Journal of Medicinal Chemistry pubs.acs.org/jmc Article 25 mL) then stirred overnight at room temperature. The solution was (Cq-7), 156.4 (Cq-4), 154.4 (Cq-8a), 125.4 (CH-5), 113.1 (CH-6), acidified to pH 5 with aqueous HCl solution (2 M), and the resulting 111.5 (Cq-4a), 103.2, 103.1 (CH-3, CH-8), 93.9 (OCH O), 59.0 2 + + precipitate was recovered by filtration, washed with HCl (0.1 M), and (CH OH), 58.9 (CH ); MS: (ESI ), m/z (%): [M + H] = 237.1 2 3 dried under vacuum. Compound 30 (0.947 g) was obtained as a green (100%). powder in a 35% yield. 1H NMR (500 MHz, (CD ) SO): δ 7.44 (d, J 7-(Methoxymethoxy)-4-(Fmoc-oxymethyl)-coumarin (34). 3 2 = 9.0 Hz, 1H, H-5), 6.69 (dd, J = 9.0 Hz, J = 2.5 Hz, 1H, H-6), 6.51 Fmoc-Cl (0.206 g, 0.8 mmol, 1.2 equiv) was added by portion over (d, J = 2.5 Hz, 1H, H-8), 5.99 (s, 1H, H-3), 3.75 (s, 2H, CH CO H), 30 min to a solution of compound 33 (0.160 g, 0.68 mmol, 1 equiv) 2 213 3.41 (q, J = 7.0 Hz, 4H, NCH ), 1.11 (t, J = 7.0 Hz, 6H, CH ); C in dry pyridine (3 mL). After stirring for 2 h at room temperature, the 2 3 NMR (126 MHz, (CD ) SO): δ 170.7 (CO H), 160.7 (CO), reaction mixture was diluted with EtOAc (20 mL) and washed with 3 2 2 155.8 (Cq-O), 150.4 (Cq-Ar), 150.1 (Cq-N), 126.3, 109.1, 108.7 brine (3 × 20 mL). The organic layer was dried, filtered, and (CH-Ar), 107.6 (Cq-Ar), 96.8 (CH-Ar), 44.0 (2 × NCH ), 37.2 evaporated under vacuum. The residue was purified by silica-gel + 2 + (CH CO H), 12.3 (2 × CH ); MS: (ESI ), m/z (%): [M + H] = column chromatography (cyclohexane/EtOAc: 9/1 to 7/3). Com- 2 2 3 + + 276.3 (100%); [2M + Na] = 573.0 (85%); [2M + H] = 550.9 pound 34 (0.108 g) was obtained as a white powder in a 34% yield. (70%). 1H NMR(500 MHz, CDCl ): δ 7.75 (d, J = 7.5 Hz, 2H, H-Ar), 7.59 2-(11-Oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido- 3 (d, J = 7.5 Hz, 2H, H-Ar), 7.40 (m, 3H, H-Ar), 7.31 (t, J = 6.5 Hz, [3,2,1-ij]quinolin-9-yl)acetic acid (31). From ClTi(OiPr)3 (2.262 2H, H-Ar), 7.03 (d, J = 2.5 Hz, 1H, H-8), 6.97 (dd, J = 9.0 Hz, J = 2.5 mL, 6.75 mmol) in toluene (9 mL), 8-hydroxyjulodin (0.851 g, 4.5 Hz, 1H, H-6), 6.38 (t, J = 1.5 Hz, 1H, H-3), 5.29 (d, J = 1.5 Hz, 2H, mmol), and dimethyl 3-oxoglutarate (681 μL, 4.5 mmol), compound CH-Coum), 5.22 (s, 2H, OCH O), 4.48 (d, J = 7.0 Hz, 2H, 31asmethylester was obtained according to general procedure A and 2 2 13 CHCH), 4.26 (t, J = 7.0 Hz, 1H, CH CH), 3.47 (s, 3H, CH ); C used without further purification in the next step. 1H NMR (500 2 2 3 NMR (126 MHz, CDCl3): δ 160.7, 160.6 (CO, Cq-O), 155.5 MHz, CDCl3): δ 6.99 (s, 1H, H-8), 5.98 (s, 1H, H-10), 3.70 (s, 3H, (OCO2), 154.7 (Cq-OMOM), 148.4 (Cq-Ar), 143.2, 141.5 (2 × Cq- CH), 3.61 (s, 2H, CH CO CH ), 3.26 (q, J = 6.0 Hz, 4H, CH -N), 3 2 2 3 2 Ar), 128.2, 127.4, 125.3 (2 × CH-Ar), 124.6 (CH-Ar), 120.3 (2 × 2.89 (t, J = 6.5 Hz, 2H, CH -Ph), 2.79 (t, J = 6.5 Hz, 2H, CH -Ph), 2 2 CH-Ar), 113.8 (CH-Ar), 111.5 (Cq-Ar), 111.1, 104.5 (CH-Ar), 94.6 1.98 (m, 4H, CH -CH -CH ). Methyl ester 31 was then dissolved in 