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BRD6688 (BRD-6688; BRD 6688) is a novel and potent HDAC1/2/3 inhibitor with IC50s of 21 nM, 100 nM, and 11.48 μM, respectively.
| Targets |
HDAC2
Histone deacetylase 2 (HDAC2); HDAC1; HDAC3. IC50 values: HDAC1 = 0.001 ± 0.001 μM, HDAC2 = 0.011 ± 0.003 μM, HDAC3 = 0.544 ± 0.205 μM (measured using human recombinant enzymes). [1] |
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| ln Vitro |
BRD6688 demonstrated preferential binding kinetics with extended half-life on HDAC2 compared to HDAC1. The residence time (T1/2) for HDAC2 was 381 minutes, while for HDAC1 it was 65 minutes, representing a 6-fold kinetic selectivity. [1]
In primary mouse forebrain neuronal cultures, treatment with BRD6688 at 10 μM for 24 hours produced significant increases in histone acetylation at H4K12 and H3K9, confirming inhibitory activity against endogenous HDACs. [1] |
| ln Vivo |
In CK-p25 mice (a model of neurodegeneration with memory deficits), daily intraperitoneal administration of BRD6688 at 1 mg/kg for 10 days rescued memory defects in a contextual fear conditioning behavioral paradigm, restoring freezing response to normal levels compared to vehicle-treated non-induced p25 littermates. [1]
In hippocampal CA1 neurons of CK-p25 mice treated with BRD6688 (1 mg/kg, i.p., daily for 10 days), increased acetylation of H4K12 was observed compared to vehicle-treated group (paired t-test significant). [1] |
| Enzyme Assay |
HDAC inhibition assays were performed using human recombinant HDACs. Compounds were tested in duplicate in a 12-point dose-response curve with 3-fold serial dilution starting from 33.33 μM. Purified HDACs were incubated with 2 μM carboxyfluorescein-labeled acetylated or trifluoroacetylated peptide substrate (Broad Substrate A and B respectively) and test compound for 60 minutes at room temperature in HDAC assay buffer containing 50 mM HEPES (pH 7.4), 100 mM KCl, 0.01% BSA, and 0.001% Tween-20. Reactions were terminated by addition of a pan-HDAC inhibitor at a final concentration of 1.5 μM. Substrate and product were separated electrophoretically, and fluorescence intensity was determined. IC50 values were calculated using a 4-parameter logistic model. [1]
Binding kinetic measurements: Slow, tight-binding kinetics of BRD6688 with HDACs 1, 2, and 3 were evaluated by reaction progression curves and dilution experiments. A series of progress curves of HDAC inhibition were generated in the presence of the compound at different concentrations. Off-rates were determined from dilution experiments. [1] |
| Cell Assay |
Measurements of increases in neuronal histone acetylation in mouse forebrain primary neuronal cultures: On the 13th day after generating the cultures, cells were treated for 24 hours with BRD6688 at 10 μM. Cells were fixed with formaldehyde, stained with antibodies to acetyl-histone H3 lysine 9 (AcH3K9) or acetyl-histone H4 lysine 12 (AcH4K12), and green fluorescent secondary antibodies. Cellular fluorescence signals were quantitated with a microcytometer. The percentage of compound-treated cells with a fluorescence signal above a baseline threshold established in vehicle (DMSO)-treated cells was calculated to determine efficacy. [1]
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| Animal Protocol |
CK-p25 mouse model: Mice at 3 months of age, CK-p25 male mice were induced for 6 weeks to obtain forebrain-specific expression of p25. Littermates lacking p25 were used as controls. All mice were heterozygous for their respective genes. [1]
Compound administration: BRD6688 was dissolved in DMSO (5% of the total resultant solution) and then diluted in 30% Cremophor / 65% physiological saline (0.9% NaCl in water), for a final dosage solution of 1 mg/kg. Vehicle solution consisted of the same solution without the compound. Solutions were prepared immediately before injection and administered daily via intraperitoneal injection for a period of 10 days prior to behavioral testing. [1] Context-dependent fear conditioning: Training consisted of habituating mice to the conditioning box for 3 minutes, followed by a foot shock (2 seconds, 0.8 mA constant current). The shock was repeated 30 seconds later, and mice remained in the box for an additional 15 seconds. A long-term memory test was performed 24 hours later by re-exposing mice for 3 minutes to the conditioning context while measuring freezing behavior. [1] Immunohistochemistry: Coronal brain slices (40 μm thickness) were permeabilized, blocked, and incubated overnight with anti-AcH2K12 antibody, then visualized with a fluorescently conjugated secondary antibody. Neuronal nuclei were stained with Hoechst 33342. Images were acquired using a confocal microscope at identical settings. Using the Hoechst signal channel, 20-40 representative non-apoptotic cells were chosen per experimental condition, and mean AcH2K12 signal intensity was measured. Quantification was performed using ImageJ by an experimenter blind to treatment groups. [1] |
