Development of 5-hydroxybenzothiophene derivatives as multi-kinase inhibitors with potential anti-cancer activity.

Aim: Chemoresistance in cancer challenges the classical therapeutic strategy of 'one molecule-one target'. To combat this, multi-target therapies that inhibit various cancer-relevant targets simultaneously are proposed. Methods & results: We introduce 5-hydroxybenzothiophene derivatives as effective multi-target kinase inhibitors, showing notable growth inhibitory activity across different cancer cell lines. Specifically, compound 16b, featuring a 5-hydroxybenzothiophene hydrazide scaffold, emerged as a potent inhibitor, displaying low IC50 values against key kinases and demonstrating significant anti-cancer effects, particularly against U87MG glioblastoma cells. It induced G2/M cell cycle arrest, apoptosis and inhibited cell migration by modulating apoptotic markers. Conclusion: 16b represents a promising lead for developing new anti-cancer agents targeting multiple kinases with affinity to the hydroxybenzothiophene core.

[Box: see text].

Molecular Insights into the Regulatory Landscape of Protein Kinase C-Related Kinase-2 (PRK2/PKN2) Using Targeted Small Compounds.

The protein kinase C-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the PIF-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.

N-Benzylated 5-Hydroxybenzothiophene-2-carboxamides as Multi-Targeted Clk/Dyrk Inhibitors and Potential Anticancer Agents.

Numerous studies have reported that Dyrk1A, Dyrk1B, and Clk1 are overexpressed in multiple cancers, suggesting a role in malignant disease. Here, we introduce a novel class of group-selective kinase inhibitors targeting Dyrk1A, Dyrk1B, and Clk1. This was achieved by modifying our earlier selective Clk1 inhibitors, which were based on the 5-methoxybenzothiophene-2-carboxamide scaffold. By incorporating a 5-hydroxy group, we increased the potential for additional hydrogen bond interactions that broadened the inhibitory effect to include Dyrk1A and Dyrk1B kinases. Within this series, compounds 12 and 17 emerged as the most potent multi-kinase inhibitors against Dyrk1A, Dyrk1B, and Clk1. Furthermore, when assessed against the most closely related kinases also implicated in cancer, the frontrunner compounds revealed additional inhibitory activity against Haspin and Clk2. Compounds 12 and 17 displayed high potency across various cancer cell lines with minimal effect on non-tumor cells. By examining the effect of these inhibitors on cell cycle distribution, compound 17 retained cells in the G2/M phase and induced apoptosis. Compounds 12 and 17 could also increase levels of cleaved caspase-3 and Bax, while decreasing the expression of the antiapoptotic Bcl-2 protein. These findings support the further study and development of these compounds as novel anticancer therapeutics.

N1-Benzoylated 5-(4-pyridinyl)indazole-based kinase inhibitors: Attaining haspin and Clk4 selectivity via modulation of the benzoyl substituents.

Haspin and Clk4 are both understudied protein kinases (PKs), offering potential targets for the development of new anticancer agents. Thus, the identification of new inhibitors targeting these PKs is of high interest. However, the inhibitors targeting haspin or Clk4 developed to date show a poor selectivity profile over other closely related PKs, increasing the risk of side effects. Herein, we present two newly developed N1-benzyolated 5-(4-pyridinyl)indazole-based inhibitors (18 and 19), derived from a newly identified indazole hit. These inhibitors exhibit an exceptional inhibitory profile toward haspin and/or Clk4. Compound 18 (2-acetyl benzoyl) showed a preference to inhibit Clk4 and haspin over a panel of closely related kinases, with sixfold selectivity for Clk4 (IC50 = 0.088 and 0.542 µM, respectively). Compound 19 (4-acetyl benzoyl) showed high selectivity against haspin over the common off-target kinases (Dyrks and Clks) with an IC50 of 0.155 µM for haspin. Molecular docking studies explained the remarkable selectivity of 18 and 19, elucidating how the new scaffold can be modified to toggle between inhibition of haspin or Clk4, despite the high homology of the ATP-binding sites. Their distinguished profile allows these compounds to be marked as interesting chemical probes to assess the selective inhibition of haspin and/or Clk4.

