Synthesis and pharmacological activity of chalcones derived from 2,4,6-trimethoxyacetophenone in RAW 264.7 cells stimulated by LPS: Quantitative structure-activity relationships

Article abstract of DOI:10.1016/j.bmc.2007.10.039

Inhibition of nitric oxide (NO) production by altering the expression of induced enzymes involved is potentially an important strategy for obtaining antiinflammatory agents. In the search for hits to obtain lead compounds for new drugs of this class, 14 s

Full text of DOI:10.1016/j.bmc.2007.10.039

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 658–667
Synthesis and pharmacological activity of chalcones derived
from 2,4,6-trimethoxyacetophenone in RAW 264.7 cells
stimulated by LPS: Quantitative structure–activity relationships
Louise Domeneghini Chiaradia,a,* Rodrigo dos Santos,a Carlos Eduardo Vitor,b
a
a
a
´
´
´
Andre Alexandre Vieira, Paulo Cesar Leal, Ricardo Jose Nunes,
Joao Batista Calixtob and Rosendo Augusto Yunesa,*
˜
aUniversidade Federal de Santa Catarina, Departamento de Quı´mica, Campus Trindade, 88040-900 Floriano´polis, SC, Brazil
bUniversidade Federal de Santa Catarina, Departamento de Farmacologia, Campus Trindade, 88040-900 Floriano´polis, SC, Brazil
Received 30 August 2007; revised 11 October 2007; accepted 15 October 2007
Available online 18 October 2007
Abstract—Inhibition of nitric oxide (NO) production by altering the expression of induced enzymes involved is potentially an
important strategy for obtaining antiinflammatory agents. In the search for hits to obtain lead compounds for new drugs of this
class, 14 synthetic chalcones derived from 2,4,6-trimethoxyacetophenone were evaluated in terms of their inhibitory action,
in vitro, in relation to NO production in murine macrophages of the line RAW 264.7 induced by bacterial lipopolysaccharides
(LPS). All the compounds were obtained by aldolic condensation between the acetophenone and corresponding aldehydes, under
basic conditions. The mean IC50 values, calculated through dose versus inhibitory effect curves, in four independent experiments,
varied between 1.34 and 27.60 lM, and were compared with the positive control, compound 1400W (IC50 = 3.78 lM), a highly selec-
tive inhibitor of iNOS (induced nitric oxide synthase). Eight chalcones gave mean IC50 values less than or equal to those obtained
for 1400W, which suggests that these molecules may act as inhibitors of inflammatory process. The QSAR study reveals that elec-
tron-withdrawing groups in the B-ring seem to increase the inhibition of nitrite production, mainly when in position 2. A substitu-
tion in the ortho position of the A-ring seems to be necessary for the activity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
muscle cells, and neuronal and endothelial cells in re-
sponse to pathological stimuli.2
In the complex reaction systems involved in the response
of a host to pathogens, chemical mediators, such as ni-
tric oxide (NO), play a pivotal role. NO is a free radical
gas produced from L-arginine and molecular oxygen by
catalytic reaction of nitric oxide synthase (NOS),1 which
exists in three different isoforms: endothelial NOS
(eNOS) and neuronal NOS (nNOS), which are constitu-
tively expressed and play an important role in physio-
logical processes including neurotransmission and
vascular homeostasis, and inducible NOS (iNOS), which
can be expressed by many cells, such as macrophages,
Kupffer cells, neutrophils, fibroblasts, vascular smooth
Most interest has been focused on the mechanism of iNOS
expression in macrophages and consequently the role of
NO production in their immune responses. NO can react
with superoxide derived from macrophages to generate
highly cytotoxic peroxynitrite which destroys the invad-
ing microorganisms.3,4 iNOS is highly expressed by lipo-
polysaccharide (LPS)-activated macrophages and this
contributes to the pathogenesis of septic shock.5 Thus,
the inhibition of NO production by iNOS is an important
target in the treatment of inflammatory diseases.
Chalcones are essential intermediate compounds in fla-
vonoid biosynthesis, and they are easily found in arbo-
real or smaller plants. They can be obtained by several
chemical methods and Claisen–Schimidt’s condensation
(aldolic condensation) is the most used one.6 Many stud-
ies have shown that they are compounds of great chem-
Keywords: Chalcones; Antiinflammatory; Nitric oxide; QSAR; iNOS
inhibitor.
*
Corresponding authors. Tel.: +55 48 3721 9759; fax: +55 48 3721
6844 (L.D.C.); e-mail addresses: louisedc@gmail.com; ryunes@qmc.
ufsc.br (R.A.Y).
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.039

L. D. Chiaradia et al. / Bioorg. Med. Chem. 16 (2008) 658–667
659
ical and pharmacological interest because they exhibit
many biological activities,7,8 such as antifungal,9,10 anti-
viral,11 antibacterial,12 antimalarial,13 anti-leishmania,14,15
antinociceptive,16 antiinflammatory,17–19 and anti-tu-
moral,18,20,21 among others. Substitutions in the A-
and B-rings of chalcones can result in compounds with
different biological activities.
with compounds 2 and 13. In the case of 13, the low
yield can be explained by the presence of a dimethyl-
amine group, which can stabilize the carbonyl group
in the aldehyde, decreasing its reactivity. Regarding
compound 2, the presence of a nitro group in position
3 in the aldehyde must increase the electropositivity of
the carbonyl group, increasing its reactivity and conse-
quently its yield; however, this was not observed and
the low yield may be due to losses during the purifica-
tion process. Compounds 4 and 5 also possess a nitro
group in positions 2 and 4, and, as the other com-
pounds, presented good yields.
Hydroxylated and chlorated chalcones have been
reported to inhibit NO synthesis and also iNOS protein
expression in RAW 264.7 cells.18,19 Other chalcones
derived from 4-dimethylaminochalcone have also been
found to inhibit nitrite production and iNOS protein
expression in the LPS-stimulated RAW 264.7 macro-
phage.17,22 20-substituted chalcones, including those
derived from 2,4,6-trimethoxyacetophenone, have been
found to be effective in inhibiting the biosynthesis of
interleukin (IL)-1 from human peripheral blood mono-
cytes stimulated with LPS.23
Compound 11 exhibited all the spectroscopic data,
including the elementary analysis, confirming the pro-
posed structure; however, its experimental melting
points was different from those reported in the literature.
Five new compounds (1, 3, 4, 6 and 14) were confirmed
1
by chemical identification data: H NMR, 13C NMR,
1
IR, and elementary analysis. H NMR spectra revealed
In this study, we evaluated the inhibition of nitrite pro-
duction in RAW 264.7 cells by some chalcones derived
that all the structures were geometrically pure and con-
figured E (JHaꢀHb = 14–17 Hz).
from 2,4,6-trimethoxyacetophenone.
A quantitative
structure–activity analysis was carried out to determine
a relationship with the pharmacological effects.
