 5974–5982
5975
Cl
N
Cl
CN
O
CN
CN
OH
Br
N
N
H
N
O
O
H
H
1
2
3
Figure 1. Various 3-cyano-2-oxopyridine derivatives with potential growth inhibitory and/or antiangiogenic actions through PIM-1 kinase inhibition (compound 1), survivin
inhibition (compound 2) or PDE3 inhibition (compound 3).
ethanone were reacted with the appropriate aldehyde, namely 2-
methoxybenzaldehyde, 2-ethoxybenzaldehyde, 4-ethoxybenzalde-
hyde, thiophene-2-carboxaldehyde and thiophene-3-carboxalde-
hyde in the presence of malononitrile or ethyl cyanoacetate and
ammonium acetate. All of the products derived from 1-(1,3-ben-
zodioxol-5-yl)ethanone showed a methylene protons as a deshield-
ed singlet peak at the downfield region d ꢀ 6.1 due to their flanking
between 2 oxygen atoms. Mass spectrometry to all products derived
from 4-bromoacetophenone showed molecular ion peaks at M+ and
M++2 due to the isotopic nature of bromine atom. In addition, the
molecular ion peaks were also the base peaks signifying their stable
character. Theinfrared(IR)spectraof allderivativesshowedbandsat
2.2. Biology
All the synthesized compounds were tested for their in vitro
ability to inhibit the growth of human HT-29 colon adenocarci-
noma tumor cells and to inhibit recombinant human PDE3A. Ini-
tially, all compounds were screened at a dose of 50 lM in
triplicate, followed by a full dose–response to calculate the exact
IC50 value. Compounds displaying percentage of inhibition >70%
was determined by testing a range of 10 concentrations with at
least two replicates per concentration.
The previous biological results showed only one compound (Id)
active as PDE3A inhibitor when cGMP was used as a substrate and
seven compounds (Ia, Ib, Id, Ie, If, Ii, Ij) displayed tumor cell
growth inhibitory activity as summarized in, Table 1.
For PDE3A inhibition, the activity of (Id) with 2-ethoxyphenyl at
position 4 and 4-bromophenyl at position 6 of the lactam ring, rel-
ative to the inactivity of Ia, Ie showed the importance of the size of
the substituent (steric factors) and positional properties upon
a stretching frequency (m
) around 3400 cmꢁ1 corresponding for the
N–H showed relatively lower values of the carbonyl stretching at
m
around 1650 cmꢁ1 than the typical carbonyl stretching at a stretch-
ing frequencyaround1700 cmꢁ1. Thismay be due tothe single-bond
character of the tautomeric enol form, leading to lower absorption
frequency.
O
O
O
H
NH4OAc
O
H
Ar
NH4OAc
O
Br
Ar
O
O
O
N
CH2(CN)2
O
N
CH2(CN)2
O
EtOH
Reflux
EtOH
Reflux
EtOH
Reflux
EtOH
Reflux
NH
NH
O
NH
NH
NC
Ar
O
NC
Ar
NH
NC
Ar
NC
Ar
NH
O
O
O
Br
O
Br
II
II
NH2
N
NH2
N
NC
Ar
NC
Ar
O
O
Br
I
I
Scheme 1. For Ar see Table 1 and Experimental.
Scheme 2. For Ar see Table 1 and Experimental.

