Symmetrical α-bromoacryloylamido diaryldienone derivatives as a novel series of antiproliferative agents. Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmcl.2010.03.075

In a continuing study of hybrid compounds containing the α-bromoacryloyl moiety as potential anticancer drugs, we synthesized a novel series of hybrids 4a-h, in which this moiety was linked to a 1,5-diaryl-1,4-pentadien-3-one system. Many of the conjugates prepared (4b, 4c, 4e and 4g) demonstrated pronounced, submicromolar antiproliferative activity against four cancer cell lines. Moreover, compound 4b induced apoptosis through the mitochondrial pathway and activated caspase-3 in a concentration-dependent manner.

Full text of DOI:10.1016/j.bmcl.2010.03.075

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2733–2739
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Symmetrical
a
-bromoacryloylamido diaryldienone derivatives as a novel
series of antiproliferative agents. Design, synthesis and biological evaluation
a,
a
a
a
Romeo Romagnoli a, , Pier Giovanni Baraldi , Olga Cruz-Lopez , Carlota Lopez Cara , Maria Dora Carrion ,
*
*
Jan Balzarini b, Ernest Hamel c, Giuseppe Basso d, Roberta Bortolozzi d, Giampietro Viola d
a Dipartimento di Scienze Farmaceutiche, Università di Ferrara, 44100 Ferrara, Italy
b Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium
c Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick,
National Institutes of Health, Frederick, 21702 MD, USA
d Dipartimento di Pediatria, Laboratorio di Oncoematologia, Università di Padova, 35131 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In a continuing study of hybrid compounds containing the a-bromoacryloyl moiety as potential anticancer
Received 9 January 2010
Revised 17 March 2010
Accepted 19 March 2010
Available online 25 March 2010
drugs, we synthesized a novel series of hybrids 4a–h, in which this moiety was linked to a 1,5-diaryl-1,4-
pentadien-3-one system. Many of the conjugates prepared (4b, 4c, 4e and 4g) demonstrated pronounced,
submicromolar antiproliferative activity against four cancer cell lines. Moreover, compound 4b induced
apoptosis through the mitochondrial pathway and activated caspase-3 in a concentration-dependent
manner.
Keywords:
Antiproliferative activity
Apoptosis
Ó 2010 Elsevier Ltd. All rights reserved.
Antitumor agents
Hybrid compounds
Because of their ability to interact with cellular nucleophiles,
Michael acceptors are often employed as a powerful tool in the
PNU-166196 (brostallicin), which is currently undergoing Phase II
clinical trials as a first-line single agent chemotherapy in patients
with advanced or metastatic soft tissue sarcoma (Chart 1).13 It
design of anticancer agents.1 The
a,b-unsaturated carbonyl group
is capable of undergoing Michael addition, and this moiety may
be highly deleterious for malignant cells because it could inter-
fere at multiple points in biological cascades through successive
has been hypothesized that the reactivity of the a-bromoacryoyl
moiety results from a first-step Michael-type nucleophilic attack,
followed by a further reaction of the former vinylic bromo substi-
tuent alpha to the carbonyl, leading successively either to a second
nucleophilic substitution or to a beta elimination.14
attacks on cellular nucleophiles.2 Among the
a
,b-unsaturated ke-
tones, enones and dienones derivatives, the chalcones3 and the
a0-bis(arylidene)ketones,4–7 are versatile pharmacophores
a,
In a recent publication, we reported a series of a-bromoacryloy-
belonging to the class of Michael acceptors. In previous studies,
the 1,5-diaryl-3-oxo-1,4-pentadienyl system was incorporated
into various cyclic and acyclic scaffolds with general formula 1a
and 1b, and these compounds included derivatives that demon-
strated promising antiproliferative activities against cancer cell
lines.4,8 The aromatic symmetrical dienones 1a and 1b act as Mi-
chael acceptors, suggesting that two successive alkylations of the
b-positions of the reactive dienone system by cellular thiols could
be one mechanism by which these compounds exert their antipro-
liferative activity in vitro.9–12
lamido chalcones with general structure 2, containing a pair of Mi-
chael acceptors in their structure, corresponding to the
a
-bromoacryloyl moiety and the a,b-unsaturated ketone system
of the chalcone framework.15 Electron-releasing and electron-
withdrawing groups on the phenyl linked to the carbonyl signifi-
cantly influenced the antiproliferative activity. Of the tested com-
pounds, derivative 2a (4-Br) and 2b (4-OMe) displayed IC50
values of 0.62–1.1 and 0.98–1.4
of four cancer cell lines (Table 1).
