Proteomic Ligandability Maps of Spirocycle Acrylamide Stereoprobes Identify Covalent ERCC3 Degraders.

Covalent chemistry coupled with activity-based protein profiling (ABPP) offers a versatile way to discover ligands for proteins in native biological systems. Here, we describe a set of stereo- and regiochemically defined spirocycle acrylamides and the analysis of these electrophilic "stereoprobes" in human cancer cells by cysteine-directed ABPP. Despite showing attenuated reactivity compared to structurally related azetidine acrylamide stereoprobes, the spirocycle acrylamides preferentially liganded specific cysteines on diverse protein classes. One compound termed ZL-12A promoted the degradation of the TFIIH helicase ERCC3. Interestingly, ZL-12A reacts with the same cysteine (C342) in ERCC3 as the natural product triptolide, which did not lead to ERCC3 degradation but instead causes collateral loss of RNA polymerases. ZL-12A and triptolide cross-antagonized one another's protein degradation profiles. Finally, we provide evidence that the antihypertension drug spironolactone─previously found to promote ERCC3 degradation through an enigmatic mechanism─also reacts with ERCC3_C342. Our findings thus describe monofunctional degraders of ERCC3 and highlight how covalent ligands targeting the same cysteine can produce strikingly different functional outcomes.

Ruthenium-Catalyzed Butadiene-Mediated Crotylation and Oxazaborolidine-Catalyzed Vinylogous Mukaiyama Aldol Reaction for The Synthesis of C1-C19 and C23-C35 of Neaumycin B.

Neaumycin B is a femtomolar inhibitor of U87 human glioblastoma. Using a newly developed anti-diastereoselective ruthenium-catalyzed butadiene-mediated crotylation of primary alcohol proelectrophiles via hydrogen auto-transfer, as well as a novel variant of the catalytic asymmetric vinylogous Mukaiyama aldol (VMA) reaction applicable to linear aliphatic aldehydes and terminally methylated dienyl ketene acetals, preparation of the key C1-C19 and C23-C35 substructures of neaumycin B is achieved in 12 and 7 steps (LLS), respectively.

Kinetic, ESI-CID-MS and Computational Studies of π-Allyliridium C,O-Benzoate-Catalyzed Allylic Amination: Understanding the Effect of Cesium Ion.

The mechanism of π-allyliridium C,O-benzoate-catalyzed allylic amination was studied by (a) reaction progress kinetic analysis (RPKA), (b) tandem ESI-MS analysis, and (c) computational studies involving density functional theory (DFT) calculations. Reaction progress kinetic analysis (RPKA) reveals a zero-order dependence on allyl acetate, first-order dependence on catalyst and fractional-order dependence on amine. These data corroborate rapid ionization of the allylic acetate followed by turnover limiting C-N bond formation. To illuminate the origins of the 0.4 kinetic order dependence on amine, ESI-MS analyses of quaternary ammonium-labelled piperazine with multistage collision induced dissociation (CID) were conducted that corroborate intervention of cesium-bridged amine dimers that dissociate to form monomeric cesium amide nucleophiles. Computational data align with RPKA and ESI-CID-MS analyses and suggest early transition states mitigate the impact of steric factors, thus enabling formation of highly substituted C-N bonds with complete levels of branched regioselectivity. Specifically, trans-effects of the iridium complex facilitate nucleophilic attack at the more substituted allyl terminus trans to phosphorus with enantioselectivity governed by steric repulsions between the chiral bisphosphine ligand and the π-allyl of a dominant diastereomer of the stereogenic-at-metal complex. Beyond defining aspects of the mechanism of π-allyliridium C,O-benzoate-catalyzed allylic amination, these data reveal that a key feature of cesium carbonate not only lies in its enhanced basicity, but also its capacity for Lewis-acid enhanced Brønsted acidification of amines.

Regio- and Enantioselective Iridium-Catalyzed Amination of Alkyl-Substituted Allylic Acetates with Secondary Amines.

Robust air-stable cyclometalated π-allyliridium C,O-benzoates modified by (S)-tol-BINAP catalyze the reaction of secondary aliphatic amines with racemic alkyl-substituted allylic acetates to furnish products of allylic amination with high levels of enantioselectivity. Complete branched regioselectivities were observed despite the formation of more highly substituted C-N bonds.

Total Synthesis of the Spliceosome Modulator Pladienolide†B via Asymmetric Alcohol-Mediated syn- and anti-Diastereoselective Carbonyl Crotylation.

The potent spliceosome modulator pladienolide B, which bears 10 stereogenic centers, is prepared in 10 steps (LLS). Asymmetric alcohol-mediated carbonyl crotylations catalyzed by ruthenium and iridium that occur with syn- and anti-diastereoselectivity, respectively, were used to form the C20-C21 and C10-C11 C-C bonds.

Novel brd4 inhibitors with a unique scaffold exhibit antitumor effects.

