ERα antagonism (IC50 = 0.28 nM); ERα downregulation (IC50 = 0.14 nM); ERα binding (IC50 = 0.82 nM)

AZD9496 demonstrates potency in ERα binding, downregulation, and antagonism, with IC50 values of 0.82 nM, 0.14 nM, and 0.28 nM, respectively. With an EC50 of 0.04 nM, AZD9496 dramatically suppresses MCF-7 cell growth[1]. The study found that AZD9496 exhibits high selectivity towards the following tested nuclear hormone receptors: progesterone receptor (PR), IC50=0.54 μM; glucocorticoid receptor (GR), IC50=9.2 μM; androgen receptor (AR), IC50=30 μM[2].

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AZD9496 is a selective ERα antagonist, downregulator, and inhibitor of ER+ tumor cell growth. AZD9496 directly targets ERα for downregulation in vitro. AZD9496 antagonizes and downregulates mutant ER in vitro. [1]

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Compound 30b (AZD9496) showed an overall property profile (Table 2) broadly comparable to progenitor 27b while maintaining excellent cross-species PK (Table 3). The significant degradation of ERα by compound 30b was confirmed in the Western blot assay with both compound 2 (fulvestrant) and estradiol (E2) as comparator compounds (Figure 7). Selectivity of compound 30b over other tested nuclear hormone receptors is high: androgen receptor (AR), IC50 = 30 μM; glucocorticoid receptor (GR), IC50 = 9.2 μM; progesterone receptor (PR), IC50 = 0.54 μM (cf. estrogen receptor (ERα), IC50 = 0.0008 μM). [2]

In the estrogen-dependent MCF-7 xenograft model, significant inhibition of tumor growth is seen at doses as low as 0.5 mg/kg. This effect is accompanied by a dose-dependent reduction in PR protein levels, indicating an effective antagonist. Compared to monotherapy alone, AZD9496 combined with CDK4/6 inhibitors and the PI3K pathway has additional growth-inhibitory effects. When AZD9496 was taken orally once a day at doses of 5 and 25 mg/kg, it increased uterine weight statistically significantly (P<0.001) in comparison to the ICI 182780 control, but not as significantly as ICI 47699 (P=0.001)[1]. AZD9496 is also tested in a long-term estrogen-deprived model (LTED) with the HCC-1428 LTED cell line, which is considered the most accurate model of aromatase inhibition due to its ability to grow in the absence of estrogen. AZD9496 has a substantial impact; in this model, tumor regressions are seen at a dose of 5 mg/kg[2].

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AZD9496 is a potent, oral inhibitor of breast tumor growth in vivo [1]
The effect of chronic, oral dosing of AZD9496 was explored in MCF-7 human breast xenografts, as a representative ER+/PR+/HER2+ breast cancer model. Good bioavailability and high clearance gave a terminal t1/2 of 5–6 hours after oral dosing in the mouse and resulted in significant dose-dependent tumor growth inhibition with 96% inhibition at 50 mg/kg and no toxicity or weight loss relative to the vehicle control group (Fig. 4A). To confirm that AZD9496 was targeting the ER pathway, PR protein levels were measured in tumor samples taken at the end of the study and a significant reduction in PR was seen, which correlated with tumor growth inhibition. A >90% reduction in PR was seen with both 10 and 50 mg/kg doses and a 75% decrease even with the 0.5 mg/kg dose, demonstrating that AZD9496 can clearly antagonize the ER pathway (Fig. 4B). Dosing 5 mg/kg of AZD9496, the minimal dose required to see significant tumor inhibition, gave greater tumor growth inhibition compared with 5 mg/mouse fulvestrant given 3 times weekly and tamoxifen given 10 mg/kg orally, daily (Fig. 4C). A series of pharmacodynamic in vivo studies were conducted to measure time taken to reach maximal inhibition of PR levels and time taken to recover back to basal levels. Three days of dosing with 5 mg/kg of AZD9496 gave 98% reduction of PR protein and continued to suppress protein levels at 48 hours (Fig. 4D), with full recovery by 72 hours (data not shown), which indicates a long pharmacologic half-life in vivo. Fulvestrant, given as 3 × 5 mg/mouse doses over one week, gave a 60% reduction in PR protein over a prolonged period with measured plasma levels approximately 8-fold higher than those achieved clinically, at steady state, with 500 mg fulvestrant (Fig. 4E). As estrogen itself is known to downregulate ER protei, we were unable to detect further decreases in ERα protein compared with control animals with AZD9496 or fulvestrant in the MCF-7 model presumably due to the high circulating plasma levels of estrogen from implanted pellets at the time tumor samples were taken (Supplementary Fig. S5). A mouse-specific metabolite of AZD9496 was detected in circulating plasma at similar levels to AZD9496 and showed a similar pharmacokinetic profile. Testing this metabolite in the in vitro MCF-7 assays resulted in approximately 5-fold lower ERα antagonism activity and 7-fold lower ERα downregulation activity than AZD9496 (data not shown). Using a pharmacokinetic/pharmacodynamic model based on PR inhibition data, at the 5 mg/kg dose in vivo, which gives 98% inhibition of PR, the inhibitory activity that could be attributed to the parent compound alone was 85% when the activity of the metabolite was discounted.

