M.M.R. Siqueira et al.
European Journal of Pharmaceutical Sciences 158 (2021) 105695
α
,β-unsaturated carbonyl (Mari n˜ o et al., 2016). This compound class has
chlorophenyl)‑prop-2-en-1-one (C15H12NOCl, hereafter named ACLO-
a yellow coloration; however, in an alkaline medium, it presents a red
color. Chalcones are present in the pigmentation of flowers, making
them attractive to birds and insects that contribute to the pollination
process of plants (Katsori and Hadjipavlou-Litina, 2009).
PHENYL). The evaluation of inhibition of the efflux pump for both
compounds was also performed. Two strains of S. aureus were used:
1199B, which overexpresses the NorA gene and the multi-drug resistant
(MDR) mutant strain K2068, which presents the MepA efflux pump.
The synthesis of chalcone compounds is essential, given that the
volume of material that can be extracted through natural processes is
usually insignificant and inappropriate for use at an industrial scale.
Among the methods of chalcones syntheses, the aldol condensation of
Claisen-Schmidt stands out (Nandedkar et al., 2013). Recently, studies
have shown different pharmacological activities for chalcones, such as
antifungal (Gupta and Jain, 2015), antibacterial (Tran et al., 2012),
anticancer (Bandeira et al., 2019; Bhat et al., 2005; Katsori and Hadji-
pavlou-Litina, 2009), antioxidant, and antidiabetic activity (Eichen-
berger et al., 2017).
2. Materials and methods
2.1. Synthesis of chalcones
The p-aminochalcones derivatives used in this study were synthe-
sized via Claisen-Schmidt condensation conducted under basic condi-
tions. A solution of p-aminoacetophenone (2 mmol) in ethanol (5 mL)
was added to a solution of benzaldehyde (2 mmol) in ethanol (5 mL)
containing 10 drops of 50% v/v sodium hydroxide, and the resulting
mixture was stirred for 48 h. The mixture was filtered under vacuum,
washed with cold water to pH 7.0, and analyzed by Thin-layer chro-
matography (TLC) (Scheme 1).
Health care-associated infections (HCAI) are considered a major
problem for public health, impacting on morbidity and mortality rates
during the hospitalization period, as well as increasing diagnostic and
therapeutic expenses (O’Neill, 2016; Oliveira et al., 2010). Antibiotic
resistance is considered a world health disorder, which impacts the
effectiveness of antibiotics for the treatment of infections (WHO, 2009).
In this context, the use of a natural or synthetic substance to modulate
bacterial resistance against an antibiotic has proven to be an alternative
to drugs that are more effective in the treatment of infectious diseases
′
Some characteristics of (2E)ꢀ 1-(4 -aminophenyl)ꢀ 3-(phenyl)‑prop-
2-en-1-one (APCHAL) are given as follows. Yellow solid (Yield:
◦
ꢀ 1
cm): 3522, 3434, 1623,
25.60%), m.p. 109.3 – 109.9 C; FT-IR (KBr,
ν
1
1578, 1554. H NMR (CD
3
OD, 300 MHz) δ: 7.40 – 7.42 (m, H-3/H-5, H-
′
′
′
′
4), 7.93 (d, H-2 /H-6 , J = 8.7 Hz), 6.73 (d, H-3 /H-5 , J = 8.7 Hz), 7.69 –
1
3
7.72 (m, H-2/H-6, H-
α, H-β). C NMR (CD
3
′
OD, 75 MHz) δ: C-1 136.8, C-
′
′
′
(
Rios et al., 1988).
2/C-6 129.6 C-3/C-5 130.1, C-4 131.4, C-1 128.2, C-2 /C-6 132.6, C-3 /
′
′
–
–
Bacterial resistance to antibiotics can be internal or obtained through
C-5 115.1, C-4 154.8, C-
α
123.3, C-β 144.4, C O 190.3. MS (EI) m/z
13NO/223 (Bandeira et al., 2019; Santiago
+
.
genetic transmission or mutation, modifying the mechanisms by which
antibiotics become incapable of combating bacterial strains (Sun et al.,
(M 223), calcd for C15
H
et al., 2018).
′
2
014). An important mechanism of bacterial resistance is the presence of
Some characteristics of (2E)ꢀ 1-(4 -aminophenyl)ꢀ 3-(4-clor-
ophenyl)‑prop-2-en-1-one (ACLOPHENYL) are given as follows. Yellow
proteins from efflux pumps in their membranes. It is known that the
efflux pump is one of the main causes of drug resistance (Webber and
Piddock, 2003). The efflux pumps act by active transport, causing the
extrusion of one or several types of antibiotics from the bacterial cyto-
plasm (Sharma et al., 2019). As examples of multi-drug resistance
pumps (MDR pumps) are NorA and MepA, analyzed in this article. In this
way, efflux pump inhibitors become an important therapeutic alterna-
tive in the treatment of infectious diseases.
