Versatile intermediates in the selective modification of the amino function of 2-amino-2-deoxy-D-mannopyranose and the 3-position of 2-acetamido-2-deoxy-D-mannose: potential membrane modifiers in neoplastic control.

A general method has been developed to selectively modify the amino group of 2-amino-2-deoxy-D-mannopyranose (D-mannosamine), a precursor of the terminal membrane sugar, sialic acid. 1,3,4,6-Tetra-O-acetyl-2-amino-2-deoxy-alpha-D-mannopyranose oxalate was prepared via two routes that allowed introduction of various acyl groups onto the amino function. These compounds were evaluated for their antineoplastic properties. The most significant preclinical therapeutic finding was the antileukemic activity found in mice for tetra-O-acetyl-2-epi-streptozotocin (the acetylated alpha-mannosamine epimer of streptozotocin). Administration of 50 mg/kg/day x 5 to leukemia L1210-bearing DBA/2Ha mice resulted in 5/5 35-day survivors. Neutralization of 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-alpha-D-mannopyranose oxalate under aqueous conditions led to 2-acetamido-1,4,6-tri-O-acetyl-2-deoxy-alpha-D-mannopyranose, the oxidation of which gave 2-acetamido-4,6-di-O-acetyl-1,5-anhydro-2-deoxy-D-erythro-hex-1-en-3- ulose. This agent demonstrated an IC50(2) of 25 microM with a murine L1210 cell culture. Administration of 100 mg/kg/day x 5 resulted in 42% ILS3 in DBA/2 mice with ip L1210 leukemia. Several other nonacetylated derivatives were also prepared by direct N-acylation, producing, for example, fluorescently tagged N-dansylmannosamine.

Fluorinated carbohydrates as potential plasma membrane modifiers. Synthesis of 3-deoxy-3-fluoro derivatives of 2-acetamido-2-deoxy-D-hexopyranoses.

Treatment of benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-alpha-D-allopyranoside with diethylaminosulfur trifluoride or of the 3-O-mesyl derivative with tetrabutylammonium fluoride gave the 2,3-unsaturated compound instead of the expected 3-deoxy-3-fluoro derivative. The latter was obtained when benzyl 2-acetamido-4,6-di-O-benzyl-2-deoxy-3-O-mesyl-alpha-D-allopyran oside was treated with potassium fluoride. Methyl 2-azido-4,6-O-benzylidene-2-deoxy-alpha-D-altropyranoside was converted into the 2-acetamido- and 2-phthalimido-3-O-mesyl derivatives; when treated with fluoride nucleophile, these gave only the 2,3-aziridine derivative. However, treatment of the 2-azido-2-deoxy derivative with diethylaminosulfur trifluoride gave methyl 2-azido-2,3-dideoxy-3-fluoro-alpha-D-mannopyranoside which, after reduction, deprotection, and acetylation, gave the acetylated derivative of methyl 2-acetamido-2,3-dideoxy-3-fluoro-alpha-D-mannopyranoside in excellent yield. These acetylated 3-fluoro derivatives exhibited inhibition of cell growth of murine L1210 leukemia cells in culture at micromolar concentrations.

Fluorinated carbohydrates as potential plasma membrane modifiers. Synthesis of 4- and 6-fluoro derivatives of 2-acetamido-2-deoxy-D-hexopyranoses.

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.

Analogs of cell surface carbohydrates. Synthesis of D-galactose derivatives having an ethynyl, vinyl or epoxy residue at C-5.

Compounds derived from D-galactose having an ethynyl, vinyl, or epoxide residue at C-5, as well as 7,7-dibromo olefinic, isomeric 7,7-gem-bromofluoro olefinic, and 6,6-gem-difluoro derivatives were obtained from 1,2:3,4-di-O-iso-propylidene-alpha-D-galacto-hexodialdo-1,5- pyranose.

General methods for modification of sialic acid at C-9. Synthesis of N-acetyl-9-deoxy-9-fluoroneuraminic acid.

