(A) Shown are synthesis strategies a and b used to generate the 35 SF-51/ML-SA1-analogous compounds shown in Figure 1—figure supplement 2. X = Cl/ Br; Y = CO/SO2 / CH2; R1 / R2 / R3 / R4=H/(cyclo)alkyl/aryl; n = 1–5; reaction conditions: a) p-toluenesulfonic acid, cyclohexane, 85°C, 15 hr. (b) H2, Pd/C, ethyl acetate, rt, 15 hr. (c) K2CO3, acetone, 0°C, 30 min, chloroacetyl chloride/bromoacetyl bromide, 0°C - rt, 15 hr. (d) LiHMDS (lithium bis(trimethylsilyl)amide), NaI, molecular sieves 4 Å, THF (dry), 70°C, 15 hr. (e) SOCl2, reflux, 1 hr. (f) Et3N, DCM, RT, 15 hr. (g) DCM, 0°C – rt, 15 hr. (B) Cartoon showing schematically the fractions of inactive, non-selective TRPML activating, TRPML2-selective, and TRPML3-selective agonists (total number = 35). (C) Fura-2 calcium imaging results showing the effect of SF-51/ML-SA1 and their analogues (10 μM) on hTRPML1(NC), hTRPML2, and hTRPML3 transfected HEK293 cells. Mean values (normalized to basal)±SEM of at least three up to >100 independent experiments with 3–10 cells per experiment are shown. With synthesis strategy a we performed numerous variations of the phthalimide residue of ML-SA1. Acetone anile was prepared following a known procedure (Chen et al., 2007) and hydrogenated according to a protocol from Venturini et al., 2010. Coupling with chloroacetyl chloride or bromoacetyl bromide respectively (Li et al., 2013) yielded haloacetamides suitable to undergo SN-reactions with a broad range of imides, lactams or sulfimides under anhydrous conditions (Escudero et al., 2011; Bansode et al., 2009). Synthesis strategy b was used both for variations in the northern acetone anile part of ML-SA1 as well as differentiations in the length of the acyl spacer. Starting from commercially available N-phthaloylglycine the acid chloride was prepared following a standard procedure (Bala et al., 2012) followed by amide coupling with different 1,2,3,4-tetrahydroisoquinolines and other cyclic and acyclic secondary amines (Volkov et al., 2014). Starting from phthalic anhydride and variable ω-aminocarboxylic acids, the southern part of the lead compounds was prepared with variable length of the acyl spacer (Tan et al., 2004) followed by chlorination with thionyl chloride (Tan et al., 2004) and amide coupling with hydrogenated acetone anile (Tagle et al., 2017) or other appropriate secondary amines.