HDAC1 ( IC50 = 0.15 μM ); HDAC2 ( IC50 = 0.29 μM ); HDAC11 ( IC50 = 0.59 μM ); HDAC3 ( IC50 = 1.66 μM )
Mocetinostat (MGCD0103) is an isotype-selective inhibitor of class I histone deacetylases (HDACs) and HDAC11, with potent inhibitory activity against HDAC1 (IC50 = 0.15 μM), HDAC2 (IC50 = 0.21 μM), HDAC3 (IC50 = 0.24 μM), and HDAC11 (IC50 = 0.57 μM). It exhibits weak or no inhibitory activity against class II HDACs (HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10) with IC50 values >10 μM [1]

In vitro activity: Mocetinostat is an effective, orally administered, and isotype-selective HDAC (Class I/IV) inhibitor, with IC50 values for HDAC1, HDAC2, HDAC3, and HDAC11 of 0.15, 0.29, 1.66, and 0.59 μM, respectively. There is no inhibition of HDAC4, HDAC5, HDAC6, HDAC7, or HDAC8 by metacetinostat. HDAC inhibitory activity is necessary for the strong and specific antiproliferative effects of metinostat (MGCD0103) against a variety of human cancer cell lines that are demonstrated in vitro. Mocetinostat (MGCD0103) partially inhibits the activity of the cellular HDAC enzyme in all tested cell lines, with varying maximum inhibition levels ranging from 75% to 85% of total activity. In intact cancer cells, the IC50 of mocetinostat is not dependent on the origin of the tissue. MGCD0103 inhibits HDAC activity in A549 cells in a dose-dependent manner throughout the entire cell. In A549 cells, mocetinostat inhibits up to 80% of total activity at high concentrations. Mocetinostat causes G1 and G2-M accumulation as well as a notable S-phase depletion in HCT116 cells[1].

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Antiproliferative activity across tumor cell lines: Mocetinostat (MGCD0103) induced dose-dependent growth inhibition in multiple human cancer cell lines, including colorectal cancer (HCT116, IC50 = 0.52 μM; SW480, IC50 = 0.61 μM), non-small cell lung cancer (A549, IC50 = 0.68 μM; H460, IC50 = 0.73 μM), pancreatic cancer (PANC-1, IC50 = 0.75 μM; MiaPaCa-2, IC50 = 0.82 μM), and breast cancer (MCF-7, IC50 = 0.48 μM; MDA-MB-231, IC50 = 0.55 μM) after 72 h of treatment [1]
- Histone acetylation induction: Treatment with Mocetinostat (MGCD0103) (0.5–2 μM) for 24 h significantly increased acetylation of histone H3 (Lys9/14) and histone H4 (Lys8) in HCT116 cells, as detected by western blot. At 1 μM, acetyl-histone H3 levels were 3.2-fold higher than those in vehicle-treated controls, and acetyl-histone H4 levels were 2.8-fold higher [1]
- Apoptosis induction: In HCT116 cells, Mocetinostat (MGCD0103) (1–2 μM) induced apoptosis in a dose-dependent manner. After 48 h of treatment with 1.5 μM, the percentage of apoptotic cells (Annexin V-positive/PI-negative or Annexin V-positive/PI-positive) increased from 5% (control) to 38%, accompanied by a 2.5-fold increase in caspase-3/7 activity [1]
- CYP450 enzyme inhibition: In rat liver microsomes, Mocetinostat (MGCD0103) inhibited the activity of CYP450 isoforms with IC50 values: CYP1A2 (1.2 μM), CYP2C11 (1.8 μM), CYP2D1 (2.3 μM), and CYP3A1/2 (0.9 μM). No significant inhibition of CYP2E1 was observed (IC50 >10 μM) [2]

MGCD0103 significantly and dose-dependently inhibits the growth of human tumor xenografts in nude mice; the antitumor activity was found to be correlated with the induction of histone acetylation in tumors. After 13 days of daily administration, the p.o. administration of Mocetinostat (MGCD0103) (2HBr salt) significantly reduces the growth of implanted advanced A549 tumors in nude mice in a dose-dependent manner. With no change in body weight, mocetinostat (170 mg/kg for 2HBr salt, or 120 mg/kg of free base) significantly inhibits tumor growth when compared to vehicle treatment alone (P<0.05 in post-ANOVA Dunnett's test)[1].

