Viscosity-Induced Emission of 5-(Diarylmethylene)imidazolone with Extended Conjugation via Attachment of N-Methylpyrrole at the 2-Position.

We have developed a series of 2-monoaryl-5-diarylmethylene analogs of the green fluorescent protein chromophore to study their viscosity-induced emission (VIE) properties. The analogs were synthesized by a condensation with methyl imidate and N-(diarylmethylene)glycinate. Among the analogs, the N-methylpyrrol-2-yl-substituted analog 1h induced the most remarkable VIE behavior in triglyceride and lipid bilayers probably due to the high π-electron-rich property of the pyrrole ring. The pyrrole substituent in imidazolone analogs can be expected to become a common template for introducing VIE behavior.

Impact of Different Attitudes toward Face-to-Face and Online Classes on Learning Outcomes in Japan.

During the coronavirus disease 2019 (COVID-19) pandemic, online-based learning has become mainstream in many countries, and its learning outcomes have been evaluated. However, various studies have shown that online-based learning needs to be optimized in the future, and the number of reports for this purpose is currently not sufficient. The purpose in this study was to determine the relationship between academic performance and attitudes toward face-to-face and remote formats among Japanese pharmacy students enrolled in a course designed for knowledge acquisition. A combination of face-to-face and remote formats was used in a practice course for sixth-year pharmacy students, designed to improve academic performance through knowledge acquisition. To evaluate learning outcomes, we used a questionnaire that was administered to the course participants and the results of examinations conducted before and after the course. Online-oriented and face-to-face-oriented groups differed in their attitudes toward the ease of asking questions of faculty and communicating with the faculty members and classmates in each format. In a knowledge acquisition course for Japanese pharmacy students, the study revealed that the same academic outcomes were achieved, regardless of the students' own perceptions of their aptitude for face-to-face or remote learning style.

Synthesis and environment-dependent fluorescence behavior of a biaryl-conjugated (diphenylmethylene)imidazolinone.

The newly designed green fluorescent protein (GFP) chromophore analog, bar-DAIN, which has a 2-biaryl-conjugated 5-(diphenylmethylene)imidazolinone structure, was effectively synthesized using the Suzuki coupling reaction. Bar-DAIN showed environment-dependent fluorescence behavior; for example, the thienyl analog emitted yellow-green fluorescence in viscous solution (λem: 535 nm), yellow-orange fluorescence in suspension (λem: 551 nm), and cyan fluorescence in a powder state (λem: 497 nm) although it showed almost no emission in common solvents such as dichloromethane. The dynamic discoloration of the fluorescence was observed by changing environmental conditions from suspension to viscous.

Synthesis and properties of geometrical 4-diarylmethylene analogs of the green fluorescent protein chromophore.

We have developed a novel analog of the GFP chromophore: geo-DAIN. Since geo-DAIN is equipped with an E/Z-photoisomerizable geometrical diarylmethylene moiety instead of benzylidene of the GFP chromophore, different-colored reversible emissions are expected. We synthesized geo-DAIN by a condensation with methyl imidate and N-(diarylmethylene)glycinate. We found the emission from geo-DAIN to be different from that of benzylidene-type analogs; in the powder state, the E- and Z-isomers of geo-DAIN emitted different fluorescence colors.

A hybrid molecule of a GFP chromophore analogue and cholestene as a viscosity-dependent and cholesterol-responsive fluorescent sensor.

Diarylmethylenated and cholestene (or -tane)-hybrid analogues of the GFP chromophore, namely, Ch-DAINs were successfully synthesised by a condensation reaction between methyl imidates and N-(diarylmethylene)glycinates. Among the Ch-DAINs synthesised, a diphenyl-type analogue showed viscosity-dependent and cholesterol-responsive fluorescent properties. It showed a nearly linear increase of the fluorescence emission in triglycerides and vesicles as the amount of cholesterol was increased.

Design and concise synthesis of a novel type of green fluorescent protein chromophore analogue.

A small molecular model compound for the green fluorescent protein chromophore was readily synthesized by a novel condensation reaction of (thio)imidate with imino-ester via an aziridine intermediate. This compound showed fluorescence in the solid and frozen solution states but not in the solution state. Its fluorescent property was successfully applied in the detection of dsDNA.

Structure-Activity Relationship for Thiirane-Based Gelatinase Inhibitors.

