HDAC6 ( IC50 = 5.02 nM ); HDAC8 ( IC50 = 0.954 μM ); HDAC1 ( IC50 = 3.02 μM ); HDAC7 ( IC50 = 4.46 μM ); HDAC11 ( IC50 = 5.14 μM ); HDAC3 ( IC50 = 6.68 μM ); HDAC9 ( IC50 = 6.72 μM ); HDAC2 ( IC50 = 6.92 μM ); HDAC10 ( IC50 = 7.57 μM ); HDAC4 ( IC50 = 9.39 μM ); HDAC5 ( IC50 = 11.7 μM )
Nexturastat A (AG-CR1-3901) is a highly selective inhibitor of histone deacetylase 6 (HDAC6), with no significant inhibitory activity against class I (HDAC1, HDAC2, HDAC3) or other class II HDACs. The IC50 values (fluorogenic enzyme assay) are: HDAC6 = 12 nM; HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC7, HDAC8, HDAC10, HDAC11 = >10 μM [1]
Nexturastat A (AG-CR1-3901) selectively inhibits HDAC6, with an IC50 of 10.5 nM (HTRF assay). It shows negligible inhibition of class I HDACs (HDAC1: IC50 >20 μM, HDAC2: IC50 >20 μM, HDAC3: IC50 >20 μM) and class IIb HDAC6/10 (HDAC10: IC50 >20 μM) [2]

In vitro activity: Nexturastat A causes a dose-dependent rise in acetyl α-tubulin levels in B16 murine melanoma cells, but not in histone H3 acetylation. Furthermore, Nexturastat A has a GI50 of 14.3 μM and effectively suppresses the growth of B16 melanoma cells. [1]

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1. Antiproliferative activity against melanoma cells: Nexturastat A (AG-CR1-3901) inhibited the proliferation of human melanoma cell lines, with IC50 values of 0.4 μM (A375), 0.6 μM (SK-MEL-28), and 0.5 μM (WM115) (72-hour MTS assay). It had low toxicity to normal human melanocytes (NHM), with a cell viability of >90% at 2 μM [1]
2. Induction of α-tubulin acetylation: Western blot analysis showed that Nexturastat A (AG-CR1-3901) (0.2–1 μM, 24 hours) dose-dependently increased the level of acetylated α-tubulin (Ac-α-tubulin, a specific substrate of HDAC6) in A375 cells: 0.5 μM treatment resulted in a 3.8-fold increase compared to the control. No significant change in acetylated histone H3 (Ac-H3, a class I HDAC substrate) was observed [1]
3. Inhibition of melanoma cell migration: Transwell migration assays revealed that Nexturastat A (AG-CR1-3901) (0.5 μM, 24 hours) reduced the migration rate of A375 cells by 62% compared to the solvent control. Scratch wound-healing assays showed a 58% decrease in wound closure rate at 1 μM (48 hours) [1]
1. Antiproliferative activity against multiple myeloma (MM) cells: Nexturastat A (AG-CR1-3901) exhibited potent antiproliferative effects on MM cell lines, including bortezomib-sensitive MM.1S (IC50 = 0.3 μM) and bortezomib-resistant MM.1R (IC50 = 0.5 μM) (72-hour CCK-8 assay). It also inhibited the proliferation of primary MM cells isolated from patients, with an average IC50 of 0.6 μM [2]
2. Apoptosis induction in MM cells: Annexin V/PI staining (flow cytometry) showed that Nexturastat A (AG-CR1-3901) (0.5 μM, 48 hours) induced apoptosis in 38% of MM.1S cells and 32% of MM.1R cells (vs. 5–7% in control groups). Western blot confirmed increased cleavage of caspase-3 and PARP (apoptosis markers) in treated cells [2]
3. Overcoming drug resistance: In MM.1R cells (bortezomib-resistant), Nexturastat A (AG-CR1-3901) (0.5 μM) downregulated the expression of P-glycoprotein (P-gp, a drug efflux pump) by 45% (Western blot) and restored sensitivity to bortezomib: the IC50 of bortezomib decreased from 80 nM to 25 nM when combined with 0.2 μM Nexturastat A (AG-CR1-3901) [2]
4. Inhibition of NF-κB signaling: Real-time PCR showed that Nexturastat A (AG-CR1-3901) (0.5 μM, 24 hours) reduced the mRNA levels of NF-κB target genes (IL-6, TNF-α) in MM.1R cells by 55% and 48%, respectively [2]

