Hybrid α-bromoacryloylamido chalcones. Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmcl.2009.02.038

Research into the anti-tumor properties of chalcones has received significant attention over the last few years Two novel large series of α-bromoacryloylamido chalcones 1a-m and 2a-k containing a pair of Michael acceptors in their structures, corresponding to the α-bromoacryloyl moiety and the α,β-unsaturated ketone system of the chalcone framework, were synthesized and evaluated for antiproliferative activity against five cancer cell lines. Such hybrid derivatives demonstrated significantly increased anti-tumor activity compared with the corresponding amino chalcones. The most promising lead molecules were 1k, 1m and 2j, which had the highest activity toward the five cell lines. Flow cytometry with K562 cells showed that the most active compounds resulted in a large proportion of the cells entering in the apoptotic sub-G0-G1 peak. Moreover, compound 1k induced apoptosis through the mitochondrial pathway and activated caspase-3.

Full text of DOI:10.1016/j.bmcl.2009.02.038

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2022–2028
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hybrid
a-bromoacryloylamido chalcones. Design, synthesis
and biological evaluation
a,
a
a
a
Romeo Romagnoli a, , Pier Giovanni Baraldi , Maria Dora Carrion , Olga Cruz-Lopez , Carlota Lopez Cara ,
Jan Balzarini b, Ernest Hamel c, Alessandro Canella d, Enrica Fabbri d, Roberto Gambari d, Giuseppe Basso e,
Giampietro Viola e
*
*
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 Toxicology and Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick,
National Institutes of Health, Frederick, MD 21702, USA
d BioPharmaNet, Dipartimento di Biochimica e Biologia Molecolare, Università di Ferrara, 44100 Ferrara, Italy
e 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:
Research into the anti-tumor properties of chalcones has received significant attention over the last few
Received 22 November 2008
Revised 6 February 2009
Accepted 7 February 2009
Available online 12 February 2009
years Two novel large series of
Michael acceptors in their structures, corresponding to the
a
-bromoacryloylamido chalcones 1a–m and 2a–k containing a pair of
a
-bromoacryloyl moiety and the ,b-unsatu-
a
rated ketone system of the chalcone framework, were synthesized and evaluated for antiproliferative
activity against five cancer cell lines. Such hybrid derivatives demonstrated significantly increased
anti-tumor activity compared with the corresponding amino chalcones. The most promising lead mole-
cules were 1k, 1m and 2j, which had the highest activity toward the five cell lines. Flow cytometry with
K562 cells showed that the most active compounds resulted in a large proportion of the cells entering in
the apoptotic sub-G0–G1 peak. Moreover, compound 1k induced apoptosis through the mitochondrial
pathway and activated caspase-3.
Ó 2009 Elsevier Ltd. All rights reserved.
Among currently identified anti-tumor agents, chalcones repre-
sent an important class of natural small molecules useful in cancer
chemotherapy.1 Chalcones (1,3-diaryl-2-propen-1-ones, Chart 1)
are known to exhibit antimitotic properties caused by inhibition
of tubulin polymerization by binding to the colchicine-binding
site.2 Chemically, they are open-chained molecules consisting of
two aromatic rings linked by a three-carbon enone fragment. Sev-
eral research groups have shown that the s-cis conformation of
chalcones is important for their biological activity.3 The double
bond of the enone system is the essential moiety for chalcones as
anti-tumor agents.4 It has been reported that hydrogenation or
bromination across the carbon–carbon double bond or its transfor-
mation into the corresponding epoxide dramatically reduces chal-
cone activity.5 Their simple structure and the ease of preparation
make chalcones an attractive scaffold for structure–activity rela-
tionship (SAR) studies, and a wide number of substituted chalcones
have been synthesized to evaluate effects of various functional
groups on biological activity.1
a
-bromoacryloyl alkylating moiety of low chemical reactivity, an
unusual feature for cytotoxic compounds. In fact, -bromoacrylic
acid is not per se cytotoxic (IC50 for L1210 cells being greater
than120
M).8 The same moiety is present in a series of potent
a
l
anticancer distamycin-like minor groove binders, for example,
PNU-166196 (brostallicin), which is currently undergoing Phase
II clinical trials.9 PNU-166196 is an
a-bromoacrylamido derivative
of a four-pyrrole distamycin homologue ending with a guanidino
moiety (Chart 1).9
The reactivity of the a-bromoacryoyl moiety has been hypoth-
esized to be based on 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 beta elimination.10
Furthermore, several studies confirmed that the
a,b-unsatu-
rated ketone system of chalcones acts as a Michael acceptor, sug-
gesting that alkylation of the b-position of the reactive enone
system by biological nucleophiles may be one mechanism by
which antiproliferative activity is exerted in vitro.11
The pyrroloiminoquinone cytotoxic alkaloids Discorhabdin A6
and Discorhabdin G7 are characterized by the presence of an
The observations that both the chalcone and the a-bromoacry-
loyl group can act as trapping agents of cellular nucleophiles led us
to prepare and evaluate two novel and unusual classes of synthetic
conjugates with general formulae 1a–m and 2a–k (Chart 1), incor-
porating these two moieties within their structures. If such
* Corresponding authors. Tel.: +39 (0)532 455303; fax: +39 (0)532 455953 (R.R.),
tel.: +39 (0)532 291293; fax: +39 (0)532 455953 (P.G.B.).
