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Purity: ≥98%
AMG-176 (AMG176; tapotoclax) is a novel, potent, selective and orally bioavailable inhibitor of MCL-1 (myeloid cell leukemia-1) with a Ki of 0.13 nM. Potential pro-apoptotic and anti-cancer effects of AMG-176 exist. MCL-1 is bound by AMG 176, which then stops it from working. This causes apoptosis in tumor cells and interferes with the formation of MCL-1/Bcl-2-like protein 11 (BCL2L11; BIM) complexes. The Bcl-2 family of proteins member MCL-1, an anti-apoptotic protein, is upregulated in cancer cells and supports the survival of tumor cells.
| Targets |
Mcl-1 (Ki = 0.13 nM)
AMG-176 targets myeloid cell leukemia 1 (MCL1) protein (Ki = 0.19 nM for human MCL1; no significant binding to BCL-2 (Ki > 1000 nM), BCL-xL (Ki > 1000 nM), BCL-w (Ki > 1000 nM), or BFL-1 (Ki > 1000 nM)) [1] |
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| ln Vitro |
Tapoclax is a compound that has the ability to stimulate cell color development and anti-tumor action. It also promotes the production of MCL-1 (Ki=0.13 nM) in inducible myeloid leukemia cells. After then, tapotoclax attaches to MCL-1 and prevents its function. It stimulates tumor cells and interferes with the formation of the MCL-1/Bcl-2-like protein 11 (BCL2L11; BIM) complex [1][2].
1. AMG-176 exhibited potent antiproliferative activity against a panel of hematological cancer cell lines, with IC50 values ranging from 0.03 μM to 1.2 μM in acute myeloid leukemia (AML) cell lines (e.g., MV4-11: 0.06 μM, MOLM-13: 0.09 μM, OCI-AML3: 0.32 μM), multiple myeloma (MM) cell lines (e.g., RPMI-8226: 0.18 μM, U266: 0.25 μM), and chronic lymphocytic leukemia (CLL) cell lines (e.g., MEC-1: 0.45 μM, HG3: 0.51 μM) [1] 2. AMG-176 induced dose-dependent apoptosis in MCL1-dependent hematological cancer cells, as demonstrated by Annexin V/PI staining via flow cytometry; in MV4-11 cells, treatment with 0.1 μM AMG-176 for 24 hours resulted in 45% apoptotic cells, while 0.5 μM led to 82% apoptotic cells [1] 3. Western blot analysis revealed that AMG-176 triggered cleavage of PARP and caspase-3/caspase-9 in AML (MV4-11) and MM (RPMI-8226) cell lines, confirming activation of the intrinsic apoptotic pathway; additionally, AMG-176 downregulated MCL1 protein levels and released BIM from MCL1-BIM complexes in a dose-dependent manner [1] 4. Clonogenic assay showed that AMG-176 (0.01-0.5 μM) significantly reduced colony formation of primary AML blasts from patient samples (n=12) with a mean IC50 of 0.12 μM, while having minimal effect on normal hematopoietic progenitor cells (CD34+ cells) with an IC50 > 5 μM [1] 5. Combination treatment of AMG-176 with standard chemotherapeutics (cytarabine, daunorubicin) or targeted agents (venetoclax, bortezomib, idelalisib) exhibited synergistic antiproliferative effects in AML, MM, and CLL cell lines (combination index < 0.8 for most pairs), with enhanced apoptotic induction compared to single-agent treatment [1] |
| ln Vivo |
1. In MV4-11 AML subcutaneous xenograft mice, AMG-176 administered intraperitoneally (IP) at 10, 30, or 50 mg/kg once daily for 14 days dose-dependently inhibited tumor growth, with tumor growth inhibition (TGI) rates of 42%, 78%, and 91%, respectively; the 50 mg/kg dose also significantly extended median overall survival (OS) from 28 days (vehicle) to 45 days [1]
2. In RPMI-8226 MM subcutaneous xenograft mice, AMG-176 (30 mg/kg IP qd × 14 days) achieved a TGI of 72% and increased median OS by 32% compared to vehicle; combination with bortezomib (0.5 mg/kg IP twice weekly) resulted in complete tumor regression in 6/8 mice and a 2.1-fold extension of median OS [1] 3. In a disseminated AML mouse model (MOLM-13-luciferase cells via tail vein injection), AMG-176 (50 mg/kg IP qd × 10 days) reduced bioluminescent signal (tumor burden) by 85% at day 14 and prolonged median OS from 22 days (vehicle) to 38 days; combination with cytarabine (100 mg/kg IP qd × 5 days) further reduced tumor burden by 96% and extended median OS to 52 days [1] 4. In primary patient-derived xenograft (PDX) models of AML (n=3) and MM (n=2), AMG-176 (30-50 mg/kg IP qd × 14 days) induced significant tumor regression (≥60% TGI) in 4/5 models and prolonged OS in all tested models, with minimal impact on normal bone marrow hematopoiesis [1] |
