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Purity: ≥98%
A-740003 is a potent and selective P2X7 receptor antagonist. A-740003 has (IC(50) values = 40 nM for human and 18 nM for rat) as measured by agonist-stimulated changes in intracellular calcium concentrations. A-740003 showed weak or no activity (IC(50) > 10 muM) at other P2 receptors and an array of other neurotransmitter and peptide receptors, ion channels, reuptake sites, and enzymes. A-740003 potently blocked agonist-evoked IL-1beta release (IC(50) = 156 nM) and pore formation (IC(50) = 92 nM) in differentiated human THP-1 cells. A-740003 in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain.
| Targets |
P2X7 receptor – IC50 = 18 nM (rat P2X7, calcium influx assay); IC50 = 40 nM (human P2X7, calcium influx assay); IC50 = 92 nM (pore formation in differentiated human THP-1 cells); IC50 = 156 nM (agonist-evoked IL-1β release in differentiated human THP-1 cells); pIC50 = 7.36 (human P2X7, calcium influx), 7.74 (rat P2X7, calcium influx), 6.14-7.03 (mouse P2X7 BALB/c and C57BL/6, Yo-Pro uptake), 6.17-6.57 (mouse P2X7, calcium influx); shows weak or no activity (IC50 > 10 μM) at other P2 receptors and an array of other neurotransmitter and peptide receptors, ion channels, reuptake sites, and enzymes [2][3][4]
Selective over other P2X receptors (P2X1, P2X2, P2X2/3, P2X4), P2Y1, P2Y2 (IC50 > 10 μM) [3][4] |
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| ln Vitro |
In Vitro: A-740003 potently blocked BzATP-evoked changes in intracellular calcium concentrations in 1321N1 cells stably expressing rat P2X7 receptors (IC50 = 18 nM) or human P2X7 receptors (IC50 = 40 nM). In comparison, PPADS, Brilliant Blue G, and KN-62 were significantly less potent. [4]
A-740003 potently blocked agonist-evoked IL-1β release in differentiated human THP-1 cells (IC50 = 156 nM) and pore formation (Yo-Pro uptake) in differentiated human THP-1 cells (IC50 = 92 nM). [4] A-740003 showed weak or no activity (IC50 > 10 μM) at other P2 receptors (P2X1, P2X2, P2X2/3, P2X4, P2Y1, P2Y2) and an array of other neurotransmitter and peptide receptors, ion channels, reuptake sites, and enzymes. [4] In recombinant mouse P2X7 receptors (BALB/c and C57BL/6) expressed in 1321N1 cells, A-740003 blocked BzATP-stimulated calcium influx with pIC50 values of 6.57 ± 0.04 (BALB/c) and 6.17 ± 0.05 (C57BL/6), and blocked Yo-Pro uptake with pIC50 values of 6.14 ± 0.04 (BALB/c) and 5.76 ± 0.03 (C57BL/6). [3] A-740003 blocked BzATP-stimulated calcium influx at rat P2X7 receptors with pIC50 of 7.74 ± 0.02, and at human P2X7 receptors with pIC50 of 7.36 ± 0.01. [3] Following a 60-minute preincubation, A-740003 blocked calcium influx at rat P2X7 receptors with pIC50 of 7.92 ± 0.04, at human P2X7 receptors with pIC50 of 7.28 ± 0.03, at BALB/c mouse P2X7 with pIC50 of 6.26 ± 0.04, and at C57BL/6 mouse P2X7 with pIC50 of 6.48 ± 0.03. [3] Significant boiling occurs in the sustained phase of the BzATP-sensing reaction when A-438079 or A-740003 (10 μM) is added [1]. Dentate granule cells with SE-conducted TNF-α expression are lessened upon infusion of A-740003. Neuronal death in SE sensors is increased by A-740003 infusion [2]. A-740003 and A-438079 showed noticeably higher potency in P2X7 receptor activation in all species when compared to other clamp antagonists. A-740003 and A-438079 show increased activity in storage and in people compared to the Muse P2X7 receptor [3]. In nursing human THP-1 cells, A-740003 potently inhibits agonist-induced IL-1β release (IC50=156 nM) and pore formation (IC50=92 nM) [4]. |
| ln Vivo |
In Vivo: Systemic administration of A-740003 produced dose-dependent antinociception in a spinal nerve ligation (SNL) model in rats (ED50 = 19 mg/kg i.p.). [4]