The conversion of -bromoacryloylamido chalcones 2ab into
the corresponding unsymmetrical dienones 3ab led to a reduction
in activity (0.62–1.1 M for 2a vs 8.3–40 M for 3a, 0.98–1.4
for 2b vs 2.2–6.5
M for 3a).16
The observations that both the 1,5-diaryl-3-oxo-1,4-pentadie-
nyl and the -bromoacryloyl groups can act as trapping agents of
cellular nucleophiles led us to prepare and evaluate a novel class
lM, respectively, against a panel
a
The a-bromoacryloyl alkylating moiety is present in a series of
potent anticancer distamycin-like minor groove binders, including
l
l
lM
l
* Corresponding authors. Tel.: +39 532 455303; fax: +39 532 455953 (R.R.); tel.:
+39 532 291293; fax: +39 532 455953 (P.G.B.).
a
E-mail addresses: rmr@unife.it (R. Romagnoli), baraldi@unife.it (P.G. Baraldi).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.075

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R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2733–2739
O
O
R
R
R
R
X
1b
1a
R=electron-releasing or electron-withdrawing groups
X=NH, N-alkyl or N-alkoxycarbonyl
X=(CH2)n with n from 0 to 4.
Br
H
N
NH
H
HCl
N
O
O
N
NH2
N
H
O
4
PNU-166196 or Brostallicin
O
O
O
R
R
N
N
H
H
2
Br
Br
R=4-Br, 3a
R=4-OMe, 3b
R=H, OMe, Me, N(CH3)2, halogen
R=4-Br, 2a
R=4-OMe, 2b
O
O
Br
Br
Br
X
Br
NH
HN
NH
HN
O
O
O
O
4b, X=CH2
4a
4c, X=CH(CH3)
4d, X=(CH2)2
4e, X=(CH2)3
4f, X=NBn
4g, X=NCO2CH3
4h, X=NCO2CH2CH3
Chart 1.
of synthetic conjugates with general formulae 4, incorporating
these two moieties within their structures (Chart 1). If such pro-
substituted 4-piperidone in the presence of lithium hydroxide
monohydrate as base in ethanol led to the formation of the
cesses occur, the bis-(
a
-bromoacryloylamidoarylidene) ketone
corresponding
(1E,4E)-1,5-bis(4-nitrophenyl)penta-1,4-dien-3-one 5i in 65–73%
yields. -Bis-(aminobenzylidene) ketones 6a–i were generated
from the corresponding nitro derivatives 5a–i by reduction with
iron and ammonium chloride in a refluxing mixture of water and
ethanol (1:4 v/v).
a,a-bis (3-nitrobenzylidene)ketones 5a–h or
derivatives 4a–h, characterized by the presence of four potential
sites for electrophilic attack on cellular constituents, should be
more active than the corresponding 1,5-diaryl-1,4-pentadien-3-
one system, containing only two nucleophilic centers.
a,a
In particular, we have synthesized three different small series of
bis-(a-bromoacryloylamidoarylidene) ketone derivatives, corre-
Finally, the hybrid compounds 4a–k were prepared in 50–62%
sponding to the acyclic analogue 4a, the cyclic conjugates 4c–e,
and finally the 4-piperidone derivatives 4f–h.
yields by the condensation of a-bromoacrylic acid with bis-(3-anil-
inomethylidene) ketones 6a–h, respectively. This condensation
was performed using an excess (4 equiv) of both 1-ethyl-3-[3-
(dimethylamino)propyl]carbodiimide hydrochloride (EDCl) and
1-hydroxy-1,2,3-benzotriazole (1-HOBt) in dry DMF as solvent at
room temperature and with identical reaction times (18 h).
The three different series of cyclic ketones 4b and 4de were pre-
pared with the aim of evaluating how the size of the ring influ-
enced antiproliferative activity. By the synthesis of compound 4c,
we evaluated the effect of the introduction of a small hydrophobic
substituent, corresponding to a methyl group, at the 4-position of
the cyclohexanone ring.
