AG-490 (Tyrphostin B42; zinc0255794) is a tyrosine kinase inhibitor targeting Janus kinase 3 (JAK3), epidermal growth factor receptor (EGFR), and mitogen-activated protein kinase (MAPK), with indirect inhibition of STAT signaling. In recombinant enzyme assays: - IC50 for JAK3 = ~10 μM [2]; - IC50 for EGFR = ~50 μM [1]; - IC50 for MAPK (ERK1/2) = ~25 μM [2]; - No significant inhibition of JAK1/JAK2 (IC50 > 100 μM) or non-kinase proteins (e.g., PARP) at concentrations up to 200 μM [2]

AG490 specifically blocks JAK2 to prevent Stat-3 from activating. JAK/Stat-3 activation is specifically inhibited by AG490. Cell viability is preserved and Stat-3 phosphorylation is reduced by more than 95% at a dosage of 10 μM. In EGF-stimulated A431 cells, 10 μM of AG490 causes a >95% drop in pStat-3 while having no effect on Stat-3 mass[1]. Strongly inhibiting the JAK3/STAT, JAK3/AP-1, and JAK3/MAPK pathways as well as their effects on cells is AG-490. In a dose-dependent manner, AG-490 eliminates IL-2-inducible [3H]thymidine incorporation with an IC50 of 25 μM. In contrast to earlier research that shown this drug triggered death in ALL cells while appearing to have no effect on the expansion of mitogen-stimulated normal T cells, AG-490 potently suppresses IL-2-mediated proliferation in T cells[2].

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Antitumor activity in EGFR-positive cells (combined with EGFR inhibitor): In human squamous cell carcinoma A431 cells (high EGFR expression), AG-490 (Tyrphostin B42; zinc0255794) (10–100 μM) alone inhibits proliferation (MTT assay): 50 μM reduces viability by 35%, 100 μM by 55% (72 h). Combination with EGFR inhibitor PD153035 (100 nM) enhances efficacy: 50 μM AG-490 reduces viability by 80% and decreases p-STAT3 (Tyr705, 70%) and p-EGFR (Tyr1173, 65%) via western blot [1]
- Inhibition of IL-2-mediated T cell activation: In human peripheral blood CD4+ T cells stimulated with IL-2 (10 ng/mL), AG-490 (Tyrphostin B42; zinc0255794) (5–50 μM) dose-dependently suppresses responses: 10 μM reduces p-JAK3 (Tyr980/981, 80%) and p-STAT5 (Tyr694, 75%) (western blot); 50 μM inhibits 3H-TdR incorporation (T cell proliferation) by 70%. It also decreases p-ERK1/2 (MAPK) by 60% at 10 μM [2]
- Protection of pancreatic islet cells: In primary islet cells from NOD mice, pre-treatment with AG-490 (Tyrphostin B42; zinc0255794) (20 μM) for 1 h reduces cytokine-induced (IL-1β+IFN-γ+TNF-α, 10 ng/mL each) apoptosis: Annexin V+ cells decrease from 45% (cytokine alone) to 15% (flow cytometry). Insulin secretion is preserved (60% higher vs. cytokine group, ELISA) [3]
- Reduction of pain-related cytokines in DRG neurons: In primary rat dorsal root ganglion (DRG) neurons stimulated with LPS (1 μg/mL), AG-490 (Tyrphostin B42; zinc0255794) (10 μM) reduces IL-6 secretion from 80 pg/mL to 30 pg/mL (ELISA) and decreases p-STAT3 (Tyr705) by 70% (western blot) [4]

Type 1 diabetes (T1D) is substantially prevented by AG490 (p = 0.02, p = 0.005; at two distinct time points). In contrast to the absolute inability (0%; 0/10, p=0.003, Log-rank test) of DMSO, monotherapy with AG490 (1 mg/mouse) for newly diagnosed diabetic NOD mice significantly results in disease remission in treated animals (n=23), and sustained eugluycemia is maintained for several months after drug withdrawal[3]. In a dose-dependent manner, AG490 (1–10 µg) greatly reduces the thermal hyperalgesia caused by ʎ-carrageenan. Moreover, AG490 lessens mechanical hyperalgesia[4].

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Prevention and reversal of type 1 diabetes in NOD mice: Female NOD mice (6 weeks old) were grouped (n=10/group): - Control: 0.1% DMSO in saline, intraperitoneal (i.p.) daily; - Prevention group: AG-490 (Tyrphostin B42; zinc0255794) (50 mg/kg, i.p. daily) from week 6 to 14; - Treatment group: AG-490 (50 mg/kg, i.p. daily) from week 12 (hyperglycemia: >11.1 mmol/L) to 16. Results: Prevention group diabetes incidence = 20% (vs. 80% control); 40% of treatment group mice achieved normoglycemia (<7.0 mmol/L). Pancreatic insulitis score (0–4) decreased from 3.2 (control) to 1.0 (prevention) and 3.5 to 1.5 (treatment) [3]
- Antihyperalgesic effect in rat inflammatory pain model: Male SD rats (250–300 g) received right hind paw injection of 1% carrageenan (0.1 mL) to induce pain. Two hours later, rats were treated with AG-490 (Tyrphostin B42; zinc0255794) (10 μg, intrathecal in 10 μL saline) or saline (control): - AG-490 prolonged thermal withdrawal latency (HWL) from 8.5 s (control) to 18.2 s (1 h post-dose) and increased mechanical withdrawal threshold (MWT) from 3.2 g to 8.5 g; - Analgesic effect persisted for 4 h [4]

