3.4.1. Crystallization of Enrofloxacin Anhydrate (1)

3.4.1. Crystallization of Enrofloxacin Anhydrate (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 *P*21/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 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 *P*21/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 in enrofloxacin (Figure 4b).

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).

**Figure 4.** (**a**) The molecular structure diagram of the product **1** (ellipsoids were drawn at the 50% probability level). (**b**) Molecular packing projections for product **1** along the [101] direction. **Figure 4.** (**a**) The molecular structure diagram of the product **1** (ellipsoids were drawn at the 50% probability level). (**b**) Molecular packing projections for product **1** along the [101] direction.

#### 3.4.2. Enrofloxacin Tartrate Trihydrate (2) 3.4.2. Enrofloxacin Tartrate Trihydrate (2)

The novel enrofloxacin salt trihydrate **2** crystallized in the triclinic *P*1 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 The novel enrofloxacin salt trihydrate **2** crystallized in the triclinic *P*1 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 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). 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).

**Figure 5.** *Cont.*

**Figure 5.** (**a**) The molecular structure diagram of the salt trihydrate **2** (ellipsoids were drawn at the 50% probability level). (**b**) Interaction between enrofloxacin and tartaric acid molecules in the crystal via O−H···O hydrogen bonds. (**c**) Molecular packing projections for salt trihydrate **2** along the [111] direction. **Figure 5.** (**a**) The molecular structure diagram of the salt trihydrate **2** (ellipsoids were drawn at the 50% probability level). (**b**) Interaction between enrofloxacin and tartaric acid molecules in the crystal via O−H···O hydrogen bonds. (**c**) Molecular packing projections for salt trihydrate **2** along the [111] direction.

#### 3.4.3. Enrofloxacin Nicotinate-EtOH Salt Solvate (3) 3.4.3. Enrofloxacin Nicotinate-EtOH Salt Solvate (3)

The novel enrofloxacin salt solvate **3** had a triclinic system and space group *P*1. Its crystal contained an enrofloxacin cation, a nicotinate anion and an EtOH molecule in the asymmetric unit (Figure 6a). The carboxylic acid group of **3** was involved in intramolecular O−H···O hydrogen bonding with the quinolone oxygen atom. The nicotinic acid was ionized by proton transfer to the enrofloxacin molecule to form N+−H···O−, while the EtOH molecule formed the O−H···O hydrogen bond with the carboxylic acid C=O group of nicotinate (Figure 6b). The quinolone moieties of the enrofloxacin molecules stacked via *π*···*π* (3.538 Å) interactions (Figure 6c). The novel enrofloxacin salt solvate **3** had a triclinic system and space group *P*1. Its crystal contained an enrofloxacin cation, a nicotinate anion and an EtOH molecule in the asymmetric unit (Figure 6a). The carboxylic acid group of **3** was involved in intramolecular O−H···O hydrogen bonding with the quinolone oxygen atom. The nicotinic acid was ionized by proton transfer to the enrofloxacin molecule to form N+−H···O−, while the EtOH molecule formed the O−H···O hydrogen bond with the carboxylic acid C=O group of nicotinate (Figure 6b). The quinolone moieties of the enrofloxacin molecules stacked via π···π (3.538 Å) interactions (Figure 6c).

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**Figure 6.** *Cont.*

**Figure 6.** (**a**) The molecular structure diagram of the salt solvate **3** (ellipsoids were drawn at the 50% probability level). (**b**) Interaction between enrofloxacin and nicotinic acid molecules in the crystal via N+−H···O<sup>−</sup> and O−H···O hydrogen bonds. (**c**) Molecular packing projections for salt solvate **3** along the [111] direction. **Figure 6.** (**a**) The molecular structure diagram of the salt solvate **3** (ellipsoids were drawn at the 50% probability level). (**b**) Interaction between enrofloxacin and nicotinic acid molecules in the crystal viaN+−H···O<sup>−</sup> and O−H···O hydrogen bonds. (**c**) Molecular packing projections for salt solvate **<sup>3</sup>** along the [111] direction.

(**c**)

#### 3.4.4. Enrofloxacin Suberate-2EtOH Salt Solvate (4) 3.4.4. Enrofloxacin Suberate-2EtOH Salt Solvate (4)

stacked via *π*···*π* (3.872 Å) interactions (Figure 7c).

The novel enrofloxacin salt solvate **4** had a triclinic system and space group *P*1 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). The carboxylic acid group of **4** was involved in intramolecular O−H···O hydrogen bonding with the quinolone oxygen atom. Enrofloxacin interactsedwith the suberate ion via N+−H···O- interactions in the crystal structure, while the EtOH molecule formed the O−H···O hydrogen bond with the carboxylic acid C=O group of the suberate ion. The quinolone moieties of the enrofloxacin molecule The novel enrofloxacin salt solvate **4** had a triclinic system and space group *P*1 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). The carboxylic acid group of **4** was involved in intramolecular O−H···O hydrogen bonding with the quinolone oxygen atom. Enrofloxacin interactsedwith the suberate ion via N+−H···O<sup>−</sup> interactions in the crystal structure, while the EtOH molecule formed the O−H···O hydrogen bond with the carboxylic acid C=O group of the suberate ion. The quinolone moieties of the enrofloxacin molecule stacked via π···π (3.872 Å) interactions (Figure 7c).

(**a**)

**Figure 7.** *Cont.*

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**Figure 7.** (**a**) The molecular structure diagram of the salt solvate **4** (ellipsoids were drawn at the 50% probability level). (**b**) Interaction between enrofloxacin and suberic acid molecules in the crystal by N+−H···O and O−H···O hydrogen bonds. (**c**) Molecular packing projections for salt solvate **4** along the [011] direction. **Figure 7.** (**a**) The molecular structure diagram of the salt solvate **4** (ellipsoids were drawn at the 50% probability level). (**b**) Interaction between enrofloxacin and suberic acid molecules in the crystal by N+−H···O<sup>−</sup> and O−H···O hydrogen bonds. (**c**) Molecular packing projections for salt solvate **4** along the [011] direction.

#### *3.5. Thermal Analysis 3.5. 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. 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.

**Figure 8.** Differential scanning calorimetry (DSC) diagrams of compounds **1**–**4**.

### **Figure 8.** Differential scanning calorimetry (DSC) diagrams of compounds **1**-**4**. *3.6. Results for Solubility Study*

*3.6. 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 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.


speculated that the solubility enhancement in the case of the salts was a result of greater ionization. **Table 3.** Solubility Data for Compounds **1–4.**

**3** 56.83 **4** 8.04 *<sup>a</sup>* Solubility measured after 24 h of equilibration. *<sup>b</sup>* Solubility of commercially available enrofloxacin.

#### *<sup>a</sup>* Solubility measured after 24 h of equilibration. *<sup>b</sup>* Solubility of commercially available enrofloxacin. **4. Discussion**

**4. 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 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.

a 57- to 406-fold higher solubility than the starting material.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4352/10/8/646/s1. The PXRD patterns with the simulated patterns obtained from the single crystal data.CIF files giving crystal data can be obtained free of charge from the Cambridge Crystallographic Data Centre via the Internet www.ccdc.cam. ac.uk/data\_request/cif.

**Author Contributions:** Writing–original draft, H.P.; Conceptualization, Y.-B.S.; Formal analysis, Y.Y., and L.-Z.C.; Funding acquisition, B.-H.F.; Investigation, J.-W.Z.; Methodology, M.-J.X.; Software, H.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Natural Science Foundation of China (No. 31872522).

**Conflicts of Interest:** There are no conflict to declare.
