AX-024 is an orally bioavailable, first-in-class TCR-Nck interaction inhibitor that selectively inhibits TCR-triggered T cell activation with IC50 of 1 nM. AX-024 regulates cell signaling by targeting the SH3 domain. AX-024 has the characteristics of low toxicity, high efficiency and high selectivity. AX-024 effectively inhibits the production of interleukin-6 (IL-6), tumor necrosis factor-α (TNFα), interferon-γ (IFN-γ), IL-10 and IL-17A.

Physicochemical Properties

Molecular Formula	C21H22FNO2
Molecular Weight	339.403289318085
Exact Mass	339.163
CAS #	1370544-73-2
Related CAS #	AX-024 hydrochloride;1704801-24-0
PubChem CID	56949412
Appearance	Light yellow to yellow solid powder
LogP	3.4
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	4
Heavy Atom Count	25
Complexity	480
Defined Atom Stereocenter Count	0
SMILES	N1(CC2=C(C3=CC=C(F)C=C3)C3=CC(OC)=CC=C3OC2)CCCC1
InChi Key	VMMKGVRPILDZML-UHFFFAOYSA-N
InChi Code	InChI=1S/C21H22FNO2/c1-24-18-8-9-20-19(12-18)21(15-4-6-17(22)7-5-15)16(14-25-20)13-23-10-2-3-11-23/h4-9,12H,2-3,10-11,13-14H2,1H3
Chemical Name	1-[[4-(4-fluorophenyl)-6-methoxy-2H-chromen-3-yl]methyl]pyrrolidine
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 it comes to blocking TCR-triggered T cell proliferation, AX-024 is more than 10,000 times more effective than AX-000. In this assay, AX-024 exhibits inhibitory effects at concentrations of 1 pM or topical, but its IC50 is 1 nM. Furthermore, at a concentration of 10 nM, AX-024 significantly inhibits the production of interleukin-6 (IL-6), tumor cytokine-α (TNF-α), interferon-γ (IFN-γ), IL-10, and IL-17A. It is also a more potent inhibitor of CD3-stimulated cytokine release from human peripheral blood mononuclear cells than AX-000. In CD8+ T cells sliced from wild-type (WT) OT1 TCR fluid (OT1Tg) OT1Tg T cells that were PRS-mutated WT, AX-024 strongly inhibited T cell proliferation at a concentration of 0.1 nM. Co-immunoprecipitation experiments in these cells show that Nck recruitment to the TCR is induced upon stimulation in the absence of the drug but inhibited in a dose-dependent manner when AX-024 is present, starting at 1 nM [1].
ln Vivo	The AX-024 treated group showed less skin thickening and fewer scales than the vehicle group. AX-024 markedly decreased the thickening of both skin layers, with the dermal layer being affected more so than the other. This result was comparable to that shown in mice given a control cream that did not include imiquimod (IMQ). In the duplex assay, AX-024 dramatically decreased the quantity of critical airway cells. Unlike mice getting vehicle, which remained later after the ataxic repair and righting crises disappeared, mice receiving AX-024 recovered quickly from neurological impairment and weight loss, with symptoms going away by day 30 [1].
References	[1]. First-in-class inhibitor of the T cell receptor for the treatment of autoimmune diseases. Sci Transl Med. 2016 Dec 21;8(370):370ra184.

Solubility Data

Solubility (In Vitro)

Ethanol : ~100 mg/mL (~294.64 mM) DMSO : ~35 mg/mL (~103.12 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.37 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 25.0 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: 2.5 mg/mL (7.37 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: ≥ 2.5 mg/mL (7.37 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 25.0 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	2.9464 mL	14.7319 mL	29.4638 mL
	5 mM	0.5893 mL	2.9464 mL	5.8928 mL
	10 mM	0.2946 mL	1.4732 mL	2.9464 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.