Eco-friendly synthesis of novel cyanopyridine derivatives and their anticancer and PIM-1 kinase inhibitory activities

Article abstract of DOI:10.1016/j.ejmech.2017.04.024

Targeting Pim-1 kinase recently proved to be profitable for conquering cancer proliferation. In the current study, we report the design, synthesis and biological evaluation of two novel series of 2-amino cyanopyridine series (5a-g) and 2-oxocyanopyridine series (6a-g) targeting Pim-1 kinase. All of the newly synthesized compounds were evaluated for their in?vitro anticancer activity against a panel of three cell lines, namely, the liver cancer cell line (HepG2), the colon cancer cell line (HCT-116) and the breast cancer cell line (MCF-7). Most of the compounds showed good to moderate anti-proliferative activity against HepG2 and HCT-116?cell lines while only few compounds showed significant cytotoxic activity against MCF-7?cell line. Further, the Pim-1 kinase inhibitory activity for the two series was?evaluated where most of the tested compounds showed marked Pim-1 kinase inhibitory activity (26%–89%). Moreover, determination of the IC₅₀ values unraveled very potent molecules in the submicromolar range where compound 6c possessed an IC₅₀ value of 0.94?μM. Moreover, apoptosis studies were conducted on the most potent compound 6c to evaluate the proapoptotic potential of our compounds. Interestingly, it induced the level of active caspase 3 and boosted the Bax/Bcl2 ratio 22704 folds in comparison to the control. Finally, a molecular docking study was conducted to reveal the probable interaction with the Pim-1 kinase active site.

Full text of DOI:10.1016/j.ejmech.2017.04.024

Accepted Manuscript
Eco-friendly synthesis of novel cyanopyridine derivatives and their anticancer and
PIM-1 kinase inhibitory activities
Khaled M. Abouzid, Ghada H. Al-Ansary, Abeer M. El-Naggar
PII:
S0223-5234(17)30281-7
10.1016/j.ejmech.2017.04.024
EJMECH 9371
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 February 2017
Revised Date: 8 April 2017
Accepted Date: 11 April 2017
Please cite this article as: K.M. Abouzid, G.H. Al-Ansary, A.M. El-Naggar, Eco-friendly synthesis of novel
cyanopyridine derivatives and their anticancer and PIM-1 kinase inhibitory activities, European Journal
of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.024.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Eco-friendly synthesis of novel cyanopyridine derivatives and
their anticancer and PIM-1 kinase inhibitory activities
Khaled M. Abouzid,a Ghada H. Al-Ansary,a Abeer M. El-Naggarb
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University,
Abbassia, 11566 Cairo , Egypt.
b
Department of Chemistry, Faculty of Science, Ain Shams University, Abbassia, 11566
Cairo, Egypt
Abstract
Targeting Pim-1 kinase recently proved to be profitable for conquering cancer
proliferation. In the current study, we report the design, synthesis and biological
evaluation of two novel series of 2-amino cyanopyridine series (5a-g) and 2-
oxocyanopyridine series (6a-g) targeting Pim-1 kinase. All of the newly
synthesized compounds were evaluated for their in vitro anticancer activity
against a panel of three cell lines, namely, the liver cancer cell line (HepG2), the
colon cancer cell line (HCT-116) and the breast cancer cell line (MCF-7). Most of
the compounds showed good to moderate anti-proliferative activity against
HepG2 and HCT-116 cell lines while only few compounds showed significant
cytotoxic activity against MCF-7 cell line. Further, the Pim-1 kinase inhibitory
activity for the two series was evaluated where most of the tested compounds
showed marked Pim-1 kinase inhibitory activity (26% - 89%). Moreover,
determination of the IC50 values unraveled very potent molecules in the
submicromolar range where compound 6c possessed an IC50 value of 0.94 µM.
Moreover, apoptosis studies were conducted on the most potent compound 6c to
evaluate the proapoptotic potential of our compounds. Interestingly, it induced
the level of active caspase 3 and boosted the Bax/Bcl2 ratio 22704 folds in
comparison to the control. Finally, a molecular docking study was conducted to
reveal the probable interaction with the Pim-1 kinase active site.
Key words: Cyanopyridine; One-pot Synthesis; Anticancer; Pim-1 kinase;
Apoptosis.

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1. Introduction
Pertaining to its role in cell-cycle progression, cell survival and tumorogenesis,
the Pim-1 kinase has spurted as an attractive pharmacologic target in cancer
therapy [1,2]. Pim-1 kinase is a member of the Pim kinases (Pim-1, Pim-2 and
Pim-3); a family of serine/threonine kinases in the CAMK group
(calcium/calmodulin-dependent protein kinase) that belongs to the proviral
insertion site in moloney murine leukemia virus (Pim) proteins family [3,4].
Lacking regulatory domains, besides being constitutively active, Pim-1 kinase is
regulated at both the transcriptional and translational levels [5]. Being a member
of the Pim kinase family, Pim-1 kinase has the privilege of possessing a unique
structure of its active site thus paving the way for designing small molecules that
confer high selectivity as Pim-1 kinase inhibitors [6]. As evidenced, high Pim-1
kinase expression levels are intimately concomitant with malignancy survival
and proliferation [7]. The underlying mechanism by which Pim-1 kinase
supports the cell survival and augments its proliferation is quite complicated
[8,9]. Upregulation of Pim-1 kinase results in phosphorylation of the Bcl-2-
associated death promoter (BAD) which in turn becomes chelated in p-BAD
protein heterodimers leading to lower levels of free BAD that is capable of
binding the antiapoptotic protein Bcl-2 and triggering the release of the
proapoptotic protein Bax [8,9]. Consequently, desregulation of Pim-1 kinase
potentiates proapoptotic proteins and ultimately induces apoptosis. Moreover,
Pim-1 kinase acts synergistically with c-Myc oncogene relieving the cell
proliferation natural constraints [8-10]. Pim-1 kinase is over expressed in many
hematological malignancies as well as solid tumors as prostate, colon, pancreatic,
hepatic and breast cancers [11-13]. Accordingly, targeting Pim-1 kinase via
designing selective small molecule inhibitors is an intriguing approach for
suppressing tumor proliferation both at apoptosis induction level and
proliferation suppression [13].
Despite the avalanche of trials to discover potent Pim-1 kinase inhibitors, none
of the disclosed molecules was approved as an anticancer drug and continuous
research is being conducted to cover the unmet medical need [14]. Endeavored

