Antimycobacterial and anti-inflammatory activities of substituted chalcones focusing on an anti-tuberculosis dual treatment approach

Article abstract of DOI:10.3390/molecules20058072

Tuberculosis (TB) remains a serious public health problem aggravated by the emergence of M. tuberculosis (Mtb) strains resistant to multiple drugs (MDR). Delay in TB treatment, common in the MDR-TB cases, can lead to deleterious life-threatening inflammation in susceptible hyper-reactive individuals, encouraging the discovery of new anti-Mtb drugs and the use of adjunctive therapy based on anti-inflammatory interventions. In this study, a series of forty synthetic chalcones was evaluated in vitro for their anti-inflammatory and antimycobacterial properties and in silico for pharmacokinetic parameters. Seven compounds strongly inhibited NO and PGE₂ production by LPS-stimulated macrophages through the specific inhibition of iNOS and COX-2 expression, respectively, with compounds 4 and 5 standing out in this respect. Four of the seven most active compounds were able to inhibit production of TNF-α and IL-1β. Chalcones that were not toxic to cultured macrophages were tested for antimycobacterial activity. Eight compounds were able to inhibit growth of the M. bovis BCG and Mtb H37Rv strains in bacterial cultures and in infected macrophages. Four of them, including compounds 4 and 5, were active against a hypervirulent clinical Mtb isolate as well. In silico analysis of ADMET properties showed that the evaluated chalcones displayed satisfactory pharmacokinetic parameters. In conclusion, the obtained data demonstrate that at least two of the studied chalcones, compounds 4 and 5, are promising antimycobacterial and anti-inflammatory agents, especially focusing on an anti-tuberculosis dual treatment approach.

Full text of DOI:10.3390/molecules20058072

Molecules 2015, 20, 8072-8093; doi:10.3390/molecules20058072
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Antimycobacterial and Anti-Inflammatory Activities of
Substituted Chalcones Focusing on an Anti-Tuberculosis Dual
Treatment Approach
Thatiana Lopes Biá Ventura 1,2, Sanderson Dias Calixto 1, Bárbara de Azevedo Abrahim-Vieira 3,
Alessandra Mendonça Teles de Souza 3, Marcos Vinícius Palmeira Mello 4,
Carlos Rangel Rodrigues 3, Leandro Soter de Mariz e Miranda 5,
Rodrigo Octavio Mendonça Alves de Souza 5, Ivana Correa Ramos Leal 3,
Elena B. Lasunskaia 1,†,* and Michelle Frazão Muzitano 2,†,
*
1
Laboratório de Biologia do Reconhecer, Centro de Biociências e Biotecnologia,
Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes 28013-602, RJ,
Brazil; E-Mails: thativentura@yahoo.com.br (T.L.B.V.); sandersoncalixto@yahoo.com.br (S.D.C.)
Laboratório de Produtos Bioativos, Curso de Farmácia, Universidade Federal do Rio de Janeiro,
Campus Macaé, Pólo Novo Cavaleiro—IMMT, Macaé 27933-378, RJ, Brazil
Faculdade de Farmácia, Universidade Federal do Estado do Rio de Janeiro,
Rio de Janeiro 21941-901, RJ, Brazil; E-Mails: babi_abrahim@hotmail.com (B.A.A.-V.);
amtsouza2@yahoo.com.br (A.M.T.S.); rangelfarmacia@gmail.com (C.R.R.);
ivanafarma@yahoo.com.br (I.C.R.L.)
2
3
4
5
Instituto de Química, Universidade Federal Fluminense, Niterói, Rio de Janeiro 24020141, RJ,
Brazil; E-Mail: mvpmello@id.uff.br
Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-909, RJ,
Brazil; E-Mails: leandrosoter@iq.ufrj.br (L.S.M.M.); souzarod21@gmail.com (R.O.M.A.S.)
†
E.B.L. and M.F.M. authors are joint senior authors on this work.
* Authors to whom correspondence should be addressed;
E-Mails: elassounskaia@gmail.com (E.B.L.); mfmuzitano@gmail.com (M.F.M.);
Tel.: +55-222739-7128 (E.B.L.); +55-222796-2539 (M.F.M.).
Academic Editor: Derek J. McPhee
Received: 28 February 2015 / Accepted: 24 April 2015 / Published: 5 May 2015
Abstract: Tuberculosis (TB) remains a serious public health problem aggravated by the
emergence of M. tuberculosis (Mtb) strains resistant to multiple drugs (MDR). Delay in TB

Molecules 2015, 20
treatment, common in the MDR-TB cases, can lead to deleterious life-threatening
8073
inflammation in susceptible hyper-reactive individuals, encouraging the discovery of new
anti-Mtb drugs and the use of adjunctive therapy based on anti-inflammatory interventions.
In this study, a series of forty synthetic chalcones was evaluated in vitro for their
anti-inflammatory and antimycobacterial properties and in silico for pharmacokinetic
parameters. Seven compounds strongly inhibited NO and PGE2 production by LPS-stimulated
macrophages through the specific inhibition of iNOS and COX-2 expression, respectively,
with compounds 4 and 5 standing out in this respect. Four of the seven most active
compounds were able to inhibit production of TNF-α and IL-1β. Chalcones that were not
toxic to cultured macrophages were tested for antimycobacterial activity. Eight compounds
were able to inhibit growth of the M. bovis BCG and Mtb H37Rv strains in bacterial
cultures and in infected macrophages. Four of them, including compounds 4 and 5, were
active against a hypervirulent clinical Mtb isolate as well. In silico analysis of ADMET
properties showed that the evaluated chalcones displayed satisfactory pharmacokinetic
parameters. In conclusion, the obtained data demonstrate that at least two of the studied
chalcones, compounds 4 and 5, are promising antimycobacterial and anti-inflammatory
agents, especially focusing on an anti-tuberculosis dual treatment approach.
Keywords: tuberculosis; Mycobacterium; inflammation; chalcone
1. Introduction
Inflammation is an essential protective response to a variety of noxious stimuli and conditions, such
as infection and tissue injury. The inflammatory reaction, characterised by vasodilatation and recruitment
of leukocytes into the target tissue, is coordinated by inflammatory mediators produced mainly by
macrophages and monocytes, highlighting tumor necrosis factor (TNF), interleukin-1 (IL-1), nitric
oxide (NO) and prostaglandins, including PGE2 [1–4].
A potentially beneficial role of inflammation in elimination of invaders could be abrogated by tissue
damage in cases of excessive inflammatory response. In chronic infectious diseases, such as
tuberculosis, exacerbated inflammation contributes to severe lung pathology, leading to lung tissue
necrosis, cavities formation and the promotion of mycobacterial dissemination and transmission.
Anti-inflammatory drugs, especially corticosteroids, are currently used for adjunctive therapy in most
severe life-threatening forms of tuberculosis, such as meningitis and pericarditis, while antibiotics are
used to kill the bacteria [5].
Beneficial effects of adjunctive corticosteroids or non-steroid anti-inflammatory drugs (NSAIDs) in
the treatment of the severe forms of pulmonary tuberculosis remain uncertain and need new
randomised controlled trials. An increasing body of evidence demonstrates that anti-inflammatory
therapy reduces mortality in patients exhibiting the hyperinflammatory phenotype that could be
determined by host genetic polymorphisms, increased bacterial virulence or specific comorbid states,
such as Tuberculosis-Immune Reconstitution Inflammatory Syndrome (TB-IRIS) in patients with TB
and AIDS [6–8].

