Investigation into the Structures and Properties of Multicomponent

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Investigation into the Structures and Properties of Multicomponent Crystals Formed from a Series of 7‑Chloroquinolines and Aromatic Acids Monica Clements, Margaret Blackie, Carmen de Kock,# Nina Lawrence,# Peter Smith,# and Tanya le Roex* Department of Chemistry and Polymer Science, University of Stellenbosch, P. Bag X1, Matieland, 7602, South Africa

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S Supporting Information *

ABSTRACT: The crystallization of a series of three triazolelinked 7-chloroquinoline antimalarials with two carboxylic acid coformers resulted in the formation of nine new multicomponent crystalline materials, with eight of these providing single crystal data. In each case, proton transfer between the carboxylic acid coformer and the nitrogen atom in the amino side chain of the 7-chloroquinoline drives salt formation. Solvent molecules are included in eight of the nine crystal structures, and in some instances can be removed, resulting in a solvent-free form. Formation of these multicomponent crystals by mechanochemistry was also investigated. Physicochemical properties, including solubility and thermal stability, and efficacy against Plasmodium falciparum of both the 7-chloroquinolines and the multicomponent crystals, were studied and compared. The work discussed herein raises key questions regarding the formation of multicomponent crystals as a viable alternative to discarding ineffective antiplasmodial agents.

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INTRODUCTION It is well-known that the physical properties of a crystalline drug stem from the packing and association of the molecules in the solid state, and that altering these intermolecular interactions through inclusion of the drug molecules in a supramolecular assembly could have a profound effect on the physicochemical properties of the drug.1−3 Exploitation of this concept has become popular in recent years as it allows for an improvement of some undesirable properties, without compromising the structure, and thus the efficacy, of the drug.1 This has led researchers to pursue the formation of salts and cocrystals using a number of drug molecules to determine the effect that this has on some of the physicochemical properties of the drug, such as solubility, thermal stability, bioavailability, and, ultimately, its pharmacokinetic profile.4−7 Despite these results, the use of this method in an attempt to improve some of the physicochemical properties of antimalarial compounds has received little attention.8 Malaria is a tropical disease that has resulted in over 584 000 global deaths in 2014.9 As a result of parasitic resistance to the majority of the current antimalarial agents, there is an urgent need for new and effective drugs that can be deployed to fight this widespread disease. The advancement of many of the recently synthesized antiplasmodial agents to clinical trials is currently hindered by their poor solubility and oral bioavailability. Given how long it takes to conceptualize, © XXXX American Chemical Society

synthesize, and test novel compounds, the formation of multicomponent forms of drug molecules could provide added value for already known compounds that have been previously discarded due to poor pharmacokinetic profiles. The work discussed herein explores the formation of multicomponent forms of a series of 7-chloroquinoline analogues, through crystallization with suitable organic coformers selected from the “Generally Regarded as Safe” (GRAS)10 or “Everything Added to Food in the United States” (EAFUS)11 lists. Furthermore, the effect that the formation of multicomponent crystals has on selected physicochemical properties such as solubility, thermal stability, and efficacy was studied, and the properties were compared to those of the parent drug molecule. The series of compounds which were synthesized and investigated in this study comprises three 7-chloroquinoline analogues, each with a 1,2,3-triazole moiety linking the 7chloroquinoline core scaffold and the amino side chain, both vital for the retention of antiplasmodial efficacy.12 These structures are shown in Figure 1 below. They were chosen due to their ease of synthesis, as well as the fact that they contain multiple sites for potential intermolecular interactions. Received: July 11, 2018 Revised: December 16, 2018

