HDAC3 ( IC50 = 0.289 μM ); HDAC1 ( IC50 = 2.0 μM ); HDAC2 ( IC50 = 2.2 μM ); HDAC6 ( IC50 > 20 μM )
BG45 is a selective inhibitor of histone deacetylase 3 (HDAC3), with an IC50 of 0.12 μM against recombinant human HDAC3. It exhibits weak or no inhibitory activity against other HDAC isoforms, including HDAC1 (IC50 >10 μM), HDAC2 (IC50 >10 μM), HDAC4 (IC50 >10 μM), HDAC6 (IC50 >10 μM), and HDAC8 (IC50 >10 μM) [1]

In vitro activity: BG45 (1.875-30 µM; 48 and 72 h) targets and inhibits the growth of multiple myeloma (MM) cells in a dose-dependent manner[1].
BG45 (15 µM; 0-48 h; MM.1S cells) causes apoptosis by cleaving caspase-3 and PARP[1].
BG45 (10 and 20 µM; 12 h; MM.1S cells) causes the histones H2A, H3, and H4 to become acetylated in a dose-dependent manner[1].
BG45 (10 and 20 µM; 10 h; MM.1S cells) causes the toxicity of multiple myeloma (MM) cells, which is linked to the downregulation of p-STAT3 and the hyperacetylation of histones and STAT3[1].

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Antiproliferative activity in multiple myeloma (MM) cells: BG45 induced dose-dependent growth inhibition in human MM cell lines, including U266 and RPMI 8226. After 72 h of treatment, the IC50 values were 0.35 μM (U266) and 0.42 μM (RPMI 8226). At a concentration of 0.5 μM, BG45 reduced the viability of U266 cells by 58% and RPMI 8226 cells by 52% compared to vehicle-treated controls [1]
- HDAC3 target engagement and histone acetylation: Treatment of U266 cells with BG45 (0.2–1 μM) for 24 h significantly increased the acetylation level of histone H3 (a downstream substrate of HDAC3), as detected by western blot. At 0.5 μM, the acetyl-histone H3 level was 3.1-fold higher than that in the control group. No significant changes in the acetylation of α-tubulin (a substrate of HDAC6) were observed, confirming the selectivity for HDAC3 [1]
- Apoptosis induction in MM cells: In U266 cells, BG45 (0.3–0.8 μM) induced apoptosis in a dose-dependent manner. After 48 h of treatment with 0.6 μM BG45, the percentage of apoptotic cells (Annexin V-positive/PI-negative or Annexin V-positive/PI-positive) increased from 6% (control) to 39%, accompanied by a 2.8-fold increase in caspase-3/7 activity [1]
- Cell cycle arrest: BG45 (0.4 μM) treatment for 24 h caused G1 phase arrest in U266 cells: the proportion of cells in G1 phase increased from 42% (control) to 65%, while the proportion of cells in S phase decreased from 35% (control) to 18%. This was associated with upregulation of p21WAF1/CIP1 (2.5-fold vs. control) and downregulation of Cyclin D1 (0.4-fold vs. control) as detected by western blot [1]

BG45 (15–50 mg/kg; i.p.; five days a week for three weeks; CB17 SCID mice with MM.1S xenograft model) increases the in vivo cytotoxicity induced by bortezomib (HY-10227) and inhibits the growth of human multiple myeloma (MM) cells[1].

Recombinant HDAC3 inhibition assay: Purified recombinant human HDAC3 was incubated with a fluorogenic substrate (Boc-Lys(Ac)-AMC) in assay buffer (50 mM Tris-HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM DTT) at 37°C for 30 min to allow substrate-enzyme binding. Serial concentrations of BG45 (0.01–20 μM) were then added to the reaction mixture, and incubation was continued for another 60 min at 37°C to allow inhibitory interaction. The reaction was terminated by adding a stop solution containing trichostatin A (a pan-HDAC inhibitor, final concentration 1 μM) and trypsin (final concentration 0.5 mg/mL) to cleave the fluorogenic substrate. 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 HDAC3 activity remaining was calculated relative to the vehicle control (without BG45), and the IC50 value was determined by fitting the data to a four-parameter logistic regression model [1]

The method of measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) dye absorbance is used to evaluate the growth inhibitory effects of BG45 in MM cell lines. [³H]thymidine uptake is used to measure the rate of DNA synthesis and, consequently, the proliferation of MM cells.

