Additional chemical experimental procedures
All reactions were carried out under an inert atmosphere with dry solvents under anhydrous conditions, unless otherwise stated. Temperatures were monitored with an IR-probe. TLC was performed on Silica Gel 60 F254 using UV light detection. Silica gel chromatography employed normal phase silica gel (60 Å, 230-400 mesh, Merck, grade 9385). Optical rotations were measured with a Perkin-Elmer 343 polarimeter at 20 °C and 589 nm. IR spectra were recorded on a Bruker Equinox 55 spectrometer equipped with an ATRdevice. The 1H and 13C NMR spectra were recorded at 298 K with a Bruker DRX-400 spectrometer and calibrated using the residual peak of solvent as internal standard [CDCl3 (CHCl3 δH 7.26 ppm, CDCl3 δC 77.16 ppm), MeOH-d4 (CD2HOD δH 3.31 ppm, CD3OD δC 49.0 ppm), DMSO-d6 (DMSO-d5 δH 2.49 ppm, DMSO-d6 δC 40.0 ppm)]. HRMS was conducted using a Micromass Q-Tof UltimaTM mass spectrometer with electrospray (ES+) ionization. Polyethylenglycol PEG-400 was used as calibration chemical. LRMS was conducted on a Micromass ZQ mass spectrometer with ES+ ionization. HPLC purifications were performed on a Beckman System Gold HPLC equipped with a250x21.2 mm Supelco Discovery® C18-column using a flow rate of 11 mL/min and detection at 254 nm.
Synthesis of KSK120 (8-Cyclopropyl-7-naphthalen-1-ylmethyl-5-oxo-5H-thiazolo[3,2-a]pyridine-3-carboxylic acid phenylamide). Compound C-10 methyl ester was oxidized at the thiazolo part by a previous method 1 and the product was hydrolyzed using LiBr and Et3N in (98/2) CH3CN/H2O. After purification by column chromatography the acid (100 mg, 0.27 mmol) was dissolved in dry dichloromethane (5 mL) and cooled to 0 0C. Oxalyl chloride (0.40 mmol) was added drop wise and the solution was allowed to reach rt and stirred for 1 h. After the evaporation of solvent to remove HCl, the residue was redissolved in dichloromethane, followed by the addition of triethylamine (0.79 mmol) and aniline (0.54 mmol). The solution was then continuously stirred for 4 h and then concentrated and purified by column chromatography on silica gel, afforded KSK120 as a white solid (90 mg, 75%). IR (u cm-1) (neat) 1637, 1597, 1541, 1466, 1443. 1H NMR (CDCl3, 400 MHz) d (ppm) 13.15 (s, 1H), 8.04 (s, 1H), 7.91-7.88 (m, 1H), 7.83-7.78 (m, 2H), 7.63 (d, 2H, J = 7.6 Hz), 7.51-7.42 (m, 3H), 7.29 (d, 1H, J = 6.8 Hz), 7.26-7.21 (m, 2H), 7.03 (t, 1H, J = 7.4 Hz), 6.03 (s, 1H), 4.58 (s, 2H), 1.91 -1.84 (m, 1H), 1.19-1.11 (m, 2H), 0.84-0.78 (m, 2H)); 13C NMR (CDCl3, 100 MHz) d (ppm) 161.3, 155.3, 153.8, 150.7, 138.8, 138.3, 134.1, 133.8, 131.9, 129.1, 128.8 (2C), 128.0, 127.8, 126.4, 125.9, 125.6, 124.3, 124.1, 123.7, 120.4 (2C), 114.6, 112.7, 36.0, 11.1, 8.4 (2C); HRMS (EI): m/z: calcd for C28H22N2O2S: 473.1300 [M+Na]; found: 473.1284.
EC364 (3R)-5-oxo-8-cyclopropyl-7-(2-(1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-8-yl)ethyl)-3,5-dihydro-2H-thiazolo[3,2-a]pyridine-3-carboxylic acid. By following the general procedure methyl ester substituted 2-pyridone (55 mg, 0.105 mmol) after purification by column chromatography on silica gel (97:3 DCM:MeOH to 90:8:2 DCM:MeOH:AcOH) gave EC364 (45 mg, 88%) as brick red solid. [a]D = -58 (c 0.1, CHCl3:MeOH 9:1). IR (u cm-1) (neat) 1720, 1629, 1548, 1492, 1308, 1197, 1157, 1078, 982. 1H NMR (DMSO, 400 MHz) d (ppm) 6.24 (s, 2H), 6.17 (s, 1H), 5.39 (d, 1H, J = 9.2 Hz), 3.78 (dd, 1H, J = 9.4, 12.2 Hz), 3.49 (d, 1H, J = 12.0 Hz), 3.33-3.18 (m, 2H), 3.01-2.82 (m, 2H), 2.41 (s, 6H), 2.35 (s, 6H), 1.60 -1.52 (m, 1H), 0.84-0.74 (m, 2H), 0.55-0.41 (m, 2H). 13C NMR (DMSO, 100 MHz) d (ppm) 170.1, 160.6, 155.6, 154.1, 148.3, 145.9, 141.5, 131.3, 122.4 (2C), 112.1, 111.5, 62.9, 32.8, 31.7, 25.5, 16.2 (2C), 14.6 (2C), 10.9, 7.9, 7.7. HRMS (EI): m/z: calcd for C26H28BF2N3O3S: 534.1810 [M+Na]; found: 534.1853.
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