Discovery of new effective N-alkyl-3,4-diarylmaleimides-based drugs for reversing the bacterial resistance to rhodamine 6G in Bacillus subtilis

Article abstract of DOI:10.1007/s11696-019-00992-7

Multidrug resistance (MDR) is a great concern worldwide. There is a great need to develop new drugs with the potential to attack target cells that show MDR phenotype. The purpose of this study was to assess the reversing effect of new N-alkyl-3,4-diarylma

Full text of DOI:10.1007/s11696-019-00992-7

Chemical Papers
https://doi.org/10.1007/s11696-019-00992-7
ORIGINAL PAPER
Discovery of new efective N‑alkyl‑3,4‑diarylmaleimides‑based drugs
for reversing the bacterial resistance to rhodamine 6G in Bacillus
subtilis
Claudia Leticia Mendoza‑Macías1 · Cesar Rogelio Solorio‑Alvarado2 · Angel Josabad Alonso‑Castro1 ·
Clara Alba‑Betancourt1 · Martha Alicia Deveze‑Álvarez1 · Felipe Padilla‑Vaca3 · Arturo Reyes‑Gualito1
Received: 3 May 2019 / Accepted: 8 November 2019
© Institute of Chemistry, Slovak Academy of Sciences 2019
Abstract
Multidrug resistance (MDR) is a great concern worldwide. There is a great need to develop new drugs with the potential to
attack target cells that show MDR phenotype. The purpose of this study was to assess the reversing efect of new N-alkyl-
3,4-diarylmaleimides on Bacillus subtilis resistant to rhodamine 6G as an indicator of its activity as modulators of efux
pumps and their additional potential as new antimicrobials. The efux pump modulator efects of N-alkyl-3-4-diarylmaleim-
ides were tested using the minimal inhibitory concentration (MIC) method on B. subtilis wild type and B. subtilis resistant
to R6G, as well as on MDR bacteria isolated from clinical samples (Escherichia coli, Staphylococcus aureus, Pseudomonas
aeruginosa, Enterococcus faecium, and Acinetobacter baumannii). In addition, their antimicrobial activity was evaluated on
clinical isolates. Five N-alkyl-3,4-diarylmaleimides showed the highest reversing activity on the resistance in the Bacillus
model as well as with the bacteria isolated from clinical samples. Antimicrobial activity was observed in N-alkyl-3,4-diar-
ylmaleimides against bacteria isolated from clinical samples. The results suggest that N-alkyl-3,4-diarylmaleimides have a
potential activity in reversing MDR phenotype and as antimicrobials and may be considered as a potentially molecules to
improve chemotherapy on resistant cells.
Keywords Bacillus subtilis · Multidrug resistance · N-alkyl-3 · 4-Diarylmaleimides · Antimicrobial
Introduction
Chemotherapy is one of the most common tools used for the
initial treatment of almost any disease. However, chemother-
apy can fail due to drug resistance developed by target cells.
Electronic supplementary material The online version of this
Acquired drug resistance can be present in eukaryotic cells
article (https://doi.org/10.1007/s11696-019-00992-7) contains
such as fungi or tumor cells, prokaryotic microorganisms
like bacteria, as well as fungi and protozoa such as Leish-
mania spp., Plasmodium falciparum, and Fasciola hepatica
(Wiese and Pajeva, 2001; Grácio et al., 2003; Teodori et al.,
2006). Multidrug-resistance (MDR) phenotype has been
described as the acquired resistance to a broad spectrum
of not related, structurally or functionally, pharmacological
targets (Fojo and Bates, 2003). The efux pump mediated
by P-glycoprotein (PgP), which reduces intracellular accu-
mulation of drugs, is the main mechanism involved in MDR
phenotype in cancer chemotherapy (Pokharel et al. 2017).
Bacterial resistance to antibiotics is the consequence of an
innate adaptation to the disfavored conditions generated by
chemotherapy or because of a response to the host defense
supplementary material, which is available to authorized users.
* Claudia Leticia Mendoza-Macías
cl.mendoza@ugto.mx
1
2
3
División de Ciencias Naturales y Exactas, Departamento
de Farmacia, Universidad de Guanajuato, Campus
Guanajuato¸Noria Alta S/N, 36050 Guanajuato, Guanajuato,
Mexico
División de Ciencias Naturales y Exactas, Departamento de
Química, Universidad de Guanajuato, Campus Guanajuato,
Cerro de la Venada S/N, 36040 Guanajuato, Guanajuato,
Mexico
División de Ciencias Naturales y Exactas, Departamento de
Biología, Universidad de Guanajuato, Campus Guanajuato,
Noria Alta S/N, 36050 Guanajuato, Guanajuato, Mexico
Vol.:(0123456789)
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Chemical Papers
mechanisms (Magiorakos et al. 2012). Bacterial MDR phe-
notype involves the participation of efux pumps, belonging
to the families MF, SMR, MATE, RND, and ABC, which
decrease the concentration of intracellular antibiotic (Du
et al. 2018). Several cell lines and bacterial models have
been used for the study of efux pumps in terms of genet-
ics, chemistry and biochemistry (Hebecker et al. 2015;
Xiong et al. 2017). Rhodamine 6G (R6G) is a small mol-
ecule with cationic and lipophilic properties, often used to
determine the transport activity of efux pumps due its abil-
ity to ingress through an active transport into cell. Because
of its toxic efects on B. subtilis at higher concentrations,
R6G induces efux pump activity in the cell, which may be
employed to obtain an MDR model on bacteria (Neyfakh
et al. 1991). The MDR model in Bacillus subtilis selected
through resistance to rhodamine 6G shows similarity to the
MDR phenotype in mammalian cells (Neyfakh, et al. 1991).
