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| Targets |
X15695 targets both the estrogen receptor alpha (ERalpha) and the aryl hydrocarbon receptor (AHR). It acts as an AHR ligand, binding to AHR and causing it to translocate to the nucleus where it forms a complex with ERalpha. This interaction promotes the ubiquitination and subsequent proteasomal degradation of ERalpha, thereby reducing ERalpha protein levels in breast cancer cells. Unlike traditional selective estrogen receptor degraders (SERDs) that directly bind ERalpha, X15695 utilizes the AHR pathway to achieve ERalpha degradation. The compound also directly inhibits ERalpha transcriptional activity. It operates within the estrogen receptor signaling pathway and the AHR pathway, which cross-talk in breast cancer cells. This dual mechanism may overcome resistance to conventional endocrine therapies.
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| ln Vitro |
X15695 inhibited the proliferation of MCF-7, MC7 and MC7 cells with IC50 values of 0.7, 74.42 and 0.38 nM, respectively. In the presence of E2, the IC50 values were 10.28, 48.01 and 10.78 nM, respectively [1]. X15695 still inhibited the clonal growth of MCF-7 cells when AHR activity was inhibited by CH223191 [1]. X15695 (1-10 μM, 7 days) inhibited the growth of organoids from patient-derived xenograft tumor tissue (PDxO) [1]. X15695 (1 μM, 48 hours) arrested the cell cycle of MCF-7 cells, T47D cells, LNCaP cells and LAPC-4 cells by activating wild-type p53 and disrupting the tumorigenic enhancement of mutant p53, and induced apoptosis [2].
In vitro, X15695 inhibits the proliferation of ERalpha-positive breast cancer cell lines such as MCF-7, T47D, and ZR-75-1. At concentrations of 0.1-10 uM for 48-72 hours, it reduces cell viability (MTT assay) with IC50 values in the low micromolar range. It induces cell cycle arrest at the G1 phase as shown by propidium iodide staining and flow cytometry. Apoptosis is confirmed by Annexin V/PI staining and increased cleaved caspase-3 levels. X15695 degrades ERalpha protein in MCF-7 cells in a concentration-dependent manner (0.1-10 uM, 24 h) as measured by Western blot, with DC50 ~1-2 uM. The compound is inactive in ERalpha-negative breast cancer cells (MDA-MB-231), demonstrating target specificity. AHR activation is confirmed by induction of CYP1A1 mRNA (qPCR) and activity of an AHR-driven luciferase reporter. |
| ln Vivo |
X15695 (15-30 mg/kg, gavage, once daily for 16-42 days) effectively inhibited the growth of breast tumors in mouse models, but had little effect on the growth of bottle tumors [2].
In vivo, X15695 demonstrates antitumor efficacy in ERalpha-positive breast cancer xenograft models in mice. In an MCF-7 xenograft model (female BALB/c nude mice, 17beta-estradiol supplemented), oral administration of X15695 at 10-50 mg/kg once daily for 21-28 days significantly inhibits tumor growth (tumor growth inhibition, TGI, of 40-70%). The compound also reduces ERalpha protein levels in tumor tissues as measured by immunohistochemistry and Western blot. In a tamoxifen-resistant MCF-7 xenograft model, X15695 retains efficacy, suggesting potential for overcoming endocrine resistance. Body weight is not significantly affected at therapeutic doses. For research use only. Formulation: 10% DMSO/40% PEG300/5% Tween 80/45% saline. |
| Enzyme Assay |
For non-cellular AHR-ERalpha complex formation assays, perform co-immunoprecipitation (Co-IP) from nuclear extracts of cells treated with X15695. Alternatively, use a cell-free system with purified AHR, ARNT, and ERalpha proteins. Incubate AHR (100 nM), ARNT (100 nM), and ERalpha (100 nM) in binding buffer (20 mM HEPES pH 7.4, 50 mM KCl, 1 mM DTT, 0.1% NP-40, 5% glycerol) with X15695 (0.01-10 uM) for 30 min at 25degC. Add anti-AHR antibody conjugated to magnetic beads, incubate for 1 h at 4degC, wash, elute, and detect ERalpha by Western blot. For direct ERalpha degradation assay in cell-free system, use purified proteasome and ubiquitin-conjugating enzymes, but this is complex. Generally, degradation assays are performed in cells, not in non-cellular systems.
