Development of a fluorous trapping reagent for rapid detection of electrophilic reactive metabolites.

A cysteine-based fluorous trapping reagent, Rf8CYS, was developed. Rf8CYS formed adducts with soft and hard electrophilic reactive metabolites. These fluorous-tagged adducts were purified via both fluorous solid-phase extraction and the direct injection method. The highly sensitive mass spectrometric detection of an unprecedented adduct of the ticlopidine metabolite was realized.

Synthesis and in Vitro Cytotoxicity Evaluation of Jadomycins.

Jadomycins, which are benzo[b]phenanthridine-type alkaloids isolated from Streptomyces venezuelae ISP5230, exhibit cytotoxic activity against multidrug-resistant breast cancer cells. We have previously achieved the total synthesis of jadomycins using the direct arylation of juglone as a key step. In this study, we achieved the total synthesis of jadomycin T and jadomycin aglycons using L-threonine and 1-amino-2-propanol as nitrogen sources. Additionally, we evaluated the cytotoxic activity of eight compounds, including glycosides, jadomycin T, and their corresponding aglycons, in eight types of tumor cells. The evaluated jadomycins tended to exhibit stronger cytotoxic activity as aglycons than as glycosides. Although the presence of a 1,3-oxazolidine ring derived from an amino acid was not essential, the presence of the 1,3-oxazolidine ring showed strong activity when the ring had a carboxyl group. Furthermore, compared to the non-natural isomer at a different position on the phenolic hydroxyl group, the naturally occurring phenanthroviridin aglycon exhibited stronger cytotoxic activity. In addition, this study suggests that jadomycins may become lead compounds for the treatment of brain tumors; however, further studies on their ability to penetrate the blood-brain barrier are required.

Discovery of a Vitamin D Receptor-Silent Vitamin D Derivative That Impairs Sterol Regulatory Element-Binding Protein In Vivo.

Vitamin D3 metabolites inhibit the expression of lipogenic genes by impairing sterol regulatory element-binding protein (SREBP), a master transcription factor of lipogenesis, independent of their canonical activity through a vitamin D receptor (VDR). Herein, we designed and synthesized a series of vitamin D derivatives to search for a drug-like small molecule that suppresses the SREBP-induced lipogenesis without affecting the VDR-controlled calcium homeostasis in vivo. Evaluation of the derivatives in cultured cells and mice led to the discovery of VDR-silent SREBP inhibitors and to the development of KK-052 (50), the first vitamin D-based SREBP inhibitor that has been demonstrated to mitigate hepatic lipid accumulation without calcemic action in mice. KK-052 maintained the ability of 25-hydroxyvitamin D3 to induce the degradation of SREBP but lacked in the VDR-mediated activity. KK-052 serves as a valuable compound for interrogating SREBP/SCAP in vivo and may represent an unprecedented translational opportunity of synthetic vitamin D analogues.

PD-L1 expression in tongue squamous cell carcinoma.

PURPOSE: Immune checkpoint proteins programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) are important therapeutic targets for head and neck cancer. This large-scale case study aimed to analyze tongue squamous cell carcinomas (SCCs) and evaluate the correlation between PD-L1 expression and clinical prognosis. So far, this study is the largest case study on PD-L1 expression in tongue SCCs. METHODS: This is a case-control study that analyzed 121 tongue SCCs. Paraffin-embedded sections and clinical data were obtained retrospectively and immunohistochemistry with PD-L1 was performed. RESULTS: 11.6% contained ≥ 50% of PD-L1-positive cells, 57.1% of these cases had a poor prognosis with nodal metastasis. Among cases of T1/2 primary lesions with nodal metastasis, cases of high PD-L1 expression had a significantly shorter disease-free survival than cases of no PD-L1 expression (p = 0.018). The hazard ratio for high PD-L1 expression was 3.21 (95 per cent CI, 1.26-8.72) compared with no PD-L1 expression after adjusting for other factors. CONCLUSIONS: These data indicate that PD-L1 upregulation in tongue SCCs is associated with a more advanced stage and shorter disease-free survival. PD-1/PD-L1 inhibitors might hence constitute potential adjuvant therapy for tongue SCCs with PD-L1 upregulation.

