General procedure for the synthesis of the α-branched carboxylic acids 42-47

Ascorbic acid derivatives as potent inhibitors of bacterial and mammalian

7.4 Potential binding modes of ascorbic acid derivatives at human Hyal-1 Hyal-1

7.6.2.7 General procedure for the synthesis of the α-branched carboxylic acids 42-47

To a solution of diethyl malonate (1 eq) in anhydrous THF was added NaH (60 % suspension, 1 eq), and the mixture was stirred for 10 min under an atmosphere of argon. The corresponding first alkyl halide (1 eq) was added followed by stirring at reflux overnight. After cooling another portion of NaH (60 % suspension, 1 eq) was added and the suspension was stirred for 1 h followed by addition of the second alkyl halide (1 eq) and stirring at reflux overnight again. After cooling, solids were removed by filtration and the solvent was evaporated. The crude product was suspended in EtOH (15 ml) and water (15 ml), KOH (10 eq) was added and the mixture was stirred at reflux overnight. The EtOH was removed under reduced pressure and the

Chapter 7 Ascorbic acid derivatives as potent hyaluronidase inhibitors

remaining aqueous solution was diluted, washed with n-hexane, acidified with 2 N HCl to pH <2 and extracted three times with diethyl ether. The combined organic layers were washed with brine, dried over MgSO4, filtered and evaporated. The crude dicarboxylic acid was decarboxylated at 170 – 200 °C in a sand bath for 5 h. The α-branched carboxylic acid was purified by flash chromatography.

2-Propyldodecanoic acid⁹¹ (7.42): The title compound was prepared according to general procedure 7.6.2.7 by alkylating diethyl malonate (10 mmol, 1.5 ml) using NaH (60 % suspension, 10 mmol, 0.40 g) and 1-bromodecane (10 mmol, 2.1 ml) in the first step and using the same amount of NaH together with 1-iodopropane (10 mmol, 1.0 ml) in the second step. The product was obtained after flash chromatography (PE/EtOAc 90/10 v/v) as a pale yellow oil (0.88 g, 36 %). ¹H-NMR (CDCl3) δ 0.90 (m, 6H, CH3), 1.23 – 1.68 (m, 22H, (CH2)9CH3, (CH2)2CH3), 2.36 (tt, 1H, ³J = 5.2 Hz, ³J = 8.8 Hz, COCH), 11.23 (bs, CO2H). CI-MS (NH3) m/z (%): 260 (100) [M+NH4]⁺. Anal. (C15H30O2) calcd. C: 74.32, H: 12.43, found C: 74.17, H: 13.12.

C15H30O2 (242.40).

2-(Hexan-1-yl)dodecanoic acid⁹² (7.43): The title compound was prepared according to general procedure 7.6.2.7 by alkylating diethyl malonate (10 mmol, 1.5 ml) using NaH (60 % suspension, 10 mmol, 0.40 g) and 1-bromodecane (10 mmol, 2.1 ml) in the first step and using the same amount of NaH together with 1-iodohexane (10 mmol, 1.5 ml) in the second step. The product was obtained after flash chromatography (PE/EtOAc 90/10 v/v) as a white foam (0.90 g, 32 %). ¹H-NMR (CDCl3) δ 0.88 (t, 6H, ³J =6.6Hz, CH2CH3), 1.26 – 1.69 (m, 28H, (CH2)9CH3, (CH2)5CH3), 2.34 (tt, 1H, ³J =5.4Hz, ³J =8.6Hz, COCH), 11.30 (bs, 1H, CO2H). EI-MS (70 eV) m/z (%): 284 (66) [M^+·]. Anal. (C18H36O2) C, H. C18H36O2 (284.48).

2-(Decan-1-yl)dodecanoic acid⁹³ (7.44): The title compound was prepared according to general procedure 7.6.2.7 by alkylating diethyl malonate (10 mmol, 1.5 ml) using NaH (60 % suspension, 10 mmol, 0.40 g) and 1-bromodecane (10 mmol, 2.1 ml) in the first step and using the same amount of NaH and 1-bromodecane in the second step. The product was obtained after flash chromatography (PE/EtOAc 90/10 v/v) as a white solid (1.33 g, 39 %). mp: 52 °C; ¹H-NMR (CDCl3) δ 0.88 (t, 6H,

3J = 6.7 Hz, CH2CH3), 1.27 – 1.68 (m, 36H, (CH2)9CH3), 2.34 (tt, 1H, ³J = 5.4 Hz, ³J = 8.6 Hz, COCH), 9.48 (s, 1H, CO2H). CI-MS (NH3) m/z (%): 359 (100) [M+NH4]⁺. Anal.

(C22H44O2) C, H. C22H44O2 (340.58).

2-(2-Phenethyl)dodecanoic acid (7.45): The title compound was prepared according to general procedure 7.6.2.7 by alkylating diethyl malonate (10 mmol, 1.5 ml) using NaH (60 % suspension, 10 mmol, 0.40 g) and 1-bromodecane (10 mmol, 2.1 ml) in the first step and using the same amount of NaH together with (2-bromoethyl)benzene (10 mmol, 1.4 ml) in the second step. The product was obtained after flash chromatography (PE/EtOAc 90/10 v/v) as a pale yellow oil (0.74 g, 24 %).

1H-NMR (CDCl3) δ 0.88 (t, 3H, ³J = 6.7 Hz, CH2CH3), 1.62 (m, 20H, CH2), 2.42 (m, 1H, COCH), 2.65 (m, 2H, CH2CH2Ph), 7.23 (m, 10H, Ph-H), 11.08 (bs, 1H, CO2H).

EI-MS (70 eV) m/z (%): 304 (16) [M^+·]. C20H32O2 (304.47).

2-(4-Methoxybenzyl)dodecanoic acid (7.46): The title compound was prepared according to general procedure 7.6.2.7 by alkylating diethyl malonate (10 mmol, 1.5 ml) using NaH (60 % suspension, 10 mmol, 0.40 g) and 1-bromodecane (10 mmol, 2.1 ml) in the first step and using the same amount of NaH together with 1-(chloromethyl)-4-methoxybenzene (10 mmol, 1.4 ml) in the second step. The product was obtained after flash chromatography (PE/EtOAc 90/10 v/v) as a pale yellow oil (0.53 g, 16 %). ¹H-NMR (CDCl3) δ 0.89 (t, 3H, ³J = 6.7 Hz, CH2CH3), 1.25 – 1.68 (m, 16H, (CH2)8CH3), 2.65 (m, 2H, OCH, CHCH2C6H4), 2.91 (dd, 1H, ³J = 7.7 Hz, ²J = 13.5 Hz, CHCH2C6H4), 3.79 (s, 3H, OCH3), 6.82 (m, 2H, Ar-H), 7.10 (m, 2H, Ar-H), 10.45 (bs, 1H, CO2H). EI-MS (70 eV) m/z (%): 320 (8) [M^+·]. Anal.

(C20H32O3·0.01C6H4CH3) C, H. C20H32O3 (320.47).

2-(Hexan-1-yl)octadecanoic acid⁹⁴ (7.47): The title compound was prepared according to general procedure 7.6.2.7 by alkylating diethyl malonate (10 mmol, 1.5 ml) using NaH (60 % suspension, 10 mmol, 0.40 g) and 1-bromohexadecane (10 mmol, 3.05 ml) in the first step and using the same amount of NaH together with 1-iodohexane (10 mmol, 1.5 ml) in the second step. The product was obtained after flash chromatography (PE/EtOAc 90/10 v/v) as a pale yellow oil (0.62 g, 17 %). ¹ H-NMR (CDCl3) δ 0.88 (t, 6H, ³J = 6.6 Hz, CH2CH3), 1.49 (m, 40H, (CH2)15CH3, (CH2)5CH3), 2.34 (tt, 1H, ³J = 5.4 Hz, ³J = 8.6Hz, COCH). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 368 (100) [M-H]^-. Anal. (C24H48O2·0.3H2O) C, H. C24H48O2

(368.64).

6-O-[6-(Phenylthio)hexanoyl]ascorbic acid (7.48): The title compound was prepared from ascorbic acid (2 mmol, 0.35 g), 7.4 (4 eq, 8 mmol, 1.79 g) and Novozyme 435^®(100 mg) in 15 ml tert-amyl alcohol according to general procedure 7.6.2.2, yielding 7.48 as a white solid (0.40 g, 54 %). mp: 108-110 °C; ¹H-NMR

Chapter 7 Ascorbic acid derivatives as potent hyaluronidase inhibitors

(DMSO-d6) δ 1.40 (m, 2H, COCH2CH2CH2), 1.56 (m, 4H, COCH2CH2, CH2CH2SPh), 2.32 (t, 2H, ³J = 7.3 Hz, COCH2), 2.95 (t, 2H, ³J = 7.2 Hz, CH2SPh), 4.02 (m, 3H, -CH2O-, CHOH) ppm 4.68 (d, 1H, ³J = 1.7 Hz, H-5), 5.31 (s, 1H, CHOH), 7.18 (m, 1H, Ph-H), 7.31 (m, 4H, Ph-H), 8.40 (bs, 1H, OH), 11.11 (bs, 1H, OH). ¹³C-NMR (DMSO-d6) δ 23.83 (-, CH2), 27.47 (-, CH2), 28.13 (-, CH2), 31.67 (-, CH2), 33.18 (-, COCH2CH2), 64.39 (-, CH2O), 65.35 (+, CHO), 74.92 (+, CH), 118.08 (quat, C-2), 125.37 (+, Ph-C), 127.81 (+, Ph-C), 128.91 (+, Ph-C), 136.32 (quat, Ph-C), 152.13 (quat, C-3), 170.22 (quat, lactone CO), 172.55 (quat, CO2CH2). ES-MS (MeCN + TFA) m/z (%): 400 (100) [M+NH4]⁺. Anal. (C18H22O7S·0.5H2O) C, H. C18H22O7S (382.43).

