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KRX-0601 (UCN-01; KRX0601; 7-hydroxystaurosporine) is a synthetic staurosporine analog with antibiotic and anticancer activity, acting by inhibiting many phosphokinases such as serine/threonine kinase AKT, calcium-dependent protein kinase C, and cyclin-dependent kinases, resulting in accumulation of cells in the G1 phase and induction of apoptosis. UCN-01 also enhances the cytotoxicity of other anti-cancer drugs, such as DNA-damaging agents and anti-metabolite drugs. This agent arrests tumor cells in the G1/S of the cell cycle and prevents nucleotide excision repair by inhibiting the G2 checkpoint kinase chk1, resulting in apoptosis.
Physicochemical Properties
| Molecular Formula | C28H26N4O4 |
| Molecular Weight | 482.53 |
| Exact Mass | 482.195 |
| Elemental Analysis | C, 69.70; H, 5.43; N, 11.61; O, 13.26 |
| CAS # | 112953-11-4 |
| Related CAS # | 112953-11-4 |
| PubChem CID | 72271 |
| Appearance | Typically exists as solid at room temperature |
| Density | 1.63g/cm3 |
| Boiling Point | 705.7ºC at 760mmHg |
| Flash Point | 380.6ºC |
| Vapour Pressure | 6.64E-21mmHg at 25°C |
| Index of Refraction | 1.827 |
| LogP | 4.564 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 5 |
| Rotatable Bond Count | 2 |
| Heavy Atom Count | 36 |
| Complexity | 935 |
| Defined Atom Stereocenter Count | 5 |
| SMILES | C[C@@]12[C@H](OC)[C@H](NC)C[C@@H](O1)N3C4=CC=CC=C4C5=C6C([C@@H](O)NC6=O)=C7C8=CC=CC=C8N2C7=C53 |
| InChi Key | GUAIZLSIUMRUNL-IHQXTNFTSA-N |
| InChi Code | InChI=1S/C28H27N4O4/c1-28-7-4-8-31-19-9-14(33)10-21(34)24(19)23-18-13-30-12-17(18)22-16-6-5-15(36-28)11-20(16)32(29-2,27(28)35-3)26(22)25(23)31/h4-7,9,12-15,27,29,33H,8,10-11H2,1-3H3/b7-4-/t14-,15-,27+,28-/m0/s1 |
| Chemical Name | 9,13-Epoxy-1H,9H-diindolo[1,2,3-3',2',1'-lm]pyrrolo[3,4-j][1,7]benzodiazonin-1-one, 2,3,10,11,12,13-hexahydro-3-hydroxy-10-methoxy-9-methyl-11-(methylamino)-, (3R,9S,10R,11R,13R)- |
| Synonyms | KW2401, UCN 01,KW-2401, 7-Hydroxystaurosporine; UCN-01; 112953-11-4; Ucn 01; UCN01; KRX-0601; UCN-02; UNII-7BU5H4V94A; KRX06017-hydroxystaurosporineUCN-01,KW 2401UCN01, Staurosporine 7-hydroxystaurosporine |
| 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) |
Biological Activity
| Targets | Phosphokinase |
| ln Vitro | Human osteosarcoma (bone cancer) is a highly malignant and the most prevalent bone tumor affecting children. Despite recent advances in the understanding of the molecular mechanism by which anticancer drugs kill osteosarcoma or block its growth, however, the mortality rate has declined only modestly. Thus, a new therapeutic approach is needed to be established. 7-hydroxystaurosporine, UCN-01, abrogates the G2 checkpoint thus enhancing the cytotoxicity of chemotherapeutic agents. In addition, it has been evaluated in clinical trials as a single antineoplastic agent in treating several cancers. However, the effects of UCN-01 on treating bone cancer has never been tested. In this study, we found that UCN-01 induced cell cycle arrest and apoptosis in the human osteosarcoma, U2-OS cells. In addition, the migration ability was also reduced, suggesting UCN-01 inhibited cell growth and migration. When U2-OS cells were treated with UCN-01, DNA damage response was triggered. The ataxia telangiectasia mutated (ATM) and the non-canonical downstream effector, ERK, was activated by UCN-01. In addition, depletion of ATM or inhibition of ERK deteriorated the cell viability in UCN-01-treated U2-OS cells. Furthermore, UCN-01 induced autophagy activation for protecting cells from apoptosis. Thus, UCN-01 might function as a single antineoplastic agent in treating human osteosarcoma.[6] |
| ln Vivo | UCN-01 causes significant, reversible arrest of normal gut epithelial cells at 24 h; this arrest persists for up to 7 days. Normal cellular proliferation returns by 2 weeks. Pre-treatment of both non-tumour-bearing and MDA-MB-468 tumour-bearing mice with UCN-01 prior to bolus 5-FU (450 mg/kg) yielded enhanced therapeutic efficacy with significantly decreased tumour volumes and increased survival. Conclusions: UCN-01 mediates a specific, reversible G1 arrest of normal cells in vivo and provides a cytoprotective strategy that decreases toxicity of cytotoxic chemotherapy without compromising efficacy.[7] |