Orally bioavailable HCV NS5A inhibitors of unsymmetrical structural class.

A novel unsymmetrical structural class of orally bioavailable hepatitis C virus (HCV) nonstructural 5A protein (NS5A) inhibitors has been generated by improving both the solubility and membrane permeability of the lead compound found in our previous work. The representative compound 14, with a 5-hydroxymethylpyrazine group and a 3-t-butylpropargyl group on each side of the molecule, exhibited the best oral bioavailability in this study, inhibiting not only the HCV genotype 1a, 1b, 2a, and 3a replicons with EC50 values in the picomolar range, but also inhibited 1a Q30 mutants induced by launched symmetrical inhibitors with EC50 values in the low nanomolar range.

Palladium-Catalyzed ß-Arylation of Cyclic α,ß-Unsaturated O-Methyl Oximes with Aryl Iodides.

We report a Pd-catalyzed ß-arylation of cyclic α,ß-unsaturated O-methyl oximes with aryl iodides. This reaction shows complete regioselectivity and excellent functional group tolerance. ß-Arylation of 2-cyclohexen-1-one O-methyl oxime (existing as 2 : 1 E/Z mixture) with certain aryl iodides such as 4-iodoanisole affords only ß-arylated (E)-O-methyl oximes.

Discovery of a novel unsymmetrical structural class of HCV NS5A inhibitors with low picomolar antiviral activity.

A novel unsymmetrical structural class of HCV NS5A inhibitors showing picomolar range antiviral activity has been identified. An unsymmetrical lead compound 2, generated from a substructure of a known symmetrical inhibitor 1, was optimized by extension of its substituents to interact with the hitherto unexplored site of the target protein. This approach afforded novel highly potent unsymmetrical inhibitor 20, which not only equally inhibited HCV genotypes1a, 1b, and 2a with EC50 values in the picomolar range, but also inhibited the 1a Q30K mutant induced by a launched symmetrical inhibitor daclatasvir with an EC50 in the low nanomolar range.

MACC1 expression is an indicator of recurrence in early-stage glottic cancer.

BACKGROUND: Metastasis-associated in colon cancer 1 (MACC1) has been reported to be an independent indicator of poor prognoses in some kinds of cancer due to disease metastasis or recurrence. We investigated the correlation between MACC1 expression and the prognosis of glottic cancer. METHODS: Paraffin-embedded, early-stage (I or II) glottic cancer specimens (n = 52) were immunohistochemically analyzed to explore MACC1 expression. The clinical records associated with each case were also examined. Recurrence-free survival (RFS) was estimated using the Kaplan-Meier method, and between-group RFS differences were assessed using the log-rank test. The multivariate analyses were evaluated using the Cox's proportional-hazard model. RESULTS: Patients were treated with only radiotherapy (RT) (n = 37, including 18 with T1 disease and 19 with T2 disease), or with chemoradiotherapy (CRT) (n = 15, including 1 with T1 disease and 14 with T2 disease). Eleven patients demonstrated local recurrence and two patients experienced cervical lymph node recurrence. Tumor specimens were MACC1-positive in 9 of the 13 (69.2%) patients with local or neck recurrence, and 7 of the 11 (63.6%) patients with local recurrence. The RFS rate of patients who were treated with only RT was significantly lower than that of patients who were treated with CRT (P = 0.0243). The RFS rate was significantly lower in cases with MACC1 expression than in those without MACC1 expression (P = 0.0003). Multivariate analysis revealed that MACC1 expression was an independent risk factor of local recurrence (P = 0.0016). CONCLUSION: MACC1 is an independent indicator of recurrence related to RFS in early-stage glottic cancer.

