Antibacterial & antitubercular activities of cinnamylideneacetophenones

Article abstract of DOI:10.3390/molecules22101685

Cinnamaldehyde is a natural product with broad spectrum of antibacterial activity. In this work, it was used as a template for design and synthesis of a series of 17 cinnamylideneacetophenones. Phenolic compounds 3 and 4 exhibited MIC (minimum inhibitory

Full text of DOI:10.3390/molecules22101685

molecules
Article
Antibacterial and Antitubercular Activities
of Cinnamylideneacetophenones
Carlos R. Polaquini 1,† ID , Guilherme S. Torrezan 1,†, Vanessa R. Santos 2, Ana C. Nazaré 1,
Débora L. Campos 3, Laíza A. Almeida 1 ID , Isabel C. Silva 3 ID , Henrique Ferreira 4
,
ID
Fernando R. Pavan 3 ID , Cristiane Duque 2
and Luis O. Regasini 1,
*
ID
1
Laboratory of Green and Medicinal Chemistry, Department of Chemistry and Environmental Sciences,
Institute of Biosciences, Humanities and Exact Sciences, São Paulo State University (Unesp),
São José do Rio Preto, SP 15054-000, Brazil; carlos_polaquini@hotmail.com (C.R.P.);
guilherme_panz@hotmail.com (G.S.T.); carolnazare@uol.com.br (A.C.N.);
laiza_araujo1@hotmail.com (L.A.A.)
2
3
4
Department of Pediatric Dentistry and Public Health, School of Dentistry,
São Paulo State University (Unesp), Araçatuba, SP 16015-050, Brazil;
vanessarodrigues_22@hotmail.com (V.R.S.); cristianeduque@yahoo.com.br (C.D.)
Department of Biological Sciences, School of Pharmaceutical Sciences, São Paulo State University (Unesp),
Araraquara, SP 14800-903, Brazil; leite.debora26@gmail.com (D.L.C.); isabelcrs10@gmail.com (I.C.S.);
fernandopavan@fcfar.unesp.br (F.R.P.)
Department of Biochemistry and Microbiology, Institute of Biosciences, São Paulo State University (Unesp),
Rio Claro, SP 13506-900, Brazil; henfer@rc.unesp.br
*
Correspondence: regasini@ibilce.unesp.br; Tel.: +55-17-3221-2362
†
These two authors contributed equally to this work.
Received: 30 August 2017; Accepted: 3 October 2017; Published: 10 October 2017
Abstract: Cinnamaldehyde is a natural product with broad spectrum of antibacterial activity. In this
work, it was used as a template for design and synthesis of a series of 17 cinnamylideneacetophenones.
Phenolic compounds
3 and 4 exhibited MIC (minimum inhibitory concentration) and MBC
(minimum bactericidal concentration) values of 77.9 to 312
µ
M against Staphylococcus aureus,
10, and 18 presented potent
70.9 µM). Hydrophilic effects caused by
Streptococcus mutans, and Streptococcus sanguinis. Compounds 2, 7,
effects against Mycobacterium tuberculosis (57.2
µM
ď
MIC
ď
substituents on ring B increased antibacterial activity against Gram-positive species. Thus, log Po/w
were calculated by using high-performance liquid chromatography-photodiode array detection
(HPLC-PDA) analyses, and cinnamylideneacetophenones presented values ranging from 2.5 to 4.1.
In addition, the effects of
toxicity (46.3 IC50
subjected to in silico prediction of pharmacokinetic properties, and did not violate Lipinski’s and
Veber’s rules, corroborating their potential bioavailability by an oral route.
3
and
4 were evaluated on pulmonary cells, indicating their moderate
µM
ď
ď
96.7 M) when compared with doxorubicin. Bioactive compounds were
µ
Keywords: cinnamaldehyde; cinnamylideneacetophenone; antimicrobial; antibacterial; antitubercular;
Mycobacterium; Claisen-Schmidt reaction
1. Introduction
Cinnamaldehyde (
for its spicy and sweet taste [
bearing an -unsaturated aromatic aldehyde. The synthetic applications of 1
1
) is the main component in cinnamon bark oil (50–70%), and it is responsible
]. Structurally, cinnamaldehyde is a natural simple phenylpropanoid,
are vast and related
1,2
α
,
β
to its abundance and low economic costs, and have led to a collection of compounds with high
chemodiversity [3].
