Regulation of π•••π Stacking Interactions in Small Molecule Cocrystals

Article Cite This: Cryst. Growth Des. 2018, 18, 1448−1458

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Regulation of π···π Stacking Interactions in Small Molecule Cocrystals and/or Salts for Physiochemical Property Modulation Published as part of a Crystal Growth and Design virtual special issue on π−π Stacking in Crystal Engineering: Fundamentals and Applications Pranita Bora, Basanta Saikia, and Bipul Sarma* Department of Chemical Sciences, Tezpur University, Napam 784028, Assam, India S Supporting Information *

ABSTRACT: The noncovalent interactions arising from solute···solute (i.e., drug···drug, drug···neutraceutical, or drug···coformer) and solute···solvent play a significant role in predicting desired properties of an active compound. We demonstrated here the role of π···π interactions in the presence of hydrogen bonding, the two important cohesive and adhesive forces in the crystallization of small molecules to regulate certain physiochemical properties in their multicomponent crystals. Acridine was employed as a representative cocrystal partner with isomeric dihydroxybenzoic acids. The choice was intentional as with a single hydrogen bond acceptor acridine provides increased surface area to favor the stacking of πframeworks at van der Waals separation (∼3.5 Å) and herringbone C−H···π interactions, and isomeric dihydroxybenzoic acids easily form COOH···Nacridine and O−H···Nacridine hydrogen bonds in competition. Structure elucidation of several cocrystals/salts underlines the influence of continuous and discrete π···π stacking and C−H···π interactions supported by other hydrogen bonds on their physiochemical properties such as solubility, cell membrane permeation, and release behavior in vitro. Experiments were performed in various pH ranges (pH = 1.2 SAL and 7.4 PBS) in order to imitate human physiological conditions. Molecular packing and interaction energies suggest a significant contribution of π···π interactions in the modulation of property. In fact coformers’ conformational energy, lipophilicity, and Log P values were found to be valued contributors. Therefore, the present study anticipates the contribution towards understanding of the impact of π···π and C−H···π interactions supported by hydrogen bonds on modulating physiochemical properties, essentially improving efficacy of a drug.

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INTRODUCTION Aryl−aryl interactions between aromatic moieties have been used to manipulate the organization of molecules in the crystal lattice. Aromatic rings can interact via edge-to-face geometry or herringbone T-motif, and face-to-face or π-stacked sandwich geometry.1 The π-rings generally orient themselves in a herringbone T-motif instead of π-stacking due to stabilization from edge-to-face C−H···π interaction.2 Among the π-stacked geometries parallel-displaced geometry with lateral offset is energetically favored, because the aromatic ring exhibits a positively charged σ-framework sandwiched orientation between two regions of negatively charged π-electron density.3 Perfectly sandwiched orientation is generally disfavored due to the dominance of π−π repulsion. The hydrogen bonding in molecular solids, π−π and C−H···π interactions, etc. are found to be crucial in protein folding and stability,4 DNA base stacking and its function, many asymmetric syntheses,5 and in modulating properties of crystalline solids majorly controlled by aromatic interactions.6 For example the complex of the enzyme acetylcholinesterase (AChE) with the drug E2020 (Aricept) to treat Alzheimer’s disease is stabilized by π−π stacking, O−H···π and cation···π interactions.7 Therefore, the π···π stacking and hydrogen bonding are two important adhesive and cohesive © 2018 American Chemical Society

forces in the crystallization of molecules irrespective of whether it is a protein or a small molecule. Representative articles by Desiraju and Gavezzotti,1a Hunter and Sanders,1b Janiak,1c Nishio,2b and Iverson8 have intricately studied the chemical and electronic factors that promote recognition between π- or phenyl rings and their adopted motifs (Figure 1). Acridine (Acr) has been considered as a cocrystal partner because of its ability to participate in hydrogen bonding (O−H···N)9 with donor molecules such as isomers of dihydroxybenzoic acids (DHBA, Scheme 1). The cocrystallization of Acr and isomeric DHBAs could also provide more than one crystalline modification because of the competition between COOHDHBA···NAcr and OHDHBA···NAcr hydrogen bonding.10 The formation of the strongest COOHDHBA···NAcr heterosynthon is expected and observed in the product cocrystals/ salts with stacking of N-heterocycles. Six cocrystals/salts were isolated and subjected to physiological property evaluation (Scheme 1) and probing of the link with π···π stacking behavior Received: September 28, 2017 Revised: December 13, 2017 Published: January 29, 2018 1448

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Figure 1. Types of face-to-face π-stacking (N-facing same and N-facing opposite) between acridines (top) and edge to face T-shaped C−H···π interactions (bottom) observed in cocrystals/salts synthesized.

