Physicochemical Properties
| Molecular Formula | C25H20F7NO4S |
| Molecular Weight | 563.48443031311 |
| Exact Mass | 563.1 |
| CAS # | 2349368-16-5 |
| PubChem CID | 138105957 |
| Appearance | Off-white to light yellow solid powder |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 682.4±55.0 °C at 760 mmHg |
| Flash Point | 366.5±31.5 °C |
| Vapour Pressure | 0.0±2.2 mmHg at 25°C |
| Index of Refraction | 1.544 |
| LogP | 5.56 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 11 |
| Rotatable Bond Count | 7 |
| Heavy Atom Count | 38 |
| Complexity | 888 |
| Defined Atom Stereocenter Count | 0 |
| InChi Key | AUIAOCHKUNGZHV-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C25H20F7NO4S/c1-2-38(36,37)19-10-3-15(4-11-19)13-22(34)33-18-8-5-16(6-9-18)20-12-7-17(14-21(20)26)23(35,24(27,28)29)25(30,31)32/h3-12,14,35H,2,13H2,1H3,(H,33,34) |
| Chemical Name | 2-(4-ethylsulfonylphenyl)-N-[4-[2-fluoro-4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]phenyl]acetamide |
| HS Tariff Code | 2934.99.9001 |
| 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) |
Biological Activity
| Targets |
XY101 targets retinoic acid receptor-related orphan receptor γ (RORγ) (Ki = 1.2 nM; GAL4 reporter gene assay IC50 = 2.5 nM) [1] XY101 shows no significant binding to other nuclear receptors (e.g., RORα, RORβ, AR, ERα) with Ki > 10 μM [1] |
| ln Vitro |
Prostate-specific antigen (PSA), AR-V7, and androgen receptor (AR) expression are all efficiently inhibited by XY101 [1]. 1. RORγ transcriptional activity inhibition: - XY101 acts as a potent RORγ inverse agonist, inhibiting RORγ-mediated transcriptional activation in GAL4-RORγ LBD transfected HEK293T cells with an IC50 of 2.5 nM [1] - It suppresses RORγ binding to its response element (RORE) in electrophoretic mobility shift assay (EMSA) at concentrations ≥5 nM [1] 2. Antiproliferative activity in CRPC cells: - XY101 inhibits proliferation of castration-resistant prostate cancer (CRPC) cell lines in a concentration-dependent manner: C4-2B (IC50 = 3.8 nM), 22Rv1 (IC50 = 5.2 nM), DU145 (IC50 = 7.1 nM) (72-hour CCK-8 assay) [1] - It exhibits minimal effect on normal prostate epithelial cells (PrEC) with IC50 > 10 μM [1] - Long-term clonogenic assay shows >85% inhibition of colony formation in C4-2B cells at 10 nM XY101 [1] 3. Downregulation of AR signaling pathway: - XY101 (5 nM, 24 hours) reduces mRNA levels of AR target genes (AR, PSA, TMPRSS2) by 65%, 72%, and 68% in C4-2B cells (RT-qPCR) [1] - Western blot analysis confirms 70% reduction in AR protein level and 80% reduction in PSA protein level in C4-2B cells treated with 10 nM XY101 for 48 hours [1] - It inhibits RORγ-AR protein-protein interaction, as demonstrated by co-immunoprecipitation (Co-IP) assay [1] 4. Induction of apoptosis and cell cycle arrest: - XY101 (10 nM) induces apoptosis in C4-2B cells, with apoptotic rate increasing from 4.5% (control) to 32.6% (48 hours, Annexin V/PI staining) [1] - Flow cytometry shows G0/G1 cell cycle arrest, with G0/G1 population increasing from 41.2% to 68.3% in C4-2B cells treated with 5 nM XY101 [1] - It upregulates pro-apoptotic proteins (Bax, cleaved caspase-3/9) and downregulates anti-apoptotic protein Bcl-2 (Western blot) [1] |
| ln Vivo |
XY101 exhibited substantial anti-tumor effectiveness during therapy, reduced tumor growth, and was well tolerated without significant weight loss [1]. 1. Antitumor activity in C4-2B CRPC xenograft model: - XY101 (10, 30 mg/kg, p.o., once daily for 21 days) inhibits C4-2B tumor growth in nude mice by 52% and 75%, respectively, compared to vehicle control [1] - The 30 mg/kg group shows significant reduction in tumor weight (from 0.82 g to 0.21 g) and no obvious body weight loss [1] - Tumor tissue analysis reveals 68% reduction in AR mRNA level, 72% reduction in PSA protein level, and 55% reduction in Ki-67 (proliferation marker) positive cells [1] 2. Antitumor activity in 22Rv1 CRPC xenograft model: - XY101 (30 mg/kg, p.o., once daily for 21 days) reduces 22Rv1 tumor volume by 70% and prolongs median survival from 35 days (control) to 58 days (drug-treated group) [1] - Immunohistochemical staining of tumor tissues shows increased apoptotic cells (TUNEL assay) and decreased RORγ/AR protein expression [1] - Serum PSA level (tumor marker) is reduced by 65% in drug-treated mice [1] |
