Design, synthesis, and evaluation of novel imidazo[1,2- a ][1,3,5]triazines and their derivatives as focal adhesion kinase inhibitors with antitumor activity

Article abstract of DOI:10.1021/jm500784e

A series of triazinic inhibitors of focal adhesion kinase (FAK) have been recently shown to exert antiangiogenic activity against HUVEC cells and anticancer efficacy against several cancer cell lines. We report herein that we further explored the heterocyclic core of these inhibitors by a fused imidazole ring with the triazine to provide imidazo[1,2-a][1,3,5]triazines. Importantly, these new compounds displayed 10^-7-10^-8 M IC₅₀ values, and the best inhibitor showed IC₅₀ value of 50 nM against FAK enzymatic activity. Several inhibitors potently inhibited the proliferation of a panel of cancer cell lines expressing high levels of FAK. Apoptosis analysis in U87-MG and HCT-116 cell lines suggested that these compounds delayed cell cycle progression by arresting cells in the G2/M phase of the cell cycle, retarding cell growth. Further investigation demonstrated that these compounds strongly inhibited cell-matrix adhesion, migration, and invasion of U87-MG cells.

Full text of DOI:10.1021/jm500784e

Article
pubs.acs.org/jmc
Design, Synthesis, and Evaluation of Novel
Imidazo[1,2‑a][1,3,5]triazines and Their Derivatives as Focal
Adhesion Kinase Inhibitors with Antitumor Activity
Pascal Dao,† Nikaia Smith,† Celine Tomkiewicz-Raulet,‡ Expedite Yen-Pon,† Marta Camacho-Artacho,§
́
́
Daniel Lietha,§ Jean-Phillipe Herbeuval,† Xavier Coumoul,‡ Christiane Garbay,† and Huixiong Chen*,†,∥
†Chemistry & Biology, Nucleo(s)tides & Immunology for Therapy (CBNIT), CNRS UMR8601, Universite
Sorbonne Paris Cite, UFR Biomedicale, 45 rue des Saints-Peres, 75270 Cedex 06 Paris, France
‡Toxicologie, Pharmacologie et Signalisation Cellulaire, INSERM, UMR S 1124, Universite
Paris Descartes, PRES Sorbonne Paris
Cite, UFR Biomedicale, 45 rue des Saints-Peres, 75270 Cedex 06 Paris, France
§Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Calle Melchor Fernan
́
Paris Descartes, PRES
́
́
̀
́
́
́
̀
́
dez
Almagro 3, Madrid 28029, Spain
∥School of Chemical Engineering and Light Industry, Guangdong University of Technology, no. 100 Waihuan Xi road, Education
Mega Center, Guangzhou, 510006, China
S
* Supporting Information
ABSTRACT: A series of triazinic inhibitors of focal adhesion
kinase (FAK) have been recently shown to exert antiangio-
genic activity against HUVEC cells and anticancer efficacy
against several cancer cell lines. We report herein that we
further explored the heterocyclic core of these inhibitors by a
fused imidazole ring with the triazine to provide imidazo[1,2-
a][1,3,5]triazines. Importantly, these new compounds dis-
played 10−7−10−8 M IC50 values, and the best inhibitor
showed IC50 value of 50 nM against FAK enzymatic activity.
Several inhibitors potently inhibited the proliferation of a panel
of cancer cell lines expressing high levels of FAK. Apoptosis analysis in U87-MG and HCT-116 cell lines suggested that these
compounds delayed cell cycle progression by arresting cells in the G2/M phase of the cell cycle, retarding cell growth. Further
investigation demonstrated that these compounds strongly inhibited cell-matrix adhesion, migration, and invasion of U87-MG
cells.
antisense oligonucleotides and siRNA to FAK led to cell
rounding, detachment, reduction of invasion, increased
apoptosis, and finally induced in vivo tumorigenesis.6,7 It was
also observed that in two transgenic mouse models there is a
strong correlation between FAK expression and late-stage
tumorigenesis, indicating that FAK is required for progression
of early tumors to late-stage adenocarcinoma.8,9 These studies
confirmed scientific rationale for further development of FAK
inhibitors as anticancer agents.
FAK has also been involved in angiogenesis as an important
modulator during development, as evidenced by the early
embryonic lethality of mice engineered to harbor an endothelial
specific deletion of this protein.10 FAK signaling pathway was
connected to increased production of vascular endothelial
growth factor (VEGF) and urokinase plasminogen activator,
known factors involved in tumor progression.11,12 It was
INTRODUCTION
■
Focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase and
scaffold protein localized to focal adhesions, is uniquely
positioned at the convergence point of integrins and receptor
tyrosine kinase signal transduction pathways, which transmit
signals from the extracellular matrix (ECM) to the cell
cytoskeleton. FAK has been identified as a key pathogenic
mediator of a variety of diseases. The most widely studied
disease in this regard is cancer. This protein is emerging as a
promising therapeutic target because it is highly expressed at
both the transcriptional and translational level in various
cancers. Ample evidence has indicated that FAK signaling
pathways can stimulate tumor progression and metastasis
through their regulation of cell migration, invasion, ECM, and
angiogenesis (for a recent review of FAK, refer to refs 1−4).
The initial attempts at inhibition focused upon down-regulation
of FAK expression, demonstrating that silencing FAK by
transfection of dominant-negative C-terminal FAK-CD de-
creased adhesion, colonization, and tumor growth in breast
cancer cells.5 Similarly, the inhibition of FAK expression with
Special Issue: New Frontiers in Kinases
Received: May 20, 2014
© XXXX American Chemical Society
A
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Chart 1
reported that FAK expression in endothelial cells is necessary
for the formation of new blood vessels, for the stability of the
vascular network, and for the survival of endothelial cells.13
Overexpression of FAK in vascular endothelial cells directly
promotes angiogenesis in transgenic mice.14 On the other hand,
endothelial FAK deletion in adult mice inhibited tumor growth
and reduced tumor angiogenesis.15 A correlation between
microvessel density on tumor specimen and FAK expression
was found in a clinical study with breast cancer patients.16
Therefore, several FAK inhibitors have been successfully
developed,17−20 and two of them were extensively examined
in preclinical or clinical studies.21,22 Their efficacy in tumor
models may be a result of their ability to potently inhibit both
tumor growth and tumor-associated angiogenesis.
Previously, we have described the generation of a series of
triazinic inhibitors of FAK, which show antiangiogenic activity
against HUVEC cells and anticancer efficacy against several
cancers in cell culture studies (glioblastoma U-87MG, colon
cancer HCT-116, breast cancer MDA-MB-231, and prostate
cancer PC-3).18,19 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-226,23 a potent ATP-
competitive inhibitor of FAK designed by Novartis Pharma AG
that stabilizes an unusual helical conformation of the DFG
motif in which the φ torsion angle of Asp564 is rotated by 113°
compared with the active kinase domain. This compound has
shown potent antiproliferative and antitumor effects in vitro and
in vivo in several types of malignancies including brain
tumors,24 esophageal cancer,25 breast cancer,26 ovarian
cancer,17 and gastrointestinal stroma tumor.22 However, TAE-
226 never entered clinical trials, because this compound also
inhibited the insulin receptor with an IC50 of 40 nM and
showed important side effects in animal studies including
severely affected glucose metabolism and glucose blood
levels.27 Interestingly our triazinic compounds did not inhibit
the insulin receptor (IR) kinase at 1 μM. Compared with TAE-
226, our compounds showed similar antiangiogenic and
anticancer activities but a poorer affinity for FAK with IC50
values in the range of 10−7 M, which could be worthy of further
development.
In an attempt to design compounds with inhibitory potency
enhancement toward FAK kinase activity, we decided to
explore the heterocyclic core by the fused imidazole ring with
the triazine to give an imidazo[1,2-a][1,3,5]triazine (Chart 1),
which we hoped to have potentially an interaction with a gate-
keeper residue Met499 in the kinase domain, and we also
expected to retain the functional selectivity against the IR
because the poorer affinity of 1,3,5-triazinic inhibitors for FAK
might be attributed to the missing chlorine atom, which in
TAE226 makes van der Waals interactions with this residue and
significantly contributes to binding capacity.
Molecular docking is an application where the lowest energy
binding mode of a small molecule bound to a protein target is
predicted based on computationally calculated ligand−protein
interactions. We first docked one imidazotriazinic compound
into FAK based on the cocrystal structure of PHM16 with the
FAK kinase domain (PDB ID 4c7t, see Chart 1). This
compound could bind to the ATP-binding site in a very similar
way to PHM16 and fits well in the nucleotide binding pocket
with the imidazotriazine ring located in the adenine pocket and
the imidazole ring oriented toward residue Met499 (3.1 Å). We
hypothesized that the imidazotriazine cycle could be used as a
good scaffold to develop novel FAK inhibitors. Recently, 1H-
pyrrolo[2,3-b]- and 3H-imidazolo[4,5-b]pyridine scaffolds were
also used to prepare FAK inhibitors with good affinity.28
This paper describes the design, the synthesis, the biological
evaluation, and the docking study of imidazotriazinic
compounds as novel FAK inhibitors. The compounds potently
suppressed the enzymatic activities of FAK with IC50 values in
the 10−7−10−8 M range. The best inhibitor of this series of
compounds showed an IC50 value of 50 nM. These compounds
also strongly suppressed the proliferation of human cancer cells
in which FAK expression was at high levels. Furthermore, the
representative compounds potently suppressed cancer cell
adhesion, migration, and invasion, indicating their potential to
serve as new lead compounds for further anticancer drug
discovery.
CHEMISTRY
■
Synthesis of imidazo[1,2-a][1,3,5]triazine scaffold is described
in Schemes 1 and 2. Starting from cyanuric chloride (Scheme
1), the first chlorine was displaced by nucleophilic substitution
B
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a
Scheme 1
a
(a) Arylamine/THF/DIEA, −20°C, and NH3/THF, TA; (b) arylamine/dioxane/Pd(OAc)2/rac-BINAP/Cs2CO3/MW, 100 °C, 10 min; (c) 2-
chloroacetaldehyde/EtOH/reflux; (d) CH3NH2/CaCl2/MW, 100 °C, 20 min; (e) Pd(PPh3)4, NaBH4, THF, TA.
with arylamines at −10 °C to produce the mono-substituted
intermediates. Intermediates 1−4 were synthesized from
cyanuric chloride (Scheme 1) by nucleophilic substitution
with arylamines at −10 °C followed by reaction with ammonia
in a one-pot procedure. The displacement of the last chlorine
by the corresponding arylamines was accomplished by a Pd-
catalyzed arylamination procedure, affording compounds 5−10
in good yields. Next, compounds 11, 13, 14, and 16−18 were
obtained in the form of two regioisomers by the condensation
of compounds 5−10 with 4.2-fold excess of 2-chloroacetalde-
hyde. These two regioisomers were isolated by column
chromatography, and their respective structures were confirmed
by NOE experiments. In theory, it was possible from this
cyclocondensation to obtain a pair of isomeric products that
differ in the position of the imidazole ring, because the starting
compounds 5−10 are differently substituted in the 4 and 6
positions. In general, the minor product is identified as the
regioisomer B. Aminolysis of esters 11, 14, and 18 was
accomplished using methylamine and CaCl2 under MW
irradiation to afford compounds 12, 15, and 19. Finally, the
protecting N-Alloc groups of compounds 18 and 19 were
removed using a palladium catalyzed reductive deprotection
procedure to produce the desired products 20 and 21,
respectively, in good yields.
The second series of imidazo[1,2-a][1,3,5]triazine derivatives
(Scheme 2) were synthesized from compound 22, which was
prepared according to a previously published method.29
Subsequent cyclization of compound 22 with methyl 2-
nitrophenylacetate or methyl 2-(2-methoxy-2-oxoethyl)-
benzoate was performed in methanolic sodium methoxide to
afford trisubstituted triazines 23 and 24. Next, compounds 25
and 26 were obtained in the form of two regioisomers by the
condensation of compounds 23 and 24 with 2-chloroacetalde-
hyde as described in the first series. Aminolysis of ester 26a led
C
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as described previously.18,19 One reported inhibitor of FAK,
TAE-226, was included to validate the screening conditions.
