PDE5; major metabolite of Sildenafil
N-Desmethyl Sildenafil targets phosphodiesterase type 5 (PDE5), the same target as its parent drug sildenafil. PDE5 is an enzyme that hydrolyzes cyclic GMP (cGMP), playing a key role in the regulation of smooth muscle tone. By inhibiting PDE5, N-Desmethyl Sildenafil prevents the breakdown of cGMP, leading to increased cGMP levels and smooth muscle relaxation. This mechanism underlies the vasodilatory effects of sildenafil and its metabolite. As the major active metabolite of sildenafil, N-Desmethyl Sildenafil contributes to the overall pharmacological activity of the parent drug.

The electroretinogram (ERG) amplitude recorded from the isolated rat retina that has acclimated to darkness is increased by N-Desmethyl Sildenafil, most likely because to an improvement in the response of the photoreceptor cells[1]. By co-expressing cytochrome b5 and human P450 oxidoreductase, CYP3A supersomes are used to investigate the synthesis of N-Desmethyl Sildenafil. CYP3A4, CYP3A5, and, to a lesser extent, CYP3A7 catalyze N-Desmethyl Sildenafil[2].

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In vitro activity data for N-Desmethyl Sildenafil are limited, but as a PDE5 inhibitor, it is expected to demonstrate similar activity to sildenafil. The compound is the major active metabolite of sildenafil and contributes to its pharmacological effects. PDE5 inhibition leads to increased cGMP levels and smooth muscle relaxation. The compound's potency and selectivity for PDE5 over other PDE isoforms would be similar to sildenafil. Further in vitro studies would be needed to fully characterize its activity profile.

Here researchers report that the active component of Viagra, Sildenafil and the first metabolite, N-desmethyl-sildenafil (UK-103, 320) increased the amplitude of flash-evoked electroretinogram (ERG) of dark-adapted albino rat retina. Effects of Sildenafil and N-desmethyl-sildenafil were comparable to those of the known phosphodiesterase inhibitor, Zaprinast. The photoreceptor cell response was isolated by blocking the glial K(+) ion-buffering and the on-bipolar components of the ERG with the use of BaCl(2) (500 microM) and the specific type VI metabotropic glutamate receptor agonist, DL-2-amino-4-phosphonobutyric acid (25 microM), respectively. Zaprinast, Sildenafil and N-desmethyl-sildenafil (1 microM each) increased the amplitude of photoreceptor cell response either. Besides, Sildenafil was significantly more effective than N-desmethyl-sildenafil. These findings suggest an increased sensitivity of photoreceptor cells in the presence of Sildenafil and it is metabolite[1].

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In vivo, N-Desmethyl Sildenafil contributes to the pharmacological effects of sildenafil. As the major active metabolite of sildenafil, it is formed in the body after sildenafil administration and contributes to the overall efficacy of the drug in treating erectile dysfunction and pulmonary arterial hypertension. The metabolite's activity is part of the overall pharmacokinetic and pharmacodynamic profile of sildenafil. Its presence in plasma after sildenafil dosing has been confirmed in clinical studies.

The aim of this study was to characterize the kinetics of metabolite formation of the phosphodiesterase type-5 (PDE5) inhibitors sildenafil and tadalafil by CYP3A4, CYP3A5, and CYP3A7 isoforms. The formations of N-desmethyl sildenafil and desmethylene tadalafil were examined using CYP3A supersomes co-expressing human P450 oxidoreductase and cytochrome b5. Both sildenafil N-demethylation and tadalafil demethylenation were catalyzed by CYP3A4, CYP3A5, and to a lesser extent by CYP3A7. The kinetics of desalkyl metabolite formation of the two drugs were well fitted to the Hill equation; however, the Hill coefficients (n) suggested CYP3A-mediated negative cooperativity. Next, we analyzed the kinetics with a two binding sites model assuming two reaction steps: reaction 1 with high-affinity and low-capacity metabolism and reaction 2 with low-affinity and high-capacity metabolism. The kinetics of desalkyl metabolite formation were also fitted to the two binding sites model. The intrinsic clearance (CLint) values of reactions 1 and 2 for sildenafil N-demethylation were 0.733 and 0.033 µL/min/pmol P450 for CYP3A4, 0.788 and 0.019 µL/min/pmol P450 for CYP3A5, and 0.079 and 0.004 µL/min/pmol P450 for CYP3A7, respectively. The CLint values of reactions 1 and 2 for tadalafil demethylenation were 0.187 and 0.014 µL/min/pmol P450 for CYP3A4, 0.050 and <0.001 µL/min/pmol P450 for CYP3A5, and 0.004 and <0.001 µL/min/pmol P450 for CYP3A7, respectively. These results may provide the basis not only for understanding the metabolic properties of the two PDE5 inhibitors, but also for one possible explanation of the mechanisms of CYP3A-mediated negative cooperativity[2].

