Chemical synthesis, molecular docking and MepA efflux pump inhibitory effect by 1,8-naphthyridines sulfonamides

Article abstract of DOI:10.1016/j.ejps.2021.105753

This study aimed to evaluate the antibacterial activity and to verify, in silico and in vitro, the inhibition of efflux mechanisms using a series of synthesized 1,8-naphthyridines sulfonamides against Staphylococcus aureus strains carrying MepA efflux pumps. The chemical synthesis occurred through the thermolysis of the Meldrum's acid adduct. The sulfonamide derivatives were obtained by the sulfonylation of 2-amino-5?chloro-1,8-naphthyridine with commercial benzenesulfonyl chloride. Antibacterial activity was assessed by the broth microdilution test. Efflux pump inhibitory capacity was evaluated in silico by molecular docking and in vitro by analyzing synergistic effects on ciprofloxacin and ethidium bromide (EtBr) and by EtBr fluorescence emission assays. The following 1,8-naphthyridines were synthesized: 4-methyl-N-(5?chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10a); 2,5-dichloro-N-(5?chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10b); 4-fluoro-N-(5?chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10c); 2,3,4-trifluoro-N-(5?chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10d); 3-trifluoromethyl-N-(5?chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10e); 4?bromo-2,5-difluoro-N-(5?chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10f). The 1,8-naphthyridines derivatives associated with sulfonamides did not show antibacterial activity. However, they showed a favorable pharmacokinetic profile with possible MepA efflux pump inhibitory action, demonstrated in molecular docking. In addition to the promising results in reducing the concentration of intracellular EtBr. 1,8-naphthyridines act as putative agents in the inhibitory action of the MepA efflux pump.

Full text of DOI:10.1016/j.ejps.2021.105753

European Journal of Pharmaceutical Sciences 160 (2021) 105753
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Chemical synthesis, molecular docking and MepA efflux pump inhibitory
effect by 1,8-naphthyridines sulfonamides
a
b
b
Cícera Datiane de Morais Oliveira-Tintino , Saulo Relison Tintino , D ´e bora Feitosa Muniz ,
b
b
c
Cristina Rodrigues dos Santos Barbosa , Raimundo Luiz Silva Pereira , I ˆe da Maria Begnini ,
Ricardo Andrade Rebelo , Luiz Everson da Silva , Sandro Lucio Mireski ,
Michele Caroline Nasato , Maria Isabel Lacowicz Krautler , Pedro Silvino Pereira ,
Tereza Cristina Leal Balbino , Jos ´e Galberto Martins da Costa ,
Fabiola Fernandes Galv a˜ o Rodrigues , Alexandre Magno Rodrigues Teixeira ,
Humberto Medeiros Barreto , Irwin Rose Alencar de Menezes ,
Henrique Douglas Melo Coutinho , Teresinha Gonçalves da Silva
c
d
c
c
c
a
e
f
f
g
h
i
b,
*
a
a
Laboratory of Pharmatoxicological Prospecting of Bioactive Products, Department of Antibiotics, Federal University of Pernambuco, UFPE, Recife, PE, Brazil
Laboratory of Microbiology and Molecular Biology, Department of Biological Chemistry, Regional University of Cariri, URCA, Crato, CE, Brazil
Department of Chemistry, Regional University of Blumenau, FURB, Itoupava Seca, 89030-903, Blumenau, SC, Brazil
Postgraduate Program in Sustainable Territorial Development, Coastal Sector, Federal University of Paran a´ , Curitiba, PR, Brazil
Department of Microbiology, Oswaldo Cruz Foundation, Aggeu Magalh a˜ es Research Center, CPqAM/Fiocruz, UFPE Campus, Recife, PE, Brazil
Laboratory of Natural Products, Department of Biological Chemistry, Regional University of Cariri, URCA, Crato, CE, Brazil
Laboratory of Simulations and Molecular Spectroscopy, Department of Biological Chemistry, Regional University of Cariri, URCA, Crato, CE, Brazil
Laboratory of Microbiology, Federal University of Piaui, UFPI, Teresina, PI, Brazil
b
c
d
e
f
g
h
i
Laboratory of Pharmacology and Molecular Chemistry, Department of Biological Chemistry, Regional University of Cariri, URCA, Crato, CE, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
This study aimed to evaluate the antibacterial activity and to verify, in silico and in vitro, the inhibition of efflux
mechanisms using a series of synthesized 1,8-naphthyridines sulfonamides against Staphylococcus aureus strains
carrying MepA efflux pumps. The chemical synthesis occurred through the thermolysis of the Meldrum’s acid
adduct. The sulfonamide derivatives were obtained by the sulfonylation of 2-amino-5‑chloro-1,8-naphthyridine
with commercial benzenesulfonyl chloride. Antibacterial activity was assessed by the broth microdilution test.
Efflux pump inhibitory capacity was evaluated in silico by molecular docking and in vitro by analyzing syner-
gistic effects on ciprofloxacin and ethidium bromide (EtBr) and by EtBr fluorescence emission assays. The
following 1,8-naphthyridines were synthesized: 4-methyl-N-(5‑chloro-1,8-naphthyridin-2-yl)-benzenesulfona-
mide (Compound 10a); 2,5-dichloro-N-(5‑chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10b);
Antibacterial agent
Bacterial resistance
Ethidium bromide
Fluorescence
SA-K2068
MepA efflux pump
4
1
2
-fluoro-N-(5‑chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10c); 2,3,4-trifluoro-N-(5‑chloro-
,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10d); 3-trifluoromethyl-N-(5‑chloro-1,8-naphthyridin-
-yl)-benzenesulfonamide (Compound 10e); 4‑bromo-2,5-difluoro-N-(5‑chloro-1,8-naphthyridin-2-yl)-benzene-
sulfonamide (Compound 10f). The 1,8-naphthyridines derivatives associated with sulfonamides did not show
antibacterial activity. However, they showed a favorable pharmacokinetic profile with possible MepA efflux
pump inhibitory action, demonstrated in molecular docking. In addition to the promising results in reducing the
concentration of intracellular EtBr. 1,8-naphthyridines act as putative agents in the inhibitory action of the MepA
efflux pump.
*
Corresponding author.
E-mail address: hdmcoutinho@gmail.com (H.D.M. Coutinho).
https://doi.org/10.1016/j.ejps.2021.105753
Received 25 December 2020; Received in revised form 27 January 2021; Accepted 8 February 2021
Available online 10 February 2021
0
928-0987/© 2021 Elsevier B.V. All rights reserved.

