Crystal Structural Analysis of DL-Mandelate Salt of Carvedilol and Its Correlation with Physicochemical Properties

A 1:1 salt of carvedilol (CVD), an anti-hypertensive drug, with DL-mandelic acid (DL-MA) was crystallized from ethanol and the structure was characterized by X-ray single-crystal diffraction, revealing salt formation by transfer of an acidic proton from the COOH group of MA to the aliphatic (acyclic) secondary amino NH group of CVD. The crystal structure is triclinic, with a P-1 space group and unit cell parameters a = 9.8416(5) Å, b = 11.4689(5) Å, c = 14.0746(7) Å, α = 108.595(8), β = 95.182(7), γ = 107.323(8), V = 1406.95(15) Å3, and Z = 2. The asymmetric unit contained one protonated CVD and one MA anion, linked via an N+–H···O ̄ strong hydrogen bond and a ratio of 1:1. As previously reported, the thermal, spectroscopic, and powder X-ray diffraction properties of the salt of CVD with DL-MA (CVD_DL-MA) differed from CVD alone. The intrinsic dissolution rate of CVD_DL-MA was about 10.7 times faster than CVD alone in a pH 6.8 buffer.


Introduction
In recent years, the use of crystal engineering to prepare multi-component crystals of pharmaceutically important drug molecules [1][2][3][4][5][6][7][8], agrochemicals [9], pigments [10,11], and explosive materials [12][13][14] has been widely investigated owing to potential applications in the modification of the physicochemical properties, such as solubility, stability, and bioavailability. Among them, pharmaceutical drug molecules are extremely significant as more than 40% of marketed drug molecules suffer solubility issues [15][16][17]. Constant and continuous efforts directed toward the development of various techniques to improve solubility of the active pharmaceutical ingredient (API) include particle size reduction [18][19][20], solid dispersion with excipient [21,22], complexation with cyclodextrin [23], polymorph screening, as well as the preparation of multi-component crystals, with generally safe coformers approved by the FDA. In recent years, related research has been widely spread in literature [24][25][26][27][28][29]. Multi-component crystals of API enhance physicochemical properties such as solubility, bioavailability, and stability, and are becoming very popular due to how they neither replace nor modify the parent API component. Carvedilol (CVD), (±)-1-(carbazol-4-yloxy)-3-[[2-(omethoxyphenoxy) ethyl] amino]-2-propanol, is widely using in the treatment of hypertension, or mild to severe heart failure [30][31][32]. CVD belongs to the Biopharmaceutics Classification System (BCS) class II and has low solubility and high permeability [33]. CVD is a remarkable potential drug to improve solubility and bioavailability, with several approaches reported in the literature [41]. Salt and cocrystal preparation remain potential methods to improve solubility; however, salt formation is considered to be superior for improving aqueous solubility [42,43]. Syntheses of various pharmaceutical salts of CVD, with pharmaceutically acceptable organic acids and physicochemical properties, have been reported in literature, indicating a 1.78-fold increase in the solubility of fumaric acid salt [40]. Interestingly, reports have indicated that the solvent depended two polymorphic forms of the mandelate salt with CVD, demonstrating a monotropic relation between them based on thermal analysis and a slurry experiment [40]. Furthermore, these polymorphs were characterized by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), thermogravimetric (TG) analysis, Fourier-transform infrared spectroscopy (FT-IR), and scanning electron microscopy [40]. However, no report has investigated the X-ray singlecrystal structure of either of the polymorphic forms of this salt. Our ongoing research is focused on improving the physicochemical properties of pharmaceutical drug molecules by inducing multicomponent crystals, such as cocrystals, salt, and its solvates [44][45][46][47][48]. Moreover, we aim to establish new crystalline solid forms of the API. In this report, we discuss our efforts to improve the solubility of CVD by preparing its crystalline salts with DL-mandelic acid (DL-MA) (Figure 1), and we evaluate the X-ray single-crystal structure of stable Form I of the DL-mandelate salt of CVD (CVD_DL-MA), crystal structural analysis, and its physiochemical characteristics.

