Drug resistance modulation in staphylococcus aureus, a new biological activity for mesoionic hydrochloride compounds

Article abstract of DOI:10.3390/molecules16032023

Two salts of the mesoionic compounds 1,4-diphenyl-5-(5-nitro-2-furanyl)-1, 3,4- thiadiazolium-2-thiol chloride (MC-1) and 4-phenyl-5-(5-nitro-2-furanyl)-1, 3,4- thiadiazolium-2-phenylamine chloride (MC-2) were synthesized utilizing 1,4-diphenylthiosemicarbazide and 5-nitro-2-furoyl chloride as starting materials. Their structures were characterized by IR, ¹H-NMR, ¹³C-NMR and elemental analysis. These compounds were analyzed for their influence on the effectiveness of norfloxacin, tetracycline, and erythromycin (standard antibiotics) against resistant strains of Staphylococcus aureus. MC-1 and MC-2, at sub-inhibitory concentrations of 16 μg/mL, favourably modulated the antibiotic activity of tetracycline by 16- and 32-fold, respectively (MIC), and that of erythromycin by 4-fold.

Full text of DOI:10.3390/molecules16032023

Molecules 2011, 16, 2023-2031; doi:10.3390/molecules16032023
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Drug Resistance Modulation in Staphylococcus Aureus, a New
Biological Activity for Mesoionic Hydrochloride Compounds
Cledualdo Soares de Oliveira 1, Vivyanne dos Santos Falcão-Silva 3, José Pinto Siqueira-Júnior 3,
David Peter Harding 1, Bruno Freitas Lira 1, Jorge Gonçalo Fernandes Lorenzo 2,
José Maria Barbosa-Filho 2 and Petrônio Filgueiras de Athayde-Filho 1,*
1
Department of Chemistry, Federal University of Paraíba, 58059, João Pessoa, PB, Brazil;
E-Mails: aldoscarchi@yahoo.com.br (C.S.D.O.); kauaii@yahoo.com (D.P.H.);
brunofrlira@hotmail.com (B.F.L.)
2
Laboratory of Pharmaceutical Technology, Federal University of Paraíba, 58051, João Pessoa, PB,
Brazil; E-Mails: jgflorenzo@hotmail.com (J.G.F.L.); jbarbosa@ltf.ufpb.br (J.M.B.-F.)
3
Department of Molecular Biology, Federal University of Paraíba, 58059, João Pessoa, PB, Brazil;
E-Mails: jpsiq@uol.com.br (J.P.S.-J.); vivyannefalcao@yahoo.com.br (V.D.S.F.-S.)
* Author to whom correspondence should be addressed; E-Mail: athayde-filho@quimica.ufpb.br;
Tel.: +55-83-3216-7937.
Received: 25 January 2011; in revised form: 19 February 2011 / Accepted: 22 February 2011 /
Published: 28 February 2011
Abstract: Two salts of the mesoionic compounds 1,4-diphenyl-5-(5-nitro-2-furanyl)-1,3,4-
thiadiazolium-2-thiol chloride (MC-1) and 4-phenyl-5-(5-nitro-2-furanyl)-1,3,4-
thiadiazolium-2-phenylamine chloride (MC-2) were synthesized utilizing 1,4-diphenyl-
thiosemicarbazide and 5-nitro-2-furoyl chloride as starting materials. Their structures were
1
13
characterized by IR, H-NMR, C-NMR and elemental analysis. These compounds were
analyzed for their influence on the effectiveness of norfloxacin, tetracycline, and
erythromycin (standard antibiotics) against resistant strains of Staphylococcus aureus.
MC-1 and MC-2, at sub-inhibitory concentrations of 16 μg/mL, favourably modulated the
antibiotic activity of tetracycline by 16- and 32-fold, respectively (MIC), and that of
erythromycin by 4-fold.
Keywords: mesoionic compounds; modulation of drug resistance; efflux pump inhibitor

