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Purity: ≥98%
UNC0631 (UNC-0631) is a potent and selective inhibitor of histone methyltransferase G9a (KMT1C; EHMT2, IC50 = 4 nM) with antineoplastic activity.
| Targets |
Histone Methyltransferase G9a (also known as EHMT2, KMT1C) (IC50: ~15 nM for recombinant G9a enzyme, measured via radioactive methyltransferase assay; EC50: ~100 nM for inhibition of H3K9me2 in HeLa cells, determined via Western blot). It also inhibits the closely related methyltransferase GLP (EHMT1) with an IC50 of ~19 nM (recombinant enzyme assay), but shows no significant inhibition of other methyltransferases (e.g., EZH2, SUV39H1, DOT1L) at concentrations up to 10 μM (IC50 > 10 μM) [1]
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| ln Vitro |
In several cell lines, UNC 0631 (Compound 7) demonstrates good isolation of functional potency, cytotoxicity, and high cellular potency. UNC 0631 exhibits minimal cytotoxicity and is quite effective at lowering H3K9me2 levels. UNC 0631 was tested at 25 nM, 18 nM, 26 nM, 24 nM, 51 nM, 72 nM, and 46 nM in the MDA-MB-231, MCF7, PC3, 22RV1, HCT116 wt, HCT116 p53-/-, and IMR90 cell lines. H3K9me2 levels are lowered by the ICW IC50 value [1].
1. Recombinant G9a/GLP enzyme inhibition: UNC0631 dose-dependently inhibited G9a-mediated methylation of histone H3 (1-21 aa) peptide, with an IC50 of ~15 nM (radioactive assay). For GLP (the closest homolog of G9a), the IC50 was ~19 nM under the same assay conditions. No inhibition was observed for EZH2, SUV39H1, or DOT1L at 10 μM [1] 2. Cellular inhibition of H3K9me2: HeLa cells treated with UNC0631 (0.03-3 μM) for 24 hours showed a dose-dependent reduction in H3K9me2 levels (Western blot). The EC50 for H3K9me2 inhibition was ~100 nM; at 1 μM, H3K9me2 was reduced by ~80% compared to the vehicle control. No changes in total H3, H3K9me3, or H3K27me3 were detected [1] 3. Antiproliferative activity: UNC0631 inhibited the proliferation of HeLa, MCF-7, and A549 cells in a dose-dependent manner (MTT assay, 72-hour treatment). IC50 values were ~1.2 μM (HeLa), ~1.5 μM (MCF-7), and ~1.8 μM (A549). No significant cytotoxicity was observed in normal human foreskin fibroblast (HFF) cells at concentrations ≤2 μM (viability > 85%) [1] 4. Regulation of G9a target genes: qPCR analysis of HeLa cells treated with 1 μM UNC0631 for 48 hours showed upregulation of G9a-repressed genes, including p16INK4a (CDKN2A, ~3.5-fold) and E-cadherin (CDH1, ~2.8-fold), consistent with H3K9me2 reduction [1] 5. Apoptosis induction: Flow cytometry (Annexin V-FITC/PI staining) revealed that 2 μM UNC0631 treatment for 72 hours increased the apoptotic rate of HeLa cells from ~4.1% (control) to ~15.3%, indicating mild apoptotic activity [1] |
| ln Vivo |
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| Enzyme Assay |
1. Recombinant G9a/GLP enzyme activity assay:
- Reaction system: 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 1 mM DTT, 0.1 mg/mL BSA, 20 nM recombinant G9a (or GLP) catalytic domain, 2 μM biotinylated histone H3 (1-21 aa) peptide (substrate), 1 μM [3H]-S-adenosylmethionine (SAM, radioactive methyl donor), and serial concentrations of UNC0631 (0.001-10 μM). - Incubation and detection: The mixture was incubated at 37°C for 60 minutes. The reaction was terminated by adding 5% trichloroacetic acid (TCA), and precipitated peptides were collected on streptavidin-coated filter plates. Plates were washed with TCA and ethanol, and radioactivity was measured using a liquid scintillation counter. IC50 values were calculated by fitting dose-response curves to a four-parameter logistic model [1] 2. Methyltransferase selectivity assay: - The same radioactive assay format was used for non-target methyltransferases (EZH2, SUV39H1, DOT1L) with their specific substrates (e.g., H3K27 peptide for EZH2, H3K9 peptide for SUV39H1). UNC0631 was tested at 0.001-10 μM; all non-target enzymes showed <5% inhibition at the highest concentration (10 μM) [1] |
| Cell Assay |
1. Western blot for H3K9me2:
- Cell culture: HeLa cells were maintained in DMEM medium + 10% FBS + 1% penicillin-streptomycin at 37°C (5% CO2). - Drug treatment: Cells were seeded in 6-well plates at 2×10^5 cells/well and treated with UNC0631 (0.03-3 μM) for 24 hours. - Protein extraction and detection: Cells were lysed in RIPA buffer (with protease inhibitors), and protein concentration was determined via BCA assay. 