New Crystal Forms for Biologically Active Compounds. Part 2: Anastrozole as N-Substituted 1,2,4-Triazole in Halogen Bonding and Lp- π Interactions with 1,4-Diiodotetraﬂuorobenzene

: For an active pharmaceutical ingredient, it is important to stabilize its speciﬁc crystal polymorph. If the potential interconversion of various polymorphs is not carefully controlled, it may lead to deterioration of the drug’s physicochemical proﬁle and, ultimately, its therapeutic e ﬃ cacy. The desired polymorph stabilization can be achieved via co-crystallization with appropriate crystallophoric excipients. In this work, we identiﬁed an opportunity for co-crystallization of anastrozole ( ASZ ), a well-known aromatase inhibitor useful in second-line therapy of estrogen-dependent breast cancer, with a classical XB donor, 1,2,4,5-tetraﬂuoro-3,6-diiodobenzene ( 1,4-FIB ). In the X-ray structures of ASZ · 1.5 ( 1,4-FIB ) co-crystal, di ﬀ erent non-covalent interactions involving hydrogen and halogen atoms were detected and studied by quantum chemical calculations and QTAIM analysis at the ω B97XD / DZP-DKH level of theory.


Introduction
The generation of a new salt form is a proven way to modify the physical and chemical properties of an active pharmaceutical ingredient (API) [1]. To be able to give rise to a new salt form, however, the API in question should be ionizable. For non-ionizable APIs, co-crystallization with a crystallophoric excipient (non-API component of the solid drug form) has become an alternative, proven way of accessing a broad range of solid forms and thus modifying various physicochemical properties and increasing API's stability [2][3][4]. An overwhelming majority of API co-crystals reported today are based on hydrogen bonding as the principal means of constructing the crystalline form. However, halogen bonds have emerged as an equally promising basis for designing new co-crystalline API forms [5][6][7][8][9][10][11][12]. However, despite the emergence of this intriguing supramolecular interaction, halogen-bonded API co-crystals remain relatively scarce. This may have to do with the limited range of pharmaceutically acceptable excipients containing polarized halogen atoms [13]. In continuation of our efforts to identify new crystalline forms for APIs that would be stabilized by halogen bonding [14,15], we turned our attention to screening of crystallization conditions for the title compound, anastrozole (IUPAC name 2,2 -(5-((1H-1,2,4-triazol-1-yl)methyl)-1,3-phenylene)bis(2-methylpropanenitrile), abbreviated as ASZ), which is an aromatase inhibitor useful in second-line therapy of estrogen-dependent breast cancer [16][17][18].  [20].
Previously, we successfully cocrystallized another API, nevirapine, with classic XB donor, 1,2,4,5-tetrafluoro-3,6-diiodobenzene (also known as 1,4-diiodotetrafluorobenzene, 1,4-FIB). Noticeably, 1,4-FIB has already been employed in the co-crystal formation for a number of biologically active compounds including nicotine [21], pyrazinamide, lidocaine, and pentoxifylline [22]. It should be noted, however, that in these studies (as well as in present work), 1,4-FIB is employed as an exploratory co-crystallization partner. For its use as an excipient for the design of solid drug forms, a further clinical investigation will be required. In this work, we found ASZ can also be cocrystallized with 1,4-FIB from their solution in MeOH, forming the 2:3 adduct. Herein, we present the results of combined single-crystal XRD experimental and theoretical studies of the adduct and noncovalent interactions found in it.

X-ray Structure Determination
Crystal of ASZ·1.5(1,4-FIB) was investigated on an Xcalibur, Eos diffractometer at 100 K (monochromated MoKα radiation with λ = 0.71073 Å). The structure was solved by the direct methods (SHELX program [23]) in the OLEX2 program package [24]. The carbon-bound H atom positions were calculated and included in the refinement in the 'riding' model approximation. U iso (H) were set to 1.5U eq (C) (for CH 3 groups) or 1.2Ueq(C) (for CH 2 and CH groups). The C-H bond lengths are 0.98 Å for CH 3 groups, 0.99 Å for CH 2 groups, and 0.95 Å for CH groups. Empirical absorption correction was applied in the CrysAlisPro [25] program. For crystallographic data and refinement parameters see Supplementary material (Table S3). Supplementary crystallographic data was deposited at Cambridge Crystallographic Data Centre (CCDC 1960975) and can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif.

Powder X-ray Diffraction Experiments
The X-ray diffraction of powder samples was measured at room temperature on a D8 Discover high-resolution diffractometer using monochromated CuKα (λ = 1.54184 Å) radiation.

Computational Details
The single point calculations based on the experimental X-ray geometry of (ASZ) 3 ·(1,4-FIB) 4 have been carried out at the DFT level of theory using the dispersion-corrected hybrid functional ωB97XD [26] with the help of the Gaussian-09 [27] program package. The Douglas-Kroll-Hess 2nd order scalar relativistic calculations requested relativistic core Hamiltonian were carried out using the DZP-DKH basis sets [28][29][30][31] for all atoms. The topological analysis of the electron density distribution with the help of the atoms in molecules (QTAIM) method developed by Bader [32] has been performed by using the Multiwfn program [33]. The Cartesian atomic coordinates of a model supramolecular cluster are presented in Supporting Information, Table S4.

