Design and synthesis of novel phenylaminopyrimidines with antiproliferative activity against colorectal cancer

Article abstract of DOI:10.1039/c9ra03359a

New phenylaminopyrimidine (PAP) derivatives have been designed and synthesised as potential tyrosine kinase inhibitors for the treatment of cancer. The synthesized compounds share a general structure and vary in the substitution pattern at position-2 of t

Full text of DOI:10.1039/c9ra03359a

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PAPER
Design and synthesis of novel
phenylaminopyrimidines with antiproliferative
Cite this: RSC Adv., 2019, 9, 21578
activity against colorectal cancer†
bc
Hanan A. Henidi,a Ahmed M. Al-Abd,
Fahad A. Al-Abbasi,a
*
Hawazen A. BinMahfouza and Ibrahim M. El-Deeb
de
*
New phenylaminopyrimidine (PAP) derivatives have been designed and synthesised as potential tyrosine
kinase inhibitors for the treatment of cancer. The synthesized compounds share a general structure and
vary in the substitution pattern at position-2 of the pyridine ring. Several derivatives have demonstrated
potent anticancer activities against HCT-116, HT-29 and LS-174T colorectal cancer cells. Furthermore,
a number of hits showed good selectivity to Src-kinase. The cytotoxic mechanisms of these compounds
were also investigated by studying their effects on cell-cycle distribution. Among all the compounds
examined, compound 8b (with a terminal pyridin-3-yl moiety at the pyridine ring) showed the highest
inhibitory selectivity towards src-kinase, which was coupled with cell cycle arrest, and apoptotic and
autophagic interference, in colorectal cancer cells. This report introduces a novel category of PAP
derivatives with promising kinase inhibitory and anticancer effects against colon cancer.
Received 5th May 2019
Accepted 4th July 2019
DOI: 10.1039/c9ra03359a
rsc.li/rsc-advances
therapeutic strategy for the treatment of many human
illnesses.4,5 A considerable number of receptor tyrosine kinases
Introduction
was found to be involved in the pathologic background of many
malignancies, and particularly colorectal cancer. Clinical
studies revealed that the overexpression/deregulation of many
receptor tyrosine kinases is of prognostic and tumour aggres-
sion predictive value in cancer patients.6
Phenylaminopyrimidines (PAPs) represent a large chemical
group of anticancer kinase inhibitors. A representative example
and a lead compound for such group is the Bcr-Abl tyrosine
kinase inhibitor, imatinib (1, Gleevec, STI-571).7 Imatinib
(Fig. 1), was the rst kinase inhibitor approved for the treatment
of cancer. Aer the successful approval of imatinib as a selective
anticancer drug for treatment of chronic myeloid leukaemia
(CML) with minimal side effects, in comparison to classical
Cancer is a major health problem worldwide and is the second
leading cause of death.1 According to the Saudi Cancer Registry
in 1994, the incidence of all cancers, including colorectal
cancer, has been showing a steady state increase over the past
two decades.2 In addition, colorectal cancer is the most
common malignancy among males and the third most common
cancer among females in Saudi Arabia, with an overall ve-year
survival rate of 44.6%.3
Targeting oncogenic receptor tyrosine kinases (RTKs) has
attracted considerable attention in development of novel cancer
therapeutics due to their advantages over classical anticancer
drugs such as selectivity, reduced toxicity, and enhanced
permeation/retention in cancer tissues.4 In addition to cancer,
several diseases' pathologic underlying reasons are connected
to protein kinase dysfunction.5 Accordingly, the ability to
modulate protein kinase activity represents an attractive
aDepartment of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah,
Saudi Arabia
bDepartment of Pharmaceutical Sciences, College of Pharmacy, Gulf Medical
University, Ajman, UAE
cPharmacology Department, Medical Division, National Research Centre, Giza, Egypt.