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A. H. Abadi et al. / Bioorg. Med. Chem. 17 (2009) 5974–5982
Table 1
Inhibitory effect of the synthesized compounds on HT-29 cells and PDE3
Compd #
Chemical structure
% Growth inhibition
Growth inhibition
IC50 M)
% PDE3 inhibition
(at 50 m)
PDE3 inhibition
IC50 M)
(at 50
lm)
(
l
l
(l
cAMP
cGMP
cAMP
cGMP
NH
NC
NH
75.03
50
16.32
ꢁ15.46
ꢁ19.78
42
ND
Ia
O
Br
NH
NC
NH
Ib
Ic
Id
Ie
98.68
35
3.57
ND
ND
ND
ND
S
Br
NH
NC
NH
ꢁ5
NDa
15
S
Br
NH
NC
O
NH
99.78
13
37.92
125.53
27
Br
NH
NH
NC
75
14.19
30
50
O
Br
NH
NH
NC
If
95
4.17
45
35
ND
O
O
O
NH
NH
NC
S
Ig
ꢁ20
ND
5
10
ND
O
O
NH
NH
NC
Ih
45
ND
18
3
ND
O
O
S

A. H. Abadi et al. / Bioorg. Med. Chem. 17 (2009) 5974–5982
5977
Table 1 (continued)
Compd #
Chemical structure
% Growth inhibition
Growth inhibition
IC50 M)
% PDE3 inhibition
(at 50 m)
PDE3 inhibition
IC50 M)
(at 50
lm)
(
l
l
(l
cAMP
cGMP
cAMP
cGMP
NH
NH
NC
O
O
91.25
3
19.04
26.25
ND
Ii
O
NH
NH
NC
Ij
87.68
13.59
ꢁ20
12
45.91
6.12
3
ꢁ8.99
ND
ND
ND
ND
ND
ND
ND
O
O
O
O
NC
NH
NH
NH
NH
IIa
IIb
IIc
IId
IIe
IIf
ND
ND
ND
ND
ND
ND
15.10
O
Br
O
NC
S
4
Br
Br
Br
O
NC
ꢁ25
2
ꢁ5
S
O
NC
2.81
13.09
4.76
30
35.25
10.79
30
O
O
NC
NH
10.65
O
Br
O
NC
NH
0.00
O
O
O
(continued on next page)

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A. H. Abadi et al. / Bioorg. Med. Chem. 17 (2009) 5974–5982
Table 1 (continued)
Compd #
Chemical structure
% Growth inhibition
(at 50 m)
Growth inhibition
IC50 M)
% PDE3 inhibition
(at 50 m)
PDE3 inhibition
IC50 M)
l
(
l
l
(l
cAMP
cGMP
cAMP
cGMP
O
NC
S
NH
6.14
ND
32.31
49.64
ND
IIg
O
O
O
NC
NH
IIh
ꢁ25
8.28
3.84
ND
ND
ND
8
20
ND
ND
ND
O
O
S
O
NC
NH
IIi
7.65
14.38
O
O
O
O
NC
NH
IIj
10.54
22.30
O
O
O
N
CN
O
Milrinone
0
>50
77
95
11.4
3.6
N
H
a
ND: Not determined.
activity, respectively. The presence of the o-ethoxy group (Id) in
the phenyl ring leads to the non-coplanarity between the o-eth-
oxyphenyl and the substituted 1,2-dihydropyridine group in the
ligand–protein complex (Fig. 2). This non-coplanarity possibly per-
mits the optimum interaction with the binding site of PDE3A. Com-
paring the active Id to its inactive congener Ii indicates the
relevance of the nature of the substituent at position 6 of the lac-
tam. The molecular electrostatic potential surface of compound
(Id) showed 2 intense red (negative) regions similar to amrinone
which is a standard PDE3A inhibitor; due to the –CO and CN func-
tions., By comparison, the inactive congener Ii showed 4 intense
red regions with extra negative areas that may lead to non-favor-
able interaction with the receptor (Fig. 3).
As per the in vitro tumor cell growth inhibitory activity, the
2-imino-1,2-dihydropyridine derivatives, namely Ia, Ib, Id, Ie, If,
Ii and Ij were more active than their 2-pyridone congeners IIa,
IIb, IId, IIe, IIe and IIj, which indicates the potential involvement
of proton of the imino in hydrogen bonding with the receptor
responsible for the anticancer activity. For the top three growth
inhibitory compounds viz Ii > If > Ij with IC50 = 3, 4.17, 12
lM,
respectively, highlights the importance of a bulky ortho substituent
upon non coplanarity. This is confirmed from the higher activity of
Id versus Ia, IC50 = 50 and 13 lM, respectively. Comparing Ic (ac-
tive) versus Ib (inactive) showed the in vitro anticancer activity in-
creases when the electronegative S atom is at 2 position rather
than 3 position of thiophenyl group.
In silico docking experiment showed Id mostly interacts with
PDE3A through H bonding of His25 with the cyano, Thr97 with
the imino N and Glu55 with the 2NHs (Fig. 2). It worthy to mention
that the –CO isostere (IId) of Id was inactive as PDE3A inhibitor,
which may be due to the lack of the proton able to afford hydrogen
bonding with Glu55. It should be noted that Id has not been able to
inhibit PDE3A utilizing cAMP as a substrate and opens the horizons
towards substrate specific inhibition of dual PDEs for example,
PDE3.
