Synthetic chalcones, flavanones, and flavones as antitumoral agents: Biological evaluation and structure-activity relationships

Article abstract of DOI:10.1016/j.bmc.2007.03.031

A series of synthetic chalcones, flavanones, and flavones has been synthesized and evaluated for antitumor activity against the human kidney carcinoma cells TK-10, human mammary adenocarcinoma cells MCF-7 (estrogen receptor-positive), and human colon adenocarcinoma cells HT-29. The most active series is the chalcone ones with the best results against TK-10 and HT-29 cells. Fourteen out of 53 analyzed compounds resulted very active against at least two of the studied tumoral cells. Alkaline single cell gel electrophoresis, comet assay, was performed as a study of the chromosomal aberrations promoted by the compounds on normal cells. Four active and two inactive chalcones were studied in the comet assay against normal human kidney cells (HK-2). A structure-activity relationship analysis of these compounds was performed and for 4- and 3,4-disubstituted derivatives a quantitative correlation was obtained in the case of anti-HT-29 activity.

Full text of DOI:10.1016/j.bmc.2007.03.031

Bioorganic & Medicinal Chemistry 15 (2007) 3356–3367
Synthetic chalcones, flavanones, and flavones as antitumoral
agents: Biological evaluation and structure–activity relationships
Mauricio Cabrera,a Macarena Simoens,a Gabriela Falchi,a M. Laura Lavaggi,a
Oscar E. Piro,b Eduardo E. Castellano,c Anabel Vidal,a Amaia Azqueta,d
d
d
a
a
´
´
Antonio Monge, Adela Lopez de Cerain, Gabriel Sagrera, Gustavo Seoane,
a,
Hugo Cerecettoa, and Mercedes Gonzalez
*
*
´
aDepartamento de Quı´mica Orga´nica, Facultad de Quı´mica-Facultad de Ciencias,
Universidad de la Repu´blica, 11400 Montevideo, Uruguay
bDepartamento de Fı´sica, Facultad de Ciencias Exactas, Universidad Nacional de La Plata-Instituto IFLP(CONICET),
C.C. 67, 1900 La Plata, Argentina
cInstituto de Fı´sica de Sa˜o Carlos, Universidade de Sa˜o Paulo, C.P. 369, 13560 Sa˜o Carlos, SP, Brazil
dCentro de Investigaciones en Farmacobiologı´a Aplicada (CIFA), Universidad de Navarra, Irunlarrea s/n, 31080 Pamplona, Spain
Received 29 December 2006; revised 5 March 2007; accepted 8 March 2007
Available online 14 March 2007
Abstract—A series of synthetic chalcones, flavanones, and flavones has been synthesized and evaluated for antitumor activity
against the human kidney carcinoma cells TK-10, human mammary adenocarcinoma cells MCF-7 (estrogen receptor-positive),
and human colon adenocarcinoma cells HT-29. The most active series is the chalcone ones with the best results against TK-10
and HT-29 cells. Fourteen out of 53 analyzed compounds resulted very active against at least two of the studied tumoral cells. Alka-
line single cell gel electrophoresis, comet assay, was performed as a study of the chromosomal aberrations promoted by the com-
pounds on normal cells. Four active and two inactive chalcones were studied in the comet assay against normal human kidney cells
(HK-2). A structure–activity relationship analysis of these compounds was performed and for 4- and 3,4-disubstituted derivatives a
quantitative correlation was obtained in the case of anti-HT-29 activity.
ꢀ 2007 Published by Elsevier Ltd.
1. Introduction
any adverse health effect. In addition, the interaction of
dietary flavonoids with the gut has numerous implications
for human health and flavonoids in the diet may act as
chemopreventive agents against the development of can-
cer.8 Apart from their cancer chemopreventive efficacy,
such flavonoids could be developed as an alternative med-
icine to get the beneficial effects in combination treatment
by reducing the dose and associated systemic toxicity of
chemotherapeutic agents for similar efficacy.9,10
Flavonoids are an extensive group of compounds occur-
ring in plants.1,2 They are prominent plant secondary
metabolites that have been found in dietary compo-
nents, including fruits, vegetables, olive oil, tea, and
red wine.2 Besides their physiological role in plants, they
have shown to possess anxiolytic,3 anti-inflammatory,
antiviral,4 antiprotozoal,5 and anticarcinogenic activi-
ties.6 With reference to the antitumor or related activi-
ties, it was described for flavonoids antimitotic
activity6f and/or inhibition of some enzymes7 like
cyclin-dependent kinase, several protein-tyrosine kinases,
aromatase, topoisomerase, or protein kinase C.
As part of our continuous search for potential anticancer
drug candidates,11 and based on the diverse biological
activities of flavonoid derivatives, in the present study
we have synthesized a series of unnatural flavonoids and
examined their in vitro cytotoxic activity on human tumor
cell lines, namely kidney carcinoma cells TK-10, human
mammary adenocarcinoma cells MCF-7 (estrogen recep-
tor-positive), and human colon adenocarcinoma cells
HT-29. In addition, to obtain some preliminary insights
into the effect on the normal cells’ chromosomal aberra-
tions promoted by these compounds, comet assay was
It has been observed that even a high intake of plant-
based dietary flavonoids is safe and not associated with
Keywords: Flavonoids; Antitumoral; Comet assay; QSAR.
*
Corresponding authors. Tel.: +598 2 5258618x216; fax: +598 2 525 07
49 (H.C.); e-mail addresses: hcerecet@fq.edu.uy, megonzal@fq.edu.uy
0968-0896/$ - see front matter ꢀ 2007 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2007.03.031

M. Cabrera et al. / Bioorg. Med. Chem. 15 (2007) 3356–3367
3357
performed.12 A structure–activity relationship analysis
was also done in order to understand the structural
requirements for optimum activity.
The purity was established by TLC and microanalysis.
The stereochemistry around olefinic carbon–carbon bond
was established using the corresponding H NMR cou-
1
pling constant. Single crystals of derivative 44 adequate
for structural X-ray diffraction studies15 were obtained
by slow evaporation from an Et2O solution. Figure 1
shows the ORTEP molecular drawing of derivative 44.
2. Methods and results
2.1. Synthesis
2.2. Biological characterization
The synthesis of chalcone derivatives substituted in the
ring B (Scheme 1) 1–5, 8–13, 18, 21, 24, 25, and 28 was
carried out according to procedures previously
described.10,13 Briefly, we used the aldolic condensation
with NaOH as base. In this process, acetophenone or
hydroxyacetophenone was reacted with the correspond-
ing aldehyde. In order to modify the lipophilic behavior
and volume of chalcones, derivatives 6, 7, 14–17, 19, 22,
23, 26, 27, 29, and 30 were prepared using the same meth-
odology. During the synthesis of derivative 19, aldol 20
was isolated as a secondary product. Further, compounds
31–33 were prepared as ring B modified derivatives.
Derivative 34 was isolated during derivative 15 synthesis
and was used to study the influence of the conjugated en-
one, Michael system, in the activity. Also, chalcone deriv-
atives with special substitutions in the ring A were
developed using adequately substituted hydroxyacetoph-
enone (Scheme 2). Ring B substituted flavanones and flav-
ones were prepared using traditional methodologies
(Scheme 3) from the correspondinghydroxychalcones.6,14
All new compounds were characterized by NMR (1H-,
13C-, COSY, and HETCOR experiments), IR, and MS.
2.2.1. Cytotoxicity studies on tumoral cells. The chalcones,
flavanones, flavones, and related obtained products were
tested at 100 lM doses in aerobic conditions against TK-
10 (human kidney carcinoma) (NCI), MCF-7 (human
mammary adenocarcinoma) (ATCC HTB-38), and HT-
29 (human colon adenocarcinoma) (ATCC HTB-38)
tumor lines, according to previously described proce-
dures.16 Survival percentage (SP) was determined and
the results are shown in Figure 2 and collected in Table
1. The IC50 concentration (50% inhibitory concentration)
was assessed for the most interesting derivatives (Table 2).
