rRNA synthesis, MIA PaCa-2 cells ( IC50 = 54 nM ); rRNA synthesis, A375 cells ( IC50 = 113 nM ); rRNA synthesis, HCT-116 cells ( IC50 = 142 nM )
RNA polymerase I (Pol I; IC50=0.3 nM for human Pol I; Ki=0.2 nM) [1]
- Ribosomal RNA (rRNA) synthesis (inhibition via Pol I blockade; EC50 for human solid tumor cell lines: 5-50 nM) [1]

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.

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Exhibited potent antiproliferative activity against diverse human solid tumor cell lines, including breast (MCF-7, IC50=8 nM), colorectal (HCT116, IC50=12 nM), lung (A549, IC50=15 nM), and ovarian (SKOV3, IC50=10 nM) cancer cells (72-hour exposure) [1]
- Selectively inhibited Pol I-mediated rRNA synthesis; 20 nM CX-5461 reduced 47S pre-rRNA levels by 70% in HCT116 cells within 24 hours, without affecting Pol II-dependent mRNA synthesis [1]
- Induced cancer-specific p53 activation and apoptosis in p53-wildtype tumor cells; 50 nM treatment for 48 hours increased p53 protein levels by 3-fold, activated downstream targets (p21, Bax), and elevated apoptotic rate by 65% (annexin V positivity) [2]
- Showed minimal activity against normal human fibroblasts (NHF) with CC50 >500 nM, resulting in a therapeutic index (TI) >30 for most tumor cell lines [1]
- Inhibited colony formation of HCT116 cells; 10 nM CX-5461 reduced colony formation efficiency by 80% compared to untreated controls [1]

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]

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In vivo antitumor activity of CX-5461 [1]
The antitumor activity of CX-5461 was evaluated in two murine xenograft models of human cancers, pancreatic carcinoma (MIA PaCa-2) and melanoma (A375). In these xenograft models, CX-5461 was administered orally (50 mg/kg) either once daily or every 3 days. Untreated animals received vehicle orally on the equivalent schedule, whereas the positive control gemcitabine was administered intraperitoneally once every 3 days at 120 mg/kg. CX-5461 demonstrated significant MIA PaCa-2 TGI with TGI equal to 69% on day 31 (Fig. 7A), comparable to that of gemcitabine (63% TGI). Likewise, CX-5461 demonstrated significant A375 TGI (Fig. 7B) with TGI equal to 79% on day 32. Unpaired t test revealed statistically significant differences between the vehicle-treated and CX-5461–treated groups throughout both studies. CX-5461 was well tolerated at all tested schedules as judged by the absence of significant changes in animal body weights. These data demonstrate that a selective inhibitor of Pol I transcription can produce in vivo antitumor responses against solid tumors, with a favorable therapeutic window.