Materials
CVD (Form II) and DL-MA were purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). All other analytical-grade solvents and reagents were commercially obtained and used without further purification.

Preparation of CVD Salts
The physical mixture of CVD and DL-MA (molar ratio = 1:1) was dissolved in ethanol and the ethanol was completely removed using a rotary evaporator. Next, the residual substance was dissolved in ethanol, and the resulting solution was maintained at ambient temperature for one week, yielding a colorless block-shaped crystal.

Single-Crystal X-Ray Diffraction
The single-crystal X-ray diffraction data were collected at 123 K for CVD_DL-MA. The measurements were carried out in ω-scan mode with an R-AXIS RAPID II (Rigaku, Tokyo, Japan) using the Cu-Kα X-ray obtained from rotating the anode source with a graphite monochromator. The integrated and scaled data were empirically corrected for absorption effects using ABSCOR [49]. The initial structure was solved using the direct method with SIR 2004 and refined on Fo 2 with SHELXL

Materials
CVD (Form II) and DL-MA were purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). All other analytical-grade solvents and reagents were commercially obtained and used without further purification.

Preparation of CVD Salts
The physical mixture of CVD and DL-MA (molar ratio = 1:1) was dissolved in ethanol and the ethanol was completely removed using a rotary evaporator. Next, the residual substance was dissolved in ethanol, and the resulting solution was maintained at ambient temperature for one week, yielding a colorless block-shaped crystal.

Single-Crystal X-Ray Diffraction
The single-crystal X-ray diffraction data were collected at 123 K for CVD_DL-MA. The measurements were carried out in ω-scan mode with an R-AXIS RAPID II (Rigaku, Tokyo, Japan) using the Cu-Kα X-ray obtained from rotating the anode source with a graphite monochromator. The integrated and scaled data were empirically corrected for absorption effects using ABSCOR [49]. The initial structure was solved using the direct method with SIR 2004 and refined on Fo 2 with SHELXL 2014 [50,51]. All non-hydrogen atoms were refined anisotropically. The hydrogen atom attached to the oxygen O5, and the nitrogen N1 atom, were located using the differential Fourier map. All other hydrogen atom positions were calculated geometrically and included in the calculation using the riding atom model. All the hydrogen atoms were refined isotropically. The molecular graphics were produced using Mercury 3.7 software [52]. CCDC 1972926 contains the supplementary crystallographic data for the CVD_DL-MA, and can be obtained free of charge from the Cambridge Crystallographic Data Centre via https://www.ccdc.cam.ac.uk/structures/.

Differential Scanning Calorimetry (DSC) and Thermogravimetric (TG) Measurements
DSC and TG measurements were carried out with Thermo plus EVO2-DSC 8230 and Thermo plus EVO2-TG8120 TG-DTA, respectively (Rigaku Co., Tokyo, Japan). The DSC sample (3 mg) was placed in an aluminum crimped pan, and the TG sample (10 mg) was placed into an aluminum open pan, and they were measured at a speed of 5 • C/min from 25 to 250 • C under nitrogen gas (flow rate = 50 mL/min). Al 2 O 3 was used as a reference.

Fourier-Transform Infrared Spectroscopy (FT-IR)
The infrared spectra of samples were obtained using FT-IR (FT-IR-4200 spectrometer, JASCO Co., Tokyo, Japan) with an attenuated total reflection (ATR) unit (ATR-PRO670H-S, JASCO Co.). The spectrum recorded represents an average of 64 scans obtained with a resolution of 4 cm −1 at room temperature. The spectra were collected in the wavenumber range from 4000-400 cm −1 . The internal reflectance element used in this study was a diamond trapezoid with 45 • entrance and exit faces.

Equilibrium Solubility Experiments
Equilibrium solubility experiments were carried out using the flask shaking method. Before the solubility test, all samples were sieved using standard mesh sieves (mesh size 150 µm) to provide powders with similar particle size distribution. Each 100 mg of CVD, DL-MA, and CVD_DL-MA (about 100 mg) mixture were added to 3 mL phosphate buffer, pH 6.8 (JP 17), and mechanically shaken (120 times/min, Personal Lt-10f, Taitec corporation, Saitama, Japan) for 24 h at 37 • C. The supernatant was filtered (pore size: 0.45 µm) and the CVD concertation was determined by HPLC. The results are expressed as the mean ± standard deviation (SD) of at least three independent experiments.