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by the previous findings, we designed and synthesized novel molecules as Pim-1
kinase inhibitors.
Cheney et al described a novel series of highly potent substituted pyridone
Pim-1 kinase inhibitors [15]. Among them, compound A; a cyanopyridone
derivative (Fig. 1) was identified as a very potent Pim-1 kinase inhibitor with
activity in the nanomolar range (IC50 = 50 nM) [15]. It could also fit snugly to
Pim-1 kinase forming favorable hydrogen bonds that further accounts for its
marked potency [15]. Thus, compound A forms a good platform for designing
Pim-1 kinase inhibitors. Our structure-based design strategy is founded on
structural diversification of compound A. This is achieved through sparing the
cyanopyridine group while substituting the 2- and 4- positions with different
aryl and heteroaryl moieties (Fig.1). Thus, two novel series were designed and
synthesized; the 2-amino cayanopyridine series (5a-g) and the 2-
oxocyanopyridine series (6a-g). All of the newly synthesized compounds were
evaluated for their anticancer activity against three cell lines; HepG2, HCT-116
and MCF-7. Moreover, all the molecules were evaluated for inhibition of Pim-1
kinase and their IC50 values were recorded. As described above, Pim-1 kinase
cancer inducing potential is achieved via apoptosis suppression. Thus, inhibition
of Pim-1 kinase implies consequent apoptosis induction. Accordingly, apoptotic
studies of the most potent compound were applied in order to evaluate the
apoptosis induction potential of our compounds. Finally, a molecular docking
study was performed to explore the probable interaction of the tested
compounds with Pim-1 kinase.

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Fig. 1: Structure-based design of cyanopyridine derivatives 5a-g and 6a-g as
Pim-1 kinase inhibitors.
2. Results and discussion
2.1. Chemistry
The diversity generating potential of multicomponent reactions (MCRs) has
been recognized and their utility in preparing libraries to screen for functional
molecules is well appreciated [16-18]. Microwave irradiation of organic
reactions has rapidly gained popularity as it accelerates a variety of synthetic
transformations [19]. The microwave-enhanced procedures are particularly eco-
friendly and the protocol has the advantages of short reaction time and high
yield [20]. In our continued interest for the synthesis of biologically active
compound [21, 22], we report herein the study of a highly efficient method for
the synthesis of two new series of 4,6-diaryl-2-imino-1,2-dihydropyridine-3-
carbonitriles (5a-g) and 4,6-diaryl-2-oxo-1,2-dihydropyridine-3-carbonitriles
(6a-g) illustrated in (Scheme 1) under microwave irradiation. Thus, we carried
out the four-component condensation of methyl aromatic ketone namely, 3-oxo-
3H-benzo[f]chromene-2-carbaldehyde, 1-(thiophen-2-yl)ethan-1-one and 1-
(naphthalen-2-yl)ethan-1-one, with the appropriate aldehyde, namely
methoxybenzaldehyde, 1-naphthaldehyde and thiophene-3-carboxaldehyde in
the presence of malononitrile (3) or (ethyl cyanoacetate, cyanoacetamide)
(4a,b) and ammonium acetate in glycol were performed under microwave
irradiation for several minutes (6-8 minutes). 2-amino-3-cyanopyridine
derivatives (5a-g) and 2-oxo-3-cyanopyridine derivatives (6a-g) were obtained

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in excellent yields (89-94 %) (Table1). The infrared (IR) spectra of all products
showed bands at a stretching frequency (ν) around 3400 cm-1 corresponding to
the N–H group. Moreover, it showed relatively lower values of the carbonyl
stretching (ν) around 1640 cm-1 than the typical carbonyl stretching which is
normally around 1700 cm-1. This may be due to the single-bond character of the
tautomeric enol form, leading to lower absorption frequency. All of the products
derived from 3-oxo-3H-benzo[f]chromene-2-carbaldehyde (5a-c), (6a-c)
exhibited absorption band at 1720 cm-1 (C=O chromene). The 1H-NMR spectra of
all of the products showed a methylene proton as a deshielded singlet peak at the
downfield region δ (6.9 - 7.3) ppm due to their being flanked between 2 oxygen
atoms. The signals observed in 13C-NMR spectra of all the products are
determined to be in accordance with the recommended molecule structures.
Mass spectrometry showed molecular ion peaks at M+, M++1, M++2 in addition, to
some of important peaks.
Scheme 1. Synthetic pathways of compounds 5(a-g) and (6a-g).

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2.2. Biological evaluation
2.2.1. In vitro cytotoxic activity
The in vitro anticancer activity of the synthesized compounds 5a-g and 6a-g
was evaluated against a panel of cell lines, namely; the liver cancer cell line
(HepG2), the colon cancer cell line (HCT-116) and the breast cancer cell line
(MCF-7). 5-FU was taken as the reference drug being widely used in tumor
management. The results were expressed as IC50 values and listed in (Table 1).
The provided data reveal that most of the tested compounds have good to
moderate antiproliferative activity against HepG2 cell line. This is evinced by the
IC50 values of compounds 5b, 5c, 5f, 5g and 6a-f (IC50 = 45.8 µM, 35.7 µM, 28.27
µM, 11.7 µM, 36.26 µM, 15.28 µM, 30.5 µM, 15.48 µM, 35.87 µM and 41.6 µM,
respectively).
Table 1
Biological evaluation of the newly synthesized compounds 5a-g and 6a-g.
Cop. No
Structure
HepG2
(µM)
HCT-116
(µM)
MCF-7
(µM)
% inhibition
of Pim-1
kinase
Pim-1
Kinase
IC50(µM)
5a
299.83
NA
NA
26.00
------
45.80
35.7
30.61
80
80.73
59.1
61.55
69.23
25.5
5b
5c
19.25
103.58
122.00
88.27
76.92
-------
5d