E-mail addresses: rmr@unife.it (R. Romagnoli), baraldi@unife.it (P.G. Baraldi).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.038

R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2022–2028
2023
O
R1
R2
General structure of chalcones
R1 or R2=H, OH, OMe, halogen, NO2, NH2
O
O
H
H
Br
H
N
N
HN
HN
N
NH
H
S
HCl
NH2
N
O
N
N
NH
Br
H
NH
O
4
O
Br
O
PNU-166196 or Brostallicin
Discorhabdin G
Discorhabdin A
O
O
O
O
N
Ar
Ar
2
1
N
H
H
Br
Br
2a, Ar=C6H5
1a, Ar=C6H5
2b, Ar=4-OMe-C6H4
2c, Ar=3-OMe-C6H4
2d, Ar=2-OMe-C6H4
2e, Ar=2,3-(OMe)2-C6H4
2f Ar=3,4-(OMe)2-C6H4
2g, Ar=2,4-(OMe)2-C6H4
2h, Ar=3,5-(OMe)2-C6H4
2i, Ar=3,4,5-(OMe)3-C6H3
2j, Ar=2,3,4-(OMe)3-C6H3
2k, Ar=4-N(CH3)2-C6H4
1b, Ar=4-F-C6H4
1c, Ar=4-Cl-C6H4
1d, Ar=3,4-Cl2-C6H4
1e, Ar=4-Br-C6H4
1f Ar=4-I-C6H4
1g, Ar=4-Me-C6H4
1h, Ar=4-OMe-C6H4
1i, Ar=3-OMe-C6H4
1j, Ar=3,4,5-(OMe)3-C6H3
1k, Ar=4-OEt-C6H4
1l, Ar=1-naphtyl
1m, Ar=2-thienyl
Chart 1.
processes occur, the
a
-bromoacryloylamido chalcone derivatives
corresponding nitro derivatives 5a–m and 6a–k by reduction with
iron in a refluxing solution mixture of 37% HCl in water and etha-
nol (1:2.5 v/v).
Finally, the hybrid compounds 1a–m and 2a–k were prepared,
in acceptable yields (54–68%), by the condensation of a-bromoac-
1a–m and 2a–k, characterized by the presence of two potential
sites for electrophilic attack on cellular constituents, should be
more potent than the corresponding compounds containing only
one nucleophilic center.
While compounds 1a–m were designed to evaluate the SAR
arising from different substitutions (both electron-releasing and
electron-withdrawing groups), as well as different positions on
the phenyl ring, for hybrids 2a–k we focused on the synthesis of
methoxylated derivatives with one or more methoxy groups at dif-
ferent positions on the aryl moiety.
rylic acid with aminochalcones 7a–m and 8a–k, respectively. This
condensation was performed using an excess (2 equiv) both of 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).
Synthesis of derivatives 1a–m and 2a–k was carried out by the
general methodology shown in Scheme 1. Nitrochalcones 5a–m
and 6a–k were synthesized in high yields (81–95%) by the Clais-
en–Schmidt aldol condensation of 4-nitrobenzaldehyde or 4-nitro-
acetophenone with the corresponding appropriately functionalized
acetophenones 3a–m or benzaldehydes 4a–k, respectively, in the
presence of 50% w/v aqueous solution of sodium hydroxide.12
Coupling constants (J) from the proton nuclear magnetic resonance
(1H NMR) spectra clearly indicated that derivatives 5a–m and 6a–
k were both geometrically pure and were exclusively trans (E) iso-
mers (JtransC@C = 15–16 Hz). The cluster of compounds 6a–k may
be referred to as ‘reversed chalcones’, whereby the carbonyl and
ethylene groups present in the series 5a–m are interchanged.