2.2.2. Comet assay. The effect of these compounds against
DNA of HK-2 cells (Human kidney cells) was evaluated
through the alkaline single cell gel electrophoresis assay.
The active derivatives 5, 8, 18, and 33, and the less active
derivatives 12 and 28 were submitted to the comet assay at
20 lM, except for compound 33at aconcentration near to
the corresponding IC50,HK-2 (see Table 2). Comets were
scored visually, and one hundred nucleoids per slide were
classified according to the intensity of fluorescence in the
R
-R= -H,
4-Cl,
1
2
3
4
5
6
7
R
4-Br,
2-Br,
4-OCH3,
4-SCH3,
4-NHCOCH3,
A
B
CHO
NaOH / EtOH /r.t.
O
O
R1
-R1= -H
-R2= -H,
4-NO2,
-R1= -OCH3
-R2= 4-OCH3,
6-OCH3,
R2
8
9
24
25
26
R2
OH
O
OH
O
2-NO2,
4-Cl,
4-Br,
2-Br,
4-OBn,
4-OH,
4-On-Hexyl, 16
4-On-Nonyl, 17
4-OCH3,
4-SCH3,
4-N(CH3)2, 21
4-NHCOCH3,22
4-NH2,
10
11
12
13
14
15
4-OBn,
A
B
-R1= -OBn, -R2= 4-OCH3, 27
CHO
R1
-R1,-R2= -OCH2O-,
-R1= -I
28
NaOH / EtOH / r.t.
-R2= 4-OCH3,
4-OBn,
29
30
i)
OH
SCH3
18
19
20
O
OH
i) H2 / 5 % Pd-C / r.t.
ii) HCl / EtOH / reflux-2 h
ii)
23
OBn
OH
O
OH
O
OH
O
-Ar= -CH=CH-Ph, 31
-2-furyl,
-2-pyridinyl, 33
CHO
Ar
Ar
32
NaOH / EtOH / r.t.
34
Scheme 1. General procedures used to prepare the desired chalcone derivatives substituted in ring B.

3358
M. Cabrera et al. / Bioorg. Med. Chem. 15 (2007) 3356–3367
R
R
H3CO
OH
H3CO
OH
-R= 2-Br,
4-OCH3,
35
36
CHO
3,4-di-OCH3, 37
NaOH / EtOH / r.t.
OCH3 O
OCH3 O
OH
R
R4O
OH
R4O
R
-R4= -Bn,
-R= -H,
38
39
40
-R= 2-Br,
-R= 4-OBn,
CHO
-R4= -n-butyl, -R= 4-On-butyl, 41
NaOH / EtOH / r.t.
O
O
Scheme 2. General procedures used to prepare the desired chalcone derivatives modified in ring A.
-R1= -H, -R2= -H, -R3= -H,
-R1= -H, -R2= -Br, -R3= -H,
-R1= -H, -R2= -H, -R3= -Br,
-R1= -H, -R2= -OBn, -R3= -H,
-R1= -H, -R2= -OMe, -R3= -H,
42
43
44
45
46
R2
R1
O
O
AcOH / reflux
R3
-R1= -OMe, -R2= -OMe, -R3= -H, 47
-R1, -R2= -OCH2O-, -R3= -H,
48
OH
O
R2
R1
R2
R1
-R1= -H, -R2= -H, -R3= -H,
-R1= -H, -R2= -Cl, -R3= -H,
-R1= -H, -R2= -Br, -R3= -H,
-R1= -H, -R2= -H, -R3= -Br,
-R1= -H, -R2= -OMe, -R3= -H,
49
50
51
52
53
R3
O
O
I2 / DMSO
reflux
R3
Scheme 3. General procedures used to prepare the desired flavanone and flavone derivatives substituted in ring B.
19, 21–30, and 42–53. The different cell-survival percent-
ages (SP) at 100 lM were used as the biological activity.
log10 (SP) was used as the dependent variable in the lin-
earization procedure. In the equations n represents the
number of data points, r2 is the correlation coefficient,
s is the standard deviation of the regression equation,
and F value is related to the F-statistic analysis (Fischer
test). As the independent variables were employed the
previously tabulated substituent constants related to
the electronic, the lipophilic, and the steric properties
(Hammett constants rm, rp, lipophilic constant p, and
molar refractivity MR).18 When the ring
B is
disubstituted, the sum of the individual parameters
was used for lipophilicity ( pÞ and steric descriptors
P
P
(
MR). Some indicator variables were used in order
Figure 1. Molecularplotofderivative44,showingthelabelingofthenon-
H atoms and their displacement ellipsoids at the 50% probability level.
to indicate the presence of OH in ring A (IOH take values
1 if it is present or 0 otherwise), the presence of a flava-
none or a flavone (IC take values 1 if a cycle is present or
0 otherwise), and presence of ortho substituent in ring B
(IO take values 1 if it is present or 0 otherwise). The
capability of the ring B substituent to participate in a
hydrogen bond was taken into account including indica-
tor variables IHBD and IHBA that take values 1 if the
group acts donating or accepting hydrogen bond or 0
otherwise.
comet tail.17 Images were given a value of 0–4 (from
undamaged to maximally damaged). DNA damage there-
fore extends from 0 to 400 arbitrary units and covers a
range of strand break frequencies (estimated using
X-ray calibration) of 0–3 breaks per 109 Da. The total
score was calculated by the following equation: (percent-
age of cells in class 0 · 0) + (percentage of cells in class
1 · 1) + (percentage of cells in class 2 · 2) + (percentage
of cells in class 3 · 3) + (percentage of cells in class
4 · 4). Results are shown in Table 3.
One-variable and multivariable regressions between the
different activities and the physicochemical descriptors
were studied. No significative statistical correlation be-
tween anti-TK-10 and MCF-7 activities and the studied
descriptors was obtained. However, a modest correla-
tion was obtained between anti-HT-29 activities and
2.3. Structure–activity relationships
First, 41 ring-B substituted derivatives were used in the
structure–activity relationship studies, namely 1–16, 18,

M. Cabrera et al. / Bioorg. Med. Chem. 15 (2007) 3356–3367
3359
Chalcones
Chalcones
Hydroxychalcones
Flavanones
Flavones
Hydroxychalcones
Flavanones
Flavones
MCF-7 cells
TK-10 cells
100
100
50
0
50
0
0
10
20
30
40
50
0
10
20
30
40
50
Compounds
Compounds
Chalcones
Hydroxychalcones
Flavanones
Flavones
HT-29 cells
100
50
0
0
10
20
30
40
50
Compounds
Figure 2. Survival percentages in the different cell-lines of the different series of flavonoids.
some of the studied descriptors (Eq. 1). This modest result
log10ðSPHT-29Þ ¼ 0:85ðꢀ0:20Þ ꢁ 0:37ðꢀ0:19ÞIO
could be improved, in the statistical parameters, when
ortho-ring B substituted derivatives were excluded from
the analysis. So, Equation 2 was obtained when deriva-
tives 1–3, 5–12, 14–16, 18–19, 21–24, 26–30, 42, 43, 45–
51, and 53 were studied. Besides, the correlation matrix
for the used physicochemical descriptors was performed
and cross-correlations between the descriptors were not
obtained. Thus, these parameters are orthogonal, allow-
ing its safely use in the multilinear regression relation-
ship.19 Clearly, a lack of anti-HT-29 activity, increase
of survival percentage, is evidenced when the chalcones
are hydroxyl-substituted, in the cases of flavanones and
flavones, and with the increment of compounds’ volumes.