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5e
5f
180.73
66.67
38.83
58.53
85.03
------
7.52
28.27
91.17
72.08
5g
11.9
20.51
24.89
19.23
36.73
86.50
83.07
1.16
10.6
6a
36.26
6b
15.28
25.91
25.23
79.54
1.47
6c
30.5
36.18
28.70
20.60
21.93
89
0.94
6d
15.48
72.95
6.70
6e
6f
35.87
41.60
220
28.6
187.06
228
27.1
248.60
236
77.85
84.82
81.05
4.8
3.16
10.04
S
CN
O
N
H
S
6g

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5-FU
8.1
9.9
7.9
-------
65.08
-------
4.3
Colchicine
------
------
------
------
Skimming the IC50 values of the tested compounds against HCT-116 colon
cancer cell line unravels that some of the compounds exert moderate
antiproliferative influence. Compounds 5b, 5g and 6a-e have relatively low IC50
values of 30.61 µM, 20.51 µM, 24.89 µM, 25.91 µM, 36.18 µM, 28.7 µM and 28.6
µM.
Figure 2: The effect of the synthesized compounds (5a-g) and (6a-g) on Pim-1 kinase activity.
Regrettably, most of the tested compounds showed low to moderate
antiproliferative activity against the breast cancer cell line MCF-7 with the
exception of compound 5e and compounds 6a-e that have good IC50 values as
compared to that of the reference drug, 5-FU (38.83 µM, 36.73 µM, 25.23 µM,
20.6 µM, 21.93 µM and 27.1 µM vss 7.9 µM, respectively).
The aforementioned findings collectively prove that compounds 6a-e possess
good antiproliferative activity against the three cell lines; HepG2, HCT-116 and
MCF-7 with relatively low IC50 values. Accordingly, this propounds that the 2-oxo
cyanopyridine scaffold is much more affirmative for the in vitro antiproliferative
activity compared to the 2-amino cyanopyridine scaffold.

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2.2.2. Evaluation of Pim-1 kinase inhibitory activity
Aiming to explore the probable antiproliferative activity mechanism of action
of the synthesized compounds, their Pim-1 kinase inhibitory activity was
evaluated where their percentage inhibition was determined and the IC50 values
were recorded (Table 1, Fig. 2). Colchicine, was taken as the reference drug.
Intriguingly, results unraveled that most of our compounds possess excellent
Pim-1 kinase inhibitory activity with percentage inhibition ranging from 26 % up
to 89 % where all of the compounds except compounds 5a, 5b and 5e (26 %,
61.55 % and 58.53 %) showed superior inhibitory activity to the reference drug
(65.08 %). Amazingly, compounds 5g, 6b and 6c showed the highest inhibitory
activity (86.5 %, 79.54 % and 89 %, respectively) at markedly low IC50 values of
1.16 µM, 1.47 µM and 0.94 µM, respectively. Further scrutinizing the activity data
reveals that compounds 6a-g of the oxo-cyanopyridine series (percentage
inhibition values ranging from 72.95 % up to 89 %) showed superior Pim-1
kinase inhibitory activity to the amino-cyanopyridine series; compounds 5a-g
(percentage inhibition values ranging from 26 % up to 86.5 %). Further
comparing the IC50 values of the two series affirms the profitability of the 2-oxo
group over the 2-amino group for Pim-1 kinase inhibitory activity. Compounds
5a-g showed IC50 values ranging from 25.5 µM down to 1.16 µM, whereas
compounds 6a-g possessed marked lower IC50 values ranging from 10.6 µM
down to 0.94 µM.
Having a closer look at the IC50 values and analyzing the data for each series,
aids us to gain useful SAR knowledge. For the amino series compounds 5a-g, it is
noticeable that the IC50 values of compounds 5a-c possessing a benzo-chromene
moiety at the 6-position and having variable lipophilic moieties at the 4-position
follow a certain detrimental pattern. This is illustrated, as compound 5a bearing
a p-methoxy phenyl moiety was inactive at experimental conditions with an
inhibitory percentage of only 26 % while compound 5b with a naphthyl group
shows an inhibition of 61.55 % and an IC50 value of 25.5 µM. Moreover,
compound 5c possessing a thienyl group revealed a better Pim-1 kinase

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inhibitory activity with 69.23 % and an IC50 value of only 19.25 µM. Analyzing
the results obtained for compounds 5d-f that bear a thienyl group in the 6-
position and diverse moieties in the 4-position following the same previous
pattern, reveals coherent SAR data. The IC50 values of compounds 5d-f markedly
decrease with grafting different lipophilic substituents in the 4-position.
Compound 5d (bearing a p-methoxyphenyl moiety at the 4-position) and
compound 5e (possessing a naphthyl group in the 4-position), their IC50 values
could not be determined under experimental conditions. On the other hand,
compound 5f bearing a theinyl group in the 4-position reveals enhanced Pim-1
kinase inhibitory activity with percentage inhibition of 85.03 % and IC50 values
of 7.52 µM. These results are harmonical with the previously discussed findings
highlighting the profitability of the theinyl moiety in the 4-position over the p-
methoxyphenyl and the naphthyl groups. Unexpectedly, compound 5g,
substituted with naphthyl rings at the 4 and 6-positions showed superior Pim-1
kinase inhibitory activity with 86.5 % inhibition and an IC50 value of 1.16 µM.
Exploring the Pim-1 kinase inhibitory activity of series 6a-g proved that
following the similar previous substitution pattern echoed the same Pim-1
kinase inhibitory activity. Compounds 6a-c that have a benzo-chromene group in
the 6-position and different lipophilic moieties in the 4-position following the
same substitution sequence (p-methoxyphenyl, naphthyl and theinyl,
respectively) exhibited marked decrease in their IC50 values while going through
the series where IC50 values of 5a-c were 10.6 µM, 1.47 µM and 0.94 µM,
respectively. The same results were gained when comparing the IC50 values of
compounds 6d-f (IC50 values 6.7 µM, 4.8 µM and 3.16 µM, respectively). These
results are in coincidence with the previous findings that the 4-theinyl group is
beneficial for Pim-1 kinase inhibitory activity over the p-methoxyphenyl moiety
and the naphthyl moiety.
2.2.3. Apoptosis studies
As previously discussed, the most conventionally approved mechanism of
tumor inducing activity of Pim-1 kinase is that it suppresses apoptosis via
phosphorylation of BAD. As our compounds successfully inhibited Pim-1 kinase,
then expectedly they will induce apoptosis. Thus, their potential to induce

