Crystal Structure and Solid-State Conformational Analysis of Active Pharmaceutical Ingredient Venetoclax

Venetoclax is an orally bioavailable, B-cell lymphoma-2 selective inhibitor used for the treatment of chronic lymphocytic leukemia, small lymphocytic lymphoma, and acute myeloid leukemia. Venetoclax’s crystal structure was until now determined only when it was bound to a B-cell lymphoma-2 (BCL-2) protein, while the crystal structure of this active pharmaceutical ingredient alone has not been reported yet. Herein, we present the first successful crystallization, which provided crystals of venetoclax suitable for X-ray diffraction analysis. The crystal structure of venetoclax hydrate was successfully determined. The asymmetric unit is composed of two crystallographically independent molecules of venetoclax and two molecules of interstitial water. Intramolecular N–H⋯O hydrogen bonding is present in both molecules, and a molecular overlay shows differences in their molecular conformations, which is also observed in respect to venetoclax molecules from known crystal structures of BCL-2:venetoclax complexes. A supramolecular structure is achieved through various N–H⋯N, O–H⋯O, C–H⋯O, C–H⋯π, C–Cl⋯π, ONO⋯π, and π⋯π interactions. The obtained crystals were additionally characterized with spectroscopic techniques, such as IR and Raman, as well as with thermal analysis.

understanding and exploitation of lesser-known interactions, which could provi efficient drug design efforts in the future [48][49][50][51][52][53][54]. In this report, we provide detai successful preparation of crystals of venetoclax suitable for single crystal X-ray di analysis, the first crystal structure of venetoclax's hydrate form, and conformation ysis of its small molecule crystal structure in comparison with the venetoclax b BCL-2.

Materials
For the purpose of this study, venetoclax was obtained from MSN Laborator derabad, India). Acetonitrile (ACN) was purchased from J. T. Baker, now part of A (Radnor, PA, USA). FTIR grade potassium bromide (KBr) and analytical grade nium bicarbonate were purchased from Merck KGaA (Darmstadt, Germany).

Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) Measu ments
ATR-FTIR spectra were collected with a Nicolet iS50FT-IR spectrometer ( Fisher Scientific, Waltham, MA, USA), using a single reflection diamond ATR cel

Raman Measurements
Raman spectra were collected with a Nicolet iS50FT-IR spectrometer (Therm Scientific, Waltham, MA, USA), equipped with the iS50 Raman accessory. Recently, crystal structures of BCL-2 and a BCL-2 mutants bound to venetoclax were reported in the literature [41], which provided the first insights into conformational preferences of venetoclax within the target protein. Surprisingly, although there are several patent literature reports on the salts, polymorphs, hydrates, and solvates of venetoclax [42][43][44], the crystal structure of active pharmaceutical ingredient venetoclax has not been described in the literature yet. This could be attributed to the well-known challenges related to the growth of single crystals of sufficient size and quality suitable for single crystal X-ray diffraction analysis [45][46][47]. Since venetoclax ( Figure 1) contains several rotatable bonds, it is reasonable to expect that venetoclax could adopt several conformation states with overall rich conformational space. Therefore, the crystal structure of venetoclax could provide new information on the conformations found in a small molecule crystal structure, which could be compared to that of the molecule bound to BCL-2. Such comparison is of high relevance because it could establish if the small molecule crystal conformations are comparable to the protein-bound conformations of venetoclax and therefore relevant to structure-based drug design in this group of compounds. In addition, increasing our knowledge of the conformations adopted by venetoclax could provide a better understanding and exploitation of lesser-known interactions, which could provide more efficient drug design efforts in the future [48][49][50][51][52][53][54]. In this report, we provide details on the successful preparation of crystals of venetoclax suitable for single crystal X-ray diffraction analysis, the first crystal structure of venetoclax's hydrate form, and conformational analysis of its small molecule crystal structure in comparison with the venetoclax bound to BCL-2.

Materials
For the purpose of this study, venetoclax was obtained from MSN Laboratories (Hyderabad, India). Acetonitrile (ACN) was purchased from J. T. Baker, now part of Avantor ® (Radnor, PA, USA). FTIR grade potassium bromide (KBr) and analytical grade ammonium bicarbonate were purchased from Merck KGaA (Darmstadt, Germany).

Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) Measurements
ATR-FTIR spectra were collected with a Nicolet iS50FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), using a single reflection diamond ATR cell.

Raman Measurements
Raman spectra were collected with a Nicolet iS50FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), equipped with the iS50 Raman accessory.

X-ray Single Crystal Analysis
Single crystal X-ray diffraction data of VEN·H 2 O were collected on an Agilent Technologies SuperNova Dual diffractometer (Agilent, UK) with an Atlas detector using monochromated Cu-Kα radiation (λ = 1.54184 Å) at 150 K. The data were processed using CrysAlis Pro [55]. The structure was solved by the SHELXT program [56] and refined by a full-matrix least-squares procedure based on F 2 with SHELXL [57] using the Olex2 program suite [58]. All non-hydrogen atoms were refined anisotropically. Water atom O16 was refined to be disordered over two positions in a ratio of 0.879(5):0.121 (5). Hydrogen atoms were readily located in different Fourier maps, except for the atoms on water oxygen atom O16 which were not included in the refinement. Hydrogen atoms bonded to carbon atoms were subsequently treated as riding atoms in geometrically idealized positions with U iso (H) = kU eq (C), where k = 1.5 for methyl groups, which were permitted to rotate but not to tilt, and 1.2 for all other H atoms. Hydrogen atoms bonded to nitrogen and oxygen atoms were refined, fixing the bond lengths and isotropic temperature factors as U iso (H) = kU eq (N,O), where k = 1.2 in case of N atoms and 1.5 in case of O atoms. The hydrogen atom H15B on water molecule O15 had to be treated, fixing the coordinates. The crystallographic data are listed in Table 1.

Powder X-ray Diffraction Analysis
Powder X-ray diffraction pattern (p-XRD) of prepared VEN·H 2 O was obtained with an X'Pert PRO diffractometer (PANalytical, Almelo, Netherlands) equipped with a Ge(111) Johannson type monochromator in reflection mode using CuKa1 radiation (λ = 1.54060 Å) and the full range of the 128 channel linear RTMS detector. The diffractogram was recorded at a tube voltage of 45 kV, tube current of 40 mA, and applying a step size of 0.034 • 2θ with an exposure time of 100 s per step in the angular range of 3 • to 50 • 2θ under ambient conditions. Since no characteristic reflections were visible above 40 • 2θ, the diffractogram is shown in the range of 3-40 • 2θ.

Synthesis and Characterization of Venetoclax Hydrate
Venetoclax (100 mg) was placed into an Erlenmeyer flask and 100 mL of ACN-NH 4 HCO 3 (10 mM solution) = 8:2 solvent mixture was added. The obtained suspension was sonicated in an ultrasonic bath for 5 minutes. The obtained turbid solution was left to stand at ambient temperature for 2 hours and then filtered through a polytetrafluoroethylene (PTFE) filter. The obtained yellow solution was placed into a glass laboratory bottle and left to stand unclosed at ambient temperature for 60 days. During this time, the solvent evaporated affording agglomerated crystals on the bottom of the bottle and needle-shaped crystals that were deposited on the walls of the glass bottle. The agglomerated crystals on the bottom of the glass bottle were discarded while the needle-like crystals suitable for the single crystal X-ray analysis obtained from the walls of the glass bottle were collected for further analysis. DSC (

Preparation of Venetoclax Hydrate
In our previous studies on the stability and liquid chromatography analytical method development for venetoclax, we observed that venetoclax formed crystals after a few days from some of the solvents used for the dissolution of venetoclax [59,60]. Therefore, we performed a targeted crystallization experiment from the most promising solvent system identified in our previous study [60]. For this purpose, venetoclax was dissolved in an ACN-NH 4 HCO 3 (10 mM solution) = 8:2 solvent mixture, and the solution was left to stand for 60 days at ambient temperature in a laboratory glass bottle. The yellow needle-shaped crystals that were formed on the walls of the glass bottle after evaporation of the solvent were collected and used for further analysis. This proved that the obtained crystalline venetoclax was suitable for single crystal X-ray diffraction. Thermal analysis and X-ray data indicated that the obtained crystals represented a venetoclax hydrate form that was previously reported in the patent literature, although it was obtained by desolvation of the venetoclax ethyl acetate solvate at ambient conditions and characterized only with a p-XRD [42].