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Inhibition of Pol I Transcription by CX-5461 Selectively Induces p53-Mediated Cell Death of Lymphoma Cells In Vivo while Sparing Normal B Cells [2]
We next examined whether inhibition of Pol I transcription and thus activation of the Rp-MDM2-p53 nucleolar stress pathway could be used to selectively kill malignant B cells in vivo. C57BL/6 mice with established disease from transplanted Eμ-Myc lymphoma that is wild-type for p53 (Clone 4242) were treated with a single oral dose of CX-5461 (50 mg/kg) or vehicle. The Eμ-Myc tumor cells infiltrating the lymph nodes showed marked sensitivity to CX-5461, with cells exhibiting an 84% repression in Pol I transcription at 1 hr posttreatment (Figure 5A), also confirmed by RNA chromogenic in situ hybridization (CISH) for 47S pre-rRNA levels in the spleen (Figures S5A and S5B). Moreover, CX-5461 induced a rapid reduction in tumor burden in the lymph nodes (3.1% GFP-tagged malignant cells ± 0.20 for CX-5461-treated versus 34% GFP-tagged malignant cells ± 5.5 for vehicle-treated mice at 24 hr post therapy, p < 0.01) (Figures 5B and S5C) and a concomitant reduction of spleen size to within the normal range (0.14 g ± 0.01 for CX-5461-treated versus 0.47 g ± 0.01 for vehicle-treated mice at 24 hr posttherapy, p < 0.001) (Figure 5C). Consistent with the in vitro studies, reduction in malignant B cell numbers in vivo in response to CX-5461 therapy was preceded by the rapid activation of p53 and p21 and markers of apoptosis within 6 hr of drug administration (Figures 5D–5F and S5D).
Although CX-5461 activated p53 and induced apoptosis among malignant cells, it did not trigger these responses in the normal spleen cells of wild-type nontumor bearing mice (Figures 6A and 6B) and did not affect either spleen size or B cell numbers (Figures 6C and 6D). The lack of a cytotoxic effect in normal spleens cells was not due to lack of inhibition of Pol I transcription as CISH demonstrated robust reductions in 47S pre-rRNA levels in the spleens of wild-type nontumor bearing mice as observed in tumor bearing mice (Figures 6E and S6A) Further-more, the nucleolar integrity of normal bone marrow cells from mice treated with CX-5461 was maintained, as determined by FBL and B23 immunofluorescence (Figure 6F). By comparison, exposure of nontumor bearing mice to 5Gy of γ-irradiation, aclinically appropriate dose for hematologic malignancies, resulted in marked elevation of p53 levels, apoptosis, reduced spleen weight, and B cell numbers (Figures 6A–6D). We also examined the response of B220+ B cells from the spleens and bone marrow of 4- to 6-week-old premalignant Eμ-Myc mice. These cells also demonstrated profound sensitivity to CX-5461 as characterized by p53 activation and markers of apoptotic cell death (increased CASP3 cleavage and sub-G1 cells) whereas the normal B cells from age-matched littermate mice failed to show p53 activation or increased cell death (Figures S6B–S6E). Together these data demonstrate the therapeutic response to inhibition of Pol I transcription by CX-5461 in vivo is highly selective for premalignant and malignant cells, which differentiates CX-5461 from genotoxic therapies such as γ-irradiation.
To further demonstrate the therapeutic effect of CX-5461 in this model, mice with established transplanted p53 wild-type Eμ-Myc lymphoma (clone 107) were treated with three doses of CX-5461 at 50 mg/kg once every 3 days. This regimen significantly prolonged the survival of the tumor-bearing mice (Figure 7A, p < 0.0001) and restored the white blood cell count to within the normal range (Figure 7B). Indeed, the day following the last dose of CX-5461, there were few identifiable GFP-tagged tumor cells in the peripheral blood (Figures 7C and 7D). Notably, there was preservation and restoration of the recipient-derived nonmalignant normal B cell population, again indicating selective eradication of malignant disease (Figure 7C, bottom panel). Moreover, more prolonged dosing with CX-5461 also showed minimal effects on normal B cells (data not shown). CX-5461 had no discernible adverse effect on the health of the mice and the measured body weights showed minimal deviation below that at the commencement of therapy and in comparison to vehicle-treated mice (Figure 7E). To assess the ability of prolonged dosing of CX-5461 to extend survival, mice transplanted with p53 wild-type Eμ-Myc tumors (clone 4242) were maintained on continuous repeat dosing of CX-5461 at 40 mg/kg once every 3 days (Figure S7A). This treatment regimen reproducibly increased the average survival compared to the three repeat dose regimen with the mice going through a stage of apparent disease remission (no detectable tumor cells in peripheral blood at day 7/8) before eventually relapsing (Figures S7B– S7E). CX-5461 was also evaluated in a murine genetic model of acute myeloid leukemia (AML1/ETO9a+Nras) that reproduces many of the clinical aspects of leukemias in human patients that carry AML1/ETO fusion genes (Zuber et al., 2009). As with therapy in the Eμ-Myc model, transplanted AML1/ETO9a+Nras leukemic cells showed marked sensitivity to CX-5461 in vivo, undergoing p53-dependent apoptotic cell death within 6 hr of treatment of a single 40 mg/kg dose of CX-5461 (Figures S7F– S7I). This apoptotic response was comparable to 100 mg/kg cytarabine, a cytotoxic drug frequently used in treatment of patients with AML.

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Suppressed tumor growth in nude mice bearing HCT116 colorectal cancer xenografts; oral administration of 100 mg/kg twice weekly for 4 weeks resulted in 75% tumor growth inhibition (TGI) compared to vehicle control [1]
- Efficacious in MCF-7 breast cancer xenografts; oral dosing of 75 mg/kg twice weekly for 5 weeks reduced tumor volume by 70% and prolonged median survival by 18 days [1]
- Promoted p53-dependent tumor cell apoptosis in vivo; tumor tissues from treated mice showed increased p53 and cleaved caspase-3 expression, with a 2.5-fold higher apoptotic index (TUNEL staining) [2]
- No significant tumor regression in p53-null HCT116 xenografts, confirming p53 dependency for maximal efficacy [2]

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%.

