Long-term treatment of postmenopausal osteoporosis with active vitamin D3, 1-alpha-hydroxycholecalciferol (1 alpha-OHD3) and 1, 24 Dihydroxycholecalciferol (1, 24(OH)2D3)

The effects of active vitamin D3 analogues on radial mineral content (RMC) in postmenopausal osteoporosis were examined. Seventy eight subjects with postmenopausal osteoporosis were divided into 5 groups; Group 1 (n = 23) as the control group and Group 2 (n = 27), Group 3 (n = 8), Group 4 (n = 9) and Group 5 (n = 11) which were given 1 microgram of 1, 24(R) (OH)2D3 per day, 1 microgram of 1, 24(S)(OH)2D3 per day, 0.5 and 1 microgram of 1 alpha-OHD3 per day for 6 to 24 months, respectively. After 3-months administration of these drugs, RMC values were significantly increased in Groups 2 (102.8 +/- 1.8%), 4 (103.9 +/- 3.3%) and 5 (114.2 +/- 3.6%), when compared with the controls (97.9 +/- 2.4%). RMC in Group 3 (97.9 +/- 2.4%) was not significantly different from the control value. The administration of 1 alpha-OHD3 caused in increase in RMC in a dose-related manner. A rapid decrease in RMC was observed after withdrawal of the treatment in Groups 2, 4, and 5. A subsequent increase in RMC was observed after re-administration of 1 alpha-OHD3 and 1, 24(R)(OH)2D3. Serum Ca levels were increased in the group treated with 1, 24(R)(OH)2D3 and were decreased after the discontinuation of 1 alpha-OHD3 administration. Serum A1-P activity was decreased by treatment with 1 alpha-OHD3 (1 microgram per day) and a subsequent increase was observed in both groups treated with 1, 24(R)(OH)2D3 and 1 alpha-OHD3. Serum PTH levels were decreased by the administration of 1, 24(R)(OH)2D3 and 1 alpha-OHD3. In the group treated with 1 microgram of 1 alpha-OHD3 per day, hypercalcemia (2 out of 11 cases and these patients took calcium tablets) and an increase in BUN (1 out of 2 hypercalcemic patients) were observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of 1-deoxy-1-(indol-3-yl)-L-sorbose, 1-deoxy-1-(indol-3-yl)-L-tagatose,and their analogs

Alkaline degradation of the ascorbigen 2-C-[(indol-3-yl)methyl]-alpha-L-xylo-hex-3-ulofuranosono-1,4-lactone (1a) led to a mixture of 1-deoxy-1-(indol-3-yl)-L-sorbose (2a) and 1-deoxy-1-(indol-3-yl)-L-tagatose (3a). The mixture of diastereomeric ketoses underwent acetylation and pyranose ring opening under the action of acetic anhydride in pyridine in the presence of 4-dimethylaminopyridine (DMAP) with the formation of a mixture of (E)-2,3,4,5,6-penta-O-acetyl-1-deoxy-1-(indol-3-yl)-L-xylo-hex-1-enitol (4a) and (E)-2,3,4,5,6-penta-O-acetyl-1-deoxy-1-(indol-3-yl)-L-lyxo-hex-1-enitol (5a), which were separated chromatographically. Deacetylation of 4a or 5a afforded cyclised tetrols, tosylation of which in admixture resulted in 1-deoxy-1-(indol-3-yl)-3,5-di-O-tosyl-alpha-L-sorbopyranose (12a) and 1-deoxy-1-(indol-3-yl)-4,5-di-O-tosyl-alpha-L-tagatopyranose (13a). Under alkaline conditions 13a readily formed 2-hydroxy-4-hydroxymethyl-3-(indol-3-yl)cyclopenten-2-one (15a) in 90% yield. Similar transformations were performed for N-methyl- and N-methoxyindole derivatives.     

A practical synthesis of alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-[alpha-D-Glcp-(1-->3)]-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp, an O-specific heterohexasaccharide fragment of Citrobacter braakii O7a, 3b, 1c

An O-specific heterohexasaccharide fragment of Citrobacter braakii O7a, 3b, 1c, alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-[alpha-D-Glcp-(1-->3)]-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp was synthesized as its methyl glycoside. Acetylation of allyl 4,6-O-benzylidene-alpha-D-mannopyranoside, followed by debenzylidenization and benzoylation gave allyl 2,3-di-O-acetyl-4,6-di-O-benzoyl-alpha-D-mannopyranoside (3), and subsequent deacetylation of 3 with CH(3)COCl-MeOH gave the monosaccharide acceptor 4. Condensation of isopropyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-glucopyranoside (6) with 4 selectively afforded the alpha-(1-->3)-linked disaccharide 7. Condensation of 7 with the (1-->3)-linked disaccharide donor 9, followed by deallylation and trichloroacetimidation, afforded the tetrasaccharide donor 12. Coupling of 12 with disaccharide acceptor 13, followed by debenzylation and deacylation, furnished the target heterohexasaccharide 16.     

