Cocrystals of Isoniazid with Polyphenols: Mechanochemical Synthesis and Molecular Structure

: Isoniazid is used as anti-tuberculosis drug which possesses functional groups capable of forming hydrogen bonds. A series of cocrystals of isoniazid (INH) with polyphenolic coformers such as catechol (CAT), orcinol (ORC), 2-methylresorcinol (MER), pyrogallol (PYR), and phloroglucinol (PLG) were prepared by solvent-assisted grinding. Powder cocrystals were characterized by infrared (IR) spectroscopy and X-ray powder di ﬀ raction. The crystal structure of the cocrystals revealed the unexpected hydration of the INH-MER cocrystal and the preference of the (phenol) O–H ··· N (pyridine) and (terminal) N-H ··· O (phenol) heterosynthons in the stabilization of the structures. The supramolecular architecture of the cocrystals is a ﬀ ected by the conformation and the substitution pattern of the hydroxyl groups of the polyphenols.


Introduction
The modification of solid forms of pharmaceutical active ingredients has led to the formation of polymorphs, salts, solvates, and cocrystals to improve physico-chemical properties, such as solubility, dissolution rate, melting point, and thermal stability [1]. Pharmaceutical cocrystals are crystalline solids formed by a pharmaceutical active ingredient and a cocrystal coformer, which remain together by noncovalent interactions-principally hydrogen bonds [2]. Crystal engineering concepts are employed in the design of pharmaceutical cocrystals considering the use of supramolecular synthons between the functional groups of the compounds involved in the formation of the cocrystal, leading to directional and geometrical well-defined hydrogen bond patterns [3].
Solvent-assisted grinding and solvent evaporation are two commonly used methods for cocrystal synthesis [4]. Solvent-assisted grinding is a fast method for obtaining powder cocrystals. The advantage of this method is that it does not depend on the solubility of the starting materials. However, not all grinding experiments lead to the formation of powder cocrystals. On the other hand, the solvent evaporation method is used to obtain single crystals of cocrystals and cocrystals that cannot be prepared by grinding experiments. The disadvantages of this method are the dependence of the solubility of the starting materials and the waiting time for the evaporation of the solvent.
Here, we report the mechanochemical synthesis, characterization, and crystal structure of cocrystals of isoniazid (INH) with the polyphenolic coformers catechol (CAT), orcinol (ORC), 2-methylresorcinol (MER), pyrogallol (PYR), and phloroglucinol (PLG) as potential anti-tuberculosis agents ( Figure 1). dependence of the solubility of the starting materials and the waiting time for the evaporation of the solvent.
Mixtures in a 1:1 ratio of INH (300 mg, 2.1 mmol) and the polyphenolic coformer (2.1 mmol, ORC = 273 mg; MER = 273 mg; CAT = 242 mg; PLG = 277 mg; PYR = 277 mg) were ground in a porcelain mortar with a pestle. Before the start of the grinding, 0.5 mL of dichloromethane was added. After 3 min of grinding, dichloromethane evaporated, and the powder was collected. The cycle of adding dichloromethane (0.5 mL) and grinding for 3 min was repeated an additional three times until 12 min of grinding was completed. After that, a polycrystalline powder was obtained. The grinding process was performed in a laboratory hood to eliminate the residual dichloromethane.
Single crystals of INH-ORC, INH-MER-H2O, INH-CAT, INH-PLG, and INH-PYR suitable for X-ray diffraction were obtained by dissolving 50 mg of the respective polycrystalline ground powder in ethanol and left to evaporate at room temperature.
Mixtures in a 1:1 ratio of INH (300 mg, 2.1 mmol) and the polyphenolic coformer (2.1 mmol, ORC = 273 mg; MER = 273 mg; CAT = 242 mg; PLG = 277 mg; PYR = 277 mg) were ground in a porcelain mortar with a pestle. Before the start of the grinding, 0.5 mL of dichloromethane was added. After 3 min of grinding, dichloromethane evaporated, and the powder was collected. The cycle of adding dichloromethane (0.5 mL) and grinding for 3 min was repeated an additional three times until 12 min of grinding was completed. After that, a polycrystalline powder was obtained. The grinding process was performed in a laboratory hood to eliminate the residual dichloromethane.
Single crystals of INH-ORC, INH-MER-H 2 O, INH-CAT, INH-PLG, and INH-PYR suitable for X-ray diffraction were obtained by dissolving 50 mg of the respective polycrystalline ground powder in ethanol and left to evaporate at room temperature.
The crystal structure of the cocrystals was analyzed in an Oxford Diffraction Gemini "A" diffractometer (Oxford Diffraction, Oxfordshire, UK) with a CCD area detector (λ MoKα = 0.71073 Å, monochromator). Crystal data, data collection, and structure refinement details are summarized in Table 1. Unit cell parameters were determined with a set of three runs of 15 frames (1 • in ω). The double-pass method of scanning was used to exclude any noise. The collected frames were integrated using an orientation matrix determined from the narrow frame scans. CrysAlisPro and CrysAlis RED software packages [18] were used for data collection and integration. Analysis of the integrated data did not reveal any decay. Final cell parameters were determined by a global refinement of 1674, 1815, 1842, 2721, and 1620 reflections (3.519 • < θ < 29.50 • ) for compounds INH-ORC, INH-MER, INH-CAT, INH-PLG, and INH-PYR respectively. Collected data were corrected for absorption effects by using an analytical numeric absorption correction [19] using a multifaceted crystal model based on expressions upon the Laue symmetry using equivalent reflections. Structure solution and refinement were carried with the programs SHELXS-2014 [20] and SHELXL-2014 [21] respectively. WinGX v2014.1, Ortep [22], and Mercury [23] software were used to prepare material for publication. Full-matrix least-squares refinement was carried out by minimizing (Fo 2 − Fc 2 ) 2 . All nonhydrogen atoms were refined anisotropically. The H atoms of the hydroxy and amine groups were located in a difference map and refined isotropically with Uiso(H) = 1.

