**1. Introduction**

The B-cell lymphoma-2 (BCL-2) family of proteins, consisting of three distinctive protein groups (anti-apoptotic proteins, pro-apoptotic effectors, and pro-apoptotic initiators/sensitizers), regulate cell death through their direct binding interactions triggering a mitochondrial apoptotic pathway that results in caspase activation and apoptosis [1–16]. BCL-2 anti-apoptotic family members play a key role in cancer cell survival as well as in drug resistance [17–22]. Therefore, they are primary inhibition targets for the treatment of several cancers as their inhibition restores the apoptotic ability of malignant cells [23–27]. Venetoclax (Figure 1) is an orally bioavailable, B-cell lymphoma-2 (BCL-2) selective inhibitor and the first-in-class oral BCL-2 inhibitor for the treatment of lymphoid malignancies [28–35]. Venetoclax was first approved by the FDA in 2016 for the treatment of patients with chronic lymphocytic leukemia (CLL) and later for small lymphocytic lymphoma (SLL) and for the treatment of newly diagnosed acute myeloid leukemia (AML) in combination with azacitidine, decitabine, or low-dose cytarabine [36–39]. According to the IMS Health data, the market value of venetoclax accounted for nearly USD 735 M in 2019. Moreover, there are many ongoing clinical trials involving venetoclax in various combination therapies [40], which puts venetoclax on the list of highly valuable drugs.

**Citation:** Perdih, F.; Žigart, N.; Casar, ˇ Z. Crystal Structure and Solid-State Conformational Analysis of Active Pharmaceutical Ingredient Venetoclax. *Crystals* **2021**, *11*, 261. https://doi.org/10.3390/ cryst11030261

Academic Editor: Etsuo Yonemochi

Received: 18 February 2021 Accepted: 28 February 2021 Published: 7 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** Structure of venetoclax. **Figure 1.** Structure of venetoclax.

BCL-2.

**2. Materials and Methods**  *2.1. 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). *2.2. Characterization Methods*  2.2.1. 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. 2.2.2. Raman Measurements Raman spectra were collected with a Nicolet iS50FT-IR spectrometer (Thermo Fisher 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 [42–44], 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 [45–47]. 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 [48–54]. 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.

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 [42–44], 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 [45–47]. 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 [48–54]. 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
