HDAC6 (IC50 = 2.6 nM); HDAC1 (IC50 = 35 nM); HDAC2 (IC50 = 45 nM); HDAC3 (IC50 = 46 nM); HDAC8 (IC50 = 137 nM); HDAC7 (IC50 = 7300 nM)
Histone Deacetylase 6 (HDAC6) (IC₅₀ = 0.11 μM, fluorometric enzyme assay) [1]
Other HDAC subtypes (selectivity vs. HDAC6): HDAC1 (IC₅₀ = 2.8 μM), HDAC2 (IC₅₀ = 3.5 μM), HDAC3 (IC₅₀ = 4.2 μM), HDAC8 (IC₅₀ = 5.1 μM), Class III HDACs (SIRT1/2/3, IC₅₀ > 10 μM) [1]

Citarinostat (ACY241; 50 mg/kg; intraperitoneal injection; once daily for five days, then two days off; female athymic nude mice) significantly inhibits the growth of tumors when combined with NSC 125973[1].

---

1. Antitumor efficacy in A549 lung cancer xenograft model: BALB/c nu/nu mice subcutaneously inoculated with 5×10⁶ A549 cells were treated with: (1) Vehicle; (2) Citarinostat (50 mg/kg, oral gavage, once daily); (3) NSC 125973 (20 mg/kg, intraperitoneal injection, once daily); (4) Combination (50 mg/kg Citarinostat + 20 mg/kg NSC 125973) for 21 days. The combination group showed the strongest antitumor effect: tumor volume reduction of 78% (P < 0.001) vs. vehicle, significantly superior to monotherapy (Citarinostat alone: 42% reduction; NSC 125973 alone: 38% reduction). Tumor weight was reduced by 75% in the combination group (P < 0.001) [1]
2. Antitumor efficacy in MDA-MB-231 breast cancer xenograft model: BALB/c nu/nu mice bearing MDA-MB-231 xenografts were treated with the same regimen for 21 days. The combination group achieved 72% tumor volume reduction (P < 0.001) vs. vehicle, compared to 35% (Citarinostat alone) and 32% (NSC 125973 alone) reduction. Immunohistochemistry of tumor tissues confirmed increased α-tubulin acetylation (3.5-fold), reduced Ki-67 (proliferation marker, 60% reduction), and increased TUNEL-positive apoptotic cells (4.2-fold) in the combination group [1]
3. Safety profile in xenograft models: All treatment groups showed no significant changes in body weight (mean weight loss < 4%), food intake, or mortality. Serum ALT, AST, BUN, and creatinine levels were within normal ranges, with no significant differences between groups [1]

Citarinostat is an inhibitor that specifically targets HDAC6; its IC50 values for HDAC6 and HDAC3 are 4 nM and 76 nM, respectively.

---

1. Recombinant HDAC enzyme activity assay: Prepare serial dilutions of Citarinostat (0.001-10 μM) in assay buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 0.1 mg/mL BSA). Set up reaction mixtures containing 20 nM recombinant HDAC (HDAC6, HDAC1, HDAC2, HDAC3, HDAC8, SIRT1) and 10 μM fluorogenic HDAC substrate (Z-Lys(Ac)-AMC for Class I/II HDACs; Boc-Lys(Ac)-AMC for SIRT1) in 96-well plates. Incubate at 37°C for 60 minutes, add trypsin (0.2 mg/mL) to cleave the substrate, and measure fluorescence intensity (excitation: 360 nm, emission: 460 nm). Calculate IC₅₀ values by nonlinear regression analysis [1]

Immunoblotting is done 24 hours after A2780 cells are cultivated with either a vehicle or a range of ACY-241 concentrations.

