Convenient synthesis of 4,6-di-O-benzyl-myo-inositol and myo-inositol 1,3,5-orthoesters

Convenient high yielding methods for the preparation of 4,6-di-O-benzyl-myo-inositol, myo-inositol 1,3,5-orthoformate and myo-inositol 1,3,5-orthoacetate, without involving chromatography are described. Myo-inositol was converted to racemic 2,4-di-O-benzoyl-myo-inositol 1,3,5-orthoformate by successive treatment with triethyl orthoformate and benzoyl chloride. The dibenzoate obtained on benzylation with benzyl bromide and silver(I) oxide gave 2-O-benzoyl-4,6-di-O-benzyl-myo-inositol 1,3,5-orthoformate. Deprotection of the benzoate and the orthoformate with isobutylamine and aqueous trifluoroacetic acid, respectively gave 4,6-di-O-benzyl-myo-inositol in an overall yield of 67%. Myo-inositol orthoformate and orthoacetate were prepared and isolated as their tribenzoates. The free orthoesters were regenerated by deprotection of the benzoates by aminolysis with isobutylamine.     

Practical synthesis of all inositol stereoisomers from myo-inositol.

Synthesis of six inositol stereoisomers was successfully carried out via conduritol intermediates prepared from myo-inositol. Dihydroxylation and epoxidation followed by ring opening of the conduritol B, C and F derivatives gave epi-, allo-, muco-, neo-, DL-chiro- and scyllo-inositol. The cis-inositol derivative, which may not be prepared by this approach, was synthesized in 5 steps via 2-O-benzoyl-myo-inositol orthoformate as the key intermediate.     

Syntheses of ferulic acid derivatives and their suppressive effects on cyclooxygenase-2 promoter activity

Novel ferulic acid derivatives in which feruloyl groups were attached to the hydroxyl groups of myo-inositol 1,3,5-orthoformate derivatives were synthesized. These feruloyl-myo-inositols suppressed the cyclooxygenase-2 (COX-2) promoter activity in a concentration-dependent manner. Among the examined compounds, compound 9 showed the highest activity. A treatment with 100 microM of compound 9 for 24 h resulted in a 50% decrease of COX-2 promoter activity without marked cytotoxicity. Both the molecular structure in which two ferulic acid moieties are facing each other and the molecular hydrophobicity may be essential for the suppression of COX-2 promoter activity.     

Appel-Lee synthesis of glycosyl inositols, substrates for inositol dehydrogenase from Bacillus subtilis

We recently reported that inositol dehydrogenase (EC 1.1.1.18) from Bacillus subtilis can catalyze the highly stereoselective oxidation of 1l-4-O-substituted myo-inositol derivatives, as well as disaccharides melibiose and isomaltose, but not gentiobiose or maltose, consistent with the requirement of an alpha-(1-->6) linkage. We believed that the enzyme might therefore catalyze efficient stereoselective oxidation of the appropriate alpha-linked glycosyl inositols. We have synthesized alpha-D-glucopyranosyl-(1-->4)-(DL)-myo-inositol and alpha-d-galactopyranosyl-(1-->4)-(DL)-myo-inositol using the Appel-Lee protocol to couple benzyl-protected glycosyl donors to protected inositols. This method failed in our hands using glycosyl donors derived from D-mannose and 2-azido-2-deoxy-D-glucose. When myo-inositol 1,3,5-monoorthoformate is used as the acceptor, the reaction is regiospecific for the 4/6-position. We report here the mildest conditions known for the removal of the orthoformate group. 2-Azido-2-deoxy-alpha-D-glucopyranosyl-(1-->4)-(DL)-myo-inositol was synthesized using the trichloroacetimidate derivative as the donor, and all three pseudo-disaccharides were substrates for inositol dehydrogenase. The glucopyranosyl and galactopyranosyl derivatives displayed apparent second-order rate constants comparable to that of myo-inositol.     

Facile syntheses of 1,2,4-triazole and s-triazine glycosides from glycosyl isothiocyanates

Reaction of glycosyl isothiocyanates (la, b or c) with acyl or aroyl hydrazine gave the corresponding glycosyl thiosemicarbazides, and which were treated with Ac2O-H3PO4 to yield 1,2,4-triazole glycosides. Similar treatment of la, b or c with amidino compounds gave glycosylisothiobiurets, followed by N-bromosuccinimide (NBS) oxidation to give 1,2,4-triazole glycosides. Treatment of glycosylisothiobiurets with triethyl orthoformate gave the corresponding s-triazine glycosides.     