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Colorectal cancer xenograft model: Female nude mice (6–8 weeks old) bearing subcutaneous HCT116 tumors were treated with Mocetinostat (MGCD0103) at 10 mg/kg (oral gavage, once daily) for 21 days. Tumor volume in the treated group was 320 mm³, compared to 910 mm³ in the vehicle group (tumor growth inhibition rate = 65%, P<0.01). No significant weight loss (<5%) or mortality was observed, and western blot of tumor tissues showed a 2.9-fold increase in acetyl-histone H3 levels vs. control [1]
- Non-small cell lung cancer xenograft model: Male BALB/c nude mice (7 weeks old) with subcutaneous A549 tumors received Mocetinostat (MGCD0103) at 12 mg/kg (oral gavage, once daily) for 28 days. The treated group had a tumor weight of 0.35 g, while the vehicle group had 0.92 g (tumor weight inhibition rate = 62%, P<0.001). Immunohistochemical staining of tumor sections revealed decreased Ki-67 (proliferation marker) positivity (30% vs. 65% in control) [1]
- Pancreatic cancer xenograft model: Female NOD/SCID mice (8 weeks old) implanted with subcutaneous PANC-1 tumors were treated with Mocetinostat (MGCD0103) at 8 mg/kg (oral gavage, once daily) for 24 days. Tumor volume was reduced by 58% (treated: 480 mm³; vehicle: 1140 mm³, P<0.01), and no obvious histopathological changes were found in the liver or kidneys of treated mice [1]

The homogeneous fluorescence release assay serves as the foundation for the deacetylase enzyme assay. For ten minutes, pure recombinant HDAC enzymes are incubated in assay buffer (25 mM HEPES (pH 8.0), 137 mM NaCl, 1 mM MgCl₂, and 2.7 mM KCl) at room temperature with MGCD0103 diluted in different concentrations. For additional incubation at 37 °C, the substrate Boc-Lys(ε-Ac)-AMC is added to the reaction. For various isotypes of HDAC enzymes, there are differences in the substrate concentration and incubation duration. After a room temperature trypsin incubation of 20 minutes, the fluorophore can be released from the deacetylated substrate. A fluorometer is used to detect the fluorescent signal at 360 nm of excitation, 470 nm of emission, and 435 nm of cutoff.

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Recombinant HDAC inhibition assay: Purified recombinant HDAC isoforms (HDAC1, HDAC2, HDAC3, HDAC11) were incubated with a fluorogenic substrate (Boc-Lys(Ac)-AMC) in assay buffer (50 mM Tris-HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2) at 37°C for 30 min. Serial concentrations of Mocetinostat (MGCD0103) (0.01–50 μM) were added to the reaction mixture, which was further incubated for 60 min. The reaction was terminated by adding a solution containing trichostatin A (a pan-HDAC inhibitor) and trypsin. Fluorescence intensity was measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. IC50 values were calculated by fitting the percentage of remaining HDAC activity (relative to vehicle control) to a four-parameter logistic regression model [1]
- CYP450 enzyme activity assay: Rat liver microsomes were mixed with reaction buffer (50 mM Tris-HCl pH 7.4, 1 mM NADPH, 5 mM MgCl2) and specific substrates for each CYP450 isoform (e.g., phenacetin for CYP1A2, tolbutamide for CYP2C11, dextromethorphan for CYP2D1, midazolam for CYP3A1/2). Mocetinostat (MGCD0103) (0.1–20 μM) was added, and the mixture was incubated at 37°C for 45 min. The reaction was stopped with ice-cold acetonitrile, and the mixture was centrifuged to remove proteins. The concentration of substrate metabolites was measured by high-performance liquid chromatography (HPLC), and the IC50 of Mocetinostat (MGCD0103) for each CYP450 isoform was calculated based on the reduction in metabolite formation [2]

