Synthesis of 3-deoxy-4-O-β-D-galactopyranosyl-D-erythro-hexos-2-ulose and of some of its derivatives from lactose

3-Deoxy-4-O-β-D-galactopyranosyl-D-erythro-hexos-2-ulose (1) was obtained from lactose by reaction with benzoylhydrazine in the presence of a slightly acidic solution of p-toluidine, followed by hydrazinolysis of the product, 3-deoxy-4-O-β-D-galactopyranosyl-D-erythro-hexos-2-ulose bis(benzoylhydrazone) (3), with benzaldehyde. A variety of derivatives of 1 and 3 was prepared. Lactose aroylhydrazones were also prepared. Quantitative determination of the oxidant during the periodate oxidation of 3 was studied. Periodate oxidation of monosaccharide arylhydrazones gave glyoxal mono(arylhydrazones) which afforded the corresponding, mixed bis(hydrazones).     

Studies on dehydro-l-ascorbic acid 2-arylhydrazone 3-oximes: Conversion into substituted triazoles and isoxazolines

l-threo-2,3-Hexodiulosono-1,4-lactone 2-(arylhydrazones) (2) were prepared by condensation of dehydro-l-ascorbic acid with various arylhydrazines. Reaction of 2 with hydroxylamine gave the 2-(arylhydrazone) 3-oximes (3). On boiling with acetic anhydride, 3 gave 2-aryl-4-(2,3-di-O-acetyl-l-threo-glycerol-l-yl)-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones (4). On treatment of 4 with liquid ammonia, 2-aryl-4-(l-threo-glycerol-l-yl)-1,2,3-triazole-5-carboxamides (5) were obtained. Acetylation of 5 with acetic anhydride-pyridine gave the triacetates, and vigorous acetylation with boiling acetic anhydride gave the tetraacetyl derivatives. Periodate oxidation of 5 gave the 2-aryl-4-formyl-1,2,3-triazole-5-carboxamides (8), and, on reduction, 8 gave the 2-aryl-4-(hydroxymethyl)-1,2,3-triazole-5-carboxamides, characterized as the monoacetates and diacetates. Controlled reaction of 2 with sodium hydroxide, followed by neutralization, gave 3-(l-threo-glycerol-l-yl)-4,5-isoxazolinedione 4-(arylhydrazones), characterized by their triacetates. Reaction of 2 with HBr-HOAc gave 5-O-acetyl-6-bromo-6-deoxy-l-threo-2,3-hexodiulosono-1,4-lactone 2-(arylhydrazones); these were converted into 4-(2-O-acetyl-3-bromo-3-deoxy-l-threo-glycerol-l-yl)-2-aryl-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones on treatment with acetic anhydride-pyridine.     

Studies on dehydro- -ascorbic acid 2-arylhydrazone 3-oximes: Conversion into substituted triazoles and isoxazolines

-threo-2,3-Hexodiulosono-1,4-lactone 2-(arylhydrazones) (2) were prepared by condensation of dehydro- -ascorbic acid with various arylhydrazines. Reaction of 2 with hydroxylamine gave the 2-(arylhydrazone) 3-oximes (3). On boiling with acetic anhydride, 3 gave 2-aryl-4-(2,3-di-O-acetyl- -threo-glycerol-l-yl)-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones (4). On treatment of 4 with liquid ammonia, 2-aryl-4-( -threo-glycerol-l-yl)-1,2,3-triazole-5-carboxamides (5) were obtained. Acetylation of 5 with acetic anhydride-pyridine gave the triacetates, and vigorous acetylation with boiling acetic anhydride gave the tetraacetyl derivatives. Periodate oxidation of 5 gave the 2-aryl-4-formyl-1,2,3-triazole-5-carboxamides (8), and, on reduction, 8 gave the 2-aryl-4-(hydroxymethyl)-1,2,3-triazole-5-carboxamides, characterized as the monoacetates and diacetates. Controlled reaction of 2 with sodium hydroxide, followed by neutralization, gave 3-( -threo-glycerol-l-yl)-4,5-isoxazolinedione 4-(arylhydrazones), characterized by their triacetates. Reaction of 2 with HBr-HOAc gave 5-O-acetyl-6-bromo-6-deoxy- -threo-2,3-hexodiulosono-1,4-lactone 2-(arylhydrazones); these were converted into 4-(2-O-acetyl-3-bromo-3-deoxy- -threo-glycerol-l-yl)-2-aryl-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones on treatment with acetic anhydride-pyridine.     

