Cytotoxic activities of substituted 3-(3,4,5-trimethoxybenzylidene)-1,3- dihydroindol-2-ones and studies on their mechanisms of action

Article abstract of DOI:10.1016/j.ejmech.2013.03.033

The synthesis of new trimethoxybenzylidene-indolinones is reported. Their cytotoxic activity was evaluated according to Developmental Therapeutics Program, National Cancer Institute, Bethesda, MD, drug screen protocols. The study of the mechanism of actio

Full text of DOI:10.1016/j.ejmech.2013.03.033

European Journal of Medicinal Chemistry 64 (2013) 603e612
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Cytotoxic activities of substituted 3-(3,4,5-trimethoxybenzylidene)-
1,3-dihydroindol-2-ones and studies on their mechanisms of action
Aldo Andreani a, Massimiliano Granaiola a, Alessandra Locatelli a, Rita Morigi a,
,
a
a
a
Mirella Rambaldi a , Lucilla Varoli , Francesco Vieceli Dalla Sega , Cecilia Prata ,
*
Tam L. Nguyen b, Ruoli Bai c, Ernest Hamel c
a Dipartimento di Farmacia e Biotecnologie FaBiT, Università di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
b Target Structure-Based Drug Discovery Group, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA
c Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick National Laboratory for
Cancer Research, Frederick, MD 21702, USA
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 trimethoxybenzylideneeindolinones is reported. Their cytotoxic activity was
evaluated according to Developmental Therapeutics Program, National Cancer Institute, Bethesda, MD,
drug screen protocols. The study of the mechanism of action suggests that inhibition of Nox4 in B1647
cells (acute myeloid leukemia) could contribute to the antiproliferative effect of some compounds.
Moreover, inhibition of tubulin assembly was observed for the most cytotoxic compound, and the
structural basis for this activity was delineated by binding models.
Received 21 January 2013
Received in revised form
14 March 2013
Accepted 20 March 2013
Available online 3 April 2013
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Oxindole
Knoevenagel reaction
Antiproliferative activity
NADPH oxidase 4
Tubulin assembly
1. Introduction
Scheme 1) in which the variations concerned primarily the indo-
linone moiety.
The 3,4,5-trimethoxyphenyl group is present, free or hindered,
in well known antitumor agents, such as combretastatin A-4
(CSA4), colchicine and podophyllotoxin. With this in mind, we
started to study the antiproliferative activity of Knoevenagel ad-
Compounds 7, 10 and 11 were prepared to evaluate the effect of
the addition of a methyl group at the 6 position (7) or at the ni-
trogen (10, 11), in order to compare their activity with those of NSC
736802 and NSC 134544. Moreover, considering that, as observed
for compound NSC 736804, the presence a bulky substituent at the
oxindole nitrogen is tolerated, substituted benzyl groups were
introduced at the nitrogen (compounds 13e15). This type of sub-
stitution gave good cytotoxicity results when performed in other
indolinone derivatives that we had studied previously [2,3].
Meanwhile, studies examining introduction of new substituents,
such as bromine, dimethylamino, or trifluoromethyl, and explora-
tion of different positions for the oxindole ring were also under-
ducts in which the oxindole moiety is linked to
a 3,4,5-
trimethoxyphenyl group by means of a methine bridge, and in
2006 we published a paper describing an initial group of com-
pounds [1]. Three derivatives (Chart 1) showed interesting activity
profiles, encouraging us to develop new analogs (3e15, 18, 19, see
Abbreviations: DTP, Developmental Therapeutics Program; NCI, National Cancer
Institute; CSA4, combretastatin A-4; Nox, NAD(P)H oxidase enzyme; ROS, reactive
oxygen species; ECs, endothelial cells; NOE, nuclear Overhauser effect; DMSO,
dimethylsulfoxide; GI, growth inhibition; TGI, total growth inhibition; LC, lethal
concentration; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide; DPI, diphenyleneiodonium; DCFH-DA, 20,70-dichlorofluorescin-diacetate;
DCF, 20,70-dichlorofluorescein; AML, human acute myeloid leukemia.
taken (compounds 3e6,
8 and 9). Finally, we took into
consideration the replacement of the methine bridge linking the
oxindole and the 3,4,5-trimethoxybenzene with an imino group
(compounds 18, 19).
The cytotoxic activities of all new compounds were evaluated
according to Developmental Therapeutics Program (DTP), National
Cancer Institute (NCI), Bethesda, MD, drug screen protocols. We
* Corresponding author. Tel.: þ39 51 2099700; fax: þ39 51 2099734.
E-mail address: mirella.rambaldi@unibo.it (M. Rambaldi).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.033

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A. Andreani et al. / European Journal of Medicinal Chemistry 64 (2013) 603e612
OCH3
OH
O
O
O
O
O
O
CH3COHN
O
O
O
O
N
H
O
HO
O
N
O
O
Chart 2. Potent Nox4 inhibitor described as 9d in Ref. [5].
N
N
H
H
NAD(P)H oxidase enzymes (Nox) constitute a family of struc-
tural homologs of phagocytic Nox (Nox2 or gp91phox) and are one
of the major sources of reactive oxygen species (ROS) in many cell
types [6,7]. It is well known that ROS can act as messengers in
cellular signaling transduction pathways and that an increase of
ROS supports cellular growth and proliferation, contributing to
cancer development [8]. In addition, accumulating evidence sug-
gests that specific Nox isoforms, in particular Nox4, are associated
with ROS production and proliferation of cancer cells. The ROS
generated by this enzyme have been implicated in numerous bio-
logical functions, including signal transduction, cell differentiation
and tumor cell growth. ROS produced by Nox4 in pancreatic cancer
has been shown to transmit cell survival signals via the AKT-ASK1
pathway, while inhibition of Nox4 activates apoptosis [9]. It has
been demonstrated that Nox4 overexpression plays an oncogenic
role in breast tumorigenesis and that treatment of Nox4 over-
expressing cells with catalase resulted in decreased tumorigenicity
[10]. In glioblastoma multiforme, cycling hypoxia triggers ROS
production via Nox4, and this is associated with increased tumor
cell growth in vitro and in mouse xenografts. Inhibition of Nox4
with siRNA or antioxidant in vitro and Nox4 knockdown in mice
were able to block cell growth or tumor progression [11]. Moreover,
it has been shown that Nox4 derived ROS are required for
NSC 736802
NSC 134544
NSC 736804
Chart 1. Most active derivatives described in Ref. [1].
also studied two possible mechanisms of action: inhibition of
tubulin assembly and inhibition of NADPH oxidase 4 (Nox4).
