Triptycene Branched Poly(aryl-co-aryl piperidinium) Electrolytes for Alkaline Anion Exchange Membrane Fuel Cells and Water Electrolyzers.

Alkaline polymer electrolytes (APEs) are essential materials for alkaline energy conversion devices such as anion exchange membrane fuel cells (AEMFCs) and water electrolyzers (AEMWEs). Here, we report a series of branched poly(aryl-co-aryl piperidinium) with different branching agents (triptycene: highly-rigid, three-dimensional structure; triphenylbenzene: planar, two-dimensional structure) for high-performance APEs. Among them, triptycene branched APEs showed excellent hydroxide conductivity (193.5 mS cm-1 @80 °C), alkaline stability, mechanical properties, and dimensional stability due to the formation of branched network structures, and increased free volume. AEMFCs based on triptycene-branched APEs reached promising peak power densities of 2.503 and 1.705 W cm-2 at 75/100 % and 30/30 % (anode/cathode) relative humidity, respectively. In addition, the fuel cells can run stably at a current density of 0.6 A cm-2 for 500 h with a low voltage decay rate of 46 µV h-1 . Importantly, the related AEMWE achieved unprecedented current densities of 16 A cm-2 and 14.17 A cm-2 (@2 V, 80 °C, 1 M NaOH) using precious and non-precious metal catalysts, respectively. Moreover, the AEMWE can be stably operated under 1.5 A cm-2 at 60 °C for 2000 h. The excellent results suggest that the triptycene-branched APEs are promising candidates for future AEMFC and AEMWE applications.

Biodegradation Studies of Polyhydroxybutyrate and Polyhydroxybutyrate-co-Polyhydroxyvalerate Films in Soil.

Due to increased environmental pressures, significant research has focused on finding suitable biodegradable plastics to replace ubiquitous petrochemical-derived polymers. Polyhydroxyalkanoates (PHAs) are a class of polymers that can be synthesized by microorganisms and are biodegradable, making them suitable candidates. The present study looks at the degradation properties of two PHA polymers: polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-polyhydroxyvalerate (PHBV; 8 wt.% valerate), in two different soil conditions: soil fully saturated with water (100% relative humidity, RH) and soil with 40% RH. The degradation was evaluated by observing the changes in appearance, chemical signatures, mechanical properties, and molecular weight of samples. Both PHB and PHBV were degraded completely after two weeks in 100% RH soil conditions and showed significant reductions in mechanical properties after just three days. The samples in 40% RH soil, however, showed minimal changes in mechanical properties, melting temperatures/crystallinity, and molecular weight over six weeks. By observing the degradation behavior for different soil conditions, these results can pave the way for identifying situations where the current use of plastics can be replaced with biodegradable alternatives.

Structure-activity relationship analysis of novel GSPT1 degraders based on benzotriazinone scaffold and its antitumor effect on xenograft mouse model.

Molecular glue degraders, such as lenalidomide and pomalidomide, bind to cereblon (CRBN) E3 ligase and subsequently recruit neosubstrate proteins, Ikaros (IKZF1) and Aiolos (IKZF3), for the ubiquitination-proteasomal degradation process. In this study, we explored structure-activity relationship analysis for novel GSPT1 degraders utilizing a benzotriazinone scaffold previously discovered as a novel CRBN binder. In particular, we focused on the position of the ureido group on the benzotriazinone scaffold, substituent effect on the phenylureido group, and methyl substitution on the benzylic position of benzotriazinone. As a result, we identified 34f (TD-522), which exhibits strong anti-proliferative effects in both KG-1 (EC50 = 0.5 nM) and TMD-8 (EC50 = 5.2 nM) cell lines. Compound 34f effectively induced GSPT1 degradation with a DC50 of 0.269 nM and Dmax of >95 % at 10 nM concentration in KG-1 cells. An in vivo xenograft study showed that compound 34f effectively suppressed TMD8-driven tumor growth, suggesting a potential role in the development of novel GSPT1 degraders.

Nanocrack-regulated self-humidifying membranes.

The regulation of water content in polymeric membranes is important in a number of applications, such as reverse electrodialysis and proton-exchange fuel-cell membranes. External thermal and water management systems add both mass and size to systems, and so intrinsic mechanisms of retaining water and maintaining ionic transport in such membranes are particularly important for applications where small system size is important. For example, in proton-exchange membrane fuel cells, where water retention in the membrane is crucial for efficient transport of hydrated ions, by operating the cells at higher temperatures without external humidification, the membrane is self-humidified with water generated by electrochemical reactions. Here we report an alternative solution that does not rely on external regulation of water supply or high temperatures. Water content in hydrocarbon polymer membranes is regulated through nanometre-scale cracks ('nanocracks') in a hydrophobic surface coating. These cracks work as nanoscale valves to retard water desorption and to maintain ion conductivity in the membrane on dehumidification. Hydrocarbon fuel-cell membranes with surface nanocrack coatings operated at intermediate temperatures show improved electrochemical performance, and coated reverse-electrodialysis membranes show enhanced ionic selectivity with low bulk resistance.

