Discovery of a cofactor-independent inhibitor of Mycobacterium tuberculosis InhA

AN12855 is a novel cofactor-independent inhibitor of Mycobacterium tuberculosis InhA. AN12855 has potent activity against M. tuberculosis, good oral bioavailability, and comparable efficacy to isoniazid in infection models.

To a mixture of compound V (1.00 g, 3.94 mmol, 1.00 eq) and compound VI (1.51 g, 7.88 mmol, 2.00 eq) in DMF (20 mL), was added K 2 CO 3 (1.09 g, 7.88 mmol, 2.00 eq) in one portion at 20 °C. The mixture was warmed to 80 °C. After 4h the mixture was cooled to RT. Water was added and the mixture was extracted with EtOAc (3x100 mL). The combined organic phase was washed with saturated brine (2x200 mL), dried with anhydrous Na 2 SO 4 , filtered and concentrated in vacuo to afford compound VII (2.00 g, crude).
To a mixture of compound VII (1.50 g, 3.52 mmol, 1.00 eq) in EtOH (30 mL To a solution of compound II (10.20 g, 41.12 mmol, 1.00 Eq) in THF/H 2 O (80/80 mL) was added NaIO 4 (43.98 g, 205.60 mmol, 5.00 Eq) and the mixture was stirred at r.t. After 3 h the reaction was filtered. The filtrate was extracted with EtOAc. The organic layer was washed with brine, dried over Na 2 SO 4 and concentrated (5.90 g, 35.56 mmol, 87% yield).
To a mixture of compound V (800.00mg, 3.15mmol, 1.00 Eq) and compound VI (907.72 mg, 4.72 mmol, 1.50 Eq) in DMF (30 mL), was added K 2 CO 3 (870.37 mg, 6.30 mmol, 2.00 Eq) in one portion at r.t. under N 2 and the reaction mixture was heated to 80 °C . After 2 hthe mixture was cooled to r.t. The residue was poured into water and the mixture was extracted with EtOAc.
The organic phase was washed with saturated brine, dried with anhydrous Na 2 SO 4 , filtered and concentrated in vacuo.
The residue was purified via silica gel chromatography to afford compound VII (900.00 mg, crude).
To a mixture of compound VII (150.00 mg, 351.97 umol, 1.00 equiv) and NH 2 OH·HCl (36.69 mg,527.95 umol,1.50 Eq) in EtOH (15 mL), was added KOAc (69.08 mg,703.94 umol,2.00 equiv) in one portion at r.t. under N 2 . Then heated to 50 °C and stirred for 1 h. HPLC showed the reaction was completed. The solvent was removed. The residue was poured into water.
The mixture was extracted with EtOAc. The organic phase was washed withbrine, dried with anhydrous Na 2 SO 4 , filtered and concentrated in vacuum. The residue was purified via prep- Reactions were performed with Platinum Taq DNA polymerase High Fidelity (Invitrogen) in the presence of 5% (v:v) DMSO and 1M betaine (pH 9) (Sigma). Regional sequence of the resultant amplicons was performed using nested primer sets. Mutations in katG were associated with isoniazid resistance for all three strains of interest. Low-level isoniazid resistance in TN5904 was associated with a previously described G944C mutation (KatG S315T ) previously shown to retain catalase-peroxidase activity in vitro (Ando AAC 2010). High level isoniazid resistance was associated with a frameshift mutation in katG (∆G803), resulting in premature termination at AA343 in strain M70. We repeatedly failed to amplify the katG region from strain M28, suggesting a partial or complete deletion or rearrangement of this region. Such deletions have been reported previously (Ando AAC 2010). No mutations in inhA or the fabGI promoter were observed for any of the strains.