Novel Organic Salts Based on Mefloquine: Synthesis, Solubility, Permeability, and In Vitro Activity against Mycobacterium tuberculosis

The development of novel pharmaceutical tools to efficiently tackle tuberculosis is the order of the day due to the rapid development of resistant strains of Mycobacterium tuberculosis. Herein, we report novel potential formulations of a repurposed drug, the antimalarial mefloquine (MFL), which was combined with organic anions as chemical adjuvants. Eight mefloquine organic salts were obtained by ion metathesis reaction between mefloquine hydrochloride ([MFLH][Cl]) and several organic acid sodium salts in high yields. One of the salts, mefloquine mesylate ([MFLH][MsO]), presented increased water solubility in comparison with [MFLH][Cl]. Moreover, all salts with the exception of mefloquine docusate ([MFLH][AOT]) showed improved permeability and diffusion through synthetic membranes. Finally, in vitro activity studies against Mycobacterium tuberculosis revealed that these ionic formulations exhibited up to 1.5-times lower MIC values when compared with [MFLH][Cl], particularly mefloquine camphorsulfonates ([MFLH][(1R)-CSA], [MFLH][(1S)-CSA]) and mefloquine HEPES ([MFLH][HEPES]).


Introduction
Tuberculosis (TB) remains a major public health concern and currently is the leading cause of human death by an infectious disease. According to the World Health Organization, around 10 million people were infected with the Mycobacterium tuberculosis in 2018 and 1.5 million deceased [1]. The emergence and spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains is particularly alarming. The conventional treatment for MDR-TB may take up to 24 months and only 56% of the cases are successfully treated [2][3][4]. Therefore, the development of new TB agents that result in shorter and more effective treatments is an urgent need.
Drug repurposing has emerged as an important tool in the fight against TB [5][6][7]. This strategy is characterized by identifying new applications for approved or underinvestigation drugs that are different from their initial scope [8][9][10][11]. The main advantage is that the pharmacological properties of the substance, such as tissue distribution, maximum serum concentration, and metabolism pathways, are already described, and the drug's the ionic species led to an improvement in the antimicrobial activities, particularly of the β-lactam-based OSILs. These salts showed activity against resistant strains of Escherichia coli and Staphylococcus aureus in comparison with the parent APIs, with minimum inhibitory concentration values similar to the ones found against the susceptible strains.
Hence, considering the potential of MFL for the development of new treatments against TB and the emergence of API-OSILs as an important strategy to achieve improved pharmaceutical properties of drugs, herein, we present our work on organic salts based on MFL as an improved formulation of this drug to tackle M. tuberculosis.

Materials and Methods
Commercially available reagents were purchased from Sigma-Aldrich, Alfa Aesar, and TCI. Solvents were purchased from LabChem. 1 H and 13 C NMR spectra in (CD 3 ) 2 SO (from Euriso-Top) were recorded on a Bruker AMX 400 spectrometer at room temperature unless specified otherwise. To perform NMR, 5 mm borosilicate tubes were used and the sample concentration was, approximately, 7 mg/mL to 1 H NMR and 30 mg/mL to 13 C NMR. Chemical shifts are reported upfield in parts per million (ppm) in reference to the residual nondeuterated solvent. The elemental analysis experiments were performed in a Thermo Finnigan-CE Instruments Flash EA 1112 CHNS series under standard conditions (T combustion reactor 900 • C, T GC column furnace 65 • C, multiseparation SS GC column, He flow 130 mL/min, and O 2 flow 250 mL/min) at the REQUIMTE Analysis Lab, Departamento de Química Faculdade de Ciências e Tecnologia. FTIR spectra of the samples were recorded on a Perkin-Elmer FT-IR Spectrometer Spectrum Two (Waltham, MA, USA), equipped with an attenuated total reflection (ATR) cell in the range of 4000-400 cm −1 . DSC thermal studies were carried out using a TA Instruments Q-series TM Q2000 DSC with a refrigerated cooling system. The sample was continuously purged with a 50 mL/min nitrogen flow. About 5 to 10 mg of each FQ-OSIL was crimped in an aluminum standard sample pan with a pinhole lid.

