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| 10mg |
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| 25mg |
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| Targets |
EGF receptor autophosphorylation (IC50 = 4 μM)
RG 13022 mainly targets and inhibits the tyrosine kinase activity of the EGFR. In cell-free assays, it inhibits EGFR autophosphorylation with an IC50 of 4 μM. In cellular assays, its IC50 against EGFR is approximately 5 μM. It can also affect downstream signaling pathways stimulated by other growth factors like IGF and TGF-α. |
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| ln Vitro |
G-13022 prevents the EGF-receptor in HER 14 cells from becoming autophosphorylated after an overnight incubation. RG-13022 has an IC50 of 5 μM. When HER 14 cells are stimulated by 50 ng/mL EGF, RG-13022 inhibits colony formation and DNA synthesis in a dose-dependent manner. For HER 14 colony formation, RG-13022's IC50 is 1 μM, while for HER 14 DNA synthesis, it is 3 μM. Additionally, EGF-stimulated MH-85 cells' colony formation and DNA synthesis are suppressed by RG-13022 in a dose-dependent manner. For MH-85 cells, the IC50 values of RG-13022 are 7 μM for colony formation and 1.5 μM for DNA synthesis.[1]
In vitro, RG 13022 effectively inhibits the proliferation of various cancer cells that overexpress EGFR. For example, it inhibits colony formation and DNA synthesis in EGF-stimulated cells like MH-85 and HER 14. Specifically, the IC50 for HER 14 colony formation is 1 μM, and for DNA synthesis is 3 μM. It can also block estrogen-induced cell proliferation signaling mediated by the EGF receptor. |
| ln Vivo |
RG-13022, at 400 μg/mouse/day, considerably suppresses the growth of MH-85 tumors. In comparison to untreated MH-85 tumor-bearing animals, animals receiving injections of RG-13022 exhibit less cachexia and hypercalcemia, eat more food, and exhibit greater levels of activity due to slower tumor growth. RG-13022 extends the life span of MH-85 tumor-bearing animals by inhibiting the growth of tumors. Significant inhibition of MH-85 growth in nude mice is observed upon administration of mAb108 i.p. (1 mg/mouse/day) at 1, 5, and 10 days following RG-13022 (400 μg/mouse/day) for 14 days as an MH-85 inoculation.[1]
In animal models, RG 13022 demonstrates in vivo anti-tumor activity. Studies show it can inhibit tumor growth in nude mice and prolong the lifespan of these tumor-bearing mice. However, one study noted that after intraperitoneal injection at a dose of 20 mg/kg, the drug is cleared very rapidly in vivo, with plasma concentrations dropping below 1 μM within 20 minutes post-injection, which may limit its sustained activity in vivo. |
| Enzyme Assay |
This assay is typically performed in a cell-free system. The procedure is as follows:
1. Immunoprecipitate the EGFR protein from cell lysates. 2. Incubate varying concentrations of RG 13022 with the immunoprecipitated EGFR protein in a reaction buffer containing ATP to induce EGFR autophosphorylation. 3. Separate proteins by SDS-PAGE and perform immunoblotting (Western Blot) using a specific anti-phosphotyrosine antibody. Compare the intensity of phosphorylation bands between drug-treated and control groups to calculate the inhibition rate of EGFR kinase activity, obtaining the IC50 value (i.e., 4 μM). |
| Cell Assay |
The following cell types are plated in complete medium: alpha MEM (100/well; 24-well plate) or HER 14 cells (200/dish; 10-cm dish), with 10% FCS added. The culture medium is changed to either DMEM supplemented with 0.5% PCS and 50 ng/mL EGF (HER14) or alpha MEM supplemented with 0.2% PCS and 50 ng/mL EGF (MH-85) after the overnight culture. For ten days, the cells are cultivated in this medium with or without increasing concentrations of RG-13022. After the culture is complete, the cells are fixed for 15 minutes at room temperature using 4% (v/v) formaldehyde in phosphate-buffered saline that is free of calcium and magnesium, and then they are stained with hematoxylin. Under a microscope, the number of colonies—which contain more than 20 cells in each well—is counted.