Raman Spectral Analysis
The Raman spectrum of venetoclax hydrate ( Figure 3) displays CH stretching of unsaturated carbons in the region above 3000 cm −1 , while CH stretching of saturated carbons populates the region from 3000 to 2840 cm −1 . The most diagnostic bands in the Raman spectrum are located at 1678 cm −1 (C=O stretch of an amide bond) and a very strong aryl C=C stretch 1607 cm −1 .

Raman Spectral Analysis
The Raman spectrum of venetoclax hydrate ( Figure 3) displays CH stretching of unsaturated carbons in the region above 3000 cm −1 , while CH stretching of saturated carbons populates the region from 3000 to 2840 cm −1 . The most diagnostic bands in the Raman spectrum are located at 1678 cm −1 (C=O stretch of an amide bond) and a very strong aryl C=C stretch 1607 cm −1 .

Raman Spectral Analysis
The Raman spectrum of venetoclax hydrate ( Figure 3) displays CH stretching of unsaturated carbons in the region above 3000 cm −1 , while CH stretching of saturated carbons populates the region from 3000 to 2840 cm −1 . The most diagnostic bands in the Raman spectrum are located at 1678 cm −1 (C=O stretch of an amide bond) and a very strong aryl C=C stretch 1607 cm −1 .

Thermal Analyses
The thermal behavior of the obtained crystalline venetoclax is shown in Figure 4. In the DSC thermogram ( Figure 4, top), two endothermic transitions were observed: the first transition at 49 • C (onset) and 64 • C (peak), which is probably associated with partial dehydration, and the second transition at 168 • C (onset) and 182 • C (peak), which is probably associated with the melting of the form obtained after dehydration. After both endothermic phenomena, an exothermic transition peak associated with decomposition was observed at temperatures above 220 • C. The TGA thermogram (Figure 4, bottom) indicates that dehydration starts above 30 • C and the mass loss is completed by 200 • C. The mass loss of 1.92% w/w is well within the expected value for a monohydrate form, i.e., 2.03% w/w. Thus, TGA and DSC data on the obtained crystalline solid venetoclax indicated that this is a hydrated form of venetoclax. The thermal behavior of the obtained crystalline venetoclax is shown in Figure 4. In the DSC thermogram ( Figure 4, top), two endothermic transitions were observed: the first transition at 49 °C (onset) and 64 °C (peak), which is probably associated with partial dehydration, and the second transition at 168 °C (onset) and 182 °C (peak), which is probably associated with the melting of the form obtained after dehydration. After both endothermic phenomena, an exothermic transition peak associated with decomposition was observed at temperatures above 220 °C. The TGA thermogram (Figure 4, bottom) indicates that dehydration starts above 30 °C and the mass loss is completed by 200 °C. The mass loss of 1.92% w/w is well within the expected value for a monohydrate form, i.e., 2.03% w/w. Thus, TGA and DSC data on the obtained crystalline solid venetoclax indicated that this is a hydrated form of venetoclax.

Powder X-Ray Diffraction Analysis
To investigate whether the analyzed crystal structure is truly representative of the bulk material, the X-ray powder diffraction (p-XRD) technique wasperformed at room temperature and compared with the pattern simulated from the crystal structure. As depicted in Figure 5, the experimental p-XRD pattern is nearly identical with the corresponding simulated one except for some differences that may be due to the preferential orientation. The studied form has a p-XRD comparable to the previously reported monohydrate form in the patent literature [42].

Powder X-ray Diffraction Analysis
To investigate whether the analyzed crystal structure is truly representative of the bulk material, the X-ray powder diffraction (p-XRD) technique wasperformed at room temperature and compared with the pattern simulated from the crystal structure. As depicted in Figure 5, the experimental p-XRD pattern is nearly identical with the corresponding simulated one except for some differences that may be due to the preferential orientation. The studied form has a p-XRD comparable to the previously reported monohydrate form in the patent literature [42].

