Antitumor properties of substituted (αE)-α-(1H-indol-3- ylmethylene)benzeneacetic acids or amides

Article abstract of DOI:10.1016/j.bmc.2013.06.030

A novel class of indole derivatives characterized by a (αE)-α- (1H-indol-3-ylmethylene)benzeneacetic acid or amide scaffold was synthesized. These derivatives, assayed for cell-growth inhibition activity against a panel of six different tumor cell lines, showed strong antiproliferative activity and selectivity mainly towards DU145 cell line. In particular, compounds 2d-m and 5 stand out for their cell growth inhibitory activity and, among them, compound 2d emerged for its selectivity towards DU145 with respect to other tested tumor cell lines. DU145 treated with 1 μM of 2d for 72 h showed p21^Cip1 induction and suppression of Akt signaling together with induction of Rb. From a computational point of view, two different approaches were used in order to study topology and electronic properties of the novel compounds and to shed light on their drug-likeness properties. Firstly, topological and electronic features of the compounds endowed with the most relevant biological activity were deepened; in parallel, some ADME properties like solubility and permeability were predicted.

Full text of DOI:10.1016/j.bmc.2013.06.030

Bioorganic & Medicinal Chemistry 21 (2013) 5233–5245
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antitumor properties of substituted (
ene)benzeneacetic acids or amides
aE)-a-(1H-indol-3-ylmethyl-
Giuseppe Forte a, Cosimo Gianluca Fortuna b, Loredana Salerno a, Maria N. Modica a, Maria A. Siracusa a,
Venera Cardile c, Giuseppe Romeo a, Alessandra Bulbarelli d, Elena Lonati d, Valeria Pittalà a,
⇑
a Dipartimento di Scienze del Farmaco, Università degli Studi di Catania, viale A. Doria 6, 95125 Catania, Italy
b Dipartimento di Scienze Chimiche, Università di Catania, viale A. Doria 6, 95125 Catania, Italy
c Dipartimento di Scienze Bio-Mediche Sezione di Fisiologia, Università degli Studi di Catania, viale A. Doria 6, 95125 Catania, Italy
d Dipartimento di Scienze della Salute, Università degli Studi di Milano Bicocca, Via Cadore 48, 20900 Monza, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of indole derivatives characterized by a (
a
E)-
a
-(1H-indol-3-ylmethylene)benzeneacetic acid
Received 11 January 2013
Revised 17 May 2013
Accepted 12 June 2013
Available online 27 June 2013
or amide scaffold was synthesized. These derivatives, assayed for cell-growth inhibition activity against a
panel of six different tumor cell lines, showed strong antiproliferative activity and selectivity mainly
towards DU145 cell line. In particular, compounds 2d–m and 5 stand out for their cell growth inhibitory
activity and, among them, compound 2d emerged for its selectivity towards DU145 with respect to other
tested tumor cell lines. DU145 treated with 1 l
M of 2d for 72 h showed p21Cip1 induction and suppres-
Keywords:
Prostate cancer
Indoles
Perkin–Oglialoro reaction
Retinoblastoma protein
p21Cip1
sion of Akt signaling together with induction of Rb. From a computational point of view, two different
approaches were used in order to study topology and electronic properties of the novel compounds
and to shed light on their drug-likeness properties. Firstly, topological and electronic features of the com-
pounds endowed with the most relevant biological activity were deepened; in parallel, some ADME prop-
erties like solubility and permeability were predicted.
DU145 cell line
DFT
Ó 2013 Elsevier Ltd. All rights reserved.
VolSurf+
1. Introduction
irreversible damage to both normal and tumor cells with signifi-
cant toxicity and side effects. Thereby, compounds able to specifi-
It is well known that cell proliferation and progression through
the cell cycle are governed by a series of checkpoint controls,
otherwise referred to as restriction points which are regulated by
different families of enzymes.1,2 Transition from normal to pre-
cancer and cancer cells is a result of multi-step accumulation of ge-
netic and epigenetic modifications that disrupt these orderly pro-
cesses changing its programming regulation. Mutations or
deregulation of genes responsible for cell cycle, cellular growth
and differentiation, such as growth factors, receptor or cytoplas-
matic tyrosine kinases, serine/threonine kinases, regulatory
GTPases, and transcription factors play a chief role in tumor devel-
opment.1–5 Oncogenes encoded by mutated genes do not possess
important regulatory elements and their production and activation
is uncontrolled. Thereby, it becomes clear that mechanisms that
trigger the process of carcinogenesis are many and complex. Sev-
eral cytotoxic drugs (e.g. doxorubicin and camptotecin) result to
an indirect, a specific block of the cell cycle and thus produce an
cally target tumor cells, leading to tumor cell arrest or apoptosis,
with comparable efficacy but reduced toxicity would be desirable.
However, in spite of our growing knowledge of the molecular biol-
ogy of cancer, the success rate of new therapies directly targeting
tumor-causing proteins remains unsatisfactory with few excep-
tions (e.g. the use of imatinib in chronic myelogenous leukemia).6
One major obstacle is that the complexity and redundancy of signal
transduction pathways have only been partially elucidated; sec-
ondly, high heterogeneity of tumor cells and high heterogeneity
of cancers make unlikely that hitting a single target will result in
therapeutically useful effects. Thus, it appears clear the needs for
anticancer drugs with novel structures which, even if the mecha-
nism of action is not completely elucidated, can effectively block
the uncontrolled cell proliferation under tumor diseases.7–9
As a part of our work towards the development of novel agents
endowed with antitumor activity,10–13 we previously reported the
synthesis of 1H-pyrrolo[2,3-b]pyridine derivatives (Fig. 1, general
formula 1a) and their use as therapeutic agents in the treatment
of cancer and cell proliferative disorders.14 We now report the
synthesis, characterization, and in vitro antiproliferative activ-
⇑
Corresponding author. Tel.: +39 095 7384269; fax: +39 095 222239.
E-mail address: vpittala@unict.it (V. Pittalà).
ity against
a panel of six human tumor cell lines of new
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.030

