mERα (IC50 = 2.3 nM); hERα (IC50 =28 nM); HBx
Estradiol benzoate acts as a prodrug of estradiol. After administration, it is hydrolyzed to release the active estrogen, 17β-estradiol, which binds to and activates estrogen receptors (ERα and ERβ). It is an estrogen receptor agonist with IC₅₀ values in the range of 22-28 nM for rat ER, human ERα, and chicken ER. Estradiol benzoate also inhibits HBx protein and androgen and hepatitis B virus (HBV) transcription and replication.

Estradiol benzoate is an asynthetic ester of the natural estrogen estradiol, more precisely its 3-benzoyl ester. The development of the female genotype during embryogenesis and puberty is regulated by estrogens. The primary estrogen that the premenopausal ovary secretes is estradiol. In order to induce estrus in domestic livestock, estradiol benzoate, an estradiol analog with a benzyl ester at the C-3 position, is frequently used in conjunction with a progestin. With IC50 values between 22 and 28 nM, this compound binds to the estrogen receptor α (ERα) in humans, mice, and chickens. When compared to estradiol, this indicates a binding affinity reduction of 6–10 fold.[1]
In vitro, estradiol benzoate is an estrogen receptor agonist with IC₅₀ values in the range of 22-28 nM for rat ER, human ERα, and chicken ER. It inhibits HBx protein and androgen and hepatitis B virus (HBV) transcription, replication. The compound shows antifertility effects and anti-Toxoplasma gondii activity. It can improve memory behavior of ovariectomized female mice.

In the Ovx female model, estradiol benzoate (20–100 μg/kg, subcutaneous injection, once daily for 4-5 weeks) enhances learning and memory behavior [1]. Estradiol benzoate (subcutaneous injection, single dose, 0.015–15000 μg/kg)
In vivo, estradiol benzoate is used in hormone therapy for the treatment of menopausal symptoms and hypoestrogenism. It is often used in combination with a progestin to induce estrus in domestic livestock. The compound increases blood coagulability and has mild anabolic and metabolic effects. It shows antifertility effects.

HBx plays a significant role in the cccDNA epigenetic modification regulating the hepatitis B virus (HBV) life cycle and in hepatocyte proliferation and carcinogenesis. By using the sleeping-beauty transposon system, we constructed a tetracycline-induced HBx-expressing stable cell line, SBHX21. HBx with a HiBiT tag can be quickly detected utilizing a NanoLuc-based HiBiT detection system. By screening a drug library using SBHX21 cells, we identified estradiol benzoate as a novel anti-HBx agent. Estradiol benzoate also markedly reduced the production of HBeAg, HBsAg, HBV pgRNA, and HBV DNA in a dose-dependent manner, suggesting that estradiol benzoate could be an anti-HBV agent. Docking model results revealed that estradiol benzoate binds to HBx at TRP87 and TRP107. Collectively, our results suggest that estradiol benzoate inhibits the HBx protein and HBV transcription and replication, which may serve as a novel anti-HBV molecular compound for investigating new treatment strategies for HBV infection.[3]
Non-cellular receptor binding assays for estradiol benzoate involve competitive radioligand binding displacement studies using estrogen receptor (ERα and ERβ) preparations. The receptor is incubated with a radiolabeled estradiol ligand and varying concentrations of estradiol benzoate. Following incubation and filtration, bound radioactivity is measured by scintillation counting. IC₅₀ values are determined from displacement curves.

Cytotoxicity Assay[3]
The cytotoxic effects of estradiol benzoate and 17β-estradiol were assessed by the Cell Counting kit-8 (CCK8) assay. The HepG2 cells were cultured in 96-well plates at 1 × 104 cells per well with three replicates per group in a concentration gradient of 1 µM, 50 µM, 100 µM, 150 µM, 200 µM, 250 µM, 300 µM, 350 µM, 400 µM, 450 µM, 500 µM, 550 µM, 600 µM, 650 µM, and 700 µM. The DMSO was normalized to 1% in all treatment groups. After 24 h, the medium was replaced with 10% CCK-8 diluted in fresh DMEM. CCK-8 solution and culture media (no cells) were added to the blank control. The absorbance (OD value) was recorded. Cell survival rate curves and IC50s were calculated using GraphPad Prism.