E-mail: ahmedmalabd@pharma.asu.edu.eg
dRoyal College of Surgeons in Ireland-Medical University of Bahrain, Bahrain
eInstitute for Glycomics, Griffith University, Gold Coast, Queensland, Australia. E-mail:
i.el-deeb@griffith.edu.au
† Electronic supplementary information (ESI) available: Complete experimental
procedures, characterisation data, 1H and 13C NMR spectra of new
intermediates and nal compounds. See DOI: 10.1039/c9ra03359a
Fig. 1 Structures of lead inhibitors, imatinib (1), CT5102 (2), and the
PAP lead 3.
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anticancer drugs, numerous kinase inhibitors targeting onco-
genic kinases have been developed. A large number of these
kinase inhibitors belong to the same chemical group of imati-
nib, PAPs. These compounds include inhibitors for several
kinases, such as Bcr-Abl,8,9 CDK,10 c-KIT,11 EGFR,9,12 Lyn9 and Src
family13 kinases. In addition to receptor tyrosine kinases, some
anticancer PAPs modulate other13 receptors such as the angio-
poietin Tie2 receptors.14
Src subfamily of tyrosine kinases consists of several
members such as c-Src, which is involved in colorectal cancer
progression and metastasis. Several small molecules targeting
Src kinases are under preclinical and clinical investigation for
cancer treatment, including the PAP derivative CT5102, 2.13 The
authors have previously reported the discovery of a new phe-
nylaminopyrimidine (PAP) lead (3) with high potency and effi-
cacy as an anticancer agent.15 This lead hit has showed good
potency over a range of cancer cell lines, combined with
remarkable efficacy expressed by low micromolar values for
total tumour growth inhibition and 50% tumour regression. In
this study, we further modied the structure of this anticancer
lead through extended substitution at the pyridin-4-yl ring. A
variety of substituted and unsubstituted aromatic structures
have been attached to this point of the molecule through
a series of Suzuki coupling reactions16,17 in order to properly
evaluate the effect of this extended substitution on compound's
activity (Fig. 2). The new inhibitors were tested for their capacity
to inhibit the growth of colon cancer cells through inhibition of
tyrosine kinase activity and the underlying mechanisms of their
tumour cell-killing properties were also investigated.
Scheme 1 Synthesis of target PAP derivatives 8a–j. Reaction condi-
tions and yields: (i) 2-chloro-4-methylpyridine, LHMDS, THF, N2, rt,
18 h, 72%; (ii) DMF-DMA, reflux, 12 h; (iii) NaOEt, guanidine-HCl, abs.
EtOH, reflux, 18 h, 58%; (iv) 3,5-bis(trifluoromethyl)phenyl bromide,
Pd(PPh3)2Cl2, xantphos, NaOtBu, toluene, reflux, 12 h, 69%; (v) aryl-
boronic acid, Pd(PPh3)2Cl2, K2CO3, N2, THF/H2O; (4 : 1), 70 ꢀC, 20 h.
heated with excess N,N-dimethylformamide dimethylacetal for
12 hours, and the resulted product was taken to the next step
without further purication, where it was allowed to react with
guanidine-hydrochloride in absolute ethanol and in the pres-
ence of sodium ethoxide, to undergo cyclization into the
pyrimidine derivative 6 in an overall yield of 58% (over two
steps).
Results
Synthesis of target compounds
The synthesis of the target compounds started with the key ester
methyl 3-methoxy-5-methylbenzoate (4), which was prepared
following reported procedures.16 As showed in Scheme 1, the
benzoate ester 4 underwent a nucleophilic attack at its carbox-
ylate carbon by the activated methylene group of 2-chloro-4-
methylpyridine. The activation of this methyl group into an
active methylene was achieved by the dropwise addition of
lithium bis(trimethylsilyl)amide (LHMDS) in dry THF at room
temperature. The resulted ketone 5 (obtained at 71% yield) was
then converted to the required pyrimidin-2-amine derivative 6
through two successive steps. In the rst step, compound 5 was
Arylation of the amino group of the resulted pyrimidine-2-
amine (6) with 3,5-bis(triuoromethyl)phenyl bromide, to
obtain the key PAP intermediate 7 was achieved in a good yield
(69%) applying optimized Buchwald–Hartwig amination
protocol.18 The produced PAP intermediate 7 was then sub-
jected to a series of Suzuki coupling reactions with a group of
arylboronic acids, in the presence of dichloro bis(-
triphenylphosphine)Pd(II) and potassium carbonate, in a mixed
ꢀ
solvent of THF and water in a (4 : 1) ratio at 70 C, to yield the
nal PAP derivatives 8a–j in good yields.
