Journal of Medicinal Chemistry pubs.acs.org/jmc Article max max 560.2402; found: 560.2424; λ = 395 nm, λ = 487 nm, ε (λ )= mL) then DIEA (785 μL, 4.5 mmol, 3 equiv) was added dropwise, −1 −1 abs em max 8870 M cm , Φ = 0.736. and the solution was stirred overnight at room temperature. After N6-((Benzyloxy)carbonyl-N2-(((7-hydroxy-2-oxo-2H-chromen-4- evaporation, the residue was purified by column chromatography on yl)methoxy)carbonyl)-L-lysine (42). 7-Hydroxy-4-hydroxy methyl- silica gel (CH Cl /MeOH/AcOH: 85/15/1). Compound 45 (0.272 coumarin 27 (0.150 g, 0.78 mmol, 1 equiv) and CDI (0.156 g, 0.93 2 2 1 g) was obtained as a white powder in a 35% yield. H NMR (500 mmol, 1.2 equiv) were added in dry DMF (2 mL), and the solution MHz, (CD ) SO): δ 12.18 (brs, 1H, CO H), 11.01 (brs, 1H, OH), was stirred for 3 h at room temperature leading to a white precipitate. 3 2 2 7.88 (d, J = 7.5 Hz, 2H, H-Ar), 7.71 (d, J = 7.0 Hz, 2H, H-Ar), 7.66 HCl·Lys(Cbz)-OMe. (0.307 g, 0.93 mmol, 1.2 equiv) and DIEA (215 (d, J = 8.5 Hz, 1H, H-5), 7.40 (brt, J = 7.5 Hz, 3H, H-Ar, NH), 7.31 μL, 1.25 mmol, 1.6 equiv) were added in solution and heated to 80 (t, J = 7.5 Hz, 2H, H-Ar), 6.78 (dd, J = 8.5 Hz, J = 2.0 Hz, 1H, H-6), °C for 1.5 h. After cooling to room temperature, the pH was adjusted 6.71 (d, J = 2.0 Hz, 1H, H-8), 6.24 (s, 1H, H-3), 4.29−4.22 (m, 3H, to 3 with aqueous HCl (1 M) and the solution was extracted with OCHCH), 4.10 (m, 1H, H-α), 3.91, 3.88 (AB, 2H, CH -Coum), EtOAc(2×30mL).TheorganiclayerwaswashedwithH O(2×20 2 2 2 2.96 (dd, J = 13.5 Hz, J = 4.0 Hz, 1H, H-β), 2.80 (dd, J = 13.5 Hz, J = mL), dried (Na SO ), filtered, and evaporated. After purification by 13 2 4 4.5 Hz, 1H, H-β); C NMR (126 MHz, (CD ) SO): δ 172.0 column chromatography on silica gel (EtOAc), the ester compound 3 2 (CO2H), 161.3 (Cq-7), 160.1 (CO-Coum), 155.8, 155.4 (Cq-8a, was diluted with a solution of aqueous LiOH (0.5 M)/THF (3 mL, NHCO), 152.4 (Cq-4), 143.8 (2 × Cq-Fmoc), 140.7 (2 × Cq- 1/1) and stirred at room temperature for 2 h. THF was removed 2 Fmoc), 127.6, 127.0 (2 × CH-Fmoc), 126.9 (CH-5), 125.2 (2 × CH- under vacuum, and the pH was adjusted to 2 with aqueous HCl Fmoc), 112.8 (CH-6), 110.3 (CH-3), 110.0 (Cq-4a), 102.3 (CH-8), solution (1 M) then extracted