II. EXPERIMENTAL

Givinostat hydrochloride monohydrate was a commercial reagent, purchased from TargetMol (Batch #T6279), and was used as received. The orange/tan powder was packed into a 0.5-mm-diameter Kapton capillary and rotated during the measurement at ~2 Hz. The powder pattern was measured at 298(1) K at the Wiggler Low Energy Beamline (Leontowich et al., Reference Leontowich, Gomez, Diaz Moreno, Muir, Spasyuk, King, Reid, Kim and Kycia2021) of the Brockhouse X-ray Diffraction and Scattering Sector of the Canadian Light Source using a wavelength of 0.819826(2) Å (15.1 keV) from 1.6 to 75.0° 2θ with a step size of 0.0025° and a collection time of 3 minutes. The high-resolution powder diffraction data were collected using eight Dectris Mythen2 X series 1K linear strip detectors. NIST SRM 660b LaB₆ was used to calibrate the instrument and refine the monochromatic wavelength used in the experiment.

The pattern was indexed using N-TREOR as incorporated into EXPO2014 (Altomare et al., Reference Altomare, Cuocci, Giacovazzo, Moliterni, Rizzi, Corriero and Falcicchio2013) on a primitive monoclinic unit cell with a = 7.99633, b = 8.20718, c = 18.22845 Å, β = 98.138°, V = 1,184.2 Å³, and Z = 2. The suggested space group was P2 ₁, which was confirmed by the successful solution and refinement of the structure. A reduced cell search of the Cambridge Structural Database (Groom et al., Reference Groom, Bruno, Lightfoot and Ward2016) yielded 15 hits, but no structures of givinostat or its derivatives.

A neutral givinostat molecule was downloaded from PubChem (Kim et al., Reference Kim, Chen, Cheng, Gindulyte, He, He and Li2023) as Conformer3D_COMPOUND_CID_9804992.sdf. It was converted to a *.mol2 file using Mercury (Macrae et al., Reference Macrae, Sovago, Cottrell, Galek, McCabe, Pidcock and Platings2020). The crystal structure was solved using Monte Carlo-simulated annealing techniques as implemented in EXPO2014 (Altomare et al., Reference Altomare, Cuocci, Giacovazzo, Moliterni, Rizzi, Corriero and Falcicchio2013), using a givinostat molecule, a Cl atom, and an O atom as fragments, with (001) preferred orientation and a bump penalty. In the best solution, the tertiary nitrogen atom N5 was 3.100 Å from the Cl atom (as expected from the patents); therefore, a hydrogen atom was added to it. The O atom was in a position to be the acceptor in a N–H···O hydrogen bond and a donor in two O–H···Cl hydrogen bonds; therefore, two approximate water hydrogen positions were generated along the O···Cl vectors using Materials Studio (Dassault Systèmes, 2023). The position of the hydroxyl proton H58 was recalculated to lie along an O···Cl vector.

Rietveld refinement was carried out with GSAS-II (Toby and Von Dreele, Reference Toby and Von Dreele2013). Only the 2.0–50.0° portion of the pattern was included in the refinements (d _min = 0.970 Å). The initial fit and the appearance of the high-angle portion of the pattern made it clear that an impurity phase was present. It was identified as NaCl, which was added to the refinement as a second phase. Its concentration refined to 1.0 wt%. The y-coordinate of O1 was fixed to define the origin. All non-H-bond distances and angles were subjected to restraints, based on a Mercury/Mogul Geometry Check (Bruno et al., Reference Bruno, Cole, Kessler, Luo, Motherwell, Purkis and Smith2004; Sykes et al., Reference Sykes, McCabe, Allen, Battle, Bruno and Wood2011). The Mogul average and standard deviation for each quantity were used as the restraint parameters. The benzene and naphthalene ring systems were restrained to be planar. The restraints contributed 2.5% to the overall χ ². The hydrogen atoms were included in calculated positions, which were recalculated during the refinement using Materials Studio (Dassault Systèmes, 2023). The U _iso values of the heavy atoms were grouped by chemical similarity. The U _iso values of the H atoms were fixed at 1.3× those of the heavy atoms to which they are attached. The thermal motion of the Cl atom was described by an anisotropic model. The peak profiles were described using the generalized (Stephens, Reference Stephens1999) microstrain model. The background was modeled using a six-term shifted Chebyshev polynomial, with peaks at 2.89 and 10.86° to model the narrow and broad scattering from the Kapton capillary and any amorphous component.

