Enzymes in cholesterol biosynthesis: oxidosqualene cyclase (OSC); sterol Δ⁸-Δ⁷ isomerase (emopamil-binding protein); desmosterol reductase (Δ²⁴-reductase) [2].
No specific IC₅₀ values were reported for U-18666A in the provided literature. Inhibition is reported as noncompetitive for OSC and desmosterol reductase [2].
Intracellular cholesterol trafficking: inhibits the egress of free cholesterol from late endosomes/lysosomes, likely by interfering with the Niemann-Pick type C1 (NPC1) protein [1, 2].
No specific IC₅₀ or EC₅₀ values were reported.
Dengue virus infection: inhibits dengue virus (DENV) entry/trafficking and replication [1].

Inhibition of sterol synthesis: U-18666A inhibits multiple steps in cholesterol biosynthesis. At low concentrations (≥0.1 μM), it inhibits oxidosqualene cyclase (OSC) and sterol Δ⁸-Δ⁷ isomerase; at higher concentrations, it also inhibits desmosterol reductase (Δ²⁴-reductase) [2].
Inhibition of cholesterol trafficking: In BHK21 cells, U-18666A (6.15 μM, 16 h) caused accumulation of free cholesterol in punctate intracellular organelles that colocalized with the late endosome/lysosome marker Lamp-1, as visualized by Filipin III staining. It also inhibited the movement of cholesterol from lysosomes to the endoplasmic reticulum and plasma membrane [1, 2].
Inhibition of cholesterol biosynthesis: In DENV-infected BHK21 cells, U-18666A (6.15 μM) treatment for 48 h reduced the level of zymosterol (an intermediate in sterol biosynthesis) by 67% (p < 0.05), while total cholesterol levels were not significantly changed [1].
Antiviral activity against dengue virus: In BHK21 cells infected with DENV (MOI=1), U-18666A (6.15 μM) treatment for 48 h reduced viral titers by >1 log (16 h pretreatment), >1 log (after entry), and >3 logs (throughout infection). In DENV replicon cell lines, U-18666A inhibited viral replication with EC₅₀ values of 6.2 μM (A549 replicon) and 2.9 μM (Huh7 replicon) [1].
Effect on viral trafficking: In U-18666A-treated BHK21 cells (6.15 μM, 16 h pretreatment), DENV particles bound to the cell surface normally but were trapped in Lamp-1-positive late endosomes/lysosomes at 4-8 h post-infection. Newly synthesized viral envelope protein was markedly reduced at 12-24 h post-infection, indicating impaired viral uncoating or fusion [1].
Induction of apoptosis: U-18666A induces apoptosis in lens epithelial cells, melanoma cells, and cultured murine cortical neurons. Apoptosis is associated with oxidative stress, activation of caspases, disruption of mitochondrial function, and accumulation of free cholesterol in lysosomes [2].
Effect on lipid raft association: In BHK21 cells infected with DENV (MOI=10) and treated with U-18666A (6.15 μM) for 24 h, the nonstructural proteins NS3 and NS4B remained associated with detergent-resistant membrane (lipid raft) fractions, indicating that replication complex formation was not disrupted [1].
Additive antiviral effect with C75: In Huh7 DENV replicon cells, combination treatment with U-18666A (1.23 μM) and the fatty acid synthase inhibitor C75 (8.3 μM) produced an 80.4% reduction in replicon activity, compared to 46.4% (U-18666A alone) and 59.7% (C75 alone), indicating an additive effect [1].

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U18666A, the antiviral effect was discovered to be induced by two events: a delay in viral trafficking in cholesterol-loaded late endosomes/lysosomes and a suppression of de novo sterol biosynthesis in treated infected cells. An additive antiviral impact of U18666A combined with C75 with medium synthase was also observed, demonstrating that dengue virus relies on amylase and medium biosynthesis for effective reproduction [1][2].