Only compound Id showed dual cancer-PDE3 inhibitory activity
with IC50 = 13 and 27 lM, for anticancer and PDE3 inhibition
(when cGMP is the substrate), respectively; while the other active
compounds possess only anticancer activity. From these experi-
ments we conclude that PDE3 inhibition is not responsible for
the tumor cell growth inhibitory activity of these milrinone
analogs.
Docking of compound (Ii) with other potential targets, namely
PIM-1 kinase showed potential H-bonding network. The apparent

A. H. Abadi et al. / Bioorg. Med. Chem. 17 (2009) 5974–5982
5979
Figure 2. Docking of PDE3A with compound (Id) in 2D diagram (a), and a MEP on a Gaussian contact surface of the binding pocket (b).
H-bonding network resulted from the interaction of the 2-imino
group and 1-NH group with the conserved water molecule that
interacts with the PIM-1 kinase catalytic residues Asp186. Addi-
tionally, the 2-imino and 3-cyano groups are making H-bonding
interactions with PIM-1 kinase catalytic residue Lys67; Figure 4.
Obviously, the docking of the most potent compound (Ii) shows
comparable interactions with the catalytic residues as compound
1 does; therefore, the docking results suggested that PIM-1 kinase
may be a potential target that mediates the tumor cell growth
inhibitory effect. On the other hand, docking of (Ii) with survivin
shows non-specific interactions (data not shown).
3. Experimental
3.1. Chemistry
All reactions were performed with commercially available re-
agents 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 out on precoated silica gel plates (ALUGRAM SIL
G/UV254) and detection of the components was made by short
and long UV light. Melting points were determined in open capil-
Figure 3. Molecular electrostatic potential on a Connolly surface of compound (Id) (a); amrinone (b); and compound (Ii) (c). A comparable electrostatic distribution of
compound (Id) and amrinone is shown. Red is negative and blue is positive.

5980
A. H. Abadi et al. / Bioorg. Med. Chem. 17 (2009) 5974–5982
Figure 4. Docking of PIM-1 kinase with compound (Ii) in 2D diagram (a) and overlay of the reference compound 1 (green) and Ii (red) in the binding pocket of PIM1 kinase
(b).
laries using a Buchi Melting Point B-540 apparatus and are uncor-
rected. 1H NMR spectra were recorded on Varian spectrometer at
300 MHz using tetramethylsilane (TMS) as internal reference.
Chemical shift values are given in ppm at room temperature using
DMSO-d6 as a solvent; chemical shifts (d) were reported in parts
per million (ppm) downfield from TMS; multiplicities are abbrevi-
ated as: s: singlet; d: doublet; q: quartet; m: multiplet; dd: doublet
of doublet; br s: broad singlet. Yields are not optimized. Elemental
analyses were performed by Institute of Organic Chemistry, Jena
University; and the Microanalytical Unit, Faculty of Science, Cairo
University; found values were within 0.4% of the theoretical ones,
unless otherwise indicated.
3.2.3. 6-(4-Bromophenyl)-2-imino-4-(thiophen-3-yl)-1,2-
dihydropyridine-3-carbonitrile (Ic)
Yield 65%; mp 204–206 °C; IR (cmꢁ1): 3357, 2209; 1H NMR
(DMSO-d6): 6.91–7.75 (m, 10H, aromatic + –NHs); Anal. Calcd for
C16H10BrN3S: C, 53.94; H, 2.83; N, 11.80; S, 9.00. Found: C, 54.09;
H, 2.82; N, 11.91; S, 9.17.
3.2.4. 6-(4-Bromophenyl)-4-(2-ethoxyphenyl)-2-imino-1,2-
dihydropyridine-3-carbonitrile (Id)
Yield 60%; mp 281–283 °C; IR (cmꢁ1): 3448, 2202; 1H NMR
(DMSO-d6): 1.41–1.46 (t, 3H, –CH2–CH3), 4.48–4.55 (q, 2H, –CH2–
CH3), 6.85–8.06 (m, 11H, aromatic + –NHs); Anal. Calcd for
C20H16BrN3O: C, 60.93; H, 4.09; N, 10.66. Found: C, 61.11; H,
4.10; N, 10.61.
