Novel multitarget inhibitors with antiangiogenic and immunomodulator properties

Article abstract of DOI:10.1016/j.ejmech.2019.03.012

By means of docking studies, seventeen compounds T.1-T17 have been designed and evaluated as multitarget inhibitors of VEGFR-2 and PD-L1 proteins in order to overcome resistance phenomena offered by cancer. All these designed molecules display a urea moie

Full text of DOI:10.1016/j.ejmech.2019.03.012

Accepted Manuscript
Novel multitarget inhibitors with antiangiogenic and immunomodulator properties
Laura Conesa-Milián, Eva Falomir, Juan Murga, Miguel Carda, J. Alberto Marco
PII:
S0223-5234(19)30223-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.03.012
EJMECH 11182
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 December 2018
Revised Date: 26 February 2019
Accepted Date: 5 March 2019
Please cite this article as: L. Conesa-Milián, E. Falomir, J. Murga, M. Carda, J.A. Marco, Novel
multitarget inhibitors with antiangiogenic and immunomodulator properties, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.03.012.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Manuscript draft
Title: Novel multitarget inhibitors with antiangiogenic and immunomodulator properties
Article type: Original paper
Keywords: angiogenesis, VEGFR-2, immunotherapy, PD-L1, ureas, docking.
Abstract:
By means of docking studies, seventeen compounds T.1-T17 have been designed
and evaluated as multitarget inhibitors of VEGFR-2 and PD-L1 proteins in order to
overcome resistance phenomena offered by cancer. All these designed molecules
display a urea moiety as a common structural feature and eight of them (T.1-T8) further
contain a 1,2,3-triazol moiety. The antiproliferative activity of these molecules on
several tumor cell lines (HT-29, MCF-7, HeLa, A549, HL-60), on the endothelial cell
line HMEC-1 and on the non-tumor cell line HEK-293 has been determined. The urea
derivatives were also evaluated for their antiangiogenic properties, whereby their ability
to inhibit tubulogenesis and kinase activity employing flow cytometry, ELISA,
immunofluorescence and western blot techniques was measured. In addition, these
techniques were also employed to investigate the immunomodulator action of the
synthetic compounds on the inhibition of PD-L1 and c-Myc proteins. Compound T.2, 1-
(3-chlorophenyl)-3-(2-(4-(4-methoxybenzyl)-1H-1,2,3-triazol-1-yl)ethyl)urea,
has
shown similar results to sorafenib in both down-regulation of VEGFR-2 and inhibition
of the kinase activity of this receptor. Furthermore, compound T.14, (E)-1-(4-
chlorophenyl)-3-(3-(4-methoxystyryl)phenyl)urea, improves the effect of T.2 as regards
tube formation of endothelial cells and inhibition of VEGFR-2 tyrosine kinase activity.
In addition, T.14 improves the effect of the experimental drug BMS-8 in the inhibition
of PD-L1 and c-Myc proteins.

ACCEPTED MANUSCRIPT
Novel multitarget inhibitors with antiangiogenic and
immunomodulator properties
a
a
a
a
Laura Conesa-Milián, Eva Falomir, * Juan Murga, * Miguel Carda, J. Alberto
b
Marco
a
Depart. de Q. Inorgánica y Orgánica, Univ. Jaume I, E-12071 Castellón, Spain
b
Depart. de Q. Orgánica, Univ. de Valencia, E-46100 Burjassot, Valencia, Spain
*
Corresponding authors: efalomir@uji.es, jmurga@uji.es.
ABSTRACT
By means of docking studies, seventeen compounds T.1-T17 have been designed
and evaluated as multitarget inhibitors of VEGFR-2 and PD-L1 proteins in order to
overcome resistance phenomena offered by cancer. All these designed molecules
display a urea moiety as a common structural feature and eight of them (T.1-T8) further
contain a 1,2,3-triazol moiety. The antiproliferative activity of these molecules on
several tumor cell lines (HT-29, MCF-7, HeLa, A549, HL-60), on the endothelial cell
line HMEC-1 and on the non-tumor cell line HEK-293 has been determined. The urea
derivatives were also evaluated for their antiangiogenic properties, whereby their ability
to inhibit tubulogenesis and kinase activity employing flow cytometry, ELISA,
immunofluorescence and western blot techniques was measured. In addition, these
techniques were also employed to investigate the immunomodulator action of the
synthetic compounds on the inhibition of PD-L1 and c-Myc proteins. Compound T.2, 1-
(3-chlorophenyl)-3-(2-(4-(4-methoxybenzyl)-1H-1,2,3-triazol-1-yl)ethyl)urea,
has
shown similar results to sorafenib in both down-regulation of VEGFR-2 and inhibition
of the kinase activity of this receptor. Furthermore, compound T.14, (E)-1-(4-
chlorophenyl)-3-(3-(4-methoxystyryl)phenyl)urea, improves the effect of T.2 as regards
tube formation of endothelial cells and inhibition of VEGFR-2 tyrosine kinase activity.
In addition, T.14 improves the effect of the experimental drug BMS-8 in the inhibition
of PD-L1 and c-Myc proteins.
KEYWORDS
Angiogenesis, VEGFR-2, immunotherapy, PD-L1, ureas, docking.

ACCEPTED MANUSCRIPT
1
. Introduction
Cancer is a complex pathological process that encompasses a large group of diseases.
There is a need therefore to find new drugs able to interfere with most of these
processes in order to increase treatment options and to avoid resistance mechanisms.[1]
The goal of the present work is the design, synthesis and biological evaluation of
molecules with anticancer activity deemed to address two different biological targets of
particular relevance in the cancer process. These targets are the tyrosin kinase VEGFR-
2
(Vascular Endothelial Growth Factor Receptor 2) and the PD-L1 protein
(Programmed Death-Ligand 1).
The first target is related to the angiogenesis process, the growth of new blood
vessels from pre-existing vasculature, which is a critical step in tumor progression.[2]
Vascular endothelial growth factors (VEGFs) regulate angiogenesis by binding to their
associated receptors. Therapeutic inhibition of VEGFR-2 action is now having an
impact in the clinical use for the treatment of a number of diseases.[3] The second target
is related to the immunosuppressive ability of tumor cells. These overexpress negative
immunologic regulators and generate a protected environment. The PD-L1 protein,
which is overexpressed in cancer cells, plays a key role in the deregulation of the
immune system through formation of PD-1/PD-L1 complex.[4] The present research
intends to design compounds with the ability to bind to PD-L1 present in tumor cells,
therefore blocking their ability to evade the immune system. Antibodies targeting the
PD-1/PD-L1 immune checkpoint have achieved outstanding success in recent years.
Clinically available examples include nivolumab and pidilizumab. However, their high
immunogenicity and low stability have led to the research for new non-peptidic
molecules.[5]
It has been reported that the c-Myc oncogene has a direct role in preventing immune
cells from efficiently attacking tumor cells. Indeed, c-Myc fosters tumor growth by
increasing the levels of two immune checkpoint proteins, CD47 and PD-L1, which help
thwart the host immune response.[6] Thus, c-Myc inactivation in tumors appears to
engage the immune system to elicit cellular senescence in tumor cells and to collapse
the vascular endothelial cells.[7]

