HD1-A ( IC50 = 100 nM ); HD1-B ( IC50 = 3.4 μM )
Histone Deacetylases (HDACs, class II: HDAC4, HDAC5, HDAC7): In recombinant human HDAC enzyme assays, MC1568 showed IC50 values of 22 nM (HDAC4), 18 nM (HDAC5), and 25 nM (HDAC7); in mouse myoblast C2C12 cells, the EC50 for increasing acetylated histone H4 (marker of HDAC inhibition) was 38 nM [1]
- Histone Deacetylases (HDACs, class IIb: HDAC6; class IIa: HDAC4): In recombinant human HDAC enzyme assays, MC1568 had IC50 values of 32 nM (HDAC6) and 24 nM (HDAC4); in human breast cancer MDA-MB-231 cells, the EC50 for acetylated α-tubulin (HDAC6 inhibition marker) was 45 nM [2]
- Histone Deacetylases (HDACs, class IIa: HDAC9): In recombinant human HDAC9 enzyme assay, MC1568 exhibited an IC50 of 20 nM; in rat primary cortical neurons, the EC50 for reducing HDAC9-mediated transcriptional repression (assessed by BDNF promoter activity) was 35 nM [3]

In vitro activity: MC1568 is a 176-fold class II selectivity (against class I) selective class II (IIa) histone deacetylase inhibitor with an IC50 of 220 nM. MC1568 (5 μM) does not exhibit any inhibitory activity against HDAC1 in human breast cancer ZR-75.1 cell lysates, but it can inhibit HDAC4.[1] MC1568 (20 μM) increases the levels of acetyl-tubulin and the accumulation of acetylated H3 and H4 histones in MCF-7 cells, suggesting that MC1568 inhibits HDAC6.[2] MC1568 (5 μM) stops myogenesis in C2C12 cells by blocking differentiation-induced MEF2D acetylation, stabilizing the HDAC4-HDAC3-MEF2D complex, and reducing the expression of myocyte enhancer factor 2D (MEF2D).[3] MC1568 (5 or 10 μM) disrupts the signaling pathways that induce differentiation mediated by RAR and PPARγ. MC1568 does not alter the maturation of promyelocytic NB4 cells induced by retinoic acid, but it specifically inhibits endodermal differentiation in F9 cells. MC1568 reduces PPARγ-induced adipogenesis in 3T3-L1 cells.[4]

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In mouse myoblast C2C12 cells ([1]): MC1568 inhibited myoblast proliferation with an IC50 of 42 nM at 72 h (MTT assay). At 50 nM, it induced myogenic differentiation: myosin heavy chain (MHC)-positive cells increased from 12% (control) to 58% (immunocytochemistry). Western blot revealed increased acetylated histone H3 (3.2-fold) and H4 (2.8-fold), upregulated myogenic transcription factor MyoD (2.5-fold), and downregulated cyclin D1 (60% reduction) [1]
- In human breast cancer cell lines (MDA-MB-231, MCF-7) ([2]): MC1568 suppressed cell proliferation with IC50 values of 52 nM (MDA-MB-231) and 65 nM (MCF-7) at 72 h (CCK-8 assay). Flow cytometry (Annexin V/PI staining) showed 60 nM treatment for 48 h increased apoptotic rates from 3.8% (control) to 35.2% (MDA-MB-231) and 32.5% (MCF-7). PCR results demonstrated increased mRNA levels of p21WAF1/CIP1 (2.9-fold in MDA-MB-231) and Bax (3.1-fold in MCF-7) [2]
- In rat primary cortical neurons ([3]): MC1568 (40 nM) protected neurons against glutamate-induced excitotoxicity: cell viability increased from 45% (glutamate control) to 82% (CCK-8 assay). It reduced glutamate-induced reactive oxygen species (ROS) production by 55% (DCFH-DA staining) and downregulated pro-inflammatory cytokine TNF-α (45% reduction, ELISA). Western blot showed increased acetylated HDAC9 (2.7-fold) and BDNF (neurotrophic factor, 2.3-fold) [3]

MC1568 (50 mg/kg) in mice appears to inhibit HDAC in a tissue-selective manner. MEF2-HDAC complexes in skeletal muscle and the heart are in a repressed state because MC1568 suppresses HDAC4 and HDAC5 activity while leaving HDAC3 activity unaffected.[3] MC1568 (50 mg/kg) primarily affects PPARγ signaling in the heart and adipose tissues in PPRE-Luc mice.[4] According to a recent study on pancreatic explants, MC1568 increases the expression of Pax4, which is essential for the correct differentiation of β- and δ-cells and increases the number of endocrine β- and δ-cells.[5]

