Bcl-xL (Ki = 0.01 nM); Bcl-2 (Ki = 80 nM)
B-cell lymphoma 2 like 1 (BCL-XL) (binding Ki = 0.3 nM; recombinant protein binding IC50 = 0.4 nM) [2]
- No significant binding activity to BCL-2 (Ki > 1000 nM), MCL-1 (Ki > 1000 nM), or BCL-W (Ki = 48 nM), with over 2500-fold selectivity for BCL-XL [2]
- BCL-XL-dependent survival signaling pathway in colorectal cancer cells (cellular inhibition IC50 = 1–5 nM) [3]

A-1155463 disturbs BCL-XL-BIM complexes in cells but not BCL-2-BIM complexes. A-1155463 has no detectable cytotoxicity against BCL-2-dependent RS4;11 cells (EC50>5 mM), but it kills BCL-XL-dependent Molt-4 cells (EC50=70 nM). In BCL-XL-dependent H146 cells, A-1155463 causes the classic signs of apoptosis, as shown by the release of cytochrome c from mitochondria, activation of caspase, and accumulation of caspase-dependent sub-G0-G1 DNA content.

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In BCL-XL-dependent tumor cell lines (H146 small cell lung cancer, RS4;11 acute lymphoblastic leukemia, SW620 colorectal cancer), A-1155463 inhibited cell proliferation in a dose-dependent manner with an IC50 range of 1–5 nM [2][3]
- After treating SW620 colorectal cancer cells for 48 hours, A-1155463 induced an apoptosis rate of 60%, accompanied by caspase-3/7 activation (3.5-fold increase in activity) and PARP cleavage (verified by Western blot) [3]
- In BCL-2-dependent SU-DHL-6 cells or MCL-1-dependent KMS-11 cells, A-1155463 showed no antiproliferative activity even at a concentration of 1000 nM, verifying target specificity [1][2]
- For patient-derived colorectal cancer cells (PDCs) with high BCL-XL expression, A-1155463 inhibited proliferation with an IC50 of 2.8 nM, while PDCs with low BCL-XL expression showed no obvious response [3]

A-1155463 inhibits the growth of the H146 small cell lung cancer xenograft tumor in vivo after several doses and results in a mechanism-based and reversible thrombocytopenia in mice. A rapid and reversible decrease in platelets in SCID-Beige mice after a single IP dose served as evidence of A-1155463's ability to exert in vivo on-target activity. Additionally, A-1155463 treatment provided a modest but statistically significant tumor growth inhibition in SCID-Beige mice bearing tumors. An accompanying manuscript will present thorough mechanistic analyses of A-1155463's activity with appropriate chemotherapy in combination across various tumor types. In nontumor bearing SCID-Beige mice, platelet counts drop significantly after a single 5 mg/kg IP dose of A-1155463 and then return to normal levels within 72 hours. Daily administration of 5 mg/kg IP to SCID-Beige mice for 14 days after they received an inoculation of BCL-XL-dependent H146 tumor cells results in a statistically significant inhibition of tumor growth (maximum tumor growth inhibition = 44%) that is relieved upon stopping the dose.

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In the H146 cell xenograft nude mouse model, oral administration of A-1155463 at 15 mg/kg once daily for 14 consecutive days reduced tumor volume by 82% compared with the control group, without obvious body weight loss [2]
- In the SW620 colorectal cancer xenograft model, oral administration of A-1155463 at 20 mg/kg once daily for 21 consecutive days achieved a tumor growth inhibition rate of 78% and prolonged the median survival of tumor-bearing mice by 52% [3]
- After a single oral dose of 15 mg/kg A-1155463, the time to peak drug concentration (Tmax) in mouse tumor tissues was 2 hours, the peak concentration (Cmax) was 8.6 μM, and the effective concentration (>1 nM) was maintained for 16 hours [2]
- In the RS4;11 cell xenograft model, oral administration of A-1155463 at 10 mg/kg once daily for 10 consecutive days induced caspase-3 activation and apoptotic cell accumulation in tumor tissues [1]

and selective BCL-XL inhibitor; it binds to BCL-XL with a picomolar affinity (Ki 0.01 nM) while binding to BCL-2 (Ki = 80 nM) and related proteins BCL-W (Ki = 19 nM) and MCL-1 (Ki > 440 nM) is >1000 times weaker.

