 Chemical Structure.png)
Physicochemical Properties
| Molecular Formula | C13H13N3OS | |
| Molecular Weight | 259.33 | |
| Exact Mass | 259.077 | |
| Elemental Analysis | C, 60.21; H, 5.05; N, 16.20; O, 6.17; S, 12.36 | |
| CAS # | 4311-88-0 | |
| Related CAS # | Necrostatin-1 (inactive control);64419-92-7 | |
| PubChem CID | 2828334 | |
| Appearance | Light yellow solid powder | |
| Density | 1.4±0.1 g/cm3 | |
| Boiling Point | 441.9±37.0 °C at 760 mmHg | |
| Melting Point | 151ºC | |
| Flash Point | 221.1±26.5 °C | |
| Vapour Pressure | 0.0±1.1 mmHg at 25°C | |
| Index of Refraction | 1.738 | |
| LogP | 1.26 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 2 | |
| Rotatable Bond Count | 2 | |
| Heavy Atom Count | 18 | |
| Complexity | 373 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | S=C1N(C([H])([H])[H])C(C([H])(C([H])([H])C2=C([H])N([H])C3=C([H])C([H])=C([H])C([H])=C23)N1[H])=O |
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| InChi Key | TXUWMXQFNYDOEZ-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C13H13N3OS/c1-16-12(17)11(15-13(16)18)6-8-7-14-10-5-3-2-4-9(8)10/h2-5,7,11,14H,6H2,1H3,(H,15,18) | |
| Chemical Name | 5-(1H-indol-3-ylmethyl)-3-methyl-2-sulfanylideneimidazolidin-4-one | |
| Synonyms |
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| 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 |
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| 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 | RIP1 kinase (EC50 = 182 nM) |
| ln Vitro | Necrostatin-1 (1-100 μM) prevents endogenous and overexpressed RIP1 from being autophosphorylated.It has been discovered that Necrostatin-1's antinecroptosis activity is primarily mediated by RIP1. [1] Necrostatin-1 effectively prevents necroptotic cell death in a range of cell types that is induced by a variety of stimuli. With an EC50 of 490 nM, necrostatin-1, previously identified as a small-molecule necroptosis inhibitor, blocks both RIP kinase and TNF-α-induced necroptosis in Jurkat cells. [2] |
| ln Vivo |
Necrostatin-1 (Nec-1) is a specific small molecule inhibitor of receptor-interacting protein kinase 1 (RIPK1) that specifically inhibits phosphorylation of RIPK1. RIPK1 regulates inflammation and cell death by interacting with receptor-interacting serine/threonine protein kinases 3(RIPK3). We hypothesized that Nec-1 may have anti-inflammatory efficacy in patients with osteoarthritis (OA), as the pathophysiology of OA involves the activation of inflammation-related signaling pathways and apoptosis. In this study, we explored the effects of Nec-1 on interleukin (IL)-1β-induced inflammation in mouse chondrocytes and the destabilised medial meniscus (DMM) mouse model. Inhibiting RIPK1 with Nec-1 dramatically suppressed catabolism both in vivo and in vitro, but did not inhibit changes in subchondral bone. Nec-1 abolished the in vitro increases in matrix metalloproteinase (MMP) and ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTs5) expression induced by IL-1β. However, adding high-mobility group box 1 (HMGB1) partially abrogated this effect, indicating the essential role of HMGB1 and Nec-1 in the protection of primary chondrocytes. Furthermore, Nec-1 decreased the expression of Toll-like receptor 4 (TLR4) and stromal cell-derived factor-1 (SDF-1), and attenuated the interaction between TLR4 and HMGB1. Western blot results suggested that Nec-1 significantly suppressed IL-1β-induced NF-κB transcriptional activity, but not MAPK pathway. Micro-computed tomography, immunohistochemical staining, and Safranin O/Fast Green staining were used in vivo to assess the degree of destruction of OA cartilage. The results show that NEC-1 can significantly reduce the degree of destruction of OA cartilage. Therefore, Nec-1 may be a novel therapeutic candidate to treat OA.