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The in vitro enzyme assay for N-Desmethyl Sildenafil involves measuring its inhibition of PDE5 enzymatic activity. Recombinant human PDE5 is expressed and purified. Enzyme activity is assessed by measuring the hydrolysis of cGMP to GMP using a scintillation proximity assay or fluorescence polarization. N-Desmethyl Sildenafil is incubated with the enzyme and substrate at various concentrations. IC50 values are determined by fitting the inhibition data to a dose-response curve. The assay buffer typically contains Tris-HCl, MgCl2, and appropriate components for enzyme activity.

In vitro cellular assays for N-Desmethyl Sildenafil would typically use smooth muscle cells or cell lines expressing PDE5. Cells are treated with N-Desmethyl Sildenafil at various concentrations. Intracellular cGMP levels are measured using ELISA. The compound's ability to increase cGMP levels is quantified. Additionally, effects on smooth muscle relaxation can be assessed in tissue-based assays. These assays confirm the compound's cellular activity.

In vivo, N-Desmethyl Sildenafil is formed after sildenafil administration. Pharmacokinetic studies in humans have confirmed the presence of the metabolite in plasma. The metabolite contributes to the overall pharmacological effects of sildenafil in treating erectile dysfunction and pulmonary arterial hypertension. Its pharmacokinetic profile, including Cmax, Tmax, and half-life, has been characterized as part of sildenafil's overall PK profile.

The pharmacokinetic properties of N-Desmethyl Sildenafil are well characterized as part of sildenafil's overall profile. It is the major active metabolite of sildenafil. After oral administration of sildenafil, N-Desmethyl Sildenafil is formed via hepatic metabolism. The metabolite has a similar pharmacokinetic profile to the parent drug, with measurable plasma concentrations contributing to the overall pharmacological effects. Its molecular weight is 460.55.

N-Desmethyl Sildenafil is the major active metabolite of sildenafil. Its safety profile is similar to that of sildenafil, as it contributes to the overall pharmacological effects of the parent drug. Sildenafil is generally well-tolerated, with known side effects including headache, flushing, and visual disturbances. As a metabolite, N-Desmethyl Sildenafil is not administered as a separate therapeutic agent and is only studied as part of sildenafil's overall safety and efficacy profile.

[1]. Sildenafil, N-desmethyl-sildenafil and Zaprinast enhance photoreceptor response in the isolated rat retina. Neurochem Int. 2003 Nov;43(6):591-5.

[2]. Contribution of CYP3A isoforms to dealkylation of PDE5 inhibitors: a comparison between sildenafil N-demethylation and tadalafil demethylenation. Biol Pharm Bull. 2015;38(1):58-65.

phosphodiesterase type 5 inhibitor; a vasodilator and urological drug used to treat erectile dysfunction and primary pulmonary hypertension.

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N-Desmethyl Sildenafil (Desmethylsildenafil; UK-103,320) is the major active metabolite of sildenafil. It is a PDE5 inhibitor that contributes to the pharmacological effects of sildenafil in treating erectile dysfunction and pulmonary arterial hypertension. The compound has a molecular weight of 460.55 and formula C21H28N6O4S. It is used as an analytical standard and in research applications. No separate clinical trials or regulatory approvals have been reported for the metabolite itself.

C21H28N6O4S

460.55

460.189

139755-82-1

N-Desmethyl Sildenafil-d8;1185168-06-2

135455980

White to off-white solid powder

1.44g/cm3

685.7ºC at 760 mmHg

158-160ºC

368.5ºC

1.17E-18mmHg at 25°C

1.683

2.616

2

8

7

32

810

0

CCCC1=NN(C2=C1N=C(NC2=O)C3=C(C=CC(=C3)S(=O)(=O)N4CCNCC4)OCC)C

UZTKBZXHEOVDRL-UHFFFAOYSA-N

InChI=1S/C21H28N6O4S/c1-4-6-16-18-19(26(3)25-16)21(28)24-20(23-18)15-13-14(7-8-17(15)31-5-2)32(29,30)27-11-9-22-10-12-27/h7-8,13,22H,4-6,9-12H2,1-3H3,(H,23,24,28)

5-(2-ethoxy-5-piperazin-1-ylsulfonylphenyl)-1-methyl-3-propyl-6H-pyrazolo[4,3-d]pyrimidin-7-one

N-Desmethyl Sildenafil; 139755-82-1; desmethylsildenafil; 5-(2-ethoxy-5-(piperazin-1-ylsulfonyl)phenyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one; desmethyl sildenafil; N-Desmethylsildenafil; UNII-L6WO34R9YG; C21H28N6O4S;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 250 mg/mL (542.83 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.52 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.52 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.52 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)