Exploring 2-methyl-substituted vitamin K3 derivatives with potent inhibitory activity against the 3CL protease of SARS-CoV-2.

Since the outbreak of the pandemic, various anti-SARS-CoV-2 drugs have been developed. In particular, 3CL protease (3C-like protease, 3CLpro) is an attractive drug target because it is an essential enzyme for viral multiplication and is present only in viruses, not in humans. To date, 3CLpro inhibitors against SARS-CoV-2 such as nirmatrelvir and ensitrelvir have been launched as oral drugs in Japan, but there is still no potent drug against SARS-CoV-2, due to issues of in vivo absorption and stability. Recently, vitamin K3 was reported to show inhibitory activity against 3CLpro of SARS-CoV-2, and the mechanism of action was predicted to be the formation of a covalent bond between the thiol group of cysteine 145, the active center of 3CLpro, and the C-3 position of vitamin K3. Therefore, we synthesized derivatives in which the 2-methyl group of the vitamin K3 was systematically converted to other substituents and examined their inhibitory activity against 3CLpro of SARS-CoV-2. The results showed that the compounds with the sulfide structure showed an approximately 4-fold increase in activity over vitamin K3. These results indicated the possibility of creating new inhibitors based on vitamin K3 and its derivatives.

Major Carotenoids of Meiothermus ruber Are Deinoxanthin Glucoside Esters, Not Meiothermoxanthin Glucoside Esters.

Meiothermus ruber DSMZ 1279T was isolated from a hot spring in Kamchatka and was red in color. The major carotenoid present was reported to be 1'-(ß-d-glucopyranosyloxy)-3,4,3',4'-tetradehydro-1',2'-dihydro-ß,ψ-caroten-2-one after saponification (Burgess et al. J. Nat. Prod. 1999, 62, 859-863). In this study, we purified the major carotenoids in this species without saponification. We then reidentified the major carotenoids present using spectroscopic data, including electronic circular dichroism (ECD), 1H NMR, rotating-frame nuclear Overhauser effect spectroscopy (ROESY), 13C NMR, heteronuclear single-quantum correlation spectroscopy (HSQC), heteronuclear multiple-bond correlation spectroscopy (HMBC), and MS, and enzymatic hydrolysis of fatty acid moieties and found deinoxanthin glucoside iso fatty acid esters. The bound fatty acids present included four iso types, and their composition differed from cellular lipids. Moreover, the previously identified carotenoid glucoside was a saponification artifact of deinoxanthin glucoside esters. Ketomyxocoxanthin glucoside esters and 1'-hydroxytorulene glucoside esters were also present. On the basis of the identification of carotenoids and the whole genome sequence of M. ruber, we propose a carotenoid biosynthetic pathway and note the corresponding genes.

A Novel Carotenoid with a Unique 2,6-Cyclo-ψ-End Group, Roretziaxanthin, from the Sea Squirt Halocynthia roretzi.

A novel carotenoid with a unique 2,6-cyclo-ψ-end group, named roretziaxanthin (1), was isolated from the sea squirt Halocynthia roretzi as a minor carotenoid along with (3S,3'S)-astaxanthin, alloxanthin, halocynthiaxanthin, mytiloxanthin, mytiloxanthinone, etc. This structure was determined to be 3-hydroxy-1',16'-didehydro-1',2'-dihydro-2',6'-cyclo-ß,ψ-carotene-4,4'-dione by UV-VIS, MS, and NMR spectral data. The formation mechanism of roretziaxanthin in the sea squirt was discussed.

Study on structure-activity relationship of vitamin K derivatives: Conversion of the naphthoquinone part into another aromatic ring and evaluation of their neuronal differentiation-inducing activity.

We synthesized novel vitamin K derivatives by converting the naphthoquinone group to benzene derivatives and benzoquinone. We evaluated their neuronal differentiation activities to investigate the effect of the quinone moiety on this process. We observed that the 1,4-quinone as well as the side chain part play important roles in neuronal differentiation. We also performed QSAR analysis to predict the compounds which would have higher differentiation activity.

Improving the Solid-State Photostability of Furosemide by Its Cocrystal Formation.

