Glycosidases have been demonstrated to be elevated in the interstitial fluid of tumors, sera of animals and patients with tumors, and in some tumor tissue as compared to normal adjacent tissue. Elevations of serum beta-N-acetylglucosaminidase and beta-glucuronidase most commonly have been found to occur and these enzymes have been shown to be secreted into the extracellular medium by many different tumor cell types in vitro. The mechanism of cellular release of these hydrolytic enzymes probably involves tumor lysosomal exocytosis. Increased tumor glycosidase levels may promote increased tumor cell shedding from primary tumors, local invasion and perhaps be responsible directly, or indirectly for structural changes in tumor cell surface glycoconjugates. These cell surface changes could facilitate tumor cell thrombus formation, secondary site implantation and attachment in the microcirculation to endothelial cells and/or subendothelial basement membrane components. Other studies have demonstrated a correlation between metastatic cell potential and increased endoglycosidase and polysaccharide lyase activity. Generally, metastatic tumor cell variants have been found to be more invasive and capable of degrading proteoglycan basement membrane components, in part due to these increased levels of degradative enzymes. Hence, it is of considerable interest to develop inhibitors against these enzymes. Initial studies with glucuronidase inhibitors in the therapy of bladder tumors have been promising and with the advent of better agents and the use of appropriate in vitro metastatic models it may be possible to design and develop agents which interfere in various metastatic events and limit tumor progression.

2-Amino-2,4-dideoxy-4-fluoro- and 2-amino-2,4,6-trideoxy-4, 6-difluoro-D-galactose, and 2-amino-2,4-dideoxy-4-fluoro- and 2-amino-4-deoxy-4, 4-difluoro-D-xylo-hexose were synthesized, as potential modifiers of tumor cell-surface glyco-conjugate, from benzyl 2-acetamido-3-O-benzyl-2-deoxy-4, 6-di-O-mesyl-alpha-D-glucopyranoside and benzyl 2-acetamido-3, 6-di-O-benzyl-2-deoxy-4-O-mesyl-alpha-D-glucopyranoside, which were converted into the corresponding 4,6-difluoro-2,4, 6-trideoxy and 2,4-dideoxy-4-fluoro derivatives. Benzyl 2-acetamido-2-deoxy-4-O-mesyl-alpha-D-galactopyranoside and benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-alpha-D-xylo-hexo-4-ulopyra noside were treated with diethylaminosulfur trifluoride to give 2-amino-2,4-dideoxy-4-fluoro-D-glucose and 2-amino-2,4-dideoxy-4, 4-di-fluoro-D-xylo-hexose derivatives, respectively, to give after deprotection the target compounds. Several of the peracetylated sugar derivatives inhibited L1210 tumor-cell growth in vitro at concentrations of 1-5 10(-5) M. The peracetylated derivative of 2-amino-2,4-dideoxy-4-fluoro-D-galactose inhibited protein and glycoconjugate biosynthesis, and also exhibited antitumor activity in mice with L1210 leukemia.

We have synthesized several potential inhibitors and/or modifiers of the carbohydrate portion of plasma membrane glycoconjugates. These include fluorinated and actylated analogs of D-glucosamine, D-galactosamine, and D-mannosamine. These compounds have been tested to determine their effects on both[14C] glucosamine and [3H] leucine incorporation into glycoconjugate and on cell growth and viability using P-288 murine lymphoma cells maintained in tissue culture. The most cytotoxic agent tested was 2-acetamido-2-deoxy-1,3,4,6-tetra-O-acetyl-beta-D-glucopyranose or simply beta-pentaacetylglucosamine which prevented cell growth at 10(-4)-10(-3) M. beta-Pentaacetylglucosamine cytotoxicity was correlated with its high lipid solubility, having an octanol/water partition coefficient of 0.424 as compared with 0.278 for the alpha-anomer and 0.017 for N-acetylglucosamine. In vitro metabolism studies with [4C]- and/or [3H]-labeled pentaacetylglucosamine have indicated intracellular de-O-acetylation leading to the biosynthesis of UDP-N-acetylglucosamine, followed by the incorporation of this sugar into cellular glycoprotein. Concomitant with the formation of increased amounts of this nucleotide sugar, intracellular UTP and CTP pools fell to one third normal within 3 h after the administration of 1 mM pentaacetylglucosamine. At present it is unclear whether the cytotoxicity of beta-pentaacetylglucosamine or other similar agents is due to alterations in nucleotide and nucleotide-sugar pools causing a decrease in energy charge and polynucleotide biosynthesis or is due to a direct effect on membrane glycoconjugate biosynthesis.

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