3.2. General procedure for the preparation of 4-aryl-6-(4-
bromophenyl)-2-imino-1,2-dihydropyridines-3-carbonitriles
and 4-Aryl-6(1,3-benzodioxol-5-yl)-2-imino-1,2-
dihydropyridines-3-carbonitriles (I a–j)
3.2.5. 6-(4-Bromophenyl)-4-(4-ethoxyphenyl)-2-imino-1,2-
dihydropyridine-3-carbonitrile (Ie)
Yield 50%; mp 173–175 °C; IR (cmꢁ1): 3435, 2206; 1H NMR
(DMSO-d6): 1.33–1.36 (t, 3H, –CH2–CH3), 4.06–4.09 (q, 2H, –CH2–
CH3) 6.85–8.30 (m, 11H, aromatic + –NHs); Anal. Calcd for
C20H16BrN3O: C, 60.93; H, 4.09; N, 10.66. Found: C, 60.76; H,
3.92; N, 11.03.
A mixture of p-bromoacetophenone or 30,40-methylenedioxy-
acetophenone (2.5 mmol), appropriate aldehyde (2.5 mmol), mal-
ononitrile (0.16 g, 2.5 mmol) and ammonium acetate (1.54 g,
20 mmol) in ethanol (50 ml) was heated under reflux for 18–
24 h. The reaction mixture was cooled and the formed precipi-
tate was filtered, washed with ethanol, then washed successively
with water, dried and crystallized from DMF/ethanol 1:2,
respectively.
3.2.6. 6-(1,3-Benzodioxol-5-yl)-2-imino-4-(2-methoxyphenyl)-
1,2-dihydropyridine-3-carbonitrile (If)
Yield 58%; mp 223–225 °C; IR (cmꢁ1): 3354, 2208; 1H NMR
(DMSO-d6): 3.80 (s, 3H, –OCH3), 6.08 (s, 2H, –O–CH2–O–), 6.98–
7.68 (m, 10H, aromatic + –NHs); Anal. Calcd for C20H15N3O3: C,
69.56; H, 4.38; N, 12.17. Found: C, 69.10; H, 4.09; N, 12.18.
3.2.1. 6-(4-Bromophenyl)-2-imino-4-(2-methoxyphenyl)-1,2-
dihydropyridine-3-carbonitrile (Ia)
Yield 50%; mp 255–257 °C; IR (cmꢁ1): 3446, 2201; 1H NMR
(DMSO-d6): 4.20 (s, 3H, –OCH3), 6.93–7.81 (m, 11H, aromatic + –
NHs); Anal. Calcd for C19H14BrN3O: C, 60.02; H, 3.71; N, 11.05.
Found: C, 60.15; H, 3.82; N, 11.12.
3.2.7. 6-(1,3-Benzodioxol-5-yl)-2-imino-4-(thiophen-2-yl)-1,2-
dihydropyridine-3-carbonitrile (Ig)
Yield 45%; mp 172–174 °C; IR (cmꢁ1): 3380, 2209; 1H NMR
(DMSO-d6): 6.14 (s, 2H, –O–CH2–O–), 6.95–7.58 (m, 9H, aro-
matic + –NHs); Anal. Calcd for C17H11N3O2S: C, 63.54; H, 3.45; N,
13.08; S, 9.97. Found: C, 63.64; H, 3.71; N,13.43; S, 9.94.
3.2.2. 6-(4-Bromophenyl)-2-imino-4-(thiophen-2-yl)-1,2-
dihydropyridine-3-carbonitrile (Ib)
Yield 60%; mp 243–245 °C; IR (cmꢁ1): 3355, 2213. 1H NMR
(DMSO-d6): 6.97–7.77 (m, 10H, aromatic + –NHs); Anal. Calcd for
C16H10BrN3S: C, 53.94; H, 2.83; N, 11.80; S, 9.00. Found: C, 54.14;
H, 2.92; N, 11.73; S, 9.23.
3.2.8. 6-(Benzodioxol-5-yl)-2-imino-4-(thiophen-3-yl)-1,2-
dihydropyridine-3-carbonitrile (Ih)
Yield 40%, buff crystals; mp 178–180 °C; IR (cmꢁ1): 3391, 2204;
1H NMR (DMSO-d6): 6.1 (s, 2H, –O–CH2–O–), 6.80–8.16 (m, 9H,

A. H. Abadi et al. / Bioorg. Med. Chem. 17 (2009) 5974–5982
5981
aromatic + –NHs); Anal. Calcd for C17H11N3O2S: C, 63.54; H, 3.45;
N, 13.08; S, 9.98. Found: C, 63.39; H, 3.44; N, 12.67; S, 9.66.