Synthesis and Structure-Activity Relationships of Aristoyagonine Derivatives as Brd4 Bromodomain Inhibitors with X-ray Co-Crystal Research.

Epigenetic regulation is known to play a key role in progression of anti-cancer therapeutics. Lysine acetylation is an important mechanism in controlling gene expression. There has been increasing interest in bromodomain owing to its ability to modulate transcription of various genes as an epigenetic 'reader.' Herein, we report the design, synthesis, and X-ray studies of novel aristoyagonine (benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one) derivatives and investigate their inhibitory effect against Brd4 bromodomain. Five compounds 8ab, 8bc, 8bd, 8be, and 8bf have been discovered with high binding affinity over the Brd4 protein. Co-crystal structures of these five inhibitors with human Brd4 bromodomain demonstrated that it has a key binding mode occupying the hydrophobic pocket, which is known to be the acetylated lysine binding site. These novel Brd4 bromodomain inhibitors demonstrated impressive inhibitory activity and mode of action for the treatment of cancer diseases.

Identification of Potent CD19 scFv for CAR T Cells through scFv Screening with NK/T-Cell Line.

CD19 is the most promising target for developing chimeric-antigen receptor (CAR) T cells against B-cell leukemic cancer. Currently, two CAR-T-cell products, Kymriah and Yescarta, are approved for leukemia patients, and various anti-CD19 CAR T cells are undergoing clinical trial. Most of these anti-CD19 CAR T cells use FMC63 single-chain variable fragments (scFvs) for binding CD19 expressed on the cancer cell surface. In this study, we screened several known CD19 scFvs for developing anti-CD19 CAR T cells. We used the KHYG-1 NK/T-cell line for screening of CD19 scFvs because it has advantages in terms of cell culture and gene transduction compared to primary T cells. Using our CAR construct backbone, we made anti-CD19 CAR constructs which each had CD19 scFvs including FMC63, B43, 25C1, BLY3, 4G7, HD37, HB12a, and HB12b, then made each anti-CD19 CAR KHYG-1 cells. Interestingly, only FMC63 CAR KHYG-1 and 4G7 CAR KHYG-1 efficiently lysed CD19-positive cell lines. In addition, in Jurkat cell line, only these two CAR Jurkat cell lines secreted IL-2 when co-cultured with CD19-positive cell line, NALM-6. Based on these results, we made FMC63 CAR T cells and 4G7 CAR T cells from PBMC. In in vitro lysis assay, 4G7 CAR T cells lysed CD19-positive cell line as well as FMC63 CAR T cells. In in vivo assay with NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, 4G7 CAR T cells eradicated NALM-6 as potently as FMC63 CAR T cells. Therefore, we anticipate that 4G7 CAR T cells will show as good a result as FMC63 CAR T cells for B-cell leukemia patients.

Induced protein degradation of anaplastic lymphoma kinase (ALK) by proteolysis targeting chimera (PROTAC).

Recently, proteolysis targeting chimera (PROTAC) technology is highlighted in drug discovery area as a new therapeutic approach. PROTAC as a heterobifunctional molecule is comprised of two ligands, which recruit target protein and E3 ligase, respectively. To degrade the anaplastic lymphoma kinase (ALK) fusion protein, such as NPM-ALK or EML4-ALK, we generated several ALK-PROTAC molecules consisted of ceritinib, one of the ALK inhibitors, and ligand of von Hippel-Lindau (VHL) E3 ligase. Among these molecules, TD-004 effectively induced ALK degradation and inhibited the growth of ALK fusion positive cell lines, SU-DHL-1 and H3122. We also confirmed that TD-004 significantly reduced the tumor growth in H3122 xenograft model.

A natural compound, aristoyagonine, is identified as a potent bromodomain inhibitor by mid-throughput screening.

