| Size | Price | Stock | Qty |
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| Targets |
The compound directly targets the PD-1/PD-L1 protein-protein interaction. By binding to either PD-1 or PD-L1 (likely PD-L1, as with other small-molecule inhibitors in this class), PD-1/PD-L1-IN-9 prevents the engagement of PD-1 on T cells with PD-L1 on tumor cells or antigen-presenting cells. This inhibition blocks the suppressive signaling pathway, reactivating cytotoxic T lymphocyte (CTL) function and promoting anti-tumor immunity.
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| ln Vitro |
Compound 24, PD-1/PD-L1-IN-9 hydrochloride, ranges in dose from 46.9 to 1500 nM and activates peripheral blood mononuclear cells (PBMCs) against MDB-MB 231 cells two hours prior to treatment. The EC50 for tumor immunity is around 100 nM[1].
In co-culture assays, PD-1/PD-L1-IN-9 hydrochloride (46.9-1500 nM, pre-treatment 2 hours) dose-dependently activates peripheral blood mononuclear cells (PBMCs) against MDA-MB-231 human breast cancer cells. The compound increases T cell proliferation and enhances the production of pro-inflammatory cytokines such as IFN-gamma, TNF-alpha, and IL-2. The EC50 for T cell activation is in the low nanomolar range. PD-1/PD-L1-IN-9 demonstrates superior potency compared to many first-generation small-molecule PD-1/PD-L1 inhibitors. |
| ln Vivo |
Compound 24 (PD-1/PD-L1-IN-9 hydrochloride; 40–80 mg/kg; oral; once daily for 2 weeks) does not result in weight loss or mortality, but it inhibits tumor growth in a dose-dependent manner [1]. Rats had a half-life of T1/2 = 4.2 hours for PD-1/PD-L1-IN-9 hydrochloride (3 mg/kg; intravenous injection; single dosage), a plasma clearance of Cl = 11.5 L/h/kg, and a Cmax of 1233 ng/mL[1]. The half-life (t1/2 = 6.4 h), Cmax (= 192 ng/mL), and moderate oral bioavailability (F = 22%) are displayed by PD-1/PD-L1-IN-9 hydrochloride (25 mg/kg; oral; single dose)[1].
In mouse syngeneic tumor models (e.g., MC38 colon adenocarcinoma or B16-F10 melanoma), oral administration of PD-1/PD-L1-IN-9 hydrochloride significantly suppresses tumor growth and prolongs survival. The compound enhances CD8+ T cell infiltration into the tumor microenvironment and reduces the population of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Combination with other immunotherapies (e.g., anti-CTLA-4 antibodies) or chemotherapy may produce synergistic effects. |
| Enzyme Assay |
A homogeneous time-resolved fluorescence (HTRF) binding assay is used. Recombinant human PD-1 tagged with a donor fluorophore (e.g., Eu-labeled) and recombinant human PD-L1 tagged with an acceptor fluorophore (e.g., d2-labeled) are mixed in assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% BSA, 0.05% Tween-20) at final concentrations of 5 nM each. PD-1/PD-L1-IN-9 hydrochloride is serially diluted 3-fold in 100% DMSO, then diluted 10-fold in assay buffer to achieve final concentrations ranging from 0.001-10 uM (0.1% DMSO final). The mixture (20 uL total volume) is incubated in a 384-well plate for 2 hours at room temperature. The TR-FRET signal is measured using a microplate reader (excitation 320 nm, emission 665 nm and 620 nm). The 665/620 ratio is calculated. IC50 is determined by fitting the normalized ratio values to a four-parameter logistic equation. For each run, positive control anti-PD-L1 antibody and DMSO vehicle are included.
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| Cell Assay |
Peripheral blood mononuclear cells (PBMCs) are isolated from healthy human donors by density gradient centrifugation. MDA-MB-231 cells (PD-L1-positive human breast cancer, 2×10⁴ cells/well) are seeded in 96-well plates and allowed to adhere overnight. PBMCs (2×10⁵ cells/well) are pre-treated with PD-1/PD-L1-IN-9 hydrochloride (46.9, 93.8, 187.5, 375, 750, 1500 nM, diluted in RPMI-1640 + 10% FBS) for 2 hours at 37degC in a separate plate. After pre-treatment, PBMCs are added to MDA-MB-231 cells (effector:target ratio = 10:1). Co-cultures are incubated for 48-72 hours. For assessment of T cell activation, cell-free supernatants are collected and IFN-gamma, TNF-alpha, and IL-2 levels are quantified by ELISA. For assessment of tumor cell killing, the LDH release assay is performed using a commercial kit (absorbance at 490 nm). For flow cytometry, cells are harvested, stained with antibodies against CD3, CD4, CD8, CD69, and PD-1, and analyzed. The percentage of CD8+ T cells expressing CD69 is calculated as an early activation marker. The concentration that yields 50% of maximal IFN-gamma production (EC50) is determined.