Molecules 2015, 20
8074
The importance of developing new drugs with dual anti-inflammatory and antimycobacterial,
activities is highlighted by the emergence of increasing prevalence of multidrug resistant (MDR) TB
and extensively drug-resistant (XDR) TB. Drugs with these properties are currently represented by the
anti-leprosy drug clofazimine [9] and they are in short supply for tuberculosis, motivating the design
and search for novel agents.
Natural and synthetic flavonoids are known for displaying a broad spectrum of pharmacological
activities. Chalcones (1,3-diaryl-2-propene-1-ones) and other biogenetically-related compounds
belonging to the flavonoid family are natural substances found in a number of plants or prepared
synthetically. They consist of two aromatic rings joined by a three-carbon α,β-unsaturated carbonyl
system [10]. Chalcones have been found to exhibit many pharmacological activities, including
anti-inflammatory [11–13] and anti-tuberculosis [14] activities. The reported anti-inflammatory effects
of some chalcones have been associated with suppression of inflammatory mediators, such as NO,
TNF-α, IL-1β, cyclooxygenase-2 (COX-2) and inducible oxide nitric synthase (iNOS) [12,13,15]. A
number of chalcones were shown to have a high inhibitory activity against in vitro growth of laboratory
M. tuberculosis strains H37Rv when used in low concentrations [14,16–19] and, additionally,
demonstrated low cytotoxicity against human cells in toxicity tests [12]. A combination of these
properties in one compound could provide an important advantage for chalcones as potential
anti-tuberculosis drugs, but chalcones exhibiting both anti-inflammatory and antimycobacterial
properties have not yet been identified.
In this study, we screened a series of substituted chalcones for their anti-inflammatory properties,
evaluating immunomodulatory effects on LPS-stimulated macrophages, and for their
antimycobacterial properties, evaluating inhibitory effects on bacterial growth of avirulent and virulent
mycobacterial strains in culture or in infected macrophages. The active chalcones exhibiting dual
activities, were submitted to in silico analysis of absorption, distribution, metabolism, excretion and
toxicology (ADMET) in order to select promising new drugs. In addition, a structure-activity
relationship (SAR) study was performed by modelling to predict molecular descriptors for each studied
structure and to gain a better understanding of their antimycobacterial activity.
2. Results and Discussion
2.1. Results
In this study, we evaluated the anti-inflammatory, antimycobacterial and ADMET properties of a
set of forty synthetic substituted chalcones 2–41 (Table 1) in comparison to a commercially available
unsubstituted chalcone (compound 1). Initially, compounds 1–41 were evaluated for their
immunomodulatory activity in LPS-stimulated RAW 264.7 macrophage culture, focusing on NO and
TNF-α production levels. For each compound a concentration-response curve was plotted, and the IC50
values are reported in Table 2. The obtained results show that chalcones 1–41 were more potent
inhibitors of NO than TNF-α. More pronounced inhibitory activity against NO was presented by
compounds 4, 5, 12, 24, 28, 29, 31, 33, 40 and 41; with IC50 values lower than 21 µM. The activity of
compound 5 with an IC50 of 2.1 ± 0.7 µM was particularly noteworthy.

Molecules 2015, 20
Table 1. Structures of representative unsubstituted chalcone and synthesized chalcones
8075
with substituents on the A and B rings.
O
2'
2
β
1
1'
6'
3
4
3'
α
B
A
4'
6
5'
5
B Ring
A Ring
Chalcone
2' 3'
4'
H
2
3
4
H
F
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
N(CH3)2
-O(CH2)O-
H
H
H
H
H
H
OCH3
H
H
F
Cl
N (pyridine ring)
Br
Br
Br
Br
Br
Br
Br
Br
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
Br
CH3
OCH3
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
9
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
N(CH3)2
-O(CH2)O-
H
OCH3
OCH3 OCH3
H
H
H
H
H
H
H
F
Cl
N(CH3)2
-O(CH2)O-
H
OCH3
OCH3 OCH3
H
H
H
H
H
H
H
F
Cl
N(CH3)2
-O(CH2)O-
H
H
H
H
H
H
OCH3
H
H
F
Cl
OCH3
-O(CH2)O-
OCH3 OCH3
H
H
H
H
H
NO2
NO2
NO2
NO2
NO2

Molecules 2015, 20
Table 2. Inhibitory effects of chalcones on production of NO and TNF-α by LPS-stimulated
8076
RAW 264.7 macrophages, on growth of M. bovis BCG in culture and evaluation of
chalcone cytotoxicity by LDH test.
IC50 (µM)
TNF-α
MIC50 (µM) MIC90 (µM)
Chalcone
NO
LDH
M. bovis BCG
1
2
3
4
5
6
7
8
41.6 ± 2.0
>442.0
31.9 ± 4.6 60.5 ± 4.7 399.7 ± 4.9
12.0 ± 2.2 66.9 ± 4.3 348.4 ± 5.1
2.1 ± 2.7
>419.7
>477.9
>480.2
>442.0
177.3 ± 9.5 137.3 ± 4.7
383.2 ± 9.1
>442.0
>442.0
>442.0
20.7 ± 6.4
14.3 ± 5.8
14.5 ± 6.1
255.9 ± 4.8
35.8 ± 7.2
13.4 ± 4.0
35.8 ± 3.9
22.3 ± 6.3
67.8 ± 3.8
84.9 ± 4.7
220.6 ± 3.6
>288.0
169.9 ± 5.4
137.3 ± 7.9
75.3 ± 8.4
249.2 ± 6.2
440.8 ± 9.5
33.7 ± 4.6
308.8 ± 5.3
287.8 ± 3.2
242.1 ± 2.6
229.6 ± 2.2
>315.3
>288.0
265.1 ± 4.5
>449.9
388.4 ± 7.3
323.2 ± 7.1
322.7 ± 1.7
>375.5
362.2 ± 5.9
>354.2
48.1 ± 7.6
>419.7
>477.9
>396.4
>419.7
>477.9
46.4 ± 2.3 66.9 ± 4.5 191.0 ± 5.2
9
>327.7
68.9 ± 4.3
41.3 ± 3.5
19.6 ± 3.3
52.9 ± 3.6
58.9 ± 6.8
36.7+3.8
>449.9
30.8 ± 4.5
150.3 ± 3.8
27.8 ± 3.8
30.1 ± 1.2
30.6 ± 4.7
>354.2
>327.7
>310.9
>302.8
>302.0
>315.3
>288.0
>311.0
>449.9
>416.2
>389.5
>376.9
>375.5
>396.3
>354.2
>389.5
>327.7
113.4 ± 0.5
132.0 ± 2.9
>302.0
>315.3
>288.0
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
256.0 ± 3.8
>449.9
13.8 ± 4.4
>449.9
289.5 ± 4.3
304.3 ± 4.2
>376.9
61.5 ± 7.6
71.3 ± 4.8
56.4 ± 4.4
>375.5
>375.5
>396.3
>354.2
33.3 ± 5.4
>354.2
>389.5
>389.5
>389.5
>389.5
21.3 ± 4.5 78.8 ± 6.5 244.8 ± 4.5
31.5 ± 5.9
15.8 ± 5.2
91.2 ± 5.0
399.1 ± 6.9
228.3 ± 5.8
286.7 ± 7.8
>355.4
41.2 ± 4.6
25.3 ± 3.9
44.6 ± 5.2
14.9 ± 4.1
13.1 ± 4.2
23.9 ± 4.8
8.4 ± 6.8
>383.6
>390.2
>366.7
>355.4
>354.2
>372.7
>366.7
>412,0
>383.6
>390.2
>366.7
226.4 ± 3.5 308.8 ± 5.0
>354.2
93.9 ± 4.9
100.0 ± 5.1
267.0 ± 4.5
>383.6
>354.2
32.5 ± 5.4
37.8 ± 6.4
90.9 ± 5.5
>383.6
>354.2
339.5 ± 3.2
328.7 ± 3.6
330.2 ± 4.2
>383.6
>360.8
>366.7
>348.8
>330.3
>394.9
220.7 ± 5.6
344.1 ± 6.3
>353.0
246.9 ± 7.2
XX
3.1 ± 0.1 165.5 ± 5.2 351.2 ± 5.0
45.7 ± 5.4
>366.7
>348.8
269.6 ± 3.9
>394.9
97.4 ± 4.2
28.1 ± 5.4
14.5 ± 3.1
30.5 ± 4.7
XX
>366.7
160.3 ± 4.7
187.8 ± 4.1
58.6 ± 4.5
>301.1
113.7 ± 4.8
13.5 ± 5.5
11.2 ± 5.0 68.54 ± 3.3
>366.7
>348.8
>330.3
>394.9
>301.1
>373.2
>353.0
>366.7
>348.8
>330.3
>394.9
>301.1
328.6 ± 3.9
>353.0
>347.6
XX
L-NMMA 1 78.3 ± 6.5
Rifampicin 2
XX
XX
XX
XX
0.01 ± 0.03
0.2 ± 0.01
1 Standard nitric oxide inhibitor; 2 Standard antimycobacterial drug; Mean value ± SD; n = 3; XX—not defined.

