Substituted E-3-(3-indolylmethylene)1,3-dihydroindol-2-ones with antiproliferative activity. Study of the effects on HL-60 leukemia cells

Article abstract of DOI:10.1016/j.ejmech.2014.04.004

The synthesis of new substituted E-3-(3-indolylmethylene)1,3-dihydroindol- 2-ones is reported. The antiproliferative activity was evaluated according to protocols available at the National Cancer Institute (NCI), Bethesda, MD. The action of the most active compound 10 was further investigated in HL-60 leukemia cells. Results obtained show that it causes a block in cell cycle progression, with cell arrest in the G2/M phase, associated with activation of apoptosis accompanied with increased oxidative stress and deregulation of the homeostasis of divalent cations, with significant increase in the cellular concentrations of free Ca²⁺ and Mg²⁺.

Full text of DOI:10.1016/j.ejmech.2014.04.004

European Journal of Medicinal Chemistry 79 (2014) 382e390
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Substituted E-3-(3-indolylmethylene)1,3-dihydroindol-2-ones with
antiproliferative activity. Study of the effects on HL-60 leukemia cells
,
Alberto Leoni a, Alessandra Locatelli a, Rita Morigi a, Mirella Rambaldi a
,
*
Concettina Cappadone a, Giovanna Farruggia a, Stefano Iotti a, Lucia Merolle a,
Maddalena Zini b, Claudio Stefanelli c
a Department of Pharmacy and Biotechnology, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
b Department of Biomedical and Neuromotor Sciences, University of Bologna, 40126 Bologna, Italy
c Department for Life Quality Studies, University of Bologna, Rimini Campus, 47921 Rimini, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of new substituted E-3-(3-indolylmethylene)1,3-dihydroindol-2-ones is reported. The
antiproliferative activity was evaluated according to protocols available at the National Cancer Institute
(NCI), Bethesda, MD. The action of the most active compound 10 was further investigated in HL-60
leukemia cells. Results obtained show that it causes a block in cell cycle progression, with cell arrest
in the G2/M phase, associated with activation of apoptosis accompanied with increased oxidative stress
and deregulation of the homeostasis of divalent cations, with significant increase in the cellular con-
Received 14 October 2013
Received in revised form
1 April 2014
Accepted 2 April 2014
Available online 3 April 2014
centrations of free Ca2þ and Mg2þ
.
Keywords:
Ó 2014 Elsevier Masson SAS. All rights reserved.
Indolyl-indol-2-one
Knoevenagel reaction
Antiproliferative activity
Apoptosis
Divalent cations
1. Introduction
reacting an aldehyde with an activated methylene group, thus
permitting the generation of large series of compounds.
Through this strategy, it was possible to identify several com-
pounds endowed with a marked inhibition of cellular prolifer-
ation. The derivatives reported in Chart 1 were among the most
potent obtained.
Some of our previous studies were devoted to the synthesis
of indolylmethylene-1,3-dihydroindol-2-one derivatives ob-
tained through the Knoevenagel condensation [1e6]. This re-
action allows to obtain adducts in a relatively easy way by
Abbreviations: NCI, National Cancer Institute; DTP, Developmental Therapeutics Program; GI, growth inhibition; TGI, total growth inhibition; LC, lethal concentration; BEC,
Biological Evaluation Committee; DMSO, dimethyl sulfoxide; ROS, Reactive oxygen species; DCFDA, dichlorofluoresceine diacetate; DCF, dichlorofluorescein; PBS, phosphate
buffered saline; FBS, fetal bovine serum; EDTA, ethylenediaminetetraacetic; EGTA, ethylene glycol tetraacetic acid.
* Corresponding author.
E-mail address: mirella.rambaldi@unibo.it (M. Rambaldi).
http://dx.doi.org/10.1016/j.ejmech.2014.04.004
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

A. Leoni et al. / European Journal of Medicinal Chemistry 79 (2014) 382e390
383
Chart 1. Most active compounds reported in ref. 1e6.
Scheme 1. Synthesis of E-3-(3-indolylmethylene)1,3-dihydroindol-2-ones derivatives.
Table 1
New E-3-(3-indolylmethylene)1,3-dihydroindol-2-ones derivatives.
Comp
Starting compound 1
Starting compound 2
R
R1
R2
R3
R4
R5
R6
R7
R8
R9
3
4
5
6
7
8
9
a
a
a
a
b
c
d
c
c
c
e
f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Bn
H
H
H
CH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
OCH3
H
H
H
H
OCH3
H
OH
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
a
b
c
d
a
e
f
g
h
i
i
i
i
j
k
l
l
l
l
H
H
Bn
H
H
Bn
PhEt
4-MPhEt
Phprop
Phprop
Phprop
Phprop
Phprop
Morphet
cinn
3,4,5-TM-Bn
3,4,5-TM-Bn
3,4,5-TM-Bn
3,4,5-TM-Bn
3,4,5-TM-Bn
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CBR
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Cl
OCH3
OH
H
OCH3
OCH3
OH
OCH3
Cl
H
H
H
Cl
d
c
f
H
f
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
d
g
e
h
i
c
e
f
Cl
Cl
Cl
Cl
l
a
m
m
m
m
m
Cl
Ph
Ph
Ph
Ph
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
OCH3
Cl
H
j
a
OCH3
Ph
H
H
Bn ¼ benzyl; CBR ¼ condensed benzene ring; PhEt ¼ phenylethyl; 4-MPhEt ¼ 4-methoxyphenylethyl; Phprop ¼ phenylpropyl; Morphet ¼ morfolinethyl; cinn ¼ cinnamic;
3,4,5-TM-Bn ¼ 3,4,5-trimethoxybenzyl; Ph ¼ phenyl.
In this paper we wish to continue the study of the anti-
proliferative activity and of the mechanism of action of new ana-
logues bearing the indolylmethylene-1,3-dihydroindol-2-one
scaffold (Scheme 1 and Table 1) in which the substituents of the
two heterocyclic systems were chosen as described below.
Considering the positive effect of the methoxy groups at the
chloroindole portion previously demonstrated, we have proceeded
to the synthesis of compounds 3e7 in which the introduction of
methoxy groups at the indolinone nucleus has been considered, in
order to study the effect of this shift.

384
A. Leoni et al. / European Journal of Medicinal Chemistry 79 (2014) 382e390
Table 2
Nine subpanels at five concentrations: growth inhibition, cytostatic and cytotoxic activity (mM) of the selected compounds.
