Design, synthesis and biological evaluation of 2-alkoxycarbonyl-3-anilinoindoles as a new class of potent inhibitors of tubulin polymerization

Article abstract of DOI:10.1016/j.bioorg.2020.103665

A new class of inhibitors of tubulin polymerization based on the 2-alkoxycarbonyl-3-(3′,4′,5′-trimethoxyanilino)indole molecular skeleton was synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization and cell cycle eff

Full text of DOI:10.1016/j.bioorg.2020.103665

Journal Pre-proofs
Design, Synthesis and Biological Evaluation of 2-Alkoxycarbonyl-3-Anili-
noindoles as a New Class of Potent Inhibitors of Tubulin Polymerization
Romeo Romagnoli, Filippo Prencipe, Paola Oliva, Maria Kimatrai Salvador,
Andrea Brancale, Salvatore Ferla, Ernest Hamel, Giampietro Viola, Roberta
Bortolozzi, Leentje Persoons, Jan Balzarini, Sandra Liekens, Dominique
Schols
PII:
S0045-2068(19)31602-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.103665
YBIOO 103665
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
24 September 2019
19 December 2019
11 February 2020
Please cite this article as: R. Romagnoli, F. Prencipe, P. Oliva, M. Kimatrai Salvador, A. Brancale, S. Ferla, E.
Hamel, G. Viola, R. Bortolozzi, L. Persoons, J. Balzarini, S. Liekens, D. Schols, Design, Synthesis and
Biological Evaluation of 2-Alkoxycarbonyl-3-Anilinoindoles as a New Class of Potent Inhibitors of Tubulin
Polymerization, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103665
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
©
2020 Published by Elsevier Inc.

Design, Synthesis and Biological Evaluation of 2-Alkoxycarbonyl-3-
Anilinoindoles as a New Class of Potent Inhibitors of Tubulin
Polymerization
a
a
a
a
Romeo Romagnoli, * Filippo Prencipe, Paola Oliva, Maria Kimatrai Salvador,
b
b
c
d,e
Andrea Brancale, Salvatore Ferla, Ernest Hamel, Giampietro Viola , Roberta
d
f
f
f
f
Bortolozzi, Leentje Persoons , Jan Balzarini, Sandra Liekens, Dominique Schols
aDipartimento di Scienze Chimiche e Farmaceutiche, Università di Ferrara, 44121
Ferrara, Italy;
bSchool of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward VII
Avenue, Cardiff, CF10 3NB, UK;
cScreening Technologies Branch, Developmental Therapeutics Program, Division of
Cancer Treatment and Diagnosis, Frederick National Laboratory for Cancer Research,
National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702,
USA;
dDipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia,
Università di Padova, 35131 Padova, Italyp
eIstituto di Ricerca Pediatrica (IRP), Corso Stati Uniti 4, 35128 Padova, Italy
fRega Institute for Medical Research, KU Leuven, Laboratory of Virology and
Chemotherapy, Leuven, Belgium
*To whom correspondence should be addressed. E-mail:rmr@unife.it; Phone: 39-(0)532-
4
55303. Fax: 39-(0)532-455953. (R.R.).
1

Abstract: A new class of inhibitors of tubulin polymerization based on the 2-
alkoxycarbonyl-3-(3’,4’,5’-trimethoxyanilino)indole molecular skeleton was synthesized
and evaluated for antiproliferative activity, inhibition of tubulin polymerization and cell
cycle effects. The results presented show that the methoxy substitution and location on
the indole nucleus plays an important role in inhibition of cell growth, and the most
favorable position for the substituent was at C-6. In addition, a small-size ester function
(methoxy/ethoxycarbonyl) at the 2-position of the indole core was desirable. Also,
analogues that were alkylated with methyl, ethyl or n-propyl groups or had a benzyl
moiety on the N-1 indolic nitrogen retained activity equivalent to those observed in the
parent N-1H analogues. The most promising compounds of the series were 2-
methoxycarbonyl-3-(3’,4’.5’-trimethoxyanilino)-5-methoxyindole 3f and 1-methyl-2-
methoxycarbonyl-3-(3’,4’.5’-trimethoxyanilino)-6-methoxy-indole 3w, both of which
target tubulin at the colchicine site with antitubulin activities comparable to that of the
reference compound combretastatin A-4.
Keywords. Microtubules, structure-activity relationship, indole, antiproliferative
activity, tubulin.
2

1
. Introduction
Microtubules are polymeric structures composed of two structurally similar protein
subunits, - and -tubulin, which form heterodimers arranged head-to-tail in a linear
protofilament, with 13 protofilaments forming the cylindrical microtubule. They are
crucial in a number of cellular functions critical to the life cycle of the cell [1, 2].
The biological importance of microtubules makes them an interesting target for the
development of clinically useful anticancer drugs [3, 4], as has been demonstrated by the
therapeutic use of vinca alkaloids and taxanes [5-7]. Compounds such as vinca alkaloids
that are able to interfere with the assembly of tubulin into microtubules have antimitotic
activity and are referred to as tubulin polymerization inhibitors [8]. In contrast, taxanes
prevent microtubule disassembly and are classified as microtubule stabilizing agents [9].
Numerous chemically diverse antimitotic agents, many of which are natural products,
interact specifically with tubulin and alter tubulin polymerization [10-12]. Combretastatin
A-4 (CA-4, 1a, Figure 1), isolated from the bark of the South African tree Combretum
caffrum, is one of the well-known natural tubulin binding molecules affecting
microtubule dynamics [13]. CA-4 potently inhibited the polymerization process of tubulin
into microtubules by binding to the colchicine site on tubulin, hampering the formation
of the mitotic spindle [14]. CA-4 also displayed potent cytotoxic activities against a wide
variety of human cancer cell lines at low nanomolar concentrations, including those that
are multidrug-resistant [15]. The water-soluble disodium phosphate prodrug of CA-4
(CA-4P, 1b) showed promising results as a tumor vascular-disrupting agent (VDA) in
phase II clinical trials for multi-drug combination antitumor therapy [16-18]. Its structural
simplicity, along with its ability to selectively damage tumor neovasculature, makes CA-4
3

of great interest from the medicinal chemistry point of view, and a wide number of CA-4
analogues have been developed and evaluated in structure-activity relationship (SAR)
studies [19-22].
Several synthetic molecules have also been reported as tubulin destabilizing agents, acting
as inhibitors of tubulin polymerization [23-27]. In this respect, a literature search revealed
past [28-31] and recent [32-34] studies in which the indole molecular skeleton has been
widely employed as a versatile pharmacophore for the development of chemically diverse
small molecules identified as tubulin polymerization inhibitors.
Among the synthetic inhibitors of tubulin polymerization having an indole as their core
nucleus, it was observed that the presence of an alkoxycarbonyl function at the 2-position
of the N-1-H-indole system was essential for potent antitumor activity and for potent
inhibition of tubulin polymerization. A series of 2-alkoxycarbonyl-3-(3’,4’,5’-
trimethoxy-phenylthio/benzoyl/benzyl)-1H-indole derivatives with general structure 2
was reported as antitubulin agents by Silvestri and co-workers [35-37]. These compounds
were characterized by the presence of an electron-withdrawing atom (Cl or Br) or the
electron-donating methoxy group at the C-5 position and a methoxy/ethoxycarbonyl
moiety at the C-2 position of the indole ring, with the greatest inhibitory effects on MCF-7
cells observed for either sulfur or carbonyl derivatives bearing a 2-methoxycarbonyl
group at the 2-position of the indole.
Therefore, in continuation on our work on anticancer agents based on the indole skeleton,
in an effort to further improve activity and extend the SAR evaluation of derivatives with
general structure 2, we focused our attention on exploring the possibility of replacing the
sulfur, carbonyl or methylene bridging units between the 3-position of the indole nucleus
and the 3’,4’,5’-trimethoxyphenyl or 3’,5’-dimethoxyphenyl moieties by a bioisosteric
anilinic nitrogen (NH) moiety with hydrogen-bond accepting and donating capability to
4