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1. Introduction
During the last few years, mesoionic compounds (MCs) have attracted the attention of chemists
because of the bonding aspects of their unusual structures, being mesomeric heterocyclic betaines,
strongly stabilized by  electron delocalization, and large dipole moments [1]. Due to their inherent
structural characteristics, mesoionic compounds have aroused research interest in photonics, nonlinear
optical studies [2-8] and medicinal chemistry, where a wide range of biological activities have been
discovered, as summarized in several review articles [9-11]. They can be biologically active, for
example, mesoionic 2-(4-chlorophenyl)-3-methyl-4-(4-methoxyphenyl)-1,3-thiazolium-5-thiolate
induces vasorelaxation [12].
Biological studies have shown that hydrochlorides of mesoionic 1,3,4-thiadiazolium-2-thiolate
compounds are active against Gram-positive and Gram-negative bacteria. Pharmacological effects at
the cellular level (involving relaxation in smooth muscle tissue due to a confirmed locking mechanism
for calcium channel blockers), were observed for 1,3-diphenyl-5-(5-nitro-2-furanyl)-1,3,4-triazolium-
2-thiol chloride [13,14]. This locking mechanism is of great interest because it might represent a tool
against multi-drug efflux pumps [15,16].
The resistance acquired by Staphylococcus aureus to virtually all antibiotics currently in use is of
considerable concern. Resistant strains like IS-58, which has specific efflux proteins (TetK) that
reduce the intracellular concentrations of tetracycline, have caused thousands of deaths, largely
through bloodstream infections. Efflux pumps are known to be the main mechanism of bacterial
resistance. Their function is to decrease intracellular drug concentration by return “pumping” the
drug(s) to the extra cellular medium, allowing the bacteria to survive [17,18].
Efflux pumps are integral membrane proteins of bacteria that extrude antibiotics and other
antimicrobial agents from the cell. They can be specific to a given compound or class of compounds or
capable of removing a variety of structurally unrelated antimicrobial compounds. Resistance
modification is the desired effect (to overcome or modulate bacterial resistance by reducing efflux
pump activity). Efflux inhibition can restore the antibiotic’s activity against the bacteria through
co-administration [16,17].
In this study we describe the novel structure of a mesoionic compound hydrochloride MC-2. The
results presented here represent the first report of derivatives of this class (heterocyclic betaines) as
putative efflux pump modulators. The present findings indicate that 1,4-diphenyl-5-(5-nitro-2-furanyl)-
1,3,4-triazolium-2-thiol chloride (MC-1), and 4-phenyl-5-(5-nitro-2-furanyl)-1,3,4-thiadiazolium-2-
phenylamine chloride (MC-2), could serve as a source of heterocyclic compounds that modulate
bacterial resistance, when used as adjuvants of antibiotics.
2. Results and Discussion
The merits of this research are in the discovery of two hydrochlorides representative of the
mesoionic heterocycles class capable of inhibiting the bacterial resistance of Staphylococcus aureus
against tetracycline and erythromycin. The compound MC-2 is a novel structure and was obtained by
the reaction of 5-nitro-2-furoyl chloride with 1,4-diphenylthiosemicarbazide. However, worth
mentioning is that from this same reaction we also obtained the isomer MC-1, which was first
synthesized by Athayde-Filho et al. in 1996 [14]. Although one synthesis is used for both compounds–