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apoptosis was evaluated by monitoring the levels of active caspase 3 and the
Bax/Bcl2 ratio that presents an unambiguous proof for induction of apoptotic
pathways.
2.2.3.1. Effects on the level of active caspase-3
Caspase 3 is the executioner caspase activated by upstream initiator caspases.
Thus the investigation of this protein expression level is indicative of apoptosis
induction. In the current study, HepG2 cells were treated with compound 6c that
proved to be potent Pim-1 kinase inhibitor with IC50 in the nanomolar range
(IC50 = 0.94 µM). Results revealed that compound 6c upregulaed the caspase 3
level 5.6 folds as compared to the control, thus proving compound 6c apoptosis
induction potential.
2.2.3.2. Effects on mitochondrial apoptosis pathway proteins Bax and Bcl-2
The Bcl-2 family of proteins is responsible for synchronizing the mitochondrial
apoptotic pathway. Among these, Bcl2 and Bax finely tune this programmed
process as Bcl2 suppresses apoptosis (anti-apoptotic) whereas Bax induces it
(pro-apoptotic). Accordingly, the tuned balance between these two distinctive
proteins is judgmental for the cell potential to undergo apoptosis. In this study,
HepG2 cells were treated with compound 6c and its effect on the levels of Bcl2
and Bax was determined as illustrated in (Table 2).
Table 2
Effect of compound 6c on the genes expression of some apoptosis key markers.
Compound No.
Caspase 3
Pg/ml
249
BAX
IU/ml
34533
59
BCL2
IU/ml
507
6c
Control
44.6
19645
Results revealed that compound 6c reduced the level of the anti-apoptotic
protein Bcl2 by 38.7 folds as compared to the control while it boosted the level of
the pro-apoptotic protein Bax by 585 folds. A rather more decisive parameter is
the ratio between the two proteins. Thus, the Bax/Bcl2 ratio for compound 6c

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was calculated to be 22704 folds as compared to the control, emphasizing that
the compound shifted the cells towards undergoing apoptosis.
2.3. Molecular Docking
Molecular docking of 2-amino cyanopyridine series (5a-g) and the 2-
oxocyanopyridine series (6a-g) to the active site of Pim-1 kinase enzyme was
performed to investigate their binding interactions and to explore their binding
modes. Moreover, our lead compound A was also docked in order to investigate
its binding pattern to the Pim-1 kinase active site (Fig. 3). The interaction of
compound A with Pim-1 kinase, illustrates hydrogen bonding between the
pyridyl OH group and the key amino acid Lys 67. Additionally, it shows good
interaction CDOCKER score (-56.70561 Kcal/mol).
Scrutinizing the binding pattern between the most active compound 6c and the
Pim-1 kinase enzyme (Fig. 4) and exploring the interaction energy, unravels that
it was able to form H-bond through its carbonyl group on the benzene
coumarinyl moiety and the key amino acid Lys 67 with CDOCKER interaction
energy score (-76.8193 Kcal/mol) that is superior to the lead compound
CDOCKER score (-56.70561 Kcal/mol). Finally, the lead compound A and
compound 6c were simultaneously docked into the Pim-1 kinase active site and
their RMSD was recorded to be 0.43 suggesting very similar binding pattern of
the two compounds with Pim-1 kinase active site (Fig. 4).
Figure 3: Binding modes of lead compound A (to the left) and of compound 6c (to the right).
Both compounds show H-bonds with the key amino acid Lys 67.

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Figure 4: 3D diagram of interaction of lead compound A (in green) and compound 6c (in grey)
showing H-bonding interaction with the key amino acid Lys 67 and excellent overlapping
between the two compounds.
3. Conclusion
Two novel series of 3- cyano pyridine derivatives 5a-g and 6a-g were designed as
Pim-1 kinase inhibitors, synthesized and their anticancer activity was evaluated
against HepG-2, HCT-116, and MCF-7 cell lines. Most of the targeted compounds
showed moderate to excellent anti-proliferative activities against the aforementioned
cell lines. Moreover, their Pim-1 kinase inhibitory activity and their IC50 values were
recorded. The results revealed that compound 6c showed potent antiproliferative
activity with high Pim-1 kinase activity with IC50 value of 0.94 µM. Furthermore,
apoptosis studies for compound 6c were conducted to evaluate the proapoptotic
potential of our compounds. Results showed that compound 6c boosted the level of
active caspase 3 by 5.6 folds as compared to the control. Additionally, it the Bax/Bcl2
ratio 22704 folds in comparison to the control.

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4. Experimental
4.1. Chemistry
All melting points were measured on a Gallen Kamp melting point apparatus
(Sanyo Gallen Kamp, UK) and were uncorrected. The Microwave reactions were
done by Microsynth instrument type MA143 (Micro wave flux). The IR spectra
were recorded on a Pye-Unicam SP-3-300 infrared spectrophotometer (KBr
1
dicks) and expressed in wave number (cm-1). HNMR spectra were run at 300
and 400 MHz, on a Varian Mercury VX-300 and Bruker Avance III NMR
spectrometer respectively, while 13C NMR spectra were run at 400 MHz. TMS
was used as an internal standard in deuterated dimethylsulphoxide (DMSO-d6).
The mass spectra were recorded on Shimadzu GCMS-QP-1000EX mass
spectrometer at 70 eV. Elemental analyses were performed on CHN analyzer and
all compounds were within ± 0.4 of the theoretical values. The reactions were
monitored by thin-layer chromatography (TLC) using TLC sheets coated with UV
fluorescent silica gel Merck 60 F254 plates and were visualized using UV lamp
and different solvents as mobile phases. All reagents and solvents were purified
and dried by standard techniques.
4. 1. 1. General procedure for 5a-g and 6a-g.
A mixture of aldehyde (1 mmol), methyl ketone (1 mmol), active methylene (1
mmol) namely malononitrile, ethylcyanoacetate, cyanoacetamide, respectively
and 2 mL of glycol / ammonium acetate (3 mmol), was added to the reaction
vessel placed into the microwave reactor and allowed to react under microwave
0
irradiation at 200 - 400 W power and 120 C for several minutes (6-8 minutes).
The automatic mode stirred helps in mixing and the uniform heating of the
reactants. The reaction vessel was cooled to room temperature. The solid
compound was collected by filtration, washed with water, and recrystallized
from ethanol / DMF mixture to give pure oxo-cyanopyridine and amino-
cyanopyridine derivatives (5a-g) and (6a-g), respectively.
4.1.2. 2-Amino-4-(4-methoxyphenyl)-6-(3-oxo-3H-benzo[f]chromen-2-yl)nicotinoni
trile (5a). Yellow crystals (yield 86 %); m.p > 300 °C. IR: 3376 (NH2), 3061 (CH-
aromatic), 2958, 2836 (CH-aliphatic), 2205 (CN), 1712 (CO chromene), 1609 (C=C).

