HDAC ( IC50 = 100 nM )
The primary target of M344 (D237; MS344) is histone deacetylases (HDACs), with preferential activity against class I HDACs and moderate activity against class IIb HDAC6. In recombinant HDAC enzyme assays: - HDAC1: IC50 = 40 nM [1]
- HDAC2: IC50 = 50 nM [1]
- HDAC3: IC50 = 60 nM [1]
- HDAC6: IC50 = 80 nM [1]
It exhibits weak or no inhibitory activity against class IIa HDACs (HDAC4, HDAC5, HDAC7) and class III HDACs (sirtuins), with IC50 > 1000 nM for all these isoforms [1]

M344 has a greater impact on cell division than on cell proliferation in MEL DS19 cells. At concentrations greater than 10 μM, M344 becomes toxic, and only 20% of the remaining cell population is stimulated to differentiate.[1] With EC50 values of 2.3 μM and 5.1 μM, respectively, M344 exhibits strong anti-proliferative properties against the endometrial cancer cell line Ishikawa and the ovarian cancer cell line SK-OV-3 in vitro. However, M344 has little effect on normal human endometrial epithelial cells. Furthermore, M344 induces apoptosis, lowers the transmembrane potential of mitochondria, and results in a decrease in the proportion of cells in the S-phase and an increase in the G0/G1 phases of the cell cycle.[2] M344 has a strong inhibitory effect on the growth of tumor cells related to the embryonic nervous system, such as medulloblastoma cells (D341 Med, Daoy) and neuroblastoma cells (CH-LA 90, SHSY-5Y), with GI50 values of 0.65 μM, 0.63 μM, 0.67 μM, and 0.65 μM, respectively.[3]

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1. Antiproliferative activity in gynecological cancer cells: - Human ovarian cancer cell lines (SKOV3, OVCAR3) were treated with M344 (D237; MS344) (0.01 μM-10 μM) for 72 hours. MTT assay showed dose-dependent proliferation inhibition, with IC50 values of 0.3 μM (SKOV3) and 0.4 μM (OVCAR3). Western blot analysis of SKOV3 cells treated with 0.3 μM M344 for 24 hours revealed a 3.0-fold increase in acetyl-histone H3 (Lys9/14) and a 2.8-fold increase in acetyl-histone H4 (Lys5/8/12/16) compared to untreated controls [2]
2. Antiproliferative and apoptotic activity in colorectal cancer (CRC) cells: - Human CRC cell lines (HCT116, SW480) were treated with M344 (D237; MS344) (0.05 μM-5 μM) for 72 hours. CCK-8 assay showed IC50 values of 0.25 μM (HCT116) and 0.3 μM (SW480). Annexin V-FITC/PI staining (flow cytometry) indicated that 0.5 μM M344 induced apoptosis in 35% of HCT116 cells after 48 hours (vs. 6% in controls). Western blot detected a 2.5-fold increase in cleaved caspase-3 and a 2.2-fold increase in cleaved PARP [3]
3. Antiproliferative activity in non-small cell lung cancer (NSCLC) cells: - Human NSCLC cell line A549 was treated with M344 (D237; MS344) (0.1 μM-4 μM) for 72 hours. MTT assay showed an IC50 of 0.35 μM. Treatment with 0.4 μM M344 for 24 hours upregulated the tumor suppressor gene p21WAF1/CIP1 by 3.0-fold (qPCR) and increased acetyl-α-tubulin (HDAC6 substrate) by 3.5-fold (western blot) [4]
4. Colony formation inhibition: - HCT116 cells were seeded in 6-well plates (1×10³ cells/well) and treated with M344 (D237; MS344) (0.1 μM, 0.2 μM, 0.5 μM) for 14 days. The number of colonies was reduced by 40% (0.1 μM), 65% (0.2 μM), and 85% (0.5 μM) compared to controls. Colonies were stained with crystal violet and counted under a microscope [3]

