Dentin Sialophosphoprotein-derived Proteins in the Dental Pulp.

Porcine dentin sialophosphoprotein (DSPP), the most abundant noncollagenous protein in dentin, is critical for proper mineralization of tooth dentin. DSPP is processed by proteases into 3 major domains: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). There are at least 2 mRNA variants expressed from the Dspp gene: one encodes the full-length DSPP protein (DSP+DGP+DPP); the other encodes only DSP. The shorter transcript is generated through the use of a polyadenylation signal within intron 4, immediately following the DSP coding region (DGP and DPP are encoded by exon 5). We fractionated DSPP-derived proteins from the dental pulp of developing porcine incisors using heparin chromatography. DSP was identified, but little DPP could be detected in any fractions. BMP-1 digestion of DSPP-derived proteins extracted from dental pulp did not generate new DPP bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (indicating an absence of intact DSPP), although the results suggested another BMP-1 cleavage site within DSP. We further purified DSPP-derived protein by reversed-phase high-performance liquid chromatography. Its amino acid composition was similar to DSP. Expression of the full-length Dspp mRNA by quantitative real-time polymerase chain reaction analysis was significantly higher in odontoblasts than in pulp, while expression of the DSP-only mRNA was almost equal in odontoblasts and in the body of the pulp. Expression of the full-length Dspp mRNA was also significantly higher than the expression of DSP-only mRNA in odontoblasts. Both the full-length and the DSP-only Dspp mRNA showed only trace expression in the pulp tip. We conclude that use of the 3' polyadenylation signal in exon 5 predominates in fully differentiated odontoblasts, while both polyadenylation signals are used throughout odontoblast differentiation.

Periostin of human periodontal ligament fibroblasts promotes migration of human mesenchymal stem cell through the αvß3 integrin/FAK/PI3K/Akt pathway.

BACKGROUND AND OBJECTIVE: The periodontal ligament (PDL) is characterized by rapid turnover, high remodeling capacity and high inherent regenerative potential compared with other connective tissues. Periostin, which is highly expressed in the fibroblasts in the PDL, has been widely discussed in relation to collagen fibrillogenesis in the PDL. Recently, several reports have indicated periostin in cell migration. The aim of this study was to examine whether human PDL fibroblasts (hPDLFs) with high levels of periostin expression promote the migration of human bone marrow mesenchymal stem cells (hMSCs). MATERIAL AND METHODS: The migration of hMSCs was examined by transwell chamber migration assay under different conditions: medium alone, hPDLFs, human dermal fibroblasts, recombinant periostin, integrin αvß3 blocking antibody (anti-CD51/61 antibody) and inhibitors of FAK (PF431396) and PI3K (LY294002). Phosphorylation of FAK and Akt in hMSCs under stimulation of periostin was examined by western blotting. RESULTS: The migration assay revealed that the number of migrated hMSCs by hPDLFs was significantly larger than those by dermal fibroblasts, periostin small interfering RNA hPDLFs and medium alone. Furthermore, recombinant periostin also strongly induced hMSC migration. The addition of anti-CD51/61 antibody, PF431396 and LY294002 caused a significant reduction in the number of migrated hMSCs respectively. The anti-CD51/61 antibody inhibited both FAK and Akt phosphorylations under periostin stimulation. PF431396 inhibited both FAK and Akt phosphorylations. LY294002 inhibited only Akt phosphorylation, and FAK phosphorylation was not influenced under periostin stimulation. CONCLUSION: Periostin expression in hPDLFs promotes the migration of hMSCs through the αvß3 integrin/FAK/PI3K/Akt pathway in vitro.

DPP and DSP are Necessary for Maintaining TGF-ß1 Activity in Dentin.

Porcine dentin sialophosphoprotein (DSPP) is the most abundant non-collagenous protein in dentin. It is processed by proteases into 3 independent proteins: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). We fractionated DPP and DSP along with TGF-ß activity by ion exchange (IE) chromatography from developing pig molars and measured their alkaline phosphatase (ALP)-stimulating activity in human periodontal (HPDL) cells with or without TGF-ß receptor inhibitor. We then purified TGF-ß-unbound or -bound DPP and DSP by reverse-phase high-performance liquid chromatography (RP-HPLC) using the ALP-HPDL system. The TGF-ß isoform bound to DPP and DSP was identified as being TGF-ß1 by both ELISA and LC-MS/MS analysis. We incubated carrier-free human recombinant TGF-ß1 (CF-hTGF-ß1) with TGF-ß-unbound DPP or DSP and characterized the binding on IE-HPLC using the ALP-HPDL system. When only CF-hTGF-ß1 was incubated, approximately 3.6% of the ALP-stimulating activity remained. DPP and DSP rescued the loss of TGF-ß1 activity. Approximately 19% and 10% of the ALP stimulating activities were retained by the binding of TGF-ß to DPP and DSP, respectively. The type I collagen infrequently bound to CF-hTGF-ß1. We conclude that both DPP and DSP help retain TGF-ß1 activity in porcine dentin.