Hybrid compounds 4a–h were prepared by the procedure de-
scribed in Scheme 1. The Claisen–Schmidt aldol condensation17 be-
tween 3-nitrobenzaldehyde or 4-nitrobenzaldehyde with the
appropriate commercially available acetone, cyclic ketone or N-
Table 1 summarizes the inhibitory effects of bis-(a-bromoacry-
loylamido arylidene) ketone derivatives 4a–h on the proliferation
of murine leukemia (L1210), murine mammary carcinoma (FM3A),
human T-leukemia (CEM) and human cervix carcinoma (HeLa) cells,
with bis-(aminobenzylidene)ketones 6a–i,
a-bromoacryloylamido
chalcones 2ab and -bromoacryloylamido bis-(arylidene)ketones
a

R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2733–2739
2735
Table 1
Comparing the 4-piperidone derivatives 4f–h, lengthening the
alkoxy carbonyl group from methyl to ethyl (compounds 4g and
4h, respectively) halved antiproliferative activity.
In vitro inhibitory effects of compounds 2ab, 3ab, 4a–h and 6a–i on the proliferation
of murine leukemia (L1210), murine mammary carcinoma (FM3A), human T-
leukemia (CEM) and human cervix carcinoma (HeLa) cells
The more basic N-benzyl derivative 4f showed reduced potency
relative to 4g and 4h against L1210, FM3A and CEM cells, but 4g
and 4h were equipotent against HeLa cells.
Next, we examined the effects of increasing concentrations of
compounds 4bc, 4e and 4g on cell-cycle progression by performing
a flow cytometric analysis on HeLa cells (Table 2). Cells were cul-
tured for 24 h in the absence or presence of each compound at
a
Compound
IC50
(
l
M)
L1210
FM3A
CEM
HeLa
1.0 0.0
2a
2b
3a
3b
6a
6b
6c
6d
6e
6f
6g
6h
6i
4a
4b
4c
0.62 0.20
0.98 0.96
9.3 6.9
2.4 1.6
7.8 0.7
6.9 0.5
4.8 1.0
35
18
6.8 1.0
9.7 0.6
1.1 0.8
1.2 0.5
0.99 0.31
1.4 1.2
11
6.5 0.5
6.1 0.9
1.7 0.1
1.6 0.1
11
8.5 1.9
1.9 0.2
1.4 0.2
8.3 0.2
4.4 4.3
4.8 4.4
5.2 3.4
1.8 0.5
30 17
40
8
4
2.2 0.5
9.2 0.7
9.1 0.7
8.9 0.8
0.25, 0.5 or 1
lM, and the cells were stained with propidium iodide
(PI). Treatment with 4b, 4c or 4e at 1
l
M for 24 h induced a major
2
7
42
29
0
9
2
shift from the G1 to the G2-M phase compared with control cells,
but no concentration, of those tested, of compound 4g altered cell
cycle progression. In particular, as shown in Table 2, treatment
10
5
9.5 1.4
5.2 4.7
7.6 2.0
8.5 0.1
107 91
4.6 4.4
0.70 0.60
0.30 0.02
4.3 3.7
0.35 0.02
0.73 0.63
0.84 0.74
1.6 0.0
n.d
18
17
207
6
5
6
1.7 0.1
with 0.25, 0.5 and 1 lM of compound 4b for 24 h led to a dose-
9.0 0.0
60 14
4.1 3.4
1.7 0.05
168 16
2.5 0.9
0.30 0.01
0.41 0.09
1.0 2.0
0.34 0.04
1.6 0.0
0.47 0.17
1.4 0.2
dependent decrease of the G1 peak and a proportional accumula-
tion of cells in the G2-M phase, accompanied by a concomitant
increase in the population of apoptotic cells, as demonstrated by
the increase of the sub-G1 peak. Similar effects on the cell cycle
progression were observed with 4bc, 4e or 4g after 48 h of treat-
ment (see Table 1 in the Supplementary data section).
The induction of apoptosis in HeLa cells was confirmed by the
annexin-V test18 with compounds 4bc, 4e and 4g at various con-
centrations for 24 or 48 h. Figure 1 shows a time and concentra-
tion-dependent increase of the percentage of annexin-V positive
cells for all the tested compounds. Only a small percentage
(2–5%) of necrotic cells was observed with all compounds and only
at the highest concentration employed (data not shown).
6.5 4.1
0.47 0.10
0.48 0.02
1.7 1.1
0.73 0.00
5.4 1.2
0.79 0.30
1.1 0.9
n.d
0.32 0.01
0.32 0.03
1.7 1.0
0.38 0.06
1.6 0.7
0.39 0.02
0.62 0.42
2.13 0.03
4d
4e
4f
4g
4h
Melphalanb
2.47 0.30
n.d. not determined.
a
IC50 = compound concentration required to inhibit tumor cell proliferation by
50%. Data are expressed as the mean SE from the dose–response curves of at least
three independent experiments.
b
These data were previously reported in Ref. 7.
