A2AR ( Ki = 3.54 nM )
Ciforadenant (CPI-444; V81444): Adenosine A2A receptor (A2AR) [1]

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.

---

1. A functional signaling assay was developed to demonstrate Ciforadenant’s blockade of A2AR; peripheral blood samples from patients treated with Ciforadenant were stimulated ex vivo with the adenosine analog NECA, and CREB phosphorylation in B and T cells was quantified by flow cytometry. After 14 days of Ciforadenant treatment, NECA-induced adenosine signaling was robustly inhibited in both single-agent and combination cohorts at the doses selected for the Phase 1b study [1]
2. Ciforadenant modulated the expression of immune checkpoints (GITR, OX40, LAG3) on tumor-infiltrating lymphocytes and circulating T cells in in vitro immune cell assays, indicating its broad role in reversing adenosine-mediated immunosuppression [1]

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.

---

1. In the syngeneic mouse MC38 tumor model, daily oral treatment with Ciforadenant (1, 10, 100 mg/kg) caused dose-dependent tumor growth inhibition, leading to tumor elimination in approximately 30% of treated mice [1]
2. Combination of Ciforadenant (100 mg/kg, qd for 14 days) with anti-PD-L1 (200 μg, 3qw for 4 doses) synergistically inhibited tumor growth in the MC38 model, with tumor elimination in 90% of treated mice [1]
3. Mice cured by Ciforadenant ± anti-PD-L1 treatment showed complete rejection of MC38 cell rechallenge (100% of challenged mice), demonstrating that Ciforadenant induced systemic anti-tumor immune memory [1]
4. Depletion of CD8⁺ T cells abrogated the anti-tumor efficacy of Ciforadenant as a single agent and in combination with anti-PD-L1, confirming that CD8⁺ T cells mediate the primary and secondary immune responses induced by the drug [1]
5. Treatment with Ciforadenant ± anti-PD-L1 was associated with increased CD8⁺ T cell infiltration and activation in MC38 tumor tissues, and a corresponding rise in PD-1 expression on CD8⁺ T cells in the spleen [1]
6. Ciforadenant modulated the levels of immune checkpoints (GITR, OX40, LAG3) on tumor-infiltrating lymphocytes and circulating T cells in vivo, suggesting a broad reversal of adenosine-mediated immunosuppression [1]

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..

---

1. To assess the A2AR-blocking effect of Ciforadenant, a functional signaling assay was established: peripheral blood samples from patients receiving Ciforadenant treatment were collected and stimulated ex vivo with NECA (an adenosine analog); CREB phosphorylation in B and T lymphocytes was quantified using flow cytometry to evaluate the inhibition of adenosine/A2AR signaling by Ciforadenant [1]

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.

---

1. Flow cytometric analysis was performed to detect the infiltration and activation status of CD8⁺ T cells in MC38 tumor tissues from mice treated with Ciforadenant ± anti-PD-L1; the expression of PD-1 on splenic CD8⁺ T cells was also quantified to assess immune cell activation [1]
2. The expression levels of immune checkpoint molecules (GITR, OX40, LAG3) on tumor-infiltrating lymphocytes and circulating T cells were measured by flow cytometry to evaluate the modulation of immune checkpoints by Ciforadenant [1]
3. CD8⁺ T cell depletion assays were conducted in vitro and in vivo: immune cells were treated with CD8⁺ T cell-depleting reagents, then co-cultured with tumor cells or transferred into tumor-bearing mice; the anti-tumor efficacy of Ciforadenant was evaluated to confirm the role of CD8⁺ T cells [1]

CPI-444 (1, 10, 100 mg/kg)
Syngeneic mouse model

---

1. Syngeneic MC38 tumor models were established in mice by implanting MC38 colorectal cancer cells into immunocompetent mice; Ciforadenant was formulated as an oral preparation and administered daily at doses of 1, 10, or 100 mg/kg for the single-agent study, with tumor volume measured regularly to assess dose-dependent anti-tumor activity [1]
2. For combination therapy studies, Ciforadenant was administered orally at 100 mg/kg daily for 14 days, while anti-PD-L1 antibody was given at 200 μg per mouse every 3 weeks for 4 doses; tumor growth was monitored to evaluate synergistic anti-tumor effects [1]
3. Tumor rechallenge experiments were performed on mice cured by Ciforadenant ± anti-PD-L1 treatment: MC38 cells were re-implanted into the cured mice, and tumor growth was monitored for up to several weeks to assess the induction of systemic anti-tumor immune memory [1]
4. CD8⁺ T cell depletion experiments were carried out by administering CD8⁺ T cell-depleting antibodies to tumor-bearing mice prior to Ciforadenant treatment (single-agent or combination); tumor volume was measured to determine the impact of CD8⁺ T cell depletion on drug efficacy [1]
5. Tumor tissues and spleens were collected from treated mice, and immune cell infiltration (CD8⁺ T cells) and activation markers (PD-1, GITR, OX40, LAG3) were analyzed by flow cytometry to explore the immunomodulatory mechanism of Ciforadenant [1]

1. Ciforadenant was well tolerated in a phase 1/1b clinical trial for a non-tumor indication[1]

[1]. Cancer Immunoly Research. September 25-28, 2016.

Ciforadenant is being investigated in the clinical trial NCT02253745 (V81444: Safety, Tolerability, Pharmacokinetics, and Efficacy Study in Attention Deficit Hyperactivity Disorder (ADHD) Volunteers). Ciforadenant is a small-molecule adenosine A2A receptor (ADORA2A) immune checkpoint inhibitor with potential antitumor activity. After 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 A2A receptors on these key immune surveillance cells, thereby eliminating adenosine-induced immunosuppression in the tumor microenvironment. This may stimulate an antitumor immune response, leading to tumor regression.

---

1. Ciforadenant (CPI-444; V81444) is a potent, oral, selective A2AR antagonist; elevated extracellular adenosine levels in the tumor microenvironment can promote tumor growth and metastasis by mediating immunosuppression on immune cells via A2AR[1]
2. Ciforadenant is the first adenosine antagonist to be shown to modulate the immunity of cancer patients, significantly inhibiting adenosine signaling in treated human lymphocytes[1]
3. A phase 1b clinical trial of Ciforadenant is underway to investigate its safety, tolerability, biomarkers, and preliminary efficacy as a monotherapy and in combination with the anti-PD-L1 antibody atezolizumab in patients with solid tumors (non-small cell lung cancer, melanoma, renal cell carcinoma, triple-negative breast cancer, head and neck cancer, MSI-H colorectal cancer, and bladder cancer)[1]
4. Peripheral blood and tumor biopsy samples will be collected before and after treatment in the phase 1b clinical trial. Used for biomarker analysis, including adenosine pathway expression activity, immune cell activation/tumor invasion, and mutational burden [1]

C20H21N7O3

407.43

407.17

C, 58.96; H, 5.20; N, 24.07; O, 11.78

1202402-40-1

1202402-40-1; 1202402-47-8 (R-isomer); 2102305-11-1 (racemic)

44537963

Solid powder

0.8

1

9

6

30

573

1

CC1=CC=C(O1)C2=C3C(=NC(=N2)N)N(N=N3)CC4=NC(=CC=C4)CO[C@H]5CCOC5

KURQKNMKCGYWRJ-HNNXBMFYSA-N

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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.

---

View More

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.)