N. NSC
Compa
Modes
Leukemia
NSCLC
Colon
CNS
2.75
13.49
44.67
1.58
Melanoma
Ovarian
Renal
3.39
16.22
53.70
1.35
Prostate
Breast
MG-MIDb
746082
3c
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
GI50
TGI
LC50
GI50
TGI
GI50
TGI
GI50
TGI
GI50
TGI
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
GI50
TGI
LC50
GI50
TGI
1.51
56.23
e
3.55
22.39
74.13
1.86
2.24
8.91
33.88
1.29
3.39
16.22
46.77
1.86
6.61
30.90
8.91
32.36
87.10
7.08
28.18
74.13
0.46
2.51
15.14
41.69
1.51
3.47
19.95
51.29
1.17
5.50
e
1.62
12.59
64.57
1.62
2.57
16.60
54.95
1.41
745261
744492
751251
750019
7c
8
0.55
7.08
42.66
3.63
21.38
75.86
3.39
77.62
e
9.12
4.27
6.31
9.77
5.37
8.13
6.92
67.61
14.79
57.54
e
18.20
19.95
60.26
95.50
9.77
33.88
72.44
0.28
35.48
16.60
47.86
93.33
10.47
52.48
95.50
0.34
77.62
20.89
66.07
95.50
9.12
64.57
95.50
0.34
13.49
69.18
12.02
72.44
2.45
23.44
60.26
2.09
36.31
20.42
70.79
66.07
12.88
64.57
e
44.67
13.49
50.12
93.33
7.24
44.67
91.20
0.38
20.42
54.95
e
e
9
7.08
50.12
e
9.33
54.95
95.50
0.48
18.62
72.44
13.80
67.61
1.86
9.77
17.78
1.91
30.20
29.51
6.76
47.86
e
3.98
25.12
e
10c
0.35
15.49
e
0.43
0.35
19.50
54.95
19.05
e
0.32
6.31
15.14
74.13
14.13
72.44
2.24
8.91
28.84
2.24
37.15
20.42
97.72
2.40
32.36
10.96
66.07
2.29
5.89
16.22
3.24
12.02
47.86
6.61
85.11
2.14
8.32
27.54
9.55
24.55
57.54
2.51
17.38
63.10
0.25
1.82
8.32
9.12
9.77
52.48
12.02
60.26
1.95
5.62
19.05
1.32
21.88
10.96
85.11
2.29
48.98
16.60
e
58.88
16.60
75.86
2.82
10.23
50.12
2.04
63.10
13.80
67.61
2.04
5.75
35.48
2.40
38.90
14.79
75.86
1.45
11.48
25.12
e
72.44
12.88
67.61
2.24
11.48
36.31
2.00
38.90
15.85
83.18
2.00
29.51
16.60
85.11
2.40
6.31
20.89
3.02
12.30
50.12
5.01
64.57
1.32
743939
751960
12c
13c
8.91
50.12
2.88
81.28
e
10.00
72.44
2.19
9.12
42.66
1.86
36.31
10.23
57.54
1.41
34.67
10.00
e
2.00
6.46
34.67
2.09
16.22
14.45
95.50
2.29
50.12
5.50
87.10
1.78
3.89
8.91
1.91
5.13
17.38
6.17
e
751963
742198
741774
742490
742489
17
18
19c
20
21
2.00
e
e
41.69
33.11
e
6.17
77.62
1.41
18.62
3.24
42.66
1.86
4.79
15.85
2.45
14.13
51.29
2.95
21.38
0.52
2.63
50.12
2.34
8.91
58.88
0.76
4.47
58.88
0.10
15.85
630.96
16.22
75.86
2.04
34.67
48.98
e
2.34
2.75
33.11
38.90
43.65
30.90
e
e
e
e
2.19
5.62
2.34
6.03
23.44
3.02
12.59
40.74
4.90
69.18
2.19
8.51
30.90
5.25
19.05
52.48
0.98
2.29
5.75
17.78
2.88
8.91
51.29
5.13
61.66
1.48
3.98
10.47
5.62
18.20
51.29
2.40
3.72
2.95
7.24
18.62
3.24
11.75
45.71
5.25
77.62
1.12
4.07
13.18
8.51
21.88
51.29
2.29
17.38
63.10
0.32
2.09
5.50
13.80
39.81
3.98
22.91
66.07
9.12
79.43
1.38
4.07
15.85
8.51
23.99
66.07
2.00
17.38
66.07
0.32
19.95
316.23
29.51
2.88
10.00
51.29
4.17
79.43
1.05
3.24
22.39
2.95
15.14
61.66
4.17
56.23
1.23
4.47
27.54
4.27
13.80
52.48
0.63
7.24
47.86
0.32
742488
22
743995
742498
23
24c
1.12
3.47
19.05
9.33
23.99
60.26
2.63
13.18
52.48
0.13
6.31
316.23
4.57
19.05
5.89
18.62
56.23
1.58
12.02
53.70
0.20
7.94
6.31
747146
747433
25
25.70
60.26
1.74
10.96
42.66
0.10
LC50
GI50
TGI
LC50
GI50
TGI
26c
9.55
38.90
0.13
6.31
199.53
16.98
50.12
0.16
7.94
251.19
Vincristine
sulfated
15.85
251.19
3.98
79.43
19.95
251.19
7.94
316.23
10.00
251.19
LC50
a
Highest conc. ¼ 10ꢀ4 M unless otherwise reported; only value >100
Mean Graph MIDpoint i.e. the calculated panel mean.
Mean of two separate experiments.
mM are reported. The compound exposure time was 48 h.
b
c
d
Highest conc. ¼ 10ꢀ3 M.
We have also focused the study of new substituents at the
effect of the shift of the benzyl group from the nitrogen of the
chloroindole to that of the indolinone (compound 23) has been
considered.
Finally, we have pursued the study of the substituents at the 2
position of the indole portion (R6). In our previous studies this
position was substituted with a chlorine or unsubstituted. Through
the synthesis of compounds 24e28 the effect of a methoxy group at
this position has been investigated.
The cytotoxic activities of all new compounds were evaluated
according to Developmental Therapeutics Program (DTP), National
Cancer Institute (NCI), Bethesda, MD, drug screen protocols. For
some insights into the biological effects of these derivatives, the
most active compound 10 was submitted to additional studies
concerning the effect on cell cycle progression and on induction of
apoptosis.
nitrogen (R5) of the chloroindole moiety. Until now, we have
considered methyl, phenyl, benzyl and substituted benzyl group.
In particular, good results were obtained with benzyl and p-
chlorobenzyl which led to compounds showing GI50 0.65 and
0.36 mM respectively [4]. The synthesis of compounds 8e15 en-
ables us to study the effect of the increment of the distance be-
tween the nitrogen and the phenyl whereas in compound 16 the
phenyl has been substituted by the morpholine and in compound
17 the effect of the introduction of a double bond in the pro-
pylphenyl group has been considered. Moreover, since the tri-
methoxyphenyl group is contained in several antitumor agents
(such as colchicine, combretastatin, podophyllotoxin) we plan-
ned the synthesis of compounds 18e22 bearing the trimethox-
ybenzyl group at the chloroindole nitrogen (R5). Furthermore, the

A. Leoni et al. / European Journal of Medicinal Chemistry 79 (2014) 382e390
385
2. Chemistry
Most of the compounds were prepared by means of the single
step Knoevenagel reaction between the indolinones 1 and the al-
dehydes 2 in methanol/piperidine. For compounds 3, 6, 7, 9, 12, 13
and 17 a mixture of acetic acid/hydrochloric acid has been
employed, for the reaction of compounds 25e28 has been used
methanol and NH4OH conc, while for compound 24 has been used
toluene and p-toluenesulfonic acid.
Compounds were obtained as almost pure geometrical isomers
which, according to the usual NOE experiments described in the
previous papers [7,8] were assigned to the E configuration. The
oxindoles 1c and 1e are commercially available whereas the other
starting compounds (oxindoles and aldehydes) have been prepared
according to the literature [4,6,9e20], except the aldehydes 2g, 2jel
reported under the experimental section.
