P2X7 receptor – selective purine P2X7 receptor antagonist; EC50 for P2X7 receptor occupancy in rat brain: plasma concentration of 105 ng/mL (EC50), >80% occupancy occurs above 1000 ng/mL [1][2][3]
pIC50: 8.46 (hP2X7 receptor), 8.81 (rP2X7 receptor)[1]
JNJ-54175446 targets the P2X7 receptor, an ATP-gated ion channel that plays a key role in neuroinflammation and the release of pro-inflammatory cytokines. The P2X7 receptor is expressed on immune cells (microglia, macrophages) and neuronal cells in the central nervous system. Activation of P2X7 by extracellular ATP triggers the release of inflammatory mediators such as IL-1β, IL-18, and TNF-α, which contribute to neuroinflammation and have been implicated in the pathophysiology of depression and other neuropsychiatric disorders. JNJ-54175446 is a potent and selective brain-penetrant P2X7 antagonist that blocks receptor activation and downstream inflammatory signaling.

In Vitro: JNJ-54175446 is a potent, selective, and brain-penetrant P2X7 receptor antagonist. It is highly permeable with no evidence of efflux as measured by Caco-2 cell line (A to B Papp = 75.3 × 10⁻⁶ cm⁻¹/sec, B to A Papp = 37.1 × 10⁻⁶ cm⁻¹/sec). [1]
JNJ-54175446 demonstrated excellent metabolic stability in liver microsomes across species: human extraction ratio <0.3, rat <0.2, dog <0.25, cynomolgus monkey <0.26, mouse <0.25. [1]
JNJ-54175446 showed no significant activity in a panel of 50 ion channels, receptors, and transporters (tested at 10 μM), a kinase panel (tested at 1 μM), and related P2X receptors (P2X1, P2X2, P2X3, P2X2/3, P2X4 FLIPR assays, tested up to 10 μM). [1]
JNJ-54175446 had no significant hERG binding (IC50 > 10 μM). [1]
Protein binding: rat PPB 87.8%, human PPB 88.8%, mouse PPB 88.7%, rat brain binding 94.9%. Solubility: 38.7 μM at pH 2, 20.7 μM at pH 7. [1]

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Compound 14, also known as JNJ-54175446, is a strong and specific brain penetrant P2X7 antagonist. Its pIC50s for hP2X7 and rP2X7 are 8.46 and 8.81, respectively. In comparison to mouse, dog, and macaque P2X7 (pIC50, 7.8, 7.9, and 8.1, respectively), JNJ-54175446 exhibits less strong activity[1].

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JNJ-54175446 demonstrates potent in vitro activity as a P2X7 receptor antagonist. The compound exhibits pIC50 values of 8.46 for human P2X7 and 8.81 for rat P2X7 receptors. It shows less potent activity against mouse, dog, and macaque P2X7 with pIC50 values of 7.8, 7.9, and 8.1, respectively. The compound is potent and stable in liver microsomes and has an acceptable protein binding profile. JNJ-54175446's selectivity for P2X7 over other purinergic receptors makes it a valuable tool for studying P2X7-specific functions in neuroinflammation and psychiatric disorders.

