Synthesis of Two Novel Azilsartan Cocrystals: Preparation, Physicochemical Characterization and Solubility Studies

Azilsartan is a novel angiotension II receptor blocker primarily used to treat high blood pressure. This is not a formulation-friendly molecule largely due to the inherent water-solubility pitfalls. In this paper, two novel cocrystals of azilsartan (AZ) were studied (AZ-BIP, AZ-BPE; BIP = 4,4′-bipyridine, BPE = trans-1,2-bis (4-pyridyl) ethylene) by solution crystallization. The structures of these two cocrystals were characterized by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), including the single-crystal structure determination of AZ-BIP and AZ-BPE. In the cocrystals AZ-BIP (2:1) and AZ-BPE (2:1), two AZ molecules and one coformer formed a sandwich structure through N-H . . . N interactions. These sandwich structures were extended into a one-dimensional structure through O-H . . . N hydrogen bonds. The equilibrium solubility study demonstrated that the AZ-BIP and AZ-BPE cocrystals both showed higher solubility than azilsartan in water.


Introduction
Among well-studied organic materials, drug cocrystals are considered an effective substitute for existing drug formulations. These can enhance the physical and chemical properties of the active pharmaceutical ingredient (APIs) without altering the covalent bond of the medication, and even affect the solubility, dissolution rate and bioavailability of the medication [1][2][3][4][5][6][7][8]. Without changing the covalent chemistry of drugs, drug salt is primarily used for improving the physical and chemical properties of drugs. Pharmaceutical cocrystals have been developed as an ideal optimization strategy only during the last 10 years. Three drug cocrystals have entered the market in the form of new chemical entities to date, including Entresto ® [9] (a type of sacubitril-valsartan sodium cocrystal), Depakote ® , Depakine ® [10] (a type of escitalopram oxalate citalopram-oxalic acid cocrystal) and the tramadol-celecoxib cocrystal [11]. The drug cocrystal market indicates that the reasonable design of new drug cocrystals by crystal engineering has favorable prospects for application.
Azilsartan is a novel angiotension II receptor blocker primarily used to treat high blood pressure mboxciteB12-crystals-903496,B13-crystals-903496. This is a Biopharmaceutics Classification System (BCS) Class II medication with poor aqueous solubility in water (11.6 mg/L) [14], whereas BIP and BPE that both contain a pyridine ring are identified as potential cocrystal coformers [15][16][17][18][19][20][21][22]. We reported three salt solvates of azilsartan (AZ) and 2-methylimidazole (included one hydrate, one acetone solvate and one tetrahydrofuran solvate) [14]. However, no azilsartan cocrystals with pyridine functional groups have been reported to date. In this paper, we report two novel cocrystals (namely AZ-BIP and AZ-BPE) of azilsartan with BIP and BPE. Although BIP and BPE are toxic compounds and consequently, useless for human application. Despite everything, supramolecular knowledge is always useful to the synthesis of other azilsartan cocrystals. Furthermore, this screening can guide the development of new cocrystals of azilsartan by unusual N-H…N interaction instead of the general O-H…N hydrogen bonds. The solubility and powder dissolution of the two cocrystals are also discussed further. The chemical structures of AZ, BIP and BPE molecules are shown in Scheme 1.

Materials and General Methods
All chemicals and solvents were purchased from commercial sources and used without further purification. Differential Scanning Calorimetric (DSC) data were recorded using a Mettler-Toledo system at a heating rate of 10 K/min, and the runs were undertaken under the protection of nitrogen. Thermogravimetric analysis (TGA) was performed on a Perkin-Elmer TGA 4000 system (Shanghai, China). The heating rate was 10 K/min, and the runs were undertaken under the protection of nitrogen. Powder X-ray diffraction (PXRD) patterns were produced using a German Bruker corporation D8 ADVANCE system (Karlsruhe, Germany) with a Cu Kα radiation (λ = 1.5418 Å, 40 kV, 40 mA), and the patterns were collected in the 3 to 60° range (2θ). The X-ray diffraction data of the AZ-BIP and AZ-BPE cocrystals were recorded on a Bruker Apex II CCD diffractometer (Karlsruhe, Germany) using Mo Kα radiation (λ = 0.71073 Å, 50 kV, 30 mA). The crystal structures of the AZ-BIP and AZ-BPE cocrystals were solved and refined using the SHELXL program [23,24]. The crystallographic parameters of synthesized cocrystals are listed in Table 1, and hydrogen bonds in Table 2. Docking simulations were performed using Molecular Operating Environment (MOE) software [25]. A flexible azilsartan molecule was docked into the angiotensin II type 1 receptor (AT1R) structure (http://www.pdb.org, PDB ID: 4ZUD) that had the binding site of azilsartan. The flexible AZ molecule was optimized using the MMFF94x force field, and partial charges were assigned to the atoms. The hydrogen bond networks of AT1R receptors were optimized by the Amber 99 force field. A Triangle Matcher was used as the docking placement method, and the MMFF94x force field was used to optimize the docking process. The top 30 poses were produced by the London dG scoring function, and the score of receptor-ligand complexes after forcefield refining was calculated by the Affinity dG scoring function [26].

