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Purity: ≥98%
GNE-0946 (GNE0946) is a novel, potent and selective RORγ( RORc) inverse agonist with the potential to be used for treating autoimmune diseases. It activates RORγ with an EC50 value of 4 nM in HEK-293 cells. The nuclear receptor (NR) retinoic acid receptor-related orphan receptor gamma (RORγ, RORc, or NR1F3) is a promising target for the treatment of autoimmune diseases. RORc is a critical regulator in the production of the pro-inflammatory cytokine interleukin-17. GNE-0946 displayed >300-fold selectivity for RORc over the other ROR family members, PPARγ, and NRs in our cellular selectivity panel. The favorable potency, selectivity, and physiochemical properties of GNE-0946 and GNE-6468, in addition to their potent suppression of IL-17 production in human primary cells, support their use as chemical biology tools to further explore the role of RORc in human biology.
ln Vitro |
We examined 2, 9/GNE-0946, 10, 18, 24, and 28/GNE-6468 in a series of HEK-293 cell Gal4-ROR construct human NR-dependent transcriptional reporter assays (Table 5). We profiled the three isoforms of human ROR (RORc, RORb, and RORa) by monitoring the suppression of their basal transcriptional activity in the absence of any exogenous agonist. In order to assess the NR cellular selectivity of the potent RORc inverse agonists, we also tested these compounds in a panel of cellular reporter assays of human farnesoid X receptor (FXR), liver X receptor (LXR)-α, LXRβ, and PXR in both agonist mode (no agonist ligand added) and antagonist mode (using T0901317 [N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide] as an exogenous ligand). All of these compounds demonstrated favorable RORc inverse agonist cellular potency values (EC50 = 4–36 nM), with no detectable activity against the other ROR family members (up to 10 μM concentration). In addition, none of these compounds demonstrated detectable activity against the other NRs in our cell assay panel in agonist or antagonist mode (up to 10 μM concentration). These results demonstrated the excellent cellular selectivity profiles of 2, 9/GNE-0946, 10, 18, 24, and 28/GNE-6468 for RORc over other NRs (>277 to >1000-fold selectivity). [1]
Based on their favorable RORc cell potency values, we progressed 2, 9/GNE-0946, 10, 18, 24, and 28/GNE-6468 into human PBMC cytokine production assays to assess their abilities to inhibit the production of IL-17 (Table 6). Compounds 2, 10, 18, and 24 displayed modest inhibition of IL-17 production in the human PBMC assay (EC50 = 230, 120, 230, and 300 nM, respectively), whereas 9/GNE-0946 and 28 displayed more potent inhibition of IL-17 production (EC50 = 17 and 30 nM, respectively). Compounds 9/GNE-0946 and 28 possessed a unique substructure in contrast to 2, 10, 18, and 24, indicating that the 1-(3′-hydroxy-4′-benzoic acid) motif present in 9/GNE-0946 and 28 may engender enhanced potency in the IL-17 production assay. It was also noteworthy that none of the compounds showed any activity in interferon (IFN)-γ or CellTiter-Glo® (CTG) counter screen assays, demonstrating that the compounds were not indiscriminately suppressing cytokine production, nor were they grossly cytotoxic. [1] Compounds 9/GNE-0946 and 28/GNE-6468 were potent inhibitors of IL-17 production in human PBMC cells (EC50 values ⩽30 nM, Table 6), yet they were no more effective in further suppressing the maximum percent inhibition of IL-17 production (%max) than compounds that were an order of magnitude less potent (i.e., 2, 10, 18, and 24). Indeed, we have noted in our previously-disclosed human PBMC data with several RORc inverse agonist chemotypes that the IL-17 PBMC %max values average 77% inhibition (±23% of the %max