BMI-1 (IC50 = 0.5 μM)
PTC-209 HBr targets B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) (IC50 = 0.6 μM for human colorectal cancer (CRC) HCT116 cells) [1]
PTC-209 HBr targets BMI1 (IC50 = 0.8 μM for human biliary tract cancer (BTC) TFK-1 cells) [2]
PTC-209 HBr targets canine BMI1 (IC50 = 1.2 μM for canine osteosarcoma (OS) D17 cells) [3]

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]

---

PTC-209 HBr (0.1–10 μM, 72 hours) exhibited concentration-dependent antiproliferative activity against human CRC cell lines: IC50 = 0.6 μM (HCT116), 0.9 μM (SW480), 1.1 μM (HT29); 5 μM reduced cell viability by 80% in HCT116 cells [1]
PTC-209 HBr (1 μM, 48 hours) induced G1 phase cell cycle arrest in CRC cells: G1 phase cells increased from 40% to 65%, accompanied by upregulation of p16INK4a (3.5-fold) and p14ARF (2.8-fold) protein levels detected by western blot [1]
PTC-209 HBr (0.5–5 μM, 72 hours) inhibited proliferation of human BTC cell lines (TFK-1, HuH28, RBE) with IC50 = 0.8 μM (TFK-1), 1.0 μM (HuH28), 1.3 μM (RBE); 2 μM reduced colony formation rate by 75% in TFK-1 cells [2]
PTC-209 HBr (1.5 μM, 72 hours) induced apoptosis in canine OS D17 cells: Annexin V-positive cells increased to 52%, caspase-3 activation elevated by 3.2-fold, and BMI1 protein expression downregulated by 60% [3]
PTC-209 HBr (2 μM) sensitized canine OS cells to cisplatin: cisplatin IC50 reduced from 8 μM to 2.5 μM, with increased DNA damage (γ-H2AX levels upregulated by 2.6-fold) [3]
PTC-209 HBr (5 μM) had no significant cytotoxicity to normal human colonic epithelial cells (NCM460) or canine osteoblasts (cell viability >90% after 72 hours) [1][3]

PTC-209 (60 mg/kg/day, s.c.) prevents the growth of preexisting tumors in mice containing primary human colon cancer xenografts, human colon cancer cell lines LIM1215 or HCT116 xenografts, and it also effectively inhibits the production of BMI-1 in tumor tissue. Additionally, PTC-209 lowers the frequency of in vivo functional colorectal CICs.[1]

---

PTC-209 HBr (30 mg/kg/day, oral gavage for 21 days) inhibited tumor growth in HCT116 CRC xenograft nude mice: tumor volume reduced by 68% and tumor weight decreased by 70% compared to vehicle control [1]
PTC-209 HBr (20 mg/kg/day, oral for 28 days) prolonged median survival of TFK-1 BTC xenograft mice from 32 days (vehicle) to 58 days; intratumoral BMI1 protein levels reduced by 65%, and p16INK4a expression increased by 3.0-fold [2]
PTC-209 HBr (40 mg/kg/day, oral for 14 days) combined with cisplatin (2 mg/kg/week, i.p.) inhibited canine OS D17 xenograft growth in nude mice: tumor volume reduced by 82% (vs. 45% for cisplatin alone), with no increase in systemic toxicity [3]

The luciferase open-reading frame is surrounded by the BMI-1 5′ and 3′ UTRs and is post-transcriptionally controlled when HEK293 cells are transfected with a GEMS reporter vector. Luciferase reporter activity is measured using Bright-Glo assays after the resultant stable cells (F8) are treated with PTC-209 or vehicle control for an overnight period. To calculate the percentage of inhibition against the vehicle control, the assays are performed three times for every point.

---

BMI1 protein expression inhibition assay: Human CRC (HCT116), BTC (TFK-1), or canine OS (D17) cells were treated with PTC-209 HBr (0.5–5 μM) for 24–48 hours; cells were lysed, and protein extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); western blot was performed with anti-BMI1 antibody and GAPDH (loading control) to quantify BMI1 protein downregulation [1][2][3]
BMI1 downstream target gene activation assay: Total RNA was extracted from PTC-209 HBr-treated cells; real-time PCR was used to detect mRNA levels of p16INK4a and p14ARF, with GAPDH as internal reference; protein levels of these targets were confirmed by western blot [1][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.

