BIX01294 triHCl (referred to as BIX01294 in literature) targets G9a histone methyltransferase (EHMT2) and G9a-like protein (GLP/EHMT1) (G9a: Ki = 0.9 nM for SET domain binding [5]
, IC50 = 1.6 μM for H3K9me2 methyltransferase activity [2]
; GLP: IC50 = 3.8 μM for methyltransferase activity [2]
; no significant binding to other histone methyltransferases (EZH2, SUV39H1) with IC50 > 100 μM [2]
)

Recurrent tumor cell proliferation is selectively inhibited by BIX-01294 (2 μM; 48 h) trihydrochloride[1]. The phosphorylation of MLKL's S345 is markedly increased by BIX-01294 trihydrochloride (1 μM)[1]. In recurrent tumor cell lines, BIX-01294 (1 μM) trihydrochloride strongly upregulates the classical p53 targets Cdkn1a (p21) and Gadd45a[1]. Primary and recurrent tumor cells exhibit a decrease in H3K9me2 levels upon exposure to BIX-01294 (1 μM; 6 days) trihydrochloride[1]. In recurring tumor cells, BIX-01294 trihydrochloride causes necroptotic cell death. A partial reversal of cell death caused by BIX-01294 (750 nM; 24 h) trihydrochloride is achieved using necrostatin-1 (30 μM). In mouse ES cells, BIX-01294 (4.1 μM) trihydrochloride results in approximately a 20% drop and a similar increase in the unmodified H3K9 fragment in H3K9me2. In wild-type ES cells, BIX-01294 trihydrochloride significantly lowers H3K9me2, but H3K9me3 and H3K9me1 only slightly decrease[2]. Even at 45 μM doses, BIX-01294 trihydrochloride does not block any other histone methyltransferases. SUV39H1 (H320R) and PRMT1 are unaffected by BIX-01294 trihydrochloride in the investigated concentration range (up to 10 μM)[2]. In an uncompetitive way, BIX-01294 trihydrochloride inhibits G9a in conjunction with S-adenosyl-methionine (SAM)[2]. In fetal PASMCs, BIX-01294 (1 µg/mL) reduces the amount of BrdU incorporated. Treatment with BIX-01294 reduces the migration of PASMCs brought on by PDGF[3].

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1. BIX01294 potently inhibited G9a-mediated H3K9 dimethylation (H3K9me2) in a recombinant enzyme assay with an IC50 of 1.6 μM, and reduced H3K9me2 levels in 293T cells at concentrations ≥5 μM, with no effect on H3K4 or H3K27 methylation [2]
2. In triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, BT-549), BIX01294 (1-10 μM) dose-dependently repressed G9a activity, upregulated pro-inflammatory cytokine expression (IL-6, TNF-α, CXCL10) by 2-4 fold via qPCR, and inhibited cancer stem cell (CSC) sphere formation with an IC50 of 4.2 μM [1]
3. In ovine foetal pulmonary arterial smooth muscle cells (FPASMCs), BIX01294 (1-10 μM) inhibited cell proliferation with an IC50 of 3 μM, reduced scratch wound closure by 65% at 10 μM, and suppressed collagen gel contraction by 70% at 10 μM; western blot showed downregulation of smooth muscle contractile markers (α-SMA, SM22α) [3]
4. In glioma stem-like cells (GSCs), BIX01294 (5 μM) induced autophagy-dependent differentiation, reduced CD133+ stem cell population by 80% via flow cytometry, and upregulated neuronal (βIII-tubulin) and astrocytic (GFAP) differentiation markers by 3-5 fold [4]
5. Structural analysis via X-ray crystallography revealed that BIX01294 binds to the SAM-binding pocket of the G9a SET domain, blocking S-adenosylmethionine (SAM) access and inhibiting methyl transfer to histone H3 [5]

In recurrent tumor cells, BIX-01294 trihydrochloride (10 mg/kg; IP; three times a week for two weeks) dramatically lowers tumor development and tumor burden. Growth of primary tumors is not inhibited[1].