| Cell Assay |
MTT assay [6] Following UCN-01 treatment, cells were washed with PBS followed by adding 1 ml MTT solution (2 mg/mL in PBS) in each well. After incubation for 3 h at 37°C, 2 mL DMSO was added and incubation in the dark for additional 30 min. Absorbance was measured at the wavelength of 570 nm. EdU incorporation assay [6] For EdU incorporation assay, EdU positive cells were stained by detecting fluorescence EdU signaling according to manufacturer's instruction. Cells were co-stained with DAPI to visualize nuclei. EdU incorporation assay was performed in asynchronized cells after U2-OS cells were treated with UCN-01 for 24 h. |
| Animal Protocol |
UCN-01/mock treatment [7] Mice were injected with UCN-01 or carrier (dimethyl sulfoxide, DMSO, Sigma Aldrich, St. Louis MO) intramuscularly (i.m.) into the right hind limb. UCN-01 was obtained from the NCI Chemotherapeutic Agents Repository and is re-suspended in DMSO at 7.5 mg/ml. Prior to injection, the UCN-01 solution is diluted 2:1 with sterile normal saline for a final concentration of 5 mg/ml. DMSO given as vehicle control was diluted in the same fashion and was given as a volume equivalent dose. To obtain the normal mouse values for flow cytometry, mice either received no treatment or were given volume equivalent PBS in the right hind limb. Tumour xenografts [7] Nu/nu mice were given 2% isoflurane to anesthetise the mice for 5 min, which was confirmed with lack of toe pinch withdrawal reflex. In all, 5 × 106 MDA-MB-436 cells were suspended in 100 µL of media and injected subcutaneously 5 mm from the 4th nipple into the mammary fat pad of each of the nu/nu female mice and observed for any complications. Mice were treated with UCN-01 or DMSO control followed by treatment with 5-FU or PBS control, similar to the treatment of non-tumour-bearing mice. Sustained release buprenorphine at 1.0 mg/kg was administered every 6–12 h by subcutaneous injection with a 28-gauge needle between shoulder blades for analgesia as needed for pain. Bromodeoxyuridine labelling [7] Mice were sacrificed at one of five time points following UCN-01/mock injection: 24 h, 48 h and 7, 14 or 28 days, and the tissue processed (Supplementary Fig. S1). On the day of sacrifice, mice were injected i.p. (as above for 5-FU) with 100 µl of 60 mg/kg BrdU 6 h prior to sacrifice (as described under Euthanasia). Various time points post-BrdU injection were analysed for Bromodeoxyuridine (BrdU) staining and 6 h post-injection was determined to be optimal (Supplementary Fig. S1). Mice were staggered by injection and sacrifice times such that each mouse had exactly 6 h of BrdU exposure. Upon sacrifice, the jejunum was dissected out and flushed with ice-cold ethanol (60% in PBS). A small piece was fixed in formalin (10%), and the remainder fixed overnight in the 60% ethanol/PBS buffer. To prepare the tissues for flow cytometry, the jejunums were opened longitudinally and placed on a glass microscope slide. A second slide was dragged across the epithelial surface to dislodge the cells; care was taken not to apply excess pressure and disrupt the basement membrane. The isolated cells were re-suspended in 5 ml of 0.04% pepsin in a 50 ml glass flask and placed in a shaking water bath at 37 °C at 90 Hz for 1 h. The cells were filtered through 35-μm mesh and centrifuged at 1200 rpm for 5 min in a swinging bucket centrifuge. Cells were re-suspended in 1.5 ml of 2 N HCl for 20 min in a 37 °C incubator. The acid was neutralised with 3 ml 0.1 M sodium borate and the cells were again centrifuged at 1200 rpm for 5 min. The cells were washed with 5 ml of PBTB (phosphate-buffered saline plus 0.5% Tween-20 and 0.5% bovine serum albumin and centrifuged at 1200 rpm for 5 min. Samples were resuspended in primary anti-BrdU antibody diluted 1:100 in PBT (phosphate-buffered saline plus 0.5% Tween-20) for 60 min. Samples were washed in 5 ml of PBTB and centrifuged at 1200 rpm for 5 min. Samples were resuspended in 0.2 ml of secondary antibody (goat anti-mouse fluorescein isothiocyanate (GAM-FITC) diluted 1:100 in PBTG (PBT with 2% normal goat serum) for 60 min, then washed in PBTB and centrifuged at 1200 rpm for 5 min. Samples were then suspended in propidium iodide solution (1 mg/ml PI) in 95% ethanol diluted 1:100 in PBT) and stored at 4 °C overnight. |
