Supramolecular Organization in Salts of Riluzole with Dihydroxybenzoic Acids—The Key Role of the Mutual Arrangement of OH Groups

Intermolecular interactions, in particular hydrogen bonds, play a key role in crystal engineering. The ability to form hydrogen bonds of various types and strengths causes competition between supramolecular synthons in pharmaceutical multicomponent crystals. In this work, we investigate the influence of positional isomerism on the packing arrangements and the network of hydrogen bonds in multicomponent crystals of the drug riluzole with hydroxyl derivatives of salicylic acid. The supramolecular organization of the riluzole salt containing 2,6-dihydroxybenzoic acid differs from that of the solid forms with 2,4- and 2,5-dihydroxybenzoic acids. Because the second OH group is not at position 6 in the latter crystals, intermolecular charge-assisted hydrogen bonds are formed. According to periodic DFT calculations, the enthalpy of these H-bonds exceeds 30 kJ·mol−1. The positional isomerism appears to have little effect on the enthalpy of the primary supramolecular synthon (65–70 kJ·mol−1), but it does result in the formation of a two-dimensional network of hydrogen bonds and an increase in the overall lattice energy. According to the results of the present study, 2,6-dihydroxybenzoic acid can be treated as a promising counterion for the design of pharmaceutical multicomponent crystals.


Introduction
The design of pharmaceutical multicomponent solids, primarily relies on the utilization of supramolecular synthons, as reliable building blocks that enable the construction of molecular assemblies with desired structural features and properties in a predictable manner [1][2][3]. Even though various types of intermolecular interactions concurrently exist in the crystal structure and contribute to its thermodynamic stability [4][5][6][7], intermolecular hydrogen bonds (H-bonds) play a more prominent role than others, due to their strength and directionality [8], allowing crystal engineering principals to be used [9][10][11][12]. However, the possibility to properly regulate or anticipate the formation of multiple supramolecular synthons in multicomponent crystals, consisting of molecules with a diverse spectrum of functional groups, remains a challenge, due to a lack of understanding of the nature and strength of intermolecular interactions responsible for competition between different synthons in organic solids [13][14][15]. Therefore, it is of great importance to provide a quantitative evaluation of the binding strength and nature of synthons, to rationalize their stabilizing and structure-directing roles in the formation of multicomponent crystals [16][17][18][19][20][21].
Riluzole (2-amino-6-trifluoromethoxy-benzothiazol) is one of the few approved options to treat amyotrophic lateral sclerosis [22]. Treatment with riluzole (RLZ) leads to slower degradation of neurons and extended survival without tracheostomy [23,24]. The Riluzole (2-amino-6-trifluoromethoxy-benzothiazol) is one of the few approved options to treat amyotrophic lateral sclerosis [22]. Treatment with riluzole (RLZ) leads to slower degradation of neurons and extended survival without tracheostomy [23,24]. The mechanism of action of riluzole is complex, involving glutamatergic neurotransmission enhancement [25], inhibition of fast and persistent sodium currents [26,27], neuroprotective activity [28], as well as neuroplasticity stimulation via stimulation of the neurotrophic factors [29,30]. RLZ was shown to exhibit neuroprotective, anticonvulsant, and antidepressant activity in multiple in vivo studies [31]. In addition, RLZ can be used for the treatment of severe mood, anxiety and impulsive disorders, including obsessive-compulsive disorder, unipolar and bipolar depression, and generalized anxiety disorder [32,33]. Recent studies have demonstrated the potential of repurposing RLZ as an antiproliferative agent for many types of cancer, including skin, breast, pancreas, colon, liver, bone, brain, lung, and nasopharynx cancers ( [34] and references therein). Because of its ability to cross the blood-brain barrier, RLZ is one of the few promising possibilities for glioma therapy [35,36] and brain metastasis in late-stage malignant melanoma [37]. The low systemic bioavailability of RLZ (60% [38,39]) is limited by its poor aqueous solubility (350 mg/L at 37 °C and pH 7 [40]), which is typical for a BCS Class II drug [41]. The potential use of RLZ in psychiatry and oncology stimulates the search for novel forms of this drug, with improved dissolution performance and reduced side effects. During its 20 years of use, many approaches for optimizing the drug delivery of RLZ have been proposed, including salt/cocrystal formation [40,42,43], encapsulation in nanoparticles [44], amorphous form stabilization [45], the use of oral suspensions [46], biodegradable sublingual foils [47], and microemulsions for intranasal delivery [48].