–CH2–CH3), 6.71–7.80 (m, 10H, aromatic + –NH–); MS (EI): m/z
396 (M+, 100%); Anal. Calcd for C20H15BrN2O2: C, 60.78; H, 3.83;
N, 7.09. Found: C, 60.48; H, 3.56; N, 6.58.
3.2.9. 6-(1,3-Benzodioxol-5-yl)-4-(2-ethoxyphenyl)-2-imino-
1,2-dihydropyridine-3-carbonitrile (Ii)
3.3.6. 6-(1,3-Benzodioxol-5-yl)-4-(2-methoxyphenyl)-2-oxo-
1,2-dihydropyridin-3-carbonitrile (IIf)
Yield 60%; mp 182–184 °C; IR (cmꢁ1): 3347, 2212; 1H NMR
(DMSO-d6): 1.25–1.30 (t, 3H, –CH2–CH3), 4.07–4.10 (q, 2H, –CH2–
CH3), 6.08 (s, 2H, –O–CH2–O–), 6.78–7.66 (m, 10H, aromatic + –
NHs); Anal. Calcd For C21H17N3O3ꢂ0.25H2O: C, 69.32; H, 4.85; N,
11.55. Found: C, 69.59; H, 4.86; N, 11.21.
Yield 50%; mp 285–287 °C; IR (cmꢁ1): 3432, 2227, 1680; 1H
NMR (DMSO-d6): 3.79 (s, 3H, –O–CH3), 6.03 (s, 2H, –O–CH2–O–),
6.12–7.49 (m, 9H, aromatic + –NH); MS (EI): m/z 346 (M+, 100%);
Anal. Calcd for C17H10N2O3ꢂ0.25H2O: C, 68.41; H, 3.99; N, 7.98.
Found: C, 68.36; H, 4.33; N, 8.07.
3.2.10. 6-(1,3-Benzodioxol-5-yl)-4-(4-ethoxyphenyl)-2-imino-
1,2-dihydropyridine-3-carbonitrile (Ij)
3.3.7. 6-(1,3-Benzodioxol-5-yl)-4-(thiophen-2-yl)-2-oxo1,2-
dihydropyridin-3-carbonitrile (IIg)
Yield 70%; mp 197–200 °C; IR (cmꢁ1): 3356, 2217; 1H NMR
(DMSO-d6): 1.32–1.37 (t, 3H, –CH2–CH3), 4.06–4.14 (q, 2H, –CH2–
CH3), 6.08 (s, 2H, –O–CH2–O–), 6.85–7.72 (m, 10H, aromatic + –
NHs); Anal. Calcd for C21H17N3O3: C, 70.18; H, 4.77; N, 11.69.
Found: C, 70.05; H, 4.91; N, 11.52.
Yield 70%; mp 312–314 °C; IR (cmꢁ1): 3440, 2213, 1637; 1H
NMR (DMSO-d6): 6.13 (s, 2H, –O–CH2–O–), 6.88–8.05 (m, 8H, aro-
matic +ꢁNH); MS (EI): m/z 322 (M+, 100%); Anal. Calcd for
C17H10N2O3Sꢂ0.25H2O: C, 62.41; H, 3.06; N, 8.58; S, 9.78. Found:
C, 62.39; H, 3.23; N, 8.59; S, 9.75.
3.3. General procedure for the preparation of 6-(4-
bromophenyl)-4-aryl-2-oxo-1,2-dihydropyridin-3-carbonitriles
and 6-(1,3-benzodioxol-5-yl)-4-aryl-2-oxo-1,2-dihydropyridin-
3-carbonitriles (IIa–j)
3.3.8. 6-(1,3-Benzodioxol-5-yl)-4-(thiophen-3-yl)-2-oxo1,2-
dihydropyridin-3-carbonitrile (IIh)
Yield 70%; mp 303–305 °C; IR (cmꢁ1): 3420, 2218, 1655; 1H
NMR (DMSO-d6): 6.12 (s, 2H, –O–CH2–O–), 6.88–8.34 (m, 8H, aro-
matic + –NH); MS (EI): m/z 322 (M+, 100%); Anal. Calcd for
C17H10N2O3Sꢂ0.25H2O: C, 62.41; H, 3.06; N, 8.56; S, 9.78. Found:
C, 62.66; H, 3.42; N, 8.55; S, 9.58.