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Western Blot[3]
To analyze the expression of HiBiT-HBx, the SBHX21 cells were induced with Dox (1 µg/mL) for 48 h in a 12-well plate. Then, the cells were treated with different concentrations of estradiol benzoate and 17β-estradiol for 24 h without Dox. We used 0.5% DMSO DMEM as the control group. After treatment, the cells were lysed with 100 µL of RIPA buffer. The cell extracts were separated on sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a nitrocellulose (NC) blotting membrane. The protein expression was analyzed using mouse monoclonal to Hepatitis B Virus X antigen and was visualized with an Odyssey CLX System.
In vitro cellular assays for estradiol benzoate involve treating cells expressing estrogen receptors (e.g., MCF-7 breast cancer cells) with the compound and measuring receptor-mediated transcriptional activity. Reporter gene assays using estrogen response element (ERE)-luciferase constructs are commonly used. Cells are treated with varying concentrations of estradiol benzoate, and luciferase activity is measured to quantify ER activation. The compound's effects on cell proliferation are also assessed.

Animal/Disease Models: Ovx mouse [1]
Doses: 20, 100 or 200 μg/kg
Route of Administration: subcutaneous injection
Experimental Results: Significant It reverses Ovx-induced decrease in uterine weight. There was no significant effect on the locomotor activity of Ovx mice. Effectively reverses Ovx-induced changes in synaptic cleft enlargement and PSD thickness reduction. Replacement with EB (100 μg/kg) for 4 weeks increased the number and density of synaptic vesicles.

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There is increasing evidence that estrogen is involved in CNS activity, particularly memory. Several studies have suggested that estrogen improves memory by altering neuronal plasticity, including increased hippocampus CA1 dendritic spine density and enhanced long-term potentiation (LTP). In the present study, we investigated the effects of estrogen on the ultrastructural modifications in cerebral frontal cortex and hippocampus of female ovariectomized mice. One week after ovariectomy (Ovx), ICR female mice received daily injection of estradiol benzoate (EB, 20, 100, 200 microg/kg, s.c.) for 4-5 weeks. Spatial memory was then tested in the water maze, and the overall locomotor activity was monitored in open field. Synaptic morphologic parameters were examined using a graph analyzer. The results from open field did not show any alterations in locomotor activity following Ovx and EB replacement. Both the latency to find the platform and the distance to reach the platform were significantly reduced in Ovx mice by EB at 20 or 100 microg/kg when compared to vehicle treated Ovx mice. The results from synaptic ultrastructural measurement and analysis did not show any differences in hemispheric or hippocampal volumes, the numeric synaptic density, the length of active zones, or the curvature of synaptic interface among Sham, Ovx, and Ovx plus EB replacement mice. However, EB replacement effectively normalized the changes induced by Ovx, reducing the width of the synaptic cleft, enlarging the thickness of postsynaptic density (PSD), and increasing the number of synaptic vesicles in the presynapse in both cerebral cortex Fr1 and hippocampus CA1 areas. These results suggest that the beneficial effects of EB on improving memory behavior of Ovx female mice are associated with the changes of some subtle structural parameters of synapses, including the width of PSD and synaptic cleft rather than some basic and permanent structure in frontal cortex and hippocampus regions.[1]

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The current project was designed to determine the dose-response relationship of the prostate gland to estradiol exposure during the developmentally critical neonatal period in the rat. Male Sprague-Dawley (SD) rats were treated on Days 1, 3, and 5 of life by s.c. injections of a 7-log range of doses (0.015 microg/kg to 15.0 mg/kg) of beta-estradiol-3-benzoate (EB) in 25 microl of peanut oil (Arachis) as vehicle. In a separate block, neonatal Fisher 344 (F344) rats received 0.15, 15.0, or 1500.0 microg EB/kg. Rats were killed on Postnatal Day (PND) 35 or 90, and the prostates were microdissected, weighed, and frozen for immunohistochemistry. Preputial separation and hepatic testosterone hydroxlase activities were monitored and measured to determine the onset of puberty. On PND 35, there was an increase in prostate weights of SD rats treated with low doses of EB and a decrease in prostate weights of SD rats treated with high doses. The low-dose effect was entirely abolished by PND 90, and only high-dose suppression of organ sizes was found. The transient nature of the effect in low-dose animals suggests an advancement of puberty as the cause for increased reproductive organ weights on PND 35. F344 rats were more sensitive than SD rats to the suppressive effects of high doses of neonatal EB on PND 90. Despite this heightened responsiveness in the F344 rats, a low-dose estrogenic effect on adult prostate weights was not observed. Thus, in the rat model a sustained effect at low doses of natural estrogens is not present in the prostate glands.[2]
In vivo animal experiments with estradiol benzoate are conducted in ovariectomized female mice to assess its effects on memory and reproductive function. Mice are treated with estradiol benzoate via injection, and memory behavior is assessed using the Morris water maze or novel object recognition test. In livestock, estradiol benzoate is used in combination with progestins to induce estrus, and reproductive outcomes are monitored.