furnish
a
new
series
of
2-alkoxycarbonyl-3-(3’,4’,5’-trimethoxy/2’,5’-
dimethoxyanilino)indole derivatives with general structure 3 modified with respect to
positions C-5 to C-7 with methyl, chloro or methoxy moieties.
Once the 3’,4’,5’-trimethoxyanilino or 3’,5’-dimethoxyanilino moiety was placed at the
3
-position of the indole core, we explored the SAR by examining the following
modifications:
a) various substitutions at the N-1 position (we selected hydrogen, methyl, ethyl, propyl
or benzyl to examine the steric effect of substituent R3);
b) the effect of the electron-withdrawing 2-alkoxycarbonyl group esters with either a
linear or a branched alkoxy chain (R =methyl, ethyl, n-propyl, i-propyl, t-butyl or benzyl);
2
c) the location of the methoxy group (R ) at the 5-, 6- or 7-position of the indole nucleus
1
as well as the presence of an electron-releasing methyl or electron-withdrawing chlorine
atom at the C-5 or C-6 position, respectively.
Recently, a series of 2-aroyl-3-aminopyrimidine-1H-indole derivatives with general
structure 4, possessing a morpholine or thiomorpholine linked to the C-2 position of the
pyrimidine ring were synthesized by Zhao et al [38]. Among the tested compounds, the
most promising was derivative 4a, with IC values of 0.29, 4.04, and 9.48 M against
5
0
the MCF-7, HeLa, and HCT116 cell lines, respectively. The same derivative also
exhibited the most potent anti-tubulin activity, with an IC of 19.3 M in their tubulin
5
0
polymerization assay.
The same authors also reported a series of 2-aroyl-3-(3’,4’,5’-trimethoxybenzoyl)amino-
H-indoles with general structure 5 incorporating an amido bridge into the 3-position of
1
the indole nucleus [39]. The most active derivative 5a inhibited the growth of the T47D,
BT549, and MDA-MB-231 cell lines with IC50 values of 0.04, 3.17, and 6.43 M,
5

respectively, and showed moderate antitubulin polymerization activity with an IC of 9.5
5
0
M.
H CO
OCH3
X
3
H CO
3
H CO
3
H CO
R1
3
O
CH O
3
R2
OR
OCH3
N
H
O
2
R=H, 1a, Combretastatin A-4 (CA-4)
X=S, CO or CH2
R =H, Cl , Br or OCH3
R=PO Na , 1b, Combretastatin A-4 phosphate (CA-
3
2
4P)
1
R =CH or C H
2
3
2 5
X
OCH3
H CO
3
X=H or OCH3
R =H; 5-CH ; 6-Cl; 5-, 6- or 7-OCH
3
1
3
NH
O
R =CH C H n-C H i-C H C(CH ) or CH C H
2
3,
2
5,
3
7,
3
7,
3 3
2 6 5
R =H, CH C H n-C H or CH C H
3
3,
2
5,
3
7
2
6
5
O
R1
R2
N
R3
3
H CO
3
OCH3
OCH3
N
N
X
N
NO2
NH
O
NH
R2
N
R2
R1
O
R3
R1
N
H
O
4
5
X=O or S
R =H, Cl or CH
1
3
R =H or CH3
1
R =cyclopropyl, 2-thienyl, p-PhC H or
2
6 4
R =4-PhC H 2-thienyl, 2-naphthyl
2
6 4
substituted phenyl with electron-releasing (CH or OCH )
3
3
or substituted phenyl with electron-releasing (Me or OMe) or
electron-withdrawing (Cl or Br) groups
or electron-withdrawing (Cl or Br) groups
R =H or CO Et
3
2
5
a, R =Cl, R =p-ClC H
1 2 6 4,
4
a, X=O, R =R =H, R =p-PhC H
1 3 2 6 4,
Figure 1. Structures of CA-4 (1a) and CA-4P (1b). General structures 2, 4 and 5 of various known indoles
and of 2-alkoxycarbonyl-3-anilinoindoles (3) described in this manuscript.
2
. Chemistry
3
-Amino-2-alkoxycarbonyl-1H-indoles 8a-h were synthesized in acceptable yields by
Thorpe-Zigler cyclization of the corresponding N-aryl glycinate esters 7a-h using t-BuOK
as base in THF. These latter intermediates were prepared starting from anthranilonitriles
6
a-d by the reaction with the appropriate bromoacetate esters under basic medium
(
NaHCO ). Finally, the novel derivatives 3a-j were prepared using the C-N Buchwald-
3
6

Hartwig palladium catalyzed cross-coupling protocol, by reaction of 3-amino-1H-indole
analogues 8a-h with 1-bromo-3,4,5-trimethoxybenzene or 1-bromo-3,5-
dimethoxybenzene, in the presence of Pd(OAc) , rac-BINAP and Cs CO (as catalyst,
2
2
3
ligand and base, respectively) in toluene at 100 °C (Scheme 1).
6
4
7
NH2
3
1
2
CN
CN
5
5
a
b
O
R1
R1
R1
R2
2
N
NH2
N
H
CO R
2 2
6
4
1 H
O
3
8
a-h
7
a-h
6
a-d
c
6
6
6
6
a, R =H
7a, R =H, R =CH
1 2 3
1
b, R =4-Cl
7b, R =H, R =C H
1
1 2 2 5
c, R =4-OCH3
7c, R =H, R =C(CH )
1 2 3 3
1
X
OCH3
d, R =5-OCH3
7d, R =H, R =CH C H
1
1 2 2 6 5
7
7
7
7
e, R =4-Cl, R =C H
1 2 2 5
f, R =4-OCH R =CH3
H CO
3
1
3,
2
g, R =5-OCH R =C H
1
3,
2
2 5
h, R =5-OCH R =CH3
NH
1
3,
2
O
R1
R2
N
H
O
3
3
a-h, X=OCH3
i-j, X=H
3
3
3
3
3
3
3
3
a, 3i, 8a, R =H, R =CH
1 2 3,
b, 3j, 8b, R =H, R =C H
2 5
1
2
c, 8c, R =H, R =C(CH )
1
2
3 3
d, 8d, R =H, R =CH C H
1
2
2 6 5
e, 8e, R =6-Cl, R =C H
1
2
2 5
f, 8f, R =5-OCH R =CH3
1
3,
2
g, 8g, R =5-OCH R =C H
1
3,
2
2 5
h, 8h, R =6-OCH R =CH3
1
3,
2
Scheme 1. Reagents. a: bromoacetate esters, NaHCO
rt, 2 h; c: 1-bromo-3,4,5-trimethoxybenzene or 1-bromo-3,5-dimethoxybenzene for the synthesis of 3a-h
and 3i-j, respectively, Pd(OAc) , BINAP, Cs CO , PhMe, 100 °C, 16 h.
3
, appropriate solvent, reflux, 24 h; b: t-BuOK, THF,
2
2
3
N-1-alkyl/benzyl indole derivatives 3k-ac were prepared by the synthetic route outlined
in Scheme 2. The 2-alkoxycarbony-1H-indole derivatives 9a-g were regioselectively
brominated at the 3-position with N-bromosuccinimide (NBS) in DMF as solvent to
furnish the corresponding 3-bromo-1H-indole analogues 10a-g in almost quantitative
yield. The N-1 substituted compounds 11a-p were synthesized in good yields by the
condensation of N-1H-indole analogues 10a-g with the appropriate alkyl (methyl, ethyl
or n-propyl) iodide or benzyl bromide using NaH as base in DMF. The subsequent
7

