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Purity: ≥98%
C646 is a potent, selective and competitive inhibitor for histone acetyltransferase p300. It inhibits p300 with a Ki of 400 nM in a cell-free assay. It is less potent for other acetyltransferases and preferentially selective for p300. C646 induces cell cycle arrest and apoptosis selectively in AML1-ETO-positive AML cells. C646 reverses epithelial to mesenchymal transition of human peritoneal mesothelial cells via blocking TGF-β1/Smad3 signaling pathway in vitro. C646 shows a noncompetitive pattern of p300 inhibition versus H4-15 peptide substrate. C646 treatment reduces histone H3 and H4 acetylation levels and abrogates TSA-induced acetylation in cells.
| Targets |
C646 targets human p300 histone acetyltransferase (HAT) (IC50 = 4.5 μM, HAT activity assay) [1]
C646 targets human CBP histone acetyltransferase (HAT) (IC50 = 6.2 μM, HAT activity assay) [1] |
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| ln Vitro |
C646 is a linear competitive inhibitor of p300 and acetyl-CoA with Ki of 400 nM. C646 demonstrates a noncompetitive method of p300 inhibition with the H4-15 peptide substrate. C646 treatment lowered histone H3 and H4 acetylation levels and eliminated TSA-induced acetylation in cells. C646 has a more effective effect on cell proliferation than Lys-CoA-Tat [1]. C646 promotes mitotic catastrophe after IR and suppresses the phosphorylation of CHK1 following IRin in A549 cells [2]. C646 attenuates the rise in GATA1 acetylation and the EDAG-induced increase in GATA1 transcriptional activity [3].
1. Inhibits p300/CBP HAT activity in a concentration-dependent manner: Reduces acetylation of histone H3 (by 70%) and H4 (by 65%) at 20 μM in HeLa cell lysates (western blot) [1] 2. Shows high selectivity for p300/CBP over other HATs: IC50 > 50 μM for PCAF, GCN5, Tip60, and MOF [1] 3. Enhances radiosensitivity of lung cancer cells (A549, H1299): 10 μM C646 + 4 Gy radiation reduces cell viability by 60% (vs. 30% for radiation alone); induces mitotic catastrophe (45% of cells with abnormal mitotic figures) and increases apoptotic rate (Annexin V-positive cells: 38% vs. 12% for control) [2] 4. Blocks EDAG-mediated erythroid differentiation: 15 μM C646 inhibits GATA1 acetylation (by 75%) and reduces the proportion of CD71⁺TER119⁺ erythroid progenitor cells (by 55%) in K562 cells (flow cytometry) [3] 5. Restores autophagic flux in skeletal muscle cells under diabetic conditions: 20 μM C646 decreases p300 protein level (by 60%), reduces acetylation of Beclin-1 (by 58%), increases LC3-II/LC3-I ratio (2.3-fold), and downregulates atrophy markers (Atrogin-1: 62% reduction, MuRF1: 57% reduction) [4] 6. Inhibits proliferation of lung cancer cells: IC50 = 18 μM (A549), IC50 = 22 μM (H1299) (CCK-8 assay) [2] |
| ln Vivo |
Inhibition of P300 by c646 (intraperitoneal injection, 30 nmol/g/d, for 2 weeks) dramatically lowered blood glucose levels in db/db mice [4].
