Satoshi Mizuta

Diastereoselective addition to N-acyliminium ions with aryl- and alkenyl boronic acids via a Petasis-type reaction

A highly diastereoselective synthesis of 2,3-disubstituted piperidines has been accomplished through nucleophilic additions to N-acyliminium ions with aryl- and alkenyl boronic acids. A reversal of stereoselectivity depending on a β-substituent on the piperidine ring was observed in the alkenylation reactions with (E)-styrylboronic acid. Our strategy was applied in the key step for the synthesis of the neurokinin NK1 receptor antagonist (±)-L-733,060.

Ionic Liquid-Mediated Hydrofluorination of o-Azaxylylenes Derived from 3-Bromooxindoles

The hydrofluorination reaction of 3-bromooxindole using mild HF reagents in an ionic liquid is described. This transformation can operate at room temperature to give a series of 3-substituted 3-fluorooxindole derivatives including racemic BMS 204352 (MaxiPost). The mechanistic study about interactions between HF and 3-butyl-1-methylimidazolium tetrafluoroborate [bmim][BF4] is also discussed on the basis of energy calculations.

Identification of small molecule inhibitors for influenza a virus using in silico and in vitro approaches

Influenza viruses have acquired resistance to approved neuraminidase-targeting drugs, increasing the need for new drug targets for the development of novel anti-influenza drugs. Nucleoprotein (NP) is an attractive target since it has an indispensable role in virus replication and its amino acid sequence is well conserved. In this study, we aimed to identify new inhibitors of the NP using a structure-based drug discovery algorithm, named Nagasaki University Docking Engine (NUDE), which has been established especially for the Destination for GPU Intensive Machine (DEGIMA) supercomputer. The hit compounds that showed high binding scores during in silico screening were subsequently evaluated for anti-influenza virus effects using a cell-based assay. A 4-hydroxyquinolinone compound, designated as NUD-1, was found to inhibit the replication of influenza virus in cultured cells. Analysis of binding between NUD-1 and NP using surface plasmon resonance assay and fragment molecular orbital calculations confirmed that NUD-1 binds to NP and could interfere with NP-NP interactions essential for virus replication. Time-of-addition experiments showed that the compound inhibited the mid-stage of infection, corresponding to assembly of the NP and other viral proteins. Moreover, NUD-1 was also effective against various types of influenza A viruses including a clinical isolate of A(H1N1)pdm09 influenza with a 50% inhibitory concentration range of 1.8–2.1 μM. Our data demonstrate that the combined use of NUDE system followed by the cell-based assay is useful to obtain lead compounds for the development of novel anti-influenza drugs.

Neutrophils and the S100A9 protein critically regulate granuloma formation

Macrophages have the potential to undergo cellular transformation into epithelioid cells, and their concentric accumulation in tissues results in the development of granulomas. Although epithelioid cells are an essential and dominant component of granulomas, other cell types have also been detected, which may contribute to the establishment of well-organized granulomas, as observed in human granulomatous diseases. We herein demonstrated that neutrophils may mediate these functions. By taking advantage of the guinea pig pulmonary granuloma model, we obtained a rat monoclonal antibody with unique reactivity to granuloma cells. This antibody, termed G213, reacted with clusters of neutrophils located in the central area of granulomas, and a biochemical analysis identified the G213-reactive antigen as S100A9, a calcium-binding protein of the S100 family, which was expressed abundantly in neutrophils. Consistent with the multifaceted functions attributed to S100A9, including its role in neutrophil extravasation and macrophage activation, the blockade of S100A9 functions with the specific inhibitor, tasquinimod, impaired the formation of organized granulomas with neutrophil cores. These results demonstrate the critical role of neutrophils and the S100A9 protein in granuloma formation. Because intragranuloma S100A9+ neutrophils were also detected in humans, these results indicate the potential of tasquinimod, a new anticancer drug candidate, for manipulating human granulomatous diseases.

Structure-based drug discovery for combating influenza virus by targeting the PA–PB1 interaction

Influenza virus infections are serious public health concerns throughout the world. The development of compounds with novel mechanisms of action is urgently required due to the emergence of viruses with resistance to the currently-approved anti-influenza viral drugs. We performed in silico screening using a structure-based drug discovery algorithm called Nagasaki University Docking Engine (NUDE), which is optimised for a GPU-based supercomputer (DEstination for Gpu Intensive MAchine; DEGIMA), by targeting influenza viral PA protein. The compounds selected by NUDE were tested for anti-influenza virus activity using a cell-based assay. The most potent compound, designated as PA-49, is a medium-sized quinolinone derivative bearing a tetrazole moiety, and it inhibited the replication of influenza virus A/WSN/33 at a half maximal inhibitory concentration of 0.47 μM. PA-49 has the ability to bind PA and its anti-influenza activity was promising against various influenza strains, including a clinical isolate of A(H1N1)pdm09 and type B viruses. The docking simulation suggested that PA-49 interrupts the PA–PB1 interface where important amino acids are mostly conserved in the virus strains tested, suggesting the strain independent utility. Because our NUDE/DEGIMA system is rapid and efficient, it may help effective drug discovery against the influenza virus and other emerging viruses.