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been included for comparative purposes. Figures were generated using POVRay.24 Powder X-ray diffraction (PXRD) was performed on a Bruker D2 Phaser diffractometer using Cu−Kα radiation (λ = 1.540598 Å) at 30 kV and 10 mA. Samples were run using a zero-background holder. Intensity data were captured with a Lynxeye detector with 2θ scans performed in the range of 5−40° with a 0.016° step size. Samples were spun at 30 rpm. Samples obtained from solution were blotted dry and ground gently until a powder was formed. Analysis of the patterns was carried out using X’Pert HighScore Plus software and images were created using Microsoft Excel. Thermal Analysis. Thermogravimetric analyses (TGA) were performed under nitrogen (flow rate: 40 mL/min) using a TA Q500 instrument. The samples (3−7 mg) were blotted dry on filter paper to remove excess mother liquor and placed in open aluminum pans, and the traces were obtained by heating at 10 °C/min under a N2 gas purge. Differential scanning calorimetry (DSC) measurements were performed using a TA Q20 instrument coupled to an RSC cooling unit. Samples (3−7 mg) were placed in nonhermetically sealed aluminum pans with a pinhole in the lid and heated at a rate of 10 °C/ min. Analysis of the traces obtained from the TGA and DSC measurements was carried out using the TA Instruments Universal Analysis program. Nuclear Magnetic Resonance (NMR). Nuclear magnetic resonance (NMR) spectroscopy was performed on a Varian 300 or 400 MHz instrument using CDCl3 or DMSO-d6 as a solvent. Spectra were analyzed using MestReNova software. Kinetic Aqueous Solubility. Using a 10 mM stock solution of each compound in 100% DMSO, dilutions were prepared to a theoretical concentration of 200 μM (n = 2) in phosphate buffered saline (PBS) pH 6.5 (in 2% DMSO), in 0.01 M HCl pH 2 (in 2% DMSO) and in biologically relevant gastrointestinal media, fasted state simulated intestinal fluid, FaSSIF (in 2% DMSO). Calibration curves (11−220 μM) were prepared in DMSO. All dilutions were equilibrated at room temperature and mixed for 2 h on an orbital shaker. The concentration of the test compounds was determined using HPLC-DAD. Biological Evaluation. The test samples were tested in triplicate against the chloroquine-sensitive (CQS) NF54 strain of Plasmodium falciparum. Continuous in vitro cultures of asexual erythrocyte stages of P. falciparum were maintained using a modified method of Trager et al.25 Quantitative assessment of antiplasmodial activity in vitro was determined via the parasite lactate dehydrogenase assay using a modified method described by Makler et al.26 The test samples were prepared to a 20 mg/mL stock solution in 100% DMSO. Samples were tested as a suspension if not completely dissolved. Stock solutions were stored at −20 °C. Further dilutions were prepared on the day of the experiment. Chloroquine (CQ) was used as the reference molecule in all experiments. A full dose−response was performed for all compounds to determine the concentration inhibiting 50% of parasite growth (IC50 value). Test samples were tested at a starting concentration of 100 μg/mL, which was then serially diluted 2-fold in complete medium to give 10 concentrations, with the lowest concentration being 0.2 μg/mL. The same dilution technique was used for all samples. The reference molecule was tested at a starting concentration of 1000 ng/mL. The highest concentration of solvent to which the parasites were exposed had no measurable effect on the parasite viability. The IC50 values were obtained using a nonlinear dose−response curve fitting analysis via Graph Pad Prism v.4.0 software.

Figure 1. Chemical structure of the three 7-chloroquinolines (1−3) used in this study.

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MATERIALS AND METHODS

All chemicals used for the synthesis of the 7-chloroquinolines, as well as the various organic linkers and solvents that were used, were obtained from Sigma Aldridge South Africa. Precursor compounds were synthesized from literature procedures.13−15 The synthesis of compounds 1−3 followed a modified procedure of Pereira et al.,15 making use of CuI and triethyl amine in DCM rather than CuSO4·H2O and sodium ascorbate in acetonitrile for the coppercatalyzed click reaction between the quinoline azide and amino alkynes. (See Supporting Information for full reaction conditions and characterization.) Each of the three 7-chloroquinoline compounds, 1−3, was then dissolved in a DCM/hexane (8:2) solvent system, and slow evaporation of the solvent resulted in the formation of diffractionquality, block-shaped crystals of 1−3 after 3 days. Formation of the multicomponent crystals was carried out by dissolving stoichiometric amounts of each 7-chloroquinoline (20 mg) and coformer in a 1:1 molar ratio in a minimum amount of a common solvent (acetonitrile/isopropyl alcohol/DMF/DMSO), using stirring and gentle heating methods. The solutions were filtered through a microfilter, and slow evaporation of the solvent resulted in diffractionquality crystals in 1−3 weeks. It should be noted that a range of molar ratios of 7-chloroquinoline/coformer was attempted, including 1:1, 1:2, and 2:1; however multicomponent crystals were only obtained from the 1:1 cocrystallizations. X-ray Diffraction. Single crystals of diffraction quality were mounted in oil, and data were collected using a Bruker SMART Apex II X-ray diffractometer equipped with a Mo fine-focus sealed tube (λ = 0.71073 Å) for the salts with salicylic acid as a coformer. A Bruker DUO Apex II X-ray diffractometer was used for crystals obtained from DMSO of the salts with pamoic acid as the coformer. A Bruker Venture D8 Apex III X-ray diffractometer was used for the crystal structures obtained from DMF of the salts with pamoic acid as the coformer. Data collections were performed at 100 K using an Oxford Cryostream cryostat (700 series Cryostream Plus) attached to the diffractometer. Collection and reduction of data were carried out using Bruker diffraction software SAINT,16 and absorption corrections were performed using SADABS.17,18 All structures were solved using SHELXS-9719 or SHELXS-1420 and refined using SHELXL-9719 or SHELXL-1621 within the X-Seed22,23 graphical user interface. Non-hydrogen atoms were refined anisotropically, and hydrogen atoms were placed on calculated positions, except those on oxygen and nitrogen atoms, which were located using electron density maps. In the structures of 3·PA·1.5H2O·DMF and 3·PA·1.5H2O· DMSO, the hydrogen atoms on the included water molecules could not be successfully modeled and were therefore not assigned. In the structures of 1·PA·2H2O·DMF, 1·PA·1.5H2O·DMSO, and 2·PA· 1.5H2O·DMF, not all hydrogen atoms on the included water molecules could be successfully modeled, and therefore some hydrogen atoms on the water molecules were not assigned. In all structures of the pamoate salts, except for 1·PA·2H2O·DMF, the water molecules are disordered. In each of these structures water molecules are observed in three positions; however due to their proximity to each other they cannot be fully occupied and were modeled with the maximum possible site occupancy of 0.5. In total there are thus one and half water molecules in each structure modeled over three positions. We were unable to obtain better quality crystals of 1·PA·2H2O·DMF or 3·PA·1.5H2O·DMF; however, the data have

