Design, synthesis, and biological evaluation of novel FAK scaffold inhibitors targeting the FAK-VEGFR3 protein-protein interaction

Article abstract of DOI:10.1016/j.ejmech.2014.04.041

Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor 3 (VEGFR3) are tyrosine kinases, which function as key modulators of survival and metastasis signals in cancer cells. Previously, we reported that small molecule chlorpyramine hydrochloride (C4) specifically targets the interaction between FAK and VEGFR3 and exhibits anti-tumor efficacy. In this study, we designed and synthesized a series of 1 (C4) analogs on the basis of structure activity relationship and molecular modeling. The resulting new compounds were evaluated for their binding to the FAT domain of FAK and anti-cancer activity. Amongst all tested analogs, compound 29 augmented anti-proliferative activity in multiple cancer cell lines with stronger binding to the FAT domain of FAK and disrupted the FAK-VEGFR3 interaction. In conclusion, we hope that this work will contribute to further studies of more potent and selective FAK-VEGFR3 protein-protein interaction inhibitors.

Full text of DOI:10.1016/j.ejmech.2014.04.041

European Journal of Medicinal Chemistry 80 (2014) 154e166
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, and biological evaluation of novel FAK scaffold
inhibitors targeting the FAKeVEGFR3 proteineprotein interaction
,
,
,d,
Priyanka N. Gogate a 1, Manivannan Ethirajan a 1, Elena V. Kurenova b
**
,
Andrew T. Magis c, Ravindra K. Pandeya , William G. Cance
,
b,d,
**
*
a Department of Cell Stress Biology/PDT Center, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
b Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
c Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
d CureFAKtor Pharmaceuticals, Orchard Park, NY 14127, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor 3 (VEGFR3) are tyrosine
kinases, which function as key modulators of survival and metastasis signals in cancer cells. Previously,
we reported that small molecule chlorpyramine hydrochloride (C4) specifically targets the interaction
between FAK and VEGFR3 and exhibits anti-tumor efficacy. In this study, we designed and synthesized a
series of 1 (C4) analogs on the basis of structure activity relationship and molecular modeling. The
resulting new compounds were evaluated for their binding to the FAT domain of FAK and anti-cancer
activity. Amongst all tested analogs, compound 29 augmented anti-proliferative activity in multiple
cancer cell lines with stronger binding to the FAT domain of FAK and disrupted the FAKeVEGFR3
interaction. In conclusion, we hope that this work will contribute to further studies of more potent and
selective FAKeVEGFR3 proteineprotein interaction inhibitors.
Received 31 March 2014
Received in revised form
10 April 2014
Accepted 12 April 2014
Available online 15 April 2014
Keywords:
Focal adhesion kinase
Vascular endothelial growth factor receptor-
3
Ó 2014 Elsevier Masson SAS. All rights reserved.
Proteineprotein interaction
Focal adhesion targeting domain
FAK scaffold inhibitor
1. Introduction
with several proteins such as p53, EGFR, c-Met, RIP, p85 subunit of
PI3K, and b-integrins, whereas the FAK-FAT domain binds to pax-
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase
that is localized to focal adhesions, and forms points of contacts
between cells and the extracellular matrix. The structural compo-
nents of FAK encompass a huge variety of binding partners, phos-
phorylation sites and signaling crosstalk with multiple extracellular
and intracellular proteins [1e3]. The FAK-FERM domain interacts
illin, talin, and Grb2 [4e6]. Thus, FAK serves as a signaling hub and
scaffolding protein to direct numerous signaling pathways to ach-
ieve specific cellular outcomes [7]. FAK is indispensable to cancer
cells, where it functions to promote the overall aggressiveness of
tumor growth by controlling cellular processes such as, adhesion,
migration, proliferation and survival [8e10]. Studies with dominant
negative FAK mutants, antisense oligonucleotides and siRNA’s for
FAK, and germline deletions in FAK, exemplify the role of FAK in
cancer cell progression and metastasis [11e15]. Massive presence of
FAK in almost all solid tumor types [16], coupled with its involve-
ment with deregulated proteins in cancer [17], offers a perfect
setting for FAK specific therapeutics. Existing FAK kinase inhibitors
such as PF-562e271, PF-04554878 and GSK2256098 have been
shown to directly inhibit FAK kinase activity and tumor growth
[18]. However, it is important to note that the aforementioned
drugs being kinase inhibitors compete for the ATP binding site, thus
raising the possibility for these drugs to exhibit non-specific kinase
inhibition and related toxicities [19].
Abbreviations: FAK, focal adhesion kinase; VEGFR, vascular endothelial growth
factor receptor; FERM, 4.1 proteineezrineradixinemoesin; FAT, focal adhesion
targeting; EGFR, endothelial growth factor receptor; c-MET, MNNG HOS Trans-
forming gene; PI3K, phosphatidylinositide 3-kinase; RIP, receptor interacting pro-
tein kinase; Grb2, Growth factor receptor bound protein 2; ATP, adenosine 50-
triphosphate; BH3, Bcl-2 homology domain; Bcl-XL, B-cell lymphoma extra-large;
MDM2, mouse double minute 2 homolog; TMRM, tetramethylrhodamine, methyl
ester.
* Corresponding author.
** Corresponding authors. Department of Surgical Oncology, Roswell Park Cancer
Institute, Buffalo, NY 14263, USA.
E-mail addresses: elena.kurenova@roswellpark.org (E.V. Kurenova), Ravindra.
pandey@roswellpark.org (R.K. Pandey), William.cance@roswellpark.org (W.
G. Cance).
VEGFR3 is part of the VEGF receptor tyrosine kinase family that
is involved in the regulation of normal and pathological endothelial
1
These authors contributed equally.
http://dx.doi.org/10.1016/j.ejmech.2014.04.041
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