Under the experimental conditions, TAE-226 inhibited the
activity of FAK with IC50 value of 7 nM (Table 1), which was
similar to previously reported data.27
a
Scheme 2
As shown in Table 1, comparison of regioisomers A and B of
imidazo[1,2-a][1,3,5]triazines revealed that, in general, the first
one is slightly more active as FAK inhibitor, especially for 19a,
which showed higher potency affinity to FAK (17.6-fold) than
19b. It is interesting to note that the introduction of the 3,4,5-
trimethoxyphenylamino group (ring B) at the position 2 of the
imidazo[1,2-a][1,3,5]triazine ring and the phenylamino moiety
(ring A) bearing an ester function at the position 4 of the same
ring (compounds 11a) resulted in a large increase in inhibitory
potency on FAK kinase activity (IC50 280 nM), compared with
the corresponding 1,3,5-triazinic FAK inhibitor (IC50 2.3
μM),18 showing specific contributions of the heterocyclic core
of imidazo[1,2-a][1,3,5]triazine. The displacement of the ester
group from ortho (compound 11a) to meta position in the
arylamine on the imidazo[1,2-a][1,3,5]triazine ring A (com-
pound 13a) was not well tolerated and led to a decreased
affinity toward FAK. This is consistent with what was observed
for related 1,3,5-triazinic inhibitors.18 However, replacing the
ester group on the arylamine (ring A for compound 11a) with
an amide moiety (compound 12a) displayed only a marginal
improvement in inhibitory activity.
Then, we investigated the replacement of 3,4,5-trimethoxy
groups on the phenyl ring (ring B) by different groups. When
the 3,4,5-trimethoxyphenyl group (compound 12a) is replaced
by 2-methoxy-4-morpholinophenyl moiety, the corresponding
compounds 15a and 16a did not display any relevant inhibitory
activity changes. In contrast, increased inhibitory potencies are
observed when the phenyl ring (ring B) is substituted by a
CH2NH2 group or CH2NHAlloc group in the para position
(19a and 21a compared with 12a), which led to a potent
inhibitor of FAK (IC50 50 nM for compound 19a). This may be
attributed to an additional interaction with the Glu506 residue
of the enzymatic site, which could further enhance the binding
capacity. The replacement of NH moiety between the phenyl
group and the triazine moiety in the first series of imidazo[1,2-
a][1,3,5]triazines by a CH2 group leading to 26, 27, and 30 did
not demonstrate an improved affinity for FAK (26a compared
with 11a and 27a compared with 12a), suggesting that the NH
group did not play an important role for the inhibitory activity
of FAK. Finally, a dihydroimidazo[1,2-a][1,3,5]triazine scaffold,
a novel heterocyclic core, was not well tolerated and led to a
decreased binding affinity toward FAK (31a compared with
30a), showing that this scaffold is not favorable for the
enzymatic site probably due to the dihydrotriazine ring, which
is not planar.
a
(a) 3,4,5-Trimethoxyaniline/dioxane/MW, 90 °C, 15 min; (b)
phenylacetate derivatives/MeONa/THF/reflux; (c) 2-chloroacetalde-
hyde/EtOH, 120 °C; (d) Al(CH3)3/NH4OH/CH2Cl2, RT, 12 h; (e)
Na2S2O4/NaHCO3/THF/EtOH/H2O, RT; (f) H2/PtO2/MeOH/
THF; (g) CH3SO2Cl/pyridine, 50 °C.
Molecular Docking Study of Compound 19a. In an
attempt to gain insight into the putative binding mode of the
potent inhibitor 19a with the FAK kinase, compared with its
regioisomer 19b, molecular docking of these compounds into
the ATP binding site of the apo-FAK kinase (PDB ID 4c7t)
was performed. The resulting 3D binding modes of compounds
19a,b to FAK are depicted in Figure S1, Supporting
Information.
to the corresponding amide derivative 27a. Finally, the
synthesis of compounds 30a and 31a was accomplished by
either chemical reduction using Na2S2O4 or catalytic hydro-
genation using PtO2 and further substitution by methanesul-
fonyl chloride as described in Scheme 2.
For compound 19a, the triazine ring, the two aniline rings (A
and B), and the carboxamide moiety take very similar positions
as observed for PHM1619 bound to FAK. The two hydrogen
bonds to the kinase hinge region made by PHM16 with its
[1,3,5]triazine ring and the aniline moiety (ring B) are
RESULTS AND DISCUSSION
■
In Vitro Activity against FAK Kinase. The in vitro
inhibition of FAK kinase activity of the newly synthesized
compounds was evaluated using a TR-FRET based kinase assay
D
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Table 1. In Vitro Enzymatic Activities of Novel Imidazo[1,2-a][1,3,5]triazines Compared with TAE-226
no.
11a
R2
3,4,5-OCH3
R1
X
regioisomer
FRET IC50 (μM)
2-CO2CH3
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
CH2
CH2
CH2
CH2
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
A
A
A
0.28 0.06
0.57 0.1
0.87 0.05
1.20 0.4
0.23 0.08
0.31 0.03
0.37 0.03
0.57 0.07
0.33 0.2
0.51 0.04
0.71 0.06
1.12 0.3
0.92 0.02
1.73 0.1
0.50 0.02
1.05 0.1
0.05 0.001
0.88 0.02
0.24 0.02
0.28 0.05
0.12 0.02
0.22 0.08
0.37 0.02
0.18 0.01
0.84 0.02
8.91 0.9
0.007 0.002
11b
13a
13b
12a
12b
14a
14b
15a
15b
16a
16b
17a
17b
18a
18b
19a
19b
20a
20b
21a
21b
26a
27a
30a
31a
TAE226
2-CO2CH3
3-CO2CH3
3-CO2CH3
2-CONHCH3
2-CONHCH3
2-CO2CH3
2-OCH3-4-morpholino
4-CN
2-CO2CH3
2-CONHCH3
2-CONHCH3
2-CONHCH3
2-CONHCH3
2-NHSO2CH3
2-NHSO2CH3
2-CO2CH3
4-CH2NHAlloc
4-CH2NH2
2-CO2CH3
2-CONHCH3
2-CONHCH3
2-CO2CH3
2-CO2CH3
2-CONHCH3
2-CONHCH3
2-CO2CH3
3,4,5-OCH3
2-CONHCH3
2-NHSO2CH3
2-NHSO2CH3
conserved in 19a; however, additional interactions are made by
the imidazo[1,2-a] ring in 19a, most notably with Met499
(Figure S1A, Supporting Information). The CO of the
carboxamide group of 19a is located near the DFG (D564-
F565-G566) motif of the activation loop of the kinase domain
and forms a hydrogen bond with the backbone nitrogen of
Asp564 of the DFG motif. This hydrogen bond together with
hydrophobic contacts between ring A and Leu567 stabilize the
short helical conformation of residues 565−568 and the typical
DFG conformation also seen in PHM16 and TAE226 bound
structures, with the side chain of Asp564 pointing up toward
the kinase N-lobe. The CH2NH on the phenyl ring B forms a
hydrogen bond with Ile-428, and the CO of the alloc group in
19a has an additional interaction with Glu506. These
interactions together with the interactions made by the
imidazo[1,2-a] ring may be responsible for the increased
binding affinity to FAK for 19a compared with PHM16.
In contrast, 19b cannot bind in the same mode as 19a,
because the imidazole ring is too close to Glu-500 in the hinge
region, leading to steric clashes with this residue (Figure S1F,
Supporting Information). As a result, the imidazo and triazine
rings are pushed further out of the pocket, preventing hydrogen
bonds between 19b and the kinase hinge and the hydrogen
bonds that are formed are much more solvent exposed and
provide lower binding energy, likely explaining the lower
affinity of 19b compared with 19a. As shown in Figure S1C,F,
Supporting Information, 19a but not 19b fits well into the
pocket as observed from a slice through the kinase just above
the binding site.
Kinase Selectivity Profile of Compound 19a. We
further profiled the best inhibitor, 19a, in vitro against a panel
of more than 30 kinases (ABL1, AKT1, ALK, CDK1, CDK2,
CDK3, CDK4, CDK5, CHK1, EGFR, ERBB2, ERK1, ERK2,
FGFR-1, FGFR-2, FGFR-3, FYN, GSK3β, IGF1R, IR, JAK3, c-
Kit, LCK, c-Met, PDGFRA, Pyk2, RAF1, c-Src, mTOR/
FRAP1, FLT1/VEGFR1, KDR/VEGFR2, and FLT4/
VEGFR3) determined by Reaction Biology Corporation,
Malvern, PA, USA to investigate its selectivity (Table S1,
Supporting Information). A radioactive in vitro kinase assay was
performed using [γ33P]ATP and human recombinant protein in
the presence of 1 μM of compound 19a. The results revealed
that compound 19a displays an excellent selectivity profile
against the tested kinases. As expected, this compound did not
inhibit the IR (insulin receptor) kinase at 1 μM in contrast to
TAE-226.27 It is also interesting to note that at 1 μM 19a did
not inhibit Pyk2, which shares a highly similar structural
organization and a high sequence homology with approximately
60% identity in the central catalytic domain and 40% identity in
E
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both the N- and C-terminal domains with FAK.30 This high
affinity binding to FAK but not Pyk2 was also observed for the
compound PF-573,228 reported by Pfizer.31,32
Inhibition of FAK Autophosphorylation and Growth
Inhibitory Activity on Cancer Cell Lines. The antiprolifer-
ative effects of several compounds, compared with TAE-226,
were investigated on human glioblastoma (U-87MG), human
colon carcinoma (HCT-116), human metastatic breast cancer
(MDA-MB-231), and human prostate cancer (PC-3), which
express high levels of FAK.33
21a with an IC50 value of 0.37 μM, significantly lower than that
of TAE-226. HCT-116 cells were as sensitive to these
compounds as to TAE-226, except for 12a, which exhibited
less cytotoxicity. PC-3 cells are more sensitive to these
compounds than to TAE-226. MDA-MB-231 cells showed
more sensitivity to compounds 19a and 21a than to 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 four cancer cell lines in a
dose-dependent manner. In general, these compounds, like
TAE-226, showed less cytotoxic effects but had stronger
antitumorigenic effects on the cancer cell lines. As shown in
Table 3, these compounds showed a similar effect on U-87MG
The inhibition of FAK autophosphorylation was first tested
in these cancer cell lines, using FACEFAK ELISA kit (Active
Motif Europe, Belgium). As shown in Figure 1, FAK
Table 3. Inhibition of Colony Formation, IC50 (μM)
no.
12a
U-87MG
HCT-116
MDA-MB-231
PC-3
0.22 0.04
1.4 0.1
0.76 0.06
0.02 0.01
0.13 0.02
0.12 0.01
0.23 0.02
3.9 0.4
>10
0.99 0.2
0.63 0.04
0.63 0.04
1.4 0.2
15a
19a
21a
0.42 0.04
0.22 0.03
0.14 0.01
0.52 0.03
1.9 0.2
TAE-226 0.19 0.01
0.26 0.1
cells as TAE-226, except for 15a. Similar results were also
observed for HCT-116 cells, except for 15a, which demon-
strated a stronger antitumorigenic effect with an IC50 value of
20 nM, compared with TAE-226. The different cellular
properties (cell permeability, intracellular stability, and
distribution) or off-target effects might account for this strong
inhibition of 15a on HCT-116 cancer cells. Concerning MDA-
MB-231 cancer cells, compounds 19a and 21a exhibited more
potent anticancer activity than TAE-226. However, these
compounds showed slightly weaker effects on PC-3 cells than
TAE-226.
Effects of Compounds 19a and 21a on Apoptosis and
on Cell Cycle in U-87MG and HCT-116 Cell Lines. To
further investigate the mechanism underlying the antiprolifer-
ative effect of these compounds, we first examined whether the
inhibitory effects of two good FAK inhibitors, 19a and 21a, on
U-87MG and HCT-116 cell lines were due to apoptosis by
examining the expression of annexin V using FACS analysis.