The photostability of three types of furosemide (FUR) cocrystal (FUR-caffeine, FUR-urea, and FUR-nicotinamide cocrystals) was studied under irradiation with a D65 fluorescent lamp. The coloration of the FUR-urea pellets was significantly faster than that of the intact FUR, whereas the coloration of FUR-nicotinamide was suppressed compared with that of intact FUR and the other cocrystals. In the case of FUR-urea, the chemical degradation of FUR increased by approximately 6.6% after irradiation for 90 d. On the other hand, FUR-nicotinamide showed better chemical stability, with only 1.3% of FUR degraded, which was significantly lower than the other cocrystals. The FUR-urea pellets showed a UV-Visible absorption spectrum similar to that of intact FUR, while the absorption range of FUR-nicotinamide shifted to a shorter wavelength. The light sensitivity of FUR-nicotinamide was improved because of the much lower emission of the D65 fluorescent lamp in the absorption range of the cocrystal.

Synthesis and In Vitro Evaluation of Novel Liver X Receptor Agonists Based on Naphthoquinone Derivatives.

We aimed to synthesize novel liver X receptor (LXR) agonists with potent agonist activity and subtype selectivity. Our synthetic scheme started with naphthoquinone derivatives, such as menadione and 2,3-dichloro-1,4-naphthoquinone. We introduced different substituents into the naphthoquinone structures, including aniline, piperidine, pyrrolidine, and morpholine, in one or two steps, and thus, we produced 14 target compounds. All 14 synthetic ligands were tested to determine whether they mediated LXR-mediated transcriptional activity. We investigated the transcriptional activity of each compound with two types of receptors, LXRα and LXRß. Among all 14 compounds, two showed weak LXRß-agonist activity, and two others exhibited potent LXRα-agonist activity. We also performed docking studies to obtain a better understanding of the modes of compound binding to LXR at the atomic level. In conclusion, we successfully synthesized naphthoquinone derivatives that act as LXRα/ß agonists and selective LXRα agonists.

Quantifying the bitter masking effect of drug-cyclodextrin complexation: NMR-ROESY mixing time approach.

Taste, especially unpleasant taste, can be key for patient compliance. In the formulation development process, drug-cyclodextrin (CD) inclusion complexes are often used to improve the solubility of a drug and/or mask its bitterness. This study aimed to evaluate the bitter masking effect of CDs on different drugs using NMR-ROESY analysis, human sensory tests, and e-tongue measurements. The strength of inclusion complex formation between drugs and CDs was investigated by NMR-ROSEY, and these results were compared to human sensory test results. In the sensory test, participants identified which drug-CD inclusion complexes were not bitter. NMR-ROSEY results aligned with the sensory tests; short magnetization transfer times corresponded to masked bitterness. The electrical tongue was not able to detect the taste of any of the drug-CD inclusion complexes. Additionally, we used NMR-ROSEY to determine which drug-CD inclusion complex formed in a system with multiple drug substances present. This research offers valuable insights into the bitter masking effect of CDs on different drugs and presents a comprehensive evaluation approach using various methods. This knowledge has significant implications for the pharmaceutical industry, clinical practice, and patient care, contributing to improved patient compliance and satisfaction with bitter medications.

Identification of Desiccation Stress-Inducible Antioxidative and Antiglycative Ultraviolet-Absorbing Oxylipins, Saclipin A and Saclipin B, in an Edible Cyanobacterium Aphanothece sacrum.

Aphanothece sacrum, a freshwater cyanobacterium, is an edible cyanobacterial strain. We identified two compounds belonging to the oxylipin family that possess UV-absorbing abilities and accumulate in the dried sample of A. sacrum. The compounds, named saclipin A and saclipin B, exhibited strong UV-absorption properties with the absorption maxima at 316 and 319 nm, respectively, and the molar extinction coefficients of 26,454 and 30,555 M-1 cm-1, respectively. The chemical structures of saclipins A and B have been elucidated, revealing that they have an all-E and a 12Z isomeric relationship within the triene structure. The saclipins could be isomerized by photoirradiation, with the cis-form saclipin B proving to be more stable in methanol, ethanol, or acetonitrile. Under drought stress conditions, the accumulation of saclipins A and B in A. sacrum was found to be increased 20- and 10-fold, respectively. Purified saclipins from A. sacrum showed biocompatibility and valuable bioactivities. Specifically, saclipins exhibited radical scavenging activity, maintaining their activity even 40 min after the reaction began. Additionally, they demonstrated inhibitory activity against glycation of elastin and collagen, which are constituents of dermal tissue. Notably, saclipins showed higher activity than the well-known glycation inhibitor aminoguanidine against collagen glycation.

Exploring Novel Vitamin K Derivatives with Anti-SARS-CoV-2 Activity.

