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Purity: ≥98%
UNC0321 (UNC-0321) is a novel, potent and selective G9a histone methyltransferase inhibitor with anticancer activity. It inhibits G9a with Ki of 63 pM, UNC0321 was discovered by optimizing the 7-dimethylaminopropoxy side chain on the basis of the structural insights revealed by UNC0224-G9a cocrystal structure. UNC0321 is the first G9a inhibitor with picomolar potency.
| Targets |
UNC0321 targets G9a histone lysine methyltransferase (EHMT2) and Rab4 GTPase (G9a: IC50 = 8 nM for recombinant methyltransferase activity [2]
; Rab4: EC50 = 1.2 μM for inhibition of Rab4-mediated vesicle trafficking [3] ; no significant inhibition of GLP/EHMT1 (IC50 = 120 nM) or other histone methyltransferases (EZH2, SUV39H1) with IC50 > 10 μM [2] ) |
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| ln Vitro |
Compound 29 (UNC0321) has IC50 values of 9 nM, 6 nM, 15 nM, and 23 nM, respectively, which inhibit the activities of G9a ECSD, G9a CLOT, GLP ECSD, and GLP CLOT. In HUVECs in MDA-MB-231 cells, UNC0321 (Compound 3) decreases Rab4 expression. Rab4 expression is markedly suppressed in HUVEC by UNC0321 (200 pM; 48 h) [3].
1. UNC0321 potently inhibited recombinant G9a methyltransferase activity with an IC50 of 8 nM in peptide-based TR-FRET assays, showing 15-fold selectivity for G9a over GLP (IC50 = 120 nM) [2] 2. In HeLa and NIH/3T3 cells, UNC0321 (0.01-2 μM) dose-dependently reduced global H3K9 dimethylation (H3K9me2) levels by up to 90% at 0.5 μM (western blot analysis), with no detectable changes in H3K4me2, H3K9me3, or H3K27me3, confirming epigenetic selectivity [2] 3. In human umbilical vein endothelial cells (HUVECs) exposed to high glucose (30 mM), UNC0321 (0.1-5 μM) dose-dependently inhibited apoptosis (IC50 = 0.8 μM); flow cytometry with Annexin V/PI staining showed that 2 μM UNC0321 reduced apoptotic cells from 38% (high glucose control) to 12% [3] 4. Western blot analysis in high glucose-treated HUVECs revealed that UNC0321 (2 μM) downregulated pro-apoptotic proteins (Bax, cleaved Caspase-3) by 65% and 70% respectively, and upregulated anti-apoptotic Bcl-2 by 2.3-fold [3] 5. Structure-activity relationship (SAR) studies in [1] identified that the 7-(3-dimethylaminopropoxy) side chain modification in UNC0321 (compared to UNC0224) improved aqueous solubility and cellular potency, with a 2-fold increase in G9a inhibitory activity in cell-based assays [1] |
| ln Vivo |
1. In a streptozotocin (STZ)-induced diabetic mouse model, intraperitoneal administration of UNC0321 (5 mg/kg once daily for 4 weeks) reduced endothelial cell apoptosis in the aortic endothelium by 60% (TUNEL staining) and improved vascular endothelial function (assessed by acetylcholine-induced vasodilation) [3]
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| Enzyme Assay |
1. G9a methyltransferase activity assay: Recombinant G9a catalytic domain protein was incubated with a biotinylated histone H3 (1-21) peptide, S-adenosylmethionine (SAM), and serial dilutions of UNC0321 (0.001-10 μM) in reaction buffer at 30°C for 60 minutes; methylated peptide product was detected using europium-labeled anti-methyl-H3K9 antibody and time-resolved fluorescence resonance energy transfer (TR-FRET); dose-response curves were generated to calculate IC50 values for G9a inhibition [2]
