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.

Nanoparticle-encapsulated P2X7 receptor antagonist in a pH-sensitive polymer as a potential local drug delivery system to acidic inflammatory environments.

We have developed nanoparticles of anti-inflammatory P2X7 receptor antagonist encapsulated in a pH-sensitive polymer, poly(tetrahydropyran-2-yl methacrylate) (poly(THPMA)), as a potential local drug delivery system to target to acidic inflammatory environments, in which P2X7 receptors are implicated in the pathology of inflammation via the activation of immune cells. The nanoparticles were prepared using single emulsion methods, also their size and shape were confirmed by microscopy and spectroscopy, etc. The profiles of the pH-dependent degradation, release of antagonist and biological activities were investigated. The nanoparticles that encapsulated the 3,5-dichloropyridine derivative (2) with poly(THPMA), were observed to be more slowly cleaved than the blank nanoparticles. Moreover, the free P2X7 receptor antagonists potently inhibited the receptor activation, whereas the nanoparticles of the 3,5-dichloropyridine derivative (2) encapsulated poly(THPMA) exhibited much lower P2X7 antagonistic activity through sustained encapsulation. Thus, the nanoparticles of the 3,5-dichloropyridine derivative (2) encapsulated poly(THPMA) may be utilized to develop a pH-sensitive local drug delivery system for controlled release of anti-inflammatory therapeutics in acidic physiological environments.

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).

Discovery of benzimidazole-indazole derivatives as potent FLT3-tyrosine kinase domain mutant kinase inhibitors for acute myeloid leukemia.

The FMS-like tyrosine kinase 3 (FLT3) gene encodes a class III receptor tyrosine kinase that is expressed in hematopoietic stem cells. The mutations of FLT3 gene found in 30% of acute myeloid leukemia (AML), leads to an abnormal constitutive activation of FLT3 kinase of the receptor and results in immature myeloblast cell proliferation. Although small molecule drugs targeting the FLT3 kinase have been approved, new FLT3 inhibitors are needed owing to the side effects and drug resistances arising from kinase domain mutations, such as D835Y and F691L. In this study, we have developed benzimidazole-indazole based novel inhibitors targeting mutant FLT3 kinases through the optimization of diverse chemical moieties substituted around the core skeleton. The most optimized compound 22f exhibited potent inhibitory activities against FLT3 and FLT3/D835Y, with IC50 values of 0.941 and 0.199 nM, respectively. Furthermore, 22f exhibited strong antiproliferative activity against an AML cell line, MV4-11 cells with a GI50 of 0.26 nM. More importantly, 22f showed single-digit nanomolar GI50 values in the mutant FLT kinase expressed Ba/F3 cell lines including FLT-D835Y (GI50 = 0.29 nM) and FLT3-F691L (GI50 = 2.87 nM). Molecular docking studies indicated that the compound exhibits a well-fitted binding mode as a type 1 inhibitor in the homology model of active conformation of FLT3 kinase.

Development of anti-coxsackievirus agents targeting 3C protease.

Peptidomimetic anti-viral agents against Coxsackievirus B3 (CVB3) were developed using a strategy involving the inhibition of 3C protease (CVB3 3C(pro)), a target for CVB3-mediated myocarditis or pericarditis. In an attempt to improve the inhibitory activity against CVB3, a variety of hetero-aromatic groups were incorporated into the α,ß-unsaturated ester as Michael acceptor moiety, which is the position of interaction with the cysteine moiety in the P1' active site of CVB3 3C(pro). Among these hetero-aromatic groups, the quinoline analogs 9c and 9e, with IC(50) values of 250 and 130 nM as determined from an enzyme assay, significantly inhibited the CVB3-mediated cell cytotoxicity, indicating parallel anti-viral activities. A comparison of the binding modes of the potent inhibitor 9e and the relatively weak inhibitor 9n was explored in a molecular docking study, which revealed that compound 9n lacked hydrogen bonds in its interactions with Gly129, 128, and 145.

