New cyanopyridine-based scaffold as PIM-1 inhibitors and apoptotic inducers: Synthesis and SARs study

Article abstract of DOI:10.1016/j.bioorg.2020.104378

Two novel series of 6-(4-benzamido-/4-phthalimido)-3-cyanopyridine derivatives were designed and synthesized as inhibitors of PIM-1 kinase. Based on cytotoxicity results via MTT assay against prostate carcinoma PC3, human hepatocellular carcinoma HepG2 and breast adenocarcinoma MCF-7 cell lines, the most potent cytotoxic cyanopyridine hits, 6, 7, 8, 12 and 13 were 1.5–3.3 times more inhibitor of cell proliferation than the reference standard, 5-FU. Selectivity profile of the latter compounds on normal human cells (WI-38), was executed, indicating that they are highly selective (IC₅₀ > 145 μM) in their cytotoxic effect. The promising compounds were further evaluated as PIM-1 kinase inhibitors. These compounds elicited remarkable inhibition of PIM-1 kinase (76.43–53.33%). Extensive studies on apoptosis were conducted for these compounds; they enhanced caspase-3 and boosted the Bax/Bcl-2 ratio 27-folds in comparison to the control. Molecular docking study of the most potent compound, 13 in PIM-1 kinase active site was consistent with the in vitro activity. Finally, prediction of chemo-informatic properties released compound 13 as the most promising ligand.

Full text of DOI:10.1016/j.bioorg.2020.104378

Journal Pre-proofs
New Cyanopyridine-Based Scaffold as PIM-1 Inhibitors and Apoptotic Induc-
ers: Synthesis and SARs Study
Amel M. Farrag, Mona H. Ibrahim, Ahmed B.M. Mehany, Magda M.F.
Ismail
PII:
S0045-2068(20)31676-X
DOI:
https://doi.org/10.1016/j.bioorg.2020.104378
YBIOO 104378
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Bioorganic Chemistry
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24 September 2020
4 October 2020
Please cite this article as: A.M. Farrag, M.H. Ibrahim, A.B.M. Mehany, M.M.F. Ismail, New Cyanopyridine-
Based Scaffold as PIM-1 Inhibitors and Apoptotic Inducers: Synthesis and SARs Study, Bioorganic Chemistry
(2020), doi: https://doi.org/10.1016/j.bioorg.2020.104378
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New Cyanopyridine-Based Scaffold as PIM-1 Inhibitors and
Apoptotic Inducers: Synthesis and SARs Study
Amel M. Farrag,a* Mona H. Ibrahim,a Ahmed B. M. Mehanyb and Magda M. F. Ismaila
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University,
Cairo, Egypt.
b Department of Zoology, Faculty of Science (Boys), Al-Azhar University, Cairo, Egypt.
ABSTRACT
Two novel series of 6-(4-benzamido-/4-phthalimido)-3-cyanopyridine derivatives were
designed and synthesized as inhibitors of PIM-1 kinase. Based on cytotoxicity results via MTT
assay against prostate carcinoma PC3, human hepatocellular carcinoma HepG2 and breast
adenocarcinoma MCF-7 cell lines, the most potent cytotoxic cyanopyridine hits, 6, 7, 8, 12 and
13 were 1.5-3.3 times more inhibitor of cell proliferation than the reference standard, 5-FU.
Selectivity profile of the latter compounds on normal human cells (WI-38), was executed,
indicating that they are highly selective (IC50>145 μM) in their cytotoxic effect. The promising
compounds were further evaluated as PIM-1 kinase inhibitors. These compounds elicited
remarkable inhibition of PIM-1 kinase (76.43-53.33%). Extensive studies on apoptosis were
conducted for these compounds; they enhanced caspase-3 and boosted the Bax/Bcl-2 ratio 27-
folds in comparison to the control. Molecular docking study of the most potent compound, 13 in
PIM-1 kinase active site was consistent with the in vitro activity. Finally, prediction of chemo-
informatic properties released compound 13 as the most promising ligand.
.
Keywords: Synthesis, Cyanopyridine, PIM-1 kinase, Cell cycle analysis, Apoptosis, Docking
1. Introduction:
PIM kinases, the proviral integration site for Moloney murine leukemia virus, are involved
in many biological processes such as cell survival, proliferation, differentiation, migration,
metabolism, and apoptosis. [1–7] PIM-1 kinase High levels are correlated with many types of
cancer such as myeloid leukemia, breast cancer and prostatic cancer, [8–11] while the deficiency
of PIM-1 kinase leads to the failure in cell survival and growth. [9,12,13] The identification of
PIM-1 role in controlling the growth of cancer stem cells and promotion of multiple drug
resistance added more to the importance of developing potent PIM-1 inhibitors as anticancer
agents that can defeat the drug resistance created by cancer stem cells.[10,14] Besides, PIM-1
was reported to act as a positive regulator of cell cycle progression at G1/S and G2/M
1

transitions.[15,16] Activation of caspases plays a central role in the execution phase of cell
apoptosis. Caspase 3 activation is required for apoptosis induction in response to
chemotherapeutic drugs e.g., taxanes, 5-fluorouracil, and doxorubicin. One of the main
regulators of cell survival is the B-cell lymphoma-2 (Bcl-2) family comprising Bcl-2, Bcl-xL and
myeloid cell leukaemia-1 (MCL-1). [17]
Inspired by these findings, and as a continuation of our previous work on PIM-1 inhibitors,
[18] the goal of this study was to design new PIM-1 kinase inhibitors; through modification of 6-
aryl moiety of the reported PIM-1 inhibitor, 6-(5-bromo-2-hydroxy) phenyl-2-oxo-4-phenyl-3-
pyridine carbonitrile (VRV). Modification of 6-aryl moiety was achieved via molecular
extension using 6-benzamidophenyl and/ rigidification by incorporation of 6-phthalimidophenyl
in pyridine core. Moreover, enhancing the cytotoxic potential of the designed compounds was
considered via synthesis of cyanopyridine derivatives bearing hydrophilic / hydrophobic moieties
at p-4, and bioisosteric replacement at p-2 of pyridine core with OH/NH2 functionality to explore
the impact of structural variation on the anticancer activity. Accordingly, two series of novel
cyanopyridines were synthesized and screened via MTT assay for cytotoxic activities against
three cancer cell lines, PC-3, HepG-2, and MCF-7 and normal Human cell line WI-38. Then,
evaluation of PIM-1 inhibition will be performed. Finally, the promising hits will be tested for
apoptosis induction via cell cycle analysis and this study will be substantiated by evaluation of
their effect on BAX, Bcl-2 genes and the crucial mediator for programmed cell death, caspase 3.
2.
Results and discussion
2.1.
Chemistry
Synthesis of two cyanopyridines series were accomplished in scheme 1. 4-
Benzamidoacetophenone 2 was obtained according to the literature [19] by benzoylation of 4-
aminoacetophenone with benzoyl chloride in DMF/Stirr. Cyclocondensation of 2 with
malononitrile/NH4OAc [20] was carried out to yield the novel 4-aryl-2-amino-6-(4-
benzamidophenyl)-3-cyanopyridines, 3-5; or with ethyl cyanoacetate by the same reaction
condition to give 4-aryl-6-(4-benzamidophenyl)-3-cyanopyridin-2-ones, [21] 6-8; (Scheme 1).
2

