hAR (IC50 = hAR )
ODM-201 and its pharmacologically active main metabolite ORM-15341 are novel and structurally distinct from any known antiandrogens including the second-generation antiandrogens enzalutamide and ARN-509. Figure 1A,B show the chemical structures of ODM-201 and ORM-15341. [1]
In competitive AR binding assays, the inhibition constant (Ki) values of ODM-201 and ORM-15341 were 11 and 8 nM, respectively, being clearly lower than those of enzalutamide (86 nM) and ARN-509 (93 nM) (Fig. 1C) that were tested under the same conditions. ODM-201 and its major metabolite ORM-15341 are also potent and full antagonists for human AR (hAR) with IC50 values of 26 and 38 nM as shown by transactivation assays in AR-HEK293 cells stably expressing full-length hAR and an androgen-responsive luciferase reporter gene construct. [1]
In contrast, in the presence of ODM-201, ORM-15341, enzalutamide, or ARN-509, AR was predominantly cytoplasmic, showing that these antiandrogens inhibit the androgen-induced nuclear translocation of overexpressed AR to same extent[1].

ODM-201 and its pharmacologically active main metabolite ORM-15341 are novel and structurally distinct from any known antiandrogens including the second-generation antiandrogens enzalutamide and ARN-509. Figure 1A,B show the chemical structures of ODM-201 and ORM-15341. [1]
In competitive AR binding assays, the inhibition constant (Ki) values of ODM-201 and ORM-15341 were 11 and 8 nM, respectively, being clearly lower than those of enzalutamide (86 nM) and ARN-509 (93 nM) (Fig. 1C) that were tested under the same conditions. ODM-201 and its major metabolite ORM-15341 are also potent and full antagonists for human AR (hAR) with IC50 values of 26 and 38 nM as shown by transactivation assays in AR-HEK293 cells stably expressing full-length hAR and an androgen-responsive luciferase reporter gene construct. [1]
In contrast, in the presence of ODM-201, ORM-15341, enzalutamide, or ARN-509, AR was predominantly cytoplasmic, showing that these antiandrogens inhibit the androgen-induced nuclear translocation of overexpressed AR to same extent[1].

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ORM-15341 is a potent and full antagonist for human wild-type androgen receptor (hAR), with an IC50 of 38 nM in a transactivation assay using AR-HEK293 cells stably expressing full-length hAR and an androgen-responsive luciferase reporter gene construct.[1]
ORM-15341 functions as a full antagonist for clinically relevant AR mutants that confer resistance to antiandrogen therapies. In transactivation assays using U2-OS cells transiently transfected with mutant AR expression vectors, it antagonized the mutant AR(F876L) (IC50 = 51 nM), AR(W741L) (IC50 = 1160 nM), and AR(T877A) (IC50 = 700 nM). In contrast, enzalutamide and ARN-509 acted as agonists for the F876L mutant.[1]
ORM-15341, like ODM-201, enzalutamide, and ARN-509, inhibited the testosterone-induced nuclear translocation of overexpressed AR in HS-HEK293 cells, as demonstrated by immunocytochemistry and high-content imaging.[1]
ORM-15341 suppressed androgen-induced proliferation of VCaP prostate cancer cells (which have endogenous AR gene amplification and overexpression) in vitro with an IC50 of 170 nM, which was more efficacious than enzalutamide (IC50 = 410 nM) and ARN-509 (IC50 = 420 nM) under the same conditions.[1]
The antiproliferative effect of ORM-15341 (and ODM-201) was specific to AR-dependent cells, as it had no effect on the viability of AR-negative DU-145 prostate cancer cells and H1581 lung cancer cells.[1]

To elucidate the in vivo efficacy of ODM-201 in a CRPC mouse model, castrated male nude mice with subcutaneously injected VCaP cells were treated orally with ODM-201 (50 mg/kg) once (qd) or twice daily (bid), or with enzalutamide (20 mg/kg, qd) for 37 days. The dose for enzalutamide was selected based on previously published in vivo studies9 and our pharmacokinetic (PK) analyses which revealed that in mice the systemic exposure (AUC0–24) for this dose of enzalutamide was 2.5 times higher than that for ODM-201 (50 mg/kg, bid). Moreover, enzalutamide exhibited a long plasma half-life (18.3 hours) while the half-life of ODM-201 in mice was not optimal (1.6 hours) supporting once daily dosing for enzalutamide and higher dose and more frequent dosing for ODM-201. PK data for enzalutamide and ODM-201 are presented in Supplementary Table S1[1].