Induction of epilepsy: Treatment of young rats with U-18666A induces chronic absence (petit mal) epilepsy. The condition is characterized by electroencephalogram (EEG) seizure patterns without major motor involvement and can persist after drug discontinuation. The mechanism may involve altered GABA receptor function and/or changes in brain sterol composition [2].
Induction of cataracts: Administration of U-18666A to young rats causes cataracts. The mechanism involves a decrease in the cholesterol-to-phospholipid ratio in lens fiber cell plasma membranes, leading to altered membrane structure, increased light scatter, and apoptosis of lens epithelial cells [2].
Induction of adrenocortical toxicity: U-18666A (also known as PD-132301 or ATR-101) causes selective toxicity to the adrenal cortex in animals, characterized by accumulation of free cholesterol in lysosomes, cortical atrophy, and apoptosis. This adrenal-specific toxicity led to its repurposing for adrenocortical carcinoma treatment [2].

Cholesterol accumulation assay (Filipin III staining): BHK21 cells were treated with U-18666A (6.15 μM) for 16 h, fixed with 4% paraformaldehyde, and stained with Filipin III (70 μg/mL) for 1 h in the dark to visualize free cholesterol. Colocalization with Lamp-1 was assessed by immunofluorescence [1].
Lipid raft isolation: BHK21 cells were infected with DENV (MOI=10) and treated with U-18666A (6.15 μM) for 24 h. Cells were homogenized in low salt buffer with 1% Triton X-100 on ice. The post-nuclear supernatant was overlaid on a 10-55-75% sucrose gradient and centrifuged at 38,000 rpm for 16 h. Fractions were collected, concentrated, and analyzed by SDS-PAGE and immunoblotting for NS3, NS4B, and caveolin-1 [1].
Sterol analysis by GC-MS: Lipids were extracted from cells with chloroform:methanol (1:2). Sterols were separated using an HPLC column and analyzed by mass spectrometry in positive atmospheric pressure chemical ionization (APCI) mode. Multiple reaction monitoring (MRM) was used for quantification of endogenous sterols, cholesterol-d6, and zymosterol-d5 [1].

Plaque assay (viral titer): BHK21 cells were seeded in 24-well plates and infected with 10-fold serial dilutions of viral supernatant. After 1 h adsorption, inoculum was replaced with 0.8% methylcellulose in RPMI-1640 with 2% FBS. After 4 days, cells were fixed with 3.7% formalin, stained with 1% crystal violet, and plaques were counted [1].
DENV replicon assay: A549 and Huh7 cells stably transfected with DENV replicon containing Renilla luciferase were seeded in 96-well plates. Compounds were added in 10-point 2-fold dilutions (0-100 μM) for 48 h. Renilla luciferase activity was measured using EnduRen substrate, and luminescence was read with a luminometer. EC₅₀ values were calculated using nonlinear regression [1].
Cell viability assay (CellTiter-Glo): Cellular ATP levels were measured using a luminescence-based kit to assess cytotoxicity. Cells were treated with compounds for 48 h, and luminescence was read to determine CC₅₀ values [1].
Immunofluorescence microscopy: Cells were fixed with 4% paraformaldehyde, permeabilized with 0.05% saponin, and labeled with primary antibodies (anti-Env 4G2, anti-EEA1, anti-Lamp-1) followed by Alexa Fluor-conjugated secondary antibodies. Stained cells were examined using a laser-scanning confocal microscope [1].
Viral binding assay: BHK21 cells (control or U-18666A-pretreated) were incubated with DENV (MOI=50) on ice for 1 h to allow binding without endocytosis. Cells were fixed and stained with anti-Env antibody (4G2) to visualize bound viral particles [1].

Induction of epilepsy in rats: Young rats were treated with U-18666A to induce chronic absence (petit mal) epilepsy. The specific dosing regimen (dose, route, frequency) was not detailed in the provided literature [2].
Induction of cataracts in rats: Young rats were treated with U-18666A to induce cataracts. The specific dosing regimen (dose, route, frequency) was not detailed in the provided literature [2].