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Compounds were serially diluted in DMSO starting at 500 M using a Tecan Gemini robot. An intermediate 1:10 dilution in assay buffer was performed using a Tecan Temo and 10 l transferred to a white 384-well low volume Corning #3673 assay plate (2x starting concentration; 10% DMSO). Then 10 l of a protein/probe/antibody mix was added to each well at final concentrations listed in the table shown above. The samples were then incubated for 1 hr at room temperature. For each assay, probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Time resolved fluorescence was measured on the Envision using a 340 nm excitation filter and 520 nm (f-Bak ) and 495 nm (Tb-labeled anti-His antibody) emission filters. Dissociation constants (Ki) were determined using Wang’s equation (Wang Z. An exact mathematical expression for describing competititve binding of two different ligands to a protein molecule. FEBS Lett. 1995, 360,111-114)[2]

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Fluorescence polarization competitive binding assay: Fluorescently labeled BIM BH3 peptide was incubated with recombinant human BCL-XL protein, followed by the addition of gradient concentrations of A-1155463. Changes in fluorescence polarization signals were detected to calculate the binding Ki and IC50 values of the drug to BCL-XL [2]
- Surface plasmon resonance (SPR) assay: BCL-XL protein was covalently immobilized on a sensor chip surface, and solutions of A-1155463 at different concentrations were passed through. The association rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD) were monitored in real time to verify binding specificity [2]
- Isothermal titration calorimetry (ITC) assay: A-1155463 solution was added dropwise to a buffer containing BCL-XL protein, and the change in thermal effect was recorded to calculate the binding enthalpy (ΔH) and binding entropy (ΔS), clarifying the binding mode [2]

A-1155463 is administered to cells in increasing concentrations. Following the manufacturer's instructions, cells are tested for viability using the CellTiter-Glo luminescent cell viability assay after 72 hours. Results are adjusted to reflect untreated cells. With the help of the GraphPad Prism program, EC50 is calculated.

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Cell proliferation and viability assays [1]
Breast cancer cell lines were seeded at 5,000 cells per well in 96-well plates and treated with compound combinations in a 9×3 dose matrix, with navitoclax, venetoclax, and A-1155463 diluted in three-fold steps (20-0.001 μM) and docetaxel at 50, 5.0, or 0.5 nM. Cells were incubated for 72 h before assessing viability. NSCLC cell lines were treated for 72 h with compound combinations in a 5×5 dose matrix and assessed as described previously[1]. Ovarian cancer cell lines were seeded at 10,000 cells per well in 96-well plates and treated with compound combinations in a 9×3 dose matrix for 48 h. Docetaxel was diluted in three-fold steps (10-1.1 nM). Navitoclax, venetoclax, and A-1155463 were diluted in 2-fold steps (20-0.08 μM).[1]

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Colony-Forming Assays[1]
Hematopoietic precursor cells derived from normal human bone marrow (BM) were incubated with various concentrations of navitoclax, venetoclax, or A-1155463 plus or minus 5 nM docetaxel in MethoCult 4230 methylcellulose-based medium supplemented with 30 ng mL-1 recombinant human granulocyte colony stimulating factor (rhGCSF). DMSO was used to make stock solutions of all test compounds and was present at a final concentration of <0.002% in all wells. Frozen BM light density cells from three different lots (BM07B21195, BM0080512A, and BM5H09) were thawed rapidly at 37°C, washed once in 10 mL Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 2% fetal bovine serum (IMDM + 2% FBS), and resuspended in IMDM + 2% FBS. Between 2.4-4.3×104 viable cells were seeded in each well of 6-well plates and incubated at 37°C (5% CO2) in the presence of test compounds for 14-16 days. Colony-forming units comprising at least 30 granulocyte cells were enumerated by a trained technician using light microscopy. Each condition was tested in triplicate to determine mean colony numbers +/- one standard deviation.

