Synthesis of novobiocin derivatives and evaluation of their antigonococcal activity and pharmacokinetics.

Gonorrhea has become a serious problem because the number of infected people is increasing and the multi-drug resistance of the causative bacteria, Neisseria gonorrhoeae, is progressing. To develop novel drugs against resistant N. gonorrhoeae, we focused on the antibiotic novobiocin (1). This natural product has a different mechanism of action from existing drugs for gonorrhea, which may make it effective against resistant strains. Actually, it was applied to resistant N. gonorrhoeae, and moderate antibacterial activity was confirmed. Based on this result, we investigated the development of an antigonococcal drug with 1 as the lead compound. The pharmacophore is thought to be the noviose sugar moiety, especially around the 3'-position, so we derivatized this part in order to improve antibacterial activity. As a result, we found that 5 with an methylpyrrole ester structure have a very potent antibacterial activity. This derivative also showed excellent antigonococcal activity against resistant strains in vitro, however it has poor water solubility and pharmacokinetics because it is the acidic lipid-soluble compound. Therefore, we considered introduction of a basic substituent into the molecule would result in an amphoteric compound with improved water solubility, and we investigated further derivatization. As a result of synthesizing various derivatives, we found 47 containing imidazole with strong antigonococcal activity and greatly improved water solubility. This derivative has also improved metabolism and blood concentration in vivo, and is expected to be orally absorbed. Based on these results, we believe that 47 is a very promising anti-gonococcal lead compound and has great potential for further development.

Anti-influenza Virus Activity of Methylthio-Formycin Distinct From That of T-705.

Seasonal influenza virus epidemics result in severe illness, and occasionally influenza pandemics cause significant morbidity and mortality, although vaccines and anti-influenza virus drugs are available. By screening an in-house library, we identified methylthio-formycin (SMeFM), an adenosine analog, as a potent inhibitor of influenza virus propagation. SMeFM inhibited the propagation of influenza A and B viruses (IC50: 34.1 and 37.9 nM, respectively) and viruses showing reduced susceptibility to baloxavir and neuraminidase inhibitors but not T-705 (Favipiravir). However, the combination of T-705 and SMeFM inhibited the propagation of the influenza virus not in an antagonistic but in a slightly synergistic manner, suggesting that SMeFM has targets distinct from that of T-705. SMeFM induced A-to-C transversion mutations in virus genome RNA, and SMeFM triphosphate did not inhibit in vitro viral RNA synthesis. Our results show that SMeFM inhibits the propagation of the influenza virus by a mechanism different from that of T-705 and is a potential drug candidate to develop for anti-influenza drug.

Synthesis and antiviral activity of formycin derivatives with anti-influenza virus activity.

In a screening using our unique natural product library, the C-nucleoside antibiotic formycin A, which exerts strong anti-influenza virus activity, was rediscovered. Aiming to develop a new type of anti-influenza virus drug, we synthesized new derivatives of formycin and evaluated its anti-influenza virus activity. Structural modifications were focused on the base moiety and sugar portion, respectively, and >40 novel formycin derivatives were synthesized. Modification of the C-7 position of the pyrazolopyrimidine ring strongly contributed to improve the activity. In particular, excellent anti-influenza virus activity was observed in the NHMe (10), SMe (12), and SeMe (15) derivatives, in which heteroatoms were introduced. In addition, in the modification of the sugar moiety, the presence of a hydroxyl group and its stereochemistry greatly affected both the expression and intensity of the activity. Furthermore, the evaluation results of the 7-SEt derivative (29) and the 2'-modified derivative (59) suggested that structural modifications may reduce cytotoxicity.

New anti-cancer chemicals Ertredin and its derivatives, regulate oxidative phosphorylation and glycolysis and suppress sphere formation in vitro and tumor growth in EGFRvIII-transformed cells.