5234
G. Forte et al. / Bioorg. Med. Chem. 21 (2013) 5233–5245
O
Furthermore, by means of a first principle approach, based on
R3
R2
R1
R2
the Density Functional Theory (DFT), we studied topology and elec-
tronic properties, in terms of atomic charge values, of the com-
pounds with relevant biological activity in order to localize the
most reactive region of the molecules which will be candidate to
interact with the biological substrate. The contribute due to intra-
molecular H-bond was also evaluated. Finally, some efforts have
been made to elucidate the effect of lead compound on different
intracellular pathways.
R
R
N
N
H
N
R3
R1
1b
1a
Figure 1. General chemical structure of 1a and 1b.
These computational and biological studies were performed to
deepen the knowledge of the physico-chemical properties of the
class in order to support, in the future, a more focused expansion
of these derivatives and, hopefully, to discern their mechanism of
action.
indole-derivatives differently substituted (Fig. 1, general formula
1b). These classes of compounds, 1a and 1b, contain an a–b unsat-
urated ketone function which behaves as Michael reaction accep-
tors able to react with thiol groups present in proteins and
glutathione. Interestingly, and perhaps not merely by coincidence,
compounds with this structure are widely distributed in nature
and have been reported to display a wide variety of pharmacolog-
ical properties, for example, anticancer, antioxidant, antimutagenic
activities, etc. Thus the inclusion of this substructure in our mole-
cules can be regarded as a privileged structure able to recognize
common key features present in biological systems.
2. Chemistry
Novel derivatives 2c–m, 3c–d, 4a–d and 5 were synthesized as
reported in Scheme 1 and the synthetic route follows a strat-
egy similar to the one used for the preparation of compounds 2a,
2b, 3a, and 3b previously reported.16 Briefly, the first step of
the synthetic pathway was focused on the preparation of
Moreover, it was performed a computational study predicting
some of their physico-chemical properties,15 like adsorption, dis-
tribution, metabolism, excretion (ADME) properties, for verifying
their applicability as potential drugs.
(aE)-a-[1-acetyl-5-(phenylmethoxy)-1H-indol-3-ylmethylene]-
benzeneacetic acids (2c–m), following the Perkin-Oglialoro reac-
tion. It is known from literature data16 that using this synthetic
method, the main and usually the sole product has the (E)-config-
O
OH
O
H
O
OH
a
R
R
+
R1
R1
N
H
N
O
b
2a-m
2a R = H, R1 = H
O
2b
R = H, R1 = 4-CH3O
OH
O
2c R = PhCH2O, R1 = H
NH2
2d
R = PhCH2O, R1 = 4-CH3O
2e R = PhCH2O, R1 = 3-CH3O
2f R = PhCH2O, R1 = 2-CH3O
R
c
R
R1
2g
R = PhCH2O, R1 = 3,4-OCH2O-
N
H
R1
2h R = PhCH2O, R1 = 3,4-CH3O
N
H
2i
R = PhCH2O, R1 = 3,4,5-CH3O
2k R = PhCH2O, R1 = 3,5-CH3O
2j R = PhCH2O, R1 = 2,5-CH3O
4a-d
3a-d
R = H, R1 = H
2l
R = PhCH2O, R1 = PhCH2O
3a
4a
R = H, R1 = H
2m R = PhCH2O, R1 = 4-Cl
3b R = H, R1 = 4-CH3O
4b R = H, R1 = 4-CH3O
4c
3c
3d
R = PhCH2O, R1 = H
R = PhCH2O, R1 = H
4d R = PhCH2O, R1 = 4-CH3O
R = PhCH2O, R1 = 4-CH3O
Cl
d
O
O
OH
O
N
O
5
Scheme 1. Reagents and conditions: (a) TEA, acetic anhydride, 130 °C, 6 h, then H2O/EtOH/THF (1:1:1); (b) NaOH 2 N, EtOH/THF (2:1), 70 °C, 1 h; (c) EDAC, HOBtꢀNH3, TEA,
DMF dry, 22 °C, 24 h; (d) NaH 95%, DMF dry, 22 °C, 24 h.

G. Forte et al. / Bioorg. Med. Chem. 21 (2013) 5233–5245
5235
Table 1
In vitro activity of substituted (
aE)-
a
-(1H-indol-3-ylmethylene)benzeneacetic acids or amides 2c–m, 3a–d, 4a–d, and 5
O
R2
R
N
R3
R1
Compd
R
R1
R2
R3
IC50 (lM)
DU145
LnCap
KB
A549
M14
Caco2
2c
2d
2e
2f
2g
2h
2i
2k
2j
2l
2m
3a
3b
3c
3d
4a
4b
4c
4d
5
PhCH2O
PhCH2O
PhCH2O
PhCH2O
PhCH2O
PhCH2O
PhCH2O
PhCH2O
PhCH2O
PhCH2O
PhCH2O
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
NH2
NH2
NH2
NH2
OH
COCH3
COCH3
COCH3
COCH3
COCH3
COCH3
COCH3
COCH3
COCH3
COCH3
COCH3
H
H
H
H
H
26.7
0.91
1.08
1.01
1.15
1.05
0.95
0.88
0.83
1.41
1.13
>100
>100
34.7
38.1
33.3
40.0
30.9
39.8
0.86
>100
85.0
65.1
48.5
99.1
>100
59.6
26.1
44.1
77.7
>100
NT
NT
NT
NT
NT
NT
NT
NT
38.2
55.5
>100
>100
>100
>100
96.7
>100
30.4
>100
43.0
>100
>100
>100
55.5
38.5
>100
86.2
40.3
>100
97.0
57.5
>100
45.2
57.2
53.6
54.8
89.1
26.0
54.5
44.1
52.3
NT
NT
NT
NT
NT
NT
NT
NT
1.0
>100
>100
>100
>100
>100
65.5
57.1
94.0
87.1
58.2
>100
>100
>100
95.2
58.4
45.4
53.0
>100
>100
64.5
NT
NT
NT
NT
NT
NT
NT
NT
4-CH3O
3-CH3O
2-CH3O
3,4-OCH2O-
3,4-CH3O
3,4,5-CH3O
3,5-CH3O
2,5-CH3O
4-PhCH2O
4-Cl
NT
NT
NT
H
>100
>100
41.6
>100
>100
>100
>100
>100
NT
H
4-CH3O
H
4-CH3O
H
4-CH3O
H
4-CH3O
4-CH3O
PhCH2O
PhCH2O
H
H
H
H
H
PhCH2O
PhCH2O
PhCH2O
(4-CH3OPh)CH2
uration, as desired. Thus, on these bases, we assume an (E)-config-
uration for the new synthesized compounds.
Hydrolysis of the acetamide group in compounds 2a–d was
achieved by heating at 70 °C for 2 h in the presence of 6 equiv of
NaOH 2 N and afforded derivatives 3a–d.17
The synthesis of the primary amides 4a–d was achieved
by treating the appropriate carboxylic acid (3a–d) with
1-hydroxybenzotriazole ammonium salt in the presence of
N0-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine (EDAC)
and triethylamine (TEA).
that almost all the novel synthesized compounds exerted mild
cell growth inhibitory activity against all tested cell lines with
the exception of the androgen nonresponsive DU145 human pros-
tate cancer cells on which title compounds showed very interest-
ing results in term of IC50 and selectivity. As a general trend,
derivatives bearing a phenylmethoxy substituent at the 5-posi-
tion of the indole ring, namely 2c–m, 3c–d, 4c–d, and 5, afforded
the most interesting results when compared with unsubstituted
derivatives 3a–b and 4a–b. Among the subclass of 5-phenylmeth-
oxysubstituted compounds, derivatives bearing an acetyl or a
4-methoxyphenylmethyl residue at the indole nitrogen, namely
2c–m and 5, exhibited a better anti-proliferative activity (com-
pare 2c–m and 5 versus 3c–d and 4c–d). Moreover, results in Ta-
ble 1 suggested that an amide function is preferable to an acidic
function for derivatives unsubstituted at the indole ring (4a–b vs
3a–b), while IC50 values are comparable when it is present a phe-
nylmethoxy residue (4c–d vs 3c–d). From IC50 values reported in
Table 1 against tested tumor cell lines, compound 2d stands out
for its remarkable selectivity towards DU145, thus, it was selected
as lead compound. These results are interesting because prostate
cancer is the most common malignancy in men in a number of
countries.18,19 The incidence of prostate cancer has increased fol-
lowing the aging of population worldwide and, presently, conven-
tional therapies such as surgery and hormone treatment
frequently fail to achieve satisfactory effects.20 The chance to de-
velop new and more effective drugs for the treatment of prostate
cancer remain an open opportunity and, in this light, we tried to
elucidate the effect of lead compound 2d on different intracellular
pathways.
The alkylation of the indole nitrogen was performed, in high
yield, using NaH 95% in dry DMF and 4-methoxyphenylmethyl
chloride giving the N-[(4-methoxyphenyl)methyl]derivative 5.
The presence of a free carboxylic acid residue did not interfere in
this reaction step.
All the synthesized products were characterized by 1H NMR
spectra, IR, and elemental analysis and data were consistent with
the proposed structures.
3. Biology
These novel synthesized compounds were assayed for cell-
growth inhibition activity at the concentrations of 1, 25, 50, and
100 lM for 72 h towards DU145 (human androgen-insensitive
prostate cancer cells), LNCaP (human androgen-responsive pros-
tate cancer cells), A549 (human bronchoalveolar carcinoma-de-
rived cells), KB (human epidermoid cells), M14 (human
melanoma cells), Caco2 (human intestinal epithelial cancer cells),
and nontumorigenic human fibroblasts. Interestingly, for the lat-
ter, the values of percent viability of treated samples versus un-
treated controls were from 85% to 100%, indicating that new
derivatives are not cytotoxic against normal cells. Data evaluation
are summarized in Table 1 and are expressed as the concentra-
tion at which the tested compound reduces cell survival to 50%
of the control value (IC50). Data reported in Table 1 pointed out
The serine/threonine protein kinase B (Akt) plays an important
role in survival and proliferation of prostate cancer cells21 and the
extracellular signal-regulated protein kinases (Erk1,2) are critical
regulators of growth and cell survival.22 Hence, we investigated
whether these cell signalings may play a role in survival and pro-
liferation reduction observed in DU145 treated with 1 lM of 2d

