**3. Results**

### *3.1. Solid State NMR Studies of HIMO*

One of the aims of the present project was the comparison of two methods of preparing cocrystals, i.e., classic cocrystallization of components with solvent (solvents) use and solvent free ball milling. For the monitoring of the processes employing the ball mill method, knowledge about the structure and properties of HIMO (1) in the solid state was crucial. In our structural studies of 1 in condensed matter, we started with a 13C CP/MAS NMR experiment performed with a spinning rate of 8 kHz at ambient temperature in a 4 mm zirconium rotor. Figure 2a shows spectrum of the sample purified by precipitation. The unexpected feature of the spectrum was its very high resolution of resonances, which is typical for highly crystalline samples with very well organized crystal lattice. Similarly, high quality spectra are usually recorded for molecules which have crystallized in multiple steps. Going further and analyzing the number of resonances both in aromatic (imidazole) and aliphatic (methyl) regions, we concluded that 1 crystallizes during precipitation in space group with Z' equal 2. The four, clearly cut methyl signals provide unambiguous proof to confirm this hypothesis.

**Figure 2.** (**a**) 13C CP/MAS spectrum recorded with a spinning rate of 8 kHz; (**b**) 15N CP/MAS spectrum recorded with a spinning rate of 8 kHz; (**c**) 1H VF/MAS NMR spectrum recorded with a spinning rate of 60 kHz.

The 15N CP/MAS spectrum (Figure 2b) confirms the presence of more than one molecule in the asymmetric part of the unit cell. Three clearly cut signals found at δ = 208.0, δ = 214.7 and δ = 216.0 ppm differed in intensity. We assumed that the larger intensity of the middle peak (ca. 215 ppm) resulted from the superposition of two resonance signals representing the A and B molecules.

The 1H NMR spectroscopy is an interesting source of information. For this measurement, we employed a unique technique called Very Fast Magic Angle Spinning (VF MAS). The "very fast" (VF) regime, i.e., at more than 50 kHz, is obtained using commercially available 1.3 mm rotors. This frequency exceeds the strength of the homonuclear 1H dipolar coupling, and is therefore expected to show a new regime for spin dynamics. In many cases, the 1H NMR spectra recorded under VF MAS conditions showed acceptable resolution and the protons positions (chemical shifts) could be correctly assigned.

The 1H VF MAS spectrum (Figure 2c) recorded with spinning rate 60 kHz displays three groups of signals representing protons of methyl residues (δ1H = 1.36 ppm), protons bonded to methine carbon C2 (δ1H = 9.17 ppm) and protons of hydroxyl residue (δ1H = 17.71 ppm). The chemical shifts of *H*C(2) and O*H* are not straightforward. In the first case, the strong deshielding effect was due to the aromatic character of imidazole strengthened by the "zwitterionic" nature of the adjacent N→O unit. In general, the chemical shift at around 9 ppm is characteristic for positions called "acidic". The value of δ1H for O*H* suggests that this residue is involved in a strong hydrogen bonding interaction.

### *3.2. Formation and Structural Studies of TBA*/*HIMO and BA*/*HIMO Cocrystals*

### 3.2.1. SS NMR Studies of TBA/HIMO Cocrystal

The TBA **2** and BA **3**, as well as their derivatives, commonly known as barbiturates, play an important role in pharmaceutical industry because of their hypnotic, sedative, anticonvulsant, antimicrobial, anesthetic, anticancer and antitumor properties [41]. Due to the unique structure of **3** and their ability to form keto–enol tautomers, it is widely used as a valuable building block in organic synthesis and advanced materials including cocrystals [42,43].

In our project, the TBA/HIMO cocrystals were obtained by the cocrystallization of equimolar amounts of TBA and HIMO dissolved in boiling methanol. When the solution was cooled down and kept at ambient temperature for 8 d in a closed vessel, the expected crystals were efficiently formed. After separation of the TBA/HIMO cocrystals, they were dried in air and measured employing 13C CP/MAS, PXRD and DSC techniques (Figure 3). All experiments confirmed the formation of new crystallographic structures. It is worth noting that in the DSC profile (Figure 3c), a strong exothermic peak corresponding to thermal decomposition, typical for high-energetic cocrystals, was observed.

