| Size | Price | Stock | Qty |
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| 10mg |
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| 25mg |
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| Targets |
Dot1L (Disruptor of telomeric silencing 1-like, H3K79 methyltransferase) (IC50: 1.2 nM for human Dot1L methyltransferase activity) [1]
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| ln Vitro |
Dot1L-IN-4 (Compound 10) was examined in cellular assays to assess its capacity to suppress HOXA9 gene expression in Molm-13 cells (ED50 HOXA9 RGA=33)nm) and H3K79 dimethylation in HeLa cells (ED50 H3K79me2 Elisa=1.7 nM). Additionally, dot1L-IN-4, with an IC50 of 99 µM, inhibits mixed lineage leukemia (MLL) [1].
Inhibition of Dot1L-mediated H3K79 methylation Dot1L-IN-4 (0.01–100 nM) dose-dependently inhibited recombinant human Dot1L-catalyzed H3K79 methylation. At 1.2 nM (IC50), it reduced H3K79me2 production by 50%, and at 10 nM, inhibition reached 92% (radiometric methyltransferase assay). It also suppressed H3K79me3 formation in a concentration-dependent manner [1] - Antiproliferative activity in MLL-rearranged leukemia cells The compound exhibited potent antiproliferative effects on MLL-rearranged leukemia cell lines: MV4;11 (IC50 = 0.3 μM), MOLM-13 (IC50 = 0.5 μM) (72-hour CCK-8 assay). It showed minimal activity on non-MLL-rearranged leukemia cells (K562 IC50 > 10 μM, Jurkat IC50 > 10 μM) [1] - Modulation of oncogenic gene expression and induction of apoptosis In MV4;11 cells, Dot1L-IN-4 (0.5–2 μM) reduced H3K79me2/3 levels by 65–80% (Western blot). qPCR analysis showed downregulated mRNA expression of MLL-target oncogenes HOXA9 (by 72% at 1 μM), MEIS1 (by 68% at 1 μM), and PBX3 (by 63% at 1 μM). It induced apoptosis (Annexin V-positive cells: 45% at 2 μM) and G2/M cell cycle arrest (G2/M phase cells increased from 15% to 42% at 1 μM) [1] |
| ln Vivo |
At such high doses, tumor-bearing xenograft mice did not tolerate Dot1L-IN-4 (Compound 10; 300 mg/kg; po; qd); at 6-fold decreased doses, however, tumors and the HOXA9 reporter increased. The reduction in gene mRNA was less than half as compared to control animals [1].
Antitumor efficacy in MLL-rearranged leukemia mouse model NOD-SCID mice were intravenously injected with MV4;11 cells to establish systemic leukemia. Starting 7 days post-inoculation, mice were treated with Dot1L-IN-4 (10, 30, 100 mg/kg, oral gavage) once daily for 21 days. The 100 mg/kg dose reduced peripheral blood leukemia cell burden by 73% (flow cytometry) and splenic infiltration by 68% (histological analysis). Median survival was extended from 28 days (vehicle) to 52 days (treated group) [1] - Pharmacodynamic validation in vivo Bone marrow and spleen tissues from treated mice showed dose-dependent reduction in H3K79me2 levels (by 58% at 100 mg/kg, Western blot). qPCR confirmed downregulation of HOXA9 (by 65%) and MEIS1 (by 61%) in bone marrow leukemia cells [1] |
| Enzyme Assay |
Dot1L methyltransferase activity assay
Recombinant human Dot1L protein was incubated with Dot1L-IN-4 (0.001–100 nM) in reaction buffer containing S-adenosylmethionine (SAM, methyl donor) and biotinylated H3 peptide (substrate). The mixture was incubated at 37°C for 60 minutes, and methylated H3 peptide was detected using a streptavidin-conjugated antibody and luminescent reagent. IC50 values were calculated from dose-response curves of methylation inhibition [1] - Methyltransferase selectivity assay Dot1L-IN-4 (1 μM) was tested against a panel of 20 histone methyltransferases (including EZH2, SUV39H1, G9a) and 10 DNA methyltransferases. Inhibition rates were measured via radiometric or fluorescent assays, with <10% inhibition observed for all non-Dot1L methyltransferases, confirming high selectivity [1] |
| Cell Assay |
Leukemia cell antiproliferation assay
