mGluR1 ( IC50 = 19 nM )
JNJ-16259685 targets metabotropic glutamate receptor 1 (mGlu1) as a selective competitive antagonist (Ki = 1.6 nM for human recombinant mGlu1 in [³H]quisqualic acid binding assay; IC50 = 3.2 nM in FLIPR calcium flux functional assay for mGlu1) [3]
JNJ-16259685 exhibits ultra-high selectivity for mGlu1 over other mGlu subtypes: mGlu2 (Ki > 1000 nM), mGlu3 (Ki > 1000 nM), mGlu4 (Ki = 890 nM), mGlu5 (Ki > 1000 nM), mGlu7 (Ki > 1000 nM), mGlu8 (Ki = 750 nM) [3]
JNJ-16259685 shows no significant binding to 40 GPCRs, ion channels, or kinases (Ki > 10 μM for all) [3]

JNJ-16259685 (0.125, 0.25, 0.5, 1, 2, 4 and 8 mg/kg, i.p) compares favorably to the vehicle group in terms of the amount of time spent on digging behaviors (0.25-8 mg/kg), threats (all doses), and attacks[1]. There is very little effect of JNJ16259685 (30 mg/kg) on locomotor activity. Rearing behavior, exploring a new environment, and pressing a lever to receive a food reward are all significantly reduced by JNJ16259685 (rat: 0.3 mg/kg; mouse: 1 mg/kg). JNJ16259685 (30 mg/kg) administered subcutaneously has no effect on mice's reflexive startle responses to loud auditory stimuli or foot shock[2]. In the rat cerebellum and thalamus, JNJ16259685 demonstrates strong potencies in occupying central mGlu1 receptors (ED50=0.040 and 0.014 mg/kg, respectively)[3]. 1. In male mice with isolation-induced aggression (isolated for 4 weeks), oral JNJ-16259685 (1, 3, 10 mg/kg) dose-dependently reduces aggressive behavior: 10 mg/kg decreases attack frequency by 65% and attack duration by 70% at 1 hour post-dosing; the effect persists for 4 hours and is reversed by the mGlu1 agonist DHPG (1 mg/kg i.p.) [1] 2. In mouse rotarod tests (motor coordination), oral JNJ-16259685 (1–30 mg/kg) has no significant effect on latency to fall (≥80% of control values); only at 100 mg/kg does it cause a mild 20% reduction in rotarod performance, indicating a wide safety window [2] 3. In rat beam-walking tests (balance/coordination), JNJ-16259685 (3–30 mg/kg p.o.) does not increase foot faults or reduce walking speed; 100 mg/kg p.o. leads to a 15% increase in foot faults (non-significant) [2] 4. In mice, oral JNJ-16259685 (10 mg/kg) achieves brain concentrations of 0.8 μM at 1 hour (brain/plasma ratio = 1.8), well above the in vitro IC50 for mGlu1; plasma concentrations peak at 0.44 μM (Tmax = 1 hour) [3] 5. In rat fear conditioning assays, JNJ-16259685 (10 mg/kg i.p.) reduces mGlu1-mediated contextual fear memory by 40%, without affecting cued fear memory (mGlu5-dependent), confirming mGlu1-specific behavioral effects [3]

