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| Targets |
DNA. Ecubectedin is a transcriptional inhibitor that binds to the minor groove of DNA, forming covalent adducts with guanine residues. This binding blocks DNA-protein interactions, particularly interfering with transcription factors and the transcription machinery, leading to inhibition of RNA synthesis (both mRNA and rRNA). This results in cell cycle arrest and apoptosis. It is a DNA/RNA synthesis inhibitor and belongs to the tetrahydroisoquinoline alkaloid class.
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| ln Vitro |
Ecubectedin exhibits potent antiproliferative activity in human solid cancer models in vitro. It inhibits the proliferation of various cancer cell lines, with IC50 values expected in the low nanomolar range (similar to other ecteinascidins). It forms DNA adducts, blocks RNA synthesis, and induces apoptosis. As a transcriptional inhibitor, it preferentially affects transcriptionally active genes. It also exhibits antimicrobial properties.
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| ln Vivo |
Ecubectedin exhibits potent antiproliferative activity in human solid cancer models in vivo. It has been shown to inhibit tumor growth in xenograft models of various solid tumors, including ovarian, breast, lung, and colon cancers. It is a synthetic analog of the DNA-binding anticancer agent trabectedin. It is in preclinical or early clinical development for solid tumors.
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| Enzyme Assay |
A typical protocol for DNA-binding studies involves incubating a DNA oligonucleotide (e.g., 50-base pair GC-rich sequence, 1 uM) with varying concentrations of Ecubectedin (1 nM-10 uM) in 20 mM Tris-HCl buffer (pH 7.5) containing 100 mM KCl, 5 mM MgCl2, and 1 mM DTT at 25degC for 2-4 hours. The formation of DNA adducts is detected by gel electrophoresis (DNA mobility shift assay) on a 1-2% agarose gel stained with ethidium bromide. Alternatively, LC-MS/MS can be used to identify the specific DNA adducts. For RNA synthesis inhibition, an in vitro transcription assay is performed. Purified RNA polymerase II (10 nM) is incubated with a DNA template (1 ug) and NTPs (100 uM each, including 3H-UTP) in transcription buffer (20 mM HEPES, pH 7.9, 60 mM KCl, 10 mM MgCl2, 2 mM DTT) at 30degC for 30 minutes. Ecubectedin (0.1-100 nM) is added at the start of the reaction. The reaction is stopped, and the RNA product is precipitated with TCA and collected on glass fiber filters. Radioactivity is counted by scintillation. The IC50 for RNA synthesis inhibition is determined by dose-response curve.
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| Cell Assay |
Human solid tumor cell lines (e.g., A549 lung cancer, HCT116 colon cancer, MCF-7 breast cancer, OVCAR-3 ovarian cancer) are seeded in 96-well plates at 3,000-5,000 cells/well in RPMI 1640 medium containing 10% FBS. After 24 hours, cells are treated with Ecubectedin at concentrations ranging from 0.01 pM to 100 nM for 72-96 hours. Cell viability is assessed using the CellTiter-Glo (ATP-based) assay or SRB (sulforhodamine B) assay. IC50 values are calculated by non-linear regression. For mechanism studies, cells are treated with Ecubectedin (0.1-10 nM) for 4-24 hours. RNA synthesis is measured by 3H-uridine incorporation into total RNA. DNA adduct formation is detected by a modified comet assay (with DNA repair enzymes) or by immunofluorescence using an anti-DNA adduct antibody. Apoptosis is measured by caspase-3/7 activity and by flow cytometry with Annexin V/PI staining. Cell cycle distribution is analyzed by propidium iodide staining, with G2/M arrest and sub-G1 accumulation indicative of apoptosis.
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| Animal Protocol |
For in vivo efficacy studies, female BALB/c nude mice (6-8 weeks old, 18-22 g) are injected subcutaneously with 5×10⁶ human cancer cells (e.g., A549 lung cancer, HCT116 colon cancer) in 100 uL of PBS mixed 1:1 with Matrigel. When tumors reach a volume of 100-200 mm3 (approximately 7-14 days post-inoculation), mice are randomized into treatment groups (n=8-10 per group). Ecubectedin is administered intravenously via tail vein at doses of 0.1-1 mg/kg, once weekly for 3-4 weeks. The vehicle control group receives saline or DMSO/PBS. Positive control groups may receive trabectedin (0.2 mg/kg) or cisplatin (5 mg/kg). Tumor volume is measured twice weekly with calipers and calculated as (length × width2)/2. Body weight is monitored twice weekly as a measure of tolerability. At the end of the study (day 28 or when tumors reach 2,000 mm3), mice are euthanized, and tumors are excised, weighed, and processed for histopathology (H&E staining), immunohistochemistry (Ki67 for proliferation, cleaved caspase-3 for apoptosis), and DNA adduct analysis by LC-MS. Survival is monitored in survival extension studies.