**Figure 3.** (**a**) 13C CP/MAS spectrum recorded with a spinning rate of 8 kHz for TBA/HIMO cocrystals obtained by cocrystallization from MeOH; (**b**) powder x-ray diffraction diffractogram for TBA/HIMO cocrystals obtained by cocrystallization from MeOH; (**c**) DSC curve of TBA/HIMO cocrystals. For a full analysis of the crystallographic forms of TBA, HIMO and TBA/HIMO based on PXRD diffractograms, see Supporting Information (Figure S1).

It is apparent that in the case of wet methods of cocrystal formation, which are based on the dissolving of starting components, the crystallographic form of applied ingredients is irrelevant. In the case of mechanochemical methods (e.g., ball milling), the structural effects (crystal structure, polymorphism, tautomerism, intermolecular contacts) can affect the process of cocrystal formation. It seems that TBA, for which a rich collection of polymorphs and one hydrated form have been isolated, is an appropriate candidate with which to test the correlation between crystallographic form and the ball milling process. In the crystal lattice, TBA exists in enol form, keto isomer, as well as a mixture of keto/enol forms, each of which can be easily recognized by 13C CP/MAS experiments (see [43]). Depending on the solvent used for the crystallization, it is possible to obtain a suitable form of TBA.

The structure, tautomerism and crystal forms of TBA were investigated by Chierotti et al. [44]. Three different forms crystallized from different solvents were also used as starting materials in the present study. TBA(I) was obtained using MeCN as a solvent for crystallization. This sample exists as tautomeric form A (Scheme 1).

**Scheme 1.** Numbering system for TBA and tautomers of TBA depending on crystallization from di fferent solvents: tautomeric form A (keto) after the crystallization from MeCN, tautomeric form B (keto/enol) after the crystallization from EtOH or water.

In this form, the C(O)NH moieties on all molecules are either involved in the typical hydrogen-bonding ring motif, with formation of a dimer, or in the formation of hydrogen-bonded chains. TBA (II) was obtained by slow evaporation of a hot ethanolic solution. TBA (II) crystallizes as a tautomer B and forms a type of hydrogen-bonded zigzag chain. Recrystallization of TBA from water leads to the formation of hydrated form III, presented as B, with 1.5 water molecules per formula unit. The acid molecules form a chain through two types of hydrogen-bonding rings involving C(O)NH and C(S)NH moieties.

Figure 4 (left column, a–c) shows the 13C CP/MAS spectra for the physical mixture of two components, i.e., TBA in di fferent forms (I-III) and HIMO, in a 1:1 ratio. It is clear that the intensity of the signals does not reflect the content of the mixture. This is because the relaxation times and e fficiency of the cross-polarization for both compounds were significantly di fferent. The TBA/HIMO cocrystals were obtained mechanochemically in the presence of MeOH added as a LAG. Figure 4 (right column, d–f) shows the 13C CP/MAS spectra after one hour of grinding in a Mixer Mill MM 200 equipped with a 5 mL agate jar and 5 mm diameter balls at an oscillation rate of 25 Hz. A comparative analysis of the spectra proved that during the grinding process, in each case, the formation of cocrystals was observed, and the final product was always very similar. The only di fference was observed for TBA (III); in that case, a small amount of unreacted HIMO was detected in the crude mixture. Apparently, the 1 h grinding was not su fficient to complete the expected transformation.

**Figure 4.** The 150.90 MHz 13C CP/MAS spectra recorded with a spinning rate of 11 or 12 kHz. The left column shows a physical mixture of HIMO and TBA in a ratio of 1:1, (**a**) HIMO and TBA crystallized from acetonitrile (tautomer A); (**b**) HIMO and TBA crystallized from EtOH (tautomer B); (**c**) HIMO and TBA crystallized from water (tautomer B). Right column displays the spectra of samples after 1 h of grinding with MeOH as a LAG, (**d**) HIMO and TBA crystallized from MeCN; (**e**) HIMO and TBA crystallized from EtOH; (**f**) HIMO and TBA crystallized from water.

The 13C CP/MAS results sugges<sup>t</sup> that during the mechanosynthesis, each TBA form yields the same product in the presence of HIMO. This hypothesis was further confirmed by 15N CP/MAS experiments. As in the case the of 13C NMR, the left column depicts the spectra of physical mixtures of TBA I-III/HIMO (Figure 5a–c). The TBA I crystallized from MeCN (Figure 5a) is characterized by two sharp peaks at δ = 171.7 and 167.2 ppm, respectively. For TBA II, the chemical shifts were found to be at δ = 169.8 ppm and 160.0 ppm, while for TBA III, the chemical shits were at δ = 174.1 and 157.2 ppm. The distinction in relative intensities of the HIMO versus the TBA 15N signals depended on differences in relaxation times of TBA. The right column (Figure 5d–f) displays the spectra for the obtained cocrystals. An inspection of the 15N CP/MAS data shows apparent resemblances. The number of resonances in the isotropic part suggests that in the asymmetric unit, at least two pairs of TBA/HIMO molecules exist (Z' = 2).