In Scheme 2, the methoxy groups in the PAP derivatives 7
and 8a–j were demethylated under the effect of BF3–dime-
thylsulde complex, in dichloromethane at room temperature,
to produce the hydroxyl analogues 9 and 10a–j, respectively in
good yields.
Tyrosine kinase inhibition assessment
The capacity of the synthesized compounds to inhibit protein
kinases was assessed using a mixture of tyrosine kinases
extracted from HT-29 cell line.
Fig. 2 General structure of the novel PAP inhibitors derived from the
structures of the lead inhibitor 3.
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Further analysis for specic Src-kinase inhibitory potency
was undertaken to determine compounds' specicity.
Compounds 7, 8a, 8b, 8g, 8j, 9 and 10g showed considerable Src
kinase inhibitions, at 10 mM concentration, by 42.3 ꢁ 10.3%, 54
ꢁ 0.6%, 77.8 ꢁ 1.3%, 28.5 ꢁ 1.2%, 54.9 ꢁ 1.6%, 33.6 ꢁ 3.3%
and 51.3 ꢁ 9.8% inhibitions, respectively. The rest of the
compounds under investigation did not show more than 14.7%
inhibition for Src kinase (Fig. 3B). As a measure for selectivity
towards Src kinase, Src kinase selectivity index was used. Src
kinase selectivity index is the ratio between Src kinase inhibi-
tions to the non-specic multi-kinase inhibitions of compounds
under investigation. Compounds 8a, 8b, 8j and 10g showed Src
kinase indices of 2.4, 2.9, 2.1 and 3.1, respectively.
Detailed dose–response curves for compounds 7, 8b, 9, 10a,
10b, 10f and 10g as Src kinase inhibitors were plotted (Fig. 4).
Compounds 8b and 10g showed potent src kinase inhibitions
with EC50's of 1.3 ꢁ 0.3 and 3.7 ꢁ 0.7 mM, respectively. On the
other hand, compounds 10a, 10b and 10f were completely
inactive in inhibiting src kinase, while compounds 7 and 9
showed moderate inhibitory activity.
Scheme 2 Synthesis of the hydroxyl analogues 9 and 10a–j. Reaction
conditions and yields: (i) BF3S(CH3)2, CHCl3, N2, rt, 48 h.
Molecular modelling
In order to predict the potential binding mode of these inhib-
itors to kinases, inhibitor 8b was used as a model. A molecular
docking simulation was applied for inhibitor 8b into the co-
crystal structure of src kinase (PDB 4MXO19), using
MOE.2008.10 soware. The compound was docked over the
ligand (bosutinib) atoms in the binding site using the soware
standard docking parameters, and the most stable docking pose
of 8b was selected as showed in Fig. 5.
As showed in the gure, the more hydrophobic part of
compound 8b skeleton (bis-3,5-(triuoromethyl)phenyl moiety)
was oriented towards the depth of the pocket, while the
terminal pyridine ring (which represents the varied part of the
skeleton within the whole series) points towards the protein
surface. Interestingly, a hydrogen bonding (HB) interaction was
predicted between the terminal pyridine nitrogen and glutamic
acid residue 353.
