Journal of Medicinal Chemistry pubs.acs.org/jmc Article exclusively caused by a single mutation, R39C,18 which method A-2 using solvent A (KH PO buffer, 50 mM, pH 5.5) and 2 4 solvent B (CH CN): 0% B for 5 min, a linear increase from 0 to 50% partially preserves the lysosomal targeting and the transport 3 6,7 B between 5 and 20 min. HPLC-MS analyses were performed on a activity of sialin. We thus applied 45 to sialin R39C- Surveyor HPLC system coupled to a LCQ Advantage Thermo transfected cells and Salla patient fibroblasts to test its potential Finnigan LCQ Advantage Instrument. HPLC was equipped with a benefits. Interestingly, this treatment partially rescued the Gemini C18 column (100 mm × 2.1 mm, 3 μm), flow: 220 μL/min. trafficking defect of sialin R39C. However, it did not rescue The dead volume was approximately 800 μL. Products were eluted sialic acid storage in patient fibroblasts, presumably because with method B using solvent A (H O/0.1% HCO H) and solvent B the rescue of the trafficking defect remained insufficient and/or 2 2 (MeOH): 40% B for 1 min, a linear increase from 40 to 100% B this effect was masked by the inhibition of sialin R39C at the between 1 and 9 min, 100% B from 10 to 13 min, and with method C lysosomal membrane. The slight increase of sialic acid storage using solvent A (H O/0.1% HCO H) and solvent B (CH CN/0.1% 2 2 3 upon 45 treatment supports the latter hypothesis. In this HCO2H): 40% B for 1 min, a linear increase from 40 to 100% B respect, the lack of competition between Neu5Ac accumulated between 1 and 6 min, 100% B from 7 to 13 min. The purity of the in the lumen of patient lysosomes and 45 binding at the tested compounds was established by analytical HPLC-MS and 1 13 cytosolic face of sialin would be a disadvantage for therapeutic HPLC and was at least 95%. Spectroscopic ( H and CNMR,MS) applications. and/or analytical data were obtained using chromatographically In summary, our study identifies a new class of cell-permeant homogeneous samples. The photophysical properties of the final inhibitors with a micromolar affinity for sialin, providing compounds were obtained by different measurements on the Greiner and Nonchuk 96-well plates using a Tecan spectrofluorometer valuable tools to study the diverse transport activities of sialin (Safire). These measurements were carried out in solution in ethanol and their physiological roles. These tools may also help at 25 °C. mechanistic and structural studies of sialin. They may also be Syntheses. General Procedure A for the Synthesis of Compounds 29, 30, and 31. A 0.5 M solution of ClTi(OiPr) (1.5 used to prevent Neu5Gc incorporation into therapeutic 3 glycoproteins produced by cell culture. In the case of Salla equiv) in dry toluene was added to aminophenol (1 equiv) and β- disease, however, further studies are needed to explore the cetoester (1 equiv) derivatives. The reaction mixture was refluxed overnight then cooled to room temperature and diluted with CH Cl potential of pharmacological chaperone therapy. 2 2 (7.5 mL/mmol). The whole solution was poured into H O (10 mL/ 2 EXPERIMENTAL SECTION mmol) and stirred for few minutes. The aqueous layer was extracted ■ twice with CH Cl (2 × 30 mL/mmol). The combined organic layers 2 2 General Chemistry Information. All the amino acids belong to were dried (Na SO ), and the solvent was removed under vacuum. 2 4 the L-series unless specified otherwise. Amino acid derivatives are Thecrude product was purified or used directly to another reaction as designated according to the three-letter code and the recommenda- mentioned in each case. tions from the IUPAC-IUB Commission on Biochemical Nomencla- General Procedure B for the Synthesis of Compounds 37−39, ture. Fmoc-Lys(Cbz)-OH 3, Fmoc-Leu-OH 13, Fmoc-Asp(tBu)-OH 41, and 44. Heteroaromatic derivatives (1 equiv), amino acid 14, Fmoc-Cys(tBu)-OH 15, Fmoc-Tyr(tBu)-OH 17, Fmoc-Phe-OH derivatives (1−1.2 equiv), and HBTU (1.1−1.2 equiv) were 18, Fmoc-Ile-OH 19, Fmoc-Cys(pMeOBzl)-OH 20, Fmoc-Thr(tBu)- suspended in CH Cl (0.1 M) then DIEA (4−6 equiv) was added 2 2 OH 21, and Fmoc-Pro-OH 23 were purchased from Novabiochem dropwise at 0 °C, and the reaction mixture was stirred for 2 h at room and used for biological