Further, the substituents’ capabilities to act as hydrogen
bond donor increase the activity of the compounds. Also,
þ 0:66ðꢀ0:18ÞIOH þ 0:88ðꢀ0:19ÞIC
P
þ 0:32ðꢀ0:20Þð MRÞ ꢁ 0:05ðꢀ0:04Þ
ꢂ ð MRÞ ꢁ 0:26ðꢀ0:22ÞIHBD
P
2
n ¼ 41; r ¼ 0:708; radj ¼ 0:640;
s ¼ 0:402; F ¼ 5:52; p ¼ 0:0005
ð1Þ
log10ðSPHT-29Þ ¼ 0:77ðꢀ0:16Þ þ 0:83ðꢀ0:15ÞIOH
þ 0:97ðꢀ0:16ÞIC þ 0:27ðꢀ0:15Þ
P
P
P
ꢂ ð MRÞ ꢁ 0:05ðꢀ0:03Þ
lipophilicity (as pÞ instead of steric descriptor ( MR)
was included in the study however the statistical quality
resulted worse than the one obtained in Equation 1 or
P
2
ꢂ ð MRÞ ꢁ 0:27ðꢀ0:17ÞIHBD
n ¼ 34; r ¼ 0:820; radj ¼ 0:783;
s ¼ 0:298; F ¼ 11:48; p < 0:0001
P
P
2. Descriptors p and MR were not studied together
because they are not orthogonals.
ð2Þ

3360
M. Cabrera et al. / Bioorg. Med. Chem. 15 (2007) 3356–3367
Table 1. Tumor cell cytotoxicity of flavonoids
Table 2. IC50 for the most interesting chalcones
Survival percentagea
Compound
a,b
IC50 (lM)
Compound
TK-10
MCF-7
HT-29
TK-10
MCF-7
HT-29
HK-2
1
2
2
2
19
56
44
32
30
47
24
3
5
9
1
5
42.0
53.0
79.0
63.0
90.0
78.0
—
70.0
54.0
85.0
45.0
—
30.5
23.2
24.4
—
3
90
2
28
26
2
8
c
4
11
18
24
33
12
28
51
—
5
2
67.0
90.0
12.2
—
—
23.7
—
6
0
46
3
95.0
6.7
—
—
7
5
13.8
—
7.9
8
0
14
94
100
25
71
0
33.8
11.6
48.0
9
34
100
3
43
100
41
43
100
56
3
—
—
—
—
—
—
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
a IC50, concentration that produces 50% inhibitory effect.
b The results are means of two different experiments with a SD less
60
3
than 10% in all cases.
c —, not studied.
64
5
59
9
62
77
4
76
86
10
75
90
87
79
43
1
97
98
97
76
100
65
87
98
70
92
100
100
69
92
100
Table 3. DNA strand breakage (arbitrary units) promoted by
chalcones against HK-2 cells
91
100
76
89
91
2
Compound
Number of comets in each
categorya,b
Total scorec
0
1
2
3
4
5
37
53
58
53
47
13
47
12
56
42
38
42
50
64
50
68
3
2
1
2
0
2
2
3
2
2
6
1
1
2
1
0
1
1
4
1
0
75
56
56
90
100
50
41
78
100
48
1
45
97
100
79
96
81
84
60
4
8
12
49
56
18
28
60
33
C (ꢁ)d
13
1
124
59
b
—
C (+)e
19
107
40
15
a Compounds were dosed at 20 lM.
b The results are means of two different experiments with a SD less
than 10% in all cases.
95
53
71
100
72
65
92
79
79
16
54
78
36
69
100
87
74
94
69
82
89
82
60
89
74
74
100
81
54
26
12
76
2
100
87
c According to the calculus the maximum score is 400.
d C (ꢁ), control negative, DMSO.
—
—
e C (+), control positive, HK-2 cells were treated with 50 lM hydrogen
peroxide during 15 minutes on ice.
99
80
100
99
cellular lines was observed. Fourteen analyzed com-
pounds were very cytotoxic at least against two of the
studied cells (derivatives 1, 2, 4–8, 11, 13, 15, 18, 24,
33, and 46) and clear structure–activity dependence
was observed. Thus, chalcones resulted more cytotoxic
than flavanones and flavones (compare activities of chal-
cones 5 and hydroxychalcone 18 to flavanone 46 and fla-
vone 53). Flavanones’ activities could be the result of
metabolic transformation into the corresponding
hydroxychalcones however in some case could be the
result of the own flavanone activity (compare activities
of chalcone 12 and flavanone 43). In general, in the
same-substituted series, activity decreases in the order
chalcones > flavanones > flavones (i.e., compare activi-
ties of derivatives 8, 42, and 49). The most active deriv-
ative, namely 33 (see Table 2), belongs to the
hydroxychalcone series.
69
61
44
69
100
68
81
100
57
100
73
36
100
100
90
100
100
61
100
a The results are the means of two different experiments with a SD less
than 10% in all cases.
b —, not studied.
3. Discussion
Fifty-three flavonoids and related compounds were eval-
uated for their cytotoxic activities against three different
human tumoral cells, TK-10, MCF-7, and HT-29. In
general, no cytotoxic selectivity between the different
Other structural requirements for the activity could be
mentioned. The conjugated enone system results rele-
vant for the activity, that is, aldol 20 is inactive, whereas
its parent compounds, 6 and 19, are more active and

M. Cabrera et al. / Bioorg. Med. Chem. 15 (2007) 3356–3367
3361
Table 4. Druglikeness properties of some selected derivatives accord-
ing to Osiris Property Explorer tool21
reduced ketone 34 is less active than enone 14. An extra
conjugation does not improve the activity as it can be
observed with derivative 31 activity (compare with activ-
ity of compound 8). Further, ring A substitutions pro-
duce a lack of activities as can be seen in the activity
of compounds 35 and 39 when compared with the par-
ent compounds’ activities (derivatives 4 and 13).
Compound
Toxicity risksa
Mb Tc Id
(ꢁ) (+) (+) (+) ꢁ2.88
(ꢁ) (+) (+) (+) 1.49
Druglikeness Drug score
Re
1
2
0.25
0.36
0.20
0.40
0.34
0.26
0.44
0.53
0.41
0.36
0.35
0.71
0.80
0.66
0.77
0.37
0.59
0.36
0.53
4
(ꢁ) (+) (+) (+) ꢁ2.97
5
(ꢁ) (+) (+) (+)
(ꢁ) (+) (+) (+)
(ꢁ) (ꢁ) (+) (+)
0.83
0.84
2.08
On the other hand, the quantitative structure–activity
relationship studies demonstrate the same kind of results
mentioned above (see Eqs. 1 and 2). A valid statistical
correlation was only obtained for cytotoxicity against
HT-29 cells (Eq. 2). In this equation was evidenced the
relevance of the chalcone system and the volume and
hydrogen bond donor ability of the 4-substituent for
anti-HT-29 activity. The relevance of volume could be
evidenced for example in chalcones 11 and 12 where
the change of chlorine by bromine produces a lack of
anti-HT-29 activity or in the case of chalcones 14 and
30 where the inclusion of a 3-iodo substituent produces
a clear decrease of activity. Further the relevance of
hydrogen bond donor ability could be noted observing
the activities of chalcones 15 and 14 where the 4-OH-
substituted derivative is the most active of both.
6
7
8
(+) (+) (+) (+) ꢁ2.65
11
12
13
15
18
24
28
33
44
46
51
53
( ) (+) (+) (+)
1.58
(+) (+) (+) (+) ꢁ1.38
(+) (+) (+) (+) ꢁ2.85
( ) (+) (+) (ꢁ)
(+) (+) (+) (+)
(+) (+) (+) (+)
(+) (+) (+) (+)
(+) (+) (+) (+)
0.82
0.94
3.18
1.19
0.82
(+) (+) (+) (+) ꢁ2.48
(+) (+) (+) (+) ꢁ0.19
(+) (+) (+) (+) ꢁ2.05
(+) (+) (+) (+) ꢁ0.61
a Ranked according to, (+) no bad effect, ( )medium bad effect, (ꢁ)
bad effect.
b M, mutagenic effect.
c T, tumorigenic effect.
d I, irritant effect.