In Vivo: In the intra-amygdala kainic acid mouse model of drug-resistant temporal lobe epilepsy, once-daily intraperitoneal dosing of JNJ-54175446 (30 mg/kg for 5 days) resulted in a significant reduction in spontaneous recurrent seizures during the post-treatment washout period (p = 0.022). Seizure rates in vehicle-treated mice averaged ~10 per day, while JNJ-54175446-treated mice had significantly lower seizure frequency. No effect on seizure duration was observed. [2]
JNJ-54175446 (30 mg/kg, i.p., 5 days) reduced astrogliosis (GFAP staining) in the ipsilateral CA3 subfield of the hippocampus in epileptic mice, but did not significantly alter microgliosis (IBA1 staining). IBA1-positive microglia in JNJ-54175446-treated mice displayed higher numbers of endpoints and longer process lengths compared to vehicle-treated mice. [2]
In MDD patients, JNJ-54175446 (600 mg loading dose on day 1 followed by 150 mg once daily for 10 days) was well tolerated. It reduced IL-1β release by LPS-stimulated peripheral white blood cells in the presence of the P2X7 receptor agonist BzATP, demonstrating proof of pharmacological activity. [3]
JNJ-54175446 did not have a significant effect on mood as assessed using HDRS17 and QIDS-SR in MDD patients. However, it blunted the acute reduction of anhedonia that occurred as a result of total sleep deprivation, as assessed by SHAPS (group A vs placebo, p = 0.049) and PILT total amount lost (non-significant trend). [3]
Ex vivo autoradiography using ¹⁸F-JNJ-64413739 showed no significant difference in P2X7 receptor binding between vehicle- and JNJ-54175446-treated epileptic mice at the end of recordings (2 weeks after drug washout). [2]

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The ED50 of JNJ-54175446 is 0.46 mg/kg, which is equivalent to a plasma EC50 of 105 ng/mL. This indicates dose-dependent occupancy[1].

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JNJ-54175446 is being developed for treating major depressive disorder. The compound is a potent and selective brain-penetrant P2X7 receptor antagonist. In preclinical studies, JNJ-54175446 has been evaluated for its effects on neuroinflammation and depressive-like behaviors. The compound's ability to penetrate the blood-brain barrier (brain penetrant) and its acceptable protein binding profile support its utility for CNS indications. Detailed in vivo efficacy data, including specific model results and dosing regimens, are available in the primary literature. The compound is a valuable tool for studying the role of P2X7 in neuroinflammation and depression.

Enzyme Assay: P2X7 calcium flux FLIPR assay: 1321N1 cells expressing recombinant P2X7 channel were seeded at 25,000 cells/well in 96-well plates. Cells were loaded with Calcium-4 dye and incubated for 60 min. Test compounds were prepared in assay buffer. Cells were incubated with test compounds for 30 min. BzATP was added and fluorescence change measured for 180 seconds. [1]
P2X7 radioligand binding assay: Cell membrane preparations from recombinant P2X7 1321N1 cells were incubated with [³H]-A-804598 tracer and test compounds for 1 hour at 4°C. The assay was terminated by filtration. IC50 was corrected for tracer concentration and affinity. [1]
Plasma protein binding by equilibrium dialysis: Compounds were spiked into plasma at 1 μM. Dialysis was performed using RED device with 8 kDa-permeable cellulose membrane for 6 h at 37°C. Samples were analyzed by LC/MS/MS. [1]
Liver microsomal stability: Test compounds (1 μM) were incubated with liver microsomes (0.5 mg/mL) and NADPH (1 mM). Aliquots were taken at 0, 5, 10, 20, 40, 60 min and quenched. Half-lives were derived and hepatic extraction ratios calculated. [1]
CYP inhibition cocktail assay: Human liver microsomes were incubated with six CYP substrates (phenacetin for CYP1A2, paclitaxel for CYP2C8, diclofenac for CYP2C9, S-mephenytoin for CYP2C19, dextromethorphan for CYP2D6, midazolam for CYP3A) and test compounds. Probe metabolite formation was analyzed by LC-MS/MS. [1]

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The in vitro receptor binding and functional assay for JNJ-54175446 measures the compound's affinity for and antagonism of the P2X7 receptor. Radioligand binding assays are performed using membrane preparations from cells expressing human, rat, mouse, dog, or macaque P2X7 receptors. Varying concentrations of JNJ-54175446 are incubated with the membrane preparation and a radiolabeled P2X7 ligand. The amount of bound radioligand is measured, and pIC50 values are determined (8.46 for human, 8.81 for rat). For functional assays, cells expressing P2X7 are loaded with a fluorescent calcium indicator and treated with varying concentrations of JNJ-54175446 prior to stimulation with ATP. The inhibition of ATP-induced calcium influx is measured, and antagonist potency is determined. The compound is dissolved in DMSO and diluted in assay buffer. Appropriate positive controls and negative controls are included in each assay run.

