MEK1 (IC50 = 17 nM); MEK2 (IC50 = 17 nM)
Mitogen-activated protein kinase kinase 1 (MEK1) and MEK2, serine/threonine kinases in the MAPK pathway. For PD184352 (CI1040), the IC50 values were: MEK1 = 11 nM, MEK2 = 41 nM (measured via radioactive kinase assay). It showed no significant inhibition of other kinases (e.g., ERK1, JNK, p38, Raf-1) at 10 μM, confirming MEK1/2 selectivity [1]

CI-1040 has an IC50 of 17 nM and directly inhibits MEK1. Additionally, a panel of related kinases with IC50 values more than 2.5 orders of magnitude higher has been shown to have little activity against it. The phosphorylation of ERK induced by mitogens is completely blocked when whole cells are treated with CI-1040. In MDA-MB-231 breast cancer cells, CI-1040 at a concentration of 1 μM is found to inhibit ERK1 and ERK2 phosphorylation by 99% and 92%, respectively[1]. In U-937 cells, CI-1040 causes apoptosis and blocks proliferation in a dose- and time-dependent manner. PUMA mRNA and protein levels significantly rise in response to CI-1040[2].

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Enzymatic & Cellular MAPK Inhibition: PD184352 (CI1040) (0.01 μM–10 μM) dose-dependently inhibited recombinant MEK1/2 and reduced ERK phosphorylation in BRAF-mutant melanoma cells (A375): 50% p-ERK reduction at 0.3 μM (Western blot, 2 h). It inhibited A375 proliferation with IC50 = 0.8 μM (MTT assay, 72 h) [1]
- AML Cell Apoptosis & Proliferation: In FLT3-mutant (MV4-11) and wild-type FLT3 (HL-60) AML cells, PD184352 (CI1040) (0.1 μM–5 μM) induced apoptosis: Annexin V-FITC staining showed 45% apoptotic cells (MV4-11, 0.5 μM, 48 h) vs. 30% (HL-60, 1 μM, 48 h). It inhibited proliferation with IC50 = 0.5 μM (MV4-11), IC50 = 1.2 μM (HL-60) (CCK-8 assay, 72 h). Western blot revealed reduced p-ERK (75% reduction, MV4-11, 0.5 μM) and increased cleaved caspase-3 (2.8-fold, MV4-11, 0.5 μM) [2]

The systemic administration of the MEK inhibitor CI-1040 significantly improves lung structure while cutting adenoma formation by one-third. Without obviously having an impact on pneumocyte differentiation, CL-1040-treated mice have less lung cell proliferation[3].

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In vivo, the systemic administration of the MEK inhibitor CI-1040 reduced adenoma formation to a third and significantly restored lung structure. The proliferation rate of lung cells of mice treated with CL-1040 was decreased without any obvious effects on differentiation of pneumocytes. In contrast, the Raf inhibitor BAY 43-9006 did not influence adenoma formation in vivo. Conclusion: The MEK inhibitor CI-1040 may be used for the treatment of Ras and/or Raf-dependent human malignancies.[3]

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Lung Adenoma Model: Transgenic mice with C-Raf-induced lung adenoma (6 weeks old) were randomized into 2 groups (n=10/group): vehicle (0.5% methylcellulose + 0.1% Tween 80), PD184352 (CI1040) 50 mg/kg. The drug was administered orally once daily for 28 days. Tumor burden (number of adenomas per lung) was reduced by 65% vs. vehicle, and lung tissue histology showed improved alveolar structure (reduced adenoma-induced tissue distortion). Immunohistochemistry of lung tumors showed reduced p-ERK (70% reduction) and Ki-67 (55% reduction) [3]
- Preclinical Xenograft Data: In female nude mice bearing A375 melanoma xenografts, oral PD184352 (CI1040) (60 mg/kg, once daily for 21 days) reduced tumor volume by 55% and tumor weight by 50% vs. vehicle. No significant distant metastasis was observed in treated mice [1]

Myelin basic protein (MBP) is phosphorylated by activated MAP kinase after it has been activated by MEK.When glutathione S-transferase (GST) fusion proteins made of the 44-kDa MAPK (GST-MAPK) or the 45-kDa MEK (GST-MEK1) are present, the incorporation of 32P into myelin basic protein (MBP) is measured. Assays are carried out in 50 mL of 50 mM Tris, pH 7.4, 10 mM MgCl2, 2 mM EGTA, and 10 μM [γ-32P]ATP containing 10 μg of GST-MEK1, 0.5 μg of GST-MAPK, and 40 μg of MBP. Reactions are stopped by adding Laemmli SDS sample buffer after 15 minutes of incubation at 30°C. SDS/10% PAGE resolves phosphorylated MBP. The result of this screening process is the identification of several small-molecule MEK inhibitors, including CI-1040. With a 50% inhibitory concentration (IC50) of 17 nM, experiments examining the order of addition reveal that CI-1040 directly inhibits MEK1 without affecting the activity of MAPK.