It is well known that anticancer agents such as colchicine and
combretastatin A-4 interfere with the dynamic equilibrium of mi-
crotubules by inhibition of tubulin polymerization. Moreover 3,4,5-
trimethoxyphenylthioindoles, along with the corresponding ke-
tone and methylene analogs, have been described as tubulin as-
sembly inhibitors [4]. The structural analogy prompted us to test
compounds 3e19 and derivatives reported in Chart 1 as inhibitors
of tubulin polymerization.
Recently, the Nox4 inhibitory activity shown by some oxindole
derivatives was described [5]. The chemical structure of the most
active compound 9d is shown in Chart 2. As in our 3-(3,4,5-
trimethoxybenzylidene)-1,3-dihydroindol-2-ones, the indolinone
moiety is linked to a substituted phenyl ring by means of a methine
bridge. We therefore decided to determine whether Nox4 was a
possible target for the compounds described here.
O
O
O
R1
CHO
O
O
R2
R3
i or ii
or iii
R1
+
O
R1
4
3
R2
R3
5
O
N
R
2
O
O
R2
R3
O
6
O
O
R4
N
1
7
N
R
R4
2
R
1
R4
3-6, 8, 9, 12-14
7, 10, 11, 15
O
O
O
NH2
O
O
R1
O
iv
N
+
O
N
+
4
R1
O
3
R1
N
R
5
O
O
2
O
N
R
O
O
6
1
N
7
R
16
17
85%
15%
18-19
aReagents and conditions: (i) methanol, piperidine; (ii) acetic acid, 37% HCl; (iii) anhydrous
sodium acetate, acetic acid; (iv) ethanol.
Scheme 1. Synthesis of 3-(3,4,5-trimethoxybenzylidene)- and 3-[(3,4,5-trimethoxyphenyl)imino]-1,3-dihydroindol-2-ones derivatives.

A. Andreani et al. / European Journal of Medicinal Chemistry 64 (2013) 603e612
605
proliferation of acute leukemia cells, and inhibition of Nox or siRNA
against Nox4 resulted in a decrease in intracellular ROS and
decreased cell viability [12]. Nox4 is also the main isoform in
endothelial cells (ECs) [13], and it plays an important role in
angiogenesis [14]. Since angiogenesis in cancer is essential for tu-
mor growth and metastasis, the inhibition of specific Nox isoforms
has been proposed as a therapeutic strategy for treatment of cancer
and other angiogenesis-dependent pathologies [15].
proton (or group) at position 4 of the indole. As an example, NOE
connectivities found in compound 5 are reported in Chart 3.
On the other hand, NOE experiments on compounds 7 and 11
showed that they were E isomers.
For compound 7, first it was necessary to perform some NOE
experiments in order to assign the aromatic singlets. Irradiation of
the methyl at position 6 of the indole (2.10 ppm) gave NOE at
6.58 ppm. This singlet was thus assigned to the indole proton at the
7 position (ind-7). Afterward, irradiation of the singlet at 7.42 ppm
gave NOE only at phenyl protons (7.00 ppm) and not at OH. As a
consequence, the singlet at 7.42 ppm was assigned to the methine
bridge and the one at 7.32 ppm to ind-4. This last NOE experiment
was also in agreement with the E configuration because it excluded
the spatial proximity of the methine bridge and ind-4. The E
configuration was also confirmed through irradiation of the phenyl
protons at 7.00 ppm. In fact, NOE was observed not only at the
methine bridge, but also at ind-4, thus revealing the spatial close-
ness of the phenyl protons and ind-4.
For compound 11, two NOE experiments were performed (see
Chart 3), both in agreement with the E configuration: i) irradiation
of ind-4 (7.30 ppm) gave NOE at 3.66 ppm (indole OCH3), at
3.81 ppm (phenyl OCH3) and at 7.07 ppm (phenyl protons); ii)
irradiation of the methine bridge (7.66 ppm) gave NOE only at the
phenyl protons (7.07 ppm).
Compd
R
R1
R2
R3
R4
3
4
5
H
H
H
OCH3
Cl
H
H
H
Br
H
H
H
OCH3
Cl
H
6
H
H
N(CH3)2
H
H
7
8
9
H
H
H
CH3
CH3
CH3
H
H
H
H
H
H
H
H
OH
H
H
OH
OCH3
Cl
H
H
OCH3
e
CH3
Cl
OCF3
H
H
H
H
H
H
e
H
H
H
H
H
H
H
H
10
11
12
13
14
15
18
19
CH2e(C6H4)epCl
CH2e(C6H4)epOCH3
CH2e(C6H4)epOCH3
H
H
OCH3
H
H
e
CH2eC6H5
e
e
e
The geometrical configuration of compounds 4, 8e10, 12, 15 was
assigned by comparing the signals of the 1H NMR spectra, since we
noticed that in E isomers protons at positions 20 and 60 of the tri-
methoxyphenyl ring gave a singlet at about 7 ppm, whereas in Z
isomers the singlet was around 8 ppm.
As far as the imino derivatives 18 and 19 are concerned, they
were obtained as mixtures of E (85%) and Z (15%) isomers by
refluxing 3,4,5-trimethoxyaniline and the appropriate isatin in
ethanol. The configuration of the prevalent isomer was determined
by means of NOE experiments. For both the compounds, irradiation
of the protons at positions 20 and 60 of the phenyl ring gave NOE not
only at the OCH3, but also at ind-4, thus confirming the E config-
uration (NOE connectivities observed in compound 18 are reported
in Chart 3).
2. Chemistry
Compounds 3e15 (Scheme 1, Table 1) were synthesized
by means of the Knoevenagel reaction between 3,4,5-
trimethoxybenzaldehyde and the appropriate oxindole. The reac-
tion was performed in methanol in the presence of piperidine
(Method A). The only exceptions were compounds 9 and 15: the
former was prepared in AcOH/HCl (Method B) and the latter in
AcOH/AcONa (Method C).
The imino-derivatives 18 and 19 were obtained by reacting the
properly substituted isatin with 3,4,5-trimethoxyaniline.
The 1H NMR spectra are in agreement with the assigned
structures.
Geometrical configuration was determined by performing NOE
experiments on some of the synthesized derivatives in order to
evaluate whether the methine bridge and the proton at the 4 po-
sition of the indole (ind-4) are close in space (Z configuration) or
not (E configuration).
