Bioactive Piperazic Acid-Bearing Cyclodepsipeptides, Lydiamycins E-H, from an Endophytic Streptomyces sp. Associated with Cinnamomum cassia.

A chemical investigation of the endophytic Streptomyces sp. HBQ95, associated with the medicinal plant Cinnamomum cassia Presl, enabled the discovery of four new piperazic acid-bearing cyclodepsipeptides, lydiamycins E-H (1-4), and one known compound (lydiamycin A). Their chemical structures, including absolute configurations, were defined by a combination of spectroscopic analyses and multiple chemical manipulations. Lydiamycins F-H (2-4) and A (5) exhibited antimetastatic activity against PANC-1 human pancreatic cancer cells without significant cytotoxicity.

A novel class of selective non-nucleoside inhibitors of human DNA methyltransferase 3A.

Screening of a small chemical library (Medicines for Malaria Venture Pathogen Box) identified two structurally related pyrazolone (inhibitor 1) and pyridazine (inhibitor 2) DNMT3A inhibitors with low micromolar inhibition constants. The uncompetitive and mixed type inhibition patterns with DNA and AdoMet suggest these molecules act through an allosteric mechanism, and thus are unlikely to bind to the enzyme's active site. Unlike the clinically used mechanism based DNMT inhibitors such as decitabine or azacitidine that act via the enzyme active site, the inhibitors described here could lead to the development of more selective drugs. Both inhibitors show promising selectivity for DNMT3A in comparison to DNMT1 and bacterial DNA cytosine methyltransferases. With further study, this could form the basis of preferential targeting of de novo DNA methylation over maintenance DNA methylation.

Design, Synthesis and Biological Evaluation of 5-amino-3-aryl-1-(6'-chloropyridazin-3'-yl)pyrazoles and their Derivatives as Analgesic Agents.

An efficient and environmental benign solvent-free synthesis of 5-amino-3-aryl-1-(6'-chloropyridazin-3'-yl)pyrazoles (4A-E): was accomplished by grinding 3-chloro-6-hydrazinopyridazine (2): and ß-ketonitriles (3A-E): in the presence of p-toulenesulfonic acid as a catalyst. Subsequently, 6'-chloro group in 4A-E: was replaced with cyclic 2° amine derivatives viz. pyrrolidine 5A: , piperidine 5B: and morpholine 5C: to obtain 6A-E: , 7A-E: , 8A-E: respectively. The newly synthesized compounds were characterized by using IR, NMR (1H and 13C), mass spectral studies, elemental analyses. All the synthesized compounds were studied for their docking interaction with target protein 6COX and screened for their in vivo analgesic mode of action against swiss albino mice (animal model) using acetic-acid induced writhing test. Consequently, docking simulations data justifies the potential of synthesized series as an analgesic and very well correlated with in vivo study. Preliminary results revealed that most of the synthesized compounds exhibited moderate to good analgesic activity as compared to reference/standard drug (s) sodium diclofenac and candidates 4D: and 7C: protrude out as a promising lead for further investigation.

Repurposing strategies on pyridazinone-based series by pharmacophore- and structure-driven screening.

We report here in silico repurposing studies on 52 new pyridazinone-based small-molecules through inverse virtual screening (iVS) methodologies. These analogues were originally designed as formyl peptide receptor (FPR) ligands. As it is sometimes the case in drug discovery programmes, subsequent biological screening demonstrated the inefficacy of the molecules in binding FPRs, failing in the identification of new hits. Through a focussed drug-repurposing approach we have defined a variety of potential targets that are suitable to interact with this library of pyridazinone-based analogues. A two-step approach has been conducted for computational analysis. Specifically, the molecules were initially processed through a pharmacophore-based screening. Secondly, the resulting features of binding were investigated by docking studies and following molecular dynamic simulations, in order to univocally confirm "pyridazinone-based ligand-target protein" interactions. Our findings propose aspartate aminotransferase as the most favourable repurposed target for this small-molecule series, worth of additional medicinal chemistry investigations in the field.

Synthesis and biological screening of some novel 6-substituted 2-alkylpyridazin-3(2H)-ones as anti-inflammatory and analgesic agents.

Some novel derivatives of 2-alkyl 6-substituted pyridazin-3(2H)-ones were synthesized by condensation of 3,6-dichloropyridazine with the sodium salt of benzyl cyanide, followed by hydrolysis and coupling with alkyl halides. The synthesized compounds were screened as cyclooxygenase (COX)-1/COX-2 inhibitors and as analgesic and anti-inflammatory agents. Among the synthesized compounds, 6-benzyl-2-methylpyridazin-3(2H)-one (4a), 6-benzoyl-2-propylpyridazin-3(2H)-one (8b), and 6-(hydroxy(phenyl)methyl)-2-methylpyridazin-3(2H)-one (9a) displayed the highest COX-2 selectivity indices of 96, 99, and 98, respectively, and analgesic efficacies of 47%, 46%, and 45% protection, respectively. Also, compounds 4a, 8b, and 9a showed anti-inflammatory activities of 65%, 60%, and 62% inhibition of edema, respectively, at a dose of 10 mg/kg, which is higher than that of diclofenac (58% inhibition of edema).