Scheme 1. Preparation of Cocrystals/Salts of Acridine with Isomeric Hydroxybenzoic Acids

component systems.20 However, functions of these interactions in regulating solubility, diffusivity, and cell membrane permeation are still obscure. Therefore, it is imperative and the present study sought to understand structure−activity relationships in multicomponent systems with the precise determination of activities played by various weak noncovalent interactions. The rational thought for choosing cocrystal systems is because the cocrystal technology is an advanced technology used in new or existing drug delivery systems for product improvement synergistically by changing molecular conformations and intermolecular interactions. Combination drug therapy is another important outcome of cocrystal technology.21 Six cocrystals/salts were isolated as demonstrated in Scheme 1 and characterized with spectroscopic techniques, thermally and structurally to understand various intermolecular interactions between Acr and DHBA. The interactions were exploited in property modulation, and during the process the role of solvent/media and pH was given careful attention. The observed differences in stacking interactions, C−H···π, and hydrogen bonding patterns were put together to predict thermodynamic and kinetic properties of the cocrystals. The

as well as C−H···π weak interactions supported by O−H···N and O−H···O hydrogen bondings. The emphasis on anti-parallel or quasi-parallel fashion due to face-to-face π···π stacking interactions has been drawn in several examples of multicomponent and host−guest complexes.11 The π···π stacking in close proximity is a prerequisite for photochemical reaction.12 The use of N-heterocycles13 such as acridine and phenazine in cocrystallization experiments is generally beneficial because they can interact with cocrystal formers via stacking, hydrogen bonding, and other interactions such as halogen bonding if halogen is present.14 A number of therapeutic agents are known based on the acridine nucleus such as quinacrine (antimalarial), acriflavine and proflavine (antiseptics), ethacridine (abortifacient), amsacrine and nitracine (anticancer), and tacrine.15 Exploiting intermolecular O− H···N hydrogen bonds and π···π stacking in supramolecular architecture,16 formation of charge-transfer complexes,17 ferroelectric organic solids,18 photochromic applications19 by phenazine, acridine molecules have been studied. Lately a few studies have appeared in the direction of understanding the role of weak interactions such as hydrogen bond, π-stacking, van der Waals forces in modulating certain properties in multi1449

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module. A total of 4−6 mg of the crystals was placed in crimped and vented pans and analyzed at a temperature range of 30−300 °C at a heating rate of 5 °C min−1 (Figure 3). Details of the melting range of

evaluated aqueous solubility, diffusivity, and/or membrane permeation at various physiological pH values shown by them were correlated to aromatic stacking, edge-to-face C−H···π interactions, and hydrogen bonding. The trade-off nature of kinetic and thermodynamic parameters was also emphasized.

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EXPERIMENTAL SECTION

Materials. Acridine and the isomeric dihydroxy benzoic acids were purchased from alfa-aesar and used as received. The phase purity of acridine was checked by powder X-ray diffraction, which matched the simulated powder pattern of acridine polymorphic form III (Figure S1).22 HPLC-grade solvents (methanol, ethanol, acetonitrile, chloroform, etc.) used for crystallization were obtained from Merck, India, and used without further purification. Double-distilled water of zero ionic strength and zero buffer capacity was used to prepare the buffer solutions resembling gastric fluids (pH 1.2) and intestinal fluids (pH 7.4) for carrying out the solubility and permeability experiments. Preparation of Cocrystals/Salts. The product materials were prepared by using the mechanochemical neat grinding method. Materials are taken in a stoichiometric ratio in a mortar pestle and ground for about 45 min. Then the materials were transferred to a 10 mL conical flask and dissolved in different organic solvents. The solutions were kept undisturbed, and suitable crystals were grown after 1−2 days for the cocrystals/salts CC-1 [1:1 ≡ Acr:23DHBA], CC-2 [1:1 ≡ Acr:24DHBA], CC-3 [1:1 ≡ Acr:25DHBA], CC-4 [1:1 ≡ Acr:26DHBA], CC-5 [Acr:34DHBA], and CC-6 [3:1 ≡ Acr:35DHBA] (Scheme 1). They were characterized using thermal analysis, spectroscopy, and X-ray diffraction techniques. Vibrational Spectroscopy (FT-IR). A PerkinElmer Frontier MIR FIR spectrophotometer was used to record the IR spectra by dispersion of the sample in KBR pellet ranging from 450 to 4000 cm−1 (Figure 2). All major vibrational frequencies (in cm−1) for

Figure 3. DSC endotherms represent melting onset of the cocrystals/ salts CC-1 to CC-6. the product materials were compared with starting compounds and summarized in the Supporting Information, Table S1. The instrument was calibrated before for temperature and heat flow accuracy using the melting of pure indium (mp 156.6 °C and ΔH of 25.45 J g−1). Powder X-ray Diffraction. Powder XRD of all samples was recorded on a Bruker D8 Focus X-ray diffractometer, Germany using Cu−Kα X-radiation (λ = 1.54056 Å) at 35 kV and 25 mA. Diffraction patterns were collected over a 2θ range of 5−50° at a scan rate of 5° min−1 (Figure 4). Rietveld refinement was performed for phase purity using Powder Cell 2.3 (overlay in Figure S2). Single Crystal X-ray Diffraction. Single crystal X-ray reflections were collected on a Bruker SMART APEX-II CCD diffractometer using Mo Kα (λ = 0.71073 Å) radiation. Bruker SAINT software23 was employed for reducing the data and SADABS for correcting the intensities of absorption. Crystal structures of CC-1, CC-2, CC-3, CC-