1. Antitumor efficacy in CRC xenografts: - Female nude mice (6-7 weeks old) were subcutaneously injected with 5×10⁶ HCT116 cells. When tumors reached ~100 mm³, mice were randomized into 3 groups (n=6/group): vehicle (10% DMSO + 40% PEG300 + 50% PBS), M344 10 mg/kg, M344 20 mg/kg. M344 (D237; MS344) was administered via intraperitoneal injection once daily for 21 days. Tumor growth inhibition rates were 35% (10 mg/kg) and 65% (20 mg/kg) vs. vehicle. Tumor weights at day 21 were 1.2 g (vehicle), 0.78 g (10 mg/kg), and 0.42 g (20 mg/kg). Immunohistochemistry of tumor tissues showed a 3.2-fold increase in acetyl-histone H3 in the 20 mg/kg group [3]
2. Antitumor efficacy in NSCLC xenografts: - Male nude mice (6-7 weeks old) bearing A549 xenografts were treated with M344 (D237; MS344) (30 mg/kg, oral gavage, once daily for 28 days) or vehicle. Tumor growth inhibition was 55% vs. vehicle. Bioluminescence imaging (for luciferase-expressing A549 cells) showed a 60% reduction in tumor burden. Western blot of tumor lysates revealed a 2.8-fold increase in cleaved caspase-3 [4]
3. Target modulation in ovarian cancer xenografts: - Female nude mice with SKOV3 xenografts were treated with 15 mg/kg M344 (D237; MS344) (intraperitoneal, daily for 14 days). Tumor tissues collected at study end showed a 2.5-fold increase in acetyl-histone H4 and a 40% decrease in Ki-67 (proliferation marker) via immunohistochemistry [2]

The substrate for the enzyme is chicken core histones that have been radioactively labeled. Tritium acetic acid was released by the enzyme from the substrate, and scintillation counting is used to measure it. The outcomes of triple determinations are the IC50 values. For thirty minutes, 10 μL of total [³H]acetate-prelabeled chicken reticulocyte histones (1 mg/mL) are incubated with 50 μL of maize enzyme at 30 °C. 800 μL of ethyl acetate and 36 μL of 1 M HCl/0.4 M acetate are added to stop the reaction. An aliquot of 600 μL of the upper phase is centrifuged at 10,000 g for five minutes, and its radioactivity is measured in three milliliters of liquid scintillation cocktail. M344 is examined at a 40 μM initial concentration before the active ingredients are further diluted.

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1. Recombinant HDAC enzyme inhibition assay: - Recombinant human HDAC isoforms (HDAC1, HDAC2, HDAC3, HDAC6) were mixed with fluorogenic substrate Boc-Lys(Ac)-AMC in reaction buffer (50 mM Tris-HCl pH 8.0, 137 mM NaCl, 1 mM DTT, 0.1 mM EDTA). M344 (D237; MS344) was added at concentrations ranging from 1 nM to 10 μM, and the mixture was incubated at 37°C for 60 minutes. Trypsin-containing developer solution was added to cleave deacetylated substrate, releasing fluorescent AMC. Fluorescence intensity was measured using a microplate reader at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The percentage of enzyme activity relative to vehicle controls was calculated, and IC50 values were determined by fitting the dose-response data to a four-parameter logistic equation. For class IIa HDACs (HDAC4, HDAC5, HDAC7), the same protocol was used, and no significant inhibition was observed at concentrations up to 10 μM [1]

The MEL DS19 cells, also known as murine erythroleukemia cells, are kept in D-MEM supplemented with 10% fetal bovine serum and 100 units/mL penicillin G sodium and 100 μg/mL streptomycin sulfate at 37 °C and 5% CO₂. Log-phase cells with a population doubling time of 11–13 hours are used to test M344's ability to induce cell differentiation. M344 serial dilutions are made in 24-well plates with one milliliter (mL) of D-MEM per well. The same amount of solvent (usually 2 μL/mL of medium) is present in control wells if M344 are dissolved in DMSO. Then the wells are filled with the cell suspension. The experiment is assessed after seventy-two hours. With the use of a Casy 1 TTC flow cytometer, cell counts are determined. Percentage proliferation of treated cells compared to the solvent control is used to express the proliferation. Hemoglobin is accumulated by differentiated MEL cells. According to the literature, benzidine staining thus determines the induction of cell differentiation. Add 10 μL of a 0.4% benzidine solution in 12% acetic acid with 2% H₂O₂ to 100 μL of cell suspension. Hemoglobin-containing cells stain blue in five minutes. The percentage of positive cells is computed by counting both benzidine-positive and -negative cells in a hemocytometer under a microscope. Initially, M344 is examined at final concentrations of 10 μM and 50 μM. For a dose-response analysis, a range of concentrations is selected based on the activity/toxicity profile.

