Pharmaceutical Salts of Enroﬂoxacin with Organic Acids

: Enroﬂoxacin is a poorly soluble antibacterial drug of the ﬂuoroquinolones class used in veterinary medicine. The main purpose of this work was to investigate the structural and pharmaceutical properties of new enroﬂoxacin salts. Enroﬂoxacin anhydrate and its organic salts with tartaric acid, nicotinic acid and suberic acid formed as pure crystalline anhydrous solids. All the crystals were grown from a mixed solution by slow evaporation at room temperature. These products were then characterized by ﬁeld-emission scanning electron microscopy, powder X-ray di ﬀ raction, Fourier transform infrared spectroscopy and di ﬀ erential scanning calorimetry. Further, X-ray single crystal di ﬀ raction analysis was used to study the crystal structure. The intermolecular interactions and packing arrangements in the crystal structures were studied, and the solubility of these salts in water was determined using high-performance liquid chromatography. The results show that the new salts of enroﬂoxacin developed in this study exhibited excellent water solubility.


Introduction
Crystal engineering through multicomponent crystals has attracted substantial attention in the pharmaceutical field in recent years [1,2] because of the possibility of improved solubility and bioavailability of newly designed drug compounds (polymorphs, salt and cocrystals) [3,4]. The formation of the salt/cocrystal is based on the crystal engineering concepts [5]. The salt/cocrystal formation provides an enormous scope for the manipulation and modification of crucial pharmaceutical physical properties such as the dissolution rate, solubility, thermodynamic stability and bioavailability [6,7]. Proton transfer is a decisive factor that distinguishes salts from cocrystals: In salt formation, proton transfer and ionization occur, while these do not occur in the formation of cocrystals [8]. Salts and cocrystals have been employed in the pharmaceutical industry because of their excellent solubility [9]. Although cocrystallization has many exciting advantages, salt formation still represents a widely accepted method to obtain higher solubility of the drug [10].
Most of the active pharmaceutical ingredients (APIs) available in the current market cause formulation difficulties because of poor water solubility, which may lead to poor oral bioavailability [11,12]. Hence, improving the solubility and bioavailability of APIs without changing their stability and other characteristics has become a challenging task. Notably, every crystal structure is the result of mutual balance between numerous noncovalent interactions, but the hydrogen

Preparation of Salts and Crystallization
Enrofloxacin and various acids in equal molar ratios were dissolved in a mixed solvent in a 50-mL conical flask, and this mixture was stirred at 50 • C until a completely clear solution was obtained. The solution was then allowed to slowly evaporate at room temperature to obtain single crystals of the products. Diffraction-quality crystals were obtained within 1-5 days. Each sample was scaled up 20 times for the solubility determination.

Field-Emission Scanning Electron Microscopy (FESEM)
The surface morphology of the samples was investigated by FESEM (Heidelberg, Germany) using a Zeiss Sigma 300.

Powder X-ray Diffraction (PXRD)
PXRD measurements of the new compounds were carried out on a Rigaku-Ultima IV X-ray powder diffractometer (Tokyo, Japan) using Cu Kα radiation (λ = 1.54178 Å) at 40 kV and 40 mA. Samples were analysed in the 2θ range from 5 • to 80 • with a scanning rate of 8 • /min. These data were collected at room temperature and analysed using Jade 6.0 software (Livermore, CA, USA).

Fourier Transform Infrared (FT-IR)
FT-IR spectra were collected using a Bruker Vertex 70 spectrometer and measured in the range of 4000−400 cm −1 with an RT-DLaTGS detector (Ettlingen, Germany). The KBr diffuse-reflectance mode was used (sample concentration: 1 mg in 100 mg of KBr) for recording the IR spectra of these samples. Data were analysed using the OPUS software (San Francisco, CA, USA).

Single Crystal X-ray Diffraction (SCXRD)
X-ray diffraction data were collected using a Rigaku Oxford Diffraction SuperNova diffractometer (Oxford, UK) equipped with a monochromator mirror for Cu Kα (λ = 1.54184 Å) radiation at 150 K. Data reduction was carried out using the CrysAlisPro software, and absorption correction was implemented in the SCALE3 ABSPACK scaling algorithm. The structure was solved (direct methods) and refined (using least squares minimization) with Olex2 [26] using SHELX [27] structure solution programs. All the non-H atoms were refined anisotropically. In addition, all the figures, including the packing and molecular structure diagrams, were drawn using Olex2 and PLATON [28]. Table 1 gives the pertinent crystal lographic data, and Table 2 gives the hydrogen-bond parameters.