6-O-[6-(Benzylthio)hexanoyl]ascorbic acid (7.49): The title compound was prepared from ascorbic acid (3.3 mmol, 0.58 g), 7.5 (4 eq, 13.2 mmol, 3.33 g) and Novozyme 435^®(165 mg) in 15 ml tert-amyl alcohol according to general procedure 7.6.2.2, yielding 7.49 as a white amorphous solid (0.53 g, 41 %). ¹H-NMR (DMSO-d6) δ 1.31 (m, 2H, COCH2CH2CH2), 1.50 (m, 4H, COCH2CH2, CH2CH2SBn), 2.31 (t, 2H,

3J = 7.4 Hz, COCH2), 2.37 (t, 2H, ³J = 7.3 Hz, CH2SCH2Ph), 3.71 (s, 2H, SCH2Ph), 4.03 (m, 3H, CH2O, CHOH), 4.68 (d, 1H, ³J = 1.6 Hz, H-5), 5.32 (d, 1H, ³J = 5.8 Hz, CHOH), 7.26 (m, 5H, Ph-H), 8.42 (s, 1H, OH), 11.13 (s, 1H, OH). ¹³C-NMR (DMSO-d6) δ 23.87 (-, CH2), 27.57 (-, CH2), 28.26 (-, CH2), 30.36 (-, CH2), 33.19 (-,COCH2CH2), 34.94 (-, SCH2Ph), 64.40 (-, CH2O), 65.40 (+, CHO), 74.93 (+, CH), 118.09 (quat, C-2), 126.61 (+, Ph-C), 128.22 (+, Ph-C), 128.70 (+, Ph-C), 138.69 (quat, Ph-C), 152.12 (quat, C-3), 170.27 (quat, lactone CO), 172.57 (quat, CO2CH2).

ES-MS (DCM/MeOH + NH4OAc) m/z (%): 395 (100) [M-H]^-. Anal.

(C19H24O7S·0.7H2O) C, H. C19H24O7S (396.45).

6-O-(6-Phenoxyhexanoyl)ascorbic acid (7.50): The title compound was prepared from ascorbic acid (3.3 mmol, 0.58 g), 7.6 (4 eq, 13.2 mmol, 2.75 g) and Novozyme 435^®(165 mg) in 15 ml tert-amyl alcohol according to general procedure 7.6.2.2, yielding 7.50 as a white solid (0.55 g, 45 %). ¹H-NMR (DMSO-d6) δ 1.43 (m, 2H, COCH2CH2CH2), 1.60 (m, 2H, CH2CH2CH2), 1.71 (m, 2H, CH2CH2CH2), 2.37 (t, 2H,

3J = 7.3 Hz, COCH2),4.03 (m, 5H, CH2O, CHOH, CH2OPh), 4.69 (d, 1H, ³J = 1.7 Hz, H-5), 5.32 (d, 1H, ³J = 6.0 Hz, CHOH), 6.91 (m, 3H, Ph-H), 7.27 (m, 2H, Ph-H), 8.40 (s, 1H, OH), 11.12 (s, 1H, OH). ¹³C-NMR (DMSO-d6) δ 24.08 (-, CH2), 25.01 (-, CH2), 28.31 (-, CH2), 33.27 (-, COCH2CH2), 64.42 (-, CH2O), 65.43 (+, CHO), 67.02 (-, CH2OPh), 74.95 (+, CH), 114.30 (+, Ph-C), 118.12 (quat, C-2), 120.26 (+, Ph-C),

129.36 (+, Ph-C), 152.10 (quat, C-3), 158.53 (quat, Ph-C), 170.26 (quat, lactone CO), 172.61 (quat, CO2CH2). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 365 (100) [M-H]^-. Anal. (C18H22O8·0.3H2O) C, H. C18H22O8 (366.36).

6-O-[6-(4-Ethylphenoxy)hexanoyl]ascorbic acid (7.51): The title compound was prepared from ascorbic acid (3.3 mmol, 0.58 g), 7.7 (4 eq, 13.2 mmol, 3.30 g) and Novozyme 435^®(165 mg) in 15 ml tert-amyl alcohol according to general procedure 7.6.2.2, yielding 7.51 as a white solid (0.65 g, 50 %). mp: 109-111 °C; ¹H-NMR (DMSO-d6) δ 1.13 (t, 3H, ³J =7.6Hz, CH2CH3), 1.41 (m, 2H, COCH2CH2CH2), 1.65 (m, 4H, COCH2CH2, CH2CH2OC6H4), 2.36 (t, 2H, ³J = 7.3 Hz, COCH2), 2.52 (q, 2H,

3J = 7.5 Hz, CH2CH3), 3.90 (t, 2H, ³J = 6.4 Hz, CH2OC6H4), 4.03 (m, 3H, CH2O, CHOH), 4.68 (d, 1H, ³J = 1.5 Hz, H-5), 5.33 (s, 1H, CHOH), 6.81 (d, 2H, ³J = 8.5 Hz, Ar-H), 7.09 (d, 2H, ³J = 8.5 Hz, Ar-H), 8.42 (bs, 1H, OH), 11.11 (bs, 1H, OH). ¹³ C-NMR (DMSO-d6) δ 15.87 (+, CH3), 24.08 (-, CH2), 25.02 (-, CH2), 27.21 (-, CH2), 28.34 (-, CH2), 33.27 (-, COCH2CH2), 64.42 (-, CH2O), 65.42 (+, CHO), 67.10 (-, CH2OC6H4), 74.94 (+, CH), 114.16 (+, Ph-C), 118.10 (quat, C-2), 128.51 (+, Ar-C), 135.44 (quat, Ar-C), 152.11 (quat, C-3), 156.59 (quat, Ar-C), 170.27 (quat, lactone CO), 172.61 (quat, CO2CH2). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 393 (100) [M-H]^-. Anal. (C20H26O8·0.3H2O) C, H. C20H26O8 (394.42).

6-O-[6-(4-Phenylphenoxy)hexanoyl]ascorbic acid (7.52): The title compound was prepared from ascorbic acid (3.3 mmol, 0.58 g), 7.8 (1.5 eq, 5.0 mmol, 1.48 g) and Novozyme 435^®(165 mg) in 15 ml tert-amyl alcohol according to general procedure 7.6.2.2, yielding 7.52 as a white solid (0.60 g, 41 %). mp: 125-126 °C; ¹H-NMR (DMSO-d6) δ 1.45 (m, 2H, COCH2CH2CH2), 1.61 (m, 2H, COCH2CH2), 1.74 (m, 2H, CH2CH2OC6H5) 2.38 (t, 2H, ³J = 7.3 Hz, COCH2), 4.06 (m, 5H, CH2O, CHOH, CH2OC6H4), 4.70 (d, 1H, ³J = 1.6 Hz, H-5), 5.33 (bs, 1H, CHOH), 7.01 (m, 2H, Ar-H), 7.30 (m, 1H, Ph-H), 7.43 (m, 2H, Ar-H), 7.59 (m, 4H, Ar-H), 8.42 (s, 1H, OH), 11.14 (s, 1H, OH). ¹³C-NMR (DMSO-d6) δ 24.08 (-, CH2), 25.01 (-, CH2), 28.30 (-, CH2), 33.28 (-, COCH2CH2), 64.43 (-, CH2O), 65.42 (+, CHO), 67.27 (-, CH2OC6H4), 74.95 (+, CH), 114.77 (+, Ar-C), 118.11 (quat, C-2), 126.06 (+, Ar-C), 126.58 (+, Ar-C), 127.64 (+, Ar-C), 128.77 (+, Ar-C), 132.31 (quat, Ar-C), 139.76 (quat, Ar-C), 152.12 (quat, C-3), 158.20 (quat, Ar-C), 170.28 (quat, lactone CO), 172.62 (quat, CO2CH2).

ES-MS (MeCN + TFA) m/z (%): 443 (100) [M+H]⁺. Anal. (C24H26O8) C, H. C24H26O8

(442.46).

Chapter 7 Ascorbic acid derivatives as potent hyaluronidase inhibitors

6-O-[6-(4-Benzyloxyphenoxy)hexanoyl]ascorbic acid (7.53): The title compound was prepared from ascorbic acid (3.3 mmol, 0.58 g), 7.9 (1.5 eq, 5.0 mmol, 1.63 g) and Novozyme 435^® (165 mg) in 15 ml tert-amyl alcohol according to general procedure 7.6.2.2, yielding 7.53 as a white solid (0.51 g, 33 %). mp: 147-148 °C; ¹ H-NMR (DMSO-d6) δ 1.41 (m, 2H, COCH2CH2CH2), 1.63 (m, 4H, COCH2CH2, CH2CH2OCH2), 2.36 (t, 2H, ³J = 7.3 Hz, COCH2CH2), 3.87 (t, 2H, ³J = 6.4 Hz, CH2OCH2Ph), 4.03 (m, 3H, -CH2O-, CHOH), 4.69 (d, 1H, ³J = 1.4 Hz, H-5), 5.02 (s, 2H, CH2Ph), 5.34 (bs, 1H, CHOH), 6.84 (m, 2H, Ar-H), 6.92 (m, 2H, Ar-H), 7.36 (m, 5H, Ph-H), 8.43 (s, 1H, OH), 11.14 (s, 1H, OH). ¹³C-NMR (DMSO-d6) δ 24.09 (-, CH2), 25.02 (-, CH2), 28.39 (-, CH2), 33.27 (-, CH2), 64.42 (-, CH2O), 65.40 (+, CHO), 67.57 CH2O), 69.52 (-, CH2Ph), 74.94 (+, CH), 115.15 (+, Ar-C), 115.57 (+, Ar-C), 118.09 (quat, C-2), 127.52 (+, Ar-C), 127.62 (+, Ar-C), 128.29 (+, Ar-C), 137.29 (quat, Ar-C), 152.12 (quat, C-3), 152.16 (quat, Ar-C), 152.72 (quat, Ar-C), 170.28 (quat, lactone CO), 172.62 (quat, CO2CH2). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 471 (100) [M-H]^-. Anal. (C25H28O9) C, H. C25H28O9 (472.48).

6-O-(6-Benzyloxyhexanoyl)ascorbic acid (7.54): The title compound was prepared from ascorbic acid (2.5 mmol, 0.44 g), 7.14 (4 eq, 10 mmol, 2.26 g) and Novozyme 435^®(125 mg) in 15 ml tert-amyl alcohol according to general procedure 7.6.2.2, yielding 7.54 as a colorless semisolid substance (0.35 g, 37 %). ¹H-NMR (DMSO-d6) δ 1.33 (m, 2H, -COCH2CH2CH2-), 1.54 (m, 4H, -COCH2CH2-, -CH2CH2OBn), 2.33 (t, 2H, ³J = 7.4 Hz, -COCH2-), 3.41 (t, 2H, ³J = 6.4 Hz, -CH2OBn), 4.04 (m, 3H, -CH2O-, -CHOH), 4.44 (s, 2H, -OCH2Ph), 4.68 (d, 1H, ³J = 1.6 Hz, H-5), 5.33 (s, 1H, -CHOH), 7.32 (m, 5H, Ph-H), 8.42 (s, 1H, -OH), 11.14 (bs, 1H, -OH). ¹³C-NMR (DMSO-d6) δ 24.14 (-, CH2), 25.17(-, CH2), 28.80 (-, CH2), 33.28 (-, COCH2CH2), 64.40 (-, CH2O), 65.40 (+, CHO), 69.35 (-, CH2OCH2Ph), 71.71 (-, CH2Ph), 74.93 (+, CH), 118.10 (quat, C-2), 127.22 (+, C), 127.32 (+, C), 128.13 (+, C), 138.61 (quat, Ph-C), 152.11 (quat, C-3), 170.27 (quat, lactone CO), 172.63 (quat, CO2CH2). ES-MS (MeCN + TFA) m/z (%): 381 (100) [M+H]⁺. Anal. (C19H24O8) C, H. C19H24O8 (380.39).

