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PKC-theta inhibitor is a novel, potent and selective PKC-θ inhibitor with an IC50 of 12 nM. It was discovered from an uHTS campaign to identify selective inhibitors of PKC-theta. Initial triaging of the hit set based on selectivity and historical analysis led to the identification of 2,4-diamino-5-nitropyrimidines as potent and selective PKC-theta inhibitors. A homology model and initial SAR is presented demonstrating that a 2-arylalkylamino substituent in conjunction with suitable 4-diamino substituent are essential for achieving selectivity over many kinases. Additional hit to lead profiling is presented on selected compounds.
| Targets |
PKC-θ (protein kinase C theta)
Other kinases screened for selectivity (IC50 values not individually listed for all, but compound 3 showed very good selectivity against VEGFR1, LYN, IR, SYK; selectivity heat map in Figure 4 for 38 analogs with PKC-θ IC50 <0.5 μM against a panel of 13 kinases representing 494 dose responses). [1] |
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| ln Vitro |
Potent inhibition of PKC-θ enzymatic activity: IC50 values for representative compounds range from 0.005 μM (compound 24) to >10 μM (compound 53). 2-Position SAR showed preference for ortho-substituted benzyl derivatives (e.g., compound 14 o-OCH3 IC50=0.017 μM; compound 22 o-SCF3 IC50=0.006 μM; compound 23 o-SCH3 IC50=0.005 μM; compound 24 o-Br IC50=0.005 μM). 4-Position SAR demonstrated importance of a terminal amine with proper spacing; compound 59 (3-(H2NCH2)-cyclohexylmethyl) gave IC50=0.003 μM. 5-Nitro series was ~10-fold more potent than 5-trifluoromethyl series (e.g., compound 6 with 3-ClPh IC50=0.067 μM vs compound 7 IC50=0.64 μM). [1]
Cellular activity: Inhibition of IL-2 production in human CD4+ T cells activated by anti-CD3/anti-CD28 mAbs. IC50 values: compound 23 = 0.08 μM, compound 24 = 0.14 μM, compound 65 = 0.19 μM, compound 57 = 0.72 μM (Table 5). [1] Selectivity: Compounds were highly selective across a panel of 13 kinases (Figure 4). Extended linker analogs (46-48) and α-Ph analog 25 showed reduced selectivity. The high selectivity is rationalized by the tight requirement for a properly positioned amino group to interact with Asp508 and the 2-benzylamino substituent controlling specificity via interactions at the glycine-rich loop, specificity surface, and hinge regions. [1] |
| ln Vivo |
Phosphoramidon at 30 mg/kg did not block the pressor response to ET-1 (1 nmol/kg) in ganglion-blocked anesthetized rats [5].
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| Enzyme Assay |
PKC-θ enzymatic activity was measured in a two-step competition fluorescence polarization (FP) assay. In the kinase reaction step, 50 nM PKC-θ diluted in assay buffer (20 mM Hepes, pH 7.6, 0.1 mM CaCl2, 10 mM MgCl2, 0.01% Chaps, 200 μM TCEP, 100 μM sodium orthovanadate, and protease inhibitor cocktail) was pre-incubated with compound dilutions at room temperature for 10 minutes. The reaction was started with a mixture of peptide substrate (RFARKGSLRQKNV) and ATP (final concentrations: 1 μM peptide and 10 μM ATP). Plates were incubated for 60 minutes at room temperature. In the FP detection step, fluorescein-labeled phosphopeptide tracer and anti-phosphoserine antibody diluted in quench buffer were added and incubated for 90 minutes at room temperature. Fluorescence polarization was measured using appropriate filters (485 nm excitation, 530 nm emission, 505 nm dichroic). [1]
ATP competition was confirmed (data not shown): Compound 3 showed IC50 = 0.29 μM at 50 μM ATP and IC50 = 0.63 μM at 100 μM ATP. [1] |
| Cell Assay |
Human CD4+ T cells were isolated from whole blood by positive selection. Purified T cells were activated through the TCR and CD28 via anti-CD3 and anti-CD28 monoclonal antibodies. Compounds were diluted in 5% DMSO to a final concentration of 0.125% DMSO at every dose. 50,000 cells were added in 100 μL of media per well, followed by 100 μL of compound or DMSO alone. Cells were incubated overnight at 37 °C, then supernatants were analyzed for IL-2 using an ELISA kit following a 1:10 dilution. [1]