2.2. Evaluation of chalcone’s ability to inhibit NO
production
Chalcones were evaluated for their ability to inhibit
NO synthesis by the RAW 264.7 macrophage stimu-
lated with LPS. Their IC50 values were calculated
for concentrations from 0.1 to 30 lM and compared
with those of the positive drug 1400W (N-(3-(amino-
methyl)benzyl)acetamidine), a highly selective inhibitor
of iNOS, which is 5000 times more selective to this
isoform than eNOS, and 200 times more than
nNOS.25,26 All compounds were able to inhibit the ni-
trite production in a concentration-dependent manner
with mean IC50 values varying from 1.34 to 27.60 lM.
Table 1 shows the yields, IC50 values, and percentage
(%) of cellular viability at the IC50 level.
2. Results and discussion
2.1. Chalcones
2,4,6-Trimethoxyacetophenone (TMA) and 14 syn-
thetic chalcones derived from it were prepared as
shown in Figure 1,6 with yields varying between 43%
and 97%. All reagents used were obtained commer-
cially (Merck, Sigma–Aldrich), except xanthoxyline,
which was prepared as previously described.24 This
method permitted the obtainment of substituted com-
pounds in the B-ring. The lowest yields were obtained
Figure 1. Synthesis of 2,4,6-trimethoxyacetophenone (TMA) and corresponding chalcones. (a) Me2SO4, NaOH/Me2CO, reflux, 24 h. (b)
Corresponding aldehyde, KOH 50% v/v, methanol, rt, 24 h. (c) 2-Naphthaldehyde, KOH 50% v/v, methanol, rt, 24 h. (d) 1-Naphthaldehyde, KOH
50% v/v, methanol, rt, 24 h. *New compounds.

660
L. D. Chiaradia et al. / Bioorg. Med. Chem. 16 (2008) 658–667
Table 1. Yield (%), IC50 values confidence intervals of inhibitory effect, and cellular viability (at IC50 level) for some selected chalcones
Chalcone
R
Yield (%)
IC50 confidence intervals (lM)
Cell viability at IC50 (%)
1
2-Cl
3-NO2
80
43
91
60
79
89
94
97
63
88
78
84
53
92
1.70 (1.44–1.97)
1.78 (0.97–3.27)
2.21 (0.38–12.9)
2.43 (2.06–2.79)
2.45 (0.46–13.20)
2.46 (2.12–2.79)
4.24 (2.02–8.93)
4.62 (2.75–6.49)
7.26 (2.01–26.30)
11.00 (8.08–14.90)
13.10 (9.11–18.70)
15.20 (10.10–22.80)
24.90 (1.77–351.00)
27.60 (5.59–136.00)
3.78 (1.87–7.63)a
100
75
2
3
3-Cl
2-NO2
100
100
70
4
5
4-NO2
3,4-Cl2
6
100
75
7
4-Cl
1-Naphthyl
2,6-Cl2
8
100
100
100
100
100
90
9
10
2-Naphthyl
3,4-OCH2–O–
4-OCH3
4-N(CH3)2
4-O(CH2)3–CH3
11
12
13
14
100
82
1400W
a Under our experimental conditions.
The structure–activity analysis demonstrated that all
chalcones with substituents that reduce the electronic
density of the B-ring, such as chlorine atoms (1, 3, 6,
7, and 9) or nitro groups (2, 4, and 5), showed the great-
est inhibition activity and selectivity in the inhibition of
nitrite production. It was possible to observe also that
the position of the chlorine atoms in the B-ring influ-
ences the inhibition of the nitrite production. In the mol-
ecules with only one chlorine atom, a proximity of this
to the carbonyl group (position 2, 1) gave the lowest
IC50 value, followed by the molecule with the chlorine
atom in position 3 (3) and then in position 4 (7). Consid-
ering the standard deviation of the experimental error,
we can observe the same effect for the molecules that
have a nitro group as the substituent, with better activity
being observed when it is in position 2 (4), followed by
the positions 3 (2) and then 4 (5). The decreases in the
activity follow the decrease in the inductive effect of
the chlorine atom and nitro group. However, the mole-
cules with nitro groups showed, in general, greater cyto-
toxicity than the molecules with chlorine atoms.
di-substituted at A-ring induced the inhibition of NO
production and the iNOS protein expression in RAW
264.7 cells stimulated by LPS.19 In our case, of the mol-
ecules that had two chlorine atoms in the B-ring, 6 (with
chlorine atoms in positions 3 and 4) was more active
than 9 (with chlorine atoms in positions 2 and 6) and
thus, the conformation also seems to be important to
the activity.
Chalcones with substituents that increase the electronic
density of the B-ring, such as methoxy (12), butoxy
(14) or dimethylamine (13) groups, did not show signif-
icant activity in the inhibition of the nitrite production.
Won et al. demonstrated that a di-methoxylated chal-
cone in positions 20 and 50 in the A-ring and 4-hydrox-
ylated in the B-ring presented inhibitory activity in
relation to NO production in RAW 264.7 cells.18 Her-
encia et al.22 and Rojas et al.17 showed that 4-dimeth-
ylaminochalcone derivatives have a high level of
inhibitory activity in relation to the induction of iNOS
in the same cells used in our study. It is interesting to
observe that in Rojas et al.17 the more active chalcones
are those which are di-methoxylated in positions 20 and
50, and 20 and 60. In our case, were tested 20,40,60-tri-
methoxylated chalcones, and 13, which has as substitu-
ent a dimethylamine group as the ring B, did not show
satisfactory activity.
Batt et al. showed that chalcones obtained from 2,4,6-
trimethoxyacetophenone (TMA), like 2, 5, 7, 8, 9, 10,
12, 13 in this study, among others, inhibited the biosyn-
thesis of IL-1 from human peripheral blood monocytes
stimulated by LPS, and a qualitative study of the
structure–activity relationship revealed that the elec-
tron-withdrawing groups in the B-ring, regardless of
the position, give enhanced potency.23 This is in accor-
dance with our results, except regarding the activity
being independent of the position of the substituents.
Furthermore, the same authors reported that an ortho
substituent in A-ring is absolutely required for activity,
because this substitution presumably induces a confor-
mational change in the molecule, forcing the A-ring to
be orthogonal to the enone.23 Our results show that
20,40,60-trimethoxylated chalcones in the A-ring, where
this conformational change in the molecule probably
occurs, also are active.
Varying the structure of the B-ring, the presence of the
methylenedioxyl group (11) makes its inhibitory activity
as poor as that of 12 (with a methoxyl group). The 1-
naphthyl group as the B-ring (8) exhibited good activity,
better than the 2-naphthyl group (10), probably due to
its spatial conformation.
Five compounds (chalcones 1, 3, 4, 6, and 8) pre-
sented IC50 and cytotoxicity values less than or equal
to those obtained for the 1400W, 3.78 (1.87–
7.63) lM, under our experimental conditions. Figure
2 shows the concentration–response curves for nitrite
production inhibition for the four most potent chal-
cones (1, 4, 6, and 8) and their respective cell
viability.