Bromodomain-containing protein 4 (Brd4) is known to play a key role in tumorigenesis. It binds acetylated histones to regulate the expression of numerous genes. Because of the importance of brd4 in tumorigenesis, much research has been undertaken to develop brd4 inhibitors with therapeutic potential. As a result, various scaffolds for bromodomain inhibitors have been identified. To discover new scaffolds, we performed mid-throughput screening using two different enzyme assays, alpha-screen and ELISA. We found a novel bromodomain inhibitor with a unique scaffold, aristoyagonine. This natural compound showed inhibitory activity in vitro and tumor growth inhibition in a Ty82-xenograft mouse model. In addition, we tested Brd4 inhibitors in gastric cancer cell lines, and found that aristoyagonine exerted cytotoxicity not only in I-BET-762-sensitive cancer cells, but also in I-BET-762-resistant cancer cells. This is the first paper to describe a natural compound as a Brd4 bromodomain inhibitor.

Preparation and Characterization of Various Poly(ether ether ketone) Containing Imidazolium Moiety for Anion Exchange Membrane Fuel Cell Application.

In this study, various poly(ether ether ketone) were synthesized using three different monomers and the imidazolium group was introduced into synthesized poly(ether ether ketone)s by using substitution reaction. Synthesized polymers were used to prepare anion exchange membranes and to evaluate its properties. Thermal, chemical and structural properties were carried out using thermogravimetric analysis, nuclear magnetic resonance. The anion exchange membranes with different imidazolium moieties were characterized by several different analytical techniques such as water up take, ion exchange capacity, hydroxide conductivity for checking the possibility to apply the anion exchange membrane fuel cell. Consequently, results of characterization were studied to understand the correlation between stabilities of the membrane and functional group and polymer backbone structures. And we confirm membrane performance was improved by increasing imidazolium cation groups.

Identification of novel ALK2 inhibitors and their effect on cancer cells.

Bone morphogenetic protein 9 (BMP9), a member of the TGF-ß superfamily, is considered a regulator of glucose homeostasis as well as a neuronal differentiation factor. BMP9 induces phosphorylation of Smad1/5 through activin receptor-like kinase 1 and 2 (ALK1 and ALK2). Recently, many studies have shown that BMP9 contributes to tumorigenesis, and aberrant ALK2 expression is involved in many diseases. To investigate the role of BMP9-ALK2 signaling in cancer cells, we used TF-1 cells that require granulocyte-macrophage colony-stimulating factor (GM-CSF) for cell proliferation. BMP9 promoted the proliferation of TF-1 cells in media lacking GM-CSF. TF-1 cells overexpressing ALK2 resulted in the autophosphorylation of Smad1/5, leading to consequent increase in cell growth. Through high-throughput screening (HTS), we found two ALK2-specific inhibitors, KRC203 and KRC360, with IC50 values of 0.9 nM and 0.3 nM. These compounds were more potent and specific for the inhibition of ALK2 when compared to LDN193189. In cell-based assays, these compounds effectively inhibited the proliferation and migration of cancer cells induced by ALK2 and BMP9. Therefore, we propose that our compounds are promising candidates for the treatment of cancer or diseases with abnormal ALK2 or BMP9 signaling.

Synthesis and biological evaluation of N9-cis-cyclobutylpurine derivatives for use as cyclin-dependent kinase (CDK) inhibitors.

A novel 6-aminopurine scaffold bearing an N9-cis-cyclobutyl moiety was designed using structure-based molecular design based on two known CDK inhibitors, dinaciclib and Cmpd-27. A series of novel 6-aminopurine compounds was prepared for structure-activity relationship (SAR) studies of CDK2 and CDK5 inhibitors. Among the compounds synthesized, compound 8l displayed potent CDK2 and CDK5 inhibitory activities with low nanomolar ranges (IC50=2.1 and 4.8nM, respectively) and showed moderate cytotoxicity in HCT116 colon cancer and MCF7 breast cancer cell lines. Here, we report the synthesis and evaluation of novel 6-aminopurine derivatives and present molecular docking models of compound 81 with CDK2 and CDK5.

Novel 2,4-diaminopyrimidines bearing fused tricyclic ring moiety for anaplastic lymphoma kinase (ALK) inhibitor.

In this study, a series of novel 2,4-diaminopyrimidines bearing fused tricyclic ring moiety was described for ALK inhibitor. The pyrazole, imidazole, 1,2,4-triazole, piperazine and phenanthridine moieties were employed at the 2-position of pyrimidine. Among the compounds synthesized, 28, 29, 36, and 42 showed promising anti-ALK activities in enzymatic- and cell-based assays. In vivo H3122 xenograft model study showed that compound 29 effectively suppressed ALK-driven tumor growth, similar to the extent of ceritinib, suggesting that it could be used for a novel ALK inhibitor development.