Molecules 2017, 22, 1685; doi:10.3390/molecules22101685
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1685
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The spectrum of antibacterial activity of
1
has been extensively reported and includes
effects against Gram-positive, Gram-negative, and mycobacteria species, such as Escherichia coli
Helicobacter pylori [ ], Pseudomonas aeruginosa [ ], Salmonella enterica [ ], Staphylococcus aureus [
Mycobacterium avium [
demonstrated selectivity for pathogenic species in comparison to commensal intestinal
[
4],
5
6
7
8],
9], Mycobacterium bovis, and Mycobacterium tuberculosis [10]. Moreover,
1
microbiota [11,12]. The mode of action of 1 involves bacterial multi-targets, including membrane
disruption [13], cell division inhibition [14], and reactive-oxygen species induction [15].
In vitro and in vivo toxicity of
1 have been clearly elucidated due to its application as a food
additive [16 17]. In this context, was classified as GRAS (generally recognized as safe) by the FDA
,
1
(Food and Drug Administration) [18]. On the other hand, cinnamaldehyde possess an aliphatic aldoxyl
group, which has undesirable functionality due to its high reactivity [19]. Thus, we designed a series
of cinnamylideneacetophenones with the replacement of the aldoxyl by an acetophenone moiety.
The resonance of an additional benzene ring leads to compounds that are chemically more stable than
cinnamaldehyde, allowing insertion of versatile substituents to derive preliminary structure-activity
relationships (Scheme 1).
O
O
reactive aldoxyl
group
stable and versatile
acetophenone moiety
Scheme 1. Design of cinnamylideneacetophenones.
In our ongoing search for new antibacterials from natural product-based synthetic compounds,
we synthesized a series of 17 cinnamylideneacetophenones (
antitubercular activities were evaluated against Gram-positive and Gram-negative species, as well
as M. tuberculosis. The two most active compounds ( and ) were investigated regarding their
2–18). Their antibacterial and
3
4
selective toxicity on eukaryotic cells by using two human pulmonary cell lineages (MRC-5 and A549).
In addition, hydrophilicity of all compounds was measured by HPLC-PDA experiments, which
enabled calculation of partition coefficients (log Po/w). In silico investigations using the Molinspiration
online property calculation toolkit contributed to the prediction of absorption, distribution, metabolism
and excretion (ADME) properties of selected bioactive cinnamylideneacetophenones.
2. Results and Discussion
2.1. Chemistry
Synthesis of cinnamylideneacetophenones 2–18 was achieved by Claisen-Schmidt condensation,
using protocol reported by Weldon and co-authors, with minor modifications [20–22] (Scheme 2).
O
O
O
NaOH, EtOH, rt
R
A
B
Scheme 2. Synthesis of cinnamylideneacetophenones.
Reactions between cinnamaldehyde and corresponding acetophenone were performed under basic
catalysis, at room temperature, resulting in yields of 33–98% (Table 1). Cinnamylideneacetophenones
exhibited substitutions at para- and meta-positions on ring B by electron-donating groups (OH, NH2,
OMe, OCH2O, and Me) and electron-withdrawing groups (F, Cl, Br, NO2, and CF3). In general, a clear

Molecules 2017, 22, 1685
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and unambiguous relationship between the stereoelectronic effect of substituents on ring B and reaction
yields cannot be established.
Table 1. Yields of cinnamylideneacetophenones.
Entry
Name
R
Yield (%)
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
(2E,4E)-1,5-diphenylpenta-2,4-dien-1-one
H
m-OH
p-OH
33
91
82
35
78
35
83
98
71
72
86
79
88
85
84
49
53
(2E,4E)-1-(3-hydroxyphenyl)-5-phenylpenta-2,4-dien-1-one
(2E,4E)-1-(4-hydroxyphenyl)-5-phenylpenta-2,4-dien-1-one
(2E,4E)-1-(3-aminophenyl)-5-phenylpenta-2,4-dien-1-one
(2E,4E)-1-(3-methoxyphenyl)-5-phenylpenta-2,4-dien-1-one
(2E,4E)-1-(4-methoxyphenyl)-5-phenylpenta-2,4-dien-1-one
(2E,4E)-1-(3,4-methylenedioxy)-5-phenylpenta-2,4-dien-1-one
