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.

Screening of Phosphodiesterase-5 Inhibitors and Their Analogs in Dietary Supplements by Liquid Chromatography-Hybrid Ion Trap-Time of Flight Mass Spectrometry.

An accurate and reliable method based on ion trap-time of flight mass spectrometry (IT-TOF MS) was developed for screening phosphodiesterase-5 inhibitors, including sildenafil, vardenafil, and tadalafil, and their analogs in dietary supplements. Various parameters affecting liquid chromatographic separation and IT-TOF detection were investigated, and the optimal conditions were determined. The separation was achieved on a reversed-phase column under gradient elution using acetonitrile and water containing 0.2% acetic acid at a flow rate of 0.2 mL/min. The chromatographic eluents were directly ionized in the IT-TOF system equipped with an electrospray ion source operating in the positive ion mode. The proposed screening method was validated by assessing its linearity, precision, and accuracy. Sequential tandem MS was conducted to obtain structural information of the references, and the fragmentation mechanism of each reference was proposed for providing spectral insight for newly synthesized analogs. Structural information, including accurate masses of both parent and fragment ions, was incorporated into the MSn spectral library. The developed method was successfully applied for screening adulterated dietary supplement samples.

Simultaneous determination and determination of sildenafil and its active metabolite in human plasma using LC-MS/MS method.

A sensitive and selective high-performance liquid chromatography-tandam mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of sildenafil and its metabolite N-desmethyl sildenafil in human plasma. Sildenafil-d8 was used as an internal standard. The analytes were extracted by precipitation extraction and chromatographed on a C18 column using mobile phase A of water (containing 0.1% formic acid) and mobile phase B of acetonitrile (containing 0.1% formic acid) with gradient elution. Quantification was done using multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 475.4 → m/z 283.3 for sildenafil, m/z 461.4 → m/z 283.2 for N-desmethyl sildenafil and m/z 483.3 → m/z 108.1 for IS in positive ionization mode. The calibration curve was established over the range of 2.00-1,000 ng/ml and the correlation coefficient was >0.99. The intra-day and inter-day relative standard deviations were <6.5% for sildenafil and 6.3% for N-desmethyl sildenafil respectively. Accuracy determinaed at four concentrations was 86.50-105.67% for sildenafil and 96.83-114.40% for N-desmethyl sildenafil. This method was successfully applied to a pharmacokinetic description of sildenafil and the effect of food intake on the pharmacokinetics of sildenafil was also demonstrated in healthy Chinese volunteers.

Isolation and identification of a new sildenafil analogue, hydroxycarbodenafil, found as an adulterant in a health supplement.

During routine screening of illegal adulterants in health supplements, a novel sildenafil analogue was discovered, and subsequently isolated by recrystallization. Its structure was elucidated by extensive analyses of high resolution mass spectrometry (HRMS), one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) data. The analogue was finally determined as hydroxycarbodenafil, featuring a hydroxyethyl group instead of an ethyl group on piperazine ring in comparison with carbodenafil.

An immunochromatographic strip sensor for sildenafil and its analogues.

In this study, a new hapten of sildenafil (Sild) was successfully synthesized and a sensitive monoclonal antibody (mAb) against Sild was produced based on this new hapten. The subclass of the mAb was IgG2a, and the half-maximal inhibitory concentration (IC50) of the mAb was 0.53 ng mL-1. Next, an immunochromatographic assay (ICA) was established for detecting Sild and its analogues in functional foods, where the visual detection limit (vLOD) and cut-off values were 0.5 and 20 µg kg-1, respectively. With the aid of a strip scan reader, the ICA can measure Sild with an LOD of 0.7 µg kg-1 and a line range of detection between 1.40 and 13.11 µg kg-1. The whole test process takes only 15 min. Therefore, the ICA provides a useful tool for the on-site detection and rapid initial screening of Sild in functional food samples.

Development of a specific fragment pattern-based quadrupole-Orbitrap mass spectrometry method to screen adulterated products of phosphodiesterase-5 inhibitors and their analogues.