Development of a High-Throughput Assay for Inhibitors of the Polo-Box Domain of Polo-Like Kinase 1 Based on Time-Resolved Fluorescence Energy Transfer.

Although enzyme-linked immunosorbent assay (ELISA) technology has been widely accepted for binding assays against the polo-box domain (PBD) of polo-like kinase-1 (Plk1), these assays have a limitation-related heterogeneous procedure, such as multiple incubations and washing steps to apply high-throughput screenings (HTSs). In the present study, a Plk1-PBD binding assay based on time-resolved fluorescence energy transfer (TR-FRET) was developed for HTS of PBD-binding inhibitors. The TR-FRET-based Plk1-PBD binding assay is sensitive and robust and can be miniaturized into the 384-well plate-based format. Compared with the ELISA-based Plk1-PBD binding assay (Z' factor, 0.53; signal-to-background ratio, 4.19), the TR-FRET-based Plk1-PBD binding assay improved the Z' factor (0.72) and signal-to-background ratio (8.16). Using TR-FRET based Plk1-PBD binding assay, pilot library screening of 1019 natural compounds was conducted and five hit compounds such as haematoxylin, verbascoside, menadione, lithospermic acid and (1,3-dioxolo[4,5-g]isoquinolinium 5,6,7,8-tetrahydro-4-methoxy-6,6-dimethyl-5-[2-oxo-2-(2-pyridinyl)ethyl]-iodide) (DITMD) were identified as Plk1-PBD inhibitor. In a functional assay to validate the hit compounds, five hit compounds exhibited suppression of HeLa cells proliferation. These results suggest that TR-FRET-based Plk1-PBD binding assay can be applied for an efficient and less time-consuming HTS of compound libraries.

Mammalian Polo-like kinase 1 (Plk1) promotes proper chromosome segregation by phosphorylating and delocalizing the PBIP1·CENP-Q complex from kinetochores.

Mammalian Plk1 is critically required for proper M phase progression. Plk1 is self-recruited to prekinetochores/kinetochores by phosphorylating and binding to the Thr-78 motif of a kinetochore scaffold protein, PBIP1 (also called CENP-U/50), which forms a stable complex with another kinetochore component, CENP-Q. However, the mechanism regulating Plk1 localization to this site remains largely unknown. Here, we demonstrate that the PBIP1·CENP-Q complex became hyperphosphorylated and rapidly delocalized from kinetochores as cells entered mitosis. Plk1 phosphorylated the CENP-Q subunit of the PBIP1·CENP-Q complex at multiple sites, and mutation of nine Plk1-dependent phosphorylation sites to Ala (9A) enhanced CENP-Q association with chromatin and prolonged CENP-Q localization to kinetochores. Conversely, mutation of the nine sites to phospho-mimicking Asp/Glu (9D/E) residues dissociated CENP-Q from chromatin and kept the CENP-Q(9D/E) mutant from localizing to interphase prekinetochores. Strikingly, both the 9A and 9D/E mutants induced a defect in proper chromosome segregation, suggesting that both timely localization of the PBIP1·CENP-Q complex to prekinetochores and delocalization from kinetochores are critical for normal M phase progression. Notably, although Plk1 did not alter the level of PBIP1 and CENP-Q ubiquitination, Plk1-dependent phosphorylation and delocalization of these proteins from kinetochores appeared to indirectly lead to their degradation in the cytosol. Thus, we propose that Plk1 regulates the timing of the delocalization and ultimate destruction of the PBIP1·CENP-Q complex and that these processes are important not only for promoting Plk1-dependent mitotic progression, but also for resetting the timing of Plk1 recruitment to prekinetochores in the next cell cycle.

Novel c-Met inhibitor suppresses the growth of c-Met-addicted gastric cancer cells.

BACKGROUND: c-Met signaling has been implicated in oncogenesis especially in cells with c-met gene amplification. Since 20 % of gastric cancer patients show high level of c-Met expression, c-Met has been identified as a good candidate for targeted therapy in gastric cancer. Herein, we report our newly synthesized c-Met inhibitor by showing its efficacy both in vitro and in vivo. METHODS: Compounds with both triazolopyrazine and pyridoxazine scaffolds were synthesized and tested using HTRF c-Met kinase assay. We performed cytotoxic assay, cellular phosphorylation assay, and cell cycle assay to investigate the cellular inhibitory mechanism of our compounds. We also conducted mouse xenograft assay to see efficacy in vivo. RESULTS: KRC-00509 and KRC-00715 were selected as excellent c-Met inhibitors through biochemical assay, and exhibited to be exclusively selective to c-Met by kinase panel assay. Cytotoxic assays using 18 gastric cancer cell lines showed our c-Met inhibitors suppressed specifically the growth of c-Met overexpressed cell lines, not that of c-Met low expressed cell lines, by inducing G1/S arrest. In c-met amplified cell lines, c-Met inhibitors reduced the downstream signals including Akt and Erk as well as c-Met activity. In vivo Hs746T xenograft assay showed KRC-00715 reduced the tumor size significantly. CONCLUSIONS: Our in vitro and in vivo data suggest KRC-00715 is a potent and highly selective c-Met inhibitor which may have therapeutic potential in gastric tumor with c-Met overexpression.