**Figure 5.** The 60.82 MHz 15N CP/MAS spectra recorded with a spinning rate of 11 or 12 kHz. The left column shows a physical mixture of HIMO and TBA in a ratio of 1:1, (**a**) HIMO and TBA crystallized from MeCN (tautomer A); (**b**) HIMO and TBA crystallized from EtOH (tautomer B); (**c**) HIMO and TBA crystallized from water (tautomer B). The right column displays the spectra of samples after 1 h of grinding with MeOH as a LAG; (**d**) HIMO and TBA crystallized from MeCN; (**e**) HIMO and TBA crystallized from EtOH; (**f**) HIMO and TBA crystallized from water.

The 1H VF/MAS measurements offer an additional source of information for the progress of cocrystal formation. Figure 6 shows the spectra for the mixture of both components (Figure 6a,c,e) and samples after 1 h grinding (Figure 6b,d,f). The 1H chemical shifts for OH and NH protons located in the region 18–10 ppm provide evidence for the very complex hydrogen bonding network in the crystal lattice. The blue vertical line represents the position of OH proton for pure, unreacted HIMO. The small shift of this signal is a probe sensing the course of grinding process. Based on the analysis of spectra for ball milled samples, one can conclude that in experiments with TBA I and TBA II, the transformation was quantitative, while in case of TBA III (crystallized from water), the part of HIMO was not involved in the formation of cocrystals. This observation is consistent with both 13C and 15N analysis.

**Figure 6.** The 600.13 MHz 1H VF/MAS spectra recorded with a spinning rate 60 kHz. Lines a, c and e show a physical mixture of HIMO and TBA in a ratio of 1:1. Lines b, d and f depict the spectra of samples after 1 h of grinding with MeOH as a LAG (**a**) HIMO and TBA crystallized from MeCN, mixture; (**b**) HIMO and TBA crystallized from MeCN, ground; (**c**) HIMO and TBA crystallized from EtOH, mixture; (**d**) HIMO and TBA crystallized from EtOH, ground; (**e**) HIMO and TBA crystallized from water, mixture; (**f**) HIMO and TBA crystallized from water, ground. The vertical blue line shows that the position of the O-H group of HIMO is not involved in interactions with TBA.

### 3.2.2. SS NMR Structural Studies of BA/HIMO Cocrystal

In the next step, employing wet and solid state methods, we tested the ability of HIMO to form cocrystals with barbituric acid (BA), an analog of TBA. As before, the cocrystallization of BA with HIMO in MeOH led to the formation of the desired BA/HIMO cocrystals. Its structure was confirmed by 13C CP/MAS, PXRD and DSC measurements (Figure 7).

**Figure 7.** (**a**) 13C CP/MAS spectrum recorded with a spinning rate of 8 kHz for BA/HIMO cocrystals obtained by cocrystallization from MeOH; (**b**) powder x-ray diffraction diffractogram for BA/HIMO cocrystal obtained by cocrystallization from MeOH; (**c**) DSC curve of BA/HIMO cocrystal. For a full analysis of the crystallographic forms of BA, HIMO and BA/HIMO based on PXRD diffractograms, see Supporting Information (Figure S3).

Analyzing the reactivity of BA in mechanochemical procedures, it has to be noted that this compound forms different polymorphs, two anhydrous forms (form I and II, see Scheme 2) and a dihydrate phase. They present a trioxo structure, as confirmed by X-ray diffraction analysis. Recently, Chieriotti and coworkers proved the existence of unstable trihydroxyl and keto-enol tautomers of BA in the crystal lattice [45].