C.D.M. Oliveira-Tintino et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105753
1
. Introduction
2.1.2. General procedure for synthesis
Scheme 1.
The Staphylococcus aureus bacterium is an opportunistic pathogen,
N-(6-aminopyridin-2-yl)-acetamide (2) (Carboni et al., 1971): 2,
6-diaminopyridine 1 (9.0 g; 82.6 mmol) and triethylamine (11.0 mL;
78.9 mmol) solutions were prepared in tetrahydrofuran (THF) (150 mL).
An acetic anhydride (7.8 mL; 82.5 mmol) solution in THF (50 mL) was
then added slowly, drop by drop, with continuous stirring. The reaction
mixture was stirred at room temperature for an additional 23 h. Water
(150 mL) was then added to the mixture, and the THF was evaporated on
a rotary evaporator, and the mixture was left in a freezer for 96 h. The
white crystals formed were filtered, washed with ice water, and dried
under vacuum without heating. The compound was purified by column
chromatography using a mixture of ethyl ether/ethyl acetate (7:3) as the
where imbalances in metabolism, use of prostheses and catheters, or
during invasive procedures, allow its entry into the circulation, causing
serious infections, especially for immunosuppressed individuals (Cluff
and Reynolds, 1965; Malanoski et al., 1995; Mathew et al., 1995; Wer-
theim et al., 2005). It is responsible for several infections such as in-
fectious endocarditis, bacteremia, osteomyelitis, scalded skin syndrome,
toxic shock syndrome, pulmonary and urinary tract infections (Car-
valho et al., 2005; Cavalcanti et al., 2005; Tong et al., 2015). The
development of resistance to multiple drugs through resistance mecha-
nisms has been an aggravating factor, provided by the efflux pumps,
responsible for the extrusion of antibiotics, such as the MepA efflux
pump (Kaatz et al., 2005; Schindler, Patel, Seo, & Kaatz, B.D. 2013). In
view of the various infections caused by this pathogen, several studies
aim to discover and synthesize new agents that try to circumvent bac-
terial resistance or restore the effectiveness of existing antibiotics.
The 1,8-naphthyridines are among these synthetic substances to be
analyzed, aiming to use them in antibacterial action. They are hetero-
cyclic compounds formed by two condensed aromatic rings, each con-
taining a nitrogen atom (Litvinov, 2004, 2006). The first naphthyridine
derivative was synthesized in 1893 by Reissert, and unsubstituted rep-
resentatives 1,5-naphthyridine and 1,8-naphthyridine, synthesized in
◦
eluent. Yield: 48%; mp: 155.5–155.7 C.
ꢀ
1
IR (KBr, cm ): 3458 (
ν
N
–
H), 3356 (
ν
N
–
H), 3224 (νN H), 1674
–
1
(
ν
C = O), 1626, 1564, 1537, 1367, 1302,1242, 1161, 987; 792 . H NMR:
(CDCl ); 5.69 (s, 2H, -NH ); 6.15 (d,
, 300 MHz, δppm): 2.02 (s, 3H, -CH
J = 9.0 Hz, 1H, CHAr); 7.20 (d, J = 9.0 Hz, 1H, CHAr); 7.32 (t, J = 6.0
Hz, 1H, CHAr); 9.83 (s, 1H, -NH). 13C NMR: (CDCl
, 75 MHz, δppm):
23.92 (-CH
); 100.79; 103.21; 138.80; 150.50; 158.42; 168.79 (-C = O).
Anal. calcd. for C O: C, 55.62; H, 6.00; N, 27.80. Found: C, 55.47;
H, 6.02; N, 27.80.
Meldrum’s Acid
3
3
2
3
3
7 9 3
H N
-
2,2-dimethyl-1,3-dioxane-4,6‑dione (3)
(Davidson and Bernhard, 1948): Concentrated sulfuric acid (0.3 mL) was
added to a malonic acid (10 g; 0.0961 mol) suspension in acetic anhy-
dride (11 mL; 0.1165 mol), under continuous stirring. The suspension
was cooled and acetone (7.6 mL; 0.1035 mol) was added during cooling
1
1
927 (Bobra n´ ski and Sucharda, 1927; Koller, 1927b; G. 1927a, G.
927c; Lowe, 2009; Reissert, 1893). The great interest in this class of
compounds can be seen by the significant number of publications, many
addressing the chemistry of these heterocycles and their pharmacolog-
ical and microbiological properties (Fu et al., 2015; Gurjar and Pal,
◦
to maintain the temperature at 0 C. The solution was stirred for 30 min.
The reaction mixture was allowed to stand for 24 h in the refrigerator,
and the resulting solid was filtered by suction and washed with ice
water. Recrystallization is conveniently effected without being heated
by dissolving this in acetone (15 mL) and slowly adding ice water (30
mL) before being allowed to stand for 24 h in the refrigerator. The
crystals were filtered by suction, washed with ice water, and dried under
2
019; Lesher et al., 1962; Litvinov, 2004; Madaan et al., 2015; Thomas
et al., 2003).
Little research exists on the antibacterial activity of 1,8-naphthyri-
dine derivatives associated with sulfonamides. In addition, they are
precursors of fluoroquinolones (FQs) (Ball and Tillotson, 1995; Hong
et al., 1997; Liliam and Jackson, 1998; Tillotson, 1996). Therefore, this
study aimed to evaluate the antibacterial activity and to verify, in silico
and in vitro, the inhibition of efflux resistance mechanisms of a series of
synthesized 1,8-naphthyridines sulfonamides against strains carrying
the MepA efflux pumps.
◦
vacuum without heating. Yield: 32%; mp: 96–96.2 C.
N-(6-{[2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-ylidene)methyl]
amino}2-pyridinyl)acetamide (5) (Graf et al., 2002; Nicoleti et al.,
2012): Meldrum’s acid 3 (2.1 g; 14.6 mmol) was refluxed with trimethyl
orthoformate (9.0 mL) for 2 h to generate the methoxymethylene deri-
vate 4 in situ. After this period the compound 2 (1.0 g; 6.6 mmol) was
added and refluxed for a further 20 min. The reaction mixture was
cooled slightly to form the crystals, while the still-hot mixture was
filtered using a Büchner funnel and washed with cold ethanol. The solid
2
. Methods
2
2
3
.1. Synthesis
◦
was dried under a vacuum. Yield: 40%; mp: 222.8–222.9 C.
ꢀ
1
.1.1. Experimental
The melting points were obtained on a K o¨ fler (Microquímica APF-
01) hot-stage apparatus and were uncorrected. Each analytical sam-
IR (KBr, cm ): 3255 (
ν
N
–
H), 1734 (
νC = O), 1691 (νC = O), 1668
(
νC = O), 1620, 1570, 1544, 1444, 1394, 1313, 1276, 1255, 1244, 1190,
1
933, 804; 786. H NMR: (CDCl
2.28 (s, 3H, -CH
), 6.72 (d, J = 9.0 Hz, 1H, CHAr), 7.74 (t, J = 6.0 Hz,
1H, CHAr), 8.05 (d, J = 9.0 Hz, 1H, CHAr), 8.10 (s, 1H, -NH), 9.26 (d, J
3
, 300 MHz, δppm): 1.76 (s, 6H, -CH
3
),
ple was homogeneous as confirmed by thin-layer chromatography (TLC)
performed on silica-gel (Kieselgel 60 F 254-Merck) plates, which were
visualized with UV light, while flash chromatography was performed
using a Merck silica-gel 60 (230–400 mesh). Infrared spectra were
determined using a Bruker Vertex 70 spectrophotometer with ATR/
FTIR. Nuclear magnetic resonance spectra were recorded with a Bruker
UltrashieldTM 300 spectrometer using tetramethylsilane as the internal
standard. Chemical shifts were recorded in ppm with the solvent reso-
nance as the internal standard. Data are reported as chemical shifts,
multiplicity (s = singlet, d = doublet, t = triplet), coupling constants
3
1
3
= 15.0 Hz, 1H, CHOlefínic); 11.17 (d, J = 15.0 Hz, 1H, -NH). C NMR:
(CDCl ); 27.09 (-CH ); 88.65; 105.29;
, 75 MHz, δppm): 24.70 (-CH
107.89; 110.95; 141.40; 147.32; 150.64; 151.16; 163.36 (-C = O);
165.35 (-C = O); 168.87 (-C = O). Anal. calcd. for C14 : C, 56.56;
H, 5.05; N, 14.14. Found: C, 56.08; H, 5.06; N, 13.97.
N-(5-oxo-5,8-dihydro-1,8-naphthyridin-2-yl)-acetamide
3
3
3
15 3 5
H N O
(6)
(Hermecz et al., 1977; Nicoleti et al., 2012): Diphenyl ether (60 mL) was
◦
refluxed at 250 C to add slowly Meldrum’s acid adduct 5 (1.45 g; 4.7
(
Hz), and integration. The 2,6-diaminopyridine (1) was obtained
commercially (Aldrich) and purified by refluxing in chloroform for 5 h.
High-resolution mass spectra were obtained with Bruker
mmol) was slowly added. Reflux was performed for a further 15 min.
Following cooling, ethyl ether (24 mL) was added, the suspension was
filtered using a Büchner funnel and washed with ethyl ether. The solid
obtained was washed with hot hexane and dried under vacuum. Yield:
a
micrOTOF-Q 10,243 apparatus using an electrospray ionization source
in the positive mode (ESI(+)) by direct injection in the CEBIME (Labo-
rat o´ rio Central de Biologia Molecular Estrutural) of the Federal Uni-
versity of Santa Catarina.
◦
84%; mp: 298–299 C.
ꢀ
1
IR (KBr, cm ): 3331 (
ν
N
–
H
); 2953 (
ν
C
–
H
); 1674 (
, 300 MHz, δ ppm):2.14
); 6.02 (d, J = 7,6 Hz, 1H, CHAr); 7.80 (d, J = 7,6 Hz, 1H,
CHAr); 8.03 (d, J = 8,8 Hz, 1H, CHAr); 8.37 (d, J = 8.4 Hz, 1H, CHAr);
νC = O); 1611 (ν
C =
1
O
); 1516; 1306; 1196; 835; 598. H NMR: (CDCl
s, 3H, -CH
3
(
3
2