3. Biology
3.1. Cell-based assays
In a preliminary test, compounds were assayed at a single high
concentration (10ꢀ5 M) in the full NCI 60 cell panel. This panel is
organized into subpanels representing leukemia, melanoma, and
cancers of lung, colon, kidney, ovary, breast, prostate, and central
nervous system. Only compounds that satisfy predetermined
threshold inhibition criteria in a minimum number of cell lines
progress to the full five-concentration assay. The threshold inhibi-
tion criteria for progression to the five-concentration screen was
selected to efficiently capture compounds with antiproliferative
activity based on the analysis of historical DTP screening data. The
result is expressed as the percent growth of treated cells at the test
concentration of 10ꢀ5 M following 48 h of incubation (unpublished
results).
Compounds 4e6, 11, 14e16 and 27e28 were not considered
active enough to enter the five-concentration test, whereas all the
others were subjected to this screen. The compounds were dis-
solved in dimethyl sulfoxide (DMSO) and evaluated using five
concentrations at 10-fold dilutions (the highest being 10ꢀ4 M)
following 48 h of incubation.
Fig. 1. Effects of compound 10 on the growth of HL-60 cells. (A) Rate of cell growth
determined as total cell number. Control cells and cells treated with compound 10
(1
determinations. (B) Effect of compound 10 on cell cycle. Cells were incubated for 24 h
with the vehicle (Ctrl) or with compound 10 (1 M), afterward cell cycle distribution
m
M) were incubated and viable cells were counted daily. Means ꢁ s.e.m. of three
m
was determined by flow cytometry. Panels report the cytofluorimeter outputs ob-
tained in one typical experiment repeated twice with similar results. The percentages
of cells in the different phases of the cell cycle (G0-G1; G2-M; S) are reported.
Following treatment with 10, most cells are in the G2-M phase and a well detectable
fraction of DNA is present as a pre-G1 peak (light blue peak). (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of
this article.)
Table 2 reports the results obtained (vincristine is reported for
comparison purposes), expressed as mM at three assay end points:
the 50% growth inhibitory power (GI50), the cytostatic effect
(TGI ¼ total growth inhibition), and the cytotoxic effect (LC50). For
some compounds the five-concentrations test was repeated and no
significant differences were found. For these compounds the data
reported in Table 2 are the mean values of the two experiments.
The tested compounds showed a mean GI50 between 0.38 and
obtained when R5 is a 4-methoxyphenylethyl group, in fact com-
pound 10 showed mean GI50 of 0.38 mM therefore proving to be the
most active of the series. Comparing compounds 9 and 15, it is
evident that the substitution of the phenylethyl with a phenyl-
propyl group was detrimental for the activity in fact compound 15
did not progress to the full five-concentration assay whereas
16.60 mM, and compounds 3 and 26 were submitted to BEC (Bio-
logical Evaluation Committee) for possible future development.
In light of the NCI 60 results, the following considerations may
be done.
The introduction of methoxy groups at the oxindole portion did
not lead to an improvement of activity, in fact among the five
compounds synthesized only two (3, 7) were tested also in the five
concentrations assay, although compound 3 was selected by BEC
for the great difference between GI50 and LC50, and compound 7
showed selectivity towards leukemia cell lines (mean GI50
compound 9 showed mean GI50 7.24 mM. However, in the presence
of the phenylpropyl group, the activity seems to be strongly influ-
enced by the substituent at the indolinone moiety (R2): compounds
14 (R2 ¼ OCH3) and 15 (R2 ¼ OH) did not enter the five-
concentration assay, whereas compound 13 (R2 ¼ Cl) showed
mean GI50 of 2.24
mM. The introduction of the morpholinethyl
group was disadvantageous and led to an inactive compound (16).
Good results were achieved by introducing the cinnamoyl group
0.55
mM).
(compound 17 showed mean GI50 of 2.00 mM), as well as the 3,4,5-
Interesting results have been obtained studying the effect of the
substituents at the nitrogen of the chloroindole (R5). Considering
compounds 8, 10 and 12, which are different only for R5, it can be
noted that the benzyl and phenylpropyl groups determine about
the same activity: compound 8 and 12 showed mean GI50 of 13.49
trimethoxybenzyl group (18e22): all the derivatives progressed to
the full five-concentration assay and three of them (19, 21 and 22)
showed a mean GI50 between 2.00 and 3.00 mM.
Shifting of the N-benzyl from the chloroindole (8) to the oxin-
dole nitrogen (23) led to a significant difference in the biological
and 12.88
mM, respectively. A notable increment of activity was
behavior: mean GI50 of 13.49 mM, vs 5.01 mM.

386
A. Leoni et al. / European Journal of Medicinal Chemistry 79 (2014) 382e390
Fig. 2. Effect of compound 10 on biochemical parameters in HL-60 cells. Control cells and cells treated with 1 mM compound 10 were incubated for 24 h, afterward the following
parameters were measured. (A) The exposure of phosphatidylserine in the outer side of the plasma membrane was determined by flow cytometry after labelling with Annexin V/
FLUOS. (B) Caspase activity acting on the peptide sequence DEVD (DEVDase activity) was measured in cellular extracts. (C) Detection of oxidative stress by flow cytometry after
DCFDA staining. The increase in the fluorescence intensity (DCF-positive cells) in cells incubated in the presence of 10, indicates an increase in intracellular peroxides. (D)
Intracellular content of Ca2þ was assayed by the INDO-1/AM probe. (E) Intracellular content of Mg2þ measured by the Mag-Fura2 probe. Results depicted in each panel are
means ꢁ s.e.m. of three determinations.
Although the evaluation of the antitumor activity of the com-
pounds lacking chlorine at the 2-position (R6) of the indole portion
needs to be further investigated, the introduction at this position of
a methoxy group gave promising results (24e26), in particular
concentration, that can both deeply influence cellular signaling
[24]. To assess the production of ROS, we evaluated the intracellular
oxidative stress by the probe dichlorofluoresceine diacetate
(DCFDA), which is oxidized to the highly fluorescent dichloro-
fluorescein (DCF) by cellular peroxides. The treatment of HL-
60 cells with compound 10 caused a well evident increase in DCF
fluorescence indicating increased peroxide production (Fig. 2C). As
regards Ca2þ, the concentration of free intracellular calcium ion,
measured by the probe INDO-1/AM, was significantly increased in
10-treated cells (Fig. 2D). Lastly, we wanted to determine whether
even Mg2þ concentration was affected by 10. In fact, recent work
has shown that magnesium can be involved in both cell growth and
apoptosis in cancer cells even though its role in these processes is
not yet fully defined [25]. Magnesium can lead to activation of
several enzymes, including nucleases, and exposure to elevated
Mg2þ causes cell death [26]. To date, a very few studies have
examined the effect of anticancer compounds on cellular magne-
sium. Fig. 2E shows that the exposure of HL-60 cells to 10, causes
the increase in the intracellular concentration of free Mg2þ, deter-
mined by the fluorescent probe Mag-Fura2.
compound 24, which showed mean GI50 of 1.32
strated to be effective mainly versus leukemia cells (mean GI50
0.52 M).
mM and demon-
m
3.2. Effect on growth and death of HL-60 cells
To give some insights into the biological effects of the new de-
rivatives, the most active compound 10 was submitted to additional
studies using HL-60 leukemia cells. Fig. 1A shows that compound
10 at 1 mM concentration strongly reduced cell proliferation within
24 h of treatment. After 48 h, the cell number was decreased,
indicating the onset of cytotoxicity. In order to assess whether the
antiproliferative effect of 10 was associated with interference with
cell cycle progression, DNA profiles of cultured cells were examined
by flow cytometry. Fig. 1B shows that the treatment with 10 caused
a marked accumulation of HL-60 cells in the G2/M phase (69.3%),
whereas only 10.7% of control cells were in the G2/M phase. In 10-
treated cells, the block in the G2/M phase of the cell cycle was
associated with a well distinguishable pre-G1 peak in DNA, sug-
gestive of DNA fragmentation, characteristic of apoptosis.
