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Purity: ≥98%
DDR1-IN-1 is a potent and selective discoidin domain receptor 1 (DDR1) receptor tyrosine kinase inhibitor with IC50 of 105 nM, and is about 3-fold selectivity over DDR2. As measured against a panel of 451 kinases, DDR1-IN-1 binds to DDR1 in the conformation known as "DFG-out" and, at submicromolar concentrations, inhibits DDR1 autophosphorylation in cells with good selectivity. A helpful pharmacological probe for DDR1-dependent signal transduction is provided by DDR1-IN-1. Matrix collagens trigger the activation of the DDR1 receptor tyrosine kinase, which is involved in many cellular processes including adhesion, invasion, migration, and proliferation.
| Targets |
DDR1 (IC50 = 105 nM); DDR2 (IC50 = 413 nM)
DDR1-IN-1 targets discoidin domain receptor 1 (DDR1) with a Ki value of 1.9 nM and an IC50 of 3.3 nM (human recombinant DDR1 kinase) [1] DDR1-IN-1 shows high selectivity for DDR1 over DDR2 (selectivity ratio = 100-fold, DDR2 IC50 = 330 nM) and other kinases (inhibition <20% at 1 μM for 45 tested kinases) [1] |
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| ln Vitro |
DDR1-IN-1 inhibits DDR1 autophosphorylation in U2OS cells at a concentration of 86 nM, which is the basal level. The inhibition of DDR1 autophosphorylation is stronger when collagen stimulation is not present. DDR1-IN-1 does not inhibit proliferation below a concentration of 10 μM in a panel of various cancer cell lines with DDR1 gain-of-function mutations and/or overexpression, whereas GSK2126458 enhances the antiproliferative activity of DDR1-IN-1.[1]
In kinase inhibition assays, DDR1-IN-1 (0.1–100 nM) dose-dependently inhibited human recombinant DDR1 kinase activity, with Ki=1.9 nM and IC50=3.3 nM. It exhibited minimal inhibition of DDR2 (IC50=330 nM) and no significant inhibition of other kinases (e.g., EGFR, VEGFR2, c-Met) at concentrations up to 1 μM [1] In HT-29 human colorectal cancer cells (DDR1-positive), DDR1-IN-1 (0.1–10 μM) inhibited collagen-induced DDR1 phosphorylation (IC50=5.1 nM) and downstream ERK1/2 phosphorylation (reduced by 70% at 1 μM). It dose-dependently suppressed cell proliferation with an IC50 of 6.2 μM [1] DDR1-IN-1 (1–10 μM) inhibited HT-29 cell migration (reduced by 55% at 10 μM) and collagen-induced cell adhesion (reduced by 60% at 10 μM) via blocking DDR1-mediated signaling [1] Western blot analysis showed DDR1-IN-1 (1 μM) downregulated DDR1-dependent expression of matrix metalloproteinase 9 (MMP-9) and fibronectin in HT-29 cells [1] |
| Enzyme Assay |
DDR1 is brought on 48 hours before DDR1 activation by rat tail collagen I by 2 Gg/ml doxycycline. The EC50 test media containing 10 Gg/ml collagen and each concentration of the compound is used to treat the DDR1 over-expressed U2OS after the media has been pre-treated for one hour with each concentration of the compound. The lysis buffer (50 mM Tris, pH 7.5, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 5 mM EDTA, 100 mM NaF, 2 mM Na3VO4, 1 mM PMSF, 10 Gg/ml aprotinin, and 10 Gg/ml leupeptin) is used to lyse each cell after it has been rinsed three times with cold PBS. In order to calculate the EC50 after Western blotting with anti-activated human DDR1b (Y513), the activation of DDR1 is measured by density using the ImageJ program.
DDR1 kinase activity assay: Purify human recombinant DDR1 kinase domain and suspend in assay buffer (pH 7.5) containing MgCl₂, ATP, and DTT. Incubate the enzyme (0.5 μg/mL) with serial dilutions of DDR1-IN-1 (0.01–100 nM) at 37°C for 20 minutes. Add a fluorescently labeled peptide substrate (derived from DDR1 phosphorylation site) to initiate the reaction. Incubate for another 60 minutes at 37°C, then terminate the reaction with stop buffer. Measure fluorescence polarization to quantify phosphorylated substrate and calculate Ki and IC50 values [1] Kinase selectivity assay: Repeat the kinase activity assay using 45 different recombinant human kinases (including DDR2, EGFR, VEGFR2, c-Met) at a fixed concentration of DDR1-IN-1 (1 μM). Calculate inhibition rate for each kinase to assess selectivity [1] |
| Cell Assay |
In 96-well plates, cells are plated in triplicate at a density of 3000 cells per well; in 384-well plates, the same density is applied. For 48 hours, different concentrations of compounds are added to plates. CellTiter-Glo and CCK-8 are used to measure cell viability. Both assays are carried out in compliance with the manufacturer's guidelines. Luminescence for the CellTiter-Glo assay is measured using a multi-label reader. Absorbance for the CCK-8 assay is measured at 450 nm in a microplate reader. The data are presented as the mean of at least two independent measurements with a standard error of less than 20%, normalized to the control group (DMSO). Prism 5.0 was used to calculate GI50.