test. Glu(OMe)-OH, DL-Leu-OH, Fmoc- temperature. The solution was diluted with CH Cl (50 mL/mmol) 2 2 Cys(Trt)-OH, Cys(Bzl)-OH, Fmoc-Lys(Boc)-OH, and HCl·Lys- and washed three times with aqueous HCl solution (0.1 M, 20 mL/ (Cbz)-OMe were purchased from Novabiochem and functionalized. mmol). Aqueous layers were extracted twice with CH Cl (50 mL/ 2 2 The other reagents were purchased from Aldrich or Acros. Prior to mmol) then the organic layers were pooled, dried (Na SO ), filtered, 2 4 use, tetrahydrofuran (THF) was distilled from sodium benzophenone and evaporated. The mixture was treated with aqueous LiOH solution and dichloromethane (CH Cl ) from CaH . All reactions were carried (0.5 M, 1.2 equiv)/THF (1/1) and stirred for 2 h at room 2 2 2 temperature. The solution was evaporated and diluted with H O then out under an argon atmosphere and monitored by thin-layer 2 chromatography with Merck 60F-254 precoated silica (0.2 mm) on acidified to pH 2 with HCl (1 M). The precipitate was filtered and, glass. Flash chromatography was performed with Merck Kieselgel 60 when necessary, purified by column chromatography on silica gel (200−500 mm); the solvent systems were given in v/v. 1H NMR (CH Cl /MeOH/AcOH: 95/5/0.1 to 85/15/1). 2 2 13 4-Carboxymethyl-7-dimethylamino-coumarin (29). General pro- (500 MHz) and C NMR (126 MHz) spectra were recorded on a Bruker AVANCEII-500 spectrometer. Chemical shifts (δ)are cedure A was followed using ClTi(OiPr) (1.791 mL, 7.5 mmol) in 3 reported in parts per million. Multiplicity was given using the toluene (12 mL), 3-dimethylaminophenol (0.685 g, 5 mmol), and following abbreviations: s (singlet), brs (broad singlet), d (doublet), dimethyl 3-oxoglutarate (1.791 mL, 5 mmol). The crude product was dd (doublet of doublets), t (triplet), q (quadruplet), and m dissolved in MeOH (12.5 mL) and aqueous NaOH solution (1 M, (multiplet). During acquisition, the spectral window covers a proton 12.5 mL) and stirred overnight at room temperature. The solution chemical shift range from −1 to +12 ppm or from −5 to +20 ppm. was acidified to pH 5 with aqueous HCl solution (2 M), and the 13 1 resulting precipitate was recovered by filtration, washed with HCl (0.1 C chemical attributions were assigned using H-decoupled spectra. For clarity, in some case, Greek letters are used as locants for NMR M), and dried under vacuum. Compound 29 (0.487 g) was obtained attribution of the side chain of the amino acid, while the heterocycle as a green-yellow powder in a 40% yield. 1H NMR (500 MHz, moiety is numbered according to the IUPAC nomenclature. Melting (CD ) SO): δ 7.47 (d, J = 9.0 Hz, 1H, H-5), 6.72 (dd, J = 9.0 Hz, J = 3 2 ̈ 2.5 Hz, 1H, H-6), 6.56 (d, J = 2.5 Hz, 1H, H-8), 6.05 (s, 1H, H-3), points were determined with a Buchi 530 apparatus and are 13 uncorrected. Mass spectra (MS) were recorded on a Thermo 3.77 (s, 2H, CH ), 3.01 (s, 6H, CH ); C NMR (126 MHz, 2 3 Finnigan LCD Advantage spectrometer and HRMS on an Exactive (CD ) SO): δ 170.7 (CO H), 160.6 (CO), 155.4 (Cq-O), 152.8 3 2 2 + (Cq-Ar), 150.2 (Cq-N), 126.0, 109.6, 109.1 (CH-Ar), 108.0 (Cq-Ar), (Thermo Scientific) spectrometer with positive (ESI ) or negative − + (ESI ) electrospray ionization. HPLC analyses were carried out on a 97.5 (CH-Ar), 40.1 (2 × NCH ), 37.2 (CH ); MS: (ESI ), m/z (%): + 3 + 2 max Prominence Shimadzu instrument with an LC20A pump, C18 column [M + H] = 248.3 (100%); [2M + Na] = 517.0 (80%); λ = 373 max −1 −1 abs (250 mm × 4.6 mm, 5 μm), flow: 1 mL/min; eluted peaks were nm, λ = 443 nm, ε (λ ) = 23,300 M cm , Φ = 0.677. em max detected by a PDA detector (SPD-M20A), and retention times are 4-Carboxymethyl-7-diethylamino-coumarin (30). General proce- reported in minutes. The dead volume is approximately 200 μL. dure A was followed using ClTi(OiPr)3 (3.582 mL, 15 mmol) in Products were eluted with method A-1 using solvent A (KH PO4 toluene (25 mL), 3-diethylaminophenol (1.652 g, 10 mmol), and 2 buffer, 50 mM, pH 5.5) and solvent B (CH3CN): 0% B for 10 min, a dimethyl 3-oxoglutarate (1.470 mL, 10 mmol). The crude product linear increase from 0 to 50% B between 10 and 25 min, or with was dissolved in MeOH (25 mL) and aqueous NaOH solution (1 M, 8241 https://dx.doi.org/10.1021/acs.jmedchem.9b02119 J. Med. Chem. 2020, 63, 8231−8249