Structure-based design and synthesis of pyrrole derivatives as MEK inhibitors

A novel series of pyrrole inhibitors of MEK kinase has been developed using structure-based drug design. Optimization of the series led to the identification of potent inhibitors with good pharmaceutical properties.     

Identification of isothiazole-4-carboxamidines derivatives as a novel class of allosteric MEK1 inhibitors

The development of potent, orally bioavailable, and selective series of 5-amino-3-hydroxy-N(1-hydroxypropane-2-yl)isothiazole-4-carboxamidine inhibitors of MEK1 and MEK-2 kinase is described. Optimization of the carboxamidine and the phenoxyaniline group led to the identification of 55 which gave good potency as in vitro MEK1 inhibitors, and good oral exposure in rat.     

MEK (MAPKK) inhibitors. Part 2: structure-activity relationships of 4-anilino-3-cyano-6,7-dialkoxyquinolines

A series of 4-anilino-3-cyano-6,7-dialkoxyquinolines with different substituents attached to the 4-anilino group has been prepared that are potent MEK (MAP kinase kinase) inhibitors. The best activity is obtained when a phenyl or a thienyl group is attached to the para-position of the aniline through a hydrophobic linker, such as an oxygen, a sulfur, or a methylene group. The most active compounds show low nanomolar IC(50)'s against MEK (MAP kinase kinase), and have potent growth inhibitory activity in LoVo cells (human colon tumor line).     

Identification of coumarin derivatives as a novel class of allosteric MEK1 inhibitors

A homogenous TR-FRET-based in vitro coupling assay for the MAP3Ks–MEK1–ERK2 kinase cascade was established and was used to screen for inhibitors of the ERK/MAPK pathway. A series of coumarin derivatives were identified from the screen. These compounds potently inhibit the activation of the unactivated human MEK1 by upstream MAP3Ks (including BRAF and COT), but do not inhibit the activity of the activated MEK1. In addition, the potency of these compounds in inhibiting MEK1 activation is not affected by varying the ATP concentration, suggesting that these inhibitors are not competitive with ATP. As expected, the coumarin compounds potently inhibit LPS-induced TNF production and ERK phosphorylation in THP-1 cells, with the most potent compound having an IC₅₀ of 90 nM. Molecular modeling studies suggest that these coumarins bind to an allosteric site in the inactive conformation of MEK1. This site has been shown to be utilized by the biarylamine series of MEK inhibitors such as PD318088. Very interestingly, the identified coumarin derivatives are almost identical to a series of inhibitors recently reported that block LPS-induced TNF production. Our findings have therefore raised the possibility that other naturally occurring or synthetic coumarins with anti-cancer and anti-inflammatory activities might exert their biological function through the inhibition of MEK1.     

4-Anilino-3-cyanobenzo[g]quinolines as kinase inhibitors

A series of 4-anilino-3-cyanobenzo[g]quinolines was prepared as potent kinase inhibitors. Compared with their bicyclic 4-anilino-3-cyanoquinoline analogues, the tricyclic 4-anilino-3-cyanobenzo[g]quinolines are less active against EGF-R kinase, equally active against MAPK kinase (MEK), and more active against Src kinase. For Src kinase inhibition, the best activity is obtained when both the 7- and 8-positions are substituted with alkoxy groups. Several of these kinase inhibitors show potent growth inhibitory activity in tumor cells.     

Synthesis and structure-activity relationships of 3-cyano-4-(phenoxyanilino)quinolines as MEK (MAPKK) inhibitors

A series of 3-cyano-4-(phenoxyanilino)cyanoquinolines has been prepared as MEK (MAP kinase kinase) inhibitors. The best activity is seen with alkoxy groups at both the 6- and 7-positions. The lead compounds show low nanomolar IC₅₀'s against MAP kinase kinase, and have potent inhibitory activity in tumor cells.     

Discovery of EBI-1051: A novel and orally efficacious MEK inhibitor with benzofuran scaffold

A novel series of benzodihydrofuran derivatives was developed as potent MEK inhibitors through scaffold hopping based on known clinical compounds. Further SAR exploration and optimization led to another benzofuran series with good oral bioavailability in rats. One of the compounds EBI-1051 (28d) demonstrated excellent in vivo efficacy in colo-205 tumor xenograft models in mouse and is suitable for pre-clinical development studies for the treatment of melanoma and MEK associated cancers. Compared to AZD6244, EBI-1051 showed superior potency in some cancer cell lines such as colon-205, A549 and MDA-MB-231.     

The discovery of the benzhydroxamate MEK inhibitors CI-1040 and PD 0325901

A novel series of benzhydroxamate esters derived from their precursor anthranilic acids have been prepared and have been identified as potent MEK inhibitors. 2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide, CI-1040, was the first MEK inhibitor to demonstrate in vivo activity in preclinical animal models and subsequently became the first MEK inhibitor to enter clinical trial. CI-1040 suffered however from poor exposure due to its poor solubility and rapid clearance, and as a result, development of the compound was terminated. Optimization of the diphenylamine core and modification of the hydroxamate side chain for cell potency, solubility, and exposure with oral delivery resulted in the discovery of the clinical candidate N-(2,3-dihydroxy-propoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide PD 0325901.     

