AS2863619 free base can convert antigen-specific effector/memory T cells into Foxp3+ regulatory T (Treg) cells to study a variety of immune diseases. AS2863619 free base is a potent, oral cyclin-dependent kinase 8 (CDK8) and CDK19 inhibitor (antagonist) with IC50 of 0.61 nM and 4.28 nM respectively. AS2863619 free base inhibits CDK8/19 to enhance the activation of STAT5, thereby activating the Foxp3 gene.

Physicochemical Properties

Molecular Formula	C16H12N8O
Molecular Weight	332.319480895996
Exact Mass	332.113
CAS #	2241300-50-3
Related CAS #	AS2863619;2241300-51-4
PubChem CID	139600292
Appearance	White to off-white solid powder
LogP	1.2
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	2
Heavy Atom Count	25
Complexity	495
Defined Atom Stereocenter Count	0
SMILES	O1N=C(C(C2=NC3C=NC=CC=3N2C2=CC=C3C(=C2)NC(C)=N3)=N1)N
InChi Key	OORKHRHPVSWORX-UHFFFAOYSA-N
InChi Code	InChI=1S/C16H12N8O/c1-8-19-10-3-2-9(6-11(10)20-8)24-13-4-5-18-7-12(13)21-16(24)14-15(17)23-25-22-14/h2-7H,1H3,(H2,17,23)(H,19,20)
Chemical Name	4-[1-(2-methyl-3H-benzimidazol-5-yl)imidazo[4,5-c]pyridin-2-yl]-1,2,5-oxadiazol-3-amine
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

ln Vitro	When mouse CD4+ T cells were treated with AS2863619 (1 μM) for 22 hours, the amount of serine phosphorylation of the STAT5b PSP motif was reduced to around 40%, whereas the amount of tyrosine phosphorylation of the C-terminal domain was increased to about 160% of the samples treated with control. %[1].
ln Vivo	AS2863619 (30 mg/kg; oral; daily; for 2 weeks; mice) Treatment after sensitization with 2,4-dinitrofluorobenzene (DNFB) inhibits the extent of secondary reactions and infiltration of inflammatory cells into the skin Less severe and reduced ratio of changes in interferon-gamma+ (IFN-γ+) cells in a skin contact hypersensitivity model compared to vehicle-treated control mice. Eliminating Tregs before evoking secondary responses eliminated AS2863619-induced suppression. KLRG1+ Foxp3+ T cells were specifically enhanced in DNFB-sensitized AS2863619-treated animals [1].
Cell Assay	Western Blot Analysis[1] Cell Types: Mouse CD4+ T cells Tested Concentrations: 1 μM Incubation Duration: 22 hrs (hours) Experimental Results: Inhibited the serine phosphorylation of the PSP motif of STAT5b to approximately 40%, while inhibiting the tyrosine phosphorylation of the PSP motif of STAT5b to about 40%, while inhibiting the tyrosine phosphorylation of the PSP motif of STAT5b. Amino acid phosphorylation was enhanced to approximately 160% of the control treated sample.
Animal Protocol	Animal/Disease Models: DNFB-induced contact skin allergy in mice [1] Doses: 30 mg/kg Route of Administration: oral; daily; lasting for 2 weeks Experimental Results: secondary reaction degree, inflammatory cells infiltration into the skin was light, interferon diminished proportion of -γ+ (IFN-γ+) cells.
References	[1]. Conversion of antigen-specific effector/memory T cells into Foxp3-expressing Treg cells by inhibition of CDK8/19. Sci Immunol. 2019 Oct 25;4(40). pii: eaaw2707.

Solubility Data

Solubility (In Vitro)

DMSO : ~250 mg/mL (~752.29 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.26 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (6.26 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (6.26 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.0091 mL	15.0457 mL	30.0915 mL
	5 mM	0.6018 mL	3.0091 mL	6.0183 mL
	10 mM	0.3009 mL	1.5046 mL	3.0091 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.