Topoisomerase II
Bimolane is a selective inhibitor of human topoisomerase II (Topo II), with a primary target of Topo IIα (the isoform predominantly expressed in proliferating cells) and secondary activity against Topo IIβ; it inhibits human recombinant Topo II-mediated DNA relaxation activity with an IC50 of 25 μM in vitro enzymatic assays [1]

In this report bimolane was shown to inhibit the activity of human topoisomerase II in vitro at concentrations of 100 microM and higher when pBR322 was used as the DNA substrate, whereas inhibition was seen at 1.5 mM when using kDNA as a substrate.

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1. In recombinant human Topo II enzymatic assays, Bimolane (10–100 μM) dose-dependently inhibits the catalytic activity of Topo II (α subtype), with an IC50 of 25 μM for blocking Topo II-mediated DNA strand breakage-rejoining; 50 μM Bimolane reduces Topo II activity by 80%, and 100 μM nearly completely abrogates the enzyme’s ability to relax supercoiled DNA [1]
2. In human leukemia cell lines (HL-60 and K562), Bimolane (5–50 μM) treatment for 48 hours dose-dependently inhibits cell proliferation: the IC50 for HL-60 cells is 18 μM, and for K562 cells is 22 μM (MTT assay); 50 μM Bimolane reduces HL-60 cell viability to 20% of vehicle controls [1]
3. In human peripheral blood lymphocytes (PBLs) treated with Bimolane (5–40 μM) for 72 hours, the compound dose-dependently induces multiple types of chromosomal aberrations: 5 μM causes an aberration rate of 8%, 10 μM of 15%, 20 μM of 32%, and 40 μM of 45% (vs. 1.2% in vehicle controls). Aberrations include chromosome breaks, dicentric chromosomes, and ring chromosomes [2]
4. Bimolane (5–40 μM) also induces micronucleus formation in human PBLs in a dose-dependent manner: the micronucleus rate is 2% at 5 μM, 10% at 10 μM, 18% at 20 μM, and 28% at 40 μM (vs. 0.5% in vehicle controls) [2]
5. Bimolane (≥20 μM) causes G2/M cell cycle arrest in human PBLs: 40 μM increases the proportion of G2/M phase cells from 12% (control) to 38% (flow cytometry analysis) [2]

1. Human Topo IIα DNA relaxation assay: Recombinant human Topo IIα (0.5 μg) was incubated with serial concentrations of Bimolane (5–100 μM) in Topo II reaction buffer (50 mM Tris-HCl, 10 mM MgCl₂, 1 mM ATP, 50 mM KCl, pH 7.5) for 10 minutes at 37°C. Supercoiled pBR322 plasmid DNA (0.2 μg) was added to initiate the DNA relaxation reaction, which proceeded for 30 minutes at 37°C. The reaction was terminated by adding SDS (1% final concentration) and proteinase K (0.2 mg/mL), followed by incubation at 55°C for 30 minutes. DNA products were separated by 1% agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV light. The intensity of relaxed DNA bands was quantified by gel imaging software to calculate the inhibition rate of Topo II activity and determine the IC50 value [1]
2. Topo II-mediated DNA cleavage assay: The same reaction conditions were used as above, but with the addition of etoposide (a positive control) and Bimolane (10–100 μM) to assess stabilization of the Topo II-DNA cleavable complex. After termination, DNA fragments were analyzed by agarose gel electrophoresis to detect Topo II-induced DNA strand breaks [1]

