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Purity: ≥98%
Darolutamide (formerly known as BAY-1841788; ODM-201; BAY1841788; Nubeqa) is a novel and potent androgen receptor (AR) antagonist with potential antitumor activity. It inhibits AR nuclear translocation with a Ki of 11 nM in competitive AR binding assays. ODM-201 inhibits VCaP cell proliferation with IC50 of 230 nM, while exhibits no effect on the viability of AR-negative cell lines tested, DU-145 prostate cancer cells and H1581 lung cancer cells. In AR-HEK293 cells stably expressing full-length human AR (hAR) and an androgen-responsive luciferase reporter gene construct. In human U2-OS osteosarcoma cells expressing wtAR or mutant AR(F876L), AR(W741L), or AR(T877A), ODM-201 and ORM-15341 also functioned as full antagonists. ODM-201 is a novel AR inhibitor that showed significant antitumor activity and a favorable safety profile in phase 1/2 studies in men with CRPC. ODM-201 is a full and high-affinity AR antagonist that, similar to second-generation antiandrogens enzalutamide and ARN-509, inhibits testosterone-induced nuclear translocation of AR. Importantly, ODM-201 also blocks the activity of the tested mutant ARs arising in response to antiandrogen therapies, including the F876L mutation that confers resistance to enzalutamide and ARN-509. In addition, ODM-201 reduces the growth of AR-overexpressing VCaP prostate cancer cells both in vitro and in a castration-resistant VCaP xenograft model. In contrast to other antiandrogens, ODM-201 shows negligible brain penetrance and does not increase serum testosterone levels in mice. In conclusion, ODM-201 is a potent AR inhibitor that overcomes resistance to AR-targeted therapies by antagonizing both overexpressed and mutated ARs. ODM-201 is currently in a phase 3 trial in CRPC.
Targets |
Androgen receptor (AR) (IC50: 26 nM in AR-HEK293 cells)
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ln Vitro |
In a competitive AR binding experiment, darolutamide (ODM-201) has an inhibitory constant (Ki) value of 11 nM. Compared to ARN-509, ODM-201 and ORM-15341 reduced androgen-induced cell proliferation more successfully. Their respective IC50 values were 230 and 170 nM for Darolutamide and ORM-15341, respectively, whereas ARN-509's was 420 nM. The anti-proliferative qualities of darolutamide and ORM-15341 are exclusive to AR-dependent PC cells, as demonstrated by the lack of effect darolutamide had on the viability of the AR-negative cell lines tested, DU-145 prostate cancer cells and H1581 lung cancer cells [1].
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ln Vivo |
At both doses, darolutamide (ODM-201) demonstrated considerable anti-tumor activity; 50 mg/kg twice day was more effective (p<0.001) than in mice that had not received treatment. It was also demonstrated to be effective against growth inhibition of tumors (p<0.05) in mice that had not received treatment. Furthermore, no symptoms of treatment-related toxicity were seen, and mice given twice daily doses of darolutamide did not significantly lose weight while receiving the medication [1].
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Enzyme Assay |
AR binding affinity [1]
AR binding affinities of test compounds were studied in cytosolic lysates obtained from ventral prostates of castrated rats by a competition binding assay as previously described. Fresh prostates were minced and homogenized with Buffer A containing protease inhibitors. The homogenates were centrifuged and the resultant supernatants were treated with a dextran-coated charcoal solution to remove endogenous steroids. The dissociation constant of the radio ligand [3H]mibolerone for isolated rat ARs was determined in a saturation binding experiment as previously described. For the determination of Ki values, prostate cytosol preparations and 1 nM [3H]mibolerone were incubated with increasing concentrations of test compounds overnight. After the incubation, bound and free steroids were separated by treatment with 100 μL of dextran-coated charcoal suspension. Bound radioactivity was determined by counting 100 μL of supernatant fraction in 200 μL of scintillation fluid (OptiPhase SuperMix, PerkinElmer) using a microbeta counter. All procedures were carried out at 0–4 °C. |
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Cell Assay |
Antagonism of ODM-201[1]
Functional activity and potency of antiandrogens to hAR were determined in AR-HEK293 cells. The cells were treated with test compounds and 0.45 nM testosterone in steroid-free assay medium supplemented with 2 nM GlutaMAX and 25 mM HEPES. After 24 hours at 37 °C with 5% CO2, cells were lysed and luciferase activity was measured with a Centro LB 960 microplate luminometer using a luciferase assay system according to manufacturer’s instructions. Mutant AR studies[1] Human U2-OS osteosarcoma cells were transiently transfected with an androgen-responsive reporter gene construct (pGV5-basic-GRE-hiv-luc) and expression vectors encoding AR mutants AR(F876L), AR(T877A), or AR(W741L) (pSG5-hAR-F876L, pSG5-hAR-T877A, or pSG5-hAR-W741L) using LipofectaminTM2000. The construction of the mutant AR expression vectors was done as previously described. For one well in a 96-well plate, 190 ng of reporter construct DNA and 10 ng of receptor construct DNA were diluted in Opti-MEM® (Gibco). Cells were treated with increasing concentrations of the test compounds in the absence or presence of a reference agonist