6-O-[6-(4-Phenylphenoxy)hexanoyl]ascorbic acid (7.55): The title compound was prepared from ascorbic acid (1.7 mmol, 0.30 g), 7.15 (1.5 eq, 2.6 mmol, 0.80 g) and Novozyme 435^®(85 mg) in 15 ml tert-amyl alcohol according to general procedure 7.6.2.2, yielding 7.55 as a white solid (0.40 g, 51 %). mp: 104 °C; ¹H-NMR (DMSO-d6) δ 1.35 (m, 2H, COCH2CH2CH2), 1.56 (m, 4H, COCH2CH2, CH2CH2OCH2), 2.34 (t, 2H, ³J = 7.3 Hz, COCH2CH2) ppm 3.44 (t, 2H, ³J = 6.4 Hz, CH2OCH2C6H4), 4.03 (m,

3H, CH2O, CHOH), 4.49 (s, 2H, OCH2C6H4), 4.69 (d, 1H, ³J = 1.6 Hz, H-5), 5.33 (bs, 1H, CHOH), 7.41 (m, 5H, Ar-H), 7.65 (m, 4H, Ar-H), 8.43 (s, 1H, OH), 11.14 (s, 1H, OH). ¹³C-NMR (DMSO-d6) δ 24.16 (-, CH2), 25.19 (-, CH2), 28.84 (-, CH2), 33.28 (-, COCH2), 64.41 (-, CH2O), 65.39 (+, CHO), 69.41 (-, CH2O), 71.39 (-, CH2C6H4), 74.94 (+, CH), 118.09 (quat, C-2), 126.47 (+, C), 126.53 (+, C), 127.30 (+, Ar-C), 127.94 (+, Ar-Ar-C), 128.85 (+, Ar-Ar-C), 137.85 (quat, Ar-Ar-C), 139.11 (quat, Ar-Ar-C), 139.84 (quat, Ar-C), 152.13 (quat, C-3), 170.29 (quat, lactone CO), 172.64 (quat, CO2CH2). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 455 (100) [M-H]^-. Anal.

(C25H28O8) C, H. C25H28O8 (456.49).

6-O-{6-[4-(Thiophen-3-yl)phenoxy]hexanoyl}ascorbic acid (7.56): The title compound was prepared from ascorbic acid (1.7 mmol, 0.30 g), 7.17 (1.5 eq, 2.6 mmol, 0.78 g) and Novozyme 435^®(85 mg) in 15 ml tert-amyl alcohol according to general procedure 7.6.2.2, yielding 7.56 as a white solid (0.40 g, 26 %). mp: 118-120

°C; ¹H-NMR (DMSO-d6) δ 1.44 (m, 2H, COCH2CH2CH2), 1.67 (m, 4H, COCH2CH2, CH2CH2OC6H4), 2.37 (t, 2H, ³J = 7.3 Hz, COCH2CH2), 4.03 (m, 5H, CH2O, CHOH, CH2OC6H4), 4.70 (d, 1H, ³J = 1.5 Hz, H-5), 5.34 (d, 1H, ³J = 4.6 Hz, CHOH), 6.95 (m, 2H, Ar-H), 7.50 (m, 1H, Ar-H), 7.62 (m, 3H, Ar-H), 7.72 (m, 1H, Ar-H), 8.43 (s, 1H, OH), 11.15 (s, 1H, OH). ¹³C-NMR (DMSO-d6) δ 24.09 (-, CH2), 25.01 (-, CH2), 28.31 (-, CH2), 33.26 (-, COCH2CH2), 64.43 (-, CH2O), 65.39 (+, CHO), 67.22 (-, CH2OC6H4), 74.94 (+, CH), 114.62 (+, Ar-C), 118.09 (quat, C-2), 119.11 (+, Ar-C), 125.94 (+, Ar-C), 126.74 (+, Ar-C), 127.14 (+, Ar-C), 127.68 (quat, Ar-C), 141.10 (quat, Ar-C), 152.14 (quat, C-3), 157.76 (quat, Ar-C), 170.30 (quat, lactone CO), 172.63 (quat, CO2CH2). ES-MS (MeCN + TFA) m/z (%): 449 (100) [M+H]⁺. Anal.

(C22H24O8S ·0.3H2O) C, H. C22H24O8S (448.49).

6-O-{6-[4-(2-Methoxyphenyl)phenyloxy]hexanoyl}ascorbic acid (7.57): The title compound was prepared from ascorbic acid (1.6 mmol, 0.28 g), 7.19 (1.5 eq, 2.4 mmol, 0.79 g) and Novozyme 435^®(80 mg) in 15 ml tert-amyl alcohol according to general procedure 7.6.2.2, yielding 7.57 after preparative HPLC (MeCN/0.1 % TFA (aq) 55/45 v/v), as a white solid (30 mg, 4 %). mp: 84-85 °C; ¹H-NMR (CD3OD) δ 1.53 (m, 2H, COCH2CH2CH2-), 1.76 (m, 4H, COCH2CH2, CH2CH2OC6H4), 2.42 (t, 2H, ³J = 7.3 Hz, COCH2CH2), 3.76 (s, 3H, OCH3), 3.99 (t, 2H, ³J = 6.3 Hz, CH2OC6H4), 4.07 – 4.30 (m, 3H, CH2O, CHOH), 4.73 (d, 1H, ³J = 2.0 Hz, H-5), 6.96 (m, 4H, Ar-H), 7.24 (m, 2H, Ar-H), 7.38 (m, 2H, Ar-H). ¹³C-NMR (CD3OD) δ 25.83 (-, CH2), 26.82 (-, CH2), 30.18 (-, CH2), 34.90 (-, COCH2CH2), 56.06 (+, OCH3), 65.95 (-,

Chapter 7 Ascorbic acid derivatives as potent hyaluronidase inhibitors

CH2O), 68.14 (+, CHO), 68.85 (-, CH2OC6H4), 77.29 (+, CH), 112.65 (+, Ar-C), 115.01 (+, Ar-C), 120.16 (quat, C-2), 121.96 (+, Ar-C), 129.29 (+, Ar-C), 131.54 (+, Ar-C), 131.63 (+, Ar-C), 131.82 (quat, Ar-C), 132.40 (quat, Ar-C), 154.08 (quat, C-3), 157.95 (quat, Ar-C), 159.53 (quat, Ar-C), 173.22 (quat, lactone CO), 175.11 (quat, CO2CH2). PI-EIMS (70 eV) m/z (%): 472 (3) [M^+·]. HRMS (PI-EIMS) calcd for C26H28O9 [M^+·], 472.1733; found, 472.1746. Anal. (C25H28O9) C, H. C25H28O9 (472.48).

6-O-{6-[4-(4-Methylphenyl)phenyloxy]hexanoyl}ascorbic acid (7.58): The title compound was prepared from ascorbic acid (1.7 mmol, 0.30 g), 7.20 (1.5 eq, 2.6 mmol, 0.81 g) and Novozyme 435^®(85 mg) in 15 ml tert-amyl alcohol according to general procedure 7.6.2.2, yielding 7.58 as a white solid (0.39 g, 50 %). mp: 147-159 °C; ¹H-NMR (DMSO-d6) δ 1.44 (m, 2H, COCH2CH2CH2), 1.68 (m, 4H, COCH2CH2, CH2CH2OC6H4), 2.32 (s, 3H, CH3), 2.37 (t, 2H, ³J = 7.3 Hz, COCH2CH2), 4.04 (m, 5H, CH2O, CHOH, CH2OC6H4), 4.70 (d, 1H, ³J = 1.6 Hz, H-5), 5.34 (d, 1H,

3J = 2.8 Hz, CHOH), 6.98 (m, 2H, Ar-H), 7.23 (m, 2H, Ar-H), 7.52 (m, 4H, Ar-H), 8.42 (s, 1H, OH), 11.14 (s, 1H, OH). ¹³C-NMR (DMSO-d6) δ 20.55 (+, CH3), 24.08 (-, CH2), 25.01 (-, CH2), 28.30 (-, CH2), 33.27 (-, COCH2CH2), 64.42 (-, CH2O), 65.40 (+, CHO), 67.24 (-, CH2OC6H4), 74.94 (+, CH), 114.72 (+, Ar-C), 118.09 (quat, C-2), 125.88 (+, Ar-C), 127.36 (+, Ar-C), 129.36 (+, Ar-C), 132.25 (quat, Ar-C), 135.74 (quat, Ar-C), 136.89 (quat, Ar-C), 152.13 (quat, C-3), 157.95 (quat, Ar-C), 170.29 (quat, lactone CO), 172.63 (quat, CO2CH2). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 455 (100) [M-H]^-. Anal. (C25H28O8) C, H. C25H28O8 (456.49).

6-O-{6-[4-(2-Methylphenyl)phenyloxy]hexanoyl}ascorbic acid (7.59): The title compound was prepared from ascorbic acid (1.5 mmol, 0.26 g), 7.21 (1.5 eq, 2.3 mmol, 0.71 g) and Novozyme 435^®(75 mg) in 15 ml tert-amyl alcohol according to general procedure 7.6.2.2, yielding 7.59 as a white solid (0.30 g, 44 %). mp: 97-99 °C; ¹H-NMR (DMSO-d6) δ 1.45 (m, 2H, COCH2CH2CH2), 1.68 (m, 4H, COCH2CH2, CH2CH2OC6H4), 2.22 (s, 3H, C6H4CH3), 2.38 (t, 2H, ³J = 7.3 Hz, COCH2CH2), 4.03 (m, 5H, CH2O, CHOH, CH2OC6H4-), 4.70 (d, 1H, ³J = 1.5 Hz, H-5), 5.35 (bs, 1H, CHOH), 6.97 (m, 2H, Ar-H), 7.21 (m, 6H, Ar-H), 8.43 (s, 1H, OH), 11.15 (s, 1H, OH). ¹³C-NMR (DMSO-d6) δ 20.21 (+, CH3), 24.09 (-, CH2), 25.04 (-, CH2), 28.35 (-, CH2), 33.28 (-, CH2OC6H4), 64.43 (-, CH2O), 65.41 (+, CHO), 67.18 (-, CH2OC6H4), 74.94 (+, CH), 114.01 (+, Ar-C), 118.09 (quat, C-2), 125.81 (+, Ar-C), 126.82 (+, Ar-C), 129.46 (+, Ar-C), 129.96(+, Ar-C), 130.20 (+, Ar-C), 133.28 (quat, C), 134.64 (quat, C), 140.88 (quat, C), 152.13 (quat, C-3), 157.53 (quat,

Ar-C), 170.29 (quat, lactone CO), 172.63 (quat, CO2CH2). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 455 (100) [M-H]^-. Anal. (C25H28O8) C, H. C25H28O8 (456.49).