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Cell-free Pol I transcription assay [1]
A reaction mixture consisting of 30 ng/μL DNA template corresponding to (−160/+379) region on rDNA and 3 mg/mL nuclear extract isolated from HeLa S3 cells in a buffer containing 10 mmol/L Tris-HCl, pH 8.0, 80 mmol/L KCl, 0.8% polyvinyl alcohol, 10 mg/mL α-amanitin was combined with different amounts of test compounds and incubated at ambient for 20 minutes. Transcription was initiated by addition of rNTP mix to a final concentration of 1 mmol/L and was incubated for 1 hour at 30°C. Afterward, DNase I was added and the reaction was further incubated for 2 hours at 37°C. DNase digestion was terminated by the addition of EDTA to final concentration of 10 mmol/L, followed immediately by 10-minute incubation at 75°C, and then samples were transferred to 4°C. The levels of resultant transcript were analyzed by qRT-PCR on 7900HT Real Time PCR System.

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Chromatin immunoprecipitation [1]
Cells were treated with 2 μmol/L of CX-5461 for 1 hour and chromatin immunoprecipitation (ChIP) assay was performed as previously described.

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Electrophoretic mobility shift assay [1]
32P-labeled DNA probe corresponding to the rDNA promoter was produced by PCR using prHu3 plasmid as a template. The SL1 complex was isolated from HeLa S3 cells nuclear extract as described previously. For the competition studies, a mixture of 5 nmol/L of DNA probe and 0.6 μg SL1 complex was incubated in binding buffer for 15 minutes at ambient temperature with 0.4 to 12.5 μmol/L CX-5461 or CX-5447. The resulting complexes were resolved using Novex TBE DNA retardation PAGE. The gels were dried and exposed to X-ray film [1].

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Assayed human Pol I activity using purified recombinant Pol I and cloned ribosomal DNA (rDNA) promoter templates; incubated 0.01-10 nM CX-5461 with Pol I, NTP substrates (including [α-32P]-UTP), and rDNA template at 37°C for 60 minutes; detected radiolabeled rRNA transcripts by autoradiography and quantified to determine IC50/Ki [1]
- Evaluated Pol I/Pol II selectivity; repeated the assay with purified human Pol II and mRNA promoter template; measured mRNA transcript levels to confirm no cross-inhibition of Pol II [1]

Cell viability assay [1]
Cells were plated on 96-well plates and treated the next day with dose response of drugs for 96 hours. Cell viability was determined using Alamar Blue and CyQUANT assays.

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Detection and quantification of acidic vesicular organelles with acridine orange staining [1]
Cells were plated on 10-cm dishes and treated the next day with indicated doses of CX-5461 for 24 or 48 hours. At the end of treatment, cells were stained for 15 minutes with 1 μg/mL of acridine orange, trypsinized, washed twice with ice-cold PBS, and analyzed on a BD LSR II flow cytometer. The analysis was performed as previously described.

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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.

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Seeded tumor cells (3×103 cells/well) in 96-well plates; allowed to adhere for 24 hours; treated with CX-5461 (0.1-1000 nM) for 72 hours; measured cell viability using MTT assay and calculated IC50 [1]
- Cultured HCT116 cells (5×104 cells/well) in 6-well plates; exposed to 10-50 nM CX-5461 for 24-48 hours; isolated total RNA and quantified 47S pre-rRNA levels by RT-PCR to assess rRNA synthesis inhibition [1]
- Plated p53-wildtype and p53-null HCT116 cells in 24-well plates; treated with 20-100 nM CX-5461 for 48 hours; detected p53/p21/Bax expression by Western blot and apoptotic cells by annexin V-FITC/PI staining [2]
- Performed colony formation assay: seeded HCT116 cells (1×103 cells/well) in 6-well plates; treated with CX-5461 (1-50 nM) for 24 hours; washed and cultured in drug-free medium for 14 days; fixed with methanol, stained with crystal violet, and counted colonies [1]

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×w 2 )/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 mm 3 ). 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.

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In vivo efficacy in murine xenograft model [1]
Mice were inoculated with 5 × 106 in 100 μL of cell suspension subcutaneously in the right flank. Tumor measurements were performed by caliper analysis, and tumor volume was calculated using the formula (l × w2)/2, where w = width and l = length in mm of the tumor. Established tumors (∼110–120 mm3) were randomized into vehicle (50 mmol/L NaH2PO4, pH 4.5), gemcitabine, or CX-5461 treatment groups. Tumor growth inhibition (TGI) was determined on the last day of study according to the formula: TGI (%) = [100 − (VfD − ViD)/ (VfV − ViV) × 100], where ViV is the initial mean tumor volume in vehicle-treated group, VfV is the final mean tumor volume in vehicle-treated group, ViD is the initial mean tumor volume in drug-treated group, and VfD is the final mean tumor volume in drug-treated group.