Materials and General Methods
All chemicals and solvents were purchased from commercial sources and used without further purification. Differential Scanning Calorimetric (DSC) data were recorded using a Mettler-Toledo system at a heating rate of 10 K/min, and the runs were undertaken under the protection of nitrogen. Thermogravimetric analysis (TGA) was performed on a Perkin-Elmer TGA 4000 system (Shanghai, China). The heating rate was 10 K/min, and the runs were undertaken under the protection of nitrogen. Powder X-ray diffraction (PXRD) patterns were produced using a German Bruker corporation D8 ADVANCE system (Karlsruhe, Germany) with a Cu Kα radiation (λ = 1.5418 Å, 40 kV, 40 mA), and the patterns were collected in the 3 to 60 • range (2θ). The X-ray diffraction data of the AZ-BIP and AZ-BPE cocrystals were recorded on a Bruker Apex II CCD diffractometer (Karlsruhe, Germany) using Mo Kα radiation (λ = 0.71073 Å, 50 kV, 30 mA). The crystal structures of the AZ-BIP and AZ-BPE cocrystals were solved and refined using the SHELXL program [23,24]. The crystallographic parameters of synthesized cocrystals are listed in Table 1, and hydrogen bonds in Table 2. Docking simulations were performed using Molecular Operating Environment (MOE) software [25]. A flexible azilsartan molecule was docked into the angiotensin II type 1 receptor (AT 1 R) structure (http://www.pdb.org, PDB ID: 4ZUD) that had the binding site of azilsartan. The flexible AZ molecule was optimized using the MMFF94x force field, and partial charges were assigned to the atoms. The hydrogen bond networks of AT 1 R receptors were optimized by the Amber 99 force field. A Triangle Matcher was used as the docking placement method, and the MMFF94x force field was used to optimize the docking process. The top 30 poses were produced by the London dG scoring function, and the score of receptor-ligand complexes after forcefield refining was calculated by the Affinity dG scoring function [26]. The AZ-BIP cocrystal was produced by acetone assisted milling of a 2:1 molar ratio of AZ and BIP. A quantity of 40 mg of the above samples was placed in 20 mL acetone solvent, and heat reflux for 2 h. The solution was left at room temperature for slow evaporation. High-quality block crystals were generated after 5-7 days.

Synthesis of the AZ-BPE Cocrystal (2:1)
The AZ-BPE cocrystal was prepared by acetone assisted milling of a 2:1 molar ratio of AZ and BPE. A quantity of 40 mg of the above samples was placed in 10 mL methanol solvent, and heat reflux for 2 h. The solution was left at room temperature for slow evaporation. High-quality block crystals were generated after 7-10 days.

Solubility and Powder Dissolution Measurement
Solubility was measured on a Shimadzu UV-2550 in water for 24 h at 37 • C. Three calibration curves of AZ, and the AZ-BIP and AZ-BPE cocrystals, were determined from aqueous solutions of known concentrations at 248 nm. The powder dissolution studies were conducted suspending 50 mg samples in 20 mL of water. These suspension liquids were stirred with magnetic force for 4 h at 37 • C. Then, specimens were drawn at a specified time (5,10,15,20,25,30,45, 60, 90, 120, 180, 240 min) and filtered with a 0.22 µm nylon filter. The concentration was measured within the range of its corresponding standard curve. Each experiment was repeated three times.