inhibition value for the 13 different human IL-17 PBMC assay results we have disclosed where the IL-17 PBMC EC50 = 0.044–3.0 μM). The lack of complete suppression of IL-17 production in human PBMCs (i.e., IL-17 PBMC %max ⩾99%) may be due to the role of RORa in the production of IL-17. Previous mouse genetic knock-out studies have demonstrated that the production of murine IL-17 is dependent on both murine RORα and RORγ. Potent and selective human RORa inverse agonists would be required to explore this hypothesis in the context of human IL-17 production. The deficiency of potent and selective human RORa tool compounds is the focus of several research programs [1]. |
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ln Vivo |
Compound 9 /GNE-0946 was profiled in a rodent in vivo pharmacokinetic (PK) experiment (Table 8) to determine if the in vitro rat hepatic clearance estimate (Table 7) was predictive of its in vivo clearance value. In a rat PK experiment, 9/GNE-0946 demonstrated a high plasma clearance value (CLp = 130 mL/min/kg) that did not correlate with its in vitro clearance value (Rat Hep CLhep = 32 mL/min/kg). The in vivo plasma clearance of 9/GNE-0946 was in excess of rodent liver blood flow (55 mL/min/kg), indicating that extrahepatic clearance mechanisms may also be participating in the metabolism of 9/GNE-0946. [1]
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Enzyme Assay |
The human PPARγ-LBD fluorescence polarization binding assay was conducted using the Life Technologies ‘Polar-Screen™ PPAR Gamma Competitor Assay, Green’ assay kit. For additional details on this assay, see: https://tools.lifetechnologies.com/content/sfs/manuals/Polar_Screen_PPARgamma_competitor_Assay_Green_PI.pdf [1].
Biochemical and Cellular Assays [1] For the detailed biochemical and cellular assay protocols, see the Supplementary Data section of: Fauber, B. P.; Boenig, G.; Burton, B.; Eidenschenk, C.; Everett, C.; Gobbi, A.; Hymowitz, S. G.; Johnson, A. R.; Liimatta, M.; Lockey, P.; Norman, M.; Ouyang, W.; René, O.; Wong, H. Bioorg. Med. Chem. Lett. 2013, 23, 6604. Aqueous Solubility Determinations [1] Compounds were dissolved in DMSO to a concentration of 10 mM. These solutions were diluted into PBS buffer (pH 7.2, composed of NaCl, KCl, Na2HPO4, and KH2PO4) to a final compound concentration of 100 µM, DMSO concentration of 2%, at pH 7.4. The samples were shaken for 24 h at 23 °C, followed by filtration. LC/CLND was used to determine compound concentration in the filtrate, with the concentration calculated by a caffeine calibration curve and the sample’s nitrogen content. An internal standard compound was spiked into each sample for accurate quantification. Metabolic Stability Studies in Human and Rat Liver Microsomes [1] The metabolic stability of tested compounds was evaluated in either pooled mixed gender human (150 donor pool) or mixed gender Sprague-Dawley rat (10 donor pool) liver microsomes. The incubation mixture (final incubation volume = 75 µL) was prepared in 0.1 M potassium phosphate buffer (pH 7.4) containing 0.5 mg/mL microsomal protein, 1 mM NADPH, and 1 µM of the tested compounds. The liver microsomes were pre warmed at 37 °C for 5 minutes before the compound was added. Reactions were initiated with the addition of the NADPH and test compound. The mixtures were incubated at 37 °C for 0, 20, 40, and 60 min. At each time point, a 15 µL aliquot of reaction was quenched with 30 µL of acetonitrile with an internal standard. Samples were centrifuged at 2000 g for 10 min. Supernatant (30 µL) was diluted in water (60 µL) before LC MS/MS analysis. The hepatic clearance (CLhep, or predicted hepatic clearance) was calculated using the well-stirred model and was scaled from the intrinsic clearance (CLint) using the equation: CLhep = Q × [(fu × CLint)/(fu × CLint/fumic + Q)], where Q is the hepatic blood flow and fu is the unbound fraction. The fu in the microsomal incubation and in blood was assumed to be equal to one. Scaling factors are listed in the table below. For additional details, see Khojasteh, S. C.; Wong, H.; Hop, C. E. C. A. Prediction of Human Pharmacokinetics. Drug Metabolism and Pharmacokinetics Quick Guide, Springer Press: New York, 2011; pp. 134-135. Plasma Protein Binding Assay [1] The extent of protein binding (n = 2) was determined in vitro in pooled human and rodent plasma by equilibrium dialysis using a Rapid Equilibrium Dialysis (RED) device in 48 well format with a molecular weight cut-off of 8,000 Daltons. Test compounds were dissolved in dimethyl sulfoxide (DMSO) and added to plasma for a final concentration of 5 µM in the plasma. The final DMSO percentage was 1%. 300 µL of plasma with the compound present were dialyzed against 500 µL PBS on a shaking platform in a humidified incubator for 4 h at 37 ºC. Post-equilibrium buffer and plasma samples were transferred to a 96-well plate and the opposing matrix was added. Plasma proteins were precipitated using 65% acetonitrile containing an internal standard (100 nM propranolol). Concentration of compounds in plasma and PBS were measured by LC-MS/MS and the percent unbound fraction (fu) was calculated using the equation: % fu = [((PA in buffer)/(IS PA))/((PA in plasma)/(IS PA))] × 100, where PA represents the analyte peak area and IS PA represents the internal standard peak area. |
Cell Assay |
Madin-Darby Canine Kidney (MDCK) Permeability Assay [1]
The Madin-Darby canine kidney (MDCK) cells were seeded at an initial concentration of 2.5 x 105 cells/mL in 24 well plates and allowed to grow for 4 d at 37 ºC, 95% humidity, and 5% CO2 in culture with Eagle’s Minimum Essential Medium Earle’s BSS (0.1% nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate) supplemented with 10% fetal bovine serum. The MDCK monolayers were equilibrated for 30 min in transport buffer (HBSS with 10 mM HEPES, pH 7.4) at 37 ºC with 5% CO2 and 95% humidity prior to the experiment. The permeability of test compounds was examined at 2 µM in the apical to basolateral (A→B) direction. The dose solutions also contained the monolayer integrity marker LY (100 µM) and a final DMSO percentage of 0.4%. The test compounds were analyzed by LC MS/MS. The apparent permeability (Papp) apical to basolateral (A→B) was calculated using the following equation: Papp(A→B) = (dQ/dt) × (1/C0) × (1/A), where dQ/dt is the rate of compound appearance in the receiver compartment (Q is the quantity of compound), C0 is the concentration in the donor compartment, and A is the surface area of the insert. |
Animal Protocol |
In Vivo Rat PK Rat Assay [1]
Compounds (e.g. GNE-0946) were co administered intravenously in a vehicle consisting of DMSO/PEG400/saline at a ratio of 25/60/15. The study compounds were dosed with other compounds (n = 9 in total) to male Sprague Dawley rats (n = 3) at a dose of 0.5 mg/kg for each compound. The same set of compounds was co administered orally to rats (n = 3) at a dose of 1.5 mg/kg in a vehicle consisting of suspension of DMSO/MCT (37/63 v/v). Dose volume was 1 mL/kg and 2 mL/kg for the intravenous and oral experiments, respectively. Blood samples were collected via the jugular vein at 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 6, and 8 h post dose for both the intravenous and oral experiments. Plasma was separated by centrifugation and stored frozen at 20 °C until analysis. Drug concentrations were determined in the plasma samples using LC MS/MS. Pharmacokinetic parameters were determined using standard non-compartmental methods.