The final refinement of 141 variables using 19,201 observations and 79 restraints yielded the residual R _wp = 0.04917. The largest peak (1.04 Å from C12) and hole (1.42 Å from O3) in the difference Fourier map were 0.37(10) and − 0.44(10) eÅ⁻³, respectively. The final Rietveld plot is shown in Figure 2. The largest features in the normalized error plot are in the asymmetry and shape of the lowest-angle peak and the shapes of some of the strong low-angle peaks. These misfits probably indicate subtle changes in the specimen during the measurement.

Figure 2. The Rietveld plot for givinostat hydrochloride monohydrate Form I. The blue crosses represent the observed data points, and the green line represents the calculated pattern. The cyan curve indicates the normalized error plot, and the red line indicates the background curve. The blue tick marks indicate the givinostat hydrochloride monohydrate peak positions, and the red tick marks indicate those of NaCl. The vertical scale has been multiplied by a factor of 5× for 2θ > 20.0̊ and by a factor of 10× for 2θ > 33.0̊.

The crystal structure of givinostat hydrochloride monohydrate was optimized (fixed experimental unit cell) with density functional techniques using VASP (Kresse and Furthmüller, Reference Kresse and Furthmüller1996) through the MedeA graphical interface (Materials Design, 2024). The calculation was carried out on 32 cores of a 144-core (768-GB memory) HPE Superdome Flex 280 Linux server at North Central College. The calculation used the GGA-PBE functional, a plane wave cutoff energy of 400.0 eV, and a k-point spacing of 0.5 Å⁻¹, leading to a 2 × 2 × 1 mesh and took approximately 2.0 hours. Single-point density functional calculations (fixed experimental cell) and population analysis were carried out using CRYSTAL23 (Erba et al., Reference Erba, Desmarais, Casassa, Civalleri, Donà, Bush and Searle2023). The basis sets for the H, C, and O atoms in the calculation were those of Gatti et al. (Reference Gatti, Saunders and Roetti1994), whereas for Cl, the basis set was that of Peintinger et al. (Reference Peintinger, Vilela Oliveira and Bredow2013). The calculations were run on a 3.5-GHz PC using eight k-points and the B3LYP functional and took approximately 3.2 hours.

III. RESULTS AND DISCUSSION

This synchrotron powder pattern of givinostat hydrochloride monohydrate (Figure 3) matches the one reported for Form I by Pinori and Mascagni (Reference Pinori and Mascagni2008) well enough to conclude that they represent the same material, and thus that our sample is Form I.

Figure 3. Comparison of the synchrotron pattern of givinostat hydrochloride monohydrate Form I (black) to that reported by Pinori and Mascagni (Reference Pinori and Mascagni2008) (green). The literature pattern (measured using Cu K_α radiation) was digitized using UN-SCAN-IT (Silk Scientific, 2013) and converted to the synchrotron wavelength of 0.819826(2) Å using JADE Pro (MDI, 2024). Image generated using JADE Pro (MDI, 2024).

The root-mean-square (rms) difference of the non-H atoms in the Rietveld-refined and VASP-optimized structures, calculated using the Mercury CSD-Materials/Search/Crystal Packing Similarity tool, is 0.097 Å. The rms Cartesian displacement of the non-H atoms in the Rietveld-refined and VASP-optimized structures of the cation, calculated using the Mercury Calculate/Molecule Overlay tool, is 0.085 Å (Figure 4). The agreements are within the normal range for correct structures (van de Streek and Neumann, Reference van de Streek and Neumann2014). The asymmetric unit is illustrated in Figure 5. The remaining discussion will emphasize the VASP-optimized structure.

Figure 4. Comparison of the Rietveld-refined (red) and VASP-optimized (blue) structures of the cation in givinostat hydrochloride monohydrate Form I. The root-mean-square Cartesian displacement is 0.085 Å. Image generated using Mercury (Macrae et al., Reference Macrae, Sovago, Cottrell, Galek, McCabe, Pidcock and Platings2020).

Figure 5. The asymmetric unit of givinostat hydrochloride monohydrate Form I, with the atom numbering. The atoms are represented by 50% probability spheroids/ellipsoids. Image generated using Mercury (Macrae et al., Reference Macrae, Sovago, Cottrell, Galek, McCabe, Pidcock and Platings2020).