BACKGROUND: EGFRvIII is a mutant form of the epidermal growth factor receptor gene (EGFR) that lacks exons 2-7. The resulting protein does not bind to ligands and is constitutively activated. The expression of EGFRvIII is likely confined to various types of cancer, particularly glioblastomas. Although an anti-EGFRvIII vaccine is of great interest, low-molecular-weight substances are needed to obtain better therapeutic efficacy. Thus, the purpose of this study is to identify low molecular weight substances that can suppress EGFRvIII-dependent transformation. METHODS: We constructed a new throughput screening system and searched for substances that decreased cell survival of NIH3T3/EGFRvIII spheres under 3-dimensional (3D)-culture conditions, but retained normal NIH3T3 cell growth under 2D-culture conditions. In vivo activity was examined using a mouse transplantation model, and derivatives were chemically synthesized. Functional characterization of the candidate molecules was investigated using an EGFR kinase assay, immunoprecipitation, western blotting, microarray analysis, quantitative polymerase chain reaction analysis, and measurement of lactate and ATP synthesis. RESULTS: In the course of screening 30,000 substances, a reagent, "Ertredin" was found to inhibit anchorage-independent 3D growth of sphere-forming cells transfected with EGFRvIII cDNA. Ertredin also inhibited sphere formation in cells expressing wild-type EGFR in the presence of EGF. However, it did not affect anchorage-dependent 2D growth of parental NIH3T3 cells. The 3D-growth-inhibitory activity of some derivatives, including those with new structures, was similar to Ertredin. Furthermore, we demonstrated that Ertredin suppressed tumor growth in an allograft transplantation mouse model injected with EGFRvIII- or wild-type EGFR-expressing cells; a clear toxicity to host animals was not observed. Functional characterization of Ertredin in cells expressing EGFRvIII indicated that it stimulated EGFRvIII ubiquitination, suppressed both oxidative phosphorylation and glycolysis under 3D conditions, and promoted cell apoptosis. CONCLUSION: We developed a high throughput screening method based on anchorage-independent sphere formation induced by EGFRvIII-dependent transformation. In the course of screening, we identified Ertredin, which inhibited anchorage-independent 3D growth and tumor formation in nude mice. Functional analysis suggests that Ertredin suppresses both mitochondrial oxidative phosphorylation and cytosolic glycolysis in addition to promoting EGFRvIII degradation, and stimulates apoptosis in sphere-forming, EGFRvIII-overexpressing cells.

Stereoselective Total Synthesis of KAE609 via Direct Catalytic Asymmetric Alkynylation to Ketimine.

A direct catalytic asymmetric alkynylation protocol is applied to provide the requisite enantioenriched propargylic α-tertiary amine, allowing for the stereoselective total synthesis of KAE609 (formerly NITD609 or cipargamin).

Direct catalytic asymmetric vinylogous conjugate addition of unsaturated butyrolactones to α,ß-unsaturated thioamides.

Soft Lewis acid/Brønsted base cooperative catalysts have enabled direct catalytic asymmetric vinylogous conjugate addition of α,ß- and ß,γ-unsaturated butyrolactones to α,ß-unsaturated thioamides with perfect atom economy. When using α-angelica lactone and its derivatives as pronucleophiles, as little as 0.5 mol% catalyst loading was sufficient to complete the reaction necessary to construct consecutive tri- and tetrasubstituted stereogenic centers in a highly diastereo- and enantioselective fashion.

Direct catalytic asymmetric alkynylation of ketoimines.

An efficient protocol for direct catalytic alkynylation of ketoimines is described. The simultaneous activation of a soft Lewis basic terminal alkyne and a ketoimine bearing a thiophosphinoyl group by soft Lewis acid Cu(I) is crucial for high conversion. The reaction can be rendered asymmetric with a chiral bisphosphine ligand (S,S)-Ph-BPE.

Epigenetic silencing of miR-335 and its host gene MEST in hepatocellular carcinoma.

MicroRNAs (miRNAs) are small non-coding RNAs that function as endogenous silencers of target genes. Some tumor-suppressive miRNAs are known to be epigenetically silenced by promoter DNA methylation in cancer. In the present study, we aimed to identify miRNA genes that are silenced by DNA hypermethylation in hepatocellular carcinoma (HCC). We screened for miRNA genes with promoter DNA hypermethylation using a genome-wide methylation microarray analysis in HCC cells. It was found that miR-335, which is harbored within an intron of its protein-coding host gene, MEST, was downregulated by aberrant promoter hypermethylation via further methylation assays, including methylation-specific PCR, combined bisulfite and restriction analysis, bisulfite sequencing analysis and 5-aza-2'-deoxycytidine treatment. The expression levels of miR-335 significantly correlated with those of MEST, supporting the notion that the intronic miR-335 is co-expressed with its host gene. The levels of miR-335/MEST methylation were significantly higher in 18 (90%) out of 20 primary HCC tumors, compared to their non-tumor tissue counterparts (P<0.001). The expression levels of miR-335 were significantly lower in 25 (78%) out of 32 primary HCC tumors, compared to their non-tumor tissue counterparts (P=0.001). Furthermore, the expression levels of miR-335 were significantly lower in HCC tumors with distant metastasis compared to those without distant metastasis (P=0.02). In conclusion, our results indicate that expression of miR-335 is reduced by aberrant DNA methylation in HCC.