| Enzyme Assay |
1. Fluorescence polarization (FP) binding assay: - Recombinant human RORγ ligand-binding domain (LBD) is diluted in binding buffer to a final concentration of 20 nM [1] - Fluorescein-labeled RORγ agonist ligand is added to the RORγ LBD solution at a final concentration of 5 nM [1] - XY101 is serially diluted (0.1 nM to 100 nM) and mixed with the above mixture, incubated at room temperature for 1 hour [1] - Fluorescence polarization is measured at excitation 485 nm and emission 535 nm, and Ki value is calculated using a competitive binding model [1] 2. GAL4-RORγ reporter gene assay: - HEK293T cells are co-transfected with GAL4-RORγ LBD expression plasmid and UAS-luciferase reporter plasmid [1] - Transfected cells are seeded in 96-well plates and cultured overnight, then treated with XY101 (0.1 nM to 50 nM) plus RORγ agonist (1 μM) for 24 hours [1] - Luciferase activity is measured using a luminometer, and IC50 is calculated by fitting the dose-response curve of activity inhibition [1] 3. Electrophoretic Mobility Shift Assay (EMSA): - Biotin-labeled RORE (ROR response element) oligonucleotide is synthesized and annealed [1] - Recombinant RORγ protein (50 ng) is incubated with XY101 (0.5-20 nM) for 30 minutes at room temperature [1] - Biotin-labeled RORE is added to the mixture and incubated for another 20 minutes, then separated by non-denaturing polyacrylamide gel electrophoresis [1] - The gel is transferred to a nylon membrane, and biotin-labeled DNA is detected using streptavidin-HRP conjugate to assess RORγ-RORE binding inhibition [1] |
| Cell Assay |
1. Cell proliferation assay (CCK-8): - CRPC cell lines (C4-2B, 22Rv1, DU145) and normal PrEC cells are seeded in 96-well plates at 3×10^3 cells per well and cultured overnight [1] - XY101 is serially diluted (0.1 nM to 20 μM) and added to the cells, incubated at 37°C with 5% CO2 for 72 hours [1] - CCK-8 reagent is added to each well, incubated for 2 hours, and absorbance at 450 nm is measured to calculate cell viability and IC50 values [1] 2. RT-qPCR for AR target genes: - C4-2B cells are seeded in 6-well plates at 2×10^5 cells per well and treated with XY101 (1, 5, 10 nM) for 24 hours [1] - Total RNA is extracted, reverse-transcribed into cDNA, and qPCR is performed using specific primers for AR, PSA, TMPRSS2, and GAPDH (internal control) [1] - Relative mRNA levels are calculated using the 2^(-ΔΔCt) method, and fold changes compared to the control group are reported [1] 3. Apoptosis and cell cycle analysis: - C4-2B cells are treated with XY101 (5, 10 nM) for 48 hours, harvested, and washed with cold PBS [1] - Apoptosis assay: Cells are stained with Annexin V-FITC and PI for 15 minutes in the dark, then analyzed by flow cytometry [1] - Cell cycle assay: Cells are fixed in 70% ethanol at -20°C overnight, stained with PI solution containing RNase A for 30 minutes, and analyzed by flow cytometry [1] 4. Western blot and Co-IP assay: - XY101-treated C4-2B cells are lysed with RIPA buffer containing protease and phosphatase inhibitors [1] - For Western blot: Equal amounts of protein are separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against AR, PSA, Bax, Bcl-2, cleaved caspase-3/9, and GAPDH [1] - For Co-IP: Cell lysates are incubated with anti-RORγ antibody overnight, then mixed with protein A/G beads for 4 hours, and the immunoprecipitates are analyzed by Western blot with anti-AR antibody [1] |
| Animal Protocol |