Figure 2. FT−IR spectra of acridine cocrystals/salts with isomeric dihydroxybenzoic acids (CC-1 to CC-6) compared with pure acridine. cocrystals/salts are assigned as follows Acr: 3041(aromatic C−H), 1512 (CN); CC-1: 3410 (O−H), 3043 (aromatic C−H), 1573 (COO−), 1406 (COO−), 1319 (C−N); CC-2: 3467 (O−H), 3044 (aromatic C−H), 1583 (COO−), 1406 (COO−), 1308 (C−N); CC-3: 3301 (O−H), 3044 (aromatic C−H), 1583 (COO−), 1445 (COO−), 1343 (C−N); CC-4: 3460 (O−H), 3050 (aromatic C−H), 1583 (COO−), 1448 (COO−), 1346(C−N); CC-5: 3247 (O−H), 3057 (aromatic C−H), 1656 (CO), 1377 (C−N); CC-6: 3496 (O−H), 3051 (aromatic C−H), 1675 (CO), 1331 (C−N). Differential Scanning Calorimetry (DSC). DSC thermograms of all the cocrystals/salts were recorded on a Mettler Toledo DSC 822e

Figure 4. PXRD patterns of pure acridine and cocrystals/salts CC-1 to CC-6. 1450

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4, and CC-6 were solved and refined using SHELXL24 with anisotropic displacement parameters for non-H atoms. Suitable crystals for single crystal X-ray data collection of CC-5 could not be isolated. In all crystal structures H atoms are located experimentally, whereas C−H atoms were fixed geometrically using the HFIX command in SHELX-TL. The figures and packing diagrams were made using X-Seed (Figure 5).25 ORTEPs were generated with 50%

Solubility Measurements. UV−vis spectroscopy was used to determine the solubility of all materials and pure Acr at room temperature considering two different pH values, i.e., pH 7.4 and 1.2 (Figure 8 and Figure 9). All experiments were performed in an Agilent Carry-60 UV−visible double beam spectrophotometer in triplicate at ambient temperature (25 °C). An excess amount of Acr and its cocrystal/salt (CC-1 to CC-6) was added to 4 mL of the buffer at ambient temperature in a jacketed water vessel connected to a circulating water bath for 12 h at a rate of 80 rpm. We maintained the temperature of syringes, pipettes, filters, vials, and needles utilized in experiments by preheating at same temperature in an incubator. The solubilities observed are an average of two determinations (Table S2). Initially, standard curves were prepared by plotting concentration vs absorbance of five standard solutions (Figure S5). The concentration of unknown solution (Cu) of the product material was evaluated from the slope and intercept of the standard curve by using the formula Cu = (Au − intercept)/slope, where Au is the absorbance of the unknown solution at infinite dilution. Permeability Measurements. The diffusion study of acridine and its cocrystals/salts was carried out through a nitrocellulose membrane obtained from HiMedia, India, using a diffusion apparatus and following the procedure reported by Desiraju et al.28a and our group.28b A suspension of 10 mg of the cocrystal/salt with 500 μL of buffer was prepared and placed in the dialysis membrane as a donor compartment and allowed to diffuse through the membrane toward the receptor compartment containing 250 mL of buffered solution (pH = 1.2 and 7.4). The release of the compounds was analyzed by UV−Vis spectrophotometry at a λmax of 255 nm after suitable dilution and at different time intervals up to 3 h. The temperature is maintained at 25 °C for carrying out the experiments. The pH remains unaltered throughout the diffusion experiments (Figures 11 and 12, Table S3). Three millilitres of the sample was withdrawn from the receptor compartment at times 0, 5, 10, 20, 30, 60, 120, and 180 min. The receptor compartment solution volume was kept constant by replacing fresh medium. The concentrations of each cocrystal/salt were measured at predetermined time intervals during the diffusion experiment. Permebility rate was obtained from an average of three determinations (Table S4). Cambridge Structure Database (CSD). A Cambridge Structural Database (ConQuest 1.19, build RC2, CSD version 5.39 May 2017 Updates, www.ccdc.cam.ac.uk) survey was performed to analyze conformational changes in positional isomers of DHBA molecules (Table S5). Probabilities were obtained only from good quality organic multicomponent crystal structures. Variation in π-stacking arrangements by aromatic systems is also analyzed using this storehouse of crystal structures and presented in Table S6.

Figure 5. Molecular packing of acridine and isomeric dihydroxybenzoic acids in (a) CC-1, (b) CC-2, (c) CC-3, (d) CC-4, (e) CC-6.