**Scheme 2.** Numbering system for BA and hydrogen bonding in the form I and II.

Figure 8a shows a mixture of BA and HIMO in a molar ratio of 1:1, crystallized from MeOH (labeled in the literature as Form II). The 13C CP/MAS spectra for BA form II have already been reported [44,45]. Two CH2 peaks located at 39.1 and at 41.1 ppm represent two independent molecules in the asymmetric unit cell where the CH2 moiety can be in or out of the plane of the ring in a half chair conformation. The resonances at 151.9, 170.4 and 171.6 (shoulder) ppm were associated with the C2, C6, and C4 carbon atoms of the carbonyl groups, respectively. The assignment of 13C NMR resonances for 1 is presented in Section 3.1. Figure 8c displays the sample prepared mechanochemically (Mixer Mill MM 200 equipped with a 5 mL agate jar and 5 mm diameter ball at an oscillation rate of 25 Hz). The 13C CP/MAS pattern shows changes and provides proof for the formation of a new species. The new signal at 79.5 ppm, which is characteristic for keto/enol-form, is apparent.

**Figure 8.** 13C CP/MAS Spectra recorded with a spinning rate of 8 kHz for (**a**) physical mixture of HIMO and BA (II) in a ratio of 1:1; (**b**) physical mixture of HIMO and BA-dihydrate in a ratio of 1:1; (**c**) HIMO and BA in a ratio of 1:1 after 2 h of grinding employing MeOH as LAG.

In extension of this study, we investigated crystals of BA containing two water molecules in the lattice, and the susceptibility of this form to yield cocrystals. Recently, King and coworkers reported on detailed structural studies on, and the unusual thermal phase behavior of, barbituric acid (BA) dihydrate [46]. Our results prove that during grinding at ambient temperature, this form is stable and does not undergo further transformation.

Figure 8b shows the 13C CP/MAS spectrum of the mixture BA·2H2O with HIMO in 1:1 molecular ratio. The peak at δ = 40 ppm is characteristic for the keto form of BA. The cocrystal was prepared by grinding in the Mixer Mill MM 200. In this case, we did not use LAG, assuming that crystalline water could be a substitute. After 1 h ball-milling, the process of BA/HIMO cocrystal formation was complete. The registered 13C NMR spectrum, like those shown in Figure 8c, confirms the structure of product.

The presence of the keto-form of BA further confirmed the validity of the 15N CP/MAS experiment. The spectrum of BA (Figure 9a) with one NMR signal located at δ = 150.5 ppm is consistent with data published by Chierotti et al. [45]. In the case of BA dihydrates, signals at δ = 150.5 and 153.6 ppm were observed (Figure 9b); Figure 9c shows the 15N CP/MAS spectrum of BA:HIMO cocrystal. Analysis of this spectrum suggests that 15N resonance representing the BA component was upfield shifted by ca. 10 ppm (δ = 140.2 ppm) compared to pure BA, while 15N representing HIMO showed a split signal at δ = 208.0 ppm. Figure 9d displays the 1H NMR VF/MAS spectrum of HIMO and BA (ratio 1:1) after 2 h of grinding recorded with a spinning rate 60 kHz. The resolution of this spectrum makes the assignment of proton signals unambiguous. The first striking difference is the lack of the OH signal at δ1H = 17.7 ppm, which, in pure sample 1, represents strong O-H···O hydrogen bonding and the appearance of new signals at δ = 15.7, 14.8 and δ = 9.6 ppm. The collected NMR data are a valuable source of information about the organization of new materials (HIMO and BA in a ratio of 1:1 after 2 h of grinding) and about the tautomeric form of BA.

**Figure 9.** 15N CP/MAS Spectra recorded with a spinning rate of 8 kHz for (**a**) barbituric acid; (**b**) barbituric acid dihydrate; (**c**) HIMO and BA in a ratio of 1:1 after 2 h of grinding; (**d**) 1H VF/MAS spectrum recorded with a spinning rate of 60 kHz for HIMO and BA in a ratio of 1:1 after 2 h of grinding.

### *3.3. X-ray Structure of HIMO and TBA*/*HIMO, BA*/*HIMO Cocrystals*

Pure HIMO crystallizes in the triclinic system in the centrosymmetric P-1 space group. The asymmetric unit contains two molecules of HIMO (Z' = 2). The crystal structure is displayed in Figure 10. Table 1 shows the experimental details and structural information.

**Figure 10.** Ortep drawing of the independent unit with a numbering system for HIMO; the position of the hydrogen atom is averaged between both oxygen acceptors at two locations with half occupancy.

The supramolecular array depicted in Figure 11 shows two structural features which are worthy of mention. Firstly, there are two strong hydrogen bonds with O···O distances equal 2.437 Å and 2.446 Å between molecules, creating asymmetric units and molecules related by a symmetry operation. These contacts are responsible for the formation of the chain structure. In both cases, the position of the hydrogen atom is averaged between both oxygen acceptors at two locations with half occupancy. The second feature seen in Figure 11 is a π–π interaction which is an additional factor stabilizing the crystal structure.