The overexpression of a protein called Bmr is responsible
for the MDR phenotype in B. subtilis. Therefore, bacteria
are also a model of MDR (Neyfakh 2002). Previously, we
showed that the 3,4-diarylmaleimide core is a promising
structure, in combination with doxorubicin, with cytotoxic
activity against primary breast cancer cells. The N-alkyl or
benzyl-substituted maleimides containing electron-donating
groups at the aryl ring showed the highest cytotoxic activ-
ity (Gutiérrez-Cano et al. 2017). The aim of this work was
to evaluate the use of new N-alkyl-3,4-diarylmaleimides
(Fig. 1) frst as modulators of efux pumps to reverse MDR
phenotype and second as potential new antimicrobials. Here,
we present reversion of resistance to rhodamine 6G induced
in Bacillus subtilis, as a bacterial model of MDR phenotype
as well as the antimicrobial activity on bacteria isolate from
clinical samples.
(MALDI and LDI). Melting points were determined using a
Büchi melting point apparatus.
The compounds 5–23 were synthesized via a cross-cou-
pling between the corresponding boronic acid to be installed
at 3,4-positions and N-alkyldibromomaleimide. For new
compounds, the spectroscopic full characterization (1H, 13
C
and HRMS) has been described. The spectroscopic data
match perfectly with those previously described (Gutiérrez-
Cano et al. 2017).
General procedure A
Alkylation of 3,4‑dibromo‑1H‑pyrrole‑2,5‑dione (1) In a
100 mL dry one-neck round bottom fask equipped with a
magnetic stir bar, 3,4-dibromomaleimide (1.00 equiv) was
added under N2 atmosphere. Acetone (0.62 M) and K2CO3
(1.10 equiv) were successively added and vigorously stirred
for 10 min. Then, the corresponding alkylating reagent
(1.20–1.40 equiv) was added to one portion. The reaction
was stirred during the overnight period and quenched by
solvent evaporation and extraction with AcOEt (3×20 mL).
The crude sample from the reaction was purifed by column
chromatography. The spectroscopic data match perfectly
with those previously described.
3,4‑Dibromo‑1H‑pyrrole‑2,5‑dione (2) Rf = 0.38 (20%
AcOEt/hexanes). Mp (°C)=113–114. IR (cm−1)=3234 (s),
3073 (w), 2696 (w), 2381 (w), 1783 (m), 1773 (m), 1727 (s),
1582 (m), 1497 (w), 1356 (w), 1329 (m), 1196 (w), 1172
(w), 1032 (s), 794 (m), 733 (m), 661 (m), 502 (m), 487 (m).
1H NMR (500 MHz, DMSO-d6) δ 11.68 (s, 1H). 13C NMR
(126 MHz, DMSO-d6) δ 165.2, 129.8. HRMS ESI (+) calc.
for C4HBr2NO2 [M+H]+: 253.8374; found: 253.8495.
3,4‑Dibromo‑1‑methyl‑1H‑pyrrole‑2,5‑dione (3) This com-
pound was synthesized in 75% using 1.40 equiv of dimethyl
sulfate according to the general procedure A, yielding a pure
yellow compound corresponding to the desired product.
Rf = 0.38 (10% AcOEt/hexanes). Mp (°C) = 114–116. IR
(cm−1)=1783 (m), 1705 (s), 1685 (w), 1595 (m), 1471 (w),
1435 (m), 1386 (m), 1266 (m), 1179 (w), 1164 (m), 996 (s),
821 (s), 728 (s), 420 (m). 1H NMR (500 MHz, CDCl3) δ 3.12
(s, 3H). 13C NMR (126 MHz, CDCl3) δ 164.1, 129.5, 25.6.
HRMS ESI (+) calc. for C5H3Br2NO2 [M+H]+: 267.8532;
found: 267.8650.
Experimental
General information of organic synthesis
All reactions were carried out under N2 in solvents dried
using a solvent purifcation system (SPS). Thin layer chro-
matography (TLC) was carried out using aluminum sheets
with 0.2 mm of silica gel (Merck Gf234). Chromatographic
purifcations were carried out using fash-grade silica gel
(SDS Chromatogel 60 ACC, 40–60 µm). Nuclear magnetic
resonance (NMR) spectra were obtained with a Bruker
Avance 400 Ultrashield and Bruker Avance 500 Ultrashield
apparatus. For some compounds, PENDANT NMR spectra
(polarization enhancement nurtured during attached nucleus
testing) are provided instead of standard 13C NMR spectra.
Mass spectra were recorded on a Waster LCT Premier Spec-
trometer (ESI and APCI) or on an Autofex Broker Daltonics
1‑Benzyl‑3,4‑dibromo‑1H‑pyrrole‑2,5‑dione (4) This com-
pound was synthesized in 77% using 1.20 equiv of benzyl
bromide according to the general procedure A, yielding a
pure yellow compound corresponding to the desired product.
Rf = 0.45 (10% AcOEt/hexanes). Mp (°C) = 112–113 °C.
IR (cm−1) = 1784 (m), 1720 (s), 1598 (s), 1437 (m), 1430
(m), 1391 (m), 1335 (m), 1102 (w), 1065 (m), 910 (m), 813
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Chemical Papers
Fig. 1 Structures of the N-alkyl-
3-4-diarylmaleimides (com-
pounds 1–24)
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Chemical Papers
(w), 753 (w), 729 (m), 700 (m), 631 (w), 501 (w). 1H NMR
(500 MHz, CDCl3) δ 7.37 (dd, J=7.8, 1.9 Hz, 2H), 7.35–
7.28 (m, 3H), 4.75 (s, 2H). 13C NMR (126 MHz, CDCl3)
δ 163.7, 135.4, 129.7, 129.0, 128.9, 128.5, 43.4. HRMS
ESI (+) calc. for C11H7Br2NO2 [M+H]+: 343.8844; found:
343.8975.