An extensive structure-activity relationship study with the template of 2-(4-phenoxyphenylsulfonylmethyl)thiirane (1), a potent and highly selective inhibitor for human gelatinases, is reported herein. Syntheses of 65 new analogs, each in multistep processes, allowed for exploration of key structural components of the molecular template. This study reveals that the presence of the sulfonylmethylthiirane and the phenoxyphenyl group were important for gelatinase inhibition. However, para- and some meta-substitutions of the terminal phenyl ring enhanced inhibitory activity, and led to improve metabolic stability. This agrees with the result from metabolism studies with compound 1 that the primary route of biotransformation is oxidation, mainly at the para position of the phenyl ring and alpha position of the sulfonyl group in the aliphatic side chain.

Anti-human immunodeficiency virus type 1 activity of novel 6-substituted 1-benzyl-3-(3,5-dimethylbenzyl)uracil derivatives.

Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are important components of current combination therapies for human immunodeficiency virus type 1 (HIV-1) infection. In screening of chemical libraries, we found 6-azido-1-benzyl-3-(3,5-dimethylbenzyl)uracil (AzBBU) and 6-amino-1-benzyl-3-(3,5-dimethylbenzyl)uracil (AmBBU) to be highly active and selective inhibitors of HIV-1 replication in vitro. To determine the resistance profiles of these compounds, we conducted a long-term culture of HIV-1-infected MT-4 cells with escalating concentrations of each compound. After serial passages of the infected cells, escape viruses were obtained, and they were more than 500-fold resistant to the uracil derivatives compared to the wild type. Sequence analysis was conducted for RT of the escape viruses at passages 12 and 24. The amino acid mutation Y181C in the polymerase domain of RT was detected for all escape viruses. Docking studies using the crystal structure of RT showed that AmBBU requires the amino acid residues Leu100, Val106, Tyr181, and Trp229 for exerting its inhibitory effect on HIV-1. Four additional amino acid changes (K451R, R461K, T468P, and D471N) were identified in the RNase H domain of RT; however, their precise role in the acquisition of resistance is still unclear. In conclusion, the initial mutation Y181C seems sufficient for the acquisition of resistance to the uracil derivatives AzBBU and AmBBU. Further studies are required to determine the precise role of each mutation in the acquisition of HIV-1 resistance.

Synthesis of 1-benzyl-3-(3,5-dimethylbenzyl)uracil derivatives with potential anti-HIV activity.

BACKGROUND: Nine novel uracil analogues were synthesized and evaluated as inhibitors of HIV-1. METHODS: Key structural modifications included replacement of the 6-chloro group of 1-benzyl-6-chloro-3-(3,5-dimethylbenzyl)uracil by other functional groups or N(1)-alkylation of 3-(3,5-dimethylbenzyl)-5-fluorouracil. RESULTS: These compounds showed only micromolar potency against HIV-1 in MT-4, though two of them; 6-azido-1-benzyl-3-(3,5-dimethylbenzyl) uracil and 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil were highly potent (half maximal effective concentration =0.067 and 0.069 µM) and selective (selectivity index =685 and 661), respectively. Structure-activity relationships among the newly synthesized uracil analogues suggest the importance of the H-bond formed between 6-amino group of 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil and amide group of HIV-1 reverse transcriptase. CONCLUSIONS: We discovered two 6-substituted 1-benzyl-3-(3,5-dimethylbenzyl) uracils, (6-azido-1-benzyl-3-(3,5-dimethylbenzyl) uracil and 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil) as novel anti-HIV agents. These compounds should be further pursued for their toxicity and pharmacokinetics in vivo as well as antiviral activity against non-nucleoside reverse transcriptase inhibitor-resistant strains.

Selective water-soluble gelatinase inhibitor prodrugs.

SB-3CT (1), a selective and potent thiirane-based gelatinase inhibitor, is effective in animal models of cancer metastasis and stroke; however, it is limited by poor aqueous solubility and extensive metabolism. We addressed these issues by blocking the primary site of metabolism and capitalizing on a prodrug strategy to achieve >5000-fold increased solubility. The amide prodrugs were quantitatively hydrolyzed in human blood to a potent gelatinase inhibitor, ND-322 (3). The arginyl amide prodrug (ND-478, 5d) was metabolically stable in mouse, rat, and human liver microsomes. Both 5d and 3 were nonmutagenic in the Ames II mutagenicity assay. The prodrug 5d showed moderate clearance of 0.0582 L/min/kg, remained mostly in the extracellular fluid compartment (Vd = 0.0978 L/kg), and had a terminal half-life of >4 h. The prodrug 5d had superior pharmacokinetic properties than those of 3, making the thiirane class of selective gelatinase inhibitors suitable for intravenous administration in the treatment of acute gelatinase-dependent diseases.

