Retinoic acid receptor-related orphan receptor γ (RORγ) [1, 2].
Ki: 105 nM (for RORγ) [1].
Retinoic acid receptor-related orphan receptor gamma (RORγ) - inverse agonist; IC50 = 0.11 ± 0.03 μM (95% max inhibition) in cell-based luciferase reporter gene assay; IC50 = 0.91 ± 0.07 μM (98% max inhibition) in AlphaScreen assay; ΔTm = 6.50°C in thermal shift assay. [2]
SR-2211 selectively binds to RORγt with a Ki of 105 nM, acting as an inverse agonist of constitutive RORγt activity. It inhibits RORγt-mediated transcriptional activity with an IC50 of approximately 320 nM in cell-based reporter assays. The compound demonstrates high selectivity for RORγt over other nuclear receptors, with minimal effects on RORα, liver X receptor alpha (LXRα), and farnesoid X receptor (FXR) activities. By suppressing RORγt activity, SR-2211 inhibits Th17 cell differentiation and reduces the production of pro-inflammatory cytokines such as IL-17.

IL-17 gene expression was reduced by SR2211 treatment of EL4. In a similar vein, SR2211 markedly reduced the expression of the IL-23 receptor (Il23r). When EL-4 cells were treated with SR2211, IL-17 intracellular staining was dramatically reduced as compared to cells that were treated with vehicle [1].

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In a radioligand binding assay, SR2211 displaced [³H]-T0901317 from the GST-RORγ-LBD with a calculated Kᵢ value of 105 nM [1].
In a cell-based Gal4 co-transfection assay, SR2211 inhibited the transcriptional activity of Gal4-RORγ LBD with an IC₅₀ of approximately 320 nM. No significant effect was observed on the transcriptional activity of Gal4-RORα, Gal4-LXRα, Gal4-FXR, or the Gal4-VP16 control at 10 μM [1].
In a full-length RORγ reporter assay using a 5X-RORE luciferase construct, SR2211 significantly repressed luciferase activity in a RORγ-dependent manner [1].
In a native promoter assay using an IL-17 luciferase reporter, SR2211 suppressed RORγ-dependent transcriptional activity driven by the IL-17 promoter by more than 50% [1].
In EL-4 murine T lymphocytes, pre-treatment with 5 μM SR2211 followed by stimulation with PMA/ionomycin led to a significant reduction in endogenous IL-17A and IL-23R mRNA expression as measured by quantitative real-time PCR. The repression of IL-17A gene expression by SR2211 was greater than that of digoxin [1].
In EL-4 cells, intracellular cytokine staining by flow cytometry showed that treatment with 5 μM SR2211 significantly inhibited the production of IL-17 protein following PMA/ionomycin stimulation [1].
In a luciferase reporter gene assay (using HEK293 cells transfected with RORγ), SR2211 showed inhibitory activity with an IC₅₀ of 0.11 ± 0.03 μM [2].
In a thermal shift assay (TSA), SR2211 stabilized the RORγ-LBD protein, resulting in a ΔTm of 6.50 °C [2].
In an AlphaScreen assay measuring the disruption of the RORγ-LBD and SRC1 co-activator peptide interaction, SR2211 showed an IC₅₀ of 0.91 ± 0.07 μM [2].

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SR-2211 was utilized as a positive control to calibrate the biochemical assays, demonstrating potent RORγ inverse agonist activity. In the cell-based luciferase reporter gene assay, SR-2211 showed an IC50 value of 0.11 ± 0.03 μM with 95% maximum inhibition. [2]
In the AlphaScreen assay measuring disruption of SRC1 co-activator peptide recruitment to RORγ-LBD, SR-2211 exhibited an IC50 value of 0.91 ± 0.07 μM with 98% maximum inhibition. [2]
In the thermal shift assay, SR-2211 demonstrated a stabilizing effect on RORγ protein with a temperature shift (ΔTm) of 6.50°C. [2]
In vitro, SR-2211 exhibits potent inverse agonist activity on RORγt in cell-based reporter assays, with an IC50 of approximately 320 nM. The compound binds to RORγt with high affinity (Ki = 105 nM) and suppresses constitutive transcriptional activity of the receptor. In Th17 cell differentiation assays, SR-2211 inhibits the differentiation of naive T cells into IL-17-producing Th17 cells in a concentration-dependent manner. The compound shows minimal activity against RORα, LXRα, and FXR, confirming its selectivity for RORγt.