Cytodifferentiation activity of synthetic human enamel sheath protein peptides.

BACKGROUND AND OBJECTIVE: Enamel sheath protein (ESP) is involved in the construction of the enamel sheath during tooth development. The 17 kDa ESP is a one-step cleavage product processed by proteolysis from the N-terminal side of sheathlin (ameloblastin/amelin), one of the porcine enamel matrix proteins. Enamel sheath protein exhibits periodontal ligament and cementum regeneration activity in a buccal dehiscence model in dogs, and promotes the cytodifferentiation of cultured human periodontal ligament (HPDL) cells. The aim of this study was to determine the peptide segment on the C-terminal side sequence of the human ESP that possesses a cytodifferentiation activity on cultured HPDL cells. MATERIAL AND METHODS: The peptides synthesized on the basis of human ESP C-terminal side sequence were tested for their ability to increase the alkaline phosphatase (ALP) and mineralization activity of cultured HPDL cells. The expressions of osteocalcin, osteopontin and bone sialoprotein were measured by semi-quantitative PCR and therefore were determined to be specific indicators of mineralized tissue differentiation. RESULTS: Multiple synthetic peptides from the human ESP increased the ALP activity and stimulated matrix mineralization in long-term cultures of HPDL cells. Semi-quantitative PCR demonstrated the osteocalcin, osteopontin and bone sialoprotein expressions to increase relative to the control values. The peptide SDKPPKPELPGVDF had the strongest cytodifferentiation activity among all the synthetic peptides tested. CONCLUSION: A specific peptide sequence derived from the C-terminal side of the human ESP promotes the cytodifferentiation and mineralization activity of HPDL cells in a cell culture system.

Long-term effects of local pretreatment with alendronate on healing of replanted rat teeth.

BACKGROUND AND OBJECTIVE: Our previous study showed that topical alendronate, an inhibitor of bone resorption, reduces root resorption and ankylosis for 21 d after replantation of rat teeth. The aim of the present study was to evaluate the long-term inhibitory effects of topical alendronate in the replanted teeth. MATERIAL AND METHODS: The rat maxillary first molars were extracted, placed in saline containing 1 mm alendronate (alendronate group) or saline (saline group) for 5 min and then replanted. The maxillae were dissected at 60 and 120 d. Microcomputed tomography horizontal sections at three root levels were analyzed for root and bone resorption, ankylosis and pulp mineralization. RESULTS: In the alendronate group at 60 and 120 d, the frequencies of resorption of roots and bone were lower than those in the saline group. The p values show statistical significances of lower frequencies in the alendronate group than in the saline group by chi-square test (see Table 1). Ankylosis and pulp mineralization occurred in the alendronate and saline groups. Bone marrow spaces were narrowed in conjunction with bone tissue expansion around the replanted teeth in the alendronate group. CONCLUSION: The inhibitory effects of topical alendronate were retained on root and bone resorption, but not on ankylosis and pulp mineralization, in the replanted teeth for 4 mo. Alendronate might also stimulate bone formation around the rat replanted teeth.

Degradation of noncollagenous components by neutrophil elastase reduces the mechanical strength of rat periodontal ligament.

BACKGROUND AND OBJECTIVE: We have previously shown that increases in neutrophil elastase in periodontal ligament with chronic periodontitis results in degradation of the noncollagenous components. The purpose of this study was to investigate whether the destruction of noncollagenous components by treatment with elastase in vitro causes changes in the mechanical properties of the periodontal ligament. MATERIAL AND METHODS: The transverse sections of mandibular first molars, prepared from male Wistar rats at 6 wk of age, were digested with 0-50 microg/mL of neutrophil elastase at 37 degrees C for 4 h. Then, their mechanical properties and morphological features were examined. RESULTS: Digestion with elastase dose-dependently decreased the maximum shear stress and failure strain energy density of the periodontal ligament (p < 0.05-0.01). The histological observations after digestion revealed marked degradation of oxytalan fibers, but no marked changes of the collagen fibers, which was confirmed by the detection of very low quantities of hydroxyproline in the digest. The light and scanning electron micrographs showed that the elastase degraded the interfibrillar substances in the periodontal ligament and exposed individual collagen fibrils. CONCLUSION: These results suggest that the increased neutrophil elastase observed in periodontal disease degrades the oxytalan fibers and interfibrillar substances in the periodontal ligament to decrease its mechanical strength.

