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Purity: ≥98%
PD: BDP9066 (BDP-9066) is a novel, potent and selective MRCK (myotonic dystrophy-related Cdc42-binding kinase) inhibitor with anticancer activities. It exhibits IC50 of 64 nM for MRCKβ in SCC12 cells, Ki values of 0.0136 nM and 0.0233 nM for MRCKα/β in house determinations, respectively. It acts by reducing substrate phosphorylation. BDP9066 leads to morphological changes in cancer cells along with inhibition of their motility and invasive character. It has therapeutic effect on skin cancer by reducing substrate phosphorylation.
| Targets |
MRCKα and MRCKβ (myotonic dystrophy-related Cdc42-binding kinases).
In vitro enzyme assays showed BDP9066 inhibited MRCKα and MRCKβ with Kᵢ values of 0.34 nM and 0.78 nM, respectively, under in-house optimized conditions. Against ROCK1 and ROCK2, Kᵢ values were >1277 nM and >2553 nM, respectively. [1] In a competitive binding assay against DMPK, BDP9066 had a Kd of 0.98 nM. [1] |
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| ln Vitro |
BDP9066 has antiproliferative properties, with blood cancer cells exhibiting the highest level of activity. BDP9066 prevents SCC12 squamous cell carcinoma motility and invasion by inhibiting MLC phosphorylation [1].
BDP9066 dose-dependently inhibited MLC2 phosphorylation in SCC12 cells with an EC₅₀ of 64 nM. [1] In SCC12 cells, BDP9066 treatment (0.5 µM for 2 hours) induced morphological changes, including increased cell length, width, and area, and decreased cell roundness, as determined by high content analysis. [1] BDP9066 (1 µM for ~18 hours) reduced the bundling of filamentous actin at cortical and cytoplasmic regions in SCC12 cells. [1] Random migration of SCC12 cells was significantly reduced by BDP9066 (0.4 µM), with mean cell velocity and accumulated distance reduced by 21%, and mean Euclidean distance reduced by 18% over 6 hours. [1] In organotypic invasion assays, BDP9066 (0.4 µM) significantly decreased the percentage of invading SCC12 cells by 50% compared to DMSO control. [1] Treatment of HEK293 cells expressing FLAG-tagged MRCKα with 1 µM BDP9066 for 2 hours blocked pS1003 immunoreactivity. [1] In an in vitro kinase assay with dephosphorylated MRCKα, BDP9066 blocked both MRCKα autophosphorylation (pS1003) and MLC2 phosphorylation. [1] BDP9066 showed anti-proliferative effects across a panel of 757 human cancer cell lines, with hematological cancers being the most sensitive. [1] |
| ln Vivo |
Topical treatment of BDP9066 significantly lowers phosphorylated MRCKα S1003 staining and tumor volume [1].
Topical application of BDP9066 (4 x 25 µg over 2 days) to mouse dorsal skin led to a significant reduction in epidermal MRCKα pS1003 positive staining. [1] In a DMBA/TPA two-stage chemical carcinogenesis mouse model of squamous cell carcinoma (SCC), topical treatment with BDP9066 (25 µg, 5 times per week for 14 weeks) significantly reduced total tumor volume and average papilloma volume per mouse compared to the DMSO vehicle control group. [1] In the same DMBA/TPA model, BDP9066 treatment was associated with significant decreases in MRCKα pS1003 immunohistochemistry staining in both treated skin and papillomas. [1] |
| Enzyme Assay |
MRCKα and MRCKβ kinase assays were performed using recombinant kinase proteins incubated with a FAM-labeled peptide substrate and ATP. After incubation, a binding reagent was added to bind the phosphorylated peptide, and fluorescence polarization was measured. Inhibition was calculated using no inhibitor (0%) or no enzyme (100%) controls. [1]
Kinase selectivity profiling was performed by an outsourced supplier using a FRET-based assay. Kinases were incubated with peptide substrate and ATP in the presence of indicated concentrations of BDP9066. [1] A competitive LanthaScreen Eu time-resolved FRET (TR-FRET) kinase binding assay was used to test BDP9066 for displacement of a fluorescent tracer binding to the DMPK ATP-binding site. The Kd for BDP9066 was calculated using the Cheng-Prusoff equation. [1] |
| Cell Assay |
SCC12 human squamous cell carcinoma cells were cultured and treated with a dose range of BDP9066 or DMSO vehicle. For western blot analysis, cells were lysed after treatment, and lysates were analyzed by SDS-PAGE. [1]
For cell viability assays, SCC12 cells were plated, treated with a dose range of BDP9066 for 24 hours, and cell viability was measured using a luminescent cell viability assay. [1] For morphology analysis, SCC12 cells were treated with BDP9066 or DMSO for 2 hours, fixed, permeabilized, and stained with phalloidin, DAPI, and a whole-cell stain. Cells were imaged and analyzed using a high content imaging system. [1] For immunofluorescence, SCC12 cells on coverslips were treated with 1 µM BDP9066 or DMSO for 1 hour, then fixed, permeabilized, and F-actin was visualized with fluorescent phalloidin. Images were taken on a confocal microscope. [1] For random migration assays, SCC12 cells were treated with 0.4 µM BDP9066 or DMSO for 1 hour, then placed in a live-cell imaging apparatus to image every 15 minutes for 6 hours. Cell tracks were analyzed using image analysis software. [1] For organotypic invasion assays, SCC12 cells were cultured on a 3D rat tail collagen matrix conditioned by cancer-associated fibroblasts. Cells were treated with 0.4 µM BDP9066 or DMSO, and the percentage of invading cells was measured. [1] HEK293 cells transfected with FLAG-MRCKα were treated with 1 µM BDP9066 for 2 hours, then lysed and analyzed by western blot for pS1003. [1] For in vitro kinase assays, immunoprecipitated MRCKα was incubated with ATP and recombinant MLC2 in kinase buffer with or without BDP9066. Reactions were stopped by adding boiling SDS, and samples were analyzed by western blot. [1] |