P.N. Gogate et al. / European Journal of Medicinal Chemistry 80 (2014) 154e166
155
cell specific responses, from embryogenesis to adulthood. Ligands
VEGF-C and VEGF-D bind to VEGFR3, which is essential for the
growth and maintenance of the lymphatic vasculature [20,21].
However, VEGFR3 [22,23] and its ligands VEGF-C [24,25] and VEGF-
D [26,27] have been shown to promote lymphangiogenesis and
metastatic spread in various tumor models. Several inhibitors of
lymphangiogenesis are being developed such as, neutralizing
monoclonal antibodies to VEGF-C and VEGF-D, peptidomimetics
based on VEGF-C and VEGF-D, and kinase inhibitors of VEGFR3 [28].
Our lab’s approach has been to understand the implications of FAK
specific proteineprotein interactions and develop inhibitors that
preferentially target FAK-protein complexes. We have shown that
VEGFR3 binds to the C-terminal FAT domain of FAK and the inter-
action between FAK and VEGFR3 is crucial for tumor cell viability
[29]. Despite the numerous challenges encountered while devel-
oping inhibitors that target proteineprotein interactions [30], in
our previous efforts, we discovered small molecule 1 (chlorpyr-
amine hydrochloride) (Fig. 1) which successfully binds and disrupts
the FAKeVEGFR3 interaction [31]. Further testing with 1 showed
reduction in tumor burden and neovascularization in mouse
models of melanoma, breast cancer, pancreatic cancer [32], and
glioblastoma.
Although compound 1 is an attractive candidate, it exerts anti-
tumor and anti-angiogenic effects at high micromolar concentra-
tions. In order to gain a detailed understanding of structure activity
relationships and improve the anti-tumor potency and physico-
chemical properties of 1, we altered the structure of 1 to address the
importance of certain chemical motifs. The newly synthesized an-
alogs were screened in vitro using several cancer cell lines and 3D
predicted binding modes of all analogs with the FAT domain of FAK
were generated through molecular modeling. We also ranked the
analogs on the basis of scoring functions. Biological studies with the
selected analogs were performed to investigate their anti-
proliferative activity, binding and disruption of the FAKeVEGFR3
complex, and mechanism of cell death. Collectively, our findings
show that analog 29, which displayed maximum potency and
specificity amongst all tested analogs, is a novel compound which
warrants further investigation in the drug development pipeline
for FAKeVEGFR3 specific inhibitors.
substituents instead of the N-p-chlorobenzyl group (B) and finally
altering the N, N-dimethyl ethyl chain (C) (Fig. 2). For our study, a
series of compounds were synthesized and are listed in Table 1. We
determined their biological efficacy and performed molecular
modeling analysis using the known crystal structure of the FAT
domain of FAK. The grid scores of the analogs from the molecular
docking study are shown in Table 1.
2.2. Chemistry
For the synthesis of the desired analogs, a three step parallel
synthesis protocol was followed, and the synthetic details are
illustrated in Schemes 1e3. For investigating the importance of the
N-p-chlorobenzyl group, a series of N-substituted analogs were
synthesized as depicted in Scheme 1. The key precursor was pre-
pared by reacting chloropyridine 6 with N, N-dimethyl ethyl-
enediamine in a sealed tube at 140ꢀ C, which gave the desired key
intermediate 7. The intermediate amine 7 was functionalized by
reacting with various alkyl/aryl halides in the presence of lithium
amide on refluxing temperature, and this gave the corresponding
pyridine derivatives 9e19 in good yields. Particularly, the benzyl
derivatives 14e18 were isolated in higher yields than the corre-
sponding N-alkyl analogs 9e13. The analog of compound 1 with an
extended carbon unit 19 was prepared by reacting with N, N-
dimethyl ethyl group with an increased carbon unit (Scheme 1).
The pyridine moiety in 1 was replaced with benzene and quinoline
on reacting the chlorobenzene 21 and 2-chloroquinoline 26 with N,
N-dimethylethylamine and thus, compounds 22 and 27 (Schemes 2
and 3) were isolated in excellent yields. By using the intermediates
22 and 27, the corresponding benzyl 23e25 and quinoline analogs
28 and 29 were synthesized (Table 1) in modest yields and the
reaction conditions were not optimized.
2.3. Comparative in vitro efficacy of 1 analogs
First, we analyzed the basal expression levels of target proteins,
FAK and VEGFR3 in A375 and C8161 (melanoma), MDA-MB-231
(breast cancer), MiaPaCa-2-luc and Panc-1-luc (pancreatic can-
cer), and U87 (glioblastoma) cell lines (Fig. 3a). Robust FAK
expression with varying expression levels of VEGFR3 was seen in all
tested cell lines. Growth inhibitory activities of 1 analogs were
determined using the MTS assay (Fig. 3b). The activities of all newly
synthesized analogs were compared to parent drug 1. Anti-
proliferative activity of the analogs was significantly affected by
the type and position of chemical modifications. Replacement of
the pyridine ring (region ‘A’) with a quinoline moiety, 29 or a
benzene ring, 25, showed improved potency in multiple cancer cell
lines. Replacement of the chloro group in the 4-chlorobenzyl
moiety (region ‘B’) with bromo, 15, iodo, 16, thio, 17, and tri-
fluoromethyl, 18, groups did not show improvement in activity over
1. Removal of the 4-chlorobenzyl moiety, analog 7 abolished ac-
tivity. Additionally, analogs 9, 10, 11, 12, and 13 share a common
theme where they all bear varying lengths of the alkyl chain. Of
these, 11, 12, and 13 showed excellent potency in all cell lines used
for screening. We found that an increase in length of the carbon
2. Results and discussion
2.1. Structure activity relationship (SAR) studies
We previously verified the potential anti-cancer activities of
commercially available compounds such as 2, 3, 4, 5, (Fig. 1) and 14
(Table 1) which are chemically similar to parent drug 1 and found
that none of these drugs showed any improvement in activity over
1. This prompted us to perform SAR studies on 1. Parent drug 1
(Fig. 2) was an excellent starting point for exploring rational drug
design and optimization, as the core template of 1 was amenable to
rapid structural modifications. To investigate the impact of various
substituents of 1 on biological efficacy, a series of novel derivatives
were obtained by replacing the pyridine moiety with other aro-
matic systems (A), or by introducing other N-alkyl or aryl
Fig. 1. Structure of parent compound 1 and commercially available compounds structurally similar to 1.

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P.N. Gogate et al. / European Journal of Medicinal Chemistry 80 (2014) 154e166
Table 1
A list of compounds related to 1 investigated for SAR studies.
Compd. number Changes at “A” Changes at “B” Changes at “C” Grid score Compd. number Changes at “A” Changes at “B” Changes at “C” Grid score
1
2
ꢁ30.4
15
16
ꢁ34.77
ꢁ32.75
ꢁ29.08
3
4
ꢁ29.91
ꢁ31.13
17
18
ꢁ35.04
ꢁ33.35
5
7
ꢁ33.29
ꢁ24.27
19
20
ꢁ32.10
ꢁ24.32
8
ꢁ29.72
ꢁ25.35
ꢁ33.69
ꢁ38.64
22
23
24
25
Not calcd.
ꢁ33.49
ꢁ40.21
ꢁ31.81
9
10
11
12
13
ꢁ43.76
ꢁ43.29
27
28
ꢁ29.25
ꢁ34.11
14
ꢁ29.36
29
ꢁ34.41
chain directly correlated with an increase in cytotoxicity and log P
values (Table S1) of these compounds, with 9 showing no activity
and 13 being the most potent in this series. Removal of the N, N-
dimethyl ethyl group (region ‘C’), 20 and introduction of 1 carbon in
the N-linker, 19 did not improve activity. Lastly, dual alterations
were made to the pyridine ring (region ‘A’) and the 4-chlorobenzyl
group (region ‘B’). Analogs 23 and 24 both have benzene rings, but
varying length of alkyl chains. 24, having the 12 carbon chain
dramatically increased cytotoxicity in some cancer cell lines
whereas 23, with the 6 carbon chain failed to show improved ac-
tivity in all tested cell lines. Overall, the trend of increased alkyl
chain length enhances cytotoxicity was seen with analogs 23 and
24 and analogs 9, 10, 11, 12, and 13. Also, analog 24 had the highest
log P of 4.8 which may not favor an optimal drug like character [33]
(Table S1). Next, we observed that removal of the 4-chlorobenzyl
group with the presence of a quinoline ring, 27, did not enhance
potency. Analog 28, with a 6 carbon chain and quinoline ring
showed no improvement in anti-proliferative activity. Lastly, when
the 4-chlorobenzyl group (region ‘B’) was removed and one extra
Fig. 2. Structure of parent compound 1 containing three functionalities A, B, and C.
Scheme 1. N-alkyl, aryl, and substituted aryl analogs of 1.

