Unconventional Knoevenagel-type indoles: Synthesis and cell-based studies for the identification of pro-apoptotic agents

Article abstract of DOI:10.1016/j.ejmech.2015.08.009

A new series of indole-based analogues were recently identified as potential anticancer agents. The Knoevenagel-type indoles herein presented were prepared via a one-pot condensation of iminium salts with active methylene reagents and were isolated as single geometric isomers. Biological evaluation in different cell-based assays revealed an antiproliferative activity for some analogues already in the nanomolar range against leukaemia, breast and renal cancer cell lines. To explain these effects, the most promising analogues of the series were engaged in further cell-based studies. Compounds 5e, l, p and 6a, b highlighted a pro-apoptotic potential being able to induce apoptosis in HL60, K562 and MCF-7 cell lines in a dose and time-dependent manner. The ability of these compounds to arrest cell cycle at the G2/M phase inspired the immunofluorescence studies which allowed us to identify tubulin as a potential target for compounds 5l and 6b.

Full text of DOI:10.1016/j.ejmech.2015.08.009

Accepted Manuscript
Unconventional Knoevenagel-type Indoles: Synthesis and Cell-based Studies for the
Identification of Pro-apoptotic Agents
Dr. Andrea Spallarossa, Chiara Caneva, Matteo Caviglia, Silvana Alfei, Stefania
Butini, Giuseppe Campiani, Sandra Gemma, Margherita Brindisi, Daniela M.
Zisterer, Sandra A. Bright, Clive D. Williams, Emmanuele Crespan, Giovanni Maga,
Giuseppina Sanna, Ilenia Delogu, Gabriella Collu, Roberta Loddo
PII:
S0223-5234(15)30193-8
10.1016/j.ejmech.2015.08.009
EJMECH 8045
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 April 2015
Revised Date: 22 July 2015
Accepted Date: 4 August 2015
Please cite this article as: A. Spallarossa, C. Caneva, M. Caviglia, S. Alfei, S. Butini, G. Campiani,
S. Gemma, M. Brindisi, D.M. Zisterer, S.A. Bright, C.D. Williams, E. Crespan, G. Maga, G. Sanna,
I. Delogu, G. Collu, R. Loddo, Unconventional Knoevenagel-type Indoles: Synthesis and Cell-based
Studies for the Identification of Pro-apoptotic Agents, European Journal of Medicinal Chemistry (2015),
doi: 10.1016/j.ejmech.2015.08.009.
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ACCEPTED MANUSCRIPT
Graphical Abstract
1

ACCEPTED MANUSCRIPT
Unconventional Knoevenagel-type Indoles: Synthesis and Cell-based
Studies for the Identification of Pro-apoptotic Agents
*,a
a
a
a
b
Andrea Spallarossa, Chiara Caneva, Matteo Caviglia, Silvana Alfei, Stefania Butini, Giuseppe
b
b
b
c
c
Campiani, Sandra Gemma, Margherita Brindisi, Daniela M. Zisterer, Sandra A. Bright, Clive D.
c
d
d
e
e
Williams, Emmanuele Crespan, Giovanni Maga, Giuseppina Sanna, Ilenia Delogu, Gabriella
e
e
Collu, Roberta Loddo.
a
Dipartimento di Farmacia, Università di Genova, Viale Benedetto XV 3, I-16132 Genova, Italy
b
European Research Centre for Drug Discovery & Development, Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena, via Aldo
Moro 2, I-53100 Siena, Italy
c
School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, 152-160 Pearse Street, Dublin 2, Ireland
d
e
.
Institute of Molecular Genetics IGM-CNR, via Abbiategrasso 207, 27100 Pavia (Italy).
Dipartimento di Scienze Biomediche, Sezione di Microbiologia e Virologia, Università di Cagliari, Cittadella Universitaria, s.p. 8, Km 0.700, I-
0
9042 Monserrato (Cagliari), Italy
*
Corresponding author:
Dr. Andrea Spallarossa
Dipartimento di Farmacia
Sezione di Chimica del Farmaco e del Prodotto Cosmetico
Università degli Studi di Genova
Viale Benedetto XV, 3
1
6132 - Genova, Italy
phone: +39 010 353 8364;
fax: +39 010 353 8358;
e-mail: andrea.spallarossa@unige.it
1

ACCEPTED MANUSCRIPT
Abstract
A new series of indole-based analogues were recently identified as potential anticancer agents. The Knoevenagel-type
indoles herein presented were prepared via a one-pot condensation of iminium salts with active methylene reagents and
were isolated as single geometric isomers. Biological evaluation in different cell-based assays revealed an
antiproliferative activity for some analogues already in the nanomolar range against leukaemia, breast and renal cancer
cell lines. To explain these effects, the most promising analogues of the series were engaged in further cell-based
studies. Compounds 5e,l,p and 5a,b highlighted a pro-apoptotic potential being able to induce apoptosis in HL60, K562
and MCF-7 cell lines in a dose and time-dependent manner. The ability of these compounds to arrest cell cycle at the
G2/M phase inspired the immunofluorescence studies which allowed us to identify tubulin as a potential target for
compounds 5l and 6b.
Keywords
Indoles
Antiproliferative agents
Pro-apoptotic agents
Tubulin immunostaining
2

ACCEPTED MANUSCRIPT
1
. Introduction
Indole is a privileged scaffold for drug discovery [1,2]. Indole derivatives have been described as useful
chemical templates in different therapeutic areas, including the treatment of neurodegenerative diseases [3-5],
psychiatric disorders [6] and inflammation [7,8]. Moreover, indole derivatives were found to be endowed with antiviral
[
9], antioxidant [10], antiplasmodial [11], antiproliferative and antitumor efficacy [12-20]. Recently induction of
methuosis, which is one of the most recently acknowledged nonapoptotic cell death phenotype involving the
accumulation of cytoplasmic vacuoles derived from macropinosomes, was identified as the mechanism of action of
conjugated 3-vinyl indoles [21]. Interestingly, Knoevenagel-type indoles, such as 3-vinyl indoles bearing two electron-
withdrawing groups at the terminal exocyclic double bond carbon, showed promising properties in different research
areas [22-27].
In previous works, the chemical reactivity of N-methyleneiminium chlorides of cyclic (thio)ureas towards active
methylene reagents (AMRs) was investigated. By reacting ethylenethiourea and ethyleneurea with benzoyl chloride in
DMF, the corresponding iminium salts were isolated and directly condensed with a series of AMRs to obtain the
stereoselective synthesis of α,β-unsaturated(thio)ureas [28]. A similar protocol was recently applied for the one-pot
stereoselective preparation of Knoevenagel-type indoles [29].
The chemical accessibility to Knoevenagel-type indoles and the above mentioned rich literature background on this
heterocyclic scaffold prompted us to evaluate the antiproliferative activity of a series of indole derivatives 2-10 (Chart 1
and Table 2).
1
2
3
2
a-g, R = R = R = H
1 2 3
Y
3a,b, R = Me; R = R = H
2 1 3
4
a-d, R = Me; R = R = H
O
N
2 1 3
R3
5a-ac, R = Ph; R = R = H
X
R2
2
1
3
O
O
6a,b, R = 4-MeO-C
6
H ; R = R = H
4
1
2
3
Me
7a,b, R = H; R = Ph; R = OMe
1
2
3
N
R
8a,b, R = H; R = 4-MeO-C
6 4
H
; R = OMe
COC
-mOMe
COC
-pC
X, Y = EWGs as in Table 2 and Scheme 2
1
2
3
1
9a,b, R = R = H; R = CH
2
6
H
4
4
2
3
1
1
0a, R = R = H; R = CH
2
6
H
H
6 5
1, PBOX-15
2-10, Title compounds
Chart 1. General structure of title compounds 2-10 and structure of reference PBOX-15
2
2
. Results and discussion
.1. Chemistry
As previously described [29], iminium salts Im11-Im17 were obtained by reacting indoles 11-17 with the DMF-
benzoyl chloride complex and directly condensed with the active methylene reagents I-XXX (Scheme 1, Table 1) in the
presence of triethylamine to afford the desired indole derivatives 2-8 (Table 2). By this two-step one-pot procedure we
were able to prepared 48 indole analogues; the series included previously described compounds (namely, 2a-e,g; 3a,b;
4
a-d; 5a,b,f,h-m,o,s-u,aa,ab,ac; 6a,b; 7a,b; 8a,b) [29-31] and 14 unreported derivatives (namely, 2f; 5c-e,g,n,p-r,v-z).
3

ACCEPTED MANUSCRIPT
Y
–
X
Y
R3
N
+
Cl
R2
C
6
H
5
COCl
R3
I-XXX
R3
X
R2
R2
N
R
DMF
DMF
1
N
R
3
Et N
N
R
1
1
120°C
1
2
3
1
1
1
1
1
1
1
1, R = R = R = H
Im11-Im17
2-8
1
2
3
2, R = Me; R = R = H
2
1
3
3, R = Me; R = R = H
2
1
3
4, R = Ph; R = R = H
2
1
3
5, R = 4-MeO-C
6 4
H
; R = R = H
1
2
3
6, R = H ; R = Ph; R = OMe
1
2
3
7, R = H ; R = 4-MeO-C
6
4
H ; R = OMe
Scheme 1. Two-step one-pot procedure for the synthesis of antiproliferative indoles 2-8
Table 1. The active methylene reagents employed in the synthesis.
AMRs
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
XV
X
Y
AMRs
XVI
XVII
XVIII
XIX
X
Y
COMe
CN CN
CN COC(Me)
CN COPh
CN CO-C
CN 2-thenoyl
CN COOEt
CN COOCH
CN CONH
CN CON[(-CH
CN CO-NHC
CN PO(OEt)
COMe
COMe
COMe
COMe
COPh
COPh
COPh
COPh
SO
SO
SO
COMe
COPh
COOEt
SO Ph
COOEt
COOEt
COCF
SO
SO
COMe
COOEt
Me
Ph
-C H -pCl
6 4
3
2
2
2
H
-pCl
6
4
XX
XXI
CH
OCH
XXII
XXIII
XXIV
XXV
XXVI
XXVII
XXVIII
XXIX
XXX
2
2
3
2
2
)
-]
-pCl
CO-C
CO-C
H
H
-pCl
-pNO
2
5
6
4
4
H
6
4
6
2
2-furoyl
COOMe
COOMe
COOEt
COOEt
2
3
i
CN PO(O C
H
)
Me
Ph
3
7
2
2
2
CN SO
CN SO
CN SO
Me
Ph
2
2
2
-C
H
-pCH
6
4
3
The condensation procedure resulted to be stereoselective in terms of E/Z isomerism at the exocyclic double bond. In
3
3
fact, products 2-8 were obtained as single stereoisomers as demonstrated by the measurement of the J and the J
C,H
P,H
coupling constants between the vinyl proton and the phosphorus (compounds 2b; 5k) or the X α-carbon (compounds
c,g; 4b,d; 5c,l,p,q,v,z,aa) or the X and Y α-carbons (compounds 2f; 5b,f,h,i,s,u,x,ab) [29]. The configurations of the
2
remaining compounds have been attributed by comparison with the configurations of the analogues bearing the same X
and Y groups. Most of the indole derivatives were isolated as E-isomers with the only exception of compounds 5s and
5
ab that were obtained as Z-isomers (Table 2).
4