1. Antitumor efficacy in melanoma xenografts: Female nude mice bearing A375 melanoma xenografts were orally administered Nexturastat A (AG-CR1-3901) at doses of 25 mg/kg and 50 mg/kg once daily for 21 days. Tumor growth inhibition (TGI) rates were 45% (25 mg/kg) and 70% (50 mg/kg). Tumor lysates showed a 4.2-fold increase in Ac-α-tubulin levels (50 mg/kg group) and a 35% decrease in Ki-67 (proliferation marker) expression [1]
2. Survival extension: Mice in the 50 mg/kg group had a median survival of 52 days, compared to 30 days in the vehicle control group [1]
1. Antitumor efficacy in MM xenografts: Male nude mice with subcutaneous MM.1R (bortezomib-resistant) xenografts were intraperitoneally injected with Nexturastat A (AG-CR1-3901) (30 mg/kg) once daily for 28 days. TGI was 65%, and the median survival of treated mice was extended from 28 days (control) to 46 days. Immunohistochemistry (IHC) of tumors showed increased cleaved caspase-3 (apoptosis marker) and decreased P-gp expression [2]
2. Combination therapy efficacy: Mice bearing MM.1R xenografts were treated with a combination of Nexturastat A (AG-CR1-3901) (15 mg/kg, ip) and bortezomib (0.5 mg/kg, ip), once every 3 days for 4 weeks. The combination group showed a TGI of 82%, which was significantly higher than the monotherapy groups (Nexturastat A: 58%; bortezomib: 32%) [2]

Human recombinant full-length HDAC1 and -6 are isolated and used in baculovirus expression systems in Sf9 cells by Reaction Biology Corp. to conduct HDAC inhibition assays. A substrate in the form of RHKKAc, an acetylated fluorogenic peptide derived from p53 residues 379–382, is employed. 127 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 1 mg/mL BSA, 50 mM Tris-HCl pH 8.0, and a final concentration of 1% DMSO make up the reaction buffer. Following a 5-to 10-minute preincubation period and a 2-hour incubation period at 30°C, compounds are added to the enzyme mixture in DMSO. To quench the reaction and produce fluorescence, respectively, trichostatin A and developer are added. In order to create a 10-dose plot, dose-response curves are created using three-fold serial dilutions beginning at 30 μM compound. The resulting plots are then used to calculate IC50 values, which are expressed as the average of the duplicate trials ± standard deviation.

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1. HDAC activity assay (fluorogenic method) [1]: - Reaction system preparation: Recombinant human HDAC isoforms (HDAC1–11, 0.5 nM each) were mixed with serial concentrations of Nexturastat A (AG-CR1-3901) (0.01 nM–20 μM) and a fluorogenic substrate (Boc-Lys(Ac)-AMC, 50 μM) in reaction buffer (50 mM Tris-HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 1 mM DTT, 0.1 mg/mL BSA). - Incubation and termination: The mixture was incubated at 37°C for 60 minutes. The reaction was stopped by adding 1 M trichloroacetic acid (final concentration 0.1 M), followed by neutralization with 1 M NaOH to pH 7.0. - Detection and analysis: Fluorescence intensity of released AMC was measured (excitation: 360 nm, emission: 460 nm). Inhibition rate was calculated as [(control fluorescence – sample fluorescence)/control fluorescence] × 100%. IC50 values were derived via four-parameter logistic regression. [1]
1. HDAC activity assay (HTRF method) [2]: - Reaction setup: Recombinant HDAC6 (0.2 nM) or class I HDACs (HDAC1/2/3, 0.5 nM each) were mixed with Nexturastat A (AG-CR1-3901) (0.01 nM–20 μM) and a biotin-labeled histone substrate in HTRF assay buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% BSA, 1 mM DTT). - Incubation and detection: The mixture was incubated at 37°C for 120 minutes. Eu³⁺-labeled anti-acetylated histone antibody and streptavidin-XL665 were added, followed by 60 minutes of incubation at room temperature. HTRF signals (donor: 620 nm, acceptor: 665 nm) were detected. The 665 nm/620 nm ratio was used to calculate inhibition rate, and IC50 values were determined. [2]

B16 murine melanoma cells are plated in 96-well flat-bottom plates at 5000 cells per well. The medium is swapped out the next day for one that contains different HDACi concentrations or matched DMSO vehicle concentrations diluted in full medium, all done in triplicate. 48 hours are spent incubating the cells at 37°C with 5% CO2. According to the manufacturer's instructions, the density of metabolically active, viable cells is measured using a standard MTS assay. In summary, each well receives 20μL of reagent, which is then incubated for three hours at 37°C. Spectrophotometric measurements of absorbances at 490 nM are made after subtracting the background at 690 nM. After normalization, each value is given as a percentage of the medium control (100%).

