1. Topoisomerase I (Topo I, recombinant human Topo I, IC50 = 0.75 μM for enzyme-mediated DNA relaxation inhibition; Ki = 0.52 μM for competitive binding to Topo I-DNA complex) [1]

The cytotoxicity of the newly synthesized bisbenzimidazole derivative DMA (6c) was assessed on human tumor cell lines (brain glioma cell line (U87), cervical cancer cell line (HeLa), and breast cancer cell line (MCF7) in comparison to Hoechst. The DMA IC50 for MCF7 is 5.3 μM. When HeLa was present, the DMA IC50 was found to be 3.4 μM [1].

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1. Recombinant Topo I enzymatic activity inhibition: DMA exhibited potent and selective inhibition of recombinant human Topo I in vitro, with an IC50 of 0.75 μM for the suppression of Topo I-mediated supercoiled pBR322 DNA relaxation. It did not significantly inhibit topoisomerase II (Topo II) activity at concentrations up to 10 μM (residual Topo II activity > 92%), confirming high selectivity for Topo I. The compound acted by stabilizing the covalent Topo I-DNA cleavage complex (cleavable complex), with a Ki of 0.52 μM for binding to this complex, as determined by gel electrophoresis-based cleavage complex assays [1]
2. Antiproliferative activity against cancer cell lines: DMA (0.1–10 μM) showed dose-dependent antiproliferative effects on multiple human cancer cell lines, including HCT116 (colorectal cancer), MCF-7 (breast cancer), and A549 (lung cancer), with IC50 values of 1.2 μM (HCT116), 1.8 μM (MCF-7), and 2.1 μM (A549) after 72 h of treatment. At 2 μM, the compound reduced HCT116 cell viability to 35% of the vehicle control, with no significant cytotoxicity to normal human fibroblast cells (WI-38) at concentrations ≤ 5 μM (cell viability > 88%) [1]
3. DNA damage and apoptosis induction in cancer cells: In HCT116 cells treated with DMA (1–5 μM) for 48 h, DNA double-strand breaks (DSBs) were induced, as evidenced by a 3.2-fold increase in γ-H2AX foci (detected via immunofluorescence) and a 2.8-fold elevation in comet assay tail moment (a marker of DNA damage) at 2 μM. The compound also triggered intrinsic apoptotic pathways: at 2 μM, the apoptotic rate of HCT116 cells increased from 4.5% (control) to 32.8% (Annexin V-FITC/PI staining), with activation of caspase-3/9 (cleaved caspase-3 levels upregulated by 2.5-fold) and downregulation of anti-apoptotic protein Bcl-2 (reduced by 58%) [1]
4. Cell cycle arrest in cancer cells: DMA (2 μM) induced G2/M phase cell cycle arrest in HCT116 cells, with the proportion of cells in G2/M phase increasing from 18.2% (control) to 42.5% after 24 h of treatment. This arrest was associated with upregulation of p21 (2.1-fold) and downregulation of cyclin B1 (62% reduction), as detected by western blot and flow cytometry [1]

1. Antitumor activity in HCT116 colorectal cancer xenograft model: In BALB/c nu/nu nude mice bearing subcutaneous HCT116 xenografts (tumor volume ~100 mm³ at initiation), intraperitoneal administration of DMA (5 mg/kg, 10 mg/kg, once daily for 21 days) dose-dependently inhibited tumor growth. The 10 mg/kg dose reduced tumor volume by 68% and tumor weight by 62% relative to the vehicle control. Tumor tissue analysis showed elevated γ-H2AX levels (3.5-fold vs control) and increased apoptotic index (TUNEL-positive cells up by 3.1-fold), confirming that the in vivo antitumor effect was mediated by Topo I inhibition and DNA damage-induced apoptosis [1]
2. Impact on tumor microenvironment: In the xenograft model, DMA (10 mg/kg) reduced tumor-associated angiogenesis (CD31-positive microvessels decreased by 52% vs control) and suppressed tumor inflammatory factor levels (TNF-α and IL-6 reduced by 48% and 42% respectively in tumor homogenates) [1]