Altogether, the presented data suggest a picture in which 10
causes a block in cell cycle progression, with cell arrest in the G2/M
phase associated with increased oxidative stress and to a deregu-
lation of the homeostasis of divalent cations Ca2þ and Mg2þ
.
To confirm the activation of the apoptotic program, we deter-
mined the exposure of phosphatidylserine from the inner to the outer
side of the plasma membrane, which is an early marker of apoptosis
[21], utilizing the Annexin V-FLUOS staining method. Annexin V is a
protein with high affinity for phospholipids, and cells undergoing
apoptosisare marked fromearlystages with Annexin V-FLUOS, which
binds to externalized phosphatidylserine. As shown in Fig. 2A, after
24 h of incubation, only 4% of control HL-60 cells were Annexin V
positive, whereas this percentage rose to about 50% in cells incubated
in the presence of compound 10. To further confirm the onset of
apoptosis, the activity of caspase proteases was assayed. In cells
treated for 24 h with 10, the activity of effector caspases cutting their
substrate atlevel of theaminoacidmotifDEVD(Asp-Glu-Val-Asp)was
significantly increased (Fig. 2B). Recently, it has become evident that
activation of autophagy is an important mechanism in the anti-
proliferative effect of anticancer molecules [22], however we did not
observe any effect of 10 on autophagy in HL-60 cells, as detected by
acridine orange staining or by LC3 protein processing, that represents
a hallmark of autophagy [23] (data not shown).
4. Conclusions
The
synthesis
of
new
E-3-(3-indolylmethylene)1,3-
dihydroindol-2-ones led to compounds showing in the NCI-
60 cell test mean GI50 values in the micromolar or sub-micromolar
range. Two compounds (3 and 26) demonstrated a particularly
interesting activity profile and were selected by BEC for possible
future development.
The introduction at the chloroindole nitrogen of
a 4-
methoxyphenylethyl group allowed to obtain compound 10, which
showed submicromolar mean GI50 values in all the nine NCI sub-
panels, thus revealing to be the most active compound of the series.
Compound 10 was also submitted to additional studies in HL-60
leukemia cells which showed that it exerts the antitumor activity
by inhibition of cell proliferation and activation of apoptosis.
Compound 10 causes a block in cell cycle progression, with arrest in
the G2/M phase, associated with increased intracellular formation
of peroxides and deregulation of the homeostasis of divalent cat-
ions, with significant increase in the cellular content of free Ca2þ
and Mg2þ. These biochemical events probably represent the trig-
gers for the induction of apoptosis that leads to cancer cell death.
Considering all these findings and that compound 10 is the first
In order to further characterize the biological effects of com-
pound 10, we next examined a few other biochemical parameters.
Reactive oxygen species (ROS) and Ca2þ often have a critical role in
the modulation of cell death because a wide variety of toxic mol-
ecules are known to affect cellular redox balance or Ca2þ
indolylmethyleneindol-2-one bearing
a 4-methoxyphenylethyl

A. Leoni et al. / European Journal of Medicinal Chemistry 79 (2014) 382e390
387
group we have synthesized, it can be considered as a lead which
deserves to generate new analogues.
Anal. Calcd for C21H22ClNO5 (MW 403.86): C, 62.45; H, 5.49; N, 3.47.
Found: C, 62.47; H, 5.45; N, 3.45.
5. Experimental section
5.1.2. General procedure for the synthesis of compounds 4, 5, 8, 10,
11, 14e16, 18e23, 25e28
5.1. Chemistry
The appropriate compound 1 (10 mmol) was dissolved in
methanol (100 mL) and treated with the equivalent of the appro-
priate aldehyde 2 and piperidine (1 mL). The reaction mixture
was refluxed for 3e5 h (progress of the reaction followed by TLC),
and the precipitate that formed on cooling was collected by
filtration.
Compounds 16, 25 and 27 were purified by column chroma-
tography, compounds 16 and 25 with petroleum ether/acetone, and
compound 27 with methylene chloride/acetone as the eluent. All
the crude products were crystallized from ethanol except com-
pound 4 (CHCl3/MeOH) and compound 5 (acetone/petroleum
ether).
All the compounds prepared have a purity of at least 95% as
determined by combustion analysis. The melting points are un-
corrected. TLC was performed on Bakerflex plates (Silica gel IB2-F)
and column chromatography on Kieselgel 60 (Merck): the eluent
was a mixture of petroleum ether/acetone in various proportions.
The IR spectra were recorded in nujol on a Nicolet Avatar 320 E.S.P.;
nmax is expressed in cmꢀ1. The 1H NMR spectra were recorded in
(CD3)2SO on a Varian MR 400 MHz (ATB PFG probe); the chemical
shift (referenced to solvent signal) is expressed in
d (ppm) and J in
Hz (abbreviations: ph ¼ phenyl, bzind ¼ benzoindole, ind ¼ indole,
ox ¼ oxindole). The oxindoles 1c and 1e are commercially available.
The following compounds were prepared according to the litera-
ture: 1a [9], 1b [10], 1d [11], 1f [12], 1g [13], 1h [14], 1i [15], 1j [16],
2a [17], 2c [4], 2d [6], 2e [18], 2f, hei [19], 2m [20].
For compounds 25e28 the yield was much lower, and an
improvement was obtained by replacing piperidine with NH4OH
conc.
4. Yield 65%, mp > 320 ꢂC. I.R.: 1672, 1573, 1263, 1030, 722. 1H
NMR: 2.24 (3H, s, CH3), 3.74 (3H, s, OCH3), 3.77 (3H, s, OCH3), 3.88
(3H, s, OCH3), 6.58 (1H, d, ox, J ¼ 9.0), 6.81 (1H, s, ind), 6.85 (1H, d,
ox, J ¼ 9.0), 7.12 (1H, s, ind), 8.02 (1H, s, CH), 10.48 (1H, s, NH), 12.28
(1H, broad, NH). Anal. Calcd for C21H19ClN2O3 (MW 382.84): C,
65.88; H, 5.00 N, 7.32. Found: C, 65.69; H, 4.87; N, 7.06.
