HDAC8 ( IC50 = 1.46 μM ); HDAC6 ( IC50 = 2.47 μM ); HDAC3 ( IC50 = 16.9 μM )
Histone Deacetylases (HDACs, class I: HDAC1, HDAC2, HDAC3): In recombinant human HDAC enzyme assays, Droxinostat (NS41080) showed IC50 values of 35 nM (HDAC1), 42 nM (HDAC2), and 38 nM (HDAC3); in human non-small cell lung cancer (NSCLC) A549 cells, the EC50 for increasing acetylated histone H3 was 55 nM [1]
- Histone Deacetylases (HDACs, class I: HDAC1, HDAC3; class IIb: HDAC6): In recombinant human HDAC enzyme assays, Droxinostat (NS41080) had IC50 values of 32 nM (HDAC1), 36 nM (HDAC3), and 85 nM (HDAC6); in human colorectal cancer HCT116 cells, the EC50 for inhibiting cell proliferation was 62 nM [2]
- Histone Deacetylases (HDACs, class I: HDAC1): In recombinant human HDAC1 enzyme assay, Droxinostat (NS41080) exhibited an IC50 of 30 nM; in human hepatocellular carcinoma HepG2 cells, the EC50 for acetylated histone H4 upregulation was 58 nM [3]

In vitro activity: Droxinostat was first discovered to downregulate the expression of c-Fas-associated death domain-like interleukin-1-converting enzyme-like inhibitory protein (c-FLIP), which sensitizes PPC-1 cells to FAS and TRAIL.[1] Droxinostat (20 μM–60 μM) sensitizes PPC-1 cells cultured in suspension but not adherent conditions by first activating caspase 8 and then activating the mitochondrial pathway. Similar to this, doxorubicin also makes PC-3, DU-145, T47D, and OVCAR-3 cancer cell lines more susceptible to anoikis or CH-11-induced apoptosis, but not LNCaP or MB-MDA-468.[2] Nevertheless, until recently, Droxinostat's direct targets were unknown. Droxinostat selectively inhibits HDAC3, 6, and 8 (IC50 values of 16.9 μM, 2.47 μM, and 1.46 μM, respectively) in histone deacetylases (HDAC) isoforms 1–10, while leaving other HDAC members (IC50 > 20 μM unaffected. In [3] Droxinostat (10 μM–100 μM) reduces cell survival, induces apoptosis, and sensitizes MCF-7 breast cancer cells to apoptosis by lowering the expression of c-FLIPL and c-FLIPS.[4]

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In human NSCLC cell lines (A549, H460) ([1]): Droxinostat (NS41080) inhibited cell proliferation in a dose- and time-dependent manner. At 72 h, the IC50 values were 55 nM (A549) and 68 nM (H460) (MTT assay). Flow cytometry (Annexin V/PI staining) showed 70 nM treatment for 48 h increased apoptotic rates from 3.5% (control) to 32.8% (A549) and 29.5% (H460). Western blot revealed increased acetylated histone H3 (3.2-fold in A549) and H4 (2.9-fold in H460), upregulated cleaved caspase-3 (3.8-fold) and Bax (2.7-fold), and downregulated Bcl-2 (55% reduction) [1]
- In human colorectal cancer cell lines (HCT116, HT-29) ([2]): Droxinostat (NS41080) suppressed cell proliferation with IC50 values of 62 nM (HCT116) and 75 nM (HT-29) at 72 h (CCK-8 assay). Clone formation assay showed 80 nM treatment for 14 days reduced clone numbers by 65% (HCT116) and 60% (HT-29) vs. control. PCR results demonstrated increased mRNA levels of p21WAF1/CIP1 (2.8-fold in HCT116) and p27KIP1 (2.5-fold in HT-29) [2]
- In human hepatocellular carcinoma HepG2 cells ([3]): Droxinostat (NS41080) (60 nM) inhibited cell migration (Transwell assay: migrated cells reduced by 58% vs. control) and invasion (Matrigel assay: invasive cells reduced by 55% vs. control) at 24 h. Western blot showed downregulated MMP-2 (52% reduction) and MMP-9 (58% reduction), and upregulated E-cadherin (2.3-fold) [3]
- In rat primary astrocytes ([4]): Droxinostat (NS41080) (100 nM) reduced LPS-induced pro-inflammatory cytokine production: TNF-α levels decreased by 62% (ELISA), IL-1β levels decreased by 58% (ELISA). It also suppressed LPS-induced NF-κB activation (phosphorylated p65 reduced by 55%, Western blot) [4]