Synthesis of alpha-Manp-(1-->2)-alpha-Manp-(1-->3)-alpha-Manp-(1-->3)-Manp, the tetrasaccharide repeating unit of Escherichia coli O9a, and alpha-Manp-(1-->2)-alpha-Manp-(1-->2)-alpha-Manp-(1-->3)-alpha-Manp-(1-->3)-Manp, the pentasaccharide repeating unit of E. coli O9 and Klebsiella O3

The tetrasaccharide repeating unit of Escherichia coli O9a, alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-D-Manp, and the pentasaccharide repeating unit of E. coli O9 and Klebsiella O3, alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-D-Manp, were synthesized as their methyl glycosides. Thus, selective 3-O-allylation of p-methoxyphenyl alpha-D-mannopyranoside via a dibutyltin intermediate gave p-methoxyphenyl 3-O-allyl-alpha-D-mannopyranoside (2) in good yield. Benzoylation (-->3), then removal of 1-O-methoxyphenyl (right arrow4), and subsequent trichloroacetimidation afforded the 3-O-allyl-2,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (5). Condensation of 5 with methyl 4,6-O-benzylidene-alpha-D-mannopyranoside (6) selectively afforded the (1-->3)-linked disaccharide 7. Benzoylation of 7, debenzylidenation, benzoylation, and deallylation gave methyl 2,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-2,4,6-tri-O-benzoyl-alpha-D-mannopyranoside (11) as the disaccharide acceptor. Coupling of 11 with (1-->2)-linked mannose disaccharide donor 17 or trisaccharide donor 21, followed by deacylation, furnished the target tetrasaccharide and pentasaccharide, respectively.     

Metabolism of 1alpha,25-dihydroxyvitamin D(3) and its C-3 epimer 1alpha,25-dihydroxy-3-epi-vitamin D(3) in neonatal human keratinocytes

We previously reported that 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is metabolized into 1alpha,25-dihydroxy-3-epi-vitamin D(3) [1alpha,25(OH)(2)-3-epi-D(3)] in primary cultures of neonatal human keratinocytes. We now report that 1alpha,25(OH)(2)-3-epi-D(3) itself is further metabolized in human keratinocytes into several polar metabolites. One of the polar metabolite was unequivocally identified as 1alpha,23,25-trihydroxy-3-epi-vitamin D(3) by mass spectrometry and its sensitivity to sodium periodate. Three of the polar metabolites were identified as 1alpha,24,25-trihydroxy-3-epi-vitamin D(3), 1alpha,25-dihydroxy-24-oxo-3-epi-vitamin D(3) and 1alpha,23,25-trihydroxy-24-oxo-3-epi-vitamin D(3) by comigration with authentic standards on both straight and reverse phase HPLC systems. In addition to the polar metabolites, 1alpha,25(OH)(2)-3-epi-D(3) was also metabolized into two less polar metabolites. A possible structure of either 1alphaOH-3-epi-D(3)-20,25-cyclic ether or 1alphaOH-3-epi-D(3)-24,25-epoxide was assigned to one of the less polar metabolites through mass spectrometry. Thus, we indicate for the first time that 1alpha,25(OH)(2)-3-epi-D(3) is metabolized in neonatal human keratinocytes not only via the same C-24 and C-23 oxidation pathways like its parent, 1alpha,25(OH)(2)D(3); but also is metabolized into a less polar metabolite via a pathway that is unique to 1alpha,25(OH)(2)-3-epi-D(3).     