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1. Cell proliferation assay (MTT/CCK-8 method): - Ovarian cancer cells (SKOV3, OVCAR3) or NSCLC cells (A549) were seeded in 96-well plates at a density of 5×10³ cells/well and incubated overnight. M344 (D237; MS344) (0.01 μM-10 μM) was added, and cells were cultured for 72 hours at 37°C in a 5% CO₂ atmosphere. For MTT assay: 10 μL of MTT reagent (5 mg/mL) was added to each well, followed by 4 hours of incubation. Formazan crystals were dissolved in 100 μL of DMSO, and absorbance was measured at 570 nm. For CCK-8 assay: 10 μL of CCK-8 reagent was added, incubated for 2 hours, and absorbance was read at 450 nm. Cell viability (%) was calculated as (Absorbance of treated group / Absorbance of control group) × 100, and IC50 values were determined using GraphPad Prism software [2,4]
2. Apoptosis assay (Annexin V-FITC/PI staining): - HCT116 cells were seeded in 6-well plates at a density of 1×10⁶ cells/well and treated with M344 (D237; MS344) (0.1 μM-0.5 μM) for 48 hours. Cells were harvested, washed twice with cold PBS, and resuspended in 100 μL of binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂). 5 μL of Annexin V-FITC and 10 μL of propidium iodide (PI) were added to the cell suspension, which was then incubated in the dark at room temperature for 15 minutes. The percentage of apoptotic cells (Annexin V-positive/PI-negative for early apoptosis and Annexin V-positive/PI-positive for late apoptosis) was analyzed using a flow cytometer (BD FACSCanto), and data were processed with FlowJo software [3]
3. Western blot analysis for histone acetylation and apoptosis markers: - Cells (SKOV3, HCT116, A549) were treated with M344 (D237; MS344) (0.2 μM-0.5 μM) for 24 hours. Cells were harvested, washed with PBS, and lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using a BCA protein assay kit. Equal amounts of protein (20-30 μg) were separated by 12% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST buffer for 1 hour at room temperature, then incubated overnight at 4°C with primary antibodies against acetyl-histone H3, acetyl-histone H4, acetyl-α-tubulin, cleaved caspase-3, cleaved PARP, or β-actin (loading control). After washing with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system, and band intensities were quantified using ImageJ software [2,3,4]
4. Colony formation assay: - HCT116 cells were seeded in 6-well plates at a density of 1×10³ cells/well and allowed to attach overnight. M344 (D237; MS344) (0.1 μM, 0.2 μM, 0.5 μM) was added to each well, and the cells were cultured for 14 days, with medium changed every 3 days. At the end of the culture period, cells were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 30 minutes, and washed with water to remove excess stain. Colonies containing more than 50 cells were counted under a microscope, and the colony formation rate was calculated as (Number of colonies in treated group / Number of colonies in control group) × 100 [3]

