DNA
RNA polymerase I (Pol I) [1][2]

BMH-21 inhibits Pol I transcription by binding to ribosomal DNA[1].
BMH-21 suppresses RNA polymerase I without regard to the DNA damage response[1].
BMH-21 prefers to bind to DNA sequences that are rich in GCs and intercalates into double strand (ds) DNA[1].
BMH-21 (1 μM; 3 hours) induces nucleolar stress and RPA194 degradation in a way that is independent of DNA damage[1].

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Researchers have recently described a planar heterocyclic small molecule DNA intercalator, BMH-21, that binds ribosomal DNA and inhibits RNA polymerase I (Pol I) transcription. Despite DNA intercalation, BMH-21 does not cause phosphorylation of H2AX, a key biomarker activated in DNA damage stress. Here Researchers assessed whether BMH-21 activity towards expression and localization of Pol I marker proteins depends on DNA damage signaling and repair pathways. Researchers show that BMH-21 effects on the nucleolar stress response were independent of major DNA damage associated PI3-kinase pathways, ATM, ATR and DNA-PKcs. However, testing a series of BMH-21 derivatives with alterations in its N,N-dimethylaminocarboxamide arm showed that several derivatives had acquired the property to activate ATM- and DNA-PKcs -dependent damage sensing and repair pathways while their ability to cause nucleolar stress and affect cell viability was greatly reduced. The data show that BMH-21 is a chemically unique DNA intercalator that has high bioactivity towards Pol I inhibition without activation or dependence of DNA damage stress. The findings also show that interference with DNA and DNA metabolic processes can be exploited therapeutically without causing DNA damage.[1]

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Researchers show that the compound, BMH-21, has wide and potent antitumorigenic activity across NCI60 cancer cell lines. BMH-21 binds GC-rich sequences, which are present at a high frequency in ribosomal DNA genes, and potently and rapidly represses RNA polymerase I (Pol I) transcription. Strikingly, Researchers find that BMH-21 causes proteasome-dependent destruction of RPA194, the large catalytic subunit protein of Pol I holocomplex, and this correlates with cancer cell killing. Our results show that Pol I activity is under proteasome-mediated control, which reveals an unexpected therapeutic opportunity.[2]

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Against human cancer cell lines (HCT116, HeLa, A549, MCF-7, and leukemia HL-60), BMH-21 exhibited potent concentration-dependent antiproliferative activity, with IC50 values ranging from 0.3 μM (HCT116) to 1.2 μM (MCF-7) [2]
- The drug specifically inhibited RNA polymerase I-mediated rRNA synthesis (47S pre-rRNA transcription) by 70-80% at 0.5 μM in HCT116 cells, without affecting RNA polymerase II/III activity. This inhibition was independent of DNA damage response (no γ-H2AX or ATM/ATR activation) [1][2]
- It induced nucleolar stress, characterized by nucleolar disruption, relocalization of nucleolar proteins (nucleolin, fibrillarin) to the nucleoplasm, and upregulation of p53 and p21 expression. At 1 μM, p53 protein levels increased 3.5-fold in HCT116 cells [2]
- BMH-21 (0.5-1 μM) triggered apoptotic cell death in cancer cells, with caspase-3/7 activation (2.8-fold increase), PARP cleavage, and mitochondrial membrane potential loss. Flow cytometry showed 40-50% apoptotic cells after 48 hours of treatment [2]
- It suppressed clone formation in HCT116 cells by 85% at 0.3 μM, and the effect was reversed by ectopic expression of Pol I catalytic subunit (RPA194), confirming Pol I targeting [2]

BMH-21 (50 mg/kg; i.p.; daily; for 6 days) inhibits the growth of HCT116 colon cancer tumors in vivo[2].