Ar`
N
Ar`
N
N
N
NH2
O
O
NH2
N
N
H
O
3-5
10-13
3, Ar`= 4-CH3-C6H4 4, Ar`= 2-OH-C6H4
5, Ar`= C4H3O
10, Ar`= 4-CH3-C6H4 12, Ar`= 2-OH-C6H4
11, Ar`= 4-CH3O-C6H4 13, Ar`= C4H3O
v
ii
O
O
O
O
O
i
iv
N
O
N
H
H2N
9
1
2
iii
vi
Ar`
Ar`
N
N
O
N
H
O
O
N
O
H
N
N
H
O
6-8
14-17
6, Ar`= 4-CH3-C6H4 7, Ar`= 2-OH-C6H4
8, Ar`= C4H3O
14, Ar`= 4-CH3-C6H4 16, Ar`= 2-OH-C6H4
15, Ar`= 4-CH3O-C6H4 17, Ar`= C4H3O
Reagents and conditions: i) C6H5COCl, DMF, Et3N / 0oC, stir, 3h; ii) Malononitrile, Ar`-CHO, NH4OAC, EtOH,
reflux, 6-8h; iii) Ethylcyanoacetate, Ar`-CHO, EtOH, NH4OAC, reflux, 4-6 h; iv) Phthalic anhydride, ACOH,
reflux, 4h; v) Malononitrile, Ar`-CHO, NH4OAC, ACOH, reflux, 12h; vi) Ethylcyanoacetate, Ar`-CHO, ACOH,
NH4OAC, reflux, 12h.
Scheme 1: Synthesis of the target cyanopyridine derivatives 3-8 and 10-17.
The structures of all 6-(4-benzamidophenyl) series, 3-8 were assigned by spectral data and
elemental analysis. Thus, IR spectrum of 3 (as an example) showed strong absorption bands at
3345 and 3300 cm-1 attributed to the NH2 and NH groups and at 2198 and 1650 cm-1 comprising
the nitrile and carbonyl of amide groups, respectively. Moreover, 1H NMR spectrum of 3
revealed a characteristic singlet at δ 2.38 ppm representing methyl protons. Also, the pyridine
proton appeared at δ 7.10 ppm as a singlet signal.
3

On the other hand, nucleophilic substitution reaction of 4-amino-acetophenone and phthalic
anhydride produced another intermediate, 2-(4-acetylphenyl) isoindoline-1,3-dione, 9. [22]
Similarly, cyclocondensation of 9 with malononitrile or ethyl cyanoacetate in NH4OAc [23] was
afforded to give both 2-amino-4-aryl-6-(4-phthalimidophenyl-3-cyanopyridines, 10-13, and 4-
aryl-6-(4-phthalimidophenyl-3-cyanopyridin-2-ones, 14-17 respectively; (Scheme 1).
The structures of 6-(4-phthalimidophenyl) series, 10-17 products were confirmed by spectral
data and elemental analysis. Consequently, IR of compound 15, for instance, exhibited
absorption bands at 3320, 2218 cm-1 due to NH and CN, respectively, and 1718, 1689 and 1663
cm-1 representing three carbonyl groups. Also, 1H NMR spectrum displayed a singlet signal at δ
3.88 ppm for methoxy protons and two doublets at δ 7.04 and 7.15 ppm for 4Hs of Ar` with J
constant equal 8.4 Hz and another two doublets resonating at δ 7.63 and 8.10 ppm due to 4 Hs of
6-phenyl with J constant equal 8.4 Hz. Mass spectrum for 15 (C27H17N3O4) showed a molecular
ion peak (M+) at m/z 447.12.
2.2.
Biological results and discussion
2.2.1. Antiproliferative screening
All newly 6-(4-benzamido-/4-phthalimido)-3-cyanopyridine derivatives 3-8 and 10-17 were
examined for their antiproliferative activities against three cancer cell lines: human prostate
carcinoma PC3, human hepatocellular carcinoma HepG2 and breast adenocarcinoma MCF-7 cell
lines, using MTT assay. 5-FU was used as a reference drug in this assay.
The most potent cyanopyridines 6-8 and 12, 13 were more active than 5-FU, and the rest of
compounds are good to moderately active cytotoxic agents. Besides, SAR analysis elucidated
that, when comparing the cytotoxicity of cyanopyridines having flexible 6-benzamidophenyl side
chain such as 2-amino analogs 3-5 with their counterparts having 2-hydroxy group, 6-8; it was
clear that 2-OH substituent has a good impact on cytotoxicity of these compounds. However,
compounds 12, 13 bearing rigid side chain (4-phthalimidophenyl) displayed higher cytotoxic
effect against all cell lines than their counterparts 16, 17 dues to the substituent effect of 2-NH2
instead of 2-OH, (Table 1).
4

Table 1: In vitro a cytotoxic activity (IC50, µM) of the tested compounds
Ar`
Ar`
N
N
O
N
H
O
O
N
NH2
Comp.
.No
N
N
H
H
6-8
3-5
IC50 (µM) ± SE
MCF-7b
Ar´
PC 3b
HEPG-2b
46.53±2.8
10.71±0.99
10.18±0.75
2.96± 0.11
5.84 ± 0.42
6.64 ± 0.53
3
4
5
6
7
8
CH3-C6H4-4 38.98±2.7
9.7 ±0.85
62.82±3.2
13.39±1.1
16.18±0.76
14.02 ± 1.1
8.94±0.77
9.18±0.82
2-OH-C6H4
C4H3O
4-CH3-C6H4
2-OH-C6H4
C4H3O
8.18 ± 0.71
2.29 ± 0.14
3.8 ± 0.24
5.51 ± 0.46
Ar`
N
Ar`
N
Comp.
.No
NH2
N
O
N
O
O
N
H
N
O
O
10-13
14-17
IC50 (µM) ± SE
HEPG-2b MCF-7b
Ar´
PC 3b
9.06±0.97
23.56±2.1
10 4-CH3-C6H4
11 2-OH-C6H4
12 4-CH3O-C6H4
13
14
15
16
17
11.41±0.97
35.04±2.4
13.37±0.97
45.24±2.4
11.22±0.97
8.17±0.83
14.38±0.85
20.44±1.3
33.76±2.1
39.31±2.2
7.76 ±0.46
5.43±0.51
C4H3O 4.82±0.36
CH3-C6H4-4 7.30±0.66
OH-C6H4-2 13.26±1.2
CH3O-C6H4-4 17.73±1.5
10.31±0.98
5.19±0.43
11.16±0.92
15.39±1.1
21.71±2.01
31.25±1.6
8.15 ±0.61
C4H3O 24.57±1.91
7.53 ±0.57
FU- 5
a Data represent the mean values of three independent determinations
b Cell lines include human prostate carcinoma (PC 3), hepatocellular carcinoma (HEPG-2) and breast
adenocarcinoma (MCF-7).
5

2.2.2. In vitro cytotoxicity screening against normal cell line WI-38
In order to evaluate the safety of the most active compounds 6, 7, 8, 12 and 13 against the
normal cell, they were tested for cytotoxicity against normal human diploid lung fibroblast cell
line WI-38 to investigate the toxicity and selectivity of these active hits. The selectivity index
was calculated using the formula SI= (IC50 of WI-38) / (IC50 of cancer cell line) (Table 2), which
is the measure of the selectivity of the drug candidate towards cancer cells rather than normal
cells. When the SI is ≥3, the drug is highly selective. [24] Interestingly, they possessed a
remarkable non cytotoxic effect on this normal cell as the concentrations needed to inhibit the
viability of WI-38 cells (145.23 - 161.25 µM) is much higher than that needed to inhibit the
viability of the various cancer cell lines (2.29 - 14.02 µM). Hence, compounds 6, 7, 8, 12 and 13
were found to be promising anticancer agents especially against prostate cancer (PC 3) with SI
values 63.41, 39.07, 27.12, 27.62 and 33.45, respectively. Moreover, compounds 6, 7, 8, 12 and
13 were selective against liver cancer cell line (HEPG-2) with SI range (14.54 – 49.06).
Table 2: cytotoxicity data (IC50, µM) and selectivity index (SI) of 6, 7, 8, 12 and 13
Compd. No.
6
Parameter
7
8
12
13
IC50 (WI-38)
SI (PC 3)
145.23± 0.48
63.41
148.45± 0.47
149.44± 0.48
149.95± 0.61
161.25± 0.45
39.07
25.42
16.61
27.12
22.51
16.28
27.62
14.54
13.36
33.45
31.07
19.74
SI (HEPG-2)
SI (MCF-7)
49.06
10.36
2.2.3. PIM-1 kinase inhibitory activity
The most active cyanopyridine analogs 6-8, 12 and 13 were evaluated for PIM-1 kinase
inhibitory activity, to discover novel PIM-1 kinase inhibitors. The percentage of PIM-1 kinase
inhibition by our hits was at the range of 76.43-53.33%. Compounds 12, 13 bearing rigid side
chains (4-phthalimidophenyl) were higher in PIM-1 kinase inhibitory activity than other analogs,
6-8 having flexible 6-benzamidophenyl side chain. It worth mentioning that, both 12, 13
displayed PIM-1 inhibitory activities in sub-micromolar range (IC50 0.76, 0.63 μM) respectively,
(Table 3, Fig. 1). PIM-1 inhibition percent of 12, 13 were 73.64 and 76.43% respectively
showing higher potencies than that of the reference standard colchicine (64.15%). From enzyme
inhibition results, it was clear that compounds 12 and 13 showed potent inhibition of PIM-1.
6

