(Rac)-Benpyrine, the racemic mixture of Benpyrine, is a potent and orally bioactive TNF-α inhibitor with the potential for research into TNF-α mediated inflammatory and autoimmune diseases.

Physicochemical Properties

Molecular Formula	C16H16N6O
Molecular Weight	308.33784198761
Exact Mass	308.14
Elemental Analysis	C, 62.32; H, 5.23; N, 27.26; O, 5.19
CAS #	1333714-43-4
Related CAS #	Benpyrine;2550398-89-3;(R)-Benpyrine
PubChem CID	53605452
Appearance	White to off-white 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	0
InChi Key	HUWOMAVUXTXEKT-UHFFFAOYSA-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)
Chemical Name	1-benzyl-4-(7H-purin-6-ylamino)pyrrolidin-2-one
Synonyms	(Rac)-Benpyrine; Benpyrine racemate
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 Note: This product requires protection from light (avoid light exposure) during transportation and storage.
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	TNF-α
ln Vitro	(Rac)-Benpyrine (Compound 001; 5-10 μM; 24 hours) treatment inhibits the level of nuclear expression of the TNF-α downstream pathway protein NF-κB in RAW264.7 macrophages[1].
ln Vivo	(Rac)-Benpyrine (Compound 001; 25-50 mg/kg; oral gavage; daily; for 3 weeks; Kunming mice) treatment clearly lowers inflammatory cytokines IFN-γ, IL-1β and IL-6 in blood serum, and clearly increases the anti-inflammatory factor IL-10. It also clearly slows the progression of foot swelling associated with arthritis, but does not reduce the weight index increase caused by arthritis[1].
Cell Assay	In a glass chamber, RAW 264.7 cells were expanded to 50–70% confluence, pretreated for 12 hours with DMSO or benpyrine, and then stimulated for 2 hours with 10 ng/mL TNF-. After PBS washing to end the cell treatments, the cells were fixed in freshly made 4% paraformaldehyde in PBS for 20 minutes. After three PBS washes, the fixed cells were permeabilized with 0.25% Triton X-100 in PBS for 10 minutes. We added the NF-B/p65 antibody at a 1:1000 dilution and incubated it at 4 °C overnight after blocking with 2 mg/mL BSA for 1 h at room temperature. We then washed the area three times with PBS and incubated it in the dark for 1 h at room temperature with a 1:500 dilution of the secondary antibody. Finally, 4′,6-diamidino-2-phenyl indole (DAPI) was used to stain the nuclei at 37 °C for 30 min in the dark.
References	[1]. Compound with TNF- α inhibitory activity and application. CN111320630A.

Solubility Data

Solubility (In Vitro)

DMSO: 10~31 mg/mL (32.4~100.5 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.