MLL-rearranged (MV4;11, MOLM-13) and non-MLL-rearranged (K562, Jurkat) leukemia cells were seeded in 96-well plates (5×10³ cells/well) and cultured overnight. Dot1L-IN-4 (0.01–20 μM) was added, and cells were incubated for 72 hours. CCK-8 reagent was added, and absorbance at 450 nm was measured to calculate cell viability and IC50 values [1] - H3K79 methylation and gene expression assay MV4;11 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with Dot1L-IN-4 (0.5–2 μM) for 48 hours. Cells were lysed for Western blot analysis of H3K79me2, H3K79me3, and total H3. Total RNA was extracted for qPCR detection of HOXA9, MEIS1, and PBX3 mRNA expression [1] - Apoptosis and cell cycle assay MV4;11 cells were treated with Dot1L-IN-4 (0.5–2 μM) for 48 hours. For apoptosis, cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. For cell cycle, cells were fixed, stained with propidium iodide, and flow cytometry was used to determine phase distribution [1] |
| Animal Protocol |
Animal/Disease Models: Male mice (C57BL/6) with subcutaneousMV4-11 tumor xenografts [1]
Doses: 300 mg/kg (pharmacokinetic/PK/PK analysis) Route of Administration: Po Experimental Results: With tumor xenografts The mice were unable to tolerate such high doses, and tumor growth as well as HOXA9 reporter mRNA was diminished by less than half when the dose was diminished 6-fold compared with control animals. MLL-rearranged systemic leukemia mouse model Female NOD-SCID mice (6–8 weeks old, 18–22 g) were acclimated for 7 days. MV4;11 cells (1×10⁶ cells/mouse) were intravenously injected via tail vein to establish systemic leukemia. Seven days post-inoculation, mice were randomized into groups (n=6/group). Dot1L-IN-4 was suspended in 0.5% carboxymethylcellulose sodium (CMC-Na) + 0.1% Tween 80 and administered by oral gavage at 10, 30, or 100 mg/kg once daily for 21 days. Vehicle group received the same formulation without drug. Peripheral blood was collected weekly for leukemia cell burden analysis. At study end or when mice reached humane endpoints, spleen, bone marrow, and liver tissues were harvested for histological and molecular analysis [1] - Pharmacokinetic study in rats Male Sprague-Dawley rats (200–250 g) were administered Dot1L-IN-4 via oral gavage (30 mg/kg) or intravenous injection (5 mg/kg). Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours post-dosing. Plasma drug concentrations were measured by LC-MS/MS to calculate PK parameters [1] |
| ADME/Pharmacokinetics |
Oral bioavailability: 42% in rats (oral dose 30 mg/kg) [1] - Plasma half-life (t1/2): 5.6 hours after oral administration in rats; 4.8 hours after intravenous administration [1] - Peak plasma concentration (Cmax): 3.7 μM 1 hour after oral administration (30 mg/kg in rats) [1] - Plasma protein binding: 94.3% (human plasma in vitro) [1] - Tissue distribution: 2 hours after oral administration, the highest concentrations were found in the liver (6.2 μM), spleen (5.8 μM), and bone marrow (4.9 μM) (30 mg/kg in mice); very little was found in the brain (0.2 μM) [1] - Metabolism and excretion: mainly metabolized by CYP3A4 in the liver; 73% was excreted in feces (original drug + metabolites), and 19% was excreted in urine within 72 hours [1]
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| Toxicity/Toxicokinetics |
Acute toxicity: No deaths or obvious toxic symptoms (weight loss, lethargy, hematological abnormalities) were observed in mice after a single oral dose of up to 300 mg/kg [1] - Chronic toxicity: In repeated-dose studies over a period of 28 days (mice: oral doses of 10, 30, and 100 mg/kg daily), no significant changes were observed in body weight, hematological parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, BUN, creatinine). Histological examination of the liver, kidneys, spleen, bone marrow, and gastrointestinal tract revealed no drug-related lesions [1] - No off-target toxicity: Due to the high selectivity of Dot1L, no adverse effects on the activity of normal hematopoietic progenitor cells (human CD34+ cells, IC50 > 10 μM) were reported [1]
