Structural characterization and antimycobacterial evaluation of a benzimidazole analogue of the antituberculosis clinical drug candidate TBA-7371

The crystal structure and in vitro antimycobacterial properties of N -(2-ﬂuoroethyl)-1-[(6-methoxy-5-methylpyrimidin-4-yl)methyl]-1 H -benzo[ d ]imida-zole-4-carboxamide (C 17 H 18 FN 5 O 2 , 1 ), a previously reported benzimidazole analogue of the 1,4-azaindole-based antituberculosis drug candidate TBA-7371, are reported. The structure determination was achieved using Hirshfeld atom reﬁnement. Compound 1 crystallizes in the triclinic system (space group P 1) with two molecules in the asymmetric unit ( Z 0

Late steps in the synthesis of 1, following the previously published route (Manjunatha et al., 2019), are sketched in Fig. 2. Benzimidazole derivative A was reacted with 4-(chloromethyl)-6-methoxy-5-methylpyrimidine to give B. It is worth mentioning that N-alkylation in part occurred at position 3 of the benzimidazole scaffold, affording side product C. Regioisomers B and C were separated by flash chromatography, resulting in an approximate 3.75:1 ratio.
Compound C was identified by 1 H and 13 C NMR spectroscopy and APCI mass spectrometry (see Supporting Information). Hydrolysis of B followed by amide coupling with 2-fluoroethanamine gave the target compound 1. X-ray crystallography unambiguously confirmed the structure.

Structural commentary
Compound 1 crystallizes in the triclinic space group P1 with two crystallographically distinct molecules (Fig. 3). In both molecules, the tilt of the 6-methoxy-5-methylpyrimidin-4-yl group of the plane out of the central benzimidazole moiety renders the conformers axially chiral. The C2-N1-C11-C12 torsion angle is 101.9 (1) in molecule 1 and 79.0 (1) in molecule 2. The enantiomeric conformers in the chosen asymmetric unit thus exhibit the same handedness, but the corresponding oppositely handed conformers are present in the centrosymmetric crystal structure. The most marked structural difference between the two unique molecules is the orientation of the 2-fluoroethyl group about the C9-C10 bond with N2-C9-C10-F1 = 68.1 (1) for molecule 1 and À61.8 (1) for molecule 2.
The plane of the amide group and the mean plane of the benzimidazole moiety are nearly co-planar in molecules 1 and 2. The angle between the two planes is 8.8 (1) in molecule 1 and 7.7 (1) in molecule 2. The amide group adopts a Z conformation in both molecules and forms an intramolecular N-HÁ Á ÁH hydrogen bond to atom N3 of the benzimidazole system (Table 1), resulting in a six-membered hydrogenbonded ring with an S(6) motif (Bernstein et al., 1995). This is in line with Etter's second hydrogen-bond rule for organic compounds, which states that intramolecular six-membered Synthesis of 1, following the published procedure (Manjunatha et al., 2019). Reagents and solvents: (a) 4-(chloromethyl)-6-methoxy-5-methylpyrimidine, Cs 2 CO 3 , NaI, DMF; (b) LiOH, MeOH; (c) HATU, 2fluoroethanamine, NMP.

Figure 3
Asymmetric unit of 1. Displacement ellipsoids are drawn at the 50% probability level. H atoms are represented by small spheres of arbitrary radius. Dashed lines represent hydrogen bonds. The number after the underscore indicates unique molecule 1 or 2.

