Spectroscopic characterization and efflux pump modulation of a thiophene curcumin derivative

Article abstract of DOI:10.1016/j.molstruc.2020.128291

Curcumin, along with its derivatives, form a large class of natural and synthetic compounds with notable biological activity. However, their highly reactive β-diketone moiety renders this type of compounds unstable at pH above 6.5. The substitution of this group for a mono-carbonyl solves this problem, while improving antibacterial and anti-inflammatory activities. A thiophene curcuminoid, (1E,4E)-1,5-Di(thiophen-2-yl)penta-1,4-dien-3-one (DB Thiophene), has been synthesised and its molecular and spectroscopic characterization is reported, as well as a complete vibrational assignment. An efflux pump inhibition assay determined that DB Thiophene exhibits a remarkable NorA and MepA efflux pump inhibition activity. Molecular docking studies were carried out in order to understand this inhibition mechanism.

Full text of DOI:10.1016/j.molstruc.2020.128291

Journal of Molecular Structure 1215 (2020) 128291
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Spectroscopic characterization and efflux pump modulation of a
thiophene curcumin derivative
a
,
*
a
,
b
a
b
Mauro M. Oliveira , Helcio S. Santos , Henrique D.M. Coutinho , Paulo N. Bandeira ,
a
a
a
a
Priscila T. da Silva , Thiago S. Freitas , Janaina E. Rocha , Jaize C. Xavier ,
a
a
a
a
Fabia F. Campina , Cristina R.S. Barbosa , Jos eꢀ B. Araújo Neto , Raimundo L.S. Pereira ,
a
a
a,
c
b
Maria M.C. Silva , D eꢀ bora F. Muniz , Alexandre M.R. Teixeira , Vanessa M. Frota ,
b
d
d, e
Tigressa H.S. Rodrigues , Ana M. Amado , Maria P.M. Marques
Luis A.E. Batista de Carvalho , Carlos E.S. Nogueira
Department of Biological Chemistry, Regional University of Cariri, Crato, CE, Brazil
Science and Technology Centre - Course of Chemistry, State University Vale Do Acaraú, Sobral, CE, Brazil
Department of Physics, Regional University of Cariri, Crato, CE, Brazil
,
d
a, c, d
a
b
c
d
e
Universidade de Coimbra, Unidade de I&D Quimica-Física Molecular (QFM-UC), Department of Chemistry, 3004-535 Coimbra, Portugal
Universidade de Coimbra, Department of Life Sciences, 3000-456, Coimbra, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 March 2020
Received in revised form
Curcumin, along with its derivatives, form a large class of natural and synthetic compounds with notable
biological activity. However, their highly reactive
unstable at pH above 6.5. The substitution of this group for a mono-carbonyl solves this problem, while
improving antibacterial and anti-inflammatory activities. thiophene curcuminoid, (1E,4E)-1,5-
b-diketone moiety renders this type of compounds
16 April 2020
A
Accepted 16 April 2020
Available online 21 April 2020
Di(thiophen-2-yl)penta-1,4-dien-3-one (DB Thiophene), has been synthesised and its molecular and
spectroscopic characterization is reported, as well as a complete vibrational assignment. An efflux pump
inhibition assay determined that DB Thiophene exhibits a remarkable NorA and MepA efflux pump in-
hibition activity. Molecular docking studies were carried out in order to understand this inhibition
mechanism.
Keywords:
Curcumin
Chalcone
Efflux pump
DFT
© 2020 Elsevier B.V. All rights reserved.
Spectroscopy
Docking
1. Introduction
treatment. In fact, it can suppress the tumorigenic activity of a
variety of carcinogens in the colon, duodenum, oesophagus,
Curcumin is a polyphenol (Scheme 1) and an active principle of
the Curcuma longa herb, also known as turmeric. Its derivatives,
called curcuminoids, form a large class of natural and synthetic
compounds with notable biological activity [1]. Curcumin interacts
with numerous targets, such as transcription and growth factors
and their receptors, as well as genes that regulate cell proliferation
and apoptosis [2]. It has thus been widely used as a chemo-
preventive agent [2] and it is a prime candidate for cancer
stomach, breast, and prostate. Curcumin and its derivatives are also
known to suppress the growth of bacteria of several genera, such as
Streptococcus, Staphylococcus, Lactobacillus and Helicobacter [3].
Additionally, it exhibits antiviral activity, including anti-HIV
through inhibition of the HIV-1 integrase, an enzyme needed for
viral replication [4].
The potential of curcumin is only hampered by its poor
bioavailability and pharmacokinetic profile, as its highly reactive
diketone moiety makes it unstable in vitro at pH above 6.5 [5].
Several studies have focused on finding synthetic derivatives with
improved pharmacokinetic profiles [6,7] by substitution of the b-
*
Corresponding author. Department of Biological Chemistry, Regional University
diketone moiety for a mono-carbonyl one. It has been shown that
this substitution not only improves stability, but also leads to a
better antibacterial and anti-inflammatory activities [8,9]. Likewise,
of Cariri, Campus Pimenta II, CEP: 63, 100-000, Crato, CE, Brazil.
E-mail addresses: mauropandi@gmail.com, mauro.oliveira@fapce.edu.br
(
M.M. Oliveira).
https://doi.org/10.1016/j.molstruc.2020.128291
022-2860/© 2020 Elsevier B.V. All rights reserved.
0

2
M.M. Oliveira et al. / Journal of Molecular Structure 1215 (2020) 128291
Scheme 1. DB Thiophene structure and atom numbering along with Curcumin structure.
modifications of the aromatic rings substitution also seems to
improve the biological activity of curcumin derivatives [10].
The use of new substances in the fight against bacterial in-
fections is often accompanied by increased resistance of these or-
ganisms. There are several mechanisms of resistance to antibiotics,
one of them being known as efflux pump. This active efflux is able
to extrude different compounds from the cell and is therefore seen
as a key element in antimicrobial resistance. Currently, more than
ten multidrug efflux pump types have been identified for S. aureus
ethidium bromide (EtBr) were acquired from Sigma Aldrich Brasil
Ltd. (S a~ o Paulo, Brazil). Brain heart infusion (BHI) was purchased
from KASVI company (S a~ o Jos eꢀ do Pinhais, Brazil).