Compounds 3, 5, 6, 13 and 14 were found to be Z isomers. In fact,
after irradiation of the methine bridge, NOE was observed not only
on protons at positions 20 and 60 of the phenyl group, but also on the
3. Biology
3.1. Cell-based assays
Compounds 3e19 were initially tested at a single high concen-
tration (10ꢀ5 M) in the full NCI 60 cell panel (NCI 60 Cell One-Dose
Screen). This panel is organized into subpanels representing leu-
kemia, melanoma and cancers of lung, colon, kidney, ovary, breast,
prostate and central nervous system. Only compounds which
satisfy predetermined threshold inhibition criteria in a minimum
number of cell lines will progress to the full 5-concentration
assay. The threshold inhibition criteria for progression to the 5-
concentration screen were selected to efficiently capture com-
pounds with antiproliferative activity based on the analysis of
historical DTP screening data. The result is expressed as the percent
growth of treated cells relative to the control following a 48 h in-
cubation. Twelve of fifteen evaluated compounds were active in the
preliminary test and progressed to the five-concentration assay.
The compounds were dissolved in dimethylsulfoxide (DMSO) and
evaluated using five concentrations at ten-fold dilutions (the
highest being 10ꢀ4 M) following a 48 h incubation. Table 2 reports
the results obtained (vincristine is reported for comparison pur-
poses), at three assay endpoints: 50% growth inhibition (GI50), total
cytostatic effect (TGI ¼ Total Growth Inhibition) and cytotoxic effect
(LC50, loss of 50% of the initial cell protein). For some compounds,
the 5-concentration test was repeated, and no significant
Table 1
Compounds 3e15, 18, 19.
Comp.
Formula
MW
Method
Mp ꢁC
3
C
C
20H21NO6
18H15Cl2NO4
371.39
380.22
390.23
354.40
341.36
345.78
395.33
341.36
355.39
359.81
435.90
431.48
461.51
342.35
402.44
A
A
A
A
A
A
B
A
A
A
A
A
C
138e140
210e212
255e256
188e190
160e163
260e262
195e197
175e180
120e124
102e105
135e137
100e105
115e120
180e182
125e130
4
5a
6
C18H16BrNO4
C20H22N2O4
C19H19NO5
C18H16ClNO4
C19H16F3NO5
C19H19NO5
C20H21NO5
7
8
9
10
11
12
13
14
15
18
19
C
19H18ClNO4
C25H22ClNO4
C26H25NO5
C27H27NO6
C18H18N2O5
C24H22N2O4
e
e
a
Compound described in Ref. [16] but not fully characterized.

606
A. Andreani et al. / European Journal of Medicinal Chemistry 64 (2013) 603e612
O
O
O
O
O
O
8.04
H
O
3.74
O
O
7.07
H
3.81
H
7.90
7.07
H
6.34
H6.10
H
7.92
H
H
N
H
3.66
7.30
H
Br
7.76
O
O
H
O
O
O
N
N
N
CH3
H
H
5
11
18 (85%)
Chart 3. NOE connectivities in compounds 5, 11 and 18.
differences were found. For these compounds, the data reported in
Table 2 are the mean values of the two experiments.
12) did not improve activity. The introduction of a benzyl ring at
position 1 (13e15) gave compounds endowed with a high cytotoxic
The tested compounds showed a mean GI50 range between 1.3
activity, with GI50 values of 2.1e2.6 mM. Among the other sub-
and 7.9
m
M, and compounds 5, 8, 13 and 15 were submitted to the
stituents on the indole system, the most interesting was chlorine at
the 6 position, since compound 8 was the most active in the whole
series.
Biological Evaluation Committee of the NCI for possible future
development.
The results obtained do not show a substantial difference in
cytotoxic activity, on the basis of the geometrical configuration
(E/Z). The substitution of the methine bridge with an imino group
(18, 19) led to lack of cytotoxic activity, and the introduction of a
methyl group at position 1 or 6 of the indolinone system (7, 10 and
3.2. Inhibition of tubulin assembly
The compounds were evaluated for potential inhibition of
tubulin assembly, in comparison with the potent colchicine site
Table 2
Nine subpanels at five concentrations: growth inhibition, cytostatic and cytotoxic activity (mM) of 12 selected compounds.
Comp.a
Modes
Leukemia
NSCLC
Colon
CNS
5.5
20.4
54.9
14.8
56.2
2.9
25.1
81.3
5.2
25.7
85.1
1.6
Melanoma
Ovarian
Renal
Prostate
Breast
MG-MIDb
3
GI50
TGI
LC50
GI50
TGI
GI50
TGI
LC50
GI50
TGI
2.6
14.1
79.4
2.1
49.0
1.1
9.3
81.3
1.8
7.1
25.1
74.1
9.5
77.6
4.2
4.0
15.5
41.7
6.2
5.2
18.6
44.7
10.2
76.0
3.5
4.6
23.4
77.6
17.0
69.2
3.2
32.4
81.3
7.1
4.5
18.6
54.9
14.1
75.9
3.8
49.0
83.2
7.1
4.3
22.9
58.9
11.0
e
2.2
19.0
64.6
3.2
45.7
2.1
4.3
19.5
58.9
7.9
67.6
2.8
32.4
89.1
4.3
20.9
69.2
1.3
5c
6c
e
2.4
2.5
43.6
e
41.7
e
34.7
81.3
4.0
18.2
53.7
0.8
42.7
e
37.1
e
7c
5.7
3.6
3.9
2.5
8.7
70.8
0.5
31.6
79.4
2.0
16.6
53.7
1.9
37.1
87.1
1.9
25.1
66.1
1.6
30.9
91.2
1.5
14.4
74.1
0.8
LC50
GI50
TGI
8c
3.6
8.3
5.4
5.0
6.8
5.4
4.9
5.4
4.1
5.4
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
56.2
2.3
27.5
e
29.5
2.9
14.1
51.3
6.3
24.6
77.6
3.7
46.8
83.2
7.4
28.2
85.1
3.4
14.4
56.2
2.0
9.3
38.9
2.3
22.9
2.8
10.2
37.1
4.3
18.2
47.9
2.6
16.6
63.1
4.0
18.2
67.6
2.2
5.9
14.4
1.8
3.6
8.3
1.9
3.8
7.9
0.1
4.0
79.43
18.6
2.6
7.4
33.9
4.3
17.4
53.7
2.4
19.0
2.6
10.7
44.7
4.9
16.2
43.6
3.1
21.9
85.1
5.0
21.4
69.2
2.9
8.9
26.9
1.7
17.4
2.9
11.2
49.0
5.5
26.3
79.4
3.5
28.2
e
14.1
3.3
12.6
34.7
5.7
22.4
70.8
3.2
24.0
e
17.0
4.6
29.5
83.2
2.8
6.3
22.9
2.6
31.6
2.1
8.7
60.3
3.2
15.1
61.7
2.7
24.0
2.7
12.3
49.0
4.4
17.4
61.7
2.7
21.9
89.1
4.5
21.4
81.3
2.6
10.7
41.7
2.1
9.3
35.5
2.3
9
10
2.2
8.3
81.3
1.4
7.6
87.1
2.3
14.4
67.6
1.5
4.9
50.1
2.2
11c
14.1
e
e
21.9
e
e
12
4.3
24.5
81.3
3.4
15.1
46.8
2.5
10.5
26.3
2.2
7.4
33.9
0.1
6.3
199.5
5.9
4.7
3.2
11.2
e
3.7
17.4
e
36.3
93.3
3.4
22.9
51.3
2.6
21.4
67.6
2.4
8.9
50.1
0.3
19.9
316.2
24.0
85.1
2.5
10.7
43.6
2.0
9.1
45.7
2.4
8.9
28.8
0.3
19.9
251.2
13c
2.2
7.6
33.1
2.7
26.3
e
2.4
12.9
95.5
2.3
17.4
93.3
2.6
13.8
79.4
0.3
14
27.5
e
4.5
19.5
2.1
5.7
23.4
0.2
15c
2.7
27.5
e
3.2
11.2
33.1
0.2
15.8
251.2
14.1
83.2
0.1
6.3
316.2
8.9
34.7
0.2
10.0
251.2
Vincristined
0.1
15.8
630.9
7.9
251.2
7.9
316.2
LC50
a
Highest conc. ¼ 10ꢀ4 M unless otherwise specified; only values <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.

