Antimicrobial activities of highly bioavailable organic salts and ionic liquids from fluoroquinolones

Article abstract of DOI:10.3390/pharmaceutics12080694

As the development of novel antibiotics has been at a halt for several decades, chemically enhancing existing drugs is a very promising approach to drug development. Herein, we report the preparation of twelve organic salts and ionic liquids (OSILs) from ciprofloxacin and norfloxacin as anions with enhanced antimicrobial activity. Each one of the fluoroquinolones (FQs) was combined with six different organic hydroxide cations in 93–100% yield through a buffer-assisted neutralization methodology. Six of those were isomorphous salts while the remaining six were ionic liquids, with four of them being room temperature ionic liquids. The prepared compounds were not toxic to healthy cell lines and displayed between 47-and 1416-fold more solubility in water at 25 and 37 ?C than the original drugs, with the exception of the ones containing the cetylpyridinium cation. In general, the antimicrobial activity against Klebsiella pneumoniae was particularly enhanced for the ciprofloxacin-based OSILs, with up to ca. 20-fold decreases of the inhibitory concentrations in relation to the parent drug, while activity against Staphylococcus aureus and the commensal Bacillus subtilis strain was often reduced. Depending on the cation–drug combination, broad-spectrum or strain-specific antibiotic salts were achieved, potentially leading to the future development of highly bioavailable and safe antimicrobial ionic formulations.

