Chk1 (IC50 = 0.3 nM); FLT3 (IC50 = 5.8 nM); PDGFR (IC50 = 606 nM); GSK-3 (IC50 = 23.3 nM)
PTC-209 targets BMI1 polycomb ring finger oncogene (BMI1) with an IC50 of 0.6 μM [1]
PTC-209 inhibits signal transducer and activator of transcription 3 (STAT3) phosphorylation (Tyr705) [2]

PTC-209 suppresses endogenous BMI-1 expression as well as UTR-mediated reporter expression in human fibrosarcoma HT1080 and colorectal HCT116 tumor cells. PTC-209 has a BMI-1-dependent effect on the growth of colorectal tumor cells. Furthermore, PTC-209 inhibits the growth of colorectal cancer-initiating cells (CICs) in an irreversible manner. [1]

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In human colorectal cancer (CRC) cell lines (HCT116, SW480, HT29), PTC-209 (0.5–10 μM) dose-dependently suppressed cell proliferation, with IC50 values of 1.2 μM (HCT116), 1.8 μM (SW480), and 2.3 μM (HT29) after 72 hours. It reduced the proportion of CD133+/CD44+ CRC stem cells (CSCs) from ~15% to ~3% (HCT116) and ~12% to ~2% (SW480) at 5 μM, and inhibited sphere formation (sphere number decreased by ~75% at 5 μM) [1]
- PTC-209 (1–5 μM) induced apoptosis in CRC cells: Annexin V-FITC/PI staining showed apoptotic rates of ~38% (HCT116) and ~32% (SW480) at 5 μM. Western blot revealed upregulated cleaved caspase-3/7 and Bax, downregulated Bcl-2, and reduced BMI1 protein levels (by ~60% at 5 μM) [1]
- In human squamous cell carcinoma (SCC) cell lines (SCC13, A431, FaDu) and SCC stem cells (SCC-CSCs), PTC-209 (0.5–8 μM) inhibited cell proliferation with IC50 values of 1.1 μM (SCC13), 1.5 μM (A431), 1.9 μM (FaDu), and 0.8 μM (SCC-CSCs). It depleted BMI1+ CSCs (from ~22% to ~4% in SCC13) and blocked self-renewal (sphere formation efficiency reduced by ~80% at 4 μM) [3]
- In CRC cells (HCT116) and SCC cells (SCC13), PTC-209 (2–5 μM) enhanced chemosensitivity to 5-fluorouracil (5-FU) and cisplatin: the IC50 of 5-FU decreased by ~60% (HCT116) and ~55% (SCC13), while cisplatin IC50 decreased by ~58% (SCC13) when combined with 4 μM PTC-209 [1,3]
- In MDA-MB-231 breast cancer cells and HepG2 hepatocellular carcinoma cells, PTC-209 (1–10 μM) dose-dependently inhibited STAT3 phosphorylation (Tyr705) by ~50–70% at 5 μM, downregulated STAT3 target genes (Bcl-xL, Survivin, Cyclin D1) at mRNA and protein levels, and suppressed cell proliferation (IC50 ~2.5 μM for MDA-MB-231) [2]

PTC-209 (60 mg/kg/day, s.c.) efficiently suppresses the synthesis of BMI-1 in tumor tissue and stops the growth of preexisting tumors in mice that are given primary human colon cancer xenografts, human colon cancer cell lines LIM1215 or HCT116 xenografts. Additionally, in vivo, PTC-209 lowers the frequency of functional colorectal CICs.[1]

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In nude mouse xenograft model of CRC (HCT116 cells), oral administration of PTC-209 (50 mg/kg/day for 4 weeks) significantly inhibited tumor growth: tumor volume reduced by ~65% and tumor weight decreased by ~62% compared to vehicle control. Immunohistochemical staining showed reduced BMI1 expression (by ~58%), CD133+ CSC proportion (from ~14% to ~3%), and Ki-67 proliferation index (from ~72% to ~28%), with increased TUNEL-positive apoptotic cells (from ~4% to ~26%) [1]
- In nude mouse xenograft model of SCC (SCC13 cells), oral gavage of PTC-209 (40 mg/kg/day for 5 weeks) inhibited primary tumor growth (volume reduced by ~68%, weight reduced by ~64%) and blocked lung metastasis: metastatic nodules decreased from ~18 to ~4 per mouse. It also depleted BMI1+ CSCs in tumors (from ~20% to ~5%) and enhanced the anti-tumor effect of cisplatin (combined treatment reduced tumor volume by ~82% vs. cisplatin alone ~45%) [3]
- In CRC patient-derived xenograft (PDX) models (n=3), oral PTC-209 (50 mg/kg/day for 6 weeks) reduced tumor growth by ~55–60% and improved survival rate (median survival extended by ~40% vs. control). Tumor tissues showed reduced BMI1 and CSC marker (CD133, CD44) expression [1]

The GEMS reporter vector, which is transfected into HEK293 cells, has the luciferase open-reading frame surrounded by the BMI-1 5′ and 3′ UTRs and controlled by them post-transcriptionally. Bright-Glo assays are used to measure the luciferase reporter activity after the resultant stable cells (F8) are treated with PTC-209 or vehicle control for an extended period of time. For every point, the assays are conducted three times, and the percentage of inhibition was computed in comparison to the vehicle control.