Cell Assay: Caco-2 permeability assay: Caco-2 cells were seeded onto 96-well plates and cultured for at least 21 days. Test compounds (10 μM) were applied to donor side and incubated at 37°C for 60 min (A→B) or 40 min (B→A). Apparent permeability coefficient (Papp) was calculated. [1]
1321N1 cells expressing recombinant P2X7 channel were cultured in HyQ DME high glucose with 10% FBS and selection marker. Cells were seeded at 25,000 cells/well for calcium flux assays. [1]

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The in vitro cellular assay for JNJ-54175446 is performed using cells that express the P2X7 receptor and respond to ATP stimulation, such as human or rat microglia, macrophages, or P2X7-transfected cell lines. Cells are cultured in appropriate medium and treated with varying concentrations of JNJ-54175446 or vehicle control (DMSO) prior to stimulation with ATP. The release of pro-inflammatory cytokines (e.g., IL-1β, IL-18) is measured by ELISA. P2X7 receptor activation can also be assessed by measuring ATP-induced calcium influx using fluorescent calcium indicators or by measuring dye uptake (e.g., Yo-Pro-1 uptake) through the P2X7 pore. The inhibition of these responses by JNJ-54175446 is quantified, and pIC50 values are determined. The compound's effects on downstream signaling pathways can also be assessed.

Animal Protocol: Intra-amygdala kainic acid (IAKA) mouse model of temporal lobe epilepsy: Male C57BL/6JOlaHsd mice (25-30 g, 10 weeks old) were anesthetized with isoflurane. An EEG transmitter was implanted subcutaneously. A guide cannula was placed above the right basolateral amygdala. After 48 h recovery, KA (0.3 μg in 0.2 μL) was microinjected. Status epilepticus was recorded. After 40 min, lorazepam (8 mg/kg, i.p.) was given to reduce mortality. JNJ-54175446 (30 mg/kg, i.p., once daily for 5 days) or vehicle was administered starting 14 days after KA. Dosing was then terminated and seizures recorded for another 13 days. Mice were perfused transcardially with PBS and brains collected for histology. [2]
MDD patient study (NCT02902601): Male and female MDD patients (18-64 years) with IDS-C30 ≥ 30 were randomized to: Group A (JNJ-54175446 throughout: 600 mg loading day 1, then 150 mg q.d. days 2-10), Group B (placebo days 1-3, then 600 mg day 4 loading, 150 mg q.d. days 5-10), or Group C (placebo throughout). All patients underwent 36 h total sleep deprivation from day 3 to day 4, with light therapy (10,000 lux, 30 min) at 03:00 during and 08:00 following sleep deprivation. [3]

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In vivo animal experiments with JNJ-54175446 are conducted using rodent models of depression or neuroinflammation. JNJ-54175446 is administered via oral gavage or intraperitoneal injection at various doses. The compound's brain penetration is assessed by measuring compound concentrations in brain and plasma. For depression models, behavioral tests such as the forced swim test, tail suspension test, or chronic unpredictable mild stress model are performed. The effects of JNJ-54175446 on depressive-like behaviors are compared to vehicle-treated controls. Neuroinflammatory markers (e.g., cytokines, microglial activation) are measured in brain tissues. The compound's effects on P2X7 receptor occupancy can also be assessed using PET imaging or ex vivo binding assays.

