ERα(EC50 = 0.2 nM)
Amcenestrant targets estrogen receptor α (ERα), a nuclear receptor that plays a critical role in the growth and proliferation of ER+ breast cancer cells. It acts as a selective estrogen receptor degrader (SERD), binding to ERα and inducing its degradation, thereby blocking estrogen-mediated signaling. It has an EC50 of 0.2 nM for ERα degradation. Amcenestrant shows selectivity for ERα over ERβ. It exhibits potent ER antagonist and degrader activity in a broad range of ER+ cells.

SAR439859 (compound 43d) exhibits the strongest in vivo anticancer activity against a variety of BC cell lines and patient-derived xenografts, including those with ERα mutations [1].
In vitro studies have demonstrated that Amcenestrant is a potent ER antagonist with ER degradation activity, with an EC50 of 0.2 nM for ERα degradation. It selectively induces degradation of ERα in MCF-7 breast cancer cells over ERβ. The compound shows potent anti-tumor activity in ER+ breast cancer cells and exhibits limited cross-resistance. It shows an outstanding overall profile with high degradation efficacy and metabolic stability. It exhibits more extensive and superior ER antagonist and degrader activity in a broad range of ER+ cells, including inhibition of ER signaling and tumor cell growth.

At a dose of 25 mg/kg/BID, SAR439859 (Compound 43d; Valve; 2.5–25 mg/kg; twice daily for 30 days) showed a significant reduction in tumor growth [1]. In the largest animal species tested, 3 mg/kg, minimum 10 mg/kg) shows low to moderate clearance (0.03-1.92 L/h?kg), low to moderate volume of distribution (Vss=0.5-6.1 L/kg), and good cross-species bioavailability (54-76%). Be aware that the T1/2 time varies depending on the species: 1.98 hours for mice, 4.13 hours for fish, and 9.80 hours for dogs, for example [1].
In vivo studies have demonstrated that Amcenestrant has potent anti-tumor efficacy in ER+ breast cancer models. As an orally available SERD, it is suitable for oral administration. Upon oral administration, it induces degradation of ERα and inhibits estrogen-mediated signaling, leading to tumor growth inhibition. The compound shows potent anti-tumor efficacy and limited cross-resistance in ER+ breast cancer. It has been evaluated in preclinical models and clinical trials for the treatment of ER+ breast cancer.

Determination of ERα Crystal Structure[1]
The ERα ligand-binding-domain (LBD)-mutated (amino acids 298–554) Tyr-536-Ser was induced in BL21 (DE3) cells and purified. The purified protein was then concentrated to ∼12 mg/mL in 50 mM Tris, pH 8, 150 mM NaCl, 50 mM β-mercapto-ethanol, 10% glycerol. The protein was incubated with the compound at 1 mM overnight at 4 °C. The protein with compound was crystallized by the hanging drop vapor diffusion method at 20 °C with a 1:1 ratio of protein solution to reservoir solution of 22.5% PEG-4000, 100 mM Cacodylate Na pH 6.5, and 0.2 M MgCl2 with 0.2 μL of the diluted seeding solution. The crystals were cryoprotected in glycerol to the mother liquor. Crystals were flash-frozen in liquid nitrogen prior to data collection. Diffraction data were collected at the SOLEIL synchrotron facility, Beamline Proxima 1, Saclay, France and were processed with programs AutoPROC and STARANISO (Global Phasing) The structure was solved using molecular replacement technique with coordinates of ER-LBD available internally and the program PHASER. The structure was refined using REFINE (Global Phasing) (X), the ligand was placed manually, and the manual rebuilds were made in COOT. The final validation checks were performed using MOLPROBITY prior to the deposition of coordinates and structure factors into the Protein Data Bank. The data set statistics of crystal structure are given in Table 8.
In vitro enzyme/receptor binding (non-cell) assays for Amcenestrant typically involve ERα binding and degradation studies. Radioligand binding assays using ³H-estradiol and recombinant ERα protein can be performed to determine binding affinity. Increasing concentrations of Amcenestrant (0.01 nM - 10 μM) are incubated with ERα and radioligand in binding buffer at 4°C for 16-24 hours. Bound and free radioligand are separated by charcoal-dextran precipitation or filtration. Radioactivity is measured by liquid scintillation counting, and Ki values are calculated. For degradation studies, ERα protein is incubated with the compound and ubiquitination/degradation components in cell-free systems to assess direct degradation activity.