Metabolism / Metabolites
The metabolic mechanism of exogenous estrogen is the same as that of endogenous estrogen. Estrogen is partially metabolized by cytochrome P450.
Estradiol benzoate has a molecular weight of 376.49 and a molecular formula of C₂₅H₂₈O₃. It is a solid powder with a purity of ≥98%. The compound is soluble in ethanol (2 mg/ml), DMSO (30 mg/ml), and DMF (30 mg/ml). It is typically stored at room temperature, protected from light and moisture. The compound is stable under recommended storage conditions.

Protein Binding
Estrogen circulates in the blood and is primarily (>95%) bound to sex hormone-binding globulin (SHBG) and albumin.
Estradiol benzoate is generally well-tolerated at therapeutic doses. As a hormone therapy, it may cause side effects similar to other estrogens, including breast tenderness, nausea, and increased risk of thromboembolic events. It should be used with caution in patients with a history of thromboembolic disorders, breast cancer, or liver disease. Standard safety precautions should be followed.

[1]. Effects of estradiol benzoate on learning-memory behavior and synaptic structure in ovariectomized mice . Life sciences, 2006, 79(16): 1553-1560.

[2]. Neonatal low-and high-dose exposure to estradiol benzoate in the male rat: I. Effects on the prostate gland . Biology of reproduction, 2001, 65(5): 1496-1505.

[3]. Identification of Estradiol Benzoate as an Inhibitor of HBx Using Inducible Stably Transfected HepG2 Cells Expressing HiBiT Tagged HBx . Molecules, 2022, 27(15): 5000.

[4]. Investigation of Antiparasitic Activity of Two Marine Natural Products, Estradiol Benzoate, and Octyl Gallate, on Toxoplasma gondii In Vitro . Frontiers in Pharmacology, 2022, 13: 841941.

[5]. Effect of estradiol benzoate on reproductive organs and fertility in the male rat . European Journal of Obstetrics & Gynecology and Reproductive Biology, 1983, 15(3): 189-198.

[6]. Macromolecular prodrugs XI. Synthesis and characterization of polymer-estradiol conjugate. Int J Pharm. 2004 Nov 5;285(1-2):35-41.

[7]. Role of sex steroid hormones in bacterial-host interactions. Biomed Res Int. 2013;2013:928290.

[8]. Estradiol benzoate.