AZD9496 is a potent and orally bioavailable, selective antagonist and downregulator of the estrogen receptor (Ki=0.7 nM).

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SILAC assays [1]
MCF-7 cells were grown for at least three passages in stable isotope labelling by amino acids (SILAC) phenol red-free RPMI media containing 13C615N4 arginine (heavy medium) to fully label peptides. Cells were then grown in heavy medium supplemented with 5% dialysed CSS for 24 hours before washing with PBS and switching to standard phenol red-free RPMI medium containing unlabelled arginine and AZD9496, fulvestrant, tamoxifen, estradiol or DMSO. Compounds were incubated over a 48 hour period before protein lysates were prepared in lysis buffer. Equal concentrations of sample proteins spiked with internal standards (lysate from MCF-7 cells labelled with 13C6 lysine only) were immunoprecipitated overnight at 4oC using an anti-ERα monoclonal antibody (SP1) and then digested with 0.4 μg trypsin in 50 mmol/L ammonium bicarbonate at 37oC overnight before analysing by mass spectrometry using relative peptide quantification by selected reaction monitoring (SRM). Degradation half-life was measured using the one-phase exponential decay equation in GraphPad PRISM (Y=Span.e-K.X+Plateau) where X is time and Y is response which starts out as Span+Plateau and decreases to Plateau with a rate constant K.

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Biacore affinity measurements [1]
For BIAcore affinity measurements a tetra-His antibody was immobilised to a biacore CM5 biosensor chip in 20 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% T20 (HBS-T) running buffer and 6His-ERα protein captured in the presence of AZD9496. The association (kass) and dissociation (kdiss) rate constants and (KD) were calculated using BIAevaluation software and used to fit the ERα LBD:AZD9496 interaction to a 1:1 Langmuir binding interaction model.

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Biochemical and in vitro cell assays [1]
Binding, ER agonism, antagonism, downregulation, and cell proliferation assays were carried out as described previously (Biomol Screen 2015;20:748–59). BIAcore affinity measurements and immunoblotting from compound-treated cells are described in Supplementary Methods.

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Protein production, crystallization, and structure determination [1]
Protein expression, purification, and crystallization of the ERα ligand-binding domain was carried out as described previously. X-ray diffraction data were collected at the ESRF on beamline ID23-1 on an ADSC detector. Data were processed using XDS as implemented within EDNA and scaled and merged using SCALA. The structure of ERα in complex with AZD9496 was solved by molecular replacement using AmoRE and an internal ERα structure as the search model. Quality checks on the protein structures were carried out using the validation tools in Coot. The final structure has been deposited in the Protein Databank with the ID code given in Supplementary Table S1.

AZD9496, ICI 182780, and ICI 47699's effects on MCF-7 cells' ERα peptide turnover. Steroid-free conditions are maintained for the indicated duration of time, and cells are grown in SILAC media containing 13C615N4 L-arginine to label ERα peptide as "heavy" (blue line). Afterwards, the culture is switched to unlabeled L-arginine to label newly synthesized protein as "normal" (red line), with 0.1% DMSO, 300 nM Tamoxife, 100 nM AZD9496, or 100 nM ICI 182780. The displayed data is an average of two separate experiments [1].

