## 3 Results and Discussion
### Interpretation of Assay Results from Previous Agonist Structures  
Compounds belonging to clusters 25, 33, 37, and 38 are characterized by their triple bond and ring structures composed of benzene and 5- and 6-membered rings (Figure 4a). Among the compounds, compound 52 (cluster 25, EC\({}_{50}\) 1.0 \(\upmu\)M, top one activity in the second assay), compound 92 (cluster 39, EC\({}_{50}\) 6.4 \(\upmu\)M, top three activity in the first assay), compound 94 (cluster 33, EC\({}_{50}\) 7.1 \(\upmu\)M, top five activity in the second assay), and compound 89 (cluster 37, EC\({}_{50}\) 24.0 \(\upmu\)M) exhibited significant activities. The EC\({}_{50}\) value of compound 52 (1 \(\upmu\)M) was the lowest in this study and was comparable with allyl isothianate (EC\({}_{50}\) 0.5 \(\upmu\)M), a natural TRPA1 agonist with high activity [10]. Compounds 52, 92, 94, and 89 have similar structures (two benzene rings, one triple bond, and one oxygen bind to a tetra-substituted carbon), hereafter called \"structure A\" (Figure 4a). In the first and second assay, 20 compounds had the structure A, and eight of them (8/12, 67%) were classified into P1, four of them (4/12, 33%) were P2, and none of them were N2 (0/12, 0%). When structure A was modified to \"one benzene ring\", one triple bond, and one oxygen binding to a tetra-substituted carbon, the compounds did not exhibit TRPA1 activities. For example, compound 79 (cluster 25) and compound 86 (cluster 31) did not show TRPA1 activity (Figure 4a). These results indicated that structure A is related to high TRPA1 activity. To the best of our knowledge, no report has reported TRPA1 activation by structure A and its similar structure. Therefore, structure A is a candidate structure rule for designing new types of TRPA1 agonists. Compounds belonging to cluster 100 have an \(\alpha\), \(\beta\)-unsaturated amide as a common structure (Figure 4b). All 13 compounds belong to cluster-100-activated TRPA1; three of  
Figure 4: Chemical structures and EC\({}_{50}\) values of library compounds and TRPA1 agonists. Compounds characterized by their triple bond and ring structures (**a**), \(\alpha\), \(\beta\)-unsaturated amide moiety (**b**), \(\alpha\), \(\beta\)-unsaturated ester moiety (**c**), and ester moiety (**d**). Chemical moieties that can contribute to TRPA1 activity of the compounds are highlighted in pink (**b**,**c**). them were classified into P1 and ten of them were P2. Compound 579 (EC\({}_{50}\) 6.6 \(\upmu\)M, top four active compounds in the second assay) was the strongest in the cluster. Sanshools (derived from Japanese pepper) and piperies (derived from pepper) are representatives of TRPA1 agonists with an \(\upalpha\), \(\beta\)-unsaturated amide structure (Figure 4b) [11,12]. Compound 579 (EC\({}_{50}\) 6.6 \(\upmu\)M) exhibited five to ten times higher affinity to TRPA1 compared with hydroxy-\(\upalpha\)-sanshool (EC\({}_{50}\) 69 \(\upmu\)M) and piperie (EC\({}_{50}\) 30 \(\upmu\)M). [PERSON] et al. investigated the contribution of an amide moiety and \(\upalpha\), \(\beta\)-unsaturated to TRPA1 activity using sanshool analogs [13]. While amide and carbonyl moieties contributed to TRPA1 activation induced by sanshools, the presence of \(\upalpha\), \(\beta\)-unsaturated did not significantly modify the TRPA1 activity. Although compounds belonging to cluster 100 have an \(\upalpha\), \(\beta\)-unsaturated amide in common, their TRPA1 activity differed, implying that substituents binding to their \(\upalpha\), \(\beta\)-unsaturated amides would modify the TRPA1 activity. Among compounds belonging to clusters 21, 22, and 120, many of them, including compound 655 (cluster 120, EC\({}_{50}\) 5.0 \(\upmu\)M, top1 activity in the first assay) and compound 81 (cluster 22, EC\({}_{50}\) 45 \(\upmu\)M), exhibited TRPA1 activity (Figure 4c). They have an \(\upalpha\), \(\beta\)-unsaturated ester in common. Several TRPA1 agonists with \(\upalpha\), \(\beta\)-unsaturated carboxyl moieties, such as cinnamaldehyde and N-methyl maleimide, have been identified (Figure 4c).