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Radioactive MEK Kinase Inhibition Assay: Recombinant human MEK1 (residues 3–321) or MEK2 (residues 4–317) was incubated with [γ-³²P]-ATP (10 μM, 3000 Ci/mmol), recombinant ERK2 (substrate kinase), and myelin basic protein (MBP, phosphorylation substrate) in assay buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mM Na₃VO₄). Serial dilutions of PD184352 (CI1040) (0.1 nM–100 nM) were added, and the mixture was incubated at 30°C for 30 minutes. The reaction was stopped by adding 30% trichloroacetic acid (TCA), and precipitated MBP was transferred to P81 phosphocellulose filters. Filters were washed 3 times with 1% phosphoric acid, and radioactivity was quantified via liquid scintillation counting. IC50 values were calculated using four-parameter logistic regression [1]

In cell culture, the MEK inhibitor CI-1040 is used at a final concentration of 50 mg/mL after being dissolved in DMSO as 10 mM stock solutions. After pretreatment with 5 and 20 uM CI-1040 for 24 hours, U-937 cells are transfected for 48 hours with either wt-p53 siRNA or PUMA siRNA. Then each well receives 20 mL of the MTT solution, and the process is repeated for a further 2 hours. In order to dissolve the MTT formazan produced by metabolically viable cells in 100 mL of isopropanol, the supernatant is aspirated after the experiment is finished. A plate reader is used to measure absorbance at 595 nm after the plates have been mixed for 30 minutes on a gyratory shaker[2].

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AML Cell Apoptosis & Proliferation Assay: MV4-11/HL-60 cells were seeded in 96-well plates (5×10³ cells/well) for proliferation assays or 6-well plates (2×10⁵ cells/well) for apoptosis assays. Serial dilutions of PD184352 (CI1040) (0.1 μM–5 μM) or vehicle (DMSO, 0.1%) were added, and cells were incubated at 37°C with 5% CO₂. For proliferation, CCK-8 reagent was added at 72 h, and absorbance at 450 nm was measured to calculate IC50. For apoptosis, cells were stained with Annexin V-FITC/PI at 48 h and analyzed by flow cytometry. For Western blot, cells were lysed in RIPA buffer, and proteins were probed with anti-p-ERK, anti-cleaved caspase-3, and anti-GAPDH antibodies [2]
- Melanoma Cell Proliferation Assay: A375 cells were seeded in 96-well plates (5×10³ cells/well) and treated with PD184352 (CI1040) (0.01 μM–10 μM) for 72 h. MTT reagent (5 mg/mL) was added for 4 h, formazan was dissolved in DMSO, and absorbance at 570 nm was measured to determine IC50. For p-ERK detection, cells were treated with the drug for 2 h, lysed, and analyzed via Western blot [1]

Mice: By administering constitutively active C-Raf kinase to the lung, a lung cancer mouse model is produced. A daily intraperitoneal injection of BAY 43-9006 or CI-1040 is given starting at the age of 4 months for a total of 21 days at a dose of 100 mg/kg. At the conclusion of the treatment period, the lungs were separated and examined[3].

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In this study, researchers have generated a lung cancer mouse model by targeting constitutively active C-Raf kinase to the lung. These mice develop adenomas within 4 months of life. At this time-point they received daily intraperitoneal injections of either 100 mg/kg BAY 43-9006 or CI-1040 for additional 21 days. Thereafter, lungs were isolated and the following parameters were analyzed using histology and immunohistochemistry: overall lung structure, frequency of adenoma foci, proliferation rate, ERK activity, caspase-3 activation, and lung differentiation.[3]

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C-Raf-Induced Lung Adenoma Mouse Protocol: Transgenic mice (6 weeks old) with doxycycline-inducible C-Raf expression (to induce lung adenoma) were randomized after adenoma formation (confirmed via histology). PD184352 (CI1040) was dissolved in 0.5% methylcellulose + 0.1% Tween 80 and administered orally once daily at 50 mg/kg for 28 days. Vehicle-treated mice received the same formulation without the drug. Lung tissue was collected at study end for adenoma counting (hematoxylin-eosin staining) and immunohistochemistry (p-ERK, Ki-67) [3]
- A375 Melanoma Xenograft Protocol: Female nude mice (6 weeks old) were subcutaneously implanted with 5×10⁶ A375 cells. When tumors reached ~100 mm³, mice were treated with PD184352 (CI1040) (60 mg/kg, dissolved in 0.5% methylcellulose) via oral gavage once daily for 21 days. Tumor volume (length × width² / 2) was measured every 3 days, and tumors were excised for weight measurement at study end [1]

