Pyrrolidine carboxamides as a novel class of inhibitors of enoyl acyl carrier protein reductase from Mycobacterium tuberculosis

Abstract

In view of the worldwide spread of multidrug resistance of Mycobacterium tuberculosis, there is an urgent need to discover antituberculosis agent with novel structures. InhA, the enoyl acyl carrier protein reductase (ENR) from M. tuberculosis, is one of the key enzymes involved in the mycobacterial fatty acid elongation cycle and has been validated as an effective antimicrobial target. We report here the discovery, through high-throughput screening, of a series of pyrrolidine carboxamides as a novel class of potent InhA inhibitors. Crystal structures of InhA complexed with three inhibitors have been used to elucidate the inhibitor binding mode. The potency of the lead compound was improved over 160-fold by subsequent optimization through iterative microtiter library synthesis followed by in situ activity screening without purification. Resolution of racemic mixtures of several inhibitors indicate that only one enantiomer is active as an inhibitor of InhA.

Figure 1

Chemical structures of InhA inhibitors (R represents various substituents).

Figure 2

Structure of lead compound d6 (left) and general structure of pyrrolidine carboxamides.

Figure 3

Microtiter amide library synthesis of amines with the core template pyrrolindine carboxylic acid.

Figure 4

Inhibitor d11 bound to the active sites of the M. tuberculosis InhA. A, Purple molecular surface shows the active site cleft in which compound d11 (in capped stick model) binds. B, conformation comparison of catalytic residue Tyr 158 before (in green) and after (in purple) the binding of d11 to InhA, showing the rotation of Tyr158 in the d11-InhA complex. C, Details of InhA-d11 interactions. Key residues within 4.5 sphere of d11 in the binding pocket are shown. The oxygen on the carbonyl group of the five member ring lactam makes hydrogen-bonding interactions with the 2’-hydroxyl moiety of the nicotinamide ribose and the hydroxyl group of Tyr158 (blue line).

Figure 5

Overlay of pyrrolidine carboxamide InhA inhibitors and triclosan in pairs showing the similar binding mode at the active site of InhA. A, Superimposition of d11 (3,5-dichloro substitution) with s1 (unsubstituted phenyl ring). B, Overlay of d11 and s4 (3-bromo substituted phenyl ring). C, Superimposition of d11 with dual triclosan molecules.

Figure 6

Overlay of compound d3 and d8 at the active site of InhA. 6A, Superimposition of d3 (2-Me, 5-Cl disubstitution) with d8 (2-Me, 3-Cl disubstitution). 6B, InhA residues within 4.1 Å of chloro atom in d8. 6C, InhA residues within 4.1 Å of chloro atom in d3.

Figure 7

Structures of pyrrolidine carboxamides with phenyl ring surrogate.

Figure 8

A. Structures of pyrrolidine carboxylic acids synthesized for the modification of pyrrolindine carboxamide Ring C section. B. Structures of amines applied for the ring C exploration of pyrrolidine carboxamide.

Figure 8

A. Structures of pyrrolidine carboxylic acids synthesized for the modification of pyrrolindine carboxamide Ring C section. B. Structures of amines applied for the ring C exploration of pyrrolidine carboxamide.

Figure 9

Racemate and enantiomers chromatograms of compounds p24 (9A), d11 (9B) and p64 (9C). Cyclobond I 2000 stationary phase; eluent, ACN/MeOH/HOAC/TEA (95/5/0.3/0.2 v/v/v/v); flow rate of 0.8 mL/min; temperature 23 °C; wavelength 260 nm (polar organic mode). 9A: A, p24 racemate, B, p24a, C, p24b, 9B: A, d11 racemate, B, d11a, C, d11b, 9C: A, p64 racemate, B, p64a, C, p64b. *: Chiral center.

Figure 9

Racemate and enantiomers chromatograms of compounds p24 (9A), d11 (9B) and p64 (9C). Cyclobond I 2000 stationary phase; eluent, ACN/MeOH/HOAC/TEA (95/5/0.3/0.2 v/v/v/v); flow rate of 0.8 mL/min; temperature 23 °C; wavelength 260 nm (polar organic mode). 9A: A, p24 racemate, B, p24a, C, p24b, 9B: A, d11 racemate, B, d11a, C, d11b, 9C: A, p64 racemate, B, p64a, C, p64b. *: Chiral center.