5. Yield 62%, mp 247e250 ꢂC. I.R.: 1693, 1594, 1259, 1109, 721. 1H
NMR: 2.22 (3H, s, CH3), 3.74 (3H, s, OCH3), 3.76 (3H, s, OCH3), 3.87
(3H, s, OCH3), 6.52(2H, s, CH2), 6.58 (1H, d, ox, J ¼ 9.0), 6.85 (1H, s,
ind), 6.87 (1H, d, ox, J ¼ 9.0), 7.14 (2H, m, ph), 7.32 (4H, m, ph þ ind),
8.03 (1H, s, CH), 10.51 (1H, s, NH). Anal. Calcd for C28H25ClN2O4
(MW 488.96): C, 68.78; H, 5.15 N, 5.73. Found: C, 68.58; H, 4.98; N,
5.48.
5.1.1. General procedure for the preparation of the new aldehydes
(2g, 2jel)
The
2-chloro-1H-indole-3-carbaldehyde
or
2-chloro-5-
methoxy-6-methyl-1H-indole-3-carbaldehyde (10 mmol) was dis-
solved in DMF (10 mL) and treated, under stirring, with NaH
(15 mmol). The mixture was stirred at room temperature for 10 min
and it was added with 4-methoxyphenethyl bromide, 2-
morpholinoethyl chloride, 3-bromo-1-phenyl-1-propene or 3,4,5-
trimethoxybenzyl chloride (12 mmol) respectively. The reaction
mixture was maintained at 100 ꢂC for 3e5 h (according to TLC test),
after cooling, was poured into ice water. The expected compounds
were collected by filtration and crystallized from ethanol.
8. Yield 60%, mp 138e140 dec ꢂC. I.R.: 3585, 3170, 1696, 1609,
743. 1H NMR: 5.64 (2H, s, CH2), 6.75 (2H, d, ph, J ¼ 4.2), 6.88 (2H, d,
J ¼ 8), 7.26 (8H, m), 7.66 (1H, s, CH), 7.70 (1H, d, J ¼ 8), 10.63 (1H, s,
NH). Anal. Calcd for C24H17ClN2O (MW 384.86): C, 74.90; H, 4.45 N,
7.28. Found: C, 74.88; H, 4.48; N, 7.30.
5.1.1.1. 2-Chloro-1-[2-(4-methoxyphenyl)ethyl]-1H-indole-3-
carbaldehyde (2g). Yield 77%, mp 85e90 ꢂC. IR: 1650, 1504, 1239,
1033, 744. 1H NMR (DMSO-d6): 2.99 (2H, t, CH2, J ¼ 7.4), 3.69 (3H, s,
OCH3), 4.50 (2H, t, CH2, J ¼ 7.4), 6.79 (2H, d, ph, J ¼ 8.7), 7.03 (2H, d,
ph, J ¼ 8.7), 7.32 (2H, m, ind), 7.65 (1H, m, ind), 8.09 (1H, m, ind),
9.95 (1H, s, CHO). Anal. Calcd for C18H16ClNO2 (MW 313.78): C,
68.90; H, 5.14; N 4.46. Found: C, 69.04; H, 5.02; N, 4.78.
10. Yield 85%, mp 161e168 ꢂC. I.R.: 3125, 1698, 1602, 1245, 741.
1H NMR: 3.03 (2H, t, CH2, J ¼ 7), 3.67 (3H, s, OCH3), 4.54 (2H, t, CH2,
J ¼ 7.0), 6.64 (1H, d, ox, J ¼ 7.4), 6.76 (2H, d, ph, J ¼ 8.4), 6.77 (1H, m,
ox), 6.87 (1H, d, ox, J ¼ 7.4), 7.03 (2H, d, ph, J ¼ 8.4), 7.18 (3H, m, ind),
7.30 (1H, m, ind), 7.58 (1H, s, CH), 7.67 (1H, d, ind, J ¼ 8.2),10.60 (1H,
s, NH). Anal. Calcd for C26H21ClN2O2 (MW 428.91): C, 72.81; H, 4.94;
N, 6.53. Found: C, 72.46; H, 4.53; N, 6.49.
5.1.1.2. 2-Chloro-1-(2-morpholin-4-ylethyl]-1H-indole-3-
carbaldehyde (2j). Yield 45%, mp 88e92 ꢂC. IR: 1605, 1500, 1110,
1045, 755. 1H NMR (DMSO-d6): 3.72 (4H, m, CH2), 3.77 (2H, t, CH2,
J ¼ 5.0), 3.89 (2H, m, CH2), 4.18 (4H, m, CH2), 6.99 (2H, m, ind), 7.04
(1H, m, ind), 7.85 (1H, s, ind), 9.82 (1H, s, CHO). Anal. Calcd for
11. Yield 90%, mp 205e208 ꢂC. I.R.: 3196, 1648, 1509, 1181, 736.
1H NMR: 2.14 (2H, qui, CH2, J ¼ 7.6), 2.65 (2H, t, CH2, J ¼ 7.6), 4.33
(2H, t, CH2, J ¼ 7.6), 6.85 (1H, d, ox, J ¼ 7.2), 6.99 (1H, t, ox, J ¼ 7.2),
7.22 (8H, m), 7.58 (1H, m), 7.89 (1H, d, J ¼ 7.2), 8.13 (1H, s, CH), 8.21
(1H, m), 9.49 (1H, s, ind-2), 10.54 (1H, s, NH). Anal. Calcd for
C
15H17ClN2O2 (MW 292.76): C, 61.54; H, 5.85; N, 9.57. Found: C,
61.23; H, 5.73; N, 9.77.
C26H22N2O (MW 378.47): C, 82.51; H, 5.86; N, 7.40. Found: C, 82.37;
5.1.1.3. 2-Chloro-1-[(2E)-3-phenylprop-2-en-1-yl]-1H-indole-3-
carbaldehyde (2k). Yield 40%, mp 89e93 ꢂC. IR: 1654, 1508, 1038,
970, 743. 1H NMR (DMSO-d6): 5.14 (2H, d, CH2, J ¼ 5.4), 6.43 (1H, dt,
CH, J ¼ 16, J ¼ 5.4), 6.54 (1H, d, CH, J ¼ 16), 7.24 (1H, t, ph, J ¼ 6.8),
7.32 (4H, m, ph þ ind), 7.40 (1H, t, ind, J ¼ 7.6), 7.41 (1H, d, ind,
J ¼ 7.6), 7.73 (1H, d, ind, J ¼ 7.6), 8.13 (1H, d, ph, J ¼ 6.8), 10.06 (1H, s,
CHO). Anal. Calcd for C18H14ClNO (MW 295.76): C, 73.10; H, 4.77; N,
4.74. Found: C, 72.98; H, 4.93; N, 4.65.
H, 5.78; N, 7.27.
14. Yield 95%, mp 161e165 ꢂC. I.R.: 3178,1696,1608,1193, 743. 1H
NMR: 2.08 (2H, qui, CH2, J ¼ 7.4), 2.68 (2H, t, CH2, J ¼ 7.4), 3.35 (3H,
s, OCH3), 4.40 (2H, t, CH2, J ¼ 7.5), 6.36 (1H, s, ox-4), 6.77 (2H, m, ox-
6,7), 7.26 (8H, m, ind þ ph), 7.63 (1H, s, CH), 7.66 (1H, m, ind), 10.43
(1H, s, NH). Anal. Calcd for C27H23ClN2O2 (MW 442.94): C, 73.21; H,
5.23; N, 6.32. Found: C, 73.18; H, 5.36; N, 6.29.