inducing a submaximal reporter gene activation (0.6 nM testosterone in case of T877A and F876L, and 10 nM DHT in case of W741L) in steroid-free assay medium and incubated for 24 hours. Luciferase activity was measured as described above. AR nuclear translocation[1] AR overexpressing HS-HEK293 cells immunolabeled with an AR-antibody were imaged either with a high-content screening (HCS) reader (Cellomics ArrayScan HCS VTI reader, Thermo) or with a confocal microscope. HS-HEK293 cells in steroid-free assay medium were plated on poly-D-lysine coated microplates (BD) (HCS reader) or on coverslips (confocal imaging). After a 48-hour incubation, the cells were treated with 0.3 (HCS reader) or 1 μM (confocal imaging) of test compounds together with 0.3 nM testosterone for 5 hours. After fixation with 3.7% PFA, the cells were washed with phosphate-buffered saline (PBS), permealized with 0.1% Triton X-100 (Sigma), and treated with 3% BSA in PBS to block unspecific staining. For HCS reader, cells were incubated with polyclonal AR antibody conjugated with Alexa Fluor® 488 (N20, Santa Cruz, dilution 1:50). Cells were washed, DNA was labeled with DAPI (Sigma, 1 μg//mL), and images were analyzed with a NucTrans. V3 assay algorithm (Thermo). For confocal imaging, polyclonal AR antibody was used as a primary and Alexa Fluor® 546 anti-rabbit IgG as a secondary antibody. Coverslips were mounted with Vectashield containing DAPI (Vector Laboratories). AR overexpressing LN-AR-C cells were treated with 3 μM of test compounds together with 0.3 nM testosterone for 4 hours, immunolabeled with the AR-antibody nd a secondary antibody and imaged with the HCS reader. VCaP proliferation assay[1] VCaP cells were treated with a submaximal concentration of mibolerone (0.1 nM) and increasing concentrations of test compounds in steroid-free assay medium supplemented with 4 mM GlutaMAX. After a 4-day incubation with the compounds, cell viability was measured using a WST-1 cell proliferation assay (Roche), according to manufacturer’s instructions. To rule out non-AR –mediated toxicity, AR-negative PC cells (DU-145) and lung cancer cells (H1581) were treated with an increasing concentration of ODM-201, and cell viability was measured as described above. |
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Animal Protocol |
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ADME/Pharmacokinetics |
Absorption, Distribution and Excretion
Darolutamide is absorbed in the gastrointestinal tract. In the fasted state, peak concentrations are reached within 3-5 hours, and within 3-8 hours in the fed state. Median Tmax is between 3-6 hours.The average darolutamide steady-state peak plasma concentration after a 600 mg twice daily dose is approximately 4.79 mg/L. The Cmax is attained approximately 4 hours after administration of a single 600 mg oral dose. The AUC 0-12h is approximately 52.82 h•μg/mL. **Effects of food** The absolute bioavailability of darolutamide is approximately 30% after fasting and taking a single 300 mg dose. Steady-state concentrations are attained between 2 and 5 days after repeated administration with food. The bioavailability of darolutamide increases by 2.0 to 2.5 times when it is given with food. In a pharmacokinetic study, a radiolabeled dose of darolutamide in an oral solution showed that 63.4% of darolutamide-related material was excreted in the urine (7% of which was unchanged drug) and 32.4% in the feces (with 30% unchanged drug). After intravenous administration, the apparent volume of distribution of darolutamide is about 119L. The clearance of darolutamide after an intravenous dose is 116 mL/min (39.7%). Metabolism / Metabolites Darolutamide is mainly metabolized by the CYP3A4 hepatic microsomal enzyme in addition to UGT1A9 and UGT1A1. The main active metabolite keto-darolutamide in found in the plasma at 2 times the concentration of darolutamide. Biological Half-Life The half-life of darolutamide and its active metabolite, keto-darolutamide is about 20 hours. A phase 1 study determined a terminal half life ranging between 10-15 hours. |
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Toxicity/Toxicokinetics |
Hepatotoxicity
In prelicensure controlled trials with 1508 patients, serum AST elevations were more common with darolutamide than placebo therapy [23% vs 14%], but were rarely above 5 times the ULN [ Likelihood score: E (unlikely cause of clinically apparent liver injury). Protein Binding The plasma protein binding for darolutamide is 92% and 99.8% for keto-darolutamide, the active metabolite. They are mainly bound to albumin. |
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References | |||
Additional Infomation |
Darolutamide is a nonsteroidal androgen receptor antagonist for the treatment of castrate-resistant, non-metastatic prostate cancer (nmCRPC). This condition occurs in the majority of patients with advanced prostate cancer who have been treated with androgen receptor antagonists. Though prior treatment for prostate cancer has been successful for these patients, the cancer eventually progresses to become resistant to existing therapies. This warrants further treatment. The goal of treatment with darolutamide is to delay the progression of prostate cancer to metastatic disease, increasing quality of life and life expectancy for those with advanced prostate cancer. Darolutamide was developed by Bayer HealthCare Pharmaceuticals Inc. and approved by the FDA on July 30th, 2019.