Indirubin derivatives as potent FLT3 inhibitors with anti-proliferative activity of acute myeloid leukemic cells.

Indirubin derivatives were identified as potent FLT3 tyrosine kinase inhibitors with anti-proliferative activity at acute myeloid leukemic cell lines, RS4;11 and MV4;11 which express FLT3-WT and FLT3-ITD mutation, respectively. Among several 5 and 5'-substituted indirubin derivatives, 5-fluoro analog, 13 exhibited potent inhibitory activity at FLT3 (IC(50)=15 nM) with more than 100-fold selectivity versus 6 other kinases and potent anti-proliferative effect for MV4;11 cells (IC(50)=72 nM) with 30-fold selectivity versus RS4;11 cells. Cell cycle analysis indicated that compound 13 induced cell cycle arrest at G(0)/G(1) phase in MV4;11 cells.

Discovery of orally active indirubin-3'-oxime derivatives as potent type 1 FLT3 inhibitors for acute myeloid leukemia.

FMS-like receptor tyrosine kinase-3 (FLT3) is expressed on acute leukemia cells and is implicated in the survival, proliferation and differentiation of hematopoietic cells in most acute myeloid leukemia (AML) patients. Despite recent achievements in the development of FLT3-targeted small-molecule drugs, there are still unmet medical needs related to kinase selectivity and the progression of some mutant forms of FLT3. Herein, we describe the discovery of novel orally available type 1 FLT3 inhibitors from structure-activity relationship (SAR) studies for the optimization of indirubin derivatives with biological and pharmacokinetic profiles as potential therapeutic agents for AML. The SAR exploration provided important structural insights into the key substituents for potent inhibitory activities of FLT3 and in MV4-11 cells. The profile of the most optimized inhibitor (36) showed IC50 values of 0.87 and 0.32 nM against FLT3 and FLT3/D835Y, respectively, along with potent inhibition against MV4-11 and FLT3/D835Y expressed MOLM14 cells with a GI50 value of 1.0 and 1.87 nM, respectively. With the high oral bioavailability of 42.6%, compound 36 displayed significant in vivo antitumor activity by oral administration of 20 mg/kg once daily dosing schedule for 21 days in a mouse xenograft model. The molecular docking study of 36 in the homology model of the DFG-in conformation of FLT3 resulted in a reasonable binding mode in type 1 kinases similar to the reported type 1 FLT3 inhibitors Crenolanib and Gilteritinib.

Synthesis and structure-activity relationships of novel, substituted 5,6-dihydrodibenzo[a,g]quinolizinium P2X7 antagonists.

Iminium quaternary protoberberine alkaloids (QPA) have been found to be novel P2X(7) antagonists. To assess their structure-activity relationships, these compounds were modified at their R(1) and R(2) groups and assayed for their ability to inhibit the 2'(3')-O-(4-benzoylbenzoyl)-ATP (BzATP)-induced uptake of fluorescent ethidium by HEK-293 cells stably expressing the human P2X(7) receptor, and their ability to inhibit BzATP-induced IL-1beta release by differentiated THP-1 cells. Compounds 15a and 15d, with alkyl groups at the R(1) position, and especially compound 19h, with the 2-NO(2)-4,5-dimethoxy-benzyl group at the R(2) position, had potent inhibitory efficacy as P2X(7) antagonists.

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.

Discovery of Potent Antiallodynic Agents for Neuropathic Pain Targeting P2X3 Receptors.