Synthesis and vasodilator activity of some pyridazin-3(2H)-one based compounds.

Aim: Hypertension is a major health problem worldwide resulting in high death rates due to its consequences and complications. Therefore, searching for new vasorelaxants is a must to find new vasodilators efficient for the treatment of different cardiovascular diseases. Methodology: Different 6-phenyl-3-pyridazinone based derivatives were synthesized and screened for their vasorelaxant activity according to the reported method using hydralazine as a standard. Results: The tested compounds revealed potent to mild activity with EC50 values 0.339-114.300 µM compared with hydralazine EC50 = 18.210 µM. Conclusion: The most active compounds were the acid 5, its ester analog 4 and 4-methoxyphenylhydrazide derivative 10c (EC50 = 0.339, 1.225 and 1.204 µM, respectively). Therefore, 6-phenylpyridazin-3(2H)-one can be a hit for structural optimization to obtain promising vasorelaxants.

Chemical Proteomic Profiling of Bromodomains Enables the Wide-Spectrum Evaluation of Bromodomain Inhibitors in Living Cells.

Bromodomains, epigenetic "readers" of lysine acetylation marks, exist in different nuclear proteins with diverse biological functions in chromatin biology. Malfunctions of bromodomains are associated with the pathogenesis of human diseases, such as cancer. Bromodomains have therefore emerged as therapeutic targets for drug discovery. Given the high structural similarity of bromodomains, a critical step in the development of bromodomain inhibitors is the evaluation of their selectivity to avoid off-target effects. While numerous bromodomain inhibitors have been identified, new methods to evaluate the inhibitor selectivity toward endogenous bromodomains in living cells remain needed. Here we report the development of a photoaffinity probe, photo-bromosporine (photo-BS), that enables the wide-spectrum profiling of bromodomain inhibitors in living cells. Photo-BS allowed light-induced cross-linking of recombinant bromodomains and endogenous bromodomain-containing proteins (BCPs) both in vitro and in living cells. The photo-BS-induced labeling of the bromodomains was selectively competed by the corresponding bromodomain inhibitors. Proteomics analysis revealed that photo-BS captured 28 out of the 42 known BCPs from the living cells. Assessment of the two bromodomain inhibitors, bromosporine and GSK6853, resulted in the identification of known as well as previously uncharacterized bromodomain targets. Collectively, we established a chemical proteomics platform to comprehensively evaluate bromodomain inhibitors in terms of their selectivity against endogenous BCPs in living cells.

Discovery of Small Molecule Splicing Modulators of Survival Motor Neuron-2 (SMN2) for the Treatment of Spinal Muscular Atrophy (SMA).

Spinal muscular atrophy (SMA), a rare neuromuscular disorder, is the leading genetic cause of death in infants and toddlers. SMA is caused by the deletion or a loss of function mutation of the survival motor neuron 1 (SMN1) gene. In humans, a second closely related gene SMN2 exists; however it codes for a less stable SMN protein. In recent years, significant progress has been made toward disease modifying treatments for SMA by modulating SMN2 pre-mRNA splicing. Herein, we describe the discovery of LMI070/branaplam, a small molecule that stabilizes the interaction between the spliceosome and SMN2 pre-mRNA. Branaplam (1) originated from a high-throughput phenotypic screening hit, pyridazine 2, and evolved via multiparameter lead optimization. In a severe mouse SMA model, branaplam treatment increased full-length SMN RNA and protein levels, and extended survival. Currently, branaplam is in clinical studies for SMA.

Depsidomycins B and C: New Cyclic Peptides from a Ginseng Farm Soil-Derived Actinomycete.

LC/MS-based chemical profiling of a ginseng farm soil-derived actinomycete strain, Streptomyces sp. BYK1371, enabled the discovery of two new cyclic heptapeptides, depsidomycins B and C (1 and 2), each containing two piperazic acid units and a formyl group at their N-terminus. The structures of 1 and 2 were elucidated by a combination of spectroscopic and chemical analyses. These new compounds were determined to possess d-leucine, d-threonine, d-valine, and S-piperazic acid based on the advanced Marfey's method and a GITC (2,3,4,6-tetra-O-acetyl-ß-d-glucopyranosyl isothiocyanate) derivatization of their hydrolysates, followed by LC/MS analysis. Depsidomycins B and C displayed significant antimetastatic activities against metastatic breast cancer cells (MDA-MB-231).