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probability ellipsoids and are available in Figure S3. No missed symmetry was observed in the final check of CIF file using PLATON.26 The detailed crystallographic data and structure refinement parameters for these crystals are summarized in Table 1, and the key hydrogen bond parameters for the crystal structures are provided in Table 2. The hydrogen bond distances are neutron-normalized. Packing Energy Calculation. The packing energy is an imperative parameter to be considered for stability and other properties of a crystalline material. It is calculated using CSD linked mercury 3.9 version using “UNI” force-field. The 6-exp potential, E = A exp(-BR)-CR−6 was used by Gavezzotti and Filippini describing medium, weak, and strong hydrogen bond interactions accurately. The factor R−6 corresponds to the Coulombic interactions, and charge parameters are also included in the force field27 (Figure 6). The same method was used for interfragment energy calculations for the crystals (Figure 10). Phase Stability. Stability of the cocrystals/salts was examined at both 1.2 and 7.4 pH media by slurry experiments. Isolated pure material was taken into 2−4 mL of buffer solution and was kept stirring (∼80 rpm) at ambient conditions (25 °C) for 24 h. Millipore water was taken for aqueous solubility determination. The PXRD patterns of the solid phases recovered from this experiment indicate the stability of these solid forms (Figure 7 for CC-1 and Figure S4 for remaining product materials).

RESULTS AND DISCUSSION Studies are obtainable that have demonstrated the role of different pH environments which can modify the mode of orientation of hydroxyl group(s) of phenol(s) and/or hydroxybenzoic acid(s).29 The mode of OH orientations could effectively interpolate the adsorption kinetics, isotherms, dissolution, surface complexation, etc. at the interface. Therefore, the conformational recognition of dihydroxybenzoic acids in constituting cocrystals/salts has significant implications in changing intermolecular interaction behavior.30 We sought to reinvestigate various syn and anti conformations of isomeric dihydroxybenzoic acids which are distinguished with respect to an arbitrary perpendicular plane cutting through the benzene ring between −OH positions. The orientation in which −OH points at the plane is named syn and the other one is anti.30 All possible isomers were extracted and the energy was computed using Gaussian 09 using B3LYP force field at the 6-311G+2(d,p) level which is listed as the most stable low energy conformation for each DHBA molecule (Scheme 2). Energy computations (Figure S6) suggest that syn, anti is the most stable 1451

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Table 1. Crystallographic Parameters of Acridine Cocrystal/Salt Materials CC-1 to CC-4 and CC-6 crystal data

CC-1

CC-2

CC-3

CC-4

CC-6

formula unit formula wt crystal system T [K] a [Å] b [Å] c [Å] α [°] β [°] γ [°] volume [Å3] space group Z Dcalc [g cm−3] μ (mm−1) reflns collected unique observed R1 [I > σ(I)] wR2 instrument X-ray CCDC no.

C20H15NO4 333.33 triclinic 298 7.5239(13) 9.4723(17) 11.580(2) 108.045(8) 93.572(9) 90.94(2) 782.6(2) P1̅ 2 1.414 0.099 4125 1517 0.0631 0.1894 Bruker APEX-II MoKα; λ = 0.71073 1577835

C20H14NO4 333.33 monoclinic 298 7.6721(8) 9.6531(9) 21.693(3) 90 95.218(7) 90 1599.9(3) P21/n 4 1.384 0.097 4787 983 0.0555 0.1762 Bruker APEX-II MoKα; λ = 0.71073 1530057

C20H15NO4 333.33 triclinic 298 7.1782(4) 8.7005(6) 13.4602(10) 73.908(6) 74.603(4) 87.739(4) 778.19(9) P1̅ 2 1.423 0.100 7299 3112 0.0638 0.2168 Bruker APEX-II MoKα; λ = 0.71073 1529961

C20H15NO4 333.33 monoclinic 298 7.4299(2) 23.1999(8) 9.6852(3) 90 101.105(2) 90 1638.21(9) P21/c 4 1.352 0.095 3325 2271 0.0428 0.1218 Bruker APEX-II MoKα; λ = 0.71073 1529962

C46H33N3O4 691.75 monoclinic 298 15.6040(10) 14.4410(9) 16.9110(11) 90 106.39(7) 90 3656(4) P21/c 1 1.257 0.081 55915 9492 0.0853 0.2549 Bruker APEX-II MoKα; λ = 0.71073 1530719

Table 2. Hydrogen Bond Parameter of Cocrystals/Salts, CC-1 to CC-4 and CC-6 cocrystal/salt

interaction

H···A/Å

D···A/Å

∠D−H···A/°

CC-1

N1−H1···O1 N1−H1···O2 O3−H3···O2 O4−H4···O1 C7−H7···O4 C9−H9···O4 C14−H14···O2 O4−H3A···O1 O6− H6A···O2 N1−H7A···O1 N1−H7A···O2 C10−H10···O3 C17−H17···O2 C31−H31···O4 O3−H1A···O2 O2−H2A···O1 N3−H3A···O4 C16−H16···O3 C22−H22···O1 O3−H3A···O5 N1−H4A···O5 O4−H6A···O2 C7−H7···O2 C19−H19···O4 N2−H2A···O2 O4−H4A···N1 C42−H42···O3