A. Andreani et al. / European Journal of Medicinal Chemistry 64 (2013) 603e612
607
agent combretastatin A-4, and for their effects at 5
m
M on the
stereochemistry relative to NSC 134544 resulted in a flip of its
indolinone motif relative to that of NSC 134544. The aryl portion of
the 8 indolinone was solvent exposed, in contrast to that of NSC
134544. However, in this alternative orientation, the indolinone of
compound 8 retained favorable contacts with tubulin. As delin-
eated in the model, the 8 indolinone moiety was packed lengthwise
against the Lys350 side chain, with the arylchloro group of 8
binding of [3H]colchicine to tubulin. In the assembly assay used, the
IC50 is defined as the concentration of compound that inhibits by
50% the extent of assembly of 10
mM tubulin after a 20 min incu-
bation at 30 ꢁC.
Compound 19 showed growth inhibition versus RXF 393 (kid-
ney cancer) cells as well as CCRF-CEM and RPMI-8226 (leukemia)
and HOP-92 (non-small cell lung cancer) cells in the NCI 60 Cell
One-Dose Screen (data not shown). Moreover, it had the greatest
making a favorable terminal interaction with the
g-carbon of
Lys350. Lastly, for compound 19, the indolinone group assumed a
similar orientation as that of NSC 134544 with its aryl ring posi-
tioned toward the interior of the colchicine site. The N-substituted
benzyl group of compound 19 was packed against the hydrophobic
Lys350 side chain, and this hydrophobic contact contributed to its
ligand binding affinity.
inhibitory effect on tubulin assembly (IC50, 3.3 ꢂ 0.3
mM vs.
1.1 ꢂ 0.1
m
M for combretastatin A-4), but it was much less effective
than combretastatin A-4 as an inhibitor of colchicine binding
(31 ꢂ 3% vs. 99% inhibition for combretastatin A-4). All other
compounds had assembly IC50 values >20
mM, except for NSC
134544 (IC50, 6.1 ꢂ 0.2
m
M with 35 ꢂ 3% inhibition of colchicine
binding) and compound 8 (IC50 15 ꢂ 0.8
m
M). These results showed
3.2.2. Structureeactivity relationship
that there was no correlation between inhibition of tubulin as-
sembly and cytotoxicity, since compound 19 gave poor inhibition of
cell growth, whereas compound 8 caused significant cell death. An
explanation for these findings with tubulin is in the modeling
section.
Besides delineating a structural basis for the antitubulin activity
of NSC 134544, 8, and 19, the binding models may also explain the
inactivity of their close congeners. Docking of compound 13, which
differs from compound 19 by the addition of a para-chlorine group
and inversion of the geometric isomer to Z, suggests that the
inactivity of compound 13 may be due, at least in part, to the
inversion of configuration of the double bond, which creates an
incompatible molecular shape for the colchicine site, as well as to
an additional para-chlorine group, which produces potentially
unfavorable steric bulk. Furthermore, the inactivity of compound
15 may also be rationalized using the binding models, which pre-
dicted a markedly lower binding affinity for 15 as compared with
19. Compound 15 has two methoxy groups that are not present in
the active congener 19. Modeling showed that the indolinone-
attached methoxy group of compound 15 shifts the indolinone
from the interior of the binding pocket relative to the analogous
moiety of compound 19. This shift in the binding pose of compound
15 reduced the binding surface contacts between the indolinone
aromatic ring and the Met257 side chain and concomitantly moves
the N-substituted arylmethoxy group into a more solvent exposed
position and, thus, lessens its binding affinity. Additionally, the
displacement of the indolinone positioned the 4-methoxy in the
benzyl ring against the amino group of Lys350, creating an
3.2.1. Binding models delineate structural basis for activity with
tubulin
As depicted in Fig. 1, the modeled binding poses of NSC 134544,
8, and 19 occupied similar conformational space in the colchicine
site of tubulin, as did the docked pose of CSA4. The trimethox-
yphenyl moieties of NSC 134544, 8, and 19 each mapped similarly
onto the trimethoxyphenyl motif of CSA4. At this segment of the
colchicine site, the trimethoxy groups formed stabilizing hydro-
phobic interactions with the Leu240 and Leu246 side chains and,
simultaneously, stabilizing polar interactions with the thiol of
Cys239. At the other segment of the colchicine site, NSC 134544, 8,
and 19 differed in their molecular components that packed onto the
hydrophobic patch formed by the side chains of Met257, Thr312,
and Lys350. For NSC 134544, its indolinone motif was directed to-
ward this hydrophobic patch with its methoxy group wedged
against the hydrophobic side chain of Met257 at the interior of the
binding pocket. In contrast, for compound 8, its inverted E/Z
Fig. 1. Binding models of CSA4, NSC 134544, 8, and 19 in the colchicine site. b-Tubulin is rendered in light blue ribbon with amino acid side chains shown in stick with carbon atoms
colored cyan. CSA4, NSC 134544, 8, and 19 are depicted in stick with carbon atoms colored green, yellow, pink and purple, respectively. Nitrogen, oxygen, sulfur and chlorine atoms
are colored blue, red, yellow, and green, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

608
A. Andreani et al. / European Journal of Medicinal Chemistry 64 (2013) 603e612
unfavorable hydrophobic-polar contact between the two func-
tionalities. While this hydrophobic-polar contact was slightly un-
favorable in the static binding model, as indicated by a carbon-to-
100
75
50
25
0
*
*
*
ꢀ
*
nitrogen distance of 4.2 A, in the native dynamics of ligand bind-
*
*
**
ing, these two functionalities may be brought closer together due to
the inherent conformational flexibility of the torsional bond angles
of the Lys350 side chain and the eOeCH3 bonds of the methoxy
group. This would result in highly unfavorable hydrophobic-polar
contacts that should preclude efficient ligand binding.
