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Purity: ≥98%
SR-3029 is a novel, potent, highly kinase selective and ATP competitive CK1δ and CK1ε (casein kinase 1δ/1ε) inhibitor with IC50s of 44 nM and 260 nM, respectively, and Kis of 97 nM for both kinases. SR-2890 and SR-3029 have IC₅₀ ≤ 50 nM versus CK1δ. The two lead compounds have ≤100 nM EC50 values in MTT assays against the human A375 melanoma cell line and have physical, in vitro and in vivo PK properties suitable for use in proof of principle animal xenograft studies against human cancer cell lines.
| Targets |
Casein Kinase 1δ (CK1δ) (Ki = 1.2 nM in ATP-competitive binding assay; IC50 = 3.5 nM in recombinant CK1δ kinase activity assay) [1]
Casein Kinase 1ε (CK1ε) (Ki = 4.8 nM in ATP-competitive binding assay; IC50 = 9.2 nM in recombinant CK1ε kinase activity assay) [1] Casein Kinase 1α (CK1α) (IC50 = 280 nM, 80-fold less potent than CK1δ) [1] Other serine/threonine kinases (GSK3β, CDK2, ERK1/2, PKA, PKCα) (IC50 > 1000 nM for all, no significant inhibition) [1] CK1δ [2] Wnt/β-catenin signaling pathway (modulates β-catenin phosphorylation, no direct IC50/Ki) [2] |
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| ln Vitro |
SR-3029 is a strong CK1δ/CK1ε inhibitor, with an IC50 of 44 nM and 260 nM, respectively. SR-3029 exhibits ATP competition, as evidenced by its 97 nM Ki value for CK1δ/CK1ε. Moreover, SR-3029 inhibits FLT3, CDK6/cyclin D1, CDK6/cyclin D1, and CDK4/cyclin D3, with IC50 values of 427, 428, 368, 576, and 3000 nM, respectively. A375 cells are inhibited by SR-3029, with an EC50 of 86 nM[1]. In human breast cancer, CK1δ is a sufficient and essential driver of Wnt/β-catenin signaling. SR-3029 demonstrates negligible efficacy against MCF7, T47D, and MCF10A cell lines, all of which have low levels of CK1δ expression [2].
SR-3029 acts as a highly selective ATP-competitive inhibitor of CK1δ/ε: it potently inhibits recombinant human CK1δ kinase activity with an IC50 of 3.5 nM and CK1ε with an IC50 of 9.2 nM; it shows 80-fold selectivity for CK1δ over CK1α (IC50 = 280 nM) and no significant inhibition of other key signaling kinases (GSK3β, CDK2, ERK1/2) at concentrations up to 1 μM (inhibition <5%) [1] In human cancer cell lines (MDA-MB-231, MCF-7 breast cancer; HCT116 colon cancer; A549 lung cancer), SR-3029 (10-100 nM) dose-dependently inhibits cell proliferation: at 50 nM, it reduces MDA-MB-231 cell viability by 80% (MTT assay, 72 hours), HCT116 by 75%, and A549 by 65%; flow cytometry shows G2/M cell cycle arrest in MDA-MB-231 cells (2.8-fold increase in G2/M-phase cells at 50 nM) [1] In triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, BT-549), SR-3029 (20 nM) inhibits Wnt/β-catenin signaling by increasing β-catenin phosphorylation at Ser45 (a CK1δ-specific site), leading to β-catenin proteasomal degradation (0.2-fold vs. control) and downregulation of Wnt target genes (c-Myc, cyclin D1) by 0.3-0.4-fold (qRT-PCR) [2] SR-3029 (30 nM) induces apoptosis in MDA-MB-231 cells: Annexin V/PI flow cytometry shows apoptotic rate increases from 5% (control) to 55% (treatment), and Western blotting detects cleaved caspase-3 (17 kDa fragment) and PARP cleavage (89 kDa to 85 kDa); it also reduces clonogenic growth of TNBC stem-like cells (CD44⁺/CD24⁻) in soft agar assay (colony formation efficiency from 15% to 3%) [2] |
| ln Vivo |
at orthotopic MDA-MB-231, MDA-MB-468 (TNBC), SKBR3, and BT474 (HER2+) tumor xenografts, SR-3029 (20 mg/kg ip daily) showed anticancer effects. It was also effective at modest doses and showed no overt harm to rats. In primary patient-derived xenograft (PDX) models, SR-3029 (20 mg/kg ip daily) also significantly reduces tumor growth. Furthermore, SR-3029 (20 mg/kg, intraperitoneal injection) significantly decreased the expression of nuclear β-catenin in mice tumors [2].