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RESULTS AND DISCUSSION Structures of the 7-Chloroquinoline Compounds. Compounds 1−3 are isostructural, with compound 1 crystallizing in the monoclinic space group P21/c and compounds 2 and 3 crystallizing in the space group P21/n, each with one molecule in the asymmetric unit. B

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Figure 2. (a) Asymmetric unit, (b) dimer, and (c) packing viewed down the a axis of 1; (d) asymmetric unit, (e) dimer, and (f) packing viewed down the a axis of 2; and (g) asymmetric unit, (h) dimer, and (i) packing viewed down the a axis of 3.

Table 1. Selected Crystallographic Data for the 7-Chloroquinoline Compounds, 1−3 molecular formula formula weight (g/mol) crystal system space group Z a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) volume (Å3) temperature (K) Rint R1 [I > 2σ(I)] wR2 GOF

1

1 (transformed)a

2

3

C16H16N5Cl 313.79 monoclinic P21/c 4 11.304(1) 11.136(1) 12.931(1) 90 115.369(1) 90 1470.9(2) 100(2) 0.021 0.032 0.086 1.04

C16H16N5Cl 313.79 monoclinic P21/n 4 13.028(3) 11.136(2) 11.304(2) 90 116.257(3) 90 1470.8(2)

C17H18N5Cl 327.82 monoclinic P21/n 4 11.719(1) 10.986(1) 13.238(2) 90 114.393(2) 90 1552.1(3) 100(2) 0.041 0.043 0.100 1.05

C16H16N5OCl 329.79 monoclinic P21/n 4 11.131(1) 11.188(1) 13.137(1) 90 113.768(1) 90 1497.3 100(2) 0.022 0.035 0.090 1.06

a

Transformation matrix (P21/c to P21/n): [1 0 1 0 1 0 1̅ 0 0].

manner, resulting in dimers with centroid-to-centroid distances of 3.63, 3.62, and 3.68 Å for compounds 1, 2, and 3, respectively. In each of the structures, these dimers pack to form a sandwich herringbone motif. In Figure 2, the

There are no hydrogen-bond donors present on these molecules, and as a result, π−π interactions play the dominant role in the association of these molecules in the solid state. In all three compounds, two molecules π−π stack in a face-to-face C