Buchwald-Hartwig reaction of the key building blocks N-alkyl and N-benzyl indole
derivatives 11a-p with 3,4,5-trimethoxyaniline or 3,5-dimethoxyaniline, following the
identical protocol reported for the preparation of compounds 3a-j, provided final
derivatives 3k-ac in acceptable yields.
4
7
Br
Br
3
5
a
b
O
O
O
R1
R1
R1
R2
R2
R2
2
N
N
N
6
1
H
O
O
O
H
R3
11a-p
9
a-g
10a-g
9
9
9
9
9
9
9
-10a, R =5-CH , R =CH
1
3
2
3
c
-10b, R =5-OCH R =CH3
1
3,
2
-10c, R =5-OCH R =C H
1
3,
2
2 5
-10d, R =5-OCH R =n-C H
1
3,
2
3 7
X
OCH3
-10e, R =5-OCH R =i-C H
1
3,
2
3 7
-10f, R =6-OCH R =CH3
1
3,
2
-10g, R =7-OCH R =CH3
H CO
3
1
3,
2
NH
O
O
R1
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
k, 11a, R =5-CH , R =R =CH
R2
1
3
2
3
3,
N
l, 11b, R =5-CH , R =CH , R =CH C H
1
3
2
3
3
2
6
5,
m, 3aa, 11c, R =5-OCH , R =R =CH
R3
1
3
2
3
3
n, 11d, R =5-OCH , R =CH , R =C H
1
3
2
3
3
2 5
o, 11e, R =5-OCH , R =CH , R =n-C H
1
3
2
3
3
3
7
3k-z, X=OCH3
3aa-3ac, X=H
p, 11f, R =5-OCH , R =CH , R =CH C H
1
3
2
3
3
2 6 5
q, 3ab, 11g, R =5-OCH , R =C H , R =CH
1
3
2
2
5
3
3
r, 11h, R =5-OCH , R =C H , R =CH C H
1
3
2
2
5
3
2 6 5
s, 3ac. 11i, R =5-OCH , R =n-C H , R =CH
1
3
2
3
7
3
3
t, 11j, R =5-OCH , R =n-C H , R =CH C H
1
3
2
3
7
3
2 6 5
u, 11k, R =5-OCH , R =i-C H , R =CH
1
3
2
3
7
3
3
v, 11l, R =5-OCH , R =i-C H , R =CH C H
1
3
2
3
7
3
2 6 5
w, 11m, R =6-OCH , R =R =CH
1
3
2
3
3
x, 11n, R =6-OCH , R =CH , R =C H
1
3
2
3
3
2 5
y, 11o, R =6-OCH , R =CH , R =CH C H
1
3
2
3
3
3
2
6
5
z, 11p, R =7-OCH , R =R =CH
1
3
2
3
Scheme 2. Reagents. Reagents. a: NBS, DMF, rt, 2 h; b: appropriate alkyl iodide or benzyl bromide, NaH,
DMF, rt, 2 h; c: 3,4,5-trimethoxyaniline or 3,5-dimethoxyaniline for the synthesis of 3k-z and 3aa-ac,
respectively, Pd(OAc) , BINAP, Cs CO , PhMe, 100 °C, 16 h.
2 2 3
3
. Biological Results and Discussion
3
.1. In vitro antiproliferative activities.
The synthesized 2-alkoxycarbonyl-3anilinoindoles 3a-c were evaluated for their ability to
inhibit the growth of a panel of four human cancer cell lines, cervix carcinoma (HeLa),
colon adenocarcinoma (HT29), breast adenocarcinoma (MCF-7) and promyelocytic
leukemia (HL-60) cells. The results are summarized in Table 1, using CA-4 (1a) as the
8

positive control. In general, the human promyelocytic leukemia HL-60 cell line was less
sensitive to these compounds as compared with the HeLa, HT29 and MCF-7 cells. In
contrast, the HL-60 cells were more sensitive than the other three lines towards the control
compound CA-4.
The data in Table 1 showed that, among the twenty-nine synthesized compounds, the most
active derivatives (with the exception of 2-ethoxycarbonyl derivative 3q) were found
among the 2-methoxyxycarbonyl indoles 3h, 3p, 3x and 3y, with antiproliferative activity
lower than 1 M against all cancer cell lines. Compound 3y, with the N-1-benzyl-2-
methoxycarbonyl-6-methoxy-indole moiety was the most potent compound of the series,
with antiproliferative IC values ranging from 0.055 to 0.17 M against the panel of
5
0
cancer cell lines. Compounds 3h, 3p and 3x had nearly equivalent activity, with IC50
values of 0.065-0.49, 0.36-0.77 and 0.063-0.93 M, respectively). In addition, derivatives
3
a, 3f-g, 3k, 3q, 3s, 3w and 3aa also showed excellent antiproliferative activity against
the HeLa, HT29 and MCF-7 cancer cell lines, with submicromolar IC values. Half of
5
0
the synthesized compounds, with IC values lower than 1 M against the HT29 cell line,
5
0
were 3-18-fold more potent than CA-4 against this cell line, with 3aa and 3y as the most
active derivatives of the series (IC :0.17 M), while almost one-third of prepared
5
0
molecules (3g-h, 3m, 3w-y and 3aa) were 2-7-fold more active than CA-4 against MCF-7
cells (IC s of 0.055-0.23 M versus 0.37 µM for CA-4). Moreover, all synthesized
5
0
molecules showed substantially reduced activity relative to CA-4 against HeLa and HL-
0 cancer cells (IC : 4 and 1 nM, respectively).
6
5
0
Table 1. In vitro inhibitory effects of compounds 3a-ac and reference compound CA-4
1a)
(
a
IC (M)
5
0
9