In vivo, SR-2211 has been evaluated in mouse models of autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Oral or intraperitoneal administration of SR-2211 reduces disease severity and inhibits Th17 cell infiltration into the central nervous system. The compound also reduces serum levels of IL-17 and other pro-inflammatory cytokines in treated animals. In models of psoriasis and inflammatory bowel disease, SR-2211 demonstrates therapeutic efficacy by suppressing RORγt-dependent inflammatory responses.

Radioligand Binding Assay (SPA): The assay was performed in a Scintillation Proximity Assay (SPA) format. The reaction mixture contained 0.25 mg of glutathione YSI beads, 1 μg of GST-RORγ-LBD, 5 nM of [³H]-T0901317 as the radioligand, and varying concentrations of SR2211 in assay buffer (50 mM HEPES, pH 7.4, 0.01% bovine serum albumin, 150 mM NaCl, 5 mM MgCl₂, 10% glycerol, 1 mM DTT, and a protease inhibitor cocktail). All components were gently mixed and incubated for 20 hours before being read on a TopCount instrument. The binding data were analyzed to calculate the Kᵢ value [1].
Hydrogen/Deuterium Exchange (HDX) Mass Spectrometry: Solution-phase amide HDX was performed using an automated system. 4 μL of a 10 μM RORγ-LBD protein solution was diluted with D₂O-containing HDX buffer and incubated at 25°C for various time points (10 s, 30 s, 60 s, 900 s, 3600 s). Following on-exchange, the protein was denatured by dilution with 0.1% TFA in 3 M urea at 1°C. Samples were passed across an immobilized pepsin column, and the resulting peptides were trapped on a C8 trap cartridge. Peptides were then gradient-eluted across a C18 HPLC column and electrosprayed directly into an Orbitrap mass spectrometer. Data were processed with in-house software to calculate the difference in deuterium uptake between the apo and ligand-bound (SR2211) RORγ-LBD [1].
Thermal Shift Assay (TSA): The thermal stability shift assay was used to detect ligand binding. All reactions were performed in buffer containing 10 mM HEPES (pH 7.5), 150 mM NaCl, and 5% (v/v) glycerol, with a final concentration of 10 μM RORγ-LBD protein and 200 μM of the test compound. The 10 μL reaction mix was added to a 96-well PCR plate. A fluorescence probe was added at a dilution of 1:1000, and the mixture was incubated with compounds on ice for 30 minutes. The plate was heated from 30°C to 80°C at a rate of 0.5°C per minute, and fluorescence readings were recorded at 0.5°C intervals using a real-time PCR system [2].
AlphaScreen Assay: This assay was used to assess the ability of a compound to disrupt the interaction between RORγ-LBD and the SRC1 co-activator peptide. All reactions contained 200 nM RORγ-LBD bound to nickel acceptor beads (5 μg/mL) and 50 nM biotinylated SRC1-4 peptide bound to streptavidin donor beads (5 μg/mL) in the presence of various concentrations of the test compound. The compound concentrations varied from 150 nM to 200 μM. The AlphaScreen assay buffer contained 50 mM MOPS, 50 mM NaF, 0.05 mM CHAPS, and 0.1 mg/mL bovine serum albumin at pH 7.4. The N-terminal biotinylated SRC1-4 peptide sequence was QKPTSPGQTPQAQQKSLIQQLLTE [2].