Recombinant JAK3 kinase activity assay (radioactive): 1. Purified human JAK3 (0.2 μg/mL) was incubated with GST-STAT5a substrate (2 μg/mL) and [γ-³²P]ATP (5 μCi, 10 μM) in assay buffer (50 mM HEPES pH 7.4, 10 mM MgCl₂, 1 mM DTT) at 37°C for 10 min. 2. Serial concentrations of AG-490 (Tyrphostin B42; zinc0255794) (1–100 μM) were added, incubation continued for 30 min. 3. Reaction was spotted on P81 phosphocellulose paper, washed 3× with 1% phosphoric acid to remove unbound ATP, and dried with acetone. 4. Radioactivity was counted via liquid scintillation; IC50 for JAK3 was calculated as ~10 μM [2]
- EGFR kinase activity assay (HTRF-based): 1. Purified human EGFR kinase domain (0.1 μg/mL) was incubated with biotinylated EGFR peptide (Y1173 motif, 1 μg/mL) and ATP (10 μM) in buffer (50 mM Tris-HCl pH 7.5, 5 mM MgCl₂) at 37°C for 15 min. 2. AG-490 (Tyrphostin B42; zinc0255794) (10–200 μM) was added, incubation extended 30 min. 3. Anti-phosphotyrosine cryptate antibody and streptavidin-europium were added; time-resolved fluorescence (665 nm/620 nm ratio) was measured. 4. IC50 for EGFR was calculated as ~50 μM [1]

A431 cell proliferation assay (MTT): 1. A431 cells (5×10³ cells/well) were seeded in 96-well plates, incubated overnight (37°C, 5% CO₂). 2. AG-490 (Tyrphostin B42; zinc0255794) (10/25/50/100 μM) or combination with PD153035 (100 nM) was added (3 replicates/group). 3. After 72 h, MTT (5 mg/mL, 10 μL/well) was added, incubated 4 h; formazan was dissolved in DMSO, absorbance at 570 nm measured to calculate viability [1]
- CD4+ T cell proliferation and signaling assay: 1. Human CD4+ T cells were isolated from PBMCs, adjusted to 1×10⁶ cells/mL, and stimulated with IL-2 (10 ng/mL). 2. AG-490 (Tyrphostin B42; zinc0255794) (5/10/25/50 μM) was added, cultured 48 h. 3. Proliferation: 3H-TdR (1 μCi/well) was added for 16 h, radioactivity counted via scintillation. 4. Signaling: Cells were lysed in RIPA buffer; 30 μg protein was analyzed for p-JAK3/p-STAT5/p-ERK1/2 via western blot [2]
- Islet cell apoptosis assay: 1. NOD mouse islets (10 islets/well) were seeded in 24-well plates, pre-treated with AG-490 (Tyrphostin B42; zinc0255794) (20 μM) for 1 h. 2. Cytokine mix (IL-1β+IFN-γ+TNF-α, 10 ng/mL each) was added, cultured 48 h. 3. Apoptosis was detected via Annexin V-FITC/PI staining (flow cytometry); insulin in supernatant was measured via ELISA [3]

Dissolved in DMSO; 0.85 mg to 0.5 mg daily; Continuous pump infusion and i.p. injection SCID mice intravenously injected with ALL cells

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NOD mouse type 1 diabetes protocol: 1. Female NOD mice (n=10/group) were assigned to control (0.1% DMSO-saline, i.p. daily), prevention (AG-490 from week 6), or treatment (AG-490 from week 12) groups. 2. AG-490 (Tyrphostin B42; zinc0255794) was prepared in 0.1% DMSO-saline, dosed at 50 mg/kg i.p. daily (prevention: 8 weeks; treatment: 4 weeks). 3. Tail vein blood glucose was measured weekly; diabetes was defined as glucose >11.1 mmol/L. 4. Mice were euthanized; pancreata were fixed in 4% formalin, paraffin-embedded, HE-stained, and scored for insulitis (0–4) [3]
- Rat inflammatory pain protocol: 1. Male SD rats (n=6/group) were grouped: normal, carrageenan+saline (control), carrageenan+AG-490. 2. Pain was induced by right hind paw injection of 1% carrageenan (0.1 mL). 3. Two hours post-induction, AG-490 (Tyrphostin B42; zinc0255794) (10 μg in 10 μL saline) was administered intrathecally; control received 10 μL saline. 4. HWL (52°C hot plate) and MWT (von Frey filaments) were measured pre-dose and at 0.5/1/2/4 h post-dose [4]

Metabolism / Metabolites
Tyrosine kinase inhibitors B42: Known human metabolites include tyrosine kinase inhibitors B42, 4-O-glucuronide, and tyrosine kinase inhibitors B42, 3-O-glucuronide.

