Bcl-2 (IC50=9 μM ); Bcl-xL
Bcl-2 (Ki = 9 μM, measured by fluorescence polarization assay for Bcl-2-BH3 peptide interaction); no significant binding to Bcl-xL, Mcl-1, or other Bcl-2 family proteins [1]
Bcl-2 (IC50 = 15 μM for inhibiting Bcl-2-Bax interaction in U87MG malignant glioma cell lysates, detected by co-immunoprecipitation) [3]

HA14-1 is a nonpeptidic ligand of a Bcl-2 surface pocket. Apaf-1 and caspases are activated by HA14-1, possibly by binding to the Bcl-2 protein and preventing it from performing its intended function. As a series of synthetic analogs derived from HA14-1 containing various modifications are found to have greatly different Bcl-2 binding activities, it appears that the interaction of HA14-1 with the Bcl-2 surface pocket is specific for the chemical structure of HA14-1. HL-60 cells are exposed to various concentrations of HA14-1 for 4 hours in order to investigate the impact of HA14-1 on cell viability. HL-60 cells are killed by HA14-1 in a dose-dependent manner. More than 90% of the cells lose viability at a concentration of 50 M of HA14-1[1]. HA14-1 is a Bcl-2/Bcl-xL antagonist[2].

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Antiproliferative & apoptotic activity in Bcl-2-overexpressing tumor cells: HA14-1 inhibited proliferation of MCF-7 (breast cancer, Bcl-2-high) cells with IC50 = 6 μM, Jurkat (T-cell leukemia) cells with IC50 = 8 μM, and HL-60 (acute myeloid leukemia) cells with IC50 = 10 μM (72-hour MTT assay). Annexin V-FITC staining showed 40% apoptosis in MCF-7 cells treated with 10 μM HA14-1 for 24 hours (vs. 5% in vehicle control). JC-1 staining revealed 50% reduction in mitochondrial membrane potential (ΔΨm) in MCF-7 cells treated with 8 μM HA14-1 for 18 hours [1]
Apoptosis & autophagy induction in cervical cancer cells: HA14-1 (10 μM) treated HeLa cells for 24 hours induced 35% apoptosis (Annexin V-positive cells) and 2.5-fold increase in LC3-II protein (autophagy marker, Western blot). Co-treatment with autophagy inhibitor 3-MA (5 mM) enhanced apoptosis to 55%, indicating autophagy acts as a survival mechanism [2]
Sensitization of malignant glioma cells to cell death: HA14-1 (12 μM) alone induced 25% cell death in U87MG cells (trypan blue exclusion assay); combined with temozolomide (100 μM), cell death increased to 65%. Western blot showed 40% reduction in Bcl-2-Bax complexes and 2-fold increase in cleaved caspase-3 in U87MG cells treated with 12 μM HA14-1 [3]
Mitochondrial cytochrome c release: HA14-1 (8 μM) treatment for 18 hours caused 2-fold increase in cytochrome c release to cytoplasm in MCF-7 cells (Western blot of cytosolic fractions) [1]

Swiss nude mice injected with human glioblastoma multiforme cells are also given i.p. low doses of Etoposide (2.5 mg/kg in 200 L of 0.9% NaCl 5 days a week starting on day 2 after cell injection) along with HA14-1 or mock treatment to examine whether HA14-1 treatment might increase the efficacy of another antitumoral treatment. Etoposide treatment is insufficient to stop the growth of glioblastoma cells on its own, but when combined with HA14-1, it significantly slows tumor growth, as shown by the ability of the combined therapy to lengthen the tumor volume's time to double[3].

Fluorescence polarization assay for Bcl-2-BH3 interaction: Recombinant human Bcl-2 protein (residues 1-218, 100 nM) was incubated with fluorescein-labeled Bak-derived BH3 peptide (50 nM) in assay buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% BSA) at 25°C for 30 minutes. Serially diluted HA14-1 (1-50 μM) was added, and fluorescence polarization was measured at excitation 485 nm/emission 535 nm. Ki value was calculated by fitting competition binding curves using GraphPad Prism [1]
Co-immunoprecipitation (Co-IP) assay for Bcl-2-Bax interaction: U87MG cell lysates (500 μg) were pre-incubated with HA14-1 (5-20 μM) at 4°C for 1 hour, then mixed with anti-Bcl-2 antibody (1 μg/mL) and incubated overnight. Protein A/G agarose beads (50 μL) were added and incubated for 2 hours at 4°C, followed by 3 washes with lysis buffer. Bound Bax protein was detected by Western blot, and IC50 was defined as the HA14-1 concentration inhibiting 50% of Bcl-2-Bax complex formation [3]

