8-Aryl xanthines potent inhibitors of phosphodiesterase 5

In clinical studies, several inhibitors of phosphodiesterase 5 (PDE5) have demonstrated utility in the treatment of erectile dysfunction. We describe herein a series of 8-aryl xanthine derivatives which function as potent PDE5 inhibitors with, in many cases, high levels of selectivity versus other PDE isoforms.     

Imidazopyridazinones as novel PDE7 inhibitors: SAR and in vivo studies in Parkinson's disease model

The synthesis and structure-activity relationship studies of a series of compounds from imidazopyridazinone scaffold as PDE7 inhibitors are disclosed. Potent analogs such as compounds 7 (31nM), 8 (27nM), and 9 (12nM) were identified. The PDE selectivity and pharmacokinetic profile of compounds 7, 8 and 9 are also disclosed. The adequate CNS penetration of compound 7 in mice allowed it to be tested in the MPTP induced PD model and haloperidol induced catalepsy model to probe the differential pharmacology of PDE7 in the striatal pathway.     

Mechanism for the emetic side effect of xanthine bronchodilators.

The usefulness of xanthine bronchodilators in the treatment of asthma is often limited by the side effects of nausea and vomiting. We investigated the mechanism of emesis induced by xanthines, by examining the roles of phosphodiesterase (PDE) inhibition and adenosine antagonism. Theophylline, enprofylline, 8-phenyltheophylline and isobutylmethylxanthine (IBMX), as well as vehicle, were given to ferrets at doses ranging from 0.1 to 150 mg/kg i.p. The potencies of these compounds in producing emetic responses were ranked IBMX greater than enprofylline greater than theophylline greater than 8-phenyltheophylline. These results correlate well with the relative potencies of the compounds as nonselective PDE inhibitors but do not correlate with their relative potencies as adenosine A1 or A2 receptor antagonists. The emetic responses also correlate well with the previously reported potencies of these xanthines as bronchodilators in guinea pigs. We conclude that the emetic side effect of xanthine bronchodilators results from the inhibition of one or more forms of PDE rather than from adenosine antagonism.     

Triazole‐Pyridine Dicarbonitrile Targets Phosphodiesterase 4 to Induce Cytotoxicity in Lung Carcinoma Cells

Phosphodiesterase 4 (PDE4) is a key enzyme involved in the hydrolysis of cyclic adenosine monophosphate (cAMP) and widely expressed in several types of cancers. The inhibition of PDE4 results in an increased concentration of intracellular cAMP levels that imparts the anti‐inflammatory response in the target cells. In the present report, two series of triazolo‐pyridine dicarbonitriles and substituted dihydropyridine dicarbonitriles were synthesized using green protocol (TBAB in refluxed water). We next evaluated the title compounds for their cytotoxicity towards lung cancer (A549) cells and identified 7′‐[4‐(methylsulfonyl)phenyl]‐5′‐oxo‐1′,5′‐dihydrospiro[cyclohexane‐1,2′‐[1,2,4]triazolo[1,5‐a]pyridine]‐6′,8′‐dicarbonitrile ( 5h ) and 7′‐(1‐methyl‐1H‐imidazol‐2‐yl)‐5′‐oxo‐1′,5′‐dihydrospiro[cyclohexane‐1,2′‐[1,2,4]triazolo[1,5‐a]pyridine]‐6′,8′‐dicarbonitrile ( 5j ) as lead analogs with the IC₅₀ values of 15.2 and 24.1 μm , respectively. Furthermore, all the new compounds were tested for PDE4 inhibitory activity and 5j showed relatively good inhibitory activity towards PDE4 with inhibition of 50.9 % at 10 μm . In silico analysis demonstrated the favorable interaction of the title compounds with the target enzyme. Taken together, the present study introduces a new scaffold for the development of novel PDE4 inhibitors to fight against inflammatory diseases.     

A family of phosphodiesterase inhibitors discovered by cocrystallography and scaffold-based drug design

Cyclic nucleotide phosphodiesterases (PDEs) comprise a large family of enzymes that regulate a variety of cellular processes. We describe a family of potent PDE4 inhibitors discovered using an efficient method for scaffold-based drug design. This method involves an iterative approach starting with low-affinity screening of compounds followed by high-throughput cocrystallography to reveal the molecular basis underlying the activity of the newly identified compounds. Through detailed structural analysis of the interaction of the initially discovered pyrazole carboxylic ester scaffold with PDE4D using X-ray crystallography, we identified three sites of chemical substitution and designed small selective libraries of scaffold derivatives with modifications at these sites. A 4,000-fold increase in the potency of this PDE4 inhibitor was achieved after only two rounds of chemical synthesis and the structural analysis of seven pyrazole derivatives bound to PDE4B or PDE4D, revealing the robustness of this approach for identifying new inhibitors that can be further developed into drug candidates.     