From the crystal engineering point of view, RLZ is known to form multicomponent crystals, with amino and carboxylic acids, aromatic amides, etc. [40,42,43]. Dihydroxybenzoic acids are promising coformers/counterions of RLZ due to their safety and biological activity. 3,4-Dihydroxybenzoic acid (syringic acid) exhibits antibacterial, antifungal, and antiprotozoal activity, 2,5-dihydroxybenzoic acid (gentisic acid) displays antioxidant properties [49], while 2,6-dihydroxybenzoic acid and other derivatives of salicylic acid display anti-inflammatory activity [50] (Scheme 1). Based on a survey of multicomponent crystals of RLZ, deposited in the Cambridge Structural Database (CSD) [51] (Supplementary Materials, Section S1), we concluded that in multicomponent crystals with salicylic acid and its hydroxyl derivatives, RLZ exists in the protonated state (as a cation), whereas acids exist in the anionic form. Depending on the position of the OH group in hydroxyl derivatives of salicylic acid, either intra-or intermolecular H-bonds can form. Because RLZ can only generate three H-bonds in the protonated state, the emergence of a new proton donor, with an excess of unshared electron pairs, may cause a dramatic shift in the H-bond network in a multicomponent Based on a survey of multicomponent crystals of RLZ, deposited in the Cambridge Structural Database (CSD) [51] (Supplementary Materials, Section S1), we concluded that in multicomponent crystals with salicylic acid and its hydroxyl derivatives, RLZ exists in the protonated state (as a cation), whereas acids exist in the anionic form. Depending on the position of the OH group in hydroxyl derivatives of salicylic acid, either intra-or intermolecular H-bonds can form. Because RLZ can only generate three H-bonds in the protonated state, the emergence of a new proton donor, with an excess of unshared electron pairs, may cause a dramatic shift in the H-bond network in a multicomponent crystal [52]. In addition, a change in the number of intra-and intermolecular H-bonds can result in a significant change in the lattice energy value [53]. Thus, the position of the second OH group in the salicylic acid derivative is likely to play an essential structure-directing role This work aims to study the effect of positional isomerism in hydroxyl derivatives of salicylic acid on the H-bond network and supramolecular organization in RLZ salts. Collaborative research, using X-ray diffraction and periodic (solid-state) density functional theory (DFT), was conducted to highlight the critical significance of the mutual arrangement of OH groups in the packing of these multicomponent crystals.

Experimental
Riluzole of 98% purity was obtained from TCI Chemicals. All the benzoic acid derivatives were purchased from Sigma-Aldrich and were used as received. The purity of materials was checked by differential scanning calorimetry (DSC). The solvents were of analytical or chromatographic grade.

Mechanochemical Experiments
The grinding experiments were performed using a Fritsch planetary micro mill, model Pulverisette 7, in 12 mL agate grinding jars with ten 5 mm agate balls at a rate of 500 rpm for 60 min. In a typical experiment, 50-60 mg of a physical mixture, with a 1:1 molar ratio, was placed into a grinding jar, and 50 µL of solvent (water, methanol, ethanol, acetonitrile, or ethyl acetate) was added with a micropipette. The resulting powder samples were analyzed by powder X-ray diffraction (PXRD), DSC, TGA, and FTIR (see Supplementary Materials, Section S2).