| ADME/Pharmacokinetics |
BRD6688 exhibited good brain penetration. The measured free fraction (fu) in brain was 0.54 (54%) based on tissue binding assays. [1]
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| Toxicity/Toxicokinetics |
BRD6688 showed low potential cardiac toxicity and high specificity against a broad panel of biological targets . [1]
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| References | |
| Additional Infomation |
BRD6688 is an ortho-aminoanilide-based, kinetically selective HDAC2 inhibitor. It incorporates an sp3-rich linker motif coupled with a 4-pyridyl 14 Å internal cavity motif. The compound demonstrates excellent thermodynamic selectivity for HDAC2 versus other Class I (>17-fold) and Class II (>500-fold) HDAC isoforms tested. [1]
Simulated target engagement profiles for BRD6688 at 10 mg/kg dose in brain showed three phases of kinetic selectivity: an initial phase (0-60 min) of good kinetic selectivity for HDAC1 (2.5-20-fold), an intermediate crossover stage with equivalent engagement of HDAC1 and HDAC2, followed by a terminal phase (t > 6 h) of high and sustained kinetic selectivity for HDAC2 (3-50-fold) while maintaining high kinetic selectivity against HDAC3 throughout. BRD6688 attains greater than 50% HDAC2 engagement for several hours. [1] BRD6688 was used to demonstrate for the first time that selective pharmacological inhibition of HDAC2 is feasible and that inhibition of its catalytic activity may serve as a therapeutic approach to enhance learning and memory processes affected in neurological and psychiatric disorders such as Alzheimer's disease, schizophrenia, and PTSD. [1] |
| Molecular Formula |
C16H18N4O
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|---|---|
| Molecular Weight |
282.34
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| Exact Mass |
282.148
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| Elemental Analysis |
C, 68.06; H, 6.43; N, 19.84; O, 5.67
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| CAS # |
1404562-17-9
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| Related CAS # |
1404562-17-9
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| PubChem CID |
66575862
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| Appearance |
Off-white to light yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
528.2±50.0 °C at 760 mmHg
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| Flash Point |
273.3±30.1 °C
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| Vapour Pressure |
0.0±1.4 mmHg at 25°C
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| Index of Refraction |
1.679
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| LogP |
0.84
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
21
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| Complexity |
350
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| Defined Atom Stereocenter Count |
0
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| SMILES |
N1(C(NC2=CC(C3C=CN=CC=3)=CC=C2N)=O)CCCC1
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| InChi Key |
YZXBMJVMSBSGMM-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H18N4O/c17-14-4-3-13(12-5-7-18-8-6-12)11-15(14)19-16(21)20-9-1-2-10-20/h3-8,11H,1-2,9-10,17H2,(H,19,21)
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| Chemical Name |
N-(2-amino-5-pyridin-4-ylphenyl)pyrrolidine-1-carboxamide
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| Synonyms |
BRD6688; BRD 6688; BRD-6688
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.5418 mL | 17.7091 mL | 35.4183 mL | |
| 5 mM | 0.7084 mL | 3.5418 mL | 7.0837 mL | |
| 10 mM | 0.3542 mL | 1.7709 mL | 3.5418 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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