Journal of Medicinal Chemistry pubs.acs.org/jmc Article a Scheme 2. Synthesis of N-Acyl and N-Carbamate Diprotected Lysine a Reagents and conditions: (i) (a) HBTU, HOBt, DMF, DIEA, RT, overnight and (b) aq LiOH, THF, RT, 2 h, 19% for 36, 8% for 40 (two steps); (ii) (a) HBTU, CH Cl , DIEA, RT, 2 h and (b) aq LiOH, THF, RT, 2 h, 50% for 37, 19% for 38, 43% for 39, 12% for 41, 5% for 44 (two steps); 2 2 (iii) (a) CDI, DMF, DIEA, RT then 80 °C, 1.5 h, 21% and (b) aq LiOH, THF, RT, 2 h 21% (two steps). Scheme 3. Synthesis of S-Alkyl Cysteinea red dotted line) or the lysine analogue 3 (brown line), showing that the length of the side chain is not critical to recognition. The most active compounds 11, 13, and 45−47 inhibited Neu5Acuptake by sialin in a concentration-dependent manner (Figure 4) in agreement with a specific interaction. Fmoc- Cys[Coum-7-OH]-OH45(LSP12-3129) was the most potent inhibitor with an IC of 2.4 ± 0.7 μM(n = 3), a value ∼400- 50 33 fold lower than the K for Neu5Ac. To assess its selectivity, M + compound 45 was tested on two other H -driven lysosomal transporters, LYAAT1 and cystinosin, measured in whole-cell assay similar to that of sialin.46,47 Interestingly, it had no effect on LYAAT1 and partially inhibited cystinosin (43.7 ± 9.1% inhibition, n = 3) at a concentration (30 μM) that fully inhibits a sialin (Figure 4C,D). Reagents and conditions: (i) from 25 and 28; DIEA, THF, RT, 16 h, Compounds 45 and 13 (Fmoc-Leu-OH) Are Non- 35 and 32%, respectively; from 26:EtN, DMF, RT, 16 h, 28%. 3 competitive Inhibitors. We next studied how 45 and, for comparison, 13 (Fmoc-Leu-OH) interact with human sialin. 8235 https://dx.doi.org/10.1021/acs.jmedchem.9b02119 J. Med. Chem. 2020, 63, 8231−8249