2.2. Synthesis
The (1E,4E)-1,5-Di(thiophen-2-yl)penta-1,4-dien-3-one was
synthesised by the ClaisenꢀSchmidt condensation method [18,19],
with thiophene-2-carbaldehyde instead of benzaldehyde. In an
Erlenmeyer flask containing 50 mL of KOH 10% (m/v) and 40 mL of
ethanol, 7 mL of thiophene-2-carbaldehyde:acetone (5:2) was
[
11], namely NorA and MepA.
NorA is a fluoroquinolone-resistant efflux pump that expels a set
of different antibiotics and other substances, such as norfloxacin,
ciprofloxacin, ethidium bromide and quaternary ammonium com-
pounds [12e14]. MepA belongs to the multidrug and toxin extru-
sion (MATE) efflux pump family. First described by Kaatz et al. [15],
it is also found in S. aureus mutant strains. Inhibition of the efflux
pumps can be achieved by reducing the pump gene expression,
interrupting pump assembly, reducing substrate binding by
competitive or non-competitive means and disrupting the pump’s
required energy source. Compounds that are capable of performing
such inhibition are called efflux pump inhibitors (EPI) [16].
slowly added, under agitation, and the temperature was kept at
ꢁ
0
C. After 30 min, the mixture was filtered: a precipitate was ob-
tained and washed with small portions of distilled water. The
compound was redissolved in hot ethanol, filtered and allowed to
cool in an ice bath. The formed crystals were washed with cold
ethanol and dried at room temperature. Scheme 2 shows the DB
Thiophene synthesis reaction.
(
1H NMR (CDCl3, 500 MHz):
H25), 7.15 (2H, m, H22, H26), 7.40 (2H,d, J ¼ 3.6 Hz, H23,H24), 6.89
2H, d, J ¼ 15.6 Hz, H17, H18), 7.92 (2H, d, J ¼ 15.6 Hz, H19, H20) 13C
NMR (CDCl3, 125 MHz):
¼ 140.3 (CH, C7, C8), 128.4 (CH, C10, C15),
28.8 (C11, C16), 135.6 (CH, C12C14), 124.4 (CH, C3, C4), 131.8 (CH,
C5, C6), 187.7 (C, C1)); EIMS m/z (Mþ. 246), calcd for: C13H10OS2/
46.
d
¼ 7.48 (2H, d, J ¼ 5.1 Hz, H21,
(
Since chalcone derivatives with a heterocyclic thiophene ring
have an increased antibacterial activity [17], a mono-carbonyl
curcumin derivative with thiophene instead the usual aromatic
rings was synthesised - the (1E,4E)-1,5-Di(thiophen-2-yl)penta-
d
1
2
1,4-dien-3-one, henceforth denominated DB thiophene (Scheme 1).
This curcuminoid was fully characterised by NMR, Raman and IR
spectroscopies, with a complete assignment of bands based on
frequency calculations using the DFT approach. In order to test the
ability of DB thiophene as an EPI, inhibition tests focused on
S. aureus strains were performed. The inhibition mechanism of the
NorA efflux pump was then studied by means of molecular docking
using a NorA protein homolog model.
2.3. Vibrational spectroscopy
The Raman spectrum was recorded using compacted powder in
the sample holder of a Bruker RAM II FT-Raman module, equipped
with a liquid nitrogen cooled high-sensitivity Ge detector, and
coupled to a Bruker Optics VERTEX 70v FTIR spectrometer. The
1064 nm line of a Nd:YAG laser was used as the excitation radiation,
with a nominal laser power of 150 mW. The spectrum was the sum
of 60 scans, at a typical resolution of ca. 4 cm .
ꢀ
1
2
. Materials and methods
The FTIR-ATR spectrum was measured at room temperature
using a spectrometer Bruker Vertex 70v equipped with a RT-
2.1. Materials
DLaTGS (Deuterated Lanthanum
phate) detector and a Wide Range MIR-FIR. The spectra were
a-Alanine doped TriGlycine Sul-
Potassium hydroxide was purchased from Din a^ mica Química
ꢀ1
Contempor ^a nea Ltd. (Indaiatuba, Brazil). Ethanol and acetone were
obtained from Vetec Química Fina Ltd (Duque de Caxias, Brazil).
Chlorpromazine (CPZ), ciprofloxacin, norfloxacin, carbonyl cyanide
m-chlorophenylhydrazone (CCCP), thiophene-2-carbaldehyde and
recorded in the region 130 to 4000 cm
with a resolution of
ꢀ1
2
cm
and accumulating 32 scans per spectrum.
A
BlackmaneHarris three-term apodization function and the Mertz
search phase correction algorithm were used.
Scheme 2. DB Thiophene synthesis reaction.

M.M. Oliveira et al. / Journal of Molecular Structure 1215 (2020) 128291
3
2
.4. NMR and GC-MS
1.57e1605.72
m
M for CPZ and 1.27e1298.50
m
M for EtBr. The trays
ꢁ
were incubated at 37 C for 24 h and bacterial growth was revealed
by staining with resazurin.
The chemical reagents were from Sigma-Aldrich. 1H and 13C
NMR spectra were obtained using a Bruker Spectrometer, model
Avance DPX - 300 and model Avance DRX-500 operating at a fre-
quency of 300 MHz and 500 MHz for hydrogen, 75 MHz and
In order to evaluate the potential inhibition of the efflux pump
by DB Thiophene, a comparative study was made assessing its
ability to decrease the MIC antibiotics and EtBr (substrate for MDR
pumps including NorA [11,13,26,27] and MepA [15,28]), by
comparing with CPZ and CCCP which are standard EPI’s [29e32].