The binding affinity of ORM-15341 for the androgen receptor was determined using a competitive binding assay. Cytosolic lysates were prepared from the ventral prostates of castrated rats. The lysates were treated to remove endogenous steroids. The assay involved incubating the prostate cytosol preparation and a fixed concentration of the radio ligand [3H]mibolerone with increasing concentrations of the test compound overnight at 0–4°C. Bound and free steroids were separated using a dextran-coated charcoal suspension. The bound radioactivity in the supernatant was measured using a liquid scintillation counter to determine the inhibition constant (Ki).[1]

The functional antagonistic activity and potency of ORM-15341 against the wild-type human AR were determined in AR-HEK293 cells stably expressing full-length hAR and an androgen-responsive luciferase reporter. Cells were treated with the test compound and a low concentration of testosterone (0.45 nM) in steroid-free assay medium. After 24 hours of incubation, cells were lysed, and luciferase activity was measured using a luminometer.[1]
The effects of ORM-15341 on mutant ARs were studied in transactivation assays. Human U2-OS osteosarcoma cells were transiently transfected with expression vectors encoding specific AR mutants (F876L, T877A, or W741L) and an androgen-responsive luciferase reporter gene construct. Cells were treated with increasing concentrations of the test compound in the presence of a reference agonist (testosterone or DHT) inducing submaximal reporter gene activation in steroid-free medium. After 24 hours of incubation, luciferase activity was measured.[1]
The effect of ORM-15341 on AR nuclear translocation was assessed using AR-overexpressing HS-HEK293 cells. Cells were plated in steroid-free medium and treated with the test compound (0.3 μM) together with testosterone (0.3 nM) for several hours. Cells were then fixed, permeabilized, and immunolabeled with an AR antibody conjugated to a fluorescent dye. The nuclear localization of AR was quantified using a high-content screening reader.[1]
The antiproliferative effect of ORM-15341 on AR-dependent prostate cancer cells was evaluated using the VCaP cell line. Cells were treated with a submaximal concentration of the synthetic androgen mibolerone (0.1 nM) and increasing concentrations of the test compound in steroid-free assay medium. After a 4-day incubation, cell viability was measured using a colorimetric cell proliferation assay (WST-1).[1]

The brain penetrance of ORM-15341 was evaluated in pharmacokinetic studies in mice. Nude male mice were orally dosed with ODM-201 (25, 50, or 100 mg/kg, twice daily) for 7 days. Blood samples were collected, and plasma was separated. Brain samples (without olfactory bulbs and medulla oblongata) were pooled and homogenized. Concentrations of ORM-15341 (as a metabolite of ODM-201) in mouse plasma and brain homogenates were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma and brain concentration versus time data were analyzed using non-compartmental methods. Brain-to-plasma ratios were calculated based on the area under the concentration-time curve from 0 to 24 hours (AUC0-24) values.[1]

ORM-15341 was identified as the major pharmacologically active metabolite of ODM-201. [1]
ORM-15341 had extremely low brain permeability in mice. The brain-plasma ratio based on AUC0-24 was 1.9%–2.8% at different dose levels of the parent compound ODM-201, and no dose-response relationship was observed. [1]

[1]. Discovery of ODM-201, a new-generation androgen receptor inhibitor targeting resistance mechanisms toandrogen signaling-directed prostate cancer therapies. Sci Rep. 2015 Jul 3;5:12007. doi: 10.1038/srep12007.

[2]. Preparation of heteroaryl carboxamides as androgen receptor modulators. From PCT Int. Appl. (2012), WO 2012143599 A1 20121026.