(2E,4E)-1-(4-hydroxy-3-methoxyphenyl)-5-phenylpenta-2,4-dien-1-one
(2E,4E)-1-(4-fluorophenyl)-5-phenylpenta-2,4-dien-1-one
(2E,4E)-1-(4-chlorophenyl)-5-phenylpenta-2,4-dien-1-one
(2E,4E)-1-(4-bromophenyl)-5-phenylpenta-2,4-dien-1-one
(2E,4E)-1-(3,4-dichlorophenyl)-5-phenylpenta-2,4-dien-1-one
(2E,4E)-1-(3-(trifluoromethyl)phenyl)-5-phenylpenta-2,4-dien-1-one
(2E,4E)-1-(3-nitrophenyl)-5-phenylpenta-2,4-dien-1-one
(2E,4E)-1-(4-nitrophenyl)-5-phenylpenta-2,4-dien-1-one
(2E,4E)-1-(3-methylphenyl)-5-phenylpenta-2,4-dien-1-one
(2E,4E)-1-(4-methylphenyl)-5-phenylpenta-2,4-dien-1-one
m-NH2
m-OMe
p-OMe
-OCH2O-
m-OMe,p-OH
p-F
p-Cl
p-Br
m,p-diCl
m-CF3
m-NO2
p-NO2
m-Me
p-Me
The structure of compounds was identified by their melting point, UV-Vis, 1H-NMR,
and 13C-NMR spectra analyses and was compared with previous data [20
23]. Out of 17 compounds,
compound was identified as a new chemical entity. UV-Vis spectra performed on HPLC-PDA
experiments indicated values ranging from 343 to 361 nm, corroborating the presence of an
–
9
λ
max
α,β,γ,δ-unsaturated ketone system in cinnamylideneacetophenone structures. For all compounds,
NMR data, including chemical shifts, integrations, multiplicities, and coupling constants, corresponded
with the proposed structures. Cinnamylideneacetophenones were analyzed by HPLC-PDA and
their purity was measured through peak area values, which ranged from 96.1 to 99.8%. Spectra and
chromatograms were presented as supplementary material.
2.2. Antibacterial, Antitubercular Activity, and Structure-Activity Relationship (SAR)
For antibacterial evaluations, three Gram-positive and two Gram-negative species were used,
including Staphylococcus aureus (ATCC 14458), Streptococcus mutans (ATCC 25175), Streptococcus
sanguinis (ATCC 10557), Pseudomonas aeruginosa (ATCC 15442) and Escherichia coli (ATCC 10536).
The antibacterial potency was demonstrated by MIC (minimum inhibitory concentration) and MBC
(minimum bactericidal concentration) values. MIC and MBC were determined by broth microdilution
method, in 96-well microtiter plates, following the criteria previously described by the National
Committee for Clinical Laboratory Standards [24]. Penicillin, vancomycin, and chlorhexidine were
used as positive controls. All cinnamylideneacetophenones were assayed in concentrations ranging
from 3.9 to 500 µM.
For antitubercular assays, Mycobacterium tuberculosis H37Rv (ATCC 27294) was used as a reference
strain. Antitubercular potency was expressed as MIC values, which were established by protocol
reported by Palomino and co-author, with minor modifications [25
,
26]. Isoniazid was used as the
reference antitubercular drug. Compounds 1–18 were evaluated in concentrations ranging from 1 to
200 µM (Table 2).

Molecules 2017, 22, 1685
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Table 2. Antibacterial and antitubercular activities and log Po/w of cinnamylideneacetophenones.
O
R
Sa *
MIC
Sm *
Ss *
Pa *
MBC MIC
Ec *
Mt *
MIC
Entry
log Po/w
R
MBC MIC MBC MIC
MIC
1
2
3
4
5
6
7
8
9
-
H
m-OH
p-OH
m-NH2
m-OMe
p-OMe
-OCH2O-
>500
>500
77.9
77.9
>500
296
-
-
>500
-
-
>500
>500
77.9
156
-
-
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
-
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
118
>200
57.2
88.8
81.9
>200
>200
66.6
>200
83.5
67.4
>200
>200
>200
>200
>200
>200
>200
70.9
-
-
>500
77.9
156
3.2
2.7
2.6
2.5
3.3
3.2
3.2
2.7
3.3
3.7
3.8
4.1
3.7
3.3
3.2
3.6
3.5
-
156
77.9
-
296
-
-
279
-
-
-
-
-
-
-
-
156
312
313
-
-
-
-
-
-
-
-
-
-
-
-
-
-
156
312
-
-
-
-
-
-
-
-
-
-
-
-
-
-
313
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
0.4
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
-
>500
>500
m-OMe,p-OH 279
10
11
12
13
14
15
16
17
18
PEN
VAN
CHX
ISO
p-F
p-Cl
p-Br
m,p-diCl
m-CF3
m-NO2
p-NO2
m-Me
p-Me
-
>500
>500
>500
>500
>500
>500
>500
>500
>500
-
-
-
6.8
-
0.4
3.3
9.5
-
-
-
-
3.3
-
-
-
2.4
-
-
4.7
-
3.3
4.7
-
-
53.8
-
-
-
-
0.3
-
-
-
19.4
-
-
* MIC = minimum inhibitory concentration; MBC = minimum bactericidal concentration. MIC and MBC in µM.