Eppendorf’s were prepared with the EPI’s and DB Thiophene at a
sub-inhibitory concentration (MIC/8), which corresponds to
1
CDCl
25 MHz for carbon respectively. The spectra were measured in
ꢁ
3
solvents at 27 C and chemical shifts are reported as
d
values
in parts per million (ppm) relative to tetramethylsilane (
d
0.00) as
the internal standard. The mass spectra were obtained with a Shi-
madzu QP201 GC-MS (Gas Chromatography coupled to Mass
Spectrometry) using RTX-5MS capillary column (30.0 m ꢂ 0.25 mm
x 0.30 mm) for compounds within the literature record.
520.32
for CPZ. Then 100
a 96-well microtiter tray with two-fold serial dilution by adding
00 L of antibiotics and EtBr with a final concentration ranging
from 1.27 to 1298.50 M for EtBr, 1.56e1603.36 M for norfloxacin
M for ciprofloxacin. The trays were incubated
mM for DB Thiophene, 625.56
mM for CCCP and 401.43 mM
mL of the Eppendorf’s content were transferred to
1
m
2.5. Computational details
m
m
and 1.50e1545.24
for 24 h at 37 C and bacterial growth was revealed by staining with
resazurin.
m
ꢁ
Density Functional Theory (DFT) calculations [20] were per-
formed using the Gaussian 09 program [21]. Initial coordinates
were generated according to the NMR results. The B3LYP exchange-
correlation functional [22] was applied, combined with the 6-31G*
basis set. All geometries were fully optimised within the Berny al-
gorithm, using redundant internal coordinates and considering the
Gaussian default convergence criteria. Vibrational wavenumber
calculations were carried out for the optimised geometries, at the
same theory level, to verify convergence to a real minimum within
the potential energy surface (no negative eigenvalues) and to assist
in the vibrational mode description.
2.7. Statistical analysis of the microbiological results
Antibacterial assays were performed in triplicate and results
were expressed as an average of the replicates. The results of the
tests are expressed as the geometric mean. Statistical hypothesis
analysis was applied using a Two-Way ANOVA followed by the
Bonferroni post hoc test (using the GraphPad Prism 5.0 [33]
software).
For an accurate comparison between the calculated and exper-
imental vibrational wavenumbers, the former were corrected for
anharmonicity and incomplete electron correlation treatment, us-
ing the scaling factors of 0.960 [23] and 0.942 for predicted values
below and above 2000 cm , respectively. Vibrational modes were
analysed, in terms of their Potential Energy Distribution (PED),
using the VEDA [24] program with default optimisation options.
2.8. Docking
The NorA sequence of S. aureus 1199 strain (Entry Q03325) was
retrieved from the Universal Protein Resource database (Uniprot).
The web-based SWISS-MODEL [34] service was used to build a
homology model of NorA. The HHblits-based automated mode was
used, resulting in 50 different templates, the best one being based
on the structure of the E. coli YajR transporter (PDB-ID: 3wdo).
The grid box for the docking procedure was defined as an
80Åx80Åx80 Å box around the geometrical center of the NorA
model. Partial Gasteiger charges were added to ligand atoms while
non-polar hydrogen atoms were mixed and rotational bonds
determined. Docking studies were carried out using the Lamarckian
genetic algorithm in Autodock 4.0 [35]. All other parameters were
kept at their default values. The ten best results were chosen by
least binding energy.
ꢀ
1
2.6. Evaluation of efflux pump inhibition by MIC reduction
The evaluation of the efflux pump inhibition was performed
according to Tintino et al. [12]. The two strains of S. aureus used
were: SA-1199 B, which overexpresses the NorA gene encoding the
NorA efflux protein and the multi-drug resistant (MDR) mutant
strain SA-K2068 which presents the MepA efflux pump. The strains
were maintained on blood agar base slants and, prior to use, the
ꢁ
cells were grown overnight at 37 C in Heart Infusion Agar slants.
Chlorpromazine (CPZ), carbonyl cyanide m-chlorophenylhy-
drazone (CCCP) and ethidium bromide (EtBr) were used for this
essay. Norfloxacin and Ciprofloxacin were the antibiotics used for
NorA and MepA efflux inhibition respectively. The antibiotics and
DB Thiophene were dissolved in dimethyl sulfoxide aqueous solu-
tion (5.1%, v/v). The CPZ and EtBr were dissolved in sterile water
3. Results and discussion
3.1. Structural and conformational analysis
Fig.1a and b and displays the H NMR and 13C NMR spectra of DB
Thiophene, respectively. The NMR profile (Fig. 1a) shows two
doublets, one at 6.89 ppm, assigned to H17 and H18 (J ¼ 15.6 Hz),
and another at 7.92 ppm, due to H19 and H20 (J ¼ 15.6 Hz). Their
coupling constants confirm the stereochemistry E of the double
bond. The signals at 7.48 (d, J ¼ 5.1 Hz), 7.40 (d, J ¼ 3.6 Hz) and
7.15 ppm (m) are ascribed to the hydrogens of the thiophene ring. In
1
and CCCP in methanol/water (1:1, v/v). All the compounds were
ꢁ
stored at ꢀ20 C, at a final concentration of 4162.60
m
M for DB
M for CPZ, 2597.00
for norfloxacin and 3090.48 mM for
Thiophene, 5004.50
for EtBr, 3206.71
ciprofloxacin.
m
M for CCCP, 3211.44
m
mM
mM
The Minimal Inhibitory Concentration (MIC) is defined as the
lowest concentration in which no growth can be observed [25]. The
MIC’s of CPZ, CCCP, EtBr and DB Thiophene were obtained in a
13
the C spectra the signal from the unsaturated carbonyl is at
187.7 ppm, while the ketone peak is seen at 203.8 ppm. However,
the presence of unsaturation causes a shift of the latter to high field.