IR Spectroscopy
IR spectroscopy is a tool used to identify the formation of new solid phases by the shift of the bands of the functional groups of the cocrystal with respect to the starting products. Characteristic bands of the functional groups of the compounds were assigned according with previous reports [14,[24][25][26]. The IR spectra of the ground powders were different with respect to the starting materials (Figures

Powder X-ray Diffraction
PXRD patterns of the polycrystalline ground mixtures were different from the starting materials (Figures S6-S10 Supplementary Materials), indicating the formation of the cocrystal. PXRD patterns of the ground products were similar to the theoretical PXRD patterns calculated with Mercury [23], indicating an adequate homogeneity between the ground mixture and the single crystal ( Figure 3). The amine N-H, carbonyl C=O, hydroxyl O-H, and pyridine C=N stretching frequencies of INH were observed to have shifted in the IR spectra of the polycrystalline ground powders and the single crystals as a consequence of the formation of intermolecular hydrogen bonds, due to the rearrangement of the crystalline structure (Table S1 Supplementary Materials). The IR spectra of INH showed the N-H stretching band at 3303 cm −1 ; meanwhile the IR spectra of the polycrystalline ground powders and the single crystals appeared shifted to higher frequencies (from 3322 cm −1 to 3397 cm −1 ). The C=O stretching band appeared at 1662 cm −1 in "free INH" while the polycrystalline ground powders and the single crystals appeared shifted from 1651 cm −1 to 1676 cm −1 . The pyridine C=N stretching band in INH also was shifted from 1553 cm −1 in "free" INH to the 1543 cm

Powder X-ray Diffraction
PXRD patterns of the polycrystalline ground mixtures were different from the starting materials (Figures S6-S10 Supplementary Materials), indicating the formation of the cocrystal. PXRD patterns of the ground products were similar to the theoretical PXRD patterns calculated with Mercury [23], indicating an adequate homogeneity between the ground mixture and the single crystal ( Figure 3). Crystals 2020, 10, x FOR PEER REVIEW 6 of 16

