Omadacycline dihydrate, C29H40N4O7·2H2O, from X-ray powder diffraction data

The crystal structure of omadacycline dihydrate has been solved and refined using synchrotron X-ray powder diffraction data.

This work was carried out as part of a project (Kaduk et al., 2014) to determine the crystal structures of large-volume commercial pharmaceuticals, and include high-quality powder diffraction data for them in the Powder Diffraction File (Gates-Rector & Blanton, 2019).

Structural commentary
The powder pattern of the hydrated omadacycline studied here is not the same as that reported for crystalline omada-cycline by Cvetovich & Warchol (2013) (Fig. 1).Our material was a commercial sample, but it is not clear how representative it is of the bulk pharmaceutical material.
The refined structure of the omadacycline molecule is different in chemical connectivity and conformation from that archived in PubChem (Kim et al., 2019;Figs. 2 and 3).In particular, C20-O3 (our numbering scheme) is a double bond, while C30-O7, C21-O5, and C18-O2 are single bonds.C21-C24 is a double bond, C20-C24 is a single bond, and several other C-C bonds in the ring system differ in order.It is unclear whether the differences represent differences between solution and the solid state, or merely the limited information content of the powder diffraction pattern of a very complex material.The bond-distance and bond-angle restraints were deliberately given low weight to gain insight into what information the diffraction data can give regarding the chemical connectivity.
It was clear from both the structure solution and refinement and a DFT calculation that the C30-O7-N10 group is oriented to form a strong intramolecular O7-H55� � �O3 hydrogen bond, and that N10 participates in intermolecular hydrogen bonds (Table 1).
There are many unusual bond distances, bond angles, and torsion angles indicated by a Mercury Mogul Geometry check (Macrae et al., 2020).Although there are some large Z-scores among the bond distances, the largest ones tend to be on the periphery of the molecule, where the chemical connectivity is not in doubt.In the ring system, the distinctions between single and double bonds seem to be clear.It is hard to make conclusions about the Z-scores of the bond angles, but some of the large Z-scores result from very small standard uncertainties on the average bond angles.Both for bond distances and bond angles, greater-and less-than-normal values tend to be correlated, probably reflecting errors in atom positions, which were restrained only modestly.Some torsion angles involving rotation about the C16-N8, C26-N9, C24-C30, C37-C36, and C31-C33 bonds are flagged as unusual.All of these reflect the orientations of peripheral groups, which do seem to be unusual in this crystal structure.

Supramolecular features
We obtained guidance on whether potential interatomic contacts were real hydrogen bonds from a DFT optimization of the anhydrous structure (without the disordered water molecules).This structure is close to that of the disordered anhydrate.A DFT optimization of an ordered dihydrate yielded a different molecular connectivity, and it is unclear Comparison of the synchrotron pattern of omadacycline dihydrate (black) to that of omadacycline (green) reported by Cvetovich & Warchol (2013).The patent pattern, measured using Cu K� radiation, was digitized using UN-SCAN-IT (Silk Scientific, 2013), and converted to the synchrotron wavelength of 0.458133 A ˚using JADE Pro (MDI, 2021).Image generated using JADE Pro (MDI, 2021).

Figure 2
The omadacycline molecule in omadacycline dihydrate, with the atom numbering.The atoms are represented by 50% probability spheres.

Figure 3
Comparison of the structure of the omadacycline molecule from this Rietveld refinement (green) to that archived in PubChem (purple).The root-mean-square Cartesian displacement of the non-H atoms is 1.08 A ˚.
how relevant such a calculation is to the disordered structure.
The differences point out that the molecular connectivity may vary depending on the state of hydration, and also in solution versus the solid state.
There are many hydrogen bonds in the structure, but (perhaps surprisingly) almost all of them are intramolecular.Only the N10-H62� � �O2 and N11-H69� � �O84 hydrogen bonds (as well as probable weak C-H� � �O and C-H� � �N hydrogen bonds) link different molecules.The intermolecular hydrogen bonds link the molecules into a three-dimensional network (Fig. 4).There are three very strong intramolecular O-H� � �O hydrogen bonds between hydroxyl and carbonyl groups.There are also short intramolecular methyl� � �methyl contacts between H49 and H52 and H66 and H63.The shortest (and only) O� � �O distances between water molecules and omadacycline molecules are 2.727 (17) and 3.119 (16) A between O84 and two symmetry-equivalent O7; the water molecules only loosely interact with the framework and it was not possible to unambiguously locate the water H atoms.
We may state that we have established the crystal structure of omadacyclic dihydrate, but the exact molecular structure is ambiguous.