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hydrogen bonding occurs between the carboxylate moiety of SA and the amino nitrogen atom of 2. Additionally, an O−H···O hydrogen bond links the water and salicylate ions, and there is also a weak hydrogen bond between the water molecule and 2 via the nitrogen atom in the quinoline ring. These units pack together, resulting in columns of 7chloroquinoline cations parallel to the a axis, held together by face-to-face π−π stacking interactions through the column (centroid−centroid distance 3.55 Å). The asymmetric unit and packing of this salt are shown in Figure 4. In addition the structure is stabilized by weak C−H···O and C−H···N interactions. Combining 3 and SA in a 1:1 ratio in isopropyl alcohol resulted in a product with a PXRD pattern that is different from those of either of the starting materials, indicating that a multicomponent crystal had been formed. Numerous attempts to obtain single crystals of this material were carried out, but all were unsuccessful in obtaining diffraction-quality crystals. Through the use of 1H NMR, IR, and thermal analysis, it could be determined that the system most likely consists of a 1:1:1 ratio of 7-chloroquinoline/SA/H2O; however, without single-crystal data, this could not be confirmed. The second set of multicomponent crystals were obtained by cocrystallization of the 7-chloroquinolines with pamoic acid from either DMF or DMSO. Six salts were obtained, and in each case, very thin, clear, plate-like crystals formed through the slow evaporation of the solvent. Each structure contains a 1:1 ratio of 7-chloroquinoline to pamoate ions, with solvent and water molecules trapped within the crystal structure. The asymmetric unit of each crystal structure is shown in Figure 5, and relevant crystallographic data can be found in Table 3. 1·PA·1.5H2O·DMSO, 2·PA·1.5H2O·DMSO, and 3·PA· 1.5H2O·DMSO crystallize from DMSO in the triclinic P1 space group and are isostructural. Each 7-chloroquinoline cation is linked to one pamoate anion through charge-assisted hydrogen bonding (between the carboxylate moiety of PA and the amino moiety of 1, 2, or 3), as shown in Figure 5a,c,e. These pairs of ions are additionally linked through an O−H··· N interaction between the second carboxylic acid moiety of PA and the nitrogen atom in the quinoline ring of a second molecule of 1, 2, or 3, resulting in the formation of a tetrameric unit that is located on a center of inversion. These tetrameric units stack along the a axis, forming columns which then pack in three dimensions, with the water and solvent molecules located between these columns in each of the structures. Interestingly, contrary to the structures formed with the salicylic acid coformer, there are no face-to-face π−π interactions between quinoline moieties, but rather face-toface π−π interactions between pairs of quinoline and pamoate naphthalene moieties (centroid to centroid distances of 3.66− 3.68 Å in the three structures). Edge-to-face π−π interactions are also present between a quinoline ring and the π cloud of one of the aromatic rings of the pamoate anion (centroid to centroid distances of 4.83−4.89 Å in the three structures). In addition, there is a DMSO molecule present in each of the structures, as well as one water molecule disordered over two positions, each with a site occupancy of 0.5, and another water molecule with a site occupancy of 0.5, which is disordered around a center of inversion. These solvent and water molecules are located between the columns of tetrameric units in each of the structures. The water molecule which is disordered over two positions, is hydrogen bonded to the DMSO molecule through an O−H···O interaction, as well as

asymmetric unit, the dimer and the packing diagram (viewed down the a axis) of 1, 2, and 3 are shown. Unit cell dimensions and other crystallographic data for compounds 1, 2, and 3 can be found in Table 1. Structures of the Multicomponent Crystals. Coformers were selected using the synthon approach, with complementary functional groups being identified on both the 7chloroquinoline and the coformer. The following coformers were chosen: oxalic acid, succinic acid, malic acid, fumaric acid, citric acid, L-ascorbic acid, salicylic acid, benzoic acid, pamoic acid, nicotinic acid, nicotinamide, caffeine, and theophylline. While many cocrystallizations were attempted with these coformers using a variety of solvents, only salicylic acid (SA) and pamoic acid (PA) yielded any multicomponent crystals with the 7-chloroquinoline compounds. The remaining combinations resulted either in no crystallization of any product or the crystallization of a mixture of starting components, as determined by PXRD. The first set of multicomponent crystals obtained includes salts which were formed when 1, 2, and 3 were combined with salicylic acid in a 1:1 molar ratio in MeOH, acetonitrile, or isopropyl alcohol, respectively. In each case crystals were formed after 3−4 days by slow evaporation of the solvent. Relevant crystallographic data for these salts can be found in Table 2. Table 2. Selected Crystallographic Data for the Salts Obtained Using Salicylic Acid As the Coformer molecular formula formula weight crystal system space group Z a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) volume (Å3) temperature (K) Rint R1 [I > 2σ(I)] wR2 GOF

1·SA

2·SA·H2O

C16H16N5Cl·C7H6O3 451.91 monoclinic P21/n 4 7.4354(9) 10.423(1) 26.584(3) 90 96.322(2) 90 2047.8(4) 100(2) 0.053 0.047 0.118 1.05

C17H18N5Cl·C7H6O3·H2O 483.96 monoclinic P21/n 4 7.106(1) 27.765(5) 11.936(2) 90 95.994(2) 90 2342.2(7) 100(2) 0.035 0.042 0.100 1.03

The combination of 1 and SA in methanol yielded crystals of a 1:1 salt, 1·SA. The structure is formed through chargeassisted hydrogen bonding between the carboxylate moiety of SA and the amino nitrogen atom of 1. The packing of these units results in columns of 7-chloroquinoline cations parallel to the a-axis, held together by offset π−π stacking interactions (centroid−centroid distances of 3.74 and 4.12 Å). These columns pack together in three dimensions resulting in a layered structure with alternating anionic and cationic layers in the ab plane, as can be seen in Figure 3. In addition, the structure is stabilized by weak C−H···O and C−H···N interactions. The combination of 2 and SA in acetonitrile yields a salt in a 1:1 ratio with a molecule of water included in the structure (2· SA·H2O). As in the structure of 1·SA, charge-assisted D

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Figure 3. (a) Asymmetric unit and (b) packing diagram viewed down the b axis of 1·SA.