INH-MER-H 2 O cocrystal.
Unexpected hydration of the INH-MER cocrystal was found after the single-crystal X-ray diffraction study. IR spectra and PXRD patterns of the INH-MER ground with dichloromethane is similar to the IR spectra and the theoretical PXRD pattern of INH-MER-H 2 O single crystal, suggesting the incorporation of water from the environment into the crystalline structure during the grinding process, obtaining the hydrated cocrystal. Polyphenols such as orcinol, phloroglucinol and pyrogallol are hygroscopic [33][34][35]; therefore, it is not rare to obtain hydrated cocrystals of polyphenols. Some examples are theophylline with resorcinol, orcinol and phloroglucinol [13]; nalidixic acid with phloroglucinol [36]; and lidocaine with phloroglucinol [14].
INH-CAT cocrystal. The isoniazid-catechol cocrystal crystallizes in the monoclinic P2/c space group, with one molecule of INH and one and a half different molecules of CAT in the asymmetric unit. A dimeric unit of CAT (synthon IX) in the syn conformation is formed via O4-H4···O3 R 2 2 (10) hydrogen bonds (CAT also form the O4-H4···O3 S(5) intramolecular hydrogen bond). INH molecules are linked to the catechol dimer by the N2-H2B···O4 (synthon III) and O3-H3···O1 (synthon II) hydrogen bonds to form an INH 2 -CAT 2 centrosymmetric tetramer depicting a set of R 3 3 (9), R 2 2 (10), S(5) hydrogen bond motifs, similar to those observed in the isoniazid-caffeic acid cocrystal [8]. The first dimensional array is given by the interlinking of tetramers by the N1-H1···N2 (synthon VIII) hydrogen bonds forming a supramolecular tape running along the c axis (Figure 8a). Tapes are connected by the second crystallographic independent molecule of CAT, which forms two O2-H2···N3 (synthon I) hydrogen bonds extending a bidimensional supramolecular sheet along the ac plane (Figure 8b). The angle between the pyridine ring and the hydroxyl group plane is 19.56 • (C4-N3-H2-O2 torsion angle). The interlinking of sheets by the N2-H2A···O1 (synthon VI), N2-H2B···O4 (synthon III) hydrogen bonds, and π-stacking (Cg1···Cg3 INH-MER-H2O cocrystal. Unexpected hydration of the INH-MER cocrystal was found after the single-crystal X-ray diffraction study. IR spectra and PXRD patterns of the INH-MER ground with dichloromethane is similar to the IR spectra and the theoretical PXRD pattern of INH-MER-H2O single crystal, suggesting the incorporation of water from the environment into the crystalline structure during the grinding process, obtaining the hydrated cocrystal. Polyphenols such as orcinol, phloroglucinol and pyrogallol are hygroscopic [33][34][35]; therefore, it is not rare to obtain hydrated cocrystals of polyphenols. Some examples are theophylline with resorcinol, orcinol and INH-CAT cocrystal. The isoniazid-catechol cocrystal crystallizes in the monoclinic P2/c space group, with one molecule of INH and one and a half different molecules of CAT in the asymmetric unit. A dimeric unit of CAT (synthon IX) in the syn conformation is formed via O4-H4•••O3 R 2 2 (10) hydrogen bonds (CAT also form the O4-H4•••O3 S(5) intramolecular hydrogen bond). INH molecules are linked to the catechol dimer by the N2-H2B•••O4 (synthon III) and O3-H3•••O1 (synthon II) hydrogen bonds to form an INH2-CAT2 centrosymmetric tetramer depicting a set of R 3 3(9), R 2 2(10), S(5) hydrogen bond motifs, similar to those observed in the isoniazid-caffeic acid cocrystal [8]. The first dimensional array is given by the interlinking of tetramers by the N1-H1•••N2 (synthon VIII) hydrogen bonds forming a supramolecular tape running along the c axis (Figure 8a). Tapes are connected by