New methodology for automated SPOT synthesis of peptides on cellulose using 1,3,5‐triazine derivatives as linkers and as coupling reagents

Two new rigid bi‐aromatic linkers for synthesis of peptide arrays by SPOT methodology were obtained from cellulose treated with 2,4‐dichloro‐6‐methoxy‐1,3,5‐triazine. Reaction with m‐phenylenediamine gave non‐cleavable TYPE I linker which enabled attachment of the peptides via resistant to harsh reaction conditions amide, ether, and amine bonds. Reaction with 3‐Fmoc‐aminobenzoic acid followed by thermal isomerization of the intermediate “superactive” ester producing an amide‐like bond gave TYPE II linker that was very stable during peptide synthesis. However, the peptide was cleavable, with fragment of the linker, in the presence of 1 M LiOH solution. The uniform loading of the cellulose and efficient synthesis of the peptide array was achieved by using N‐(4,6‐dimethoxy‐1,3,5‐triazin‐1‐yl)‐N‐methylmorpholinium 4‐toluenesulfonate as the coupling reagent.     

Use of pressurized liquid extraction (PLE)/gas chromatography-electron capture detection (GC-ECD) for the determination of biodegradation intermediates of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in soils

A rapid, sensitive, and reproducible method was developed for quantitative determination of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and its biodegradation intermediates, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) in soils. RDX, MNX, DNX, or TNX was extracted from soil by pressurized liquid extraction (PLE), followed by cleanup using florisil. Instrumental analysis was performed using gas chromatography with electron capture detection (GC-ECD), which was highly sensitive to the parent explosive and its metabolites. The method detection limits (MDLs) were 0.243, 0.095, 0.138, and 0.057 ng/g for RDX, MNX, DNX, and TNX, respectively. The method gave high recovery (98-102%), good precision (0.22-5.14%), and reproducibility, and proved to be suitable for real world sample analysis.     

Synthesis of all possible regioisomers of scyllo-inositol phosphate

scyllo-Inositol is the all equatorial stereoisomer of myo-inositol. All possible 12 regioisomers of scyllo-inositol phosphate were synthesized for the first time via a scyllo-inositol benzoate intermediate, which was derived from a myo-inositol derivative. The stereoinversion of myo-inositol into scyllo-inositol was accomplished by Mitsunobu reaction of the vicinal cis-diol. The requisite intermediates, scyllo-inositol benzoates were obtained by benzoyl migration or random benzoylation, and phosphorylated to give scyllo-IPn.     

Microwave-assisted synthesis of imidazoles: reaction of p-toluenesulfonylmethyl isocyanide and polymer-bound imines

A convenient method for the synthesis of 1,5-disubstituted imidazoles has been developed on a polymeric support using base-promoted 1,3-dipolar cycloaddition reaction of p-toluenesulfonylmethyl isocyanide (TOSMIC) with immobilized imines under microwave irradiation. The immobilized imines were synthesized by the reaction of various primary benzyl amines with 4-formyl-3-methoxyphenoxymethyl polystyrene in the presence of trimethyl orthoformate at room temperature. Cleavage from the polymeric support using trifluoroacetic acid gave the desired 1,5-disubstituted imidazoles with excellent yield and high purity.     

Synthesis of methyl dl-(1,3/2,4,5)- and dl-(1,3,4/2,5)-2,3,4,5-tetrahydroxycyclohexane-1-carboxylates

Two new diastereoisomers of the pseudo-hexuronate methyl 2,3,4,5-tetrahydroxycyclohexane-1-carboxylate, having (1,3/2,4,5)- (8) and (1,3,4/2,5)-configurations (16), have been synthesised from the readily available bromo-lactone (1) of the endo-adduct of furan and acrylic acid. Treatment of 1 with 20% hydrogen bromide in acetic acid at 80° resulted in regioselective cleavage of the 1,4-anhydro ring to give dl-(1,3,5/2,4)-2,3-diacetoxy-4,5-dibromocyclohexane-1-carboxylic acid (2). Debromination of 2 with zinc dust gave dl-(1,3/2)-2,3-diacetoxycyclohex-4-ene-1-carboxylic acid (5). The methyl ester (6) of 5 was oxidised with osmium tetraoxide and hydrogen peroxide, followed by acetylation, to give the tetra-acetate of 8. Epoxidation of 6 gave two isomeric epoxides, each of which gave 16 on hydrolysis followed by O-deacetylation.     