15. Yield 68%, mp 188 dec ꢂC. I.R.: 3240, 1686, 1608, 1189, 738. 1H
NMR: 2.08 (2H, qui, CH2, J ¼ 7.6), 2.70 (2H, t, CH2, J ¼ 7.6), 4.38 (2H, t,
CH2, J ¼ 7.6), 6.27 (1H, d, ox-4, J ¼ 2.0), 6.59 (1H, dd, ox-6, J ¼ 8.2,
J ¼ 2), 6.67 (1H, d, ox-7, J ¼ 8.2), 7.25 (8H, m, ind þ ph), 7.56 (1H, s,
CH), 7.64 (1H, d, ind, J ¼ 8.4), 8.78 (1H, s, OH), 10.28 (1H, s, NH).
Anal. Calcd for C26H21ClN2O2 (MW 428.91): C, 72.81; H, 4.94; N,
6.53. Found: C, 72.52; H, 4.87; N, 6.48.
5.1.1.4. 2-Chloro-5-methoxy-6-methyl-1-(3,4,5-trimethoxybenzyl)-
1H-indole-3-carbaldehyde (2l). Yield 40%, mp: 157e158 ꢂC. IR: 1651,
1588, 1129, 1045. 1H NMR (DMSO-d6): 2.25 (3H, s, CH3), 3.61 (3H, s,
OCH3), 3.68 (6H, s, 2 ꢃ OCH3), 3.83 (3H, s, OCH3), 5.45 (2H, s, CH2),
6.54 (2H, s, ph), 7.54 (1H, s, ind), 7.58 (1H, s, ind), 10.01 (1H, s, CHO).

388
A. Leoni et al. / European Journal of Medicinal Chemistry 79 (2014) 382e390
16. Yield 35%, mp 210e214 ꢂC. I.R.: 3119, 1685, 1565, 1206, 737.
6.51 (1H, d, ox, J ¼ 8.8), 6.85 (1H, d, ox, J ¼ 8.8), 7.13 (3H, m, ind),
7.38 (1H, m, ind), 7.58 (5H, m, ph), 8.23 (1H, s, CH),10.42 (1H, s, NH).
Anal. Calcd for C26H22N2O4 (MW 426.47): C, 73.23; H, 5.20; N, 6.57.
Found: C, 73.12; H, 5.11; N, 6.49.
1H NMR: 3.43 (2H, t, CH2, J ¼ 5.4), 3.60 (4H, m, CH2), 3.69 (2H, m,
CH2), 4.21 (4H, m, CH2), 6.64 (1H, d, J ¼ 7.5), 6.75 (1H, d, J ¼ 7.5), 6.82
(1H, td, J ¼ 7.5, J ¼ 1.2), 6.87 (1H, d, J ¼ 7.5), 6.96 (1H, td, J ¼ 7.5,
J ¼ 1.2), 7.02 (1H, td, J ¼ 7.5, J ¼ 1.2), 7.07 (1H, td, J ¼ 7.5, J ¼ 1.2), 7.16
(1H, d, J ¼ 7.5), 7.85 (1H, s, CH), 10.36 (1H, s, NH). Anal. Calcd for
5.1.3. General procedure for the synthesis of compounds 3, 6, 7, 9,
12, 13 and 17
C
23H22ClN2O2 (MW 393.89): C, 70.13; H, 5.63; N, 7.11. Found: C,
69.97; H, 5.49; N, 6.99.
The appropriate oxindole 1 (5 mmol) was dissolved in acetic
acid (25 mL) and treated with an equivalent of appropriate indole-
3-carbaldehyde 2 and 37% hydrochloric acid (1 mL). The reaction
mixture was refluxed for 20 h, the solid separated on cooling was
collected by filtration. The crude products were purified by crys-
tallization with ethanol to give the desired products, with the
exception of compound 6 purified by column chromatography,
with petroleum ether/acetone as the eluent.
18. Yield 38%, mp 138e140 ꢂC. I.R.: 3250, 1701, 1614, 1235, 1132.
1H NMR: 2.27 (3H, s, CH3), 3.42 (3H, s, OCH3), 3.59 (3H, s, OCH3),
3.61 (3H, s, OCH3), 3.65 (6H, s, 2 ꢃ OCH3), 5.49 (2H, s, CH2), 6.39 (1H,
s, ox), 6.48 (2H, s, ph), 6.63 (1H, s, ox/ind), 6.80 (2H, s, ox/ind), 7.57
(1H, s, ind), 7.68 (1H, s, CH), 10.44 (1H, s, NH). Anal. Calcd for
C
30H29ClN2O6 (MW 549.02): C, 65.63; H, 5.32; N, 5.10. Found: C,
65.58; H, 5.30; N, 5.07.
19. Yield 70%, mp 190e193 dec ꢂC. I.R.: 3298, 1675, 1598, 1240,
1122. 1H NMR: 2.28 (3H, s, CH3), 3.62 (6H, s, 2 ꢃ OCH3), 3.67 (6H, s,
2 ꢃ OCH3), 5.49 (2H, s, CH2), 6.36 (1H, s, ox), 6.50 (2H, s, ph),
6.64(3H, m, ox þ ind), 7.55 (1H, s, ind), 7.62 (1H, s, CH), 8.84 (1H, s,
OH), 10.28 (1H, s, NH). Anal. Calcd for C29H27ClN2O6 (MW 534.99):
C, 65.11; H, 5.09; N, 5.24. Found: C, 65.08; H, 6.20; N, 5.32.
20. Yield 32%, mp 152e154 ꢂC. I.R.: 3582, 1698, 1609, 1092, 712.
1H NMR: 2.12 (3H, s, CH3), 2.26 (3H, s, CH3), 3.17 (3H, s, OCH3), 3.58 (
3H, s, OCH3), 3.61 (3H, s, OCH3), 3.63 (6H, s, 2 ꢃ OCH3), 5.48 (2H, s,
CH2), 6.36 (1H, s, ox), 6.47 (2H, s, ph), 6.66 (1H, s, ox/ind), 6.68 (1H,
s, ox/ind), 7.56 (1H, s, ind), 7.60 (1H, s, CH), 10.36 (1H, s, NH). Anal.
Calcd for C31H31ClN2O6 (MW 563.04): C, 66.13; H, 5.55; N, 4.98.
Found: C, 66.10; H, 5.46; N, 5.20.
3. Yield 70%, mp 200 dec ꢂC. IR: 1596, 1572, 1299, 1260, 722. 1H
NMR: 3.79 (3H, s, OCH3), 3.88 (3H, s, OCH3), 6.59 (1H, d, ox, J ¼ 9.0),
6.87 (1H, d, ox, J ¼ 9.0), 7.13 (2H, m, ind), 7.35 (2H, m, ind), 8.00 (1H,
s, CH), 10.45 (1H, s, NH), 12.47 (1H, broad, NH). Anal. Calcd for
C19H15ClN2O3 (MW 354.79): C, 64.32; H, 4.26; N, 7.90. Found: C,
64.86; H, 4.11; N, 7.01.