Synthetic studies of the lichen macrolide lepranthin. Stereoselective synthesis of the diolide framework based on regioselective epoxide-opening reactions.

Stereoselective synthesis of the 16-membered diolide 27, a fully functionalized congener of lepranthin (1), is described. The requisite five asymmetric carbon centers in monomer 23 were constructed in a highly stereoselective manner by using different epoxide-opening reactions of α,ß-unsaturated γ,δ-epoxy esters and epoxy alcohol derivatives as the key steps. The monomer 23 was successfully transformed into the MOM protected diolide 27 by Yamaguchi macrolactonization.

Efficient strategy for controlling postoperative hemorrhage in total knee arthroplasty.

PURPOSE: The objective of the present study was to compare the intraoperative use of tranexamic acid (TNA) plus intra-articular diluted-epinephrine (DEP) with preoperative autologous blood donations and transfusions in reducing an allogeneic blood transfusion (ABT) in primary unilateral total knee arthroplasty (TKA). METHODS: Patients (n=133) treated with unilateral primary TKA were divided into three groups retrospectively: patients administered autologous blood transfusions were assigned to group A (n=51); patients administered preoperative injections of TNA and postoperative intra-articular injections of DEP were assigned to group B (n=42); and patients treated with the drain-clamp method in addition to injections of TNA and DEP were assigned to group C (n=40). The rate of avoidance of ABTs, postoperative blood loss, and complications (DVT/PE, skin problems) were examined. RESULTS: The differences among the three groups were not significant in terms of the proportion of patients requiring no ABTs (94% in group A, 93% in group B and 95% in group C, n.s.). The total blood loss calculated was 1,140±451 ml, 852±343 ml, and 850±296 ml, respectively (group B>A, group C>A, P=0.0009). The significant complications were not observed in three groups. CONCLUSION: The results of the study showed that the TNA plus DEP combination exerted a comparable effect with preoperative autologous blood transfusion in avoiding ABTs in unilateral primary TKA. Considering several problems of preoperative autologous blood donation, the use of TNA plus DEP is recommended. In addition, it is highly possible that allogeneic blood transfusions can be avoided for patients with preoperative Hb values≥10.5 using the method described in this study, and the need for preoperative autologous blood donations can be decreased.

Attenuation of N-nitrosodiethylamine-induced liver fibrosis by high-molecular-weight fucoidan derived from Cladosiphon okamuranus.

BACKGROUND AND AIM: Liver fibrosis is closely associated with the progression of various chronic liver diseases. Fucoidan exhibits different biological properties such as anti-inflammatory, anti-oxidant and anti-fibrotic activities. The aim of this study was to determine whether oral fucoidan administration inhibits N-nitrosodiethylamine (DEN)-induced liver fibrosis. METHODS: Liver fibrosis was induced in rats by injecting DEN (50 mg/kg). Rats were given 2% of crude fucoidan solution or 2% of high-molecular-weight (HMW) fucoidan solution. They were divided into a crude fucoidan group, an HMW fucoidan group, a DEN alone group, a DEN + crude fucoidan group, a DEN + HMW fucoidan group and a control group. RESULTS: Liver fibrosis and hepatic hydroxyproline levels were significantly more decreased in the DEN + HMW fucoidan group than in the DEN-alone group. Anti-fibrogenesis was unremarkable in the DEN + crude fucoidan group. Hepatic messenger RNA levels and immunohistochemistry of transforming growth factor beta 1 were markedly increased by DEN. This increase was attenuated by HMW fucoidan. Hepatic chemokine ligand 12 expression was increased by DEN. This increase was suppressed by HMW fucoidan. HMW fucoidan significantly decreased the DEN-induced malondialdehyde levels. Also, fucoidan markedly increased metallothionein expression in the liver. Fucoidan was clearly observed in the liver by immunohistochemical staining in HMW fucoidan-treated rats, while it was faintly stained in the livers of crude fucoidan-treated rats. CONCLUSION: These findings suggest that the HMW fucoidan treatment causes anti-fibrogenesis in DEN-induced liver cirrhosis through the downregulation of transforming growth factor beta 1 and chemokine ligand 12 expressions, and that scavenging lipid peroxidation is well-incorporated in the liver.

