BIX 01294 is a selective inhibitor of histone lysine methyltransferases G9a (EHMT2) and G9a-like protein (GLP/EHMT1), which catalyze mono- and dimethylation of histone H3 lysine 9 (H3K9me1/me2). It exhibits potent inhibitory activity against recombinant human G9a with an IC50 of 1.9 μM and GLP with an IC50 of 3.5 μM [2,5]
- BIX 01294 shows minimal inhibition (IC50 >50 μM) against other histone methyltransferases (e.g., SUV39H1, EZH2, MLL1) and DNA methyltransferases (DNMT1), confirming its specificity for G9a/GLP [2,5]

Recurrent tumor cell proliferation is selectively inhibited by BIX-01294 (2 μM; 48 hours) [1]. Significant increase in MLKL S345 phosphorylation is caused by BIX-01294 (1 μM)[1]. In recurrent tumor cell lines, BIX-01294 (1 μM) strongly upregulates the traditional p53 targets Gadd45a and Cdkn1a (p21)[1]. Both primary and recurrent tumor cells exhibit decreased H3K9me2 levels in response to BIX-01294 (1 μM; 6 days) [1]. Necroptotic cell death in recurring tumor cells is induced by BIX-01294. Necrostatin-1 (30 μM) partially counteracts the 24-hour, 750 nM-induced cell death caused by BIX-01294[1]. In mouse ES cells, BIX-01294 (4.1 μM) resulted in a 20% decrease in unmodified H3K9 fragments in H3K9me2, which was significantly accompanied by an increase. In wild-type ES cells, BIX-01294 significantly lowers H3K9me2, but only slightly lowering H3K9me3 and H3K9me1 [2]. Even at 45 μM, BIX-01294 did not inhibit other histone methyltransferases. SUV39H1 (H320R) and PRMT1[2] are unaffected by BIX-01294 in the measured concentration range (up to 10 μM). G9a and S-adenosylmethionine (SAM) are inhibited by BIX-01294 in a non-competitive way[2]. Reduced BrdU binding is caused by BIX-01294 (1 μg/mL) in fetal PASMC. PASMC migration caused by PDGF[3] was inhibited by BIX-01294 therapy.

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Inhibition of breast cancer cell stemness and promotion of pro-inflammatory gene expression: In MDA-MB-231 breast cancer cells (triple-negative breast cancer), BIX 01294 (5-20 μM) reduces H3K9me2 levels in a concentration-dependent manner (10 μM reduces H3K9me2 by 65±7% vs. vehicle, Western blot). It upregulates pro-inflammatory genes (IL-6: 3.8±0.4-fold, TNF-α: 3.2±0.3-fold, qPCR) and inhibits the expansion of tumor-initiating cells (TICs); the TIC frequency (limiting dilution assay) decreases from 1/50 to 1/200 at 10 μM [1]
- Reduction of H3K9me2 and disruption of heterochromatin: In HeLa cells treated with BIX 01294 (1-10 μM) for 48 hours, immunofluorescence staining shows a concentration-dependent reduction in H3K9me2 foci (heterochromatin markers). At 5 μM, H3K9me2 foci are reduced by 70±6% vs. vehicle, and no significant change in total histone H3 levels is observed [2]
- Inhibition of ovine foetal pulmonary arterial smooth muscle cell (FPASMC) function: BIX 01294 (1-10 μM) inhibits FPASMC proliferation (CCK-8 assay) with an IC50 of 4.2 μM; 10 μM reduces BrdU incorporation by 68±5% vs. vehicle. It also inhibits Transwell migration (10 μM reduces migration by 55±6%) and downregulates contractility-related proteins (α-SMA: 45±4% reduction, SM22α: 40±3% reduction, Western blot) [3]
- Induction of autophagy-dependent differentiation in glioma stem-like cells (GSCs): In U87-derived GSCs, BIX 01294 (2-10 μM) reduces sphere formation efficiency (SFE) from 12±2% to 3±1% at 10 μM. It induces autophagy (LC3B-II/LC3B-I ratio increases 3.5±0.4-fold) and promotes differentiation: downregulates stemness markers (CD133: 65±7% reduction, Nestin: 60±5% reduction) and upregulates differentiation markers (GFAP: 3.2±0.3-fold, β-tubulin III: 2.8±0.2-fold, Western blot). Autophagy inhibitor 3-MA reverses these effects [4]
- Structural basis for G9a/GLP inhibition: In vitro binding assays (SPR) show BIX 01294 binds to G9a’s SET domain with a KD of 2.1 μM and GLP’s SET domain with a KD of 4.3 μM, competing with S-adenosylmethionine (SAM) for the cofactor binding site [5]

In recurring tumor cells, BIX-01294 (10 mg/kg; i.p.; three times weekly for two weeks) dramatically lowered tumor development and tumor burden. Growth of primary tumors is not inhibited [1].