P.N. Gogate et al. / European Journal of Medicinal Chemistry 80 (2014) 154e166
157
chains with the groove of the FAT protein. While analog 29 had a
modest score (ꢁ34.4 kcal/mol), it was chosen for additional
computational analysis due to its anti-proliferative activity in
MiaPaCa-2-luc, Panc-1-luc, and U87 cell lines (Fig. 3b) and impor-
tantly, its target specificity for the FAKeVEGFR3 interaction
(Fig. 3c). Also, analog 29 had a slightly favorable grid score than the
parent compound 1 (ꢁ30.4 kcal/mol). Based on the surface repre-
sentation of the FAT protein, there appears to be a pocket on the FAT
protein which could favor drug binding. This pocket is comprised of
hydrophobic residues Tyr 925, Val 928, Val 932, and Leu 1035
(Fig. 4a). The predicted docking pose of 29 suggests that it occupies
this pocket on the FAT protein. Analog 29 sits in a charged cavity
surrounded by residues Ser 910, Thr 929, Lys 1032, and Asp 1036
Scheme 2. Synthesis of phenyl analogs of 1.
carbon was introduced to the N, N-dimethyl ethyl group (region
‘C’), analog 8 did not show enhanced activity.
On the basis of in vitro screening results, it appears that retaining
the hetero-aromatic moiety in region ‘A’ plays an important role in
biological efficacy. Replacing the chloro group from the p-chlor-
obenzyl functionality in region ‘B’ of 1 with bromo-, 15, or iodo-, 16,
reduced efficacy probably due to their bulkier nature. Diminished
activity of 3, 5-bis-(trifluoromethyl) benzyl analog, 18, again sug-
gests the importance of the chlorobenzyl group in 1. Interestingly,
replacing the chlorobenzyl group with long alkyl side chains, ana-
logs 11, 12, 13 and 24 due to their flexible nature might show
improved activity due to increased hydrophobic contacts with the
FAT protein and could also contribute toan increase in toxicity due to
non-specific protein binding. Any modification made to region ‘C’
failed toimproveactivity, which suggests that retaining this groupin
parent compound 1 is important. Based on these results, we further
investigated the effects of analogs 25 and 29 on FAKeVEGFR3
disruption in MCF7-VEGFR3 cells [34], which overexpress both FAK
and VEGFR3, and performed immunoprecipitation with FAK anti-
body (Fig. 3c). 1 was used as a positive control based on previous
reports [31]. Analog 7 was included as a negative control, since it
showed no anti-proliferative activity in all cell lines used for
screening (Fig. 3b) and also had the lowest grid score from modeling
analysis (Table 1). Changes in VEGFR3 protein levels co-precipitated
with FAK were analyzed after addition of the selected analogs for
24 h. Decrease in VEGFR3 protein levels was seen following treat-
(Fig. 4b). A cation-
and Lys 1032 along with a potential
p
interaction between the quinoline ring of 29
stacking between the 4-
p
chlorobenzyl group of 29 and the aromatic side chain of Tyr 925
are the key interactions observed. The N, N-dimethyl ethyl group
might have a secondary effect of orienting the two other groups to
this hydrophobic pocket. It is worthwhile exploring the importance
of this functionality to promote hydrogen bonding with the nearby
residues. The proximity of analog 29 to Tyr 925 is of utmost
importance and could be the main reason for its enhanced activity,
since Tyr 925 is a key residue that has been shown to be involved in
the activation of the Grb2-Ras-MAPK pathway which accelerates
tumor progression and angiogenesis [35,36]. To further investigate
the importance of the 4-chlorobenzyl group, we analyzed the
predicted binding modes of analogs 27 and 28 to the FAT protein
since both these analogs are structurally similar to analog 29
(Table 1). Analogs 27 (Fig. S21) and 28 (Fig. S22) occupy a binding
region slightly above from that of analog 29. Analog 27 does not
appear to make any key interactions with its surrounding residues,
Ile 936, Ser 939, His 1025, and Ala 1028. This is also reflected by its
relatively low grid score (ꢁ29.25). Although, the quinoline ring of
analog 28 participates in a hydrogen bond formation with Lys 1032
and the alkyl chain sits in the hydrophobic groove (Val 932, Ile 936,
and Ala 1028) of FAT, this analog showed no improvement in anti-
proliferative activity. In summary, the docking analysis strongly
suggests that retaining a heteroaromatic moiety and the 4-
chlorobenzyl group are critical for activity and that Tyr 925 and
the residues which form the druggable pocket on FAT (Fig. 4a) could
play an important role in the binding and specificity of 1 analogs. To
further validate the specificity of 29 for the FAT domain of FAK, we
evaluated its binding efficacy compared to parent compound 1 by
using the Octet RED Assay based on the principle of bio-layer
interferometry [37]. Binding constants obtained from this assay
revealed that the Kd (M) of 29 (Kd (M) ¼ 1.8 ꢂ 10ꢁ8) was signifi-
cantly greater than 1 (Kd (M) ¼ 5.7 ꢂ 10ꢁ7) and analog 7 which was
included as a negative control did not show any binding to FAT
protein.
ment with 10 mM of 29, confirming that it successfully inhibits the
FAKeVEGFR3 interaction. We also analyzed the effect of analog 29
on kinase specificity (Table 2). A panel of 10 kinases was tested for
inhibition caused by analog 29 at 1 mM. Select kinases were included
in this assay as they are closely related to FAK or VEGFR3 proteins or
are involved in FAK downstream signaling cascades. We found that
29 did not significantly inhibit (<40%) any of the tested kinases. This
data demonstrates that analog 29 does not cause FAKeVEGFR3
disruption by targeting the kinase activity of both these proteins and
proteins involved in the FAK signaling pathway. Analog 29 retained
the target specific propertiesof parent compound 1 andhence, in the
rest of the study we have focused our efforts on evaluating the
biological efficacy of analog 29.
2.4. Molecular modeling and binding analysis
2.5. Cellular activity of analog 29
In silico docking was performed on all analogs and the grid
scores were computed (Table 1). The compound with the highest
predicted affinity for the FAT domain was analog 12 (ꢁ43.7 kcal/
mol), followed by 13 (ꢁ43.3 kcal/mol), 24 (ꢁ40.2 kcal/mol), and 11
(ꢁ38.6 kcal/mol). The high grid scores of these compounds are
most likely due to the hydrophobic interactions of the long alkyl
From the in vitro screening, we found that analog 29 showed
maximum anti-proliferative effects in pancreatic cancer cell lines.
Hence, we conducted further analyses of this analog using a
pancreatic cancer model system. We first analyzed the expression
levels of FAK and VEGFR3 in a panel of six pancreatic cancer cell
lines, SU8686, AsPc, Panc 213, Panc 10.5, Panc-1-luc and MiaPaCa-
2-luc (Fig. 5a). The MCF7-VEGFR3 cell line was included as a posi-
tive control cell line. High levels of FAK and varying levels of
VEGFR3 were found in all these cell lines, with MCF7-VEGFR3
showing very high levels of both FAK and VEGFR3. Next, we
determined the IC50 of 29 in all these cell lines and found that Panc-
1-luc and MiaPaCa-2-luc cell lines were particularly sensitive to
drug treatment (Fig. 5b). MCF7-VEGFR3 cells showed maximum
Scheme 3. Synthesis of quinoline analogs of 1.

Fig. 3. In vitro screening of 1 and its analogs. (A) Basal expression levels of FAK and VEGFR3 protein were analyzed in the indicated cancer cell lines. GAPDH was used as a loading
control. (B) Anti-proliferative effects of 1 analogs in the indicated cell lines that were treated with 50
M of the each analog for 72 h. Error bars represent ꢃSD. (C) Immuno-
precipitation with FAK antibody in MCF7-VEGFR3 cells after treatment with the selected analogs for 24 h followed by immunoblotting with total VEGFR3 and total FAK antibodies.
m

P.N. Gogate et al. / European Journal of Medicinal Chemistry 80 (2014) 154e166
159
Table 2
susceptibility to cytotoxic effects of 29. We then assessed the effects
of 29 on colony formation using MiaPaCa-2-luc and MCF-VEGFR3
cells (Fig. 5c). 29 inhibited colony formation in both cell lines at
Kinase inhibitory profile of analog 29.
Kinase tested
% Inhibitiona
10
m
M compared to compound 1 which achieved the same effect at
M. Analog 7 was also tested for colony for-
AKT1 (PKB
a
)
7
4
1
a concentration >10
mation as a negative control.
m
EGFR (ErbB1)
FLT1 (VEGFR1)
FLT4 (VEGFR3)
IGF1R
KDR (VEGFR2)
PDGFRA (PDGFR
PTK2 (FAK)
PTK2B (FAK2)
SRC
4
We further evaluated the ability of 29 to disrupt the FAKe
VEGFR3 interaction in MiaPaCa-2-luc cells. 1 was included as a
positive control. Immunoprecipitation of FAK was carried out and
levels of FAK pan-phosphorylated tyrosine and co-precipitated
VEGFR3 were evaluated after drug treatment for 24 h (Fig. 6a).
Levels of VEGFR3 and phosphorylated tyrosine residues associated
with FAK protein decreased in a dose dependent manner following
drug exposure. Similarly, reverse immunoprecipitation using
VEGFR3 antibody, confirmed a dose dependent increase in FAKe
VEGFR3 disruption along with reduction in VEGFR3 pan-
phosphorylated tyrosine levels after 24 h treatment of 1 and 29
(Fig. 6b). Densitometry analysis of the protein levels obtained from
the immunoprecipitation results confirmed a dose dependent
decrease after treatment with 1 and 29 in both directions, using
FAK (Fig. 6a) and VEGFR3 antibodies (Fig. 6b). Together, these re-
sults confirm that 29 enhanced target selectivity and specifically
11
14
8
13
0
a
)
3
a
Percent inhibition was calculated according to the Z0-LYTEÒ
Data Analysis protocol (Life Technologies).
inhibited the FAKeVEGFR3 interaction in
a dose dependent
manner.
2.6. Mechanism of cell death of analog 29
Given that 29 exert cytotoxic effects in multiple cancer cell lines,
we investigated the involvement of caspase-3/7 in the cell death
mechanism induced in MiaPaCa-2-luc cells. We used two dyes,
TMRM, a cell permeable red-orange fluorescent dye which is used
to measure the membrane potential of mitochondria and
CellEventÔ, a cell permeable bright green fluorescent dye which
exhibits fluorescence only in the presence of caspase-3/7 activa-
tion. Staurosporine, a well-studied activator of apoptosis [38] was
included as a positive control in this experiment. Increasing doses
of 29 led to increased apoptosis, which was marked by a loss of
TMRM signal with a concomitant increase in the CellEventÔ signal
(Fig. 7a). Apoptotic cells with bright green (in the web version)
staining were further counted for each condition and data were
quantified (Fig. 7b). We found a statistically significant increase in
apoptosis at 70 mM of 29 compared to control cells. We further
tested the dose dependent apoptotic effects of 29 on caspase-3 and
PARP cleavage in MiaPaCa-2-luc and MCF-VEGFR3 cell lines after
24 h drug treatment (Fig. 7c). In both cell lines, an increase in
caspase-3 and PARP cleavage product was seen with increasing
doses of 29. In summary, our results confirm that analog 29 induces
dose dependent apoptotic cell death involving caspase-3/7 and
PARP activation. The apoptotic cell death caused by analog 29 is
consistent with the effects seen with compound 1 which support
our previous conclusions that the FAKeVEGFR3 interaction exerts a
pro-survival effect on cancer cells [29,31].
3. Conclusions
In this report, we investigated the importance of specific
chemical motifs inherent in 1 with an aim to develop small mole-
cules with improved anti-cancer activity, enhanced potency, and
target specificity for the FAKeVEGFR3 interaction. Our SAR
approach was to first divide molecule 1 into three parts in order to
understand the importance of the chemical groups for anti-cancer
activity. Next, we designed and synthesized novel analogs of parent
Fig. 4. Predicted binding mode of analog 29. (A) Surface representation of the FAT
domain of FAK (gray) showing docking of compound 29 (magenta stick). Potentially
druggable pocket on FAT is indicated in orange. (B) Ribbon representation of the FAT
domain (gray) showing the binding pose of analog 29 (magenta stick) and residues
within 5 Å of 29 (green stick). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
compound
1 and subsequently tested them using an anti-
proliferative screening assay along with molecular modeling with
an aim to filter out potential FAKeVEGFR3 inhibitors for further