All of the bond distances, bond angles, and torsion angles fall within the normal ranges indicated by a Mercury Mogul Geometry check (Macrae et al., Reference Macrae, Sovago, Cottrell, Galek, McCabe, Pidcock and Platings2020). Quantum chemical geometry optimizations of isolated givinostat cations (DFT/B3LYP/6-31G*/water) using Spartan ‘24 (Wavefunction, Inc., 2023) indicated that the observed conformation is 5.7 kcal/mol higher in energy than a local minimum, but very similar in geometry. The global minimum-energy conformation (MMFF force field) is only 3.2 kcal/mol lower in energy but is very different than the observed conformation (rms displacement = 1.916 Å). Intermolecular interactions are thus important in determining the solid-state conformation.

The crystal structure (Figure 6) consists of layers of cations and anions/water molecules parallel to the ab-plane. The cations stack along the a-axis, with the phenyl and naphthalene rings alternating in the stacks. Hydrogen bonds (discussed below) link the cations, anions, and water molecules. The mean planes of the phenyl and naphthalene ring systems are approximately (1, −13, 17) and (2, 7, 13). The Mercury Aromatics Analyser indicates one strong interaction between the phenyl and naphthalene rings, with a distance of 4.02 Å, as well as several moderate interactions with distances ranging from 5.36 to 6.11 Å.

Figure 6. The crystal structure of givinostat hydrochloride monohydrate Form I, viewed down the a-axis. Image generated using Diamond (Crystal Impact, 2023).

Analysis of the contributions to the total crystal energy of the structure using the Forcite module of Materials Studio (Dassault Systèmes, 2023) indicates that the intramolecular energy is fairly small, and that bond, angle, and torsion distortion terms contribute significantly. The intermolecular energy is dominated by electrostatic attractions, which, in this force field-based analysis, also include hydrogen bonds. The hydrogen bonds are better discussed using the results of the density functional theory (DFT) calculation.

The water molecule, the hydroxylamino group, and the protonated N atom all form strong hydrogen bonds to the chloride anion (Table I). Both amino groups N7 and N6 act as donors in N–H···O hydrogen bonds, to the water molecule and carbonyl groups, respectively. The energies of the N–H···O hydrogen bonds were calculated using the correlation of Wheatley and Kaduk (Reference Wheatley and Kaduk2019). The N6–H52···O3 hydrogen bonds link the cations into a chain with a graph set C1,1(8) (Etter, Reference Etter1990; Bernstein et al., Reference Bernstein, Davis, Shimoni and Chang1995; Motherwell et al., Reference Motherwell, Shields and Allen2000). The other hydrogen bonds are discrete but result in two-dimensional networks of hydrogen bonds linking the cations, anions, and water molecules parallel to the ab-plane. There are several C–H···O hydrogen bonds from the cations to the water molecules, as well as between cations. C–H···Cl and one C–H···C interactions also contribute to the lattice energy.

Table I. Hydrogen bonds (CRYSTAL23) in givinostat hydrochloride monohydrate

^a Intramolecular.

The Hirshfeld surface for givinostat hydrochloride monohydrate (Hirshfeld, Reference Hirshfeld1977; Spackman et al., Reference Spackman, Turner, McKinnon, Wolff, Grimwood, Jayatilaka and Spackman2021) is shown in Figure 7. The volume is 581.56 Å³, 98.27% of half the unit cell volume. The packing density is thus typical. The only significant close contacts (red in Figure 7) involve the hydrogen bonds. The volume/non-hydrogen atom is typical at 17.9 Å³.

Figure 7. The Hirshfeld surface of givinostat hydrochloride monohydrate Form I. Intermolecular contacts longer than the sums of the van der Waals radii are colored blue, and contacts shorter than the sums of the radii are colored red. Contacts equal to the sums of radii are white. Image generated using CrystalExplorer (Spackman et al., Reference Spackman, Turner, McKinnon, Wolff, Grimwood, Jayatilaka and Spackman2021).

The Bravais–Friedel–Donnay–Harker (Bravais, Reference Bravais1866; Friedel, Reference Friedel1907; Donnay and Harker, Reference Donnay and Harker1937) algorithm suggests that we might expect platy morphology for givinostat hydrochloride monohydrate, with {001} as the major faces. A second-order spherical harmonic model was included in the refinement. The texture index (calculated from the refined harmonic coefficients) was 1.007(0), indicating that the preferred orientation was insignificant in this rotated capillary specimen.