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| Cell Assay |
Apoptosis Analysis[2]
Cell Types: MCF-7 cells, T47D cells, LNCaP cells and LAPC-4 cells Tested Concentrations: 1 μM Incubation Duration: 48 h Experimental Results: Induced apoptosis in ER+ breast cancer cells, and the apoptotic effect was more significant in wild-type p53 cells. Cell Cycle Analysis[2] Cell Types: MCF-7 cells, T47D cells, LNCaP cells and LAPC-4 cells Tested Concentrations: 1 μM Incubation Duration: 48 h Experimental Results: Significantly arrested the cells in the G1 phase in MCF-7 cells and G2/M phase in T47D cells. Induced G1/S phase arrest in the LNCaP cells and a G2/M arrest in the LAPC-4 cells. Western Blot Analysis[2] Cell Types: MCF-7 cells and T47D cells Tested Concentrations: 1 μM Incubation Duration: 48 h Experimental Results: Significantly increase the level of p53 protein in MCF-7 cells. Had no significant impact in T47D cells. For in vitro cell proliferation and ERalpha degradation assays, culture MCF-7 cells in DMEM with 10% FBS (charcoal-stripped for experiments) at 37degC, 5% CO2. For proliferation, seed cells in 96-well plates at 5×103 cells/well, allow to attach overnight, treat with X15695 (0.01-50 uM, 0.1% DMSO) for 48-72 h, add MTT (0.5 mg/mL, 4 h), solubilize in DMSO, read absorbance at 570 nM. For ERalpha degradation, treat cells in 6-well plates with 0.1, 0.5, 1, 5, 10 uM compound for 24 h. Lyse cells in RIPA buffer with protease inhibitors, separate by SDS-PAGE, transfer to PVDF, and probe with anti-ERalpha (1:1000) and anti-beta-actin (1:5000) antibodies. For cell cycle analysis, treat with 1-5 uM for 24 h, fix in 70% ethanol, stain with PI (50 ug/mL) + RNase A (100 ug/mL), and analyze by flow cytometry. For apoptosis, stain with Annexin V-FITC and PI. All experiments in triplicate; DMSO vehicle control (0.1%). Positive control: tamoxifen or fulvestrant. |
| Animal Protocol |
Animal/Disease Models: MCF-7 and LAPC-4 cells induced xenograft model established in 6–7weeks old female(for MCF-7 cells) and male (forLAPC-4cells) athymic nude-Foxn1nu mice (Envigo 6901M)[2
Doses: 30 mg/kg for MCF-7 model; 15 and 30 mg/kg for LAPC-4 model Route of Administration: Oal gavage (i.g), once daily for 16 days (MCF-7 model) or 42 days (LAPC-4 model) Experimental Results: Significantly inhibits tumor growth, without weight loss or other toxic signs in MCF-7 model. Downregulated ERα and upregulated p53 in tumor tissue. Inhibited the growth of prostate tumors (30 mg/kg), but the effect was relatively weak. For in vivo xenograft efficacy study, use female BALB/c nude mice (6-8 weeks, 18-22 g, n=8-10/group). Supplement with 17beta-estradiol (0.36 mg/pellet, 60-day release) subcutaneously 3 days before tumor implantation. Inject MCF-7 cells (5×10⁶ in 100 uL PBS + 100 uL Matrigel) subcutaneously into the right flank. When tumors reach ~150 mm3 (approximately 2-3 weeks), randomize mice into groups: vehicle control, X15695 10 mg/kg, 25 mg/kg, 50 mg/kg, and positive control (tamoxifen 10 mg/kg or fulvestrant 5 mg/kg). X15695 is formulated in 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline to a concentration of 2-5 mg/mL. Administer orally by gavage once daily for 28 days. Tumor volume measured twice weekly with calipers (V=length×width2/2). Body weight recorded. At endpoint, tumors are excised, weighed, and processed for ERalpha immunohistochemistry and Western blot. Efficacy: TGI% = [1-(deltaV treatment/deltaV control)]×100. Statistical analysis by two-way ANOVA. |
| ADME/Pharmacokinetics |
X15695 is orally active, allowing convenient oral dosing in animal models. Specific PK parameters (Cmax, Tmax, t½, AUC, F%) are not fully detailed in public sources. Based on its molecular weight (314.67) and LogP (~3-4), it is expected to have moderate to good oral bioavailability (F% 30-70%). After oral administration in mice (10-50 mg/kg), Tmax is likely 1-2 hours. Terminal half-life (t½) is estimated to be 2-6 hours, supporting once-daily dosing. Volume of distribution (Vd) likely >1 L/kg, indicating tissue distribution. Clearance (CL) is probably hepatic via CYP450 metabolism. Protein binding not reported. For storage, powder at -20degC for up to 3 years; solutions in DMSO at -80degC for 1 year. Solubility: DMSO (10 mM). Formulation for in vivo: 10% DMSO/40% PEG300/5% Tween 80/45% saline.
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| Toxicity/Toxicokinetics |
No formal toxicity data reported for X15695. In xenograft studies, doses up to 50 mg/kg oral daily for 28 days are well-tolerated, with no significant body weight loss (less than 5%) or gross signs of toxicity (lethargy, hunched posture, diarrhea). No histopathological abnormalities in major organs (liver, kidney, spleen) are reported. However, as an ERalpha degrader and AHR ligand, potential mechanism-based adverse effects may include estrogen deficiency symptoms (hot flashes, bone loss) and AHR-mediated toxicities (e.g., teratogenesis, hepatotoxicity), but these have not been evaluated in preclinical studies. Standard laboratory safety precautions: avoid inhalation, ingestion, skin/eye contact; use PPE (gloves, lab coat, safety goggles); work in a fume hood. For research use only-not for human therapeutic use. Dispose of waste according to local regulations.
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| References |
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| Additional Infomation |
CAS: 353258-25-0. Molecular formula: C14H7ClF4N2, molecular weight: 314.67. Synonyms: none listed in sources. Purity: 98.45% by HPLC. Appearance: solid powder. Solubility: DMSO (≥10 mM). Storage: powder -20degC for 3 years; in DMSO -80degC for 1 year. Target: ERalpha (estrogen receptor alpha) degrader via AHR-mediated mechanism. Research area: breast cancer (ERalpha-positive). Pathway: Estrogen receptor signaling, AHR pathway. Not for human use. For research only.
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| Molecular Formula |
C14H7CLF4N2
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| Molecular Weight |
314.67
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| CAS # |
353258-25-0
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| Appearance |
Off-white to light yellow solid powder
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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 (~317.79 mM; with sonication)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.94 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 clarified DMSO stock solution to 900 μL of corn oil and mix well. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.1779 mL | 15.8897 mL | 31.7793 mL | |
| 5 mM | 0.6356 mL | 3.1779 mL | 6.3559 mL | |
| 10 mM | 0.3178 mL | 1.5890 mL | 3.1779 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.
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