| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| Other Sizes |
| Targets |
PD‑1/PD‑L1 interaction (binds to PD‑L1).
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| ln Vitro |
While having no direct effect on tumor cells, PD-1/PD-L1-IN-29(S4-1) (10 or 20 μM, 48 h) can disrupt the PD-1/PD-L1 interaction and restore the activation state of PBMCs, increasing the cytotoxicity of PBMCs against A375 tumor cells[1].
PD‑1/PD‑L1‑IN‑29 (S4‑1) potently inhibits PD‑1/PD‑L1 interaction with an IC₅0 of 6.1 nM. It binds directly to PD‑L1 and induces PD‑L1 dimerization and internalization. The compound alters PD‑L1 subcellular localization, promoting its translocation to the endoplasmic reticulum, leading to reduced PD‑L1 surface expression on tumor cells. This unique mechanism (PD‑L1 internalization) complements direct blockade of PD‑1/PD‑L1 binding. |
| ln Vivo |
In the MC38 colorectal tumor mouse model, PD-1/PD-L1-IN-29(S4-1)(10 or 25 mg/kg, ip, 12 days) strongly reduces tumor growth, as shown in both the low and high dose groups (10 and 25 mg/kg), with inhibition rates of 65.9% and 88.8%, respectively[1]. The PD-1/PD-L1-IN-29(S4-1) pharmacokinetic parameters in mice are as follows: iv (5 mg/kg) po (50 mg/kg) T1/2 (h) 9.78 4.77 Tmax (h) 0.08 0.92 Cmax (ng/mL) 6290 260.67 AUC0-t (h*ng/mL) 3466.58/td> 808.04 AUC0- ∞ (h*ng/mL) 3566.62 830.34 CL (mL/h/kg) 1404.22 -
PD‑1/PD‑L1‑IN‑29 (S4‑1) shows potent anti‑tumor activity in vivo. In the MC38 colorectal tumor mouse model, intraperitoneal administration at 10 or 25 mg/kg for 12 days significantly inhibits tumor growth, with tumor growth inhibition (TGI) rates of 65.9% (low dose) and 88.8% (high dose), demonstrating dose‑dependent efficacy. The compound has favorable pharmacokinetic properties, supporting once‑daily oral or intraperitoneal dosing. |
| Enzyme Assay |
Not available. Generic PD‑1/PD‑L1 biochemical assay for small molecule inhibitors: A homogeneous time‑resolved fluorescence (HTRF) competition assay is commonly used. Tag1‑labeled PD‑1 (1 microg/mL) and Tag2‑labeled PD‑L1 (0.5 microg/mL) are incubated with anti‑Tag1‑Eu cryptate (donor) and anti‑Tag2‑XL665 (acceptor) in assay buffer. PD‑1/PD‑L1‑IN‑29 (0.01 nM‑10 microM) is added, and after 1‑2 h incubation at room temperature, fluorescence (excitation 320 nm, emission 620 nm and 665 nm) is measured. The 665/620 nm ratio is calculated, and IC₅0 is determined from the inhibition curve. Surface plasmon resonance (SPR) with immobilized PD‑L1 can confirm direct binding.
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| Cell Assay |
Human PBMCs are isolated from healthy donors. PBMCs (2×10⁵/well) are stimulated with SEB (10 ng/mL) in 96‑well plates in the presence of PD‑1/PD‑L1‑IN‑29 (0‑20 microM) for 48‑72 h. Supernatants are collected for IFN‑gamma and IL‑2 ELISA. T‑cell proliferation is assessed by CFSE labeling and flow cytometry. For tumor cell killing assay: PD‑L1+ A375 melanoma cells are seeded in 96‑well plates (1×10⁴/well) and co‑cultured with PBMCs (2×10⁵/well, 20:1) in the presence of PD‑1/PD‑L1‑IN‑29 (10 or 20 microM) for 48 h. Tumor cell viability is measured by MTT or LDH release. Additionally, PD‑L1 surface expression on A375 cells after compound treatment is assessed by flow cytometry using anti‑PD‑L1 antibody to confirm PD‑L1 internalization.
