Mitosis inhibitor (R)‐litronesib (LY2523355) is a 1,3,4‐thiadiazoline‐bearing phenyl and N‐(2‐ethylamino)ethanesulfonamido‐methyl substituents on tetrahedral C5. Chiral instability has been observed at pH 6 and above with the rate of racemization increasing with pH. A positively charged trigonal intermediate is inferred from the fact that p‐methoxy substituent on the phenyl accelerated racemization, whereas a p‐trifluoromethyl substituent had the opposite effect. Racemization is proposed to occur through a relay mechanism involving intramolecular deprotonation of the sulfonamide by the side chain amino group and attack of the sulfonamide anion on C5, cleaving the C5–S bond, to form an aziridine; heterolytic dissociation of the aziridine yields an ylide. This pathway is supported by (1) a crystal structure providing evidence for a hydrogen bond between the sulfonamide NH and the amino group, (2) effects of substituents on the rate of racemization, and (3) computational studies. This racemization mechanism results from neighboring group effects in this densely functionalized molecule. Of particular novelty is the involvement of the side‐chain secondary amino group, which overcomes the weak acidity of the sulfonamide by anchimeric assistance. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:2797–2808, 2014