Unesbulin (PTC596; 20-200 nM; for 48 hours) causes apoptosis in AML cells in a p53-independent way. Overexpression of BMI-1 renders AML cells insensitive to PTC596-induced apoptosis [1]. Unesbulin (200 nM; for 10 hours) promotes accumulation of cells in G2/M phase [1]. Unesbulin (0.012-1 μM; for 20 hours) dramatically lowers BMI-1 protein levels [1]. Unesbulin inhibits APC/CCDC20 activity, resulting to persistent activation of CDK1 and CDK2, ultimately causing hyperphosphorylation of BMI1 [2].

Unesbulin (PTC596; 20-200 nM; for 48 hours) causes apoptosis in AML cells in a p53-independent way. Overexpression of BMI-1 renders AML cells insensitive to PTC596-induced apoptosis [1]. Unesbulin (200 nM; for 10 hours) promotes accumulation of cells in G2/M phase [1]. Unesbulin (0.012-1 μM; for 20 hours) dramatically lowers BMI-1 protein levels [1]. Unesbulin inhibits APC/CCDC20 activity, resulting to persistent activation of CDK1 and CDK2, ultimately causing hyperphosphorylation of BMI1 [2].

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Treatment with PTC596 (20-200 nM, 20 hours) reduced BMI-1 protein expression in MOLM-13 and U-937 acute myeloid leukemia (AML) cells in a dose-dependent manner. [1]
PTC596 exhibited nanomolar potency against AML cell lines, with an average IC50 (concentration inhibiting 50% cell growth) of 30.7 ± 4.1 nM and an average ED50 (effective concentration inducing 50% annexin V positivity) of 60.3 ± 6.7 nM at 48 hours. [1]
Apoptosis induced by PTC596 was p53-independent, as p53 mutant cell lines (U-937, HL-60) were as sensitive as wild-type p53 cell lines, and stable p53 knockdown in OCI-AML3, MOLM-13, and MV4-11 cells did not significantly alter their sensitivity to PTC596. [1]
PTC596 did not increase p53 protein levels in AML cell lines, and BMI-1 knockdown by siRNA also did not increase p53 levels, suggesting p53-mediated signaling is not involved in PTC596-induced apoptosis. [1]
Overexpression of BMI-1 in MOLM-13 and K562 cells significantly desensitized them to PTC596-induced apoptosis, indicating on-target BMI-1 inhibition. [1]
Treatment with PTC596 (200 nM, 10 hours) induced G2/M cell cycle arrest in both p53 wild-type (MOLM-13) and p53-defective (U-937) AML cells, associated with morphological features of mitotic arrest. [1]
Western blot analysis showed that PTC596 treatment (≥110 nM, 20 hours) in MOLM-13 cells reduced levels of BMI-1 and its downstream target ubiquitinated histone H2A, increased levels of phosphorylated CDK1 (T161) and phosphorylated CDK2 (T160), and elevated levels of cyclin B1 and securin. [1]
PTC596 induced mitochondrial apoptosis, as evidenced by BAX conformational change, caspase-3 cleavage, and loss of mitochondrial membrane potential (Δψm) in MOLM-13 and U-937 cells. [1]
PTC596 treatment decreased the protein levels of the anti-apoptotic protein MCL-1 in a dose- and time-dependent manner in MOLM-13 and U-937 cells, without affecting mRNA levels. Levels of BCL-2, BCL-XL, and BAX were not significantly changed. [1]
The reduction of MCL-1 by PTC596 was not significantly affected by the protein synthesis inhibitor cycloheximide but was blocked by the proteasome inhibitor MG132 and the pan-caspase inhibitor Z-VAD-FMK, suggesting PTC596 inhibits new MCL-1 protein synthesis and enhances its proteasome- and caspase-mediated degradation. [1]
In primary AML cells from 13 patients, PTC596 induced dose-dependent apoptosis (annexin V positivity), with >30% specific increase observed in 10 of 13 samples at 500 nM. [1]
Immature CD34+CD38low/– AML stem/progenitor cells were more susceptible to PTC596-induced apoptosis than mature AML cells. [1]
Mass cytometry (CyTOF) analysis of a primary AML sample showed that PTC596 reduced MCL-1 expression, most prominently in the CD34+CD38low/– stem/progenitor population. It also reduced levels of phosphorylated AKT (Ser473, 42.8% reduction), among other MCL-1 inducers. [1]