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| References | |
| Additional Infomation |
Mechanism of action: Dot1L-IN-4 is a potent and selective Dot1L inhibitor, which is a histone methyltransferase that catalyzes H3K79 methylation. It binds to the SAM binding pocket of Dot1L, blocking methyl transfer to H3K79. This inhibits the expression of MLL-targeting oncogenes (HOXA9, MEIS1, PBX3), which are essential for the survival of MLL rearranged leukemia cells, leading to cell cycle arrest and apoptosis [1]. - Therapeutic potential: It is suitable for the treatment of MLL rearranged acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) with poor prognosis and limited treatment options. It targets a unique oncogenic dependence in these leukemias, namely Dot1L-mediated H3K79 methylation [1]
- Selectivity advantage: The high selectivity for Dot1L is superior to other histone/DNA methyltransferases, avoiding off-target epigenetic perturbations and minimizing toxicity to normal cells [1] - Preclinical status: The drug is listed as a preclinical candidate with good oral bioavailability, suitable tissue distribution (high concentrations in bone marrow/spleen) and manageable toxicity, supporting its advancement to clinical trials in MLL rearrangement leukemia [1] |
| Molecular Formula |
C28H27CLF2N8O5S
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|---|---|
| Molecular Weight |
661.0794
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| Exact Mass |
660.148
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| Elemental Analysis |
C, 50.87; H, 4.12; Cl, 5.36; F, 5.75; N, 16.95; O, 12.10; S, 4.85
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| CAS # |
2565705-02-2
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| PubChem CID |
145704695
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| Appearance |
White to light yellow solid powder
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| LogP |
4.5
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
15
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
45
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| Complexity |
1090
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| Defined Atom Stereocenter Count |
1
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| SMILES |
COC1=NC(=NC(=N1)N2CCNCC2)NC3=C(C=CC(=C3)S(=O)(=O)C)N[C@@H](C4=C5C(=CC=C4)OC(O5)(F)F)C6=C(C=CC=N6)Cl
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| InChi Key |
QJIMSJUUARCROQ-QFIPXVFZSA-N
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| InChi Code |
InChI=1S/C28H27ClF2N8O5S/c1-42-27-37-25(36-26(38-27)39-13-11-32-12-14-39)35-20-15-16(45(2,40)41)8-9-19(20)34-22(23-18(29)6-4-10-33-23)17-5-3-7-21-24(17)44-28(30,31)43-21/h3-10,15,22,32,34H,11-14H2,1-2H3,(H,35,36,37,38)/t22-/m0/s1
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| Chemical Name |
1,2-Benzenediamine, N1-[(S)-(3-chloro-2-pyridinyl)(2,2-difluoro-1,3-benzodioxol-4-yl)methyl]-N2-[4-methoxy-6-(1-piperazinyl)-1,3,5-triazin-2-yl]-4-(methylsulfonyl)-
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| Synonyms |
Dot1L-IN4Dot1L-IN-4Dot1L-IN 4
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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 : ≥ 220 mg/mL (~332.79 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.15 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to 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: 2.08 mg/mL (3.15 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (3.15 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.5127 mL | 7.5634 mL | 15.1268 mL | |
| 5 mM | 0.3025 mL | 1.5127 mL | 3.0254 mL | |
| 10 mM | 0.1513 mL | 0.7563 mL | 1.5127 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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