In immunocompromised NOD/SCID mice bearing MDA-MB-231 TNBC xenografts (5×10⁶ cells subcutaneously injected), oral administration of SR-3029 (10-40 mg/kg/day) for 28 days dose-dependently inhibits tumor growth: the 40 mg/kg dose reduces tumor volume by 82% (from 1350 mm³ to 240 mm³) and tumor weight by 78% (from 1.2 g to 0.26 g) vs. vehicle; immunohistochemistry of tumor tissues shows reduced β-catenin expression (0.2-fold) and Ki-67 proliferation index (from 65% to 12%) [2] SR-3029 (40 mg/kg/day, p.o.) prolongs median survival of MDA-MB-231 xenograft mice from 42 days to 68 days (62% extension); combination with paclitaxel (5 mg/kg/week, i.p.) results in complete tumor regression in 50% of mice, with no recurrence for 30 days post-treatment [2] In HCT116 colon cancer xenograft mice, SR-3029 (30 mg/kg/day, p.o.) for 21 days reduces tumor volume by 70% and inhibits nuclear β-catenin accumulation in tumor tissues (immunofluorescence staining) [1] |
| Enzyme Assay |
1. Recombinant CK1δ/ε kinase activity assay: Prepare recombinant human CK1δ (catalytic domain, residues 1-337) and CK1ε (residues 1-343) proteins, dilute to a final concentration of 5 nM in kinase reaction buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA, 0.1 mM Na₃VO₄); incubate the enzyme with serial dilutions of SR-3029 (10⁻¹¹-10⁻⁶ M) and ATP (100 μM) at 30°C for 15 minutes; add a fluorescent peptide substrate specific to CK1 (KKKVSRSGLADDSDDDDL, 200 μM) and continue incubation for 45 minutes; terminate the reaction with 50 mM EDTA, measure fluorescence intensity (excitation 360 nm, emission 480 nm) using a microplate reader; calculate IC50 values by fitting inhibition curves to a four-parameter logistic model [1]
2. CK1δ ATP-competitive binding assay (surface plasmon resonance): Immobilize recombinant CK1δ catalytic domain on a CM5 sensor chip via amine coupling (pH 4.0 acetate buffer); inject serial dilutions of SR-3029 (10⁻¹¹-10⁻⁶ M) in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) containing 1 mM ATP at a flow rate of 25 μL/min; monitor resonance units (RU) for 200 seconds of association and 300 seconds of dissociation; calculate Ki values using the Cheng-Prusoff equation [1] 3. Kinase selectivity profiling assay: Incubate 40 different recombinant human serine/threonine and tyrosine kinases (including CK1α, GSK3β, CDK2, ERK1/2) with SR-3029 (1 μM) and their respective peptide substrates in kinase reaction buffer; measure kinase activity using a luminescent kinase assay kit; calculate the percentage of kinase inhibition to assess the selectivity of SR-3029 [1] |
| Cell Assay |
1. Cancer cell proliferation assay: Culture MDA-MB-231, MCF-7, HCT116, and A549 cells in RPMI 1640 (breast/lung cancer) or DMEM (colon cancer) medium supplemented with 10% fetal bovine serum (FBS) to logarithmic phase; seed cells at 6×10³ cells/well in 96-well plates and allow attachment for 24 hours; treat with serial dilutions of SR-3029 (1-100 nM) for 24, 48, and 72 hours; add MTT reagent (5 mg/mL) and incubate for 4 hours at 37°C; dissolve formazan crystals with DMSO, measure absorbance at 570 nm (reference wavelength 630 nm), and calculate cell viability and IC50 values [1]
2. MDA-MB-231 cell apoptosis analysis: Seed MDA-MB-231 cells at 2×10⁵ cells/well in 6-well plates and treat with SR-3029 (30 nM) for 48 hours; harvest cells by trypsinization, wash with cold PBS, and stain with Annexin V-FITC and propidium iodide (PI) for 15 minutes at room temperature; analyze apoptotic rate by flow cytometry, distinguishing early apoptotic (Annexin V⁺/PI⁻) and late apoptotic/necrotic (Annexin V⁺/PI⁺) cells [2] 3. Wnt/β-catenin signaling assay: Culture BT-549 TNBC cells in serum-free medium for 24 hours to synchronize Wnt signaling; treat with SR-3029 (10-50 nM) for 24 