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, displaying classical hallmarks of apoptosis that are dependent on caspases and BAX/BAK-mediated mitochondrial outer membrane permeabilisation. Compared to mouse MCL-1, it has a 6 fold higher affinity for human MCL-1[1]. S63845 is effective in vitro, in vivo, and on AML samples as well as haematological cancer-derived cell lines, but it is not very effective on normal human haematopoietic progenitor cells[2].

---

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].

---

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/

---

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]

Running buffer is composed of 10 mM HEPES pH 7.4, 175 mM NaCl, 25 μM EDTA, 1 mM TCEP, 0.01% P20, and 1% DMSO. Proteins that have been double His-tagged are used to create the ligand surface. The compound is diluted serially in buffer and injected onto the protein surface. The flow rate used for all sample measurements is 30 μL per minute (injection time: 120 s, dissociation time: 360 s). By repeatedly injecting 0.35 M EDTA pH 8.0 with 0.1 mg/mL trypsin, 0.5 M imidazole, and 45% DMSO (60 s, 15 μL per min), the sensor surface is restored.

---

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]

Before using anti-FLAG antibody for immunoprecipitation, HeLa cells transduced with Flag-BCL-XL, Flag-BCL-2, or Flag-MCL1 expression constructs are treated for 4 hours with increasing concentrations of S63845. Immunoblotting is used to examine immunoprecipitates and total inputs for FLAG-tagged proteins as well as the related BAK and BAX proteins.

---

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]

Human multiple myeloma (H929 and AMO1) xenografted mice; Intravenously injected (i.v.), 25 mg/kg

---

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]

Xenograft efficacy: 5 × 10⁶ tumor cells were subcutaneously implanted into NSG mice. Treatment began when the tumor volume reached 100-200 mm³. S63845 was dissolved in 10% DMSO/40% PEG300/50% PBS solution and administered intraperitoneally daily (25-50 mg/kg) for 14-21 days. [2] PDX model: Primary AML patient cells were transplanted into mice. Treatment began when the concentration of human CD45+ cells in the blood reached 1%. [2] Tolerance: Dose escalation (12.5-75 mg/kg intraperitoneally daily). The maximum tolerated dose (MTD) was defined as a weight loss of ≤20%. [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] Journal of Xiangya Medicine. 2017.

[2] 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]

---

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 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 has the potential to become an antitumor target. This study chose to investigate the effect of the small molecule inhibitor S63845, which targets MCL-1, on the normal hematopoietic system. https://pubmed.ncbi.nlm.nih.gov/37111571/

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

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

S63845 Trifluoroacetic acid; S-63845 TFA; S 63845

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)

DMSO: >41.45mg/mL
Water: >10mg/mL
Methanol: >20mg/mL

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.

---

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.

---

View More

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.

---

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