1. C4-2B CRPC subcutaneous xenograft model: - Male BALB/c nude mice (6-8 weeks old) are subcutaneously injected with 5×10^6 C4-2B cells suspended in Matrigel (1:1 with PBS) [1] - When tumors reach a volume of ~100 mm³, mice are randomized into vehicle control and XY101 treatment groups (n=8 per group) [1] - XY101 is dissolved in 0.5% carboxymethyl cellulose (CMC) + 0.1% Tween 80, administered orally at 10 or 30 mg/kg once daily for 21 days [1] - Tumor volume is measured every 3 days using calipers, and body weight is monitored weekly [1] - At the end of treatment, mice are euthanized, tumors are excised, weighed, and stored for mRNA, protein, and immunohistochemical analysis [1] 2. 22Rv1 CRPC xenograft model: - Male NOD-SCID mice (6-8 weeks old) are subcutaneously injected with 1×10^7 22Rv1 cells [1] - When tumors reach ~150 mm³, mice are randomized into control and XY101 groups (n=10 per group) [1] - XY101 is administered orally at 30 mg/kg once daily for 21 days, with the same vehicle as the C4-2B model [1] - Survival time is recorded daily, and serum PSA level is measured by ELISA every 7 days [1] - Mice are euthanized when tumors exceed 1500 mm³ or show distress, and tumor tissues are collected for TUNEL and immunohistochemical staining [1] |
| ADME/Pharmacokinetics |
- XY101 exhibits oral bioavailability of 45% in rats (10 mg/kg p.o.) and 42% in mice (30 mg/kg p.o.) [1] - In rats, intravenous administration (5 mg/kg) shows a plasma half-life (t1/2) of 4.8 hours, volume of distribution (Vd) of 1.5 L/kg, and total clearance (CL) of 210 ml/kg/h [1] - Peak plasma concentration (Cmax) of 2.1 μg/ml is achieved at 1 hour after oral administration of 30 mg/kg in mice, with AUC₀-24h of 18.6 μg·h/ml [1] - Plasma protein binding rate is 93% (human plasma) and 91% (mouse plasma) [1] - It shows good stability in human liver microsomes (t1/2 > 2 hours) and is mainly metabolized via CYP3A4 [1] |
| Toxicity/Toxicokinetics |
- Acute toxicity: No mortality or adverse effects observed in mice at single oral doses up to 200 mg/kg [1] - Subacute toxicity: Mice treated with 30 mg/kg/day XY101 (once daily for 28 days) show no significant changes in body weight, food intake, or hematological parameters (WBC, RBC, platelets) [1] - Serum ALT, AST, creatinine, and urea nitrogen levels are within normal ranges in drug-treated mice [1] - No histopathological abnormalities detected in liver, kidney, heart, lung, or prostate tissues of treated mice [1] |
| References |
[1]. Discovery and Characterization of XY101, a Potent, Selective, and Orally Bioavailable RORγ Inverse Agonist for Treatment of Castration-Resistant Prostate Cancer. J Med Chem. 2019 May 9;62(9):4716-4730. |
| Additional Infomation |
- XY101 is a potent, selective, and orally bioavailable RORγ inverse agonist developed for the treatment of castration-resistant prostate cancer (CRPC) [1] - Its binding mode involves occupying the ligand-binding pocket of RORγ, forming hydrogen bonds with key residues (Tyr502, His479) and hydrophobic interactions with the pocket’s hydrophobic residues [1] - It exerts antitumor effects by inhibiting RORγ-mediated transcriptional activation of androgen receptor (AR) target genes, thereby suppressing CRPC cell proliferation and inducing apoptosis [1] - CRPC is characterized by persistent AR signaling despite androgen deprivation therapy, and XY101 targets this key pathway, providing a potential new therapeutic strategy for CRPC [1] |
Solubility Data
| Solubility (In Vitro) | DMSO : ~250 mg/mL (~443.67 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.69 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 (3.69 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 20.8 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (3.69 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 | 1.7747 mL | 8.8734 mL | 17.7469 mL | |
| 5 mM | 0.3549 mL | 1.7747 mL | 3.5494 mL | |
| 10 mM | 0.1775 mL | 0.8873 mL | 1.7747 mL |