Use of structure based design to increase selectivity of pyridyl-cinnoline phosphodiesterase 10A (PDE10A) inhibitors against phosphodiesterase 3 (PDE3)

We report our successful effort to increase the PDE3 selectivity of PDE10A inhibitor pyridyl cinnoline 1 using a combination of computational modeling and structural–activity relationship investigations. An analysis of the PDE3 catalytic domain compared to the co-crystal structure of cinnoline analog 1 in PDE10A revealed two areas of structural differences in the active sites and suggested areas on the scaffold that could be modified to exploit those unique structural features. Once SAR established the cinnoline as the optimal scaffold, modifications on the methoxy groups of the cinnoline and the methyl group on the pyridine led to the discovery of compounds 33 and 36. Both compounds achieved significant improvement in selectivity against PDE3 while maintaining their PDE10A inhibitory activity and in vivo metabolic stability comparable to 1.     

A Yeast-Based Chemical Screen Identifies a PDE Inhibitor That Elevates Steroidogenesis in Mouse Leydig Cells via PDE8 and PDE4 Inhibition

A cell-based high-throughput screen (HTS) was developed to detect phosphodiesterase 8 (PDE8) and PDE4/8 combination inhibitors. By replacing the Schizosaccharomyces pombe PDE gene with the murine PDE8A1 gene in strains lacking adenylyl cyclase, we generated strains whose protein kinase A (PKA)-stimulated growth in 5-fluoro orotic acid (5FOA) medium reflects PDE8 activity. From our previously-identified PDE4 and PDE7 inhibitors, we identified a PDE4/8 inhibitor that allowed us to optimize screening conditions. Of 222,711 compounds screened, ∼0.2% displayed composite Z scores of >20. Additional yeast-based assays using the most effective 367 compounds identified 30 candidates for further characterization. Among these, compound BC8-15 displayed the lowest IC₅₀ value for both PDE4 and PDE8 inhibition in in vitro enzyme assays. This compound also displays significant activity against PDE10A and PDE11A. BC8-15 elevates steroidogenesis in mouse Leydig cells as a single pharmacological agent. Assays using BC8-15 and two structural derivatives support a model in which PDE8 is a primary regulator of testosterone production by Leydig cells, with an additional role for PDE4 in this process. BC8-15, BC8-15A, and BC8-15C, which are commercially available compounds, display distinct patterns of activity against PDE4, PDE8, PDE10A, and PDE11A, representing a chemical toolkit that could be used to examine the biological roles of these enzymes in cell culture systems.     

Design and synthesis of xanthine analogues as potent and selective PDE5 inhibitors

We have discovered potent and selective xanthine PDE5 inhibitors. Compound 25 (PDE5 IC(50)=0.6 nM, PDE6/PDE5=101) demonstrated similar functional efficacy and PK profile to Sildenafil (PDE5 IC(50)=3.5 nM, PDE6/PDE5=7).     

Potent and selective xanthine-based inhibitors of phosphodiesterase 5

Inhibitors of PDE5 are useful therapeutic agents for treatment of erectile dysfunction. A series of novel xanthine derivatives has been identified as potent inhibitors of PDE5, with good levels of selectivity against other PDE isoforms, including PDE6. Studies in the dog indicate excellent oral bioavailability for compound 21.     

Highly potent and selective chiral inhibitors of PDE5: an illustration of Pfeiffer's rule

A series of potent chiral PDE5 inhibitors are described that are based on the sildenafil architecture but exhibit much greater selectivity over PDE6. Eudismic analysis of the SAR in this series provided a clear illustration of Pfeiffer's rule and indicated that the chiral motif was involved in a highly-stereoselective interaction with PDE5. This PDE5 specificity translated to levels of selectivity over PDE6 that were hitherto unprecedented in the sildenafil scaffold. UK-371,800 (compound 8) was identified as a development candidate from this series that married sildenafil-like molecular properties with high selectivity over PDE6. Clinical data confirm that UK-371,800 has markedly superior human pharmacokinetics to a previously-described higher molecular weight achiral analogue in this template (compound 1).     