Intrinsic Dissolution Experiment
The dissolution studies were carried out using the paddle method with a dissolution tester (NTR-3000, TOYAMA SANGYO CO., LTD., Osaka, Japan), and the paddle rotating speed was 100 rpm. Prior to the dissolution test, CVD and CVD_DL-MA were sieved using standard mesh sieves (mesh size 150 µm) and then the excess from the samples was added to a vessel filled with 500 mL PBS (pH 6.8) at 37 • C. Samples (9 mL) were withdrawn and filtered (0.45 µm) for analysis at specified time points, and assessed for CVD content by the HPLC method. Results are expressed as the mean ± standard deviation (SD) of at least three independent experiments.

High-Performance Liquid Chromatography (HPLC) Conditions
The HPLC comprises a PU-plus intelligent HPLC pump, a UV-intelligent UV/VIS detector, a CO-2060 plus intelligent column oven, an AS-2055 plus intelligent sampler, and a ChromNAV chromatography data system Ver. 1.08 (all from JASCO, Tokyo, Japan). The analytical column, a J-Pak Vario XBP C8-T (250 × 4.6 mm i.d., particle size 5 µm, from JASCO) was used at 55 • C. The mobile phase consisted of 0.05 M of phosphate buffer (pH 5.0) and acetonitrile (70:30, v/v) at a flow rate of 1.2 mL/min. The injection volume was 10 µL. The column eluate was monitored using a visible wavelength of 240 nm.

Crystal Structure
CVD was presented as a flexible molecule, with the central aliphatic chain attached to one terminal by carbazol-4-yloxy and other by the 2-methoxyphenoxy moiety as seen in Figure 1. As CVD has hydrogen acceptor and donor sites, the probability of forming a multi-component crystal was higher. From the crystal engineering point of view, CVD could be a potential candidate for exploring various conformations in its different crystalline forms, such as polymorph, salt, cocrystal, and solvates. In the present study, the API was CVD, and DL-MA was the coformer. The salt formation was as expected based on the basic rule of three, the ∆pKa difference between the CVD (pka 7.8) and DL-MA (pka 3.41) was more than three, and its experimental validation in the formation and characterization of salt has been reported [40]. However, the single-crystal structure was not determined. The X-ray single-crystal structure determination and the study of non-covalent interaction within is important, as this reflects the physical properties, and the structural information generated encourages further modification in achieving the desired properties. The X-ray single-crystal structure confirmed the formation of salt with approximately similar C-O bond lengths (1.247(2), 1.266(2) Å) of the (COO − ) carboxylate group of DL-MA. These similarities in the bond length of C-O confirmed the transfer of an acidic proton from DL-MA to the aliphatic (acyclic) secondary amino group of CVD. CVD_DL-MA crystalized in the triclinic P-1 space group contains one molecule of each in the asymmetric unit and is linked by strong N + -H···O − hydrogen bonds, revealing the molecular salt in the 1:1 molar ratio ( Figure 2). The crystallographic information and geometrical parameters for the hydrogen bonding interaction are summarized in Tables 1 and 2. The HPLC comprises a PU-plus intelligent HPLC pump, a UV-intelligent UV/VIS detector, a CO-2060 plus intelligent column oven, an AS-2055 plus intelligent sampler, and a ChromNAV chromatography data system Ver. 1.08 (all from JASCO, Tokyo, Japan). The analytical column, a J-Pak Vario XBP C8-T (250 × 4.6 mm i.d., particle size 5 μm, from JASCO) was used at 55 °C. The mobile phase consisted of 0.05 M of phosphate buffer (pH 5.0) and acetonitrile (70:30, v/v) at a flow rate of 1.2 mL/min. The injection volume was 10 μL. The column eluate was monitored using a visible wavelength of 240 nm.