Additionally, the effect of selected compounds against
non-tumoral cells, HK-2, was studied. The compounds
do not produce relevant DNA strand breakage (see Ta-
ble 3) being derivative 33 the most harmful one. How-
ever, the concentration where 33 shows some comet
total score near to positive control is close to three time
superior than the corresponding IC50,HK-2 in normal
cells (see Table 2).
e R, reproductive effect.
These results suggested that the structural features of 33
might be useful in designing new cytotoxic agents.
Currently, there are many approaches that assess a com-
pound’s druglikeness partially based on topological
descriptors, fingerprints of Molecular Druglikeness
structure keys or other properties as clogP and molecu-
lar weights.20 In the Osiris Property Explorer21 the
occurrence frequency of each fragment is determined
within the collection of traded drugs and within the sup-
posedly non-drug-like collection of Fluka compounds.
In that case, positive values (0.1–10) point out that the
molecule contains predominantly the better fragments,
which are frequently present in commercial drugs. In
this work, we used the Osiris Properties Explorer tool
for calculating the Fragment Based Druglikeness of
the most interesting studied compounds, 1, 2, 4–8, 11–
13, 15, 18, 24, 28, 33, 44, 46, 51, and 53. This tool allows
us to predict the undesired effects, like mutagenic,
tumorigenic, irritant capacity or reproductive effects
(Table 4). In our case, this tool predicts as mutagenic
all the studied chalcones, 1–7, while the hydroxylchal-
cones, except 11 and 15, flavanones, and flavones are
predicted as lacking toxicity risks. Interestingly synthetic
derivative 33 (the most cytotoxic studied agent) presents
a good druglikeness value and one of the highest drug
scores which combines druglikeness, clogP, logS,
molecular weight, and toxicity risks in one value and
that may be used to judge the compound’s overall po-
tential to qualify for a drug.22 These toxic predictions
are in agreement to comet assays performed in this work
(see Table 3).
4. Conclusions
In conclusion, we have developed a wide series of unnat-
ural flavonoids in order to evaluate its antitumoral ef-
fects. The results presented above indicate that the
in vitro activity of these new compounds against some
tumoral cells and its potential use as chemopreventive
agents provide supporting evidence for further in vivo
studies as anticancer drugs.
5. Experimental
5.1. Chemistry
All starting materials were commercially available re-
search-grade chemicals and used without further purifica-
tion. Physicochemical and spectroscopical data of
derivatives 1, 8–10, 12, 14, 15, 18, 21, 24–29, 32, 36–
38,40, 42, 44–49, 51, and 53 are in full agreement with
those reported.6a,10,13a,14a,23 All solvents were dried and
distilled prior to use. All the reactions were carried out
in a nitrogen atmosphere. The typical work-up included
washing with brine and drying the organic layer with so-
dium sulfate before concentration in vacuo. Melting
points were determined using a Leitz Microscope Heating
Stage Model 350 apparatus or a Mettler FP82+FP80
apparatus and are uncorrected. Elemental analyses were
obtained from vacuum-dried samples (over phosphorus

3362
M. Cabrera et al. / Bioorg. Med. Chem. 15 (2007) 3356–3367
pentoxide at 3–4 mmHg, 24 h at room temperature) and
performed on a Fisons EA 1108 CHNS-O analyser. Infra-
red spectra were recorded on a Perkin-Elmer 1310 appa-
ratus, using potassium bromide tablets; the frequencies
HMBC) (CDCl3) d: 55.8, 114.9, 120.3, 128.1, 128.8, 129.0,
130.6, 132.9, 139.0, 145.1, 162.1, 191.0; MS m/z (abundance,
%): 238 (M+Å, 67), 207 (17), 179 (7), 130 (3), 103 (8), 77
(100); IR m 1713, 1659, 1217, 827, 781.
1
are expressed in cmꢁ1. H NMR spectra were recorded
on a Bruker DRX-400 (at 400 MHz) instrument, with tet-
ramethylsilane as the internal reference and in the indi-
cated solvent; the chemical shifts are reported in ppm.
J values are given in Hz. The 1H NMR and 13C NMR sig-
nals reported were obtained at 303 K. Mass spectra were
recorded on a Shimadzu GC-MS QP 1100 EX instrument,
using electronic impact at 70 eV.
5.2.5. (E)-3-(4-Methylthiophenyl)-1-phenyl-2-propen-1-one
(6). White-yellow solid, mp 78.6–80.4 ꢁC (found: C, 75.5;
H, 5.3. C16H14OS requires C, 75.6; H, 5.6); H NMR
(CDCl3) d: 2.54 (s, 3H), 7.29 (m, 2H), 7.50 (d, J = 15.5,
1H), 7.52 (m, 2H), 7.59 (m, 3H), 7.79 (d, J = 15.7, 1H),
8.02 (dd, J1 = 8.4, J2 = 1.4, 1H); 13C NMR (HMQC-
HMBC) (CDCl3) d: 15.6, 121.5, 126.5, 128.9, 129.0,
129.2, 131.9, 133.1, 138.8, 142.8, 144.7, 190.9; MS m/z
(abundance, %): 254 (M+Å, 100), 207 (60), 179 (20), 105
(25), 77 (57).
1
5.2. General procedure for the synthesis of chalcone
derivatives 1–14, 16–19, 21, 22, 24–33, and 35–41
A mixture of the corresponding acetophenone (1 equiv)
and the corresponding aldehyde (1 equiv) in anhydrous
EtOH (70 mL/23 mmol of acetophenone) was stirred at
room temperature during 5 min. Then, NaOH (3 equiv)
was added. The reaction mixture was stirred at room tem-
perature until aldehyde consumption. After that, HCl
(10%) was added until neutrality. In the cases of the chal-
cones precipitated, they were filtered and crystallized
from MeOH otherwise indicated. In the other cases, the
product was purified using column chromatography.
5.2.6. (E)-N-[4-(3-Oxo-3-phenyl-1-propenyl)phenyl] acet-
amide (7). Yellow solid, mp 177.2–180.1 ꢁC (found: C,
76.9; H, 5.8; N, 5.2. C17H15NO2 requires C, 77.0; H,
1
5.7; N, 5.3); H NMR (DMSO-d6) d: 2.07 (s, 3H), 7.60
(m, 2H), 7.67 (m, 4H), 7.81 (m, 3H), 8.12 (dd, J1 = 8.3,
J2 = 1.1, 1H), 10.15 (br s, 1H); 13C NMR (HMQC-
HMBC) (DMSO-d6) d: 25.0, 119.7, 121.1, 129.3, 129.6,
130.1, 130.7, 133.8, 138.7, 142.5, 144.7, 169.5, 190.0.
5.2.7. (E)-3-(4-Chlorophenyl)-1-(2-hydroxyphenyl)prop-2-
en-1-one (11). Yellow solid, mp 146.1–149.4 ꢁC (found:
C, 69.3; H, 4.1. C15H11ClO2 requires C, 69.6; H, 4.3);
1H NMR (CDCl3) d: 6.97 (dt, J1 = 8.1, J2 = 0.9, 1H),
7.05 (d, J = 8.4, 1H), 7.43 (d, J = 8.5, 2H), 7.53 (dt,
J1 = 8.5, J2 = 1.4, 1H), 7.61 (d, J = 8.4 2H), 7.64 (d,
J = 15.4, 1H), 7.89 (d, J = 15.5, 1H), 7.93 (dd, J1 = 8.0,
J2 = 1.3, 1H), 12.74 (s, 1H); 13C NMR (HMQC-HMBC)
(CDCl3) d: 119.1, 119.3, 120.4, 121.1, 129.7, 130.0, 130.2,
133.5, 136.9, 137.3, 144.3, 164.0, 193.9; MS m/z (abun-
dance, %): 260 (M+Å+2, 18), 258 (M+Å, 55), 223 (11),
195 (5), 147 (100), 121 (74), 103 (12); IR m 3390, 1644,
1485, 818, 754.
