The fragment molecular orbital (FMO) method is a fast quantum-mechanics method that divides systems into pieces of fragments and performs ab initio calculations. The method has been expected to improve the accuracy of describing protein-ligand interactions by incorporating electronic effects. In this article, FMO calculation with solvation methods were applied to the affinity prediction at the ATP-binding site of PDHK4. As the ionized aspartic acid lies at the center and is involved in the complex hydrogen bond networks, this system has turned out to be a difficult target to describe by traditional molecular-mechanics method. In the FMO calculation with the polarizable continuum model (PCM) solvation method, a considerable amount of charge (−0.27e) was transferred from the ionized aspartate to the surrounding residues. We found that using FMO with the PCM solvation method was important to increase the correlation, and by incorporating the ligand deformation energy, the correlation was improved to R = 0.81 for whole twelve compounds and R = 0.91 without one outlier compound.

The purpose of the present study was to provide the experimental and theoretical basis of bioequivalence (BE) dissolution test criteria for formulation development of high solubility-low permeability drugs. According to the biowaiver scheme based on the biopharmaceutics classification system (BCS), for BCS class III drugs, a test formulation and a reference formulation are predicted to be BE when 85% of the drug dissolves within 15 min (T_85% < 15 min) in the compendial dissolution test. However, previous theoretical simulation studies have suggested that this criterion may possibly be relaxed for use in practical formulation development. In the present study, the dissolution profiles of 14 famotidine formulations for which BE has been clinically confirmed were evaluated by the compendial dissolution test at pH 1.2 and 6.8. The plasma concentration–time profiles of famotidine formulations were simulated using the dissolution data. In addition, virtual simulations were performed to estimate the range of dissolution rates to be bioequivalent. The fastest and slowest dissolution rates among the famotidine formulations were T_85% = 10 min and T_85% = 60 min at pH 6.8, respectively. The virtual simulation BE study suggested that famotidine formulations can be bioequivalent when T_85% < 99 min. In the case of BCS III drugs, the rate-limiting step of oral drug absorption is the membrane permeation process rather than the dissolution process. Therefore, a difference in the dissolution process has less effect on BE. These results contribute to a better understanding of the biowaiver approach and would be of great help in the formulation development of BCS class III drugs.

It is generally accepted that the orexin 2 receptor (OX₂R) plays a critical role in the arousal-promoting function, and in vivo imaging of OX₂R is expected to contribute to elucidation of orexin systems and the development of drugs to treat sleep disorder. In this study, we newly synthesized and characterized a radioiodinated triazole-pyrolidine derivative ([¹²⁵I]TPI) to detect OX₂R in the brain. In vitro studies using OX₁R or OX₂R expression cells showed selective binding of [¹²⁵I]TPI to OX₂R. In addition, in vitro autoradiography using rat brain sections showed high accumulation of radioactivity in the OX₂R expression region. However, [¹²⁵I]TPI showed low brain uptake in normal mice. These results suggest that [¹²⁵I]TPI has a fundamental character to detect OX₂R in vitro, but further structural modification to improve brain pharmacokinetics is required to use it for in vivo detection of OX₂R.

Amphipathic peptides composed of cationic amino acids and hydrophobic amino acids have cell-penetrating ability and are often used as a delivery tool for membrane-impermeable compounds. Small interfering RNA (siRNAs) are one of the delivery targets for such cell-penetrating peptides (CPPs). Cationic CPPs can associate with anionic siRNAs by electrostatic interactions resulting in the formation of nano-sized complexes, which can deliver siRNAs intracellularly. CPPs containing unnatural amino acids offer promising tools to siRNA delivery. However, the detailed structure–activity relationship in siRNA delivery has been rarely studied. In the current study, we designed peptides containing dipropylglycine (Dpg) and explored the cellular uptake and cytotoxicity of peptide/siRNA complexes. The amphipathic structure of the peptides played a key role in complexation with siRNAs and intracellular siRNA delivery. In the amphipathic peptides, cellular uptake of siRNA increased with increasing peptide length, but cytotoxicity was reduced. A peptide containing four Dpg exhibited an effective gene-silencing effect with small amounts of peptides without cytotoxicity in medium containing serum. These findings will be helpful for the design of novel CPPs for siRNA delivery.