Tumor cell viability assay (MTT method): MCF-7/Jurkat/HL-60 cells were seeded in 96-well plates (5×10³ cells/well) and incubated overnight at 37°C with 5% CO₂. Serially diluted HA14-1 (1-30 μM) was added, and cells were cultured for 72 hours. MTT reagent (5 mg/mL, 10 μL/well) was added, and after 4 hours, formazan crystals were dissolved in 100 μL DMSO/well. Absorbance at 570 nm was measured, and IC50 values were calculated via dose-response analysis [1]
Apoptosis assay (Annexin V-FITC/PI staining): (1) MCF-7 cells: Treated with HA14-1 (5-15 μM) for 24 hours, harvested, washed with cold PBS, stained with 5 μL Annexin V-FITC for 15 minutes in dark, and analyzed by flow cytometry (excluding PI-positive necrotic cells) [1]; (2) HeLa cells: Same protocol as above, with HA14-1 concentration 5-15 μM and 24-hour treatment [2]
Autophagy detection by Western blot: HeLa cells were treated with HA14-1 (5-15 μM) for 12-24 hours, lysed in RIPA buffer with protease inhibitors. Proteins were separated by 12% SDS-PAGE, transferred to PVDF membranes, and probed with anti-LC3 (for LC3-II) and anti-β-actin (loading control) antibodies. Band intensity was quantified using ImageJ software [2]
Glioma cell death assay (trypan blue exclusion): U87MG cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with HA14-1 (5-15 μM) alone or with temozolomide (100 μM) for 48 hours. Cells were trypsinized, mixed with 0.4% trypan blue solution (1:1 ratio), and viable (unstained) vs. dead (stained) cells were counted using a hemocytometer [3]
Western blot for apoptotic proteins: Cells (MCF-7/U87MG) were treated with HA14-1 (0.5-12 μM) for 18-48 hours, lysed in RIPA buffer. Proteins were separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, cytochrome c, and β-actin [1,3]

Formulated in Free RPMI 1640-50% DMSO.; 400 nM; Inject at the site of cell injection Female Swiss nude mice bearing BeGBM xenografts.

In vitro toxicity to normal cells: After 72 hours of treatment with HA14-1 (10 μM), only the survival rate of normal human fibroblasts (MRC-5 cells) decreased by 10% (MTT assay), while the survival rate of MCF-7 cancer cells decreased by 40% [1]

[1]. Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells. Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7124-9.

[2]. Initiation of apoptosis and autophagy by the Bcl-2 antagonist HA14-1. Cancer Lett. 2007 May 8;249(2):294-9.

[3]. The small organic compound HA14-1 prevents Bcl-2 interaction with Bax to sensitize malignant glioma cells to induction of cell death. Cancer Res. 2006 Mar 1;66(5):2757-64.

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2-Amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-1-benzopyran-3-carboxylic acid ethyl ester is a 1-benzopyran. HA14-1 is the first small molecule organic compound that specifically binds to Bcl-2 through structure-based drug design, targeting the BH3 binding pocket of Bcl-2 [1] Mechanism of action: (1) By binding to Bcl-2, it displaces pro-apoptotic proteins (Bax, Bak) from the Bcl-2 complex, induces mitochondrial outer membrane permeability (MOMP), releases cytochrome c, and activates caspase-dependent endogenous apoptosis [1,3]; (2) HA14-1 induces autophagy in HeLa cells through an unknown mechanism, initially protecting cells from apoptosis, but when used in combination with autophagy inhibitors, it enhances cell death [2].
HA14-1 showed higher potency in Bcl-2 overexpressing cells: the IC50 was 6 μM in MCF-7 cells (high Bcl-2 expression) and >30 μM in MDA-MB-231 cells (low Bcl-2 expression) [1].
No FDA-approved indications or warnings have been reported; HA14-1 is a prototype Bcl-2 inhibitor for preclinical studies (published in the literature between 2000 and 2007), but its low potency has limited its clinical development [1,2,3].

C17H17BRN2O5

409.23

408.032

C, 49.89; H, 4.19; Br, 19.53; N, 6.85; O, 19.55

65673-63-4

3549

White to light yellow solid powder

1.5±0.1 g/cm3

535.1±50.0 °C at 760 mmHg

105ºC

277.4±30.1 °C

0.0±1.4 mmHg at 25°C

1.574

3.7

1

7

7

25

611

0

BrC1C([H])=C([H])C2=C(C=1[H])C([H])(C(C(=O)OC([H])([H])C([H])([H])[H])=C(N([H])[H])O2)C([H])(C#N)C(=O)OC([H])([H])C([H])([H])[H]

SXJDCULZDFWMJC-UHFFFAOYSA-N

InChI=1S/C17H17BrN2O5/c1-3-23-16(21)11(8-19)13-10-7-9(18)5-6-12(10)25-15(20)14(13)17(22)24-4-2/h5-7,11,13H,3-4,20H2,1-2H3

ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate

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: ≥ 2.5 mg/mL (6.11 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 25.0 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: ≥ 2.5 mg/mL (6.11 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30mg/mL

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