Tubulin at the colchicine-binding site [1]

In HA22T, Hep3B, and HepG2 cells, CHM-1 (0-100 μM; 24 hours) significantly inhibits growth in a concentration-dependent manner, with HA22T cells showing the strongest effect (IC50 = 0.75 μM)[1]. In HA22T cells, CHM-1 (0-10 μM; 24 hours) dramatically boosts cyclin B1's binding to Cdc2 [1].

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CHM-1 induced concentration-dependent growth inhibition in HA22T, Hep3B, and HepG2 human hepatocellular carcinoma cells, with IC50 values of 0.75 ± 0.11 μmol/L for HA22T cells after 24 h treatment [1]

CHM-1 was less toxic to normal cells: human fibroblasts (MRC5) and mouse hepatocytes (AML12) showed higher IC50 values compared to cancer cells [1]

CHM-1 (1 μmol/L) caused accumulation of cells in G2-M phase with concomitant loss in G0-G1 phase, maximum effect at 24 h; hypodiploid cells (apoptotic cells) increased at 48 h [1]

CHM-1 induced apoptosis as shown by positive TUNEL staining and concentration-dependent oligonucleosomal DNA fragmentation after 24-48 h treatment [1]

CHM-1 (3 μmol/L for 24 h) disrupted microtubule organization, causing microtubule depolymerization similar to colchicine, as visualized by immunofluorescence microscopy [1]

CHM-1 inhibited tubulin polymerization in a concentration-dependent manner (3, 10, 30 μmol/L) in an in vitro microtubule assembly assay [1]

CHM-1 (3 μmol/L for 24 h) inhibited in vivo microtubule assembly: polymerized tubulin level was 20.8 ± 6.2% compared to 40.5 ± 2.0% in control [1]

CHM-1 (1-10 μmol/L) did not affect stathmin expression but induced concentration-dependent inhibition of MAP4 expression [1]

CHM-1 sustained high levels of cyclin B1, increased Thr161-phosphorylated Cdc2, and did not change total Cdc2 or Tyr15-phosphorylated Cdc2 [1]

CHM-1 significantly increased the binding of cyclin B1 to Cdc2 and increased Cdc2 kinase activity [1]

Roscovitine (a cyclin-dependent kinase inhibitor) significantly abolished CHM-1-induced Cdc2 activity, growth inhibition, and elevation of MPM2 phosphopeptides [1]

CHM-1 did not induce activation of effector caspases (caspase-3, -6, -7) or initiator caspases (caspase-8, -9), nor did it alter cIAP1, cIAP2, XIAP, or survivin levels [1]

CHM-1 did not increase caspase-3, -8, or -9 activity; the pan-caspase inhibitor z-VAD-fmk did not prevent CHM-1-induced cell death [1]

CHM-1 induced translocation of apoptosis-inducing factor (AIF) from mitochondria to cytosol and then to nucleus, but not endonuclease G [1]

Confocal microscopy showed AIF translocation into the nucleus and nuclear condensation after 24 h treatment with CHM-1 (3 μmol/L) [1]

Roscovitine alone triggered AIF release and did not prevent CHM-1-induced AIF translocation [1]

siRNA targeting AIF substantially attenuated CHM-1-induced AIF translocation to the cytosol [1]

The growth of HA22T tumors is dose-dependently inhibited by CHM-1 (10 mg/kg; IP) [1].

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CHM-1 (5 and 10 mg/kg, i.p., twice weekly) inhibited tumor growth in severe combined immunodeficiency mice bearing subcutaneous HA22T xenografts; tumor weights were significantly reduced at the end of treatment (day 70) [1]

CHM-1 did not significantly affect body weights of treated mice compared to doxorubicin [1]

CHM-1 prolonged the lifespan of mice intraperitoneally inoculated with HA22T cells, with a T/C value of 167% (P < 0.01); doxorubicin-treated mice all died on day 53 [1]

Immunohistochemical analysis of tumor specimens from CHM-1-treated animals showed increased translocation of AIF to nuclei and accumulation of MPM2 in nuclei of apoptotic cells [1]

In vitro microtubule assembly assay: Tubulin proteins (>99% purity) were suspended in G-PEM buffer plus glycerol in the absence (DMSO) or presence of CHM-1 (3, 10, or 30 μmol/L), paclitaxel (10 μmol/L), or vincristine (10 μmol/L). The polymerization of tubulin/microtubule was detected using a commercial kit [1]

In vivo microtubule assembly assay: After 24 h treatment with vehicle or antimitotic agents (3 μmol/L each), cells were lysed and the cytosolic and cytoskeletal fractions (containing soluble and polymerized tubulin, respectively) were separated by centrifugation. The fractions were resolved by SDS-PAGE and immunoblotted with an antibody against β-tubulin [1]

Cdc2 kinase assay: Cdc2 kinase activity was determined using a commercial assay kit according to the manufacturer's instructions. Cells were treated with CHM-1 with or without roscovitine, and activity was measured [1]

Caspase activity assay: Caspase protease activity in cell lysates was assayed by spectrophotometric detection using a commercial kit [1]

Cell Viability Assay[1]
Cell Types: HA22T, Hep3B and HepG2 Cell
Tested Concentrations: 0-100 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induction of cell cycle G2-M arrest, followed by apoptosis.

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Western Blot Analysis [1]
Cell Types: HA22T Cell
Tested Concentrations: 0-10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induced changes in the expression and phosphorylation status of G2-M regulatory factors in human hepatocellular carcinoma cells.

