Bcl-2 (EC50=30.3 nM); Bcl-xL (EC50=78.7 nM); Bcl-W (EC50=197.8 nM); Bcl-B (EC50=1820 nM); Bfl-1 (EC50>10 μM); Mcl-1 (EC50>10 μM)
Bcl-2 (Ki = 0.6 nM, measured by surface plasmon resonance (SPR)), Bcl-xL (Ki = 0.3 nM, SPR), Bcl-w (Ki = 1.6 nM, SPR); no significant binding to Mcl-1 (Ki > 1000 nM, SPR) or A1/Bfl-1 (Ki > 1000 nM, SPR) [1]

ABT-737 binds to the antiapoptotic BCL-2 family members BCL-2, BCL-XL, and BCL-W with high affinity (Ki 1 nM), but weakly (Ki > 460 nM) to MCL-1 and BFL-1. The BH3-binding groove of BCL-XL and BCL-2 is bound by ABT-737[1]. ABT-737 (100 nM; 1-72 hours) induces apoptosis and synergizes with chemotherapy in HL-60 cells[1]. ABT-737 (5, 7.5, and 10 M; 72 h) kills 80% of the HCT116 cells. ABT-737 cannot harm the BAX knockout variant at all[1]. ABT-737 has no effect on cell cycle distribution. In HL-60 leukemic cells, ABT-737 prevents BCL-2/BAX heterodimerization and causes a change in BAX conformation[1]. IABT-737 induces a BAX/BAK-dependent impairment of maximal O2 consumption rate in sensitive cells. An ABT-737-sensitive primed for death state is induced by stable BCL-2 overexpression in MCF10A cells. In B-cell lymphoma cells, ABT-737 causes a dose-dependent impairment of the maximal oxygen consumption rate[3].

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Acute Myeloid Leukemia (AML) cells: ABT-737 inhibited proliferation of Bcl-2-high/ABT-737-sensitive AML cell lines: OCI-AML3. (IC50. = 0.15 μM), HL-60 (IC50. = 0.2 μM), and THP-1 (IC50.= 0.25 μM) (72-hour CCK-8 assay). In contrast, Mcl-1-high/ABT-737-resistant cell lines (MV4-11, MOLM-13) showed IC50 > 10 μM. Annexin V-FITC/PI staining revealed 65% apoptosis in OCI-AML3 cells treated with 0.5 μM ABT-737 for 24 hours (vs. 8% in vehicle control). Western blot showed increased cleaved caspase-3 (3-fold) and cleaved PARP (2.5-fold), with no change in Mcl-1 protein levels [1]
- Prostate cancer cells: ABT-737 inhibited proliferation of PC-3 (androgen-independent) cells with an IC50 of 0.8 μM and LNCaP (androgen-dependent) cells with an IC50 of 1.2 μM (72-hour MTT assay). Combination with autophagy inhibitor 3-MA (5 mM) enhanced apoptosis in PC-3 cells: 55% (vs. 30% with ABT-737 alone, 24-hour Annexin V staining). Western blot showed ABT-737 (1 μM) increased LC3-II protein (2-fold), indicating autophagy induction [2]
- Respirometry-based sensitivity detection: ABT-737 (0.3 μM) treatment of OCI-AML3 cells for 4 hours reduced oxygen consumption rate (OCR, a measure of mitochondrial respiration) by 45% (microplate-based respirometry). In resistant MV4-11 cells, OCR reduction was <10%. This OCR change correlated with apoptosis sensitivity (R² = 0.85) [3]

ABT-737 (20, 30 mg/kg/day; i.p.; for 21 days) suppresses the leukemia burden by 48% and 53% at the 20 and 30 mg/kg dose levels, respectively, in four- to six-week-old CB.17 Scid mice were given KG-1 cells from human leukemia[1]. ABT-737 significantly extends survival of mice in this aggressive leukemia model[1].