Estrogen Receptor Degradation Activity[1]
The measurements on the protein abundance of ER were made by fluorescence detection in the MCF7 BC cell line. MCF7 cells (ATCC) were seeded in a 384-well microplate at a concentration of 10 000 cells/30 μL per well in red-phenol-free MEM alpha medium containing 5% charcoal-dextran-stripped fetal bovine serum (FBS). The following day, compounds were added to the cells in a 10-point dose response with a 1:3 fold serial dilution in 2.5 μL at final concentrations ranging from 300 to 0.15 nM or 0.1 μM for fulvestrant (used as a positive control). At 4 h post-compound-addition, the cells were fixed by adding 25 μL of formalin (final concentration 5% formalin containing 0.05% triton) for 15 min at room temperature and then washed twice with PBS-1×. Then, cells were incubated overnight in a cold room with 25 μL of anti-ER rabbit monoclonal antibody diluted at 1:500 in Power Block buffer. Wells were washed three times with PBS-1× and incubated at 37 °C for 60 min in Power Block buffer containing goat antirabbit antibody Alexa 488 (1:1000) and Syto-64 a DNA dye (1.5 μM final concentration). Cells were then washed three times in PBS-1× and scanned in an ACUMEN explorer apparatus. Integrated intensities in the green fluorescence and red fluorescence were measured to determine the levels of ERα and DNA, respectively. The degradation potency is determined as the concentration of compound that achieves 50% degradation of the estrogen receptor (or IC50) in nanomoles. The percent ERα level decrease was calculated as follows: % inhibition = 100*(1 – (sample – fulvestrant)/(DMSO – fulvestrant)).
For in vitro cell-based assays, MCF-7 breast cancer cells or other ER+ cell lines are cultured in appropriate media. Cells are treated with Amcenestrant at concentrations ranging from 0.01 nM - 10 μM for 24-72 hours. ERα degradation is measured by Western blot or by quantifying ERα protein levels using ELISA-based methods. ER signaling inhibition is assessed by measuring the expression of estrogen-responsive genes (e.g., pS2, GREB1) by qRT-PCR. Cell viability and proliferation are assessed by MTT, CellTiter-Glo, or colony formation assays. Selectivity for ERα over ERβ can be confirmed using ERβ-expressing cells.

Animal/Disease Models: Nu/nu mouse MCF7 tumor xenograft model [1]
Doses: 2.5, 5, 12.5, 25 mg/kg
Doses: Oral;. twice (two times) daily for 30 days
Experimental Results: Demonstrated significant tumor growth inhibition and demonstrated tumor regression at a dose of 25 mg/kg/BID.

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Animal/Disease Models: Mice, rats and dogs [1]
Doses: 3 mg/kg (iv) and 10 mg/kg (oral) (pharmacokinetic/PK/PK analysis)
Route of Administration: intravenous (iv) (iv)or oral
Experimental Results: In three Shows low to moderate clearance among animal species tested (0.03-1.92 L/h·kg), low to moderate volume of distribution (Vss=0.5-6.1 L/kg), and good bioavailability across species degree (54-76%).

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MCF-7 Xenograft Studies[1]
0.72 mg of 17-β estradiol pellets (90 days release) was subcutaneously implanted into nu/nu mice 2 days prior to MCF7 cell implantation. MCF7 cells were grown in Eagle’s minimum essential medium supplemented with 10% FBS and 0.01 mg/mL human insulin in 5% CO2 at 37 °C. The cells were harvested and resuspended in DPBS with 50% matrigel and subcutaneously implanted into the right flank of female nude mice. The tumor volume (length × width2/2) was monitored biweekly. When tumors reached an average volume of 200 mm3, animals were randomized and treatment was started. Animals were treated with the vehicle or the 43d compound twice daily. The tumor volume and body weight were monitored biweekly throughout the study.
In vivo animal studies with Amcenestrant typically use mouse xenograft models of ER+ breast cancer. Immunodeficient mice are subcutaneously implanted with human ER+ breast cancer cells (e.g., MCF-7). When tumors reach a certain size, mice are treated with Amcenestrant orally at doses determined from pharmacokinetic studies. Tumor volume and body weight are monitored regularly. At study endpoint, tumors are excised, weighed, and processed for histopathological and molecular analyses (e.g., immunohistochemistry for ERα, Ki-67, cleaved caspase-3). Blood samples are collected for pharmacokinetic analysis and assessment of estrogen levels. The compound's anti-tumor efficacy and limited cross-resistance have been demonstrated in these models.