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1HNMR (400 MHz, DMSO-d6) δ ppm: 3.85 (s, 3H, OCH3), 7.07 (s, 1H, Ar-H of
pyridine ring), 7.13 (d, 2H, Ar-H, J = 8 Hz), 7.84-7.69 (m, 2H, Ar-H), 8.10 (d, 2H,
Ar-H, J = 8 Hz), 8.28 (d, 2H, Ar-H, J = 8 Hz), 8.54 (d, 2H, Ar-H, J = 8.8 Hz), 9.69 (s,
13
1H, CH chromene), 11.98 (s, 2H, NH2, D2O exchangeable). C NMR (400 MHz,
DMSO-d6) δ 55.2, 98.5, 104.0, 113.5, 114.4(2), 116.7, 118.9, 124.3, 127.3, 128.5,
129.5, 129.8, 130.0, 130.9, 131.1, 132.0, 132.4, 132.9, 134.1, 147.4, 157.3, 160.4,
162.2, 162.9, 163.8. Anal. Calcd. for C26H17N3O3 (419.44): C, 74.45; H, 4.09; N,
10.02. Found: C, 74.19; H, 3.98; N, 9.97.
4. 1. 3. 2-Amino-4-(naphthalen-2-yl)-6-(3-oxo-3H-benzo[f]chromen-2-yl)nicotinon
itril (5b). Yellow crystals (yield 87 %); m.p. > 300°C. IR: 3376, NH2, 3051 (CH-
aromatic), 2209 (CN), 1723 (CO chromene), 1619 (C=N), 1568 (C=C). 1HNMR (400
MHz, DMSO-d6) δ ppm: 7.01 (s, 1H, Ar-H of pyridine ring), 7.08-8.62 (m, 11H, Ar-
H), 9.24 (s, 1H, CH chromene), 9.71 (d, 2H, Ar-H, J = 6.1 Hz) 11.95 (s, 2H, NH2,
13
D2O exchangeable). C NMR (400 MHz, DMSO-d6) δ 85.4, 116.6, 116.7, 121.9,
122.4, 124.9, 125.0, 125.2, 126.4, 126.5, 127.0, 127.1, 127.3 (2), 128.6, 128.9, 129.1,
129.3, 129.9, 130.1, 130.4, 130.6, 133.7, 134.7, 139.6, 141.0, 145.2, 149.5, 159.1.
Anal. Calcd. for C29H17N3O2 (439.47): C, 79.26; H, 3.90; N, 9.56. Found: C, 78.97;
H, 3.80; N, 9.49.
4. 1. 4. 2-Amino-6-(3-oxo-3H-benzo[f]chromen-2-yl)-4-(thiophen-2-yl)nicotinonitri-
le (5c). Yellow crystals (yield 86 %); m.p. > 300 °C. IR: 3355, NH2, 3062 (CH-
aromatic), 2210 (CN), 1722 (CO chromene), 1628 (C=N), 1567 (C=C). 1HNMR (400
MHz, DMSO-d6) δ ppm: 6.91 (s, 1H, Ar-H of pyridine ring), 7.18 (s, 2H, NH2, D2O
exchangeable), 7.28 (m, 1H, Ar-H of thiophene ring), 7.62-8.30 (m, 4H, Ar-H), 8.52
(d, 2H, Ar-H, J = 8.4 Hz), 8.61 (d, 2H, Ar-H, J = 8 Hz), 9.21 (s, 1H, CH chromene).
13C NMR (400 MHz, DMSO-d6) δ 82.3, 103.7, 117.7 (2), 123.4, 124.7, 126.4, 128.6,
128.9, 129.2, 129.4(2), 130.1, 130.3, 134.6, 139.6, 139.8, 140.9, 142.1, 154.0, 154.2,
159.0, 160.7. Anal. Calcd. for C23H13N3O2S (395.44): 69.86; H, 3.31; N, 10.63. Found
69.59; H, 3.20; N, 10.56.
4. 1. 5. 2-Amino-4-(4-methoxyphenyl)-6-(thiophen-2-yl)nicotinonitrile (5d). Yellow
crystals (yield 89 %); m.p. > 300 °C. IR: 3424 (NH2), 3102 (CH-aromatic), 2924,
1
2843 (CH-aliphatic), 2206 (CN), 1632 (C=N), 1507 (C=C). HNMR (400 MHz,