From our compound library of vitamin K derivatives, we found that some compounds exhibited anti-SARS-CoV-2 activity in VeroE6/TMPRSS2 cells. The common structure of these compounds was menaquinone-2 (MK-2) with either the m-methylphenyl or the 1-naphthyl group introduced at the end of the side chain. Therefore, new vitamin K derivatives having more potent anti-SARS-CoV-2 activity were explored by introducing various functional groups at the ω-position of the side chain. MK-2 derivatives with a purine moiety showed the most potent antiviral activity among the derivatives. We also found that their mechanism of action was the inhibition of RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2. The chemical structures of our compounds were completely different from those of nucleic acid derivatives such as remdesivir and molnupiravir, clinically approved RdRp inhibitors for COVID-19 treatment, suggesting that our compounds may be effective against viruses resistant to these nucleic acid derivatives.

Derivatization-assisted enzyme-linked immunosorbent assay for identifying hallucinogenic mushrooms with enhanced sensitivity.

A sensitive immunochemical method for identifying hallucinogenic mushrooms (magic mushrooms) is required for regulating their illicit use. We have previously generated a monoclonal antibody (mAb) that targets psilocin (Psi), the major psychoactive compound in hallucinogenic mushrooms, and developed an enzyme-linked immunosorbent assay (ELISA). However, this ELISA failed to achieve the expected low-picomole-range sensitivity, as a result of insufficient affinity of the mAb to Psi. It is recognized that haptenic antigens with a larger molecular mass tend to induce antibodies with higher affinities. Thus, we herein report a "derivatization-assisted ELISA," in which the "real analyte" Psi was determined as a "surrogate analyte," the tert-butyldimethylsilyl ether analog thereof (TBS/Psi) having a 1.6-fold greater molecular mass (Mr 318.53) than Psi. A novel mAb against TBS/Psi, prepared by immunizing mice with a TBS/Psi-albumin conjugate showed a 69-fold higher affinity to TBS/Psi residues (Ka = 3.6 × 107 M-1 as IgG) than that of our previous mAb against Psi. This mAb consequently enabled a competitive ELISA for measuring TBS/Psi with the desired sensitivity: the dose-response curve midpoint (12.1 pmol per assay) was >100-fold lower than that of the previous ELISA for determining Psi. Extracts of dried mushroom powders were mixed with TBS triflate for 30 min at room temperature, converting Psi into TBS/Psi in approximately 50% yield. The reaction mixture was then subjected to an ELISA using the anti-TBS/Psi mAb to determine TBS/Psi. Psilocybe cubensis, a species of hallucinogenic mushrooms, gave rise to positive signals, indicating the presence of Psi therein in the expected quantity, while no detectable response was observed for four kinds of edible mushrooms available in the markets.

Dried Blood Spot Screening System for Spinal Muscular Atrophy with Allele-Specific Polymerase Chain Reaction and Melting Peak Analysis.

Background and Aim: Spinal muscular atrophy (SMA) is a lower motor neuron disease with autosomal recessive inheritance caused by homozygous SMN1 deletions. Although SMA has been considered as incurable, newly developed drugs improve life prognoses and motor functions of patients. To maximize the efficacy of the drugs, SMA patients should be treated before symptoms become apparent. Thus, newborn screening for SMA is strongly recommended. In this study, we aim to establish a new simple screening system based on DNA melting peak analysis. Materials and Methods: A total of 124 dried blood spot (DBS) on FTA® ELUTE cards (51 SMN1-deleted patients with SMA, 20 carriers, and 53 controls) were punched and subjected to direct amplification of SMN1 and CFTR (reference gene). Melting peak analyses were performed to detect SMN1 deletions from DBS samples. Results: A combination of allele-specific polymerase chain reaction (PCR) and melting peak analyses clearly distinguished the DBS samples with and without SMN1. Compared with the results of fresh blood samples, our new system yielded 100% sensitivity and specificity. The advantages of our system include (1) biosafe collection, transfer, and storage for DBS samples, (2) obviating the need for DNA extraction from DBS preventing contamination, (3) preclusion of fluorescent probes leading to low PCR cost, and (4) fast and high-throughput screening for SMN1 deletions. Conclusion: We demonstrate that our system would be applicable to a real-world newborn screening program for SMA, because our new technology is efficient for use in routine clinical laboratories that do not have highly advanced PCR instruments.

Chiral liquid chromatography-circular dichroism-NMR for estimating separation conditions of chiral HPLC without authentic samples.