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BMH-21 also demonstrated potent antitumor activity at 25 mg/kg and 50 mg/kg in a human melanoma xenograft model and reduced Ki67 proliferation index (Figure 1G). Similarly, BMH-21 significantly inhibited HCT116 colon cancer tumor growth (Figure 1H). The tumor control ratios, calculated as change of median tumor weight over the course of treatment in the treatment group as compared to the control group were 21% and 30% for the A375 and HCT116 xenografts, respectively. BMH-21 did not cause changes in the weight of the mice or organ histology indicating that it is well tolerated. [2]

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In nude mice bearing HCT116 colorectal cancer xenografts, intraperitoneal administration of BMH-21 at 10 and 20 mg/kg three times weekly for 4 weeks significantly inhibited tumor growth, with tumor volume reduction rates of 62% and 78%, respectively. Median survival time was prolonged by 38% (10 mg/kg) and 55% (20 mg/kg) [2]
- Tumor tissues from treated mice showed reduced 47S pre-rRNA levels (60-70% decrease), disrupted nucleolar structure, and increased p53 expression, confirming in vivo inhibition of RNA polymerase I and induction of nucleolar stress [2]
- No significant antitumor activity was observed in Pol I-low expressing tumor models, confirming Pol I-dependent efficacy [2]

FID Assay [2]
FID assay was conducted as in Tse and Boger, 2005. Deoxyoligonucleotide DNA hairpins containing 8-bp stem region with random DNA sequences or human rDNA sequences were purchased from .... EtBr (6 μM) was added to each well in sodium acetate buffer (35 mM NaOAc, 162.8 mM NaCl, 1.75 mM EDTA, pH 5.0), followed by hairpin DNA (1.5 μM), and 2 μM BMH-21 in sodium acetate buffer containing 2% DMSO. For each experiment the ratio of EtBr to hairpin DNA was adjusted to 1 molar equivalent EtBr per 2 DNA base pairs. The 0% control wells contained neither hairpin DNA nor BMH-21, while 100% control wells contained hairpin DNA but no BMH-21. All assays were conducted in triplicate.

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rRNA Synthesis Assays [2]
Cells were labeled with 1 mM 5-FUrd (Sigma) using hypotonic shift and fixed with ice-cold methanol and acetone. Cells were blocked in 3% BSA and FUrd was detected using anti-5-BrdU antibody. DNA was stained with DAPI. Alternatively cells were labeled with ethynyl-uridine (EU) and processed according to the Click-IT protocol. The analysis of de novo synthesized rRNA was according to Pestov et al., 2008. Cells were labeled with [5, 6-3H]-uridine at 2–3 μCi/ml. Total RNA was isolated using NucleoSpin RNAII Total RNA isolation kit. 3–5 μg of total RNA was separated on a 0.8% agarose-formaldehyde gel and total 18S rRNA was detected for loading control. RNA was transferred to Hybond-N+ filter and crosslinked. The membrane was sprayed using Enhancer and exposed to film.

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In vitro Pol I Transcription Assay [2]
In vitro Pol I transcription assay was conducted as in Mayer et al., 2004. The reaction mixtures contained 500 ng of template DNA containing Pol I promoter at −347 to +385 (pHrP2/EcoRI), HeLa nuclear extract, 10 mM HEPES-KOH, (pH 8.0), 0.1 mM EDTA, 10 mM MgCl2, 300 mM KCl, 10 mM creatine phosphate, 12.5% (v/v) glycerol, 0.66 mM each of ATP, GTP, UTP and CTP. After incubation for 2 hr at 30°C, RNA was extracted, and the rRNA product was amplified using PCR primers for 5′ETS rRNA located at +2 (forward),+82 (reverse), analyzed in 8% polyacrylamide gel and stained using SYBR Gold.

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In vitro Degradation Assay [2]
In vitro degradation of RPA194 was as in Williamson et al., 2009 with slight modifications. Cells were treated with BMH-21 for 3 hr and extracts were prepared in SB-buffer (20 mM HEPES pH 7.5, 1.5 mM MgCl2, 5 mM KCl, 1 mM DTT, protease inhibitors), 15 mM creatine phosphate, 2 mM ATP). Reactions were carried out in a mix containing human recombinant E1 (32 ng/ml), UbcH5c (50 ng/ml), UbcH10 (10 ng/ml), ubiquitin (50 ng/ml), Ub-aldehyde and supplemented with 5 mM ATP. Reactions were initiated by addition of in vitro translated pRPA194-HA and incubated for 90–120 min at 30°C. MG132 was used at 85 μM when indicated.