| Toxicity/Toxicokinetics | mouse LD50 intraperitoneal 25 mg/kg Journal of Antibiotics., 42(571), 1989 [PMID:2656615] |
| References |
1. Cancer Res. 2000.60: 2108-2112. PMID: 10786669 7. Br J Cancer. 2020 Mar;122(6):812-822. |
| Additional Infomation |
UCN-02 is an indolocarbazole. 7-Hydroxystaurosporine has been reported in Streptomyces and Streptomyces longisporoflavus with data available. 7-Hydroxystaurosporine is a synthetic derivative of staurosporine with antineoplastic activity. 7-hydroxystaurosporine inhibits many phosphokinases, including the serine/threonine kinase AKT, calcium-dependent protein kinase C, and cyclin-dependent kinases. This agent arrests tumor cells in the G1/S of the cell cycle and prevents nucleotide excision repair by inhibiting the G2 checkpoint kinase chk1, resulting in apoptosis. (NCI04) In this study, we tested the hypothesis that UCN-01 is a cytoprotective agent temporarily inhibits the cell cycle progression of normal proliferating cells and improving the survival and health of mice receiving high-dose chemotherapy. Our in vivo findings suggest that UCN-01 meets both criteria for efficacy as a cytoprotective strategy: (1) the arrest of normal small bowel epithelial cells was reversible, and (2) the cell cycle arrest was specific to normal proliferating cells and did not protect tumour cells from the cytotoxic effects of 5-FU chemotherapy. Using a tumour-bearing mouse model, we have also shown that pre-treatment with UCN-01 prior to chemotherapy improves tolerance and prolongs survival.[7] In summary, we show that treatment of mice with low-dose UCN-01 induces a reversible, post-mitotic G1 arrest in normal cells of the small bowel and that this arrest improves tolerance to bolus 5-FU in non-tumour-bearing mice. In addition, in tumour-bearing mice we show that pre-treatment with low-dose UCN-01 prior to 5-FU decreased chemotoxicity and allowed for dose escalation of 5-FU to enhance its therapeutic efficacy.[7] In this study, we shown that UCN-01 inhibited U2-OS cell growth by blocking S phase entry and inducing apoptosis. In addition, the ability of cell migration was also reduced by UCN-01 treatment. The DNA damage response was also triggered by UCN-01 for maintaining cell survival. Furthermore, we also observed the activation of autophagy by UCN-01 treatment and inhibition of autophagy accelerated cell death under UCN-01 treatment. Thus, we uncovered the effects of UCN-01 on the growth and migration of human osteosarcoma U2-OS cell line. [6] UCN-01 treatment induced DNA damage response. The ATM is the upstream activator when cells suffer from DNA double-strand breaks. The canonical downstream effector of ATM is checkpoint kinase 2 (CHK2) or checkpoint kinase 1 (CHK1). However, neither CHK1 nor CHK2 were activated, only ERK1/2 were activated by ATM upon UCN-01 treatment. Under ionizing radiation, a dose-dependent AKT phosphorylation is observed and its activation leads to ERK phosphorylation. In addition, AKT was critical for activating ERK as reduced ERK phosphorylation is observed in dominant negative AKT overexpressing cells. These data suggest that ATM-activated ERK depends on AKT. However, other study shows that ATM binds and phosphorylates ERK directly independent of AKT when cells suffer from etoposide or cisplatin treatment. In our model, AKT was not activated by UCN-01 treatment, but only ERK activation was observed. In addition, depletion of ATM alleviated UCN-01-induced ERK activation in osteosarcoma cells. Thus, UCN-01 triggered an AKT-independent ERK activation by ATM.[6] UCN-01-treatment inhibited U2-OS cell migration. During normal migration, the centrosome locates in the anterior region of the nucleus. When checking the nuclear-centrosomal axis upon UCN-01 treatment, centrosome did not localize to the anterior margin of the nucleus and disorganized Golgi ribbon was observed. It has been suggested that microtubule nucleation activity is important for the maintaining the Golgi ribbon and the nuclear-centrosomal axis. By using microtubule regrowth assay, we found that UCN-01 inhibited microtubule nucleation. Thus, we speculated that UCN-01 reduced the microtubule nucleation activity followed by disrupted nuclear-centrosomal