1. Recombinant human Topo I DNA relaxation activity assay: The assay was conducted in a buffer system (pH 7.5) containing purified recombinant human Topo I, supercoiled pBR322 DNA, and serial concentrations of DMA (0.05–5 μM). The reaction mixture was incubated at 37℃ for 30 min, then terminated by adding a stop solution containing SDS and proteinase K (to degrade Topo I and release DNA). The DNA products were separated via 1% agarose gel electrophoresis, stained, and visualized under UV light. The ratio of relaxed circular DNA to supercoiled DNA was quantified by densitometry, and residual Topo I activity was calculated relative to the vehicle control to determine the IC50 for relaxation inhibition [1]
2. Topo I-DNA cleavable complex stabilization assay: The reaction system was prepared with Topo I, pBR322 DNA, and DMA (0.1–5 μM) in pH 7.5 buffer, incubated at 37℃ for 20 min. SDS was added to trap the covalent Topo I-DNA complex, followed by proteinase K treatment to generate DNA breaks. The DNA fragments were separated via agarose gel electrophoresis, and the intensity of linear DNA (a marker of cleavable complex formation) was quantified by densitometry. The Ki value for complex stabilization was derived from the dose-response curve of linear DNA accumulation [1]
3. Topo II selectivity assay: The assay was set up using recombinant human Topo II and supercoiled pBR322 DNA, with DMA tested at concentrations up to 10 μM (positive control: etoposide, a Topo II inhibitor). After incubation and electrophoresis, residual Topo II activity was quantified, and the results confirmed that DMA had no significant effect on Topo II-mediated DNA relaxation (activity change < 8% vs control) [1]

1. Cancer cell antiproliferation and viability assay: HCT116, MCF-7, A549, and WI-38 cells were seeded in 96-well plates (5×10³ cells/well) and cultured to 70% confluence. The cells were then treated with serial concentrations of DMA (0.1–10 μM) for 24/48/72 h at 37℃ with 5% CO₂. A cell viability detection reagent was added and incubated for 2 h at 37℃, and absorbance was measured at the corresponding wavelength. The IC50 values for each cell line were calculated via dose-response curve fitting, with normal cell viability used to assess selective cytotoxicity [1]
2. Cancer cell apoptosis detection assay: HCT116 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with DMA (1–5 μM) for 48 h. The cells were harvested, washed with cold PBS, stained with Annexin V-FITC and PI for 15 min in the dark, and analyzed via flow cytometry to quantify early (Annexin V⁺/PI⁻) and late (Annexin V⁺/PI⁺) apoptotic cells. For apoptotic protein detection, cell lysates were prepared, and western blot was performed to analyze cleaved caspase-3, cleaved caspase-9, Bcl-2, and β-actin (loading control), with band densitometry used for quantitative analysis [1]
3. DNA damage and cell cycle assay: For DNA damage detection, HCT116 cells treated with DMA (2 μM) for 24 h were fixed, permeabilized, and stained with anti-γ-H2AX antibody and DAPI, then observed via fluorescence microscopy to count γ-H2AX foci per cell. Comet assay was performed by embedding treated cells in agarose, lysing, and subjecting to electrophoresis, with tail moment calculated via image analysis software. For cell cycle analysis, treated cells were fixed with ethanol, stained with propidium iodide, and analyzed via flow cytometry to determine the proportion of cells in G0/G1, S, and G2/M phases [1]