Table 3: PIM-1 percentage inhibition and IC50 values of 6-8, 12 and 13
PIM-1
Comp. No.
Inhibition %
(Con.10µM)
53.33
IC50 (µM)
6
7
8
12
13
7.04±0.37
2.83±0.14
8.09±0.46
0.76±0.03
0.63±0.02
3.98±0.15
60.07
51.98
73.64
76.43
Colchicine
64.15
a
CN
-
0.05
Br
N
O
H
OH
a was declared as a potent PIM-1 kinase inhibitor. [25]
Fig. 1: Pim 1 % inhibition of promising Hits and Colchicine
80
60
40
Pim 1 % inhibition
20
0
Cpd No.
2.2.4. Cell cycle analysis
Appropriate investigating to the potential mechanistic pathways responsible for cell
proliferation inhibition by 6- 8, 12 and 13, cell cycle analysis was tested in the most sensitive
cancer cell line, PC 3. As shown in Table 4 and Fig. 2, compounds 6- 8, 12 and 13 increased the
cell proportion of G2/M phase and Pre-G1phase, while the cell proportions in G1 and S phases
were markedly reduced.
7

Table 4: Percentages of the PC 3 cell cycle phases of 6- 8, 12, 13, DMSO and 5-FU.
Comp. No.
Conc. (µM)
G0-G1%
S%
G2/M%
Pre-G1%
DMSO / PC-3
(negative control)
0
54.77
36.41
8.82
1.76
FU / PC-3-5
(positive control)
7.53
29.88
27.96
42.16
15.22
6
7
0.93
1.55
2.1
21.39
33.92
26.52
37.26
33.72
27.41
23.89
25.41
27.22
34.29
51.2
42.19
48.07
35.52
31.99
27.03
19.26
22.81
13.75
19.64
8
12
13
2.35
1.96
8

Fig. 2: Cell cycle analysis of PC 3 cells treated with compounds: DMSO (A),
5-FU (B), 6 (C), 7 (D), 8 (E), 12 (F) and 13 (G)
2.2.5. Annexin V-FITC apoptosis assay
To confirm whether the Pre-G1phase cells induced by 6-8, 12 and 13 were due to apoptosis
or necrosis, these treated compounds with PC 3 cells were further investigated with PI and FITC-
Annexin V staining for apoptosis identification. Results declared that all the tested compounds
induced apoptosis dependent death. Compounds 7, 8 and 13 exhibited the highest significant
potential for induction of apoptosis (>90), while 6 and 12 displayed 89.6% and 89.3%
respectively when compared with 5-FU (85.8%), (Table 5, Fig. 3).
9

Table 5: Percentages of the apoptotic PC 3cells populations in of 6, 7, 8, 12, 13, DMSO and 5-
FU
% Apoptosis
Necrosis
Comp. No.
Total
1.76
Early
Late
0.43
8.12
DMSO / PC-3
(negative control)
FU / PC-3-5
(positive control)
6
0.82
4.94
0.51
2.16
15.22
27.03
19.26
22.81
13.75
19.64
4.82
8.29
12.14
5.19
19.4
9.3
8.56
7.09
12.05
2.81
1.67
2.11
1.47
1.71
7
8
12
13
5.88
10

Fig. 3: Histograms display the percentage of PC 3 cell apoptosis distribution of
compounds DMSO (A), 5-FU (B), 6 (C), 7 (D), 8 (E), 12 (F) and 13 (G)
2.2.6. Apoptosis studies
2.2.6.1.
Effects on the level of active caspase 3
Activation of the caspases’ pathway which consider as a hallmark of apoptosis and can be
used in cellular assays to quantify activators and inhibitors of the “death cascade.” Among
caspases, caspase 3 is the vital player that cleaves multiple proteins in the cells, leading to
apoptotic cell death. [26] Cyanopyridine analogs 6- 8, 12 and 13 were evaluated for their effect on
caspase 3, which is considered as a marker for apoptosis. They showed an increase in the level of
11

active caspase 3 at the range of 266.0-371.9 Pg/ml compared to that of control (55.3 Pg/ml),
(Table 6).
Table 6: Caspase 3, BAX and Bcl-2 assays results of 6, 7, 8, 12 and 13
Caspase 3
Pg/ml
BAX
Pg/ml
193.2
Bcl-2
Pg/ml
1.657
BAX/Bcl-2
ratio
Comp. No.
6
305.7
6..116
7
8
12
266.0
319.4
371.9
352.9
55.3
154.7
141.90
230.6
192.9
18.14
2.502
2.648
1.151
1.668
5.418
61.83
53.59
200.35
115.65
3.35
13
Control
2.2.6.2.
Effects on mitochondrial apoptosis pathway proteins Bax and Bcl-2
The B-cell lymphoma protein 2 (Bcl-2) families plays a crucial role in tumor progression or
inhibition of the intrinsic apoptotic pathway triggered by mitochondrial dysfunction, with Bcl-2
itself and BAX as pro-apoptotic proteins. [27] Several direct BAX activators have been identified
to hold promise for cancer therapy with the advantages of specificity and the potential of
overcoming chemo- and radioresistance. It is possible that Bcl-2 levels in the mitochondrial
membrane must decrease if BAX levels in the mitochondria are to rise. The effect of the most
active cyanopyridine analogs 6- 8, 12 and 13 on the expression levels of BAX and Bcl-2 were
determined. Analyzing the results disclose that all the tested derivatives caused upregulation in
the level of the proapoptotic protein; BAX while it markedly downregulated the levels of the
antiapoptotic proteins Bcl-2, (c.f. Table 6). As of literature study, there is a strong indication that
the ratio of pro-apoptotic BAX versus antiapoptotic Bcl-2 proteins indicates the susceptibility of
cancer cells to undergo apoptosis more accurately. [28] Consistent with our results, compound
12 increased the BAX/Bcl-2 ratio by approximately 60 folds, as well as compounds 6 and 13
boosted the BAX/Bcl-2 ratio by almost 35 folds, while 7 and 8 enhanced the ratio by around 16
and 18 folds, respectively, as compared to the control. These findings support the effectiveness
as apoptosis induction effect of compounds 6- 8, 12 and 13.
2.3.
Molecular modeling and in silico predictions
2.3.1. Molecular docking study of ligand with PIM-1
Molecular docking was performed to understand the binding and interactions of derivative
13 inside the known crystal structure of PIM-1 kinase protein (PDB ID: 2OBJ), [25] using the
Molecular Operating Environment (MOE 2010) software. VRV was initially re-docked into the
active site of PIM-1 (Fig. 4) to validate docking procedures. The result revealed superimposition
of the redocked ligand above native VRV with RMSD of 0.88 Å.
Analysis of docking results of compound 13 showed that it had comparable docking score
energy with that of the reference ligand (VRV). Also, its binding mode to PIM-1 protein was like
12

that of the VRV in that it occupied the ATP-binding pocket, showing hydrogen bonding
interaction with the hinge region residue (Fig. 5). As we know, Lys67 is critical to PIM-1
catalytic activity and in ATP-bound structure, where the nitrogen of CN group acts as hydrogen
bond acceptor to the highly conserved residue, Lys67, in addition to arene-H interaction of the
pyridine core to Val52. The central phenyl was recognized by Leu44 (NTD), making
hydrophobic interaction and binds through arene-H interaction to Leu174 (CTD). Moreover, the
phthalimido moiety contributes to PIM-1 affinity via making additional HBA via carbonyl
oxygen of phthalimido moiety with Asp128, arene-H bonding to Gln127 and hydrophobic
interaction with Val126 (Hinge region) and Asp131. The hydrophobic interactions described
above determine the affinity of inhibitor for the ATP-binding pocket.
13

Fig. 4: Docking and binding pattern of the ligand, (VRV), into PIM-1 kinase active site (PDB
2OBJ) in 3D (upper panel) and 2D (lower panel)
14

Fig. 5: Docking and binding pattern of compound 13 into PIM-1 kinase active site
(PDB 2OBJ) in 3D (upper panel) and 2D (lower panel)
15