1.51 2.51 1.55 1.97 2.48 2.56 2.31 1.38 1.49 2.58 1.60 2.54 2.53 2.57 1.91 1.47 1.37 2.47 2.56 1.59 1.59 1.64 2.51 2.55 1.60 1.78 1.75

2.615(3) 3.191(3) 2.528(3) 2.856(3) 3.395(4) 3.405(4) 3.170(4) 2.586(3) 2.567(3) 3.420(4) 2.645(4) 3.341(4) 3.359(4) 3.489(4) 2.736(19) 2.499(18) 2.574(18) 3.392(2) 3.307(2) 2.520(2) 2.633(19) 2.551(19) 3.274(2) 3.408(3) 2.574(5) 2.751(5) 2.779(6)

177 118 151 142 167 152 155 177 153 135 164 144 148 172 162 161 172 173 138 155 175 154 140 153 160 171 157

CC-2

CC-3

CC-4

CC-6

conformation in general, though anti, anti and syn, syn is most stable in the case of 35DHBA and 26DHBA respectively. Despite its higher energy, the anti, anti and syn, syn conformations of 24DHBA and 35DHBA respectively are observed in two systems viz. CC-2 and CC-6, whereas remaining systems have adopted only the stable conformations of DHBA molecules. The presence of high energy con-

symmetry code

−1 + x, y, z 1 + x, y, z −x, −y, −z 1 − x, 1 − y, 1 − z 1/2 − x, 1/2 + y, 1/2 − z 1 + x, 1 + y, z 1 + x, 1 + y, z −x, 1 − y, −z −1/2 − x, 1/2 + y, 1/2 − z −1 + x, y, z

1 + x, 1 + y, −1 + z 1 − x, −y, 2 − z

−x, 1 − y, 1 − z 1 + x, y, z x, 1/2 − y, 1/2 + z −x, 1/2 + y, 1/2 − z

formations of DHBA essentially contributed toward the overall packing energy of the system (Figure 6). As support we have extracted the occurrence probabilities for possible OH conformations in DHBA molecules in organic crystal structures reported in the Crystal Structure Database (CSD version 5.39, update May 2017). Inclusion of high energy conformations in the crystal lattice renders changes in kinetic properties. 1452

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Figure 6. Contribution of packing energy calculated in all the product materials, CC-1 to CC-4 and CC-6. Figure 9. Solubility comparisons between Acr and cocrystal/salts at pH 1.2 medium.

Scheme 2. Stable Conformations of Dihydroxybenzoic Acid Isomersa

a

Figure 7. Phase stability test of CC-1 confirms (by slurry experiment) its stability by the obtainment of identical powder X-ray patterns at pH 1.2 and 7.4 up to not less than 12 h. Tests for remaining materials are described in Supporting Information, Figure S4.

Details are given in the Supporting Information.

be explored. Herein we have examined these important factors systematically by considering a model system of acridine-based cocrystals/salts. The reason for choosing Acr is because it readily offers intermolecular hydrogen bonds and π-stacking structures, provided two stacking possibilities in the solid states (Figure 1), where N acceptors orient in parallel or anti-parallel fashion. The energetically favored anti-parallel stack motif has been observed in higher occurrence probability of 63.4%, whereas the parallel stack is present in only 9.6% organic crystals containing an acridine moiety extracted from CSD analysis (Table S6). The electrostatic interactions between πcharge distributions as a function of orientation prefer face-toface or edge-on or T-shaped geometry by aromatic rings when they orient orthogonally (Figure 1) and the C−H···π hydrogen bond prevails. In contrast, the herringbone T-shaped motif is present in 2.4% of crystals for acridine-containing systems. Thus, the anti-parallel stack of acridine could play an essential role in arranging molecules in the crystal lattice. The Nacceptor of Acr with pKa = 5.58 renders formation of strong hydrogen bonds with functional groups such as COOH and OH. The DHBA molecules have three hydrogen bond donors viz., one COOH and two OH groups and one acceptor, i.e., CO of the COOH group. Thus, O−H···N and O−H···O are two important strong hydrogen bonds in these multicomponent structures. Since the pKa of COOH hydrogen is low, the COOH···NAcr hydrogen bond is the most directional

Figure 8. Solubility comparisons between Acr and cocrystal/salts at pH 7.4 medium.

