Quantitative NMR (qNMR) is applied to determine the absolute quantitative value of analytical standards for HPLC-based quantification. We have previously reported the optimal and reproducible sample preparation method for qNMR of hygroscopic reagents, such as saikosaponin a, which is used as an analytical standard in the assay of crude drug section of Japanese Pharmacopoeia (JP). In this study, we examined the absolute purity determination of a hygroscopic substance, indocyanine green (ICG), listed in the Japanese Pharmaceutical Codex 2002, using qNMR for standardization by focusing on the adaptation of ICG to JP. The purity of ICG, as an official non-Pharmacopoeial reference standard (non-PRS), had high variation (86.12 ± 2.70%) when preparing qNMR samples under non-controlled humidity (a conventional method). Additionally, residual ethanol (0.26 ± 0.11%) was observed in the non-PRS ICG. Next, the purity of non-PRS ICG was determined via qNMR when preparing samples under controlled humidity using a saturated sodium bromide solution. The purity was 84.19 ± 0.47% with a lower variation than that under non-controlled humidity. Moreover, ethanol signal almost disappeared. We estimated that residual ethanol in non-PRS ICG was replaced with water under controlled humidity. Subsequently, qNMR analysis was performed when preparing samples under controlled humidity in a constant temperature and humidity box. It showed excellent results with the lowest variation (82.26 ± 0.19%). As the use of a constant temperature and humidity box resulted in the lowest variability, it is recommended to use the control box if the reference ICG standard is needed for JP assays.

Herein, we describe two counterexamples of the previously reported β/α-selectivity of 96/4 for glycosylation using ethyl 2-O-[2,3,4-tris-O-tert-butyldimethylsilyl (TBS)-α-L-rhamnopyranosyl]-3,4,6-tris-O-TBS-thio-β-D-glucopyranoside as the glycosyl donor. Furthermore, we investigated the effects of protecting group on the rhamnose moieties in the glycosylation with cholestanol and revealed that β-selectivity originated from the two TBS groups at the 3-O and 4-O positions of rhamnose. In contrast, the TBS group at the 2-O position of rhamnose hampered the β-selectivity. Finally, the β/α-selectivity during the glycosylation was enhanced to ≥99/1. The results obtained herein suggest that the protecting groups on the sugar connected to the 2-O of a glycosyl donor with axial-rich conformation can control the stereoselectivity of glycosylation.

A novel series of 4-(4-methoxynaphthalen-1-yl)-5-arylpyrimidin-2-amines were designed, synthesized, and evaluated for their anticancer activities. Most of the synthesized compounds exhibited moderate to high antiproliferative activity in comparison to the standard drug cisplatin. Among them, 5i bearing ethoxy at the 4-position of the phenyl was found to be the most active on MCF-7 and HepG2 cancer cell lines, with IC₅₀ values of 3.77 ± 0.36 and 3.83 ± 0.26 µM, respectively. Further mechanism study shown that 5i potently inhibited tubulin polymerization, induced cell cycle arrest at G2/M phase and cell apoptosis in MCF-7 cell line. Furthermore, molecular modeling study suggested that 5i probably binds to the colchicine site of tubulin.

Regioselectivity for intramolecular Diels–Alder (IMDA) reactions of 6-acetoxy-6-alkenylcyclohexa-2,4-dien-1-ones that were formed by oxidation of 2-alkenylphenols with lead tetraacetate in acetic acid were studied. Bridged regioselectivity was observed in the IMDA reactions of 6-acetoxy-6-alkenylcyclohexa-2,4-dien-1-ones having a dienophile part which could conjugate with an aromatic group. Bridged seven- and eight-membered rings and bicyclo[2.2.2]octane skeletons were constructed by the present IMDA reactions. Density functional theory (DFT) calculations suggested that conjugation of the dienophile with neighboring aromatic groups lowered the highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) energy gap and preceded bridged [4 + 2] adducts.