Ko et al. observed that, substitutions in chalcones by
chlorine atoms in positions 3 and 4 of the B-ring and

L. D. Chiaradia et al. / Bioorg. Med. Chem. 16 (2008) 658–667
661
Figure 2. Effect of chalcones 1, 4, 6, and 8 on nitrite production (a–d) and cell viability (e–h) in vitro. RAW 264.7 macrophages were plated at
1 · 10ꢀ6 cells/ml for 2 h. The non-adherent cells were removed by washing the plates twice with PBS. Chalcones were then added to the plates at
various concentrations, as indicated in the graphs, for 24 h. Nitrite concentration was measured by the Gries method, while cell viability was
measured by MTT assay. The graphs represent means ( SEM) of four independent experiments. ###P < 0.001 compared with non-stimulated cells
*
(first column); P < 0.05, ***P < 0.001 compared with cells stimulated with LPS (second column). One-way ANOVA followed by Bonferroni’s test.
2.3. Theoretical quantitative structure–activity
relationship
trend with other classes of compounds has been noted
in a theoretical study on the antispasmodic activity of
xanthoxyline derivatives; Santos et al.27 found a rela-
tionship between this activity and the reactivity indices
of HOMO and LUMO, as well as their sums over two
distinct parts of the molecules. Also, Galeazzi
et al.28,29 found the same trend in recent studies on
the herbicide activity of sulfonylureas and imidazoli-
nones. These observations prompted us to try a simi-
lar study on the chalcones.
Preliminary calculations of the HOMO and LUMO
surfaces for the chalcones 1–14 of Table 2 suggested
some relationship between the activity and the loca-
tion of these orbitals in distinct parts of the molecules.
In the case of chalcones, higher potency seemed to be
related to a higher contribution of HOMO in the
TMA moiety and LUMO in the R moiety. A similar

662
L. D. Chiaradia et al. / Bioorg. Med. Chem. 16 (2008) 658–667
Table 2. Biological activity [Log(1/C), C = IC50], activity calculated from equation N [EQ(N)] and quantum chemical descriptors found to correlate
with biological activity of the chalcones
P
P
No.
R groups
Log(1/C)
EQ(1)
EQ(2)
RHðTMAÞ
9.108
RLðRÞ
9.109
1
2
R1, X = 2-Cl
R1, X = 3-NO2
5.77
5.75
5.65
5.61
5.61
5.61
5.37
5.34
5.14
4.96
4.88
4.82
4.60
4.56
5.391
5.658
5.521
5.632
5.651
5.728
5.352
4.986
5.204
4.972
4.789
4.710
4.648
4.690
5.473
4.229
4.849
4.659
4.918
4.745
4.702
4.698
5.159
5.650
5.568
4.177
4.186
4.768
4.832
5.500
5.574
4.691
4.690
4.536
5.369
5.517
5.757
5.453
5.421
5.600
5.087
4.640
5.104
5.767
4.650
4.551
4.550
5.100
5.668
5.773
5.564
5.794
5.766
5.370
5.075
4.816
5.804
4.825
4.833
4.851
4.823
4.843
5.315
4.799
4.906
4.848
4.896
4.819
4.852
4.829
4.972
5.393
5.205
4.815
4.818
4.843
4.832
5.767
5.727
4.830
4.828
4.808
5.771
5.758
5.776
5.755
5.721
5.627
4.803
4.828
4.932
5.749
4.842
4.849
4.840
4.827
10.206
8.018
10.430
10.132
5.986
2.892
0.178
10.535
0.272
0.353
0.539
0.248
0.461
5.406
0.001
1.125
0.511
1.014
0.206
0.552
0.310
1.808
6.224
4.259
0.165
0.198
0.458
0.346
10.148
9.730
0.319
0.298
0.095
10.190
10.050
10.240
10.025
9.667
8.682
0.041
0.302
1.391
9.958
0.454
0.526
0.431
0.290
11.964
9.197
12.076
11.835
10.124
9.552
11.276
9.445
11.010
9.673
8.822
8.960
8.829
8.595
6.737
8.704
8.543
9.334
9.672
8.745
9.168
9.659
9.597
9.752
6.080
6.074
9.340
9.961
10.873
10.888
9.107
9.141
8.532
10.079
10.872
12.642
10.427
9.831
10.120
12.198
8.809
9.926
12.379
8.588
7.818
7.988
11.802
3
R1, X = 3-Cl
R1, X = 2-NO2
R1, X = 4-NO2
4
5
6
R1, X = 3,4-Cl2
R1, X = 4-Cl
R2, X = H
7
8
9
R1, X = 2,6-Cl2
R3
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
R1, X = 3,4-OCH2O–
R1, X = 4-OCH3
R1, X = 4-N(CH3)2
R1, X = 4-O(CH2)3CH3
R1, X = 3-CH2NH2
R1, X = 2,3-(OH)2
R1, X = 4-CH3
R1, X = 3-OCH3
R1, X = 2,6-(OCH3)2
R1, X = 2,5-(OCH3)2
R1, X = 2,4,6-(OCH3)3
R1, X = 3-OCH3-4-OH
R1, X = 4-F
R1, X = 2-F
R1, X = 4-Br
R1, X = 2-OH
R1, X = 2-OH-4-OCH3
R1, X = 3,4,5-(OCH3)3
R1, X = 2,4,5-(OCH3)3
R1, X = 2-COOH
R1, X = 4-COOH
R1, X = 4-NH2
R1, X = 3-CH3-4-N(CH3)2
R1, X = 3-N(CH3)2
R1, X = 3-CF3
R1, X = 3-CF3-4-Cl
R1, X = 3-CF3-4-NO2
R1, X = 4-CF3
R1, X = 3,5-Cl2
R1, X = 2,4-Cl2
R2, X = OH
R1, X = 3-OCH3-4-OCH2C6H6
R4, Y = S, X = H
R4, Y = S, X = NO2
R4, Y = O, X = CH3
R5, X = H
R5, X = CH3
R6
The R groups are those shown in Figure 3.
In order to evaluate the effect of various substituents on
the activity, QSARs were calculated based on chalcones
1–14 of Table 2, and the predictions for another 34
non-synthesized chalcones were carried out. Attempts
to correlate the activity with LogP, surface area, molecu-
larvolume,theenergiesoftheHOMOandLUMO,EHOMO
and ELUMO, and their difference, DE (=ELUMO ꢀ EHOMO),
were not successful. The best equations were found to cor-
relate the activity with the sums of the reactivity indices of
HOMO and LUMO. These descriptors encode informa-
tion that quantifies the magnitude of the frontier orbitals
in distinct parts of the molecules. Concerning the drug–
receptor interactions, it can be stated that regions of the
molecules that contain a greater HOMO contribution
tend to be electron-donor ‘anchors’ to the receptor site,
and the regions that contain a greater LUMO contribu-
tion tend to be electron-acceptor ‘anchors’. In the equa-
P
tions below,
indices of the HOMO of the TMA moiety (Fig. 3),
RHðTMAÞ is the sum of the reactivity
P
RLðRÞ is the sum of reactivity indices of the LUMO
of the R moiety, the number in parentheses is the 95% con-
fidence interval of the coefficient, n is the number of com-
pounds taken into account in the regression, r is the
multiple correlation coefficient, s is the standard devia-
tion, and F is the ratio of regression to residual. In the con-
struction of each equation, the two most discrepant

L. D. Chiaradia et al. / Bioorg. Med. Chem. 16 (2008) 658–667
663
Figure 3. The 2,4,6-trimethoxyacetophenone moiety (TMA) and R groups of the chalcones.
compounds were removed from the data set in order to
achieve optimal statistical quality.
which Log(1/IC50) = 5.42. All compounds with activity
values higher than 5.42 can be considered more active
than 1400W. Thus, as can be seen in Table 2, chalcones
1–6 are more active than 1400W, and chalcones 7–14 are
less active. For the non-synthesized chalcones, it is pos-
sible to predict that the presence of electron-withdraw-
ing substituents (like CF3, NO2, and COOH) in the R
moiety must increase the activity.