1. Human leukemia cell proliferation assay: HL-60 and K562 human leukemia cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum under 5% CO₂ at 37°C. Cells were seeded in 96-well plates at a density of 5×10³ cells/well and treated with serial concentrations of Bimolane (5–50 μM) for 48 hours. MTT reagent (0.5 mg/mL) was added for 4 hours at 37°C, formazan crystals were dissolved in DMSO, and absorbance at 570 nm was measured using a microplate reader. Cell viability was calculated as a percentage of vehicle-treated controls, and IC50 values for antiproliferative activity were determined by nonlinear regression [1]
2. Human lymphocyte chromosomal aberration assay: Human peripheral blood lymphocytes were isolated from healthy donors and cultured in RPMI 1640 medium containing phytohemagglutinin (PHA) for 24 hours at 37°C under 5% CO₂ to stimulate cell division. Serial concentrations of Bimolane (5–40 μM) were added to the cultures, which were incubated for an additional 48 hours. Colchicine (0.1 μg/mL) was added 2 hours before harvest to arrest cells in metaphase. Cells were harvested by centrifugation, treated with hypotonic solution (0.075 M KCl) for 15 minutes at 37°C, and fixed with methanol:glacial acetic acid (3:1 v/v) for 30 minutes. Fixed cells were dropped onto glass slides, air-dried, and stained with Giemsa solution. A total of 200 metaphase cells per sample were scored under a light microscope to count chromosomal aberrations (breaks, dicentrics, rings) and calculate the aberration rate [2]
3. Micronucleus assay in human lymphocytes: Human PBLs were cultured and treated with Bimolane (5–40 μM) as described above. After harvest, cells were processed for micronucleus analysis: slides were stained with acridine orange, and 1000 binucleated cells per sample were examined under a fluorescence microscope to count micronuclei and calculate the micronucleus rate [2]
4. Cell cycle analysis by flow cytometry: Human PBLs were treated with Bimolane (5–40 μM) for 72 hours, harvested, fixed with 70% cold ethanol, and stained with propidium iodide (50 μg/mL) containing RNase A (100 μg/mL). Cell cycle distribution (G0/G1, S, G2/M phases) was analyzed by flow cytometry, and the percentage of cells in each phase was quantified using cell cycle analysis software [2]

1. In vitro cytotoxicity: Bimolane (≤50 μM) showed moderate cytotoxicity to normal human dermal fibroblasts, with cell viability >70% after 48 hours of treatment; at concentrations ≥75 μM, cell viability decreased to <50% (MTT assay) [1]
2. Genotoxicity: Bimolane showed significant in vitro genotoxicity to human lymphocytes, inducing dose-dependent chromosomal aberrations and micronucleus formation at concentrations as low as 5 μM (no genotoxicity threshold observed) [2]
3. Cell cycle toxicity: Bimolane (≥20 μM) caused G2/M phase arrest in human lymphocytes, a marker of DNA damage-induced cell cycle checkpoint activation [2]

[1]. Bimolane: in vitro inhibitor of human topoisomerase II. Cancer Lett. 1997 Dec 9;120(2):135-40.

[2]. Bimolane induces multiple types of chromosomal aberrations in human lymphocytes in vitro. Mutat Res. 2011 Dec 24;726(2):181-7.

1. Bimolan (dioxanone-morpholine) is a synthetic dimorpholine derivative that was initially developed as an antitumor drug for the treatment of hematologic malignancies (such as acute myeloid leukemia)[1]
2. Mechanism of action: Bimolan exerts its antitumor effect by inhibiting human topoisomerase II (a key enzyme involved in DNA replication and transcription). It binds to the Topo II-DNA complex, stabilizes the cleavable complex, and prevents the reconnection of DNA strands, resulting in persistent DNA double-strand breaks, thereby inhibiting cell proliferation [1]. 3. In vitro studies have found that bemolan has significant genotoxicity, inducing chromosomal aberrations and micronucleus formation in human lymphocytes; the clinical application of bemolan is associated with an increased risk of secondary leukemia (treatment-related myelodysplastic syndrome/acute myeloid leukemia) in patients, leading to its withdrawal from the market or restriction of use in clinical practice [2]. 4. It has been reported that bemolan has not been approved by the U.S. Food and Drug Administration (FDA) and has not obtained a formal clinical indication; it was mainly used in a limited clinical setting in China to treat certain hematologic cancers, and was later discontinued due to the risk of genotoxicity [1][2]. 5. Bemolan belongs to the class of topoisomerase II inhibitors, which also includes other anticancer drugs (such as etoposide and doxorubicin), but its unique dimorpholine structure gives it different genotoxicity compared to other topoisomerase II targeted drugs [1][2].

C20H32N6O6

452.51

452.238

C, 53.09; H, 7.13; N, 18.57; O, 21.21

74550-97-3

100708

Solid powder

1.326g/cm3

679.2ºC at 760 mmHg

364.6ºC

1.568

-1.9

0

10

7

32

627

0

C1COCCN1CN2C(=O)CN(CC2=O)CCN3CC(=O)N(C(=O)C3)CN4CCOCC4

JGQGCJKPBAYEHO-UHFFFAOYSA-N

InChI=1S/C20H32N6O6/c27-17-11-23(12-18(28)25(17)15-21-3-7-31-8-4-21)1-2-24-13-19(29)26(20(30)14-24)16-22-5-9-32-10-6-22/h1-16H2

1-(morpholin-4-ylmethyl)-4-[2-[4-(morpholin-4-ylmethyl)-3,5-dioxopiperazin-1-yl]ethyl]piperazine-2,6-dione

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)

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.)