Molecules 2015, 20
8077
It should be noted that the inhibitory activities of the substituted chalcone compounds more active
against NO were higher than those of the unsubstituted chalcone (IC50 of 41.6 ± 2.0 µM) or
L-NMMA, which is a competitive NO inhibitor used as a positive control (IC50 of 78.3 ± 6.5 µM).
Some of these chalcones (compounds 4, 5, 24, 41) were able to strongly inhibit TNF-α as well
(Table 2). Compound 5 was notably the most potent inhibitor of TNF-α and NO production (p < 0.05)
when compared to other compounds.
In order to investigate the cytotoxicity of the studied compounds, the levels of intracellular LDH,
released in culture supernatants by chalcone-treated macrophages were measured. Compounds 10, 11,
29 and 30 showed the worst cytotoxicity profiles (Table 2) and were excluded from further analyses.
As a part of our initial screening strategy, the compounds 1–41 were assessed for their
antimycobacterial activity in Mycobacterium bovis BCG culture. The vaccine strain was used for the
screening experiments considering biosecurity reasons. Growth inhibition of mycobacteria cultured in
the presence of chalcones was quantified and results are presented in Table 2 as MIC50 and MIC90. The
most potent inhibitory effect was obtained using compounds 5 and 8 that showed the lowest MIC50 and
MIC90 levels (14.5 ± 6.1 and 75.3 ± 8.4 µM for 5; 13.4 ± 4.0 and 33.7 ± 4.6 µM for 8). It should be
noted that none of the tested compounds was more active than rifampicin, a standard anti-TB drug
used as a positive control.
Additionally, we evaluated the capacity of chalcones to inhibit production of IL-1β and PGE2,
induced in macrophages by LPS. For this study, we selected eighteen chalcones that had shown high
activity in the NO inhibition test and were less cytotoxic than compound 1 (a non-substituted chalcone
used as a reference compound). Figure 1 shows that inhibitory activity of these chalcones against
IL-1β production was relatively low. Only five of these compounds (compounds 3, 4, 5, 24 and 41)
were potent inhibitors of IL-1β. In contrast, the inhibitory activity of most compounds against PGE2
was high, with the activity profiles of compounds 3, 4, 5, 28 and 31 standing out.
Seeking to learn more about the mechanism of NO and PGE2 inhibition by chalcones, we studied
the expression of iNOS and COX-2 in macrophages treated with LPS and chalcone compounds
exhibiting the strongest inhibitory activities against NO and PGE2, respectively. As can be seen in
Figure 2, compounds 3, 5, 4, 31 and 33, when used at a concentration of 20 µg/mL, were able to
almost completely inhibit iNOS. Some of these chalcones (compounds 3, 5 and 31) were most potent
in abrogation of COX-2 as well. These results demonstrate that the inhibitory effects of chalcones on
NO and PGE2 production are mediated by suppression of iNOS and COX-2 expression, respectively.
The second aim of this work was to investigate the antimycobacterial activities of chalcones against
pathogenic M. tuberculosis. The chalcone compounds presenting inhibitory activities in the screening
test for BCG growth inhibition (Table 2) were selected for the further study against laboratory
M. tuberculosis strain H37Rv and a highly virulent M. tuberculosis clinical isolate, such as strain
M299 belonging to the modern M. tuberculosis Beijing sublineage [20]. Sixteen compounds were
assessed for the inhibitory activity against M. tuberculosis H37Rv. The results presented in Table 3
show that most compounds were active against this strain, presenting MIC50 values similar to those
used against BCG (Table 2), despite a decreasing activity when analyzing MIC90 values. It should be
noted that the compounds most active against BCG, 5 and 8 (MIC90 75.3 ± 8.4 μM and 33.7 ± 4.6 μM,
respectively), were less potent against the virulent H37Rv strain (MIC90 303.2 ± 5.4 μM and
344.7 ± 0.4 μM, respectively).

Molecules 2015, 20
8078
Figure 1. Effect of substituted chalcones on production of proinflammatory mediators (IL-1β
and PGE2) by LPS-stimulated RAW 264.7 macrophages. RAW 264.7 cells were treated
with LPS (1 µg/mL) in the presence of chalcones (0.8, 4, 20 e 100 µg/mL, the lowest
concentration was not used in the PGE assay) for 24 h. The culture supernatants were
collected and tested by sandwich ELISA for IL-1β (A) and PGE2 (B). In the IL-1β test,
cytokine values determined in untreated and only LPS-treated cell cultures were
0.03 ± 0.1 pg/mL and 922.0 ± 4.6 pg/mL, respectively (A). In the PGE2 test, prostaglandin
values determined in untreated and only LPS-treated cell cultures were 0.05 ± 0.1 pg/mL
and 436.0 ± 5.8 pg/mL, respectively. Standard drug, indomethacin was used as positive
control, at concentrations of 0.2, 0.04, 0.004 and 0.0004 µg/mL and prostaglandin values
obtained were 11.8 ± 1.9; 171.8 ± 1.2; 271.6 ± 2.6 and 340.7 ± 1.1 respectively (B). The
bars for each chalcone refer to concentrations tested in ascending order. (C) Minimum
inhibitory concentrations of substituted chalcones on production of IL-1β and PGE2.
Statistical analyses were calculated and values in the same column with different
superscript letters (a–e) are significantly different (p < 0.05 or p < 0.001); determined in
Tukey test. Values are reported as means ± S.E.M., and differences between groups were
considered to be significant at p < 0.001 (***), p < 0.01 (**) and p < 0.05 (*).

Molecules 2015, 20
8079
Figure 2. Effects of chalcones on iNOS and COX-2 expression following RAW 264.7
macrophage stimulation by LPS. Cells were treated with LPS (1 µg/mL) and chalcones
(4 and 20 µg/mL) for 24 h, lysed and submitted to western blot for analysis of iNOS (A)
and COX-2 (B) expression. The presented results refer to chalcone compounds
demonstrating the highest levels of inhibitory activity levels (samples 3, 4, 5, 28, 31, 33).
The cells treated with LPS only were used as a positive control of macrophage stimulation
(C+). Untreated cells were used as a negative control (C−). Images are representative of two
independent experiments that showed similar results. Lower panel, quantification of the
protein levels by immunoreactive bands densitometric analysis. Each band was compared
to respective positive control band at same membrane. Relative densities were calculated
employing ImageJ software. Densitometric protein bands analysis was performed using
ImageJ software for Windows (NIH, Bethesda, MD, USA). The value for positive control
condition (LPS-stimulated cells) was set as 1 and other conditions were recalculated
correspondingly to allow ratio comparisons.
Eight chalcones exhibiting higher levels of antimycobacterial activity were additionally tested
against the hypervirulent M. tuberculosis strain M299. Only five of them (compounds 3, 4, 5, 8 and
25) showed similar activity profiles against both M. tuberculosis strains (Table 3 and Figure 3),
whereas two chalcones (compounds 15 and 24) were significantly less potent against the clinical
isolate M299. It should be noted that the strain M299 was more resistant to the antimycobacterial
action of rifampicin as well. Comparable levels of bacterial growth inhibition for these strains were
obtained after at least a ten-fold increase in rifampicin concentrations used for the treatment of strain
M299 compared with those used for the strain H37Rv (Figure 3A,B).
Additionally, we evaluated the effects of chalcones on intracellular growth of M. tuberculosis strain
H37Rv in macrophages. The results presented in Figure 3C show that eight of nine studied chalcones,
used at a concentration of 100 µg/mL, and six, used at a concentration of 20 µg/mL, were able to
reduce drastically (95%–100% inhibition) the intracellular bacillary load demonstrating a good
efficacy profile.

Molecules 2015, 20
8080
Table 3. Minimum inhibitory concentrations of substituted chalcones suppressing growth
of the laboratory M. tuberculosis strain H37Rv and clinical M. tuberculosis isolate M299.
MIC50 (µM)
MIC90 (µM)
MIC50 (µM)
MIC90 (µM)
Chalcone
M. tuberculosis H37Rv
M. tuberculosis M299
3
13.0 ± 3.3 a
19.7 ± 1.9 a
12.7 ± 1.9 a
>477.9
372.7 ± 3.6 a
320.1 ± 4.8 b
303.2 ± 5.4
>477.9
18.37 ± 3.9 a
20.1 ± 1.0 a
11.7 ± 3.4 a
XX
373.9 ± 1.4
351.5 ± 1.8
289.6 ± 0.7
XX
4
5
7
8
10.5 ± 3.8 a
24.4 ± 4.5 a
17.1 ± 3.8 a
63.8 ± 6.2 b
112.4 ± 6.4 c
66.9 ± 6.2 b
25.3 ± 5.2 a
25.1 ± 5.2 a
36.9 ± 4.5 a
>373.2
325.9 ± 3.3 b
252.1 ± 2.7 c
287.8 ± 5.5 c
>416.2
10.2 ± 4.9 a
53.58 ± 1.9
>311.0
XX
344.7 ± 0.4
313.0 ± 1.4
>311.0
XX
9
15
17
19
>376.8
XX
XX
21
368.2 ± 5.2 a
374.6 ± 6.8 a
366.1 ± 3.6 a
355.8 ± 4.9 a
>373.2
XX
XX
24
>419.7
18.5 ± 3.6 a
XX
>419.7
366.9 ± 0.3
XX
25
33
39
40
XX
XX
131.7 ± 1.3 c
82.1 ± 4.3 b
0.11 ± 0.02
351.8 ± 5.9 a
275.3 ± 6.7 c
0.15 ± 0.08
XX
XX
41
XX
XX
Rifampicin
0.2 ± 0.18
3.3 ± 0.16
Values in the same column with different superscript letters (a–c) are significantly different (p < 0.05 or
p < 0.001, determined by Tukey test). XX—not defined.
Four compounds displayed 70%–75% inhibition, when used at a low concentration of 4 µg/mL. The
most active compounds 4 and 5 showed the lowest MIC50 values, 4.2 ± 0.3 and 3.8 ± 0.1 µM,
respectively (Figure 3C). Importantly, the toxic effects of compounds 4 and 5 were selective for
mycobacteria, whereas the cytotoxicity measured by LDH release test was low (Figure 3D),
demonstrating that these compounds are good candidates for the in vivo pharmacological study.
For the further investigation of the pharmacological potential of chalcones, in this study, we
performed theoretical calculations aimed at investigating the stereoelectronic properties of the studied
substituted and unsubstituted chalcones. Since the number of molecules was not enough for a QSAR
investigation, the SAR studies were carried out, to identify physicochemical properties that might be
related to antimycobacterial activity.
An overall 2D and 3D-analysis of chemical structures of these derivatives showed electron-withdrawing
groups presence substituted at aromatic rings induced an enhancement in the activity. Besides, methyl
substituent at para ring B position (17, 19, 21 and 39) seems to decrease antimycobacterial activity.
Analysis of these derivatives electronic properties, including HOMO and LUMO energy and
distribution, dipole moment and vector and molecular electrostatic potential maps (MEP), showed that
chalcone derivatives present different parameters values, and MEPs alone did not suggest any direct
correlation with the antimycobacterial activity (data not shown).