Sa = Staphylococcus aureus; Sm = Streptococcus mutans; Ss = Streptococcus sanguinis; Pa = Pseudomonas aeruginosa;
Ec = Escherichia coli; Mt = Mycobacterium tuberculosis; PEN = penicillin; VAN = vancomycin; CHX = chlorhexidine;
ISO = isoniazid; - not determined.
Cinnamaldehyde (
P. aeuruginosa, and E. coli. These results corroborated other investigations, which have described MIC
values higher than 500 M. Domadia and collaborators reported MIC values of 1000 mg/L (7566 µM
1) displayed MIC > 500 µM against S. aureus, S. mutans, S. sanguinis,
µ
)
against E. coli [14]. Ferro and co-authors described MIC values of 0.25 mg/mL (1891 µM) against
the S. aureus strains [27]. On the other hand, our result against M. tuberculosis (MIC > 200 µM) was
opposite to that described by Ochoa and colleagues, who reported MIC value 3.12
We used unsubstituted cinnamylideneacetophenone ( ) as a framework to investigate the
relationship between substitutions on ring B and antibacterial/antitubercular activities. Firstly, was
compared with phenolic compounds and , which suggested the hydroxyl group improved
antibacterial activity, mainly against Gram-positive species. Compound demonstrated MIC and
MBC values of 77.9 and 156 M against S. aureus, S. mutans and S. sanguinis, respectively. Compound
displayed similar antibacterial potency with MIC and MBC values ranging from 77.9 to 312 M. On the
other hand, the hydroxyl group reduced antitubercular activity, because unsubstituted compound
(MIC = 57.2 M) was more potent than phenolics (MIC = 88.8 M) and (MIC = 81.9 M). Also,
aminoderivative exhibited activity against S. mutans (MIC = MBC = 313 M) and was not able to act
against S. aureus, S. sanguinis, nor M. tuberculosis. These results indicate that electron-donating and
µg/mL (23 µM) [10].
2
2
3
4
3
µ
4
µ
2
µ
3
µ
4
µ
5
µ
hydrophilic groups interfere with bioactivity, and weak acidic compounds 3 and 4 were more active
than basic compound 5.
Second, comparisons were made to confirm the importance of hydrophilic, acidic,
and electron-donating groups for bioactivity. Methoxylated compounds and were less active
than their hydroxylated congeners and against S. aureus, S. mutans, and S. sanguinis,
6
7
3
4

Molecules 2017, 22, 1685
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with MIC values ě 296 µM. 3,4-Methylenedioxylated compound
8
, which is closely similar to
6 and
7
, was inactive with MIC values > 500 µM. Vanillyl compound 9, which possess meta-methoxyl and
para-hydroxyl groups, was active effect against S. aureus (MIC = MBC = 279 µM).
Third, comparisons were made to establish if hydrophilicity or electron donation is pivotal
for bioactivity. Cinnamylideneacetophenones carrying hydrophobic and electron-withdrawing
substituents (F, Cl, Br, CF3, and NO2) (10–16) were inactive against S. aureus, S. mutans, and S. sanguinis.
In addition, methylated compounds 17 and 18, which are compounds substituted by hydrophobic and
electron-donating groups, were also inactive. Thus, the electronic effect was not crucial for antibacterial
activity, but hydrophilicity increased it. In contrast, antitubercular assays indicated para-fluorinated
and para-methylated compounds 10 (MIC = 67.4 µM) and 18 (MIC = 70.9 µM), respectively, were active
against M. tuberculosis. This comparison suggested hydrophobic groups reduced the antibacterial
activity and the electronic nature of the substituent was irrelevant for antitubercular activity.
Fourth, we evaluated the effect of the position of the substituent on bioactivity. For pairs
of regioisomers
were essential for antitubercular activity. Para-substituted compounds
of 66.6 and 70.9 M, respectively, against M. tuberculosis, while their respective meta-substituted
compounds were inactive (MIC > 200 M). The position of hydroxyl and nitro substituents of
6
/7
and 17
/
18, the position of the methoxyl and methyl groups, respectively,
7
and 18 presented MIC values
µ
µ
regioisomer pairs 3/4 and 15/16 did not interfere with antitubercular activity.
Interestingly, cinnamylideneacetophenones that were bioactive against Gram-positive species
were not able to inhibit Gram-negative species growth, such as P. aeruginosa and E. coli, therefore,
they exhibited reduced spectrum. Gram negative bacteria demonstrate a complex barrier system,
which makes them less susceptible to action of antibacterial agents. As part of this system, the cell wall
is mainly responsible for impermeability due to the presence of an external envelope, composed of
lipopolysaccharides, phospholipids, and proteins [28,29].