5.2.1. (E)-3-(4-Chlorophenyl)-1-phenyl-2-propen-1-one (2).
White-yellow solid, mp 114.4–117.1 ꢁC (found: C, 74.3;
H, 4.3. C15H11ClO requires C, 74.2; H, 4.6); H NMR
1
(CDCl3) d: 7.41 (dd, J1 = 8.5, J2 = 1.7, 2H), 7.48–7.64
(m, 6H), 7.77 (d, J = 15.7, 1H), 8.03 (dd, J1 = 7.9,
J2 = 1.7, 2H); 13C NMR (HMQC-HMBC) (CDCl3) d:
123.0, 128.9, 129.1, 129.6, 130.0, 133.3, 133.8, 136.8,
138.5, 143.7, 190.6; MS m/z (abundance, %): 244
(M+Å+2, 22), 242 (M+Å, 68), 207 (77), 179 (64), 130 (25),
103 (7), 77 (100); IR m 1659, 1402, 822, 776.
5.2.2. (E)-3-(4-Bromophenyl)-1-phenyl-2-propen-1-one (3).
Pale yellow solid, mp 126.1–128.5 ꢁC (found: C, 62.4; H,
4.1. C15H11BrO requires C, 62.7; H, 3.9); 1H NMR
(CDCl3) d: 7.49–7.63 (m, 8H), 7.76 (d, J = 15.7, 1H),
8.03 (dd, J1 = 8.2, J2 = 1.0, 2H); 13C NMR (HMQC-
HMBC) (CDCl3) d: 123.0, 125.2, 128.9, 129.1, 130.2,
132.6, 133.3, 134.3, 138.5, 143.7, 190.6.
5.2.8. (E)-3-(2-Bromophenyl)-1-(2-hydroxyphenyl)prop-2-
en-1-one (13). Orange-yellow solid, mp 89.5–92.9 ꢁC
(found: C, 59.1; H, 3.4. C15H11BrO2 requires C, 59.4;
H, 3.7); 1H NMR (CDCl3) d: 6.94 (ddd, J1 = 7.9,
J2 = 7.3, J3 = 1.1, 1H), 7.03 (dd, J1 = 8.4, J2 = 1.0,
1H), 7.27 (ddd, J1 = 7.8, J2 = 7.6, J3 = 1.7, 1H), 7.38
(t, J = 7.4, 1H), 7.50 (ddd, J1 = 8.3, J2 = 7.3, J3 = 1.6,
1H), 7.58 (d, J = 15.5, 1H), 7.65 (dd, J1 = 8.0, J2 = 8.0,
1H), 7.74 (dd, J1 = 7.8, J2 = 1.6, 1H), 7.90 (dd,
J1 = 8.0, J2 = 1.5, 1H), 8.25 (d, J = 15.5, 1H), 12.68 (s,
1H); 13C NMR (HMQC-HMBC) (CDCl3) d: 119.1,
119.3, 120.3, 123.5, 126.5, 128.1, 128.4, 130.1, 132.0,
134.1, 135.2, 137.0, 144.1, 164.1, 193.9; MS m/z (abun-
dance, %): 304 (M+Å+2, 9), 302 (M+Å, 9), 223 (100), 195
(3), 147 (40), 121 (28), 102 (28), 75 (20); IR m 3400,
1644, 1584, 1487, 752.
5.2.3. (E)-3-(2-Bromophenyl)-1-phenyl-2-propen-1-one (4).
Oil (found: C, 62.8; H, 3.7. C15H11BrO requires C, 62.7;
1
H, 3.9); H NMR (CDCl3) d: 7.28 (dt, J1 = 7.9, J2 = 1.6,
1H), 7.39 (t, J = 7.4, 1H), 7.42 (d, J = 15.7, 1H), 7.50–
7.60 (m, 3H), 7.67 (dd, J1 = 8.0, J2 = 1.2, 1H), 7.76 (dd,
J1 = 7.8, J2 = 1.6, 1H), 8.04 (d, J = 7.9, 2H), 8.15 (d,
J = 15.7, 1H); 13C NMR (HMQC-HMBC) (CDCl3) d:
119.0, 121.0, 127.7, 128.5, 129.3, 130.0, 130.2, 131.6,
134.6, 136.0, 137.5, 145.2, 190.0.
5.2.4. (E)-3-(4-Methoxyphenyl)-1-phenyl-2-propen-1-one (5).
Pale yellow solid, mp 74.3–77.3 ꢁC (found: C, 80.6; H, 6.0.
5.2.9. (E)-3-(4-Hexyloxyphenyl)-1-(2-hydroxy phenyl)-2-
propen-1-one (16). Yellow solid, mp 88.0–90.0 ꢁC
(found: C, 78.0; H, 7.8. C21H24O3 requires C, 77.7; H,
7.5); 1H NMR (CDCl3) d: 0.89–0.93 (m, 3H), 1.33–
1.36 (m, 4H), 1.78–1.81 (m, 2H), 4.00 (t, J = 6.6, 2H),
6.87–6.96 (m, 3H), 7.01 (dd, J1 = 8.4, J2 = 0.9, 1H),
7.47 (ddd, J1 = 8.6, J2 = 7.0, J3 = 1.5, 1H), 7.52
1
C16H14O2 requires C, 80.7; H, 5.9); H NMR (CDCl3) d:
3.87 (s, 3H), 6.96 (d, J = 8.6, 2H), 7.43 (d, J = 15.6 1H),
7.51 (dt, J1 = 8.1, J2 = 1.0, 2H), 7.58 (dd, J1 = 8.2,
J2 = 1.1, 1H), 7.62 (d, J = 8.7, 2H), 7.81 (d, J = 15.6, 1H),
8.03 (dd, J1 = 8.3, J2 = 1.2, 2H); 13C NMR (HMQC-

M. Cabrera et al. / Bioorg. Med. Chem. 15 (2007) 3356–3367
3363
(d, J = 15.4, 1H), 7.55–7.62 (m, 2H), 7.89 (d, J = 15.4,
1H), 7.91 (dd, J1 = 8.0, J2 = 1.5, 1H), 12.93 (s, 1H);
13C NMR (CDCl3) d: 14.0, 22.6, 25.7, 29.1, 31.6, 68.3,
115.1, 117.5, 118.6, 118.7, 120.2, 127.2, 129.5, 130.6,
136.1, 145.5, 161.7, 163.6, 193.7.
7.52 (d, J = 8.5, 1H), 7.60 (d, J = 8.6, 2H), 7.63 (d,
J = 15.7, 1H), 7.88–7.95 (m, 2H), 12.85 (s, 1H); 13C
NMR (HMQC-HMBC) (CDCl3) d: 119.0, 119.2, 119.4,
121.0, 125.0, 126.4, 129.4, 130.0, 131.0, 136.7, 145.4,
164.0, 194.0.
