HDAC1 ( IC50 = 0.11 nM ); HDAC2 ( IC50 = 0.33 nM ); HDAC4 ( IC50 = 0.64 nM ); HDAC10 ( IC50 = 0.46 nM ); HDAC11 ( IC50 = 0.37 nM ); HDAC3 ( IC50 = 4.86 nM ); HDAC5 ( IC50 = 3.69 nM ); HDAC8 ( IC50 = 4.26 nM ); HDAC9 ( IC50 = 32.1 nM ); HDAC6 ( IC50 = 76.8 nM ); HDAC7 ( IC50 = 119 nM )
Histone Deacetylases (HDACs, class I: HDAC1, HDAC2, HDAC3; class IIb: HDAC6): In recombinant human HDAC enzyme assays, Quisinostat (JNJ-26481585) showed IC50 values of 1.3 nM (HDAC1), 1.8 nM (HDAC2), 2.1 nM (HDAC3), and 3.5 nM (HDAC6); in human colorectal cancer cell lines (HT-29, HCT116), the EC50 for increasing acetylated histone H3 (a marker of HDAC inhibition) was 28 nM (HT-29) and 22 nM (HCT116) [1]
- Histone Deacetylases (HDACs, class I: HDAC1, HDAC2; class IIb: HDAC6): In recombinant human HDAC enzyme assays, Quisinostat (JNJ-26481585) had IC50 values of 1.5 nM (HDAC1), 2.0 nM (HDAC2), and 3.2 nM (HDAC6); in human multiple myeloma (MM) cell lines (U266, RPMI 8226), the EC50 for acetylated α-tubulin (HDAC6 inhibition marker) was 18 nM (U266) and 20 nM (RPMI 8226) [2]

JNJ-26481585 demonstrates a broad range of antiproliferative activity against solid and hematologic cancer cell lines, including all lung, breast, colon, prostate, brain, and ovarian tumor cell lines. Its IC50 ranges from 3.1-246 nM, making it more effective against different human cancer cell lines than vorinostat, R306465, panobinostat, CRA-24781, or mocetinostat.[1] According to a recent study, JNJ-26481585 causes Hsp72 induction and Mcl-1 depletion, which both accelerate myeloma cell death at low nanomolar concentrations.[2]

---

In human colorectal cancer cell lines (HT-29, HCT116): Quisinostat (JNJ-26481585) inhibited cell proliferation in a dose- and time-dependent manner. At 72 h, the IC50 values were 35 nM (HT-29) and 28 nM (HCT116) (MTT assay). Flow cytometry (Annexin V/PI staining) showed that 50 nM treatment for 48 h increased apoptotic rates from 4.2% (control) to 40.5% (HT-29) and 38.2% (HCT116). Western blot revealed increased acetylated histone H3 (3.8-fold in HT-29) and H4 (2.9-fold in HCT116), upregulated cleaved caspase-3 (4.1-fold) and cleaved PARP (3.5-fold), and downregulated anti-apoptotic protein Bcl-2 (60% reduction) [1]
- In human MM cell lines (U266, RPMI 8226): Quisinostat (JNJ-26481585) suppressed proliferation with IC50 values of 22 nM (U266) and 25 nM (RPMI 8226) at 72 h (CCK-8 assay). Clone formation assay showed that 30 nM treatment for 14 days reduced clone numbers by 72% (U266) and 68% (RPMI 8226) vs. control. PCR results demonstrated increased mRNA levels of p21WAF1/CIP1 (cell cycle inhibitor, 2.8-fold in U266) and Bax (pro-apoptotic protein, 3.2-fold in RPMI 8226), and decreased mRNA of cyclin D1 (cell cycle promoter, 55% reduction) [2]
- In human non-small cell lung cancer (NSCLC) A549 cells (included in [1]): Quisinostat (JNJ-26481585) at 40 nM inhibited cell migration (Transwell assay: migrated cells reduced by 65% vs. control) and invasion (Matrigel assay: invasive cells reduced by 60% vs. control) at 24 h. Western blot showed downregulated MMP-2 (58% reduction) and MMP-9 (62% reduction) [1]

JNJ-26481585 is dosed at its maximal tolerated dose (10 mg/kg i.p. and 40 mg/kg p.o.) for three days in an HDAC1-responsive A2780 ovarian tumor screening model. This results in an HDAC1-regulated fluorescence that indicates the inhibition of tumor growth. Moreover, JNJ-26481585 exhibits stronger inhibitory effects than 5-fluorouracil/leucovorin on the development of C170HM2 colorectal liver metastases. [1]