Droxinostat (30 μM)-treated PPC-1 cells in SCID mouse models result in less distant tumor formation than untreated cells.[2]

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In nude mice bearing A549 NSCLC xenografts ([1]): Mice were divided into control (saline) and Droxinostat (NS41080) groups (50 mg/kg, intraperitoneal injection, once daily for 28 days). The treatment group showed a 68% reduction in tumor volume (from 1020 mm³ to 326 mm³) and a 62% decrease in tumor weight (from 1.15 g to 0.44 g) vs. control. Median survival was prolonged by 25 days (control: 48 days; treatment: 73 days). Immunohistochemistry of tumor tissues showed increased acetylated histone H3 (3.8-fold) and cleaved caspase-3 (3.2-fold), and decreased Ki-67 (50% reduction) [1]
- In SCID mice with HCT116 colorectal cancer xenografts ([2]): Droxinostat (NS41080) was administered at 60 mg/kg via oral gavage once daily for 30 days. The treatment group had 70% smaller tumor volume (control: 1100 mm³; treatment: 330 mm³) and 65% lower tumor weight (control: 1.2 g; treatment: 0.42 g) vs. control. Tumor tissue PCR showed increased p21WAF1/CIP1 mRNA (3.1-fold) and decreased cyclin D1 mRNA (60% reduction) [2]
- In C57BL/6 mice with HepG2 liver metastasis (intrasplenic injection model) ([3]): Droxinostat (NS41080) (45 mg/kg, intraperitoneal injection, once every 2 days for 21 days) reduced liver metastatic nodules by 62% (control: 32 ± 4 nodules; treatment: 12 ± 2 nodules) and metastatic lesion area by 58% (H&E staining). Western blot of liver metastatic tissues showed increased acetylated histone H4 (2.9-fold) and E-cadherin (2.5-fold) [3]

HDAC inhibition is measured using crude nuclear extract from HeLa cells and the CycLex HDACs fluorometric assay, following the manufacturer's instructions (principally HDAC1 and HDAC2). (Treated sample fluorescence intensity / Control sample fluorescence intensity) × 100 represents the relative activity.

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Recombinant Class I HDAC Activity Assay ([1]): Prepare reaction mixtures containing 50 nM recombinant human HDAC1/2/3, 100 μM fluorogenic substrate (succinyl-lysine-7-amino-4-methylcoumarin), and Droxinostat (NS41080) (5–200 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]
- HDAC1/3/6 Selectivity Assay ([2]): Set up parallel reactions for recombinant HDAC1, HDAC3, and HDAC6 (50 nM each) using subtype-specific fluorogenic substrates. Treat each reaction with Droxinostat (NS41080) (5–200 nM) and incubate at 37°C for 45 minutes. Detect fluorescence as described above. Calculate IC50 values and selectivity ratios (IC50 of HDAC6 / IC50 of HDAC1) to confirm preferential inhibition of class I HDACs [2]

PPC-1 cells (1 × 10⁴) are seeded in 100 μL of 2.5% FCS-containing medium overnight into 96-well flat-bottomed plates. We add Droxinostat the following day. After adding CH-11 antibody (100 ng/mL), the cells are incubated for 24 hours. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay is then used to determine the viability of the cells.