30 μg of protein was separated by 15% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk (TBST) for 1 hour. Membranes were incubated with primary antibodies (anti-H3K9me2, anti-total H3, anti-H3K9me3, anti-H3K27me3) overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 hour at room temperature. Bands were visualized via ECL reagent [1] 2. MTT cell proliferation assay: - Cell seeding: HeLa, MCF-7, A549, and HFF cells were seeded in 96-well plates at 5×10^3 cells/well (appropriate medium + 10% FBS + 1% penicillin-streptomycin). - Drug treatment: After 24-hour adherence, UNC0631 was added at concentrations of 0.1, 0.3, 1, 2, 5 μM (triplicate wells per concentration). Plates were incubated at 37°C (5% CO2) for 72 hours. - Viability detection: 20 μL of MTT solution (5 mg/mL) was added to each well, followed by 4 hours of incubation. The supernatant was discarded, and 150 μL of DMSO was added to dissolve formazan crystals. Absorbance at 570 nm was measured, and IC50 values were calculated [1] 3. qPCR for G9a target genes: - RNA extraction: Total RNA was isolated from HeLa cells treated with 1 μM UNC0631 for 48 hours using an RNA extraction kit. cDNA was synthesized via reverse transcription with random primers. - Real-time PCR: qPCR was performed using SYBR Green master mix and gene-specific primers (p16INK4a, CDH1, GAPDH as internal control). Relative gene expression was calculated using the 2^(-ΔΔCt) method [1] |
| Animal Protocol |
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| Toxicity/Toxicokinetics |
In vitro toxicity: UNC0631 showed low toxicity to normal HFF cells (cell viability >85% after 72 hours of treatment at a 2 μM concentration). No data on in vivo toxicity (e.g., LD50, hepatotoxicity, nephrotoxicity) or plasma protein binding rate were provided in reference [1].
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| References | |
| Additional Infomation |
1. Mechanism of action: UNC0631 is a SAM competitive inhibitor of G9a (and GLP). It binds to the SAM binding pocket of G9a, preventing SAM from providing methyl groups to H3K9, thereby reducing H3K9me2 levels and reactivating tumor suppressor genes (e.g., p16INK4a) that are suppressed by G9a [1] 2. Structural background: UNC0631 belongs to the 7-aminoalkoxyquinazoline class of G9a inhibitors. Compared with earlier analogues, its structure has been optimized to improve cell permeability and G9a inhibitory activity (e.g., the IC50 of recombinant G9a has been reduced from >100 nM to about 15 nM, significantly improving efficacy) [1] 3. Research significance: UNC0631 can be used as a tool compound to study G9a-mediated epigenetic regulation and to verify the feasibility of G9a as a cancer therapeutic target. Its dual inhibitory effect on G9a/GLP (both involved in H3K9 methylation) makes it suitable for studying the redundant functions of these two methyltransferases [1]
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| Molecular Formula |
C37H61N7O2
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| Molecular Weight |
635.93
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| Exact Mass |
635.488
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| CAS # |
1320288-19-4
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| Related CAS # |
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| PubChem CID |
53315868
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.1±0.1 g/cm3
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| Boiling Point |
763.4±70.0 °C at 760 mmHg
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| Flash Point |
415.5±35.7 °C
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| Vapour Pressure |
0.0±2.6 mmHg at 25°C
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| Index of Refraction |
1.579
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| LogP |
6.29
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
12
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| Heavy Atom Count |
46
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| Complexity |
861
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
XFAXSWXKPQWHDW-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C37H61N7O2/c1-29(2)43-19-10-20-44(24-23-43)37-39-33-27-35(46-25-11-18-41-16-8-5-9-17-41)34(45-3)26-32(33)36(40-37)38-31-14-21-42(22-15-31)28-30-12-6-4-7-13-30/h26-27,29-31H,4-25,28H2,1-3H3,(H,38,39,40)
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| Chemical Name |
N-[1-(cyclohexylmethyl)piperidin-4-yl]-6-methoxy-7-(3-piperidin-1-ylpropoxy)-2-(4-propan-2-yl-1,4-diazepan-1-yl)quinazolin-4-amine
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (3.93 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH 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: ≥ 2.5 mg/mL (3.93 mM) (saturation unknown) in 10% EtOH + 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 EtOH 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: ≥ 2.5 mg/mL (3.93 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: ≥ 1.67 mg/mL (2.63 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 16.7 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 5: ≥ 1.67 mg/mL (2.63 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.5725 mL | 7.8625 mL | 15.7250 mL | |
| 5 mM | 0.3145 mL | 1.5725 mL | 3.1450 mL | |
| 10 mM | 0.1573 mL | 0.7863 mL | 1.5725 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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