Radioimmunoassay of contraceptive steroids. I. Synthesis of 6-oxomestranol 6-(O-carboxymethyl) oxime

A synthesis of 6-(O-carboxymethyl) oxime of 6-oxo-mestranol (3-methoxy-17-ethinyl-17β -hydroxy-1,3,5 (10)-estratrien-6-one) which was required for coupling with bovine serum albumin in order to produce a specific anti-sera for mestranol (3-methoxy-17-ethinyl-1,3,5 (10)-estratrien-17β-ol) has been described. 6-Oxoestradiol-17β 3-methyl ether was prepared from estradiol-17β 3,17-diacetate by chromic acid oxidation, followed by hydrolysis and methylation. It was converted to its O-carboxymethyloxime derivative which was smoothly oxidized by Jones reagent to the corresponding es estrone derivative. This was easily ethinylated with lithium acetylide-ethylenediamine complex to the desired compound. In an alternate approach to the desired compound, it was found that 6-oxoestradiol-17β 3- methyl ether could not be converted to its ketal under any of a variety of conditions. Ethinylation of 6-oxoestrone 3-methyl ether with limited amount of lithium acetylide reagent probably gave the 17α -ethinyl derivative as was indicated from IR and UV spectra, but its identity could not be further confirmed due to its extremely poor yield.     

Synthesis of feruloyl-myo-insitol derivatives and their inhibitory effects on phorbol ester-induced superoxide generation and Esptein-Barr virus activation

We prepared 14 feruloyl-myo-inositol derivatives, and evaluated the relationships between their stereostructure and inhibitory activity toward the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced superoxide (O(2)(-)) generation. And further, their suppressive effect on the TPA-induced Epstein-Barr virus (EBV) activation was examined in order to estimate their anti-carcinogenic potentials. Among the derivatives tested, 1,6-O-bis[3-(4'-hydroxy-3'-methoxyphenyl)-2-propenoyl]-myo-inositol (6b) showed an excellent suppressive activity on the O(2)(-) generation at a concentration of 20 microM. For the suppressive effects on the EBV activation, 2,4,6-O-tris[3-(4'-hydroxy-3'-methoxyphenyl)-2-propenoyl]-myo-inositol 1,3,5-orthoformate (9b) showed the highest activity at a concentration of 100 microM among the derivatives tested. These results suggest that the inhibitory potencies of feruloyl-myo-inositol derivatives depend on the stereostructure of molecules rather than the hydrophobicity of molecules.     

Microbial degradation of 19-hydroxy-sterol side chains

Experimental evidence is herein presented to show that C₂₂ acids are key intermediates in the microbial degradation of cholesterol and campesterol (β-sitosterol) side chains. Exposure of 19-hydroxy-sterols to Rhodococcus mutant K-3 gave four new steroid carboxylic acids in addition to that known as estrone (P1); the chemical structures of these metabolites were characterized as 2(3-hydroxy-1,3,5(10)-estratrien-17-yl)-propionic acid (P2), 2-methyl-6(3-hydroxy-1,3,5(10)-estratrien-17-yl)-heptanoic acid (P3), 2,3-dimethyl-6(3-hydroxy-1,3,5(10)-estratrien-17-yl)-heptanoic acid (P4), and 2(3-hydroxy-1,3,5(10), 17-estratetraen-17-yl)-propionic acid (P5). We propose a degradation pathway of 19-hydroxy-cholesterol and campesterol (β-sitosterol) side chains.     

Transport of cyclitols by a proton symport in Klebsiella aerogenes.

The respiration and the ATP content of Klebsiella aerogenes in the presence of various inhibitors were compared to the transport of scyllo-inositol. The ATPase was found to be inhibited by dicyclohexyl carbodiimide. The transport has been tested in anaerobiosis and aerobiosis. From the results obtained it is concluded that either ATP or respiration can sustain the transport activity in independent manner. 2. The energy derived from the respiratory chain reactions or the ATP hydrolysis results in electrogenic extrusion of protons. The electrochemical potential created drives the accumulation of scyllo-inositol, as shown by an increase of pH of the medium on addition of the substrate to cells in anaerobiosis. With non-induced cells no change in pH occurs, which demonstrates that proton flow is really linked to the transport. No H+/Na+ or K+ exchange is observed and the proton conductor carbonylcyanide m-chlorophenylhydrazone abolishes the pH shift caused by substrate addition. The stoichiometry of the symport H+/cyclitol is 1 and the half-maximum value of the pH variation as a function of the amount of scyllo-inositol added corresponds to a concentration of scyllo-inositol very close to the KT of influx.     