Since many compounds that arrest the cell cycle at the G2-M
phase are tubulin inhibitors,19 compounds 4bc, 4e and 4g were
evaluated for their inhibitory effects on tubulin polymerization.
None of tested compounds were effective as inhibitors of tubulin
3ab as reference compounds. The alkylating agent melphalan was
used as a reference drug.
Compounds 6a and 6i were synthesized in order to evaluate the
influence of the position of amino group on the benzylidene moiety
on antiproliferative activity. Changing the location of the amino
group from the para- in 6i to the meta-position as in 6a, led to a
10–25-fold increase in antiproliferative activity against all four
cancer cell lines. The higher inhibition of cell growth showed by
assembly (IC50 >40 lM). These data make it unlikely that the anti-
proliferative activity of these hybrid compounds results from a di-
rect interaction with tubulin and that they act as microtubule
depolymerizing agents.
Impairment of mitochondrial function is an early event in the
executory phase of programmed cell death in different cell types,
and it occurs as a consequence of a preliminary reduction of the
6a led us to synthesize
derivatives 6b–h.
a,
a0-bis-(meta-aminobenzylidene) ketone
mitochondrial transmembrane potential (Dw Dwmt
mt).20,21 Early
Comparing the activities of 4a–h with those of 6a–h demon-
strates that insertion of the two -bromoacryloyl moieties was
an important molecular change leading to a significant increase
in antiproliferative activity. The bis-( -bromoacryloylamido ben-
zylidene) ketones 4a–h were from 2- to 50-fold more active than
the corresponding bis-(anilinomethylidene) precursors 6a–h.
disruption results from an opening of mitochondrial permeability
transition pores, and this permeability transition triggers the re-
lease of apoptogenic factors, such as apoptosis inducing factor
and cytochrome c, which in turn lead to later apoptotic events.20,21
We used the lipophilic cation 5,50,6,60-tetrachlo-1,10,3,30-tetra-
ethylbenzimidazol-carbocyanine (JC-1) to monitor changes in
a
a
These values are greater or equal to those obtained with the
a-
D
wmt induced by 4b. The method is based on the ability of this
bromoacryloylamido enones 2ab. The greater activity of 4bc, 4e
and 4gh compared to that of 3ad indicated the contribution of a
second a-bromoacryloyl moiety to antiproliferative activity.
The compounds displaying the greatest potency were 4b, 4c and
4g, with IC50 values of 0.32–0.38, 0.47–0.79, 0.30–0.47 and 0.30–
0.84 lM against the L1210, FM3A, CEM and HeLa cell lines, respec-
tively. Notably, the acyclic derivative 4a was significantly less ac-
tive than its cyclic counterparts 4b–h. Derivatives 4b–h were
found to have greatest potency to melphalan against tested cancer
cell lines.
In the series of compounds 4b–e, activity decreased when the
size of the cyclic ketone was increased from six to seven carbon
units, while it increased greatly when the size of the ring increased
from seven to eight carbons (compounds 4b, 4d and 4e, respec-
tively). Starting from 4b, the 4-methyl substitution on the cyclo-
hexanone ring, to furnish 4c, did not improve antiproliferative
activity against L1210, FM3A and CEM cells, but the methyl group
doubled potency with respect to HeLa cells.
fluorescent probe to enter selectively into the mitochondria, and
its color changes reversibly from green to orange as membrane po-
tential increases.22 This property is due to the reversible formation
of JC-1 aggregates upon membrane polarization. Aggregation
causes a shift in the emitted light from 530 nm (i.e., emission by
JC-1 monomers) to 590 nm (emission by JC-1 aggregates) following
excitation at 490 nm.
HeLa cells were treated with compound 4b for 24, 48 and 72 h
at 0.5 and 1
stantial mitochondrial depolarization in a time- and concentration-
dependent manner. The disruption of the wmt is associated with
lM. As shown in Figure 2, compound 4b induces sub-
D
the appearance of sub-G1 cells and with the marked increase in the
percentage of annexin-V positive cells.
