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| Targets |
Programmed cell death 1 (PD-1) / Programmed cell death ligand 1 (PD-L1) interaction - inhibitor (IC50 = 12.5 nM).
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| ln Vitro |
Compound NP19, also known as PD-1/PD-L1-IN-NP19 (0.37-10 μM; 72 h), significantly and dose-dependently increases the amount of IFN-γ produced by T cells co-cultured with tumor cells[1]. The activity of PD-1/PD-L1-IN-NP19 to inhibit mice is substantially decreased. inhibition of the human PD1/PD-L1 connection (IC50=12.5 nM) is smaller than that of the PD-1/PD-L1 interaction, with an IC50 in the micromolar range (>1µM)[1]. When PD-1/PD-L1-IN-NP19 (10 µM; 48 h) is added to A549, MCF-7, and B16-F10 cells, no discernible cytotoxic effects are seen[1].
In vitro studies demonstrate that NP19 is a potent inhibitor of the PD-1/PD-L1 protein-protein interaction with an IC50 of 12.5 nM in homogeneous time-resolved fluorescence (HTRF) binding assays. PD-1/PD-L1 is a key immune checkpoint pathway exploited by tumors to evade immune surveillance. By disrupting PD-1/PD-L1 binding, NP19 enhances T cell activation and restores antitumor immune responses. The compound activates the immune microenvironment in tumors, which may contribute to its antitumor effects. NP19 serves as a valuable tool for investigating checkpoint blockade mechanisms and for exploring alternatives to monoclonal antibody therapies. |
| ln Vivo |
Compound NP19, also known as PD-1/PD-L1-IN-NP19 (25-100 mg/kg; intragastric gavage once daily for 15 days) significantly suppresses the formation of melanoma tumors in mice[1]. In an H22 hepatoma mouse model, PD-1/PD-L1-IN-NP19 (25 mg/kg; intraperitoneally every day for 14 days) exhibits strong anticancer activity with a tumor growth inhibition (TGI) of 76.5% and is well tolerated[1]. Rats' half time (t1/2=1.5±0.5 h), clearance rate (CL=0.9±0.2 L/h/kg), and apparent distribution volume (Vss = 2.1±0.5 L/kg) are displayed in PD-1/PD-L1-IN-NP19 (1 mg/kg; iv)[1]. Rats' oral absorption (Tmax=0.6±0.2 h), long half-life (t1/2=10.9±7.7 h), and oral bioavailability (F=5%) are demonstrated by PD-1/PD-L1-IN-NP19 (10 mg/kg; po)[1].
In vivo studies have demonstrated that NP19 reduces tumor growth in various cancer models. In the B16/F10 murine melanoma model, treatment with NP19 at doses of 25, 50, and 100 mg/kg resulted in significant tumor growth inhibition. Similar antitumor efficacy was observed in the H22 murine hepatocellular carcinoma model. The compound's ability to activate the immune microenvironment in tumors, as evidenced by increased T cell infiltration and activation, contributes to its antitumor effects. These findings support the potential of NP19 as a small-molecule alternative to antibody-based PD-1/PD-L1 checkpoint inhibitors for cancer immunotherapy. |
| Enzyme Assay |
PD-1/PD-L1 binding inhibition assays are performed using homogeneous time-resolved fluorescence (HTRF) technology. Recombinant PD-1 and PD-L1 proteins, labeled with appropriate donor and acceptor fluorophores, are incubated with varying concentrations of NP19. The proximity of the two proteins brings the donor and acceptor into close proximity, resulting in fluorescence resonance energy transfer (FRET). Inhibition of the protein-protein interaction by NP19 reduces FRET signal. IC50 values are calculated from dose-response curves. Binding affinity is confirmed using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC).
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| Cell Assay |
Cellular assays for NP19 typically involve co-culture systems of immune cells and cancer cells to assess T cell activation. Primary human T cells or T cell lines are co-cultured with cancer cells that express PD-L1 in the presence of NP19 at various concentrations. T cell activation is assessed by measuring interleukin-2 (IL-2) production, interferon-γ (IFN-γ) production, or T cell proliferation (by CFSE dilution or [3H]-thymidine incorporation). The compound's ability to restore T cell function in the presence of PD-L1-expressing cancer cells is quantified, and EC50 values are calculated. Cytotoxicity against mammalian cells is assessed in parallel.