1‑Methyl‑3,4‑di‑p‑tolyl‑1H‑pyrrole‑2,5‑dione (10) 1H
NMR (500 MHz, CDCl3) δ 7.38 (d, J = 8.2 Hz, 4H), 7.15
(d, J = 8.0 Hz, 4H), 3.14 (s, 3H), 2.36 (s, 6H). 13C NMR
(126 MHz, CDCl3) δ 171.3, 140.1, 135.9, 129.9, 129.4,
126.2, 24.3, 21.6.
1‑Methyl‑3,4‑di(naphthalen‑2‑yl)‑1H‑pyrrole‑2,5‑dione
1
(11) H NMR (500 MHz, CDCl3) δ 8.22 (s, 2H), 7.85 (d,
General procedure B
J=7.9 Hz, 2H), 7.80 (d, J=7.9 Hz, 2H), 7.70 (d, J=8.6 Hz,
2H), 7.56–7.48 (m, 4H), 7.38 (dd, J=8.6, 1.6 Hz, 2H), 7.26
(s, 1H), 3.23 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 171.2,
136.2, 133.9, 133.3, 130.9, 129.1, 128.2, 127.9, 127.6,
126.7, 126.6, 25.5, 24.5.
The Suzuki cross‑coupling reaction
A dry Shlenck tube adapted with a magnetic stir bar was
successively charged with 3,4-dibromo-1H-pyrrole-
2,5-dione 1 (0.050 g, 0.186 mmol, 1.00 equiv), the corre-
sponding boronic acid (0.465 mmol, 2.50 equiv), K3PO4
(0.197 g, 0.93 mmol, 5 equiv) and Pd(PPh3)2Cl2 (0.0052 g,
0.0074 mmol, 4 mol%) under N2 atmosphere. Deoxygenated
1,4-dioxane (5.3 mL, 0.035 M) was added and the reaction
was heated to 90 °C by 3.5 h. Once the reaction was com-
plete judging by TLC, the reaction was quenched adding
10 mL of brine. The mixture was extracted with AcOEt
(3×15 mL). The collected organic phases were dried over
magnesium sulfate, evaporated to dryness and purifed by
column chromatography.
3,4‑Bis(4‑fluorophenyl)‑1‑methyl‑1H‑pyrrole‑2,5‑dione
1
(12) H NMR (500 MHz, CDCl3) δ 7.50–7.43 (m, 4H),
7.09–7.04 (m, 4H), 3.15 (s, 3H). 13C NMR (126 MHz,
CDCl3) δ 170.8, δ 163.7 (d, J=252.5 Hz), 135.2, 132.1(d,
J = 8.5 Hz), 124.7(d, J = 3.5 Hz), 116.21(d, J = 21.8 Hz),
24.47.
3,4‑Bis(3,4‑difuorophenyl)‑1‑methyl‑1H‑pyrrole‑2,5‑dione
1
(13) H NMR (500 MHz, CDCl3) δ 7.35 (ddd, J=11.1, 7.6,
2.0 Hz, 2H), 7.23–7.15 (m, 4H), 3.16 (s, 3H). 13C NMR
(126 MHz, CDCl3) δ 170.0, 152.77–150.58 (m), 151.70–
149.51 (m), 134.9, 126.7(dd, J = 6.6, 3.8 Hz), 125.0(d,
J = 6.4 Hz), 119.3(d, J = 18.9 Hz), 118.2(d, J = 17.7 Hz),
24.60.
1
1‑Methyl‑3,4‑diphenyl‑1H‑pyrrole‑2,5‑dione (5) H NMR
(500 MHz, CDCl3) δ 7.47 (d, J = 6.8 Hz, 4H), 7.40–7.32
(m, 6H), 3.16 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 171.0,
136.5, 129.98, 129.95, 128.8, 128.7, 24.4.
3,4‑Bis(3‑chloro‑4‑fluorophenyl)‑1‑methyl‑1H‑pyr‑
1
role‑2,5‑dione (14) H NMR (500 MHz, CDCl3) δ 7.60 (dd,
3,4‑Bis(4‑methoxyphenyl)‑1‑methyl‑1H‑pyrrole‑2,5‑dione
J = 7.0, 2.2 Hz, 2H), 7.31 (ddd, J = 8.6, 4.6, 2.2 Hz, 2H),
7.15 (t, J=8.6 Hz, 2H), 3.16 (s, 3H). 13C NMR (126 MHz,
CDCl3) δ 170.0, δ 159.2 (d, J = 255.0 Hz), 134.8, 132.4,
130.0 (d, J = 7.6 Hz), 125.3 (d, J = 4.2 Hz), 122.2 (d,
J=18.2 Hz), 117.4, 24.62.
1
(6) H NMR (500 MHz, CDCl3) δ 7.47 (d, J=8.9 Hz, 4H),
6.88 (d, J=8.9 Hz, 4H), 3.83 (s, 6H), 3.13 (s, 3H). 13C NMR
(126 MHz, CDCl3) δ 171.6, 160.9, 134.5, 131.5, 121.6,
114.3, 55.4, 24.3.
