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| Targets |
(Z)-4-Hydroxy Tamoxifen-d5 targets the estrogen receptor (ER) as it is a labeled analog of the nonsteroidal antiestrogen (Z)-4-Hydroxy Tamoxifen. This deuterated compound binds with high affinity to estrogen receptors (ERalpha and ERbeta), functioning as a selective estrogen receptor modulator (SERM). Its primary mechanism involves antagonizing the effects of estradiol in breast tissue, thereby inhibiting estrogen-dependent cell proliferation. Due to its structural identity with its non-labeled counterpart, the isotope-labeled version mimics the same receptor binding characteristics. This allows it to serve as an accurate tracer for receptor-binding studies in vitro and in vivo without altering the pharmacological target. The use of this labeled compound enables precise quantification and tracking of drug-receptor interactions using mass spectrometry techniques.
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| ln Vitro |
Drug compounds have included stable heavy isotopes of carbon, hydrogen, and other elements, mostly as quantitative tracers while the drugs were being developed. Because deuteration may have an effect on a drug's pharmacokinetics and metabolic properties, it is a cause for concern [1].
The in vitro biological activity of (Z)-4-Hydroxy Tamoxifen-d5 is presumed to be equivalent to that of its unlabeled form, (Z)-4-Hydroxy Tamoxifen. The unlabeled compound displays potent antiestrogenic activity in human breast cancer cell lines, including MCF-7 and T47D cells. It functions by competitively binding to estrogen receptors (ERs) and inhibiting estrogen-induced transcriptional activation of target genes. This leads to cell cycle arrest in the G0/G1 phase and the induction of apoptosis in hormone-sensitive cells. In addition to its ER-mediated actions, it can also activate alternative signaling pathways involving protein kinase C (PKC) and calmodulin. While the deuterated version is not typically used directly in efficacy experiments, it serves as an internal standard to accurately measure the parent compound and its metabolites in in vitro assay systems using LC-MS, thereby improving assay robustness and data accuracy. |
| ln Vivo |
The in vivo biological activity of (Z)-4-Hydroxy Tamoxifen-d5 is inferred from the established pharmacological profile of its unlabeled counterpart, (Z)-4-Hydroxy Tamoxifen. The unlabeled compound exhibits potent antiestrogenic effects in animal models, primarily by blocking estrogen receptors in target tissues such as the mammary gland and uterus. It is known to inhibit estrogen-induced tumor growth in ovariectomized athymic mice implanted with MCF-7 human breast cancer cells. Due to the absence of significant isotope effects, the deuterated version is expected to possess identical in vivo disposition and pharmacological activity, making it an ideal internal standard for pharmacokinetic and pharmacodynamic studies. Its use in vivo is focused on improving the accuracy of quantification via mass spectrometry, enabling researchers to precisely determine the exposure-response relationship of Tamoxifen and its metabolites.
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| Enzyme Assay |
Since (Z)-4-Hydroxy Tamoxifen-d5 is an isotope-labeled compound, a generic non-cell-based assay for the unlabeled ligand can be referenced. An example protocol involves a competitive radioligand binding assay using estrogen receptor (ER) protein. First, prepare serial dilutions of the test compound in assay buffer (e.g., 10 mM Tris-HCl, 1.5 mM EDTA, 10% glycerol, pH 7.4). Incubate the ER protein (e.g., recombinant human ERalpha or ERbeta) with a fixed concentration of a high-affinity radioligand (e.g., 0.5 nM 3H-Estradiol) and increasing concentrations of the unlabeled (Z)-4-Hydroxy Tamoxifen (1 pM to 10 uM) for 2-4 hours at 4degC. Following incubation, separate bound from free radioligand using a charcoal-dextran suspension or by filtration through a glass fiber filter. Quantify the retained radioactivity using a scintillation counter. The half-maximal inhibitory concentration (IC₅0) and binding affinity (Ki) of the unlabeled compound are then calculated via nonlinear regression analysis using GraphPad Prism or similar software.
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| Cell Assay |
For (Z)-4-Hydroxy Tamoxifen-d5, a standard reference protocol for the unlabeled counterpart is as follows: Culture MCF-7 human breast cancer cells (estrogen receptor-positive) in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Before the assay, switch the cells to phenol red-free DMEM containing 10% charcoal-dextran stripped FBS to remove estrogens. Seed the cells in 96-well plates at a density of 5,000-10,000 cells per well and allow them to attach overnight. Treat the cells with various concentrations of unlabeled (Z)-4-Hydroxy Tamoxifen (1 nM to 10 uM) or vehicle control for 24-72 hours at 37degC in a 5% CO2 incubator. After treatment, add 10 uL of WST-8 or MTT solution to each well and incubate for 1-4 hours. Measure the absorbance at 450 nm (for WST-8) or 570 nm (for MTT) using a microplate reader. Calculate the half-maximal inhibitory concentration (IC₅0) for cell viability inhibition based on dose-response curves. The deuterated compound serves as the LC-MS internal standard to validate exposure levels in treated samples.
