SARMs/selective androgen receptor modulators
Androgen Receptor (AR) (Binding pKi = 8.3 ± 0.2; R-SAT™ assay pEC50 = 7.9 ± 0.3, % Efficacy = 67 ± 9; Luciferase reporter gene assay pEC50 = 8.8 ± 0.1, % Efficacy = 72 ± 9) [1]

AC-262536 dose-dependently prevents the proliferation of LNCaP that is induced by dihydroxytestosterone (DHT). The effects peak at 1 μM treatment (%inhibition 50.7±7.6) and are statistically significant at 100 nM AC-262536 (%inhibition 47.2±12.2). In prostate cells, AC-262536 can therefore function as a functional antagonist[1].

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AC-262536 exhibited potent agonist activity at the androgen receptor, with an affinity in the low nanomolar range (1–10 nM) as demonstrated in two functional assays and binding assay. This prototype lead has no detectable agonist activity at other nuclear receptors including the steroid receptors ERa, ERb, GR, MR and PR. In contrast to steroidal androgens and published SARMs, AC-262536 was found to be a partial agonist relative to testosterone, both in cell proliferation and transcriptional assays. Its activity was about 70–75% that of DHT. Additionally, a number of AR mutants were generated to further confirm the partial agonist nature of AC-262536. The mutations were chosen because they impair the response of AR to testosterone. Thus, such receptor mutants being less sensitive to the full agonist testosterone, should only respond marginally or not at all to a partial agonist. Indeed, AC-262536 was weakly responsive further supporting its partial agonist nature. [1]

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In a functional cell-based R-SAT™ assay, AC-262536 demonstrated potent agonist activity at the AR with a pEC50 of 7.9 ± 0.3. It exhibited partial agonist properties, achieving 67 ± 9% efficacy relative to the natural androgen testosterone. [1]
In a luciferase reporter gene assay using MDA-kb2 cells, AC-262536 modulated AR-dependent transcriptional activity with low nanomolar potency (pEC50 = 8.8 ± 0.1) and displayed partial agonism (72 ± 9% efficacy relative to testosterone). [1]
In binding assays using cell extracts expressing the AR, AC-262536 showed a physical interaction with the receptor, exhibiting a binding pKi of 8.3 ± 0.2. [1]
In selectivity profiling, AC-262536 was extremely selective for AR, showing no significant affinity for 47 other human nuclear receptors, including the steroid receptors estrogen receptors (ERα, ERβ), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and progesterone receptor (PR), at doses as high as 10 µM. [1]
Using AR mutants, the partial agonist nature of AC-262536 was further characterized. Its activity was markedly reduced in the T877A mutant (37% relative to testosterone at WT) and was completely inactive at the G743A and A748T mutants. [1]
In the human prostate cancer cell line LNCaP, AC-262536 dose-dependently inhibited dihydroxytestosterone (DHT, 10 nM)-induced proliferation. The inhibition was 47.2 ± 12.2% at 100 nM (p = 0.006) and reached a plateau at 1 µM with 50.7 ± 7.6% inhibition (p = 0.002). [1]

In castrated rats, AC-262536 (3, 10, 30 mg/kg) reverses the luteinizing hormone (LH) spike[1].

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A 2-week study in castrated male rats was conducted to evaluate and compare the effects of AC-262536 to testosterone. AC-262536 suppressed castration-induced elevations in plasma LH. These effects were similar in potency and efficacy to that of testosterone. Additionally, robust anabolic effects were evident on muscle weight. AC-262536 restored the weight of levator ani to about 70% of the maximum effect seen with testosterone. At doses where AC-262536 displayed significant anabolic effects on muscle, the androgenic effects on prostate and seminal vesicles were weak. Furthermore, it appeared that the maximum effects of AC-262536 produced in these tissues were much less relative to testosterone. These effects are consistent with the results observed in the prostate cancer cell line LNCaP, where AC-262536 functionally antagonized the proliferative properties of the full agonist DHT.[1]