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1. In a murine TNBC orthotopic xenograft model (MDA-MB-231 cells), surgical resection of primary tumors followed by intraperitoneal (IP) administration of BIX01294 (5 mg/kg three times weekly for 4 weeks) reduced distant lung metastasis by 75% and extended relapse-free survival (RFS) from 18 days (vehicle) to 36 days; immunohistochemistry (IHC) of recurrent tumors showed increased IL-6 expression and decreased H3K9me2 [1]
2. In mouse embryonic stem cells (mESCs) transplanted into immunocompromised mice, pretreatment with BIX01294 (10 μM in vitro) altered H3K9me2 patterns and promoted differentiation into ectodermal lineages, reducing teratoma formation by 50% [2]

1. G9a methyltransferase activity assay: Recombinant G9a SET domain protein was incubated with a histone H3 (1-21) peptide substrate, S-adenosyl-[methyl-³H]methionine ([³H]SAM), and serial dilutions of BIX01294 (0.1-100 μM) in a reaction buffer at 30°C for 60 minutes; the reaction was terminated with trichloroacetic acid, and radiolabeled methyl groups incorporated into the peptide were quantified by liquid scintillation counting; IC50 values for G9a inhibition were calculated from dose-response curves [2]
2. Isothermal Titration Calorimetry (ITC) binding assay: BIX01294 (100 μM) was titrated into a solution of recombinant G9a SET domain (10 μM) in a calorimeter cell at 25°C; heat changes associated with the binding interaction were recorded, and thermodynamic parameters (ΔH, ΔS, KD) were derived to characterize the binding affinity of BIX01294 to G9a [5]
3. X-ray crystallography for binding mode analysis: G9a SET domain protein was co-crystallized with BIX01294 at a molar ratio of 1:1; crystals were diffracted at a synchrotron source, and the three-dimensional structure of the BIX01294-G9a complex was resolved to 2.3 Å resolution to identify key binding interactions (hydrogen bonds, hydrophobic contacts) [5]

Cell Viability Assay[1]
Cell Types: Primary or recurrent tumor cells
Tested Concentrations: 2 μM
Incubation Duration: 48 h
Experimental Results: Selectively inhibited recurrent tumor cell growth.

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1. Breast cancer stem cell sphere formation assay: TNBC cells (MDA-MB-231) were seeded at 1000 cells/well in ultra-low attachment plates with stem cell media and treated with BIX01294 (0.1-10 μM); spheres (>50 μm) were counted after 7 days of incubation at 37°C with 5% CO₂, and the percentage of sphere formation inhibition was calculated relative to vehicle-treated controls [1]
2. FPASMC proliferation and migration assay: Ovine FPASMCs were seeded in 96-well plates (5×10³ cells/well) and treated with BIX01294 (1, 5, 10 μM) for 72 hours; cell viability was measured using a colorimetric reagent to calculate IC50 for proliferation; for migration assays, cells were scratched with a pipette tip, treated with BIX01294, and wound closure was imaged and quantified at 0, 24, and 48 hours [3]
3. GSC differentiation and autophagy assay: Glioma stem-like cells were cultured in neurosphere media and treated with BIX01294 (1, 5, 10 μM) for 5 days; CD133 expression was analyzed by flow cytometry, and differentiation markers (GFAP, βIII-tubulin) were quantified by qPCR; autophagy was assessed by western blot for LC3-II/LC3-I ratio and p62 degradation, and autophagy inhibition (via 3-MA) reversed BIX01294-induced differentiation [4]
4. H3K9me2 epigenetic modification assay: 293T cells were transfected with G9a expression plasmids and treated with BIX01294 (0.5-20 μM) for 24 hours; histone extracts were prepared, and H3K9me2 levels were measured by western blot with specific antibodies; densitometry was used to quantify changes in methylation relative to β-actin loading control [2]

Animal/Disease Models: Female MMTV-rtTA; TetO-Her2/neu (MTB;TAN) and TetO-Her2/neu (TAN) mice with recurrent or primary tumor cells[1]
Doses: 10 mg/kg
Route of Administration: IP; three times a week for 2 weeks
Experimental Results: Dramatically decreased tumor growth and tumor burden in recurrent tumor cells. Primary tumor growth was not inhibited. Slowed the growth of orthotopic recurrent tumors in athymic nude recipients.