The role of hydrogen bonds in predicting physiochemical activities of cocrystals is quite comprehensive, supported by a good number of studies appearing in recent years including our work on various drug systems.31 However, the robustness and reliability of π···π interactions in order to alter cocrystal properties (in the presence of directional hydrogen bonds with the functionalities such as COOH, phenolic OH etc.) are yet to 1453

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Table 3. ΔpKa Value for All the Cocrystal/Salt Materials and the N···H Distance (a) and O···H Distance (b) Are Shown

than O−H···NAcr, and thus more reliable. Single crystal X-ray crystal structure determination reveals the presence of robust COOH···NAcr hydrogen bonds in all structures. Prior to X-ray structure determination, we did preliminary investigations in order to judge whether the pure phase of cocrystals have formed. Fourier transform infrared (FT-IR) spectroscopy for all cocrystal/salt materials showed the O−H stretching vibrations in the range 3300−3500 cm−1, while the O−H vibration for pure dihydroxy acid appears at 3400−3600 cm−1. The lowering and broadening of O−H bands in cocrystals/salts could be attributed to the presence of intermolecular hydrogen bonding between Acr and DHBA coformers (Figure 2). The CO absorption band for CC-1, CC-2, CC-3,and CC-4 was observed at a lower region (∼1590 cm−1 and ∼1460 cm−1) suggesting proton transfer from COOH to NAcr. The salt is defined by the location of a proton at the end of the spectrum of the salt-cocrystal continuum, while at the cocrystal end proton transfer is absent. The CO absorption band at 1656 and 1675 cm−1 for CC-5 and CC-6 respectively nullifies the possibility of proton transfer. All major IR stretching vibrations for CC-1 to CC-6 are listed in the Experimental Section. Single endothermic onset melting of each cocrystal/salt in the DSC thermogram signifies absence of starting materials and formation of single phase pure product (Figure 3, Table S1). Powder X-ray patterns of bulk materials further confirmed the phase purity supported by Rietveld refinement using Powder Cell suite 2.3 which equated the simulated PXRD pattern extracted from single crystal structure. The experimental PXRD patterns (Figure 4) of CC-1 to CC-4 exactly resembled the PXRD patterns generated from single crystal structures, whereas due to the growth of poor quality single crystals we could not successfully determine the structure of CC-5. Differential scanning calorimetry and powder X-ray patterns confirm the formation of pure CC-5. The Rietveld refined overlaid PXRD patterns of each product material are presented in the Supporting Information (Figure S2). An interesting observation while isolating CC-6 was that they generally afford solvated crystal forms. Indeed we have isolated a few solvated forms of CC-6, but those crystal forms do not pertain to the present study. Dissolving Acr and 3,5-DHBA in dry methanol followed by a controlled rate of solvent evaporation resulted in desolvated CC-6. The nonsolvated CC-6 was further confirmed by DSC and PXRD pattern compared with the simulated PXRD pattern extracted from the crystal structure (Figure S2, Supporting Information). The ΔpKa rule of three suggests an unpredictable zone for salt or cocrystal formation in the product materials.32 In accordance, we observed either complete proton transfer or salt-cocrystal continuum by the location of the proton between the acid and the base. The proton location in each product material was determined by single crystal X-ray structure determination to confirm its phase behavior. They exist in a salt-cocrystal continuum and distance parameters are listed in Table 3. We further learned from the crystal structures that the C−N−C bond angle and C−N bond distances in the pyridine ring of Acr, carboxylic acid O−C−O bond angles, and C−O bond distances in DHBA are in accordance with the predicted parameters for a salt and/or cocrystal. Details are in the Supporting Information, Table S7. The guest -free crystalline modification of CC-1 was isolated from ethanol, and single crystal data were solved and refined in triclinic P1̅ space group with one Acr and one 23DHBA symmetry independent molecules. The crystal structure reveals the formation of salt

as the carboxylic acid proton is transferred to the N of Acr. The 2,3-dihydroxy benzoates form a molecular tape via O−H···O hydrogen bonds along the [100] axis. Protonated acridine are perpendicular to this molecular tape through N+−H···O− hydrogen bonds. Two such molecular tapes intercalate each other through π···π stacking of acridines completing a sheetlike structure (Figure 5a). They are further arranged via C−H···π and C−H···O interactions to complete 2D packing. Such 2D molecular sheets are arranged via C−H···π to complete 3D packing of molecules. Thus, weak interactions like π···π, C− H···π, and C−H···O interactions show significant roles in predicting the molecular arrangement in the lattice. Single crystal data for CC-1 to CC-4 and CC-6 are summarized in Table 1. ORTEPs are shown in Figure S3. Hydrogen bond matrices are displayed in Table 2. Similarly, the crystal structure of CC-2 was solved and refined in P21/n space group with one Acr and one 24DHBA molecules. The acid proton has been located closer to Acr N suggesting N+−H···O− hydrogen bond synthon formation between Acr and 24DHBA. In CC-2, the 24DHBA forms a helix via O−H···O hydrogen bond from para OH to COO− along the crystallographic [010] axis (Figure 5b). The ortho OH is involved in intramolecular O−H···O hydrogen bonds. Proton transfer occurs also in CC-3 and CC-4 cocrystals, whereas CC-6 was isolated in its neutral form (Figure 5c). In CC-4, strong π···π interactions between Acr molecules and weak C−H···π are the primary driving force in building up the crystal lattice as both OH groups 26DHBA are involved in intramolecular hydrogen bonding (Figure 5d). The single crystal structure for CC-6 suggests that two out of three OH donors of 35DHBA are involved in strong hydrogen bonding with three molecules of Acr resulting in 1:3 cocrystals. Surprisingly one OH from DHBA and one Acr (N center) do not participate in strong hydrogen bonding (Figure 5e); rather, π···π and C−H···π interactions prevail. After complete characterization of all product materials, it is desirable to understand the contribution of various intermolecular interactions in the solid states by quantitative measures. The packing energy calculation has been carried out to analyze the energy contributions from various intermolecular interactions, and a histogram is generated to signify the quantitative amount of packing energies in CC-1 to CC-4 and CC-6 (Figure 6). The packing energy was calculated using “UNI” force-field popularized by Gavezzotti and Filippini (details in the Experimental Section). The single crystal structure of CC-5 could not be determined due to crystal quality, and thus packing energy comparison is not possible. The higher the negative packing energy value, the higher the stabilization of the cocrystal/salt system. From Figure 6 it is observed that CC-2 and CC-6 shows the higher stabilization energies despite having high energy DHBA conformations. This is because π-stacking (Figure 10) contribution is prominent in these structures. 1454