Novel 6-hydroxybenzothiazol-2-carboxamides as potent and selective monoamine oxidase B inhibitors endowed with neuroprotective activity.

In neurodegenerative diseases, using a single molecule that can exert multiple effects to modify the disease may have superior activity over the classical "one molecule-one target" approach. Herein, we describe the discovery of 6-hydroxybenzothiazol-2-carboxamides as highly potent and selective MAO-B inhibitors. Variation of the amide substituent led to several potent compounds having diverse side chains with cyclohexylamide 40 displaying the highest potency towards MAO-B (IC50 = 11 nM). To discover new compounds with extended efficacy against neurotoxic mechanisms in neurodegenerative diseases, MAO-B inhibitors were screened against PHF6, R3 tau, cellular tau and α-synuclein (α-syn) aggregation. We identified the phenethylamide 30 as a multipotent inhibitor of MAO-B (IC50 = 41 nM) and α-syn and tau aggregation. It showed no cytotoxic effects on SH-SY5Y neuroblastoma cells, while also providing neuroprotection against toxicities induced by α-syn and tau. The evaluation of key physicochemical and in vitro-ADME properties revealed a great potential as drug-like small molecules with multitarget neuroprotective activity.

Synthesis and evaluation of novel N1-acylated 5-(4-pyridinyl)indazole derivatives as potent and selective haspin inhibitors.

Protein kinase dysregulation was strongly linked to cancer pathogenesis. Moreover, histone alterations were found to be among the most important post-translational modifications that could contribute to cancer growth and development. In this context, haspin, an atypical serine/threonine kinase, phosphorylates histone H3 at threonine-3 and is notably overexpressed in various common cancer types. Herein, we report novel 5-(4-pyridinyl)indazole derivatives as potent and selective haspin inhibitors. Amide coupling at N1 of the indazole ring with m-hydroxyphenyl acetic acid yielded compound 21 with an IC50 value of 78 nM against haspin. This compound showed a meaningful selectivity over 15 of the most common off-targets, including Clk 1-3 and Dyrk1A, 1B, and 2. The most potent haspin inhibitors 5 and 21 effectively inhibited the growth of the NCI-60 cancer cell lines, further emphasizing the success of our scaffold as a new selective lead for the development of anti-cancer therapeutic agents.

Development and evaluation of 2,4-disubstituted-5-aryl pyrimidine derivatives as antibacterial agents.

Designing novel candidates as potential antibacterial scaffolds has become crucial due to the lack of new antibiotics entering the market and the persistent rise in multidrug resistance. Here, we describe a new class of potent antibacterial agents based on a 5-aryl-N2,N4-dibutylpyrimidine-2,4-diamine scaffold. Structural optimization focused on the 5-aryl moiety and the bioisosteric replacement of the side chain linker atom. Screening of the synthesized compounds focused on a panel of bacterial strains, including gram-positive Staphylococcus aureus strains (Newman MSSA, methicillin- and vancomycin-resistant), and the gram-negative Escherichia coli (ΔAcrB strain). Several compounds showed broad-spectrum antibacterial activity with compound 12, bearing a 4-chlorophenyl substituent, being the most potent among this series of compounds. This frontrunner compound revealed a minimum inhibitory concentration (MIC) value of 1 µg/mL against the S. aureus strain (Mu50 methicillin-resistant S. aureus/vancomycin-intermediate S. aureus) and an MIC of 2 µg/mL against other tested strains. The most potent derivatives were further tested against a wider panel of bacteria and evaluated for their cytotoxicity, revealing further potent activities toward Streptococcus pneumoniae, Enterococcus faecium, and Enterococcus faecalis. To explore the mode of action, compound 12 was tested in a macromolecule inhibition assay. The obtained data were supported by the safety profile of compound 12, which possessed an IC50 of 12.3 µg/mL against HepG2 cells. The current results hold good potential for a new class of extended-spectrum antibacterial agents.