**Figure 11.** Molecular packing of HIMO.

The crystal structure and molecular packing of TBA/HIMO is shown in Figure 12. The experimental details and structural information are collected in Table 1. The TBA/HIMO crystallizes in the triclinic system in the centrosymmetric P-1 space group. The independent unit contains two molecules of TBA and two molecules of HIMO. Both components in the crystal lattice are connected by hydrogen bonds forming a unique supramolecular structure. The leading motif is created by the planary located TBA molecules forming ribbons (flat chains), rotated such that the thiocarbonyl groups point into the opposite directions. These chains are bonded by strong hydrogen bridges between CC(2)=OC(1)···HD(2)–ND(2) and CC(4)=OC(2)···HD(1)–ND(1), and further by CD(4)=OD(2)···HC(1)–NC(1) and CD(2)=OD(1)···HC(2)–NC(2). TBA chains are connected by the HIMO molecules to create a plane structure. The methyl groups of HIMO from two layers are pointing into the plane interface. The interaction in other interfaces between planes is created by π stacking interactions.

**Figure 12.** Ortep drawing of the independent unit in TBA/HIMO crystal.

Figure 13 shows the supramolecular pattern for TBA/HIMO in the crystal lattice. Clearly visible characteristic motifs are sheets formed by TBA molecules. These planes are created from the ribbons described above. The distances between OC(1)···ND(2) and OC(2)···ND(1) were found to be 2.854 Å and 2.839 Å, respectively. The analogous OD(2)···NC(1) and OD(1)···NC(2) lengths are equal to 2.850 Å and 2.886 Å. The oxygen atoms O1 and O2 of molecules A and B are involved in bifurcated hydrogen bonding with neighboring HIMO molecules. The strength of these bonds is defined by short O···O contacts equal 2.432 Å, 2.598 Å and 2.503 Å, 2.595 Å, respectively. The imidazole *N*-oxide molecules act as a link between adjoining TBA sheets during the formation of hydrogen bridges. As we postulated, at this stage, one of the hydrogens of the methylene group is transferred to the oxygen, then to HIMO oxygen forming intermolecular bonding. The second oxygen acts as an acceptor by interacting with OH residue in HIMO. In such an arrangement, carbonyl C=O groups, which are involved in strong hydrogen bonding, disturb the tautomeric nature of TBA. The TBA loses its pure keto tautomeric form and exists in keto-enol C–O···H–O–N form. Such a complex transfer process makes it difficult to refine the position of the hydrogen in the bridges but position of peak on electron density map shows that is predominantly attached to oxygen in HIMO.

**Figure 13.** Supramolecular structure of TBA/HIMO cocrystal.

Finally, the supramolecular structure of TBA/HIMO is supplemented by CH···<sup>π</sup> interactions between the methyl groups of HIMO and the ring of TBA. The average distance between the planes of the HIMO and TBA sheets, measured as the distance between the CH3 atom and the center of the TBA ring, is ca. 4 Å. The imidazole rings are in a stacked arrangement, and are twisted with respect to the TBA plane. It is worth noting that the C=S moiety, in this crystal form, interacts with the π-electrons of imidazole. The distance between the sulfur and the center of the imidazole ring is 3.5 Å.

Figure 14 shows the crystal structure and molecular packing of the BA/HIMO cocrystals. The experimental details and structural information are collected in Table 1. The BA/HIMO sample crystallizes in the triclinic system in the centrosymmetric P-1 space group. The asymmetric unit contains one molecule of BA and one of HIMO (Z = 2). The supramolecular structure is depicted in Figure 15. The motifs seen in the TBA/HIMO structure are also observed for BA/HIMO. The first similar pattern is the ribbon structure (flat chains) formed by barbituric acid. The hydrogen bonding network is similar to the one observed before. The layers are formed by BA ribbons located perpendicularly to the plane and stabilized by π stacking interactions. The HIMO molecules bridge the layers of BA such that protonated oxygens create hydrogen bonds with keto groups of BA. The lengths of N-H···O=C bonds were found to be 2.869 Å and 2.912 Å, respectively. The C(1)=O(1) and C(4)=O(3) groups are involved in the formation of bifurcated hydrogen bonds. The counter partner in these bridges is HIMO, which acts as a staple connecting the BA sheets located in parallel planes. The O···O contacts are equal to 2.474 Å and 2.593 Å. The keto-enol form is created according to the mechanism described above for TBA, and is also effective for BA/HIMO cocrystals. The evidence confirming the hypothesis that the tautomeric form is forced by hydrogen bonds is found within the analysis of the C-O bond lengths for BA. For C(2)–O(2), this length is 1.222 Å, while for C(1)=O(1) and C(4)=O(3), it is 1.283 Å and 1.276 Å, respectively. It is interesting to note that for pure anhydrous BA, existing in keto form, these bond lengths are 1.229 Å, 1.222 Å, 1.189 Å for C(2)=O(2), C(1)=O(1) and C(4)=O(3), respectively.