2.3.2. In silico prediction of physicochemical and ADME properties and pharmacokinetic
profile
In silico ADME data (absorption, distribution, metabolism, and excretion), provide first key
insights into how the human body will eventually treat a drug. Poor pharmacokinetic parameters
constitute a significant reason for the lack of therapeutic activity of some drug candidates.
Consequently, evaluation of the pharmacokinetic parameters of drug candidates at an early stage
of screening is a vital step in the drug development process that can directly lead optimization
efforts into improved hits [29] and reduce the late-stage attrition in drug discovery programmes.
Lipinski has emerged in a set of rules focusing attention on the importance of lipophilicity
(octanol-water partition or LogP), the number of hydrogen bond acceptors (HBA) and donors
(HBD), and molecular weight (MW). These rules considered as basic assistance to relate
physicochemical properties with effective drug development by examination of the structures of
orally administered drugs, and drug candidates. An orally available drug candidate is compatible
with Lipinski's rule if LogP is no more than 5, MW is less than 500, number of HBD is less than
5 and number of HBA is less than 10. [30] Additionally, Veber [31] rule defines drug-likeness
constraints as rotatable bond count ≤ 10; as the more flexible the molecule, the less likely it is to
be orally active, while polar surface area (TPSA) could be used as a factor instead of the number
of hydrogen bonding groups. TPSA of less than 140 Å2 should present high oral bioavailability
in rats.
In this, Molinspiration, [32] Molsoft, [33] Pre-ADMET, [34] Data warrior, [35] PAINS-
Remover, [36] and SwissADME, [37] software were used to evaluate the pharmacokinetic
parameters of the most active compounds 6, 7, 8, 12 and 13. It was found that the
physicochemical properties (Table7) of all the active compounds have Lipinski zero violation
except for one compound 6 which has only one violation LogP value 5.02 (>5), but it still stays
in the orally bioavailable compounds. Furthermore, they all showed NROTB values of 3 and
4(<10), and TPSA values range of 85.75 and 120.01 Å2 (<140 Å2). Additionally, absorption (%
ABS) was estimated by using the equation % ABS = 109 – (0.345 x TPSA), [38] founding that
the calculated % ABS of all these hits ranged between 67.60% and 79.42%, demonstrating that
these synthesized derivatives may have the required cell membrane permeability and
bioavailability. All compounds have rotatable bonds between 3 and 4 which indicating molecular
flexibility to their bio target.
Alternatively, Molsoft software was used to evaluate the solubility and drug-likeness model
score for the active derivatives. Aqueous solubility is a well-known parameter that, affect
absorption and distribution qualities significantly. 6, 7, 8, 12 and 13 achieved the requirements of
solubility with values hits ranged between of 8.38 and 62.71 mg/L (more than 0.0001 mg/L).
Compounds showing positive drug-likeness model scores are recognized as drug-like and can
behave as drug molecules. A positive model-score was predicted for compound 7 and 8 (0.49
and 0.04, respectively) while that for compound 6, 12 and 13 was negative (-0.14, -0.21 and -
0.51, respectively), as well as 5-FU (-1.07). Likewise, in silico assessment of the following
16

pharmacokinetic parameters was performed using Pre-ADMET software: Caco2 (human colon
adenocarcinoma) permeability coefficient, Human intestinal absorption (HIA), Brain-blood
barrier partition coefficient (BBB) and human plasma protein binding (PPB) (Table 7). 6, 7, 8,
12 and 13 showed medium cell permeability in the Caco- 2 cell model with values between
21.01 and 21.52 nm/s. They showed high human intestinal absorption values (93.7107 - 98.3290
%) indicating very well-absorbed compounds, which is better than that of 5-FU (75.9253 %).
Furthermore, they demonstrated low BBB penetration capability (0.0251 - 0.0886). All the
biologically active derivatives were found to be highly bound to human plasma proteins (92.7812
- 95.3666 %).
Toxicity risk assessment tries to locate substructures within the chemical structure being
indicative of a toxicity risk within one of four major toxicity classes (Mutagenicity,
Tumorigenicity, Irritant, and Reproductive Effective). The overall toxicities of the most active
synthesized compounds and compared them with three standard anticancer agents, that is, 5-FU,
Colchicine and Sorafenib, were determined using the Datawarrior software. Datawarrior
predicted that derivatives 6 and 7 had tumorigenic toxicity. While 8 was predicted to have high
mutagenic and tumorigenic effect. On the other hand, 12 and 13 were found to pass the toxicity
risk assessment. Conversely, 5-FU was predicted to have high mutagenic, tumorigenic, irritant
and reproductive toxicities, whereas colchicine was predicted to have a high reproductive effect.
Another important consideration during preclinical analysis trial was to analyse the P-
glycoprotein (P-gp) non-substrate candidature. P-gp works as efflux transporter which pumps
drugs and other compounds and its substrate out of the cell, which was found to be one of
explanation for its resistance to various chemotherapeutics for cancer. It has been found that P-
gp is a critical transporter of drug which consequently results in resistance to anticancer drugs
like Imatinib, Lonafarnib, and Taxanes. [39] Therefore, the hits had been analyzed using the
SwissADME website. We found that all the synthesized compounds are not substrates of P-gp
protein (Table 7), indicating that these hits have very less chance to efflux out of the cell, thus
resulting in a maximum effect. Bioavailability is an index of the amount of drug present in the
plasma and is considered as the most crucial factor affecting absorption. Interestingly, all the
synthesized derivatives were found to have high bioavailability scores equal to that of 5-FU.
SwissADME Synthetic Accessibility (SA) Score is based primarily on the assumption that
the frequency of molecular fragments in ‘really’ obtainable molecules correlates with the ease of
synthesis, the score is normalized to range from 1 (very easy) to 10 (very difficult to synthesize) .
[37] SA scores of all the analogs were found to be between 2.99 and 3.22, indicating that they
can be readily synthesized on a large scale.
Pan-assay interference compounds (PAINS) are chemical compounds that often give false
positive results in high-throughput screens. [40] PAINS tend to react non-specifically with
numerous biological targets rather than specifically affecting one desired target . [41] So, it is
17

essential to check any PAINS alert of the newly synthesized derivatives. PAINS was performed
by SwissADME, [[37]] and PAINS-Remover [36] displayed zero alerts to all the hits.
Collectively and based on the estimated ligand efficiency, drug likeness and
pharmacokinetic predictors, these active compounds considered as a pharmacologically active
framework that should be considered on progressing further potential hits.
Table 7: In silico physicochemical properties, ADME and ligand efficiency data of compounds 6-8,
12 and 13
Compd no
Property
6
7
8
12
13
FU-5
Colchicine
Molinspiration
logPa
5.02
405.46
4.31
407.43
3.71
381.39
4.44
432.44
3.85
406.40
0.59-
130.08
1.10
399.44
MWb
HBAc
HBDd
Lipinski’s violation
TPSAe
5
2
1
6
3
0
4
2
0
6
2
0
5
1
0
7
3
0
1
7
0
85.75
79.42
4
105.98
72.44
4
98.89
74.88
4
120.01
67.60
3
114.92
69.35
3
65.72
86.33
0
83.11
80.33
5
ABSf%
NROTBg
Molsoft
S (mg/L) h
8.38
41.80
0.49
62.71
0.04
55.64
0.21-
62.51
0.51-
5809.82
1.07-
6013.76
0.90
Drug likeness
model score
Pre-ADME
0.14-
Caco2I
21.49 21.01
93.7107
0.0375 0.0251
PPBl 95.3666 94.4651
92.7812
Datawarrior
21.52
21.01
96.2023 98.3290
0.0346 0.0886
21.15
17.25
75.9253
37.47
HIAJ 95.77
95.4748
96.8889
BBBK
0.0335
0.1995
8.3130
0.0127
39.3768
94.7186 92.8878
Mutagenicitym
Tumorigenicitym
Irritantm
None
High
None
None
None
High
None
None
High
High
None
None
None
None
None
None
None
High
High
High
High
None
None
None
High
None
None
None
Reproductive effectivem
SWISS ADME
Pgp substrate
No
0.55
3.01
No
0.55
2.99
No
0.55
3.17
No
0.55
3.05
No
0.55
3.22
No
0.55
1.52
Yes
0.55
3.87
Bioavailability Score
Synthetic Accessibility
PAINS-Removen
0
Pains alert
0
0
0
0
0
0
a LogP: logarithm of compound partition coefficient between n-octanol and water.
b MW: molecular weight.
c HBA: number of hydrogen bond acceptors.
d HBD: number of hydrogen bond donors.
e TPSA: topological polar surface area.
f %ABS: percentage of absorption.
g NROTB: number of rotatable bonds.
18