X-Ray Single Crystal Analysis
• Molecular Geometry Needle-shaped crystals of venetoclax, suitable for single crystal X-ray diffraction, were prepared by crystallization from an ACN-aqueous ammonium bicarbonate buffer system. Thermal analysis and X-ray data indicated that the crystals obtained represented a venetoclax hydrate form [42] that was previously described only in the patent literature and characterized solely with a p-XRD analysis. Crystallographic data are listed in Table  1. The compound VEN·H2O crystalizes in triclinic space group P-1 with two crystallographically independent molecules of venetoclax (A and B) and two molecules of interstitial water in the asymmetric unit, with one (O16) being disordered over two positions (Figure 6a). In each molecule (A and B), intramolecular N-H···ONO hydrogen bonding between the amine group (N3, N10) and nitro group, as well as intramolecular N-H···O hydrogen bonding between the amide group (N1, N8) and phenyl oxygen atom (O7, O14), are present and stabilize the molecular structure (Table 2, Figure 6b,c). (a)

X-ray Single Crystal Analysis
• Molecular Geometry Needle-shaped crystals of venetoclax, suitable for single crystal X-ray diffraction, were prepared by crystallization from an ACN-aqueous ammonium bicarbonate buffer system. Thermal analysis and X-ray data indicated that the crystals obtained represented a venetoclax hydrate form [42] that was previously described only in the patent literature and characterized solely with a p-XRD analysis. Crystallographic data are listed in Table 1 (see supplementary material for further details). The compound VEN·H 2 O crystalizes in triclinic space group P-1 with two crystallographically independent molecules of venetoclax (A and B) and two molecules of interstitial water in the asymmetric unit, with one (O16) being disordered over two positions (Figure 6a). In each molecule (A and B), intramolecular N-H· · · ONO hydrogen bonding between the amine group (N3, N10) and nitro group, as well as intramolecular N-H· · · O hydrogen bonding between the amide group (N1, N8) and phenyl oxygen atom (O7, O14), are present and stabilize the molecular structure (Table 2, Figure 6b,c).  (3) 166.0(2) Symmetry codes: (i) −x, −y + 1, −z; (ii) x − 1, y, z; (iii) x + 1, y, z; (iv) x + 1, y − 1, z; (v) x, y + 1, z; (vi) −x, −y + 1, −z + 1; (vii) x − 1, y + 1, z; a venetoclax hydrate form [42] that was previously described only in the patent literature and characterized solely with a p-XRD analysis. Crystallographic data are listed in Table  1. The compound VEN·H2O crystalizes in triclinic space group P-1 with two crystallographically independent molecules of venetoclax (A and B) and two molecules of interstitial water in the asymmetric unit, with one (O16) being disordered over two positions (Figure 6a). In each molecule (A and B), intramolecular N-H···ONO hydrogen bonding between the amine group (N3, N10) and nitro group, as well as intramolecular N-H···O hydrogen bonding between the amide group (N1, N8) and phenyl oxygen atom (O7, O14), are present and stabilize the molecular structure (Table 2, (Figure 7). Some difference in the inclination of the chlorophenylcyclohexenyl moiety is also evident with the N-C-C-C torsion angle (Φ3) being 110.2(2)°(A) and 113.7(2)°(B), with the quaternary atom C35 (molecule A) being oriented away from the 1H-pyrrolopyridine moiety, while atom C80 (molecule B) is being oriented toward to this moiety. Furthermore, the difference observed between the two conformations is also due to the inclination of the 1H-pyrrolopyridine-containing substituent in respect of the benzamide scaffold with the C-C-O-C torsion angle (Φ4) being 18.1(3)° for A and 51.6(3)° for B. In molecule B, the 1H-pyrrolopyridine moiety is thus in close proximity of the nitrobenzenesulfonyl and tetrahydropyranyl rings.  (Figure 7). Some difference in the inclination of the chlorophenylcyclohexenyl moiety is also evident with the N-C-C-C torsion angle (Φ 3 ) being 110.2(2) • (A) and 113.7(2) • (B), with the quaternary atom C35 (molecule A) being oriented away from the 1H-pyrrolopyridine moiety, while atom C80 (molecule B) is being oriented toward to this moiety. Furthermore, the difference observed between the two conformations is also due to the inclination of the 1H-pyrrolopyridine-containing substituent in respect of the benzamide scaffold with the C-C-O-C torsion angle (Φ 4 ) being 18.1(3) • for A and 51.6(3) • for B. In molecule B, the 1H-pyrrolopyridine moiety is thus in close proximity of the nitrobenzenesulfonyl and tetrahydropyranyl rings. Molecule A forms a hydrogen bonded centrosymmetric dimer via N5-H5···N4 i interactions between adjacent 1H-pyrrolopyridine moieties with the