At 10 mM concentration, compounds 7, 8a, 8b, 8g, 8j, 9, 10a,
10f, 10g and 10j showed multi-kinase inhibitory effects by 37.4
ꢁ 3.7%, 23 ꢁ 0.4%, 26.6 ꢁ 2.4%, 33.1 ꢁ 0.4%, 26 ꢁ 0.5%, 30.4 ꢁ
2.3%, 24.7 ꢁ 4.6%, 23.9 ꢁ 7.4%, 39 ꢁ 0.4% and 30.2 ꢁ 0.4%,
respectively. The rest of the series did not show more than
16.5% overall inhibition against multi-kinase cellular extract
(Fig. 3A).
Fig. 3 The effect of test compounds at 10 mM concentration against Fig. 4 Dose–response relationship of selected compounds against
the activity of total intracellular tyrosine kinase (A) and purified Src the activity of purified Src kinase enzyme. Data are expressed as mean
kinase (B). Data are expressed as mean ꢁ SD; n ¼ 3.
ꢁ SD; n ¼ 3.
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to control cells (Fig. 6A). Furthermore, compounds, 8f and 9
exhibited more than 50% drop in viability at 10 mM concen-
tration, while at this concentration, compounds 8b, 8g, 10a,
10b, 10c, 10f, 10g and 10j showed viability drop by 30–45%
compared to control cells.
In HT-29 cells, all compounds under investigation, except for
8h and 10h, reduced cell viability, at 100 mM concentration, by
more than 50% compared to control cells (Fig. 6B). At 10 mM
concentration, compounds, 7, 8f, 8g, 9, 10a, 10b and 10f
induced more than 50% drop in viability, while compounds 8b,
8c, 10c, 10d and 10j reduced cell viability by 35–45%, compared
to control cells.
LS-174T was the most resistant cell line among the colorectal
cancer cells used for evaluation, where at 100 mM; only
compounds 7, 8f, 8g, 10c, 10d, 10g and 10j induced viability
drop by more than 50%, compared to control cells (Fig. 6C). In
addition, only compounds 8f, 9, 10c, 10d and 10j were able to
reduce viability by more than 50% at 10 mM concentration.
Detailed dose–response relationship against colorectal cancer
cells
Aer evaluation of multi-tyrosine kinase inhibition, selective
Src kinase inhibition and the preliminary antiproliferative/
cytotoxic effects of the synthesized compounds, it was impor-
tant to construct dose–response curves of the most potent
compounds 7, 8b, 9, 10a, 10b, 10f and 10g to calculate accurate
IC50 values against HCT-116 and HT-29 cell lines. Furthermore,
Fig. 5 Molecular docking simulation of compound 8b into src kinase
co-crystal protein structure 4MXO. Upper panel: a 3D presentation of
the most stable docking pose of 8b showing proposed HB interaction
between the terminal pyridine nitrogen and glutamate 353. Lower
panel: a 2D presentation for the docked structure.
Antiproliferative/cytotoxicity assessment
Tyrosine kinase is an intracellular enzyme and inhibiting the
enzyme in cell free system is not necessarily interpreted as
a cytotoxic effect. Therefore, preliminary assessment of the
potential antiproliferative/cytotoxic properties of the whole
series under investigation is a key step in short-listing these
compounds for more detailed biological assessment. Accord-
ingly, HCT-116, HT-29 and LS-174T colorectal cancer (CRC) cell
lines were exposed to 10 and 100 mM concentrations of the
compounds under investigation for 72 h and viabilities were
compared to control untreated cells. Only compounds 8h and
10h failed to show any considerable antiproliferative/cytotoxic
effects even at 100 mM against all colorectal cancer cell lines
used (Fig. 6A–C).
Fig. 6 Effect of compounds under investigation on the viability of
HCT-116 (A), HT-29 (B) and LS-174T colorectal cancer cells (C). Cells
were treated with 10 mM and 100 mM of test compounds for 72 h and
viability was determined using SRB assay. Data are expressed as mean
ꢁ SD; n ¼ 6.