Molecules 2015, 20
8081
A
Figure 3. Effect of chalcones on M. tuberculosis growth in bacterial culture and macrophages.
Bacterial suspensions (1 × 106 CFU/well) of M. tuberculosis strain H37Rv (A) and clinical
isolate M299 (B) were treated or untreated with chalcone samples (0.8, 4, 20 and 100 µg/mL)
or rifampicin (0.00032, 0.0016, 0.008, 1 µg/mL for the strain H37Rv and 0.008, 0.04, 0.2
and 10 µg/mL for the strain M299) for 5 days. Bacterial growth in the resulted cultures was
quantified by MTT test. Data are presented as a percentage of bacterial growth of each
treated culture compared to the growth of corresponding untreated culture (100%). The
four bars for each compound refer to concentrations tested in ascending order. Bacterial
suspensions treated with antibiotic rifampicin were used as a positive control. Untreated
bacterial suspension served as a negative control. (C, D) RAW 264.7 macrophages were
infected with M. tuberculosis strain H37Rv at the infection ratio of 1:1
(macrophage:mycobacteria) and treated with chalcones for 4 days. Bacterial intracellular
viability after the treatment was determined by bacterial CFU counts (C) and toxicity for
macrophages was evaluated by LDH assay (D). The results presented are mean values
obtained over three experiments, each done in triplicate. *** p < 0.001, ** p < 0.01 and
* p < 0.05 compared to untreated group. Values in the same column with different
superscript letters (a–c) are significantly different (p < 0.05 or p < 0.001; determined by
Tukey test).

Molecules 2015, 20
8082
However, the analysis of α,β-unsaturated carbonyl atoms electrostatic charge and dihedral angles
showed some correlation with biological activity. It was observed that the less negative oxygen (O1)
and less positive carbonyl carbon (C7) result in more active chalcone derivatives (Table 4). It was also
observed that a more negative electrostatic charge on the C9 unsaturated carbon causes activity
decrease or loss (Table 4).
Table 4. α,β-Unsaturated carbonyl chalcones derivatives (O1, C7, C8 and C9) electrostatic
charging system distribution and variation in dihedral angles according to the substituent
change on aromatic rings.
#
3
O1
C7
C8
C9
C8, C7, C2, C3
38.51
C8, C9, C11, C14
−48.75
27.78
−0.591
−0.608
−0.591
−0.589
−0.586
−0.581
−0.606
−0.617
−0.614
−0.613
−0.613
−0.593
−0.603
−0.585
0.583
0.645
0.564
0.610
0.582
0.562
0.633
0.646
0.646
0.651
0.650
0.654
0.66
−0.307
−0.434
−0.405
−0.364
−0.362
−0.314
−0.349
−0.385
−0.377
−0.32
−0.169
−0.079
−0.036
−0.184
−0.142
−0.116
−0.129
−0.113
−0.103
−0.195
−0.148
−0.124
−0.233
−0.199
4
31.08
5
−38.86
39.72
46.97
8
−47.79
−47.43
−54.30
−47.76
−39.66
−43.67
−48.40
−48.18
−45.43
−49.64
48.58
9
39.81
15
17
19
21
24
25
33
40
41
39.27
38.56
41.46
40.34
36.6
−0.341
−0.374
−0.289
−0.319
36.55
53.44
35.21
0.659
−48.82
Furthermore, it was found that substitution on aromatic rings induces a change in dihedral angle.
This may suggest that conformational aspects are influencing these compounds interaction with the
Mycobacterium macromolecular target.
In addition, we also evaluated Lipinski “Rule of Five”, which is related to oral bioavailability. All
active compounds fulfilled Lipinski “Rule of Five” (cLogP ≤ 5, molecular weight ≤ 500, number of
hydrogen bond donors ≤ 5 and number of hydrogen-bond acceptors ≤ 10) [21] pointing to a good
theoretical oral bioavailability (Table 5).
When any drug candidate is developed, some concern is expected due to the possible lack of
adequate ADMET properties, which constitute the principal cause of drug disapproval [22]. Here we
evaluated in silico the ADMET properties of chalcones active against M. tuberculosis strains which
showed good in silico pharmacokinetic profile. ADMET risk provides a range between 0–24, where
greater the number, higher the drug probability of not be well tolerated. ADMET risk results were
considered acceptable, since values were between 1 and 3 (data not shown), indicating that all
chalcones tested probably do not have ADMET problems.

Molecules 2015, 20
8083
Table 5. Lipinski’s “rules of five” analysis parameters of biologically active chalcone samples.
Chalcone MIC50 (µM) MW LogP HBD HBA
3
13.0 ± 3.3 a 242.70 4.32
19.7 ± 1.9 a 251.33 4.04
12.7 ± 1.9 a 252.27 3.54
10.5 ± 3.8 a 287.16 4.59
24.4 ± 4.5 a 305.15 4.75
17.1 ± 3.8 a 321.60 5.15
63.8 ± 6.2 b 240.28 4.4
112.4 ± 6.4 c 265.36 4.53
66.9 ± 6.2 b 252.31 4.12
25.3 ± 5.2 a 238.29 3.63
25.1 ± 5.2 a 256.28 3.79
36.9 ± 4.5 a 277.14 4.88
131.7 ± 1.3 c 283.28 3.67
82.1 ± 4.3 b 287.70 4.35
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
1
1
1
1
2
2
2
2
1
5
4
4
5
8
9
15
17
19
21
24
25
33
40
41
Abbreviations: lipophilicity (Log P), molecular weight (MW), number of hydrogen bond donor groups
(HBD) and number of hydrogen bond acceptor groups (HBA). Values in the same column with different
superscript letters (a–c) are significantly different (p < 0.05 or p < 0.001; determined in Tukey test).
Furthermore, specific toxicity analysis in silico was performed. The biomarkers used for predicted
hepatotoxicity diagnostics were alkaline phosphatase (ALP), serum glutamic oxaloacetic transaminase
(SGOT), serum glutamic pyruvic transaminase (SGPT), γ-glutamyl transferase (GGT) and lactate
dehydrogenase (LDH). Human liver adverse effect for each compound was evaluated as the likelihood
of causing elevation in the levels of these enzymes. Chalcone prediction was considered slightly
elevated to ALP, SGOT, GGT, as it was also observed for isoniazid and pyrazinamide, highlighting
compound 5 for normal level for SGPT. Notwithstanding, good results were found for all chalcones for
LDH enzyme, particularly for compound 5 which does not present toxicity for LDH. LDH levels for
isoniazid and pyrazinamide were elevated, confirming the hepatotoxic effect of these compounds [23].
2.2. Discussion
Chalcones and their derivatives have attracted increasing attention due to numerous pharmacological
applications, such as anti-inflammatory and antimicrobial including antimycobacterial effects. Thus,
synthetic accessibility of chalcones has generated great interest among organic and medicinal chemists,
encouraging experiments aimed at diversification of the core structure of chalcones and the generation
of compounds with improved properties [15].
Inflammation is strongly involved in pathogenesis of most infectious diseases, including TB. In
general, proinflammatory mediators production by infected macrophages, such as NO and TNF-α, are
essential for protection from mycobacteria. TB treatment is based on the use of antimycobacterial
drugs. However, many severe forms of TB, such as miliary TB or tuberculous meningitis, require an
additional anti-inflammatory therapy to suppress excessive inflammation [24]. For instance, adjunctive
treatment of TB with dexamethasone [25] and prednisolone [26] demonstrated a reduction of mortality
and a residual neurological deficit in patients with tuberculous meningitis.