[3] To examine the involvement of necroptosis in ischemic brain injury, we determined the effect of Nec-1 on ischemic damage resulting from middle cerebral artery occlusion (MCAO) in mice. Intracerebroventricular administration of Nec-1 significantly (P < 0.05) reduced the infarct volume after MCAO, which suggested that necroptosis could be involved in this form of pathologic death (Fig. 6a). For more detailed analyses, we switched to 7-Cl-Nec-1, which showed greater activity in vitro. 7-Cl-Nec-1 also provided a significant (P < 0.05) and dose-dependent reduction in the infarct volume and a proportionate improvement in the neurological score after MCAO (Fig. 6b).[2] Necrostatin-1 (Nec-1) is a specific small molecule inhibitor of receptor-interacting protein kinase 1 (RIPK1), which prevents RIPK1's phosphorylation. |
| Enzyme Assay |
In vitro kinase assay[1] The assay was performed essentially as described13. 293T cells were transfected with pcDNA3-FLAG-RIP1 vector, vectors encoding RIP1 mutant proteins or pcDNA3-RIP2-Myc and pcDNA3-FLAG-RIP3 vectors using standard Ca3(PO4)2 precipitation procedure. Culture medium was replaced 6 h after the transfection and cells were lysed 48 h later in the TL buffer consisting of 1% Triton X-100, 150 mM NaCI, 20 mM HEPES, pH 7.3, 5 mM EDTA, 5 mM NaF, 0.2 mM NaVO3 (ortho) and complete protease inhibitor cocktail. Immunoprecipitation was carried out for 16 h at 4 °C using anti-FLAG M2 agarose beads, followed by three washes with TL buffer and two washes with 20 mM HEPES, pH 7.3. Beads were incubated in 15 μl of the reaction buffer containing 20 mM HEPES, pH 7.3, 10 mM MnCl2 and 10 mM MgCl2 for 15 min at 23–25 °C in the presence of different concentrations of necrostatins. For these assays, compound stocks (in DMSO) were diluted to appropriate concentrations in DMSO before the addition to the reactions to maintain final concentration of DMSO for all samples at 3%. Kinase reaction was initiated by addition of 10 μM cold ATP and 1 mCi of [γ-32P] ATP, and reactions were carried out for 30 min at 30 °C. Reactions were stopped by boiling in SDS-PAGE sample buffer and subjected to 8% SDS-PAGE. RIP1 band was visualized by analysis in a Storm 8200 Phosphorimager. Similar protocol was used for endogenous RIP1 kinase reactions, except mouse monoclonal RIP1 antibody and protein magnetic beads or rabbit RIP1 antibody-coupled agarose beads were used. For recombinant baculovirally expressed RIP1, protein was expressed in Sf9 cells according to manufacturer's instructions and purified using glutathione-sepharose bead. Protein was eluted in 50 mM Tris-HCl, pH 8.0 supplemented with 10 mM reduced glutathione, and eluted protein was used in the kinase reactions, supplemented with 5 × kinase reaction buffer (100 mM HEPES, pH 7.3, 50 mM MnCl2, 50 mM MgCl2, 50 μM cold ATP and 5 μCi of [γ-32P]ATP). Phosphorylation of RIP1 requires its kinase activity. RIP1 kinase assays are carried out as outlined in the Methods in the presence of [γ-32P]ATP for 30 min at 30°C using expression constructs of FLAGtagged wild-type (WT) or a kinase-inactive pointmutant of RIP1 (K45M) or FLAGtagged wild-type (WT) or K45M. Following SDS-PAGE, samples are subjected to autoradiography to identify the RIP1 band. This autoradiograph, along with all others, displays the ratio-based relative intensities of radioactive bands. To make sure that there are equal amounts of protein in kinase reactions, a sample of beads is put through a western blot analysis using anti-RIP1 antibody. |
| Cell Assay |
Cell Viability Assay[3] The CCK-8 assay (Boster) was used to assess cell proliferation and viability. Briefly, primary chondrocytes were seeded at a density of 10,000 cells per well in 96-well plates. The next day, culture medium containing DMSO (vehicle) or an equal volume of Necrostatin-1 (Nec-1) (30 μmol/L) was added every day. The culture medium was replaced after 24, 48, and 72 h with 100 μL of medium containing a 10% CCK-8 solution, then incubated for 1 h in the dark at 37°C. Absorbance was measured at 450 nm with an ELX800 microplate reader. Apoptosis Analysis[3] An Annexin V-FITC/propidium iodide (PI) kit was used to detect