Synthesis and crystallization
Omadacycline was a commercial reagent, purchased from TargetMol (Batch #132019), and was used as received.

Refinement
Crystal data, data collection and structure refinement details are summarized in Table 2.

Figure 5
The Rietveld plot for the refinement of omadacycline dihydrate.The blue crosses represent the observed data points, and the green line is the calculated pattern.The cyan curve is the normalized difference plot.The vertical scale has been multiplied by a factor of 20� for 2� > 8.0 � .
sdf.It was converted into a *.mol2 file using Mercury (Macrae et al., 2020), and into a Fenske-Hall Z-matrix file using OpenBabel (O'Boyle et al., 2011).The structure was solved using FOX (Favre-Nicolin & C ˇerny ´, 2002) using (sin �/�) max = 0.4 A ˚À 1 .Visualization of the structure revealed the presence of several voids.By placing oxygen atoms (possibly water molecules) into the voids and refining their positions and occupancies (some refined to less than 0, and were removed from the model), four potential sites, corresponding to 18.1 H 2 O/cell, or 2.0 H 2 O/omadacycline (i.e., a dihydrate) were identified.NMR analysis of the omadacycline sample was performed using a 400 MHz Bruker Avance spectrometer equipped with a multinuclear probe.The 1 H NMR of the pharmaceutical sample was performed in d 6 DMSO, which was stored over flame-dried 3 A ˚molecular sieves.The 1 H NMR analysis of the sample indicated the presence of water in addition to omadacycline (Gottlieb et al., 1997).By comparing the water signal at 3.33 ppm to the signal at 7.41 ppm, which belongs to the arene C-H group of the pharmaceutical moleucle, the water content was estimated to be approximately 1.5 water molecules to 1 omadacycline.Moreover, the 1 H NMR spectrum of the omadacycline sample indicated that there was no observable trace of residual organic solvent or tosylate counter-ion.The NMR data therefore indicate that the species in the pores in the crystal structure is water.After evaporation of the DMSO solvent, the solid was discolored.
Rietveld refinement (Fig. 5) was carried out using GSAS-II (Toby & Von Dreele, 2013).Only the 2.0-25.0� portion of the pattern was included in the refinement (d min = 1.058A ˚).The z-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 (Sykes et al., 2011;Bruno et al., 2004).The Mogul average and standard deviation for each quantity were used as the restraint parameters.The weight of the restraints was gradually decreased during the refinement.The restraints contributed 3.8% to the final � 2 .The hydrogen atoms were included in calculated positions, which were recalculated during the refinement using Materials Studio (Dassault, 2021).The U iso values were grouped by chemical similarity.The U iso for the H atoms were fixed at 1.3 � the U iso of the heavy atoms to which they are attached.A second-order spherical harmonic model was included in the refinement to account for preferred orientation and the refined texture index is 1.001 (0).The peak profiles were described using the generalized microstrain model.The background was modeled using a six-term shifted Chebyshev polynomial, plus a peak at 5.63 � 2� to model the scattering from the Kapton capillary and any amorphous component.

Figure 1
Figure 1 Polymorphs of crystalline omadacycline tosylate are claimed in US Patent 8,383,610 B2 (Cvetovich & Warchol, 2013; Paratek Pharmaceuticals).A powder pattern of the parent compound is also provided.A reduced cell search in the Cambridge Structural Database (CSD, version 5.45 November 2023; Groom et al., 2016) combined with C, H, N, and O only, yielded two hits, but no structures of omadacycline derivatives.

Figure 4
Figure 4The crystal structure of omadacycline dihydrate, viewed down the c-axis direction showing the voids occupied by disordered water molecules.

Table 2
Experimental details.