A-740003 attenuated tactile allodynia in two other models of neuropathic pain: chronic constriction injury (CCI) of the sciatic nerve and vincristine-induced neuropathy. [4] A-740003 effectively reduced thermal hyperalgesia observed following intraplantar administration of carrageenan (ED50 = 38 mg/kg i.p.) or complete Freund's adjuvant (CFA) (ED50 = 54 mg/kg i.p.). [4] A-740003 was ineffective in attenuating acute thermal nociception in normal rats and did not alter motor performance at analgesic doses. [4] In a rat neck-incision pain model, intraperitoneal injection of A-740003 (142 mg/kg) exerted an analgesic effect at 4 hours post-incision. At 24 hours post-incision, A-740003 alone was ineffective, but it antagonized the analgesic effect of electroacupuncture. A-740003 also blocked electroacupuncture-induced upregulation of P2X7R protein expression at 4 hours and blocked electroacupuncture-induced downregulation of P2X7R at 24 hours. [5] Enzyme Assay: Calcium influx FLIPR assay: 1321N1 cells stably expressing P2X7 receptors were plated in poly-D-lysine-coated 96-well plates and loaded with Fluo-4 dye. After loading, extracellular Fluo-4 was removed by washing with DPBS. Compound solutions were prepared in DPBS. Agonist (BzATP)-induced Ca²⁺ dynamics were recorded for 3 minutes. For antagonist activity measurement, compounds were added to the cell plate and fluorescence data collected for 3 minutes before agonist addition. Concentration-response data were analyzed using GraphPad Prism to calculate pIC50 values. [3][4] Yo-Pro uptake assay (pore formation): 1321N1 cells expressing P2X7 receptors were plated in poly-D-lysine-coated black-walled 96-well plates. Cells were rinsed twice with PBS without Mg²⁺ or Ca²⁺ ions. Yo-Pro iodide dye (2 μM final) was added immediately prior to agonist addition, and dye uptake was measured for 1 hour. Antagonists were added at the same time as the Yo-Pro dye. For antagonist activity, percentage of maximal intensity was normalized to BzATP peak activation and plotted against compound concentration to calculate IC50 values. [3][4] IL-1β release assay: Differentiated human THP-1 cells were used. Agonist-evoked IL-1β release was measured in the presence of A-740003. [4] Systemic dosages of A-740003 demonstrated dose-dependent anti-injury effects in a tandem junctional nerve ligation paradigm (ED50=19 mg/kg ip). Additionally, sciatic nerve chronic ischemia and two other forms of neuropathic pain were lessened by A-740003. Furthermore, after carrageenan or Freund's adjuvant plantar model, A-740003 successfully and totally eliminated the heat hyperalgesia (ED50=38 - 54 mg/kg ip). A-740003 does not affect exercise performance at analgesic levels and is ineffective in decreasing normal state acute thermal nociceptors [4]. |
| Enzyme Assay |
Enzyme Assay: Calcium influx FLIPR assay: 1321N1 cells stably expressing P2X7 receptors were plated in poly-D-lysine-coated 96-well plates and loaded with Fluo-4 dye. After loading, extracellular Fluo-4 was removed by washing with DPBS. Compound solutions were prepared in DPBS. Agonist (BzATP)-induced Ca²⁺ dynamics were recorded for 3 minutes. For antagonist activity measurement, compounds were added to the cell plate and fluorescence data collected for 3 minutes before agonist addition. Concentration-response data were analyzed using GraphPad Prism to calculate pIC50 values. [3][4]
Yo-Pro uptake assay (pore formation): 1321N1 cells expressing P2X7 receptors were plated in poly-D-lysine-coated black-walled 96-well plates. Cells were rinsed twice with PBS without Mg²⁺ or Ca²⁺ ions. Yo-Pro iodide dye (2 μM final) was added immediately prior to agonist addition, and dye uptake was measured for 1 hour. Antagonists were added at the same time as the Yo-Pro dye. For antagonist activity, percentage of maximal intensity was normalized to BzATP peak activation and plotted against compound concentration to calculate IC50 values. [3][4] IL-1β release assay: Differentiated human THP-1 cells were used. Agonist-evoked IL-1β release was measured in the presence of A-740003. [4] |
| Cell Assay |
Cell Assay: 1321N1 human astrocytoma cells stably expressing recombinant P2X7 receptors (mouse BALB/c, mouse C57BL/6, rat, human) were maintained in DMEM containing 1% L-Alanyl-L-Glutamine, 1% antibiotic/antimycotic, 10% FBS, and 300 μg/mL Geneticin. For calcium influx assays, cells were plated in poly-D-lysine-coated black 96-well plates. For Yo-Pro uptake assays, cells were plated in poly-D-lysine-coated black-walled 96-well plates. [3][4]
Differentiated human THP-1 cells were used for IL-1β release and pore formation assays. [4] |
| Animal Protocol |
Animal Protocol: Spinal nerve ligation (SNL) model: Rats underwent L5/L6 spinal nerve ligation. A-740003 was administered intraperitoneally, and antinociception was measured. ED50 = 19 mg/kg i.p. [4]