h S: aqueous solubility.
i Caco2: permeability through cells derived from human colon adenocarcinoma; Caco2 values < 4 nm/s (low
permeability), values ranged from 4 to 70 nm/s (medium permeability) and values > 70 nm/s (high permeability).
J HIA: percentage human intestinal absorption; HIA values ranged from 0 to 20% (poorly absorbed), values ranged
from 20 to 70% (moderately absorbed) and ranged from 70 to 100% (well absorbed).
KBBB: blood-brain barrier penetration; BBB values < 0.1 (low CNS absorption), values ranged from 0.1 to 2
(medium CNS absorption) and values > 2 (high CNS absorption).
l PPB: plasma protein binding; PPB values < 90% (poorly bound) and values > 90% (strongly bound).
m Toxicity Risk Assessment.
n
PAINS Alerts were performed by SwissADME and PAINS-Remover.
3.
Conclusion
All the examined cyanopyridine hits are potent to moderate cytotoxic on the three cancer cell
lines, prostate carcinoma PC3, human hepatocellular carcinoma HepG2 and breast
adenocarcinoma MCF-7 compared to the reference standard 5-FU. For PIM-1 inhibition,
compounds bearing 6-phthalimidophenyl is more favored than those with 6-benzamidophenyl
side chain. The most potent compounds were further selected to investigate its apoptotic inducing
effect. They showed an increase in the level of active caspase 3 by 7-folds, an upregulation in
BAX and downregulation in Bcl-2 levels, compared to the control cells. These compounds cause
cell cycle arrest at G2/M and induce apoptosis via enhancement of Pre-G in cell cycle analysis.
Finally, molecular modeling studies were carried out on compound 13, showed perfect fitting in
PIM-1 active site. Furthermore, drug-likeness assessment via Molinspiration, Molsoft, Pre-
ADMET, Data warrior, SWISS ADME and PAINS-remover software and websites elucidated
their full compliance with Lipinski's rule, favorable physicochemical properties, and convenient
predicted pharmacokinetic parameters. These results have improved the understanding of the
molecular mechanism of tumor genesis of prostate cancer to provide novel insights for the
treatment of prostate cancer.
4.
Experimental section
4.1.
Chemistry
Reagents and solvents were purchased from usual commercial suppliers and were used
without further purification. Yields reported refer to purified products. All reactions were
routinely checked by thin-layer chromatography (TLC) on Merck Silica Gel 60 F254 (0.25mm
thick) and visualization was performed with UV lamp. Melting points were measured in open
capillary tubes using Stuart SMP3 apparatus. IR spectra (KBr) were measured on a Shimadzu
FT/IR 1650 (Perkin Elmer) spectrometer. 1H and 13C NMR spectra were recorded on a Brüker
Advance-400 instrument (400 MHz for 1H and 100 MHz for 13C) in DMSO-d6 Chemical shifts
(δ) are reported in ppm relative to TMS as an internal standard, or to the solvent in which the
spectrum was recorded. Mass spectra were performed on a Shimadzu GS/MS-QP 2010 plus
spectrometer at 70 eV.
19

4.1.1. N-(4-acetylphenyl)benzamide (2)
To a solution of p-aminoacetophenone (1.35g, 0.01mol) in dimethyl formamide (20 ml) an
equimolar amount of benzoyl chloride (1.40g, 0.01mol) was added. The mixture was stirred at 0
̊
obtained, which was filtered, washed with water and after drying it was crystallized out from
ethanol. The yield was 95 % and the melting temperature was 200-202 ̊C. [19]
4.1.2. General procedure for the synthesis of N-(4-(6-Amino-5-cyano-4-arylpyridin-2-
yl)phenyl) benzamides (3-5)
A mixture of 2 (2.39g, 0.01 mol), different aryl aldehydes (0.01 mol), ammonium acetate (6.16g,
0.08 mol) and malononitrile (0.66 g, 0.01 mol) were refluxed in ethanol (20 ml) for 6-12 h. After
completion of reaction, it was further cooled and filter the solid. The solid was purified by
ethanol with 85 - 95% yields.
4.1.2.1. N-(4-(6-Amino-5-cyano-4-(p-tolyl)pyridin-2-yl)phenyl) benzamide(3). Yield 88 %; m.p.
100-102 oC; IR (KBr, cm-1): 3345, 3300 (NH, NH2) , 2198 (C≡N), 1650 (C=O), 1587 (C=N); 1H
NMR (DMSO-d6) δ (ppm): 2.38 (s, 3H, CH3), 7.10 (s, H, pyridine- H), 7.36 (d, 2H, H3.5 Ar`-H),
7.51 - 7.60 (m, 4H, H2,6 Ar`-H, H3,5Phenyl-H), 7.77 -7.79 (t, H, H4 phenyl-H), 7.92 – 8.01 (m, 4H,
H2,6 Ar-H, H2,6 Phenyl-H), 8.18 (d, 2H, H3,5 Ar-H), , 10.42, 10.51 (2s, 3H, NH, NH2; exchangeable
with D2O); 13C NMR (DMSO-d6) δ (ppm): 21.46, 90.12, 114.17, 115.52, 119.91, 127.53, 128.08,
128.24, 128.92, 129.68, 132.08, 132.27, 133.85, 135.20, 154.87, 158.58, 164.13, 166.40; MS,
m/z: 404 (M+, 100 %), 405 [(M+1)+, 31.4 %], 406 [(M+2)+, 5.25 %]; Anal. Calcd. For C26H20N4O
(404.16): C, 77.21; H, 4.98; N, 13.85, Found: C, 77.43; H, 5.12; N, 14.08.
4.1.2.2.N-(4-(6-amino-5-cyano-4-(2-hydroxyphenyl)pyridin-2-yl)phenyl) benzamide (4). Yield 85
%; m.p. 140 -142 oC; IR (KBr, cm-1): 3429, 3321, 3240 (OH, NH, NH2) , 2195 (C≡N), 1656
(C=O), 1603 (C=N), 1562 (C=C); 1H NMR (DMSO-d6) δ (ppm): 6.89 -7.00 (m, 2 H, H3,5 Ar`-H),
7.14 (s, H, pyridine- H), 7.28 - 7.31 (t, H, H4 Ar`-H), 7.62 - 7.75 (m, 4H, H6 Ar`-H, H3,4,5 Phenyl-
H), 7.91- 8.03 (m, 4H, H2,6 Ar-H , H2,6 phenyl-H), 8.39 (d, H, H3,5 Ar-H), 10.45, 10.53, 12.19 (3s,
4H, NH, NH2, OH; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 90.89, 111.44,
115.63, 117.99, 119.37, 122.89, 128.27, 128.92, 129.96, 131.28, 133.97, 134.87, 136.52, 154.55,
158.59, 161.35, 164.30; MS, m/z: 406 (M+, 1.02 %), 407 [(M+1)+, 8.03 %], 408 [(M+2)+, 3.54
%]; Anal. Calcd. For C25H18N4O2 (406.14): C, 73.88; H, 4.46; N, 13.78, Found: C, 74.19; H, 4.57;
N, 14.01.
4.1.2.3.N-(4-(6-amino-5-cyano-4-(furan-2-yl)pyridin-2-yl)phenyl)benzamide (5). Yield 95 %;
m.p. 304-306 oC; IR (KBr, cm-1): 3440 - 3353 (NH, NH2) , 2204 (C≡N), 1652 (C=O), 1597
(C=N), 1521 (C=C); 1H NMR (DMSO-d6) δ (ppm): 6.76 -6.77 (m, 1H, H4 Ar`-H), 7.08 (d, 1H,
H5 Ar`-H), 7.14 (s, H, pyridine- H), 7.53 -7.62 (m, 3 H, H3,4,5 Phenyl-H), 7.89 – 7.99 (m, 3H, H3
Ar`-H, H2,6 Ar-H), 8.08 – 8.14 (m, 4H, H2,6 Phenyl-H, H3,5 Ar-H), 10.36, 10.44 (2s, 3H, NH, NH2;
exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 89.27, 107.37, 111,49 113.47, 120.11,
20

