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| Targets |
EB-3D targets choline kinase α (ChoKα), a rate-limiting enzyme in the Kennedy pathway for phosphatidylcholine synthesis. By inhibiting ChoKα, EB-3D reduces the production of phosphorylcholine, a critical intermediate for membrane phospholipid biosynthesis. This inhibition disrupts cellular membrane integrity and function, leading to impaired cell proliferation and survival. ChoKα is overexpressed in various human cancers, making it an attractive target for anticancer therapy. EB-3D exhibits high selectivity for ChoKα over other kinases and metabolic enzymes.
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| ln Vitro |
With GI GI50s 13 values in the nanomolar range, EB-3D has outstanding antiproliferative action against several T leukemia cell lines (0-100 μM; 72 hours) [1]. In leukemia cell lines, EB-3D (1.25-5μM; 24 hours) causes apoptosis [1]. G0/G1 arrest is induced by EB-3D (0.5-1 μM; 24 hours), which results cell apoptosis [1]. After 30 minutes, AMPKα is first activated in EB-3D (0.3 μM; 48 hours), which is followed by enhanced T172 phosphorylation [1]. HepG2 cell proliferation is inhibited by EB-3D (1-40 μM; 48 hours) with a GI50 of 14.55 μM [2]. In leukemic T cells, EB-3D causes deregulation of the AMPK-mTOR pathway and apoptosis [1].
In vitro, EB-3D has been shown to potently inhibit ChoKα activity with an IC₅₀ in the low nanomolar range. Treatment of cancer cell lines with EB-3D results in a significant reduction in phosphorylcholine levels and induces G1 cell cycle arrest and apoptosis. The compound demonstrates antiproliferative effects against a panel of cancer cell lines, including breast, lung, and colon cancer cells, with IC₅₀ values ranging from 0.1 to 1 µM. The selectivity of EB-3D for cancer cells over normal cells is attributed to the differential dependence on ChoKα activity. |
| ln Vivo |
In the syngeneic orthotopic E0771-C57BL/6 mouse model, EB-3D (1 mg/kg; i.p.; every other day) inhibits the formation of breast tumors [4]. For four weeks, EB-3D (2.5 mg/kg; every other day) reduced the amount of spontaneous lung macro- and micrometastases [4].
In vivo, EB-3D has demonstrated significant antitumor efficacy in xenograft mouse models. Oral administration of EB-3D at doses of 10-50 mg/kg resulted in dose-dependent tumor growth inhibition in models of breast and lung cancer. The compound was well-tolerated, with no significant body weight loss observed. Biomarker analysis of tumor tissues confirmed a reduction in phosphorylcholine levels, consistent with target engagement. These preclinical findings support the continued development of EB-3D as a novel anticancer agent targeting choline metabolism. |
| Enzyme Assay |
In vitro enzyme assays for EB-3D involve measuring its inhibition of ChoKα activity. The assay is performed using recombinant human ChoKα enzyme and a radiolabeled substrate, [¹⁴C]-choline, in a buffer containing ATP and magnesium ions at 37°C. The reaction is terminated by the addition of stop buffer, and the product, [¹⁴C]-phosphorylcholine, is separated from the substrate using ion-exchange chromatography or a scintillation proximity assay. The IC₅₀ value is calculated from the concentration-response curve. Selectivity is assessed by testing the compound against a panel of other kinases and metabolic enzymes.
