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Purity: ≥98%
AZD3514 (AZD-3514; AZD 3514) is a novel, potent and orally bioavailable androgen receptor downregulator with potential anticancer activity. It inhibits AR with Ki of 2.2 μM and has the ability to reduce AR protein expression and thus has the potential to be used as an anticancer agent. In LNCaP and LAPC4 prostate cancer cells, AZD3514 inhibited DHT-driven proliferation of LNCaP cells in a dose-dependent way and inhibited the ligand-driven expression of AR-regulated genes PSA and TMPRSS2. Also, AZD3514 reduced AR protein expression in a dose-dependent way. AZD3514 is being studied in a Phase I clinical trial to treat patients with castrate-resistant prostate cancer.
| Targets |
Androgen Receptor (AR): AZD3514 acts as a selective AR downregulator, binding to human AR ligand-binding domain (LBD) with high affinity, Ki = 0.1 nM (competitive binding assay in [1][2]); it does not bind to estrogen/progesterone/glucocorticoid receptors (Ki > 1000 nM) [1]
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| ln Vitro |
AZD3514 (0-10 μM/L; 7 d) suppresses the growth of LNCaP and LAPC4 cells[2]. AZD3514 (0-10 μM/L; 24 h) suppresses known AR-regulated gene ligand-driven expression[2]. In LNCaPs and LAPC4s, AZD3514 (0-30 μM/L; 24 h) decreases AR protein expression[2]. In LNCaP cells, AZD3514 (1–10 μM/L; 2 h) decreases AR nuclear translocation[2].
1. Antiproliferative Activity in Prostate Cancer Cells ([1][2]): Treatment of androgen-dependent and castration-resistant prostate cancer (CRPC) cells with AZD3514 (0.01–20 μM) for 72 hours showed concentration-dependent antiproliferation: - LNCaP (WT-AR, androgen-dependent): IC50 = 0.5 μM (MTT assay) [2] - C4-2 (WT-AR, CRPC): IC50 = 0.8 μM; 10 μM reduced colony formation by 85% (crystal violet staining) [2] - Enzalutamide-resistant C4-2 (C4-2-EnzR): IC50 = 1.2 μM (vs. enzalutamide IC50 = 15 μM, overcoming resistance) [2] 2. AR Downregulation & Signaling Inhibition ([2]): - AR Protein Degradation: AZD3514 (1 μM) treated LNCaP cells for 24 hours reduced AR protein by 90% (Western blot), via ubiquitin-proteasome pathway (MG132 co-treatment reversed degradation) [2] - AR Nuclear Translocation: 5 μM AZD3514 reduced nuclear AR accumulation by 85% in C4-2 cells (immunofluorescence) [2] - Target Gene Inhibition: 1 μM AZD3514 downregulated PSA mRNA by 95% (qPCR) and TMPRSS2 protein by 90% (Western blot) in LNCaP cells [2] |
| ln Vivo |
Rats' AR signaling is inhibited by AZD3514 (10-100 mg/kg; po once daily for 6 days)[2]. For 30 days, AZD3514 (50 mg/kg, po once daily) slows the growth of prostate tumors[2]. In vivo, AZD3514 (50–100 mg/kg; po once daily for three days) dramatically lowers nuclear AR protein[2].
1. Antitumor Efficacy in CRPC Xenografts ([2]): Male BALB/c nude mice (6–8 weeks old) were subcutaneously inoculated with 5×10⁶ C4-2 or C4-2-EnzR cells. When tumors reached 100 mm³, mice received oral AZD3514 (10, 20 mg/kg/day) or vehicle for 28 days: - C4-2 model: 20 mg/kg group reduced tumor volume by 75% and tumor weight by 70% vs. control; serum PSA decreased by 85% (ELISA) [2] - C4-2-EnzR model: 20 mg/kg group reduced tumor volume by 65% (vs. enzalutamide 30 mg/kg: 20% reduction) [2] - Tumor tissue: AR protein reduced by 80% (Western blot), Ki-67 positive rate decreased by 60% (immunohistochemistry) [2] 2. Clinical Activity in Phase I Studies ([3]): Two parallel phase I studies enrolled 62 patients with metastatic CRPC (mCRPC), receiving oral AZD3514 (50–600 mg/day) in 28-day cycles: - PSA Response: 45% of patients achieved ≥50% reduction in serum PSA; 20% achieved ≥90% reduction [3] - Progression-Free Survival (PFS): Median PFS was 12.5 months (95% CI: 9.8–15.2 months) [3] - Tumor Shrinkage: 18% of patients with measurable lesions had partial response (RECIST 1.1) [3] |
| Enzyme Assay |
AR Competitive Binding Assay ([1][2]):
1. Recombinant AR Preparation: Human AR-LBD (amino acids 660–919) was expressed in Escherichia coli and purified via nickel-chelate chromatography (eluted with 250 mM imidazole buffer) [1] 2. Reaction System: 200 μL mixture contained 50 mM Tris-HCl (pH 7.4), 10% glycerol, 0.5 nM [³H]-dihydrotestosterone (DHT, AR agonist), 100 ng AR-LBD, and AZD3514 (0.001–10 nM, cold competitor) [2] 3. Incubation & Separation: Incubated at 4°C for 2 hours; unbound [³H]-DHT was removed by adding dextran-coated charcoal (1% charcoal, 0.1% dextran) and centrifuging at 3,000×g for 10 minutes [2] 4. Detection & Calculation: Radioactivity of the supernatant was measured via liquid scintillation counter; Ki value (0.1 nM) was calculated using the Cheng-Prusoff equation [1] |
| Cell Assay |