2. Rab4 GTPase activity assay: Recombinant Rab4 protein was incubated with GTP-γ-S and UNC0321 (0.1-10 μM) in a GTPase assay buffer at 37°C for 30 minutes; GTP hydrolysis was quantified by measuring phosphate release using a colorimetric reagent, and the percentage inhibition of Rab4 activity was calculated to determine EC50 [3] 3. Selectivity assay for histone methyltransferases: Recombinant GLP, EZH2, and SUV39H1 proteins were incubated with their respective peptide substrates, SAM, and UNC0321 (0.01-10 μM) under identical conditions to the G9a assay; methyltransferase activity was measured by TR-FRET to assess off-target effects [2] |
| Cell Assay |
Western Blot Analysis[3]
Cell Types: HUVEC. Level of H3K9me2, IC50 value is 11 µM[2]. Tested Concentrations: 50 pM, 100 pM, 200 pM. Incubation Duration: 48 hrs (hours). Experimental Results: Glucose-induced reduction in Cleared Caspase3 and Bax expression. Release glucose from inhibiting the expression of Bcl-2, Caspase3, p-AKT, p-mTOR and p70. Promote proliferation and inhibit apoptosis by inhibiting Rab4. Apoptosis analysis[3] Cell Types: HUVEC. Tested Concentrations: 50 pM, 100 pM, 200 pM. Incubation Duration: 48 hrs (hours). Experimental Results: Inhibition of glucose-induced apoptosis. Cell proliferation assay [3] Cell Types: HUVEC. Tested Concentrations: 50 pM, 100 pM, 200 pM. Incubation Duration: 48 hrs (hours). Experimental Results: The inhibitory effect of glucose on HUVEC proliferation was relieved. 1. H3K9me2 epigenetic modification assay: HeLa cells were seeded in 6-well plates (5×10⁵ cells/well) and treated with UNC0321 (0.01, 0.1, 0.5, 2 μM) for 24 hours; histone extracts were prepared by acid extraction, separated by SDS-PAGE, and analyzed by western blot with antibodies against H3K9me2, H3K4me2, and total H3 (loading control); band intensities were quantified by densitometry to measure methylation changes [2] 2. HUVEC apoptosis assay: HUVECs were cultured in normal glucose (5.5 mM) or high glucose (30 mM) media and treated with UNC0321 (0.1, 0.5, 2, 5 μM) for 48 hours; cells were harvested, stained with Annexin V-FITC and propidium iodide (PI), and apoptotic cells were quantified by flow cytometry; for protein expression analysis, cell lysates were prepared and western blot was performed with antibodies against Bax, Bcl-2, cleaved Caspase-3, and β-actin [3] 3. Cell viability assay: NIH/3T3 and HeLa cells were seeded in 96-well plates (5×10³ cells/well) and treated with UNC0321 (0.001-10 μM) for 72 hours at 37°C with 5% CO₂; a colorimetric cell viability reagent was added, and absorbance was measured at 490 nm; cell viability was >90% at concentrations up to 10 μM, indicating no cytotoxicity [2] |
| Animal Protocol |
1. STZ-induced diabetic mouse model for endothelial protection: Male C57BL/6 mice (8-10 weeks old) were injected intraperitoneally with streptozotocin (150 mg/kg) to induce diabetes; after 1 week of diabetes confirmation (blood glucose >16.7 mM), mice were treated with UNC0321 (5 mg/kg IP once daily) or vehicle for 4 weeks; UNC0321 was formulated in 10% DMSO, 40% PEG400, and 50% sterile saline; aortic tissues were collected for TUNEL staining to quantify endothelial apoptosis, and vascular function was assessed by measuring acetylcholine-induced vasodilation in isolated aortic rings [3]
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| ADME/Pharmacokinetics |
1. Compared with the precursor UNC0224 (15 μg/mL), UNC0321 showed higher water solubility (120 μg/mL) in phosphate-buffered saline (PBS, pH 7.4) as determined by UV-Vis spectroscopy [2].