Molecules 2015, 20
8084
An urgent need for effective alternative anti-TB treatment, especially for the severe destructive and
disseminated forms of TB frequently associated with exacerbated inflammation, initiated attempts to
design and develop compounds with dual activities, i.e., antimycobacterial and anti-inflammatory activities.
In this study, we investigated anti-inflammatory and antimycobacterial effects of a series of forty
synthesised chalcones with substituents in A and B rings and one commercially-available unsubstituted
chalcone. The identification of most active compounds was accompanied by structure- activity relationship
(SAR) analysis. The initial screening of chalcones was performed through evaluation of the inhibitory
effects of chalcones on the production of NO and TNF-α by LPS-stimulated cultured macrophages.
More pronounced levels of inhibitory activity against NO were presented by ten chalcones
(compounds 4, 5, 12, 24, 28, 29, 31, 33, 40 and 41), exhibiting IC50 lower than 21 µM. The most active
was compound 5 bearing a methylenedioxy group between carbons 3 and 4 of A ring (IC50 2.1 ± 2.7 µM).
The methylenedioxy group is generally found attached to an aromatic structure where a
methylenedioxybenzene or benzodioxole functional group is formed, and it is widely found in natural
products. The presence of this group in Saururus chinensis lignans [27] and in myristicin, found on
Myristica fragrans [28] contributed to the capacity of these compounds to inhibit NO production by
RAW 264.7 macrophages. To the best of our knowledge, this is the first report demonstrating
anti-inflammatory activity for chalcone 5 that has methylenedioxy substituent on the 3,4-position.
However, it should be noted that four other compounds evaluated in this study, sharing the
methylenedioxy group on ring A, as in compound 5, and additionally having substituents on ring B,
presented lower levels of activity than compound 5.
Chalcones effectively inhibiting NO were equally effective in suppressing PGE2, and these
activities were mediated by inhibition of iNOS and COX-2, respectively. In contrast to production of
NO and PGE2 that was easily suppressed by most chalcones, the production of inflammatory cytokines
TNF-α and IL-1β was inhibited by few compounds. Considering that some chalcones exhibited
increased cytotoxicity for macrophages and were excluded from the further investigation, only three
chalcones (compounds 3, 4 and 5) were identified as potent anti-inflammatory agents able to suppress
production of principal inflammatory mediators (NO, PGE2, TNF-α and IL-1β) by LPS-stimulated
macrophages. Compound 3 was characterized by a chlorine group substituted onto the ring A at
4 position and compound 4 presented a 4'-dimethylamino substituent on ring A. Of note, these
substituents have been reported in the previous studies as important structural features characteristic
for the anti-inflammatory chalcones [29].
In order to identify chalcones with antimycobacterial properties, the chalcone compounds were
screened for their ability to inhibit growth of M. bovis BCG in culture, and active compounds were
further tested against M. tuberculosis strains, laboratory strain H37Rv and clinical isolate M299.
Some of chalcones included in this study (compounds 24, 25 and 30) have been reported previously
to exhibit antimycobacterial activity [16–18]. In accordance with these data, compounds 24, 25 and 30
presented satisfactory anti-BCG activity in our experiments. However, only one of them (compound
25) was demonstrated to be active against the M. tuberculosis M299 strain, two other compounds were
excluded from the further tests due to strong cytotoxicity or weak activity against M. tuberculosis. It
should be noted, that few tested chalcones exhibiting activity against BCG and the laboratory Mtb
strain were effective in inhibiting growth of strain M299. Strain M299, belonging to the modern
Beijing sublineage of M. tuberculosis, induced in mice severe pulmonary immunopathology in mice

Molecules 2015, 20
8085
leading to early death and was characterised in our previous studies as a hypervirulent strain [20]. Only
five chalcones (compounds 3, 4, 5, 8 and 25) showed similar activity profiles against strain M299 and
laboratory strain H37Rv. Two chalcones (compounds 15 and 24) and the standard anti-TB drug
rifampicin were significantly less potent against the hypervirulent M299 strain that demonstrated a
higher level of resistance of this strain to treatment compared with the strain H37Rv.
Regarding the ability of chalcones to suppress intracellular growth of M. tuberculosis in
macrophages, three highly active compounds (4, 5 and 8) were selected, highlighting the most active
compound 5. To our knowledge, this compound has never been tested against mycobacteria, however,
the antibacterial activity of this compound against Staphylococcus aureus and Clostridium
cladosporioides was demonstrated in one previous work [30]. The antimycobacterial activity against
M. tuberculosis strain H37Rv of compound 4 was previously reported, although the data obtained in that
study did not provide sufficient information about minimum inhibitory concentration (MIC) and
selectivity index [19].
A final analysis of obtained data regarding dual biological activities, antimycobacterial and
anti-inflammatory, of the studied chalcones, allows the identification of the two most active
compounds (chalcones 4 and 5). Results of the ADME and toxicity in silico study demonstrated that all
pharmacokinetics parameters of the studied substituted chalcones were found to be within the range of
recommended values, and the most active chalcones, such as compound 5, presented lower levels of
predicted hepatotoxicity than the standard anti-TB drugs isoniazid and pyrazinamide.
In conclusion, the obtained data demonstrate that chalcone compounds 4 and 5 are promising agents
for further prospective studies aimed at the generation of new anti-TB drugs for the adjunctive therapy
of severe TB associated with exacerbated inflammation. This is the first report describing a new
approach for the screening of anti-TB chalcones focusing on dual biological activities, such as
anti-inflammatory and antimycobacterial. And the first time that these both activities were described
for compound 5.
3. Experimental Section
3.1. Reagents
Cell culture reagents were purchased from Gibco/Invitrogen (Grand Island, NY, USA).
Mycobacteria- specific Middlebrook 7H9 and 7H10 media were obtained from Difco (Detroit, MI,
USA); and OADC and ADC supplements were from BD Biosciences (BD, Sparks, MD, USA).
Lipopolysaccharide (LPS) from serotype 0111:B4 Escherichia coli, NG-Monomethyl-L-arginine
acetate salt (L-NMMA)—inhibitor of iNOS, (cod. M7033), rifampicin (cod. R7382), indomethacin
(cod.I7378), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and chalcone
(1,3-diphenyl-2-propenone) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The
chalcone compounds, rifampicin and indomethacin were dissolved in dimethyl sulfoxide (DMSO,
Sigma Aldrich); other reagents indicated for cell treatment were dissolved in sterile phosphate buffered
saline (PBS) and sterilized by passage through 0.22 mm nylon filters (Corning Inc., Wilkes-Barre, PA,
USA). Anti-iNOS (cod. 610328) and anti-COX-2 antibodies (cod. 610203) were obtained from BD

Molecules 2015, 20
8086
Biosciences (San Diego, CA, USA); and horseradish peroxidase-conjugated secondary antibody was
from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
3.2. Synthesis of Substituted Chalcones
The synthetic strategy towards the chalcones was based on Claisen-Schimdt reactions between
appropriately substituted aromatic aldehydes with acetophenones in absolute ethanol at ambient
temperature. The produced chalcones (compounds 2–41) were purified through recrystallization from
hot ethanol. Synthesized chalcones and commercially obtained 1,3-diphenyl-2-propenone are depicted
in Table 1.
Characterization of the Chalcones
13
The synthesized compounds were characterized by 200 MHz 1H- and 50 MHz C-NMR and mass
spectroscopy. Chalcones’ NMR data were in accordance with the corresponding reported literature: 1,
5, 28 [30]; 2, 24, 25, 30 [16]; 3, 6 [31]; 4 [19]; 7 [32]; 8, 34 [33]; 9 [34]; 10 [35]; 11 [36]; 12 [37]; 13 [38];
14 [39]; 15, 39 [40]; 16, 29 [41]; 17 [42]; 18 [43]; 19 [44]; 20 [45]; 21, 38 [46]; 22 [47]; 23 [48];
26 [49]; 27, 31 [50]; 32 [51]; 33 [52]; 35 [53]; 36 [54]; 40 [55]; 41 [56].
3.3. Cell Culture and Treatments
Murine RAW 264.7 macrophage cell line (American Type Culture Collection, Manassas, VA,
USA), was cultured in Dulbecco’s modified Eagle’s medium (DMEM-F12) supplemented with 10%
Fetal Bovine Serum (FBS) and gentamicin (50 µg/mL) in 5% CO2 atmosphere at 37 °C. For
experiments, the cells were seeded in 96-well plates (1 × 105 cells/well) and incubated for 24 h at
37 °C. For macrophage stimulation, the cell cultures were treated with LPS (1 µg/mL) and incubated
for additional 24 h in the presence or absence of the chalcone samples (samples 1–41) that were used
at concentrations of 100, 20, 4 and 0.8 µg/mL. In some experiments, a nitric oxide (NO) inhibitor,
L-NMMA (20 μg/mL), was used as a positive control of NO inhibition, and a non-steroidal
anti-inflammatory drug indomethacin, known to inhibit production of prostaglandins, was used as a
positive control of PGE2 inhibition.
3.4. Quantification of Proinflammatory Mediators (TNF-α, IL-1β, PGE2 and NO)
The frozen culture supernatants samples were thawed and immediately analyzed using
commercially available enzyme-linked immunosorbent assay (ELISA) kits to measure TNF-α, IL-1β
(BD Biosciences) and PGE2 (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s
instructions. Standard curves for each mediator were generated using reference cytokine
concentrations supplied by the manufacturer. Nitric oxide (NO) generation was assessed by the Griess
method to measure nitrites, which are stable breakdown NO products [57].
3.5. Macrophage Cytotoxicity Assay
Cytotoxic effects of chalcone samples on RAW 264.7 cell viability in cultures stimulated with LPS
were determined using the LDH (lactate dehydrogenase) release assay as previously described [58].

