Exploiting Supramolecular Synthons in Cocrystals of Two Racetams with 4-Hydroxybenzoic Acid and 4-Hydroxybenzamide Coformers

: Structures of three cocrystals of nootropic racetams were studied. They included two cocrys-tals of phenylpiracetam ( PPA ) with 4-hydroxybenzoic acid ( HBA ) with different stoichiometries, PPA · HBA and PPA · 2HBA , and cocrystal of 2-(4-phenyl-2-oxopyrrolidin-1-yl)-N’-isopropylideneace tohydrazide ( PPAH ) with 4-hydroxybenzamide ( HBD ), PPAH · HBD · (acetone solvate). X-ray study of the pure forms of PPA and PPAH was also carried out to identify variations of molecular synthons under the inﬂuence of conformers. The cocrystal structures revealed the diversity of supramolecular synthons namely, amide-amide, amide-acid, acid-acid, and hydroxyl-hydroxyl; however, very similar molecular chains were found in PPA and PPA · 2HBA , and similar molecular dimers in PPAH and PPAH · HBD . In addition, conformational molecular diversity was observed as disorder in PPA · 2HBA as it was observed earlier for rac -PPA that allows for the consideration that cocrystal as an example of partial solid solution. Quantum chemical calculations of PPA and PPAH conformers demonstrated that for most conformers, energy differences do not exceed 2 kcal/mol that suggests the inﬂuence of packing conditions (in this case R - and S -enantiomers intend to occupy the same molecular position in crystal) on molecular conformation.


Introduction
Currently there is a significant interest in pharmaceutical materials that can be used for treatment of central nervous system (CNS) disorders. One of the groups of such materials is nootropics that are prescribed as drugs or supplements to improve memory, focus, and cognitive performance. Racetams (piracetam and its derivatives) are nootropic agents known since the 1970s [1][2][3][4]. Depending on their substituents, they have different degrees of pharmacological activity.
Nootropil (2-oxo-1-pyrrolidinyl-acetamide, piracetam, PA, Scheme 1) was marketed by the United Collection Bureau (UCB) to treat memory and balance problems [5]. The physiological action and structural peculiarities of PA have been studied thoroughly. This compound of one hydrate and five anhydrous crystalline forms was documented by Fabbiani et al. [6].
In search for nootropics with improved physicochemical properties, significant efforts were addressed to the PA cocrystals [7][8][9][10][11]. Zaworotko at al. [11] demonstrated the importance of supramolecular homo-and heterosynthons in crystal engineering of active pharmaceutical ingredients (APIs) for an explanation and prediction of polymorphic and cocrystalline forms of APIs. This paradigm looks significantly important for nonionized

Chemicals and Solvents
Phenylpiracetam powder (100%) was purchased from Purenootropics (Purenootropics.net), and Phenylpiracetam as a supplement in capsules was purchased from BrainMedics. 4-Hydroxybenzoic acid, 4-hydroxybenzamide, and acetone solvent were purchased from Sigma Aldrich and used without purification.
To mask their bitter taste, some racetams including PA and PPA were cocrystallized with saccharine and zinc saccharinate [7]. Particularly for PPA, these attempts resulted in preparation of two crystalline complexes, ZnSac 2 Car (Car = carphedon in [7], refcode NEFLOB) as a 1D coordination chain where both carbonyl oxygens of pyrrolidone and amide groups coordinate to two different zinc atoms, and [ZnSac 2 (H 2 O) 2 ]·Car·EtOH·H 2 O (refcode NEFMOE) as a discrete inclusion compound with carphedon interacting with the [ZnSac 2 (H 2 O) 2 ] complex through hydrogen bonding [7]. Apart from the PPA pure forms, these are the only two known examples of structurally studied multicomponent PPA crystals.
The goal of this study was a search for new forms of nootropics PPA and PPAH, and a comparison of molecular synthons found in pure materials, different cocrystalline, and polymorphic forms. As coformers for PPA and PPAH, we used compounds with molecular structures similar to those which were successfully used for formation of PA cocrystals [10,11], such as 4-hydroxybenzoic acid (HBA) and 4-hydroxybenzamide (HBD). The HBA in particular has performed well in cocrystal design with APIs [11,[23][24][25][26]. Structural, and computational studies were carried out for the following pure and cocrystalline materials: PPA, PPA·HBA, PPA·2HBA, PPAH, and PPAH·HBD·(acetone solvate).