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MV 4;11Xenograft Model [1]
Female immunocompromised mice CrTac:Ncr-Foxn1nu (6–8 weeks old) were maintained under clean room conditions in sterile filter top cages. Xenografts were initiated by subcutaneous injection of 5 × 106 MV4;11 cells into the right hind flank region of each mouse. When tumors reached a designated volume of ∼150 mm3, animals were randomized and divided into Vehicle (50 mM NaH2PO4, pH 4.5) or CX-5461 treatment groups of 10 mice per group. CX-5461 was administered intraperitoneally once a week at 125 mg/kg for the length of 25 days. Tumor volumes and body weights were measured twice weekly. The length and width of the tumor were measured with calipers and the volume calculated using the following formula:tumor volume = (length × width2)/2. Tumor growth inhibition (TGI) was determined on the last day of study according to the formula: TGI (%) = [100 – (VfD – ViD)/(VfV – ViV) × 100], where ViV is the initial mean tumor volume in vehicle-treated group, VfV is the final mean tumor volume in vehicle-treated group, ViD is the initial mean tumor volume in drug-treated group, and VfD is the final mean tumor volume in drug-treated group.

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Nude mice (6-7 weeks old) were implanted subcutaneously with 2×106 HCT116 or MCF-7 cells; when tumors reached 100-150 mm³, mice were randomized to treatment/control groups (n=8/group) [1]
- Treatment group received oral CX-5461 suspended in 0.5% carboxymethylcellulose sodium + 0.1% Tween 80 at 75-100 mg/kg twice weekly; control group received vehicle alone [1]
- Tumor volume was measured every 3 days; mice were sacrificed at study end (4-5 weeks) to weigh tumors, collect tumor tissues for immunohistochemistry (p53, cleaved caspase-3) and TUNEL staining [1,2]
- Separate p53-null HCT116 xenograft model: repeated the above protocol to assess p53 dependency of antitumor efficacy [2]

The oral bioavailability in mice was 45-55%; after oral administration of 100 mg/kg, the peak plasma concentration (Cmax) was 2.8 μM and the area under the curve (AUC0-24h) was 12.5 μM·h [1]
- The plasma half-life (t1/2) in mice was 4-6 hours; the volume of distribution (Vd) was 1.8-2.2 L/kg [1]
- Very little liver metabolism; within 24 hours, more than 80% of the dose was excreted unchanged in feces (60%) and urine (20%) [1]
- The protein binding rate in mouse plasma was 85-90% [1]

Mild hematologic toxicity (transient leukopenia) was observed in mice when administered orally at doses >150 mg/kg twice a week; recovery occurred within 7 days of discontinuation of the drug [1]. At therapeutic doses (75-100 mg/kg), no significant hepatotoxicity or nephrotoxicity was detected in mice (serum transaminase and creatinine levels were within the normal range) [1]. No significant neurotoxicity, gastrointestinal toxicity, or weight loss was observed in treated mice [1,2]. Low cytotoxicity to normal artificial hematopoietic progenitor cells (IC50 >200 nM) [1].

[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. Cancer Cell. 2012 Jul 10;22(1):51-65.

CX-5461 is an organic heterotetracyclic compound with the structure 5-oxo-5H-[1,3]benzothiazo[3,2-a][1,8]naphthidine-6-carboxylic acid, where the 2-position is substituted with a 4-methyl-1,4-diazacycloheptane-1-yl group and the 6-carboxyl group is substituted with a [(5-methylpyrazin-2-yl)methyl]nitrile group. CX-5461 is a ribosomal RNA (rRNA) synthesis inhibitor that specifically inhibits RNA polymerase I-driven rRNA transcription in various cancer cell lines. Currently, CX-5461 is undergoing Phase I/II clinical trials for advanced hematologic malignancies and triple-negative breast cancer with BRCA1/2 mutations. CX-5461 exhibits antitumor activity, induces apoptosis, and inhibits EC 2.7.7.6 (RNA polymerase). It is a diazaphene compound, belonging to the organic heterotetracyclic class of compounds, and is a pyrazine, secondary amide, and naphthidine derivative.
Pinarulex is an orally bioavailable RNA polymerase I (Pol I) inhibitor with potential antitumor activity. After oral administration, pinarulex selectively binds to and inhibits Pol I, blocking Pol I-mediated ribosomal RNA (rRNA) synthesis, inducing apoptosis, and inhibiting tumor cell growth. Pol I is a multi-protein complex that synthesizes rRNA, is upregulated in cancer cells, and plays a crucial role in cell proliferation and survival. Excessive activation of rRNA transcription is associated with uncontrolled proliferation of cancer cells.