Synthesis of New Isoxazole-, Pyridazine-, Pyrimidopyrazines and Their Anti-Inflammatory and Analgesic Activity.

BACKGROUND: Isoxazoles, pyridazines, and pyrimidopyrazines have recently attracted attention due to their potent pharmacological activities. They exhibited anticancer, neuroprotective, analgesic and anti-inflammatory effects. OBJECTIVE: The study aimed to synthesize novel isoxazoles, pyridazines, and pyrimidopyrazines through efficient high yield protocol for evaluating their analgesics and anti-inflammatory activities. METHOD: A series of novel isoxazole-, pyridazine-, pyrimidopyrazine derivatives was prepared from 5,8-alkyl-1,3-dimethyl-5,6-dihydropyrimido[5,6-e]pyrazine-2,4,7-trione (1a,b) as the starting material. RESULTS: The prepared derivatives were synthesized in moderate to good yields (60-75%) in a stepwise efficient protocol under mild condition. These new compounds have been proven by several spectroscopic techniques as IR, 1D and 2D NMR techniques and mass analysis. The in vivo anti-inflammatory was assessed for the synthesized compounds using carrageenan-induced rat hind paw edema model. Also, the in vivo analgesic activity for these products was examined utilizing hot-plate and acetic acid-induced writhing response assays. CONCLUSION: The isoxazole derivatives (3a-f) showed the most forceful anti-inflammatory and analgesic activities. Pyrimidopyrazines (4a-f) demonstrated weaker but comparable antiinflammatory and analgesic activities to the positive controls.

Challenges in the development of an M4 PAM preclinical candidate: The discovery, SAR, and biological characterization of a series of azetidine-derived tertiary amides.

Herein we describe the continued optimization of M4 positive allosteric modulators (PAMs) within the 5-amino-thieno[2,3-c]pyridazine series of compounds. In this letter, we disclose our studies on tertiary amides derived from substituted azetidines. This series provided excellent CNS penetration, which had been challenging to consistently achieve in other amide series. Efforts to mitigate high clearance, aided by metabolic softspot analysis, were unsuccessful and precluded this series from further consideration as a preclinical candidate. In the course of this study, we found that potassium tetrafluoroborate salts could be engaged in a tosyl hydrazone reductive cross coupling reaction, a previously unreported transformation, which expands the synthetic utility of the methodology.

Discovery of potent and efficacious pyrrolopyridazines as dual JAK1/3 inhibitors.

A series of potent dual JAK1/3 inhibitors have been developed from a moderately selective JAK3 inhibitor. Substitution at the C6 position of the pyrrolopyridazine core with aryl groups provided exceptional biochemical potency against JAK1 and JAK3 while maintaining good selectivity against JAK2 and Tyk2. Translation to in vivo efficacy was observed in a murine model of chronic inflammation. X-ray co-crystal structure determination confirmed the presumed inhibitor binding orientation in JAK3. Efforts to reduce hERG channel inhibition will be described.

Synthesis and Biological Evaluation of Novel Neuroprotective Pyridazine Derivatives as Excitatory Amino Acid Transporter 2 (EAAT2) Activators.

LDN-212320 (3) was found to be a potent EAAT2 activator at a translational level, restoring the normal clearance of glutamate and providing neuronal protection. Since the pharmacologic activation of EAAT2 represents a valuable strategy to relieve neuropathic pain, we synthesized novel activators (4a-f) of EAAT2. Among them 4f, analyzed in comparison with 3 by different paradigms in a rat model of oxaliplatin-induced neuropathic pain, showed the better antihypersensitive profile being able to fully counteract the oxaliplatin-induced neuropathy.

Kinase Inhibitors in Multitargeted Cancer Therapy.

The old-fashioned anticancer approaches, aiming at arresting cancer cell proliferation interfering with non-specific targets (e.g. DNA), have been replaced, in the last decades, by more specific target oriented ones. Nonetheless, single-target approaches have not always led to optimal outcomes because, for its complexity, cancer needs to be tackled at various levels by modulation of several targets. Although at present, combinations of individual singletarget drugs represent the most clinically practiced therapeutic approaches, the modulation of multiple proteins by a single drug, in accordance with the polypharmacological strategy, has become more and more appealing. In the perspective of a multi-target approach, the closely related evolutionary members of the tyrosine kinase family are ideal candidates. Indeed, tyrosine kinase activities are not only critical in tumor phenotype maintenance, but also modulate several functions in the tumor microenvironment. Consequently, several multikinase inhibitors were approved in the last decade, and many new molecules are currently in preclinical or clinical development. In the present review we report on the most widely FDA-approved multitargeted drugs, discussing about their mechanism of action and outlining the clinical trials that have brought them to approval.