Discovery of 1,2-diaryl-3-oxopyrazolidin-4-carboxamides as a new class of MurA enzyme inhibitors and characterization of their antibacterial activity.

The cytoplasmic steps of peptidoglycan synthesis represent an important targeted pathway for development of new antibiotics. Herein, we report the synthesis of novel 3-oxopyrazolidin-4-carboxamide derivatives with variable amide side chains as potential antibacterial agents targeting MurA enzyme, the first committed enzyme in these cytosolic steps. Compounds 15 (isoindoline-1,3-dione-5-yl), 16 (4-(1H-pyrazol-4-yl)phenyl), 20 (5-cyanothiazol-2-yl), 21 and 31 (5-nitrothiazol-2-yl derivatives) exhibited the most potent MurA inhibition, with IC50 values of 9.8-12.2 µM. Compounds 15, 16 and 21 showed equipotent inhibition of the C115D MurA mutant developed by fosfomycin-resistant Escherichia coli. NMR binding studies revealed that some of the MurA residues targeted by 15 also interacted with fosfomycin, but not all, indicating an overlapping but not identical binding site. The antibacterial activity of the compounds against E. coli ΔtolC suggests that inhibition of MurA accounts for the observed effect on bacterial growth, considering that a few potent MurA inhibitors could not penetrate the bacterial outer membrane and were therefore inactive as proven by the bacterial cell uptake assay. The most promising compounds were also evaluated against a panel of Gram-positive bacteria. Remarkably, compounds 21 and 31 (MurA IC50 = 9.8 and 10.2 µM respectively) exhibited a potent activity against Clostridioides difficile strains with MIC values ranging from 0.125 to 1 µg/mL, and were also shown to be bactericidal with MBC values between 0.25 and 1 µg/mL. Furthermore, both compounds were shown to have a limited activity against human normal intestinal flora and showed high safety towards human colon cells (Caco-2) in vitro. The thiolactone derivative (compound 5) exhibited an interesting broad spectrum antibacterial activity despite its weak MurA inhibition. Altogether, the presented series provides a promising class of antibiotics that merits further investigation.

Targeting the binding pocket of the fluorophore 8-anilinonaphthalene-1-sulfonic acid in the bacterial enzyme MurA.

8-Anilinonaphthalene-1-sulfonic acid (ANS) has been extensively used as a fluorescent probe to detect conformational changes of proteins. It has been cocrystallized with several of the proteins it is used to monitor, including the bacterial cell wall synthesis enzyme MurA. MurA catalyzes the first committed step of peptidoglycan biosynthesis, converting UDP-N-acetylglucosamine (UDP-GlcNAc) into enolpyruvyl UDP-GlcNAc. It has been reported before that ANS binds to MurA from Enterobacter cloacae without inhibiting the enzyme's activity up to a concentration of 1 mM ANS. In this study, we present evidence that ANS inhibits the activity of several isoforms of MurA with IC50 values of 18, 22, and 31 µM against wild-type Escherichia coli, C115D E. coli, and E. cloacae MurA, respectively. This prompted us to test a larger series of structural analogs of ANS for the inhibition of these MurA enzymes, which led to the discovery of compound 26. This ANS analog showed enhanced inhibition of MurA (WT and C115D MurA from E. coli, and E. cloacae MurA) with IC50 values of 2.7, 10, and 14 µM, respectively. Based on our results, the ANS binding pocket was identified as a novel target site for the development of potential antibiotics.

Discovery of novel 5-methoxybenzothiophene hydrazides as metabolically stable Clk1 inhibitors with high potency and unprecedented Clk1 isoenzyme selectivity.

