Discovery of 3-Phenyl Indazole-Based Novel Chemokine-like Receptor 1 Antagonists for the Treatment of Psoriasis.

Chemokine-like receptor 1 (CMKLR1)─a G protein-coupled receptor─has functional roles in the immune system and related diseases, including psoriasis and metabolic diseases. Psoriasis is a chronic inflammatory disease characterized by skin redness, scaliness, and itching. In this study, we sought to develop novel CMKLR1 antagonists by screening our in-house GPCR-targeting compound library. Moreover, we optimized a phenylindazole-based hit compound with antagonistic activities and evaluated its oral pharmacokinetic properties in a murine model. A structure-based design on the human CMKLR1 homology model identified S-26d as an optimized compound that serves as a potent and orally available antagonist with a pIC50 value of 7.44 in hCMKLR1-transfected CHO cells. Furthermore, in the imiquimod-induced psoriasis-like mouse model, oral administration of S-26d for 1 week significantly alleviated modified psoriasis area and severity index scores (severity of erythema, scaliness, skin thickness) compared with the control group.

Development of LpxH Inhibitors Chelating the Active Site Dimanganese Metal Cluster of LpxH.

Despite the widespread emergence of multidrug-resistant nosocomial Gram-negative bacterial infections and the major public health threat it brings, no new class of antibiotics for Gram-negative pathogens has been approved over the past five decades. Therefore, there is an urgent medical need for developing effective novel antibiotics against multidrug-resistant Gram-negative pathogens by targeting previously unexploited pathways in these bacteria. To fulfill this crucial need, we have been investigating a series of sulfonyl piperazine compounds targeting LpxH, a dimanganese-containing UDP-2,3-diacylglucosamine hydrolase in the lipid A biosynthetic pathway, as novel antibiotics against clinically important Gram-negative pathogens. Inspired by a detailed structural analysis of our previous LpxH inhibitors in complex with K. pneumoniae LpxH (KpLpxH), here we report the development and structural validation of the first-in-class sulfonyl piperazine LpxH inhibitors, JH-LPH-45 (8) and JH-LPH-50 (13), that achieve chelation of the active site dimanganese cluster of KpLpxH. The chelation of the dimanganese cluster significantly improves the potency of JH-LPH-45 (8) and JH-LPH-50 (13). We expect that further optimization of these proof-of-concept dimanganese-chelating LpxH inhibitors will ultimately lead to the development of more potent LpxH inhibitors for targeting multidrug-resistant Gram-negative pathogens.

Synthesis and evaluation of cyclopentane-based muraymycin analogs targeting MraY.

Antibiotic resistance is one of the most challenging global health issues and presents an urgent need for the development of new antibiotics. In this regard, phospho-MurNAc-pentapeptide translocase (MraY), an essential enzyme in the early stages of peptidoglycan biosynthesis, has emerged as a promising new antibiotic target. We recently reported the crystal structures of MraY in complex with representative members of naturally occurring nucleoside antibiotics, including muraymycin D2. However, these nucleoside antibiotics are synthetically challenging targets, which limits the scope of medicinal chemistry efforts on this class of compounds. To gain access to active muraymycin analogs with reduced structural complexity and improved synthetic tractability, we prepared and evaluated cyclopentane-based muraymycin analogs for targeting MraY. For the installation of the 1,2-syn-amino alcohol group of analogs, the diastereoselective isocyanoacetate aldol reaction was explored. The structure-activity relationship analysis of the synthesized analogs suggested that a lipophilic side chain is essential for MraY inhibition. Importantly, the analog 20 (JH-MR-23) showed antibacterial efficacy against Staphylococcus aureus. These findings provide insights into designing new muraymycin-based MraY inhibitors with improved chemical tractability.

Synthesis and evaluation of sulfonyl piperazine LpxH inhibitors.

The UDP-2,3-diacylglucosamine pyrophosphate hydrolase LpxH is essential in lipid A biosynthesis and has emerged as a promising target for the development of novel antibiotics against multidrug-resistant Gram-negative pathogens. Recently, we reported the crystal structure of Klebsiella pneumoniae LpxH in complex with 1 (AZ1), a sulfonyl piperazine LpxH inhibitor. The analysis of the LpxH-AZ1 co-crystal structure and ligand dynamics led to the design of 2 (JH-LPH-28) and 3 (JH-LPH-33) with enhanced LpxH inhibition. In order to harness our recent findings, we prepared and evaluated a series of sulfonyl piperazine analogs with modifications in the phenyl and N-acetyl groups of 3. Herein, we describe the synthesis and structure-activity relationship of sulfonyl piperazine LpxH inhibitors. We also report the structural analysis of an extended N-acyl chain analog 27b (JH-LPH-41) in complex with K. pneumoniae LpxH, revealing that 27b reaches an untapped polar pocket near the di-manganese cluster in the active site of K. pneumoniae LpxH. We expect that our findings will provide designing principles for new LpxH inhibitors and establish important frameworks for the future development of antibiotics against multidrug-resistant Gram-negative pathogens.