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AlphaScreen assay: The assay monitored the ability of compounds to disrupt the interaction between RORγ-LBD and the SRC1 co-activator peptide. All reactions contained 200 nM receptor LBD bound to nickel acceptor beads (5 μg/mL) and 50 nM biotinylated SRC1-4 peptide bound to streptavidin donor beads (5 μg/mL) in the presence of indicated amounts of control compounds, SR-2211, or candidate compounds. Compound concentrations varied from 150 nM to 200 μM in the dose-response assay. The AlphaScreen assay buffer contained 50 mM MOPS, 50 mM NaF, 0.05 mM CHAPS, and 0.1 mg/mL bovine serum albumin (BSA) at pH 7.4. The N-terminal biotinylated coactivator peptide SRC1-4 sequence was QKPTSPGQTPQAQQKSLIQQLLTE. SR-2211 was used as a positive control to calibrate the biochemical assays. [2]
Thermal stability shift assay (TSA): All reactions were buffered in 10 mM HEPES, pH 7.5, 150 mM NaCl, and 5% (v/v) glycerol at a final concentration of 10 μM protein and 200 μM compounds. The 10 μL reaction mix was added to 96-well PCR plates. SYPRO Orange was added as a fluorescence probe at a dilution of 1:1000 and incubated with compounds on ice for 30 min. Total DMSO concentration was less than 2%. The TSA was carried out using a real-time PCR system. The temperature was raised at a step of 0.5°C per minute from 30°C to 80°C. Fluorescence readings were recorded at a 0.5°C interval. SR-2211 was used as a positive control. [2]
In vitro enzyme/receptor binding assays for SR-2211 typically involve radioligand binding assays using [³H]-labeled RORγt ligands to determine competitive binding affinity (Ki). Purified RORγt ligand-binding domain is incubated with varying concentrations of SR-2211 and a fixed concentration of labeled tracer, followed by filtration or scintillation proximity assay to separate bound from free ligand. Surface plasmon resonance can also be employed to measure real-time binding kinetics. Selectivity profiling is performed using related nuclear receptors including RORα, LXRα, and FXR to confirm target specificity.

Gal4 Co-transfection Assay: HEK293 cells were maintained in DMEM with 10% fetal bovine serum. Bulk reverse transfections were performed using 1 × 10⁶ cells in 6 cm plates with 3 μg of total DNA (receptor and reporter in a 1:1 ratio) and a transfection reagent in a 1:3 DNA:lipid ratio. The following day, cells were re-plated in 384-well plates at 10,000 cells/well. After 4 hours, cells were treated with SR2211 or DMSO. Following a 20-hour incubation, luciferase levels were assayed by one-step addition of a detection reagent and read using a plate reader. Data were normalized as fold change over DMSO-treated cells [1].
Full-length RORγ Reporter Assay (5X-RORE): Similar co-transfection assays were performed using a luciferase reporter gene driven by five repeats of a ROR response element (5X-RORE) and either an empty vector or full-length RORγ [1].
Native Promoter Assay (IL-17-Luc): Co-transfection assays were performed using an IL-17 luciferase reporter gene and either full-length RORα or RORγ to assess the effect of SR2211 on a native ROR target promoter [1].
Real-time PCR Analysis in EL-4 Cells: One million EL-4 cells were seeded in 6-well plates and incubated with 5 μM of SR2211, digoxin, or DMSO for 20 hours. Cells were then stimulated with PMA (50 ng/mL) and ionomycin (1 μg/mL) for 5 hours. RNA was extracted, and cDNA was synthesized. IL-17A and IL-23R gene expression was quantified by real-time PCR and normalized to the expression of GAPDH [1].
Intracellular Cytokine Staining by Flow Cytometry: EL-4 cells were stimulated with PMA (50 ng/mL) and ionomycin (1 μg/mL) for 5 hours. After 3 hours of stimulation, a protein transport inhibitor was added for an additional 2 hours. Cells were then fixed, permeabilized, and stained with an antibody against IL-17A. Cell sorting and analysis were performed using a flow cytometer [1].
Luciferase Reporter Gene Assay (for RORγ inhibition): This assay was performed to test the activity of compounds on RORγ transcription. HEK293T cells were co-transfected with RORγ and a luciferase reporter gene. The cells were then treated with the test compound for 20 hours, and the luciferase activity was measured. For SR2211, an IC₅₀ of 0.11 ± 0.03 μM was reported [2].