6-O-{6-[4-(4-Hydroxyphenyl)phenyloxy]hexanoyl}ascorbic acid (7.60): The title compound was prepared from ascorbic acid (1.6 mmol, 0.28 g), 7.22 (1.5 eq, 2.4 mmol, 0.75 g) and Novozyme 435^®(80 mg) in 15 ml tert-amyl alcohol according to general procedure 7.6.2.2, yielding 7.60 as a white solid (0.40 g, 55 %). mp: 158-161 °C; ¹H-NMR (DMSO-d6) δ 1.44 (m, 2H, COCH2CH2CH2), 1.67 (m, 4H, COCH2CH2, CH2CH2OC6H4), 2.37 (t, 2H, ³J = 7.3 Hz, COCH2CH2), 4.03 (m, 5H, CH2O, CHOH, CH2OC6H4), 4.70 (d, 1H, ³J = 1.5 Hz, H-5), 5.35 (bs, 1H, CHOH), 6.81 (m, 2H, Ar-H), 6.95 (m, 2H, Ar-H), 7.44 (m, 4H, Ar-H), 8.44 (bs, 1H, OH), 9.45 (s, 1H, C6H4OH), 11.15 (s, 1H, -OH). ¹³C-NMR (DMSO-d6) δ 24.08 (-, CH2), 25.01 (-, CH2), 28.32 (-, CH2), 33.27 (-, COCH2CH2), 64.42 (-, CH2O), 65.40 (+, CHO), 67.20 (-, CH2OC6H4), 74.94 (+, CH), 114.64 (+, Ar-C), 115.53 (+, Ar-C), 118.09 (quat, C-2), 126.89 (+, Ar-C), 127.10 (+, Ar-C), 130.65 (quat, Ar-C), 132.52 (quat, Ar-C), 152.13 (quat, C-3), 156.38 (quat, Ar-C), 157.39 (quat, Ar-C), 170.29 (quat, lactone CO), 172.63 (quat, CO2CH2). ES-MS (MeCN + TFA) m/z (%): 459 (100) [M+H]⁺. Anal.

(C24H26O9) C, H. C24H26O9 (458.46).

6-O-{6-[4-(3-Hydroxyphenyl)phenyloxy]hexanoyl}ascorbic acid (7.61): The title compound was prepared from ascorbic acid (1.5 mmol, 0.26 g), 7.23 (1.5 eq, 2.3 mmol, 0.71 g) and Novozyme 435^®(75 mg) in 15 ml tert-amyl alcohol according to general procedure 7.6.2.2, yielding 7.61 as a white solid (0.30 g, 44 %). mp: 143-144 °C; ¹H-NMR (DMSO-d6) δ 1.45 (m, 2H, COCH2CH2CH2), 1.68 (m, 4H, COCH2CH2, CH2CH2OC6H4), 2.37 (t, 2H, ³J = 7.3 Hz, COCH2CH2), 4.04 (m, 5H, CH2O, CHOH, CH2OC6H4), 4.70 (d, 1H, ³J = 1.6 Hz, H-5), 5.33 (bs, 1H, CHOH), 6.70 (m, 1H, Ar-H), 6.98 (m, 4H, Ar-H), 7.21 (m, 1H, Ar-H), 7.50 (m, 2H, Ar-H), 8.42 (s, 1H, OH), 9.45 (s, 1H, C6H4OH), 11.13 (s, 1H, OH). ¹³C-NMR (DMSO-d6) δ 24.08 (-, CH2), 25.01 (-, CH2), 26.76 (-, CH2), 28.30 (-, CH2), 33.28 COCH2CH2), 64.43 (-, CH2O), 65.42 (+, CHO), 67.26 (-, CH2OC6H4), 74.95 (+, CH), 112.88 (+, Ar-C), 113.60 (+, Ar-C), 114.69 (+, Ar-C), 116.87 (+, Ar-C), 118.11 (quat, C-2), 127.52 (+, Ar-C), 129.73 (+, Ar-C), 132.46 (quat, Ar-C), 141.20 (quat, Ar-C), 152.12 (quat, C-3), 157.66 (quat, Ar-C), 158.14 (quat, Ar-C), 170.28 (quat, lactone CO), 172.62 (quat, CO2CH2). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 457 (100) [M-H]^-. Anal.

(C24H26O9) C, H. C24H26O9 (458.46).

Chapter 7 Ascorbic acid derivatives as potent hyaluronidase inhibitors

6-O-(11-Phenoxyundecanoyl)ascorbic acid (7.62): The title compound was prepared from ascorbic acid (3.3 mmol, 0.58 g), 7.33 (1.5 eq, 5.0 mmol, 1.45 g) and Novozyme 435^®(165 mg) in 15 ml tert-amyl alcohol according to general procedure 7.6.2.2, yielding 7.62 as a white solid (0.69 g, 50 %). mp: 100-103 °C; ¹H-NMR (DMSO-d6) δ 1.19 – 1.58 (m, 14H, COCH2(CH2)7), 1.69 (m, 2H, CH2CH2OPh), 2.32 (t, 2H, ³J = 7.4Hz, COCH2CH2), 3.91 – 4.11 (m, 5H, CH2O, CHOH, CH2OPh), 4.68 (d, 1H, ³J = 1.6 Hz, H-5), 5.32 (d, 1H, ³J = 6.4 Hz, CHOH), 6.90 (m, 3H, Ph-H), 7.27 (m, 2H, Ph-H), 8.42 (s, 1H, OH), 11.13 (s, 1H, OH). ¹³C-NMR (DMSO-d6) δ 24.30 (-, CH2), 25.45 (-, CH2), 28.38 (-, CH2), 28.62 (-, CH2), 28.68 (-, CH2), 28.77 (-, CH2), 28.87 (-, CH2), 33.30 (-, COCH2CH2), 64.37 (-, CH2O), 65.39 (+, CHO), 67.12 (-, CH2OC6H4), 74.92 (+, CH), 114.27 (+, Ph-C), 118.09 (quat, C-2), 120.22 (+, Ph-C), 129.35 (+, Ph-C), 152.10 (quat, C-3), 158.55 (quat, Ph-C), 170.27 (quat, lactone CO), 172.66 (quat, CO2CH2). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 437 (100) [M+H]⁺. Anal. (C23H32O8) C, H. C23H32O8 (436,50).

6-O-[11-(4-Phenylphenoxy)undecanoyl]ascorbic acid (7.63): The title compound was prepared from ascorbic acid (3.0 mmol, 0.53 g), 7.34 (1.5 eq, 4.5 mmol, 1.66 g) and Novozyme 435^® (150 mg) in 15 ml tert-amyl alcohol according to general procedure 7.6.2.2, yielding 7.63 as a white solid (0.40 g, 26 %). mp: 148-149 °C; ¹ H-NMR (DMSO-d6) δ 1.26 – 1.55 (m, 14H, COCH2(CH2)7), 1.72 (m, 2H, CH2CH2OC6H4), 2.32 (t, 2H, ³J = 7.4 Hz, COCH2CH2), 4.03 (m, 5H, CH2O, CHOH, CH2OC6H4), 4.68 (d, 1H, ³J = 1.6 Hz, H-5), 5.33 (bs, 1H, CHOH), 7.00 (m, 2H, Ar-H), 7.30 (m, 1H, Ph-H), 7.42 (m, 2H, Ar-H), 7.59 (m, 4H, Ar-H), 8.43 (s, 1H, OH), 11.14 (s, 1H, OH). ¹³C-NMR (DMSO-d6) δ 24.31 (-, CH2), 25.45 (-, CH2), 28.39 (-, CH2), 28.63 (-, CH2), 28.70 (-, CH2), 28.78 (-, CH2), 28.89 (-, CH2), 33.30 (-, COCH2CH2), 64.37 (-, CH2O), 65.39 (+, CHO), 67.38 (-, CH2OC6H4), 74.93 (+, CH), 114.75 (+, Ar-C), 118.08 (quat, C-2), 126.04 (+, Ar-Ar-C), 126.57 (+, Ar-Ar-C), 127.63 (+, Ar-Ar-C), 128.76 (+, Ar-C), 132.26 (quat, Ar-C), 139.76 (quat, Ar-C), 152.11 (quat, C-3), 158.23 (quat, Ar-C), 170.28 (quat, lactone CO), 172.67 (quat, CO2CH2). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 513 (30) [M+H]⁺. Anal. (C29H36O8) C, H. C29H36O8 (512,59).

5,6-O-Isopropylidene-L-ascorbic acid (7.64)³⁹: To L-ascorbic acid (100 g, 568 mmol), suspended in absolute acetone (1 l), was added acetyl chloride (8.0 ml, 113 mmol) under vigorous stirring. After stirring overnight at room temperature, the solid was collected by filtration, washed with EtOAc and dried over potassium carbonate to yield the title compound as white solid (89,85 g, 73 %). mp: 193-194 °C

(ref.⁹⁵: 201-203 °C); ¹H-NMR (DMSO-d6) δ 1.26 (s, 6H, C(CH3)2), 3.88 (dd, 1H, ³J = 6.3 Hz, ²J = 8.4 Hz, CH2O), 4.10 (dd, 1H, ³J = 7.1 Hz, ²J = 8.3 Hz, CH2O), 4.26 (ddd, 1H, ³J = 7.1 Hz, ³J = 5.6 Hz, ³J = 2.5 Hz, CHO), 4.71 (d, 1H, ³J = 2.9 Hz, H-5), 8.48 (bs, 1H, OH), 11.30 (s, 1H, OH). C9H12O6 (216.19).