The charge delocalisation is either caused by the aromatic ring
(thiophene) or by the double bond, rendering the carbonylic carbon
less electron deficient. The olefinic carbons C3 and C4 are observed
at 124.4 ppm, while C5 and C6 appear at 131.8 ppm. The carbon
atoms belonging to the thiophene rings are at 140.3, 135.6, 128.8
and 128.4 ppm. The chromatogram (Fig. S1) confirms the presence
microdilution assay using 150
solution, corresponding to a final concentration of 10 colony-
forming units/mL, followed by addition of 1350 L of brain heart
infusion (BHI) broth. Aliquots of 100 L were transferred to a 96-
well multiplate with two-fold serial dilutions by adding 100 L of
each compound solutions with final concentrations ranging from
.03 to 2081.00 M for DB Thiophene, 2.44e2502.25 M for CCCP,
mL of each strain suspended in saline
5
m
m
m
2
m
m

4
M.M. Oliveira et al. / Journal of Molecular Structure 1215 (2020) 128291
Fig. 1. (a) 1 H NMR Spectrum of DB thiophene (b) 13 C NMR Spectrum of DB Thiophene.
of only one compound, thereby indicating the analytical purity of
chalcone. ESIMS and a proposed fragmentation scheme are pro-
vided as supplementary material as Fig. S2 and Scheme S1,
respectively.
Rotations around the single bonds of the enone groups lead to
three possible conformational isomers, namely: cis-cis, cis-trans
and trans-trans isomers, henceforth denominated C0, C1 and C2,
respectively (Fig. 2). DFT calculations performed on the isolated
molecule show that the trans-trans conformation, i.e. the C2
conformer, is the preferred geometry (at least in vacuum). The
calculated SCF (self-consistent field method) energies for the C0
and C1 isomers are 10.0 and 5.0 kJ/mol higher, respectively. This is
consistent with the crystal structure reported by Murugavel et al.
[
36], in which the asymmetric unit of the compound was found to
be in trans-trans conformation.
3.2. Vibrational analysis
The DB thiophene molecule contains 26 atoms, yielding 72
vibrational modes. The Raman and IR spectra of the compound, as
well as the calculated spectra for the considered conformers are
shown in Fig. 3a and b. Comparison of spectra for wavenumbers
ꢀ1
higher than 2000 cm are given in Figs. S3 and S4 (Supplementary
information).
The calculated Raman profile of C1 is the one which matches
more closely the experimental data, although the three conformers
of the compound exhibit almost the same modes at roughly the
ꢀ
1
same frequencies, within 10 cm of each other, which hardly af-
fects the overall assignment. These differences will be discussed
Fig. 2. Representation of the three conformers of DB thiophene.

M.M. Oliveira et al. / Journal of Molecular Structure 1215 (2020) 128291
5
Table 1
Observed (Raman and Infrared) and calculated vibrational wavenumbers (cmꢀ1) for
Thiophene (C1).
u
IR
u
Raman
uCalc Assignment
3
3
3
3
088 3088
071 3074
044
3081
3079
3047
3034
2900
1673
1599
1573
1517
1426
1417
1355
1351
1307
1299
1258
1232
1203
1185
1115
1079
1034
1025
977
950
881
867
837
825
801
727
716
679
675
584
559
480
478
375
303
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
(CH)ring
(CH)ring
(CH)ring
(CH)ring
(CH)enone
(OC)enone
(CC)enone
(OC)enone
013 3012
2990
1
1
1
1
662 1658
602 1604
562 1567
512 1512
þ
þ
n
(CC)enone
(CC)enone
n
(CC)ring
(CC)ring
(CC)ring
(CC)ring
(CC)ring
þ
d
d
(HCC)ring
(HCC)ring
(HCC)ring
(HCS)ring
(HCS)ring
1425
þ
þ
þ
þ
1
1
1
1
1
1
1
1
1
1
1
1
1
9
9
8
8
8
8
419 1420
376 1376
359 1359
319
307 1309
270 1271
224 1225
199 1199
169
106 1095
095 1088
045 1047
038 1041
67
59
73
56
36
14
d
d
þ
d(HCC)ring
d
(CC)enone
(CC)enone
þ
þ
d
(HCC)enone
(HCC)enone
(HCC)ring
d
d
(HCC)enone
þ
d
d
d
(HCS)ring
(CC)enone
þ
(HCC)ring
(CCC)ring
n
n
n
n
n
n
þ
d
(CC)ring
(CC)ring
(CC)enone
(CC)ring
(CC)ring
þ
d
(HCC)enone
þ
d
(HCC)ring
(CCC)ring
(HCC)ring
þ
n
þ
d
(CC)enone
þ
d
d
(HCS)ring
þ
d
þ
(HCC)ring
(CCC)ring
þ
d
þ
d
(HCC)ring
(HCenoneCringCring)
t
(HCenoneCenoneCring) þ
t
963
n
t
t
(CC)enone
þ
d
(CCC)enone
(HCCC)ring
(OCCC)enone
(CCC)ring
(HCCC)enone (HCCC)ring
(CCC)enone
(HCSC)ring
þ
t
þ
t(CCCC)ring
(HCCC)enone
(SC)ring
þ
g
n
þ
d
815
t
d
þ
t
788
(OCC)enone
þ
d
727
711
681
666
579
564
503
477
397
314
264
191
n
(SC)ring
(SC)ring
þ
d
(CCC)ring
(CCC)ring
705
680
n
þ
d
d
(OCC)enone
þ
d
(CCC)ring
(OCCC)enone
(SCC)ring
(SCCC)ring
þ
d(SCC)ring
t
(HCSC)ring
þ
g
n(SC)ring
þ
d
(CCC)ring
þ
d
t
d
(CCCC)ring
þ
t
504
(CCC)enone
(CCCC)ring
(CCC)enone
t
d
þ
þ
t
(CringCringCringCenone) þ
(CringCringCenone) þ (CenoneCringS)
(CCCC)enone (SCCC)ring
(CenoneCringS)
t(SCCC)ring
d
d
t
d
(CringCringCringCenone) þ
t
þ
t
263
196
(OCC)enone
þ
d
(CCC)enone
þ
d
197
t
(CCCC)enone
71
88
lattice
ꢀ
1
Fig. 3. Raman (a) and IR (b) spectra of DB thiophene.
strong Raman band at 1567 cm was assigned to a combination of
the carbonyl and C]C stretchings. For curcumin, the intense band
ꢀ1
related to the diketone stretching mode is recorded at 1626 cm
38]. On the IR spectrum, said bands appear at 1662, 1602 and
below. A conformation consistent with C1 model, shown by
calculated Raman profile, is corroborated by the fact that the
experimental dipole moment measured previously [37] (3.19D) is
closer to the theorical calculated C1 value (3.11D) than C0 (4.15D)
and C2 (1.98D).
