CX-5461

Alias: CX5461; CX 5461; CX-5461

Cat No.:V1463 Purity: ≥98%

COA Product Data Instructions for use SDS

CX-5461 (CX 5461; CX5461) is a novel, selective andorally bioavailable inhibitor of rRNA synthesis and rDNA transcription inhibitor with potential antitumor activity.

CX-5461 Chemical Structure CAS No.: 1138549-36-6
Product category: DNA(RNA) Synthesis

This product is for research use only, not for human use. We do not sell to patients.

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Citations by Top Journals: Nature, Cell, Science, etc.

Top Publications Citing lnvivochem Products

Nature 2021, 597(7874):119-125.

Cell 2020,183:1202-1218.e25.

Science Adv 2022: 8, eabm9427.

Nature 597, 119–125 (2021)

Cell Stem Cell 2020,26(6):845-861.e12.

Science Trans Med 2023,15(701):eadd7872.

STTT 2023,8(1):91.

Cell Reports 2023,42(4):112313

Science Trans Med 2023, 15: eadd7872.

Cancer Cell 2021,39(4):529-547.e7.

Cancer Discov 2022,12(4):1106-1127.

Immunity 2023,56(3):620-634.e11.

J Am Chem Soc 2022,144:21831-21836.

Cell 2020,183:1202-1218.e25.

Science Adv 2022:8,eabm9427.

Nature 2021,597(7874):119-125.

Cell 2020,183:1202-1218.e25.

PNAS Nexus 2022,1(3):pgac063.

ACS Nano 2023,17(10):9110-9125.

Biomaterial 2023 Sep16:302:122333.

Purity & Quality Control Documentation

Current batch：

Purity: ≥98%

COA

MSDS

NMR

Instructions

Product Description
Bioactivity & Protocols
Physicochemical Properties
Solubility Data
Calculators
Clinical Trial Information
Biological Data

Product Description

CX-5461 (CX 5461; CX5461) is a novel, selective and orally bioavailable inhibitor of rRNA synthesis and rDNA transcription inhibitor with potential antitumor activity. It inhibits rRNA transcription driven by Pol I in a variety of cell types, including MIA PaCa-2, A375, and HCT-116 cells, with an IC50 of 142 nM. CX-5461 exhibits strong in vivo antitumor efficacy against solid human tumors in models of murine xenograft.

Biological Activity I Assay Protocols (From Reference)

Targets

rRNA synthesis, MIA PaCa-2 cells ( IC50 = 54 nM ); rRNA synthesis, A375 cells ( IC50 = 113 nM ); rRNA synthesis, HCT-116 cells ( IC50 = 142 nM )

ln Vitro

CX-5461 is found to selectively inhibit rRNA synthesis (Pol I IC50 = 142 nM; Pol II IC50 > 25 μM; selectivity ~200-fold) in the HCT-116 cells. In two additional human solid tumor cell lines, pancreatic carcinoma MIA PaCa-2 (Pol I IC50=54 nM; Pol II IC50 ~25 mM) and melanoma A375 (Pol I IC50 = 113 nM; Pol II IC50 > 25 μM), selective inhibition of rRNA synthesis by CX-5461 is verified. CX-5461 exhibits a 250–300 fold increase in selectivity when it comes to inhibiting rRNA transcription as opposed to DNA replication and protein translation. CX-5461 has little effect on the viability of nontransformed human cells but demonstrates broad antiproliferative potency in a panel of cancer cell lines in human cancer cell lines. While all normal cell lines have EC50 values of roughly 5,000 nM, the median EC50 for all tested cell lines is 147 nM. The HCT-116, A375, and MIA PaCa-2 cell lines' antiproliferative dose response evaluation yielded EC50 values of 167, 58, and 74 nM. Through a mechanism that is p53-independent, CX-5461 causes autophagy and senescence in solid tumor cancer cells as opposed to apoptosis.

ln Vivo

CX-5461 exhibits in vivo antitumor activity against solid tumors of the human body and is orally bioavailable in murine xenograft models. On day 31, CX-5461 exhibits significant MIA PaCa-2 TGI, with a TGI of 69%, which is similar to gemcitabine's TGI of 63%. A positive control, gemcitabine, is given intraperitoneally once every three days at a dose of 120 mg/kg. Similarly, on day 32, CX-5461 exhibits significant A375 TGI, with TGI equal to 79%.[1]

Enzyme Assay

CX-5461-related effects on transcription are measured by qRT-PCR, which quantifies two short-lived RNA transcripts (half-lives ~20-30 minutes), one produced by Pol I and another by Pol II. As the Pol I transcript, the 45S pre-rRNA functioned, while the comparator Pol II transcript was the protooncogene c-myc mRNA. Cell stress in general is known to impact both Pol I and Pol II transcription. Cells are only exposed to test agents for a brief amount of time (2 hours) in order to reduce any possible effects of such stress. There is enough time for CX-5461 to impact the synthesis of these transcripts, resulting in a reduction of more than 90%.