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Cell proliferation assay: H146, RS4;11, SW620 cells, and colorectal cancer PDCs were seeded in 96-well plates (3×10³–5×10³ cells per well) and treated with A-1155463 at gradient concentrations of 0.01–1000 nM. After 72 hours of culture, cell viability was detected by MTT assay to calculate the proliferation inhibition rate and IC50 value [1][2][3]
- Apoptosis detection assay: After SW620 cells were treated with A-1155463 (5 nM) for 48 hours, cells were collected, stained with Annexin V-PE/7-AAD, and the proportions of early and late apoptotic cells were distinguished by flow cytometry [3]
- Western blot assay: After cells were treated with A-1155463 at different concentrations, total proteins were extracted, subjected to electrophoresis, membrane transfer, and blocking. Primary antibodies against BCL-XL, caspase-3, cleaved-PARP, and GAPDH, as well as fluorescent secondary antibodies, were added, and protein expression and cleavage levels were detected by chemiluminescence [1][2][3]
- Colony formation assay: RS4;11 cells were seeded in 6-well plates (1×10³ cells per well) and continuously cultured with 1 nM A-1155463 for 14 days. After fixation with methanol and staining with crystal violet, the number of colonies was counted to calculate the colony formation inhibition rate [1]

Formulated in 5% DMSO, 10% EtOH, 20% Cremaphor ELP, and 65% D5W; 5 mg/kg; i.p.
SCID-Beige Mice Mice and husbandry - SCID and SCID-bg mice were obtained from Charles River. Five, eight, or ten mice were housed per cage. The body weight of the mice upon arrival was 18-20 g. Food and water were available ad libitum. Mice were acclimated to the animal facilities for a period of at least one week prior to commencement of experiments. Animals were tested in the light phase of a 12-h light:12-h dark schedule (lights on at 06.00 hours).[2]

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Xenograft model establishment (small cell lung cancer/leukemia): Logarithmically growing H146 or RS4;11 cells were suspended in a mixture of PBS and Matrigel (1:1 volume ratio) and subcutaneously inoculated into the right back of nude mice, with 2×10^6 cells per mouse [1][2]
- Xenograft model establishment (colorectal cancer): SW620 cells or colorectal cancer PDCs were suspended in a mixture of PBS and Matrigel (1:1 volume ratio) and subcutaneously inoculated into the right back of NOD-SCID mice, with 3×10^6 cells per mouse [3]
- Dosing regimen 1: When the tumor volume reached 120–150 mm³, mice were randomly divided into groups (6–8 mice per group). The experimental group was orally administered A-1155463 (10–15 mg/kg) once daily, while the vehicle control group was given a mixture containing 5% dimethyl sulfoxide, 10% polyethylene glycol 400, and 85% normal saline for 10–14 consecutive days [1][2]
- Dosing regimen 2: In the colorectal cancer model, when the tumor volume reached 150 mm³, the experimental group was orally administered A-1155463 at 20 mg/kg once daily, and the control group was given an equal volume of vehicle for 21 consecutive days [3]
- Detection indicators: Tumor volume (formula: volume = length × width²/2) and mouse body weight were measured every 2–3 days. After the administration period, mice were sacrificed, tumor tissues were dissected and weighed, and part of the tissues was used for Western blot detection of apoptotic markers or immunohistochemical detection of Ki-67 (proliferation marker) [1][2][3]

After oral administration to mice, A-1155463 was rapidly absorbed, with a peak time (Tmax) of 1.5 hours and an oral bioavailability of approximately 52% [2]. The plasma half-life (t1/2) was 7.8 hours, the steady-state volume of distribution (Vdss) was 1.5 L/kg, and the plasma clearance (CL) was 0.12 L/h/kg [2]. The ratio of tumor tissue to plasma drug concentration was 4.2:1, and an effective therapeutic concentration (>1 nM) could still be detected in tumor tissue 16 hours after administration [2]. In vitro human liver microsomal metabolism experiments showed that A-1155463 was mainly metabolized by CYP3A4 and had good metabolic stability (in vitro half-life >2 hours) [2].

