Pyrazolopyrimidinone Based Selective Inhibitors of PDE5 for the Treatment of Erectile Dysfunction

Continuing research with our earlier finding of sildenafil based analogs in the search of new inhibitors of PDE5 for erectile dysfunction suggested that there is a scope of modifications at N-methylpiperazine ring with hydrophobic region followed by hydrogen bond donor or acceptor region. However, the leads identified earlier had some limitations like poor pharmacokinetic (PK) profile, low aqueous solubility and poor bioavailability. In this direction, a new series of sildenafil based analogs were designed, synthesized and screened for their PDE5 inhibitory activity. In this series compound 18 was found to have excellent in vitro activity with selectivity towards PDE5 isozyme, also the in vivo activity and pharmacokinetic profile was excellent. The cyp inhibition and CaCO₂ permeability was also excellent for compound 18 .     

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).     

Vardenafil: structural basis for higher potency over sildenafil in inhibiting cGMP-specific phosphodiesterase-5 (PDE5)

Phosphodiesterase-5 (PDE5) inhibitors act by competing with the substrate, cGMP, for the catalytic site of the enzyme. Two commercialized PDE5 inhibitors, sildenafil and vardenafil, are being used to treat erectile dysfunction. These two compounds differ in the heterocyclic ring system used to mimic the purine ring of cGMP. They also differ in the substituent (ethyl/methyl) of a piperazine side chain. Although these are the only two structural differences, vardenafil has more than 20-fold greater potency than sildenafil for inhibiting purified PDE5. The molecular structural basis for the difference in potency of the two compounds was investigated by synthesizing an analog of sildenafil ("methyl-sildenafil") that contained the sildenafil ring system but with the appended ethyl group found in vardenafil, and an analog of vardenafil ("demethyl-vardenafil") that contained the vardenafil ring system but with the appended methyl group found in sildenafil. The IC50 of methyl-sildenafil for inhibiting PDE5 indicated that it was 64 times less potent than demethyl-vardenafil, which was similar to the finding that, based on IC50, sildenafil was 40 times less potent than vardenafil. Similarly, the EC50 of methyl-sildenafil for inhibiting [3H]vardenafil binding to PDE5 indicated that it was 84 times less potent than demethyl-vardenafil, while the EC50 for sildenafil indicated that it was 31 times less potent than vardenafil. It is concluded that the methyl/ethyl appended group on the piperazine moiety plays very little role in the difference in potency between sildenafil and vardenafil for inhibiting PDE5, whereas the differences in the ring systems play a critical role in higher potency of vardenafil over sildenafil.     

Quinazolines as potent and highly selective PDE5 inhibitors as potential therapeutics for male erectile dysfunction

In an effort to minimize side effects associated with low selectivity against PDE isozymes, we have successfully identified a series of 6,7,8-substituted quinzaolines as potent inhibitors of PDE5 with high level of isozyme selectivity, especially against PDE6 and PDE11. PDE5 potency and isozyme selectivity of quinazolines were greatly improved with substitutions both at 6- and 8-position. The synthesis, structure-activity relationships and in vivo efficacy of this novel series of potent PDE5 inhibitors are described.     

Investigation of PDE5/PDE6 and PDE5/PDE11 selective potent tadalafil-like PDE5 inhibitors using combination of molecular modeling approaches,molecular fingerprint-based virtual screening protocols and structure-based pharmacophore development