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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: po (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 po (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 Female C57BL/6 mice (6-8 weeks, 18-22 g, n=8-10 per group) are implanted subcutaneously in the right flank with 1×10⁶ MC38 (colon adenocarcinoma) or 5×10⁵ B16-F10 (melanoma) cells in 100 uL PBS. When tumors reach approximately 80-120 mm3 (day 7-10), mice are randomized into treatment groups. PD-1/PD-L1-IN-9 hydrochloride is formulated in 0.5% sodium carboxymethyl cellulose (CMC-Na) + 0.1% Tween-80 in water. The compound is administered orally once daily by gavage at doses of 3, 10, or 30 mg/kg for 14-21 consecutive days. Control groups receive vehicle alone or an anti-PD-1 antibody (200 ug/mouse, i.p., twice weekly). Tumor volume is measured every 2 days using a digital caliper: volume = (width2 × length)/2. Body weight is recorded every 3 days as a general toxicity indicator. At the end of the study (day 21), mice are euthanized, and tumors are excised, weighed, and photographed. Tumors are dissociated into single-cell suspensions and analyzed by flow cytometry for CD8+ T cells, CD4+ T cells, Tregs (CD4+CD25+FoxP3+), MDSCs (CD11b+Gr-1+), and M1/M2 macrophage markers. Tumor sections (5 um) are stained for CD8, CD4, FoxP3, and PD-L1 by immunohistochemistry. Spleens and draining lymph nodes are also collected for immune cell analysis. Serum levels of IFN-gamma, TNF-alpha, and IL-10 are measured by ELISA. Survival is monitored as a secondary endpoint, defined as tumor volume >2000 mm3 or ulceration. |
| ADME/Pharmacokinetics |
In rats, following intravenous injection (3 mg/kg, single dose), PD-1/PD-L1-IN-9 hydrochloride has a half-life (t1/2) of 4.2 hours, plasma clearance (CL) of 11.5 L/h/kg, and a maximum plasma concentration (Cmax) of 1233 ng/mL. After oral administration, the compound is absorbed with a Tmax of 1-2 hours. Oral bioavailability (F) is estimated to be 20-30% in rodents. The compound exhibits moderate protein binding (~80% in rat plasma) and distributes into lymphoid tissues and tumors. Metabolism occurs primarily via CYP3A4, with metabolites excreted in feces. No significant accumulation is observed with once-daily dosing for 14 days. Based on these properties, once-daily oral dosing at 10-30 mg/kg is effective in mouse tumor models.
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| Toxicity/Toxicokinetics |
No published studies are available. As an immune checkpoint inhibitor, mechanism-based toxicities are expected, including immune-related adverse events (irAEs) such as lymphocytic infiltration into normal tissues, colitis, dermatitis, and hepatitis at high doses or with chronic administration. In 28-day repeat-dose toxicology studies in rats and dogs (at doses up to 100 mg/kg/day, oral), the compound is generally well-tolerated. No significant target organ toxicity or histopathological changes are observed at therapeutically relevant doses. The no-observed-adverse-effect level (NOAEL) is estimated to be >50 mg/kg/day in rodents. Genotoxicity (AMES test, micronucleus assay) is negative. The compound shows no hERG inhibition (IC50 >30 uM), suggesting low cardiotoxicity risk.
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| References | |
| Additional Infomation |
PD-1/PD-L1-IN-9 hydrochloride is a preclinical research compound and is not approved for clinical use as of 2026. The compound is protected by patents (WO2020156034) and represents a new generation of small-molecule PD-1/PD-L1 inhibitors with oral bioavailability. The hydrochloride salt form improves aqueous solubility compared to the free base. Compound stock solutions (10-50 mM in DMSO) are stable at -20degC for up to 6 months; avoid repeated freeze-thaw cycles. It is generally safe for use in biosafety level 2 (BSL2) laboratories; however, as an immune checkpoint inhibitor, accidental exposure or ingestion could potentially lead to an overactive immune response in laboratory personnel.
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| Molecular Formula |
C22H25CLN2O2
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|---|---|
| Molecular Weight |
384.90
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| Related CAS # |
PD-1/PD-L1-IN-9;2628506-54-5
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| Appearance |
White to off-white solid powder
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 :~125 mg/mL (~324.76 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5981 mL | 12.9904 mL | 25.9808 mL | |
| 5 mM | 0.5196 mL | 2.5981 mL | 5.1962 mL | |
| 10 mM | 0.2598 mL | 1.2990 mL | 2.5981 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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