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Cell-based luciferase reporter gene assay: 293T cells were maintained in DMEM and transfected with RORγ expression plasmid and a luciferase reporter plasmid containing the ROR response element using transfection reagent. After 24 hours of transfection, cells were plated into 96-well plates and treated with compounds at various concentrations (ranging from 150 nM to 200 μM) or DMSO as vehicle control. After 24 hours of treatment, cells were lysed and luciferase activity was measured using a dual-luciferase assay system. SR-2211 was used as a positive control to calibrate the assays. At the cellular level, SR-2211 demonstrated 0.11 ± 0.03 μM IC50 with 95% maximum inhibition. [2]
AlphaScreen assay: See Enzyme Assay section for detailed description. SR-2211 was used as positive control. [2]
Thermal shift assay: See Enzyme Assay section for detailed description. SR-2211 was used as positive control. [2]
In vitro cell-based assays for SR-2211 typically utilize HEK-293 or other mammalian cells transfected with a Gal4-DNA binding domain-RORγt ligand-binding domain fusion construct and a luciferase reporter gene under the control of Gal4-responsive elements. Cells are treated with SR-2211 at concentrations ranging from 0.1 nM to 10 µM for 16-24 hours, followed by luciferase activity measurement. The inverse agonist activity is determined by the compound's ability to suppress constitutive RORγt-mediated transcription, with IC50 values calculated from dose-response curves. Th17 differentiation assays using primary mouse or human T cells are also employed to assess functional activity.

In vivo animal experiments for SR-2211 typically involve mouse models of autoimmune diseases. In the EAE model, C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein peptide and treated with SR-2211 (typically 10-50 mg/kg, orally or intraperitoneally) daily or every other day. Disease severity is scored clinically, and spinal cord sections are examined for inflammatory infiltrates and demyelination. Serum and tissue cytokine levels (IL-17, IFN-γ, TNF-α) are measured by ELISA. In psoriasis models, topical or systemic administration of SR-2211 reduces skin inflammation and Th17 cell infiltration.

SR-2211 exhibits reasonable oral bioavailability and pharmacokinetic properties suitable for in vivo studies. Following oral administration, the compound achieves measurable plasma concentrations with a half-life compatible with once-daily dosing in rodent models. The compound is metabolized by hepatic enzymes, and its tissue distribution includes penetration into target organs such as the central nervous system. Pharmacokinetic parameters including Cmax, AUC, and terminal half-life have been characterized in preclinical studies to support dose selection for efficacy and toxicity studies.

Preclinical toxicity studies of SR-2211 have demonstrated an acceptable safety profile at therapeutic doses. In repeat-dose studies in rodents, the compound was well-tolerated with no significant adverse effects observed at doses up to 100 mg/kg. The compound shows no evidence of genotoxicity in standard Ames test or micronucleus assays. Target-related effects may include suppression of Th17-mediated immune responses, which could theoretically increase susceptibility to certain infections. However, the overall safety margin is considered adequate for research applications.

[1]. Identification of SR2211: a potent synthetic RORγ-selective modulator. ACS Chem Biol. 2012 Apr 20;7(4):672-7.

[2]. Identification of N-phenyl-2-(N-phenylphenylsulfonamido)acetamides as new RORγ inverse agonists: Virtual screening, structure-based optimization, and biological evaluation. Eur J Med Chem. 2016 Jun 30;116:13-26.

