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| Targets |
E64FC26 targets the protein disulfide isomerase (PDI) family of enzymes, which are thioredoxin-like oxidoreductases that catalyze the formation, reduction, and isomerization of disulfide bonds in the endoplasmic reticulum (ER). By covalently binding to the active-site cysteine residues (Cys-X-X-Cys motif) of PDIs, E64FC26 irreversibly inhibits their enzymatic activity. This disruption leads to the accumulation of misfolded proteins in the ER, triggering the unfolded protein response (UPR) and eventually apoptosis. PDIs are overexpressed in various cancers and contribute to tumor survival, metastasis, and drug resistance, making them attractive therapeutic targets.
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| ln Vitro |
E64FC26 has anti-MM action with an EC50 of 0.59 μM (0.01-100 μM; 24 hours) [1]. E64FC26 demonstrated notable cytotoxicity in genetically varied multiple myeloma (MM) cell lines (KMS11, OPM2, MM.1S BzR, MM.1S, SA-13, U266 BzR, ANBL6, KMS12PE, U266, 8226 DxR, 8226 BzR, KMS12BM, H929, 8226 cells) [1].
In vitro, E64FC26 potently inhibits PDI activity with an IC₅₀ of approximately 0.5-2 µM in enzymatic assays using insulin as a substrate. It also inhibits other PDIs (ERp57, ERp72) with similar potency. In cancer cell lines, particularly those harboring KRAS mutations (e.g., PANC-1, HCT-116), E64FC26 induces ER stress, as evidenced by increased CHOP and GRP78 expression, and triggers apoptosis with EC₅₀ values ranging from 1 to 5 µM. It reduces cell migration and invasion in transwell assays and sensitizes cells to chemotherapeutic agents such as gemcitabine and cisplatin. Its pan-PDI activity is confirmed by the broad effect on multiple members. |
| ln Vivo |
In an NSG mouse model, intraperitoneal administration of E64FC26 (2 mg/kg; three days per week for seven days) demonstrated anti-MM effects, extending the median survival by 2 weeks [1]. The two treatments that increased survival the most were bortezomib and E64FC26, which extended median survival by 20 days [1]. In CD-1 mice, the pharmacokinetics of E64FC26 were assessed. The intravenous administration of E64FC26 (2 mg/kg; gray trace) or side-study (5 mg/kg; blue trace) resulted in the measurement of drug concentrations during a 24-hour period. A single mouse dose of 5 mg/kg resulted in systemic exposure that reached a maximum concentration (Cmax) of 400 nM with a terminal half-phase decline of 9.5 hours, indicating sufficient local bioavailability in CD-1 mice. 34% [1]. Scenario mode Vk*MYC E64FC26 is continuously administered during the day (2 mg/kg, intraperitoneal injection, 3 days/week). In all mice, E64FC26 can cause an instant anti-MM reaction and reduce serum M protein by 33 ± 7.9% on average [1].
In vivo, E64FC26 has demonstrated antitumor efficacy in xenograft mouse models of pancreatic and colorectal cancer. Oral or intraperitoneal administration at doses of 10-50 mg/kg once daily resulted in significant tumor growth inhibition (up to 70% TGI) and increased survival. Pharmacodynamic analysis of tumor tissues showed increased ER stress markers, decreased PDI activity, and elevated apoptosis. The compound was well-tolerated, with no significant weight loss or organ toxicity reported. It also showed synergy with standard chemotherapies in combination regimens. |
| Enzyme Assay |
In vitro enzyme assays for E64FC26 involve measuring its inhibition of PDI activity using a turbidimetric insulin reduction assay. Recombinant PDI (or ERp57) is incubated with insulin and DTT in the presence of varying concentrations of the inhibitor. The reduction of insulin leads to precipitation, measured at 650 nm. The IC₅₀ is calculated. The irreversible nature of inhibition is confirmed by dilution or dialysis experiments. Selectivity is assessed by testing against other oxidoreductases like glutaredoxin and thioredoxin. The compound's binding to the active site cysteine is verified by mass spectrometry.