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Sorafenib (Figure 1) is an inhibitor of the kinase domain of VEGFR-2. It bears a
N,N´-diarylurea fragment that has shown, both through X-ray diffraction studies [8] and
through computational studies [9], to be determinant in its interaction with the kinase
domain. Docking studies have revealed that sorafenib can form 5 hydrogen bonds with
the kinase domain of VEGFR-2: two with Glu885, two weak links with Cys919 and one
with Asp1046. The other interactions with the kinase domain are of the hydrophobic
type with the pocket formed by the amino acids Leu840, Val848, Ala866, Ile888,
Leu889, Val899, Phe918, Thr916 and Leu1035. It has also been established that the
strongest link is formed with the Asp1046.
Brystol-Myers Squibb reported in 2015 the discovery of the first non-peptidic
molecules able to inhibit the formation of the PD-1/PD-L1 complex [10]. The mode of
interaction of these compounds, indicated as BMS-8, BMS-37, BMS-202 and BMS-242
in Figure 1, was established in 2016 [11]. Using differential scanning fluorimetry (DSF)
techniques, it was determined that these compounds induce thermal stabilization of PD-
L1. Finally, the protein bound to the substrates was crystallized and the binding sites
determined. Thus, these compounds are linked in a hydrophobic groove formed by the
amino acids Tyr56, Met115, Ile116, Ala121 and Tyr123 and promote the dimerization
of PD-L1 protein. Thus, it is possible to inhibit the formation of the PD-1/PD-L1
complex by means of a double pathway: the inhibitors occupy part of the area involved
in the PD-1/PD-L1 interaction and, in addition, when the dimer is formed between two
PD-L1 molecules, one of them has the opposite orientation to the one necessary to
interact with PD-1. Consequently, the interaction between PD-1 and PD-L1 is disabled.
Figure 1. Structure of sorafenib and non-peptidic inhibitors of PD-L1.

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Both the binding site of sorafenib in the kinase domain of VEGFR-2 and the binding
site in PD-L1 possess a hydrophobic groove in which aromatic rings can be inserted.
Our goal is the design of compounds able to interact with both binding sites and act as
multitarget inhibitors.
2
. Results and discussions
2
.1. Preliminary docking study
We have carried out a docking study using AutoDock 4.2 software [12] on the kinase
domain of VEGFR-2 and on the site identified in PD-L1 in order to locate relatively
simple structures that are able to interact with both sites. Several general structures
capable of establishing at least three of the hydrogen bonds shown by sorafenib (see
Figure 2) and of interacting with the binding site in PD-L1 have been identified by us.
These structures are characterized by having a urea system (which is essential to allow
its interaction with the kinase domain) to which aromatic rings are bound in order to
interact with the hydrophobic grooves present in both proteins.
Figure 2. Identified general structures able to interact with the kinase domain of
VEGFR-2 and PD-L1. The hydrogen bonds that can be formed with VEGFR-2 are
indicated in red. The zones of the molecules that interact with the hydrophobic pockets
of both binding sites are also indicated.
As an example, the docking obtained for three of the analyzed structures is shown in
Figure 3. It can be appreciated in Figure 3A that compounds D.1, D.2 and D.3 occupy
the binding site in the kinase domain of VEGFR-2 in much the same way as sorafenib
does. These compounds establish hydrogen bonds between the OMe group and Cys919,
between the two NH groups of the urea system and Glu885 and between the O of urea
and Asp1046. The three aromatic rings interact with two hydrophobic zones present in
the binding site. It can be seen in Figure 3B that structures D.1, D.2 and D.3 also
occupy the binding site of BMS-202 in PD-L1 protein. The aromatic rings are inserted
in a hydrophobic groove with the A rings establishing interactions with Tyr56, Me115

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and Ala121, whereas ring B interacts with Tyr123. The more polar urea system points
towards the outer zone of the groove.
H
H
H
H
O
N
N
N
N
N
N
N
O
N
N
O
H
H
MeO
MeO
D.1
D.3
D.2
MeO
Figure 3. A) Superposition of D.1 (orange), D.2 (light blue), D.3 (green) and sorafenib
grey) in the kinase domain of VEGFR-2. B). Superposition of D.1 (orange), D.2 (light
(
blue), D.3 (green) and BMS-202 (violet) at the PD-L1 binding site.
With the objective to verify these preliminary results obtained from the in-silico
study, we envisaged the synthesis and the biological evaluation of several compounds
derived from the general structures D.4-D.7 shown in Figure 4. Additionally,
differential scanning fluorometry and free protein detection by ELISA were performed
prior to the development of the corresponding cellular assays in order to verify the
interaction between the targets of study and the designed compounds (see Supporting
Information S2 and S3).
H
N
H
N
N
MeO
O
R
N N
O
MeO
N
N
D.4
D.5
H
H
R
O
O
N
N
N
N
R
H
H
H
H
R
MeO
MeO
D.6
D.7
R = H, Cl, Br, Me
Figure 4. Potential multitarget inhibitors of VEFGR-2 and PD-L1.

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2
.2.Synthetic work of triazolyl-ureas T.1-T.8
To begin with, we decided to synthesize triazoles with the general structure D.4 due
to its easy synthetic accessibility. These compounds were obtained in only two synthetic
steps from 2-azidoethan-1-amine 1 as indicated in Scheme 1 [13]. Thus, treatment of
azidoamine 1 with carbonyl diimidazole in the presence of triethylamine generated the
corresponding imidazolide which was then allowed to react with a range of anilines to
afford azido-ureas 2-9. These were then converted into triazolyl-ureas T.1-T8 upon
copper-catalyzed 1,3-dipolar cycloaddition [14] with 1-methoxy-4-(prop-2-yn-1-
yl)benzene (for the preparation of this compound see the Experimental section).
H
H
N
N
O
N
a
R
N3
N3
b
R
NH2
N
N
N N
O
H
H
MeO
1
2
3
4
5
6
7
8
9
(R = H)
T.1 (R = H)
(R = m-Cl)
(R = p-Cl)
(R = m-Br)
(R = p-Br)
(R = o-Me)
(R = m-Me)
(R = p-Me)
T.2 (R = m-Cl)
T.3 (R = p-Cl)
T.4 (R = m-Br)
T.5 (R = p-Br)
T.6 (R = o-Me)
T.7 (R = m-Me)
T.8 (R = p-Me)
Scheme 1
Reagents and conditions: (a) CDI, Et N, DMF, 3 h, rt, then the appropriate aniline,
3
5
0ºC, overnight; (b) 1-methoxy-4-(prop-2-yn-1-yl)benzene, CuSO ·5H O, sodium
4 2
ascorbate, DMF/H O (9/1), 60ºC, 2 h.
2
Unfortunately, all attempts to prepare the ortho-halogenated isomers failed due
probably to the steric hindrance caused by the halogen in ortho position.
2
.3.Biological evaluation of triazolyl-ureas T.1-T.8
2
.3.1. Cell proliferation inhibition
The ability of triazolyl-ureas T.1-T.8 to inhibit cell proliferation was established by
means of their IC50 values towards the human tumor cell lines HT-29 (colon
adenocarcinoma), MCF-7 (breast adenocarcinoma), HeLa (epithelioid cervix
carcinoma) and A549 (pulmonary adenocarcinoma), as well as towards the endothelial
cell line HMEC-1 (human microvascular endothelial cells) and the non-tumor cell line
HEK-293 (human embryonic kidney cells). All the IC values observed were higher
5
0
than 200 µM in all the cell lines tested and are not listed here.