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ADME/Pharmacokinetics |
We also evaluated 9/GNE-0946 and 28/GNE-6468 in a suite of in vitro ADME assays (Table 7).43, 44 Compound 9/GNE-0946 displayed moderate predicted hepatic clearance (CLhep) values in human and rodent hepatocyte metabolic stability assays (CLhep = 9 and 32 mL/min/kg, respectively), whereas 28/GNE-6468 displayed high predicted clearance values in human and rodent hepatocytes (CLhep = 17 and 42 mL/min/kg, respectively). Further profiling of the compounds in human and rat plasma-protein binding (PPB) assays demonstrated that GNE-0946/9 and 28/GNE-6468 were highly protein bound in both species (%bound = >99% and 99%, respectively). Compounds 9 and 28/GNE-6468 displayed favorable apparent permeability (Papp(A→B) = 12 and 8 × 10−6 cm/s, respectively) with minimal efflux (Papp(A→B)/Papp(B→A) < 2) in a Madin–Darby canine kidney (MDCK) cellular permeability assay. Both 9 and 28/GNE-6468 were highly soluble as assessed by a kinetic aqueous solubility assay (45–86 μM). Thus, 9 and 28/GNE-6468 were highly potent and selective RORc inverse agonists with favorable properties and moderate-to-high clearance values. [1]
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References | |
Additional Infomation |
The nuclear receptor (NR) retinoic acid receptor-related orphan receptor gamma (RORγ, RORc, or NR1F3) is a promising target for the treatment of autoimmune diseases. RORc is a critical regulator in the production of the pro-inflammatory cytokine interleukin-17. We discovered a series of potent and selective imidazo[1,5-a]pyridine and -pyrimidine RORc inverse agonists. The most potent compounds displayed >300-fold selectivity for RORc over the other ROR family members, PPARγ, and NRs in our cellular selectivity panel. The favorable potency, selectivity, and physiochemical properties of GNE-0946 (9) and GNE-6468 (28), in addition to their potent suppression of IL-17 production in human primary cells, support their use as chemical biology tools to further explore the role of RORc in human biology. [1]
A very recent set of patent applications from Glenmark Pharmaceuticals describe two series of compounds related to 1. It is unknown if the compounds in the Glenmark patent applications possess similar metabolic stabilities as those observed with 9/GNE-0946 and 28/GNE-6468. In conclusion, we evolved a literature series of RORc inverse agonists (i.e., 1) into a series of imidazo[1,5-a]pyridine and -pyrimidines with improved selectivity for RORc over PPARγ. Several compounds within the imidazo[1,5-a]pyridine and -pyrimidine series demonstrated potent RORc inverse agonist activity in biochemical and cellular assays. The most potent compounds also displayed >300-fold selectivity for RORc over the other ROR family members and NRs in our cellular selectivity panel. The favorable potency, selectivity, and physiochemical properties of GNE-0946 (9) and GNE-6468 (28), in addition to their potent suppression of IL-17 production in human primary cells, support their use as chemical biology tools to further explore the role of RORc in human biology. [1] |
Molecular Formula |
C22H12CLF3N2O4
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Molecular Weight |
460.789895057678
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Exact Mass |
460.043
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Elemental Analysis |
C, 57.35; H, 2.63; Cl, 7.69; F, 12.37; N, 6.08; O, 13.89
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CAS # |
1677667-24-1
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PubChem CID |
91664317
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Appearance |
Light yellow to green yellow solid powder
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LogP |
6.4
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Hydrogen Bond Donor Count |
2
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Hydrogen Bond Acceptor Count |
8
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Rotatable Bond Count |
4
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Heavy Atom Count |
32
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Complexity |
724
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Defined Atom Stereocenter Count |
0
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SMILES |
ClC1=CC=CC(C(F)(F)F)=C1C(C1=NC(C2C=CC(C(=O)O)=C(C=2)O)=C2C=CC=CN21)=O
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InChi Key |
UAAUZNSONHHQQI-UHFFFAOYSA-N
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InChi Code |
InChI=1S/C22H12ClF3N2O4/c23-14-5-3-4-13(22(24,25)26)17(14)19(30)20-27-18(15-6-1-2-9-28(15)20)11-7-8-12(21(31)32)16(29)10-11/h1-10,29H,(H,31,32)
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Chemical Name |
4-[3-[2-chloro-6-(trifluoromethyl)benzoyl]imidazo[1,5-a]pyridin-1-yl]-2-hydroxybenzoic acid
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Synonyms |
GNE-0946; 1677667-24-1; 4-[3-[2-chloro-6-(trifluoromethyl)benzoyl]imidazo[1,5-a]pyridin-1-yl]-2-hydroxybenzoic acid; CHEMBL3598148; SCHEMBL18064640; UAAUZNSONHHQQI-UHFFFAOYSA-N; CSC66724;
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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 : ~100 mg/mL (~217.02 mM)
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Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.43 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 | 2.1702 mL | 10.8509 mL | 21.7019 mL | |
5 mM | 0.4340 mL | 2.1702 mL | 4.3404 mL | |
10 mM | 0.2170 mL | 1.0851 mL | 2.1702 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.