As shown in Figure 2, we tested apoptosis caused by these
two compounds and compared them with TAE-226. In U-
87MG cells, 3 μM 19a and 0.3 μM 21a did not induce
significant apoptosis. This finding is similar to that reported in
the literature19,27 and suggests that these two compounds may
disrupt cell cycle progression. Interestingly, when the HCT-116
cell line was exposed to 19a at 3 μM, it resulted in a significant
increase in apoptosis. Indeed, cell apoptosis was increased by
25.9% for 19a compared with control. This effect is less potent
than that observed with TAE-226 (41.8%). Low concentrations
of 19a and TAE-226 were also able to induce significant
apoptosis in HCT-116 cell lines (Figure S2, Supporting
Information). In contrast, 21a did not induce significant
apoptosis.
Figure 1. Cells (U-87MG, HCT-116, MDA-MB-231, and PC-3) were
treated for 48 h with 0.1, 1, and 10 μM of compound 19a 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 19a and for TAE-226.
autophosphorylation was significantly inhibited by treatment
with the best inhibitor, 19a, compared with TAE-226.
Consistent with its inhibitory activity shown against the FAK
kinase, compound 19a blocked tyrosine 397 phosphorylation in
a dose-dependent manner in these cancer cell lines, suggesting
that these inhibitors are able to effectively inhibit cellular FAK
autophosphorylation and phosphorylation of kinase targets at
low concentrations.
Then, we investigated the inhibition of cancer cell growth
with several imidazo[1,2-a][1,3,5]triazine compounds using the
WST-1 assay. As shown in Table 2, the IC50 values of these
compounds against different tumor cells ranged between 0.27
and 4.2 μM except for compounds 12a and 15a to which
MDA-MB-231 cells are not sensitive in terms of decrease in cell
survival. Among them, U-87MG cells were the most sensitive to
Table 2. Antiproliferation Activity, IC50 (μM)
Next, we wished to determine whether 19a- or 21a-induced
decrease of FAK phosphorylation would result in cell cycle
arrest in U-87MG and HCT-116 cell lines, compared with
TAE-226. Cell cycle analysis was performed using flow
cytometry (Figure 3). Cell lines were treated with 19a at 3
μM, 21a at 0.4 μM, or TAE-226 at 1 μM. When the U-87MG
cell line was exposed to 19a or 21a for 24 h, the DNA contents
no.
12a
U-87MG
HCT-116
MDA-MB-231
PC-3
4.2 0.3
0.95 0.2
0.34 0.08
0.52 0.21
0.31 0.18
0.4 0.08
>10
0.35 0.19
0.27 0.08
0.29 0.17
1.27 0.28
1.6 0.2
15a
19a
21a
2.31 0.2
2.89 0.3
0.37 0.04
>10
0.80 0.27
1.08 0.45
2.8 0.2
TAE-226 1.2 0.3
F
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Figure 2. Two cancer cell lines (U87-MG and HCT-116) were plated at 105 cells per well in a 6-well culture dish, incubated overnight, and then
cultured with 19a, 21a, or TAE-226 for 24 h. Cells were stained with annexin V and 7AAD and analyzed by FACS calibur. Cells were then classified
as alive with no apoptosis (i.e., 7AAD negative, annexin V negative), dead (i.e., 7AAD positive), or apoptotic (i.e., annexin V positive). (A) U87-MG
cells were treated with 3 μM 19a, 0.3 μM 21a, or 1 μM TAE-226; (B) HCT-116 cells were treated with 0.5 μM 19a, 0.4 μM 21a, or 0.3 μM TAE-
226. One representative experiment is shown.
Figure 3. Cell cycle distribution was studied by flow cytometry: cells (U87-MG and HCT-116) were plated at 105 per well in a 6-well culture dish,
incubated overnight, and then exposed to 19a, 21a, or TAE-226 along with a vehicle treatment control for 24 h. Cell cycle distribution was
represented by histograms. Asterisks denote statistically significant differences (*p < 0.01; **p < 0.001) as determined by unpaired Student’s t test.
(A) U87-MG cells were treated with 3 μM 19a, 0.3 μM 21a, or 1 μM TAE-226; (B) HCT-116 cells were treated with 0.5 μM 19a, 0.4 μM 21a, or
0.3 μM TAE-226. One representative experiment is shown.
of the live population were 61.7%, 16.0%, and 20.0% for
untreated cells, 34.3%, 5.1%, and 58.8% for 19a-treated cells,
and 47.6%, 9.8%, and 40.7% for 21a-treated cells, respectively,
for G0/G1, S, and G2/M phase. This finding is similar to that
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Figure 4. Effects of compounds 12a, 15a, 19a, and 21a on the cell attachment, migration, and invasion in U-87MG cell lines. (A) Cells were treated
for 30 min in the presence of 12a, 15a, 19a, 21a, or TAE226. The attached cells were removed and counted. (B) Comparative xCELLigence analysis
of the effect of different treatments on the migration of U87-MG human cancer cell lines. CI values are plotted on the graph. The slopes (1/h),
representing the cell migrating ability, were calculated based on CI values in the time interval indicated on the plots by the boxes. The mean slopes
calculated with DMSO (control) were set to 100. The experiment was repeated two times and was consistent. (C) Comparative xCELLigence
analysis of the effect of different treatment on the invasion of U87-MG human cancer cell lines. CI values are plotted on the graph. The slopes (1/h),
representing the cell invasion ability, were calculated based on CI values in the time interval indicated on the plots by the boxes. The mean slopes
calculated with DMSO (control) were set to 100. The experiment was repeated two times and was consistent.
H
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observed with TAE-226. Indeed, treatment with 3 μM 19a or
0.4 μM 21a resulted in a 2.9-fold and 2-fold, respectively
increase in the fraction of cells arrested at G2. Similar results
were also observed for the HCT-116 cell line. When these cells
were treated with 19a at 3 μM or 21a at 0.4 μM, the number of
cells in G2−M phase increased from 14.6% to 54.7% and
61.0%, whereas cells in G0/G1 phase decreased from 63.8% to
29.9% and 24.4%, respectively, compared with the controls.
Low concentrations of 19a, 21a, and TAE-226 were also able to
induce significant cell cycle arrest in U-87MG and HCT-116
cell lines (Figure S3, Supporting Information). Taken together,
these results suggest that these compounds delayed cell cycle
progression by arresting cells in the G2/M phase of the cell
cycle, retarding U-87MG and HCT-116 cell growth.
Effects of Compounds 12a, 15a, 19a, and 21a on the
Adherence, Migration, and Invasion of U-87MG and
HCT-116 Cell Lines. Metastasis is multistep process that
results from the convergence of a variety of cellular processes.
Some of these processes include cell migration, invasion,
adhesion into the surrounding stroma and through vessel walls,
and survival in a foreign environment. Furthermore, the ability
of tumor cells to migrate, invade, and metastasize is associated
with increased FAK expression.33 Indeed, a role for FAK in cell
migration was first suggested by observations of its increased
expression or activation in the migrating keratinocytes in
epidermal wound healing or ECs migrating into the wounded
monolayer in vitro, respectively.34,35 Interference with FAK
function via antisense oligonucleotides or by FRNK expression
inhibits the motility of various cell types.7,36 FAK−/−
fibroblasts derived from FAK KO mouse embryo showed a
significant decrease in cell migration compared with the cells
from wild-type mice.37 Because FAK is critical for cell
attachment, migration, and invasion, we fist treated the U-
87MG cells with different doses of our best inhibitors of FAK
(12a, 15a, 19a, and 21a) for the adhesion assay. We observed
that these compounds caused dose-dependent decrease of
attachment (Figure 4A). Among them, 21a and 19a showed
strong potencies for adhesion inhibition. Indeed, at low dose of
0.5 μM 21a or 3 μM 19a, the attachment decreased about 50%,
compared with that treated with TAE-226, which only
decreased 36% of attachment at 1 μM (Figure S4, Supporting
Information).
Next, we performed measurements of U-87MG cell
migration and invasion using modified 16-well plates on the
xCELLigence DP device form Roche Diagnostics. This assay is
based on real time monitoring of cell migration or invasion,
which captures cell responses during the entire course of an
experiment. The cell index (CI) reflects the electrical
impedance across the interdigitated microelectrodes integrated
on the lower side of the 8 μm pore membrane, caused by the
cells invading through the Matrigel or collagen, across the
membrane and spreading on the lower side of the membrane.
As shown in Figure 5B, treatment with 4 μM 12a, 2 μM 15a, 3
μM 19a, and 0.4 μM 21a significantly decreased the number of
cells migrating through the collagen-coated Transwell mem-
brane chambers after 12 h. Interestingly, these compounds were
even more potent for the cell mobility reduction than TAE-226.
Among them, 19a and 21a showed strong inhibition of U87-
MG cell migration. Indeed, as shown by the slopes (1/h),
representing the cell migration ability, the migration of U87-
MG cells across collagen-coated Transwell membrane was
reduced by 66.4% in the presence of 0.4 μM 21a and 69.5% in
the presence of 3 μM 19a compared with the reduction of only
25.5% in the presence of 1 μM TAE-226. This result is
consistent with that observed in the adhesion inhibition and
also confirmed by Boyden chamber assay, which showed that
our compounds inhibited cell migration in a dose-dependent
manner (Figure S5A, Supporting Information).
Cell invasion is a prerequisite for establishment of metastases
at secondary sites requiring movement through basement
membranes.38 To allow for measurement of cellular invasion,
the membrane separating the upper and lower compartment of
the CIM-plate 16 was coated with a layer of Matrigel, forcing
the cell to digest the Matrigel prior to moving the bottom side
of the membrane. As shown in Figure 4C, these compounds
significantly reduced the invasive propensity of U87-MG cells
compared with the vehicle control. Quantification of these
results showed that compounds 19a and 21a could inhibit U87-
MG cell invasion by at least 50% of untreated control,
supporting their potent anti-invasion activity. As observed in
the inhibition of cell migration, these compounds were more
potent than TAE-226 in inhibiting cell invasion, except for
compound 15a, which showed the similar result as TAE-226.
This result is consistent with that observed in the cellular
migration and also confirmed by Boyden chamber assay, which
showed that these compounds inhibited cell invasion in a dose-
dependent manner (Figure S5B, Supporting Information).
Taken together, these compounds can prevent cell adhesion,
migration, and invasion of human U87-MG cells in vitro, which
suggest that inhibition of FAK may uncouple adhesions from
the substrate while still allowing them to be connected to the
mobile actin network in the cell and this uncoupling would lead
to defects in the generation or maintenance of tension at sites
of adhesion, affecting thus cell migration, invasion, and tumor
metastasis.39
CONCLUSION
■
In summary, a series of novel imidazo[1,2-a][1,3,5]triazines
and their derivatives were found to be selective FAK inhibitors
against the tested kinases. These compounds displayed IC50
values in the range of 10−7−10−8 M and the best inhibitor
showed IC50 values of 50 nM against FAK. Several inhibitors
potently inhibited the proliferation of a panel of cancer cell
lines expressing high levels of FAK, including U87-MG, HCT-
116, MDA-MB-231, and PC-3. Apoptosis analysis in U87-MG
and HCT-116 cell lines suggested that these compounds (19a
and 21a) may disrupt cell cycle progression but do not induce
apoptosis as measured by 7AAD and annexin V, except for 19a
in the HCT-116 cell line. Indeed, using flow cytometry analysis,
when the U-87MG and HCT-116 cell lines were exposed to
these two compounds, the number of cells in G2−M phase
increased and those in G0/G1 phase decreased compared with
the controls. These observations suggest that they inhibited the
cell growth through a mechanism associated with preventing
progression through the G2−M phase of the cell cycle. Further
investigation demonstrated that these compounds (12a, 15a,
19a, and 21a) strongly inhibited cell-matrix adhesion,
migration, and invasion in U87-MG cells in a dose-dependent
manner. The careful evaluation of pharmacokinetics and
pharmacodynamics in vivo will be required to fully understand
the role of FAK inhibition in the regulation of tumorigenesis
and metastatic progression. These compounds may provide
useful tools to assess the role of adhesion signaling in the
regulation of cellular proliferation and migration and may have
important therapeutic applications in the prevention or
treatment of cancer. More importantly, they will offer possible
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organic phase was dried over Na2SO4, reduced to a small volume, and
purified by silica gel column chromatography eluted with a mixture of
dichloromethane and methanol.
alternative therapeutic approaches for the currently difficult
treatment of distant metastases.