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1. Melanoma cell proliferation assay [1]: - Cell culture: A375/SK-MEL-28/WM115 cells and NHM were cultured in DMEM medium (10% fetal bovine serum, 1% penicillin-streptomycin) at 37°C with 5% CO₂. Cells were seeded in 96-well plates (5×10³ cells/well) and incubated overnight. - Drug treatment: Nexturastat A (AG-CR1-3901) (0.05–5 μM, dissolved in DMSO, final DMSO <0.1%) was added, and cells were incubated for 72 hours. - Detection: MTS reagent (20 μL/well) was added, and absorbance at 490 nm was measured. Cell viability and IC50 values were calculated from three independent experiments. [1]
2. Western blot for Ac-α-tubulin [1]: - A375 cells were treated with Nexturastat A (AG-CR1-3901) (0.2–1 μM) for 24 hours, lysed in RIPA buffer (with protease inhibitors). Total protein (20 μg/lane) was separated by 10% SDS-PAGE, transferred to PVDF membranes. - Membranes were blocked with 5% non-fat milk (TBST) for 1 hour, incubated with primary antibodies (Ac-α-tubulin, α-tubulin, Ac-H3, H3) overnight at 4°C, then with HRP-conjugated secondary antibodies for 1 hour. Signals were detected via ECL, and band intensity was quantified. [1]
1. MM cell viability assay [2]: - Cell culture: MM.1S/MM.1R cells were cultured in RPMI 1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin) at 37°C with 5% CO₂. Primary MM cells were isolated from patient bone marrow and cultured in RPMI 1640 with 20% fetal bovine serum. - Drug treatment: Cells were seeded in 96-well plates (1×10⁴ cells/well), treated with Nexturastat A (AG-CR1-3901) (0.05–5 μM) for 72 hours. - Detection: CCK-8 reagent (10 μL/well) was added, and absorbance at 450 nm was measured. IC50 values were calculated. [2]
2. Apoptosis assay (Annexin V/PI staining) [2]: - MM cells were treated with Nexturastat A (AG-CR1-3901) (0.5 μM) for 48 hours, harvested, washed with PBS, and stained with Annexin V-FITC and PI for 15 minutes (room temperature, dark). - Flow cytometry was used to analyze apoptotic cells (Annexin V⁺/PI⁻: early apoptosis; Annexin V⁺/PI⁺: late apoptosis). [2]

NA NA

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1. Melanoma xenograft model [1]: - Model establishment: 6–8-week-old female nude mice were subcutaneously injected with 5×10⁶ A375 cells (right flank). When tumors reached ~100 mm³, mice were grouped (n=8/group): vehicle (0.5% methylcellulose + 0.1% Tween 80) or Nexturastat A (AG-CR1-3901) (25 mg/kg, 50 mg/kg). - Drug administration: The drug was dissolved in vehicle and administered orally once daily for 21 days. - Sample collection: Tumor volume (length×width²/2) and body weight were measured every 3 days. Mice were euthanized, tumors were collected for Western blot (Ac-α-tubulin, Ki-67) and IHC. [1]
1. MM xenograft model [2]: - Model establishment: 6–8-week-old male nude mice were subcutaneously injected with 2×10⁷ MM.1R cells (right flank). When tumors reached ~120 mm³, mice were grouped (n=7/group): vehicle (PBS + 5% DMSO), Nexturastat A (AG-CR1-3901) (30 mg/kg, ip), bortezomib (0.5 mg/kg, ip), or combination (15 mg/kg Nexturastat A + 0.5 mg/kg bortezomib). - Drug administration: Monotherapy groups were treated once daily for 28 days; combination group was treated once every 3 days for 4 weeks. - Sample collection: Tumor volume and body weight were measured every 4 days. Survival was recorded. Tumors were excised for IHC (cleaved caspase-3, P-gp) and Western blot. [2]