Synthesis and Biological Evaluation of Novel σ1 Receptor Ligands for Treating Neuropathic Pain: 6-Hydroxypyridazinones.

By use of the 6-hydroxypyridazinone framework, a new series of potent σ1 receptor ligands associated with pharmacological antineuropathic pain activity was synthesized and is described in this article. In vitro receptor binding studies revealed high σ1 receptor affinity (Ki σ1 = 1.4 nM) and excellent selectivity over not only σ2 receptor (1366-fold) but also other CNS targets (adrenergic, µ-opioid, sertonerigic receptors, etc.) for 2-(3,4-dichlorophenyl)-6-(3-(piperidin-1-yl)propoxy)pyridazin-3(2H)-one (compound 54). Compound 54 exhibited dose-dependent antiallodynic properties in mouse formalin model and rats chronic constriction injury (CCI) model of neuropathic pain. In addition, functional activity of compound 54 was evaluated using phenytoin and indicated that the compound was a σ1 receptor antagonist. Moreover, no motor impairments were found in rotarod tests at antiallodynic doses and no sedative side effect was evident in locomotor activity tests. Last but not least, good safety and favorable pharmacokinetic properties were also noted. These profiles suggest that compound 54 may be a member of a novel class of candidate drugs for treatment of neuropathic pain.

Hybrid chemistry. Part 4: Discovery of etravirine-VRX-480773 hybrids as potent HIV-1 non-nucleoside reverse transcriptase inhibitors.

A novel series of etravirine-VRX-480773 hybrids were designed using structure-guided molecular hybridization strategy and fusing the pharmacophore templates of etravirine and VRX-480773. The anti-HIV-1 activity and cytotoxicity was evaluated in MT-4 cell cultures. The most active hybrid compound in this series, N-(2-chlorophenyl)-2-((4-(4-cyano-2,6-dimethylphenoxy)pyrimidin-2-yl)thio)acetamide 3d (EC50=0.24 , SI>1225), was more potent than delavirdine (EC50=0.66 µM, SI>67) in the anti-HIV-1 in vitro cellular assay. Studies of structure-activity relationships established a correlation between anti-HIV activity and the substitution pattern of the acetanilide group.

New platelet aggregation inhibitors based on pyridazinone moiety.

New series of pyridazinone derivatives (4, 5 and 6) were synthesized in good yields following a synthetic strategy based on singlet oxygen oxidation of alkyl furans, in which a suitable ß(α)-substituted γ-hydroxybutenolide (10 or 11) or a bicyclic lactone (12 or 13) was the key intermediate. The synthesized compounds were tested in vitro as antiplatelet agents and some of them (compounds 4b, 4d and 5b) exhibited potent inhibitory effects on collagen-induced platelet aggregation with IC50 values in the low µM range. Studies performed with the most active compound of these series (4b) demonstrated its lack of activity as inhibitor of platelet aggregation induced by other agonists as thrombin, ionomycin or U-46619 suggesting a selective action on the biochemical mechanisms triggered by collagen in the platelets.

Discovery of substituted 6-pheny-3H-pyridazin-3-one derivatives as novel c-Met kinase inhibitors.

We report a series of phenyl substituted pyridazin-3-ones substituted with morpholino-pyrimidines. The SAR of the phenyl was explored and their c-Met kinase and cell-based inhibitory activity toward c-Met driven cell lines were evaluated. Described herein is a potent c-Met inhibitor by structural modification of the parent morpholino-pyridazinone scaffold, with particular focus on the phenyl and pyrimidine substituents.

Discovery of (1R,6S)-5-[4-(1-cyclobutyl-piperidin-4-yloxy)-phenyl]-3,4-diaza-bicyclo[4.1.0]hept-4-en-2-one (R,S-4a): histamine H(3) receptor inverse agonist demonstrating potent cognitive enhancing and wake promoting activity.

A series of fused cyclopropyl-4,5-dihydropyridazin-3-one (3,4-diaza-bicyclo[4.1.0]hept-4-en-2-one) phenoxypiperidine analogs was designed and synthesized, leading to the identification of (1R,6S)-5-[4-(1-cyclobutyl-piperidin-4-yloxy)-phenyl]-3,4-diaza-bicyclo[4.1.0]hept-4-en-2-one (R,S-4a) as a second-generation pyridazin-3-one H3R antagonist. Compound R,S-4a was a potent H3R functional antagonist in vivo in the rat dipsogenia model, demonstrated potent wake activity in the rat EEG/EMG model, and enhanced short-term memory in the rat social recognition memory model at doses as low as 0.03-0.3 mg/kg po.