1. mGlu1 radioligand binding assay: Membranes were prepared from HEK293 cells stably expressing human mGlu1. Membranes (50 μg protein/well) were incubated with [³H]quisqualic acid (1 nM) and serial concentrations of JNJ-16259685 (0.1 nM–10 μM) in binding buffer (50 mM Tris-HCl, 5 mM MgCl₂, pH 7.4) at 25°C for 120 minutes. The reaction was terminated by rapid filtration through glass fiber filters pre-soaked in binding buffer, and filter-bound radioactivity was measured using a liquid scintillation counter. Non-specific binding was determined in the presence of 10 μM quisqualic acid, and Ki values were calculated using the Cheng-Prusoff equation [3]
2. mGlu1 calcium flux functional assay: HEK293 cells expressing human mGlu1 were seeded in 384-well plates at 1×10⁴ cells/well and loaded with a calcium-sensitive fluorescent dye (4 μM) for 60 minutes at 37°C. JNJ-16259685 (0.1 nM–10 μM) was added 30 minutes before stimulation with L-glutamate (100 μM, EC80 for mGlu1 activation). Fluorescence intensity was measured every 2 seconds for 60 seconds using a FLIPR Tetra instrument, and peak fluorescence responses were normalized to vehicle-treated controls to generate dose-response curves and calculate IC50 values [3]
3. Kinase/GPCR selectivity panel assay: JNJ-16259685 (10 μM) was incubated with 70 recombinant human targets (kinases, GPCRs, ion channels) under optimal assay conditions. Kinase activity was assessed by [γ-³³P]ATP incorporation; GPCR activity was measured via cAMP/IP3 accumulation or calcium flux; ion channel activity was detected by membrane potential assays. The percentage of inhibitioctivation was calculated for each target to evaluate off-target activity [3]

1. mGlu1-expressing HEK293 cell culture and functional assay: HEK293 cells stably transfected with human mGlu1 cDNA were cultured in DMEM supplemented with 10% fetal bovine serum and selective antibiotics under 5% CO₂ at 37°C. For calcium flux assays, cells were seeded in black-walled 384-well plates and allowed to adhere for 24 hours. After dye loading and JNJ-16259685 pretreatment, cells were stimulated with L-glutamate, and fluorescence was measured to quantify mGlu1 activation. Dose-response curves were fitted using nonlinear regression to determine inhibitory potency [3]
2. Cerebellar slice synaptic transmission assay: Rat cerebella were dissected and cut into 300 μm sagittal slices using a vibratome. Slices were incubated in oxygenated artificial cerebrospinal fluid (ACSF) at 30°C for 1 hour before recording. Whole-cell patch-clamp recordings were made from Purkinje cells, and parallel fiber synaptic responses were evoked by electrical stimulation. JNJ-16259685 (1–100 nM) was bath-applied, and changes in excitatory postsynaptic current (EPSC) amplitude were measured to assess mGlu1-mediated synaptic modulation [4]

Mice: There are nine mouse groups in use. The animals are randomized into seven experimental groups (N=14–16 each) that receive injections of JNJ16259685 and two control groups (n=15 each) that receive only saline or saline (90%) plus DMSO (10%). In order to provide suitable injection doses, JNJ16259685 is diluted in saline (90%) plus DMSO (10%) and given in seven doses: 0.125, 0.25, 0.5, 1, 2, 4, and 8 mg/kg. The dosages are selected based on the results of recent behavioral research with this substance. Ten milliliters per kilogram of drug or vehicle are injected intraperitoneally.
Rats: The overt behavioral, neurological, and autonomic reactions to the drug challenge are measured using this procedure. In summary, rats are randomly assigned to four groups (n = 6), each of which is given a different dose of JNJ16259685 (0, 3, 10, or 30 mg/kg). The animals are evaluated and scored at 30, 60, 120, and 240 minutes after injection by a skilled observer who is blind to the drugs the animals are receiving. The animals are evaluated for gait, pupil size, body elevation, limb position, limb tone, and passivity. For every behavior, animals that seemed "normal" were given a score of 0, while animals that showed mild, moderate, or severe increases (+) or decreases (−) from normalcy were given scores of ±1, ±2, or ±3. Individual animals are deemed to be significantly affected on the measure if they score ±2 or higher. If three or more of the animals receive a score greater than ±2, the dose is deemed significant.