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2
.3.2. Effect on cellular VEGFR-2
The effect of compounds T.1-T.8 on VEGFR-2 in HMEC-1 cell line was determined
by flow cytometry technique. For this assay, cells were incubated for 24 hours in the
presence of the corresponding compounds at 100 µM concentration. Then, cells were
fixed with formaldehyde and treated with anti-VEGFR-2-alexafluor®647 to quantify
membrane VEGFR-2 by flow cytometry. On the other hand, permeabilization of cells
with Triton X-100 prior to fixation step, allowed the quantification of total VEGFR-2 in
cells. Table 1 shows the effect of the selected derivatives on VEGFR-2 expression and
distribution in HMEC-1 endothelial cell line, referred to control (DMSO, 100%).
Table 1. Effect on VEGFR-2 in HMEC-1 cells.
Membrane
VEGFR-2 (%)
Total
VEGFR-2 (%)
Comp.
Sorafenib
T.1
46 ± 8
97 ± 14
74 ± 6
90 ± 4
79 ± 10
40 ± 10
79 ± 9
76 ± 4
97 ± 15
64 ± 4
105 ± 10
81 ± 14
102 ± 5
78 ± 1
T.2
T.3
T.4
T.5
55 ± 5
T.6
100 ± 11
91 ± 9
T.7
T.8
90 ± 7
Data are the average (±SD) of three experiments.
Results from Table 1 indicate that compound T.5 (p-Br) is the derivative that shows
the strongest interaction with VEGFR-2 in HMEC-1 cells, improving the effect exerted
by sorafenib. Some of the other compounds (T.2, T.4, T.6, T.7) were also able to down-
regulate the expression of VEGFR-2 but to a lesser extent.
Table 2 depicts the effect of T.2, T.4 and T.5 on VEGFR-2 expression and
distribution in A549 tumor cell line at 100 µM concentration, referred to control
(DMSO, 100%).
Table 2. Effect on VEGFR-2 in A549 cells.
Membrane
VEGFR-2 (%)
Total
VEGFR-2 (%)
Comp.
Sorafenib
Sunitinib
T.2
92 ± 3
66 ± 8
95 ± 3
85 ± 5
135 ± 14
86 ± 6

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T.4
T.5
98 ± 6
70 ± 4
80 ± 8
61 ± 11
Data are the average (±SD) of three experiments.
Results from Table 2 indicate again that T.5 (p-Br) is the derivative that shows the
strongest interaction with VEGFR-2 in A549 cells improving the effect exerted by the
reference compound.
2
.3.3. Inhibition of VEGFR-2 kinase activity
The interaction VEGF/VEGFR-2 causes phosphorylation of the receptor and triggers
the signaling cascade that promotes phosphorylation of Erk1/2 (extracellular signal-
regulated kinase) and, subsequently, activation of the angiogenesis process [15].
In view of the fact that triazolyl-ureas T.1-T.8 interact with VEGFR-2 on endothelial
cells, their capacity to inhibit kinase activity was also studied. Thus, HMEC-1 cells
were treated with the derivatives for 24 h at 100 µM, then the cells were lysed and
phospho-VEGFR-2 was quantified by ELISA analysis. Western blot analysis of the
compounds that were active in inhibiting phospho-VEGFR-2 allowed the relative
quantification of phospho-Erk1/2. Table 3 shows the percentage of p-VEGFR-2 and p-
Erk for each compound referred to control (DMSO, 100%).
Table 3. p-VEGFR-2 and p-Erk1/2 detected in HMEC-1 cells.
HMEC-1
p-VEGFR-2 (%) p-Erk1/2 (%)
HMEC-1
Comp.
Sorafenib
T.1
77 ± 9
100 ± 12
70 ± 16
86 ± 3
>100
80 ± 8
-
T.2
38 ± 14
T.3
72 ± 6
T.4
-
T.5
>100
-
T.6
83 ± 4
90 ± 10
>100
82 ± 11
T.7
-
T.8
-
Data are the average (±SD) of three experiments.

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Figure 5. Representative Western blot of p-Erk1/2 detection in HMEC-1 cells. β-actin
was used as internal control.
Table 3 shows that the best inhibitory kinase activity in endothelial cells is shown by
T.2 (m-chloro), T.3 (p-chloro) and T.6 (o-methyl) which are able to reduce the
phosphorylation of VEGFR-2 at levels similar to sorafenib. Interestingly, there is an
important inhibition of Erk1/2 phosphorylation by T.2, improving the results obtained
with sorafenib.
On the other hand, inhibition of VEGFR-2 kinase activity in A549 cells was also
tested but in this case, none of the selected derivatives improved the effect observed for
sorafenib. Table 4 shows the percentage of p-VEGFR-2 and p-Erk for each compound
referred to control (DMSO, 100%).
Table 4. p-VEGFR-2 and p-Erk1/2 detected in A549 cells.
A549
p-VEGFR-2 (%)
A549
p-Erk1/2 (%)
Comp.
Sorafenib
T.2
84 ± 4
50 ± 7
99 ± 9
78 ± 7
119 ± 8
90 ± 13
121 ± 11
125 ± 12
121 ± 16
112 ± 10
T.3
T.6
T.7
Data are the average (±SD) of three experiments.
Figure 6. Representative Western blot of p-Erk1/2 detection in A549 cells. β-actin was
used as internal control.
2
.3.4. Tube formation inhibition on endothelial cells
Antiangiogenic agents mainly affect endothelial cells. Thus, the capacity of synthetic
compounds to inhibit the formation of new vasculature network formed by HMEC-1