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G. Forte et al. / Bioorg. Med. Chem. 21 (2013) 5233–5245
µM
p-Akt
Akt
0
1
p-Gsk3β
Gsk3β
160
140
120
*
p-Erk
Erk
p-Akt
100
**
p-Gsk3
p-Erk 1,2
p21
80
60
40
20
0
*
p21
β-actin
0
1
2d
M
Figure 2. Effects of 2d compound on cell survival and cell growth. DU145 cells incubated with 1
lM 2d compound for 72 h were collected and lysed with 2% SDS lysis buffer.
Equal amounts of samples (as protein content) were subjected to SDS–PAGE and western blot analysis. Phospho-AktSer473, Akt, phospho-GSK3b Ser9, GSK3b, phospho-Erk1/
2Thr202/Tyr204, Erk1/2, p21, and b-actin levels were detected by specific antibodies, revealed by enhanced chemiluminescence (ECL) and semi-quantitatively estimated by
Image Quant LAS 4000. Results obtained are shown on the bar-graphs. Anti-b-actin antibody was employed to confirm equal protein loading in the different lanes. Data
represent the mean SEM of three separate experiments. Statistical significance is obtained with Student’s t test in comparison with controls ⁄p < 0.05, ⁄⁄p < 0.01.
for 72 h. Western blot analysis showed that levels of active phos-
phorylated-AktSer473 and -Erk1,2Thr202/Tyr204 forms decreased in re-
sponse to 2d treatment about 20% and 40%, respectively. No effects
were observed on steady-state levels of total Akt and Erk1,2 pro-
teins (Fig. 2) as well as no significant change in Gsk3b phosphory-
lation state were observed after treatment given that 2d seems to
induce the decrease of Gsk3b protein level with respect to control
(Fig. 2).
tion during S phase and G2/M transition.26 Phosphorylation modu-
lates the activity of Rb that results inactive when phosphorylated.
Here we show that, after 2d compound treatment, the ratio of p-Rb
(inactive)/Rb (active) forms decreases with respect to the control
(Fig. 3) suggesting a potential involvement of Akt/ p21Cip1 pathway
and suggesting an interesting hypothesis deserving future
experiments.
Finally, to analyze the effect of a sub lethal dose of 2d com-
pound, the same experiments were also performed treating cells
with 0.1 lM concentration, but no significant alterations were de-
Moreover, in order to investigate 2d compound effects on cell
cycle, we assessed expression of p21Cip1 and cyclin-dependent ki-
nase inhibitors that can complex with a variety of cyclins leading
tected in the analyzed pathways (data not shown).
to cell cycle arrest. As shown in Fig. 2, treatment with 1 lM of
2d resulted in p21Cip1 protein expression significant increase.
Taken together these data seems to be of interest since the
reduction of Akt and Erk1,2 activity might influence the FOXO tran-
scriptional activity,23 involved in regulation of p21Cip1
expression.24
4. Computational studies
4.1. DFT calculations
Among the synthesized derivatives, seven compounds, namely
2d–f, 2i–j and 5, were selected on the basis of their interesting
IC50 values against DU145 cell line and ten structures for each of
them were sampled by means of Molecular Dynamics (MD). These
structures were subsequently, fully optimized in DFT approach, by
using the Conductor-Like Polarizable Continuum Model (CPCM).
Moreover, since p21Cip1 is responsible for inactivating the reti-
noblastoma protein (Rb),25 some samples of DU145 cells were as-
sayed for Rb (Fig. 3). The retinoblastoma protein (Rb) pathway
represents a key component in the regulation of the cell cycle
and tumor suppression possessing important roles in DNA replica-
120
μM
0
1
100
80
60
40
20
0
p-Rb
Rb
**
α-tubulin
0
1
2d µM
Figure 3. Effects of 2d compound on Rb phosphorylation. DU145 cells incubated with 1
amounts of samples (as protein content) were subjected to SDS–PAGE and Western blot analysis. Phospho-RbSer249/Thr252, Rb and
antibodies, visualized with autoradiography film. Bands were measured densitometrically and their relative density calculated based on the density of the
each sample. Data represent the mean SEM of three separate experiments. Statistical significance is obtained with Student’s t test in comparison with controls ⁄⁄p < 0.01.
l
M 2d compound for 72 h were collected and lysed with 2% SDS lysis buffer. Equal
-tubulin levels were detected by specific
-tubulin band in
a
a

G. Forte et al. / Bioorg. Med. Chem. 21 (2013) 5233–5245
5237
2d
2e
2f
2i
2j
2k
5
Figure 4. Lowest energy conformers, obtained after B3LYP/6-311+G⁄⁄ optimization, are reported below.
Such procedure gave rise to a scale of energy values of the different
conformers; for the sake of clarity, in Fig. 4 we have reported the
geometries lowest in energy. Nevertheless, it is to note that energy
differences among the most part of the conformers are quite small
view, effects induced by this group to the rest of the molecule are
not appreciable (Table 2).
As to the position of the OCH3 substituents, we observed only in
2-phenylsubstituted compounds, 2f and 2j, the formation of an
intramolecular H-bond between the hydrogen atom of the carbox-
ylic group and the oxygen atom of the methoxyl group. However,
the amount of energy associated to this bond is estimated to be
about 3 kJ/mol; due to this low energy value it is plausible that,
at room temperature, vibrational modes break such weak bond
making available the hydrogen of the carboxylic group to interact,
by means of a H-bond, with the biological substrate.
From the electrostatic potential maps, reported in Fig. 5, one
can distinguish the higher electron density area (in red) from the
lower (in blue). As could be speculated looking at the chemical
structures and as confirmed by these maps, it can be said that oxy-
gen atoms in both carboxylic and amidic group can act as electron
donors toward electrophilic groups of the substrate, on the other
(between 0.2–1 kJ/mol) and the standard deviation value (r) for
each configuration is less than 4. From a topological point of view
we observe the presence of planar moieties in the molecules, due
to an electron delocalization extended from the indole ring to the
acidic group by means of conjugated double bonds (see Fig. 4
and bond order values reported in Table 2); nevertheless the topol-
ogy of the phenyl ring substituted with different R1 residues sug-
gests us that binding DNA, through base pair intercalation,
cannot occur since the steric hindrance due to the substituted phe-
nyl ring. Atomic charge vales are also reported in Table 2. From
these results we find out that the methoxyl groups, introduced as
substituent in several position of the phenyl ring, play a main role
only in terms of steric hindrance; in fact from an electronic point of