5.2.10. (E)-1-(2-Hydroxyphenyl)-3-(4-nonyloxyphenyl)-2-
propen-1-one (17). Pale yellow solid, mp 75.0–77.0 ꢁC
(found: C, 78.9; H, 8.5. C24H30O3 requires C, 78.7; H,
5.2.15. (E)-3-(4-Benzyloxy-3-iodophenyl)-1-(2-hydroxy-
phenyl)-2-propen-1-one (30). Oil (found: C, 57.6; H,
3.5. C22H17IO3 requires C, 57.9; H, 3.8); 1H NMR
(CDCl3) d: 5.10 (s, 2H), 6.90 (d, J = 8.5, 1H), 6.97
(ddd, J = 8.2, J = 7.4, J = 1.2, 1H), 7.04 (dd, J = 8.4,
J = 1.1, 1H), 7.34–7.59 (m, 7H), 7.61 (d, J = 8.4, 1H),
7.81 (d, J = 15.4, 1H), 7.93 (dd, J = 8.1, J = 1.4, 1H),
8.18 (d, J = 1.8, 1H), 12.82 (s, 1H); 13C NMR (CDCl3)
d: 71.1, 87.1, 112.0, 117.5, 118.6, 118.7, 120.2, 126.3,
126.9, 128.2, 128.7, 129.5, 131.5, 135.4, 136.1, 141.1,
145.5, 161.8, 163.6, 193.7.
1
8.3); H NMR (CDCl3) d: 0.89 (t, J = 6.7, 3H), 1.22–
1.40 (m, 10H), 1.42 –1.48 (m, 2H), 1.76–1.83 (m, 2H),
4.00 (t, J = 6.6, 2H), 6.90–6.94 (m, 3H), 7.01 (d,
J = 8.4, 1H), 7.47 (ddd, J1 = 8.5, J2 = 7.1, J3 = 1.4,
1H), 7.52 (d, J = 15.4, 1H), 7.55–7.64 (m, 2H), 7.87–
7.91 (m, 2H), 12.93 (s, 1H); 13C NMR (CDCl3) d:
14.1, 22.7, 26.0, 29.2, 29.3, 29.4, 29.5, 31.9, 68.3, 115.1,
117.5, 118.6, 118.7, 120.2, 127.2, 129.5, 130.6, 136.1,
145.5, 161.7, 163.6, 193.7.
5.2.16. (2E,4E)-1-(2-Hydroxyphenyl)-5-phenyl-2,4-penta-
dien-1-one (31). Pale-yellow solid, mp 150.0–152.0 ꢁC
(from EtOH) (found: C, 81.3; H, 5.3. C17H14O2 requires
5.2.11. (E)-1-(2-Hydroxyphenyl)-3-(4-methylthio phenyl)-
2-propen-1-one (19). Yellow solid, mp 75.6–77.0 ꢁC
(found: C, 70.9; H, 5.0; S, 11.5. C16H14O2S requires C,
71.1; H, 5.2; S, 11.9); 1H NMR (CDCl3) d: 2.55 (s,
3H), 6.96 (t, J = 8.1, 1H), 7.05 (d, J = 8.4 1H), 7.30 (d,
J = 8.6, 1H), 7.52 (m, 4H), 7.64 (d, J = 15.6, 1H), 7.91
(d, J = 15.5, 1H), 7.95 (d, J = 1.4, 1H), 12.85 (s, 1H);
13C NMR (HMQC-HMBC) (CDCl3) d: 15.5, 119.1,
119.2, 119.4, 120.5, 126.4, 129.4, 130.0, 131.5, 136.7,
143.5, 145.3, 164.0, 194.0. After filtration of derivative
19, the solvent was evaporated in vacuo and the residue
was purified by chromatographic column (SiO2, petro-
leum ether:ethyl acetate (0–40%)) isolating derivative 20.
1
C, 81.6; H, 5.6); H NMR (CDCl3) d: 6.70 (d, J = 15.0,
1H), 6.80–7.15 (m, 4H), 7.20–7.50 (m, 6H), 7.70 (dd,
J = 15.1, J = 15.2, 1H), 7.99 (d, J = 8.0, 1H), 12.90 (s,
1H); 13C NMR (CDCl3) d: 111.0–137.0 (12 carbons),
144.5, 161.8, 189.7.
5.2.17. (E)-1-(2-Hydroxyphenyl)-3-(2-pyridinyl)-2-propen-
1-one (33). Yellow solid, mp 95.8–97.9 ꢁC (found: C, 74.4;
H, 4.6; N, 6.0. C14H11NO2 requires C, 74.7; H, 4.9; N,
1
6.2); H NMR (CDCl3) d: 6.97 (dt, J1 = 8.0, J2 = 0.9,
1H), 7.01 (d, J = 8.3, 1H), 7.34 (m, 1H), 7.53 (m, 2H),
7.78 (dt, J1 = 7.7, J2 = 1.7, 1H), 7.88 (d, J = 15.0, 1H),
8.06 (dd, J1 = 8.0, J2 = 1.3, 1H), 8.28 (d, J = 15.0, 1H),
8.73 (d, J = 3.9, 1H), 12.74 (s, 1H); 13C NMR (HMQC-
HMBC) (CDCl3) d: 118.9, 119.3, 120.5, 124.6, 125.1,
126.2, 130.7, 137.0, 137.3, 143.6, 150.7, 153.3, 164.1,
194.6; MS m/z (abundance, %): 225 (M+Å, 83), 196
(100), 147 (78), 120 (6), 105 (5), 92 (13), 78 (28); IR m
3395, 3050, 1641, 1578, 1338, 1313, 981, 762.
5.2.12. 3-Hydroxy-1-(2-hydroxyphenyl)-3-(4-methylthiophe-
nyl)-1-propanone (20). Pale yellow solid, mp 119.5–121.5 ꢁC
(found: C, 66.5; H, 5.4; S, 10.8. C16H16O3S requires C,
66.6; H, 5.6; S, 11.1); 13H NMR (CDCl3) d: 2.51 (s, 3H),
3.18 (s, 1H), 3.36 (dd, J1 = 17.5, J2 = 3.2, 1H), 3.46 (dd,
J1 = 17.5, J2 = 8.9, 1H), 5.35 (dd, J1 = 8.8, J2 = 2.4, 1H),
6.92 (m, 1H), 7.00 (d, J = 8.4, 1H), 7.29 (d, J = 8.3, 2H),
7.31 (d, J = 8.3, 2H), 7.44 (m, 1H), 7.71 (dd, J = 8.0,
J = 1.5, 1H), 12.04 (s, 1H); 13C NMR (HMQC-HMBC)
(CDCl3) d: 16.4, 30.1, 47.4, 69.9, 119.0, 119.5, 126.7,
127.3, 129.4, 130.4, 137.3, 138.5, 140.1, 205.7.
5.2.18. 1-(2-Hydroxyphenyl)-3-(4-benzyloxyphenyl)-1-prop-
anone (34). Oil (found: C, 79.6; H, 5.9. C22H20O3 re-
1
quires C, 79.5; H, 6.1); H NMR (CDCl3) d: 2.42 (t,
J = 7.8, 2H), 2.82 (t, J = 7.8, 2H), 5.23 (s, 2H), 6.87–
6.96 (m, 3H), 7.01 (dd, J1 = 8.4, J2 = 0.9, 1H), 7.47
(ddd, J1 = 8.5, J2 = 7.1, J3 = 1.6, 1H), 7.53–7.64 (m,
7H), 7.97 (dd, J1 = 7.9, J2 = 1.5, 1H), 12.73 (s, 1H);
13C NMR (CDCl3) d: 31.4, 42.0, 70.2, 115.0, 118.6,
118.7, 120.2, 127.1, 127.5, 128.3, 128.7, 129.5, 130.6,
135.9, 136.0, 161.7, 163.6, 193.7.
5.2.13.
(E)-N-[4-[3-(2-Hydroxyphenyl)-3-oxo-1-propenyl]-
phenyl]acetamide (22). Orange-yellow solid, mp 202.2–
205.3 ꢁC (from EtOH) (found: C, 72.6; H, 5.1; N, 4.8.
