Synthesis of novel 1,2,4-triazine scaffold as FAK inhibitors with antitumor activity

Article abstract of DOI:10.1016/j.bmcl.2017.02.072

A series of 1,3,5-triazinic inhibitors of focal adhesion kinase (FAK) has recently been shown to exert antiangiogenic activity against HUVEC cells and anticancer efficacy against several cancer cell lines. In this report, we designed and synthesized a series of new compounds containing a 1,2,4-triazine core as novel scaffold for FAK inhibitors. These compounds displayed 10^?7?M IC₅₀ values, and the best one showed IC₅₀ value of 0.23?μM against FAK enzymatic activity. Among them, several inhibitors potently inhibited the proliferation of glioblastoma (U-87MG) and colon (HCT-116) cancer cell lines. Docking of compound 10 into the active site of the FAK kinase was performed to explore its potential binding mode.

Full text of DOI:10.1016/j.bmcl.2017.02.072

Accepted Manuscript
Synthesis of novel 1,2,4-triazine scaffold as FAK inhibitors with antitumor ac-
tivity
Pascal Dao, Daniel Lietha, Mélanie Etheve-Quelquejeu, Christiane Garbay,
Huixiong Chen
PII:
S0960-894X(17)30215-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.02.072
BMCL 24744
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 January 2017
25 February 2017
27 February 2017
Please cite this article as: Dao, P., Lietha, D., Etheve-Quelquejeu, M., Garbay, C., Chen, H., Synthesis of novel
1,2,4-triazine scaffold as FAK inhibitors with antitumor activity, Bioorganic & Medicinal Chemistry Letters (2017),
doi: http://dx.doi.org/10.1016/j.bmcl.2017.02.072
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Bioorganic & Medicinal Chemistry Letters
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Synthesis of novel 1,2,4-triazine scaffold as FAK inhibitors with antitumor
activity
Pascal Daoa, Daniel Liethab, Mélanie Etheve-Quelquejeua, Christiane Garbaya, and Huixiong Chen*,a
aCNRS UMR8601, Université Paris Descartes, PRES Sorbonne Paris Cité, UFR Biomédicale,45 rue des Saints-Pères, 75270 Paris Cedex 06, France
bCell Signalling and Adhesion Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Calle Melchor Fernández
Almagro 3, Madrid 28029, Spain
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
A series of 1,3,5-triazinic inhibitors of focal adhesion kinase (FAK) has recently been shown to
exert antiangiogenic activity against HUVEC cells and anticancer efficacy against several cancer
cell lines. In this report, we designed and synthesized a series of new compounds containing a
Accepted
1,2,4-triazine core as novel scaffold for FAK inhibitors. These compounds displayed 10−7
M
Available online
IC50 values, and the best one showed IC50 value of 0.23 µM against FAK enzymatic activity.
Among them, several inhibitors potently inhibited the proliferation of glioblastoma (U-87MG)
and colon (HCT-116) cancer cell lines. Docking of compound 10 into the active site of the FAK
kinase was performed to explore its potential binding mode.
Keywords:
FAK inhibitors
Synthesis
1,2,4-triazines
Anti-cancer activity
Molecular docking
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Crystal structures of FAK kinase domain have been published
and their studies have been helpful for structure-based design
Focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase
and scaffold protein localized to focal adhesions, is an important
functional regulator of cell signaling within the tumor
microenvironment.1,2 This protein is emerging as a promising
therapeutic target because it is over-expressed at both the
transcriptional and translational level in a large variety of
cancers, including pancreatic, ovarian and cervical cancers,
prostate neoplasm, head and neck squamous cell carcinoma,
glioblastoma, colorectal, breast, lung and kidney cancers.3–5
Ample evidence has indicated that FAK signaling pathways can
stimulate tumor progression and metastasis formation through
their regulation of cell migration, invasion and angiogenesis.6–8
However, some of FAK’s functions in tumorigenesis remain
under investigation.
