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Purity: ≥98%
JNJ-31020028 (JNJ31020028) is a potent, high-affinitiy, selective and brain penetrant small molecule antagonist of the neuropeptide Y(2) receptor with pIC50=8.07 (human); pIC50=8.22 (rat); it is >100-fold selective versus human Y1/Y4/Y5 receptors. JNJ-31020028 was >100-fold selective against human Y(1), Y(4), and Y(5) receptors and bound with high affinity (pIC(50) = 8.07 +/- 0.05, human, and pIC(50) = 8.22 +/- 0.06, rat). Functional assays showed that JNJ-31020028 is an antagonist (pK(B) = 8.04 +/- 0.13). After being administered subcutaneously to rats, JNJ-31020028 occupied 90% of the binding sites for the Y(2) receptor at a dose of 10 mg/kg. In line with the colocalization of norepinephrine and neuropeptide Y, JNJ-31020028 enhanced norepinephrine release in the hypothalamus. It was discovered that JNJ-31020028 was ineffective in a number of anxiety models, but that it prevented stress-induced increases in plasma corticosterone without changing basal levels and restored basal food intake in stressed animals.
| Targets |
rat Y2 receptor ( pIC50 = 8.22 ); human Y2 receptor ( pIC50 = 8.07 )
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| ln Vitro |
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| ln Vivo |
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| Enzyme Assay |
In functional assays, JNJ-31020028 was shown to be an antagonist (pK(B) = 8.04 +/- 0.13).
Radioligands binding assays[1] Competition binding assays were performed as previously described (Bonaventure et al. 2004) using [125I]PYY for Y1, Y2, and Y5 receptor and [125I]PP for Y4 receptor. Cells used in the radioligand binding experiments with NPY receptor subtypes were SK-N-MC endogenously expressing Y1 receptors, KAN-Ts endogenously expressing Y2 receptors, Chinese hamster ovary (CHO) cells transfected with human Y4 cDNA for Y4 receptors, and HEK-293 transfected with human Y5 cDNA for Y5 receptors. Membranes from rat and mouse hippocampus were also prepared and assayed for [125I]PYY binding as previously described (Bonaventure et al. 2004). IC50 values (i.e., concentration of unlabeled antagonist required to compete for 50% of specific binding to the radioligand) were calculated using the GraphPad Prism software with a fit to a sigmoidal dose–response curve. Data were expressed as pIC50 values where pIC50 = −log IC50. In addition, the selectivity of JNJ-31020028 was evaluated in a large variety of ion channels, transporters, receptor binding, and kinase assays.[1] |
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| Cell Assay |
Calcium mobilization assays[1]
A calcium mobilization assay was established by stably expressing a chimeric G protein Gqi9 in KAN-Ts cells endogenously expressing Y2 receptors as previously described (Dautzenberg 2005). Briefly, dye-loaded cells were plated on to 96-well ViewPlates and incubated at 37°C, 5% CO2 for 1 h. For antagonist potency determinations, cells were pre-incubated with the compounds (diluted in Dulbecco’s modified Eagle medium/F-12) for 10 min before agonist (PYY 10 nM) stimulation. Ligand-induced calcium release was measured using a Fluorometric Imaging Plate Reader. Functional responses were measured as peak fluorescence intensity minus basal. The concentration of agonist that produced a half-maximal response is represented by the EC50 value. Antagonistic potency values were converted to apparent pKB values using a modified Cheng–Prusoff correction. Apparent pKB = −log IC50/1 + [conc agonist/EC50]. Data are expressed as mean ± SEM. |
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| Animal Protocol |
Dissolved in 20% 2-hydroxypropyl-beta-cyclodextrin for systemic treatment[2]; or dissolved in 1% DMSO and sterile saline for i.c.v. studies[2];
0, 15, 30, and 40 mg/kg, subcutaneousRats [olfactory bulbectomized (OBX) rat] Ex vivo receptor occupancy[1] Male Sprague-Dawley rats (300–350 g) were treated by s.c. administration of vehicle or JNJ-31020028 (three animals per dose and three animals per time point). JNJ-31020028 was formulated at 1–15 mg/ml in 40% 2-hydroxypropyl-beta-cyclodextrin or 5% pharmasolve, 19% 2-hydroxypropyl-beta-cyclodextrin, and delivered in a volume of 1 or 2 ml/kg. The animals were euthanized at various time points after drug administration using carbon dioxide and brain tissues were collected. Ex vivo receptor binding autoradiography was performed on brain sections as previously described (Bonaventure et al. 2004). Twenty-micron-thick coronal sections at the level of the hypothalamic regions were collected and incubated as described in the in vitro autoradiography section but with the following modification: The sections were not washed prior to incubation and were incubated 10 min with 100 pM [125I]PYY in the presence of 1 μM BIBP-3226 (R-N2-(diphenylacetyl)-N-(4-hydroxyphenyl)-methyl argininamide) for Y1 receptor occlusion.