with EtOAc (2 × 15 mL). Organic 65.6 (OCH CH), 54.2 (C-α), 46.6 (OCH CH), 33.4 (C-β), 31.3 layers were dried (Na SO ), filtered, and evaporated. The residue was 2 2 2 4 (CH2-Coum); HPLC (method A-1): tR = 20.3 min; HPLC (method purified by column chromatography on silica gel (CH Cl /MeOH/ A-2): t = 16.2 min; HPLC-MS (method B): t = 16.6 min; HPLC- 2 2 R R AcOH: 85/15/1). Compound 42 (0.080 g) was obtained as a white + + MS (method C): t = 5.8 min; MS: (ESI ), m/z (%): [M + H] = gumina21%yield.mp:72−73°C;1HNMR(500MHz,CD OD):δ R − − 3 518.2 (100%); MS: (ESI ), m/z (%): [M − H] = 516.1 (100%); 7.44 (d, J = 9.0 Hz, 1H, H-5), 7.28 (m, 5H, H-Ph), 6.76 (dd, J = 8.5 − − HRMS (ESI ) calcd for [C H NO S − H] : 516.1122, found: Hz, J = 2.5 Hz, 1H, H-6), 6.68 (d, J = 2.5 Hz, 1H, H-8), 6.24 (s, 1H, max max 28 23 7 −1 −1 516.1126; λ =328nm,λ =399nm,ε(λ )=5960M cm ,Φ abs em max H-3), 5.25 (s, 2H, CH2-Ph), 5.02 (s, 2H, CH2-Coum), 4.18 (m, 1H, = 0.0922. H-α), 3.11 (t, J = 6.5 Hz, 2H, H-ε), 1.87, 1.72 (2 m, 2H, H-β), 1.54− N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-S-((7-methoxy-2-oxo- 13 2H-chromen-4-yl)methyl)-L-cysteine (46). 4-Chloromethyl-7-me- 1.42 (m, 4H, H-γ,H-δ); C NMR (126 MHz, CD OD): δ 175.3 3 (CO2H), 163.5, 163.0, 158.9 (Cq-O), 157.7 (Cq-Ar), 156.7 thoxy-coumarin 26 (0.224 g, 1.0 mmol, 1 equiv) and Fmoc-Cys- (NHCO), 153.2, 138.4 (Cq-Ar), 129.5 (2 × CH-Ar), 128.9 (CH- OH16(0.343g,1.0mmol,1equiv)weredilutedindryDMF(8mL) 2 then Et N (278 μL, 2.0 mmol, 2 equiv) was added dropwise, and the Ar), 128.7 (2 × CH-Ar), 126.4 (CH-Ar), 114.6 (CH-Ar), 111.0 (Cq- 3 Ar), 108.9 (CH-Ar), 103.9 (CH-Ar), 67.4 (OCH -Ph), 62.0 (CH - solution was stirred overnight at room temperature. After evaporation, 2 2 Coum), 52.9 (C-α), 41.6 (C-ε), 32.4 (C-β), 30.4 (C-δ), 24.1 (C-γ); the residue was purified by column chromatography on silica gel HPLC (method A-2): t = 13.9 min; HPLC-MS (method B): t = (CH Cl /MeOH/AcOH: 85/15/1). Compound 46 (0.152 g) was R R 2 2 1 + + obtained as a white powder in a 28% yield. H NMR (500 MHz, 12.9 min; MS: (ESI ), m/z (%): [M + H] = 499.1 (50%); [2M + + − − (CD ) SO): δ 7.88 (d, J = 7.5 Hz, 2H, H-Ar), 7.75 (d, J = 8.0 Hz, 1H, Na] = 1019.0 (100%); MS: (ESI ), m/z (%): [M − H] = 497.2 3 2 − − H-Ar), 7.75 (d, J = 8.0 Hz, 1H, H-Ar), 7.70 (m, 2H, H-Ar), 7.40 (t, J (100%); HRMS (ESI ) calcd for [C H N O − H] : 497.1565; max max 25 26 2 9 −1 found: 497.1567; λ =324nm,λ =395nm,ε(λ )=11,200M =7.5 Hz, 2H, H-Ar), 7.30 (t, J = 7.5 Hz, 3H, H-Ar, NH), 6.96 (s, 1H, −1 abs em max H-Ar), 6.89 (d, J = 8.0 Hz, 1H, H-Ar), 6.32 (s, 1H, H-3), 4.33−4.17 cm , Φ = 0.12. 2 6 (m, 3H, OCH CH), 4.05 (m, 1H, NHCH), 3.92 (m, 2H, CH - N -(((9H-Fluoren-9-yl)methoxy)carbonyl)-N -(2-(7-(dimethylami- 2 2 no)-2-oxo-2H-chromen-4-yl)acetyl)-L-lysine (44). From 7-dimethy- Coum), 3.82 (s, 3H, CH ), 2.98 (dd, J = 13.5 Hz, J = 3.0 Hz, 1H, H- 3 13 lamino-4-carboxymethyl-coumarin 29 (0.247 g, 1.0 mmol, 1 equiv), β), 2.81 (dd, J = 12.5 Hz, J = 8.5 Hz, 1H, H-β); CNMR(126MHz, Fmoc-Lys-OMe43(0.442g,1.0mmol,1equiv),HBTU(416mg,1.1 (CD ) SO): δ 172.1 (CO H), 162.2 (CO Coum), 159.9 (Cq- mmol, 1.1 equiv), and DIEA (865 μL, 5.0 mmol, 5 equiv), compound 3 2 2 OMe),155.6, 155.3 (Cq-O, NHCO ), 152.3 (Cq-Ar), 143.8 (2 × Cq- 2 44 (0.030 g) was obtained after purification by column chromatog- Ar), 140.6 (2 × Cq-Ar), 127.5, 127.0 (4 × CH-Ar), 126.7 (CH-Ar), raphy on silica gel (CH Cl /MeOH/AcOH: 85/15/1) as a yellow 125.2, 120.0 (4 × CH-Ar), 111.8 (CH-Ar), 111.3 (CH-Ar), 111.2 2 2 1 powder in a 5% yield, according to general procedure B. H NMR (Cq-Ar), 100.9 (CH-Ar), 65.6 (OCH CH), 55.8, 55.7 (C-α, OCH ), (500 MHz, CD OD): δ 7.76 (d, J = 7.5 Hz, 2H, H-Ar), 7.64 (d, J = 2 3 3 46.6 (OCH CH), 33.7 (C-β), 31.3 (CH -Coum); HPLC (method A- 2 2 7.5 Hz, 2H, H-Ar), 7.51 (d, J = 9.0 Hz, 1H, H-5), 7.36 (t, J = 7.5 Hz, 2): tR = 18.0 min; HPLC-MS (method B): tR = 18.0 min; HPLC-MS 2H, H-Ar), 7.28 (t, J = 7.5 Hz, 2H, H-Ar), 6.71 (dd, J = 9.0 Hz, J = + + (method C): t = 10.8; MS: (ESI ), m/z (%): [M + H] = 532.2 −R − − 2.5 Hz, 1H, H-6), 6.48 (d, J = 2.5 Hz, 1H, H-8), 6.02 (s, 1H, H-3), (100%); (ESI ), m/z (%): [M − H] = 530.2 (60%), [2M − H] = 4.33 (d, J = 6.0 Hz, 2H, OCH CH), 4.18 (t, J = 6.0 Hz, 1H, − − 2 1061.3 (100%); HRMS (ESI ) calcd for [C H NO S − H] : OCHCH),4.09(m,1H,H-α),3.64(s,2H,CH -Coum),3.21(t,J = max max 29 25 7 2 2 530.1279, found: 530.1277; λ = 324 nm, λ = 396 nm, ε (λ )= −1 −1 abs em max 6.5 Hz, 2H, H-ε), 3.01 (m, 6H, N(CH ) ), 1.84, 1.68 (2m, 2H, H-β), 8570 M cm , Φ = 0.0428. 