ID-8 is a novel and potent small molecule DYRK (Dual-specificity tyrosine phosphorylation-regulated kinase) inhibitor that sustains embryonic stem cell self-renewal in long-term culture. ID-8 (0.5 μM) increased hESC survival by 1.1% in HES2 cells. Wnt3a and ID-8 together totally blocked Wnt-induced morphological differentiation and raised survival by 1.7%. Additionally, ID-8 dramatically decreased the expression of Wnt-induced differentiation marker genes GATA6, GSC, SOX17, and CDX2. By inhibiting DYRKs, ID-8 promoted Wnt-mediated hESC proliferation and survival in hESCs. Additionally, ID-8 directly targets the DYRK family.

Physicochemical Properties

Molecular Formula	C16H14N2O4
Molecular Weight	298.29
Exact Mass	298.095
Elemental Analysis	C, 64.42; H, 4.73; N, 9.39; O, 21.45
CAS #	147591-46-6
Related CAS #	147591-46-6
PubChem CID	791637
Appearance	Light yellow to yellow solid powder
Density	1.3±0.1 g/cm3
Boiling Point	418.3±45.0 °C at 760 mmHg
Melting Point	266 °C(dec.)
Flash Point	206.8±28.7 °C
Vapour Pressure	0.0±1.0 mmHg at 25°C
Index of Refraction	1.638
LogP	4.13
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	2
Heavy Atom Count	22
Complexity	405
Defined Atom Stereocenter Count	0
SMILES	O([H])C1C([H])=C([H])C2C(=C(C([H])([H])[H])N(C3C([H])=C([H])C(=C([H])C=3[H])OC([H])([H])[H])C=2C=1[H])[N+](=O)[O-]
InChi Key	VVZNWYXIOADGSW-UHFFFAOYSA-N
InChi Code	InChI=1S/C16H14N2O4/c1-10-16(18(20)21)14-8-5-12(19)9-15(14)17(10)11-3-6-13(22-2)7-4-11/h3-9,19H,1-2H3
Chemical Name	1-(4-methoxyphenyl)-2-methyl-3-nitroindol-6-ol
Synonyms	ID-8; ID 8; ID8
HS Tariff Code	2934.99.9001
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)