ACCEPTED MANUSCRIPT
Table 2. Yields, E/Z ratio and MT-4 cell-based cytotoxicity of Knoevenagel-type indoles 2-8.
Y
R3
X
R2
N
1
R
1
2
3
a
IDs
R
H
H
H
H
H
H
H
R
H
H
H
H
H
H
H
H
H
R
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
X
Y
CN
PO(OEt)
SO
SO
COOEt
COOEt
SO
SO
SO
Isomer Yield EC µM
50
2
2
2
2
2
2
2
3
3
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
6
6
7
7
8
8
a
b
c
d
e
CN
CN
CN
CN
COPh
CO-C
COOMe
CN
CN
CN
CN
CN
COOMe
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
COMe
COMe
COMe
COMe
COPh
COPh
COPh
COPh
-
E
E
E
E
E
E
E
E
E
E
E
E
-
E
E
E
E
E
E
E
E
E
E
E
E
E
-
72
28
73
78
50
32
53
45
60
77
40
45
39
74
51
72
83
40
91
89
55
60
62
36
78
69
72
42
48
4
12
40
14
36
17
47
22
10
43
42
45
13
70
62
58
78
73
53
36±2
80±3
>100
>100
>100
100±5
>100
>100
>100
56±2
>100
>100
2
Me
Ph
2
2
f
6
H
4
-pNO
2
g
a
b
a
b
c
d
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
2
2
2
Ph
Me
Ph
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
CN
SO
SO
SO
CN
COCMe
COPh
CO-C H -pCl
2-thenoyl
COOEt
COOCH
CONHb
COPip
CO-NHC
PO(OiPr)
Me
Ph
Ph
2
2
2
31±1.5
0.4±0.05
0.5±0.05
0.5±0.02
0.4±0.01
0.4±0.01
1.7±0.02
1.2±0.05
6.0±0.4
9.6±0.6
1.7±0.08
11±0.4
0.3±0.06
0.4±0.03
0.4±0.02
1.5±0.04
0.5±0.02
0.4±0.03
>100
2.0±0.04
8.9±0.5
7.0±0.9
3.0±0.06
9.0±0.5
1.5±0.08
0.8±0.01
0.4±0.03
1.0±0.03
0.6±0.01
2.3±0.07
0.3±0.01
0.2±0.06
1.0±0.05
1.3±0.02
0.3±0.01
0.2±0.04
0.03±0.001
3
6 4
CH OMe
2 2
2
H -pCl
4
6
2
SO
SO
SO
Me
Ph
2
2
2
-C
6
H
-pCH
-pCl
4
4
3
COMe
SO
SO
SO
COMe
COPh
COOEt
SO Ph
COOEt
COOEt
COCF
SO
SO
COMe
COOEt
CN
SO
CN
Me
Ph
E
E
E
Z
-
2
2
2
-C
6
H
s
t
u
v
w
x
y
z
aa
ab
ac
a
b
a
b
a
b
E
E
E
E
n.d.
E
E
Z
-
2
CO-C
CO-C
H
H
-pCl
-pNO
6
4
4
6
2
2-furoyl
COOMe
COOMe
COOEt
COOEt
CN
3
Me
Ph
2
2
4-MeO-C
4-MeO-C
Ph
Ph
4-MeO-C
4-MeO-C
H
H
-
E
-
E
-
E
6
4
4
CN
Ph
Ph
Ph
6
2
MeO CN
MeO CN
MeO CN
MeO CN
SO
CN
SO
2
H
H
6
4
4
6
2
Doxorubicin
a
Compound concentration (µM) required to reduce the viability of mock-infected MT-4 cells by 50%, as determined by
the MTT method, under conditions allowing untreated controls to undergo at least three consecutive rounds of
multiplication. Data represent mean values (±SD) for three independent determinations.
5

ACCEPTED MANUSCRIPT
Moreover, two of the obtained products (derivatives 2d and 2e) were N-alkylated to afford compounds 9a,b and 10a via
deprotonation of the indole nitrogen with sodium hydride and subsequent reaction of the corresponding salts with
different ω-bromoacetophenones (Scheme 2).
R4
Y
Br
Y
O
X
NaH
X
R4
dry DMF
N
N
H
O
4
2d,e
9a, X = CN; Y = SO
9
1
2
Ph; R = 3-OMe
4
b, X = COPh; Y = COOEt; R = 3-OMe
0a, X = CN; Y = SO2Ph; R = 4-Ph
4
Scheme 2. Synthesis of compounds 9a,b and 10a
2
.2. Biological evaluation
Compounds 2-10 were preliminary tested in MT-4 cell-based assay in order to evaluate their cytotoxic activity (Table
). The antiproliferative activity was found to be affected by both the nature of the substituent of the indole scaffold and
2
the X and Y groups (Table 2). Thus, all the compounds showing submicromolar EC50 values bear at position 2 of the
indole nucleus either a phenyl (derivatives 5a-e,g,l-n,p,q,y,z,ab) or a p-anisyl (derivatives 6, 7 and 8) group.
Conversely, the unsubstituted indole compounds 2 emerged to be poorly active or inactive. Similarly, N-alkylation led
to inactive compounds (derivatives 3, 9 and 10) and also the introduction of a 2-methyl group was found to be
detrimental for activity (derivatives 4 vs 5 and 6). The activity of Knoevenagel-type indoles appears to be influenced
also by the nature of the X and Y groups as highlighted by the different potencies of the 2-phenylindole derivatives 5
(
Table 2). Thus, when X is a cyano group and Y is a cyano, keto, ester, N-phenylamido or sulphonyl substituent we
registered a good antiproliferative activity (EC50 value range: 0.4-1.7 µM, see derivatives 5a-g,j,l,m,n); conversely
h,i,k, sharing the X cyano group and characterized by an amide or a phosphonic ester as Y substituents were poorly
5
active (EC50 value range: 6.0-11.0 µM). The presence of a benzoyl substructure as X substituent seemed to correlate
with poor activity (see compounds 5t-v) even if derivative 5s emerged to be active at micromolar concentrations.
Furthermore the introduction of a p-NO group at the benzoyl moiety led to a 5-fold increase in activity (5u vs 5x),
2
while the p-Cl substitution caused a slight reduction in potency (5u vs 5w). Finally, derivatives 5y-ac bearing as X
groups a 2-furoyl or an ester substituent showed EC values in the (sub)micromolar concentration range.
50
In order to define the safety profile of the most interesting compounds, their cytotoxic activity for tumour cells
haematological and solid human tumour-derived cell lines) was compared to that for non-neoplastic cell lines (Tables 3
(
and 4). Doxorubicin was used as reference drug. This analysis revealed a cytotoxic potential for compounds 6b and 8b
against all the human leukaemia-lymphoma-derived cell lines evaluated. Notably, compounds 5g, 6b and 8b showed a
good activity against lung squamous, prostate and hepatocellular human carcinoma accompanied by a favourable
selectivity index.
6

ACCEPTED MANUSCRIPT
Table 3. Cytotoxic activity of selected indole compounds against human leukaemia-lymphoma-derived (CCRF-CEM,
WIL-2NS, CCRF-SB) cell lines
a
EC (µM)
5
0
b
c
d
IDs
CCRF-CEM WIL-2NS
CCRF-SB
5
5
5
5
5
5
5
5
5
6
7
8
d
e
g
s
1.1±0.1
1.2±0.2
1.6±0.1
8.1±0.3
12±0.5
4.5±0.4
6.4±0.7
2.2±0.1
12±0.9
72±4
0.6±0.001
0.6±0.004
2.2±0.1
11±0.8
12±0.8
9.6±0.9
2.2±0.3
1.8±0.1
1.6±0.1
t
u
x
y
ab
b
b
b
8±0.3
12±1
1.8±0.2
1.9±0.09
1.4±0.1
0.3±0.1
1.5±0.2
0.3±0.001
2.4±0.2
2.3±0.1
2.7±0.1
0.4±0.008
2.8±0.2
0.5±0.003
0.3±0.005
1.9±0.09
0.3±0.003
Doxorubicin 0.02±0.001
0.04±0.002 0.01±0.001
a
Compound concentration (µM) required to reduce cell proliferation by 50%, as determined by the MTT method, under
conditions allowing untreated controls to undergo at least three consecutive rounds of multiplication. Data represent
b
+
c
mean values (± SD) for three independent determinations. CD4 human acute T-lymphoblastic leukaemia. Human
splenic B-lymphoblastoid cells. Human acute B-lymphoblastic leukaemia.
d
Table 4. Cytotoxic activity of selected indole compounds against solid tumour-derived (SK-MEL 28, SK-MES-1, DU-
1
45, Hep-G2, MCF-7) and normal control (MRC-5 and CRL 7065) cell lines.
a
EC (µM)
5
0
b
c
d
e
f
g
h
IDs
MRC-5 CRL-7065 SK-MEL28 SK-MES 1 DU-145 Hep-G2 MCF-7
5
5
5
5
5
5
5
5
5
6
7
8
d
e
g
s
14±1.5
20±2.1
>100
>100
>100
>100
>100
12
31±3
73±5
14±2.8
22±2
7.4±2.4
45±6
76±3
>100
>100
>100
>100
8.1±0.7
83±2
>100
15±2.5
28±6
9.3±2
>100
>100
>100
>100
33±5
30±1.5
>100
10±0.3
>100
19±4
4±0.5
5±0.05
>100
>100
>100
19±1
27±1
23±1
43±4
11±2.2
11±0.8
4.2±1
>100
>100
68±2
11±0.9
9.2±0.6
11±2
2±0.3
10±0.2
3±1
37±14
25±1
85±3
79±2
>100
31±3
28±3
16±3
19±2
28±3.5
29±1.2
12±0.7
>100
>100
43±5
t
u
x
y
ab
b
b
b
40±2
10±2.6
32±2.5
>100
12±0.5
40±3
11±2.5
9.6±1.9
6.4±0.4
11±0.1
>100
12±1.3
>100
Doxorubicin 0.3±0.01
0.9±0.02
0.7±0.01
0.1±0.009
0.2±0.03 0.3±0.01 0.3±0.02
a
Compound concentration (µM) required to reduce cell proliferation by 50%, as determined by the MTT method, under
conditions allowing untreated controls to undergo at least three consecutive rounds of multiplication. Data represent
b
c
mean values (±SD) for three independent determinations. Human normal lung fibroblasts. Human normal foreskin
fibroblasts. Human skin melanoma. Human lung squamous carcinoma. Human prostate carcinoma. Human
hepatocellular carcinoma. Human breast adenocarcinoma.
d
e
f
g
h
Derivatives 2-8 were submitted to the NCI (Bethesda MA-USA) developmental therapeutics program for
antiproliferative testing against different human tumour cell lines, organized into 9 disease-oriented panels; the fifteen
derivatives reported in Tables 5-7 were selected and screened by NCI. The results are expressed as GI50 (measure the
growth inhibitory power), TGI (measure of the cytostatic activity) and LC50 (measure of the cytocidal effect). Table 5
summarizes the number of cell lines against which each compound was screened, the number of lines against which it
7

ACCEPTED MANUSCRIPT
gave a positive (inferior to 100 µM) GI , TGI or LC values and the corresponding concentration range. The GI
50
5
0
50
values of the fifteen molecules are reported in Tables 6 and 7.
Table 5. GI , TGI and LC values ranges of selected compounds from NCI’s in vitro screening.
5
0
50
a
b
c
GI (µM)
TGI (µM)
LC (µM)
50
No
35
5
0
d
d
d
IDs No
Range
No
47
Range
5.2-96.7
0.698-90.4 10
4.1-86.9
4.7-96.3
5.6-88.1
2.0-46.6
-
Range
22.2-98.7
12.1-98.4
58.2
7.6-86.7
17.3-67.7
4.7-81.4
-
4
d
a
c
m
p
s
56
59
54
60
59
59
31
58
1.1-17.4
5
5
5
5
5
5
5
5
6
6
7
7
8
8
0.246-43.2 50
0.370-65.3 13
0.288-23.6 52
0.415-43.8 21
0.295-7.2
3.1-97.1
1
12
3
39
-
56
-
v
z
0.671-19.6 46
0.219-23.9 60
0.0173-2.8 50
<0.01-19.3 52
0.607-44.2 20
4.0-98.5
12.7-66.0
3.2-80.7
4.7-96.3
3.3-57.6
7.1-85.5
5.6-88.1
16
37
5
9
2
-
-
4
8.2-69.8
33.2-99.1
1.9-23.9
6.8-90.8
55.4-75.0
-
ac 60
a
b
a
b
a
b
60
60
60
58
47
58
1.1-37.3
18
0.075-23.0 21
0.276-46.1 50
-
3.2-80.7
8.5-47.0
a
b
Compound concentration that gives 50% growth inhibition. Compound concentration that gives total growth
c
d
inhibition. Compound concentration that produces 50% lethal (cytocidal) effect. The table shows the number of cell
lines on which each compound gave a positive (i.e. lower than 100 µM) GI50 or TGI or LC50 value and the
corresponding concentration ranges.
With the exception of derivatives 4d, 5v and 7b, all the tested compounds showed submicromolar GI50 values against
specific tumour cell lines. Derivatives 6a,b and 8a,b were endowed with a widespread submicromolar activity against
the tested tumour panels and proved to be particularly effective against selected cell lines [6a: GI50 (leukaemia-SR) =
1
7.3 nM; 6b: GI (breast cancer-MDA-MB-468) < 10 nM; 8a: GI (renal cancer-A498) = 75 nM].
50 50
8