apoptosis in chondrocytes in the presence of IL-1β (5 ng/mL) with or without Necrostatin-1 (Nec-1) (30 μmol/L). The chondrocytes were collected after treatment, washed three times in ice-cold PBS and resuspended in binding buffer. Then, 5 μL PI and 5 μL Annexin V were added to the buffer for 15 min at 4°C in the dark. A FACS Calibur flow cytometer was used to analyze the apoptotic cells in the early and late phases according to the manufacturer’s instructions. Cellular EC50 determination of necrostatins[1] Determination of EC50 was performed in FADD-deficient Jurkat cells treated with human TNFα as previously described. Briefly, cells were seeded into 96-well plates and treated with a range of necrostatin concentrations (30 nM to 100 μM, 11 dose points) in the presence and absence of 10 ng ml–1 human TNFα for 24 h. For these and all other cellular assays, compound stocks (in DMSO) were diluted to appropriate concentrations in DMSO before addition to the cells to maintain final concentration of DMSO for all samples at 0.5%. Cell viability was determined using CellTiter-Glo luminescent cell viability assay. Ratio of luminescence in compound and TNF-treated wells to compound-treated, TNF-untreated wells was calculated (viability, %) and used to calculate EC50 by nonlinear regression in GraphPad Prizm. |
| Animal Protocol |
For drug administration, we dissolved 7-Cl-Necrostatin-1 (Nec-1) or other derivatives in 4% methyl-β-cyclodextrin (Sigma) solution in PBS and administered it through intracerebroventricular administration at the time points indicated. Typically, we performed two 2-μl injections of 4 mM stock solution (unless otherwise indicated). For preocclusion delivery, we performed injections 5 min before the onset of 2-h MCAO occlusion and immediately after the cessation of the occlusion, at the time of the reperfusion. For postocclusion delivery, we performed injections at the time of reperfusion after 2 h of MCAO as well as 2 h after the onset of reperfusion. In the case of infusion, we infused 20 μl of compound over a 30-min time period. In the case of injection 6 h after occlusion, we injected a single 4-μl dose. In the case of zVAD.fmk administration, we added it to the Necrostatin-1 (Nec-1) formulation and administered a total dose of 160 ng.[2] Twelve-week-old male C57BL/6 mice were housed in a light- and temperature-controlled room and fed a standard diet. The body weight of animals was presented in the Supplementary Table I. The destabilised medial meniscus (DMM) surgical model of OA was produced on the right knee according to a previously published protocol (Glasson et al., 2007). Forty mice were divided into four groups (n = 10 per group): (1) sham group: sham-operated mice administered 15 μL vehicle (5% DMSO, 45% PEG300, and ddH2O), (2) the sham + Necrostatin-1 (Nec-1) group: sham-operated mice treated with 15 μL Necrostatin-1 (Nec-1) (0.0468 mg/Kg), (3) the DMM group: DMM surgery mice administered 15 μL vehicle, and (4) the DMM + Necrostatin-1 (Nec-1) group: DMM surgery mice administered 15 μL Nec-1. The Necrostatin-1 (Nec-1) solution or vehicle was injected intra-articularly twice weekly for 8 weeks before sacrifice.[3] |
| References |
[1].Nat Chem Biol . 2008 May;4(5):313-21. [2]. Nat Chem Biol . 2005 Jul;1(2):112-9. [3]. Front Pharmacol . 2018 Nov 27;9:1378. |
| Additional Infomation |
5-(1H-indol-3-ylmethyl)-3-methyl-2-sulfanylidene-4-imidazolidinone is an organonitrogen compound and an organooxygen compound. It is functionally related to an alpha-amino acid. Necroptosis is a cellular mechanism of necrotic cell death induced by apoptotic stimuli in the form of death domain receptor engagement by their respective ligands under conditions where apoptotic execution is prevented. Although it occurs under regulated conditions, necroptotic cell death is characterized by the same morphological features as unregulated necrotic death. Here we report that necrostatin-1, a previously identified small-molecule inhibitor of necroptosis, is a selective allosteric inhibitor of the death domain receptor-associated adaptor kinase RIP1 in vitro. We show that RIP1 is the primary cellular target responsible for the antinecroptosis activity of necrostatin-1. In addition, we show that two other necrostatins, necrostatin-3 and necrostatin-5, also target the RIP1 kinase step in the necroptosis pathway, but through mechanisms distinct from that of necrostatin-1. Overall, our data establish necrostatins as the first-in-class inhibitors of RIP1 kinase, the key upstream kinase involved in the activation of necroptosis.