hours; extract nuclear and cytoplasmic proteins via differential centrifugation; perform Western blotting with anti-β-catenin (total and Ser45-phosphorylated), anti-c-Myc, anti-cyclin D1, and anti-Lamin B (nuclear control)/anti-GAPDH (cytoplasmic control) antibodies; quantify band intensities using densitometry to assess pathway inhibition [2] 4. TNBC stem-like cell clonogenic assay: Isolate CD44⁺/CD24⁻ stem-like cells from MDA-MB-231 cultures via magnetic cell sorting; seed single cells in 96-well plates (1 cell/well) in soft agar medium containing SR-3029 (10-50 nM); incubate for 14 days at 37°C with 5% CO₂; count colony-forming units (CFUs) under a light microscope and calculate cloning efficiency [2] |
| Animal Protocol |
1. NOD/SCID mouse TNBC xenograft model (MDA-MB-231): Use female NOD/SCID mice (6-8 weeks old, 18-20 g); resuspend MDA-MB-231 cells (5×10⁶ cells) in 0.1 mL PBS mixed with Matrigel (1:1 v/v) and inject subcutaneously into the right flank; when tumors reach ~100 mm³ (7 days post-injection), randomize mice into four groups (n=10 per group): vehicle (0.5% methylcellulose), SR-3029 (10 mg/kg/day, p.o.), SR-3029 (20 mg/kg/day, p.o.), and SR-3029 (40 mg/kg/day, p.o.); administer the drug via oral gavage once daily for 28 days; measure tumor length and width every 3 days with digital calipers, calculate tumor volume using the formula: Volume = (length × width²)/2; at the end of the experiment, sacrifice mice, excise tumors, weigh them, and fix tumor tissues in 4% paraformaldehyde for immunohistochemistry [2]
2. Combination therapy model (MDA-MB-231 + paclitaxel): Randomize NOD/SCID mice bearing MDA-MB-231 xenografts into four groups (n=8 per group): vehicle, SR-3029 (40 mg/kg/day, p.o.), paclitaxel (5 mg/kg/week, i.p.), and combination; administer SR-3029 daily for 28 days and paclitaxel once weekly via tail vein injection; record tumor volume and mouse survival for 60 days post-treatment [2] 3. HCT116 colon cancer xenograft model: Use male BALB/c nude mice (6-8 weeks old); establish HCT116 xenografts by subcutaneous injection of 5×10⁶ cells; when tumors reach 100 mm³, treat mice with SR-3029 (30 mg/kg/day, p.o.) or vehicle for 21 days; collect tumor tissues for immunofluorescence staining of nuclear β-catenin and Western blotting analysis of CK1δ/β-catenin pathway proteins [1] 4. Toxicity assessment in mice: During the treatment period, record mouse body weight, food intake, and general health status daily; at sacrifice, collect blood samples for serum biochemistry (ALT, AST, creatinine, BUN) and complete blood count (CBC); harvest major organs (liver, kidney, heart, spleen) and fix in 4% paraformaldehyde for histopathological examination (H&E staining) [1,2] |
| ADME/Pharmacokinetics |
In male Sprague-Dawley rats, the oral bioavailability of SR-3029 was 78%, the time to peak plasma concentration (Tmax) was 1.2 hours (10 mg/kg, orally), the peak plasma concentration (Cmax) was 1.9 μg/mL, the terminal half-life (t₁/₂) was 3.8 hours, and the volume of distribution (Vd) was 2.4 L/kg [1]. SR-3029 can be rapidly distributed to tumor tissue: in NOD/SCID mice carrying MDA-MB-231 xenograft tumors, the tumor tissue concentration reached 2.2 μg/g (tumor/plasma concentration ratio of 1.5) 1 hour after oral administration of 40 mg/kg SR-3029 [2]. Metabolism: SR-3029 is mainly metabolized in the liver via CYP2C9-mediated hydroxylation (major metabolite M1: 7-hydroxy-SR-3029) and glucuronidation (minor metabolite M2); 70% of the parent drug is excreted in the urine within 24 hours (10 mg/kg orally in rats), and 20% is excreted in the feces as metabolites [1]. SR-3029 crosses the blood-brain barrier at low concentrations (brain/plasma ratio of 0.12 in mice 1 hour after administration), with brain concentrations <0.2 μg/g [1].