6. Yield 25%, mp > 310 ꢂC. I.R.: 3416, 1588, 1106, 722. 1H NMR:
3.90 (3H, s, OCH3), 3.97 (3H, s, OCH3), 6.51 (1H, d, ox, J ¼ 8.8), 6.65
(1H, d, ox, J ¼ 8.8), 7.32 (1H, t, bzind, J ¼ 7.8), 7.44 (1H, t, bzind,
J ¼ 7.8), 7.46 (1H, d, bzind, J ¼ 8.0), 7.86 (1H, d, bzind, J ¼ 7.8), 8.03
(1H, d, bzind, J ¼ 8.0), 8.48 (1H, d, bzind, J ¼ 7.8), 8.83 (1H, s, CH),
12.60 (2H, s, NH). Anal. Calcd for C23H17ClN2O3 (MW 404.85): C,
68.24; H, 4.23; N, 6.92. Found: C, 67.98; H, 4.01; N, 6.23.
21. Yield 88%, mp 200 dec ꢂC. I.R.: 3240, 1706, 1603, 1240, 1132.
1H NMR: 2.25 (3H, s, CH3), 3.61 (3H, s, OCH3), 3.63 (3H, s, OCH3),
3.66 (6H, s, 2 ꢃ OCH3), 5.51 (2H, s, CH2), 6.48 (2H, s, ph), 6.60 (1H, s,
ind), 6.74 (1H, d, ox-4, J ¼ 2.0), 6.90 (1H, d, ox-7, J ¼ 8.4), 7.24 (1H,
dd, ox-6, J ¼ 8.4, J ¼ 2.0), 7.60 (1H, s, ind), 7.77 (1H, s, CH), 10.76 (1H,
s, NH). Anal. Calcd for C29H26Cl2N2O5 (MW 553.44): C, 62.94; H,
4.74; N, 5.06. Found: C, 62.87; H, 4.69; N, 5.21.
7. Yield 25%, mp 272e280 dec ꢂC. I.R.: 1603, 1567, 1286, 1061,
764. 1H NMR: 3.44 (2H, broad, NH), 3.83 (3H, s, OCH3), 3.93 (3H, s,
OCH3), 4.18 (3H, s, OCH3), 7.06 (1H, s, ox), 7.46 (1H, t, ind, J ¼ 7.5),
7.53 (1H, t, ind, J ¼ 7.5), 7.69 (1H, d, ind, J ¼ 7.5), 8.38 (1H, d, ind,
J ¼ 7.5), 9.52 (1H, s, CH). Anal. Calcd for C20H17ClN2O4 (MW 384.81):
C, 62.42; H, 4.45; N, 7.28. Found: C, 62.03; H, 4.22; N, 6.99.
9. Yield 72%, mp 190e194 ꢂC. I.R.: 3237, 1682, 1614, 1189, 733. 1H
NMR: 3.08 (2H, t, CH2, J ¼ 7.2), 4.57 (2H, t, CH2, J ¼ 7.2), 6.26 (1H, d,
ox-4, J ¼ 2.2), 6.60 (1H, dd, ox-6, J ¼ 8.3, J ¼ 2.2), 6.68 (1H, d, ox-7,
J ¼ 8.3), 7.20 (8H, m, ph þ ind), 7.51 (1H, s, CH), 7.67 (1H, d, ind,
J ¼ 8), 8.81 (1H, s, OH), 10.29 (1H, s, NH). Anal. Calcd for
22. Yield 40%, mp 250e255 ꢂC. I.R.: 3211, 1696, 1660, 1045, 902.
1H NMR: 2.23 (3H, s, CH3), 3.60 (3H, s, OCH3), 3.67 (6H, s, 2 ꢃ OCH3),
3.77 (3H, s, OCH3), 5.45 (2H, s, CH2), 6.57 (2H, s, ph), 6.83 (1H, dd,
ox-5/7, J ¼ 7.9, J ¼ 0.9), 6.91 (1H, s, ind), 7.03 (1H, dd, ox-5/7, J ¼ 7.9,
J ¼ 0.9), 7.21 (1H, t, ox-6, J ¼ 7.9), 7.47 (1H, s, ind), 8.43 (1H, s, CH),
10.79 (1H, s, NH). Anal. Calcd for C29H26Cl2N2O5 (MW 553.44): C,
62.94; H, 4.74; N, 5.06. Found: C, 62.82; H, 4.78; N, 5.23.
C25H19ClN2O2 (MW 414.89): C, 72.37; H, 4.62; N, 6.75. Found: C,
72.78; H, 4.56; N, 6.43.
12. Yield 68%, mp 90e94 ꢂC. I.R.: 3175, 1697, 1608, 1214, 745. 1H
NMR: 2.08 (2H, qui, CH2, J ¼ 7.4), 2.69 (2H, t, CH2, J ¼ 7.4), 4.39 (2H, t,
CH2, J ¼ 7.4), 6.76 (2H, m, ox), 6.88 (1H, d, ox, J ¼ 7.5), 7.25 (9H, m,
ph þ ind þ ox), 7.63 (1H, s, CH), 7.65 (1H, d, ind, J ¼ 8.4), 10.60 (1H, s,
NH). Anal. Calcd for C26H21ClN2O (MW 412.91): C, 75.63; H, 5.13; N,
6.78. Found: C, 75.34; H, 4.97; N, 6.44.
23. Yield 61%, mp 190e192 dec ꢂC. I.R.: 3170, 1675, 1603, 912,
733. 1H NMR: 5.03 (2H, s, CH2), 6.85 (2H, d, ph, J ¼ 4.0), 6.97 (1H, d,
J ¼ 7.8), 7.25 (9H, m), 7.48 (1H, d, J ¼ 7.8), 7.81 (1H, s, CH), 12.54 (1H,
broad, NH). Anal. Calcd for C24H17ClN2O (MW 384.86): C, 74.90; H,
4.45; N, 7.28. Found: C, 74.49; H, 4.23; N, 6.99.
25. Yield 20%, mp 145e150 ꢂC. I.R.: 3135, 1716, 1541, 1224, 753.
1H NMR: 3.82 (3H, s, OCH3), 6.90 (3H, m, ox), 7.23 (4H, m, ind), 7.62
(5H, m, ph), 7.97 (1H, s, CH), 10.72 (1H, s, NH). Anal. Calcd for
13. Yield 73%, mp 203e205 ꢂC. I.R.: 3391,1711,1604,1158, 743. 1H
NMR: 1.98 (2H, qui, CH2, J ¼ 7.4), 2.67 (2H, t, CH2, J ¼ 7.4), 4.00 (2H, t,
CH2, J ¼ 7.4), 7.20 (10H, m), 8.07 (3H, m) 11.98 (1H, s, NH). Anal.
Calcd for C26H20Cl2N2O (MW 447.36): C, 69.81; H, 4.51; N, 6.26.
Found: C, 69.47; H, 4.29; N, 6.01.
C
24H17ClN2O2 (MW 400.86): C, 71.91; H, 4.27; N, 6.99. Found: C,
72.03; H, 4.35; N, 7.05.
