 Chemical Structure.png)
Physicochemical Properties
| Molecular Formula | C13H7CLF2INO2 |
| Molecular Weight | 409.55 |
| Exact Mass | 408.918 |
| Elemental Analysis | C, 38.13; H, 1.72; Cl, 8.66; F, 9.28; I, 30.99; N, 3.42; O, 7.81 |
| CAS # | 303175-44-2 |
| PubChem CID | 10112191 |
| Appearance | Off-white to gray solid powder |
| LogP | 4.737 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 5 |
| Rotatable Bond Count | 3 |
| Heavy Atom Count | 20 |
| Complexity | 363 |
| Defined Atom Stereocenter Count | 0 |
| InChi Key | XCNBGWKQXRQKSA-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C13H7ClF2INO2/c14-8-5-6(17)1-4-10(8)18-12-7(13(19)20)2-3-9(15)11(12)16/h1-5,18H,(H,19,20) |
| Chemical Name | 2-(2-chloro-4-iodoanilino)-3,4-difluorobenzoic acid |
| Synonyms | zapnometinib; 2-(2-chloro-4-iodophenylamino)-3,4-difluorobenzoic acid; ATR-002; Zapnometinib [INN]; PD-0184264; Benzoic acid, 2-[(2-chloro-4-iodophenyl)amino]-3,4-difluoro-; 4RZD8LK83V; |
| 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 Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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 | MEK (IC50 = 5.7 nM) |
| ln Vitro | In A549, MDCK cells, and human PBMC, zapnometinib (0.1 nM-1 μM) suppresses MEK with IC50s of 30.96 nM, 357 nM, and 15 nM, respectively[1]. In human PBMC, zapnometinib (100 μM; 4 hours) suppresses the phosphorylation of ERK1/2 caused by ionomycin (PMA/I) [1]. H3N2 and IV H1N1pdm09 virus titers are decreased by zapnometinib (1-100 μM) [1]. |
| ln Vivo | Zapnometinib (8.4–75 mg/kg/day; orally administered three times daily) decreases lung virus titers and increases mice survival following a fatal H1N1pdm09 infection [1]. Zapnometinib (150 mg/kg) administered intravenously or orally to mice resulted in AUC values of 860.02 and 1953.68 μg?h/mL, respectively [1]. |
| Enzyme Assay |
Cell free kinase assay: [1] A 96-well microtiter plate was coated overnight with the monoclonal anti-ERK (extracellular signal-regulated kinase) antibody in a dilution 1:1000 (100 μl/well) in TBST (50 mM Tris-HCl, 0.9% NaCl, 0.05% Tween 20). After washing (3 × 5 min) with 100 μl/well TBST, wells were blocked with 100 μl/well of 1% BSA in PBS for 60 min at RT. Prior to the addition of the kinase reaction sample (see below), plates were washed again. To determine the IC50 value of the inhibitors, the final test concentrations (1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, and 0.1 nM in DMSO) were prepared. Active GST c-Raf1-DD was expressed in Baculovirus infected SF9 cells, while GST-MEK1 and His-ERK2 were expressed in Escherichia coli. GST-tagged proteins were purified via binding to glutathione sepharose beads and His-tagged ERK2 was purified on a Ni-chelate column. The three purified recombinant kinases were mixed in a 3:2:3 ratio in kinase buffer (10 mM MgCl2, 25 mM beta-glycerophosphate, 25 mM HEPES; pH 7.5, 5 mM benzamidine, 0.5 mM dithiothreitol, 1 mM sodium vanadate) and DMSO/inhibitor and was pre-incubated in a dark at RT followed by the addition of 10 mM ATP and further incubation for 30 min. To stop the kinase reaction, a 20% SDS solution was added for 10 min at 50°C and further diluted with blocking buffer (1% BSA in TBST). Next, 100 μl of each sample was added to the anti-ERK coated 96-well microtiter plate. The kinase reaction samples were incubated for 60 min at RT in the anti-ERK coated 96-well microtiter plate blocked with BSA. For the detection of phosphorylated ERK, the samples were incubated overnight at 4°C with an anti-phospho ERK (p44/p42) antibody followed by Horseradish Peroxidase anti-mouse IgG specific antibody diluted in TBST buffer for 60 min at RT. Afterwards, the plate was washed and incubated for 30 min with ABTS substrate solution. The reaction