| Enzyme Assay |
1. Fluorescence polarization (FP) binding assay for MCL1: Recombinant human MCL1 protein was incubated with a fluorescently labeled BH3 peptide (BIM BH3) and serial dilutions of AMG-176 in a buffer system at room temperature for 60 minutes; polarization values were measured using a microplate reader, and competition binding curves were generated to calculate the Ki value of AMG-176 for MCL1; specificity was confirmed by incubating AMG-176 with recombinant BCL-2, BCL-xL, BCL-w, and BFL-1 proteins under identical conditions [1]
2. Isothermal titration calorimetry (ITC) assay: AMG-176 was titrated into a solution of recombinant MCL1 protein in a calorimeter cell at 25°C; heat changes associated with the binding interaction were recorded, and the data were analyzed to determine the binding affinity (KD) and thermodynamic parameters (ΔH, ΔS) of the AMG-176-MCL1 complex [1] 3. Homogeneous time-resolved fluorescence (HTRF) assay: MCL1 protein was incubated with a biotinylated BH3 peptide and Eu-labeled anti-MCL1 antibody, along with increasing concentrations of AMG-176; HTRF signals were measured at 665 nm and 620 nm, and the inhibition of MCL1-BH3 peptide binding was quantified to confirm the antagonistic activity of AMG-176 [1] |
| Cell Assay |
1. Cell viability assay: Hematological cancer cell lines (AML, MM, CLL) and normal CD34+ hematopoietic progenitor cells were seeded in 96-well plates at a density of 5×10³ cells/well; serial dilutions of AMG-176 (0.001-10 μM) were added, and cells were incubated for 72 hours at 37°C with 5% CO₂; a cell viability reagent was added, and absorbance/fluorescence was measured to calculate IC50 values for antiproliferative activity; for combination studies, AMG-176 was co-administered with cytarabine, bortezomib, or venetoclax at fixed dose ratios, and combination indices were calculated using the Chou-Talalay method [1]
2. Apoptosis detection assay: Cancer cells were treated with AMG-176 (0.01-1 μM) for 24-48 hours, then harvested and stained with Annexin V-FITC and propidium iodide (PI); flow cytometry was used to quantify the percentage of early (Annexin V+/PI-) and late (Annexin V+/PI+) apoptotic cells; for primary AML blasts, cells were isolated from patient bone marrow samples, treated with AMG-176, and apoptotic rates were measured using the same staining protocol [1] 3. Western blot analysis for apoptotic markers: Cells were lysed in a buffer containing protease and phosphatase inhibitors 24-48 hours after AMG-176 treatment; protein concentrations were quantified, and equal amounts of protein were separated by SDS-PAGE and transferred to membrane; the membrane was probed with antibodies against MCL1, BIM, PARP, caspase-3, caspase-9, and β-actin (loading control); bound antibodies were detected using a secondary antibody conjugate, and band intensities were quantified by densitometry [1] 4. Colony formation assay: Primary AML blasts and normal CD34+ cells were plated in semi-solid media containing growth factors and serial dilutions of AMG-176 (0.01-5 μM); colonies were counted after 14 days of incubation at 37°C with 5% CO₂, and the percentage of colony formation inhibition was calculated relative to vehicle-treated controls [1] |
| Animal Protocol |
1. Subcutaneous xenograft model (AML/MV4-11): Female NOD/SCID mice (6-8 weeks old) were injected subcutaneously with 5×10⁶ MV4-11 cells into the right flank; tumors were allowed to reach 100-150 mm³ before treatment initiation; AMG-176 was formulated in a vehicle of 10% DMSO, 40% PEG400, and 50% sterile saline, and administered intraperitoneally (IP) at 10, 30, or 50 mg/kg once daily for 14 days; tumor volume was measured every 2 days using calipers (volume = length × width² / 2), and body weight was monitored to assess toxicity; mice were euthanized when tumors exceeded 2000 mm³ or showed signs of distress [1]
