Ciforadenant (formerly known as CPI-444; V-81444) is a potent, selective and orally bioactive small molecule inhibitor of the adenosine-A2a receptor (A2AR) on T-lymphocytes with anticancer and immunomodulatory activities and the potential to be used in cancer immunotherapy. Ciforadenant is presently being tested against a variety of solid tumors in a Phase I clinical trial both as a single agent and in conjunction with the PD-L1 inhibitor atezolizumab. It is possible to elicit antitumor responses with ciporadenant. When taken orally, ciporadenant binds to adenosine A2A receptors expressed on the surface of immune cells, such as DCs, T-lymphocytes, macrophages, and natural killer (NK) cells. This stops tumor-released adenosine from interacting with these important immune surveillance cells' A2A receptors, thereby reversing the immunosuppressive effects of adenosine in the tumor microenvironment.

Physicochemical Properties

Molecular Formula	C20H21N7O3
Molecular Weight	407.43
Exact Mass	407.17
Elemental Analysis	C, 58.96; H, 5.20; N, 24.07; O, 11.78
CAS #	1202402-40-1
Related CAS #	1202402-40-1; 1202402-47-8 (R-isomer); 2102305-11-1 (racemic)
PubChem CID	44537963
Appearance	Solid powder
LogP	0.8
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	6
Heavy Atom Count	30
Complexity	573
Defined Atom Stereocenter Count	1
SMILES	CC1=CC=C(O1)C2=C3C(=NC(=N2)N)N(N=N3)CC4=NC(=CC=C4)CO[C@H]5CCOC5
InChi Key	KURQKNMKCGYWRJ-HNNXBMFYSA-N
InChi Code	InChI=1S/C20H21N7O3/c1-12-5-6-16(30-12)17-18-19(24-20(21)23-17)27(26-25-18)9-13-3-2-4-14(22-13)10-29-15-7-8-28-11-15/h2-6,15H,7-11H2,1H3,(H2,21,23,24)/t15-/m0/s1
Chemical Name	7-(5-methylfuran-2-yl)-3-[[6-[[(3S)-oxolan-3-yl]oxymethyl]pyridin-2-yl]methyl]triazolo[4,5-d]pyrimidin-5-amine
Synonyms	Ciforadenant; CPI-444; V81444; CPI 444; V-81444; CPI444; V 81444
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	A2AR ( Ki = 3.54 nM )
ln Vitro	In vitro activity: Ciforadenant (also known as CPI-444 and V81444) is a potent, selective and orally bioactive small molecule inhibitor of the adenosine-A2a receptor (A2AR) on T-lymphocytes. It is also referred to as CPI-444 and V81444. A Phase I clinical trial is presently investigating the use of ciporadenant alone and in combination with PD-L1 inhibitor atezolizumab to treat a range of solid tumors. It is possible to elicit antitumor responses with ciporadenant. When taken orally, ciporadenant binds to adenosine A2A receptors expressed on the surface of immune cells, such as DCs, T-lymphocytes, macrophages, and natural killer (NK) cells. This stops tumor-released adenosine from interacting with these important immune surveillance cells' A2A receptors, thereby reversing the immunosuppressive effects of adenosine in the tumor microenvironment.
ln Vivo	CPI-444 is being studied as a single agent and in combination with the anti-PD-L1 antibody atezolizumab in solid tumors as part of a Ph 1b study. In preclinical investigations, CPI-444 (1, 10, 100 mg/kg) administered daily to the syngeneic mouse model MC38 resulted in dose-dependent inhibition of tumor growth, which in approximately 30% of treated mice, led to tumor elimination. In MC38 models, the combined administration of CPI-444 (100 mg/kg, qd, 14 days) and anti-PD-L1 (200 μg, 3qw, 4 doses) resulted in a synergistic inhibition of tumor growth, with 90% of treated mice showing tumor elimination. Tumor growth was rejected in 100% of challenged mice when MC38 cells were re-introduced to cured mice, suggesting that CPI-444 induced systemic anti-tumor immune memory. Depletion of CD8+ T cells rendered CPI-444 treatment ineffective when used alone or in conjunction with anti-PD-L1, indicating that CD8+ T cells are involved in mediating both primary and secondary immune responses. Increased CD8+ cell infiltration and activation in MC38 tumor tissues, along with a corresponding rise in PD-1 expression on CD8+ T cells in the spleen, were linked to the anti-tumor efficacy of CPI-444 ± anti-PD-L1.
Enzyme Assay	Ciforadenant (also known as CPI-444 and V81444) is a potent, selective and and orally bioactive small molecule inhibitor of the adenosine-A2a receptor (A2AR) on T-lymphocytes..
Cell Assay	CPI-444 is a potent, oral, selective antagonist of A2AR. The effectiveness of CPI-444 treatment both alone and in conjunction with anti-PD-L1 is negated by CD8+ T cell depletion, suggesting a function for CD8+ T cells in mediating both primary and secondary immune responses. CPI-444±anti-PD-L1'santitumorefficacy is linked to elevated CD8+ cell infiltration and activation in MC38 tumor tissues, as well as an increase in PD-1 expression on CD8+ T cells in the spleen. Furthermore, treatment with CPI-444 modifies the levels of immune checkpoints on tumor infiltrating lymphocytes and circulating T cells, such as GITR, OX40, and LAG3. This suggests a broad role for adenosine-mediated immunosuppression.
Animal Protocol	CPI-444 (1, 10, 100 mg/kg) Syngeneic mouse model
References	[1]. Cancer Immunoly Research. September 25-28, 2016.
Additional Infomation	Ciforadenant is under investigation in clinical trial NCT02253745 (Safety, Tolerability, PK & Efficacy of V81444 in Volunteers With Attention Deficit/ Hyperactivity Disorder (ADHD)). Ciforadenant is a small molecule immune checkpoint inhibitor of the adenosine A2A receptor (ADORA2A) with potential antineoplastic activity. Upon oral administration, ciforadenant binds to adenosine A2A receptors expressed on the surface of immune cells, including T-lymphocytes, natural killer (NK) cells, macrophages and dendritic cells (DCs). This prevents tumor-released adenosine from interacting with the A2A receptors on these key immune surveillance cells, thereby abrogating adenosine-induced immunosuppression in the tumor microenvironment. This may stimulate anti-tumor immune responses, resulting in tumor regression.

Solubility Data

Solubility (In Vitro)

DMSO: > 75 mg/mL Water: N/A Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.25 mg/mL (5.52 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 22.5 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 2: ≥ 2.08 mg/mL (5.11 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 3: ≥ 2.08 mg/mL (5.11 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	2.4544 mL	12.2720 mL	24.5441 mL
	5 mM	0.4909 mL	2.4544 mL	4.9088 mL
	10 mM	0.2454 mL	1.2272 mL	2.4544 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.