120.44, 127.99, 128.20, 128.29, 128.94, 132.89, 134.20, 134.89, 141.67, 151.27, 156.53, 161.63,
166.22; MS, m/z: 380 (M+, 85.37 %), 381 [(M+1)+, 11.53%]; Anal. Calcd. For C23H16N4O2
(380.13): C, 72.62; H, 4.24; N, 14.73, Found: C, 72.80; H, 4.41; N, 14.97.
4.1.3. General procedure for the synthesis of N-(4-(5-Cyano-6-oxo-4-(aryl)-1,6-
dihydropyridin-2-yl)phenyl)benzamides (6-8)
A mixture of 2 (2.39g, 0.01 mol), different aryl aldehydes (0.01 mol), ammonium acetate (6.16g,
0.08 mol) and ethyl cyanoacetate (1.13g, 0.01 mol) were refluxed in ethanol (20 ml) for 6-12 h.
After completion of reaction, it was further cooled and filter the solid. The solid was purified by
ethanol with 80 - 90% yields.
4.1.3.1. N-(4-(5-Cyano-6-oxo-4-(p-tolyl)-1,6-dihydropyridin-2-yl)phenyl) benzamide (6). Yield
80%; m.p. 255 - 257 oC; IR (KBr, cm-1): 3430, 3356 (2 NH) , 2218 (C≡N), 1723, 1674 (2C=O),
1598 (C=C); 1H NMR (DMSO-d6) δ (ppm): 2.41 (s, 3H, CH3-Hs), 7.15 (d, 2H, H3,5 Ar`-H, J = 8
Hz), 7.41 (d, 2H, H2,6 Ar`-H, J = 8 Hz), 7.56 – 7.63 (m, 5H, H2,6 Ar-H, H3,4,5 Phenyl-H), 7.94 –
7.99 (m, 4H, H2,6 Phenyl-H, H3,5 Ar-H), 8.36 (s, H, pyridine- H) , 10.51, 10.56 (2s, 2H, NH;
exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 21.39, 101.62, 116.27, 116.89, 120.43,
128.17, 128.23, 128.27, 128.66, 128.91, 132.35, 132.48, 133.40, 135.04, 136.86, 155.42, 157.08,
159.88, 162.48, 166.54; MS, m/z: 405 (M+, 3.57 %), 406 [(M+1)+, 1.73%], 407 [(M+2)+, 1.47%];
Anal. Calcd. For C26H19N3O2 (405.15): C, 77.02; H, 4.72; N, 10.36, Found: C, 76.89; H, 4.87; N,
10.59.
4.1.3.2.N-(4-(5-Cyano-4-(2-hydroxyphenyl)-6-oxo-1,6-dihydropyridin-2-yl)phenyl) benzamide
(7). Yield 90%; m.p. 321 - 323 oC; IR (KBr, cm-1): 3474, 3349, 3299 (OH, 2 NH) , 2203 (C≡N),
1706, 1675 (2C=O), 1611 (C=C); 1H NMR (DMSO-d6) δ (ppm): 7.33 – 7.46 (m, 4H, Ar`-Hs),
7.51 – 7.72 (m, 5H, , H2,6 Ar-H, H3,4,5 Phenyl-H,), 7.99 – 8.08 ( m, 4H, H3,5 Ar-H, H2,6 Phenyl-H),
8.33 (H, pyridine-H), 10.48, 10.55, 11.65 (3s, 3H, 2 NH, OH; exchangeable with D2O); 13C NMR
(DMSO-d6) δ (ppm): 104.64, 115.55, 116.72, 117.38, 120.38, 121.76, 126.52, 127.06, 128.47,
128.93, 129.55, 130.95, 132.36, 134.08, 135.07, 155.34, 160.50, 161.36, 164.03, 166.41; MS,
m/z: 407 (M+, 9.74 %), 408 [(M+1)+, 3.81%]; Anal. Calcd. For C25H17N3O3 (407.13): C, 73.70;
H, 4.21; N, 10.31; Found: C, 73.94; H, 4.37; N, 10.48.
4.1.3.3.N-(4-(5-Cyano-4-(furan-2-yl)-6-oxo-1,6-dihydropyridin-2-yl)phenyl) benzamide (8) Yield
85%; m.p. 104 - 106 oC; IR (KBr, cm-1): 3404, 3353 (2 NH) , 2222 (C≡N), 1717, 1675 (2C=O),
1620 (C=C); 1H NMR (DMSO-d6) δ (ppm): 6.84 (d, H, H4 Ar`-H), 7.50 – 7.60 (m, 6H, H5 Ar`-H,
H2,6 Ar-H, H3,4,5 Phenyl-H ), 7.93 – 7.96 (m, 4H, H3,5 Ar-H, H2,6 Phenyl-H ), 8.12 ( s, H, pyridine-
H), 8.20 ( d, H, H3 Ar`-H), 10.52, 10.53 (2s, 2H, 2NH; exchangeable with D2O); 13C NMR
(DMSO-d6) δ (ppm): 104.38, 110.05, 114.80, 115.75, 119.90, 120.03, 125.14, 126.11, 126.99,
128.27, 128.92, 132.36, 135.04, 139.60, 144.08, 150.58, 159.05, 160.53, 162.68, 166.46; MS,
21

m/z: 381 (M+, 8.62 %); Anal. Calcd. For C23H15N3O3 (381.11): C, 72.43; H, 3.96; N, 11.02;
Found: C, 72.70; H, 4.11; N, 11.34.
4.1.4. 2-(4-acetylphenyl)isoindoline-1,3-dione (9)
Phthalic anhydride (1.48 g, 0.01 mole) and p-aminoacetophenone (1.35g, 0.01 mol) were refluxed
in (50 ml) acetic acid for 4 hour. The reaction mixture was filtered off while hot and the solvent
was evaporated. The solid separated was filtered and recrystallized from ethanol. The yield was
90 % and the melting temperature was 248-250 ̊C. [42]
4.1.5. General procedure for the synthesis of 2-Amino-6-(4-(1,3-dioxoisoindolin-2-yl)phenyl)-
4-(aryl)nicotinonitriles (10-13)
A mixture of 9 (2.65g, 0.01 mol), different aryl aldehydes (0.01 mol), ammonium acetate (6.16g,
0.08 mol) and and malononitrile (0.66 g, 0.01 mol) were refluxed in glacial acetic acid (15 ml) for
12 h. After completion of reaction, it was further cooled and filter the solid. The solid was
purified by ethanol with 70 - 89% yields.
4.1.5.1.2-Amino-6-(4-(1,3-dioxoisoindolin-2-yl)phenyl)-4-(p-tolyl) nicotinonitrile (10). Yield
84%; m.p. 189 - 191 oC; IR (KBr, cm-1): 3390, 3330 (NH2) , 2206 (C≡N), 1717, 1679 (2C=O),
1658 (C=N), 1599 (C=C); 1H NMR (DMSO-d6) δ (ppm): 2.39 (s, 3H, CH3-Hs), 5.58 (s, 2H,
NH2; exchangeable with D2O), 7.27– 7.39 (m, 4H, Ar`-Hs), 7.63 (d, 2H, H2,6 Ar-H, J = 8.4 Hz),
7.83 ( s, H, pyridine-H), 7.86 – 8.01 (m, 4H, Phthalimide-Hs ), 8.10 ( d, 2H, H3,5 Ar-H, J = 8.4
Hz); 13C NMR (DMSO-d6) δ (ppm): 21.71, 105.65, 114.33, 118.60, 123.40, 124.03, 127.51,
129.24, 129.78, 129.92, 130.32, 130.61, 131.97, 133.07, 134.79, 136.49, 155.26, 156.78, 169.70;
MS, m/z: 430.34 (M+, 19.31 %), 431.26 [(M+1)+, 11.43%], 432.00 [(M+2)+, 2.67%]; Anal.
Calcd. For C27H18N4O2 (430.17): C, 75.34; H, 4.21; N, 13.02; Found: C, 75.43; H, 4.15; N, 13.04.
4.1.5.2.2-Amino-6-(4-(1,3-dioxoisoindolin-2-yl)phenyl)-4-(4-methoxy phenyl) nicotinonitrile (11).
Yield 70%; m.p. 182 - 184 oC; IR (KBr, cm-1): 3406, 3347 (NH2) , 2219 (C≡N), 1719, 1690
(2C=O), 1620 (C=N), 1595 (C=C); 1H NMR (DMSO-d6) δ (ppm): 3.74 (s, 3H, CH3O-Hs), 5.75
(s, 2H, NH2; exchangeable with D2O), 7.13 (d, 2H, H3,5 Ar`-H), 7.68 – 7.74 (m, 4H, H2,6Ar`-H,
H2,6 Ar-H), 7.83 ( s, H, pyridine-H), 7.91 – 8.02 (m, 4H, Phthalimide-Hs ), 8.11 ( d, 2H, H3,5 Ar-
H); 13C NMR (DMSO-d6) δ (ppm): 55.93, 105.20, 114.97, 115.30, 123.40, 127.76, 128.10,
129.84, 130.54, 132.06, 132.92, 133.08, 134.80, 155.30, 156.59, 162.24, 163.83, 169.71; MS,
m/z: 446.49 (M+, 5.53 %), 447.91 [(M+1)+, 1.17%]; Anal. Calcd. For C27H18N4O3 (446.14): C,
72.64; H, 4.06; N, 12.55; Found: C, 72.89; H, 4.24; N, 12.71.
4.1.5.3.2-Amino-6-(4-(1,3-dioxoisoindolin-2-yl)phenyl)-4-(2-hydroxyphenyl) nicotinonitrile (12).
Yield 89%; m.p. 375 - 377 oC; IR (KBr, cm-1): 3438, 3300 (OH, NH2) , 2199 (C≡N), 1707, 1669
(2C=O), 1606 (C=N), 1558 (C=C); 1H NMR (DMSO-d6) δ (ppm): 5.59 (s, 2H, NH2;
exchangeable with D2O), 7.31 – 7.37 (m, 3H, H3,4,5 Ar`-H), 7.43 (d, H2,6 Ar-H, J = 8.4 Hz), 7.60 (
22