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P.N. Gogate et al. / European Journal of Medicinal Chemistry 80 (2014) 154e166
Fig. 5. In vitro cytotoxicity profile of analog 29 in pancreatic cancer cell lines. (A) Analysis of FAK and VEGFR3 protein expression in six pancreatic cancer cell lines and MCF7-VEGFR3
cell line. GAPDH was used as a loading control. (B) IC50 values of 29 in the indicated cell lines. Error bars represent ꢃSD. (C) MiaPaCa-2-luc or MCF7-VEGFR3 cells were treated with
29 at the indicated concentrations. After 14 days, colonies were stained with 0.25% of 1, 9-dimethyl-methylene blue in 50% methanol for 1 h. Representative images from three
independent experiments have been shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

P.N. Gogate et al. / European Journal of Medicinal Chemistry 80 (2014) 154e166
161
Fig. 6. Dose dependent disruption of FAKeVEGFR3 binding by analog 29 in MiaPaCa-2-luc cells. (A) Immunoprecipitation with FAK antibody or (B) VEGFR3 antibody after 24 h
treatment with 1 and 29 followed by immunoblotting with pan phosphorylated tyrosine, total FAK and total VEGFR3 antibodies. Isotype and no antibody lanes indicate negative
controls.
studies. Our results highlight the importance of the heteroaromatic
moiety and p-chlorobenzyl group being critical for activity. Analog
29 caused an increase in cytotoxicity in multiple cancer cell lines
compared to parent drug 1. We have shown that analog 29 shows
target specificity for the FAKeVEGFR3 interaction site using both, a
cell based assay and a label free binding assay. Importantly, analog
29 showed greater selectivity for MCF7-VEGFR3 cells that over-
express both target proteins, FAK and VEGFR3, as compared to
MiaPaCa-2-luc cells that have moderate levels of both proteins.
Compound 1 and the selected analog 29 are unconventional
anti-cancer drugs, as they are not kinase inhibitors but in fact
disrupt proteineprotein interactions, in this case between FAK and
VEGFR3. A few examples of well characterized proteineprotein
interaction inhibitors are SP4206 that disrupts the interaction
plastic sheet (20 cm ꢂ 20 cm) strips (POLYGRAM SIL N-HR). 1H NMR
spectra were recorded on Varian 400 spectrometers at 303 K in
CDCl3 or CD3OD. Proton chemical shifts (d) are reported in parts per
million (ppm) relative to CDCl3 (7.26 ppm), CD3OD (3.34 ppm) or
TMS (0.00 ppm). Coupling constants (J) are reported in Hertz (Hz)
and s, d, t, q, p, m and br refer to singlet, doublet, triplet, quartet,
pentet, multiplet and broad respectively. Mass spectral data (Elec-
tro Spray Ionization, ESI by fusion) were obtained from Biopolymer
Facility, Roswell Park Cancer Institute, Buffalo, NY, HRMS data were
obtained from the Mass Spectrometry Facility, Michigan State
University, East Lansing, MI.
4.2. N, N-dimethyl-N0-(pyridine-2-yl)ethane-1,2-diamine (7)
between interleukin 2 (IL-2) and the
a-chain of the IL-2 receptor
(IL-2R ) [39], BH3-mimetic ABT-737 [40] which possesses high-
a
In a dry 25 mL sealed tube, 2-chloropyridine 6 (10 eq, 1.13 g) and
N, N-dimethyl ethylenediamine (11 eq, 1.20 g) were taken and
heated the entire mixture to 140 ꢀC over a period of 12 h with
constant stirring. Then, the reaction mixture was cooled down to
room temperature and the resulting crude was purified on a silica
gel column by eluting (5% MeOH-DCM) as a solvent mixture to
afford the desired derivative 7 (0.676 g) in 41% yield. 1H NMR
affinity binding to the hydrophobic BH3-binding site of recombi-
nant Bcl-XL, ABT-737, and Nutlins that were found to disrupt
MDM2ep53 complexes [41]. By inhibiting the interplay between
FAK and VEGFR3, we have previously shown that 1 downregulated
major FAK and VEGFR3 tyrosine phosphorylation sites that in turn
affect key protein signaling networks associated with cancer cell
survival, angiogenesis, and metastasis [32,34]. Here, we have
discovered that 29 successfully disrupted the FAKeVEGFR3 inter-
action and induced apoptotic cancer cell death, thus we anticipate
29 to mimic the overall anti-cancer effects of 1, albeit at much
lower concentrations. Analog 29 shows much promise as a specific
FAKeVEGFR3 inhibitor and could be used to treat a variety of solid
cancers. Further development and optimization of 29 is currently
underway.
400 MHz (CDCl3)
d
: 8.08 (dd, J ¼ 4.8 Hz, J ¼ 0.8 Hz, 1H); 7.38 (dt,
J ¼ 7.2 Hz, J ¼ 2 Hz, 1H); 6.54 (dt, J ¼ 5.6 Hz, J ¼ 0.8 Hz, 1H); 6.40 (d,
J ¼ 8.4 Hz, 1H); 5.04 (brS, NH, 1H); 3.36 (q, J ¼ 5.6 Hz, 2H); 2.55 (t,
J ¼ 6.0 Hz 2H); 2.27 (s, 6H); 13C NMR (CDCl3)
d: 158.7, 147.9, 137.1,
112.5, 107.4, 58.0, 45.1, 39.2; MASS (ESI): m/z 166 ([MH]þ, 100%);
HRMS: C9H15N3 [M]þ Calcd 165.1266, Obsd. 165.1275.
4.3. N, N-dimethyl-N0-(pyridine-2-yl)propane-1,2-diamine (8)
Compound 8 was synthesized by following the procedure for
4. Experimental section
4.1. Chemical synthesis
compound 7. Yield is 48%. 1H NMR 400 MHz (CDCl3)
d: 8.03 (dd,
J ¼ 4.8 Hz, J ¼ 0.8 Hz, 1H); 7.38 (td, J ¼ 7.2 Hz, J ¼ 2 Hz, 1H); 6.48e
6.54 (m, 2H); 5.64 (brS, NH, 1H); 3.50 (t, J ¼ 6.4 Hz, 2H); 2.89 (t,
J ¼ 6.4 Hz 2H); 2.64 (s, 6H); 2.05 (quin, J ¼ 6.0 Hz, 2H); 13C NMR
All chemicals were of reagent grade and used as such. All re-
agents were purchased from Aldrich chemical company and were
used as received. Solvents were dried using standard methods
unless stated otherwise. Reactions were carried out under nitrogen
atmosphere and were monitored by pre-coated (0.20 cm) silica TLC
(CDCl3) d: 147.0, 137.5, 112.7, 109.0, 56.1, 43.3, 38.5, 30.9, 24.7; MASS
(ESI): m/z 180 ([MH]þ, 100%); HRMS: C10H17N3 [M]þ Calcd 179.1422,
Obsd. 179.1488.