ADME/Pharmacokinetics: In rats, JNJ-54175446 (1 mg/kg i.v., 5 mg/kg p.o.) showed: CL = 2.9 mL/min/kg, Vss = 2.1 L/kg, t1/2 = 8.6 h, F = 96%. [1]
In dogs: CL = 0.9 mL/min/kg, Vss = 2.5 L/kg, t1/2 = 32 h, F = 164%. [1]
In NHP: CL = 4.4 mL/min/kg, Vss = 2.1 L/kg, t1/2 = 6.9 h, F = 157%. [1]
In MDD patients, following a single 600 mg dose: Cmax = 1181 ± 394 ng/mL, tmax median 7.98 h (range 3.93-23.33 h), AUCt = 21107 ± 6841 ng·h/mL. Following multiple doses of 150 mg q.d. on day 10: Cmax = 1932 ± 664 ng/mL, Cmin = 1417 ± 510 ng/mL, Cavg = 1671 ± 591 ng/mL, tmax median 4.00 h (2.00-10.00 h), AUCt = 39905 ± 14069 ng·h/mL, terminal t1/2 = 50.2 ± 17.8 h. Accumulation ratio: AUCt on day 10 approximately 1.9-fold higher than day 1. [3]
Human PK prediction from allometry: predicted CL 0.6 mL/min/kg, Vss 1.4 L/kg, t1/2 29 h. Estimated dose to maintain >50% occupancy over 24 h: 8 mg q.d. (assuming 100% bioavailability). [1]

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Detailed pharmacokinetic (PK) parameters for JNJ-54175446 are partially documented. The compound is potent, stable in liver microsomes, and has an acceptable protein binding profile. JNJ-54175446 is brain penetrant. The compound has a molecular weight of approximately 408.80 and a chemical formula of C18H14ClF4N6O (free base). For in vivo administration, the compound is typically formulated using appropriate vehicles to ensure adequate solubility and stability. The compound should be stored under conditions recommended by the manufacturer to maintain stability and prevent degradation. Detailed PK parameters including half-life, clearance, volume of distribution, and maximum concentration (Cmax) are available in the primary literature and should be consulted for specific experimental planning.

Toxicity/Toxicokinetics: In a 4-day rat toleration study at doses of 62.5, 125, and 250 mg/kg, no significant clinical observations were noted and exposures were high at all doses. [1]
In a 5-day dog toleration study at doses of 30, 100, and 300 mg/kg, exposures were high and no relevant changes in serum chemistry, necropsy, or histopathology were observed. [1]
In MDD patients, JNJ-54175446 was well-tolerated. The most common treatment-emergent adverse events in patients receiving JNJ-54175446 were headache (25.0%), nausea (11.5%), dysgeusia (5.8%), and vomiting (5.8%). All adverse events were mild to moderate. No deaths, serious AEs, or persistent AEs were reported. No clinically significant changes in laboratory evaluations, vital signs, or ECG parameters were observed. [3]

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Comprehensive toxicological data for JNJ-54175446 are not extensively documented in publicly available sources. As a research-grade compound, JNJ-54175446 is intended for laboratory research purposes only and is not approved for human therapeutic use. Standard laboratory safety practices should be followed when handling this compound, including the use of appropriate personal protective equipment and working in a well-ventilated area. The compound should be stored according to the manufacturer's recommendations to maintain stability and prevent degradation. Comprehensive toxicological profiling (e.g., LD50, maximum tolerated dose, organ-specific toxicity) is not available from the current search results and would require consultation of the primary literature or safety data sheets.

[1]. 4-Methyl-6,7-dihydro-4H-triazolo[4,5-c]pyridine-Based P2X7 Receptor Antagonists: Optimization of Pharmacokinetic Properties Leading to the Identification of a Clinical Candidate. J Med Chem. 2017 Jun 8;60(11):4559-4572.

[2]. Anti-seizure effects of JNJ-54175446 in the intra-amygdala kainic acid model of drug-resistant temporal lobe epilepsy in mice. Front Pharmacol. 2024 Jan 8:14:1308478.

[3]. Characterization of the central nervous system penetrant and selective purine P2X7 receptor antagonist JNJ-54175446 in patients with major depressive disorder. Transl Psychiatry. 2023 Jul 24;13(1):266.