Mechanistic target of rapamycin (mTOR) is an effective anti-tumor drug target. Several mTOR kinase inhibitors have entered clinical research, but there are still challenges of potential toxicity. As a new type of targeted drug, proteolysis targeting chimeras (PROTACs) have features of low dosage and low toxicity. However, this approach has been rarely reported to involve mTOR degradation. In this study, the mTOR kinase inhibitor MLN0128 was used as the ligand to the protein of interest and conjugated with pomalidomide by diverse intermediate linkage chains. Several potential small molecule PROTACs for the degradation of mTOR were designed and synthesized. PROTAC compounds exhibited mTOR inhibitory activity and suppressed MCF-7 cell proliferation. The representative compound P1 could inhibit the expression of mTOR downstream proteins and the growth of cancer cells by inducing autophagy but not affecting the cell cycle and not inducing apoptosis.

Epidermal growth factor receptor (EGFR) C797S mutation leads to Osimertinib drug resistance by disturbing the covalent biding of Michael acceptor group to the Cys797 residue in the ATP biding cleft. In this manuscript, a class of 2-amine-4-oxyphosaniline pyrimidine derivatives were designed, synthesized and evaluated as new noncovalent reversible EGFR inhibitors against L858R/T790M/C797S (CTL) triple mutant. The kinases inhibitiory activity evaluation showed that four compounds exhibited significant inhibitory activities against CTL (IC₅₀ < 30 nM). In particularly, the most promising compound 7a showed excellent enzymatic inhibitory activity against CTL with IC₅₀ value of 9.9 nM, which was more potent than control compound Osimertinib. Moreover, cell proliferation assays indicated that 7a effectively inhibited H1975-EGFR L858R/T790M/C797S with IC₅₀ value of 0.33 µM. Furthermore, compound 7a displayed good metabolic stabilities in human, rat and mouse liver microsomes, and the putative biding mode of compound 7a with ATP was revealed by molecular docking study. These findings strongly indicated that compound 7a was a promising L858R/T790M/C797S mutant EGFR inhibitor.

In the present study, we conducted a detailed evaluation of the effects of humidification on the quality of five types of commercial magnesium oxide (MgO) tablet formulations. When near-IR spectroscopy was performed, a peak derived from the first overtone of the stretching vibration of the hydroxyl group was observed at approximately 7200 cm⁻¹ in a humidified MgO tablet formulation. To visually evaluate the effect of this humidification, a mapping image was created using microscopic IR spectroscopy. In the IR spectrum, a peak derived from the stretching vibration of the hydroxyl group appears at approximately 3700 cm⁻¹, so we created a mapping image using the absorbance ratio of 3700 and 3400 cm⁻¹ as an index. In the mapping image of humidified MgO tablet formulations, many areas had a higher absorbance ratio than the dried tablet formulations. From these results, it is qualitatively confirmed that the MgO was changed to magnesium hydroxide (Mg(OH)₂) by humidification. Although these results were observed in the four types of MgO tablet formulations, only one type of tablet formulation was less affected by humidification. In addition, although most tablet formulations tended to prolong disintegration time due to humidification, there was almost no effect of humidification on the disintegration time in one type of tablet formulation, which had little change in the above evaluation. Thus, in most commercial MgO tablet formulations, humidification prolongs the disintegration time, and Mg(OH)₂ significantly contributes to this factor.

Palladium-catalyzed, hydroxy-group-directed C–H arylation of [1,1′-biphenyl]-2-ols with chloroarenes was performed. The reaction showed a broad substrate scope and was successfully applied to pharmaceuticals containing a chloro group. Using 2-heteroarylphenols instead of [1,1′-biphenyl]-2-ols also yielded the desired products. The arylated product was further transformed into a triphenylene derivative.