Evaluation of Manassantin A Tetrahydrofuran Core Region Analogues and Cooperative Therapeutic Effects with EGFR Inhibition.

Tumors adapt to hypoxia by regulating angiogenesis, metastatic potential, and metabolism. These adaptations mediated by hypoxia-inducible factor 1 (HIF-1) make tumors more aggressive and resistant to chemotherapy and radiation. Therefore, HIF-1 is a validated therapeutic target for cancer. In order to develop new HIF-1 inhibitors for cancer chemotherapy by harnessing the potential of the natural product manassantin A, we synthesized and evaluated manassantin A analogues with modifications in the tetrahydrofuran core region of manassantin A. Our structure-activity relationship study indicated that the α,α'-trans-configuration of the central ring of manassantin A is critical to HIF-1 inhibition. We also demonstrated that a combination of manassantin A with an epidermal growth factor receptor inhibitor shows cooperative antitumor activity (∼80% inhibition for combination vs ∼30% inhibition for monotherapy). Our findings will provide important frameworks for the future therapeutic development of manassantin A-derived chemotherapeutic agents.

Structure-Activity Relationship of Sulfonyl Piperazine LpxH Inhibitors Analyzed by an LpxE-Coupled Malachite Green Assay.

The UDP-2,3-diacylglucosamine pyrophosphatase LpxH in the Raetz pathway of lipid A biosynthesis is an essential enzyme in the vast majority of Gram-negative pathogens and an excellent novel antibiotic target. The 32P-radioautographic thin-layer chromatography assay has been widely used for analysis of LpxH activity, but it is inconvenient for evaluation of a large number of LpxH inhibitors over an extended time period. Here, we report a coupled, nonradioactive LpxH assay that utilizes the recently discovered Aquifex aeolicus lipid A 1-phosphatase LpxE for quantitative removal of the 1-phosphate from lipid X, the product of the LpxH catalysis; the released inorganic phosphate is subsequently quantified by the colorimetric malachite green assay, allowing the monitoring of the LpxH catalysis. Using such a coupled enzymatic assay, we report the biochemical characterization of a series of sulfonyl piperazine LpxH inhibitors. Our analysis establishes a preliminary structure-activity relationship for this class of compounds and reveals a pharmacophore of two aromatic rings, two hydrophobic groups, and one hydrogen-bond acceptor. We expect that our findings will facilitate the development of more effective LpxH inhibitors as potential antibacterial agents.

Synthesis and structure-activity relationships of quinolinone and quinoline-based P2X7 receptor antagonists and their anti-sphere formation activities in glioblastoma cells.

Screening a compound library of quinolinone derivatives identified compound 11a as a new P2X7 receptor antagonist. To optimize its activity, we assessed structure-activity relationships (SAR) at three different positions, R1, R2 and R3, of the quinolinone scaffold. SAR analysis suggested that a carboxylic acid ethyl ester group at the R1 position, an adamantyl carboxamide group at R2 and a 4-methoxy substitution at the R3 position are the best substituents for the antagonism of P2X7R activity. However, because most of the quinolinone derivatives showed low inhibitory effects in an IL-1ß ELISA assay, the core structure was further modified to a quinoline skeleton with chloride or substituted phenyl groups. The optimized antagonists with the quinoline scaffold included 2-chloro-5-adamantyl-quinoline derivative (16c) and 2-(4-hydroxymethylphenyl)-5-adamantyl-quinoline derivative (17k), with IC50 values of 4 and 3 nM, respectively. In contrast to the quinolinone derivatives, the antagonistic effects of the quinoline compounds (16c and 17k) were paralleled by their ability to inhibit the release of the pro-inflammatory cytokine, IL-1ß, from LPS/IFN-γ/BzATP-stimulated THP-1 cells (IC50 of 7 and 12 nM, respectively). In addition, potent P2X7R antagonists significantly inhibited the sphere size of TS15-88 glioblastoma cells.