graph-set motif R2 2 (8) [61] (Table 2, Figure 8). Dimers are further connected into a chain along the a-axis via C27-H27A···O4 iii interactions between the piperazine moiety and nitro group as well as via C39-H39A···O1 iii interactions between the chlorophenyl ring and the amide oxygen atom forming a graph-set motif R2 2 (19). This interaction is supported by almost parallel π···π interactions between each ring of the 1H-pyrrolopyridine moiety and the nitrophenyl ring of the adjacent molecule with a centroid-to-centroid distance of 3.8869(13) and 3.8873(12) Molecule A forms a hydrogen bonded centrosymmetric dimer via N5-H5· · · N4 i interactions between adjacent 1H-pyrrolopyridine moieties with the graph-set motif R 2 2 (8) [61] (Table 2, Figure 8). Dimers are further connected into a chain along the a-axis via C27-H27A· · · O4 iii interactions between the piperazine moiety and nitro group as well as via C39-H39A· · · O1 iii interactions between the chlorophenyl ring and the amide oxygen atom forming a graph-set motif R 2 2 (19). This interaction is supported by almost parallel π· · · π interactions between each ring of the 1H-pyrrolopyridine moiety and the nitrophenyl ring of the adjacent molecule with a centroid-to-centroid distance of 3.8869(13) and 3.8873(12) Å and ring slippage of 2.037 and 2.016 Å, respectively. Moreover, ONO· · · π interactions are present between the nitro group and the pyridine ring of the 1H-pyrrolopyridine moiety with an O· · · π distance of 3.0931 (19) Å. The chains are further connected into a layer along the ab-plane via C29-H29A· · · O6 iv interactions between the piperazine moiety and the tetrahydropyrane oxygen atom of the adjacent molecule.
Crystals 2021, 11, x FOR PEER REVIEW 9 of 17 Å and ring slippage of 2.037 and 2.016 Å, respectively. Moreover, ONO···π interactions are present between the nitro group and the pyridine ring of the 1H-pyrrolopyridine moiety with an O···π distance of 3.0931 (19) Å. The chains are further connected into a layer along the ab-plane via C29-H29A···O6 iv interactions between the piperazine moiety and the tetrahydropyrane oxygen atom of the adjacent molecule.  (14) 156 (5)  In contrast to molecules of A, where the centrosymmetric hydrogen bonded dimer between the adjacent 1H-pyrrolopyridine units is formed, molecules of B form a chain along the b-axis through N12-H12···O11 v interactions with the 1H-pyrrolopyridine moiety acting as a hydrogen bond donor and the nitro group of the adjacent molecule as a hydrogen bond acceptor (Table 2, Figure 9a). Two such chains are connected into a belt via centrosymmetric C61-H61···O10 vi interactions between the tetrahydropyranyl methylene group and the sulfonyl oxygen atom, forming a graph-set motif R2 2 (22). The belt structure is supported by π···π interactions between the pyrrole ring of the 1H-pyrrolopyridine moiety and the nitrophenyl ring of the adjacent molecule with a centroid-to-centroid distance of 3.860(1) Å and an angle between both rings of 25.4(1)°. The belts are further connected into a layer along the ab-plane via C61-H61A···π interactions between the tetrahydropyranyl methylene group and the C46-C51 aromatic system (Figure 9b). (a) Hydrogen bonded dimers formed via N5-H5· · · N4 i interactions connected into a chain along the a-axis via C27-H27A· · · O4 iii , π· · · π, and ONO· · · π interactions; (b) layer formation via C29-H29A· · · O6 iv interactions; and (c) view of a layer along the a-axis. Hydrogen bonds are drawn by dashed blue lines and π· · · π and ONO· · · π interactions by dashed green lines (presented only in (a) for clarity). Hydrogen atoms not involved in the motif shown have been omitted for clarity.
In contrast to molecules of A, where the centrosymmetric hydrogen bonded dimer between the adjacent 1H-pyrrolopyridine units is formed, molecules of B form a chain along the b-axis through N12-H12· · · O11 v interactions with the 1H-pyrrolopyridine moiety acting as a hydrogen bond donor and the nitro group of the adjacent molecule as a hydrogen bond acceptor (Table 2, Figure 9a). Two such chains are connected into a belt via centrosymmetric C61-H61· · · O10 vi interactions between the tetrahydropyranyl methylene group and the sulfonyl oxygen atom, forming a graph-set motif R 2 2 (22). The belt structure is supported by π· · · π interactions between the pyrrole ring of the 1H-pyrrolopyridine moiety and the nitrophenyl ring of the adjacent molecule with a centroid-to-centroid distance of 3.860(1) Å and an angle between both rings of 25.4(1) • . The belts are further connected into a layer along the ab-plane via C61-H61A· · · π interactions between the tetrahydropyranyl methylene group and the C46-C51 aromatic system (Figure 9b). •