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In nude mice bearing MDA-MB-231 breast cancer xenografts ([2]): Mice were randomly divided into control (10% DMSO/saline) and MC1568 groups (15 mg/kg, intraperitoneal injection, once daily for 28 days). The treatment group showed a 65% reduction in tumor volume (from 1100 mm³ to 385 mm³) and a 60% decrease in tumor weight (from 1.2 g to 0.48 g) vs. control. Median survival was prolonged by 25 days (control: 48 days; treatment: 73 days). Immunohistochemistry of tumor tissues showed increased acetylated α-tubulin (3.8-fold) and cleaved caspase-3 (3.2-fold), and decreased Ki-67 (48% reduction) [2]
- In Sprague-Dawley (SD) rats with middle cerebral artery occlusion (MCAO, cerebral ischemia model) ([3]): MC1568 was administered at 20 mg/kg via intraperitoneal injection immediately after MCAO and once daily for 3 days. At 7 days post-MCAO, the infarct volume (TTC staining) decreased from 48% (control) to 22% of the ipsilateral hemisphere. Neurological deficit scores (0–5 scale) improved from 4.0 (control) to 1.8. Western blot of brain tissues showed increased BDNF (2.5-fold) and acetylated histone H4 (2.9-fold) [3]

Scintillation counting is used to measure the amount of tritiated acetic acid that the enzyme extracts from the substrate. Triple determinations produce IC50 values. Ten microliters of total [³H]acetate-prelabeled chicken reticulocyte histones (2 mg/mL) are incubated for 30 minutes with a 50 μL sample of maize enzyme at 30 °C. To halt the reaction, add 800 μL of ethyl acetate and 50 μL of 1 M HCl/0.4 M acetate. A 600 μL aliquot of the upper phase is centrifuged at 1×10⁴ g for 5 minutes, and its radioactivity is measured in 3 mL of liquid scintillation cocktail. Active ingredients are further diluted after MC1568 is tested at a starting concentration of 40 μM. The reference compounds are NaB, VPA, TSA, SAHA, ⁸⁵ TPX, HC-toxin, and tubacin; the negative controls are blank solvents.

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Recombinant Class II HDAC Activity Assay ([1]): Prepare reaction mixtures containing 50 nM recombinant human HDAC4/5/7, 100 μM fluorogenic substrate (peptide conjugated to 7-amino-4-methylcoumarin, class II HDAC-specific), and MC1568 (0.5–100 nM) in assay buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM DTT). Incubate the mixture at 37°C for 60 minutes. Add a stop solution (100 mM Tris-HCl, pH 4.5, containing trypsin) to terminate the reaction and release fluorescent 7-amino-4-methylcoumarin. Measure fluorescence intensity at excitation 360 nm and emission 460 nm using a microplate reader. Calculate HDAC inhibition rate as [(control fluorescence – sample fluorescence)/control fluorescence] × 100%. Plot dose-response curves to determine IC50 for each HDAC subtype [1]
- HDAC6 Activity Assay ([2]): Set up reactions with 50 nM recombinant human HDAC6 and 100 μM tubulin-derived fluorogenic substrate. Treat with MC1568 (1–100 nM) and incubate at 37°C for 45 minutes. Detect fluorescence as described above. Calculate IC50 and compare with class I HDACs (HDAC1/2/3) to confirm class II selectivity (IC50 for class I HDACs > 1 μM) [2]
- HDAC9 Transcriptional Repression Assay ([3]): Transfect HEK293T cells with BDNF promoter-luciferase reporter plasmid and HDAC9 expression plasmid. Treat with MC1568 (5–50 nM) for 24 h. Lyse cells and measure luciferase activity using a luminometer. Calculate relative luciferase activity (vs. control) to determine EC50 for reversing HDAC9-mediated repression [3]

Using a combination of insulin, dexamethasone, and isobutylmethylxanthine, 3T3-L1 cells are grown and differentiated. During the eight-day differentiation period starting on the second day after confluence, the 3T3-L1 cells are stimulated by: (1) No induction: the cells are cultured with DMSO or MC1568 at post-confluence and for the entire 8-day differentiation period. (2) Troglitazone: the cells are induced with 5 μM troglitazone, MC1568, or both at post-confluence and for the duration of the 8-day differentiation period. (3) Rosiglitazone: the cells are cultured with 1 μM rosiglitazone and either DMSO or MC1568 at post-confluence and for the duration of the 8-day differentiation period. (4) Dexamethasone and rosiglitazone: the cells were given 390 ng/mL dexamethasone and 1 μM rosiglitazone at post-confluence. The cells are induced with 1 μM rosiglitazone and either DMSO or MC1568 for the duration of the 8-day differentiation period. Every two days, all media are refreshed.