Design and synthesis of a library of tertiary amides: evaluation as mimetics of the melanocortins' active core

Two hundred and ten tertiary amides were prepared on solid phase. Diamines were coupled to activated carboxylated Wang polymer, and the polymeric substituted benzyloxycarbonyl protected diamines obtained were reacted with aldehydes or ketones in trimethyl orthoformate giving resin attached Schiff bases. Coupled resins were then reduced to secondary amines by sodium cyanoborohydride in 4% acetic acid/trimethyl orthoformate, followed by acylation with the carboxylic acid in the presence of PyBroP and diisopropylethylamine. Cleavage of tertiary amides from the resin was made by trifluoroacetic acid in the presence of scavengers (mainly 1,2-ethanedithiol). When indole derivatives were prepared, parallel alkylation with the linker fragment occurred, giving derivatives of 2-(4-hydroxybenzyl)-indole as side products. Solution synthesis or mixed liquid/solid phase preparation of title substances proved to be advantageous in cases when the above method did not give acceptable results. According to this approach an efficient formation of Schiff bases was achieved in the presence of TiCl(4). Substances were isolated by reversed phase chromatography; in some cases isomers were additionally separated by chiral chromatography on Chirobiotic T. When tested on human recombinant melanocortin receptors all the tertiary amides showed some binding affinities; for the highest affinity compounds the K(i)s reached 400 nM on MC(1), 2 microM on MC(3) and 1 microM on MC(4) and MC(5) receptors. cAMP assays of some of the title compounds showed that the tertiary amides are melanocortin receptor antagonists on the four MC receptor subtypes.     

Allyl deprotection of galacturonic acid derivatives: mechanistic aspects of mercuric-catalyzed prop-1-enyl acetal cleavage

Different deallylation methods were assayed for selective deprotection of allyl galactopyranosiduronic acid derivatives. A two-step procedure using DABCO and (Ph(3)P)(3)RhCl followed by mercuric-assisted cleavage gave quantitative yields. Reaction in the presence of [(18)O]water allowed us to obtain evidence about the mechanism of prop-1-enyl cleavage.     

Synthesis of glycosides in which the aglycon is an N-(Hydroxymethyl)amino-1,3,5-triazine derivative

The synthesis of analogues of the anti-tumour drug 2-[N-(hydroxymethyl)methylamino]-4,6-bis (dimethylamino)-1,3,5-triazine (HMPMM) in which the OH or a dimethylamino group is replaced by a carbohydrate has been explored. Triazinyl β-glycosides were readily prepared by reaction of sugars with trimethyl-triazinylammonium salts. These were made with one or two methylamino groups on the triazine for reaction with formaldehyde to give the cytotoxic NMeCH2OH group. However, reaction of the triazinyl glycosides with formaldehyde gave complex intractable mixtures. When the carbohydrate portion was changed to the fully protected 2,3,4,6-tetra-O-acetyl glucose a good yield of the 2-[N-(hydroxymethyl)methylamino]-4-(dimethylamino)-1,3,5-triazin-2-yl tetra-O-acetyl β-glucoside was obtained. However, de-acetylation using sodium methoxide also removed the N–CH2OH group. We are investigating protection of the base-sensitive N–CH2OH group as trialkylsilyl and benzyl ethers and are looking at de-acetylation methods that are more selective. We have prepared glycosides in which the sugar is joined through the oxygen of the NMeCH2OH group. Coupling of acetobromoglucose with HMPMM catalysed by silver salts was not successful. Although methyl and cyclohexyl derivatives of HMPMM may be produced in high yields by reaction of HMPMM with methyl and cyclohexyl alcohols under acidic catalysis, production of glycosides in this way gave poor yields. MNDO calculations on reactions of HMPMM helped us devise improved reaction conditions for the condensation of 2,3,4,6-tetra-O-acetyl glucose with HMPMM and its derivatives. The best procedure to generate one of the target glycosides is to react 2,3,4,6-tetra-O-acetyl glucose and formaldehyde with 2-methylamino- 4,6-bis(dimethylamino)-1,3,5-triazine. The β-glycoside product was de-acetylated using potassium carbonate in dry methanol. Abbreviations: HMM, hexamethylmelamine (2) or 2,4,6-tris(dimethylamino)-1,3,5-triazine; HMPMM, hydroxymethylpentamethylmelamine or 2-[N-(hydroxymethyl)-methylamino]-4,6-bis(dimethylamino)-1,3,5-triazine; PMM, Pentamethylmelamine or 2-methylamino-4,6-bis(dimethylamino)-1,3,5-triazine; TBMS, t-Butyldimethylsilyl; p-TSA, p-Toluenesulphonic acid This revised version was published online in November 2006 with corrections to the Cover Date.     