Moyamoya disease presenting with an acute confusional state in an elderly patient.

Moyamoya disease is the angiographic diagnosis of a clinical syndrome showing bilateral stenosis or occlusion of the distal internal carotid arteries and their major branches with extensive parenchymal, leptomeningeal, or transdural anastomoses. The clinical features normally present as reversible ischemic neurologic deficits, sensory-motor attacks with acute hemiplegia, and motor convulsion. An acute confusional state (ACS) among hospitalized patients is a frequent and serious problem. It is characterized by an acute neurologic deficit with a fluctuating course of impaired attention span, unorganized thinking, and altered levels of consciousness. We report a case of 66-year-old woman who presented with an ACS in the emergency department. The subsequent workups including a neuroradiological examination revealed a rare case of moyamoya disease with bifrontal ischemic infarction. The recognition of an ACS as a manifestation of moyamoya disease should therefore be included in the differential diagnosis of elderly patients who present with an acutely altered neuropsychiatric state. A prompt diagnosis may help to select the most appropriate therapy for this rare disorder especially in elderly patients.

Evaluation of soft-tissue balance during total knee arthroplasty.

PURPOSE: To evaluate soft-tissue balance during versus after total knee arthroplasty (TKA). METHODS: 18 men and 75 women aged 52 to 85 (mean, 68) years who had moderate-to-severe varus deformity underwent TKAs using the Scorpio non-restrictive geometry posterior-stabilised system (Stryker Howmedica Osteonics; Allendale, [NJ], USA). All surgeries were performed by a single surgeon using the medial parapatellar approach. After the bony and soft-tissue procedures, soft-tissue balance was measured intra-operatively using a tensor/balancer device. The coronal laxity--angles between the cut surfaces of the femur and tibia--were measured at 0 degree (in extension) and 90 degrees (in flexion). The central gap was also measured. Immediate postoperative soft-tissue balance was measured using an arthrometer, while anteroposterior stress radiographs were being taken. A valgus or varus force was applied just above the knee on the lateral or medial side, with the knee counter-supported and at 15 degrees flexion. RESULTS: Intra-operatively, the mean coronal laxity at 0 degree (in extension) and 90 degrees (in flexion) was 2.1 degrees and -1.6 degrees, and the mean central gaps were 21.2 and 23.5 mm, respectively. Immediate postoperative mean coronal laxity was 2.9 degrees, indicating that lateral laxity was greater than medial laxity. The postoperative coronal laxity was positively corrected to the intra-operative coronal laxity at 0 degree (r=0.304, p=0.003), but not to the intra-operative coronal laxity at 90 degrees (r= -0.07, p=0.47). CONCLUSION: Slightly greater lateral laxity was observed after TKA, although equal medial-lateral balance was achieved intra-operatively.

Evaluation of autofluorescence colonoscopy for diagnosis of superficial colorectal neoplastic lesions.

BACKGROUND AND AIM: Autofluorescence (AF) imaging, which can potentially differentiate tissue types based on differences in fluorescence emission, may be useful in the diagnosis and treatment of colorectal malignancies. This study was designed to assess the potential usefulness of AF colonoscopy for evaluating superficial colorectal neoplastic lesions. METHODS: A total of 49 colorectal lesions in 43 patients were investigated. All superficial colorectal neoplastic lesions were identified with white light (WL) colonoscopy. Each detected lesion was investigated by WL colonoscopy, AF colonoscopy, and chromoendoscopy using 0.2% indigo carmine dye. Three endoscopists, blinded to each patient's history, evaluated the still images (as obtained with these three methods) in random order and evaluated their influence on the assessment of lesion visualization. All the lesions were biopsied or resected endoscopically, with the pathological results used as the gold standard. RESULTS: For visualization of the surface appearance and differences in color of the lesions compared with the surrounding mucosa, AF colonoscopy was superior to WL colonoscopy (p < 0.01) and comparable to chromoendoscopy (Mann-Whitney U test). For visualization of the circumferential margin, AF colonoscopy was superior to WL colonoscopy (p < 0.05) but inferior to chromoendoscopy (p < 0.01). CONCLUSIONS: AF colonoscopy may be a valuable tool for detection and diagnosis of superficial colorectal neoplastic lesions.

Tissue factor pathway inhibitor 2 (TFPI2) is frequently silenced by aberrant promoter hypermethylation in gastric cancer.