Methyl 2-[2-(3,4,5-Trimethoxyphenylamino)imidazo[1,2-a]-
[1,3,5]triazin-4-ylamino]benzoate (11a) and Methyl 2-[4-(3,4,5-
Trimethoxyphenylamino)imidazo[1,2-a][1,3,5]triazin-2-ylamino]-
EXPERIMENTAL SECTION
■
All commercial materials were used without further purification.
Microwave irradiation was carried out with a microwave monomode
reactor (infrared detector for temperature). Melting points were
determined on a Kofler apparatus as uncorrected values. Analytical
thin-layer chromatography was performed on precoated 250 μm layer
thickness silica gel 60 F254 plates and visualized with UV light.
Column chromatography was performed using silica gel 60 (40−63
1
benzoate (11b). White solid, mp 196 °C, 70 mg (62%). H NMR
(250 MHz, DMSO): δ 10.47 (s, 1H), 9.11 (dd, J = 8.0, 1.5 Hz, 1H),
7.94 (dd, J = 8.0, 1.5 Hz, 1H), 7.70 (d, J = 1.5 Hz, 1H), 7.5 (td, J = 8.0,
1.5 Hz, 1H), 7.1 (d, J = 1.5 Hz, 1H), 7.01 (br, 2H), 6.95 (td, J = 8.0,
1.5 Hz, 1H), 3.87 (s, 3H), 3.80 (s, 6H), 3.67 (s, 3H). 13C NMR (500
MHz, DMSO): δ 168.2, 156.5, 152.3, 151.4, 147.5, 143.6, 133.9, 132.3,
130.5, 129.2, 119.3, 118.9, 112.7, 106.9, 101.0, 60.1, 55.5, 52.0. MS
(ESI) m/z 451.2 [M + 1]+. White solid, mp 198 °C, 37 mg (33%). 1H
NMR (250 MHz, DMSO): δ 10.82 (s, 1H), 10.05 (br, 1H), 8.95 (dd, J
= 8.0, 1.5 Hz, 1H), 8.01 (d, J = 1.0 Hz, 1H), 8.0 (dd, J = 8.0, 1.5 Hz,
1H), 7.62 (td, J = 8.0, 1.5 Hz, 1H), 7.38 (d, J = 1.0 Hz, 1H), 7.23 (br,
2H), 7.08 (td, J = 8.0, 1.5 Hz, 1H), 3.87 (br, 9H), 3.71 (s, 3H). 13C
NMR (500 MHz, DMSO): δ 168.4, 157.5, 153.6, 148.9, 141.3, 133.5,
130.2, 128.4, 124.3, 119.7, 107.5, 101.1, 60.2, 55.7, 52.0. MS (ESI) m/z
451.2 [M + 1]+.
1
μm). H NMR and 13C NMR spectra were measured on 250 or 500
MHz spectrometer in DMSO-d6 or CDCl3 with chemical shift (δ)
given in parts per million (ppm) relative to TMS as internal standard
and recorded at 23 °C. MS (ESI) was determined by using a Q-Tof1
spectrometer with Z-spray source. High-resolution mass spectra
(HRMS) were performed on Q-TOF Micro micromass positive ESI
(CV = 30 V).
General Method for the Synthesis of Compounds 6−10. A
solution of the corresponding compound (2−4,40 0.18 mmol, 1 equiv)
with arylamine (0.18 mmol, 1 equiv) and Cs2CO3 (0.27 mmol, 1.5
equiv) was introduced into a 10 mL microwave vial. The mixture was
purged with argon before the addition of Pd(OAc)2 (7.2 nmol, 0.04
equiv) and BINAP (7.2 nmol, 0.04 equiv). The mixture was subjected
to microwave irradiation for 10 min at 100 °C using irradiation power
of 100 W. The solvent was evaporated under vacuum, and the crude
residue was purified by silica gel column chromatography eluted with
mixture of dichloromethane and methanol.
Methyl 3-[2-(3,4,5-Trimethoxyphenylamino)imidazo[1,2-a]-
[1,3,5]triazin-4-ylamino]benzoate (13a) and Methyl 3-[4-(3,4,5-
Trimethoxyphenylamino)imidazo[1,2-a][1,3,5]triazin-2-ylamino]-
1
benzoate (13b). White solid, mp 154 °C, 65 mg (58%). H NMR
(250 MHz, DMSO): δ 9.9 (br, 1H), 9.64 (s, 1H), 8.38 (d, J = 2.0 Hz,
1H), 8.17 (dd, J = 7.5, 1.5 Hz, 1H), 7.93 (s, 1H), 7.56 (dd, J = 7.5, 1.5
Hz, 1H), 7.4 (td, J = 7.5, 1.5 Hz, 1H), 7.33 (d, J = 2.0 Hz, 1H), 7.02
(br, 2H), 3.84 (s, 3H), 3.80 (s, 6H), 3.70 (s, 3H). 13C NMR (500
MHz, DMSO): δ 52.0, 55.5, 55.5, 60.1, 101.0, 101.0, 106.9, 112.7,
118.9, 119.3, 129.2, 129.2, 130.5, 132.3, 133.9, 143.6, 147.5, 151.4,
152.3, 152.3, 156.5, 168.2. MS (ESI) m/z 451.2 [M + 1]+. White solid,
mp 164 °C, 30 mg (26%). 1H NMR (250 MHz, DMSO): δ 10.11 (br,
1H), 9.32 (s, 1H), 8.39 (d, J = 2.0 Hz, 1H), 8.15 (td, J = 7.5, 1.5 Hz,
1H), 7.93 (dd, J = 7.5, 1.5 Hz, 1H), 7.75 (td, J = 7.5, 1.5 Hz, 1H), 7.57
(dd, J = 7.5, 1.5 Hz, 1H), 7.30 (d, J = 2.0 Hz, 1H), 7.20 (br, 2H), 3.87
(s, 3H), 3.68 (s, 6H), 3.62 (s, 3H). 13C NMR (500 MHz, DMSO): δ
52.0, 55.7, 55.7, 60.2, 101.1, 101.1, 107.5, 119.7, 119.7, 124.3, 128.4,
130.2, 130.2, 130.2, 133.5, 133.5, 141.3, 148.9, 148.9, 153.6, 157.5,
168.4. MS (ESI) m/z 451.2 [M + 1]+.
Methyl 3-[4-Amino-6-(3,4,5-trimethoxyphenylamino)-1,3,5-tria-
zin-2-ylamino]benzoate (6). White solid, mp 150 °C, 124 mg
1
(87%). H NMR (250 MHz, DMSO): δ 9.25 (s, 1H), 8.97 (s, 1H),
8.33 (dd, J = 7.5 Hz, 1.5 Hz, 1H), 8.16 (s, 1H), 7.57 (dd, J = 7.5, 1.5
Hz, 1H), 7.42 (td, J = 7.5, 1.5 Hz, 1H), 7.16 (s, 2H), 6.68 (s, 2H), 3.85
(s, 3H), 3.74 (s, 6H), 3.64 (s, 3H).
Methyl 2-[4-Amino-6-(2-methoxy-4-morpholinophenylamino)-
1,3,5-triazin-2-ylamino]benzoate (7). Violet solid, mp 174 °C,
1
132.5 mg (88%). H NMR (250 MHz, DMSO): δ 10.27 (s, 1H),
7.95 (dd, J = 8.0, 1.5 Hz, 1H), 7.85 (s, 1H), 7.61 (d, J = 8.5 Hz, 1H),
7.5 (td, J = 7.5, 1.5 Hz, 1H), 7.03 (td, J = 7.5, 1.5 Hz, 1H), 6.72 (bs,
2H), 6.65 (d, J = 2.0 Hz, 1H), 6.50 (dd, J = 8.5, 2.0 Hz, 1H), 3.88 (s,
3H), 3.81 (s, 3H), 3.77 (t, J = 4.5 Hz, 4H), 3.13 (t, J = 4.5 Hz, 4H).
2-[4-Amino-6-(4-cyanophenylamino)-1,3,5-triazin-2-ylamino]-N-
methylbenzamide (8). Pale yellow solid, mp 163 °C, 107 mg
(89%).1H NMR (250 MHz, DMSO): δ 9.52 (s, 1H), 8.41 (s, 1H),
8.04 (d, J = 4.5 Hz, 1H), 7.96 (d, J = 9.0, 2H), 7.94 (dd, J = 7.5, 1.5
Hz, 1H), 7.65 (d, J = 9 Hz, 2H), 7.41 (td, J = 7.5, 1.5 Hz, 1H), 7.35
(dd, J = 7.5, 1.5 Hz, 1H), 7.12 (td, J = 7.5, 1.5 Hz, 1H), 6.87 (bs, 2H),
2.65 (d, J = 4.5 Hz, 3H).
Methyl 2-[2-(2-Methoxy-4-morpholinophenylamino)imidazo-
[1,2-a][1,3,5]triazin-4-ylamino]benzoate (14a) and Methyl 2-[4-(2-
Methoxy-4-morpholinophenylamino)-imidazo[1,2-a][1,3,5]triazin-
2-ylamino]benzoate (14b). Violet solid, mp 168 °C, 67 mg (56%).1H
NMR (250 MHz, DMSO): δ 10.40 (s, 1H), 9.76 (s, 1H), 8.76 (d, J =
8.6 Hz, 1H), 7.97 (d, J = 1.6 Hz, 1H), 7.93 (d, J = 1.6 Hz, 1H), 7.5
(ddd, J = 8.5, 7.0, 1.6 Hz, 1H), 7.35 (s, 1H), 7.3 (d, J = 8.5 Hz, 1H),
7.03 (t, J = 8 Hz, 1H), 6.75 (d, J = 2.5 Hz, 1H), 6.63 (dd, J = 8.5, 2.5
Hz, 1H), 3.86 (s, 3H), 3.84−3.75 (m, 7H), 3.27−3.20 (m, 4H). 13C
NMR (500 MHz, DMSO): δ 168.7, 157.3, 155.0, 148.8, 148.2, 147.1,
141.5, 131.6, 131.5, 131.3, 124.3, 123.5, 123.2, 122.8, 122.4, 115.4,
107.0, 102.3, 65.5, 56.7, 52.0, 47.3. MS (ESI) m/z 476.2 [M + 1]+.
Violet solid, mp 152 °C, 33 mg (28%).1H NMR (250 MHz, DMSO):
δ 10.40 (s, 1H), 9.76 (s, 1H), 8.76 (d, J = 8.6 Hz, 1H), 7.97 (d, J = 1.6
Hz, 1H), 7.93 (d, J = 1.6 Hz, 1H), 7.5 (ddd, J = 8.5, 7.0, 1.6 Hz, 1H),
7.35 (s, 1H), 7.3 (d, J = 8.5 Hz, 1H), 7.03 (t, J = 8 Hz, 1H), 6.75 (d, J
= 2.5 Hz, 1H), 6.63 (dd, J = 8.5, 2.5 Hz, 1H), 3.86 (s, 3H), 3.84−3.75
(m, 7H), 3.27−3.20 (m, 4H). 13C NMR (500 MHz, DMSO): δ 168.8,
156.8, 155.0, 148.9, 148.9, 146.9, 141.8, 131.6, 131.5, 131.3, 124.1,
123.9, 123.6, 122.6, 115.1, 107.1, 101.8, 65.6, 56.8, 52.1, 47.3. MS
(ESI) m/z 476.2 [M + 1]+.
2-[2-(4-Cyanophenylamino)imidazo[1,2-a][1,3,5]triazin-4-ylami-
no]-N-methylbenzamide (16a) and 2-[4-(4-Cyanophenylamino)-
imidazo[1,2-a][1,3,5]triazin-2-ylamino]-N-methylbenzamide (16b).