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Immunoblotting [1]
Cells were lysed in 25 mmol/L Tris/HCL pH6.8, 3 mmol/L EDTA, 3 mmol/L EGTA, 50 mmol/L NaF, 2 mmol/L sodium orthovanadate, 270 mmol/L sucrose, 10 mmol/L -glycerophosphate, 5 mmol/L sodium pyrophosphate and 0.5% Triton X-100 supplemented with protease inhibitors and phosphatase inhibitors and proteins run on 4% to 12% Tris-HCl precast gels. Membranes were probed overnight with primary antibodies followed by incubation with HRP-tagged secondary antibodies and visualized on a Syngene ChemiGenius with Super-Signal West Dura Chemiluminescence Substrate.

Mice: The effectiveness of AZD9496 in an MCF-7 xenograft model in vivo. PEG/captisol (vehicle) or AZD9496 (0.02, 0.1, 0.5, 10, and 50 mg/kg, p.o., q.d.) are the daily doses given to MCF-7 xenografts cultivated in male SCID mice. Every few months, the growth of the tumor is measured with a caliper, and the mean tumor volumes for every dose group are plotted.

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Rat uterine and xenograft studies [1]
MCF-7 cells (5 x 106 ) were implanted subcutaneously in the hind flank of immuno-compromised (SCID) male mice the day after each mouse was surgically implanted with a 0.5 mg/21 day estrogen pellet. HCC1428 LTED (10 x 106 cells) were implanted transdermally into the fourth mammary fat pad of immuno-compromised (NSG) female mice 7 days after they were surgically ovariectomized. CTC-714 PDX model was derived from patient CTC cultures. EpCAM+CD44+ cells were suspended in phosphate buffered saline (PBS) mixed with high concentration matrigel (BD Biosciences) at 10 mg/ml and ~ 650 cells were injected into the third mammary fat pad of a NOD/SCID (Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mouse. In tumour transplantation study, 2 × 2 mm pieces of tumour tissue from CTC-derived tumour xenografts were implanted in the mammary fad pad of Beige Nude XID mice. Tumour growth was calculated weekly by bilateral caliper measurement (length x width) and mice randomised into vehicle or treatment groups with approximate mean start size of 0.2 to 0.4 cm3 for efficacy studies or 0.5 to 0.8 cm3 for PD studies. Mice were dosed once daily by oral gavage or subcutaneous (s.c.) injection for fulvestrant at the times and doses indicated for the duration of the treatment period. Tumour growth inhibition from start of treatment was assessed by comparison of the mean change in tumour volume for the control and treated groups. Statistical significance was evaluated using a one-tailed Student t test. Tumours were excised at specific time points and fragments either fixed in 10% buffered formalin or snap-frozen in liquid nitrogen and stored at -80 oC and terminal bleeds plasma PK samples collected. Measurement of estradiol levels in mouse plasma from the MCF-7 xenograft model with implanted estrogen pellets was done using a custom-made immunoassay kit from Meso scale Discovery (MSD).

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Rat Uterine Model [1]
Sexually immature female Han Wistar rats were randomised and dosed either with vehicle, AZD9496 or tamoxifen once daily for 3 days by oral gavage or a single s.c dose of fulvestrant. At 24 hours after the final dose of each agent was given, rats were euthanised, terminal plasma samples collected and uterine tissue removed with both horns intact, blotted dry and weighed. Protein extracts were prepared for immunoblot analysis as described for xenograft studies.

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Pharmacokinetic studies [1]
The pharmacokinetics of AZD9496 and its active mouse metabolite was investigated using combined data from multiple studies and analysed via population PK modelling. Specific PK studies consisted of IV bolus and PO doses of the parent, and IV bolus dose of the metabolite with multiple time points per animal, 2 animals per time point and were designed to establish a parent-metabolite pharmacokinetic model. The concentration of AZD9496 and its active metabolite in plasma samples was determined within AstraZeneca Oncology DMPK. Samples were analysed for parent and metabolite using LC-MS/MS detection using analytical standards over a final concentration range of 1 nM – 10,000 nM before being analysed using Masslynx and processed using Quanlynx.

Oral bioavailability (F) = 63%, 128%, 79% in rat, mouse and dog, respectively. [2]

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The effect of chronic, oral dosing of AZD9496 was explored in MCF-7 human breast xenografts, as a representative ER+/PR+/HER2+ breast cancer model. Good bioavailability and high clearance gave a terminal t1/2 of 5–6 hours after oral dosing in the mouse and resulted in significant dose-dependent tumor growth inhibition with 96% inhibition at 50 mg/kg and no toxicity or weight loss relative to the vehicle control group. [1]

[1]. AZD9496: An Oral Estrogen Receptor Inhibitor That Blocks the Growth of ER-Positive and ESR1-Mutant Breast Tumors in Preclinical Models. Cancer Res. 2016 Jun 1;76(11):3307-18.