Additionally, the binding model for compound 8 may explain
the weaker activity of its close congener compound 9, which has a
eOCF3 substituent at the 6-position rather than the eCl group of 8.
With 9, this eOCF3 group is positioned at the solvent front, making
it possible to accommodate the additional four heavy atoms relative
to the eCl moiety. However, the binding space of the eOCF3 group
is restricted by the Lys350 side chain. Highly unfavorable in-
teractions between this side chain and the eOCF3 moiety can result
from bond rotation between the oxygen atom and the CF3 group
during binding dynamics. Thus, the Lys350 side chain may be a
stereoelectronic hindrance to binding by 9 in the colchicine site.
**
l
M
M
M
M
M
M
M
M
M
1
M
M
M
ro
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
0
nt
5
I 5
4 5
5 5
6 5
7 5
I 10
4 10
5 10
6 10
7 10 13
Co
13 DP
DP
Fig. 3. Effect of compounds 4, 5, 6, 7, 13 and DPI on intracellular ROS level in B1647 cells.
After a 30 min treatment with each compound dissolved in DMSO, cells (1 ꢃ 106/mL)
were washed twice in PBS and incubated with 5 m
M 20,70-dichlorofluorescin-diacetate
(DCFH-DA) for 20 min at 37 ꢁC. DCFH-DA is a small nonpolar, nonfluorescent molecule
that diffuses into the cells where it is enzymatically deacetylated by intracellular es-
terases to the polar nonfluorescent compound, which is then oxidized to the fluorescent
20,70-dichlorofluorescein (DCF). The fluorescence of oxidized probes was measured in a
multiwell plate. Results are averages of four independent experiments. *P < 0.05;
**P < 0.01 significantly different from control.
3.3. Nox4 inhibition
Compounds active against different NCI leukemia cell lines were
tested for their ability to inhibit Nox4 in B1647 cells, a human acute
myeloid leukemia cell line [12]. Compound 8 was not examined
since measurement of ROS levels was not possible due to its
intrinsic fluorescence. Nox4 is known to be constitutively active
[17] and to play the major role in the generation of ROS required for
sustaining cellular growth and proliferation of the B1647 cell line
[12]. Initially, the cells were incubated with four compounds among
the most effective against the NCI cell lines (5, 6, 7, and 13), and
with compound 4 as a negative control, in order to assay the effect
on viability after a 24 h treatment with the compounds at 5 or
RNA silencing of Nox2. The data showed that both compounds 5
and 7 inhibited ROS production to the same extent either in Nox2
silenced cells or in the scrambled cells used as the control. These
data confirmed the hypothesis that the ROS decrease was due to
Nox4 inhibition (Fig. 4).
Finally, since recent evidence suggests that Nox4 could modu-
late its own expression and that a natural extract endowed with
antioxidant activity is also able to downregulate Nox4, we inves-
tigated the potential effect on Nox4 expression exerted by com-
pound 5 and 7 [19,20]. Western blotting analysis was performed
with B1647 cells after a 24 h treatment with the two compounds.
Data showed that treatments with compounds 5 and 7 were able to
downregulate Nox4 expression (Fig. 5). Most notably, compound 7
10 mM. The MTT assay confirmed the antiproliferative activity of the
5 compounds (Fig. 2). Then, ROS production was measured in B1647
cells after a 30 min treatment with each compound at a concen-
tration of 5 or 10 mM. We found that ROS generation was partially
inhibited by the treatment with compounds 5 and 7 in a dose-
dependent manner (Fig. 3). As B1647 cells express Nox2 and
Nox4 [18], in order to rule out the role of Nox2-derived ROS, the
acute inhibition of ROS production was also measured following
100
*
*
*
§
§
**
***
§§§
***
75
50
25
0
100
*
*
*
*
*
**
**
75
50
25
0
***
**
I
2
5
7
5
7
2
P
2
ed
D
DPI
2
mbl
ra
Sc
A Nox
A Nox A Nox
A Nox
mbled mbled
a
a
r
r
mbled RN
a
si
RN
si
RN
si
r
Sc
Sc
RN
si
Sc
l
M
M
M
M
M
M
M
M
M
M
ro
nt
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
0
5
Fig. 4. Effects of compounds 5, 7 and DPI on intracellular ROS after Nox2 silencing in
B1647 cells. RNA interference experiments were performed as described in the
Experimental section. Briefly, for transient siRNA transfection, B1647 cells were
nucleofected with siRNA against Nox2 or nonspecific RNA (scrambled) at a final con-
centration of 50 nM. Intracellular ROS levels were measured as described in Fig. 3. The
nonspecific control (scrambled) did not give a significant effect compared with the
electroporated sample and was considered as control. Results are averages of four
independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 significantly different
from scrambled. xP < 0.05; xxxP < 0.001 significantly different from siRNA Nox2.
4 5
5 5
6 5
7 5
1
4 10
5 10
6 10
7 10 13
Co
13
Fig. 2. Effect of compounds 4, 5, 6, 7 and 13 on viability of acute leukemia B1647 cells.
Viable cells were evaluated by the MTT test. Cells (106 per well in 1 mL) were pre-
treated for 24 h with each compound dissolved in DMSO prior to adding 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). MTT reduction was
measured in a microplate reader. Results are averages of four independent experi-
ments. *P < 0.05; **P < 0.01; ***P < 0.001 significantly different from the control.