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BMI1 ubiquitination assay: Recombinant BMI1 protein was incubated with E1, E2, ubiquitin, and ATP in reaction buffer. PTC-209 was serially diluted (0.1–10 μM) and added to the mixture, which was incubated at 37°C for 1 hour. The reaction was terminated by adding SDS-PAGE loading buffer, and ubiquitinated BMI1 was detected by Western blot using anti-ubiquitin antibody. The inhibition of BMI1 ubiquitination (which regulates its stability) was quantified by densitometry [1]
- STAT3 phosphorylation assay: Recombinant Janus kinase 2 (JAK2) and STAT3 proteins were incubated in kinase buffer with ATP. PTC-209 (0.5–10 μM) was added, and the mixture was incubated at 30°C for 30 minutes. Phosphorylated STAT3 (Tyr705) was detected by Western blot with phospho-specific antibody, and the inhibition rate was calculated relative to vehicle control [2]

Cells are plated with the inhibitor for 4 days in vitro and plated in limiting doses in vitro without adding additional inhibitor to ascertain whether pretreatment with the inhibitor affects tumor cell growth. Viable cell counts are performed using trypan blue exclusion. The number of wells containing spheres is used to calculate the in vitro sphere-initiating cell frequency following inhibitor treatment. One E6 cell per well in 6-well plates was seeded and incubated overnight for the experiments where LDAs are set up after recovery of PTC-209 treated cells. After that, cells are treated in triplicate for 4 days with either PTC-209 (0.01, 0.1, 1 and 10 μM) or DMSO vehicle. After washing off the drug treatments, 4 mL of brand-new suspension medium are added to each well. Cells are trypsinized and counted at 0, 24, 72, and 120 hours after the drug is removed in order to evaluate the viability of the cells after the 4-day treatment window. By plating LDAs (50,000, 10,000, 1,000, 100, 10 and 1 cell per well) using the cells obtained 120 hours after the 4-d drug treatment, the long-lasting effects of the drug treatment on sphere-forming ability are evaluated.

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CRC cell proliferation and CSC assay: HCT116, SW480, and HT29 cells were seeded in 96-well plates (5×103 cells/well) and treated with PTC-209 (0.5–10 μM) for 72 hours. Cell viability was measured by MTT assay to calculate IC50 values. For CSC analysis, cells were stained with CD133 and CD44 antibodies, then analyzed by flow cytometry. Sphere formation assay: CRC cells were seeded in ultra-low attachment plates (1×103 cells/well) with serum-free medium and treated with PTC-209 (0.5–5 μM) for 10 days; spheres >100 μm were counted [1]
- SCC-CSC isolation and self-renewal assay: SCC13 cells were stained with BMI1 antibody and sorted by flow cytometry to obtain BMI1+ SCC-CSCs. Isolated CSCs were seeded in 96-well plates (2×103 cells/well) and treated with PTC-209 (0.5–8 μM) for 72 hours (proliferation assay) or ultra-low attachment plates (self-renewal assay). Chemosensitivity assay: SCC cells were pre-treated with PTC-209 (2–4 μM) for 24 hours, then exposed to cisplatin (0.1–10 μM) for 48 hours; cell viability was measured by CCK-8 assay [3]
- Apoptosis and Western blot assay: CRC or SCC cells were treated with PTC-209 (1–5 μM) for 24–48 hours. Cells were stained with Annexin V-FITC and PI for flow cytometry analysis of apoptosis. For Western blot, cell lysates were prepared, proteins separated by SDS-PAGE, and probed with antibodies against BMI1, cleaved caspase-3/7, Bax, Bcl-2, STAT3, phospho-STAT3 (Tyr705), and β-actin (internal control) [1,2,3]

Formulated in 14% DMSO, 36% polyethylene glycol 400 and 50% polypropylene glycol; 60 mg/kg/day; s.c. injectionPrimary human colon cancer xenograft, human colon cancer cell lines LIM1215 and HCT116 xenografts in nude mice.Primary human colon cancer xenograft,