2 2 2 (OCHO), 70.7 (CH CH), 64.7 (CH -Coum), 56.6 (CH ), 46.9 2 2 2 3 MeOH(15mL)andaqueousNaOHsolution(1M,15mL),andthe + + (OCHCH); MS: (ESI ), m/z (%): [M + H] = 459.3 (100%). mixture was stirred overnight at room temperature. The pH was 2 7-Hydroxy-4-(Fmoc-oxymethyl)-coumarin (35). Compound 34 adjusted to 2 with aqueous HCl solution (1 M), and the resulting (0.090 g, 0.2 mmol) was suspended in CH Cl (1.5 mL) at 0 °C, and precipitate was recovered by filtration and washed with aqueous HCl 2 2 TFA (1.5 mL) was added. The solution was stirred 1.5 h at room solution (0.1 M, 30 mL). Compound 31 (0.890 g) was obtained as a temperature. After evaporation of the solvents, the residue was yellow powder in a 66% yield. mp: 186−187 °C; 1H NMR (500 MHz, purified by silica gel column chromatography (cyclohexane/EtOAc: (CD ) SO): δ 12.67 (brs, 1H, CO H), 7.05 (s, 1H, H-8), 5.94 (s, 1H, 3 2 2 9/1 to 7/3). Compound 35 (0.069 g) was obtained as a white powder H-10), 3.71 (s, 2H, CH CO H), 3.24 (m, 4H, CH -N), 2.71 (m, 4H, 1 2 2 2 in an 83% yield. H NMR (500 MHz, (CD ) SO): δ 10.63 (s, 1H, CH-Ph), 1.88 (m, 4H, CH -CH -CH ); 13C NMR (126 MHz, 3 2 2 2 2 2 OH), 7.89 (d, J = 7.5 Hz, 2H, H-Ar), 7.66 (d, J = 7.5 Hz, 2H, H-Ar), (CD ) SO): δ 170.8 (CO H), 160.7 (CO), 150.7, 150.1, 145.5 3 2 2 7.51 (d, J = 8.5 Hz, 1H, H-5), 7.41 (t, J = 7.5 Hz, 2H, H-Ar), 7.32 (t, J (Cq-Ar), 122.0 (CH-Ar), 117.7 (Cq-Ar), 108.3 (CH-Ar), 107.4, =7.5 Hz, 2H, H-Ar), 6.81 (dd, J = 8.5 Hz, J = 2.5 Hz, 1H, H-6), 6.75 105.6 (Cq-Ar), 49.2, 48.6 (CH -N), 37.2 (CH CO H), 27.0, 20.9, 2 2 2 (d, J = 2.5 Hz, 1H, H-8), 6.08 (s, 1H, H-3), 5.33 (s, 2H, CH -Coum), + + 2 20.0, 19.9 (CH2); MS: (ESI ), m/z (%): [M + H] = 300.1 (80%); 4.61 (d, J = 6.0 Hz, 2H, OCH CH), 4.34 (t, J = 6.0 Hz, 1H, [2M + Na]+ = 621.1 (100%). 13 2 OCHCH); CNMR(126MHz,(CD)SO):δ161.4,159.9, 155.0 3-(3,6-Dihydroxy-xanthen-9-ylidene)propanoic Acid (32). A 2 3 2 (Cq-O), 153.9 (OCO ), 149.7 (Cq-Ar), 143.2 (2 × Cq-Ar), 140.8 (2 stirred mixture of succinic anhydride (2.50 g, 25 mmol, 0.5 equiv) 2 ×Cq-Ar), 127.7, 127.1 (2 × CH-Ar), 125.9 (CH-Ar), 124.8, 120.2 (2 and resorcinol (2.75 g, 25 mmol, 1 equiv) in aqueous H SO solution 2 4 × CH-Ar), 113.0 (CH-Ar), 108.9 (Cq-4a), 108.4 (CH-3), 102.4 (70%, 30 mL) was heated to 140 °C for 4 h. The reaction mixture was (CH-8), 69.2 (OCH CH), 64.6 (CH -Coum), 46.3 (OCH CH); then cooled to room temperature and poured into H O (500 mL). 2 2 + 2 2 HPLC-MS (method B): tR = 14.1 min; MS: (ESI ), m/z (%): [M + The stirred solution was alkalinized to pH 12 with aqueous NaOH + + − H] = 415.1 (100%); [2M + Na] = 850.9 (60%); HRMS (ESI ) solution (50%), while the temperature was kept at 0 °C. Acetic acid − max calcd for [C H O − H] : 413.1030; found: 413.1040; λ = 324 was added to the solution until pH 4, and the resulting brown max 25 18 6 −1 −1 abs nm, λ = 399 nm, ε (λ ) = 4760 M cm , Φ = 0.243. precipitate was filtered. The filtrate was washed with H O(3× 25 6 em max 2 2 N -((Benzyloxy)carbonyl-N -(2-(7-hydroxy-2-oxo-2H-chromen-4- mL) and acetone (15 mL) and dried under reduced pressure to give yl)acetyl)-L-lysine (36). 