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Cell viability assay: Cells were inoculated in 96-well plates at 10⁴ per well, treated with various concentrations of CHM-1 for 24 h, and cell survival was assessed using MTT colorimetric assay [1]

Apoptosis detection: Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) immunofluorescence staining was used to detect apoptotic cells. Quantitative assessment of oligonucleosomal DNA fragmentation was performed using a Cell Death ELISA kit [1]

Cell cycle analysis: Cells were treated with CHM-1 (1 μmol/L) for indicated times, stained with propidium iodide, and cell cycle distribution was determined using flow cytometry [1]

Immunocytochemistry and confocal microscopy: After treatment, cells were fixed with methanol, blocked with bovine serum albumin, stained with anti-β-tubulin or anti-AIF monoclonal antibody, then FITC-conjugated secondary antibody. Mitochondria were stained with Mitotracker Red CMXRos (100 mmol/L) before fixation. Nuclear staining was done with DAPI. Cells were imaged with a confocal system [1]

Immunoprecipitation and Western blot: Total protein was immunoprecipitated with anti-Cdc2 antibody, mixed overnight at 4°C, then protein A/G PLUS-agarose beads were added. Immunoblotting was done using anti-cyclin B1 and anti-Cdc2 antibodies. For Western blot, equal amounts of protein were separated by SDS-PAGE and immunoblotted with specific primary antibodies [1]

RNA interference: Stealth siRNA targeting AIF mRNA (sequence: 5'-GGCCAGGGUACUGAUUGUAUUCUGAA-3') or control siRNA (5'-GGCCGUGCAGUUAGUAUUCUUACGAA-3') was transfected into HA22T cells at 50% confluency with 200 nmol/L siRNA duplexes using lipofectamine. After 24 h, cells were treated with CHM-1 and analyzed by Western blot [1]

Animal/Disease Models: Male severe combined immunodeficiency mice (HA22T) [1]
Doses: 10 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: Induced dose-dependent HA22T tumor growth inhibition.

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Subcutaneous xenograft model: CHM-1 was dissolved in a vehicle mixture of DMSO/Cremophor EL/saline. Doxorubicin (positive control) was diluted with sterile saline. Male severe combined immunodeficiency mice (4-6 weeks old) were injected subcutaneously with 6 × 10⁶ HA22T cells. When tumors reached approximately 50 mm³, mice were treated with CHM-1 (5 and 10 mg/kg) or doxorubicin (1 mg/kg) by intraperitoneal injection twice weekly for 1 month. Tumor volumes were calculated as V = lw²/2 [1]

Intraperitoneal antitumor activity assay: HA22T cells (10⁷ per mouse) were injected intraperitoneally on day 0. CHM-1 and doxorubicin were delivered intraperitoneally twice weekly starting on day 1 for 7 consecutive weeks. Antitumor activity was assessed as T/C (%) = (median survival time of drug-treated group / median survival time of control group) × 100 [1]

CHM-1 in its original capsule formulation displayed low plasma concentrations relative to oral doses, indicating low bioavailability, possibly due to low aqueous solubility. A water‑soluble prodrug CHM-1-P was synthesized to address this problem. Phosphate prodrugs are generally freely soluble in water and readily hydrolyzed in vivo by alkaline phosphatase [2].

CHM-1 was less toxic to normal cells (MRC5 human fibroblasts and AML12 mouse hepatocytes) compared to doxorubicin, with broader IC50 range between cancer and normal cells [1]

In vivo, body weights of mice were not significantly affected by CHM-1 treatment compared to doxorubicin [1]

[1]. CHM-1, a novel synthetic quinolone with potent and selective antimitotic antitumor activity against human hepatocellular carcinoma in vitro and in vivo. Mol Cancer Ther. 2008 Feb;7(2):350-60.

[2]. CHM-1, a novel microtubule-destabilizing agent exhibits antitumor activity via inducing the expression of SIRT2 in human breast cancer cells. Chem Biol Interact. 2018 Jun 1;289:98-108.

[3]. CHM-1, a new vascular targeting agent, induces apoptosis of human umbilical vein endothelial cells via p53-mediated death receptor 5 up-regulation. J Biol Chem. 2010 Feb 19;285(8):5497-506.

CHM-1 (2'-fluoro-6,7-methylenedioxy-2-phenyl-4-quinolone) is a synthetic 6,7-substituted 2-phenyl-4-quinolone derivative initially identified as an anti-invasive agent in hepatocellular carcinoma cells (inhibiting MMP-9) [1]

CHM-1 exhibits a novel antimitotic antitumor activity against human hepatocellular carcinoma via a caspase-independent pathway, involving AIF translocation from mitochondria to nucleus [1]

CHM-1 is proposed as a promising chemotherapeutic agent worthy of further development into a clinical trial candidate for treating cancer, especially hepatocellular carcinoma [1]

C16H10FNO3

283.25

283.064

154554-41-3

375860

Off-white to light yellow solid powder

3.062

1

5

1

21

466

0

ZMYDAPJHGNEFGQ-UHFFFAOYSA-N

InChI=1S/C16H10FNO3/c17-11-4-2-1-3-9(11)12-6-14(19)10-5-15-16(21-8-20-15)7-13(10)18-12/h1-7H,8H2,(H,18,19)

6-(2-fluorophenyl)-[1,3]dioxolo[4,5-g]quinolin-8(5H)-one

CHM-1 CHM1CHM 1 NSC-656158 NSC656158 NSC 656158

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 : ~5 mg/mL (~17.65 mM)
H2O : < 0.1 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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