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Purity: ≥98%
JNJ-61432059 is a novel, potent, oral bioactive and selective negative modulator of AMPAR (α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors) associated with trans-membrane AMPAR regulatory protein (TARP) γ-8, with a pIC50 of 9.7 for GluA1/γ-8. It exhibits time- and dose-dependent AMPAR/γ-8 receptor occupancy in mouse hippocampus, resulting in robust seizure protection in corneal kindling and pentylenetetrazole (PTZ) anticonvulsant models.
| Targets |
When tested at 10 μM, JNJ-61432059 did not inhibit glutamate-induced calcium-flux in heterologous cells that coexpressed AMPARs with any TARP other than γ-8 (Supplementary Table 1). In addition, no cross-reactivity was noted when JNJ-61432059 was screened against a panel of 52 receptors, ion channels, and transporters using radioligand displacement assays (<50% inh @ 1 μM; Eurofins/Cerep, Poitiers, France).
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| ln Vitro |
When tested at 10 μM, JNJ-61432059 did not inhibit glutamate-induced calcium-flux in heterologous cells that coexpressed AMPARs with any TARP other than γ-8 (Supplementary Table 1). In addition, no cross-reactivity was noted when JNJ-61432059 was screened against a panel of 52 receptors, ion channels, and transporters using radioligand displacement assays (<50% inh @ 1 μM; Eurofins/Cerep, Poitiers, France).
In a FLIPR assay using HEK-293 cells expressing a human GluA1o-γ-8 fusion protein, JNJ-61432059 potently inhibited glutamate-induced calcium flux with pIC₅₀ = 9.7 ± 0.5[1] It showed >1000-fold selectivity for γ-8 over γ-2 (pIC₅₀ < 5.0)[1] At 10 µM, it did not inhibit glutamate-induced calcium flux in cells co-expressing AMPARs with TARPs other than γ-8[1] Whole-cell electrophysiology on a related early lead (imidazopyrazine 5) in acutely dissociated rat hippocampal neurons showed that a saturating concentration (10 µM) partially reduced peak currents (~50% inhibition) and completely blocked steady-state currents evoked by 10 mM L-glutamate; no effect was observed in rat cerebellar Purkinje neurons[1] |
| ln Vivo |
Following oral administration, JNJ-61432059 exhibited time- and dose-dependent AMPAR/γ-8 receptor occupancy in mouse hippocampus, which resulted in robust seizure protection in corneal kindling and pentylenetetrazole (PTZ) anticonvulsant models.
Following oral administration in mice, JNJ-61432059 exhibited dose-dependent receptor occupancy in the hippocampus, with maximal occupancy exceeding 90% at 1 h post-dose (10 mg/kg)[1] In the mouse corneal kindling model, it provided robust, dose-dependent protection against seizures with ED₅₀ = 1.3 ± 0.1 mg/kg po, and protection was nearly complete at saturating doses[1] No attenuation of seizure protection was observed after 5 days of continued once-daily oral dosing at 5 mg/kg/day, indicating no development of tolerance[1] In the intravenous pentylenetetrazole (PTZ) test in mice, a single oral dose of 5 mg/kg increased the threshold amount of PTZ required to induce twitch and clonus[1] Anticonvulsant efficacy in the PTZ test was maintained after chronic oral administration (5 mg/kg/day for 5 days)[1] No compound-related effects on motor function (ataxia) were observed in the rotarod test at doses providing full seizure protection in the corneal kindling model[1] |
| Enzyme Assay |
Cytochrome P450 inhibition assays were conducted. JNJ-61432059 inhibited CYP2C8 (IC₅₀ = 3.0 µM) and CYP2C9 (IC₅₀ = 1.9 µM)[1]
A hERG binding assay was performed using [³H]dofetilide. At concentrations up to 10 µM, JNJ-61432059 did not displace the radioligand[1] |
| Cell Assay |
Calcium mobilization (FLIPR) assay: HEK-293 cells expressing a human GluA1o-γ-8 fusion construct or co-transfected with human GluA1o and TARP γ-2 were used. Cells were loaded with a calcium-sensitive fluorescent dye. Glutamate was added to induce calcium flux, and inhibition by serial dilutions of JNJ-61432059 was measured as a decrease in fluorescence signal[1]
Metabolic stability assay: Stability was assessed by incubating the compound (1 µM) with human or rat liver microsomes. Samples were taken over time, and the amount of parent compound remaining was quantified to determine the extraction ratio[1] Efflux assay: Apparent permeability and efflux ratio were determined using MDCK-MDR1 cells. The compound was applied to the apical (A) or basolateral (B) side, and the amount transported to the opposite compartment was measured over time to calculate the B-to-A / A-to-B ratio[1] |
| Animal Protocol |
After oral dosing at 10 mg/kg in rats, JNJ-61432059 distributed into the brain (Kpu,u = 0.4) despite low plasma exposures (Cmax = 26 ng/mL) and high clearance (Cl = 57 mL/min/kg). [1]