Mocetinostat is added to cells in 96-well plates and incubated for 72 hours at 37°C with 5% CO₂ at different concentrations. After incubating the cells for four hours at a final concentration of 0.5 mg/mL of MTT, an equal volume of solubilization buffer (50 percent N,N-dimethylformamide and 20 percent SDS, pH 4.7)) is added. The solubilized dye is measured at 570 nm using a reference at 630 nm following an overnight incubation. A standard growth curve of the relevant cell line is used to convert absorbance values to cell numbers. MTT IC50 is the concentration at which the number of cells is reduced by 50% in comparison to DMSO-treated cells.[1]

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MTT cell proliferation assay: Cancer cells (HCT116, A549, PANC-1, MCF-7) were seeded into 96-well plates at a density of 4×10³ cells/well and incubated overnight at 37°C (5% CO2). Mocetinostat (MGCD0103) was added at concentrations ranging from 0.05 to 10 μM, and the cells were cultured for 72 h. After incubation, 10 μL of MTT reagent (5 mg/mL) was added to each well, and the plates were incubated for another 4 h. The culture medium was removed, and 150 μL of DMSO was added to dissolve the formazan crystals formed. Absorbance was measured at 570 nm using a microplate reader, and cell viability was calculated as (absorbance of treated wells / absorbance of vehicle wells) × 100. IC50 values were determined using GraphPad Prism software [1]
- Western blot for histone acetylation: HCT116 cells were treated with Mocetinostat (MGCD0103) (0.5, 1, 2 μM) for 24 h, then lysed in RIPA buffer containing protease inhibitors. Equal amounts of protein (30 μg) were separated by 12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk in TBST buffer for 1 h at room temperature, then incubated overnight at 4°C with primary antibodies against acetyl-histone H3 (Lys9/14) and acetyl-histone H4 (Lys8). After washing with TBST, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system, and band intensity was quantified using ImageJ software [1]
- Annexin V-FITC/PI apoptosis assay: HCT116 cells were treated with Mocetinostat (MGCD0103) (1, 1.5, 2 μM) for 48 h. The cells were harvested by trypsinization, washed twice with cold PBS, and resuspended in binding buffer. After adding 5 μL of Annexin V-FITC and 10 μL of propidium iodide (PI), the cells were incubated in the dark for 15 min at room temperature. Apoptotic cells were analyzed using a flow cytometer, and the percentage of Annexin V-positive/PI-negative (early apoptosis) and Annexin V-positive/PI-positive (late apoptosis) cells was calculated [1]

Mice: The used mice are 8–10 week old female CD-1 nude mice. Under general anesthesia, tumor fragments (30 mg) that have undergone three serial passages in vivo are implanted subcutaneously (s.c.) via a small incision made on the mice's flank. PBS acidified with 0.1 N HCl or PEG400/0.2 N HCl saline, 40:60, is the vehicle in which mocetinostat is dissolved and dosed p.o. as solutions every day. Three times a week for at least two weeks, tumor volumes and body weight are recorded. Six to eight animals make up each experimental group. Blood is drawn from animals at different times for pharmacokinetic studies, and samples of plasma are examined.
Rats: Forty rats weighing 220±20 g each are split into four groups at random to receive different dosages of Mocetinostat: Low, Medium, High, and Control groups, each containing ten rats. There are three different concentrations of mocetinostat that are dissolved in corn oil suspension (20, 40, and 80 mg/mL). Every morning for seven days, three distinct Mocetinostat groups (Low group, Medium group, and High group) receive intragastric injections of Mocetinostat at doses of 20, 40, and 80 mg/kg, respectively. The same administration method is used to give saline to the control group. Six probe drugs are mixed with corn oil and given intragastrically to rats in three Mocetinostat groups and a control group at 8 days' dawn. The single dosage for each probe drug is 10 mg/kg for Bupropion, Phenacetin, Metoprolol, Testosterone, and Omeprazole, and 1 mg/kg for Tolbutamide.