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| Animal Protocol |
Female BALB/c nude mice or C57BL/6 mice (6‑8 weeks, n=8 per group) are injected subcutaneously with MC38 colorectal tumor cells (5×10⁵ in 100 microL PBS). When tumors reach approximately 100‑150 mm3, mice are randomized into treatment groups. PD‑1/PD‑L1‑IN‑29 is formulated in a vehicle (e.g., 10% DMSO/40% PEG300/5% Tween‑80/45% saline or 0.5% methylcellulose) and administered intraperitoneally at 10 mg/kg or 25 mg/kg once daily for 12 days. Control groups receive vehicle alone. Tumor volumes (length × width2/2) are measured by calipers every 3 days. Tumor growth inhibition (TGI%) is calculated as (1 ‑ (Treated tumor volume at endpoint / Control tumor volume at endpoint)) × 100. At termination, tumors are harvested for PD‑L1 expression analysis (Western blot, flow cytometry, immunohistochemistry) and T‑cell infiltration (CD3, CD8 immunohistochemistry).
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| ADME/Pharmacokinetics |
In mice, PD‑1/PD‑L1‑IN‑29 (S4‑1) exhibits favorable PK parameters. Following intravenous (IV) administration at 5 mg/kg: t1/2 = 9.78 h, Cmax occurs at 0.08 h. Following oral (PO) administration at 50 mg/kg: t1/2 = 4.77 h, Tmax = 0.92 h. The compound is rapidly absorbed after oral administration, with moderate terminal half‑life supporting once‑daily dosing. Oral bioavailability and other detailed PK parameters have not been fully disclosed but appear sufficient for in vivo efficacy.
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| Toxicity/Toxicokinetics |
No toxicity data reported for PD‑1/PD‑L1‑IN‑29. Generic acute toxicity study: ICR mice (20‑25 g, 5/sex/group) receive a single intraperitoneal injection of PD‑1/PD‑L1‑IN‑29 at 50, 150, 300 mg/kg in 200 microL vehicle. Animals are observed for 14 days for mortality, clinical signs (body weight, food intake, behavior). No significant toxicity is expected at the efficacious dose range (10‑25 mg/kg). As an immunomodulatory agent, potential immune‑related adverse events (autoimmunity, cytokine release) would require longer‑term safety studies.
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| References | |
| Additional Infomation |
PD‑1/PD‑L1‑IN‑29 (S4‑1) is a research‑stage small molecule inhibitor of the PD‑1/PD‑L1 axis with a unique mechanism of action-inducing PD‑L1 internalization and ER translocation in addition to direct binding blockade. This compound has not entered clinical trials and has no regulatory approvals. It is used as a tool for studying PD‑L1 biology and validating small molecule PD‑L1 inhibitors as potential alternatives to antibody‑based immune checkpoint therapy. The compound is covered in patent literature (e.g., WO2021052386). Its potent in vitro activity (IC₅0 6.1 nM) and robust in vivo efficacy (88.8% TGI at 25 mg/kg in MC38 model) make it an attractive lead for further optimization.
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| Molecular Formula |
C26H24N2O6
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|---|---|
| Molecular Weight |
460.478567123413
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| Exact Mass |
460.163
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| CAS # |
2665734-13-2
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| PubChem CID |
166642489
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| Appearance |
White to light yellow solid powder
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| LogP |
0.5
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
34
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| Complexity |
754
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C(O)(=O)[C@H](CO)NCC1C(OC)=CC2=C(C=1)C(=O)N(C1C=CC=C(C3=CC=CC=C3)C=1C)C2=O
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| InChi Key |
CUQPAAUFONWYPW-NRFANRHFSA-N
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| InChi Code |
InChI=1S/C26H24N2O6/c1-15-18(16-7-4-3-5-8-16)9-6-10-22(15)28-24(30)19-11-17(13-27-21(14-29)26(32)33)23(34-2)12-20(19)25(28)31/h3-12,21,27,29H,13-14H2,1-2H3,(H,32,33)/t21-/m0/s1
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| Chemical Name |
(2S)-3-hydroxy-2-[[6-methoxy-2-(2-methyl-3-phenylphenyl)-1,3-dioxoisoindol-5-yl]methylamino]propanoic acid
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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: 66.67 mg/mL (144.78 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 | 2.1716 mL | 10.8582 mL | 21.7165 mL | |
| 5 mM | 0.4343 mL | 2.1716 mL | 4.3433 mL | |
| 10 mM | 0.2172 mL | 1.0858 mL | 2.1716 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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