In HCT-116 cells, all test compounds, except for 8h and 10h,
induced viability drop by more than 50% at 100 mM, compared
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detailed cytotoxic parameters were also deduced. Compounds 9,
10a and 10f showed the most potent cytotoxic effects against
both cell lines with IC50's ranging from 7.2 ꢁ 1.1 mM to 10.3 ꢁ
0.6 mM and resistance fraction less than 1% (Table 1, Fig. 7C, D
and F).
Compounds 7, 8b and 10b have also showed good
antiproliferative/cytotoxic effects in HT-29 with IC50's of 9.4 ꢁ
0.5, 11.3 ꢁ 0.5 and 6.4 ꢁ 0.5 mM, respectively (Fig. 7A, B and E).
The resistance fractions of HT-29 cells towards these
compounds were less than 5% (Table 1). On the other hand,
compounds 7, 8b and 10b showed signicantly weaker
antiproliferative/cytotoxic effects against HCT-116 cells with
IC50's higher than 25 mM (Fig. 7A, B and E). Interestingly,
compound 10g showed the weakest antiproliferative/cytotoxic
prole among the selected compounds with both high IC50
value against HT-29 cells (24.2 ꢁ 2.1 mM) and very high resis-
tance fraction (41.6 ꢁ 3.2%) within HCT-116 cells (Fig. 7F)
(Table 1).
Inuence of test compounds on cell cycle distribution of
colorectal cancer cells
In order to inspect in further details the potential anti-
proliferative properties, cell cycle distribution was assessed
aer exposure of HCT-116 and HT-29 cells to 10 mM of test
compounds for 48 h. For this test and the following tests, the six
compounds that showed the most potent inhibitions on both
cell lines (compounds 7, 8b, 9, 10a, 10b and 10f) were used.
In HCT-116 cells, only compound 8b induced signicant
antiproliferative effect in terms of accumulating cells in G0/G1-
phase (50.2 ꢁ 1.0%) with reciprocal decrease of cells entering S-
phase (22.8 ꢁ 2.3%) compared to 39.5 ꢁ 4.6% and 36.4 ꢁ 4.1%
of control cells in G0/G1-phase and S-phase, respectively
(Fig. 8A).
Fig. 7 Dose–response assessment for compounds 7 (A), 8b (B), 9 (C),
10a (D), 10b (E), 10f (F) and 10g (G) against HCT-116 (solid lines) and
HT-29 cells (dotted lines). Cells were treated with test compounds for
In HT-29 cells, both compounds 8b and 9 induced signi- 72 h and viability was determined using SRB assay. Data are expressed
as mean ꢁ SD; n ¼ 6.
cant antiproliferative effects, increasing cell population in G0/
G1-phase from 53.4 ꢁ 2.7% to 58.8 ꢁ 1.8% and 59.7 ꢁ 0.73%,
respectively. Reciprocally, compounds 8b and 9 signicantly
decreased cells in S-phase from 27.3 ꢁ 0.6% to 21.0 ꢁ 0.9% and
22.0 ꢁ 1.2%, respectively. Compound 10a induced mild but
signicant cell cycle arrest in S-phase and G2/M-phase
increasing their cell populations from 27.3 ꢁ 0.6% and 19.3 ꢁ
1.6% to 29.8 ꢁ 0.6% and 24.8 ꢁ 0.4%, respectively. This cell
cycle arrest induced by compound 10a resulted in reciprocal
decrease in cell population in G0/G1-phase to 45.4 ꢁ 0.6%
(compared to 53.4 ꢁ 2.7% in control). Compound 10b selec-
tively arrested cells in G2/M-phase increasing its cell population
from 19.3 ꢁ 1.6% to 25.1 ꢁ 0.6%; which resulted in reciprocal
decrease of S-phase cells from 27.3 ꢁ 0.6% to 23.0 ꢁ 0.5%
(Fig. 8B).
