Bcl-2 (Ki = 1.3 nM); Bcl-xL (Ki = 520 nM); Bcl-2 (Ki = 3.9 nM); Bcl-xL (Ki = 186 nM)
S55746 is located in BCL-2's hydrophobic groove. S55746 causes the externalization of phosphatidylserine, caspase-3 activation, and PARP cleavage in a panel of hematological cell lines, which are signs of apoptosis. S55746 exhibits much weaker activity in H146 (IC50 = 1.7 μM), a BCL-XL-dependent cell line, which expresses high levels of BCL-XL and low levels of BCL-2. S55746 potently induces RS4;11 cell killing after 72 hours of treatment with an IC50 of 71.6 nM. On BCL-XL-dependent cells, like platelets, it has no cytotoxic activity[1].

S55746 is located in BCL-2's hydrophobic groove. S55746 causes the externalization of phosphatidylserine, caspase-3 activation, and PARP cleavage in a panel of hematological cell lines, which are signs of apoptosis. S55746 exhibits much weaker activity in H146 (IC50 = 1.7 μM), a BCL-XL-dependent cell line, which expresses high levels of BCL-XL and low levels of BCL-2. S55746 potently induces RS4;11 cell killing after 72 hours of treatment with an IC50 of 71.6 nM. On BCL-XL-dependent cells, like platelets, it has no cytotoxic activity[1].

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S55746 potently induced cell death in the BCL-2-dependent acute lymphoblastic leukemia cell line RS4;11 with an IC50 of 71.6 nM after 72 hours of treatment. In contrast, it exhibited much weaker activity (IC50 1.7 µM) in the BCL-XL-dependent small cell lung cancer cell line H146.[1]
S55746 induced apoptosis in RS4;11 cells in a concentration-dependent manner, as evidenced by phosphatidylserine externalization, caspase-3 activation, and PARP cleavage.[1]
Co-immunoprecipitation experiments demonstrated that S55746 disrupted the BCL-2/BAX complex in RS4;11 cells and in BCL-2-overexpressing HeLa cells in a concentration-dependent manner, without affecting the BCL-XL/BAX complex in BCL-XL-overexpressing cells. In contrast, the dual BCL-2/BCL-XL inhibitor ABT-263 disrupted both complexes.[1]
Apoptosis induction by S55746 was dependent on the pro-apoptotic effector proteins BAX and BAK. BAX-deficient RS4;11 cells showed markedly reduced PARP cleavage and a ~50-fold decrease in sensitivity to S55746 (IC50 = 2.7 µM vs. 0.057 µM in control cells).[1]
In IL-3-dependent murine FL5.12 cells, S55746 strongly induced apoptosis upon IL-3 withdrawal in BCL-2-expressing cells but had only a minor effect in BCL-XL-expressing cells.[1]
Isolated platelets from healthy volunteers, which are dependent on BCL-XL for survival, were insensitive to S55746 (EC50 > 3 µM).[1]
In a panel of non-Hodgkin lymphoma cell lines, S55746 showed activity (IC50 < 1 µM) in 6 out of 11 Diffuse Large B-Cell Lymphoma (DLBCL) lines and 2 out of 5 Mantle Cell Lymphoma (MCL) lines. All Burkitt Lymphoma cell lines tested had IC50 values above 10 µM.[1]
In primary Chronic Lymphocytic Leukemia (CLL) cells from 7 patients, S55746 induced rapid apoptosis with EC50 values in the low nanomolar range (4.4 to 47.2 nM after 4 hours). Treatment led to caspase-9 and caspase-3 processing and PARP cleavage.[1]
In primary Mantle Cell Lymphoma (MCL) cells from 8 patients, S55746 potently induced apoptosis with EC50 values ranging from 2.5 to 110 nM after 24 hours.[1]

S55746 exhibits strong anti-tumor efficacy in two hematological xenograft models (RS4;11 and Toledo models) when given orally daily to mice, with no weight loss and no behavioral changes[1].