7.6.2.8 General procedure for the synthesis of 2,3-di-O-alkylated 5,6-O-isopropylidene-L-ascorbic acid derivatives 65 and 129

A solution of 5,6-O-isopropylidene-L-ascorbic acid (7.64) (1 eq), potassium carbonate (1.1 eq) and the pertinent alkyl halide (2.2 eq) in anhydrous DMF was heated under stirring at 40 °C for 4 h. The solvent was removed under reduced pressure, the oily residue was taken up in water and extracted tree times with dichloromethane. The combined organic phases were dried over magnesium sulfate and evaporated in vacuo. The remaining raw product was recrystallized or subjected to flash chromatography.

2,3-Di-O-benzyl-5,6-O-isopropylidene-L-ascorbic acid⁹⁶ (7.65): The title compound was prepared from 7.64 (100 mmol, 21.60 g), potassium carbonate (110 mmol, 15.20 g) and benzylbromide (220 mmol, 37.6 g) in anhydrous DMF (300 ml) according to general procedure 7.6.2.8. Recrystallization from methanol yielded a white solid (29.10 g, 73 %). mp: 116-118 °C (ref.⁴⁰: 123-125 °C); ¹H-NMR (DMSO-d6): δ 1.28 (s, 3H, C(CH3)2), 1.29 (s, 3H, C(CH3)2), 3.87 (dd, 1H, ²J = 8.5 Hz, ³J = 5.9 Hz, CH2O), 4.08 (dd, 1H, ²J = 8.5 Hz, ³J = 7.1 Hz, CH2O), 4.27 (ddd, 1H, ³J = 7.1 Hz,

3J = 5.9 Hz, ³J = 2.5 Hz, CHO), 4.94 (d, 1H, ²J = 11.2 Hz, CH2Ph), 4.95 (d, 1H, ³J = 2.5 Hz, H-5), 5.01 (d, 1H, ²J =11.2 Hz, CH2Ph), 5.16 (d, 1H, ²J = 11.8 Hz, CH2Ph), 5.25 (d, 1H, ²J = 11.8 Hz, CH2Ph), 7.27-7.43 (m, 10H, Ph-H). CI-MS (NH3) m/z (%):

414 (100) [M+NH4]⁺. C23H24O6 (396.43).

2,3-Di-O-benzyl-L-ascorbic acid⁹⁶ (7.66): A solution of 7.65 (50 mmol, 19.8 g), methanol (200 ml) and aqueous acetic acid (50 %, 500 ml) was stirred at 90 °C for 5 h. The solvent was evaporated and the oily yellow residue was dissolved in ethyl acetate (400 ml). After washing three times with a saturated solution of sodium carbonate, the organic layer was washed with water, dried over magnesium sulfate and evaporated under reduced pressure to obtain the title compound as a colorless oil which solidified over time (15.1 g, 85 %). mp: 65 °C (ref.⁴⁰: 67-69 °C); ¹H-NMR (DMSO-d6) δ 3.36-3.51 (m, 2H, CH2OH), 3.66-3.73 (m, 1H, CHOH), 4.88 (dd, 1H, ³J

= 6.1 Hz, ³J = 4.8 Hz, CH2OH), 4.90 (d, 1H, ³J = 1.4 Hz, H-5), 4.94 (d, 1H, ²J = 11.3

Chapter 7 Ascorbic acid derivatives as potent hyaluronidase inhibitors

Hz, CH2Ph), 4.98 (d, 1H, ²J = 11.3 Hz, CH2Ph), 5.15 (d, 1H, ³J = 6.3 Hz, CHOH), 5.20 (d, 1H, ²J = 11.5 Hz, CH2Ph), 5.26 (d, 1H, ²J = 11.5 Hz, CH2Ph), 7.29-7.45 (m, 10H, Ph-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 374 (100) [M+NH4]⁺. C20H20O6

(356.37).

7.6.2.9 General procedure for the synthesis of 6-O-acylated 2,3-di-O-protected ascorbic acids 67-83, 77a, 78a, 80a-83a, 104, 107-109, 114, 131 and 143-145

To a solution of the corresponding 2,3-di-O-protected ascorbic acid (1 eq), the pertinent carboxylic acid (1 eq) and DMAP (1.2 eq) in anhydrous DMF was added EDAC (1.1 eq) portion wise under an atmosphere of argon at 0 °C. The mixture was stirred overnight at room temperature and the solvent was removed under reduced pressure. The remaining raw product was taken up in water and extracted tree times with EtOAc. The combined organic layers were washed with 1 N HCl and brine, dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The acylated products were purified by flash chromatography.

2,3-Di-O-benzyl-6-O-(12-tert-butoxy-12-oxododecanoyl)-L-ascorbic acid (7.67):

The title compound was prepared from 7.66 (4.0 mmol, 1.42 g), 7.35 (4.0 mmol, 1.14 g), DMAP (4.4 mmol, 0.54 g) and EDAC (4.2 mmol, 0.80 g) in anhydrous DMF (15 ml) according to general procedure 7.6.2.9. Compound 7.67 was obtained after flash chromatography (PE/EtOAc 90/10 to 80/20 v/v) as a pale yellow oil (0.63 g, 25 %).

1H-NMR (CDCl3) δ 1.22 (m, 12H, COCH2CH2(CH2)6), 1.39 (s, 9H, C(CH3)3), 1.53 (m, 4H, COCH2CH2), 2.15 (t, 2H, ³J = 7.5 Hz, COCH2), 2.28 (t, 2H, ³J = 7.6 Hz, COCH2), 4.01 (ddd, 1H, ³J = 1.9 Hz, ³J = 5.1 Hz, ³J = 6.7 Hz, CHOH), 4.15 (dd, 1H, ³J = 5.0 Hz, ²J = 11.6 Hz, CH2O), 4.27 (dd, 1H, ³J = 6.8 Hz, ²J = 11.6 Hz, CH2O), 4.62 (d, 1H,

3J = 2.1 Hz, H-5), 5.04 (d, 2H, ²J = 11.5 Hz, CH2Ph), 5.08 (d, 1H, ²J = 11.6 Hz, CH2Ph), 5.17 (d, 1H, ²J = 11.7 Hz, CH2Ph), 7.17 (m, 2H, Ph-H), 7.32 (m, 8H, Ph-H).

ES-MS (DCM/MeOH + NH4OAc) m/z (%): 642 (100) [M+NH4]⁺. C36H48O9 (624.76).

2,3-Di-O-benzyl-6-O-[2-(decan-1-yloxy)benzoyl]-L-ascorbic acid (7.68): The title compound was prepared from 7.66 (2.0 mmol, 0.71 g), 7.39 (2.0 mmol, 0.56 g), DMAP (2.4 mmol, 0.29 g) and EDAC (2.2 mmol, 0.42 g) in anhydrous DMF (10 ml) according to general procedure 7.6.2.9. Compound 7.68 was obtained after flash chromatography (PE/EtOAc 90/10 to 70/30 v/v) as colorless oil (0.43 g, 35 %). ¹ H-NMR (CDCl3) δ 0.87 (t, 3H, ³J = 6.7 Hz, CH2CH3), 1.31 (m, 12H, (CH2)6CH3), 1.78 (m,

2H, OCH2CH2CH2), 4.00 (t, 1H, ³J = 6.8 Hz, COCH2CH2), 4.20 (m, 1H, CHOH), 4.42 (dd, 1H, ³J = 5.4 Hz, ²J = 11.4 Hz, CH2O), 4.52 (dd, 1H, ³J = 6.8 Hz, ²J = 11.4 Hz, CH2O), 4.79 (d, 1H, ³J = 2.1 Hz, H-5), 5.10 (s, 2H, CH2Ph), 5.13 (d, 1H, ²J = 11.9 Hz, CH2Ph), 5.22 (d, 1H, ²J = 11.7 Hz, CH2Ph), 6.95 (m, 2H, Ar-H) 7.20 (m, 2H, Ar-H), 7.39 (m, 9H, Ar-H), 7.79 (dd, 1H, ⁴J =1.8Hz, ³J =8.0Hz, Ar-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 617 (100) [M+H]⁺. Anal. (C37H44O8) C, H. C37H44O8 (616.74).

2,3-Di-O-benzyl-6-O-[3-(decan-1-yl)benzoyl]-L-ascorbic acid (7.69): The title compound was prepared from 7.66 (2.0 mmol, 0.71 g), 7.40 (2.0 mmol, 0.56 g), DMAP (2.4 mmol, 0.29 g) and EDAC (2.2 mmol, 0.42 g) in anhydrous DMF (10 ml) according to to general procedure 7.6.2.9. The title compound was obtained after flash chromatography (PE/EtOAc 90/10 to 70/30 v/v) as a colorless oil (0.39 g, 32 %). ¹H-NMR (CDCl3) δ 0.88 (t, 3H, ³J = 6.7 Hz, CH2CH3), 1.26 – 1.50 (m, 12H, (CH2)6CH3), 1.78 (m, 2H, OCH2CH2CH2), 3.98 (t, 2H, ³J = 6.5 Hz, COCH2CH2), 4.21 (m, 1H, CHOH), 4.45 (dd, 1H, ³J = 5.2 Hz, ²J = 11.6 Hz, CH2O), 4.55 (dd, 1H, ³J = 6.7 Hz, ²J = 11.6 Hz, CH2O), 4.76 (d, 1H, ³J = 2.1 Hz, H-5), 5.11 (s, 2H, CH2Ph), 5.13 (d, 1H, ²J = 12.7 Hz, CH2Ph), 5.22 (d, 1H, ²J = 11.7 Hz, CH2Ph), 7.21 (m, 1H, Ar-H), 7.35 (m, 9H, Ar-H), 7.53 (dd, 1H, ⁴J = 1.5 Hz, ⁴J = 2.4 Hz, Ar-H), 7.60 (m, 1H, Ar-H).

ES-MS (DCM/MeOH + NH4OAc) m/z (%): 634 (100) [M+NH4]⁺. Anal. (C37H44O8) C, H. C37H44O8 (616.74).

2,3-Di-O-benzyl-6-O-[2-(hexan-1-yl)decanoyl]-L-ascorbic acid (7.70): The title compound was prepared from 7.66 (2.0 mmol, 0.71 g), commercially available 2-hexyldecanoic acid (2.0 mmol, 0.59 ml), DMAP (2.4 mmol, 0.29 g) and EDAC (2.2 mmol, 0.42 g) in anhydrous DMF (10 ml) according to general procedure 7.6.2.9.