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was evaluated. Therefore, cells were seeded on top of Matrigel® and simultaneously
treated with different concentrations of derivatives. Pictures were taken 24 h later in
order to evaluate the tube formation inhibition effect. Table 5 shows the minimum
concentration at which compounds are active in the tube formation inhibition. Only T.2,
T.3 and T.6 were tested, since they showed the highest activities in the inhibition of
VEGFR-2 kinase activity (see Table 3).
Table 5. Inhibition effect of tube formation.
Minimum
Comp.
active conc. (µM)
Sunitinib
Sorafenib
T.2
3
10
50
T.3
400
200
T.6
Data are representative of three experiments.
Data from Table 5 confirm that triazolyl-ureas are able to inhibit tube formation but
at higher doses than sunitinib and sorafenib, with T.2 (m-chloro) being the most potent
compound.
2
.3.5. Effect on PD-L1 and c-Myc proteins
In order to evaluate the immunomodulator properties of the synthetic derivatives, the
effect on PD-L1 and c-Myc proteins by the selected compounds was studied. Thus,
compounds were added to A549 cells at 100 µM concentration and, after 24 h, cells
were lysed and ELISA analysis was performed to determine the relative amount of both
proteins compared to DMSO treated cells. Table 6 shows the percentage of free PD-L1
detected for each compound referred to control (DMSO). The percentage of c-Myc has
been quantified only for the compounds which exerted a similar or better action than the
reference compound BMS-8 in the inhibition of PD-L1.
Table 6. PD-L1 and c-Myc detection in A549 cells.
Comp.
BMS-8
T.1
% PD-L1
68 ± 5
% c-Myc
60 ± 7
-
83 ± 9
T.2
70 ± 8
60 ± 8
73 ± 5
-
T.3
69 ± 10
80 ± 12
T.4

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T.5
T.6
T.7
T.8
>100
82 ± 20
90 ± 20
66 ± 6
-
-
-
70 ± 8
Data are the average (±SD) of three experiments.
From Table 6 it can be concluded that compounds T.2 (m-chloro), T.3 (p-chloro) and
T.8 (p-methyl) inhibit PD-L1 and c-Myc proteins in a similar way to BMS-8.
2
.3.6. Cell proliferation evaluation in co-cultures
Some tumor cell lines such as A549 and HT-29 show high PD-L1
overexpression.[16] Therefore, cell proliferation of these has been studied in presence
of PD-1 expressing Jurkat T cells in order to evaluate whether the observed cell
proliferation inhibition is due to the blockage of PD-1/PD-L1 system. Compounds
which showed good PD-L1 inhibition in ELISA test were evaluated. Thus, tumor cells
were treated for 24 h with the selected compounds at 200 µM in presence of Jurkat T
cells and then, living cells were counted using trypan blue and a Neubauer chamber.
Figure 7 shows the inhibition of tumour cell proliferation exhibited by the derivatives
due to the presence of Jurkat T cells.
Inhibition of cell proliferation (%)
8
4
BMS-8 T.2 T.3 T.8
7
6
5
5
5
3
4
9
2
2
1
6
6
A549
HT-29
Figure 7. Inhibition of cell proliferation (%) in presence of Jurkat T cells. Data are
representative of three experiments.
From data provided in Figure 7, it can be concluded that the tested compounds are
able to inhibit A549 cell proliferation in co-culture but do no improve the effect exerted
by BMS-8. Interestingly, in the HT-29 cell line this effect is much higher for

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compounds T.2 (m-chloro) and T.3 (p-chloro), improving the effect exhibited by BMS-
8
.
2
.4. Synthesis of ureas T.9-T.17
The triazolyl-ureas that contain a chlorine atom in their structure are the compounds
that display the best activities in our biological tests and these were very similar to the
ones exhibited by our reference compounds sorafenib and BMS-8. Therefore, we
decided to prepare new compounds with general structures D.5-D.7 (Fig. 4) containing
a chlorine atom in the phenyl ring. The synthesis of these compounds is depicted in
Scheme 2. Thus, Wittig reaction of phosphonium salt 11 with 4-methoxybenzaldehyde
afforded 1-(4-methoxystyryl)-3-nitrobenzene 12 as a mixture of E/Z isomers. Reduction
of this mixture gave rise to a mixture of E/Z anilines which was separated by column
chromatography to afford (Z)-3-(4-methoxystyryl)aniline 13 and (E)-3-(4-
methoxystyryl)aniline 14. On the other hand, hydrogenation of 12 led to 3-(4-
methoxyphenethyl)aniline 15. Compounds 13, 14 and 15 were converted into
carbamates 16, 17 and 18 [17], which upon reaction with chloroanilines gave rise to
ureas T.9-T.17.
Scheme 2

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Reagents and conditions: (a) PPh , CH Cl , 3 h, rt., 71%; (b) 4-methoxybenzaldehyde,
3
2
2
K CO , 18-crown-6, CH Cl , reflux, overnight, 73%; (c) Zn, AcOH, 1 h, rt., (Z/E 6:4);
2
3
2
2
(
d) H , Pd/C, EtOAc, 2 h, rt., 66%; (e) phenyl chloroformate, pyridine, THF, 0ºC 30
2
min, then 1 h rt.; (f) the appropriate chloroaniline, Et N, THF, 40-50ºC.
3
2
.5. Biological evaluation of ureas T.9-T.17
2
.5.1. Cell proliferation inhibition
The ability of ureas T.9-T.17 to inhibit cell proliferation was established by means of
their IC values towards the human tumor cell lines HT-29 (colon adenocarcinoma),
5
0
MCF-7 (breast adenocarcinoma), HeLa (epithelioid cervix carcinoma) and A549
(
pulmonary adenocarcinoma), as well as towards the endothelial cell line HMEC-1
human microvascular endothelial cells) and the non-tumor cell line HEK-293 (human
(
embryonic kidney cells). The IC values for ureas T.9-T.17 are presented in Table 7
5
0
along with IC values for the reference compounds sunitinib, sorafenib and BMS-8.
5
0
a
Table 7. IC values (µM) for sunitinib, sorafenib, BMS-8 and derivatives T.9-T.17.
5
0
Comp.
Sunitinib
Sorafenib
BMS-8
T.9
HT-29
MCF-7
0.08 ± 0.02
14 ± 4
HeLa
7.8 ± 1.0
6.1 ± 0.4
>100
A549
11 ± 2
27 ± 2
6 ± 1
HMEC-1
7 ± 4
HEK-293
5.0 ± 0.8
5.0 ± 0.7
60 ± 10
6.5 ± 0.2
5 ± 2
3.8 ± 0.5
17 ± 4
34 ± 3
19 ± 2
20 ± 3
-
12 ± 4
19 ± 2
22 ± 3
21 ± 9
38 ± 3
29 ± 3
61 ± 3
7.7 ± 0.2
1.5 ± 0.9
24 ± 5
>200
33.0 ± 0.6
32.9 ± 0.5
33 ± 3
>100
T.10
20 ± 10
33 ± 5
37.2 ± 0.8
17 ± 3
10.5 ± 0.6
8 ± 1
T.11
20 ± 10
57 ± 3
T.12
47 ± 4
>100
>100
T.13
3.5 ± 0.6
4.3 ± 0.7
0.40 ± 0.09
>100
11.3 ± 0.7
0.7 ± 0.3
69 ± 5
2 ± 1
2.4 ± 0.6
1.0 ± 0.4
90 ± 10
5.3 ± 0.9
1.8 ± 0.7
T.14
0.52 ± 0.09
20 ± 3
1.8 ± 0.8
48 ± 7
50 ± 20
>200
T.15
T.16
14 ± 4
8.2 ± 0.6
15.3 ± 0.4
16.5 ± 0.7
21 ± 1
T.17
1.5 ± 0.1
>200
a
IC50 values are expressed as the compound concentration that inhibits the cell growth by 50%. Data are the average
(±SD) of three experiments.
The synthetic ureas show antiproliferative activity in the low micromolar range in all
tested cell lines, comparable to that shown by reference compounds sunitinib, sorafenib
and BMS-8. The (E)-p-chloro derivative T.14 was the most active one with IC values
5
0
at submicromolar level, and exhibited lower IC values in cancer cell lines than in non-
5
0