efforts aimed at improving selectivity and potency of FAK
inhibitors.14–16 Structural scaffolds of FAK inhibitors described as
type I inhibitors, generally bearing nitrogen heterocyclic ring,
occupy the nucleotide binding pocket in a manner to bind the
hinge region of the kinase domain. Nitrogen(s) in the ring can
form hydrogen bonds with the carbonyl group of Cys502/Glu500
residues of the kinase hinge region. The aromatic ring makes
hydrophobic contacts with Ala452 and Leu553 in the catalytic
site. It was observed that pyrimidine derivatives, specifically 2,4-
pyrimidine derivatives, are the major scaffolds found in
compounds described by pharmaceutical companies and
academic research.11,17–21
As part of our ongoing efforts to identify new chemical
classes of FAK inhibitors associated with antiproliferative
activity, we previously described a series of 1,3,5-triazinic
inhibitors, which show antiangiogenic activity against HUVEC
cells and antiproliferative efficacy against several cancers in cell
growth studies (glioblastoma U-87MG, colon cancer HCT-116,
breast cancer MDA-MB-231, and prostate cancer PC-3).22,23 X-
ray crystallographic analysis of the cocrystal structure of these
compounds in the FAK kinase domain revealed that the mode of
interaction is highly similar to that observed in the complex of
TAE-226, a potent ATP competitive inhibitor of FAK, designed
by Novartis Pharma AG.15 As TAE-226, compound 3422
stabilizes an unusual helical conformation of the DFG motif
(Asp-Phe-Gly) in which the φ torsion angle of Asp564 is rotated
by 113° compared with the active kinase domain. Despite the
In the past few years, phase I and II clinical trials have been
initiated with small FAK inhibitor molecules.9–11 They decrease
tumor growth and metastasis development in several preclinical
models and possess initial clinical activity in patients, even if
some adverse events have been reported.12 The trials are studied
either individually or in combination to prevent diseases related
to FAK signaling or overcome resistance related to FAK
inhibition. The progress in evaluation of FAK inhibitors in both
preclinical models and human clinical trials holds good
promise.12,13 Thus, extensive research efforts are currently
invested in the discovery of novel FAK inhibitors for developing
highly specific therapeutic drugs for particular tumor phenotypes.

Table 1. In Vitro Enzymatic Activities of Novel 1,2,4-
diarylaminotriazines compared with TAE-226.
very similar interaction mode of these compounds, the in vitro
potency of 1,3,5-triazinic compound 34, is approximately 45-fold
lower than that of TAE-226. From X-ray structure we proposed
that one major reason could be due to the missing chlorine atom
in our 1,3,5-triazinic compound, which in TAE226 makes van
der Waals interactions with Met499. It has been shown
previously that filling binding cavities to improve geometric fits
and hence van der Waals contacts can significantly contribute to
binding affinity.24 The aim of the work reported herein was to
develop 1,2,4-triazinic compounds as novel scaffold of FAK
inhibitors, which can contain one chlorine atom in position 6,
able to provide van der Waals interactions with Met499.