Compound 7.70 was obtained after flash chromatography (PE/EtOAc 90/10 v/v) as a white solid (0.6 g, 50 %). mp: 43 °C; ¹H-NMR (CDCl3) δ 0.87 (m, 6H, CH2CH3), 1.23 (m, 20H, (CH2)4CH3, (CH2)6CH3), 1.51 (m, 4H, COCHCH2), 2.35 (tt, 1H, ³J = 5.5 Hz,

3J = 8.7 Hz, COCH), 4.02 – 4.36 (m, 3H, CH2O, CHOH), 4.66 (d, 1H, ³J = 2.0 Hz, H-5), 5.10 (s, 2H, CH2Ph), 5.14 (d, 1H, ²J = 11.7 Hz, CH2Ph), 5.22 (d, 1H, ³J = 11.7 Hz, CH2Ph), 7.22 (m, 2H, Ph-H), 7.36 (m, 8H, Ph-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 613 (100) [M+NH4]⁺. Anal. (C36H50O7) C, H. C36H50O7 (594.78).

2,3-Di-O-benzyl-6-O-[2-(propan-1-yl)dodecanoyl]-L-ascorbic acid (7.71): The title compound was prepared from 7.66 (2.0 mmol, 0.71 g), 7.42 (2.0 mmol, 0.49 mg), DMAP (2.4 mmol, 0.29 g) and EDAC (2.2 mmol, 0.42 g) in anhydrous DMF (10 ml) according to general procedure 7.6.2.9. After flash chromatography (PE/EtOAc 80/20

Chapter 7 Ascorbic acid derivatives as potent hyaluronidase inhibitors

v/v) compound 7.71 was obtained as a colorless oil (0.49 g, 42 %). ¹H-NMR (CDCl3) δ 0.89 (m, 6H, CH2CH3), 1.18 – 1.68 (m, 22H, (CH2)9CH3, (CH2)2CH3), 2.37 (m, 1H, COCH), 4.05 (ddd, 1H, ³J = 1.6 Hz, ³J = 4.8 Hz, ³J = 6.6 Hz, CHOH), 4.17 (ddd, 1H,

3J = 2.9 Hz, ³J = 5.1 Hz, ²J = 11.6 Hz, CH2O), 4.32 (ddd, 1H, ³J = 3.4 Hz, ³J = 6.9 Hz,

2J = 11.4 Hz, CH2O), 4.65 (d, 1H, ³J = 2.1 Hz, H-5), 5.07 – 5.25 (m, 4H, CH2Ph), 7.22 (m, 2H, Ph-H), 7.36 (m, 8H, Ph-H).ES-MS (DCM/MeOH + NH4OAc) m/z (%): 598 (100) [M+NH4]⁺. Anal. (C35H48O7) C, H. C35H48O7 (580.75).

2,3-Di-O-benzyl-6-O-[2-(hexan-1-yl)dodecanoyl]-L-ascorbic acid (7.72): The title compound was prepared from 7.66 (2.0 mmol, 0.71 g), 7.43 (2.0 mmol, 0.49 mg), DMAP (2.4 mmol, 0.29 g) and EDAC (2.2 mmol, 0.42 g) in anhydrous DMF (10 ml) according to general procedure 7.6.2.9. Flash chromatography (PE/EtOAc 80/20 v/v) yielded compound 7.72 as a colorless oil (0.52 g, 42 %). ¹H-NMR (CDCl3) δ 0.86 (m, 6H, CH2CH3), 1.24 – 1.65 (m, 28H, (CH2)9CH3, (CH2)5CH3), 2.35 (m, 1H, COCH), 4.06 (ddd, 1H, ³J = 1.9 Hz, ³J = 4.5 Hz, ³J = 7.1 Hz, CHOH), 4.18 (ddd, 1H, ³J = 1.3 Hz, ³J = 5.3 Hz, ²J = 11.5 Hz, CH2O), 4.33 (ddd, 1H, ³J = 1.5 Hz, ³J = 6.9 Hz, ²J = 11.5 Hz, CH2O), 4.66 (d, 1H, ³J = 2.1 Hz, H-5), 5.10 (s, 2H, CH2Ph), 5.14 (d, 1H, ²J = 11.7 Hz, CH2Ph), 5.22 (d, 1H, ²J = 11.7 Hz, CH2Ph), 7.21 (m, 2H, Ph-H), 7.36 (m, 8H, Ph-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 640 (100) [M+NH4]⁺. Anal.

(C38H54O7) C, H. C38H54O7 (622.83).

2,3-Di-O-benzyl-6-O-[2-(decan-1-yl)dodecanoyl]-L-ascorbic acid (7.73): The title compound was prepared from 7.66 (2.0 mmol, 0.71 g), 7.44 (2.0 mmol, 0.68 mg), DMAP (2.4 mmol, 0.29 g) and EDAC (2.2 mmol, 0.42 g) in anhydrous DMF (10 ml) according to general procedure 7.6.2.9. Flash chromatography (PE/EtOAc 90/10 v/v) gave compound 7.73 as a pale yellow oil (0.45 g, 33 %). ¹H-NMR (CDCl3) δ 0.88 (t, 6H, ³J = 6.7 Hz, CH2CH3), 1.24 – 1.65 (m, 36H, (CH2)9CH3), 2.35 (m, 1H, COCH), 4.06 (ddd, 1H, ³J = 2.0 Hz, ³J = 5.3 Hz, ³J = 7.1 Hz, CHOH), 4.18 (dd, 1H, ³J = 5.3 Hz, ²J = 11.5 Hz, CH2O), 4.32 (dd, 1H, ³J = 7.0 Hz, ²J = 11.5 Hz, CH2O), 4.66 (d, 1H,

3J = 2.0 Hz, H-5), 5.10 (s, 2H, CH2Ph), 5.14 (d, 1H, ²J = 11.7 Hz, CH2Ph), 5.22 (d, 1H, ²J = 11.7 Hz, CH2Ph), 7.22 (m, 2H, Ar-H), 7.36 (m, 8H, Ar-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 697 (100) [M+NH4]⁺. C42H62O7 (678.94).

2,3-Di-O-benzyl-6-O-[2-(2-phenylethyl)dodecanoyl]-L-ascorbic acid (7.74): The title compound was prepared from 7.66 (2.0 mmol, 0.71 g), 7.45 (2.0 mmol, 0.61 mg), DMAP (2.4 mmol, 0.29 g) and EDAC (2.2 mmol, 0.42 g) in anhydrous DMF (10 ml) according to general procedure 7.6.2.9. Flash chromatography (PE/EtOAc 90/10 to

80/20 v/v) yielded 7.74 as a colorless oil (0.30 g, 23 %). ¹H-NMR (CDCl3) δ 0.87 (t, 3H, ³J = 6.7 Hz, CH2CH3), 1.23 – 2.02 (m, 20H, CH2), 2.40 (m, 1H, COCH), 2.58 (m, 2H, CH2CH2Ph), 4.02 – 4.36 (m, CH2O, CHOH), 4.65 (m, 1H, H-5), 5.09 (d, 1H, ²J = 11.4 Hz, OCH2Ph), 5.13 (d, 1H, ²J = 11.5 Hz, OCH2Ph), 5.15 (d, 1H, ²J = 11.7 Hz, OCH2Ph), 5.22 (d, 1H, ²J = 11.7 Hz, OCH2Ph), 7.26 (m, 15H, Ph-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 661 (100) [M+NH4]⁺. Anal. (C40H50O7) C, H.

C40H50O7 (642.82).

2,3-Di-O-benzyl-6-O-[2-(4-methoxybenzyl)dodecanoyl]-L-ascorbic acid (7.75):

The title compound was prepared from 7.66 (1.0 mmol, 0.36 g), 7.46 (1.0 mmol, 0.32 mg), DMAP (1.2 mmol, 0.15 g) and EDAC (1.1 mmol, 0.21 g) in anhydrous DMF (5 ml) according to general procedure 7.6.2.9. Flash chromatography (PE/EtOAc 85/15 to 75/35 v/v) yielded 7.75 as a colorless oil (0.26 g, 39 %). ¹H-NMR (CDCl3) δ 0.88 (t, 3H, ³J = 6.7 Hz, CH2CH3), 1.20 – 1.69 (m, 18H, (CH2)9CH3), 2.73 (m, 3H, COCHCH2C6H4), 3.73 – 4.68 (m, 7H, OCH3, CH2O, CHOH, H-5), 5.16 (m, 4H, CH2Ph), 6.77 (m, 2H, Ar-H), 7.05 (m, 2H, Ar-H), 7.22 (m, 2H, H), 7.36 (m, 8H, Ph-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 676 (100) [M+NH4]⁺. Anal.

(C40H50O8·0.3H2O) C, H. C40H50O8 (658.82).

2,3-Di-O-benzyl-6-O-[2-(hexan-1-yl)octadecanoyl]-L-ascorbic acid (7.76): The title compound was prepared from 7.66 (1.5 mmol, 0.54 g), 7.47 (1.5 mmol, 0.55 mg), DMAP (1.8 mmol, 0.22 g) and EDAC (1.65 mmol, 0.32 g) in anhydrous DMF (8 ml) according to general procedure 7.6.2.9. Compound 7.76 was obtained after flash chromatography (PE/EtOAc 90/10 to 80/20 v/v) as a white solid (0.29 g, 27 %). mp:

53-54 °C; ¹H-NMR (CDCl3) δ 0.87 (m, 6H, CH2CH3), 1.24 – 1.65 (m, 40H, (CH2)15CH3, (CH2)5CH3), 2.35 (tt, 1H, ³J = 5.5 Hz, ³J = 8.6 Hz, COCH), 4.05 (ddd, 1H,

3J = 2.0 Hz, ³J = 5.3 Hz, ³J = 7.1 Hz, CHOH), 4.18 (ddd, 1H, ³J = 1.4 Hz, ³J = 5.3 Hz,

2J = 11.5 Hz, CH2O), 4.32 (ddd, 1H, ³J = 1.6 Hz, ³J = 7.0 Hz, ²J = 11.5 Hz, CH2O), 4.66 (d, 1H, ³J = 2.1 Hz, H-5), 5.10 (s, 2H, CH2Ph), 5.15 (d, 1H, ²J = 11.7 Hz, CH2Ph), 5.23 (d, 1H, ²J = 11.7 Hz, CH2Ph), 7.22 (m, 2H, Ph-H), 7.36 (m, 8H, Ph-H).

ES-MS (DCM/MeOH + NH4OAc) m/z (%): 725 (100) [M+NH4]⁺. Anal. (C44H66O7) C, H. C44H66O7 (706.99).