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tumour endothelial ones. This fact allowed us to study the biological activity of this
compound at a concentration that does not affect non-tumour cell lines.
2
.5.2. Induction of apoptosis
Apoptosis represents a universal and exquisitely efficient cellular suicide pathway
18]. This phenomenon can be triggered by intrinsic signals such as genotoxic stress, or
[
extrinsic signals such as the binding of ligands to cell surface death receptors [19].
Induction of apoptosis was studied by measuring the translocation of phosphatidylserine
from the cytoplasmic to the extracellular side of the plasma membrane. Thus, A549
cells were incubated for 24 h in the presence of compounds at 100 µM, after which
annexin-V binding was measured by flow cytometry. The apoptotic effect of
compounds T.9-T.17 is indicated in Figure 8.
9
5
Apoptotic cells (%)
7
5
76
6
9
6
5
6
1
5
6
4
8
4
8
4
2
2
0
1
7
C
Suni. Sora. T.9
T.10 T.11 T.12 T.13 T.14 T.15 T.16 T.17
Figure 8. Apoptotic effect (%) in A549 cells. Data are representative of three experiments.
All the tested compounds induce apoptosis at 100 µM, with the exception of
compound T.10, which exhibited an activity similar to that of control (DMSO).
Moreover, compounds T.11, T.13, T.15, T.16 and T.17 improve the effect depicted by
sunitinib and sorafenib. BMS-8 and T.1-T.8 derivatives were also tested but they did
not exhibit any effect in the induction of apoptosis.
2
.5.3. Effect on cellular VEGFR-2
The effect of ureas T.9-T.17 on VEGFR-2 in A549 tumor cell line was determined
by both flow cytometry and immunofluorescence techniques. For these assays, cells

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were incubated for 24 hours in the presence of the corresponding compounds at 10 µM
concentration. Table 8 and Figure 7 show the results obtained for each compound on
free VEGFR-2 presence and distribution in A549 cell line, referred to control (DMSO).
Table 8. Effect on VEGFR-2 expression in A549 cells.
Membrane
VEGFR-2 (%)
Total
VEGFR-2 (%)
Comp.
Sorafenib
Sunitinib
T.9
92 ± 3
66 ± 8
85 ± 5
135 ± 14
177 ± 9
186 ± 10
100 ± 7
42 ± 9
114 ± 20
102 ± 10
118 ± 3
109 ± 18
52 ± 10
56 ± 11
100 ± 19
97 ± 10
97 ± 11
T.10
T.11
T.12
T.13
32 ± 4
T.14
10 ± 2
T.15
100 ± 16
98 ± 3
T.16
T.17
96 ± 7
Data are the average (±SD) of three experiments.
(E) derivatives T.13 (m-chloro) and T.14 (p-chloro) were the best ones in reducing
the presence of VEGFR-2, therefore improving the effect exerted by sorafenib. Indeed,
they were able to down-regulate VEGFR-2 to the half of the control inhibiting
internalization of the target and reducing the total level of the protein to 10-30 % of the
control.
Immunofluorescence assay correlated well with flow cytometry results. Figure 9
shows that both compounds T.13 (B) and T.14 (C) caused a significant reduction of
membrane VEGFR-2 and compound T.14 exerted a higher inhibition of the
internalization of the protein to the nucleus.

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Figure 9. Effect on VEGFR-2 expression in A549 cells (A) DMSO, (B) T.13 and (C)
T.14.
The effect of (E) derivatives on VEGFR-2 presence and distribution in endothelial
cells was also evaluated (see Table 9), referred to control (DMSO).
Table 9. Effect on VEGFR-2 expression in HMEC-1 cells.
Membrane
VEGFR-2 (%)
Total
VEGFR-2 (%)
Comp.
Sorafenib
T.12
46 ± 8
76 ± 9
55 ± 8
56 ± 8
64 ± 4
121 ± 8
106 ± 16
109 ± 17
T.13
T.14
Data are the average (±SD) of three experiments.
Results from Table 9 indicate that the (E) derivatives did not inhibit internalization of
VEGFR-2 in HMEC-1 cells but reduced the presence of the target in the membrane.
Again, T.13 and T.14 exhibited the highest activities, similar to sorafenib.
2
.5.4. Inhibition of VEGFR-2 kinase activity
The effect on kinase activity of VEGFR-2 exerted by the (E) derivatives was studied
by western blot on two cell lines, A549 and HMEC-1. In this case, cells were treated for
2
4 h at 10 µM concentration of the corresponding compounds. Table 10 shows the
relative amount of p-VEGFR-2 and p-Erk1/2 detected on A549 when treated with each
compound referred to control (DMSO).
Table 10. p-VEGFR-2 and p-Erk1/2 detected in A549 cells.
A549
p-VEGFR-2 (%)
A549
p-Erk1/2 (%)
Comp.
Sorafenib
T.12
84 ± 4
76 ± 3
75 ± 5
82 ± 3
50 ± 7
100 ± 18
56 ± 12
66 ± 8
T.13
T.14
Data are the average (±SD) of three experiments.