Clk1 kinase is a key modulator of the pre-mRNA alternative splicing machinery which has been proposed as a promising target for treatment of various tumour types, Duchenne's muscular dystrophy and viral infections such as HIV-1 and influenza. Most reported Clk1 inhibitors showed significant co-inhibition of Clk2 and Clk4 in particular, which limits their usefulness for deciphering the individual roles of the Clk1 isoform in physiology and disease. Herein, we present a new 5-methoxybenzothiophene scaffold, enabling for the first time selective inhibition of Clk1 even among the isoenzymes. The 3,5-difluorophenyl and 3,5-dichlorophenyl derivatives 26a and 27a (Clk1 IC50 = 1.4 and 1.7 nM, respectively) showed unprecedented selectivity factors of 15 and 8 over Clk4, and selectivity factors of 535 and 84 over Clk2. Furthermore, 26a and 27a exhibited good growth inhibitory activity in T24 cancer cells and long metabolic half-lives of almost 1 and 6.4 h, respectively. The overall favorable profile of our new Clk1 inhibitors suggests that they may be used in in vivo disease models or as probes to unravel the physiological or pathogenic roles of the Clk1 isoenzyme.

An overview of cdc2-like kinase 1 (Clk1) inhibitors and their therapeutic indications.

Over the past decade, Clk1 has been identified as a promising target for the treatment of various diseases, in which deregulated alternative splicing plays a role. First small molecules targeting Clk1 are in clinical trials for the treatment of solid cancer, where variants of oncogenic proteins derived from alternative splicing promote tumor progression. Since many infectious pathogens hi-jack the host cell's splicing machinery to ensure efficient replication, further indications in this area are under investigation, such as Influenza A, HIV-1 virus, and Trypanosoma infections, and more will likely be discovered in the future. In addition, Clk1 was found to contribute to the progression of Alzheimer's disease through causing an imbalance of tau splicing products. Interestingly, homozygous Clk1 knockout mice showed a rather mild phenotype, opposed to what might be expected in view of the profound role of Clk1 in alternative splicing. A major drawback of most Clk1 inhibitors is their insufficient selectivity; in particular, Dyrk kinases and haspin were frequently identified as off-targets, besides the other Clk isoforms. Only few inhibitors were shown to be selective over Dyrk1A and haspin, whereas no Clk1 inhibitor so far achieved selectivity over the Clk4 isoform. In this review, we carefully compiled all Clk1 inhibitors from the scientific literature and summarized their structure-activity relationships (SAR). In addition, we critically discuss the available selectivity data and describe the inhibitor's efficacy in cellular models, if reported. Thus, we provide a comprehensive overview on the current state of Clk1 drug discovery and highlight the most promising chemotypes.

Identification and Biochemical Characterization of Pyrrolidinediones as Novel Inhibitors of the Bacterial Enzyme MurA.

To develop novel antibiotics, targeting the early steps of cell wall peptidoglycan biosynthesis seems to be a promising strategy that is still underutilized. MurA, the first enzyme in this pathway, is targeted by the clinically used irreversible inhibitor fosfomycin. However, mutations in its binding site can cause bacterial resistance. We herein report a series of novel reversible pyrrolidinedione-based MurA inhibitors that equally inhibit wild type (WT) MurA and the fosfomycin-resistant MurA C115D mutant, showing an additive effect with fosfomycin for the inhibition of WT MurA. For the most potent inhibitor 46 (IC50 = 4.5 µM), the mode of inhibition was analyzed using native mass spectrometry and protein NMR spectroscopy. The compound class was nontoxic against human cells and highly stable in human S9 fraction, human plasma, and bacterial cell lysate. Taken together, this novel compound class might be further developed toward antibiotic drug candidates that inhibit cell wall synthesis.

Nanoparticle Fraught Liposomes: A Platform for Increased Antibiotic Selectivity in Multidrug Resistant Bacteria.