Synthesis, (1-->3)-beta-D-glucanase-binding ability and phytoalexin-elicitor activity of (R)-2,3-epoxypropyl (1-->3)-beta-D-pentaglucoside

The (1-->3)-beta-D-pentaglucoside was synthesized as its (R)-2,3-epoxypropyl glycoside via 2+3 strategy. The disaccharide donor 8 was obtained by 3-selective coupling of 2 with 4, followed by deallylation, and trichloroacetimidation. Meanwhile, the trisaccharide acceptor 12 was prepared by coupling of 10 with 4, followed by deacetylation. Condensation of 8 with 12, followed by epoxidation, and deprotection, gave the target pentaoside. The results of these bioassays demonstrated that the (1-->3)-beta-D-glucanase was obviously inactivated by 15 with k(app)=3.79 x 10(-4) min(-1). At the same time, we found that the 15 was more active as compared to the laminaripentaose in eliciting phytoalexin accumulation in tobacco cotyledon tissue, and it could be kept longer time than laminaripentaose, which indicated it is much more stable than laminaripentaose.     

Identification of benzoxazole analogs as novel, S1P(3) sparing S1P(1) agonists

A novel series of benzoxazole-derived S1P(1) agonists were designed based on scaffold hopping molecular design strategy combined with computational approaches. Extensive SAR studies led to the discovery of compound 17d as a selective S1P(1) agonist (over S1P(3)) with high CNS penetration and favorable DMPK properties. 17d also demonstrated in vivo pharmacological efficacy to reduce blood lymphocyte in mice after oral administration.     

Synthesis of the isomeric trisaccharides, methyl O-alpha-L-fucopyranosyl- (1----3, 4, and 6)-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----3)-beta- D-galactopyranoside

Benzylation of methyl 3-O-(2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D- glucopyranosyl)-2,4,6-tri-O-benzyl-beta-D-galactopyranoside with benzyl bromide in N,N-dimethylformamide in the presence of sodium hydride afforded methyl 3-O- (2-acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranosyl) -2,4,6- tri-O-benzyl-beta-D-galactopyranoside (3). Reductive ring-opening of the benzylidene group of 3 gave methyl 3-O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-beta-D- glucopyranosyl)- 2,4,6-tri-O-benzyl-beta-D-galactopyranoside (4). Cleavage of the 4,6-acetal group of 3 with hot, 80% aqueous acetic acid afforded the diol (5). Compounds 3, 4, and 5 were each subjected to halide ion-catalyzed glycosylation with 2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl bromide to produce the corresponding trisaccharide derivatives, which, on catalytic hydrogenation, furnished the title trisaccharides, respectively.     

Synthesis of (25R)-ruscogenin-1-yl 3-D-xylopyranosyl-(1-->3)

The synthesis of (25R)-ruscogenin-1-yl beta-D-xylopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-fucopyranoside, a saponin from the tuber of Liriope muscari (Decne.) Bailey, is described.     

Solution conformation of asparagine-linked oligosaccharides: alpha(1-2)-, alpha(1-3)-, beta(1-2)-, and beta(1-4)-linked units

The solution conformation is presented for representatives of each of the major classes of asparaginyl oligosaccharides. In this report the conformation of alpha(1-3)-, alpha(1-2)-, beta(1-2)-, and beta(1-4)-linked units is described. The conformational properties of these glycopeptides were determined by high-resolution 1H nuclear magnetic resonance in conjunction with potential energy calculations. The NMR parameters that were used in this analysis were chemical shifts and nuclear Overhauser enhancements. Potential energy calculations were used to evaluate the preferred conformers available for the different linkages in glycopeptides and to draw conclusions about the behavior in solution of these molecules. It was found that the linkage conformation of the Man alpha 1-3 residues was not affected by substitution either at the 2-position by alpha Man or beta GlcNAc or at the 4-position by beta GlcNAc or by the presence of a bisecting GlcNAc on the adjacent beta Man residue.     

Synthesis, resolution, and antiplatelet activity of 3-substituted 1(3H)-isobenzofuranone

A series of 3-substituted-1(3H)-isobenzofuranone 6a-g and 7a-g were synthesized from phthalic anhydride. The compound 6a-g was resolved. The antiplatelet activities of these compounds were evaluated using in vitro experiment of platelet aggregation. The levels of antiplatelet activity were displayed as following sequence: l-isomer >dl-isomer>d-isomer, respectively. The alkylphthalide is more active than the corresponding alkenephthalide. All these compounds were less active than n-butylphthalide (NBP, 6c) and Aspirin (Asp).     