ACCEPTED MANUSCRIPT
Table 6. GI values for compounds 4d and 5a,c,m,p,q,v,z,ac.
5
0
a
Panel / cell line
GI (µM)
50
4
d
5a
5c
5m
2.6
1.8
5p
4.7
2.6
5q
1.4
1.7
5v
5z
7.6
3.4
5ac
2.6
b
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
n.d. 2.4
n.d. 1.8
7.3 b
b
n.d.
5.0
4.4
c
Leukaemia
0.478
43.2
0.582 2.4 b
0.348 0.679 0.442 6.5 b n.t.
3.6
3.9
0.219
2.1
6.4
12.4
5.5
8.8
11.4
2.9
12.7
3.8
3.4
4.9
10.1
4.3
4.1
4.6
3.5
5.6
b
1.9
3.2
1.1
4.9
5.0
7.7
4.1
4.3
4.8
8.8
3.0
3.2
7.5
4.9
3.8
3.1
5.2
2.4
5.2
3.3
3.1
3.4
7.6
3.1
3.4
2.0
7.3
4.4
2.9
n.d.
2.2
4.4
1.6
2.2
n.tc
7.2
3.3
1.2
5.9
1.9
1.6
6.1
1.1
1.3
1.7
1.4
n.d. 4.7
7.6
9.9
0.714 3.0
6.2
8.1
c
n.d.
7.6
10.0
16.3
4.5
6.8
16.0
3.8
4.5
4.0
17.3
3.7
6.2
9.3
10.3
4.1
4.7
9.2
2.7
4.6
1.4
10.9
2.0
1.1
n.t.
7.3
6.4
4.6
5.8
11.3
7.7
4.1
4.7
11.4
2.1
9.6
1.8
10.4
2.3
0.971 3.5
0.526 3.4
0.684 2.0
0.539 3.0
13.4
1.6
2.1
5.7
2.0
1.2
3.3
26.0
1.9
35.0 5.3
4.6
5.0
9.6
5.9
8.9
Non-Small
Cell
12.1
3.8
27.1
4.0
9.6
2.8
3.6
3.0
8.0
2.9
2.0
10.7
6.6
5.0
8.0
7.4
3.5
Lung Cancer
91.8 3.1
7.8
5.0
14.5
5.3
4.4
8.8
0.754 4.9 b 4.2
Colon Cancer
CNS Cancer
0.836 n.d. 2.4
0.595 1.6
0.652 2.1
0.570 2.1
0.589
1.0
0.778 n.d. 2.9
3.6
5.1 b 1.5
5.7
3.0
6.0
16.8
3.2
4.7
7.4
12.3
4.8
4.5
2.4
2.5
8.3
1.7
2.6
4.6
8.4
3.8
4.2
3.9
1.9
3.4
1.5
2.9
3.4
4.8
1.6
3.4
2.4
1.3
6.1
1.7
1.2
1.4
2.1
4.3
3.1
3.1
8.7
6.8
7.6
14.5
23.9
7.0
4.9
5.6
8.9
3.5
10.1 2.3
8.6 3.6
74.7 5.1
12.2
0.521 15.6 3.3
4.2
0.246 0.370 0.288 0.415 0.295 3.9
0.671 3.4
11.8
6.0
n.t
11.2
3.3
1.7
1.9
6.2
19.6
6.3
Melanoma
10.6 27.6
5.8c 5.1
35.2
12.0
n.t.
10.9
3.6
12.7
8.3
1.82
18.2
2.6
3.3
1.4
5.5
13.9
6.0
1.1
6.8
9.2
11.0
7.0
5.5
3.6
5.2
5.6
6.7
3.4
2.9
2.2
3.8
23.6
11.6
3.8
8.6
3.3
1.9
43.8
3.2
8.3
2.3
3.8
16.1
5.2
2.1
4.4
3.5
3.9
5.8
5.6
2.6
6.9
13.5
5.8
4.5
3.5
2.7
5.5
1.6
2.4
2.3
1.8
1.4
2.0
0.832 9.0
2.2
4.7
7.7
3.8
7.5
4.6
5.7
4.2
10.5
18.5
4.1
c
c
n.t.
7.0
2.8
1.5
2.7
5.7
1.1
28.1
2.6
0.757 2.5
1.8
5.2
1.0
1.5
1.6
3.7
2.0
2.5
0.520
2.9
1.6
2.0
1.8
2.4
1.3
1.9
3.8
1.6
2.5
3.0
9.0
2.4
65.3
Ovarian
Cancer
6.6
17.4 19.7
3.9
4.2
9.3
8.2
5.5
5.5
3.3
3.6
2.6
8.1
4.4
5.6
2.9
2.8
2.7
2.9
5.4
3.2
6.3
6.0
0.826 3.1
2.2
3.3
12.1
7.5
2.9
6.0
8.6
3.0
7.3
6.1
3.3
6.8
5.3
11.1
3.0
3.3
1.1
1.8
2.3
9.4
4.6
1.0
15.2
7.6
13.6
12.9
16.9
4.4
10.3
3.7
5.1
13.6
4.0
3.3
9.7
4.5
5.8
7
86-0
16.5
15.3
7.2
2.5
9.6
b
A498
n.d. 6.6
6.7
3.1
97.1 7.2
3.1
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
PC-3
DU-145
MCF7
MDA-MB-
9.2
1.8
Renal Cancer
5.7
11.1
30.5
3.5
5.2
4.0
13.2
6.3
3.0
5.3
5.6
7.7
5.5
4.2
2.5
2.9
2.4
Prostate
Cancer
5.0
0.945 5.0
1.7
5.0
1.9
1.4
1.7
Breast Cancer HS 578T
39.5 3.5
4.3
3.4
4.2
3.8
5.3
2.7
3.1
BT-549
T-47D
MDA-MB-468
2.8
1.9
5.4
a
c
b
Compound concentration that gives 50% growth inhibition (Only the values < 100 ꢀM are reported). Not determined.
Not tested. GI values lower than 1.0 µM are reported in bold
.
50
9

ACCEPTED MANUSCRIPT
Table 7. GI values for compounds 6a,b, 7a,b and 8a,b.
5
0
a
Panel / cell line
GI (µM)
50
6
a
6b
1.3
0.294 2.5
0.535 2.1
0.871 3.7
7a
3.4
7b
8a
8b
1.3
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
0.352
0.271
0.343
0.578
0.490
6.5
2.4
1.4
4.7
n.t.
0.363 0.899
0.345 0.508
0.369 0.765
Leukaemia
c
2.1
2.6
5.1
1.3
0.0173 0.350 0.865 2.2
0.152 0.305
A549/ATCC
0.700
0.656
1.2
1.2
1.1
0.803 7.0
6.0
6.9
8.3
6.1
9.4
8.0
3.8 b
n.d.
3.3
23.0
1.5
2.8
EKVX
1.3
1.0
2.3
2.8
4.1
5.3
2.4
3.5
3.3
HOP-62
Non-Small Cell HOP-92
3.1
0.698
3.7
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
Lung Cancer
0.600
0.490
0.359
0.798
0.223
0.751
0.411
0.593
0.354
0.374
0.440
0.709
0.336
0.263
1.8
0.418
0.483
1.8
0.874
0.295
0.128
0.763
1.2
0.932 4.1
1.5 7.4
0.404 3.6
3.0 4.6
0.254 2.1
1.2 6.1
0.399 4.4
0.554 3.2
0.314 3.1
0.374 3.5
0.506 3.6
2.0
0.946
30.1 0.380 4.3
4.6
4.0
7.4
0.472 0.687
1.7
0.291 0.396
b
18.7 n.d.
2.0
3.7
4.0
3.5
3.0
4.0
13.3 1.7
2.8
5.5
0.487 0.539
0.778 0.632
0.407 0.435
0.590 0.654
0.579 0.540
Colon Cancer
1.1
8.3
1.6
0.500 4.2
0.299 2.9
0.863 6.0
0.379 2.8
0.473 3.5
0.306 0.760
0.774 0.510
CNS Cancer
Melanoma
13.5 13.7
3.2
SNB-75
U251
5.9
4.6
7.4
19.4
3.7
0.593 0.612
0.635 0.563
1.2
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
1.3
1.2
4.9
4.4
3.2
0.383 2.3
0.203 0.607 1.1
0.308 0.500
0.122 0.276
5.9
1.7
18.1
1.8
5.9
4.4
3.7
6.1
3.1
4.9
3.8
4.8
5.0
8.3
37.3
7.8
3.8
7.1
6.1
9.3
12.9
4.5
9.0
0.787
0.350
0.424
0.357
1.4
0.265
0.798
1.8
0.344 2.9
0.324 0.319
10.7
30.0
46.1
0.597 2.5
0.357 0.660
5.8
0.424 0.359
19.3
8.4
10.6
0.244 3.1
0.578 5.4
2.1
4.6
3.1
Ovarian Cancer
Renal Cancer
1.9
1.3
44.2
7.7
1.2
0.343
0.445
0.720
0.458
2.3
0.679
0.518
1.2
0.428 2.8
0.528 4.8
0.403 0.494
0.892 0.901
14.3
0.075 n.t.
1.8
0.392 4.2
5.5
15.9
7
86-0
1.2
1.1
1.9
6.6
2.0
7.4
2.1
c
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
0.997 4.1
c
1.0
1.5
4.8
4.7
9.8
4.7
5.1
6.4
n.t.
7.8
17.5
4.0
2.3
5.0
3.1
0.925
2.8
Prostate Cancer PC-3
DU-145
MCF7
0.547
0.484
0.347
0.557 5.4
0.450 6.5
0.398 2.2
0.649 3.8
0.682 2.4
8.3
0.342 0.534
10.1 0.917 0.875
3.9
7.5
7.6
11.6
5.2
4.2
0.385 0.851
1.3 1.4
0.513 0.538
MDA-MB-231/ATCC 0.562
Breast Cancer
HS 578T
BT-549
0.241
0.769
0.481
0.418
1.6
2.2
4.8
0.905
T-47D
MDA-MB-468
0.533 2.3
<0.01 2.1
2.6
a
c
0.503 1.3b
Compound concentration that gives 50% growth inhibition (Only the values < 100 ꢀM are reported). Not determined.
Not tested. GI values lower than 1.0 µM are reported in bold
.
50
1
0

ACCEPTED MANUSCRIPT
With the aim of identifying the mechanism behind the Knoevenagel-type indoles antiproliferative activity and
considering the recent literature on the pro-apoptotic properties of indole derivatives [32], the most promising
compounds (5e,l,p and 5a,b) were selected for further cellular studies concerning their antimitotic and pro-apoptotic
efficacy. Time-dependent G2/M cell cycle arrest and time-dependent apoptosis induction were evaluated on various
cancer cell lines. MCF-7, a breast carcinoma cell line, K562, a chronic myeloid leukaemia cell line and HL-60, a
promyelocytic leukaemia cell line, were thus used as representative solid and haematological malignancies to evaluate
the antitumor properties of these novel compounds. The obtained results are shown in Figures 1 and 2 while Figure 3
describes the immunofluorescence studies performed on MCF-7 cells.
Figure 1. Compounds 5e,l,p and 6a,b potently reduce cell viability and induce apoptosis in HL-60, K562 and MCF-7
a
cell lines. A. Cellular viability; Compound concentration that gives 50% growth inhibition. Cells were analyzed for
apoptotic cell death B. HL-60 cells, C. K562 cells and D. MCF-7 cells. Values represent the mean ± S.E.M. of three
independent experiments performed in triplicate.
Results showed that the five compounds tested were able to dose-dependently and significantly reduce cell viability in
all three cell lines used following a 48 h treatment (Figure 1A). Further analysis showed the compounds to actively
induce apoptosis in these cells in a time dependent manner (Figure 1B-D). Our results highlighted 5e and 6b as the most
potent compounds in HL-60 cells, inducing 61.3 ± 4.2 and 65.5 ± 9.1% apoptosis, while 5l and 6a were most potent in
K562 cells inducing 47.5 ± 4.8 and 44.4 ± 4.0% apoptosis, respectively. For MCF-7 cells, 5l and 5e induced 28.4 ± 6.0
and 34.1 ± 7.9% apoptosis.
These results prompted us to study cell cycle progression for the most potent pro-apoptotic agents identified.
Accordingly, compounds 5l and 6b were chosen for further testing. Cell cycle analysis of cells treated with these two
compounds showed apoptosis to be preceded by a G2/M arrest (Figure 2). 5l induced a G2/M arrest of 39.7 ± 5.0, 46.8
±
0.9 and 41.3 ± 3.5% in HL-60, K562 and MCF-7 cells after 18 h (HL-60, MCF-7) or 24 h (K562), respectively.
Similarly, 6b induced a G2/M arrest of 47.3 ± 3.0, 42.4 ± 7.6 and 40.4 ± 7.0% in the same cell lines.
1
1

