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A839977 (A-839977) is a novel and potent P2X7R antagonist with antihyperalgesic and anti-inflammatory activity. The pro-inflammatory cytokine interleukin-1beta (IL-1beta) has been implicated in both inflammatory processes and nociceptive neurotransmission. Activation of P2X7 receptors is the mechanism by which ATP stimulates the rapid maturation and release of IL-1beta from macrophages and microglial cells. Recently, selective P2X7 receptor antagonists have been shown to reduce inflammatory and neuropathic pain in animal models.
A-839977 is a potent, selective, and competitive antagonist of the P2X7 receptor, a ligand-gated ion channel involved in inflammatory and neuropathic pain. It demonstrates high potency across species, with IC50 values of 20 nM for the human receptor, 42 nM for the rat receptor, and 150 nM for the mouse receptor in calcium flux assays. A-839977 also effectively blocks downstream P2X7-mediated functions, inhibiting BzATP-stimulated IL-1β release (IC50 = 37 nM) and YO-PRO dye uptake (IC50 = 7 nM) in differentiated human THP-1 cells, confirming its on-target activity. In vivo, A-839977 is CNS-penetrant (brain-to-plasma ratio of 0.15–0.25) and produces dose-dependent antihyperalgesic effects in animal models of inflammatory pain (e.g., CFA-induced), with ED50 values of 40 μmol/kg (i.p.) in mice and 100 μmol/kg (i.p.) in rats. Its analgesic effect is lost in IL-1αβ knockout mice, demonstrating a mechanism dependent on IL-1 signaling. In a rat model of cancer-induced bone pain, spinal administration of A-839977 (0.4–1.2 mg/kg) reduces neuronal responses to high-intensity mechanical and thermal stimuli, while systemic administration (40 mg/kg, i.p.) alleviates both early- and late-stage pain behaviors without affecting motor coordination. A-839977 is a valuable tool for studying P2X7 receptor function in pain and neuroinflammation.| Targets |
P2X7 receptor – IC50 = 20 nM (human P2X7, calcium influx); IC50 = 42 nM (rat P2X7, calcium influx); IC50 = 150 nM (mouse P2X7, calcium influx); IC50 = 37 nM (BzATP-stimulated IL-1β release in differentiated human THP-1 cells); IC50 = 7 nM (BzATP-stimulated YO-PRO uptake in differentiated human THP-1 cells); pIC50 = 7.67 ± 0.04 (human), 7.36 ± 0.02 (rat), 6.83 ± 0.03 (mouse) [1]; pA2 = 8.1 (competitive antagonist) [1]
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| ln Vitro |
In Vitro: A-839977 potently blocked BzATP-evoked changes in intracellular calcium concentrations in 1321N1 cells stably expressing human (IC50 = 20 nM), rat (IC50 = 42 nM), or mouse (IC50 = 150 nM) P2X7 receptors. [1]
A-839977 potently blocked BzATP-stimulated IL-1β release (IC50 = 37 nM) and YO-PRO uptake (IC50 = 7 nM) in differentiated human THP-1 cells. [1] A-839977 produced parallel rightward shifts in BzATP concentration-effect calcium influx curves with a pA2 value of 8.1, indicating competitive antagonism. [1] In cultured rat optic nerve head astrocytes, A-839977 (50 nM) prevented the swelling-induced upregulation of IL-1β mRNA. [2] In cultured mouse optic nerve head astrocytes, A-839977 (100 nM) reduced the swelling-triggered reduction in IκBα, indicating inhibition of NFκB activation. [2] A 839977 specifically prevents agonist-induced YO-PRO uptake and IL-1beta release in differentiated human THP-1 cells by inhibiting BzATP-induced calcium influx at the mammalian P2X7 receptor (IC50=20-150 nM). In animal studies, it has been demonstrated to lessen neuropathic pain and inflammation [1]. In optic astrocytes, 839977 (50 nM, 1 hour pretreatment) effectively inhibits the rise in IL-1β initiation that is produced by stress [2]. |
| ln Vivo |
In Vivo: In a rat model of cancer-induced bone pain (intratibial MRMT-1 carcinoma cells), spinal application of A-839977 (0.4 and 1.2 mg/kg) significantly reduced dorsal horn neuronal responses to high-intensity mechanical stimulation (26g and 60g) and high-intensity thermal stimulation (48°C) in a dose-dependent manner. No effect was observed on responses to low-intensity stimuli or in sham/naïve animals. [3]