Antagonism of the P2X3 receptor is one of the potential therapeutic strategies for the management of neuropathic pain because P2X3 receptors are predominantly localized on small to medium diameter C- and Aδ-fiber primary afferent neurons, which are related to the pain-sensing system. In this study, 5-hydroxy pyridine derivatives were designed, synthesized, and evaluated for their in vitro biological activities by two-electrode voltage clamp assay at hP2X3 receptors. Among the novel hP2X3 receptor antagonists, intrathecal treatment of compound 29 showed parallel efficacy with pregabalin (calcium channel modulator) and higher efficacy than AF353 (P2X3 receptor antagonist) in the evaluation of its antiallodynic effects in spinal nerve ligation rats. However, because compound 29 was inactive by intraperitoneal administration in neuropathic pain animal models due to low cell permeability, the corresponding methyl ester analogue, 28, which could be converted to compound 29 in vivo, was investigated as a prodrug concept. Intravenous injection of compound 28 resulted in potent antiallodynic effects, with ED50 values of 2.62 and 2.93 mg/kg in spinal nerve ligation and chemotherapy-induced peripheral neuropathy rats, respectively, indicating that new drug development targeting the P2X3 receptor could be promising for neuropathic pain, a disease with high unmet medical needs.

Potent Suppressive Effects of 1-Piperidinylimidazole Based Novel P2X7 Receptor Antagonists on Cancer Cell Migration and Invasion.

The P2X7 receptor (P2X7R) has been reported as a key mediator in inflammatory processes and cancer invasion/metastasis. In this study, we report the discovery of novel P2X7R antagonists and their functional activities as potential antimetastatic agents. Modifications of the hydantoin core-skeleton and the side chain substituents of the P2X7R antagonist 7 were performed. The structure-activity relationships (SAR) and optimization demonstrated the importance of the sulfonyl group at the R1 position and the substituted position and overall size of R2 for P2X7R antagonism. The optimized novel analogues displayed potent P2X7 receptor antagonism (IC50 = 0.11-112 nM) along with significant suppressive effects on IL-1ß release (IC50 = 0.32-210 nM). Moreover, representative antagonists (12g, 13k, and 17d) with imidazole and uracil core skeletons significantly inhibited the invasion of MDA-MB-231 triple negative breast cancer cells and cancer cell migration in a zebrafish xenograft model, suggesting the potential therapeutic application of these novel P2X7 antagonists to block metastatic cancer.

Structure-activity relationship studies of pyrimidine-2,4-dione derivatives as potent P2X7 receptor antagonists.

As an optimization strategy, the flexible structure of KN-62, a known P2X7 receptor antagonist, was converted into conformationally constrained derivatives using pyrimidine-2,4-dione as the core skeleton. Various modifications at the 4-position of the piperazine moiety of the new lead compound were performed to improve P2X7 receptor antagonistic activities, which were evaluated in HEK293 cells stably expressing the human P2X7 receptor (EtBr uptake assay) and in THP-1 cells (IL-1ß ELISA assay). According to the results, polycycloalkyl acyl or di-halogenated benzoyl substituents were much more favorable than the original phenyl group of KN-62. Among these compounds, the trifluoromethyl-chloro benzoyl derivative 18 m and adamantyl carbonyl derivatives 19 g-19 i and 19k showed potent antagonistic effects, with IC50 values ranging from 10 to 30 nM. In addition, the in vitro adsorption, distribution, metabolism, excretion, and toxicity (ADMET) profile of 18 m was determined to be in acceptable ranges in terms of metabolic stability and cytotoxicity. These results suggest that pyrimidine-2,4-dione derivatives may be promising novel P2X7 receptor antagonists for the development of anti-inflammatory drugs.

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.

Discovery of novel 2,5-dioxoimidazolidine-based P2X(7) receptor antagonists as constrained analogues of KN62.

Novel 2,5-dioxoimidazolidine-based conformationally constrained analogues of KN62 (1) were developed as P2X7 receptor (P2X7R) antagonists using a rigidification strategy of the tyrosine backbone of 1. SAR analysis of the 2,5-dioxoimidazolidine scaffold indicated that piperidine substitution at the N3 position and no substitution at N1 position were preferable. Further optimization of the substituents at the piperidine nitrogen and the spacer around the skeleton resulted in several superior antagonists to 1, including 1-adamantanecarbonyl analogue 21i (IC50 = 23 nM in ethidium uptake assay; IC50 = 14 nM in IL-1ß ELISA assay) and (3-CF3-4-Cl)benzoyl analogue (-)-21w (54 nM in ethidium uptake assay; 9 nM in IL-1ß ELISA assay), which was more potent than the corresponding (+) isomer. Compound 21w displayed potent inhibitory activity in an ex vivo model of LTP-induced pain signaling in the spinal cord and significant anti-inflammatory activity in in vivo models of carrageenan-induced paw edema and type II collagen-induced joint arthritis.