O
N
Cl
N
N
N
Cl
N
R1
N
N
N
H
A
R2
B
H
O
N
H
N
H
O
N
H
TAE-226
N°
5
R1
R2
IC50 (µM)
2.5 0.3
3,4,5-OCH3
3,4,5-OCH3
4-CH2NH2
3-NHSO2CH3
2-CONHCH3
2-CONHCH3
2-CONHCH3
2-CONHCH3
2-CONHCH3
0.64 0.06
0.56 0.06
0.87 0.04
0.34 0.03
0.23 0.03
0.007 0.002
6
7
2-OCH3-4-Cl-5-CH3
4-morpholino
2-OCH3-4-morpholino
8
9
10
TAE-226
The synthetic route of new 1,2,4-triazinic compounds 5-10
was outlined in Scheme 1. Starting from commercially available
1,2,4-triazine-3,5(2H,4H)-dione (Scheme 1), the electrophilic
bromine was introduced in position 6 of the triazinic ring at room
temperature to obtain the intermediate 1 (91%), which was
converted to 1,2,4-trichlorotriazine 2 in presence of N, N-
In an attempt to gain insight into the putative binding mode of
these inhibitors with the FAK kinase, compound 10 was docked
into the apo FAK kinase structure (PDB ID 2jkk with TAE226
removed). The kinase receptor was kept rigid, whereas for the
ligand 7 rotatable bonds were defined. Docking was performed
with Autodock 4.2, using a standard protocol. In brief, the protein
structure was screened within a cubic box with size 25 × 25 × 25
Å centered on the ATP binding site, applying a space grid of
0.375 Å. The lowest energy and most populated cluster was
selected after 100 cycles of running a Lamarckian genetic
algorithm with a population size of 150. A maximum of 2.5
million energy evaluations was applied, and results were
clustered using a tolerance of 2.0 Å. Autodock computed a mean
binding energy of -9.46 kcal/mol for the lowest energy cluster,
which contained 57 of the 100 docked structures. The best pose
in the cluster is shown in Fig.1 and has a calculated binding
energy of -9.96 kcal/mol. The next best cluster was only singly
populated with a calculated binding energy of -8.58 kcal/mol.
diethylaniline,
phosphorus
pentachloride
and
phosphoryltrichloride at 130 °C with a high yield of 81%.25 The
first chlorine atom in position 5 of compound 2 is displaced by
substituted anilines in the presence of triethylamine in THF at
reflux to afford monosubstituted compounds 3-4 in good yields
(64-68%). The chlorine atom in position 3 of compound 3-4 was
further substituted to the compounds 5-10 in the presence of
camphorsulfonic acid (CSA) in isopropanol through the agency
of the corresponding arylamines in a range of yield of 48-55%,
except for compound 7 which was obtained after deprotection of
the amino protecting Boc group. Final products 5-10 were fully
characterized by NMR and mass spectroscopy.
N
N
Cl
N
N
Cl
N
N
N
N
Br
O
N
N
Cl
Cl
HN
a
HN
b
c
d
N
Cl
NH
HN
NH
or d,e
O
N
H
O
O
N
H
Cl
R1
R2
R1
2
3-4
5-10
1
Scheme 1. a) Br2, b) PCl5/N,N-diethylaniline/POCl3/130°C, c) arylamine/
Et3N/THF/reflux, d) arylamine/CSA/isopropanol/reflux, e) TFA/CH2Cl2
All compounds were evaluated for their ability to inhibit FAK
kinase activity using a TR-FRET based kinase assay as described
previously.26 TAE-226 was used as a control. In our test
conditions, TAE-226 inhibited the activity of FAK with IC50
value of 7 nM (Table 1), which was similar to previously
reported data.27
As presented in Tables 1, tested compounds demonstrated
potencies with IC50 values in the 10−7 M range. The choice of the
different substituents at R1 on the ring A and R2 on the ring B
was based on the results obtained in the previous 1,3,5-
diarylaminotriazinic series.22 We have chosen amide in the ortho
position of the ring B and varied the R2 substituents on the ring
A, including 3,4,5-OCH3, 4-CH2NH2 and 2-OCH3-4-morpholino
groups. These different pharmacomodulations showed effects
similar to what had been obtained in the diarylamino-1,3,5-
triazine series. Indeed, compound 10 with 2-OCH3-4-morpholino
group shows the best FAK inhibitory activity with IC50 of 0.23
µM, compared with its diaryl-1,3,5-aminotriazinic analogue with
IC50 of 0.32 µM.22 It seems that methoxy in the 2 position of the
ring A in compound 10 can make slightly better interactions with
the hinge than the compound 9 without this group, since
compound 9 has a somewhat higher IC50 value. Unfortunately,
the addition of chlorine in position 6 on the 1,2,4-triazine ring
(compound 10) failed to gain affinity, as compared to TAE226
(IC50 = 7 nM).