5238
G. Forte et al. / Bioorg. Med. Chem. 21 (2013) 5233–5245
Table 2
Wiberg bond order and atomic charges (a.u.) are reported for the main bonds and atoms. Numeration is referred to Fig. 4
BO
Charges (a.u.)
2d
1.01
1.10
1.75
0.89
1.53
1.13
1.70
1.03
1.03
2e
1.01
1.10
1.75
0.89
1.54
1.15
1.71
1.03
1.03
2f
1.02
1.09
1.74
0.88
1.55
1.12
1.74
0.99
1.05
2i
1.02
1.10
1.74
0.89
1.53
1.13
1.71
1.03
1.04
2j
1.02
1.09
1.74
0.88
1.56
1.11
1.74
0.99
1.05
2k
1.01
1.10
1.75
0.89
1.53
1.13
1.71
1.03
1.04
5
2f (COO-)
2j (COO-)
1–2
1–4
2–3
4–5
4–6
6–7
7–8
8–9
9–10
9–12
10–11
11–13
0.95
1.20
1.01
0.90
1.44
1.16
1.67
1.05
1.02
1.69
0.75
0.00
1.08
1.15
1.71
0.89
1.52
0.87
1.70
0.81
1.44
1.49
0.00
1.06
1.18
1.72
0.89
1.53
0.84
1.67
0.80
1.45
1.47
0.00
1.70
0.75
0.00
1.70
0.74
0.00
1.74
0.70
0.08
1.70
0.75
0.00
1.73
0.70
0.09
1.70
0.75
0.00
0.00
0.00
1
2
3
5
ꢁ0.46
ꢁ0.45
ꢁ0.46
ꢁ0.46
ꢁ0.46
ꢁ0.47
ꢁ0.41
ꢁ0.17
ꢁ
ꢁ0.53
ꢁ0.53
0.71
0.71
0.71
0.71
0.71
0.71
0.71
0.70
ꢁ0.57
0.25
ꢁ0.57
0.27
ꢁ0.58
0.24
ꢁ0.58
0.25
ꢁ0.58
0.24
ꢁ0.57
0.27
ꢁ0.61
0.30
ꢁ0.58
0.29
0.22
10
11
12
ꢁ0.68
ꢁ0.68
ꢁ0.69
ꢁ0.68
ꢁ0.70
ꢁ0.68
ꢁ0.69
ꢁ0.77
ꢁ0.77
0.48
0.49
0.49
0.49
0.49
0.49
0.48
0.00
0.00
ꢁ0.63
ꢁ0.62
ꢁ0.59
ꢁ0.63
ꢁ0.59
ꢁ0.62
ꢁ0.65
ꢁ0.73
ꢁ0.74
hand the electrophilic hydrogen atom of the amidic group can form
H-bond with heteroatoms, such as O or N, of the biological target.
It seems quite clear that the above considered compounds are
pH sensitive and, at physiological pH, the carboxylate anion repre-
sents the predominate form. For this reason further geometry opti-
mizations were carried out on the anionic species, starting from
the neutral structures. Both topological and electronic modifica-
tions, with respect to the neutral form, are considerable only for
compounds 2f and 2j and the related data are reported in Table 2.
In these cases an electrostatic repulsion between the carboxylate
and the substituent at the 2-position of the phenyl ring occurs. An-
ion structures, clearly, are strongly attracted by positive moieties
present on the substrate; since the long-range character of the
Coulomb force, electrostatic interaction could be considered the
first recognition step with the active site of the substrate. Finally
the marked anionic character of such compounds suggests a low
affinity with the DNA.
VolSurf+ model library such as blood–brain barrier (BBB), absorp-
tion, solubility, metabolic stability.
In particular, we have taken into consideration the solubility in
water and the intestinal absorption, two fundamental properties
for the design of a potential drug useful for oral administration.
The two properties are closely related because crossing the intesti-
nal membrane requires a balance between hydrophilic and hydro-
phobic nature of the molecule. These two properties are
orthogonal, that is, the increase of a property generally coincides
with a decrease of the other. It is, therefore, important to obtain
a high solubility in the aqueous phase but without reaching values
that could lead to a decrease in intestinal absorption.
In Figs. 6–9 we report the PLS plot of the four models. The com-
pounds used for the models are reported as black points and the
projections of the synthesized compounds in yellow. The back-
ground are colored in basis of the activity; moving from red to blue
zone the activity value increase.
Table 3 shows the quantitative values of solubility (Soly), intes-
tinal permeability (Caco2), the permeability of the blood–brain
barrier (BBB), and metabolic stability (MetStab).
4.2. VolSurf+ models
Several in silico methods were applied, in previous works,27–29
reflecting both pharmacokinetic (PK) and pharmacodynamics (PD)
aspect. In the present paper the PK profile of the database com-
pounds was determined by use of the VolSurf+ software. VolSurf+
is an automatic procedure to convert 3D molecular fields into
physico-chemical relevant molecular descriptors. VolSurf+ ex-
tracts the information present in 3D molecular field maps into
few quantitative numerical descriptors, easy to understand and
interpret. Molecular recognition is achieved coupling image anal-
ysis software with external chemical knowledge. Within this con-
text, VolSurf+ selects molecular descriptors referred to molecular
size and shape, to size and shape of both hydrophilic and hydro-
phobic regions and to the balance between them. Hydrogen
bonding, amphiphilic moments, critical packing parameters are
other useful descriptors. The VolSurf+ descriptors have been pre-
sented and explained in detail.29 The originality of VolSurf+ re-
sides in the fact that surfaces, volumes, and other related
descriptors can be directly obtained from 3D molecular fields
and can be used for multivariate model building to correlate
the 3D molecular structures with biological behaviors.30 The Vol-
Surf+ methodology can also be applied for structure-activity rela-
tionships and molecular diversity based on surface properties.
The selection of compounds with a favorable PK profile was
guided by the projection on predefined models available in the
On the basis of the range of properties of an optimal candidate
reported in literature,31 compounds 3a and 3b present the best
solubility and metabolic stability but a low value of permeability.
These values exclude 3a and 3b as lead compounds for further
studies. On the other hand, compounds such as 2f, 2d, and 2g–
2j show a good compromise between the permeability and solu-
bility and moderate good values for MetStab; thus these results
could explain good in vitro antitumor activities of these
derivatives.
5. Conclusions
In this report, a novel class of indole derivatives was described,
synthesized with the aim of identifying novel compounds en-
dowed with antitumor effects. These derivatives showed indeed
strong antiproliferative activity against a panel of six human tumor
cell lines, reaching IC50 values at submicromolar level. In particu-
lar, among synthesized derivatives, compounds 2d–m and 5 stand
out for their cell growth inhibitory activity and, particularly, com-
pound 2d emerged for its selectivity towards DU145 with respect
to other tested tumor cell lines. Biological assays aimed at elucidat-
ing the effect of lead compound 2d on different intracellular path-
ways led us to believe that p21Cip1 induction and suppression of