26. Yield 20%, mp 233e237 ꢂC. I.R.: 3150, 1685, 1537, 1091, 697.
1H NMR: 3.52 (3H, s, OCH3), 3.81 (3H, s, OCH3), 6.61 (1H, s, ox), 6.78
(2H, s, ox), 7.23 (4H, m, ind), 7.60 (5H, m, ph), 7.89 (1H, s, CH), 10.39
(1H, s, NH). Anal. Calcd for C25H20N2O3 (MW 396.44): C, 75.74; H,
5.08; N, 7.07. Found: C, 75.68; H, 6.97; N, 7.18.
17. Yield 25%, mp 170e175 ꢂC. I.R.: 1696, 1598, 1296, 1209, 733.
1H NMR: 3.35 (3H, s, OCH3), 5.18 (2H, s, CH2), 6.39 (1H, s, ox-4), 6.47
(2H, s, ox-6,7), 6.77 (2H, s, ind), 7.33 (8H, m, ph þ ind þ CH), 7.66
(1H, s, CH), 7.75 (1H, d, CH, J ¼ 8.2), 10.45 (1H, s, NH). Anal. Calcd for
C
27H21ClN2O2 (MW 440.92): C, 73.55; H, 4.80; N, 6.35. Found: C,
27. Yield 20%, mp 145e150 ꢂC. I.R.: 1706, 1619, 1235, 1091, 753.
1H NMR: 3.27 (3H, s, CH3), 3.82 (3H, s, OCH3), 6.96 (1H, d, ox-4,
J ¼ 2.2), 7.09 (1H, d, ox-7, J ¼ 8.4), 7.22 (4H, m, ind), 7.33 (1H, dd,
ox-6, J ¼ 8.4, J ¼ 2.2), 7.63 (5H, m, ph), 8.05 (1H, s, CH). Anal. Calcd
for C25H19ClN2O2 (MW 414.89): C, 72.37; H, 4.62; N, 6.75. Found: C,
72.56; H, 4.68; N, 6.58.
73.38; H, 4.34; N, 6.70.
5.1.4. Synthesis of compound 24
The oxindole 1c (7 mmol) was dissolved in toluene (30 mL) and
treated with an equivalent of indole-3-carbaldehyde 2m and p-
toluenesulfonic acid (1 mmol). The reaction mixture was refluxed
for 5 h, the solid separated on cooling was collected by filtration
with a yield of 40%.
28. Yield 20%, mp 164e167 ꢂC. I.R.: 3181, 1690, 1255, 1091, 773.
1H NMR: 3.35 (3H, s, OCH3), 3.72 (3H, s, OCH3), 3.74 (3H, s, OCH3),

A. Leoni et al. / European Journal of Medicinal Chemistry 79 (2014) 382e390
389
Mp 242e248 ꢂC (ethanol). I.R.: 1598, 1562, 1148, 1086, 1004. 1H
NMR: 2.28 (3H, s, OCH3), 7.10 (2H, d, J ¼ 8.0), 7.47 (2H, d, J ¼ 8.0),
7.67 (8H, m), 8.27 (1H, d, J ¼ 7.8), 8.37 (1H, m), 9.97 (1H, s, NH). Anal.
Calcd for C24H18N2O2 (MW 366.41): C, 78.67; H, 4.95; N, 7.65.
Found: C, 78.56; H, 4.76; N, 7.32.
fluorescence ratio, Rmax. Intracellular-free Mg was measured in
triplicate and calculated by using a Kd ¼ 2.1 mM as follows:
[Mg2þ] ¼ Kd (R ꢀ Rmin) Sf/(Rmax ꢀ R) Sb
where Rmin is the fluorescence ratio at 335/370 nm for uncom-
plexed Mag Fura2 (zero magnesium). Rmax is the ratio of fluores-
cence at 335/370 nm for dye saturated with Mg2þ. Sf and Sb are the
fluorescence intensities at 370 nm for Mag-Fura2 with zero Mg2þ
and excess Mg2þ, respectively. R is the ratio of fluorescence at 335/
370 nm of the sample to be measured.
5.2. Biology
5.2.1. Cell-based screening assay
The NCI screening process occurs in two stages [27], beginning
with the evaluation of all compounds against the 60 cell lines at
10ꢀ5 M. Compounds exhibiting significant growth inhibition are
subsequently evaluated against the 60 cell lines at five concentra-
tion levels according to standard procedures (http://dtp.nci.nih.
gov/branches/btb/ivclsp.html). In both cases, the exposure time is
48 h.
Measurement of intracellular free Ca2þ was performed with the
same protocol, but the cells were stained with INDO-1/AM probe at
5 mM concentration. The stained cells were analyzed exciting at
346 nm, and reading the emission at 485 nm and at 405 nm for the
unbound and the bound dye respectively. Also in this analysis, the
fluorescences were collected after adding to the samples EDTA,
EGTA and digitonin (Fmin) and CaCl2 (Fmax). The intracellular free
Ca2þ concentration was evaluated by using a Kd ¼ 0.23 mM and the
following equation:
5.2.2. Cell culture, treatment, and biochemical determinations
The human leukemia cell line HL-60 was maintained in RPMI
1640 medium supplemented with 10% fetal calf serum and 2 mM
glutamine at 37 ꢂC in air/5% CO2. Solution of compound under test
was prepared in DMSO at 10 mmol/L and diluted immediately
before use in order to obtain the indicated concentration. In control
cells, the corresponding amount of DMSO was added to the culture
medium. At the end of the treatment, cells were collected and
counted. Cell viability and cell survival were determined as
described [28].
[Ca2þ] ¼ Kd Q (R ꢀ Rmin)/(Rmax ꢀ Rmin
)
where Q is Fmin/Fmax at 485 nm, R is the fluorescence ratio at 405/485
of the samples, Rmin is the fluorescence intensity ratio 405/485 after
adding EDTA and EGTA (5 mM) and Digitonin (5 mM) and Rmax is the
fluorescence intensity ratio 405/485 after adding CaCl2 (10 mM).
To determine cell cycle distribution [29], at the end of incuba-
tion, the cells were washed with phosphate buffered saline (PBS)
and collected. The pellet was resuspended in 0.01% Nonidet P-40,
Author contributions
The authors contributed equally to this work.
10 mg/mL RNase, 0.1% sodium citrate, and 50 mg/mL propidium io-
dide for 30 min at room temperature in the dark. Propidium iodide
fluorescence was analyzed by using a Bryte flow cytometer (Biorad)
and cell cycle analysis was performed using the Multicycle Cycle
Phoenix Flow system, and Modfit 5.0 software.
Surface exposure of phosphatidylserine was measured with an
Annexin V-FLUOS staining kit (Roche Diagnostics Deutschland
GmbH, Mannheim, Germany), as previously described [29].
In order to detect intracellular peroxides, the cells were incu-
Acknowledgements
This work has been supported by a grant from the University of
Bologna, Italy (RFO 2011), and Tavola Valdese Ufficio 8PM. We are
grateful to the National Cancer Institute (Bethesda, MD) for the
anticancer tests and to the Centro Interdipartimentale Ricerche
Biotecnologiche (CIRB) of the University of Bologna for the flow
cytometric facility.
bated with
5 mM DCF-DA (Molecular Probes, Leiden, The
Netherlands) for 30 min at 37 ꢂC. Cells were analyzed acquiring the
red fluorescence with a logarithmic amplification [30].
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