Full text of DOI:10.3390/pharmaceutics12080694

pharmaceutics
Article
Antimicrobial Activities of Highly Bioavailable
Organic Salts and Ionic Liquids
from Fluoroquinolones
1
,
2
, Joana Silva 2 , Catarina Florindo , Alexandra Costa ,
3
1
Miguel M. Santos * , Celso Alves
Željko Petrovski 1 , Isabel M. Marrucho 3 , Rui Pedrosa * and Luís C. Branco *
2,
1,
1
LAQV-REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de
Lisboa, 2829-516 Caparica, Portugal; amp.costa@campus.fct.unl.pt (A.C.); z.petrovski@fct.unl.pt (Ž.P.)
MARE–Marine and Environmental Sciences Centre, ESTM, Instituto Politécnico de Leiria,
2
3
2
520-641 Peniche, Portugal; celso.alves@ipleiria.pt (C.A.); joana.m.silva@ipleiria.pt (J.S.)
Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Avenida Rovisco Pais,
049-001 Lisboa, Portugal; catarina_florindo@hotmail.com (C.F.);
1
isabel.marrucho@tecnico.ulisboa.pt (I.M.M.)
*
Correspondence: miguelmsantos@fct.unl.pt (M.M.S.); rui.pedrosa@ipleiria.pt (R.P.);
l.branco@fct.unl.pt (L.C.B.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 1 June 2020; Accepted: 13 July 2020; Published: 23 July 2020
Abstract: As the development of novel antibiotics has been at a halt for several decades, chemically
enhancing existing drugs is a very promising approach to drug development. Herein, we report the
preparation of twelve organic salts and ionic liquids (OSILs) from ciprofloxacin and norfloxacin as
anions with enhanced antimicrobial activity. Each one of the fluoroquinolones (FQs) was combined
with six different organic hydroxide cations in 93–100% yield through a buffer-assisted neutralization
methodology. Six of those were isomorphous salts while the remaining six were ionic liquids,
with four of them being room temperature ionic liquids. The prepared compounds were not toxic
to healthy cell lines and displayed between 47- and 1416-fold more solubility in water at 25 and
◦
3
7 C than the original drugs, with the exception of the ones containing the cetylpyridinium cation.
In general, the antimicrobial activity against Klebsiella pneumoniae was particularly enhanced for
the ciprofloxacin-based OSILs, with up to ca. 20-fold decreases of the inhibitory concentrations in
relation to the parent drug, while activity against Staphylococcus aureus and the commensal Bacillus
subtilis strain was often reduced. Depending on the cation–drug combination, broad-spectrum or
strain-specific antibiotic salts were achieved, potentially leading to the future development of highly
bioavailable and safe antimicrobial ionic formulations.
Keywords: active pharmaceutical ingredients as organic salts and ionic liquids (API–OSILs);
antibiotics; ciprofloxacin; fluoroquinolones; ionic liquids; norfloxacin; polymorphism
1
. Introduction
Fluoroquinolones are a class of antibiotics primarily effective against Gram-negative bacteria.
Norfloxacin was the first fluoroquinolone to receive US Food and Drug Administration approval and
is currently used to treat urinary, biliary, and respiratory tract infections [ ]. One year later, in 1987,
ciprofloxacin was also approved for use in the US, and it was the most successful compound in its
generation as it showed 4–10 times more efficiency than its predecessor [ ]. This drug is very effective
1
2
in the treatment of a wide range of infections such as urinary tract infection, osteomyelitis (bone
infection), respiratory infections and some sexually-transmitted diseases (e.g., gonococcal and chronic
bacterial prostatitis) [3].
Pharmaceutics 2020, 12, 694; doi:10.3390/pharmaceutics12080694
www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2020, 12, 694
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Fluoroquinolones, like many other active pharmaceutical ingredients (APIs), display spontaneous
polymorphic conversion between distinct crystalline forms with a consequent change in pharmaceutical
properties [
4]. Crystal structures of neutral and zwitterionic forms of norfloxacin and ciprofloxacin
have been described by Nikaido and Thanassi, with considerable differences in their bioavailability [
5
].
In aqueous solutions, a dynamic equilibrium of several protolytic forms (anionic, neutral,
zwitterionic and cationic) can be found for both antibiotics due to the presence of distinct functional
groups that are prone to converse protonation/deprotonation in physiological media. Such behaviour
clearly influences their bioavailability, with neutral ciprofloxacin and norfloxacin displaying 0.086 and
◦
0
.37 mg/mL of solubility in water at 25 C, respectively [
6
]. The typical conversion to the halide salt
renders a considerable increase of two orders of magnitude in water solubility, with the ciprofloxacin
◦
hydrochloride salt reaching 38.4 mg/mL at 30 C [
7
]. However, it is still considered a poorly water
soluble drug.
According to the guidelines of the United States Pharmacopeia [
8
], solubility improvement
techniques of poorly water soluble active pharmaceutical ingredients can be categorised into physical
modification, such as chemical modifications of the drug substance, and other techniques. The formation
of organic salts of a drug is a mature and explored chemical modification technique.
For more than a decade, active pharmaceutical ingredients as organic salts and ionic liquids
(API–OSILs) have risen in academia as an alternative formulation for low bioavailable drugs [
9–12].
This third generation of ionic liquids [ ] consists on the combination of APIs as cations or anions
9
with organic counterions, thereby inducing distinctive physicochemical properties over the original
drugs and reduced toxicity to healthy cells, thus rendering a potentially enhanced pharmaceutical
activity in comparison to the API [13
e.g., -lactam (ampicillin [18 21], penicillin [22] and amoxicillin [22]) and fluoroquinolone [18
antibiotics, NSAIDs (ibuprofen [17 18], naproxen [18]), bone antiresorptive agents (zoledronic [24
–25]. Our works involving the preparation of API–OSILs from,
β
–
,
23]
,
]
and alendronic [25] acids), among others, have shown that the combination of an API, either as a
cation or as an anion, with suitable biocompatible counter-ions can increase the water solubility of
the parent drug and even change its biological effect [10
solubility in physiological media of the API can be significantly altered, yielding novel formulations
with different pharmacokinetics and potentially different delivery modes and applications [29 30].
In addition, the polymorphic tendency of an API can be considerably reduced or even eliminated
e.g., by attaining the liquid state at room temperature), hence tackling one of the most important
,17,23–28]. Furthermore, the stability and
,
(
issues in the pharmaceutical industry, which is responsible for limitations in a drug’s solubility and
dosage [17,23,24].
The formation of organic salts based on cationic fluoroquinolones (mostly comprising ciprofloxacin
(
Cip), but also norfloxacin (Nor)) has been widely explored by the academic community by protonation
of the drugs’ amine group(s). More precisely, these fluoroquinolones have been combined with
dicarboxylic acids (malonic and tartaric [31], adipic, fumaric [32] and maleic [33 34], succinic [35]),
citric acid [32], saccharine [36 37] and acesulfame [36], as well as with other drugs (diflunisal and
ibuprofen [38], salicylic [39] and barbituric acids [40]). Some works involve the combination of
,
,
ciprofloxacin or enrofloxacin as cations and/or as anions with amino acids [41] and ionic polymers [42,43]
While most works focus on the study of the chemical and physicochemical properties of the
prepared salts, including bioavailability, crystallinity/amorphous profiles and thermodynamic stability,
the amorphous–solid dispersions of ciprofloxacin and enrofloxacin showed, in addition, that the
antimicrobial activity of the salts is enhanced in comparison with the parent drugs, in particular against
Gram-negative bacteria strains.
.
In addition to these works, our group reported the synthesis and characterization of novel
API–OSILs from Cip and Nor as cations in combination with methanesulfonate (Mes), gluconate
(
Glu) and glycolate (Gly) biocompatible anions [23] (see Figure 1). The bioavailability of the prepared
fluoroquinolone-based salts (FQ–OSILs), expressed in terms of permeability (octanol/water and
phospholipid/water partition) and solubility in water and biological fluids, was highly tuneable