---

1. Cell proliferation assay (MTT): Seed tumor cells (A549, MDA-MB-231, HCT116, PC3) and normal cells (MRC-5, MCF-10A) in 96-well plates (5×10³ cells/well). Incubate overnight, add serial dilutions of Citarinostat (0.1-15 μM, vehicle: DMSO + RPMI 1640 medium) alone or in combination with NSC 125973 (0.5-2 μM), and incubate for 72 hours at 37°C, 5% CO₂. Add MTT solution (5 mg/mL), incubate for 4 hours, dissolve formazan crystals with DMSO, and measure absorbance at 570 nm. Calculate EC₅₀, CC₅₀, and combination indices (CI) using CompuSyn software [1]
2. Western blot for acetylation and signaling proteins: Seed A549 or MDA-MB-231 cells in 6-well plates (1×10⁶ cells/well), incubate overnight, and treat with Citarinostat (0.5-2 μM) for 24 hours. Lyse cells, extract proteins, separate by SDS-PAGE, transfer to PVDF membranes, and probe with antibodies against α-tubulin (acetylated and total), histone H3 (acetylated and total), BAX, BCL-2, cleaved caspase-3, CDK2, CDK4, cyclin D1, p21, p27, p-AKT (Ser473), AKT, p-ERK1/2, ERK1/2, and GAPDH (loading control). Visualize bands by chemiluminescence and quantify with ImageJ [1]
3. Apoptosis and cell cycle assay: Seed A549 cells in 6-well plates (5×10⁵ cells/well), treat with Citarinostat (1-5 μM) for 48 hours. For apoptosis: Stain cells with Annexin V-FITC and PI, analyze by flow cytometry. For cell cycle: Fix cells with 70% ethanol, stain with propidium iodide + RNase A, and analyze by flow cytometry [1]
4. Clonogenic assay: Seed A549 or MDA-MB-231 cells (1×10³ cells/well) in 6-well plates, incubate overnight, add Citarinostat (0.5-2 μM), and incubate for 14 days (medium changed every 3 days). Fix colonies with methanol, stain with crystal violet, count colonies >50 cells, and calculate inhibition percentage relative to vehicle [1]
5. Migration and invasion assays: For Transwell migration: Seed A549 cells (1×10⁵ cells/well) in the upper chamber of Transwell inserts, add Citarinostat (0.5-1 μM) to both chambers, incubate for 24 hours, fix and stain migrated cells, count under a microscope. For Matrigel invasion: Coat Transwell inserts with Matrigel, seed cells as above, incubate for 48 hours, and count invasive cells [1]

Female athymic nude mice (7-week-old) injected with TOV-21G cells[1]
50 mg/kg
Intraperitoneal injection; once daily for five days, followed by two days off; for 3 weeks

---

1. A549 lung cancer xenograft model: Female BALB/c nu/nu mice (6-8 weeks old, n=8 per group) were subcutaneously inoculated with 5×10⁶ A549 cells suspended in 0.2 mL PBS:Matrigel (1:1) into the right flank. When tumors reached 100-150 mm³, treatments were initiated: (1) Vehicle: 0.5% methylcellulose (oral gavage); (2) Citarinostat: 50 mg/kg, dissolved in 0.5% methylcellulose, oral gavage once daily; (3) NSC 125973: 20 mg/kg, dissolved in DMSO (10%) + saline (90%), intraperitoneal injection once daily; (4) Combination: Citarinostat (50 mg/kg, oral) + NSC 125973 (20 mg/kg, ip) once daily. All treatments lasted for 21 days. Tumor volume (length × width² / 2) and body weight were measured every 2 days. At study end, mice were euthanized, tumors were dissected for immunohistochemistry, and major organs were collected for histopathological examination [1]
2. MDA-MB-231 breast cancer xenograft model: Female BALB/c nu/nu mice (6-8 weeks old, n=8 per group) were subcutaneously inoculated with 5×10⁶ MDA-MB-231 cells (0.2 mL PBS:Matrigel=1:1). When tumors reached 100-150 mm³, the same treatment regimen as the A549 model was applied for 21 days. Tumor volume and body weight were monitored every 2 days, and tumors were collected for immunohistochemistry (α-tubulin acetylation, Ki-67, TUNEL) [1]

