The synthesis and biological evaluation of nucleobases/tetrazole hybrid compounds: A new class of phosphodiesterase type 3 (PDE3) inhibitors.

Spired by the chemical structure of Cilostazol, a selective phosphodiesterase 3A (PDE3A) inhibitor, several novel hybrid compounds of nucleobases (uracil, 6-azauracil, 2-thiuracil, adenine, guanine, theophylline and theobromine) and tetrazole were designed and successfully synthesized and their inhibitory effects on PDE3A as well as their cytotoxicity on HeLa and MCF-7 cancerous cell lines were studied. Obtained results show the linear correlation between the inhibitory effect of synthesized compounds and their cytotoxicity. In some cases, the PDE3A inhibitory effects of synthesized compounds are higher than the Cilostazol. Besides, compared to a standard anticancer drug methotrexate, some of the synthesized compounds showed the higher cytotoxicity against the HeLa and MCF-7 cancerous cell lines.

Pharmacokinetic modeling analysis of cilostazol and its active metabolites (OPC-13015 and OPC-13213) after multiple oral doses of cilostazol in healthy Korean volunteers.

Cilostazol is a selective inhibitor of phosphodiesterase III (PDE III), which is prescribed for patients with peripheral arterial disease, especially intermittent claudication. The purpose of the study was to investigate the pharmacokinetic (PK) of cilostazol and its metabolites on the immediate (IR) formulation of cilostazol in healthy Korean male volunteers by population PK modeling analysis implemented using NONMEM software.A 2 × 2 crossover study comparing multiple oral doses of IR and SR formulations of cilostazol were conducted. Serial plasma concentrations of cilostazol and its active metabolites were used in this analysis.The PK was best depicted by one-compartment model, with absorption kinetics of cilostazol having mixed first- and zero-order kinetics with a time delay at the beginning of absorption. The introduction of interoccasion variabilities into zero-order (D1), first-order (Ka), and relative bioavailability (F1) significantly improved the model fit, and total body water (TBW) was identified as a significant covariate positively affecting the clearance of cilostazol. The model validation suggested that the model constructed in this study predicted the plasma concentration of cilostazol and its two active metabolites reasonably well.The PK model we developed explored the PK characteristics of cilostazol in Korean male subjects, and may be useful for identifying optimal individual dosing regimens of cilostazol.

One step N-glycosylation by filamentous fungi biofilm in bioreactor of a new phosphodiesterase-3 inhibitor tetrazole.

An efficient and rapid process for N-glycosylation of 5-(1-(3-fluorophenyl)-1H-pyrazol-4-yl)-2H-tetrazole-LQFM 021 (1), a new synthetic derivative of pyrazole with phosphodiesterase-3 (PDE-3) inhibitory action, vasorelaxant activity and low toxicity catalyzed by filamentous fungi biofilm in bioreactor was successfully developed. A maximum N-glycosyl yield of 68% was obtained with Cunninghamella echinulata ATCC 9244 biofilm in bioreactor with conditions of 25mgml(-1) of 1 in PDSM medium at 28°C for 96h. After extraction with ethyl acetate, the derivative was identified by Ultrahigh Resolution Mass Spectrometry and (1)H-(13)C HSQC/HMBC.

Design and discovery of 2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(4-phenyl)thiazole derivatives as cardiotonic agents via inhibition of PDE3.

A series of novel 2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(4-phenyl)thiazole derivatives, 6(a-o) were designed, synthesized and evaluated for inhibitory activity against human PDE3A and PDE3B. In PDE3 assay, entire set of targeted analogs showed considerable inhibition of PDE3A (IC50=0.24 ± 0.06-16.42 ± 0.14 µM) over PDE3B (IC50=2.34 ± 0.13-28.02 ± 0.03 µM). Among the synthesized derivatives, compound 6d exhibited most potent inhibition of PDE3A with IC50=0.24 ± 0.06 µM than PDE3B (IC50=2.34 ± 0.13 µM). This compound was further subjected for evaluation of cardiotonic activity (contractile and chronotropic effects) in comparison with Vesnarinone. Results showed that, it selectively modulates the force of contraction (63%± 5) rather than frequency rate (23% ± 2) at 100 µM. Docking study of above compound was also carried out in the active site of PDE3 protein model to give proof to the mechanism of action of designed inhibitor. Further, in sub-acute toxicity experiment in Swiss-albino mice, it was found to be non-toxic up to 100mg/kg dose for 28days.

Repurposing de novo designed entities reveals phosphodiesterase 3B and cathepsin L modulators.

Using computational bioactivity prediction models we identified phosphodiesterase 3B (PDE3B) and cathepsin L as macromolecular targets of de novo designed compounds. By disclosing the most potent cathepsin L activator known to date, small molecule repurposing by target panel prediction represents a feasible route towards innovative leads for chemical biology and molecular medicine.