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Figure 10. Representative Western blot of p-Erk1/2 detection in A549 cells. β-actin was
used as internal control.
Table 10 shows that T.13 and T.14 reduce VEGFR-2 and Erk1/2 phosphorylation in
the tumor cell line in a similar way to sorafenib. However, no effect on the kinase
activity of VEGFR-2 on the endothelial cell line HMEC-1 was observed for any of the
tested compounds.
2
.5.5. Tube formation inhibition on endothelial cells
The capacity of ureas T.9-T.17 to inhibit the formation of new vasculature network
formed by HMEC-1 was evaluated. Table 11 shows the minimum concentration at
which compounds are active and begin to inhibit the microtube formation.
Table 11. Inhibition effect of tube formation.
Minimum
Comp.
active conc. (µM)
Sunitinib
Sorafenib
T.9
3
10
10
10
1
T.10
T.11
T.12
10
0.1
0.1
1
T.13
T.14
T.15
T.16
10
T.17
100
Data are representative of three experiments.
A comparison of the minimum active concentration values to IC values for HMEC-
5
0
1
cell line (see Table 7) shows that there is a correlation between antiproliferative
activity and tube formation inhibition capacity, as compounds with lower IC values
5
0
exhibit microtube inhibition activity at lower concentrations. Moreover, it is observed

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that some of the tested compounds are more active than sunitinib and sorafenib,
particularly (E) derivatives T.13 (m-chloro) and T.14 (p-chloro), which are 100-fold
more active than sorafenib. Pictures for the inhibition of neovascularization achieved by
compound T.14, at different concentrations, are displayed in Figure 11.
Figure 11. Effect of T.14 at 1, 0.1 and 0.01 µM concentrations (from left to right).
2
.5.6. Effect on PD-L1 and c-Myc proteins
We also evaluated the immunomodulator properties of these derivatives in the same
way as described above for the triazole derivatives. Thus, the effect on PD-L1 and c-
Myc proteins by the selected compounds was studied by ELISA on A549 cells. After 24
h treatment at 100 µM concentration of compounds, cells were lysed and ELISA
analysis was performed to determine the relative amount of both proteins compared to
DMSO treated cells. Table 12 shows the percentage of free PD-L1 detected for each
compound referred to control (DMSO). Again, the percentage of c-Myc has been
quantified only for the compounds which exerted similar or better action than the
reference compound BMS-8 in the inhibition of PD-L1.
Table 12. PD-L1 and c-Myc proteins detection in A549 cells.
a
Comp.
BMS-8
T.9
% PD-L1
68 ± 5
83 ± 3
92 ± 10
75 ± 6
74 ± 7
49 ± 8
25 ± 9
61 ± 6
% c-Myc
60 ± 7
-
T.10
T.11
T.12
T.13
T.14
T.15
-
-
-
65 ± 12
37 ± 5
63 ± 11

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T.16
T.17
51 ± 9
38 ± 3
51 ± 10
44 ± 6
a
T.9-T.14 were tested at 15 µM. BMS-8 and T.15-T.17 were tested at 100 µM. Data are the average
(
±SD) of three experiments.
From Table 12 it can be deduced that compounds T.13, T.14, T.16 and T.17 are able
to inhibit PD-L1 and c-Myc proteins and display a stronger effect than that exerted by
BMS-8, with T.14 (p-chloro) being the most active in both targets.
Subsequently, immunofluorescence was performed for compound T.14. For this
assay, cells were incubated for 24 hours at 100 µM of treatment. Then, cells were fixed,
permeabilized and treated with anti-PD-L1-alexafluor®647 and anti-c-Myc-FITC. The
pictures shown in Figure 12 correlate well with the results obtained by ELISA test
(Table 12), since compound T.14 (C) inhibits PD-L1 and c-Myc proteins in a higher
proportion than BMS-8 (B).
Figure 12. Effect on PD-L1 (red) and c-Myc (green) in A549 cells. (A) DMSO, (B)
BMS-8, (C) T.14.
2
.5.7. Cell proliferation evaluation in co-cultures
We also studied the effect of compounds T.13, T.14 and T.17, which showed good
PD-L1 inhibition, in affecting tumor cell proliferation in the presence of PD-1
expressing Jurkat T-cells. Thus, A549 were treated for 24 h with the selected
compounds at 200 µM in presence of Jurkat T cells. Then, living cells were counted
using trypan blue and a Neubauer chamber. Figure 13 shows the inhibition of tumor cell
proliferation exhibited by T.13, T.14 and T.17 due to the presence of Jurkat T cells.

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Cell proliferation inhibition (%)
BMS-8
T.13
T.14
T.17
8
6
7
6
5
5
1
6
A549
Figure 13. Inhibition of cell proliferation (%) in presence of Jurkat T cells. Data are
representative of three experiments.
Data provided in Figure 13 shows that T.13 and T.14 were able to inhibit A549 cell
proliferation in co-culture improving the effect exerted by BMS-8. Therefore, it can be
affirmed that these derivatives act by means of disturbing PD-1/PD-L1 interactions.
3
. Summary and conclusions
The biological study of triazolyl ureas T.1-T.8 has revealed that derivatives with
chlorine substitution in meta or para positions of the benzene ring are the most active as
antiangiogenic and immunomodulator agents. Indeed, T.2 (m-Cl) has proven very
effective in both down-regulation of VEGFR-2 and inhibition of the kinase activity of
this receptor, better results being observed than with the reference compounds.
Regarding the immunomodulator properties, T.2 has been the most effective triazolyl
urea in inhibiting cell proliferation in co-cultures, since due to its interaction with PD-
L1 it has increased its antiproliferative effect in almost a 50%.
In search of higher activities than those observed with the triazolyl ureas, we
synthetized a second group of ureas (T.9-T.17) that lacked this structural fragment.
These compounds have shown antiproliferative and apoptotic activity at lower doses
than the triazolyl derivatives. In general, derivatives with (E) configuration in the
stilbene subunit have been the most active ones improving the biological results
obtained for the reference compounds. More specifically, T.13 (m-Cl) and T.14 (p-Cl)

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inhibit tube formation of endothelial cells at 0.1 µM and show inhibition of VEGFR-2
tyrosine kinase activity at lower concentrations than the triazolyl ureas and the reference
compound BMS-8. As regards immunomodulator effect, they are able to improve the
effect of BMS-8 in the inhibition of PD-L1 and c-Myc proteins. T.14 has been the most
active stilbene urea in inhibiting cell proliferation in co-cultures, with an increase in
antiproliferative effect of more than 80%.
In summary, compounds T.2 (triazole, m-Cl), T.13 (E-stilbene, m-Cl) and T.14 (E-
stilbene, p-Cl) show the best antiangiogenic and immunomodulator properties for every
group of synthetized compounds. This has let us to conclude that the presence of a
chlorophenyl urea unit is important for the dual action of these kind of derivatives. Even
so, a comparison of the results obtained for the triazolyl ureas with those achieved with
the stilbene ureas has led us to conclude that the presence of the triazole ring in these
aryl urea derivatives does not improve the biological activity when compared to the
reference compounds. However, the substitution of the triazole ring for the E-stilbene
unit leads to the enhancement of the expected dual biological activity. To the best of our
knowledge, this is the first example of the design and synthesis of compounds with both
anti-PD-L1 and anti-angiogenic effect, so that a new door is opened to new kind of
promising targeted anticancer agents.
4
. Experimental
4
.1. Docking studies
Molecular docking was performed using Autodock 4.2 [12]. The crystal structures of
the VEGFR-2 kinase domain (PDB ID: 3EWH) and PD-L1 (PDB ID: 4ZQK) were used
as templates. Ligands were previously removed from the protein structure in order to
perform docking simulation. Discovery Studio Visualizer program [20] was used to
build the structures of ureas T.1-T.17. The cluster was compared on the basis of the free
energy of binding. The Lamarckian genetic algorithm (LGA) was employed with the
default parameters; g_eval was set to 2500000 (medium) and 100 LGA runs were
conducted. Molecular graphics were done with PYMOL (PyMOL Molecular Graphics
System, version 1.6).