Cell Assay

The following day, cells are treated with CX-5461 dose response for 96 hours after being plated on 96-well plates. Alamar Blue and CyQUANT assays are used to measure cell viability.

Animal Protocol

Mice: Female Balb/c athymic mice (NCr nu/nu fisol) aged 5 to 6 weeks are used in animal experiments. Hypnic (NCr nu/nu fisol) mice are injected subcutaneously in the right flank of the mice with 100 μL of cell suspension. Calculating the tumor volume involves applying the formula (l×w²)/2, where w represents the tumor's width and l its length in millimeters. Tumor measurements are carried out through caliper analysis. randomized to treatment groups with either vehicle (50 mM NaH₂PO₄, pH 4.5), NSC 613327, or CX-5461 for established tumors (approx 110-120 mm³). On the last day of the study, tumor growth inhibition (TGI) is calculated using the following formula: TGI (%)=[100 − (Vf^D− Vi^D)/ (Vf^V − Vi^V) × 100], where Vi^V represents the initial mean tumor volume in the vehicle-treated group, Vf^V represents the final mean tumor volume in the vehicle-treated group, Vi^D represents the initial mean tumor volume in the drug-treated group, and Vf^D represents the final mean tumor volume in the drug-treated group on the study day.

References

[1]. Cancer Res . 2011 Feb 15;71(4):1418-30.

These protocols are for reference only. InvivoChem does not independently validate these methods.

Physicochemical Properties

Molecular Formula

C27H27N7O2S

Molecular Weight

513.61

Exact Mass

513.19

Elemental Analysis

C, 63.14; H, 5.30; N, 19.09; O, 6.23; S, 6.24

CAS #

1138549-36-6

Related CAS #

1138549-36-6; 2101314-20-7 (HCl)

Appearance

White solid powder

SMILES

CC1=CN=C(C=N1)CNC(=O)C2=C3N(C4=CC=CC=C4S3)C5=C(C2=O)C=CC(=N5)N6CCCN(CC6)C

InChi Key

XGPBJCHFROADCK-UHFFFAOYSA-N

InChi Code

InChI=1S/C27H27N7O2S/c1-17-14-29-18(15-28-17)16-30-26(36)23-24(35)19-8-9-22(33-11-5-10-32(2)12-13-33)31-25(19)34-20-6-3-4-7-21(20)37-27(23)34/h3-4,6-9,14-15H,5,10-13,16H2,1-2H3,(H,30,36)

Chemical Name

2-(4-methyl-1,4-diazepan-1-yl)-N-[(5-methylpyrazin-2-yl)methyl]-5-oxo-[1,3]benzothiazolo[3,2-a][1,8]naphthyridine-6-carboxamide

Synonyms

CX5461; CX 5461; CX-5461

HS Tariff Code

2934.99.9001

Storage

Powder -20°C 3 years

4°C 2 years

In solvent -80°C 6 months

-20°C 1 month

Shipping Condition

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

Solubility Data

Solubility (In Vitro)

DMSO: <1 mg/mL

Water: <1 mg/mL

Ethanol: <1 mg/mL

Solubility (In Vivo)

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.

Injection Formulations
(e.g. IP/IV/IM/SC)

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)

Oral Formulations

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

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.

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

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.9470 mL	9.7350 mL	19.4700 mL
	5 mM	0.3894 mL	1.9470 mL	3.8940 mL
	10 mM	0.1947 mL	0.9735 mL	1.9470 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

Calculator

Mass

Concentration

Volume

Molecular Weight*

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Molarity Calculator allows you to calculate the mass, volume, and/or concentration required for a solution, as detailed below:

Calculate the Mass of a compound required to prepare a solution of known volume and concentration
Calculate the Volume of solution required to dissolve a compound of known mass to a desired concentration
Calculate the Concentration of a solution resulting from a known mass of compound in a specific volume

See Example

An example of molarity calculation using the molarity calculator is shown below:
What is the mass of compound required to make a 10 mM stock solution in 5 ml of DMSO given that the molecular weight of the compound is 350.26 g/mol?