[
ꢀ
1
1
562 cm , respectively.
A strong Raman band at 1419 cm was assigned to a combi-
nation of (CC) and (HCC) bands of the thiophene rings. Accord-
ꢀ
1
n
d
ꢀ
1
ingly, this mode appears at 1425 cm in thiophene substituted
chalcone [39]. In the IR spectrum, the same mode is seen as a
medium intensity absorption at 1419 cm
Table 1 comprises the Raman and IR data for DB thiophene, as
well as the corresponding assignments. The calculated vibrational
wavenumbers and Potential Energy Distribution (PED) analysis, for
all three conformers of the compound, are comprised in Tables S1,
S2 and S3 (Supplementary information).
ꢀ
1
.
In the Raman spectrum, two very weak modes occur at 1359 and
ꢀ
1
1
309 cm , that were assigned as a combination of
of the thiophene rings, and as (HCC) of the enone spacers,
(CC)þ
respectively. The three very weak bands at 1271, 1225 and
n(CC) and d(HCS)
n
d
ꢀ1
The IR absorptions in the 3020-3100 cm region arise from the
CH stretching vibrations. In this region the Raman spectrum shows
ꢀ
1
1
199 cm are not visible for the C0 conformer, appearing as very
ꢀ
1
two bands, at 3088 and 3074 cm , both assigned to CH stretching
modes of the thiophene rings. Two other Raman bands at 3012 and
weak bands for the other two. The first one was assigned as HCC
bending modes of both enone and thiophene rings, while the sec-
ꢀ
1
2
990 cm are due to the antisymmetric and symmetric olefinic CH
ond is a combination of
The third band was assigned as
d
(HCS) and
d
(HCC) of the thiophene atoms.
(CC) of the enone spacer and
(CCC) of the rings. Three medium intensity features appear at 873,
stretching modes, respectively.
n
In the fingerprint region, there are two weak Raman bands at
d
ꢀ
1
ꢀ1
1
658 and 1604 cm . The former was assigned as a combination of
8
56 and 836 cm : the first two are torsional modes from the rings
stretching modes of the carbonyl group and of the C]C bonds,
while the latter was attributed to stretching of CC bonds. A very
and the enones, while the last was assigned to a SeC stretching and
a CeCeC bending modes. Finally, an isolated weak Raman band at

6
M.M. Oliveira et al. / Journal of Molecular Structure 1215 (2020) 128291
ꢀ
5
04 cm 1 was ascribed to the bending of the enone group. The
Similar compounds containing the thiophene rings were already
tested as inhibitors of the SA-1199 B NorA efflux pump and showed
a significant enhancement in the activity of ciprofloxacin, with an
increase in the intracellular level of the antibiotic, confirming the
mode of action as an EPI [40e42]. In the present experiment, DB
thiophene led to an effective reduction of norfloxacin MIC
compared to CCCP. When used in conjunction with EtBr, DB thio-
phene was effective as CPZ, while there was no statistical difference
compared to CCCP. To sum up, the essays indicated that DB thio-
phene seems to be an inhibitor of NorA when co-administered with
norfloxacin, and it is more effective than CCCP (Fig. 4a).
ꢀ
1
equivalent IR band is a medium absorption at 503 cm
.
3
.3. Efflux pump modulation
Unlike the standard inhibitors, DB Thiophene did not reveal
clinically relevant antibacterial activity, with a MIC ꢃ4162.60 M.
However, when used in association with norfloxacin against SA-
199 B, significant synergistic effects were noted - DB thiophene
was able to reduce the MIC of norfloxacin by 30%. This synergic
effect against NorA efflux pump is due to the inhibition of the efflux
pump mechanism. That could be confirmed by examining the as-
sociation of this curcumin derivative with EtBr, since an identical
behaviour was observed.
m
1
The mutant SA-K2068 is recognized as having a multi-drug
resistance that is mediated by MepA instead of NorA. The essays
show the ability of DB thiophene to inhibit the MepA efflux pump,
Fig. 4. (a) MIC of Norfloxacin and EtBr, in association with the standard inhibitors and in association with DB thiophene against the strain S. aureus 1199 B. a1,a2p < 0.0001 vs control,
b1,b2
c1,c2
p < 0.0001 vs CPZ,
p < 0.0001 vs CCCP. (b) MIC of Ciprofloxacin and EtBr, in association with the standard inhibitors and in association with DB thiophene against the strain
a1,a2
b1,b2
c1,c2
S. aureus K2068.
p < 0.0001 vs control,
p < 0.0001 vs CPZ,
p < 0.0001 vs CCCP (bottom).

M.M. Oliveira et al. / Journal of Molecular Structure 1215 (2020) 128291
7
place of the antibiotic.
All these results suggest that DB Thiophene is likely to function
as a competitive inhibitor of NorA efflux protein.
4. Conclusions
The compound (1E,4E)-1,5-Di(thiophen-2-yl)penta-1,4-dien-3-
one was synthesised and fully characterised. A study on its vibra-
tional properties was performed using FTIR and FT-Raman spec-
troscopies as well as quantum chemical calculations at the DFT
level (for the three possible conformers of the compound). The
experimental and calculated (scaled) vibrational spectra were
compared and showed a very good correspondence. These results
and the description of the normal modes according to the PED,
were used to elucidate the vibrational wavenumber assignments of
this synthetic substance. It was shown that this curcuminoid not
only inhibits the NorA efflux pump, but also the MepA, thus being a
potential new EPI. DB Thiophene interacts with the binding site of
efflux pump models and it’s likely that DB Thiophene acts as a
competitive inhibitor of efflux pumps.
Author’s Contribution
Mauro M. Oliveira e Investigation, Writing, original draft,
Formal analysis, Helcio S. Santos e Investigation, conducted the
spectroscopic analysis by NMR, Henrique D. M. Coutinho e
Conceptualization, conceived the experimental design of the
microbiological studies., Paulo N. Bandeira e Investigation, per-
formed the synthesis of the compound, Priscila T. da Silva e
Investigation, performed the synthesis of the compound, Thiago S.