Intercellular lipids fill the interstices of corneocytes and serve a barrier function. The amount of transdermal water evaporation varies depending on the packing structure of intercellular lipids, as this structure is important for maintaining barrier efficacy. This packing structure consists of a mixture of crystals (orthorhombic and hexagonal) and liquid crystals (fluid phase), and the proportion of these phases is thought to affect barrier function. However, there have been no methods to visualize the actual distribution of the domains formed by packing structure in intercellular lipids. In this study, the planar distribution of intercellular lipid structures was determined using focal plane array (FPA)-based Fourier transform (FT) IR imaging analysis of stratum corneum cell units obtained by grid stripping. The lipid composition of ceramides was revealed by electrospray ionization tandem mass spectrometry (ESI-MS/MS)-based shotgun lipidomics. The distribution of domains formed by packing structures and the lipid composition of ceramides was compared in skin with high- or low-transepidermal water loss (TEWL). The orthorhombic proportion was lower in high-TEWL skin than in low-TEWL skin. ESI-MS/MS-based shotgun lipidomics analysis showed that the alpha-hydroxyceramide content in the low- and high-TEWL groups differed regarding the distribution of fatty acid chain lengths. The evaluation of stratum corneum cell units using FPA-based FTIR imaging is an innovative technology that can visualize the distribution of domains formed by intercellular lipid-packing structures. Increased proportions of alpha-hydroxyceramide subclasses such as alpha-hydroxy-sphingosine ceramide and alpha-hydroxy-phytosphingosine ceramide were associated with a reduced proportion of the orthorhombic packing structure domain.

Globalization of pharmaceutical supply chains has expanded and manufacturers are required to manufacture products in compliance with the pharmacopoeial standards used in all exporting countries/regions to ensure product quality. International harmonization has been facilitated by the Pharmacopoeial Discussion Group consisting of the Japanese Pharmacopoeia, the United States Pharmacopeia, and the European Pharmacopoeia. However, since the pharmacopoeias have been developed individually under the regulatory framework of each country/region, differences exist between these pharmacopoeias. When using pharmacopoeias, an understanding of common pharmacopoeial rules is essential. Clarifying the similarities and differences in the General Notices of the pharmacopoeias widely referenced worldwide is considered valuable for those already using one or two of them to access the remaining pharmacopoeias. In this study, we compared the existence of items and the contents described in the General Notices of the three pharmacopoeias to clarify the differences. Investigation of the existence of items revealed that more than 70% of the 105 items in General Notices in the three pharmacopoeias were in the entire pharmacopoeias (for Japan, including Japanese laws and notifications). Furthermore, investigating contents revealed that approximately 20% of the 105 items have some differences such as numerical values and test conditions. However, it was shown that most of the items did not have major differences. It is expected that the three pharmacopoeias will be utilized simultaneously by understanding the similarities and differences shown in this study.

Understanding the characteristics of crystal polymorphism of active pharmaceutical ingredients and analyzing them with high sensitivity is important for quality of drug products, appropriate characterization strategies, and appropriate screening and selection processes. However, there are few methods to measure intra- and intermolecular correlations in crystals other than X-ray crystallography, for which it is sometimes difficult to obtain suitable single crystals. Recently, solid-state NMR has been recognized as a straightforward method for measuring molecular correlations. In this study, we selected ranitidine hydrochloride, which is known to exist in two forms, 1 and 2, as the model drug and investigated each form using solid-state NMR. In conducting the analysis, rotating the sample tube, which had a 1-mm inner diameter, increased the solid-state NMR resolution at 70 kHz. The ¹H–¹⁴N dipolar-based heteronuclear multiple quantum coherence (D-HMQC) analysis revealed the intermolecular correlation of Form 1 between the N atom of the nitro group and a proton of the furan moiety, which were closer than those of the intramolecular correlation reported using single X-ray crystal analysis. Thus, ¹H–¹⁴N D-HMQC analysis could be useful for characterizing intermolecular interaction in ranitidine hydrochloride crystals. In addition, we reassigned the ¹³C solid-state NMR signals of ranitidine hydrochloride according to the liquid-state and multiple solid-state NMR experiments.