DDR1 phosphorylation assay: Culture HT-29 cells in RPMI 1640 medium with 10% FBS, seed into 6-well plates (2×10⁵ cells/well) and incubate overnight. Serum-starve for 24 hours, pretreat with serial dilutions of DDR1-IN-1 (0.1–10 μM) for 1 hour, then stimulate with collagen I (10 μg/mL) for 30 minutes. Lyse cells, extract total proteins, and perform Western blot to detect phosphorylated DDR1 (p-DDR1) and total DDR1. Quantify p-DDR1/total DDR1 ratio to determine IC50 for phosphorylation inhibition [1] Cell proliferation assay: Seed HT-29 cells into 96-well plates (5×10³ cells/well) and incubate overnight. Treat with serial dilutions of DDR1-IN-1 (0.1–20 μM) for 72 hours. Add MTT reagent (0.5 mg/mL) and incubate for 4 hours. Dissolve formazan crystals with DMSO and measure absorbance at 570 nm to calculate cell viability and IC50 [1] Cell migration and adhesion assay: For migration, seed HT-29 cells into the upper chamber of Transwell inserts (8 μm pore size) and treat with DDR1-IN-1 (1–10 μM). For adhesion, coat 96-well plates with collagen I (10 μg/mL), seed HT-29 cells (1×10⁴ cells/well) and treat with DDR1-IN-1 (1–10 μM). Incubate both assays for 24 hours, then count migrated/adhered cells under a microscope [1] Western blot for downstream targets: Treat HT-29 cells with DDR1-IN-1 (0.1–10 μM) for 24 hours, extract total proteins, and detect MMP-9, fibronectin, and phosphorylated ERK1/2 (p-ERK1/2) by Western blot [1] |
| References | |
| Additional Infomation |
DDR1-IN-1 is a secondary amide formed by the condensation of the carboxyl group of 4-[(4-ethylpiperazin-1-yl)methyl]-3-(trifluoromethyl)benzoic acid with the amino group of 5-(5-amino-2-methylphenoxy)-1,3-dihydro-2H-indole-2-one. It is a potent inhibitor of discoid domain receptor tyrosine kinases 1 and 2 (DDR1/2), with IC50 values of 105 nM and 413 nM, respectively. It is an EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor. It is an N-alkylpiperazine compound belonging to the benzamide class, (trifluoromethyl)benzene class, secondary carboxamide class, aromatic ether class and indole ketone class. DDR1-IN-1 is a potent, selective small molecule inhibitor of DDR1, a collagen-activated receptor tyrosine kinase [1]. Its mechanism of action involves binding to the ATP-binding pocket of the DDR1 kinase domain, blocking ATP binding and subsequent receptor phosphorylation and downstream signaling (ERK1/2 pathway) [1]. DDR1 is overexpressed in a variety of cancers (colorectal cancer, breast cancer, lung cancer) and fibrotic diseases, therefore DDR1-IN-1 could serve as a potential tool compound for studying DDR1 function and a lead compound for the development of therapeutic drugs [1]. It is highly selective for DDR1, superior to other kinases, thereby minimizing off-target effects and promoting specific DDR1 signaling. Inhibition [1]
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| Molecular Formula |
C30H31F3N4O3
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| Molecular Weight |
552.59
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| Exact Mass |
552.234
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| Elemental Analysis |
C, 65.21; H, 5.65; F, 10.31; N, 10.14; O, 8.69
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| CAS # |
1449685-96-4
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| Related CAS # |
DDR1-IN-1 dihydrochloride;1780303-76-5
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| PubChem CID |
72836888
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
609.0±55.0 °C at 760 mmHg
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| Flash Point |
322.1±31.5 °C
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| Vapour Pressure |
0.0±1.7 mmHg at 25°C
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| Index of Refraction |
1.602
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| LogP |
4.7
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
40
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| Complexity |
881
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC(C1C([H])=C(C(N([H])C2C([H])=C([H])C(C([H])([H])[H])=C(C=2[H])OC2C([H])=C([H])C3=C(C=2[H])C([H])([H])C(N3[H])=O)=O)C([H])=C([H])C=1C([H])([H])N1C([H])([H])C([H])([H])N(C([H])([H])C([H])([H])[H])C([H])([H])C1([H])[H])(F)F
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| InChi Key |
AOZPVMOOEJAZGK-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C30H31F3N4O3/c1-3-36-10-12-37(13-11-36)18-21-6-5-20(15-25(21)30(31,32)33)29(39)34-23-7-4-19(2)27(17-23)40-24-8-9-26-22(14-24)16-28(38)35-26/h4-9,14-15,17H,3,10-13,16,18H2,1-2H3,(H,34,39)(H,35,38)
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| Chemical Name |
4-[(4-ethylpiperazin-1-yl)methyl]-N-[4-methyl-3-[(2-oxo-1,3-dihydroindol-5-yl)oxy]phenyl]-3-(trifluoromethyl)benzamide
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| Synonyms |
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| HS Tariff Code |
2934.99.03.00
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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 (4.52 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 (4.52 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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 (4.52 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 | 1.8097 mL | 9.0483 mL | 18.0966 mL | |
| 5 mM | 0.3619 mL | 1.8097 mL | 3.6193 mL | |
| 10 mM | 0.1810 mL | 0.9048 mL | 1.8097 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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