C.D.M. Oliveira-Tintino et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105753
1
0.69 (s, 1H, -NH); 11.64 (d, J = 4.8 Hz, 1H, -NH).13C NMR:(CDCl
3
, 75
(=CH). 1H NMR: (DMSO‑d
6
, 300 MHz, δ ppm): 2,35 (s, 3 H, -CH
3
), 7,36 (d,
MHz, δ ppm): 24.91 (-CH
1
3
); 110.50; 111.09; 117.53; 137.68; 140.47;
J = 8,2 Hz, 2 H, CHAR), 7,62 (d, J = 5,1 Hz, 2 H CHAR), 7,83 (s, 2 H,
CHAR), 8,39 (d, J = 9,3 Hz, 1 H, CHAR), 8,68 (s, 1 H, CHAR), 13,59 (s, 1 H,
50.20; 154.78; 170.76 (-C = O); 177.59 (-C = O).
-acetamido-5‑chloro-1,8-naphthyridine (7) (Carboni et al.,
971; Nicoleti et al., 2012): Under atmosphere N , N-(5-oxo-5,8-dihy-
2
N
H). 13C NMR: (DMSO‑d
–
6
, 75 MHz, δ ppm: 21,44 (-CH
3
); 121,10;
1
2
123,79; 129,43; 129,70; 129,92; 132,63; 136,85; 139,51; 142,39;
145,09; 153,62; 156,32.HR-MS (ESI, TOF): C15 SCl, theoretical
dro-1,8-naphthyridin-2-yl) acetamide 6 (0.6952 g, 0.0034 mol) was
12 3 2
H N O
+
+
added to 14 mL of dimethylformamide under continuous stirring, then
[M] m/z = 334,0411; experimental [M] m/z = 334,0412.
dichloromethane (21 mL) was added to the mixture. The mixture was
2,5-dichloro-N-(5‑chloro-1,8-naphthyridin-2-yl)-benzenesulfo-
namide (10b): 1.5 h reaction time as determined by TLC. Extraction
was performed with ethyl acetate, and the product was purified with a
◦
ice-cooled (ꢀ 15 C) under stirring for 15 min. Then the POCl
3
(1.1 mL)
was added, and the mixture remained in an ice bath for another 10 min.
The ice bath was removed, and the mixture remained for another two
2 2
CH Cl /MeOH (98:2) mixture. Yellow copper solid. Yield: 71%, mp:
◦
¡1
hours under continuous stirring. Then, 18 mL of NH
4
OH were added to
Cl (3
O. The organic phase was filtered over
247.5–248 C. Unpublished compound. IR (ATR/FTIR, cm ): 3222
neutralize the reaction, and the solution was extracted using CH
2
2
(N
–
H), 3046 (=CH), 1621 (N
–
H), 1591, 1530 e 1402 (C = C), 1289 (as S = O)
,
1
×
10 mL) and washed with H
2
1131(s S = O), 1107 (C
–
N), 1041 (Ar-Cl), 827 (=CH). H NMR: (DMSO‑d6,
Na
2
SO4, and the solvent was concentrated on a rotary evaporator. Yield:
300 MHz, δ ppm): 7,59–7,72 (m, 3 H, CHAR,); 7,75 (d, J = 9,7 Hz, 1 H,
◦
7
0%, mass: 1.7 g; mp: 251–253 C.
CHAR); 8,14 (d, J = 2,0 Hz, 1 H, CHAR); 8,42 (d, J = 9,7 Hz, 1 H, CHAR);
ꢀ 1
13
IR (KBr, cm ): 3387 (
535; 1491; 1400; 1306; 1240; 1136 (
00 MHz, δ ppm): 2.31 (s, 3H, -CH ); 7.47 (d, J = 3.0 Hz, 1H, CHAr); 8.58
ν
N
–
H
); 3051 (
ν
=C
–
H
); 1709 (
ν
C = O); 1609;
8,67 (d, J = 5,1 Hz, 1 H, CHAR); 13,84 (s, 1 H, N H). C NMR: (75
–
1
1
3
ν
C
–
Cl); 995; 835. H NMR: (CDCl
3
,
MHz, DMSO‑d
6
, δ ppm): 114,97; 118,82; 121,39; 129,42; 130,32;
3
132,43; 133,65; 133,89; 137,53; 142,08; 142,61; 149,95; 153,71;
+
(
d, J = 9.0 Hz, 1H, CHAr); 8.64 (d, J = 9.0 Hz, 1H, CHAr); 8.88 (d, J = 6.0
157,51. HR-MS (ESI, TOF): C14
H
N
8 3
O
2
3
SCl , theoretical [M] m/z =
1
3
+
Hz, 1H, CHAr); 9.47 (s, 1H, -NH). C NMR: (CDCl
3
, 75 MHz, δ ppm):
); 116.24; 119.34; 120.84; 136.26; 142.85; 153.16; 154.61;
55.39; 169.86 (-C = O).
-amino-5‑chloro-1,8-naphthyridine (8) (Luigi Ferrarini, Manera,
Mori, Badawneh, and Saccomanni, 1998): A suspension of 7 (1.7 g; 7.6
mmol) in H SO 10% (18 mL) was heated and refluxed for 10 min. After
cooling, NH OH was added until complete neutralization was achieved.
The solution was transferred to an extraction funnel and extracted using
CH Cl
(3 × 10 mL). The organic phases obtained were combined,
filtered over anhydrous Na SO4, and the solvent was concentrated on a
387,9472; experimental [M] m/z = 387,9476.
2
1
4.99 (-CH
3
4-fluoro-N-(5‑chloro-1,8-naphthyridin-2-yl)-benzenesulfona-
mide (10c): 1.5 h reaction time as determined by TLC. Extraction was
performed with ethyl acetate, and the product was purified with a
2
CH
196.9–197.9 C. Unpublished compound. IR (ATR/FTIR, cm ): 3042
H), 1378 (as S = O), 1142(s S = O), 1595, 1529, 1484 e
2 2
Cl /MeOH (98:2) mixture. Yellow copper solid. Yield: 34.71%, mp:
◦
¡1
2
4
4
(
ν
=CH), 1631 (δ N
–
1
1409
(
ν
C = C), 1085 (Ar-Cl), 833 (=CH)
6
. H NMR (DMSO‑d , 300 MHz,
2
2
δppm): 7.39 (t, J = 8.8 Hz, 2 H, CHAR); 7.60 (d, J = 5.1 Hz, 1 H, CHAR);
7.67 (d, J = 9.3 Hz, 1 H, CHAR); 8.02 (dd, J = 8.3, 5.4 Hz, 2 H, CHAR);
2
rotary evaporator, resulting in a pale-yellow solid. Yield: 91%; mp:
8.39 (d, J = 9.5 Hz, 1 H, CHAR); 8.67 (d, J = 5.1 Hz, 1 H, CHAR); 13.45 (s,
◦
13
1
74–176 C.
6
1 H, N H). C NMR (DMSO‑d , 75 MHz, δppm): 115.18; 116.43;
–
IR (KBr, cm 1): 3354 (
431; 1383; 1140 ( Cl); 968; 826; 675. H NMR: (CDCl
); 7.34 (d, J = 4.8
Hz, 1H, CHAr); 8.16 (d, J = 9.2 Hz, 1H, CHAr); 8.63 (d, J = 4.4 Hz, 1H,
ꢀ
ν
); 3169 (
ν
); 1651; 1622; 1584; 1549;
116.73; 117.93; 120.58; 121.16; 127.73; 128.39; 129.81; 137.06;
N
–
H
N
–
H
1
1
ν
C
–
3
, 300 MHz, δ
139.36; 142.46; 151.51; 153.62; 156.8137; 162.80; 166.12.
ppm): 6.97 (d, J = 8.8 Hz, 1H, CHAr); 7.21 (s, 2H, -NH
2
2,3,4-trifluoro-N-(5‑chloro-1,8-naphthyridin-2-yl)-benzene-
sulfonamide (10d): 3.5 h reaction time as determined by TLC.
Extraction was performed with ethyl acetate, and the product was pu-
1
3
CHAr). C NMR: (CDCl
3
, 75 MHz, δ ppm): 114.53; 114.77; 117.30;
1
33.33; 140.98; 151.86; 157.51; 161.22.
rified with a CH
2
Cl
2
/MeOH (98:2) mixture. Orange solid. Yield: 57%,
◦
¡1
mp: 198.6–199.6 C. Unpublished compound. IR (ATR/FTIR, cm ):
2
.1.3. The general methodology for the synthesis of sulfonamides derived
3200 (N
–
H), 1639 (N
–
H), 1599, 1506, 1459 e 1405 (C = C), 1314 (as S = O)
,
1
from N-(6-aminopyridin-2-yl)-acetamide (10a–f)
1149 (s S = O), 1123 (Ar-F), 1037 (Ar-Cl), 826 (=CH). H NMR(Acetonitrile-
d3,300 MHz, δ ppm): 7.25 (ddd, J = 9.5, 2.38, 2.20 Hz, 1 H, CHAR);
The syntheses of the compounds (10a–f) were conducted and
adapted according to the methodology described by Saupe et al. (1991).
In a round bottom flask, 0.1 g of the 2-amino-5‑chloro-1,8-naphthyr-
idine 8 (0.6 mmol), 40.0 mL THF and 0.8 g NaH (33.0 mmol) were added
7.33 (d, J = 9.5 Hz, 1 H, CHAR); 7.53 (d, J = 5.1 Hz, 1 H, CHAR
)
7.77–7.86 (m, 1 H, CHAR); 8.34 (d, J = 9.5 Hz, 1 H, CHAR); 8.60 (d, J =
1
3
6
5.1 Hz, 1 H, CHAR); 11.62 (s, 1 H, N H). C NMR (DMSO‑d , 75 MHz,
–
under an inert atmosphere (N
2
). The reaction mixture was placed under
δppm): 111.33; 111.38; 111.42; 111.57; 111.62; 113.30; 114.00;
114.06; 114.25; 114.30; 115.97; 116.66; 119.33; 119.68; 122.36;
125.15; 135.98; 138.67; 140.97; 143.64; 143.70; 148.04; 150.70;
◦
continuous stirring and heated at 60 C for 1 h. Then, benzenesulfonyl
chloride 9 (4.2 mmol) was added, and the reaction was kept under
◦
continuous stirring at 60 C for 1.5–3.5 h, depending on the benzene-
7 3 2 3
151.94; 154.59; 155.90; 158.55. HR-MS (ESI, TOF): C14H N O SClF ,
+
+
sulfonyl chloride derivative used (as determined by the TLC). The so-
lution was allowed to cool to room temperature, transferred to an
extraction funnel, and extracted with EtOAc (3 × 10 mL). The organic
phases were pooled, transferred to an extraction funnel, and washed
theoretical [M] m/z = 373.9975; experimental [M] m/z = 373.9972.
3-trifluoromethyl-N-(5‑chloro-1,8-naphthyridin-2-yl)-benzene-
sulfonamide (10e): 3.5 h reaction time as determined by TLC. Extrac-
tion was performed with ethyl acetate, and the product was purified
with a saturated NH
4
Cl solution (3 × 10 mL). The organic phase was
SO , concentrated on a rotary
2 2
with a CH Cl /MeOH (98:2) mixture. Light yellow solid. Yield: 53%,
◦
¡1
separated and filtered over anhydrous Na
2
4
mp: 222.2–224 C. Unpublished compound. IR (ATR/FTIR, cm ):
evaporator, and dried under vacuum at room temperature. The residual
solid obtained was purified on a silica gel chromatography column
3265 (N
–
H), 3066 (=CH), 1645 (N
–
H), 1600, 1534, 1468 e 1410 (C = C)
,
1
1323 (as S = O), 1122 (s S = O), 1100 (Ar-Cl), 826 (=CH). H NMR (DMSO‑d
6
,
(
(
(
0.063–0.200 mm; 70–30 mesh) using pure CH
2
Cl
2
, CH
2
Cl
2
/MeOH
300 MHz, δ ppm): 7.33 (d, J = 9.72 Hz, 1 H, CHAR); 7.50 (d, J = 5.1 Hz,
1 H, CHAR); 7.71–7.78 (m, 1 H, CHAR); 7.88–7.93 (m, 1 H, CHAR);
8.19–8.27 (m, 2 H, CHAR); 8.30 (d, J = 9.7 Hz, 1 H, CHAR); 8.59 (d, J =
98:2), or pure AcOEt, depending on the compound. The compounds
10a–f) listed below were obtained:
1
3
4
-methyl-N-(5‑chloro-1,8-naphthyridin-2-yl)-benzenesulfona-
6
5.1 Hz, 1 H, CHAR); 11.47 (s, 1 H, N H). C NMR (DMSO‑d , 75 MHz,
–
mide (10a): 2 h reaction time as determined by TLC. Extraction was
δ ppm): 107.83; 112.65; 112.88; 113.26; 121.36; 121.97; 124.67;
performed with ethyl acetate, and the product was purified with a
125.58; 129.51; 129.94; 130.37; 130.81; 131.29; 133.65; 136.65;
CH
2
Cl
2
/MeOH (98:2) mixture. Yellow solid. Yield: 84%, mp: 249–250
137.50; 140.86; 144.10; 149.05; 154.98; 172.95. 19F NMR (DMSO‑d
,
6
◦
ꢀ 1
C. IR (ATR/FTIR, cm ): 3413 (N
–
H), 3062 (=CH), 1638 (N
–
H), 1408,
282 MHz,
δ
ppm): ꢀ 58.5
(
ν
Ar-CF3)
.
HR-MS (ESI, TOF):
+
+
1
480, 1531 e 1596 (C = C), 1301 (as S = O), 1143 (s S = O), 1085 (Ar-Cl), 828
C
15
H
9
N
3
O
2
SClF
3
, theoretical [M] m/z = 388.0130; experimental [M]
3