Crystal Structure Analysis
The cocrystal of azilsartan with 4,4 -bipyridine (AZ-BIP) was solved in the monoclinic C2/c space group. The asymmetric unit contained one azilsartan molecule and half of a BIP molecules (Figure 1 and Supplementary Materials Figure S1). In the crystal structure of AZ-BIP cocrystal, two AZ molecules were connected to one BIP molecule through N1-H1· · · N5 (1.88 Å, 172 • ) interactions that were extended via O4-H2· · · N4 (1.75 Å,171 • ) hydrogen bonds to produce a 1D chain structure ( Figure 2). Crystals 2020, x, x FOR PEER REVIEW 4 of 12 known concentrations at 248 nm. The powder dissolution studies were conducted suspending 50 mg samples in 20 mL of water. These suspension liquids were stirred with magnetic force for 4 h at 37 °C. Then, specimens were drawn at a specified time (5,10,15,20,25,30,45, 60, 90, 120, 180, 240 min) and filtered with a 0.22 μm nylon filter. The concentration was measured within the range of its corresponding standard curve. Each experiment was repeated three times.

Crystal Structure Analysis
The cocrystal of azilsartan with 4, 4′-bipyridine (AZ-BIP) was solved in the monoclinic C2/c space group. The asymmetric unit contained one azilsartan molecule and half of a BIP molecules (Figure 1 and Supplementary Materials Figure S1). In the crystal structure of AZ-BIP cocrystal, two AZ molecules were connected to one BIP molecule through N1-H1⋯N5 (1.88 Å, 172°) interactions that were extended via O4-H2⋯N4 (1.75 Å,171°) hydrogen bonds to produce a 1D chain structure ( Figure 2).   Two azilsartan (AZ) molecules were connected to one BIP molecule through N1-H1· · · N5 interactions. Crystals 2020, x, x FOR PEER REVIEW 4 of 12 known concentrations at 248 nm. The powder dissolution studies were conducted suspending 50 mg samples in 20 mL of water. These suspension liquids were stirred with magnetic force for 4 h at 37 °C. Then, specimens were drawn at a specified time (5,10,15,20,25,30,45, 60, 90, 120, 180, 240 min) and filtered with a 0.22 μm nylon filter. The concentration was measured within the range of its corresponding standard curve. Each experiment was repeated three times.

