Synthesis and β-Glucuronidase Mediated Cleavage of an Alcohol Prodrug Incorporating a Double Spacer Moiety

Abstract

An alcohol prodrug has been prepared by incorporation of two spacer groups between the trigger (glucuronic acid) and nitroveratryl alcohol, used as a model. Release of the latter has been observed after hydrolysis of the glycoside by β-glucuronidase. Such prodrugs may find application in ADEPT or PMT protocols in cancer chemotherapy.

prodrug - spacer - glycoside - cleavage - enzyme

References

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16

Preparation of 8: 6-Nitroveratryl chloroformate (85 mg, 0.30 mmol) and pyridine (0.075 mL, 0.9 mmol) were added to a stirred solution of 7 (100 mg, 0.15 mmol) in CH₂Cl₂ (0.65 mL) at r.t. After 3 h the mixture was worked up with a saturated solution of NaHCO₃ and the aqueous layer was washed with CH₂Cl₂. The combined organic layers were dried (MgSO₄), filtered, concentrated to dryness and the remaining yellow solid was purified by flash chromatography (petroleum ether-EtOAc 1:1) to give 8 (116 mg, 0.13 mmol, 87%). ¹H NMR (300 MHz, CD₃COCD₃), δ: 2.04, 2.05, 2.07 (3 × s, 9 H, CH₃COO), 3.26
(s, 3 H, CH₃N), 3.50 (s, 3 H, OCH₃), 3.70 (s, 3 H, COOCH₃), 3.87 (s, 6 H, OCH₃), 4.37 (d, 2 H, J = 6 Hz, CH₂N), 4.65
(d, 1 H, J = 9.5 Hz, H-5), 5.00 (s, 2 H, CH₂O), 5.18-5.69 (m, 6 H, H-1,2,3,4, CH₂O), 6.48 (s, 1 H, H_arom), 7.02 (s, 1 H, NH), 7.30-7.84 (m, 8 H, H_arom) ppm; ¹³C NMR (75 MHz, CD₃COCD₃), δ: 37.9 (CH₃N), 41.3 (CH₂N), 53.0 (COOCH₃), 56.6 (OCH₃), 65.0-65.1 (CH₂O), 69.9, 71.1, 72.1, 72.8 (C-2,3,4,5), 100.0 (C-1), 121.2-149.6 (C_arom), 155.0-156.1 (C_arom), 167.7 (COOMe), 169.5, 170.0, 170.2 (CH₃CO) ppm; mp 100 °C.

17

Data for 9: [α_D] = +6,4 (c 0.95, CHCl₃); ¹H NMR (300 MHz, CD₃COCD₃), δ: 3.51 (s, 3 H, CH₃N), 3.59-3.62 (m, 3 H,
H-2,3,4), 3.71 (s, 3 H, COOCH₃), 3.87 (s, 6 H, OCH₃), 4.20 (d, 1 H, J = 9.5 Hz, H-5), 4.37 (d, 2 H, J = 6 Hz, CH₂N), 5.00 (s, 2 H, CH₂O), 5.33 (s, 2 H, CH₂O), 5.51 (d, 1 H, J = 16 Hz, H-1), 7.00 (broad s, 1 H, HNH), 7.31-7.83 (m, 9 H, H_arom) ppm; ¹³C NMR (75 MHz, CD₃COCD₃), δ: 37.9 (CH₃N), 41.3 (CH₂N), 52.5 (COOCH₃), 56.5, 56.6 (OCH₃), 65.0, 65.1 (CH₂O), 72.3, 74.1, 76.4, 76.9 (C-2,3,4,5), 101.9 (C-1), 118.1-150.0 (C_arom), 155.2, 157.1 (C_carbamate), 169.5 (COOMe) ppm; mp 90 °C. The highly polar compound 10 does not lead to well resolved NMR spectra (this is observed for all glucuronylated prodrugs).

18

HPLC conditions : C-16 reverse phase column : Discovery RP amide, 5 m (15 cm × 4.6 mm) and pre-column Discovery, 5 µm, UV detection (λ 254 nm), mobile phase (acetonitrile-0.065 M acetate ammonium solution, 30:70, 1 mL min^-1). Retention time: 10: 7.8 min; 4-hydroxy-3-nitrobenzyl alcohol: 3.6 min; 3 (R¹ = CH₃, R² = H): 4.1 min; 6-nitroveratryl alcohol: 5.2 min; 1 (R¹ = CH₃, R² = H, R³ = 6-nitroveratryl alcohol): 6.3 min.

19 We have recently shown that 2-nitro-quinone-methides are not irreversible inhibitors of bovine β-glucuronidase: Azoulay M. Chalard F. Gesson J.-P. Florent J.-C. Monneret C. Carbohydr. Res.  2001,  332:  151