Darolutamide is a third generation, oral nonsteroidal antiandrogen used to treat nonmetastatic castration-resistant prostate cancer. Darolutamide is associated with a low rate of serum enzyme elevation during therapy, but has not been linked to cases of clinically apparent liver injury with jaundice. Darolutamide is a formulation containing an androgen receptor (AR) antagonist with potential antineoplastic activity. Darolutamide binds to ARs in target tissues; subsequently, inhibiting androgen-induced receptor activation and facilitating the formation of inactive complexes that cannot translocate to the nucleus. This prevents binding to and transcription of AR-responsive genes that regulate prostate cancer cell proliferation. This ultimately leads to an inhibition of growth in AR-expressing prostate cancer cells. Drug Indication Darolutamide is indicated for the treatment of adults with non-metastatic castration-resistant prostate cancer (nmCRPC) and metastatic hormone-sensitive prostate cancer (mHSPC) in combination with [docetaxel]. NUBEQA is indicated for the treatment of adult men with- non metastatic castration resistant prostate cancer (nmCRPC) who are at high risk of developing metastatic disease (see section 5. 1). - metastatic hormone sensitive prostate cancer (mHSPC) in combination with docetaxel and androgen deprivation therapy (see section 5. 1). Mechanism of Action The actions of androgens on androgen receptors (AR) potentiate the growth and survival of prostate cancer cells. Darolutamide competitively inhibits androgens from binding to their receptors, inhibiting AR nuclear translocation, as well as AR-mediated transcription. The end result of these processes is a decrease in prostate cancer cell proliferation and tumor size. Its main metabolite, keto-darolutamide, shows similar pharmacological activity to the parent drug, darolutamide. Darolutamide has been found to bind more tightly to the AR receptor than [apalutamide] and [enzalutamide], which are other androgen receptor antagonists. Darolutamide can act as a progesterone receptor (PR) antagonist in the laboratory setting with approximately 1% activity when compared to its actions at the androgen receptor. The clinical relevance is not known at this time. Pharmacodynamics Darolutamide, through its downstream effects on cancer cell growth, treats castrate-resistant prostate cancer. It inhibits cancer cell growth and markedly lowers prostate specific antigen (PSA) levels through potent androgen receptor antagonism. |
Molecular Formula |
C19H19CLN6O2
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Molecular Weight |
398.85
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Exact Mass |
398.125
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Elemental Analysis |
C, 57.22; H, 4.80; Cl, 8.89; N, 21.07; O, 8.02
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CAS # |
1297538-32-9
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Related CAS # |
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PubChem CID |
67171867
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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 |
719.5±60.0 °C at 760 mmHg
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Flash Point |
388.9±32.9 °C
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Vapour Pressure |
0.0±2.4 mmHg at 25°C
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Index of Refraction |
1.681
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LogP |
-0.04
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Hydrogen Bond Donor Count |
3
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Hydrogen Bond Acceptor Count |
5
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Rotatable Bond Count |
6
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Heavy Atom Count |
28
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Complexity |
598
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Defined Atom Stereocenter Count |
1
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SMILES |
ClC1=C(C#N)C=CC(C2=NN(C[C@H](C)NC(C3=NNC(C(O)C)=C3)=O)C=C2)=C1
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InChi Key |
ANGUXJDGJCHGOG-UHFFFAOYSA-N
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InChi Code |
InChI=1S/C25H29N5O/c1-19-8-9-22-23(27-19)6-3-7-24(22)29-16-14-28(15-17-29)12-10-20-4-2-5-21(18-20)30-13-11-26-25(30)31/h2-9,18H,10-17H2,1H3,(H,26,31)
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Chemical Name |
N-((S)-1-(3-(3-chloro-4-cyanophenyl)-1H-pyrazol-1-yl)propan-2-yl)-5-(1-hydroxyethyl)-1H-pyrazole-3-carboxamide
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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: ≥ 2.08 mg/mL (5.21 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 (5.21 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (5.21 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 | 2.5072 mL | 12.5360 mL | 25.0721 mL | |
5 mM | 0.5014 mL | 2.5072 mL | 5.0144 mL | |
10 mM | 0.2507 mL | 1.2536 mL | 2.5072 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.