[Identification of Descarbonsildenafil in an Adulterated Dietary Supplement and Evaluation of Its Inhibitory Activity for Phosphodiesterase Type 5 (PDE5)].

Some illegal dietary supplements contain phosphodiesterase type 5 (PDE5) inhibitors, such as sildenafil, for exerting "therapeutic" effects in erectile dysfunction. This is apparently dangerous, and thus, should be appropriately regulated. Identification of descarbonsildenafil was first reported in Singapore in a coffee sample labeled to exert male sexual performance enhancement effects. However, it is unclear whether the compound possesses PDE5 inhibitory activity. We encountered during our survey of dietary supplements, a sexual enhancement product commercially available in Tokyo, in which a peak presumed to be of descarbonsildenafil was detected by LC-UV and electrospray ionization-tandem MS (ESI-MS/MS). The compound was isolated and identified as descarbonsildenafil with liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS), NMR, and X-ray crystal structural analysis. In addition, descarbonsildenafil showed PDE5 inhibitory activity in PDE5 inhibition assay, and its IC50 value for PDE5A1 was found to be 30 nmol/L. The results of INADEQUATE NMR and X-ray crystal structural analysis in this study provide information for the identification of descarbonsildenafil. Since this study indicates that this compound is a PDE5 inhibitor having adequate activity, it is regulated as a drug component in Japan.

Variable temperature-nuclear magnetic resonance experiment and high-resolution MS/MSn measurement of hydroxycarbodenafil, and its PDE5 inhibitory activity.

Hydroxycarbodenafil, an analogue of carbodenafil, was detected in a dietary supplement in China in 2020. However, previous reports have not identified some carbon signals from the piperazine ring in nuclear magnetic resonance (NMR) experiments. Because the compound contains an amide bond, the reaction was suggested to be characteristic of compounds with rotational isomers. Variable-temperature NMR is used to determine the rotational barrier between different conformations by changing the measurement temperature. Using this technique, we succeeded in obtaining the first distinct data, including the carbon signals of the piperazine ring in the NMR spectrum of hydroxycarbodenafil. We also confirmed that this technique could be applied to other carbodenafil analogues. Multi-stage mass spectrometry (MSn) measurements with a high-resolution mass spectrometer specific to the substructures were performed to develop a protocol for the structural determination of the carbodenafil analogues. In addition, hydroxycarbodenafil was analysed using X-ray crystallography, and its inhibitory activity against phosphodiesterase type 5 (PDE5) was measured. The IC50 value of the inhibitory activity of hydroxycarbodenafil for PDE5A1, a PDE5 isoform, of 2.9 nM was lower than the 4.5 nM for sildenafil, a positive control.

Development of a detector tube for screening tadalafil and its analogues in adulterated sexual enhancement products.

Sexual enhancement products adulterated with phosphodiesterase 5 inhibitors (PDE-5i) pose a serious public health concern. Tadalafil and its analogues (Tds) are PDE-5i frequently detected as adulterants. In this study, a Td detector tube for the rapid detection of Tds was developed based on the color change reaction between sulfuric acid and Tds. The specificity of this test method was evaluated using 13 Tds, all of which elicited positive results. Additionally, 30 commonly found adulterants in dietary supplements, 11 active pharmaceutical ingredients of psychotropic drugs and 18 food ingredients were tested and obtained no false-positive results, except levomepromazine. The test tube accurately detected the presence or absence of Tds in 54 commercially available products. The visual detection limit was 2-50 and 5-20 µg/ml for Tds and tadalafil-spiked samples with matrix, respectively. The applicability of the developed detector tube to a semiquantitative test using digital image analyses were investigated using red, green, and blue color values. The results of the recovery test suggested that the tube test was affected by the dark-colored matrix. The results of semiquantitative analyses of tadalafil for five marketed products were consistent with the liquid chromatographic quantification results, except for the blue value. The detector tube developed in this study can facilitate with the rapid screening of Tds in adulterated sexual enhancement products.

Structure elucidation of a PDE5 inhibitor detected as an illegal adulteration in a libido-boosting dietary supplement.

A compound with potent inhibitory activity for phosphodiesterase type 5 (PDE5) was identified as an illegal adulteration in a libido-boosting dietary supplement being sold at a store in Tokyo. This compound was identified as 5,6-diethyl-2-{5-[(4-methylpiperazin-1-yl)sulphonyl]-2-propoxyphenyl}pyrimidin-4(3H)-one using liquid chromatography-diode array detector (LC-DAD), liquid chromatography-tandem mass spectrometer (LC-MS), LC-HRMS, nuclear magnetic resonance (NMR), and X-ray crystallography. The IC50 value of the inhibitory activity for PDE5A1 (one of the PDE5 isoforms) was 2.0 nM (sildenafil IC50 value was 4.5 nM). This compound was previously synthesised as a PDE5 inhibitor by Shanghai Institute of Materia Medica. The dietary supplement contained 85 mg of this compound in a capsule, which was about 26% of the capsule content (320 mg).