Recently, adulterated supplements with phosphodiesterase-5 inhibitors (PDE-5i) have frequently observed. New synthetic analogues obtained from the chemical modification of parent compounds are frequently found in illicit products despite continuous efforts to inspect for these adulterants. A rapid and accurate method based on quadrupole-Orbitrap mass spectrometry was developed for simultaneously confirming and quantifying 85 PDE-5i and derived analogues present in illicit products for erectile dysfunction (ED). Common ions of PDE-5i according to their similar structures were proposed based on MS/MS fragmentations. These common ions could be an important diagnosis of their presence targets or new emerging analogues in supplements. Several validation parameters were employed, resulting in a limit of detection and quantification of 0.09-8.55 ng/mL and 0.24-17.10 ng/mL, respectively. The linear correlation coefficient (r2) was higher than 0.995, and mean recoveries of target compounds were in the range of 82-118%. A total of 187 illicit products, obtained from on/offline markets over a period of 3 years (2015-2017), were screened by the established method. Approximately 53% of them were adulterated with PDE-5i or derived analogues at concentrations of 0.1-726.0 mg/g in the illicit products. In the interests of public health, this study describes a rapid and accurate method to determine PDE-5i and new emerging analogues in adulterated products.

Isolation and characterisation of a novel sildenafil analogue adulterant, desmethylpiperazinyl propoxysildenafil, in a dietary supplement.

A new sildenafil analogue was detected during routine screening of dietary supplements suspected to be adulterated with an erectile dysfunction drug(s) using HPLC-DAD. The UV spectrum of this compound was highly similar to that of sildenafil and almost identical to that of desmethylpiperazinyl sildenafil. The analogue was purified by using semi-preparative HPLC and structurally elucidated by performing mass spectrometric and NMR spectroscopic experiments. The spectral data revealed that this sildenafil analogue bears an n-propoxy group instead of an ethoxy group and possesses no methylpiperazinyl moiety. The isolated compound, structure of which was further confirmed by spectral comparison with synthetic one, was thus named as desmethylpiperazinyl propoxysildenafil.

Characterization of a new illicit phosphodiesterase-type-5 inhibitor identified in the softgel shell of a dietary supplement.

A new sildenafil analog has been identified in the softgel shell of a dietary supplement. The compound was investigated by UV spectroscopy and high-resolution MS analysis, leading to the proposed structure 1-methyl-5-{5-[2-(4-methylpiperazin-1-yl)acetyl]-2-propoxyphenyl}-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one. A synthetic reference compound with the proposed structure was prepared, and the two sets of analytical data were compared, confirming the structure of the new compound. The compound was named propoxyphenyl noracetildenafil from its structure and similarity with the known compound.

Identification and structural elucidation of a new sildenafil analogue, dithiopropylcarbodenafil, from a premixed powder intended as a dietary supplement.

A new sildenafil analogue was detected during the monitoring of a premixed powder intended as a dietary supplement. The ultraviolet (UV) spectrum of the unknown compound was similar to that of dithiodesmethylcarbodenafil and dithiodesethylcarbodenafil, although their corresponding HPLC peaks were observed at different retention times. The chemical structure of the unknown compound was characterized by liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-Q-TOF/MS), followed by nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. The comparison of its structure with that of dithiodesmethylcarbodenafil, revealed that the N-methyl group on the piperazine ring is replaced by a propyl group. This new sildenafil analogue was identified as 5-(2-ethoxy-5-(4-propylpiperazine-1-carbonothioyl)phenyl)-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidine-7-thione and designated as a dithiopropylcarbodenafil. To the best of our knowledge, this is the first study reporting the identification and characterization of dithiopropylcarbodenafil.

New metabolites of hongdenafil, homosildenafil and hydroxyhomosildenafil.