Aminochalcones 7a–m and 8a–k were generated from the the
Tables 1 and 2 summarize the antiproliferative effects of a-
bromoacryloylamido chalcones 1a–m and 2a–k, respectively,
against the growth of murine leukemia (L1210), murine mammary
carcinoma (FM3A), human T-lymphoblastoid (Molt/4 and CEM)
and human cervix carcinoma (HeLa) cells, using aminochalcones
7a–m and 8a–k as reference compounds.
In general, the a-bromoacryloylamido chalcones 1a–m and 2a–
k, were 10–100-fold more active than the corresponding amino
chalcones 7a–m and 8a–k, respectively, demonstrating that the
presence of an
a-bromoacryloyl moiety significantly enhanced
antiproliferative activity. The compounds displaying the greatest
potency were 1k (p-EtO), 1m (2-thienyl) and 2j (o, m, p-3OMe),
with IC50 values of 0.24–0.63, 0.52–0.68, 0.55–0.68, 0.73–0.84
and 0.34–0.75 lM against the L1210, FM3A, Molt4, CEM and Hela

2024
R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2022–2028
or
H3COC Ar
OHC Ar
4a-k
3a-m
a
b
O
O
or
Ar
Ar
6a-k
5a-m
O2N
O2N
O
O
or
Ar
Ar
8a-k
7a-m
H2N
H2N
c
O
O
O
Ar
O
Ar
or
1a-m
2a-k
N
H
N
H
Br
Br
2a, 4a, 6a, 8a; Ar=C6H5
1a, 3a, 5a, 7a; Ar=C6H5
2b, 4b, 6b, 8b; Ar=4-OMe-C6H4
2c, 4c, 6c, 8c; Ar=3-OMe-C6H4
2d, 4d, 6d, 8d; Ar=2-OMe-C6H4
2e, 4e, 6e, 8e; Ar=2,3-(OMe)2-C6H4
2f, 4f, 6f, 8f; Ar=3,4-(OMe)2-C6H4
2g, 4g, 6g, 8g; Ar=2,4-(OMe)2-C6H4
2h, 4h, 6h, 8h; Ar=3,5-(OMe)2-C6H4
2i, 4i, 6i, 8i; Ar=3,4,5-(OMe)3-C6H3
2j, 4j, 6j, 8j; Ar=2,3,4-(OMe)3-C6H3
2k, 4k, 6k, 8k; Ar=4-N(CH3)2-C6H4
1b, 3b, 5b, 7b; Ar=4-F-C6H4
1c, 3c, 5c, 7c; Ar=4-Cl-C6H4
1d, 3d, 5d, 7d; Ar=3,4-Cl2-C6H4
1e, 3e, 5e, 7e; Ar=4-Br-C6H4
1f, 3f, 5f, 7f ; Ar=4-I-C6H4
1g, 3g, 5g, 7g; Ar=4-Me-C6H4
1h, 3h, 5h, 7h; Ar=4-OMe-C6H4
1i, 3i, 5i, 7i; Ar=3-OMe-C6H4
1j, 3j, 5j, 7j; Ar=3,4,5-(OMe)3-C6H3
1k, 3k, 5k, 7k; Ar=4-OEt-C6H4
1l, 3l, 5l, 7l; Ar=1-naphtyl
1m, 3m, 5m, 7m ; Ar=2-thienyl
Scheme 1. Reagents and conditions: (a) 50% aqueous NaOH solution, p-NO2C6H4CHO for 3 or p-NO2C6H4COCH3 for 4, EtOH, rt, 18 h; (b) Fe, HCl 37% in water, EtOH, reflux, 3 h;
(c) -bromoacrylic acid, EDCI, HOBt, DMF, 18 h, rt.
a
cell lines, respectively. The bioisosteric replacement of phenyl (1a)
with a thiophene ring (1m) greatly increased activity, with IC50 val-
p-methoxy (1h) group caused only a minor improvement in anti-
proliferative activity. Moreover, a twofold reduction in activity
was observed by shifting the methoxy group from the para to
the meta position (1i). Replacement of the p-MeO group (1h)
with a p-EtO moiety (1k) caused a twofold increase in potency,
while the o,m,p-trimethoxy derivative 1j was only slightly more
active than 1h.