In vivo toxicity in NOD mice: AG-490 (tyrosine kinase inhibitor B42; zinc 0255794) (50 mg/kg, intraperitoneal injection, 8 weeks) did not cause significant weight loss (<5% vs. control group). Serum ALT (23±4 U/L vs. 25±3 U/L control group), AST (42±6 U/L vs. 45±5 U/L control group) and creatinine (0.48±0.1 mg/dL vs. 0.5±0.1 mg/dL control group) were all within the normal range. No histopathological changes were observed in the pancreas, liver and kidneys [3] - In vivo toxicity in rats: Intrathecal injection of AG-490 (tyrosine kinase inhibitor B42; zinc 0255794) (10 μg) did not cause neurotoxicity (e.g., seizures, ataxia) within 48 hours. No changes were observed in body weight or serum liver and kidney function [4]
- In vitro safety of normal cells: Human peripheral blood mononuclear cells (PBMCs) treated with AG-490 (tyrosine kinase inhibitor B42; zinc 0255794) (≤50 μM) for 72 hours showed >85% survival (MTT assay) [2]

[1]. Combined inhibition of epidermal growth factor receptor and JAK/STAT pathways results in greater growth inhibition in vitro than single agent therapy. Mol Cancer Ther. 2004 Apr;3(4):459-63.

[2]. JAK3, STAT, and MAPK signaling pathways as novel molecular targets for the tyrphostin AG-490 regulation ofIL-2-mediated T cell response. J Immunol. 1999 Apr 1;162(7):3897-904.

[3]. The tyrphostin agent AG490 prevents and reverses type 1 diabetes in NOD mice. PLoS One. 2012;7(5):e36079.

[4]. Anti-hyperalgesic effects of AG490, a Janus kinase inhibitor, in a rat model of inflammatory pain. Biomed Rep. 2015 Sep;3(5):703-706.

Tyrphostin B42 is a monocarboxylic acid amide formed by the condensation of the carboxyl group of (2E)-2-cyano-3-(3,4-dihydroxyphenyl)prop-2-enoic acid with the amino group of benzylamine. It possesses a variety of activities, including as an EC 2.7.10.2 (non-specific protein tyrosine kinase) inhibitor, antioxidant, STAT3 inhibitor, anti-inflammatory agent, apoptosis inducer, and anti-aging agent. It is an enamide, monocarboxylic acid amide, nitrile compound, catechol compound, and secondary amide. Tyrphostin B42 belongs to the tyrosine kinase inhibitor family and can inhibit epidermal growth factor receptors, blocking the growth of leukemia cells in vitro and in vivo by inducing programmed cell death. It also inhibits constitutive activation of STAT-3 DNA binding and IL-2-induced growth of mycosis fungoides tumor cells. (NCI)

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Mechanism of action: AG-490 (tyrosine kinase inhibitor B42; zinc0255794) competitively binds to the ATP-binding pocket of JAK3/EGFR/MAPK, inhibiting its kinase activity. This blocks the downstream JAK/STAT (IL-2-driven T cell activation) and EGFR/MAPK (tumor cell proliferation) pathways and reduces pro-inflammatory cytokines (IL-6) in pain models [1,2,3,4]
-Therapeutic potential: Preclinical data support its use in EGFR-positive cancers (synergistic with EGFR inhibitors), type 1 diabetes (islet protection), and inflammatory pain (anti-hyperalgesia) [1,3,4]
-Limitations: Limited efficacy as a monotherapy against cancer; EGFR inhibition requires high concentrations (≥50 μM), which limits its clinical translation in cancer monotherapy [1]

C17H14N2O3

294.30

294.1

133550-30-8

(E/Z)-AG490;134036-52-5

5328779

Light yellow to yellow solid powder

1.3±0.1 g/cm3

615.2±55.0 °C at 760 mmHg

215°C(lit.)

325.9±31.5 °C

0.0±1.8 mmHg at 25°C

1.679

2.11

3

4

4

22

460

0

C1=CC=C(C=C1)CNC(=O)/C(=C/C2=CC(=C(C=C2)O)O)/C#N

TUCIOBMMDDOEMM-RIYZIHGNSA-N

InChI=1S/C17H14N2O3/c18-10-14(8-13-6-7-15(20)16(21)9-13)17(22)19-11-12-4-2-1-3-5-12/h1-9,20-21H,11H2,(H,19,22)/b14-8+

(E)-N-benzyl-2-cyano-3-(3,4-dihydroxyphenyl)acrylamide

Zinc-0255794; Tyrphostin AG490; Zinc0255794; Zinc 0255794; Tyrphostin AG-490; AG 490; AG-490; AG490; Tyrphostin AG 490

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.07 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.

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Solubility in Formulation 2: 2.08 mg/mL (7.07 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 20.8 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.

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Solubility in Formulation 3: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)