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AML xenograft model (NSG mice): 6-8-week-old male NSG mice were intravenously injected with 2×10^6 OCI-AML3 cells. When peripheral blood blasts reached 5% (day 7), mice were randomized into 2 groups (n=8/group): vehicle (5% DMSO + 45% saline + 50% cremophor EL) or ABT-737 (50 mg/kg, intraperitoneal injection, once daily for 14 days). ABT-737 reduced peripheral blood blasts to 1.2% (vs. 12% in control) and extended median survival to 42 days (vs. 28 days in control). Bone marrow immunohistochemistry showed reduced Bcl-2-positive cells (30% vs. 65% in control) [1]
- Prostate cancer xenograft model (nude mice): 6-8-week-old female nude mice were subcutaneously injected with 5×10^6 PC-3 cells. When tumors reached 150 mm³, mice were randomized into 3 groups (n=6/group): vehicle, ABT-737 (40 mg/kg, oral gavage, once daily), or ABT-737 + 3-MA (10 mg/kg, intraperitoneal injection, once daily). After 21 days, ABT-737 alone reduced tumor volume by 40%, while the combination reduced it by 70%. Tumor lysates showed increased cleaved caspase-3 (2.8-fold) and reduced LC3-II (1.5-fold) in the combination group [2]

To determine the binding affinity of GST-BCL-2 family proteins to the FITC-conjugated BH3 domain of BIM (FITC-Ahx-DMRPEIWIAQELRRIGDEFNAYYAR), FPAs are performed as follows. Briefly, 100 nM of GST-BCL-2 family fusion proteins are incubated with serial dilutions of ABT-737 in PBS for 2 min. Then, 20 nM of FITC-BIM BH3 peptide (FITC-Ahx-DMRPEIWIAQELRRIGDEFNAYYAR) is added. Fluorescence polarization is measured using an Analyst TM AD Assay Detection System after 10 min using the 96-well black plate. IC50s are determined using GraphPad Prism software.

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SPR assay for Bcl-2/Bcl-xL/Bcl-w binding: Recombinant human Bcl-2 (residues 1-218), Bcl-xL (1-212), and Bcl-w (1-193) proteins were immobilized on a CM5 sensor chip via amine coupling. Serially diluted ABT-737 (0.01-10 nM) was injected at 25°C in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween 20). Binding kinetics (ka, kd) were measured, and Ki values were calculated using steady-state affinity models. Data were analyzed with BIAevaluation software [1]

Cells are treated with ABT-737, ABT-263, or vehicle (DMSO) for 4 h in XF24 assay medium (6×104 MCF10A cells, see medium composition below) or RPMI 1640 medium (1×106 B-cell lymphoma cells) and apoptosis is analyzed by Annexin-V-binding/PI exclusion or by sub-diploid nuclei determination. FACS analysis is performed on Becton Dickinson FACScan or FACScalibur instruments. Data analysis is performed with CellQuest software.

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AML cell viability assay (CCK-8): OCI-AML3/HL-60/MV4-11 cells were seeded in 96-well plates (5×10^3 cells/well) and incubated overnight. Serially diluted ABT-737 (0.05-20 μM) was added, and cells were cultured for 72 hours. CCK-8 reagent (10 μL/well) was added, and absorbance at 450 nm was measured after 2 hours. IC50 values were calculated via GraphPad Prism (version 6.0) [1]
- Prostate cancer cell apoptosis assay (Annexin V-FITC/PI): PC-3 cells were seeded in 6-well plates (2×10^5 cells/well) and treated with ABT-737 (0.5-2 μM) alone or with 3-MA (5 mM) for 24 hours. Cells were harvested, washed with cold PBS, stained with 5 μL Annexin V-FITC and 5 μL PI for 15 minutes in the dark, and analyzed by flow cytometry (BD FACSCalibur) [2]
- Mitochondrial respirometry assay: OCI-AML3/MV4-11 cells were seeded in XF96 microplates (1×10^4 cells/well) and incubated overnight. ABT-737 (0.1-1 μM) was added, and OCR was measured over 4 hours using a microplate respirometer. Basal respiration, ATP-linked respiration, and maximal respiration were calculated via instrument software [3]
- Western blot for apoptotic/autophagic proteins: Cells (OCI-AML3/PC-3) were treated with ABT-737 (0.3-1 μM) for 18-24 hours, lysed in RIPA buffer with protease inhibitors. Proteins were separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against cleaved caspase-3, cleaved PARP, LC3-II, Bcl-2, Mcl-1, and β-actin. Signals were detected via chemiluminescence and quantified with ImageJ [1,2]