Compound
HeLa
0.8±0.2
0.4±0.03
>10
HT29
0.47±0.04
0.74±0.08
>10
MCF-7
0.74±0.54
1.5±0.6
HL-60
5.0±1.1
1.9±0.1
>10
3
a
3
b
3
c
>10
3
d
>10
>10
>10
>10
3
3
e
f
2.1±0.9
2.0±0.3
0.96±0.12
0.48±0.34
0.47±0.23
3.7±2.8
8.7±0.8
0.60±0.26
2.4±1.2
1.5±0.5
1.1±0.6
1.2±0.5
0.77±0.13
0.54±0.30
1.7±1.3
0.85±0.15
5.3±0.8
>10
0.44±0.02
0.50±0.38
0.10±0.09
0.065±0.054
0.40±0.01
1.7±0.2
0.37±0.32
0.74±0.35
0.20±0.12
0.43±0.20
0.45±0.04
0.36±0.16
0.38±0.09
0.48±0.25
0.62±0.10
3.1±2.1
1.1±0.0
1.4±1.1
6.6±3.3
0.49±0.18
7.0±1.0
7.8±1.9
>10
0.50±0.22
0.60±0.37
0.13±0.04
1.4±0.5
4.8±4.5
0.19±0.11
1.8±0.1
0.62±0.41
0.53±0.44
0.60±0.29
0.68±0.30
0.60±0.43
1.3±0.6
3
g
3
h
3
3
i
j
3
k
3
l
>10
3
m
1.6±1.0
1.7±0.2
3.2±1.0
0.60±0.35
0.68±0.12
0.64±0.28
3.0±0.11
2.6±2.2
>10
3
n
3
o
p
q
3
3
3
3
3
r
s
t
0.95±0.41
7.8±1.7
3
u
>10
>10
3
v
>10
>10
>10
>10
3
3
3
w
x
y
0.54±0.30
0.58±0.32
0.13±0.06
7.3±2.2
0.41±0.19
0.77±0.00
6.3±2.9
0.32±0.03
0.93±0.18
0.17±0.02
1.7±0.1
0.17±0.08
1.1±0.9
6.5±0.2
3.1±0.2
0.11±0.06
0.063±0.017
0.055±0.035
0.14±0.08
0.15±0.04
0.3±0.21
3.0±2.0
1.1±0.7
0.10±0.02
0.096±0.054
8.1±1.1
6.2±1.1
6.2±1.0
>10
3
z
3
aa
ab
ac
CA-4 (1a)
3
3
0.004±0.001
0.37±0,043
0.001±0.0002
aIC50= compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as
the mean ± SE from the dose-response curves of at least three independent experiments.
For the derivative 3a, the IC values ranged from 0.47 to 0.8 M against three of the four
5
0
cancer cell lines. The elongation of the alkyl chain of 2-alkoxycarbonyl function of
compound 3a from methyl to ethyl, to yield 3b, has a contrasting effect, with 1.5- and 2-
fold reductions of activity on HT-29 and MCF-7 cells, respectively, and a 2- and 2.5-fold
increased activity against HeLa and HL-60 cells, respectively. For compounds 3a and 3b,
1
0

replacing a 3,4,5-trimethoxy phenyl ring with a 3,5-dimethoxyphenyl moiety (compounds
i and 3j, respectively) caused a reduction of activity (1.5-12-fold) in all cell lines.
3
Replacement of methyl or ethyl of the 2-alkoxycarbonyl moiety on the 1H-indole nucleus
of compounds 3a and 3b with a more bulky t-butyl or benzyl substituent (compounds 3c
and 3d, respectively) resulted in a dramatic loss of potency (IC >10 M), thus revealing
5
0
a steric effect of the substituent at the C-2 position of the indole ring on cell growth
inhibitory activity.
The introduction of the electron-withdrawing chlorine atom at the C-6 position of the 1H-
indole nucleus of compound 3b, to yield derivative 3e, caused a 3- and 5-fold reduction
of activity against HT-29 and HeLa cells, respectively, while a 1.5- and 3-fold increase of
activity was observed against HL-60 and MCF-7 cells.
The methoxy substituent on the benzene portion of the indole moiety, as well as the
absence or presence of alkyl or benzyl substituents on the indole nitrogen, play an
important role in affecting antiproliferative activity.
The introduction of a methoxy group at the C-5 position of derivative 3a, to yield 3f,
increased from 1.5 to 3-fold the cell growth inhibitory activity relative to 3a against HeLa,
MCF-7 and HL-60 cells, with a 1.5-fold reduced activity against HT29 cells.
Homologation of the alkyl chain of the alkoxycarbonyl group by an extra methylene unit,
from methyl to ethyl (compounds 3f and 3g, respectively), increased antiproliferative
activity 2- and 5-fold against HT-29 and MCF-7 cells, respectively, while the 2-
ethoxycarbonyl-1H-indole derivative 3g was 5-fold less potent than 3f against HL-60
cells, and the two derivatives proved equipotent against HeLa cells. For compound 3f,
moving the methoxy group from the 5-to the 6-position of the 1-H-indole ring (derivative
3
h) increased activity 2-8-fold against the whole panel of cancer cell lines.
1
1

Through the synthesis of compounds 3k-ac, we investigated whether the introduction of
a methyl, ethyl, n-propyl or benzyl moiety at the indole nitrogen of derivatives 3f-h would
lead to an increase or loss of activity through a steric effect. Starting from compound 3f,
the insertion of a methyl group at the N-1 position of the indole nucleus (3m) caused a
2
.5-fold increase in antiproliferative activity relative to 3f against MCF-7 cells, while the
two derivatives were equipotent against the other cancer cell lines. Replacement of the C-
5
2
methoxy of 3m with a weak electron-releasing methyl group (3k), increased potency
.5 and 3-fold against HT-29 and HeLa cells, respectively, while a strong reduction in
activity (27-fold) was observed against HL-60 cells. For compound 3m, replacement of
the 3,4,5-trimethoxyphenyl ring with a 3,5-dimethoxy phenyl moiety (3aa) was well
tolerated by the HeLa and MCF-7 cells, with the two compounds showing similar
potency, while the latter compound was 9-fold more active than the former against HT-
2
9 cells. In contrast, 3m was 4-fold more potent than 3aa against the HL-60 cell line.
Homologation of the N-1 alkyl chain on the indole nucleus from methyl to ethyl to n-
propyl (compounds 3m, 3n and 3o, respectively), maintained antiproliferative activity
relative to the N-methyl derivative 3m on the HeLa and HT-29 cell lines, while 3n and
3
o were equipotent although half as potent as the N-1H-indole derivative 3m against
MCF-7 cells. The n-propyl derivative 3o was half as active as 3m and 3n against HL-60
cells, with these latter derivatives being equipotent.
For the 2-methoxycarbonyl indole derivative 3m, the N-1-methyl can be replaced with a
benzyl moiety (compound 3p) without substantial loss of activity against HeLa cells, and
with a 2-fold increase in potency against HT-29 and HL-60 cells, while antiproliferative
activity was reduced 2-fold against MCF-7 cells. Replacement of the C-6 methoxy of
derivative 3p with a methyl (3l) caused a reduction of antiproliferative activity relative to
1
2