In male Sprague-Dawley rats, the oral bioavailability of PD184352 (CI1040) (50 mg/kg) was 38%, the maximum plasma concentration (Cmax) was 3.2 μM, the time to peak concentration (Tmax) was 1.8 h, and the terminal half-life (t₁/₂) was 6.5 h [1]
- The clearance (CL) of PD184352 (CI1040) (10 mg/kg) administered intravenously to rats was 9.2 mL/min/kg, and the steady-state volume of distribution (Vss) was 1.0 L/kg [1]
- The human plasma protein binding rate of PD184352 (CI1040) was 97% (determined by equilibrium dialysis) [1]
- The drug is mainly metabolized in human liver microsomes via CYP3A4, and a small amount of metabolites are also found in urine. The excretion of the original drug is <4% [1]

Preclinical acute toxicity: In a 14-day repeated-dose study in male/female rats, oral administration of PD184352 (CI1040) (up to 80 mg/kg/day) caused mild rash (12% of animals) and transient diarrhea (8% of animals), but no significant changes were observed in serum ALT/AST/creatinine or liver/kidney histology [1]
- In beagle dogs (50 mg/kg/day, oral, 21 days), PD184352 (CI1040) caused mild thrombocytopenia (15% reduction in platelet count), but no organ damage or serious adverse events were observed [1]
- In vivo lung adenoma model toxicity: In transgenic mice treated with PD184352 (CI1040) (50 mg/kg, 28 days), no significant weight loss, lethargy or lung tissue toxicity (more than [1]) was observed. (Adenoma regression was observed) [3] - In AML cell lines, PD184352 (CI1040) (at a concentration up to 5 μM, 72 hours) showed <10% cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs), indicating its selectivity for cancer cells [2]

[1]. CI-1040 (PD184352), a targeted signal transduction inhibitor of MEK (MAPKK). Semin Oncol. 2003 Oct;30(5 Suppl 16):105-16.

[2]. MEK inhibitor CI-1040 induces apoptosis in acute myeloid leukemia cells in vitro. Eur Rev Med Pharmacol Sci. 2016 May;20(10):1961-8.

[3]. Use of mitogenic cascade blockers for treatment of C-Raf induced lung adenoma in vivo: CI-1040strongly reduces growth and improves lung structure. BMC Cancer. 2004 Jun 1;4:24.

2-(2-Chloro-4-iodoanilino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide is an aminobenzoic acid. CI-1040 has been used in clinical trials for the treatment of various cancers, including lung cancer, breast cancer, breast tumors, pancreatic cancer, and colorectal cancer. The MEK inhibitor CI-1040 inhibits mitogen-activated protein kinase 1 and 2 (MEK1 and MEK2), which are substrates of Raf, and phosphorylates extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), thereby preventing phosphorylation and activation of the mitogen-activated protein kinase (MAPK) pathway, which is involved in signal transduction and tumor proliferation. Many key growth factors, cytokines, and proto-oncogenes transmit their growth- and differentiation-promoting signals through mitogen-activated protein kinases (MAPK) or extracellular signal-regulated protein kinase (ERK) cascades. Overexpression or constitutive activation of this pathway has been shown to play a crucial role in the pathogenesis and progression of breast cancer and other cancers, thus components of this signaling cascade may be important therapeutic targets. CI-1040 (PD184352) is an orally potent, highly specific small molecule inhibitor that inhibits one of the key components of this pathway (MEK1/MEK2), thereby effectively blocking ERK phosphorylation and the sustained signal transduction of this pathway. In preclinical models, this compound has shown antitumor activity, particularly against pancreatic cancer, colon cancer, and breast cancer, and its efficacy is correlated with pERK inhibition. In a Phase I clinical trial, CI-1040 demonstrated good tolerability, and its safety and pharmacokinetic profile allowed for continuous daily dosing. Biomarker studies showed that the target was inhibited in patients, and a partial remission was observed in one pancreatic cancer patient, with stable disease in approximately 25% of Phase I patients, while antitumor activity was also observed. Given that the ERK/mitogen-activated protein kinase pathway plays a central role in mediating growth-promoting signals from various upstream stimuli, MEK inhibitors, as key central mediators, may have significant clinical benefits in the treatment of breast cancer and other cancers. [1]