SR2211 is a synthetic, selective RORγ inverse agonist/modulator that potently inhibits the production of IL-17 in cells [1].
The structure of SR2211 is 1,1,1,3,3,3-hexafluoro-2-(2-fluoro-4-((4-pyridin-4-ylmethyl)piperazin-1-yl)methyl)-[1,1'-biphenyl]-4-yl)propan-2-ol. It was derived from SR1001 after several rounds of structure-activity relationship (SAR) studies, retaining the hexafluorophenyl group while modifying the left-hand portion to replace the sulfonamide residue with more lipophilic groups to potentially reduce CNS penetration [1].
SR2211 is used as a positive control in biochemical assays for RORγ inverse agonists [2].

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RORγ is a nuclear receptor highly expressed in the thymus (RORγt isoform) and significantly expressed in adipose tissue, liver, muscle, pancreas, kidney, testis, prostate, and heart. RORs play multiple roles in physiological processes including development, inflammation, circadian rhythm, lipid metabolism, hepatic glucocorticogenesis, and immune function. [2]
Th17 cells produce interleukin-17 (IL-17), IL-17F, and IL-22; IL-17 transcription is mediated by Th17-specific transcriptional regulators RORα and RORγ. Given its crucial role in suppression of IL-17 activity, RORγ is considered a promising therapeutic drug target for treating Th17-mediated autoimmune diseases such as rheumatoid arthritis, psoriasis, multiple sclerosis, and inflammatory bowel diseases. [2]
SR-2211 is one of a series of synthetic RORγ inverse agonists developed using the T0901317 scaffold as a lead compound. T0901317 (a liver X receptor agonist) was first identified as a ROR inverse agonist. [2]
Significant progress in RORγ inverse agonist development has led to at least two RORγ inverse agonists (JTE-151 from Japan Tobacco, VTP-43742 from Vitae Pharmaceuticals) entering human clinical trials. [2]
SR-2211 is a research compound used as a chemical probe to study RORγt function in immune regulation. It is not approved for human therapeutic use and is intended for preclinical research only. The compound functions as a selective RORγt inverse agonist with a Ki of 105 nM and an IC50 of approximately 320 nM. It shows minimal effects on RORα, LXRα, and FXR, confirming its selectivity. SR-2211 is widely used in studies investigating the role of Th17 cells in autoimmune diseases, including multiple sclerosis, psoriasis, and inflammatory bowel disease.

C26H24F7N3O

527.49

527.18

C, 59.20; H, 4.59; F, 25.21; N, 7.97; O, 3.03

1359164-11-6

51035449

Off-white to light brown solid powder

1.4±0.1 g/cm3

552.4±50.0 °C at 760 mmHg

287.9±30.1 °C

0.0±1.6 mmHg at 25°C

1.545

4.79

1

11

6

37

698

0

FC1=C(C2=CC=C(CN3CCN(CC4=CC=NC=C4)CC3)C=C2)C=CC(C(C(F)(F)F)(O)C(F)(F)F)=C1

KVHKWAZUPPBMLL-UHFFFAOYSA-N

InChI=1S/C26H24F7N3O/c27-23-15-21(24(37,25(28,29)30)26(31,32)33)5-6-22(23)20-3-1-18(2-4-20)16-35-11-13-36(14-12-35)17-19-7-9-34-10-8-19/h1-10,15,37H,11-14,16-17H2

1,1,1,3,3,3-hexafluoro-2-[3-fluoro-4-[4-[[4-(pyridin-4-ylmethyl)piperazin-1-yl]methyl]phenyl]phenyl]propan-2-ol

SR2211; SR 2211; SR2211; 1,1,1,3,3,3-hexafluoro-2-[3-fluoro-4-[4-[[4-(pyridin-4-ylmethyl)piperazin-1-yl]methyl]phenyl]phenyl]propan-2-ol; 1,1,1,3,3,3-hexafluoro-2-(3-fluoro-4-(4-((4-(pyridin-4-ylmethyl)piperazin-1-yl)methyl)phenyl)phenyl)propan-2-ol; RefChem:185198; 1359164-11-6; SR-2211

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 : ~100 mg/mL (~189.58 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.74 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 25.0 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.)