The essential biological function of phosphodiesterase (PDE) type enzymes is to regulate the cytoplasmic levels of intracellular second messengers, 3′,5′-cyclic guanosine monophosphate (cGMP) and/or 3′,5′-cyclic adenosine monophosphate (cAMP). PDE targets have 11 isoenzymes. Of these enzymes, PDE5 has attracted a special attention over the years after its recognition as being the target enzyme in treating erectile dysfunction. Due to the amino acid sequence and the secondary structural similarity of PDE6 and PDE11 with the catalytic domain of PDE5, first-generation PDE5 inhibitors (i.e. sildenafil and vardenafil) are also competitive inhibitors of PDE6 and PDE11. Since the major challenge of designing novel PDE5 inhibitors is to decrease their cross-reactivity with PDE6 and PDE11, in this study, we attempt to identify potent tadalafil-like PDE5 inhibitors that have PDE5/PDE6 and PDE5/PDE11 selectivity. For this aim, the similarity-based virtual screening protocol is applied for the “clean drug-like subset of ZINC database” that contains more than 20 million small compounds. Moreover, molecular dynamics (MD) simulations of selected hits complexed with PDE5 and off-targets were performed in order to get insights for structural and dynamical behaviors of the selected molecules as selective PDE5 inhibitors. Since tadalafil blocks hERG1 K channels in concentration dependent manner, the cardiotoxicity prediction of the hit molecules was also tested. Results of this study can be useful for designing of novel, safe and selective PDE5 inhibitors.     

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.     

Synthesis and biological activities of novel beta-carbolines as PDE5 inhibitors

A series of N(2)-furoyl and N(2)pyrimidinyl beta-carbolines was discovered to possess potent inhibitory activity against PDE5. During the synthesis we developed a tandem resin quenching protocol, which allowed us to synthesize large number of target compounds in a rapid fashion. Representative compounds exhibit superior selectivity to sildenafil versus other isozymes of PDEs, and demonstrated in vivo efficacy in increasing introcavernosal pressure in dogs.     

Effects of PDE5 Inhibitors and sGC Stimulators in a Rat Model of Artificial Ureteral Calculosis

Urinary colics from calculosis are frequent and intense forms of pain whose current pharmacological treatment remains unsatisfactory. New and more effective drugs are needed to control symptoms and improve stone expulsion. Recent evidence suggested that the Nitric Oxide (NO) / cyclic guanosine monophosphate (cGMP) / phosphodiesterase type 5 (PDE5) system may contribute to ureteral motility influencing stone expulsion. We investigated if PDE5 inhibitors and sGC stimulators influence ureteral contractility, pain behaviour and stone expulsion in a rat model of ureteral calculosis. We investigated: a)the sex-specific PDE5 distribution in the rat ureter; b)the functional in vitro effects of vardenafil and sildenafil (PDE5 inhibitors) and BAY41-2272 (sGC stimulator) on induced ureteral contractility in rats and c)the in vivo effectiveness of vardenafil and BAY41-2272, alone and combined with ketoprofen, vs hyoscine-N-butylbromide alone or combined with ketoprofen, on behavioural pain indicators and stone expulsion in rats with artificial calculosis in one ureter. PDE5 was abundantly expressed in male and female rats’ ureter. In vitro, both vardenafil and BAY41-2272 significantly relaxed pre-contracted ureteral strips. In vivo, all compounds significantly reduced number and global duration of “ureteral crises” and post-stone lumbar muscle hyperalgesia in calculosis rats. The highest level of reduction of the pain behaviour was observed with BAY41-2272 among all spasmolytics administered alone, and with the combination of ketoprofen with BAY41-2272. The percentage of stone expulsion was maximal in the ketoprofen+BAY41-2272 group. The NO/cGMP/PDE5 pathway is involved in the regulation of ureteral contractility and pain behaviour in urinary calculosis. PDE5 inhibitors and sGC stimulators could become a potent new option for treatment of urinary colic pain.     

Expression, activity, and pro-hypertrophic effects of PDE5A in cardiac myocytes

Cyclic GMP-selective phosphodiesterase type 5 (PDE5) has been traditionally thought to play a little role in cardiac myocytes, yet recent studies using selective inhibitors such as sildenafil suggest it can potently modulate acute and chronic cardiac stress responses. To date, evidence for myocyte PDE5 expression and regulation has relied on small-molecule inhibitors and anti-sera, leaving open concerns regarding non-specific immune-reactivity, and off-target drug effects. To directly address both issues, we engineered a robust PDE5-gene silencing shRNA (inserted into miRNA-155 cassette) and DsRed–PDE5 fusion protein, both coupled to a CMV promoter and incorporated into adenoviral vectors. PDE5 mRNA and protein knock-down eliminated anti-sera positivity on immunoblots and fluorescent immuno-histochemistry in neonatal and adult cardiomyocytes, and suppressed PDE5 enzyme activity. Stimulation of myocyte hypertrophy by phenylephrine was blunted by PDE5 gene silencing in a protein kinase G dependent manner, and this effect was similar to that from sildenafil with no additive response by both combined. DsRed–PDE5 fusion protein expression showed normal z-band localization in adult myocytes but was diffused in eNOS^−/− myocytes; echoing reported findings with anti-sera. PDE5 overexpression increased enzyme activity and amplified natriuretic peptide gene expression from phenylephrine stimulation. These data confirm PDE5 expression, activity, and targeted inhibition by sildenafil in cardiomyocytes, as well as the role of this PDE in cardiomyocyte hypertrophy modulation.     