Gray solid, mp 164 °C, 40 mg (42%). 1H NMR (250 MHz, DMSO): δ
11.49 (br, 1H), 10.89 (s, 1H), 10.11 (s, 1H), 8.64 (d, J = 2.0 Hz, 1H),
7.93 (d, J = 9.0 Hz, 2H), 7.85−7.63 (m, 4H), 7.51 (d, J = 2.0 Hz, 1H),
7.48 (td, J = 7.5, 1.5 Hz, 1H), 7.19 (dd, J = 7.5, 1.5 Hz, 1H), 3.06 (d, J
= 4.5 Hz, 3H). 13C NMR (500 MHz, DMSO): δ 171.5, 154.6, 148.9,
148.3, 141.1, 140.6, 132.2, 131.6, 130.6, 127.6, 126.9, 124.6, 123.8,
122.6, 119.1, 107.1, 103.3, 26.2. MS (ESI) m/z 385.1 [M + 1]+. Gray
N-{2-[4-Amino-6-(4-cyanophenylamino)-1,3,5-triazin-2-
ylamino]phenyl}methanesulfonamide (9). White solid, mp 184 °C,
1
95 mg (72%). H NMR (250 MHz, DMSO): δ 9.69 (s, 1H), 8.43 (s,
1H), 8.15 (bs, 1H), 8.0 (d, J = 9.0 Hz, 2H), 7.95 (dd, J = 7.5, 1.5 Hz,
1H), 7.66 (d, J = 9 Hz, 2H), 7.35 (td, J = 7.5, 1.5 Hz, 1H), 7.30 (dd, J
= 7.5, 1.5 Hz, 1H), 7.17 (td, J = 7.5, 1.5 Hz, 1H), 6.93 (bs, 2H), 2.94
(s, 3H).
Methyl 2-{4-Amino-6-[4-((benzyloxycarbonylamino)methyl)-
phenylamino]-1,3,5-triazin-2-ylamino}benzoate (10). White solid,
mp 192 °C, 104 mg (69%). 1H NMR (250 MHz, DMSO): δ 10.38 (s,
1H), 9.30 (s, 1H), 8.95 (d, J = 7.5 Hz, 1H), 7.98 (dd, J = 7.5, 1.5 Hz,
1H), 7.73 (d, J = 7.5 Hz, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.57 (td, J =
7.5, 1.5 Hz, 1H), 7.18 (d, J = 8.0 Hz, 2H), 7.06 (td, J = 7.5, 1.5 Hz,
1H), 6.89 (br, 2H), 6.03−5.83 (m, 1H), 5.38−5.13 (m, 2H), 4.51 (dt,
J = 5.3, 1.5 Hz, 2H), 4.16 (d, J = 6.0 Hz, 2H), 3.89 (s, 3H).
General Method for the Synthesis of Compounds 11, 13, 14,
16, 17, 18, 25, and 26. A solution of the corresponding compound
(540−10 and 23, 0.25 mmol, 1 equiv) and 2-chloroacetaldehyde (50%
in H2O, 0.07 mL, 1.05 mmol, 4.2 equiv) in DMSO was stirred at 120
°C for 5−7 h. The solvent was evaporated under vacuum, and the
crude residue was treated with NaHCO3 saturated aqueous solution.
This alkaline solution was extracted with dichloromethane. The
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solid, mp 158 °C, 26 mg (27%). 1H NMR (250 MHz, DMSO): δ 9.72
(br, 1H), 9.57 (s, 1H), 8.04 (dd, J = 8.0, 1.5 Hz, 1H), 7.95−7.75 (m,
4H), 7.68 (d, J = 8.0 Hz, 1H), 7.54 (td, J = 8.0, 1.5 Hz, 1H), 7.34 (d, J
= 1.5 Hz, 1H), 7.25 (td, J = 8.0, 1.5 Hz, 1H), 6.84 (dd, J = 8.0, 1.5 Hz,
1H), 3.01 (s, 3H). 13C NMR (500 MHz, DMSO): δ 171.5, 155.0,
148.9, 148.8, 141.1, 132.6, 131.6, 129.7, 129.5, 126.3, 124.8, 124.2,
122.2, 119.1, 107.1, 105.6, 26.2. MS (ESI) m/z 385.1 [M + 1]+.
N-{2-[2-(4-Cyanophenylamino)imidazo[1,2-a][1,3,5]triazin-4-
ylamino]phenyl}methanesulfonamide (17a) and N-{2-[4-(4-
Cyanophenylamino)imidazo[1,2-a][1,3,5]triazin-2-ylamino]-
phenyl}methanesulfonamide (17b). Gray solid, mp 166 °C, 46 mg
(44%). 1H NMR (250 MHz, DMSO): δ 9.77 (s, 1H), 7.92 (d, J = 9.0
Hz, 2H), 7.83 (d, J = 2.0 Hz, 1H), 7.62 (d, J = 9.0 Hz, 2H), 7.56−7.50
(m, 2H), 7.38 (d, J = 2.0 Hz, 1H), 7.30−7.19 (m, 2H), 2.97 (s, 3H).
13C NMR (500 MHz, DMSO): δ 154.6, 148.9, 148.3, 141.1, 131.6,
= 7.5, 1.5 Hz, 1H), 7.63 (dd, J = 7.5, 1.5 Hz, 1H), 7.62 (d, J = 2 Hz,
1H), 7.54 (td, J = 7.5, 1.5 Hz, 1H), 7.37 (td, J = 7.5, 1.5 Hz, 1H), 7.34
(d, J = 2.0 Hz, 1H), 6.14 (s, 2H), 4.11 (s, 2H), 3.87 (s, 3H), 3.80 (s,
6H). 13C NMR (500 MHz, DMSO): δ 175.0, 167.3, 165.1, 152.6,
147.5, 138.4, 134.6, 132.9, 132.8, 132.0, 131.9, 130.1, 130.0, 126.7,
106.9, 100.3, 60.0, 55.8, 51.8, 42.9. MS (ESI) m/z 450.2 [M + 1]+.
General Method for the Synthesis of Compounds 12, 15,
and 19. A solution of the corresponding compound (11, 14, 18, and
26a, 0.05 mmol, 1 equiv), methylamine (2 M in MeOH, 10 equiv, 0.5
mmol), and CaCl2 (0.15 mmol, 3equiv) in MeOH was introduced into
a 10 mL microwave vial. The mixture was subjected to microwave
irradiation for 20 min at 80 °C using irradiation power of 100 W. The
solvent was evaporated under vacuum and the crude residue was
purified by silica gel column chromatography eluted with mixture of
dichloromethane and methanol.
131.4, 130.6, 128.9, 127.5, 126.7, 123.8, 122.2, 121.6 119.1, 107.1,
N-Methyl-2-[2-(3,4,5-trimethoxyphenylamino)imidazo[1,2-a]-
103.3, 42.2. MS (ESI) m/z 421.1 [M + 1]+. Gray solid, mp 162 °C, 32
[1,3,5]triazin-4-ylamino]benzamide (12a). White solid, mp 186 °C,
1
mg (31%). H NMR (250 MHz, DMSO): δ 8.74 (s, 1H), 7.87−7.65
1
18 mg (82%). H NMR (250 MHz, DMSO): δ 11.37 (s, 1H), 9.98
(m, 5H), 7.37 (td, J = 7.5, 1.5 Hz, 1H), 7.34 (d, J = 2.0 Hz, 1H), 7.27
(dd, J = 7.5, 1.5 Hz, 1H), 7.19 (td, J = 7.5, 1.5 Hz, 1H), 2.92 (s, 3H).
13C NMR (500 MHz, DMSO): δ 155.0, 148.9, 148.8, 141.1, 132.5,
(br, 1H), 8.86 (dd, J = 8.5, 1.5 Hz, 1H), 8.71 (q, J = 4.5 Hz, 1H), 8.03
(d, J = 2.0 Hz, 1H), 7.72 (dd, J = 8.0, 1.5 Hz, 1H), 7.5 (td, J = 8.0, 1.5
Hz, 1H), 7.35 (d, J = 2.0 Hz, 1H), 7.27 (br, 2H), 7.05 (td, J = 8.0, 1.5
Hz, 1H), 3.88 (s, 6H), 3.71 (s, 3H), 2.81 (d, J = 4.5 Hz, 3H). 13C
NMR (500 MHz, DMSO): δ 168.2, 156.5, 152.3, 151.4, 147.5, 143.6,
133.9, 132.3, 130.5, 129.2, 119.3, 118.9, 112.7, 106.9, 101.0, 60.1, 55.5,
26.2. MS (ESI) m/z 450.2 [M + 1]+.
131.6, 129.5, 129.0, 128.2, 126.3, 124.8, 122.7, 121.6, 119.3, 107.2,
105.6, 42.3. MS (ESI) m/z 421.1 [M + 1]+.
Methyl 2-((2-((4-((((Vinyloxy)carbonyl)amino)methyl)phenyl)-
amino)imidazo[1,2-a][1,3,5]-triazin-4-yl)amino)benzoate (18a)
and Methyl 2-((4-((4-((((Vinyloxy)-carbonyl)amino)methyl)phenyl)-
amino)imidazo[1,2-a][1,3,5]triazin-2-yl)amino)benzoate (18b).
White solid, mp 174 °C, 61 mg (52%). 1H NMR (250 MHz,
DMSO): δ 10.45 (s, 1H), 9.30 (s, 1H), 8.92 (dd, J = 7.5, 1.5 Hz, 1H),
8.05−7.95 (m, 4H), 7.55 (d, J = 2.0 Hz, 1H), 7.3 (d, J = 8.0 Hz, 2H),
7.03 (td, J = 7.5, 1.5 Hz, 1H), 7.18 (d, J = 8.0 Hz, 2H), 7.06 (td, J =
7.5, 1.5 Hz, 1H), 6.89 (br, 2H), 6.06−5.82 (m, 1H), 5.39−5.12 (m,
2H), 4.53 (dt, J = 5.3, 1.5 Hz, 2H), 4.23 (d, J = 6.0 Hz, 2H), 3.87 (s,
3H). 13C NMR (500 MHz, DMSO): δ 168.8, 158.6, 154.6, 148.9,
148.3, 141.6, 138.2, 134.9, 134.1, 131.6, 131.5, 131.3, 128.4, 123.2,
122.9, 122.4, 120.2, 117.4, 107.1, 66.0, 52.1, 43.5. MS (ESI) m/z 474.2
[M + 1]+. White solid, mp 166 °C, 37 mg (31%). 1H NMR (250 MHz,
DMSO): δ 11.37 (s, 1H), 9.26 (s, 1H), 8.14 (dd, J = 7.5, 1.5 Hz, 1H),
7.81 (d, J = 2.0 Hz, 1H), 7.67−7.54 (m, 2H), 7.42 (td, J = 7.5, 1.5 Hz,
1H), 7.23 (d, J = 8.0, 1.5 Hz, 2H), 7.20 (dd, J = 7.5, 1.5 Hz, 1H), 7.03
(d, J = 8.0, 1.5 Hz, 2H), 6.52 (s, 1H), 5.96−5.77 (m, 1H), 5.29−5.10
(m, 2H), 4.63 (dt, J = 5.3, 1.5 Hz, 2H), 4.32 (d, J = 6.0 Hz, 2H), 3.90
(s, 3H). 13C NMR (500 MHz, DMSO): δ 168.8, 158.6, 155.0, 148.9,
148.8, 141.8, 137.2, 135.4, 134.1, 131.6, 131.5, 131.3, 128.0, 124.1,
122.6, 121.7, 117.4, 107.1, 66.0, 52.1, 43.5. MS (ESI) m/z 474.2 [M +
1]+.