was terminated with a 20% SDS solution followed by measurement of the Optical Density at 405 nm with an ELISA reader. Kinase assays in A549, MDCK cells and human PBMCs: [1] A549 and MDCK II cells were cultured in IMDM containing 10% FBS (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.), 100U/mL Penicillin, 100 µl/mL Streptomycin and 1% L-Glutamine. Next, 4×105 A549 or MDCK II cells were seeded in a 24-well plate overnight and then starved for 24 hrs in IMDM containing 0% FBS. After starvation the cells were stimulated with 20 ng/ml tumor necrosis factor-(TNF) for 30 minutes and then treated with different concentrations of either CI-1040 or Zapnometinib (PD0184264; ATR-002) dissolved in DMSO, for 1 h. To determine the IC50 value for CI-1040 and Zapnometinib (PD0184264; ATR-002) on MDCK II cells, 12 test concentrations in two-fold dilution steps were used (CI-1040: 6.25 μM, to 3 nM; Zapnometinib (PD0184264; ATR-002) : 25 µM to 12 nM. To determine the IC50 value for CI-1040 and Zapnometinib (PD0184264; ATR-002) on A549 cells, 12 test concentrations in two-fold dilution steps were prepared (CI-1040: 3.07 μM to 1.5 nM; Zapnometinib (PD0184264; ATR-002) : 30.72 µM to 15 nM). Treatments were stopped immediately after the incubation time by washing the cells twice with ice-cold PBS. Cells were lysed on ice in ice-cold modified-RIPA buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl) supplemented with Complete Protease Inhibitor Cocktails and Phosphatase Inhibitor Cocktails. The lysates were sonicated briefly and centrifuged at 18,000 × g for 5 minutes at 4°C. Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood by Ficoll density gradient separation and then cultured in RPMI 1640-medium containing 10% autologous serum, 100 U/mL Penicillin, 100 µg/mLStreptomycin and 1% L-Glutamine. Then 5x106 of the cultured PBMCs were seeded in a 24-well plate and stimulated with 100 ng/mL Phorbol 12-myristate 13-acetate (PMA) and 1 µg/mL ionomycin (I) for 4 hrs. To determine the IC50 value for CI-1040 and Zapnometinib (PD0184264; ATR-002) on human PBMCs, 12 test concentrations in two-fold dilution steps were prepared (CI-1040: 200 nM to 0.097 nM; Zapnometinib (PD0184264; ATR-002) : 780 nM to 0.38 nM). Treatments were stopped and procedure was continued as described for A549/MDCK cells. All samples were kept for western blot analysis. |
| Cell Assay |
Western Blot Analysis[1] Cell Types: human PBMCs Tested Concentrations: 100 μM Incubation Duration: 4 h Experimental Results: Inhibited the Ionomycin (PMA/I )-increased pERK1/2. Progeny virus inhibition assay[1] A549 cells were inoculated with the aforementioned IAV strains at the respective MOI prepared in phosphate buffered saline (PBS) supplemented with 0.2% (w/v) bovine serum albumin, 1 mM MgCl2, 0.5 mM CaCl2, 100 U/mL penicillin, 100 μg/mL streptomycin for 45 min at 37 °C and 5% CO2. Inocula were removed, cells were rinsed with PBS, and supplemented with 1 mL IV infection media (Dulbecco's Modified Eagle Medium; DMEM supplemented with 0.2% BSA, 1 mM MgCl2, 0.5 mM CaCl2, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 μg/mL TPCK-treated Trypsin, containing different concentrations of either CI-1040 or ATR-002 (100 μM, 50 μM, 10 μM, 5 μM, 1 μM, 0.5 μM, and 0.1 μM, final DMSO concentration 1% for 24 h at 37 °C in 5% CO2). Cell viability assay (WST-Assay)[1] A549 cells, MDCK cells and human PBMCs were seeded in a 96-well flat-bottom tissue plate and were grown over-night as described in section 2.2. Thereafter, cells were treated with different concentrations of ATR-002 (100 μM, 50 μM, 10 μM, 5 μM, 1 μM, 0.5 μM, and 0.1 μM) dissolved in 100 μl IMDM supplemented with 5% FBS with a final DMSO concentration of 1% (v/v). Cells were cultured at 37 °C with 5% CO2 for 24 h. Thereafter, 10 μl WST-1 reagent was added to the culture medium and incubated for 4 h. Thereafter, the formazan dye formed was quantified on an ELISA plate reader at 405 nm. |