Crystal Packing
The supramolecular structure of VEN·H2O is achieved through C-H···O, C-H···π, and C-Cl···π interactions between layers of molecules of A and layers of molecules of B (Table 2, Figure 10). Molecules of A act as hydrogen bond donors and molecules of B as acceptors through C15-H15···O13 ii interactions connecting the methine group of a tetrahydropyranyl ring with the oxygen atom of tetrahydropyranyl and through C31-H31A···O12 interactions connecting the methylene unit attached to the piperazine moiety and the nitro group. Furthermore, C37-H37···π interactions connect the methylene unit of the cyclohexenyl ring of molecules of A with the pyrrole ring of molecules of B, while C41-Cl1···π interactions are present between molecules of A and the benzene ring C46-C51 of molecules of B. Furthermore, C87-H87···π interactions connect the chlorobenzene moiety of molecules of B with the benzene ring C1-C6 of molecules of A. Venetoclax crystalizes in a form of monohydrate with two water molecules in the asymmetric unit. These water molecules are also involved in supramolecular aggregation. Water molecule O15 acts as a hydrogen bond donor in the interaction with the disordered water molecule O16 (O15-H15A···O16A, O15-H15A···O16B) and in the interaction with the sulfonyl O9 atom of molecule B as well as a hydrogen bond acceptor in the C71-H71···O15 interaction with the pyrrole ring of molecule B. Hydrogen atoms on the disordered water molecule O16 were not found in the Fourier maps; however, O16B···O9 and O16A···N11 separations of 2.79 and 2.94 Å, respectively, indicate hydrogen bonding interactions with molecules of B. In addition, water molecule O16B is a hydrogen bond acceptor in C29-H29B···O16B, C45-H45A···O16B, and C51-H51···O16B interactions. axis formed via N12-H12· · · O11 v , C61-H61B· · · O10 vi , and π· · · π interactions. (b) Layer formation along the ab-plane via C61-H61A· · · π interactions. Hydrogen bonds are drawn by dashed blue lines and π· · · π and C-H· · · π interactions by dashed green lines. Hydrogen atoms not involved in the motif shown have been omitted for clarity.

•
Crystal Packing The supramolecular structure of VEN·H 2 O is achieved through C-H· · · O, C-H· · · π, and C-Cl· · · π interactions between layers of molecules of A and layers of molecules of B (Table 2, Figure 10). Molecules of A act as hydrogen bond donors and molecules of B as acceptors through C15-H15· · · O13 ii interactions connecting the methine group of a tetrahydropyranyl ring with the oxygen atom of tetrahydropyranyl and through C31-H31A· · · O12 interactions connecting the methylene unit attached to the piperazine moiety and the nitro group. Furthermore, C37-H37· · · π interactions connect the methylene unit of the cyclohexenyl ring of molecules of A with the pyrrole ring of molecules of B, while C41-Cl1· · · π interactions are present between molecules of A and the benzene ring C46-C51 of molecules of B. Furthermore, C87-H87· · · π interactions connect the chlorobenzene moiety of molecules of B with the benzene ring C1-C6 of molecules of A. Venetoclax crystalizes in a form of monohydrate with two water molecules in the asymmetric unit. These water molecules are also involved in supramolecular aggregation. Water molecule O15 acts as a hydrogen bond donor in the interaction with the disordered water molecule O16 (O15-H15A· · · O16A, O15-H15A· · · O16B) and in the interaction with the sulfonyl O9 atom of molecule B as well as a hydrogen bond acceptor in the C71-H71· · · O15 interaction with the pyrrole ring of molecule B. Hydrogen atoms on the disordered water molecule O16 were not found in the Fourier maps; however, O16B· · · O9 and O16A· · · N11 separations of 2.79 and 2.94 Å, respectively, indicate hydrogen bonding interactions with molecules of B. In addition, water molecule O16B is a hydrogen bond acceptor in C29-H29B· · · O16B, C45-H45A· · · O16B, and C51-H51· · · O16B interactions. Crystals 2021, 11, x FOR PEER REVIEW 12 of 17 Figure 10. Packing of layers of molecules of A (green) and B (blue). •