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In a 14-day chronic study in castrated male rats, daily subcutaneous administration of AC-262536 (3, 10, 30 mg/kg) significantly suppressed the castration-induced elevation in plasma luteinizing hormone (LH). At 10 and 30 mg/kg, the effect was stronger than testosterone propionate (TP, 1 mg/kg), reducing LH levels to 1.91 ± 0.32 ng/ml and 1.53 ± 0.34 ng/ml, respectively (vs. 3.12 ± 0.69 ng/ml for TP). The ED50 for this effect was 2.8 mg/kg. [1]
AC-262536 dose-dependently increased the weight of the levator ani muscle, a measure of anabolic effect. At the highest dose of 30 mg/kg, it increased muscle weight to 192 ± 12 mg, achieving 66% of the maximal effect seen with TP (255 ± 14 mg). The ED50 for the anabolic effect was 17.3 mg/kg. [1]
In contrast to its anabolic effects, AC-262536 exhibited weak androgenic effects on the prostate and seminal vesicles. At 10 and 30 mg/kg, prostate weights were 55 ± 6 mg and 54 ± 5 mg, respectively, reaching only 27.0 ± 2.5% of the TP-induced increase (223 ± 14 mg). Similarly, seminal vesicle weights at the highest dose (208 ± 15 mg) were only 29.9 ± 2.2% of the TP-induced increase (748 ± 48 mg). [1]

Binding assays[1]
Hamster DDT cells were grown in DMEM 5% charcoal-stripped FBS in presence of 10 nM testosterone. Cells were plated at 25,000 cells/well in a 24-well plate (500 μl/well) and grown to 80% confluency (typically 3 days). At that stage, the media was replaced and 3H-DHT added to a final concentration of 2 nM, along with varying concentrations of the test ligands. Cells were incubated for an additional 24-h period. Cells were then washed multiple times with ice-cold HBSS (Hank's balanced salt solution) then resuspended in 100% ethanol (100 μl/well). Plates were then sealed and shaken for 6 h. Extracts were then quantified in a Beckman scintillation counter.

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Androgen Receptor Binding Assay: Cells expressing the androgen receptor were lysed to obtain extracts. These extracts were incubated with varying concentrations of AC-262536 to determine its binding affinity. The binding constant (pKi) was calculated from the competition binding data, with results showing a high affinity interaction. [1]
Nuclear Receptor Selectivity Profiling: AC-262536 was screened against a panel of 47 human nuclear receptors, including other steroid receptors like ERα, ERβ, GR, MR, and PR. The compound was tested at concentrations up to 10 µM to assess any significant binding or agonist activity, confirming its high selectivity for the androgen receptor. [1]

DHT/dihydroxytestosterone-induced proliferation of LNCaP[1]
Human prostate cancer LNCaP cells were plated at 30,000 cells per 96-well overnight. Media was then replaced by DMEM 2% charcoal-stripped FBS in presence of 10 nM DHT. AC-262536 was added at the indicated doses. Media was replenished every other day. After 5 days, the media was removed and cell proliferation measured by incubating with the tetrazolium salt WST-1 according to manufacturer's instructions (Roche Diagnostics). Absorbance at 405 nM was measured using a microplate reader after a 1-h incubation at 37 °C.[1]

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R-SAT™ Assay: Mouse NIH-3T3 fibroblasts were plated and transiently transfected with expression vectors for the androgen receptor, β-galactosidase reporter gene, and co-activators (SRC1, DRIP205, GRIP1). Post-transfection, cells were incubated with various concentrations of AC-262536 for 5 days. The receptor-dependent proliferative response was measured by adding a β-galactosidase substrate (ONPG), and the absorbance was read at 420 nm to generate dose-response curves. [1]
Luciferase Reporter Gene Assay: Human breast carcinoma MDA-kb2 cells, which stably express AR and contain an MMTV-luciferase construct, were plated. The following day, the medium was replaced with fresh medium containing varying concentrations of AC-262536. After 24 hours, cells were lysed, and luciferase activity was measured as a readout of AR-mediated transcriptional activity. [1]
DHT-Induced Proliferation Assay in LNCaP Cells: Human prostate cancer LNCaP cells were plated. After 24 hours, the medium was replaced with medium containing 10 nM DHT to induce proliferation, along with various doses of AC-262536. The medium was replenished every other day. After 5 days, cell proliferation was measured by adding the tetrazolium salt WST-1. Absorbance was measured at 405 nm after a 1-hour incubation. [1]

Male Sprague-Dawley rats (200-225 g)[1]
3, 10 and 30 mg/kg
Administered subcutaneously; once daily for 14 consecutive days

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Male Sprague–Dawley rats (200–225 g) were either castrated or sham-operated and allowed to recover for 5 days. Thereafter, castrated animals (n = 8 per group) were treated once daily with vehicle, testosterone propionate (TP 1 mg/kg) or AC-262536 (3, 10 and 30 mg/kg) administered subcutaneously for 14 consecutive days. Twenty-four hours following the last injection, animals were sacrificed. The plasma samples were then obtained and the organs harvested. The levator ani muscle, the prostate glands and the seminal vesicles were dissected free of adipose tissue, blotted dry and their respective weights determined. Plasma levels of luteinizing hormone (LH) were quantified using a commercially available ELISA kit according to manufacturer's instructions [1].