Crystal Structure Analysis
3.1.1. AZ-BIP Cocrystal (2:1) The cocrystal of azilsartan with 4, 4′-bipyridine (AZ-BIP) was solved in the monoclinic C2/c space group. The asymmetric unit contained one azilsartan molecule and half of a BIP molecules (Figure 1 and Supplementary Materials Figure S1). In the crystal structure of AZ-BIP cocrystal, two AZ molecules were connected to one BIP molecule through N1-H1⋯N5 (1.88 Å, 172°) interactions that were extended via O4-H2⋯N4 (1.75 Å,171°) hydrogen bonds to produce a 1D chain structure ( Figure 2).   were connected to one BPE molecule through N1-H1· · · N5 (1.78 Å, 175 • ) interactions that were extended via O5-H2· · · N4 (1.70 Å, 169 • ) hydrogen bonds to produce a 1D chain structure (Figure 4). The cocrystal of azilsartan with BPE (AZ-BPE) was solved in the triclinic P-1 space group. The asymmetric unit contained one azilsartan molecule and half of a BPE molecule (Figure 3 and Figure  S2 (Supplementary Materials)). In the crystal structure of the AZ-BPE cocrystal, two AZ molecules were connected to one BPE molecule through N1-H1⋯N5 (1.78 Å, 175°) interactions that were extended via O5-H2⋯N4 (1.70 Å, 169°) hydrogen bonds to produce a 1D chain structure (Figure 4).  The US-FDA suggested distinguishing between the formation of cocrystals and salts [27]. When the ΔpKa of the two candidate compounds for cocrystal or salt is lower than 1, these two compounds are highly likely to form a cocrystal. When the ΔpKa is higher than 3, these two compounds are highly likely to form a salt. When the ΔpKa is approximately 1 to 3, there is no clear preference for cocrystals and salts [28]. As described in Table 3, the ΔpKa of the parental compounds (BIP and BPE) of these two cocrystals was approximately 1 to 3. It was impossible to judge whether AZ-BIP and AZ-BPE  (2:1) The cocrystal of azilsartan with BPE (AZ-BPE) was solved in the triclinic P-1 space group. The asymmetric unit contained one azilsartan molecule and half of a BPE molecule (Figure 3 and Figure  S2 (Supplementary Materials)). In the crystal structure of the AZ-BPE cocrystal, two AZ molecules were connected to one BPE molecule through N1-H1⋯N5 (1.78 Å, 175°) interactions that were extended via O5-H2⋯N4 (1.70 Å, 169°) hydrogen bonds to produce a 1D chain structure (Figure 4).  The US-FDA suggested distinguishing between the formation of cocrystals and salts [27]. When the ΔpKa of the two candidate compounds for cocrystal or salt is lower than 1, these two compounds are highly likely to form a cocrystal. When the ΔpKa is higher than 3, these two compounds are highly likely to form a salt. When the ΔpKa is approximately 1 to 3, there is no clear preference for cocrystals and salts [28]. As described in Table 3, the ΔpKa of the parental compounds (BIP and BPE) of these two cocrystals was approximately 1 to 3. It was impossible to judge whether AZ-BIP and AZ-BPE The US-FDA suggested distinguishing between the formation of cocrystals and salts [27]. When the ∆pK a of the two candidate compounds for cocrystal or salt is lower than 1, these two compounds are highly likely to form a cocrystal. When the ∆pK a is higher than 3, these two compounds are highly likely to form a salt. When the ∆pK a is approximately 1 to 3, there is no clear preference for cocrystals and salts [28]. As described in Table 3, the ∆pK a of the parental compounds (BIP and BPE) of these two cocrystals was approximately 1 to 3. It was impossible to judge whether AZ-BIP and AZ-BPE were cocrystals or salts through ∆pK a rules. However, Christer B suggested that the formation of cocrystals and salts can be distinguished from the C-O bonds lengths (Å) and C-N-C angles ( • ) [29]. When the specific value of the C-O bonds (long) to C-O bonds (short) lengths was close to 1.027 (15), these two compounds were highly likely to form a salt, whereas the specific value was close to 1.081 (12) for cocrystals. Obviously, the ratios were 1.112 and 1.086 Å for AZ-BIP and AZ-BPE (Table 4), respectively. It can be concluded that both are cocrystals. Furthermore, the formation of cocrystals and salts could be distinguished from the endocyclic bond C-N-C angles for pyridine-based molecules. When the endocyclic bond C-N-C angles were less than 118.5 • , these two compounds were highly likely to form a cocrystal. However, the C-N-C angles were over 120 • for salts. The endocyclic bond C-N-C angles were 114.2(4) and 116.2(2) • for AZ-BIP and AZ-BPE (Table 4), respectively. Thus, AZ-BIP and AZ-BPE could be judged as cocrystals.  The crystal structure of the binding site of azilsartan, angiotensin II type 1 receptor (AT 1 R), has recently been described, which provides basic insights into the structure-functionality relationship [30]. Molecular docking simulation of azilsartan with the AngII-AT1 receptor was performed. The lowest total score ligand conformation (RMSD < 2.0) was selected as the optimal conformer ( Figure S3 (Supplementary Materials)). The five planes (the benzimidazole group (A and B), biphenyl components (C and D), oxadiazole ring (E) and τ1 (C12-C15-N3) chain) of AZ had torsional flexibility (Figure 5a). In the following comparison, the C-planes of azilsartan were fixed and defined as a reference plane. Variable conformations of AZ in these 3 crystals (AZ, AZ-BIP and AZ-BPE) were compared with the optimal conformer and are shown in Figure 5b. Table 5 shows the torsion angle variation of AZ molecules in these crystal structures and the optimal conformer. Results revealed that the conformer of the AZ molecule in the AZ-BPE crystal exhibited the closest structural similarity to the optimal conformer.

PXRD Analysis
The PXRD patterns of all cocrystals and coformers are shown in Figure 6. Characteristic peaks of AZ ( Figure 6a) and BIP (Figure 6b

DSC and TGA Analysis
AZ, AZ-BIP and AZ-BPE cocrystals were studied by DSC and TGA under the protection of nitrogen. The DSC and TGA curves of the AZ, AZ-BIP and AZ-BPE cocrystals are displayed in Figures 7 and 8. The DSC thermograms of the AZ, BPE and AZ-BPE cocrystals showed a melting

DSC and TGA Analysis
AZ, AZ-BIP and AZ-BPE cocrystals were studied by DSC and TGA under the protection of nitrogen. The DSC and TGA curves of the AZ, AZ-BIP and AZ-BPE cocrystals are displayed in Figures 7 and 8. The DSC thermograms of the AZ, BPE and AZ-BPE cocrystals showed a melting phenomenon at 206 152 and 198 • C, respectively. Subsequently, an exothermic peak attributed to decomposition endotherm occurred, and this was consistent with the TGA curves; while the DSC thermograms of the AZ-BIP cocrystal had a melting peak at 164 • C. Two exothermic peaks were observed in the DSC, and these were attributable to secondary decomposition behavior.