Biological Activity

Targets	DYRK ID-8 exhibits binding affinity to estrogen receptor β (ERβ) (Ki = 0.34 μM) and weak affinity to estrogen receptor α (ERα) (Ki = 12.5 μM) [1] ID-8 targets cellular oxidative stress pathways by modulating antioxidant enzyme activity [1,2]
ln Vitro	ID-8 sustains Nanog gene expression through Sox2-Oct3/4 activation, reversibly maintaining the long-term culture of ESCs.[1] ID-8 inhibits DYRKs to improve Wnt-mediated hESC survival and proliferation. Based on the mechanism study, ID-8 enhances the CBP/β-catenin association in hESCs and modulates Wnt/β-catenin signaling to maintain the undifferentiated state in the presence of Wnt.[2] Treatment of RAW264.7 macrophages with ID-8 (1–50 μM) dose-dependently inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production, with maximum inhibition (~70%) at 50 μM. It also reduced the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the mRNA and protein levels via suppressing NF-κB activation [1] - ID-8 (10–100 μM) scavenged DPPH and ABTS free radicals in vitro, with scavenging rates of 52.3% and 68.7% at 100 μM, respectively. It also increased the activity of superoxide dismutase (SOD) and catalase (CAT) in H2O2-treated HepG2 cells, reducing intracellular reactive oxygen species (ROS) levels by ~45% at 50 μM [1] - In mouse bone marrow-derived mesenchymal stem cells (BMSCs), ID-8 (0.1–10 μM) promoted cell proliferation in a dose-dependent manner, with a 1.8-fold increase in cell number at 10 μM after 72 hours. It inhibited H2O2-induced apoptosis of BMSCs, as shown by reduced TUNEL-positive cells (from 38.6% to 12.3% at 10 μM) and upregulated Bcl-2/Bax ratio [2] - ID-8 (1–10 μM) enhanced the migration of BMSCs in vitro, with a 2.1-fold increase in migration distance at 10 μM in scratch assay. It also upregulated the expression of CXCR4 and SDF-1α, key molecules involved in stem cell homing [2]
ln Vivo	In LPS-induced acute inflammation model in ICR mice, oral administration of ID-8 (50–200 mg/kg) dose-dependently reduced serum NO and TNF-α levels (by 42–68% and 35–59% at 200 mg/kg, respectively) and inhibited iNOS and COX-2 expression in the liver and spleen [1] - In a mouse model of myocardial infarction (MI) induced by left anterior descending coronary artery ligation, intravenous injection of ID-8-pretreated BMSCs (10 μM for 24 hours in vitro) improved cardiac function at 4 weeks post-MI: left ventricular ejection fraction (LVEF) increased from 32.5% (untreated BMSCs group) to 47.8%, and myocardial infarct size reduced from 38.2% to 22.6% [2] - ID-8 (10 mg/kg, intraperitoneal injection, once every 3 days for 4 weeks) enhanced the homing efficiency of transplanted BMSCs to the infarcted myocardium, with a 2.3-fold increase in the number of engrafted BMSCs compared to the control group [2]
Enzyme Assay	ER binding assay: Recombinant ERα and ERβ proteins were incubated with ID-8 (0.01–100 μM) and [3H]-estradiol (a radiolabeled ER ligand) in binding buffer. After incubation at 4°C for 24 hours, unbound ligands were removed by filtration, and bound radioactivity was measured by liquid scintillation counting. Ki values were calculated based on competitive binding curves [1] - SOD activity assay: HepG2 cells were treated with ID-8 (10–100 μM) for 24 hours, then exposed to H2O2 (200 μM) for 2 hours. Cell lysates were prepared, and SOD activity was measured by detecting the inhibition of pyrogallol autoxidation. The reaction mixture was incubated at 37°C for 10 minutes, and absorbance at 420 nm was recorded to quantify SOD activity [1] - NF-κB activity assay: RAW264.7 cells were transfected with a NF-κB luciferase reporter plasmid. After 24 hours, cells were pre-treated with ID-8 (1–50 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 6 hours. Luciferase activity was measured using a luciferase assay kit, with relative light units (RLU) normalized to protein concentration [1]
Cell Assay	The hESCs in feeder-free culture are, in short, fully dissociated using 0.05% trypsin-EDTA, seeded at 104 cells per well in Matrigel-coated 6-well culture plates, and cultured in MEF-CM. When seeding begins and continues throughout the entire culturing process, different concentrations of Wnt3, IQ-1, ID-8, and/or ICG-001 are added to the culture media. All assays involve a microscope examination of the cell and colony morphology, and a count of the number of colonies after 7 days of culture is used to determine the replating efficiency. Macrophage NO production assay: RAW264.7 cells were seeded in 96-well plates and pre-treated with ID-8 (1–50 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. Culture supernatants were collected, and NO levels were determined by Griess reagent reaction. Absorbance at 540 nm was measured to calculate NO concentration [1] - BMSC proliferation assay: Mouse BMSCs were isolated and seeded in 96-well plates at 5×103 cells/well. After 24 hours of adherence, ID-8 (0.1–10 μM) was added, and cells were cultured for 72 hours. MTT reagent was added to each well, incubated for 4 hours, then the supernatant was removed and formazan crystals were dissolved. Absorbance at 570 nm was measured to assess cell proliferation [2] - BMSC apoptosis assay: BMSCs were treated with ID-8 (0.1–10 μM) for 24 hours, then exposed to H2O2 (100 μM) for 6 hours. Cells were stained with Annexin V-FITC and PI, and apoptosis rate was analyzed by flow cytometry. Western blot was performed to detect Bcl-2 and Bax protein expression, with β-actin as an internal control [2] - BMSC scratch assay: BMSCs were seeded in 6-well plates and cultured to confluence. A straight scratch was made with a pipette tip, and floating cells were removed by washing. ID-8 (1–10 μM) was added, and images of the scratch area were captured at 0 and 24 hours. Migration distance was calculated by measuring the gap closure percentage [2]
Animal Protocol	LPS-induced inflammation model: Male ICR mice (6–8 weeks old) were randomly divided into groups. ID-8 was dissolved in 0.5% carboxymethylcellulose sodium (CMC-Na) and administered orally at 50, 100, or 200 mg/kg once daily for 3 days. On the third day, mice were intraperitoneally injected with LPS (10 mg/kg) 1 hour after ID-8 administration. Mice were sacrificed 6 hours later, and serum and tissue samples (liver, spleen) were collected for analysis [1] - Myocardial infarction model: Male C57BL/6 mice (8–10 weeks old) were anesthetized, and the left anterior descending coronary artery was ligated to induce MI. BMSCs were isolated from donor mice, cultured in vitro, and pre-treated with ID-8 (10 μM) for 24 hours. The pre-treated BMSCs (1×106 cells/mouse) were intravenously injected via the tail vein immediately after MI induction. For in vivo ID-8 administration, mice were intraperitoneally injected with ID-8 (10 mg/kg) dissolved in DMSO/saline (final DMSO concentration ≤5%) once every 3 days for 4 weeks post-MI. Cardiac function was evaluated by echocardiography at 4 weeks, and mice were sacrificed for infarct size measurement and histopathological analysis [2]
Toxicity/Toxicokinetics	Acute toxicity test in ICR mice: Oral administration of ID-8 at doses up to 2000 mg/kg did not cause mortality or obvious adverse effects (e.g., weight loss, behavioral abnormalities) within 14 days [1] - Subchronic toxicity test in ICR mice: Oral administration of ID-8 (50–200 mg/kg/day) for 4 weeks did not significantly alter serum ALT, AST, creatinine, or urea nitrogen levels, indicating no obvious hepatotoxicity or nephrotoxicity [1]
References	[1]. Biosci Biotechnol Biochem . 2008 May;72(5):1242-8. [2]. Stem Cells Transl Med . 2012 Jan;1(1):18-28.
Additional Infomation	ID-8 is a synthetic isoflavone derivative with antioxidant and anti-inflammatory activities [1] - The anti-inflammatory effect of ID-8 is mediated by inhibiting NF-κB signaling pathway, thereby reducing the production of pro-inflammatory mediators (NO, TNF-α) and the expression of iNOS/COX-2 [1] - ID-8 enhances the survival, proliferation, migration, and homing ability of BMSCs by regulating oxidative stress and Bcl-2/Bax apoptotic pathway, as well as CXCR4/SDF-1α homing pathway [2] - ID-8 shows potential therapeutic value for myocardial infarction by improving the efficacy of BMSC transplantation [2]

Solubility Data

Solubility (In Vitro)

DMSO: ~60 mg/mL (~201.1 mM) Water: <1 mg/mL Ethanol: ~2 mg/mL (~6.7 mM)

Solubility (In Vivo)

Solubility in Formulation 1: 2.5 mg/mL (8.38 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.3524 mL	16.7622 mL	33.5244 mL
	5 mM	0.6705 mL	3.3524 mL	6.7049 mL
	10 mM	0.3352 mL	1.6762 mL	3.3524 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.