[1] Pharmacokinetics and bioanalysis[1] One group of nine male Sprague-Dawley rats (three animals per route, ~400 g) was used. The pharmacokinetic parameters of JNJ-31020028 were determined after acute bolus oral, subcutaneous, and intravenous administration. For oral administration, JNJ-31020028 was formulated at 5 mg/ml in 40% sulfobutyl-ether-beta-cyclodextrin and delivered in a volume of 2 ml/kg. For subcutaneous administration, JNJ-31020028 was formulated at 10 mg/ml in 40% sulfobutyl-ether-beta-cyclodextrin and delivered in a volume of 1 ml/kg. For intravenous administration, JNJ-31020028 was formulated at 1 mg/ml in 40% sulfobutyl-ether-beta-cyclodextrin and delivered in a volume of 1 ml/kg. Blood samples (250 μl) were taken from the lateral tail vein into heparinized Natelson blood collection tubes and expelled into 1.5 ml microcentrifuge tubes. The blood samples were centrifuged for 5 min at 18,000×g in a microcentrifuge. Plasma was retained and kept in a −20°C freezer until analysis by liquid chromatography–mass spectrometry (LC–MS)/MS. [1] View More
Microdialysis and HPLC[1] Tests for anxiolytic activity[1] The elevated plus maze test was performed by Porsolt and Partners Pharmacology in male Wistar rats (Elevage Janvier, France; 180–260 g). The plus maze consisted of four arms (50 × 10 cm) elevated 65 cm from the floor. JNJ-31020028 (1–10 mg/kg, s.c.) or vehicle (20% 2-hydroxypropyl-beta-cyclodextrin at 5 ml/kg) was injected 30 min before the 5-min test. Time spent in the open arms and total number of entries were recorded and analyzed with a one-way ANOVA and Newman–Keuls post hoc when appropriate. In the Vogel lick suppression test, male Sprague-Dawley rats (300–350 g) were used. Animals were water-deprived for 24 h before the test and, 30 min before the test, were injected with JNJ-31020028 (3–10 mg/kg, s.c.) or vehicle (20% 2-hydroxypropyl-beta-cyclodextrin at 1 ml/kg). During the test, animals were able to lick a waterspout to obtain water, and after the completion of every 20 licks, a 0.4-mA intensity shock was delivered through the waterspout. A separate group of control animals received no shocks. The total number of licks during the 5-min test was analyzed with a Kruskal–Wallis test followed by Dunn’s multiple comparison tests. The light–dark test was performed by Porsolt and Partners Pharmacology using male NMRI mice (Elevage Janvier, France; 20–30 g). The testing apparatus consisted of two compartments, one lit and open (25 × 27 × 27 cm) and the other dark and closed (20 × 27 × 27 cm). JNJ-31020028 (1–10 mg/kg, s.c.) or vehicle (20% 2-hydroxypropyl-beta-cyclodextrin at 10 ml/kg) was injected 15 min before the 3-min test. The time spent in the light compartment and the number of crossings were analyzed with a one-way ANOVA and Newman–Keuls post hoc when appropriate. The stress-induced hyperthermia test was conducted by Porsolt Partners Pharmacology using male NMRI mice (20–30 g) using the procedure previously described by Lecci et al. (1990). Briefly, on the day of the test, three mice from a cage of three were removed, weighed, and injected with the same treatment, either JNJ-31020028 (3–10 mg/kg, s.c.) or vehicle (20% 2-hydroxypropyl-beta-cyclodextrin at 10 ml/kg), and then placed in a new cage without bedding. The next 12 mice were only handled and placed in a new cage without bedding. The final three mice were removed, weighed, injected with the same treatment as the first three mice, and then placed into a new cage without bedding. After this, all mice were returned to the homecage. Sixty minutes after manipulating the first cage of mice, all the mice were removed in the same order, and rectal temperature was measured in the first and last set of three mice (Physitemp Instruments: Digital Laboratory Thermometer Model BAT-12). This procedure was repeated to obtain an N of 9 per group. Drug effects on basal body temperature were determined by comparing body temperatures of the first set of mice, while drug effects on stress-induced body temperature were determined by comparing the body temperature of the last set of mice, using a one-way ANOVA and Newman–Keuls post hoc test when appropriate. [1] Stress-induced corticosterone levels[1] Male Sprague-Dawley rats (240 ± 5 g) were used. Animals were injected with JNJ-31020028 (3–20 