the second crystallographic independent molecule of CAT, which forms two O2-H2•••N3 (synthon I) hydrogen bonds extending a bidimensional supramolecular sheet along the ac plane (Figure 8b). The angle between the pyridine ring and the hydroxyl group plane is 19.56°   [38,39]. The interconnection of supramolecular sheets is given by the N2-H2A···O2 (synthon III), O3-H3···N3, and O4-H4···N6 (synthon I) hydrogen bonds, generating the 3D supramolecular array. Crystals 2020, 10, x FOR PEER REVIEW 11 of 16  INH-PYR cocrystal. The isoniazid-pyrogallol cocrystal crystallizes in the triclinic P-1 group with one molecule of INH and one molecule of PYR in the anti conformation in the asymmetric unit (PYR form the O2-H2···O4 S(5) intramolecular hydrogen bond). PYR dimerization (synthon IX) occurs via O2-H2···O4 hydrogen bonds R 2 2 (10) motif in a similar way to the INH-CAT cocrystal. The PYR dimer is linked to two INH molecules by the N1-H1···O2 (synthon IV) and O4-H4A···N2 (synthon V) hydrogen bonds, depicting a R 3 3 (7) motif, forming an INH 2 -PYR 2 centrosymmetric tetramer and showing a set of R 3 3 (7), R 2 2 (10), S(5) hydrogen bond motifs. A bidimensional supramolecular sheet, extended along the (1 1 1) plane, is formed by the propagation of the INH 2 -PYR 2 by the O3-H3···N3 (synthon I) and N2-H2B···O1 (synthon VI) hydrogen bonds, forming C 2 2 (14) chains (Figure 10a). The 1,2,3-trisubstituted PYR allowed for the formation of a finite dimer assembly like CAT, as well as the formation of an infinite chain as in ORC and MER. The angle between the pyridine ring and the hydroxyl group plane is −98.55 • (C4-N3-H3A-O3). The third dimensional supramolecular array is given by the interlinking of sheets by the N2-H2A···O3 (synthon III) hydrogen bond depicting a C 2 2 (8) motif (Figure 10b). An analysis of the supramolecular synthons found in the cocrystals ( Figure 5) revealed that the phenol-pyridine heterosynthon (synthon I) is present in the five cocrystals, indicating the hierarchy of this synthon [38,40,41]   Polyphenol conformation affects the supramolecular arrangement. In the bis-phenols (ORC, MER, and CAT) when the hydroxyl groups adopt an anti conformation, a linear chain is formed as in INH-MER-H 2 O cocrystal. On the other hand, a syn conformation leads to the formation of a finite motif as in the INH-ORC cocrystal or dimerization as CAT. In the case of the INH-PLG cocrystal, two of the hydroxyl groups point in the same direction (C 3h conformation), forming a finite motif. In the INH-PYR cocrystal two hydroxyl groups point in the same direction, generating the dimerization of PYR; meanwhile, the third hydroxyl group points to the opposite direction, leading to the formation of a linear chain.

Conclusions
Cocrystals of isoniazid with polyphenolic coformers catechol, orcinol, 2-methyl resorcinol, phloroglucinol, and pyrogallol were obtained by the solvent-assisted grinding method. IR spectra and powder X-ray diffraction patterns of the polycrystalline ground powders were different from the starting materials and similar to those obtained for the single crystals. Incorporation of water into the crystalline structure of INH-MER was discovered. The molecular structure of the cocrystals showed the preference for the (phenol) O-H···N (pyridine) and (terminal) N-H···O (phenol) hydrogen bond synthons. The supramolecular architecture of the cocrystals is affected by the conformation and the substitution pattern of the hydroxyl groups in the polyphenols.  Table S1. Characteristic IR frequencies (cm −1 ) of INH, the polyphenols, the polycrystalline ground powder, and the single crystals.