A. Andreani et al. / European Journal of Medicinal Chemistry 64 (2013) 603e612
609
available, whereas 1-benzyl-isatin [21] and the other indolinones
[22e31] were prepared according to the literature.
5.1.1. Synthesis of the 3-(3,4,5-trimethoxybenzylidene)-1,3-
dihydroindol-2-ones
5.1.1.1. Method
A (3e8, 10e14). 3,4,5-Trimethoxybenzaldehyde
(10 mmol) was dissolved in methanol (100 mL) and treated with
the equivalent of the appropriate indolinone and piperidine (1 mL).
The reaction mixture was refluxed for 1e6 h (according to a TLC
test), and the precipitate formed on cooling was collected by
filtration. Compounds 3e6, 8,11e13 were crystallized from ethanol,
7 from acetone/petroleum ether and 10 from toluene (yield 20e
40% for 3, 4, 6, 7, 10e12, 14, 60e70% for 5, 8, 13). Compound 14 was
purified by column chromatography and crystallized from ethanol
(yield 25%).
Fig. 5. Nox4 expression in B1647 cells after a 24 h treatment with 5, 7 or DPI.
Representative immunoblots showing Nox4 expression level in B1647 cells after a 24 h
treatment with compounds 5, 7 or DPI (10 mM). 40 mg of protein per lane were elec-
trophoresed and immunoblotted, as described in the Experimental section. Tubulin
immunoblots confirm that each lane was loaded with the same amount of protein. The
data shown are representative of three independent experiments. Relative amounts
determined by scanning densitometry are expressed in arbitrary units. *P < 0.05;
**P < 0.01 significantly different from control.
3. I.R.: 3165, 1690, 1573, 1112. 1H NMR: 3.70 (3H, s, OCH3), 3.75
(3H, s, OCH3), 3.81 (6H, s, 2ꢃ OCH3), 3.87 (3H, s, OCH3 ind-4), 6.57
(1H, d, ind, J ¼ 9), 6.85 (1H, d, ind, J ¼ 9), 7.77 (2H, s, ph), 8.04 (1H, s,
CH), 10.59 (1H, s, NH). Anal. Calcd for C20H21NO6 (MW 371.39): C,
64.68; H, 5.70; N, 3.77. Found: C, 64.57; H, 5.76; N, 4.02.
4. I.R.: 3181, 1685, 1568, 994. 1H NMR: 3.76 (3H, s, OCH3), 3.84
(6H, s, 2ꢃ OCH3), 7.05 (1H, d, ind, J ¼ 8.8), 7.29 (1H, d, ind, J ¼ 8.8),
7.78 (2H, s, ph), 8.47 (1H, s, CH), 11.23 (1H, s, NH). Anal. Calcd for
C18H15Cl2NO4 (MW 380.22): C, 56.86; H, 3.98; N, 3.68. Found: C,
56.94; H, 4.05; N, 3.55.
inhibited ROS production and downregulated Nox4 more effec-
tively than the nonspecific Nox inhibitor diphenyleneiodonium
(DPI). In conclusion, our results indicate that Nox4 could be a target
for compounds 5 and 7 and that Nox4 inhibition seems to
contribute to their antiproliferative activity in acute leukemia
B1647 cells. These compounds thus may have promise as a novel
antileukemic therapy that attacks an unexploited target.
5. I.R.: 3145, 1701, 1573, 1127. 1H NMR: 3.76 (3H, s, OCH3), 3.85
(6H, s, 2ꢃ OCH3), 6.80 (1H, d, ind-7 J ¼ 8), 7.35 (1H, dd, ind-6, J ¼ 8,
J ¼ 1.6), 7.90 (1H, s, CH), 7.92 (1H, d, ind-4, J ¼ 1.6), 8.04 (2H, s, ph),
10.71 (1H, s, NH).
4. Conclusions
6. I.R.: 3155, 1685, 1578, 1122. 1H NMR: 2.87 (6H, s, 2ꢃ CH3), 3.75
(3H, s, OCH3), 3.85 (6H, s, 2ꢃ OCH3), 6.67 (2H, m, ind), 7.20 (1H, d,
ind-4, J ¼ 2), 7.74 (1H, s, CH), 8.05 (2H, s, ph), 10.22 (1H, s, NH). Anal.
Calcd for C20H22N2O4 (MW 354.40): C, 67.78; H, 6.26; N, 7.90.
Found: C, 68.03; H, 6.03; N, 8.02.
Biological data indicate that the growth-inhibiting activity of the
compounds investigated was associated with both cytotoxic and
cytostatic effects. Data obtained in the acute leukemia B1647 cell
line revealed that Nox4 could be a target for compounds 5 and 7
contributing to their antiproliferative effect and suggested a po-
tential role of these compounds in a novel multitarget antileukemic
therapy. The antitubulin activity of the previously synthesized NSC
134544 was only observed in compounds 8 and 19, but cellular
evidence (increased proportion of cells in G2/M) for this mecha-
nism of action (data not presented) was only obtained for the more
cytotoxic compound 8, which was less active than 19 with purified
tubulin.
We conclude that Nox4 and tubulin assembly inhibition could
explain, at least in part, the cytotoxic and cytostatic effects of the
described compounds, which, however, probably act by means of
further targets and, indeed, could have multiple targets. Molecules
acting by means of multiple mechanisms could be useful for tack-
ling the multifactorial nature of cancer.
7. I.R.: 3365,1680,1624,1127. 1H NMR: 2.10 (3H, s, CH3), 3.75 (3H,
s, OCH3), 3.82 (6H, s, 2ꢃ OCH3), 6.58 (1H, s, ind-7), 7.00 (2H, s, ph),
7.32 (1H, s, ind-4), 7.42 (1H, s, CH), 8.94 (1H, broad, OH),10.19 (1H, s,
NH). Anal. Calcd for C19H19NO5 (MW 341.36): C, 66.85; H, 5.61; N,
4.10. Found: C, 67.02; H, 5.79; N, 3.94.
8. I.R.: 3135e3120, 1690, 1573. 1H NMR: 3.76 (3H, s, OCH3), 3.85
(6H, s, 2ꢃ OCH3), 6.86 (1H, s, ind-7), 7.05 (1H, d, ind, J ¼ 8), 7.69 (1H,
d, ind, J ¼ 8), 7.81 (1H, s, CH), 8.00 (2H, s, ph), 10.73 (1H, s, NH). Anal.
Calcd for C18H16ClNO4 (MW 345.78): C, 62.52; H, 4.66; N 4.05.
Found: C, 62.77; H, 4.82; N, 3.97.