Solubility and Powder Dissolution Rate Analysis
Solubility and powder dissolution results are shown in Figure 9 and Table 6. From Table 6, solubilities of the AZ-BIP and AZ-BPE cocrystals were both higher than that of azilsartan, indicating that the solubility of azilsartan can be increased by building a drug cocrystal. PXRD results of the residual samples after 24 h solubility experiments showed that the AZ-BIP and AZ-BPE cocrystals

Solubility and Powder Dissolution Rate Analysis
Solubility and powder dissolution results are shown in Figure 9 and Table 6. From Table 6, solubilities of the AZ-BIP and AZ-BPE cocrystals were both higher than that of azilsartan, indicating that the solubility of azilsartan can be increased by building a drug cocrystal. PXRD results of the

Solubility and Powder Dissolution Rate Analysis
Solubility and powder dissolution results are shown in Figure 9 and Table 6. From Table 6, solubilities of the AZ-BIP and AZ-BPE cocrystals were both higher than that of azilsartan, indicating that the solubility of azilsartan can be increased by building a drug cocrystal. PXRD results of the residual samples after 24 h solubility experiments showed that the AZ-BIP and AZ-BPE cocrystals were stable ( Figures S4 and S5 (Supplementary Materials)). The powder dissolution behavior for AZ and the AZ-BIP and AZ-BPE cocrystals in water at 37 • C are shown in Figure 9. The AZ-BIP and AZ-BPE cocrystals both showed higher powder dissolution rates than AZ. However, the powder dissolution results showed that the AZ-BIP cocrystal took two hours to reach equilibrium, indicating that the process could take more time to approach dissolution equilibrium.
Crystals 2020, x, x FOR PEER REVIEW 10 of 12 and the AZ-BIP and AZ-BPE cocrystals in water at 37 °C are shown in Figure 9. The AZ-BIP and AZ-BPE cocrystals both showed higher powder dissolution rates than AZ. However, the powder dissolution results showed that the AZ-BIP cocrystal took two hours to reach equilibrium, indicating that the process could take more time to approach dissolution equilibrium.

Conclusions
Two novel cocrystals of an antihypertensive drug azilsartan were synthesized using the solution crystallization method. In the cocrystals AZ-BIP (2:1) and AZ-BPE (2:1), crystal structure analysis revealed that two AZ molecules and the coformer connected through N-H⋯N H-bonds and formed a sandwich structure. Compared with AZ, the AZ-BIP and AZ-BPE cocrystals both showed improved solubility in water. After 24 h solubility experiments, PXRD results of the residual samples showed that the AZ-BIP and AZ-BPE cocrystals were stable. More profoundly, the powder dissolution profiles of these two cocrystals of azilsartan were studied. The two cocrystals of AZ showed better dissolution rates than that of pure AZ. The optimal conformer exhibited the best structural similarity to the AZ-BPE cocrystal via the angiotensin receptor AT1R.

Conclusions
Two novel cocrystals of an antihypertensive drug azilsartan were synthesized using the solution crystallization method. In the cocrystals AZ-BIP (2:1) and AZ-BPE (2:1), crystal structure analysis revealed that two AZ molecules and the coformer connected through N-H· · · N H-bonds and formed a sandwich structure. Compared with AZ, the AZ-BIP and AZ-BPE cocrystals both showed improved solubility in water. After 24 h solubility experiments, PXRD results of the residual samples showed that the AZ-BIP and AZ-BPE cocrystals were stable. More profoundly, the powder dissolution profiles of these two cocrystals of azilsartan were studied. The two cocrystals of AZ showed better dissolution rates than that of pure AZ. The optimal conformer exhibited the best structural similarity to the AZ-BPE cocrystal via the angiotensin receptor AT 1 R.