Supplementary Materialsml8b00591_si_001. ASICs, caught our attention specifically. An optical technology based on membrane potential detection by voltage-sensitive dyes (VSDs)22?25 was proposed. This original assay format showed an adequate throughput performance, and the ability to efficiently measure the inhibitory effect of ASICs-targeted NCEs. Moreover, additional evidence suggested the use of optic based assays, owing to the membrane potential sensitivity of ASIC1a binding affinity to small molecules.20 The preliminary structural elaboration of DA, in three sequential steps, enabled the identification of LRCH1 1 1,4-diaryl, 3C5-dimethylpyrazole derivative?2a (Chart 1), an early lead compound that was fully characterized in terms of ASICs inhibition. In particular, the linear triazene linker present in DA was initially replaced by a 1,3-disubstituted five-membered heterocycle, with the aim to rigidify the molecular core. Then, to reduce the basic character of the molecule, with the aim to improve VX-765 (Belnacasan) its drug-like character and possibly secure BBB permeability, one of the two amidine VX-765 (Belnacasan) functions was successfully removed. Finally, an initial exploration was made on the effect of the substitution of terminal phenyl rings. As shown in Table 1, compound 2a exhibited greater activity than compound 1 and comparable activity on ASIC2a and ASIC1a/2a (IC50 = 18.9 and 10.9 M, respectively), while being inactive on ASIC1a. Based on these preliminary encouraging results, the rapid 4 points analoging exploration strategy?depicted in?Chart 2?was envisioned. Namely, we focused on the sequential elaboration of the two aryl moieties (pink and green), the heterocycle core (fuchsia), and the suitable rigidification/masking of the potentially metabolically labile terminal benzyl function (cyan). Open in a separate window Chart 2 Structural Optimization of Early Lead2a Table 1 Compounds 2aCi: ASICs Inhibitiona = 3 independent experiments SEM. At first, following the synthetic strategy shown in Scheme 1, nine pink analogues 2aCi were synthesized. In particular, = 3 independent experiments SEM. To acquire additional SAR information, a methylene spacer was introduced between N1 of the pyrazole moiety (fuchsia compound 2k, Scheme 3) by bromination and Suzuki coupling on 3,5-dimethyl pyrazole 8, followed by alkylation at the N1 position of 3,5-dimethyl pyrazole derivative 10a with to position of the phenyl ring (compound 2l, Scheme 4), by introducing a methyl group at the benzylic position (compound 2m, Scheme 4), or by C-1 homologation (compound 2n, Scheme 4). Open in a separate window Scheme 4 Variations on the Benzylamine (Cyan): Compounds 2lCnReagents and conditions: (a) 3-cyanophenylboronic acid, VX-765 (Belnacasan) Cu(OAc)2H2O, pyridine, DMF, 125 C, 3 h, 11%; (b) LiAlH4, THF, r.t, 3 h, 31%; (c) MeMgBr, THF, r.t, 5 h, then LiAlH4, THF, 0 to r.t, 13%; (d) 4-bromobenzyl cyanide, CuI,?L-proline, K2CO3, DMSO, 140 C, 26 h, 33%; (e) NaBH4, CoCl2, MeOH, r.t, 1 h, 25%. In particular, as for the synthesis of 2l, 4-(activity and complete ASIC2a isoform selectivity. Conversely, compound 2q inhibited both ASIC2a and ASIC1a/2a isoforms (IC50 = 9.9 and 11.3 M, respectively). Notably, the corresponding indoline derivatives 2p and 2r were inactive. Finally, the phenyl ring bearing the benzyl amine function was replaced (green exploration) by a 2-pyridine (2s) and a 3-pyridine (2t) ring, whereas dihydropyrrolo[3,4-PK characterization of 2u over 2o in mice was given priority, owing to its more drug-like physicochemical features (cLogP = 2.6 and 4.1; TPSA = 56 and 29 ?2 for 2u and 2o, respectively). The summary of both the and characterization studies performed on 2a, 2o, and 2u? is reported in Table 3. Table 3 ADME Profiling: Compounds 2a, 2o, and 2u PK studies were performed at 1 mg/kg, i.v. and at 3 mg/kg, p.o.; fBrain penetration studies were performed at 1 mg/kg, i.v. and B/P was VX-765 (Belnacasan) calculated from 2 to 8 h after dosing. As for physicochemical descriptors, compound 2u was significantly less.