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| Targets |
IC50: 3.8 nM (PD-1/PD-L1)[1]
PD-L1 (Programmed death-ligand 1) is a transmembrane protein expressed on the surface of tumor cells and antigen-presenting cells. The interaction between PD-L1 on tumor cells and PD-1 on T cells leads to T cell exhaustion, anergy, and immune evasion. PD-1/PD-L1-IN-9 binds to dimeric PD-L1 with high affinity and inhibits the PD-1/PD-L1 interaction with an IC50 of 3.8 nM. The compound blocks the PD-1/PD-L1 immune checkpoint axis, thereby enhancing the ability of immune cells to recognize and kill tumor cells. The aminoethanol tail of the compound forms critical hydrogen bonds with AAsp122 and BGln66 on the PD-L1 dimer, stabilizing the binding interaction. |
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| ln Vitro |
Compound 24 (PD-1/PD-L1-IN-9; 46.9-1500 nM; 2 h pretreatment) dose-dependently stimulates the anti-tumor immunity of peripheral blood mononuclear cells (PBMCs) against MDB-MB 231 cells; the EC50 is roughly 100 nM[1].
In vitro, PD-1/PD-L1-IN-9 inhibits the PD-1/PD-L1 interaction with an IC50 of 3.8 nM. In co-culture systems, the compound (46.9-1500 nM; pretreated for 2 h) dose-dependently activates the antitumor immunity of PBMCs against MDA-MB-231 breast cancer cells, with an EC50 of approximately 100 nM. The compound enhances the killing activity of immune cells against tumor cells, as measured by increased tumor cell lysis and cytokine production. PD-1/PD-L1-IN-9 shows no significant cytotoxicity against healthy cells at concentrations up to 1500 nM. |
| ln Vivo |
PD-1/PD-L1-IN-9 (Compound 24; 40-80 mg/kg; PO; once daily for 2 weeks) does not result in weight loss or death[1], but it does limit tumor growth in a dose-dependent manner. PD-1/PD-L1-IN-9 (3 mg/kg; intravenous injection; single dosage) had a half-life of 4.2 hours, a plasma clearance of 11.5 L/h/kg, and a Cmax of 1233 ng/mL in rats[1]. Rats with Cmax = 192 ng/mL show moderate oral bioavailability (F = 22%) and half-life (t1/2 = 6.4 h) for PD-1/PD-L1-IN-9 (25 mg/kg; oral; single dose)[1].
In vivo, PD-1/PD-L1-IN-9 demonstrates significant antitumor activity in a CT26 mouse colon carcinoma model. Oral administration of the compound at 40-80 mg/kg once daily for 2 weeks inhibits tumor growth in a dose-dependent manner. The compound does not cause any body weight loss or mortality of mice, indicating a favorable safety profile at efficacious doses. The antitumor efficacy of PD-1/PD-L1-IN-9 is comparable to that of other small-molecule PD-L1 inhibitors and supports its further development as an oral immune checkpoint inhibitor. |
| Enzyme Assay |
The in vitro enzyme/receptor binding (cell-free) assay for PD-1/PD-L1-IN-9 typically involves a homogeneous time-resolved fluorescence (HTRF) assay or surface plasmon resonance (SPR) to measure the inhibition of PD-1/PD-L1 binding. In the HTRF assay, tagged PD-1 and PD-L1 proteins are incubated with varying concentrations of the compound, and the fluorescent signal is measured to determine the IC50. SPR is used to measure the binding affinity of the compound to immobilized PD-L1 protein, providing kinetic parameters including association rate (ka), dissociation rate (kd), and equilibrium dissociation constant (Kd).
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| Cell Assay |
In vitro cellular assays for PD-1/PD-L1-IN-9 use human peripheral blood mononuclear cells (PBMCs) co-cultured with PD-L1-expressing tumor cells such as MDA-MB-231 breast cancer cells. The compound is added at concentrations ranging from 46.9 to 1500 nM for 2 hours. The activation of PBMCs is assessed by measuring the release of IFN-γ and other cytokines using ELISA. Tumor cell killing is evaluated by measuring the viability of tumor cells using lactate dehydrogenase (LDH) release assays or flow cytometry-based cytotoxicity assays. The EC50 for immune activation is approximately 100 nM.