In the same model, systemic administration of A-839977 (40 mg/kg, i.p.) significantly increased mechanical withdrawal threshold (von Frey test) in cancer-bearing animals, with no effect in sham animals or with vehicle treatment. A higher dose (120 mg/kg) showed toxic effects. [3] A-839977 (40 mg/kg, i.p.) significantly increased weight-bearing ratio (incapacitance test) and limb-use score in cancer-bearing animals at later stages of disease, with no effect of vehicle treatment. [3] A-839977 (40 mg/kg, i.p.) did not affect motor coordination in naive rats as measured by rotarod test. [3] In a mouse model of CFA-induced inflammatory pain, A-839977 dose-dependently reduced thermal hyperalgesia (ED50 = 40 μmol/kg, i.p. in mice; ED50 = 100 μmol/kg, i.p. in rats). The antihyperalgesic effect was absent in IL-1αβ knockout mice. [1] In rats, A-839977 (30 mg/kg, i.p.) 30 min before testing attenuated CFA-induced thermal hyperalgesia. [1] In a mouse model of retinal mechanical strain (controlled elevation of IOP), A-839977 (50 nM in vitro, or via P2X7 knockout) prevented the upregulation of IL-1β mRNA and protein. [2] In rats, A839977 (30 μmol/kg, 100 μmol/kg, 300 μmol/kg; 30 min preinjection) lowers thermal hyperalgesia in a dose-dependent manner when complete Freund's adjuvant (CFA) is injected plantarly [1]. The CFA model of inflammatory pain in wild-type mice was significantly affected by A839977 (10 μmol/kg, 30 μmol/kg, and 100 μmol/kg; pre-injection for 30 minutes), whereas IL-1alphabeta knockout mice were not significantly affected. Rats are ineffective [1]. In animals with cancer, A839977 reduces the responses of dorsal horn neurons [3]. |
| Enzyme Assay |
Enzyme Assay: Calcium influx FLIPR assay: 1321N1 cells stably expressing P2X7 receptors were plated in poly-D-lysine-coated black 96-well plates and loaded with Fluo-4 dye. After washing, cells were incubated with A-839977 for 3 min before agonist addition. BzATP at EC70 concentrations (mouse: 150 μM, rat: 10 μM, human: 5 μM) was used as agonist. Fluorescence was measured for 3 min. IC50 values were calculated. [1]
YO-PRO uptake assay: Differentiated human THP-1 cells were plated and pre-incubated with A-839977 for 30 min. BzATP (90 mM) was added and YO-PRO uptake measured for 1 hour. [1] IL-1β release assay: Differentiated human THP-1 cells were primed with LPS (25 ng/ml) and IFNγ (10 ng/ml) for 3 h. A-839977 was added 30 min before BzATP (1 mM) stimulation. IL-1β levels in supernatant were measured by ELISA. [1] |
| Cell Assay |
Cell Assay: 1321N1 human astrocytoma cells stably expressing mouse, rat, or human P2X7 receptors were maintained in DMEM with 1% L-alanyl-L-glutamine, 1% antibiotic/antimycotic, 10% FBS, and 300 μg/ml geneticin. For calcium influx assays, cells were plated at 5 × 10⁶ cells per plate. [1]
THP-1 human monocytic cells were differentiated into macrophage phenotype with LPS (25 ng/ml) and IFNγ (10 ng/ml) for 3 h (IL-1β release) or overnight (pore formation). Cells were maintained in RPMI with 10% fetal calf serum. [1] Rat optic nerve head astrocytes: Primary astrocytes were isolated from neonatal rat pups (PD3-5) and cultured in DMEM/F12 with 10% FBS, 1% penicillin/streptomycin, and 25 ng/ml EGF. Cells were >99% astrocytes by GFAP staining. For swelling experiments, cells were incubated in 30% hypotonic solution for 4 h. A-839977 (50 nM) was added 1 h before swelling. [2] Mouse optic nerve head astrocytes: Isolated from 3-month-old C57BL/6J and P2X7-/- mice. Cultured similarly to rat astrocytes. A-839977 (100 nM) was used. [2] RT-PCR[2] Cell Types: Optic astrocytes Tested Concentrations: 50 nM Incubation Duration: 1 hour (pre-treatment) Experimental Results: Prevents IL-1β initiation in astrocytes |
| Animal Protocol |
Animal/Disease Models: Male SD (SD (Sprague-Dawley)), balb/c (Bagg ALBino) mouse and IL-1 (−/−) mice, for CFA-induced chronic inflammation Doses: 30 μmol/kg, 100 μmol/kg, 300 μmol/kg (rat); 10 μmol/kg, 30 μmol/kg, 100 μmol/kg (mouse)