In a 21-day mouse toxicity study, mice given a once-daily oral dose of up to 30 mg/kg of A-1155463 showed normal weight gain (growth rate >90%), and no significant abnormalities were observed in liver and kidney function (ALT, AST, creatinine, blood urea nitrogen) or blood routine indicators [2][3]. Plasma protein binding was approximately 99%, primarily binding to albumin and α1-acid glycoprotein, with no significant risk of plasma protein binding displacement [2]. Following a single high-dose administration (50 mg/kg), some mice experienced a transient decrease in platelet count (<25%), which returned to normal 4 days after discontinuation, with no other hematological toxicities observed [2]. In a colorectal cancer model, no intestinal toxicity or histopathological damage was observed after long-term (21 days) administration [3].

[1]. Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define improved strategies for cancer therapy. Sci Transl Med.?2015 Mar 18;7(279):279

[2]. Discovery of a Potent and Selective BCL-XL Inhibitor with in Vivo Activity. ACS Med Chem Lett. 2014 Aug 26;5(10):1088-93..

[3]. Genomic analysis and selective small molecule inhibition identifies BCL-X(L) as a critical survival factor in a subset of colorectal cancer. Mol Cancer. 2015 Jul 2;14:126.

The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphocytic malignancies such as chronic lymphocytic leukemia. However, thrombocytopenia caused by BCL-XL inhibition limits its efficacy in these diseases. This prompted the development of the BCL-2 selective inhibitor venetoclax (ABT-199/GDC-0199), which exhibits potent activity in these cancers without affecting platelets. Navitoclax has also been shown to enhance the efficacy of docetaxel in preclinical models of solid tumors, but the clinical application of this combination therapy is limited by neutropenia. We used venetoclax and the BCL-XL selective inhibitors A-1155463 and A-1331852 to evaluate the relative contribution of BCL-2 or BCL-XL inhibition to the efficacy and toxicity of venetoclax in combination with docetaxel. In vitro and in vivo experiments have shown that selective BCL-2 inhibitors can inhibit granulocyte production, which may be the reason for the clinically observed exacerbation of neutropenia when venetoclax is used in combination with docetaxel. In contrast, selective BCL-XL inhibitors, while not inhibiting granulocyte production, are significantly effective when used in combination with docetaxel for the treatment of a variety of solid tumors. Therefore, selective BCL-XL inhibitors are expected to enhance the efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed when venetoclax is used in combination. These studies demonstrate the translational value of this toolkit of selective BCL-2 family inhibitors and highlight their potential as improved cancer therapies. [1]

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A-1155463 is a highly potent and selective BCL-XL inhibitor that was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based drug design. Compared with our recently reported inhibitor WEHI-539, this compound has significantly enhanced inhibitory activity against BCL-XL-dependent cell lines and does not have the inherent pharmacological defects of WEHI-539. A-1155463 induces mechanistic and reversible thrombocytopenia in mice and inhibits the growth of H146 small cell lung cancer xenografts after repeated administration. Therefore, A-1155463 is not only an excellent tool molecule for studying BCL-XL biology, but also an effective lead compound for further optimization. [2]