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In RS4;11 xenograft-bearing mice, a single oral dose of S55746 at 25 and 100 mg/kg induced caspase-3 activity in tumors approximately 11 and 28 times higher than in vehicle-treated animals, respectively, 16 hours post-dosing.[1]
S55746 treatment at 25 and 100 mg/kg did not induce platelet loss in vivo, whereas ABT-263 treatment at 100 mg/kg caused a strong decrease in platelet counts.[1]
In the RS4;11 xenograft model, daily oral treatment with S55746 for 7 consecutive days at 25, 50, and 100 mg/kg induced significant dose-dependent anti-tumor activity and tumor growth inhibition. Complete regression was observed in all animals treated at 100 mg/kg.[1]
In the Toledo (DLBCL) xenograft model, oral treatment with S55746 five times a week for 3 weeks at 200 and 300 mg/kg induced significant tumor growth inhibition (13% and 2% T/C, respectively), with efficacy similar to ABT-199 at 200 mg/kg.[1]
Body weight did not significantly differ in treatment groups compared to the vehicle control group in both xenograft models, even at doses up to 300 mg/kg.[1]

Fluorescence Polarization (FP) assays were performed in low-binding 384-well plates in a buffer containing HEPES, NaCl, Tween 20, and DMSO, in the presence of a fluorescein-labeled PUMA peptide. Proteins (BCL-2, BCL-XL, MCL-1, BFL-1) were used at final concentrations of 10-20 nM. Assay plates were incubated for 2 hours at room temperature, and fluorescence polarization was measured. Inhibition curves were generated, and IC50 values were determined using a 4-parameter logistic model, from which inhibition constants (Ki) were calculated.[1]
Surface Plasmon Resonance Affinity In Solution (SPR AIS) experiments were performed at 30°C in HBS-EP buffer supplemented with TCEP, glycerol, and DMSO. A PUMA BH3 peptide was immobilized on a sensor chip. Compounds were pre-incubated with BCL-2 protein and then injected over the chip surface. Affinity (KD) was evaluated using specific analysis software.[1]
Isothermal Titration Calorimetry (ITC) experiments were performed by titrating BCL-2 protein from a syringe into a cell containing the compound. Both protein and compound solutions were carefully matched in buffer (HEPES, NaCl, glycerol, MTG) containing DMSO. The experiment consisted of 13 injections at 25°C. Data were analyzed using single-site binding models to determine binding enthalpy (ΔH) and stoichiometry (N).[1]

Apoptosis induction in RS4;11 cells subjected to a 2-hour treatment with S55746 at various concentrations. PI and annexin V-FITC labeled cells were examined using flow cytometry.

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For apoptosis analysis in cell lines (e.g., RS4;11, THP-1), cells were treated with compounds, then washed and incubated with Annexin V-FITC (or Annexin V-APC) and propidium iodide (or DAPI) in binding buffer. After incubation, samples were analyzed by flow cytometry to determine the percentage of apoptotic cells.[1]
For primary CLL cells, cells were treated with S55746 for 4 hours, then stained with Annexin V-FITC and propidium iodide and analyzed by flow cytometry to calculate EC50 values.[1]
For primary MCL cells, CD19+ cells were cultured with S55746 for 24 hours. Cell death was assessed by Annexin V-FITC staining combined with analysis of altered cellular morphology (lower forward scatter) via flow cytometry.[1]
For platelet apoptosis, isolated human platelets were treated with S55746 for 3 hours, stained with CD41-PE and Annexin-FITC, and analyzed by flow cytometry.[1]
Cell viability was assessed using the CellTiter-Glo luminescent assay. Cells were seeded in 96-well plates, treated with serial dilutions of compounds, and after incubation, CellTiter-Glo reagent was added. Luminescence was measured, and IC50 values were calculated.[1]
For co-immunoprecipitation assays, cells were treated with compounds, lysed, and immunoprecipitated with an anti-BCL-2 or anti-Flag antibody. Immunoprecipitates and cell lysates were then analyzed by western blotting for proteins like BAX and BCL-2.[1]
Western blotting was used to analyze protein expression and cleavage (e.g., PARP, caspases, BCL-2 family proteins). Cells were lysed, proteins were separated by SDS-PAGE, transferred to membranes, and probed with specific antibodies.[1]