Supramolecular features
The most significant supramolecular feature of the title compound's solid-state structure is a short C-HÁ Á ÁN contact between the amidine C2-H2 group of the benzimidazole moiety in molecule 2 and N5 of the pyrimidine ring in molecule 1 (Fig. 3), which provides structural evidence for a C-HÁ Á ÁN weak hydrogen bond ( Table 1). The amidine C2-H2 group in molecule 1 forms a short C-HÁ Á ÁO contact to the amide carbonyl group of molecule 2. The geometric parameters including a D-HÁ Á ÁA angle >140 (Wood et al., 2009) are characteristic of a weak hydrogen bond (Thakuria et al., 2017). FÁ Á ÁF interactions are not encountered in the crystal structure, but parallel arrangements between the pyrimidine ring of molecule 1 and the benzimidazole moiety of a neighbouring molecule 2 (Fig. 4) and between the benzimidazole moieties of two molecules 1 about a center of symmetry are notable features (Fig. 5). The latter and the stacking of these units with the pyrimidine rings of molecule 2 in the b * -axis direction no doubt contribute to the 040 reflection having by far the strongest intensity in the diffraction data set. A packing index of 71.9% (Kitaigorodskii, 1973), as calculated with PLATON (Spek, 2020), suggests that the solid-state structure appears to be mainly governed by close packing. In PUMXAX, the amide group likewise forms an intramolecular N-HÁ Á ÁN hydrogen bond to N3 of the benzimidazole system with an S(6) motif, and the 2,6-difluorobenzyl group and the benzimidazole moiety adopt an orientation to one another similar to that of the 6-methoxy-5-methylpyrimidin-4-yl group and the benzimidazole system in 1.

Figure 4
Section of the crystal structure of 1, showingstacking between the 6methoxy-5-methylpyrimidin-4-yl moiety and the benzimidazole system in adjacent molecules. The distance between the centroids of the two sixmembered rings (thin dashed line) is 3.6164 (11) Å . Thick dashed lines represent hydrogen bonds. Carbon-bound H atoms have been omitted for clarity. Symmetry code: (i) Àx + 1, Ày + 1, Àz.

Figure 5
View of the triclinic unit cell of 1, showing the stacking of benzimidazole and 6-methoxy-5-methylpyrimidin moieties in an AABB fashion in the b * -axis direction. Dashed lines represent hydrogen bonds. H atoms have been omitted for clarity, except for amide and amidine H atoms.
The non-tuberculous Mycobacterium abscessus is an opportunistic pathogen, which can cause difficult-to-treat skin, soft tissue and pulmonary infections, in particular in patients with structural lung diseases such as cystic fibrosis (Boudehen & Kremer, 2021). Screening of antitubercular agents for activity against M. abscessus has been proposed (Ganapathy & Dick, 2022). Mechanism-based covalent DprE1 inhibitors with potent activity against M. tuberculosis and other mycobacteria like M. smegmatis form covalent adducts with the thiol group of Cys387 on the FAD substrate binding domain (Shetye et al., 2020). These compounds are usually inactive against M. abscessus, since the M. abscessus DprE1 has an alanine residue in the corresponding amino-acid position, which prevents covalent linkage. Testing of non-covalent DprE1 inhibitors against M. abscessus, however, could be a promising approach to identifying potential lead structures. Therefore, we also tested 1 against M. abscessus ATCC19977 in vitro. In both Middlebrook 7H9 medium supplemented with 10% ADS and 0.05% polysorbate 80 and Mueller Hinton II Broth with 0.05% polysorbate 80, however, no growth inhibition could be detected (MIC 90 > 100 mM). While this work was in progress, the same observation was reported for the parent 1,4-azaindole TBA-7371 (Sarathy et al., 2022). It is worth noting, however, that Sarathy et al. (2022) found moderate in vitro activity against several M. abscessus strains and clinical isolates for the 3,4-dihydrocarbostyril-based non-covalent DprE1 inhibitor and Phase 2b/c clinical antituberculosis drug candidate OPC-167832.

Synthesis and crystallization
Compound 1 was synthesized as described by Manjunatha et al. (2019). Analytical data for A, B, C and 1 can be found in the supporting information. Crystals of 1 suitable for X-ray diffraction were grown from a solution in ethyl acetate/n-heptane (1:1) by slow evaporation of the solvents at room temperature.

Special details
Experimental. Crystal mounted on a MiTeGen loop using Perfluoropolyether PFO-XR75. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > 2sigma(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.