Freitas e Investigation, conducted the efflux pump inhibition as-
says, Janaina E. Rocha e Investigation, conducted the efflux pump
inhibition assays, Jaize C. Xavier e Investigation, carried out the
antibacterial assays, Fabia F. Campina e Investigation, carried out
the antibacterial assays, Cristina R. S. Barbosa e Investigation, car-
Fig. 5. (a) The best five poses of DB Thiophene (green) and the best pose of norfloxacin
blue) on the binding site of the NorA model. Regions on the binding site are marked 1
and 2. (b) 2D ligand-protein interaction diagram of DB Thiophene and the NorA model
on regions 1 and 2.
(
ried out the antibacterial assays, Jos
eꢀ B. Araújo Neto e Investiga-
with a two-fold reduction in MIC of ciprofloxacin. This is a
remarkable result since DB thiophene performed better than CPZ,
although it was not as effective as CCCP (Fig. 4b). We can’t make
direct comparisons with other curcumin derivatives, since, to the
best of our knowledge, there are no studies on NorA or MepA efflux
pump inhibition involving other curcuminoids. The mechanism by
which DB thiophene inhibits the NorA or MepA efflux pumps can
be explored by molecular docking (to be discussed further on).
tion, carried out the antibacterial assays, Raimundo L. S. Pereira e
Investigation, performed modulatory antimicrobial activity assays,
Maria M. C. Silva e Investigation, performed modulatory antimi-
crobial activity assays, D eꢀ bora F. Muniz e Investigation, performed
modulatory antimicrobial activity assays, Alexandre M.R. Teixeira e
Investigation, carried out the Raman spectroscopy studies, Vanessa
M. Frota e Investigation, conducted the analysis by CG-MS, Tigressa
H. S. Rodrigues e Investigation, conducted the analysis by CG-MS,
Ana M. Amado e writing and Investigation, reviewed the manu-
script and conducted the Raman analysis, Maria P. M. Marques e
overviewed the writing, reviewed the manuscript and aided on the
Conceptualization, Luis A. E. Batista de Carvalho e supervised the
experiments, reviewed manuscript, Carlos E. S. Nogueira e Inves-
tigation, carried out the DFT calculations, supervised and wrote the
original draft
3.4. Docking results
Fig. 5a shows the five best poses of DB Thiophene, inserted in
the binding site of the NorA model, with energies ranging
from ꢀ26.527 to ꢀ26.276 kJ/mol. For comparison, Li et al. [43]
found that the affinity of tetracycline to the same protein
was ꢀ28.033 kJ/mol. In our simulations, norfloxacin best pose en-
ergy was ꢀ32.677 kJ/mol. Docking calculations showed that there
are two preferred regions in the binding site of the NorA model,
labeled regions 1 and 2 (Fig. 5a).
Fig. 5b displays a 2D ligand-protein interaction diagram, with
the contact distances in green. The best pose of DB Thiophene is in
region 1. In region 2, the C]O group is favorably oriented to form a
hydrogen bond with Arg310 (Fig. 5b). Norfloxacin best pose over-
laps with poses of DB thiophene in both regions, as can be seen in
Fig. 5a. Additionally, and confirming these results, Dantas et al. [44]
described the binding site of norfloxacin of a NorA model identical
to the one reported here, with interactions with residues 340, 310,
Declaration of competing interests
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
Financial support is acknowledged: from the Portuguese Foun-
dation for Science and Technology (FCT) (UIDB/00070/2020).
CENAUREMN - Northeastern Center for the Application and Use
of Nuclear Magnetic Resonance and EMBRAPA AGROINDÚSTRIA
51 and 16. It can thus be argued that DB thiophene hinders the
binding of norfloxacin to NorA, being expelled from the bacteria in

8
M.M. Oliveira et al. / Journal of Molecular Structure 1215 (2020) 128291
TROPICAL-Laborat oꢀ rio Multiusu aꢀ rio de Química de Produtos
Naturais.
Caricato, A. Marenich, J. Bloino, B. G. Janesko, R. Gomperts, B. Mennucci, H. P.
Hratchian, J. V. Ort, Gaussian 09, Revision A.02, ([s.d.]).
22] P.J. Stephens, F.J. Devlin, C.F. Chabalowski, M.J. Frisch, Ab initio calculation of
vibrational absorption and circular dichroism spectra using density functional
force fields, J. Phys. Chem. 98 (1994) 11623e11627, https://doi.org/10.1021/
j100096a001.
[
[
Appendix A. Supplementary data
23] NIST Computational Chemistry Comparison and Benchmark Database, ([s.d.])
doi:10.18434/T47C7Z.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molstruc.2020.128291.
[24] M.H. Jamr
oꢀ z, Vibrational energy distribution analysis (VEDA): scopes and
limitations, spectrochim, Acta Part A Mol. Biomol. Spectrosc. 114 (2013)
20e230, https://doi.org/10.1016/J.SAA.2013.05.096.
2
References
[
25] M.A. Wikler, Methods for dilution antimicrobial susceptibility tests for bac-
teria that grow aerobically: approved standard, CLSI 26 (2006). M7eA7.
[
1] A.N. Nurfina, M.S. Reksohadiprodjo, H. Timmerman, U.A. Jenie, D. Sugiyanto,
H. Van Der Goot, Synthesis of some symmetrical curcumin derivatives and
their antiinflammatory activity, Eur. J. Med. Chem. 32 (1997) 321e328,
https://doi.org/10.1016/S0223-5234(97)89084-8.
[26] H. Yoshida, M. Bogaki, S. Nakamura, K. Ubukata, M. Konno, Nucleotide
sequence and characterization of the Staphylococcus aureus norA gene, which
confers resistance to quinolones, J. Bacteriol. 172 (1990) 6942e6949, https://
doi.org/10.1128/jb.172.12.6942-6949.1990.