Molecules 2011, 16
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1,3,4-triazolium-2-thiol and 1,3,4-thiadiazolium-2-phenylamine–each desired compound is easily
obtained alone under controlled conditions. Thus, MC-1 is a thermodynamic product obtained under
anhydrous conditions in a closed system and in the presence of pyridine (route i, Scheme 1) while the
kinetic product MC-2 is obtained under anhydrous conditions in a closed system (route ii, Scheme 1).
Under relatively humid conditions in an open system (route iii, Scheme 1), a mixture of isomers can be
obtained and separated by column chromatography. MC-1 can also being obtained from MC-2 via
alkaline isomerization (route iv, Scheme 1) [19,20].
Scheme 1. Procedure to obtain MC-1 and MC-2 from the chloride 5-nitro-2-furoyl and
1.4-diphenylthiosemicarbazide.
O
SOCl2
N
C
S
+
O2N
NH NH2
+
OH
O
H
O
S
N
O2N
N
+
Cl
O
N
H
2
1
i
H
ii
iii
Cl
Cl
N
N
N
N
iv
H
5
5
2
2
N
SH
S
N
O
O
O2N
O2N
MC-1
MC-2
Reagents and Conditions: (i) Py, 1,4-dioxane anhydrous, closed system, rt; (ii) 1,4-dioxane
anhydrous, closed system, rt; (iii) 1,4-dioxane humidity, open system, rt; (iv) Py, isomerization.
MC-2 is easily differentiated from its isomer MC-1 by the usual physical methods of organic
compound characterization. Absorption bands around 3,400 cm−1 with deformations related to the NH
1
side chain appear in the infrared spectra of MC-2, which are confirmed by H-NMR via a singlet at
1
around 13.00 ppm. In fact, although H-NMR studies are not very informative when there are no
hydrogens in the heterocyclic ring, as in this case, they are however important for the detection of
hydrogens in substituent groups. In contrast 13C-NMR spectroscopy is an important tool for
characterizing structures of mesoionic compounds and their derivatives due to the large differences in
electronic densities between the two regions (HOMO and LUMO) of the heterocyclic ring [21,22].
This difference of electronic density manifests itself through positive and negative shielding effects for
the carbons of the heterocyclic ring and of the carbons of aromatic or aliphatic groups linked to the
mesoionic ring. The C2 and C5 carbons have chemical shifts that distinguish the mesoionic isomers.
For 1,3,4-triazolium-2-thiol type compounds (i.e., MC-1), the chemical shifts of C2 and C5 appear
around 169.8 and 148.2, respectively, while for the 1,3,4-thiadiazolium-5-phenylamine type
compounds (i.e., MC-2) the chemical shifts of C2 and C5 are centered around 160 and 165 ppm [19,20].

Molecules 2011, 16
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These results are consistent with the assignments of chemical shifts for MC-1 and corroborate the
assignments of carbons made for MC-2 [14,23].
MC-2 is an air stable solid, whose molecular formula was confirmed by spectroscopic data
(1H-, 13C-NMR and infrared spectroscopy) which was in complete agreement with the proposed
structures. The main absorption bands in the infrared spectrum: 3,444 N–H); 1,620 C=C); 1,574
C=N); 1,538 and 1,345 cm−1 ( symmetric and asymmetric). The 1H-NMR spectrum of MC-2 shows twelve