162
P.N. Gogate et al. / European Journal of Medicinal Chemistry 80 (2014) 154e166
Fig. 7. Analog 29 induced cell death via activation of the apoptotic pathway. (A) MiaPaCa-2-luc cells were treated with analog 29 at the indicated concentrations. After 24 h cells
were stained with CellEventÔ, a caspase-3, 7 dye and TMRM, a mitochondrial dye. Representative images are shown from three independent experiments. (B) Apoptotic cells were
counted from multiple microscopic fields for each condition. Error bars represent ꢃSEM. *p < 0.05 (C) Addition of analog 29 to MiaPaCa-2-luc and MCF-VEGFR3 cells at the indicated
concentrations for 24 h. Antibodies for caspase-3 and PARP were used to detect apoptosis. GAPDH serves as a loading control.

P.N. Gogate et al. / European Journal of Medicinal Chemistry 80 (2014) 154e166
163
4.4. N, N-dimethyl-N0-(phenyl)ethane-1,2-diamine (22)
4.6.4. N-[decyl]-N-[2-(dimethylamino)ethyl]pyridin-2-amine (12)
Compound 12 was obtained by following the general procedure
mentioned above; Yield 53%. 1H NMR 400 MHz (CD3OD)
: 8.09 (dd,
Compound 22 was synthesized by following the procedure for
d
compound 7. Yield is 40%. 1H NMR 400 MHz (CD3OD)
d
: 7.07e7.11
J ¼ 5.4 Hz, J ¼ 1.2 Hz, 1H), 7.54 (td, J ¼ 8.8 Hz, J ¼ 2.0 Hz, 1H); 6.69 (d,
J ¼ 8.8 Hz,1H); 6.61 (t, J ¼ 6.4 Hz,1H); 3.78 (t, J ¼ 6.0 Hz, 2H); 3.42 (t,
J ¼ 7.6 Hz, 2H); 3.08 (t, J ¼ 6.0 Hz, 2H); 2.75 (s, 6H); 1.62 (m, 2H);
(m, 2H); 6.59e6.64 (m, 3H); 6.48e3.21 (t, J ¼ 6.8 Hz, 2H); 2.57 (t,
J ¼ 7.2 Hz, 2H); 2.29 (s, 6H); 13C NMR (CD3OD)
d: 148.5, 129.1, 117.1,
112.8, 58.0, 45.1, 41.1; MASS (ESI): m/z 165 ([MH]þ, 100%); HRMS:
1.26e1.42 (m, 14H), 0.89 (t, J ¼ 7.2 Hz, 3H); 13C NMR (CD3OD)
d:
C
10H16N2 [M]þ Calcd 164.1313, Obsd. 164.1374.
157.7, 147.0, 137.4, 111.2, 106.2, 50.0, 48.8, 45.6, 43.8, 31.6, 29.3, 29.2,
29.0, 26.9, 26.5, 22.2, 12.9; MASS (ESI): m/z 306 ([MH]þ, 100%);
HRMS: C19H35N3 [M]þ Calcd 305.2831, Obsd. 305.2612.
4.5. N, N-dimethyl-N0-(quinolin-2-yl)ethane-1,2-diamine (27)
Compound 27 was obtained by following the procedure for
4.6.5. N-[dodecyl]-N-[2-(dimethylamino)ethyl]pyridin-2-amine
(13)
compound 7. Yield is 41%. 1H NMR 400 MHz (CD3OD)
d: 7.82 (d,
Compound 13 was obtained by following the general procedure
J ¼ 8.8 Hz, 1H); 7.58 (t, J ¼ 8.4 Hz, 2H), 7.47 (t, J ¼ 8.0 Hz, 1H); 7.16 (t,
mentioned above; Yield 42%. 1H NMR 400 MHz (CD3OD)
d: 8.02 (d,
J ¼ 8.0 Hz, 1H); 6.72 (d, J ¼ 8.8 Hz, 1H); 3.60 (t, J ¼ 6.8 Hz, 2H); 2.64
J ¼ 4.4 Hz, 1H), 7.43 (td, J ¼ 8.0 Hz, J ¼ 1.6 Hz, 1H); 6.61 (t, J ¼ 6.0 Hz,
1H); 6.55 (d, J ¼ 8.4 Hz, 1H); 3.81 (t, J ¼ 6.4 Hz, 2H); 3.52 (t,
J ¼ 6.4 Hz, 2H); 3.36e3.42 (m, 2H); 3.16 (s, 6H); 1.78 (m, 2H); 1.21e
(t, J ¼ 6.8 Hz 2H); 2.34 (s, 6H); 13C NMR (CD3OD)
d: 156.9, 148.0,
136.9, 129.4, 127.4, 126.0, 123.3, 121.8, 112.1, 58.0, 45.1, 38.7; MASS
(ESI): m/z 216 ([MH]þ,100%); HRMS: C13H17N3 [M]þ Calcd 215.3000,
Obsd. 215.3226.
1.34 (m, 18H), 0.89 (t, J ¼ 6.4 Hz, 3H); 13C NMR (CD3OD)
d: 157.8,
146.7, 138.2, 112.5, 107.1, 58.3, 49.5, 48.2, 44.8, 42.7, 31.6, 29.33,
29.31, 29.2, 29.1, 29.0, 26.57, 26.51, 22.3, 13.0; MASS (ESI): m/z 334
([MH]þ, 100%); HRMS: C21H39N3 [M]þ Calcd 333.3144, Obsd.
333.3210.
4.6. General procedure for the synthesis of 1 analogs
The compound of type 7 (1eq) and dry LiNH2 (1.2 eq) were taken
in a dry RB flask and was added dry CH3CN (5 mL) as a solvent. After
stirring for 5 min under Ar atm, the alkyl/benzyl halide (1.2 eq) was
added drop by drop via syringe. The stirring was continued for 8 h
at reflux temperature. The TLC indicated the formation of the new
product spot. The reaction mixture was filtered and the filtrate was
evaporated under reduced pressure. The crude obtained was pu-
rified on a silica gel column. Elution of 3% MeOH-DCM solvent
mixture delivered the expected alkylated derivatives in 40e68%
yield.
4.6.6. N-[(4-bromophenyl)methyl]-N-[2-(dimethylamino)ethyl]
pyridin-2-amine (15)
Compound 15 was obtained by following the general procedure