◦
ꢀ 1
solid (Yield: 53.59%), m.p. 162,9 - 163,3 C; FT-IR (KBr,
ν
cm): 3555,
1
3350, 1621, 1570, 1550, 1490. H NMR (CD
3
OD, 300 MHz): δ 7.91 (d,
′
H-2/H-6, J = 8.73 Hz), 7.69 (d, H-3/H-5, J = 8.79 Hz), 7.42 (d, H-2 /H-
′
′
3
′
6 , J = 8.46 Hz), 6.68 (d, H-3 /H-5 , J Hz), 7.70 (d, H-
α
, J = 14.28 Hz),
1
7.74 (d, H-β, J = 15.60 Hz). C NMR (CD
3
OD, 75 MHz): δ C-1 135.6, C-
′
′
′
2/C-6 132.7, C-3/C-5 130.9, C-4 137.5, C-1 127.6, C-2 /C-6 130.8, C-
′
′
′
–
–
O 189.9. MS (EI) m/z
3 /C-5 114.6, C-4 155.8, C-
α
124.3, C-β 142.9, C
+
.
Previous studies had reported that some compounds can inhibit
efflux pumps, such as certain compounds derived from indole (Lepri
et al., 2016). Derivatives of 2-arylquinoline (Felicetti et al., 2020) and
boronic acid derivatives have the ability to inhibit NorA efflux pumps
(M 257.5), calcd for: C15H12NOCl/257.5 (Bandeira et al., 2019).
2.2. Microorganisms
(
Fontaine et al., 2014), while compounds derived from 2-phenylquino-
In the studies related to the modification of antibiotic activity, the
standard bacterial strains used were Escherichia coli ATCC 25922 and
Staphylococcus aureus ATCC 25923, while the resistant strains were
E. coli 06 and Staphylococcus aureus 10. The E. coli 06 strain is resistant to
several β-lactams such as cephalothin, cephalexin, and ceftriaxone,
while the Staphylococcus aureus 10 strain is resistant to β-lactams, fluo-
roquinolones, and macrolides.
line showed the ability to inhibit NorA and MepA efflux pumps (Feli-
cetti et al., 2018). Previous studies had shown that chalcones can exhibit
antibacterial properties via efflux pump inhibition (Dan and Dai, 2020;
Holler et al., 2012; Thai et al., 2015). Holler et al. screened a library of
1
17 chalcones for efflux pump inhibition activity against NorA (Holler
et al., 2012). In this study, twenty chalcones were shown to be inhibitors
of the NorA efflux pump in everted membrane vesicles. It was also
shown that two chalcones exhibited potential activity equal to the
known efflux pump inhibitor reserpine. These results lead them to
suggest that chalcones can be transformed into drugs for overcoming
multi-drug resistance based on efflux transporters of microorganisms.
Thai et al. carried out a virtual screening and molecular docking in a
series of forty-seven natural compounds, including chalcones, to inves-
tigate the novel Staphylococcus aureus NorA efflux pump inhibitors (Thai
et al., 2015). They observed that seven of them were predicted as NorA
In the efflux pump tests, the bacterial strains of Staphylococcus aureus
1199B (which overexpresses the NorA gene encoding the NorA efflux
protein) and Staphylococcus aureus K2068 (which presents the MepA
efflux pump) were used. All the bacterial strains were cultivated in heart
infusion agar (HIA) during 24 h. Brain heart infusion (BHI) broth was the
medium used at the time of the test.
2.3. Antimicrobial activity and effect
′
inhibitors, among them the (2E)ꢀ 1-(4 -aminophenyl)- 3-(4-chlor-
2.3.1. Drugs
ophenyl)‑prop-2-en-1-one, which is one of the chalcones that will be
investigated in this work.
For the evaluation of the antibiotic potentiating activity of the
APCHAL and ACLOPHENYL chalcones, two antibiotics were used: the
aminoglycoside antibiotic gentamicin, the β-lactam antibiotic penicillin.
In tests with efflux pumps, the antibiotics chosen act as specific
substrates for each one of the bacterial strains: norfloxacin for SA 1199B,
and ciprofloxacin for SA K2068.
An attempt to contain the bacterial resistance by efflux pump inhi-
bition happens through the association of antibiotics with substances
that are capable of inhibiting these pumps. In this study, we investigated
the antimicrobial and antibiotic potentiating activity of the chalcones
′
(
2E)ꢀ 1-(4 -aminophenyl)ꢀ 3-(phenyl)‑prop-2-en-1-one
(C15
H
13NO,
The solutions were prepared on the basis of recommendations
established in CLSI (CLSI, 2008) and diluted in sterile water to reach a
′
hereafter named APCHAL) and (2E)ꢀ 1-(4 -aminophenyl)ꢀ 3-(4-
2