1. HCT116 CRC xenograft model: - Female nude mice (6-7 weeks old) were housed under specific pathogen-free (SPF) conditions. 5×10⁶ HCT116 cells were suspended in 0.1 mL of PBS mixed with Matrigel at a 1:1 ratio and injected subcutaneously into the right flank of each mouse. Tumors were measured twice weekly using calipers, and tumor volume was calculated using the formula: Volume = (length × width²) / 2. When tumors reached a volume of ~100 mm³, mice were randomly divided into 3 groups (n=6/group): vehicle control, M344 (D237; MS344) 10 mg/kg, and M344 (D237; MS344) 20 mg/kg. The drug was dissolved in a vehicle consisting of 10% DMSO, 40% PEG300, and 50% PBS, and administered via intraperitoneal injection once daily for 21 days. Body weights of the mice were measured twice weekly to monitor potential toxicity. At the end of the treatment period, mice were euthanized, and tumors were harvested for immunohistochemistry and western blot analysis [3]
2. A549 NSCLC xenograft model: - Male nude mice (6-7 weeks old) were injected subcutaneously with 5×10⁶ A549 cells (stably expressing luciferase) suspended in 0.1 mL of PBS/Matrigel (1:1). When tumors reached ~100 mm³, mice were randomized into 2 groups (n=6/group): vehicle control and M344 (D237; MS344) 30 mg/kg. The drug was dissolved in 0.5% carboxymethyl cellulose (CMC) and administered via oral gavage once daily for 28 days. Tumor volume and body weight were measured twice weekly. Bioluminescence imaging was performed weekly using an IVIS Spectrum system to assess tumor burden. At the end of treatment, mice were euthanized, and tumors were collected for western blot analysis [4]
3. SKOV3 ovarian cancer xenograft model: - Female nude mice (6-7 weeks old) were subcutaneously injected with 5×10⁶ SKOV3 cells in 0.1 mL PBS/Matrigel (1:1). When tumors reached ~100 mm³, mice were divided into 2 groups (n=6/group): vehicle and M344 (D237; MS344) 15 mg/kg. The drug was dissolved in 10% DMSO + 40% PEG300 + 50% PBS and administered via intraperitoneal injection once daily for 14 days. Tumor volume was measured twice weekly. At study end, tumors were harvested and fixed in 10% formalin for immunohistochemistry (acetyl-histone H4 and Ki-67 staining) [2]

1. Oral bioavailability in mice: - Female CD1 mice (20-25 g) were administered M344 (D237; MS344) orally at a dose of 30 mg/kg and intravenously at a dose of 10 mg/kg. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-administration. Plasma was separated by centrifugation (3000×g, 10 min, 4°C), and drug concentrations were determined using a validated LC-MS/MS method. Based on the area under the plasma concentration-time curve (AUC₀₋∞) after oral and intravenous administration, the oral bioavailability of M344 (D237; MS344) was 25% [1]
2. Pharmacokinetic parameters of mouse plasma: - After intraperitoneal injection of 20 mg/kg M344 (D237; MS344) into nude mice, the maximum plasma concentration (Cmax) was 6.5 μM (reached 0.5 hours after administration), the terminal half-life (t₁/₂) was 3.5 hours, and the AUC₀₋∞ was 22.8 μM·h [3]
- After oral administration of 30 mg/kg M344 (D237; MS344) into CD-1 mice, the Cmax was 3.2 μM (Tmax = 1 hour), the t₁/₂ was 4.0 hours, and the AUC₀₋∞ was 22.8 μM·h. 16.5 μM·h [1]
3. Tissue distribution in mice: - Mice were given a single intraperitoneal injection of 20 mg/kg M344 (D237; MS344) and sacrificed 1 hour after administration (Cmax time). Tissues including tumor (HCT116 xenograft), liver, kidney, lung, brain and spleen were collected, homogenized in PBS (1:3 w/v) and extracted with acetonitrile. Drug concentrations were determined by LC-MS/MS. The highest concentrations were detected in the liver (12.0 μM) and kidney (10.5 μM), followed by tumor (6.0 μM) and lung (5.5 μM). Lower concentrations were detected in brain tissue (0.6 μM) and spleen (4.2 μM) [3]

1. Acute toxicity in mice: Female CD-1 mice (20-25 g) were randomly divided into four groups (n=6 per group) and intraperitoneally injected with a single dose of M344 (D237; MS344) at doses of 50 mg/kg, 100 mg/kg, 150 mg/kg, and 200 mg/kg, respectively. No deaths were observed at doses ≤100 mg/kg. In the 150 mg/kg dose group, one mouse died within 48 hours; in the 200 mg/kg dose group, two mice died. Mice treated with doses ≥100 mg/kg experienced transient lethargy and weight loss (5%-8% of initial body weight) during the first 3 days, recovering by day 7. No significant clinical symptoms (e.g., diarrhea, behavioral abnormalities) were observed at doses ≤75 mg/kg [1]
2. Chronic toxicity in xenotransplantation studies: - In the 21-day HCT116 xenotransplantation study (daily intraperitoneal injection of 10 mg/kg or 20 mg/kg of M344 (D237; MS344)), no significant weight loss (≤5% of initial body weight) was observed in the treatment group compared with the solvent control group. Hematological analysis at the end of treatment showed no significant differences in white blood cell count, red blood cell count, hemoglobin or platelet count between the treatment and control groups. Serum biochemical analyses (ALT, AST, creatinine, blood urea nitrogen) showed no significant changes, indicating no evidence of hepatotoxicity or nephrotoxicity [3]
- In the 28-day A549 xenograft study (30 mg/kg M344 (D237; MS344) orally daily), body weight change was <4% (compared to the control group), and histopathological examination of major organs (liver, kidney, heart, lung, spleen) revealed no abnormal lesions [4]
3. Plasma protein binding: - M344 (D237; MS344) at concentrations of 1 μM and 10 μM was added to human plasma, incubated at 37°C for 30 minutes, and then ultrafiltered using a 30 kDa filtration membrane. The concentration of free drug in the filtrate and the total drug concentration in the plasma were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). At both concentrations, the plasma protein binding rate of M344 (D237; MS344) was greater than 95% [1].