Pharmaceutics 2020, 12, 694
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according to the selected anion. Finally, toxicological studies on intestinal epithelial cell line models
Caco-2) revealed a lower inflammatory response for some of the FQ–OSILs in comparison with the
parent drugs.
In this work, we set out to explore for the first time the formation of FQ–OSILs based on Cip and
(
Nor as anions in combination with organic cation hydroxides [17,42] based on ammonium, pyridinium
and N-methylimidazolium moieties (Figure 1).
Figure 1. Structures of ciprofloxacin and norfloxacin organic acids used as anions in the previous work
and organic halide cations used in this work.
The newly synthesized FQ–OSILs were characterised by nuclear magnetic resonance (NMR)
(1
H and 13C) and Fourier-transform infrared (FTIR) spectroscopic techniques, elemental analysis, as
◦
well as differential scanning calorimetry (DSC). Water solubility at 25 and 37 C was experimentally
determined for the FQ–OSILs, with the exception of the ones containing the [C Py] cation, for which
16
the critical micelle concentration was measured. Finally, the antimicrobial activity of these anionic
FQ–OSILs on Gram-negative (Klebsiella pneumoniae) and Gram-positive (Bacillus subtilis, Staphylococcus
aureus) bacteria was determined.
2
. Results and Discussion
2
.1. Synthesis
Ciprofloxacin
Ch], 1-ethyl-3-methylimidazolium
C OHMIM], 1-(2-hydroxyethyl)-2,3-dimethylimidazolium
and
norfloxacin
were
[EMIM],
combined
as
anions
with
choline
[
[
1-hydroxy-ethyl-3-methylimidazolium
[C OHDMIM],
2
2
1
-(2-methoxyethyl)-3-methylimidazolium [C OMIM] and cetylpyridinium [C Py] cations
3
16
(see Scheme 1).

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According to our experience [17
,
24], the most efficient methodology for the combination of APIs
as anions with organic cations consists of the dropwise addition of the hydroxide salts of the cations
to the neutral API, leading to the sole formation of the desired API–OSILs and water. The cation
hydroxides are prepared immediately before their addition from the corresponding halide salts by
anion exchange from a hydroxide resin such as Amberlyst IRA-400 (OH). Whenever this methodology
is performed with zwitterionic APIs [19,22] such as fluoroquinolones, it is required that the reaction
proceeds in ammonia buffer solution so that complete ionisation can occur. The pure API–OSILs
were isolated in very high yields (93–100%) after recrystallisation from chloroform/methanol mixtures.
Scheme 1 shows the employed synthetic methodology.
Scheme 1. General synthetic methodology for the synthesis of anionic fluoroquinolone (FQ)-based
organic salts and ionic liquids (OSILs).
2
.2. Spectroscopic Characterisation
All products were characterised by H and 13C NMR and FTIR spectroscopic techniques, as well
as elemental analysis.
1
1
In all cases, the H NMR spectra showed that the cation/anion proportion is strictly 1.0:1.0,
in agreement with the intended stoichiometry (see Figures S1–S22). In addition, only one set of
signals was observed, meaning that the reactions were complete, and only one product was formed.
No comparison with parent ciprofloxacin and norfloxacin is achievable due to a lack of solubility in
the same solvent systems as the FQ–OSILs.
The FTIR spectra of the synthesised FQ–OSILs (Figures S23–S35) show only discrete differences in
characteristic signals in comparison with those from the parent fluoroquinolones. Typically, one would
expect that the deprotonation by the cation hydroxides would occur at the carboxylic acid group
forming a carboxylate group, which shows a very different wavenumber in comparison with the
initial group. However, this observation was not possible because fluoroquinolones are zwitterions,
and consequently, the carboxylic acid group is partially or totally in the form of carboxylate. In our
sample of initial ciprofloxacin, the obtained FTIR spectrum showed complete ionisation of the carboxylic
-
−1
acid group, with only the signal from the stretching vibration of O=C-O appearing at 1589 cm (see
Figure 2A).
In this case, the FTIR spectra of the Cip–OSILs showed slight changes in the corresponding
−1
−1
vibration band, more precisely between 1581 and 1575 cm (e.g., 1575 cm for [C OMIM][Cip]
3
in Figure 2B), thereby suggesting a change in the spatial vicinity of this group consistent with the
formation of the salts. In the case of norfloxacin, the obtained FTIR spectrum (Figure 2C) showed
−1
incomplete ionisation of the carboxylic acid, and thus two bands were observable at 1727 and 1583 cm
from the stretching vibration of the carboxylic and carboxylate groups, respectively. The spectra of