Crystal Structure
CVD was presented as a flexible molecule, with the central aliphatic chain attached to one terminal by carbazol-4-yloxy and other by the 2-methoxyphenoxy moiety as seen in Figure 1. As CVD has hydrogen acceptor and donor sites, the probability of forming a multi-component crystal was higher. From the crystal engineering point of view, CVD could be a potential candidate for exploring various conformations in its different crystalline forms, such as polymorph, salt, cocrystal, and solvates. In the present study, the API was CVD, and DL-MA was the coformer. The salt formation was as expected based on the basic rule of three, the ΔpKa difference between the CVD (pka 7.8) and DL-MA (pka 3.41) was more than three, and its experimental validation in the formation and characterization of salt has been reported [40]. However, the single-crystal structure was not determined. The X-ray single-crystal structure determination and the study of non-covalent interaction within is important, as this reflects the physical properties, and the structural information generated encourages further modification in achieving the desired properties. The X-ray singlecrystal structure confirmed the formation of salt with approximately similar C-O bond lengths  Table 1 and Table 2.

Crystal Structural Analysis and Its Correlation with Physicochemical Properties
Single-crystal XRD showed the CVD_DL-MA salt crystalized in the triclinic P-1 space group containing one molecule of each in the asymmetric unit, suggesting the molecular salt had a 1:1 molar ratio. The proton was transferred from the COOH group of DL-MA to the aliphatic (acyclic) secondary NH group of CVDs. The conformation of protonated CVD in CVD_DL-MA salt is shown in Figure 2. In the crystal structure of salt, two inversion-symmetry related protonated CVD molecules form dimeric units, bridged by D-MA and L-MA's anions, by using the strong N + -H···O¯hydrogen bonds in R 2 4 (8) ring motif that involve two acceptor and four donor atoms. In this association, the O7 oxygen atom of both D and L-MA's anion is bifurcated and involved in N + -H···O¯hydrogen bonding, namely N2-H2A···O7, N2-H2B···O7, with two inversion-symmetry related protonated CVD molecules. Similarly, another oxygen O6 atom of both D and L-MA anions was involved in O-H···O¯hydrogen bonding with a hydroxyl group O2-H2B of protonated CVD molecules, namely a O2-H2B···O6 in R 4 4 (18) ring motif involving four donors and four acceptors ( Figure 3). Further, this dimeric association also supported by weak C-H··· π contact, namely C13-H13A···Cg4 and C15-H15A···Cg8 interactions.  Furthermore, such dimeric units linked via N1-H1AA•••O5 hydrogen bonding to the adjacent dimeric units formed a 1D chain, as shown in Figure 4. In this association, the O5 oxygen atom of the mandelate anion formed short and linear hydrogen bonds with N1-H1AA hydrogen atom of the cyclic secondary amino group of carbazol-4-yloxy moiety in protonated CVD. Furthermore, such dimeric units linked via N1-H1AA···O5 hydrogen bonding to the adjacent dimeric units formed a 1D chain, as shown in Figure 4. In this association, the O5 oxygen atom of the mandelate anion formed short and linear hydrogen bonds with N1-H1AA hydrogen atom of the cyclic secondary amino group of carbazol-4-yloxy moiety in protonated CVD.
lines indicate the non-covalent interaction (hydrogen atoms not involved in the hydrogen bonding were removed for clarity).
Furthermore, such dimeric units linked via N1-H1AA•••O5 hydrogen bonding to the adjacent dimeric units formed a 1D chain, as shown in Figure 4. In this association, the O5 oxygen atom of the mandelate anion formed short and linear hydrogen bonds with N1-H1AA hydrogen atom of the cyclic secondary amino group of carbazol-4-yloxy moiety in protonated CVD.    Neighboring 1D-dimeric chains assembled along the c-axis by weak C-H···O hydrogen bonding interaction. In this arrangement, the O2 oxygen atom of protonated CVD formed dimeric hydrogen bonds (C9-H9···O2) with the H9 hydrogen atom of carbazol-4-yloxy moiety of protonated CVD molecules, with the opposite configuration from neighboring chain along the c-axis, and further supported by C-H···O¯interaction between the O-6 oxygen atom of the MA anion with the H8 hydrogen atom of carbazol-4-yloxy moiety of protonated CVD (C8-H8···O6), with the resulting packing shown in Figure 6.