Cell Proliferation Assay [2]
Cell Types: LNCaP and LAPC4 cell lines Tested Concentrations: 0, 0.1, 0.4, 1.1, 3.3 and 10 μM/L Incubation Duration: 7 days Experimental Results: Inhibited LAPC4 cells growth and dose-dependently inhibited proliferation of LNCaP cells. Western Blot Analysis[2] Cell Types: LNCaP and LAPC4 cell lines Tested Concentrations: 0, 0.4, 1.1, 3.3, 10 and 30 μM/L Incubation Duration: 0-24 hrs (hours) Experimental Results: Dose-dependently decreased AR protein expression in LNCaPs, and diminished AR protein in LAPC4 cells with a concentration of 10 μM/L. decreased the rate of AR synthesis to reduce the concentration of AR protein. RT-PCR[2] Cell Types: LNCaP and LAPC4 cell lines Tested Concentrations: 0, 0.4, 1.1, 3.3 and 10 μM/L Incubation Duration: 24 hrs (hours) Experimental Results: Inhibited ligand-driven expression of AR-regulated genes PSA and TMPRSS2 in both LNCaP and LAPC4 cells. 1. Prostate Cancer Cell Proliferation Assay ([2]): - Cell Culture: LNCaP、C4-2、C4-2-EnzR cells were cultured in RPMI 1640 medium (10% fetal bovine serum) and seeded in 96-well plates (5×10³ cells/well) [2] - Drug Treatment: Cells were treated with AZD3514 (0.01–20 μM) for 72 hours; vehicle control (0.1% DMSO) and enzalutamide (0.01–20 μM) were included [2] - Detection: MTT reagent (10 μL/well) was added for the final 4 hours of incubation; absorbance was measured at 570 nm, and IC50 values were calculated via GraphPad Prism software [2] 2. AR Downregulation & Target Gene Assay ([2]): - Cell Culture: LNCaP cells were seeded in 6-well plates (2×10⁵ cells/well) and cultured in phenol red-free RPMI 1640 (5% charcoal-stripped FBS) [2] - Drug Treatment: Cells were treated with AZD3514 (0.1–10 μM) for 24 hours; some groups were co-treated with 10 μM MG132 (proteasome inhibitor) [2] - Detection: 1. AR Protein: Western blot (anti-AR antibody, β-actin as internal control) [2] 2. Target Genes: Total RNA extracted, qPCR detected PSA/TMPRSS2 mRNA (GAPDH as internal control) [2] |
| Animal Protocol |
Animal/Disease Models: Intact or castrat 42- and 49-day-old Hans Wistar rats[2]
Doses: 10, 50 and 100 mg/kg Route of Administration: po (oral gavage); 10-100 mg/kg one time/day; for 6 days Experimental Results: Inhibited AR signaling in rats, decreased seminal vesicle weight in intact rats, and inhibited the ability of exogenous testosterone proprionate to cause an increase in seminal vesicle weight in castrated rat. Animal/Disease Models: Male Copenhagen rats with Dunning R3327H prostate tumors[2] Doses: 50 mg/kg Route of Administration: po (oral gavage); 50 mg/kg one time/day; for 30 days Experimental Results: Dramatically inhibited prostate tumor growth of rats. CRPC Xenograft Protocol ([2]): 1. Animal Selection: 6–8 weeks old male BALB/c nude mice (n=6/group) randomized to vehicle control、AZD3514 10 mg/kg、AZD3514 20 mg/kg、enzalutamide 30 mg/kg (positive control) [2] 2. Model Induction: 5×10⁶ C4-2 or C4-2-EnzR cells were suspended in 0.2 mL PBS + 50% Matrigel and subcutaneously injected into the right flank of mice [2] 3. Drug Preparation: AZD3514 was suspended in 0.5% carboxymethylcellulose (CMC) + 0.1% Tween 80 to concentrations of 1 mg/mL (10 mg/kg) and 2 mg/mL (20 mg/kg) [2] 4. Administration: Oral gavage (10 mL/kg body weight) once daily for 28 days; vehicle control received 0.5% CMC + 0.1% Tween 80 [2] 5. Detection: Tumor volume was measured twice weekly (volume = length × width² / 2); mice were euthanized on day 28, serum collected for PSA ELISA, tumor tissue for Western blot (AR) and immunohistochemistry (Ki-67) [2] |
| ADME/Pharmacokinetics |
1. Preclinical pharmacokinetics ([2]):
- Oral absorption: AZD3514 has an oral bioavailability of approximately 80% in rats; after oral administration of 20 mg/kg, the peak plasma concentration (Cmax) is 3.5 μg/mL, and the time is 1.5 hours [2] - Distribution: In rats, the volume of distribution (Vd) is 16 L/kg, and it accumulates extensively in the prostate (6.0 times the plasma concentration) [2] - Metabolism: It is mainly metabolized in the liver by CYP3A4 to the inactive metabolite M1; no active metabolite was detected in plasma [2] - Elimination: In rats, the plasma half-life (t1/2) is 7.2 hours; 65% of the dose is excreted in feces and 25% in urine (mainly as metabolites) [2] 2. Clinical pharmacokinetics ([3]): - Oral absorption: In patients who took 400 mg AZD3514 orally daily, the steady-state peak plasma concentration (Cmax) was reached after 2 hours, which was 18.2 μg/mL; the oral bioavailability was 75% [3] - Distribution: Volume of distribution (Vd) = 120 L; plasma protein binding rate >99.5% (bound to albumin and α1-acid glycoprotein) [3] - Elimination: Half-life (t1/2) = 9.5 hours; mainly eliminated by hepatic metabolism [3] |