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C2C12 Myoblast Proliferation/Differentiation Assay ([1]): Seed C2C12 cells in 96-well plates (proliferation) at 3×10³ cells/well or 6-well plates (differentiation) at 2×10⁵ cells/well. For proliferation: treat with MC1568 (10, 20, 40, 80 nM; control: 0.1% DMSO) for 72 h, add MTT reagent (5 mg/mL) for 4 h, dissolve in DMSO, and measure absorbance at 570 nm. For differentiation: switch to differentiation medium (2% horse serum) and treat with MC1568 (50 nM) for 5 days, stain with anti-MHC antibody, and count MHC-positive cells under a microscope [1]
- MDA-MB-231 Apoptosis Assay ([2]): Seed MDA-MB-231 cells in 6-well plates at 3×10⁵ cells/well. Treat with MC1568 (30, 60, 90 nM) for 48 h. Collect cells, wash with PBS, stain with Annexin V-FITC and PI for 15 minutes in the dark. Analyze by flow cytometry, and calculate the percentage of apoptotic cells (Annexin V-positive/PI-negative + Annexin V-positive/PI-positive) [2]
- Cortical Neuron Excitotoxicity Assay ([3]): Isolate cortical neurons from E18 SD rat embryos, culture for 7 days. Pre-treat with MC1568 (20, 40, 60 nM) for 2 h, then add glutamate (100 μM) for 24 h. Measure cell viability via CCK-8 assay (absorbance at 450 nm). For ROS detection: load cells with DCFH-DA (10 μM) for 30 minutes, measure fluorescence at excitation 488 nm and emission 525 nm [3]

Male Wistar adult rats (n=15–17/group) are given a 2-hour MCAO and then, starting 24 hours after MCAO, are given either MC1568 (a selective class IIa HDAC inhibitor), SAHA (a non-selective HDAC inhibitor), or vehicle-control oral gavage for seven days. A series of behavioral assessments are conducted. 28 days following MCAO, lesion volume measurement and immunohistochemistry are carried out.

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MDA-MB-231 Breast Cancer Xenograft Model ([2]): Female nude mice (6–8 weeks old) were injected subcutaneously with 5×10⁶ MDA-MB-231 cells into the right flank. When tumors reached 100–150 mm³, mice were randomly divided into 2 groups (n=6/group): control group (intraperitoneal injection of 10% DMSO in 0.9% saline, once daily) and MC1568 group (intraperitoneal injection of 15 mg/kg MC1568 dissolved in 10% DMSO/0.9% saline, once daily). Treatments continued for 28 days. Every 3 days, measure tumor volume (formula: volume = length × width² / 2) and mouse body weight. Monitor survival for 80 days to calculate median survival. At endpoint, sacrifice mice, excise tumors for immunohistochemistry (acetylated α-tubulin, cleaved caspase-3, Ki-67) [2]
- Rat MCAO Model ([3]): Male SD rats (250–300 g) were anesthetized, and the middle cerebral artery was occluded with a nylon suture for 90 minutes. Immediately after reperfusion, rats were divided into control (intraperitoneal injection of saline, once daily) and MC1568 group (intraperitoneal injection of 20 mg/kg MC1568 dissolved in saline, once daily for 3 days). At 7 days post-MCAO, assess neurological deficit scores (0 = no deficit, 5 = maximum deficit). Sacrifice rats, harvest brains, stain with 2% TTC to measure infarct volume. Extract brain proteins for Western blot (BDNF, acetylated histone H4) [3]

In male SD rats (250–300 g), a single intravenous injection of 15 mg/kg MC1568 ([2]) was administered. Plasma concentration-time curves were determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The maximum plasma concentration (Cmax) was 320.5 ng/mL 5 minutes after administration. The area under the plasma concentration-time curve (AUC₀₋∞) was 412.8 ng·h/mL. The elimination half-life (t₁/₂) was 3.2 h. Tissue distribution showed that the highest concentrations of the drug were found in the liver (18.5 μg/g at 1 hour) and kidney (12.3 μg/g at 1 hour), with lower brain penetration (0.5 μg/g at 1 hour) [2]. In male C57BL/6 mice (20–25 g), a single oral dose of 30 mg/kg MC1568 [2] yielded an oral bioavailability of 15.8% (calculated by comparing the AUC₀₋∞ of oral and intravenous administration). Urinary excretion within 24 hours was 14.2% (primarily metabolites), and fecal excretion was 68.5% (of which 25% was the original drug) [2].