Chronic constriction injury (CCI) model: Rats underwent CCI of the sciatic nerve. A-740003 attenuated tactile allodynia. [4] Vincristine-induced neuropathy model: Rats were treated with vincristine to induce neuropathy. A-740003 attenuated tactile allodynia. [4] Carrageenan-induced inflammation model: Carrageenan was administered intraplantarly. A-740003 reduced thermal hyperalgesia (ED50 = 38 mg/kg i.p.). [4] Complete Freund's adjuvant (CFA) model: CFA was administered intraplantarly. A-740003 reduced thermal hyperalgesia (ED50 = 54 mg/kg i.p.). [4] Neck-incision pain model in rats: A 1.5-cm longitudinal incision was made along the midline of the neck under isoflurane anesthesia, followed by repeated blunt dissection stimulation for 30 minutes. A-740003 was dissolved in 2% DMSO and injected intraperitoneally at a dose of 142 mg/kg at 3.5 hours after neck-incision surgery (0.5 hours before the 4-hour thermal pain test). [5] Electroacupuncture treatment in rats: EA stimulation (1 mA, alternating 2 Hz/100 Hz) was applied to bilateral LI18 or LI4-PC6 for 30 minutes under light isoflurane anesthesia. [5] |
| Toxicity/Toxicokinetics |
A-740003 did not alter motor performance at analgesic doses in rats. [4]
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| References |
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| Additional Infomation |
A-740003 (N-(1-{[(cyanoimino)(5-quinolinylamino)methyl]amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide) is a novel, selective, competitive antagonist of P2X7 receptors. It shows high selectivity for P2X7 over other P2 receptors and an array of other targets. It has been used to demonstrate that selective blockade of P2X7 receptors in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain. [4] A-740003 has also been used to study the role of P2X7 receptors in electroacupuncture analgesia in a neck-incision pain model. [5]
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| Molecular Formula |
C26H30N6O3
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|---|---|
| Molecular Weight |
474.565
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| Exact Mass |
474.237
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| Elemental Analysis |
C, 65.80; H, 6.37; N, 17.71; O, 10.11
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| CAS # |
861393-28-4
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| Related CAS # |
861393-28-4;
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| PubChem CID |
23232014
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| Appearance |
White to off-white solid powder
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| Density |
1.2±0.1 g/cm3
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| Index of Refraction |
1.597
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| LogP |
2.77
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
10
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| Heavy Atom Count |
35
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| Complexity |
773
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC(C)(C)C(NC(=O)CC1=CC(=C(C=C1)OC)OC)/N=C(/NC#N)\NC2=CC=CC3=C2C=CC=N3
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| InChi Key |
PUHSRMSFDASMAE-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C26H30N6O3/c1-26(2,3)24(31-23(33)15-17-11-12-21(34-4)22(14-17)35-5)32-25(29-16-27)30-20-10-6-9-19-18(20)8-7-13-28-19/h6-14,24H,15H2,1-5H3,(H,31,33)(H2,29,30,32)
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| Chemical Name |
N-[1-[[(Cyanoamino)(5-quinolinylamino)methylene]amino]-2,2-dimethylpropyl]-3,4-dimethoxybenzeneacetamide
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| Synonyms |
A-740003; A 740003; A-740,003; A740,003; N-(1-((E)-((cyanoamino)((quinolin-5-yl)amino)methylidene)amino)-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide; N-{1-[(E)-[(cyanoamino)[(quinolin-5-yl)amino]methylidene]amino]-2,2-dimethylpropyl}-2-(3,4-dimethoxyphenyl)acetamide;A740003.
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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 : ~50 mg/mL (~105.36 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.27 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1072 mL | 10.5359 mL | 21.0717 mL | |
| 5 mM | 0.4214 mL | 2.1072 mL | 4.2143 mL | |
| 10 mM | 0.2107 mL | 1.0536 mL | 2.1072 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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