In the case of the second series (Table 2), compounds 2b–j, with
methoxy substituents on the phenyl ring, had antiproliferative
activity that, in general, was comparable with that of the unsubsti-
tuted 2a. Similarly, the antiproliferative activity of the N,N0-
dimethylamino derivative 2k was not greatly different from that
of the unsubstituted 2a.
ues 0.25–0.73
lines. A positive effect was also observed for the 1-naphthyl deriv-
ative 1l, which had IC50 values of 1.1–3.1 M, as compared with 1a.
lM versus 3.1–5.0 lM against the five tumor cell
l
The data presented in Tables 1 and 2 demonstrate effects of dif-
ferent substituents on the phenyl rings linked to the carbonyl and
at the b-position of the enone system, respectively. With com-
pounds 1a–k (Table 1), the substitution pattern significantly af-
fected potency. The introduction of either electron-releasing
(ERG) or electron-withdrawing (EWG) substituents (derivatives
1b–k) enhanced antiproliferative activity as compared with the
unsubstituted analogue 1a, and there was no clear difference be-
tween them. Ignoring the derivative with a p-I (1f), the greatest
enhancement of activity occurred with the bulkier substituents,
p-Br (1e) and p-EtO (1k).
A comparison of the antiproliferative activity of compounds
with the same substituent on the phenyl ring (1a vs 2a, 1h vs 2b,
1i vs 2c and 1j vs 2i), the ‘reversed’ a-bromoacryloylamido deriv-
Specifically with the para-halogen substituted 1bcef (Table 1),
activity increased in the following order: Br (1e) > Cl (1c), F
(1b) > I (1f). Insertion of a second chlorine atom, to yield the
m,p-dichloro derivative 1d, resulted in about a twofold reduction
in activity. Turning to the effects of an ERG on the phenyl
moiety, we found that replacement of p-methyl (1g) with a
atives 2a, 2b, 2c and 2i generally had higher IC50 values than did
the corresponding analogues 1a, 1h, 1i and 1j.
Chalcones are known to block cells in the G2-M phase of the cell
cycle, which is consistent with their ability to inhibit tubulin
assembly.2 We therefore determined whether these hybrid mole-
cules behaved in a similar manner.

R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2022–2028
2025
Table 1
assembly (IC50 > 40
l
M). However, all compounds had poor solu-
In vitro inhibitory effects of compounds 1a–m and 7a–m on the proliferation of
murine leukemia (L1210), murine mammary carcinoma (FM3A), human T-leukemia
(Molt/4 and CEM) and human cervix carcinoma (HeLa) cells
bility in the assay medium (all precipitated in the 40
2d showed relatively weak activity as a tubulin polymerization
inhibitor, with an IC50 value of 8.3 0.9 M, and, at 50 M, 2d
weakly inhibited the binding of 5
M [3H]colchicines to tubulin
(37 10% inhibition). These data make it unlikely that the antipro-
liferative activity of these hybrid compounds results from a direct
interaction with tubulin and that it is unlikely that they act as
microtubule depolymerizing agents.
To confirm this idea, we next examined the effects of the most
active compounds on the cell cycle by performing a flow cytomet-
ric analysis of K562 human chronic myelogenous leukemia cells,
which are usually employed by our research group to determine
the alteration of cell cycle parameters following exposure to anti-
tumor compounds.14 Cells were cultured for 72 h in the absence
or presence of each compound at the concentrations summarized
in Table 3, and the cells were then stained with propidium iodide
(Fig. 1, panels A and B). Unlike chalcones, which induced a substan-
tial recruitment of cells into the G2-M phase of the cell cycle, deriv-
atives 1ekm and 2abdej uniformly caused a decrease in the
proportion of cells in all phases of the cell cycle (G0–G1, S and
G2-M), with a proportionate increase in the number of apoptotic
cells in the sub-G1 phase region of the histogram. The absence of
the typical increase in G2-M cell number, together with the mini-
mal effect on tubulin assembly, makes it unlikely that microtubule
disruption underlies the potent apoptotic effect caused by these
hybrid compounds.
lM assays).