Solubility Analyses
The solubility of these products was determined according to the shake-flask method [29]. Excess amounts (100 mg) of the crystals were added to the screw-capped glass vials containing Crystals 2020, 10, 646 6 of 18 2.5 mL of ultrapure water, and the resulting suspensions was shaken at room temperature. After 24 h, the suspensions were filtered through 0.22-µm polycarbonate filters, and the compounds' concentrations were determined using the Agilent 1260 Infinity II HPLC system (Palo Alto, CA, USA) equipped with a 1260 Multi λ Fluorescence detector with the wavelength of the excited and emitted spectra of crystals being 280 nm and 450 nm, respectively. The C18 HPLC column (Poroshell 120 EC-C18, 4.6 mm × 100 mm, 2.7 µm) was employed, and acetonitrile, methanol and triethylamine phosphate (pH = 2.5) were used as the mobile phase (1:24:75, v:v::v) with a flow rate of 1 mL/min. Each solubility test was performed in triplicate.

Field-Emission Scanning Electron Microscopy (FESEM)
The FESEM (Heidelberg, Germany) images provide information about the crystal morphology of several new crystals ( Figure 1). Needle-shaped or long rod-shaped forms are seen for the products 1 and 2, whereas 3 and 4 displayed rectangular blocks and fragmentary crystals. The solubility of these products was determined according to the shake-flask method [29]. Excess amounts (100 mg) of the crystals were added to the screw-capped glass vials containing 2.5 mL of ultrapure water, and the resulting suspensions was shaken at room temperature. After 24 h, the suspensions were filtered through 0.22-μm polycarbonate filters, and the compounds' concentrations were determined using the Agilent 1260 Infinity II HPLC system (Palo Alto, CA, USA) equipped with a 1260 Multi λ Fluorescence detector with the wavelength of the excited and emitted spectra of crystals being 280 nm and 450 nm, respectively. The C18 HPLC column (Poroshell 120 EC-C18, 4.6 mm × 100 mm, 2.7 μm) was employed, and acetonitrile, methanol and triethylamine phosphate (pH = 2.5) were used as the mobile phase (1:24:75, v:v::v) with a flow rate of 1 mL/min. Each solubility test was performed in triplicate.

Field-Emission Scanning Electron Microscopy (FESEM)
The FESEM (Heidelberg, Germany) images provide information about the crystal morphology of several new crystals ( Figure 1). Needle-shaped or long rod-shaped forms are seen for the products 1 and 2, whereas 3 and 4 displayed rectangular blocks and fragmentary crystals.

PXRD Analysis
PXRD enables the study and characterization of novel crystalline materials [30]. PXRD patterns of the four crystal samples are shown in Figure 2. The patterns for 1 were different from those of the starting material and from the previously published PXRD patterns for the polymorphic form [31]. The purity of all products was confirmed by comparing the PXRD patterns with the simulated patterns obtained from the single crystal data (see the Supplementary Materials, Figures S1−S4). No apparent peaks corresponding to impurities were observed, so the obtained powder compounds were considered to be of high chemical purity. Further, these PXRD patterns of enrofloxacin compounds 2-4 differ significantly from the patterns of individual APIs, proving the formation of the new crystalline phases.

PXRD Analysis
PXRD enables the study and characterization of novel crystalline materials [30]. PXRD patterns of the four crystal samples are shown in Figure 2. The patterns for 1 were different from those of the starting material and from the previously published PXRD patterns for the polymorphic form [31]. The purity of all products was confirmed by comparing the PXRD patterns with the simulated patterns obtained from the single crystal data (see the Supplementary Materials, Figures S1−S4). No apparent peaks corresponding to impurities were observed, so the obtained powder compounds were considered to be of high chemical purity. Further, these PXRD patterns of enrofloxacin compounds 2-4 differ significantly from the patterns of individual APIs, proving the formation of the new crystalline phases. Crystals 2020, 10, x FOR PEER REVIEW 7 of 18

Spectroscopic Characterization
The FTIR analysis results for the products are given in Figure 3. The absorption band of crystalline enrofloxacin was located at 1737 cm −1 because of the carbonyl stretching of its unionized carboxylic acid C=O group [32]. The C=O band in 1 was at 1736.4 cm −1 , which indicates that the enrofloxacin exists as a neutral molecule. Slight peak shifts were also seen in compounds 2-4, in particular, for the carboxylic acid C=O stretching, which shifted to 1728.8 cm −1 , 1728.5 cm −1 and 1729.3 cm −1 , respectively. This can be attributed to intermolecular interactions such as the formation of hydrogen bonds [33]. The terminal amino group of the piperazine ring was protonated in the process of crystallization, and this is proved by the presence of broad bands from 2600 cm −1 to 3000 cm −1 . However, confirming this is difficult because the broad IR absorption bands possibly overlap with others, e.g., in the case of the C−H stretching [34]. However, Karanam et al. reported FTIR analysis results that showed that enrofloxacin salts were protonated in the process of crystallization [31]. The medium-intensity broad band in the region of 3300-3500 cm −1 is attributed to the O−H stretching of the water molecule 2 and ethanol molecule 3-4. The existence of enrofloxacin as a neutral molecular and in the ionic state in the crystal structures was confirmed by SCXRD analysis.