Structural comparison between conformations of VEN·H2O and protein:venetoclax complexes
The crystal structure of VEN·H2O possesses two crystallographically independent venetoclax molecules with distinctly different conformations. A variety of conformations are possible due to the composition of the molecule containing several rings connected primarily in para positions by flexible linkers. Free rotation along the Ar-NH-CH2-R, Ar-CO-NH-SO2-Ar, Ar-O-Ar, and N-CH2-R linkers enables the molecules to adjust to different chemical spaces especially in protein binding sites. We decided to extend our research in order to compare conformations of molecules in VEN·H2O with the structures of venetoclax molecules from known crystal structures of protein:venetoclax complexes. VEN·H2O was compared with venetoclax molecules in complexes with a BCL-2 antagonist (two crystallographically independent molecules), G101V mutant (two crystallographically independent molecules), G101A mutant, and F104L mutant (two crystallographically independent molecules) [41] since hydrogen bonding and other non-covalent interactions, as well as packing effects, can have a marked influence on the conformation of the venetoclax molecule (Figure 11a). In all the venetoclax structures studied, the intramolecular hydrogen bond N-H···ONO between the amine group (N3, N10 in VEN·H2O) and the nitro group is present showing the robustness of this structural motif. On the other hand, the intramolecular hydrogen bond N-H···O between the amide group (N1, N8 in VEN·H2O) and the phenyl oxygen atom (O7, O14 in VEN·H2O) can be observed only in VEN·H2O with a N(H)-C(=O)-C-C(-O) dihedral angle of -5.32 and -7.06° (Figure 11b), respectively, while in all protein:venetoclax complexes, the amide NH group is directed away from the phenyl oxygen atom with dihedral angles in the range 140.7-155.4° in five protein complexes (Figure 11c) and with a dihedral angle of 52.1 and 68.5° in two protein complexes (Figure 11d). The 1H-pyrrolopyridine unit in VEN·H2O is involved in hydrogen bonding with adjacent molecules, while in all protein:venetoclax structures, it is involved in N-H···O interactions with the carboxylate side arm of the aspartic acid unit of the protein chain. In most protein:venetoclax structures, the carbonyl oxygen atom of the