Structure-activity relationships and optimization of 3,5-dichloropyridine derivatives as novel P2X(7) receptor antagonists.

Screening of a library of chemical compounds showed that the dichloropyridine-based analogue 9 was a novel P2X(7) receptor antagonist. To optimize its activity, we assessed the structure-activity relationships (SAR) of 9, focusing on the hydrazide linker, the dichloropyridine skeleton, and the hydrophobic acyl (R(2)) group. We found that the hydrazide linker and the 3,5-disubstituted chlorides in the pyridine skeleton were critical for P2X(7) antagonistic activity and that the presence of hydrophobic polycycloalkyl groups at the R(2) position optimized antagonistic activity. In the EtBr uptake assay in hP2X(7)-expressing HEK293 cells, the optimized antagonists, 51 and 52, had IC(50) values of 4.9 and 13 nM, respectively. The antagonistic effects of 51 and 52 were paralleled by their ability to inhibit the release of the pro-inflammatory cytokine, IL-1ß, by LPS/IFN-γ/BzATP stimulation of THP-1 cells (IC(50) = 1.3 and 9.2 nM, respectively). In addition, 52 strongly inhibited iNOS/COX-2 expression and NO production in THP-1 cells, further indicating that this compound blocks inflammatory signaling and suggesting that the dichloropyridine analogues may be useful in developing P2X(7) receptor targeted anti-inflammatory agents.

Design and synthesis of 1,4-dihydropyridine derivatives as BACE-1 inhibitors.

BACE-1 has been shown to be an attractive therapeutic target in Alzheimer's disease (AD). Using a 1,4-dihydropyridine (DHP) scaffold, we synthesized new inhibitors of BACE-1 by modifying the known BACE inhibitor 2 containing a hydroxyethylamine (HEA) motif. Using structure-based drug design based on computer-aided molecular docking, the isophthalamide ring of 2 was replaced with a 1,4-dihydropyridine ring as a brain-targeting strategy. Several of the new dihydropyridine derivatives were synthesized and their BACE-1-inhibitory activities were evaluated using a cell-based, reporter gene assay system that measures the cleavage of alkaline phosphatase (AP)-APP fusion protein by BACE-1. Most of the 1,4-DHP analogs showed BACE-1-inhibitory activities with IC50 values in the range 8-30 microM, suggesting that the 1,4-DHP skeleton may be utilized to develop brain-targeting BACE-1 inhibitors.

5,5'-substituted indirubin-3'-oxime derivatives as potent cyclin-dependent kinase inhibitors with anticancer activity.

To enhance the ability of indirubin derivatives to inhibit CDK2/cyclin E, a target of anticancer agents, we designed and synthesized a new series of indirubin-3'-oxime derivatives with combined substitutions at the 5 and 5' positions. A molecular docking study predicted the binding of derivatives with OH or halogen substitutions at the 5' position to the ATP binding site of CDK2, revealing the critical interactions that may explain the improved CDK2 inhibitory activity of these derivatives. Among the synthesized derivatives, the 5-nitro-5'-hydroxy analogue 3a and the 5-nitro-5'-fluoro analogue 5a displayed potent inhibitory activity against CDK2, with IC(50) values of 1.9 and 1.7 nM, respectively. These derivatives also showed antiproliferative activity against several human cancer cell lines, with IC(50) values of 0.2-3.3 microM. A representative analogue, 3a, showed greater than 500-fold selectivity for CDK relative to selected kinase panel and potent in vivo anticancer activity.