W.-Q. Liu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4254–4259
4255
structural modifications in the benzimidazole and/or the phenolic
and/or the benzodioxane moieties, are synthesized using
S
O
a
convergent procedure (Scheme 1). On one hand, the reductive
condensation of substituted o-phenylenediamine (X = NH2) with
4-methyl-3-nitrobenzaldehyde or 3-nitrobenzaldehyde in pres-
ence of sodium metabisulfite in refluxing DMF afforded an amino-
phenylbenzimidazole (2) with a 50–65% yield.26–28 Interestingly,
following the same synthetic route, benzothiazole is obtained
using 2-aminobenzenethiol (X = SH) as the starting material with
a 50% yield.28 On the other hand, different acyl isothiocyanates
(3) are quantitatively prepared from corresponding carboxylic
acids in two steps through acyl chloride and subsequent thiocya-
nate substitution.29 Lastly, the nucleophilic addition of aminophe-
nylbenzimidazoles (2) to acyl isothiocyanates (3) in refluxing dry
acetone leads to compound 1 and its derivatives 4a–n with an
average 45% yield (scheme 1).29
N
O
O
N
H
N
H
NH
Compound 1
Chart 1. Structure of compound 1.
respectively KI = 1.2
l
M
and IC50 = 8
lM
(measured with
125I-VEGF-A165 and porcine aortic endothelial cells expressing
NRP-1). In addition, EG00229 reduces the viability of human lung
carcinoma A549 cells and strengthens the potency of paclitaxel.20
Nevertheless, pseudo-peptidic compounds rarely provide pharma-
cological agents, and therefore we focused our efforts on the iden-
tification of new VEGF-A165/NRP-1 non-peptidic antagonists.
By an in silico/in vitro screening procedure, our team reported
recently the very first and original fully non-peptidic VEGF-A165/NRP
antagonist (compound 1, chart 1).21 In vitro, this ‘hit’ bound
NRP-1 and NRP-2 with IC50 in the micromolar range and exhibited
anti-angiogenic and pro-apoptotic activities in cellular assays.
Moreover, in vivo studies on MDA-MB-231 NOG-xenografted mice
pointed out that compound 1 increased animal survival and
reduced cell tumor growth. Its in vivo anti-angiogenic and
pro-apoptotic potencies were also demonstrated by CD34, CD31
and Ki-67 immunostaining.21
The newly synthesized antagonists were firstly evaluated on
endothelial cells (HUVEC) and on breast invasive tumor cells
(MDA-MB-231). Cell adhesion was evaluated by a colorimetric test
using cristal violet adhesion,30 while cytotoxicity assays (cell via-
bility) were based on tetrazolium salt cleavage by viable cells.31
They were performed after three days of cell incubation at 37 °C
with various concentrations of each antagonist. The results are
given as IC50 values and are reported in Table 1. In a second step,
eight molecules were evaluated as antagonists of the biotinylated
(bt)-VEGF-A165/NRP-1 and/or (bt)-VEGF-A165/VEGF-R1 binding as
control at the unique concentration of 10 l
M by a binding assay.32
The percentage of displaced (bt)-VEGF-A165 by the compounds are
The structure of compound 1 might be divided into four sec-
tions: a benzimidazole core connected to a methylbenzene linked
to a benzodioxane motif through an unusual carboxythiourea
spacer. Taking advantage of the known X-ray structure of tuftsin
bound to NRP-1, compound 1 was docked in the NRP-1 extracellu-
lar b1 coagulation factor domain, and in the tuftsin arginine bind-
ing pocket.22,23 This docking, performed using ICM-version-3.424
suggested that compound 1 acts as a staple and pointed out that
the benzimidazole core mimics an arginine guanidinium, in order
to respect the N-terminal arginine rule.21 We next performed
preliminary energy-minimization studies using the Accelrys soft-
ware25 and the CFF91 force-field. Two interesting energy-minima
candidates were found.