C.D.M. Oliveira-Tintino et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105753
m/z = 388.0129.
definition of pharmacokinetics and drug-like properties, as well as other
ADME properties.
4
-Bromo-2,5-difluoro-N-(5‑chloro-1,8-naphthyridin-2-yl)-ben-
zenesulfonamide (10f): 1.5 h reaction time as determined by TLC.
Extraction was performed with ethyl acetate, and the product was pu-
2.2.2. Bioactivity predictions using molinspiration
rified with a CH
2
Cl
2
100%. Yellow copper solid. Yield: 69.06%, mp:
Molinspiration is a cheminformatics software tool that calculates the
bioactivity of G protein-coupled receptors ligand (GPCR ligands), ki-
nase, ion channel blockers/modulators, protease, nuclear receptor li-
gands, and enzyme inhibition based on fragment contribution, such that
the sum of activity contributions and molecular activity scores are
processed in an online interface from the Molinspiration Chem infor-
matics server (http://www.molinspiration.com).
◦
¡1
2
22.7–223.5 C. Unpublished compound. IR (ATR/FTIR, cm ): 3250
(
(
ν
N
–
H), 3040
(ν
=CH), 1633 (δ N
–
H), 1597, 1531, 1473 e 1407
(
ν
C = C), 1312
1
as S = O), 1132(s S = O), 1132
(
ν
Ar-F)
.
H NMR (DMSO‑d
6
, 300 MHz,
δppm): 7.68 (d, J = 5.1 Hz, 1 H, CHAR); 7.75 (d, J = 10.0 Hz, 1 H, CHAR);
7
.92 – 8.00 (m, 2 H, CHAR); 8.46 (d, J = 9.7 Hz, 1 H, CHAR); 8.71 (d, J =
13
5
N H 6
–
.1 Hz, 1 H, CHAR); 14.02 (s, 1 H, ). C NMR (DMSO‑d , 75 MHz,
δppm): 115.09; 116.58; 116.69; 116.80; 116.96; 117.82; 118.51;
1
1
1
1
19.27; 119.64; 121.21; 121.50; 122.77; 123.12; 124.22; 125.47;
25.83; 132.35; 137.89; 140.61; 142.68; 145.42; 152.54; 152.84;
53.04; 153.83; 155.55; 155.72; 156.29; 156.53; 157.75; 158.9277;
60.40.
2.3. Substances and drugs used
The ethidium bromide (EtBr) and ciprofloxacin were obtained from
Sigma Aldrich Co. Ltda. All 1,8-naphthyridines used were synthesized
and donated by researchers from the Federal University of Paran a´ and
Regional University of Blumenau. All 1,8-naphthyridines, the cipro-
floxacin, and EtBr were dissolved in dimethyl sulfoxide (DMSO) and
2
.2. Molecular modeling and docking studies
The three-dimensional (3D) structure of MepA was elucidated based
further diluted in water to a concentration of 1024 g/mL.
μ
on sequence identity with a high score, lower e-value, highest resolu-
tion, and the R-factor of the template structure by performing BLASTP
with an S. aureus NCTC 8325 input sequence obtained from S. aureus
2.4. Bacterial strains
8
325–4 and SH1000 genome sequence data. The coordinates for the
For the assays, strain SA-K2068 was used, which codes for the MepA
efflux protein, the basis for the majority of the multiple drug resistance
(MDR) phenotype in this mutant strain, and is responsible for extruding
DNA-intercalating dyes, hydrophilic fluoroquinolones, and other drugs
(Kaatz et al., 2005; Schindler, Patel, et al., B.D. 2013). The strain, kindly
provided by Prof. Glenn Kaatz (Wayne State University), was grown
query structure were obtained by Clustal X alignment. Phyre2 (Protein
Homology/Analogy Recognition Engine - http://www.sbg.bio.ic.ac.
uk/phyre2) was used to predict the three-dimensional structure of
MepA, while I-TASSER (http://zhanglab.ccmb.med.umich.edu/
I-TASSER/) and MODELLER 9.14 were used to confirm the Phyre2
prediction. The final model was evaluated by the Ramachandran plot
using PROCHECK to check the stereochemical quality of the protein
structure. The root-mean-square deviation (RMSD) of the model was
calculated by the superimposition of the 3D model with the template
using the Swiss-PDB Viewer (PDB ID: 4LZ9). The topographic structure
of the pockets and cavities can be intermediated by the CASTp server
◦
overnight at 37 C in Heart Infusion Agar slants (HIA, Difco) before use
(Gibbons et al., 2003).
2.5. Antibacterial activity and efflux pump inhibition
2.5.1. Antibacterial activity test by minimum inhibitory concentration
(MIC)
(
http://cast.engr.uic.edu) with a standard value of 1.4 Å for the radius.
The 3DLigandSite is a web server for the prediction of ligand-binding
sites.
The minimum inhibitory concentration of 1,8-naphthyridines was
determined in a microdilution assay. 100
suspended in saline solution was used, corresponding to 0.5 on the
McFarland scale, followed by the addition of 900 L of brain heart
μL of the bacterial inoculum
Virtual screening and docking were performed by the SwissDock web
server (http://www.swissdock.ch/) (Grosdidier et al., 2011). SwissDock
is a protein-small molecule docking web service based on EADock DSS.
Nucleic acids research, 39 (suppl_2), W270-W277, and conformed by
AutoDock Vina. The 3D coordinates of ligands were generated using the
CORINA online server (Molecular Networks. CORINA online Demo:
http://www.molecularnetworks.com/online_demos/corina_demo), and
μ
infusion (BHI) culture media in Eppendorf to obtain a volume of 1 mL.
An aliquot of this medium with the bacteria was distributed in 96-
well microtiter plates. Serial dilutions were performed using the cipro-
floxacin, or ethidium bromide and 1,8-naphthyridines with concentra-
tions ranging from 0.5 to 512 µg/mL (1:1). The last well was left to
◦
energy was minimized with
a
universal force field using
a
ensure the control of growth. The plates were incubated at 37 C for 24
conjugate-gradient algorithm with 200 run iterations. Docking runs
were performed blinded by covering the entire protein and defining the
protein coordinates of a region of interest as binding pockets to ensure
blind docking. The best-docked ligand conformations were saved, and
the bond angles, bond lengths, and hydrogen bonding interactions were
analyzed using UCSF Chimera visualization program. The Discovery
Studio (DS) program was utilized in detail. The binding energy score was
used to calculate the inhibition constant (Ki value) based on the equa-
tion by Onawole et al. (2018) and Ligand efficiency (LE) measurement
using the equation determined by Nissink (2009).
h, and resazurin (7‑hydroxy-3H-phenoxazin-3-one-10-oxide) was added
to assess bacterial growth. The minimum inhibitory concentration was
the lowest concentration in which there was no bacterial growth, ac-
cording to Tintino et al. (2017).
2.5.2. Efflux pump inhibition tests
To assess whether 1,8-naphthyridines were a potential efflux pump
inhibitor, their ability to decrease the minimum inhibitory concentra-
tion of the antibiotic and ethidium bromide were evaluated. The sub-
strates for the MepA pump used were ethidium bromide, a substrate for
many MDR pumps, and the antibiotic ciprofloxacin, according to the
methodology by Tintino et al. (2017).
2
.2.1. The measure of drug likeliness, Lipinski rule analysis, and ADME
predictions
The therapeutic target in the organism is dependent on pharmaco-
2.5.3. Evaluation of efflux pump inhibition by antibiotic and ethidium
bromide mic reduction
kinetic parameters, such as absorption, distribution, metabolism, and
excretion (ADME). Moreover, several parameters can influence several
structural parameters. Thus, an understanding of this helps screen out
weak candidates in the early stages of drug development from potential
drug candidates. The SwissADME server (http://www.swissadme.ch)
generated the prediction of physical-chemical descriptors and the
Efflux pump inhibition was tested using a sub-inhibitory 1,8-naph-
thyridine concentration (MIC/8). In preparing the controls, 150 L of
μ
each bacterial inoculum was suspended in saline, corresponding to 0.5
on the McFarland scale, and transferred to Eppendorf along with 1350
μL of brain and heart infusion (BHI). In the test solutions, a solution was
4