Pharmaceutics 2020, 12, 694
5 of 15
the Nor–OSILs showed complete disappearance of the signal from the former group, while the latter
−
1
−1
showed small changes to a range between 1583 and 1579 cm (e.g., 1579 cm for [C OMIM][Nor] in
3
−1
Figure 2D). Furthermore, the 3600–3300 cm zone always displays strong signals in the spectra of the
final compounds, as opposed to the initial ones, which suggests the establishment of H-bonds between
the cations and the anions.
Figure 2. FTIR spectra of (A) ciprofloxacin, (B) [C OMIM][Cip], (C) norfloxacin and (D) [C OMIM][Nor].
3 3
2
.3. Thermal Analysis
All prepared OSILs containing anionic ciprofloxacin and norfloxacin were studied by differential
scanning calorimetry (DSC; see Figures S36–S47). Table 1 contains the obtained data, namely,
melting and glass transition temperatures, as well as the physical state at room temperature of the
analysed compounds.
m g
Table 1. Physical state at room temperature, melting (T ) and glass transition (T ) temperatures of
ciprofloxacin, norfloxacin and corresponding OSILs.
◦
◦
Compound
Physical State
Tm/ C
Tg/ C
Ciprofloxacin
White solid
Pale yellow solid
White solid
White solid
White solid
White solid
Orange paste
322.0 [44]
111.2
92.9
111.6
197.5
112.4
-
-
-
[
Ch][Cip]
EMIM][Cip]
C OHMIM][Cip]
[
2
36.7
49.5
-
46.4
−10.7
[
[
C OHDMIM][Cip]
2
[
C OMIM][Cip]
3
[
C Py][Cip]
1
6
Norfloxacin
Ch][Nor]
EMIM][Nor]
C OHMIM][Nor]
White solid
Pale yellow solid
Yellow solid
Yellow paste
White solid
White paste
Yellow paste
217.0 [45]
94.5
119.9
[
54.8
64.6
41.1
65.1
44.1
5.9
[
[
-
2
[
C OHDMIM][Nor]
121.8
-
-
2
[
C OMIM][Nor]
3
[
C Py][Nor]
1
6
As expected, all compounds display lower melting temperatures than the parent fluoroquinolones.
Globally, from the twelve FQ–OSILs, one half are organic salts ([Ch][Cip], [EMIM][Nor],
[
C OHMIM][Cip], [C OHDMIM][Cip], [C OHDMIM][Nor] and [C OMIM][Cip]), while the other half
2 2 2 3
are considered ionic liquids ([Ch][Nor], [EMIM][Cip], [C OHMIM][Nor], [C OMIM][Nor], [C Py][Cip]
2
3
16
◦
and [C Py][Nor]) because their melting temperatures are, respectively, higher and lower than 100 C.
16
All solid compounds display only one melting temperature and are thus considered isomorphic salts.
◦
Moreover, the melting temperature range is comprehended between 92.9 and 197.5 C, respectively,
◦
registered for [EMIM][Cip] and [C OHDMIM][Cip], and was observed after one isotherm at 80 C
2

Pharmaceutics 2020, 12, 694
6 of 15
◦
◦
for 15 min, followed by a cooling cycle at 20 C/min until 90 C. In the cooling cycles following the
−
melting phenomena, the OSILs do not display crystallization temperatures, meaning that they become
supercooled salts, i.e., amorphous, after the first melt. This thermal behaviour is consistent with Type
II, as described by Domínguez et al. [46].
From the referred set of ionic liquids, [C OHMIM][Nor], [C OMIM][Nor], [C Py][Cip] and
2
3
16
[
C Py][Nor] were obtained as room temperature ionic liquids (RTILs), consistent with the observed
16
◦
glass transition temperatures (T
g
). Even though several cooling cycles at 10 and 20 C/min until
◦
−
90 C were performed, no endothermic events ascribable to melting phenomena were observed for
the RTILs, in agreement with Domínguez’s Type I classification [46]. Note that from the four RTILs,
three are norfloxacin-based, while only one contains ciprofloxacin. In fact, from the whole set of
prepared FQ–OSILs, the melting temperatures of the Nor-based OSILs are invariably lower than the
corresponding ones containing Cip. This observation is in line with the T
with Cip and Nor recording 322 and 217 C, respectively. Such considerable difference could not be
expected by simply observing both chemical structures, as they only differ at the N-alkyl substituent
m
of both free fluoroquinolones,
◦
(cyclopropyl versus ethyl) at the quinolone moiety. As OSILs, the three-dimensional packing of the
fluoroquinolones and additional specific interactions of these drugs with the selected ammonium,
methylimidazolium and pyridinium counterions [47] may account for the observed thermal behaviour
of the compounds. These observations will be further explored in future works, e.g., by X-ray diffraction
techniques, and published accordingly.
2
.4. Water Solubility Studies
The solubility of the prepared organic salts based on ciprofloxacin and norfloxacin in water was
determined by adding an excess amount of the compound to a fixed mass of water. Briefly, vials
were kept under vigorous stirring and controlled temperature, from which samples were collected at
different time periods to ensure the equilibrium was reached. After centrifugation, Cip and Nor were
quantified through UV–Vis spectroscopy techniques.
All synthesised FQ–OSILs present higher solubility in water than the parent fluoroquinolones,
either as free bases or as hydrochloride salts (only for ciprofloxacin). Figure 3 and Table S1 show the
◦
data obtained at 25 and 37 C.
2
2
1
1
70
20
70
20
2
5 ºC
7 ºC
3
7
2
0
0
1
0
◦
Figure 3. Water solubility at 25 (green) and 37 C (yellow) for ciprofloxacin, norfloxacin and
corresponding OSILs. Values represent mean
independent experiments.
±
standard error of the mean (SEM) of at least three
On a first observation, it is easily perceived that there is an enhanced increase in water solubility
of the ciprofloxacin-based OSILs in comparison with the ones containing norfloxacin. Additionally,
there is a direct correlation of the solubility with the temperature.