PXRD Measurements
PXRD profiles were used for the confirmation of the newly formed crystalline phase in the solid state, as well as to determine the purity of the generated form by comparing it with the simulated pattern from the X-ray single-crystal structure (Figure 8). Every crystalline phase of a compound Crystals 2020, 10, 53 9 of 14 displayed its own characteristic PXRD pattern. PXRD profiles of the CVD, DL-MA, and CVD_DL-MA (experimental and simulated) were recorded, and they confirmed the formation of a 1:1 salt of CVD and DL-MA. Furthermore, the overlay of the experimental PXRD pattern of these crystals matched the simulated PXRD pattern obtained from the single-crystal X-ray data, confirming the homogeneity of the sample and ruling out the possibility of the involvement of another phase (Form I). This form is considered as Form I, according to reported results from the previous result [40].
Crystals 2020, 10, x FOR PEER REVIEW 9 of 14 PXRD profiles were used for the confirmation of the newly formed crystalline phase in the solid state, as well as to determine the purity of the generated form by comparing it with the simulated pattern from the X-ray single-crystal structure (Figure 8). Every crystalline phase of a compound displayed its own characteristic PXRD pattern. PXRD profiles of the CVD, DL-MA, and CVD_DL-MA (experimental and simulated) were recorded, and they confirmed the formation of a 1:1 salt of CVD and DL-MA. Furthermore, the overlay of the experimental PXRD pattern of these crystals matched the simulated PXRD pattern obtained from the single-crystal X-ray data, confirming the homogeneity of the sample and ruling out the possibility of the involvement of another phase (Form I). This form is considered as Form I, according to reported results from the previous result [40].

FT-IR Spectrum
FT-IR is a powerful tool for detecting molecular complexes since the vibrational changes serve as probes for intermolecular interactions in solid materials. A comparison of the FT-IR spectra of the obtained CVD_DL-MA, CVD, and DL-MA ( Figure 9) showed numerous changes, confirming that new multi-component crystals were generated. FT-IR analysis was also used to differentiate the salt formation compared to other multi-component crystals (cocrystals), as distinguished by the proton location between the acid and the base [53][54][55]. In the formation of a salt species, typical carboxylate anions which have a carbonyl stretching band were demonstrated: a strong asymmetrical band below 1600 cm −1 , and the appearance of a shoulder between 1505 cm −1 and 1610 cm −1 where the ionized carboxyl group can be observed [56], not present in the spectra of the individual components. On the other hand, when the frequency of the carbonyl group in carboxylic acid shifted to the higher energy (approximate frequency range of 1700-1730 cm −1 ), a cocrystal species formed [57]. Examination of the FT-IR spectrum indicated a proton transfer from the salt form to CVD, confirming the salt formation between CVD and DL-MA.

FT-IR Spectrum
FT-IR is a powerful tool for detecting molecular complexes since the vibrational changes serve as probes for intermolecular interactions in solid materials. A comparison of the FT-IR spectra of the obtained CVD_DL-MA, CVD, and DL-MA ( Figure 9) showed numerous changes, confirming that new multi-component crystals were generated. FT-IR analysis was also used to differentiate the salt formation compared to other multi-component crystals (cocrystals), as distinguished by the proton location between the acid and the base [53][54][55]. In the formation of a salt species, typical carboxylate anions which have a carbonyl stretching band were demonstrated: a strong asymmetrical band below 1600 cm −1 , and the appearance of a shoulder between 1505 cm −1 and 1610 cm −1 where the ionized carboxyl group can be observed [56], not present in the spectra of the individual components. On the other hand, when the frequency of the carbonyl group in carboxylic acid shifted to the higher energy (approximate frequency range of 1700-1730 cm −1 ), a cocrystal species formed [57]. Examination of the FT-IR spectrum indicated a proton transfer from the salt form to CVD, confirming the salt formation between CVD and DL-MA. Crystals 2020, 10, x FOR PEER REVIEW 10 of 14

Thermal Properties
The thermal properties of the salt were evaluated by DSC and TG measurements. DSC revealed a single sharp endotherm at 169.4 °C, corresponding to melting. This suggested the non-involvement of any phase change before the melting point. TG data revealed that no weight loss before melting confirmed the absence of any solvent or hydrate in the crystal lattice ( Figure 10). Finally, PXRD, FT-IR, DSC, and TG data of CVD_DL-MA demonstrated good agreement with previously reported data for a Form I [40] stable polymorph. Hence, it was confirmed that the obtained salt was stable Form I.