C.D.M. Oliveira-Tintino et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105753
Table 1
1
,8-naphthyridines sulfonamides structures and molecular mass.
–1
◦
Compound
Structure
Theoretical molecular mass (g.mol
)
Available mass (g)
Yield (%)
M.P. ( C)
–
Compound 6
–
–
298–299
Compound 10a
Compound 10b
Compound 10c
Compound 10d
Compound 10e
Compound 10f
333.82
388.67
337.78
373.76
387.79
434.66
0.0282
0.0213
0.0105
0.0247
0.0531
0.0095
84.0
70.8
34.7
56.5
52.6
69.1
249–250
247.5–248
196.9–197.9
198.6–199.6
222.2–224
222.7–223.5
Compound 6 = N-(5-oxo-5,8-dihydro-1,8-naphthyridin-2-yl)-acetamide; Compound 10a ¼ 4-methyl-N-(5-chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide;
Compound 10b ¼ 2,5-Dichloro-N-(5-chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide; Compound 10c ¼ 4-fluoro-N-(5-chloro-1,8-naphthyridin-2-yl)-benzene-
sulfonamide; Compound 10d ¼ 2,3,4-trifluoro-N-(5-chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide; Compound 10e ¼ 3-trifluoromethyl-N-(5-chloro-1,8-
naphthyridin-2-yl)-benzenesulfonamide; Compound 10f ¼ 4‑bromo-2,5-difluoro-N-(5-chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide.
prepared to contain 150
saline, 1,8-naphthyridines (MIC/8), and BHI. The solutions were trans-
ferred to 96-well microtiter plates. 100 L of ciprofloxacin or ethidium
μ
L of each bacterial inoculum suspended in
ethidium bromide, and 2,3,4-trifluoro-N-(5‑chloro-1,8-naphthyridin-2-
yl)-benzenesulfonamide (compound 10d), as a test group; bacterial
inoculum, ethidium bromide, and carbonyl cyanide 3-chlorophenylhy-
drazone (CCCP), as a positive control. All substances in a MIC/8 con-
centration. Fluorescence spectra were obtained on a Shimadzu RF6000
spectrofluorometer with a 150 W xenon lamp for sample excitation. The
samples were packed in a quartz cuvette (10 mm optical path) of four
polished faces and subjected to scattered light excitation with a
maximum wavelength of 530 nm, scanning speed of 600 nm/min, and 3
nm spectral resolution. The emission was monitored from 400 to 800 nm
with 0.5 nm increments (Tintino et al., 2018).
μ
bromide was used for micro dilutions (1:1). The incubation and reading
followed the same pattern as the previous item. The minimum inhibitory
concentration of the control was assessed using ciprofloxacin or
ethidium bromide alone (Tintino et al., 2017).
2
.5.4. Evaluation of mepA efflux pump inhibition by ethidium bromide
fluorescence emission
◦
The strains were grown in HIA for 24 h, at a temperature of 37 C.
Subsequently, the inoculum was prepared by suspending the strains in
saline solution, corresponding to 0.5 on the McFarland scale. Then the
test solutions were prepared, containing 500 µL of the inoculum, the
substances, and the final volume supplemented with BHI until a volume
2.6. Statistical analysis of microbiological results
Antibacterial assays were performed in triplicates, and the results
were expressed as the geometric mean of replicates. Statistical hypoth-
esis analysis was performed using a two-way ANOVA, followed by
Tukey’s post hoc test, using GraphPad Prism 5.0 software.
◦
of 2 mL was obtained, kept in a greenhouse at 37 C in incubation for 6 h.
Then, the samples were centrifuged at 3000 rpm for 3 min, and the pellet
obtained was resuspended in 3 mL saline solution to read the fluores-
cence emission. The following groups were tested: bacterial inoculum
and ethidium bromide, as a negative control; bacterial inoculum,
5

C.D.M. Oliveira-Tintino et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105753
Fig. 1. Final MepA receptor structure. A) The blue ball shows the most probable active sites of interactions and the green grid box used in docking simulations. B)
Ramachandran plot for the MepA structure. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)
◦
3
. Results and discussion
(Scheme 1), obtaining a 70% yield and mp > 250 C. The compound 7
2 4
was then deacetylated in a reflux system with 10% H SO for 10 min to
3
.1. Synthesis
generate 2-amino-5‑chloro-1,8-naphthyridine 8 with a 91% yield and
◦
mp 174.5–176.5 C.
The synthesis of 1,8-naphthyridines was performed using the meth-
The 1,8-naphthyridine sulfonamide 10a–10f preparation was con-
ducted based on the methodology described by Saupe et al. (1991),
where 2-amino-5‑chloro-1,8-naphthyridine 8 was reacted with different
commercial benzenesulfonyl chlorides. Reactions were performed in
odology described by Silva et al. (2008), through the thermolysis of
Meldrum’s acid adduct, with some modifications made in the synthesis
process. Initially, monoacetylation of 2,6-diaminopyridine 1 was carried
out by reacting 2,6-diaminopyridine 1 with acetic anhydride in THF and
using TEA as the base, yielding the monoacetylated product 2 with a
◦
THF at a temperature of 60 C using NaH as the base.
The time for the reactions varied from 1 h to 3.5 h for each sulfon-
amide. Thus, the synthetic route tested proved to be very interesting due
to the significant decrease in reaction time compared to the similar
methodologies described in the literature (Davidson and Bernhard,
1948). The reactions were accompanied by TLC (thin layer chroma-
tography), and the obtained compounds listed in Table 1 were charac-
terized by their melting point, IR spectroscopy, 13C, and H NMR, and
high-resolution mass spectrometry.
4
8% yield and mp 155–157 (Yield 49% and mp 159–160 ◦C) (Bernar-
dino et al., 2012). The synthesis of Meldrum acid 3 was then performed
by the reaction between malonic acid, acetic anhydride, and concen-
trated sulfuric acid as the catalyst, followed by the addition of acetone,
◦
slowly at 0 C, according to the methodology described by Davidson and
1
Bernhard (Davidson and Bernhard, 1948). A yield of 32% and an mp of
9
6–96.2 ◦C were obtained [(literature: yield 34% and mp 94–95 ◦C)]
◦
(
Bernardino et al., 2012) (91–92 C) (Davidson and Bernhard, 1948).
After Meldrum’s acid 3 was obtained, the methoxymethylenic derivative
of Meldrum’s acid 4 was obtained by the reaction of Meldrum’s acid 3 in
trimethyl orthoformate, which is not isolated and reacted with 2-acet-
amido-6-aminopyridine 2 to provide the Meldrum’s acid adduct 5.
3
3
.2. Molecular modeling and docking studies
.2.1. Receptor homology
The phyre2-predicted MepA model included 98% of MepA residues,
The yield obtained for the adduct 5 was 40% and the mp was
◦
with the calculated confidence of the model being 100%. I-TASSER and
Modeller generated a highly similar model using 99% and 97% of MepA
residues, with a predicted accuracy of 95% and 93%, respectively. The
final model was corroborated by Ramachandran plot calculations using
PROCHECK which revealed 361 residues (90.9%) were aligned within
the most favored region, 30 residues (7.6%) were located within the
additionally allowed regions, three residues (0.8%) were plotted within
the generously allowed region, and three residues (0.8%) were aligned
within the disallowed region. Quadrant II shows the largest region in the
graph with the most favorable atom conformations, sterically allowed
conformations for beta-strands. However, quadrant III shows the
2
22.8–222.9 C. The Meldrum’s acid adduct 5 was then thermolyzed in
◦
diphenyl ether at 250 C for 15 min to give naphthyridinone, or com-
◦
pound 6 (Scheme 1) with an 84% yield and mp 297.5–299 C (Litera-
ture: mp > 300 C) (Bernardino et al., 2012).
◦
Chlorination of compound 6 was carried out, using an adaptation of
the methodology described by Salvio et al. (2014) to obtain 2-acet-
amido-5‑bromo-1,8-naphthyridine using DMF, CH
the halogenating agent was replaced, and the procedure was main-
tained. Chlorination was performed using DMF, CH Cl2, and POCl in an
2 3
Cl2, and PBr . Thus,
2
3
ice bath at ꢀ 15 ◦C to form 2-acetamido-5‑chloro-1,8-naphthyridine 7
6