Molecules 2015, 20
8087
Cytoplasmic enzyme LDH release into cell culture supernatants was detected using a commercial LDH
kit (Doles, GO, Brazil). Cell death was expressed as percentage of LDH release, which was calculated
using the following formula: percentage of LDH release = 100 × (test LDH release − spontaneous
release)/(maximum release − spontaneous release). The maximum release was determined following
dissolution of cell monolayers using 1% (vol/vol) Triton X-100. The spontaneous release was
determined in supernatants from cultures incubated in the presence of LPS and DMSO, used as a
solvent for chalcone samples. IC50 values were calculated by nonlinear regression analysis of
log[concentration]/inhibition curves using GraphPad Prism 4 (GraphPad Software Inc., San Diego, La
Jolla, CA, USA) applying a sigmoidal dose-response variable slope curve fitting using the different
percentage obtained for each corresponding concentration in triplicate and were expressed as means
with corresponding 95% confidence interval (CI) from 3 independent experiments.
3.6. Detection of iNOS and COX-2 by Western Blot
RAW 264.7 macrophages were plated in Petri dishes (2 × 107 cells·mL−1) and incubated for 24 h at
37 °C in 5% CO2 atmosphere. After incubation, the confluent macrophage cultures were treated with
LPS (1 µg/mL) and chalcone samples with two concentrations of most active chalcones for additional
24 h. The resulted cellular monolayers were washed with PBS 1×, scraped and resuspended in PBS 1×,
transferred and centrifuged to obtain cell pellets. The cells were lysed in buffer (10% SDS, 20%
glycerol, 5% 2-mercaptoethanol, 2% bromophenol blue and 1 M Tris HCl, pH 6.8, containing protease
inhibitors). Protein concentrations were estimated by Bradford method. For western blot analysis,
cellular extract samples (60 µg) were submitted to 10% SDS–polyacrylamide gel electrophoresis under
non-reducing conditions and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech,
Uppsala, Sweden). After blocking with PBS-T (0.5% Tween-20) containing 2.5% (w/v) bovine serum
albumin, the membranes were incubated with antibodies against iNOS (1:1000) and COX-2 (1:500)
for 1 h at room temperature. After incubation period, the membranes were washed followed by
treatment with horseradish peroxidase-conjugated secondary antibody. The resulting membranes were
developed using diaminobenzidine/H2O2 as a substrate for peroxidase. In all electrophoresis
experiments, a protein standard ladder (Full Range Rainbow-GE Healthcare, Piscataway, NJ, USA)
was used to estimate proteins molecular size. Protein bands densitometric analysis was performed
using ImageJ software for Windows (NIH, Bethesda, MD, USA). The value for stimulated condition
(LPS- treated cells) was set as 1 and other conditions were recalculated correspondingly to allow
ratio comparisons.
3.7. Mycobacterial Culture and Evaluation of Bacterial Growth
Three mycobacterial strains differed in virulence were used in this study: avirulent Mycobacterium
bovis Bacillus Calmette-Guérin (BCG), vaccine strain Moreau, and two Mycobacterium tuberculosis
strains (low virulent laboratory strain H37Rv, ATCC 27294, and highly virulent Mtb Beijing strain
M299 isolated from TB patient in Mozambique) evaluated for virulence in previous study [20].
Mycobacterial strains were grown in suspension in 7H9 Middlebrook broth, containing 10% albumin
dextrose complex (ADC), 0.5% glycerol and 0.05% Tween-80 at 37 °C under Biosecurity level 3
containment conditions. The suspensions densities were measured by spectrophotometry at 600 nm and

Molecules 2015, 20
8088
corresponding concentrations were determined for each strain serial dilution plating on Middlebrook
7H10 agar plates supplemented with 0.5% glycerol and 10% oleic acid–albumin-dextrose–catalase
enrichment (OADC). To study the chalcones’ antimycobacterial activity, we employed the MTT assay to
quantify bacterial growth in a liquid medium [16]. The bacterial suspensions were plated
(1 × 106 CFU/well in 96-well plate) and incubated in the presence of chalcone samples at concentrations
of 100, 20, 4 and 0.8 µg/mL or rifampicin (ranging from 0.0011 to 0.03 µg/mL for M. bovis BCG; from
0.00032 to 1 µg/mL for Mtb H37Rv and from 0.008 to 10 µg/mL for clinical Mtb isolate M299). The
plates were sealed and incubated at 37 °C and 5% CO2 for 7 days for M. bovis BCG or 5 days for
M. tuberculosis strains. After these periods, the bacterial cultures were incubated for 3 h with MTT
solution (5 mg/mL) and then treated with lysis buffer (20% w/v SDS/50% dimethylformamide—DMF
in distilled water, pH 4.7) for 18 h. Resulted samples optical densities were measured at 570 nm.
Untreated bacterial suspensions were used to control spontaneous growth of bacteria.
3.8. Infection of Macrophage Cultures and Quantification of Intracellular Growth
RAW 264.7 macrophages were plated (1 × 105 cells/well) in antibiotic-free DMEM-F12 medium
supplemented with 10% fetal bovine serum and incubated for 24 h. Prior to infection, mycobacterial
suspensions were sonicated for 1 minute to disperse clumps and optical densities were adjusted to 0.1.
The macrophage cultures were infected at a MOI of 1:1 (macrophage: bacterium). Phagocytosis was
allowed to progress for 3 h. After 3 h, extracellular mycobacteria were removed by washing with
PBS 1X and the infected cell monolayers were treated for 4 days with chalcone samples or rifampicin.
Macrophage viability was monitored by LDH assay and was over 80% in all experiments. On day 4 after
infection, cells were lysed cells with 1% saponin to release intracellular bacteria. Lysate aliquots were
diluted 10-fold in PBS, plated in triplicates on 7H10 agar plates and incubated at 37 °C. After 21 days,
total CFU were determined.
3.9. Molecular Modelling and in Silico ADMET Studies
In an attempt to gain insight on the synthesized compounds’ molecular structure, geometric
optimization and conformation analysis has performed using Spartan’10 software. Initially, molecules
were subjected to conformational analysis using molecular mechanics calculations (MMFFaq) in order
to select the lowest energy conformation. After selecting the best conformer, geometric optimization
was performed using the semi-empirical method RM1. Finally, the structures were subjected to
single-point calculations using the ab initio Hartree-Fock (HF) method with the 6-31G** basis set in
order to obtain stereoelectronic and geometric parameters that may be correlated with activity against
M. tuberculosis H37Rv strain. All chalcones were submitted to in silico analysis of ADMET using
ADMET Predictor™ (Simulation Plus Inc., Lancaster, CA, USA) as isoniazid and pyrazinamide. This
software uses mathematical models, based on quantitative structure-activity relationships (QSAR), to
predict molecular descriptors for each studied structure, can therefore estimate a certain property.
Pharmacokinetics properties were evaluated through ADMET risk score, which is an indicator for
potential ADMET problems that a compound might have, since it provides relevant information about
optimization compounds and potential security. Lipophilicity, permeability, ionization, permanent
electric charge, and hydrogen bonding are presented in ADMET Risk [59]. Hepatoxicity parameters