2. Subcutaneous xenograft model (MM/RPMI-8226): NOD/SCID mice were injected subcutaneously with 1×10⁷ RPMI-8226 cells; once tumors reached 150-200 mm³, mice were randomized to receive AMG-176 (30 mg/kg IP qd ×14), bortezomib (0.5 mg/kg IP twice weekly ×3), or the combination; tumor volume and body weight were measured twice weekly, and overall survival was recorded for 60 days [1] 3. Disseminated AML model (MOLM-13-luciferase): NOD/SCID mice were injected via tail vein with 1×10⁶ MOLM-13 cells stably expressing luciferase; 7 days post-injection, bioluminescent imaging (BLI) was performed to confirm tumor engraftment; mice were treated with AMG-176 (50 mg/kg IP qd ×10), cytarabine (100 mg/kg IP qd ×5), or the combination; BLI was conducted every 7 days to quantify tumor burden, and survival was monitored for 60 days [1] 4. Patient-derived xenograft (PDX) models: Primary AML/MM cells were isolated from patient bone marrow aspirates and injected into NOD/SCID gamma (NSG) mice (subcutaneously for MM, intravenously for AML); once tumors were detectable (AML: BLI signal; MM: palpable tumors), mice were treated with AMG-176 (30-50 mg/kg IP qd ×14); tumor growth was assessed by BLI (AML) or caliper measurement (MM), and peripheral blood/bone marrow was analyzed by flow cytometry to evaluate normal hematopoiesis [1] |
| ADME/Pharmacokinetics |
1. In male CD-1 mice, the terminal half-life (t1/2) of intravenously administered (IV) AMG-176 (5 mg/kg) was 2.8 h, the volume of distribution (Vd) was 0.9 L/kg, and the total clearance (CL) was 0.25 L/h/kg; the bioavailability of oral administration (20 mg/kg) was low (F = 8.2%), with a peak plasma concentration (Cmax) of 0.32 μM and a time to peak concentration of 1 hour (Tmax) [1]
2. In female NOD/SCID mice, the Cmax of intraperitoneally administered AMG-176 (30 mg/kg) was 2.1 μM (Tmax = 0.5 h), and the 24-hour AUC₀ was 6.8 μM·h; the drug showed good tissue penetration, with bone marrow (1.8 μM·h) 2 hours after administration. The concentrations in the spleen (2.3 μM) and spleen (2.3 μM) were higher than the in vitro IC50 values of most MCL1-dependent cancer cells [1]. 3. AMG-176 is primarily metabolized by CYP3A4 in human liver microsomes, with smaller contributions from CYP2C9 and CYP2D6; less than 5% of the drug was excreted unchanged in mouse urine and feces within 48 hours [1]. |
| Toxicity/Toxicokinetics |
1. AMG-176 showed high plasma protein binding rates in mouse, rat, and human plasma (97.5%, 98.2%, and 99.1%, respectively) [1]
2. Acute toxicity studies in CD-1 mice showed that no death or significant toxicity was observed at intraperitoneal injection doses up to 200 mg/kg; subchronic toxicity tests (intraperitoneal injection, 50 mg/kg/day, for 14 days) showed mild weight loss (<10%), and no significant changes in liver and kidney function indicators (ALT, AST, BUN, creatinine) or hematological parameters (white blood cells, red blood cells, platelets) [1] 3. In vitro CYP450 inhibition assays showed that AMG-176 did not inhibit CYP1A2, CYP2C19, or CYP2D6 at concentrations up to 10 μM, and did not inhibit CYP3A4 (IC50 = 8.7 μM) and CYP2C9 (IC50 = 9.2 μM). The inhibitory effect was weak, indicating a low risk of drug interaction [1]. 4. In non-human primates (cynomolgus monkeys), repeated intravenous injection of AMG-176 (10-30 mg/kg once a week for 4 weeks) resulted in transient lymphopenia and neutropenia, which returned to normal within 7 days after drug withdrawal; no histopathological changes were observed in the liver, kidneys, bone marrow or other major organs [1]. |
| References | |
| Additional Infomation |
Tapotoclax is an inhibitor of MCL-1 (myeloid leukemia-1), a protein that induces differentiation in myeloid leukemia cells, and possesses potential pro-apoptotic and anti-tumor activities. After administration, tapotoclax binds to MCL-1 and inhibits its activity. This disrupts the formation of the MCL-1/Bcl-2-like protein 11 (BCL2L11; BIM) complex and induces tumor cell apoptosis. MCL-1 is an anti-apoptotic protein belonging to the Bcl-2 protein family, upregulated in cancer cells, and promotes tumor cell survival.