Neutrophil elastase is involved in the initial destruction of human periodontal ligament.

BACKGROUND AND OBJECTIVE: It has been reported that noncollagenous proteins may provide mechanical strength to the periodontal ligament. Several proteolytic activities, including that of neutrophil elastase, are reported to increase significantly in periodontal disease. The aim of this study was to investigate the function of neutrophil elastase in the initial destruction of periodontal ligament at early stages of periodontal disease. MATERIAL AND METHODS: The detection and identification of proteinases in chronic periodontitis and healthy periodontal ligament were examined by zymographic and zymo-Western analysis. The morphological changes of periodontal ligament, digested with or without authentic proteinases, were observed using scanning electron microscopy. RESULTS: Increases in neutrophil elastase, plasminogen, and matrix metalloproteinase-9 were detected in periodontal ligament from chronic periodontitis, compared with healthy periodontal ligament. Among these proteinases, only neutrophil elastase digested the intact noncollagenous proteins of periodontium. When human healthy periodontal ligament was directly digested by neutrophil elastase in an in vitro system, the morphological features were quite similar to that of the periodontal ligament in chronic periodontitis . In healthy periodontal ligament, the collagen fibrils are covered with noncollagenous proteins containing 110 kDa acidic glycoprotein, which was degraded initially by the neutrophil elastase. CONCLUSION: It was concluded that neutrophil elastase is involved in the degradation of noncollagenous protein-covered collagen fibrils in the early destructive stages of periodontal disease.

Enamel matrix derivative gel stimulates signal transduction of BMP and TGF-{beta}.

It has been shown that Emdogain Gel (Emd-Gel) containing enamel matrix proteins promotes biomineralization, such as osteogenesis and cementogenesis, during the regeneration of periodontal tissues. However, the growth factors involved in these activities of Emd-Gel remain unclear. In this study, Emd-Gel was fractionated into 22 sub-fractions by size exclusion chromatography. The osteoinductive factors, TGF-beta and BMP, were examined by a specific luciferase reporter gene assay. In the unfractionated Emd-Gel, TGF-beta-like activity was detected, while BMP activity was not. In contrast, in the fractionated Emd-Gel samples, TGF-beta-like activity was detected from fractions 8 to 13, and BMP-like activity was detected from fractions 4 to 6. Also, it was confirmed that the BMP-like activity in Emd-Gel was inhibited by authentic TGF-beta1 and TGF-beta-like activity. These results indicate that Emd-Gel contains both TGF-beta- and BMP-like growth factors that contribute to the induction of biomineralization during periodontal regeneration.

Porcine amelogenin is expressed from the X and Y chromosomes.

Amelogenin is the major enamel matrix component in developing teeth. In eutherian mammals, amelogenin is expressed from the X chromosome only, or from both the X and Y chromosomes. Two classes of porcine amelogenin cDNA clones have been characterized, but the chromosomal localization of the gene(s) encoding them is unknown. To determine if there are sex-based differences in the expression of porcine amelogenin, we paired PCR primers for exons 1a, 1b, 7a, and 7b, and amplified enamel organ-derived cDNA separately from porcine males and females. The results show that exons 1a/2a and 7a are always together and can be amplified from both males (XY) and females (XX). Exons 1b/2b and 7b are also always paired, but can be amplified only from females. We conclude that porcine amelogenin is expressed from separate genes on the X and Y chromosomes, and not, as previously proposed, from a single gene with two promoters.

Effects of alendronate on restoration of biomechanical properties of periodontium in replanted rat molars.