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| Animal Protocol |
Animal/Disease Models: balb/c (Bagg ALBino) mouse (6-8 weeks) with B16-F10 melanoma tumors[1]
Doses: 25 , 50, 100 mg/kg Route of Administration: intragastric (po) gavage one time/day for 15 days Experimental Results: Inhibited the growth of melanoma tumors up to 51.1, 75 and 80.9% at dose of 25, 50, 100 mg/kg, respectively. demonstrated normal physical activity and increased body weights slightly. Animal/Disease Models: Male SD (Sprague-Dawley) rats[1] Doses: 1 mg/kg for iv and 10 mg/kg for po (pharmacokinetic/PK Analysis) Route of Administration: Iv and po Experimental Results: Iv: t1/2=1.5 h; Cmax=1751 μg/L; CL=0.9 L/h/kg. Po: t1/2=10.9 h; Cmax=69.5 μg/L; CL=23.1 L/h/kg. In vivo efficacy of NP19 is evaluated in syngeneic mouse models of cancer, including the B16/F10 murine melanoma model and the H22 murine hepatocellular carcinoma model. Mice are implanted with cancer cells and then treated with NP19 via oral or intraperitoneal administration at doses ranging from 25 to 100 mg/kg. Tumor volume is monitored over time. Immune cell infiltration and activation in tumors are assessed by flow cytometry (for T cell markers, activation markers, and exhaustion markers) and immunohistochemistry. Cytokine levels in serum and tumor tissues are measured by ELISA. Survival is monitored in some studies. These studies help establish the compound's potential as a cancer immunotherapeutic. |
| ADME/Pharmacokinetics |
Pharmacokinetic properties of NP19 have been characterized to support its use as a research tool and potential therapeutic agent. The compound's molecular weight of 555.06 and chemical properties influence its absorption, distribution, metabolism, and excretion (ADME) characteristics. Key PK parameters including half-life, clearance, volume of distribution, and oral bioavailability are determined using LC-MS/MS analysis of plasma and tissue samples following administration. The compound's ability to reach tumor tissues and maintain therapeutic concentrations is important for its antitumor efficacy. The compound's favorable pharmacokinetic profile supports its potential for further development.
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| Toxicity/Toxicokinetics |
Toxicological evaluation of NP19 is typically conducted in parallel with efficacy studies in animal models. Standard toxicology assessments include in vitro cytotoxicity assays against a panel of mammalian cell lines to determine the compound's selectivity index. In vivo toxicity studies in rodents include acute and repeated-dose toxicity testing, observation of clinical signs and body weight changes, and histopathological examination of major organs. As an immune checkpoint inhibitor that activates T cells, potential immune-related adverse events such as autoimmunity, cytokine release syndrome, and organ inflammation are carefully monitored. The compound's safety profile is established to define the therapeutic window for research and development.
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| References | |
| Additional Infomation |
NP19 is a research tool compound and potential therapeutic agent used for studying PD-1/PD-L1 immune checkpoint blockade and for developing small-molecule alternatives to antibody-based cancer immunotherapy. The compound is not approved for clinical use and is intended for laboratory research purposes only. Its mechanism of action involves inhibition of the PD-1/PD-L1 protein-protein interaction, which enhances T cell activation and restores antitumor immune responses. This compound is valuable for investigating checkpoint blockade mechanisms, for studying the tumor immune microenvironment, and for exploring the therapeutic potential of small-molecule PD-1/PD-L1 inhibitors in cancer.
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| Molecular Formula |
C33H31CLN2O4
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|---|---|
| Molecular Weight |
555.0632
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| Exact Mass |
554.197
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| CAS # |
2377916-66-8
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| PubChem CID |
154573771
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| Appearance |
White to off-white solid powder
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| LogP |
4.5
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
40
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| Complexity |
853
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1=C(C([H])=C(C(=C1[H])C([H])([H])N([H])C(C(=O)O[H])(C([H])([H])[H])C([H])([H])[H])OC([H])([H])C1C([H])=C([H])C([H])=C(C#N)C=1[H])OC([H])([H])C1C([H])=C([H])C([H])=C(C2C([H])=C([H])C([H])=C([H])C=2[H])C=1C([H])([H])[H]
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| InChi Key |
YZPKZNFXWGTUGO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C33H31ClN2O4/c1-22-26(13-8-14-28(22)25-11-5-4-6-12-25)21-40-31-17-30(39-20-24-10-7-9-23(15-24)18-35)27(16-29(31)34)19-36-33(2,3)32(37)38/h4-17,36H,19-21H2,1-3H3,(H,37,38)
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| Chemical Name |
2-((5-Chloro-2-((3-cyanobenzyl)oxy)-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)-2-methylpropanoic acid
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| Synonyms |
NP19 NP-19 NP 19
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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 : ~2 mg/mL (~3.60 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 | 1.8016 mL | 9.0080 mL | 18.0161 mL | |
| 5 mM | 0.3603 mL | 1.8016 mL | 3.6032 mL | |
| 10 mM | 0.1802 mL | 0.9008 mL | 1.8016 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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