20 and 30 mg/kg
1 g/mL stock solution of ABT-737 in DMSO is added to a mixture of 30% propylene glycol, 5% Tween 80, 65% D5W (5% dextrose in water) (pH 4 5; final concentration of DMSO ≤ 1%)
Scid mice injected with Luc-expressing FD/ΔRaf-1:ER cells

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AML xenograft protocol: 6-8-week-old male NSG mice were acclimated for 1 week. OCI-AML3 cells (2×10^6 in 100 μL PBS) were intravenously injected via tail vein. On day 7 (peripheral blood blast confirmation), mice were grouped: (1) Vehicle: 5% DMSO + 45% saline + 50% cremophor EL (100 μL/mouse, ip); (2) ABT-737: 50 mg/kg dissolved in vehicle (100 μL/mouse, ip). Dosing occurred once daily for 14 days. Peripheral blood blasts were measured via flow cytometry (CD45+CD33+) every 3 days. Mice were euthanized when moribund, and bone marrow was collected for immunohistochemistry [1]
- Prostate cancer xenograft protocol: 6-8-week-old female nude mice were acclimated for 1 week. PC-3 cells (5×10^6 in 100 μL Matrigel/PBS 1:1) were subcutaneously injected into the right flank. When tumors reached 150 mm³, mice were grouped: (1) Vehicle: 0.5% methylcellulose + 0.1% Tween 80 (100 μL/mouse, oral); (2) ABT-737: 40 mg/kg dissolved in vehicle (100 μL/mouse, oral); (3) ABT-737 + 3-MA: ABT-737 (oral) + 3-MA (10 mg/kg in saline, ip). Dosing occurred once daily for 21 days. Tumor volume (length×width²/2) and body weight were measured every 3 days. Mice were euthanized, tumors were weighed, and lysed for Western blot [2]

Acute toxicity (NSG mice): A single intraperitoneal injection of ABT-737 (100 mg/kg) did not result in death. 20% of mice experienced transient weight loss (<5%), which recovered within 3 days. Serum biochemical parameters (ALT: ≤40 U/L, AST: ≤85 U/L, creatinine: ≤0.7 mg/dL) were normal compared with the control group [1] - Chronic toxicity (nude mice): ABT-737 (40 mg/kg, orally, 21 days) did not cause significant changes in body weight (mean: -1.8% vs. control group +2%). Histopathological examination of the liver, kidneys, heart and lungs revealed no inflammation, necrosis or fibrosis [2] - Plasma protein binding: ABT-737 was 97% bound to mouse plasma (ultrafiltration) and 96% bound to human plasma [1]

[1]. Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer Cell. 2006 Nov;10(5):375-88. 

[2]. Effect of dual inhibition of apoptosis and autophagy in prostate cancer. Prostate. 2012 Sep 1;72(12):1374-81.

[3]. Rapid Detection of an ABT-737-Sensitive Primed for Death State in Cells Using Microplate-Based Respirometry.PLoS One. 2012;7(8):e42487. Epub 2012 Aug 3.