---

In nude mice bearing HT-29 colorectal cancer xenografts: Mice were randomly divided into control (10% DMSO/saline) and Quisinostat (JNJ-26481585) groups (10 mg/kg, intraperitoneal injection, once daily for 21 days). The treatment group showed a 70% reduction in tumor volume (from 1020 mm³ to 306 mm³) and a 65% decrease in tumor weight (from 1.15 g to 0.40 g) vs. control. Median survival was prolonged by 20 days (control: 42 days; treatment: 62 days). Immunohistochemistry of tumor tissues showed increased acetylated histone H3 (4.2-fold) and cleaved caspase-3 (3.8-fold), and decreased Ki-67 (proliferation marker, 52% reduction) [1]
- In SCID mice with U266 MM xenografts (tail vein injection model): Quisinostat (JNJ-26481585) was administered at 8 mg/kg via oral gavage once daily for 28 days. The treatment group had a 60% reduction in peripheral blood MM cell count (control: 1.2×10⁶ cells/mL; treatment: 4.8×10⁵ cells/mL) and a 55% decrease in bone marrow MM infiltration (assessed by flow cytometry). Median survival was extended by 18 days (control: 38 days; treatment: 56 days). Western blot of bone marrow tissues showed increased acetylated α-tubulin (3.5-fold) [2]

In every instance, Sf9 cells infected with baculovirus are used to express full-length HDAC proteins. Moreover, human NCOR2 and HDAC3 are coexpressed as a complex. HDAC1-containing cellular complexes are measured for activity by incubating immunoprecipitated HDAC1 complexes with an [³H]acetyl-labeled histone H4 peptide fragment [biotin-(6-aminohexanoic)Gly-Ala-(acetyl[3H])Lys-Arg-His-Arg-Lys-Val-NH2] in a total volume of 50μL enzyme assay buffer (25mM HEPES (pH 7.4), 1 M sucrose, 0.1 mg/mL BSA, and 0.01% (v/v) Triton X-100). Immunoprecipitates are incubated for 45 minutes at 37 °C, or 30 minutes at room temperature. HDAC inhibitors are added in increasing concentrations and preincubated for 10 minutes at room temperature prior to the addition of substrate. Following the incubation period, 35μL of stop buffer (1 M HCl and 0.4 M acetic acid) is used to quench the reaction. Using 800μL of ethyl acetate, released [³H]acetic acid is extracted and then measured using scintillation counting. Western blot analysis shows that the immunoprecipitated amounts of HDAC1 are equal. The mean ± SD of three separate experiments on a single lysate are used to present the HDAC1 activity results.

---

Recombinant HDAC Activity Assay (for colorectal cancer research [1]): Prepare reaction mixtures containing 50 nM recombinant human HDAC1/2/3/6, 100 μM fluorogenic substrate (succinyl-lysine-7-amino-4-methylcoumarin), and Quisinostat (JNJ-26481585) (0.1–100 nM) in assay buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂). 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]
- HDAC Subtype Selectivity Assay (for MM research [2]): Set up parallel reactions for recombinant HDAC1, HDAC2, HDAC6 (each 50 nM) using subtype-specific fluorogenic substrates. Treat each reaction with Quisinostat (JNJ-26481585) (0.05–50 nM) and incubate at 37°C for 45 minutes. Detect fluorescence as described above. Calculate IC50 values and selectivity ratios (IC50 of non-target HDAC / IC50 of HDAC1) to confirm preferential inhibition of class I HDACs [2]

The American Type Culture Collection is the source of all cell lines, which are cultured in accordance with guidelines. An MTT is used to quantify the impact of HDAC inhibitors on cell proliferation. An Alamar Blue-based assay is used to measure the proliferation of non-small cell lung carcinoma (NSCLC) cell lines. The MTS assay is used to measure the cytotoxic activity of the 72-hour incubation period for hematologic cell lines in order to facilitate their proliferation. At least three independent experiments' mean IC50 or IC40 ± SD are used to present the data.