Single Crystal XRD
Single crystal diffraction data were collected using a Bruker D8 Venture diffractometer (Bruker AXS, Karlsruhe, Germany) with graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å). It was found that crystals [RLZ+2,4DHBA] degrade below 170 K and thus reflection intensities were collected at 175 K. Absorption corrections, based on measurements of equivalent reflections, were applied [54]. The structures were solved by direct methods and refined by full matrix least-squares on F 2 with anisotropic thermal parameters for all non-hydrogen atoms [55]. In the structure [RLZ+2,6DHBA], the trifluoromethoxy group is disordered over two positions, with occupancy ratio 0.785(3)/0.225 (3), and all fluorine atoms were refined with restrained C-F and F···F distances (SADI instruction). All hydrogen atoms were found from difference Fourier synthesis and refined isotropically. Experimental details are listed in  [56,57] and PBE-D3 [58,59] are the most popular functionals in periodic (solidstate) DFT calculations of molecular crystals [60,61]. B3LYP and PBE-D3 provide a grounded trade-off between the accuracy and the rate of calculations of experimentally observed properties of molecular crystals [62][63][64][65][66][67][68]. Computations were conducted using the CRYS-TAL17 package [69]. The 6-31G** basis set was used. The space groups and the unit cell parameters of the crystal obtained from the X-ray diffraction experiment were fixed, and the structural relaxations were limited to the positional parameters of the atoms [70,71]. Tolerance on energy controlling the self-consistent field convergence for geometry optimizations and frequency computations was set to 10 −10 and 10 −11 Hartree, respectively. The shrinking factor of the reciprocal space net was set to three. The structures optimized at the PBE-D3/6-31G** level were found to correspond to the potential energy surface's minimum point. Therefore, PBE-D3 was used in estimating the lattice energy using Equation (3).

Enthalpy of the Intermolecular H-Bonds
The enthalpy ∆H HB of the intermolecular H-bond in the considered crystals was estimated using the Rozenberg approach [72]: where the R(H···O) is the H···O distance (nm). Equation (1) describes −∆H HB varying from 10 to 80 kJ·mol −1 [72]. The R(H···O) values were obtained as a result of geometry optimization.

Estimation of the Crystal Lattice Energy
The energy of intermolecular noncovalent interaction E int in the considered crystal was evaluated according to ref. [73] as follows: where G b is the positively defined local electronic kinetic energy density at the (3,−1) bond critical point, corresponding to the considered intermolecular noncovalent interaction [74]. The methodology of the calculation is presented elsewhere [75]. Periodic wave functions computed at the PBE-D3/6-31G** level were used in Topond14 [76]. Equation (2) overestimates energies for the short (strong) intermolecular H-bonds [77]. Therefore, the use of Equation (1) is preferable for systems with short H-bonds, both in crystals [77] and in solutions [78].
On the other hand, Equation (2) enables one to estimate the lattice energy of a twocomponent organic crystal using the following approach [75]: where indices j and i denote the atoms belonging to different molecules. Equation (3) is BSSE-free. Details of the E latt computations in the two-component crystals using Equation (3) are given elsewhere [75]. In the present study, we were interested in the relative E latt values estimated for riluzole salts. Hence, overestimating the values of E int given by Equation (2) does not play a significant role.

Molecular Electrostatic Potential
The molecular electrostatic potential surfaces (MEPs) [79] of the RLZ and selected (di)hydroxybenzoic acids were generated at the B3LYP/def2-tzvp level of theory, with the latest version of the D3 dispersion correction, using Gaussian09 [80]. Calculations were carried out using the optimized molecular geometries. The local maxima and minima sites on the molecular electrostatic potential surfaces were extracted using the Multiwfn software [81].
