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

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-ca​rboxamide(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

Cross-resistance of <i>M. bovis</i> BCG mutants resistant to IP compounds.

<p><i>M. bovis</i> BCG resistant mutants raised against IP <b>1</b>, <b>3</b> and <b>4</b> were plated at 5× MIC of each of the respective compounds. A wild-type control demonstrates the resistant nature of the mutants.</p

Compounds from the IP series active against <i>M. tuberculosis</i>.

<p>Structures of the initial IP hits (<b>1</b> and <b>2</b>) identified in the HTS campaign against <i>M. bovis</i> BCG (activity later confirmed in <i>M. tuberculosis</i>) and of optimized compounds <b>3</b> and <b>4</b>.</p

Primers used for the construction of pMV261::<i>qcrB</i>, pMV261::<i>qcrCAB</i> and pMV261::<i>ctaE-qcrCAB.</i>

a<p>Restriction sites used for cloning are underlined (Bam<i>HI,</i> Hind<i>III</i>).</p

<i>M. bovis</i> BCG genome region harboring the genes for the cytochrome <i>bc</i>₁ complex and <i>ctaE</i>.

<p>The DNA regions cloned into the pMV261 vector are indicated.</p

Whole blood pharmacokinetic profile and main parameters of IP 3 after oral administration of a 50 mg/kg suspension in 1% aqueous methylcellulose.

<p>Main pharmacokinetic parameters were established after non-compartmental analysis: Cmax (maximum concentration observed in whole blood), 3.4 µg/ml; AUC (Area Under the Curve) (0–8 hours), 4.36 µg.h/ml; F (percentage bioavailability), 61%.</p

Single nucleotide polymorphisms detected in <i>M. bovis</i> BCG spontaneous mutants resistant to IP compounds.

<p>Wild-type alleles are denoted by a ‘−’ character. Where mixtures of alleles were seen in the population this is indicated, e.g. A/V demonstrates both the genotype coding for alanine and the genotype coding for valine is seen.</p>a<p>Genomic positions are relative to <i>M. bovis</i> BCG str. Pasteur 1173P2 chromosome (Genbank accession: NC_008769.1).</p

Preliminary anti-tubercular activity, cytotoxicity and microsomal stability profile of the HTS hits identified in the IP series.

<p>A small representative set of Gram-positive and Gram-negative organisms were analyzed in addition to the <i>mycobacteria</i>: <i>Enterococcus faecium</i>, <i>Enterococcus faecalis</i>, <i>Haemophilus influenzae</i>, <i>Moraxella catarrhalis</i>, <i>Streptococcus pneumoniae</i>, <i>Escherichia coli</i> and <i>Streptococcus pyogenes</i>.</p>a<p>L1210, HepG2, NEURO 2A, MDCK and H9C2(2-1).</p>b<p><i>M. tuberculosis</i> strains.</p>c<p>ND, Not determined.</p

Effect on the MIC of IP 3 during the over-expression of QcrB in <i>M. bovis</i> BCG.

<p>The over-expression constructs pMV261, pMV261::<i>qcrB</i>, pMV261::<i>qcrCAB</i> and pMV261::<i>ctaE-qcrCAB</i> were electroporated into <i>M. bovis</i> BCG and the MIC of IP <b>3</b> was evaluated. The plate lay-out is shown (<i>M. bovis</i> BCG containing the construct detailed) and the MIC of IP <b>3</b> with reference to wild-type <i>M. bovis</i> BCG is stated along with the corresponding concentration analyzed. Kanamycin was present at 25 µg/ml to select for the pMV261 vector.</p

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

<p>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.</p