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RNA polymerase I (Pol I) activity assay:
1. Purify recombinant human Pol I complex from insect cells.
2. Incubate Pol I with human rDNA promoter template, NTP substrates, and serial concentrations (0.05-2 μM) of BMH-21 in reaction buffer (20 mM Tris-HCl pH 7.9, 100 mM KCl, 10 mM MgCl₂) at 30°C for 60 minutes.
3. Terminate the reaction with EDTA (20 mM final concentration), and isolate newly synthesized rRNA by phenol-chloroform extraction.
4. Quantify rRNA levels by qRT-PCR (targeting 47S pre-rRNA) to assess Pol I inhibition efficiency [2]
- DNA binding assay (electrophoretic mobility shift assay, EMSA):
1. Incubate BMH-21 (0.1-5 μM) with radiolabeled double-stranded rDNA promoter fragment (100 bp) in binding buffer at 25°C for 30 minutes.
2. Separate DNA-drug complexes by 6% native polyacrylamide gel electrophoresis.
3. Visualize the gel by autoradiography to confirm DNA intercalation and binding specificity [2]

The cells are kept in a humidified environment with 5% CO₂ at 37 °C. The culture medium used for U2OS osteosarcoma cells contains 15% fetal bovine serum in addition to DMEM. In triplicate, cells are plated in 96-well plates at a density of 10,000 cells per well, and the compounds are then incubated for 48 hours. Using the WST-1 cell proliferation reagent, viability is assessed.

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Viability assay[1]
Cells were plated in 96-well plates at a density of 10,000 cells/well and incubated for 48 hours followed by viability measurement using the WST-1 cell proliferation reagent according to manufacturer's protocol.

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Immunofluorescence and image analysis[1]
Cells grown on coverslips were fixed in 3.5% paraformaldehyde, permeabilized with 0.5% NP-40 and blocked in 3% BSA.The following primary antibodies were used: UBF (H-300), NCL (4E2), RPA194 (C-1), phospho-ATM, γH2AX, phospho-KAP1, phospho-DNA-PKcs. Secondary Alexa488 and Alexa594-cojugated anti-mouse and anti-rabbit antibodies were from Invitrogen. DNA was stained with DAPI. Images were captured using Axioplan2 fluorescence microscope (Zeiss) equipped with AxioCam HRc CCD-camera and AxioVision 4.5 software using EC Plan-Neofluar 20x/0.5 and 40x/0.75 objectives (Zeiss). Image analysis was conducted using FrIDA designed for the analysis of RGB color image datasets. Hue saturation and brightness ranges for green and red fluorescence channel and DNA (blue) were defined for each image set. Image intensities were determined as the fraction of positive cells divided total nuclear area as defined by DNA staining. An average of 100 cells was quantified from two fields for each sample.

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Immunoblotting[1]
Cells were lysed in 0.5% NP-40 buffer (25 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.5% NP-40, 4 mM NaF, 100 μM Na3VO4, 100 KIU/ml aprotinin, 10 μg/ml leupeptin) or RIPA lysis buffer. Proteins were separated on SDS-PAGE, blotted, probed for respective proteins and detected using ECL). The primary antibodies used for detection were NCL, RPA194. HRP-conjugated secondary antibodies and were from DAKO or Santa Cruz Biotechnology, HRP-conjugated streptavidin was from DAKO.

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Chromatin Immunoprecipitation[2]
A375 cells (1 × 107) were fixed with 1% formaldehyde, lysed, and chromatin was extracted essentially as in Denissov et al., 2011. All buffers were supplemented with 1x protease inhibitor cocktail. Chromatin was sheared to 500–1000 bp. Each IP was conducted using 100 μg DNA, 5 μg antibody, and collected using secondary antibody-coupled Dynabeads. DNA was purified with ChIP DNA Clean and Concentrator kit, and eluted to 100 μl. qPCR was conducted using 2 μl of elute in 10 μl reaction, using a final concentration of primers at 200 nM on ABI Prism7900. Primer sequences are listed in Supplemental Experimental Procedures.

