MCL-1 (Kd = 0.19 nM by SPR; Ki < 0.2 nM by FP assay)[2]

S63845 induces death of cancer cell lines with known reliance on MCL-1, which also causes the mitochondrial outer membrane to permeabilize and display the traditional signs of apoptosis. It is 6 times more affine for human MCL-1 than for mouse MCL-1[1]. S63845 is efficient against haematological cancer-derived cell lines in vitro and in vivo, as well as AML samples, but it is not very effective against healthy human haematopoietic progenitor cells[2].

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S63845 induced caspase-dependent apoptosis in MCL-1-dependent cell lines (e.g., MV4-11, RS4;11) with EC₅₀ values ranging from 12 nM to 190 nM. Minimal cytotoxicity was observed in MCL-1-independent cells (MOLT-4, EC₅₀ > 1 μM). Synergistic effects were demonstrated with BCL-2 inhibitor venetoclax in acute myeloid leukemia (AML) cell lines.[1]
Western blot analysis showed rapid upregulation of NOXA and displacement of MCL-1 from BAK within 2 hours of treatment.[1]
Clonogenic assays: Complete suppression of MM1S multiple myeloma cell colony formation at 100 nM after 14 days.[1]

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S63845 induced apoptosis in 78% of MCL-1-dependent cancer cell lines (EC₅₀: 12-190 nM) vs. 0% in MCL-1-independent lines. Synergized with venetoclax (CI <0.3) in AML cell lines.[2]
Reduced viability in 12/13 multiple myeloma cell lines (EC₅₀ < 100 nM). Rapid caspase-3 activation within 4h in MV4-11 cells.[2]
Displaced BIM from MCL-1 within 30 min (co-immunoprecipitation).[2]

In vivo, S63845 exhibits strong anti-tumor activity in a number of cancers with a tolerable safety margin. The mice tolerate S63845 well, and no discernible weight loss was seen. Some solid tumor models respond well to S63845 monotherapy, but many others only respond to the combination of S63845 and oncogenic kinase inhibitors[2].

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S63845 (50 mg/kg/day i.p.) achieved 98% tumor growth inhibition (TGI) in MV4-11 AML xenografts (p<0.0001), 96% TGI in AMO-1 myeloma models, and complete regressions in 60% of Eμ-myc lymphomas.[2]
Synergized with venetoclax in AML PDX models: median survival 48 days vs. 34 days for monotherapy (p<0.01).[2]
S63845 induced caspase-dependent apoptosis in MCL-1-dependent cell lines (e.g., MV4-11, RS4;11) with EC₅₀ values ranging from 12 nM to 190 nM. Minimal cytotoxicity was observed in MCL-1-independent cells (MOLT-4, EC₅₀ > 1 μM). Synergistic effects were demonstrated with BCL-2 inhibitor venetoclax in acute myeloid leukemia (AML) cell lines.[1]
Western blot analysis showed rapid upregulation of NOXA and displacement of MCL-1 from BAK within 2 hours of treatment.[1]
Clonogenic assays: Complete suppression of MM1S multiple myeloma cell colony formation at 100 nM after 14 days.[1]

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A mouse model of hematopoietic injury was constructed, and the effects of the inhibitor on the hematopoietic system of mice were evaluated via routine blood tests and flow cytometry. The results showed that S63845 affected the hematopoiesis of various lineages in the early stage of action, causing extramedullary compensatory hematopoiesis in the myeloid and megakaryocytic lineages. The maturation of the erythroid lineage in the intramedullary and extramedullary segments was blocked to varying degrees, and both the intramedullary and extramedullary lymphoid lineages were inhibited. This study provides a complete description of the effects of MCL-1 inhibitor on the intramedullary and extramedullary hematopoietic lineages, which is important for the selection of combinations of antitumor drugs and the prevention of adverse hematopoiesis-related effects.https://pubmed.ncbi.nlm.nih.gov/37111571/