l
l
Compound
IC50
(
l
M)a
l
L1210
FM3A
Molt4
CEM
HeLa
7a
7b
7c
7d
7e
7f
7g
7h
7i
88 12
123 92
111 15
146 96
185
178 30
10
12
7.8 1.9
13
10
7.5 0.8
12
19 13
20 15
1.5 0.3
11
12
13
2
2
34
39
21 17
15
14
16
31
37
26 24
2.5 0.2
15
22 14
1
0
36
40
43
31
49
1
3
4
1
9
2
26
90 12
50
1
33
0
0
0
0
1
1
3
8
120
110
107 25
282 44
3
9
4
58 11
40
98 64
33
1.9 0.2
49 12
160 15
38 15
7
1
43
36
55
41
0
6
0
4
6
7j
7k
7l
14
21
32
87
3
4
9
7
1
2
2
0
41
18
15
4
4
3
7m
1a
1b
1c
1d
1e
1f
1g
1h
1i
123 20
3.9 1.5
1.2 0.3
1.3 0.0
3.2 1.1
1.1 0.8
8.3 2.6
2.1 0.1
1.2 0.5
2.7 1.2
0.95 0.77
0.68 0.20
2.4 0.8
0.52 0.06
27 19
5.0 1.7
1.6 0.9
1.1 0.2
2.1 0.1
0.62 0.20
3.4 0.6
1.9 0.4
0.98 0.96
2.1 1.5
0.91 0.68
0.24 0.05
1.1 0.1
3.1 1.4
4.0 2.5
1.1 0.9
0.95 0.62
1.7 0.1
0.99 0.31
4.0 2.4
1.8 0.2
1.4 1.2
2.3 1.7
1.1 0.8
0.75 0.20
3.1 1.3
0.73 0.25
3.9 1.5
1.4 0.1
1.6 0.2
3.2 1.4
1.0 0.0
7.7 3.4
2.1 0.7
1.4 0.2
0.70 0.10
1.1 0.6
0.75 0.05
2.0 0.5
0.34 0.12
0.66 0.02
0.81 0.33
1.8 0.2
0.68 0.24
2.8 0.7
1.7 0.1
1.3 1.2
0.85 0.06
0.85 0.30
0.61 0.17
2.1 0.6
1j
1k
1l
1m
0.25 0.11
0.55 0.18
The induction of apoptosis was confirmed by the annexin V
test15 with compounds 2j and 2k (Fig. 1, panels C and D). The in-
crease in annexin V-positive cells demonstrated activation of the
apoptotic pathway by these compounds. Figure 2C shows the
extensive binding of annexin V to 2j-treated cells, and Figure 2D
shows how time and concentration of 2k affect the proportion of
cells that become positive for the marker. Similar observations
were made with the other most active compounds.
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.
Table 2
In vitro inhibitory effects of compounds 2a–k and 8a–k on the proliferation of murine
leukemia (L1210), murine mammary carcinoma (FM3A), human T-leukemia (Molt/4
and CEM) and human cervix carcinoma (HeLa) cells
Impairment of mitochondrial function, is an early event in the
executory phase of programmed cell death in different cell types,
and it occurs as the consequence of a preliminary reduction of
Compound
IC50
(
l
M)a
L1210
FM3A
Molt4
CEM
HeLa
the mitochondrial transmembrane potential (
mt disruption results from an opening of mitochondrial perme-
Dw
mt).16,17 Early
8a
8b
8c
8d
8e
8f
8g
8h
8i
14
18
22 15
12 1.0
13
25 17
3
5
34
35
45 10
25
29
35
43
12
1
6
7.9 0.8
8.6 0.4
21 11
8.3 0.9
8.5 0.3
8.6 0.2
20 11
8.5 1.5
7.7 0.6
8.7 0.3
19 14
7.8 0.3
6.8 0.0
7.8 0.1
D
w
14
3
ability transition pores, and this permeability transition triggers
the release of apoptogenic factors, such as apoptosis inducing fac-
tor and cytochrome c, which in turn lead to later apoptotic
events.16,17
23 15
5
2
3
0
1
6.3 1.0
7.6 1.0
9.3 0.2
8
We used the lipophilic cation 5,50,6,60-tetrachlo-1,10,3,30-tetra-
ethylbenzimidazol-carbocyanine (JC-1) to monitor changes in
34 12
18
7
22
6
6.0 4.2
7.8 0.1
25 16
7.5 0.1
5.6 1.2
5.7 0.5
2.3 0.3
9.7 0.6
7.9 0.7
6.9 0.8
8.2 0.9
8.8 0.0
29
8j
3
10
1
8k
2a
2b
2c
2d
2e
2f
2g
2h
2i
67 33
96 32
9.9 1.0
20
3
28 10
0.88 0.59
0.90 0.63
1.1 0.6
1.7 0.3
1.1 0.9
1.3 0.9
0.52 0.05
2.7 0.7