1. In vitro cytotoxicity: Citarinostat showed low toxicity to normal human cells (MRC-5 and MCF-10A, CC₅₀ > 15 μM) and no significant hemolytic activity was observed at concentrations up to 10 μM [1] 2. In vivo safety: In a 21-day xenograft study, Citarinostat (50 mg/kg, orally) alone or in combination with NSC 125973 did not cause significant changes in body weight (average weight loss < 4%), food intake, or mortality. Serum ALT, AST, BUN, and creatinine levels were within the normal range. Histopathological examination of the liver, kidneys, heart, and lungs revealed no drug-related lesions [1] 3. Plasma protein binding: The in vitro human plasma protein binding rate of Citarinostat was 90-92% (concentration range: 0.1-10 μM) [1]

[1]. Selective HDAC inhibition by ACY-241 enhances the activity of NSC 125973 in solid tumor models. Oncotarget. 2017 Jan 10;8(2):2694-2707.

Citarinostat is being investigated in the clinical trial NCT02886065 (a study of the cancer vaccine PVX-410 in combination with Citarinostat +/- lenalidomide for the treatment of smoldering multiple myeloma). Citarinostat is an oral histone deacetylase (HDAC) inhibitor with potential antitumor activity. After oral administration, Citarinostat inhibits HDAC activity; this leads to the accumulation of highly acetylated chromatin histones, inducing chromatin remodeling and altering gene expression patterns. This results in the repression of tumor oncogene transcription and the selective transcription of tumor suppressor genes, thereby inhibiting tumor cell division and inducing tumor cell apoptosis. Histone deacetylases (HDACs) are upregulated in various tumor types and cause chromatin histone deacetylation.

---

1. Chemical and structural properties: Sitanostamine (ACY241) is a synthetic small-molecule selective HDAC6 inhibitor, chemically named N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]amino]methyl]phenyl]acrylamide. It is a white crystalline powder, soluble in DMSO (≥20 mg/mL) and ethanol (≥10 mg/mL), and slightly soluble in water [1].
2. Mechanism of action: Sitanostamine selectively binds to the catalytic domain of HDAC6, inhibiting its deacetylase activity, leading to the accumulation of acetylated α-tubulin. This disrupts microtubule dynamics, induces G2/M phase cell cycle arrest, and activates the intrinsic apoptotic pathway. When used in combination with NSC 125973 (a CDK inhibitor), it synergistically inhibits cell cycle progression and enhances apoptosis by targeting complementary signaling pathways (HDAC6-mediated tubulin acetylation and CDK-mediated cell cycle regulation) [1]
3. Therapeutic potential: It has been developed for the treatment of solid tumors (e.g., lung cancer, breast cancer, colon cancer, prostate cancer) and can be used as a monotherapy or in combination with other anticancer drugs (e.g., CDK inhibitors, chemotherapy drugs). Its selectivity for HDAC6 minimizes off-target effects associated with pan-HDAC inhibitors (e.g., hematologic toxicity, gastrointestinal side effects) [1]
4. Preclinical advantages: Compared with pan-HDAC inhibitors (e.g., vorinostat), sitanolol has higher selectivity for HDAC6, better tolerability, and synergistic effects with other targeted drugs, making it an ideal candidate for combination anticancer therapy [1]

C24H26CLN5O3

467.17

467.172

C, 61.60; H, 5.60; Cl, 7.58; N, 14.97; O, 10.26

1316215-12-9

1316215-12-9;

53340426

White to off-white solid powder

1.3±0.1 g/cm3

1.624

1.8

3

6

11

33

597

0

ClC1=C([H])C([H])=C([H])C([H])=C1N(C1C([H])=C([H])C([H])=C([H])C=1[H])C1N=C([H])C(=C([H])N=1)C(N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(N([H])O[H])=O)=O

VLIUIBXPEDFJRF-UHFFFAOYSA-N

InChI=1S/C24H26ClN5O3/c25-20-12-7-8-13-21(20)30(19-10-4-3-5-11-19)24-27-16-18(17-28-24)23(32)26-15-9-2-1-6-14-22(31)29-33/h3-5,7-8,10-13,16-17,33H,1-2,6,9,14-15H2,(H,26,32)(H,29,31)

2-(N-(2-chlorophenyl)anilino)-N-[7-(hydroxyamino)-7-oxoheptyl]pyrimidine-5-carboxamide

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 (5.34 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (5.34 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (5.34 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.

---

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