Eq. 1 best quantifies the appearance of HOMO and
LUMO in the series of chalcones experimentally tested.
In this case, the two most discrepant compounds were
chalcones 1 and 8 of Table 2. This equation indicates
that the electronic properties play an important role in
the interactions of chalcones with the receptor site.
P
According to this model,
optimal value for maximum activity, and
should be as high as possible. Apparently, this effect
can be achieved by electron-withdrawing substituents
in the R moiety (Table 2). The calculated correlation
coefficient for these variables is 0.154.
RHðTMAÞ should have an
P
RLðRÞ
3. Conclusions
In summary, of the 14 synthesized and tested molecules,
eight (1–8) gave a mean IC50 value less than or equal to
that obtained for the positive control drug, compound
1400W, a highly selective inhibitor of iNOS. The struc-
ture–activity analysis demonstrates that chalcones with
substituents that reduce the electronic density in the B-
ring, such as chlorine atoms or nitro groups, show the
greatest activity and selectivity in the inhibition of nitrite
production. However, molecules with nitro groups
show, in general, a slightly higher cytotoxicity than mol-
ecules with chlorine atoms. The inhibitory activity is
also dependent on the position of the electron-with-
drawing group in the B-ring, and position 2 seems to
be the more important, with chalcone 1 being the most
active.
h
i
X
X
2
Logð1=CÞ ¼ ꢀ0:024ðꢁ0:007Þ
RHðTMAÞ
X
þ 0:307ðꢁ0:081Þ
þ 0:154ðꢁ0:073Þ
þ 3:185ðꢁ0:711Þ
RHðTMAÞ
RLðRÞ
ð1Þ
n ¼ 12; r ¼ 0:980; s ¼ 0:103; F 3;8 ¼ 63:55;
X
RHðTMAÞ
¼ 6:356
OPTIMAL
Attempts were also made to construct parabolic and lin-
ear models with one variable. Eq. 2 was the best equation
with only one variable. This equation was obtained by
removing compounds 1 and 9 (Table 2) from the regres-
sion. Despite its good statistical quality, the absence of a
descriptor that counterpoises
tion may give predictions that are too optimistic. This
According to the QSAR studies, the presence of elec-
tron-withdrawing substituents (like CF3, NO2, and
COOH) in the R moiety of chalcones would increase
their antiinflammatory activity. Such substituents exert
an influence on the location of HOMO and LUMO in
the molecules and a correlation with the activity is sug-
gested. These results, together with the results of earlier
studies,27 demonstrate that the reactivity indices of
HOMO and LUMO encode important information
about the compounds, in relation to their activity.
P
RHðTMAÞ in this equa-
trend can be observed in Table 2.
X
Logð1=CÞ ¼ 0:095ðꢁ0:028Þ
þ 4:799ðꢁ0:185Þ
n ¼ 12; r ¼ 0:919; s ¼ 0:193; F 1;10 ¼ 54:08
RHðTMAÞ
ð2Þ
Thus, the results showed that these chalcones are prob-
ably active in inflammatory processes, since NO produc-
tion is an important step of the inflammation. The
inhibition of the production of NO and PGE2, mainly
The antiinflammatory potency of the chalcones can be
assessed by comparing the values of Table 2 with the
activity value of the standard inhibitor, 1400W, for

664
L. D. Chiaradia et al. / Bioorg. Med. Chem. 16 (2008) 658–667
in macrophages, which affects the expression of the
induced enzymes involved, may be an important strat-
egy for obtaining new drugs.17,18
followed by heat application as the developing agent
and purified by recrystallization from hexane and
dichloromethane. 1H NMR spectra revealed that all
the structures were geometrically pure and configured
E (JHaꢀHb = 14–17 Hz).
4. Experimental
4.1. Synthesis
4.2.1. TMA: 2,4,6-trimethoxyacetophenone. C11H14O4,
creamy solid; mp = 100–102 ꢁC. 1H NMR (CDCl3) d
2.45 (s, 3H, CH3), 3.81 (s, 3H, p-OCH3), 3.78 (s, 6H,
o-OCH3), 6.09 (s, 2H, H3, H5). RMN 13C (CDCl3) d
32.41 (CH3), 55.31 (m-OCH3), 55.71 (p-OCH3), 90.49
(C3, C5), 113.60 (C1), 158.25 (C2, C6), 162.25 (C4),
201.61 (C@O). Yield = 84%.
The 2,4,6-trimethoxyacetophenone (TMA) and the chal-
cones were synthesized as shown in Figure 1. All reagents
used were obtained commercially (Merck, Sigma–Al-
drich), except xanthoxyline, which was prepared as pre-
viously described.24 The 2,4,6-trimethoxyacetophenone
(TMA) was obtained by magnetic agitation of a solution
of xanthoxyline (7.50 g; 38.25 mmol), NaOH (2.62 g;
65.5 mmol in 50 ml of water), acetone (300 ml), and
Me2SO4 (5.24 ml; 38.25 mmol), under reflux for 24 h.
After evaporation of the solvent, the solid formed was
recrystallized in hexane, obtaining the 2,4,6-trimethoxy-
acetophenone (TMA) crystals. This methodology of
synthesis was used for the obtainment of 3-bromine-
2,4,6-trimethoxyacetophenone from monobromide
xanthoxyline in a previous study,24 however, the synthe-
sis of 2,4,6-trimethoxyacetophenone (TMA) from xanth-
oxyline has not been previously described. The
substituted chalcones (1–14) were prepared by magnetic
agitation of TMA (0.25 g; 1.2 mmol), methanol (20 ml),
KOH 50% w/v (5 ml), and the corresponding aldehyde
(1.2 mmol), at room temperature for 24 h. Distilled
water and hydrochloric acid (10%) were added to the
reaction for total precipitation of the compounds. The
compounds were then obtained by vacuum filtration
and later recrystallized in dichloromethane and hexane.
Chalcones 11 and 13 were previously described, respec-
tively, by Phrutivorapongkul et al.11 and by Edwards
et al.20 Compounds 2, 5, 7, 8, 9, 10, 12, and 13 were cited
by Batt et al.,23 and structures 1, 3, 4, 6, and 14 are new
chalcones.