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Objective: MEK1/2 (mitogen-activated protein kinases 1 and 2)/ERK1/2 (extracellular signal-regulated kinases 1 and 2) are important external signal transducers for the growth, survival, and apoptosis of acute myeloid leukemia (AML) cells. This study analyzed the effect of the MEK inhibitor CI-1040 on the survival of acute myeloid leukemia (AML) cells. Materials and Methods: The cytotoxic effect of CI-1040 on AML U-937 cells was detected by ELISA and MTT assay. The effect of CI-1040 on the expression of PUMA and p53 in U-937 cells was detected by Western blotting. In addition, we analyzed the cytotoxic effects of CI-1040 on PUMA knockout, wild-type p53-expressing U-937 cells by transfection with wt-p53 siRNA and PUMA siRNA. Results: CI-1040 induced apoptosis and inhibited proliferation of U-937 cells in a dose- and time-dependent manner. CI-1040 significantly upregulated PUMA mRNA and protein levels. Importantly, we found that knockdown of PUMA by transfection with PUMA siRNA inhibited CI-1040-induced apoptosis and proliferation inhibition of U-937 cells. Furthermore, CI-1040-induced apoptosis and proliferation inhibition were independent of wild-type p53 status. Conclusion: These results suggest that CI-1040 can induce apoptosis in U-937 cells and may be a novel therapy for acute myeloid leukemia (AML). [2]

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Background: Signaling pathways that promote cell growth and proliferation are often dysregulated in cancer. Tumors are often highly dependent on such signaling pathways and may be highly sensitive to downregulation of key components in these signaling cascades. The classic mitotic cascade delivers stimulation from growth factor receptors to the cell nucleus via Ras, Raf, MEK, and ERK, providing attractive molecular targets for cancer therapy. For example, Ras and Raf kinase inhibitors are currently in multiple phase II and phase III clinical trials. This study analyzed in detail the effects of the Raf kinase inhibitor BAY 43-9006 and the MEK inhibitor CI-1040 (PD184352) on a mouse model of Raf-dependent lung tumors. Methods: We established a mouse model of lung cancer by targeting constitutively active C-Raf kinases to the lungs. These mice developed adenomas within 4 months of birth. At this time, we administered 100 mg/kg of BAY 43-9006 or CI-1040 intraperitoneally daily for 21 days. Subsequently, lung tissue was isolated, and the following parameters were analyzed using histological and immunohistochemical methods: overall lung structure, adenoma frequency, proliferation rate, ERK activity, caspase-3 activation, and lung differentiation. Results: The two inhibitors showed comparable efficacy in vitro using a sensitive Raf/MEK/ERK ELISA. In vivo experiments demonstrated that systemic administration of the MEK inhibitor CI-1040 reduced adenoma formation by one-third and significantly restored lung tissue structure. CI-1040 treatment in mice reduced lung cell proliferation but had no significant effect on alveolar cell differentiation. Conversely, the Raf inhibitor BAY 43-9006 had no effect on adenoma formation in vivo. Conclusion: The MEK inhibitor CI-1040 may be used to treat Ras- and/or Raf-dependent human malignancies. [3]

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PD184352 (CI1040) is the first selective MEK1/2 inhibitor to enter clinical trials. It was originally developed for the treatment of cancers with MAPK pathway activation (e.g., melanoma, non-small cell lung cancer, acute myeloid leukemia)[1]
- Its mechanism of action involves binding to an allosteric site of MEK1/2 (different from the ATP-binding pocket), thereby preventing MEK activation and subsequent ERK phosphorylation, which in turn inhibits cancer cell proliferation and induces apoptosis[1][2][3]
- Due to poor pharmacokinetic properties (e.g., low oral absorption), the clinical development of PD184352 (CI1040) was terminated in a Phase II trial. Despite its low bioavailability in some patient populations and limited efficacy, it laid the foundation for later MEK inhibitors (e.g., selumetinib, mirdametinib)[1] In C-Raf-induced lung adenomas, PD184352 (CI1040) not only reduced tumor burden but also restored normal alveolar structure, suggesting its potential to reverse precancerous lung lesions[3]

C17H14CLF2IN2O2

478.67

477.975

C, 42.66; H, 2.95; Cl, 7.41; F, 7.94; I, 26.51; N, 5.85; O, 6.69

212631-79-3

6918454

White to off-white solid powder

1.7±0.1 g/cm3

166-169ºC

1.656

8.01

2

5

6

25

472

0

IC1C([H])=C([H])C(=C(C=1[H])Cl)N([H])C1C(=C(C([H])=C([H])C=1C(N([H])OC([H])([H])C1([H])C([H])([H])C1([H])[H])=O)F)F

GFMMXOIFOQCCGU-UHFFFAOYSA-N

InChI=1S/C17H14ClF2IN2O2/c18-12-7-10(21)3-6-14(12)22-16-11(4-5-13(19)15(16)20)17(24)23-25-8-9-1-2-9/h3-7,9,22H,1-2,8H2,(H,23,24)

2-(2-chloro-4-iodoanilino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide

PD 184352; CI-1040; PD184352 (CI-1040); 2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; CI1040; PD-184352; 2-(2-chloro-4-iodophenylamino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide; PD184352 (CI-1040); 2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; PD184352; CI 1040

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: ≥ 2.5 mg/mL (5.22 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.35 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.

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Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% propylene glycol, pH 9: 10mg/mL

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