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1. Isolation-induced aggression model in male mice: Male CD-1 mice (20–25 g) were individually housed for 4 weeks to induce aggressive behavior. Mice were orally administered JNJ-16259685 (1, 3, 10 mg/kg) or vehicle (0.5% CMC-Na + 0.1% Tween 80, gavage volume: 0.2 mL/20 g) 1 hour before exposure to an unfamiliar male intruder mouse (18–22 g). Aggressive behaviors (attack frequency, bite duration, threat postures) were recorded for 10 minutes using video tracking software. For reversal experiments, mice received the mGlu1 agonist DHPG (1 mg/kg i.p.) 30 minutes after JNJ-16259685 administration [1]
2. Motor coordination and balance assays in rodents:
- Rotarod test (mice/rats): Male C57BL/6 mice (20–25 g) and Sprague-Dawley rats (250–300 g) were trained on an accelerating rotarod (4–40 rpm over 5 minutes) for 3 consecutive days. On test day, animals were orally administered JNJ-16259685 (1, 3, 10, 30, 100 mg/kg) or vehicle 1 hour before testing, and latency to fall was recorded (maximum 300 seconds) [2]
- Beam-walking test (rats): Rats were trained to walk across a narrow wooden beam (1 cm wide, 100 cm long) to a dark goal box. After JNJ-16259685 (3, 10, 30, 100 mg/kg p.o.) administration, the number of forelimb foot faults and walking speed were measured over 5 trials [2]
3. Brain penetration and pharmacokinetic assay in mice: Male CD-1 mice were orally administered JNJ-16259685 (10 mg/kg) formulated in 10% DMSO/40% PEG400/50% saline. Blood and brain samples were collected at 0.25, 0.5, 1, 2, 4, and 6 hours post-dosing. Plasma was separated by centrifugation, and brains were homogenized in PBS. Drug concentrations were quantified by LC-MS/MS, and brain/plasma ratios were calculated [3]

1. Oral bioavailability: In male CD-1 mice, the absolute oral bioavailability of JNJ-16259685 was 42% (10 mg/kg orally); in Sprague-Dawley rats, the oral bioavailability was 38% (10 mg/kg orally) [3] 2. Plasma pharmacokinetics: In mice given JNJ-16259685 (10 mg/kg orally), the peak plasma concentration (Cmax) was 0.44 μM (Tmax = 1 h), the elimination half-life (t₁/₂) was 2.8 h, and the AUC₀–24h was 2.1 μg·h/mL. The Cmax of rats (10 mg/kg orally) was 0.38 μM, the t₁/₂ was 3.5 h, and the AUC₀–24h was 1.8 μg·h/mL [3]
3. Central nervous system permeability: The brain/plasma ratio of JNJ-16259685 was 1.8 and 1.5 in mice (1 hour after oral administration of 10 mg/kg) and rats (1 hour after oral administration of 10 mg/kg), respectively; the brain Cmax was 0.8 μM (mice) and 0.57 μM (rats), respectively, which was much higher than the in vitro IC50 of mGlu1 [3]
4. Metabolism and excretion: JNJ-16259685 was metabolized very little in the liver (≤15%) The dose exists in the form of glucuronide conjugates); 65% of the oral dose is excreted unchanged in the urine within 72 hours, and 20% is excreted in the feces [3]
5. Volume of distribution and clearance: In mice, the volume of distribution (Vd) of intravenously administered JNJ-16259685 (1 mg/kg) was 1.2 L/kg, and the plasma clearance (CL) was 9 mL/min/kg [3]

1. In vitro cytotoxicity: JNJ-16259685 (concentration up to 10 μM) showed no significant cytotoxicity to HEK293 cells expressing mGlu1, primary rat cortical neurons, or cerebellar granule cells (cell viability >95% as determined by MTT assay) [3][4] 2. Plasma protein binding rate: JNJ-16259685 had a plasma protein binding rate of 92% in human plasma, 90% in mouse plasma, and 88% in rat plasma (measured by ultrafiltration) [3] 3. Acute in vivo toxicity: No death or behavioral abnormalities (ataxia, somnolence) were observed in mice after a single oral administration of JNJ-16259685 (500 mg/kg) within 7 days; a single intraperitoneal injection (300 mg/kg) in rats resulted in a transient decrease in activity (recovering within 24 hours), with an LD50 > 500 mg/kg (oral) and >300 mg/kg (intraperitoneal injection) [3]
4. Chronic toxicity: After 28 days of oral administration of JNJ-16259685 (30 mg/kg/day) to rats, no changes were observed in serum liver function (ALT/AST) or kidney function (creatinine/urea); histopathological analysis of the brain, liver, kidney and testes showed no abnormalities [2]
5. Drug interactions: JNJ-16259685 (≤10 μM) does not inhibit human CYP450 enzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) in vitro, and no pharmacokinetic interactions with diazepam or fluoxetine were observed in mice [3]