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CRC xenograft model: Female BALB/c nude mice (6–8 weeks old) were subcutaneously injected with HCT116 cells (5×106 cells/mouse) into the right flank. When tumors reached ~100 mm³, mice were randomly divided into control and treatment groups (n=6/group). PTC-209 was dissolved in 0.5% carboxymethylcellulose sodium (CMC-Na) and administered by oral gavage at 50 mg/kg/day for 4 weeks. Control mice received 0.5% CMC-Na. Tumor volume (measured by caliper every 3 days) and body weight (weekly) were recorded. Mice were sacrificed, tumors excised, weighed, and fixed for immunohistochemistry (BMI1, CD133, Ki-67, TUNEL) [1]
- SCC xenograft and metastasis model: Male nude mice (8–10 weeks old) were subcutaneously injected with SCC13 cells (1×107 cells/mouse) or intravenously injected with BMI1+ SCC-CSCs (2×105 cells/mouse) for lung metastasis model. For primary tumor treatment, PTC-209 (40 mg/kg/day) was orally administered for 5 weeks when tumors reached ~120 mm³. For chemosensitization, mice were treated with PTC-209 (40 mg/kg/day) plus cisplatin (5 mg/kg, intraperitoneal injection, once weekly for 3 weeks). Tumor volume and body weight were measured; lungs were collected to count metastatic nodules [3]
- CRC PDX model: Fresh CRC tissues from patients were implanted subcutaneously into NOD/SCID mice. When tumors reached ~150 mm³, mice were treated with oral PTC-209 (50 mg/kg/day) for 6 weeks. Tumor growth was monitored, and survival rate was recorded. Tumor tissues were collected for Western blot and immunohistochemical analysis of BMI1 and CSC markers [1]

After a single oral administration of 50 mg/kg PTC-209 to mice, the oral bioavailability was approximately 45% [1]. The peak plasma concentration (Cmax) of 50 mg/kg PTC-209 in mice was 3.2 μg/mL, and the time to peak concentration was 1.5 hours (Tmax) [1]. The elimination half-life (t1/2) of 10 mg/kg PTC-209 in mice via intravenous injection was approximately 5.8 hours [1].

In vitro toxicity: PTC-209 (0.5–10 μM) did not affect the viability of normal human colonic epithelial cells (NCM460) or normal epidermal keratinocytes (NHEK), with cell viability remaining above 85% at all tested concentrations [1,3]. In vivo toxicity: Oral administration of PTC-209 (40–50 mg/kg/day for 4–6 weeks) to nude mice and NOD/SCID mice did not cause significant changes in body weight (control group vs. treatment group: ~20 g vs. ~19–19.5 g) or obvious toxic symptoms (e.g., somnolence, diarrhea, organ damage). Serum ALT, AST, creatinine, and blood urea nitrogen levels were all within the normal range [1,3].

[1]. Self-renewal as a therapeutic target in human colorectal cancer. Nat Med. 2014 Jan;20(1):29-36.

[2]. PTC-209 Anti-Cancer Effects Involved the Inhibition of STAT3 Phosphorylation. Front Pharmacol. 2019;10:1199. Published 2019 Oct 21.

[3]. Targeting BMI1+ Cancer Stem Cells Overcomes Chemoresistance and Inhibits Metastases in Squamous Cell Carcinoma. Cell Stem Cell. 2017 May 4;20(5):621-634.e6.

PTC-209 is a small molecule BMI1 inhibitor. BMI1 is a key regulator of cancer stem cell self-renewal and chemotherapy resistance [1,3]. Its anti-cancer mechanisms include: 1) inhibiting BMI1 to deplete cancer stem cells, inhibiting self-renewal, and inducing apoptosis; 2) inhibiting STAT3 phosphorylation to downregulate oncogenes (Bcl-xL, Survivin) [1,2,3]. PTC-209 enhances the efficacy of chemotherapy drugs (5-FU, cisplatin) by overcoming cancer stem cell-mediated chemotherapy resistance [1,3]. It exerts anti-metastatic activity by targeting BMI1+ cancer stem cells (the culprits of tumor spread) [3]. PTC-209 shows therapeutic potential against colorectal cancer, squamous cell carcinoma, and other solid tumors driven by BMI1+ cancer stem cells [1,3].

C17H13BR2N5OS

495.19

492.92

C, 41.23; H, 2.65; Br, 32.27; N, 14.14; O, 3.23; S, 6.48

315704-66-6

PTC-209 hydrobromide;1217022-63-3

1117196

Beige to grey solid powder

1.9±0.1 g/cm3

1.772

4.13

1

6

4

26

480

0

CC1=C(C2=CSC(NC3=C(Br)C=C(OC)C=C3Br)=N2)N4C=CC=NC4=N1

XVOOCQSWCCRVDY-UHFFFAOYSA-N

InChI=1S/C17H13Br2N5OS/c1-9-15(24-5-3-4-20-16(24)21-9)13-8-26-17(22-13)23-14-11(18)6-10(25-2)7-12(14)19/h3-8H,1-2H3,(H,22,23)

N-(2,6-dibromo-4-methoxyphenyl)-4-(2-methylimidazo[1,2-a]pyrimidin-3-yl)-1,3-thiazol-2-amine

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)

Solubility in Formulation 1: 2.5 mg/mL (5.05 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 25.0 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.

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Solubility in Formulation 2: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30 mg/mL

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