Live Cell Labeling with Terpyridine Derivative Proligands to Measure Cytotoxicity Mediated by Immune Cells

Immunotherapy using immune checkpoint inhibitors and CAR‐T cells has revolutionized treatment for patients with malignant tumors. However, measuring tumor cell cytotoxicity mediated by immune effector cells in clinical laboratories has been difficult due to the requirement for radioactive substances. In this study, a series of novel terpyridine derivative proligands were synthesized, and a non‐radioactive cellular cytotoxicity assay using the newly synthesized compounds was developed for use in preclinical and clinical studies for cancer immunotherapy. Once internalized into target cells, the compounds are hydrolyzed by esterases, resulting in the intracellular accumulation of the negatively charged terpyridine derivatives. When the labeled target cells are recognized and killed by immune effector cells, the integrity of the cell membrane is disrupted, and the terpyridine derivatives are released. Upon combining the culture supernatant with europium (Eu3+), the cytotoxicity of immune effector cells for the target cells can be quantitatively determined by measuring the intensity of the Eu3+/ligand‐derived time‐resolved fluorescence. Thus, the assay developed in this study would facilitate the development of novel cancer immunotherapies.

Activity of N,N′-dialkyl-2-trifluoromethylthioimidazolium salts as phase-transfer catalyst for the alkylation of active methylene compounds

We herein report the synthesis of N,N′-dialkyl-2-trifluoromethylthioimidazolium salts from thioureas using nucleophilic trifluoromethylating reagents. The activity of imidazolium salts as phase-transfer catalysts under solid–liquid phase conditions was investigated for reactions such as the alkylation of active methylene compounds.

3,3-Dibromo-2-trifluoromethyl acrylic acid ethyl ester: a versatile platform for the stereoselective preparation of functionalized-α-trifluoromethyl α,β-unsaturated lactones and trifluoromethyl pyrazolinones

We herein describe a method for synthetic routes to multi-functionalized-α-trifluoromethyl α,β-unsaturated lactones and trifluoromethyl pyrazolinones. This involves a tandem stereoselective functionalization of 3,3-dibromo-2-trifluoromethyl acrylic acid ethyl ester and intramolecular cyclization reaction to afford precursors for a Suzuki–Miyaura cross-coupling reaction with arylboronic acids.

Fragment Molecular Orbital Study of the Interaction between Sarco/Endoplasmic Reticulum Ca2+-ATPase and its Inhibitor Thapsigargin toward Anti-Malarial Development

Plasmodium falciparum, the causative agent of malignant malaria, is insensitive to thapsigargin (TG), a well-known inhibitor of the human sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). To understand the key factor causing the difference of the sensitivity, the molecular interaction of TG and each SERCA was analyzed by the fragment molecular orbital (FMO) method. While the major component of the interaction energy was the nonpolar interaction, the major difference in the molecular interaction arose from the polar interaction, namely, the hydrogen bonding interaction with a hydroxyl group of TG. Additionally, we successfully confirmed these FMO calculation results by measuring the inhibitory activity of a synthesized TG derivative. Our calculations and experiments indicated that, by replacing the hydroxyl group of TG with another functional group, the sensitivities of TG to human and P. falciparum SERCAs can be reversed. This study provides important information to develop antimalarial compounds targeting P. falciparum SERCA.

Synthesis of Trifluoromethyl-α,β-unsaturated Lactones and Pyrazolinones and Discovery of Influenza Virus Polymerase Inhibitors

To explore the potential biological activities of trifluoromethyl heterocycles, we recently developed a synthetic approach to access a series of α‐trifluoromethyl‐α,β‐unsaturated lactones and trifluoromethyl pyrazolinones. The compounds were tested for their antimicrobial activity, and we found that some compounds had anti‐influenza viral activity. The β‐aryl‐α‐trifluoromethyl α,β‐unsaturated lactone derivatives 5 g (5‐(4‐chlorophenyl)‐5‐methyl‐4‐phenyl‐3‐(trifluoromethyl)furan‐2‐one), 7 b (4‐(4‐methoxyphenyl)‐3‐(trifluoromethyl)spiro[furan‐5,1′‐indane]‐2‐one), and the trifluoromethyl pyrazolinone 12 c (1‐(6‐methoxy‐2‐naphthyl)‐2‐(trifluoromethyl)‐5,6,7,8‐tetrahydropyrazolo[1,2‐a]pyridazin‐3‐one) were found to possess promising inhibitory activity against influenza virus type A, strain A/WSN/33 (H1N1). These three hit compounds were successfully optimized, and we identified that the most potent compound 5 h (5‐(4‐chlorophenyl)‐4‐(6‐methoxy‐2‐naphthyl)‐5‐methyl‐3‐(trifluoromethyl)furan‐2‐one) showed inhibitory activity against various types of influenza A and B viruses in the low‐micromolar range without showing cytotoxicity. Moreover, 5 h was more effective against the clinical isolate A/California/7/2009 (H1N1pdm) strain than the influenza viral polymerase inhibitor, favipiravir (T‐705). We also delineated the structure–activity relationship and obtained mechanistic insight into inhibition of the viral polymerase.