4.2.2. 1: (2E)-3-(2-Chloro-phenyl)-1-(20,40,60-trimethoxy-
phenyl)-2-propen-1-one. C18H17ClO4, light yellow solid;
mp = 112–114 ꢁC; IR mmax/cmꢀ1 1666, 1206 (C@O),
1585 (C@C), 1231, 1019 (C–O), 3015, 2971, 2942,
2844, 1602, 1458, 1440, 1416, 1081, 972, 821, 766 (Ar)
1
(KBr). H NMR (CDCl3) d 3.78 (s, 6H, OCH3), 3.86
(s, 3H, OCH3), 6.16 (s, 2H, H30, H50), 6.91 (d, 1H,
J = 16.0 Hz, Ha), 7.27–7.28 (m, 2H, H4, H5), 7.38 (d,
1H, J = 8.0 Hz, H3), 7.66 (d, 1H, J = 8.0 Hz, H6), 7.77
(d, 1H, J = 16.0 Hz, Hb). 13C NMR (CDCl3) d 55.69
(p0-OCH3), 56.13 (o0-OCH3), 90.97 (C30, C50), 111.66
(C10), 127.27 (Ca), 128.04 (C5), 130.28 (C6), 131.08
(C4), 131.48 (C3), 133.52 (C2), 135.28 (C1), 140.17
(Cb), 159.21 (C20, C60), 162.82 (C40), 194.30 (C@O).
Anal. Calcd for C18H17ClO4: C, 64.97; H, 5.15. Found:
C, 64.20; H, 5.52. Yield = 80%.
4.2.3. 2: (2E)-3-(3-Nitro-phenyl)-1-(20,40,60-trimethoxy-
phenyl)-2-propen-1-one. C18H17NO6, dark yellow solid;
mp: 144–146 ꢁC (lit. p.f.: 146–148 ꢁC); IR mmax/cmꢀ1
1675, 1206 (C@O), 1588 (C@C), 1528, 1352, 855
(N@O), 1231, 1017 (C–O), 2965, 2943, 2842, 1608,
1
1467, 1434, 1413, 1081, 972, 917, 817 (Ar) (KBr). H
NMR (CDCl3) d 3.79 (s, 6H, OCH3), 3.87 (s, 3H,
OCH3), 6.18 (s, 2H, H30, H50), 7.06 (d, 1H,
J = 16.0 Hz, Ha), 7.43 (d, 1H, J = 16.0 Hz, Hb), 7.56
(dd, 1H, J = 7.9 Hz, H5), 7.84 (d, 1H, J = 7.7 Hz, H6),
8.20 (d, 1H, J = 7.9 Hz, H4), 8.35 (s, 1H, H2). 13C
NMR (CDCl3) d 51.80 (p0-OCH3), 52.26 (o0-OCH3),
87.02 (C30, C50), 107.62 (C10), 119.00 (C2), 120.58
(C4), 126.15 (Ca), 127.69 (C5), 130.11 (C6), 133.23
(C1), 136.48 (Cb), 144.90 (C3), 155.42 (C20, C60),
159.18 (C40), 189.35 (C@O). Yield = 43%.
4.2. Physico-chemical data on synthesized compounds
The purified chalcones were obtained in yields between
43% and 97%. The structures were identified using melt-
1
ing points (mp), infrared spectroscopy (IR), H and 13
C
nuclear magnetic resonance (NMR) spectroscopy, and,
for the unpublished ones, also elementary analysis.
´
Melting points were determined with a Microquımica
MGAPF-301 apparatus and are uncorrected. IR spectra
were recorded with an Abb Bomen FTLA 2000 spec-
trometer on KBr disks. NMR (1H and 13C NMR) spec-
tra were recorded on a Brucker Ac-200F (200 MHz) or
Varian Oxford AS-400 (400 MHz) instrument, using tet-
ramethylsilane as an internal standard. Elementary anal-
4.2.4. 3: (2E)-3-(3-Chloro-phenyl)-1-(20,40,60-trimethoxy-
phenyl)-2-propen-1-one. C18H17ClO4, yellow solid;
mp = 133–135 ꢁC; IR mmax/cmꢀ1 1644, 1202 (C@O),
1602 (C@C), 1231, 1022 (C–O), 3014, 2940, 2841,
1
1458, 1412, 969, 888, 825, 792 (Ar) (KBr). H NMR
(CDCl3) d 3.75 (s, 6H, OCH3), 3.83 (s, 3H, OCH3),
6.13 (s, 2H, H30, H50), 6.92 (d, 1H, J = 16.0 Hz, Ha),
7.25–7.30 (m, 2H, H5, H6), 7.27 (d, 1H, J = 16.0 Hz,
Hb), 7.37 (d, 1H, H4), 7.46 (s, 1H, H2). 13C NMR
(CDCl3) d 55.69 (p0-OCH3), 56.14 (o0-OCH3), 90.95
(C30, C50), 111.77 (C10), 126.65 (Ca), 128.29 (C6),
130.14 (C2), 130.27 (C4), 130.35 (C5), 134.99 (C3),
137.16 (C1), 142.18 (Cb), 159.17 (C20, C60), 162.87
(C40), 193.89 (C@O). Anal. Calcd for C18H17ClO4: C,
64.97; H, 5.15. Found: C, 64.67; H, 5.63. Yield = 91%.
ysis was carried out using
a CHNS EA 1110.
Percentages of C and H were in agreement with the
product formula (within 0.4% of theoretical values
for C). The purity of the synthesized chalcones was ana-
lyzed by thin-layer chromatography (TLC) using Merck
silica pre-coated aluminum plates of 200 lm thickness,
with several solvent systems of different polarities. Com-
pounds were visualized with ultraviolet light (k = 254
and 360 nm) and using sulfuric anisaldehyde solution

L. D. Chiaradia et al. / Bioorg. Med. Chem. 16 (2008) 658–667
665
4.2.5. 4: (2E)-3-(2-Nitro-phenyl)-1-(20,40,60-trimethoxy-
phenyl)-2-propen-1-one. C18H17NO6, yellow solid;
mp = 153–155 ꢁC; IR mmax/cmꢀ1 1674, 1207 (C@O),
1603 (C@C), 1233, 1018 (C–O), 1522, 1337, 855
(N@O), 2968, 2943, 2844, 1470, 1439, 1415, 970, 949,
814 (Ar) (KBr). 1H NMR (CDCl3) d 3.80 (s, 6H,
OCH3), 3.85 (s, 3H, OCH3), 6.15 (s, 2H, H30, H50),
6.81 (d, 1H, J = 16.0 Hz, Ha), 7.52 (d, 1H, J = 8.0 Hz,
H6), 7.65 (d, 1H, J = 16.0 Hz, Hb), 7.65–7.72 (m, 2H,
H4, H5), 8.01 (d, 1H, J = 8.0 Hz, H3). 13C NMR
(CDCl3) d 55.68 (p0-OCH3), 56.08 (o0-OCH3), 90.81
(C30, C50), 110.62 (C10), 125.12 (C3), 129.53 (Ca),
130.30 (C6), 131.58 (C4), 133.73 (C1), 133.77 (C5),
140.14 (Cb), 148.51 (C2), 159.18 (C20, C60), 162.98
(C40), 194.69 (C@O). Anal. Calcd for C18H17NO6: C,
62.97; H, 4.99; N, 4.08. Found: C, 62.94; H, 5.65; N,
3.94. Yield = 59%.
(C1), 142.54 (Cb), 159.18 (C20, C60), 162.81 (C40),
194.05 (C@O). Yield = 94%.