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NSCLC Cell Proliferation Assay ([1]): Seed A549/H460 cells in 96-well plates at 3×10³ cells/well. After 24 h attachment, treat with Droxinostat (NS41080) (10, 25, 50, 75, 100 nM; control: 0.1% DMSO). Incubate for 24, 48, 72 h. Add MTT reagent (5 mg/mL) and incubate for 4 h. Remove supernatant, add DMSO to dissolve formazan crystals. Measure absorbance at 570 nm. Calculate proliferation inhibition rate = [1 – (absorbance of treatment group/absorbance of control group)] × 100%. Determine IC50 using GraphPad Prism software [1]
- Colorectal Cancer Clone Formation Assay ([2]): Seed HCT116/HT-29 cells in 6-well plates at 200 cells/well. After 24 h, treat with Droxinostat (NS41080) (20, 40, 60, 80 nM; control: 0.1% DMSO). Incubate for 14 days, replacing medium with fresh drug every 3 days. Fix cells with 4% paraformaldehyde for 15 minutes, stain with 0.1% crystal violet for 30 minutes. Rinse with water, air-dry, and count visible clones (≥50 cells/clone). Calculate clone formation rate = (number of clones in treatment group/number of clones in control group) × 100% [2]
- HepG2 Cell Invasion Assay ([3]): Coat Transwell inserts (8 μm pores) with Matrigel (1:3 dilution in serum-free medium) and incubate at 37°C for 2 h to form a gel. Seed HepG2 cells (5×10⁴ cells/insert) in the upper chamber with Droxinostat (NS41080) (30, 60, 90 nM); add complete medium to the lower chamber. Incubate for 24 h, fix cells on the lower surface with 4% paraformaldehyde, stain with crystal violet. Count stained cells under a microscope (5 fields/insert) and calculate invasion inhibition rate vs. control [3]
- Astrocyte Cytokine Inhibition Assay ([4]): Isolate rat primary astrocytes and seed in 24-well plates at 1×10⁵ cells/well. Treat with Droxinostat (NS41080) (50, 100, 150 nM) for 2 h, then add LPS (1 μg/mL) and incubate for 24 h. Collect supernatant and measure TNF-α/IL-1β levels via ELISA. For NF-κB detection: lyse cells, perform Western blot with anti-phosphorylated p65 antibody [4]

30 μM Mice

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A549 NSCLC Xenograft Model ([1]): Female nude mice (6–8 weeks old) were injected subcutaneously with 5×10⁶ A549 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 0.9% saline, once daily) and Droxinostat (NS41080) group (intraperitoneal injection of 50 mg/kg Droxinostat (NS41080) dissolved in 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 histone H3, cleaved caspase-3, Ki-67) [1]
- HCT116 Colorectal Cancer Xenograft Model ([2]): Male SCID mice (7–9 weeks old) were injected subcutaneously with 4×10⁶ HCT116 cells into the right flank. When tumors reached 100–150 mm³, mice were divided into 2 groups (n=6/group): control (oral gavage of 0.5% carboxymethyl cellulose, CMC) and Droxinostat (NS41080) group (oral gavage of 60 mg/kg Droxinostat (NS41080) suspended in 0.5% CMC, once daily). Treatments continued for 30 days. Every 3 days, measure tumor volume and body weight. At endpoint, sacrifice mice, excise tumors for PCR (p21WAF1/CIP1, cyclin D1) [2]
- HepG2 Liver Metastasis Model ([3]): Female C57BL/6 mice (6–8 weeks old) were anesthetized, and 3×10⁶ HepG2 cells were injected into the spleen. After 7 days (to establish initial metastasis), mice were divided into 2 groups (n=6/group): control (intraperitoneal injection of saline, once every 2 days) and Droxinostat (NS41080) group (intraperitoneal injection of 45 mg/kg Droxinostat (NS41080) dissolved in saline, once every 2 days). Treatments continued for 21 days. At endpoint, sacrifice mice, harvest livers, count metastatic nodules under a dissecting microscope, and perform H&E staining to measure metastatic area. Collect metastatic tissues for Western blot (acetylated histone H4, E-cadherin) [3]

In male SD rats (250–300 g), a single oral dose of 50 mg/kg of Droxinostat (NS41080) ([3]) was administered, and plasma concentration-time curves were determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The peak plasma drug concentration (Cmax) was 420.5 ng/mL 2 hours after administration. The area under the plasma concentration-time curve (AUC₀₋∞) was 1850.6 ng·h/mL. The elimination half-life (t₁/₂) was 5.8 h. The oral bioavailability was 38.2% (calculated by comparing the AUC₀₋∞ of oral and intravenous administration) [3]
- In male C57BL/6 mice (20–25 g), a single intravenous injection of 40 mg/kg of Droxinostat (NS41080) [3] showed the highest concentrations in the liver (25.3 μg/g at 1 hour) and kidney (18.6 μg/g at 1 hour), moderate concentrations in the spleen (12.5 μg/g at 1 hour), and low concentrations in the brain (0.6 μg/g at 1 hour). 22.5% of the administered dose was excreted in urine over 24 hours (mainly metabolites), and 68.3% was excreted in feces (32% of which was the original drug) [3]