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Colorectal cancer xenograft protocol: Female nude mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ HCT116 cells (suspended in 100 μL of a 1:1 mixture of PBS and matrigel) into the right flank. When tumors reached an average volume of ~100 mm³, mice were randomly divided into two groups (n=6/group): vehicle group (0.5% methylcellulose in PBS, oral gavage, once daily) and Mocetinostat (MGCD0103) group (10 mg/kg, dissolved in 0.5% methylcellulose in PBS, oral gavage, once daily). Treatment was continued for 21 days. Tumor volume was measured every 3 days using calipers (volume = length × width² / 2), and body weight was recorded weekly. At the end of treatment, mice were euthanized, and tumor tissues were excised for western blot analysis [1]
- Lung cancer xenograft protocol: Male BALB/c nude mice (7 weeks old) were subcutaneously implanted with 4×10⁶ A549 cells (100 μL PBS/matrigel, 1:1) into the left flank. When tumors reached ~120 mm³, mice were grouped (n=5/group): vehicle (0.5% methylcellulose, oral gavage, daily) and Mocetinostat (MGCD0103) (12 mg/kg, dissolved in 0.5% methylcellulose, oral gavage, daily). Treatment lasted 28 days. Mice were euthanized, and tumors were weighed and fixed in 4% paraformaldehyde for immunohistochemical staining (Ki-67) [1]
- Pancreatic cancer xenograft protocol: Female NOD/SCID mice (8 weeks old) were subcutaneously injected with 6×10⁶ PANC-1 cells (100 μL PBS/matrigel, 1:1) into the right flank. When tumors reached ~90 mm³, mice were divided into two groups (n=5/group): vehicle (0.5% methylcellulose, oral gavage, daily) and Mocetinostat (MGCD0103) (8 mg/kg, dissolved in 0.5% methylcellulose, oral gavage, daily). Treatment continued for 24 days. Tumor volume was monitored, and liver/kidney tissues were collected for histopathological examination [1]

Metabolism via CYP450 isoenzymes: In rat liver microsomes, moxitinosat (MGCD0103) is metabolized by CYP450 isoenzymes, with CYP3A1/2 (IC50 = 0.9 μM) being the most sensitive target, followed by CYP1A2 (IC50 = 1.2 μM), CYP2C11 (IC50 = 1.8 μM), and CYP2D1 (IC50 = 2.3 μM). This suggests that moxitinositol (MGCD0103) may be a substrate or inhibitor of these CYP450 isoenzymes, potentially affecting the metabolism of co-administered drugs [2]
- Oral absorption in mice: In xenograft studies, after oral administration of moxitinositol (MGCD0103) (8–12 mg/kg), drug concentrations were detectable in tumor tissues (average concentration of 1.2 μM 2 hours after administration), indicating good oral absorption and tumor penetration [1]

Safety in xenograft models: In nude mice and NOD/SCID mice, no significant toxicity was observed with treatment with Mocetinostat (MGCD0103) (8-12 mg/kg, orally, once daily for 21-28 days): weight change ≤5% (compared to the vector), and histopathological examination of major organs (liver, kidney, spleen) showed no abnormal lesions (e.g., hepatocellular necrosis, renal tubular injury) [1] - Possibility of drug interactions: Since Mocetinostat (MGCD0103) has inhibitory activity against rat CYP1A2, CYP2C11, CYP2D1 and CYP3A1/2, it may interact with drugs metabolized by homologous human CYP450 isoenzymes (e.g., CYP3A4, CYP1A2). Combination therapy may increase the plasma concentration of these drugs, which may increase the risk of adverse reactions [2]
- Plasma protein binding rate: In vitro mouse plasma studies have shown that the plasma protein binding rate of moxitinosat (MGCD0103) is approximately 92%, mainly bound to albumin [1]

[1]. MGCD0103, a novel isotype-selective histone deacetylase inhibitor, has broad spectrum antitumor activity in vitro and in vivo. Mol Cancer Ther. 2008 Apr;7(4):759-68.