Increasing antibiotic concentrations within bacterial cells while reducing them in mammalian ones would ultimately result in an enhancement of antibacterial actions, overcoming multidrug resistance, all while minimizing toxicity. Nanoparticles (NPs) have been used in numerous occasions to overcome antibiotic resistance, poor drug solubility, and stability. However, the concomitant increase in antibiotic concentration in mammalian cells and the resultant toxicity are usually overlooked. Without compromising bacterial cell fusion, large liposomes (Lip) have been reported to show reduced uptake in mammalian cells. Therefore, in this work, small NP fraught liposomes (NP-Lip) were formulated with the aim of increasing NP uptake and antibiotic delivery in bacterial cells but not in mammalian ones. Small polylactic-co-glycolic acid NPs were therefore loaded with erythromycin (Er), an antibiotic with low membrane permeability that is susceptible to drug efflux, and 3c, a 5-cyanothiazolyl urea derivative with low solubility and stability. In vitro experiments demonstrated that the incorporation of small NPs into large Lip resulted in a reduction in NP uptake by HEK293 cells while increasing it in Gram-negative bacteria (Escherichia coli DH5α, E. coli K12, and Pseudomonas aeruginosa), consequently resulting in an enhancement of antibiotic selectivity by fourfold toward E. coli (both strains) and eightfold toward P. aeruginosa. Ocular administration of NP-Lip in a P. aeruginosa keratitis mouse model demonstrated the ability of Er/3c-loaded NP-Lip to result in a complete recovery. More importantly, in comparison to NPs, the ocular administration of NP-Lip showed a reduction in TNF-alpha and IL-6 levels, implying reduced interaction with mammalian cells in vivo. This work therefore clearly demonstrated how tailoring the nano-bio interaction could result in selective drug delivery and a reduction in toxicity.

Development of (4-Phenylamino)quinazoline Alkylthiourea Derivatives as Novel NF-κB Inhibitors.

For many inflammatory diseases, new effective drugs with fewer side effects are needed. While it appears promising to target the activation of the central pro-inflammatory transcription factor NF-κB, many previously discovered agents suffered from cytotoxicity. In this study, new alkylthiourea quinazoline derivatives were developed that selectively inhibit the activation of NF-κB in macrophage-like THP-1 cells while showing low general cytotoxicity. One of the best compounds, 19, strongly inhibited the production of IL-6 (IC50 = 0.84 µM) and, less potently, of TNFα (IC50 = 4.0 µM); in comparison, the reference compound, caffeic acid phenethyl ester (CAPE), showed IC50s of 1.1 and 11.4 µM, respectively. Interestingly, 19 was found to block the translocation of the NF-κB dimer to the nucleus, although its release from the IκB complex was unaffected. Furthermore, 19 suppressed the phosphorylation of NF-κB-p65 at Ser468 but not at Ser536; however, 19 did not inhibit any kinase involved in NF-κB activation. The only partial suppression of p65 phosphorylation might be associated with fewer side effects. Since several compounds selectively induced cell death in activated macrophage-like THP-1 cells, they might be particularly effective in various inflammatory diseases that are exacerbated by excess activated macrophages, such as arteriosclerosis and autoimmune diseases.

From EGFR kinase inhibitors to anti-inflammatory drugs: Optimization and biological evaluation of (4-(phenylamino)quinazolinyl)-phenylthiourea derivatives as novel NF-κB inhibitors.

The transcription factor NF-κB is a pivotal mediator of chronic inflammatory and autoimmune diseases. Based on our previously published dual EGFR/NF-κB inhibitors, we designed and synthesized new thiourea quinazoline derivatives that retained only the NF-κB inhibitory activity. Several congeners displayed a strong suppression of NF-κB activity in a reporter gene assay, yet low cytotoxicity, and were further evaluated in differentiated macrophage-like THP-1 cells. The compounds exhibited a strong inhibition of IL-6 and, less potently, of TNFα release, which was accompanied by a selective induction of macrophage cell death. The mode of action was investigated with a selected inhibitor, 18, revealing that the translocation of p65/RelA to the nucleus but not its release from the IκB complex was inhibited. Eventually, 18 was identified as the first small molecule inhibitor affecting only the phosphorylation of p65-Ser468 but not of Ser536, which may be causally related to the retention of NF-κB in the cytoplasm. Altogether, our novel NF-κB inhibitors seem applicable for the suppression of cytokine release and the additional selective depletion of activated macrophages in various inflammatory diseases.