Since bromodomain containing 4 (brd4) has been considered as a prominent cancer target, numerous attempts have been made to develop potent brd4 bromodomain inhibitors. The present study provided a novel chemical scaffold which inhibited brd4 activity. Mid-throughput screening against brd4 bromodomain was performed using alpha-screen and homogeneous time-resolved fluorescence assays. Furthermore, cell cytotoxicity and xenograft assays were performed to examine if the compound was effective both in vitro and in vivo. As a result, it was revealed that compounds having naphthalene-1,4-dione scaffold inhibited the binding of bromodomain to acetylated histone. The compounds with naphthalene-1,4-dione had cytotoxic effects against the Ty82 cell line, a NUT midline carcinoma cell line, whose proliferation is dependent on brd4 activity. A10, one of the compounds with naphthalene-1,4-dione scaffold, also exhibited tumor growth inhibition effects in the xenograft assay. In addition, the compounds exhibited cytotoxic effects against gastric cancer cell lines which were resistant to I-BET-762, a BET bromodomain inhibitor. In conclusion, the novel scaffold to suppress brd4 activity was effective against cancer cells both in vitro and in vivo.

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.

Enantioselective Iridium-Catalyzed Allylation of Acetylenic Ketones via 2-Propanol-Mediated Reductive Coupling of Allyl Acetate: C14-C23 of Pladienolide†D.

Highly enantioselective catalytic reductive coupling of allyl acetate with acetylenic ketones occurs in a chemoselective manner in the presence of aliphatic or aromatic ketones. This method was used to construct C14-C23 of pladienolide D in half the steps previously required.

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.

Synthesis and biological evaluation of indazole-4,7-dione derivatives as novel BRD4 inhibitors.

Bromodomain-containing protein 4 (BRD4) is known to regulate the expression of c-Myc to control the proliferation of cancer cells. Therefore, development of small-molecule inhibitors targeting the bromodomain has been widely studied. However, some clinical trials on BRD4 inhibitors have shown its drawbacks such as toxicity including the loss of organ weight. Here, we report the development of the novel and promising scaffold, 1H-indazol-4,7-dione, as a bromodomain inhibitor and synthesized derivatives for the inhibition of binding of bromodomain to acetylated histone peptide. Through this effort, we obtained 6-chloro-5-((2,6-difluorophenyl)amino)-1H-indazole-4,7-dione (5i), which showed a highly potent activity with a half-maximal inhibitory concentration (IC50) of 60 nM. The in vivo xenograft assay confirmed that the 1H-indazol-4,7-dione compound reduced the tumor size significantly. These results show that the 1H-indazol-4,7-dione scaffold is highly potent against bromodomain.

Synthesis and biological evaluation of thiazole derivatives as GPR119 agonists.

A series of 4-(phenoxymethyl)thiazole derivatives was synthesized and evaluated for their GPR119 agonistic effect. Several 4-(phenoxymethyl)thiazoles with pyrrolidine-2,5-dione moieties showed potent GPR119 agonistic activities. Among them, compound 27 and 32d showed good in vitro activity with an EC50 value of 49 nM and 18 nM, respectively with improved human and rat liver microsomal stability compare with MBX-2982. Compound 27 &32d did not exhibit significant CYP inhibition, hERG binding, and cytotoxicity. Moreover, these compounds lowered the glucose excursion in mice in an oral glucose-tolerance test.

Antihelminthic drug niclosamide inhibits CIP2A and reactivates tumor suppressor protein phosphatase 2A in non-small cell lung cancer cells.

Protein phosphatase 2A (PP2A) is a critical tumor suppressor complex responsible for the inactivation of various oncogenes. Recently, PP2A reactivation has emerged asan anticancer strategy. Cancerous inhibitor of protein phosphatase 2A (CIP2A), an endogenous inhibitor of PP2A, is upregulated in many cancer cells, including non-small cell lung cancer (NSCLC) cells. We demonstrated that the antihelminthic drug niclosamide inhibited the expression of CIP2A and reactivated the tumor suppressor PP2A in NSCLC cells. We performed a drug-repurposing screen and identified niclosamide asa CIP2A suppressor in NSCLC cells. Niclosamide inhibited cell proliferation, colony formation, and tumor sphere formation, and induced mitochondrial dysfunction through increased mitochondrial ROS production in NSCLC cells; however, these effects were rescued by CIP2A overexpression, which indicated that the antitumor activity of niclosamide was dependent on CIP2A. We found that niclosamide increased PP2A activity through CIP2A inhibition, which reduced the phosphorylation of several oncogenic proteins. Moreover, we found that a niclosamide analog inhibited CIP2A expression and increased PP2A activity in several types of NSCLC cells. Finally, we showed that other well-known PP2A activators, including forskolin and FTY720, did not inhibit CIP2A and that their activities were not dependent on CIP2A. Collectively, our data suggested that niclosamide effectively suppressed CIP2A expression and subsequently activated PP2A in NSCLC cells. This provided strong evidence for the potential use of niclosamide asa PP2A-activating drug in the clinical treatment of NSCLC.