Pharmacophore elucidation and molecular docking studies on phosphodiesterase-5 inhibitors

cGMP-binding cGMP-specific PDE, PDE5 plays a key role in the hydrolysis of cyclic guanidine monophosphate. Because cGMP mediates vascular functions, a PDE5 inhibitor that elevates cGMP level is an attractive means for vasodilatation and treatment of erectile dysfunction. In this paper we report the elucidation of the common pharmacophore hypothesis of different classes of PDE5 inhibitors. Using LigandScout program, pharmacophore modelling studies were performed on prior reported potent PDE5 inhibitors with a variety of scaffolds in order to identify one common set of critical chemical features of these PDE5 inhibitors 1-52. The best pharmacophore model, model-1, characterized by four chemical features: one aromatic ring, one hydrophobe, one hydrogen acceptors and one hydrogen donor. Using Dock6 program, docking studies were performed in order to investigate the mode of binding of these compounds. The molecular docking study allowed confirming the preferential binding mode of different classes of PDE5 inhibitors inside the active site. The obtained binding mode was as same as that of vardenafil, X-ray ligand with different orientation with varied PDE5 inhibitors׳ scaffold.     

The discovery of novel, potent and selective PDE5 inhibitors.

The design and synthesis of a novel scaffold for potent and selective PDE5 inhibitors are described. Compound 3a was more potent (PDE5 IC50=0.31 nM) and selective (>10,000-fold vs PDE1 and 160-fold selective vs PDE6) PDE5 inhibitor than sildenafil.     

Design,synthesis and biological evaluation of pyrazolopyrimidinone based potent and selective PDE5 inhibitors for treatment of erectile dysfunction

Our previous discovery of series of pyrazolopyrimidinone based PDE5 inhibitors led to find potent leads but with low aqueous solubility and poor bioavailability, and low selectivity. Now, a new series of same pyrazolopyrimidinone scaffold is designed, synthesized and evaluated for its PDE5 inhibitory potential. In this study, some of the molecules are found more potent and selective PDE5 inhibitors in vitro than sildenafil. The studies revealed that compound 5 is 20 fold selective to PDE5 against PDE6. As PDE6 enzyme is involved in the phototransduction pathway in the retina and creates distortion problem, the selectivity for PDE5 specifically against PDE6 enzyme is preferred for any development candidate and in present study, compound 5 has been found to be devoid of this liability of selectivity issue. Moreover, compound 5 has shown excellent in vivo efficacy in conscious rabbit model, it's almost comparable to sildenafil. The preclinical pharmacology including pharmacokinetic and physicochemical parameter studies were also performed for compound 5, it was found to have good PK properties and other physicochemical parameters. The development of these selective PDE5 inhibitors can further lead to draw strategies for the novel preclinical and/or clinical candidates based on pyrazolopyrimidinone scaffold.     

Quinolines as extremely potent and selective PDE5 inhibitors as potential agents for treatment of erectile dysfunction

In a continuing effort to discover novel chemotypes as potent and selective PDE5 inhibitors for the treatment of male erectile dysfunction (ED), we have found that 4-benzylaminoquinoline derivatives are very potent and selective PDE5 inhibitors. Some compounds in this series had PDE5 IC(50)'s as low as 50 pM. While an electron withdrawing group at the C6-position of the quinoline substantially improved PDE5 potency, an ethyl group at the C8-position not only improved the PDE5 potency but also the isozyme selectivity. Substitutents at the C3-position can incorporate a variety of different groups. The synthesis and primary structure-activity relationship of this new series of potent PDE5 inhibitors are described.     

Scanning peptide array analyses identify overlapping binding sites for the signalling scaffold proteins, beta-arrestin and RACK1, in cAMP-specific phosphodiesterase PDE4D5

The cAMP-specific phosphodiesterase PDE4D5 can interact with the signalling scaffold proteins RACK (receptors for activated C-kinase) 1 and beta-arrestin. Two-hybrid and co-immunoprecipitation analyses showed that RACK1 and beta-arrestin interact with PDE4D5 in a mutually exclusive manner. Overlay studies with PDE4D5 scanning peptide array libraries showed that RACK1 and beta-arrestin interact at overlapping sites within the unique N-terminal region of PDE4D5 and at distinct sites within the conserved PDE4 catalytic domain. Screening scanning alanine substitution peptide arrays, coupled with mutagenesis and truncation studies, allowed definition of RACK1 and beta-arrestin interaction sites. Modelled on the PDE4D catalytic domain, these form distinct well-defined surface-exposed patches on helices-15-16, for RACK1, and helix-17 for beta-arrestin. siRNA (small interfering RNA)-mediated knockdown of RACK1 in HEK-293 (human embryonic kidney) B2 cells increased beta-arrestin-scaffolded PDE4D5 approx. 5-fold, increased PDE4D5 recruited to the beta2AR (beta2-adrenergic receptor) upon isoproterenol challenge approx. 4-fold and severely attenuated (approx. 4-5 fold) both isoproterenol-stimulated PKA (protein kinase A) phosphorylation of the beta2AR and activation of ERK (extracellular-signal-regulated kinase). The ability of a catalytically inactive form of PDE4D5 to exert a dominant negative effect in amplifying isoproterenol-stimulated ERK activation was ablated by a mutation that blocked the interaction of PDE4D5 with beta-arrestin. In the present study, we show that the signalling scaffold proteins RACK1 and beta-arrestin compete to sequester distinct 'pools' of PDE4D5. In this fashion, alterations in the level of RACK1 expression may act to modulate signal transduction mediated by the beta2AR.     