Methods
All MFL organic salts were prepared according to the following general procedure: to a magnetically stirred solution of mefloquine hydrochloride in ethanol (50 mg/mL), the organic acid sodium salt (1.0 equivalent with regard to mefloquine hydrochloride) was added dissolved in ethanol. After 16 h at room temperature, the reaction mixture was filtered to remove the formed sodium chloride and the solvent was subsequently removed under reduced pressure to obtain the desired product. Following the general procedure, sodium tosylate (0.196 g; 0.73 mmol) was added to a solution of mefloquine hydrochloride. White solid (75%). T m = 250.6 • C. 1 Following the general procedure, sodium HEPES (0.198 g; 0.76 mmol) was added to a solution of mefloquine hydrochloride. White solid (85%). T m = 134.2 • C with decomposition. 1 13  Between 2 and 10 mg of the MFL salts was added to 1 mL of Milli-Q water and left to stir for 24 h at 37 • C. After filtration through a microporous (45 µm) syringe filter, an adequate dilution of the solution was performed and analyzed in a UV/Vis spectrophotometer at 284 nm. The absorbance value correlated with the concentration of MFL in water through the following calibration curve.

General Procedure for the Permeability (P), Diffusion (D), and Partition Coefficient (K d ) Measurements
The permeability measurements were conducted using a glass Franz-type diffusion cell (PermeGear) with an 8 mL reactor compartment (effective mass transfer area of 1 cm 2 ). The polyethersulphone (PES-U) membranes of 150 µm thickness and 0.45 µm pore size (Sartorius Stedim Biotech, Gottingen, Germany) were placed between the two compartments and secured with a stainless-steel clamp. The receptor compartment was entirely filled with water (no bubbles) while the donor compartment contained a saturated solution of the MFL salts. Aliquots of 200 µL were removed from the donor compartment at the determined time periods (10, 20, and 30 min, and 1 and 2 h) and replenished with distilled water. The amount of MFL salts was measured by absorbance at a wavelength of 284 nm using a microplate reader (Synergy HT, Bio-TEK, Winooski, VT, USA). The experiments were performed at 37 • C, and the receptor compartment was stirred at 300 rpm using a magnetic bar to eliminate the boundary layer effect.
The permeability (P) of the MFL organic salts was calculated by the equation: where C t is the concentration in the receptor compartment at time t, C 0 is the initial concentration in the donor compartment, V is the solution volume in the two compartments, and A is the effective area of permeation. The permeability coefficient can be calculated from the slope of the curve − The diffusion coefficient (D) (cm 2 .s −1 ) of solutes across the membrane was calculated according to Fick's law of diffusion: where C i and C f are the initial and final concentrations, respectively; C t is the concentration at time t of the solute in the receptor side. V 1 and V 2 correspond to the volume of the liquid in the donor compartment and in the receptor compartment (cm 3 ), respectively. h is the thickness of the membrane (cm) and A is the effective diffusion area of the membrane (cm 2 ). The partition coefficient (K d ) is defined as a measure of the solubility of the solute in the membrane and is calculated by the equation: where P is the permeability, h is the thickness of the membrane, and D is the diffusion coefficient.

General Procedure for the Antimycobacterial Activity Studies
Briefly, 200 µL of sterile deionized water was added in all outer-perimeter wells of sterile 96-well plates (falcon, 3072: Becton Dickinson, Lincoln Park, Chicago, NJ, USA) to minimize evaporation of the medium in the test wells during incubation. The 96 plates received 100 µL of the Middlebrook 7H9 broth containing the mycobacterial cells (Difco laboratories, Detroit, MI, USA). The tested compounds were dissolved in DMSO (Sigma-Aldrich) and a serial dilution of the MFL salts was made directly on the plate. The final drug concentrations tested were 3.12-100 µg/mL. Plates were covered and sealed with parafilm and incubated at 37 • C for five days. After this time, 25 µL of a freshly prepared 1:1 mixture of Alamar Blue (Accumed International, Westlake, OH, USA) reagent and 10% Tween 80 was added in the plate and incubated for 24 h. A blue color in the well was interpreted as no bacterial growth, and a pink color was scored as growth. The MIC (minimal inhibition concentration) was defined as the lowest drug concentration, which prevented a color change from blue to pink.