N-Acetyl-7-nitroindoline has a characteristic reaction in that its acetyl group is photo-activated to acetylate amines to form amides. In this study, the N-acetyl-7-nitroindoline part was connected to the 2′-deoxyribose part at the 3- or 5-position or to a glycerol unit at the 3-position through an ethylene linker (1, 2, and 3, respectively). They were incorporated into the oligodeoxynucleotides, and their photo-reactivities toward the complementary RNA were evaluated. The acetyl group of 1 was photo-activated to form the deacelylated nitroso derivative without affecting the RNA strand. The photoreaction with 2 suggested acetylation of the RNA strand. In contrast, compound 3 formed the photo-cross-linked adduct with the RNA. These results have shown the potential application of N-acetyl-7-nitroindoline unit in aqueous solutions.

The nitrolactonization of alkenyl carboxylic acids mediated by Fe(NO₃)₃·9H₂O has been developed. Nitrolactones were obtained in up to 93% yield by treatment of alkenyl carboxylic acids with Fe(NO₃)₃·9H₂O. Mechanistic studies disclosed that the reaction proceeded through a radical intermediate generated from addition of NO₂ to alkenyl carboxylic acids.

Proteins incorporating artificial moieties such as fluorophores and drugs have enjoyed increasing use in chemical biology and drug development research. Preparation of such artificial protein derivatives has relied mainly on native chemical ligation in which peptide/protein thioesters chemoselectively react with N-terminal cysteine (Cys) peptides to afford protein molecules. The protein thioesters derived from expressed proteins represent thioesters that are very useful for the preparation of artificial proteins by native chemical ligation with synthetic peptides with N-terminal Cys. We recently have developed a traceless thioester-producing protocol using carboxypeptidase Y (CPaseY) which is compatible with an expressed protein. The traceless character is ensured by CPaseY-mediated hydrazinolysis of C-terminal Xaa (X)-Cys-proline (Pro)-leucine (Leu)-OH sequence followed by an auto-processing of the Cys-Pro (CP) dipeptide unit, affording the corresponding X-thioester (X-SR). However, hydrazinolysis of the amide bond in the prolyl leucine junction depends significantly on the nature of X. In the case of hydrophobic X residues, the hydrazinolysis overreacts to give several hydrazides while the reaction of hydrophilic X residues proceeds slowly. In this research, we attempted to develop an X-independent CPaseY-mediated protocol and found that the incorporation of a triple CP sequence into the C-terminal end (X-(CP)₃-Leu-OH) allows for efficient X-SR formation in a manner that is independent of X.

Molecular imprinting technology is a new analytical method that is highly selective and specific for certain analytes in artificial receptor design. The renewal possibilities of this technology make it an ideal material for sundry application fields. Molecularly imprinted polymers (MIPs) are polymeric matrices that have molecules printed on their surfaces; these surfaces can chemically interact with molecules or follow the pattern of the available template cavities obtained using imprinting technology. A MIP is useful for separating and analysing complex samples, such as biological fluids and environmental samples, because it is a strong analytical recognition element that can mimick natural recognition entities like biological receptors and antibodies. The MIP components consist of the target template, functional monomer, crosslinker, polymerisation initiator, and porogen. The effectiveness and selectivity of a MIP are greatly influenced by variations in the components. This review will provide an overview of the effect of MIP component ratio on analytical performance to each target analyte; it will also provide a strategy to obtain the best MIP performance. For every MIP, each template : monomer : crosslinker ratio shows a distinct performance for a specific analyte. The effects of the template : monomer : crosslinker ratio on a MIP’s analytical performances—measured by the imprinting factor, sorbent binding capacity, and sorbent selectivity—are briefly outlined.