ACCEPTED MANUSCRIPT
Figure 2. Apoptosis is preceded by a G2/M arrest following treatment with 5l and 6b. Values represent the mean ±
S.E.M. of three independent experiments.
In the majority of cell proliferation and apoptosis assays performed, compound 6b demonstrated the highest potency in
terms of apoptosis and G2/M arrest, while 5e and 5p were somehow the less effective. Overall, the data so far acquired
confirmed a pro-apoptotic character of the five indoles tested.
As microtubule targeting agents (MTAs) are associated with eliciting a G2/M arrest [33], MCF-7 cells were stained for
tubulin and DNA and subjected to immunofluorescence. Cells treated with paclitaxel, a tubulin polymeriser and PBOX-
1
5, a tubulin depolymeriser [34-36] were used as positive controls for highlighting the effect of MTAs on tubulin. In
comparison to these MTAs, the tested analogues (5l and 6b) had, in analogy with PBOX-15, a partial depolymerising
effect on tubulin suggesting that the molecular target of the 5l and 6b compounds might be tubulin (Figure 3).
Tubulin
Nuclei
Merge
Vehicle
5l
6
b
PBOX-15
Paclitaxel
Figure 3. Effect of 5l and 6b on tubulin. DNA is colored red and microtubules in green. Bar represents 20 µm. Results
are representative of three independent experiments
1
2

ACCEPTED MANUSCRIPT
As a further analysis we also explored the possibility that 5l could behave as a DNA intercalating agent. To this purpose
we incubated saturating amounts of the inhibitor in the presence of double stranded plasmid DNA. Our experiments
showed that 5l does not intercalate DNA since the electrophoretic mobility properties of the plasmid in the presence of
5
3
l are not different from those of the plasmid alone (see figure S1 of the Supplementary Information).
. Conclusions
The synthesized indole derivatives showed an overall good antiproliferative activity, which was specifically dependent
on the presence of aryl substituents at position 2. Some of the compounds resulted selective for tumor cells, ensuring a
good level of safety for normal cells. Moreover, activity data generated by NCI further confirmed the anti-proliferative
potential of different indole compounds, in particular 6a,b and 8a,b are able to inhibit proliferation at submicromolar
concentrations in most of the cancer cell lines tested. Further studies on selected compounds demonstrated their
pro-apoptotic efficacy and tubulin was identified as their potential biological target.
1
3

ACCEPTED MANUSCRIPT
4
4
. Experimental section
.1. Chemistry
All reactions were carried out in dried glassware. The commercially available building blocks were purchased from
Alfa Aesar and Aldrich Chemical (Milan, Italy) and were used without further purification. 2-(4-methoxyphenyl)indole
1
5, 5-methoxy-2-phenyl-1H-indole 16, 5-methoxy-2-(4-methoxyphenyl)-1H-indole 17 were prepared according
literature procedures [37].
Solvents were reagent grade. DMF was dried on molecular sieves (5Å 1/16" inch pellets). Organic solutions were dried
over anhydrous sodium sulfate and evaporated using a rotatory evaporator operating at reduced pressure of about 10-20
Torr. Thin layer chromatography system for routine monitoring the course of reactions and confirming the purity of
analytical samples employed aluminium-backed silica gel plates (Merck DC-Alufolien Kieselgel 60 F254); chloroform
and methanol were used as developing solvents and detection of spots was made by UV light and/or by iodine vapours.
Melting points were determined on a Fisher-Johns apparatus and are uncorrected. IR spectra were recorded on a Perkin
1
Elmer 398 spectrometer. H NMR spectra were collected on a BRUKER DPX-300 (300 MHz) or on a Varian Gemini
1
3
2
00 (200 MHz) instrument. C NMR spectra were acquired on a BRUKER DPX-300 at 75.5 MHz. TMS was used as
1
internal reference and DMSO-d or CDCl were used as solvents. H NMR spectra were assigned with the help of
6
3
homo-decoupling, COSY and NOE-difference experiments. Coupling constant values were given in Hertz. Fully
1
3
3
13
decoupled C NMR spectra are reported. For the determination of J , fully coupled C NMR spectra were acquired
CH
in the gated-decoupled mode with at least 0.5 Hz/pt digital resolution. Some selective proton-decoupling experiments
were also needed. Chemical shifts were reported in δ (parts per million) units relative to the internal standard
tetramethylsilane, and the splitting patterns were described as follows: s (singlet), d (doublet), t (triplet) and m
(
multiplet). Elemental analyses were performed by an EA1110 Analyser, Fison Instruments (Milan) and were within
0.4% of theoretical values. UV analysis was carried out by a Thermo Scientific Evolution 300 spectrophotometer.
±
4
.1.1. General one-pot procedure for the preparation of compounds 2-8.
To a stirred solution of the suitable indole 11-17 (10 mmol) in dry DMF (5 mL), benzoyl chloride (1.41 g, 10 mmol)
was added in a single portion. After stirring at rt for 1 h, the reaction mixture was allowed to stand at 0 °C for 18-48 h.
Then, a solution of the suitable active methylene reagent (11 mmol) and triethylamine (1.21 g, 12 mmol) in dry DMF (8
mL) pre-warmed at 80 °C, were added in a single portion. The reaction mixture was stirred for 45 min at 90 °C (for 5b
and 5u: 1.5 h at 105 °C; for 5v: 1.5 h at 120 °C). After cooling to rt, Et N·HCl precipitated and the resulting suspension
3
was treated with water (50 mL). Then, work-ups 1-4 were carried out, as follows:
Work-up 1 (for 2a,c-g, 3b, 4a,b, 5a-h,j,l-p,r,s,u,x,z, 6a,b, 7a,b, 8a,b): the precipitate obtained was filtered off, dried
and crystallized from the suitable solvent or solvent mixture.
Work-up 2 (for 4d, 5i,w,y): the precipitate was filtered off and dissolved in CH Cl . The organic layer was washed with
2
2
water (3 × 20 mL), dried over anhydrous Na SO and filtered through a pad of Florisil. After evaporation of the solvent,
2
4
the crude product was purified by crystallization from ethanol.
Work-up 3 (for 4c, 5k,q,t): the precipitate was filtered off, air dried, purified by chromatography (eluents: petroleum
ether/ethyl acetate) and then crystallized from the suitable solvent or solvent mixture.
Work-up 4 (for 2b, 3a, 5v,aa-ac): the reaction mixture was extracted with ethyl acetate (2b, 3a, 5ac) or CH Cl
2
2
(
5v,aa,ab) (3×35 mL). The combined organic extracts were washed with water (5×30 mL), dried over anhydrous
Na SO and filtered through a pad of Florisil. After evaporation of the filtrate, the crude products 3a and 5v,ac
2
4
were crystallized from the proper solvent(s). Conversely, the crude products of 2b and 5z,aa were purified by
chromatography (eluent: petroleum ether/ethyl acetate) and then crystallized from the proper solvent mix.
1
4

ACCEPTED MANUSCRIPT
.1.1.1. 1H-(Indol-3-ylmethylene)malononitrile (2a) [29].
4
4
4
4
4
4
.1.1.2. Diethyl (E)-1-cyano-2-(1H-indol-3-yl)vinylphosphonate (2b) [29].
.1.1.3. (2E)-3-(1H-Indol-3-yl)-2-(methylsulfonyl)acrylonitrile (2c) [29].
.1.1.4. (2E)-3-(1H-Indol-3-yl)-2-(phenylsulfonyl)acrylonitrile (2d) [29].
.1.1.5. Ethyl (2E)-2-benzoyl-3-(1H-indol-3-yl)acrylate (2e) [29].
.1.1.6. Ethyl (2E)-3-(1H-indol-3-yl)-2-(4-nitrobenzoyl)acrylate (2f). Yellow solid; Yield 32%; mp: 161-162°C; IR
-1
1
(
KBr, υmax cm ): 3228 (NH), 1698 (COOEt), 1675 (COAr), 1603 (C=C), 1523 (NO ), 1345 (NO ), 1249 (COOEt); H
2 2
NMR (300 MHz, DMSO-d ): δ 1.07 (t, J = 7.0 Hz, 3H, CH ), 4.12 (q, J = 7.0 Hz, 2H, CH ), 7.14-7.18 (m, 2H, Ar-H),
6
3
2
7
.39-7.42 (m, 2H, Ar-H), 7.71-7.75 (m, 1H, Ar-H), 8.12 (d, J = 8.4 Hz, 2H, Ar-H), 8.25 (s, 1H, CH=C), 8.29 (d, J = 8.4
1
3
Hz, 2H, Ar-H), 11.87 (br s, 1H, NH); C NMR (75.5 MHz, DMSO-d ): δ 13.95, 60.61, 108.90, 112.43, 118.18, 121.11,
6
3
1
22.30, 122.83, 124.23, 124.23, 126.81, 129.75, 129.94, 129.94, 135.58, 136.02, 140.79, 150.25, 164.70, 195.16; JC,H
=
10.2 Hz (CO); 7.5 Hz (COO); Anal. Calcd for C H N O : C, 65.93; H, 4.43; N, 7.69; Found: C, 65.84; H, 4.34; N,
20 16 2 5
7
4
4
4
4
4
4
4
4
4
4
.64.
.1.1.7. Methyl (2E)-3-(1H-indol-3-yl)-2-(phenylsulfonyl)acrylate (2g) [29].
.1.1.8. (2E)-3-(1-Methyl-1H-indol-3-yl)-2-(methylsulfonyl)acrylonitrile (3a) [29].
.1.1.9. (2E)-3-(1-methyl-1H-indol-3-yl)-2-(phenylsulfonyl)acrylonitrile (3b) [29].
.1.1.10. [(2-Methyl-1H-indol-3-yl)methylene]malononitrile (4a) [29].
.1.1.11. (2E)-3-(2-Methyl-1H-indol-3-yl)-2-(methylsulfonyl)acrylonitrile (4b) [29].
.1.1.12. (2E)-3-(2-Methyl-1H-indol-3-yl)-2-(phenylsulfonyl)acrylonitrile (4c) [29].
.1.1.13. Methyl (2E)-3-(2-methyl-1H-indol-3-yl)-2-(phenylsulfonyl)acrylate (4d) [29].
.1.1.14. [(2-Phenyl-1H-indol-3-y)lmethylene]malononitrile (5a) [29].
.1.1.15. (2E)-2-(2,2-Dimethylpropanoyl)-3-(2-phenyl-1H-indol-3-yl)acrylonitrile (5b) [29].
.1.1.16. (2E)-2-Benzoyl-3-(2-phenyl-1H-indol-3-y)acrylonitrile (5c). Yellow solid; Yield: 72%; mp: 254-256 °C; IR
-1
1
(
KBr, υmax cm ): 3323 (NH), 2209 (CN), 1622 (CO), 1549 (C=C); H NMR (300 MHz, DMSO-d ): δ 7.23-7.40 (m, 2H,
6
Ar-H), 7.48-7.60 (m, 9H, Ar-H), 7.73-7.78 (m, 2H, Ar-H), 8.08 (s, 1H, CH=C), 8.22-8.31 (m, 1H, indole H-4), 12.97
1
3
(
br s, 1H, NH); C NMR (75.5 MHz, DMSO-d ): δ 103.25, 108.42, 112.65, 118.92, 121.78, 122.92, 123.95, 124.71,
6
1
28.37, 128.37, 128.44, 128.44, 128.89, 128.89, 129.53, 130.01, 130.05, 130.05, 131.91, 137.13, 137.27, 149.32,
1
5