General Procedure for the Cytotoxicity Assays
Murine macrophage Raw 264.7 cells were cultured in Dulbecco's modified Eagle's medium (ThermoFisher Scientific, São Paulo, Brazil) supplemented with 10% (v/v) heatinactivated fetal bovine serum, 100 IU/mL of penicillin, and 100 µg/mL of streptomycin in flat-bottomed 96-well microtiter plates (Techno Plastic Products, São Paulo, Brazil) for 24 h in 5% CO 2 at 37 • C. At confluence, nonadherent cells were removed by washing with sterile 0.15 M phosphate-buffered saline, pH 7.2. The medium containing the compounds was added to each well, and the plate was incubated for 24 h as above. Tests containing medium alone and medium plus 1% DMSO served as controls. Cell viability was determined by the dimethylthiazol dimethyl tetrazolium bromide (MTT, Sigma-Aldrich, Burlington, MA, USA) reduction assay as recommended by the manufacturer. The assays were carried out in triplicate and performed on two separate occasions.

Synthesis of the MFL Salts
MFL was combined with several low-toxicity sulfonate and sulfiminium anions in order to provide a wide range of physical and chemical properties to the pharmaceutical salts. For example, dioctyl sulfosuccinate (AOT) has previously shown to display significant activity against methicillin-resistant Staphylococcus aureus when combined with organic cations such as ethyl glycine [56]. Moreover, saccharinate (Sac) and HEPES have yielded salts with improved solubility in water [56], while mesylate (MsO) and the (1R)-and (1S)camsylates (CSA) have been extensively used in pharmaceutical drug formulations [57]. In fact, camphor and several derivatives are known for their bioactive properties, particularly against the M. tuberculosis [58,59]. Finally, tosylate (TsO) can correlate with MsO on the role of the carbon atom chain length and intramolecular interactions in the physicochemical and pharmacological properties of the MFL salts.
The MFL salts were obtained through a metathesis reaction (Scheme 1), a straightforward and well-described method for the preparation of this type of compound [60,61]. and (1S)-camsylates (CSA) have been extensively used in pharmaceutical drug formulations [57]. In fact, camphor and several derivatives are known for their bioactive properties, particularly against the M. tuberculosis [58,59]. Finally, tosylate (TsO) can correlate with MsO on the role of the carbon atom chain length and intramolecular interactions in the physicochemical and pharmacological properties of the MFL salts.
The MFL salts were obtained through a metathesis reaction (Scheme 1), a straightforward and well-described method for the preparation of this type of compound [60,61]. Briefly, mefloquine hydrochloride [MFLH][Cl] reacted with the respective sodium salt of the selected anion in ethanol as a solvent, at room temperature, during 16 h. Subsequently, the reaction media was filtered to remove the produced sodium chloride and the desired products were obtained in good to excellent yields and high purity after concentration under reduced pressure.
The obtained MFL salts were characterized by 1 H and 13 C NMR, FTIR-ATR, and elemental analysis. A strict 1.0:1.0 proportion was observed in the 1 H NMR spectra of all MFL salts, in consonance with the desired stoichiometry, and the elemental analysis was in agreement with the theoretical values for the expected products (see ESI). The spectroscopic data, particularly NMR and FTIR-ATR spectra, were important to conclude the desired chemical structure of each MFL salt and the protonation of the MFL scaffold. In order to check the efficacy of protonation, it is possible to observe a chemical shift in the signals from the MFL cation compared to the starting compound as well as the presence of sulfonate anions in the FTIR-ATR signals compared to original sulfonic acids.
The thermal properties were determined through DSC experiments (see Figures S22-S27). The obtained melting and glass transition temperatures are gathered in Table 1, alongside the physical state of the salts at room temperature. Briefly, mefloquine hydrochloride [MFLH][Cl] reacted with the respective sodium salt of the selected anion in ethanol as a solvent, at room temperature, during 16 h. Subsequently, the reaction media was filtered to remove the produced sodium chloride and the desired products were obtained in good to excellent yields and high purity after concentration under reduced pressure.
The obtained MFL salts were characterized by 1 H and 13 C NMR, FTIR-ATR, and elemental analysis. A strict 1.0:1.0 proportion was observed in the 1 H NMR spectra of all MFL salts, in consonance with the desired stoichiometry, and the elemental analysis was in agreement with the theoretical values for the expected products (see ESI). The spectroscopic data, particularly NMR and FTIR-ATR spectra, were important to conclude the desired chemical structure of each MFL salt and the protonation of the MFL scaffold. In order to check the efficacy of protonation, it is possible to observe a chemical shift in the signals from the MFL cation compared to the starting compound as well as the presence of sulfonate anions in the FTIR-ATR signals compared to original sulfonic acids.
The thermal properties were determined through DSC experiments (see Figures S22-S27). The obtained melting and glass transition temperatures are gathered in Table 1, alongside the physical state of the salts at room temperature.