1
3,4‑Bis(3,4‑dimethoxyphenyl)‑1‑methyl‑1H‑pyrrole‑2,5‑di‑
one (7) H NMR (500 MHz, CDCl3) δ 7.21 (dd, J = 8.4,
1‑Benzyl‑3,4‑diphenyl‑1H‑pyrrole‑2,5‑dione (15) H NMR
1
(500 MHz, CDCl3) δ 7.49–7.43 (m, 6H), 7.38–7.29 (m, 9H),
4.81 (s, 2H). 13C NMR (126 MHz, CDCl3) δ 170.6, 136.6,
136.4, 130.1, 130.0, 129.0, 128.9, 128.8, 128.7, 128.0, 42.2.
2.0 Hz, 2H), 7.05 (d, J= 1.9 Hz, 2H), 6.86 (d, J= 8.4 Hz,
2H), 3.90 (s, 6H), 3.72 (s, 6H), 3.14 (s, 3H). 13C NMR
(126 MHz, CDCl3) δ 171.4, 150.5, 148.8, 134.4, 123.6,
121.6, 112.7, 111.1, 56.0, 55.9, 24.3.
1‑Benzyl‑3,4‑bis(4‑methoxyphenyl)‑1H‑pyrrole‑2,5‑dione
(16) H NMR (500 MHz, CDCl3) δ 7.49–7.46 (m, 4H),
1
3,4‑Bis(4‑chlorophenyl)‑1‑methyl‑1H‑pyrrole‑2,5‑dione
7.45 (d, J = 7.3 Hz, 2H), 7.33 (t, J = 7.3 Hz, 2H), 7.28 (t,
J = 5.0 Hz, 1H), 6.89–6.83 (m, 4H), 4.78 (s, 2H), 3.82 (s,
6H). 13C NMR (126 MHz, CDCl3) δ 171.1, 160.9, 136.8,
134.3, 131.6, 128.9, 128.8, 127.9, 121.51, 114.2, 55.4, 42.0.
1
(8) H NMR (500 MHz, CDCl3) δ 7.42–7.41 (d, 4H), 7.36–
7.34 (d, 4H), 3.16 (s, 3H). 13C NMR (126 MHz, CDCl3) δ
170.5, 136.5, 135.6, 131.3, 129.3, 127.0, 24.5.
3,4‑Bis(4‑bromophenyl)‑1‑methyl‑1H‑pyrrole‑2,5‑dione
(9 )1H NMR (500 MHz, CDCl3) δ 7.52–7.50 (d, 4H), 7.35–
7.33 (d, 4H), 3.15 (s, 3H). 13C NMR (126 MHz, CDCl3) δ
170.4, 136.7, 132.2, 131.4, 127.4, 125.0, 24.5.
1‑Benzyl‑3,4‑bis(3,4‑dimethoxyphenyl)‑1H‑pyrrole‑2,5‑di‑
1
one (17) H NMR (500 MHz, CDCl3) δ 7.46 (d, J=7.3 Hz,
2H), 7.37–7.28 (m, 3H), 7.20 (dd, J=8.4, 1.5 Hz, 2H), 7.05
(d, J=1.3 Hz, 2H), 6.85 (d, J=8.4 Hz, 2H), 4.79 (s, 2H),
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Chemical Papers
3.90 (s, 6H), 3.71 (s, 6H). 13C NMR (126 MHz, CDCl3)
δ 171.0, 150.6, 148.9, 136.8, 134.3, 128.9, 128.8, 127.9,
123.8, 121.7, 112.9, 111.2, 56.1, 57.0, 42.0.
three and N-alkyldibromomaleimide. The spectroscopic data
match perfectly with those previously described (Gutiérrez-
Cano et al. 2017).
1‑Benzyl‑3,4‑bis(4‑chlorophenyl)‑1H‑pyrrole‑2,5‑dione
(18) H NMR (500 MHz, CDCl3) δ 7.47–7.43 (m, 2H),
General procedure for the addition of amines
to 3,4‑dibromo‑1H‑pyrrole‑2,5‑dione 1
1
7.43–7.39 (m, 4H), 7.35 (d, J=1.8 Hz, 6H), 7.32–7.29 (m,
1H), 4.80 (s, 2H). 13C NMR (126 MHz, CDCl3) δ 170.0,
136.6, 136.3, 135.4, 131.3, 129.2, 129.0, 128.9, 128.1,
126.9, 42.3.
1‑Benzyl‑3‑(benzylamino)‑4‑bromo‑1H‑pyrrole‑2,5‑dione
1
(24) H NMR (500 MHz, CDCl3) δ 7.42–7.25 (m, 10H),
5.61 (s, 1H), 4.83 (d, J=6.4 Hz, 2H), 4.67 (s, 2H). 13C NMR
(126 MHz, CDCl3) δ 167.5, 165.8, 143.0, 137.0, 136.2,
129.1, 128.8, 128.6, 128.3, 128.0, 127.7, 47.0, 42.3.
1
1‑Benzyl‑3,4‑bis‑p‑tolyl‑1H‑pyrrole‑2,5‑dione (19) H NMR
(500 MHz, CDCl3) δ 7.47–7.44 (m, 2H), 7.39–7.36 (m, 4H),
7.35–7.31 (m, 2H), 7.32–7.28 (m, 2H), 7.14 (d, J=8.0 Hz,
4H), 4.79 (s, 2H), 2.35 (s, 6H). 13C NMR (126 MHz, CDCl3)
δ 170.9, 140.2, 136.7, 135.6, 129.9, 129.4, 129.0, 128.8,
127.9, 126.1, 42.1, 21.6.