C.D.M. Oliveira-Tintino et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105753
Table 2
Molecular docking results for different 1,8-naphthyridines investigated with the MepA efflux pump, presenting the structure, MIC of the substances associated with
EtBr and ciproflocacin, Energy of interactions, Ki value and size-independent ligand efficiency (SILE).
Compound
Structure
MICSubstance + EtBr
µg/mL)
MIC Substance + Cipro
(µg/mL)
Energy of interactions
kcal/mol
Calculated Ki
SILE
(
(µM)
Ethidium
bromide
256
–
ꢀ 8.6
0.51
0.19
–
Ciprofloxacin
64
ꢀ 8.2
ꢀ 6.5
0.99
0.39
CCCP
50.8
16
17.44
7.90
Compound 6
128
8
ꢀ 7.1
ꢀ 8.4
6.33
2.82
Compound 10a
64
16
0.71
0.28
Compound 10b
32
2
ꢀ 8.2
0.99
0.39
Compound 10d
32
1
ꢀ 8.8
0.36
0.14
Compound 10e
128
8
ꢀ 8.6
0.51
0.19
CCCP = carbonyl cyanide 3-chlorophenylhydrazone; Compound 6 = N-(5-oxo-5,8-dihydro-1,8-naphthyridin-2-yl)-acetamide; Compound 10a = 4-methyl-N-(5-
chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide; Compound 10b = 2,5-Dichloro-N-(5-chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide; Compound 10d =
2
,3,4-trifluoro-N-(5-chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide;
Compound
10e
=
3-trifluoromethyl-N-(5-chloro-1,8-naphthyridin-2-yl)-
benzenesulfonamide.
7

C.D.M. Oliveira-Tintino et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105753
Fig. 2. The 3D structure of the binding pocket of the efflux pump MepA and map of amino acid residues. A) Binding pocket for the best binding with the highest
stability for ethidium bromide and MepA. B) Map of amino acids residues within the ethidium bromide binding pocket of MepA. C) Binding pocket for the best
binding with the highest stability for CCCP and MepA. D) Map of amino acid residues within the CCCP binding pocket of MepA. E) Binding pocket for the best binding
with the highest stability for compound 10d interaction with MepA. F) Map of amino acid residues interactions with the compound 10d binding pocket of MepA.
8

C.D.M. Oliveira-Tintino et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105753
Table 3
Physicochemical and Pharmacokinetic Predictions by Swiss ADME of 1,8-naphthyridines sulfonamides.
Molecular Properties of the ligands
Pharmacokinetics parameters
Compound
NRB
2
NHba
0
NHbd
2
MR
TPSA
55.92
CLog
Po/w
GI
BBB
P-gp
CYP1A2
inhibitor
CYP2C19
inhibitor
CYP2C9
inhibitor
CYP2D6
inhibitor
CYP3A4
inhibitor
RL-
5
substrate
Ethidium
bromide
CCCP
113.02
2.17
Hight
Yes
Yes
No
No
No
Yes
No
No
2
3
2
3
3
3
4
3
5
3
4
4
7
7
1
2
2
1
1
1
1
53.55
92.55
56.68
86.63
91.68
81.54
86.66
71.97
74.57
74.85
80.33
80.33
80.33
80.33
1.83
0.98
0.67
2.88
3.49
3.33
3.74
Hight
Hight
Hight
Hight
Hight
Hight
Hight
Yes
No
No
No
No
No
No
No
Yes
No
No
No
No
No
Yes
No
No
No
No
No
No
Yes
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Ciprofloxacin
6
No
Yes
Yes
Yes
No
No
1
1
1
1
0a
0b
0d
0e
Yes
Yes
Yes
Yes
Yes
BBB = blood-brain barrier permeation; CCCP = carbonyl cyanide 3-chlorophenylhydrazone; CLog Po/w = the arithmetic mean of the values predicted by the five
proposed methods; Compound 6 = N-(5-oxo-5,8-dihydro-1,8-naphthyridin-2-yl)-acetamide; Compound 10a = 4-methyl-N-(5-chloro-1,8-naphthyridin-2-yl)-ben-
zenesulfonamide; Compound 10b = 2,5-Dichloro-N-(5-chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide; Compound 10d = 2,3,4-trifluoro-N-(5-chloro-1,8-
naphthyridin-2-yl)-benzenesulfonamide; Compound 10e = 3-trifluoromethyl-N-(5-chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide; GI = human gastrointestinal
absorption (HIA); MR = Molar refractivity; NRB = Number of rotatable bonds; NHba = Number of hydrogen-bond acceptors; NHbd = Number of hydrogen-bond
donors; RL-5 = Violation in Lipinski’s Rule of five; TPSA = Topological polar surface area.
majority of amino acids are present in this largest region of the graph,
where right-handed alpha helices are shown (Fig. 1).
acids but only three types for ethidium bromide. The differences in the
interactions between the receptor-ligands may explain the affinity and
stability of naphthyridine.
In the evaluation of receptor homology, it was shown in the Ram-
achandran plot that most of the amino acids present in the protein
structure appear as alpha helices that are hallmark characteristics of the
transmembrane segments. The results presented herein corroborate with
the study by Schindler et al. (B.D. 2013).
3.2.3. In silico pharmacokinetic studies and bioactivity predictions
In the search for new compounds with antimicrobial activity that
cause a reduction in bacterial resistance, it is essential to understand the
pharmacokinetic parameters such as absorption, distribution, and
metabolism for oral bioavailability, potency, and selectivity. The results
are shown in Table 3 obtained from in silico runs using SwissADME
indicate a possible effective permeation of intestinal epithelium in oral
absorption for all ligands. Results from the partition coefficient indicate
the presence of halogenic groups influence the lipophilicity of all com-
pounds, but they do not cross the blood-brain barrier. The compounds
showed an ideal Log P (<5).
3
.2.2. Efflux pump molecular docking
Docking simulations were carried out for ligand-bound protein
complexes to minimize energy and stabilize the docked complex. The
best-docked ligand conformations were saved in output clusters 0. The
docking studies revealed that 1,8-naphthyridine derivatives were
recorded with the most favorable link energy in the range varying from
ꢀ
8.2 to ꢀ 8.8 kcal/mol for MepA (Table 2). The results corroborate with
the experimental MIC, in which the compounds with better binding and
interaction energy stability obtained lower MIC values.
Other parameters such as molar refractivity, total polar surface area
(TPSA), hydrogen bond donors, and acceptors are favorable indications
to classify virtual drug-like molecules. The compounds showed <10
rotatable bonds representing lower molecular flexibility. The TPSA for
all proposed compounds showed a range of 71.97–80.33, indicating that
the compounds could easily enter a cell. Lower TPSA is a parameter for
drug-likeness and corroborates signifying with good oral bioavailability.
Molar refractivity is associated not only with the volume of the mole-
cules but also represent the London dispersive forces that can be influ-
enced in drug-receptor interactions. Finally, they presented a number of
hydrogen bond acceptors <10, and number of hydrogen bond donors
<5. The results have shown no violations of Lipinski’s rule of five, which
indicate drug-like characteristics, with adverse or toxic effects, miti-
gated. The chemical properties present a positive correlation with the
docking results, where, characteristics such as hydrogen bonds, molar
refractivity, and lipophiles can be represented in interactions maps by
different types of hydrogen bonds, Van der Walls, Pi-alkyl, and alkyl
interactions.
The fact of having a small MIC value to inhibit bacterial growth in-
dicates a greater efficiency of the compound. The excellent energy
interaction values suggest that possibly naphthyridine interacts effi-
ciently with the MepA protein and, therefore, acts with an inhibitor by
the following possible mechanisms: directly obstructing the protein’s
duct (Zeng et al., 2010); or inhibiting the efflux pump in a competitive or
non-competitive way, reducing the binding of the substrate to the pro-
tein (Bhardwaj and Mohanty, 2012; Lomovskaya and Bostian, 2006;
Mahamoud et al., 2007; Marquez, 2005; Nakashima et al., 2013;
Nguyen et al., 2015; Opperman et al., 2014; Opperman and Nguyen,
2
015; Pages; Amaral; Fanning, 2011).
Fig. 2 shows the 3D structure of both efflux pump binding pockets
and interaction maps. To understand the underlying mechanism behind
the potential of 1,8-naphthyridines to promote efflux inhibition against
bacterial resistance, we investigated the binding modes of 1,8-naphthyr-
idine derivatives by molecular docking experiments.
The MepA interaction map for ethidium bromide and compound 10d
show similar Van der Waals interactions with amino acids Tyr35,
Val176, Thr201, and Tyr138 in addition to Pi-alkyl interactions with
Ile40 and Leu59. Ethidium bromide is stabilized by hydrogen bonds with
Ash183 (4.47 Å) and Ala197 (3.35 Å), while compound 10d shows in-
teractions with Arg281 (6.56 Å) and Asn39 (4.68 Å). The better in-
teractions energy of compound 10d may be explained by other types of
interactions, such as carbon-hydrogen bonds with Ser175 (3.91 Å) and
Gly36 (3.64 Å), as well as halogen interactions with Asn179 (4.41 Å) and
Asn205 (4.66 Å) and different alkyl and Pi-alkyl connections with 5 Å
distances, approximately. Thus, the sum of all interactions and their
diversity resulted in 12 amino acids and five types of interactions for
CCCP; 17 amino acids, and six types for compound 10d; and 16 amino
For metabolism, the results showed that the compound 10d was the
one that least inhibited cytochrome P450 (CYP) isoforms involved in
metabolism, this being an relevant factor for their relationship with
drug-drug interactions. It is also observed that they cannot be substrates
of P-glycoprotein (P-gp). The P-gp suggests an influence in ATP-binding
cassette transporters or ABC-transporters, but only ethidium bromide
and ciprofloxacin were predicted to be the substrates for this protein.
The results were promising because the efflux of the drug by the P-gp of
the gastrointestinal lumen, interferes in its bioavailability and in the
ADME properties (Beaulieu et al., 1997; Edwards et al., 2005; Ma et al.,
2010; Raghava and Lakshmi, 2012).
Predictions of bioactivity scores were made for six different protein
structures viz, the GPCR ligand, ion channel modulator, kinase inhibitor,
9