In nucleic acid drug discovery, it is extremely important to develop a technology to understand the distribution in target organs and to trace the degradation process in the body in order to optimize the structure and improve the efficiency of the clinical trial process. Since nucleic acid drugs are essentially metabolically degraded into numerous fragments, labeling at the internal position is preferable to that at the terminus. Due to the high molar specific activity of tritium, various approaches for tritium-labeling have been studied for nucleic acid drugs. Nevertheless, a generally-applicable method for tritium labeling of the internal position of a nucleic acid has not been established. In this study, we have demonstrated a new and efficient method for site-specific tritium labeling of the cytosine base at a predefined internal position in nucleic acid drugs. This method was developed by the chemical modification of the cytosine 4-amino group with the pyridinyl vinyl keto group by the functionality-transfer reaction using the reactive oligodeoxynucleotide (ODN), followed by reduction with NaBT₄. Applicability to a variety of chemical structures, such as 5-methyl cytosine, 2′-O-methyl, 2′-fluoro ribose derivatives, Locked/Bridged nucleic acid (LNA/BNA) derivatives, as well as phosphorothioate bonds, has been evidenced using nine oligoribonucleic acid (ORN) substrates. It has been clearly demonstrated that this method is an excellent method for tritium-labeling of nucleic acid with an average conversion efficiency of 74%, an average isolated labeling yield of 60%, and an average specific activity of 61 GBq/mmol. This method is expected to contribute to the preclinical absorption, distribution, metabolism, excretion (ADME) studies of nucleic acid drug candidates.

A new coumarin derivative (1) and 30 known compounds were isolated from Mammea siamensis and Andrographis paniculata, guided by B cell-specific Moloney murine leukemia virus insertion region 1 (BMI1) promoter inhibitory activity. Among the isolated compounds, 15 compounds showed BMI1 promoter inhibitory activity, and five compounds were found to be cytotoxic. 14-Deoxy-11,12-dehydroandrographolide (18) was highly cytotoxic to DU145 cells with an IC₅₀ value of 25.4 µM. Western blotting analysis of compound 18 in DU145 cells suggested that compound 18 suppresses BMI1 expression.

Quantitative ¹H-NMR (¹H-qNMR) is useful for determining the absolute purity of organic molecules; however, it is sometimes difficult to identify the target signal(s) for quantitation because of their overlap and complexity. Therefore, we focused on the ³¹P nucleus because of the simplicity of its signals and previously reported ³¹P-qNMR in D₂O. Here we report ³¹P-qNMR of an organophosphorus compound, sofosbuvir (SOF), which is soluble in organic solvents. Phosphonoacetic acid (PAA) and 1,4-bis(trimethylsilyl)benzene-d₄ (1,4-BTMSB-d₄) were used as reference standards for ³¹P-qNMR and ¹H-qNMR, respectively, in methanol-d₄. The purity of SOF determined by ³¹P-qNMR was 100.63 ± 0.95%, whereas that determined by ¹H-qNMR was 99.07 ± 0.50%. The average half bandwidths of the ³¹P signal of PAA and SOF were 3.38 ± 2.39 and 2.22 ± 0.19 Hz, respectively, suggesting that the T₂ relaxation time of the PAA signal was shorter than that of SOF and varied among test laboratories. This difference most likely arose from the instability in the chemical shift due to the deuterium exchange of the acidic protons of PAA, which decreased the integrated intensity of the PAA signal. Next, an aprotic solvent, dimethyl sulfoxide-d₆ (DMSO-d₆), was used as the dissolving solvent with PAA and sodium 4,4-dimethyl-4-silapentanesulfonate-d₆ (DSS-d₆) as reference standards for ³¹P-qNMR and ¹H-qNMR, respectively. SOF purities determined by ³¹P-qNMR and ¹H-qNMR were 99.10 ± 0.30 and 99.44 ± 0.29%, respectively. SOF purities determined by ³¹P-qNMR agreed with the established ¹H-qNMR values, suggesting that an aprotic solvent is preferable for ³¹P-qNMR because it is unnecessary to consider the effect of deuterium exchange.

From the less polar fraction of the MeOH extract of the leaves and twigs of Omphalea oppositifolia, five new ent-rosane-type diterpenoids, named omphalines A–E (1–5), were isolated together with one known compound, 7-keto-ent-kaurane-16β,17-diol (6), by a combination of various kinds of chromatography. The structure of omphaline A (1) was elucidated to be 19-nor-ent-rosane-4,15-diene-2β,6α-diol-3-one. Omphalines B (2), C (3), D (4), and E (5) possessed two double bonds at 5- and 15-positions, and hydroxy functional groups at 3β-, 2α,3α-, 2α,3β-, and 2α,19-positions, respectively. The absolute configuration of 1 was determined by the comparison of the experimental electronic circular dichroism (ECD) spectrum and calculated ECD spectra.