4-(2-Nitrobenzyl)-N-(3,4,5-trimethoxyphenyl)imidazo[1,2-a]-
[1,3,5]triazin-2-amine (25a) and 2-(2-Nitrobenzyl)-N-(3,4,5-
trimethoxyphenyl)imidazo[1,2-a][1,3,5]triazin-4-amine (25b). Yel-
low solid, mp 129 °C, 62 mg (57%). 1H NMR (250 MHz, DMSO) δ
9.48 (s, 1H), 8.23 (dd, J = 7.5 Hz, 1.5 Hz, 1H), 7.90 (d, J = 2.0 Hz,
1H), 7.85 (td, J = 7.5 Hz, 1.5 Hz, 1H), 7.74 (dd, J = 7.5 Hz, 1.5 Hz,
1H), 7.69 (td, J = 7.5 Hz, 1.5 Hz, 1H), 7.43 (d, J = 2.0 Hz, 1H), 7.21
(br, 2H), 4.89 (s, 2H), 3.70 (s, 6H), 3.62 (s, 3H). Gray solid, mp126
°C, 25 mg (22%).1H NMR (250 MHz, DMSO) δ 10.11 (s, 1H), 8.11
(d, J = 2.0 Hz, 1H), 8.05 (dd, J = 7.5 Hz, 1.5 Hz, 1H), 7.72 (td, J = 7.5
Hz, 1.5 Hz, 1H), 7.62 (dd, J = 7.5 Hz, 1.5 Hz, 1H), 7.57 (td, J = 7.5
Hz, 1.5 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.07 (br, 2H), 4.89 (s, 2H),
3.74 (s, 6H), 3.68 (s, 3H).
N-Methyl-2-[4-(3,4,5-trimethoxyphenylamino)imidazo[1,2-a]-
[1,3,5]triazin-2-ylamino]benzamide (12b). White solid, mp 172 °C,
1
17 mg (78%). H NMR (250 MHz, DMSO): δ 11.32 (s, 1H), 8.87
(dd, J = 8.0, 1.5 Hz, 1H), 8.71 (q, J = 4.5 Hz, 1H), 7.96 (d, J = 1.5 Hz,
1H), 7.71 (dd, J = 8.0, 1.5 Hz, 1H), 7.49 (td, J = 8.0, 1.5 Hz, 1H), 7.33
(d, J = 1.5 Hz, 1H), 7.24 (br, 2H), 7.04 (t, J = 7.5, 1.5 Hz, 1H), 3.88 (s,
6H), 3.71 (s, 3H), 2.81 (d, J = 4.7 Hz, 3H). 13C NMR (500 MHz,
DMSO): δ 171.4, 156.6, 156.7, 155.0, 148.9, 148.8, 141.1, 134.5, 134.4,
132.6, 131.6, 129.7, 126.3, 124.2, 122.2, 107.1, 102.8, 102.9, 60.6, 56.8,
56.9, 26.2. MS (ESI) m/z 450.2 [M + 1]+.
2-[2-(2-Methoxy-4-morpholinophenylamino)imidazo[1,2-a]-
[1,3,5]triazin-4-ylamino]-N-methylbenzamide (15a). White solid,
1
mp 192 °C, 16 mg (68%). H NMR (250 MHz, DMSO): δ 10.72
(s, 1H), 8.66 (d, J = 8.0 Hz, 1H), 8.61 (q, J = 4.5 Hz, 1H), 7.89 (s,
1H), 7.86 (d, J = 2.0 Hz, 1H), 7.62 (dd, J = 8.0, 1.5 Hz, 1H), 7.37 (td,
J = 8.0, 1.5 Hz, 1H), 7.29 (s, 1H), 7.27 (d, J = 8.0 Hz, 1H), 6.99 (td, J
= 8.0, 1.5 Hz, 1H), 6.61 (dd, J = 8.0, 1.5 Hz, 1H), 3.84−3.73 (m, 7H),
3.24 (m, 4H), 2.76 (d, J = 4.5 Hz, 3H). 13C NMR (500 MHz,
DMSO): δ 171.5, 157.3, 155.0, 148.9, 148.3, 147.1, 140.6, 132.2, 131.6,
127.6, 126.9, 124.6, 124.3, 123.6, 122.6, 115.5, 107.1, 102.3, 65.6, 56.8,
47.3, 26.2. MS (ESI) m/z 475.2 [M + 1]+.
2-[4-(2-Methoxy-4-morpholinophenylamino)imidazo[1,2-a]-
[1,3,5]triazin-2-ylamino]-N-methylbenzamide (15b). White solid,
1
mp 187 °C, 17 mg (72%). H NMR (250 MHz, DMSO): δ 11.06
(s, 1H), 8.76 (q, J = 4.5 Hz, 1H), 8.40 (dd, J = 7.5, 1.5 Hz, 1H), 7.72
(td, J = 7.5, 1.5 Hz, 1H), 7.14 (d, J = 2.0 Hz, 1H), 7.10 (dd, J = 7.5, 1.5
Hz, 1H), 7.07 (td, J = 7.5, 1.5 Hz, 1H), 7.02 (d, J = 2.0 Hz, 1H), 6.89
(dd, J = 8.0, 1.5 Hz, 1H), 6.60 (dd, J = 8.0, 1.5 Hz, 1H), 6.05 (s, 1H),
3.86−3.70 (m, 7H), 3.42−3.20 (m, 4H), 2.86 (d, J = 4.5 Hz, 3H). 13C
NMR (500 MHz, DMSO): δ 171.5, 156.8, 155.0, 148.9, 148.3, 146.9,
141.1, 132.6, 131.6, 129.7, 126.3, 124.2, 123.9, 123.6, 122.2, 115.1,
107.1, 101.8, 65.6, 56.8. MS (ESI) m/z 475.2 [M + 1]+.
Vinyl 4-((4-((2-(Methylcarbamoyl)phenyl)amino)imidazo[1,2-a]-
[1,3,5]triazin-2-yl)amino)benzylcarbamate (19a). White solid, mp
187 °C, 12 mg (52%). 1H NMR (250 MHz, DMSO): δ 11.24 (s, 1H),
9.85 (q, J = 4.5 Hz, 1H), 7.82 (dd, J = 7.5, 1.5 Hz, 1H), 7.79 (d, J = 2.0
Hz, 2H), 7.67 (d, J = 2.0 Hz, 2H), 7.47 (td, J = 7.5, 1.5 Hz, 1H), 7.26
(dd, J = 7.5, 1.5 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 7.15 (td, J = 7.5, 1.5
Hz, 1H), 7.03 (d, J = 8.0 Hz, 1H), 6,97 (s, 1H), 5.99−5.82 (m, 1H),
5.46−5.16 (m, 2H), 4.73 (d, J = 6.0 Hz, 2H), 4.50 (dt, J = 5.3, 1.5 Hz,
2H), 2.82 (d, J = 4.5 Hz, 3H). 13C NMR (500 MHz, DMSO): δ 171.5,
158.6, 154.6, 148.9, 148.3, 140.6, 138.2, 134.9, 134.1, 132.2, 131.6,
128.4, 127.6, 126.9, 124.6, 122.6, 120.2, 117.4, 107.1, 66.0, 43.5, 26.2.
MS (ESI) m/z 473.5 [M + 1]+.
Methyl 2-((2-(3,4,5-Trimethoxyphenylamino)imidazo[1,2-a]-
[1,3,5]triazin-4-yl)methyl)-benzoate (26a) and Methyl 2-((4-
((3,4,5-Trimethoxyphenyl)amino)imidazo[1,2-a][1,3,5]triazin-2-yl)-
1
methyl)benzoate (26b). White solid, mp 162 °C, 62 mg (55%). H
NMR (250 MHz, CDCl3): δ 8.15 (dd, J = 7.5, 1.5 Hz, 1H), 7.61 (td, J
= 7.5, 1.5 Hz, 1H), 7.51 (td, J = 7.5, 1.5 Hz, 1H), 7.50 (d, J = 2.0 Hz,
1H), 7.43 (d, J = 2.0 Hz, 1H), 7.39 (dd, J = 7.5, 1.5 Hz, 1H), 7.06 (s,
2H), 4.72 (s, 2H), 3.88 (s, 6H), 3.83 (s, 3H), 3.78 (s, 3H). 13C NMR
(500 MHz, DMSO): δ 167.2, 158.2, 155.0, 153.3, 150.3, 134.8, 134.6,
134.1, 134.0, 132.8, 132.3, 131.4, 129.6, 128.1, 107.0, 97.0, 61.0, 56.2,
52.3, 38.5. MS (ESI) m/z 450.2 [M + 1]+. White solid, mp 154 °C, 27
mg (24%). 1H NMR (250 MHz, CDCl3): δ 10.86 (s, 1H), 7.89 (dd, J
K
dx.doi.org/10.1021/jm500784e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Vinyl 4-((2-((2-(Methylcarbamoyl)phenyl)amino)-imidazo[1,2-a]-
70 °C using irradiation power of 100 W. On cooling to room
temperature, the mixture was evaporated under vacuum, and the
residue was subjected to flash chromatography (silica gel, 5%
methanol/CH2Cl2) to afford the desired product as a white solid.
6-(2-Nitrobenzyl)-N2-(3,4,5-trimethoxyphenyl)-1,3,5-triazine-2,4-
diamine (23). White solid, mp 157 °C, 197 mg (48%). 1H NMR (250
MHz, DMSO): δ 9.20 (s, 1H), 8.04 (dd, J = 7.5, 1.5 Hz, 1H), 7.71 (td,
J = 7.5, 1.5 Hz, 1H), 7.59 (dd, J = 7.5, 1.5 Hz, 1H), 7.55 (td, J = 7.5,
1.5 Hz, 1H), 7.12 (br, 2H), 6.99 (br, 2H), 4.22 (s, 2H), 3.70 (s, 6H),
3.59 (s, 3H).
Methyl 2-((4-Amino-6-(3,4,5-trimethoxyphenylamino)-1,3,5-tria-
zin-2-yl)methyl)-benzoate (24). White solid, mp 168 °C, 234 mg
(55%). 1H NMR (250 MHz, CDCl3): δ 7.97 (dd, J = 7.5, 1.5 Hz, 1H),
7.50 (td, J = 7.5, 1.5 Hz, 1H), 7.36 (td, J = 7.5, 1.5 Hz, 1H), 7.35 (dd, J
= 7.5, 1.5 Hz, 1H), 6.79 (s, 2H), 4.35 (s, 2H), 3.82 (s, 6H), 3.79 (s,
3H).
N-Methyl-2-((2-(3,4,5-trimethoxyphenylamino)imidazo[1,2-
a][1,3,5]triazin-4-yl)methyl)benzamide (27a). A solution of
methylamine (2.5 M in hexane, 0.25 mmol, 1 equiv) and NH4OH
(28% in H2O, 0.25 mmol, 1 equiv) in CH2Cl2 was stirred for 15 min
under argon at room temperature, and compound 26a (0.25 mmol, 1
equiv) was added. The solvent was evapored under vacuum, the crude
residue was extracted with dichloromethane, and the organic phase
was purified by silica gel column chromatography eluted with a
mixture of dichloromethane and methanol. White solid, mp 152 °C, 72
mg (64%). 1H NMR (250 MHz, DMSO): δ 9.52 (s, 1H), 8.29 (q, J =
4.5 Hz, 1H), 7.79 (d, J = 1.5 Hz, 1H), 7.56 (d, J = 7.0 Hz, 1H), 7.50−
7.39 (m, 3H), 7.34 (d, J = 1.5 Hz, 1H), 7.26 (s, 2H), 4.60 (s, 2H), 3.74
(s, 6H), 3.63 (s, 3H), 2.63 (d, J = 4.5 Hz, 3H). MS (ESI) m/z 449.2
[M + 1]+.