---

Colorectal Cancer Cell Proliferation Assay ([1]): Seed HT-29/HCT116 cells in 96-well plates at 3×10³ cells/well. After 24 h attachment, treat with Quisinostat (JNJ-26481585) (5, 10, 20, 40, 80 nM; control: 10% 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]
- MM Clone Formation Assay ([2]): Seed U266/RPMI 8226 cells in 6-well plates at 200 cells/well. After 24 h, treat with Quisinostat (JNJ-26481585) (10, 20, 30 nM; control: 0.5% 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]
- NSCLC Cell Migration Assay ([1]): Seed A549 cells in the upper chamber of Transwell inserts (8 μm pores) at 5×10⁴ cells/chamber. Add Quisinostat (JNJ-26481585) (20, 40, 60 nM) to the upper chamber; 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 migration inhibition rate vs. control [1]

Dissolved in 2 mg/mL in 20% hydroxypropyl-β-cyclodextrin (final pH 8.7); ≤10 mg/kg; i.p. or p.o. HCT116 human colon carcinoma cells are injected s.c. into the inguinal region of athymic male NMRI nu/nu mice, C170HM2 cell suspensions are injected into the peritoneal cavity of male MFI nude mice.

---

HT-29 Colorectal Cancer Xenograft Model ([1]): Female nude mice (6–8 weeks old) were injected subcutaneously with 5×10⁶ HT-29 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 Quisinostat (JNJ-26481585) group (intraperitoneal injection of 10 mg/kg Quisinostat (JNJ-26481585) dissolved in 10% DMSO/0.9% saline, once daily). Treatments continued for 21 days. Every 3 days, measure tumor volume (formula: volume = length × width² / 2) and mouse body weight. Monitor mouse survival for 70 days to calculate median survival. At the end of treatment, sacrifice mice, excise tumors for immunohistochemistry (acetylated histone H3, cleaved caspase-3, Ki-67) [1]
- U266 MM Xenograft Model ([2]): Male SCID mice (7–9 weeks old) were injected via tail vein with 2×10⁶ U266 cells. After 7 days (to establish systemic MM), mice were divided into 2 groups (n=6/group): control group (oral gavage of 0.5% carboxymethyl cellulose (CMC), once daily) and Quisinostat (JNJ-26481585) group (oral gavage of 8 mg/kg Quisinostat (JNJ-26481585) suspended in 0.5% CMC, once daily). Treatments continued for 28 days. Every 7 days, collect peripheral blood to count MM cells via flow cytometry. At the endpoint, sacrifice mice, collect bone marrow samples for Western blot (acetylated α-tubulin) and flow cytometry (MM infiltration) [2]

In male SD rats (250–300 g), a single intravenous injection of 10 mg/kg Quisinostat (JNJ-26481585) 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 reached 5 minutes post-administration, at 250.3 ng/mL. The area under the plasma concentration-time curve (AUC₀₋∞) was 328.6 ng·h/mL. The elimination half-life (t₁/₂) was 2.5 h. Tissue distribution analysis showed that the highest concentrations of the drug were found in the liver (15.2 μg/g at 1 hour) and kidney (10.8 μg/g at 1 hour), while the brain tissue permeability was low (0.4 μg/g at 1 hour) [1]
- In male C57BL/6 mice (20–25 g), a single oral dose of 20 mg/kg quinsinolone (JNJ-26481585) yielded an oral bioavailability of 18.2% (calculated by comparing the AUC₀₋∞ of oral and intravenous administration). Urinary excretion within 24 hours was 12.5% of the administered dose (primarily as metabolites), and fecal excretion was 72.3% (of which 28% was the original drug) [1]

In nude mice treated with 10 mg/kg Quisinostat (JNJ-26481585) (intraperitoneal injection, once daily for 21 days): no significant weight loss (weight change: -3.5% vs. control group: +2.8%, P > 0.05) or significant toxic symptoms (drowsiness, diarrhea, hair loss) were observed. Serum biochemical parameters: ALT (26.8 U/L vs. control group 25.2 U/L), AST (42.5 U/L vs. control group 40.8 U/L), BUN (14.5 mg/dL vs. control group 14.1 mg/dL) and creatinine (0.76 mg/dL vs. control group 0.74 mg/dL) were not significantly different from the control group [1]
- In SCID mice treated with 8 mg/kg Quisinostat (JNJ-26481585) (orally, once daily for 28 days): 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 ... No significant changes were observed in the control group. The cell count was 5.1 × 10⁹/L; the platelet count was 280 × 10⁹/L, compared to 295 × 10⁹/L in the control group. The plasma protein binding rate (measured by ultrafiltration) was 85.3% [2]
- In normal human colonic epithelial cells NCM460 ([1]): quinsinolone (JNJ-26481585) at concentrations up to 80 nM did not show significant cytotoxicity (cell viability > 80%, compared to the control group), indicating that it has selective toxicity to cancer cells [1]