Synthetic Chemical Probes That Dissect Vitamin D Activities.

Vitamin D3 metabolites are capable of controlling gene expression in mammalian cells through two independent pathways: vitamin D receptor (VDR) and sterol regulatory element-binding protein (SREBP) pathways. In the present study, we dissect the complex biological activity of vitamin D by designing synthetic vitamin D3 analogs specific for VDR or SREBP pathway, i.e., a VDR activator that lacks SREBP inhibitory activity, or an SREBP inhibitor devoid of VDR activity. These synthetic vitamin D probes permitted identification of one of the vitamin D-responsive genes, Soat1, as an SREBP-suppressed gene. The chemical probes developed in the present study may prove useful in dissecting the intricate interplay of vitamin D actions, thereby providing insights into how vitamin D target genes are regulated.

KRAS mutations in tongue squamous cell carcinoma.

Background: p16INK4a (p16) expression in tongue cancer (TC) is reportedly not associated with human papilloma virus (HPV). Mutations of KRAS in cancer cells are most frequently observed within codon 12. However, few reports have investigated the association between KRAS mutations and p16 status in TC. Objectives: This study aimed to evaluate the influence of KRAS mutations on TC. Methods: Clinical records and surgically resected specimens of 85 TC patients were analyzed. Tumor samples were analyzed for mutations of KRAS located within codons 12 and 13. p16 staining was performed and considered positive in cases with moderate to strong nuclear and cytoplasmic staining. Results: Positive p16 staining was observed in 10 cases (11.8%). A KRAS mutation was detected in one case (1.2%). The case with KRAS mutation showed negative p16 staining. Despite being at an early stage, the patient died of lung metastasis at 43 months from initial treatment. Conclusions and Significance: KRAS mutations are not associated with p16 expression in TC and may predict poor prognosis in TC patients. Further analysis of mutations in regions other than codons 12 and 13 of KRAS will be necessary to determine the relationship between KRAS mutations and prognosis of this disease.

Stereoselective Synthesis of Four Calcitriol Lactone Diastereomers at C23 and C25.

(23 S,25 R)-Calcitriol lactone is a major metabolite of vitamin D3, but its synthesis has been far less well investigated than that of 1α,25(OH)2 vitamin D3, the active form of vitamin D3, even though the lactone is present at a significant level in serum. This paper describes stereoselective syntheses of natural calcitriol lactone and its diastereomers at C23 and C25. This work features (i) the diastereoselective Reformatsky-type crotylation of aldehyde 25 in the presence of chiral ligand L2 to construct the stereochemistry at C23 and (ii) the diastereoselective epoxidation of homoallylic-allylic alcohol 31 to control the stereochemistry at C25. These key reactions allowed us to synthesize CD-ring synthon 30 with all four stereoisomers, and these were further converted into calcitriol lactones 3a-3d by reaction with ene-yne-type A-rings 33 in the presence of a palladium (0) catalyst.

Young adult patients with squamous cell carcinoma of the tongue strongly express p16 without human papillomavirus infection.

BACKGROUND: Long-term smoking and drinking are known to contribute to the onset of tongue cancer (TC). However, the increasing incidence of TC in younger adults has been suggested to be associated with other factors. OBJECTIVES: The present study investigated the relationship between TC and human papillomavirus (HPV) infection. MATERIAL AND METHODS: Clinical records and surgically resected specimens from 86 patients (<40-years-old, n = 12; ≥40-years-old, n = 74) with TC were analyzed. Strong nuclear and cytoplasmic p16 staining was considered positive. HPV DNA (high-risk subtypes: 16, 18, 31, 33, 35, 52b, and 58; low-risk subtypes: 6 and 11) was detected using consensus primer-mediated polymerase chain reaction. RESULTS: Strong p16 expression was observed in 10 (11.6%) patients. HPV DNA was detected in 9 (10.5%) patients (high-risk subtypes, n = 2; low-risk subtypes, n = 7). Strong p16 expression was observed more frequently among younger adults than among older adults (33.3% vs. 8.1%; p = .045). p16 staining did not correlate with the detection of HPV DNA (correlation coefficient, 0.113; p = .300). CONCLUSIONS AND SIGNIFICANCE: In TC, p16 expression was not associated with HPV infection, suggesting that it may be caused by a different mechanism.