Chemicals and Solvents
Phenylpiracetam powder (100%) was purchased from Purenootropics (Purenootropics.net), and Phenylpiracetam as a supplement in capsules was purchased from BrainMedics. 4-Hydroxybenzoic acid, 4-hydroxybenzamide, and acetone solvent were purchased from Sigma Aldrich and used without purification. An amount of 44.0 mg (0.2 mmol) of PPA and 42.0 mg (0.3 mmol) of HBA were dissolved in 5 mL of acetone at room temperature in a test tube covered with parafilm with a hole. After a week the solvent had evaporated and crystals were observed.

Synthesis of PPAH·HBD Cocrystal
Phenylpiracetam produced by BrainMedics was removed from capsules and used for cocrystallization. After cocrystal was obtained and characterized with single crystal X-ray diffraction analysis, we realized that instead of PPA we had its derivative, 2-(4-phenyl-2oxopyrrolidin-1-yl)-N -isopropylideneacetohydrazide (PPAH) presented in Scheme 1. To be sure that powder from capsules was indeed PPAH, we recrystallized it from acetone and the single crystal X-ray study unambiguously confirmed that the substance was PPAH not PPA. In addition, HPLC analysis was performed to check the purity of the substance from capsules. It demonstrated only one peak that stated the compound from capsules did not have any contaminations.

Synthesis of PPAH·HBD Cocrystal
To obtain the cocrystal, PPAH (27.0 mg, 0.1 mmol) and HBD (14.0 mg, 0.1 mmol) in 1:1 molar ratio were separately dissolved in 5 mL of acetone, filtered, and mixed. The resulting solution was covered with parafilm with small holes for slower evaporation and left at room temperature. Small colorless needles showed up on the vial walls after 24 h. Melting points for PPAH (144 • C), HBD (161 • C) and 1:1 cocrystal PPAH·HBD(acetone solvate) (108 • C) were measured on SRS MeltTemp apparatus. All samples were heated with a heat rate 1 • C/min until the substance was completely liquid.

Single-Crystal X-ray Diffraction Analysis
X-ray diffraction data for PPA, PPAH, and PPA·2HBA cocrystal, were collected on a SMART APEX II CCD diffractometer (Bruker AXS, Madison, WI [27] using graphitemonochromatized Mo Kα radiation (λ = 0.71073 Å). For PPA·HBA and PPAH·HBD cocrys-tals, X-ray diffraction data were collected on a Bruker D8 VENTURE diffractometer with microfocus sealed tube using graphite-monochromatized Cu Kα radiation (λ = 1.54178 Å). SADABS program was used for absorption correction and scaling of observed data [28]. The structures were solved by direct methods and refined by full-matrix least-squares on F 2 for all data, using SHELXS97 [29] and OLEX 2.0 [30] software suites. The nonhydrogen atoms were refined in anisotropic approximation. The disorder was resolved for PPA in the pure form and in the 1:1 cocrystal PPA·HBA. The C-H atoms were fixed at idealized positions and refined with a riding-model approximation: C-H = 0.95-1.00 Å with Uiso(H) = 1.5 Ueq (C-methyl) and 1.2 Ueq (C) for other H atoms. The N-H and O-H hydrogens were located from the difference Fourier map or constrained. In cocrystal PPAH·HBD acetone solvent was disordered and the SQUEEZE procedure [31] was used to treat the diffused solvent in voids. The electron counts and the volume of associated voids indicated approximately one acetone solvent molecule per asymmetric unit. The sum formula reflected this ration. A check of the final CIF files (CCDC no.2247133-2247137) using PLATON did not show any missed symmetry. MERCURY2022.3.0 program (Cambridge Crystallographic Data Centre, Cambridge, UK)was used to make the molecular graphics [32].