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Structural comparison between conformations of VEN·H 2 O and protein:venetoclax complexes The crystal structure of VEN·H 2 O possesses two crystallographically independent venetoclax molecules with distinctly different conformations. A variety of conformations are possible due to the composition of the molecule containing several rings connected primarily in para positions by flexible linkers. Free rotation along the Ar-NH-CH 2 -R, Ar-CO-NH-SO 2 -Ar, Ar-O-Ar, and N-CH 2 -R linkers enables the molecules to adjust to different chemical spaces especially in protein binding sites. We decided to extend our research in order to compare conformations of molecules in VEN·H 2 O with the structures of venetoclax molecules from known crystal structures of protein:venetoclax complexes. VEN·H 2 O was compared with venetoclax molecules in complexes with a BCL-2 antagonist (two crystallographically independent molecules), G101V mutant (two crystallographically independent molecules), G101A mutant, and F104L mutant (two crystallographically independent molecules) [41] since hydrogen bonding and other non-covalent interactions, as well as packing effects, can have a marked influence on the conformation of the venetoclax molecule (Figure 11a). In all the venetoclax structures studied, the intramolecular hydrogen bond N-H· · · ONO between the amine group (N3, N10 in VEN·H 2 O) and the nitro group is present showing the robustness of this structural motif. On the other hand, the intramolecular hydrogen bond N-H· · · O between the amide group (N1, N8 in VEN·H 2 O) and the phenyl oxygen atom (O7, O14 in VEN·H 2 O) can be observed only in VEN·H 2 O with a N(H)-C(=O)-C-C(-O) dihedral angle of -5.32 and -7.06 • (Figure 11b), respectively, while in all protein:venetoclax complexes, the amide NH group is directed away from the phenyl oxygen atom with dihedral angles in the range 140.7-155.4 • in five protein complexes (Figure 11c) and with a dihedral angle of 52.1 and 68.5 • in two protein complexes (Figure 11d). The 1H-pyrrolopyridine unit in VEN·H 2 O is involved in hydrogen bonding with adjacent molecules, while in all protein:venetoclax structures, it is involved in N-H· · · O interactions with the carboxylate side arm of the aspartic acid unit of the protein chain. In most protein:venetoclax structures, the carbonyl oxygen atom of the amide unit interacts with the arginine side arm of the adjacent protein and/or water molecule, while sulfonyl oxygen atoms are mostly connected to water molecules and to the glycine NH amide group of the protein chain. Piperazine nitrogen atoms in VEN·H 2 O are not involved in hydrogen bonding; however, in all protein:venetoclax complexes, one nitrogen atom interacts with a water molecule. Since venetoclax molecules in protein complexes are in a markedly different environment with respect to VEN·H 2 O, adopted conformations vary greatly. However, the 1H-pyrrolopyridine and nitrobenzene moieties are in close proximity, as is also observed in molecule B of VEN·H 2 O. On the other hand, the chlorobenzene ring in both molecules of VEN·H 2 O is directed in the opposite direction compared to in all of the protein complexes.

Conclusions
In this report, we present the first crystal structure of venetoclax, a B-cell lymphoma-2 selective inhibitor used for the treatment of chronic lymphocytic leukemia, small lymphocytic lymphoma, and acute myeloid leukemia. The X-ray single crystal structural analysis revealed the formation of venetoclax hydrate (VEN·H2O) crystalizing in triclinic space group P-1 with two crystallographically independent molecules of venetoclax (A and B) and two molecules of interstitial water in the asymmetric unit. Two intramolecular N-H···O hydrogen bonds are present in both molecules, and a molecular overlay shows differences in their molecular conformations. Differences are also shown in respect to venetoclax molecules from known crystal structures of protein:venetoclax complexes with BCL-2 antagonist and BCL-2 mutants. In VEN·H2O, molecules of A form hydrogen bonded layers via a series of N-H···N, C-H···O, ONO···π, and π···π interactions, as well as molecules of B via N-H···N, C-H···O, C-H···π, and π···π interactions. The supramolecular structure of VEN·H2O is achieved through various C-H···O, C-H···π, and C-Cl···π inter-

Conclusions
In this report, we present the first crystal structure of venetoclax, a B-cell lymphoma-2 selective inhibitor used for the treatment of chronic lymphocytic leukemia, small lymphocytic lymphoma, and acute myeloid leukemia. The X-ray single crystal structural analysis revealed the formation of venetoclax hydrate (VEN·H 2 O) crystalizing in triclinic space group P-1 with two crystallographically independent molecules of venetoclax (A and B) and two molecules of interstitial water in the asymmetric unit. Two intramolecular N-H· · · O hydrogen bonds are present in both molecules, and a molecular overlay shows differences in their molecular conformations. Differences are also shown in respect to venetoclax molecules from known crystal structures of protein:venetoclax complexes with BCL-2 antagonist and BCL-2 mutants. In VEN·H 2 O, molecules of A form hydrogen bonded layers via a series of N-H· · · N, C-H· · · O, ONO· · · π, and π· · · π interactions, as well as molecules of B via N-H· · · N, C-H· · · O, C-H· · · π, and π· · · π interactions. The supramolecular structure of VEN·H 2 O is achieved through various C-H· · · O, C-H· · · π, and C-Cl· · · π interactions between layers of molecules of A and layers of molecules of B as well as through the O-H· · · O and C-H· · · O interactions involving the hydrate molecules. The obtained crystals were additionally characterized with spectroscopic techniques, such as IR and Raman, as well as with thermal analysis.