Synthesis and Enzymatic Evaluation of Substrates and Inhibitors of β‐Glucuronidases

Abstract

Abstract The phosphono and the tetrazolyl analogues 4 and 5 of 4‐methylumbelliferyl β‐ D ‐glucuronide (=(4‐methyl‐2‐oxo‐2 H ‐1‐benzopyran‐7‐yl β‐ D ‐glucopyranosid)uronic acid; 6 ) were synthesized and evaluated as substrates of β‐glucuronidases. Similarly, the phenylcarbamate 7 and its phosphono analogue 8 were prepared and evaluated as inhibitors. To examine the diastereoselectivity of the phosphorylation, we also synthesized the protected L ‐ ido ‐ D ‐ gluco ‐, and D ‐ galacto ‐configurated phospha‐glycopyranuronates 12, 13, 21, 22, 34 and 35 . Two strategies were followed. In the first one, the glucuronic acid 19 was decarboxylated to 11 and further transformed, via 20 , into the trichloroacetimidate 10 ( Scheme 2 ). Phosphorylation of 10 with (MeO) 3 P yielded the diastereoisomers 12 and 13 , the diastereoselectivity depending on the solvent. In MeCN, 12 and 13 were obtained in a ratio of 1:1, while in non‐participating solvents the L ‐ ido 12 was by far the major diastereoisomer. The acetate 11 was inert to (MeO) 3 P, but reacted with (PhO) 3 P to the anomeric mixture 21/22 , in keeping with a stabilizing 1,3‐interaction in the intermediate phosphonium salt. Similarly, the phospha‐galacturonates 34 and 35 were prepared from the galactoside 23 via the enol ether 26 , the lactone 27 , and the acetates 28/29 that were also transformed into the trichloroacetimidate 33 ( Scheme 3 ). In the second, higher‐yielding strategy, phosphorylation of the pentodialdehyde 39 to 40/41 was followed by hydrolysis and acetylation to the phospha‐glucuronates 43/44 ( Scheme 4 ). Transesterification to 45/46 , selective deacetylation to 48/49 , and formation of the trichloroacetimidates 50/51 were followed by glycosidation and deprotection to 4 . The tetrazole 5 was prepared from the lactones 54/55 via the N ‐benzylamides 57/58 that were treated with TfN 3 to give the N ‐benzyltetrazoles 59/60 ( Scheme 4 ). These were transformed into the trichloroacetimidates 63/64 , glycosylated to 65 , and deprotected. The O ‐carbamoylhydroximo‐lactone 7 derived from the glucuronate 67/68 , and the phosphonate analogue 8 were prepared by established methods. The phosphonate 4 is slowly hydrolyzed by the E. coli β‐glucuronidase, but neither 4 nor the tetrazole 5 are affected by the bovine liver β‐glucuronidase ( Table 4 ). The phenylcarbamate 7 of D ‐glucarhydroximo‐1,5‐lactone, but not its phosphonate analogue 8 , is an inhibitor ( K I = 8 m̈ M ) of the E. coli β‐glucuronidase. The bovine liver β‐glucuronidase is inhibited strongly by 7 ( IC 50 = 0.2 m̈ M ) and weakly by 8 ( IC 50 = 2m M ).