Spectroscopic Characterization
The FTIR analysis results for the products are given in Figure 3. The absorption band of crystalline enrofloxacin was located at 1737 cm −1 because of the carbonyl stretching of its unionized carboxylic acid C=O group [31]. The C=O band in 1 was at 1736.4 cm −1 , which indicates that the enrofloxacin exists as a neutral molecule. Slight peak shifts were also seen in compounds 2-4, in particular, for the carboxylic acid C=O stretching, which shifted to 1728.8 cm −1 , 1728.5 cm −1 and 1729.3 cm −1 , respectively. This can be attributed to intermolecular interactions such as the formation of hydrogen bonds [32]. The terminal amino group of the piperazine ring was protonated in the process of crystallization, and this is proved by the presence of broad bands from 2600 cm −1 to 3000 cm −1 . However, confirming this is difficult because the broad IR absorption bands possibly overlap with others, e.g., in the case of the C−H stretching [33]. However, Karanam et al. reported FTIR analysis results that showed that enrofloxacin salts were protonated in the process of crystallization [34]. The medium-intensity broad band in the region of 3300-3500 cm −1 is attributed to the O−H stretching of the water molecule 2 and ethanol molecule 3-4. The existence of enrofloxacin as a neutral molecular and in the ionic state in the crystal structures was confirmed by SCXRD analysis.   (1) Single crystals suitable for X-ray diffraction were obtained to determine the structures of the anhydrous form with a neutral molecule (no proton transfer from carboxylic acid to piperazinyl N atom; Figure 4a). The enrofloxacin anhydrate crystallized in the monoclinic P21/n space group with one molecule in the asymmetric unit. Its space group and cell parameters are different from those previously reported [31]. Combined with the PXRD patterns observed in this work, this indicates that 1 is a polymorph. In addition, the piperazine ring in enrofloxacin usually exhibits a stable chair conformation with a torsion angle of C12-N2-C15-C16 = 158.25°. The carboxylic acid group participated in intramolecular O−H···O hydrogen bonding with the carbonyl oxygen atom of the quinolone moiety. The crystal structure was stacked with π···π (3.598 Å) interactions and was further stabilized by weak C−H···O and C−H···F hydrogen bonds Table 2 in enrofloxacin (Figure 4b).   (1) Single crystals suitable for X-ray diffraction were obtained to determine the structures of the anhydrous form with a neutral molecule (no proton transfer from carboxylic acid to piperazinyl N atom; Figure 4a). The enrofloxacin anhydrate crystallized in the monoclinic P2 1 /n space group with one molecule in the asymmetric unit. In addition, the piperazine ring in enrofloxacin usually exhibits a stable chair conformation with a torsion angle of C12-N2-C15-C16 = 158.25 • . It has the same crystal structure compared with the previous published [34]. The carboxylic acid group participated in intramolecular O−H···O hydrogen bonding with the carbonyl oxygen atom of the quinolone moiety. The crystal structure was stacked with π···π (3.598 Å) interactions and was further stabilized by weak C−H···O and C−H···F hydrogen bonds Table 2

Enrofloxacin Tartrate Trihydrate (2)
The novel enrofloxacin salt trihydrate 2 crystallized in the triclinic 1 P space group. The 1:1 salt structure of enrofloxacin tartrate trihydrate contains an enrofloxacin cation, a tartrate anion and three H2O molecules in the asymmetric unit (Figure 5a). One of the carboxylic acid groups of tartaric acid transferred one proton to the piperazinyl-ring N atom of the enrofloxacin molecule, resulting in a tartrate anion and enrofloxacin cation in the crystal structure (Figure 5b). The

Enrofloxacin Tartrate Trihydrate (2)
The novel enrofloxacin salt trihydrate 2 crystallized in the triclinic P1 space group. The 1:1 salt structure of enrofloxacin tartrate trihydrate contains an enrofloxacin cation, a tartrate anion and three H 2 O molecules in the asymmetric unit (Figure 5a). One of the carboxylic acid groups of tartaric acid transferred one proton to the piperazinyl-ring N atom of the enrofloxacin molecule, resulting in a tartrate anion and enrofloxacin cation in the crystal structure (Figure 5b). The carboxylic acid group of enrofloxacin was involved in intramolecular O−H···O hydrogen bonding with the quinolone oxygen atom. The product 2 displayed a unique hydrogen-bonding pattern with the formation of multicomponent crystals. The enrofloxacin cation interacted with the tartrate ion via N + −H···O interactions to form the crystal structure, rather than forming hydrogen bonds with ionized oxygen atoms. Further, one of the water molecules connected the enrofloxacin cation and tartrate anion via O−H···O interactions (Figure 5c).