C.D.M. Oliveira-Tintino et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105753
Table 4
Molinspiration bioactivity score of 1,8-naphthyridines sulfonamides.
Compound
GPCR ligand
Ion channel modulator
Kinase inhibitor
Nuclear receptor ligand
Protease inhibitor
Enzyme inhibitor
Ethidium bromide
CCCP
0.08
ꢀ 1.02
0.20
ꢀ 0.33
0.02
0.09
0.07
0.13
0.72
ꢀ 0.19
ꢀ 1.26
0.01
ꢀ 0.90
ꢀ 1.85
ꢀ 0.03
ꢀ 1.15
ꢀ 0.52
ꢀ 0.44
ꢀ 0.47
ꢀ 0.26
ꢀ 0.33
ꢀ 1.34
ꢀ 0.27
ꢀ 0.82
ꢀ 0.27
ꢀ 0.23
ꢀ 0.13
ꢀ 0.10
0.69
ꢀ 0.64
0.17
0.12
0.14
0.20
0.22
0.17
ꢀ 0.80
–0.08
ꢀ 0.07
ꢀ 0.25
ꢀ 0.17
ꢀ 0.28
ꢀ 0.10
Ciprofloxacin
Compound 6
Compound 10a
Compound 10b
Compound 10d
Compound 10e
0.08
0.00
0.04
ꢀ 0.02
0.08
CCCP = carbonyl cyanide 3-chlorophenylhydrazone; Compound 6 = N-(5-oxo-5,8-dihydro-1,8-naphthyridin-2-yl)-acetamide; Compound 10a = 4-methyl-N-(5-
chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide; Compound 10b = 2,5-Dichloro-N-(5-chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide; Compound 10d =
2
,3,4-trifluoro-N-(5-chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide;
Compound
10e
=
3-trifluoromethyl-N-(5-chloro-1,8-naphthyridin-2-yl)-
benzenesulfonamide.
Fig. 3. Ability of compounds 6, 10a,
1
0b, 10d, and 10e of inhibiting MepA
efflux pump in association with cipro-
floxacin and ethidium bromide, against
the SA-K2068 multiresistant strain. The
values represent the geometric mean ±
S.E.M. (standard error of the mean).
Two-way ANOVA, followed by Bonfer-
roni’s test. A) Associated with anti-
biotic. B) Associated with ethidium
bromide. a4 = P < 0.0001 vs control;
CCCP = carbonyl cyanide 3-chlorophe-
nylhydrazone; Cipro
Compound 10a = 4-methyl-N-(5-chloro-
,8-naphthyridin-2-yl)-benzenesulfona-
mide; Compound 10b = 2,5-Dichloro-N-
5-chloro-1,8-naphthyridin-2-yl)-benze-
=
ciprofloxacin;
1
(
nesulfonamide; Compound 10d = 2,3,4-
trifluoro-N-(5-chloro-1,8-naphthyridin-
2
1
1
-yl)-benzenesulfonamide; Compound
0e 3-trifluoromethyl-N-(5-chloro-
,8-naphthyridin-2-yl)-benzenesulfona-
=
mide; EtBr = ethidium bromide.
nuclear receptor ligand, protease, and enzyme inhibition. The values
attained indicated the binding affinity of the selected candidates to the
mentioned enzymes and receptors (the larger the score value, the more
probability the molecule is to be active). The results shown in Table 4
demonstrate that all compounds, as well as ciprofloxacin, present sig-
nificant enzyme inhibitory activity and lower interaction capacity with
GPCR or inhibition with Kinase enzymes. These results corroborate with
the docking analysis that showed a favorable interaction with MepA
depicting its potential for inhibition.
1
0

C.D.M. Oliveira-Tintino et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105753
Table 5
3.4. Efflux pump inhibition by antibiotic and ethidium bromide mic
reduction
Minimum inhibitory concentration (MIC) values of ciprofloxacin and ethidium
bromide alone and in combination with of compounds 6, 10a, 10b, 10d, and 10e
in subinhibitory concentration, against the SA-K2068 multiresistant strain.
In the present study the naphthyridines 4-methyl-N-(5‑chloro-1,8-
Compound
MIC of
MIC Cipro +
MIC EtBr +
Compound (µg/
mL)
naphthyridin-2-yl)-benzenesulfonamide (compound 10a), 2,5-dichloro-
substances (µg/
mL)
Compound (µg/mL)
N-(5‑chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide
(compound
1
0b), 2,3,4-trifluoro-N-(5‑chloro-1,8-naphthyridin-2-yl)-benzenesulfo-
Ethidium
256
–
–
namide (compound 10d), and 3-trifluoromethyl-N-(5‑chloro-1,8-naph-
thyridin-2-yl)-benzenesulfonamide (compound 10e) showed an unique
ability to reduce the MICs, when associated with both the ciprofloxacin
and ethidium bromide, against SA-K2068 (Fig. 3A and 3B and Table 5).
According to the data published so far, possible inhibition of the
efflux pump can be assessed by the occurrence of a reduction of twice or
more in the MIC of antibiotics that are the substrate of the pump and
ethidium bromide in comparison with the control, and checking the
inhibition of ethidium bromide fluorescence within the cell (Davies and
Wright, 1997; El-Baky et al., 2019; Kaatz et al., 2003; Kristiansen et al.,
bromide
Ciprofloxacin
Compound 6
Compound 10a
Compound 10b
Compound 10d
Compound 10e
64
–
8
–
≥ 1024
≥ 1024
≥ 1024
≥ 1024
≥ 1024
128
64
32
32
128
16
2
1
8
Compound 6 = N-(5-oxo-5,8-dihydro-1,8-naphthyridin-2-yl)-acetamide; Com-
pound 10a 4-methyl-N-(5-chloro-1,8-naphthyridin-2-yl)-benzenesulfona-
mide; Compound 10b 2,5-Dichloro-N-(5-chloro-1,8-naphthyridin-2-yl)-
=
=
benzenesulfonamide; Compound 10d = 2,3,4-trifluoro-N-(5-chloro-1,8-naph-
thyridin-2-yl)-benzenesulfonamide; Compound 10e = 3-trifluoromethyl-N-(5-
chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide.
2
010; Patel et al., 2010; Piddock, 2006; Ramalhete et al., 2011;
Trzcinski et al., 2000). This result was observed in the present study,
both in association with the control antibiotic and with ethidium
bromide.
3
.3. Minimum inhibitory concentration (MIC)
MepA is encoded by the chromosomal gene of the same name, mepA,
and was the first multidrug transporter of the MATE family to be
described in S. aureus, conferring resistance to this strain, being
responsible for the multidrug resistance (MDR) phenotype in the SA-
K2068. This protein has 451 amino acids and 12 transmembrane seg-
ments (Kaatz et al., 2005; McAleese et al., 2005). Its source of energy for
1
,8-Naphthyridides did not show clinically relevant antibacterial
g/mL. However, significant results were
activity, with all MICs ≥ 1024
μ
obtained concerning its efflux pump inhibitory potential by reducing the
antibiotic and ethidium bromide minimum inhibitory concentration. In
addition, one of the advantages of efflux inhibitors is that they are free of
any antibacterial activity, reducing their likelihood of future bacterial
resistance against new drugs (Ashima K. Bhardwaj & Priyabrata
Mohanty, 2012).
substrate transport is the proton gradient, through the sodium ions
+
(
Na ) antiport, considering that it belongs to the MATE family, whose
proteins work by this mechanism (He et al., 2004; Kuroda and Tsuchiya,
009). The substrates of this protein are hydrophilic fluoroquinolones
2
such as ciprofloxacin, norfloxacin; hydrophobic fluoroquinolones such
as moxifloxacin, sparfloxacin; glycylcyclines; biocides; and dyes such as
Fig. 4. Fluorescence emission of the ethidium bromide retained within the bacterial cell. 1: Peak corresponding to the emission caused by bacterial cellular contents;
: Enlarged image of the emission peak for ethidium bromide. CCCP = carbonyl cyanide 3-chlorophenylhydrazone; Compound 10d = 2,3,4-trifluoro-N-(5-chloro-1,8-
naphthyridin-2-yl)-benzenesulfonamide; EtBr = ethidium bromide.
2
1
1