3
ACCEPTED MANUSCRIPT
24 16 2
1
51,18, 190.89; J = 13.5 Hz (CN); Anal. Calcd for C H N O: C, 82.74; H, 4.63; N, 8.04; Found: C, 83.02; H, 4.94;
C,H
N, 8.04.
4
.1.1.17. (E)-2-(4-Chlorobenzoyl)-3-(2-phenyl-1H-indol-3-yl)prop-2-enenitrile (5d). Yellow solid; Yield: 83%; mp:
-1
1
2
32-234 °C; IR (KBr, υmax cm ): 3219 (NH), 2210 (CN), 1632 (CO), 1558 (C=C); H NMR (200 MHz, DMSO-d ): δ
6
7
.15-7.65 (m, 10H, Ar-H), 7.73-7.81 (m, 2H, Ar-H), 8.07 (s, 1H, CH=C), 8.28-8.33 (m, 1H, indole H-4), 13.01 (br s,
13
1
H, NH); C NMR (50.3 MHz, DMSO-d ): δ 97.25, 102.41, 108.24, 112.37, 118.55, 121.56, 122.61, 123.73, 124.53,
6
1
28.17, 128.59, 129.14, 129.77, 130.03, 135.65, 136.39, 136.95, 149.38, 150.99, 189.47; Anal. Calcd for C H ClN O:
24
15
2
C, 75.29; H, 3.95; N, 7.32; Found: C, 75.29; H, 4.18; N, 7.25.
4
2
7
.1.1.18. (2E)-3-(2-Phenyl-1H-indol-3-yl)-2-(thien-2-ylcarbonyl)acrylonitrile (5e). Orange solid; Yield: 40%; mp: 206-
-1
1
07 °C; IR (KBr, υmax cm ): 3232 (NH), 2206 (CN), 1587 (CO), 1561 (C=C); H NMR (200 MHz, DMSO-d ): δ 7.27-
6
1
3
.42 (m, 3H, Ar-H), 7.58-7.66 (m, 6H, Ar-H), 8.06-8.29 (m, 3H, Ar-H), 8.40 (s, 1H, CH=C), 13.02 (br s, 1H, NH);
C
NMR (50.3 MHz, DMSO-d ): δ 100.83, 108.46, 112.34, 119.08, 121.48, 122.61, 123.66, 124.40, 128.28, 128.67,
6
1
3
29.38, 129.79, 130.38, 135.02, 136.89, 141.68, 149.16, 150.51, 178.99; Anal. Calcd for C H N OS: C, 74.55; H,
22 14 2
.98; N, 7.90; S, 9.05; Found: C, 74.64; H, 4.34; N, 7.90; S, 8.88.
4
.1.1.19. Ethyl (2E)-2-cyano-3-(2-phenyl-1H-indol-3-yl)acrylate (5f) [29].
4
.1.1.20. 2-Methoxyethyl (2E)-2-cyano-3-(2-phenyl-1H-indol-3-yl)acrylate (5g). Yellow solid; Yield: 89%; mp: 198-
-1
1
2
00 °C; IR (KBr, υmax cm ): 3445 (NH), 2218 (CN), 1716 (CO), 1577 (C=C); H NMR (200 MHz, DMSO-d ): δ 3.28
6
(
s, 3H, CH ), 3.58-3.63 (m, 2H, CH ), 4.32-4.37 (m, 2H, CH ), 7.30-7.39 (m, 2H, Ar-H), 7.56-7.64 (m, 6H, Ar-H),
3 2 2
1
3
8
6
1
.21-8.25 (m, 1H, indole H-4), 8.30 (s, 1H, CH=C), 12.94 (br s, 1H, NH); C NMR (50.3 MHz, DMSO-d ): δ 57.82,
6
4.31, 69.30, 93.77, 107.57, 112.28, 117.31, 121.38, 122.20, 123.53, 124.44, 128.64, 129.38, 129.74, 136.78, 148.45,
49.47, 163.43; Anal. Calcd for C H N O : C, 72.82; H, 5.24; N, 8.09; Found: C, 72.95; H, 5.54; N, 8.03.
2
1
18
2
3
4
4
4
4
4
4
.1.1.21. (2E)-2-Cyano-3-(2-phenyl-1H-indol-3-yl)acrylamide (5h) [29].
.1.1.22. (2E)-3-(2-Phenyl-1H-indol-3-yl)-2-(piperidin-1-ylcarbonyl)acrylonitrile (5i) [29].
.1.1.23. (E)-N-(4-Chlorophenyl)-2-cyano-3-(2-phenyl-1H-indol-3-yl)prop-2-enamide (5j) [29].
.1.1.24. Diisopropyl (E)-1-cyano-2-(2-phenyl-1H-indol-3-yl)vinylphosphonate (5k) [29].
.1.1.25. (2E)-2-(Methylsulfonyl)-3-(2-phenyl-1H-indol-3-yl)acrylonitrile (5l) [29].
.1.1.26. (2E)-3-(2-Phenyl-1H-indol-3-yl)-2-(phenylsulfonyl)acrylonitrile (5m) [29].
.1.1.27. (E)-3-(2-Phenyl-1H-indol-3-yl)-2-(p-tolylsulfonyl)prop-2-enenitrile (5n). Orange solid; Yield: 72%; mp: 234-
4
-1
1
2
35 °C; IR (KBr, υmax cm ): 3262 (NH), 2209 (CN), 1566 (C=C), 1327 (SO ), 1150 (SO ); H NMR (200 MHz,
2 2
DMSO-d ): δ 2.44 (s, 3H, CH ), 7.29-7.37 (m, 2H, Ar-H), 7.51-7.71 (m, 8H, Ar-H), 7.82-7.86 (m, 2H, Ar-H), 8.05 (s,
6
3
1
6

ACCEPTED MANUSCRIPT
13
1
1
1
4
H, CH=C), 8.11-8.16 (m, 1H, indole H-4), 13.09 (br s, 1H, NH); C NMR (50.3 MHz, DMSO-d ): δ 20.74, 104.10,
6
06.48, 112.58, 115.28, 121.65, 121.93, 123.91, 124.32, 127.03, 128.78, 128.95, 129.82, 130.04, 130.11, 136.17,
36.89, 144.67, 146.14, 149.95; Anal. Calcd for C H N O S: C, 72.34; H, 4.55; N, 7.03; S, 8.05; Found: C, 72.39; H,
24
18
2
2
.68; N, 7.02; S, 7.84.
4
.1.1.28. 3-[(2-Phenyl-1H-indol-3-yl)methylene]pentane-2,4-dione (5o) [29].
4
.1.1.29. (3E)-3-(Methylsulfonyl)-4-(2-phenyl-1H-indol-3-yl)but-3-en-2-one (5p). Orange solid; Yield: 48%; mp: 229-
-1
1
2
30 °C; IR (KBr, υmax cm ): 3248 (NH), 1666 (CO), 1579 (C=C), 1346 (SO ), 1131 (SO ); H NMR (300 MHz,
2 2
DMSO-d ): δ 2.17 (s, 3H, CH ), 3.25 (s, 3H, CH ), 7.12-7.32 (m, 4H, Ar-H), 7.51-7.63 (m, 5H, Ar-H), 8.01 (s, 1H,
6
3
3
1
3
CH=C), 12.58 (br s, 1H, NH); C NMR (75.5 MHz, DMSO-d ): δ 30.85, 44.21, 105.70, 112.63, 118.96, 121.55,
6
3
1
23.31, 126.18, 129.01, 129.01, 129.49, 129.49, 129.59, 130.13, 135.19, 136.77, 139.24, 144.12, 198.87; JC,H = 9.9 Hz
(
CO); Anal. Calcd for C H NO S: C, 67.24; H, 5.05; N, 4.13; S, 9.45; Found: C, 67.34; H, 5.24; N, 3.77; S, 9.10.
19 17 3
4
.1.1.30. (3E)-4-(2-Phenyl-1H-indol-3-yl)-3-(phenylsulfonyl)but-3-en-2-one (5q). Yellow solid; Yield: 4%; mp: 172-
-1
1
1
73 °C; IR (KBr, υmax cm ): 3257 (NH), 1672 (CO), 1568 (C=C), 1347 (SO ), 1137 (SO ); H NMR (300 MHz,
2 2
DMSO-d ): δ 2.13 (s, 3H, CH ), 7.07-7.18 (m, 2H, Ar-H), 7.24-7.29 (m, 1H, Ar-H), 7.53-7.74 (m, 9H, Ar-H), 7.89-7.92
6
3
13
(
m, 2H, Ar-H), 8.23 (s, 1H, CH=C), 12.61 (br s, 1H, NH); C NMR (75.5 MHz, DMSO-d ): δ 30.86, 106.19, 112.66,
6
1
1
4
18.98, 121.64, 123.35, 125.99, 127.46, 127.46, 129.02, 129.02, 129.22, 129.22, 129.59, 129.59, 129.67, 130.10,
3
33.19, 134.71, 136.83, 139.97, 141.49, 144.74, 197.38; J = 9.6 Hz (CO); Anal. Calcd for C H NO S: C, 71.80; H,
C,H
24 19
3
.77; N, 3.49; S, 7.99; Found: C, 71.76; H, 4.48; N, 3.49; S, 7.63.
4
.1.1.31. (E)-3-(4-Chlorophenyl)sulfonyl-4-(2-phenyl-1H-indol-3-yl)but-3-en-2-one (5r). Yellow solid; Yield: 12%; mp:
-1
1
1
73-174 °C; IR (KBr, υmax cm ): 3261 (NH), 1666 (CO), 1576 (C=C), 1346 (SO ), 1133 (SO ); H NMR (200 MHz,
2 2
DMSO-d ): δ 2.14 (s, 3H, CH ), 7.09-7.32 (m, 3H, Ar-H), 7.53-7.72 (m, 8H, Ar-H), 7.71-7.94 (m, 2H, Ar-H), 8.25 (s,
6
3
1
3
1
1
H, CH=C), 12.69 (br s, 1H, NH); C NMR (50.3 MHz, DMSO-d ): δ 30.53, 105.85, 112.39, 118.60, 121.44, 123.12,
6
25.65, 128.71, 129.04, 129.18, 129.33, 129.44, 129.68, 133.65, 136.50, 137.89, 140.10, 140.23, 144.74, 197.15; Anal.
Calcd for C H ClNO S: C, 66.13; H, 4.16; N, 3.21; S, 7.36; Found: C, 66.06; H, 4.23; N, 3.18; S, 7.52.
2
4
18
3
4
4
.1.1.32. (2Z)-1-Phenyl-2-[(2-phenyl-1H-indol-3-yl)methylene]butane-1,3-dione (5s) [29].
.1.1.33. 1,3-Diphenyl-2-[(2-phenyl-1H-indol-3-yl)methylene]propane-1,3-dione (5t) [30]. Yellow solid; Yield: 14%;
-1
1
mp: 223-224 °C; IR (KBr, υmax cm ): 3228 (NH), 1661 (CO), 1563 (C=C); H NMR (200 MHz, DMSO-d ): δ 6.88-
6
6
.96 (m, 1H, Ar-H), 7.10-7.24 (m, 2H, Ar-H), 7.38-7.50 (m, 12H, Ar-H), 7.76-7.78 (m, 3H, Ar-H), 7.92-7.96 (m, 2H,
1
3
Ar-H and CH=C), 12.32 (br s, 1H, NH); C NMR (50.3 MHz, DMSO-d ): δ 107.75, 111.72, 120.29, 120.37, 122.50,
6
1
1
3
24.91, 128.11, 128.28, 128.42, 128.48, 128.81, 129.09, 130.12, 131.52, 132.94, 133.25, 136.33, 137.06, 137.79,
41.11, 143.82, 194.88, 196.63; Anal. Calcd for C H NO : C, 84.29; H, 4.95; N, 3.28; Found: C, 84.01; H, 5.02; N,
30
21
2
.20.
4
.1.1.34. Ethyl (2E)-2-benzoyl-3-(2-phenyl-1H-indol-3-yl)acrylate (5u) [29].
1
7