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| Animal Protocol |
Animal/Disease Models: Male balb/c (Bagg ALBino) mouse (5-6 weeks) were inoculated CT26 cells[1]
Doses: 40 mg/kg, 80 mg/kg Route of Administration: Oral gavage; one time/day, for 2 weeks Experimental Results: Dramatically diminished the final tumor weight, with TGI values of 60 and 67% at the dose of 40 and 80 mg/kg, respectively. Animal/Disease Models: pharmacokinetic/PK analysis in SD (Sprague-Dawley) rats[1] Doses: 3 mg/kg and 25 mg/kg Route of Administration: intravenous (iv) injection or oral gavage; single dose Experimental Results: Route Dose (mg/kg) AUC(0-t) (ng·h/mL) Cmax (ng/mL) t1/2 (h) Tmax Cl (L·h /kg) Vz (L/kg) F (%) iv 3 430.5 1233 4.2 0.03 11.5 78.6 / po 25 787.4 192 6.4 0.69 28.8 249.3 22 In vivo animal experiments for PD-1/PD-L1-IN-9 are conducted in CT26 tumor-bearing mice. The compound is administered orally once daily for 2 weeks at doses of 40, 60, or 80 mg/kg. Tumor volumes are measured every 2-3 days using calipers, and tumor growth inhibition is calculated relative to vehicle-treated controls. Body weights are monitored to assess tolerability. At the end of the study, tumors are excised for histopathological analysis and immune cell infiltration is assessed by immunohistochemistry for CD8+ T cells and other immune markers. |
| ADME/Pharmacokinetics |
In rats, intravenous administration of PD-1/PD-L1-IN-9 (3 mg/kg, single i.v.) exhibits a half-life (t1/2) of 4.2 hours, plasma clearance (Cl) of 11.5 L/h/kg, and maximum plasma concentration (Cmax) of 1233 ng/mL. Oral administration (25 mg/kg, single p.o.) shows moderate oral bioavailability (F=22%), a half-life of 6.4 hours, and Cmax of 192 ng/mL. The compound has a molecular weight of 348.44 g/mol and exhibits high solubility in DMSO (100 mg/mL).
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| Toxicity/Toxicokinetics |
In preclinical studies, PD-1/PD-L1-IN-9 does not cause body weight loss or mortality in mice at efficacious doses of 40-80 mg/kg, indicating a favorable safety profile. No significant off-target toxicities have been reported. The compound is well tolerated upon oral administration, and no dose-limiting toxicities were observed in the dose range studied. The favorable safety profile of PD-1/PD-L1-IN-9 supports its continued development as an oral immune checkpoint inhibitor for cancer immunotherapy.
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| References | |
| Additional Infomation |
PD-1/PD-L1-IN-9 (CAS: 2628506-54-5) is a research compound developed as an orally available small-molecule inhibitor of the PD-1/PD-L1 immune checkpoint pathway. The compound has a molecular formula of C22H24N2O2 and a molecular weight of 348.44. It is available as the hydrochloride salt with a molecular weight of 384.9 g/mol. PD-1/PD-L1-IN-9 is not approved for human use and is intended for research purposes only. The compound represents a promising lead for the development of oral PD-1/PD-L1 inhibitors as an alternative to monoclonal antibody-based immunotherapies.
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| Molecular Formula |
C22H24N2O2
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|---|---|
| Molecular Weight |
348.438165664673
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| Exact Mass |
348.183
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| CAS # |
2628506-54-5
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| Related CAS # |
PD-1/PD-L1-IN-9 hydrochloride
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| PubChem CID |
155925913
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| Appearance |
White to off-white solid powder
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| LogP |
3.5
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
26
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| Complexity |
400
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O(C)C1=C(C=CC(C2C=CC=C(C3C=CC=CC=3)C=2C)=N1)CNCCO
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| InChi Key |
ANLIWBJMRQBWBM-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H24N2O2/c1-16-19(17-7-4-3-5-8-17)9-6-10-20(16)21-12-11-18(15-23-13-14-25)22(24-21)26-2/h3-12,23,25H,13-15H2,1-2H3
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| Chemical Name |
2-[[2-methoxy-6-(2-methyl-3-phenylphenyl)pyridin-3-yl]methylamino]ethanol
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
DMSO: 100 mg/mL (286.99 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.17 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 (7.17 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (7.17 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8699 mL | 14.3497 mL | 28.6993 mL | |
| 5 mM | 0.5740 mL | 2.8699 mL | 5.7399 mL | |
| 10 mM | 0.2870 mL | 1.4350 mL | 2.8699 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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