Mice survival can be markedly prolonged by unesbulin (PTC596; 5 mg/kg; oral gavage; once every 3 days for 13 days) [1]. Compared to control SCID mice with K562 cells, unesbulin (20 mg/kg; oral gavage; once weekly for 15 days) produced noticeably smaller tumor volumes [1]. The oral gavage of unesbulin (10 or 12.5 mg/kg) twice weekly until death was found to significantly extend the survival of NOD-SCID mice bearing HL-60 cells compared to the vehicle-treated group [1].

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In an aggressive MOLM-13 xenograft model using NOD-SCID/IL2Ry-KO (NSG) mice, oral administration of PTC596 (5 mg/kg every 3 days) starting one day after leukemia cell injection significantly reduced the percentage of circulating human CD45+ leukemia cells in peripheral blood by day 13 and significantly prolonged median survival (16.5 days vs. 13.0 days for vehicle) compared to the control group. [1]
In a K562 subcutaneous xenograft model in CB17 SCID mice, weekly oral administration of PTC596 (20 mg/kg) after tumor establishment significantly reduced mean tumor volume compared to vehicle control. [1]
In an HL-60 intravenous xenograft model in NOD-SCID mice, oral administration of PTC596 (10 or 12.5 mg/kg, twice weekly) starting three days after cell injection significantly prolonged mouse survival in a dose-dependent manner compared to vehicle control. [1]
A spleen colony-forming unit (CFU-S) assay indicated that PTC596 treatment (two oral doses of 20 mg/kg, one week apart) in donor mice did not statistically reduce the number of macroscopic spleen colonies formed by transplanted bone marrow cells in lethally irradiated recipient mice, compared to vehicle treatment, suggesting it spares normal hematopoietic stem/progenitor cells. In contrast, 5-FU treatment severely reduced colony numbers. [1]

Apoptosis analysis[1]
Cell Types: AML cell lines (MOLM-13, OCI-AML3, MOLM-14, MV4-11, U-937, HL-60)
Tested Concentrations: 20, 50, 100, 200 nM
Incubation Duration: For 48 hrs (hours)
Experimental Results: Induced apoptosis in a dose- and time-dependent manner, with average IC50 and ED50 values of 30.7 nM and 60.3 nM, respectively, across the six cell lines.

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Cell cycle analysis [1]
Cell Types: MOLM-13 and U-937 Cell
Tested Concentrations: 200 nM
Incubation Duration: 10 hrs (hours)
Experimental Results: Result in accumulation of cells in G2/M phase, while the percentage of cells in G1 phase decreases.

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Western Blot Analysis[1]
Cell Types: MOLM-13 Cell
Tested Concentrations: 0.012, 0.037, 0.11, 0.33, 1 μM
Incubation Duration: 20 hrs (hours)
Experimental Results: Significant protein levels of BMI-1 and its downstream target ubiquitinated histone H2A reduce. Cyclin B1 and securin levels are increased.