Quantum Chemical Calculations
For the comparison of different enantiomers and their conformers quantum chemical calculations of total energy of PPA molecules in pure form (4 conformers), and in two cocrystals with HBA, PPAH in pure form and in cocrystal with HBD were carried out using GAUSSIAN 09 program package [33] and the B3LYP/6-311++G(d,p) level of theory [34]. The experimental X-ray coordinates were used for calculations. Correction on zero-point energy (ZPE) was included into molecular energy. A summary of results is presented in Table S1.

Results
Single crystal X-ray diffraction data, data collection, and structure refinement details are summarized in Table 1. Hydrogen bonding interactions were analyzed and finally calculated using the HTAB instruction in SHELXL [29], see Table 2.     Figure S1). To estimate if purchased PPA material represents a racemic mixture or enantiopure compound we carried out its diffraction study. It was found that this material was a racemate with two symmetrically independent molecular positions in the unit cell, each of which was occupied by either Ror S-enantiomers with almost equal~50% occupation factor (Figure 1a). These results are very close to data previously published by Rekis et al. for rac-PPA [20]. The rac-PPA structure allowed the authors of the above mentioned paper to describe the molecular arrangement in this crystal as a solid solution of enantiomers. Such a situation is common for structures of enantiomers when Rand S-enantiomers are interchangeable due to their isosterism, or significant similarity of their molecular shapes [35][36][37][38][39]. It was also mentioned that conformational flexibility of enantiomers helps them to adjust to their positions in crystal lattice. For room-temperature crystal structure PPA·HBA, the disorder was not resolved during structure refinement ( Figure 1b); however, thermal ellipsoids of the phenyl and pyrrolidone rings demonstrate hints of such disorder. For low-temperature structure PPA·2HBA the disorder model was found (Figure 1c) with occupancy factors of 0.510 (6) and 0.490(6) for two disordered positions similar to the PPA pure form.  Figure S1). To estimate if purchased PPA material represents a racemic mixture or enantiopure compound we carried out its diffraction study. It was found that this material was a racemate with two symmetrically independent molecular positions in the unit cell, each of which was occupied by either R-or S-enantiomers with almost equal ~50% occupation factor (Figure 1a). These results are very close to data previously published by Rekis et al. for rac-PPA [20]. The rac-PPA structure allowed the authors of the above mentioned paper to describe the molecular arrangement in this crystal as a solid solution of enantiomers. Such a situation is common for structures of enantiomers when R-and S-enantiomers are interchangeable due to their isosterism, or significant similarity of their molecular shapes [35][36][37][38][39]. It was also mentioned that conformational flexibility of enantiomers helps them to adjust to their positions in crystal lattice. For room-temperature crystal structure PPA·HBA, the disorder was not resolved during structure refinement ( Figure 1b); however, thermal ellipsoids of the phenyl and pyrrolidone rings demonstrate hints of such disorder. For low-temperature structure PPA·2HBA the disorder model was found (Figure 1c) with occupancy factors of 0.510(6) and 0.490(6) for two disordered positions similar to the PPA pure form.  Figure (a,c). Colorings scheme: oxygen-red; nitrogen-blue; carbon-grey; hydrogen-white, as used here and further.
Cocrystals PPA·HBA and PPA·2HBA were grown using the different ratios of starting materials (1:1 and 2:3, see Experimental Section). It is worth mentioning that variable stoichiometric ratios are not uncommon for API cocrystals [11,40,41].
The PPA molecule in PPA, PPA·HBA, and PPA·2HBA crystals demonstrates conformational flexibility. Figure 2 presents three examples of overlay of the molecular structures of the same enantiomers found in the above mentioned crystals. Absolute and relative molecular energies of the enantiomers with different conformations are given in Table  S1. It is possible to see that energy differences between these structures are not large and related mostly to different orientations of amide and phenyl groups attached to the oxopyrrolidine ring.  Figure (a,c). Colorings scheme: oxygen-red; nitrogen-blue; carbon-grey; hydrogen-white, as used here and further.
Cocrystals PPA·HBA and PPA·2HBA were grown using the different ratios of starting materials (1:1 and 2:3, see Experimental Section). It is worth mentioning that variable stoichiometric ratios are not uncommon for API cocrystals [11,40,41].