On the ring transformation of hydrazine derivatives of l-ascorbic acid into nitrogen heterocyclic derivatives

l-hreo-2,3-hexodiulosono-1,4-lactone 2-(p-methoxyphenylhydrazone) (1) was condensed with arylhydrazines to give mixed bishydrazones, whose acetylation gave the corresponding di-O-acetyl derivatives. The hydrazone 1 undergoes elimination of one molecule of water per molecule during, the acetylation, and gives 4-(2-acetoxy- ethylidene)-4-hydroxy-2,3-dioxobutano-1,4-lactone 2-(p-methoxyphenylhydrazone), which reacts with methylhydrazine, via a ring transformation process, to give 1-methyl-3-(L-methylpyrazolin-3-yl)-4,5-pyrazoledione 4-(p-methoxyphenylhydrazone). Alkali rearranged the mixed bishydrazones to 1-aryl-3-(l-threo-glycerol-1-yl)-4,5- pyrazoledione 4-(p-methoxyphenylhydrazones), which gave triacetyl and tribenzoyl derivatives, and, upon periodate oxidation, afforded 1-aryl-3-formyl-4,5- pyrazolediones 4-(p-methoxyphenylhydrazones) that gave the corresponding phenylhydrazones. The n.m.r. and mass spectra of some of these derivatives have been investigated.     

Facile synthesis and rearrangement of L-threo-2,3-hexodiulosono-1,4-lactone 2-(2-arylhydrazones)

Controlled reaction of L-threo-2,3-hexodiulosono-1,4-lactone with substituted phenylhydrazines gave the 2-(monoarylhydrazones) (2), which underwent dehydrative acetylation to 4-(2-acetoxyethylidene)-4-hydroxy-2,3-dioxohutyro-1,4-lactone 2-(2-arylhydrazones) (3). The latter reacted with methylhydrazine to give 1-methyl-3-(1-methylpyrazolin-3-yl)-4,5-pyrazoledione 4-(2-arylhydrazones) (4). Reaction of the monoarythydrazones (2) with phenylhydrazine gave the mixed bishydrazones (5), which were rearranged by alkali and acidification to the pyrazolediones (6). Compounds 6 gave triacetyl (7) and tribenzoyl derivatives (8), and, on periodite oxidation, the aldehydes (9), which afforded the monohydrazones (10). The i.r.. n.m.r.. and mass-spectral data of some of the compounds were investigated.     

The cations and anions of cyclobutanetetraone poly(phenylhydrazones)

Six cyclobutanetetraone poly(arylhydrazones) have been treated with acids and bases, and the structures of the resulting anions and cations studied by UV-Vis absorption and NMR spectroscopy. In acid media, all the hydrazones studied formed cations, which exhibited bathochromic shifts due to the extension of their resonance systems. However, in bases, only some (those which could enolize) formed anions that exhibited hypsochromic shifts; the others were unaltered.     

(7-Chloroquinolin-4-yl)arylhydrazones: Candida albicans enzymatic repression and cytotoxicity evaluation,Part 2

Objective: This work describes the anti-enzymatic activity of (7-chloroquinolin-4-yl)arylhydrazones against Candida albicans and examines their cytotoxicity.

Material and methods: Ten C. albicans strains [nine isolates and one azole-resistant standard strain (ATCC 62342)] were used to assess the anti-enzymatic activity. Fifteen compounds at sub-antifungal concentrations ranging from 12.5 to 100?µg/ml were assessed after a 30-min exposure. The strains were seeded onto petri dishes with selective agar media for aspartyl proteases (Saps) and phospholipases (PLs). Enzymatic inhibition was measured by the reduction of the precipitation zone (Pz) against untreated strains (positive control). A colorimetric MTT assay was used with 3T3/NIH mouse fibroblasts to evaluate cytotoxicity. Cells were exposed to 15 compounds in concentrations from 6.25 to 100?µg/ml for 24 and 48?h.

Results: Four hydrazones showed enzymatic repression values over 40% to Pl and three over 20% to Saps. The cell viability was over 50% at hydrazone concentrations of 25–100?µg/ml.