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Cell viability and apoptosis were assessed using trypan blue dye exclusion and annexin V binding by flow cytometry. Cells were seeded at a density of 1–2 × 10^5 cells/ml, exposed to various concentrations of compounds for specified durations (e.g., 48 or 72 hours), and then analyzed. For annexin V assay, cells were harvested, stained with annexin V conjugate, and analyzed by flow cytometry to determine the percentage of apoptotic cells. [1]
For apoptosis mechanism studies, mitochondrial membrane potential loss (Δψm) was assessed by staining cells with a fluorescent dye (e.g., DiOC6) and analyzing by flow cytometry. BAX conformational change was detected using a conformation-specific antibody against active BAX, followed by flow cytometry. Caspase-3 cleavage was analyzed by flow cytometry using an antibody specific for cleaved/active caspase-3. [1]
Cell cycle analysis was performed using the Click-IT EdU incorporation kit. Cells were incubated with EdU to label newly synthesized DNA, then fixed, permeabilized, and stained with a fluorescent azide conjugate via click chemistry. DNA content was stained with propidium iodide (PI). Cell cycle distribution (G1, S, G2/M phases) was determined by flow cytometry based on EdU incorporation versus DNA content. [1]
Intracellular protein levels (e.g., BMI-1, p53) were analyzed by flow cytometry. Cells were fixed, permeabilized, stained with specific primary antibodies and fluorescently labeled secondary antibodies, and the mean fluorescence intensity was measured by flow cytometry. [1]
Western blot analysis was used to assess protein expression. Cells were lysed, proteins were separated by SDS-PAGE, transferred to membranes, and probed with specific primary antibodies (e.g., against BMI-1, MCL-1, BCL-2 family proteins, cell cycle regulators, phosphorylated proteins). After incubation with horseradish peroxidase-conjugated secondary antibodies, signals were detected using chemiluminescence. [1]
Quantitative real-time PCR (qRT-PCR) was used to measure mRNA levels. Total RNA was extracted, reverse transcribed into cDNA, and target gene expression (e.g., BMI1, MCL1) was quantified using TaqMan gene expression assays on a real-time PCR system. GAPDH served as the reference gene for normalization. [1]
For gene knockdown, cells were transfected with small interfering RNA (siRNA) oligonucleotides targeting BMI-1 or a negative control using a nucleofection device and specific nucleofector kits. Cells were then cultured and analyzed 48-72 hours post-transfection. [1]
For stable overexpression, cells were transduced with lentiviruses harboring BMI-1 cDNA or empty vector control. Stable populations were selected and validated for BMI-1 overexpression by Western blot or flow cytometry. [1]
Mass cytometry (CyTOF) was used for single-cell analysis of primary AML samples. Cells were stained with a panel of metal-conjugated antibodies targeting surface markers (e.g., CD34, CD38) and intracellular proteins (e.g., MCL-1, phosphorylated signaling proteins). Cells were analyzed by time-of-flight mass cytometry, and data were processed using specialized software. [1]

Animal/Disease Models: NOD-SCID/IL2Rγ-KO (NSG) mice with MOLM-13 cells [1]
Doses: 5 mg/kg
Route of Administration: po (oral gavage); once every 3 days for 13 days
Experimental Results: vs. Mice survived Dramatically longer than vehicle-treated mice in a dose-dependent manner.

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For the MOLM-13 intravenous xenograft model, NSG mice were injected intravenously with 1 × 10^6 MOLM-13 cells. One day later (day 1), mice were randomized into treatment groups. PTC596 was administered by oral gavage at a dose of 5 mg/kg, suspended in a vehicle of 0.5% hydroxypropyl methylcellulose and 0.1% Tween 80 in distilled water. Dosing occurred every 3 days. Peripheral blood was collected on day 13 to analyze circulating leukemia cells (human CD45+) by flow cytometry. Survival was monitored as the primary endpoint. [1]
For the K562 subcutaneous xenograft model, CB17 SCID mice were injected subcutaneously in the flank with 2 × 10^7 K562 cells mixed 1:1 with Matrigel. After tumors were established (11 days later), mice were randomized into groups matched for tumor volume. PTC596 was administered orally at a dose of 20 mg/kg, once a week. Tumor sizes were measured regularly. [1]
For the HL-60 intravenous xenograft model, NOD-SCID mice were injected intravenously with 1 × 10^7 HL-60 cells. Three days after injection, mice were randomized into groups. PTC596 was administered orally at doses of 10 or 12.5 mg/kg, twice a week. Survival was monitored as the primary endpoint. [1]
For the spleen colony-forming unit (CFU-S) assay to assess effects on normal hematopoiesis, donor C57BL/6 mice received either a single oral dose of vehicle, two oral doses of 20 mg/kg PTC596 given one week apart, or a single intraperitoneal injection of 150 mg/kg 5-fluorouracil (5-FU). Two weeks after the last dose, bone marrow cells were harvested from donor mice. A total of 5 × 10^4 bone marrow cells per mouse were transplanted intravenously into lethally irradiated (10 Gy) recipient mice. Eleven days after transplantation, spleens were harvested, fixed in Carnoy's solution, and macroscopic colonies on the spleen surface were counted. [1]