In line with the recent globalization of the drug supply chain and promotion of the use of generic drugs worldwide, quality assurance is required for drugs globally. In particular, controlling impurities is one of the biggest areas of interest regarding pharmaceutical quality, and it is desirable that the latest scientific standards harmonized in the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) are not only implemented in approval applications but also incorporated in pharmacopoeias which are public standards to ensure pharmaceutical quality more widely. However, incorporation into a pharmacopoeia takes time because careful consideration is required owing to the characteristics of a pharmacopoeia that is widely used for drugs, including those already on the market. To consider a smooth approach for the incorporation, we retrospectively examined approaches to incorporate the concepts of the ICH Q3C, Q3D, and M7 guidelines covering residual solvents, elemental impurities, and mutagenic impurities which are particularly toxic impurities into the European Pharmacopoeia, United States Pharmacopeia-National Formulary, and Japanese Pharmacopoeia, with approaches to implement these guidelines into approval processes in Europe, the U.S., and Japan. We also identified barriers and facilitators to this goal via cause and effect analysis. Moreover, we developed a logic model for the smooth incorporation of the concepts of impurity-related ICH guidelines. We expect that our proposed approach will be applied as a framework to smoothly incorporate the results of international harmonization activities for controlling impurities into each pharmacopoeia.

In clinical practice, a thickening solution is frequently used to allow easy swallowing of tablets by patients suffering from dysphagia. This study investigated the effect of the thickening solution on tablet disintegration. Model tablets containing different disintegrants were prepared and their disintegration times (DTs) measured using standard methods. We also performed an additional disintegration test on the model tablets after immersing them for 1 min in thickening solution containing xanthan gum (XTG-SOL) (“modified disintegration test”). The DTs of the test tablets were substantially prolonged by immersion in XTG-SOL. Furthermore, the effect of the XTG-SOL on the DTs differed depending on the type of disintegrant contained in the tablets. To investigate in more detail this prolongation of tablet disintegration, we examined the contribution of tablet properties to their DTs. The properties analyzed included contact angle, T₂ relaxation time, wetting time, and water absorption ratio. The contributions of these properties to the DTs were analyzed using multiple regression analysis. This analysis clarified that the tablet properties affecting DTs changed after immersion in XTG-SOL: wetting time significantly affected the DTs measured in the normal disintegration test, while T₂ was crucial for the DTs of tablets immersed in XTG-SOL. These findings provide valuable information for design of tablet formulations, and for clinical medication management for older patients with dysphagia.

Fragment-based approach combined with electrophilic reactive compounds is a powerful strategy to discover novel covalent ligands for protein target. However, the promiscuous reactivity often interferes with identification of the fragments possessing specific binding affinity to the targeted protein. In our study, we report the fragment-based covalent drug discovery using the chemically tuned weak reactivity of chlorofluoroacetamide (CFA). We constructed a small fragment library composed of 30 CFA-appended compounds and applied it to the covalent ligand screening for cysteine protease papain as a model protein target. Using the fluorescence enzymatic assay, we identified CFA-benzothiazole 30 as a papain inhibitor, which was found to irreversibly inactivate papain upon enzyme kinetic analysis. The formation of the covalent papain-30 adduct was confirmed using electrospray ionization mass spectrometry analysis. The activity-based protein profiling (ABPP) experiment using an alkynylated analog of 30 (i.e., 30-yne) revealed that 30-yne covalently labeled papain with high selectivity. These data demonstrate potential utility of the CFA-fragment library for de novo discovery of target selective covalent inhibitors.

Oxygen atoms have a lone pair of electrons, so they have high chelation ability, high nucleophilic ability, stabilizing ability of adjacent cations, and take a chelate or oxocarbenium ion structure with Lewis acids and metals. I took advantage of these properties to develop three new reactions, 1) asymmetric synthesis of chiral quaternary carbon centers, 2) asymmetric synthesis using acetal functions, and 3) organic chemistry using acetal-type reactive salt chemical species, and applied them to biologically active natural products synthesis. New reactions described here are all innovative and useful for natural products synthesis. In particular, the first asymmetric synthesis of fredericamycin A, and concise asymmetric synthesis of anthracycline antibiotics, scyphostatin, (+)-Sch 642305, (−)-stenine, clavolonine, (+)-rubrenolide, (+)-rubrynolide, (+)-centrolobine, and decytospolide A and B, etc., are noteworthy. Furthermore, since reactions using acetal-type reactive salt chemical species allow the coexistence of functional groups that normally cannot coexist, the reactions using reactive salts have potential to change the retrosynthesis planned based on conventional reactions.