2.3. Determination of Partition Coefficient Log Po/w
Preliminary structure-activity relationship (SAR) indicated the essentiality of the hydrophilicity
for antibacterial and antitubercular activities. Thus, we calculated the partition coefficient
(n-octanol/water) (log Po/w) by using the HPLC-PDA method suggested by Organisation for
Economic Co-operation and Development (OECD) protocols [30]. Log Po/w values of the
cinnamylideneacetophenones 2–18 are summarized in Table 2. The reference compounds, aniline,
phenol, acetophenone, benzoic acid, benzene, toluene, benzophenone, and thymol, were used to
construct a log K versus log Po/w curve. The capacity factor (log K) of the cinnamylideneacetophenones
was determined from retention times. Anti-Staphylococcus and anti-Streptococcus compounds exhibited
log Po/w values ranging from 2.5 to 3.3. Among these, compounds
with log Po/w values of 2.7 and 2.6, respectively. The four most active anti-Mycobacterium compounds
(MIC 70.9 M) presented log Po/w values ranging from 3.2 to 3.5. Highly hydrophobic compounds
3 and 4 were the most potent,
ď
µ
displayed log Po/w > 3.5 (11
and M. tuberculosis.
–14 and 17) and were not able to act against S. aureus, S. mutans, S. sanguinis,
2.4. Evaluation of Cytotoxicity and Determination of Selectivity Index
The two most potent compounds ( and ) were investigated for their selective toxicity against
3
4
eukaryotic cells. Human lung fibroblast (MRC-5, ATCC, CCL-171) and human lung adenocarcinoma
cells (A549, ATCC, CCL-185) were used for cytotoxicity evaluations. The choice for pulmonary cells
was based on their importance as host site infection of S. aureus and M. tuberculosis [26
assays were performed using protocol described by our group [32]. Compounds
similar effects against both human cells, demonstrating values of IC50 (concentration able to inhibit
50% of cell growth) ranging from 46.3 to 96.7 M (Table 3). These compounds were moderately toxic
,
31]. The cytotoxic
3
and displayed
4
µ
against pulmonary cells when compared with doxorubicin which exhibited IC50 values of 0.7 and
1.8 µM against MRC-5 and A549, respectively. In order to compare the effect towards prokaryotic cells

Molecules 2017, 22, 1685
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versus eukaryotic cells, the selectivity indexes (SI) of
3
and
4
were calculated as the ratio of cytotoxicity
to antimicrobial activities (SI = IC50 eukaryotic cell/MIC prokaryotic cells) (Table 3). The effectiveness
of a compound is indicated by SI 10. However, SI 1 indicates toxicity of that compound for
eukaryotic cells and low selectivity for the prokaryotic cells. Despite and demonstrating promising
ě
ď
3
4
antibacterial and antitubercular activities, these compounds showed SI values ranging from 0.5 to 1.2.
Table 3. Toxicity against human lung cells and selectivity index of selected cinnamylideneacetophenones.
MRC-5
SI
A549
SI
Entry
IC50
*
Sa
Mt
IC50
*
Sa
Mt
3
4
46.3
53.1
0.7
0.6
0.7
-
0.5
0.6
-
96.7
77.5
1.8
1.2
1.0
-
1.2
0.9
-
doxorubicin
* IC50 in µM.
Antimicrobial agents with in vitro toxicity against human cells should not be ignored. Although SI
values are relevant as a general orientation, this ratio cannot be used as the unique parameter to decide
if a compound can continue to preclinical trials [33
indicated to capture the potential of 3 and 4 as valuable molecules.
The low selectivity of the cinnamylideneacetophenones and
,34]. In this context, in vivo toxicity trials are highly
3
4 can be correlated with their
phenolic nature. Studies report the ability of phenolic compounds to act on widespread radical
processes shared by eukaryotic and prokaryotic cells [35]. We hypothesized this behavior may be
responsible by simultaneous activity toward bacteria and pulmonary cells. Therefore, we evaluated
the radical scavenging ability of
Compounds and were evaluated at 1600, 800, 400, and 200
DPPH scavenging ability below 8% (Figure 1), suggesting their low efficacy on radical processes.
Altogether, low selectivity of and cannot be strongly related to radical processes. Quercetin and
gallic acid were used as positive controls due to their phenolic nature and potent radical scavenging
ability [38 39]. These compounds were tested in the same concentrations used for compounds and
3
and
4
using DPPH (2,2-diphenyl-1-picrylhydrazyl) protocol [36
,37].
