Tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide and N-benzyl-6',7'-dihydrospiro[piperidine-4,4'-thieno[3,2-c]pyran] analogues with bactericidal efficacy against Mycobacterium tuberculosis targeting MmpL3

Abstract

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (THPP) and N-benzyl-6',7'-dihydrospiro[piperidine-4,4'-thieno[3,2-c]pyran] (Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This 'genetic phenotype' was further confirmed by a 'chemical phenotype', whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice.

Figure 1. Chemical structures of 1 and 2.

Absolute configuration of 1 was determined by circular dichroism (Protocol S3). Racemic mixture was separated by high performance liquid chromatography.

Figure 2. M. tuberculosis killing kinetics of 1 and 2 at 20x MIC.

The number of cfus were quantified after incubation with the different compounds at different times in 10 ml of 7H9 10%ADC 0,05% Tween 80 medium containing 5 µM of 1, 7.5 µM of 2, 9.4 µg/ml of Linezolid, 1.2 µg/ml of Moxifloxacine and also for internal growth control. The mean and the standard deviations of at triplicate cultures of each point are shown.

Figure 3. 2D-TLC analysis of [¹⁴C]-labelled lipids from M. bovis BCG grown in the presence of MmpL3 inhibitors.

Cultures were grown in the absence or presence of inhibitor (3× MIC) for 8 hours, and then and labelled using [¹⁴C]acetate for 8 hours. Chloroform-methanol extracts (polar lipids) were separated using (i) System D: chloroform:methanol:water (100∶14∶0.8) in direction 1 and chloroform:acetone:methanol:water (50∶60∶2.5∶3) in direction 2 with the position of TMM is indicated by the solid arrows; (ii) System E: chloroform:methanol:water (60∶30∶6) in direction 1 and chloroform:acetic acid:methanol:water (40∶25∶3∶6) in direction 2 with the position of phosphatidylinositol (PI) and phosphatidylinositol mannosides (PIMs) indicated. Lipids were visualized by 48 h exposure on X-ray films by autoradiography (Kodak Biomax MR film).

Figure 4. Profile of lead compounds 3 and 4.

Structure, in vitro antimycobacterial activity against M. tuberculosis H37Rv, intracellular activity against M. tuberculosis H37Rv (RAW264.7 macrophages), cytotoxicity in HepG2 cells, ClogP, clearance in mouse microsomes, % PPB and areas under the curve versus time (AUC) after oral administration (po 50 mg/kg) of compounds 3 and 4.

Figure 5. Whole blood pharmacokinetic profile and main parameters of compounds 3 and 4.

Compounds were given orally at 50 mg/Kg suspension in 1% aqueous methylcellulose. Main pharmacokinetic parameters were established after non-compartimental analysis. AUC: Area Under the Curve; Cmax: Maximum concentration observed in whole blood; %F: percentage bioavailability.

Figure 6. Therapeutic efficacy of Compound 3 (A) and Compound 4 (B) against H37Rv in vivo.

B6 mice were infected by intratracheal instillation with 10⁵ CFU H37Rv per mouse. The mice were treated orally once a day from day 1 to day 8 and sacrificed on day 9. Every point represents data from lungs of one mouse. Moxifloxacin (30 mg/kg) was used as a quality control of the assay. DL: Limit of detection.