ACCEPTED MANUSCRIPT
.1.1.35. (2E)-1-Phenyl-3-(2-phenyl-1H-indol-3-yl)-2-(phenylsulfonyl)prop-2-en-1-one (5v). Yellow solid; Yield: 17%;
4
-1
1
mp: 212-214 °C; IR (KBr, υmax cm ): 3325 (NH), 1655 (CO), 1575 (C=C), 1360 (SO ), 1138 (SO ); H NMR (300
2
2
MHz, CDCl ): δ 6.95-7.27 (m, 6H, Ar-H), 7.38-7.71 (m, 11H, Ar-H), 7.98-8.01 (m, 2H, Ar-H), 8.53 (s, 1H, CH=C),
3
1
3
8
1
1
7
.59 (br s, 1H, NH); C NMR (75.5 MHz, DMSO-d ): δ 106.37, 112.11, 119.55, 120.95, 123.00, 125.55, 127.64,
6
27.64, 128.39, 128.39, 128.88, 128.88, 129.04, 129.04, 129.22, 129.22, 129.58, 129.58, 130.10, 131.51, 133.25,
3
33.76, 136.43, 136.43, 136.94, 139.45, 141.44, 144.47, 192.89; JC,H = 9.8 Hz (CO); Anal. Calcd for C H NO S: C,
29
21
3
5.14; H, 4.57; N, 3.02; S, 6.92; Found: C, 74.82; H, 4.98; N, 2.93; S, 6.60.
4
.1.1.36. Ethyl (E)-2-(4-chlorobenzoyl)-3-(2-phenyl-1H-indol-3-yl)prop-2-enoate (5w). Yellow solid; Yield: 47%; mp:
-1
1
1
54-156 °C; IR (KBr, υmax cm ): 3299 (NH), 1659 (CO), 1569 (C=C); H NMR (200 MHz, DMSO-d ): δ 1.07 (s, 3H,
6
CH ), 4.01-4.21 (m, 2H, CH ), 6.87-6.95 (m, 1H, Ar-H), 7.05-7.17 (m, 2H, Ar-H), 7.37-7.70 (m, 8H, Ar-H), 7.85-7.90
3
2
13
(
m, 2H, Ar-H), 8.08 (s, 1H, CH=C), 12.32 (br s, 1H, NH); C NMR (50.3 MHz, DMSO-d ): δ 13.59, 60.20, 107.09,
6
1
1
7
11.76, 120.09, 120.29, 122.44, 123.81, 124.76, 128.49, 128.56, 128.92, 129.33, 130.20, 130.27, 135.82, 136.21,
36.55, 137.90, 138.64, 143.91, 165.25, 194.11; Anal. Calcd for C H ClNO : C, 72.64; H, 4.69; N, 3.26; Found: C,
26
20
3
2.43; H, 4.62; N, 3.27.
4
.1.1.37. Ethyl (2E)-2-(4-nitrobenzoyl)-3-(2-phenyl-1H-indol-3-yl)acrylate (5x). Red solid; Yield: 22%; mp: 175-177
-1
°
C; IR (KBr, υmax cm ): 3298 (NH), 1678 (COOEt), 1656 (COAr), 1570 (C=C), 1523 (NO ), 1346 (NO ), 1238 and
2 2
1
1
7
221 (COOEt); H NMR (300 MHz, DMSO-d ): δ 0.99 (t, J = 7.0 Hz, 3H, CH ), 4.07 (q, J = 7.0 Hz, 2H, CH ), 6.93-
6 3 2
.17 (m, 3H, Ar-H) 7.39 (d, J = 6.0 Hz. 1H, Ar-H), 7.53-7.59 (m, 5H, Ar-H), 7.97-8.03 (m, 3H, 2 Ar-H + CH=C), 8.24
(
d, J = 8.6 Hz, 2H, Ar-H), 12.40 (br s, 1H, NH); Anal. Calcd for C H N O : C, 70.90; H, 4.58; N, 6.36; Found: C,
26 20 2 5
7
0.86; H, 4.91; N, 5.96.
4
.1.1.38. (2E)-4,4,4-Trifluoro-1-(2-furyl)-2-[(2-phenyl-1H-indol-3-yl)methylene]butane-1,3-dione (5y). Yellow Solid;
-1
-1
1
Yield: 10%; mp: 192-194°C; IR (KBr, υmax cm ): 3370 (NH), 1671 (COCF ), 1633 cm (CO), 1556 (C=C); H NMR
3
(
200 MHz, DMSO-d ): δ 6.54-6.57 (m, 1H, furan H-4), 7.03-7.72 (m, 10H, Ar-H), 7.95 (s, 1H, furan H-5) 8.13 (s, 1H,
6
1
3
CH=C), 12.87 (br s, 1H, NH); C NMR (50.3 MHz, DMSO-d ): δ 40.95, 58.07, 110.50, 112.46, 115.45, 118.70,
6
1
1
22.12, 122.90, 126.98, 128.57, 128.92, 129.58, 130.72, 130.74, 132.58, 139.48, 147.17, 149.70, 150.55, 180.65,
80.80; Anal. Calcd for C H F NO : C, 67.48; H, 3.45; N, 3.42; Found: C, 67.18; H, 3.78; N, 3.49.
2
3
14
3
3
4
.1.1.39. Methyl (2E)-2-(methylsulfonyl)-3-(2-phenyl-1H-indol-3-yl)acrylate (5z). Yellow solid; Yield: 43%; mp: 201-
-1
2
02 °C; IR (KBr, υmax cm ): 3247 (NH), 1693 (CO), 1578 (C=C), 1348 (SO ), 1291 and 1248 (COOMe), 1138 (SO );
2 2
1
H NMR (300 MHz, DMSO-d ): δ 3.29 (s, 3H, CH ), 3.62 (s, 3H, CH ) 7.18-7.23 (m, 3H, Ar-H), 7.48-7.58 (m, 6H, Ar-
6
3
3
13
H), 7.95 (s, 1H, CH=C), 12.50 (br s, 1H, NH); C NMR (75.5 MHz, DMSO-d ): δ 43.58, 52.25, 106.09, 112.47,
6
1
1
4
19.61, 121.36, 123.15, 125.62, 126.58, 128.99, 128.99, 129.45,129.45, 129.57, 130.18, 136.73, 140.53, 145.04,
3
64.47; J = 11.0 Hz (CO); Anal. Calcd for C H NO S: C, 64.21; H, 4.82; N, 3.94; S, 9.02; Found: C, 64.15; H,
C,H
19 17
4
.89; N, 3.89; S, 8.80.
4
4
.1.1.40. Methyl (2E)-3-(2-phenyl-1H-indol-3-yl)-2-(phenylsulfonyl)acrylate (5aa) [29].
.1.1.41. Ethyl (2Z)-2-acetyl-3-(2-phenyl-1H-indol-3-yl)acrylate (5ab) [29].
1
8

ACCEPTED MANUSCRIPT
.1.1.42. Diethyl [(2-phenyl-1H-indol-3-yl)methylene]malonate (5ac) [31]. Yellow solid; Yield: 13%; mp: 148-150 °C;
4
-1
1
IR (KBr, υmax cm ): 3262 (NH), 2980 (CH CH ), 1722 (COOEt), 1599 (C=C), 1262 and 1242 (COOEt); H NMR (300
2
3
MHz, DMSO-d ): δ 1.09 (t, J = 7.0 Hz, 3H, CH ), 1.23 (t, J = 7.0 Hz, 3H, CH ), 4.09-4.24 (m, 4H, 2CH ), 7.12-7.27
6
3
3
2
13
(
m, 3H, Ar-H), 7.39-7.64 (m, 6H, Ar-H), 7.84 (s, 1H, CH=C), 12.32 (br s, 1H, NH); C NMR (75.5 MHz, DMSO-d6):
δ 13.63, 14.05, 60.67, 60.72, 107.29, 112.17, 119.64, 120.43, 122.87, 125.65, 128.90, 129.17, 129.36, 130.70, 136.54,
1
3
38.40, 143.33, 164.85, 166.60; Anal. Calcd for C H NO : C, 72.71; H, 5.82; N, 3.85; Found: C, 72.79; H, 6.18; N,
22 21 4
.90.
4
4
4
4
4
4
4
.1.1.43. {[2-(4-Methoxyphenyl)-1H-indol-3-yl]methylene}malononitrile (6a) [29].
.1.1.44. (2E)-3-[2-(4-Methoxyphenyl)-1H-indol-3-yl]-2-(phenylsulfonyl)acrylonitrile (6b) [29].
.1.1.45. [(5-Methoxy-2-phenyl-1H-indol-3-yl)methylene]malononitrile (7a) [29].
.1.1.46. (2E)-3-[(5-Methoxy-2-phenyl-1H-indol-3-yl)-2-(phenylsulfonyl)acrylonitrile (7b) [29].
.1.1.47. {[5-Methoxy-2-(4-methoxyphenyl)-1H-indol-3-yl]methylene}malononitrile (8a) [29].
.1.1.48. (2E)-3-[5-Methoxy-2-(4-methoxyphenyl)-1H-indol-3-yl]-2-(phenylsulfonyl)acrylonitrile (8b) [29].
.1.2. General procedure for the preparation of N-alkylindoles 9 and 10.
A solution of 2d (0.771, 2.5 mmol) or 2e (0.798 g, 2.5 mmol) in dry DMF (5 mL) was added dropwise to a stirred
suspension of sodium hydride (60% dispersion in mineral oil, 0.110 g, 2.75 mmol) in dry DMF (5 mL) at 0°C. After
stirring at 0°C for 10 min and at rt for 20 min, the reaction mixture was cooled again to 0°C and a solution of the
suitable ω-bromoacetophenone (2.5 mL) in dry DMF (2 mL) was added dropwise. After stirring at rt for 18 h, a
saturated aqueous NH Cl solution (15 mL) was added. For 9a and 9b, the mixture was extracted with CH Cl (3×20
4
2
2
mL); the combined extracts were washed with water (5×20 mL) and dried over anhydrous Na SO ; after evaporation of
2
4
the solvent, the crude product was purified by crystallization from ethanol. For 10a, the precipitate obtained was filtered
off, dried and crystallized from ethyl acetate.
4
.1.2.1. (2E)-3-{1-[2-(3-Methoxyphenyl)-2-oxoethyl]-1H-indol-3-yl}-2-(phenylsulfonyl)acrylonitrile (9a). Yellow solid;
-1
1
Yield: 34%; mp: 212-213 °C; IR (KBr, υmax cm ): 2206 (CN), 1688 (CO), 1571 (C=C), 1324 (SO ), 1152 (SO ); H
2
2
NMR (200 MHz, CDCl ): δ 3.85 (s, 3H, OCH ), 6.21 (s, 2H, CH ), 7.33-7.37 (m, 3H, Ar-H), 7.55-7.85 (m, 7H, Ar-H),
3
3
2
13
8
.03-8.08 (m, 3H, Ar-H), 8.63 (s, 1H, CH=C); C NMR (50.3 MHz, DMSO-d ): δ 53.28, 55.10, 102.73, 108.33,
6
1
11.44, 112.47, 114.80, 118.68, 119.63, 120.28, 122.42, 123.63, 126.57, 127.09, 129.55, 129.72, 133.87, 135.35,
36.83, 137.11, 139.22, 144.07, 159.11, 192.41; Anal. Calcd for C H N O S: C, 68.41; H, 4.42; N, 6.14; S, 7.02;
1
26
20
2
4
Found: C, 68.20; H, 4.65; N, 6.20; S, 6.89.
4
.1.2.2. Ethyl (2E)-2-Benzoyl-3-{1-[2-(3-methoxyphenyl)-2-oxoethyl]-1H-indol-3-yl}acrylate (9b). Yellow solid; Yield:
-1
1
2
2%; mp: 123-125 °C; IR (KBr, υmax cm ): 1700 (COOEt), 1692 (COAr), 1669 (COPh), 1582 (C=C); H NMR (300
MHz, CDCl ): δ 1.10 (t, J = 7.1 Hz, 3H, CH ), 3.83 (s, 3H, OCH ), 4.16 (q, J = 7.1 Hz, 2H, CH ), 5.92 (s, 2H, NCH ),
3
3
3
2
2
13
7
.19-7.66 (m, 11H, Ar-H), 7.81-7.92 (m, 3H, Ar-H), 8.21 (s, 1H, CH=C); C NMR (75.5 MHz, DMSO-d ): δ 13.97,
6
1
9