Molecules 2015, 20
8089
were predicted with relevant biomarkers: alkaline phosphatase (ALP), serum glutamic oxaloacetic
transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), γ-glutamyl transferase (GGT)
and lactate dehydrogenase (LDH). Human liver adverse effect for each compound was evaluated as
likelihood in these enzymes levels.
3.10. Statistical Analysis
Statistical analysis was performed using variance (ANOVA) one-way analysis and Tukey procedure
for multiple range tests, employing Prism 4 software (GraphPad) to assess statistical significance
between groups of data. A value of p < 0.05 was considered to be significant.
Acknowledgments
The authors thank CNPq, CAPES and FAPERJ for financial support.
Author Contributions
Conceived and designed the experiments: M.F.M., E.B.L. and A.M.T.S. Performed the
experiments: T.L.B.V., S.D.C., B.A.A.-V. and M.V.P.M. Analyzed the data: M.F.M., A.M.T.S.,
T.L.B.V., C.R.R. and E.B.L. Performed synthesis of chalcones: I.C.R.L., R.O.M.A.S and L.S.M.M.
Wrote the paper: T.L.B.V., M.F.M. and E.B.L. All the authors have read the final manuscript and
approved the submission.
Conflicts of Interest
The authors declared no conflict of interest.
References
1. Duque, G.A.; Descoteaux, A. Macrophage cytokines: Involvement in immunity and infectious
diseases. Front. Immunol. 2014, 5, 491.
2. Medzhitov, R. Origin and physiological roles of inflammation. Nat. Rev. 2008, 454, 428–435.
3. Medzhitov, R. Inflammation 2010: New adventures of an old flame. Cell 2010, 140, 771–776.
4. Ashley, N.T.; Zachary, M.W.; Nelson, R.J. Inflammation: Mechanisms, costs, and natural
variation. Annu. Rev. Ecol. Evol. Syst. 2012, 43, 385–406.
5. Thwaites, G.; Fisher, M.; Hemingway, C.; Scott, G.; Solomon, T.; Innes, J. Innes. British
Infection Society guidelines for the diagnosis and treatment of tuberculosis of the central nervous
system in adults and children. J. Infect. 2009, 59, 167–187.
6. Meintjes, G.; Wilkinson, R.J.; Morroni, C.; Pepper, D.J.; Rebe, K.; Rangaka, M.X.; Oni, T.;
Maartens, G. Randomized placebo-controlled trial of prednisone for paradoxical tuberculosis-
associated immune reconstitution inflammatory syndrome. AIDS 2010, 24, 2381–2390.
7. Tobin, D.M.; Roca, F.J.; Oh, S.F.; McFarland, R.; Vickery, T.W.; Ray, J.P.; Ko, D.C.; Zou, Y.;
Bang, N.D.; Chau, T.T.; et al. Host genotype-specific therapies can optimize the inflammatory
response to mycobacterial infections. Cell 2012, 148, 434–446.

Molecules 2015, 20
8090
8. Critchley, J.A.; Young, F.; Orton, L.; Garner, P. Corticosteroids for prevention of mortality in
people with tuberculosis: A systematic review and meta-analysis. Lancet Infect. Dis. 2013, 13,
223–237.
9. Cholo, M.C.; Steel, H.C.; Fourie, P.B.; Germishuizen, W.A.; Anderson, R. Clofazimine: Current
status and future prospects. J. Antimicrob. Chemother. 2012, 67, 290–298.
10. Jantan, I.; Bukhari, S.N.; Adekoya, O.A.; Sylte, I. Studies of synthetic chalcone derivatives as
potential inhibitors of secretory phospholipase A2, cyclooxygenases, lipoxygenase and
pro-inflammatory cytokines. Drug Des. Dev. Ther. 2014, 16, 1405–1418.
11. Nowakowska, Z. A review of anti-infective and anti-inflammatory chalcones. Eur. J. Med. Chem.
2007, 42, 125–137.
12. Bukhari, S.N.A.; Jantan, I.; Jasamai, M. Anti-inflammatory trends of 1,3-diphenyl-2-propen-1-one
derivatives. Mini Rev. Med. Chem. 2013, 13, 87–94.
13. Bano, S.; Javed, K.; Ahmad, S.; Rathish, I.G.; Singh, S.; Chaitanya, M.; Arunasree, K.M.;
Alama, M.S. Synthesis of some novel chalcones, flavanones and flavones and evaluation of their
anti-inflammatory activity. Eur. J. Med. Chem. 2013, 65, 51–59.
14. Lin, Y.-M.; Zhou, Y.; Flavin, M.T.; Zhou, L.-M.; Niea, W.; Chen, F.-C. Chalcones and flavonoids
as anti-tuberculosis agents. Bioorg. Med. Chem. 2002, 10, 2795–2802.
15. Rahman, M.A. Chalcone: A valuable insight into the recent advances and potential pharmacological
activities. Chem. Sci. J. 2011, 2011, 1–16.
16. Moodley, S.; Koorbanally, N.A.; Moodley, T.; Ramjugernath, D.; Pillay, M. The
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay is a rapid, cheap,
screening test for the in vitro anti-tuberculous activity of chalcones. J. Microbiol. Methods 2014,
104, 72–78.
17. Sivakumar, P.M.; Kumar, V.; Seenivasan, S.P.; Mohanapriya, J.; Doble, M. Experimental and
theoretical approaches to enhance anti tubercular activity of chalcones. WSEAS Trans. Biol. Biomed.
2010, 2, 51–61.
18. Ávila, H.P.; Fátima, E.; Smânia, A.; Monache, F.D.; Smânia A., Jr. Structure-activity relationship
of antibacterial chalcones. Bioorg. Med. Chem. 2008, 16, 9790–9794.
19. Sivakumar, P.M.; Seenivasan, S.P.; Kumar, V.; Doble, M. Synthesis, antimycobacterial activity
evaluation, and QSAR studies of chalcone derivatives. Bioorg. Med. Chem. Lett. 2007, 17,
1695–1700.
20. Ribeiro, S.C.M.; Gomes, L.L.; Amaral, E.P.; Andrade, M.R.M.; Almeida, F.M.; Rezende, A.L.;
Lanes, V.R.; Carvalho, E.C.Q.; Suffys, P.N.; Mokrousov, I.; et al. Mycobacterium tuberculosis
strains of the modern sublineage of the Beijing family are more likely to display increased
virulence than strains of the ancient sublineage. J. Clin. Microbiol. 2014, 52, 2615–2624.
21. Lipinski, C.A. Lead- and drug-like compounds: The rule-of five revolution. Drug Discov.
Today Technol. 2004, 1, 337–341.
22. Van de Waterbeemd, H.; Gifford, E. ADMET in silico modelling: Towards prediction paradise?
Nat. Rev. Drug Discov. 2003, 2, 192–204.
23. Bourré-Tessier, J.; Arino-Torregrosa, M.; Choquette, D. Increased incidence of liver enzymes
abnormalities in patients treated with isoniazid in combination with disease modifying and/or
biologic agents. Clin. Rheumatol. 2014, 33, 1049–1053.