Specifically, when JNJ-61432059 was incubated with hepatocytes for 4 h at 37 °C, the O-glucuronide was detected as the major metabolite in rat, but only as a minor metabolite in human, mouse, dog, and monkey hepatocytes. [1] This species-specific metabolism was further supported by mouse PK studies, in which JNJ-61432059 displayed improved clearance (40 mL/min/kg) and ∼80-fold higher plasma exposures (Cmax = 2037 ng/mL) compared to an equivalent dose in rat. Furthermore, when administered orally at 10 mg/kg,JNJ-61432059 showed high target engagement in mouse hippocampus, as measured by ex vivo autoradiography,5 with maximal receptor occupancy exceeding 90% at 1 h. [1] Mouse pharmacokinetics and receptor occupancy study: JNJ-61432059 was formulated in a suitable vehicle (specific vehicle not detailed for mouse) and administered orally at doses ranging from 0.3 to 30 mg/kg. Blood and brain samples were collected at various time points for exposure analysis. Receptor occupancy in the hippocampus was measured ex vivo using quantitative autoradiography[1] Mouse corneal kindling model: Mice were kindled via repeated corneal electrical stimulation until stable seizures were established. On the test day, JNJ-61432059 was administered orally (vehicle not specified) at various doses (e.g., 0.3-10 mg/kg) 1 hour before seizure challenge. Seizure severity was scored using the Racine scale. Immediately prior to seizure challenge, motor function was assessed using a rotarod test[1] Mouse pentylenetetrazole (PTZ) test: Mice received a single oral dose of JNJ-61432059 (5 mg/kg) or vehicle. For chronic studies, mice were dosed once daily for 5 days. Two hours post-dose, PTZ was infused intravenously at a constant rate. The thresholds for the first twitch and generalized clonus were recorded[1] Rat pharmacokinetics study: JNJ-61432059 was administered intravenously (1 mg/kg) and orally (5 mg/kg) to Sprague-Dawley rats. The formulation for intravenous dosing was a solution in 20% HP-β-CD. Blood samples were collected over time for plasma concentration analysis[1] |
| ADME/Pharmacokinetics |
In vitro metabolic stability: human liver microsomal extraction rate was 0.6; rat liver microsomal extraction rate was 0.5[1]
In vitro efflux: apparent permeability (Papp) A to B was 13 x 10⁻⁶ cm/s; in MDCK-MDR1 cells, the migration rate from B to A was 3.3 x 10⁻⁶ cm/s, and the efflux ratio was 0.25[1] Rat pharmacokinetics (intravenous injection, 1 mg/kg): clearance (CL) = 57 mL/min/kg[1] Rat pharmacokinetics (oral, 5 mg/kg): maximum plasma concentration (Cmax) = 26 ng/mL[1] Mouse pharmacokinetics (oral, 10 mg/kg): Cmax = 2037 ng/mL, clearance (CL) = 40 mL/min/kg[1] Rat brain permeability (oral, 10 mg/kg) mg/kg): Free brain partition coefficient (Kp,uu) = 0.4[1] Mouse brain permeability: Excellent, brain concentration reaches target binding level (receptor occupancy EC50 = 77 ± 7 ng/g)[1] Species-specific metabolism: The main metabolite of rat hepatocytes after 4 hours of incubation is O-glucuronide conjugate; the content of this metabolite in human, mouse, dog and monkey hepatocytes is extremely low[1] |
| Toxicity/Toxicokinetics |
JNJ-61432059 inhibited CYP2C8 (IC₅₀ = 3.0 µM) and CYP2C9 (IC₅₀ = 1.9 µM) in vitro [1]
In the [³H]dofetiricil replacement assay, no significant binding to hERG channels was observed at concentrations up to 10 µM [1] In vivo, assessed by rotarod assay, no ataxia or motor dysfunction was observed in mice at effective doses (up to 10 mg/kg, orally) in epilepsy models [1] |
| References | |
| Additional Infomation |
JNJ-61432059 is a pyrazolo[1,5-c]pyrimidine derivative that has been found to be a selective negative regulator of the AMPA receptor associated with the hippocampal-enriched accessory subunit TARPγ-8[1]. Its mechanism of action involves partially disrupting the protein-protein interaction between TARPγ-8 and the AMPA receptor pore-forming subunit, thereby inhibiting receptor function, but not completely blocking it[1]. It is being developed as a potential antiepileptic drug with a higher therapeutic index, designed to selectively inhibit hippocampal hyperexcitability while preserving cerebellar function to minimize motor side effects[1]. Preclinical data support its efficacy in acute and chronic epilepsy models without the development of tolerability[1].
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| Molecular Formula |
C25H22FN5O2
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|---|---|
| Molecular Weight |
443.472888469696
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| Exact Mass |
443.175
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| CAS # |
2035814-50-5
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| PubChem CID |
122656119
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| Appearance |
White to off-white solid powder
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| LogP |
3
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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 |
3
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| Heavy Atom Count |
33
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| Complexity |
711
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC1C=CC(=CC=1)C1C(C2C=CC3=C(CC(N3)=O)C=2)=C2C=CN=C(N2N=1)N1CCC(CC1)O
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| InChi Key |
UWIJVELUZWBFEU-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H22FN5O2/c26-18-4-1-15(2-5-18)24-23(16-3-6-20-17(13-16)14-22(33)28-20)21-7-10-27-25(31(21)29-24)30-11-8-19(32)9-12-30/h1-7,10,13,19,32H,8-9,11-12,14H2,(H,28,33)
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| Chemical Name |
5-[2-(4-fluorophenyl)-7-(4-hydroxy-1-piperidyl)pyrazolo[1,5-c]pyrimidin-3-yl]indolin-2-one.
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| Synonyms |
JNJ-61432059; JNJ 61432059; JNJ61432059;
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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 (~112.75 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.69 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.69 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2549 mL | 11.2747 mL | 22.5494 mL | |
| 5 mM | 0.4510 mL | 2.2549 mL | 4.5099 mL | |
| 10 mM | 0.2255 mL | 1.1275 mL | 2.2549 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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