s, H, pyridine-H), 7.85 (d, H, H6 Ar`-H), 8.00 – 8.19 (m, 4H, Phthalimide-Hs ), 8.29 ( d, 2H, H3,5
Ar-H, J = 8.4 Hz), 11.66 (s, H, OH; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm):
105.88, 111.05, 116.00, 118.08, 123.96, 124.87, 127.43, 128.99, 129.21, 131.03, 132.02, 133.97,
135.17, 152.50, 157.55, 159.99, 161.90, 165.67; MS, m/z: 432.18 (M+, 36.61 %), 434.31
[(M+2)+, 55.20%]; Anal. Calcd. For C26H16N4O3 (432.12): C, 72.21; H, 3.73; N, 12.96; Found: C,
72.35; H, 3.90; N, 13.24.
2.1.5.4.2-Amino-6-(4-(1,3-dioxoisoindolin-2-yl)phenyl)-4-(furan-2-yl) nicotinonitrile (13). Yield
75%; m.p. 330 - 332 oC; IR (KBr, cm-1): 3420, 3347 (NH2), 2208 (C≡N), 1716, 1660 (2C=O),
1599 (C=N), 1557 (C=C); 1H NMR (DMSO-d6) δ (ppm): 6.16 (s, 2H, NH2; exchangeable with
D2O), 6.79 – 6.82 (m, H, H4 Ar`-H), 7.04 (d, H, H5 Ar`-H), 7.56 (d, H, H3 Ar`-H), 7.61 (d, H2,6 Ar-
H, J = 8.4 Hz), 7.83 ( s, H, pyridine-H), 7.92 – 8.04 (m, 4H, Phthalimide-Hs ), 8.26 ( d, 2H, H3,5
Ar-H, J = 8.4 Hz); 13C NMR (DMSO-d6) δ (ppm): 105.07, 107.21, 113.29, 113.73, 117.68,
123.96, 127.69, 128.00, 132.00, 133.97, 135.27, 141.90, 149.16, 155.08, 158.44, 162.79, 167.33;
MS, m/z: 406.32 (M+, 100 %), 407.27 [(M+1)+, 30.28%], 408.25 [(M+2)+, 7.29%]; Anal. Calcd.
For C24H14N4O3 (406.11): C, 70.93; H, 3.47; N, 13.79; Found: C, 71.08; H, 3.68; N, 13.88.
4.1.6. General procedure for the synthesis of 6-(4-(1,3-Dioxoisoindolin-2-yl)phenyl)-2-oxo-
4-(aryl)-1,2-dihydro pyridine-3-carbonitriles (14-17)
A mixture of 9 (2.65g, 0.01 mol), different aryl aldehydes (0.01 mol), ammonium acetate (6.16g,
0.08 mol) and ethyl cyanoacetate (1.13g, 0.01 mol) were refluxed in glacial acetic acid (15 ml) for
12 h. After completion of reaction, it was further cooled and filter the solid. The solid was
purified by ethanol with 63 - 91% yields.
4.1.6.1.6-(4-(1,3-Dioxoisoindolin-2-yl)phenyl)-2-oxo-4-(p-tolyl)-1,2-dihydropyridine-3-
carbonitrile (14). Yield 63%; m.p. 260 - 262 oC; IR (KBr, cm-1): 3300 (NH) , 2222 (C≡N), 1720,
1680, 1665 (3C=O), 1614 (C=C); 1H NMR (DMSO-d6) δ (ppm): 2.42 (s, 3H, CH3-Hs), 6.98 (d,
2H, H3,5 Ar`-H, J = 6 Hz), 7.09 (d, 2H, H2.6 Ar`-H, J = 6 Hz), 7.35 (d, 2H, H2,6 Ar-H, J = 8.4 Hz),
7.54 – 7.81 (m, 4H, Phthalimide-Hs ), 8.00 ( s, H, pyridine-H), 8.29 ( d, 2H, H3,5 Ar-H, J = 8.4
Hz), 10.31 (s, H, NH; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 22.02, 99.98,
116.08, 117.93, 123.55, 124.65, 126.02, 127.67, 128.28, 130.93, 132.55, 133.13, 135.94, 137.02,
157.63, 159.56, 161.21, 167.44; MS, m/z: 431.42 (M+, 33.55 %), 432.40 [(M+1)+, 8.76%], 433.44
[(M+2)+, 8.72%]; Anal. Calcd. For C27H17N3O3 (431.13): C, 75.16; H, 3.97; N, 9.74; Found: C,
75.37; H, 4.28; N, 9.81.
4.1.6.2.6-(4-(1,3-Dioxoisoindolin-2-yl)phenyl)-4-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-
3-carbonitrile (15). Yield 90%; m.p. 253 - 255 oC; IR (KBr, cm-1): 3320 (NH) , 2218 (C≡N),
1718, 1689, 1663 (3C=O), 1602 (C=C); 1H NMR (DMSO-d6) δ (ppm): 3.88 (s, 3H, CH3O-Hs),
7.04 (d, 2H, H3,5 Ar`-H, J = 8.4 Hz), 7.15 (d, 2H, H2,6Ar`-H, J = 8.4 Hz), 7.63 (d, 2H, H2,6 Ar-H, J
= 8.4 Hz), 7.88 – 8.01 (m, 4H, Phthalimide-Hs ), 8.10 ( d, 2H, H3,5 Ar-H, J = 8.4 Hz), 8.32 ( s, H,
pyridine-H), 10.39 (s, H, NH; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 55.99,
23