Amcenestrant is an orally bioavailable compound. It has a molecular weight appropriate for small molecule oral administration. The compound is a nonsteroidal SERD with remarkable metabolic stability. Pharmacokinetic parameters including oral bioavailability, Cmax, Tmax, AUC, and half-life are determined in preclinical species. The compound is metabolized primarily by hepatic enzymes, with elimination via biliary and renal routes. Clinical pharmacokinetic data are available from clinical trials.

In preclinical and clinical studies, Amcenestrant has shown a manageable safety profile. As a SERD, the compound is expected to have side effects similar to other endocrine therapies, including hot flashes, fatigue, and musculoskeletal symptoms. The compound's selectivity for ERα over ERβ may reduce certain side effects. In clinical trials, Amcenestrant has been evaluated for safety and efficacy in ER+ breast cancer patients. The compound is not yet FDA-approved and is in clinical development.

[1]. Discovery of 6-(2,4-Dichlorophenyl)-5-[4-[(3S)-1-(3-fluoropropyl)pyrrolidin-3-yl]oxyphenyl]-8,9-dihydro-7H-benzo[7]annulene-2-carboxylic acid (SAR439859), a Potent and Selective Estrogen Receptor Degrader (SERD) for the Treatment of Estrogen-Receptor-Positive Breast Cancer. J Med Chem. 2019 Nov 27.

[2]. Abstract 943: SAR439859, an orally bioavailable selective estrogen receptor degrader (SERD) that demonstrates robust antitumor efficacy and limited cross-resistance in ER+ breast cancer.

Ancinant is an orally administered, nonsteroidal selective estrogen receptor degrader/downregulator (SERD) with potential antitumor activity. After oral administration, ancinant specifically targets and binds to the estrogen receptor (ER), inducing conformational changes and promoting ER degradation. This blocks ER-mediated signaling and inhibits the growth and survival of ER-expressing cancer cells.
Amcenestrant (SAR-439859) is a potent, orally available, nonsteroidal selective estrogen receptor degrader (SERD) with potential anticancer activity. It is an effective ER antagonist with ER degradation activity, with an EC50 of 0.2 nM for ERα degradation. The compound shows potent anti-tumor efficacy in ER+ breast cancer and exhibits limited cross-resistance. Amcenestrant has been evaluated in clinical trials for the treatment of ER+ breast cancer. It is also known as SAR439859 and has the INN name Amcenestrant. The compound is not yet FDA-approved.

C₃₁H₃₀CL₂FNO₃

554.48

553.16

C, 67.15; H, 5.45; Cl, 12.79; F, 3.43; N, 2.53; O, 8.66

2114339-57-8

130232326

White to off-white solid powder

1.297±0.06 g/cm3(Predicted)

676.7±55.0 °C(Predicted)

5.3

1

5

8

38

832

1

ClC1C([H])=C(C([H])=C([H])C=1C1C([H])([H])C([H])([H])C([H])([H])C2C([H])=C(C(=O)O[H])C([H])=C([H])C=2C=1C1C([H])=C([H])C(=C([H])C=1[H])O[C@@]1([H])C([H])([H])C([H])([H])N(C([H])([H])C([H])([H])C([H])([H])F)C1([H])[H])Cl

KISZAGQTIXIVAR-VWLOTQADSA-N

InChI=1S/C31H30Cl2FNO3/c32-23-8-12-27(29(33)18-23)28-4-1-3-21-17-22(31(36)37)7-11-26(21)30(28)20-5-9-24(10-6-20)38-25-13-16-35(19-25)15-2-14-34/h5-12,17-18,25H,1-4,13-16,19H2,(H,36,37)/t25-/m0/s1

6-(2,4-dichlorophenyl)-5-[4-[(3S)-1-(3-fluoropropyl)pyrrolidin-3-yl]oxyphenyl]-8,9-dihydro-7H-benzo[7]annulene-2-carboxylic acid

SAR439859; SAR439859; Amcenestrant [INN]; Amcenestrant [USAN]; TBF1NHY02O; (S)-8-(2,4-Dichlorophenyl)-9-(4-((1-(3-fluoropropyl)pyrrolidin-3-yl)oxy)phenyl)-6,7-dihydro-5H-benzo[7]annulene-3-carboxylic acid; SAR-439859

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 : ~83.33 mg/mL (~150.28 mM)

Solubility in Formulation 1: ≥ 8.33 mg/mL (15.02 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 83.3 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.

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Solubility in Formulation 2: ≥ 8.33 mg/mL (15.02 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 83.3 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.

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Solubility in Formulation 3: ≥ 8.33 mg/mL (15.02 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 83.3 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.)