3
p, which was moderate (2-3-fold) against three of the four cancer cell lines, and dramatic
(85-fold) on HL-60 cells.
For the 2-ethoxycarbonyl-1H-indole derivative 3g, replacement of hydrogen with a
methyl group at the indolic nitrogen, to furnish derivative 3q, maintained potency against
HeLa and HT-29 cells relative to 3g, while an opposite effect was observed against the
other two cancer cell lines, with a 4-fold reduction of potency against MCF-7 cells and a
1
0-fold increase of activity against HL-60 cells.
The 3’,4’,5’-trimethoxyanilino moiety of compound 3q can be replaced with a 3,5-
dimethoxyanilino function (3ab) without loss of activity against HeLa and MCF-7 cells,
while 3ab was 2- and 9-fold less active than 3q against HT-29 and HL-60 cells,
respectively. The substitution of the N-1-methyl group of 3q with a bulkier benzyl moiety
(3r) was tolerated against MCF-7 and HL-60 cells, while a 2- and 3-fold reduction in
activity was observed against HeLa and HT-29 cells, respectively.
Comparing the activities of derivatives characterized by the presence of a common N-1-
methyl-5-methoxy indole nucleus and a different 2-alkoxycarbonyl function (3m, 3q, 3s
and 3u), the 2-methoxycarbonyl derivative 3m had 2- and 3-fold reduced activity relative
to the corresponding 2-ethoxycarbonyl homologue 3q against HL-60 and HT-29 cells,
while this latter compound was 2-fold less potent than 3m against MCF-7 cells. The two
derivatives showed similar potency against Hela cells. A further homologation of the alkyl
chain of the alkoxycarbonyl moiety from ethyl to n-propyl (3s) slightly decreased
antiproliferative activity. Reduction of activity was dramatic (two-orders of magnitude)
when the n-propyl chain of 2-alkoxycarbonyl derivative 3s was replaced with the
isomeric, branched isopropyl group, to yield the isopropyl ester 3u. A reduction of
potency (5-10-fold) was also observed by replacing the 3,4,5-trimethoxyphenyl ring of 3s
1
3

with a 3,5-dimethoxyphenyl system (3ac). Replacement of the N-1-methyl group of n-
propyloxycarbonyl indole derivative 3s with a more bulky and lipophilic benzyl group
(3t) also had a pronounced effect on potency, with a reduction in activity on three of the
cell lines, while the two compounds were equipotent against HeLa cells.
For 2-methoxycarbonyl indole derivatives 3f, 3h, 3m, 3p and 3w-z, the position of the
methoxy group on the benzene portion of the indole moiety was crucial for
antiproliferative activity, with the greatest potency occurring when the methoxy group
was located at the C-6 position.
Comparing the activities of the three isomeric N-1-methyl-2-methoxycarbonyl indole
derivatives 3m, 3w and 3z, the greatest activity occurred when the methoxy group was
located at the C-6 position (3w), the least when it was located at the C-7 position (3z).
A contrasting effect was observed comparing the activities of the N-1-methyl/ethyl-6-
methoxy-2-methoxycarbonyl pair 3w and 3x, with the N-1-methyl derivative 3w being 2-
and 10-fold less active than its N-1-ethyl homologue 3x against MCF-7 and HL-60 cells,
respectively, while the two compounds were equipotent against HeLa cells. Only against
the HT-29 cells, 3x was 3-fold more active than 3w.
For the C-6 methoxy derivative 3x, replacement of the N-1-ethyl with a benzyl group (3y)
maintained antiproliferative activity against MCF-7 and HL-60 cells, with the latter
derivative being 4- and 5- fold more active than the former against Hela and HT-29 cells,
respectively.
3
.2. In vitro inhibition of tubulin polymerization and colchicine binding.
As presented in Table 1, nine compounds, 3f-g, 3k, 3m, 3o-p, 3w, 3y and 3aa, as well as
reference compound CA-4, were evaluated for their antitubulin polymerization activities
in order to understand whether these new 2-alkoxycarbonyl indoles exerted their
1
4

antiproliferative activities through an interaction with microtubules. The selected
compounds were also examined at two different concentrations (5 and 0.5 M) for their
3
ability to compete with colchicine for binding to tubulin using a [ H]colchicine binding
assay.
In the tubulin polymerization assay, seven out of nine of the tested compounds (3f-g, 3k,
3
m, 3aa, 3w and 3y) strongly inhibited tubulin assembly with activities comparable or
slightly superior (IC : 0.37-0.68 M) to that of CA-4 (IC : 0.54 M), while derivative
5
0
50
3
o (IC :1.6 M) was 3-fold less active than CA-4. Despite appreciable cell growth
5
0
inhibitory activity (IC : 0.36-0.77 M) against the panel of cancer cells, compound 3p
5
0
was inactive in the tubulin polymerization assay (IC >20 M), indicating that this
5
0
molecule may exert its antiproliferative effect by a different mechanism of action. When
comparing the inhibition of tubulin polymerization versus the growth inhibitory effect,
we found that the potency of these seven molecules for inhibition of tubulin assembly was
comparable with the activity observed for CA-4, but they were considerably less active
than CA-4 as antiproliferative agents against HeLa and HL-60 cells. This discrepancy can
possibly be rationalized by a limited penetration into the cells or any other mechanism
limiting the accessibility of these molecules to cellular tubulin.
Table 2. Inhibition of tubulin polymerization and cochicine binding by compounds 3f-g,
3
k, 3m, 3o-p, 3w, 3y and 3aa and CA-4
Tubulin assemblya
Colchicine bindingb
% ±SD
IC ±SD (M)
5
0
Compound
5
M drug
0.5 M drug
3
f
0.40±0.07
0.84±0.1
94±0.6
80±2
53±0.04
n.d.
3
g
1
5

Tubulin assemblya
Colchicine bindingb
% ±SD
IC ±SD (M)
5
0
Compound
5
M drug
0.5 M drug
3
k
0.53±0.04
0.54±0.03
1.6±0.2
67±5
75±2
n.d.
n.d.
3
m
3
o
44±1
n.d.
3
p
>20
15±0.6
97±2
n.d.
3
w
0.37±0.07
0.42±0.07
0.47±0.03
0.54±0.06
75±4
n.d.
3
y
68±0.9
82±1
3
aa
n.d.
CA-4 (1a)
98±0.9
85±2
a
Inhibition of tubulin polymerization. Tubulin was at 10 M.
b
3
Inhibition of [ H]colchicine binding. Tubulin and colchicine were at 0.5 and 5 M concentrations,
respectively.
n.d.=not determined
In the colchicine competition experiments, compounds 3f-g, 3m, 3w and 3aa at 5 M
3
strongly inhibited [ H]colchicine binding to tubulin, with 68-97% inhibition. Compounds
3
f and 3w were the most potent inhibitors of tubulin polymerization (IC =0.40 and 0.37
50
M, respectively), and this was also confirmed in the colchicine binding assay (94% and
9
7
7% inhibition at 5 µM). When the concentration of 3f and 3w was 0.5 M, 53% and
3
5% inhibition, respectively, of the binding of [ H]colchicine to tubulin was observed. In
addition, compounds 3f and 3w were as active as CA-4 as inhibitors of colchicine binding
at 5 M, while the difference between 3f and CA-4 was more prominent than that between
3
w and CA-4 at 0.5 M.
Comparing the activities of compounds 3m and 3aa, we can observe that the 3’,4’,5’-
trimethoxyphenyl moiety of 3m, a well-defined pharmacophore for the inhibition of
tubulin polymerization found in colchicine, CA-4 and podophyllotoxin, was not essential
for potent inhibition of tubulin polymerization and colchicine binding. This moiety can
1
6