3
4
µM and exhibited all percentages of
3
4
,
3
4,
demonstrating percentages of DPPH scavenging ability ě95%.
10
1600 μM
800 μM
400 μM
200 μM
8
6
4
2
0
3
4
Figure 1. Radical scavenging ability of selected cinnamylideneacetophenones. DPPH = 2,2-diphenyl-
1-picrylhydrazyl.
2.5. In Silico Prediction of ADME of Cinnamylideneacetophenones
Several reasons contribute to the attrition of drug candidates, including pharmacokinetic
difficulties, insufficient efficacy, and adverse reactions in animal and humans. In the development
phase of oral drugs, nearly 30% of drug candidates fail due to poor ADME properties [40]. Bioactive

Molecules 2017, 22, 1685
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cinnamylideneacetophenones were also subjected to pharmacokinetic filters. An in silico study was
performed for prediction of ADME properties using the Molinspiration property explorer toolkits [41].
In Table 4, we summarize the computed molecular properties of the selected compounds, such as
absorption (%ABS), topological polar surface area (TPSA), lipophilicity (milog P), molecular weight
(MW), number of hydrogen bond donors (HBD), number of hydrogen bond acceptors (HBA), violations
of Lipinski’s rule of five (Ro5), and number of rotatable bonds (NROTB).
Table 4. Lipinski’s and Veber’s parameters of selected cinnamylideneacetophenones.
TPSA (Å2)
Entry
%ABS
milog P
MW
HBD
HBA
Lipinski’s Violations
NROBT Veber’s Violations
Ro5
Vb
1
2
3
4
5
6
7
-
-
-
ď5.00
<500
-
ď5
ď10
1
-
-
ď10
2
4
4
4
4
5
5
-
ď140
17.07
17.07
37.30
37.30
43.09
26.30
26.30
46.53
17.07
17.07
-
-
-
0
0
0
0
0
0
0
0
0
0
0
100
100
96.1
96.1
94.1
99.9
99.9
92.9
100
100
2.48
4.33
3.83
3.85
3.38
4.36
4.39
3.67
4.49
4.78
132.16
234.30
250.30
250.30
249.31
264.32
263.32
280.32
252.29
248.32
1
1
2
2
2
2
2
3
0
0
1
1
2
0
0
1
0
0
0
0
0
0
0
0
0
0
9
10
18
5
4
4
1
1
0
0
%ABS = percent absorption; TPSA = topological polar surface area; milog P = lipophilicity; MW = molecular weight;
HBD = number of hydrogen bond donors; HBA = number of hydrogen bond acceptors; NROBT = number of
rotatable bonds; Ro5 = Boundaries of Lipinski’s rules; Vb = Boundaries of Veber’s rules.
TPSA has been used as descriptor for characterizing absorption and passive transportation
properties through biological membranes, allowing a good prediction of transport of candidate drugs
in the intestines and through the blood-brain barrier (BBB). TPSA is sum of van der Waals surface
areas of electronegative atoms (oxygen and nitrogen). Erlt and co-authors described TPSA values
useful for prediction of drug transport properties [42]. Compounds with TPSA values in the range
ď
150 Å2 have good intestinal absorption and
exhibited TPSA values
46.53 Å2. The four more potent antitubercular cinnamylideneacetophenones
10, and 18) displayed TPSA values ranging from 17.07 to 26.30 Å2, suggesting their efficient BBB
penetration, which is a crucial property to combat tuberculous meningitis [43]. TPSA was used to
calculate the percentage of absorption (%ABS) according to the equation: %ABS = 109 0.345 TPSA.
All selected compounds demonstrated high %ABS, ranging from 92.9 to 100. The three most potent
anti-Mycobacterium compounds ( 10, and 18) exhibited a %ABS value of 100, which is relevant for
ď
90 Å have BBB penetration. All selected compounds
ď
(2, 7,
´
ˆ
2
,
their oral absorption, as well as for penetration through the highly hydrophobic mycolic acid layer of
M. tuberculosis [44].
In order to achieve the optimum properties intended for oral administration, Lipinski and
collaborators established a series of rules for drug candidates such as: (a) a log P less than 5, (b) a
molecular weight less than 500 Da, (c) less than five hydrogen bond donors (sum of OH and NH
groups), and (d) less than 10 hydrogen bond acceptors (sum of oxygen and nitrogen) [45]. Selected
cinnamylideneacetophenones did not violate Lipinski’s parameters, corroborating their oral drug
likeness properties.