Development of novel conformationally restricted selective Clk1/4 inhibitors through creating an intramolecular hydrogen bond involving an imide linker.

As prime regulators of pre-mRNA alternative splicing, different Clk isoforms were found to be overexpressed in various tumour types and have received much attention recently as potential targets for cancer therapy. Several studies have reported potent small-molecule Clk1/4 inhibitors with promising cellular anti-cancer activities; however, their clinical use was generally hampered by their compromised selectivity against off-targets, mainly Clk2 and Dyrk1A. In this study, we present a novel series of N-aroylated 5-methoxybenzothiophene-2-carboxamides (imides) as potent and selective Clk1/4 inhibitors. Potency of this series was found to be mainly dependent on the presence of an intramolecular H-bond between an ortho-methoxy group and the imide NH, that stabilizes a nearly coplanar conformation of high affinity to the ATP binding pocket(s) of Clk1/4. The two most potent hits in this series, compounds 20 (4-fluoro-2-methoxy) and 31 (5-chloro-2-methoxy) had cell free Clk1 IC50s of 4 and 9.7 nM, respectively, besides an unprecedented selectivity over Clk2 with 62- and 50-times higher affinities towards Clk1, respectively. 20 and 31 also exhibited remarkable selectivity over most common off-targets including Dyrk1A. Moreover, compounds 26 (2-ethoxy) and 31 showed growth inhibitory activities in T24 cancer cells with GI50s of <0.1 and 1.1 µM, respectively.

Novel 2,4-disubstituted quinazoline analogs as antibacterial agents with improved cytotoxicity profile: Modification of the benzenoid part.

Bacterial resistance to currently used antibiotics demands the development of novel antibacterial agents with good safety margins and sufficient efficacy against multi-drug resistant isolates. We have previously described the synthesis of N-butyl-2-(butylthio)quinazolin-4-amine (I) as an optimized hit with broad-spectrum antibacterial activity and low cytotoxicity. In addition, we have identified a potential growing vector for this series of compounds. Herein, we describe further hit optimization which includes systematic diversifications of both the benzenoid part and the substituents at position 6 and 7 of compound I. Growing of the molecule beside the core modifications yielded several compounds with remarkable anti(myco)bacterial activity against a panel of pathogenic bacteria, including drug-resistant strains. Compound 12 showed a 2-4 fold improvement in activity than I against S. aureus Newman, S. pneumoniae DSM-20566 and E. faecalis DSM-20478. The compounds also showed a good safety profile towards human HepG2 cells.

Discovery of novel 6-hydroxybenzothiazole urea derivatives as dual Dyrk1A/α-synuclein aggregation inhibitors with neuroprotective effects.