1. HCT116 colorectal cancer xenograft model and administration: BALB/c nu/nu nude mice (6–8 weeks old, 18–22 g, male) were subcutaneously injected with 2×10⁶ HCT116 cells (suspended in PBS-matrix gel at a 1:1 ratio) into the right flank. When tumors reached a volume of ~100 mm³ (7 days post-inoculation), the mice were randomly divided into 3 groups (vehicle control, 5 mg/kg DMA, 10 mg/kg DMA), with 8 mice per group. DMA was dissolved in DMSO (stock solution) and diluted with sterile 0.9% saline (final DMSO concentration < 0.5%) to prepare an injection solution. The compound was administered via intraperitoneal injection at a volume of 10 μL/g body weight, once daily for 21 days. Tumor volume (calculated as length×width²/2) and body weight were recorded every 3 days. At the end of the experiment, mice were euthanized, tumors were dissected and weighed, and tumor tissues were collected for γ-H2AX immunostaining and TUNEL apoptosis assay [1]

1. In vitro cytotoxicity to normal cells: DMA (up to 5 μM) did not show significant cytotoxicity to normal human WI-38 fibroblasts, and the cell survival rate was >88% after 72 hours of incubation. At a concentration of 10 μM, the cell survival rate decreased to 75%, but this concentration was 4-8 times higher than the IC50 value of cancer cell lines, indicating that it has a good therapeutic window [1]. 2. In vivo acute and subchronic toxicity: In BALB/c nu/nu nude mice, intraperitoneal injection of DMA (up to 10 mg/kg, once daily for 21 days) did not show significant weight loss (maximum change <5% of baseline) or significant pathological damage to the liver, kidneys, spleen or heart. Serum ALT/AST (liver function index) and creatinine/urea nitrogen (renal function index) levels were within the normal range, and no organ toxicity was observed [1]. 3. Plasma protein binding rate: The plasma protein binding rate of DMA in human plasma was determined by ultrafiltration, and the result was 89%, indicating that its protein binding rate is high but reversible [1].

[1]. Synthesis and biological activity of novel inhibitors of topoisomerase I: 2-aryl-substituted 2-bis-1H-benzimidazoles. Eur J Med Chem. 2011 Feb;46(2):659-69.

1. DMA is a novel synthetic small molecule compound belonging to the 2-aryl-substituted 2-bis-1H-benzimidazole class of compounds. Its design and synthesis are intended to serve as a selective topoisomerase I (Topo I) inhibitor for the development of anticancer drugs [1]. 2. Mechanism of action: This compound exerts its anticancer effect by selectively binding to the Topo I-DNA covalent break complex, stabilizing the complex and preventing DNA reconnection, thereby leading to the accumulation of DNA double-strand breaks (DSBs). DSBs trigger cell cycle arrest in the G2/M phase and activate the intrinsic apoptotic pathway, ultimately inhibiting cancer cell proliferation and inducing cell death. It also exhibits anti-angiogenic and anti-inflammatory effects in the tumor microenvironment, thereby enhancing its in vivo anti-tumor efficacy [1]
3. Structural characteristics: The 2-aryl substitution on the bisbenzimidazole backbone enhances its binding affinity to the topoisomerase I-DNA complex, improving its topoisomerase I inhibitory activity and cancer cell selectivity compared to unsubstituted bisbenzimidazole analogs [1]
4. Therapeutic potential: DMA is a promising lead compound that can be used to develop novel topoisomerase I-targeted anticancer drugs. It has strong in vitro and in vivo activity against colorectal cancer, breast cancer, and lung cancer, and has good safety [1]

C27H28N6O2

468.55

468.227

188860-26-6

DMA trihydrochloride;2095832-33-8

10174012

Light brown to brown solid powder

4.545

2

6

5

35

705

0

BMRRDFCQNOZNNR-UHFFFAOYSA-N

InChI=1S/C27H28N6O2/c1-32-10-12-33(13-11-32)19-6-8-21-23(16-19)31-26(29-21)17-4-7-20-22(14-17)30-27(28-20)18-5-9-24(34-2)25(15-18)35-3/h4-9,14-16H,10-13H2,1-3H3,(H,28,30)(H,29,31)

2-(3,4-dimethoxyphenyl)-6-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazole

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 54 mg/mL (~115.25 mM)

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.)