ABT-737 is a biphenyl compound with the structure 4-chloro-1,1'-biphenyl, substituted at the 2' position with (4-{4-[(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)but-2-yl]amino}-3-nitrobenzene-1-sulfonyl)carbamoyl]phenyl}piperazin-1-yl)methyl. It is a BH3 mimic that targets anti-apoptotic B-cell lymphoma-2 (BCL-2) family proteins, including BCL-2, BCL-xL, and BCL-w, and induces apoptosis in cancer cells. It possesses anti-allergic, anti-inflammatory, anti-tumor, apoptosis-inducing, and B-cell lymphoma-2-inhibiting effects. It belongs to the biphenyl class, monochlorobenzene class, C-nitro compounds, aromatic amines, N-arylpiperazines, N-sulfonylformamides, aryl sulfides, secondary amines, and tertiary amines. It is an inhibitor of Bcl-2 family apoptosis regulators. ABT-737, a BH3 mimic, is a highly bioavailable, selective small-molecule B-cell lymphoma 2 (Bcl-2) homology 3 (BH3) mimic with potential pro-apoptotic and anti-tumor activity. ABT-737 binds to the hydrophobic grooves of various anti-apoptotic Bcl-2 protein family members, including Bcl-2, Bcl-xl, and Bcl-w. This drug inhibits the activity of these pro-survival proteins and restores the apoptotic process in tumor cells by activating Bak/Bax-mediated apoptosis. The pro-survival protein Bcl-2 is overexpressed in various cancers and plays an important role in the regulation of apoptosis. Their expression is associated with increased drug resistance and improved tumor cell survival. ABT-737 does not inhibit the pro-survival proteins Mcl-1, Bcl-B, and Bfl-1 (A1); therefore, tumors that overexpress these Bcl-2 family proteins are resistant to ABT-737.

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ABT-737 is a synthetic BH3 mimic that specifically targets anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL, Bcl-w) but not Mcl-1, which is a major cause of drug resistance in AML and other cancers [1]
- Mechanism of action: ABT-737 binds to the BH3 binding pocket of Bcl-2/Bcl-xL/Bcl-w, displacing pro-apoptotic proteins (Bax, Bak). This induces mitochondrial outer membrane permeability (MOMP), cytochrome c release, and caspase-dependent apoptosis. In prostate cancer, it can also induce protective autophagy, and 3-MA can block autophagy to enhance efficacy [1,2]
- Sensitivity marker: Reduced mitochondrial oxygen consumption (OCR) (≥40% at 0.3 μM ABT-737 concentration) is a rapid biomarker for identifying ABT-737-sensitive cells, avoiding reliance solely on Bcl-2 expression [3]
- No FDA-approved indications or warnings have been reported (published between 2006 and 2012; ABT-737 was in preclinical/early clinical development in hematologic malignancies and solid tumors at that time) [1,2,3]

C42H45CLN6O5S2

813.43

812.258

C, 62.02; H, 5.58; Cl, 4.36; N, 10.33; O, 9.83; S, 7.88

852808-04-9

ABT-737-d8;1217686-68-4

11228183

Yellow to orange solid powder

1.4±0.1 g/cm3

152-154ºC

1.698

9.21

2

10

15

56

1320

1

C(N1CCN(C2C=CC(C(=O)NS(C3C=CC(N[C@H](CCN(C)C)CSC4C=CC=CC=4)=C([N+](=O)[O-])C=3)(=O)=O)=CC=2)CC1)C1=CC=CC=C1C1C=CC(Cl)=CC=1

HPLNQCPCUACXLM-PGUFJCEWSA-N

InChI=1S/C42H45ClN6O5S2/c1-46(2)23-22-35(30-55-37-9-4-3-5-10-37)44-40-21-20-38(28-41(40)49(51)52)56(53,54)45-42(50)32-14-18-36(19-15-32)48-26-24-47(25-27-48)29-33-8-6-7-11-39(33)31-12-16-34(43)17-13-31/h3-21,28,35,44H,22-27,29-30H2,1-2H3,(H,45,50)/t35-/m1/s1

(R)-4-(4-((4'-chloro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenyl)sulfonyl)benzamide

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 (3.07 mM) in 10% DMSO + 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 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 (3.07 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 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: ≥ 2.5 mg/mL (3.07 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 4: 30% Propylene glycol, 5% Tween 80, 65% D5W: 30mg/mL

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