Figure 4. (a) Asymmetric unit and (b) packing diagram viewed down the c axis of 2·SA·H2O.

Figure 5. Asymmetric unit of (a) 1·PA·1.5H2O·DMSO, (b) 1·PA·2H2O·DMF, (c) 2·PA·1.5H2O·DMSO, (d) 2·PA·1.5H2O·DMF, (e) 3·PA· 1.5H2O·DMSO, and (f) 3·PA·1.5H2O·DMF showing the hydrogen-bonding interactions between the components.

E

DOI: 10.1021/acs.cgd.8b01049 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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Table 3. Selected Crystallographic Data for the Salts Formed by the 7-Chloroquinoline and Pamoic Acid molecular formula formula weight crystal system space group Z a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) volume (Å3) temperature (K) Rint R1 [I > 2σ(I)] wR2 GOF

1·PA·1.5H2O·DMSO

1·PA·2H2O·DMF

2·PA·1.5H2O·DMSO

2·PA·1.5H2O· DMF

3·PA·1.5H2O·DMSO

C16H16N5Cl· C23H16O6·1.5(H2O)· C2H6OS 805.31 triclinic P1̅ 2 8.7897(8) 10.4028(9) 21.425(2) 79.750(4) 88.229(5) 88.317(5) 1926.3(3) 100(2)

C16H17N5Cl· C23H15O6·2(H2O)· C3H7NO 810.26 monoclinic P21/n 4 10.946(1) 8.7835(7) 40.830(3) 90 94.182(2) 90 3915.2(6) 100(2)

C17H19N5Cl· C23H15O6·1.5(H2O)· C2H6OS 820.34 triclinic P1̅ 2 8.9591(6) 10.5640(7) 21.066(1) 99.166(1) 90.623(1) 91.020(1) 1967.8(2) 100(2)

C17H19N5Cl·C23H15O6· 1.5(H2O)·C3H7NO 814.80 triclinic P1̅ 2 8.833(2) 10.629(2) 21.280(4) 99.033(2) 90.229(3) 91.903(3) 1971.8(7) 100(2)

C16H17N5OCl· C23H15O6·1.5(H2O)· C2H6OS 821.31 triclinic P1̅ 2 8.5856(2) 10.6183(3) 21.4338(6) 80.608(2) 87.134(2) 87.965(2) 1924.66(9) 100(2)

3·PA·1.5H2O·DMF

815.26 triclinic P1̅ 2 8.4951(3) 10.5678(3) 22.0366(7) 100.150(2) 95.684(2) 90.836(2) 1936.7(1) 100(2)

0.050 0.057 0.156 1.03

0.092 0.059 0.146 1.02

0.054 0.053 0.136 1.02

0.067 0.052 0.128 1.03

0.047 0.049 0.132 1.07

0.181 0.067 0.193 1.03

C16H17N5OClC23H15O6· 1.5(H2O)·C3H7NO

Figure 6. (a) Tetramer formed by 1 and PA with solvent and water molecules omitted and (b) the packing of 1·PA·2H2O·DMF shown down the b axis.

centroid distances of 3.62−3.71 Å in the three structures), and the same edge-to-face π−π interactions are also found between a quinoline ring and an aromatic ring of the pamoate anion (centroid to centroid distances of 4.82−4.85 Å in the three structures). The hydrogen bonding linking the 7-chloroquinoline and pamoate ions in all three salts is analogous to that in the structures of the DMSO solvates described above, resulting in the formation of similar tetrameric units (Figure 6a) that pack down the b axis (in 1·PA·2H2O·DMF) or down the a axis (in 2·PA·1.5H2O·DMF and 3·PA·1.5H2O·DMF), forming columns. These columns then pack in three dimensions, resulting in the formation of small channels in the case of 1· PA·2H2O·DMF and 2·PA·1.5H2O·DMF, down either the b or a axis, in which the water molecules are located (see Figure 6b). In the structure of 3·PA·1.5H2O·DMF, the morpholinyl ring of 3 is disordered over two positions, with site occupancies which refine to 0.67 and 0.33. Due to this disorder the water molecules are located in pockets between the columns of tetrameric units, rather than in channels. The hydrogen