4.2.9. 8: (2E)-3-(1-Naphthyl)-1-(20,40,60-trimethoxy-phe-
nyl)-2-propen-1-one. C22H20O4, creamy solid; mp = 172–
174 ꢁC (lit. p.f.: 176–178 ꢁC); IR mmax/cmꢀ1 1640, 1205
(C@O), 1586 (C@C), 1227, 1025 (C–O), 3003, 2942,
2840, 1605, 1466, 1454, 1412, 1082, 974, 818, 798, 771
1
(Ar) (KBr). H NMR (CDCl3) d 3.81 (s, 6H, OCH3),
3.88 (s, 3H, OCH3), 6.20 (s, 2H, H30, H50), 7.05 (d,
1H, J = 16.0 Hz, Ha), 7.47–7.55 (m, 3H, H3, H6, H7),
7.81 (d, 1H, J = 6.8 Hz, H2), 7.86–7.89 (m, 2H, H5,
H8), 8.07 (d, 1H, J = 8.0 Hz, H4), 8.25 (d, 1H,
13
J = 16.0 Hz, Hb). C NMR (CDCl3) d 55.73 (p0-
OCH3), 56.19 (o0-OCH3), 91.02 (C30, C50), 112.04
(C10), 123.64 (Ca), 125.43 (C2, C8), 125.75 (C3),
126.35 (C6), 126.94 (C7), 128.96 (C4), 130.59 (C5),
131.78 (C9), 132.63 (C10), 133.93 (C1), 140.94 (Cb),
159.32 (C20, C60), 162.83 (C40), 194.08 (C@O).
Yield = 97%.
4.2.6. 5: (2E)-3-(4-Nitro-phenyl)-1-(20,40,60-trimethoxy-
phenyl)-2-propen-1-one. C18H17NO6, light yellow solid;
mp = 173–175 ꢁC (lit. p.f.: 176–178 ꢁC); IR mmax/cmꢀ1
1683, 1209 (C@O), 1591 (C@C), 1223, 1019 (C–O),
1513, 1341, 847 (N@O), 2938, 2836, 1613, 1465,
1411, 980, 950, 806 (Ar) (KBr). 1H NMR (CDCl3)
d 3.79 (s, 6H, OCH3), 3.87 (s, 3H, OCH3), 6.16 (s,
2H, H30, H50), 7.06 (d, 1H, J = 16.4 Hz, Ha), 7.43
(d, 1H, J = 16.4 Hz, Hb), 7.66 (d, 2H, H2, H6),
8.23 (d, 2H, H3, H5). 13C NMR (CDCl3) d 55.75
(p0-OCH3), 56.21 (o0-OCH3), 90.97 (C30, C50),
110.99 (C10), 124.31 (C3, C5), 129.05 (C2, C6),
132.65 (Ca), 140.05 (C1), 141.71 (Cb), 148.51 (C4),
159.47 (C20, C60), 163.21 (C40), 193.01 (C@O).
Yield = 79%.
4.2.10. 9: (2E)-3-(2,6-Dichloro-phenyl)-1-(20,40,60-tri-
methoxy-phenyl)-2-propen-1-one. C18H16Cl2O4, beige so-
lid; mp = 115–117 ꢁC (lit. p.f.: 119–121 ꢁC); IR mmax
/
cmꢀ1 1649, 1206 (C@O), 1590 (C@C), 1231, 1028 (C–
O), 3009, 2943, 2842, 1604, 1556, 1458, 1414, 1080,
1
975, 820, 776 (Ar) (KBr). H NMR (CDCl3) d 3.79 (s,
6H, OCH3), 3.85 (s, 3H, OCH3), 6.15 (s, 2H, H30,
H50), 7.08 (d, 1H, J = 16.4 Hz, Ha), 7.15 (dd, 1H,
J = 8.0 Hz, H4), 7.33 (d, 2H, J = 8.0 Hz, H3, H5), 7.49
(d, 1H, J = 16.4 Hz, Hb). 13C NMR (CDCl3) d 55.69
(p0-OCH3), 56.12 (o0-OCH3), 90.88 (C30, C50), 111.44
(C10), 128.97 (C3, C5), 129.77 (Ca), 132.94 (C4),
135.28 (C2, C6), 136.91 (C1), 137.13 (Cb), 159.53 (C20,
C60), 163.02 (C40), 193.80 (C@O). Yield = 63%.
4.2.7. 6: (2E)-3-(3,4-Dichloro-phenyl)-1-(20,40,60-trimeth-
oxy-phenyl)-2-propen-1-one. C18H16Cl2O4, light yellow
solid; mp = 125–127 ꢁC; IR mmax/cmꢀ1 1672, 1205
(C@O), 1587 (C@C), 1229, 1018 (C–O), 2940, 2800,
1603, 1467, 1412, 969, 948, 839, 808 (Ar) (KBr). 1H
NMR (CDCl3) d 3.77 (s, 6H, OCH3), 3.86 (s, 3H,
OCH3), 6.15 (s, 2H, H30, H50), 6.92 (d, 1H,
J = 16.0 Hz, Ha), 7.27 (d, 1H, J = 16.0 Hz, Hb), 7.35
(d, 1H, H5), 7.43 (d, 1H, H6), 7.58 (s, 1H, H2). 13C
NMR (CDCl3) d 55.71 (p0-OCH3), 56.18 (o0-OCH3),
90.98 (C30, C50), 111.76 (C10), 127.44 (Ca), 130.12
(C6), 130.65 (C2), 131.01 (C5), 133.32 (C4), 134.11
(C3), 135.46 (C1), 140.84 (Cb), 159.27 (C20, C60),
162.97 (C40), 193.54 (C@O). Anal. Calcd for
C18H16Cl2O4: C, 58.87; H, 4.39. Found: C, 59.51; H,
5.04. Yield = 89%.
4.2.11. 10: (2E)-3-(2-Naphthyl)-1-(20,40,60-trimethoxy-
phenyl)-2-propen-1-one. C22H20O4, light yellow solid;
mp = 139–141 ꢁC (lit. p.f.: 145–146 ꢁC); IR mmax/cmꢀ1
1639, 1206 (C@O), 1588 (C@C), 1231, 1027 (C–O),
3002, 2945, 2845, 1622, 1605, 1459, 1415, 1083, 980,
1
859, 826 (Ar) (KBr). H NMR (CDCl3) d 3.78 (s, 6H,
OCH3), 3.87 (s, 3H, OCH3), 6.18 (s, 2H, H30, H50),
7.07 (d, 1H, J = 16.4 Hz, Ha), 7.48–7.55 (m, 4H, H3,
H6, H7, Hb), 7.70 (d, 1H, J = 8.4 Hz, H4), 7.81–7.83
(m, 2H, H5, H8), 7.90 (s, 1H, H1). 13C NMR (CDCl3)
d 55.71 (p0-OCH3), 56.19 (o0-OCH3), 91.01 (C30, C50),
112.11 (C10), 124.09 (Ca), 126.86 (C3), 127.39 (C6),
128.00 (C7), 128.75 (C1), 128.80 (C5), 129.49 (C8),
130.49 (C4), 132.83 (C10), 133.55 (C9), 134.45 (C2),
144.49 (Cb), 159.14 (C20, C60), 162.68 (C40), 194.53
(C@O). Yield = 88%.