A mixture of p-bromoacetophenone or 30,40-methylenedioxy-
acetophenone (2.5 mmol), appropriate aldehyde (2.5 mmol), ethyl
cyanoacetate (0.28 g, 2.5 mmol) and ammonium acetate (1.54 g,
20 mmol) in ethanol (50 ml) was heated under reflux for 10–
20 h. the reaction mixture was cooled and the formed precipitate
was filtered, washed with ethanol, then washed successively with
water, dried and crystallized from DMF/ethanol 1:2, respectively.
3.3.9. 6-(1,3-Benzodioxol-5-yl)-4-(2-ethoxyphenyl)-2-oxo1,2-
dihydropyridin-3-carbonitrile (IIi)
Yield 40%; mp 274–276 °C; IR (cmꢁ1): 3444, 2220, 1652; 1H
NMR (DMSO-d6): 1.27–1.31 (t, 3H, –CH2–CH3), 4.09–4.12 (q, 2H,
–CH2–CH3), 6.12 (s, 2H, –OCH2O–), 7.03–7.47 (m, 9H, aromatic
+ꢁNH); MS (EI): m/z 360 (M+, 100%); Anal. Calcd for C21H16N2O4:
C, 69.99; H, 4.48; N, 7.77. Found: C, 69.77; H, 4.23; N, 6.96.
3.3.1. 6-(4-Bromophenyl)-4-(2-methoxyphenyl)-2-oxo-1,2-
dihydropyridin-3-carbonitrile (IIa)
Yield 55%; mp 310–312 °C; IR (cmꢁ1): 3390, 2220, 1647; 1H
NMR (DMSO-d6): 3.81 (s, 3H, –OCH3), 7.07–7.83 (m, 10H, aro-
matic + –NH–), Anal. Calcd for C19H13BrN2O2: C, 59.86; H, 3.44; N,
7.35. Found: C, 59.73; H, 3.40; N, 7.34.
3.3.10. 6-(1,3-Benzodioxol-5-yl)-4-(4-ethoxyphenyl)-2-oxo1,2-
dihydropyridin-3-carbonitrile (IIj)
Yield 60%; mp 288–290 °C; IR (cmꢁ1): 3430, 2219, 1680; 1H NMR
(DMSO-d6): 1.44–1.56 (t, 3H, –CH2–CH3), 4.11–4.13 (q, 2H, –CH2–
CH3), 6.09 (s, 2H, –O–CH2–O–), 6.61–7.68 (m, 9H, aromatic +); MS
(EI): m/z 360 (M+; 100%); Anal. Calcd for C21H16N2O4ꢂ0.5H2O: C,
68.23; H, 4.33; N, 7.58. Found: C, 68.54; H, 4.44; N, 7.56.
3.3.2. 6-(4-Bromophenyl)-4-(thiophen-2-yl)-2-oxo-1,2-
dihydropyridin-3-carbonitrile (IIb)
Yield 40%; mp 330–332 °C; IR (cmꢁ1): 3446, 2220, 1653; 1H
NMR (DMSO-d6): 7.30–8.06 (m, 9H, aromatic + –NH–); Anal. Calcd
for C16H9BrN2OS: C, 53.80; H, 2.54; N, 7.84; S, 8.98. Found: C,
53.66; H, 2.65; N, 7.96; S, 8.90.
3.4. Biology
3.3.3. 6-(4-Bromophenyl)-4-(thiophen-3-yl)-2-oxo-1,2-
dihydropyridin-3-carbonitrile (IIc)
3.4.1. Cell cultures
HT-29 tumor cells were obtained from ATCC. They were grown
under standard cell culture conditions at 37 °C in a humidified
atmosphere with 5% CO2. Cells were grown in RPMI 1640 contain-
ing 5% fetal bovine serum. Cell count and viability was determined
by Trypan blue staining followed by hemocytometry. Only cultures
displaying >95% cell viability were used for experiments.
