3.2.3. 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.

*Crystals* **2021**, *11*, x FOR PEER REVIEW 6 of 17

**Figure 4.** DSC and TGA thermograms of **VEN·H2O**. **Figure 4.** DSC and TGA thermograms of **VEN**·**H2O**.

#### 3.2.4. Powder X-Ray Diffraction Analysis 3.2.4. 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]. 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].

**Figure 5.** Simulated (blue) and experimental (red) powder X-ray diffraction pattern of **VEN·H2O**. **Figure 5.** Simulated (blue) and experimental (red) powder X-ray diffraction pattern of **VEN**·**H2O**.

3.2.5. X-Ray Single Crystal Analysis 3.2.5. X-ray Single Crystal Analysis

#### • Molecular Geometry • 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). 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**·**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).


**Table 2.** Hydrogen bonds for **VEN**·**H2O** [Å and ◦ ].

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*; (viii) −*x* + 1, −*y*, −*z* + 1.

are present and stabilize the molecular structure (Table 2, Figure 6b,c).

**Figure 5.** Simulated (blue) and experimental (red) powder X-ray diffraction pattern of **VEN·H2O**.

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),

3.2.5. X-Ray Single Crystal Analysis

• Molecular Geometry

**Figure 6.** (**a**) Thermal ellipsoid figure of an asymmetric unit of **VEN·H2O** drawn at the 30% probability level. Asymmetric unit contains two crystallographically independent molecules of venetoclax and two water molecules (O15, O16 in disorder). (**b**) Molecule A and (**c**) molecule B of **VEN·H2O** with an atom numbering scheme. Intramolecular hydrogen bonds are drawn with dashed blue lines. **Figure 6.** (**a**) Thermal ellipsoid figure of an asymmetric unit of **VEN**·**H2O** drawn at the 30% probability level. Asymmetric unit contains two crystallographically independent molecules of venetoclax and two water molecules (O15, O16 in disorder). (**b**) Molecule A and (**c**) molecule B of **VEN**·**H2O** with an atom numbering scheme. Intramolecular hydrogen bonds are drawn with dashed blue lines.

The molecular overlay shows that the main difference between molecules A and B is in the orientation of the nitrobenzenesulfonyl moiety with C–S–N–C torsion angle (*Φ*1) of 57.09(19)° (molecule A) vs. –57.7(2)° (molecule B) with the additional difference in C–N– C–C torsion angle (*Φ*2) of the terminal tetrahydropyranyl substituent of –170.15(18)° (A) vs. 97.6(3)° (B) (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 1*H-*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 1*H*-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 1*H*-pyrrolopyridine moiety is thus in close proximity of the nitrobenzenesulfonyl and tetrahydropyranyl rings. The molecular overlay shows that the main difference between molecules A and B is in the orientation of the nitrobenzenesulfonyl moiety with C–S–N–C torsion angle (*Φ*1) of 57.09(19)◦ (molecule A) vs. –57.7(2)◦ (molecule B) with the additional difference in C–N–C–C torsion angle (*Φ*2) of the terminal tetrahydropyranyl substituent of –170.15(18)◦ (A) vs. 97.6(3)◦ (B) (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 1*H*-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 1*H*-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 1*H*-pyrrolopyridine moiety is thus in close proximity of the nitrobenzenesulfonyl and tetrahydropyranyl rings.

N

Molecule A forms a hydrogen bonded centrosymmetric dimer via N5–H5···N4i

actions between adjacent 1*H*-pyrrolopyridine moieties with the graph-set motif R22(8) [61] (Table 2, Figure 8). Dimers are further connected into a chain along the *a*-axis via C27– H27A···O4iii interactions between the piperazine moiety and nitro group as well as via C39–H39A···O1iii interactions between the chlorophenyl ring and the amide oxygen atom forming a graph-set motif R22(19). This interaction is supported by almost parallel π···π interactions between each ring of the 1*H*-pyrrolopyridine moiety and the nitrophenyl ring of the adjacent molecule with a centroid-to-centroid distance of 3.8869(13) and 3.8873(12)

3

N

O

4

O N H S O

O

1

N H N

O O

O

2

inter-

N NH

**Figure 7.** (**a**) Superposition showing the difference in conformation of venetoclax molecules A (orange) and B (light green). For clarity, hydrogen atoms are omitted, and Cl and S atoms are drawn as small spheres. (**b**) Selected torsion angles high-

Cl

(**a**) (**b**)

lighted.

are drawn with dashed blue lines.

