DESIGN, SYNTHESIS, AND EVALUATION OF MITOMYCIN-TETHERED PHOSPHOROTHIOATE OLIGODEOXYNUCLEOTIDES

Mitomycin C (1) is the prototypical bioreductive alkylating agent. Stu dies have shown that mitomycin C and its derivatives selectively alkyl ate guanine residues within di- and trinucleotide DNA sequences. This investigation sought to improve the selective DNA bonding properties o f the mitomycins by coupling them with antisense oligodeoxynucleotides . Two procedures were developed that allowed the attachment of a phosp horothioate oligodeoxynucleotide containing a hexylamino spacer at the 5' terminus with a C(10)-activated mitomycin. In the first procedure, decarbamoylation of 1 (NaOCH3/benzene) benzene) afforded 10-decarbamo ylmitomycin C (10), which was treated with either dimethyl sulfate or methylthiochloroformate and base to yield 10-decarbamoylporfiromycin ( 11) and 1a)-[(methylthio)carbonyl]-10-decarbamoylmitomycin C (12), res pectively. Activation of the C(10) site in 11 and 12 with 1,1'-carbony ldiimidazole or with 1,1'-thiocarbonyldiimidazole provided the N(1a)-s ubstituted mitomycin 10-decarbamoyl-10-O-carbonylimidazoles (5, 7) and 10-decarbamoyl-10-O-thiocarbonylimidazoles (6, 8), respectively. Comp ounds 5-8 were reacted with glycine methyl ester hydrochloride (17) an d base in both methylene chloride and aqueous buffered solutions to de termine the ease and efficiency in which these C(10)-activated mitomyc in derivatives coupled to amines. It was found that 5-8 all reacted wi th 17 in methylene chloride to give the coupled products 18-21 but tha t improved amine coupling yields in water were observed for the 10-dec arbamoyl-10-O-thiocarbonylimidazoles 6 and 8 as compared with the 10-d ecarbamoyl-10-O-carbonylimidazoles 5 and 7. This finding led to the co upling of the phosphorothioate oligo deoxynucleotide, H2N(CH2)(6)-P(S) (OH)-GGCCCCGTG-GTGGCTCCAT (22) to 8. Compound 22 complemented a 19-bas e sequence in the translation initiation region of the human A-raf-1 g ene. Use of excess 8 (28 equiv) with 22 gave only a 36% yield of the c oupled product 23, which proved difficult to separate from 22. In the second procedure, phosphorothioate oligodeoxynucleotides that containe d a hexylamino spacer at the 5' termini were coupled to 10-des(carbamo yloxy)-10-isothiocyanatoporfiromycin (9). Compound 9 was prepared in f our steps from 11. Mesylation (methanesulfonyl chloride/pyridine) of 1 1 gave the C(10) mesylate 13, which was then treated with NaN3 (dimeth ylformamide, 90 degrees C) to give 10-des(carbamoyloxy)-10-azidoporfir omycin (14). Catalytic reduction (PtO2, H-2) of 14 in pyridine afforde d C(10) amine 15. Treatment of 15 with di-2-pyridyl thionocarbonate pr ovided the desired 10-des(carbamoyloxy)-10-isothiocyanatoporfiromycin (9). Compound 9 readily coupled with 17 and base in both methylene chl oride and aqueous buffered solutions to give 25. Use of the 5' hexylam inophosphorothioate oligodeoxynucleotides 32-35 in place of 17 gave th e conjugated adducts 28-31, respectively, in a 12% to near-quantitativ e yield. The products were purified by semipreparative HPLC. Antisense agents 28-31 were designed to target a 30-base-long region from the c oding region of the human FGFR1 gene. One adduct, 29, reduced the numb er of FGFR1 receptors in human aortic smooth cells for bFGF on the cel l surface, which suggested down-regulation of FGFR1 gene expression. F urther, 29 inhibited cultured human aortic smooth muscle cell prolifer ation and was less cytotoxic than porfiromycin (2). The biological ass ay data suggest that the phosphorothioate oligodeoxynucleotide porfiro mycin conjugates may be more target selective and less toxic than eith er mitomycin or porfiromycin and thus be promising therapeutic agents.