2,3-Di-O-benzyl-6-O-{6-[4-(4-methoxyphenyl)phenoxy]hexanoyl}-L-ascorbic acid (7.77): The title compound was prepared from 7.66 (2.0 mmol, 0.71 g), 7.29 (2.0 mmol, 0.63 g), DMAP (2.4 mmol, 0.29 g) and EDAC (2.2 mmol, 0.42 g) in anhydrous DMF (15 ml) according to general procedure 7.6.2.9. Compound 7.77 was obtained

Chapter 7 Ascorbic acid derivatives as potent hyaluronidase inhibitors

after flash chromatography (PE/EtOAc 80/20 to 60/40 v/v) as a white solid (0.39 g, 30 %). ¹H-NMR (CDCl3) δ 1.51 (m, 2H, COCH2CH2CH2), 1.76 (m, 4H, COCH2CH2, CH2CH2OCH2), 2.38 (t, 2H, ³J = 7.4 Hz, COCH2CH2), 3.83 (s, 3H, OCH3), 3.98 (t, 2H,

3J = 6.3 Hz, CH2OC6H4), 4.14 (m, 2H, CH2O, CHOH), 4.33 (dd, 1H, ³J = 6.7 Hz, ²J = 11.6 Hz, CH2O), 4.67 (d, 1H, ³J = 2.0 Hz, H-5), 5.09 (s, 2H, CH2Ph), 5.09 (d, 1H, ²J = 12.8 Hz, CH2Ph), 5.20 (d, 1H, ²J = 11.7 Hz, CH2Ph), 6.94 (m, 4H, Ar-H), 7.21 (m, 2H, Ar-H), 7.35 (m, 8H, Ar-H) 7.46 (m, 4H, Ar-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 670 (100) [M+NH4]⁺. Anal. (C39H40O9) C, H. C39H40O9 (652.73).

2,3-Di-O-benzyl-5,6-O-bis{6-[4-(4-methoxyphenyl)phenoxy]hexanoyl}-L-ascorbic acid (7.77a): Compound 7.77a was obtained as a byproduct in the synthesis of 7.77 as a colorless oil (0.31 g, 16 %). ¹H-NMR (CDCl3) δ 0.88 (t, 6H, ³J = 6.7 Hz, CH2CH3), 1.27 – 1.49 (m, 24H, (CH2)6CH3), 1.78 (m, 4H, OCH2CH2CH2), 3.99 (m, 4H, COCH2CH2), 4.62 (m, 2H, CH2O), 4.82 (d, 1H, ²J = 11.2 Hz, CH2Ph), 4.99 (m, 2H, H-5, CH2Ph), 5.08 (d, 1H, ²J = 11.1 Hz, CH2Ph), 5.14 (d, 1H, ²J = 11.4 Hz, CH2Ph), 5.77 (ddd, 1H, ³J = 1.8 Hz, ³J = 6.4 Hz, ³J = 6.0 Hz, CHO), 6.88 (m, 4H, Ar-H), 7.24 (m, 10H, Ar-Ar-H), 7.93 (m, 4H, Ar-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 895 (100) [M+NH4]⁺. C54H68O10 (877.11).

2,3-Di-O-benzyl-6-O-{6-[4-(4-naphthalen-1-yl)phenoxy]hexanoyl}-L-ascorbic acid (7.78): The title compound was prepared from 7.66 (0.67 mmol, 0.24 g), 7.30 (0.67 mmol, 0.22 g), DMAP (0.80 mmol, 0.10 g) and EDAC (0.73 mmol, 0.14 g) in anhydrous DMF (5 ml) according to general procedure 7.6.2.9. Compound 7.78 was obtained after flash chromatography (PE/EtOAc 80/20 to 60/40 v/v) as a pale yellow oil (0.16 g, 35 %). ¹H-NMR (CDCl3) δ 1.55 (m, 2H, COCH2CH2CH2), 1.80 (m, 4H, COCH2CH2, CH2CH2OC6H4), 2.41 (t, 2H, ³J = 7.4 Hz, COCH2CH2), 4.04 (m, 3H, CH2OC6H4, CHOH), 4.23 (dd, 1H, ³J = 4.9 Hz, ²J = 11.6 Hz, CH2O), 4.35 (dd, 1H, ³J

= 6.8 Hz, ²J = 11.6 Hz, CH2O), 4.68 (d, 1H, ³J = 2.1 Hz, H-5), 5.12 (m, 3H, CH2Ph), 5.22 (d, 1H, ²J = 11.6 Hz, CH2Ph), 7.01 (m, 2H, Ar-H), 7.22 (m, 2H, Ar-H), 7.32 – 7.53 (m, 14H, Ar-H), 7.88 (m, 3H, Ar-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%):

690 (100) [M+NH4]⁺. Anal. (C42H40O8·0.2H2O) C, H. C42H40O8 (672.76).

2,3-Di-O-benzyl-5,6-O-bis{6-[4-(4-napthalen-1-yl)phenoxy]hexanoyl}-L-ascorbic acid (7.78a): Compound 7.78a was obtained as byproduct in the synthesis of 7.78 as a pale yellow oil (0.11 g, 17 %). ¹H-NMR (CDCl3) δ 1.49 – 2.40 (m, 16H), 4.02 (m, 4H), 4.28 (dd, 1H, ³J = 7.0 Hz, ²J = 11.6 Hz, CH2O), 4.39 (dd, 1H, ³J = 5.5 Hz, ²J = 11.6 Hz, CH2O), 4.83 (d, 1H, ³J = 2.1 Hz, H-5), 5.14 (m, 4H, CH2Ph), 5.42 (ddd, 1H,

3J = 2.1 Hz, ³J = 5.6 Hz, ³J = 7.5 Hz, CHO), 6.99 (m, 4H, Ar-H), 7.24 (m, 2H, Ar-H), 7.44 (m, 22H, Ar-H), 7.88 (m, 4H, Ar-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%):

1007 (100) [M+NH4]⁺. Anal. (C64H60O10) calc C:77.81, H:6.11 found C:77.29 H:6.67.

C64H60O10 (989.16).

2,3-Di-O-benzyl-6-O-{6-[4-(4-tert-butoxycarbonylphenyl)phenoxy]hexanoyl}-L-ascorbic acid (7.79): The title compound was prepared from 7.66 (0.94 mmol, 0.33 g), 7.31 (0.94 mmol, 0.36 g), DMAP (1.12 mmol, 0.14 g) and EDAC (1.03 mmol, 0.20 g) in anhydrous DMF (5 ml) according to general procedure 7.6.2.9. Compound 7.79 was obtained after flash chromatography (PE/EtOAc 80/20 to 70/30 v/v) as a white solid (0.21 g, 31 %). mp: 81-83 °C; ¹H-NMR (CDCl3) δ 1.55 (m, 11H, COCH2CH2CH2, C(CH3)3), 1.77 (m, 4H, COCH2CH2, CH2CH2OC6H4), 2.39 (t, 2H, ³J = 7.4 Hz, COCH2CH2), 4.00 (t, 2H, ³J = 6.3 Hz, CH2OC6H4), 4.06 (ddd, 1H, ³J = 2.0 Hz, ³J = 5.0 Hz, ³J = 6.8 Hz, CHOH), 4.21 (dd, 1H, ³J = 5.0 Hz, ²J = 11.6 Hz, CH2O), 4.34 (dd, 1H, ³J = 6.7 Hz, ²J = 11.6 Hz, CH2O), 4.67 (d, 1H, ³J = 2.1 Hz, H-5), 5.10 (s, 2H, CH2Ph), 5.12 (d, 1H, ²J = 11.9 Hz, CH2Ph), 5.20 (d, 1H, ²J = 11.7 Hz, CH2Ph), 6.97 (m, 2H, Ar-H), 7.21 (m, 2H, Ar-H), 7.36 (m, 8H, Ar-H), 7.56 (m, 4H, Ar-H), 8.02 (m, 2H, Ar-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 740 (100) [M+NH4]⁺. Anal.

(C43H46O10) C, H. C43H46O10 (722.82).

2,3-Di-O-benzyl-6-O-{6-[4-(3-tert-butoxycarbonylphenyl)phenoxy]hexanoyl}-L-ascorbic acid (7.80): The title compound was prepared from 7.66 (1.20 mmol, 0.43 g), 7.32 (1.20 mmol, 0.46 g), DMAP (1.44 mmol, 0.18 g) and EDAC (1.32 mmol, 0.25 g) in anhydrous DMF (5 ml) according to general procedure 7.6.2.9. Compound 7.80 was obtained after flash chromatography (PE/EtOAc 80/20 to 70/30 v/v) as a colorless oil (0.27 g, 31 %). ¹H-NMR (CDCl3) δ 1.53 (m, 2H, COCH2CH2CH2), 1.61 (s, 9H, C(CH3)3), 1.77 (m, 4H, COCH2CH2, CH2CH2OC6H4), 2.39 (t, 2H, ³J = 7.4 Hz, COCH2CH2), 4.00 (t, 2H, ³J = 6.3 Hz, CH2OC6H4), 4.07 (ddd, 1H, ³J = 2.0 Hz, ³J = 5.0 Hz, ³J = 6.8 Hz, CHOH), 4.21 (dd, 1H, ³J = 4.9 Hz, ²J = 11.6 Hz, CH2O), 4.34 (dd, 1H, ³J = 6.7 Hz, ²J = 11.6 Hz, CH2O), 4.67 (d, 1H, ³J = 2.1 Hz, H-5), 5.10 (s, 2H, CH2Ph), 5.12 (d, 1H, ²J = 12.0 Hz, CH2Ph), 5.21 (d, 1H, ²J = 11.7 Hz, CH2Ph) 6.96 (m, 2H, Ar-H), 7.21 (m, 2H, Ar-H), 7.43 (m, 11H, Ar-H), 7.70 (m, 1H, Ar-H), 7.92 (m, 1H, Ar-H), 8.17 (m, 1H, Ar-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 740 (100) [M+NH4]⁺. Anal. (C43H46O10) C, H. C43H46O10 (722.82).