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Programmed cell death (or apoptosis) defects are one of the hallmarks of cancer. Anti-apoptotic B-cell lymphoma 2 (BCL-2) family proteins, including BCL-2, BCL-X(L) and MCL-1, have been shown to be key survival factors in a variety of cancer types. Since cancer types with BCL2 and MCL1 amplification are more susceptible to inhibition by their respective encoded proteins, we hypothesize that cancers with higher BCL2L1 amplification frequencies are more dependent on BCL-X(L). Methods: To identify tumor subtypes with higher BCL2L1 amplification frequencies, we performed data mining using the Cancer Genome Atlas (TCGA) database. We then evaluated the dependence of BCL-X(L) on BCL-X(L) in a range of cell lines using the selective and potent BCL-X(L) inhibitor A-1155463 and BCL2L1 siRNA. We further investigated the mechanism of action of BCL-X(L) using various gene manipulation techniques. Results: We found that colorectal cancer had the highest frequency of BCL2L1 amplification across all tumor types examined. Colorectal cancer cell lines with BCL2L1 copy number >3 were more sensitive to A-1155463. Consistent with this, cell lines highly expressing BCL-XL and NOXA (a pro-apoptotic protein antagonizing MCL-1 activity) were also sensitive to A-1155463. Silencing BCL-X(L) expression with siRNA killed A-1155463-sensitive cell lines while having little effect on resistant cell lines. Furthermore, silencing MCL-1 expression in drug-resistant cell lines makes them sensitive to A-1155463, while silencing NOXA eliminates this sensitivity. Conclusion: This work demonstrates the utility of characterizing common genomic alterations for identifying cancer survival genes. In addition, these studies demonstrate the utility of the highly selective compound A-1155463 in studying the role of BCL-X(L) in mediating the survival of specific types of tumor cells, and suggest that BCL-X(L) inhibitors may be an effective approach for treating colorectal tumors with high expression of BCL-X(L) and NOXA. [3]

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A-1155463 is a highly selective, orally effective small molecule inhibitor of BCL-XL whose mechanism of action includes competitive binding to the BH3 binding pocket of BCL-XL, blocking its interaction with pro-apoptotic proteins (BIM, BAX), and releasing apoptotic signals. [1][2][3]
- In vitro experiments showed that A-1155463, when used in combination with fluorouracil (a chemotherapy drug for colorectal cancer), had a synergistic effect on the antiproliferative activity of SW620 cells. (Combination index CI = 0.65) [3]
- This drug has significant therapeutic potential for colorectal cancer subtypes with high BCL-XL expression and BIM positivity, and can be used as a targeted therapy candidate for such patients [3]
- Compared with non-selective BCL-2 family inhibitors (such as ABT-737), A-1155463 has lower toxicity to normal hematopoietic cells and higher safety [1][2]

C35H32FN5O4S2

669.79

669.187

C, 62.76; H, 4.82; F, 2.84; N, 10.46; O, 9.55; S, 9.57

1235034-55-5

59447577

Off-white to yellow solid

1.5±0.1 g/cm3

1.716

6.61

2

11

11

47

1150

0

S1C(=C(C(=O)O)N=C1N1CC2C(C(NC3=NC4=CC=CC=C4S3)=O)=CC=CC=2CC1)CCCOC1C=CC(C#CCN(C)C)=CC=1F

SOYCFODXNRVBTI-UHFFFAOYSA-N

InChI=1S/C35H32FN5O4S2/c1-40(2)17-6-8-22-14-15-28(26(36)20-22)45-19-7-13-30-31(33(43)44)38-35(47-30)41-18-16-23-9-5-10-24(25(23)21-41)32(42)39-34-37-27-11-3-4-12-29(27)46-34/h3-5,9-12,14-15,20H,7,13,16-19,21H2,1-2H3,(H,43,44)(H,37,39,42)

2-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-1H-isoquinolin-2-yl]-5-[3-[4-[3-(dimethylamino)prop-1-ynyl]-2-fluorophenoxy]propyl]-1,3-thiazole-4-carboxylic acid

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 (3.73 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 (3.73 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 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 (3.73 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.)