| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
|
||
| 5mg |
|
||
| Other Sizes |
| Targets |
G9a 23-27 nM (IC50)
RK-701 primarily targets the histone methyltransferases G9a (EHMT2) and GLP (EHMT1), with IC50 values of 23‑27 nM for G9a and 53 nM for GLP. It acts as a reversible, substrate‑competitive inhibitor that competes with the histone H3 substrate at the enzyme active site. Additionally, it inhibits the transcriptional repressors BCL11A and ZBTB7A, which are crucial in fetal hemoglobin regulation. |
|---|---|
| ln Vitro |
HbF and γ-globin mRNA expression levels were upregulated by RK-701 (0.01-3 µM; 4 d) without compromising cell viability or erythroid differentiation. RK-701 raises the percentage of cells that express HbF in a concentration-dependent way. RK-701 also selectively increases the expression level of BGLT3 and decreases the percentage of H3K9me2 in β-globin in HUDEP-2 cells and human primary CD34+ cells [1]. In the absence of BCL11A or ZBTB7A, RK-701 (1 µM; 4 d) cannot increase the expression of BGLT3 in HUDEP-2 cells and significantly reduces the proportions of BCL11A and ZBTB7A in BGLT3 [1].
In vitro, RK-701 (0.01‑3 uM; 4 days) up‑regulates HbF and gamma‑globin mRNA levels without affecting cell viability or erythroid differentiation, and increases HbF‑expressing cells in a concentration‑dependent manner. In HUDEP‑2 cells and human primary CD34+ cells, it selectively up‑regulates BGLT3 and reduces H3K9me2 in the beta‑globin locus. At 1 uM, it reduces BCL11A and ZBTB7A proportions. It also exhibited tumor growth inhibition in MOLT‑4 cells. |
| ln Vivo |
RK-701 selectively enhances mouse embryonic epsilon globin and dramatically downregulates the expression level of H3K9me2 in peripheral blood cells (20 mg/kg and 50 mg/kg; intraperitoneal injection; 5 weeks, 5 consecutive days per week). RK-701 is not very harmful [1].
In vivo, RK-701 (20 mg/kg and 50 mg/kg; intraperitoneal injection; 5 weeks, 5 consecutive days/week) selectively increases mouse embryonic ε‑globin and significantly down‑regulates H3K9me2 expression in peripheral blood cells. Its low toxicity profile supports its use in animal studies. No detailed tumor xenograft efficacy in mice has been reported, but its target engagement has been confirmed in vivo. |
| Enzyme Assay |
For substrate‑competitive enzyme assays: Incubate recombinant human G9a (full‑length or catalytic domain) with 3H‑SAM and peptide substrate (histone H3) in assay buffer (50 mM Tris‑HCl pH 8.5, 10 mM MgCl2, 5 mM DTT) containing varying concentrations of RK-701 (0.1‑1000 nM) for 1‑2 hours. Spot onto P81 filters, wash, and measure radioactivity. Alternatively, use a fluorescence‑based or mass spectrometry assay to monitor H3K9 methylation.
|
| Cell Assay |
Cell Viability Assay[1]
Cell Types: HUDEP-2 cells and CD34+-derived cells. Tested Concentrations: 0.01, 0.03, 0.1, 0.3, 1 and 3 µM. Incubation Duration: 4 d. Experimental Results: demonstrated activation for HbF, γ- Globin and BGLT3. Cell Viability Assay[1] Cell Types: HUDEP-2 cells. Tested Concentrations: 1 µM. Incubation Duration: 4 d. Experimental Results: demonstrated inhibition for BCL11A and ZBTB7A. For cell‑based HbF expression assays: Culture HUDEP‑2 or human primary CD34+‑derived erythroid cells in expansion medium. Treat cells with RK-701 (0.01, 0.03, 0.1, 0.3, 1, and 3 uM) for 4 days. Harvest cells for RNA extraction and qRT‑PCR for HbF and gamma‑globin mRNA. For protein analysis, fix and permeabilize cells, stain with anti‑HbF antibody, and analyze by flow cytometry. Include a cell viability assay (e.g., trypan blue or MTT) to confirm no cytotoxicity. |
| Animal Protocol |
For in vivo efficacy in a mouse model: Administer RK-701 to C57BL/6 mice via intraperitoneal injection at 20 mg/kg or 50 mg/kg, 5 consecutive days per week for 5 weeks. Collect peripheral blood at various time points for analysis of ε‑globin (qRT‑PCR) and H3K9me2 levels (western blot). Monitor body weight and general behavior for toxicity. No standard tumor xenograft protocol is established, but IP administration can be used for pharmacokinetic or pharmacodynamic studies.