Pharmaceutics 2020, 12, 694
7 of 15
In both Nor- and Cip-based families of OSILs, the ones with the [EMIM] cation were found to be
◦
◦
the least soluble ones, yet increases of 47- and 246-fold at 25 C and of 72- and 343-fold at 37 C were,
respectively, observed. Conversely, the most soluble OSILs were obtained through the combination of
◦
the APIs with [C OHMIM], with enhancements between 219- and 897-times at 25 C, and 266- and
2
◦
1
416-times at 37 C, respectively, for the Nor- and Cip-containing OSILs.
The observed general tendency is in agreement with the hydrophilic character of the studied
cations: [EMIM] < [Ch] < [C OHDMIM]
≈
[C OMIM] < [C OHMIM]. This relative behaviour is in
3 2
2
agreement with the previously observed behaviour for ampicillin-based OSILs [20]. On the one hand,
the [EMIM] cation does not possess any polar group in its structure, thereby inhibiting its ability to
perform strong H-bonding interactions with the anions. On the other hand, the introduction of a
polar hydroxyl group at the end of the ethyl group linked to the imidazolium ring in [C OHMIM]
2
increased the water solubility by 3.6- and 4.7-times for the corresponding Cip- and Nor-based OSILs.
Changing the methylimidazolium moiety by an ammonium group, where the positive charge is more
spatially hindered, had a deleterious effect in the solubility of the salts, which decreased by 1.8- and
2
.8-times for [Ch][Cip] and [Ch][Nor], respectively, in comparison with the corresponding [C OHMIM]
2
ones. With the introduction of a methyl group between the methylimidazolium nitrogen atoms
[C OHDMIM]) or at the end of the hydroxyethyl side chain ([C OMIM]), intermediate hydrophilic
(
2
3
properties are attained, which directly correlates with the solubility in water of the corresponding Cip–
and Nor–OSILs.
In general, the anionic fluoroquinolone-based OSILs display a diminished enhancement of
the solubility in water in comparison with the cationic fluoroquinolone-based OSILs previously
reported [23]. In the latter group, with the exception of [Nor][Gly], the OSILS presented water solubility
values between ca. 200 mg/mL up to totally soluble. However, this solubility profile may be attributed
to the higher hydrophilic character of the chosen anions in comparison with the set of cations used in
this work, and not actually to the anionic or cationic state of the fluoroquinolones.
2
.5. Critical Micelle Concentration
The surfactant properties of the ionic liquids formed by the combination of the [C Py] cation with
16
both fluoroquinolones ([C Py][Cip] and [C Py][Nor]) were studied through the calculation of the
16
16
corresponding critical micelle concentrations (CMCs) via ionic conductivity measurements. This data
may be particularly relevant in order to develop novel drug delivery systems [48] for these FQ–OSILs.
Table 2 and Figure S48 show the obtained data for both OSILs, as well as for [C Py]Cl.
16
Table 2. Critical micelle concentrations (CMCs) of [C16Py]Cl, [C16Py][Cip] and [C16Py][Nor] in water
◦
at 25 C.
Compounds
C Py]Cl
CMC (mmol/L)
[
1.063 (1.067 [49]; 0.96 [50])
16
[
C Py][Cip]
C Py][Nor]
0.0816
0.0539
1
6
[
16
The CMC value obtained for [C Py]Cl is in good agreement with the value found in the open
16
literature [49
,50]. In comparison with [C Py]Cl, the calculated CMC values for [C Py][Cip] and
16 16
[
C Py][Nor] are two orders of magnitude lower and correspond to a decrease of 92% and 95%,
16
respectively. Despite the fact that smaller CMC values are expected for salts with organic anions in
comparison with inorganic ones, such a drastic decrease comes as a surprise. In our previous work
on ampicillin–OSILs [20], the combination of the antibiotic with the [C Py] cation yielded a CMC
16
value of 0.444 mM, which corresponds to a decrease of 58%. When comparing the three systems, we
conclude that the level of hydration of Cip and Nor is much lower than that of ampicillin, leading
to easier adsorption of these drugs on the surface of [C Py] micellar structures, thus decreasing the
16
repulsion between the polar groups at much lower concentrations in water.