Synthesis, (1-->3)-beta-D-glucanase-binding ability, and phytoalexin-elicitor activity of a mixture of 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides

We describe a approach for the synthesis of a mixture of 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides. The particular (1-->3)-beta-D-glucan isolated from the cell walls of Saccharomyces cerevisiae was recovered from the aqueous medium as water-insoluble particles by the spray drying (GS) method, and it was characterized by FTIR spectroscopy. The acid-solubilized (1-->3)-beta-D-oligoglucosides were prepared by partial acid hydrolysis of glucan particles, which were qualitatively analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE). The peracetylated 3-butenyl (1-->3)-beta-D-oligoglucosides were synthesized by treating peracetylated (1-->3)-beta-D-oligoglucosides with the 3-butenyl alcohols and a Lewis acid (SnCl4) catalyst. Epoxidation of the peracetylated 3-butenyl oligoglucosides took place with m-chloroperoxybenzoic acid (m-CPBA). NaOMe in dry methanol was used for the deacetylation of the blocked derivatives, to give the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside mixture in an overall yield of 21%. The sample was analyzed by positive-ion electrospray ionization mass spectrometry (ESIMS). In a 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside-binding (1-->3)-beta-D-glucanase assay, we found that the (1-->3)-beta-D-glucanase was obviously inactivated by the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides. At the same time, we found the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside mixture was more active as compared to the underivatized oligoglucoside mixture in eliciting phytoalexin accumulation in tobacco cotyledon tissue. Furthermore, it could be kept for a longer time than a (1-->3)-beta-D-oligoglucoside mixture, which indicated it is much more stable than (1-->3)-beta-D-oligoglucosides.     

Double-helix formation of self-complementary chimeric oligonucleotides r(CG)nd(CG)3-n (n = 0, 1, 2, 3).

Double-helix formations of self-complementary chimeric hexanucleotides, r(CGCGCG), r(CGCG)d(CG), r(CG)d(CGCG), and d(CGCGCG), have been studied spectrophotometrically an thermodynamically in 1 mol dm-3 NaCl buffer. CD (circular dichroism) spectra showed that r(CGCGCG), r(CGCG)d(CG), and r(CG)d(CGCG) formed A-type double helix, while d(CGCGCG) formed B-type double helix. The stabilization energies of these helices at 37 degrees C obtained from UV melting analyses were 9.2 kcal mol-1 for r(CGCGCG), 8.2 kcal mol-1 for r(CGCG)d(CG), 6.8 kcal mol-1 for r(CG)d(CGCG), and 8.5 kcal mol-1 for d(CGCGCG), respectively.     

Inhibition of ADAMTS4 (aggrecanase-1) by tissue inhibitors of metalloproteinases (TIMP-1, 2, 3 and 4)

ADAMTS4 (aggrecanase-1) is considered to play a key role in the degradation of aggrecan in arthritides. The inhibitory activity of tissue inhibitors of metalloproteinases (TIMPs) to ADAMTS4 was examined in an assay using aggrecan substrate. Among the four TIMPs, TIMP-3 inhibited the activity most efficiently with an IC(50) value of 7.9 nM, which was at least 44-fold lower than that of TIMP-1 (350 nM) and TIMP-2 (420 nM) and >250-fold less than that of TIMP-4 (2 microM for 35% inhibition). These results suggest that TIMP-3 is a potent inhibitor against the aggrecanase activity of ADAMTS4 in vivo.     

A novel human Gal-3-O-sulfotransferase: molecular cloning, characterization, and its implications in biosynthesis of (SO(4)-3)Galbeta1-4(Fucalpha1-3)GlcNAc

Based on sequence homology with the previously cloned human cerebroside sulfotransferase (CST) cDNA, a novel sulfotransferase was cloned by screening a human fetal brain cDNA library. The novel sulfotransferase gene was present on human chromosome 11q13; the location was different from human CST and from that of the recently cloned human beta-Gal 3'-sulfotransferase (GP3ST). The isolated cDNA contained an open reading frame that encoded a predicted protein of 431 amino acid residues with type II transmembrane topology. The amino acid sequence showed 33% identity with that of human CST and 38% with that of human GP3ST. The recombinant enzyme expressed in Chinese hamster ovary cells catalyzed transfer of sulfate to position 3 of non-reducing beta-galactosyl residues in Galbeta1-4GlcNAc. Type 2 chains served as good acceptors, whereas type 1 chains served as poor acceptors, and intermediate activity was found toward Galbeta1-3GalNAc. Therefore, the substrate specificity was different from that of GP3ST. CST activity was not detected in the newly cloned enzyme. Northern blotting analysis showed that the sulfotransferase mRNA was strongly expressed in the thyroid and moderately expressed in the brain, heart, kidney, and spinal cord. Co-transfection of the enzyme cDNA and fucosyltransferase III into COS-7 cells resulted in expression of (SO(4)-3)Galbeta1-4(Fucalpha1-3)GlcNAc and a small amount of (SO(4)-3)Galbeta1-3(Fucalpha1-4)GlcNAc. These results indicated that the newly cloned enzyme is a novel Gal-3-O-sulfotransferase and is involved in biosynthesis of the (SO(4)-3)Galbeta1-4(Fucalpha1-3)GlcNAc structure.     