All the crystals under consideration contain a robust amino-N-heterocycle carboxylate supramolecular heterosynthon, of 2 2 (8) topology. This synthon is structurally identical to 2-aminopyridinium carboxylate [82], which is known to play a structure-directing role in various multicomponent crystals [83][84][85][86][87][88]. The mutual orientation of molecules in the synthon is determined by the side H-bonds, and varies from coplanar in  1). Because the second OH group is absent in the sixth position, intermolecular charge-assisted hydrogen bonds are formed in the latter crystals (Table 1). This isomerism leads to the appearance of a two-dimensional network of H-bonds, c.f. Figure 1 and   As can be seen from the crystal structure data, different positions of the hydroxyl group in the counterion lead to variations in the H-bond topology and mutual orientation of H-bonded units, without significant changes in the interaction energy (Table 1). A structural similarity analysis, performed with the XPac program [89,90], revealed that the salts of RLZ with salicylic and isomeric dihydroxybenzoic acids have no common packing motifs that could be associated with any kind of non-covalent interaction. Such a variety of crystal structures of structurally related compounds, based on a single "basic" supramolecular heterosynthon, highlights the gap between the topology of H-bonds and the actual packing architecture. The differences in the packing of heterodimers observed in the [RLZ+2,4DHBA] salt, and its solvate with methanol [42], are explained by the participation of the solvent molecule in the H-bond network. No similarity was found between [RLZ+2,6DHBA] and the reported [RLZ+2HBA] crystal [43], even taking into account the fact that the number and topology of H-bonds in these crystals are very similar. In the latter structure, the RLZ-acid dimer is twisted from planarity, which greatly affects the packing. The non-planar structure of the dimer allows the keto oxygen atom to participate in a side N-H···O H-bond with the amide group of RLZ. For [RLZ+2,6DHBA], this site is inaccessible for intermolecular contacts due to the strong O-H···O intramolecular bond.
The studied crystals share the same T-shaped mutual orientation of heterodimers in the hydrogen-bonded chain, despite having varying symmetry and packing. In contrast to the two-centered 2 2 (8) motif, that facilitates the coplanar orientation of molecules within the heterodimer, the side N-H···O interaction is single-centered, so the heterodimers can rotate around the bond. An indicative parameter of the T-packing is the dihedral angle between the two nearest heterodimers, which varies from 67° in [RLZ+2,6DHBA] to 87° in [RLZ+2HBA]. The difference in packing angles reflects the difference in the mutual positions of donors and acceptors of side H-bonds within the heterodimer and the influence of weak interactions on the packing architecture.
The variety of H-bonds formed in the salts under study can be rationalized based on the comparison of the minima on the MEP surface of RLZ and its counterions (Figure 3). The relative strength of the acceptor sites at the hydroxyl oxygen atom in the ortho-position (O2), is larger than that of hydroxyl oxygens in other positions (O4 and O5), which makes it a preferred target for H-bonding with the Na-Hc donor group remaining after  Table 1. The difference between the calculated (B3LYP/6-31G** and PBE-D3/6-31G**) and experimental values of the distances between heavy atoms ∆D in the considered crystals. The enthalpies ∆H HB (kJ·mol −1 ) of these H-bonds, evaluated using the Rozenberg approach, see Equation (1)  As can be seen from the crystal structure data, different positions of the hydroxyl group in the counterion lead to variations in the H-bond topology and mutual orientation of H-bonded units, without significant changes in the interaction energy (Table 1). A structural similarity analysis, performed with the XPac program [89,90], revealed that the salts of RLZ with salicylic and isomeric dihydroxybenzoic acids have no common packing motifs that could be associated with any kind of non-covalent interaction. Such a variety of crystal structures of structurally related compounds, based on a single "basic" supramolecular heterosynthon, highlights the gap between the topology of H-bonds and the actual packing architecture. The differences in the packing of heterodimers observed in the [RLZ+2,4DHBA] salt, and its solvate with methanol [42], are explained by the participation of the solvent molecule in the H-bond network. No similarity was found between [RLZ+2,6DHBA] and the reported [RLZ+2HBA] crystal [43], even taking into account the fact that the number and topology of H-bonds in these crystals are very similar. In the latter structure, the RLZ-acid dimer is twisted from planarity, which greatly affects the packing. The non-planar structure of the dimer allows the keto oxygen atom to participate in a side N-H···O H-bond with the amide group of RLZ. For [RLZ+2,6DHBA], this site is inaccessible for intermolecular contacts due to the strong O-H···O intramolecular bond.