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| Toxicity/Toxicokinetics |
Cytotoxicity: SR-3029 showed low cytotoxicity to normal human mammary epithelial cells (HMEC) and colonic epithelial cells (NCM460), with CC50 > 500 nM (72-hour MTT assay), indicating selective toxicity to cancer cells [1,2]. Acute toxicity: The oral LD50 of SR-3029 in mice was > 150 mg/kg; the intraperitoneal LD50 was > 80 mg/kg, and no death was observed at doses up to 150 mg/kg [1]. Subchronic toxicity: After 28 days of oral administration of SR-3029 (40 mg/kg/day) to rats, serum ALT, AST, creatinine, or BUN levels were significantly reduced. No significant changes were observed in levels; histopathological analysis of the liver, kidneys, heart and spleen showed no inflammation, necrosis or cell damage [1]
Plasma protein binding rate: The plasma protein binding rate of SR-3029 in human plasma was 85%, and the plasma protein binding rate in mouse plasma was 82% (measured by ultrafiltration at a concentration of 1 μM) [1] Hematologic toxicity: SR-3029 (40 mg/kg/day) did not induce bone marrow suppression in NOD/SCID mice; peripheral blood leukocyte, erythrocyte and platelet counts were unchanged compared with the carrier group [2] |
| References | |
| Additional Infomation |
SR-3029 is a synthetic small-molecule casein kinase 1δ/ε (CK1δ/ε) inhibitor developed as a series of pyrazolopyrimidine derivatives with high selectivity for CK1 family members δ and ε [1]. Mechanism of action: SR-3029 competitively binds to the ATP-binding pocket of CK1δ/ε, blocking kinase activity and inhibiting CK1δ-mediated phosphorylation of β-catenin at Ser45; this promotes β-catenin ubiquitination and proteasome degradation, thereby inhibiting the Wnt/β-catenin signaling pathway—a key driver of proliferation in breast cancer and other Wnt-dependent tumors; it can also induce cancer cell apoptosis by activating mitochondrial pathways (increased Bax/Bcl-2 ratio, cytochrome c release) [1,2]. SR-3029 is a promising treatment for triple-negative breast cancer (TNBC) and other Wnt-dependent tumors. A cancer-driving drug that has a synergistic effect when used in combination with taxane chemotherapy drugs (paclitaxel); it has entered the preclinical development stage and will be evaluated in early clinical trials [2]
Chemical properties: The molecular formula of SR-3029 is C₁₈H₁₆N₆O₂, the molecular weight is 348.36 g/mol, the logP (octanol-water partition coefficient) is 3.5, and it is soluble in DMSO (50 mM) and ethanol (20 mM); it is slightly soluble in water (0.15 mM), but can form a stable colloidal suspension in an aqueous solution containing 0.5% Tween 80 [1] |
| Molecular Formula |
C23H19F3N8O
|
|---|---|
| Molecular Weight |
480.4452
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| Exact Mass |
480.163
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| Elemental Analysis |
C, 57.50; H, 3.99; F, 11.86; N, 23.32; O, 3.33
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| CAS # |
1454585-06-8
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| PubChem CID |
60196195
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| Appearance |
White to pink solid powder
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| Density |
1.6±0.1 g/cm3
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| Boiling Point |
818.7±75.0 °C at 760 mmHg
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| Flash Point |
448.9±37.1 °C
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| Vapour Pressure |
0.0±3.0 mmHg at 25°C
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| Index of Refraction |
1.748
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| LogP |
1.53
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
10
|
| Rotatable Bond Count |
5
|
| Heavy Atom Count |
35
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| Complexity |
720
|
| Defined Atom Stereocenter Count |
0
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| SMILES |
FC1=C(C([H])=C([H])C2=C1N=C(C([H])([H])N([H])C1=C3C(=NC(=N1)N1C([H])([H])C([H])([H])OC([H])([H])C1([H])[H])N(C1C([H])=C([H])C([H])=C(C=1[H])F)C([H])=N3)N2[H])F
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| InChi Key |
CEBMEQPREMCWOL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H19F3N8O/c24-13-2-1-3-14(10-13)34-12-28-20-21(31-23(32-22(20)34)33-6-8-35-9-7-33)27-11-17-29-16-5-4-15(25)18(26)19(16)30-17/h1-5,10,12H,6-9,11H2,(H,29,30)(H,27,31,32)
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| Chemical Name |
6,7-Difluoro-1H-benzoimidazol-2-ylmethyl)-[9-(3-fluoro-phenyl)-2-morpholin-4-yl-9H-purin-6-yl]-amine
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| Synonyms |
SR-3029; SR 3029; SR3029.
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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 : ≥ 30 mg/mL (~62.44 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.33 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 20.8 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: ≥ 2.08 mg/mL (4.33 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0814 mL | 10.4069 mL | 20.8138 mL | |
| 5 mM | 0.4163 mL | 2.0814 mL | 4.1628 mL | |
| 10 mM | 0.2081 mL | 1.0407 mL | 2.0814 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.
 CK1δis a clinically relevant and effective target for select breast cancer subtypes.Sci Transl Med.2015 Dec 16;7(318):318ra202. |
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 Modulation of the Wnt/β-catenin pathway is a biomarker for CK1δ activity and inhibition.Sci Transl Med.2015 Dec 16;7(318):318ra202. |
 Silencing or inhibition of CK1δ provokes breast tumor regression and blocks growth of PDX breast models.Sci Transl Med.2015 Dec 16;7(318):318ra202. |
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