mentioned above; Yield 64%. 1H NMR 400 MHz (CD3OD)
d: 8.02 (d,
J ¼ 4.4 Hz, 1H); 7.68 (d, J ¼ 8.4 Hz, 2H); 7.51 (d, J ¼ 8.4 Hz, 2H), 7.42
(td, J ¼ 8.0 Hz, J ¼ 1.6 Hz, 1H); 6.62 (t, J ¼ 6.8 Hz, 1H); 6.56 (d,
J ¼ 8.4 Hz, 1H); 4.60 (s, 2H); 3.90 (t, J ¼ 6.8 Hz, 2H); 3.53 (t,
J ¼ 6.4 Hz, 2H); 3.12 (s, 6H); 13C NMR (CD3OD)
d: 157.9, 146.9, 137.3,
134.5, 132.1, 126.5, 125.1, 113.0, 109.3, 67.2, 62.9, 49.4, 35.0; MASS
(ESI): m/z 335 ([MH]þ, 100%); HRMS: C16H20BrN3 [M]þ Calcd
333.0841, Obsd. 333.0698.
4.6.1. N-[propyl]-N-[2-(dimethylamino)ethyl]pyridin-2-amine (9)
Compound 9 was obtained by following the general procedure
mentioned above; Yield 51%. 1H NMR 400 MHz (CDCl3)
d: 8.01 (d,
4.6.7. N-[(4-iodophenyl)methyl]-N-[2-(dimethylamino)ethyl]
pyridin-2-amine (16)
J ¼ 4.4 Hz,1H), 7.35 (t, J ¼ 7.6 Hz,1H); 6.70 (d, J ¼ 8.4 Hz,1H); 6.57 (t,
Compound 16 was obtained by following the general procedure
J ¼ 6.0 Hz, 1H); 3.98 (m, 2H); 3.82 (m, 2H); 3.38e3.60 (m, 2H), 3.39
mentioned above; Yield 61%. 1H NMR 400 MHz (CD3OD)
d: 8.01 (dd,
(s, 6H); 1.80 (m, 2H), 0.98 (t, J ¼ 7.2 Hz, 3H); 13C NMR (CDCl3)
d:
J ¼ 5.2 Hz, J ¼ 01.2 Hz, 1H); 7.87 (d, J ¼ 8.0 Hz, 2H); 7.42 (td,
J ¼ 9.6 Hz, J ¼ 2.0 Hz,1H); 7.35 (d, J ¼ 8.4 Hz, 2H), 6.60 (td, J ¼ 7.2 Hz,
J ¼ 0.8 Hz, 1H); 6.56 (d, J ¼ 8.4 Hz, 1H), 4.58 (s, 2H); 3.90 (t,
J ¼ 6.8 Hz, 2H); 3.53 (t, J ¼ 6.4 Hz, 2H); 3.11 (s, 6H); 13C NMR
157.5, 147.2, 137.3, 113.5, 110.1, 66.8, 62.7, 52.3, 36.0, 16.4, 10.5; MASS
(ESI): m/z 208 ([MH]þ, 100%); HRMS: C12H21N3 [M]þ Calcd 207.1735,
Obsd. 207.1797.
(CD3OD) d: 157.9, 146.9, 138.2, 137.3, 134.5, 126.9, 113.0, 109.3, 96.9,
4.6.2. N-[hexyl]-N-[2-(dimethylamino)ethyl]pyridin-2-amine (10)
Compound 10 was obtained by following the general procedure
67.4, 62.9, 49.5, 35.0; MASS (ESI): m/z 382 ([MH]þ, 100%); HRMS:
16H20IN3 [M]þ Calcd 381.0702, Obsd. 381.0672.
C
mentioned above; Yield 54%. 1H NMR 400 MHz (CD3OD)
d: 7.96 (d,
J ¼ 4.0 Hz,1H), 7.31 (t, J ¼ 6.8 Hz,1H); 6.68 (d, J ¼ 7.2 Hz,1H); 6.52 (t,
J ¼ 6.8 Hz, 1H); 3.94 (m, 2H); 3.76 (m, 2H); 3.55 (m, 2H); 3.37 (s,
6H); 1.67 (m, 2H); 1.21e1.30 (m, 6H), 0.81 (t, J ¼ 5.6 Hz, 3H); 13C
4.6.8. N-[(4-methylthiophenyl)methyl]-N-[2-(dimethylamino)
ethyl]pyridin-2-amine (17)
Compound 17 was obtained by following the general procedure
NMR (CD3OD) d: 157.6, 148.0, 137.0, 111.2, 105.6, 56.5, 49.2, 46.3,
mentioned above; Yield 64%. 1H NMR 400 MHz (CDCl3)
d: 8.01 (dd,
45.3, 31.7, 27.4, 26.7, 22.6, 14.0; MASS (ESI): m/z 250 ([MH]þ, 100%);
J ¼ 5.2 Hz, J ¼ 1.2 Hz,1H); 7.52 (d, J ¼ 8.4 Hz, 2H); 7.33 (td, J ¼ 8.8 Hz,
J ¼ 2.0 Hz, 1H); 7.21 (d, J ¼ 8.4 Hz, 2H), 6.69 (d, J ¼ 8.4 Hz, 1H); 6.54
(t, J ¼ 6.8 Hz,1H); 4.91 (s, 2H); 4.05e4.09 (m, 2H); 3.84 (t, J ¼ 5.2 Hz,
HRMS: C15H27N3 [M]þ Calcd 249.2205, Obsd. 249.2211.
4.6.3. N-[octyl]-N-[2-(dimethylamino)ethyl]pyridin-2-amine (11)
Compound 11 was obtained by following the general procedure
2H); 3.29 (s, 6H); 2.45 (s, 3H); 13C NMR (CDCl3)
d: 157.6, 147.0, 142.9,
137.3, 133.4, 126.0, 122.8, 113.3, 110.3, 67.6, 63.7, 50.5, 36.3, 14.9;
MASS (ESI): m/z 302 ([MH]þ, 100%); HRMS: C17H23N3S [M]þ Calcd
301.1613, Obsd. 301.1596.
mentioned above; Yield 51%. 1H NMR 400 MHz (CDCl3)
d: 8.08 (dd,
J ¼ 5.4 Hz, J ¼ 1.2 Hz, 1H), 7.52 (td, J ¼ 8.6 Hz, J ¼ 2.2 Hz,1H); 6.59 (d,
J ¼ 8.6 Hz, 1H); 6.56 (t, J ¼ 6.6 Hz, 1H); 4.04 (t, J ¼ 6.0 Hz, 2H); 3.82
(t, J ¼ 6.0 Hz, 2H); 3.56e3.62 (m, 2H); 3.46 (s, 6H); 1.60 (m, 2H);
4.6.9. N-[(3,5-bis-trifluoromethyl-phenyl)methyl]-N-[2-
(dimethylamino)ethyl]pyridin-2-amine (18)
1.26e1.42 (m, 10H), 0.87 (t, J ¼ 7.2 Hz, 3H); 13C NMR (CD3OD)
d:
157.1, 147.4, 137.4, 112.2, 106.2, 50.5, 48.7, 41.7, 31.5, 29.1, 29.0, 28.8,
Compound 18 was obtained by following the general procedure
26.6, 25.9, 22.2, 12.9; MASS (ESI): m/z 278 ([MH]þ, 100%); HRMS:
mentioned above; Yield 63%. 1H NMR 400 MHz (CD3OD)
d
: 8.25 (s,
C
17H31N3 [M]þ Calcd 277.2518, Obsd. 277.2529.
2H), 8.19 (s, 1H), 8.02 (d, J ¼ 4.4 Hz, 1H); 7.43 (td, J ¼ 8.0 Hz,