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Essentially the energy contributions from the high energy DHBA conformations are compensated by the crystal packing energy. The better packing energy contributions can have an impact on lowering aqueous solubility and are observed in the present system. Before we conduct experiments for solubility determination and membrane permeation studies, it is a precondition that all cocrystal/salt materials should be stable at various pH conditions. As pH 1.2 and 7.4 appear like the physiological pH in stomach and intestine, we examined studies including a stability test at these two pH conditions maintained by saline (SAL) and phosphate (PBS) buffer solution, respectively. An aliquot was taken out at the time interval 3 and 12 h from the slurry at pH 1.2, and then the sample was dried and PXRD was recorded. The PXRD patterns of the solid phases recovered from slurry indicates a constancy up to 12 h shown by these solid forms. A parallel set of experiments was carried out at pH 7.4, and results showed that materials are stable even at pH 7.4. A representative example of CC-1 is demonstrated in Figure 7 and the remaining is in the Supporting Information (Figure S4). As desired the solubility factors were determined for powder materials at pH 1.2 and pH 7.4. The results are demonstrated in Figure 8 for pH 7.4 and Figure 9 for pH 1.2. A close look into the molecular packing extracted from single crystal structure allowed us to understand the regulation of intermolecular interactions such as hydrogen bonding and π···π stacking in modifying solubility at different pH conditions. It is apparent that the π off-stacking behavior of Acr molecules in the crystals structures is not consistent from CC-1 to CC-6 (Figure 10). The higher solubility of CC-1 could be attributed to the fact

that it shows the lowest packing energy (−124.9 kJ/mol) and π···π stabilization interactions (−51.2 kJ/mol and −30.4 kJ/ mol) that leads to lattice instability. Appropriate off-stacking πinteraction along with higher packing energy in CC-2 (−218.5 kJ/mol) and CC-6 (−318.9 kJ/mol) renders stability of the crystals leading to lower solubility. Solubility parameters of CC3 and CC-4 are determined at nominal differences and thus follow the trend with the packing energies of CC-3 (−202.4 kJ/ mol) and CC-4 (−196.3 kJ/mol). This observation was further corroborated with minimal difference in the π-stabilization energies. Therefore, from the collective investigation of both the energy parameters it could be concluded that better the packing and off-stacking interactions, the higher is the stability which essentially affected the solubility of the system. A second set of experiments was carried out at pH 1.2 buffers, and improved solubility was observed for all materials. In fact it shows better solubility behavior while compared with solubility at pH 7.4 medium except CC-4. Due to the ionic nature (except CC-6) a partial charge will be generated which favors π-stacking interactions and afforded relatively stable crystals. Moreover, the solubility of the material in acidic pH depends on the acidity of the coformer. The stronger the acidity of the coformers, the higher will be its tendency to remain in ionized form in acidic medium. Hence, the ionic salt tries to retain in its ionic form at pH 1.2 resulting in lower solubility in polar media. All other interaction patterns will remain same, whereas the solubility trend is also followed like in pH 7.4 as the cocrystal/ salt materials are found to be stable at pH 1.2 media. Phase stability was confirmed employing a slurry test by analyzing product materials at regular intervals up to 12 h (Figure S4). Apart from the contribution of weak intermolecular interactions toward property modulation, the role of various other parameters like packing index, density, morphology, etc. cannot be overruled. Intrinsic membrane permeation rate measurements for all solid materials were carried out at pH 1.2 and 7.4 buffer medium and demonstrated in Figures 11 and 12. The cumulative release amount of the products with respect to time and their flux density measurements showed improved release kinetics which increases with time until it reaches the equilibrium. Instantaneous release behaviors say up to 30 min depicts the fastest release of CC-5 and CC-6 at pH condition 1.2. On the other hand the release behavior at pH 7.4 is not so encouraging within a 30 min time window. The permeability rate percentage slowly increases over time for CC-6, CC-1, and CC-3. The key factors for such changes are the lipophilic nature obtained by the cocrystal/salt unit, and noncovalent interactions exist in the lattice and interaction with the media. Since lipophilicity of the coformer may also be a concern in changing the solution kinetics, we looked into these parameters which is nothing but the partition coefficient (Log P), when one of the solvents is water and the other is a nonpolar solvent. A higher log P value (lipophilicity) indicates better thermodynamic activity.33 The corresponding log P values of the respective coformers are 23DHBA (1.20), 24DHBA (1.63), 25DHBA (1.56), 26DHBA (2.24), 34DHBA (1.15), 35DHBA (1.11), although the log P value is not only the deciding factor for permeability studies of these cocrystal/salt systems. Studies suggested the better the π···π off-stacking the better is the thermodynamic stability, which implies a slower rate of permeation. We further investigated the packing of the molecules in the crystal lattice in view of the involvement of π··π stacking and C−H···π interactions, which