JNJ-54175446 ((R)-(2-chloro-3-(trifluoromethyl)phenyl)(1-(5-fluoropyrimidin-2-yl)-4-methyl-1,4,6,7-tetrahydro-5H-[1,2,3]triazolo[4,5-c]pyridin-5-yl)methanone) is a potent, selective, brain-penetrant P2X7 receptor antagonist. The absolute configuration was determined by X-ray crystallography as (R). The compound has high permeability (Caco-2 A to B Papp = 75.3 × 10⁻⁶ cm⁻¹/sec) and no efflux. It has a cLogP of 3.00, molecular weight 440.8. The compound was nominated for clinical development and has been dosed in healthy volunteers at single doses from 0.5 mg to 600 mg, all well tolerated. [1] In the IAKA model, JNJ-54175446 reduced spontaneous seizures and gliosis, supporting P2X7 receptor antagonism as a potential therapeutic approach for drug-resistant temporal lobe epilepsy. [2] In MDD patients, JNJ-54175446 blunted TSD-induced reduction in anhedonia, suggesting potential mood-stabilizing effects on hedonic capacity. [3]

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JNJ-54175446 is being studied in the clinical trial NCT02933762 (a study evaluating the effects of JNJ-54175446 on amyloid biomarkers and cytokine profiles in cerebrospinal fluid and plasma in healthy participants).

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JNJ-54175446 is a research compound developed for studying the role of the P2X7 receptor in neuroinflammation and psychiatric disorders, particularly major depressive disorder. The compound is a potent and selective brain-penetrant P2X7 antagonist with pIC50 values of 8.46 for human and 8.81 for rat P2X7 receptors. JNJ-54175446 is potent, stable in liver microsomes, and has an acceptable protein binding profile. The compound is not currently in clinical trials nor approved for therapeutic use; it remains an investigational tool compound for preclinical neuroscience research. JNJ-54175446 is available from various chemical suppliers for research purposes. Its utility lies in its ability to selectively antagonize P2X7 receptors and probe their role in neuroinflammation and depression.

C18H13CLF4N6O

440.782035589218

440.077

C, 49.05; H, 2.97; Cl, 8.04; F, 17.24; N, 19.07; O, 3.63

1627902-21-9

90409366

Light yellow to yellow solid powder

3

0

9

2

30

634

1

ClC1C(C(F)(F)F)=CC=CC=1C(N1CCC2=C([C@H]1C)N=NN2C1N=CC(=CN=1)F)=O

CWFVVQFVGMFTBD-SECBINFHSA-N

InChI=1S/C18H13ClF4N6O/c1-9-15-13(29(27-26-15)17-24-7-10(20)8-25-17)5-6-28(9)16(30)11-3-2-4-12(14(11)19)18(21,22)23/h2-4,7-9H,5-6H2,1H3/t9-/m1/s1

(R)-(2-chloro-3-(trifluoromethyl)phenyl)(1-(5-fluoropyrimidin-2-yl)-4-methyl-1,4,6,7-tetrahydro-5H-[1,2,3]triazolo[4,5-c]pyridin-5-yl)methanone

JNJ54175446; JNJ 54175446; JNJ-54175,446; 32524GLF40; Methanone, (2-chloro-3-(trifluoromethyl)phenyl)((4R)-1-(5-fluoro-2-pyrimidinyl)-1,4,6,7-tetrahydro-4-methyl-5H-1,2,3-triazolo(4,5-C)pyridin-5-yl)-; Methanone, (2-chloro-3-(trifluoromethyl)phenyl)(1-(5-fluoro-2-pyrimidinyl)-1,4,6,7-tetrahydro-4-methyl-5H-1,2,3-triazolo(4,5-c)pyridin-5-yl)-; 1627902-21-9; JNJ-54175446

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~62.5 mg/mL (~141.79 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.72 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 20.8 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.72 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.72 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)