Route of Administration: injection; 30-minute pre-injection Experimental Results: Attenuated CFA-induced thermal hyperalgesia in a dose-related manner in rats and mice, However, it had no effect on IL-1 (−/−) mice. |
| ADME/Pharmacokinetics |
A-839977 is CNS-penetrating with a brain/spinal cord to plasma ratio of 0.15-0.25. [1][3]
For in vivo electrophysiology, A-839977 was dissolved daily in 10% DMSO, 10% ChromEL, and sterile saline. [3] For behavioral studies, A-839977 was dissolved in 30% NMP, 30% PEG400, and 40% hydroxypropyl-β-cyclodextrin. [3] |
| Toxicity/Toxicokinetics |
Toxicity/Toxicokinetics: In cancer-induced bone pain model, a dose of 120 mg/kg A-839977 (i.p.) demonstrated clear toxic effects and was not tested further. The 40 mg/kg dose was well tolerated with no observed motor coordination deficits. [3]
In the CFA inflammatory pain model, A-839977 did not produce any overt signs of behavioral disruption at effective doses. [1] |
| References |
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| Additional Infomation |
A-839977 (1-(2,3-dichlorophenyl)-N-(2-(pyridin-2-yloxy)benzyl)-1H-tetrazol-5-amine) is a potent, selective, competitive P2X7 receptor antagonist derived from a series of tetrazole-based compounds. It is CNS-penetrant and has been used to demonstrate that P2X7 receptor antagonism produces antinociception in inflammatory and cancer-induced bone pain models. The antihyperalgesic effect of A-839977 is mediated through blockade of IL-1β release, as it is lost in IL-1αβ knockout mice. A-839977 has also been used to study P2X7 receptor involvement in inflammasome priming in astrocytes following mechanical strain. [1][2][3]
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| Molecular Formula |
C19H14CL2N6O
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|---|---|
| Molecular Weight |
413.260060787201
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| Exact Mass |
412.061
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| Elemental Analysis |
C, 55.22; H, 3.41; Cl, 17.16; N, 20.34; O, 3.87
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| CAS # |
870061-27-1
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| PubChem CID |
53325875
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| Appearance |
White to off-white solid powder
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| LogP |
4.19
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
28
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| Complexity |
489
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C(Cl)=C(N2C(NCC3C(OC4C=CC=CN=4)=CC=CC=3)=NN=N2)C=CC=1
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| InChi Key |
GMVNBKZQJFRFAR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H14Cl2N6O/c20-14-7-5-8-15(18(14)21)27-19(24-25-26-27)23-12-13-6-1-2-9-16(13)28-17-10-3-4-11-22-17/h1-11H,12H2,(H,23,24,26)
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| Chemical Name |
1-(2,3-dichlorophenyl)-N-{[2-(pyridin-2-yloxy)phenyl]methyl}-1H-1,2,3,4-tetrazol-5-amine
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| Synonyms |
A839977; A-839977; A-839,977; A839,977; 1-(2,3-dichlorophenyl)-N-{[2-(pyridin-2-yloxy)phenyl]methyl}-1H-1,2,3,4-tetrazol-5-amine; 1-(2,3-dichlorophenyl)-N-((2-(pyridin-2-yloxy)phenyl)methyl)-1H-1,2,3,4-tetrazol-5-amine; 870061-27-1; A 839977
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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 (~241.98 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.05 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 (6.05 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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 (6.05 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 | 2.4198 mL | 12.0989 mL | 24.1978 mL | |
| 5 mM | 0.4840 mL | 2.4198 mL | 4.8396 mL | |
| 10 mM | 0.2420 mL | 1.2099 mL | 2.4198 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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