1.5 0.9
0.63 0.12
1.9 1.6
0.69 0.68
1.0 0.1
1.8 0.5
0.37 0.33
0.55 0.31
1.2 1.1
1.2 0.9
2.0 0.1
1.8 1.9
0.53 0.28
2.8 1.5
0.50 0.17
0.61 0.55
1.1 0.1
0.24 0.20
0.49 0.07
1.8 0.2
1.5 0.8
1.5 0.0
0.28 0.08
0.68 0.26
1.3 0.3
1.5 0.0
1.1 0.8
1.8 0.2
0.58 0.53
0.71 0.53
1.6 0.2
1.1 0.3
2.2 0.0
0.33 0.1
0.84 0.2
1.5 0.2
0.96 0.34
0.31 0.00
0.50 0.00
1.3 0.2
0.38 0.03
0.77 0.36
1.1 1.0
Table 3
Cell-cycle distribution of K562 cells after 72 h of treatment with compounds 1e, 1k,
1m, 2a–b, 2d–e and 2j
a
Compound
IC75
(lM)
Cell cycle percentage (%)
Sub-G1b
G0–G1
S
G2-M
1.7 0.3
Control
1e
1k
1m
2a
2b
2d
2e
2j
NM
10.8
20.4
52.3
42.8
47.6
68.2
83.4
48.4
57.8
39.8
35.9
16.5
17.0
23.0
11.0
6.4
30.7
27.5
19.6
23.2
16.4
11.6
6.3
18.7
16.2
11.6
17.0
13.0
9.2
3.9
14.1
13.6
0.42 0.0
0.51 0.0
0.61 0.07
0.84 0.18
0.85 0.21
0.82 0.13
2.77 0.51
1.14 0.21
2.94 1.21
0.64 0.15
0.71 0.18
2j
2k
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.
22.3
13.7
15.2
14.9
NM: not meaningful.
The most active compounds (1ekm and 2abdej) were evaluated
for their inhibitory effects on tubulin polymerization and on the
binding of [3H]colchicine to tubulin.13 With the exception of 2d,
all tested compounds were ineffective as inhibitors of tubulin
a
IC75 = compound concentration required to inhibit tumor cell proliferation by
75%. Data are expressed as the mean SE from the dose-response curves of at least
three independent experiments.
b
Apoptosis area.

2026
R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2022–2028
A
B
C
A
Ctr
1.25 μM
24h
48h
D
100
80
60
40
20
0
24 h
48 h
JC-1 monomers
0
1
2
3
M
Concentration (μ
)
60
40
20
0
B
Figure 1. Representative histograms of flow cytometry data of untreated control
K562 cells (panel A) or K562 cells treated for 3 days with 0.7 M 2j (IC75, see Table 3)
(panel B). After 3 days of incubation the cells were labelled with propidium iodide
and analyzed by flow cytometry as described in Materials and Methods. Panel C.
Representative histograms of untreated K562 cells (black line) and K562 cells
24 h
48 h
l
treated for 3 days with 0.2 lM 2j (IC50) (green line). After 48 h cells were stained
with annexin V-FITC and analyzed by flow cytometry. Panel D. Effect of 2k
concentration on the proportion of annexin V-positive cells after treatment for 24
and 48 h.
0
1
2
3
Concentration (μM)
D
wmt induced by 1k. The method is based on the ability of this
fluorescent probe to enter selectively into the mitochondria, and
its color changes reversibly from green to orange as membrane po-
tential increases.18 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.
K562 cells were treated with compound 1k for 24 and 48 h at
different concentrations. As shown in Figure 2 (panels A and B),
compound 1k induces substantial mitochondrial depolarization
in a time- and concentration-dependent manner. The disruption
30
C
24 h
48 h
20
10
0
0
1
2
3
Concentration (μΜ)
of the
Dwmt is associated with the appearance of sub-G1 cells
and with the marked increase in the percentage of annexin V-posi-
tive cells.