164
P.N. Gogate et al. / European Journal of Medicinal Chemistry 80 (2014) 154e166
J ¼ 2.0 Hz, 1H); 6.57e6.63 (m, 2H), 4.84 (s, 2H); 3.94 (t, J ¼ 6.4 Hz,
J ¼ 8.8 Hz, 1H), 7.61e7.64 (m, 4H), 7.48e7.54 (m, 3H); 7.23 (t,
J ¼ 6.8 Hz, 1H); 6.80 (d, J ¼ 8.8 Hz, 1H); 4.69 (s, 2H), 4.10 (t,
J ¼ 6.8 Hz, 1H); 3.67 (t, J ¼ 6.8 Hz, 1H); 3.19 (s, 6H); 13C NMR
2H); 3.63 (t, J ¼ 6.4 Hz, 2H); 3.18 (s, 6H); 13C NMR (CD3OD)
d: 157.9,
146.9, 137.3, 133.3, 130.5, 124.4, 124.3, 121.6, 113.1, 109.4, 66.1, 63.6,
49.5, 35.1; MASS (ESI): m/z 392 ([MH]þ, 100%); HRMS: C18H19F6IN3
[M]þ Calcd 391.1483, Obsd. 391.1446.
(CD3OD) d: 156.4, 147.4, 137.2, 136.8, 134.4, 129.1, 129.0, 127.3, 126.1,
125.4, 123.6, 122.1, 112.5, 67.1, 62.7, 49.5, 34.8; MASS (ESI): m/z 340
([MH]þ, 100%); HRMS: C20H22ClN3 [M]þ Calcd 339.1502, Obsd.
339.1494.
4.6.10. N-[(4-chlorophenyl)methyl]-N-[2-(dimethylamino)propyl]
pyridin-2-amine (19)
Compound 19 was obtained by following the general procedure
4.7. N-(4-chlorobenzyl)pyridin-2-amine (20)
mentioned above; Yield 68%. 1H NMR 400 MHz (CD3OD)
d: 7.96 (d,
J ¼ 4.4 Hz, 1H); 7.45e7.53 (m, 5H); 6.58e6.32 (m, 2H); 4.54 (s, 2H);
Compound 20 was obtained by following the known procedure;
3.44 (t, J ¼ 6.4 Hz, 2H); 3.30e3.45 (m, 2H); 3.05 (s, 6H); 2.13e2.20
Yield 40%. 1H NMR 400 MHz (CDCl3)
d: 8.10 (m, 1H); 7.40 (dt,
(m, 2H); 13C NMR (CD3OD)
d: 158.0, 145.3, 138.0, 136.7, 134.2, 129.0,
J ¼ 7.2 Hz, J ¼ 2 Hz, 1H); 7.29 (s, 4H); 6.60 (dt, J ¼ 5.6 Hz, J ¼ 1.2 Hz,
1H); 4.48 (d, J ¼ 6.0 Hz, 1H); 4.86 (brS, NH, 1H). MASS (ESI): m/z 219
([MH]þ). (Known compound).
126.0, 112.2, 109.5, 66.5, 62.1, 49.2, 37.7, 22.6; MASS (ESI): m/z 304
([MH]þ, 100%); HRMS: C17H22ClN3 [M]þ Calcd 303.1502, Obsd.
303.1598.
4.8. Cell culture
4.6.11. N-[hexyl]-N-[2-(dimethylamino)ethyl]phenylamine (23)
Compound 23 was obtained by following the general procedure
MiaPaCa-2-luc and Panc-1-luc cell lines were transfected with
retroviral expression vector pMSCV-LucSh (provided by Dr. Andrew
M. Davidoff, St. Jude Children’s Research Hospital), which contains a
luciferase and zeocin resistance fusion gene that was used to create
cell lines stably expressing luciferase. The C8161 human melanoma
line was kindly provided by Dr. Bernard Weissman, University of
North Carolina, Chapel Hill, North Carolina. MDA-MB-231, U87,
A375, SU8686, AsPc, Panc 213 and Panc 10.5 were purchased from
American Type Culture Collection (ATCC, Rockville, MD, USA). The
MCF7-VEGFR3 cell line has been previously developed and char-
acterized [34]. All cell lines were maintained in DMEM supple-
mented with 10% fetal bovine serum and incubated at 37 ꢀC in 5%
CO2.
mentioned above; Yield 44%. 1H NMR 400 MHz (CDCl3)
d: 7.20e7.23
(m, 2H), 6.34e7.19 (m, 3H); 3.42e3.46 (m, 2H); 3.28 (t, J ¼ 7.6 Hz,
1H); 2.48e2.50 (m, 2H); 2.31 (s, 6H); 1.57e1.61 (m, 2H); 1.31e1.36
(m, 6H); 0.92 (t, J ¼ 5.6 Hz, 3H); 13C NMR (CDCl3)
d: 147.8, 129.2,
115.5, 111.7, 56.3, 51.3, 49.2, 45.8, 31.7, 27.2, 26.7, 22.6, 14.0; MASS
(ESI): m/z 249 ([MH]þ, 100%); HRMS: C16H28N2 [M]þ Calcd
248.2252, Obsd. 248.2266.
4.6.12. N-[dodecyl]-N-[2-(dimethylamino)ethyl]phenylamine (24)
Compound 24 was obtained by following the general procedure
mentioned above; Yield 42%. 1H NMR 400 MHz (CDCl3)
d: 7.12e7.16
(m, 2H), 6.67 (d, J ¼ 7.6 Hz, 2H); 6.58 (t, J ¼ 7.6 Hz, 1H); 3.43 (t,
J ¼ 8.0 Hz, 2H); 3.27 (t, J ¼ 8.2 Hz, 2H); 2.48 (t, J ¼ 8.4 Hz, 2H); 2.29
(s, 6H); 1.56 (p, J ¼ 7.2 Hz, 2H); 1.28e1.32 (m, 18H); 0.89 (t,
4.9. Antibodies and reagents
J ¼ 6.8 Hz, 3H); 13C NMR (CDCl3)
d: 147.9, 129.2, 115.4, 111.6, 56.4,
51.2, 49.4, 45.9, 31.9, 29.66, 29.6, 29.53, 29.50, 29.3, 27.5, 27.1, 22.6,
14.1; MASS (ESI): m/z 333 ([MH]þ, 100%); HRMS: C22H40N2 [M]þ
Calcd 332.3191, Obsd. 332.3114.
FAK mouse monoclonal antibody (clone 4.47), VEGFR3 clone
9D9, phosphorylated tyrosine 4G10 (Millipore, MA). VEGFR3 and
phosphorylated VEGFR3 rabbit polyclonal antibodies (Santa Cruz
Biotechnology Inc, Santa Cruz, CA). Caspase-3 and PARP rabbit
polyclonal antibodies (Cell Signaling Technology, Danvers, MA).
GAPDH (Invitrogen, Grand Island, NY). Chlorpyramine hydrochlo-
ride (Sigma). Staurosporine (EMD Millipore Corporation, Billerica,
MA), CellEventÔ Caspase-3/7 Green Detection Reagent and Tetra-
methylrhodamine, Methyl Ester, Perchlorate (TMRM) (Life Tech-
nologies, Grand Island, NY).
4.6.13. N-[(4-chlorophenyl)methyl]-N-[2-(dimethylamino)ethyl]
phenylamine (25)
Compound 25 was obtained by following the general procedure
mentioned above; Yield 52%. 1H NMR 400 MHz (CDCl3)
d: 7.23 (dd,
J ¼ 22.8 Hz, J ¼ 8.4 Hz, 4H); 7.13 (t, J ¼ 7.6 Hz, 2H); 6.69 (d, J ¼ 8.0 Hz,
2H); 6.63 (t, J ¼ 7.2 Hz, 1H); 4.53 (s, 2H); 3.55 (t, J ¼ 7.6 Hz, 2H); 2.55
(t, J ¼ 7.6 Hz, 2H); 2.28 (s, 6H). 13C NMR (CDCl3)
d: 147.9, 129.2,115.4,
111.6, 56.4, 51.2, 49.4, 45.9, 31.9, 29.66, 29.6, 29.53, 29.50, 29.3, 27.5,
4.10. Cell viability assay
27.1, 22.6, 14.1; MASS (ESI): m/z 289 ([MH]þ, 100%); HRMS:
C
17H21ClN2 [M]þ Calcd 288.1393, Obsd. 288.1412.
Cancer cell lines were seeded at a density of 5000 cells/well in 96
well plates and allowed to attach overnight. Drugs were added at
the indicated concentrations and plates were incubated at 37 ꢀC for
72 h. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium compound from Promega
(Madison, WI) was added to each well and plates were incubated at
37 ꢀC for approximately 1 h. Absorbance was read on a microplate
reader (BioTek) at 570 nm. The absorbance values were used to
calculate the viability and data was normalized to control values.
Each experiment was repeated three times independently with 3e5
replicate wells for each drug concentration or DMSO/untreated
controls.
4.6.14. N-[hexyl]-N-[2-(dimethylamino)ethyl]quinolin-2-amine
(28)
Compound 28 was obtained by following the general procedure
mentioned above; Yield 49%. 1H NMR 400 MHz (CD3OD)
d: 7.91 (d,
J ¼ 8.8 Hz, 1H), 7.63 (d, J ¼ 8.8 Hz, 2H), 7.51 (t, J ¼ 8.0 Hz, 1H), 7.23 (t,
J ¼ 7.6 Hz, 1H); 6.76 (d, J ¼ 9.2 Hz, 1H); 3.98 (t, J ¼ 6.0 Hz, 2H); 3.64
(t, J ¼ 6.4 Hz, 2H); 3.43e3.47 (m, 2H); 3.21 (s, 6H); 1.83 (m, 2H);
1.26e1.36 (m, 6H), 0.87 (t, J ¼ 6.4 Hz, 3H); 13C NMR (CD3OD)
d:
156.4, 147.2, 137.3, 129.1, 127.3, 125.3, 123.5, 122.1, 112.4, 64.3, 61.6,
50.8, 34.9, 30.9, 25.6, 22.2, 22.0, 12.8; MASS (ESI): m/z 300 ([MH]þ,
100%); HRMS: C19H29N3 [M]þ Calcd 299.2361, Obsd. 299.2380.
4.11. Biolayer interoferometry
4.6.15. N-[(4-chlorophenyl)methyl]-N-[2-(dimethylamino)ethyl]
quinolin-2-amine (29)
The binding studies were performed by Lampire Biological
Laboratories, PA. The human FAT domain protein was biotinylated
and the interaction between the small molecules and the FAT
Compound 29 was obtained by following the general procedure
mentioned above; Yield 58%. 1H NMR 400 MHz (CD3OD)
d: 7.89 (d,