3‑Bromo‑1‑phenethyl‑4‑(phenethylamino)‑1H‑pyr‑
role‑2,5‑dione (25) H NMR (500 MHz, CDCl3) δ 7.37–7.26
1
(m, 5H), 7.22 (dd, J=17.9, 7.5 Hz, 5H), 5.40 (s, 1H), 3.87
(q, J=7.0 Hz, 2H), 3.77–3.69 (m, 2H), 2.93 (t, J=7.2 Hz,
2H), 2.91–2.85 (m, 2H). 13C NMR (126 MHz, CDCl3) δ
165.8, 138.1, 137.5, 129.0, 128.9, 128.7, 127.2, 126.8, 64.6,
44.3, 40.0, 37.2, 34.9.
1‑Benzyl‑3,4‑bis(4‑fluorophenyl)‑1H‑pyrrole‑2,5‑dione
(20) 1H NMR (500 MHz, CDCl3) δ 7.47 (tt, J = 7.6,
2.1 Hz, 6H), 7.36–7.29 (m, 3H), 7.08–7.03 (m, 4H), 4.80
(s, 2H). 13C NMR (126 MHz, CDCl3) δ 170.4, δ 132.14
(d, J = 8.4 Hz), 164.76, 162.76, 136.43, 135.12, 128.95
(d, J=13.7 Hz), 128.12, 124.63 (d, J=3.8 Hz), 116.14 (d,
J=21.8 Hz), 42.26.
3‑Bromo‑1‑(3,4‑dimethoxyphenethyl)‑4‑((3,4‑dimethoxy‑
phenethyl)amino)‑1H‑pyrrole‑2,5‑dione (26) Rf=0.12 (30%
AcOEt/hexanes). 1H NMR (500 MHz, CDCl3) δ 6.86–6.68
(m, 6H), 5.41 (s, 1H), 3.87 (d, J = 1.7 Hz, 6H), 3.86 (d,
J=1.9 Hz, 3H), 3.84 (d, J=6.5 Hz, 3H), 2.90–2.78 (m, 4H).
13C NMR (126 MHz, CDCl3) δ 165.8, 149.4, 150.0, 148.3,
147.9, 130.5, 129.9, 120.98, 120.95, 112.1, 113.0, 111.8,
111.5, 56.1, 57.0, 56.0, 44.3, 41.0, 36.6, 34.3.
1‑Benzyl‑3,4‑bis(3,4‑difuorophenyl)‑1H‑pyrrole‑2,5‑dione
1
(21) H NMR (500 MHz, CDCl3) δ 7.47–7.42 (m, 2H),
7.39–7.35 (m, 3H), 7.34–7.30 (m, 2H), 7.22–7.14 (m, 4H),
4.79 (s, 2H). 13C NMR (126 MHz, CDCl3) δ 169.6, 152.77–
150.56 (m), 151.70–149.43 (m), 136.12, 134.71, 129.02,
128.96, 128.27, 126.75 (dd, J=6.5, 3.8 Hz), 125.05–124.88
(m), 119.33 (d, J=18.9 Hz), 118.16 (d, J=17.7 Hz), 42.43.
Bacterial strains and culture conditions
Bacillus subtilis (ATCC 33608), used as wild strain, was
grown in antibiotic medium no. 3 (Difco, Detroit, MI), sup-
plemented with 0.4% glucose at 37° C and 180 rpm. The
resistant cultures were selected with rhodamine 6G (R6G)
(Sigma, St. Louis, MO) as previously described by Neyfakh,
et al. (1991). Briefy, bacteria were grown in liquid antibi-
otic medium no. 3 supplemented with R6G at 0.5, 1, 2, 3,
and 4 μg/mL, consecutively. The cultures were diluted 1:100
with fresh medium and R6G every 24–48 h. The concentra-
tion of R6G was increased every four changes of medium.
MDR bacteria isolated from clinical samples (Escherichia
coli, Staphylococcus aureus, Pseudomonas aeruginosa,
Enterococcus faecium, and Acinetobacter baumannii) were
collected from a High Specialty General Hospital of Bajío
(Leon, State of Guanajuato, Mexico) and were stored in
nutritive medium with 10% of glycerol at −70 °C until fur-
ther use. For experiments, clinical isolates were grown in
blood agar base (Difco, Detroit, MI) for 18 h at 37 °C.
After 18-h incubation, bacterial cultures were washed
with saline solution, centrifuged at 1200 rpm for 5 min, and
the optical density (OD) was adjusted at 0.04 at 630 nm in
1‑Benzyl‑3,4‑bis(3‑chloro‑4‑fuorophenyl)‑1H‑pyrrole‑2,5‑di‑
1
one (22) H NMR (500 MHz, CDCl3) δ 7.61 (dd, J=7.0,
2.2 Hz, 2H), 7.46–7.42 (m, 2H), 7.35 (dd, J=8.1, 6.2 Hz,
2H), 7.32–7.29 (m, 3H), 7.13 (t, J = 8.6 Hz, 2H), 4.79 (s,
2H). 13C NMR (126 MHz, CDCl3) δ 169.6, δ 159.3 (d,
J = 255.0 Hz), 136.1, 134.6, 132.4, 130.0(d, J = 7.6 Hz),
128.99, 128.96, 128.3, 125.3(d, J = 4.3 Hz), 122.2(d,
J=18.1 Hz), 117.3(d, J=21.6 Hz), 42.4.
1‑Benzyl‑3,4‑di(naphthalen‑2‑yl)‑1H‑pyrrole‑2,5‑dione
1
(23) H NMR (500 MHz, CDCl3) δ 8.26–8.21 (m, 2H),
7.87–7.78 (m, 4H), 7.69 (d, J = 8.6 Hz, 2H), 7.52 (dtt,
J=9.4, 7.9, 1.3 Hz, 6H), 7.40–7.32 (m, 5H), 4.88 (s, 2H).