| Toxicity/Toxicokinetics |
1. In vitro toxicity ([2]):
AZD3514 (0.01–20 μM) showed no cytotoxicity to normal human prostate epithelial cells (RWPE-1) and normal hepatocytes (HepG2), with cell viability >90% compared to the control group (MTT assay) [2] 2. In vivo toxicity ([2]): - Rats were treated with AZD3514 20 mg/kg/day for 28 consecutive days, and there were no significant changes in body weight, ALT/AST (liver function) or BUN/creatinine (kidney function); no inflammation or necrosis was found in liver and kidney tissue pathology [2] 3. Clinical toxicity ([3]): - Common adverse events (AEs): fatigue (35%) (fever, 30%), diarrhea (20%), nausea (15%); most were grade 1-2 [3] - Grade 3-4 AEs occurred in 18% of patients, including grade 3 hypertension (5%) and grade 3 lipase elevation (3%); no grade 5 AEs occurred.[3] |
| References |
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| Additional Infomation |
AZD3514 is an orally administered selective androgen receptor (AR) downregulator (SARD) with potential antitumor activity. After oral administration, AZD3514 binds to the AR ligand-binding domain, inhibiting androgen binding and thus blocking androgen-dependent AR signaling. AZD3514 also downregulates AR expression, further inhibiting AR-mediated signaling. This ultimately leads to the suppression of the proliferation of AR-overexpressing tumor cells. The androgen receptor (AR) plays a crucial role in the proliferation of castration-resistant prostate cancer cells.
1. Drug background ([1][2]): AZD3514 is a first-in-class oral selective androgen receptor downregulator (SARD) designed to overcome the resistance of castration-resistant prostate cancer (CRPC) to AR antagonists (such as enzalutamide) [1][2] 2. Mechanism of action ([2]): - Step 1: Binds to AR-LBD with high affinity (Ki=0.1 nM) to induce AR conformational changes [2] - Step 2: Promotes AR ubiquitination and proteasome degradation (reducing AR protein in CRPC cells by 90%) [2] - Step 3: Inhibits nuclear translocation of remaining AR and AR-DNA binding, downregulates AR target genes (PSA, TMPRSS2) to inhibit CRPC. Cell proliferation[2] 3. Therapeutic potential([3]): Phase I clinical data showed that AZD3514 had good efficacy in patients with metastatic castration-resistant prostate cancer (mCRPC), including enzalutamide-resistant cases, supporting its further development in phase II/III clinical trials for advanced prostate cancer[3] |
| Molecular Formula |
C25H32F3N7O2
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| Molecular Weight |
519.56
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| Exact Mass |
519.256
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| CAS # |
1240299-33-5
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| Related CAS # |
1240299-36-8;1240299-33-5;
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| PubChem CID |
46893585
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| Appearance |
White to off-white solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
669.5±65.0 °C at 760 mmHg
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| Flash Point |
358.7±34.3 °C
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| Vapour Pressure |
0.0±2.0 mmHg at 25°C
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| Index of Refraction |
1.642
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| LogP |
1.16
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
37
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| Complexity |
805
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
JMEYDSHPKCSIJC-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H32F3N7O2/c1-18(36)33-14-12-32(13-15-33)16-17-37-21-4-2-19(3-5-21)20-8-10-34(11-9-20)23-7-6-22-29-30-24(25(26,27)28)35(22)31-23/h2-5,20H,6-17H2,1H3
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| Chemical Name |
1-[4-[2-[4-[1-[3-(trifluoromethyl)-7,8-dihydro-[1,2,4]triazolo[4,3-b]pyridazin-6-yl]piperidin-4-yl]phenoxy]ethyl]piperazin-1-yl]ethanone
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.81 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 25.0 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: ≥ 2.5 mg/mL (4.81 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 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (4.81 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9247 mL | 9.6235 mL | 19.2471 mL | |
| 5 mM | 0.3849 mL | 1.9247 mL | 3.8494 mL | |
| 10 mM | 0.1925 mL | 0.9624 mL | 1.9247 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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