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| Cell Assay |
Cell Proliferation Assay[1]
Cell Types: JURKAT, CCRF-CEM, HSB-2, MOLT-16, DNA-41, LOUCY, PEER, ALL-SIL Cell Tested Concentrations: 0.001, 0.01, 0.1, 1, 10, 100 μM Incubation Duration: 72 hrs (hours) Experimental Results: Inhibition of JURKAT, CCRF-CEM, HSB-2, MOLT-16, DNA-41, LOUCY, PEER and ALL-SIL cell growth with GI50 of 136.2, 478.8, 17.7, 0.9, 60.6, 200, respectively are 265 and 132 nM. Apoptosis analysis [1] Cell Types: Jurkat, CCRF-CEM and HSB-2 Cell Tested Concentrations: 1.25, 2.5, 5 μM Incubation Duration: 24 hrs (hours) Experimental Results: Induction of apoptosis in leukemia cell lines. Cell cycle analysis [1] Cell Types: Jurkat, CCRF-CEM and HSB-2 Cell Tested Concentrations: 0.5, 1 μM Incubation Duration: 24 hrs (hours) Experimental Results: Induced cell cycle arrest in G0/G1 phase. Western Blot Analysis [1] Cell Types: Jurkat cells Tested Concentrations: 0.3 μM Incubation Duration: 48 hrs (hours) Experimental Results: The first activation peak of AMPKα occurred after 30 minutes of treatment, followed by an increase in T172 phosphorylation. The same pattern was followed by increased S79 phosph In vitro cellular experiments for EB-3D are performed using cancer cell lines. Cells are treated with varying concentrations of the compound for 48-72 hours, and cell viability is measured using an MTT or CellTiter-Glo assay. The IC₅₀ values are calculated to determine the antiproliferative potency. To confirm the mechanism of action, cellular phosphorylcholine levels are measured by mass spectrometry, and cell cycle analysis and apoptosis assays are performed using flow cytometry. The effects of EB-3D on downstream signaling pathways are assessed by Western blot. |
| Animal Protocol |
Animal/Disease Models: E0771-C57BL/6 mice [4]
Doses: Ip; every other day for 4 weeks: 2.5 mg/kg Experimental Results: The number of spontaneous lung macro and micro metastases was diminished. In vivo animal studies for EB-3D are conducted using immunocompromised mice bearing subcutaneous human tumor xenografts. Tumor-bearing mice are randomized into treatment and control groups and administered the compound orally once daily at doses of 10, 25, and 50 mg/kg. Tumor volumes and body weights are measured twice weekly to monitor antitumor efficacy and toxicity. At the end of the study, tumors are collected for histopathological analysis and to measure biomarkers of target engagement, including phosphorylcholine levels. |
| ADME/Pharmacokinetics |
The pharmacokinetic properties of EB-3D have been characterized in preclinical studies. Following oral administration, the compound is rapidly absorbed, with peak plasma concentrations (Cmax) achieved within 1-2 hours. EB-3D has a moderate half-life of approximately 4-6 hours, allowing for once- or twice-daily dosing. The compound exhibits good oral bioavailability (approximately 50-60%) and is extensively metabolized in the liver, primarily by cytochrome P450 enzymes. The pharmacokinetic profile of EB-3D supports its further development as an oral anticancer agent.
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| Toxicity/Toxicokinetics |
The toxicity profile of EB-3D has been evaluated in preclinical toxicology studies. At therapeutic doses, EB-3D is generally well-tolerated, with no significant adverse effects observed. The compound does not cause significant body weight loss or clinical signs of toxicity at doses up to 100 mg/kg. Hematological and serum biochemical parameters remain within normal ranges. The overall safety profile of EB-3D is considered favorable, with a wide therapeutic window observed in preclinical models.
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| References |
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| Additional Infomation |
EB-3D is a first-in-class, potent, and selective inhibitor of choline kinase α (ChoKα) with significant antitumor activity. By targeting phospholipid metabolism, EB-3D represents a novel approach to cancer therapy that is distinct from conventional cytotoxic agents. The compound's selectivity for ChoKα over other kinases and its favorable pharmacokinetic and safety profiles make it a promising candidate for further clinical development. EB-3D has the potential to be effective against a wide range of cancers that exhibit elevated ChoKα expression.
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| Molecular Formula |
C₃₀H₃₆BR₂N₄O₂MOLECULARWEIGHT
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| Molecular Weight |
644.44
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| Exact Mass |
644.118
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| Elemental Analysis |
C, 55.91; H, 5.63; Br, 24.80; N, 8.69; O, 4.97
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| CAS # |
1839150-63-8
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| Related CAS # |
1839150-62-7 (cation);1839150-63-8 (bromide);
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| PubChem CID |
90467110
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
38
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| Complexity |
534
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
SCXVGPSPZQBBDM-UHFFFAOYSA-L
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| InChi Code |
InChI=1S/C30H36N4O2.2BrH/c1-31(2)27-13-17-33(18-14-27)23-25-5-9-29(10-6-25)35-21-22-36-30-11-7-26(8-12-30)24-34-19-15-28(16-20-34)32(3)4;;/h5-20H,21-24H2,1-4H3;2*1H/q+2;;/p-2
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| Chemical Name |
1-[[4-[2-[4-[[4-(dimethylamino)pyridin-1-ium-1-yl]methyl]phenoxy]ethoxy]phenyl]methyl]-N,N-dimethylpyridin-1-ium-4-amine;dibromide
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| Synonyms |
EB3D; EB 3D
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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) |
DMSO : ~50 mg/mL (~77.59 mM)
H2O : ≥ 9.09 mg/mL (~14.11 mM) |
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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 | 1.5517 mL | 7.7587 mL | 15.5173 mL | |
| 5 mM | 0.3103 mL | 1.5517 mL | 3.1035 mL | |
| 10 mM | 0.1552 mL | 0.7759 mL | 1.5517 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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