Computed Tomography Findings for Diagnosing Follicular Thyroid Neoplasms.

Since no diagnostic method has been established to distinguish follicular thyroid carcinoma (FTC) from follicular thyroid adenoma (FTA), surgery has been the only way to reach a diagnosis of follicular neoplasm. Here we investigated the computed tomography (CT) features of follicular neoplasms, toward the goal of being able to identify specific CT features allowing the preoperative differentiation of FTC from FTA. We retrospectively analyzed the cases of 205 patients who underwent preoperative CT of the neck and were histopathologically diagnosed with FTC (n=31) or FTA (n=174) after surgery between January 2002 and June 2016 at several hospitals in Japan. In each of these 205 cases, non-enhanced and contrast-enhanced CT images were obtained, and we analyzed the CT features. On univariate analysis, inhomogeneous features of tumor lesions on contrast-enhanced CT were more frequently observed in FTC than in FTA (p=0.0032). A multivariate analysis identified inhomogeneous features of tumor lesions on contrast-enhanced CT images as an independent variable indicative of FTC (p=0.0023). CT thus offers diagnostic assistance in distinguishing FTC from FTA.

Laryngeal squamous cell papilloma is highly associated with human papillomavirus.

OBJECTIVE: To delineate the association between characteristics of adult-onset laryngeal squamous cell papilloma and human papillomavirus (HPV) infection. METHODS: Clinical records and paraffin-embedded specimens of 77 papilloma patients who had been treated between 1998 and 2014 were collected. Of the 77 cases, 34 were identified in the larynx, 28 in the oral cavity and 15 in the oropharynx. Specimens were investigated by polymerase chain reaction (PCR) to detect HPV 6, 11, 16, 18, 31, 33, 35, 52b and 58, and immunohistochemical (IHC) staining for anti-p16INK4a antibody. RESULTS: In 21 cases (61.8%) with laryngeal squamous cell papilloma, various types of HPV were detected: 14 cases (41.2%) were positive of high-risk HPV, 18 (52.9%) were positive of low-risk HPV and 11 (32.4%) were positive of both high-risk HPV and low-risk HPV. Younger patients (<60 years) showed a higher rate of HPV infection than older patients. Among the 34 cases with laryngeal papilloma, no malignant transformation was observed during the study period. With IHC staining, positive expression of p16 was observed in 20 cases (58.8%). HPV infection and p16-expression were associated with the pathological finding of koilocytosis. Only four cases (14.3%) showed HPV-positivity in the oral cavity, and none of the 15 oropharyngeal cases were positive for HPV, and none of the oral cavity and oropharyngeal cases showed koilocytosis. Results of HPV-PCR and p16-IHC staining were significantly correlated each other. CONCLUSIONS: HPV infection is frequently associated with laryngeal squamous cell papilloma, and koilocytosis is a characteristic pathological finding. To the best of our knowledge, this is the first report which have described infections with multiple HPV types in laryngeal papilloma.

Synthetic studies of (23S,25R)-1α,25-dihydroxyvitamin D3 26,23-lactone (calcitriol lactone) and its derivatives.

(23S,25R)-1α,25-Dihydroxyvitamin D3 26,23-lactone (calcitriol lactone) is a major metabolite of 1α,25-dihydroxyvitamin D3 that binds to vitamin D receptor (VDR) and exhibits various biological activities. This lactone and its derivatives are considered to have potential as drug candidates to treat VDR-related diseases, but their biological activities have not yet been fully characterized, mainly because of their limited availability by chemical synthesis. This review deals with synthetic studies of calcitriol lactone, and its derivatives, i.e., methylene lactones (TEI-9647 and its derivatives) and calcitriol lactams (DLAMs). We also discuss their biological activities, VDR-binding affinity and structure-activity relationships.