Figure 10. Pictorial representations of the off-stacking π···π interactions present in the cocrystal/salt systems. The distance between two stacked ring centroids {Cg(1)−Cg(1)} is represented by d. 1455

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Figure 11. Plots of (i) membrane permeability profiles and (ii) flux with respect to time of Acr and their cocrystals/salts at pH 1.2.

could play an important role in altering the kinetic and thermodynamic parameters of the product materials. Variations in the π···π off-stacking behavior extracted from the crystal structure are displayed in Figure 10. The lowest release rate of CC-4 at both 1.2 and 7.4 pH media is due to its more ionic character (Table 3), which could produce micellization in polar media, resulting in decreased free fraction for permeation. In both the pH media, CC-6 and CC-5 show a higher permeation rate because all the free OH groups present on the neutral coformer (from FT-IR experiment) are well connected with neighboring molecules through hydrogen bonding. This entails the hydrophobic moiety to be exposed to the polar media for interaction ensuring a higher rate of permeation. The pH 1.2 suggests a highly acidic and ionic environment for drug molecule to permeate across the membrane. However, CC-2 shows a lower diffusion rate due to stabilized off-stacking π−π interaction which is further supported by lattice energy of the system (Figure 6).

Figure 12. Plots of (i) Membrane permeability profiles and (ii) flux with respect to time for CC-1 to CC-6 at pH 7.4.

to understand the absorption behavior of a drug, we transformed a representative system, acridine, into a multicomponent cocrystal or salt using crystal engineering principles, which is an enabling technology to modulate the hydrophilic or hydrophobic nature of the drug compounds. Six cocrystals/salts were isolated with antioxidant isomeric dihydroxybenzoic acid coformers and systematically characterized and subjected to solubility and membrane permeation in vitro at different pH buffers viz. pH 1.2 and 7.4. The outcome of physiochemical property studies of multicomponent crystals of small molecules allowed us to establish a link to adhesive and cohesive intermolecular forces such as hydrogen bonds and most importantly π···π interactions. Essentially this study demonstrated how these noncovalent interactions especially π···π interactions play a crucial role in modifying molecular packing energies in the multicomponent drug formulation. This could effectively predict the net bioavailability and efficacy of the drug. Moreover it is a systematic study of a promising class of model cocrystals/salts designed based on adhesive and cohesive forces to facilitate modified transport mechanism of drug

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CONCLUSIONS Cell membranes play significant roles in the metabolic equilibrium in organisms. Generally a concentration gradient facilitates the partition of small hydrophobic molecules. However, hydrophilic molecules require additional supports for their transport mechanism such as lipid bilayers. Therefore, 1456

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molecules across the membranes for a better formulation and offers a guidance for formulation and process development in the pharmaceutical industry.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.7b01377. Rietveld refinement of compounds with simulated PXRD patterns; table of melting temperatures of the parent components and DSC endothermic transitions; ORTEP diagrams of the product materials; phase stability test; solubility of Acr and its cocrystals/salts; calibration curves for determination of solubility of all the cocrystal/ salt materials; pH monitoring during the diffusion experiment; permeability rate % of Acr and its cocrystals/salt; Crystal Structure Database analysis; different isomers of DHBAs, along with the possible conformations and their respective conformational notations; ΔpKa values for all the cocrystal/salt materials (PDF) Accession Codes

CCDC 1529961−1529962, 1530057, 1530719, and 1577835 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 data_request@ccdc. cam.ac.uk, 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] or [email protected]. ORCID

Bipul Sarma: 0000-0002-7704-1121 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank CSIR India ((02(0327)/17/EMR-II) and SERB India (EMR/2014/000214) for financial support, and Department of Chemical Sciences and Tezpur University for laboratory facilities.

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REFERENCES

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