Enter 350.26 in the Molecular Weight (MW) box
Enter 10 in the Concentration box and choose the correct unit (mM)
Enter 5 in the Volume box and choose the correct unit (mL)
Click the “Calculate” button
The answer of 17.513 mg appears in the Mass box. In a similar way, you may calculate the volume and concentration.

Concentration (Start)

Volume (Start)

Concentration (End)

Volume (End)

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Dilution Calculator allows you to calculate how to dilute a stock solution of known concentrations. For example, you may Enter C1, C2 & V2 to calculate V1, as detailed below:

What volume of a given 10 mM stock solution is required to make 25 ml of a 25 μM solution?
Using the equation C1V1 = C2V2, where C1=10 mM, C2=25 μM, V2=25 ml and V1 is the unknown:

Enter 10 into the Concentration (Start) box and choose the correct unit (mM)
Enter 25 into the Concentration (End) box and select the correct unit (mM)
Enter 25 into the Volume (End) box and choose the correct unit (mL)
Click the “Calculate” button
The answer of 62.5 μL (0.1 ml) appears in the Volume (Start) box

Molecular formula

Total molecular weight

g/mol

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Molecular Weight Calculator allows you to calculate the molar mass and elemental composition of a compound, as detailed below:

Note: Chemical formula is case sensitive: C12H18N3O4 c12h18n3o4
Instructions to calculate molar mass (molecular weight) of a chemical compound:

To calculate molar mass of a chemical compound, please enter the chemical/molecular formula and click the “Calculate’ button.

Definitions of molecular mass, molecular weight, molar mass and molar weight:

Molecular mass (or molecular weight) is the mass of one molecule of a substance and is expressed in the unified atomic mass units (u). (1 u is equal to 1/12 the mass of one atom of carbon-12)
Molar mass (molar weight) is the mass of one mole of a substance and is expressed in g/mol.

Volume (to add to vial)

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Reconstitution Calculator allows you to calculate the volume of solvent required to reconstitute your vial.

Enter the mass of the reagent and the desired reconstitution concentration as well as the correct units
Click the “Calculate” button
The answer appears in the Volume (to add to vial) box

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)

Dosage mg/kg

Average weight of animals g

Dosing volume per animal μL

Number of animals

Step 2: Enter in vivo formulation (This is only a calculator, not the exact formulation for a specific product. Please contact us first if there is no in vivo formulation in the solubility section.)

% DMSO

% Tween 80

% ddH₂O

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Calculation results

Working concentration： mg/mL；

Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.

Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH₂O，mix and clarify.

Note： (1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.

Clinical Trial Information

NCT Number	Recruitment	interventions	Conditions	Sponsor/Collaborators	Start Date	Phases
NCT04890613	Recruiting	Drug: CX-5461	COVID-19	Senhwa Biosciences, Inc.	September 8, 2021	Phase 1
NCT02719977	Completed	Drug: CX5461	Cancer	Canadian Cancer Trials Group	June 13, 2016	Phase 1

Biological Data

BJ-T and BJ-T p53sh cells exhibit G1 arrest, S-phase delay and G2 cell cycle arrest in response to CX-5461.Oncotarget.2016 Aug 2;7(31):49800-49818.
Inhibition of Pol I transcription initiation by CX-5461 activates the ATM/ATR signaling pathway.Oncotarget.2016 Aug 2;7(31):49800-49818.
Combination treatment of ATM and ATR inhibitors with CX-5461 induces cell death in the absence of p53.Oncotarget.2016 Aug 2;7(31):49800-49818.
CX-5461 combination with a dual CHK1/CHK2 inhibitor induces cancer cell death ofTp53-null (Tp53−/−)Eμ-Myclymphoma cellsin vitroandin vivo.Oncotarget.2016 Aug 2;7(31):49800-49818.
CX-5461 activates ATM signaling within the nucleoli in the absence of DNA damage.Oncotarget.2016 Aug 2;7(31):49800-49818.

CX-5461

Inhibition of Pol I transcription initiation by CX-5461 results in rDNA repeats that are devoid of Pol I, but maintain an exposed chromatin state that associates with ATM/ATR pathway activation.Oncotarget.2016 Aug 2;7(31):49800-49818.