axis, leading to poor migration activity. Although we do not have direct evidences support the inter-correlation between these two events, however, according to the published results, proper microtubule nucleation is required for establishing the nucleus-centrosome-Golgi back-to-front axis, and disruption of this cellular polarity affects cell migration directionality.41 Thus, we speculate that UCN-01 treatment affects microtubule nucleation followed by reducing the cell migration ability. [6] Our data demonstrate for the first time UCN-01 treatment abrogates microtubule nucleation in the centrosome, however, it is still unclear how UCN-01 affects microtubule nucleation. It has been suggested that protein phosphatase 4 (Ppp4) is a serine/threonine phosphatase that is highly enriched at centrosomes. It forms a trimeric protein complex with the regulatory subunits, R2 and R3A, to mediate centrosome maturation and cell migration. Further study shows that the cyclin B/CDK1 complex phosphorylates Ppp4-R2-R3A (Ser159 and Thr173 of R2 and Ser741 of R3A) hetero-trimer thus allowing the γ-tubulin ring complex to nucleate microtubule growth. Inhibition of cyclin B/CDK1 reduces the phosphorylation of Ppp4-R2-R3A complex thus deucing the ability of γ-tubulin ring complex to nucleate microtubule growth. As UCN-01 also behaves as a CDK pan inhibitor. We thus speculate that UCN-01 might inhibit CDK1 activity followed by reducing Ppp4-R2-R3A phosphorylation, thus leading to poor microtubule nucleation and cell migration, but this hypothesis still needs to be tested. [6] In our study, we found that UNC-01 induced DNA damage responses via ATM activation. It is well known that ATM-activated DNA damage response triggers cell cycle arrest and DNA repair. However, little is known about the role of DNA damage response in regulating cell migration. Recent study shows that under hydrogen peroxide-induced oxidative stress, ATM-depended DNA damage response is activated. Meanwhile, reduced cell migration and invasion are also observed. However, there is no direct evidence support the reduced cell migration is caused by DNA damage response. One pilot study demonstrates that the anti-cancer drug, doxorubicin, activates p53 followed by repressing microtubule-associated protein 4 (MAP4) in C127 breast cancer cells. MAP4 is the major microtubule-associated protein in almost all tissues except neurons and it functions in regulating the microtubule polymerization. Proper microtubule nucleation is required for the centrosome positioning and cell migration. Thus, we speculate that UCN-01 treatment triggers DNA damage response followed by activating p53, leading to downregulation of MAP4 and poor microtubule nucleation, and cell migration. This hypothesis and the correlation between DNA damage response and cell migration still needs to be deeply studied.[6] Autophagy is a cellular process for the maintenance of metabolic homeostasis. In addition, it is also known as a protective mechanism under stresses. Here we found that UCN-01 activated autophagy for maintaining osteosarcoma survival. It has long been suggested that when DNA damages cannot be repaired, cellular death, or senescence will be triggered to prevent tumorigenesis. However, recent studies suggest that the transcriptional regulation of several autophagy related genes are regulated by FOXO3A, which is activated and promotes cell cycle arrest, DNA repair, or even apoptosis during DNA damages. In addition, FOXO3A directly interacts with ATM and facilitates its activity. As we observe the activation of ATM and autophagy in UCN-01-treated U2-OS cells, we speculate that UCN-01-induced DNA damage response activates either ATM or FOXO3A, and activated-FOXO3A positively facilitates the ATM activation and also promotes autophagic flux. However, this hypothesis still need to be tested.[6] |
Solubility Data
| Solubility (In Vitro) | May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples |
| 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). 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). 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 | 2.0724 mL | 10.3621 mL | 20.7241 mL | |
| 5 mM | 0.4145 mL | 2.0724 mL | 4.1448 mL | |
| 10 mM | 0.2072 mL | 1.0362 mL | 2.0724 mL |