Figure 1. (a) Docking of Compound 10 into the ATP binding pocket of FAK.
(b-c) Crystal structures of compound 34 (PDB ID: 4brx) and TAE226 (PDB
ID: 2jkk) are shown bound to the active site of the FAK kinase. Key side
chains and the inhibitors (yellow) are shown in stick representation. For
clarity residues 429–431 in the P-loop are rendered transparent. Hydrogen
bond interactions between receptor and ligand are shown as orange dashes
lines. (d) The lowest energy docking pose of compound 10 fits well into the
ATP binding pocket of FAK.

The resulting 3D binding modes of this compound to FAK is
depicted in Figure 1a. Hydrogen bond interactions between
receptor and ligand are shown as orange dashes lines. As shown
in Fig.1d, compound 10 fits well into the ATP binding pocket.
Comparison of compound 10 with TAE-226 (PDB ID 2jkk)15 and
a 1,3,5-triazine compound 34 that we reported previously (PDB
ID 4brx)22 indicates a very similar binding mode to the hinge
region and the DFG motif of the FAK kinase (Figs 1a-c).
Table 2. Cytotoxicity on U-87MG and HCT-116 cell lines.
N°
R1
R2
U-87MG
IC50 (µM)
12.5 0.8
17.0 2.1
2.4 0.2
HCT-116
IC50 (µM)
14.8 0.5
14.9 1.4
1.8 0.09
5.8 0.6
0.5 0.02
0.19 0.03
0.39 0.04
3,4,5-OCH3
3,4,5-OCH3
4-CH2NH2
3-NHSO2CH3
2-CONHCH3
2-CONHCH3
2-CONHCH3
2-CONHCH3
2-CONHCH3
5
6
7
8
2-OCH3-4-Cl-5-CH3
4-morpholino
2-OCH3-4-morpholino
9.2 0.6
As shown in Fig 1a, two hydrogen bonds are formed between
the carbonyl group of Cys502 of the kinase hinge with nitrogens
in the 1,2,4-triazine and 2-methoxyaniline moieties. The carbonyl
group on the ring B forms a hydrogen bond with the backbone
nitrogen of Asp564 of the DFG motif. The carbon atoms in the
1,2,4-triazine ring A make hydrophobic contacts with Ala452 and
Leu553, whereas carbons of the 2-methoxyaniline ring interact
with Ile428 and Gly505. The chlorine atom at the C6 position of
the 1,2,4-triazine ring penetrates deepest into the ATP binding
pocket and is located near the gatekeeper residue Met499. Our
data shows that the van der Waals interactions between the
chlorine atom and Met499 did not play an important role for
binding affinity. Therefore, the reason that the in vitro potency of
compound 10 is lower than that of TAE-226 is likely due to the
extra nitrogen in the 1,2,4-triazine ring. Sharing electrons
between three electronegative nitrogens in the 1,2,4-triazine ring,
compared to two nitrogens in the pyrimidine ring of TAE226,
result in less electrons being available in the triazine ring for
hydrogen bonding with the hinge backbone of FAK, hence
weakening the interaction.
10.1 0.9
13.3 1.2
1.3 0.07
9
10
TAE-226
The effects of these compounds and TAE-226 on the
tumorigenicity of these cancer cell lines were also examined
using an in vitro colony formation assay. These compounds
inhibited colony formation of the two cancer cell lines in a dose-
dependent manner. In general, comparison of table 2 and 3,
shows that these compounds, like TAE-226, have less cytotoxic
effects but demonstrate stronger antitumorigenic effects on the
cancer cell line growth. As shown in Table 3, these compounds
showed a similar effect on U-87MG cells as TAE-226, except for
compounds
5
and 7, which demonstrated
a
stronger
antitumorigenic effect with an IC50 value of 70 and 14 nM,
compared with TAE-226 (170 nM). Similar results were also
observed for HCT-116 cells, since compound 5 showed a
stronger antitumorigenic effect with an IC50 value of 60 nM,
compared with TAE-226 (270 nM). The different cellular
properties (cell permeability, intracellular stability, and
distribution) or off-target effects might account for these strong
growth inhibitions on the two cancer cell lines.