We have developed a simple and accurate method for quantifying sugars in herbal medicines, which have hitherto been difficult to quantify. Using ultra performance liquid chromatography-quadrupole-time-of-flight (UPLC-Q-TOF)-MS and two types of columns with different chemical properties, we determined the optimum conditions for separating nine sugars (fructose, galactose, glucose, mannitol, sucrose, melibiose, raffinose, manninotriose, and stachyose) commonly found in herbal medicines. Separation was completed within 10 min when an apHera NH₂ HPLC column was used, although galactose and glucose could not be separated. On the other hand, the nine sugars were completely separated within 16 min when a hydrophilic interaction chromatography (HILIC)pak VG-50 2D column was used. The calibration curves obtained using those two columns gave good linearity for the sugar standards, and the coefficient of determination was 0.995 or higher. Both columns showed excellent performance with short analysis time and high sensitivity. Using our developed method, we were able to quantify sugars in galactose-free herbal medicines within 10 min and in herbal medicines containing galactose within 16 min. We revealed that our method could be used for the analysis of sugars in Angelica acutiloba and Rehmannia glutinosa roots.

Here, we synthesized three acetogenin analogs containing pyrimidine moieties linked by amine bonds, which represent the skeleton structure of pyrimidifen, a mitochondrial complex I-inhibiting insecticide. Replacing the pyrimidine moiety linked by the amine bond remarkably enhanced growth-inhibitory activity of the analogs against several human cancer cell lines. Moreover, these analogs selectively and potently inhibited the growth of these human cancer cell lines regardless of the pyrimidine substituents. Furthermore, COMPARE analyses suggested that these analogs inhibited cancer growth by inhibiting mitochondrial complex I. Our study provides insights into the design of acetogenin analogs as novel antitumor agents.

Chemically modified nucleic acids are essential for the therapeutic application of oligonucleotides. In this study, 6′-C-spiro-thymidine exhibiting a fixed torsion angle γ was designed, synthesized, and incorporated into oligonucleotides. The conformational analysis of the 6′-C-spiro-thymidine monomer revealed that its torsion angle γ was in the +synclinal range (approx. 60°), which is similar to that in a natural RNA duplex, as expected. On the other hand, the sugar conformation of the RNA duplex is known to be predominantly an N-type, whereas that of the synthesized monomer was an S-type. The results of the UV melting analysis demonstrated that the duplex-forming ability of 6′-C-spiro-thymidine was inferior to that of natural DNA. Contrarily, 6′-C-spiro-thymidine could enhance the stability of oligonucleotides toward nucleases. Particularly, the incorporation of 6′-C-spiro-thymidine on the 3′-ends of the oligonucleotides significantly increased the nuclease resistance of the oligonucleotides.

Proteins modified in a controlled manner with artificial moieties such as fluorophores or affinity tags have been shown to be a powerful tool for functional or structural analysis of proteins. A reliable way to prepare proteins with a well-defined modification is protein synthesis. Although many successful syntheses have been reported, the poor aqueous solubility of synthetic intermediates causes difficulty in the chemical synthesis of proteins. Here we describe a solubilizing strategy for poorly soluble peptides which uses chemoselective incorporation of a hydrophilic tag onto a hydrazide in a peptide. We found that a hydrophilic tag possessing a dialkoxybenzaldehyde moiety can react with peptide hydrazides through reductive N-alkylation. No protecting groups are required for this reaction, and peptides modified in this way show enhanced solubility and consequently good peak separation during HPLC purification. The tag can be removed subsequently by treatment with trifluoroacetic acid to generate a free hydrazide, which can be converted in a one-pot reaction to a thioester for further modification. This method was validated by synthesis of a Lys₆₃-linked ubiquitin dimer derivative. This late-stage solubilization can be applied in principal to any peptide and opens the possibility of the synthesis of proteins that have previously been considered inaccessible due to their poor solubility.

Computational chemistry is useful in synthetic organic chemistry, as it can be used not only to analyze reaction mechanisms, but also to calculate biosynthetic pathways and to plan and evaluate strategies for total syntheses. Here we report the computation-guided total synthesis of vitisinol G, a resveratrol dimer.