13C NMR (126 MHz, CDCl3) δ 170.7, 136.6, 136.0, 133.9,
133.2, 131.0, 129.1, 129.0, 128.9, 128.2, 128.1, 127.9,
127.6, 126.7, 126.6, 126.3, 42.3.
The compounds 24–26 were synthesized between the
corresponding alkylamines to be installed at the position
1 3

Chemical Papers
antibiotic medium 3 (Bacillus subtilis) or Mueller–Hinton
broth (Difco, Detroit, MI) (bacteria isolated from clinical
samples). For assays of inhibitor/modulator compounds,
bacteria received a pretreatment for 15 min at room tem-
perature in D3G medium or Mueller–Hinton broth with
N-alkyl-3,4-diarylmaleimides, verapamil (25 μg/mL), or
reserpine (5 μg/mL).
isolates. All experiments were performed in triplicate and
MICs were determined as mentioned above.
Results and discussion
N‑alkyl‑3,4‑diarylmaleimides modulation of R6G
resistance in B. subtilis
R6G sensitivity assay
To assess N-alkyl-3,4-diarylmaleimides as modulators of
bacterial resistance, sub-inhibitory concentrations of these
compounds were used as inhibitors of R6G resistance in
B. subtilis. Almost all compounds, with exception of 8, 13,
14, 16, 17, and 25, have potential to reverse and/or modu-
late the resistance to R6G on B. subtilis. Some compounds
(5–7, 9–12, 15, 18, 24, and 26) decreased in a concentration-
dependent manner the MIC values, from 8 μg/mL to 4 or 2
μg/mL (Table 1), on B. subtilis resistant to rhodamine 6G.
All compounds exerted (5–7, 9–12, 15, 18, 24, and 26)
promising reversal ability (Table 2). The highest efect was
observed with compounds 5, 9, 10, 11, and 21, with the
Fresh cultures of resistant strain of B. subtilis were inocu-
lated for 18 h, washed three times with saline solution, and
the OD was adjusted at 0.04 at 630 nm in the antibiotic
medium no. 3. Bacterial suspensions were inoculated into
96-well plates containing serial double dilutions in D3G
medium of R6G (1.0–64.0 µg/mL) The minimal concentra-
tion of the drug with inhibitory efect on bacterial growth
(MIC) was determined after 18 h of incubation at 37 °C,
180 rpm and the OD at 0.630 nm. The assays were per-
formed two times by duplicate.
Determination of antimicrobial activity
Table 1 The efect of N-alkyl-3,4-diarylmaleimides derivatives on
rhodamine 6G resistance on B. subtilis
The antibacterial activity for ciprofloxacin or N-alkyl-
3,4-diarylmaleimides was determined as the MIC by the
microplate method specified in the M07-A8 document,
using a cation-adjusted Mueller–Hinton broth (Clinical and
Laboratory Standard Institute (CLSI) 2012). Briefy, bac-
terial strains were grown overnight, and the inoculum of
the test organisms was prepared in normal saline (9 g/L)
and adjusted to 5×105 UFC/mL. A stock solution of cipro-
foxacin or N-alkyl-3,4-diarylmaleimides was prepared in
DMSO (Sigma) and further diluted in Mueller–Hinton Broth
to reduce DMSO concentration to 1%. One hundred micro-
liters of stock solution was dispensed into the frst well and
serially diluted into each well with 100 µL of Mueller–Hin-
ton Broth added with 100 µL of the standardized bacterial
inoculum to give a fnal concentration that ranged between
0.03125 and 2.0 µg/mL for ciprofoxacin and 4–256 µg/mL
for N-alkyl-3,4-diarylmaleimides. All procedures were per-
formed in triplicate. The plates were incubated at 37 °C for
18 h. The MIC was recorded as the lowest concentration at
which no visible growth was observed.
Compound
Rhodamine 6G MIC (μg/mL)
Treatment (μg/mL)
0
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
2.5
8
8
8
8
2
2
2
4
8
8
8
8
8
8
4
4
4
4
8
4
8
4
5
8
8
8
8
2
2
2
4
8
8
8
8
8
4
4
4
2
4
8
4
8
4
10
2
4
8
8
2
ND
2
4
8
8
4
8
8
4
4
4
2
20
2
4
4
8
2
ND
2
4
8
8
4
8
8
ND
4
4
2
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Bacterial resistance modulation assay
4
8
ND
8
4
4
ND
8
For evaluation of the N-alkyl-3,4-diarylmaleimides as modu-
lators of the R6G resistance or antibiotic resistance, MICs of
R6G and ciprofoxacin were determined in the presence or
absence of sub-inhibitory concentrations of N-alkyl-3,4-dia-
rylmaleimides: 2.5–20 µg/mL for R6G resistance and 10 µg/
mL for ciprofoxacin resistance against B. subtilis or clinical
4
Ethanol was used as solvent control
ND not determined
1 3

Chemical Papers
Table 2 Drug-resistance reversal ability of selected N-alkyl-3,4-diar-
ylmaleimides in Bacillus subtilis
recognize largely overlapping spectra of substrates and
share many common inhibitors (Neyfakh et al. 1991), thus
N-alkyl-3,4-diarylmaleimides derivatives may potentially act
on ABC transporter P-glycoprotein and contribute to revert
the resistance in a broad spectrum of molecules mediated
by this efux pump, as shown in R6G resistance in Bacillus.