[2]. Optimization of a Novel Binding Motif to (E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic Acid (AZD9496), a Potent and Orally Bioavailable Selective Estrogen.

AZD-9496 is under investigation in clinical trial NCT02248090 (AZD9496 First Time in Patients Ascending Dose Study).
Selective Estrogen Receptor Degrader AZD9496 is an orally available selective estrogen receptor degrader (SERD), with potential antineoplastic activity. Upon administration, SERD AZD9496 binds to the estrogen receptor (ER) and induces a conformational change that results in the degradation of the receptor. This prevents ER-mediated signaling and inhibits the growth and survival of ER-expressing cancer cells.

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Fulvestrant is an estrogen receptor (ER) antagonist administered to breast cancer patients by monthly intramuscular injection. Given its present limitations of dosing and route of administration, a more flexible orally available compound has been sought to pursue the potential benefits of this drug in patients with advanced metastatic disease. Here we report the identification and characterization of AZD9496, a nonsteroidal small-molecule inhibitor of ERα, which is a potent and selective antagonist and downregulator of ERα in vitro and in vivo in ER-positive models of breast cancer. Significant tumor growth inhibition was observed as low as 0.5 mg/kg dose in the estrogen-dependent MCF-7 xenograft model, where this effect was accompanied by a dose-dependent decrease in PR protein levels, demonstrating potent antagonist activity. Combining AZD9496 with PI3K pathway and CDK4/6 inhibitors led to further growth-inhibitory effects compared with monotherapy alone. Tumor regressions were also seen in a long-term estrogen-deprived breast model, where significant downregulation of ERα protein was observed. AZD9496 bound and downregulated clinically relevant ESR1 mutants in vitro and inhibited tumor growth in an ESR1-mutant patient-derived xenograft model that included a D538G mutation. Collectively, the pharmacologic evidence showed that AZD9496 is an oral, nonsteroidal, selective estrogen receptor antagonist and downregulator in ER(+) breast cells that could provide meaningful benefit to ER(+) breast cancer patients. AZD9496 is currently being evaluated in a phase I clinical trial. [1]

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The discovery of an orally bioavailable selective estrogen receptor downregulator (SERD) with equivalent potency and preclinical pharmacology to the intramuscular SERD fulvestrant is described. A directed screen identified the 1-aryl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole motif as a novel, druglike ER ligand. Aided by crystal structures of novel ligands bound to an ER construct, medicinal chemistry iterations led to (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (30b, AZD9496), a clinical candidate with high oral bioavailability across preclinical species that is currently being evaluated in phase I clinical trials for the treatment of advanced estrogen receptor (ER) positive breast cancer.[2]

C29H29F3N2O6

558.5456

442.19

C, 67.86; H, 5.70; F, 12.88; N, 6.33; O, 7.23

1639042-28-6

AZD9496;1639042-08-2

92044376

Light yellow to brown solid powder

4

10

7

40

824

2

C[C@@H]1CC2=C([C@H](N1CC(C)(C)F)C3=C(C=C(C=C3F)/C=C/C(=O)O)F)NC4=CC=CC=C24.C(=C\C(=O)O)\C(=O)O

GYRUZXFUFGWTAG-DMSKNVGDSA-N

InChI=1S/C25H25F3N2O2.C4H4O4/c1-14-10-17-16-6-4-5-7-20(16)29-23(17)24(30(14)13-25(2,3)28)22-18(26)11-15(12-19(22)27)8-9-21(31)32;5-3(6)1-2-4(7)8/h4-9,11-12,14,24,29H,10,13H2,1-3H3,(H,31,32);1-2H,(H,5,6)(H,7,8)/b9-8+;2-1-/t14-,24-;/m1./s1

(Z)-but-2-enedioic acid;(E)-3-[3,5-difluoro-4-[(1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-1,3,4,9-tetrahydropyrido[3,4-b]indol-1-yl]phenyl]prop-2-enoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.75 mg/mL (4.92 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 27.5 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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 (Please use freshly prepared in vivo formulations for optimal results.)