Formation of [3H]Inositol Metabolites in Rat Hippocampal Formation Slices Prelabelled with [3H]Inositol and Stimulated with Carbachol

Rat hippocampal formation slices were prelabelled with [3H]inositol and stimulated with carbachol for times between 7 s and 3 min. The [3H]inositol metabolites in an acid extract of the slices were resolved with anion-exchange HPLC. Carbachol dramatically increased the concentration of [3H]inositol monophosphate, [3H]inositol bisphosphate (two isomers), [3H]inositol 1,3,4-trisphosphate, [3H]inositol 1,4,5-trisphosphate, and [3H]inositol 1,3,4,5-tetrakisphosphate. The levels of [3H]inositol 1,4,5-trisphosphate rose most rapidly; they were maximally elevated after only 7 s and declined toward control levels in 1 min followed by a more sustained elevation in levels for up to 3 min. When [3H]inositol 1,4,5-trisphosphate was incubated with hippocampal formation homogenates in an ATP-containing buffer it was very rapidly metabolised. After 5 min [3H]inositol 1,4-bisphosphate, [3H]inositol 1,3,4-trisphosphate, and [3H]inositol 1,3,4,5-tetrakisphosphate could be detected in the homogenates. Under similar experimental conditions [3H]inositol 1,3,4,5-tetrakisphosphate is metabolised to [3H]inositol 1,3,4-trisphosphate and an inositol bisphosphate isomer that is not [3H]inositol 1,4-bisphosphate. We conclude that like other tissues the primary event in the hippocampus following carbachol stimulation is the activation of phosphatidylinositol 4,5-bisphosphate selective phospholipase C.     

Omega-pyridiniumalkylethers of steroidal phenols: new compounds with potent antibacterial and antiproliferative activities

Novel omega-pyridiniumalkylethers of two steroidal phenols were synthesized as compounds with potential antimicrobial activity. 3-Hydroxy-estra-1,3,5(10)-triene-17-one and 1-hydroxy-4-methyl-estra-1,3,5(10)-triene-17-one were reacted with omega,omega'-dibromoalkanes to omega-bromoalkoxy-estra-1,3,5(10)-trienes followed by reaction with pyridine to obtain the desired steroidal omega-pyridiniumalkoxy compounds as bromides. Their antimicrobial activity against strains of multiresistant Staphylococcus aureus (MRSA), a vancomycin resistant Enterococcus faecalis and fast growing mycobacteria depends clearly on the length of the alkyl chain. A strong broadband activity has been found for the compounds with eight or 10 C-atoms; in some cases better than ciprofloxacin or cetylpyridinium salts. In addition, the antiproliferative and cytotoxic activity depends on the chain length, too. The differentiation between antibacterial and cytotoxic activity is better for the steroid hybrid molecules than the cetylpyridinium salts. These new compounds can serve as lead compounds for further optimization.     

Allantoxanamide: A potent new uricase inhibitor in vivo

Allantoxanamide (2,4-dihydroxy-6-carboxamide-1,3,5-triazine) was studied as a uricase inhibitor in the rat. Uricase activity $\text{in vitro}$ was inhibited 50% by allantoxanamide at 9 × 10- M concentration. A single 250 mg/kg i.p. dose in the rat gave rise to a serum uric acid level of 14 mg/dl 6 hr after dosing; serum uric acid was still elevated (10 mg/dl) after 24 hr. At this dose level, deposition of uric acid in kidney tubules was observed. Studies with [8-¹⁴ C] uric acid indicated that the effect of allantoxanamide on serum uric acid was due to inhibition of uricase. The allantoxanamide-treated rat may serve as a useful animal model for the study of problems related to purine biosynthesis, drug-induced hyperuricemia and hyperuricosuria, and associated nephropathy.