ACCEPTED MANUSCRIPT
5
2.95, 55.40, 60.45, 108.63, 110.96, 112.73, 118.16, 119.82, 120.50, 121.16, 122.75, 123.99, 127.38, 128.66, 128.87,
1
29.95, 133.00, 133.36, 133.76, 135.80, 136.12, 159.41, 164.95, 193.33, 196.05; Anal. Calcd for C H NO : C, 75.50;
29
25
5
H, 5.39; N, 3.00; Found: C, 75.41; H, 5.71; N, 3.11.
4
.1.2.3. (2E)-3-{1-[2-(1,1'-Biphenyl-4-yl)-2-oxoethyl]-1H-indol-3-yl}-2-(phenylsulfonyl)acrylonitrile (10a). Yellow
-1
solid; Yield: 32%; mp: 222-223 °C; IR (KBr, υmax cm ): 2212 (CN), 1701 (CO), 1574 (C=C), 1350 (SO ), 1156 (SO );
2
2
1
13
H NMR (300 MHz, CDCl ): δ 6.24 (s, 2H, CH ), 7.31-7.82 and 7.92-8.18 (m, 19H, Ar-H), 8.62 (s, 1H, CH=C);
C
3
2
NMR (75.5 MHz, DMSO-d ): δ 53.55, 60.23, 103.01, 108.69, 111.84, 114.13, 115.16, 119.05, 122.79, 124.00, 126.93,
6
1
26.97, 127.03, 127.43, 128.57, 128.94, 129.12, 129.91, 133.19, 134.23, 137.52, 138.68, 139.60, 145.34, 192.45; Anal.
Calcd for C H N O S: C, 74.08; H, 4.41; N, 5.57; S, 6.38; Found: C, 74.17; H, 4.62; N, 5.56; S, 6.01.
3
1
22
2
3
4
4
.2. Biological studies
.2.1 Cytotoxicity assays (MTT method).
Compounds were dissolved in DMSO at 100 mM and then diluted in culture medium. Cell lines were purchased from
American Type Culture Collection (ATCC). Haematological tumour-derived cells were grown in RPMI-1640 medium
supplemented with 10% fetal calf serum (FCS), 100 units/mL penicillin G and 100 µg/mL streptomycin. Solid tumour-
derived cells were grown in their specific media supplemented with 10% FCS and antibiotics. Cell cultures were
incubated at 37 °C in a humidified 5% CO atmosphere. The absence of mycoplasma contamination was checked
2
periodically by the Hoechst staining method. Exponentially growing cells derived from human haematological tumours
+
5
[
CD4 human T-cells containing an integrated HTLV-1 genome (MT-4)] were seeded at an initial density of 1x10
cells/mL in 96 well plates in RPMI-1640 medium supplemented with 10% FCS, 100 units/mL penicillin G and 100
µg/mL streptomycin. Cell cultures were then incubated at 37 °C in a humidified 5% CO atmosphere in the absence or
2
presence of serial dilutions of test compounds. Cell viability was determined after 96 hrs at 37 °C by the by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method [37]. The compound concentrations resulting in
5
0% cell growth inhibition in comparison with untreated controls and expressed as EC50 values were determined by
linear regression analysis. The most cytotoxic derivatives were also evaluated against a panel of cell lines derived from
+
human haematological [CD4 acute T-lymphoblastic leukaemia (CCRF-CEM); splenic B-lymphoblastoid cells (WIL-
2
NS) and acute B-lymphoblastic (CCRF-SB) and human solid tumours [skin melanoma (SKMEL-28); lung squamous
carcinoma (SK-MES-1); prostate carcinoma (DU-145); hepatocellular carcinoma (Hep-G2) and breast adenocarcinoma
MCF-7)] and against normal tissues [lung fibroblasts (MRC-5) and foreskin fibroblasts (CRL-7065)]. Cell viability
was determined after 96 hrs at 37 °C by the MTT method.
(
4
.2.2. Evaluation of anticancer activity at NCI
The NCI high-flux anticancer drug screen [39-41] utilized a panel of 60 human tumor cell lines in culture derived from
nine cancer types (lung, colon, CNS, ovarian, renal, prostate and breast cancer, leukemia and melanoma). The
-4
-8
compound were tested at 10-fold dilutions of five concentrations ranging from 10 to 10 M. According to the NCI
protocol, cell lines were exposed to test agents in 96-well plates for the last 48 of a 72 h incubation and a
sulforhodamine B (SRB) protein assay was used to estimate cell viability or growth. For each compound, the drug
concentration required to produce 50% (GI ) and total (TGI) growth inhibition, and 50% cytocidal effect (LC ) were
5
0
50
2
0

ACCEPTED MANUSCRIPT
obtained for up to 60 cell lines. Values were calculated for each of these parameters if the level activity was reached; if
the effect was not reached or was exceeded, the value is expressed as greater or lesser than the maximum or minimum
concentration tested.
4
.2.3. Evaluation of antiproliferative activity in HL-60, K562 and MCF-7 cells (Alamar blue method).
Antiproliferative of HL-60, K562 and MCF-7 was determined [cells from European Cell Culture Collection (Salisbury,
UK). grown in RPMI 1640 (HL-60, K562) or DMEM (MCF-7) containing 10% foetal calf serum (FCS), 100 UI/mL
4
4
penicillin G and 100 µg/mL streptomycin]. Briefly, 200 µL of 4x10 (HL-60, K562) or 2x10 (MCF-7) cells were added
to each well of flat-bottomed microtiter trays and serial dilutions of test compounds. After a 3 day incubation at 3 7°C
the number of viable cells was determined by the Alamar blue method. Alamar blue (20 µL) was added to each well and
plates were incubated at 37°C in the dark for 3 h. Plates were then read on a fluorescent plate reader (SpectraMax
Gemini, Molecular Devices) with excitement and emission wavelengths of 544 nm and 590 nm respectively. The
compound concentrations resulting in 50% cell growth inhibition in comparison with untreated controls and expressed
as IC values were determined by linear regression analysis.
5
0
4
.2.4. Flow cytometry.
Following treatment with the desired compound/s, cells were harvested by centrifugation and resuspended in 100µL
ice-cold phosphate buffered saline (PBS) and fixed in ice-cold 70% ethanol (1 mL) at 4°C. Samples were then
centrifuged at 800xg for 10 mins and resuspended in 200 µL PBS containing 10µg/mL RNase A and 100 µg/mL
propidium iodide. Samples were incubated for 30 min at 37 °C in the dark. Following sample preparation, cell cycle
analysis was performed at 488 nm using a Becton Dickinson fluorescent activated cell sorting analysis (FACS) Calibur
flow cytometer (Becton Dickinson, California, U.S). The Macintosh-based application CellQuest was then used to
analyze the data of 10,000 gated cells once cell debris had been excluded. The data was stored as frequency histograms.
4
.2.5. Immunofluorescence.
MCF-7 cells were grown and treated on chamber well slides. Cells were then fixed in 100% ice-cold methanol, washed
in PBS-T (0.1% (v/v) Triton X-100 in PBS) and blocked for 30 min in 5% (w/v) BSA in PBS-T. Cells were then
incubated with a mouse anti-tubulin antibody for 1 h, washed 3 times with PBS-T and incubated for a further 1 h in the
secondary antibody Alexa 488. Cells were again washed 3 times in PBS-T, after which the DNA was stained with 0.2
µg/mL propidium iodide in blocking buffer for 2 min. Slides were mounted with phenylene diamine (2 µL/mL) in 50:50
PBS/glycerol. Projected images from a z-series of images were captured using the OLYMPUS 1X81 microscope
coupled with Olympus Fluoview Ver 1.5 software. All images were collected on the same day using identical
parameters.
4
.2.6. Electrophoretic mobility assay.
The compound to be tested was dissolved in MetOH and incubated with a commercial double strand plasmid DNA
vector (pQE-30, Qiagen) for 10 min at 37 °C. Gel loading buffer (0.04% bromophenol blue, 0.04% xylene cyanol, 10%
sucrose) was added and the samples were run onto a 0.7% Agarose gel to separate the closed circular (CC) and
supercolied (SC) forms. The gel was stained with EtBr to visualize the corresponding DNA bands.
2
1

ACCEPTED MANUSCRIPT
Acknowledgments
This work was financially supported by the University of Genova (PRA 2013) and MIUR (Ministero dell’Istruzione,
dell’Università e della Ricerca, PRIN # 20105YY2HL_004). The authors thank Mr. O. Gagliardo for the microanalyses,
Mr. F. Tuberoni, Dr. M. Anzaldi and Dr. R. Raggio for the IR and NMR spectra. University of Genova is gratefully
acknowledged for financially supporting C.C.
2
2

ACCEPTED MANUSCRIPT
References and notes
[
[
[
1]
2]
3]
F.R. De Sá Alves, E.J. Barreiro, C.A. Fraga, From nature to drug discovery: the indole scaffold as a 'privileged
structure', Mini Rev. Med. Chem. 9 (2009) 782-793.
S. Lal, T. J. Snape, 2-Arylindoles: a privileged molecular scaffold with potent, broad-ranging pharmacological
activity, Curr. Med. Chem. 19 (2012) 4828-4837.
M.J. Thompson, V. Borsenberger, J.C. Louth, K.E. Judd, B. Chen, Design, synthesis, and structure−activity
relationship of indole-3-glyoxylamide libraries possessing highly potent activity in a cell line model of prion
disease, J. Med. Chem. 52 (2009) 7503-7511.
[
[
[
4]
5]
6]
B. López-Iglesias, C. Pérez, J.A. Morales-García, S. Alonso-Gil, A. Pérez-Castillo, A. Romero, M.G. López,
M. Villarroya, S. Conde, M.I. Rodríguez-Franco, New melatonin-N,N-dibenzyl(N-methyl)amine hybrids:
potent neurogenic agents with antioxidant, cholinergic, and neuroprotective properties as innovative drugs for
Alzheimer's disease, J. Med. Chem. 57 (2014) 3773-3785.
L.C. Klein Jr., C.S. Passos, A.P. Moraes, V.G. Wakui, E.L. Konrath, A. Nurisso, P.A. Carrupt, C.M. de
Oliveira, L. Kato, A.T. Henriques, Indole alkaloids and semisynthetic indole derivatives as multifunctional
scaffolds aiming the inhibition of enzymes related to neurodegenerative diseases--a focus on Psychotria L.
Genus, Curr. Top Med. Chem. 14 (2014) 1056-1075.
R. Chakrabarty, J. Rao, A. Anand, A.D. Roy, R. Roy, G. Shankar, P.R. Dua, A.K. Saxena, Rational design,
*
*
synthesis
and
evaluation
of
(6aR ,11bS )-1-(4-fluorophenyl)-4-{7-[4-(4-fluorophenyl)-4-
oxobutyl]1,2,3,4,6,6a,7,11b,12,12a(RS)-decahydropyrazino[2′,1′:6,1]pyrido[3,4-b]indol-2-yl}-butan-1-one as a
potential neuroleptic agent, Bioorg. Med. Chem. 15 (2007) 7361-7367.
[
[
7]
8]
M. Amir, K. Somakala, S. Ali, p38 MAP kinase inhibitors as anti inflammatory agents, Mini Rev. Med. Chem.
1
3 (2013) 2082-2096.
A.S. Guerra, D.J. Malta, L.P. Laranjeira, M.B. Maia, N.C. Colaҫo, M. do C. de Lima, S.L. Galdino, I. da R.
Pitta, T. Gonҫalves-Silva, Anti-inflammatory and antinociceptive activities of indole-imidazolidine derivatives,
Int. Immunopharmacol. 11 (2011) 1816-1822.
[
[
[
9]
M.-Z. Zhang, Q. Chen, G.-F. Yang, A review on recent developments of indole-containing antiviral agents,
Eur. J. Med. Chem. 89 (2015) 421-441.
10]
11]
C. Karaaslan, H. Kadri, T. Coban, S. Suzen, A.D. Westwell, Synthesis and antioxidant properties of substituted
2
-phenyl-1H-indoles, Bioorg. Med. Chem. Lett. 23 (2013) 2671-2674.
C. Passemar, M. Saléry, P.N. Soh, M.-D. Linas, A. Ahond, C. Poupat, F. Benoit-Vical, Indole and
aminoimidazole moieties appear as key structural units in antiplasmodial molecules, Phytomedicine 18 (2011)
1
118-1125.
[
[
[
12]
13]
14]
Shaveta, P. Singh, Structural optimization of indole based compounds for highly promising anti-cancer
activities: Structure activity relationship studies and identification of lead molecules, Eur. J. Med. Chem. 74
(
2014) 440-450.
S.-H. Zhuang, Y.-C. Lin, L.-C. Chou, M.-H. Hsu, H.-Y. Lin, C.-H. Huang, J.-C. Lien, S.-C. Kuo, L.-J. Huang,
Synthesis and anticancer activity of 2,4-disubstituted furo[3,2-b]indole derivatives, Eur. J. Med. Chem. 66
(
2013) 466-479.
A.E. Shchekotikhin, V.A. Glazunova, L.G. Dezhenkova, Y.N. Luzikov, V.N. Buyanov, H.M. Treshalina, N.A.
Lesnaya, V.I. Romanenko, D.N. Kaluzhny, J. Balzarini, K. Agama, Y. Pommier, A.A. Shtil, M.N.
2
3