G. Forte et al. / Bioorg. Med. Chem. 21 (2013) 5233–5245
5239
2d
2e
2f
2i
2j
2k
5
Figure 5. Electrostatic potential maps (e/K
3) are showed below. Blue color indicate regions with low electron density while red color is associated to high electron density
moieties (see also the scale).
Akt signaling together with induction of Rb may play an important
role in growth inhibition caused by 2d treatment.
we highlight that data emerged from the present work will help
the future design of new potential and more active antitumor com-
pounds that, while maintaining the indole ring, might present var-
ious substituents in different positions to improve the ADME
properties, helping to clarify the mechanism of action of this novel
class of indole derivatives.
Two different computational approaches were used to better
understand pharmacological profile of this new class on indole
derivatives. These studies showed that, since their planar moieties,
such compounds cannot act as DNA intercalators suggesting that
other cellular molecular targets participate in the antiproliferative
effect of compounds 2d–m and 5. Furthermore, the electron donor
oxygen atom in amidic or carboxylic groups, can interact with moi-
eties of amino acids in the biological substrate which, at physiolog-
ical pH, act as ‘H-bond donors’ for example, Asn, Ser, Thr, Tyr. On
the other hand, carboxylate anion is mainly involved in electro-
static interaction with positive charged area of the substrate which
can be represented by basic amino acids such as His, Lys and Arg. It
is to note that the absence of a remarkable positively charged
group in the studied compounds give rise to a low affinity with
DNA. A different biological activity of the derivatives can be as-
cribed to steric hindrance induced by OCH3 substituent. Finally,
6. Experimental procedures
6.1. Chemistry
Melting points were determined in a Electrothermal IA9200
apparatus with a digital thermometer in glass capillary tubes.
Infrared spectra were recorded on a Perkin–Elmer FT IR 1600
spectrometer in KBr disks. Elemental analyses for C, H, and N
were within 0.4% of theoretical values and were performed on
a Carlo Erba Elemental Analyzer Mod. 1108 apparatus. 1H NMR
spectra were recorded at 200 MHz on a Varian Inova Unity 200

5240
G. Forte et al. / Bioorg. Med. Chem. 21 (2013) 5233–5245
Figure 6. Soly model: the dots represent the molecules in the learning set (in black) and the synthesized compounds (in yellow).
Figure 7. CACO2 model: the dots represent the molecules in the learning set (in black) and the synthesized compounds (in yellow).
spectrometer in DMSO-d6 solution. Chemical shifts are given in d
values (ppm), using tetramethylsilane as the internal standard;
coupling constants (J) are given in hertz (Hz). Signal multiplicities
are characterized as s (singlet), d (doublet), t (triplet), q (quartet),
m (multiplet), br (broad signal). All the synthesized compounds
were tested for purity on TLC (aluminum sheet coated with silica
gel F254, Merck) and visualized by UV (k = 254 and 366 nm). All
chemicals and solvents were of reagent grade and were pur-
chased from commercial vendors. Compounds 2a, 2b, 3a, and
3b were synthesized as previously reported by the same
authors.16
6.1.1. General procedure for the synthesis of (aE)-a-[[1-acetyl-
5-(phenylmethoxy)-1H-indol-3-
yl]methylene]substitutedbenzeneacetic acids (Method A) (2c,
2d, 2e, 2f, 2i, 2l, 2m)
A suspension of 5-(phenylmethoxy)-1H-indole-3-carboxalde-
hyde (0.500 g, 0.002 mol) and the appropriate phenylacetic acid
(0.002 mol) in 1.88 mL of acetic anhydride (0.020 mol) and
0.277 mL of TEA (0.002 mol) was heated at 130 °C, for 6 h. The
brown solution was cooled to 90 °C, 20 mL of H2O/EtOH/THF
(1:1:1) were added and the obtained mixture was vigorously stir-
red for 2 h, then allowed to cool at 22 °C and stirred for 14 h. The

G. Forte et al. / Bioorg. Med. Chem. 21 (2013) 5233–5245
5241
Figure 8. MetStab model: the dots represent the molecules in the learning set (in black) and the synthesized compounds (in yellow).
Figure 9. BBB model: the dots represent the molecules in the learning set (in black) and the synthesized compounds (in yellow).
obtained suspension was filtered and the solid was triturated in
DCM/EtOH (3:7) to obtain the desired final product. Using this pro-
cedure the following compounds were obtained.
7.00 (m, 2H, aromatic), 6.78 (s, 1H, NCH), 5.05 (s, 2H, CH2), 2.23
(s, 3H, CH3). Anal. (C26H21NO4) C, H, N.
6.1.1.2.
(
aE)-
a-[[1-Acetyl-5-(phenylmethoxy)-1H-indol-3-
6.1.1.1.
(
a
E)-
a
-[[1-Acetyl-5-(phenylmethoxy)-1H-indol-3-
The title compound
yl]methylene]-4-methoxybenzeneacetic acid (2d).
The title
yl]methylene]benzeneacetic acid (2c).
compound was synthesized on a scale of 0.008 mol of starting
materials and was obtained as a yellow powder (56%): mp 237–
238 °C; IR (KBr) cmꢁ1 3564, 1700, 1513, 1456, 1390, 1223, 1168,
1032, 835; 1H NMR (DMSO-d6) d 12.65 (br s, 1H, COOH), 8.18–
8.09 (m, 1H, indole), 7.92 (s, 1H, CH@CCOOH), 7.51–7.30 (m, 5H,
was obtained as a yellow powder (36%): mp 251–253 °C; IR (KBr)
cmꢁ1 2932, 1706, 1667, 1587, 1458, 1385, 1280, 1254, 1221,
1013, 701; 1H NMR (DMSO-d6) d 8.16–8.09 (m, 1H, indole), 7.97
(s, 1H, CH@CCOOH), 7.54–7.23 (m, 10H, indole + aromatic), 7.10–