Halogen Bonding in ASZ·1.5(1,4-FIB)
Slow evaporation of a MeOH solution of ASZ with 1,4-FIB taken in a 1:1 ratio leads to the formation on single crystals of ASZ·1.5(1,4-FIB) suitable for the X-ray diffraction experiment. It is notable that we also tried to synthesize the ASZ·1.5(1,4-FIB) pure phase both by mechanical grinding of 2:3 ASZ + 1,4-FIB mixture with MeOH additions during the process or by crystallization of the same 2:3 mixture from methanol with the following grinding of obtained crystalline material. Powder X-ray diffraction experiments for both cases show that ASZ·1.5(1,4-FIB) coexists with some other unidentified phases (see Figures S3 and S4 in SI). For details on the powder x-ray diffraction experiments see also Section 2.3.
According to the single-crystal XRD data, the cocrystallization of ASZ with 1,4-FIB does not lead to any relevant changes, considering the 3σ criterion, in covalent bond lengths of ASZ [20] and 1,4-FIB [34].

Hydrogen Bonding in ASZ·1.5(1,4-FIB)
As well as in the structure of free ASZ, cyano N atoms are involved in weak hydrogen bonding (theoretically estimated strength of appropriate contacts vary from 0.9 to 1.9 kcal/mol) ( Figure 6 and Table 3). Apart from methyl H atoms, the hydrogen atom in the methylene group is also an HB donor, which was not observed in the ASZ structure previously.

Hydrogen Bonding in ASZ·1.5(1,4-FIB)
As well as in the structure of free ASZ, cyano N atoms are involved in weak hydrogen bonding (theoretically estimated strength of appropriate contacts vary from 0.9 to 1.9 kcal/mol) ( Figure 6 and Table 3). Apart from methyl H atoms, the hydrogen atom in the methylene group is also an HB donor, which was not observed in the ASZ structure previously.

Theoretical Study of Different Non-covalent Interactions in ASZ·1.5(1,4-FIB)
The supramolecular structure of ASZ·1.5(1,4-FIB) is formed by various non-covalent contacts (viz. lp-π interactions, hydrogen, and halogen bonding). We performed quantum chemical calculations and QTAIM analysis [32] to study the nature and energies of these non-covalent contacts in a model supramolecular cluster (ASZ) 3 ·(1,4-FIB) 4 based on the appropriate X-ray diffraction data (Supporting Information, Table S4). This approach depends very slightly on the basis set [81,82] or method [83,84] used and it was already successfully used by us previously for similar chemical systems [14,15,79,85,86] and upon studies of different non-covalent interactions (e.g., hydrogen/chalcogen/halogen bonds, stacking interactions, metallophilic interactions) in other organic and inorganic compounds [14,15,[87][88][89][90][91][92]. The results of QTAIM analysis are presented in Table 4 and visualized in Figure 7. Table 4. Values of the density of all electrons-ρ(r), Laplacian of electron density-∇ 2 ρ(r), energy density-H b , potential energy density-V(r), and Lagrangian kinetic energy-G(r) (a.u.) at the bond critical points (3, −1), corresponding to different non-covalent interactions in (ASZ) 3 ·(1,4-FIB) 4 , bond lengths-l (Å), as well as energies for these contacts E int (kcal/mol), defined by two approaches.* .   The QTAIM analysis reveals the existence of bond critical points (3, −1) (BCPs) for all non-covalent interactions listed in Table 4. The properties of electron density, Laplacian of electron density and energy density in these BCPs are common for non-covalent interactions. Energies for these non-covalent contacts (vary from 0.9 to 6.0 kcal/mol) were defined according to the procedures developed by Espinosa et al. [93] and Vener et al. [94] using the equations Eint = 0.5(−V(r)) or Eint = 0.429G(r), respectively. The balance between the potential energy density V(r) and Lagrangian kinetic energy G(r) at the BCPs reveals that a covalent contribution is absent in all supramolecular contacts listed in Table 4, except I1S···N3 halogen bonding [96].

Conclusions
In combination with 1,2,4,5-tetrafluoro-3,6-diiodobenzene, a classical XB donor, we have identified a new halogen-bonded solid for anastrozole, an anticancer aromatase inhibitor drug. These findings continue to provide proof-of-principle for the productive employment of halogen bonds in the design and discovery of stable crystalline forms of important drug substances. Moreover, these results suggest that the range of potential XB donors for co-crystallization with basic nitrogen-rich molecular frameworks can potentially be expanded beyond the classical ones. The distinctive features of the crystal structures obtained and characterized in detail in this work are the presence of XBs with both triazole N atoms, firstly found for anastrazole. Apart from that, The QTAIM analysis reveals the existence of bond critical points (3, −1) (BCPs) for all non-covalent interactions listed in Table 4. The properties of electron density, Laplacian of electron density and energy density in these BCPs are common for non-covalent interactions. Energies for these non-covalent contacts (vary from 0.9 to 6.0 kcal/mol) were defined according to the procedures developed by Espinosa et al. [93] and Vener et al. [94] using the equations E int = 0.5(−V(r)) or E int = 0.429G(r), respectively. The balance between the potential energy density V(r) and Lagrangian kinetic energy G(r) at the BCPs reveals that a covalent contribution is absent in all supramolecular contacts listed in Table 4, except I1S· · · N3 halogen bonding [96].

Conclusions
In combination with 1,2,4,5-tetrafluoro-3,6-diiodobenzene, a classical XB donor, we have identified a new halogen-bonded solid for anastrozole, an anticancer aromatase inhibitor drug. These findings continue to provide proof-of-principle for the productive employment of halogen bonds in the design and discovery of stable crystalline forms of important drug substances. Moreover, these results suggest that the range of potential XB donors for co-crystallization with basic nitrogen-rich molecular frameworks can potentially be expanded beyond the classical ones. The distinctive features of the crystal structures obtained and characterized in detail in this work are the presence of XBs with both triazole N atoms, firstly found for anastrazole. Apart from that, the adduct structure demonstrates the lp(I)· · · π(triazole) attractive interactions, which may also be important for the adduct formation. The findings encourage us to continue searching for yet novel opportunities to detect XBs as indispensable forces leading to the formation of a new crystal. The results of these studies will be reported in due course.