Recently, illegal sildenafil analogues have emerged, causing serious social issues. In spite of the importance of sildenafil analogues, their metabolic profiles or clinical effects have not been reported yet. In this study, new metabolites of illegal sildenafil analogues such as hongdenafil, homosildenafil, and hydroxyhomosildenafil were determined using liquid chromatography quadrupole-time of flight mass spectrometry (LC-Q-TOF-MS) and tandem mass spectrometry (LC-Q-TOF-MS/MS). To prepare metabolic samples, in vitro and in vivo studies were performed. For in vivo metabolites analysis, urine and feces samples of rats treated with sildenafil analogues were analyzed. For in vitro metabolites analysis, human liver microsomes incubated with sildenafil analogues were extracted and analyzed. All metabolites were characterized by LC-Q-TOF-MS and LC-Q-TOF-MS/MS. As a result, five, six, and seven metabolites were determined in hongdenafil, homosildenafil, and hydroxyhomosildenafil treated samples, respectively. These results could be applied to forensic science and other analytical fields. Moreover, these newly identified metabolites could be used as fundamental data to determine the side effect and toxicity of illegal sildenafil analogues.

Palladium-Catalyzed 2,2,2-Trifluoroethoxylation of Aromatic and Heteroaromatic Chlorides Utilizing Borate Salt and the Synthesis of a Trifluoro Analogue of Sildenafil.

A simple and convenient method was developed for the introduction of a 2,2,2-trifluoroethoxy group to various aromatic and heteroaromatic systems. The novel process utilizes aromatic chlorides as substrates, and tetrakis(2,2,2-trifluoroethoxy) borate salt as an inexpensive and readily available fluoroalkoxy source in a palladium-catalyzed cross-coupling reaction. The power of the developed methodology was demonstrated in the synthesis of a fluorous derivative of Sildenafil.

Solid-phase extraction assisted dispersive liquid-liquid microextraction based on solidification of floating organic droplet to determine sildenafil and its analogues in dietary supplements.

A novel analytical method for the simultaneous determination of the concentration of sildenafil and its five analogues in dietary supplements using solid-phase extraction assisted reversed-phase dispersive liquid-liquid microextraction based on solidification of floating organic droplet combined with ion-pairing liquid chromatography with an ultraviolet detector was developed. Parameters that affect extraction efficiency were systematically investigated, including the type of solid-phase extraction cartridge, pH of the extraction environment, and the type and volume of extraction and dispersive solvent. The method linearity was in the range of 5.0-100 ng/mL for sildenafil, homosildenafil, udenafil, benzylsildenafil, and thiosildenafil and 10-100 ng/mL for acetildenafil. The coefficients of determination were ≥0.996 for all regression curves. The sensitivity values expressed as limit of detection were between 2.5 and 7.5 ng/mL. Furthermore, intraday and interday precisions expressed as relative standard deviations were less than 5.7 and 9.9%, respectively. The proposed method was successfully applied to the analysis of sildenafil and its five analogues in complex dietary supplements.

Non-allowed Pharmacologically Active Substances in Physical and Sexual Performance Enhancing Products.

BACKGROUND: Recently, a large amount of physical and sexual performance enhancing products have started to be freely sold mainly on internet web sites as dietary supplements. However, there a high suspicion that pharmacologically active substance, prohibited in these products, can be present to provide the expected effect. METHODS: A simple and rapid systematic toxicological analysis by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry has been applied after a liquidliquid extraction at acidic, neutral and alkaline pH with chloroform-isopropanol (9:1 v/v). The assays were validated in the range from 10 mg to 250 mg/g products showing a good linearity for the calibration curves (r2 ≥0.99). Mean extraction recoveries of analytes from different products were always higher than 90% and intra-assay and inter-assay precision and accuracy were always better than 15%. RESULTS: The developed method was applied to the analysis of products with a high percentage of sales in websites and smart and sexy shops. In twelve of eighty supplements, anabolic steroids, antiestrogenic drugs, psychoactive substances and sildenafil and analogs were identified and quantified. CONCLUSION: Eventual health hazards caused by the hidden presence of pharmacologically active substances in physical and sexual performance enhancing products are reported.

A Case Report of Fatal Desmethyl Carbodenafil Toxicity.