[1]. The novel BMI-1 inhibitor PTC596 downregulates MCL-1 and induces p53-independent mitochondrial apoptosis in acute myeloid leukemia progenitor cells. Blood Cancer J. 2017 Feb 17;7(2):e527.

[2]. BMI1 inhibitor PTC596.

Unesbulin is an orally potent inhibitor of the multicomb ring finger oncogene BMI1 (B-cell specific Moloney murine leukemia virus integration site 1) with potential antitumor activity. After oral administration, unesbulin targets BMI1 expressed in tumor cells and cancer stem cells (CSCs), inducing BMI1 hyperphosphorylation and leading to its degradation. This inhibits BMI1-mediated signal transduction pathways, thereby reducing the proliferation of BMI1-expressing tumor cells. BMI1 is a key protein of the multicomb inhibitory complex 1 (PRC1), overexpressed in certain tumor cell types, and plays a crucial role in CSC survival, proliferation, and chemotherapy resistance. Its expression is associated with increased tumor invasiveness and poor prognosis. PTC596 is a novel small molecule discovered through high-throughput screening of BMI-1 inhibitors, exhibiting superior pharmacokinetic properties compared to the previous inhibitor PTC-209. [1]
Its mechanism of action includes inducing CDK1 to bind to BMI-1 and CDK1-mediated phosphorylation of BMI-1 at a new N-terminal site, leading to BMI-1 degradation. [1]
PTC596 downregulates MCL-1 protein levels through posttranscriptional mechanisms, including inhibiting the synthesis of new proteins and enhancing proteasome and caspase-mediated degradation, leading to mitochondrial membrane potential instability and induction of mitochondrial apoptosis. [1]
The antileukemic activity of PTC596 is independent of p53, so PTC596 may be a potential treatment option for AML patients with poor complex karyotypes or treatment-related AML, who often have p53 mutations and poor prognosis. [1]
PTC596 exhibits potent activity against CD34+CD38low/– AML stem/progenitor cells, which are the cause of relapse and chemotherapy resistance, while appearing not to affect normal hematopoietic stem/progenitor cells in CFU-S assays. [1]
As of the time of this publication, PTC596 has entered a Phase I clinical trial (NCT02404480) for patients with advanced solid tumors. [1]

C₁₉H₁₃F₅N₆

420.3387

420.112

1610964-64-1

1610964-64-1;Unesbulin HCl;

74223469

White to off-white solid powder

1.6±0.1 g/cm3

583.9±60.0 °C at 760 mmHg

307.0±32.9 °C

0.0±1.6 mmHg at 25°C

1.645

3.68

2

10

3

30

586

0

TWLWOOPCEXYVBE-UHFFFAOYSA-N

InChI=1S/C19H13F5N6/c1-9-26-13-7-4-11(20)8-14(13)30(9)18-28-16(25)15(21)17(29-18)27-12-5-2-10(3-6-12)19(22,23)24/h2-8H,1H3,(H3,25,27,28,29)

5-Fluoro-2-(6-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)-N4-(4-(trifluoromethyl)phenyl)pyrimidine-4,6-diamine

PTC596 PTC-596 PTC 596

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)

DMSO : ~16.67 mg/mL (~39.66 mM)

Solubility in Formulation 1: ≥ 1.67 mg/mL (3.97 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 16.7 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: ≥ 1.67 mg/mL (3.97 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 16.7 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.)