Tissue distribution: In rats, U-18666A distributes to the brain, lens, and other tissues. The half-life and clearance were not reported in the provided literature [2].
Binding to membranes: U-18666A binds with high specificity to intact cell membranes, likely intercalating into the hydrocarbon core and increasing membrane order [2].

Adrenocortical toxicity: U-18666A causes selective toxicity to the adrenal cortex, characterized by accumulation of free cholesterol in lysosomes, cortical atrophy, and apoptosis. This effect is dose-dependent and specific to steroidogenic tissues [2].
Cataract induction: U-18666A is cataractogenic in rats, causing lens opacification due to altered cholesterol-to-phospholipid ratios in lens fiber cell membranes and apoptosis of lens epithelial cells [2].
Neurotoxicity: U-18666A induces apoptosis in cultured murine cortical neurons, associated with oxidative stress, mitochondrial dysfunction, and caspase activation. Pravastatin (an HMG-CoA reductase inhibitor) can protect against U-18666A-induced cell death, suggesting that intermediates in the sterol pathway may contribute to cytotoxicity [2].
Cardiotoxicity: U-18666A was originally reported to have coronary vasodilator activity, but this claim was not substantiated in the literature [2].

[1]. U18666A, an intra-cellular cholesterol transport inhibitor, inhibits dengue virus entry and replication. Antiviral Res. 2012 Jan;93(1):191-8.

[2]. Cholesterol synthesis inhibitor U18666A and the role of sterol metabolism and trafficking in numerous pathophysiological processes. Lipids. 2009 Jun;44(6):477-87.

3β-(2-Diethylaminoethoxy)androst-5-en-17-one hydrochloride is prepared by reacting 3β-(2-diethylaminoethoxy)androst-5-en-17-one with an equivalent amount of hydrochloric acid. It is an inhibitor of cholesterol synthesis and transport. It has various activities, including as an EC 1.3.1.72 (Δ(24)-sterol reductase) inhibitor, nicotinic receptor antagonist, sterol biosynthesis inhibitor, antiviral agent, and Hedgehog signaling pathway inhibitor. It contains 3β-(2-diethylaminoethoxy)androst-5-en-17-one (1+).

C25H42CLNO2

424.06

423.29

C, 70.81; H, 9.98; Cl, 8.36; N, 3.30; O, 7.55

3039-71-2

2855-62-1;3039-71-2 (HCl);

9954082

White to off-white solid powder

6.047

1

3

6

29

624

6

Cl[H].O=C1CC[C@]2([H])[C@]1(C)CC[C@]1([H])[C@@]3(C)CC[C@@H](CC3=CC[C@]12[H])OCCN(CC)CC

GZFYZYBWLCYBMI-MYZJJQSMSA-N

InChI=1S/C25H41NO2.ClH/c1-5-26(6-2)15-16-28-19-11-13-24(3)18(17-19)7-8-20-21-9-10-23(27)25(21,4)14-12-22(20)24;/h7,19-22H,5-6,8-17H2,1-4H3;1H/t19-,20-,21-,22-,24-,25-;/m0./s1

(3S,8R,9S,10R,13S,14S)-3-[2-(diethylamino)ethoxy]-10,13-dimethyl-1,2,3,4,7,8,9,11,12,14,15,16-dodecahydrocyclopenta[a]phenanthren-17-one;hydrochloride

U18666A; U 18666A; 3039-71-2; U-18666-A; NSC-70801; 3beta-(2-Diethylaminoethoxy)androst-5-en-17-one hydrochloride; ...; U18666-A; U-18666A

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: 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)

DMSO : ~20.83 mg/mL (~49.12 mM)
H2O : ~5.56 mg/mL (~13.11 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.90 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.90 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.90 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 5 mg/mL (11.79 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)