OBJECTIVE: We examined the effect of the pretreatment of roots with alendronate on the restoration of the support function of the healing periodontal ligament in replanted rat molars. METHODS: The left maxillary first molars were extracted, placed in 0.9% NaCl containing 1 mm alendronate (alendronate group) or 0.9% NaCl (control group) for 5 min, and were replanted into their sockets. Groups of animals were killed at 7, 14, and 21 days after replantation. Normal control rats were also killed on the same days. The force required to extract the replanted or normal tooth from its socket was measured, and a load-deformation curve was developed and analyzed. Micro-computed tomography and histologic analyses were also made. RESULTS: The mechanical properties of the healing periodontal ligament in the alendronate group were gradually restored from 7 to 21 days. However, fractures of the roots and bones during mechanical testing occurred in most of the replanted teeth in the control group at 21 days. The rates of restoration of the mechanical strength, extensibility, stiffness, and toughness for the alendronate group at 21 days were 67, 98, 74, and 68% of the normal controls, respectively. Micro-computed tomography and histologic observations revealed that bone-like structures within the pulp and ankylosis between the roots and socket bones occurred commonly in the control group, but were uncommon in the alendronate group. CONCLUSIONS: Our findings suggest that the pretreatment with alendronate inhibits the formation of abnormal mineralized tissues and results in better restoration of the support function of the healing periodontal ligament in replanted teeth.

Relative levels of mRNA encoding enamel proteins in enamel organ epithelia and odontoblasts.

Amelogenin, enamelin, sheathlin (ameloblastin/ amelin), enamelysin (MMP-20), and KLK4 (EMSP-1) are the major structural proteins and proteinases in developing tooth enamel. Recently, odontoblasts were reported to express amelogenin, the most abundant enamel protein. In this study, we hypothesized that odontoblasts express all enamel proteins and proteases, and we measured their relative mRNA levels in enamel organ epithelia and odontoblasts associated with porcine secretory- and maturation-stage enamel by RT-PCR, using a LightCycler instrument. The results showed that amelogenin mRNA in secretory-stage EOE is 320-fold higher than in odontoblasts beneath secretory-stage enamel, and over 20,000-fold higher than in odontoblasts under maturation-stage enamel. Similar results were obtained for enamelin and sheathlin. Enamelysin mRNA levels were equivalent in these two tissues, while KLK4 mRNA was higher in odontoblasts than in secretory-stage EOE. These results support the conclusion that odontoblasts are involved in the formation of the enamel layer adjacent to enamel-dentin junction.

Synergistic effect of fibroblast growth factor-4 in ectopic bone formation induced by bone morphogenetic protein-2.

Bone morphogenetic protein family members (BMPs) are essential signaling molecules during limb development and, in this process, fibroblast growth factor family members (FGFs) cooperate with BMPs. FGFs also exert anabolic effects in bone when systemically or locally applied. Thus, it is likely that the cooperation with FGFs also occurs in BMP-induced ectopic bone formation and that the exogenous FGF application would promote this bone formation. In the present study, after subcutaneously implanting recombinant human BMP-2 (rhBMP-2) in rats, we examined the expression of FGF-4 and FGF receptors (FGFRs) mRNAs and the effect of exogenous recombinant human FGF-4 (rhFGF-4) on bone formation. Three days after implantation, the pellets containing rhBMP-2 were surrounded by fibroblastic mesenchymal cells; on day 7, cartilage tissue appeared; on day 10, hypertrophic chondrocytes and a small amount of mineralized tissue were observed; and, on day 14, the amount of mineralized tissue increased. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that FGF-4 expression appeared at early stages (days 3 and 7) and its expression decreased at later stages (days 10, 14, and 21), whereas FGFRs were expressed continuously. In situ hybridization revealed that, on days 3 and 7, FGF-4, and FGFR subtypes 1 and 2 (FGFR-1 and FGFR-2) were expressed in mesenchymal cells and chondrocytes, and in the area of alkaline phosphatase (ALP) expression. On day 10, FGF-4 was not detected, whereas the expression of FGFR-1 and FGFR-2 was detectable in the area of alkaline phosphatase (ALP) expression. Injection of rhFGF-4 on days 2, 3, and 4 enhanced the mineralized tissue formation induced by rhBMP-2; however, neither rhFGF-4 treatment on days 6, 7, and 8 nor rhFGF-4 treatment on days 9, 10, and 11 influenced the amount of rhBMP-2-induced mineralization. Our results indicate that FGF-4 and FGFR signals play important roles during rhBMP-2-induced bone formation. We further suggest that the combination of rhBMP-2 and rhFGF-4 would be useful for bone augmentation.