For efficacy and pharmacodynamic studies, female SCID/beige mice were implanted subcutaneously with RS4;11 or Toledo tumor cells suspended in a 1:1 mixture of cold DPBS and Matrigel.[1]
When tumors reached approximately 200 mm³ (efficacy) or 300 mm³ (pharmacodynamics), mice were randomized into treatment groups.[1]
S55746 was formulated in PEG300/ethanol/water (40/10/50, v/v/v). The compound was administered via oral gavage at a volume of 10 ml/kg.[1]
In the RS4;11 efficacy study, mice were treated orally with S55746 daily for 7 consecutive days at doses of 25, 50, and 100 mg/kg.[1]
In the Toledo efficacy study, mice were treated orally with S55746 five times a week for 3 weeks at doses of 200 and 300 mg/kg.[1]
For pharmacodynamic studies (caspase-3 activity, platelet counts), mice received a single oral dose of S55746 (25 or 100 mg/kg) or vehicle. Tumors and blood were collected 16 hours post-dosing for analysis.[1]
Body weights and tumor volumes were measured regularly throughout the studies.[1]

In vivo, in xenograft models, daily oral doses of S55746 up to 300 mg/kg did not cause significant weight loss or behavioral changes, with no significant difference compared to the vector control group. [1] Unlike ABT-263, which causes severe thrombocytopenia, S55746 did not induce thrombocytopenia in mice at doses of 25 and 100 mg/kg. [1] In vitro, S55746 did not induce apoptosis in isolated platelets from healthy volunteers (EC50 > 3 µM), indicating that it does not have BCL-XL-mediated platelet toxicity. [1]

[1]. S55746 is a novel orally active BCL-2 selective and potent inhibitor that impairs hematological tumor growth. Oncotarget. 2018 Apr 13;9(28):20075-20088.

Bcl-2 inhibitor BCL201 is a selective anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) inhibitor with potential pro-apoptotic and anti-tumor activity. After administration, Bcl-2 inhibitor BCL201 binds to Bcl-2 and inhibits its activity, thereby restoring the apoptotic process of tumor cells. Bcl-2 protein is overexpressed in a variety of cancers and plays an important role in the negative regulation of apoptosis; its expression is associated with increased drug resistance and increased tumor cell survival.

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S55746 is a novel, orally bioavailable BH3 mimic compound that selectively inhibits the anti-apoptotic protein BCL-2. [1]
It occupies the hydrophobic groove (S1/S2/S3 pocket) of BCL-2 and forms a hydrogen bond with the carbonyl group of the main chain of Ala149 residues. Its binding mode is different from that of ABT-199 (Veneclax), but partially overlaps. [1]
This drug is intended to treat BCL-2-dependent hematologic malignancies, such as certain lymphomas and leukemias. [1]
S55746 is currently in Phase I clinical trials for hematologic malignancies (ClinicalTrials.gov registration numbers: NCT02920697, NCT02920441, NCT02603445). [1]

C43H42N4O6

710.82

710.31

C, 72.66; H, 5.96; N, 7.88; O, 13.50

1448584-12-0

S55746 hydrochloride;1448525-91-4

71654876

White to off-white solid powder

5.8

1

7

7

53

1250

1

C1CCN2C(=C(C=C2C3=CC4=C(C=C3C(=O)N5CC6=CC=CC=C6C[C@H]5CN7CCOCC7)OCO4)C(=O)N(C8=CC=CC=C8)C9=CC=C(C=C9)O)C1

VYXJULKGMXJVGI-XIFFEERXSA-N

InChI=1S/C43H42N4O6/c48-34-15-13-32(14-16-34)47(31-10-2-1-3-11-31)43(50)37-23-39(45-17-7-6-12-38(37)45)35-24-40-41(53-28-52-40)25-36(35)42(49)46-26-30-9-5-4-8-29(30)22-33(46)27-44-18-20-51-21-19-44/h1-5,8-11,13-16,23-25,33,48H,6-7,12,17-22,26-28H2/t33-/m0/s1

N-(4-hydroxyphenyl)-3-[6-[(3S)-3-(morpholin-4-ylmethyl)-3,4-dihydro-1H-isoquinoline-2-carbonyl]-1,3-benzodioxol-5-yl]-N-phenyl-5,6,7,8-tetrahydroindolizine-1-carboxamide

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: 5.75 mg/mL (8.09 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 sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 57.5 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 2: ≥ 2.5 mg/mL (3.52 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 3: ≥ 2.5 mg/mL (3.52 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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 (Please use freshly prepared in vivo formulations for optimal results.)