C.D.M. Oliveira-Tintino et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105753
et al., 1962). It is suggested that, in this case, 1,8-naphthyridine removal
does not occur, but a disruption of MepA function, through substituent
groups binding to the active site, occurs.
Silva et al. (2020) demonstrated that Meldrum’s Acid, a precursor of
,8-naphthyridines presented in the current work, did not show direct
1
antibacterial activity, but inhibited bacterial growth when associated
with fluoroquinolones, corroborating our results. It is known in the
literature that various modified antibiotics function as efflux pump in-
hibitors (Anderson et al., 2005b, 2005a; Chollet et al., 2004; Chopra and
Roberts, 2001; Rice et al., 2005; Sabatini et al., 2011; Schindler, Jacinto,
&
Kaatz, B.D. 2013). Substances of chemically similar characteristics
have also been reported inhibiting efflux pumps for cancer cells, for
example, pyronaridine, a type of naphthyridine that is capable of
inhibiting the P-gp pump of carcinogenic cells. But there are hardly any
reports of these compounds in action against bacterial cell efflux pumps
(
Amaral et al., 2012; Kim et al., 2003; Misbahi et al., 2002; Qi et al.,
2
004, 2002).
3
.5. Evaluation of pump inhibition by ethidium bromide fluorescence
emission
In the fluorescence emission assessment, a subtle increase in fluo-
rescence intensity is observed when comparing the EtBr peak associated
with 2,3,4-trifluoro-N-(5‑chloro-1,8-naphthyridin-2-yl)-benzenesulfo-
namide relative to EtBr alone (Fig. 4).
Ethidium bromide retention within the cell enables its binding to
bacterial DNA such that the greater the amount of ethidium bromide
retained within the cell, the greater the amount of possible DNA binding
exists, thus the greater the fluorescence emission. When efflux pumps
are inhibited, ethidium bromide is trapped inside the cell resulting in
increased fluorescence emission (Blair and Piddock, 2016; Gibbons
et al., 2003; M. Viveiros et al., 2008; Miguel Viveiros et al., 2008).
Studies have been published showing the ability of various sub-
stances in acting as efflux pump inhibitors by assessing increased fluo-
rescence emission (Pannek et al., 2006). However, despite not showing
significant results, this study is innovative in that it does not evaluate
this effect at different times but at an exact time point, and thus evalu-
ates the final result of the efflux pump action.
Scheme 1. General scheme for the preparation of 1,8-naphthyridines
sulfonamides 10a–10f. R = 4-methyl; 2,5-dichloro; 4-fluoro; 2,3,4-trifluoro;
3
-trifluoromethyl; 4‑bromo-2,5-difluoro.
4
. Conclusions
ethidium bromide (Kaatz et al., 2006; Kaatz et al., 2005; McAleese et al.,
005). Reserpine, a commonly used efflux pump inhibitor (EPI), has
The sum of the results found that 4-methyl-N-(5‑chloro-1,8-naph-
2
thyridin-2-yl)-benzenesulfonamide, 2,5-dichloro-N-(5‑chloro-1,8-naph-
been shown to decrease the MIC of substances tested in strains con-
taining MepA, as well as block the flow of ethidium bromide (Kaatz
et al., 2005).
thyridin-2-yl)-benzenesulfonamide,
2,3,4-trifluoro-N-(5‑chloro-1,8-
and 3-trifluoromethyl-N-
naphthyridin-2-yl)-benzenesulfonamide
(
5‑chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide, possess a favor-
The 1,8-naphthyridines studied herein have in their radical struc-
tures capable of binding to the efflux pump structure, according to the in
silico data obtained. Compound 10d, among others analyzed, possesses a
larger number of substituent groups capable of binding MepA protein
active sites due to the presence of three fluorine substituent groups. It is
also important to emphasize that fluorine is the most electronegative of
the substituents and part of fluoroquinolones and quinolones composi-
tion, suggesting that this element may be responsible for the greatest
inhibitory effect of Compound 10d (Hooper, 2001; Pham et al., 2019).
Another compound that stood out was the Compound 10b. It is
possible to observe in its structure the presence of chloride, an element
that distinguishes it from other derivatives. It is known that chloride is
responsible for significant antibacterial activity. Therefore, it is sug-
gested that this ion may have influenced the observed action of com-
pound 10b (Dasari TP and Y, 2015; Gahtori et al., 2012; Share et al.,
able pharmacokinetic profile and interaction energy. Numerous and
diverse bonds have been established in the protein-naphthyridine
complex, and satisfactory ADME properties have been observed. The
in silico data corresponded to the microbiological data. Despite not
showing antibacterial activity, these heterocyclic compounds showed
synergistic effects when associated with antibiotics and ethidium bro-
mide, inducing a reduction in the minimum inhibitory concentration
and indicating inhibitory effects of the MepA efflux pumps. The naph-
thyridine was also able to increase the intensity of fluorescence emission
of ethidium bromide, reinforcing the hypothesis that these heterocyclic
compounds act as MepA inhibitors. Further tests involving antibacterial
activity and inhibition of different efflux pumps from other bacterial
strains are being developed. In addition, in vivo studies are necessary to
ascertain the safety and therapeutic window of these compounds.
2
016; Sławi n´ ski et al., 2013).
,8-naphthyridines are precursors of fluoroquinolones, considering
Author statement
1
that FQs are synthesized through modification of 1-alkyl-1,8-naphthyr-
idin-4-one3-carboxylic acid, which makes its use as an efflux pump in-
hibitor even more advantageous (Bryskier and Chantot, 1995; Lesher
Cícera Datiane de Morais Oliveira-Tintino, Saulo Relison Tintino,
D ´e bora Feitosa Muniz, Cristina Rodrigues dos Santos Barbosa and Rai-
mundo Luiz Silva Pereira – Methodology (bacterial assays);
1
2

C.D.M. Oliveira-Tintino et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105753
I ˆe da Maria Begnini, Ricardo Andrade Rebelo, Luiz Everson da Silva,
Sandro Lucio Mireski, Michele Caroline Nasato and Maria Isabel Laco-
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https://doi.org/10.1016/0002-9343(65)90100-2.
Alexandre Magno Rodrigues Teixeira, Humberto Medeiros Barreto
and Irwin Rose Alencar de Menezes – Methodology (Statistical and in
silico analysis)
Da Silva, L.E., Joussef, A.C., Nunes, R.J., De Oliveira, K.N., 2008. Synthesis of 1,8-
naphthyridine-4(1H)-one sulfonamides by thermolysis of 2-aminopyridinemethylene
(
Meldrum’s acid) derivative. Synth. Commun. https://doi.org/10.1080/
Pedro Silvino Pereira, Tereza Cristina Leal Balbino, Jos ´e Galberto
Martins da Costa and Fabiola Fernandes Galv ˜a o Rodrigues - Resources
Henrique Douglas Melo Coutinho and Teresinha Gonçalves da Silva –
Supervision and coordination of the project
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Conflict of interest
The authors declare no conflicts of interest.
El-Baky, R.M.A., Sandle, T., John, J., Abuo-Rahma, G.E.D.A., Hetta, H.F., 2019. A novel
mechanism of action of ketoconazole: inhibition of the nora efflux pump system and
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Acknowledgments
We thank Prof. Glenn Kaatz of Wayne State University for kindly
giving the strains. This study was funded, by the Foundation for Support
of Science and Technology of the State of Pernambuco (FACEPE), pro-
cess number IBPG-1241-4.03/16; National Council of Scientific and
Technological Development (CNPq) process 150456/2018-2 and
1
0.1021/co500120b.
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SAR and antibacterial activity of hybrid chloro, dichloro-phenylthiazolyl-s-triazines.
Saudi Pharm. J. https://doi.org/10.1016/j.jsps.2011.05.003.
4
06685/2018-5; Coordination for the Improvement of Higher Education
Personnel (CAPES), finance code 001; and Financier of Studies and
Projects (FINEP). The authors have no other relevant affiliations or
financial involvement with any organization or entity with a financial
interest in or financial conflict with the subject matter or materials
discussed in the manuscript apart from those disclosed.
Gibbons, S., Oluwatuyi, M., Kaatz, G.W., 2003. A novel inhibitor of multidrug efflux
pumps in Staphylococcus aureus. J. Antimicrob. Chemother. https://doi.org/
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2002. The synthesis of aromatic diazatricycles from phenylenediamine-bis
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