Binding assays are widely used to study the estrogenic activity of compounds targeting the estrogen receptor (ER). The fluorescence properties of benzofurazan (BD), an environmentally sensitive fluorophore, are affected by solvent polarity. In this study, we synthesized BD-labeled estradiol (E2) derivatives hoping to develop a fluorescent ligand to be used in ER binding assays, without the separation of free- from bound-ligand. Three fluorescent ligands with a BD skeleton were obtained and their fluorescence properties were investigated. Analysis of the fluorescent ligands and human recombinant ERα (hr-ERα) interactions revealed that the fluorescence intensity increased in hydrophobic environments, such as the receptor-binding site. In saturation binding assays, ABD-E2 derivative 2c showed positive cooperative binding, and its dissociation constant (K_d) and Hill coefficient were 23.4 nM and 1.34, respectively. The estrogenic compounds affinity, assessed by competitive binding assays was well correlated with the results obtained by conventional studies, using the fluorescence polarization method. Overall, the developed assay using BD-labeled ligands was a simple, rapid, and reliable method for the evaluation of ER binding affinity.

Dantrolene capsule, an effective therapeutic agent for the treatment of spasticity, is administered to children who cannot swallow the capsule after reformulation into a powder. The powdered drug can alter the specified dosage and it is also difficult to dispense the powdered formulation because of its bulky and sticky nature. To resolve these problems, we reformulated dantrolene capsules into granules using a centrifugal planetary mixer in the pharmacy. The granules containing lactose-cornstarch, D-mannitol, or microcrystalline cellulose as a diluent were examined to determine particle size distribution, flowability, drug content uniformity, and disintegration time. The granules with microcrystalline cellulose were superior to the other forms, owing to their smaller size, good drug content uniformity, and rapid disintegration. We further investigated the usability of the granules in the dispensing procedure (dividing and packing) and in the dosing process (retrieval from package) using the powders as controls. The deviation of the divided amount and loss on dosing were reduced relative to the powders. In addition, drug dissolution properties and storage stability for 12 months were the same as those of the powders. Therefore, we concluded that dantrolene granules are excellent alternatives as an extemporaneous preparation in pharmacies.

By comparing the two types of activated carbon fibers (ACFs), characteristics of adsorption sites for nitrate ion other than quaternary nitrogen (N-Q) were investigated. Using phenol resin as precursors, activation with ZnCl₂ was performed, and then heat treatment at 950 °C was carried out to prepare ACFs without N-Q, while ACFs with N-Q was prepared in the same method using polyacrylonitrile-based carbon fiber as precursors. We assessed the amount of functional groups, elemental composition, porous properties, and model of unit crystal size of graphene. For both ACFs with N-Q and without N-Q, equilibrium adsorption amount was not always simply proportional to surface area, but to the average number of benzene rings (Bz-rings) of graphene universally. PhR-5.0Z4 had only 20 benzene rings per graphene unit, but after heat treatment at 950 °C, the number drastically increased to 1088 (PhR-5.0Z4-9.5HT30). However, when the ACFs contained a large amount of oxygen, the number of Bz-rings was limited to 792 (PhR-5.0Z4-Ox-9.5HT30) even after heat treatment at 950 °C, and did not increase sufficiently. Cπ sites are more susceptible to oxygen inhibition than N-Q in adsorbing nitrate ions. For ACFs having Cπ sites as main adsorption sites, the heat treatment at 950 °C without oxidation can enhance the nitrate ion adsorption capacity.