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| ADME/Pharmacokinetics |
CYP inhibition IC50 values (Table 5): For compound 23: CYP2C9 = 1.0 μM, CYP2D6 = 4.3 μM, CYP3A4 = 0.71 μM; compound 24: 2C9 = 1.4 μM, 2D6 = 4.7 μM, 3A4 = 6.9 μM; compound 65: 2C9 = 8.4 μM, 2D6 = 1.7 μM, 3A4 = 1.7 μM; compound 57: 2C9 = 17.6 μM, 2D6 = 26 μM, 3A4 = >30 μM. [1]
Human liver microsome (HLM) stability half-life (Table 5): Compound 23 = 4.3 min, compound 24 = 4.7 min, compound 65 = 1.7 min, compound 57 = 26 min. [1] Caco-2 permeability (AB/BA, 10-6 cm/s, Table 5): Compound 23 = 0.71/1.44, compound 24 = 6.9/>30, compound 65 = 1.7/13, compound 57 = 26/>30. [1] Compounds were shown to be ATP competitive (data not shown). [1] |
| Toxicity/Toxicokinetics |
The compounds contain a nitro group, which is a potential structural alert; however, the overall profile was acceptable for further advancement in the hit-to-lead process. No specific toxicity data provided. [1]
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| References | |
| Additional Infomation |
PKC-θ is a member of the novel PKC subfamily (nPKCδ, ε, η, θ) requiring DAG and PS but calcium independent. Upon T cell activation, PKC-θ translocates upon antigenic stimulation of TCR. The knockout phenotype indicates that a PKC-θ inhibitor would be an immunosuppressive agent without altering T cell development. Autoimmune diseases such as psoriasis and transplant complications are characterized by pathogenic responses from inappropriate T cell activation. Present treatments include cyclosporine, FK506, and steroids, which have side effects; thus an unmet medical need exists for an immunosuppressive drug with a safer side-effect profile. [1]
The uHTS screen identified two classes: diaminopyrimidines and indolinones. The pyrimidine class (highlighted in green tabs in Figure 2) showed a trend toward selectivity, while indolinones had no exploitable SAR for selectivity. The pyrimidine class was therefore advanced. [1] General synthetic method (Scheme 1): 2,4-dichloro-5-nitropyrimidine was treated with potassium thiocyanate in AcOH to react selectively at the 4-position. Displacement of the 2-chloro with arylalkylamines proceeded smoothly, followed by displacement of the thiocyanate with an appropriate amine. Excess diamines or mono-protection/deprotection sequences were used to avoid di-addition. [1] |
| Molecular Formula |
C20H25F3N6O3
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| Molecular Weight |
454.45
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| Exact Mass |
454.194
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| CAS # |
736048-65-0
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| Related CAS # |
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| PubChem CID |
10253430
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| Appearance |
White to off-white solid powder
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| LogP |
4.7
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
32
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| Complexity |
586
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1CC(CN)CCC1CNC1=NC(NCC2=C(C=CC=C2)OC(F)(F)F)=NC=C1[N+](=O)[O-]
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| InChi Key |
HKOWATVSFKRXRW-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H25F3N6O3/c21-20(22,23)32-17-4-2-1-3-15(17)11-26-19-27-12-16(29(30)31)18(28-19)25-10-14-7-5-13(9-24)6-8-14/h1-4,12-14H,5-11,24H2,(H2,25,26,27,28)
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| Chemical Name |
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.50 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 (5.50 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 (5.50 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2005 mL | 11.0023 mL | 22.0046 mL | |
| 5 mM | 0.4401 mL | 2.2005 mL | 4.4009 mL | |
| 10 mM | 0.2200 mL | 1.1002 mL | 2.2005 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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