2,3-Di-O-benzyl-5,6-O-bis{6-[4-(3-tert-butoxycarbonylphenyl)phenoxy]-hexanoyl}-L-ascorbic acid (7.80a): Compound 7.80a was obtained as a byproduct

Chapter 7 Ascorbic acid derivatives as potent hyaluronidase inhibitors

in the synthesis of 7.80 as a pale yellow oil (0.23 g, 18 %). ¹H-NMR (CDCl3) δ 1.45 – 1.85 (m, 30H, C(CH3)3, COCH2(CH2)3), 2.29 (m, 4H, COCH2CH2), 3.97 (m, 4H, CH2OC6H4), 4.26 (dd, 1H, ³J = 7.0 Hz, ²J = 11.5 Hz, CH2O), 4.36 (dd, 1H, ³J = 5.6 Hz, ³J = 11.5 Hz, CH2O), 4.81 (d, 1H, ³J = 2.1 Hz, H-5), 5.05 – 5.18 (m, 4H, CH2Ph), 5.40 (ddd, 1H, ³J = 2.1 Hz, ³J = 5.6 Hz, ³J = 7.4 Hz, CHO), 6.94 (m, 4H, Ar-H), 7.22 (m, 2H, Ar-H), 7.32 – 7.54 (m, 14H, Ar-H), 7.69 (m, 2H, Ar-H), 7.91 (m, 2H, Ar-H), 8.17 (m, 2H, Ar-H). ES-MS (MeOH + NH4OAc) m/z (%): 909 (100) [M+NH4]⁺. Anal.

(C66H72O14·0.2H2O) C, H. C66H72O14 (1089.27).

2,3-Di-O-benzyl-6-O-[6-(hexan-1-yloxy)hexanoyl]-L-ascorbic acid (7.81): The title compound was prepared from 7.66 (1.5 mmol, 0.53 g), 7.13 (1.5 mmol, 0.32 g), DMAP (1.8 mmol, 0.22 g) and EDAC (1.68 mmol, 0.32 g) in anhydrous DMF (10 ml) according to general procedure 7.6.2.9. Flash chromatography (PE/EtOAc 80/20 to 70/30 v/v) yielded compound 7.81 as a colorless oil (0.21 g, 25 %). ¹H-NMR (CDCl3) δ 0.88 (t, 3H, ³J = 6.8 Hz, CH2CH3), 1.32 (m, 8H, (CH2)3CH3, COCH2CH2CH2), 1.60 (m, 6H, COCH2CH2, CH2CH2OCH2CH2), 2.34 (t, 2H, ³J = 7.5 Hz, COCH2CH2), 3.38 (m, 4H, CH2OCH2), 4.05 (ddd, 1H, ³J = 2.0 Hz, ³J = 5.0 Hz, ³J = 6.8 Hz, CHOH), 4.20 (dd, 1H, ³J = 4.9 Hz, ²J = 11.6 Hz, CH2O), 4.31 (dd, 1H, ³J = 6.9 Hz, ²J = 11.6 Hz, CH2O), 4.66 (d, 1H, ³J = 2.1 Hz, H-5), 5.11 (s, 2H, CH2Ph), 5.13 (d, 1H, ²J = 11.7 Hz, CH2Ph), 5.21 (d, 1H, ²J = 11.7 Hz, CH2Ph), 7.21 (m, 2H, Ph-H), 7.36 (m, 8H, Ph-H).

ES-MS (DCM/MeOH + NH4OAc) m/z (%): 555 (100) [M+H]⁺. Anal. (C32H42O8) C, H.

C32H42O8 (554.67).

2,3-Di-O-benzyl-5,6-O-bis[6-(hexan-1-yloxy)hexanoyl]-L-ascorbic acid (7.81a):

The title compound was obtained as byproduct in the synthesis of 7.81 as a colorless oil (0.17 g, 15 %). ¹H-NMR (CDCl3) δ 0.88 (t, 6H, ³J = 6.7 Hz, CH2CH3), 1.32 (m, 16H, (CH2)3CH3, COCH2CH2CH2), 1.57 (m, 12H, COCH2CH2, CH2CH2OCH2CH2), 2.24 (m, 4H, COCH2CH2), 3.37 (m, 8H, CH2OCH2), 4.22 (dd, 1H, ³J = 7.1 Hz, ²J = 11.6 Hz, CH2O), 4.32 (dd, 1H, ³J = 5.7 Hz, ²J = 11.5 Hz, CH2O), 4.80 (d, 1H, ³J = 2.1 Hz, H-5), 5.07 – 5.18 (m, 4H, CH2Ph), 5.36 (ddd, 1H, ³J = 2.1 Hz, ³J = 5.7 Hz, ³J = 7.1 Hz, CHO), 7.23 (m, 2H, Ph-H), 7.36 (m, 8H, Ph-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 771 (100) [M+NH4]⁺. C44H64O10 (752.97).

2,3-Di-O-benzyl-6-O-[5-(heptan-1-yloxy)pentanoyl]-L-ascorbic acid (7.82): The title compound was prepared from 7.66 (1.5 mmol, 0.53 g), 7.14 (1.5 mmol, 0.32 g), DMAP (1.8 mmol, 0.22 g) and EDAC (1.68 mmol, 0.32 g) in anhydrous DMF (10 ml) according to general procedure 7.6.2.9. Flash chromatography (PE/EtOAc 80/20 to

70/30 v/v) yielded compound 7.82 as a colorless oil (0.22 g, 26 %). ¹H-NMR (CDCl3) δ 0.87 (t, 3H, ³J = 6.7 Hz, CH2CH3), 1.27 (m, 8H, (CH2)4CH3), 1.61 (m, 4H, CH2CH2OCH2CH2), 2.37 (t, 2H, ³J = 7.3 Hz, COCH2CH2), 3.39 (m, 4H, CH2OCH2), 4.05 (ddd, 1H, ³J = 1.9 Hz, ³J = 4.9 Hz, ³J = 6.8 Hz, CHOH), 4.20 (dd, 1H, ³J =4.9Hz,

2J =11.6Hz, CH2O), 4.32 (dd, 1H, ³J = 6.9 Hz, ²J = 11.6 Hz, CH2O), 4.67 (d, 1H, ³J = 2.1 Hz, H-5), 5.10 (s, 2H, CH2Ph), 5.12 (d, 1H, ²J = 11.7 Hz, CH2Ph), 5.21 (d, 1H, ²J

= 11.7 Hz, CH2Ph), 7.21 (m, 2H, Ph-H), 7.36 (m, 8H, Ph-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 572 (100) [M+NH4]⁺. Anal. (C32H42O8) C, H. C32H42O8 (554.67).

2,3-Di-O-benzyl-5,6-O-bis[5-(heptan-1-yloxy)pentanoyl]-L-ascorbic acid (7.82a):

The title compound was obtained as byproduct in the synthesis of 7.82 as a colorless oil (0.16 g, 12 %). ¹H-NMR (CDCl3) δ 0.87 (t, 6H, ³J = 6.2 Hz, CH2CH3), 1.28 (m, 16H, (CH2)4CH3), 1.60 (m, 8H, CH2CH2OCH2CH2), 2.28 (m, 4H, COCH2CH2), 3.38 (m, 8H, CH2OCH2), 4.22 (dd, 1H, ³J = 7.0 Hz, ²J = 11.5 Hz, CH2O), 4.33 (dd, 1H, ³J = 5.7 Hz, ²J = 11.5 Hz, CH2O), 4.80 (d, 1H, ³J = 2.1 Hz, H-5), 5.07 – 5.22 (m, 4H, CH2Ph), 5.36 (ddd, 1H, ³J = 2.1 Hz, ³J = 5.9 Hz, ³J = 7.7 Hz, CHO), 7.23 (m, 4H, Ph-H), 7.36 (m, 8H, Ph-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 771 (100) [M+NH4]⁺. Anal. (C44H64O10) C, H. C44H64O10 (752.97).

2,3-Di-O-benzyl-6-O-[4-(decan-1-yloxy)benzoyl]-L-ascorbic acid (7.83): The title compound was prepared from 7.66 (2.0 mmol, 0.71 g), 7.41 (2.0 mmol, 0.56 g), DMAP (2.4 mmol, 0.29 g) and EDAC (2.2 mmol, 0.42 g) in anhydrous DMF (10 ml) according to general procedure 7.6.2.9. Compound 7.83 was obtained after flash chromatography (PE/EtOAc 90/10 to 70/30 v/v) as a white solid (0.34 g, 28 %). mp:

78 °C; ¹H-NMR (CDCl3) δ 0.88 (t, 3H, ³J = 6.7 Hz, CH2CH3), 1.26 – 1.50 (m, 12H, (CH2)6CH3), 1.79 (m, 2H, OCH2CH2CH2), 3.99 (t, 2H, ³J = 6.6 Hz, COCH2CH2), 4.20 (m, 1H, CHOH), 4.42 (dd, 1H, ³J = 5.2 Hz, ²J = 11.6 Hz, CH2O), 4.53 (dd, 1H, ³J = 6.7 Hz, ²J = 11.6 Hz, CH2O), 4.75 (d, 1H, ³J = 2.1 Hz, H-5), 5.11 (s, 2H, CH2Ph), 5.13 (d, 1H, ²J = 12.3 Hz, CH2Ph), 5.22 (d, 1H, ²J = 11.7 Hz, CH2Ph), 6.89 (m, 2H, Ar-H), 7.21 (m, 2H, Ar-H), 7.35 (m, 8H, Ar-H), 7.96 (m, 2H, Ar-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 618 (100) [M+H]⁺. Anal. (C37H44O8) C, H. C37H44O8 (616.74).

2,3-Di-O-benzyl-5,6-O-bis[4-(decan-1-yloxy)benzoyl]-L-ascorbic acid (7.83a):

The title compound was obtained as byproduct in the synthesis of 7.83 as a colorless oil (0.18 g, 10 %). ¹H-NMR (CDCl3) δ 0.88 (t, 6H, ³J = 6.7 Hz, CH2CH3), 1.27 – 1.49 (m, 24H, (CH2)6CH3), 1.78 (m, 4H, OCH2CH2CH2), 3.99 (m, 4H, COCH2CH2), 4.62 (m, 2H, CH2O), 4.82 (d, 1H, ²J = 11.2 Hz, CH2Ph), 4.99 (m, 2H, H-5, CH2Ph), 5.08 (d,

Chapter 7 Ascorbic acid derivatives as potent hyaluronidase inhibitors

1H, ²J = 11.1 Hz, CH2Ph), 5.14 (d, 1H, ²J = 11.4 Hz, CH2Ph), 5.77 (ddd, 1H, ³J = 1.8 Hz, ³J = 6.4 Hz, ³J = 6.0 Hz, CHO), 6.88 (m, 4H, Ar-H), 7.24 (m, 10H, Ar-H), 7.93 (m, 4H, Ar-H). ES-MS (DCM/MeOH + NH4OAc) m/z (%): 895 (100) [M+NH4]⁺. C54H68O10

(877.11).

7.6.2.10 General procedure for the synthesis of carboxylic acids 84-86, 86a

Im Dokument Inhibitors of Bacterial and Mammalian Hyaluronidases: Design, Synthesis and Structure-Activity Relationships with Focus on Human Enzymes (Seite 167-186)