Chiral separation by high performance liquid chromatography (Chiral HPLC) is one of the most powerful methods for estimating optical and chemical purity of chiral compounds. However, it has a weakness in that much time and effort are required to prepare authentic samples. A novel chiral liquid chromatography-circular dichroism-NMR (LC-CD-NMR) technique, on the other hand, requires only crude chiral compounds that include enantiomers as minor impurities. In this study, chiral LC-CD-NMR was constructed by connecting a conventional LC-NMR system with a CD detector. A pyridylalanine derivative mixture was prepared to mimic technical grade material in an early phase of development. By chiral LC-CD-NMR, the enantiomer peak is identified by an opposite sign of the CD Cotton effect curve and an identical (1)H NMR spectrum to that of the main component. Using NMR as a detector, this method is superior in ability to discriminate enantiomers from other isomers indistinguishable by MS. Furthermore, this method is also applicable for selecting the best separation conditions of chiral HPLC. The degrees of separation (Rs) between the main component and its enantiomer in several chiral columns were compared. Even with modern chromatographic methods, establishing the best chiral HPLC conditions in an early phase of development is difficult: chiral LC-CD-NMR is a suitable solution.

Phosphoethanolamine Elevation in Plasma of Spinal Muscular Atrophy Type 1 Patients.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by degeneration or loss of lower motor neurons. The survival of motor neuron (SMN) 1 gene, which produces the SMN protein, has been identified as a responsible gene for the disease. SMN is ubiquitously expressed in any tissue and may play an important role on the metabolism in the human body. However, no appropriate biomarkers reflecting the alteration in the metabolism in SMA have been identified. METHODS: Low-molecular-weight metabolites were extracted from plasma of 20 human infants (9 SMA type 1 patients and 11 controls) and 9 infant mice (5 SMA-model mice, 4 control mice), and derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide. Finally, the derivatized products were applied to Gas Chromatography/Mass Spectrometry apparatus. To confirm the metabolite abnormality in SMA type 1 patients, we performed SMN-silencing experiment using a hepatocyte-derived cell line (HepG2). RESULTS: We performed a comprehensive metabolomics analysis of plasma from the patients with SMA type 1 and controls, and found that phosphoethanolamine (PEA) was significantly higher in the patients than in the controls. HepG2 experiment also showed that SMN-silencing increased PEA levels. However, comprehensive metabolomics analysis of plasma from SMA-model mice and control mice showed different profile compared to human plasma; there was no increase of PEA even in the SMA-model mice plasma. CONCLUSION: Our data suggested that PEA was one of the possible biomarkers of human SMA reflecting metabolic abnormalities due to the SMN protein deficiency.

Carotenoid synthesis: retrospect and recent progress.

Carotenoid synthesis during the past approximately 30 years is summarized. Total syntheses of peridinin, fucoxanthin and halocynthiaxanthin, and biomimetic total syntheses of mytiloxanthin, crassostreaxanthin B, capsanthin, capsorubin, cucurbitaxanthin A, cycloviolaxanthin and capsanthin 3,6-epoxide are discussed.

Enantiomeric separation of racemic 1-benzyl-N-methyltetrahydroisoquinolines on chiral columns and chiral purity determinations of the O-methylated metabolites in plant cell cultures by HPLC-CD on-line coupling in combination with HPLC-MS.

Effective enantiomeric separations of 1-benzyl-N-methyltetrahydroisoquinolines were achieved using commercially available Chiralcel OD-H and OJ-H columns. Online LC-CD analysis allowed for the establishment of a correlation between the absolute configuration of the separated enantiomers and their characteristic CD transitions. LC-MS combined with LC-CD analysis permitted chiral purity determinations of O-methylated metabolites of nine phenolic 1-benzyl-N-methyltetrahydroisoquinolines in cell cultures of Corydalis, Macleaya, and Nandina species.

Development of an LC-ESI-MS/MS method for the determination of histamine: application to the quantitative measurement of histamine degranulation by KU812 cells.

A rapid, simple, and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the identification and quantification of histamine without a previous derivatization step or the addition of general ion-pairing reagents to the mobile phase. This method was used to measure histamine release following degranulation of KU812 human basophilic cells, using pyrazol as an internal standard. Analyses were performed on an LC system employing a Cosmosil 5C(18) PAQ column and an isocratic elution with methanol-0.005% trifluoroacetic acid (1:1) at a flow rate of 0.2 mL/min. A triple-quadrupole mass spectrometer, equipped with an electrospray ionization interface was employed, operating in the positive ion mode. The retention time of histamine and the internal standard were 4.0 and 5.0 min, respectively. The relative standard deviations (R.S.D.s) of the retention time and peak area were between 0.47% and 2.03%. Micropipette tip solid-phase extraction (SPE) using LooseTip C(18) allowed for not only rapid sample preparation, but also decreased suppression effects, improving peak shape. This method was used to evaluate the anti-allergic effects of compounds contained in Taxus yunnanensis extracts. Four constituents that were isolated from the wood extracts of T. yunnanensis and sodium cromoglicate, which is used as a first line anti-allergic drug, were tested in an in vitro histamine release inhibition assay. Of these compounds, taxiresinol and isotaxiresinol were more inhibitory than sodium cromoglicate.