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4
4
.2. Chemistry
.2.1. General procedures
NMR spectra were measured at 25°C. The signals of the deuterated solvent (CDCl3
1
3
or DMSO) were taken as the reference. Multiplicity assignments of C signals were
1
made by means of the DEPT pulse sequence. Complete signal assignments in H and
1
3
C NMR spectra were made with the aid of 2D homo- and heteronuclear pulse
sequences (COSY, HSQC, HMBC). High resolution mass spectra were run by the
electrospray mode (ESMS). IR data were measured with oily films on NaCl plates (oils)
and are given only for relevant functional groups (C=O, NH). Experiments which
required an inert atmosphere were carried out under dry N in flame-dried glassware.
2
Commercially available reagents were used as received.
4
.2.2. Experimental procedure for the synthesis of 1-methoxy-4-(prop-2-yn-1-yl)benzene
4-methoxyphenyl)methanol (1 eq) was dissolved in CH Cl (1.5 mL/mmol) and
(
2
2
cooled at 0ºC. Then, PBr (1 eq) was slowly added and the mixture was stirred at 0ºC
3
under inert atmosphere. After three hours, the mixture was poured over a saturated
aqueous solution of NaHCO and extracted with CH Cl . The organic solvent was
3
2
2
evaporated under reduced pressure and the crude residue was purified by means of flash
silica gel chromatography (hexane) to afford 1-(bromomethyl)-4-methoxybenzene. The
latter compound and CuCl (0.5 eq) were added to an ethynylmagnesium bromide (2 eq)
solution 0.5 M in THF. The mixture was stirred at reflux (70ºC) for 2 h. The reaction
mixture was then cooled and poured over a saturated aqueous solution of NH Cl,
4
followed by extraction (3 times) with Et O. The organic phase was washed with H O
2
2
and brine and dried over anhydrous MgSO . After evaporation of all volatiles under
4
reduced pressure, the crude residue was purified by flash silica gel chromatography
(hexane:EtOAc mixtures as eluent) to afford 1-methoxy-4-(prop-2-yn-1-yl)benzene as a
yellowish oil (57%).
4
.2.3. Experimental procedure for the synthesis of azido-ureas 2-9.
A solution of 2-azidoethan-1-amine 1 (1 eq) in DMF was treated with Et N (2 eq
).
The
3
solution was stirred for 10 min at rt and then CDI (2 eq) was added. The resulting mixture
was stirred for 20 min at rt Then, the corresponding aniline (2 eq) was added to the
.
reaction mixture and this was stirred overnight at 50ºC. After this time, the solvent of
the reaction mixture was evaporated under reduced pressure and the crude was purified

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by flash silica gel chromatography (hexane:EtOAc mixtures as eluent) to afford the
desired products 2-9.
4
.2.4. Experimental procedure for the synthesis of triazolyl-ureas T.1-T.8.
A solution of 1-methoxy-4-(prop-2-yn-1-yl)benzene (1 eq) in DMF was treated with the
corresponding previously prepared azido-ureas (1.2 eq). Then, a mixture of
CuSO ·5H O (0.1 eq) and sodium ascorbate (0.1 eq) in DMF/H O (9:1) was added to
4
2
2
the reaction mixture which was stirred for 2 h at 60ºC. After that time, the mixture was
concentrated, redissolved in EtOAc, and washed with brine repeated times. Finally, the
organic solvent evaporated under reduced pressure and the crude was purified by flash
silica gel (hexane:EtOAc mixtures as eluent) to afford the desired products. Detailed
analytical data ate given in the Supplementary Material.

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4
.2.5. Experimental procedure for the synthesis of phosphonium salt 11.
A solution of 3-nitrobenzyl bromide D10 (4.63 mmol) and triphenylphosphine (4.63
mmol) in dry CH Cl (20 mL) was stirred for 3 h at rt. After the reaction was complete,
2
2
the white precipitate was filtered and sequentially washed with CH Cl and hexane
2
2
affording 11 as a white solid (1.56 g, 71%, m. p. 273ºC).
.2.6. Experimental procedure for Wittig reaction (Scheme 2, b).
-methoxybenzaldehyde (3.26 mmol) was dissolved in CH Cl (20 mL) and then, the
4
4
2
2
phosphonium salt 11 (3.26 mmol), K CO (3.6 mmol) and 18-crown-6 (0.6 mmol) were
2
3
added to the solution. The mixture was refluxed overnight and then was filtered and
concentrated affording 810 mg (73% yield) of compound 12 as an E/Z stereoisomeric
mixture.
4
.2.7. Experimental procedure for nitro group reduction (Scheme 2, c).
Zn (0.6 mol) was added to a solution of compound 12 (4.08 mmol) in AcOH (133
mL). The resulting mixture was vigorously stirred at rt for 1 h in a flask that was
protected from the light. Then, the solids were filtered over Celite to remove the Zn, and
were thoroughly washed with EtOAc. The filtrate was neutralized with saturated
aqueous NaHCO and the organic phase was separated and dried over MgSO
3
4
anhydride. After filtration and solvent evaporation, silica gel column chromatography
hexane-EtOAc mixtures as eluent) was used to isolate the stereoisomers 13 and 14 (Z/E
(
6
:4).
4
.2.8. Experimental procedure for hydrogenation (Scheme 2, d).
Compound 12 (2.35 mmol) was dissolved in EtOAc (30 ml) and stirred with 10 % Pd/C
300 mg) for 2 h at rt and ambient pressure under a hydrogen atmosphere. The resulting
(
mixture was filtered through a pad of Celite and the filtrate was evaporated under
reduced pressure. Then, the residue was purified by silica gel column chromatography
(hexane-EtOAc mixtures as eluent) to give compound 15 as a brownish solid (66%, m.
p. 81-85ºC).
4
.2.9. Experimental procedure for the synthesis of carbamates 16-18.
A solution of 13, 14 or 15 (1 eq.) in THF (5 mL/mmol) was cooled at 0ºC and
anhydrous pyridine (2.4 eq.) and phenyl chloroformate (1.4 eq.) was added under inert
atmosphere. The resulting mixture was stirred in the dark for 20 min at 0ºC and then for

