Structural characterization of the azoxy derivative of an antitubercular 8-nitro-1,3-benzothiazin-4-one 1

(Z)-1,2-Bis[4-oxo-2-(piperidin-1-yl)-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-8-yl]diazene oxide, C28H24F6N6O3S2, was obtained and its structure determined while attempting to crystallize and structurally characterize 8-nitro-2-(piperidin-1-yl)-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one, a simplified analogue of the antituberculosis clinical drug candidate BTZ043. X-ray crystallography revealed the structure of the azoxy compound to be comprised of two benzothiazinone moieties linked by a Z-configured azoxy group in an almost coplanar arrangement. In the crystal, the molecules are densely packed, revealing a herringbone pattern.


Chemical context
8-Nitro-1,3-benzothiazin-4-ones (BTZs) are a class of covalently binding inhibitors of decaprenylphosphoryl--d-ribose-2 0 -epimerase (DprE1), an enzyme crucial for cell-wall synthesis in Mycobacterium tuberculosis, the primary pathogen causing tuberculosis (Chikhale et al., 2018). BTZ043 ( Fig. 1; Makarov et al., 2009) is one of the most advanced candidates and has recently completed a Phase Ib/IIa clinical study (ClinicalTrials.gov Identifier: NCT04044001). Compound 1 (Fig. 1) represents a simplified analogue of BTZ043, lacking the spiroketal moiety (Richter et al., 2018). The generally accepted mechanism of action of BTZs is a reduction of the nitro group to a nitroso group by FADH 2 , followed by a semimercaptal formation with Cys387 (Trefzer et al., 2010(Trefzer et al., , 2012Neres et al., 2012;Richter et al., 2018). Tiwari et al. (2013) suggested an alternative mechanism in which the reduction to the nitroso form is initiated by nucleophilic addition of thiolate to C-7 of the BTZ system. Subsequent formation of the azoxy form was postulated, but no proof of the structure is available. Liu et al. (2019) reported detection of the BTZ043 azoxy form by LC/MS in a reaction mixture. To the best of our knowledge, an azoxy derivative of an antitubercular BTZ has not been structurally characterized thus far.
The azoxy derivative of 1 was obtained unintentionally during an attempt to grow crystals of 1 for X-ray crystallography by leaving a dimethylformamide (DMF) solution of 1 at ambient conditions and allowing the solvent to evaporate slowly. Fig. 2 shows a possible reaction pathway to the azoxy derivative. Compound 1 is reduced to the nitroso congener 2 and then to the hydroxylamine 3, which reacts with excess of 2 in a condensation reaction to yield the azoxy compound 4. Although it remains unclear how the reduction of the nitro group in 1 was induced in the absence of an intended reducing agent, this pathway has some plausibility (Chen et al., 2017;Cole et al., 2017). Possibly DMF acted as a reducing agent here (Heravi et al., 2018). Moreover, DMF usually contains small amounts of water, which causes partial hydrolysis (Meglitskii & Kvasha, 1972). Thus, trace amounts of dimethylamine often contained in DMF may have initiated reduction of 1 by nucleophilic addition to C-7 of the BTZ system. A related reaction of BTZs with nucleophilic attack by thiolates on C-7 was postulated by Tiwari et al. (2013).
The identification and structural characterization of 4 could be relevant for drug stability assessment of BTZs. To the best of our knowledge, targeted synthesis of an azoxy derivative of an antitubercular BTZ and antimycobacterial testing has not been reported so far. In this context, it is interesting to note that a variety of azoxy compounds occur naturally and have various biological effects, including potent growth inhibition of M. tuberculosis in vitro exerted by the compound elaiomycin (Dembitsky et al., 2017;Wibowo & Ding, 2020). Fig. 3 shows the molecular structure of 4 in the crystal. The two benzothiazinone moieties and the Z-configured azoxy linkage exhibit a nearly planar structure. The dihedral angles between the mean plane of the azoxy group (i.e. N1 0 , N1 and O2) and the mean planes of the attached benzene rings are 6.7 (1) for the ring C4A-C8A and 5.4 (1) for the ring C4A 0 -C8A 0 . The tilt angle between the mean planes of the two benzene rings is 4.15 (6) . The planar conformation is assumed to be the ground state, possibly stabilized by intramolecular C-SÁ Á ÁO and C-SÁ Á ÁN chalcogen bonds (Scilabra et al., 2019). Additional stabilization, however, does not appear to be necessary, considering that (Z)-azoxybenzene (diphenyldiazene oxide) is planar in the gas phase, as revealed by electron diffraction and ab initio calculations (Tsuji et al., 2000) but not in the crystal (vide infra). The piperidine rings attached to C-2 of the BTZ system both adopt a low-energy chair conformation with slight distortions from the ideal tetrahedral angle ( Table 1). The azoxy oxygen atom O2 has a significant effect on an otherwise symmetrical hypothetical azo-BTZ structure, with the N1 0 -C8 0 distance at 1.394 (1) Å being notably shorter than the N1-C8 distance of 1.459 (1) Å and a clear geometry change at the C-8 position. The difference between the two parts of the molecule is highlighted in Fig. 4, which shows a superposition of the benzene rings of the BTZ moieties of two identical molecules.