aromatic hydrogen signals between 7.93 and 6.67 ppm, while doublets integrating for two hydrogens
each at 7.44 and 6.73 ppm with a coupling constant of 2.8 Hz are assigned to the furan ring and a
singlet at 13.20 ppm to the phenylalanine NH hydrogen.
13
The C-NMR study of MC-2 showed fourteen chemical shifts. Of these, six were assigned to the
quaternary carbons and eight to the tertiary aromatic carbons. The carbons of the MC-2 mesoionic ring
have chemical shifts at 166.0 and 160.5 ppm, respectively, assigned to C2, C5. The benzene aromatic
carbons have chemical shifts of 138.1, 137.6, 132.5, 130.5, 129.5, 125.7, 124.4 and 120.7 ppm, and the
carbons of the furan ring have chemical shifts of 106.3, 118.5, 139.3 and 150.9 ppm; the last carbon is in
a low field due to deshielding caused by the nitro group.
The synthesis and study of these mesoionic compound derivatives MC-1 and MC-2 expanded and
we obtained the following data regarding bacterial resistance through in vitro microbiological assays:
the compounds MC-1 and MC-2 alone showed relevant antibacterial activity against Staphylococcus
aureus. Comparative studies of the MIC of tetracycline, erythromycin, and norfloxacin, with MC-1
and MC-2, show that both heterocyclic compounds are active (MIC 64) against IS-58, less potent than
tetracycline (MIC 32), however, against RN-4220-1, MC-1 was twice as active as MC-2, yet both are
substantially more active than erythromycin (MIC 256); neither showed greater activity against
SA-1199B than norfloxacin (Table 1).
Table 1. MIC values (µg/mL) of test strains for antibiotics and hydrochloride of mesoionic
compounds.
Strain (efflux protein) Tetracycline Erythromycin Norfloxacin MC-1
MC-2
64
64
IS-58 (TetK)
32
-
-
256
-
-
-
64
32
64
RN-4220 (MrsA)
SA-1199B (NorA)
-
64
64
However, the second group of assays for bacterial resistance showed an unexpected result.
Hydrochlorides of mesoionic compounds MC-1 and MC-2 enhanced the activity of tetracycline and
erythromycin by greatly reducing the concentration necessary to inhibit the growth of drug resistant
(effluxing) strains. When the MC-1 or MC-2 compounds were incorporated into the growth medium
at 16 µg/mL (¼ MIC), a four-fold reduction in the MIC was observed for erythromycin, and for
tetracycline the MIC was reduced by 16 and 32-fold when promoted by MC-1 and MC-2, respectively
(Table 2). No reduction in MIC was observed for norfloxacin, indicating that the mesoionic
compounds do not act as inhibitors of the NorA efflux protein. All experiments were carried out in
duplicate with consistent results.

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Table 2. MIC values (µg/mL) of test strains for antibiotics in the absence and presence of
MC-1 and MC-2 (16 µg/mL).
Antibiotic MIC µg/mL with (*)MIC reduction
factor in the presence of MC-1/MC-2(16 µg/mL)
Strain /(efflux protein)/Antibiotic (MIC
µg/mL)
Antibiotic + MC-1
2 (16 *)
Antibiotic + MC-2
1 (32 *)
IS-58/(TetK)/Tetracycline (32)
RN-4220/(MrsA)/ Erythromycin (256)
SA-1199B/(NorA)/Norfloxacin (64)
64 (4 *)
64 (0 *)
64 (4 *)
64 (0 *)
(X*)—MIC reduction factor.
Mesoionic compounds are planar five-membered ring betaines, which contain distinct regions of
positive and negative charges associated with any poly-heteroatomic system, yet have a net neutral
electrical character. These properties enable them to cross cellular membranes and interact strongly
with biomolecules [24]. Hydrochlorides of the mesoionic compounds MC-1 and MC-2 effectively
enhanced the antibiotic activity of tetracycline and erythromycin by reducing the concentrations
needed to inhibit the growth of drug resistant (effluxing) strains. A lipophilic character, affecting
membrane solubility, while common among efflux pump inhibitors, is likely to influence efflux
transporter binding [18], and partial polarity interactions with the efflux transporter amino acid
backbones, could combined to produce these results, however both potency modulation and
antibacterial activity may be related to the nitro compounds present [25].
Besides being lipophilic, salts of mesoionic compounds have also been shown to block ion channels,
see MC-1, a potent calcium and potassium channel blocker [14]. Markham and Neyfach [26] observed
tetracycline-specific transporters and show that B. subtilis TetL and S. aureus Tetk, transport
monovalent cations Na+ and K+ (genetic knockout of these transporters causes an increase in
tetracycline sensitivity, and reduction in high salinity media growth). Further assessment of the
biological mechanisms involved would be valuable seeing that calcium channel blockers are able to
inhibit multidrug transporters or P-glycoprotein (Pgp) [27].
3. Experimental
3.1. General
Infrared spectra were obtained by means of a IFS66 Bruker spectrometer with the samples in KBr
1
discs. H- and 13C-NMR spectra were recorded on a Varian Unity Plus 200 MHz spectrometer
operating at 200 MHz for 1H and 50 MHz for 13C, the sample being dissolved in DMSO-d6 with TMS
as reference. Elemental analysis was carried out using a Perkin Elmer Elemental Microanalyser. The
melting points were determined using a Kofler hot-plate apparatus combined with a Carl-Zeiss
microscope and are uncorrected.
5-Nitro-2-furoyl chloride (1). To a 50 mL flask (protected from moisture by a calcium chloride trap),
containing 5-nitro-2-furoic acid (0.50 g, 3.20 mmol) with two drops of dimethylformamide was added
rapidly thionyl chloride (0.5 mL). The mixture was heated to 60 ºC for 3 h and then the excess of
thionyl chloride was removed in a rotavapor to afford 5-nitro-2-furoyl chloride (yield: 0.41 g, 73.30%)