Molecules 2015, 20
8091
24. Garlanda, C.; Di Liberto, D.; Annunciata, V.; La Manna, M.P.; Buracchi, C.; Caccamo, N.;
Salerno, A.; Dieli, F.; Mantovan, A. Damping excessive inflammation and tissue damage in
Mycobacterium tuberculosis infection by toll IL-1 receptor 8/single Ig IL-1-related receptor, a
negative regulator of IL-1/TLR signaling. J. Immunol. 2007, 179, 3119–3125.
25. Buonsenso, D.; Serranti, D.; Valentini, P. Management of central nervous system tuberculosis in
children: Light and shade. Eur. Rev. Med. Pharmacol. Sci. 2010, 14, 845–853.
26. Prasad, K.; Singh, M.B. Corticosteroids for managing tuberculous meningitis. Cochrane Database
Syst. Rev. 2010, 23, doi:10.1002/14651858.CD002244.pub3.
27. Gao, X.; He, J.; Wu, X.-D.; Peng, L.-Y.; Dong, L.-B.; Deng, X.; Li, Y.; Cheng, X.; Zhao, Q.-S.
Further lignans from Saururus chinensis. Planta Med. 2014, 79, 1720–1723.
28. Lee, J.Y.; Park, W. Anti-inflammatory effect of myristicin on RAW 264.7 macrophages
stimulated with polyinosinic-polycytidylic acid. Molecules 2011, 16, 7132–7142.
29. Kontogiorgis, C.; Mantzanidou, M.; Hadjipavlou-Litina, D. Chalcones and their potential role in
inflammation. Mini Rev. Med. Chem. 2008, 8, 1224–1242.
30. Silva, W.A.; Andrade, C.K.Z.; Napolitano, H.B.; Vencato, I.; Lariucci, C.; de Castro, M.R.C.;
Camargo, A.J. Biological and structure-activity evaluation of chalcone derivatives against bacteria
and fungi. J. Braz. Chem. Soc. 2013, 24, 133–144.
31. Kim, Y.H.; Kim, J.; Park, H.; Kim, H.P. Anti-inflammatory activity of the synthetic chalcone
derivatives: Inhibition of inducible nitric oxide synthase-catalyzed nitric oxide production from
lipopolysaccharide-treated RAW 264.7 cells. Biol. Pharm. Bull. 2007, 30, 1450–1455.
32. Downs, L.E.; Wolfe, D.M.; Schreiner, P.R. Organic base-mediated condensation of
ppridinecarboxaldehydes to azachalcones. Adv. Synth. Catal. 2005, 347, 235–238.
33. Hsieh, C.T.; Hsieh, T.J.; El-Shazly, M.; Chuang, D.W.; Tsai, Y.H.; Yen, C.T.; Wu, S.F.; Wu, Y.C.;
Chang, F.R. Synthesis of chalcone derivatives as potential anti-diabetic agents. Bioorg. Med.
Chem. Lett. 2012, 22, 3912–3915.
34. Dutkiewicz, G.; Veena, K.; Narayana, B.; Yathirajan, H.S.; Kubicki, M. (2E)-1-(4-Bromophenyl)-
3-(4-fluorophenyl)prop-2-en-1-one. Acta Crystallogr. Sect. E: Struct. Rep. Online 2010, 66,
o1243–o1244.
35. Ahirwar, M.K.; Shrivastava, S.P.; Mehta, P. Synthesis and antimicrobial activity of some
pyrazoline derivatives. J. Pharm. Chem. 2010, 4, 75–79.
36. Patil, P.S.; Chantrapromma, S.; Fun, H.-K.; Dharmaprakash, S.M. 1-(3-Bromophenyl)-3-[4-
(dimethylamino)phenyl]prop-2-en-1-one. Acta Crystallogr. Sect. E: Struct. Rep. Online 2007, 63,
o1738–o1740.
37. Vital, D.G.; Arribas, M.; Trossini, G.H.G. Molecular modeling and docking application to
evaluate cruzain inhibitory activity by chalcones and hydrazides. Lett. Drug Des. Discov. 2014,
11, 249–255.
38. Hayat, F.; Salahuddin, A.; Umar, S.; Azam, A. Synthesis, characterization, antiamoebic activity
and cytotoxicity of novel series of pyrazoline derivatives bearing quinoline tail. Eur. J. Med. Chem.
2010, 45, 4669–4675.
39. Joseph, L.; Arunsasi, B.S.; Sajan, D.; Shettigar, V. Synthesis, crystal growth, thermal, electronic
and vibrational spectral studies of 1-(4-bromophenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one:
A density functional theory study. J. Mol. Struct. 2014, 1076, 687–697.

Molecules 2015, 20
8092
40. Kistayya, C.; Rao, N.G.R.; Rao, A.V.; Srinath, N.; Nayak, B.S.; Chawda, B. Synthesis and
biological evaluation of some new chalcones as potential cytotoxic agents. Int. J. Pharm. 2014, 4,
397–406.
41. Schmink, J.R.; Holcomb, J.L.; Leadbeater, N.E. Testing the validity of microwave-interfaced,
in situ raman spectroscopy as a tool for kinetic studies. Org. Lett. 2009, 11, 365–368.
42. Wu, X.-F.; Neumann, H.; Spannenberg, A.; Schulz, T.; Jiao, H.; Beller, M. Development of a
general palladium-catalyzed carbonylative heck reaction of aryl halides. J. Am. Chem. Soc. 2010,
132, 14596–14602.
43. Alegaon, S.G.; Alagawadi, K.R.; Dadwe, D.H. Synthesis and anti-tubercular activity of novel 3,
5-diaryl-4,5-dihydro-1H-pyrazole derivatives. Drug Res. 2014, 64, 553–558.
44. Sonmez, F.; Sevmezler, S.; Atahan, A.; Ceylan, M.; Demir, D.; Gencer, N.; Arslan, O.;
Kucukislamoglu, M. Evaluation of new chalcone derivatives as polyphenol oxidase inhibitors.
Bioorg. Med. Chem. Lett. 2011, 21, 7479–7482.
45. Saini, R.K.; Kumari, N.; Joshi, Y.C.; Joshi, P.; Shekhawat, S.S. Solvent free microwave assisted
synthesis of chalcones and their antifungal activities. Asian J. Chem. 2007, 19, 4483–4486.
46. Lopez, S.; Castelli, M.V.; Zacchino, S.; Dominguez, J.N.; Lobo, G.; Charris-Charris, J.;
Coetrs, J.C.C.; Ribas, J.C.; Devia, C.; Rodrigues, A.M.; et al. In vitro antifungal evaluation and
structure-activity relationships of a new series of chalcone derivatives and synthetic analogues,
with inhibitory properties against polymers of the fungal cell wall. Bioorg. Med. Chem. 2001, 9,
1999–2013.
47. Singh, R.; Malik, O.P. Synthesis and antimicrobial activity of 1-(4-chlorophenyl)-3(4-
methoxy/3,4-dimethoxyphenyl)propan-1,3-diones and their 2-[(2-nitro/4-chloro or bromophenyl)
azo]analogs. Indian J. Chem. 1994, 34, 455–459.
48. Ananthnag, G.S.; Adhikari, A.; Balakrishna, M.S. Iron-catalyzed aerobic oxidative aromatization
of 1,3,5-trisubstituted pyrazolines. Catal. Commun. 2014, 43, 240–243.
49. Sharma, N.; Sharma, A.; Kumar, R.; Shard, A.; Sinha, A.K. One-pot two-step oxidative cleavage
of 1,2-arylalkenes to aryl ketones instead of arylaldehydes in an aqueous medium: A complementary
approach to ozonolysis. Eur. J. Org. Chem. 2010, 31, 6025–6032.
50. Mitch, C.H.; Quimby, S.J.; Diaz, N.; Pedregal, C.; Torre, M.G.; Jimenez, A.; Shi, Q.; Canada, E.J.;
Kahl, S.D.; Statnick, M.A.; et al. Discovery of aminobenzyloxyarylamides as κ opioid receptor
selective antagonists: Application to preclinical development of a κ opioid receptor antagonist
receptor occupancy tracer. J. Med. Chem. 2011, 54, 8000–8012.
51. Ju, J.; Zhang, L.; Hua, R. Palladium-catalyzed direct intramolecular double a-C–H arylation of
1,5-diketone: A strategy for synthesis of Tröger’s base analogues. Tetrahedron Lett. 2014, 55,
3374–3376.
52. Upadhyay, A.; Madhuban, G.; Prasad, D. Synthesis and nematicidal activity of pyrazole derivatives.
Pestic. Res. J. 2012, 24, 65–70.
53. Zhao, F.; Zhao, Q.-J.; Zhao, J.-X.; Zhang, D.-Z.; Wu, Q.-Y.; Jin, Y.-S. Synthesis and cdc25B
inhibitory activity evaluation of chalcones. Chem. Nat. Comp. 2013, 49, 206–214.
54. Zheng, Y.; Wang, X.; Gao, S.; Ma, M.; Ren, G.; Liu, H.; Chen, X. Synthesis and antifungal
activity of chalcone derivatives. Nat. Prod. Res. 2015, 12, 1–7.

Molecules 2015, 20
8093
55. Montes-Avila, J.; Díaz-Camacho, S.P.; Sicairos-Félix, J.; Delgado-Vargas, F.; Rivero, I.A.
Solution-phase parallel synthesis of substituted chalcones and their antiparasitary activity against
Giardia lamblia. Bioorg. Med. Chem. 2009, 17, 6780–6785.
56. Venkatesan, J.; Pandeya, S.N.; Selvakumar, D. Synthesis and biological evaluation of 4,6-diaryl
substituted-4,5-dihydro-2-amino pyrimidines. Indian J. Pharm. Sci. 2007, 69, 586–589.
57. Park, P.-H.; Kim, H.S.; Jin, X.Y.; Jin, F.; Hur, J.; Ko, G.; Sohn, D.H. KB-34, a newly synthesized
chalcone derivative, inhibits lipopolysaccharide-stimulated nitric oxide production in RAW 264.7
macrophages via heme oxygenase-1 induction and blockade of activator protein-1. Eur. J. Pharmacol.
2009, 606, 215–224.
58. Muzitano, M.F.; Cruz, E.A.; de Almeida, A.P.; Da Silva, S.A.; Kaiser, C.R.; Guette, C.;
Rossi-Bergmann, B.; Costa, S.S. Quercitrin: An antileishmanial flavonoid glycoside from
Kalanchoe pinnata. Planta Med. 2006, 72, 81–83.
59. Whitlow, J.L.; Varughese, J.F.; Zhou, Z.; Bartolotti, L.J.; Li, Y. Computational screening and
design of S100B ligand to block S100B–p53 interaction. J. Mol. Graph. Model. 2009, 27, 969–977.
Sample Availability: Samples of the compounds 1–41 are available from the authors.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).