P.N. Gogate et al. / European Journal of Medicinal Chemistry 80 (2014) 154e166
165
domain protein was determined by biolayer interferometry using
an Octet Red 96 instrument (FortéBio Inc.). Super streptavidin
biosensor tips (FortéBio, Inc) were pre-wetted with buffer (For-
téBio) to establish a baseline before protein immobilization. Asso-
ciation and dissociation measurements were carried out over 210 s.
Two concentrations of the various small molecules were used. Ki-
netic parameters (Kon, Koff, and Kd) measurements were calculated
using the FortéBio data analysis software.
AMBER molecular mechanics force field. The final grid score was
computed as the sum of van der Waals, electrostatic, and ligand
internal energy terms for each derivative. Images depicting the
predicted binding poses of various analogs to the FAT domain of
FAK (surface and ribbon representation) were created using PyMOL
v.1.3.
4.15. Colony formation
4.12. Western blotting
700 cells/well were seeded in 6 well plates, immediately fol-
lowed by the addition of the compounds at specified concentra-
tions. Plates were then incubated at 37 ꢀC for 10e14 days or until
colonies appeared. Cells were rinsed with PBS, 1% methylene blue
staining solution was added to each well and plates were allowed to
sit at room temperature for 1 h. The staining solution was aspirated,
cells were washed with phosphate buffered saline (PBS) and
distilled water and the plates were allowed to dry overnight. Plates
with stained colonies were scanned using HP Scanjet G3110. Each
experiment was done in duplicates and was repeated three times
independently.
Indicated cancer cell lines were seeded at a density such that
they were w80% confluent after 24 h. Drugs were added and cells
were incubated at 37 ꢀC for 24 h. Cells were washed twice with ice
cold PBS followed by cell lysis using 1% NP40 (Nonidet P-40) lysis
buffer containing PhosSTOP and Complete inhibitor cocktail tablets
(Roche). After centrifugation of the cell lysate, protein concentra-
tion of the supernatant fraction was determined using Pierce BCA
Protein Assay Reagent A and B (Thermo Scientific). Briefly, protein
extracts (30 mg) were separated on 7.5% or 4e20% mini-PROTEAN
TGX gels (Bio-Rad). Proteins were transferred to a PVDF (poly-
vinylidene fluoride) membrane. Non-specific interactions were
blocked using 5% BSA in 0.1% Tween 20/PBS solution for 1 h at room
temperature. Specific primary antibodies were added to the
membrane and allowed to incubate overnight at 4 ꢀC. Peroxidase
conjugated secondary antibodies were then exposed to the mem-
brane for 1 h at room temperature, followed by enhanced chem-
iluminescence detection (ECL, GE Healthcare) using BioBlot BXR
film for western blot (LPS, Inc) developed by Kodak X-OMAT Pro-
cessor (SourceOne Healthcare Technologies).
4.16. Kinase specificity
The Z0-LYTE SelectScreenÒ Single Point biochemical assay was
performed by Life Technologies. Percent inhibition was calculated
as per the Z’-LYTEÒ Data Analysis protocol (Life Technologies).
Concentration of analog 29 used in the reaction mixture was 1
mM
and ATP was 100 M. Inhibition data represent mean kinase inhi-
m
bition from two independent runs.
4.17. Detection of apoptosis
4.13. Immunoprecipitation
MiaPaCa-2-luc or MCF7-VEGFR3 cells were plated at a density of
10,000 cells/well and allowed to adhere overnight at 37 ꢀC. Drugs
were added and 24 h later, cells were rinsed with ice cold PBS,
Protein A/Plus Protein G Sepharose beads (Calbiochem) were
added to the cell lysate suspension (500
mg or 800 mg protein) and
the volume was made up to 500 l using ice cold immunoprecip-
m
followed by addition of 5 mM CellEvent caspase-3/7 dye and 2.5 mM
itation buffer (NP40 containing protease and phosphatase in-
hibitors) to pre-clear by constant rotation for 1 h at 4 ꢀC. After
centrifugation at 3000 rpm at 4 ꢀC, the supernatant was collected
and appropriate antibodies, FAK 4.47, VEGFR3 clone 9D9 or control
IgG were added to facilitate protein-antibody binding. Next, after
rotating the lysate containing antibodies for 2 h at 4 ꢀC, beads were
added and the resultant lysate mixture was placed on a rotator
overnight at 4 ꢀC. Beads were washed three times with ice cold
immunoprecipitation buffer before boiling the samples with
Laemmli Sample Buffer (Bio-Rad) for 5 min at 95 ꢀC. Samples were
loaded onto a 7.5% Mini-PROTEAN TGX gels (Bio-Rad) followed by
the western blotting procedure described above.
mitochondrial specific TMRM dye in PBS for 30 min at 37 ꢀC. Plates
were protected from light during incubation and subsequent steps.
Cells were washed with ice cold PBS and images were captured on
camera using a Zeiss Axio Observer A1 microscope. Images from
multiple fields were taken across each sample. Apoptotic cells,
whose nuclei were bright green, were counted from 6 to 8 fields for
each condition. Percent apoptosis was calculated and data were
normalized to controls. Representative images are shown from
three independent experiments.
4.18. Statistical analysis
Comparisons between groups were made using a Students t test.
Data were considered significant when p < 0.05.
4.14. Molecular modeling
Derivatives of 1 were built using ChemDraw 11.0 Suite (Cam-
bridgeSoft), and saved in MOL2 format. Structures were prepped for
molecular docking using the “Dock Prep” function in the UCSF
Chimera software [42]. As part of the docking preparations, partial
charges were calculated using the AMBER module ANTECHAMBER
using the Gasteiger method. All ligand structures were energy
minimized following partial charge calculations. In silico scoring of
the 1 analogs was performed with UCSF DOCK 6.6 (dock.compio.
ucsf.edu). The target of the molecular docking was the FAT
domain of FAK (PDB ID: 1K05) [43]. The docking parameters
included testing up to 10,000 orientations of each derivative while
allowing for ligand flexibility, followed by energy minimization of
the docked pose. The scoring function used was the DOCK 6.6 grid
scoring method, which consists of the non-bonded terms of the
Disclosure of potential conflicts of interest
William Cance and Elena Kurenova are stockholders and foun-
ders of CureFAKtor Pharmaceuticals, LLC. University of Florida and
Roswell Park Cancer Institute have patents pending and awarded
that are based on their work developing FAK inhibitors.
Acknowledgments
This work was supported by the National Cancer Institute grant
RO1-CA65910 (W.G.C), Photolitec Grant (R.K.P), and Roswell Park
Cancer Institute. This work utilized core resources supported by the
NCI Cancer Center Support Grant CA016156 (Trump, DL).

166
P.N. Gogate et al. / European Journal of Medicinal Chemistry 80 (2014) 154e166
[23] Y. He, I. Rajantie, K. Pajusola, M. Jeltsch, T. Holopainen, S. Yla-Herttuala,
Appendix A. Supplementary data
T. Harding, K. Jooss, T. Takahashi, K. Alitalo, Vascular endothelial cell growth
factor receptor 3-mediated activation of lymphatic endothelium is crucial for
tumor cell entry and spread via lymphatic vessels, Cancer Research 65 (2005)
4739e4746.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.041.
[24] T. Karpanen, M. Egeblad, M.J. Karkkainen, H. Kubo, S. Yla-Herttuala,
M. Jaattela, K. Alitalo, Vascular endothelial growth factor C promotes tumor
lymphangiogenesis and intralymphatic tumor growth, Cancer Research 61
(2001) 1786e1790.
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