Introducing a new category of activity cliffs with chemical modifications at multiple sites and rationalizing contributions of individual substitutions.

Activity cliffs (ACs) are formed by structurally similar active compounds with large potency differences. In medicinal chemistry, ACs are of high interest because they reveal structure-activity relationship (SAR) information and SAR determinants. Herein, we introduce a new type of ACs that consist of analog pairs with different substitutions at multiple sites (multi-site ACs; msACs). A systematic search for msACs across different classes of bioactive compounds identified more than 4000 of such ACs, most of which had substitutions at two sites (dual-site ACs; dsACs). A hierarchical analog data structure was designed to analyze contributions of individual substitutions to AC formation. Single substitutions were frequently found to determine potency differences captured by dsACs. Hence, in such cases, there was redundancy of AC information. In instances where both substitutions made significant contributions to dsACs, additive, synergistic, and compensatory effects were observed. Taken together, the results of our analysis revealed the prevalence of single-site ACs (ssACs) in analog series, followed by dsACs, which reveal different ways in which paired substitutions contribute to the formation of ACs and modulate SARs.