Blockade of the ERK pathway markedly sensitizes tumor cells to HDAC inhibitor-induced cell death

Constitutive activation of the extracellular signal-regulated kinase (ERK) pathway is associated with the neoplastic phenotype of a large number of human tumor cells. Although specific blockade of the ERK pathway by treating such tumor cells with potent mitogen-activated protein kinase/ERK kinase (MEK) inhibitors completely suppresses their proliferation, it by itself shows only a modest effect on the induction of apoptotic cell death. However, these MEK inhibitors markedly enhance the efficacy of histone deacetylase (HDAC) inhibitors to induce apoptotic cell death: such an enhanced cell death is observed only in tumor cells in which the ERK pathway is constitutively activated. Co-administration of MEK inhibitor markedly sensitizes tumor cells to HDAC inhibitor-induced generation of reactive oxygen species, which appears to mediate the enhanced cell death induced by the combination of these agents. These results suggest that the combination of MEK inhibitors and HDAC inhibitors provides an efficient chemotherapeutic strategy for the treatment of tumor cells in which the ERK pathway is constitutively activated.     

Structure-based design and synthesis of bicyclic fused-pyridines as MEK inhibitors

The MAPK pathway is identified as one of the most important pathways involved in cell proliferation and differentiation. A key kinase in the pathway, the Mitogen-activated protein kinase kinase (MEK) is recognized as a promising target for antitumor drugs. Structure-based design and optimization of known MEK inhibitors resulted in identification of compound 10a as a potent non-ATP competitive MEK inhibitor in both in vitro and in vivo tests.     

Discovery of 3-hydroxy-4-carboxyalkylamidino-5-arylamino-isothiazoles as potent MEK1 inhibitors

3-Hydroxy-4-carboxyalkylamidino-5-arylamino-isothiazoles were discovered as potent in vitro MEK1 inhibitors.     

Discovery of MEK/PI3K dual inhibitor via structure-based virtual screening

Mitogen/extracellular signal-regulated kinase (MEK) and phosphoinositide 3-kinase (PI3Kα) are considered to be promising targets for the development of anticancer therapeutics. We report the first example of the successful application of structure-based virtual screening to identify novel inhibitors of MEK with IC(50) values ranging from 1 to 25 μM. One of the four newly identified MEK inhibitors was found to be also a potent inhibitor of PI3Kα with submicromolar inhibitory activity (IC(50)=0.3 μM). Because this dual inhibitor was screened for having desirable physicochemical properties as a drug candidate as well as the high inhibitory activities against MEK and PI3Kα, it warrants further development through structure-activity relationship (SAR) studies to optimize the inhibitory and anticancer activities. Structural features relevant to the stabilization of the dual inhibitor in the ATP-binding sites of MEK1 and PI3Kα are addressed in detail.     

Thermodynamics of nucleotide and non-ATP-competitive inhibitor binding to MEK1 by circular dichroism and isothermal titration calorimetry

MEK1 is a member of the MAPK signal transduction pathway that responds to growth factors and cytokines. A wealth of information about the enzymatic activity of MEK1, its domain functions, and inhibitor action is available; however, the thermodynamic properties of the interaction between MEK1 and ligands, such as nucleotides and non-ATP-competitive inhibitors, have not been reported. This study describes the thermodynamic parameters for the binding interactions of MEK1, nucleotides, and non-ATP-competitive inhibitor complexes using temperature-dependent circular dichroism (TdCD) and isothermal titration calorimetry (ITC). Non-phosphorylated MEK1 (npMEK1) has a high affinity for both AMP-PNP and ADP (Kd approximately 2microM). The binding is enthalpically favored and Mg-dependent. The active, phosphorylated form of MEK1 (pMEK1) bound nucleotides with a similar high affinity (Kd approximately 2muM) and had a thermodynamic profile and Mg-dependence similar to that of the non-phosphorylated form. The non-ATP-competitive MEK1 inhibitors, U0126 and PD0325901, showed no preference for npMEK1 and pMEK1 by TdCD. TdCD results also showed that these inhibitors are more potent in the presence of the nucleotide than in its absence. The ternary complex, MEK1.PD0325901.nucleotide, showed synergistic binding as evidenced by a large, non-additive shift in the midpoint of the protein unfolding transition (Tm). This was apparent for both npMEK1 and pMEK1 using either ADP or AMP-PNP. ITC binding studies confirmed the synergistic binding effect. The ITC-determined affinity of nucleotide (AMP-PNP, ADP) binding to the npMEK1.PD0325901 complex was enhanced nearly 5-fold compared to nucleotide binding to npMEK1 alone. In addition, the affinity of PD0325901 binding to npMEK1.nucleotide complexes was increased nearly 10-fold relative to the affinity of PD0325901 for npMEK1 alone. These are the first thermodynamic binding studies that characterize the affinity of the allosteric non-ATP-competitive inhibitors U0126 and PD0325901 with and without the nucleotide. The results indicate these allosteric inhibitors have a dynamic range in the type of MEK1 activation states and nucleotide complexes that they can bind.     