Apoptosis/necrosis assessment
Table 1 Detailed cytotoxicity parameters of selected compounds with
potential killing effects against HCT-116 and HT-29 cells
Annexin V-FITC/PI staining coupled with ow cytometry was
used to differentially assess proportion of cells dying via
necrosis versus cells undergoing apoptosis in colorectal cancer
cell lines. HCT-116 and HT-29 cells were treated with 10 mM of
selected test compounds for 48 h prior to apoptosis/necrosis
differential quantication by ow cytometry. In HCT-116 cells,
compounds 8b, 9, 10a, 10b and 10f induced signicant
apoptosis, increasing apoptotic cell population by 3.8, 2.1, 7.5,
4.7 and 15.2 folds, respectively.
HCT-116
HT-29
IC50 (mM)
R (%)
IC50 (mM)
R (%)
Cpd # 7
Cpd # 8b
Cpd # 9
Cpd # 10a
Cpd # 10b
Cpd # 10f
Cpd # 10g
83.3 ꢁ 5.8
32.4 ꢁ 2.8
9.4 ꢁ 1.2
10.3 ꢁ 0.6
25.9 ꢁ 1.3
9.2 ꢁ 0.8
9.4 ꢁ 0.9
N/A
9.4 ꢁ 0.5
11.3 ꢁ 0.5
7.5 ꢁ 0.5
9.9 ꢁ 0.3
6.4 ꢁ 0.5
7.2 ꢁ 1.1
24.2 ꢁ 2.1
3.9 ꢁ 1.3
3.7 ꢁ 0.9
<1
<1
<1
<1
<1
<1
<1
<1
<1
In addition, compounds, 7, 8b, 10a, 10b and 10f induced
signicant necrosis, increasing necrotic cell population by 3.4,
41.6 ꢁ 3.2
12.9 ꢁ 3.9
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Fig. 9 Apoptosis/necrosis assessment in HCT-116 (A) and HT-29 (B)
cells after exposure to test compounds. Cells were exposed to 10 mM
of compounds 7, 8b, 9, 10a, 10b, and 10f for 48 h and were stained
with annexin V-FITC/PI. Different cell populations were plotted as
percentage of total events. Data is presented as mean ꢁ SD; n ¼ 3. (*):
Significantly different from control group.
Fig. 8 The effect of compounds 7, 8b, 9, 10a, 10b, and 10f on the cell
cycle distribution of HCT-116 (A) and HT-29 (B) cells. Cells were
exposed to 10 mM of test compounds for 48 h and cell cycle distri-
bution was determined using DNA cytometry analysis and different cell
phases were plotted as percentage of total events. Data is presented as
mean ꢁ SD; n ¼ 3. (*): Significantly different from control group.
(chloroquine) increased autophagic signal in HCT-116 cells by
65% (Fig. 10A). In contrast to what was observed at HCT-116,
compounds 8b, 9 and 10a inhibited autophagy in HT-29 cells
2.9, 4.0, 2.4 and 3.7 folds, respectively. This explains the cell
killing effects attributed to these compounds against HCT-116
cells; and shows that the calculated IC50 values of compounds
7, 8b, 9, 10a, 10b and 10f are indicative of cytotoxic effects with/
without moderate antiproliferative activity. In addition, the
cytotoxic effects of compounds 9, 10a, 10b and 10f were found to
be attributed more to apoptosis rather than non-specic
necrosis (Fig. 9A).
With respect to HT-29 cells, compounds 8b, 10b and 10f were
found to induce apoptosis, increasing apoptotic cell population
by 2.5, 2.3 and 1.7 folds, respectively. The rest of the tested
compounds induced apparent apoptotic cell population
increase; but this increase was not signicant. On the other
hand, none of these compounds induced any signicant non-
specic necrosis. In other words, cell-killing effects of these
compounds against HT-29 cells are mainly attributed to
apoptosis rather than non-specic necrosis (Fig. 9B).