4.2.8. 7: (2E)-3-(4-Chloro-phenyl)-1-(20,40,60-trimethoxy-
phenyl)-2-propen-1-one. C18H17ClO4, light yellow solid;
mp = 128–130 ꢁC (lit. p.f.: 132–133 ꢁC); IR mmax/cmꢀ1
1672, 1208 (C@O), 1606 (C@C), 1229, 1017 (C–O),
3005, 2965, 2961, 2835, 1568, 1472, 1451, 1402, 1088,
4.2.12. 11: (2E)-3-(1,3-Benzodioxol-5-yl)-1-(20,40,60-tri-
methoxy- phenyl)-2-propen-1-one. C19H18O6, creamy so-
lid; mp = 124–126 ꢁC (lit. p.f.: 143–144 ꢁC); IR mmax
/
1
972, 952, 809 (Ar) (KBr). H NMR (CDCl3) d 3.77 (s,
cmꢀ1 1639, 1206 (C@O), 1599 (C@C), 1264, 1034 (C–
O), 3009, 2938, 2839, 1503, 1467, 1451, 1417, 976, 925,
6H, OCH3), 3.85 (s, 3H, OCH3), 6.16 (s, 2H, H30,
H50), 6.92 (d, 1H, J = 16.0 Hz, Ha), 7.32 (d, 1H,
J = 16.0 Hz, Hb), 7.33 (d, 2H, H2, H6), 7.44 (d, 2H,
1
824, 807 (Ar) (KBr). H NMR (CDCl3) d 3.79 (s, 6H,
OCH3), 3.86 (s, 3H, OCH3), 5.99 (s, 2H, –OCH2O–),
6.10 (s, 1H, H2), 6.15 (s, 2H, H30, H50), 6.80 (d, 1H,
J = 15.4 Hz, Ha), 6.98 (d, 2H, J = 8.0 Hz, H5), 7.05
(d, 2H, H6), 7.28 (d, 1H, J = 14.8 Hz, Hb). 13C NMR
13
H3, H5). C NMR (CDCl3) d 55.70 (p0-OCH3), 56.18
(o0-OCH3), 90.98 (C30, C50), 111.94 (C10), 129.31 (Ca),
129.66 (C3, C5), 129.74 (C2, C6), 133.82 (C4), 136.23

666
L. D. Chiaradia et al. / Bioorg. Med. Chem. 16 (2008) 658–667
(CDCl3) d 55.70 (p0-OCH3), 56.16 (o0-OCH3), 90.91
(C30, C50), 101.76 (–OCH2O–), 107.02 (C2), 108.74
(C5), 112.12 (C10), 125.08 (C6), 127.51 (Ca), 129.68
(C1), 144.33 (Cb), 148.51–149.81 (C3, C4), 159.01
(C20, C60), 162.56 (C40), 194.44 (C@O). Anal. Calcd
for C19H18O6: C, 66.66; H, 5.30. Found: C, 65.62; H,
5.97. Yield = 78%.
Janeiro, RJ, Brazil) were maintained at 37 ꢁC in DMEM
(GIBCO BRL, USA) supplemented with 10% heat-inac-
tivated calf serum (GIBCO BRL, USA), glucose (4.5 g/l),
sodium bicarbonate (1.5 g/l), L-glutamine (2 mM), strep-
tomycin (100 lg/ml), and penicillin (100 U/ml) in a
humidified atmosphere of 5% CO2.
4.3.2. Nitrite quantification. Cells were plated at
1 · 106 cells/ml in 96-well culture plates. After 2 h of
incubation at 37 ꢁC non-adherent cells were removed
by washing the plates repeatedly. Compounds in various
concentrations (as indicated in the graphs) were then
added to the plates. Thirty minutes later cells were stim-
ulated with LPS (100 gg/ml) for 24 h. The isolated
supernatants (100 ll) were mixed with 100 ll of Gries re-
agent (1% sulfanilamide, 0.1% naphthylethylenediamine
dihydrochloride in 2% phosphoric acid) and incubated
at room temperature for 5 min. The concentration of ni-
trite was measured by OD reading at 550 nm using a
NaNO2 standard curve. CI50 was calculated using
Graphpad Prism Software.
4.2.13. 12: (2E)-3-(4-Methoxy-phenyl)-1-(20,40,60-tri-
methoxy-phenyl)-2-propen-1-one. C19H20O5, creamy
solid; mp = 112–114 ꢁC (lit. p.f.: 116–118 ꢁC); IR mmax
/
cmꢀ1 1673, 1203 (C@O), 1598 (C@C), 1256, 1026
(C–O), 2961, 2938, 2837, 1635, 1512, 1467, 1420, 1081,
1
979, 820, 804 (Ar) (KBr). H NMR (CDCl3) d 3.76 (s,
6H, OCH3), 3.84 (s, 3H, OCH3), 3.86 (s, 3H, OCH3),
6.16 (s, 2H, H30, H50), 6.84 (d, 1H, J = 14.8 Hz, Ha),
6.89 (d, 2H, H3, H5), 7.31 (d, 1H, J = 14.8 Hz, Hb),
7.47 (d, 2H, H2, H6). 13C NMR (CDCl3) d 55.62
(p-OCH3), 55.69 (p0-OCH3), 56.16 (o0-OCH3), 90.92 (C30,
C50), 112.15 (C10), 114.50 (C3, C5), 127.21 (Ca), 127.91
(C1), 130.35 (C2, C6), 144.56 (Cb), 158.96 (C4), 161.62
(C20, C60), 162.48 (C40), 194.73 (C@O). Yield = 84%.
4.3.3. Cell viability. MTT (3-[4,5-dimethyl-2-thiazolyl]-
2,5-diphenyl tetrazolium bromide) is a substrate which
produces a dark blue formazan product when incubated
with living cells by cleavage of the MTT ring in active
mitochondria. An MTT assay was used to measure cell
viability following treatment with indicated compounds.
After removal of the supernatants, the cells were incu-
bated with MTT (0.05 mg/ml) at 37 ꢁC for at least 4 h.
Supernatants were then gently removed and 100 ll of
dimethylsulfoxide (DMSO) was added to the wells.