Synthesis and phosphodiesterase 5 inhibitory activity of new 5-phenyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one derivatives containing an N-acylamido group phenyl ring.

New sildenafil analogues with an N-acylamido group at the 5'-position of the phenyl ring, 6a--e, were prepared from the readily available starting compound 2 in four straightforward steps. Enzyme assays demonstrated that all the target compounds 6a-e showed higher PDE5 inhibitory activities than sildenafil. It was observed that the PDE5 inhibitory activity was enhanced as the chain length of R group increased, but introduction of the branched alkyl groups such as isopropyl (6d) and cyclohexyl (6e) resulted in the drop of potency compared with 6c. In particular the N-butyrylamido derivative 6c exhibited the highest PDE5 inhibitory activity, and was about 6-fold more potent than sildenafil. However, all the compounds exhibited somewhat weak selectivity (1--3-fold) over PDE6, indicating that the compounds 6a--e have intrinsically lower selectivity than sildenafil.     

The cGMP-binding, cGMP-specific phosphodiesterase (PDE5): intestinal cell expression, regulation and role in fluid secretion

The expression and regulation of the cGMP-binding, cGMP-specific phosphodiesterase, PDE5, was studied in intestinal cells. Both PDE5A1 and PDE5A2 splice forms were cloned from the cDNA prepared from human colonic T84 cells, and PDE5 activity was dependent on increases in intracellular cGMP levels which correlated with increased phosphorylation of the enzyme. PDE5 expression was monitored in different regions of the gastrointestinal tract and nearly 50% of the phosphodiesterase activity in the duodenum, jejunum, ileum and colon was inhibited by sildenafil citrate. Administration of the stable toxin to intestinal loops resulted in activation of PDE5. Inhibition of PDE5 by sildenafil citrate led to fluid accumulation in loops, suggesting a possible explanation for the side effect of diarrhoea observed in individuals administered sildenafil citrate. Our results therefore represent the first study on the expression and regulation of PDE5 in intestinal tissue, and indicate that mechanisms to control its activity may have important consequences in intestinal physiology.     

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.     

Discovery of selective PDE4B inhibitors

In this study the first PDE4B selective inhibitor is described. Optimization of lead 2-arylpyrimidine derivatives afforded a series of potent PDE4B inhibitors with >100-fold selectivity over the PDE4D isozyme. With a good pharmacokinetic profile, a selected compound exhibited potent anti-inflammatory effects in vivo and showed less emesis compared with Cilomilast.     

Conformational conversion of PDE5 by incubation with sildenafil or metal ion is accompanied by stimulation of allosteric cGMP binding