| Animal Protocol |
Animal/Disease Models: Female C57BL/6 mice (8 weeks; 21-24 g) were infected with H1N1pdm09[1] Doses: 8.4, 25, 75 mg/ kg/day (2.8, 8.4, 25 mg/kg) Route of Administration: Po three times a day Experimental Results: Dramatically diminished the virus titer at the dose of either 75 mg/kg/day or 25 mg/kg/day. Preparation of compounds for pharmacokinetic analysis[1] For i.v. administration, 30.65 mg CI-1040 was dissolved in 0.075 mL DMSO and further diluted with 0.225 mL Cremophor EL and 2.7 mL PBS. Next, 34.88 mg ATR-002 was dissolved in 0.075 mL DMSO and further diluted with 0.225 mL Cremophor EL and 2.7 mL PBS. For oral administration 202.5 mg CI-1040 was dissolved in 0.5 mL DMSO, 1.5 mL Cremophor EL, and 8.0 mL PBS. Accordingly, 81.0 mg Zapnometinib (PD0184264; ATR-002) was dissolved in 0.2 mL DMSO, 0.6 mL Cremophor EL, and 3.2 mL PBS. Preparation of compounds to investigate virus titer reduction in the lungs of mice[1] For oral application of 25 mg Zapnometinib (PD0184264; ATR-002) /kg bodyweight, 10 mg of ATR-002 was dissolved in 50 μl DMSO and further diluted with 0.15 mL Cremophor EL and 0.8 mL PBS. For application of 8.4 mg/kg and 2.8 mg/kg, 3.36 mg or 1.12 mg ATR-002 was dissolved in 50 μl DMSO and further diluted with 0.15 mL Cremophor EL and 0.8 mL PBS. Additionally, 202.5 mg CI-1040 was dissolved in 0.5 mL DMSO, 0.15 mL Cremophor EL, and 0.8 mL PBS. |
| References |
[1]. Antiviral efficacy against influenza virus and pharmacokinetic analysis of a novel MEK-inhibitor, ATR-002, in cell culture and in the mouse model. Antiviral Res. 2020 Jun;178:104806. [2]. Improved in vitro Efficacy of Baloxavir Marboxil Against Influenza A Virus Infection by Combination Treatment With the MEK Inhibitor ATR-002. Front Microbiol. 2021 Feb 12;12:611958. [3]. Metabolic conversion of CI-1040 turns a cellular MEK-inhibitor into an antibacterial compound. Sci Rep. 2018 Jun 14;8(1):9114. |
| Additional Infomation | Zapnometinib is an orally bioavailable, small molecule inhibitor of mitogen-activated protein kinase kinase (MAP2K; MAPK/ERK kinase; MEK), with potential anti-viral and anti-inflammatory activities. Upon oral administration, zapnometinib selectively binds to and inhibits the activity of MEK. This may prevent the export of the viral genome protein complexes from the nucleus to the cytoplasm, thereby inhibiting the formation of new viral particles and the replication of various RNA viruses, including influenza virus, hantavirus, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In addition, the inhibition of the activity of MEK may also decrease gene expression and inhibit the production of proinflammatory cytokines and chemokines, including tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1-beta, IL-8, C-X-C motif chemokine 10 (interferon gamma-induced protein 10; IP-10), C-C motif chemokine 2 (monocyte chemoattractant protein-1; MCP-1) and C-C motif chemokine 3 (macrophage inflammatory protein 1-alpha; MIP-1a), thereby reducing inflammation. MEK, a dual-specificity threonine/tyrosine kinase family, plays a key role in the activation of the RAS/RAF/MEK/ERK signaling pathway. Many RNA viruses rely on this pathway inside human cells to replicate. |
Solubility Data
| Solubility (In Vitro) | DMSO : 62.5 mg/mL (152.61 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.08 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 (5.08 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 | 2.4417 mL | 12.2085 mL | 24.4170 mL | |
| 5 mM | 0.4883 mL | 2.4417 mL | 4.8834 mL | |
| 10 mM | 0.2442 mL | 1.2209 mL | 2.4417 mL |