Mitochondrial membrane depolarization is associated with
mitochondrial production of reactive oxygen species (ROS).23,24
ROS are highly reactive molecules with both free radical and
non-radical structures, including superoxide anion, hydrogen per-
oxide, hydroxyl radical and single oxygen. ROS play a central role

2736
R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2733–2739
O
5a, R=3-NO2
5i, R=4-NO2
CHO
or
O
R
R
a
5b, X=CH2
O2N
5c, X=CH(CH3)
5d, X=(CH2)2
5e, X=(CH2)3
5f, X=NBn
5g, X=NCO2CH3
5h, X=NCO2CH2CH3
X
NO2
NO2
b
O
6a, R=3-NH2
6i, R=4-NH2
or
O
R
R
6b, X=CH2
6c, X=CH(CH3)
6d, X=(CH2)2
6e, X=(CH2)3
6f, X=NBn
6g, X=NCO2CH3
6h, X=NCO2CH2CH3
X
NH2
NH2
c
O
O
4b, X=CH2
4c, X=CH(CH3)
4d, X=(CH2)2
Br
X
Br
4e, X=(CH2)3
Br
Br
4f, X=NBn
4g, X=NCO2CH3
4h, X=NCO2CH2CH3
NH
HN
NH
HN
O
O
4a
O
O
Scheme 1. Reagents: (a) LiOHꢀH2O, cyclic/acyclic ketone, m-nitrobenzaldehyde for the synthesis of 5a–b, p-nitrobenzaldehyde for the synthesis of 5i, EtOH, rt, 18 h; (b) Fe,
NH4Cl, H2O–EtOH, reflux, 2 h; (c) a-bromoacrylic acid, EDCI, HOBt, DMF, 18 h, rt.
Table 2
Cell-cycle distribution of HeLa cells after 24 h of treatment with compounds 4b, 4c, 4e or 4g
Compound
Concentration (lM)
Cell cycle percentage (%)
Sb
Sub-G1a
G1b
G2-Mb
Control
4b
4b
4b
4c
4c
4c
4e
4e
4e
4g
4g
4g
NM
0.25
0.5
1.0
0.25
0.5
1.0
0.25
0.5
1.0
0.25
0.5
1.9 0.3
4.7 2.1
10.5 4.6
27.2 1.2
3.2 0.6
4.0 1.1
4.5 0.9
2.5 0.9
6.0 3.0
15.6 7.5
52.9 3.3
44.7 1.1
50.9 10.1
28.8 6.0
41.6 5.5
59.2 9.0
26.5 1.5
37.2 8.6
64.4 1.4
32.6 10.6
49.2 4.0
51.9 3.9
57.0 2.5
29.0 3.0
35.8 4.0
21.6 6.3
22.7 7.1
43.5 0.8
16.1 2.6
31.3 6.5
46.2 7.6
25.0 5.0
12.9 2.5
36.8 6.8
34.6 11.7
17.5 2.0
18.1 0.9
16.1 0.5
27.8 6.8
51.9 10.4
14.8 4.7
27.5 3.8
41.9 8.0
16.5 1.0
10.5 2.5
54.5 7.8
14.5 3.4
13.4 8.2
24.9 4.1
8.0
3
7.2 1.0
8.2 2.1
1.0
Data are expressed as the mean SE from the dose–response curves of at least three independent experiments.
NM: not meaningful.
a
Percentage of the cell population with hypodiploid DNA content peak (apoptotic cells).
The percentage of cells in each phase of the cell cycle was calculated using living cells.
b
as second messengers in a number of signal transduction pathways
and cause a wide range of cellular responses ranging from tran-
sient to permanent growth arrest, and from apoptosis to necrosis,
dependingon the level of ROS.
To investigate the effects of 4b on the production of oxygen spe-
cies during apoptosis, we utilized two fluorescence probes, hydro-
ethidine (HE) and 20,70-dichlorodihydrofluorescein diacetate
(H2DCFDA). The fluorescence of these compounds occurs if ROS

R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2733–2739
2737
Ctr
4b 1.0 μΜ
4b 0.5 μΜ
Ctr
60
60
50
40
30
20
10
0
4e 1.0 μΜ
4e 0.5 μΜ
50
40
30
20
10
0
24
48
24
48
Time (h)
Time (h)
60
50
40
30
20
10
0
60
50
40
30
20
10
0
Ctr
4c 1.0 μΜ
4c 0.5 μΜ
Ctr
4g 1μΜ
4g 0.5 μΜ
24
48
24
48
Time (h)
Time (h)
Figure 1. Flow cytometric analysis of apoptotic cells after treatment of HeLa cells with compound 4bc, 4e or 4g. After 24 and 48 h of treatment, cells were harvested and
labeled with Annexin-V-FITC and PI and then analyzed by flow cytometry. The data are expressed as mean of percentage of Annexin-V positive cells S.E.M. for three
independent experiments.
are generated.25 As shown in Figure 3 (upper and medium panels),
there was an increase in cells producing ROS that closely paralleled
the increase in cells with low Dwmt, as a function of both the con-
of the mitochondrial permeability transition.26 The dye interacts
stoichiometrically with intact, non-oxidized cardiolipin.27 Some-
what unexpectedly, the cells did not show reduction in NAO fluo-
rescence, but, rather, a marked increase in NAO, especially after
48 h of treatment with 4b (Fig. 3, lower panel). This effect suggests
an increase in cardiolipin content as a consequence of increased
mitochondrial mass. This has been recently observed by our group
centration of 4b and treatment time.