Conclusion: These results revealed that select (7-chloroquinolin-4-yl)arylhydrazones may be potential antifungal agents for the control of C. albicans infections.     

Aminyl and iminyl radicals from arylhydrazones in the photo-induced DNA cleavage

Photolytic cleavage of the nitrogen-nitrogen single bond in benzaldehyde phenylhydrazones produced aminyl (R2N*) and iminyl (R2C=N*) radicals. This photochemical property was utilized in the development of hydrazones as photo-induced DNA-cleaving agents. Irradiation with 350 nm UV light of arylhydrazones bearing substituents of various types in a phosphate buffer solution containing the supercoiled circular phiX174 RFI DNA at pH 6.0 resulted in single-strand cleavage of DNA. Attachment of the electron-donating OMe group to arylhydrazones increased their DNA-cleaving activity. Results from systematic studies indicate that both the aminyl and the iminyl radicals possessed DNA-cleaving ability.     

Action of strong and weak nitrogenous bases on respiratory chain enzymes of mitochondria

The action of nitrogenous basis--electroneutral hydrazides (pK less than 7,50 and positive charged arylhydrazones (pK greater than 8)--on the respiratory chain enzymes and the influence of the electric charge and the size of alkoxylic group on biological activity compounds have been investigated. It has been shown that the size of alkoxylic group defines the selective action of nitrogenous basis on the enzymes of mitochondrial respiratory chain. The nitrogenous basis with a long alkoxylic group is shown to be inhibitors of NADH-dehydrogenase, their action is similar to rotenone. At the same time compounds with a short group are more effective in the inhibition of the enzymes of the initial segment in the respiratory chain mitochondria. The affinity of the organic cations of arylhydrazones to NADH-dehydrogenase is 100-1000 times higher than the affinity of electric neutral compounds.     

Novel pyrazolopyrimidine derivatives as GSK-3 inhibitors

A series of [1-aryl-1H-pyrazolo[3,4-d]pyrimidin-4-yl]arylhydrazones were discovered as novel inhibitors glycogen synthase kinase-3 (GSK-3). Based on initial modeling a detailed SAR was constructed. Modification of the interior binding aryl ring (Ar(1)) determined this to be a tight binding region with little room for modification. As predicted from the model, a large variety of modifications could be incorporated into the hydrazone aryl ring. This work led to GSK-3 inhibitors in the low nano-molar range.     

Preparation and characterization of well defined d-erythro sphingomyelins

A simple semisynthetic procedure for the preparation of various d-erythro sphingomyelins (SPMs), differing in their acyl chains, is described. They were prepared by one-step condensation of the desired free fatty acid with sphingosyl phosphorylcholine (SPC) using dicyclohexylcarbodiimide. The d-erythro SPMs were obtained in high purity, high yields and resemble bovine brain SPM in their chromatographic behavior, infrared, circular dichroism (CD) and proton NMR (PMR) spectra as well as in their rate of hydrolysis by Staphylococcus aureus sphingomyelinase. Multilamellar vesicles can be prepared from the semisynthetic SPMs. Their thermotropic behavior is dependent mainly on the acyl chain though it is also affected by the heterogeneity of the sphingosine base composition. Intact sealed small unilamellar vesicles (SUV) cannot be prepared from a single semisynthetic saturated SPM but can be prepared from their mixtures. This acylation procedure can also be applied for preparing simple neutral glycosphingolipids. The sphingolipids prepared by this method can be used to study metabolism, enzymology and physicochemical properties of d-erythro well defined simple sphingolipids.     

Novel GSK-3 inhibitors with improved cellular activity

A novel series of [1-(1H-benzimidazol-7-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl] arylhydrazones was synthesized and shown to potently inhibit glycogen synthase kinase-3 (GSK-3). In light of detailed structure-activity relationships and structural knowledge of the GSK-3 binding pocket, a benzimidazole substituent was incorporated onto the pyrazolopyrimidine core resulting in improved potency over previous analogs. More importantly, these derivatives show low nanomolar efficacy for stimulating glycogen synthesis in vitro and therefore may be useful in the treatment of type 2 diabetes mellitus.     