[1]. JNJ16259685, a selective mGlu1 antagonist, suppresses isolation-induced aggression in male mice. Eur J Pharmacol. 2008 May 31;586(1-3):217-20.

[2]. Characterization of the selective mGluR1 antagonist, JNJ16259685, in rodent models of movement and coordination. Pharmacol Biochem Behav. 2011 Apr;98(2):181-7.

[3]. JNJ16259685, a highly potent, selective and systemically active mGlu1 receptor antagonist. Neuropharmacology. 2004 Dec;47(7):961-72.

[4]. Potent and Specific Action of the mGlu1 Antagonists YM-298198 and JNJ16259685 on Synaptic Transmission in Rat Cerebellar Slices. Br J Pharmacol. 2007 Jul;151(6):870-6.

3,4-Dihydro-2H-pyrano[2,3-b]quinoline-7-yl-(4-methoxycyclohexyl) ketone is an organic nitrogen heterocyclic compound, an oxygen heterocyclic compound, and an organic heterotricyclic compound.

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1. JNJ-16259685 is a highly efficient, selective, and systemically active metabolite glutamate receptor 1 (mGlu1) antagonist developed by Johnson & Johnson Pharmaceutical Research and Development Company[3]
2. JNJ-16259685 is a competitive antagonist of the ortho-binding site of mGlu1, blocking glutamate-mediated receptor activation and downstream Gq/PLC signaling pathways (calcium mobilization, IP3 generation)[3][4]
3. mGlu1 is a Gq-coupled receptor that regulates synaptic plasticity, motor coordination, aggression, fear memory, and pain perception in the cerebellum, striatum, and hippocampus; JNJ-16259685 is a key research tool for studying the physiological and pathological effects of mGlu1[1][2][3][4]
4. JNJ-16259685 has a broad therapeutic window, and the effective behavioral dose (1-10 mg/kg, orally) is much lower than the dose that causes motor disorders (≥100 mg/kg, orally) [2]
5. JNJ-16259685 is the first oral mGlu1 antagonist with high bioavailability and the ability to cross the blood-brain barrier. It has sub-nanomolar potency and excellent selectivity, overcoming the limitations of early mGlu1 inhibitors (poor solubility and low brain exposure) [3]

C20H23NO3

325.41

325.168

C, 73.82; H, 7.12; N, 4.30; O, 14.75

409345-29-5

11313361

White to off-white solid powder

1.21g/cm3

502.5ºC at 760 mmHg

257.7ºC

1.604

3.947

0

4

3

24

446

0

O=C(C1=CC=C2N=C3C(CCCO3)=CC2=C1)[C@H]4CC[C@@H](OC)CC4

QOTAQTRFJWLFCR-UHFFFAOYSA-N

InChI=1S/C20H23NO3/c1-23-17-7-4-13(5-8-17)19(22)14-6-9-18-16(11-14)12-15-3-2-10-24-20(15)21-18/h6,9,11-13,17H,2-5,7-8,10H2,1H3

3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl-(4-methoxycyclohexyl)methanone

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)

Solubility in Formulation 1: ≥ 2.75 mg/mL (8.45 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 27.5 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: ≥ 2.75 mg/mL (8.45 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 27.5 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: ≥ 2.75 mg/mL (8.45 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 27.5 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.)