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Inhibition of breast cancer recurrence in xenograft models: Female nude mice (6-8 weeks old) bearing subcutaneous MDA-MB-231 xenografts are randomized into 2 groups (n=8/group): vehicle (10% DMSO in PBS) and BIX 01294 25 mg/kg. Drugs are administered via intraperitoneal injection every 2 days for 21 days. After tumor resection, BIX 01294 reduces recurrence rate from 80% (vehicle) to 30% and prolongs recurrence-free survival from 25±3 days to 45±5 days. Tumor tissues show reduced H3K9me2 (55±6% reduction, IHC) and increased IL-6 expression (2.8±0.3-fold, qPCR) [1]

G9a activity assay (radioactivity-based): Recombinant human G9a (catalytic domain) is incubated in reaction buffer (50 mM Tris-HCl pH 8.0, 5 mM MgCl2, 0.1 mM DTT) containing 10 μM biotinylated H3(1-20) peptide, 2 μM [3H]-SAM (methyl donor), and BIX 01294 (0.1-50 μM). The mixture is incubated at 37°C for 60 minutes. The reaction is terminated by adding 20% TCA, and precipitated peptides are transferred to a filter membrane. Radioactivity (3H-methyl incorporation) is measured via liquid scintillation counting. Inhibition rate is calculated vs. vehicle, and IC50 is derived via nonlinear regression [2]
- GLP activity assay (HTRF-based): Recombinant human GLP is incubated with 50 mM Tris-HCl pH 7.5, 2 mM α-ketoglutarate, 10 μM fluorescently labeled H3K9me1 peptide, and BIX 01294 (0.1-50 μM). After 90 minutes at 37°C, an anti-H3K9me2 Eu-labeled antibody and streptavidin-APC are added. Fluorescence intensity (excitation 320 nm, emission 665 nm) is measured, and IC50 is calculated based on reduced H3K9me2 formation [5]
- SPR binding assay: G9a/GLP SET domains are immobilized on a CM5 sensor chip. BIX 01294 (0.1-20 μM) is injected at a flow rate of 30 μL/min. Binding curves are fitted using a 1:1 Langmuir model to calculate equilibrium dissociation constants (KD) [5]

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

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Breast cancer TIC frequency assay (limiting dilution): MDA-MB-231 cells are treated with BIX 01294 (10 μM) for 72 hours, then serially diluted (100, 50, 20, 10 cells/well) in 96-well plates (Matrigel-coated). After 14 days, wells with tumor spheres (>100 μm) are counted. TIC frequency is calculated using Extreme Limiting Dilution Analysis (ELDA) [1]
- FPASMC migration assay (Transwell): FPASMCs (5×104 cells/well) are serum-starved for 24 hours, resuspended in medium containing BIX 01294 (1-10 μM), and added to the upper chamber of a Transwell insert (8 μm pore). Medium with 10% FBS is added to the lower chamber. After 24 hours, cells on the upper surface are removed; lower surface cells are fixed, stained with 0.1% crystal violet, and counted [3]
- GSC sphere formation assay: U87-derived GSCs (1×103 cells/well) are seeded in ultra-low attachment 6-well plates with stem cell medium (DMEM/F12 + 20 ng/mL EGF + 20 ng/mL bFGF) containing BIX 01294 (2-10 μM). Medium is replaced every 3 days. After 7 days, spheres (>50 μm) are counted, and SFE = (number of spheres/number of seeded cells) × 100% [4]
- H3K9me2 immunofluorescence: HeLa cells are grown on coverslips, treated with BIX 01294 (1-10 μM) for 48 hours, fixed with 4% formaldehyde, and permeabilized with 0.1% Triton X-100. Cells are incubated with anti-H3K9me2 antibody (1:500) overnight at 4°C, followed by Alexa Fluor 488-conjugated secondary antibody (1:1000). Nuclei are stained with DAPI. Fluorescence foci are quantified using ImageJ [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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MDA-MB-231 breast cancer xenograft recurrence model: Female nude mice (6-8 weeks old) are acclimated for 1 week. MDA-MB-231 cells (5×106 cells) are suspended in 50% Matrigel and subcutaneously injected into the right flank. When tumors reach 200-250 mm³, mice are randomized into 2 groups (n=8/group): 1. Vehicle group: Intraperitoneal injection of 0.2 mL 10% DMSO in PBS every 2 days for 21 days; 2. BIX 01294 group: Intraperitoneal injection of BIX 01294 (25 mg/kg, dissolved in 10% DMSO in PBS) every 2 days for 21 days. On day 21, tumors are surgically resected. Mice are monitored for recurrence (tumor >50 mm³) for 60 days. Recurrence-free survival is recorded, and residual tumor tissues are collected for H3K9me2 IHC and IL-6 qPCR [1]
- No animal protocols for BIX 01294 are provided in Literatures [2,3,4,5] [2,3,4,5]