Phosphodiesterase-5 (PDE5) is a dimer containing a cGMP-specific catalytic domain and an allosteric cGMP-binding subdomain (GAF A) on each subunit. PDE5 exhibits three conformational forms that can be separated by Native PAGE and are denoted as Bands 1, 2, and 3 in decreasing order of mobility. A preparation comprised mainly of Band 2 PDE5 was partially converted to Band 3 PDE5 by 1 h incubation with cGMP or the PDE5-specific inhibitors sildenafil, vardenafil, or tadalafil, but not with cAMP, milrinone (PDE3-specific), or rolipram (PDE4-specific). Band 2 PDE5 was converted almost entirely to Band 3 PDE5 by overnight incubation with sildenafil at 30 °C. This time-dependent conversion was accompanied by a 7-fold increase in allosteric cGMP-binding activity, suggesting that Band 3 PDE5 is a much more active form than Band 2 PDE5 for allosteric cGMP binding. Conversion of Band 2 PDE5 to Band 3 PDE5 occurred faster by pre-incubation with cGMP, which binds to both the allosteric and catalytic sites of PDE5, than with catalytic site-specific sildenafil. Overnight incubation of a Band 2/Band 3 PDE5 mixture with EDTA caused time-dependent conversion to Band 1 PDE5 (apoenzyme), and this conversion was accompanied by a 50% loss in cGMP-binding activity. After incubation with EDTA, addition of Mn⁺⁺ or Mg⁺⁺ caused reversion of Band 1 to a Band 2/Band 3 PDE5 mixture in which Band 3 PDE5 predominated. This reversion was accompanied by a 3-fold increase in allosteric cGMP-binding activity. The combination of results implied that physiological conversion of Band 2 to Band 3 PDE5 by cGMP and/or divalent metal ion occupancy of the catalytic domain would increase allosteric cGMP binding to the enzyme. This conversion would produce a greater negative feedback effect on cGMP action by increasing sequestration of cGMP at the allosteric cGMP-binding site of PDE5 and by increasing cGMP degradation at the catalytic site of the enzyme. This conversion would also increase PDE5 inhibitor binding to the enzyme.     

Effect of PDE5 inhibition on coronary hemodynamics in pacing-induced heart failure

Inhibition of phosphodiesterase type 5 (PDE5) can relax systemic and coronary vessels by causing accumulation of cGMP. Both the endothelial dysfunction with decreased nitric oxide production and increased natriuretic peptide levels in congestive heart failure (CHF) have the potential to alter cGMP production, thereby influencing the response to PDE5 inhibition. Consequently, this study examined the effects of PDE5 inhibition with sildenafil in dogs with CHF produced by rapid ventricular pacing. CHF resulted in decreases of left ventricular (LV) systolic pressure, coronary blood flow, and the maximal first time derivative of LV pressure (LV dP/dt(max)) at rest and during treadmill exercise compared with normal, whereas resting LV end-diastolic pressure increased from 10 +/- 1.4 to 23 +/- 1.4 mmHg. Sildenafil (2 and 10 mg/kg per os) caused a 5- to 6-mmHg decrease of aortic pressure (P < 0.05), with no change of heart rate, LV systolic pressure, or LV dP/dt(max). Sildenafil caused no change in coronary flow or myocardial oxygen consumption in animals with CHF at rest or during exercise. In contrast to findings in normal animals, sildenafil did not augment endothelium-dependent coronary vasodilation in response to acetylcholine in animals with CHF. Furthermore, Western blotting showed decreased PDE5 protein expression in myocardium from failing hearts. These findings demonstrate that PDE5 contributes little to regulation of coronary hemodynamics in CHF.     

Expression of three isoforms of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in human penile cavernosum

Inhibition of cGMP-specific phosphodiesterase type V (PDE5) has been shown to improve penile erection in patients with erectile dysfunction. We report here the cloning of three PDE5 isoforms from human penile tissues. Two of the isoforms were identical to PDE5A1 and PDE5A2, respectively, which had been isolated from nonpenile tissues. The third isoform was novel and hence called PDE5A3. The deduced amino acid sequence of PDE5A3 was the same as the C-terminal 823-residue sequence of PDE5A1 and PDE5A2. While PDE5A1 and A2 isoforms were expressed in all tissues examined, the A3 isoform was confined to tissues with a smooth muscle or cardiac muscle component. When expressed in COS-7 cells, PDE5A1, A2, and A3 isoforms had similar cGMP-catalytic activities with K(m) of 6.2, 5.75, and 6.06 microM, respectively. Their cGMP-catalytic activities were inhibited by zaprinast with IC(50) values of 3.2 microM, 1.3 microM, and 1.6 microM, respectively, and by sildenafil with IC(50) of 28, 14, and 13 nM, respectively.     