A protocol commonly used to evaluate the compound's effect on cancer cell proliferation, using HER 14 cells as an example: 1. Plate HER 14 cells in culture plates and allow them to attach overnight in medium containing 10% serum. 2. Remove the old medium and add fresh medium containing 0.5% serum and 50 ng/mL EGF (as a growth stimulant), along with different concentrations of RG 13022 (typically diluted from a 40 mM DMSO stock). Incubate for 10 days. 3. After incubation, fix the cells with 4% formaldehyde and stain with hematoxylin. 4. Count the number of colonies formed (typically counting colonies containing >20 cells) under a microscope and compare to the control group to calculate the inhibition rate of colony formation, thereby assessing the anti-proliferative activity. |
| Animal Protocol |
Mice: MH-85 tumors 5 mm in diameter are injected subcutaneously (s.c.) into the right dorsal region of male BALB/c nu/nu mice that are 4–6 weeks old. I.p. injections of RG-13022 or RG-14620 in 0.1 ml 100% DMSO are given twice a day starting one day following the inoculation of the MH-85 tumor. The vehicle is given to control animals. Under nembutal anesthesia (0.05 mg/g body weight, i.p.), tumor sizes are measured once a week and computed.
An example protocol to evaluate its anti-tumor efficacy in a mouse xenograft model: 1. Establish a xenograft tumor model by subcutaneously (s.c.) injecting tumor cells (e.g., MH-85 cells) into nude mice (e.g., BALB/c nu/nu). 2. Once the tumor reaches a certain size (e.g., approximately 5mm in diameter), start intraperitoneal (i.p.) injection administration. The typical dose used is 400 μg/mouse/day, though doses like 20 mg/kg have also been studied. 3. Measure tumor dimensions (length and width) regularly (e.g., weekly) to calculate tumor volume. Monitor and record body weight changes and survival status of the mice to evaluate the in vivo efficacy of the drug. |
| ADME/Pharmacokinetics |
RG 13022 exhibits pharmacokinetic properties characterized by rapid biexponential elimination. Key parameters include:
Elimination Half-Life: 50.4 minutes. Plasma Exposure: Following i.p. injection of 20 mg/kg in mouse models, plasma concentration drops rapidly below 1 micromole (μM) within 20 minutes. Solubility: The compound shows good solubility in DMSO (approximately 49-53 mg/mL). |
| Toxicity/Toxicokinetics |
Based on current preclinical data, RG 13022 shows relatively low cellular toxicity.
Cytotoxicity: In vitro, neither the drug substance nor its geometrical isomer, (E)-RG13022, displayed significant cytotoxicity. In Vivo Toxicity: No significant drug-related toxicity or lethality was observed in animal studies. While its rapid clearance limits sustained in vivo anti-tumor activity, this is considered a pharmacokinetic issue rather than a toxicity concern. Thus, researchers have indicated that improved formulations or dosing schedules are required to enhance its in vivo exposure. |
| References |
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| Additional Infomation |
3-(3,4-Dimethoxyphenyl)-2-(3-pyridyl)-2-acrylonitrile is a dimethoxybenzene. RG 13022 is a tyrosine kinase inhibitor that selectively inhibits epidermal growth factor receptor (EGFR), EGFR-stimulated HER14 cell proliferation, and tumor growth in vivo. (NCI)
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| Molecular Formula |
C16H14N2O2
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|---|---|
| Molecular Weight |
266.3
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| Exact Mass |
266.106
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| Elemental Analysis |
C, 72.17; H, 5.30; N, 10.52; O, 12.02
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| CAS # |
136831-48-6
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| Related CAS # |
136831-48-6
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| PubChem CID |
5468216
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| Appearance |
White to off-white solid powder
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| Density |
1.172g/cm3
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| Boiling Point |
423.8ºC at 760mmHg
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| Melting Point |
118 °C
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| Flash Point |
210.1ºC
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| Vapour Pressure |
2.18E-07mmHg at 25°C
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| Index of Refraction |
1.605
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| LogP |
3.162
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
20
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| Complexity |
385
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| Defined Atom Stereocenter Count |
0
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| SMILES |
N#C/C(C1=CC=CN=C1)=C/C2=CC=C(OC)C(OC)=C2
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| InChi Key |
DBGZNJVTHYFQJI-ZSOIEALJSA-N
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| InChi Code |
InChI=1S/C16H14N2O2/c1-19-15-6-5-12(9-16(15)20-2)8-14(10-17)13-4-3-7-18-11-13/h3-9,11H,1-2H3/b14-8-
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| Chemical Name |
(E)-3-(3,4-dimethoxyphenyl)-2-pyridin-3-ylprop-2-enenitrile
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| Synonyms |
RG13022; RG-13022; RG 13022; Tyrphostin; RG 13022
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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: ~100 mg/mL (~375.5 mM)
Ethanol: ~5 mg/mL (~18.8 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (9.39 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 (9.39 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 (9.39 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 | 3.7552 mL | 18.7758 mL | 37.5516 mL | |
| 5 mM | 0.7510 mL | 3.7552 mL | 7.5103 mL | |
| 10 mM | 0.3755 mL | 1.8776 mL | 3.7552 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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