| Animal Protocol |
For pharmacokinetic and pharmacodynamic studies, FVB mice were treated topically with BDP9066 (25 µg in 50 µL 80% DMSO) or vehicle (50 µL 80% DMSO) four times over 2 days. Mice were culled 2 hours after the final treatment, and skin and blood were collected for concentration measurement and immunohistochemistry. [1]
To study dosing regimens, mice were treated topically once with 10 µg or 25 µg, or four times daily with 25 µg, or eight times daily (4 days on, 2 days off, 4 days on) with 25 µg BDP9066 in 80% DMSO. Mice were culled at various time points (2, 4, 8, 24 hours) after the final treatment. [1] For the DMBA/TPA skin cancer model, FVB mice were treated topically with 25 µg DMBA on day 1. From day 5, 4.7 µg TPA was applied three times weekly. Also from day 5, mice were treated with 25 µg BDP9066 in 50 µL 80% DMSO or vehicle five times per week for 14 weeks. Mice weights and general conditions were monitored, and tumor sizes and numbers were recorded weekly. Experimenters were blinded to treatment groups. Mice were culled when papillomas reached 12 mm in diameter. [1] |
| ADME/Pharmacokinetics |
Topical application of 25 µg BDP9066 (4 times over 2 days) on mouse skin resulted in a mean concentration of 26 µM in the skin and 0.04 µM in the blood. [1]
After a single 10 µg dose, skin and blood concentrations were measurable. Repeated 25 µg doses (4 times over 2 days) resulted in 2.8-fold higher skin concentrations and 4-fold higher blood concentrations than the single 10 µg dose, though this was less than the 10-fold difference in total dose. [1] Following a single 25 µg dose or 8 doses (4 days on, 2 days off, 4 days on) of 25 µg, >16 µM BDP9066 was detected in the skin 24 hours after the final dose, while blood levels were undetectable at 24 hours. [1] In the DMBA/TPA efficacy study at endpoint, topical BDP9066 application resulted in undetectable compound in blood and a mean concentration >1 µM in the skin. [1] |
| Toxicity/Toxicokinetics |
BDP9066 was relatively non-toxic in SCC12 cells at concentrations that profoundly inhibited substrate phosphorylation. SCC12 cell viability was unaffected by concentrations up to 0.5 µM after 24 hours of treatment, with only a 25% decrease in viability observed at 1 µM. [1]
In the DMBA/TPA mouse model, topical application of BDP9066 (25 µg, 5 times per week for 14 weeks) did not result in observable systemic toxicity, as blood compound concentrations were undetectable at the experimental endpoint, and mouse weights and general conditions were monitored. [1] |
| References | |
| Additional Infomation |
BDP9066 is a potent and selective MRCK inhibitor discovered through structure-guided fragment elaboration from a 7-azaindole-3-carbonitrile hit. [1]
The compound inhibits MRCK by binding to the ATP-binding site, forming hydrogen bonds with the hinge region (Asp154 and Tyr156) and interacting with pocket waters. The spiro moiety provides a 'plug' for the ATP-binding site opening. [1] Inhibition of MRCK by BDP9066 leads to reduced MLC2 phosphorylation, changes in cell morphology, and reduced cell motility and invasion without significant effects on cell viability at lower concentrations. [1] The study provides pre-clinical proof-of-concept that MRCK inhibition with BDP9066 is a valid therapeutic strategy for skin cancer. [1] |
| Molecular Formula |
C20H24N6
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|---|---|
| Molecular Weight |
348.444763183594
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| Exact Mass |
348.21
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| Elemental Analysis |
C, 68.94; H, 6.94; N, 24.12
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| CAS # |
2226507-04-4
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| Related CAS # |
(R)-BDP9066;2284549-25-1
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| PubChem CID |
132275018
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| Appearance |
White to light brown solid powder
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| LogP |
2.2
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
26
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| Complexity |
487
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| Defined Atom Stereocenter Count |
1
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| SMILES |
N1CCCC[C@@]21CN(C1C=CN=C3C=1C(C1C=CN=CN=1)=CN3)CCC2
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| InChi Key |
UELSMLDRSQFVHG-FQEVSTJZSA-N
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| InChi Code |
InChI=1S/C20H24N6/c1-2-8-25-20(6-1)7-3-11-26(13-20)17-5-10-22-19-18(17)15(12-23-19)16-4-9-21-14-24-16/h4-5,9-10,12,14,25H,1-3,6-8,11,13H2,(H,22,23)/t20-/m0/s1
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| Chemical Name |
(6S)-8-(3-pyrimidin-4-yl-1H-pyrrolo[2,3-b]pyridin-4-yl)-1,8-diazaspiro[5.5]undecane
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| Synonyms |
BDP9066; BDP-9066; BDP 9066
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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) |
DMSO : ~10 mg/mL (~28.70 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1 mg/mL (2.87 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 1 mg/mL (2.87 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 10.0 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8699 mL | 14.3497 mL | 28.6993 mL | |
| 5 mM | 0.5740 mL | 2.8699 mL | 5.7399 mL | |
| 10 mM | 0.2870 mL | 1.4350 mL | 2.8699 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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