Odontoblasts enhance the maturation of enamel crystals by secreting EMSP1 at the enamel-dentin junction.

The temporal expression patterns and activity distributions of enamelysin and EMSP1, which are the major proteinases in immature enamel, were characterized. Extracellular matrix fractions from developing porcine incisors, individually comprised of predentin, dentin, and four secretory-stage enamel samples, including the highly mineralized enamel (HME) at the enamel-dentin junction (EDJ), were isolated, and their resident proteinases were identified by zymography. Soft-tissue fractions, which included cells from the extension site of enamel formation (ESEF), secretory- and maturation-stage ameloblasts, and odontoblasts, were characterized histologically and by RT-PCR for their expression of enamelysin and EMSP1. A significant finding was that EMSP1, expressed by odontoblasts, concentrates in the HME, but is not detected in predentin or dentin. We conclude that odontoblasts deposit EMSP1 via their cell processes into the deepest enamel layer, which facilitates the hardening of this layer and contributes significantly to the functional properties of the EDJ.

Noggin blocks osteoinductive activity of porcine enamel extracts.

Enamel extracts induce biomineralization such as osteogenesis and cementogenesis, but the molecular component responsible for this activity remains uncertain. We fractionated enamel extracts from developing pig teeth and isolated the osteoinductive fraction. Proteins from pig enamel scrapings were extracted under alkaline conditions (pH 10.8) and fractionated with the use of a Sephadex G-100 (size exclusion) column. The ability of each fraction to enhance alkaline phosphatase (ALP) activity was assayed in ST2 cells, a mouse bone marrow stromal cell line. The osteoinductive fraction of enamel extracts (OFE) was found in fractions 44 and 45, which induced ST2 cells to express the phenotype of bone-forming osteoblasts, and to form mineralized nodules. Furthermore, the ALP activity of ST2 cells exposed to OFE was reduced by noggin, an antagonist of BMPs, and OFE reacted with BMP-2/4 antibody in dot-blot analysis. These results indicate that OFE contains BMPs that contribute to the induction of biomineralization.

Amelogenin gene expression in porcine odontoblasts.

Amelogenin is the major organic component in the enamel matrix of developing teeth and plays an important role in enamel biomineralization. Amelogenin has been reported to be a specific secretory product of ameloblasts. In this study, we examined amelogenin gene expression in various cell layers prepared from a porcine permanent tooth germ using reverse transcription-polymerase chain-reaction (RT-PCR). Amelogenin amplification products were detected only in the secretory ameloblast layer after 20 cycles of PCR. After 30 cycles of PCR, amelogenin amplification products were detected in secretory and maturation-stage ameloblasts and in odontoblasts. The relative levels of amelogenin gene expression in secretory and maturation-stage ameloblasts and odontoblasts were determined. Secretory ameloblasts expressed over 1000 times the level of amelogenin mRNA found in odontoblasts. Amelogenin gene expression in odontoblasts was confirmed in an erupted porcine permanent first molar, which has no ameloblasts. Amelogenin PCR amplification products were identified from 4 different alternatively spliced transcripts in the ameloblast samples, and the same spliced forms were detected in the odontoblast samples.

Calcium binding of enamel proteins and their derivatives with emphasis on the calcium-binding domain of porcine sheathlin.

Dental enamel is believed to form by the transfer of ions from solution, primarily calcium, phosphate, hydroxyl and carbonate, to the surface of solid-state mineral. Such precipitation phenomena can be controlled by regulating the degree of saturation of the solution with respect to the potential solid phases that can form. The concentration of free calcium is the factor that most affects the degree of saturation for calcium hydroxyapatite, and its buffering by calcium-binding proteins has been proposed as the mechanism that determines the enamel mineral structure. In this study, Stains-all staining was used to identify and isolate calcium-binding proteins from the enamel matrix, and determine their structures and association constants for calcium. Proteolytic cleavage fragments derived from the C-terminus of sheathlin, having apparent molecular weights of 13, 15, 27 and 29 kDa, were characterized by amino-terminal protein sequencing, amino acid analysis, and sugar, phosphate and sulphate determinations. Sheathlin C-terminal cleavage products were shown to have no N-linked glycosylations or phosphorylated amino acids, but Pro(350) was hydroxylated, and there was one sulphated O-linked glycosylation at Thr(386), containing galactose and N-acetylgalactosamine. The calcium-binding association constants for enamel proteins ranged from a high of 1.2 x 10(4) M(-1) to a low of 4.4x10(1) M(-1). The relative strengths of binding in order of decreasing affinity were: 13 and 15 kDa calcium-binding domain of sheathlin >27 and 29 kDa calcium-binding proteins >32 kDa enamelin >89 kDa enamelin >6.5 kDa, 25 kDa, 23 kDa, 20 kDa, 13 kDa, 5.3 kDa amelogenins. It is concluded that if enamel proteins have similar calcium-binding properties in vivo as have been measured in vitro, they would tend to buffer the free calcium ion concentration in enamel fluid.