We present the case report of a 34-year-old Hispanic male who was found unresponsive in the carport of his residence. Surveillance video footage from a security camera showed that he collapsed as he was walking to his vehicle. The decedent had no medical history and no history of illicit drug use. Initial toxicology testing revealed no alcohol or illicit drugs. Autopsy findings indicated a need for additional toxicological analysis due to a lack of trauma and the paucity of pathophysiologically significant natural disease. Liquid chromatography time-of-flight mass spectrometry of postmortem blood revealed the presence of two large peaks corresponding to desmethyl carbodenafil, an unapproved sildenafil analogue and its hydroxy metabolite. Species that are probable desmethyl and hydroxydesmethyl metabolites of desmethyl carbodenafil were also found. The mass and retention time of the parent compound in the decedent's sample were matched to those of a commercial standard. Based on this preliminary match, a method was developed and validated to quantify desmethyl carbodenafil in human blood. This is the first known case of fatal intoxication by desmethyl carbodenafil, a phosphodiesterase-5 inhibitor that is not approved for use in the United States. Over the past several years, retailers have issued voluntary recalls for dietary supplements marketed as sexual performance enhancers on the basis that these supplements may contain undeclared desmethyl carbodenafil.

Isolation and identification of a novel sildenafil analogue adulterant in herbal products.

A novel sildenafil analogue found in herbal products by a routine drug-adulteration screening programme was isolated by column chromatography. On the basis of extensive 1D- and 2D-nuclear magnetic resonance (NMR) spectroscopy and mass spectral analysis, the structure of a new compound YJ-07 was established as 1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl) benzenesulfonamide. It common name is aminosildenafil.

Identification of a new sildenafil analogue adulterant, desethylcarbodenafil, in a herbal supplement.

In a maca-containing herbal supplement claimed to remedy erectile dysfunction, a new sildenafil analogue was found using adulterant screening with TLC, GC-MS and LC-MS/MS. This compound was isolated by column chromatography and HPLC, and identified by extensive 1D- and 2D-NMR and mass spectral analyses. The structure of this new compound was established as 5-[2-ethoxy-5-(piperazine-1-carbonyl) phenyl]-1-methyl-3-propyl-1,6-dihydro-pyrazolo[4,3-d]pyrimidin-7-one, and was named desethylcarbodenafil.

Development and evaluation of an immunochromatographic strip for rapid screening of sildenafil-type compounds as illegal additives in functional foods.

Sildenafil is a phosphodiesterase-5 inhibitor (PDE-5) for the treatment of erectile dysfunction. Undeclared sildenafil and related analogues adulterated in functional foods are a threat to public health. To screen these illegal drugs rapidly in herbal samples, an immunochromatographic (IC) assay was developed based on polyclonal antibodies specific to both sildenafil and its analogues. A group that is pharmacological necessary for sildenafil and its analogues was employed as a representative hapten for the generation antibodies against the target compounds. The desired antisera showed satisfactory specificities to sildenafil and major analogues with IC50 values ranging from 19.3 to 34.6 ng ml(-1) in a referring enzyme-linked immunosorbent assay (ELISA). The optimised IC assay showed detection thresholds in the range 5.0-20 µg g(-1) for sildenafil and major analogues in herbal samples. Sixty herbal food supplements were screened and six were found to be positive using the IC strip. It was confirmed by ELISA and UPLC-PDA-MS/MS that positive samples contain target illegal additives in levels of 10-40 mg g(-1) (1-4%). In this range, sensitivity of the IC strip is adequate to screen sildenafil-type compounds in herbal commodities under a dilution ratio of 1:10(3). Thus, the current IC assay is a suitable tool for screening sildenafil and its analogues as illegal additives in herbal food supplements.

Single dose sublingual testosterone and oral sildenafil vs. a dual route/dual release fixed dose combination tablet: a pharmacokinetic comparison.