Among pharmacokinetic difficulties to drug candidates, low oral bioavailability has been
considered a central reason for interrupting further drug development phases. In this context, Veber
and collaborators suggested a filter for drug candidates, containing two essential criteria for oral
bioavailability in rats: (a) TPSA equal or less than 140 Å2 and (b) ten or fewer rotatable bonds [46].
All selected compounds exhibited NROTB
bioavailable by an oral route.
ď5 and TPSA values
ď
46.53 Å2, making them potentially

Molecules 2017, 22, 1685
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3. Materials and Methods
3.1. Synthesis of Cinnamylideneacetophenones
All starting reagents were purchased from Sigma-Aldrich. Compounds were synthesized
by Claisen-Schmidt condensation, according to the method reported by Weldon and co-authors,
with minor modifications [20–22]. Cinnamaldehyde (6 mmol) was added to solution of respective
acetophenone (5 mmol) in ethanol (5 mL). The solution was stirred at room temperature for 30 min,
followed by dropwise addition of 5 mL ethanolic solution of NaOH (1.0 M). The progress of reaction
was checked by thin-layer chromatography (TLC) analyses. Reaction medium was poured into
crushed ice. Precipitated crude products were filtered, washed with cold water, and dried at room
temperature. Soluble crude products were partitioned with ethyl acetate (3
ˆ
25 mL). Organic phase
was concentrated under reduced pressure. Compounds and 10 were recrystallized in ethanol.
2,
7,
8
–7
Compounds 3–6, 9 and 18 were recrystallized in mixtures of hexane and chloroform.
3.2. Antibacterial Activity of Cinnamylideneacetophenones
Microorganisms used in this study were Staphylococcus aureus (ATCC 14458), Streptococcus mutans
(ATCC 25175), Streptococcus sanguinis (ATCC 10557), Pseudomonas aeruginosa (ATCC 15442),
and Escherichia coli (ATCC 10536). MIC and MBC values were determined by broth microdilution
method, in 96-well microtiter plates, following the criteria previously described by the National
Committee for Clinical Laboratory Standards [24]. Bacterial cell cultures in Brain Heart Infusion broth
(BHI) at the mid-log phase were harvested by centrifugation for 10 min at 3000
discarded and the pellet resuspended in Mueller-Hinton broth (Difco Laboratories, Detroit, MI, USA).
The final bacterial suspension was 5–10
105 CFU (colony forming units) per mL. Compounds
were diluted in sterile deionized water in concentrations ranging from 500 to 3.9 M. Bacterial
suspension was inoculated in each well. The plates were incubated at 37 C for 24 h in 5% CO2.
Thus, 15 L of 0.01% resazurin staining was added to each well and incubated for 4 h to determine
ˆ
g. Supernatant was
ˆ
µ
˝
µ
cell viability. Wells corresponding to MIC and at least three previous wells were homogenized,
serial˝ly diluted, and plated on Mueller-Hinton agar to determine MBC. The plates were incubated
at 37 C for 24 h in 5% CO2. Number of colonies forming units per mL (CFU/mL) was determined.
The MBC was considered when the compounds killed more than 99% of the tested bacterial culture.
Penicillin G, vancomycin, chlorhexidine, and culture medium were used as positive and negative
controls, respectively. Three independent assays were performed.
3.3. Antitubercular Activity of Cinnamylideneacetophenones
The antitubercular activity of compounds was determined by the REMA (Resazurin Microtiter
Assay) method [25
and diluted in Middlebrook 7H9 broth (Difco) supplemented with oleic acid, albumin, dextrose,
and catalase (OADC), to obtain the final concentration range from 100 to 1 M. Suspension of
,26]. Stock solutions of compounds were prepared in DMSO (dimethyl sulfoxide)
µ
Mycobacterium tuberculosis H37Rv strain (ATCC 27294) was cultured in Middlebrook 7H9 broth
supplemented with OADC. When the culture obtained a turbidity of McFarland standard No. 1,
it was adjusted by 3–5 ˆ 105 CFU per mL. Thus, 100
µL of inoculum were added to each well in a
96-well microplate with 100 µ
L of the compound solution. Plates were incubated for 7 days at 37 ˝C
and 5% CO2. Resazurin was added (30 µL of 0.01%). The fluorescence of the wells was measured after
24 h on Cytation 3 (BioTek, Winooski, VT, USA) equipment. MIC value was defined as the lowest
concentration resulting in 90% inhibition of growth of M. tuberculosis. Isoniazid and culture medium
were used as positive and negative controls, respectively. Three independent assays were performed.