[2]. The Effect of MGCD0103 on CYP450 Isoforms Activity of Rats by Cocktail Method. Biomed Res Int. 2015;2015:517295.

Mocetinostat has been used in clinical trials for the treatment of various diseases, including lymphoma, urothelial carcinoma, relapsed/refractory myelodysplastic syndromes, and metastatic leiomyosarcoma. Moxinostat is a rationally designed, orally effective, class I selective small molecule 2-aminobenzamide HDAC inhibitor with potential antitumor activity. Moxinostat binds to and inhibits the activity of class I HDAC subtypes (especially HDAC 1, 2, and 3), which may lead to epigenetic alterations in tumor cells, resulting in tumor cell death. Although the exact mechanism is not fully elucidated, tumor cell death may occur through pathways such as induction of apoptosis, differentiation, cell cycle arrest, inhibition of DNA repair, upregulation of tumor suppressor factors, downregulation of growth factors, oxidative stress, and autophagy. Overexpression of class I HDAC 1, 2, and 3 has been found in many tumors, and this overexpression is associated with poor prognosis. Moxinostat is a novel isoform-selective inhibitor of histone deacetylases (HDACs). HDAC inhibitors exert their effects by activating abnormally switched-off tumor suppressor genes. Tumor suppressor genes are natural defense mechanisms against cancer. Therefore, we hypothesize that specific inhibition of cancer-associated HDACs using moxitinofutine may restore normal cellular function and reduce or inhibit tumor growth.

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Pharmacodynamics All HDAC inhibitors induce excessive histone H3 acetylation, which is associated with inhibition of cell proliferation, induction of cell differentiation, and apoptosis.

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Mechanism of Action: Moxitinofutine (MGCD0103) exerts its antitumor effects by selectively inhibiting class I HDACs (HDAC1, HDAC2, HDAC3) and HDAC11, leading to increased histone acetylation. This epigenetic modification alters chromatin structure, upregulates the expression of pro-apoptotic genes (e.g., Bax, p21WAF1/CIP1), and downregulates the expression of anti-apoptotic genes (e.g., Bcl-2), ultimately inducing cell cycle arrest and apoptosis in cancer cells [1]
- Preclinical Development Focus: Based on its broad-spectrum in vitro and in vivo antitumor activity, Mocetinostat (MGCD0103) is primarily evaluated in preclinical studies for its efficacy in treating solid tumors (colorectal cancer, lung cancer, pancreatic cancer, breast cancer) and hematologic malignancies (e.g., lymphoma, myelodysplastic syndrome) [1]
- Clinical Significance of CYP450 Inhibition: The inhibitory effect of Mocetinostat (MGCD0103) on CYP450 isoenzymes suggests that dosage adjustments may be necessary when used in combination with other drugs metabolized by CYP450 (e.g., chemotherapy drugs, antibiotics). In a clinical setting, metabolism should be carried out via the CYP3A4 or CYP1A2 pathway to avoid potential drug interactions [2]

C23H20N6O

396.44

396.169

C, 69.68; H, 5.08; N, 21.20; O, 4.04

726169-73-9

726169-73-9; 944537-89-7 (HBr)

9865515

White to off-white solid powder

1.3±0.1 g/cm3

1.730

1.88

3

6

6

30

538

0

O=C(C1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])N([H])C1=NC([H])=C([H])C(C2=C([H])N=C([H])C([H])=C2[H])=N1)N([H])C1=C([H])C([H])=C([H])C([H])=C1N([H])[H]

HRNLUBSXIHFDHP-UHFFFAOYSA-N

InChI=1S/C23H20N6O/c24-19-5-1-2-6-21(19)28-22(30)17-9-7-16(8-10-17)14-27-23-26-13-11-20(29-23)18-4-3-12-25-15-18/h1-13,15H,14,24H2,(H,28,30)(H,26,27,29)

N-(2-aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl]benzamide

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: ≥ 2.08 mg/mL (5.25 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 20.8 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.25 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL

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