10. I.R.: 1690, 1634, 1578, 1009. 1H NMR: 3.14 (3H, s, CH3), 3.75
(3H, s, OCH3), 3.82 (6H, s, 2ꢃ OCH3), 6.71 (1H, dd, ind-6, J ¼ 8.4,
J ¼ 2.2), 6.85 (1H, d, ind-7, J ¼ 8.4), 7.04 (2H, s, ph), 7.33 (1H, d, ind-4,
J ¼ 2.2), 7.60 (1H, s, CH), 9.18 (1H, s, NH). Anal. Calcd for C19H19NO5
(MW 341.36): C, 66.85; H, 5.61; N, 4.10. Found: C, 67.01; H, 5.73; N,
4.27.
5. Experimental section
11. I.R.: 1716, 1588, 1127, 717. 1H NMR: 3.17 (3H, s, CH3), 3.66 (3H,
s, OCH3), 3.74 (3H, s, OCH3), 3.81 (6H, s, 2ꢃ OCH3), 6.94 (1H, dd, ind-
6, J ¼ 8.8, J ¼ 2.4), 6.97 (1H, d, ind-7, J ¼ 8.8), 7.07 (2H, s, ph), 7.30
(1H, d, ind-4, J ¼ 2.4), 7.66 (1H, s, CH). Anal. Calcd for C20H21NO5
(MW 355.39): C, 67.59; H, 5.96; N, 3.94. Found: C, 67.89; H, 6.03; N,
4.04.
5.1. Chemistry
All compounds synthesized had 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 eluting
solution 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
12. I.R.: 1701, 1603, 1096, 815. 1H NMR: 3.21 (3H, s, CH3), 3.76
(3H, s, OCH3), 3.85 (6H, s, 2ꢃ OCH3), 7.01 (1H, d, ind-7, J ¼ 8.4), 7.32
(1H, dd, ind-6, J ¼ 8.4, J ¼ 2.2), 7.83 (1H, d, ind-4, J ¼ 2.2), 7.93 (1H, s,
CH), 8.06 (2H, s, ph). Anal. Calcd for C19H18ClNO4 (MW 359.81): C,
63.42; H, 5.04; N, 3.89. Found: C, 63.27; H, 4.98; N, 4.01.
13. I.R.: 1701, 1573, 1132, 723. 1H NMR: 3.76 (3H, s, OCH3), 3.87
(6H, s, 2ꢃ OCH3), 5.02 (2H, s, CH2), 6.93 (1H, d, ind-7, J ¼ 7.6), 7.07
(1H, t, ind, J ¼ 7.6), 7.23 (1H, t, ind, J ¼ 7.6), 7.34 (2H, d, bz, J ¼ 9), 7.39
(2H, d, bz, J ¼ 9), 7.77 (1H, d, ind-4, J ¼ 7.6), 7.91 (1H, s, CH), 8.07 (2H,
expressed in
bz benzyl, ind
6-chloro-2-indolinone and 5-methoxy-isatin are commercially
d
(ppm) and J in Hz (abbreviations: ph ¼ phenyl,
¼
¼
indole). 3,4,5-Trimethoxybenzaldehyde,

610
A. Andreani et al. / European Journal of Medicinal Chemistry 64 (2013) 603e612
s, ph). Anal. Calcd for C25H22ClNO4 (MW 435.90): C, 68.88; H, 5.09;
N, 3.21. Found: C, 69.01; H, 4.98; N, 3.44.
(http://dtp.nci.nih.gov/branches/btb/ivclsp.html). In both cases the
exposure time was 48 h.
14. I.R.: 1685, 1568, 1127, 717. 1H NMR: 3.70 (3H, s, OCH3), 3.76
(3H, s, OCH3), 3.87 (6H, s, 2ꢃ OCH3), 4.94 (2H, s, CH2), 6.88 (2H, d,
bz, J ¼ 8.6), 6.95 (1H, d, ind-7, J ¼ 7.4), 7.05 (1H, t, ind-5, J ¼ 7.4), 7.22
(1H, t, ind-6, J ¼ 7.4), 7.27 (2H, d, bz, J ¼ 8.6), 7.74 (1H, d, ind-4,
J ¼ 7.4), 7.89 (1H, s, CH), 8.07 (2H, s, ph). Anal. Calcd for
C26H25NO5 (MW 431.48): C, 72.37; H, 5.84; N, 3.25. Found: C, 72.60;
H, 5.69; N, 3.36.
5.2.2. Inhibition of tubulin assembly
Combretastatin A-4 was a generous gift of Dr. G. R. Pettit, Ari-
zona State University. Bovine brain tubulin was purified as
described previously [33]. The tubulin assembly assay was per-
formed with 10 mM tubulin and varying compound concentrations
as described previously [34]. The IC50 is the compound concen-
tration that inhibits extent of assembly after 20 min at 30 ꢁC. The
5.1.1.2. Method B (9). 3,4,5-Trimethoxybenzaldehyde (10 mmol)
was dissolved in acetic acid (50 mL) and treated with one equiva-
lent of 6-trifluoromethoxy-2-indolinone and 37% HCl (1 mL). The
reaction mixture was refluxed for 20 h, and the precipitate formed
on cooling was collected by filtration (yield 20%) and crystallized
from ethanol.
colchicine binding assay was performed with 1.0
m
M tubulin,
5.0
m mM inhibitor. Incubation was for
M [3H]colchicine and 5.0
10 min at 37 ꢁC. At this time point, about 40e60% maximum
colchicine binding occurs in the control reactions. Details of the
method were described previously [35].
9. I.R.: 3093, 1696, 1511, 994. 1H NMR: 3.75 (3H, s, OCH3), 3.85
(6H, s, 2ꢃ OCH3), 6.77 (1H, s, ind), 6.99 (1H, d, ind, J ¼ 8.3), 7.79 (1H,
d, ind, J ¼ 8.3), 7.86 (2H, s, ph), 8.01 (1H, s, CH), 10.79 (1H, s, NH).
Anal. Calcd for C19H16F3NO5 (MW 395.33): C, 57.72; H, 4.08; N, 3.54.
Found: C, 57.88; H, 3.99; N, 3.26.
5.2.2.1. Molecular modeling. The scientific program Maestro
9 (Schrödinger, LLC, New York) was used in this modeling
study. Computer simulations were performed using the OPLS 2005
force-field, and a distance-dependent dielectric with a nonbonded
ꢀ
interaction limited to within 13 A. Minimizations involved up to
500 steps of Polak-Ribiere conjugate gradient.