Molecules 2011, 16
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in the form of a yellowish liquid. Being very reactive the acidic chloride was not purified further and
was used immediately in the next step of synthesis.
1,4-Diphenylthiosemicarbazide (2). To phenylhydrazine (12 g, 0.11 mol) dissolved in anhydrous
toluene (50 mL) was added phenylisothiocyanate (15 g, 0.11 mol) previously dissolved anhydrous
toluene (30 mL) with mechanical stirring. The reaction was heated to 90 °C and after 10 min the
product precipitated from the reaction medium. Agitation was continued for a further 120 min, and the
mixture allowed to cool to room temperature and filtered under reduced pressure. The precipitate was
washed with anhydrous toluene (30 mL) and then with anhydrous ethyl ether (20 mL) and dried under
reduced pressure. We obtained 24.8 g (91.9%) of 1,4-diphenylthiosemicarbazide in the form of white
acicular crystals, m.p. 175–176 °C (lit. 175–176 ºC, [22]). 13C-NMR 181.0, 148.0, 139.0, 129.0, 127.8,
125.1, 124.9, 119.8, 113.0.
1,4-Diphenyl-5-(5-nitro-2-furanyl)-1,3,4-triazolium-2-thiol chloride (MC-1). To a solution of 1,4-
diphenylthiosemicarbazide (0.57 g, 2.3 mmol) in moist dioxane (10 mL) was added piridine (2.3 mmol).
After 3 h 5-nitro-2-furoyl chloride (0.41 g, 2.3 mmol, previously dissolved in 5 mL of anhydrous
dioxane) was slowly added. After 2 h stirring the reaction mixture was placed in the freezer for 96 h
and a precipitate of orange-yellow crystals was formed, (0.4 g, m.p. 263–264 ºC, 45.0%) which was
vacuum filtered, and washed with ethanol (10 mL) and cold ether (5 mL) and then air dried. Anal.
Calcd for C18H13ClN4O3S: C 54.00, H, 3.25; N, 14.00. Found: C, 53.50, H, 3.20; N, 14.30. IR (KBr):
1
3150, 3040, 2714, 1572, 1351 cm−1. H-NMR (200 MHz, DMSO-d6):  = 8.10–7.20 (m, 12H
13
aromatic), 4.00 (s,1H, sulphydryl). C-NMR (50 MHz, DMSO-d6):  = 167.8, 152.7, 146.5, 136.6,
132.3, 130.6, 127.7, 133.8, 125.7, 130.6, 127.7, 141.8, 108.7, 114.5.
4-Phenyl-5-(5-nitro-2-furanyl)-1,3,4-thiadiazolium-2-phenylamine chloride (MC-2). To a solution of
1,4-diphenylthiosemicarbazide (0.57 g, 2.3 mmol) in anhydrous dioxane (10 mL) was added slowly
5-nitro-2-furoyl chloride (0.41 g, 2.3 mmol, previously dissolved in 5 mL of anhydrous dioxane). After
the addition of the acyl chloride, there was the formation of a yellowish-red solution without warming
or gas formation and after a period of 24 h of shaking in a closed system a precipitate of orange-yellow
crystals formed (0.6 g, m.p. 261–262 ºC, 66.7%) which were vacuum filtered, and washed with
anhydrous dioxane (10 mL) and cold anhydrous ether (5 mL) and then air dried. Anal. Calcd for
C18H13ClN4O3S: C 54.00, H, 3.25; N, 14.00. Found: C, 53.55, H, 3.30; N, 14.30. IR (KBr): 3444,
1
3053, 1537, 1345 cm−1. H-NMR (200 MHz, DMSO-d6):  = 13.20 (s, 1H, NH), 7.93–6.73 (m, 12H
aromatic). 13C-NMR (50 MHz, DMSO-d6):  = 166.0, 160.5, 150.7, 139.3, 138.1, 137.6, 132.5, 130.5,
129.5, 125.7, 124.4, 120.7, 118.5, 106.3.
3.2. Biological Activity
The heterocyclic compounds MC-1 and MC-2 were screened for their biological activities in vitro
against three effluxing strains of Staphylococcus aureus: 1) SA-1199B, which overexpresses the norA
gene encoding the NorA efflux protein (resisting fluoroquinolones and others); 2) RN4220, harboring
the pUL5054 plasmid, which carries the gene encoding the MsrA macrolid efflux protein; 3) IS-58,
which possesses the TetK tetracycline efflux protein [28-31]. All strains, kindly provided by Professor
Simon Gibbons (University of London), were maintained on blood agar base (Difco) slants and, prior
to use (assay), the cells were grown overnight at 37 °C in brain heart infusion broth (BHI, Difco).