Ketodarolutamide is a nonsteroidal antiandrogen (NSAA) and its major active metabolite. Androgen receptor (AR) activation is crucial for prostate cancer growth. Notably, castration-resistant prostate cancer (CRPC) also depends on functional AR, and several mechanisms have been proposed to explain this dependence. Known etiologies of CRPC include AR gene amplification and overexpression, as well as point mutations. This article reports the pharmacological characteristics of a novel AR inhibitor, ODM-201. This inhibitor demonstrated significant antitumor activity and a favorable safety profile in a phase I/II study of CRPC patients. ODM-201 is a complete and high-affinity AR antagonist, similar to second-generation antiandrogens enzalutamide and ARN-509, inhibiting testosterone-induced AR nuclear translocation. Importantly, ODM-201 also blocks the activity of mutant androgen receptors (AR) induced by antiandrogen therapy, including the F876L mutation, which confers resistance to enzalutamide and ARN-509. In addition, ODM-201 inhibited the growth of AR-overexpressing VCaP prostate cancer cells in vitro and in castration-resistant VCaP xenograft models. Unlike other antiandrogen drugs, ODM-201 has very low brain penetration and does not increase serum testosterone levels in mice. In summary, ODM-201 is a potent AR inhibitor that overcomes resistance to AR-targeted therapies by antagonizing overexpression and mutation of AR. ODM-201 is currently undergoing a phase III clinical trial for CRPC. [1] In summary, ODM-201 is a highly active next-generation androgen receptor (AR) inhibitor that antagonizes known AR mutants AR(F876L), AR(W741L), and AR(T877A) that mediate resistance to first- and second-generation antiandrogens. ODM-201 also exerts an antagonistic effect in AR-overexpressing cells and inhibits nuclear translocation of the receptor. In nonclinical in vitro and in vivo models of CRPC, ODM-201 was more effective than other tested antiandrogens and did not stimulate the androgen feedback loop of the hypothalamic-pituitary-gonadal axis. In summary, these results suggest that ODM-201 has unique properties that may be superior to first- and second-generation antiandrogens in the treatment of CRPC. These nonclinical results have translated into significant antitumor activity and good tolerability and safety observed in phase 1 and phase 2 clinical trials in male patients with metastatic CRPC.28 In these trials, 86% of treatment-naïve patients receiving 700 mg twice daily achieved a PSA response (a decrease of 50% or more). [1]

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ORM-15341 is a novel compound whose structure is different from any known antiandrogen, including second-generation antiandrogens such as enzalutamide and ARN-509. [1]
ORM-15341 is a complete antagonist of the AR(F876L) mutation, which confers resistance by converting enzalutamide and ARN-509 into agonists. [1]
ORM-15341 (and ODM-201) have extremely low brain penetration, suggesting a low risk of inducing seizures through central nervous system mechanisms, a common side effect of some other antiandrogens. [1]

C₁₉H₁₇CLN₆O₂

396.83

396.11

C, 57.51; H, 4.32; Cl, 8.93; N, 21.18; O, 8.06

1297537-33-7

1297537-33-7;

52919826

White to off-white solid powder

3.21

2

5

6

28

637

1

ClC1=C(C#N)C=CC(=C1)C1C=CN(C[C@H](C)NC(C2=CC(C(C)=O)=NN2)=O)N=1

GMBPVBVTPBWIKC-NSHDSACASA-N

InChI=1S/C19H17ClN6O2/c1-11(22-19(28)18-8-17(12(2)27)23-24-18)10-26-6-5-16(25-26)13-3-4-14(9-21)15(20)7-13/h3-8,11H,10H2,1-2H3,(H,22,28)(H,23,24)/t11-/m0/s1

3-acetyl-N-[(2S)-1-[3-(3-chloro-4-cyanophenyl)pyrazol-1-yl]propan-2-yl]-1H-pyrazole-5-carboxamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.30 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.

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Solubility in Formulation 2: 2.5 mg/mL (6.30 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.30 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 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.)