Synthesis and anti-HCV activity of 2',5'-deoxy-5'-phenacyladenosine analogs.

Several nucleoside analogs containing a methylene group instead of a 5'-O atom were synthesized to study the effect of the 5'-modification of nucleoside analogs on their anti-HCV activity. Among the analogs, a 5'-phenacyl analog exhibited good anti-HCV activity with an EC(50) of 15.1 muM. This compound is hypothesized to function via a novel type of mechanism that does not involve the conventional 5'-O-triphosphorylation process.

Synthesis and evaluation of 5'-modified 2'-deoxyadenosine analogues as anti-hepatitis C virus agents.

In order to study the effect of 5'-modification of 2'-deoxynucleoside on its anti-HCV activity, several analogues were synthesized and evaluated. Among the analogues, a 5'-deoxy-5'-phenacylated analogue exhibited a good anti-HCV activity with an EC(50) of 15.1 microM. This compound is expected to operate via a type of mechanism that does not involve a generally known 5'-O-triphosphorylation process.

Total synthesis of leustroducsin B.

Leustroducsin B was synthesized via a convergent route based on division of the leustroducsin molecule into three segments A, B, and C. Two coupling reactions (Julia coupling reaction and Nozaki-Hiyama-Kishi (NHK) reaction) were employed for coupling of segments A and B: segment A1 for the Julia coupling reaction was prepared by a combination of Sharpless asymmetric epoxidation and an epoxide-cleavage reaction with an organoaluminum reagent, while segment A2 for the NHK reaction was synthesized from optically active alcohol that had previously been prepared by lipase-catalyzed kinetic resolution. Segment B, whose structure was modified with some functional groups, was synthesized from (R)-malic acid by a combination of Wittig reaction and Sharpless asymmetric dihydroxylation, and segment C, containing a cyclohexane moiety, was prepared by asymmetric Diels-Alder reaction. Segment B was first coupled with segment A1 via the Julia coupling reaction, but the yield was low due to unexpected epimerization. The NHK reaction of segment A2 proceeded to give the coupling product in good yield. This product was coupled with segment C via Wittig and Stille coupling reactions, and finally, phosphorylation was carried out by partial hydrolysis of a cyclic phosphate to give leustroducsin B.

Suppression of subgenomic hepatitis C virus replication by 5'-O-masked analogues of 6-chloropurine-2'-deoxyriboside.

A series of nucleoside analogues whose 5'-hydroxyl groups are masked by various protective groups were synthesized and evaluated to develop novel anti- hepatitis C virus (HCV) agents. Among the several analogues that showed anti-HCV potency, a 5'-O-benzoyl-2'-deoxyribonucleoside analogue exhibited high anti-HCV activity with an EC(50) of 6.1 microM.

5'-O-masked 2'-deoxyadenosine analogues as lead compounds for hepatitis C virus (HCV) therapeutic agents.

On the basis of our previous study on antiviral agents against the severe acute respiratory syndrome (SARS) coronavirus, a series of nucleoside analogues whose 5'-hydroxyl groups are masked by various protective groups such as carboxylate, sulfonate, and ether were synthesized and evaluated to develop novel anti-hepatitis C virus (HCV) agents. Among these, several 5'-O-masked analogues of 6-chloropurine-2'-deoxyriboside (e.g., 5'-O-benzoyl, 5'-O-p-methoxybenzoyl, and 5'-O-benzyl analogues) were found to exhibit effective anti-HCV activity. In particular, the 5'-O-benzoyl analogue exhibited the highest potency with an EC(50) of 6.1 microM in a cell-based HCV replicon assay. Since the 5'-O-unmasked analogue (i.e., 6-chloropurine-2'-deoxyriboside) was not sufficiently potent (EC(50)=47.2 microM), masking of the 5'-hydroxyl group seems to be an effective method for the development of anti-HCV agents. Presently, we hypothesize two roles for the 5'-O-masked analogues: One is the role as an anti-HCV agent by itself, and the other is as a prodrug of its 5'-O-demasked (deprotected) derivative.