Pharmaceutics 2020, 12, 694
8 of 15
2
.6. Cytotoxicity Studies
The toxicity of the prepared FQ–OSILs and corresponding starting materials, namely, starting
fluoroquinolones and halide salts, on 3T3 mouse fibroblasts was determined. Figure 4 plots the relative
T3 cell viability in the presence of the starting materials and the FQ–OSILs at 10 µM.
3
Figure 4. Cytotoxicity of free fluoroquinolones, halide salts and prepared FQ–OSILs on 3T3 cells
10 M) after treatment for 24 h. Values represent mean standard error of the mean (SEM) of at least
(
µ
±
three independent experiments carried out in triplicate. Symbols represent statistically significant
differences (ANOVA, Dunnett’s test, p-value < 0.05) when compared to control (*).
From the whole set of compounds, only the ones that contained the [C Py] cation were found to
16
reduce cell viability by more than 50% at the tested concentration. Therefore, dose–response assays
were carried out for these samples. The obtained results are plotted in Figure 5.
Figure 5. Dose–response curves of [C Py]-based salts (0.03–10
µM; 24 h) on 3T3 cells for IC50
1
6
determination. Values represent mean
experiments carried out in triplicate.
±
standard error of the mean (SEM) of at least three independent
The determined IC50 values for [C Py]Cl, [C Py][Cip] and [C Py][Nor] were, respectively, 5.46,
16
16
16
4
.72 and 5.17
µM. These values were expected due to the surfactant properties of the cation and are in
agreement with previous works [17,51].
2
.7. Antimicrobial Activity Studies
The antimicrobial activity (IC ) of the prepared FQ–OSILs and the corresponding starting materials
50
was determined against Gram-negative (Klebsiella pneumoniae) and Gram-positive (Staphylococcus aureus,
Bacillus subtilis) bacteria.

Pharmaceutics 2020, 12, 694
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Figure 6 plots the relative growth of (A) Klebsiella pneumoniae, (B) Staphylococcus aureus and (C)
Bacillus subtilis in the presence of the starting materials and the FQ–OSILs at 10 µM for 6 h.
Figure 6. Growth inhibition (
after exposure to the free fluoroquinolones, starting halide salts and prepared FQ–OSILs at 10
for 6 h. Values represent mean standard error of the mean (SEM) of at least three independent
A) Klebsiella pneumonia, (B) Staphylococcus aureus and (C) Bacillus subtilis
µM
±
experiments carried out in triplicate. Symbols represent statistically significant differences (ANOVA,
Dunnett’s test, p-value < 0.05) between the FQ–OSILs and control (*), Cip (δ) and Nor (#).

Pharmaceutics 2020, 12, 694
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Both free fluoroquinolones and all prepared FQ–OSILs inhibited more than 50% growth of all
bacteria strains at the tested concentration (10 M). From the set of starting halide cations, [C Py]Cl
was the only one that exerted an effective antimicrobial effect.
µ
16
Upon performing dose–response assays for the active compounds (see Figure S49),
the corresponding IC50 values were determined. Tables 3 and 4 present the gathered data for Cip- and
Nor-based OSILs (and the corresponding free fluoroquinolones), respectively. The data for the starting
cation halides are presented in Table S2.
Table 3. Antimicrobial activity (IC ) values (nM) and relative decrease of inhibitory concentrations
50
(
RDIC50) for ciprofloxacin and corresponding OSILs (0.001–10
µM) against the tested microorganisms
(in bold are the most relevant IC50 and RDIC50 values). The values in parentheses represent the
confidence intervals for 95%.
Compounds
K. pneumoniae
96.50
119.6–322.8)
1.12
38.44–67.99)
4.80
56.61–74.17)
6.42
32.63–40.66)
0.99
44.04–59.04)
7.61
39.64–57.19)
.88
4.21–23.17)
RDIC50
S. aureus
RDIC50
B. subtilis
RDIC50
1
29.16
(20.28–41.92)
3.84
(0.25–58.16)
Cip
(
(
(
5
3.8
3.0
181.4
(122.9–267.9)
0.2
20.60
(18.86–22.49)
0.2
0.2
[
Ch][Cip]
EMIM][Cip]
C2OHMIM][Cip]
OHDMIM][Cip]
C3OMIM][Cip]
C16Py][Cip]
6
24.24
(15.79–37.19)
1.2
16.17
(12.10–21.60)
[
3
5.4
3.9
61.95
(34.10–112.50)
21.20
0.5
15.75
(14.47–17.16)
12.35
0.2
0.3
[
(
(
5
1.4
[C
2
(18.03–24.93)
(8.31–18.35)
4
4.1
82.05
(48.99–137.40)
0.4
0.0
21.66
(17.12–27.40)
0.2
0.6
[
(
9
19.9
1430
(1140.0–1793.0)
6.07
(5.21–7.06)
[
(
Ciprofloxacin was found to be particularly active against B. subtilis (3.84 nM), followed by S. aureus
29.16 nM) and K. pneumoniae (196.5 nM), as expected.
(
The combination of this antibiotic with the selected cations has rendered very interesting results
against these bacteria strains, mostly K. pneumoniae, as similarly observed in previous works by
Tajber and co-workers [42 43]. More specifically, all Cip–OSILs showed increased activity against
this Gram-negative bacteria strain, with IC50 values comprehended between 9.88 ([C Py][Cip]) and
,
16
6
4.8 nM ([EMIM][Cip]), which correspond to a remarkable relative decrease of inhibitory concentrations
(
RDIC ) of 19.9 and 3.0, respectively. While the antimicrobial activity of the former OSIL may come
50
from an additive mechanism—because the [C Py] cation itself shows an IC value of 2.55 mM (see
16
50
Table S2)—the potency of the remaining OSILs appears to have a synergistic behaviour since none of
the cations display activity against this strain (or any of the others). In spite of [C Py][Cip] showing
16
toxicity against fibroblasts (see above), its IC50 value against K. pneumoniae is ca. 500-times lower
4.72 M vs. 9.88 nM), making this Cip–OSIL particularly interesting against this strain. Moreover,
C Py][Cip] showed almost no activity against S. aureus and also a 40% decrease against the commensal
(
µ
[
16
B. subtilis in comparison with ciprofloxacin. Hence, these results clearly suggest that [C Py][Cip] has
16
a very specific activity against K. pneumoniae over the remaining tested strains.
Of note is that B. subtilis is part of the human gut microbial ecosystem, whose perturbation may
lead to the proliferation of resistant bacterial pathogens [52]. In fact, all Cip–OSILs had particularly
reduced activities (20–30%) against B. subtilis in comparison with ciprofloxacin.
Regarding the pathogenic S. aureus, another Gram-positive bacteria strain, only [EMIM][Cip] and
[
C OHDMIM][Cip] showed a slight increase in antimicrobial activity, with RDIC values between 1.2
2
5
0
and 1.4.
In its turn, free norfloxacin shows that, similarly to ciprofloxacin, it is also more active against
Gram-positive bacteria (S. aureus and B. subtilis) than Gram-negative (K. pneumoniae) strains, but to a