Autophagy assessment
Besides apoptosis, autophagy represents
a big research
controversy; whether it is a programmed cell death mechanism
or an escape-death mechanism. Herein, we further investigated
the effect of test compounds on autophagy process within Fig. 10 Autophagic cell death assessment in HCT-116 (A) and HT-29
(B) cells after exposure to test compounds. Cells were exposed to 10
mM of compounds 7, 8b, 9, 10a, 10b, and 10f for 48 h. Cells were
stained with Cyto-ID autophagosome tracker. Net fluorescent inten-
sity (NFI) were plotted and compared to basal fluorescence of control
colorectal cancer cell lines using Cyto-ID autophagy detection
dye coupled with ow cytometry.
In HCT-116, compounds 8b, 10b and 10f signicantly
induced autophagic signal higher than control cells by 54%,
group. Data is presented as mean ꢁ SD; n ¼ 3. (*): Significantly
30% and 99%, respectively. Positive control treatment different from control.
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by 21%, 15% and 24%, respectively. On the other hand and in
Further assessment of antiproliferative/cytotoxicity activity
a similar fashion to what was observed with HCT-116 cells, showed that LS-174T cells were the most resistant to the
positive control treatment (chloroquine) induced autophagy in compounds under investigation. The other two colorectal
HT-29 cells (Fig. 10B).
cancer (CRC) cell lines (HCT-116 and HT-29) showed consider-
able susceptibilities to the tyrosine kinase inhibitors under
investigations. It is worthy to mention that LS174T and HCT-15
cells were reported to show resistance to the selective MEK1/2
Discussion
The design of the new series aimed at extending the modica- tyrosine kinase inhibitor (selumetinib).20 In contrast to the
tions at the terminal pyridine ring in the potent PAP inhibitor 3 effect of the methoxy group in maintaining good src-kinase
to yield the methoxy series (7 and 8a–j) and their hydroxy inhibition selectivity, series 10 (compounds with
a free
analogues (9 and 10a–j). The initial screening of the synthesized phenolic OH) demonstrated in general more antiproliferative/
series over multiple kinases and on different colorectal cancer cytotoxic effects than their methoxy group counterpart (series
cells demonstrated that this new design was successful in 8). This observation suggests that the overall cytotoxic effects of
producing potent anticolorectal cancer agents that are capable these compounds might be attributed in part to kinase inhibi-
of targeting and inhibiting protein kinases. From the whole tion, but does not exclude the involvement of other potential
synthesized series, several compounds showed considerable underlying mechanisms. In studying the effect of substitution
non-specic multi-tyrosine kinase inhibitory activity. However, at the terminal pyridine ring on the overall antiproliferative
compounds 8a, 8b and 8j showed the highest src-kinase selec- effect of the compound, it was interesting to see that
tivities. On the other hand, compounds 10a and 10f did not compounds 8h and 10h, with the bulkiest substituent on the
exert any signicant src-kinase inhibition despite their consid- pyridine ring (N,N-diphenylamino group) have showed the least
erable multi-kinase inhibition. Additionally, compounds 7, 9 anticancer activities on all the tested cell-lines. This indicates
and 10b non-selectively inhibited the multi-tyrosine kinases that there is a size limit for the extended modications within
panel at an equivalent level to the inhibition of src-kinase.
this new series of compounds, and that this particular modi-
These results show that the new skeleton provides a good cation has exceeded this limit, resulting in completely dimin-
template for the development of novel kinase inhibitors. ishing the anticancer activity of the compound.
However, the substitution pattern plays a critical role in
To further dissect the antiproliferative/cytotoxic effects of the
dening the kinase inhibition selectivity prole of the inhibitor. tyrosine kinase inhibitors under investigation, the inuence of
At the terminal pyridine ring, the attachment of an unsub- these compounds on cell cycle distribution and apoptosis/
stituted phenyl (as in 8a) or 3-pyridyl (as in 8b) resulted in good autophagy induction was examined. In this testing, only
inhibitory selectivities towards src kinase. On the other hand, compound 8b induced signicant antiproliferative effect and
decorating the phenyl ring with either electron donating or accumulation of both HCT-116 and HT-29 cells in G0/G1-phase.