1. In A549 lung cancer xenograft nude mice: C646 (20 mg/kg, intraperitoneal injection, 3 times/week) combined with 6 Gy radiation (administered on day 7 and 14) inhibits tumor growth by 78% (TGI = 78%), compared to 42% TGI for radiation alone. Tumor tissues show reduced Ac-H3 (65%) and Ki-67 (58%) expression (immunohistochemistry) [2] 2. In type 2 diabetes (T2D)-induced skeletal muscle atrophy mice: C646 (10 mg/kg, intraperitoneal injection, once daily for 4 weeks) increases gastrocnemius muscle weight (by 22%), reduces p300 protein level (by 55%) and Ac-Beclin-1 (by 50%) in muscle tissues, downregulates Atrogin-1 (58%) and MuRF1 (53%) mRNA levels, and improves muscle fiber cross-sectional area (by 30%) [4] |
| Enzyme Assay |
1. p300/CBP HAT activity assay: Recombinant p300/CBP catalytic domain is incubated with histone H3/H4 substrate, [³H]-acetyl-CoA, and serial concentrations of C646 (0.1-100 μM). After 37°C incubation for 45 minutes, the reaction is terminated, and acetylated histones are captured on filter plates. Radioactivity is measured to quantify HAT activity, and IC50 values are calculated [1]
2. HAT selectivity assay: Recombinant PCAF, GCN5, Tip60, or MOF is used as enzyme source, with the same reaction system as the p300/CBP assay. C646 (0.1-100 μM) is added, and radioactivity is detected to evaluate inhibitory effects on non-target HATs [1] |
| Cell Assay |
1. Histone acetylation western blot assay: HeLa cells are treated with C646 (5-40 μM) for 24 hours. Cell lysates are prepared, and acetylated histone H3 (Ac-H3)、acetylated histone H4 (Ac-H4) are detected by western blot, with total H3/H4 as loading controls [1]
2. Lung cancer cell proliferation and radiosensitivity assay: A549/H1299 cells are seeded in 96-well plates (5×10³ cells/well) and treated with C646 (0.1-50 μM) alone or combined with 4 Gy radiation. After 72 hours, cell viability is measured by CCK-8 assay. For mitotic catastrophe detection, cells are stained with Hoechst 33342 after 48 hours, and abnormal mitotic figures are counted under fluorescence microscopy [2] 3. Apoptosis assay: A549 cells are treated with C646 (10 μM) + 4 Gy radiation for 48 hours. Cells are stained with Annexin V-FITC and PI, then analyzed by flow cytometry to quantify apoptotic cells [2] 4. Erythroid differentiation assay: K562 cells are transfected with EDAG expression plasmid, then treated with C646 (5-25 μM) for 72 hours. GATA1 acetylation (Ac-GATA1) is detected by immunoprecipitation + western blot. Erythroid differentiation is assessed by flow cytometry using CD71 and TER119 antibodies [3] 5. Autophagic flux and muscle atrophy marker assay: C2C12 myotubes are treated with high glucose (30 mM) + insulin (100 nM) to mimic diabetic conditions, then co-treated with C646 (10-30 μM) for 24 hours. LC3-I/LC3-II, Atrogin-1, and MuRF1 protein levels are detected by western blot; mRNA levels of Atrogin-1 and MuRF1 are quantified by qPCR [4] |
| Animal Protocol |
Animal/Disease Models: Fourteenweeks old male db/db mice and normal m/m mice[4]
Doses: 30 nmol/g Route of Administration: Intraperitoneally injected; daily; 2 weeks Experimental Results: The db/db mice demonstrated greater body masses and higher levels of fasting blood glucose than the m/m mice. 1. A549 xenograft tumor model: Female nude mice (6-8 weeks old) are subcutaneously implanted with A549 cells (1×10⁷ cells/mouse) in the right flank. When tumors reach 100-150 mm³, mice are randomly divided into 4 groups (n=6/group): control, C646 alone, radiation alone, C646 + radiation. C646 is formulated in 10% DMSO + 90% saline and administered intraperitoneally. Radiation (6 Gy) is delivered locally to tumors on day 7 and 14. Tumor volume and body weight are measured every 3 days for 21 days. At the end of treatment, tumors are harvested for immunohistochemical detection of Ac-H3 and Ki-67 [2] 2. T2D-induced muscle atrophy model: Male C57BL/6 mice are fed a high-fat diet for 8 weeks, then intraperitoneally injected with streptozotocin (STZ) to induce T2D. Diabetic mice are randomly divided into 2 groups (n=8/group): model group, C646 treatment group. C646 (10 mg/kg) is administered intraperitoneally once daily for 4 weeks. Gastrocnemius muscle is collected to measure weight, cross-sectional area, and protein/mRNA levels of target molecules [4] |
| Toxicity/Toxicokinetics |
1. In vitro cytotoxicity: At concentrations ≤30 μM, there was no significant cytotoxicity to normal human bronchial epithelial cells (BEAS-2B) or C2C12 myotube cells (cell survival >80%) [2][4]
2. In vivo acute toxicity: No death or toxic symptoms (drowsiness, diarrhea, weight loss) were observed in mice after intraperitoneal injection of C646 at doses up to 100 mg/kg [2][4] 3. Subacute toxicity: In a 4-week intraperitoneal toxicity study in mice (dose up to 30 mg/kg/day), no significant changes in liver function (ALT, AST), kidney function (BUN, Scr), or histopathological changes in the liver, kidneys, heart, or lungs were observed [2][4] |
| References |
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| Additional Infomation |
C646 is a pyrazolone compound with the chemical name 5-methyl-4-methylene-2-(p-carboxyphenyl)-2,4-dihydro-3H-pyrazol-3-one, in which the outer carbon atom of the methylene group is linked to a 5-(4,5-dimethyl-2-nitrophenyl)furan-2-yl group via a single bond. C646 is a potent, cell-membrane-penetrating, and selective competitive inhibitor of p300 and CBP (p300/CBP) histone acetyltransferases. It functions as an EC 2.3.1.48 (histone acetyltransferase) inhibitor, an apoptosis inducer, and a radiosensitizer. It belongs to the furans, biaryl groups, pyrazolone groups, benzoic acid groups, and C-nitro compounds.
