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] Intravenous injection in mice (10 mg/kg): clearance (CL) = 32 mL/min/kg, steady-state volume of distribution (Vss) = 2.9 L/kg, half-life (t1/2) = 4.3 h. [2] Oral bioavailability: 23% (mouse 10 mg/kg). [2] Brain permeability: brain/plasma ratio = 0.07. [2]

Maximum tolerated dose (MTD): 50 mg/kg daily via intraperitoneal injection (reversible weight loss ≤10%). [2]
No hepatotoxicity (ALT/AST unchanged) or nephrotoxicity (BUN/creatinine normal). [2]
hERG IC₅₀ >30 μM; inhibition of major CYP450 isoenzymes IC₅₀ >10 μM. [2]
May induce myeloid hyperplasia at effective 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 apoptosis mechanisms have long been considered a crucial factor in cancer development and progression, and also a significant mechanism by which tumor cells develop chemoresistance. Resistance to apoptosis in various malignant tumors is attributed to increased levels of multiple pro-survival BCL-2 family members (e.g., BCL-2, BCL-XL, MCL-1, BCL-W, BFL-1, and BCL-B), which can prevent BAX/BAK-mediated alterations in mitochondrial outer membrane permeability. Inhibitors targeting these BCL-2 family members have attracted considerable attention, with the most promising lead compound being the BH3 mimic Venetoclax (also known as ABT-199, marketed as Venclexta™ and Venclyxto™), a selective BCL-2 protein inhibitor recently approved for the treatment of 17p deletion chronic lymphocytic leukemia (CLL). In a phase I clinical trial of venetoclax in relapsed or refractory chronic lymphocytic leukemia (CLL), a response rate of 79% (1) was achieved, prompting researchers to conduct further trials in other hematologic malignancies. Despite the success of venetoclax in CLL, its response rate as a monotherapy in other hematologic malignancies is low (2), mainly because cell survival in these cancers depends on other members of the BCL-2 family, such as MCL-1. Indeed, studies using gene knockout models and RNA interference have shown that MCL-1 is essential for the development and progression of acute myeloid leukemia (AML) (3), MYC-driven lymphoma (4), and multiple myeloma (5), and is one of the mechanisms of venetoclax resistance in these cancers (6). In recent years, researchers have developed methods to indirectly target MCL-1, such as transcriptional repression (7,8) or post-translational degradation (9). However, direct targeting with the MCL-1, BCL-2, and BCL-XL inhibitor obatoclax can induce neuronal toxicity (10,11). Recently, a selective MCL-1 inhibitor called A-1210477 (12) has shown activity against multiple myeloma cells in vitro (13); however, these anticancer effects may be the result of a combination of MCL-1 targeting and off-target effects (14). [1]

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Avoiding apoptosis is crucial for tumor development and sustained growth. The pro-survival protein myeloid leukemia 1 (MCL1) is overexpressed in a variety of cancers, but developing small molecule drugs that target this protein for clinical trials has been challenging. This paper describes S63845, a small molecule that binds specifically to the MCL1 BH3 binding groove with high affinity. Our mechanistic studies showed that S63845 effectively kills MCL1-dependent cancer cells, including multiple myeloma, leukemia, and lymphoma cells, by activating the BAX/BAK-dependent mitochondrial apoptosis pathway. In vivo experiments showed that S63845, as a single drug, exhibited potent antitumor activity and acceptable safety in a variety of cancers. In addition, MCL1 inhibitors, whether used alone or in combination with other anticancer drugs, were effective against a variety of solid tumor cell lines. These results suggest that MCL1 can be a potential target for the treatment of a variety of tumors. [2] Traditional chemotherapy drugs use cytotoxic drugs to kill cancer cells, but they have low selectivity, high toxicity, and a narrow therapeutic index. High-specificity targeted drugs, on the other hand, achieve precise killing of tumors by inhibiting molecular pathways that are crucial for tumor growth. Myeloid leukemia 1 (MCL-1) is an important pro-survival protein in the BCL-2 family and is a promising antitumor target. This study chose to investigate the effects of the small molecule inhibitor S63845, which targets MCL-1, on the normal hematopoietic system. https://pubmed.ncbi.nlm.nih.gov/37111571/

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BH3 mimics specifically occupy the hydrophobic trench of MCL-1 (selectivity for BCL-2/BCL-xL >1000-fold). [2]
Overcomes resistance to BCL-2 inhibitors in BIM defective models. [1]
Has the potential to treat hematologic malignancies (acute myeloid leukemia, 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.)