CX-5461 dihydrochloride

Alias: CX5461 dihydrochloride; CX 5461 2HCl; CX-5461 diHCl

Cat No.:V4349 Purity: ≥98%

COA Product Data Instructions for use SDS

CX-5461 dihydrochloride Chemical Structure 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

Instructions

Product Description
Bioactivity & Protocols
Physicochemical Properties
Solubility Data
Calculators
Biological Data

Product Description

CX-5461 dihydrochloride is a novel, potent, selective and orally bioavailable inhibitor of rRNA synthesis, it selectively inhibits Pol I-driven transcription of rRNA with IC50 of 142 nM in HCT-116, A375, and MIA PaCa-2 cells, has no effect on Pol II, and possesses 250- to 300-fold selectivity for inhibition of rRNA transcription versus DNA replication and protein translation. It was discovered that CX-5461 inhibits Pol I transcription by encouraging p53 stabilization. Furthermore, it has been shown that CX-5461 causes autophagy and senescence in MIA Paca-2 and A375 cell lines, but not apoptosis. In vivo p53-mediated tumor cell apoptosis can be selectively induced by CX-5461's inhibition of Pol I. CX-5461 is presently being tested in a clinical trial for individuals with advanced cancers of the blood system (Peter Mac, Melbourne). In the absence of DNA damage, CX-5461 also induces p53-independent cell cycle checkpoints mediated by ATM/ATR signaling. Furthermore, p53-null tumors in vivo, which are typically resistant to each drug alone, can be treated more effectively when medications targeting ATM/ATR signaling and CX-5461 are combined. Through a unique chromatin structure that CX-5461 induces, ATM signaling is activated within the nucleoli. This structure is caused by transcriptionally competent relaxed rDNA repeats that lack transcribing Pol I. In order to increase the effectiveness of treatment, CX-5461's acute suppression of Pol transcription initiation causes a novel nucleolar stress response that can be targeted.

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 inhibits Pol I-mediated rRNA synthesis potently and orally (IC50s of 142 nM in HCT-116, 113 nM in A375, and 54 nM in MIA PaCa-2 cells). It has negligible or no effect on Pol II (IC50, ≥25 μM). CX-5461 has a slight inhibitory effect on the translation of proteins and DNA. At a mean EC50 of 147 nM, CX-5461 demonstrates extensive antiproliferative activity against a panel of human cancer cell lines. However, its minimal impact on the viability of nontransformed human cells is evident, as its EC50 values are approximately 5000 nM. The CX-5461 EC50 values for the HCT-116, A375, and MIA PaCa-2 cell lines are 167, 58, and 74 nM, in that order. Solid tumor cancer cells are subjected to a p53-independent process by CX-5461 that causes autophagy and senescence instead of apoptosis[1]. With an IC50 of 27.3 nM ± 8.1 nM for Pol I transcription after 1 hour and IC50 of 5.4 nM ± 2.1 nM for cell death after 16 hours, Eμ-Myc lymphoma cells from tumor-bearing mice exhibit exceptional sensitivity to CX-5461. Through the nucleolar stress response in Eμ-Myc Lymphoma Cells, CX-5461 activates p53[2].

ln Vivo

CX-5461 exhibits antitumor activity against solid tumors of humans in murine xenograft models. Significant MIA PaCa-2 growth inhibition is demonstrated by CX-5461 (50 mg/kg, p.o. ), with TGI equal to 69% on day 31 and 79% on day 32 on A375[1]. At one hour after treatment, CX-5461 (50 mg/kg, p.o.) inhibits Eμ-Myc tumor cells with 84% repression in Pol I transcription in C57BL/6 mice. Additionally, CX-5461 causes the tumor burden in the lymph nodes to rapidly decrease, and it also causes the spleen's size to decrease to within normal limits[2].

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[1].

Animal Protocol

Mice: Female athymic Balb/c mice, aged 5 to 6 weeks, are used in animal experiments (NCr nu/nu fisol). Athymic (NCr nu/nu fisol) mice are subcutaneously injected with 100 μL of cell suspension in the right flank of the mice. Tumor measurements are made using caliper analysis, and the formula (l×w²)/2 is used to calculate the tumor volume, where w is the tumor's width and l is its length in millimeters. Established tumors (110–120 mm³) are randomly assigned to treatment groups with CX-5461, NSC 613327, or vehicle (50 mM NaH₂PO₄, pH 4.5). 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.

References

[1]. Targeting RNA polymerase I with an oral small molecule CX-5461 inhibits ribosomal RNA synthesis and solid tumor growth. Cancer Res. 2011 Feb 15;71(4):1418-30.

[2]. Inhibition of RNA Polymerase I as a Therapeutic Strategy to Promote Cancer-Specific Activation of p53.

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

Physicochemical Properties

Molecular Formula

C27H29CL2N7O2S

Related CAS #

N/A

Appearance

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.Cl.Cl

InChi Key

LXNKBUVQVKWAHI-UHFFFAOYSA-N

InChi Code

InChI=1S/C27H27N7O2S.2ClH/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);2*1H

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;dihydrochloride

Synonyms

CX5461 dihydrochloride; CX 5461 2HCl; CX-5461 diHCl

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

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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: N/A

Water: gt;20 mg/mL

Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: 6.25 mg/mL (10.66 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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

Calculator

Mass

Concentration

Volume

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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?

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The answer of 17.513 mg appears in the Mass box. In a similar way, you may calculate the volume and concentration.

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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:

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The answer of 62.5 μL (0.1 ml) appears in the Volume (Start) box

Molecular formula

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g/mol

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

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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

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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.)

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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.

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 DIHYDROCHLORIDE

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.