[
[
[
2] A. Goel, A.B. Kunnumakkara, B.B. Aggarwal, Curcumin as “Curecumin”: from
[27] G.W. Kaatz, S.M. Seo, C.A. Ruble, Efflux-mediated fluoroquinolone resistance
in Staphylococcus aureus, Antimicrob. Agents Chemother. 37 (1993)
1086e1094, https://doi.org/10.1128/AAC.37.5.1086.
kitchen to clinic, Biochem. Pharmacol. 75 (2008) 787e809, https://doi.org/
10.1016/j.bcp.2007.08.016.
3] I. Chattopadhyay, K. Biswas, U. Bandyopadhyay, R.K. Banerjee, Turmeric and
curcumin: biological actions and medicinal applications, Curr. Sci. 87 (2004)
[28] P. Petersen, A. Ruzin, S.J. Projan, E. Murphy, P.A. Bradford, F. McAleese,
P.M. Dunman, A novel MATE family efflux pump contributes to the reduced
susceptibility of laboratory-derived Staphylococcus aureus mutants to tige-
cycline, Antimicrob. Agents Chemother. 49 (2005) 1865e1871, https://
doi.org/10.1128/aac.49.5.1865-1871.2005.
44e53.
4] A. Mazumder, K. Raghavan, J. Weinstein, K.W. Kohn, Y. Pommier, Inhibition of
human immunodeficiency virus type-1 integrase by curcumin, Biochem.
Pharmacol. 49 (1995) 1165e1170, https://doi.org/10.1016/0006-2952(95)
[29] J.M. Pag eꢁ s, M. Masi, J. Barbe, Inhibitors of efflux pumps in Gram-negative
9
8514-A.
bacteria, Trends Mol. Med. 11 (2005) 382e389, https://doi.org/10.1016/
j.molmed.2005.06.006.
[
5] M.A. Tomren, M. M aꢀ sson, T. Loftsson, H.H. Tønnesen, Studies on curcumin and
curcuminoids. XXXI. Symmetric and asymmetric curcuminoids: stability, ac-
tivity and complexation with cyclodextrin, Int. J. Pharm. 338 (2007) 27e34,
https://doi.org/10.1016/j.ijpharm.2007.01.013.
[30] M. Mall eꢀ a, J. Chevalier, A. Eyraud, J.M. Pag eꢁ s, Inhibitors of antibiotic efflux
pump in resistant Enterobacter aerogenes strains, Biochem. Biophys. Res.
Commun. 293 (2002) 1370e1373, https://doi.org/10.1016/S0006-291X(02)
00404-7.
[
6] G. Liang, L. Shao, Y. Wang, C. Zhao, Y. Chu, J. Xiao, Y. Zhao, X. Li, S. Yang,
Exploration and synthesis of curcumin analogues with improved structural
stability both in vitro and in vivo as cytotoxic agents, Bioorg. Med. Chem. 17
[31] S. Sabatini, G.W. Kaatz, G.M. Rossolini, D. Brandini, A. Fravolini, From
phenothiazine to 3-phenyl-1,4-benzothiazine derivatives as inhibitors of the
Staphylococcus aureus NorA multidrug efflux pump, J. Med. Chem. 51 (2008)
4321e4330, https://doi.org/10.1021/jm701623q.
[32] G.W. Kaatz, V.V. Moudgal, S.M. Seo, J.E. Kristiansen, Phenothiazines and thi-
oxanthenes inhibit multidrug efflux pump activity in Staphylococcus aureus,
Antimicrob. Agents Chemother. 47 (2003) 719e726, https://doi.org/10.1128/
AAC.47.2.719-726.2003.
(
2009) 2623e2631, https://doi.org/10.1016/j.bmc.2008.10.044.
[
[
[
7] V. Basile, E. Ferrari, S. Lazzari, S. Belluti, F. Pignedoli, C. Imbriano, Curcumin
derivatives: molecular basis of their anti-cancer activity, Biochem. Pharmacol.
78 (2009) 1305e1315, https://doi.org/10.1016/j.bcp.2009.06.105.
8] G. Liang, S. Yang, L. Jiang, Y. Zhao, L. Shao, J. Xiao, F. Ye, Y. Li, X. Li, Synthesis
and anti-bacterial properties of mono-carbonyl analogues of curcumin, Chem.
Pharm. Bull. (Tokyo) 56 (2008) 162e167, https://doi.org/10.1248/cpb.56.162.
9] C. Zhao, Y. Zhang, P. Zou, J. Wang, W. He, D. Shi, H. Li, G. Liang, S. Yang,
Synthesis and biological evaluation of a novel class of curcumin analogs as
anti-inflammatory agents for prevention and treatment of sepsis in mouse
model, Drug Des. Dev. Ther. 9 (2015) 1663e1678, https://doi.org/10.2147/
DDDT.S75862.
10] B.W. Ho, W.S. Shin, S. Lee, M.A. Chan, Synthesis of novel curcumin mimics
with asymmetrical units and their anti-angiogenic activity, Bioorg. Med.
Chem. Lett 15 (2005) 3782e3786, https://doi.org/10.1016/j.bmcl.2005.05.064.
11] S.S. Costa, M. Viveiros, L. Amaral, I. Couto, Multidrug efflux pumps in Staph-
ylococcus aureus: an update, Open Microbiol. J. 7 (2013) 59e71, https://
doi.org/10.2174/1874285801307010059.
[33] GraphPad Software, La Jolla California USA, ([s.d.]).
[34] A. Waterhouse, M. Bertoni, S. Bienert, G. Studer, G. Tauriello, R. Gumienny,
F.T. Heer, T.A.P. de Beer, C. Rempfer, L. Bordoli, R. Lepore, T. Schwede, SWISS-
MODEL: homology modelling of protein structures and complexes, Nucleic
Acids Res. 46 (2018) W296eW303., https://doi.org/10.1093/nar/gky427.
[35] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
A.J. Olson, AutoDock4 and AutoDockTools4: automated docking with with
selective receptor flexibility, J. Comput. Chem. 30 (2010) 2785e2791, https://
doi.org/10.1002/jcc.21256.
[
[
[
[36] S. Murugavel, G. Ganesh, A. SubbiahPandi, R. Murugan, S. SrimanNarayanan,
(E,E)-1,5-Di-2-thienylpenta-1,4-dien-3-one, International Union of Crystal-
lography, 2008, https://doi.org/10.1107/S1600536808026603.