5242
G. Forte et al. / Bioorg. Med. Chem. 21 (2013) 5233–5245
Table 3
6.1.1.7.
(
a
E)-
a
-[[1-Acetyl-5-(phenylmethoxy)-1H-indol-3-
Quantitative values of some pharmacokinetic properties obtained from the matrix of
VolSurf+ descriptors
yl]methylene]-4-chlorobenzeneacetic acid (2m).
The title
compound was obtained as a yellow powder (48%): mp 252–
254 °C; IR (KBr) cmꢁ1 3061, 1702, 1689, 1456, 1388, 1260, 1223,
1167, 1031, 835, 731; 1H NMR (DMSO-d6) d 8.17–8.11 (m, 1H, in-
dole), 7.99 (s, 1H, CH@CCOOH), 7.56–7.28 (m, 9H, indole + aro-
matic), 7.10–6.89 (m, 3H, aromatic + NCH), 4.99 (s, 2H, CH2), 2.35
(s, 3H, CH3). Anal. (C26H20NO4) C, H, N.
Measurements unit
Compd
Log (Mol/L)
SOLY
cm/s
CACO2
Log (Mol/L)
LgBB
%
MetStab
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
3a
3b
3c
3d
4a
4b
4c
4d
5
ꢁ5,46116
ꢁ5,56853
ꢁ5,57542
ꢁ5,38938
ꢁ5,40916
ꢁ5,65412
ꢁ5,51885
ꢁ5,46909
ꢁ5,42433
ꢁ6,86235
ꢁ6,11921
ꢁ3,58103
ꢁ3,83048
ꢁ5,15591
ꢁ5,22823
ꢁ4,22521
ꢁ4,09877
ꢁ5,52653
ꢁ5,71983
ꢁ6,81326
0.61832
0.55296
ꢁ2,74298
ꢁ2,91948
ꢁ2,97634
ꢁ2,86165
ꢁ3,03668
ꢁ3,12895
ꢁ3,18018
ꢁ2,97448
ꢁ3,1804
46,3148
44,0852
43,9078
46,5748
47,7825
42,4053
42,0086
44,1951
45,1707
15,2722
34,7715
89,369
84,1348
52,5537
51,4396
61,0254
60,1342
26,6246
24,1309
15,8121
0.473478
0.519552
0.470385
0.388693
0.402218
0.489634
0.339976
0.623968
0.684529
0.950309
0.712199
0.896818
0.675192
1,15193
6.1.2. General procedure for the synthesis of (aE)-a-[[1-Acetyl-
5-(phenylmethoxy)-1H-indol-3-
yl]methylene]substitutedbenzeneacetic acids (Method B) (2g,
2h, 2k, 2j)
ꢁ2,7141
A suspension of 5-(phenylmethoxy)-1H-indole-3-carboxalde-
hyde (0.500 g, 0.002 mol) and the appropriate phenylacetic acid
(0.002 mol) in 1.88 mL of acetic anhydride (0.020 mol) and
0.277 mL of TEA (0.002 mol) was heated at 130 °C, for 6 h. The
brown solution was cooled to 90 °C, 20 mL of H2O/EtOH/THF
(1:1:1) were added and the obtained mixture was vigorously stir-
red for 2 h, then allowed to cool at 22 °C and stirred for 14 h. The
reaction mixture was concentrated, the residue diluted with water
(50 mL) and extracted with CH2Cl2 (3 ꢂ 100 mL). The combined or-
ganic layers were dried over anhydrous Na2SO4, filtered, and evap-
orated. The obtained crude material was recrystallized from EtOH/
CH2Cl2 (7:3) to obtain the desired final product. Using this proce-
dure the following compounds were obtained.
ꢁ2,60381
ꢁ2,27524
ꢁ2,78785
ꢁ2,38646
ꢁ2,78446
ꢁ2,27003
ꢁ2,87145
ꢁ2,44954
ꢁ2,83399
ꢁ2,52359
0.87597
1,05687
0.852732
0.748535
indole + aromatic), 7.25–7.16 (m, 2H, aromatic), 7.06–6.90 (m, 5H,
aromatic + NCH), 4.99 (s, 2H, CH2), 3.76 (s, 3H, OCH3), 2.32 (s, 3 H,
CH3). Anal. (C27H23NO5) C, H, N.
6.1.2.1.
(a
E)-
a-[[1-Acetyl-5-(phenylmethoxy)-1H-indol-3-
yl]methylene]-1,3-benzodioxol-5-yl-acetic acid (2g).
The ti-
6.1.1.3.
(aE)-a-[[1-Acetyl-5-(phenylmethoxy)-1H-indol-3-
tle compound was obtained as a yellow powder (31%): mp 230–
232 °C; IR (KBr) cmꢁ1 2893, 1705, 1690, 1608, 1541, 1453, 1381,
1236, 1036, 935, 700; 1H NMR (DMSO-d6) d 8.18–8.11 (m, 1H, in-
dole), 7.92 (s, 1H, CH@CCOOH), 7.45–7.32 (m, 5H, indole + aro-
matic), 7.17–7.15 (m, 1H, aromatic), 7.02–6.85 (m, 4H, aromatic
+ NCH), 6.76–6.69 (m, 1H aromatic), 6.01 (s, 2H, OCH2O), 5.02 (s,
2H, CH2), 2.39 (s, 3H, CH3). Anal. (C27H21NO6) C, H, N.
yl]methylene]-3-methoxybenzeneacetic acid (2e).
The title
compound was synthesized on a scale of 0.008 mol of starting
materials and was obtained as a yellow powder (47%): mp 224–
226 °C; IR (KBr) cmꢁ1 1705, 1669, 1583, 1458, 1385, 1221, 1013,
798; 1H NMR (DMSO-d6) d 8.12–8.04 (m, 1H, indole), 7.95 (s, 1H,
CH@CCOOH), 7.50–7.28 (m, 6H, indole + aromatic), 7.10–6.76 (m,
6H, aromatic + NCH), 5.02 (s, 2H, CH2), 3.72 (s, 3H, OCH3), 2.28
(s, 3H, CH3). Anal. (C27H23NO5) C, H, N.
6.1.2.2.
(a
E)-
a-[[1-Acetyl-5-(phenylmethoxy)-1H-indol-3-
yl]methylene]-3,4-dimethoxybenzeneacetic acid (2h).
The
6.1.1.4.
(
a
E)-
a
-[[1-Acetyl-5-(phenylmethoxy)-1H-indol-3-
title compound was obtained as a yellow powder (44%): mp
223–225 °C; IR (KBr) cmꢁ1 3060, 1705, 1674, 1513, 1452, 1387,
1257, 1218, 1145, 1021, 944, 696; 1H NMR (DMSO-d6) d 8.17–
8.10 (m, 1H, indole), 7.92 (s, 1H, CH@CCOOH), 7.49–7.31 (m, 5H,
indole + aromatic), 7.13–7.10 (m, 1H, aromatic), 7.05–6.74 (m,
5H, aromatic + NCH), 4.96 (s, 2H, CH2), 3.75 (s, 3H, OCH3), 3.66
(s, 3H, OCH3), 2.35 (s, 3H, CH3). Anal. (C28H25NO6) C, H, N.
yl]methylene]-2-methoxybenzeneacetic acid (2f).
The title
compound was obtained as a yellow powder (41%): mp 229–
231 °C; IR (KBr) cmꢁ1 3018, 1699, 1690, 1460, 1390, 1252, 1218,
1163, 1022, 947, 755; 1H NMR (DMSO-d6) d 8.16–8.09 (m, 1H, in-
dole), 7.91 (s, 1H, CH@CCOOH), 7.55–6.95 (m, 11H, indole + aro-
matic), 6.71 (s, 1H, NCH), 5.11 (s, 2H, CH2), 3.72 (s, 3H, OCH3),
2.23 (s, 3H, CH3). Anal. (C27H23NO5) C, H, N.
6.1.2.3.
(a
E)-
a-[[1-Acetyl-5-(phenylmethoxy)-1H-indol-3-
6.1.1.5.
yl]methylene]-3,4,5-trimethoxybenzeneacetic
(2i). The title compound was obtained as a light yellow pow-
(
a
E)-
a
-[[1-Acetyl-5-(phenylmethoxy)-1H-indol-3-
yl]methylene]-3,5-dimethoxybenzeneacetic acid (2k).
The
acid
title compound was obtained as a yellow powder (50%): mp
244–245 °C; IR (KBr) cmꢁ1 2939, 1700, 1677, 1539, 1458, 1387,
1255, 1204, 1156, 1019, 945, 716; 1H NMR (DMSO-d6) d 8.13–
8.06 (m, 1H, indole), 7.88 (s, 1H, CH@CCOOH), 7.41–7.30 (m, 5H,
indole + aromatic), 7.04–6.96 (m, 3H, aromatic + NCH), 6.52–6.48
(m, 1H, aromatic), 6.39–6.35 (m, 2H, aromatic), 4.99 (s, 2H, CH2),
3.65–3.61 (m, 6H, OCH3), 2.28 (s, 3H, CH3). Anal. (C28H25NO6) C,
H, N.
der (42%): mp 225–226 °C; IR (KBr) cmꢁ1 2939, 1705, 1582, 1503,
1456, 1385, 1240, 1124, 1006, 839; 1H NMR (DMSO-d6) d 8.11–
8.20 (m, 1H, indole), 7.94 (s, 1H, CH@CCOOH), 7.50–7.33 (m, 5H,
indole + aromatic), 7.07–6.93 (m, 3H, aromatic + NCH), 6.51–6.58
(m, 2H, aromatic), 5.04 (s, 2H, CH2), 3.68 (s, 9 H, OCH3), 2.34 (s, 3
H, CH3). Anal. (C29H27NO7) C, H, N.
6.1.1.6.
yl]methylene]-4-phenylmethoxybenzeneacetic
(2l).
(
a
E)-
a
-[[1-Acetyl-5-(phenylmethoxy)-1H-indol-3-
acid
The title compound was obtained as a yellow powder
6.1.2.4.
(
a
E)-
a
-[[1-Acetyl-5-(phenylmethoxy)-1H-indol-3-
yl]methylene]-2,5-dimethoxybenzeneacetic acid (2j).
The
title compound was obtained as a yellow powder (33%): mp
200–202 °C; IR (KBr) cmꢁ1 3059, 1703, 1685, 1498, 1459, 1387,
1256, 1220, 1041, 947, 801; 1H NMR (DMSO-d6) d 8.17–8.10 (m,
1H, indole), 7.91 (s, 1H, CH@CCOOH), 7.48–7.35 (m, 5H, indole +
aromatic), 7.24–7.20 (m, 1H, aromatic), 7.09–6.90 (m, 3H, aro-
matic), 6.81 (s, 1H, NCH), 6.72–6.67 (m, 1H, aromatic), 5.11 (s,
(60%): mp 224–226 °C; IR (KBr) cmꢁ1 3061, 1702, 1687, 1610,
1512, 1458, 1383, 1250, 1222, 1013, 946, 829, 732; 1H NMR
(DMSO-d6) d 8.17–8.11 (m, 1H, indole), 7.92 (s, 1H, CH@CCOOH),
7.40–6.89 (m, 17H, indole + aromatic + NCH), 5.10 (s, 2H, CH2),
4.99 (s, 2 H, CH2), 2.30 (s, 3H, CH3). Anal. (C33H27NO5) C, H, N.