Drug Indications Treatment of acute myeloid leukemia 1. AMG-176 is a first-in-class selective small molecule MCL1 inhibitor. MCL1 is a pro-survival member of the BCL-2 protein family, often overexpressed in hematologic malignancies and associated with chemotherapy resistance [1]. 2. The mechanism of action of AMG-176 is to bind to the BH3 binding slot of MCL1, disrupting its interaction with pro-apoptotic proteins (BIM, BAK, BAX), thereby triggering the mitochondrial intrinsic apoptosis pathway [1]. 3. AMG-176 is undergoing a phase I/II clinical trial (NCT02675452, NCT03672695) for the treatment of relapsed/refractory AML, MM and CLL, and preliminary data show clinical activity and manageable safety [1]. 4. [2] pointed out that AMG-176 represents a major advance in targeting MCL1, a member of the BCL-2 family, which has been difficult to treat due to its flexible BH3 binding pocket; its targeting of MCL1 The high selectivity relative to other BCL-2 family proteins minimizes targeting toxicity (e.g., thrombocytopenia associated with BCL-2 inhibitors such as venetoclax) [2] 5. Preclinical data suggest that AMG-176 is particularly effective against MCL1-dependent cancers, including FLT3-mutant AML, t(11;14)-positive MM, and del(17p) CLL, and can overcome adaptive resistance to MCL1 inhibition when used in combination with other drugs [1] |
| Molecular Formula |
C33H41CLN2O5S
|
|---|---|
| Molecular Weight |
613.207047224045
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| Exact Mass |
612.24
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| Elemental Analysis |
C, 64.64; H, 6.74; Cl, 5.78; N, 4.57; O, 13.05; S, 5.23
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| CAS # |
1883727-34-1
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| Related CAS # |
1883727-34-1
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| PubChem CID |
118910268
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| Appearance |
White to off-white solid powder
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| LogP |
6.8
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| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
6
|
| Rotatable Bond Count |
1
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| Heavy Atom Count |
42
|
| Complexity |
1110
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| Defined Atom Stereocenter Count |
6
|
| SMILES |
C[C@H]1C/C=C/[C@@H]([C@@H]2CC[C@H]2CN3C[C@@]4(CCCC5=C4C=CC(=C5)Cl)COC6=C3C=C(C=C6)C(=O)NS(=O)(=O)[C@@H]1C)OC
|
| InChi Key |
JQNINBDKGLWYMU-GEAQBIRJSA-N
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| InChi Code |
InChI=1S/C33H41ClN2O5S/c1-21-6-4-8-30(40-3)27-12-9-25(27)18-36-19-33(15-5-7-23-16-26(34)11-13-28(23)33)20-41-31-14-10-24(17-29(31)36)32(37)35-42(38,39)22(21)2/h4,8,10-11,13-14,16-17,21-22,25,27,30H,5-7,9,12,15,18-20H2,1-3H3,(H,35,37)/b8-4+/t21-,22+,25-,27+,30-,33-/m0/s1
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| Chemical Name |
(3'R,4S,6'R,7'S,8'E,11'S,12'R)-7-chloro-7'-methoxy-11',12'-dimethyl-13',13'-dioxospiro[2,3-dihydro-1H-naphthalene-4,22'-20-oxa-13lambda6-thia-1,14-diazatetracyclo[14.7.2.03,6.019,24]pentacosa-8,16(25),17,19(24)-tetraene]-15'-one
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| Synonyms |
tapotoclaxum; tapotoclax; AMG-176; AMG176; AMG 176
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: ~62.5 mg/mL (~101.9 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2 mg/mL (3.26 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 20.0 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. Solubility in Formulation 2: 2 mg/mL (3.26 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 20.0 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. View More
Solubility in Formulation 3: 2 mg/mL (3.26 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6308 mL | 8.1538 mL | 16.3076 mL | |
| 5 mM | 0.3262 mL | 1.6308 mL | 3.2615 mL | |
| 10 mM | 0.1631 mL | 0.8154 mL | 1.6308 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT05209152 | Recruiting | Drug: AMG 176 Drug: Azacitidine |
Higher Risk Myelodysplastic Syndrome Chronic Myelomonocytic Leukemia |
Amgen | September 1, 2019 | Phase 1 |
| NCT02675452 | Recruiting | Drug: AMG 176 Drug: Azacitidine |
Relapsed or Refractory Multiple Myeloma Relapsed or Refractory Acute Myeloid Leukemia |
Amgen | June 13, 2016 | Phase 1 |
| NCT03797261 | Terminated | Drug: Venetoclax Drug: AMG 176 |
Acute Myeloid Leukemia Non-Hodgkin's Lymphoma |
AbbVie | March 18, 2019 | Phase 1 |
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