Towards a Novel Class of Multitarget-Directed Ligands: Dual P2X7-NMDA Receptor Antagonists.

Multi-target-directed ligands (MTDLs) offer new hope for the treatment of multifactorial complex diseases such as Alzheimer's Disease (AD). Herein, we present compounds aimed at targeting the NMDA and the P2X7 receptors, which embody a different approach to AD therapy. On one hand, we are seeking to delay neurodegeneration targeting the glutamatergic NMDA receptors; on the other hand, we also aim to reduce neuroinflammation, targeting P2X7 receptors. Although the NMDA receptor is a widely recognized therapeutic target in treating AD, the P2X7 receptor remains largely unexplored for this purpose; therefore, the dual inhibitor presented herein-which is open to further optimization-represents the first member of a new class of MTDLs.

Escape from adamantane: Scaffold optimization of novel P2X7 antagonists featuring complex polycycles.

The adamantane scaffold, despite being widely used in medicinal chemistry, is not devoid of problems. In recent years we have developed new polycyclic scaffolds as surrogates of the adamantane group with encouraging results in multiple targets. As an adamantane scaffold is a common structural feature in several P2X7 receptor antagonists, herein we report the synthesis and pharmacological evaluation of multiple replacement options of adamantane that maintain a good activity profile. Molecular modeling studies support the binding of the compounds to a site close to the central pore, rather than to the ATP-binding site and shed light on the structural requirements for novel P2X7 antagonists.

Discovery and structure-activity relationship studies of quinolinone derivatives as potent IL-2 suppressive agents.

The quinolinone skeleton has been utilized to develop various mechanism-based immune modulators. However, the effects of quinolinone derivatives on the release of T cell-associated interleukin-2 (IL-2) have not been established. In this study, a series of novel quinolinone derivatives was synthesized, and their immunosuppressive activity was evaluated by measuring suppression of IL-2 release from activated Jurkat T cells. Optimizing the three side chains around the quinolinone skeleton revealed the most active compound: 11l. This compound exhibits potent inhibitory activity toward IL-2 release in both 12-o-tetradecanoylphorbol-13-acetate (PMA)/A23187 (ionomycin) (IC50=80±10nM) and anti-CD3/CD28-stimulated Jurkat T cells (83% inhibition at 10µM) without cytotoxic effects. Further investigation into the underlying mechanism of 11l indicated the suppression of NF-κB and nuclear factor of activated T cells (NFAT) promoter activities in Jurkat T cells.

Discovery of novel purine-based heterocyclic P2X7 receptor antagonists.

The pyridine core skeleton of the previously reported dichloropyridine-based potent hP2X7 receptor antagonist 5 (IC50 = 13 nM in hP2X7-expressing HEK293 cells) was modified with various heterocyclic scaffolds. Among the derivatives with quinoline, quinazoline, acridine, and purine scaffolds, the chloropurine-based analog 9o exhibited the most potent antagonistic activity, with an IC50 value of 176 ± 37 nM in an ethidium bromide uptake assay. In addition, 9o significantly inhibited IL-1ß release in THP-1 cells stimulated with LPS/IFN-γ/BzATP (IC50 = 120 ± 15 nM). Although 9o was less active than the previous antagonist 5, 9o exhibited greatly improved metabolic stability in the in vitro evaluation (71.4% in human, 72.3% in mouse).

Solid-phase synthesis of quinolinone library.

Quinolinones have various biological activities, including antibacterial, anticancer, and antiviral properties. The 3-substituted amide quinolin-2(1H)-ones not only show antibacterial activity, but also act as immunomodulators, 5-HT4 receptor agonists, cannabinoid receptor inverse agonists, and AchE and, BuchE inhibitors. To investigate the potent biological activity of 3-substituted amide quinolin-2(1H)-ones, a large number of 3,5-amide substituted-2-oxoquinolinones were prepared by parallel solid-phase synthesis. The compound 5-amino-1-(4-methoxybenzyl)-2-oxo-1,2-dihydroquinoline-3-carboxylic acid was loaded onto 4-formyl-3,5-dimethoxyphenoxy (PL-FDMP) resin by reductive amination with high efficiency. Various building blocks were attached to the 3 and 5 positions to yield 3,5-disubstituted-2-oxoquinolinones with high purity and good yield. The ability some of these compound to inhibit the release of IL-1ß, a cytokine involved in the immune response was measured, and they showed about 50% inhibition at 10 µM.