ACCEPTED MANUSCRIPT
Preobrazhenskaya, Synthesis and evaluation of new antitumor 3-aminomethyl-4,11-dihydroxynaphtho[2,3-
f]indole-5,10-diones, Eur. J. Med. Chem. 86 (2014) 797-805.
[
[
15]
16]
J.-P. Liou, N. Mahindroo, C.-W. Chang, F.-M. Guo, S.W.-H. Lee, U.-K. Tan, T.-K. Yeh, C.-C. Kuo, Y.-W.
Chang, P.-H. Lu, Y.-S. Tung, K.-T. Lin, J.-Y. Chang, H.-P. Hsieh, Structure-activity relationship studies of 3-
aroylindoles as potent antimitotic agents, ChemMedChem. 1 (2006) 1106-1118.
G. La Regina, M.C. Edler, A. Brancale, S. Kandil, A. Coluccia, F. Piscitelli, E. Hamel, G. De Martino, R.
Matesanz, J.F. Díaz, A.I. Scovassi, E. Prosperi, A. Lavecchia, E. Novellino, M. Artico, R. Silvestri,
Arylthioindole inhibitors of tubulin polymerization. 3. Biological evaluation, structure-activity relationships
and molecular modeling studies, J. Med. Chem. 50 (2007) 2865-2874.
[
[
17]
18]
S.N. Baytas, N. Inceler, A. Yilmaz, A. Olgac, S. Menevse, E. Banoglu, E. Hamel, R. Bortolozzi, G. Viola,
Synthesis, biological evaluation and molecular docking studies of trans-indole-3-acrylamide derivatives, a new
class of tubulin polymerization inhibitors, Bioorg. Med. Chem. 22 (2014) 3096-3104.
M.-J. Lai, H.-L. Huang, S.-L. Pan, Y.-M. Liu, C.-Y. Peng, H.-Y. Lee, T.-K. Yeh, P.-H. Huang, C.-M. Teng,
C.-S. Chen, H.-Y. Chuang, J.-P. Liou, Synthesis and biological evaluation of 1-Arylsulfonyl-5-(N-
hydroxyacrylamide)indoles as potent histone deacetylase inhibitors with antitumor activity in vivo, J. Med.
Chem. 55 (2012) 3777-3791.
[
[
[
[
19]
20]
21]
22]
A. Brancale, R. Silvestri, Indole, a core nucleus for potent inhibitors of tubulin polymerization, Med. Res. Rev.
2
7 (2006) 209-238.
S.A. Patil, R. Patil, D.D. Miller, Indole molecules as inhibitors of tubulin polymerization: potential new
anticancer agents, Future Med. Chem. 16 (2012) 2085-2115.
W.A. Maltese, J.H. Overmeyer, Methuosis: nonapoptotic cell death associated with vacuolization of
macropinosome and endosome compartments, Am. J. Pathol. 184 (2014) 1630-1642.
Y. Lv, Y. Guo, J. Xu, S. Shao, Simple indole-based colorimetric sensors with electron-withdrawing
chromophores: Tuning selectivity in anion sensing, J. Fluorine Chem. 132 (2011) 973-977 and references
therein.
[
[
23]
24]
A. Gazit, H. App, G. McMahon, J. Chen, A. Levitzki, F.D. Bohmer, Tyrphostins. 5. Potent inhibitors of
Platelet-Derived Growth Factor Receptor tyrosine kinase: structure−activity relationships in quinoxalines,
quinolines, and indole tyrphostins, J. Med. Chem. 39 (1996), 2170-2177.
(a) M. Matsuoka, M. Takao, T. Kitao, T. Fujiwara, K. Nakatsu, Cyanovinylheteroaromatics for organic
nonlinear optics, Mol. Cryst. Liq. Cryst. Inc. Nonlinear Opt. 182 (1990) 71-79; (b) L. Zhengdong, W. Kechen,
S. Genbo, H. Youping, Nonlinear optical properties of some derivatives of stilbene and vinyl cyanide, Opt.
Mater. 20 (2002) 295-299.
[
[
[
25]
26]
27]
T.A., Hill, C.P. Gordon, A.B. McGeachie, B. Venn-Brown, L.R. Odell, N. Chau, A. Quan, A. Mariana, J.A.
Sakoff, M. Chircop, P.J. Robinson, A. McCluskey, Inhibition of Dynamin Mediated Endocytosis by the
Dynoles—Synthesis and Functional Activity of a Family of Indoles, J. Med. Chem. 52 (2009) 3762–3773.
M. Pojarová, D. Kaufmann, R. Gastpar, T. Nishino, P. Reszka, P.J. Bednarski, E. von Angerer, [(2-
Phenylindol-3-yl)methylene]propanedinitriles inhibit the growth of breast cancer cells by cell cycle arrest in
G /M phase and apoptosis, Bioorg. Med. Chem. 15 (2007) 7368-7379.
2
(a) B. Hansen, T. Hansen, S. Tullin, M. Colding-Jorgensen, Chemical uncouplers for the treatment of obesity,
US Pat. Appl. (2004) US20040138301 A1. (b) L.B. Christiansen, T.K. Hansen, P.H. Olesen, T.M. Tagmose,
Indole derivatives for use as chemical uncoupler, Int. Pat. Appl. (2005) WO2005051908 A1.
2
4

ACCEPTED MANUSCRIPT
[
28]
A. Ranise, S. Cesarini, A. Spallarossa, F. Sancassan, F. Bondavalli, O. Bruno, S. Schenone, G. Menozzi, P.
Fossa, L. Mosti, Unprecedented one-pot stereoselective synthesis of Knoevenagel-type derivatives via in situ
condensation of N-Methyleniminium Salts of Ethylenethiourea and Ethyleneurea with active methylene
reagents, Synthesis 16 (2007) 2495-2502.
[
[
29]
30]
A. Ranise, F. Lucchesini, M. Caviglia, S. Alfei, A. Spallarossa, C. Caneva, Unconventional stereoselective
one-pot synthesis of Knoevenagel-type indoles via in situ condensation of iminium salts with active methylene
reagents, Tetrahedron 69 (2013) 10858-10868.
J. Liu, X. Wang, H. Guo, X. Shi, X. Ren, G. Huang, A novel NH OAc and CuBr(PPh ) -induced
4
3 3
intermolecular Pummerer-type reaction between free (NH)-indoles and dimethylsulfoxide: facile synthesis of
-methylthiomethyl substituted indoles, Tetrahedron 68 (2012) 1560-1565.
3
[
[
31]
32]
C.H. Oh, H.S. Park, N. Park, S.Y. Kim, L. Piao, Scandium-Catalyzed Intramolecular Friedel-Crafts reactions
of 2-[(2-Alkyl-1H-indol-3-yl)methylene]malonates, Synlett 25 (2014) 579-585.
Z. Shiokawa, K. Hashimoto, B. Saito, Y. Oguro, H. Sumi, M. Yabuki, M. Yoshimatsu, Y. Kosugi, Y. Debori,
N. Morishita, D.R. Dougan, G.P. Snell, S. Yoshida, T. Ishikawa, Design, synthesis, and biological activities of
novel hexahydropyrazino[1,2-a]indole derivatives as potent inhibitors of apoptosis (IAP) proteins antagonists
with improved membrane permeability across MDR1 expressing cells, Bioorg. Med. Chem. 21 (2013) 7938-
7
954.
[
[
33]
34]
E. Pasquier, M. Kavallaris, Microtubules: a dynamic target in cancer therapy, IUBMB Life 60 (2008) 165-170.
Greene, L. M.; Campiani, G.; Lawler, M.; Williams, D. C.; Zisterer, D. M.; BubR1 is required for a sustained
mitotic spindle checkpoint arrest in human cancer cells treated with tubulin-targeting pyrrolo-1,5-
benzoxazepines. Mol. Pharmacol. 73 (2008) 419-430
[
[
35]
36]
J.M. Mulligan, L.M. Greene, S. Cloonan, M.M. Mc Gee, V. Onnis, G. Campiani, C. Fattorusso, M. Lawler,
D.C. Williams, D.M. Zisterer, Identification of tubulin as the molecular target of proapoptotic pyrrolo-1,5-
benzoxazepines, Mol. Pharmacol. 70 (2006) 60-70
McElligott, A. M.; Maginn, E. N.; Greene, L. M.; McGuckin, S.; Hayat, A.; Browne, P. V.; Butini, S.;
Campiani, G.; Catherwood, M. A.; Vandenberghe, E.; Williams, D. C.; Zisterer, D. M.; Lawler, M.; The novel
tubulin-targeting agent pyrrolo-1,5-benzoxazepine-15 induces apoptosis in poor prognostic subgroups of
chronic lymphocytic leukemia. Cancer Res. 69 (2009) 8366-8375.
[
[
[
37]
38]
39]
S, Mahboobi, E. Eichhorn, M. Winkler, A. Sellmer, U. Möllmann, Antibacterial activity of a novel series of 3-
bromo-4-(1H-3-indolyl)-2,5-dihydro-1H-2,5-pyrroledione derivatives
- An extended structure–activity
relationship study, Eur. J. Med. Chem. 43 (2008) 633-656.
R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, D. Schols, P. Herdewijn, J. Desmyter, E. De Clercq, Rapid and
automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds, J. Virol. Methods 20
(
1988) 309-321.
A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose, J. Langley, P. Cronise, A.
Vaigro-Wolff, M. Gray-Goodrich, H. Campbell, J. Mayo, M. Boyd, Feasibility of a High-Flux anticancer drug
screen using a diverse panel of cultured human tumor cell lines, J. Natl. Cancer Inst. 83 (1991) 757-766.
M.R. Grever, S.A. Shepartz, B.A. Chabner, The National Cancer Institute: cancer drug discovery and
development program, Semin. Oncol. 19 (1992) 622-638.
[
[
40]
41]
J.N. Weinstein, T.G. Myers, P.M. O'Connor, S.H. Friend, A. J. Fornace Jr., K.W. Kohn, T. Fojo, S.E. Bates,
L.V. Rubinstein, N.L. Anderson, J.K. Buolamwini, W.W. van Osdol, A.P. Monks, D.A. Scudiero, E.A.
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Sausville, D.W. Zaharevitz, B. Bunow, V.N. Viswanadhan, G.S. Johnson, R.E. Wittes, K.D. Paull, An
Information-Intensive Approach to the Molecular Pharmacology of Cancer, Science 275 (1997) 343-349.
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Highlights
•
•
•
Synthesis of Knoevenagel-type indoles via one-pot procedure
Selected compounds showed antiproliferative activities in cell-based assays
Some derivatives caused apoptosis and partially depolymerised tubulin
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ACCEPTED MANUSCRIPT
Supplementary data
Unconventional Knoevenagel-type Indoles: Synthesis and Cell-based
Studies for the Identification of Pro-apoptotic Agents
*,a
a
a
a
b
Andrea Spallarossa, Chiara Caneva, Matteo Caviglia, Silvana Alfei, Stefania Butini, Giuseppe
b
b
b
c
c
Campiani, Sandra Gemma, Margherita Brindisi, Daniela M. Zisterer, Sandra A. Bright, Clive D.
c
d
d
e
e
Williams, Emmanuele Crespan, Giovanni Maga, Giuseppina Sanna, Ilenia Delogu, Gabriella
e
e
Collu, Roberta Loddo.
a
Dipartimento di Farmacia, Università di Genova, Viale Benedetto XV 3, I-16132 Genova, Italy
b
European Research Centre for Drug Discovery & Development, Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena, via Aldo
Moro 2, I-53100 Siena, Italy
c
School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, 152-160 Pearse Street, Dublin 2, Ireland
d
.
Institute of Molecular Genetics IGM-CNR, via Abbiategrasso 207, 27100 Pavia (Italy).
e
Dipartimento di Scienze Biomediche, Sezione di Microbiologia e Virologia, Università di Cagliari, Cittadella Universitaria, s.p. 8, Km 0.700, I-
0
9042 Monserrato (Cagliari), Italy
*
Corresponding author:
Dr. Andrea Spallarossa
Dipartimento di Farmacia
Sezione di Chimica del Farmaco e del Prodotto Cosmetico
Università degli Studi di Genova
Viale Benedetto XV, 3
1
6132 - Genova, Italy
phone: +39 010 353 8364;
fax: +39 010 353 8358;
e-mail: andrea.spallarossa@unige.it
S1