[1,3,5]triazin-4-yl)amino)benzylcarbamate (19b). White solid, mp
1
179 °C, 11 mg (48%). H NMR (250 MHz, DMSO): δ 9.76 (q, J =
4.5 Hz, 1H), 8.58 (s, 1H), 7.76 (dd, J = 7.7, 1.4 Hz, 2H), 7.65 (d, J =
7.5 Hz, 2H), 7.34 (td, J = 7.4, 1.4 Hz, 1H), 7.28 (d, J = 7.5 Hz, 2H),
7.13 (d, J = 2.2 Hz, 1H), 7.08 (dd, J = 7.5, 1.5 Hz, 1H), 7.07 (td, J =
7.5, 1.5 Hz, 1H), 7.01 (d, J = 7.5 Hz, 2H), 6.71 (s, 1H), 6.04 (s, 1H),
5.96−579 (m, 1H), 5.23−5.12 (m, 2H), 4.75 (d, J = 6.0 Hz, 2H), 4.33
(s, 2H), 2.79 (d, J = 4.5 Hz, 3H). 13C NMR (500 MHz, DMSO): δ
171.5, 158.6, 155.0, 148.9, 148.8, 141.1, 137.2, 135.4, 134.1, 132.6,
131.6, 129.7, 128.0, 126.3, 124.2, 122.2, 121.7, 117.4, 107.1, 66.0, 43.5,
26.2. MS (ESI) m/z 473.2 [M + 1]+.
General Method for the Synthesis of Compounds 20 and 21.
A solution of the corresponding compound (18 or 19, 0.2 mmol, 1
equiv) with morpholine (1 mmol, 5 equiv), triphenylphosphine (0.04
mmol, 0.2 equiv), and tetrakis(triphenylphosphine)palladium(0) (0.01
mmol, 0.05 equiv) in dichloromethane was stirred at room
temperature for 4 h. The solvent was evaporated under vacuum, and
the crude residue was purified by silica gel column chromatography
eluted with a mixture of dichloromethane and methanol.
Methyl 2-{2-[4-(Aminomethyl)phenylamino]imidazo[1,2-a]-
[1,3,5]triazin-4-ylamino}-benzoate (20a). White solid, mp 186 °C,
1
63 mg (82%). H NMR (250 MHz, DMSO): δ 11.25 (s, 1H), 10.12
(s, 1H), 7.90 (dd, J = 7.5, 1.5 Hz, 1H), 7.65 (d, J = 2.0 Hz, 1H), 7.45
(td, J = 7.5, 1.5 Hz, 1H), 7.24 (d, J = 8.0 Hz, 2H), 7.22 (dd, J = 7.5, 1.5
Hz, 1H), 7.16 (d, J = 2.0 Hz, 1H), 7.10 (td, J = 7.5, 1.5 Hz, 1H), 7.00
(d, J = 8.0 Hz, 2H), 5.92 (br, 2H), 4.02 (s, 2H), 3.80 (s, 3H). 13C
NMR (500 MHz, DMSO): δ 168.8, 154.6, 148.9, 148.3, 141.6, 137.0,
136.2, 131.6, 131.5, 131.3, 127.4, 123.2, 122.9, 122.4, 120.2, 107.1,
52.1, 46.2. MS (ESI) m/z 390.2 [M + 1]+.
4-(2-Aminobenzyl)-N-(3,4,5-trimethoxyphenyl)imidazo[1,2-
a][1,3,5]triazin-2-amine (28a). A solution of compound 25a40 (0.2
mmol, 1 equiv) in THF (5 mL), water (2.5 mL), and EtOH (5 mL)
with sodium dithionite (0.4 mmol, 2 equiv) and sodium bicarbonate
(0.4 mmol, 2 equiv) was stirred at room temperature for 24 h. The
mixture was evapored under vacuum, and the crude residue was
purified by silica gel column chromatography and eluted with a
mixture of dichloromethane and methanol to give 44 mg (54%), light-
Methyl 2-{4-[4-(Aminomethyl)phenylamino]imidazo[1,2-a]-
[1,3,5]triazin-2-ylamino}benzoate (20b). White solid, mp 164 °C,
1
60 mg (77%). H NMR (250 MHz, DMSO): δ 11.06 (s, 1H), 10.65
(s, 1H), 7.82 (dd, J = 7.5, 1.5 Hz, 1H), 7.70 (d, J = 2.0 Hz, 1H), 7.52
(d, J = 2.0 Hz, 1H), 7.42 (td, J = 7.5, 1.5 Hz, 1H), 7.24 (d, J = 8.0 Hz,
2H), 7.08 (dd, J = 7.5, 1.5 Hz, 1H), 7.05 (td, J = 7.5, 1.5 Hz, 1H), 6.92
(d, J = 8.0 Hz, 2H), 5.75 (br, 2H), 4.00 (s, 2H), 3.88 (s, 3H). 13C
NMR (500 MHz, DMSO): δ 168.8, 155.0, 148.9, 148.8, 141.8, 136.5,
136.2, 131.6, 131.5, 131.3, 127.2, 124.1, 122.6, 121.7, 107.1, 52.1, 46.2.
MS (ESI) m/z 390.2 [M + 1]+.
1
yellow solid, mp 234 °C. H NMR (250 MHz, DMSO): δ 12.80 (s,
1H), 9.59 (s, 1H), 7.18 (d, J = 2.0 Hz, 1H), 7.01 (td, J = 7.5, 1.5 Hz,
1H), 6.96 (d, J = 1.5 Hz, 1H), 6.78−6.66 (m, 4H), 6.52 (td, J = 7.5, 1.5
Hz, 1H), 5.98 (t, J = 5 Hz, 2H), 3.76 (s, 6H), 3.63 (s, 3H), 3.22−2.94
(m, 2H).
2-{2-[4-(Aminomethyl)phenylamino]imidazo[1,2-a][1,3,5]triazin-
4-ylamino}-N-methylbenzamide (21a). White solid, mp 171 °C, 63
1
mg (81%). H NMR (250 MHz, DMSO): δ 11.60 (s, 1H), 10.05 (s,
1H), 8.82 (q, J = 4.5 Hz, 1H), 7.87 (dd, J = 7.5, 1.5 Hz, 1H), 7.60 (d, J
= 2.0 Hz, 1H), 7.42 (td, J = 7.5, 1.5 Hz, 1H), 7.32 (dd, J = 7.5, 1.5 Hz,
1H), 7.20 (d, J = 8.0 Hz, 2H), 7.18 (td, J = 7.5, 1.5 Hz, 1H), 7.12 (d, J
= 2.0 Hz, 1H), 6.94 (d, J = 8.0 Hz, 2H), 5.70 (br, 2H), 3.95 (s, 2H),
2.82 (d, J = 4.5 Hz, 3H). 13C NMR (500 MHz, DMSO): δ 171.5,
154.6, 148.9, 148.3, 140.6, 137.0, 136.2, 132.2, 131.6, 127.6, 127.4,
126.9, 124.6, 122.6, 120.2, 107.1, 46.2, 26.2. MS (ESI) m/z 389.2 [M +
1]+.
4-(2-Aminobenzyl)-N-(3,4,5-trimethoxyphenyl)-3,4-
dihydroimidazo[1,2-a][1,3,5]triazin-2-amine (29a). To a solution
of compound 25a (0.2 mmol, 1 equiv) in methanol (10 mL), 10%
platinum oxide (8 mg) is added under a nitrogen atmosphere. The
reaction vessel is charged with hydrogen and evacuated three times
until the reaction is under a hydrogen atmosphere. The reaction is
stirred overnight. The reaction mixture is filtered through a pad of
Celite and washed with methanol. The filtrate is concentrated to a
small volume and purified by flash chromatography to give 78 mg
(96%), light-yellow solid, mp 260 °C. 1H NMR (250 MHz, DMSO): δ
9.75 (s, 1H), 7.67 (d, J = 2.0 Hz, 1H), 7.51 (dd, J = 7.5, 1.5 Hz, 1H),
7.32 (s, 2H), 7.30 (s, 2H), 7.27 (d, J = 2.0 Hz, 1H), 7.13 (td, J = 7.5,
1.5 Hz, 1H), 7.04 (dd, J = 7.5, 1.5 Hz, 1H), 6.82 (td, J = 7.5, 1.5 Hz,
1H), 4.41 (s, 2H), 3.76 (s, 6H), 3.64 (s, 3H).
General Method for the Synthesis of Compounds 30a and
31a. A solution of the corresponding compound (28a or 29a, 0.2
mmol, 1 equiv) in pyridine (2 mL) with acetic anhydride (0.4 mmol, 2
equiv) was stirred at 50 °C for 5 h. The solvent was evapored under
vacuum, and the crude residue was purified by silica gel column
chromatography eluted with a mixture of dichloromethane and
methanol.
2-{4-[4-(Aminomethyl)phenylamino]imidazo[1,2-a][1,3,5]triazin-
2-ylamino}-N-methylbenzamide (21b). White solid, mp 154 °C,
1
261.1 mg (82.6%). H NMR (250 MHz, DMSO): δ 12.06 (s, 1H),
11.22 (s, 1H), 8.50 (q, J = 4.5 Hz, 1H), 7.78 (dd, J = 7.5, 1.5 Hz, 1H),
7.77 (d, J = 2.0 Hz, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.41(td, J = 7.5, 1.5
Hz, 1H), 7.22 (d, J = 8.0 Hz, 2H), 7.14 (td, J = 7.5, 1.5 Hz, 1H), 7.09
(dd, J = 7.5, 1.5 Hz, 1H), 6.88 (d, J = 8.0 Hz, 2H), 5.87 (br, 2H), 3.90
(s, 2H), 2.77 (d, J = 4.5 Hz, 3H). 13C NMR (500 MHz, DMSO): δ
171.5, 155.0, 148.9, 148.8, 141.1, 136.5, 136.2, 132.6, 131.6, 129.7,
127.2, 126.3, 124.2, 122.2, 121.7, 107.1, 46.2. MS (ESI) m/z 389.2 [M
+ 1]+.
General Method for the Synthesis of Compounds 23 and 24.
A mixture of sodium methoxide (1.5 mmol, 1.5 equiv) prepared from
Na and methanol, 3,4,5-trimethoxyphenylbiguanide chlorohydrate (1
mmol, 1 equiv), and methyl 2-(2-nitrophenyl)acetate or methyl 2-(2-
methoxy-2-oxoethyl)benzoate (3 mmol, 3 equiv) in dry THF (3 mL)
was introduced into a 50 mL round-bottomed flask equipped with a
condenser and a magnetic stirring bar. The flask was placed in the
microwave cavity and exposed to microwave irradiation for 20 min at
N-{2-[4-(3,4,5-Trimethoxyphenylamino)imidazo[1,2-a][1,3,5]-
triazin-2-ylmethyl]phenyl}methanesulfonamide (30a). Light yellow
1
solid, mp 204 °C, 76 mg (79%). H NMR (250 MHz, DMSO): δ
11.54 (br, 1H), 9.01 (s, 1H), 8.87 (dd, J = 7.5, 1.5 Hz, 1H), 8.03 (d, J
= 2.0 Hz, 1H), 7.37 (td, J = 7.5, 1.5 Hz, 1H), 7.36 (d, J = 2.0 Hz, 1H),
7.28 (td, J = 7.5, 1.5 Hz, 1H), 7.26 (dd, J = 7.5, 1.5 Hz, 1H), 7.16 (s,
L
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2H), 4.30 (s, 2H), 3.68 (s, 9H), 2.97 (s, 3H). 13C NMR (500 MHz,
DMSO): δ 41.0, 56.7, 56.7, 60.5, 101.5, 101.5, 127.8, 130.1, 130.7,
134.3, 134.8, 135.2, 136.3, 148.9, 157.5, 157.5, 161.3, 165.9, 169.6. MS
(ESI) m/z 485.2 [M + 1]+.
the cell viability; the reference wavelength was 620 nm. Data were
calculated as the ratio of the values obtained for the treated cells to
those for the controls (DMSO). The results are expressed as the mean
of three independent experiments with three determinations per tested
concentration per experiment. For each compound, the IC50 values
were calculated using nonlinear regression model (logarithmic
inhibitor vs normalized response-variable slope) in Graph-Pad Prism
(GraphPad Software, San Diego, CA, USA).
Colony Assays. Cancer cell lines were seeded in 6-well plates at a
density of 250 cells per well for HCT-116 and 300 cells per well for U-
87MG, MDA-MB-231, and PC-3 and then incubated overnight. The
medium was then removed before treatment at various concentrations
of different compound for 2 weeks. The colonies were washed with
PBS and fixed with 4% paraformaldehyde solution at room
temperature for 30 min, stained with 0.1% crystal violet for 10 min,
and finally, washed with water. Positive colony formation (more than
50 cells/colony) was counted, and colony formation rate was
calculated. For each compound, the IC50 values were calculated
using nonlinear regression model (logarithmic inhibitor vs normalized
response-variable slope) in Graph-Pad Prism (GraphPad Software, San
Diego, CA, USA).