mg/kg, s.c.) or vehicle (5% pharmasolve, 19% 2-hydroxypropyl-beta-cyclodextrin) 60 min before blood collection for corticosterone analysis. In some cases, animals were subjected to a 20-min restraint stress in clear, plexiglass tubes starting 40 min after injection. Corticosterone was analyzed with the Coat-A-Count Rat Corticosterone (DPC TKRC-1) kit per the manufacturer’s instructions. Briefly, 50 μl of rat plasma was added to the antibody-coated tubes followed by 1 ml of [125I]-rat corticosterone. The samples were incubated for 2 h at room temperature. The solution was decanted and the tubes were counted in a gamma counter. The level of corticosterone was calculated from a standard curve generated with the calibrators supplied in the kit. Corticosterone levels were analyzed with a one-way ANOVA and Newman–Keuls post hoc. Stress-induced anorexia[1] Male Sprague-Dawley rats weighing 250–275 g at the start of the study and food-deprived for 18 h prior to the refeeding test were used. On the day of the experiment, animals were habituated for 1 h to cage racks lined with photobeams (a grid of 4 × 8; Hamilton-Kinder, San Diego, CA, USA) that automatically recorded locomotor activity in the home cage. Animals were injected with JNJ-31020028 (3–20 mg/kg, s.c. in 20% 2-hydroxypropyl-beta-cyclodextrin at 1 ml/kg 1 h before the start of restraint stress), diazepam (3 mg/kg, s.c. in 10% ethanol, 40% propylene glycol at 2 ml/kg 10 min before the start of the restraint stress), or the respective vehicles. Animals that received a pharmacological treatment then underwent 1 h of restraint stress in clear, plexiglass tubes. Another set of animals that served as an additional control received no pharmacological treatment or stress but remained in the home cage without food or water for 1 h. Immediately following the restraint stress, animals were placed back into the home cage, at which time all animals received a premeasured amount of standard rodent chow. Locomotor activity was recorded during the test, and the amount of food remaining was weighed 1 h later. In a parallel experiment, JNJ-31020028 (20 mg/kg, s.c.) or vehicle was given to nonstressed animals, either food-deprived or nonfood-deprived, 2 h before feeding using a similar protocol. Food intake was analyzed as the weight difference in food before and after the test (in grams) and locomotor activity as the total distance traveled (in centimeters) with an ANOVA and Duncan’s post hoc test when appropriate. |
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| ADME/Pharmacokinetics |
Pharmacokinetics of JNJ-31020028 in rat [1]
JNJ-31020028 was administered to rats by the oral (10 mg/kg), intravenous (1 mg/kg), and subcutaneous (10 mg/kg) route, and pharmacokinetic parameters were determined (Fig. 5; Table 2). The compound had poor oral bioavailability (6%) but high subcutaneous bioavailability (100%). Subcutaneous dosing was the preferred route for several of the models described in this paper and yielded good plasma levels (C max 4.35 μmol/l) and fast absorption with a 0.83-h half-life. |
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| References | ||
| Additional Infomation |
Rationale: The lack of potent, selective, brain penetrant Y(2) receptor antagonists has hampered in vivo functional studies of this receptor.[1]
Objective: Here, we report the in vitro and in vivo characterization of JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a novel Y(2) receptor antagonist.[1] Methods: The affinity of JNJ-31020028 was determined by inhibition of the PYY binding to human Y(2) receptors in KAN-Ts cells and rat Y(2) receptors in rat hippocampus. The functional activity was determined by inhibition of PYY-stimulated calcium responses in KAN-Ts cells expressing a chimeric G protein Gqi5 and in the rat vas deferens (a prototypical Y(2) bioassay). Ex vivo receptor occupancy was revealed by receptor autoradiography. JNJ-31020028 was tested in vivo with microdialysis, in anxiety models, and on corticosterone release. [1] Results: JNJ-31020028 bound with high affinity (pIC(50) = 8.07 +/- 0.05, human, and pIC(50) = 8.22 +/- 0.06, rat) and was >100-fold selective versus human Y(1), Y(4), and Y(5) receptors. JNJ-31020028 was demonstrated to be an antagonist (pK(B) = 8.04 +/- 0.13) in functional assays. JNJ-31020028 occupied Y(2) receptor binding sites (approximately 90% at 10 mg/kg) after subcutaneous administration in rats. JNJ-31020028 increased norepinephrine release in the hypothalamus, consistent with the colocalization of norepinephrine and neuropeptide Y. In a variety of anxiety models, JNJ-31020028 was found to be ineffective, although it did block stress-induced elevations in plasma corticosterone, without altering basal levels, and normalized food intake in stressed animals without affecting basal food intake.[1] Conclusion: These results suggest that Y(2) receptors may not be critical for acute behaviors in rodents but may serve modulatory roles that can only be elucidated under specific situational conditions.[1] |
