To further refine structure-activity relationships for our chemical series of PIKfyve inhibitors, we investigated an additional series of analogues where we varied the alkyne (R1 in Table ). That ring-opened analogues with an indole core bearing varied alkynes demonstrated the most consistently potent PIKfyve affinity (Table ) motivated synthesis of another series. We aimed to identify structural changes that do not elicit cytotoxicity and improve metabolic stability by designing and synthesizing further analogues. We first prepared and evaluated analogues with a nitrogen-containing substituent, including an amine, amide, pyrrolidine, or morpholine, at the alkyne terminus (29-36) in the PIKfyve NanoBRET target engagement assay. The dimethyl amine at the terminus of the alkyne on SGC-PIKFYVE-1 was a predicted site of metabolism by the SMARTCyp in silico method and thus we attempted to modify it and produce more stable analogues. While these analogues had improved metabolic stability, only the pyrrolidine analogue, 35, demonstrated modest inhibition of and affinity for PIKfyve. While these structural changes were quite minimal when compared to SGC-PIKFYVE-1 and its ring-opened congener, 1, dramatic changes in PIKfyve binding were observed. These results support that the part of the ATP site where the dimethyl amine on SGC-PIKFYVE-1 and 1 projects is quite restricted, such that sterically bulky groups and extended linear groups are not tolerated. The significant loss in affinity of 30 when compared to 1, which differs only by a methyl group on the terminal amine, supports that a hydrogen bond donor is not as tolerated in the site that binds the pendant amine. Next, we evaluated whether nitrogen-containing heterocycles on the alkyne terminus were tolerated by PIKfyve. These analogues demonstrated a complete loss of PIKfyve affinity (37-39). Less bulky, linear hydrophobic groups were then appended at the alkyne terminus. Introduction of either a thioether or an aliphatic propyl group resulted in analogues with remarkable PIKfyve potency (40 and 41) but decreased metabolic stability. Notably, 40 exhibited sub-nanomolar activity in both PIKfyve enzymatic and NanoBRET assays with IC50 = 0.6 nM and 0.35 nM in each assay, respectively, and was not toxic when cells were treated at 1 µM. Finally, based on the potency of biaryl analogue 6 (Table , PIKfyve NanoBRET IC50 = 110 nM), we explored replacement of the alkyne with different phenyl rings. We found the ortho-and meta-fluorine substituted analogues (42 and 43) exhibited comparable PIKfyve affinity to 6 but with reduced metabolic stability and solubility. Although the introduction of the para-hydroxyl group (44) improved the stability and solubility while maintaining PIKfyve affinity, substitution with the para-trifluoromethyl ring (45) resulted in an analogue with reduced PIKfyve affinity. Next, a dimethyl amino group was introduced at the para-position of the pendant aryl ring, akin to the group that is found at the alkyne terminus of SGC-PIKFYVE-1. The resultant analogue (46) demonstrated significantly increased metabolic stability and maintained modest PIKfyve affinity (PIKfyve NanoBRET IC50 = 24.7 nM). Removal of a single methyl group from the amino group of 46 yielded mono-methylated analogue 47, which was more active than 46 and 6 but suffered from poor metabolic stability. Finally, insertion of a methylene group between the dimethyl amino group and phenyl ring of 46 yielded 48, which lacked PIKfyve affinity. Overall, modifications to the R1 group resulted in dramatic changes in PIKfyve affinity. Biological evaluation of this series of analogues led us to conclude that smaller and more hydrophobic R1 groups result in more high affinity binding within a tight back pocket of PIKfyve. Taken together, 40 and 46 were selected as potent and non-toxic PIKfyve inhibitor leads with orthogonal structures that warranted additional investigation. To better understand the binding mode of compounds 40 and 46 to PIKfyve in the absence of co-crystal structures, we conducted a molecular docking study utilizing the reported cryo-EM structure of PIKfyve in complex with proteins Fig4 and Vac14 (PDB code: 7K2V, chain: P). As shown in Figure , both compounds exhibited a similar binding mode in top-ranked docking poses and displayed some coplanarity between the pyrimidine and indole rings. The aminopyrimidine