Tissue distribution: In MDA-MB-231 xenograft mice, the tumor/plasma concentration ratio was 2.8 ± 0.3 4 hours after intraperitoneal injection of BIX 01294 (25 mg/kg) (detected by HPLC-MS/MS) [1] - Plasma protein binding: Balanced dialysis showed that the plasma protein binding rate of BIX 01294 was 90 ± 2% (human) and 88 ± 3% (mice), mainly bound to albumin (75%) and α1-acid glycoprotein (15%) [2]

In vitro cytotoxicity to normal cells: BIX 01294 (1-30 μM) showed low toxicity to normal cells: human mammary epithelial cells (MCF-10A, CC50 >30 μM), normal human astrocytes (NHA, CC50 = 28±4 μM), and normal sheep lung endothelial cells (OECs, CC50 = 32±5 μM) showed low toxicity to normal cells after 72 hours of treatment [1,3,4]
- In vivo acute toxicity: BALB/c mice intraperitoneally injected with BIX 01294 (50 mg/kg/day for 7 days) did not experience death or serious toxicity (e.g., lethargy, ataxia). Body weight change was -2±1% (compared to -1±1% in the control group), and serum ALT, AST, BUN, and creatinine levels were all within the normal range [1]. Chronic toxicity in xenograft mice: In the MDA-MB-231 relapse model, BIX 01294 (25 mg/kg every 2 days for 21 days) did not cause histopathological damage to the liver, kidneys, or spleen. Peripheral blood cell counts (white blood cells, platelets) were normal [1].

[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.

6,7-Dimethoxy-2-(4-methyl-1,4-diazacycloheptane-1-yl)-N-[1-(benzyl)-4-piperidinyl]-4-quinazolinamine is a member of the piperidine class of compounds. Mechanism of action: BIX 01294 exerts its biological effects by inhibiting G9a/GLP-mediated H3K9 dimethylation. Reduction of H3K9me2 (a repressive histone marker) relieves the inhibition of target genes: pro-inflammatory genes in breast cancer (IL-6, TNF-α), contractile genes in smooth muscle cells (α-SMA), and differentiation genes in GSCs (GFAP)—ultimately inhibiting tumor stemness/recurrence, smooth muscle proliferation, and promoting GSC differentiation [1,3,4] - Structural basis of inhibition: BIX 01294 binds to the SAM binding pocket of the SET domain of G9a/GLP, blocking the entry of methyl donors. X-ray crystallography shows that BIX 01294 forms hydrogen bonds with Asp1083 (G9a) and Asp1560 (GLP) residues, stabilizing the enzyme's inactive conformation [5]. Preclinical potential: BIX 01294 has shown preclinical application value in the following areas: 1) preventing breast cancer recurrence by targeting TICs; 2) treating pulmonary hypertension by inhibiting smooth muscle proliferation; 3) treating glioma by inducing differentiation of glioma stem cells. Its low toxicity to normal cells supports its further development [1,3,4].

C28H38N6O2

490.640326023102

490.305

935693-62-2

BIX-01294 trihydrochloride;1392399-03-9

25150857

White to off-white solid powder

178.44° C

6.22

1

8

7

36

656

0

OSXFATOLZGZLSK-UHFFFAOYSA-N

InChI=1S/C28H38N6O2/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)

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

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

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)

Solubility in Formulation 1: ≥ 2.75 mg/mL (5.60 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 27.5 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.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (5.60 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 27.5 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.

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Solubility in Formulation 3: ≥ 2.75 mg/mL (5.60 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 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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