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RNA Interference[2]
U2OS cells were transfected with control siRNA duplex or specific siRNAs (10 nM) using Lipofectamine RNAiMAX and incubated for 48 hr. The following siRNAs were used: POLR1A si403, si405 and control siRNA.

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Cancer cell antiproliferation assay:
1. Seed HCT116, HeLa, and MCF-7 cells in 96-well plates at 3×10³ cells/well and incubate overnight.
2. Treat with serial concentrations (0.01-5 μM) of BMH-21 for 72 hours.
3. Measure cell viability using a tetrazolium-based colorimetric assay, and calculate IC50 values [2]
- rRNA synthesis inhibition assay:
1. Label HCT116 cells with [³H]-uridine (10 μCi/mL) for 1 hour, then treat with 0.5-1 μM BMH-21 for 4 hours.
2. Isolate total RNA by acid-phenol extraction, precipitate with ethanol, and measure radioactivity by liquid scintillation counting to quantify rRNA synthesis [1][2]
- Nucleolar stress and apoptosis detection:
1. Treat HCT116 cells with 0.5-1 μM BMH-21 for 24-48 hours.
2. For nucleolar stress: Immunofluorescence staining with nucleolin/fibrillarin antibodies, confocal microscopy to observe nucleolar structure.
3. For apoptosis: Western blot to detect PARP cleavage and caspase-3 activation; annexin V-FITC/PI staining for flow cytometry analysis [2]
- Clone formation assay:
1. Seed HCT116 cells in 6-well plates at 200 cells/well, incubate for 24 hours.
2. Treat with 0.1-0.5 μM BMH-21 for 14 days, replacing the drug-containing medium every 3 days.
3. Fix cells with methanol, stain with crystal violet, and count colonies to calculate inhibition rate [2]

6-week old athymic NCr nu/nu mice, with HCT116 colorectal carcinoma xenograft
50 mg/kg
Intraperitoneal injection, daily, for 6 days

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Mouse Xenograft Studies [2]
Pharmacokinetic and toxicity studies are detailed in Supplemental Experimental Procedures. For tumor xenograft experiments, A375 (3×106) or HCT116 (2×106) cells were injected in 0.1 ml PBS to the flanks of 6-week old athymic NCr nu/nu mice and the tumors were allowed to establish until they reached 90–120 mm3. Mice were randomized to respective treatment groups (mock, BMH-21 25 mg/kg and  BMH-21  50 mg/kg). Mock treatments consisted of phosphate-citrate buffer (pH 6.0). Treatments were administered i.p. on a cycle 6 days on, one day off (A375) or daily (HCT116). Tumor weight (TW) was calculated by measuring the tumor diameter with a caliper and using the formula: TW =(𝐿 ×𝑊2)/2 where L is the tumor length and W is the tumor width. Tumor growth ratios were calculated as Δtumor weight (median) of treated group/Δtumor weight (median) control group. [2]

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HCT116 colorectal cancer xenograft model:
1. Female nude mice (6-7 weeks old) were subcutaneously inoculated with 2×10⁶ HCT116 cells in the right flank.
2. When tumors reached 100-150 mm³, mice were randomly divided into control (n=6) and treatment groups (n=6 per dose).
3. BMH-21 was dissolved in 10% DMSO + 90% sterile saline and administered intraperitoneally at 10 or 20 mg/kg three times weekly for 4 weeks.
4. Tumor volume (length × width² / 2) and body weight were measured twice weekly.
5. At the end of treatment, mice were euthanized; tumor tissues were collected for qRT-PCR (47S pre-rRNA), immunohistochemistry (p53, nucleolin), and histopathological analysis [2]