Therefore, further experiments were carried out with
these fve compounds. There are two main characteristics
to be highlighted with regard to the chemical structures of
the compounds 5, 9, 10, 11 and 21. The frst one is that they
have a highly non-polar character based upon their aromatic
substituents (phenyl, p-bromophenyl, p-tolyl, 2-naphthyl
and 3,4-difuorophenyl, respectively) at positions 3,4 of the
maleimide core. Remarkably, this low-polar characteris-
tic enables the lipophilicity of the molecule allowing the
penetration into the cell membrane. The second important
characteristic is that the oxygen atoms of the two-carbonyl
groups in the lactamic moiety of maleimide acts as a Brøn-
sted base, displaying the ability to react with Brønsted acids,
mainly protons.
Treatment
MIC (μg ml−1
)
RF
Rhodamine 6G
8
2
4
4
2
2
2
4
4
4
4
4
4
2
4
4
4
4
4
4
1
4
2
2
4
4
4
2
2
2
2
2
2
4
2
2
2
2
2
2
+10 μg ml−1
+10 μg ml−1
+20 μg ml−1
+2.5 μg ml−1
5
6
7
9
+2.5 μg ml−1 10
+2.5 μg ml−1 11
+2.5 μg ml−1 12
+10 μg ml−1 15
+5 μg ml−1 18
+2.5 μg ml−1 19
+2.5 μg ml−1 20
+2.5 μg ml−1 21
+5 μg ml−1 21
+2.5 μg ml−1 22
+20 μg ml−1 23
+2.5 μg ml−1 24
+2.5 μg ml−1 26
+5 μg ml−1 reserpine
+25 μg ml−1 verapamil
Antibacterial activity on clinical isolates of bacteria
The potential use of 5, 9, 10, 11, and 21 was assessed on
reversal resistance and antibacterial activity on clinical iso-
lates of MDR bacteria, such as Escherichia coli, Staphy-
lococcus aureus, Pseudomonas aeruginosa, Acinetobacter
baumannii, and Enterococcus faecium. Compound 9 showed
good antibacterial activity against S. aureus and E. faecium
with lower MIC values, compared to those found with cip-
rofoxacin (Table 3). Compound 10 showed discreet anti-
bacterial activity against P. aeruginosa, but showed a lower
MIC value on S. aureus, compared to that found with cipro-
foxacin (Table 3). MDR in clinical isolates is an important
health problem to solve and it is a priority for World Health
Organization (Cabibbe and Cirillo 2016). All clinical iso-
lates evaluated here represent bacteria with MDR phenotype
(data no show) obtained from a tertiary-care hospital and are
The reversal fold (RF) value was calculated as the ratio between the
R6G MIC with and without treatment
lowest concentration in the treatment (2.5 µg/mL) and the
highest reversal fourfold (Table 2). The reversal efect found
with these compounds was comparable with that found for
verapamil and reserpine, which are PgP inhibitors (Table 2),
highly suggesting a modulation efect on Bacillus efux
pump Bmr. It was previously reported that PgP inhibitors
should decrease in threefold their MIC value in the reversal
assay (Chang et al. 2006). The compounds 5, 9, 10, 11 and
21 met this criterion. The multidrug transporters in Gram-
positive bacteria Bmr and ABC transporter P-glycoprotein
Table 3 Antibacterial efect of
Compound
MIC (μg/mL)
EC-291-URO PA- 255-URO SA-107-EXP SA-176-HQ AB-44-PLEU EF-68-URO
N-alkyl-3,4-diarylmaleimides
derivatives on clinically isolated
bacteria
5
9
10
11
>128
>128
>128
>128
>128
16
>128
>128
32
>128
>128
16
>128
>128
16
>128
>128
0.5
>128
32
>128
>128
>128
>128
>128
64
>128
8
>128
>128
>128
128
2
>128
>128
256
21
Ciprofoxacin
EC-291-URO Escherichia coli from urine, PA-255-URO Pseudomonas aeruginosa from urine, SA-
107-EXP Staphylococcus aureus from expectoration, SA-176-HQ Staphylococcus aureus from surgical
wound, AB-44-PLEU Acinetobacter baumannii from pleural liquid, EF-68-URO Enterococcus faecium
from urine
1 3

Chemical Papers
considered in the global priority list of antibiotic-resistant
bacteria to guide research, discovery, and development of
new antibiotics according to the WHO (Bush et al., 2011).
An important antimicrobial activity was observed for com-
pounds 9 and 10 on bacteria from clinical isolates. A com-
pound with an MIC value less than 10 μg/mL is considered
as an excellent antimicrobial agent (Gibbons et al. 2003).
The MIC values obtained for S. aureus (compound 10) and
E. faecalis (compound 9) are under 10 μg/mL, which rep-
resent potentially antimicrobial compounds. Further inves-
tigation is necessary to establish clinical correlation with
in vitro susceptibility observed in this work. The compound
9, 3,4-bis(4-bromophenyl)-1-methyl-1H-pyrrole-2,5-dione,
was previously reported as a modulator of cell proliferation
inhibition (2.4 mM and RF=2.77) in the presence of doxo-
rubicin (Gutiérrez-Cano et al. 2017). These data agree with
the results presented in this work. The results here presented
are in agreement with the antimicrobial activity shown
on substituents on phenyl rings with for 3,4-bis(arylthio)
maleimides (Panov, et al. 2019). Therefore, the fndings sug-
gest a potentially inhibitory activity of compound 9 on efux
pumps.