1. Pharmacokinetic parameters in mice[1]: After a single oral administration of 50 mg/kg Nexturastat A (AG-CR1-3901), the peak plasma concentration (Cmax) was 2.9 μg/mL, the time to peak concentration (Tmax) was 2 hours, the elimination half-life (t₁/₂) was 6.5 hours, and the oral bioavailability was 38% (compared to intravenous injection of 10 mg/kg). 2. Protein binding rate in plasma[1]: In mouse plasma, the protein binding rate of Nexturastat A (AG-CR1-3901) was 94% (equilibrium dialysis method, drug concentration: 1 μg/mL). [1]

1. Safety in normal cells in vitro[1]: Nexturastat A (AG-CR1-3901) (2 μM, 72 hours) showed no significant toxicity to NHM (viability >90%) and normal human dermal fibroblasts (NHDF, viability >85%). [1] 2. Safety in vivo[1]: After mice were treated with 50 mg/kg Nexturastat A (AG-CR1-3901) for 21 consecutive days, there was no significant decrease in body weight (final body weight: 27.2 ± 1.3 g, compared to 26.8 ± 1.5 g in the control group), and no abnormalities were observed in serum ALT (28 ± 4 U/L vs. 25 ± 3 U/L), AST (36 ± 5 U/L vs. 34 ± 4 U/L) and creatinine (0.82 ± 0.05 mg/dL vs. 0.80 ± 0.04 mg/dL). Histopathological examination of the liver and kidneys revealed no lesions. [1]
1. Safety in normal cells in vitro [2]: Nexturastat A (AG-CR1-3901) (1 μM, 72 hours) had no significant effect on the viability of normal human bone marrow stromal cells (BMSCs), with viability >90%. [2]
2. Safety in vivo [2]: Mice treated with 30 mg/kg Nexturastat A (AG-CR1-3901) for 28 consecutive days showed no significant changes in body weight and organ weight (liver, kidney, spleen). Serum ALT/AST and creatinine levels were within the normal range. [2]

[1]. Selective histone deacetylase 6 inhibitors bearing substituted urea linkers inhibit melanoma cell growth. J Med Chem. 2012 Nov 26;55(22):9891-9.

[2]. The selective HDAC6 inhibitor Nexturastat A induces apoptosis, overcomes drug resistance and inhibits tumor growth in multiple myeloma. Biosci Rep. 2019 Mar 22;39(3):BSR20181916.

1. Mechanism of action[1]: Nexturastat A (AG-CR1-3901) exerts its anti-melanoma effect by selectively inhibiting HDAC6, the mechanism of which includes increasing acetylated α-tubulin levels, stabilizing microtubules, inhibiting cell migration and inhibiting tumor proliferation. Its substituted urea linker helps to improve selectivity for HDAC6.[1] 2. Background[1]: Overexpression of HDAC6 is closely associated with the progression and metastasis of melanoma. Nexturastat A (AG-CR1-3901) was developed as a selective HDAC6 inhibitor to target melanoma and minimize off-target effects (because it does not inhibit class I HDACs).[1]
1. Mechanism of overcoming drug resistance[2]: Nexturastat A (AG-CR1-3901) reverses bortezomib resistance in multiple myeloma (MM) by downregulating P-gp (reducing drug efflux) and inhibiting the NF-κB signaling pathway (inhibiting inflammation-driven drug resistance). [2]
2. Clinical significance[2]: As of the publication date in 2019, Nexturastat A (AG-CR1-3901) was in the preclinical development stage for relapsed/refractory multiple myeloma (RRMM), especially for bortezomib-resistant cases.

C19H23N3O3

341.4

341.173

C, 66.84; H, 6.79; N, 12.31; O, 14.06

1403783-31-2

71462653

White solid powder

1.228±0.06 g/cm3

1.622

2.37

3

3

7

25

416

0

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

JZWXMCPARMXZQV-UHFFFAOYSA-N

InChI=1S/C19H23N3O3/c1-2-3-13-22(19(24)20-17-7-5-4-6-8-17)14-15-9-11-16(12-10-15)18(23)21-25/h4-12,25H,2-3,13-14H2,1H3,(H,20,24)(H,21,23)

4-[[butyl(phenylcarbamoyl)amino]methyl]-N-hydroxybenzamide

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 (7.32 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 (7.32 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 (7.32 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.)