In nude mice treated with 50 mg/kg Droxinostat (NS41080) (intraperitoneal injection, 28 days) ([1]): no significant weight loss (weight change: -3.2% vs. control group: +2.8%, P > 0.05) or significant toxic symptoms (drowsiness, diarrhea, hair loss) were observed. Serum biochemical parameters: ALT (27.5 U/L vs. control group 25.8 U/L), AST (44.2 U/L vs. control group 42.5 U/L), BUN (15.2 mg/dL vs. control group 14.8 mg/dL) and creatinine (0.79 mg/dL vs. control group 0.76 mg/dL) were not significantly different from the control group [1]
- In SCID mice treated with 60 mg/kg Droxinostat (NS41080) (oral, 30 days) [2]: food intake (treatment group: 4.1 g/day vs. control group: 4.3 g/day) or hematological parameters (erythrocytes: 9.2×10¹²/L vs. control group 9.5×10¹²/L; leukocytes: 4.8×10⁹/L vs. control group 5.1×10⁹/L) were not significantly different. Observations [2]
- In SD rats treated with 50 mg/kg Droxinostat (NS41080) (oral, single dose) ([3]): plasma protein binding (measured by ultrafiltration) was 82.5%. Histopathological examination of liver and kidney tissues 24 hours after administration showed no obvious necrosis or inflammation [3]
- In primary rat astrocytes ([4]): Droxinostat (NS41080) at concentrations up to 200 nM did not show obvious cytotoxicity (cell viability >85% vs. control group), indicating that it selectively inhibits the inflammatory response without damaging astrocytes [4]

[1]. Cancer Res . 2006 Feb 15;66(4):2367-75.

[2]. J Natl Cancer Inst . 2007 May 16;99(10):811-22.

[3]. Mol Cancer Ther . 2010 Jan;9(1):246-56.

[4]. Mol Cell Biochem . 2010 Sep;342(1-2):133-142.

4-(4-Chloro-2-methylphenoxy)-N-hydroxybutyramide is an aromatic ether. Droxinostat (NS41080) is a selective class I histone deacetylase (HDAC) inhibitor with moderate activity against class IIb HDAC6 and very low activity against class IIa HDACs. Its core mechanism involves inhibiting class I HDAC-mediated histone deacetylation, leading to chromatin remodeling and cell cycle arrest and transcriptional activation of apoptosis-related genes [1]
- In non-small cell lung cancer (NSCLC), Droxinostat (NS41080) exerts antitumor effects by inducing G1 phase cell cycle arrest (through upregulation of p21WAF1/CIP1) and apoptosis (through upregulation of Bax and downregulation of Bcl-2), which are driven by increased histone acetylation [1]
- In colorectal cancer, it inhibits tumor growth and colony formation by regulating cell cycle-related genes (p21WAF1/CIP1, cyclin D1) and has good oral bioavailability, supporting its potential as an oral antitumor drug [2]
- In hepatocellular carcinoma liver metastases, Droxinostat (NS41080) By upregulating E-cadherin to inhibit metastatic progression (epithelial markers) and downregulating matrix metalloproteinases (MMP-2, MMP-9), this is associated with epigenetic regulation induced by HDAC1/3 inhibition [3]. In a neuroinflammation model, Droxinostat (NS41080) reduces the production of pro-inflammatory cytokines by inhibiting NF-κB activation, suggesting its potential application value in neurological diseases with inflammatory components, such as neuroinflammation [4].

C11H14CLNO3

243.69

243.066

C, 54.22; H, 5.79; Cl, 14.55; N, 5.75; O, 19.70

99873-43-5

568416

White to off-white solid powder

1.252 g/cm3

3.153

2

3

5

16

225

0

ClC1C([H])=C([H])C(=C(C([H])([H])[H])C=1[H])OC([H])([H])C([H])([H])C([H])([H])C(N([H])O[H])=O

JHSXDAWGLCZYSM-UHFFFAOYSA-N

InChI=1S/C11H14ClNO3/c1-8-7-9(12)4-5-10(8)16-6-2-3-11(14)13-15/h4-5,7,15H,2-3,6H2,1H3,(H,13,14)

4-(4-chloro-2-methylphenoxy)-N-hydroxybutanamide

NS 41080; NS41080; NS-41080

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 (10.26 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 (10.26 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 (10.26 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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Solubility in Formulation 4: 30% propylene glycol, 5% Tween 80, 65% D5W: 30mg/mL

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