|
| ADME/Pharmacokinetics |
No detailed PK data is publicly available for RK-701. In vitro solubility studies indicate it is soluble in DMSO (100 mg/mL, 223.94 mM) and ethanol (89 mg/mL), but insoluble in water. In vivo solubility data suggests it can be formulated as a suspension (CMC‑Na) or a clear solution (5% DMSO / 40% PEG300 / 5% Tween‑80 / 50% ddH2O) for injection. Oral bioavailability has not been reported; IP administration is used in published studies.
|
| Toxicity/Toxicokinetics |
Preclinical studies characterize RK-701 as a non‑genotoxic and low‑toxicity G9a inhibitor. In the 5‑week mouse study at 20‑50 mg/kg IP, no obvious toxicity was noted. It does not affect cell viability in erythroid cells at 0.01‑3 uM in vitro. No organ toxicity, weight loss, or behavioral changes have been reported at these doses. Higher doses have not been systematically evaluated. As an epigenetic probe, it should still be handled with care.
|
| References | |
| Additional Infomation |
RK-701 is a research‑grade compound for laboratory use only, not for clinical therapy. It is supplied as a light yellow to yellow solid powder, and should be stored at -20degC (powder stable for 3 years) protected from light. It is soluble in DMSO (100 mg/mL) and ethanol, insoluble in water. The compound has been cited in publications (e.g., Takase S, et al. Nat Commun. 2023;14(1):23; Nishigaya Y, et al. J Med Chem. 2023;66(6):4059‑4085). No clinical trials or approved status exist.
|
| Molecular Formula |
C26H30N4O3
|
|---|---|
| Molecular Weight |
446.541406154633
|
| Exact Mass |
446.231
|
| CAS # |
2648855-18-7
|
| Related CAS # |
RK-0133114
|
| PubChem CID |
156745798
|
| Appearance |
Light yellow to yellow solid powder
|
| LogP |
4.1
|
| Hydrogen Bond Donor Count |
3
|
| Hydrogen Bond Acceptor Count |
4
|
| Rotatable Bond Count |
7
|
| Heavy Atom Count |
33
|
| Complexity |
819
|
| Defined Atom Stereocenter Count |
1
|
| SMILES |
N1C2=C(C(=O)CC(C)(C)C2)C(C)=C1C(N[C@H](C(NC1=CC=C(C#N)C=C1)=O)CCC1CC1)=O
|
| InChi Key |
OWORSODZTADOQX-IBGZPJMESA-N
|
| InChi Code |
InChI=1S/C26H30N4O3/c1-15-22-20(12-26(2,3)13-21(22)31)29-23(15)25(33)30-19(11-8-16-4-5-16)24(32)28-18-9-6-17(14-27)7-10-18/h6-7,9-10,16,19,29H,4-5,8,11-13H2,1-3H3,(H,28,32)(H,30,33)/t19-/m0/s1
|
| Chemical Name |
N-[(2S)-1-(4-cyanoanilino)-4-cyclopropyl-1-oxobutan-2-yl]-3,6,6-trimethyl-4-oxo-5,7-dihydro-1H-indole-2-carboxamide
|
| HS Tariff Code |
2934.99.9001
|
| 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)
|
| Solubility (In Vitro) |
DMSO: ~100 mg/mL (~223.9 mM; with ultrasonication)
|
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (5.60 mM) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), Clear solution; with ultrasonication and warming and heat to 80°C.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of DMSO stock solution (25.0 mg/mL) to 400 μL of PEG300 and mix well; then add 50 μL of Tween-80 and mix well; finally add 450 μL of physiological saline and adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 2.5 mg/mL (5.60 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), Clear solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of DMSO stock solution (25.0 mg/mL) to 900 μL of corn oil and mix well. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2394 mL | 11.1972 mL | 22.3944 mL | |
| 5 mM | 0.4479 mL | 2.2394 mL | 4.4789 mL | |
| 10 mM | 0.2239 mL | 1.1197 mL | 2.2394 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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