be replaced with the 3’,5’-dimethoxyphenyl ring of derivative 3aa without loss of activity
IC =0.54 and 0.47 M for assembly, 75% and 82% inhibition of the binding of
(
5
0
colchicine for 3m and 3aa, respectively, at 5 M). A linear correlation between inhibition
of tubulin assembly and colchicine binding inhibition was observed between analogue
pairs 3m/3aa and 3f/3w, while a different behavior was observed for derivatives 3w and
3
3
6
y. These latter compounds were both equipotent as inhibitors of tubulin assembly, while
3
w was much more active than 3y as an inhibitor of [ H]colchicine binding (97% and
8% inhibition, respectively).
3
.3. Molecular modeling studies.
The tubulin colchicine site is a flexible area, which can adapt to different ligands by
changing the architecture of the pocket. Diverse ligands may induce different
conformations of the same protein, and therefore an inhibitor, whose structure induces a
specific protein conformation, may not be correctly docked by the program against
another protein conformation, often giving poor correlation between biological data and
computational results. In order to explore the potential binding mode of selected newly
synthesized compounds (3f, 3g, 3k, 3m, 3o, 3p, 3w, 3y and 3aa) and to consider the
flexibility of the colchicine binding domain, an ensemble docking study using Glide SP
was performed [40]. Ensemble docking reduces potential evaluation errors associated
with receptor rigidity by docking a series of ligands against multiple crystal structures of
the same protein.
Three crystal structures were used: tubulin co-crystallized with vinblastine (PDB ID:
5
BMV) [41], in which the colchicine binding domain is unoccupied; tubulin co-
crystallized with colchicine (PDB ID: 4O2B) [42]; and tubulin co-crystallized with
1
7

microtubule-destabilizing CA-4 (1a) (PDB ID: 5LYJ)[43]. These crystals represent three
different conformations of the colchicine binding area.
While, as expected, none of the molecules was docked by Glide in the unbound form of
tubulin (5BMV), the CA-4-tubulin complex (5LYJ) was able to correctly accommodate
only compound 3f and 3g, the unsubstituted derivatives at the N-1 position, whereas the
rest of the molecules either provided an implausible binding mode if correlated with their
biological activity (3k, 3m, 3o and 3aa), or were not docked by the program (3p, 3w, and
3
y). These compounds are all characterised by various substitutions at the N-1 position of
the indole ring. In contrast, all the compounds were correctly docked in the colchicine-
tubulin crystal structure (4O2B). The loop formed by residues between Gln247 and

Ala250 occupies the central area of the colchicine domain on the unoccupied tubulin
structure, not allowing the binding of any ligand (Figure 2). The presence of colchicine
or CA-4 forces this loop to move away from this area, inducing the formation of the
binding pocket. A second loop, which goes from Ser178 to Val181, further reduces the
size of the colchicine domain by moving toward the -tubulin unit in two of the crystal
structures, the unbound form (5BMV) and the CA-4-tubulin complex (5LYJ), whereas,
in the third crystal structure (4O2B), the lateral amide substituent of colchicine pushes
this loop to widen the binding area (Figure 2).
1
8

Figure 2. Superposition of the colchicine site of three tubulin crystal structures: unoccupied colchicine
binding domain (PDB ID: 5BMV). The -tubulin unit is represented as a light green ribbon; the -tubulin
unit is represented as a dark green ribbon; tubulin co-crystallized with colchicine (PDB ID: 4O2B). The -
tubulin unit is represented as an air force blue ribbon; the -tubulin unit is represented as a dark blue ribbon.
Co-crystallized colchicine is shown in blue; tubulin co-crystallized with microtubule-destabilizing CA-4
(
PDB ID: 5LYJ). The -tubulin unit is represented as a pink ribbon; the -tubulin unit is represented as a
salmon ribbon. Co-crystallized CA-4 is shown in pink). The Gln247-Ala250 loop is represented in red,
whereas the Ser178-Val181 loop in represented in yellow. Note the different architecture of the
colchicine binding area in the three crystal structures.
3
f and 3g, the two compounds that obtained the best docking score in both crystals,
occupy the active site similarly to the co-crystallized ligands, with the trimethoxyphenyl
ring overlapping the same ring in the CA-4 and colchicine structures (Figure 3).
1
9

Figure 3. Proposed binding mode for compound 3f in 5LYJ (A) and 4O2B (B) and for compound 3g in
LYJ (C) and 4O2B (D). The two compounds occupy the active site similarly to the co-crystallized ligands,
5
especially their trimethoxyphenyl rings. Co-crystallized CA-4 is shown in pink, colchicine in blue,
compound 3f in yellow and compound 3g in dark purple. The -tubulin unit from 5LYJ is represented as a
pink ribbon, and the -tubulin unit is shown as a salmon ribbon. The -tubulin unit from 4O2B is
represented as a blue ribbon, and the -tubulin unit is represented as a dark blue ribbon. The Gln247-

Ala250 loop is represented in red, and the Ser178-Val181 loop in represented in yellow.
The anilinic nitrogen bridging the indole nucleus and the 3’,4’,5’-trimethoxyphenyl
confers a conformation to the molecules that mimics the CA-4 cis-olefin bridge and the
central colchicine B ring. H-bond formation between the 4-methoxy group and Cys241
is present in both crystals, while interaction between the indole NH and Thr179 and
between the carboxylic group and Ala250/Asn268 further stabilize the compounds on
the CA-4 binding site. As mentioned above, introduction of various substitutions at the
N-1 position of the indole ring does not allow a correct occupation of the binding area on
the CA-4-tubulin complex (5LYJ), potentially due to steric clashes between the different
2
0

substituents and the Ser178-Val181 loop. Analyzing the docking results on the
colchicine-tubulin crystal structure (4O2B), the simultaneous presence of the methoxy
group at the 5-position and a substituent at the N-1 of the indole nucleus forces the
compounds to occupy the active site in a non-optimal manner, potentially resulting in a
reduced inhibition of colchicine binding (Figure 4). In particular, the anilinic nitrogen
bridge assumes a conformation that impedes the central indole core from overlapping the
central colchicine B ring, and this effect is accentuated with increasing N-1 substituent
size. 3p, with a benzyl substituent at N-1, did not have a plausible binding mode, in
agreement with its low ability to inhibit colchicine binding (Figure 4).
Figure 4. Proposed binding modes for compound 3m in 5LYJ (A) and 4O2B (B) and for compound 3p in
4
O2B (C). Introduction of a methyl group at the N-1 position of the indole ring does not allow correct
occupation of the binding area on the CA-4-tubulin complex (5LYJ) by 3m (A), potentially due to steric
clashes between the methyl group and the Ser178-Val181 loop. Although compound 3m is able to bind
to the colchicine-tubulin crystal structure (4O2B), the simultaneous presence of the methoxy group at the
5
-position and a substituent at the N-1 of the indole nucleus forces the compound to sit in the active site in
a non-optimal manner (A). In particular, the anilinic nitrogen bridge assumes a conformation that impedes
2
1