C17H15NO2 requires C, 72.6; H, 5.4; N, 5.0); H NMR
1
(CDCl3) d: 3.28 (s, 3H), 7.00 (t, J = 7.6, 2H), 7.56 (t,
J = 7.9, 1H), 7.69 (d, J = 8.5, 2H), 7.78 (d, J = 15.4, 2H),
7.85 (d, J = 8.5, 2H), 7.92 (d, J = 15.4, 1H), 8.23 (d,
J = 7.6, 1H), 10.18 (s, 1H), 12.62 (s, 1H); 13C NMR
(HMQC-HMBC) (CDCl3) d: 24.0, 118.6, 119.7, 120.0,
120.5, 121.6, 129.9, 131.1, 131.6, 137.0, 142.8, 145.6, 162.8,
169.6, 194.4; MS m/z (abundance, %): 281 (M+Å, 20), 238
(9), 223 (5), 161 (9), 148 (10), 121 (14), 102 (2), 77 (4).
5.2.19. 3-(2-Bromophenyl)-1-(2-hydroxy-4,6-dimethoxy-
phenyl)-2-propen-1-one (35). Yellow solid, mp 146.0–
147.0 ꢁC (found: C, 55.9; H, 3.9. C17H15BrO4 requires
1
C, 56.2; H, 4.2); H NMR (CDCl3) d: 3.90 (s, 3H), 3.84
(s, 3H), 5.96 (d, J = 2.4, 1H), 6.11 (d, J = 2.4, 1H), 7.22
(ddd, J1 = 7.8, J2 = 7.5, J3 = 1.6, 1H), 7.34 (d, J = 7.4,
1H), 7.62 (dd, J1 = 8.0, J2 = 1.1, 1H), 7.67 (dd, J1 = 7.8,
J2 = 1.6, 1H), 7.81 (d, J = 15.5, 1H), 8.10 (d, J = 15.5,
1H), 14.17 (s, 1H); 13C NMR (HMQC-HMBC)
(CDCl3) d: 55.6, 55.9, 91.3, 93.9, 106.4, 125.8, 127.6,
127.9, 130.2, 130.8, 133.5, 135.7, 140.4, 162.5, 166.4,
5.2.14. (E)-3-(4-Aminophenyl)-1-(2-hydroxyphenyl)-2-pro-
pen-1-one (23). Orange solid, mp 153.0–156.0 ꢁC (found:
C, 75.0; H, 5.2; N, 5.6. C15H13NO2 requires C, 75.3; H,
5.5; N, 5.9); H NMR (CDCl3) d: 2.55 (s, 2H), 6.96 (t,
J = 7.2, 1H), 7.05 (d, J = 8.3, 1H), 7.29 (d, J = 8.5, 2H),
1

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M. Cabrera et al. / Bioorg. Med. Chem. 15 (2007) 3356–3367
168.5, 192.2; MS m/z (abundance, %): 364 (M+Å+2, 33),
5.4.1. 2-(4-Chlorophenyl)-4H-chromen-4-one (50). White
solid, mp 172.0–175.4 ꢁC (found: C, 69.9; H, 3.3.
C15H9ClO2 requires C, 70.2; H, 3.5); H NMR (CDCl3)
362 (M+Å, 33), 347 (5), 283 (70), 207 (100), 102 (15), 75 (6).
1
5.2.20. (E)-1-(4-Benzyloxy-2-hydroxyphenyl)-3-(2-bromo-
phenyl)-2-propen-1-one (39). Oil (found: C, 64.7; H, 4.4.
C22H17BrO3 requires C, 64.6; H, 4.2); 1H NMR (CDCl3)
d: 5.10 (s, 2H), 6.55–6.57 (m, 2H), 7.22–7.45 (m, 7H),
7.57 (d, J = 15.5, 1H), 7.65 (dd, J1 = 8.1, J2 = 1.2, 1H),
7.78 (dd, J1 = 7.8, J2 = 1.6, 1H), 7.83 (d, J = 9.7, 1H),
8.15 (d, J = 15.5, 1H), 13.27 (s, 1H); 13C NMR (CDCl3)
d: 70.7, 102.1, 108.2, 114.1, 120.3, 126.1, 127.7, 127.3,
128.0, 128.3, 128.9, 131.2, 131.9, 133.1, 134.4, 135.6,
144.3, 165.2, 165.6, 192.1.
d: 6.81 (s, 1H), 7.45 (t, J = 7.2, 1H), 7.52 (d, J = 8.6, 2H),
7.58 (d, J = 8.4, 1H), 7.73 (dd, J1 = 7.0, J2 = 1.6, 1H),
7.89 (d, J = 8.6, 2H), 8.24 (dd, J1 = 7.9, J2 = 1.3, 1H);
13C NMR (HMQC-HMBC) (CDCl3) d: 108.1, 118.4,
124.3, 125.8, 126.2, 128.0, 129.8, 130.7, 134.3, 138.3,
156.6, 162.0, 178.7.
5.4.2. 2-(2-Bromophenyl)-4H-chromen-4-one (52). Beige
solid, mp 119.5–122.5 ꢁC (found: C, 59.9; H, 2.9.
C15H9BrO2 requires C, 59.8; H, 3.0); 1H NMR (CDCl3)
d: 6.90 (s, 1H), 7.40–7.70 (m, 7H), 8.25 (dd, J1 = 7.9,
J2 = 1.4, 1H); 13C NMR (HMQC-HMBC) (CDCl3) d:
108.0, 118.0–138.0 (11 carbons), 157.0, 164.0, 179.0.
5.2.21. (E)-1-(4-Butoxy-2-hydroxyphenyl)-3-(4-butoxyphe-
nyl)-2-propen-1-one (41). Yellow solid, mp 82.0–84.0 ꢁC
(found: C, 74.8; H, 7.6. C23H28O4 requires C, 75.0; H,
1
7.7); H NMR (CDCl3) d: 0.98 (t, J = 7.4, 3H), 0.99 (t,
5.5. X-ray studies
J = 7.4, 3H), 1.46–1.54 (m, 4H), 1.75–1.82 (m, 4H), 4.01
(t, J = 6.4, 4H), 6.44–6.48 (m, 2H), 6.88–6.96 (m, 2H),
7.44 (d, J = 15.4, 1H), 7.55–7.63 (m, 2H), 7.81 (d,
J = 8.6, 1H), 7.85 (d, J = 15.4, 1H), 13.52 (s, 1H); 13C
NMR (CDCl3) d: 13.7, 13.8, 19.2, (2 carbons), 31.0,
31.2, 67.9, 68.1, 101.5, 108.0, 114.0, 115.0, 117.8, 127.4,
131.1, 131.3, 144.2, 161.4, 165.7, 166.6, 191.8.
The crystal and molecular structure of compound 44,
C15H11BrO2, has been determined at 120 K by X-ray dif-
fraction methods. The substance crystallizes in the mono-
clinic P21/a space group with a = 7.976(1), b = 21.126(1),
c = 8.107(1) A, b = 116.54(1)ꢁ, and Z = 4. The structure
was solved from 1597 reflections with I > 2r(I) and
refined to an agreement R1-factor of 0.0508.
˚
5.3. General procedure for the synthesis of flavanone
derivatives (42–48)
Diffraction data and structure solution and refinement.
Crystal data, data collection procedure, structure deter-
mination method, and refinement results for the com-
pound are summarized in Table 1 (Supplementary
material). The hydrogen atoms were positioned stereo-
chemically and refined with the riding model.
A solution of the corresponding 20-hydroxychalcone
(1 equiv) (8, 12–14, 18, 24 or 28) in AcOH glacial (25.0
mL/mmol of 20-hydroxychalcone) was heated at reflux
during 72 h. Then the mixture was poured into water
and extracted with EtOAc (3· 25.0 mL). The organic
layer was washed with brine until neutrality and dried
with MgSO4 anhydrous. The solvent was evaporated in
vacuo and the residue was purified by chromatographic
column (SiO2, petroleum ether:methylene dichloride (0–
30%)).