AIM: The aim was to compare the pharmacokinetic profiles of two formulations of a combination drug product containing 0.5 mg testosterone and 50 mg sildenafil for female sexual interest/arousal disorder. The prototype (formulation 1) consists of a testosterone solution for sublingual administration and a sildenafil tablet that is administered 2.5 h later. The dual route/dual release fixed dose combination tablet (formulation 2) employs a sublingual and an oral route for systemic uptake. This tablet has an inner core of sildenafil with a polymeric time delay coating and an outer polymeric coating containing testosterone. It was designed to increase dosing practicality and decrease potential temporal non-adherence through circumventing the relatively complex temporal dosing scheme. METHODS: Twelve healthy premenopausal subjects received both formulations randomly on separate days. Blood was sampled frequently to determine the pharmacokinetics of free testosterone, total testosterone, dihydrotestosterone, sildenafil and N-desmethyl-sildenafil. RESULTS: Formulation 2 had a higher maximum concentration (Cmax ) for testosterone, 8.06 ng ml(-1) (95% confidence interval [CI] 6.84, 9.28) and higher area under the plasma concentration-time curve (AUC), 7.69 ng ml(-1)  h (95% CI 6.22, 9.16) than formulation 1, 5.66 ng ml(-1) (95% CI 4.63, 6.69) and 5.12 ng ml(-1)  h (95% CI 4.51, 5.73), respectively. Formulation 2 had a lower Cmax for sildenafil, 173 ng ml(-1) (95% CI 126, 220) and a lower AUC, 476 ng ml(-1)  h (95% CI 401, 551) than formulation 1, 268 ng ml(-1) (95% CI 188, 348) and 577 ng ml(-1)  h (95% CI 462, 692), respectively. Formulation 2 released sildenafil after 2.75 h (95% CI 2.40, 3.10). CONCLUSIONS: The dual route/dual release fixed dose combination tablet fulfilled its design criteria and is considered suitable for further clinical testing. WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Female sexual interest/arousal disorder (FSIAD) is a significant problem impacting psychological well-being, but the pharmacotherapeutic options for this problem are lacking. The combined, on-demand, sublingual administration of low dose sublingual testosterone and oral administration of sildenafil is a novel pharmacotherapeutic option under development for FSIAD. In proof-of-concept trials, these compounds were successfully administered via different dosage forms (sublingual and oral) at different time points (separated by 2.5 h) because of their markedly different pharmacokinetic-pharmacodynamic profiles. For future larger scale studies and the clinical practice, this raises obvious adherence issues. WHAT THIS STUDY ADDS: A newly developed dual route/dual release fixed dose combination tablet containing testosterone and sildenafil mimics the pharmacokinetic profile of these components when they are administered as different dosage forms, 2.5 h apart. This combination tablet is a suitable final pharmaceutical drug product that will be used in future studies.

Relaxant effects of selected sildenafil analogues in the rat aorta.

A new series of sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazine with chiral amino group has been synthesized and characterized. The compounds were tested for their relaxant effects in the rat aorta. Evaluation of prepared derivatives demonstrated that compound (8a) is probably a non-selective phosphodiesterase (PDE) inhibitor, as it induced aortic relaxation through endothelium-independent mechanism.

Simultaneous Detection of Three Phosphodiesterase Type 5 Inhibitors and Eight of Their Analogs in Lifestyle Products and Screening for Adulterants by High-Performance Thin-Layer Chromatography.

An HPTLC method is proposed to permit effective screening for the presence of three phosphodiesterase type 5 inhibitors (PDE5-Is; sildenafil, vardenafil, and tadalafil) and eight of their analogs (hydroxyacetildenafil, homosildenafil, thiohomosildenafil, acetildenafil, acetaminotadalafil, propoxyphenyl hydroxyhomosildenafil, hydroxyhomosildenafil, and hydroxythiohomosildenafil) in finished products, including tablets, capsules, chocolate, instant coffee, syrup, and chewing gum. For all the finished products, the same simple sample preparation may be applied: ultrasound-assisted extraction in 10 mL methanol for 30 min followed by centrifugation. The Rf values of individual HPTLC bands afford preliminary identification of potential PDE5-Is. Scanning densitometry capabilities enable comparison of the unknown UV spectra with those of known standard compounds and allow further structural insight. Mass spectrometric analysis of the material derived from individual zones supplies an additional degree of confidence. Significantly, the proposed screening technique allows focus on the already known PDE5 Is and provides a platform for isolation and chemical categorization of the newly-synthesized analogs. Furthermore, the scope could be expanded to other therapeutic categories (e.g., analgesics, antidiabetics, and anorexiants) that are occasionally coadulterated along with the PDE5-Is. The method was successfully applied to screening of 45 commercial lifestyle products. Of those, 31 products tested positive for at least one illegal component (sildenafil, tadalafil, propoxyphenyl hydroxyhomosildenafil, or dimethylsildenafil).