PD-1/ PD-L1
PD-1-IN-17 targets the programmed cell death-1 (PD-1) receptor. It functions as a PD-1/PD-L1 interaction inhibitor, blocking the binding between PD-1 on T cells and PD-L1 on tumor cells or antigen-presenting cells. By disrupting this immune checkpoint, the compound prevents the PD-1/PD-L1-mediated suppression of T-cell activation, thereby enhancing antitumor immunity.

PD-1-IN-17, an analogue of CA-170, is a programmed cell death-1 (PD-1) inhibitor.

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In vitro, PD-1-IN-17 inhibits 92% of splenocyte proliferation at a concentration of 100 nM, demonstrating potent immunosuppressive reversal activity. This indicates that the compound can effectively block the PD-1/PD-L1 axis and restore T-cell proliferation and function. The compound's activity is comparable to other small-molecule PD-1/PD-L1 inhibitors in its class.

In vivo, PD-1-IN-17 has the potential to exhibit antitumor activity by reactivating T-cell-mediated immune responses against tumors. As a small-molecule PD-1/PD-L1 inhibitor structurally similar to CA-170, it offers the advantages of oral bioavailability and potential for better tumor penetration compared to biologics. Preclinical studies are expected to demonstrate its efficacy in various tumor models.

In vitro binding assays typically involve ELISA-based methods or surface plasmon resonance to measure the inhibition of PD-1/PD-L1 interaction. Recombinant PD-1 protein is immobilized, and PD-L1 is incubated with varying concentrations of PD-1-IN-17. The binding signal is measured to calculate the IC50 for inhibition of the protein-protein interaction.

Cellular assays are performed using splenocyte proliferation assays. Mouse splenocytes are stimulated with anti-CD3 antibody in the presence of PD-L1-expressing cells or recombinant PD-L1 protein. PD-1-IN-17 is added at various concentrations, and T-cell proliferation is assessed by [³H]-thymidine incorporation or CFSE dilution. The percentage inhibition of proliferation is calculated to determine the compound's potency.

In vivo studies are typically conducted in syngeneic mouse tumor models (e.g., MC38 colon carcinoma or B16 melanoma) bearing tumors expressing PD-L1. PD-1-IN-17 is administered orally or intraperitoneally at various doses. Tumor volume is measured periodically, and immune cell infiltration into tumors is assessed by flow cytometry. Serum cytokine levels are measured to evaluate systemic immune activation.

PD-1-IN-17 (molecular weight 374.35, formula C₁₃H₂₂N₆O₇) is a small-molecule compound. It is soluble in DMSO (~125 mg/mL) and water (~100 mg/mL). The compound is typically stored as a powder at -20°C (3 years) or 4°C (2 years). Its physicochemical properties support oral bioavailability and favorable drug-like characteristics.

Preclinical toxicity studies of PD-1-IN-17 have been limited, as the compound is primarily used as a research tool. As a PD-1/PD-L1 inhibitor, its toxicity profile is expected to be related to immune-related adverse events. No significant toxicity has been reported in the available literature.

[1]. 1,3,4-oxadiazole and 1,3,4-thiadiazole derivatives as immunomodulators. WO2015033301A1.

CA-170 is a selective small molecule PD-L1 inhibitor. The PD-L1/PD-L2/VISTA antagonist CA-170 is an orally bioavailable small molecule inhibitor that inhibits the immune checkpoint regulatory proteins programmed death-ligand-1 (PD-L1; B7-H1; CD274), PD-L2, and V-domain immunoglobulin (Ig) T-cell activation inhibitor (VISTA; programmed death-1 homolog; PD1H; PD-1H), exhibiting potential negative immune checkpoint modulation and antitumor activity. After oral administration, the PD-L1/PD-L2/VISTA antagonist CA-170 targets and binds to PD-L1, PD-L2, and VISTA. This drug inhibits the PD-L1/PD-L2/VISTA-mediated signaling pathway, eliminates the suppression of T lymphocyte immune responses induced by PD-L1, PD-L2, and VISTA, enhances the proliferation and activation of cytotoxic T cells, promotes T cell cytokine production, and inhibits tumor cell growth. PD-L1, PD-L2, and VISTA are negative checkpoint molecules of immune activation, playing a crucial role in suppressing T cell function. CA-170 is currently the only small molecule modulator undergoing clinical trials, targeting PD-L1 and VISTA proteins—important regulators of negative checkpoints of immune activation. Reported treatment results are somewhat similar to those of FDA-approved monoclonal antibodies, overcoming the limitations of the latter's high production costs and significant side effects. However, there is currently no conclusive biophysical evidence demonstrating that CA-170 binds to hPD-L1. Using established in vitro methods, including NMR binding assays, HTRF assays, and cell activation assays, we have clearly demonstrated that there is no direct binding between CA-170 and PD-L1. To further validate our findings, we conducted control experiments on the CA-170 precursor, the 29-peptide AUNP-12. Positive controls included well-reported small molecule PD-L1 inhibitors: BMS-1166 and peptide-57. [Molecules. 2019 Aug 1;24(15):2804.]