Preparation and characterization of microemulsion of cilostazol for enhancement of oral bioavailability.

Cilostazol is a promising drug for antiplatelet combination therapy that is very important for treatment for various cardiovascular disorders. However, oral delivery of this drug is greatly impeded by the poor solubility in aqueous solutions. The aim of this study was to develop microemulsion (ME) delivery system capable of improving the drug bioavailability. In this study, Capmul MCM C8 (glycerol monocaprylate) based MEs containing Tween 20(polysorbate 20) and/or Labrafil M 1944(poly oxyglycerides) as surfactant(S) and Transcutol P(diethyl glycol monoethyl ether) as cosurfactant(CoS) were studied as potential delivery systems of cilostazol. A number of such systems were prepared containing different S:CoS ratios(1:1, 2:1 and 3:1) based on phase diagrams. Loading of cilostazol was selected as per solubilization capacity and was characterized for pH, viscosity, conductivity, particle size, zeta potential and % transmittance. The MEs systems were further investigated for chemical stability, diffusion and bioavailability. Cilostazol displayed high solubility in microemulsions with particle size up to 70 nm. It was also stable at ambient temperature up to 6 months without significant change in particle size, zeta potential, and % transmittance. Dilution up to 100 fold with aqueous medium observed a visible cloudiness having a particle size up to 104 nm. The in vitro release, and ex vivo intraduodenal diffusion, and in vivo study indicated the capacity of developed ME to improve the bioavailability (1.43 fold) via oral route administration when compared with commercially available tablets (Pletoz-50).

Selective regulation of cyclic nucleotide phosphodiesterase PDE3A isoforms.

Inhibitors of cyclic nucleotide phosphodiesterase (PDE) PDE3A have inotropic actions in human myocardium, but their long-term use increases mortality in patients with heart failure. Two isoforms in cardiac myocytes, PDE3A1 and PDE3A2, have identical amino acid sequences except for a unique N-terminal extension in PDE3A1. We expressed FLAG-tagged PDE3A1 and PDE3A2 in HEK293 cells and examined their regulation by PKA- and PKC-mediated phosphorylation. PDE3A1, which is localized to intracellular membranes, and PDE3A2, which is cytosolic, were phosphorylated at different sites within their common sequence. Exposure to isoproterenol led to phosphorylation of PDE3A1 at the 14-3-3-binding site S312, whereas exposure to PMA led to phosphorylation of PDE3A2 at an alternative 14-3-3-binding site, S428. PDE3A2 activity was stimulated by phosphorylation at S428, whereas PDE3A1 activity was not affected by phosphorylation at either site. Phosphorylation of PDE3A1 by PKA and of PDE3A2 by PKC led to shifts in elution on gel-filtration chromatography consistent with increased interactions with other proteins, and 2D electrophoresis of coimmunoprecipitated proteins revealed that the two isoforms have distinct protein interactomes. A similar pattern of differential phosphorylation of endogenous PDE3A1 and PDE3A2 at S312 and S428 is observed in human myocardium. The selective phosphorylation of PDE3A1 and PDE3A2 at alternative sites through different signaling pathways, along with the different functional consequences of phosphorylation for each isoform, suggest they are likely to have distinct roles in cyclic nucleotide-mediated signaling in human myocardium, and raise the possibility that isoform-selective inhibition may allow inotropic responses without an increase in mortality.

Modulating the cyclic guanosine monophosphate substrate selectivity of the phosphodiesterase 3 inhibitors by pyridine, pyrido[2,3-d]pyrimidine derivatives and their effects upon the growth of HT-29 cancer cell line.

Analogues with the scaffolds of 3-cyano-4-alkoxyphenyl-6-bromoaryl-2-pyridone and 2-amino-3-cyano-4-alkoxyphenyl-6-bromoarylpyridine were synthesized. Cyclization of the 2-amino derivatives with formic acid and formamide gave the corresponding pyrido[2,3-d]pyrimidin-4(3H)-one and the pyrido[2,3-d]-pyrimidin-4-amine derivatives, respectively. Active phosphodiesterase 3 (PDE3) inhibitors were identified from each of the four aforementioned scaffolds. This is the first report that pyrido[2,3-d]pyrimidin-4(3H)-one and pyrido[2,3-d]pyrimidin-4-amine derivatives can inhibit PDE3. The analogues with the pyridone and pyrido[2,3-d]pyrimidin-4(3H)-one scaffolds inhibited both cAMP and cyclic guanosine monophosphate (cGMP) hydrolysis by PDE3, while the amine containing scaffolds were more selective for cGMP hydrolysis. This observation may set the base for substrate-selective pharmacological modulation of this important class of drug targets and with less side effects, particularly tachcardia. The dual inhibitors of PDE3 were more potent inhibitor towards the growth of HT-29 cancer cell lines.