17β-Estetrol-3-benzoate is a benzoate formed by the condensation of benzoic acid and the phenolic hydroxyl group of 17β-Estetrol. It is an isoestrogen and estrogen receptor agonist. It is a benzoate and a 17β-hydroxy steroid, functionally related to 17β-Estetrol. Estetrol benzoate is a prodrug ester of [DB00783], Estetrol being a naturally occurring circulating hormone in the human body. Estetrol is the most potent form of estrogen steroids in all mammals and is the primary female sex hormone. As a prodrug of Estetrol, Estetrol benzoate produces the same downstream effects as Estetrol in vivo by binding to estrogen receptors (ER). These receptors include ERα and ERβ subtypes and are distributed in a variety of tissues, such as the mammary glands, uterus, ovaries, skin, prostate, bone, fat, and brain. [DB00783] typically has an ester side chain because the oral bioavailability of endogenous Estetrol is extremely low (2-10%). Estetrol molecules are rapidly degraded by first-pass metabolism in the intestines and liver before entering systemic circulation to exert their estrogenic effects. Esterification of Estetrol aims to improve absorption and bioavailability after oral administration by increasing lipophilicity (e.g., Estetrol valerate) or by increasing release from intramuscularly injected sustained-release formulations (e.g., Estetrol cyclopentylpropionate). Upon absorption, the ester group is cleaved, releasing endogenous Estetrol or 17β-Estetrol. Therefore, Estetrol ester prodrugs are considered biocompatible estrogens. Estetrol benzoate is currently not marketed in Canada or the United States. Estetrol benzoate is a synthetic benzoic ester of Estetrol, a steroid sex hormone essential for maintaining female fertility and secondary sexual characteristics. As the primary and most potent estrogen produced by the ovaries, Estetrol binds to and activates specific nuclear receptors. This drug has mild anabolic and metabolic effects and may increase blood clotting. (NCI04)
See also: Estetrol (with active moiety); Estetrol benzoate; Progesterone (component); Estetrol benzoate; Testosterone propionate (component)...See more...
Drug Indications
Currently, Estetrol benzoate is not included in any products approved by the U.S. Food and Drug Administration (FDA) or Health Canada.
Mechanism of Action
Estetrol can freely enter target cells (e.g., female organs, breasts, hypothalamus, pituitary gland) and interact with target cell receptors. When estrogen receptors bind to their ligands, they enter the target cell nucleus, regulating gene transcription to form messenger RNA (mRNA). mRNA interacts with ribosomes to produce specific proteins that express the effects of Estetrol on target cells. Estrogen increases the liver's synthesis of sex hormone-binding globulin (SHBG), thyroid-binding globulin (TBG), and other serum proteins, and inhibits the anterior pituitary gland's secretion of follicle-stimulating hormone (FSH).

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Pharmacodynamics
Estetrol is the main intracellular estrogen, and its activity at the cellular level is much higher than that of its metabolites estrone and estriol.
Estradiol benzoate (CAS 50-50-0) is a synthetic ester prodrug of 17β-estradiol used in hormone therapy for menopausal symptoms and hypoestrogenism. It is an estrogen receptor agonist with IC₅₀ values of 22-28 nM. Estradiol benzoate is used in combination with progestins to induce estrus in livestock. The compound is available from various commercial suppliers for research applications.

C25H28O3

376.49

376.203

C, 79.76; H, 7.50; O, 12.75

50-50-0

Alpha-Estradiol;57-91-0;Estradiol (Standard);50-28-2;Estradiol-d3;79037-37-9;Estradiol-d4;66789-03-5;Estradiol-d5;221093-45-4;Estradiol-13C2;82938-05-4;Estradiol (cypionate);313-06-4;Estradiol benzoate;50-50-0;Estradiol enanthate;4956-37-0;Estradiol hemihydrate;35380-71-3;Estradiol-d2;53866-33-4;Estradiol-13C6;Estradiol-d2-1;3188-46-3;rel-Estradiol-13C6; 979-32-8 (valerate); 113-38-2 (dipropionate); 57-63-6 (ethinyl); 172377-52-5 (sulfamate); 3571-53-7 (undecylate)

222757

White to off-white solid powder

1.2±0.1 g/cm3

531.2±50.0 °C at 760 mmHg

191-198 °C(lit.)

212.0±22.9 °C

0.0±1.5 mmHg at 25°C

1.604

6.24

1

3

3

28

582

5

O([H])[C@@]1([H])C([H])([H])C([H])([H])[C@@]2([H])[C@]3([H])C([H])([H])C([H])([H])C4C([H])=C(C([H])=C([H])C=4[C@@]3([H])C([H])([H])C([H])([H])[C@@]21C([H])([H])[H])OC(C1C([H])=C([H])C([H])=C([H])C=1[H])=O

UYIFTLBWAOGQBI-BZDYCCQFSA-N

InChI=1S/C25H28O3/c1-25-14-13-20-19-10-8-18(28-24(27)16-5-3-2-4-6-16)15-17(19)7-9-21(20)22(25)11-12-23(25)26/h2-6,8,10,15,20-23,26H,7,9,11-14H2,1H3/t20-,21-,22+,23+,25+/m1/s1

[(8R,9S,13S,14S,17S)-17-hydroxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-3-yl] benzoate

Estradiol monobenzoate; Benzo-Gynoestryl; Ovasterol-B; Oestradiol benzoate; Oestradiol benzoate; Benovocylin; Estradiol 3-benzoate; Estradiol monobenzoate; Benzo-Gynoestryl; Estradiol Benzoate

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: 19~100 mg/mL (265.61~50.5 mM)
H2O: ~0.1 mg/mL (~0.3 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.64 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.)