Apoptosis Determination. Cell apoptosis was evaluated on U-
87MG and HCT-116 cells using annexin V-PE (BD Pharmingen) in
conjunction with the vital dye 7-amino-actinomycin D (7-AAD) to
differentiate early apoptosis (annexin V+ 7-AAD−) from late
apoptosis/necrosis (annexin V+ 7-AAD+). The cells were seeded in
24-well plates at 5 × 104 cells per well and incubated overnight before
treatment for 48 h. The treated cells were collected and incubated in
annexin V binding buffer with annexin V-PE and 7-AAD for 30 min at
room temperature. The cells were then washed, resuspended in
annexin V binding buffer, and analyzed by flow cytometry immediately
on a BD LSR II (Becton Dickinson, San Jose, CA). Results were
analyzed using FlowJo software (Treestar, Ashland, OR).
Flow Cytometer Analysis of Cell Cycle. Cell cycle was
analysized on U-87MG or HCT-116 cells using propidium iodide.
The cells were seeded in 24-well plates at 5 × 104 cells per well and
incubated overnight before treatment for 48 h. The treated cells were
collected, resuspended, and incubated for 15 min at 37 °C with PI/
triton buffer. Cells were finally acquired by flow cytometer, and cell
cycle was analyzed using Dean−Jett−Fox methods on a FACS calibur
(Becton Dickinson, San Jose, CA).
Cell Adhesion Assay. Cells cultured in media containing 10%
serum were collected by trypsinization and diluted to a concentration
of 100 000 cells/mL in media containing 10% serum and then treated
for 30 min with DMSO and each of compounds 12a, 15a, 19a, and
21a at various concentrations. Next, 200 μL of the treated cells with
inhibitor (20 000 cells) was added to a 96-well plate and incubated for
1 h to allow attachment. Cells were fixed with paraformaldehyde 4%
for 30 min and then stained by Toluidine Blue 0.1% in PFA 4% for 30
min. After washing with PBS, the optical density was analyzed with a
microplate reader (Bio-Rad) at 620 nm to determine the attached
cells.
xCELLigence Real-Time Cell Analysis (RTCA) of Migration
and Invasion. Cell migration and invasion experiments were carried
out using the xCELLigence RTCA DP instrument (Roche Diagnostics
GmbH, Mannheim, Germany), which was placed in a humidified
incubator at 37 °C and 5% CO2. The experiments were performed
using modified 16-well plates (CIM-16, Roche Diagnostics GmbH,
Mannheim, Germany) with each well consisting of an upper and a
lower chamber separated by a microporous membrane. Micro-
electrodes were attached to the underside of the membrane for
impedance-based detection of migrated cells.
N-{2-[2-(3,4,5-Trimethoxyphenylamino)-3,4-dihydro-imidazo-
[1,2-a][1,3,5]triazin-4-ylmethyl]phenyl}acetamide (31a). Yellow
1
solid, mp 187 °C, 60 mg (62%). H NMR (250 MHz, DMSO): δ
10.27 (s, 1H), 9.70 (s, 1H), 7.72 (d, J = 2.0 Hz, 1H), 7.54 (dd, J = 7.5,
1.5 Hz, 1H), 7.34 (s, 2H), 7.32 (d, J = 2.0 Hz, 1H), 7.10 (td, J = 7.5,
1.5 Hz, 1H), 7.04 (dd, J = 7.5, 1.5 Hz, 1H), 6.80 (td, J = 7.5, 1.5 Hz,
1H), 4.38 (s, 2H), 3.76 (s, 6H), 3.61 (s, 3H), 3.08 (s, 3H). 13C NMR
(500 MHz, DMSO): δ 161.0, 156.7, 152.5, 139.7, 134.5, 134.2, 132.0,
131.7, 131.2, 126.8, 125.2, 121.3, 110.9, 101.5, 101.0, 61.3, 56.9, 56.6.
42.3, 36.3. MS (ESI) m/z 487.2 [M + 1]+.
Ligand Docking. Compounds 19a and 19b were docked into the
apo-FAK inactive conformation (PDB ID 4c7t), which is similar to the
TAE-226 bound conformation with the “DFG motif” adopting a
helical conformation. The kinase receptor was kept rigid, while the
flexible ligands were positioned in the binding pocket according to the
PHM16 location. A standard protocol using Autodock 4.241 was
carried out. In brief, the protein structure was screened within a 50 ×
50 × 50 Å3 cube centered at the “DFG motif” applying a space grid of
0.375 Å. The lowest energy and most populated clusters were 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.
In Vitro Kinase Assay. Ten microliters of assay mixture containing
100 nM FAK substrate ULight-poly GT, 0.1 nM FAK, 25 μM ATP,
and 1 μL of compounds at desired concentrations in kinase buffer (50
mM Tris HCl, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, 0.01%
Tween 20, pH 7.4) was added into a 384-well plate. After incubation at
30 °C for 1.6 h, the kinase reaction was stopped by the addition of 5
μL of 40 mM EDTA in LANCE detection buffer 10× (Perkin Elmer)
for 5 min and then 5 μL of the Eu-labeled antibody, 8 nM in detection
buffer. The plate was incubated at 30 °C for 1 h, and the TR-FRET
signal was detected with an Envision plate reader. For each compound,
the IC50 value was determined from a sigmoid dose−response curve
using Graph-Pad Prism (GraphPad Software, San Diego, CA, USA).
FACE FAK ELISA Assay. Cells (U-87MG, HCT-116, MDA-MB-
231, and PC-3) were cultured in 96-well plates at 3 × 103 cells per well
and treated with the compounds at different concentrations. Following
treatment, the cells were fixed using 4% formaldehyde in PBS. Each
well was then incubated with a primary antibody that recognizes either
phosphorylated FAK or total FAK as suggested by the manufacturer.
The phospho-FAK antibody recognizes FAK only when it is
phosphorylated at Tyr397. The total-FAK antibody recognizes FAK
regardless of its phosphorylation state. Subsequent incubation with
secondary HRP-conjugated antibody and developing solution
provided an easily quantified colorimetric response at 450 nm. The
relative cell number in each well is then determined using the provided
Crystal Violet solution and quantified at 595 nm. The levels of FAK
phosphorylation were normalized by both the levels of total FAK
protein and total cell number in each well.
Cell Culture and Reagents. Human glioma U-87MG (ATCC
HTB-14), colorectal carcinoma HCT-116 (ATCC CCL-247), breast
adenocarcinoma MDA-MB-231 (ATCC CRM-HTB-26), and prostate
adenocarcinoma PC-3 (ATCC CRL-1435) were purchased from
American Type Culture Collection (ATCC) and cultured at 37 °C in a
5% CO2 humidified incubator and maintained in DMEM supple-
mented with 10% fetal bovine serum and 5% penicillin−streptomycin.
Cell Viability Assay. Cell viability was determined in 96-well
plates by using the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-
tetrazolio]-1,3-benzene disulfonate (WST-1) (Roche Diagnostics,
Penzberg, Germany). The exponentially growing cells were seeded
at 5 × 103 cells per well in 96-well plates and incubated overnight. The
medium was then removed, and the cells were treated for 48 h with
200 μL of medium containing various concentrations of different
compounds prepared in DMSO. Then, WST-1 was added to each well,
and the cells were further incubated for 2 h. The optical density was
analyzed with a microplate reader (Bio-Rad) at 450 nm to determine
Prior to each experiment, U-87MG cells were cultured in 24-well
plates at 5 × 104 cells per well, incubated overnight, and then exposed
to different compounds or DMSO in 3% FBS for 24 h. Initially, 165
and 15 μL of media were added to the lower and upper chambers,
respectively, and the CIM-16 plate was locked in the RTCA DP device
at 37 °C and 5% CO2 during 60 min to obtain equilibrium according
to the manufacturer’s guidelines. After this incubation period, a
measurement step was performed as a background signal, generated by
M
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cell-free media. To initiate an experiment, cells were harvested,
resuspended in 3% FBS-medium, counted, and seeded in the upper
chamber applying 4 × 104 cells in 100 μL. After cell addition, CIM-16
plates were incubated during 30 min at room temperature to allow the
cells to settle onto the membrane according to the manufacturer’s
guidelines, they were locked in the RTCA DP device in the incubator,
and the impedance value of each well was automatically monitored by
the xCELLigence system and expressed as a cell index value (CI).
Lower chambers contained media with 10% FBS in order to assess
chemotactic migration when exposed to FBS. Each condition was
performed in duplicate with a programmed signal detection schedule
of every 5 min during 10 h of incubation.
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An identical protocol was performed to follow the invasion, with the
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and dynamic process follow-up during 12 hours. All data have been
recorded by the supplied RTCA software (vs 1.2.1).
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ASSOCIATED CONTENT
■
S
* Supporting Information
In vitro profile of compound 19a against a panel of ten kinases,
purity data of tested compounds (HPLC), apoptosis in U-
87MG and HCT-116 cells assessed by annexin V/7-AAD
double staining, cell cycle distribution studied by flow-
cytometry, effects of TAE-226 on the cell attachment in U-
87MG cell lines, effects of compounds TAE-226, 12a, 15a, 19a,
and 21a on the migration/invasion in U-87MG cell lines, using
Boyden chamber assays, and experimental details. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +331-428-640-85. Fax: +331-428-640-82. E-mail:
huixiong.chen@parisdescartes.fr.
Notes
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Wang, R. Endothelial FAK Is Essential for Vascular Network Stability,
Cell Survival, and Lamellipodial Formation. J. Cell Biol. 2006, 172,
151−162.
(14) Peng, X.; Ueda, H.; Zhou, H.; Stokol, T.; Shen, T.-L.; Alcaraz,
A.; Nagy, T.; Vassalli, J.-D.; Guan, J.-L. Overexpression of Focal
Adhesion Kinase in Vascular Endothelial Cells Promotes Angiogenesis
in Transgenic Mice. Cardiovasc. Res. 2004, 64, 421−430.
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Kostourou, V.; Hart, I.; Fruttiger, M.; Parsons, M.; Hodivala-Dilke, K.
M. Endothelial FAK Is Required for Tumour Angiogenesis. EMBO
Mol. Med. 2010, 2, 516−528.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Agence Nationale Recherche (Grant RNA-
NTO5-2_42590) and Visiotac Pharma.
ABBREVIATIONS
■
FAK, focal adhesion kinase; HUVEC, human umbilical vein
endothelial cell; ECM, extracellular matrix; siRNA, small
interfering RNA; VEGF, vascular endothelial growth factor;
ATP, adenosine triphosphate; DFG, aspartate-phenylalanine-
glycine; IR, insulin receptor; NOE, nuclear Overhauser effect;
MW, microwave; RT, room temperature; TR-FRET, fluo-
rescence resonance energy transfer; EGFR, epidermal growth
factor receptor; Pyk2, proline-rich tyrosine kinase 2; FGFR2,
fibroblast growth factor receptor 2; FLT1/VEGFR1, Fms-
related tyrosine kinase 1/vascular endothelial growth factor
receptor 1; IGF1R, insulin-like growth factor 1 receptor; KDR/
VEGFR2, kinase insert domain receptor/vascular endothelial
growth factor receptor 2; PDGFRA, α-type platelet-derived
growth factor receptor; EGTA, ethylene glycol tetraacetic acid;
DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid;
PBS, phosphate buffered saline; DMEM, Dulbecco’s modified
Eagle medium
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M.; Bornmann, W.; Fernandez, A.; Lopez-Berestein, G.; Sood, A. K.
Therapeutic Efficacy of a Novel Focal Adhesion Kinase Inhibitor
TAE226 in Ovarian Carcinoma. Cancer Res. 2007, 67, 10976−10983.
(18) Dao, P.; Jarray, R.; Le Coq, J.; Lietha, D.; Loukaci, A.;
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