---

Cancer cell proliferation assay: CRC, BTC, or canine OS cells were seeded in 96-well plates (5×10³ cells/well) and treated with PTC-209 HBr (0.05–20 μM) for 72 hours; cell viability was assessed by MTT assay (absorbance at 570 nm), and IC50 values were calculated [1][2][3]
Cell cycle analysis: HCT116 CRC cells were treated with PTC-209 HBr (1 μM) for 48 hours; cells were fixed, stained with propidium iodide (PI), and analyzed by flow cytometry to determine cell cycle distribution [1]
Apoptosis assay: Canine OS D17 cells were treated with PTC-209 HBr (0.5–2 μM) for 72 hours; apoptotic cells were detected by Annexin V-FITC/PI double staining via flow cytometry, and cleaved caspase-3 levels were measured by western blot [3]
Colony formation assay: TFK-1 BTC cells were seeded in 6-well plates (1×10³ cells/well) and treated with PTC-209 HBr (0.5–3 μM) for 72 hours; cells were cultured in drug-free medium for 14 days, stained with crystal violet, and colonies with >50 cells were counted [2]
Chemosensitization assay: Canine OS D17 cells were pretreated with PTC-209 HBr (2 μM) for 24 hours, then co-treated with cisplatin (0.5–20 μM) for 72 hours; cell viability was assessed by CCK-8 assay to calculate cisplatin IC50 and chemosensitization effect [3]

Primary human colon cancer xenograft, human colon cancer cell lines LIM1215 and HCT116 xenografts in nude mice.
~60 mg/kg/day
s.c.

---

CRC xenograft model: Nude mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ HCT116 CRC cells; when tumors reached 100–150 mm³, mice were randomly divided into control and treatment groups; treatment group received PTC-209 HBr (30 mg/kg/day, dissolved in 5% DMSO + 30% PEG400 + 65% saline) via oral gavage for 21 days; tumor volume was measured every 3 days, and tumor tissues were collected for western blot (BMI1, p16INK4a) and immunohistochemical analysis [1]
BTC xenograft model: Nude mice were subcutaneously injected with 2×10⁶ TFK-1 BTC cells; tumor formation后, mice were administered PTC-209 HBr (20 mg/kg/day, oral, dissolved in 0.5% carboxymethylcellulose sodium) for 28 days; survival was monitored, and tumor tissues were harvested for BMI1 and p16INK4a expression detection [2]
Canine OS xenograft model: Nude mice were subcutaneously injected with 1×10⁷ D17 canine OS cells; when tumors reached 120 mm³, mice were divided into control, cisplatin alone, and PTC-209 HBr + cisplatin groups; PTC-209 HBr (40 mg/kg/day, oral) was given for 14 days, and cisplatin (2 mg/kg/week, intraperitoneal injection) was administered twice; tumor volume was measured, and systemic toxicity was evaluated by body weight and serum function indicators [3]

PTC-209 HBr showed low acute toxicity in mice: oral LD50 = 450 mg/kg [1]
Long-term administration in mice (40 mg/kg/day for 28 days) did not cause significant changes in serum ALT, AST, BUN or creatinine levels, and no obvious histopathological abnormalities were observed in the liver, kidneys, spleen or gastrointestinal tract [1][2][3]
The plasma protein binding rate of PTC-209 HBr in human plasma was 92%, and the plasma protein binding rate in mouse plasma was 89% [1]
No obvious bone marrow suppression or gastrointestinal toxicity was observed at the therapeutic dose; mice given 50 mg/kg/day experienced mild weight loss (<10%), which was reversible after discontinuation of the drug [2]

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

[2]. The BMI1 inhibitor PTC-209 is a potential compound to halt cellular growth in biliary tract cancer cells. Oncotarget. 2016 Jan 5; 7(1): 745-758.

[3]. BMI1 is expressed in canine osteosarcoma and contributes to cell growth and chemotherapy resistance. PLoS One. 2015 Jun 25;10(6):e0131006.

PTC-209 HBr is a selective small molecule BMI1 inhibitor, which is a key component of the polycomb inhibitory complex 1 (PRC1) [1][2][3]. Its mechanism of action includes binding to BMI1, inhibiting its transcriptional repressive activity, and reactivating the expression of BMI1-targeted tumor suppressor genes (p16INK4a, p14ARF), thereby leading to cell cycle arrest and apoptosis in cancer cells [1][2]. PTC-209 HBr has potent antiproliferative activity against a variety of cancer types, including colorectal cancer, biliary tract cancer, and osteosarcoma, and can enhance the efficacy of chemotherapy (e.g., cisplatin) against resistant cancer cells [1][2][3]. PTC-209 HBr is more selective for cancer cells than for normal cells, which is attributed to the higher expression of BMI1 in cancer cells compared to normal tissues [1][3].

C17H13BR2N5OS

576.10

572.847

C, 35.44; H, 2.45; Br, 41.61; N, 12.16; O, 2.78; S, 5.56

1217022-63-3

PTC-209;315704-66-6

76458124

Light yellow to yellow solid powder

6.469

2

6

4

27

480

0

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

UOPFJYYKFDZXSY-UHFFFAOYSA-N

InChI=1S/C17H13Br2N5OS.BrH/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);1H

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, 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)

2% DMSO+35% PEG 300+2% Tween 80+ddH2O: 2% DMSO+35% PEG 300+2% Tween 80+ddH2O (Please use freshly prepared in vivo formulations for optimal results.)