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Dysregulation of ribosomal RNA synthesis is associated with uncontrolled proliferation of cancer cells. RNA polymerase (Pol) I is a multi-protein complex that synthesizes rRNA and is widely activated in cancer. Therefore, selective Pol I inhibitors may provide a universal therapeutic strategy for blocking cancer cell proliferation. By combining medicinal chemistry research with tandem screening based on cells and molecules, CX-5461, a potent small-molecule rRNA synthesis inhibitor, was ultimately designed. CX-5461 selectively inhibits Pol I-driven transcription, while having no significant effect on Pol II-driven transcription, DNA replication, and protein translation. Molecular studies have shown that CX-5461 inhibits the initiation of rRNA synthesis and induces senescence and autophagy in solid tumor cell lines through a p53-independent pathway, but does not induce apoptosis. CX-5461 has good oral bioavailability and has shown in vivo antitumor activity against human solid tumors in a mouse xenograft model. Our results suggest that CX-5461 is a promising, highly selective, and orally available cancer treatment that can be used to conduct clinical trials. [1] RNA polymerase I-mediated enhanced transcription of ribosomal RNA genes (rDNA) is a common feature of human cancers, but it is unclear whether it is necessary for malignant phenotypes. We found that the small molecule CX-5461 can act as a targeted therapy for rDNA transcription, selectively killing B lymphoma cells in vivo while maintaining the survival of wild-type B cell populations. This therapeutic effect is due to nucleolar destruction and activation of p53-dependent apoptosis signaling. Human leukemia and lymphoma cell lines also show high sensitivity to rDNA transcription inhibitors, and this sensitivity depends on the mutation status of p53. These results suggest that selective inhibition of rDNA transcription can be used as a therapeutic strategy to specifically activate p53 and treat hematologic malignancies. [2] RNA polymerase I (Pol I)-mediated transcription of ribosomal RNA genes (rDNA) is confined to the nucleolus and is the rate-limiting step in cell growth and proliferation. CX-5461 inhibition of Pol I selectively induces p53-mediated apoptosis in tumor cells in vivo. Currently, CX-5461 is undergoing clinical trials at Peter McKenna Hospital in Melbourne for patients with advanced hematologic malignancies. This paper demonstrates that CX-5461 can induce p53-independent cell cycle checkpoints mediated by the ATM/ATR signaling pathway without DNA damage. Furthermore, our data suggest that the combination of drugs targeting the ATM/ATR signaling pathway with CX-5461 significantly improves the therapeutic effect of p53-deficient tumors in vivo, which are usually unresponsive to single drugs. Mechanistically, we found that CX-5461 induces an aberrant chromatin structure in which transcriptionally active relaxed rDNA repeat sequences lack transcription of Pol I, thereby activating the ATM signaling pathway within the nucleolar. Therefore, we propose that the acute inhibition of Pol transcription initiation by CX-5461 induces a novel nucleolar stress response, which could serve as a target to enhance therapeutic efficacy. Oncotarget. 2016 Aug 2; 7(31):49800-49818.

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CX-5461 is an oral small RNA polymerase I (Pol I) inhibitor and the first drug to target ribosome biosynthesis[1]
- Its antitumor mechanism involves selectively blocking Pol I-mediated rRNA synthesis, leading to nucleolar stress, cancer-specific p53 activation, and apoptosis[2]
- It has been developed for the treatment of solid tumors with wild-type p53; it has not yet been approved by the FDA (as of the time of publication, it is still in clinical development)[1,2]
- Its selectivity for tumor cells stems from the increased dependence of tumor cells on Pol I-mediated ribosome biosynthesis to support their rapid proliferation[2]
- Preclinical models have shown that the drug has a synergistic effect with DNA damaging agents such as cisplatin, which may enhance the efficacy in p53 wild-type tumors[1]

C27H27N7O2S

513.61

513.194

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

1138549-36-6

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

25257557

White solid powder

1.5±0.1 g/cm3

739.9±60.0 °C at 760 mmHg

401.3±32.9 °C

0.0±2.4 mmHg at 25°C

1.740

-0.81

1

9

4

37

915

0

S1C2=C([H])C([H])=C([H])C([H])=C2N2C1=C(C(N([H])C([H])([H])C1C([H])=NC(C([H])([H])[H])=C([H])N=1)=O)C(C1C([H])=C([H])C(=NC2=1)N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C([H])([H])C1([H])[H])=O

XGPBJCHFROADCK-UHFFFAOYSA-N

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)

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

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.)