ꢀ
5.1.1.3. Method C (15). 3,4,5-Trimethoxybenzaldehyde (15 mmol)
was treated with one equivalent of 5-methoxy-1-(4-methoxy-
benzyl)-2-indolinone, 30 mmol of anhydrous sodium acetate and
130 mL of acetic acid. The reaction mixture was stirred at room
temperature for 7 days, evaporated under reduced pressure and
poured into ice water. The resulting precipitate was collected by
filtration, purified by column chromatography and crystallized
from ethanol (yield 30%).
The 3.58 A 1SA0 crystal structure of ab-tubulin in complex with
DAMA-colchicine was selected as the template for docking studies.
The
ted, and the amino acid sequence of
using as the basis the Uniprot code Q6B856 for bovine
b
-tubulin subunit with bound DAMA-colchicine was extrac-
-tubulin was renumbered
-tubulin,
b
b
which was the construct for the crystal structure. This was fol-
lowed by transformation of bound DAMAecolchicine to colchicine.
The b-tubulinecolchicine complex was refined using the standard
protein preparation protocol in Maestro that allowed for energy
15. I.R.: 1690, 1240, 1122, 717. 1H NMR: 3.63 (3H, s, OCH3), 3.70
(3H, s, OCH3), 3.74 (3H, s, OCH3), 3.81 (6H, s, 2ꢃ OCH3), 4.87 (2H, s,
CH2), 6.89 (4H, m, bz), 7.09 (2H, s, ph), 7.28 (3H, m, ind), 7.38 (1H, s,
CH). Anal. Calcd for C27H27NO6 (MW 461.51): C, 70.27; H, 5.90; N,
3.03. Found: C, 69.98; H, 4.98; N, 2.89.
ꢀ
minimization with a maximal deviation limited to 0.3 A. Manual
adjustment of the torsional bond angles of the Lys350 side chain
optimized the colchicine site for docking studies. Subsequently,
the proteineligand complex was energy minimized with tethered
restraints of 100 kcal to the polypeptide backbone. A docking grid
5.1.2. Synthesis of the 3-[(3,4,5-trimethoxyphenyl)imino]-1,3-
dihydroindol-2-ones 18, 19
for b-tubulin was created using bound colchicine as the reference
molecule, and this grid was used as the template for modeling the
reported binding poses.
3,4,5-Trimethoxyaniline (10 mmol) was dissolved in ethanol
(100 mL) and refluxed for 3 h with one equivalent of the appro-
priate isatin. The precipitate formed on cooling was collected by
filtration and crystallized from ethanol (18, yield 40%) or acetone/
petroleum ether (19, yield 50%).
As a reference, CSA4 was docked into the b-tubulin docking grid
using the Glide program in Maestro. Among the binding poses,
CSA4 assumed the expected conformation in the colchicine site,
with its trimethoxyphenyl motif mapped onto that of colchicine
and its arylhydroxyl motif mapped onto the tropolone functionality
of colchicine. CSA4 gave a Glide XP docking score of ꢀ6.3, which
indicates favorable binding. The docked poses of the active com-
pounds 19 and 8 gave scores of ꢀ5.2 and ꢀ4.1, respectively. Addi-
tionally, consistent with experimental results, the docking program
Glide gave a binding pose for compound 15 with a docking score
of ꢀ3.7, which indicates markedly less binding affinity. Further-
Data for 18. I.R.: 3140e3100, 1731, 1132. 1H NMR: 3.47 (3H, s,
OCH3), 3.68 (3H, s, OCH3), 3.74 (6H, s, 2ꢃ OCH3), 6.10 (1H, d, ind-4,
J ¼ 2.4), 6.34 (2H, s, ph), 6.82 (1H, d, ind-7, J ¼ 8.4), 6.98 (1H, dd,
ind-6, J ¼ 8.4, J ¼ 2.4), 10.77 (1H, s, NH). Anal. Calcd for C18H18N2O5
(MW 342.35): C, 63.15; H, 5.30; N, 8.18. Found: C, 63.03; H, 5.54;
N, 8.03.
Data for 19. I.R.: 1711, 1578, 1127, 728. 1H NMR: 3.71 (3H, s,
OCH3), 3.74 (6H, s, 2ꢃ OCH3), 4.99 (2H, s, CH2), 6.41 (2H, s, ph),
6.72 (1H, d, ind, J ¼ 7.6), 6.86 (1H, t, ind, J ¼ 7.6), 7.03 (1H, d, ind,
J ¼ 7.6), 7.35 (6H, m, 5bz þ 1ind). Anal. Calcd for C24H22N2O4
(MW 402.44): C, 71.63; H, 5.51; N, 6.96. Found: C, 71.45; H, 5.67; N,
7.02.
more, the Glide program quantified
a highly unfavorable
docking score (>1000) for the binding pose of the inactive com-
pound 13 that was modeled on the binding pose of the active
congener 19.
5.3. Nox4 inhibition
5.2. Biology
5.3.1. Cell culture
Human acute myeloid leukemia (AML) B1647 cells were
cultured in a humidified atmosphere (37 ꢁC, 5% CO2) in IMDM (PAA,
Cölbe, Germany) supplemented with 5% human serum (PAA) [12].
5.2.1. Cell-based screening assay
The NCI screening is a two stage process [32], beginning
with the evaluation of all compounds against the 60 cell lines at a
single concentration of 10ꢀ5 M. Compounds exhibiting significant
growth inhibition were evaluated against the 60 cell panel at five
concentration levels by the NCI according to standard procedures
5.3.2. Cell viability and proliferation
Viable cells were evaluated by the MTT assay. Cells were
incubated with 0.5 mg/mL MTT (SigmaeAldrich, St. Louis, MO,

A. Andreani et al. / European Journal of Medicinal Chemistry 64 (2013) 603e612
611
USA) for 4 h at 37 ꢁC. At the end of the incubation, purple for-
mazan salt crystals were formed and dissolved by adding the
solubilization solution (10% SDS, 0.01 M HCl), then the plates
were incubated overnight in a humidified atmosphere (37 ꢁC, 5%
CO2). Absorption at 570 nm was measured in a multiwell plate
reader (Wallac Victor2, PerkineElmer).
suggestions and comments, which have contributed to the perfor-
mance of this study.
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Author contributions
The authors contributed equally to this work.
Acknowledgments
This work has been supported by a grant from the University of
Bologna, Italy (RFO), and from MIUR (PRIN 2009). We are grateful to
the National Cancer Institute (Bethesda, MD) for the anticancer
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