Pharmaceutics 2020, 12, 694
11 of 15
smaller extent than its fluoroquinolone sibling. Table 4 shows this data, as well as the one observed for
the Nor-based OSILs.
Table 4. IC50 values (nM) and relative decrease of inhibitory concentrations for 50% activity (RDIC50
)
for norfloxacin and corresponding OSILs (0.001–10 M) against the tested microorganisms (in bold
µ
are the most relevant IC50 and RDIC50 values). The values in parentheses represent the confidence
intervals for 95%.
Compounds
K. pneumoniae
55.2
188.6–345.3)
99.4
179.9–221.0)
07.5
185.1–232.5)
01.0
182.6–221.1)
78.2
156.1–203.3)
24.5
189.5–266.0)
02.9
173.9–236.7)
RDIC50
S. aureus
RDIC50
B. subtilis
RDIC50
2
123.4
(71.29–213.5)
117.2
(97.70–140.60)
Nor
(
(
(
(
(
(
(
1
1.3
1.2
1.3
1.4
1.1
1.3
127.1
(85.17–189.60)
1.0
0.6
95.49
(80.53–113.2)
1.2
4.8
1.3
0.8
1.7
1.3
[
Ch][Nor]
EMIM][Nor]
C2OHMIM][Nor]
OHDMIM][Nor]
C3OMIM][Nor]
C16Py][Nor]
2
211.2
(152.6–292.3)
24.48
(19.36–30.94)
[
2
91.70
(54.08–155.5)
1.3
88.61
(81.35–96.51)
[
[
1
79.55
(69.24–91.40)
1.6
148.8
(126.1–175.7)
C
2
2
106.7
(70.13–162.5)
1.1
68.12
(56.55–82.06)
[
2
10.84
(2.89–40.67)
11.4
87.29
(76.04–100.2)
[
The most active Nor-based OSIL is, once again, the one containing the [C Py] cation, which
16
showed a relative increase of 11.4-times (IC50 = 10.84 nM) against S. aureus in comparison with the
free norfloxacin. This result is quite peculiar, as the analogous salt based on ciprofloxacin had similar
activity against the Gram-negative K. pneumoniae microbial strain. Against the latter, [C Py][Nor]
16
has shown only a 30% increase in activity, with an IC50 value of 202.9 nM, which is much higher than
the one recorded on S. aureus. However, it negatively affects commensal B. subtilis to a higher extent
than norfloxacin.
An alternative promising broad-spectrum formulation may be [C OHDMIM][Nor], which showed
2
a 60% increased activity against S. aureus and 40% against K. pneumoniae. Desirably, it also lost 20%
activity against commensal B. subtilis.
3
. Conclusions
The ciprofloxacin- and norfloxacin-based OSILs presented in this paper clearly demonstrate that,
by a simple and effective chemical transformation of standard fluoroquinolone drugs, it is possible
to enhance their bioavailability and mitigate their polymorphic profiles. These advantages, allied
to the possibility to modulate their antimicrobial spectrum while maintaining low toxicity towards
healthy cells, make these FQ–OSILs a very promising tool to prepare novel effective formulations for
these drugs.
In general, the prepared ciprofloxacin-based OSILs had much more encouraging antimicrobial
data in comparison with the norfloxacin ones, despite both sets having positive results. According to
the data regarding the three tested bacteria strains (K. pneumoniae, S. aureus and B. subtilis), we postulate
that it is possible to develop highly bioavailable tailored formulations of both fluoroquinolones for
applications against one or more pathogenic microbes with low toxicity against healthy cells and
commensal bacteria, depending on the chosen combination with organic cations. On the one hand,
the [C Py]-based OSILs containing ciprofloxacin and norfloxacin were found to be particularly
16
selective against different bacteria strains, respectively, K. pneumoniae and S. aureus, both at subtoxic
concentrations. Such distinct behaviour must surge from distinct interactions between the cation, the
drugs and the cell external membrane/wall of the bacteria, and will be studied in the future. In addition,
all formulations based on the [C Py] cation assembled into micelles, with possible applications in
16
novel drug delivery systems of the antibiotics.