[1]. J Med Chem . 1999 Nov 4;42(22):4669-79.

[2]. Gynecol Oncol . 2006 Apr;101(1):108-13.

[3]. Int J Cancer . 2007 Apr 15;120(8):1787-94.

[4]. Cancer Cell Int . 2010 Sep 9:10:32.

4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxohepyl]benzamide is a benzamide formed by the condensation of the carboxyl group of 4-(dimethylamino)benzoic acid and the amino group of 7-amino-N-hydroxyheptamide. It is a potent histone deacetylase inhibitor that can induce cell cycle arrest and apoptosis in various human cancer cell lines. It possesses dual activities as an apoptosis inducer, antitumor agent, and EC 3.5.1.98 (histone deacetylase) inhibitor. It belongs to the benzamide class, hydroxamic acid class, secondary amide class, and tertiary amine class.
A hydroxamic acid and aniline derivative, it can act as a histone deacetylase inhibitor. It is used to treat cutaneous T-cell lymphoma and Cezari syndrome. 1. Mechanism of Action: M344 (D237; MS344) exerts its anticancer effect by inhibiting HDACs, leading to increased acetylation levels of histones and non-histone proteins (e.g., α-tubulin). This epigenetic modification relaxes chromatin structure, promotes the transcription of tumor suppressor genes (e.g., p21WAF1/CIP1), and inhibits the expression of oncogenes. Furthermore, increased α-tubulin acetylation stabilizes microtubules, inhibits cancer cell proliferation, and induces apoptosis [1,3,4]. 2. Preclinical Relevance to Gynecologic Cancers: M344 (D237; MS344) showed good activity against ovarian cancer cell lines (SKOV3, OVCAR3) and xenograft tumors, significantly inhibiting tumor growth with low toxicity. This supports its potential as a treatment for ovarian cancer, a disease with a high degree of unmet medical need due to frequent relapses and resistance to chemotherapy [2]
3. Comparison with other HDAC inhibitors: Compared with the pan-HDAC inhibitor trichostatin A (TSA), M344 (D237; MS344) has a longer half-life (3.5 hours vs. 2.0 hours in mice) and higher oral bioavailability (25% vs. TSA <10%), making it more suitable for oral administration in long-term treatment regimens [1]
4. Potential clinical applications: Based on preclinical data, M344 (D237; MS344) has the potential to treat solid tumors, including colorectal cancer, non-small cell lung cancer and ovarian cancer. It can simultaneously inhibit class I HDAC and HDAC6 with low toxicity, thus it can be considered as a candidate drug for combination therapy with other anticancer drugs (such as chemotherapy and targeted therapy) [2,3,4]

C16H25N3O3

307.39

307.189

251456-60-7

3994

White to off-white solid powder

1.1±0.1 g/cm3

161℃

1.558

1.06

3

4

9

22

340

0

MXWDSZWTBOCWBK-UHFFFAOYSA-N

InChI=1S/C16H25N3O3/c1-19(2)14-10-8-13(9-11-14)16(21)17-12-6-4-3-5-7-15(20)18-22/h8-11,22H,3-7,12H2,1-2H3,(H,17,21)(H,18,20)

4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]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: ≥ 2.5 mg/mL (8.13 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.13 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.13 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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