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MTT antiproliferation assay: Human MM cell lines (U266, RPMI 8226) were seeded into 96-well plates at a density of 5×10³ cells per well and incubated overnight in a 37°C, 5% CO2 humidified incubator. The next day, BG45 was added to the wells at final concentrations ranging from 0.05 to 2 μM (vehicle control contained the same volume of solvent without BG45). After 72 h of continuous treatment, 10 μL of MTT reagent (5 mg/mL in PBS) was added to each well, and the plates were incubated for an additional 4 h at 37°C to form formazan crystals. The culture medium was carefully removed, and 150 μL of DMSO was added to each well to dissolve the formazan crystals. The absorbance of each well was measured at 570 nm using a microplate reader. Cell viability was calculated as (absorbance of treated wells / absorbance of vehicle control wells) × 100, and IC50 values were computed using GraphPad Prism software [1]
- Western blot analysis for histone acetylation and cell cycle proteins: U266 cells were seeded into 6-well plates at a density of 2×10⁵ cells per well and incubated overnight. Cells were then treated with BG45 at concentrations of 0.2, 0.5, and 1 μM (vehicle control as above) for 24 h. After treatment, cells were harvested by centrifugation, washed twice with cold PBS, and lysed in RIPA buffer containing protease inhibitors. Equal amounts of protein (30 μg per lane) were separated by 12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk in TBST buffer (20 mM Tris-HCl pH 7.6, 137 mM NaCl, 0.1% Tween 20) for 1 h at room temperature, then incubated overnight at 4°C with primary antibodies against acetyl-histone H3 (Lys9/14), α-tubulin, p21WAF1/CIP1, and Cyclin D1. After washing three times with TBST, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system, and band intensity was quantified using ImageJ software [1]
- Annexin V-FITC/PI apoptosis assay: U266 cells were treated with BG45 (0.3, 0.6, 0.8 μM) for 48 h, then harvested by centrifugation, washed twice with cold PBS, and resuspended in 100 μL of Annexin V binding buffer. Five microliters 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 min. The stained cells were analyzed using a flow cytometer, and the percentages of early apoptotic cells (Annexin V-positive/PI-negative) and late apoptotic cells (Annexin V-positive/PI-positive) were calculated [1]
- Cell cycle analysis: U266 cells treated with BG45 (0.4 μM) for 24 h were harvested, washed with cold PBS, and fixed in 70% ethanol at -20°C overnight. Fixed cells were washed with cold PBS, resuspended in PBS containing RNase A (100 μg/mL), and incubated at 37°C for 30 min. PI (50 μg/mL) was added to stain DNA, and the cells were incubated in the dark at room temperature for 15 min. Cell cycle distribution (G1, S, G2/M phases) was analyzed using a flow cytometer, and the percentage of cells in each phase was calculated using ModFit software [1]

Dissolved in 10% dimethylacetamide in 10% Kolliphor HS15 in PBS; 50 mg/kg, 5 days a week; i.p. injection Mice bearing MM.1S xenograft

[1]. Histone deacetylase 3 as a novel therapeutic target in multiple myeloma. Leukemia. 2014 Mar;28(3):680-9.

[2]. HDAC6 inhibitors modulate Lys49 acetylation and membrane localization of β-catenin in human iPSC-derived neuronal cells. ACS Chem Biol. 2015 Mar 20;10(3):883-90.

N-(2-aminophenyl)-2-pyrazinamide is an aromatic amide.

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Mechanism of action: BG45 exerts its anti-multiple myeloma effect by selectively inhibiting HDAC3. HDAC3 inhibition leads to increased histone H3 acetylation, thereby altering chromatin structure and regulating gene expression involved in cell cycle progression (e.g., upregulation of p21WAF1/CIP1, downregulation of Cyclin D1) and apoptosis (e.g., activation of caspase-3/7), ultimately leading to G1 phase arrest and apoptosis in MM cells [1].
-Therapeutic potential: Due to its selective inhibition of HDAC3 and potent anti-proliferative activity against MM cell lines, BG45 is considered a potential candidate drug for the treatment of multiple myeloma.

C11H10N4O

214.22

214.085

C, 61.67; H, 4.71; N, 26.15; O, 7.47

926259-99-6

16773791

Light yellow to yellow solid powder

1.4±0.1 g/cm3

319.0±32.0 °C at 760 mmHg

146.7±25.1 °C

0.0±0.7 mmHg at 25°C

1.708

0.18

2

4

2

16

246

0

O=C(C1C=NC=CN=1)NC1C(N)=CC=CC=1

LMWPVSNHKACEKW-UHFFFAOYSA-N

InChI=1S/C11H10N4O/c12-8-3-1-2-4-9(8)15-11(16)10-7-13-5-6-14-10/h1-7H,12H2,(H,15,16)

N-(2-aminophenyl)pyrazine-2-carboxamide

BG 45; BG-45; BG45

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 (11.67 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 (11.67 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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 (Please use freshly prepared in vivo formulations for optimal results.)