13 3 2 1.56−1.38 (m, 4H, H-γ,H-δ); C NMR (126 MHz, CD OD): δ N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-S-((6-chloro-7-hydroxy- 3 2-oxo-2H-chromen-4-yl)methyl)-L-cysteine (47). 4-Chloromethyl-6- 177.4 (CO H), 167.5 (CONH), 160.7 (CO-Coum), 155.5, 155.3 2 chloro-7-hydroxy-coumarin 28 (0.049 g, 0.2 mmol, 1 equiv) and (Cq-O, NHCO ), 152.7 (Cq-Ar), 151.4 (Cq-N(Me)2), 143.8, 140.6 2 Fmoc-Cys-OH 16 (0.068 g, 0.2 mmol, 1 equiv) were diluted in dry (2 × Cq-Fmoc), 127.5, 127.0 (2 × CH-Fmoc), 126.0 (CH-Ar), 125.2 (2 × CH-Fmoc), 120.0 (2 × CH-Fmoc), 109.3 (CH-Ar), 108.9 (CH- DMF (2 mL) then DIEA (108 μL, 0.6 mmol, 3 equiv) was added Ar), 108.2 (Cq-Ar), 97.4 (CH-Ar), 65.2 (OCH CH), 52.00 (C-α), dropwise, and the solution was stirred overnight at room temperature. 2 After evaporation, the residue was purified by column chromatog- 46.7 (OCH CH), 40.1 (C-ε), 40.0 (2 × NCH ), 38.7 (CH -Coum), 2 3 2 raphy on silica gel (CH Cl /MeOH/AcOH: 90/10/1). Compound 32.0 (C-β), 28.9 (C-δ), 22.7 (C-γ); HPLC (method A-2): t = 17.7 2 2 R 1 min; HPLC-MS (method B): tR = 15.2 min; MS: (ESI+), m/z (%): 47(0.035 g) was obtained as a white powder in a 32% yield. H NMR + − − (500 MHz, (CD ) SO): δ 12.85 (brs, 1H, CO H), 11.38 (brs, 1H, [M+H] =598.4(100%);MS:(ESI ),m/z(%):[M−H] =596.3 3 2 2 − − OH-Ar), 7.88 (m, 2H, H-Ar), 7.75 (d, J = 8.0 Hz, 1H, NH), 7.71 (d, J (100%); HRMS (ESI ) calcd for [C H N O − H] : 596.24022, 34 35 3 7 found: 596.24084; λmax= 373 nm, λmax= 447 nm, ε (λ ) = 17,600 = 7.5 Hz, 2H, H-Ar), 7.41 (t, J = 7.5 Hz, 2H, H-Ar), 7.31 (t, J = 7.5 −1 −1 abs em max Hz, 2H, H-Ar), 6.91 (s, 1H, H-Ar), 6.31 (s, 1H, H-3), 4.33−4.15 (m, M cm ,Φ=0.599. N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-S-((7-hydroxy-2-oxo- 4H, OCH2CH, H-α), 3.96 (m, 2H, CH2-Coum), 2.95 (dd, J = 13.5 2H-chromen-4-yl)methyl)-L-cysteine (45). 4-Chloromethyl-7-hy- Hz, J = 4.0 Hz, 1H, H-β), 2.76 (dd, J = 13.5 Hz, J = 10.0 Hz, 1H, H- droxy-coumarin 25 (0.316 g, 1.5 mmol, 1 equiv) and Fmoc-Cys- 13 β); CNMR(126MHz,(CD)SO):δ172.0(CO H),159.6 (CO- 3 2 2 OH16(0.515 g, 1.5 mmol, 1 equiv) were diluted in dry THF (7.5 Coum),156.2, 155.9, 155.8, 153.6, 151.4, 143.8, 143.7 (Cq-Ar), 140.7 8244 https://dx.doi.org/10.1021/acs.jmedchem.9b02119 J. Med. Chem. 2020, 63, 8231−8249