Supramolecular features
In the crystal structure, the molecules are densely packed, as revealed by a packing index of 73.0% (Kitaigorodskii, 1973), which was calculated with PLATON (Spek, 2020). A view of the crystal structure along the [101] direction reveals a herringbone pattern (Fig. 5). The separation between the planes of stacked molecules is ca 3.31 Å , similar to the interplanar distance in graphite (3.35 Å ; Delhaes, 2001). As can be seen in the crystal structure, the trifluoromethyl groups of adjacent molecules are in close proximity to one another, but no intermolecular FÁ Á ÁF contacts shorter than the sum of the corresponding van der Waals radii (Bondi, 1964) are encountered.  (1) Å . This can be compared with the C8-N1 bond length of 1.459 (1) and the C8 0 -N1 0 bond length of 1.394 (1) Å in 4, which highlights the short C8 0 -N1 0 bond length resulting from O2 being bonded to N1. A substructure search for variously substituted acyclic azoxybenzene moieties yielded more than a hundred hits. Almost half of these have dihedral angles between the phenyl rings of less than 20 , although there are exceptions such as 1,3-dimethoxy-2-(phenylazoxy)benzene (VUNSII; Zhang et al., 2015) with a dihedral angle between the aromatic rings of ca 90 , illustrating that packing and steric effects are sufficient to disturb the ground-state conformation. The simplest azoxybenzene structure is that of (Z)-azoxybenzene (TIHTEK; Gonzá les Martínez & Bernè s, 2007). The structure most related to that of 4, containing bicycles with fused sixmembered rings, appears to be that of (Z)-1,2-bis[2-(2,2,2trifluoroacetyl)naphthalen-1-yl]diazene oxide (XOZHUS; Belligund et al., 2019). In contrast to 4, in both TIHTEK and XOZHUS the aromatic rings are not coplanar and are significantly tilted out of the plane of the azoxy group. This can be reasonably attributed to effects of crystal packing in TIHTEK and steric effects of the substituents in ortho-position to the azoxy group in XOZHUS.

Synthesis and crystallization
The synthesis of 1 is described elsewhere (Richter et al., 2018). DMF was of reagent-grade quality. Crystals of 4 suitable for single-crystal X-ray diffraction were obtained from a solution of 1 in DMF at room temperature, when the solvent was allowed to evaporate slowly over a period of several weeks.

Refinement
The crystal structure was initially refined to convergence by standard independent atom model (IAM) refinement with SHELXL (Sheldrick, 2015b). The final structure refinement was performed with Hirshfeld atom refinement (HAR), using aspherical scattering factors with NoSpherA2 Midgley et al., 2021) partitioning in OLEX2 (Dolomanov et al., 2009) based on electron density from iterative single determinant SCF single-point DFT calculations using ORCA (Neese et al., 2020) with a B3LYP functional (Becke, 1993;Lee et al., 1988) and a def2-TZVPP basis set. Fig. 6 depicts the F calc (HAR)-F calc (IAM) deformation density map, showing the modelled deformation of the electron density as a result of bonding between independent spherical atoms. Crystal data, data collection and structure refinement details are summarized in Table 2.  Superposition of the benzene rings of the benzothiazinone moieties of two identical molecules (green and orange), illustrating the difference in the attachment of the azoxy group to C8 and C8 0 in the two parts of 4.