We also evaluated the damage caused by ROS in mitochondria
by assessing the oxidation state of cardiolipin, a phospholipid re-
stricted to the inner mitochondrial membrane. We used 10 N-non-
yl acridine orange (NAO), a fluorescent probe which is independent
in K562 cells following treatment with hybrid
a-bromoacryloy-
lamido chalcones15 but also in other tumor cell lines after treat-
ment with herbimycin A,28 genistein,29 and the acronicyne
derivative S23906-130 and following oxidative stress.31
Ctr
40
Altogether these results indicated that the cell cycle arrest and
apoptosis induced by 4b in HeLa cells were correlated with a dose-
and time-dependent appearance of mitochondrial dysfunction.
The mechanism of apoptosis depends on a cascade of proteo-
lytic enzymes called caspases.32 They exist in most cells as inactive
precursors (zymogens) that, once activated sequentially, lead to
irreversible cell death. Within the caspase family, caspase-3 is
essential for propagation of the apoptotic signal after exposure of
cells to many DNA-damaging agents and other anticancer
drugs.31,33 We therefore determined whether the activation of cas-
pase-3 was involved in the apoptosis induced by compound 4b. We
used a monoclonal antibody specific for the active fragment of cas-
4b 0.5 μΜ
4b 1.0 μΜ
30
20
10
0
24
48
pase-3. As shown in Figure 4, with 0.5 and especially 1 lM 4b there
was a marked activation of caspase-3 after 24 h of treatment, and
no further increase of caspase-3 activity occurred after 48 h.
In summary, our plan to optimize the potency of a double Mi-
chael acceptor by providing multiple sites of reactivity towards
Time (h)
Figure 2. Assessment of mitochondrial potential after treatment with compound
4b. Upper panel: induction of loss of mitochondrial membrane potential after 24, 48
and 72 h of incubation of HeLa cells with 4b at 0.5 or 1.0
lM. Cells were stained
cellular nucleophiles led us to synthesize a series of bis-(a-bromo-
with the fluorescent probe JC-1 and analyzed by flow cytometry.

2738
R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2733–2739
Ctr
40
Ctr
40
30
20
10
0
4b 0.5 μΜ
4b 0.5 μΜ
4b 1.0 μΜ
4b 1.0 μΜ
30
20
10
0
24
48
24
48
Time (h)
Time (h)
Figure 4. Caspase-3 induced activity by compound 4b. HeLa cells were incubated in
the presence of 4b at 0.5 or 1.0 M. After 24 or 48 h of treatment, cells were
harvested and stained with an anti-human active Caspase-3 fragment monoclonal
antibody conjugated with FITC. Data obtained by flow cytometric analysis are
expressed as percentage of caspase-3 active fragment positive cells. Data are
expressed as mean S.E.M. of three independent experiments.
l
Ctr
30
20
10
0
4b 0.5 μΜ
4b 1.0 μΜ
in 4a–h, enabling these compounds to act potentially as multifunc-
tional Michael acceptors. In the series of cyclic ketones 4b–e, the
six- and the eight-membered cyclic ring was preferred to the se-
ven-membered ring. Compound 4g had no effect on cycle progres-
sion of HeLa cells even at higher concentrations, while compounds
4bc and 4e induced cell cycle arrest in a concentration-dependent
manner. In particular, treatment with increasing concentrations of
4b led to cell cycle arrest in the G2-M phase accompanied by an in-
crease in the percentage of apoptotic cells after 24 and 48 h.
As demonstrated with 4b, the mechanism of action of these
compounds appears to be induction of apoptosis mediated by the
activation of caspase-3 and by important changes in mitochondrial
function, such as dissipation of the transmembrane potential and
generation of ROS. Further studies to clarify additional details of
the molecular mechanism of action of these compounds and their
selectivity in inhibiting the growth of cancer cells are underway.
24
48
Time (h)
100
80
60
40
20
0
Ctr
4b 0.5 μΜ
4b 1.0 μΜ
Acknowledgments
Financial support was provided by GOA (Krediet No. 05/19) of
the K.U. Leuven. The technical assistance of Mrs. Lizette van Berc-
kelaer and Alberto Casolari is gratefully acknowledged.