Yield 65%; mp 347–349 °C; IR (cmꢁ1): 2222, 1653; 1H NMR
(DMSO-d6): 6.98–8.35 (m, 9H, aromatic + –NH–); MS (EI); m/z
356 (M+, 100%), m/z 358 (M++2
, 99.5%); Anal. Calcd for
C16H9BrN2OS: C, 53.80; H, 2.54; N, 7.84; S, 8.98. Found: C, 53.60;
H, 2.75; N, 7.64; S, 8.71.
3.3.4. 6-(4-Bromophenyl)-4-(2-ethoxyphenyl)-2-oxo-1,2-
dihydropyridin-3-carbonitrile (IId)
3.4.2. 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 treat-
ment by the Cell Titer Glo Assay (Promega), which is a luminescent
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.10
Yield 70%; mp 281–283 °C; IR (cmꢁ1): 3442, 2221, 1647; 1H
NMR (DMSO-d6): 1.40–1.44 (t, 3H, –CH2–CH3), 4.15–4.17 (q, 2H,
–CH2–CH3), 6.70–7.71 (m, 10H, aromatic + –NH–); MS (EI): m/z
394 (M+, 100%), m/z 396 (M+2+, 83%); Anal. Calcd for C20H15BrN2O2:
C, 60.78; H, 3.83; N, 7.09. Found: C, 60.71; H, 3.73; N, 6.96.
3.3.5. 6-(4-Bromophenyl)-4-(4-ethoxyphenyl)-2-oxo-1,2-
dihydropyridin-3-carbonitrile (IIe)
3.4.3. Phosphodiesterase inhibitory activity
Yield 60%; mp 269–271 °C; IR (cmꢁ1): 3446, 2220, 1656; 1H
NMR (DMSO-d6): 1.45–2.50 (t, 3H, –CH2–CH3), 4.10–4.17 (q, 2H,
PDE activity was measured using an adaptation of the IMAPÒ
fluorescence polarization phosphodiesterase assay (Molecular

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A. H. Abadi et al. / Bioorg. Med. Chem. 17 (2009) 5974–5982
Devices). PDE hydrolysis of the fluorescent-labeled substrate al-
lows it to bind the IMAPÒ reagent, which increases fluorescence
polarization (FP). The assay used fluorescein (Fl)-cAMP and tetram-
ethylrhodamine (TAMRA)-cGMP as substrates. The different exci-
tation 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
200 retained structures; the binding site was identified from the
abundance of 30 top score conformers; the default docking pro-
cedure is done for milrinone as standards for validation in the
identified binding site and then for the selected synthesized
compounds.
Docking procedure of PIM-1 kinase was as following: (a) the co-
crystallized compound 1 was identified; therefore, the binding site
was identified with its residues. (2) Ligand interactions were com-
puted for the X-ray co-crystallized compound 1 to reveal the differ-
ent types of interaction as a validation for the coming docking
procedure. (3) The co-crystallized compound 1 is then removed
and the selected synthesized compounds are used instead. (4)
The docking was done with the default settings of the MOE–DOCK
as following: a. the option: Rotate Bonds was selected to give flex-
ible ligand-rigid receptor docking; b. the scoring function was Lon-
don dG with a replacement of Alpha Triangle; c. 30 conformers of
the ligand were retained with highest and best score by default; d.
the top score ligand-receptor docking was then demonstrated by
2D ligand-receptor interactions and by 3D MEP (Molecular Electro-
static Potential) on a Gaussian contact surface of the binding
pocket.
Docking procedure of survivin was as following: (1) Survivin is
an identical dimeric protein; therefore, the unnecessary chain was
deleted. (2) ZnSO4 moieties of co-crystallization were deleted. (3)
Then, the binding site was selected by selecting the residues L6,
L96, L98, L102, L104, F86, F93, F101, V89 and K15. These residues
were reported to form the binding site of survivin inhibitors of
compound 1.2 (4) Then the docking procedure was done by the de-
fault settings of MOE–DOCK as previously mentioned.
0.1% phosphate-free BSA as the carrier. Each well contained 20
ll
of recombinant enzyme (BPS Biosciences, San Diego, CA) and
10
10
l
l
l inhibitor. The reaction was initiated by the addition of
l of a substrate solution containing 50 nM Fl-cAMP and/or
TAMRA-cGMP. After incubating at room temperature for 60 min,
the reaction was terminated by adding 120
FP was measured with a BioTek Synergy 4.
ll of binding solution.