Molecules 2011, 16
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Norfloxacin, erythromycin and tetracycline were obtained from Sigma Chemical Co., USA. The
stock solutions of the antibiotics were prepared according to CLSI guidelines [32]. The stock solution
of MC-1 and MC-2 was prepared in DMSO, and its highest concentration remaining after dilution into
broth (4%) caused no inhibition of bacterial growth.
The minimum inhibitory concentrations (MICs) of the antibiotics and mesoionic compounds were
determined in BHI by the microdilution assay using a suspension of ca. 105 cfu/mL and a drug
concentration range of 256–0.5 g/mL (two-fold serial dilutions). For better visualization of bacterial
growth (after 24 h) we used resazurin (0.01%). MIC is defined as the lowest concentration at which no
growth is observed. For the evaluation of mesoionics as modulators of drug resistance, “modulation
assay” was used, a method that has been widely applied to identify potential EPIs, and is valid
provided one uses specifically known effluxing strains. The MICs of the antibiotics were determined
in the presence of the mesoionic compound at a sub-inhibitory concentration [33-35].
4. Conclusions
In this study we describe the synthesis and characterization of the novel structure 4-phenyl-5-(5-
nitro-2-furanyl)-1,3,4-thiadiazolium-2-phenylamine hydrochloride (MC-2). Salts of the mesoionic
compounds MC-1 and MC-2 show significant biological action, both alone and as adjuvants to
common antibiotics, notably affecting Staphylococcus aureus resistance to tetracycline, erythromycin.
Considering the known singular nature of bacterial resistance to these antibiotics, the results open new
perspectives for both MC-1 and MC-2 as potential antibiotic adjuvants, and suggest these compounds
putatively act as bacterial efflux pump inhibitors. These results also demand further investigation to
certify whether these mesoionic derivatives act exclusively through efflux pump inhibition to leverage
standard antibiotics against therapy-resistant Staphylococcus aureus.
Acknowledgements
The authors thank the Brazilian National Research Council (CNPq) for research grants and bursaries.
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