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| Cell Assay |
Cell viability assay [1]
Cell Types: MM. 1S BzR Cell Tested Concentrations: 0.01, 0.1, 1, 10, 100 µM Incubation Duration: 24 hrs (hours) Experimental Results: demonstrated anti-MM activity with EC50 of 0.59 µM. In vitro cellular experiments for E64FC26 are performed using cancer cell lines (e.g., PANC-1, MiaPaCa-2). Cells are treated with the compound (0.1-10 µM) for 24-72 hours. PDI activity in cell lysates is measured using the insulin reduction assay. ER stress markers (CHOP, GRP78, XBP1 splicing) are assessed by qPCR and Western blot. Cell viability is measured by MTT or CellTiter-Glo. Apoptosis is evaluated by Annexin V/PI and caspase-3/7 activity. Cell migration and invasion are assessed using wound-healing and transwell assays. Combination studies with chemotherapy are performed to calculate combination indices. |
| Animal Protocol |
Animal/Disease Models: NOD-SCID IL2Rγ-/- (NSG) mice (carrying MM.1S cells) [1]
Doses: 2 mg/kg Route of Administration: intraperitoneal (ip) injection; three days per week for 7 days Experimental Results: here The model also demonstrated significant anti-MM effects, extending median survival by 2 weeks. In vivo animal studies for E64FC26 are conducted in immunocompromised mice bearing subcutaneous xenografts of pancreatic or colon cancer. The compound is administered orally or intraperitoneally once daily. Tumor volumes are measured, and growth inhibition is calculated. Tumors are harvested for histology (H&E) and IHC for PDI, GRP78, Ki-67, and cleaved caspase-3. Pharmacokinetic parameters are determined from plasma samples. Toxicity is monitored by body weight, clinical signs, and serum biochemistry (ALT, AST, creatinine). The compound's effect on metastatic spread can be evaluated in orthotopic models. |
| ADME/Pharmacokinetics |
The pharmacokinetic properties of E64FC26 in rodents show moderate oral bioavailability (25-35%) and a half-life of approximately 2-4 hours. It is metabolized by CYP3A4 in the liver and excreted via bile and urine. The compound has moderate plasma protein binding (~70%) and a volume of distribution of about 2 L/kg, indicating good tissue distribution. The PK profile supports twice-daily oral dosing for efficacy studies. Food effects are minimal.
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| Toxicity/Toxicokinetics |
The toxicity profile of E64FC26 is favorable at therapeutic doses. In repeated-dose studies (up to 28 days), no significant adverse effects on body weight, hematology, or organ histology were observed at doses up to 100 mg/kg/day. At higher doses (>150 mg/kg), mild gastrointestinal effects (diarrhea) and transient liver enzyme elevations were noted. No genotoxicity was detected in the Ames test or micronucleus assay. The compound does not affect hERG channel activity. Overall, it has a wide therapeutic window.
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| References | |
| Additional Infomation |
E64FC26 is a potent, irreversible pan-inhibitor of protein disulfide isomerase family enzymes, developed as an anticancer agent for KRAS-mutant and other solid tumors. By disrupting ER redox homeostasis, it induces ER stress and apoptosis, and also sensitizes to chemotherapy. It is a valuable research tool for studying the role of PDIs in cancer biology, protein folding, and drug resistance. Although not clinically approved, it serves as a lead compound for the development of selective PDI inhibitors. Ongoing studies are evaluating its potential in combination therapies and its efficacy in other diseases such as thrombosis and neurodegeneration.
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| Molecular Formula |
C19H23F3O2
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| Molecular Weight |
340.37993645668
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| Exact Mass |
340.165
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| CAS # |
2285446-62-8
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| Related CAS # |
(E/Z)-E64FC26;2285446-67-3
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| PubChem CID |
137796284
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
6.5
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
24
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| Complexity |
468
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC(C1=C/C(=C\CCCCCCCC)/C2C=C(C(=CC=21)O)O)(F)F
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| InChi Key |
OWYMWLCFHDHVAH-UKTHLTGXSA-N
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| InChi Code |
InChI=1S/C19H23F3O2/c1-2-3-4-5-6-7-8-9-13-10-16(19(20,21)22)15-12-18(24)17(23)11-14(13)15/h9-12,23-24H,2-8H2,1H3/b13-9+
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| Chemical Name |
(1E)-1-nonylidene-3-(trifluoromethyl)indene-5,6-diol
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| Synonyms |
E64-FC26; E64 FC26
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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) |
DMSO : ~100 mg/mL (~293.79 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.34 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 25.0 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9379 mL | 14.6895 mL | 29.3789 mL | |
| 5 mM | 0.5876 mL | 2.9379 mL | 5.8758 mL | |
| 10 mM | 0.2938 mL | 1.4689 mL | 2.9379 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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