3.4.4. Experimental design and data analysis
Drug effects on tumor cell growth and PDE activity were mea-
sured and potency was expressed by an IC50 value (50% inhibitory
concentration). For growth assays, the IC50 value was determined
by testing a range of 8 concentrations with at least four replicates
per concentration. For enzyme assays, the IC50 value was deter-
mined by testing a range of 10 concentrations with at least two
replicates per concentration. Dose response curves were analyzed
using PrismTM 4 software (GraphPad) to calculate IC50 values using
a four parameter logistic equation.
3.5. Molecular modeling
The compounds with the correct stereochemistry and subjected
to energy minimization using Force Field-MMFF94x by Molecular
Operating Environment (MOE) software.11
Acknowledgments
The first author is grateful to the Alexander von Humboldt
foundation, Germany for sponsoring a postdoctoral research stay
in Germany. The second author is indebted to the Faculty of post-
graduate studies, German University in Cairo, for partial finance of
this research.
3.5.1. Source of target proteins
The Crystal structure of Human PIM-1 kinase complexed with
its inhibitor compound 1 (PDB ID code: 20BJ),1 survivin (PDB ID
code: 1F3H),12 and PDE3A a theoretical homology model (PDB ID
code: 1LRC) were downloaded from the Protein data bank and
opened with MOE software. The complexed inhibitor-if any-was
removed when using the synthesized compounds as ligands for
docking. MOE was also used to calculate the best score between
the ligands and the enzymes’ binding sites as given in the software
manual. MOE–Dock is used to search for favorable binding config-
urations between the ligand and the macromolecular target.
References and notes
1. Cheney, I. W.; Yan, S.; Appleby, T.; Walker, H.; Vo, T.; Yao, N.; Hamatake, R.;
Hong, Z.; Wu, J. Z. Bioorg. Med. Chem. Lett. 2007, 17, 1679.
2. Wendt, M. D.; Sun, C.; Kunzer, A.; Sauer, D.; Sarris, K.; Hoff, E.; Yu, L.;
Nettesheim, D. G.; Chen, J.; Jin, S.; Comess, K. M.; Fan, Y.; Anderson, S. N.; Isaac,
B. Bioorg. Med. Chem. Lett. 2007, 17, 3122.
3. Aqui, N. A.; Vonderheide, R. H. Cancer Biol. Ther. 2008, 7, 1888.
4. Ambrosini, G.; Adida, C.; Altieri, D. C. Nat. Med. 1997, 3, 917.
5. Gary, P.; Soh, J.-W.; Mao, Y.; Kim, M.-G.; Pamukcu, R.; Li, H.; Thompson, W. J.;
Weinstein, I. B. Clin. Cancer Res. 2000, 6, 4136.
6. Cheng, J.; Grande, J. P. Exp. Biol. Med. 2007, 232, 38.
7. Murata, T.; Shimizu, K.; Narita, M.; Manganiello, V.; Tagawa, T. Anticancer Res.
2002, 22, 3171.
8. Murata, T.; Sugatani, T.; Shimizu, K.; Manganiello, V.; Tagawa, T. Anticancer
Drugs 2001, 12, 79.
9. Moon, E.; Lee, R.; Near, R.; Weintraub, L.; Wolda, S.; Lerner, A. Clin. Cancer Res.
2002, 8, 589.
3.5.2. Energy minimization procedure
Selected compounds for docking were drawn in mol format and
all hydrogens are added, they are energy minimized using Hamil-
tonian-Force Field-MMFF94x and the partial charges are also
calculated.
10. Liu, L.; Li, H.; Underwood, T.; Lloyd, M.; David, M.; Sperl, G.; Pamukcu, R.;
Thompson, W. J. J. Pharmacol. Exp. Ther. 2001, 299, 583.
11. MOE, Chemical Computing Group Inc.: Montreal, http://www.chemcomp/com.
12. Verdecia, M. A.; Huang, H.; Dutil, E.; Kaiser, D. A.; Hunter, T.; Noel, J. P. Nat.
Struct. Biol. 2000, 7, 602.
3.5.3. Docking procedure
Milrinone was drawn and energy minimized and docked to
the PDE3A homology model (PDB ID code 1LRC) using the whole
protein as the binding site to identify the binding pocket. For the