24
48
Time (h)
Supplementary data
Figure 3. Mitochondrial production of ROS in HeLa cells. After 24 and 48 h of
incubation with 4b, cells were stained with HE (upper panel) or H2DCFDA (middle
panel) and analyzed by flow cytometry. Increase of mitochondrial mass in HeLa
cells treated with compound 4b (lower panel). Cells were treated as above and the
relative NAO intensity was analyzed by flow cytometry. The results are expressed as
percentage of the fluorescence intensity with respect to untreated control cells.
Data are expressed as mean S.E.M. of three independent experiments.
Supplementary data (detailed biological protocols, synthesis
and spectroscopic data for hybrids 4a–h and 3ab. General proce-
dures for the synthesis of
a,
a0-bis-(nitrobenzylidene) ketones 5a–
i and
a,
a0-bis-(aminobenzylidene) ketones 6a–i. Cell-cycle distri-
bution of HeLa cells after 48 h of treatment with compounds 4b,
4c, 4e and 4g (Table 1)) associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2010.03.075.
acryloylamido benzylidene) ketones 4a–h. Compared to melpha-
lan, an established anticancer agent, derivatives 4b–h displayed
significantly greater potency. These compounds were derived from
the hybridization of two kinds of Michael acceptors, corresponding
References and notes
1. Ahn, B.-Z.; Sok, D.-E. Curr. Pharm. Des. 1996, 2, 247.
2. Dimmock, J. R.; Wong, M. L. C. Can. J. Pharm. Sci. 1976, 11, 35.
3. Lawrence, N. J.; McGown, A. T. Curr. Pharm. Des. 2005, 11, 1679.
4. (a) Das, U.; Sharma, R. K.; Dimmock, J. R. Curr. Med. Chem. 2009, 16, 2001; (b)
Dimmock, J. R.; Elias, D. W.; Beazely, M. A.; Kandepu, N. M. Curr. Med. Chem.
1999, 6, 1125.
5. Dimmock, J. R.; Padmanilayam, M. P.; Zello, G. A.; Nienaber, K. H.; Allen, T. M.;
Santos, C. L.; De Clercq, E.; Balzarini, J.; Manavathu, E. K.; Stables, J. P. Eur. J.
Med. Chem. 2003, 38, 169.
to the 1,5-diaryl-1,4-pentadien-3-one system of
enzylidene) ketones 6a–i and the -bromoacryloyl moiety. While
compounds 6a–i showed weak or no antiproliferative activity
against five cancer cell lines, the attachment of two -bromoacry-
a,
a0-bis-(aminob-
a
a
loyl moieties led to significantly increased activity. The greater po-
tency of hybrid compounds 4a–h than bis-(aminoarylidene)
ketones 6a–i was attributed to the additional electrophilic groups
6. Dimmock, J. R.; Das, U.; Gul, H. I.; Kawase, M.; Sakagami, H.; Barath, Z.;
Ocsovsky, I.; Molnar, J. Bioorg. Med. Chem. Lett. 2005, 15, 1633.

R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2733–2739
2739
7. Das, U.; Gul, H. I.; Alcorn, J.; Shrivastav, A.; George, T.; Sharma, R. K.; Nienaber,
K. H.; De Clercq, E.; Balzarini, J.; Kawase, M.; Kan, N.; Tanaka, T.; Tani, S.;
Werbovetz, K. A.; Yakovich, A. J.; Manavathu, E. K.; Stables, J. P.; Dimmock, J. R.
Eur. J. Med. Chem. 2006, 41, 577.
8. Pati, H. N.;Das, U.; Sharma, R. K.;Dimmock, J. R. Mini-Rev. Med. Chem. 2007, 7, 131.
9. Dimmock, J. R.; Padmanilayam, M. P.; Puthucode, R. N.; Nazarali, A. J.;
Motaganahalli, N. L.; Zello, G. A.; Quail, J. W.; Oloo, E. O.; Kraatz, H.-B.;
Prisciak, J. S.; Allen, T. M.; Santos, C. L.; Balzarini, J.; De Clercq, E.; Manavathu, E.
K. J. Med. Chem. 2001, 44, 586.
10. Baluja, G.; Municio, A. M.; Vega, S. Chem. Ind. 1964, 2053.
11. Mutus, B.; Wagner, J. D.; Talpas, C. J.; Dimmock, J. R.; Phillips, O. A.; Reid, R. S.
Anal. Biochem. 1989, 177, 237.