Crystal Structures of PPAH Pure Form and PPAH·HBD Cocrystal
Asymmetric units of PPAH and PPAH·HBD (the disordered acetone solvent molecule was excluded) crystals and overlapping diagram for PPAH from these two solids are shown in Figure 5a-c (for numbering see Figure S1). The geometrical parameters of the PPAH molecule are similar in the pure form and in the cocrystal. These similarities are evident from the overlay of these molecules presented in Figure 5c. Molecular energies of the PPAH molecule in pure crystal and in cocrystal are very close (Table S2).
Chemistry 2023, 5, FOR PEER REVIEW 9 chains in the layer in cocrystal via CH···O short contacts and π-π stacking interactions between the phenyl rings. (d) Packing of HBA chains in stacking layer.

Crystal Structures of PPAH Pure Form and PPAH·HBD Cocrystal
Asymmetric units of PPAH and PPAH·HBD (the disordered acetone solvent molecule was excluded) crystals and overlapping diagram for PPAH from these two solids are shown in Figure 5a-c (for numbering see Figure S1). The geometrical parameters of the PPAH molecule are similar in the pure form and in the cocrystal. These similarities are evident from the overlay of these molecules presented in Figure 5c. Molecular energies of the PPAH molecule in pure crystal and in cocrystal are very close (Table S2). PPAH crystallizes in the monoclinic P21/c space group with one molecule in the asymmetric unit. The expected dimerization of molecules occurs via amide-amide homosynthon, N···O = 2.948(2) Å (Figure 6a). The dimers pack in stacks along the shortest a-axis with only weak intermolecular CH···O interactions between stacks (Table 2, Figure 5b). In the PPAH·HBD cocrystal, the self-association of each component occurs via amide-amide homosynthon (Figure 7a,b). Similar to the pure form, the PPAH molecules in cocrystal form the centrosymmetric dimers (N···O = 2.924(4) Å), while HBD coformers PPAH crystallizes in the monoclinic P2 1 /c space group with one molecule in the asymmetric unit. The expected dimerization of molecules occurs via amide-amide homosynthon, N···O = 2.948(2) Å (Figure 6a). The dimers pack in stacks along the shortest a-axis with only weak intermolecular CH···O interactions between stacks (Table 2, Figure 5b).
Chemistry 2023, 5, FOR PEER REVIEW 9 chains in the layer in cocrystal via CH···O short contacts and π-π stacking interactions between the phenyl rings. (d) Packing of HBA chains in stacking layer.

Crystal Structures of PPAH Pure Form and PPAH·HBD Cocrystal
Asymmetric units of PPAH and PPAH·HBD (the disordered acetone solvent molecule was excluded) crystals and overlapping diagram for PPAH from these two solids are shown in Figure 5a-c (for numbering see Figure S1). The geometrical parameters of the PPAH molecule are similar in the pure form and in the cocrystal. These similarities are evident from the overlay of these molecules presented in Figure 5c. Molecular energies of the PPAH molecule in pure crystal and in cocrystal are very close (Table S2). In the PPAH·HBD cocrystal, the self-association of each component occurs via amide-amide homosynthon (Figure 7a,b). Similar to the pure form, the PPAH molecules in cocrystal form the centrosymmetric dimers (N···O = 2.924(4) Å), while HBD coformers In the PPAH·HBD cocrystal, the self-association of each component occurs via amideamide homosynthon (Figure 7a Table 2) identical with that found for (PPA) 2 (HBA) 2 tetramers (Figure 2a). The discrete PPAH dimers and roughly planar HBD chains interconnect through HBD hydroxyl and PPAH carbonyl groups (O···O = 2.665(4) Å) resulting in the H-bonded layer (Figure 7c). An interesting feature is that the stacking pattern of PPAH in the pure form was conserved in cocrystal strengthening by the PPAH···HBD coformer H-bond ( Figure S3). The packing of the layers remains voids of 241 Å 3 filled by the disordered acetone solvent ( Figure S4).
form the H-bonded double chain where the amide-amide homosynthons are further interlinked via synthon's sequence …R2 2 (8)R4 2 (8)R2 2 (8)… (N···O = 2.940(4), 3.064(4) Å, Table  2) identical with that found for (PPA)2(HBA)2 tetramers (Figure 2a). The discrete PPAH dimers and roughly planar HBD chains interconnect through HBD hydroxyl and PPAH carbonyl groups (O···O = 2.665(4) Å) resulting in the H-bonded layer (Figure 7c). An interesting feature is that the stacking pattern of PPAH in the pure form was conserved in cocrystal strengthening by the PPAH···HBD coformer H-bond ( Figure S3). The packing of the layers remains voids of 241 Å 3 filled by the disordered acetone solvent ( Figure S4). Quantum chemical calculations of PPA and PPAH conformers found in the pure materials and cocrystals (see SI) demonstrated that energy differences for most conformers do not exceed 2 kcal/mol. Such a difference suggests that conformational variations of these molecules are mostly defined by influence of packing conditions (in this case R-and S-enantiomers intend to occupy same molecular position in crystal) on molecular conformation.

