DNA gyrase is the only type II topoisomerase in Mycobacterium tuberculosis (Mtb), unlike other bacteria and its absence in human being makes it a clinically validated target for developing anti-tubercular leads against Mtb. In the present study, our effort was to optimize and synthesize a series of compounds by a combination of molecular docking, and synthetic chemistry approach for better activity. A series of twenty eight substituted 2-amino-5-phenylthiophene-3-carboxamide derivatives were designed based on our earlier reported Mtb GyrB inhibitor lead. Hit expansion of the previously identified lead by chemical synthesis led to improved inhibitor with an IC50 value of 0.86±0.81μM against Mtb DNA gyrase supercoiling and Mycobacterium smegmatis GyrB IC50 of 1.35±0.58μM. Further a biophysical investigation using differential scanning fluorimetry experiments re-ascertained the affinity of these molecules towards the GyrB domain.