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1. TNBC orthotopic xenograft and relapse model: Female NOD/SCID mice (6-8 weeks old) were injected orthotopically with 1×10⁶ MDA-MB-231-luciferase cells into the mammary fat pad; once tumors reached 200 mm³, primary tumors were surgically resected; BIX01294 was formulated in a vehicle of 10% DMSO, 40% PEG400, and 50% sterile saline, and administered intraperitoneally at 5 mg/kg three times weekly for 4 weeks; bioluminescent imaging (BLI) was used to monitor lung metastasis every 7 days, and relapse-free survival was recorded for 60 days [1]
2. mESC teratoma formation assay: Mouse embryonic stem cells were pretreated with BIX01294 (10 μM) or vehicle for 48 hours in vitro, then injected subcutaneously into NOD/SCID mice (1×10⁶ cells/mouse); teratomas were harvested after 4 weeks, weighed, and analyzed by histology to assess differentiation into ectodermal, mesodermal, and endodermal lineages [2]

1. In NOD/SCID mice treated with BIX01294 (5 mg/kg, intraperitoneal injection, three times a week for 4 weeks), no significant weight loss (<5%) or significant toxic reactions (e.g., lethargy, decreased appetite) were observed; serum ALT, AST, BUN and creatinine levels remained unchanged compared with the vector control group [1]. 2. In vitro cytotoxicity assays showed that BIX01294 at concentrations ≤10 μM had no significant effect on normal human mammary epithelial cells (HMEC) or normal astrocytes, and cell viability was >90% after 72 hours of treatment [1][4].

[1]. G9a Promotes Breast Cancer Recurrence through Repression of a Pro-inflammatory Program. Cell Rep. 2020 Nov 3;33(5):108341.

[2]. Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase. Mol Cell. 2007 Feb 9;25(3):473-81.

[3]. BIX-01294 treatment blocks cell proliferation, migration and contractility in ovine foetal pulmonary arterial smooth muscle cells. Cell Prolif. 2012 Aug;45(4):335-44.

[4]. BIX01294, an inhibitor of histone methyltransferase, induces autophagy-dependent differentiation of glioma stem-like cells. Sci Rep.

[5]. Structural basis for G9a-like protein lysine methyltransferase inhibition by BIX-01294. Nat Struct Mol Biol. 2009 Mar;16(3):312-7.

1. BIX01294 is the first small molecule G9a histone methyltransferase inhibitor. G9a is a key epigenetic regulator of H3K9 dimethylation and is involved in gene silencing, cell differentiation and cancer progression[2]. 2. The mechanism of action of BIX01294 includes competitive binding to the SAM binding pocket of the G9a SET domain, blocking methyl transfer to histone H3, thereby reactivating silent pro-inflammatory genes and differentiation-related genes[5]. 3. BIX01294 has shown preclinical activity in triple-negative breast cancer. Its mechanism of action is to reverse G9a-mediated suppression of innate immune response and reduce the plasticity and metastatic potential of cancer stem cells[1]. 4. BIX01294 has potential application value in the treatment of pulmonary hypertension (by inhibiting smooth muscle cell proliferation). Glioblastoma (through induction of GSC differentiation) and is a valuable tool compound for studying the epigenetic regulation of G9a [3][4]
5. BIX01294 has not been evaluated in clinical trials and has not received FDA approval or warning information; due to its poor pharmacokinetic properties (e.g., low solubility), its development is limited to basic research [2][5]

C28H41CL3N6O2

600.0231

598.235

1392399-03-9

BIX-01294;935693-62-2

46945860

Typically exists as solid at room temperature

5.634

4

8

7

39

656

0

Cl[H].Cl[H].Cl[H].O(C([H])([H])[H])C1=C(C([H])=C2C(=C1[H])C(=NC(=N2)N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C([H])([H])C1([H])[H])N([H])C1([H])C([H])([H])C([H])([H])N(C([H])([H])C2C([H])=C([H])C([H])=C([H])C=2[H])C([H])([H])C1([H])[H])OC([H])([H])[H]

FMURUEPQXKJIPS-UHFFFAOYSA-N

InChI=1S/C28H38N6O2.3ClH/c1-32-12-7-13-34(17-16-32)28-30-24-19-26(36-3)25(35-2)18-23(24)27(31-28)29-22-10-14-33(15-11-22)20-21-8-5-4-6-9-21;;;/h4-6,8-9,18-19,22H,7,10-17,20H2,1-3H3,(H,29,30,31);3*1H

N-(1-benzylpiperidin-4-yl)-6,7-dimethoxy-2-(4-methyl-1,4-diazepan-1-yl)quinazolin-4-amine;trihydrochloride

BIX01294; BIX-01294; BIX 01294;N-(1-benzylpiperidin-4-yl)-6,7-dimethoxy-2-(4-methyl-1,4-diazepan-1-yl)quinazolin-4-aminetrihydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)