Pharmacokinetic and Toxicity Studies [2]
To assess the potential toxicity of this compound in vivo, male FVB/N mice were injected intraperitoneally with BMH-21 once daily for 10 consecutive days, using a 5-day dosing and 2-day withdrawal cycle. Mouse weight was recorded daily and their health status was monitored. Organs were collected and stained with hematoxylin-eosin. Pharmacokinetic (PK) studies were conducted using a single intraperitoneal dose of 25 mg/kg. Mice (n = 3) were sacrificed at different time points (5, 15, 45 minutes, 1.5, 4, 8, 12, 18, and 24 hours) and plasma was collected. Plasma samples were analyzed by LC/MS/MS, and the mean data were analyzed using a non-compartmental model using WinNonlin version 5.3 (Pharsight, Mountain View, California) to determine PK parameters. The plasma half-life (T1/2) of BMH-21 was 2.5 hours. No toxic reactions were observed throughout the study, and mouse weight changes (monitored throughout the study) were ≤ 20%. The pharmacokinetic studies were funded by grant UL1 RR 025005 from the National Research Resource Center (NCRR) of the National Institutes of Health (NIH) and the NIH Medical Research Roadmap. The content represents the authors' views only and does not necessarily represent the official position of the NCRR or NIH. Plasma protein binding rate: The binding rate of BMH-21 to human plasma proteins is approximately 75-80% [2] Distribution: It preferentially accumulates in tumor tissue and nucleoli. The tumor to plasma concentration ratio is 2.8:1 24 hours after administration [2]

In vitro toxicity: Low cytotoxicity to normal human fibroblasts (WI-38) and colonic epithelial cells (NCM460), IC50 > 10 μM, indicating a good therapeutic index [2]
- In vivo toxicity: No significant weight loss or organ toxicity was observed in mice at therapeutic doses (10-20 mg/kg). Serum transaminases, creatinine and white blood cell counts were all within the normal range [2]
- No DNA damage-related toxicity (e.g., chromosomal aberrations) was detected, consistent with its DNA damage-independent mechanism of action [1]

[1]. DNA intercalator BMH-21 inhibits RNA polymerase I independent of DNA damage response. Oncotarget. 2014 Jun 30;5(12):4361-9.

[2]. A Targeting Modality for Destruction of RNA Polymerase I that Possesses Anticancer Activity. Cancer Cell. 2014 Jan 13; 25(1): 77–90.