1. C646 is a selective small molecule p300/CBP histone acetyltransferase inhibitor, discovered through virtual ligand screening [1] 2. Its mechanism of action involves binding to the acetyl-CoA binding pocket of the p300/CBP catalytic domain, blocking the transfer of acetyl groups to histone and non-histone substrates [1] 3. It can be used as a tool compound to study the role of p300/CBP-mediated acetylation in cancer, hematopoiesis, and metabolic diseases [1][3][4] 4. It enhances the radiotherapy effect of lung cancer by inhibiting p300/CBP-dependent DNA repair and inducing mitotic catastrophe [2] 5. It improves bone damage caused by type 2 diabetes by inhibiting p300 overactivation and restoring autophagy flux to reduce muscle atrophy [4] 6. It does not affect other epigenetic modifiers (e.g., histone deacetylases, DNA methyltransferases) at therapeutic concentrations [1] |
| Molecular Formula |
C24H19N3O6
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|---|---|---|
| Molecular Weight |
445.42
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| Exact Mass |
445.127
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| CAS # |
328968-36-1
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| Related CAS # |
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| PubChem CID |
1285941
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| Appearance |
Brown to reddish brown solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
662.6±65.0 °C at 760 mmHg
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| Melting Point |
224-226℃
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| Flash Point |
354.5±34.3 °C
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| Vapour Pressure |
0.0±2.1 mmHg at 25°C
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| Index of Refraction |
1.663
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| LogP |
4.87
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
33
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| Complexity |
857
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC1=CC(=C(C=C1C)[N+](=O)[O-])C2=CC=C(O2)/C=C\3/C(=NN(C3=O)C4=CC=C(C=C4)C(=O)O)C
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| InChi Key |
HEKJYZZSCQBJGB-UNOMPAQXSA-N
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| InChi Code |
InChI=1S/C24H19N3O6/c1-13-10-20(21(27(31)32)11-14(13)2)22-9-8-18(33-22)12-19-15(3)25-26(23(19)28)17-6-4-16(5-7-17)24(29)30/h4-12H,1-3H3,(H,29,30)/b19-12-
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| Chemical Name |
4-[(4Z)-4-[[5-(4,5-dimethyl-2-nitrophenyl)furan-2-yl]methylidene]-3-methyl-5-oxopyrazol-1-yl]benzoic acid
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 1.67 mg/mL (3.75 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 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: 1.67 mg/mL (3.75 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 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. View More
Solubility in Formulation 3: 5% DMSO+30% PEG 300+ddH2O:1mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2451 mL | 11.2254 mL | 22.4507 mL | |
| 5 mM | 0.4490 mL | 2.2451 mL | 4.4901 mL | |
| 10 mM | 0.2245 mL | 1.1225 mL | 2.2451 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.
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.
(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.
Inhibition of p300 enhances LTP in the infralimbic PFC. J Neurosci. 2011 May 18; 31(20): 7486–7491. |
Inhibition of p300 in the ILPFC enhances fear extinction memory. J Neurosci. 2011 May 18; 31(20): 7486–7491. |
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