Synthesis and biological evaluation of nucleoside analogues having 6-chloropurine as anti-SARS-CoV agents.

Nucleoside analogues that have 6-chloropurine as the nucleobase were synthesized and evaluated for anti-SARS-CoV activity by plaque reduction and yield reduction assays in order to develop novel anti-SARS-CoV agents. Among these analogues, two compounds, namely, 1 and 11, exhibited promising anti-SARS-CoV activity that was comparable to those of mizoribine and ribavirin, which are known anti-SARS-CoV agents. Moreover, we observed several SAR trends such as the antiviral effects of the 6-chloropurine moiety, unprotected 5'-hydroxyl group and benzoylated 5'-hydroxyl group.

Anti-SARS-CoV activity of nucleoside analogs having 6-chloropurine as a nucleobase.

Nucleoside analogs that have 6-chloropurine as a nucleobase were synthesized and evaluated to develop novel anti-SARS-CoV agents. Two compounds among them showed promising activity that was comparable to mizoribine and ribavirin. Moreover, one of the compounds showed a structurally unique property.

Synthesis and triplex-forming ability of 2',4'-BNAs bearing imidazoles as a nucleobase.

To expand the sequence of double-stranded DNA (dsDNA) targets in a triplex formation, 2',4'-BNAs (2'-O,4'-C-methylene bridged nucleic acids) having imidazoles as a nucleobase were synthesized and incorporated into oligonucleotides. Triplex-forming ability of the modified oligonucleotides was evaluated by using melting temperature (Tm) measurements.

Design, synthesis, and evaluation of a mechanism-based inhibitor for gelatinase A.

[structure: see text] Matrix metalloproteinases (MMPs), of which 26 are known, have been implicated in a number of pathological conditions, including tumor metastasis. We have previously described the first mechanism-based inhibitor for MMPs (J. Am. Chem. Soc. 2000, 122, 6799-6800), which in chemistry mediated by the active site zinc ion selectively and covalently inhibits MMP-2, -3, and -9. Computational analyses indicated that this selectivity in inhibition of MMPs could be improved by design of new variants of the inhibitor class. We report herein the syntheses of methyl 2-(4-{4-[(2-thiiranylpropyl)sulfonyl]phenoxy}phenyl)acetate (3) and 2-(4-{4-[(2-thiiranylpropyl)sulfonyl]phenoxy}phenyl)acetic acid (4), and show that compound 3 serves as a mechanism-based inhibitor exclusively for MMP-2. This molecule should prove useful in delineating the functions of MMP-2 in biological systems.

Potent mechanism-based inhibitors for matrix metalloproteinases.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play important roles in physiological and pathological conditions. Both gelatinases (MMP-2 and -9) and membrane-type 1 MMP (MMP-14) are important targets for inhibition, since their roles in various diseases, including cancer, have been well established. We describe herein a set of mechanism-based inhibitors that show high selectivity to gelatinases and MMP-14 (inhibitor 3) and to only MMP-2 (inhibitors 5 and 7). These molecules bind to the active sites of these enzymes, initiating a slow binding profile for the onset of inhibition, which leads to covalent enzyme modification. The full kinetic analysis for the inhibitors is reported. These are nanomolar inhibitors (Ki) for the formation of the noncovalent enzyme-inhibitor complexes. The onset of slow binding inhibition is rapid (k(on) of 10(2) to 10(4) M(-1) s(-1) and the reversal of the process is slow (k(off) of 10(-3) to 10(-4) s(-1)). However, with the onset of covalent chemistry with the best of these inhibitors (e.g. inhibitor 3), very little recovery of activity (<10%) was seen over 48 h of dialysis. We previously reported that broad spectrum MMP inhibitors like GM6001 enhance MT1-MMP-dependent activation of pro-MMP-2 in the presence of tissue inhibitor of metalloproteinases-2. Herein, we show that inhibitor 3, in contrast to GM6001, had no effect on pro-MMP-2 activation by MT1-MMP. Furthermore, inhibitor 3 reduced tumor cell migration and invasion in vitro. These results show that these new inhibitors are promising candidates for selective inhibition of MMPs in animal models of relevant human diseases.