| Molecular Formula |
C34H36FN5O2
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| Molecular Weight |
565.68
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| Exact Mass |
565.285
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| Elemental Analysis |
C, 72.19; H, 6.41; F, 3.36; N, 12.38; O, 5.66
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| CAS # |
1094873-14-9
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| Related CAS # |
(R)-JNJ-31020028; 1094873-17-2; 1094873-16-1 (S-isomer); 1094873-14-9 (recemate)
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| PubChem CID |
25134625
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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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| Boiling Point |
677.5±55.0 °C at 760 mmHg
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| Flash Point |
363.5±31.5 °C
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| Vapour Pressure |
0.0±2.1 mmHg at 25°C
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| Index of Refraction |
1.628
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| LogP |
5.23
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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 |
9
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| Heavy Atom Count |
42
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| Complexity |
857
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| Defined Atom Stereocenter Count |
0
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| SMILES |
0
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| InChi Key |
OVUNRYUVDVWTTE-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C34H36FN5O2/c1-3-38(4-2)34(42)32(25-11-6-5-7-12-25)40-21-19-39(20-22-40)31-17-16-27(23-30(31)35)37-33(41)29-15-9-8-14-28(29)26-13-10-18-36-24-26/h5-18,23-24,32H,3-4,19-22H2,1-2H3,(H,37,41)
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| Chemical Name |
N-[4-[4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl]-3-fluorophenyl]-2-pyridin-3-ylbenzamide
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| Synonyms |
JNJ 31020028; JNJ31020028; 73F8XED6YP; N-[4-[4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl]-3-fluorophenyl]-2-pyridin-3-ylbenzamide; CHEMBL1823342; N-(4-(4-(2-(diethylamino)-2-oxo-1-phenylethyl)piperazin-1-yl)-3-fluorophenyl)-2-(pyridin-3-yl)benzamide; JNJ-31020028
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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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.42 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 (4.42 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 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 (4.42 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 | 1.7678 mL | 8.8389 mL | 17.6778 mL | |
| 5 mM | 0.3536 mL | 1.7678 mL | 3.5356 mL | |
| 10 mM | 0.1768 mL | 0.8839 mL | 1.7678 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.
(A and C) Digitized computer images of distribution of125I-PYY (A) andN-11C-methyl-JNJ-31020028 (C) binding sites in 20-μm sagittal cryosections of pig brain. Anterior part of brain has been removed so that sections could fit on double-sized microscope slides. (B and D) Inhibition plots of125I-PYY (B) orN-11C-methyl-JNJ-31020028 (D) binding in presence of JNJ-31020028.J Nucl Med.2014 Apr;55(4):635-9. |
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Parametric images ofN-11C-methyl-JNJ-31020028 BPNDin living pig brain. Image is mean of 4 separate determinations, resampled into MR imaging–based common stereotactic space for pig brain and shown superimposed on MR imaging atlas.
Time course of radioactivity concentration studied by PET in living pig brain after intravenous injection ofN-11C-methyl-JNJ-31020028.J Nucl Med.2014 Apr;55(4):635-9. |
Metabolism ofN-11C-methyl-JNJ-31020028 measured in blood plasma of pigs after intravenous injection. Symbols are as follows:N-[11C]methyl-JNJ-31020028 (●), first-eluting metabolite (○), second-eluting metabolite (□).
Whole-body distribution ofN-11C-methyl-JNJ-31020028 in pig.J Nucl Med.2014 Apr;55(4):635-9. |
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