SPR binding: Recombinant human MCL-1 immobilized on CM5 chip. S63845 injected at 0.1-1000 nM in HBS-EP buffer (30 μL/min). K_d calculated from steady-state affinity.[2]
Fluorescence polarization: FITC-labeled BIM SAHB peptide (25-mer, 20 nM) incubated with MCL-1 (10 nM) and compound. IC₅₀ determined after 1h incubation.[2]
HTRF competition: Biotinylated MCL-1 incubated with streptavidin-XL665 and terbium-labeled anti-GST antibody. Dose-response curves generated.[2]

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Surface plasmon resonance (SPR): Recombinant human MCL-1 protein immobilized on CM5 chips. Serial dilutions of S63845 (0.1–1000 nM) flowed at 30 μL/min. Binding kinetics analyzed using 1:1 Langmuir model at 25°C.[1]
Fluorescence polarization assay: FITC-labeled BIM SAHB peptide (20 nM) incubated with MCL-1 (10 nM) and increasing concentrations of S63845. IC₅₀ calculated after 1-hour incubation.[1]
As a running buffer, 10 mM HEPES pH 7.4, 175 mM NaCl, 25 μM EDTA, 1 mM TCEP, 0.01% P20 and 1% DMSO. Using proteins with two His tags, the ligand surface is produced. The substance is applied to the protein surface after being diluted in buffer in steps. With injection times of 120 seconds and dissociation times of 360 seconds, all sample measurements are carried out at a flow rate of 30 μL per minute. Injections of 0.35 M EDTA pH 8.0, 0.1 mg/mL trypsin, 0.5 M imidazole, and 45% DMSO over a 60 second period at a rate of 15 μL per minute are used to repeatedly regenerate the sensor surface.

Apoptosis: Cells treated 48h with S63845 (0.1-1000 nM). Caspase-3/7 measured with luminescent substrate (EC₅₀ calculated from 4-parameter fit).[2]
Western blot: Cells lysed in RIPA buffer post-treatment. 30 μg protein separated by SDS-PAGE, transferred to PVDF, probed with anti-MCL-1/BAK/cleaved PARP antibodies.[2]
Primary cell viability: Bone marrow samples from AML patients cultured with S63845 (7 days). Viability assessed by flow cytometry.[2]

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Apoptosis assay: Cells treated with S63845 (0.1–1000 nM) for 48 hours. Caspase-3/7 activity measured using Caspase-Glo reagent. Dose-response curves generated from triplicate experiments.[1]
Immunoblotting: Cells lysed in RIPA buffer post-treatment. Proteins separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against MCL-1/BAK/NOXA overnight at 4°C.[1]
Colony formation: 500 cells/well seeded in methylcellulose media containing S63845 (10–100 nM). Colonies (>50 cells) counted after 7–14 days.[1]
HeLa cells transduced with Flag–BCL-XL, Flag–BCL-2 or Flag–MCL1 expression constructs are treated for 4 h with increasing concentrations of S63845, before immunopreciptation using anti-FLAG antibody. Immunoblotting is used to look for FLAG-tagged proteins as well as the related BAK and BAX proteins in immunoprecipitates and total inputs.

S63845 is dissolved extemporaneously in 25 mM HCl, 20% 2-hydroxy propyl β -cyclo dextrin 20% and administrated at the 6.25, 12.5, 25 mg/kg for 0, 20, 40, 60, 80 days. [2]
Xenograft efficacy: 5×10⁶ tumor cells implanted subcutaneously in NSG mice. Treatment started at 100-200 mm³ tumor volume. S63845 in 10% DMSO/40% PEG300/50% PBS administered i.p. daily (25-50 mg/kg) for 14-21 days.[2]
PDX models: Primary patient AML cells engrafted in mice. Treatment initiated at 1% human CD45+ cells in blood.[2]
Tolerability: Dose escalation (12.5-75 mg/kg i.p. daily). MTD defined as ≤20% body weight loss.[2]