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DMSO-d6) δ ppm: 3.89 (s, 3H, OCH3), 7.21 (d, 2H, Ar-H, J = 8 Hz), 7.35 (s, 1H, CH
of pyridine ring), 7.40- 7.61 (m, 1H, Ar-H of thiophene ring), 8.10 (d, 2H, Ar-H, J = 8
Hz), 8.75 (d, 1H, Ar-H of thiophene ring, J = 6.6 Hz), 9.42 (d, 1H, Ar-H of thiophene
ring, J = 6.8 Hz), 11.54 (s, 2H, NH2, D2O exchangeable.13CNMR (400 MHz, DMSO-
d6) δ 56.4, 98.5, 113.7, 117.4(2), 128.5, 128.9, 129.7(2), 132.4, 135.4, 139.4, 145.1,
155.0, 157.2, 160.0, 163.7. Anal. Calcd for C17H13N3OS (307.37): C, 66.43; H, 4.26;
N, 13.67. Found: C, 66.15; H, 4.18; N, 13.59.
4. 1. 6. 2-Amino-4-(naphthalen-1-yl)-6-(thiophen-2-yl)nicotinonitrile (5e). Yellow
crystals (yield 90 %); m.p. > 300 °C. IR: br 3221, NH2, 3096 (CH-aromatic), 2219
1
(CN), 1613 (C=N), 1607 (C=C); HNMR (400 MHz, DMSO-d6) δ ppm: 4.15 (s, 2H,
NH2, D2O exchangeable), 6.96 (s, 1H, CH of pyridine ring), 7.26 (d, 1H, Ar-H, J =
8.4 Hz), 7.27-7.87 (m, 7H, Ar-H), 8.02 (d, 1H, Ar-H, J = 8.4 Hz), 8.03-8.07 (m, 2H,
13
Ar-H), 8.18 (d, 1H, Ar-H, J = 7.6 Hz). C NMR (400 MHz, DMSO-d6) δ 99.3,
114.7, 117.7, 125.4, 126.1, 127.7, 129.4 (2), 130.6 (2), 131.2, 132.8, 134.2, 134.6,
136.3, 139.4, 145.1, 156.8, 158.2, 162.7 Anal. Calcd for C20H13N3S (327.41): C,
73.37; H, 4.00; N, 12.83. Found: C, 73.08; H, 3.90; N, 12.74.
4. 1. 7. 2-Amino-4,6-di(thiophen-2-yl)nicotinonitrile (5f).Yellow crystals (yield 91
%); m.p. > 300 °C. IR: 3371, 3428 (NH2), 3121 (CH-aromatic), 2209 (CN), 1626
1
(C=N), 1571 (C=C). HNMR (400 MHz, DMSO-d6) δ ppm: 7.01 (s, 2H, NH2, D2O
exchangeable), 7.19-7.25 (m, 1H, Ar-H), 7.26-7.36 (m, 1H, Ar-H), 7.37 (s, 1H, CH
of pyridine ring), 7.73 (d, 1H, Ar-H, J = 7.4 Hz), 7.83 (d, 1H, Ar-H, J = 8.4 Hz),
7.86 (d, 1H, Ar-H, J = 7.4 Hz), 7.97 (d, 1H, Ar-H, J = 8.4 Hz). 13C NMR (400 MHz,
DMSO-d6) δ 94.3, 113.7, 115.7, 128.6 (2), 128.9 (2), 129.4(2), 140.1, 143.3, 150.6,
156.0, 164.7. Anal. Calcd for C14H9N3S2 (283.37): C, 59.34; H, 3.20; N, 14.83;
Found: C, 59.14; H, 3.09; N, 14.74.
4. 1. 8. 2-Amino-4-(naphthalen-1-yl)-6-(naphthalen-2-yl)nicotinonitrile (5g).
Yellow crystals (yield 93 %); m.p. > 300 °C. IR: br 3371 NH2, 3007 (CH-aromatic),
2207 (CN), 1626 (C═N), 1571 (C=C); 1HNMR (400 MHz, DMSO-d6) δ ppm: 6.91 (s,
1H, CH of pyridine ring), 7.22 (d, 1H, Ar-H, J = 8.4 Hz), 7.50- 8.30 (m, 11H, Ar-H)
8.32 (1H, Ar-H, J = 8 Hz), 8.58 (1H, Ar-H, J = 8.4 Hz, 8.92 (s, 2H, NH2, D2O

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exchangeable),13C NMR (400 MHz, DMSO-d6) δ 92.3, 112.7, 115.7, 125.4, 126.7,
126.9, 127.6, 128.3, 128.4(2), 128.5(2), 128.9, 129.2, 129.5(2), 130.1, 131.3, 134.4,
139.2, 139.1, 142.9, 143.1, 156.0, 159.0, 163.7. Anal. Calcd. for C26H17N3 (371.14):
C, 84.07; H, 4.61; N, 11.31. Found: C, 83.87; H, 4.43; N, 11.20.
4. 1. 9. 4-(4-methoxyphenyl)-2-oxo-6-(3-oxo-3H-benzo[f]chromen-2-yl)-1,2-dihyd-
ropyridi-ne-3-carbonitrile (6a). Yellow crystals (yield 93 %); m.p. > 300 °C. IR: br
3377 (NH), 3060 (CH-aromatic), 2934, 2838 (CH-aliphatic), 2216 (CN), 1724 (CO
chromene), 1646 (CO), 1602 (C=N), 1563 (C=C). 1HNMR (400 MHz, DMSO-d6) δ
ppm: 3.85 (s, 3H, OCH3), 7.00 (s, 1H, CH of pyridine ring), 7.21 (d, 2H, Ar-H, J = 8
Hz), 7.44- 7.99 (m, 3H, Ar-CH) 8.03 (d, 2H, Ar-H, J = 8 Hz), 8.14-8.44 (m, 3H, Ar-
13
CH), 9.43 (s, 1H, CH chromene), 9.73 (s, 1H, NH, D2O exchangeable). C NMR
(400 MHz, DMSO-d6) δ 56.2, 62.5, 102.0, 115.4(2), 116.2, 116.9, 117.3, 120.1,
122.8, 123.4, 124.4, 124.7, 126.1, 127.4, 128.5(2), 129.6, 131.2, 133.9, 138.5, 143.8,
152.3, 154.4, 164.2, 164.2. MS (m/z): 422 (M++2, 2 %), 214 (48%) 158 (100 %).
Anal. Calcd. for C26H16N2O4 (420.42): C, 74.28; H, 3.84; N, 6.66. Found: C, 73.96;
H, 3.64; N, 6.56.
4. 1. 10. 4-(Naphthalen-1-yl)-2-oxo-6-(3-oxo-3H-benzo[f]chromen-2-yl)-1,2-dihydr-
opyridin- e-3-carbonitrile (6b). Yellow crystals (yield 91 %); m.p. > 300 °C. IR: br
3418 NH, 3051 (CH-aromatic), 2219 (CN), 1720 (CO chromene), 1643 (CO
1
pyridine). HNMR (400 MHz, DMSO-d6) δ ppm: 7.30 (s, 1H, CH of pyridine ring),
7.60- 8.14 (d, 9H, Ar-H), 8.32 (d, 2H, Ar-H, J = 8.2 Hz), 8.69 (d, 2H, Ar-H, J = 8.8
Hz), 9.49 (s, 1H, CH chromene), 12.85 (s, H, NH, D2O exchangeable). 13C NMR (400
MHz, DMSO-d6) δ 116.5, 116.9, 117.9, 121.7, 122.0, 123.1, 124.7, 124.9, 125.0,
125.4, 125.9, 126.2, 126.4, 127.3, 127.8, 128.5, 128.8, 129.1, 129.4, 129.6, 130.4,
130.6, 133.8, 134.0, 148.8, 158.7, 159.4. MS m/z: 375 (19 %), 250 (80 %), 206 (100
%). Anal. Calcd for C29H16N2O3 (440.12): C, 79.08; H, 3.66; N, 6.36. Found: C,
78.79; H, 3.54; N, 6.29.
4. 1. 11. 2-Oxo-6-(3-oxo-3H-benzo[f]chromen-2-yl)-4-(thiophen-2-yl)-1,2-dihydro-
pyridine-3-carbonitrile (6c). Yellow crystals (yield 92 %); m.p. > 300 °C. IR: br 3420
NH, 3057 (CH-aromatic), 2951, 2838 (CH-aliphatic), 2219 (CN), 1720 (CO
1
chromene), 1643 (CO pyridine). HNMR (400 MHz, DMSO-d6) δ ppm: 7.13- 8.30
(m, 9H, Ar-H + s, 1H, CH of pyridine ring), 8.60 (d, H, Ar-H, J = 8.8 Hz of