Thermal Properties
The thermal properties of the salt were evaluated by DSC and TG measurements. DSC revealed a single sharp endotherm at 169.4 • C, corresponding to melting. This suggested the non-involvement of any phase change before the melting point. TG data revealed that no weight loss before melting confirmed the absence of any solvent or hydrate in the crystal lattice ( Figure 10).

Thermal Properties
The thermal properties of the salt were evaluated by DSC and TG measurements. DSC revealed a single sharp endotherm at 169.4 °C, corresponding to melting. This suggested the non-involvement of any phase change before the melting point. TG data revealed that no weight loss before melting confirmed the absence of any solvent or hydrate in the crystal lattice ( Figure 10). Finally, PXRD, FT-IR, DSC, and TG data of CVD_DL-MA demonstrated good agreement with previously reported data for a Form I [40] stable polymorph. Hence, it was confirmed that the obtained salt was stable Form I. Finally, PXRD, FT-IR, DSC, and TG data of CVD_DL-MA demonstrated good agreement with previously reported data for a Form I [40] stable polymorph. Hence, it was confirmed that the obtained salt was stable Form I.

Dissolution Studies
The equilibrium solubility of CVD and CVD_DL-MA were 1.2 ± 0.13 and 1.6 ± 0.03 µg/mL, respectively. The equilibrium solubility of the salt was almost 1.3 times as that of CVD alone. Thus, this work should be emphasized as an example that salt formation can improve the solubility of drugs. Furthermore, this result will be more valuable if it is accompanied by a kinetic aspect, represented by the dissolution rate. As shown in Figure 11, the intrinsic dissolution rate of the CVD_DL-MA was approximately 10.7 times faster than that of CVD alone. Overall, dissolution testing indicated higher CVD in salt samples, emphasizing the importance of the solid state of the investigated formulations, as well as the presence of an excipient that potentially creates a favorable pH environment for the drug upon dissolution as seen for the CVD_DL-MA.
Crystals 2020, 10, x FOR PEER REVIEW 11 of 14 The equilibrium solubility of CVD and CVD_DL-MA were 1.2 ± 0.13 and 1.6 ± 0.03 μg/mL, respectively. The equilibrium solubility of the salt was almost 1.3 times as that of CVD alone. Thus, this work should be emphasized as an example that salt formation can improve the solubility of drugs. Furthermore, this result will be more valuable if it is accompanied by a kinetic aspect, represented by the dissolution rate. As shown in Figure 11, the intrinsic dissolution rate of the CVD_DL-MA was approximately 10.7 times faster than that of CVD alone. Overall, dissolution testing indicated higher CVD in salt samples, emphasizing the importance of the solid state of the investigated formulations, as well as the presence of an excipient that potentially creates a favorable pH environment for the drug upon dissolution as seen for the CVD_DL-MA.

Conclusions
The X-ray structural analysis of the CVD_DL-MA salt revealed the presence of a strong association between the CVD and DL-MA. Protonated CVD formed a dimeric unit bridged with DL-MA anions by strong N + -H•••O¯, O-H•••O¯ hydrogen bonds. Furthermore, such dimeric units were linked through N-H•••O hydrogen bonding to form a 1D-dimeric chain. Neighboring 1D-dimeric chain assembled along the a, b, and c-axis by weak non-covalent interaction. Thermal and powder XRD studies confirmed that the CVD_DL-MA salt was stable Form I. The intrinsic dissolution rate of the CVD_DL-MA was approximately 10.7 times faster than that of CVD alone.