7-Hydroxy-4-carboxymethyl-coumarin (0.220 pure compound 32 (3.28 g) as an orange solid in a 92% yield from g, 1 mmol, 1 equiv), HBTU (0.417 g, 1.1 mmol, 1.1 equiv), and resorcinol. 1H NMR (500 MHz, (CD ) SO): δ 7.51 (d, 1H, J = 8.5 HOBt(0.148g,1.1mmol,1.1equiv) werediluted in anhydrous DMF 3 2 Hz, H-Ar), 7.41 (d, 1H, J = 8.5 Hz, H-Ar), 6.65−6.47 (m, 4H, H-Ar), (3 mL). DIEA (380 μL, 2.2 mmol, 2.2 equiv) was added dropwise, 5.87 (t, 1H, J = 7.0 Hz, CCH), 3.31 (d, 2H, J = 7.0 Hz, CH2-CH); and the resulting solution was stirred for a few minutes then a solution 13 C NMR (126 MHz, (CD ) SO): δ 173.5 (CO H), 158.2, 157.9 of HCl·Lys(Cbz)-OMe (0.331 g, 1 mmol, 1 equiv) in DMF (1 mL) 3 2 2 (Cq-OH), 153.0, 151.3 (Cq-Ar), 128.6 (CH-Ar), 125.7 (Cq-Ar), was added. The reaction mixture was stirred overnight at room 124.3, 116.1 (CH-Ar), 114.0, 113.1 (Cq-Ar), 112.0 (CH-Ar), 110.8 temperature, and DMF was evaporated. The residue was diluted in + (CH), 102.6, 102.3 (CH-Ar), 36.3 (CH ); MS: (ESI ), m/z (%): EtOAc (50 mL), washed with brine (3 × 40 mL), dried (Na SO ), + max 2 max 2 4 [M + H] = 285.1 (100%); λ = 455 nm, λ = 516 nm, ε (λ )= filtered, and evaporated. The ester compound was diluted with THF −1 −1 abs em max 5140 M cm , Φ = 0.243. (4 mL) and a solution of aqueous LiOH (0.5 M, 4 mL) and stirred 2 4-Hydroxymethyl-7-methoxymethoxy-coumarin (33). Com- h at room temperature. After evaporation of THF, the pH was pound 27 (0.576 g, 3 mmol, 1 equiv) was suspended in CH Cl at adjusted to 2 with aqueous HCl solution (1 M) and extracted with 2 2 0°C.DIEA(272μL,3.6mmol,1.2equiv)andMOM-Cl(627μL,3.6 EtOAc (2 × 40 mL). Organic layers were dried (Na SO ), filtered, 2 4 mmol, 1.2 equiv) were added dropwise, and the solution was stirred and evaporated. The residue was purified by column chromatography for 45 min at 0 °C. After evaporation under reduced pressure, H O on silica gel (CH Cl /MeOH/AcOH: 85/15/1). Compound 36 2 2 2 (10 mL) was added to the residue and the resulting precipitate was (0.090 g) was obtained as a white powder in a 19% yield. mp: 78−79 1 recovered by filtration and dried under vacuum. Compound 33 °C; HNMR(500MHz,CD OD):δ7.47(d,J=8.5Hz,1H,H-Ar), 1 3 (0.511 g) was obtained as a white solid in a 71% yield. H NMR (500 7.17 (m, 5H, H-Ph), 6.64 (d, J = 8.5 Hz, 1H, H-Ar), 6.54 (s, 1H, H- MHz, (CD ) SO): δ 7.63 (d, J = 8.5 Hz, 1H, H-5), 7.04 (d, J = 2.5 Ar), 6.06 (s, 1H, H-Ar), 4.89 (s, 2H, CH -Ph), 4.19 (m, 1H, H-α), 3 2 2 Hz, 1H, H-8), 7.00 (dd, J = 8.5 Hz, J = 2.5 Hz, 1H, H-6), 6.32 (s, 1H, 3.60 (s, 2H, CH2-Coum), 2.93 (m, J = 6.5 Hz, 2H, H-ε), 1.70, 1.56 13 H-3), 5.30 (s, 2H, OCH O), 4.72 (s, 2H, CH OH), 3.39 (s, 3H, (2m, 2H, H-β), 1.34−1.25 (m, 4H, H-γ,H-δ); CNMR(126MHz, 13 2 2 CH); C NMR (126 MHz, (CD ) SO): δ 160.3 (CO), 159.4 CDOD): δ 175.2 (CO H), 170.8 (CONH), 163.1, 163.0, 158.9 3 3 2 3 2 8242 https://dx.doi.org/10.1021/acs.jmedchem.9b02119 J. Med. Chem. 2020, 63, 8231−8249