Mutasynthesis of C17- and C21-Substituted Ansamitocins for Use as ADC Payloads

Anna E Stanley-Smith; Nigel Coates; James C Fox; Frank Delfino; Sylvia Degrado; Arthur Kunz; Shu Mao; Feng Zhao; Ben Garforth; Adam Hold; Hui Hong; John Kendall; Jessica Lane; Coline Meï; Annabel Murphy; Joanna Pogwizd; Celia Ruiz Escobar; Benjamin Wertz; Steven J Moss; Matthew A Gregory; Thomas Nittoli

doi:10.1021/acsomega.5c05529

Mutasynthesis of C17- and C21-Substituted Ansamitocins for Use as ADC Payloads

ACS Omega. 2025 Sep 11;10(37):42993-42998. doi: 10.1021/acsomega.5c05529. eCollection 2025 Sep 23.

Authors

Anna E Stanley-Smith¹, Nigel Coates¹, James C Fox¹, Frank Delfino², Sylvia Degrado³, Arthur Kunz², Shu Mao², Feng Zhao², Ben Garforth¹, Adam Hold¹, Hui Hong¹, John Kendall¹, Jessica Lane¹, Coline Meï¹, Annabel Murphy¹, Joanna Pogwizd¹, Celia Ruiz Escobar¹, Benjamin Wertz³, Steven J Moss¹, Matthew A Gregory¹, Thomas Nittoli²

Affiliations

¹ Isomerase, Newnham Building, Chesterford Research Park, Cambridge CB10 1XL, U.K.
² Regeneron, 777 Old Saw Mill River Road, Tarrytown, New York 10591, United States.
³ Abzena, 360 George Patterson Blvd, Bristol, Pennsylvania 19007, United States.

Abstract

Strain engineering and process improvement were used to improve the titer of mutasynthetically generated ansamitocins generated by feeding 3-amino-5-hydroxybenzoic acid (AHBA) analogs to cultures of Actinosynnema pretiosum inactivated in AHBA biosynthesis. Ansamitocin analogs with fluorine and bromine substituents at C17 and C21 were then generated by feeding hydroxylated AHBA analogs. Fully processed C17 and C21 fluoro and bromo ansamitocins had cytotoxic activity similar to that of Ansamitocin P3. The C21 fluoro derivative was converted to a cytotoxic payload and an antibody drug conjugate (ADC).