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14-Day Efficacy Study in Castrated Rats:** Male Sprague-Dawley rats (200-225 g) were either castrated (ORX) or sham-operated. After a 5-day recovery period, castrated animals (n=8 per group) were treated once daily for 14 consecutive days with subcutaneous injections. Treatment groups included: vehicle, testosterone propionate (TP, 1 mg/kg), and AC-262536 at 3, 10, and 30 mg/kg. Twenty-four hours after the final injection, animals were sacrificed. Plasma samples were collected, and the levator ani muscle, prostate glands, and seminal vesicles were dissected, blotted dry, and weighed. Plasma levels of luteinizing hormone (LH) were quantified using an ELISA kit. [1]

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14-Day Efficacy Study in Castrated Rats: Male Sprague-Dawley rats (200-225 g) were either castrated (ORX) or sham-operated. After a 5-day recovery period, castrated animals (n=8 per group) were treated once daily for 14 consecutive days with subcutaneous injections. Treatment groups included: vehicle, testosterone propionate (TP, 1 mg/kg), and AC-262536 at 3, 10, and 30 mg/kg. Twenty-four hours after the final injection, animals were sacrificed. Plasma samples were collected, and the levator ani muscle, prostate glands, and seminal vesicles were dissected, blotted dry, and weighed. Plasma levels of luteinizing hormone (LH) were quantified using an ELISA kit. [1]

[1]. Pharmacological characterization of AC-262536, a novel selective androgen receptor modulator. J Steroid Biochem Mol Biol. 2008 Mar;109(1-2):129-37.

A selective androgen receptor modulator.

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AC-262536 (4-(3-Hydroxy-8-aza-bicyclo[3.2.1]octyl)-naphthalene-1-carbonitrile) is a novel, non-steroidal selective androgen receptor modulator (SARM). It was identified using a functional cell-based assay (R-SAT™). Its structure is distinct from other known SARMs. [1]
The partial agonist nature of AC-262536 allows it to function as a functional antagonist in prostate cells, as demonstrated by its inhibition of DHT-induced proliferation in LNCaP cells. This property may contribute to its favorable tissue selectivity. [1]
The in vivo data from the 14-day castrated rat study indicates that AC-262536 effectively separates anabolic effects (increasing levator ani muscle weight, suppressing LH) from androgenic effects (prostate and seminal vesicle growth), classifying it as a SARM with a favorable safety and efficacy profile. [1]

C18H18N2O

278.36

278.142

C, 77.67; H, 6.52; N, 10.06; O, 5.75

870888-46-3

870888-46-3

44512434

White to off-white solid powder

3.268

1

3

1

21

428

2

O[C@H]1C[C@H]2CC[C@H](N2C2C=CC(C#N)=C3C=CC=CC=23)C1

ATKWLNSCJYLXPF-YIONKMFJSA-N

InChI=1S/C18H18N2O/c19-11-12-5-8-18(17-4-2-1-3-16(12)17)20-13-6-7-14(20)10-15(21)9-13/h1-5,8,13-15,21H,6-7,9-10H2/t13-,14+,15?

4-[(1R,5S)-3-hydroxy-8-azabicyclo[3.2.1]octan-8-yl]naphthalene-1-carbonitrile

AC-262536; AC262536; 870888-46-3; U8VS41J5O6; 4-(3-endo-Hydroxy-8-azabicyclo(3.2.1)oct-8-yl)naphthalene-1-carbonitrile; AC 262536; 4-[(3-endo)-3-hydroxy-8-azabicyclo[3.2.1]oct-8-yl]-1-naphthalenecarbonitrile; 4-[(1R,5S)-3-hydroxy-8-azabicyclo[3.2.1]octan-8-yl]naphthalene-1-carbonitrile; 4-(3-hydroxy-8-azabicyclo[3.2.1]oct-8-yl)naphthalene-1-carbonitrile; AC 262536

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: ~50 mg/mL (~179.6 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.98 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.98 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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Solubility in Formulation 3: ≥ 0.56 mg/mL (2.01 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 5.6 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.)