Aberrant methylation of promoter CpG islands is associated with transcriptional inactivation of tumor-suppressor genes in cancer. TFPI2, a Kunitz-type serine proteinase inhibitor, has been identified as a putative tumor-suppressor gene from genome-wide screening for aberrant methylation, using a microarray combined with the methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dCyd) in various types of tumors. We assessed the methylation status of TFPI2 and investigated its expression pattern in human primary gastric cancer (GC) tissues and in GC cell lines. Hypermethylation of the promoter CpG island, which was observed in more or less all of GC cell lines, was prevalent in a high proportion of primary GC tissues (15/18, or 83%), compared with noncancerous (4/18, or 22%) or normal (0/3, or 0%) stomach tissues, and expression of TFPI2 mRNA was reduced in 7 of the 17 primary GC tissues (41%). Moreover, immunohistochemical analyses showed decreased levels of TFPI-2 protein, compared with adjacent noncancerous tissues in 8 of the 20 primary GC tissues examined (40%). TFPI2 mRNA expression was restored in gene-silenced GC cells after treatment with 5-aza-dCyd. Aberrant methylation of TFPI2 promoter CpG island occurred not only in GC cells but also in primary GC tissues at a high frequency, suggesting that epigenetic silencing of TFPI2 may contribute to gastric carcinogenesis.

Epigenetic silencing of RELN in gastric cancer.

RELN (Reelin) is an extracellular glycoprotein that plays a critical role in neuronal migration. Here we show that the RELN gene is frequently silenced in gastric cancers (GCs) by aberrant promoter hypermethylation. Although RELN was strongly expressed in non-tumor gastric epithelia, its expression was weak, or absent, in GC cell lines and primary GC tumors. Absence of RELN expression significantly correlated with a more advanced stage of GC. Methylation of the RELN promoter was frequently found in GC cell lines and in primary GC tumors. These findings suggest that disruption of the RELN pathway may be involved in gastric carcinogenesis.

Inhibitory effect of fucoidan on Huh7 hepatoma cells through downregulation of CXCL12.

The aim of this study is to assess whether fucoidan modulates the expression of chemokine ligand 12 (CXCL12)/chemokine receptor 4 (CXCR4) and exerts antitumor activity toward Huh7 hepatoma cells. According to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, fucoidan inhibited the growth of Huh7 cells and HepG2 cells in a dose-dependent manner, with a 50% inhibition of cell growth (IC50) of 2.0 and 4.0 mg/ml, respectively. alpha-fetoprotein levels in medium collected from fucoidan-treated cells were significantly decreased in Huh7 cells but not in HepG2 cells. Western blotting revealed that the amount of alpha-fetoprotein was decreased by 1.0 mg/ml of fucoidan in Huh7 cells, whereas it was unchanged in HepG2 cells. In Huh7 cells, CXCL12 mRNA expression was significantly downregulated by 1.0 mg/ml of fucoidan, whereas CXCR4 mRNA expression was unchanged by fucoidan. CXCL12 and CXCR4 mRNA were barely expressed in HepG2 cells. In addition, 1.0 mg/ml of fucoidan mildly arrested the cell cycle and induced apoptosis in Huh7 cells. The findings suggest that fucoidan exhibits antitumor activity toward Huh7 cells through the downregulation of CXCL12 expression.

Induction of type 1 interferon receptor by zinc in U937 cells.

This study aims to determine whether zinc enhances interferon (IFN)-alpha activity in U937 cells. Type 1 IFN2 receptor (IFNAR2) protein in U937 cells was measured by flow cytometry. After 24h of exposure to zinc chloride or polaprezinc (a chelate of zinc and L-carnosine) at concentrations ranging from 50 to 200 microM, histograms showing anti-IFNAR2 antibody-positive cells shifted to a higher FITC intensity. Zinc chloride and polaprezinc increased IFNAR2 mRNA levels approximately 30% and 40%, respectively, compared to the control. L-carnosine alone did not alter IFNAR2 mRNA or protein levels. Cellular levels of 2'-5' oligoadenylate synthetases (OAS) were markedly increased by IFN-alpha, and the increase was significantly accelerated by polaprezinc. However, polaprezinc alone did not increase 2'-5'OAS levels. The finding suggests that zinc, especially polaprezinc, enhances the expression of INFAR2 in U937 cells, thereby inducing production of the anti-viral protein 2'-5'OAS.