to the pamoate anion via the carboxylate group. The water molecule disordered around the center of inversion links the other water molecules via hydrogen bonding. In addition the structures are stabilized by a number of weak C−H···O interactions between the amino side chain and the included water molecules. 2·PA·1.5H2O·DMF and 3·PA·1.5H2O·DMF crystallize in the triclinic space group P1̅ and are isostructural to a large extent to the pamoate salts formed from DMSO. Both of these salts have one DMF molecule as well as one and a half water molecules included in the structure, with the water molecules disordered over three positions, each with a site occupancy of 0.5. 1·PA·2H2O·DMF crystallizes in the monoclinic space group P21/n, and has one DMF molecule and two water molecules included in the structure, however many of the same structural features that are observed in the other pamoate salts are also preserved in this structure. As in the structures formed from DMSO, all of the pamoate salts formed from DMF also have face-to-face π−π interactions present between pairs of quinoline and pamoate naphthalene moieties (centroid to F

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corresponds to the evaporation of the water from the system, rather than a melt. This was confirmed visually using a melting point apparatus. A similar observation was made for the crystals formed by the combination of 3 and SA, which also showed two endotherms in the DSC trace which are likely to correspond to the dissolution and subsequent loss of water from the system. The pamoate salts all exhibited DSC traces with multiple endotherms. TGA was used as a complementary method to determine whether the endotherms corresponded to solvent loss or a melt. As an example, an overlay of the TGA and DSC trace of 2·PA·1.5H2O·DMF is shown in Figure 8, where two mass losses of 4.7% and 8.1% (total 12.8%) can be observed in the TGA trace. These percentages correspond to the calculated mass loss for one and a half water molecules and one DMF molecule (calculated mass loss = 12.3%), which corresponds to a ratio of 1:1:1.5:1 of 7-chloroquinoline/PA/H2O/solvent as confirmed by single crystal X-ray diffraction. The first endotherm at 87.5 °C in the DSC trace correlates with the loss of water from the system, while the second endotherm at 128.9 °C correlates with the loss of the DMF solvent from the system. The sharp endotherm at 240.3 °C would then correspond to the simultaneous melt and decomposition of the system, which is over 2-fold higher than the melting point of the 7-chloroquinoline. Similar TGA and DSC traces were obtained for the remaining salts, which can be found in the Supporting Information. The TGA traces for each of the pamoate salts showed an initial solvent loss, well before decomposition of the material at 240 °C. We thus attempted to remove the solvent in order to determine whether another solvent-free form of the salt could be identified. This was achieved by heating the powdered sample to the respective temperatures that solvent loss is observed at during TGA analysis. Subsequent PXRD indicated that a new phase had been obtained that did not match that of the salt nor a mixture of the individual components. The same solvent-free forms are obtained from desolvation of either the DMSO or DMF solvate of the pamoate salts of 1 and 3; however in the case of desolvation of the salts of 2, the PXRD patterns after desolvation were inconclusive. Hot stage microscopy was used to determine whether the loss of solvent occurred in a single-crystal-to-single crystal fashion; however, this was not the case as the crystals became opaque and no longer diffracted once the solvent had been lost. Unfortunately, single crystals of these new forms could not be obtained as they do not dissolve in any solvent except DMSO and DMF. Simultaneous melt and decomposition made melt recrystallization methods impractical for these samples. In Vitro Properties. Kinetic Solubility. Overall, the kinetic solubilities of compounds 1−3 were excellent, with all values greater than 150 μM. In most cases values were higher in an acidic medium than in a more neutral medium. In the two cases of 1 and 2, the solubility values decreased at pH 6.5 when cocrystallized with salicylic acid, while in the case of 3, a slight increase was observed. Similar results can be seen in FaSSIF media, where the cocrystallization either had no effect, or reduced the solubility of the system. For the multicomponent crystals with pamoic acid, the solubility results were irregular as a result of two peaks in the HPLC chromatogram. Due to the presence of two carboxylic acid moieties on the coformer, as well as the presence of multiple proton acceptor sites on the 7-chloroquinoline, solution effects could cause a double deprotonation of the