The inhibition of FAK autophosphorylation was first tested on
human glioblastoma (U-87MG), human colon carcinoma (HCT-
116), using FACEFAK ELISA kit (Active Motif Europe,
Belgium). As shown in Figure 2, FAK autophosphorylation was
significantly inhibited by treatment with the best inhibitor 10,
compared with TAE-226. Consistent with its inhibitory activity
shown against the FAK kinase, compound 10 blocked tyrosine
397 phosphorylation in a dose-dependent manner in these two
cancer cell lines, suggesting that these inhibitors are able to
effectively inhibit cellular FAK autophosphorylation and
phosphorylation of kinase targets.
Table 3. Inhibition of Colony Formation on U-87MG and HCT-
116 cell lines.
N°
R1
R2
U-87MG
IC50 (µM)
HCT-116
IC50 (µM)
0,06 0.004
2.1 0.2
3,4,5-OCH3
3,4,5-OCH3
4-CH2NH2
3-NHSO2CH3 0.07 0.006
2-CONHCH3 1.7 0.1
2-CONHCH3 0.014 0.001
5
6
7
1.1 0.1
2-OCH3-4-Cl-5-CH3
4-morpholino
2-OCH3-4-morpholino 2-CONHCH3
2-CONHCH3
2-CONHCH3
0.5 0.02
0.7 0.03
1.5 0.09
0.17 0.04
4.5 0.4
1.7 0.2
1.0 0.1
0.27 0.04
8
9
10
TAE-226
In summary, we have synthesized a series of novel 1,2,4-
diarylaminotriazines as FAK inhibitors. Molecular docking of the
most potent inhibitor 10 into binding site of FAK was performed,
and the results showed compound 10 could bind in the FAK
active site as compound 34 belonging to 1,3,5-triazinic series and
TAE-226 to the pyrimidine series. These results clearly show the
importance of the number and location of nitrogens beared by the
aromatic ring inserted in the catalytic pocket as well for H
bonding as for hydrophobic interactions. Moreover, the potential
effect of polarizable water molecules inside the active site may
have an important role in ligand-macromolecule interactions.28
Otherwise, the 1,2,4-triazinic inhibitors, like TAE-226, showed
less cytotoxic effects but had stronger antitumorigenic effects on
the cancer cell lines (U87-MG and HCT-116). Especially,
compounds 5 and 7 exhibited higher antitumorigenic effects in
U-87MG cells with IC50 values of 70 and 14 nM, respectively,
compared with TAE-226. Finally, taking account of the activity
data and binding information of these novel scaffolds as FAK
inhibitors it should provide a reliable tool for reasonable design
of FAK inhibitors in the future.
Figure 2. Cells (U-87MG and HCT-116) were treated for 48 h with 0.1, 1
and 10 µM of compound 10 or TAE-226 as control. Data obtained by the
Face method are expressed as the fold decrease relative to the control and
represent mean of triplicate readings of three independent experiments. A
decrease of FAK tyrosine 397 phosphorylation in a dose-dependent manner
is shown for 10 and for TAE-226.
Then, inhibition of cancer cell growth was investigated with
the 1,2,4-triazinic compounds 5-10 using the WST-1 assay. As
shown in Table 2, IC50 values of these compounds against two
tumor cell lines (U87MG and HCT-116) ranged between 0.19
and 17 µM except for compounds 9 to which two cells are not
sensitive in terms of decrease in cell survival. Among them, U-
87MG cells were the most sensitive to compound 7 with an IC50
value of 2.4 µM, similar to that of TAE-226. HCT-116 cells
showed more sensitivity to compounds 10 than to TAE-226.

Acknowledgements
The authors thank Agence Nationale de la Recherche
(RNANT05-2_42590) and Worldwide Cancer Research (15-
1177) for financial support.
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