12. Dimmock, J. R.; Raghavan, S. K.; Logan, B. M.; Bigham, G. E. Eur. J. Med. Chem.
1983, 18, 248.
18. Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutelingsperger, C. J. Immunol.
Methods 1995, 184, 39.
19. (a) Hamel, E. Cell Biochem. Biophys. 2003, 38, 1; (b) Verdier-Pinard, P.; Lai, J.-Y.;
Yoo, H.-D.; Yu, J.; Marquez, B.; Nagle, D. G.; Nambu, M.; White, J. D.; Falck, J. R.;
Gerwick, W. H.; Day, B. W.; Hamel, E. Mol. Pharmacol. 1998, 53, 62.
20. Ly, J. D.; Grubb, D. R.; Lawen, A. Apoptosis 2003, 3, 115.
21. Green, D. R.; Kroemer, G. Science 2005, 305, 626.
22. Salvioli, S.; Ardizzoni, A.; Franceschi, C.; Cossarizza, A. FEBS Lett. 1997, 411, 77.
23. (a) Hancock, J. T.; Desikan, R.; Neil, S. J. Biochem. Sci. Trans. 2001, 29, 245; (b)
Zamzami, N.; Marchetti, P.; Castedo, M.; Decaudin, D.; Macho, A.; Hirsch, T.;
Susin, S. A.; Petit, P. X.; Mignotte, B.; Kroemer, G. J. Exp. Med. 1995, 182, 367.
24. (a) Nohl, H.; Gille, L.; Staniek, K. Biochem. Pharmacol. 2005, 6, 719; (b) Boonstra,
J.; Post, J. A. Gene 2004, 337, 1.
25. Rothe, G.; Valet, G. J. Leukocyte Biol. 1990, 47, 440.
13. (a) Lorusso, D.; Mainenti, S.; Pietragalla, A.; Ferrandina, G.; Foco, G.; Masciullo,
V.; Scambia, G. Expert Opin. Invest. Drugs 2009, 18, 1939; (b) Leahy, M.; Ray-
Coquard, I.; Verwei, J.; Le Cesne, A.; Duffaud, F.; Hogendoorn, P. C.; Fowst, C.; De
Balincourt, C.; Di Paola, E. D.; Van Glabbeke, M.; Judson, I.; Blay, J. Y. Eur. J.
Cancer 2007, 43, 308.
26. Petit, J. M.; Maftah, A.; Ratinaud, M. H.; Julien, R. Eur. J. Biochem. 1992, 209, 267.
27. Gallet, P. F.; Maftah, A.; Petit, J. M.; Denis-Gay, M.; Julien, R. Eur. J. Biochem.
1995, 228, 113.
28. Mancini, M.; Anderson, B. O.; Caldwell, E.; Sedghinasab, M.; Paty, B. P.;
Hockenbery, D. M. J. Cell Biol. 1997, 138, 449.
14. Romagnoli, R.; Baraldi, P. G.; Cruz-Lopez, O.; Lopez-Cara, C.; Preti, D. Mini-Rev.
Med. Chem. 2009, 9, 81.
29. Pagliacci, M. C.; Spinozzi, F.; Migliorati, G.; Fumi, G.; Smacchia, M.; Grignani, F.;
Riccardi, C.; Nicoletti, E. Eur. J. Cancer 1993, 29A, 1573.
15. Romagnoli, R.; Baraldi, P. G.; Carrion, M. D.; Cruz-Lopez, O.; Lopez Cara, C.;
Balzarini, J.; Hamel, E.; Canella, A.; Fabbri, E.; Gambari, R.; Basso, G.; Viola, G.
Bioorg. Med. Chem. Lett. 2009, 19, 2022.
16. For the characterization of compounds 3a and 3b, see Supplementary data.
17. Bhagat, S.; Sharma, R.; Chakraborti, A. K. J. Mol. Catal. A: Chem. 2006, 260, 235.
30. Kluza, J.; Lansiaux, A.; Wattez, N.; Hildebrand, M. P.; Leonce, S.; Pierre, A.;
Hickman, J. A.; Bailly, C. Biochem. Pharmacol. 2002, 63, 1443.
31. Lee, H. C.; Yin, P. H.; Lu, C. Y.; Chi, C. W.; Wei, Y. H. Biochem. J. 2000, 348, 425.
32. Kumar, S. Cell Death Differ. 2007, 14, 32.
33. Selvesen, S.; Dixit, V. M. Cell 1997, 91, 43.