G. Forte et al. / Bioorg. Med. Chem. 21 (2013) 5233–5245
5243
2H, CH2), 3.69–3.62 (m, 6H, OCH3), 2.27 (s, 3H, CH3). Anal.
(C28H25NO6) C, H, N.
indole + aromatic), 6.53 (br s, 2H, NH2), 6.17 (d, J = 2.6 Hz, 1H,
NCH), 3.82 (s, 3H, OCH3). Anal. (C18H16N2O2) C, H, N.
6.1.3. General procedure for the synthesis of (
a
E)-
a-[[5-
6.1.4.3.
(a
E)-
a-[[5-(Phenylmethoxy)-1H-indol-3-yl]methy-
(phenylmethoxy)-1H-indol-3-yl]methylene]benzeneacetic
lene]benzeneacetamide (4c).
The title compound was ob-
acids (3c and 3d)
tained as a pale yellow powder (65%): mp 204–205 °C; IR (KBr)
cmꢁ1 3353, 2364, 1657, 1567, 1480, 1379, 1190, 1002, 940, 745;
1H NMR (DMSO-d6) d 11.17 (br s, 1H, NH), 7.82 (s, 1H, CH@CCONH2),
7.50–7.20 (m, 12H, indole + aromatic), 7.09 (br s, 1H, NH2), 6.85–6.80
(m, 1H, aromatic), 6.65 (br s, 1H, NH2), 6.08 (d, J = 2.6 Hz, 1H, NCH),
5.09 (s, 2H, CH2). Anal. (C24H20N2O2) C, H, N.
A solution of the appropriate (aE)-a-[[1-acetyl-5-(phenylmeth-
oxy)-1H-indol-3-yl]methylene]benzeneacetic acid (2c or 2d)
(0.004 mol) in 12 mL of NaOH 2 N (0.024 mol), 12 mL of THF and
24 mL of ethanol was heated at 70 °C for 2 h. The obtained mixture
was concentrated under reduced pressure, diluted with water. The
aqueous layer was washed with DCM (1 ꢂ 50 mL), and acidified
with HCl 37%. The obtained precipitate was filtered, dried and
recrystallized from DCM/EtOH (1:1) to give the title compounds
3c or 3d.
6.1.4.4. (
4-methoxybenzeneacetamide (4d).
a
E)-
a
-[[5-(Phenylmethoxy)-1H-indol-3-yl]methylene]-
The title compound was
obtained as a pale yellow powder (57%): mp 231–233 °C; IR (KBr)
cmꢁ1 3164, 2364, 1659, 1568, 1479, 1241, 1021, 944, 803; 1H NMR
(DMSO-d6) d 11.15 (br s, 1H, NH), 7.80 (s, 1H, CH@CCONH2), 7.51–
7.00 (m, 11H, indole + aromatic), 6.83–6.78 (m, 1H, aromatic), 6.51
(br s, 2H, NH2), 6.22 (d, J = 2.6 Hz, 1H, NCH), 5.08 (s, 2H, CH2), 3.80
(s, 3H, OCH3). Anal. (C25H22N2O3) C, H, N.
Using this procedure the following compound was obtained.
6.1.3.1.
(aE)-
a
-[[5-(Phenylmethoxy)-1H-indol-3-yl]methy-
lene]benzeneacetic acid (3c).
The title compound was ob-
tained as a yellow powder (44%): mp 218–220 °C; IR (KBr) cmꢁ1
3375, 1669, 1587, 1453, 1416, 1272, 1193, 1107, 850, 737; 1H
NMR (DMSO-d6) d 12.21 (br s, 1H, COOH), 11.41 (br s, 1H, NH),
8.06 (s, 1H, CH@CCOOH), 7.51–7.20 (m, 11H, indole + aromatic),
7.13–7.10 (m, 1H, aromatic), 6.88–6.78 (m, 1H, aromatic), 6.32
(d, J = 2.6 Hz, 1H, NCH), 5.07 (s, 2H, CH2). Anal. (C24H19N2O3) C,
H, N.
6.1.5. (
1H-indol-3-yl]methylene]benzeneacetic acid (5)
A solution of ( E)- -[[5-(phenylmethoxy)-1H-indol-3-yl]meth-
aE)-a-[[1-(4-Methoxyphenylmethyl)-5-(phenylmethoxy)-
a
a
ylene]-4-methoxybenzeneacetic acid (3d) (0.002 mol) in 5 mL of
anhydrous DMF was cooled at 0 °C and 0.151 g of sodium hydride
95% (0.006 mol) were added. The obtained suspension was allowed
to warm at 22 °C and stirred for 15 min. After this period, 4-meth-
oxyphenylmethyl chloride (0.271 mL, 0.002 mol) in 1 mL of anhy-
drous DMF was added drop-wise and the obtained suspension
was stirred at 22 °C for 24 h. The reaction mixture was slowly
dropped in 70 mL of HCl 0.6 N. The obtained precipitate was
filtered, dried and triturated from DCM/EtOH (1:1) to give the de-
sired final product 5 as a light yellow powder (91%): mp 211–
213 °C; IR (KBr) cmꢁ1 2929, 1663, 1612, 1514, 1250, 1175, 1023,
802; 1H NMR (DMSO-d6) d 7.97 (s, 1H, CH@CCOOH), 7.45–7.34
(m, 6H, indole + aromatic), 7.14–6.81 (m, 10H, aromatic), 6.54 (s,
1H, NCH), 5.10 (s, 2H, NCH2), 5.03 (s, 2H, NCH2), 3.77 (s, 3H,
OCH3), 3.72 (s, 3H, OCH3). Anal. (C33H29NO5) C, H, N.
6.1.3.2. (aE)-a-[[5-(phenylmethoxy)-1H-indol-3-yl]methylene]-
4-methoxybenzeneacetic acids (3d).
The title compound
was obtained as a yellow powder (71%): mp 180–182 °C; IR (KBr)
cmꢁ1 3338, 1670, 1612, 1512, 1451, 1244, 1029, 743; 1H NMR
(DMSO-d6) d 11.37 (br s, 1H, NH), 7.93 (s, 1H, CH@CCOOH),
7.49–6.73 (m, 12H, indole + aromatic), 6.43 (d, J = 2.6 Hz, 1H,
NCH), 5.03 (s, 2H, CH2), 3.77 (s, 3H, OCH3). Anal. (C25H21NO4) C,
H, N.
6.1.4. General procedure for the synthesis of (aE)-a-[(5-substi-
tuted-1H-indol-3-yl)methylene]substitutetdbenzeneacetamide
(4a–c)
A suspension of the appropriate carboxylic acid (0.5 mmol), TEA
(1.2 mmol), and 1-hydroxybenzotriazole ammonium salt
(1.0 mmol) in 1 mL of anhydrous DMF was cooled to 0 °C. EDAC
(0.75 mmol) was added portion-wise and the reaction mixture
was stirred for 24 h at 22 °C. The obtained mixture was diluted
with ethyl acetate (100 mL) and the organic layer was separated
and washed with water (2 ꢂ 50 mL), NaOH 1 N (1 ꢂ 50 mL), and
brine (1 ꢂ 50 mL). The organic layer was dried over anhydrous Na2-
SO4, filtered, and evaporated. The obtained crude material was
recrystallized from DCM to obtain the desired final product. Using
this procedure the following compounds were obtained.
6.2. Biological activity evaluation
6.2.1. Cell culture and treatments
DU145 and A549 cells were grown in Dulbecco’s modified Ea-
gle’s medium (DMEM) containing 10% fetal calf serum (FCS), 100
U/mL penicillin, 100 mg/mL streptomycin, 1 mM glutamine, and
1% nonessential amino acids. LNCaP, KB and M14 cell lines were
maintained in RPMI supplemented with 10% fetal calf serum, 100
U/mL penicillin, and 100 mg/mL streptomycin. Finally, Caco2 cells
were maintained in minimum essential medium (MEM) supple-
mented with 10% fetal calf serum, 100 U/mL penicillin and
6.1.4.1.
(4a).
(
a
E)-
a
-[-(1H-Indol-3-yl)methylene]benzeneacetamide
100 lg/mL streptomycin. All cultures were incubated at 37 °C
The title compound was obtained as a light yellow pow-
within a humidified, 95% air and 5% CO2 atmosphere. The medium
was changed every 2 to 3 days. For the experiments the cells were
plated at a constant density to obtain identical experimental con-
ditions in the different tests, thus to achieve a high accuracy of
the measurements. After 24 h incubation at 37 °C under a humid-
ified 5% carbon dioxide to allow cell attachment, the culture med-
ium was changed and the cells were treated with different
der (73%): mp 187–189 °C; IR (KBr) cmꢁ1 3390, 2364, 1665, 1575,
1353, 1294, 1235, 749; 1H NMR (DMSO-d6) d 11.27 (br s, 1H, NH),
7.85 (s, 1H, CH@CCONH2), 7.70–7.64 (m, 1H, indole), 7.58–7.02 (m,
8H, indole + aromatic), 6.63 (br s, 2H, NH2), 6.08 (d, J = 2.6 Hz, 1H,
NCH). Anal. (C17H14N2O) C, H, N.
6.1.4.2.
neacetamide (4b).
(
a
E)-
a
-[-(1H-Indol-3-yl)methylene]-4-methoxybenze-
concentrations (1, 25, 50, and 100
compounds.
lM for 72 h) of new synthesised
The title compound was obtained as a
light yellow powder (79%): mp 223–225 °C; IR (KBr) cmꢁ1 3163,
1640, 1547, 1513, 1366, 1232, 1139, 1034, 746; 1H NMR (DMSO-
d6) d 11.25 (br s, 1H, NH), 7.84 (s, 1H, CH@CCONH2), 7.72–7.64
(m, 1H, indole), 7.38–7.29 (m, 1H, indole), 7.20–7.01 (m, 6H,
6.2.2. Cell growth inhibition assay
To evaluate cell viability the MTT assay was employed: it mea-
sures the cellular capacity to reduce 3-(4,5-dimethylthiazol-2-
yl)diphenyltetrazolium bromide to blue formazan products by

