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.

Physicochemical Properties

Molecular Formula	C27H28CLN7O2S
Molecular Weight	550.074922561646
Exact Mass	549.171372
Elemental Analysis	C, 58.95; H, 5.13; Cl, 6.44; N, 17.82; O, 5.82; S, 5.83
CAS #	2101314-20-7
Related CAS #	1138549-36-6
Appearance	Light yellow to yellow solid powder
SMILES	C(C1C(C2=CC=C(N3CCCN(C)CC3)N=C2N2C3=CC=CC=C3SC=12)=O)(=O)NCC1=NC=C(C)N=C1.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)

Biological Activity

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 − (VfD− ViD)/ (VfV − ViV) × 100], where ViV represents the initial mean tumor volume in the vehicle-treated group, VfV represents the final mean tumor volume in the vehicle-treated group, ViD represents the initial mean tumor volume in the drug-treated group, and VfD 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.
Additional Infomation	CX-5461 is an organic heterotetracyclic compound that is 5-oxo-5H-[1,3]benzothiazolo[3,2-a][1,8]naphthyridine-6-carboxylic acid substituted by a 4-methyl-1,4-diazepan-1-yl group at position 2 and in which the carboxy group at position 6 is substituted by a [(5-methylpyrazin-2-yl)methyl]nitrilo group. An inhibitor of ribosomal RNA (rRNA) synthesis which specifically inhibits RNA polymerase I-driven transcription of rRNA in several cancer cell lines. It is currently in phase I/II clinical trials for advanced hematologic malignancies and triple-negative breast cancer with BRCA1/2 mutation. It has a role as an antineoplastic agent, an apoptosis inducer and an EC 2.7.7.6 (RNA polymerase) inhibitor. It is a diazepine, an organic heterotetracyclic compound, a member of pyrazines, a secondary carboxamide and a naphthyridine derivative. Pidnarulex is an orally bioavailable inhibitor of RNA polymerase I (Pol I), with potential antineoplastic activity. Upon oral administration, pidnarulex selectively binds to and inhibits Pol I, prevents Pol I-mediated ribosomal RNA (rRNA) synthesis, induces apoptosis, and inhibits tumor cell growth. Pol I, the multiprotein complex that synthesizes rRNA, is upregulated in cancer cells and plays a key role in cell proliferation and survival. Hyperactivated rRNA transcription is associated with uncontrolled cancer cell proliferation. Deregulated ribosomal RNA synthesis is associated with uncontrolled cancer cell proliferation. RNA polymerase (Pol) I, the multiprotein complex that synthesizes rRNA, is activated widely in cancer. Thus, selective inhibitors of Pol I may offer a general therapeutic strategy to block cancer cell proliferation. Coupling medicinal chemistry efforts to tandem cell- and molecular-based screening led to the design of CX-5461, a potent small-molecule inhibitor of rRNA synthesis in cancer cells. CX-5461 selectively inhibits Pol I-driven transcription relative to Pol II-driven transcription, DNA replication, and protein translation. Molecular studies demonstrate that CX-5461 inhibits the initiation stage of rRNA synthesis and induces both senescence and autophagy, but not apoptosis, through a p53-independent process in solid tumor cell lines. CX-5461 is orally bioavailable and demonstrates in vivo antitumor activity against human solid tumors in murine xenograft models. Our findings position CX-5461 for investigational clinical trials as a potent, selective, and orally administered agent for cancer treatment.[1] Increased transcription of ribosomal RNA genes (rDNA) by RNA Polymerase I is a common feature of human cancer, but whether it is required for the malignant phenotype remains unclear. We show that rDNA transcription can be therapeutically targeted with the small molecule CX-5461 to selectively kill B-lymphoma cells in vivo while maintaining a viable wild-type B cell population. The therapeutic effect is a consequence of nucleolar disruption and activation of p53-dependent apoptotic signaling. Human leukemia and lymphoma cell lines also show high sensitivity to inhibition of rDNA transcription that is dependent on p53 mutational status. These results identify selective inhibition of rDNA transcription as a therapeutic strategy for the cancer specific activation of p53 and treatment of hematologic malignancies.[2] RNA polymerase I (Pol I)-mediated transcription of the ribosomal RNA genes (rDNA) is confined to the nucleolus and is a rate-limiting step for cell growth and proliferation. Inhibition of Pol I by CX-5461 can selectively induce p53-mediated apoptosis of tumour cells in vivo. Currently, CX-5461 is in clinical trial for patients with advanced haematological malignancies (Peter Mac, Melbourne). Here we demonstrate that CX-5461 also induces p53-independent cell cycle checkpoints mediated by ATM/ATR signaling in the absence of DNA damage. Further, our data demonstrate that the combination of drugs targeting ATM/ATR signaling and CX-5461 leads to enhanced therapeutic benefit in treating p53-null tumours in vivo, which are normally refractory to each drug alone. Mechanistically, we show that CX-5461 induces an unusual chromatin structure in which transcriptionally competent relaxed rDNA repeats are devoid of transcribing Pol I leading to activation of ATM signaling within the nucleoli. Thus, we propose that acute inhibition of Pol transcription initiation by CX-5461 induces a novel nucleolar stress response that can be targeted to improve therapeutic efficacy. Oncotarget. 2016 Aug 2;7(31):49800-49818.

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.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.8179 mL	9.0896 mL	18.1792 mL
	5 mM	0.3636 mL	1.8179 mL	3.6358 mL
	10 mM	0.1818 mL	0.9090 mL	1.8179 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.