Ethenzamide (Energy Chemical Co., Ltd., China), Trimesic acid (TMA) (Energy Chemical Co., Ltd., China), Methanol (Shanghai Lingfeng Chemical Reagent Co., Ltd., China) and toluene (Shanghai Lingfeng Chemical Reagent Co., Ltd., China).

Fourier Transform-Infrared Spectroscopy (FT-IR) (VERTEX 70, Bruker, America), Powder X-Ray Diffraction (PXRD) (Rigaku, Tokyo, Japan), Single Crystal X-Ray diffraction (SXRD) (Bruker APEX-II, Bruker, America), High-performance Liquid Chromatography (HPLC) (Waters-e2695, Waters, America).

Equimolar quantities of ETZ (0.20 mol) and TMA (0.20 mol) were added to a mixed solvent of 2 mL of methanol and 3 mL of toluene in a 10 mL glass vial. The slurry was stirred at 60˚C for 10 min. The obtained clear solution was cooled down slowly to room temperature for 2 days.

The infrared spectra of samples were evaluated by an FT-IR spectrometer (Bruker Alpha FT-IR spectrometer). The measurements were performed in a range of 4000 - 400 cm⁻¹ with a resolution of 4 cm⁻¹. Powder samples (about 2 mg) were manually mixed with 100 mg of dry KBr in an agate mortar and pressed into thin pellets. Data were analyzed by Spectrum software.

The diffraction patterns were measured on Rigaku Ultima IV X-ray Diffractometer (Rigaku, Tokyo, Japan) using Cu Kα X-ray (λ = 1.5406 Å) and the generator operating at 40 kV and 40 mA. The scans ran from 5.0˚ to 50.0˚ (2θ), with an increasing step size of 0.02˚ and a scan rate of 5˚ min⁻¹.

A single crystal of suitable size and good quality was measured by using an area detector on a Bruker APEX-II CCD diffractometer with graphite monochromatic Ga-Kα radiation (λ = 1.34138 Å). Absorption corrections were applied by using a multi-scan program. Using Olex2, the structure was solved with the ShelXT structure solution program using Intrinsic Phasing and refined with the ShelXL refinement package using Least Squares minimization.

The dissolution studies of ETZ and ETA∙2TMA∙MeOH were measured by using equal molar samples at 37˚C for oscillation. Aliquots of 1 mL were withdrawn at predetermined time points (5, 10, 15, 30, 45, 60, 120, 180, 240 and 1380 min) substituting the same with an equal quantity of fresh dissolution media.

The solubility and dissolution rate of ETZ and ETA∙2TMA∙MeOH were quantified by High Performance Liquid Chromatography. A Waters e2695 series HPLC equipped with a UV detector (2489) and an automated injector. Methanol-water (80:20) was used as mobile phase at a flow rate of 1 mL・min⁻¹ in an isocratic mode on an Agilent Zorbax SB-C18 (4.6 mm × 250 mm, 5 μm) column at 25˚C. After suitable dilution, 10 µL of the sample was injected, and the absorbance of elute was recorded at 235 nm.

Both the Hirshfeld surface image and 2D fingerprint are generated by Crystal Explorer software.

The ETZ∙2TMA∙MeOH cocrystal was obtained from a mixed solvent of methanol and toluene at room temperature by the solvent evaporation method. The single crystal belongs to the orthorhombic crystal system with space group P2₁2₁2₁ (Table 1). In the unit cell of the cocrystal, the main forces are the formation of intermolecular hydrogen bonds between the amide groups on ETZ and the carboxyl groups on TMA and the hydroxyl group on methanol (Figure 1 and Table 2).

¹1 − X, −1/2 + Y, −1/2 − Z; ²+X, +Y, 1 + Z; ³1 − X, 1/2 + Y, −1/2 − Z; ⁴+X, +Y, −1 + Z; ⁵+X, −1 + Y, +Z; ⁶+X, 1 + Y, +Z.

On the one hand, the O atom on the amide group of the ETZ molecule is a hydrogen bond acceptor, and the H atom on the carboxyl group (O-H) of the TMA molecule is a hydrogen bond donor, forming an intermolecular hydrogen bond O-H…O (symmetry code: +X, +Y, −1 + Z) [length 2.523 (4) Å, angle 165.4˚]. On the other hand, the O atom on the hydroxyl group of the methanol molecule is a hydrogen bond acceptor, and the H atom on the molecular carboxyl group (O-H) of the ETZ molecule is a hydrogen bond donor, forming an intermolecular hydrogen bond O-H…O (symmetry code: +X, +Y, −1 + Z) [length 2.515 (4) Å, angle 170.2˚]. The methanolate cocrystal of ETZ and TMA were formed. Views of the calculated ETA∙2TMA∙MeOH Hirshfeld surface and 2D fingerprint were shown in Figure 2. The force of H… O/O… H is 34.5%, and the force of H… N/N… H is 0.7%. The force of the hydrogen bond between ETZ and TMA mainly depends on H… O/O… H force.

The crystal structures of ETZ, TMA, and ETA∙2TMA∙MeOH were measured by PXRD (Figure 3). The peaks at 2θ = 9.64˚, 14.54˚, 14.70˚, 19.36˚, 25.32˚, and 33.78˚, which are the characteristic peaks of ETZ, disappeared in the PXRD result of ETA∙2TMA∙MeOH. Meanwhile, a number of new peaks belonging to ETA∙2TMA∙MeOH emerged at 2θ = 26.84˚. Thus, the formation of ETA∙2TMA∙ MeOH can be confirmed by the notable changes through the PXRD.

The synthesized ETA∙2TMA∙MeOH was analyzed by FT-IR spectroscopy. FT-IR spectra of ETZ, TMA and ETA∙2TMA∙MeOH were compared in order to confirm the cocrystal formation. All the molecular cocrystals exhibited a change in the carbonyl stretching frequency of the acid group with respect to their starting material.

In the ETA∙2TMA∙MeOH, a significant shift of carboxylate (−C=O) and amino (−NH₂) stretching vibration was observed (Figure 4). The characteristic peak of N-H in the molecular structure of ETZ was shifted from 3370.51 cm⁻¹ to 3453.93 cm⁻¹, the characteristic absorption peak of C=O in the molecular structure of TMA was shift from 1644.27 cm⁻¹ to 1705.51 cm⁻¹.

It is well known that the solid form of APIs has a substantial impact on the solubility and dissolution profiles of drug. Therefore, it is important to select an appropriate API solid state form for successful drug development. According to the Biopharmaceutical Classification System (BCS), ETZ was classified as a low solubility drug (BCS class II). Solubility can be enhanced either by salt formation or by cocrystal formation. The ETZ molecule does not have any ionization site for salt formation; therefore, improvement in the solubility of ETZ drug molecule can only be done by cocrystallization. The solubility of ETZ and ETA∙ 2TMA∙MeOH in 37˚C water was determined. The results in Figure 5 clearly show that the solubility of ETA∙2TMA∙MeOH decreased to 19.30% of pure ETZ.