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CA-170 is the first small molecule inhibitor targeting PD-L1 to enter Phase I and Phase II clinical trials. However, we definitively determined that there is no actual direct binding between CA-170 and its peptide precursor AUNP-12 and PD-L1. In NMR binding assays, neither CA-170 nor AUNP-12 interacted with human or mouse single-domain or double-domain PD-L1, nor with human PD-1. In in vitro systems of HTRF assays and cell assays simulating in vivo conditions, CA-170 and AUNP-12 also failed to dissociate the complex. All measurements were cross-validated using the positive control peptide-57 and the small molecule BMS-1166. Therefore, the mechanism by which CA-170 directly blocks the hPD-1/hPD-L1 interaction needs to be revised. [Molecules. Aug 1, 2019;24(15):2804.]

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PD-1-IN-17 is a small-molecule PD-1/PD-L1 interaction inhibitor extracted from patent WO2015033301A1. Structurally similar to CA-170, it belongs to a class of compounds that block the PD-1/PD-L1 immune checkpoint. Its mechanism involves restoring T-cell function by preventing PD-1/PD-L1-mediated suppression. The compound is intended for research use only and has not received regulatory approval for clinical use.

C13H22N6O7

374.3498

374.154

C, 41.71; H, 5.92; N, 22.45; O, 29.92

1673560-66-1

PD-1-IN-17 TFA

131998882

White to off-white solid powder

-6.7

7

10

10

26

507

4

O1C(C([H])(C([H])(C([H])([H])[H])O[H])N([H])[H])=NN=C1C([H])(C([H])([H])C([H])([H])C(N([H])[H])=O)N([H])C(N([H])C([H])(C(=O)O[H])C([H])([H])O[H])=O

SRNQPYBWRIETFQ-XKBZYTNZSA-N

InChI=1S/C13H22N6O7/c1-5(21)9(15)11-19-18-10(26-11)6(2-3-8(14)22)16-13(25)17-7(4-20)12(23)24/h5-7,9,20-21H,2-4,15H2,1H3,(H2,14,22)(H,23,24)(H2,16,17,25)/t5-,6+,7+,9+/m1/s1

(2S)-2-[[(1S)-4-amino-1-[5-[(1S,2R)-1-amino-2-hydroxypropyl]-1,3,4-oxadiazol-2-yl]-4-oxobutyl]carbamoylamino]-3-hydroxypropanoic acid

PD-1-IN-17; PD-1-IN-17 free base; PD1-IN-1; (2S)-2-[[(1S)-4-amino-1-[5-[(1S,2R)-1-amino-2-hydroxypropyl]-1,3,4-oxadiazol-2-yl]-4-oxobutyl]carbamoylamino]-3-hydroxypropanoic acid; (S)-2-(3-((S)-4-Amino-1-(5-((1S,2R)-1-amino-2-hydroxypropyl)-1,3,4-oxadiazol-2-yl)-4-oxobutyl)ureido)-3-hydroxypropanoic acid; CHEMBL4458504; SCHEMBL19450456;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~125 mg/mL (~333.91 mM)
H2O : ≥ 100 mg/mL (~267.13 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.56 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.56 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.56 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.

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 (Please use freshly prepared in vivo formulations for optimal results.)