Synthesis of glycosyl esters of oleanolic acid

The trisaccharide, O-(2,3,4-tri-O-benzoyl-β-L-rhamnopyranosyl)-(1→4)-O-(2,3,6-tri-O-benzoyl-β-D-glucopyranosyl)-(1→6)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranose has been prepared by two different routes. Condensation of this trisaccharide with oleanolic acid afforded the corresponding 1,2-trans glycosyl ester. Some other glycosyl esters of oleanolic acid were also prepared by the same method.     

Preparation of 1-thioglycosides having ω-aldehydo aglycons useful for attachment to proteins by reductive amination

A series of 1-thioglycosides containing an ω-aldehydo group (as the dimethyl acetal) on the aglycon were prepared by reaction of O-acetyl-1-thioaldoses with N-(chloroacetyl)aminoacetaldehyde dimethyl acetal, a compound readily prepared by the action of chloroacetyl chloride or chloroacetic anhydride on 2-aminoacetaldehyde dimethyl acetal. O-Deacetylation of the 1-thioglycosides, followed by deacetalation, yielded the desired products. An analogous 1-thioglycoside having a longer aglycon was prepared by reaction of 1-thio-D-galactose with (6-aminohexanoyl)-aminoacetaldehyde dimethyl acetal, obtained by condensing 6-bromohexanoic acid and aminoacetaldehyde with 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide. These glycosides were found to be useful for modification of proteins to yield neoglyco-proteins.     

Hémisynthèse de nouveaux glycosides analogues de la daunorubicine

Seven daunorubicin analogs containing α-l-, α-d-, and β-d-glycosidic linkages, in which the natural occurring sugar (l-daunosamine) was replaced by diastereo-isomeric 3-amino-2,3,6-trideoxyhexoses (3-epi-l-daunosamine, d-acosamine, d-daunosamine, d-ristosamine, and 3-epi-d-daunosamine), were prepared. In all cases, glycosidation with daunomycinone was performed in the presence of p-toluene-sulfonic acid starting from 1-O-acetyl-2,3,6-trideoxy-4-O-p-nitrobenzoyl-3-trifluoroacetamidohexopyranoses (prepared from the corresponding methyl 3-amino-2,3,6-trideoxyhexopyranosides) or from 1,5-anhydro-2,3,6-trideoxy-4-O-p-nitrobenzoyl-3-trifluoroacetamidohex-1-enitols (prepared from glycals or pseudoglycals, the 3-amino group being introduced by substitution with sodium azide and subsequent reduction). Glycosidation was followed by removal of the protecting groups.     

Synthesis,characterization and biological activity of ring-substituted 6-benzylamino-9-tetrahydropyran-2-yl and 9-tetrahydrofuran-2-ylpurine derivatives

In an attempt to improve specific biological functions of cytokinins routinely used in plant micropropagation, 33 6-benzylamino-9-tetrahydropyran-2-ylpurine (THPP) and 9-tetrahydrofuran-2-ylpurine (THFP) derivatives, with variously positioned hydroxy and methoxy functional groups on the benzyl ring, were prepared. The new derivatives were prepared by condensation of 6-chloropurine with 3,4-dihydro-2H-pyran or 2,3-dihydrofuran and then by the condensation of these intermediates with the corresponding benzylamines. The prepared compounds were characterized by elemental analyses, TLC, HPLC, melting point determinations, CI+ MS and ¹H NMR spectroscopy. The cytokinin activity of all the prepared derivatives was assessed in three classical cytokinin bioassays (tobacco callus, wheat leaf senescence and Amaranthus bioassay). The derivatives 6-(3-hydroxybenzylamino)-9-tetrahydropyran-2-ylpurine (3) and 6-(3-hydroxybenzylamino)-9-tetrahydrofuran-2-ylpurine (23) were selected, because of the high affinity of their parent compound meta-topolin (mT, 6-(3-hydroxybenzylamino)purine) to cytokinin receptors, as model compounds for studying their perception by the receptors CRE1/AHK4 and AHK3 in a bacterial assay. Both receptors perceived these two derivatives less well than they perceived the parent compound. Subsequently, the susceptibility of several new derivatives to enzyme degradation by cytokinin oxidase/dehydrogenase was studied. Substitution of tetrahydropyran-2-yl (THP) at the N⁹ position decreased the turnover rates of all new derivatives to some extent. To provide a practical perspective, the cytotoxicity of the prepared compounds against human diploid fibroblasts (BJ) and the human cancer cell lines K-562 and MCF-7 was also assayed in vitro. The prepared compounds showed none or marginal cytotoxicity compared to the corresponding N⁹-ribosides. Finally, the pH stability of the two model compounds was assessed in acidic and neutral water solutions (pH 3–7) by high-performance liquid chromatography (HPLC).     