Enrofloxacin Nicotinate-EtOH Salt Solvate (3)
The novel enrofloxacin salt solvate 3 had a triclinic system and space group P1. Its crystal contained an enrofloxacin cation, a nicotinate anion and an EtOH molecule in the asymmetric unit (Figure 6a

Enrofloxacin Suberate-2EtOH Salt Solvate (4)
The novel enrofloxacin salt solvate 4 had a triclinic system and space group P1 with an enrofloxacin cation, a suberate anion and two EtOH molecules in the asymmetric unit. The carboxylic acid and cyclopropyl groups of the enrofloxacin molecule were observed to exhibit disorder in this crystal structure (Figure 7a). One of the carboxylic acid groups of the suberic acid transferred one proton to the piperazinyl-ring N atom of the enrofloxacin molecule, thereby forming a suberate anion and enrofloxacin cations in the crystal structure (Figure 7b

Thermal Analysis
DSC curves showing the thermal behaviour of the products 1-4 are shown in Figure 8. The endothermic peak for melting of 1 was found at 228 °C. In the case of 2, the endothermic melting peak was at 228 °C, and this was followed by a phase transition. The endothermic transitions between 75 °C and 125 °C in the DSC thermograms for 2 showed the loss of water molecules from the crystal structure. In the DSC thermogram for 3, several steps of small endothermic transitions at about 120-180 °C due to solvent loss were observed, followed by a four-step endothermic melting transition of the salt solvate Further, 4 was found to melt at 112 °C.

Thermal Analysis
DSC curves showing the thermal behaviour of the products 1-4 are shown in Figure 8. The endothermic peak for melting of 1 was found at 228 • C. In the case of 2, the endothermic melting peak was at 228 • C, and this was followed by a phase transition. The endothermic transitions between 75 • C and 125 • C in the DSC thermograms for 2 showed the loss of water molecules from the crystal structure. In the DSC thermogram for 3, several steps of small endothermic transitions at about 120-180 • C due to solvent loss were observed, followed by a four-step endothermic melting transition of the salt solvate Further, 4 was found to melt at 112 • C. Crystals 2020, 10, x FOR PEER REVIEW 16 of 18

Results for Solubility Study
The solubility data are presented in Table 3. The solubility of commercially available enrofloxacin in water was 0.14 mg/mL, which is similar to the previously reported values [21]. The solubilities of 1 and the commercially available enrofloxacin are similar. As expected, the enrofloxacin salts showed a considerable improvement in solubility: 2-4 were found to be 57-to 406-times more soluble than pure enrofloxacin. Correlating the solubility with crystal structures is difficult because the limited experimental/calculated data on crystal packing, etc. However, it can be speculated that the solubility enhancement in the case of the salts was a result of greater ionization.

Discussion
Enrofloxacin anhydrate was crystallized, and the crystal structure was determined. Further, new salts formed using tartaric acid, nicotinic acid and suberic acid have been reported for the first time. The novel salts were prepared efficiently via evaporation using a mixed solvent has been found to be highly rewarding. These compounds formed a layered structure, and these layers were stacked via hydrogen bonds and π···π interactions. In the structure of salts, the piperazinyl moieties of enrofloxacin interacted with the carboxylate ions. These carboxylate ions connected the H2O and EtOH molecules formed a stacking structure. The enrofloxacin salts prepared in this study showed a 57-to 406-fold higher solubility than the starting material.

Results for Solubility Study
The solubility data are presented in Table 3. The solubility of commercially available enrofloxacin in water was 0.14 mg/mL, which is similar to the previously reported values [21]. The solubilities of 1 and the commercially available enrofloxacin are similar. As expected, the enrofloxacin salts showed a considerable improvement in solubility: 2-4 were found to be 57-to 406-times more soluble than pure enrofloxacin. Correlating the solubility with crystal structures is difficult because the limited experimental/calculated data on crystal packing, etc. However, it can be speculated that the solubility enhancement in the case of the salts was a result of greater ionization.

Discussion
Enrofloxacin anhydrate was crystallized, and the crystal structure was determined. Further, new salts formed using tartaric acid, nicotinic acid and suberic acid have been reported for the first time. The novel salts were prepared efficiently via evaporation using a mixed solvent has been found to be highly rewarding. These compounds formed a layered structure, and these layers were stacked via hydrogen bonds and π···π interactions. In the structure of salts, the piperazinyl moieties of enrofloxacin interacted with the carboxylate ions. These carboxylate ions connected the H 2 O and EtOH molecules formed a stacking structure. The enrofloxacin salts prepared in this study showed a 57-to 406-fold higher solubility than the starting material.