Identification of purine inhibitors of phosphodiesterase 7 (PDE7)

A series of purine based inhibitors of PDE7 has been derived from screening lead 1a. The synthesis, structure-activity relationships (SAR), and selectivity against several other PDE family members are described.     

Metal ion stimulators of PDE5 cause similar conformational changes in the enzyme as does cGMP or sildenafil

Purified PDE5 preparations exhibited variable proportions of two mobility forms (Bands 2 and 3) by native PAGE. Treatment of recombinant or native PDE5 with either cGMP or a substrate analog such as sildenafil, each of which is known to produce stimulatory effects on enzyme functions, caused a similar native PAGE band-shift to the lower mobility form (shift of Band 2 to Band 3). Incubation of PDE5 with Mg⁺⁺ or Mn⁺⁺, which is known to stimulate activity, caused a similar shift of the enzyme from Band 2 to Band 3 as did cGMP or sildenafil, but incubation with EDTA caused a time- and concentration-dependent shift to higher mobility (shift of Bands 2 and 3 to Band 1). A slow time course of the EDTA-induced band-shift suggested removal of a pre-bound metal ion (Me⁺⁺) with affinity of ~ 0.1 nM, which was similar to the previously determined affinity of PDE5 for Zn⁺⁺. The EDTA-treated enzyme (Band 1) could be shifted to Bands 2 and 3 by addition of cGMP, sildenafil, or Me⁺⁺; however, the cGMP- or sildenafil-induced shift was inhibited and the Me⁺⁺-induced shift was facilitated by treatment with EDTA. Results suggested that Me⁺⁺ removal from PDE5 produces a unique apoenzyme form (Band 1, more globular, negatively charged, or both) of PDE5 that can be partially converted to forms (Band 2, less globular or negatively charged, or both; and Band 3, more elongated/positively charged, or both) by addition of Me⁺⁺, substrate, or substrate analog. It is concluded that Me⁺⁺ causes conversion of PDE5 to similar conformational forms as caused by substrate or inhibitor binding to the catalytic site.     

Three-dimensional structures of PDE4D in complex with roliprams and implication on inhibitor selectivity

Selective inhibitors against the 11 families of cyclic nucleotide phosphodiesterases (PDEs) are used to treat various human diseases. How the inhibitors selectively bind the conserved PDE catalytic domains is unknown. The crystal structures of the PDE4D2 catalytic domain in complex with (R)- or (R,S)-rolipram suggest that inhibitor selectivity is determined by the chemical nature of amino acids and subtle conformational changes of the binding pockets. The conformational states of Gln369 in PDE4D2 may play a key role in inhibitor recognition. The corresponding Y329S mutation in PDE7 may lead to loss of the hydrogen bonds between rolipram and Gln369 and is thus a possible reason explaining PDE7's insensitivity to rolipram inhibition. Docking of the PDE5 inhibitor sildenafil into the PDE4 catalytic pocket further helps understand inhibitor selectivity.     

Expression of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in mouse tissues and cell lines using an antibody against the enzyme amino-terminal domain

We have produced a polyclonal antibody that specifically recognizes cGMP-binding cGMP-specific phosphodiesterase (PDE5). The antibody was raised in rabbit using as immunogen a fusion protein, in which glutathione S-transferase was coupled to a 171 amino acid polypeptide of the N-terminal region of bovine PDE5. The antibody is able to immunoprecipitate PDE5 activity from mouse tissues and neuroblastoma extracts while it has no effect on all other PDE isoforms present in the extracts. PDE5 activity recovered in the immunoprecipitates retains its sensitivity to specific inhibitors such as zaprinast (IC(50)=0.6 microM) and sildenafil (IC(50)=3.5 nM). Bands of the expected molecular mass were revealed when solubilized immunoprecipitates were analysed in Western blots. The antibody selectively stained cerebellar Purkinje neurones, which are known to express high levels of PDE5 mRNA. Western blot analysis of mouse tissues revealed the highest expression signal in mouse lung, followed by heart and cerebellum, while a lower signal was evident in brain, kidney and a very low signal was present in the liver. In the hybrid neuroblastoma-glioma NG108-15 cells the antibody revealed a high PDE5 induction after dibutyryl-cAMP treatment.