bonding of the water molecules in 2·PA·1.5H2O·DMF is the same as that observed in the pamoate salts obtained from DMSO. In 1·PA·2H2O·DMF and 3·PA·1.5H2O·DMF the hydrogen bonding of the water molecules is also very similar, with the only differences being that in 1·PA·2H2O·DMF the water molecules are not disordered and in 3·PA·1.5H2O·DMF the positions of the water molecules are slightly different. A number of weak C−H···O interactions are also present in all three of these structures between the amino side chain and the included water and solvent molecules. The fact that only two coformers resulted in multicomponent crystal formation could possibly be due to the fact that the 7-chloroquinoline molecules already pack in a space-efficient manner and that the incorporation of the coformer into the crystal lattice is simply not a preferable alternative as a packing arrangement in the solid state. Only the use of these particular organic acids appeared to give rise to hydrogen-bonding interactions that were favorable enough to result in heteromeric molecules assembling in the solid state. Mechanochemistry. The use of mechanochemistry (neat and liquid-assisted grinding using a mortar and pestle) was investigated as an alternative method of synthesizing these multicomponent crystals. Stoichiometric equivalents of each component were ground together (either with or without 1 drop of solvent) for 5 min before obtaining a PXRD pattern (see Supporting Information). The liquid-assisted grinding experiments were successful for the synthesis of the salicylic acid salts. In the case of the pamoic acid salts, the addition of even one small drop of solvent resulted in a paste that could not be analyzed by PXRD. In an attempt to synthesize the desolvated forms of the salts of pamoic acid, neat grinding of the two components was attempted, but even after longer grinding times (10−15 min), only a mixture of the starting components was obtained in all cases. Thermal Analysis. The thermal properties of the parent 7chloroquinoline compounds as well as the corresponding salts were investigated and compared. TGA and DSC traces for each system can be found in the Supporting Information. A summary of the melting points of the individual components as well as each salt can be found in Table 4. Table 4. Summary of the Melting Points (°C) of the Individual Components and Salts 1·SA 2·SA·H2O 3·SA·H2O 1·PA·2H2O·DMF 2·PA·1.5H2O·DMF 3·PA·1.5H2O·DMF 1·PA·1.5H2O·DMSO 2·PA·1.5H2O·DMSO 3·PA·1.5H2O·DMSO

7-chloroquinoline

coformer

salts

100.8 112.4 160.4 100.8 112.4 160.4 100.8 112.4 160.4

160.5

152.9

325.5

242.0 238.9 232.6 246.1 239.8 228.7

325.5

The DSC trace of 1·SA displays a melting point between that of the two starting components, 52 °C higher than that of the original 7-chloroquinoline. For the case of 2·SA·H2O, two endotherms are seen in the DSC trace between 75 and 110 °C (Figure 7). By studying the TGA of this system the first endotherm in the DSC trace at 82.7 °C can be attributed to the loss of water from the crystals and into the surroundings, causing dissolution of the system. The second endotherm G

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Figure 7. (a) Comparison of the DSC of 2 (blue), SA (green), and 2·SA·H2O (red). (b) Overlay of the TGA (blue) and DSC (red) of 2·SA·H2O.

Figure 8. (a) Comparison of the DSC of 2 (blue), PA (green), and 2·PA·1.5H2O·DMF (red). (b) Overlay of the TGA (blue) and DSC (red) of 2· PA·1.5H2O·DMF.

Why, then, is only one form (singly protonated) seen in the solid state? This is likely either because a different solvent was used, or that the singly protonated form is favored in the solid state, and once crystallization of this form occurs, it drives the equilibrium toward the crystallization of this form. Consequently, a mixture of both species is not obtained in the solid state, and hence the single crystal data and PXRD patterns correlate. Because of the formation of multiple species in solution, this method could not be used to assess the kinetic solubility for these multicomponent crystalline materials, and care should be taken in future when using this method, especially when more than one proton donor and acceptor site is present within the system. Efficacy against P. falciparum. The efficacy against P. falciparum of the parent molecules was compared to that of the corresponding multicomponent form with salicylic acid and pamoic acid as coformers. In the case of the pamoate salts, the

Table 5. Kinetic Solubility Results for the Multicomponent Crystals with SA kinetic solubility (μM) compound

pH 6.5

FaSSIF

pH 2

1 1·SA 2 2·SA·H2O 3 3·SA·H2O

192.4 176.5 200.0 196.1 178.3 184.7

199.9 161.3 200.0 200.0 200.0 185.4

198.2 188.6 198.2 200.0 198.0 192.3

coformer and subsequent protonation of the 7-chloroquinoline. This is especially true in protic solvents such as water. Consequently, the pH values used in the solubility assay could result in the formation of a mixture of singly and doubly protonated forms of the multicomponent system, resulting in two peaks observed on the HPLC chromatogram.