Methyl 5-acetamido-3,5-dideoxy-2-O-methyl-D-glycero-D-galacto-2-nonulopyrano sate was converted into the 9-O-trityl derivative and the remaining hydroxyl groups were protected as benzyl ethers. Removal of the trityl group, followed by treatment with diethylaminosulfur trifluoride gave the 9-deoxy-9-fluoro derivative, and deprotection N-acetyl-9-deoxy-9-fluoroneuraminic acid (8). In another procedure, coupling of 2-acetamido-2,6-dideoxy-6-fluoro-D-glucopyranose with potassium di(tert-butyl) oxaloacetate, followed by hydrolysis and decarboxylation gave 8. Some of the derivatives were active as inhibitors of growth of mouse mammary adenocarcinoma (TA3) and L1210 cells in culture.

Fluorinated carbohydrates as potential plasma membrane modifiers and inhibitors. Synthesis of 2-acetamido-2,6-dideoxy-6-fluoro-D-galactose.

Reaction of benzyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-mesyl-alpha-D-galactopyran oside with cesium floride gave benzyl 2-acetamido-3,6-anhydro-4-O-benzyl-2-deoxy-alpha-D-galactopyranoside instead of the desired 6-fluoro derivative. Acetonation of benzyl 2-acetamido-2-deoxy-6-O-mesyl-alpha-D-galactopyranoside gave the corresponding 3,4-O-isopropylidene derivative. The 6-O-mesyl group was displaced by fluorine with cesium fluoride in boiling 1,2-ethanediol, and hydrolysis and subsequent N-acetylation gave the target compound. In another procedure, treatment of 2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-alpha-D-galactose with N-(diethylamino)sulfur trifluoride gave 2-acetamido-1,3,4-tri-O-acetyl-2,6-dideoxy-6-fluoro-D-galactose which, on acid hydrolysis followed by N-acetylation, gave 2-acetamido-2,6-dideoxy-6-fluoro-D-galactose.

Therapeutic and diabetogenic potential of two newly synthesized nitrosoureido sugars.

Two newly synthesized nitrosoureido sugars have been evaluated for their antitumor activity and diabetogenic potential in a number of in vitro and in vivo preclinical tumor model systems. 2-Amino-2-deoxy-N'-methyl-N'-nitrosoureido-1,3,4,6-tetra-O-acetyl-alpha- D- mannopyranose (MAZ), a lipophilic mannosamine derivative, and ethyl-6-deoxy-3,5-di-O-methyl-6-(3-methyl-3-nitrosoureido)-alph a- D-glucofuranoside (EDOMEN or CGP 6'809), were both found to inhibit L1210 leukemia cell growth in vitro by 50% at approximately 5.0 X 10(-5) M. At these concentrations, little effect was noted immediately on L1210 cell radiolabeled precursor incorporation; however, at higher concentrations, EDOMEN inhibited [3H]leucine and [3H]mannose incorporation, while MAZ specifically decreased L1210 cell [3H]thymidine and [3H]leucine incorporation. Inhibition of Lewis lung carcinoma and B16 melanoma cell growth by 50% in vitro was achieved at higher concentrations of these agents (10(-4) to 10(-3) M). Since the currently available nitrosoureido sugars, streptozotocin and chlorozotocin, have been observed by us to be diabetogenic, EDOMEN and MAZ were evaluated for their specific toxicity to rat pancreatic beta-cells in vitro. Cytotoxicity in beta-cell cultures was monitored both by phase-contrast microscopy and the release of insulin into the culture medium. beta-Cells were found to be 10-fold more sensitive to the toxic effects of MAZ than were pancreatic fibroblasts. EDOMEN, on the other hand, did not damage beta-cells preferentially and therefore was not considered diabetogenic. Both MAZ and EDOMEN had moderate activity as antileukemic agents in mice. At 50 mg/kg/day i.p. for 5 days, MAZ increased the life span of female DBA/2J mice with L1210 leukemia by over 50%. Similarly, doses of EDOMEN at 125 to 250 mg/kg/day i.p. for 5 days increased L1210 leukemic life span by nearly 60%. At these doses, no effect of MAZ was observed on primary Lewis lung carcinoma growth or life span of tumor-bearing C57BL/6 mice. EDOMEN, however, increased life span in Lewis lung carcinoma mice by up to 33% and caused an apparent antimetastatic effect. These studies indicate that EDOMEN may have enhanced value as a cancer chemotherapeutic agent due to its therapeutic effectiveness, lack of diabetogenic potential, and other favorable formulation properties (water solubility) as compared with other clinically available nitrosoureas.

