Evixapodlin (GS-4224) is an inhibitor (blocker/antagonist) of human PD-1/PD-L1 protein/protein interaction with IC50 of 0.213 nM. Evixapodlin has anticancer and antiviral properties.

Physicochemical Properties

Molecular Formula	C34H36CL2N8O4
Molecular Weight	691.6068
Exact Mass	690.223
CAS #	2374856-75-2
PubChem CID	139415912
Appearance	White to yellow solid powder
LogP	2.2
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	10
Rotatable Bond Count	13
Heavy Atom Count	48
Complexity	985
Defined Atom Stereocenter Count	2
SMILES	ClC1=C(C([H])=C([H])C([H])=C1C1=C([H])N=C(C(=N1)OC([H])([H])[H])C([H])([H])N([H])C([H])([H])[C@]1([H])C([H])([H])C([H])([H])C(N1[H])=O)C1C([H])=C([H])C([H])=C(C=1Cl)C1=C([H])N=C(C(=N1)OC([H])([H])[H])C([H])([H])N([H])C([H])([H])[C@]1([H])C([H])([H])C([H])([H])C(N1[H])=O
InChi Key	OIIOPWHTJZYKIL-PMACEKPBSA-N
InChi Code	InChI=1S/C34H36Cl2N8O4/c1-47-33-27(15-37-13-19-9-11-29(45)41-19)39-17-25(43-33)23-7-3-5-21(31(23)35)22-6-4-8-24(32(22)36)26-18-40-28(34(44-26)48-2)16-38-14-20-10-12-30(46)42-20/h3-8,17-20,37-38H,9-16H2,1-2H3,(H,41,45)(H,42,46)/t19-,20-/m0/s1
Chemical Name	(5S)-5-[[[5-[2-chloro-3-[2-chloro-3-[6-methoxy-5-[[[(2S)-5-oxopyrrolidin-2-yl]methylamino]methyl]pyrazin-2-yl]phenyl]phenyl]-3-methoxypyrazin-2-yl]methylamino]methyl]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

ln Vitro	Evixapodlin (compound 139) increases the production of granzyme B and IFN-γ in CD8+ and CD4+ T cells with chronic hepatitis B (CHB). Additionally, in HBV-specific CD8+ and CD4+ T cells, evixapodlin enhanced the frequency of GrB+ cells. Durvalumab and evixapodlin both have the same capacity to increase the antiviral activity of HBV-specific CD8+ and CD4+ T cells in vitro [1].
ln Vivo	Evixapodlin (Compound 139) administered intraperitoneally once daily for six days at a dose of 10–50 mg/kg, demonstrated >90% PD-L1 target occupancy (TO) on tumor cells. Evixapodlin significantly affects female C57BL/6 mice inoculated with MC38 tumor cells, an animal model that expresses human PD-L1 [1].
Animal Protocol	Animal/Disease Models: Female C57BL/6 mice injected with MC38 tumor cells[1] Doses: 10 mg/kg, 25 mg/kg, and 50 mg/kg Route of Administration: intraperitoneal (ip) injection, daily, for 6 days Experimental Results:demonstrated greater than 90% TO on the tumors and inhibited tumor growth in vivo.
References	[1]. Pd-1/pd-l1 inhibitors. WO2019160882A1.

Solubility Data

Solubility (In Vitro)

DMSO : ~50 mg/mL (~72.30 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.61 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 (3.61 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 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 (3.61 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	1.4459 mL	7.2295 mL	14.4590 mL
	5 mM	0.2892 mL	1.4459 mL	2.8918 mL
	10 mM	0.1446 mL	0.7230 mL	1.4459 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.