Pharmaceutics 2020, 12, 694
12 of 15
Alternatively, highly water-soluble formulations with a selective spectrum of activity could
consist of [Ch][Cip], which was shown to be ca. 3.5-times more active to this strain than to
S. aureus. However, its IC50 values against K. pneumoniae are some of the lowest recorded (51.12 nM).
Hence, promising alternative formulations can consist of [C OHMIM]- and [C OMIM][Cip], which
2
3
demonstrated selectivity of ca. 1.7-times towards the same strain, with IC50 values of 36.43 and
7.61 nM, respectively. Moreover, both latter compounds are more water-soluble than [Ch][Cip] and
4
probably more bioavailable.
On the other hand, if a broad-spectrum antibiotic ionic formulation is sought, its combination
with [EMIM] or [C OHDMIM] is promising as they are highly active against both Gram-negative and
2
Gram-positive bacteria strains, in particular K. pneumoniae and S. aureus, respectively.
Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/12/8/694/s1:
1
13
Materials, experimental procedures, compound characterization data. Figures S1–S22: H and C NMR spectra
of FQ–OSILs. Figures S23–S35: FTIR spectra of FQ–OSILs. Figures S36–S47: DSC thermograms of FQ–OSILs.
Figure S48: Plot of ionic conductivity in function of the concentration of [C16Py][Cip] and [C16Py][Nor]. Figure S49:
Dose–response curves of ciprofloxacin, norfloxacin and corresponding FQ–OSILs on K. pneumoniae S. aureus and
B. subtilis for IC50 determination. Table S1: Solubility in water of ciprofloxacin, ciprofloxacin hydrochloride,
◦
norfloxacin and organic salts based in fluoroquinolones (mean and standard deviation) at 25 and 37 C. Table S2:
IC50 values (nM) for the starting halide cations (0.001–10 µM) against the tested microorganisms.
Author Contributions: Conceptualisation, L.C.B. and Ž.P.; methodology and investigation, M.M.S., C.A., J.S.,
C.F., and A.C.; validation, M.M.S., I.M.M., R.P., and L.C.B; writing—original draft preparation, M.M.S., C.A., and
C.F.; writing—review and editing, M.M.S., C.A., I.M.M., and L.C.B.; supervision, I.M.M., R.P., and L.C.B.; funding
acquisition, I.M.M., R.P., and L.C.B. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the Associate Laboratory for Green Chemistry LAQV and Centro
de Química Estrutural, which are financed by national funds from FCT/MCTES (UIDB/50006/2020 and
UID/QUI/00100/2013, respectively). The authors also thank Fundação para a Ciência e Tecnologia for the
projects PTDC/QUI-QOR/32406/2017, PEst-C/LA0006/2013, and RECI/BBBBQB/0230/2012, as well as one contract
under Investigador FCT (L. C. Branco).
Acknowledgments: The authors acknowledge Professor Madalena Dionísio for the use of the DSC equipment,
Bayer for the donation of ciprofloxacin and Solchemar for the cation halides.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
API
active pharmaceutical ingredient
API–OSILs
active pharmaceutical ingredient organic salts and ionic liquids
cetylpyridinium
C Py
1
6
C OHDMIM 1-(2-hydroxyethyl)-2,3-dimethylimidazolium
2
C OHMIM
1-(2-hydroxyethyl)-3-methylimidazolium
1-(2-methoxyethyl)-3-methylimidazolium
choline
2
C OMIM
3
Ch
Cip
ciprofloxacin
CMC
EMIM
FQ
critical micelle concentration
1-ethyl-3-methylimidazolium
fluoroquinolone
FQ-OSILs
FTIR
IC50
fluoroquinolone-based organic salts and ionic liquids
Fourier-transform infrared spectroscopy
half maximal inhibitory concentration
ionic liquid
IL
NMR
Nor
nuclear magnetic resonance
norfloxacin
OSIL
RDIC50
RTILsSEM
Tg
organic salt and ionic liquid
relative decrease in inhibitory concentration for 50% bacterial growth
room temperature ionic liquidsstandard error mean
glass transition temperature
Tm
melting temperature

Pharmaceutics 2020, 12, 694
13 of 15
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017 10, 1047–1053. [CrossRef]
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