Novel 2,4-diaminopyrimidines bearing tetrahydronaphthalenyl moiety against anaplastic lymphoma kinase (ALK): Synthesis, in vitro, ex vivo, and in vivo efficacy studies.

A series of novel 2,4-diaminopyrimidines bearing tetrahydronaphthalenyl moiety were synthesized and evaluated for their anti-anaplastic lymphoma kinase (ALK) activities using enzymatic and cell-based assays. Among the compounds synthesized, compound 17b showed promising pharmacological results in in vitro, ex vivo, and pharmacokinetic studies. An in vivo efficacy study with compound 17b demonstrated highly potent inhibitory activity in H3122 tumor xenograft model mice. A series of kinase assays showed that compound 17b inhibited various kinases including FAK, ACK1, FGFR, RSK1, IGF-1R, among others, thus demonstrating its potential for synergistic anti-tumor activity and development as a multi-targeted non-small cell lung cancer (NSCLC) therapy.

Discovery of novel tetrahydroisoquinoline-containing pyrimidines as ALK inhibitors.

Exploration of the two-position side chain of pyrimidine in LDK378 with tetrahydroisoquinolines (THIQs) led to discovery of 8 and 17 as highly potent ALK inhibitors. THIQs 8 and 17 showed encouraging in vitro and in vivo xenograft efficacies, comparable with those of LDK378. Although THIQ analogs (8a-o and 17a-i) prepared were not as active as their parent compounds, both 8 and 17 have significant inhibitory activities against various ALK mutant enzymes including G1202R, indicating that this series of compounds could be further optimized as useful ALK inhibitors overcoming the resistance issues found from crizotinib and LDK378.

Development of potent ALK inhibitor and its molecular inhibitory mechanism against NSCLC harboring EML4-ALK proteins.

Here, we show the newly synthesized and potent ALK inhibitor having similar scaffold to KRCA-0008, which was reported previously, and its molecular mechanism against cancer cells harboring EML4-ALK fusion protein. Through ALK wild type enzyme assay, we selected two compounds, KRCA-0080 and KRCA-0087, which have trifluoromethyl instead of chloride in R2 position. We characterized these newly synthesized compounds by in vitro and in vivo assays. Enzyme assay shows that KRCA-0080 is more potent against various ALK mutants, including L1196M, G1202R, T1151_L1152insT, and C1156Y, which are seen in crizotinib-resistant patients, than KRCA-0008 is. Cell based assays demonstrate our compounds downregulate the cellular signaling, such as Akt and Erk, by suppressing ALK activity to inhibit the proliferation of the cells harboring EML4-ALK. Interestingly, our compounds induced strong G1/S arrest in H3122 cells leading to the apoptosis, which is proved by PARP-1 cleavage. In vivo H3122 xenograft assay, we found that KRCA-0080 shows significant reduction in tumor size compared to crizotinib and KRCA-0008 by 15-20%. Conclusively, we report a potent ALK inhibitor which shows significant in vivo efficacy as well as excellent inhibitory activity against various ALK mutants.

Stereoselective construction of 2,6-cis-disubstituted tetrahydropyrans via intramolecular amide enolate alkylation: total synthesis of (-)-centrolobine.

A highly stereoselective construction of 2,6-cis-disubstituted tetrahydropyrans was achieved by using an intramolecular amide enolate alkylation with KHMDS. The efficiency and practicality of this methodology was successfully demonstrated in the total synthesis of (-)-centrolobine (1).

Synthesis and evaluation of novel 2,4-diaminopyrimidines bearing bicyclic aminobenzazepines for anaplastic lymphoma kinase (ALK) inhibitor.

A series of novel 2,4-diaminopyrimidine compounds bearing bicyclic aminobenzazepine were synthesized and evaluated for their anti-ALK activities. The activities of these compounds were confirmed in both enzyme- and cell-based ALK assays. Amongst compounds synthesized, KRCA-0445 showed very promising results in pharmacokinetic study and in vivo efficacy study with H3122 xenograft mouse model.