Water Solubility and Permeability Studies
In vitro bioavailability studies were performed for all synthesized MFL salts. Typically, the formation of organic salts from pharmaceutical drugs leads to a modulation of the water solubility and partition coefficients between hydrophobic and hydrophilic media, according to the established cation-anion interactions. In the search for new lead compounds or formulations, an improved water solubility or partition through biological membranes can lead to a higher therapeutic efficiency and decreased side-effects. In fact,

Water Solubility and Permeability Studies
In vitro bioavailability studies were performed for all synthesized MFL salts. Typically, the formation of organic salts from pharmaceutical drugs leads to a modulation of the water solubility and partition coefficients between hydrophobic and hydrophilic media, according to the established cation-anion interactions. In the search for new lead compounds or formulations, an improved water solubility or partition through biological membranes can lead to a higher therapeutic efficiency and decreased side-effects. In fact, it is known that MFL possesses poor solubility in water, a long elimination half-life, and variations in the oral bioavailability. This substance is classified in the Biopharmaceutics Classification System (BCS) as a class II or IV drug because, associated with its low solubility, there is a lack of data related to its permeability [62][63][64]. Several strategies have been described in the literature aiming to improve MFL solubility, including the formulation of Pheroid vesicles containing MFL [65], liposome encapsulation [66], cocrystallization with different cocrystal formers [67], and the development of formulations using oil-in-water emulsions [68]. Table 2 resumes the obtained water solubility, diffusion, permeability, and corresponding partition coefficient data at 37 • C. Table 2. Solubility, diffusion (D), and permeability (P) in water, and partition coefficients (K d ) of the MFL salts.

Almost all MFL salts presented improved in vitro bioavailability in comparison with the original [MFLH][Cl]. In terms of solubility in water (24 h shake-flask method), only [MFLH]
[MsO] showed improvement, probably due to the small size and solvability of the anion. Unfortunately, we were unable to grow suitable single crystals for X-ray diffraction studies, which could provide valuable insights into the crystal packing and water solvation. In addition to this enhancement in water solubility, [MFLH][MsO] also showed a slight increase in the partition coefficient (0.19), spawned from improved diffusion (0.62 cm 2 /s) and permeability (0.79 cm/s) through the synthetic membrane.
Although all remaining salts presented diminished water solubility, their diffusion and permeability parameters were improved in comparison with the original drug. The highest partition coefficient was obtained for [MFLH][TsO] (0.52), which is the least soluble studied MFL salt. These data correlate well with its excellent permeability across the membrane (4.23 cm/s) and good diffusion (1.21 cm 2 /s), which lead to the highest partition coefficient obtained.
Moreover, the two salts based on the combination of MFL with the isomers of camsylate showed very distinct results. Most probably related with very distinct interactions between the cation, the anions, and the membrane, The salts containing these two anions, but also [Sac] and [HEPES], are probably constituted through strong hydrogen bonds between the cation and the anion. More precisely, the oxygen atom of the carbonyl group in the structures of the camsylates and saccharinate can act as an hydrogen bond acceptor. This can also be the case with the nitrogen atoms of the piperazine ring in [HEPES]. Moreover, in this anion, the hydroxyl group can act as both hydrogen bond donor and acceptor. In this case, the partition coefficient is decreased in relation with the starting drug, despite its permeability and diffusion values being improved, ca., 10 times. Finally, the combination of MFL with [AOT] led to a highly hydrophobic salt, which precluded the execution of the partition studies.