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| Animal Protocol |
A typical in vivo experimental protocol for the unlabeled counterpart of (Z)-4-Hydroxy Tamoxifen-d5 involves using a xenograft mouse model. For this protocol, 6-8 week old female athymic nude mice are ovariectomized and allowed to recover for one week. MCF-7 human breast cancer cells (5×10⁶ cells) are then implanted subcutaneously into the flank in 100 uL of Matrigel. When tumors reach approximately 100-150 mm3, mice are randomized (n=8-10 per group) and treated daily via oral gavage with vehicle (corn oil) or the unlabeled (Z)-4-Hydroxy Tamoxifen (10-50 mg/kg). Tumor volumes and body weights are measured twice weekly. At the end of the experiment (e.g., 4-6 weeks), blood samples are collected via cardiac puncture, and tumors are excised, weighed, and flash-frozen in liquid nitrogen. The deuterated compound (Z)-4-Hydroxy Tamoxifen-d5 is then used as the internal standard in the LC-MS analysis of these collected plasma and tumor samples to accurately quantify the levels of the administered drug and its metabolites.
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| ADME/Pharmacokinetics |
As a stable isotope-labeled compound, (Z)-4-Hydroxy Tamoxifen-d5 is designed to exhibit almost identical absorption, distribution, metabolism, and excretion (ADME) properties as its non-labeled counterpart due to the minor isotope effect of deuterium. The unlabeled (Z)-4-Hydroxy Tamoxifen is well absorbed after oral administration. It undergoes extensive hepatic metabolism primarily by CYP3A4, with an elimination half-life of approximately 7-14 days in humans. It is highly bound to plasma proteins (>99%) and accumulates in tissues such as the breast, uterus, and liver. Due to its high lipophilicity, it exhibits a large volume of distribution. The deuterated version is not intended for therapeutic use, but rather serves as an internal standard for quantitative analysis in PK studies. This allows for more accurate assessment of the parent drug's PK profile by minimizing matrix effects and ion suppression in mass spectrometry assays.
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| Toxicity/Toxicokinetics |
(Z)-4-Hydroxy Tamoxifen-d5 itself is used as an analytical reference standard and is not intended for therapeutic human use; thus, its toxicological profile is derived from the non-labeled version, (Z)-4-Hydroxy Tamoxifen. The unlabeled compound has demonstrated genotoxic potential in some standard assays. It exhibits estrogenic activity in the rodent uterus and has shown the capacity to form DNA adducts in rat liver. Tamoxifen is associated with an increased risk of endometrial cancer in humans, although this is a species-specific effect requiring metabolic activation. Acute toxicity studies in rodents have reported an oral LD₅0 of over 1000 mg/kg for Tamoxifen. For researchers handling this compound, standard precautions for handling potentially carcinogenic chemicals should be taken, including the use of personal protective equipment (PPE) and proper containment to prevent dermal or inhalation exposure.
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| References | |
| Additional Infomation |
The deuterium-labeled (Z)-4-Hydroxy Tamoxifen-d5 is exclusively intended for research applications and not for human therapeutic use. It serves as a critical internal standard for mass spectrometry assays, including LC-MS/MS, enabling the precise quantification of Tamoxifen and its active metabolites in plasma, tissue, and other biological samples. The presence of five deuterium atoms provides a mass shift of +5 Da, allowing clear differentiation from the endogenous analyte. This compound is invaluable for method validation studies, Abbreviated New Drug Applications (ANDAs), and quality control processes during the manufacture of Tamoxifen. (Z)-4-Hydroxy Tamoxifen itself is classified as a selective estrogen receptor modulator (SERM) and is being investigated for its clinical potential in breast cancer chemoprevention and other estrogen-dependent disorders.
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| Molecular Formula |
C26H24D5NO2
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| Molecular Weight |
392.54
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| Exact Mass |
392.251
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| CAS # |
164365-20-2
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| PubChem CID |
45039530
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| Appearance |
White to off-white solid powder
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| LogP |
5.701
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
29
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| Complexity |
493
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| Defined Atom Stereocenter Count |
0
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| SMILES |
[2H]C([2H])([2H])C([2H])([2H])/C(=C(\C1=CC=C(C=C1)O)/C2=CC=C(C=C2)OCCN(C)C)/C3=CC=CC=C3
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| InChi Key |
TXUZVZSFRXZGTL-FUYVPVGLSA-N
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| InChi Code |
InChI=1S/C26H29NO2/c1-4-25(20-8-6-5-7-9-20)26(21-10-14-23(28)15-11-21)22-12-16-24(17-13-22)29-19-18-27(2)3/h5-17,28H,4,18-19H2,1-3H3/b26-25-/i1D3,4D2
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| Chemical Name |
4-[(Z)-3,3,4,4,4-pentadeuterio-1-[4-[2-(dimethylamino)ethoxy]phenyl]-2-phenylbut-1-enyl]phenol
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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 |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5475 mL | 12.7376 mL | 25.4751 mL | |
| 5 mM | 0.5095 mL | 2.5475 mL | 5.0950 mL | |
| 10 mM | 0.2548 mL | 1.2738 mL | 2.5475 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.
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