99.70, 114.55, 115.98, 116.55, 123.39, 124.02, 127.50, 129.24, 131.97, 133.07, 134.78, 135.32,
136.29, 136.49, 157.85, 159.88, 161.98, 167.11; MS, m/z: 447.71 (M+, 8.38 %); Anal. Calcd. For
C27H17N3O4 (447.12): C, 72.48; H, 3.83; N, 9.39; Found: C, 72.90; H, 3.71; N, 9.48.
4.1.6.3.6-(4-(1,3-Dioxoisoindolin-2-yl)phenyl)-4-(2-hydroxyphenyl)-2-oxo-1,2-dihydropyridine-3-
carbonitrile (16). Yield 85%; m.p. 181 - 183 oC; IR (KBr, cm-1): 3429, 3201 (OH, NH) , 2220
(C≡N), 1709, 1699, 1667 (2C=O), 1601 (C=C); 1H NMR (DMSO-d6) δ (ppm): 6.94 – 6.99 (m,
2H, H3,5 Ar`-H), 7.27 - 7.36 (m, 2H, H4,6 Ar`-H), 7.50 (d, H2,6 Ar-H, J = 8.4 Hz), 7.87 (d, H, H3,5
Ar-H, J = 8.4 Hz), 7.99 – 8.01 (m, 4H, Phthalimide-Hs ), 8.29 ( s, H, pyridine-H), 10.00, 10.99
(2s, 2H, OH, NH; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 100.02, 116.70,
117.37, 117.72, 121.74, 123.39, 123.92, 124.74, 126.06, 126.46, 126.72, 129.05, 130.74, 132.01,
132.32, 133.07, 156.37, 158.95, 160.70, 165.34, 167.44; MS, m/z: 433.60 (M+, 1.92 %), 434.31
[(M+1)+, 0.85 %]; Anal. Calcd. For C26H15N3O4 (433.11): C, 72.05; H, 3.49; N, 9.70; Found: C,
72.31; H, 3.72; N, 9.89.
4.1.6.4.6-(4-(1,3-dioxoisoindolin-2-yl)phenyl)-4-(furan-2-yl)-2-oxo-1,2-dihydropyridine-3-
carbonitrile (17). Yield 91%; m.p. 150 - 152oC; IR (KBr, cm-1): 3356 (NH) , 2221 (C≡N), 1716,
1695, 1650 (3C=O), 1596 (C=C); 1H NMR (DMSO-d6) δ (ppm): 6.90 – 7.08 (m, H, H3,4 Ar`-H),
7.26 (d, H2,6 Ar-H, J = 8.4 Hz), 7.59 – 7.73 (m, 4H, Phthalimide-Hs), 7.83 ( s, H, pyridine-H),
7.91 (d, H, H5 Ar`-H), 8.26 ( d, 2H, H3,5 Ar-H, J = 8.4 Hz), 10.48 (s, 2H, NH2; exchangeable with
D2O); 13C NMR (DMSO-d6) δ (ppm): 97.37, 110.73, 114.80, 115.75, 121.34, 123.56, 125.13,
126.04, 127.56, 131.25, 132.37, 135.65, 139.59, 148.65, 159.27, 162.68, 167.36, 169.55; MS,
m/z: 407.28 (M+, 42.70 %); Anal. Calcd. For C24H13N3O4 (407.09): C, 70.76; H, 3.22; N, 10.31;
Found: C, 70.94; H, 3.41; N, 10.58.
4.2. Biological evaluation
4.2.1. Anticancer screening
Cancer cells from different cancer cell lines; Human prostate carcinoma cell lines (PC-3),
hepatocellular carcinoma cell lines (HEPG-2) and human breast adenocarcinoma cell line (MCF-
7) were obtained from American Type Cell Culture Collection (ATCC, Manassas, USA) and
grown on the appropriate growth medium Dulbecco’s modified Eagle’s medium (DMEM/Life
Technologies) supplemented with 10% FBS (fetal bovine serum) (Hyclone), 10 ug/ml of insulin
(Sigma) and 1% penicillin–streptomycin. All the other chemicals and reagents were from Sigma,
or Invitrogen. Plate cells (cells density 1.2 – 1.8 _ 10,000 cells/well) in a volume of 100 ml
complete growth medium + 100 ul of the tested compound per well in a 96-well plate for 24 h
before the MTT assay.
24

4.2.2. PIM-1 kinase inhibitory activity
The effects of the synthesized compounds 6, 7, 8, 12 and 13 on the activity of PIM-1 kinase
were measured using Human proto-oncogene serine/threonine- protein kinase PIM-1 (PIM-1)
ELISA Kit (catalog #MBS701210). The cells were centrifuged for 15 min at 1000x g, 2-8
assayed immediately according to the manufacturer's instructions. Shortly, the assay was
performed using 100 mL of the supernatant of the cell, which were incubated for 2 h at 37
̊C and
̊C
before starting the assay procedure. Finally, the optical density of each well was determined
within 5 min using a microplate reader set at 450 nm.
4.2.3. Cell cycle analysis
PC-3 cells were seeded in a 6-well plate at a concentration of 1 × 105 cells per well, then
incubated for 24 h. The cells were treated with (0.1% DMSO) vehicle or 2.04 μM of 6, 7, 8, 12
and 13 compounds for 24 h. After that, cells were harvested and fixed for 12 h using ice-cold
70% ethanol at 4 °C. Removal of ethanol and washing cells with cold PBS was done; then,
incubated for 30 min at 37 °C in 0.5 ml of PBS containing 1 mg/mL Rnase. The cells were
stained for 30 min with propidium iodide in the dark. Then flow cytometer was used to detect
DNA contents. [43]
4.2.4. Annexin V-FITC apoptosis assay
PC-3 cells were seeded in a 6-well plate (1 X105 cell/well), incubated for 24 h; then treated
with vehicle (0.1% DMSO) or 2.04 μM of compounds 6, 7, 8, 12 and 13 for 24 h. The cells were
then harvested, washed using PBS and stained for 15 min at room temperature in the dark using
annexin V-FITC and PI in binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2 at
pH 7.4), then analyzed by the flow cytometer. [44]
4.2.5. Apoptosis studies
4.2.5.1. Effects on the level of active caspase 3
To determine the effect of 6, 7, 8, 12 and 13 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 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 before the assay. At
the end of the assay the optical density of each well was determined within 30 min using a
microplate reader set at 450 nm.
4.2.5.2. Effects on mitochondrial apoptosis pathway proteins Bax and Bcl-2
Cells were obtained from American Type Culture Collection, cells were grown in RPMI
1640 containing 10% fetal bovine serum at 37°C, stimulated with the compounds to be tested for
BAX or Bcl-2 and lysed with Cell Extraction Buffer. This lysate was diluted in Standard Diluent
Buffer over the range of the assay and measured for human active BAX or Bcl-2 content. (cells
25

are Plated in a density of 1.2 – 1.8 × 10,000 cells/well in a volume of 100µl complete growth
medium + 100 ul of the tested compound per well in a 96-well plate for 24 hours before
measured for human active BAX or Bcl-2).
4.3.
Molecular modeling and in silico predictions
4.3.1. Molecular docking study
The molecular modeling study was performed using the Molecular Operating
Environment (MOE 2010) software. The three-dimensional structures and conformations of the
enzymes were acquired from the Protein Data Bank (PDB) website using PIM-1 kinase (PDB ID
code 2OBJ). The ligand molecules were constructed in MOE using the builder module and
collected in a database. The database was prepared by using the option “Protonate 3D” to add
hydrogens, calculate partial charges and minimize energy (using Force Field MMFF94x). In
addition, the protein structure was prepared by deleting the repeated chains, water molecules and
any surfactants, hydrogens were also added to the atom of the receptor and the partial charges
were calculated. MOE was used to calculate the best score between the ligands and the enzymes’
binding sites. Scoring was determined using alpha HB as a scoring function. The resulted
database contained the score between the ligands' conformers and the enzyme binding sites in
kcal/mol. To confirm the credibility of docking results, self-docking was used to validate the
adopted docking protocol in which co-crystallized ligand (VRV) were drawn in MOE, prepared
as the targeted compounds (hydrogens addition, partial charges calculation and energy
minimization), and then docked into the active site of the protein using the same protocol. The
top ranked pose exhibited Root mean square deviation (RMSD) value of less than 1.5 Å from the
experimental crystal structure. This result indicated that the Molecular Operating Environment
(MOE) docking could reliably predict docking pose for the studied compounds to an enzyme. It
was reported that values less than 1.5 or 2 Å were a sign of a successful and reliable docking
protocol.
4.3.2. In silico prediction of physicochemical and ADME properties and pharmacokinetic
profile
Compounds 6, 7, 8, 12 and 13 were subjected to molecular properties prediction by
Molinspiration online property calculation toolkit, drug-likeness, and solubility parameter
calculation by MolSoft software, ADME profiling by Pre-ADMET calculator. Toxicity risk
assessment and Ligand efficiency metrics calculation by Data warrior software. Pgp substrate,
bioavailability score and synthetic accessibility prediction were evaluated by SWISSADME
online website. While PAINS prediction was performed by SWISSADME online website and
PAINS-Remover, to evaluate and analyse their suitability to qualify for a drug candidate.
Declaration of competing interest
Authors have no conflict of interest to declare.
26

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