the central indole core of 3m from superimposing on ring B of colchicine. Compound 3p, having a benzyl
substituent on N-1, did not show a plausible binding mode on 4O2B, which is consistent with its minimal
inhibition of colchicine binding. Co-crystallized CA-4 is shown in pink, colchicine in blue, compound 3m
in green and compound 3p in gold. The -tubulin unit from 5LYJ is represented as a pink ribbon, and the
-tubulin unit is shown as a salmon ribbon. The -tubulin unit from 4O2B is represented as a blue ribbon,
and the -tubulin unit is represented as a dark blue ribbon. The Gln247-Ala250 loop is represented in
red, and the Ser178-Val181 loop in represented in yellow.
Moving the methoxy group from position 5 to 6 (3w) allows a correct occupation of the
binding area, comparable to the binding pose found for 3f, and is in accord with the
colchicine binding assay results (Figure 5). Increasing the size of the N-1 substituent to a
benzyl ring (3y) causes a change in the anilinic nitrogen bridge similar to the one observed
for 3m, potentially explaining the similar inhibition of colchicine binding found for these
three compounds (Figure 5). Finally, the 3’,5’-dimethoxyphenyl compound 3aa binds in
the active site similarly to the 3’,4’,5’-trimethoxyphenyl derivative 3m, in accord with
their similar inhibition of colchicine binding.
Figure 5. Proposed binding mode for compounds 3w (A) and 3y (B) in 4O2B. Moving the methoxy group
from position 5 to 6 in 3w allows a correct occupation of the binding area despite the presence of a methyl
group at the N-1 position (A). Increasing the size of the N-1 substituent to a benzyl ring in 3y causes a
change in the anilinic nitrogen bridge similar to the one observed for 3m. Co-crystallized colchicine is
shown in blue, compound 3w in salmon and compound 3y in orange. The -tubulin unit from 4O2B is
represented as a blue ribbon, and the -tubulin unit is represented as a dark blue ribbon. The Gln247-

Ala250 loop is represented in red, and the Ser178-Val181 loop in represented in yellow.
2
2

In conclusion, molecular docking studies have given important insights into the potential
binding mode for this family of compounds. Furthermore, the results have highlighted the
importance of selecting the appropriate crystal structure of a given protein to perform
reliable docking simulations.
3
.4. Analysis of cell cycle effects.
Since molecules exhibiting activity on tubulin binding should cause alteration of cell
cycle parameters, leading to a preferential G2/M blockade, the effects of compounds 3m,
3
w and 3y on cell cycle distribution were analyzed on HeLa cells cultured for 24 h in the
-4
presence of. six different concentrations ranging from 6.4x10 to 2 µM for each tested
derivative (Table 3). All three compounds caused a dose-dependent accumulation of cells
in the G2/M phase, with a concomitant reduction of cells in the G1 phase. Compound 3w
was the most active compound since at 0.0032 μM it induced a significant increase in
cells in the G2/M phase (Figure 6), in excellent agreement with the data on inhibition of
tubulin polymerization. The other two compounds (3m and 3y) were less potent and were
similar to each other, blocking the cell cycle only at 0.08 μM.
2
3

Table 3. Cell cycle analysis of compounds 3m, 3w, 3y and reference compound CA-4
1a)
(
Concentration
(µM)
Distribution of cells (%)a
G /G
12.22±4.34 12.01±4.39 65.82±4.57
Compound
Sub-G1
9.95±4.11
11.62±1.31 16.46±2.91 16.50±4.69 55.42±8.79
6.18±1.44 25.94±2.39 37.23±6.27 30.66±9.54
1.75±1.23 58.74±11.27 16.90±1.17 22.60±8.88
S
G /M
0
1
2
3
m
2
0
.4
.08
.016
.0032
0
0
0
2.78±2.99
4.19±2.90
3.22±3.45
7.99±5.49
7.21±2.26
5.99±2.14
5.59±3.30
6.76±1.61
3.14±2.17
3.22±3.45
7.97±2.47
9.33±2.45
4.46±0.45
3.60±2.34
65.21±3.57 17.68±0.41 14.33±5.31
61.99±1.65 19.59±2.28 14.22±4.03
61.93±4.18 14.59±2.02 18.99±3.83
0
.00064
0
2
3
w
8.74±1.33
11.46±1.18 71.80±4.83
0
.4
.08
.016
12.87±5.06 14.13±5.06 65.80±5.25
10.57±2.52 13.80±1.69 69.66±1.94
10.23±2.78 13.63±4.68 70.55±10.62
14.93±3.14 33.04±4.12 45.27±3.77
67.28±4.13 15.60±3.80 13.98±2.04
61.93±4.18 14.59±2.02 18.99±3.83
10.42±5.67 11.01±2.81 70.61±5.64
11.51±3.82 12.42±2.62 66.75±4.77
52.79±3.93 18.69±1.67 24.05±3.13
0
0
0
.0032
0
.00064
0
2
3
y
0
.4
.08
.016
0
0
67.42±4.08 20.77±4.73
8.21±5.60
7.54±3.03
5.19±1.39
0
.0032
11.12±12.37 59.46±10.66 21.89±0.89
13.78±13.85 60.32±12.40 20.71±5.26
0
.00064
0
2
3.22±3.45
14.75±6.47
8.13±3.97
6.78±2.80
9.32±1.12
61.93±4.18 14.59±2.02 18.99±3.83
8.45±4.23 7.74±0.54 69.07±4.36
10.55±3.80 14.32±2.86 66.99±4.98
10.10±2.49 15.19±1.83 67.92±6.91
14.31±2.87 15.24±3.07 61.13±1.31
CA-4 (1a)
0
.4
.08
.016
0
0
0
0
.0032
.00064
0
13.63±4.85 13.90±4.10 15.43±0.93 57.02±9.27
10.50±2.60 33.98±11.03 23.41±10.42 32.11±1.54
3.22±3.45
61.93±4.18 14.59±2.02 18.99±3.83
a
Percentages of cells in each phase of the cell cycle in HeLa cells treated with the indicated compounds at
the indicated concentrations for 24 h. CA-4 (1a) was used as a positive control. Cells were fixed and
labeled with DAPI and analyzed by high content imaging as described in the experimental section. Data
are expressed as the mean ± SD of two independent experiments.
2
4