Glycosidases in cancer and invasion.

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.

S-, N-, and O-glycosyl derivatives of 2-acetamido-2-deoxy-D-glucose with hydrophobic aglycons as potential chemotherapeutic agents and N-acetyl-beta-D-glucosaminidase inhibitors.

S-, N-, and O-Glycosyl derivatives of 2-acetamido-2-deoxy-D-glucose with hydrophobic aglycons have been obtained as potential, plasma-membrane active agents. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-beta-D-glucopyranose (6) was converted into benzyl, diphenylmethyl, triphenylmethyl, and other thioglycosides. Acylation of 6 gave adamantoyl and haloacetyl derivatives. A similar series of N- and O-glycosyl derivatives was obtained from the corresponding NH2-1 and OH-1 analogs of 6, such as O- and N-dinitrophenyl, O- and N-adamantoyl, and N-4-methylbenzylidene derivatives. Several N- and S-glycosyl derivatives were found to inhibit mouse mammary adenocarcinoma (TA3) cells in vitro as well as N-acetyl-beta-D-glucosaminidase from beef liver.

A high-performance liquid chromatography method for the assay of cytidine monophosphate-sialic acid synthetase.

An HPLC method has been developed for the assay of cytidine monophosphate-sialic acid synthetase (EC 2.7.7.43) using ion-pair chromatography and gradient elution. This procedure permits the assay of alternative substrates and inhibitors of the enzyme and is not subject to the limitation of the colorimetric method. The newly synthesized N-acetyl-9-deoxy-9-fluoro-D-neuraminic acid was found to be a good substrate of the enzyme with a Km of 6.35 mM as compared to 1.84 mM for N-acetylneuraminic acid.

Effects of a membrane sugar analogue, 6-deoxy-6-fluoro-D-galactose, on the L1210 leukemic cell ectosialyltransferase system.

In L1210 leukemia cells, 6-deoxy-6-fluoro-D-galactose specifically inhibited the incorporation of [3H]-D-galactose, while that of other precursors of glycoconjugate biosynthesis, including mannose and glucosamine, was unaffected. The activation of [6-3H]-6-deoxy-6-fluoro-D-galactose to a nucleotide sugar was similar to that found for [3H]-D-galactose. The incorporation of either sugar after 1 hr was visualized by electron microscopic autoradiography to be in the Golgi region. Treatment of L1210 cells with 6-deoxy-6-fluoro-D-galactose in vitro or in vivo resulted in a specific, dose- and time-dependent decrease in the activity of cell surface sialyltransferase (ectosialyltransferase) but not of 5'-nucleotidase, a plasma membrane marker enzyme. The decrease in ectosialyltransferase activity appeared to be selective and is suggested to be due to structural modification of the cell surface galactoprotein acceptors for this enzyme. The data indicate that 6-deoxy-6-fluoro-D-galactose is an effective modifier of cellular glycoconjugate in that its incorporation into certain cell surface components results in a modification of plasma membrane structure and function.

Spectroscopic studies of complexes between pyridoxamine (pyridoxine)-5'-phosphate oxidase and pyridoxyl 5'-phosphate compounds differing at position 4'.

The absorbance spectrum of pyridoxamine (pyridoxine)-5'-phosphate oxidase (EC 1.4.3.5) is altered upon the binding of pyridoxal 5'-phosphate or analogs with different substituents at position 4'. The absorbance difference spectra are similar for complexes of the oxidase and pyridoxal 5'-phosphate, 4'-desoxypyridoxine 5'-phosphate, and 4-ethynyl-4-deformylpyridoxal 5'-phosphate; hence, these perturb the flavoprotein absorbance by similar interactions primarily involving the pyridoxyl 5'-phosphate moiety, and not specifically the 4-formyl group or other relatively small and uncharged functions at this position. A different type of spectral perturbation is caused by analogs with larger substituents at position 4' (i.e. 4'-methoxypyridoxine 5'-phosphate, 4-methyl-vinyl-4-deformylpyridoxal 5'-phosphate, and pyridoxal 5'-phosphate oxime and hydrazone). These analogs impose bulky groups in a region of the active site that critically influences the environment of the flavin, and, thus, may reflect positioning of this portion of the substrates close to the flavin ring, as is required for their redox interaction.