Immunoblot detection and expression of enamel proteins at the apical portion of the forming root in porcine permanent incisor tooth germs.

There have been many immunohistochemical studies of enamel proteins during root formation. In the present article, the detection and expression of enamel proteins in tissue samples prepared from the apical portion of the forming root (APFR) in porcine permanent incisor tooth germs were studied. Amelogenin, enamelin, and sheathlin were detected by immunoblot analysis, but only in small amounts. The detection of their derivatives indicated their degradation. It is, at present, unclear as to which proteinases are involved in these degradations, because activity of enamel matrix serine proteinase 1 and enamelysin was not detected on gelatin and casein zymograms. The expression of enamel proteins was also proved in the APFR sample by the detection of polymerase chain reaction products of their cDNAs, and this may be related to cells of fragmentized Hertwig's epithelial root sheath. Amelogenin expression was not greater than that of enamelin and sheathlin. It was different from the expression pattern of secretory ameloblasts involved in enamel matrix formation. These results suggest that the amelogenins found in the APFR do not form a three-dimensional structure of amelogenin micelles, which has been proposed for the secretory enamel matrix structure. In this case, the enamel proteins could spread out easily following degradation into the matrix of future cementum. Some of their derivatives may play a role in the formation of the cementum.

Identification of a novel frameshift mutation (383insT) in the RUNX2 (PEBP2 alpha/CBFA1/AML3) gene in a Japanese patient with cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder due to mutations in runt-related gene 2 (RUNX2)/polyomavirus enhancer-binding protein 2alphaA (PEBP2alphaA)/core-binding factor A1 (CBFA1)/acute myeloid leukemia 3 (AML3). To investigate the RUNX2 mutations in a Japanese patient with classic CCD, we analyzed the RUNX2 gene using polymerase chain reaction (PCR)-single-strand conformation polymorphism and PCR-restriction fragment length polymorphism. The patient had hypoplasia of the clavicles, patent fontanelles, short stature, supernumerary teeth, and retention of deciduous dentition. We identified a 1-bp insertion (383insT) at codon 128 of the RUNX2 gene. The 383T insertion affects the conserved residue in the runt domain and results in premature termination in the runt domain.

Synthesis and in vitro antifungal activities of novel triazole antifungal agent CS-758.

Synthesis and in vitro antifungal activities of a novel triazole antifungal agent CS-758 (former name, R-120758) are described. The minimum inhibitory concentrations (MICs) of a series of dioxane-triazole compounds related to R-102557 were examined. Variation of the length of the chain between the dioxane ring and the phenyl ring revealed that the linkage with two double bonds is the most preferable. When a cyano group was introduced to the C4 position on the benzene ring, MICs improved further. A fluorine atom was introduced to obtain CS-758. The MICs of CS-758 surpassed those of fluconazole and itraconazole against Candida, Aspergillus and Cryptococcus species. The precursor (E,E)-aldehyde was synthesized stereoselectively from 3-fluoro-4-methylbenzonitrile using the Horner-Wadsworth-Emmons reaction.

Synthesis and antifungal activities of R-102557 and related dioxane-triazole derivatives.

Novel triazole compounds with a dioxane ring were synthesized. Condensation of the diol precursor 10 with various aromatic aldehydes 11-13 under acidic conditions afforded a series of dioxane-triazole compounds 14-16. The antifungal activities of the compounds 14-16 were evaluated in vivo in mice infection models against Candida and Aspergillus species. High activities were seen for the derivatives with one or two double bond(s) and an aromatic ring substituted with an electron-withdrawing group in the side chain. Among the derivatives, R-102557 (16R: Ar=4-(2,2,3,3-tetrafluoropropoxy)phenyl) showed excellent in vivo activities against Candida, Aspergillus and Cryptococcus species. It also showed high tolerance in a preliminary toxicity study in rats.