**Figure 14.** Ortep drawing of the independent unit in BA/HIMO cocrystal.

**Figure 15.** Supramolecular structure of BA/HIMO cocrystal.

The sheets formed by BA are located in parallel planes with a distance of ca. 4 Å. The HIMO clips next to the C(1)=O(1) and C(4)=O(3) BA positions, responsible for creating of structural network, are oriented in opposite directions. Such an orientation allows the BA sheets to join in a "zipper" type mechanism.

Concluding this part, the presented X-ray results are consistent with a publication by Braga and coworkers, who, while studying the gas–solid reactions between the different polymorphic modifications of barbituric acid and amines, reported similar structural motifs with complex hydrogen bonding networks [47].

### *3.4. Cell Cytotoxicity and Solubility of Tested Compound (HIMO and Its Cocrystals)*

As highlighted in the Introduction, 1-hydroxy-4,5-dimethyl-imidazole 3-oxide (**1**) has never been considered as a coformer in the formation of pharmaceutical cocrystals. Among the main problems were a lack of biological tests and limited knowledge about its cytotoxicity.

In view of its potential medical application, cell viability should to be tested to exclude potential toxicity. The cytotoxicity of the studied compounds was measured by the MTT assay method against HeLa (cervical cancer), K562 (chronic myelogenous leukemia) and noncancerous, 293T (derived from human embryonic kidney). Figure 16 shows the cellular viability after concentration-dependent treatment; the results indicate no significant toxicity in the case of incubation with the tested compounds in the 1–100 μM concentration range. The viability of cells was at a level of 80–100 percent, and only in the case of the highest concentration of the first cocrystal (BA/HIMO) did the survival rate decrease to 70% with HeLa cells. The results for noncancerous 293T were similar to those for HeLa cells. The obtained data indicate that all the tested compounds are suitable candidates for use in live cancer and noncancerous cells at concentrations of 1 μM to 100 μM.

**Figure 16.** The viability (%) of K562 and HeLa cells after 48 h incubation with the tested compounds in a concentration range from 1 μM (blue bar) to 10 μM, 50 μM and 100 μM (red, green violet bars) respectively. The results represent the mean ± standard error.

Figure 17 shows the solubility of pure TBA and BA samples versus the solubility of the studied cocrystals. The measurements were carried out in three media: water, SGFsp and EtOH. It can be concluded that in water (pH 5.7), the solubility of BA/HIMO cocrystal is slightly better compared to that of BA. In the case of TBA and TBA/HIMO, the relationship is reversed. The solubility of the cocrystals is reduced by over 30%. A cocrystal analysis in simulated gastric fluid without pepsin (SGFsp, pH 1.2) revealed a decrease in solubility for both binary systems compared to TBA and BA. For TBA/HIMO, solubility is reduced by ca. 17%, while for BA/HIMO, it was reduced by about 33% compared to the same cocrystals in water. In EtOH, the solubility of BA is higher compared to that of the cocrystal, while that of TBA and TBA/HIMO is comparable. Comparing the APIs and cocrystals, an increase in solubility was not observed. Usually, increased solubility is the first indication to begin the preparation of cocrystals, because this parameter has an influence on drug pharmacodynamics and the efficiency of treatment. In the case of TBA/HIMO and BA/HIMO, only a minute increase of solubility was noticed for BA/HIMO cocrystals in water. However, while solubility is an important aspect, this is not an arbitrary factor justifying the use of cocrystals in drug delivery. Other functions, for instance, the protection of sensitive APIs from the environmental effects or controlling the delivery of drugs to specific points in the body are important as well. Also relevant from a drug delivery point of view is providing more than one active pharmaceutical ingredient. This seems to be an attractive feature in the case of imidazole *N*-oxide 1 (HIMO).

**Figure 17.** Results of solubility measurements of TBA and BA and their cocrystals with HIMO.