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thiophene), 8.65 (d, H, Ar-H of thiophene ring, J = 8 Hz), 9.45 (s, 1H, CH chromene),
11.88 (s, H, NH, D2O exchangeable). 13C NMR (400 MHz, DMSO-d6) δ 98.3, 104.7,
117.7 (2), 124.7, 126.4, 127.3, 128.4, 128.9, 129.2, 130.1, 131.3, 133.3, 134.6, 139.6,
139.8, 140.9, 142.1(2), 144.5, 158.2, 159.0, 162.4. MS m/z: 397 (M++1, 19 %), 352
(34 %), 310 (100 %). Anal. Calcd. for C23H12N2O3S (396.42): C, 69.69; H, 3.05; N,
7.07. Found: C, 69.45; H, 2.93; N, 6. 96.
4. 1. 12. 4-(4-methoxyphenyl)-2-oxo-6-(thiophen-2-yl)-1,2-dihydropyridine-3-carbo-
nitrile (6d). Yellow crystals (yield 94 %). m.p. > 300 °C. IR: br 3129 (NH), 3034
1
(CH-aromatic), 2906, 2833 (CH-aliphatic), 2215 (CN), 1688 (CO). HNMR (400
MHz, DMSO-d6) δ ppm: 3.85 (s, 3H, -OCH3), 7.86 (s, 1H, CH of pyridine ring), 7.12
(d, 2H, Ar-H of phenyl, J = 8.4 Hz ), 7.26 (m, 1H, Ar-H of thiophene ring), 7.50 (d,
1H, Ar-H, J = 8 Hz of thiophene), 7.79 (d, 1H, Ar-H, J = 8 Hz of thiophene), 8.29 (d,
2H, Ar-H of phenyl ring, J = 8.8 Hz), 12.60 (s, H, NH, D2O exchangeable). 13C NMR
(400 MHz, DMSO-d6) δ 55.4, 108.4, 114.3, 115.7, 124.5, 129.1(2), 130.4, 137.5(2),
134.7, 138.7, 145.6, 149.3, 158.2, 159.9, 160.4. MS m/z: 309 (M++1, 22 %), 308 (100
%), 307 (28 %).s Anal. Calcd for C17H12N2O2S (308.36): C, 66.22; H, 3.92; N, 9.08.
Found: C, 66.02; H, 3.81; N, 9.00.
4. 1. 13. 4-(naphthalen-1-yl)-2-oxo-6-(thiophen-2-yl)-1,2-dihydropyridine-3-carbon-
itrile (6e). Yellow crystals (yield 86 %). m.p. > 300 °C. IR: br 3334 (NH), 3097 (CH-
1
aromatic), 2930, 2825 (CH-aliphatic), 2221 (CN), 1647 (CO); HNMR (400 MHz,
DMSO-d6) δ ppm: 7.24 (s, 1H, CH of pyridine ring), 7.57– 7.86 (m, 8H, Ar-H), 8.06
(d, 1H, Ar-H, J = 8.4 Hz), 8.10 (d, 1H, Ar-H, J = 8 Hz), 12.92 (s, H, NH, D2O
13
exchangeable). C NMR (400 MHz, DMSO-d6) δ 101.9, 107.4, 115.0, 124.7, 125.3,
126.3, 126.6, 127.3, 128.8, 129.9(2), 130.5(2), 130.8, 133.7, 134.1(2), 144.2, 161.4,
163.65. MS m/z: 329 (M++1, 12 %), 328 (89 %), 311 (100 %), 302 (65 %). Anal.
Calcd for C20H12N2OS (328.07): C, 73.15; H, 3.68; N, 8.53. Found: C, 72.87; H, 3.55;
N, 8.44.
4. 1. 14. 2-oxo-4,6-di(thiophen-2-yl)-1,2-dihydropyridine-3-carbonitrile (6f). Yellow
crystals (yield 90 %); m.p. > 300 °C. IR: br 3275 (NH), 3043 (CH-aromatic), 2211
1
(CN), 1650 (CO); HNMR (400 MHz, DMSO-d6) δ ppm: 7.25 (d, 1H, Ar-H, J = 8
Hz), 7.32 (d, 1H, Ar-H, J = 8.4 Hz), 7.88 - 8.06 (m, 5H, Ar-H), 12.76 (s, H, NH, D2O

