# Targeting Mycobacterium tuberculosis: The Role of Alkyl Substitution in Pyrazinamide Derivatives

## TL;DR

This paper explores how modifying pyrazinamide with alkyl groups improves its effectiveness against tuberculosis, especially drug-resistant strains.

## Contribution

The study introduces new pyrazinamide derivatives with 5-alkyl and 5-alkanamido modifications that show enhanced antimycobacterial activity.

## Key findings

- 5-alkyl chain length correlates with antimycobacterial activity, with heptyl substituents showing maximal potency.
- Compound 23 (5-hexyl-N-(3-trifluoromethylphenyl)pyrazine-2-carboxamide) showed strong activity against MDR TB strains with low cytotoxicity.
- 5-alkyl derivatives are not hydrolyzed by PncA, suggesting a different mechanism of action than PZA.

## Abstract

Tuberculosis (TB) remains a significant global health
challenge
due to the rapid emergence of drug resistance. Despite substantial
progress in anti-TB drug development, effective treatment options
are limited. In this study, we report the synthesis and biological
evaluation of pyrazinamide (PZA) derivatives with 5-alkyl and 5-alkanamido
modifications, designed to enhance antimycobacterial activity by increasing
lipophilicity and improving penetration of the lipid-rich mycobacterial
cell wall. A positive correlation between the length of the 5-alkyl
chain and antimycobacterial activity was observed, with maximal potency
achieved with the heptyl substituent (4: 5-heptylpyrazine-2-carboxamide,
MIC_M. tuberculosis H37Rv = 3.13 μg/mL).
In series C with phenyl substitution on the C-2 carboxamide, different
simple substituents were tolerated on the benzene ring (both electron-donating
and electron-withdrawing, both lipophilic and hydrophilic), and the
length of the alkyl chain was the main determinant of the antimycobacterial
activity. Compound 23 (5-hexyl-N-(3-trifluoromethylphenyl)­pyrazine-2-carboxamide)
exerted MIC = 3.13 μg/mL and selectivity index (SI, compared
to HepG2 cells) >25. Notably, the tested compounds exhibited significant
activity against multidrug-resistant (MDR) Mycobacterium
tuberculosis strains while maintaining favorable selectivity
profiles and low cytotoxicity. In contrast, 5-alkanamido derivatives
(series B and D) were devoid of antimycobacterial activity. Mechanistic
investigations revealed that unlike PZA, the 5-alkyl pyrazinamide
derivatives are not hydrolyzed by mycobacterial pyrazinamidase (PncA),
indicating a distinct mode of action. While molecular modeling initially
suggested enoyl-ACP reductase (InhA) as a potential target of series
C, subsequent experimental validation disproved this hypothesis; thus,
the precise mechanism of action remains to be elucidated.