Conclusions
Two cocrystals of phenylpiracetam (PPA) with 4-hydroxybenzylcarboxylic acid (HBA) coformer with different stoichiometry and a cocrystal of 2-(4-phenyl-2-oxopyrrolidin-1-yl)-N′-isopropylideneacetohydrazide (PPAH) with 4-hydroxybenzamide (HBD) coformer were obtained by cocrystallization experiments. Despite the fact that both racetams contain a primary amide as a main functional group for hydrogen bonding, the structures of reported cocrystals are significantly different and dependent on the interplay of amide-amide, amide-carboxylic acid, carboxylic acid-carboxylic acid supramolecular homo-and heterosynthons. Otherwise, the hydroxyl-carbonyl heterosynthon was found in all three cocrystal structures with both coformers. The H-bonded homomeric chain found in the PPA rac-and enantiopure forms, was also present in the 1:2 cocrystal resulting in alternation of PPA and HBA homomeric regions. The dimerization of PPAH via amide-amide supramolecular homosynthon led to the identical stacking of this homodimer as a dominating motif both in the cocrystal and in the racetam pure form. PPA in two cocrystals, PPA·HBA and PPA·2HBA demonstrated conformational freedom originating from the lack of strong intermolecular interactions with participation of phenyl and oxopyrrolidine rings that was reflected in disorder registered for these moieties in the crystals. The described situation allowed us to consider these cocrystals as examples of partial solid solutions. Quantum chemical calculations of PPA and PPAH conformers found in the pure materials and cocrystals (see Supplementary Materials) demonstrated that energy differences for most conformers do not exceed 2 kcal/mol. Such a difference suggests that conformational variations of these molecules are mostly defined by influence of packing conditions (in this case R-and S-enantiomers intend to occupy same molecular position in crystal) on molecular conformation.

Conclusions
Two cocrystals of phenylpiracetam (PPA) with 4-hydroxybenzylcarboxylic acid (HBA) coformer with different stoichiometry and a cocrystal of 2-(4-phenyl-2-oxopyrrolidin-1yl)-N -isopropylideneacetohydrazide (PPAH) with 4-hydroxybenzamide (HBD) coformer were obtained by cocrystallization experiments. Despite the fact that both racetams contain a primary amide as a main functional group for hydrogen bonding, the structures of reported cocrystals are significantly different and dependent on the interplay of amideamide, amide-carboxylic acid, carboxylic acid-carboxylic acid supramolecular homo-and heterosynthons. Otherwise, the hydroxyl-carbonyl heterosynthon was found in all three cocrystal structures with both coformers. The H-bonded homomeric chain found in the PPA racand enantiopure forms, was also present in the 1:2 cocrystal resulting in alternation of PPA and HBA homomeric regions. The dimerization of PPAH via amide-amide supramolecular homosynthon led to the identical stacking of this homodimer as a dominating motif both in the cocrystal and in the racetam pure form. PPA in two cocrystals, PPA·HBA and PPA·2HBA demonstrated conformational freedom originating from the lack of strong intermolecular interactions with participation of phenyl and oxopyrrolidine rings that was reflected in disorder registered for these moieties in the crystals. The described situation allowed us to consider these cocrystals as examples of partial solid solutions.