Reactions of chitosan: 4. Preparation of organosoluble derivatives of chitosan

Fully subtituted di-O-acetyl-N-acetylchitosan (chitin diacetate) has been prepared by a route in which the hydroxyl groups are acetylated prior to N-acetylation. This overcomes the previously reported intramolecular steric hindrance to esterification caused by the N-acetamido group. The resultant products were of high viscosity but had a limited solubility range. Di-O-acrylcarbamate derivatives of N-acetylchitosan (chitin) have been produced by a similar route, whilst di-O-arylcarbamate-N-arylureidochitosans have been prepared directly from chitosan. These products also have limited solubility ranges and have inherent viscosities similar to that of di-O-acetylchitosan prepared from the same batch of chitosan.     

Synthesis of α-l-rhamnosyl ceramide and evaluation of its binding with anti-rhamnose antibodies

An α-l-rhamnosyl ceramide (1, α-l-RhaCer) has been prepared that was recognized by anti-l-rhamnose (anti-Rha) antibodies. During these studies we explored the use of an α-l-rhamnosyl thioglycoside and a trichloroacetimidate as a glycosyl donors. Subsequently, the acceptors desired for glycosylation, 3-O-benzoylazidosphingosine or 3-O-alloxycarbonylsphingosine, were prepared from d-xylose. The thioglycoside donor, 2,3,4-tri-O-acetyl-1-(4-tolyl)thio-α-l-rhamnopyranoside, and the trichloroacetimidate donor, 2,3,4-tri-O-acetyl-1-(2,2,2-trichloroethanimidate)-α-l-rhamnopyranoside, were synthesized in 50% and 78% yield overall, respectively. The synthesis of the glycosylation acceptor employed an addition–fragmentation olefination that was successfully carried out in 53% yield. With the successful synthesis of key intermediates, α-l-RhaCer (1) was prepared without any insurmountable obstacles. Anti-Rha antibodies were prepared in BALB/c mice by immunizing them with rhamnose-ovalbumin (Rha-Ova) with Sigma Adjuvant System (SAS) and the anti-l-Rha antibodies were isolated from the blood sera. Liposomes and EL4 tumor cells were used as model systems to demonstrate the ability of 1 to insert into a lipid bilayer. The interaction of the liposomes or the EL4 cells with α-l-RhaCer (1) and anti-Rha antibodies were investigated by fluorescence microscopy and flow cytometry, respectively, to confirm the ability of glycolipid 1 to be displayed on the tumor cell surface as well as the ability to be recognized by anti-Rha antibodies.     

Distribution of antigens in established cell lines of Drosophila melanogaster

Antibodies prepared against two Drosophila cell lines together with antibodies prepared against other Drosophila extracts were used to study the distribution of antigens in 10 cell lines. The results suggest: (1) that cultured cells express differentiated functions; (2) that each cell line, including sub-clones of a clone, is unique; and (3) that the cell lines are derived from the lateral ectoderm.     

Cultivation of Mycoplasmas on Glass

Eight Mycoplasma species of human origin were successfully cultivated on glass. Complement-fixing (CF) antigens prepared from glass-adherent mycoplasmas were potent, specific, and free from anticomplementary activity. PPLO broth medium supplemented with 1 to 5% PPLO serum fraction (bovine), 2.5% fresh yeast extract, and 1% glucose (glycolytic species) or 1% arginine (arginine-utilizing species) supported moderate to luxuriant growth of mycoplasmas on glass. The potency of CF antigens prepared from glass-adherent mycoplasmas varied with the species of Mycoplasma tested and the duration of incubation. When the potency of CF antigens prepared from glass-adherent mycoplasmas was compared with that material sedimented from the broth phase of the same culture, three patterns of growth were observed: M. hominis and M. orale type 2 grew preferentially in the broth phase; M. salivarium, M. orale types 1 and 3, M. pneumoniae, and M. lipophilum preferentially adhered to the glass; and M. fermentans was biphasic. The growth of mycoplasmas on glass provides a simple means of concentrating and purifying such organisms for immunological and biochemical studies.