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| ADME/Pharmacokinetics |
No detailed pharmacokinetic data is publicly available for Ecubectedin. As a synthetic analog of trabectedin, it is expected to have a similar PK profile. Trabectedin has a large volume of distribution (Vd ~1800-4500 L/m2 in humans), indicating extensive tissue binding, and is highly bound to plasma proteins (>95%). It is primarily metabolized by CYP3A4 in the liver. The terminal half-life is long (approximately 80-100 hours). Ecubectedin is administered intravenously due to poor oral bioavailability.
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| Toxicity/Toxicokinetics |
No specific toxicity data has been reported. As a DNA-binding transcriptional inhibitor, Ecubectedin is expected to have a toxicity profile similar to that of trabectedin, which includes hepatotoxicity (elevated transaminases), hematological toxicity (neutropenia, thrombocytopenia), fatigue, nausea, and vomiting. These effects are dose-limiting but generally reversible. The compound is a potent cytotoxic agent and must be handled with appropriate safety precautions (biosafety cabinet, protective equipment). Not for human use outside of clinical trials.
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| Additional Infomation |
Ecubectedin is a synthetic analogue of the DNA minor groove binding agent rubintidine and possesses potential antitumor activity. After administration, Ecubectedin specifically inhibits RNA synthesis, thereby preventing the transcription of protein-coding genes. This may kill tumor cells.
Ecubectedin (PM14) is a synthetic analog of ecteinascidins, a family of tetrahydroisoquinoline alkaloids with broad antitumor and antimicrobial activities. It is a DNA-binding transcriptional inhibitor that forms DNA adducts, blocking RNA synthesis. It is for research use only, not for human therapeutic use outside of clinical trials. Ecubectedin is being developed as an anticancer agent for solid tumors. The molecular formula is C41H44N4O10S, and the molecular weight is 784.87. |
| Molecular Formula |
C41H44N4O10S
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| Molecular Weight |
784.873869895935
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| Exact Mass |
784.277
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| CAS # |
2248127-53-7
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| PubChem CID |
146175071
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
3.3
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
14
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
56
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| Complexity |
1530
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| Defined Atom Stereocenter Count |
8
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| SMILES |
CC1=CC2=C([C@@H]3[C@@H]4[C@H]5C6=C(C(=C7C(=C6[C@@H](N4[C@H]([C@H](C2)N3C)O)COC(=O)[C@@]8(CS5)C9=C(C[C@H](N8)CO)C2=CC=CC=C2N9)OCO7)C)OC(=O)C)C(=C1OC)O
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| InChi Key |
PVXBRBBFWHHDFS-DJQPDYNTSA-N
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| InChi Code |
InChI=1S/C41H44N4O10S/c1-17-10-20-11-25-39(49)45-26-14-52-40(50)41(38-23(12-21(13-46)43-41)22-8-6-7-9-24(22)42-38)15-56-37(31(45)30(44(25)4)27(20)32(48)33(17)51-5)29-28(26)36-35(53-16-54-36)18(2)34(29)55-19(3)47/h6-10,21,25-26,30-31,37,39,42-43,46,48-49H,11-16H2,1-5H3/t21-,25-,26-,30+,31+,37+,39-,41+/m0/s1
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| Chemical Name |
[(1R,2R,3R,3'S,11S,12S,14R,26R)-5,12-dihydroxy-3'-(hydroxymethyl)-6-methoxy-7,21,30-trimethyl-27-oxospiro[17,19,28-trioxa-24-thia-13,30-diazaheptacyclo[12.9.6.13,11.02,13.04,9.015,23.016,20]triaconta-4(9),5,7,15,20,22-hexaene-26,1'-2,3,4,9-tetrahydropyrido[3,4-b]indole]-22-yl] acetate
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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 | 1.2741 mL | 6.3705 mL | 12.7410 mL | |
| 5 mM | 0.2548 mL | 1.2741 mL | 2.5482 mL | |
| 10 mM | 0.1274 mL | 0.6370 mL | 1.2741 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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