This review summarizes the recent progress in research on the non-heme Fe(II)- and 2-oxoglutarate-dependent dioxygenases, which are involved in the biosynthesis of pharmaceutically important fungal meroterpenoids. This enzyme class activates a selective C–H bond of the substrate and catalyzes a wide range of chemical reactions, from simple hydroxylation to dynamic carbon skeletal rearrangements, thereby significantly contributing to the structural diversification and complexification of the molecules. Structure–function studies of these enzymes provide an excellent platform for the development of useful biocatalysts for synthetic biology to create novel molecules for future drug discovery.

Rubbing actions are often conducted to apply topical formulations onto the skin. Although rubbing was found to increase the skin permeation of drugs, few reports have revealed whether rubbing enhanced either drug permeation through the stratum corneum (SC) or hair follicles (HFs) pathways, or through both. In the present study, we investigated the effects of rubbing on caffeine (CAF) distribution in the SC and HFs. The effect of rubbing direction on the skin penetration of CAF was also investigated. The skin concentration of CAF and its cumulative permeation amount were increased clearly by rubbing. More than six times higher CAF concentrations in the viable epidermis and dermis were observed 5 min after rubbing application compared with no rubbing. On the other hand, slightly increased CAF concentrations were observed in the SC, suggesting that CAF was delivered through the HF pathway by rubbing. Rubbing against the natural hair direction provided the highest skin permeation as well as skin concentrations. Changes in the morphology of the HF opening area might be related to the enhancement effect. These results may provide useful information to understand the effect of rubbing on the skin permeation of applied drugs.

Two new macrolides, iriomoteolides-14a (1) and 14b (2) have been isolated from the marine dinoflagellate Amphidinium species (strain KCA09057). Compounds 1 and 2 are 15-membered macrolides, which are structural analogs of amphidinolides O (3) and P (4). The structures of 1 and 2 were assigned on the basis of detailed NMR analyses and chemical conversion studies. Compounds 1 and 2 showed moderate cytotoxic activity against human cervix adenocarcinoma HeLa cells.

NMR spectroscopy has recently been utilized to determine the absolute amounts of organic molecules with metrological traceability since signal intensity is directly proportional to the number of each nucleus in a molecule. The NMR methodology that uses hydrogen nucleus (¹H) to quantify chemicals is called quantitative ¹H-NMR (¹H qNMR). The quantitative method using ¹H qNMR for determining the purity or content of chemicals has been adopted into some compendial guidelines and official standards. However, there are still few reports in the literature regarding validation of ¹H qNMR methodology. Here, we coordinated an international collaborative study to validate a ¹H qNMR based on the use of an internal calibration methodology. Thirteen laboratories participated in this study, and the purities of three samples were individually measured using ¹H qNMR method. The three samples were all certified via conventional primary methods of measurement, such as butyl p-hydroxybenzoate Japanese Pharmacopeia (JP) reference standard certified by mass balance; benzoic acid certified reference material (CRM) certified by coulometric titration; fludioxonil CRM certified by a combination of freezing point depression method and ¹H qNMR. For each sample, ¹H qNMR experiments were optimized before quantitative analysis. The results showed that the measured values of each sample were equivalent to the corresponding reference labeled value. Furthermore, assessment of these ¹H qNMR data using the normalized error, E_n-value, concluded that statistically ¹H qNMR has the competence to obtain the same quantification performance and accuracy as the conventional primary methods of measurement.

Tyrosyl radical generation is one of the major factors for hemin/peroxide-induced oxidative stress. A method for trapping tyrosyl radical directly was developed using N-methyl luminol derivative, a tyrosine labeling reagent. N-Methyl luminol derivative selectively forms a covalent bond with a tyrosine residue under the single-electron oxidation condition. This reaction labels oxidative stress hotspots not only at the protein level but also at the level of tyrosine residues undergoing oxidation. Human serum albumin complexed with hemin was labeled at Tyr138, the tyrosine residue closest to the hemin binding site and most strongly subjected to oxidative stress caused by hemin/H₂O₂. Oxidatively damaged proteins were visualized in protein mixtures.