Biological Activity
The antimycobacterial activity of the developed MFL salts was assessed against the susceptible M. tuberculosis H37RV strain (ATCC 27294) [69], using the micro-plate Alamar Blue assay (MABA) [70], a nontoxic methodology that employs a thermally stable reagent and shows good correlation with proportional and BACTEC radiometric methods [71,72]. The assays were performed in triplicate and bacteria were exposed to MFL salts in 100, 50, 25, 12.5, 6.25, and 3.12 µg/mL concentrations. The minimal inhibitory concentration (MIC) was defined as the lowest drug concentration that prevented a color change from blue to pink, and was expressed in µg/mL and µM.
The anti-mycobacterial activities and the relative decrease in inhibitory concentrations (RDIC) of the MFL salts are shown in Table 3. With the exception of [MFLH][AOT], all salts presented the same MIC (in µg/mL). However, in molar concentrations, these salts present a slightly improved antimycobacterial activity in comparison with the starting drug. All of the starting anion sodium salts were inactive against this strain of M. tuberculosis (MIC > 350 µM, see Table S1), indicating that the observed biological activity originates from a synergistic effect between the MFL cation and the different organic anions.

Cell Viability Assay
Finally, the cytotoxicity of the MFL salts was assessed in the monocyte/macrophagelike cells Raw 264.7.
During TB infection, macrophages are the most important immune cells in the early immune response to M. tuberculosis once they participate in the elimination of infecting mycobacteria. However, several times, they are incapable of this and the mycobacteria colonize, survive, and grow inside macrophages [73]. Thus, due to this important role in TB pathogenesis, it is relevant to assay the cytotoxicity of anti-TB drug candidates using in vitro macrophage models. We have chosen to use Raw 264.7 cells that are monocyte/macrophagelike cells, originating from the Abelson leukemia virus-transformed cell line derived from BALB/c mice. These are the most commonly used myeloid cell line for the in vitro model for at least 40 years and they proved to be phenotypically and functionally stable until 30 passages [74].
The cellular viability of the MFL salts was determined by the MTT assay at three different concentrations (15 µM, 30 µM, and 60 µM) within the range of the MIC values (20.27-38.80 µM). The results are expressed as percentage of cell viability (%CV) (Figure 2 and Table S2). According to the %CV exhibited at each concentration, the compounds could be classified as noncytotoxic if the CV is as low as 95%, as moderately cytotoxic if the CV is between 70% and 94%, and as cytotoxic if the CV is lower than 50%. Moreover, we also evaluated the cytotoxicity of the sodium salts used for the preparation of their corresponding MFL salts (Table S2). In general, the results indicate that these salts were not cytotoxic in all tested concentrations, except for [Na][Sac] that was cytotoxic at 60 µM. These data suggest that the association of the MFL cation with the selected organic anions tends to increase its cytotoxicity toward macrophages but also to an increase in the anti-mycobacterial activity.

Conclusions
In this work, different MFL salts based on direct protonation of the original drug by the use of organic sulfonic acids have been developed. The spectroscopic techniques such as NMR, FTIR-ATR, and elemental analysis prove the desired chemical structures. It is important to note that mefloquine mesylate ( Moreover, we also evaluated the cytotoxicity of the sodium salts used for the preparation of their corresponding MFL salts (Table S2). In general, the results indicate that these salts were not cytotoxic in all tested concentrations, except for [Na][Sac] that was cytotoxic at 60 µM. These data suggest that the association of the MFL cation with the selected organic anions tends to increase its cytotoxicity toward macrophages but also to an increase in the anti-mycobacterial activity.

Conclusions
In this work, different MFL salts based on direct protonation of the original drug by the use of organic sulfonic acids have been developed. The spectroscopic techniques such as NMR, FTIR-ATR, and elemental analysis prove the desired chemical structures. It is important to note that mefloquine mesylate (