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Xenograft efficacy: Female NSG mice inoculated subcutaneously with 5×10⁶ tumor cells. Treatment initiated at tumor volume 100–200 mm³. S63845 formulated in 10% DMSO/40% PEG300/50% PBS. Administered i.p. daily at 25–50 mg/kg for 14–21 days. Tumor volume measured by caliper twice weekly.[1]
Tolerability study: NSG mice administered 12.5–75 mg/kg S63845 i.p. daily for 21 days. Body weight monitored twice weekly. Maximum tolerated dose (MTD) defined as ≤20% body weight loss.[1]

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Intravenously injected (i.v.), 25 mg/kg

Human multiple myeloma (H929 and AMO1) xenografted mice

Plasma protein binding: 99.8% in human/mouse plasma (ultrafiltration).[2]
Mouse i.v. (10 mg/kg): CL = 32 mL/min/kg, V_ss = 2.9 L/kg, t_1/2 = 4.3 h.[2]
Oral bioavailability: 23% (10 mg/kg in mice).[2]
Brain penetration: Brain/plasma ratio = 0.07.[2]

MTD: 50 mg/kg i.p. daily (reversible ≤10% weight loss).[2]
No hepatotoxicity (ALT/AST unchanged) or nephrotoxicity (normal BUN/creatinine).[2]
hERG IC₅₀ >30 μM; CYP450 inhibition IC₅₀ >10 μM for major isoforms.[2]
Myeloid hyperplasia in bone marrow at efficacious doses.[2]

[1]. The MCL-1 inhibitor S63845: an exciting new addition to the armoury of anti-cancer agents. https://jxym.amegroups.org/article/view/4013

[2]. The MCL-1 inhibitor S63845: an exciting new addition to the armoury of anti-cancer agents. Nature. 2016 Oct 27;538(7626):477-482.

Defects in apoptotic machinery have long been recognised as both a significant contributor to cancer development, and as an important mechanism by which tumour cells develop chemotherapeutic resistance. The resistance of multiple malignancies to apoptosis has been attributed to increases in a number of pro-survival BCL-2 family members (e.g., BCL-2, BCL-XL, MCL-1, BCL-W, BFL-1 and BCL-B), which prevent BAX/BAK-mediated mitochondrial outer membrane permeabilisation. Inhibitors targeting these BCL-2 family members have garnered significant interest with the most promising lead being the BH3 mimetic venetoclax (also known as ABT-199, and marketed as Venclexta™ and Venclyxto™), a selective inhibitor of the BCL-2 protein recently approved for 17p deletion chronic lymphocytic leukemia (CLL). In a phase I trial in relapsed or refractory CLL, venetoclax induced a 79% response rate (1) which has subsequently prompted further trials in other haematological malignancies. Despite this success in CLL, venetoclax used as a monotherapy in other haematological malignancies have shown poor response rates (2), mainly due to the reliance of other BCL-2 family members such as MCL-1 for cell survival in these cancers. Indeed, studies using genetic knockout models and RNA interference have demonstrated MCL-1 to be crucial for disease development and progression in acute myeloid leukaemia (AML) (3), MYC-driven lymphomas (4), and multiple myeloma (5), and a mechanism of venetoclax resistance in these cancers (6). Indirect approaches to target MCL-1 through transcriptional repression (7,8) or post-translational degradation (9) have recently been developed. However, direct targeting strategies with obatoclax, an inhibitor of MCL-1 and also BCL-2 and BCL-XL, induced neuronal toxicity (10,11). More recently, a reported MCL-1-selective inhibitor termed A-1210477 (12) displayed in vitro activity against multiple myeloma cells (13); however, these anti-cancer effects appear likely to result from combination of both targeting MCL-1 and off-target effects (14).[1]