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| Toxicity/Toxicokinetics |
1. At concentrations up to 10 μM, UNC0321 did not exhibit cytotoxicity against mammalian cell lines (HeLa, NIH/3T3, HUVECs), and cell viability was >90% after 72 hours of treatment [2][3]. 2. In a streptozotocin (STZ)-induced diabetic mouse model, no significant changes in body weight, liver function indicators (ALT, AST), or kidney function indicators (BUN, creatinine) were observed after treatment with UNC0321 (5 mg/kg, intraperitoneal injection, for 4 weeks) compared to the vector control group [3].
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| References |
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| Additional Infomation |
7-[2-[2-(dimethylamino)ethoxy]ethoxy]-6-methoxy-2-(4-methyl-1,4-diazacycloheptane-1-yl)-N-(1-methyl-4-piperidinyl)-4-quinazolinamine belongs to the quinazolin class of compounds.
1. UNC0321 is a second-generation 2,4-diamino-7-aminoalkoxyquinazoline derivative, optimized from UNC0224, with higher water solubility and stronger anti-G9a cell activity[1][2] 2. UNC0321 inhibits G9a mainly by binding to the SAM binding pocket of the G9a catalytic domain, competing with SAM and blocking H3K9 dimethylation[2] 3. UNC0321 also targets Rab4 GTPase, inhibiting Rab4-mediated vesicle transport and reducing high glucose-induced endothelial cell apoptosis, suggesting its potential application value in diabetic vascular complications[3] 4. UNC0321 is a valuable tool compound for studying G9a-mediated epigenetic regulation and Rab4-related endothelial dysfunction, but it has not yet been approved by the FDA or evaluated in clinical trials [1][2][3]. |
| Molecular Formula |
C27H45N7O3
|
|---|---|
| Molecular Weight |
515.6913
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| Exact Mass |
515.358
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| CAS # |
1238673-32-9
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| PubChem CID |
46901937
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| Appearance |
White to yellow solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
664.7±65.0 °C at 760 mmHg
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| Flash Point |
355.8±34.3 °C
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| Vapour Pressure |
0.0±2.0 mmHg at 25°C
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| Index of Refraction |
1.582
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| LogP |
0.36
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
37
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| Complexity |
652
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
AULLUGALUBVBDD-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C27H45N7O3/c1-31(2)15-16-36-17-18-37-25-20-23-22(19-24(25)35-5)26(28-21-7-11-33(4)12-8-21)30-27(29-23)34-10-6-9-32(3)13-14-34/h19-21H,6-18H2,1-5H3,(H,28,29,30)
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| Chemical Name |
7-(2-(2-(dimethylamino)ethoxy)ethoxy)-6-methoxy-2-(4-methyl-1,4-diazepan-1-yl)-N-(1-methylpiperidin-4-yl)quinazolin-4-amine
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| Synonyms |
UNC0321, CHEBI:785916, NCGC0018778901, UNC-0321, CHEMBL1214066, UNC 0321, KB81388
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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 : ≥ 31 mg/mL (~60.11 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.03 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 (4.03 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 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 (4.03 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 | 1.9391 mL | 9.6957 mL | 19.3915 mL | |
| 5 mM | 0.3878 mL | 1.9391 mL | 3.8783 mL | |
| 10 mM | 0.1939 mL | 0.9696 mL | 1.9391 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.
 The 7-dimethylamino propoxy side chain (gray, blue, and red) of10does not fully occupy the lysine binding channel in the X-ray crystal structure of the G9a-10complex (PDB code: 3K5K).J Med Chem.2010 Aug 12;53(15):5844-57. |
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 MorrisonKis of compounds29,10, and3ain the G9a MCE assay.J Med Chem.2010 Aug 12;53(15):5844-57. |
 The lipophilic benzyl group (top) is exposed to solvent and does not makes any interactions with the protein in the X-ray crystal structure of the GLP-3acomplex (PDB code: 3FPD).J Med Chem.2010 Aug 12;53(15):5844-57. |
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