Injection of basic fibroblast growth factor for unilateral vocal cord paralysis.

OBJECTIVE: Unilateral vocal cord paralysis (UVCP) not only induces severe dysphonia, but aspiration as well. Although laryngeal framework surgery is usually performed to treat this condition, the procedure is not tolerated by some patients. In the previous study, basic fibroblast growth factor (bFGF) injections for vocal cord scarring and sulcus have been reported to provide favorable outcomes while being minimally invasive. In this study, the authors retrospectively investigated phonological outcomes after bFGF injection in patients with UVCP. METHODS: This study was registered in University hospital Medical Information Network - Clinical Trials Registry (UMIN000019347). Nineteen patients with unilateral cord paralysis were treated with bFGF injection. The treatment regimen involved a single injection of 50 µg of bFGF into the muscle layer. More than six months after the injection, aerodynamic and acoustic outcomes were examined. RESULTS: The voice handicap index, maximum phonation time, mean airflow rate, and pitch range improved significantly after injection of bFGF. No sex-related differences were observed in any phonological parameter. CONCLUSION: bFGF injection, an easy method and suitable as an office procedure, significantly improved the hoarseness caused by UVCP. It is expected to be widely adopted and effective adjunctive drugs, and procedures are anticipated to be developed.

A-Ring Synthons of 19-Nor Type Vitamin D Derivatives.

Various 19-nor vitamin D analogs, which lack the methylene group at C19, exhibit significant vitamin D (VD)-related biological activities, but generally show a reduced calcemic side-effect compared with VD itself. Among them, paricalcitol is already used clinically for treatment and prevention of secondary hyperparathyroidism associated with chronic renal failure. Therefore, considerable synthetic efforts have been directed towards 19-nor VD analogs, focusing especially on A-ring synthons suitable for use in convergent synthetic strategies based on coupling of CD-ring synthons and A-ring synthons. For example, we have recently developed a new synthetic route to A-ring synthons from linear dienes based upon a ring-closing olefin metathesis strategy. Here, we review recent synthetic approaches to A-ring synthons for 19-nor VD derivatives.

Synthesis of 24,24-Difluoro-1,3-cis-25-dihydroxy-19-norvitamin D3 Derivatives and Evaluation of Their Vitamin D Receptor-Binding Affinity.

Two vitamin D3 derivatives, namely 24,24-difluoro-1ß,3ß,25-dihydroxy-19-norvitamin D3 (6a) and 24,24-difluoro-1α,3α,25-dihydroxy-19-norvitamin D3 (6b), were synthesized via a convergent route employing Julia-Kocienski olefination as a key step. Compounds 6a and b bound to vitamin D receptor (VDR) with IC50 values of 64.8 and 57.6 nM, respectively, exhibiting about 400- and 30-fold greater binding affinity than the corresponding non-fluorinated derivatives 5a and b.

Synthesis of Diastereomers of 1,3-cis-25-Dihydroxy-19-norvitamin D3.

1ß,3ß,25-Dihydroxy-19-norvitamin D3 (4a) and 1α,3α,25-dihydroxy-19-norvitamin D3 (4b) were synthesized by employing a new A-ring synthon, (1R,3S)-3-((tert-butyldimethylsilyl)oxy)-5-oxocyclohexyl benzoate (19), which was derived from D-(-)-quinic acid in 12 steps. The A-ring was coupled with the circular dichroism (CD) ring by means of Julia-Kocienski olefination to construct the diene unit. The structures of the products were confirmed by (1)H-NMR and nuclear Overhauser effect (NOE) experiments.