Benpyrine is a specific orally bioactive TNF-α inhibitor (antagonist) with a Kd of 82.1 μM. Benpyrine binds tightly to TNF-α and blocks its interaction with TNFR1 with IC50 of 0.109 µM. Benpyrine may be utilized in the study of TNF-α-mediated inflammation and autoimmune diseases.

Physicochemical Properties

Molecular Formula	C16H16N6O
Molecular Weight	308.3378
Exact Mass	308.138
CAS #	2550398-89-3
Related CAS #	(Rac)-Benpyrine;1333714-43-4;(R)-Benpyrine
PubChem CID	95327594
Appearance	White to yellow solid powder
LogP	1.2
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	4
Heavy Atom Count	23
Complexity	427
Defined Atom Stereocenter Count	1
SMILES	O=C1C([H])([H])[C@@]([H])(C([H])([H])N1C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])N([H])C1C2=C(N=C([H])N2[H])N=C([H])N=1
InChi Key	HUWOMAVUXTXEKT-LBPRGKRZSA-N
InChi Code	InChI=1S/C16H16N6O/c23-13-6-12(8-22(13)7-11-4-2-1-3-5-11)21-16-14-15(18-9-17-14)19-10-20-16/h1-5,9-10,12H,6-8H2,(H2,17,18,19,20,21)/t12-/m0/s1
Chemical Name	(4S)-1-benzyl-4-(7H-purin-6-ylamino)pyrrolidin-2-one
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	KD: 82.1 μM (TNF-α)[1]
ln Vitro	Benpyrine (5–20 μM; 14 hours; RAW264.7 cells) pretreatment causes a dose-dependent reduction in RAW264's IκBα phosphorylation.Ten milligrams of TNF-α or one microgram of LPS per milliliter were used to stimulate seven cells. In RAW264, benpyrine reverses the nuclear translocation of NF-κB/p65 caused by TNF-α.7 cells [1]. Benpyrine raised cell survival to 80% and only prevented TNF-α-induced cell death in WT and Y119A. Benpyrine has no discernible effect on the cytotoxicity of L929 induced by Y59L and L57A [1].
ln Vivo	Benpyrine treatment (25–50 mg/kg; oral gavage; daily; for 2 weeks; Balb/c mice) markedly reduced the symptoms of arthritis produced by collagen. IFN-γ, IL-1β, and IL-6 are examples of pro-inflammatory cytokines whose levels are dose-dependently decreased by benpyrine, while the concentration of the anti-inflammatory cytokine IL-10 is increased [1]. Benpyrine (25 mg/kg) was found to lessen TNF-α-induced inflammation in a mouse model of endotoxemia, which in turn lessened liver and lung injury [1].
Cell Assay	Western Blot Analysis[1] Cell Types: RAW264.7 cells Tested Concentrations: 5 µM, 10 µM, 20 µM Incubation Duration: 14 hrs (hours) Experimental Results: Resulted in a dose-dependent decrease in the phosphorylation of IκBα.
Animal Protocol	Animal/Disease Models: balb/c (Bagg ALBino) mouse: (18-20 g) injected with collagen[1] Doses: 25 mg/kg, 50 mg/kg Route of Administration: po (oral gavage); daily; for 2 weeks Experimental Results: Relieved the symptoms of collagen-induced arthritis in mice.
References	[1]. Discovery of an Orally Active Small Molecule TNF-α Inhibitor. J Med Chem. 2020 Jul 15.

Solubility Data

Solubility (In Vitro)

DMSO : 12.5 mg/mL (40.54 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 1.25 mg/mL (4.05 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 12.5 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. Solubility in Formulation 2: ≥ 1.25 mg/mL (4.05 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 12.5 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. Solubility in Formulation 3: ≥ 1.25 mg/mL (4.05 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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.2432 mL	16.2159 mL	32.4317 mL
	5 mM	0.6486 mL	3.2432 mL	6.4863 mL
	10 mM	0.3243 mL	1.6216 mL	3.2432 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.