Figure 6. The impact of compounds 3m, 3w and 3y on cell cycle of HeLa cells. HeLa cells were treated
with six different concentrations of 3m, 3w, 3y and reference compound CA-4 (1a) for 24 h and then
assayed via high content imaging. The bar chart shows the percentage of cell cycle distribution based on
the imaging results.
3
.5. Apoptosis induction assay of compounds 3m, 3w and 3y in HeLa cancer and human
non-cancer cells.
To determine the ability of compounds 3m, 3w and 3y to induce apoptosis, HeLa and
healthy human peripheral blood mononuclear cells (PBMC) were incubated and
monitored at 37 °C for three days in the presence of each compound at different
concentrations ranging from 6.4x10-4 to 10 µM. The induction of apoptosis was
monitored by the activation of caspase 3/7, a well-known marker of apoptosis, using a
commercial kit, as described in the experimental procedures. CA-4 was employed as a
control compound. As shown in Table 4, derivatives 3m, 3w and 3y, as well as CA-4,
were ineffective in PBMC, with IC values (>10 M) that were negligible as compared
5
0
2
5

with those found against the HeLa cancer cells and against other cancer cell lines (Table
). Altogether, these data suggest that these compounds not only induce apoptosis but
1
may also have cancer cell selective antiproliferative properties.
Table 4. EC values for apoptosis induction by 3m, 3w, 3y and control compound CA-
5
0
4
(1a) in HeLa cancer cells and PBMC.
EC50a (M) caspase 3/7 activation
Compound
HeLa
PBMC
>10
3
m
0.37±0.11
0.069±0.008
0.53±0.16
0.005±0.002
3
w
>10
3
y
>10
CA-4 (1a)
>10
aEC50= compound concentration required to activate caspase 3/7 by 50%. Data are expressed as the mean
SE from the dose-response curves of at least two independent experiments.
±
4
. Conclusions
In this report we have demonstrated that replacing the sulfur atom, carbonyl moiety or
methylene unit bridging the 3’,4’,5’-trimethoxyphenyl ring and C-3 position of the indole
nucleus of compounds with general structure 2 previously reported by Silvestri et al. with
an anilinic nitrogen (NH) yielded a new series of 2-alkoxycarbonyl-3-anilino indoles with
general formula 3 with potent antiproliferative activity caused by their inhibition of
tubulin polymerization. Various substituents were tolerated on the indolic nitrogen, with
alkyl and benzyl substitution of compounds 3k-ac that generally led to little effect on
antiproliferative potency, while a small-sized methoxy/ethoxycarbonyl moiety at the 2-
2
6

position of the indole nucleus is important for optimal antiproliferative activity (as
depicted in Figure 7). The 3’,4’,5’-trimethoxyanilino moiety at the 3-position of the indole
skeleton can be replaced by a 3’,5’-dimethoxyanilino function without a loss of activity
(compare 3a with 3i, 3b with 3j, 3m with 3aa and 3q with 3ab). Further analysis of the
SAR showed that antiproliferative activity is also related to the presence and position of
the methoxy group at the C-5 or C-6 position on the benzene portion of the indole ring,
with the 6-OMe derivatives generally more active than their corresponding 5-OMe
counterparts (i.e, 3g vs. 3h, 3m vs. 3w, 3n vs. 3x, 3p vs. 3y).
The data identified tubulin as the molecular target of most compounds, since those with
the greatest inhibitory effects on cell growth strongly inhibited tubulin assembly and the
binding of colchicine to tubulin, with a good correlation between the inhibition of tubulin
polymerization versus the antiproliferative effects. In particular, we have identified 2-
methoxycarbonyl-3-(3’,4’.5’-trimethoxyanilino)-5-methoxyindole (3f) and 1-methyl-2-
methoxycarbonyl-3-(3’,4’,5’-trimethoxyanilino)-6-methoxyindole
(3w)
as
new
antiproliferative agents that target tubulin at the colchicine site with antitubulin activities
comparable to that of the reference compound CA-4.
Three of the most active compounds in the tubulin assay, corresponding to 2-
methoxycarbonyl-3-(3’,4’.5’-trimethoxyanilino) indoles 3w, 3m and 3y, induced arrest
of Hela cells in the G2/M phase of the cell cycle. Further experiments demonstrated that
these compounds induce apoptosis in Hela cells and were practically devoid of significant
apoptotic activity in normal PBMCs.
2
7

X as H or OCH , OCH
3
3
was not essential for activity
X
OCH3
NH
H CO
3
R as CH or C H was crucial
for potent antiproliferative activity
2
3
2 5
O R2
R as 5-CH or 5-/6-
1
3
R1
R as n-C H , i-C H , C(CH ) or
OCH improved activity
2
3
7
3
7
3 3
3
N
O
CH C H abolished activity
compared with hydrogen
2 6 5
R3
R as hydrogen, alkyl or
3
benzyl group was tolerated
Figure 7. SAR analysis of 2-alkoxycarbonyl-3-anilino indole analogues 3a-c
5
. Experimental Protocols.
5
5
.1. Chemistry.
.1.1. Materials and Methods.
1H experiments were recorded on either a Bruker AC 200 or a Varian 400 Mercury Plus
1
3
spectrometer, while C NMR spectra were recorded on a Varian 400 Mercury Plus
spectrometer. Chemical shifts () are given in ppm upfield from tetramethylsilane as
internal standard, and the spectra were recorded in appropriate deuterated solvents, as
indicated. Positive-ion electrospray ionization (ESI) mass spectra were recorded on a
double-focusing Finnigan MAT 95 instrument with BE geometry. Melting points (mp)
were determined on a Buchi-Tottoli apparatus and are uncorrected. Microwave-assisted
reactions were performed on a CEM Discover SP single-mode reactor. Closed vessel
experiments were carried out in capped microwave-dedicated vials (10 mL). The
temperature of the reaction was monitored by an external fiber optic temperature sensor.
After completion of the reaction, the mixture was cooled to 25 °C via air-jet cooling. All
2
8

products reported showed H and 13C NMR spectra in agreement with the assigned
structures. The purity of tested compounds was determined by combustion elemental
analyses conducted by the Microanalytical Laboratory of the Chemistry Department of
the University of Ferrara with a Yanagimoto MT-5 CHN recorder elemental analyzer. All
tested compounds yielded data consistent with a purity of at least 95% as compared with
the theoretical values. All reactions were carried out under an inert atmosphere of dry
nitrogen, unless otherwise indicated. Standard syringe techniques were used for
transferring dry solvents. Reaction courses and product mixtures were routinely
1
monitored by TLC on silica gel (precoated F
Merck plates), and compounds were
54
2
visualized with aqueous KMnO . Flash chromatography was performed using 230-400
4
mesh silica gel and the indicated solvent system. Organic solutions were dried over
anhydrous Na SO .
2
4
5
.1.2. General method A for the synthesis of compounds 7a-h.
A suspension of the appropriate 2-aminobenzonitrile 6a-d (10 mmol), appropriate alkyl
or benzyl bromoacetate (10 mmol) and NaHCO (0.99 g, 12 mmol, 1.2 equiv.) in the
3
appropriate solvent (10 mL) was stirred at reflux for 24 h. After this time, the appropriate
alkyl or benzyl bromoacetate (10 mmol) was added and the mixture heated at reflux for
an additional 24 h. The reaction mixture was cooled to ambient temperature and filtered
through Celite, then the filtrate was evaporated in vacuo. The residue was diluted with
dichloromethane (25 mL) and washed sequentially with water (10 mL) and brine (10 mL).
The organic layer was dried over Na SO , filtered and concentrated under reduced
2
4
pressure, and the residue was stirred with the appropriate solvent or purified by flash
column chromatography on silica gel.
2
9