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13
exchangeable). C NMR (400 MHz, DMSO-d6) δ 114.5, 116.9, 123.0, 128.1, 128.9,
129.4, 129.7, 131.9, 133.4, 138.1(2), 139.8, 158.7, 168.4. MS m/z: (285 M++1, 12 %),
241 (100 %). Anal. Calcd for C14H8N2OS2 (284.35): C, 59.14; H, 2.84; N, 9.85.
Found: C, 58.98; H, 2.99; N, 9.92.
4. 1. 15. 4-(naphthalen-1-yl)-6-(naphthalen-2-yl)-2-oxo-1,2-dihydropyridine-3-car-
bonitrile (6g). Yellow crystals (yield 90 %); m.p. > 300 °C. IR: br 3178 NH, 3007
1
(CH-aromatic), 2220 (CN), 1641 (CO). HNMR (400 MHz, DMSO-d6) δ ppm: 7.03
(s, 1H, CH of pyridine ring), 7.58 - 7.68 (m, 5 H, Ar-H), 7.82 (d, 1H, Ar-H, J = 8 Hz),
8.12 (d, 1H, Ar-H, J = 8 Hz), 7.98 – 8.08 (m, 6 H, Ar-H), 8.58 (d, 1H, Ar-H, J = 8
Hz), 13.03 (s, 1H, NH exchangeable). 13C NMR (400 MHz, DMSO-d6) δ 94.3, 110.7,
115.7 (2), 124.4, 125.7 (2), 126.4, 127.3, 128.6, 128.9, 129.2, 129.4(2), 130.1, 132.3,
134.6, 135.2, 139.6, 139.8, 140.9, 142.1, 154.0, 153.2, 158.0, 161.7. MS m/z: 373
(M++1, 12 %), 357 (89 %), 329 (100 %). Anal. Calcd for C26H16N2O (372.43): C,
83.85; H, 4.33; N, 7.52. Found: C, 83.65; H, 4.21; N, 7.44.
4.2. Biological Evaluation
4.2.1. Determination of PIM kinase activity
The effects of the synthesized compounds on the activity of PIM kinase were
measured using Human proto-oncogene serine/threonine-protein kinase pim-1 (PIM1)
ELISA Kit (catalog #MBS701210). The cells were centrifuged for 15 minutes at
1000x g, 2 – 8 °C and assayed immediately according to the manufacturer’s
instructions. Shortly, the assay was performed using 100µL of the cells supernatant in
ο
each well, which were incubated for 2 hours at 37 C before starting the assay
procedure. Finally, the optical density of each well was determined within 5 minutes
using a microplate reader set at 450 nm.
4.2.2. Determination of the active caspase-3
To determine the effect of the synthesized compounds on apoptosis, the active
caspase-3 level was measured by using Quantikine-Human active Caspase-3
Immunoassay (R&D Systems, Inc. Minneapolis, USA) according to the
manufacturer protocol. Briefly, after washing the cells with PBS, the cells were

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collected and lysed by adding it to the extraction buffer containing protease
inhibitors (1 mL per 1 x 107 cells.) then the lysate was diluted immediately prior
to the assay. At the end of the assay the optical density of each well was
determined within 30 minutes using a microplate reader set at 450 nm.
4.2.3. RNA extraction, Real time PCR analysis and quantification of gene
expression.
The gene expression of Bax and Bcl-2 was assessed by total RNA extraction
from cells using RNeasy Mini Kit® (Qiagen Inc. Valencia, CA, USA). cDNA library
was constructed from different treatments using High-Capacity cDNA Reverse
Transcription Kit (Applied Biosystems, Foster City, CA). The archived cDNA
libraries were then subjected to quantitative real time PCR reactions [23] using
cyber green fluorophore (Fermentas Inc., Glen Burnie, MD, USA). Primer
sequences for each gene were as follows: Bcl-2 forward primer GGG-TAC-GAT-
AAC-CGG-GAGAT and reverse primer CTG-AAG-AGC-TCC-TCC-ACC-AC; Bax
forward primer TCT-GAC-GGC-AAC-TTC-AAC-TG and reverse primer TGG-GTG-
TCCCAAAGT-AGG-AG; GAPDH was used as reference housekeeping gene with
forward primer TGC-ACC-ACC-AAC-TGC-TTAG and reverse primer GAT-GCA-
GGG-ATG-ATG-TTC.
4.3. Molecular docking
Molecular docking study was conducted in order to elucidate interactions of
investigated compounds with the target protein; Pim-1 kinase. First, the crystal
structure of the Pim-1 kinase co-crystallized with the lead compound A was retrieved
from the Protein Data Bank (PDB ID: 5KZI). Docking study was performed using
Accelerys software (Discovery Studio 2.5) provided by the computer drug design lab
in pharmaceutical chemistry department, faculty of pharmacy, Ain Shams University.
Protein preparation for docking study was performed following the standard protein
preparation procedure integrated in Accelry's discovery studio 2.5 and prepared by
prepare protein protocol. Lead compound A, and our ligands were drawn as a
database and prepared by prepare ligand protocol to generate 3D structure and refined

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using CHARMM force field with full potential. Docking simulations were run using
CDOCKER protocol where maximum bad orientations were 800 and orientation vdW
energy threshold was 300. Simulated annealing simulation would then be carried out
consisting of a heating phase 700 K with 2000 steps and a cooling phase back to 5000
steps. In order to rank the docking poses, the binding energy was calculated as a
score. The top 10 docking poses were finally saved. Docking poses were ranked
according to their CDOCKER interaction energy, and the top pose was chosen for
analysis of interactions for each compound.
Acknowledgment
The authors would like to extend their sincere appreciation to Ghada A. Abdellatif,
PhD (Department of pharmacology & toxicology, Faculty of pharmacy, Misr
International university) for contributing in the writing of the experimental
methodology of PIM-kinase.

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Highlights
•
Fourteen compounds of novel 3-cyano pyridine derivatives were
designed and synthesized.
•
•
Molecular docking was carried out against Pim-1 kinase.
In vitro assay of anti-proliferative activity against HePG-2, HCT- 116and
MCF-7 cell lines, enzymatic inhibition of Pim-1 kinase and apoptosis
studies were carried out.
•
Compounds 5g, 6c, 6b, 6e and 6f were the most potent anticancer agents
against the three human cell lines and showed the highest inhibitory
activity. Compound 6c increases the level of active caspase 3 and boosted
the Bax/Bcl2 ratio.

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