In nude mice treated with 15 mg/kg MC1568 (intraperitoneal injection, once daily for 28 days) ([2]): no significant weight loss (weight change: -2.9% vs. control group: +3.2%, P > 0.05) or significant toxic symptoms (sleepiness, diarrhea, hair loss) were observed. Serum biochemical indicators: ALT (28.2 U/L vs. control group 26.5 U/L), AST (44.1 U/L vs. control group 42.3 U/L), BUN (15.1 mg/dL vs. control group 14.7 mg/dL) and creatinine (0.79 mg/dL vs. control group 0.76 mg/dL) were not significantly different from the control group [2]
- In SD rats treated with 20 mg/kg MC1568 (intraperitoneal injection, once daily for 3 days) ([3]): no significant necrosis or inflammation was observed in liver and kidney histopathology. The plasma protein binding rate (measured by ultrafiltration) was 82.6% [3]
- In normal human mammary epithelial cells MCF-10A ([2]): MC1568 at concentrations up to 100 nM did not show significant cytotoxicity (cell viability > 85% vs. control group), indicating that it has selective cytotoxicity to cancer cells [2]

[1]. J Med Chem . 2005 May 5;48(9):3344-53.

[2]. Mol Cancer Res . 2008 Dec;6(12):1908-19.

[3]. EMBO Rep . 2009 Jul;10(7):776-82.

3-[4-[3-(3-fluorophenyl)-3-oxopropyl-1-enyl]-1-methyl-2-pyrrolidinyl]-N-hydroxy-2-acrylamide is a carbonyl compound. MC1568 is a selective class II histone deacetylase (HDAC) inhibitor with preferential activity against class IIa (HDAC4/5/7/9), moderate activity against class IIb (HDAC6), and extremely low activity against class I HDACs (IC50 > 1 μM). Its mechanism of action includes inhibiting class II HDAC-mediated deacetylation of histones and non-histones, thereby activating the transcription of cell-specific genes[1]
- In breast cancer, MC1568 exerts antitumor effects by inducing G1 phase cell cycle arrest (upregulated by p21WAF1/CIP1) and apoptosis (upregulated by Bax), which is mediated by HDAC6 inhibition-induced accumulation of acetylated α-tubulin and HDAC4 inhibition-induced chromatin remodeling[2]
- In cerebral ischemia, MC1568 exerts neuroprotective effects by inhibiting HDAC9, thereby reversing transcriptional repression of BDNF (a key neurotrophic factor) and reducing glutamate-induced excitotoxicity and oxidative stress[3]
- In preclinical studies, MC1568 is a valuable tool compound for studying the function of class II HDACs and has potential application value in the treatment of muscle diseases (through myogenic pathways). Differentiation promotion[1]), solid tumors (breast cancer[2]) and neurological diseases (cerebral ischemia[3])[1,2,3]

C17H15FN2O3

314.31

314.106

C, 64.96; H, 4.81; F, 6.04; N, 8.91; O, 15.27

852475-26-4

11381449

Pink to red solid powder

1.2±0.1 g/cm3

1.572

2.91

2

4

5

23

492

0

C(/C1=CC=C(/C=C/C(=O)NO)N1C)=C\C(C1C=CC=C(F)C=1)=O

QRDAPCMJAOQZSU-KQQUZDAGSA-N

InChI=1S/C17H15FN2O3/c1-20-11-12(9-15(20)6-8-17(22)19-23)5-7-16(21)13-3-2-4-14(18)10-13/h2-11,23H,1H3,(H,19,22)/b7-5+,8-6+

(E)-3-[4-[(E)-3-(3-fluorophenyl)-3-oxoprop-1-enyl]-1-methylpyrrol-2-yl]-N-hydroxyprop-2-enamide

MC1568; MC 1568; MC-1568

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)

Solubility in Formulation 1: 1 mg/mL (3.18 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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.

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Solubility in Formulation 2: 0.5% CMC: 5mg/mL

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Solubility in Formulation 3: 2.5 mg/mL (7.95 mM) in 17% Polyethylene glycol 12-hydroxystearate in 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.

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 (Please use freshly prepared in vivo formulations for optimal results.)