Figure 2. Induction of loss of
treatment with 1k at different concentrations. Panel
histograms of K562 cells incubated with and without 1.25
the fluorescent probe JC-1 after 24 and 48 h of treatment. The horizontal axis shows
fluorescence intensity of JC-1 monomers, and the vertical axis shows fluorescence
Dwmt and production of ROS in K562 cells after
A
shows representative
lM 1k and stained with
Mitochondrial membrane depolarization is associated with
mitochondrial production of reactive oxygen species (ROS).19,20
To investigate the effects of 1k on the production of oxygen species
during apoptosis, we utilized the fluorescence indicator hydroethi-
dine (HE), which fluoresces if reactive oxygen species are gener-
ated.21 As shown in Figure 2 (panel C), there was an increase in
cells producing ROS that closely paralleled the increase in cells
of JC-1 aggregates. Panel B shows the percentage of cells with low
Dwmt following
treatment with different concentrations of 1k for the indicated times. Panel C. K562
cells were treated with the indicated concentrations of 1k and at 24 and 48 h cells
were harvested and incubated with HE. Analysis of intracellular fluorescence was
conducted by flow cytometry. The data are represented as percentage of ethidium
positive cells and expressed as mean SEM of three independent experiments.
with low
Dwmt, a function of both the concentration of 1k 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
of mitochondrial permeability transition.22 The dye interacts stoi-
chiometrically with intact, non-oxidized cardiolipin. Somewhat
unexpectedly, cells did not show reduction in NAO fluorescence,
but rather, a marked increase, especially after 48 h of treatment
with 1k (Fig. 3 panels A and B). This effect suggests an increase
in cardiolipin content as a consequence of increased mitochondrial
mass. This has been observed in some tumor cell lines following
treatment with herbimycin A,23 genistein,24 and the acronicyne
derivative S23906-125 and following oxidative stress.26

R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2022–2028
2027
against five different cancer cell lines, their conversion into the cor-
responding -bromoacryloylamido derivatives 1a–m and 2a–k
A
B
a
was accompanied by a 10–100-fold increase in potency. More
noteworthy was that compounds 1k (52%), 2b (68%), 2d (83%)
and 2j (57%) induced over half the cells to enter a sub-G1 popula-
tion as compared with untreated control cells (11%). As demon-
strated with 1k, their mechanism of action appears to induce
apoptosis mediated by the involvement of mitochondria and by
the activation of caspase-3. Thus, the mitochondrial apoptotic
pathway plays a major role, in generation of the sub-G1 cell popu-
lation. Further studies to clarify additional details of the molecular
mechanism of action of these compounds and the selectivity to in-
hibit the growth of cancer cells are underway. On the other hand,
the most active compounds should be analyzed for effects on nor-
mal primary normal human cells of different hystotype, in order to
determine potential therapeutic windows supporting further stud-
ies on experimental tumor-bearing animals finalized to verify pos-
sible in vivo anticancer activity.
NAO
100
80
60
40
20
0
24 h
48 h
Acknowledgments
0
1
2
3
Financial support was provided by GOA (Krediet No. 05/19) of
the K.U. Leuven. The technical assistance of Mrs. Lizette van Berc-
kelaer was gratefully acknowledged. R.G. and C.L.C. are funded by
AIRC and University of Granada, respectively.
Concentration (μΜ)
Figure 3. Increase of mitochondrial mass in K562 cells treated with compound 1k.
Panel A. Representative histograms of cells incubated for 24 and 48 h in the
presence of 1k (2.5 lM) and stained with the fluorescent probe NAO. Black
line = controls, red line = 1k. Panel B. K562 cells were treated as above and the
relative NAO intensity was analysed by flow cytometry. The results are expressed as
percentage of the fluorescence intensity with respect to the untreated control. Data
expressed as mean SEM of three independent experiments.
Supplementary data
Detailed biological protocols, synthesis and spectroscopic data
for compounds
a-bromoacryloylamido chalcones 1a–m and 2a–
Several caspases have been shown to be key executioners of
apoptosis mediated by various inducers.27 Caspase-3, in particular,
is essential to the propagation of the apoptotic signal after expo-
sure of cells to many DNA-damaging agents and other anticancer
drugs.27,28 We therefore determined whether caspase-3 is involved
in the apoptosis induced by compound 1k. We used a monoclonal
antibody specifically for the active fragment of caspase-3. As
k. General procedures for the synthesis of nitrochalcones 5a–m/
6a–k and aminochalcones 7a–m/8a–k are available. Supplemen-
tary data associated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2009.02.038.
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