In the first minimized case (Fig. 1A), the benzimidazole is
stacked in parallel between Tyr-353 and Tyr-297 and in a perpen-
dicular fashion to Trp-301. Its N–H is engaged in an H-bond with
the Asp-320 carboxylate. The carboxythiourea entity binds to
Lys-351 through H-bonds involving both its carbonyl and thiocar-
bonyl groups. The Tyr-353 hydroxyl proton appears to interact
with the electron-rich ring of the benzene of benzodioxane, while
the hydroxyl proton of Tyr-297 appears to interact with the elec-
tron-rich methylbenzene ring. The dioxane ring is partially stacked
over the methyl group of Thr-413. The benzene ring of benzodiox-
ane is stacked perpendicular to methylbenzene and benzimidazole.
The overall conformation of the hit is stabilized by a hammer-like
stacking interaction of the benzene ring of benzimidazole over the
aromatic ring of the methylbenzene.
shown in Table 2.
Cellular assays demonstrate a good correlation between results
obtained on cell adhesion and cytotoxicity. In terms of selectivity,
the newly synthesized molecules seem to be more potent against
HUVEC than against MDA-MB-231 breast cancer cells (e.g., 4d
reduced viability/proliferation of HUVEC twenty times higher than
the same assay results obtained for MDA-MB-231). Moreover, 4a,
4c and 4d are more selective than compound 1 in the cytotoxic
assay for HUVEC, which has a stronger ability for angiogenesis,
compared to MDA-MB-231 cells.
Compounds 1, 4a, 4c,d show sub-micromolar cellular activities
on HUVEC, and antagonize at 10 lM significantly the (bt)-VEGF-
A165 binding to NRP-1. These results confirm that the removal of
the methyl group of the benzene (4a, 4d) and the substitution of
the dioxane ring by a dioxolane ring are not detrimental in terms
of NRP-1-b1 binding (4a and 4d respective binding inhibition at
10 lM are 38 2% and 46 3% compared to 34 3% for 1).
Moreover, 4a and 4d show a rather good selectivity for NRP-1,
since they are respectively three and seven times more potent to
antagonize (bt)-VEGF-A165/NRP-1 than the (bt)-VEGF-A165/VEGF-
R1 interaction (shown in Table 2).
Conversely, compounds in which the benzimidazole ring was
switched into a benzothiazole exhibited some lower cellular activ-
ity and also lower receptor binding affinity (4m). Thus, the most
potent benzothiazole derivative, compound 4m, showed three-fold
lower activity against HUVEC proliferation when compared to 4d
(IC50 = 0.9 0.03 lM) and it partly antagonized (bt)-VEGF-A165
In the second minimized case (Fig. 1B), the benzimidazole core is
again stacked in a parallel fashion between Tyr-297 and Tyr-353 and
is perpendicular to Trp-301. Asp-320 now interacts with the partly
acidic C–H hydrogen of the benzimidazole benzene instead of the
benzimidazole N–H. The thiocarbonyl sulfur accepts a proton from
the N–H indole of Trp-301, while the carbonyl oxygen is involved
in an intramolecular H-bond with the thiocarbonyl N–H group.
In order to select one of these two structure–activity
relationship hypotheses, we report here synthesis and biological
evaluations of new antagonists structurally related to compound
1. Thus, compound 1 and its derivatives 4a–n, which encompass
binding to NRP-1 (14 1%). According to the docking hypothesis,21
the benzimidazole ring is deeply inserted into the arginine binding
pocket and mimics the arginine guanidinium motif in order to
fulfill the NRPs binding C-end rule.33 These results demonstrated
the structural relevance of the benzimidazole nitrogen H-bond
donor, missing in the benzothiazole ring. This appeared to be very
detrimental in terms of receptor binding. Consequently, it might be
suggested that the first energy-minimization model (Fig. 1A) might
be more accurate than the second (Fig. 1B).
Finally, the introduction of a substituent in the benzimidazole
ring (even a fluorine, 4f, yet a small atom known to increase global
Liu, Wang-Qing