Enzyme kinetics and binding studies on inhibitors of MEK protein kinase

Inhibition of the protein kinase, MEK1, is a potential approach for the treatment of cancer. Inhibitors may act by prevention of activation (PoA), which involves interfering with phosphorylation of nonactivated MEK1 by the upstream kinase, B-RAF. Modulation also may occur by inhibition of catalysis (IoC) during phosphorylation of the downstream substrate, ERK2, by activated MEK1. Here, five MEK inhibitors are characterized in terms of binding affinity, PoA, and IoC. The compounds are a butadiene (U-0126), an N-alkoxy amide (CI-1040), two CI-1040 analogues (an anthranilic acid and an N-alkyl amide), and a cyanoquinoline. Some compounds give different mechanisms of inhibition (ATP-competitive, noncompetitive, or uncompetitive) in PoA compared to IoC or show a change in potency between the assays. The inhibitors also exhibit different shifts in potency when either PoA or IoC is compared with binding to nonactivated MEK. The inhibitor potency ranking, therefore, is dependent upon the assay format. When the ATP concentration equals K m, IoC IC 50 increases in the order CI-1040 approximately cyanoquinoline < anthranilic acid approximately U-0126 < alkyl amide. Conversely, the K d from nonactivated MEK1 for four of the compounds varies between more than 6-fold lower and over 18-fold higher than this IC 50, with U-0126 having the lowest K d and CI-1040 having the highest. In PoA when the ATP concentration equals K m, U-0126 has the lowest IC 50, becoming more potent than CI-1040, the cyanoquinoline, and the anthranilic acid. These observations have implications for understanding structure-activity relationships of MEK inhibitors and illustrate how assays can be designed to favor different compounds.     

Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo.

The mitogen-activated protein kinase pathway is thought to be essential in cellular growth and differentiation. Here we report the discovery of a highly potent and selective inhibitor of the upstream kinase MEK that is orally active. Tumor growth was inhibited as much as 80% in mice with colon carcinomas of both mouse and human origin after treatment with this inhibitor. Efficacy was achieved with a wide range of doses with no signs of toxicity, and correlated with a reduction in the levels of activated mitogen-activated protein kinase in excised tumors. These data indicate that MEK inhibitors represent a promising, noncytotoxic approach to the clinical management of colon cancer.     

MEK inhibitors block AICAR-induced maturation in mouse oocytes by a MAPK-independent mechanism

The present study was carried out to assess the possible role of mitogen-activated protein kinase (MAPK) in the meiosis-inducing action of the AMP-activated protein kinase (AMPK) activator, 5-aminoimidazole-4-carboxamide 1-beta-ribofuranoside (AICAR). Cumulus cell-enclosed oocytes (CEO) or denuded oocytes (DO) from immature, eCG-primed mice were cultured 4 hr in Eagle's minimum essential medium containing dbcAMP plus increasing concentrations of AICAR or okadaic acid (OA). OA is a phosphatase inhibitor known to stimulate both meiotic maturation and MAPK activation and served as a positive control. Both OA and AICAR were potent inducers of meiotic resumption in mouse oocytes and brought about the phosphorylation (and thus, activation) of MAPK, but by different kinetics: MAPK phosphorylation preceded GVB in OA-treated oocytes, while that resulting from AICAR treatment appeared only after GVB. The MEK inhibitors, PD98059 and U0126, blocked the meiotic resumption induced by AICAR but not that induced by OA. Although the MEK inhibitors suppressed MAPK phosphorylation in both OA- and AICAR-treated oocytes, meiotic resumption was not causally linked to MAPK phosphorylation in either group. Furthermore, AICAR-induced meiotic resumption in Mos-null oocytes (which are unable to stimulate MAPK) was also abrogated by PD98059 treatment. A non-specific effect of the MEK inhibitors on AICAR accessibility to the oocyte was discounted by showing that they failed to suppress either nucleoside uptake or AICAR-stimulated phosphorylation of acetyl CoA carboxylase (ACC), a substrate of AMPK. The suppression of AICAR-induced maturation by MEK inhibitors must, therefore, be occurring by actions unrelated to MEK stimulation of MAPK; consequently, it would be prudent to consider this possible non-specific action of the inhibitors when they are used to block MAPK activation in mouse oocytes.     

Pyrrolopyridazine MEK inhibitors

The synthesis and SAR of a series of pyrrolopyridazine MEK inhibitors are reported. Optimal activity was achieved by incorporation of a 4-phenoxyaniline substituent at C4 and an acylated amine at C6.     

Fused thiophene derivatives as MEK inhibitors

A number of novel fused thiophene derivatives have been prepared and identified as potent inhibitors of MEK. The SAR data of selected examples and the in vivo profiling of compound 13 h demonstrates the functional activity of this class of compounds in HT-29 PK/PD models.