*Crystals* **2021**, *11*, x FOR PEER REVIEW 9 of 17

(**b**) (**c**)

**Figure 6.** (**a**) Thermal ellipsoid figure of an asymmetric unit of **VEN·H2O** drawn at the 30% probability level. Asymmetric unit contains two crystallographically independent molecules of venetoclax and two water molecules (O15, O16 in disorder). (**b**) Molecule A and (**c**) molecule B of **VEN·H2O** with an atom numbering scheme. Intramolecular hydrogen bonds

**Figure 7.** (**a**) Superposition showing the difference in conformation of venetoclax molecules A (orange) and B (light green). For clarity, hydrogen atoms are omitted, and Cl and S atoms are drawn as small spheres. (**b**) Selected torsion angles highlighted. **Figure 7.** (**a**) Superposition showing the difference in conformation of venetoclax molecules A (orange) and B (light green). For clarity, hydrogen atoms are omitted, and Cl and S atoms are drawn as small spheres. (**b**) Selected torsion angles highlighted. C29–H29A···O6iv 0.99 2.48 3.456(3) 166.9 C29–H29B···O16Bii 0.99 2.51 3.393(13) 148.8 C31–H31A···O12 0.99 2.50 3.292(3) 136.8

Molecule A forms a hydrogen bonded centrosymmetric dimer via N5–H5···N4i interactions between adjacent 1*H*-pyrrolopyridine moieties with the graph-set motif R22(8) [61] (Table 2, Figure 8). Dimers are further connected into a chain along the *a*-axis via C27– H27A···O4iii interactions between the piperazine moiety and nitro group as well as via C39–H39A···O1iii interactions between the chlorophenyl ring and the amide oxygen atom forming a graph-set motif R22(19). This interaction is supported by almost parallel π···π interactions between each ring of the 1*H*-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<sup>i</sup> interactions between adjacent 1*H*-pyrrolopyridine moieties with the graph-set motif R<sup>2</sup> 2 (8) [61] (Table 2, Figure 8). Dimers are further connected into a chain along the *a*-axis via C27– H27A· · · O4iii interactions between the piperazine moiety and nitro group as well as via C39–H39A· · · O1iii interactions between the chlorophenyl ring and the amide oxygen atom forming a graph-set motif R<sup>2</sup> 2 (19). This interaction is supported by almost parallel π· · · π interactions between each ring of the 1*H*-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 1*H*-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· · · O6iv interactions between the piperazine moiety and the tetrahydropyrane oxygen atom of the adjacent molecule. C31–H31A···N9 0.99 2.61 3.310(3) 128.1 C39–H39···O1iii 0.95 2.55 3.244(2) 130.5 C45–H45A···O16B 0.98 2.48 3.429(14) 163.8 N8–H8···O14 0.880(17) 1.90(2) 2.650(3) 141(3) N10–H10···O12 0.879(18) 2.05(3) 2.665(3) 127(3) N12–H12A···O11v 0.870(17) 2.25(2) 3.070(3) 157(3) C51–H51···O16B 0.95 2.47 3.328(13) 150.3 C61–H61B···O10vi 0.99 2.58 3.330(3) 132.5 C71–H71···O15vii 0.95 2.55 3.483(3) 168.8 O15–H15A···O16A 0.871(10) 2.18(3) 2.890(4) 139(4) O15–H15A···O16B 0.871(10) 2.03(3) 2.843(14) 156(5) O15–H15B···O9viii 0.858(3) 2.330(2) 3.170(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)

C27–H27A···O4iii 0.99 2.43 3.368(3) 157.1

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 1*H*-pyrrolopyridine moi-

**Figure 8.** *Cont*.

*Crystals* **2021**, *11*, x FOR PEER REVIEW 10 of 17

(**c**)

**Figure 8.** Crystal architecture formed by molecules of A in **VEN·H2O**. (**a**) Hydrogen bonded dimers formed via N5– H5···N4i interactions connected into a chain along the *a*-axis via C27–H27A···O4iii, π···π, and ONO···π interactions; (**b**) layer formation via C29–H29A···O6iv 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. **Figure 8.** Crystal architecture formed by molecules of A in **VEN**·**H2O**. (**a**) Hydrogen bonded dimers formed via N5– H5· · · N4<sup>i</sup> interactions connected into a chain along the *<sup>a</sup>*-axis via C27–H27A· · · O4iii , π· · · π, and ONO· · · π interactions; (**b**) layer formation via C29–H29A· · · O6iv interactions; and (**c**) view of a layer along the *<sup>a</sup>*-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 1*H*-pyrrolopyridine units is formed, molecules of B form a chain along the *b*-axis through N12–H12···O11v interactions with the 1*H*-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···O10vi interactions between the tetrahydropyranyl methylene group and the sulfonyl oxygen atom, forming a graph-set motif R22(22). The belt structure is supported by π···π interactions between the pyrrole ring of the 1*H*-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). In contrast to molecules of A, where the centrosymmetric hydrogen bonded dimer between the adjacent 1*H*-pyrrolopyridine units is formed, molecules of B form a chain along the *<sup>b</sup>*-axis through N12–H12· · · O11<sup>v</sup> interactions with the 1*H*-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· · · O10vi interactions between the tetrahydropyranyl methylene group and the sulfonyl oxygen atom, forming a graph-set motif R<sup>2</sup> 2 (22). The belt structure is supported by π· · · π interactions between the pyrrole ring of the 1*H*-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).