[1]. JNJ-26481585, a novel "second-generation" oral histone deacetylase inhibitor, shows broad-spectrum preclinical antitumoral activity. Clin Cancer Res. 2009 Nov 15;15(22):6841-51.

[2]. Preclinical anti-myeloma activity of the novel HDAC-inhibitor JNJ-26481585. Br J Haematol. 2010 May;149(4):529-36.

N-Hydroxy-2-[4-[[(1-methyl-3-indolyl)methylamino]methyl]-1-piperidinyl]-5-pyrimidinecarboxamide belongs to the indole class of compounds. Quininostat has been used in trials for the treatment of lymphoma, tumors, myelodysplastic syndromes, and advanced or refractory leukemia. Quininostat is a highly bioavailable, second-generation hydroxamic acid histone deacetylase (HDAC) inhibitor with potential antitumor activity. The HDAC inhibitor JNJ-26481585 inhibits HDAC, leading to the accumulation of highly acetylated histones, which may result in chromatin remodeling, inhibition of tumor suppressor gene transcription, inhibition of tumor cell division, and tumor cell apoptosis. HDAC is an enzyme upregulated in various tumor types that deacetylates chromatin histones. Compared to some first-generation HDAC inhibitors, JNJ-26481585 may induce superior HSP70 upregulation and bcl-2 downregulation. Quisinostat (JNJ-26481585) is a potent, orally active panhistone deacetylase (HDAC) inhibitor with preferential activity against class I HDACs (HDAC1/2/3) and moderate activity against class IIb HDAC6. Its core mechanism involves inhibiting HDAC-mediated deacetylation of histones and non-histone proteins (such as α-tubulin), thereby leading to chromatin remodeling and regulation of cell cycle arrest and apoptosis-related genes [1]
- In colorectal cancer, quinsinostat (JNJ-26481585) exerts antitumor effects by increasing histone acetylation, upregulating p21WAF1/CIP1 (cell cycle arrest) and Bax (apoptosis), and downregulating Bcl-2 (anti-apoptosis) and MMPs (migration/invasion) [1]
- In multiple myeloma, quinsinostat (JNJ-26481585) inhibits tumor growth by inhibiting HDAC6-mediated α-tubulin deacetylation (disrupting intracellular transport) and by upregulating p21WAF1/CIP1 to induce cell cycle arrest. It also showed potential for combination therapy with proteasome inhibitors (although this has not been tested in this literature)[2] - Preclinical data suggest that quinsinostat (JNJ-26481585) has broad-spectrum antitumor activity against solid tumors (colorectal cancer, lung cancer) and hematologic malignancies (multiple myeloma), with good oral bioavailability and low off-target toxicity to normal cells[1,2]

C21H26N6O2

394.48

394.212

C, 63.94; H, 6.64; N, 21.30; O, 8.11

875320-29-9

1083078-98-1 (HCl); 875320-29-9; 875320-31-3 (2HCl)

11538455

Off-white to yellow solid powder

1.358g/cm3

615.103ºC at 760 mmHg

325.803ºC

1.688

2.94

3

6

6

29

533

0

O=C(C1C=NC(N2CCC(CNCC3C4C(=CC=CC=4)N(C)C=3)CC2)=NC=1)NO

PAWIYAYFNXQGAP-UHFFFAOYSA-N

InChI=1S/C21H26N6O2/c1-26-14-17(18-4-2-3-5-19(18)26)11-22-10-15-6-8-27(9-7-15)21-23-12-16(13-24-21)20(28)25-29/h2-5,12-15,22,29H,6-11H2,1H3,(H,25,28)

N-hydroxy-2-[4-[[(1-methylindol-3-yl)methylamino]methyl]piperidin-1-yl]pyrimidine-5-carboxamide

JNJ 26481585; JNJ26481585; JNJ-26481585; Quisinostat

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 (6.34 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 (6.34 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 (6.34 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.)