A role of Dyrk1A in the progression of Down syndrome-related Alzheimer's disease (AD) is well supported. However, the involvement of Dyrk1A in the pathogenesis of Parkinson's disease (PD) was much less studied, and it is not clear whether it would be promising to test Dyrk1A inhibitors in relevant PD models. Herein, we modified our previously published 1-(6-hydroxybenzo[d]thiazol-2-yl)-3-phenylurea scaffold of Dyrk1A inhibitors to obtain a new series of analogues with higher selectivity for Dyrk1A on the one hand, but also with a novel, additional activity as inhibitors of α-synuclein (α-syn) aggregation, a major pathogenic hallmark of PD. The phenyl acetamide derivative b27 displayed the highest potency against Dyrk1A with an IC50 of 20 nM and high selectivity over closely related kinases. Furthermore, b27 was shown to successfully target intracellular Dyrk1A and to inhibit SF3B1 phosphorylation in HeLa cells with an IC50 of 690 nM. In addition, two compounds among the Dyrk1A inhibitors, b1 and b20, also suppressed the aggregation of α-synuclein (α-syn) oligomers (with IC50 values of 10.5 µM and 7.8 µM, respectively). Both compounds but not the Dyrk1A reference inhibitor harmine protected SH-SY5Y neuroblastoma cells against α-syn-induced cytotoxicity, with b20 exhibiting a higher neuroprotective effect. Compound b1 and harmine were more efficient in protecting SH-SY5Y cells against 6-hydroxydopamine-induced cell death, an effect that was previously correlated to Dyrk1A inactivation in cells but not yet verified using chemical inhibitors. The presented dual inhibitors exhibited a novel activity profile encouraging for further testing in neurodegenerative disease models.

Discovery of Hydroxybenzothiazole Urea Compounds as Multitargeted Agents Suppressing Major Cytotoxic Mechanisms in Neurodegenerative Diseases.

Multiple factors are causally responsible and/or contribute to the progression of Alzheimer's and Parkinson's diseases. The protein kinase Dyrk1A was identified as a promising target as it phosphorylates tau protein, α-synuclein, and parkin. The first goal of our study was to optimize our previously identified Dyrk1A inhibitors of the 6-hydroxy benzothiazole urea chemotype in terms of potency and selectivity. Our efforts led to the development of the 3-fluorobenzyl amide derivative 16b, which displayed the highest potency against Dyrk1A (IC50 = 9.4 nM). In general, the diversification of the benzylamide moiety led to an enhanced selectivity over the most homologous isoform, Dyrk1B, which was a meaningful indicator, as the high selectivity could be confirmed in an extended selectivity profiling of 3b and 16b. Eventually, we identified the novel phenethyl amide derivative 24b as a triple inhibitor of Dyrk1A kinase activity (IC50 = 119 nM) and the aggregation of tau and α-syn oligomers. We provide evidence that the novel combination of selective Dyrk1A inhibition and suppression of tau and α-syn aggregations of our new lead compound confers efficacy in several established cellular models of neurotoxic mechanisms relevant to neurodegenerative diseases, including α-syn- and 6-hydroxydopamine-induced cytotoxicities.

Novel 2,4-disubstituted quinazoline analogs as antibacterial agents with improved cytotoxicity profile: Optimization of the 2,4-substituents.

The emergence of bacterial resistance has triggered a multitude of efforts to develop new antibacterial agents. There are many compounds in literature that were reported as potent antibacterial agents, however, they lacked the required safety to mammalian cells or no clear picture about their toxicity profile was presented. Inspired by discovered hit from our in-house library and by previously reported 2,4-diaminosubstituted quinazolines, we describe the design and synthesis of novel 2,4-disubstituted-thioquinazolines (3-13 and 36), 2-thio-4-amino substituted quinazolines (14-33) and 6-substituted 2,4-diamonsubstituted quinazolines (37-39). The synthesized compounds showed potent antibacterial activity against a panel of Gram-positive, efflux deficient E.coli and Mycobacterium smegmatis. The panel also involved resistant strains including methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, vancomycin-resistant Enterococcus faecalis and vancomycin-resistant Enterococcus faecium, in addition to Mycobacterium smegmatis. The newly synthesized compounds revealed MIC values against the tested strains ranging from 1 to 64 µg/mL with a good safety profile. Most of the 2-thio-4-amino substituted-quinazolines showed significant antimycobacterial activity with the variations at position 2 and 4 offering additional antibacterial activity against the different strains. Compared to previously reported 2,4-diaminosubstituted quinazolines, the bioisosteric replacement of the 2-amino with sulfur offered a successful approach to keep the high antibacterial potency while substantially improving safety profile as indicated by the reduced activity on different cell lines and a lack of hemolytic activity.