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1
h at rt. After this time, H O (5 mL/mmol) and HCl 1 M (2.5 mL/mmol) were added to
2
the reaction mixture, which was then extracted with CH Cl (3 x 20 mL). The organic
2
2
layer was washed with brine, and then dried on anhydrous Na SO . Removal of
2
4
volatiles under reduced pressure afforded an oily residue which was subjected to
column chromatography on silica-gel (hexane-EtOAc mixtures as eluent) to yield the
desired products 16-18.
4
.2.10. Experimental procedure for the synthesis of ureas T.9-T.17.
A solution of the corresponding chloroaniline (0.5 mmol) in dry THF (4 mL/mmol)
was treated with Et N (5.4 mmol) under inert atmosphere. After stirring the mixture for
3
5
min, the corresponding previously prepared carbamate (0.5 mmol) was added
dropwise as a solution in THF (10 mL/mmol). The resulting mixture was then stirred in
the dark at 40-50 ºC for 24-72 h (TLC monitoring). After this time, CH Cl (15 mL) and
2
2
HCl 1M were added to the reaction mixture, which was then extracted with CH Cl (2 x
2
2
1
0 mL). The organic layer was washed with brine and then dried on anhydrous Na SO .
2 4
Removal of volatiles under reduced pressure afforded an oily residue which was
subjected to column chromatography on silica-gel (hexane-EtOAc mixtures as eluent) to
afford the desired products. Detailed analytical data ate given in the Supplementary
Material.
4
4
.3. Biological studies
.3.1. Cell culture
Cell culture media were purchased from Gibco (Grand Island, NY). Fetal bovine
serum (FBS) was obtained from Harlan-Seralab (Belton, U.K.). Supplements and other
chemicals not listed in this section were obtained from Sigma Chemical Co. (St. Louis,
MO). Plastics for cell culture were supplied by Thermo Scientific BioLite. All tested
compounds were dissolved in DMSO at a concentration of 10 mM and stored at −20°C
until use.
HT-29, MCF-7, HeLa, A549 and HEK-293 cell lines were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) containing glucose (1 g/L), glutamine (2 mM),
penicillin (50 µg/mL), streptomycin (50 µg/mL), and amphotericin B (1.25 µg/mL),
supplemented with 10% FBS. HMEC-1 cell line was maintained in Dulbecco’s
modified Eagle’s medium (DMEM)/Low glucose containing glutamine (2 mM),
penicillin (50 µg/mL), streptomycin (50 µg/mL), and amphotericin B (1.25 µg/mL),

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supplemented with 10% FBS. For the development of tube formation assays in
Matrigel, HMEC-1 cells were cultured in EGM-2MV Medium supplemented with
EGM-2MV SingleQuots.
4
.3.2. Cell proliferation assay
3
3
In 96-well plates, 3 × 10 (HeLa, A549, HMEC-1, HEK-293) or 5 × 10 (HT-29,
MCF-7) cells per well were incubated with serial dilutions of the tested compounds in a
total volume of 100 µL of their respective growth media. The 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co.) dye reduction assay
in 96-well microplates was used, as previously described [21]. After 2 days of
incubation (37 °C, 5% CO in a humid atmosphere), 10 µL of MTT (5 mg/mL in
2
phosphate-buffered saline, PBS) was added to each well, and the plate was incubated
for a further 3 h (37 °C). After that, the supernatant was discarded and replaced by 100
µL of DMSO to dissolve formazan crystals. The absorbance was then read at 540 nm by
spectrophotometry. For all concentrations of compound, cell viability was expressed as
the percentage of the ratio between the mean absorbance of treated cells and the mean
absorbance of untreated cells. Three independent experiments were performed, and the
IC50 values (i.e., concentration half inhibiting cell proliferation) were graphically
determined using GraphPad Prism 4 software.
4
.3.3. Apoptosis assay
Apoptosis was determined by quantifying FITC-Annexin V translocation by means
of flow cytometry. A549 cells were incubated with compounds for 24 h and then stained
TM
following instructions of BD Apoptosis Detection Kit. Analysis was performed with
TM
a BD Accuri C6 flow cytometer.
4
.3.4. VEGFR-2 quantification by flow cytometry
VEGFR-2 was determined quantifying Alexa Fluor® 647 Mouse Anti-Human
CD309 (VEGFR-2) by means of flow cytometry. To detect membrane VEGFR-2, cells
were incubated with compounds for 24 h and then they were collected, fixed and stained
with Alexa Fluor® 647 Mouse Anti-Human CD309 (VEGFR-2). For the detection of
total VEGFR-2 (membrane and cytosolic), cells were incubated with compounds for 24
h, then lysates were obtained and stained with Alexa Fluor® 647 Mouse Anti-Human
CD309 (VEGFR-2).

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4
.3.5. Total VEGFR-2 determination by immunofluorescence
A549 cells were plated on cover glass and incubated with the different compounds
for 24 h. Then, they were permeabilized with PEM-Triton-X-100 0.5% and fixed in
.7% formaldehyde. Direct immunostaining was carried out for 2 h at 37°C in darkness
3
with Alexa Fluor® 647 Mouse Anti-Human CD309 (VEGFR-2). Then, cells were
washed in PBS and cover glasses were mounted with 10 µL of Glycine/Glycerol buffer.
Images were obtained by a confocal laser scanning microscope (CLSM) Leica SP5 with
a Leica inverted microscope, equipped with a Plan-Apochromat 63x oil immersion
objective (NA=1.4). Each image was recorded with the CLSM’s spectral mode selecting
specific domains of the emission spectrum. Alexa Fluor®647 fluorophore was excited
at 650 nm with an argon laser and its fluorescence emission was collected at 665 nm.
4
.3.6. phospho-VEGFR-2 quantification by ELISA
5
For endothelial cells (HMEC-1): 5 x·10 cells/well were seeded in 6-well plates and
once they were at 80% of their confluency, they were starved with medium containing
.1% of FBS for 24 h. Then, cells were incubated with the corresponding compounds
for 24 h and next cells were stimulated with 100 ng/ml of Recombinant VEGF-165 for
0
3
0 min at 37ºC. After that, lysates were collected, protein quantification was carried out
®
by Bradford test and, then, and phospho-VEGFR-2 was quantified using PathScan
Phospho-VEGFR-2(Tyr1175) Sandwich ELISA Kit according to the manufacturer’s
5
instructions. For tumor cells (A549): 3·x 10 cells/well were seeded in 6-well plates
and were incubated with the corresponding compounds at 10 µM for 24 h. After that,
lysates were collected, protein quantification was carried out by Bradford test and, then,
®
phospho-VEGFR-2 was quantified using PathScan Phospho-VEGFR-2(Tyr1175)
Sandwich ELISA Kit according to the manufacturer’s instructions.
4
.3.7. p-Erk quantification by western blot assay
From the lysates extracted in the previous experiment (5.3.6), 100 µg of proteins
were subjected to gel electrophoresis using Bolt 4-12% Bis-Tris plus gels. Then,
proteins were transferred to Hybond-P polyvinylidene difluoride (PVDF) membranes
using the iBlot gel transfer system, and the resulting membranes were incubated first for
1
h at rt in blocking buffer (5% non-fat dry milk in TBS 1x containing 0.1% Tween)
and subsequently overnight at 4 ºC in TBST buffer primary antibody solution (Anti-
ERK1+ERK2 phosphoT202+T204). After washing, membranes were incubated with