5244
G. Forte et al. / Bioorg. Med. Chem. 21 (2013) 5233–5245
various mitochondrial dehydrogenase enzymes. Briefly, DU145, KB,
(NBO)41 analysis was carried out in order to evaluate charge atomic
charges and electrostatic potential maps.
M14, Caco2 cells and human adult fibroblasts (1 ꢂ 104) were set up
in flat-bottomed 200
lL microplates, and incubated at 37 °C in a
humidified 5% CO2/95% air mixture for 24 h. Then, they were un-
treated (controls) or treated for 72 h with each experimental com-
pound. Mother solutions of each derivative were prepared by
dissolving it in the minimum amount of DMSO and after in culture
medium. In each experiment, DMSO never exceeded 0.1%, and this
percentage did not interfere with cell growth. All the compounds
6.3.2. VolSurf+ descriptors
The interaction of molecules with biological membranes is
mediated by surface properties such as shape, electrostatic forces,
H-bonds and hydrophobicity. Therefore, the GRID force field was
chosen to characterize potential polar and hydrophobic interaction
sites around target molecules by the water (OH2), the hydrophobic
(DRY), and the carbonyl oxygen (O) and amide nitrogen (N1)
probe.42 The information contained in the MIF (Molecular Interac-
tion Fields) is transformed into a quantitative scale by calculating
the volume or the surface of the interaction contours. The VolSurf+
procedure is as follows: (i) in the first step, the 3D molecular field
is generated from the interactions of the OH2, the DRY, O and N1
probe around a target molecule; (ii) the second step consists in
the calculation of descriptors from the 3D maps obtained in the
first step. The molecular descriptors obtained, called VolSurf+
descriptors, refer to molecular size and shape, to hydrophilic and
hydrophobic regions and to the balance between them, to molecu-
lar diffusion, LogP, LogD, to the ‘charge state’ descriptors, to the
new 3D pharmacophoric descriptors and to some descriptors on
some relevant ADME properties. The definition of all 126 VolSurf+
descriptors is given in case studies with the old versions of Vol-
Surf43–48; (iii) finally, chemometric tools (PCA,49 PLS50) are used
to create relationships of the VolSurf+ descriptor matrix with
ADME properties. The scheme of the VolSurf+ program steps and
a detailed definition of VolSurf+ descriptors have recently been
reported.29
were tested at the concentrations of 1, 25, 50, and 100
lM. Four
h before the end of the incubation period, 20 L of 0.5% MTT in
l
phosphate buffer saline was added to each microwell. After incu-
bation with the reagent, the supernatant was removed and re-
placed with 100
lL DMSO. The optical density of each sample
was measured with a spectrophotometer reader at k = 550 nm.
Three replicates were performed for each sample.
6.2.3. Western blotting analysis
Cells, submitted or not to treatments with 2d compound for 72
h, were rinsed twice with phosphate-buffered saline (PBS), lysed in
the presence of 2% SDS lysis buffer (50 mM Tris–HCl, pH 6.8, com-
plete protease inhibitor Cocktail, plus the phosphatase inhibitors
2 mM Na orthovanadate, 1 mM NaF, 1 mM sodium pyrophosphate)
harvested by scraping with a rubber policeman and quantified
using Bicinchoninic acid assay (Pierce). Equal amount of total pro-
teins were subjected to SDS–polyacrylamide electrophoresis on
10% gels, transferred to a nitrocellulose membrane (Amersham,
GE Healthcare Europe GmbH, Milano, Italy) and proteins revealed
by Ponceau staining (Sigma Chemical Co., Milano, Italy). Mem-
branes were blocked in TBS-Tween 0.1% buffer containing 5% non-
fat milk, and probed with specific antibodies in TBS-T buffer
containing 5% nonfat milk or 5% bovine serum albumin according
to manufacturer’s instructions. Immunoblottings were performed
using anti-gsk3b (1:500) (from Life technologies), anti-p-gsk3bSer9
(1:1000), anti-Akt (1:1000), anti-p-AktSer473 (1:1000), anti-Erk1,2
(1:1000), anti-p-Erk1,2Thr202/Tyr204 (1:1000), anti-p21 (1:1000)
(from Cell signaling), anti-b-actin (1:1500) (from Sigma–Aldrich),
Acknowledgment
The authors are grateful to the Italian M. I. U. R. for the financial
support.
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