Application of LC-NMR to analysis of carotenoids in foods.

LC-NMR has been applied to componential analysis of carotenoids in several foods, specifically, tomato juice, palm oil, and satsuma mandarin orange juice. The crude carotenoids extracted with organic solvent from these foodstuffs were analyzed after simple pre-processing. Three, four, and two kinds of carotenoids were identified for tomato juice, palm oil, and satsuma mandarin orange, respectively, primarily by comparing their NMR spectra with those of pure standard.

Newborn Screening for Spinal Muscular Atrophy: DNA Preparation from Dried Blood Spot and DNA Polymerase Selection in PCR.

BACKGROUND: Polymerase chain reaction (PCR) analysis using DNA from dried blood spot (DBS) samples on filter paper is a critical technique for spinal muscular atrophy (SMA) newborn screening. However, DNA extraction from DBS is time-consuming, and elimination of PCR inhibitors from DBS is almost impossible. METHODS: Exon 7 of the two homologous SMA-related genes, survival motor neuron (SMN) 1 and SMN2, of five SMA patients and five controls were amplified by PCR with a punched-out circle of the DBS paper. Two types of DNA preparation methods were tested; DNA-extraction (extracted DNA was added in a PCR tube) and non-DNA-extraction (a punched-out DBS circle was placed in a PCR tube). As for the DNA polymerases, two different enzymes were compared; TaKaRa Ex Taq™ and KOD FX Neo™. To test the diagnostic quality of PCR products, RFLP (Restriction fragment length polymorphism) analysis with DraI digestion was performed, differentiating SMN1 and SMN2. RESULTS: In PCR using extracted DNA, sufficient amplification was achieved with TaKaRa Ex Taq™ and KOD FX Neo™, and there was no significant difference in amplification efficiency between them. In direct PCR with a punched-out DBS circle, sufficient amplification was achieved when KOD FX Neo™ polymerase was used, while there was no amplification with TaKaRa Ex Taq™. RFLP analysis of the direct PCR products with KOD FX Neo™ clearly separated SMN1 and SMN2 sequences and proved the presence of both of SMN1 and SMN2 in controls, and only SMN2 in SMA patients, suggesting that the direct PCR products with KOD FX Neo™ were of sufficient diagnostic quality for SMA testing. CONCLUSION: Direct PCR with DNA polymerases like KOD FX NeoTM has potential to be widely used in SMA newborn screening in the near future as it obviates the DNA extraction process from DBS and can precisely amplify the target sequences in spite of the presence of PCR inhibitors.

Online structural elucidation of alkaloids and other constituents in crude extracts and cultured cells of Nandina domestica by combination of LC-MS/MS, LC-NMR, and LC-CD analyses.

The combination of NMR, MS, and CD data permitted the structural elucidation including the absolute configuration of the known alkaloids and unknown components in the extract matrix solution of Nandina domestica without isolation and sample purification prior to the coupling experiments. Unstable natural stereoisomers were identified by LC-NMR and LC-MS. Five known alkaloids, (S)-isoboldine, (S)-domesticine, (S)-nantenine, sinoacutine, and menispermine, were identified from N. domestica. O-Methylpallidine and (E, E)-, (E, Z)-, and (Z, Z)-terrestribisamide were also characterized for the first time from this plant. Known jatrorrhizine, palmatine, and berberine and unknown (R)-carnegine and (E, E)-, (E, Z)-, and (Z, Z)-terrestribisamide were identified in the callus of N. domestica.

Synthesis of Novel Synthetic Vitamin K Analogues Prepared by Introduction of a Heteroatom and a Phenyl Group That Induce Highly Selective Neuronal Differentiation of Neuronal Progenitor Cells.

We synthesized novel vitamin K2 analogues that incorporated a heteroatom and an aromatic ring in the side chain and evaluated their effect on the selective differentiation of neuronal progenitor cells into neurons in vitro. The results showed that a menaquinone-2 analogue bearing a p-fluoroaniline had the most potent activity, which was more than twice as great as the control. In addition, the neuronal selectivity was more than 3 times greater than the control.