related to the multidrug-resistance phenotype in A. bauman-
nii: the RND superfamily, as well as the SMR, MATE, and
MFS families. Particularly, the quinolone resistance shown
in A. baumannii is mainly mediated by the RND superfam-
ily (Leus et al. 2018). Reversing ciprofoxacin resistance in
A. baumannii using efux pump inhibitors such as carbonyl
cyanide m-chlorophenylhydrazone has been previously
reported (Ardebili et al. 2014). A. baumannii resistant to
multiple drugs causes infections that are extremely dif-
cult to treat. In consequence, new efux pump inhibitors
that produce a reduction in MIC for antibiotics may result
in an alternative to treat these bacteria. The efux pump
in A. baumannii belongs to the resistance-nodulation-cell
division (RND) family and has a three-component struc-
ture: AdeA is the membrane fusion protein, AdeB is the
multidrug transporter, and AdeC is the outer membrane
protein. The adeABC operon is strongly regulated by a two-
component system (AdeR–AdeS): AdeS is a sensor kinase
(Ardebili et al. 2014). Compound 10 and 11 may inhibit
phosphorylation thorough AdeS, responsible for regulation
of adeABC operon.
The putative mechanisms in the inhibition of RND efux
pumps by compounds 10 and 11 may be due to their activ-
ity as protonophores. These two compounds contain in their
structure activated oxygen atoms at the carbonyls of the
maleimide fragment. Compounds 9 and 10 may transport
protons through the cellular membrane against the concen-
tration gradient, inactivating the energy source of the efux
pump, or by their putative activity as kinase inhibitors. Com-
pounds 10 and 11 are aromatic compounds, in consequence
hydrophobic and able to penetrate the cellular membrane.
Nevertheless, they can catch and stabilize protons in both
carbonyl groups at maleimide moiety.
Modulation of ciprofoxacin resistance on clinical
isolates of bacteria
Only compounds 10 and 11 decreased the MIC value in
Acinetobacter baumannii, resistant to ciprofoxacin, from
64 µg/mL to 32 µg/mL (Fig. 2). The MIC value found in
these two compounds was lower compared to that found
with reserpine.
A. baumannii has become one of the most emergent
pathogens in the healthcare environment due its impressive
ability to acquire high resistance to almost any antimicro-
bial agent clinically available (Lee et al. 2017). As in other
Gram-negative bacteria, four classes of efux pumps are
Molinspiration analysis
The Molinspiration analysis of the theoretical, physico-
chemical and bioactive properties for compounds 5, 10,
11, and 21 is presented in Table 4. The physicochemical
properties and drug likeness were analyzed as previously
reported (Gutiérrez-Cano et al. 2017) and compound 5 and
10 showed the highest scores in terms of prediction of good
membrane penetration, good drug absorption including
intestinal absorption, blood–brain barrier penetration as well
as favorable bioavailability. All predicted data highly suggest
that compounds 5 and 10 would be potentially inhibitors of
efux pumps such as PgP through their putative activity as
kinase inhibitors.
70
64
64
64
64
64
60
50
40
30
20
10
0
32
32
no
treatment
reserpine
5
9
10
11
21
TREATMENT
Nevertheless, experimental evidence is necessary to dem-
onstrate their activity as kinase inhibitors as predicted in the
Molinspiration analysis and as a protonophores to clarify
their mechanism of action in modulation of resistance. Com-
pounds 9 and 10 have a predicted physicochemical property
Fig. 2 Efect of N-alkyl-3-4-diarylmaleimides on ciprofoxacin resist-
ance on clinical isolate of Acinetobacter baumannii. MIC (µg/mL) for
Acinetobacter baumannii after treatment with compound 5, 9, 10, 11,
and 21 (10 µg/mL) and reserpine (25 µg/mL)
1 3

Chemical Papers
Table 4 Molinspiration analysis
of theoretical physicochemical
and bioactive properties of
Comp
MW (g/mol)
Physicochemical properties
Drug-likeness score
O+N
OH+NH
cLog P
TPSA
Nviol
KI
NRL
EI
N-alkylmaleimide derivatives
5
263.3
3
3
3
3
0
0
0
0
3.23
4.13
5.6
39.08
39.08
39.08
39.08
0
0
1
1
0.27
0.24
0.33
0.23
0.05
0.08
0.15
0.15
0.05
−0.02
0.09
10
11
21
291.35
363.42
411.35
5.04
0.13
MW molecular weight, O+N number of hydrogen bond acceptors, OH+NH number of hydrogen bond
donors, cLog P calculated log p value, TPSA total polar surface area, Nviol number of violation, KI kinase
inhibitor, NRL nuclear receptor ligand, EI enzyme inhibitor
baumannii clinical isolates. Ann Lab Med 34:433–438. https://
doi.org/10.3343/alm.2014.34.6.433
compatible with a good lipophilicity and good membrane
penetration (Table 4). However, no antimicrobial activity
was observed for Gram-negative bacteria with an outer
membrane.
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a promising biological activity as efux pump inhibitors
against eukaryotic and prokaryotic efux pump, such as
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structural factor and may infuence the biological activities
shown in these compounds. Prediction of physicochemical
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Acknowledgements The authors thank the Directorate for Research
Support and Postgraduate Programs (University of Guanajuato) for the
fnancial support granted to Dra. Claudia Leticia Mendoza Macías and
for the grant CIIC-054 -2018 of Convocatoria Institucional de Investi-
gación Científca 2018. We gratefully acknowledge Aidee Guadalupe
Aguilar Granados, Claudia Teresa Carranza López and Sarahí Mont-
serrat García Miranda for their contribution in antimicrobial assays
for this work.
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Compliance with ethical standards
Conflict of interest There are no conficts of interest to declare.
Hebecker S, Krausze J, Hasenkampf T, Schneider J, Groenewold M,
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bacterial resistance factors mediating tRNA-dependent ami-
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