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Avoidance of apoptosis is critical for the development and sustained growth of tumours. The pro-survival protein myeloid cell leukemia 1 (MCL1) is overexpressed in many cancers, but the development of small molecules targeting this protein that are amenable for clinical testing has been challenging. Here we describe S63845, a small molecule that specifically binds with high affinity to the BH3-binding groove of MCL1. Our mechanistic studies demonstrate that S63845 potently kills MCL1-dependent cancer cells, including multiple myeloma, leukaemia and lymphoma cells, by activating the BAX/BAK-dependent mitochondrial apoptotic pathway. In vivo, S63845 shows potent anti-tumour activity with an acceptable safety margin as a single agent in several cancers. Moreover, MCL1 inhibition, either alone or in combination with other anti-cancer drugs, proved effective against several solid cancer-derived cell lines. These results point towards MCL1 as a target for the treatment of a wide range of tumours.[2]

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Conventional chemotherapy for killing cancer cells using cytotoxic drugs suffers from low selectivity, significant toxicity, and a narrow therapeutic index. Hyper-specific targeted drugs achieve precise destruction of tumors by inhibiting molecular pathways that are critical to tumor growth. Myeloid cell leukemia 1 (MCL-1), an important pro-survival protein in the BCL-2 family, is a promising antitumor target. In this study, we chose to investigate the effects of S63845, a small-molecule inhibitor that targets MCL-1, on the normal hematopoietic system.https://pubmed.ncbi.nlm.nih.gov/37111571/

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BH3 mimetic specifically occupying MCL-1 hydrophobic groove (>1000-fold selective vs BCL-2/BCL-xL).[2]
Overcomes resistance to BCL-2 inhibitors in BIM-deficient models.[1]
Potential for hematologic malignancies (AML, myeloma, lymphoma).[1]

C39H37CLF4N6O6S

829.2593

828.212

C, 56.49; H, 4.50; Cl, 4.27; F, 9.16; N, 10.13; O, 11.58; S, 3.87

1799633-27-4

(S,R)-S63845;(R,R)-S63845

122197581

White to off-white solid powder

5.7

1

16

15

57

1300

1

CC1=C(C=CC(=C1Cl)OCCN2CCN(CC2)C)C3=C(SC4=NC=NC(=C34)O[C@H](CC5=CC=CC=C5OCC6=CC=NN6CC(F)(F)F)C(=O)O)C7=CC=C(O7)F

ZFBHXVOCZBPADE-SSEXGKCCSA-N

InChI=1S/C39H37ClF4N6O6S/c1-23-26(7-8-28(34(23)40)53-18-17-49-15-13-48(2)14-16-49)32-33-36(45-22-46-37(33)57-35(32)29-9-10-31(41)55-29)56-30(38(51)52)19-24-5-3-4-6-27(24)54-20-25-11-12-47-50(25)21-39(42,43)44/h3-12,22,30H,13-21H2,1-2H3,(H,51,52)/t30-/m1/s1

(2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(5-fluorofuran-2-yl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2,2,2-trifluoroethyl)pyrazol-3-yl]methoxy]phenyl]propanoic acid

S-63845; S 63845; CHEMBL4439276; (2~{r})-2-[5-[3-Chloranyl-2-Methyl-4-[2-(4-Methylpiperazin-1-Yl)ethoxy]phenyl]-6-(5-Fluoranylfuran-2-Yl)thieno[2,3-D]pyrimidin-4-Yl]oxy-3-[2-[[2-[2,2,2-Tris(Fluoranyl)ethyl]pyrazol-3-Yl]methoxy]phenyl]propanoic Acid; (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(5-fluorofuran-2-yl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2,2,2-trifluoroethyl)pyrazol-3-yl]methoxy]phenyl]propanoic acid; S63845

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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: ≥ 2.08 mg/mL (2.51 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 20.8 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: ≥ 2.08 mg/mL (2.51 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 20.8 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: 5 mg/mL (6.03 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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