Table 6. Comparison of Efficacy Results for the Individual Compounds, Their Respective Multicomponent Forms, and Known Antimalarial Agents Chloroquine and Artesunate sample code

NF54: IC50 (μg/mL)

sample code

NF54: IC50 (μg/mL)

sample codea

NF54: IC50 (μg/mL)

1 2 3 chloroquine artesunate

4.56 ± 0.64 8.62 ± 1.4 66.7 ± 8.8 0.003 ± 0.001 100 ± ND 91.7 ± 9.4 >100 ± ND

a

In each case, a sample of the desolvated pamoate salt obtained from DMSO was sent for testing. H

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The results of this study show that the inclusion of antiplasmodials in multicomponent crystals may certainly be useful, and that further investigation into the use of multicomponent systems is warranted; however, further study regarding testing of some of their properties is required, and explicit connections need to be made with in vivo pharmacokinetic data. This work indicated that the choice of coformer can certainly have an influence on the efficacy of a drug molecule, either through synergistic or antagonistic effects or that the coformer can alter some physicochemical aspect of the drug molecule that, in turn, has an effect of the efficacy of the drug against P. falciparum.

desolvated forms were tested in each case, rather than the solvated forms, in order to avoid the results being influenced by the solvent. A comparison of the results obtained are summarized in Table 6. All compounds that were tested have poor efficacies in comparison to the two standards, chloroquine and artesunate. However, in all three cases where salicylic acid is used as a coformer, the efficacy of the multicomponent crystals have improved significantly, with the most significant improvement being in the case of the salt formed by 3 and SA. It was also found that the use of pamoic acid as a coformer has a detrimental effect on the efficacy of the compounds. This suggests that while salicylic acid may have synergistic effects, pamoic acid could in fact be acting as an antagonist for these 7chloroquinolines.

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ASSOCIATED CONTENT

S Supporting Information *

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.8b01049. Relevant data pertaining to the synthesis of the 7chloroquinolines, PXRD, thermal analysis, and in vitro work (PDF)

CONCLUSIONS The formation of multicomponent crystals with 1,2,3-triazolecontaining 7-chloroquinolines was investigated using a variety of coformers that can all be found on the GRAS or EAFUS lists. Both linear and aromatic carboxylic acids were chosen based on their capabilities as both hydrogen bond donors and acceptors; however only the aromatic acids yielded any multicomponent products. Although there are many possible hydrogen acceptor sites on the 7-chloroquinolines, it seems that the charge-transfer hydrogen bond between the carboxylate and amino moieties ultimately drives the packing of the molecules in the solid state. Crystal structures of 1 and 2 with salicylic acid showed the formation of a charge-assisted hydrogen bond between the carboxylate moiety of the coformer and the nitrogen atom in the amino side chain of the 7-chloroquinoline. Additional π−π interactions play a role in the three-dimensional packing of the molecules in the solid state. It was found that these salicylic acid salts can also all be formed by liquid-assisted grinding. The crystal structures of the salts formed with pamoic acid displayed the same charge-assisted hydrogen bonds observed in the salts formed with salicylic acid. Additionally, a second O−H···N hydrogen bond between the remaining carboxylic acid moiety on the coformer and the nitrogen in the quinoline ring of the 7-chloroquinoline links the molecules into tetrameric units that stack in columns in three dimensions. Different π−π interactions are also observed in these structures compared to those in the salicylate salt structures. In each of these systems, both water and solvent (DMSO or DMF) molecules are present in the crystal structures, and it was found that these can be removed by heating, resulting in a new crystalline form. These new forms are most likely the 1:1 salt of the 7-chloroquinoline to coformer; however, no crystal structures could be obtained. Thermal analysis revealed that the melting point of each new multicomponent crystal was significantly higher than that of the parent 7-chloroquinoline. While the kinetic solubilities of the parent compounds were excellent, the use of salicylic acid as a coformer decreased the solubility over both pH values; however, the salicylic acid salts are still considered to have good aqueous solubility. The use of pamoic acid, however, shed light on the fact that care should be taken when choosing coformers, especially those with multiple proton donor and acceptor sites. The question of whether the methodology used is suitable for the determination of the aqueous solubility, especially in cases where proton transfer occurs between the components, is valid.

Accession Codes

CCDC 1852929−1852939 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; tel: +27 21 808 3343. ORCID

Tanya le Roex: 0000-0002-1874-1996 Present Address #

(C.d.K., N.L., P.S.) Division of Clinical Pharmacology, Department of Medicine, University of Cape Town, Observatory, 7925, South Africa.

Notes

Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors, and therefore the NRF does not accept any liability in regard thereto. The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the National Research Foundation of South Africa and Stellenbosch University for funding. REFERENCES

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