The studied crystals share the same T-shaped mutual orientation of heterodimers in the hydrogen-bonded chain, despite having varying symmetry and packing. In contrast to the two-centered R 2 2 (8) motif, that facilitates the coplanar orientation of molecules within the heterodimer, the side N-H···O interaction is single-centered, so the heterodimers can rotate around the bond. An indicative parameter of the T-packing is the dihedral angle between the two nearest heterodimers, which varies from 67 According to the results of the MEP analysis, the molecules have several donor and acceptor sites, that may be rated by strength in accordance with their electrostatic potential values. The isolated RLZ molecule has three acceptors and two H-bond donors, with According to the results of the MEP analysis, the molecules have several donor and acceptor sites, that may be rated by strength in accordance with their electrostatic potential values. The isolated RLZ molecule has three acceptors and two H-bond donors, with the heterocyclic nitrogen being considerably stronger than the ether oxygen and the amine nitrogen. 2HBA has one strong donor and two acceptor sites at the carbonyl oxygen and hydroxyl oxygen atoms, while dihydroxybenzoic acids have three strong acceptor sites. Due to the formation of the second intramolecular hydrogen bond, the number of available donors and acceptors in the 2,6DHBA molecule is lower by one than in 2,4DHBA and 2,5DHBA. The competition between acceptor sites with close MEP values at the carbonyl oxygen atom of the carboxylic group and the oxygen atom of the side hydroxyl groups, is observed in the (di)hydroxybenzoic acids. In 2,4DHBA and 2,5DHBA, the hydroxyl oxygen in the ortho-position is stronger than the oxygen atoms of the free hydroxyl groups.
Due to steric factors, the preferred interaction in the crystal is the carboxyl-amino-N-heterocycle heterosynthon [82,91] with the two strongest donor/acceptor sites in the RLZ molecule, e.g., N a -H and N p -H (for atom labeling see Figures 1 and 2). Compared to (di)hydroxybenzoic acids, RLZ has only one good acceptor site (the N atom of the thiazole ring), which becomes a donor after protonation. After the main amino-N-heterocycle carboxylate heterosynthon is formed, RLZ can only participate in the H-bonding, since its remaining acceptors (the lone pairs at the -OCF 3 oxygen and the -NH 2 nitrogen) have lower absolute electrostatic potential values than the acceptor sites of the hydroxybenzoic acids. The formation of H-bonds occurs in accordance with Etter's third rule, as the strongest donors bind to the strongest available acceptors [92]. The remaining strong donor in the RLZ molecule (N a -H c ) is then attached to the stronger of two competing H-bond acceptors: the O2 atom in the salts with 2,4DHBA and 2,5DHBA, and the O6 atom in the salt with 2,6DHBA.
The enthalpies, ∆H HB , of the intermolecular H-bonds in the considered crystals, evaluated using Equation (1), are given in Table 1. As follows from the presented data, the total enthalpy of the amino-N-heterocycle carboxylate supramolecular heterosynthon (65-70 kJ·mol −1 ) does not change significantly depending on the number of OH groups in the substituted benzoic acid or the number of intramolecular hydrogen bonds. The obtained values of the total enthalpy are in good agreement with the available literature data [42,52,93]. On the other hand, the [RLZ+2,4DHBA] (1:1) and [RLZ+2,5DHBA] (1:1) salts feature an additional relatively short O-H···O − bond, with an enthalpy above 30 kJ·mol −1 ( Table 1)

Discussion
The formation of salts in the RLZ-2,6DHBA and RLZ-2,4DHBA systems supports our hypothesis on the influence of the pK a of components on governing the ionization state. As seen from Table S1, the ∆pK a values for RLZ-2,6DHBA and RLZ-2,4DHBA pairs are positive, which agrees with the trend observed for the known RLZ crystals. In brief, hydroxybenzoic acids with -OH groups in positions other than 2 and 6 have pK a values larger than protonated RLZ, and form cocrystals, whereas ortho-hydroxybenzoic acids result in positive ∆pK a with RLZ, and form salts.