A prenylated flavonol,sophoflavescenol: a potent and selective inhibitor of cGMP phosphodiesterase 5

During the search for naturally occurring cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5) inhibitors, it was found that the extracts from Sophora flavescens exhibit potent inhibitory activity against cGMP PDE5 prepared from rat diaphragm. Therefore, the inhibitory activities of five flavonoids, kushenol H (1), kushenol K (2), kurarinol (3), sophoflavescenol (4) and kuraridine (5), isolated from S. flavescens were measured against cGMP PDE5 to identify potent cGMP PDE5 inhibitory constituents. Among tested compounds, sophoflavescenol (4), a C-8 prenylated flavonol, showed the most potent inhibitory activity (IC(50)=0.013 microM) against cGMP PDE5 with 31.5- and 196.2-fold selectivity over PDE3 and PDE4, respectively. Kinetic analysis revealed that sophoflavescenol was a mixed inhibitor of PDE5 with a K(i) value of 0.005 microM.     

Cyclic nucleotides and neuroblastoma differentiation

We have shown that intracellular cGMP levels increase during retinoic acid- and mycophenolic acid-induced neuroblastoma differentiation and that a 6 days treatment with 1 mM dbcGMP lead LAN5 cell to elaborate a network of neuritic processes suggesting an involvement of cGMP in neuroblastoma differentiation. We have also investigated the effects of some specific inhibitors of phosphodiesterases (PDE1, PDE3, PDE4 and PDE5) on human neuroblastoma (LAN5 and SHEP) growth and differentiation. After six days of incubation in the presence of each specific inhibitor at 10 x IC50 levels a cytostatic and differentiating effect was only observed with the PDE5 inhibitors Zaprinast and MY-5445. The cytostatic effect of these compounds increased increasing their concentrations far above their IC50 levels for PDE5, suggesting that these compounds could act by interfering with other molecular events than direct cGMP-PDE inhibition. No appreciable effect was observed using Dipyridamole, another specific PDE5 inhibitor.     

Discovery of new inhibitor for PDE3 by virtual screening

In this work, we tried to find a new scaffold for a PDE3 using virtual screening for the obesity treatment. We first analyzed structural features for the known PDE3 inhibitors based on the PDE3B-ligand complex structure, and then carried out a docking study based on PDE3B 3D structure. We obtained a compound as potent PDE3 inhibitor stimulating lipolysis in murine adipocytes and human adipocytes.     

Cyclic Nucleotides and Neuroblastoma Differentiation

We have shown that intracellular cGMP levels increase during retinoic acid‐ and mycophenolic acid‐induced neuroblastoma differentiation and that a 6 days treatment with 1 mM dbcGMP lead LAN5 cell to elaborate a network of neuritic processes suggesting an involvement of cGMP in neuroblastoma differentiation. We have also investigated the effects of some specific inhibitors of phosphodiesterases (PDE1, PDE3, PDE4 and PDE5) on human neuroblastoma (LAN5 and SHEP) growth and differentiation. After six days of incubation in the presence of each specific inhibitor at 10 × IC₅₀ levels a cytostatic and differentiating effect was only observed with the PDE5 inhibitors Zaprinast and MY‐5445. The cytostatic effect of these compounds increased increasing their concentrations far above their IC₅₀ levels for PDE5, suggesting that these compounds could act by interfering with other molecular events than direct cGMP‐PDE inhibition. No appreciable effect was observed using Dipyridamole, another specific PDE5 inhibitor.     

Identification of novel isocytosine derivatives as xanthine oxidase inhibitors from a set of virtual screening hits

In recent years, xanthine oxidase has emerged as an important target not only for gout but also for cardiovascular and metabolic disorders involving hyperuricemia. Contrary to popular belief, recent clinical trials with uricosurics have demonstrated that enhanced excretion of uric acid is, by itself, not adequate to treat hyperuricemia; simultaneous inhibition of production of uric acid by inhibition of xanthine oxidase is also important. Virtual screening of in-house synthetic library followed by in vitro and in vivo testing led to the identification of a novel scaffold for xanthine oxidase inhibition. In vitro activity results corroborated the results from molecular docking studies of the virtual screening hits. The isocytosine scaffold maintains key hydrogen bonding and pi-stacking interactions in the deep end of the xanthine-binding pocket, which anchors it in an appropriate pose to inhibit binding of xanthine and shows promise for further lead optimization using structure-based drug design approach.