After 15 min of gently mixing the absorbance was deter-
mined at 550 nm. Naive cells were considered as 100% of
viability.
4.2.14. 13: (2E)-3-[4-(Dimethylamino)phenyl]-1-(20,40,60-
trimethoxy-phenyl)-2-propen-1-one. C20H23NO4, yellow
solid; mp = 148–150 ꢁC (lit. p.f.: 153–155, 149–150 ꢁC);
IR mmax/cmꢀ1 2994, 2937, 2839, 1628, 1210 (C@O),
1597 (C@C), 1230, 1029 (C–O), 1304 (C–N), 1529,
1
1453, 1442, 1413, 1084, 968, 945, 813 (Ar) (KBr). H
NMR (CDCl3) d 3.01 (s, 6H, N–CH3), 3.76 (s, 6H,
OCH3), 3.86 (s, 3H, OCH3), 6.16 (s, 2H, H30, H50),
6.65 (d, 2H, H3, H5), 6.78 (d, 1H, J = 16.0 Hz, Ha),
7.27 (d, 1H, J = 16.0 Hz, Hb), 7.41 (d, 2H, H2, H6).
13C NMR (CDCl3) d 40.38 (N–CH3), 55.68 (p0-OCH3),
56.15 (o0-OCH3), 90.91 (C30, C50), 111.99 (C3, C5),
112.49 (C10), 122.84 (Ca), 124.67 (C1), 130.47 (C2,
C6), 146.20 (Cb), 152.05 (C4), 158.80 (C20, C60),
162.19 (C40), 194.95 (C@O). Yield = 53%.
4.3.4. Statistical analysis. All data are expressed as
means SEM. Statistical significance of differences be-
tween the groups was determined by One-way ANOVA
followed by the Bonferroni test. Statistical analysis and
calculation of IC50 values were performed using Graph-
Pad Prism 4 software (GraphPad Software Inc., San
Diego, CA).
4.2.15. 14: (2E)-3-(4-Butoxy-phenyl)-1-(20,40,60-trimeth-
oxy-phenyl)-2-propen-1-one. C22H26O5, creamy solid;
mp = 110–112 ꢁC; IR mmax/cmꢀ1 1667, 1204 (C@O),
1587 (C@C), 1228, 1021 (C–O), 2946, 2871, 2840,
1562, 1508, 1457, 1413, 1062, 980, 947, 824 (Ar)
1
(KBr). H NMR (CDCl3) d 0.93–1.02 (m, 3H, –CH3),
4.4. Quantitative structure–activity relationships—com-
putational methods
1.43–1.57 (m, 2H, –CH2–CH3), 1.63–1.83 (m, 2H,
–CH2–CH2–CH3), 3.76 (s, 6H, OCH3), 3.85 (s, 3H,
OCH3), 3.98 (t, 2H, –O–CH2–), 6.16 (s, 2H, H30, H50),
6.83 (d, 1H, J = 16.7 Hz, Ha), 6.87 (d, 2H, J = 8.7 Hz,
H3, H5), 7.30 (d, 1H, J = 16.7 Hz, Hb), 7.45 (d,
2H,J = 8.7 Hz, H2, H6). 13C NMR (CDCl3) d 14.05
(–CH3), 19.43 (–CH2–CH3), 31.42 (–CH2–CH2–CH3),
55.68 (p0-OCH3), 56.15 (o0-OCH3), 68.06 (–O–CH2–),
90.97 (C30, C50), 112.23 (C10), 115.01 (C3, C5), 127.07
(Ca), 127.67 (C1), 130.33 (C2, C6), 144.68 (Cb),
158.96 (C20, C60), 161.28 (C4), 162.48 (C40), 194.72
(C@O). Anal. Calcd for C22H26O5: C, 71.33; H, 7.07.
Found: C, 67.54; H, 7.36. Yield = 92%.
Initially, a conformational search was performed for all
chalcones by means of the ‘conformer distribution’ re-
source implemented in the Spartan’04 package30 with
a semi-empirical AM1 level of theory.31 Chalcones 1–
13 were submitted to the systematic method. For chal-
cone 14, the Monte Carlo method had to be employed,
since the systematic method has been shown to be more
time-consuming. The default settings were used for chal-
cones 15–48 (in this case, the choice of the systematic or
Monte Carlo method depended on the complexity of the
molecule). After optimization of all conformers, the
lowest energy conformers of each chalcone were opti-
mized using the AM1 method and a gradient tolerance
of 8.4 · 10ꢀ6 Hartrees Bohrꢀ1. The eigenvalues and
eigenvectors, printed in log files, were used in the calcu-
lation of the reactivity indices of HOMO and LUMO
4.3. Biological assays
4.3.1. Cell culture. RAW 264.7 cells (ATCC TIB71) pur-
´
chased from Banco de Celulas do Rio de Janeiro (Rio de

L. D. Chiaradia et al. / Bioorg. Med. Chem. 16 (2008) 658–667
667
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over each atom ‘i’, RH(i) and RL(i), respectively. These
are calculated as follows
frEðiÞ
X
2
RHðiÞ ¼
RLðiÞ ¼
ꢂ 100 frEðiÞ ¼
ðCHOMO;nÞ
ꢀEHOMO
frNðiÞ
X
2
ꢂ 100 frNðiÞ ¼
ðCLUMO;nÞ
ELUMO ꢀ EHOMO
10. Boeck, P.; Leal, P. C.; Yunes, R. A.; Cechinel-Filho, V.;
where frE and frN are the electrophilic and nucleophilic
atomic frontier electron densities, respectively, and CHO-
MO,n and CLUMO,n are the coefficients of the atomic orbi-
tal Xn in the HOMO and LUMO, respectively. In order
to better represent quantitatively the contributions of
these orbitals in two distinct parts of the molecules,
´
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P
P
the sums of the indices RH and RL,
were calculated over the 2,4,6-trimethoxyacetophenone
RH and
RL,
moiety (TMA, Fig. 3) and the variable radical moiety
RHðTMAÞ,
P
P
(R, Fig. 3), which gave
RLðTMAÞ,
P
P
RHðRÞ, and
RLðRÞ. These summations were used
as QSAR descriptors, since high correlations between
RH and RL arise for various atoms of the same moiety.
Other descriptors taken into account were LogP, sur-
face area, molecular volume, the energies of the HOMO
and LUMO in electron volt, EHOMO and ELUMO, and
their difference, DE (=ELUMO ꢀ EHOMO). The QSAR
equations and statistics were calculated with the BuildQ-
SAR program.32
´
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Acknowledgments
This study was supported by grants and fellowships
from CNPq (Conselho Nacional de Desenvolvimento
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´
Cientıfico e Tecnologico). This is part of the research
of L.D. Chiaradia who is a doctorate student in Chem-
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tively, a doctorate in Chemistry and master’s degree in
Pharmacology at UFSC. We thank the Department of
Chemistry of UFSC (Universidade Federal de Santa
Catarina) for the chemical analysis.
´
´
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