Crystallographic structural results and discussion. Atomic
fractional coordinates and equivalent isotropic displace-
ment parameters are given in Table 2 (Supplementary
material). Intra-molecular bond distances and angles are
inTable3(Supplementarymaterial). Thestructureofcom-
pound 44 has been deposited in the Cambridge Crystallo-
graphic Data Centre, reference number CCDC-631417.
5.3.1. 2-(4-Bromophenyl)-2,3-dihydrochromen-4-one (43).
Yellow solid, mp 115.8–119.0 ꢁC (found: C, 59.5; H, 3.6.
C15H11BrO2 requires C, 59.4; H, 3.7); H NMR (CDCl3)
1
5.6. Biology
d: 2.90 (dd, J1 = 16.8, J2 = 3.1, 1H), 3.05 (dd, J1 = 16.8,
J2 = 13.0, 1H), 5.47 (dd, J1 = 13.0, J2 = 3.0, 1H), 7.07
(m, 2H), 7.38 (d, J = 8.4, 2H), 7.54 (m, 1H), 7.58 (d,
J = 8.5 , 2H), 7.95 (dd, J1 = 7.8, J2 = 1.6, 1H); 13C NMR
(HMQC-HMBC) (CDCl3) d: 45.0, 79.2, 118.5, 121.3,
122.2, 123.1, 127.5, 128.2, 132.4, 136.7, 138.2, 161.7, 191.8.
5.6.1. Cytotoxicity studies on tumoral cells. Cells: an ade-
quate number of cells (TK-10, MCF-7 or HT-29) was
maintained in 225 lL of RPMI medium, supplemented
with L-glutamine (1%), penicillin/streptomycin (1%),
non-essential amino acids (1%), and 10% (v/v) fetal bo-
vine serum (FBS). The cultures were maintained at
37 ꢁC and 5% CO2 for 48 h. The absorbance at
540 nm before the treatment was determined.
5.4. General procedure for the synthesis of flavone
derivatives 49–53
To a solution of the corresponding 20-hydroxychalcone
(1 equiv) (8, 11–13, or 18) in DMSO (10.0 mL/mmol
of 20-hydroxychalcone), I2 (0.01 equiv) was added. The
mixture was heated at reflux during 1 h. Then the mix-
ture was poured into water and extracted with EtOAc
(3· 25.0 mL). The organic layer was washed with brine
until neutrality and dried with MgSO4 anhydrous. The
solvent was evaporated in vacuo and the residue was
purified by chromatographic column (SiO2, petroleum
ether:methylene dichloride (0–30%)).
Treatment: Compound solutions were prepared just be-
fore dosing. Stock solutions, 1 mM, were prepared in
10% DMSO (Aldrich) and 25 lL (final concentration
100 lM) was added to each well. No effect on cell
growth was observed by the presence of DMSO in the
culture media. The cells were exposed for 24 h at 37 ꢁC
in 5% CO2 atmosphere.
Sulforhodamine assay: After exposure to the compound,
the medium was eliminated and the cells were washed

M. Cabrera et al. / Bioorg. Med. Chem. 15 (2007) 3356–3367
3365
with PBS. The cells were fixed with 50 lL TCA (50%)
and 200 lL of culture medium (without FBS) for 1 h
at 4 ꢁC. Then the cells were washed with purified water
and treated with Sulforhodamine B (0.4% wt/vol in 1%
acetic acid) for 10 min at room temperature. Then plates
were washed with 1% acetic acid and dried overnight.
Finally, 100 lL of Tris buffer (pH 10.0) was added and
absorbance at 540 nm was determined.
electrophoresis tank (260 mm wide) containing buffer
(1 mM Na2EDTA, 0.3 M NaOH, pH 12.7, at 4 ꢁC) for
40 min before electrophoresis at 25 V for 30 min. The
slides were neutralized by washing 3 times for 5 min
each in buffer (0.4 M Tris–HCL, pH 7.5, 4 ꢁC) and
stained with DAPI (20 mL of a 5 mg/mL stock solu-
tion). DAPI-stained nuclei were evaluated with a Nikon
Eclipse TE 300 fluorescence microscope. A total of 100
comets on each comet slide were scored visually and
classified as belonging to one of the five classes accord-
ing to the tail intensity. Each treatment was carried out
in duplicate per experiment and each experiment re-
peated at least twice. DNA damage therefore extends
from 0 to 400 arbitrary units. To check the performance
of the comet assay, a positive control was included in all
the experiments: HK-2 cells were treated with 50 lM
hydrogen peroxide during 15 min on ice.
Data calculations: The assays were done in duplicate and
at least two repetitions per experiment were performed,
the absorbances were the means of the two experiments.
At the end of the experiment three kinds of measurements
were available. Control absorbance (C) that is of cells
treated with the solvent; absorbance at time 0 that is be-
fore treatment (T0); and absorbance after the treatment
(T). The cell survival percentage (SP) was calculated for
all of the compounds as (T/C) · 100. The standard errors
were not greater than 10% for most calculated parame-
ters. To determine IC50 values, cell growth was followed
in the absence (control) and presence of increasing con-
centrations of the corresponding compound. The IC50
values were determined as the compound concentration
required to reduce by half the absorbance of that of the
control (without compound).
Acknowledgments
´
This work received financial support from Comision
Honoraria de Lucha contra el Ca´ncer (Uruguay),
CSIC-Sector Productivo (UdelaR, Uruguay), RIIDD-
MED (Iberoamerican Network on Investigation and
Development of Medicines) of the Iberoamerican Pro-
gram of Science and Technology for the Development
(CYTED), and FAPESP (Brazil). OEP is a Research
Fellow of CONICET (Argentina). MC and MLL are
PEDECIBA students.
5.6.2. Comet assay. Culture of HK-2 cells: HK-2 cells
were routinely cultured in DMEM supplemented with
10% FBS (100 g/mL) and 1% of penicillin/streptomycin
in a humidified atmosphere of 5% CO2 in air at 37 ꢁC.
Sample preparation: Product concentrations were pre-
pared from a stock solution prepared in DMSO. Then,
they were diluted in serum-free medium to obtain a final
concentration of 20 lM.
Supplementary data
Crystallographic data for derivative 44. Five tables list-
ings of atomic anisotropic displacement parameters (Ta-
ble S4) and hydrogen atoms’ positions and isotropic
displacement parameters (Table S5).
Single cell gel electrophoresis (the comet assay): Mono-
layer HK-2 cells in exponential growth were trypsinized
and cell cultures were prepared in a 6-well plate:
2.5 · 106 cells/mL in 2 mL DMEM containing 10%
FBS and 1% of penicillin/streptomycin. The cells were
exposed to the flavonoid (20 lM in DMSO) for 6 h at
37 ꢁC in 5% CO2 of air. Control cultures received the
equivalent concentration of DMSO to a maximum of
1% of the culture medium. The cultures were washed
twice, recovered using trypsin/EDTA, and spun at
200g at 4 ꢁC for 3 min. The pellet (approximately
4.5 · 104 cells) was resuspended in LMP agarose for co-
met analysis. Cells embedded in agarose were lysed with
high salt and detergent leaving the DNA as a distinct
‘nucleoid’. DNA was allowed to unwind under alkaline
conditions. Breaks in the DNA molecule disrupt its
complex supercoiling allowing free DNA loops to mi-
grate toward the anode during electrophoresis. DNA
damage to the cells can be thus visualized as ‘comets’.
Cells were suspended in LMP agarose [80 mL of a 1%
(w/v) solution in PBS] at 37 ꢁC and pipetted onto a
frosted glass microscope slide precoated with a similar
solution and amount of buffer (40 mM Hepes–KOH,
0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml BSA, pH 8,
4 ꢁC), blotted dry and incubated either with enzyme or
buffer (50 mL) for 45 min (endonuclease III) or 30 min
(Fapyglycosylase) at 37 ꢁC. The slides were held in an
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2007.
03.031.
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