**Figure 9.** Crystal architecture formed by molecules of B in **VEN·H2O**. (**a**) Belt formation along the *b*-axis formed via N12– H12···O11v, C61–H61B···O10vi, 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. **Figure 9.** Crystal architecture formed by molecules of B in **VEN**·**H2O**. (**a**) Belt formation along the *b*-axis formed via N12–H12· · · O11<sup>v</sup> , C61–H61B· · · O10vi, and <sup>π</sup>· · · <sup>π</sup> interactions. (**b**) Layer formation along the *ab*-plane via C61–H61A· · · <sup>π</sup> 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 • 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···O13ii 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. 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· · · O13ii 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). **Figure 10.** Packing of layers of molecules of A (green) and B (blue).

The crystal structure of **VEN·H2O** possesses two crystallographically independent • Structural comparison between conformations of VEN·H2O and protein:venetoclax complexes

• Structural comparison between conformations of VEN·H2O and protein:venetoclax complexes

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 1*H*-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 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 1*H*-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 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**·**H2O** 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**·**H2O**, adopted conformations vary greatly. However, the 1*H*-pyrrolopyridine and nitrobenzene moieties are in close proximity, as is also observed in molecule B of **VEN**·**H2O**. On the other hand, the chlorobenzene ring in both molecules of **VEN**·**H2O** is directed in the opposite direction compared to in all of the protein complexes. *Crystals* **2021**, *11*, x FOR PEER REVIEW 14 of 17

**Figure 11.** (**a**) Superposition showing the difference in conformation of venetoclax molecules in **VEN·H2O** (A—orange, B—light green) from this work and venetoclax molecules from the BCL-2:venetoclax complex (A—blue, B—light blue), BCL-2 G101V:venetoclax complex (A—red, B—pink), BCL-2 G101A:venetoclax complex (green), and BCL-2 F104L:venetoclax complex (A—magenta, B—light magenta) from Birkinshaw and Czabotar [41]. For clarity, hydrogen atoms are omitted, and Cl and S atoms are drawn as small spheres. Differences in the orientation of the sulfonylamide moiety in (**b**) **VEN·H2O** versus (**c**) most of the protein:venetoclax complexes and (**d**) molecules of B in BCL-2 and in BCL-2 G101A. **Figure 11.** (**a**) Superposition showing the difference in conformation of venetoclax molecules in **VEN**·**H2O** (A—orange, B—light green) from this work and venetoclax molecules from the BCL-2:venetoclax complex (A—blue, B—light blue), BCL-2 G101V:venetoclax complex (A—red, B—pink), BCL-2 G101A:venetoclax complex (green), and BCL-2 F104L:venetoclax complex (A—magenta, B—light magenta) from Birkinshaw and Czabotar [41]. For clarity, hydrogen atoms are omitted, and Cl and S atoms are drawn as small spheres. Differences in the orientation of the sulfonylamide moiety in (**b**) **VEN**·**H2O** versus (**c**) most of the protein:venetoclax complexes and (**d**) molecules of B in BCL-2 and in BCL-2 G101A.

#### In this report, we present the first crystal structure of venetoclax, a B-cell lymphoma-**4. Conclusions**

as well as with thermal analysis.

**4. Conclusions** 

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 ve-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

netoclax 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

structure of **VEN·H2O** 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,

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 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.

**Supplementary Materials:** CCDC 2063224 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data\_request/cif or by emailing data\_request@ccdc.cam.ac.uk or by contacting The Cambridge Crystallography Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

**Author Contributions:** Conceptualization, Z.C.; methodology, N.Ž., Z. ˇ C., and F.P.; validation, Z. ˇ C., ˇ and F.P.; formal analysis, F.P. and Z.C.; investigation, N.Ž., Z. ˇ C., and F.P.; resources, Z. ˇ C.; data curation, ˇ Z.C. and F.P.; writing—original draft preparation, Z. ˇ C. and F.P.; writing—review and editing, N.Ž., ˇ Z.C., and F.P.; visualization, Z. ˇ C. and F.P.; supervision, Z. ˇ C.; project administration, Z. ˇ C.; funding ˇ acquisition, Z.C. All authors have read and agreed to the published version of the manuscript. ˇ

**Funding:** This research was funded by Lek Pharmaceuticals d.d. The APC was funded by Lek Pharmaceuticals d.d.

**Data Availability Statement:** All the data supporting the findings of this study are available within the article and supplementary materials.

**Acknowledgments:** Authors gratefully acknowledge D. Lipovec for technical assistance in crystallization experiments; H. Cimerman for the acquisition of IR and Raman spectra as well as for the DCS and TGA measurements; and the EN-FIST Centre of Excellence, Ljubljana, Slovenia, for using the SuperNova diffractometer.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