[1] The mechanism of apoptosis has been extensively characterized over the past decade, but little is known about alternative forms of regulated cell death. Although stimulation of the Fas/TNFR receptor family triggers a canonical 'extrinsic' apoptosis pathway, we demonstrated that in the absence of intracellular apoptotic signaling it is capable of activating a common nonapoptotic death pathway, which we term necroptosis. We showed that necroptosis is characterized by necrotic cell death morphology and activation of autophagy. We identified a specific and potent small-molecule inhibitor of necroptosis, necrostatin-1, which blocks a critical step in necroptosis. We demonstrated that necroptosis contributes to delayed mouse ischemic brain injury in vivo through a mechanism distinct from that of apoptosis and offers a new therapeutic target for stroke with an extended window for neuroprotection. Our study identifies a previously undescribed basic cell-death pathway with potentially broad relevance to human pathologies.[2] Necrostatin-1 (Nec-1) is a specific small molecule inhibitor of receptor-interacting protein kinase 1 (RIPK1) that specifically inhibits phosphorylation of RIPK1. RIPK1 regulates inflammation and cell death by interacting with receptor-interacting serine/threonine protein kinases 3(RIPK3). We hypothesized that Nec-1 may have anti-inflammatory efficacy in patients with osteoarthritis (OA), as the pathophysiology of OA involves the activation of inflammation-related signaling pathways and apoptosis. In this study, we explored the effects of Nec-1 on interleukin (IL)-1β-induced inflammation in mouse chondrocytes and the destabilised medial meniscus (DMM) mouse model. Inhibiting RIPK1 with Nec-1 dramatically suppressed catabolism both in vivo and in vitro, but did not inhibit changes in subchondral bone. Nec-1 abolished the in vitro increases in matrix metalloproteinase (MMP) and ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTs5) expression induced by IL-1β. However, adding high-mobility group box 1 (HMGB1) partially abrogated this effect, indicating the essential role of HMGB1 and Nec-1 in the protection of primary chondrocytes. Furthermore, Nec-1 decreased the expression of Toll-like receptor 4 (TLR4) and stromal cell-derived factor-1 (SDF-1), and attenuated the interaction between TLR4 and HMGB1. Western blot results suggested that Nec-1 significantly suppressed IL-1β-induced NF-κB transcriptional activity, but not MAPK pathway. Micro-computed tomography, immunohistochemical staining, and Safranin O/Fast Green staining were used in vivo to assess the degree of destruction of OA cartilage. The results show that NEC-1 can significantly reduce the degree of destruction of OA cartilage. Therefore, Nec-1 may be a novel therapeutic candidate to treat OA.[3] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (9.64 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (9.64 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. Solubility in Formulation 3: 5% DMSO+45% PEG 300+ddH2O: 10mg/mL Solubility in Formulation 4: 12.5 mg/mL (48.20 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 5: 1.67 mg/mL (6.44 mM) in 10% (50% EtOH 50% Cremophor EL) 90% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.8561 mL | 19.2805 mL | 38.5609 mL | |
| 5 mM | 0.7712 mL | 3.8561 mL | 7.7122 mL | |
| 10 mM | 0.3856 mL | 1.9280 mL | 3.8561 mL |