electron withdrawing groups resulted in disturbance of this This could be explained on the basis of selective src-kinase
kinase selectivity prole. Surprisingly, the only substitution that inhibition, which is usually coupled with antiproliferative
proved to be effective in maintaining the selectivity towards src activity and accumulation of cells in G0/G1-phase. Several
kinase was that with a bulky 4-phenoxy group at the terminal reports showed that interference with receptor tyrosine kinases
phenyl ring (compound 8j). The effect of the terminal aryl such as src-kinase results in cell cycle arrest at G0/G1-phase.21,22
substituent on inhibitor's src kinase inhibition corresponds This arrest is partly attributed to interrupting the function of
with the molecular modelling simulations carried out on cyclin-dependent kinases (CDK) and other cell cycle controlling
compound 8b. The docking simulations have showed that the kinases.23 On the other hand, the non-src-kinase inhibitors
terminal pyridine moiety of 8b orients towards the surface of (compounds 10a and 10b) resulted in accumulation of HT-29
the binding pocket where it engages, through its heterocyclic cells in S-phase and G2/M-phase. This was manifested in the
nitrogen, in HB interaction with GLU353. This hydrogen form of blocking DNA replication and eventually resulted in
bonding interaction can account for the higher selectivity and altered cell morphology and induction of apoptosis.24 Further-
potency of 8b towards src kinase, relative to other inhibitors more, compounds 8b (selective src-kinase inhibitor), 10b and
within the series. This observed orientation could also explain 10f induced signicant apoptosis in both HCT-116 and HT-29
why excessive bulkiness at this part of the skeleton can result in cells. Apoptosis induction by compound 8b might be attrib-
decreased potency of the compound (as in inhibitors 8h and uted to accumulation of cells at G0/G1-phase and failure of
10h), where excessive bulkiness may result in clashing with transition to S-phase.21,22 Yet, it is reported that defects in G2-M-
certain surface residues in the protein, such as GLU 353.
phase checkpoint might allow a damaged cell to enter mitosis
Another important observation is the effect of the methoxy and undergo apoptosis.25 In addition, cell cycle arrest can
group in the (3-methoxy-5-methylphenyl) ring on src-kinase explain proliferation inhibition and is known to trigger
inhibitory selectivity in these three compounds (8a, 8b and apoptosis in cancer cells.26 On the other hand, compounds 7,
8j). Removal of this O-methyl group from these inhibitors to 8b, 9, 10a, 10b and 10f induced programmed as well as non-
yield a free phenolic OH in their hydroxy counterparts (10a, 10b programmed necrotic cell death in HCT-116 cells.
and 10j, respectively) resulted in a drop in src-kinase inhibitory
Autophagy is another programmed cell death mechanism. It
potency, which emphasized the importance of this O-methyl is highly controversial and complicated to conclude whether it
group in maintaining good inhibitory potency for src-kinase.
is a pro-death mechanism or an escape-death mechanism in
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cancer cell death.27 Herein, HCT-116 cells underwent autophagy
in response to treatment with compounds 8b, 10b and 10f;
while autophagy was suppressed in HT-29 cells treated with
compounds 8b, 9 and 10a. Putting the killing prole of these
compounds (Table 1) in consideration, we suggest that, HCT-
116 and HT-29 CRC cells are escaping apoptosis via auto-
phagy shunting. In other words and similar to what was
observed in our previous study on breast cancer cells,28 auto-
phagy is considered here to be an apoptosis escape shelter.27,29,30
Acknowledgements
This research was supported by the Royal College of Surgeons in
Ireland-Medical University of Bahrain (Grant number BR00063).
The authors are grateful for Dr Marc Devocelle and Ms Siobhan
O'Flaherty for their assistance in characterising the synthesized
compounds using spectroscopy facility in RCSI (Royal College of
Surgeons in Ireland).
´
Notes and references
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Conflicts of interest
The authors declare no conict of interests.
This journal is © The Royal Society of Chemistry 2019
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