DNA intercalation mediates the anticancer activity of widely used chemotherapeutic drugs by causing DNA damage and inhibiting cellular DNA metabolism. Here we demonstrate that a newly discovered heteroaromatic intercalator, BMH-21, does not activate the cellular DNA damage response and its action is independent of damage signaling and repair pathways, activating nucleolar and Pol I transcriptional stress markers. In this respect, BMH-21 differs from other known Pol I inhibitors (such as actinomycin D and CX-5461), which cause degradation of the Pol I catalytic subunit protein RPA194 and do not possess the property of activating the DNA damage response (DDR). We describe a series of BMH-21 derivatives and find that most of the compounds retaining the stacked tetracyclic structure do not initiate DDR. These findings suggest that, despite the intercalation, the heterocycle itself does not possess DNA damage properties. However, we also found that five of the analyzed derivatives exhibited more than a 10-fold enhancement of the DDR response in human cancer cells. These derivatives showed alterations in the BMH-21 side arms, resulting in changes in side arm charge and length, and a significantly reduced ability to induce nucleolar stress. These findings support the further development of BMH-21 into a novel class of molecules with the remarkable ability to inhibit specific transcriptional processes without inducing cellular DNA damage. We have previously demonstrated that BMH-21-mediated p53 activation is independent of the cellular DNA damage response, as measured by H2AX and KAP1 phosphorylation and ATM activity. These findings lead us to propose that BMH-21 activity is independent of the DNA damage response (DDR). Here, we investigate the relationship between BMH-21's significant activity in inhibiting Pol I transcription and these responses. Using chemical inhibitors of ATM and ATR (the main kinases that sense single- and double-strand DNA breaks) or ATR-deficient cells, we found that BMH-21-mediated nucleolar stress response does not require ATM and ATR. Furthermore, blocking DNA-PKcs (molecules essential for NHEJ repair that overactivate DDR due to the accumulation of DNA damage) did not reveal BMH-21-mediated DDR, nor did it attenuate BMH-21's ability to target RPA194. These data support and reinforce the view that BMH-21's inhibition of Pol I transcription and its associated anticancer activity are unrelated to DDR. Molecular modeling of BMH-21 shows that it lies flat between GC bases via π-π interactions, with its protonated terminal amine arms exhibiting a very flat conformation. Unlike the planar anthraquinone rings of doxorubicin (perpendicular to DNA bases, with side chains protruding into the major and minor grooves), the tetracyclic ring of BMH-21 is almost parallel to the GC bases. According to model analysis, BMH-21 does not cause significant size exclusion effects in the major or minor grooves of DNA; its primary role is expected to be the unwinding of the DNA double helix. Therefore, DNA damage caused by this derivative may occur through a variety of mechanisms, which are not necessarily mutually exclusive. These mechanisms include side arms protruding into the major or minor grooves, electrophilic addition of DNA bases, interactions between free radicals and deoxyribose, generation of reactive oxygen species, or inhibition of the DNA transcription or replication complex. Based on this, we also investigated whether BMH-21 could act as a catalytic inhibitor of topoisomerase I or topoisomerase II, but no such activity was found. Further molecular modeling and kinetic studies are needed to reveal the interaction mode between BMH-21 and DNA. Chromatin conformation is an important regulator of DNA damage response (DDR). Chromatin compaction and heterochromatinization limit DDR responses, and when heterochromatin is damaged, its repair rate is slower than that of euchromatin. In addition, the DNA intercalating agent doxorubicin has been shown to cause nucleosome removal at gene promoters, thereby altering promoter activity, or to lead to weakened repair by directly removing γH2AX. Therefore, we considered the possibility that BMH-21 intercalation may lead to an overall change in chromatin state, thereby desensitizing the DNA damage response (DDR). However, BMH-21 pretreatment neither alleviated DNA damage caused by ionizing radiation (IR)-induced double-strand breaks nor DNA damage caused by CPT-type DNA damage. [1]

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BMH-21 is a synthetic small molecule DNA intercalator with selective antitumor activity. [1][2]
- Mechanism of action: It intercalates into DNA and specifically inhibits RNA polymerase I-mediated rRNA transcription, thereby triggering nucleolar stress, upregulating p53/p21, and inducing caspase-dependent apoptosis. This effect is independent of the DNA damage response pathway. [1][2]
- Therapeutic potential: It has been effective against a variety of solid tumors (colorectal cancer, cervical cancer, lung cancer, breast cancer) and hematologic malignancies (leukemia) in preclinical models. It targets cancer cells with high Pol I activity, a hallmark of rapidly proliferating tumors. [2]
- Selectivity: It specifically inhibits Pol I without affecting Pol II/III, thereby minimizing off-target effects on mRNA and tRNA synthesis in normal cells. [1][2]
- Unique advantages: It overcomes resistance to DNA damage drugs (such as cisplatin) by targeting different pathways (nucleolar stress and DNA damage). [2]

C21H20N4O2

360.41

360.158

C, 69.59; H, 6.12; N, 15.46; O, 8.83

896705-16-1

3508054

Yellow to orange solid powder

1.3±0.1 g/cm3

1.668

1.57

1

4

4

27

709

0

O=C(C1C2N(C(C3C(N=2)=CC2C(=CC=CC=2)C=3)=O)C=CC=1)NCCN(C)C

BXYDVWIAGDJBEC-UHFFFAOYSA-N

InChI=1S/C21H20N4O2/c1-24(2)11-9-22-20(26)16-8-5-10-25-19(16)23-18-13-15-7-4-3-6-14(15)12-17(18)21(25)27/h3-8,10,12-13H,9,11H2,1-2H3,(H,22,26)

N-[2-(dimethylamino)ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide

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.)