3.4. Partition Coefficient (log Po/w) of Cinnamylideneacetophenones
Values of log Po/w were determined using the HPLC method according to OECD-Guidelines for
the Testing of Chemicals [30], on Shimadzu (Kyoto, Japan) HPLC-PDA (model CBM 20-A) equipped

Molecules 2017, 22, 1685
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with a UV-Vis detector (model SPD-20A), binary pumping system (model LC-20AD), solvent degasser
(model DGU-20A3r), and a Phenomenex Gemini C-18 column (Torrance, CA, USA) (250 mm 4.6 mm;
m; 110 Å). Analysis was performed on isocratic mode in MeOH:H2O (3:1) and 1.0 mL/min. Injected
volume of sample was 20.0 L and the wavelength in the detector was adjusted according to max of
each compound. Reference compounds phenol, acetophenone, benzoic acid, toluene, and thymol were
used to construct the curve log K log Po/w. Capacity factor of the cinnamylideneacetophenones
ˆ
5
µ
µ
λ
ˆ
was determined from their retention times and interpolated in linearity curve log K
ˆ
log Po/w.
Experiments were performed in duplicate.
3.5. Toxic Effect on Lung Cells of Selected Cinnamylideneacetophenones
Cytotoxicity of compounds was evaluated according to Silva and colleagues [32]. The cell
lineages used were MRC-5 (human lung fibroblast cell line—ATCC, CCL-171) and A549 (human
lung adenocarcinoma epithelial cell line—ATCC, CCL-185). Cells were routinely maintained in
complete medium supplemented ˝with 10% fetal bovine serum (FBS) plus amphotericin B (2 mg/L)
and gentamicin (50 mg/L) at 37 C in a humidified 5% CO2 atmosphere. For cytotoxicity assay,
2.5 ˆ 104 cells/mL were seeded into a 96-well cell culture plate with a total volume of 100
µL for 24 h.
Cells were treated with compounds at concentrations ranging from 100 to 1 µM. After 24 h incubation,
medium was removed, and 50 µL of resazurin (0.01% w/v) was added to each well, and the plates
were incubated at 37 ˝C for 3 h. Fluorescence of wells was measured on a Synergy H1 microplate
reader (BioTek, Winooski, VT, USA) in which were used excitation (530 nm) and emission (590 nm)
filters. The IC50 values were calculated from a calibration curve by regression analysis. Doxorubicin
and untreated cells constituted the positive and negative controls, respectively. Three independents
assays were performed.
3.6. DPPH Scavenging Ability of Selected Cinnamylideneacetophenones
Radical scavenging activity of compounds
1-picrylhydrazyl (DPPH) [36 37]. Aliquots of 10
gallic acid) at concentrations 1600, 800, 400, and 200
190 L DPPH (100
M). The mixtures were incubated in the dark at 25 ˝C for 30 min. Remaining
3
and
4
was determined using 2,2-diphenyl-
,
µL of
3, 4, and positive controls (quercetin and
µM (in EtOH) were individually added in
µ
µ
DPPH was determined spectroscopically at 490 nm by blanking against absolute ethanol. Radical
scavenging ability was expressed by using mean values obtained from triplicates as the percentage of
radicals reduced by calculating the following equation: DPPH scavenging % = [1
where A is test absorbance without sample (only EtOH and DPPH) and Asample is test absorbance
´
(Asample/A)
ˆ
100],
with compounds.
4. Conclusions
In summary, a series of cinnamylideneacetophenones containing several substituents on ring
B were designed and synthesized as part of our ongoing search for antibacterial and antitubercular
compounds based on natural products. Five compounds ( ) exhibited potent antibacterial
3–
6
and
9
activity against Gram-positive species. Seven compounds (2–4, 7, 9, 10 and 18) were able to
inhibit M. tuberculosis growth. Preliminary SAR investigations suggested hydrophilicity is the central
parameter for antibacterial and antitubercular activities. Furthermore, our results corroborated the
potential of cinnamaldehyde as a privileged starting material and template for synthetic collection of
hits; it did not violate Lipinski’s and Veber’s boundaries.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: The authors gratefully acknowledge financial support from the Coordination for the
Improvement of Higher Education Personnel (CAPES), Brazilian Council for Scientific and Technological
Development (CNPq) (Grants 471129/2013-5 and 306251/2016-7), and the São Paulo Research Foundation
(FAPESP) (Grant 2014/18330-0). LAA thanks FAPESP research fellowship (2016/06401-5).

Molecules 2017, 22, 1685
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Author Contributions: L.O.R., C.D., F.R.P., and H.F. conceived and designed the experiments; C.R.P., G.S.T.,
V.R.S., A.C.N., D.L.C., L.A.A., and I.C.S. performed the experiments; everybody contributed to data analysis.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2017 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
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).