12] S.R. Tintino, C.D.M. Oliveira-Tintino, F.F. Campina, R.L.P. Silva, M. do S. Costa,
I.R.A. Menezes, J.T. Calixto-Júnior, J.P. Siqueira-Junior, H.D.M. Coutinho,
T.C. Leal-Balbino, V.Q. Balbino, Evaluation of the tannic acid inhibitory effect
against the NorA efflux pump of Staphylococcus aureus, Microb. Pathog. 97
[37] S.V. Tseukerman, V.M. Nikitchenko, V.D. Orlov, V.F. Lavrushin, Dipole mo-
ments OF thiophene OF chalcones and their VINYLOGS, Chem. Heterocycl.
Compd. 3 (1967) 232e238.
[38] P.R.K. Mohan, G. Sreelakshmi, C.V. Muraleedharan, R. Joseph, Water soluble
complexes of curcumin with cyclodextrins: characterization by FT-Raman
spectroscopy, Vib. Spectrosc. 62 (2012) 77e84, https://doi.org/10.1016/
j.vibspec.2012.05.002.
[39] T.A. de Toledo, R.C. da Costa, H.M. Al-Maqtari, J. Jamalis, P.S. Pizani, Temper-
ature dependence of the Raman spectrum of 1-(4-chlorophenyl)-3-(2-thienyl)
prop-2-en-1-one, Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 180 (2017)
9e17, https://doi.org/10.1016/j.saa.2017.02.051.
[40] J. Fournier dit Chabert, B. Marquez, L. Neville, L. Joucla, S. Broussous,
P. Bouhours, E. David, S. Pellet-Rostaing, B. Marquet, N. Moreau, M. Lemaire,
Synthesis and evaluation of new arylbenzo[b]thiophene and diarylthiophene
derivatives as inhibitors of the NorA multidrug transporter of Staphylococcus
aureus, Bioorg. Med. Chem. 15 (2007) 4482e4497, https://doi.org/10.1016/
j.bmc.2007.04.023.
[41] F. Liger, P. Bouhours, C. Ganem-Elbaz, C. Jolivalt, S. Pellet-Rostaing,
F. Popowycz, J.M. Paris, M. Lemaire, C2 arylated benzo[b]thiophene de-
rivatives as Staphylococcus aureus NorA efflux pump inhibitors, Chem-
MedChem 11 (2016) 320e330, https://doi.org/10.1002/cmdc.201500463.
[42] S. Alibert, J. Pag eꢁ s, Efflux pump inhibitors in bacteria, Expert Opin. Ther. Pat.
17 (2007) 883e888, https://doi.org/10.1517/13543776.17.8.883.
[43] Y. Li, Z.T. Cao, X.Y. Wang, X.Z. Ge, Expression of the TetA gene encoding TetA
efflux protein in E. coli contributes to its increased bacterial resistance toward
berberine, J. Asian Nat. Prod. Res. 20 (2018) 374e384, https://doi.org/10.1080/
10286020.2017.1384818.
(
2016) 9e13, https://doi.org/10.1016/j.micpath.2016.04.003.
[
[
[
13] A.A. Neyfakh, C.M. Borsch, G.W. Kaatz, Fluoroquinolone resistance protein
NorA of Staphylococcus aureus is a multidrug efflux transporter, Antimicrob.
Agents Chemother. 37 (1993) 128e129, https://doi.org/10.1128/AAC.37.1.128.
14] G.W. Kaatz, S.M. Seo, C.A. Ruble, Mechanisms of fluoroquinolone resistance in
staphylococcus aureus, J. Infect. Dis. 163 (1991) 1080e1086, https://doi.org/
10.1093/infdis/163.5.1080.
15] G.W. Kaatz, F. McAleese, S.M. Seo, Multidrug resistance in Staphylococcus
aureus due to overexpression of a novel multidrug and toxin extrusion
(
1
MATE) transport protein, Antimicrob. Agents Chemother. 49 (2005)
857e1864, https://doi.org/10.1128/AAC.49.5.1857-1864.2005.
[
[
16] B.D. Schindler, P. Jacinto, G.W. Kaatz, Inhibition of drug efflux pumps in
Staphylococcus aureus: current status of potentiating existing antibiotics,
Future
Microbiol.
8
(2013)
491e507,
https://doi.org/10.11250/
chemotherapy1953.16.843.
17] P. Singh, A. Anand, V. Kumar, Recent developments in biological activities of
chalcones: a mini review, Eur. J. Med. Chem. 85 (2014) 758e777, https://
doi.org/10.1016/j.ejmech.2014.08.033.
[
18] L. Claisen, A. Clapar eꢁ de, Condensationen von Ketonen mit Aldehyden, Berichte
der Dtsch. Chem. Gesellschaft. 14 (1881) 2460e2468.
[
19] J.G. Schmidt, Ueber die Einwirkung von Aceton auf Furfurol und auf
Bittermandel o€ l bei Gegenwart von Alkalilauge, Berichte der Dtsch. Chem.
Gesellschaft.
14
(1881)
1459e1461,
https://doi.org/10.1002/
cber.188101401306.
[44] N. Dantas, T.M. de Aquino, J.X. de Araújo-Júnior, E. da Silva-Júnior, E.A. Gomes,
A.A.S. Gomes, J.P. Siqueira-Júnior, F.J.B. Mendonça Junior, Aminoguanidine
hydrazones (AGH’s) as modulators of norfloxacin resistance in Staphylococcus
aureus that overexpress NorA efflux pump, Chem. Biol. Interact. 280 (2018)
8e14, https://doi.org/10.1016/j.cbi.2017.12.009.
[
[
20] W. Kohn, L.J. Sham, Self-consistent equations including exchange and corre-
lation effects, Phys. Rev. 140 (1965) A1133eA1138, https://doi.org/10.1103/
PhysRev.140.A1133.
21] D.J.F. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R.
Cheeseman, G. Scalmani, V. Barone, G. A. Petersson, H. Nakatsuji, X. Li, M.