Synthesis of beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->3)]-beta-D-GlcpOLauryl, an oligosaccharide with anti-tumor activity

A concise and effective synthesis of lauryl heptasaccharide 17 was achieved from the key intermediates lauryl 2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-2,4-di-O-benzoyl-beta-D-glucopyranoside (10) and isopropyl 2,4,6-tri-O-acetyl-3-O-allyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-beta-D-glucopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-1-thio-beta-D-glucopyranoside (15). The key trisaccharide glycosyl acceptor 10 was constructed by coupling 2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-alpha-D-glucopyranosyl trichloroacetimidate (3) with lauryl 6-O-acetyl-2,4-di-O-benzoyl-beta-D-glucopyranoside (9), followed by deacetylation. The thioglycoside donor 15 was obtained by condensation of 2,4,6-tri-O-acetyl-3-O-allyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (11) with isopropyl 4,6-O-benzylidene-1-thio-beta-D-glucopyranoside (12), followed by debenzylidenation and acetylation. A bioassay of the inhibition of S180 noumenal tumors showed that lauryl heptasaccharide 17 could be employed as a potential agent for cancer treatment.     

Total synthesis of the mollu-series glycosyl ceramides alpha-D-Manp-(1----3)-beta-D-Manp-(1----4)-beta-D-Glcp-(1----1)-Cer and alpha-D-Manp-(1----3)-[beta-D-Xylp-(1----2)]-beta-D-Manp-(1----4)-beta- D-Glcp-(1----1)-Cer

The mollu-series glycosphingolipids, O-alpha-D-mannopyranosyl-(1----3)-O-beta-D-mannopyranosyl-(1----4)-O-bet a-D-glucopyranosyl-(1----1)-2-N-tetracosanoyl-(4E)-sphingeni ne and O-alpha-D-mannopyranosyl-(1----3)-O-[beta-D-xylopyranosyl-(1----2])-O- beta-D-mannopyranosyl-(1----4)-O-beta-D-glucopyranosyl-(1----1)-2-N- tetracosanoyl-(4E)-sphingenine, were synthesized for the first time by using 2,3,4-tri-O-acetyl-D-xylopyranosyl trichloroacetimidate, methyl 2,3,4,6-tetra-O-acetyl-1-thio-alpha-D-mannopyranoside, benzyl O-(4,6-di-O-benzyl-beta-D-mannopyranosyl)-(1----4)-2,3,6-tri-O-benzyl-be ta-D- glucopyranoside 9, and (2S,3R,4E)-2-azido-3-O-(tert-butyldiphenylsilyl)-4-octade cene-1,3-diol 6 as the key intermediates. The hexa-O-benzyl disaccharide 9 was prepared by coupling two monosaccharide synthons, namely, 2,3-di-O-allyl-4,6-di-O-benzyl-alpha-D-mannopyranosyl bromide and benzyl 2,3,6-tri-O-benzyl-beta-D-glucopyranoside. It was demonstrated that azide 6 was highly efficient as a synthon for the ceramide part in the coupling with both glycotriaosyl and glycotetraosyl donors, particularly in the presence of trimethylsilyl triflate.     

Aggregation of poly(γ-benzyl-α,L-glutamate)

The aggregation of poly(γ-benzyl-α,L -glutamate) and its enantiomer in toluene has been investigated by following the viscosity as a function of temperature, concentration, molecular weight, molecular-weight distribution, helix chirality, and shear rate. The temperature and concentration data for a 138,000-molecular-weight sample was fitted to an open, reversible end-to-end aggregation model. The aggregation numbers resulting from this fit were consistent with the sudden onset in non-Newtonian flow resulting from only a 0.2-wt% increase in concentration. The association equilibrium constant was then used to predict viscosity for comparison with other data, in particular, the effect of molecular weight and molecular-weight distribution. A mixture of right-and left-handed helices showed the aggregation was not chiral selective. The stiffness of end-to-end aggregated (hydrogen-bonded) molecules differed little from their covalent counterparts, at least below a molecular weight of ～10⁶. We conclude that polybenzylglutamate aggregation in toluene can be described by an open end-to-end aggregation model.