Although the solubility of a single-component organic compound can be estimated using the Yalkowsky equation [95], or other QSAR models [96,97], with a fair degree of precision, forecasting the solubility of pharmaceutical salts containing different types of counterions is not a trivial task. In fact, only a handful of studies have looked at this issue in depth, examining the effect of structural and physicochemical features of organic salts on their solubility in a systematic manner [98][99][100][101]. The generally accepted view on this topic, implies that the change in the free energy of salt dissolution can be represented as the sum of contributions from the values of the free energy of hydration of the cation and anion, as well as the free energy of the crystal [101,102]: In the case of salts of riluzole with hydroxyl derivatives of salicylic acid, the first term on the right side of Equation (4) is a constant, and the second term can also be considered a constant in the first approximation. Thus, the differences in the solubility of the considered salts are mainly due to the third contribution on the right side of Equation (4). From the point of view of the "enthalpy-determined drug solubility processes" [103], ∆ latt G(salt) can be replaced by ∆ latt H(salt). The latter value is proportional to the crystal lattice energy of the salt. Given that [RLZ+2,6DHBA] has the lowest crystal lattice energy among all the salts under consideration, it might be reasonable to assume that [RLZ+2,6DHBA] is likely to have a better solubility compared to other salts. This conclusion is consistent with the literature data. For example, the solubility of the ethionamide salt with 2,6DHBA, was found to be higher than that with 2,3-dihydroxybenzoic and 2,4-dinitrobenzoic acids [104]. To summarize, 2,6DHBA appears to be a promising compound to consider during cocrystal or salt screening trials, due to its ability to modify the crystalline environment of the target drug without compromising total lattice energy gain and, thus, solubility.

Conclusions
In this work, specific features of hydrogen bond topology, induced by the introduction of a second OH group into the counterion molecule, were revealed through a detailed structural analysis of multicomponent crystals of riluzole with 2-hydroxybenzoic acid and 2,4-, 2,5-, and 2,6-dihydroxybenzoic acids. In the solid forms containing 2-hydroxybenzoic acid and 2,6-dihydroxybenzoic acid, the hydrogen bonding network is sustained solely by the N-H groups of riluzole, resulting in the formation of 1D chains. The competition between the acceptor centers of the counterion causes a change in the topology of hydrogen bonds and differences in the packing arrangements of salts with 2-hydroxybenzoic and 2,6-dihydroxybenzoic acids. The presence of an additional H-bond donor at position 4 or 5, leads to the formation of charge-assisted H-bonds and the combination of isolated hydrogenbonded chains into 2D sheets.
According to periodic DFT calculations, the number and position of the OH-groups in the counterion molecule appear to have little effect on the enthalpy of the primary supramolecular synthon (65-70 kJ·mol −1 ). A novel intermolecular charge-assisted H-bond in the salts containing 2,4-and 2,5-dihydroxybenzoic acids, stabilizes the corresponding crystal structure and increases the lattice energy by about 30 kJ·mol −1 . When compared to riluzole salts with 2,4-and 2,5-dihydroxybenzoic acids, the salt with 2,6-dihydroxybenzoic acid lacks an intermolecular charged-assisted O-H···O − H-bond, resulting in a lower lattice energy and expected higher solubility. In addition, 2,6-dihydroxybenzoic acid is preferred over salicylic acid, because the lattice energy of riluzole salt with 2,6-dihydroxybenzoic acid is lower than that of riluzole salt with 2-hydroxybenzoic acid. We do hope that this prediction encourages experimentalists to conduct further investigations of riluzole multicomponent crystals with dihydroxybenzoic acids, especially 2,6-dihydroxybenzoic acid.